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Molybdopterin-Modeling: The Synthesis of Pterin Dithiolene Ligands
I n a u g u r a l d i s s e r t a t i o n
zur
Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften (Dr. rer. nat.)
der
Mathematisch-Naturwissenschaftlichen Fakultät
der
Universität Greifswald vorgelegt von Ivan Trentin
Greifswald 15. April 2019
Dekan: Prof. Dr. Werner Weitschies 1. Gutachter : Prof. Dr. Carola Schulzke 2. Gutachter: Prof. Dr. Albrecht Berkessel Tag der Promotion: 15. April 2019
Table of contents 1. Pteridines and pterins ................................................................................................................................... 1
1.1 Historical background .............................................................................................................................. 1
1.2 Nomenclature .......................................................................................................................................... 5
1.3 Pteridines chemistry ................................................................................................................................ 6
1.3.1 Aromaticity, π-excessive and π-deficient .......................................................................................... 6
1.3.2 Stability of pteridines and pterins ..................................................................................................... 8
1.3.3 Covalent hydration reaction ............................................................................................................. 9
1.3.4 Reactivity of pteridine ..................................................................................................................... 11
2. Biological Background ................................................................................................................................ 15
2.1 Molybdenum dependent enzymes ......................................................................................................... 15
2.2 Molybdenum cofactor natural synthesis and maturation ..................................................................... 18
2.2.1 Cyclization of GTP ........................................................................................................................... 18
2.2.2 Sulfuration of cPMP ........................................................................................................................ 19
2.2.3 Molybdenum uptake ....................................................................................................................... 20
2.2.4 Insertion of the metal ..................................................................................................................... 21
2.2.5 Cofactor insertion into apoenzyme ................................................................................................. 22
2.3 Molybdenum cofactor and Human diseases-MoCD and ISOD .............................................................. 24
2.3.1 Synthetic cofactor, drug for a possible cure ................................................................................... 25
3. Results and discussion ................................................................................................................................ 26
3.1 Modelling molybdopterin (MPT)............................................................................................................ 26
3.2 Protection of Pterins .............................................................................................................................. 31
3.3 First attempt for the synthesis of pterin-dithiolene ligand, preparation of 6-acyl ................................ 35
pterins by condensation .............................................................................................................................. 35
3.4 Synthesis of pterin-dithiolene ligands through the “Minisci reaction” .................................................. 57
3.5 Acylation protocol variations, targeting MPT ....................................................................................... 81
3.5.1 Pyran ring variation ........................................................................................................................ 81
3.5.2 Phosphate variation, northern and southern functionalization ..................................................... 94
4 .Conclusion ................................................................................................................................................... 95
Experimental section ...................................................................................................................................... 98
IR and UV-VIS spectra ................................................................................................................................... 115
Molecular structures and X-ray data ........................................................................................................... 124
References ..................................................................................................................................................... 131
Eigenständigkeitserklärung .......................................................................................................................... 135
Lebenslauf - Ivan Trentin .............................................................................................................................. 136
Acknowledgement ........................................................................................................................................ 139
«The period of fifteen years of investigation in two laboratories on the structure of the butterfly
pigments indicates already some principal difficulties encountered with pteridines which are
attributed to their incomplete combustibility in elemental analysis, their high melting point and
decomposition point, their poor solubilities in water and most organic solvent and hence problem
purification.
Nevertheless the natural pteridines reveal so many exceptional physical and extraordinary chemical
properties that we cannot only learn basic principles of heterocyclic chemistry, in general, but also
get detailed knowledge and under-standing why special structures are prerequisite for specific
biochemical and enzymatic reaction.
The fascination to discover the unexpected and anomaly is one of the reasons why I have devoted
already forty years of research efforts to a large extent to pteridine chemistry».
“Chemistry and Biology of Pteridines and Folates
Natural Pteridines – A Chemical Hobby”
Prof. Dr. Wolfgang Eugen Pfleiderer
1
1. Pteridines and pterins
1.1 Historical background
The interesting story of the pteridines compounds family has been described in different books and by different
scientists all around the word. The best narrative remains the one written by the main characters of this tale who could
directly have influenced the development of this attractive field of research. The reading of books like “Chemistry and
Biology of Pteridine and Folates” or “Fused Pyrimidines” offers not only amazing reports of experimental data but mainly
the ideas and passion of the authors and the researchers [1]. Therefore, if we want to fully describe the story of
pteridines, we can’t but emphasize the devotion of the researchers and the spirit of a topic that W. Pfleiderer defined
as a “chemical hobby“ [2].
The development of pteridine research can be ascribed to the organic chemistry of the second half of the 19th century.
The first publications came from two different fields — synthetical chemistry and isolation of natural products. It is
interesting to observe how different scientists were able to isolate some products but due to the particular chemical
features of pteridines, the complete elucidations of the structures were obtained only many years later at the beginning
of the 20th century. Exemplary are the publications of F. Wöhler [3] and H. Hlasiwetz [4] in 1857, which describe the
formation of a yellow product by heating uric acid in water; eighty-five years later in 1942 F. G. Hopkins repeated the
experiment and suggested the formation of a compound analogous to the wings’ pigments of the sulfur-yellow butterfly
“Pieridae” [5]. The final elucidation of the reaction was achieved only in 1959 by W. Pfleiderer [6] with the isolation and
characterization of twelve different substances of which six are main products, represented in Figure 1.
FIGURE 1 – FORMATION OF PTERIDINES FROM URIC ACID DESCRIBED BY W. PFLEIDERER : 2,4,7-TRIHYDROXY PTERIDINE (I); 2,4,7-TRIHYDROXY-6-METHYL PTERIDINE (II); 2,4,6-TRIHYDROXY PTERIDINE (III); 2,4,7-TRIHYDROXY-6- PTERIDINECARBOXYLIC ACID (IV); 2,4,6,8-TETRAHYDROXY
PYRIMIDO-[4.5-G]-PTERIDIN (V); 2,4,5,7-TETRAHYDROXY-PYRIMIDO[5.4-G]- PTERIDIN (VI)
2
The summary of the first directed synthesis of pteridines does not disclose the difficulties faced by the pioneers of this
field (Scheme 1); in fact, despite the development of the organic chemistry between the end of the 19th and beginning
of the 20th century, the analysis of particular compounds like pteridines represented a tripping stone for the
researchers.
The first rational protocol to obtain a pteridine was reported by O. Kühling [7] in 1894. He synthesized through an
oxidation and decarboxylation of alloxazine a 2,4(1H,3H)-pteridinedione named alloxazin. In 1907 S. Gabriel and A. Sonn
[8] in Berlin obtained the same product of O. Kühling starting from 2,3-pyrazinedicarboxyamide and in 1937 R. Kuhn
with A. Cook [9] prepared the same pteridine, this time condensing a 2,4-dihydroxy-5,6-diaminopyrimidine with a
glyoxal molecule and changing its name from alloxazin to lumazine (Scheme 1).
SCHEME 1 – FIRST SYNTHESIS OF PTERIDINES FROM THE BOOK “FUSED PYRIMIDINES”
In 1901 S. Gabriel with J. Colman prepared the first pteridine from a pyrimidine ring, condensing a 4,5-
pyrimidinediamine with diphenylglyoxal and calling the final product “das Azin” (6,7-diphenyl-pteridine) [10]. After 5
years in the same laboratory the same reaction was curiously described and it was used for the synthesis of various
“Azin-purin” derivatives [10, 11] (Scheme 1).
Despite the analytical issues faced by the synthesis of pteridines, at the beginning of the 20th century the basic protocols
of this chemistry were already delineated; many research groups started to work on this topic and the number of
respective publications increased. Still the main authors of this field refer the origins of pteridine chemistry to the
3
natural products; it is in fact the isolation of the natural occurring pterins which had the largest impact on the scientific
community.
Surely the first and most representative author in this field is F. G. Hopkins, who between 1889 and 1895 described the
isolation of two rich fractions of butterfly pigments – the first from the English brimstone butterfly and the second from
the white cabbage butterfly (Figure 2) [12]. However, as other contemporary chemists, he did not manage to give any
structural elucidations, mainly due to the unusual physical and chemical properties of the substances, like poor solubility
and high melting point.
In 1924 C. Schöpf, a young butterfly collector and junior professor in Freiburg, persuaded his professor H. Wieland to
work on the publication of Hopkins and tried to identify the structure of the interesting pigments. The two scientist
worked many years on the characterization of the molecules isolated by Hopkins, they named the class of pigments
“pterins” referring to their origin (from Greek pteron = wing) and the two substances according to their colouration –
xanthopterin (yellow) and leucopterin (white, colourless). Two years later they managed to isolate a third
isoxanthopterin pigment but unfortunately the composition of all the pigments remained unknown until 1940 when R.
Purrmann, one of Wieland’s PhD student, was able to elucidate the structure of the three pigments represented in
Figure 2 [13-16].
FIGURE 2 – PIGMENTS ISOLATED BY C. SCHÖPF AND H. WIELAND
Representative of many years of research is the opening address to the third Pteridine Symposium in Stuttgart in 1962,
when C. Schöpf described the practical and chemical difficulties encountered in almost fifteen years of research:
«However, I want to call attention to a most paradoxical situations. The chemistry of those pyrimidopyrazines, which to-
day we call pteridines, had been very successfully investigated in Berlin from 1884 onwards; in Munich, it was not realized
that the butterfly pigments belonged to this family of compounds until 1940, nearly half a century later. Why did natural
products cause so many difficulties? Collection was fitful, because of the season, and the material precious. The lack of
melting points removed an important criterion for purity. Not until 1940 was xanthopterin freed from an impurity that
gave a red colour with hydrogen peroxide. The poor solubility in all common solvents diminished ease of purification and
no molecular weights were obtainable. The resistance to complete combustion, leading to nitrogenous coke, produced
errors in the analytical results. Particularly misleading was the supposed analogy with purines based on the fact that
pterins and purines gave similar substances upon degradation» [17].
Schöpf ’s lecture expressed in detail the challenges faced by the research group and emphasizes the interpretative
mistake to consider pterins purine-like molecules.
4
Figure 3 shows the proposed structure of xanthopterin during the fifteen years of research until 1940 when finally R.
Purrmann managed to discover its correct composition and conformation [17].
FIGURE 3 – PROPOSED MOLECULAR STRUCTURES OF XANTHOPTERIN FROM THE BOOK “CHEMISTRY AND BIOLOGY OF PTERIDINES”
5
1.2 Nomenclature
For most chemical compounds, their nomenclature is influenced by the historical development of their chemistry. As
previously described, the pteridine family grew up in two different fields of research — the synthetical chemistry and
the isolation of natural compounds. Due to this pteridines literature is overcrowded with trivial names and various
numbering system.
The discovery of many natural pteridines or biomolecules containing the pteridine scaffold, has introduced into the
entire scientific literature a variety of different terminology, among which the term “pterins” represents the first
historical example. It was in 1941, after the structure elucidations achieved by R. Purrmann, that H. Wieland firstly used
the term “pteridine” to describe a pyrazine-pyrimidine ring system. In 1963 W. Pfleiderer suggested to use the name
“pterins” only for the pteridines 2-amino-4(3H)-pteridinone derivatives [11], this rule is nowadays almost universally
accepted (Figure 4).
Two numbering systems have been proposed by the scientific community – the first was used by F. Sachs and G.
Mayerheim at the beginning of the 20th century to define the “azine-purine” products and it beholds the idea of a
purine-like structure (1, Figure 4); the second was described by R. Kuhn and his A. Cook in 1937 and is nowadays the
most commonly accepted nomenclature (2, Figure 4) [11, 18].
FIGURE 4 – NOMENCLATURES
6
1.3 Pteridines chemistry
In the book “Fused Pyrimidines”, D. J. Brown describes pteridines as a: «semiaromatic system with no great stability
towards ring fission, prone to nucleophilic but not electrophilic reactions and subject to covalent addition reaction». In
the following respective aspects will be detailed with a focus on the particular subclasses “pterins” [1].
1.3.1 Aromaticity, π-excessive and π-deficient
In order to better understand the π-deficiency of pteridines, it is useful to recall the concept of “aromaticity” and the
meaning of “aromatic character”.
The most of the organic chemistry books contain a chapter dedicated to benzene and its low reactivity compared to a
normal alkene system; a typical example is the bromination reaction which leads to a monosubstituted benzene and
not to an addition product (Scheme 2).
SCHEME 2 – BROMINATION ALKENE AND AROMATIC SYSTEM
The benzene ring has six carbon atoms, each bearing three sp2 and one unhybridized p orbitals. Two of these sp2 orbitals
are used for σ bonding with the adjacent carbons and the third one binds with an hydrogen atom. The remaining p
orbital, which is perpendicular to the planar ring, participates to a π-electron delocalization that makes the ring more
stable and provides it with particular features making it an “aromatic system” [18].
The aromaticity exemplified for the case of benzene is then generalized for all compounds showing a similar
delocalization and defined by the Hückel rule: «an aromatic compound contains a cyclic system of atoms whit a cyclic
conjugated, π bond system of (4n+2) π-electrons (Hückel number)» [19].
The effect of the aromaticity on reactivity and physical properties of a molecule can be summarized in few points [20].
1. Aromatics tend to undergo ring substitution reactions rather than addition reactions which are
characteristics of alkenes.
2. Cyclic system are more stable to oxidation and reduction than typical alkenes.
3. The existence of a “delocalization energy”.
4. Bond length in aromatic rings are intermediate, between normal σ bond and normal π bonded atoms.
5. Characteristic absorbance in the U.V. or visible region of the spectrum.
6. The exhibition of a ring current when an aromatic compound is placed in a magnetic field.
The so called “aromatic character” described above is an ideal situation and it is possible to find many aromatic
compounds deviating from this. An example are the heterocycles containing nitrogen compounds characterized by a
loss in aromaticity directly proportional to the N/C ratio [18].Three representative cases are pyrrole, pyridine and
pteridine heterocycles shown in the Figure 5. All these molecules are aromatic and the nitrogen atoms can be described
with exactly the same terms as benzene.
7
In the case of the pyridine and pteridine, nitrogen carries the lone pare on one of the sp2 orbitals and only one electron
is localized in the p orbital perpendicular to the plane. The pyrrole ring is a different system which by binding hydrogen
atoms carries its lone pare, two electrons, on the unhybridized p orbital responsible for the π-electron delocalization.
The higher electronegativity of nitrogen, compared to carbon, effects directly the electron density distribution in the
ring, resulting in a distortion of the π-orbitals and a consequent loss of aromaticity [20]. In the case of the pyrrole this
effect is compensated by the higher number of electrons participating to the delocalization, which produces a higher
electron density on the carbon close to the nitrogen, also compared to the benzene model. That’s why these systems
are termed π-excessive. For pyridines or pteridines the vicinal carbon atoms are partially positive charged and the π-
delocalization is drastically reduced, therefore those heterocycles are termed π-deficient [20].
Based on this D.J. Brown describes this particular π-deficiency as a “semi-aromatic character”, meaning that all chemical
and physical properties of these heterocycles are strongly influenced. Pteridines represent among the heterocycles the
most effected class of compounds.
π – deficient π – excessive
FIGURE 5 – AROMATICITY IN HETEROCYCLES
8
1.3.2 Stability of pteridines and pterins
The pteridine ring has 10 π-electrons contributing to the aromaticity like for instance naphthalene but, the presence of
nitrogen atoms results in a loss in stabilization energy, which makes the system susceptible to cleavage [20, 21]. The
carbon atoms of the pyrimidine moiety carriyng a partial positive charge easily undergo hydrolytic cleavage, acid or base
catalysed, resulting in the formation of substituted pyrazines (Figure 6) [20]. This instability can be softened by the
introduction of an electron donating group. For example the xanthopterin natural product carries an amino group and
is stable to boiling in 7 M hydrochloric acid and only slightly effected by boiling with barium hydroxide for 20 hrs [22]
(Figure 6).
FIGURE 6 – HYDROLYTIC CLEAVAGE PRODUCT AND XANTHOPTERIN
The photosensitivity of pteridines and pterins [23-25] is another important aspect that must be taken into account
during the synthesis and characterization of these compounds; reactions and work-up with pteridines has to be carried
out as prophylaxis in dark-room. The photodegradation of pteridines has been investigated and in most cases they
proceed through a Norrish-type II mechanism. One of the first examples in literature is the irradiation of biopterin under
aerobic conditions (Scheme 3) [26-28].
SCHEME 3 – PHOTOCHEMISTRY OF BIOPTERIN IN AQUEOUS SOLUTION FROM [26-28]
9
1.3.3 Covalent hydration reaction
The addition of water to a double bound is a typical reaction in organic chemistry but few examples of them have a
covalent character. In most of the cases the stability of the final product and the nature of the double bond play the
main role. Many studies on a large range of aldehydes revealed almost fifty per cent of their respective hydrated form
in aqueous solution, while the reaction with alkenes has a high activation energy and to be overcome it is necessary the
application of a catalyst like hydrogen or hydroxy ions [29].
A different scenario is represented by the C=N bound which has been investigated for a long time and results in most
of the cases in a broken bond between carbon and nitrogen, like for the hydration of benzylidene-aniline to a Dimroth
base shown in Scheme 4 [29].
SCHEME 4 – ADDITION OF WATER TO C=N BOND
Pteridines are one of the few cases in which the formation of the hydration product does not lead to the expected
scission. This peculiarity is even more interesting if we consider that the additional reaction is associated to a further
loss in aromaticity, which as previously described, has already been pauperized by the distortion of the electron density.
A possible explanation was postulated by professor A. Albert from the University of Canberra. He attributes the easy
covalent addition to pteridines to two main factors — the first is the high electron affinity of the nitrogen that produces
an exposed polarized double bond and the second is an extra stabilization energy arisen from the formation of
resonance structures [29].
It was A. Albert who in 1951 observed for the first time the hydration of a pteridine. The professor and his co-worker
were investigating the pKa of different organic bases like pteridines and purines, when a strange phenomenon was
observed (Scheme 5); the titration curve of 6-hydroxypteridine with alkali revealed a weak acidity with a pKa of 9.7 as
value, but the back-titration with acids exhibited the formation of a new curve with a stronger acidity around a pKa of
6.7 [29].
SCHEME 5 – COVALENT HYDRATION
The interesting behaviour of this pteridine found an explanation in 1955 with the proposal of a covalent hydration
mechanism on one of the free carbon atoms; the exact structure of the neutral hydrated product 6,7-dihydroxy-7,8-
dihydropteridine (Scheme 5) was proven one year later by D.J. Brown and S.F. Mason [30] through their UV
spectroscopic studies on the species formed by titrations.
10
Afterwards, the covalent hydration became an important parameter for the characterization of pteridines, since this
kind of reaction influences conformation, redox potential and pKa value in aqueous solution, which are fundamental
features in the biological system. The observation of this phenomenon had stimulated a large number of studies in
spectrometric and potentiometric research with pteridines. Therefore, it became necessary to establish a method to
reveal the presence of hydration. With this exact purpose A. Albert in cooperation with F. Reich in 1961 used the methyl
derivatives of 6-hydroxypteridine to confirm the attack of the water at position 7 (Figure 7) [31]. In their method the
presence of the bulky methyl-group on the carbon atom blocks the addition of water and other nucleophilic attacks.
This allows us to determine which of the derivative does not give the hydrated form and to localise the position involved
in the reaction mechanism (Figure 7).
FIGURE 7 – METHOD WITH METHYL SUBSTITUENT
Pterins are a particular subclass of pteridines and as previously described, the presence of an electron donating group
stabilizes the entire ring system. Nevertheless, examples of covalent addition are reported in the literature, like the
natural product xanthopterin which partially reacts with water and methanol (Scheme 6) [32].
SCHEME 6 – COVALENT ADDITION WITH METHANOL OF XANTHOPTERIN
The role of this mechanism in biology becomes extremely relevant for pterins, which are a class of compound occurring
in a huge variety of natural products and consequently having an echo in medicine, physiology, biotechnology and many
other disciplines. It is therefore always suggested the study of a possible addiction mechanism on pterins and, as
recommended by A. Albert «any substances which obstinately retains a molecule of water (on the evidence of
elementary analysis) should be examined for covalent hydration» [29].
11
1.3.4 Reactivity of pteridine
The main topic of this dissertation is the chemistry of pterins and particularly the synthesis of the pterin-dithiolene
systems. It is not therefore my intention to describe each reaction about pteridines, but to focus on the most relevant
reactivity of this compounds’ family. Nevertheless, it is important to stress the utility of pteridine chemistry in
understanding the behaviour of pterins and its derivatives in chemical and biological fields [11].
Many reactions have been tested on pteridine ring systems resulting in a clear preference towards nucleophilic attack.
It is indeed the specific high depauperate π-electron layer that suppresses the typical affinity of aromatic system for
electrophiles; this reactivity is amplified by the introduction of an electron withdrawing group and repressed by electron
donating substituent.
Pteridines participate in four main classes of reactions — nucleophilic addition, nucleophilic metatheses, reductive
reactions and oxidative reactions. The homolytic c-acylation will be fully discussed later in a following chapter, since it
is the core of the synthetical strategy reported in this dissertation.
Nucleophilic addition
The covalent hydration and its implications on the biology for the natural occurring pteridines was already discussed. It
is, hence, likely that there will be the same mechanism with other nucleophiles, stronger then water, like alcohols,
amines and in general Michael reagents [33].
The link between the nucleophilic addition reaction on pteridine and the Michael reaction is interesting. They are both
deactivated by the introduction of a methyl group, for electronical and steric reasons and most of the Michael reagents
attack the pteridine ring (Scheme 7).
There is a general tendency to prefer positions 4,7 and in few cases position 6, but the introduction of substituents
make any substrate a particular case. The best example are pterin derivatives with their have two positions blocked and
an amino and hydroxy group that make the substrate more stable against nucleophilic additions [11].
SCHEME 7 – DIMEDONE ADDUCT FORM ADDITION OF A MICHAEL REAGENTS
12
Nucleophilic metatheses
The presence of a leaving group on the pteridine ring makes the nucleophilic substitution easier. It is in fact present in
the literature a large number of nucleophilic metathesis; common leaving group are halogen, sulfo and alkylsulfonyl
(Scheme 8). Chloro is the most often used living group and has been employed in many syntheses as part of starting
material or intermediate product [34].
SCHEME 8 – NUCLEOPHILIC SUBSTITUTION ON PTERIDINES
Other substituents can be replaced when working with harsher conditions, a typical example is the reaction from
pteridinone to chloropteridines (Scheme 9), which requires high temperatures but at the same time permits obtaining
an extremely versatile intermediate product [35].
SCHEME 9 – NUCLEOPHILIC SUBSTITUTION
The nature of other substituents influences the reactivity of the substrate. An electron donating group like an amino
function makes the replacement of a good leaving group like chloro more difficult (Scheme 10).
13
SCHEME 10 – NUCLEOPHILIC SUBSTITUTION
Oxidative reactions
While pyrimidines can be further oxidized to alloxan, the pteridines are already in a high oxidation state. The oxidative
nuclear reactions therefore usually involve the di- or tetra derivatives obtained by chemical reduction; a strong oxidant
reagent like potassium permanganate but also normal oxygen in basic condition can be successfully adopted. The
chemistry involved in those reactions is not really interesting, but it becomes fundamental for the study of biomolecules
like tetrahydrobiopterin (Scheme 11).
SCHEME 11 – OXIDATION OF PTERIDINES
The oxidation of pteridines with peroxide or peroxycarboxylic acid results in the formation of N-oxide derivatives, like
for the other heterocycles this product can be used as versatile intermediate. The regioselectivity of this reaction is
influenced by steric factors, like the introduction of a bulky group adjacent or in peri position, also the choice between
two different peroxycarboxylic acids has an effect. The 2,4(1H,3H)-pteridinedione and its 6,7 dimethyl derivatives are
oxidized at position 5 with trifluoroacetic acid and at position 8 using peroxide and formic acid. The targeted
regioselectivity is not always possible and in many case the product is a mixture (Scheme 12).
SCHEME 12 – FORMATION OF N-OXIDE
The N-oxide form has been largely used for the synthesis of 6-substituted pterins. The best example is the synthesis of
6-chloropterin (Scheme 13), in this case the formation of the N(8)-oxide actives the carbon 6 and permits introducing a
chloro substituent only in one position.
14
SCHEME 13 – REGIOSELECTIVE INTRODUCTION OF CHLORO ON THE N-OXIDE DERIVATIVE
Reductive reactions
The reduction of the pteridine ring is in general an easy reaction. Depending on the stability of substrate and product is
possible to use sodium borohydride, sodium dithionite, catalytic hydrogenation, or metal amalgams (Scheme 14). The
reaction will preferably take place at the carbon atom without substituent [36].
SCHEME 14 – REDUCTION OF PTERIDINES
Also pterins have been successfully reduced using Platinum and hydrogen gas [37]. The synthesis of pterin molecules in
their reduced form is of particular interest for the biological application; the pterin-motive is present in different
important natural systems, with particular implications in the medicine and enzymology [38]. Three important examples
are the folic acid, the tetrahydrobiopterin and ultimately the molybdenum cofactor.
15
2. Biological Background
2.1 Molybdenum dependent enzymes
Among the metals of biological relevance, molybdenum represents an interesting and valuable example for the
bioinorganic field. From the simplest bacterial to the human organism, this 4d transition metal has been selected by
evolution as cofactor constituent of many enzyme families. Despite the low percentage of molybdenum in the earth’s
crust, this metal is bioavailable as molybdate, a high water soluble form that makes molybdenum the most abundant
transition metal presence in sea water (MoO42-, molybdate 10 μg/L) [39]. The access to different oxidation states and
its redox-activity also under physiological condition make molybdenum a perfect and flexible candidate for a biological
system [40].
Depending on the nature of the active site, molybdenum dependent enzymes are divided in two groups – the
nitrogenase and the oxidoreductases. The first group is characterized by an iron-sulfur cluster (Figure 8), an unique
molybdenum complex that was centre of an intensive crystallographic discussion from 1992 to 2011 [41-43]; in
particularly the central atom was at the beginning unidentified than postulated as nitrogen and finally assigned as
hypervalent carbide.
FIGURE 8 – ATOMIC STRUCTURE OF FEMOCO OF NITROGENASE
The strong interest of the scientific community toward this enzymes is motivated by its peculiar and powerful ability to
catalyse the conversion of atmospheric nitrogen to ammonia, a reaction known as nitrogen fixation. The analogue
industrial conversion, called Haber-Bosch process requires extremely high temperature and pressure, while the
enzymatic way can be conducted at room temperature and atmospheric pressure [44].
The oxidoreductase group are characterized by the Molybdenum cofactor (Figure 9), which is probably the most
relevant discovery in the recent history of pterin chemistry and biochemistry. Indeed, the finding of pterin natural
products or derivatives had always stimulated great interest on this class of compounds, as for biopterin and folic acid
a huge number of publications, studies and stimulating ideas have been reported. The structural elucidation of Moco
was achieved by K.V. Rajagopalan, J.L. Johnson in 1992 [45], when the two scientists postulated the structure of the
molybdenum cofactor to be a pterin derivative.
From then on, the synthesis of Moco model compounds became an important field of research and after more than
twenty years of studies some good models have been synthetized [21], nevertheless many aspects of the cofactor need
to be clarified.
The structure described by K.V. Rajagopalan, J.L. Johnson was a 6-substituted fully reduced pterin with a phosphorylated
dihydroxybutyl side chain and carrying a cis-dithiolene functionality attached to the Molybdenum (Figure 9). This
composition was in 1995 largely confirmed with the first reported crystal structure of a molybdoenzyme, the aldehyde
ferrodoxin oxidoreductase (AOR) from Pyrococcus furiosus [46].
16
The proposal of K.L. Rajagopalan was corresponding with the exception of the additional pyran ring, in half-chair
conformation, formed by the hydroxy group of the sidechain attacking the position 7 of the pterin ring (Figure 9). Further
crystal structures have later accredited the tricyclic form as representing the actual natural cofactor structure.
All these enzyme families catalyse oxygen and two electron transfer reactions from or to a substrate playing a key role
in the metabolic process of carbon, nitrogen and sulfur manly with detoxification purposes. A malfunction in the
biosynthesis of Moco or in the enzyme’s maturation causes two severe diseases the molybdenum cofactor deficiency
(MoCD) and the isolated sulfite oxidase deficiency (ISOD), described in the following.
Escherichia coli represent the best characterized system and it was chosen for the biological experimentation of the
Moco model compounds. It is therefore of utility to have a general overview of the enzymes characterized in this system.
The Molybdenum cofactor has been determined in three different configuration in Escherichia coli [40] and therefore
historically divided in the families — xanthine oxidase (XO), DMSO reductase (DMSOR) and the sulfite oxidase (SO) as
reported in Figure 10.
The active sites of these three families are characterized by the presence of the MPT ligand system and differ each other
by the coordination sphere of the metal centre. The supplement of a cytosine monophosphate in case of xanthine
oxidase and of guanine monophosphate in DMSO reductase constitutes an additional variation (Figure 10).
The xanthine oxidase family bears an oxidized MPT- MoVIOS(OH) nucleus with one equivalent of metal and MPT, they
are participating in a two electron transfer hydroxylation with water as source of oxygen. The sulfite oxidase family is
characterized by an MPT- MoVIO2 nucleus with one equivalent of metal to MPT and a further cysteine coordinated to
the meatal centre. Oxo transfer reactions are catalysed by these enzymes, with atom translocation occurring potentially
in both direction, to or from the substrate.
The DMSO reductase family is the most numerous group of proteins in Escherichia coli, it is characterized by a two
equivalents of MPT for one metal centre. The MPT2-MoVIO(X) core catalyses mainly oxygen atom transfer and
dehydrogenation reactions. In absence of the substrate they are active as terminal reductases, sulfur or proton transfer
catalyst, and non-redox catalyst. The oxygen in the coordination sphere is substituted by a sulfur in formate
dehydrogenase H, while the extra ligand X can be a cysteine, serine, selenocysteine and aspartate; in many cases they
are also involved in the respiratory system [47].
FIGURE 9 – MOCO STRUCTURE HYPOTHESIS AND MOLYBDOPTERIN (MPT)
17
FIGURE 10 – DIFFERENT STRUCTURE OF MOCO EXPRESSED IN ESCHERICHIA COLI, SCHEME FROM REF. [40]
18
2.2 Molybdenum cofactor natural synthesis and maturation
The biosynthesis of Moco may appear not directly of interest for the synthesis of Moco model compounds, but the
biological mechanism, intermediates and the proteins involved open the research view and permit the expression of
concepts and ideas for the understanding of the working principals in the molybdenum dependent enzymes. Therefore
the Moco synthesis in Escherichia coli is here shortly summarized with the intention to emphasize the possibilities and
the value of the synthetic protocol reported in this scientific research.
The natural synthesis of Moco is basically divided in three parts — cyclization of GTP) [48] (Scheme 15) to form the
cyclopyranopterin monophosphate (cPMP), sulfuration of cPMP to give the mature pyranopterin and finally the
coordination of molybdate to the cis-dithiolene anticipated by molybdenum uptake. The complete maturation of this
enzyme, namely the insertion of MPT into the apoenzyme will be described in the following text as an important
mechanism for the biological investigation on the Moco model compounds.
2.2.1 Cyclization of GTP
The first step involves a radical abstraction of a proton at the position 3’ of GTP carried out by MoaA, an enzyme which
belongs to the radical SAM family (S-adenosylmethionine) [49, 50], subsequent radical migration leads to the
intermediate 3,8-cH2GTP that was isolated and fully characterized. MoaC-catalyses the hydrolytic ring opening and
formation of the pyran cyclophosphate ring resulting in the intermediate cPMP [51] as reported in Scheme 15 (other
possible mechanism are reported in literature) [52].
SCHEME 15 – CYCLIZATION OF GTP, SCHEME FROM [40]
19
2.2.2 Sulfuration of cPMP
The second step is catalysed by the MPT synthase, this enzyme is a heterotetramer constituting two small subunits-
MoaD and two larger MoaE. As shown in the Scheme 16 below two sulfur equivalents are required for the complete
sulfuration of the substrate. These sulfur atoms are carried by the smaller MoaD subunit while MoaE is binding the
cPMP intermediate.
SCHEME 16 – SULFURATION OF CPMP IMAGE TAKEN FROM REF. [40]
20
2.2.3 Molybdenum uptake
While most of the first-row transition metals are already available as cation in the cell, molybdenum with its anionic
form access the cellular membrane via a highly specific transporter called ModABC [53]. This system belongs to the ATP-
binding cassete superfamily of membrane transporters, and it consists of two integral membrane proteins (ModB, B-
subunit), two ATP-binding peripheral membrane protein (ModC) and a periplasmic binding protein (ModA) (Figure 11)
[53-55].
Briefly summarised, the external periplasmic A-protein scavenges the oxoanion molybdate as tetrahedral complex,
which binds to a pair of translocating, transmembrane B-subunits (ModB), these prepare the cavity for the transport
and deliver to the cytoplasmic pair C-subunits with ATPase activity. The molybdate is finally released into the cytoplasm
[53]. The interaction of ModABC in molybdate-bound form with another protein called ModE [56] produces an
enhancement in the transcription of molybdenum enzymes like DMSO reductase, nitrate reductase [53].
FIGURE 11 – CELLULAR UPTAKE OF MOLYBDENUM AND TUNGSTEN, IMAGE TAKEN FROM REF. [16]
21
2.2.4 Insertion of the metal
Once the molybdate is transported into the cytoplasm, as described above, the final step for the complete construction
of the molybdenum cofactor is the combination with the biosynthesized molybdopterin (MPT). In Escherichia coli this
task is manged by two enzymes MogA and MoeA, both participate in the process with different function. It has been
shown in vitro reaction that MoeA is able to mediate the anchoring of molybdate to MPT; MogA in absence of ATP
inhibits the reaction and accelerates if in presence of the same [40].
The phenomenon has been explained based on a crystal structure of a MPT-AMP product. MogA catalyses the
adenylation of the MPT substrate preliminary to the coupling with the molybdenum oxoanion (Scheme 17). The role of
MogA with the formation of the adenylated MPT is of relevant at physiological concentration of the metal, but it has
been proven that in concentrated solution the molybdenum insertion is accomplished by the MoeA alone [47, 57].
SCHEME 17 – METAL INSERTION SCHEME FROM [40]
22
2.2.5 Cofactor insertion into apoenzyme
The insertion of the cofactor into the apoenzyme represents the final process of maturation for the molybdoenzymes,
namely the acquisition of the catalytic activity specific to the protein’s family. Therefore it is of extreme interest for the
researchers, which want to understand the mechanism of function in molybdenum enzymes carrying Moco. It is useful
to have a simplified introduction of this process looking at the acting proteins, but keeping in mind that many aspects
are postulated and many questions are still open (Figure 12) [40].
The synthetic address developed in this dissertation targets the synthesis of bis-dithiolene molybdenum complexes, i.e.
compounds that would act as model for a bis-MGD cofactor. Hence, it is appropriate to describe the maturation of a
DMSO reductase family ‘s enzyme, in particular the most explored TMAO reductase (TorA) with its chaperon TorD.
FIGURE 12 – IMAGE TAKEN FROM REF. [40]
The biosynthesis of the molybdenum cofactor is a highly conserved process in all organisms employing molybdenum
enzymes. In Escherichia coli after Moco-basic formation, depending on the family of enzymes, further modifications are
needed before the complete maturation takes place (Figure 12). To xanthine oxidase is added a CMP (cytosine
monophosphate, from CTP) via pyrophosphate bond and also a further sulfuration of the Mo=O bond. In the DMSO
reductase family the cofactor receives a GMP always via pyrophosphate bond, but it couples itself with another MPT,
resulting in a bis-MGD form. Different case is the sulfite oxidase family where the cofactor is directly inserted into the
apoenzyme and it does not require any other steps [40].
As discussed above after the coordination of molybdate to MPT, such Moco basic form is involved in structural
modifications by two proteins MobA and MobB, while the first plays definitely an important role, MobB has probably
some functions as adapter protein working in concert with MobA. However the maturation process has been
reproduced in vitro also in absence of MobB.
MobA is a highly specific GTP binding protein and as first step combines Moco with a nucleotide resulting in a MGD,
which is later coupled with another molecule MPT to produce bis-MGD. This passage (step 2) represented in Figure 12
which culminates in the insertion of bis-MGD into ApoTorA, can occur direct on MobA (which has enough space) or on
23
the surface of the enzyme (Apo-Tor A, TorA). In both cases this happens in collaboration with the chaperon TorD [40,
57]. The mechanism adopted is still an enigma and requires more in depth studies and experimentation, possibly with
the employment of model compounds.
TorD is probably the protein that fulfils the majority of the tasks in the entire maturation process. It belongs to a family
of molecular chaperons [58] present in bacterial and in archaea cells. Its ability to interact has been proven with most
of the players of this natural process. First and foremost it binds with the molybdenum enzyme TorA, which in absence
of it decompose (70-80%) under thermal stress; thus it is reasonable to assume that the first job of TorD is to protect
the enzyme and to carry it to the Tat-machinery for the periplasmic release.
Furthermore TorD is involved in the maturation of bis-MGD. First he interacts with MobA and Mo-MPT for the addition
of a GMP from GTP and later most probably he works as platform for the formation of bis-MGD. It has also been proven
that the chaperone influences the maturation level of ApoTorA in vitro with the source of the molybdenum cofactor,
which in presence of TorD increases from twenty at basal level to eighty percent. Thus, the chaperone somehow
facilitates the insertion of bis-MGD in ApoTorA in a short time in order to avoid the proteolytic attack on the holoprotein
(Figure 12).
The last step of the maturation process involves preTorA and TorD, which remains bound to the protein until the Tat-
machinery is prompted to transport the molybdenum enzyme to the periplasm. This passage is till now not clear, but
most probably the release of TorD plays also in this case a role for the activation of the Tat-machinery.
24
2.3 Molybdenum cofactor and Human diseases-MoCD and ISOD
The molybdenum dependent enzymes, count around one-hundred of components, present in all kingdoms of life. As
previously described except for the nitrogenase carrying the FeMoco active site, the majority harbours the molybdenum
cofactor, a unique biological motif central for the carbon, sulfur, and nitrogen cycles [59].
So far four molybdenum dependent enzymes are formed in the human — sulfite oxidase (SO), xanthine oxidoreductase
(XOR), aldehyde oxidase (AO) and only recently discovered was the mitochondrial amidoxime-reducing component
(mARC) [60, 61]. These proteins are mainly involved in redox reactions using water as acceptor or donor of oxygen; in
particular SO and XOR have been intensely studied for their important role in the catabolic reactions in cysteine and
purine metabolism whereas AO and mARC require an “in depth-analysis” about their exact physiological function in the
human organism.
Two important pathologies are related to Moco — the molybdenum cofactor deficiency (MoCD) and the isolated sulfite
oxidase deficiency (ISOD). Both are autosomal recessive disorders and lead to a severe impairment or directly to the
death of the patients. The molybdenum cofactor deficiency was reported for the first time in 1978 when a baby of a
Danish family died after it had shown the typical symptomatology of MoCD, namely therapy-resistant seizures,
neurological abnormalities, lens dislocation, dysmorphic shape of the head, feeding difficulties, occurring during the
neonatal period (0-28 days of life) [59]; most probably the other siblings, which had previously died, were also affected
but no analysis was reported. Since this first case, about one hundred patients have been recorded, this is a quite rare
pathology that counts around 1 case in 100.000 to 200.000 live births; nevertheless many studies have been started and
also some treatments addressed [59].
The biosynthesis of Moco is a highly conserved biological process, meaning that the basic pathway is the same in all
kingdoms of life with the employment of related enzymes specific for the living organism. At the beginning of this
chapter a detailed description of the Moco biosynthesis is given. It is therefore sufficient to show the main passages
constituting the formation of the cofactor and consequently maturation in order to understand at which steps MoCD
and ISOD are induced (Scheme 18).
SCHEME 18 – STEPS OF THE BIOLOGICAL SYNTHESIS OF MOCO AND MATURATION OF THE ENZYMES
Four main passages delineate the natural synthesis and maturation of the enzymes — cPMP synthesis, MPT synthesis,
coordinatoion to the molybdenum ( synthesis of Moco) and final insertion into the apoenzymes [59].
MoCD is caused by biallilic phatogenic variants in the enzymes MOCS1, MOCS2 or GEPH [62] involved in the formation
of Moco, and is clasified in types A, B or C depending on which step of the process is impaired; the ISOD is localized in
the last step of coordination with the apoezyme. In all types of pathology the accumulation of sulfite, taurine, thiosulfate
25
and all the related toxins is the most dangerous and deleterious effect. It causes permanent damage to the patients or
directly their death.
2.3.1 Synthetic cofactor, drug for a possible cure
The MoCD type A desease has been already succefully treated by the employment of a biothechnology prepared cPMP.
This method was developed by Professor G. Schwarz at the University of Cologne [63] and it helps many patients every
year.
Unfortunately, there is not yet a treatment for the MoCD type B, C and the ISOD; in particular the instability of Moco
and of the enzyme does not permit their employment as drugs.
The final goal of the work reported in this dissertation is the preparation of a synthetic cofactor, which can be used as
drug for the MoCD type B,C and ISOD. The synthetical strategy developed in this research makes possible the
preparation of model compounds of MPT and in the future also of Moco. Indeed, the study of these models allows a
deeper comprehension of the natural cafactor and its enzymes families.
The general features of our hypothetical cofactor can be summarized in four points:
✓ The cofactor has to be of about the same size as the biological cofactor or smaller and able to bind inside the
pocket.
✓ The cofactor has to be able to catalyse the oxygen transfer reactions.
✓ Because MPT bind to molybdenum by its dithiolene function this feature will be part of every model.
✓ The synthetised cofactor has to be more stable than the natural cofactor.
In these context the chemistry reported in the following are basic studies for the development of possible treatment
for MoCD and ISOD.
26
3. Results and discussion
3.1 Modelling molybdopterin (MPT)
The modelling of natural products is of utmost importance for the medical research and in general for fundamental
biological investigations. Succeeding the elucidation of the Moco structure by K.V. Rajagopalan, J.L. Johnson in 1992
[64] the literature output on MPT, its chemistry and biology increased. Many studies were centred on MPT models, but
many aspects of the fundamental behaviour of the Moco enzymes have to be clarified still. In the last quarter of the
20th century different synthetical approaches were developed and some good model compounds were synthesized.
The numerous and rich portfolio of results make a comprehensive schematic résumé extremely difficult, but some
important works are worthy to be mentioned.
The first is the entire synthesis of the masked form of MPT reported by B. Bradshaw, C. D. Garner and J. A. Joule et al.
in 2001 [65]. This publication is the closure of an intense research started in the 90’s with the aim to develop a protocol
for the synthesis of MPT [66-68]. Initially, the quinoxaline-halogen derivative II was investigated as an easier platform
for the coupling reaction and later they prepared pterin substrate III comprising all the necessary protections (Figure
13).
The laborious protocol permits the preparation of product VI, which represents the closest synthetic system to the MPT
natural product. Many issues were encountered during the development of the coupling reaction, in particular low
yield, no reactivity and homocoupling of the dithiolene partner I. Finally, it was optimized a Stille cross-coupling with
copper thiophene-2-carboxylate as a stochiometric mediator with sixty percent of yield [69].
Complexation reactions of this ligand have been heralded, but never published, probably due to the long and laborious
procedure that makes the following step extremely costly and difficult. Speculations are not helping at all, rather, it is
useful to contemplate this huge work as the first big effort in the synthesis of the natural product MPT and extract all
the information necessary for future research.
FIGURE 13 – REAGENTS OF THE COUPLING REACTION OF B. BRADSHAW
27
In this publication are reported, in fact, many important mechanisms besides the cross-coupling reaction. First of all,
the formation of the pyran ring was carried out by an elegant reduction-protection, protocol introduced by F.W. Fowler
in 1972 as a protected-stabilized form of reduced pyridines using sodium borohydride in the presence of methyl
chloroformate [70]. Interestingly, in the case of the quinoxaline derivative (Scheme 19) the reaction with benzyl
chloroformate in absence of solvent or added-base and at room temperature proceeds toward the formation of the
pyran ring without reductant; moreover only one diastereoisomer was found [68].
SCHEME 19 – REDUCTION-PROTECTION PROTOCOL WITH SIMULTANEOUS RING CLOSING STEP
In the case of MPT synthesis, the ring protection-reduction leads to the formation of two diastereoisomers, among
which the unwanted one comes as minor product and can be recycled by separation through chromatography column.
Another important aspect is the second reduction of the pyrazine moiety with cyanoborohydride, producing only one
diastereoisomer as for quinoxaline, without cleaving the dithiolene masked form, promising a sufficient stability also
for the model compounds synthesized in this dissertation.
The final step is the introduction of the protected phosphate unit carried out with N,N-diisopropyl-bis-
[2(methylsulfonyl)ethyl]phosphoramidite in the presence of triazole [71]; also this reaction could be used to implement
the compounds reported in the following.
28
The best model-compound for the molybdenum cofactor was synthesized by Burgmayer Group in 2007, supported even
by crystal structures [72]. The synthetic approach described in Scheme 20 employs the direct reaction between a
molybdenum polysulfide with an unsaturated bond, inserted through Pd C-C coupling reaction at the 6 position of a
pterin-substrate [21]. This protocol is of extreme utility and permitted the production of different molybdenum
complexes; the only limitation is the necessary presence of an electron-withdrawing group at the alkyne functionality.
The exceptionality of this model is even more amplified by the observation of an interesting solvent-dependent
pyranopterin cyclization; it has been, in fact, proven an interconversion between tricyclic and bicyclic forms, thought
the insertion of the hydroxy group close to the 7 position of the pterin moiety. This phenomenon, reminiscent of the
covalent addition as classical behaviour of pteridines has been hypothesized to being part of the catalytic mechanism
of Moco [73]. Presumably the only downside of this model compound is the water insolubility (not favoured by the huge
organic moiety), which is a critical point for the planning of biological studies.
The last example is the most relevant for this dissertation and it permits the preparation of pterin-dithiolene ligands.
The most common protocol for the synthesis of dithiolene in masked form starts from a carbonyl functionality [74]. On
this basis, J.A. Joule et. al. have optimised a protocol for the synthesis of asymmetrical dithiolene ligands from a carbonyl
attached on the 6 position of pterin. Moreover they proved the feasibility of this method for the preparation of bis-
dithiolene complexes [75] (Figure 14). Besides the great achievement, the synthesis of the pterin substrate remains
quite complicated starting from 6-formylpterin, prepared with a fifty percent of yield by the hydrobromic acid
degradation of commercial folic acid; furthermore the construction of the acetic groups requires different synthetical
passages [76].
SCHEME 20 – SYNTHETICAL SCHEME TAKEN FROM REF.[72]
29
FIGURE 14 – COMPLEXES BY J.A. JOULE ET AL WITH PTERIN-DITHIOLENE LIGANDS
Like for the model compound of Burgmayer’s group, the poor water solubility remains a disadvantage. In fact, the
isolation of the molybdenum complex in pure form had required, the exchange of the counter cation with consequent
decrease of the water solubility.
The main observation arising from these three approaches are the particular hurdles in Moco modelling research; the
topic requires high familiarity with the organic synthesis of natural products, one of the most challenging fields in organic
synthesis and last but not least high competence in handling the complexation reactions.
This dissertation is part of a wider project centred exactly on this problematic, with the scope to put aside the
complicated task of the entire MPT synthesis and focus its attention on the subunits composing Moco; namely three
important moieties are targeted – the pterin rings, the pyrazine-pyran bicyclic system and the phosphate lateral chain
(Figure 15). Each of these subunits has a particular influence on the cofactor activity and the study of relative complexes
with molybdenum represents a possible key to a better understanding of the Moco principals of functions.
Theoretical calculations have suggested a partial influence of the pyrazine ring on the active site; the extension with
also the pyran unit which has been already indicated as participating in the activity with its scission-cyclization
mechanism, constitutes a desirable model compound [77]. The phosphate functionality does not have an electronic
influence on the main structure, however it is surely able to create hydrogen bonding and keep the cofactor tightly
inside of the apoenzyme’s pocket.
The pterin moiety has been also exonerated from any electronic role in Moco [77], but it’s great ability to create
intermolecular hydrogen bonding plays most probably a role; recent vibrational studies have suggested a mechanism
of fine-tuning Moco for electron and atom-transfer reactivity in catalysis [78]. Finally, the dithiolene moiety is of extreme
importance, since it is directly connected to the metal centre and well known as a non-innocent ligand system [79].
In this research work, the pterin unit has been investigated and the results are discussed in the following text, moreover
the cooperation with a co-worker has permitted the synthesis of new interesting structure combinations of pterin with
the phosphate subunit.
Further biological characterization is planned, namely the study of a possible interaction between proteins involved in
the maturation of the molybdenum dependent enzymes and the apoenzymes (see chapter 2 “Biological Background”).
30
FIGURE 15 – BREAK DOWN APPROACH FOR MPT
31
3.2 Protection of Pterins
The synthesis of pterin-dithiolene ligands has already been successfully achieved by the two approaches described
above and in both cases the researchers had to face the solubility problems of pterins. Mother issue has been the
selection of suitable protection for the amine and amide functionalities in order to increase the solubility in organic
solvents. [80].
The protecting group was chosen taking into account the stability during the synthetical pathway and also the potential
cleavage at the end of the process, which permit the modelling of the natural free form. The literature is rich of possible
pterin derivatives with blocked functionality, and in most of the cases an increment of solubility has been described,
but a detailed respective evaluation of the large assortment is not reported. Thus, the most often used protections with
relative good stability were chosen. Two different pterins were prepared according to literature carrying two different
protecting groups concomitant – methanimidamide N'-(6-chloro-3,4-dihydro-4-oxo-2-pteridinyl)-N,N-dimethyl-(5) and
2-pteridinamine, 4-(pentyloxy)-(10) (Figure 16).
Substrate 5 is the starting material of the coupling protocols developed by C. D. Garner and J.A. Joule (Figure 13) for the
synthesis of the masked form of MPT previously describe [65]. This product was protected at the amino group employing
a particular product, called Bredereck’s reagent, usually used in α-methylation, α-amination of various carbonyl systems,
ring-opening polymerization and indole synthesis [81]. Substrate 10 was prepared following a procedure by D. Mohr, Z.
Kazimierczuk and W. Pfleiderer for the synthesis of 6-thioxanthopterin and 7-thioisoxanthopterin [82].
Both protecting groups are cleaved under strong basic conditions, which is typical step of a complexation reaction with
molybdenum, thus, removable at the end of the entire synthetic pathway. Product 10 can be also hydrolysed by acid,
albeit with lower efficiency [82].
As illustrated in Scheme 21, the synthesis of 5 starts with the formation of the pyrazine ring 1 and builds up the
pyrimidine by condensation with a guanidine salt. After hydrolysis of the amino group product 3 is obtained. The
interesting and critical transformation is the insertion of the chlorine atom (see chapter 1 “Pteridine and Pterins”). The
use of the N(8)-oxide form induces a stereospecific insertion at position 6 of the pyrazine ring and yields the chemical
structure 4. While the chemistry of N-oxide heterocycles would suggest an α-rearrangement, in this case the position 6
is preferred by a faster β-rearrangement accompanied by the release of the oxide group [83]. In the last step, the amino
group reacts with Bredereck’s reagent with a consequent increase of the solubility.
FIGURE 16 – 5 BREDERECK’S REAGENT AS PROTECTION, 10 PENTHYLOXY
PROTECTION
32
SCHEME 22 – CARBONYL PROTECTION, PENTYLOXY
The preparation of substrate 10 is an Isay-Gabriel condensation [80], that is the reaction between a pre-protected
pyrimidine 7 with a glyoxal molecule. Using the previous N(8)-oxide protocol a chlorine atom is then stereospecifically
attached at position 6 (Scheme 22).
SCHEME 21 – AMINO PROTECTION, BREDERECK’S REAGENT
33
Comparison of the two products reveals a higher solubility of 10 in a larger variety of solvent, in particular in those with
low polarity, while product 5 surprisingly exibits a better solubility in more polar solvents. This led us to first attempting
to use the pentyloxy as protecting group.
Moreover, the high solubility of the substrate in a broad range of solvents allowed the application of different crystal-
growing techniques and eventually x-ray characterization which hasn’t been avalable before (Figures 17,18,19).
FIGURE 19 – MOLECULAR STRUCTURE OF PRODUCT 10
BASED ON NOT ENTIRELY RELIABLE STRUCTURAL DATA
FIGURE 17 – MOLECULAR STRUCTURE OF PRODUCT 6
FIGURE 18 – MOLECULAR STRUCTURE OF PRODUCT 9
34
In this dissertation the major part of the synthetic work is focused on the synthesis of 6-acyl pterins as starting point for
the construction of the dithiolene ring. Nevertheless, some attempts to prepare also a coupling partner were
undertaken, e.g. the reaction reported below in Scheme 23 for the preparation of the iodine derivative was tested with
the undesired cleavage of the pentyloxy protection as result.
SCHEME 23 – ATTEMPT TO EXCHANGE THE CHLORINE BY IODINE USING HYDROIODIC ACID
35
3.3 First attempt for the synthesis of pterin-dithiolene ligand, preparation of 6-acyl
pterins by condensation
The first attempt for the synthesis of a pterin-dithiolene ligand started with a stereoselective Timmis condensation
reaction [80, 84] as reported by W. Pfleiderer and G. Heizmann [85]. This condensation allows the preparation of 6-
acetyl-4-cyclohexyloxy-7-methylpterin (Scheme 24).
SCHEME 24 – SYNTHESIS OF A 6-ACYL PTERIN REPORTED BY PFLEIDERER
Encouraged by the positive crystallographic results and increased solubility due to employing the pentyloxy protecting
group, it was decided to modify the reaction and use the nitroso pyrimidine intermediate 6 and proceed with the
condensation with acetylacetone (2,4-pentandione, Scheme 25).
SCHEME 25 – SYNTHESIS OF 4 PENTYLOXY-6-ACYL-7 METHYL PTERIN BY A MODIFIED PROTOCOL OF W.PFLEIDERER
The targeted condensation product 11 was obtained with a yield of eighty percent. Growing crystals by the slow
evaporation method gave, as desired, a good and reliable crystal structure reported in Figure 20 below.
36
FIGURE 20 – MOLECULAR STRUCTURE OF PRODUCT 11
The successive transformations, following the procedure for the masked form of dithiolenes of C. D. Garner, comprise
bromination in α-position of the carbonyl and substitution with isopropyl xanthate. Among a large number of
brominating agents it was decided to use PHT (2‐Pyrrolidone hydrotribromide) as mild reagent that permits maintening
the protecting group; moreover PHT is described in the literature as highly selective for ketones [86, 87].
In particular, the combination of PHT with an excess of 2-pyrrolidone prevents any acidic or basic hydrolysis due to the
formation of a hemihydrobromide salt with the leaving hydrobromic acid as described in Scheme 26.
SCHEME 26 – BROMINATION WITH PHT
As a precautionary measure the bromination of the synthesized 6-acyl pterin 11 was carried out in dried tetrahydrofuran
since the brominating agent is sensitive to water. Moreover, this avoids the possible involvement of an acidic or basic
environment and also the possible substitution of the bromine atom. After bromination the crude product was treated
with potassium isopropyl dithiocarbonate as a source of the protected dithiolene sulfur and oxygen atoms. This reaction
proceeds at room temperature in acetone with the precipitation of potassium bromide as clear white solid. The product
obtained and purified was supposed to be the expected product with a xanthate moiety in α-position of the carbonyl
attached to position 6 (Scheme 27).
SCHEME 27 – BROMINATION WITH PHT AND SUBSTITUTION WITH ISOPROPYL XANTHATE
37
The elemental analysis and 1H and 13C NMR supported the success of the synthesis of the targeted structure. However
results obtained later revealed a misinterpretation of the analytical data and an unexpected synthetical side reaction
(Table 1).
TABLE 1 – ELEMENTAL ANALYSIS OF THE EXPECTED PRODUCT
Even though the clarification of this synthetic struggle is not the topic of this dissertation, the problems faced are of
value for the next person working with these structures and of analytical interest for the understanding of future studies.
In particular for the interpretation of NMR spectra, described below, previous publications reporting pterins carrying
the pentyloxy protecting group [88-92] were taken into considerations, together with the analysis of the new products
prepared in this dissertation. A mechanistic assumption about the result and a crystal structure is provided in the final
part of this section, with the confidence that the substrate obtained is not suitable for the main aim of this dissertation,
or at least not with the applied protocols. Therefore, after really intense and long work dedicated to this product, it was
decided to shelve it and focus the efforts on the acylation protocol described in the following.
Expected product N C H S
Calculated 16.53 51.04 5.95 15.14
Measured 16.31 50.53 5.64 16.31
38
The original assignment of the 1H NMR signals of the pentyloxy protected addresses the typical overlap of the CH2 peaks
on the alkyl chain B – C and the broad signal of the amino function H, which is in many cases not visible. The other
signals were easily assigned as shown in Spectrum 1, except for the singlets E and F which belong either to the CH3 at
the 7 position of the pterin or to the CH3 in α-position of the carbonyl (Spectrum 1).
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
No
rma
lize
d In
ten
sity
3.024.062.182.993.002.011.94
0.9
30
.96
0.9
8
1.3
81
.391.4
1
1.4
51
.461.4
71
.48
1.4
81
.49
1.5
01
.51
1.5
51
.56
1.9
11
.93
1.9
6
2.7
6
2.9
2
4.5
34
.55
4.5
8
6.0
0
7.2
8
Spectrum 1 – 1H NMR of product 11 1H NMR (300MHz ,CHLOROFORM-d) δ = 6.00 (br. s., 1 H), 4.55 (t, J = 6.8 Hz, 2 H), 2.92 (s, 3 H), 2.76 (s, 3 H),
2.00 - 1.85 (m, 2 H), 1.60 - 1.35 (m, 4 H), 1.04 - 0.88 (m, 3 H) ppm
The 13C NMR spectra were assigned based on the precedent literature and the additional application of a DEPT 135 13C
NMR experiment, which clearly shows the presence of the CH2 negative signals, the CH/CH3 positive signals and the
disappearance of the quaternary carbons. Like for the proton NMR, the exact assignment of the C7-CH3 and CH3-C=O
signals at 27.47 and 25.15 ppm is not possible using this method (Spectrum 2).
D
A
C B
D
E or F
G
H
A
B – C
H F
E G
39
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
13
.96
22
.33
25
.13
27
.46
28
.06
68
.50
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
No
rma
lize
d In
ten
sity
13
.97
22
.32
25
.1427
.47
28
.07
68
.49
Spectrum 2 – 13C NMR DEPT 135, product 11
13C NMR (75 MHz): δ = 14.0 (CH3), 22.3 (CH2), 25.1 (CH3), 27.5 (CH3), 28.1 (CH2, CH2 ), 68.5 (CH2) ppm
The original interpretation of the 13C NMR spectrum assigned an overlap of the γ and δ carbon atoms at 22.32 ppm. Nevertheless, the structure C’ which will be described later, reveals the presence of two almost overlapped signals around 28 ppm. This belongs most probably to the β and γ CH2 of the extranuclear alkyl chain (Spectrum 2).
ε
δ β
α γ
C7-CH3 / CH3-C=O
β
δ
α
γ
ε
CH3-C=O
C7-CH3
40
200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
12
0.6
1
14
2.6
6
15
7.0
6
16
1.3
7
16
3.0
0
16
7.8
7
19
9.8
9
Spectrum 3 – 13C NMR of product 11, quaternary carbons 13C NMR (75MHz ,CHLOROFORM-d) δ= 199.9, 167.9, 163.0, 161.4, 157.1, 142.7, 120.6 ppm
The bromination and substitution with sodium isopropyl xanthate is accompanied by the appearance of three important
signals – the peaks L and I belonging to the O-isopropyl and a singlet at 4.9 ppm (E’ or F’) with integration 2, which is
ascribable to a CH2 that has been brominated first and then received the xanthate unit. Indeed, one of the CH3 has
disappeared (Spectrum 4).
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.016.114.041.992.612.002.011.042.00
DMSO
Water
0.8
80
.90
0.9
3
1.2
91
.31
1.4
11
.42
1.4
6
1.7
91
.821.8
41
.86
1.8
9
2.6
4
4.4
94
.51
4.5
3
4.9
0
5.5
45.5
65
.58
5.6
05
.62
5.6
45
.677
.68
7.8
0
7.9
3
8.0
6
Spectrum 4 – 1H NMR product of bromination and substitution with xanthate (expected product) 1H NMR (DMSO-d6) δ = 7.80 (br. s., 1 H), 7.68 (br. s., 1 H), 5.60 (td, J = 6.3, 12.3 Hz, 1 H), 4.90 (s, 2 H), 4.51
(t, J = 6.6 Hz,2 H), 2.64 (s, 3 H), 1.84 (quin, J = 6.9 Hz, 2 H), 1.52 - 1.35 (m, 4 H), 1.30 (d, J = 6.4 Hz, 7 H), 0.94 - 0.85 (m, 3 H) ppm
L
I
E ’or F’
C=O
C-2 C-8A
C-4
C-6
C-7
C-4a
A
C B
D
E or F
G
H
I
E’ or F’
L
C=O
41
The relative good matching of the analytical with the expected data encouraged us to proceed to the second and final
step for the synthesis of a pterin-dithiolene ligand. This step was therefore carried out using concentrated sulfuric acid,
according to the method described by C. D. Garner et al. [76]. Unfortunately, applying the reported protocol did not
give any result; In particular, the only effect observed was the hydrolysis of the protecting group within two or three
days in pure sulfuric acid.
Subsequently sodium diethyldithiocarbamate was used in the nucleophilic substitution of bromine.The typical signals
of the carbamate chain N and O arrised coherently with the previous product. After purification and characterization
the same protocol was applied with the hope of a different outcome, but unfortunately also in this case no reaction was
observed.
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.143.283.194.012.042.562.202.392.032.172.04
DMSO
Water
0.8
80
.90
0.9
31
.12
1.1
41
.24
1.2
61
.38
1.4
21
.44
1.4
61.8
21.8
41
.86
2.6
4
3.7
53
.77
3.9
03
.93
4.4
84
.51
4.5
3
4.9
9
7.6
57
.70
7.7
8
Spectrum 5 – 1H NMR product of bromination and substitution with carbamate (experimental section, B’- carbamate
derivative) 1H NMR (300MHz ,DMSO-d6) δ = 7.65 (s, 1 H), 7.70 (s, 1 H), 4.99 (s, 2 H), 4.50 (t, J = 6.6 Hz, 2 H), 3.91 (q, J = 6.5 Hz, 2 H), 3.76 (q, J =
6.9 Hz, 2 H), 2.64 (s, 3 H), 1.84 (quin, J = 6.9 Hz, 2 H), 1.53 - 1.33 (m, 4 H), 1.24 (t, J = 7.0 Hz, 3 H), 1.20 - 1.09 (m, 3 H), 0.97 - 0.85 (m,
3 H) ppm
The protonation of the amino group, which leads to a change in the electronic distribution of the pterin ring has been
postulated to be the cause of the unreactivity of the substrate. The previous examples in the literature were in fact
either protected or proposed to have a different basicity. Therefore it was also tried to reduce the acidic strength and
concentration using a diluted solution of sulfuric acid, hydrochloric acid, perchloric acid and acetic acid. Later, it was
also tried to reduce the contact surface between the reagent by the employment of HCl in gas form, with the
understanding that the ring closing is an acid catalyzed reaction.
The hydrochloric acid in gas form was obtained by the controlled reaction between sodium chloride and concentrated
sulfuric acid. The apparatus was constructed by joining two schlenks, one containing sodium chloride and the other
equippedt with a filter carrying the substrate and an outlet directed to a basic water solution (sodium hydroxide). During
the dropwise introduction of sulfuric acid, the developed HCl(g) was transported through the filter with the help of a soft
nitrogen gas flow. The experiment resulted in a color change of the powder product, but successive 1H NMR analysis
shows the intact isopropyl xanthate chain and only a shifting of one of the aminic protons to high-field, probably due to
the lower shielding caused by protonation (Figure 21, Spectrum 6).
H
i
A
C B
D
G
N
O
N
O
E’ or F’
E or F
E’ or F’
42
FIGURE 21 – PROTONATION OF THE AMINO FUNCTION WITH HYDROCHLORIC ACID GAS
10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.053.183.524.142.023.122.092.091.982.040.880.93
0.9
30
.95
0.9
81
.20
1.3
01
.32
1.3
41
.47
1.4
91
.50
1.5
21.9
31.9
61
.98
2.0
0
2.8
2
3.6
93
.71
3.7
43
.85
3.8
73
.90
4.7
04
.72
4.7
54
.90
7.7
2
9.8
2
Spectrum 6 – 1H NMR of protonation of the amino group by reaction with hydrochloric acid in gas form(Experimental section, B’-
Carbamate derivative protonated) 1H NMR (300MHz ,CHLOROFORM-d) δ = 9.82 (br. s., 1 H), 7.72 (br. s., 1 H), 4.90 (s, 2 H), 4.72 (s, 2 H), 3.86 (q, J = 6.9 Hz, 2 H), 3.73 (q,
J = 6.7 Hz, 2 H), 2.82 (s, 3 H), 1.96 (quin, J = 6.8 Hz, 2 H), 1.57 - 1.37 (m, 4 H), 1.32 (t, J = 7.0 Hz, 3 H), 1.18 (t, J = 7.2 Hz, 3 H), 1.02 - 0.89 (m, 3 H) ppm
In order to avoid protonation, the amino group was protected by a mixture of acetic acid and acetic anhydride
(AcOH/AcO2), a typical method for pterins and previously described in different pubblications [93]. After three hours of
refluxing the reaction mixture, two products were isolated. The first, a white solid was characterized by 1H NMR and
recognised to be the protected form of the starting material by the formation of the CH3 signal M and the shifting of
one of the aminic protons H’ (Spectrum 7).
The second product, a deep red solid, shows in the 1H NMR the protection of the amino group, the disappearance of
the xanthogenate unit and, most importantly a singlet signal P, at 7.78 ppm, that was first wrongly assigned to a proton
of the dithiolene ring (Spectrum 8).
The hydrolysis of the protecting group in the red powder product was carried out in methanol at room temperature
overnight (experimental section- red solid). As aspected the amino group signal H reappeared around 5.7 ppm and the
methyl group of the protection disappeared (Spectrum 9). Aside the formation of a small broad signal at 2.04 ppm was
observed and initially assigned to some impurities due to variation of the integration values. Later, the position,
stabilized integration of 1 and the characteristic broadness of this new signal was ascribed to a hydroxy or thioxy
functionality and the appearance over time was attributed to the general instability or reactivity of the targeted product
(Spectrum 9).
H
H
A
C
B
D
G
N
O
E or F
E’ or F’
8.3 8.2 8.1 8.0 7.9 7.8 7.7 7.6 7.5 7.4 7.3 7.2 7.1 7.0 6.9
Chemical Shift (ppm)
2.00
7.6
5
7.7
0
7.7
7
43
11.0 10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00N
orm
aliz
ed
Inte
nsi
ty
3.006.104.342.013.012.821.961.941.000.95
DMSO
Water
0.8
80
.91
0.9
3
1.3
01
.32
1.4
31
.45
1.4
6
1.8
61
.88
1.9
11
.93
2.3
5
2.7
1
4.6
14
.63
4.6
5
4.9
8
5.5
45.5
65.5
85.6
05
.62
5.6
45
.66
10
.92
Spectrum 7 – 1H NMR of protection of the amino group, first product isolated (experimental section, B’ - white solid) 1H NMR (300MHz ,DMSO-d6) δ = 10.92 (s, 1 H), 5.60 (spt, J = 6.2 Hz, 1 H), 4.98 (s, 2 H), 4.63 (t, J = 6.6 Hz, 2 H), 2.71 (s, 3 H), 2.35 (s, 3
H), 1.88 (quin, J = 6.9 Hz, 2 H), 1.51 - 1.36 (m, 4 H), 1.32 (s, 3 H), 1.30 (s, 3 H), 0.94 - 0.88 (m, 3 H) ppm
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.104.382.153.003.012.091.001.01
0.9
30
.95
0.9
7
1.3
81.4
01
.46
1.4
71
.48
1.5
0
1.8
61.8
81
.91
2.6
62
.68
4.4
84
.50
4.5
2
7.2
67.7
8
8.1
6
Spectrum 8 – 1H NMR of protection of the amino group, second product isolated (experimental section, C’ - red solid) 1H NMR (300MHz ,CHLOROFORM-d) δ = 8.16 (s, 1 H), 7.78 (s, 1 H), 4.50 (t, J = 6.8 Hz, 2 H), 2.68 (s, 3 H), 2.66 (s, 3 H),
1.95 - 1.83 (m, 2 H), 1.53 - 1.36 (m, 4 H), 1.03 - 0.87 (m, 3 H) ppm
L
H’
I
i
M
A
C
B
D
G
E or F
I
M
H’
A
C
B
D
G
P
E or F
E’ or F’
H’
M
H’
M
P
44
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.034.062.021.062.952.001.810.96
0.9
20
.95
0.9
7
1.3
81.4
21
.44
1.4
51
.46
1.4
71
.81
1.8
31.8
61
.88
2.0
4
2.6
2
4.4
14
.43
4.4
6
5.7
1
7.7
0
Spectrum 9 – 1H NMR of hydrolysis of protection, second product isolated (experimental section - C’)
1H NMR (300MHz ,CHLOROFORM-d) δ = 7.70 (s, 1 H), 5.71 (br. s., 2 H), 4.43 (t, J = 6.8 Hz, 2 H), 2.62 (s, 3 H),
1.86 (quin, J = 7.1 Hz, 2 H), 1.52 - 1.38 (m, 4 H), 0.99 - 0.90 (m, 3 H) ppm
The elemental analysis was partially fitting except for the sulfur content, which was lower than expected. Also, the
deprotected derivative obtained by hydrolysis with hydrochloric acid, exhibited the same decrease in sulfur content
(Scheme 28, Table 2, experimental section, C’- C’ without pentyloxy chain).
TABLE 2 – ELEMENTAL ANALYSIS OF THE EXPECTED PRODUCT (THEORETICAL) AND OF THE ISOLATED PRODUCT C’ AND THE DEPROTECTED FORM
Mass spectrometry showed a peak at 348.3 m/z and not the expected 364(H+) m/z. Nevertheless, considering possible
fragmentation and the basic instability of the compound, the achievment of the targeted structure could not be
excluded and the complexation reaction with K3Na[(MoO2)(CN)4] was tried as shown in Scheme 29.
Red powder h. N C H S
Theoretical 19.27 49.57 4.71 17.64
Experimental 19.54 51.57 4.68 9.19
Expected C’ dep. N C H S
Theoretical 23.88 40.95 2.41 21.86
Experimental 21.90 41.27 3.22 10.06
A
C
B
D
G
H
– CH – OH
– SH
E or F
– CH
SCHEME 28 – ABOVE DEPROTECTION WITH HYDROCHLORIC ACID(YIELD 42%)
45
SCHEME 29 – COMPLEXATION REACTION WITH THE ISOLATED PRODUCT
TABLE 3 – POSSIBLE ELEMENTAL ANALYSIS OF THE COMBINATION WITH THE COUNTER CATIONS
The protocol for the complexation with molybdenum contemplates a final purification step of the dianionic product.
This can be achieved by exchange of the counter cations with tetraphenylphosphonium chloride, by precipitation with
crown ether or by extraction with acetonitrile in case of enough solubility in organic solvent. Employment of all these
methods did not give any results and the presence of inorganic salts, mainly cyanide, was always detected by IR
spectroscopy. The best result was obtained by washing the inorganic salts with a small amount of methanol
(experimental section, C’- complexation product).
The NMR spectra did not give interpretable results and the elemental analysis, coherently with the previous step,
showed a lower content of sulfur. The IR spectrum shows two bands in the range of the double bond of the dithiolene
C=C at 1506 cm-1 and the characteristic Mo=O at 835 cm-1 typical of the bis-dithiolene complexes [75] (Spectrum 10).
Complexation N C H S
theoretical with 2 Na+
20.34 31.40 2.05 18.63
theoretical with 2 K+
19.43 29.99 1.96 17.80
experimental 18.91 32.11 2.73 4.78
SPECTRUM 10 – IR OF THE COMPLEXATION PRODUCT
46
An explanation of the collected data, incongruent with the synthetical aim, arises from two crystal structures obtain
from the complexation mixture and from the red powder product after reaction with the acetic acid and acetic
anhydride mixture (Figure 23, 23).
FIGURE 23 – COLOURLESS CRYSTALS FROM COMPLEXATION MIXTURE
Both structures may result from the decomposition of the desired product. However the presence of a totally intact
methyl group on position 6 of the pterin, points toward a failure already during the bromination reaction. Most
probably, the reaction has accurred on the methyl group at position 7 of the pterin, and this apparently with a high
selectivity since no other side products were isolated or observed. Therefore this 6-acyl pterin can aparently not be used
as a substrate in this specific protocol and the high selectivity for the 7 methyl position over the α-position must be
taken into account for future work. Further attempts to brominate the α-carbonyl position with bromine (and acetic
acid as catalyst) leads to the cleavage of the protecting group and it was eventually not contemplated as a possible
solution any further.
A comprehensive explanation of the side reaction can be derived from a study reported in 2004 by A. Kalinin and V. A.
Mamedov for the synthesis of thiazole-quinoxaline from chloroderivatives with the use of carbon disulfide [94]. The
group observed the formation of thiazole C by the reaction of the quinaxoline-chloride substrate in basic condition in
presence of carbon disulfide (Scheme 30)
This reaction is similar to a Cook–Heilbron synthesis [95] and has been postulated to go through a xanthogenate
derivative in the reported work. Therefore, in order to confirm their hypothesis, they synthesized product B and
recovered the same product C with an acid catalysed ring closing reaction (Scheme 30)
Notably the applied reaction conditions were very similar to our protection protocol (AcOH/Ac2O at reflux). Even
through TFA (trifluoroacetic acid) is far stronger than acetic acid, the prolonged refluxing time may eventually lead to
the same product. It is, thus, possible to propose a similar pathway (Scheme 31).
FIGURE 22 – CRYSTAL STRUCTURE FROM THE RED POWDER
47
SCHEME 30 – SYNTHESIS OF THIAZOLE-QUINOXALINE [94]
As depicted in Scheme 31, the first step presumably is the bromination of the methyl group at 7 position (instead of the
α-position) and formation of the product A’ followed by substitution with isopropyl xanthate that leads to product B’.
Finally the reflux in acetic acid reults in the formation of the five membered cyclic thiazole between position 7 and 8.
SCHEME 31 – PROPOSED MECHANISM AS DERIVED FROM THE SYNTHESIS OF THIAZOLE QUINOXALINE
48
The proposed final product C’ is in perfect accordance with the elemental analysis of the C’, including the sulfur content (Table 4).
TABLE 4 – ELEMENTAL ANALYSIS OF PRODUCT C’
Also MS-APCI spectrometry shows the exact mass peak and a fitting fragmentation, i.e. corrisponding to a loss of the
pentyloxy chain , as observed for the majority of the 6-acyl pterins synthesized in this dissertation (Figure 24).
FIGURE 24 – MASS SPECTRA OF THE PRODUCT C’ POSITIVE AND NEGATIVE MODUS
Product-C’ N C H S
Theoretical 20.16 51.86 4.93 9.23
Experimental 19.54 51.57 4.68 9.19
348.3 [M + H]+
278.2 [M1 + H]+
[M1] = [M] =
346.2 [M - H]-
49
Also the 1H and 13C NMR are in good accordance with the proposed structure of C’ (Spectra 11, 12, 13)
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.034.062.021.062.952.001.810.96
0.9
20
.95
0.9
7
1.3
81.4
21
.44
1.4
51
.46
1.4
71
.81
1.8
31.8
61
.88
2.0
4
2.6
2
4.4
14
.43
4.4
6
5.7
1
7.7
0
Spectrum 11 – 1H NMR product C’
1H NMR (300MHz ,CHLOROFORM-d) δ = 7.70 (s, 1 H), 5.71 (br. s., 2 H), 4.43 (t, J = 6.8 Hz, 2 H), 2.62 (s, 3 H), 1.86 (quin, J = 7.1 Hz, 2 H), 1.52 - 1.38 (m, 4 H), 0.99 - 0.90 (m, 3 H) ppm
200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
No
rma
lize
d In
ten
sity
10
8.2
9
12
4.9
5
14
1.4
8
14
9.4
1
16
2.0
0
16
6.1
71
67
.05
19
8.0
1
Spectrum 12 – 13C NMR of C’ quaternary carbons only
13C NMR (75MHz ,CHLOROFORM-d) δ = 198.0, 167.0, 166.2, 162.0, 149.4, 141.5, 125.0, 108.3 ppm
D
A
C B
D
E
F
G
A
B
G
E F
H H
C
C=O
C-2
C-8a C- 4
C-6
C-7
C-4a
S-C=O
C=O
S-C=O
50
105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
14
.00
22
.35
25
.65
28
.08
28
.26
67
.94
76
.57
76
.997
7.4
1
10
3.3
5
105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
No
rma
lize
d In
ten
sity
13
.99
22
.33
25
.65
28
.06
28
.25
67
.92
10
3.3
4
Spectrum 13 – 13C NMR, DEPT 135, product C’
13C NMR (75 MHz): δ = 14.0 (CH3), 22.3 (CH2), 25.7 (CH3), 28.1 (CH2), 28.2 (CH2), 67.9 (CH2), 103.3 (CH)ppm
The 13C NMR spectrum, as previously mentioned, shows an overlap of signals β and γ carbon atoms of two CH2 groups
of the pentyloxy chain. Taking into account the proposted structure of C’ the signal at 25.65 ppm is assigned to CH3-C=O
(Spectrum 13).
Product B’ is a constitutional isomer of the expected product. Therefore the elemental analysis and the mass peak are
the same. However, some interesting differences can be observed between the carbamate and the xanthogenate
derivatives. The spectrum with the quaternary carbons shows the appearance of a peak at 212.5 ppm of the thio
carbonyl S–C=S and the shifting of C–7 to high field from 163 ppm to 157 ppm, according to the reaction occurring close
to this position (Spectrum 15).
β
γ
α δ
ε
β
δ
α
γ
ε
C-H
CH3-C=O
CH3-C=O
51
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.016.114.041.992.612.002.011.042.00
DMSO
Water
0.8
80
.90
0.9
3
1.2
91
.31
1.4
11
.42
1.4
6
1.7
91
.821.8
41
.86
1.8
9
2.6
4
4.4
94
.51
4.5
3
4.9
0
5.5
45.5
65
.58
5.6
05
.62
5.6
45
.677
.68
7.8
0
7.9
3
8.0
6
Spectrum 14 – 1H NMR of product B’ 1H NMR (DMSO-d6) δ = 7.80 (br. s., 1 H), 7.68 (br. s., 1 H), 5.60 (td, J = 6.3, 12.3 Hz, 1 H), 4.90 (s, 2 H), 4.51
(t, J = 6.6 Hz,2 H), 2.64 (s, 3 H), 1.84 (quin, J = 6.9 Hz, 2 H), 1.52 - 1.35 (m, 4 H), 1.30 (d, J = 6.4 Hz, 7 H), 0.94 - 0.85 (m, 3 H) ppm
210 205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
No
rma
lize
d In
ten
sity
12
0.3
9
13
9.8
6
15
6.8
615
7.1
6
16
3.3
8
16
6.7
6
19
8.9
3
21
2.5
0
Spectrum 15 – 13C NMR of B’, quaternay carbons only
13C NMR (DMSO-d6) δ = 212.5, 198.9, 166.8, 163.4, 157.2, 156.9, 139.9, 120.4 ppm
L
I
E
L
I
E
F’
F’ A
B
C
D
G
H
A C
B
D
G
H
C – 4a
C=O C – 2
C – 8A
C – 4 C – 6
C – 7
C=S
C=S
C=O
52
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
13
.88
20
.88
21
.80
26
.79
27
.61
67
.56
78
.29
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
13
.88
20
.88
21
.79
26
.79
27
.60
40
.22
67
.56
78
.29
Spectrum 16 – 13C NMR DEPT 135 of B’
13C NMR (DMSO-d6) δ = 13.9 (CH3), 20.9 (CH3, CH3), 21.8 (CH2), 26.8 (CH3), 27.6 (CH2, CH2), 40.2 (CH2), 67.6 (CH2), 78.3 (CH) ppm
The 13C NMR and DEPT 135 experiments support the assignment of the CH3 -C=O moiety similar to the structure of C’
at 26.70 ppm. Also, the S-CH2 carbon atoms can be ascribed to the signal at 40.22 ppm (Spectrum 16).
CH3 -C=O
ε
Xa-(CH3)2
Xa-CH
Xa-(CH3)2
Xa-CH
S-CH2
δ β σ
γ
S-CH2
ε
δ
β γ
α
CH3 -C=O
53
Following this hypothesis also the carbamate derivative can be identified unambiguously by its carbon and proton NMR (Spectra 17, 18, 19).
10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.143.283.194.012.042.562.202.392.032.172.04
DMSO
Water
0.8
80
.90
0.9
31
.12
1.1
41
.24
1.2
61
.38
1.4
21
.44
1.4
61.8
21.8
41
.86
2.6
4
3.7
53
.77
3.9
03
.93
4.4
84
.51
4.5
3
4.9
9
7.6
57
.70
7.7
8
Spectrum 17 – 1H NMR of carbamate derivative 1H NMR (300MHz ,DMSO-d6) δ = 7.65 (s, 1 H), 7.70 (s, 1 H), 4.99 (s, 2 H), 4.50 (t, J = 6.6 Hz, 2 H), 3.91 (q, J = 6.5 Hz, 2 H), 3.76 (q, J = 6.9 Hz, 2 H), 2.64 (s, 3 H), 1.84 (quin, J = 6.9 Hz, 2 H), 1.53 - 1.33 (m, 4 H), 1.24 (t, J = 7.0 Hz, 3 H), 1.20 - 1.09 (m, 3 H), 0.97 - 0.85 (m,
3 H) ppm
200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
No
rma
lize
d In
ten
sity
11
9.8
6
14
1.1
5
15
6.8
81
58
.01
16
3.1
6
16
6.7
6
19
3.6
3
19
8.9
7
Spectrum 18 – 13C NMR of carbamate derivative, quaternary carbons
13C NMR (75MHz ,DMSO-d6) δ = 199.0, 193.6, 166.8, 163.2, 158.0, 156.9, 141.1, 119.9 ppm
N
H
i
A
C
B
D
G
N
O
F’
E
F’
O
E
C-4a C=O
C-2 C-8A
C-4 C-6 C-7 N-C=S
N-C=S
C=O
54
The quaternary carbon spectrum reveals a similar situation with the C-7 shifted to 158 ppm. The N-C=S of the carbamate
unit, appears at higher field compared to the other derivative, probably because of the inductive effect of the two alkyl
substituents and the lower electronegativity of nitrogen compared to oxygen (Spectrum 18).
The carbamate derivative shows a similar S-CH2 signal as the xanthate and the signals which belong to Car-(CH2)2 and
Car-(CH3)2 (Spectrum 19).
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
.31
12
.3813
.87
21
.79
26
.902
7.6
1
41
.79
46
.65
49
.04
67
.52
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
No
rma
lize
d In
ten
sity
11
.30
12
.38
13
.88
21
.80
26
.91
27
.60
41
.79
46
.64
49
.03
67
.51
Spectrum 19 – 13C NMR DEPT 135 of carbamate derivative 13C NMR (75MHz ,DMSO-d6) δ= 11.3 (CH3), 12.4 (CH3), 13.9 (CH3), 21.8 (CH2), 26.9 (CH3), 27.6 (CH2, CH2), 41.8 (CH2),
46.6 (CH2), 49.0 (CH2), 67.5 (CH2) ppm
Car-(CH3)2
Car-(CH2)
CH3 -C=O
ε – CH3
γ
σ S-CH2
δ
β
Car-(CH3)2
Car-(CH2)2
S-CH2
CH3 -C=O
ε
δ
γ
β
α
55
The signal at 2.04 ppm, in the 1H NMR of C’, appearing over time may arise from S-oxidation, which occurs in solution
and results in the formation of the sulfur bearing an -OH functionality. This is, in fact, a possible mechanism leading to
the chemical structure shown in Scheme 32, accompanied by the re-aromatization of the pyrazine ring. Keeping the
product in solution with methanol or chloroform for a prolonged time does not change the MS peak and no other signals
arise, indicating a chemical species not stable enough to be observed or the presence of an equilibrium only in solution.
Possibly by a light driven mechanism during data collection with the diffractometer or due to long exposure to light a
transformation to species E’ occured. This would be in accordance with pterins being natural photosensitizer [25].
SCHEME 32 – POSSIBLE STEPS OF S-OXIDATION AND ACTUAL RESULT OF THE COMPLEXATION ATTEMPT
Intrestingly, a similar reaction is described as first step in the metabolism of the thiazolidinedione (TZD) ring, catalyzed
by cytochrome P450 [96, 97]. The employment of this structure as model compounds represents an interesting research
opportunity.
Refering to structures C and C’, respectively, the presence of a phenyl substituent, a typical electron-pushing group,
instead of a proton plays most probably a role in the stabilization of the thioazole ring. Indeed, the distinct aromaticity
of the quinoxaline compare to the pteridine rings must be taken into account.
The attempted complexation reaction in – basic condition under the false presumption that the targeted ligand
precursor had been made, leads to the formation of product F’ by hydrolisis of the nitrogen at postion 8 and formation
of a new five membered ring by the attack of sulfur on carbons (Scheme 32). Similar structure arises from the oxidation
of the natural Moco [98] and they are quite common in other research work on pterins [99]. Further speculations
regarding the mechanism of this side reaction would require more in depht-studies, which are not inherent to the main
goal of this dissertation. Therefore it was decided to proceed to the synthesis of other 6-acyl pterins by the acylation
protocol described in the following. Beside the formation of colorless crystal of F’ a brownish powder was isolated G’
(experimental section,C’- complexation product, code IT 114, Scheme 32). Interestingly, its incubation with the protein
apoTor A indicates in the respective UV spectrum the formation of specific binding compared to the unspecific binding
56
on the external protein suface developed with BSA (bovine serum albumin). The content of molybdenum measured by
ICP-OES was close to zero, meaning that the species effectively bound to the protein was most probably neither a
complex nor molybdate.
This observation does not prove any specific association between pterin and protein. Nevertheless, the particularity of
this result should be emphasized, i.e it was not observed with many other model compounds as of yet. In this context
it is important to consider the purification process after the incubation with protein, which is a size exclusion
chromatography as shown in Figure 26. With this method the molecules are separated by size and if the product binds
to the protein they are collected in the same fraction; therefore it is possible to assume the formation of a kind of
interaction between the protein and the not unambiguously identified pterin product, which most likely bears free
amino and amide fuctionalities as shown in the crystal structure of F’ (Scheme 32). During the experiment the product
has shown signs of instability, which suggests a certain sensitivity towards oxygen and light. This should be taken into
account for future studies.
300 400 500 600
0
400
800
1200
1600
Ab
sorp
tio
n, a
.u.
Wavelength, nm
IT 114 with apoTorA
IT 114 with BSA
FIGURE 25 – UV STUDIES AFTER INCUBATION WITH BSA AND APOTOR A
FIGURE 26 - GEL FILTRATION, SIZE-EXCLUSION
57
3.4 Synthesis of pterin-dithiolene ligands through the “Minisci reaction”
The particular π-deficient character of heteroaromatic bases was already described above In the chapter “Pteridines
and Pterins”. Pteridines are a prototype of this electronic condition and possess, hence, a characteristic chemical
behaviour compared to other aromatic cycles. The high reactivity toward nucleophilic reagents is the best example of
this characteristic reactivity and the covalent hydration a direct consequence [100].
The presence of electron donating substituents on the pteridine ring like amino and hydroxy group does not change this
behaviour; therefore, subclasses like pterins and lumazines maintain this reactivity and the nucleophilic attack remains
a powerful method for their derivatization. The protonation of heteroaromatic bases enhances this reactivity, but
usually most of the nucleophiles act as deprotonating agent instead of proceeding to substitution. This inconvenience
does not occur if the nucleophile is a radical species and exactly in this perimeter plays the so called “Minisci reaction”,
a chemical protocol that has been developed mainly for C-C bond formation [101].
This reaction has various interesting aspects — high reactivity rates, good selectivities and specular synthetical
applications as the Friedel-Craft reaction [102]. The reaction mechanism in Scheme 33 can be divided in three parts —
the formation of the radicals (for example with a Fenton reaction [103]), the addition to the heteroaromatic ring and
the re-aromatization. While the first two transformations are quite easily understood, the final step has not been clearly
deciphered [104].
SCHEME 33 – HOMOLYTIC ACYLATION MECHANISM
58
The nucleophilic character of the acyl radicals is due to the stability of its cations [105] and it influences the reaction
rate together with the so called polar effect [106], namely the high reactivity increment due to the presence of an
electron withdrawing group or in the case reported above the protonation of the substrate [104]. The synthetic interest
for this reaction is also related to its selectivity; in fact, the presence of other activated positions does not represent an
issue, since the introduction of the first acyl activates further the substrate but also decreases the basic character of the
same changing the condition for the protonation.
The reaction has the same synthetic capability as the Friedel-Crafts aromatic alkylation but with opposite reactivity and
selectivity [102, 106]. All electrophilic species used in the aromatic electrophilic substitution are suitable, as
corresponding radicals, for the homolytic acylation, since more stable carbonium ions give more nucleophilic radicals
[101, 102].
Pterins have been used as substrate for this protocol in a few examples in the literature, in particular by W. Pfleiderer
in the chemical synthesis of deoxysepiapterin [35] (Scheme 34) and the derivatization of Lumazine [18]; also in the book
“Fused Pyrimidines” the reaction is described as not completely explored on pteridines with a stress for wider future
employment [11].
SCHEME 34 – CLOSELY FOLLOWING THE PROCEDURE INTRODUCED BY W. PFLEIDERER FOR THE SYNTHESIS OF 6-ACYL PTERIN
59
Among a large number of methods for the generation of acyl radicals W. Pfleiderer used a Fenton reagent with TBHP
(tert-Butyl hydroperoxide solution 70%), iron sulfate, 17 equivalents of aldehyde (propionaldehyde) and as solvent a 3:1
acetic/water mixture [107]. Product 6 is the same as used before for the synthesis of the 6 chloro derivative 10 with the
pentyloxy chain as protection.
The acylation reaction is particularly facile and, as shown in Scheme 34 requires only 2 minutes of reaction time. The
quenching and isolation of the product is carried out by adding water and consequent precipitation of the pterin
substrate. In most of the cases no further purification is necessary except for washing with water and n-hexane or
recrystallization from a methanol and water mixture. The synthetic work of W. Pfleiderer describes also other pterin
substrates and aldehydes with consequent variation of the reaction conditions [35]. Nevertheless the work-up and
general procedure remain simple and do not require particular modifications [35]. The main aim of this dissertation is
only indirectly connected to this protocol, therefore the reaction parameters have not been further optimized for the
synthesis of the other 6-acyl pterins reported in this work.
Reliable crystallographic data was obtained of the intermediates 14, 15 and 16 (Figure 27, 28, 29); the orientation of
the carbonyl is also in this molecular structures toward the substituent at position 7, exactly as for the previously
described product 11.
FIGURE 27– MOLECULAR STRUCTURE OF INTERMEDIATE 14
FIGURE 28 – MOLECULAR STRUCTURE OF INTERMEDIATE 15
60
The reaction using acetyl aldehyde as reactant also gave with good yield the expected product 17, which shows the
same orientation of the carbonyl like in the previous molecular structures above. This derivative was synthesized in
order to appreciate the possible differences in stability of dithiolene-pterin ligands or complexes, related to the variation
in steric hindrance of the dithiolene substituents (Scheme 35). This aspect requires further engagement in this topic.
FIGURE 29 – MOLECULAR STRUCTURE OF INTERMEDIATE 16
SCHEME 35 – ACYLATION REACTION WITH ACETYL ALDEHYDE
FIGURE 30 – MOLECULAR STRUCTURE PRODUCT 17
61
The signals in the NMR spectra of product 17 are assigned by comparison with those of 16, which are already reported
in the literature (Spectra 20, 21, 22).
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.063.054.131.252.152.002.942.002.011.80
0.9
1
1.0
4
1.3
91.4
11
.44
1.4
8
1.6
31
.70
1.7
31
.75
1.8
51
.87
1.9
01
.92
2.6
9
3.1
83.2
13
.23
4.4
74
.49
4.5
1
5.6
8
7.2
2
Spectrum 20 – 1H NMR of product 17
1H NMR (300MHz ,CHLOROFORM-d) δ = 5.68 (br. s., 1 H), 4.49 (t, J = 6.8 Hz, 2 H), 3.27 - 3.11 (m, 2 H), 2.69 (s, 3 H), 1.86 (d, J = 7.2
Hz, 2 H), 1.81 - 1.65 (m, 2 H), 1.52 - 1.31 (m, 4 H), 1.04 (t, J = 7.4 Hz, 3 H), 0.98 - 0.81 (m, 3 H) ppm
200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
7.7
3
14
0.1
1
15
6.5
116
3.2
6
16
5.5
5
16
8.1
9
19
8.7
8
Spectrum 21 – 13C NMR of product 17, quaternary carbons 13C NMR (75MHz ,CHLOROFORM-d) δ = 198.8, 168.2, 165.5, 163.3, 156.5, 140.1, 117.7 ppm
E
C
H
I
L
C – D
G
L
F
E
I
A
C
B
C
C
C
D
C
F
C
G
A
C
B
C
H
C=O C-2 C-8A
C-4 C-6 C-7
C-4a
C=O
62
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
13
.77
13
.98
21
.31
22
.33
26
.43
28
.08
28
.11
32
.32
68
.46
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
No
rma
lize
d In
ten
sity
13
.77
13
.99
21
.31
22
.35
26
.43
28
.08
32
.32
68
.47
Spectrum 22 – 13C NMR DEPT 135, product 17 13C NMR (75 MHz): δ = 13.8 (CH3), 14.0 (CH3), 21.3 (CH2), 22.3 (CH2), 26.4 (CH3), 28.1 (CH2), 28.1 (CH2), 32.3 (CH2), 68.5 (CH2) ppm
The bromination and substitution reactions were conducted with PHT and 2 pyrrolidone in order to prevent the cleavage
of the protection (Scheme 36). The achievement of the wanted product was confirmed by the collection of x-ray data
of both derivatives 18 and 19 (Figures 31, 32).
β
δ
α
γ
ζ
ϑ
ι
ε
ι
ζ
β
γ α
ϑ
δ
η
ε
η
ζ
63
SCHEME 36 – BROMINATION AND SUBSTITUTION WITH XANTHATE AND CARBAMATE
FIGURE 31 – MOLECULAR STRUCTURE OF PRODUCT 18
64
FIGURE 32 – MOLECULAR STRUCTURE OF PRODUCT 19
The molecular structure, 13C NMR DEPT-135, mass and IR spectra constitute novel information about the product 16.
The signal I (CH2) of the thiol chain is overlapped with the peak of CH2 H of the α-position of the carbonyl at around 3.26
ppm; the amino group is always squashed around 6 ppm and difficult to integrate (Spectrum 23).
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.013.013.014.052.272.044.002.001.83
0.9
30
.96
0.9
81
.07
1.0
91
.11
1.2
01
.22
1.2
5
1.4
11
.44
1.4
61
.47
1.4
81
.49
1.5
01
.50
1.5
21
.73
1.7
61
.78
1.8
01
.88
1.9
21
.95
1.9
7
3.2
03
.23
3.2
53
.27
4.5
24
.54
4.5
6
5.9
2
7.2
8
Spectrum 23 – 1H NMR of product 16 1H NMR (300MHz ,CHLOROFORM-d) δ = 5.92 (br. s., 2 H), 4.54 (t, J = 6.8 Hz, 2 H), 3.35 - 3.13 (m, 4 H), 1.92 (quin, J = 7.0 Hz, 2 H),
1.79 (sxt, J = 7.3 Hz, 2 H), 1.54 - 1.40 (m, 4 H), 1.23 (t, J = 7.4 Hz, 3 H), 1.09 (t, J = 7.4 Hz, 3 H), 1.00 - 0.92 (m, 3 H) ppm
The DEPT 135 experiment shows the expected signals and the separation between the peaks β and γ is like in the
previous derivative C’ (Spectrum 24).
A
D
G
B
H C
E F
I
L
M
C
D
H
A
F
G
B
E
I
L
M
65
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
7.9
0
13
.75
13
.98
21
.33
22
.33
28
.06
28
.10
31
.58
32
.32
68
.41
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
No
rma
lize
d In
ten
sity
7.8
813
.74
13
.96
21
.33
22
.33
28
.05
28
.09
31
.58
32
.31
68
.41
Spectrum 24 – 13C NMR, DEPT 135, product 16 13C NMR (75 MHz): δ = 7.9 (CH3), 13.7 (CH3), 14.0 (CH3), 21.3 (CH2), 22.3 (CH2), 28.1 (CH2, CH2), 31.6 (CH2), 32.3 (CH2), 68.4 (CH2) ppm
β
δ
α
γ
ε
η ζ
ϑ
ι
κ
ε
κ
η
δ
β
γ α
ζ ι
ϑ
66
205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
7.6
2
13
9.9
0
15
6.4
8
16
3.2
3
16
5.4
616
8.1
5
20
1.4
2
Spectrum 25 – 13C NMR product 16, quaternary carbons 13C NMR (75MHz ,CHLOROFORM-d) δ = 201.4, 168.2, 165.5, 163.2, 156.5, 139.9, 117.6 ppm
C=O
C-2 C-8A
C-4
C-6 C-7
C-4a
C=O
67
The NMR spectra of product 19 are in accordance with the proposed structures (Spectra 26, 27, 28).
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
0.003.003.136.004.123.612.092.142.162.011.391.34
0.9
30
.96
1.0
61
.08
1.1
11
.29
1.3
11
.40
1.4
51.4
71
.49
1.5
31
.63
1.6
61
.75
1.7
81
.80
1.8
71
.91
1.9
41
.96
3.2
43
.27
3.2
9
4.4
94
.51
4.5
14
.53
4.5
44
.55
4.5
64
.57
5.5
95.6
15
.635
.65
5.6
75
.81
5.8
35
.86
5.8
8
Spectrum 26 – 1H NMR product 19 1H NMR (300MHz ,CHLOROFORM-d) δ = 5.84 (q, J = 7.2 Hz, 1 H), 5.65 (td, J = 6.2, 12.5 Hz, 1 H), 4.53 (dt, J = 2.0, 6.7 Hz, 2 H), 3.27 (t, J
= 7.2 Hz, 2 H), 1.91 (quin, J = 6.9 Hz, 2 H), 1.84 - 1.72 (m, 2 H), 1.65 (d, J = 7.2 Hz, 3 H), 1.56 - 1.37 (m, 4 H), 1.30 (d, J = 6.2 Hz, 6 H),
1.08 (t, J = 7.3 Hz, 3 H), 1.00 - 0.90 (m, 3 H) ppm
210 205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
7.7
6
13
8.2
7
15
6.5
916
3.4
6
16
6.1
9
16
8.2
2
19
6.2
5
21
1.8
1
Spectrum 27 – 13C NMR of product 19, quaternary carbons 13C NMR (75MHz ,CHLOROFORM-d) δ = 211.8, 196.3, 168.2, 166.2, 163.5, 156.6, 138.3, 117.8 ppm
A
C’
B
D
F
A
B
H’ E
G
C’ G
F
I
I
L
L
H’
M
M
N
N
E
D
R
C=O
C-2
C-8A C-4
C-6 C-7
C-4a
C=O C=S
C=S
68
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
13
.72
14
.00
16
.60
21
.09
21
.1921
.31
22
.35
28
.11
28
.18
32
.28
49
.21
68
.43
78
.18
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
13
.76
14
.04
16
.64
21
.13
21
.23
21
.35
22
.392
8.1
42
8.2
232
.32
49
.25
68
.47
78
.22
Spectrum 28 – 13C-NMR DEPT 135, product 19
13C NMR (75 MHz) δ = 13.8 (CH3), 14.0 (CH3), 16.6 (CH3), 21.1 (CH3), 21.2 (CH3), 21.3 (CH2), 22.4 (CH2), 28.1 (CH2), 28.2 (CH2), 32.3
(CH2), 49.2 (CH), 68.5 (CH2), 78.2 (CH) ppm
The signals for the two μ are overlapped at 21.13 and 21.23 and the typical chemical shift of the signal for CH λ is proof
of the attached xanthogenate at the α-position of the carbonyl of the 6-acyl pterin. The carbon of C=S has a chemical
shift of 211.3, exactly as in the structure of B’, meaning any electronic differences between the positions 6 and 7 and
the carbonyl or alkyl chain are negligible. Also the C-7 carbon this time does not exhibit any particular shifting with
respect to the starting material 16 (Spectrum 25).
β
γ α
ϑ
ζ
δ
ι
ε
μ
λ
β γ λ
μ α
ϑ
ι'
κ'
ε δ
ζ η
κ'
ι'
69
The protocol described by Garner at al. for the ring closing step worked properly and product 21 was synthesized
(Scheme 37) [75]. The employment of concentrated sulfuric acid permitted, at the same time, the cleavage of the
protecting group.
SCHEME 37 – RING CLOSING STEP AND SIMULTANEOUS DEPROTECTION BY SULFURIC ACID: FORMATION OF PRODUCT 21
The amidic proton O at lower field relative to the normal amine function confirms the cleavage of the protection
together with the disappearing of the pentyloxy chain (Spectrum 29). The good solubility is probably due to the thioether
chain which is still attached.
7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
No
rma
lize
d In
ten
sity
3.002.002.872.191.40
DMSO
0.9
50
.97
1.0
0
1.6
51
.67
1.7
01
.72
1.7
5
2.1
2
3.1
43
.17
3.1
9
3.6
1
7.3
4
Spectrum 29 – 1H NMR of product 21
1H NMR (300MHz ,DMSO-d6) δ = 7.34 (br. s., 1 H), 3.17 (t, J = 7.2 Hz, 2 H), 2.12 (s, 3 H), 1.69 (sxt, J = 7.3 Hz, 2 H), 0.97 (t, J = 7.3 Hz, 3
H) ppm
C’’
C’’
B
I
F
B
F
I
O
O
70
Also the DEPT 135 NMR spectrum confirms the preparation of compound 21 (Spectrum 30).
110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
0.19
No
rma
lize
d In
ten
sity
13
.26
15
.35
21
.77
31
.40
115 110 105 100 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
13
.26
15
.34
21
.76
31
.39
Spectrum 30 – 13C NMR DEPT 135, product 21
13C NMR (75 MHz): δ = 13.3 (CH3), 15.3 (CH3), 21.8 (CH2), 31.4 (CH2) ppm
ϑ
κ
κ'’
ϑ
ζ
κ'’
ζ
η
η
71
195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
-0.01
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
0.14
0.15
0.16
0.17
0.18
No
rma
lize
d In
ten
sity
12
2.1
9
12
4.1
613
1.5
9
13
6.1
1
15
4.5
51
55
.46
16
0.1
5
16
1.8
6
19
0.5
5
Spectrum 31 – 13C NMR of product 21, quaternary carbons 13C NMR (75MHz ,DMSO-d6) δ = 190.5, 161.9, 160.1, 155.5, 154.5, 136.1, 131.6, 124.2, 122.2 ppm
C-2
C-8A C-4
C-6
C-7
C-4a
S2C=O
S2C=O C=C
C=C
72
SCHEME 38 – COMPLEXATION REACTION AND THE ELEMENTAL ANALYSIS OF THE BROWNISH POWDER OBTAINED
The reaction of product 21 with the molybdenum precursor NaK3[MoO2(CN)4]6 *H20 [108] has given a dark-red powder
product, i.e. exhibiting a coloration typical of a molybdenum-(IV) complex, but a complete and unambiguous
characterization of the complex was not achieved yet (Scheme 38). The elemental analysis reports a lower content for
most of the elements probably because of salt impurities, which are side product of the reaction. Neither the
precipitation by various counter cations nor the extraction by acetonitrile did improve the analysis.
The possible formation of a product with a 1:1 ratio molybdenum/pterin by concomitant direct coordination with the
carbonyl and nitrogen donors as anchor, which has been already reported in the literature [109] cannot be excluded
without crystallographic data; nevertheless the metal in oxidation state IV should prefer the dithiolene ligand [110, 111].
Mass spectrometry was also inconclusive, most probably by the interference of salt impurities. It is in fact mentioned
by the device’s manual that this kind of impurities are problematic. The only low resolution peak obtained carrying a
molybdenum isotopic pattern has a molecular weight which is almost half of the expected complex including the counter
cations (Spectrum 32).
TABLE 5 – ELEMENTAL ANALYSIS OF THE COMPLEXATION REACTION
Complexation Product
N C H S
theoretical with 2 Na+
16.78 34.53 2.90 23.05
theoretical with 2 K+
16.15
33.25
2.79
22.19
theoretical with Na+, K+
16.46 33.88 2.84 22.61
experimental 11.23 23.98 2.92 10.99
73
SPECTRUM 32 – ESI MASS PEAK WITH MOLYBDENUM ISOTOPIC PATTERN
The basic solution condition of the mass measurement may cause the formation of a bi-charged species as reported
below in Figure 33 obtained by deprotonation of the OH involved in the keto-enol tautomerism (Table 6).
TABLE 6 – POSSIBLE INTERPRETATION OF THE MASS PEAK
The signal obtained by ESI-MASS in a diluted methanol solution does not clearly demonstrate the formation of the target
complex. The 95Mo NMR of the product was measured showing a signal at -2.22 ppm which lies inside of the range of a
molybdenum (IV) [110]. Unfortunately no other related example is reported in the literature and it is not possible to
reference it (Spectrum 33).
Notably, the complexation reaction with another pterin-dithiolene ligand synthesized by the ERC-project team’s
member Nicolas Chrysochos, obtained also with the acylation protocol, reveals a similar chemical shift on the 95Mo NMR
(see Nicolas Chrysochos thesis).
Possible fragments
K Na K, Na
Complex 867.87 835.92 853.91
Complex1- 828.9 812.93
Complex2- 394.97 394.97
[M] =433.7 867.87/2 = 433.93
[M] =433.7
FIGURE 33 – POSSIBLE FRAGMENTATIONS AND STRUCTURE
74
8 6 4 2 0 -2 -4 -6 -8 -10 -12 -14 -16 -18
Chemical Shift (ppm)
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
-2.2
2
SPECTRUM 33 – 95MO NMR, COMPLEXATION PRODUCT 95Mo NMR (20MHz ,Deuterium Oxide) δ = -2.22 ppm
75
The carbamate derivative was not completely characterized in the course of this dissertation because of the difficulties
in isolating the pure product. The final compound is a salt (Scheme 39) and it was not possible to be purified by column
chromatography. The yield of reaction was only estimated to be around 40%.
Some interesting NMR spectra of 20 were obtained after deprotection with the mixture DCM/Et2O/H2SO4 and are
reported below (Spectra 34, 35, 36).
The NMR spectra of product 22 (Spectra 37, 38, 39) were measured from the same NMR tube of product 20 after around
24 hrs. The ring closing reaction was probably catalyzed by the presence of traces of sulfuric acid from the previous
reaction step (from 19 to 20).
SCHEME 39 – DEPROTECTION AND SPONTANEOUS RING CLOSING STEP
76
6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.002.973.082.622.222.164.100.84
DMSO
Water
0.9
50
.98
1.0
01
.10
1.1
21
.14
1.2
51
.27
1.5
01
.53
1.6
01
.62
1.6
41
.67
1.6
91
.72
3.0
43
.06
3.0
9
3.7
13
.73
3.7
53
.82
3.8
73
.89
3.9
13
.94
3.9
65.8
25
.84
5.8
75
.89
Spectrum 34 – 1H NMR of product 20
1H NMR (300MHz ,DMSO-d6) δ = 5.96 - 5.74 (m, 1 H), 4.03 - 3.70 (m, 4 H), 3.06 (t, J = 7.2 Hz, 2 H), 1.66 (sxt, J = 7.3 Hz, 2 H), 1.51 (d, J
= 7.3 Hz, 3 H), 1.25 (t, J = 7.0 Hz, 3 H), 1.12 (t, J = 6.9 Hz, 3 H), 0.98 (t, J = 7.3 Hz, 3 H) ppm
195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
No
rma
lize
d In
ten
sity
12
2.8
3
13
6.7
2
15
5.9
01
56
.84
16
0.1
0
16
3.3
6
19
2.4
8
19
5.9
2
Spectrum 35 – 13C-NMR product 20, quaternary carbons
13C NMR (75MHz ,DMSO-d6) δ = 195.9, 192.5, 163.4, 160.1, 156.8, 155.9, 136.7, 122.8 ppm
B
B
C’
F I
H’ M
N
M
N
H’
C’
F
I
C=O C-2
C-8A
C-4 C-6
C-7
C-4a
C=S
C=O
C=S
77
90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
.30
12
.42
13
.50
16
.4521
.45
31
.16
47
.11
48
.81
50
.62
90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
11
.29
12
.42
13
.50
16
.45
21
.45
31
.16
47
.10
48
.80
50
.62
Spectrum 36 – 13C NMR, DEPT 135, product 20
13C NMR (75 MHz): δ = 11.3 (CH3), 12.4 (CH3), 13.5 (CH3), 16.5 (CH3), 21.5 (CH2), 31.2 (CH2), 47.1 (CH2), 48.8 (CH2), 50.6 (CH) ppm
λ
μ
ϑ
ι'
κ'
η ζ
ι'
λ
ζ η
ϑ
κ'
μ
78
The signal C’’ in product 22 is unfortunately exactly overlapped with the DMSO signal; the product showed the best
solubility in this solvent (Spectrum 37).
8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.006.062.001.883.44
DMSO
0.9
70
.99
1.0
21.2
31
.26
1.2
81
.631.6
51.6
71
.70
1.7
21
.75
3.1
23.1
53
.17
3.6
13
.63
3.6
53
.68
4.8
8
7.2
1
8.4
6
Spectrum 37 – 1H NMR product 22
1H NMR (300MHz ,DMSO-d6) δ = 3.64 (q, J = 7.2 Hz, 4 H), 3.15 (t, J = 7.2 Hz, 2 H), 1.69 (sxt, J = 7.3 Hz, 2 H), 1.26 (t, J = 7.2 Hz, 6 H),
0.99 (t, J = 7.4 Hz, 3 H) ppm
190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
No
rma
lize
d In
ten
sity
12
2.5
1
13
1.5
8
15
6.5
31
57
.13
15
9.0
3
16
4.8
7
18
0.3
7
18
8.3
9
Spectrum 38 – 13C NMR of product 22, quaternary carbons
13C NMR (75MHz ,DMSO-d6) δ = 188.4, 180.4, 164.9, 159.0, 157.1, 156.5, 131.6, 122.5 ppm
C’’
B
I
F
C’’ M’
N’
N’
M’
I
F
B
2.65 2.60 2.55 2.50 2.45 2.40 2.35 2.30
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
DMSO
C-2
C-8A
C-4 C-6 C-7
C-4a C=N C=C
79
The signal ν at 55 ppm could not be assigned and it may belong to some impurities. The presence of a positive charge
influences the entire molecule. In fact, a difference in the chemical shifts in comparison with the previous product 21
can be observed. For example, the chemical shift of C-4 appeared at lower field.
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
No
rma
lize
d In
ten
sity
11
.66
13
.40
21
.52
28
.27
31
.05
47
.70
55
.00
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
No
rma
lize
d In
ten
sity
11
.41
13
.16
21
.27
28
.02
30
.80
47
.44
Spectrum 39 – 13C-NMR DEPT 135, product 22
13C NMR (75 MHz) δ = 11.4 (CH3, CH3), 13.2 (CH3), 21.3 (CH2), 28.0 (CH3), 30.8 (CH2), 47.4 (CH2, CH2) ppm
ϑ
κ'’
ζ η
ϑ
κ'’
ζ
η λ'
μ'
ν ?
λ' μ'
80
The formation of the dithiolene precursor was also confirmed by the mass spectrum reported below. In particular, the
peak appeared in the positive ESI-MASS mode since, as salt, the target molecule is already positively charged (Spectrum
40)
SPECTRUM 40 – ESI MS OF CARBAMATE DERIVATIVE 22
[M] =
[M]
81
3.5 Acylation protocol variations, targeting MPT
The initial good results obtained with the acylation protocol encouraged us to include further subunits of MPT. In
particular through collaboration with other members of the project two variations were pursued — the “pyrane ring
variation” and the “northern/-southern phosphate variation”. The results , which are from joint work combining the
different expertise and strategies from coplementary approaches, are reported partly in this text as well as in the
dissertations of the other two group members Nicolas Chrysochos and Mohsen Ahmadi.
3.5.1 Pyran ring variation
In this mutual work the substrate 15 and 15’ were prepared in order to allow for a synthetic comparison between the
substituents at position 7 of the pterin scaffold (Scheme 40). These alkyl-thio and alkyl-oxo chains protect this position
during the acylation reaction with the aldehydes (H, I). In later steps they work as potential leaving group during the
ring closing mechanism or can be removed as described in the following for pterin 15 (Schema 41). The derivatization
of 15 is described in this dissertation while the work on substrate 15’ is reported by Nicolas Chrysochos. Preparation of
the aldehyde I was carried out by Mohsen Ahmadi as the project member with more expertise in the handling of
phosphate/phosphnate functionalities; in fact, the preparation of the 6-alky pterin 24 precedes not only upon
deprotection, the formation of the pyran ring but also adds a functionality ready for O-P bond formation with a
phosphate moiety as described by Taylor in 2001 [65]. The deveplopment of new pterin-dithiolene ligand precursors
from the 6-acyl pterins 23 and 24 requires further synthetic work, which was not possible to carry out in the course of
this dissertation.
SCHEME 40 – PYRAN VARIATION
Product 23 was isolated after acylation with the aldehyde H; a coordinated molecule of acetic acid was revealed by the
NMR spectra (signal P,Spectrum 41) and the elemental analysis (experimental section). This phenomenon was observed
also in the molecular structure of product 24 reported in the following (Figure 34).
82
Upon prolonged exposure of product to acetic acid the cleavage of the silane protection was observed. In particular a
complete loss of solubility in n-hexane is a clear signal of such side reaction. Al the signals were assigned to the targeted
structure, except for the amino function which could not be integrated (Spectrum 41).
7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0 -0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
6.099.133.083.124.152.102.022.872.022.002.002.00
0.0
5
0.8
5
0.9
61
.06
1.0
81
.11
1.4
51
.46
1.4
71
.48
1.7
41.7
61
.79
1.8
91
.91
1.9
42
.11
3.1
83
.20
3.2
33
.41
3.4
33
.45
4.0
64
.08
4.1
0
4.5
24
.54
4.5
6
7.2
7
Spectrum 41 – 1H NMR of product 23 1H NMR (300MHz ,CHLOROFORM-d) δ = 4.54 (t, J = 6.6 Hz, 2 H), 4.08 (t, J = 6.8 Hz, 2 H), 3.43 (t, J = 6.6 Hz, 2 H), 3.25 - 3.16 (m, 2H),
2.11 (s, 3H), 1.91 (quin, J = 7.0 Hz, 2 H), 1.77 (sxt, J = 7.4 Hz, 2 H), 1.56 - 1.36 (m, 4 H), 1.08 (t, J = 7.4 Hz, 3 H), 0.99 - 0.92 (m, 3 H),
0.85 (s, 9 H), 0.05 (s, 6 H) ppm
205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110 105 100
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
11
7.3
8
13
9.9
5
15
5.7
716
3.2
1
16
5.6
9
16
8.1
7
17
6.3
319
9.0
8
Spectrum 42 – 13C NMR product 23, quaternary carbons 13C NMR (75MHz ,CHLOROFORM-d) δ = 199.1, 176.3, 168.2, 165.7, 163.2, 155.8, 140.0, 117 ppm
N
D
P B
D
C
F
N O
L
E
G
G
A
1
B
P
A
I C H
O
F
G
L
E
I
H
C
C=O
C-2 C-8A C-4 C-6 C-7
C-4a CH3-C=O
CH3-C=O
83
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00N
orm
aliz
ed
Inte
nsi
ty
-5.4
0
13
.66
13
.98
18
.22
21
.08
21
.752
2.3
3
25
.83
28
.06
28
.11
32
.26
41
.495
9.2
9
68
.57
95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 -5 -10
Chemical Shift (ppm)
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
-5.3
9
13
.66
13
.97
21
.07
21
.74
22
.33
25
.82
28
.06
28
.11
32
.25
41
.50
59
.29
68
.56
Spectrum 43 – 13C NMR DEPT 135, product 23 and quaternary carbon C-9 13C NMR (75 MHz) δ = -5.4 (CH3), 13.7 (CH3), 14.0 (CH3), 21.1 (CH3), 21.7 (CH2), 22.3 (CH2), 25.8 (CH3), 28.1 (CH2), 28.1
(CH2), 32.3 (CH2), 41.5 (CH2), 59.3 (CH2), 68.6 (CH2) ppm
The signal of the carbon C-9 appears at high field in accordance with the literature references [112]. The presence of
the silicon atom permitted the measurement of the 29Si NMR (Spectrum 44).
β
δ
γ
ζ
α
ε
ϑ
κ
ι
λ
μ
η
λ
μ
ϑ
ε
β
γ α ζ
η δ κ ι
ν
ν
λ
C-9
84
27 26 25 24 23 22 21 20 19 18 17 16 15 14 13 12 11 10 9
Chemical Shift (ppm)
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
No
rma
lize
d In
ten
sity
19
.88
Spectrum 44 – 29Si NMR of product 23 29Si NMR (60MHz ,CHLOROFORM-d) δ = 19.88 ppm
85
The synthesis of product 24 was carried out with a racemic mixture of aldehyde H with the intention to test the stability
of the acetone protection during the acylation reaction using a cheaper reagent. The product was isolated with a
coordinated molecule of acetic acid (Spectrum 45, H), as confirmed by its molecular structure shown in Figure 34.
6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.803.9811.492.492.263.012.001.131.000.991.202.001.070.010.73
0.8
80
.96
1.0
9
1.2
6
1.3
91
.45
1.7
41
.76
1.7
91
.891
.91
1.9
3
2.1
1
3.1
83
.20
3.2
3
3.3
33
.363
.71
3.7
93
.83
3.8
54.2
84.3
04
.32
4.5
44
.654.6
74
.69
Spectrum 45 – 1H NMR of product 24 1H NMR (300MHz ,CHLOROFORM-d) δ = 4.75 - 4.61 (m, 1 H), 4.54 (t, J = 6.0 Hz, 2 H), 4.30 (dd, J = 6.2, 8.0 Hz, 1 H), 3.81 (dd, J = 5.6,
17.4 Hz, 1 H), 3.71 (t, J = 7.5 Hz, 1 H), 3.32 (dd, J = 7.6, 17.3 Hz, 1 H), 3.20 (t, J = 7.4 Hz, 2 H), 2.11 (s, 3 H), 1.97 - 1.85 (m, 2 H), 1.83 -
1.70 (m, 2 H), 1.34 – 1.54 (m, 10 H), 1.09 (t, J = 7.3 Hz, 3 H), 0.96 (t, J = 6.8 Hz, 3 H) ppm
FIGURE 34 – MOLECULAR STRUCTURE OF PRODUCT 24
A
F
I D
E
C
G
G
B
H
C
H
I
F
C
L
V
M
V L
V
M
V
N
V
A
B
C D
O
V
E
C
L
M
V
N
V
O
V
86
The description of the carbon 13C NMR is not reported, since the assignment of the signals requires further analysis.
Nevertheless the APCI-MASS (Spectrum 46) and the crystal structure strongly support the preparation of the targeted
product (Figure 34).
SPECTRUM 46 – APCI MS OF PRODUCT 24
[M] =
450.0 [M + H]+
87
The removal of the alkyl-thioether substituent from position 7 of the pterin was established following a general protocol
described in 2011 for nitrogen containing heterocycles [113]. Even though the reaction’s yield was quite low (Scheme
41), the method is milder than the one described by W. Pfleiderer and it does not provoke a partial exchange of the
pentyloxy protection.
SCHEME 41 – REDUCTIVE SCISSION
During the first step the amino group reacted also with the triethyl silane giving intermediate 25 but by simple hydrolysis
overnight in methanol the desired product 26 is obtained. Also, the unreacted starting material can be recovered by
column chromatography.
A prolonged reaction does not improve the yield and increases the risk of side reactions. In particular, the reduction of
the pteridine ring was suggested by a APCI-MASS TCL-Express measurement (Spectra 47, 48). The spectra show the
formation of two new compounds with the molecular weight of reduced derivatives 26 and 25 and different Rf on the
TLC, meaning they are different compounds and do not result from a fragmentation process.
SPECTRUM 47 – APCI MS PRODUCT 26
[M26 red +H] =291.9
[M26 red]
88
SPECTRUM 48 – APCI MS OF PRODUCT 26 AND A POTENTIAL REDUCED PRODUCT
Isolation and comprehensive characterization of these species was not achieved as of yet and requires future in-depth
studies in continuity of this dissertation.
The singlet for P at 9.45 ppm in the 1H NMR confirms the free position 7 of product 26 (Spectrum 49).
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.053.384.193.472.002.012.001.800.95
0.9
40
.97
0.9
91.2
31
.25
1.2
81
.47
1.5
11
.62
1.9
01.9
31
.95
1.9
72
.003
.24
3.2
63
.28
3.3
14.5
64
.58
4.6
0
5.7
4
9.4
5
Spectrum 49 – 1H NMR of product 26 1H NMR (300MHz ,CHLOROFORM-d) δ = 9.45 (s, 1 H), 5.78 (br. s., 2 H), 4.58 (t, J = 6.8 Hz, 2 H), 3.27 (q, J = 7.3 Hz, 2 H), 2.02 -
1.89 (m, 2 H), 1.57 - 1.40 (m, 4 H), 1.25 (t, J = 7.2 Hz, 3 H), 1.01 - 0.92 (m, 3 H) ppm
A
C
D
H
P
A
L
E G
D
E
C
P
L
R
R
H
G
[M26] =
[M26+H] =289.9
[M25 red ] =
[M25 red+H] = 405.9
89
Also the 13C NMR (Spectrum 51) and DEP 135 experiment (Spectrum 50) show the comparison of signal C-7 that belongs
to the CH at position 7.
144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
7.7
4
14
.00
22
.362
8.0
62
9.6
83
0.7
9
68
.76
11
0.8
7
14
2.8
7
14
9.8
0
152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16 8
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
No
rma
lize
d In
ten
sity
7.7
3
13
.98
22
.35
28
.05
29
.67
30
.78
68
.76
77
.19
14
9.7
9
Spectrum 50 – 13C NMR DEPT 135, product 26 13C NMR (75 MHz): δ = 7.7 (CH3), 14.0 (CH3), 22.4 (CH2), 28.1 (CH2, CH2), 29.7 (CH2), 30.8 (CH2), 68.8 (CH2), 149.8 (CH) ppm
β γ
ι κ
α
ε
δ
C-7 κ
ε
β γ
α
δ ι
C-7
90
200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115 110
Chemical Shift (ppm)
0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
No
rma
lize
d In
ten
sity
11
0.8
7
14
2.8
7
14
9.8
0
15
8.5
2
16
2.7
0
16
8.2
4
20
0.9
4
Spectrum 51 – 13C NMR product 26, quaternary carbons and C-7 13C NMR (75MHz ,CHLOROFORM-d) δ = 200.9, 168.2, 162.7, 158.5, 149.8, 142.9, 110.9 ppm
C=O
C-2 C-8A
C-4 C-6
C-4a
C-7
C=O
91
Bromination and susbstitution with isopropyl xanthate gave product 27 confimed also by the collection of the crystal
structural date (Scheme 42, Figure 35).
SCHEME 42 – XANTHATE DERIVATIVE OF PRODUCT 27
Interestingly the bromination of substrate 26 was achieved in a one day reaction while for substrate 16 the complete bromination was achieved only in 3 days (experimental section). Thepresence of the substituent at position 7 has probably either a steric and/or electronic infuence on this reaction.
9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
3.106.074.463.022.132.111.171.240.891.00
0.9
40
.96
0.9
9
1.2
81
.30
1.3
21
.43
1.4
81
.54
1.6
51
.68
1.8
91
.911
.94
1.9
61
.98
4.5
54
.57
4.6
0
5.6
25
.67
5.6
9
5.8
8
7.5
2
9.4
1
Spectrum 52 – 1H NMR of product 27
1H NMR (300MHz ,CHLOROFORM-d) δ = 9.41 (s, 1 H), 5.86 (q, J = 7.2 Hz, 1 H), 5.67 (quind, J = 6.5, 12.5 Hz, 1 H), 4.65 - 4.49 (m, 2 H), 1.94 (quin, J = 7.0 Hz, 2 H), 1.66 (d, J = 7.2 Hz, 3 H), 1.58 - 1.38 (m, 4 H), 1.35 - 1.26 (m, 6 H), 1.01 - 0.91 (m, 3 H) ppm
A
C’ G
L
H’ M
N
E
D
R
R
S
S
A
M
D
E
C’
L
G N
H’
R
92
152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
13
.98
16
.21
21
.10
22
.33
28
.08
48
.25
68
.75
78
.31
12
2.2
114
1.1
5
15
0.2
0
152 144 136 128 120 112 104 96 88 80 72 64 56 48 40 32 24 16
Chemical Shift (ppm)
-1.0
-0.9
-0.8
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
No
rma
lize
d In
ten
sity
13
.99
16
.22
21
.10
22
.33
28
.07
48
.25
68
.75
78
.32
15
0.2
0
Spectrum 53 – 13C NMR DEPT 135 of product 27
13C NMR (75 MHz): δ = 14.0 (CH3), 16.2 (CH3), 21.1 (CH3), 22.3 (CH2), 28.1 (CH2, CH2), 48.3 (CH), 68.8 (CH2), 78.3 (CH), 150.2 (CH) ppm
β γ κ' ι'
α
δ
λ
μ
ε
ν
ν ι'
λ μ
β
γ δ α
κ'
93
215 210 205 200 195 190 185 180 175 170 165 160 155 150 145 140 135 130 125 120 115
Chemical Shift (ppm)
0
0.05
0.10
0.15
0.20
0.25
0.30
0.35
0.40
0.45
0.50
0.55
0.60
0.65
0.70
0.75
0.80
0.85
0.90
0.95
1.00
No
rma
lize
d In
ten
sity
12
2.2
114
1.1
5
15
0.2
0
15
8.4
116
3.1
4
16
8.1
019
6.1
4
21
1.5
1
Spectrum 54 – 13C NMR of product 27, quaternary carbons and C-7
13C NMR (75MHz ,CHLOROFORM-d) δ = 211.5, 196.1, 168.1, 163.1, 158.4, 150.2, 141.1, 122.2 ppm
FIGURE 35 – MOLECULAR STRUCTURE OF PRODUCT 27
C=O
C-2 C-8A
C-4
C-4a
C=S C=O
C=S
C-7
C-6
94
3.5.2 Phosphate variation, northern and southern functionalization
Investigations on the phosphate functionality by project member Mohsen Ahmadi had supported the possibility to
adopt a C-P bond instead of the O-P bond in MPT models. In particular this alternative resulted in more stable compound
which in addition were easier to prepare. Therefore it was decided to synthesize the products 28 and 29, using as
reactants the aldehyde M and the sodium salt L (Scheme 43).
The northern functionalization (nicknamed for P-functionalization on the pre-dithiolene moiety) was carried out using
substrate 15 according to the acylation protocols described above. The C-P coordination did not show any instability
during the reaction and product 29 was obtained; a partial hydrolysis of the phosphate-ethoxy protection was the only
obdserved side reaction.
The southern functionalization (nicknamed for P-functionalization on the pyrazine moiety) requires an initial first
installation of the phosphate derivative L and the subsequent acylation with acetaldehyde. Product 28 was obtained
and it represents the first example of a pterin carrying a phosphonate moiety modeling the phosphate anchor to the
peptide of MPT.
SCHEME 43– PHOSPHATE VARIATION
The complete characterization of the products, yields of the reactions and the unique crystal structure of 28 is
reported in the dissertation of Mohsen Ahmadi.
95
4 .Conclusion
The synthesis of a pterin-dithiolene ligand system was achieved, in particular the employment of the acylation protocol
had largely simplified the precedent method reported by Garner [75]. Moreover, the final model bears free amino and
amide functionalities, an aspect of extreme importance for the biological application and characterization (Figure 36).
FIGURE 36 – PTERIN DITHIOLENE LIGAND WITH FREE AMINO AND AMIDE FUNCTIONS
The intensive cooperation with the project members Nicolas Chrysochos and Mohsen Ahmadi resulted in two variations
of the acylation protocol with focus on the formation of the pyran ring and the introduction of the phosphate subunit.
The synthesis of these 6-acyl pterin derivatives (Figure 38) redrafted this synthetic protocol and further approached the
real natural cofactor with the intention of modelling MPT (Figure 37) and, perhaps, in the future MoCo.
FIGURE 37 – TARGETED SUBSTRUCTURES IN MPT
Through the preparation of these structure also the stability of the alternative C-P coordination of the phosphate
anchor with respect to the acylation protocol has been tested and verified (Figure 38, product 28 and 29).
96
FIGURE 38 – STRUCTURE OBTAINED BY THE DEVELOPMENT OF THE ACYLATION PROTOCOL
A method for the removal of the alkyl-thioether chain of product 16 was optimized towards milder and easier conditions
and the xanthate derivative 27 was synthesized (Scheme 44).
SCHEME 44 – REMOVAL OF THE ALKYL-THIOETHER CHAIN
A complete characterization of the molybdenum complex with the prepared ligands was not achieved, either because
of the many encountered issues during the isolation and purification steps, or because of the formation of a different
species with a 1:1 ratio molybdenum-pterin, which could not be excluded unambiguous. The biological test with G’ (IT
114) has shown a kind of specific binding with the ApoTorA compare to the BSA ( as evidenced by comparison with BSA
bovine serum albumin). Nevertheless, further in-depth studies would be necessary prior to drawing final definite
conclusions.
Even though the first synthetic approach for the synthesis of a pterin-dithiolene ligands did not give the expected result,
the side reaction occurring during the bromination has been eventually understood and was described for the benefit
of future research along these lines (Schema 39).
The final product C’ is not the desired structure, but it is a new red coloured thiazole-pterin which could be used as
model for the S-Oxidation in the metabolism of the thiazolidinedione (TZD) ring, a reaction catalysed by cytochrome
P450 [96, 97].
97
FIGURE 39 – C’ AS MODEL FOR S-OXIDATION OF TZD
98
Experimental section
General
All reactions and manipulations were carried out using standard Schlenk and glove box techniques under an atmosphere
of high purity nitrogen or argon. The inert gas stream was first passed through a copper catalyst tube (from company
BASF with specification R3 / 11) at a reaction temperature of 200 °C followed by P4O10 and KOH columns and finally
the stream of inert gas was dried by a molecular sieve column (3 Å and 4 Å) before entering the Schlenk line.
Elemental Analysis (C, H, N and S) were carried out with an Elementar Vario micro elemental analyzer. Calculations of
the theoretical molar masses was based on the relative atomic masses obtained from IUPAC tables.
Nuclear Magnetic Resonance NMR measurements were recorded on a Bruker Avance II-300 MHz. All samples were
dissolved in deuterated solvents and chemical shifts (δ) are given in parts per million (ppm) using solvent signals as the
reference (CDCl3 1H: δ = 7.24 ppm; 13C: δ = 77.0 ppm; d6-DMSO 1H: δ = 2.49 ppm; 13C: δ = 39.5 ppm; CD3CN 1H: δ = 1.94
ppm; 13C: δ = 1.3 ppm) related to external tetramethylsilane (δ = 0 ppm). 1H decoupled 13C C NMR spectra were recorded.
Coupling constants (J) are reported in Hertz (Hz).
Infrared spectra were recorded on a Shimadzu IR Affinity-1 FTIR-spectrophotometer in the range of 4000–400 cm-1
using KBr pellets.
Single-crystal X-ray diffraction suitable single crystals of compounds were mounted on a thin glass fibre coated with
paraffin oil. X-ray single-crystal structural data were collected using a STOEIPDS 2T diffractometer equipped with a
normal-focus, 2.4 kW, sealed-tube X-ray source with graphite-monochromated MoKα radiation (λ =0.71073 Å).
The molecular structure C’-IT 70 was measured by Dr. G. J. Palm, the crystal was mounted in a 0.1 mm loop in paraffin
oil and frozen and stored in liquid nitrogen in April 2017. Data were collected at DESY on beamline P11 on 02-
03.07.2017.The wavelength was 20 keV. 720 0.5° oscillation images were taken. The exposure time was 300 ms.
Transmission was set to 100% (full intensity beam). The crystal-detector distance was 207.7 mm and gave a maximum
resolution of 0.8 A. A 50 μm beam size was used. The detector was a Pilatus detector (2463 x 2527 pixel). Data were
collected at 100 K in a N2 stream.
2-Amino-3-cyanopyrazine 1-oxide 1
Method B as reported in a publication [114] was followed exactly: a suspension of powdered glyoxime (17.3 g, 0.196
mol) and aminomalononitrile tosylate (49.8 g, 0.19 mol) in 160 ml of water was stirred at RT for 17 hrs. During this time
all starting materials passed into solution and a new precipitate formed. The reaction product was collected by filtration,
washed with a small amount of cold water and then with ethanol. After drying, the crude product was crystallized from
a mixture of DMF/EtOH. Data code = IT 3; yield 26%; elemental analysis: calculated C-44.12, H-2.96, N-41.16; found C-
42.14, H-3.039, N-39.3 ; 1H NMR (300MHz, DMSO-d6) δ= 8.48 (d, J = 3.8 Hz, 1 H), 7.98 (s, 3 H), 7.85 (d, J = 3.8 Hz, 1 H)
ppm.
99
2,4-Diaminopteridine 8-oxide 2
The procedure as reported in a publication [114] was followed exactly: guanidine hydrochloride (2.4 g, 39.2 mmol) was
added to a solution of sodium (1.5 g, 65 mmol) in 200 ml of absolute methanol, and the precipitated sodium chloride
was removed by filtration. To the filtrate was added product 1 (2.9 g, 21.3 mmol), and the resulting mixture was heated
under reflux with stirring for 16 hrs. A yellow precipitate started to separate from the orange solution after
approximately 1 hr. The mixture was cooled and filtered and the collected solid washed well with methanol and dried.
The analytical sample was prepared by crystallization from a large volume of DMF. Data code = IT 4; yield 89%; 1H NMR
(300MHz , DMSO-d6) δ = 8.42 (d, J = 3.8 Hz, 1 H), 8.05 (d, J = 3.8 Hz, 1 H), 7.85 (br. s., 2 H) ppm.
4(3H)-Pteridinone, 2-amino-, 8-oxide 3
It was followed the exactly procedure reported in the publication [83]: was followed exactly: a suspension of 1.00 g of
2 (5.6 mmol) in 100 ml of 5% NaOH was heated under reflux until completely dissolved; then for additional 5 min (total
heating time 30 min). After that, the yellow solution was brought to pH 3 with 6 N HCl and allowed to stand for 30 min
before filtering, washing thoroughly with water and drying to yield a bright yellow powder. Data code = IT 5; yield 95%;
elemental analysis: calculated C-40.23, H-2.81, N-39.10 found C-36.53, H-2.65, N-35.26; IR (KBr pellet) cm-1 : C=O
1714,74 cm-1.
2-Amino-6-chloro-4(3H)-pteridinone(6-chloropterin) 4
It was followed the exactly procedure reported in the publication [115] was followed exactly: a suspension of product
3 (0.5 g, 28 mmol) in 2.5 mL of acetyl chloride was cooled to -60°C (chloroform/N2 bath) and 2.5 mL TFA (trifluoroacetic
acid), precooled to -60°C was added. The mixture was sealed in a glass pressure bottle, and gradually warmed to RT
(overnight, the glass must be leaved in the bath). The product 3 slowly dissolved, and after about 2 min at RT a pale
yellow solid started to precipitate from the reaction mixture, It was stirred at RT for additional 45 min, the pressure
bottle opened (careful !HCl is released), the suspension diluted with dry ether, and a pale yellow solid, collected by
filtration and dried in vacuo. Product 4 was prepared by dissolution of the above hydrochloride (0.5 g) in 5 mL of 5 %
cold sodium hydroxide solution. After 15 min stirring at RT, the clear pale yellow solution was filtered and the filtrate
acidified with glacial acetic acid. After stirring for 30 min, the resulting suspension was filtered and the collected solid
washed thoroughly with water and then with acetone. Data code = IT 5; yield 90% ; as hydrochloric salt 1H NMR (300MHz
,DMSO-d6) δ = 8.90 (s, 1 H), 8.49 (br. s., 2 H) ppm ; 13C NMR (75MHz ,DMSO-d6) δ = 172.0, 158.0, 152.9, 149.1, 142.8,
128.3 ppm; 13C NMR (75 MHz, DEPT-135): δ = 149.1 (CH) ppm.
100
6-Chloro-2-{[(dimethylamino)methylene]amino}pteridin-4-one 5
It was followed the exactly procedure reported in the publication [65] was followed exactly: to a stirred solution of
product 4 (3.8 g, 0.019 mol) in DMF (8 mL) at 25°C was added t-BuO(Me2N)2CH (Bredereck’s reagent) (4.7 ml, 21.7
mmol) in one portion. After 24 hrs, Et2O (30 ml) was added and the resulting precipitate collected by filtration. The
precipitate was washed with Et2O then dried by heating (100 °C) under vacuum to yield product 5 as a fine yellow
powder. Data code = IT 163.2; yield 84%; 1H NMR (300MHz , CHLOROFORM-d) δ = 8.98 (s, 1 H), 8.72 (s, 1 H), 3.27 (s, 3
H), 3.21 (s, 3 H) ppm.
2,4-Pyrimidinediamine, 5-nitroso-6-(pentyloxy) 6
A procedure as reported in publications [93, 116-118] was followed exactly : sodium (10 g, 0.43 mol, 1.5 equivalent)
was added to 600 mL of n-penthanol in a two-neck round bottom-flask and the resulting mixture was stirred and heated
at reflux for 2 hrs, than 4-chloro-2,6-diaminopyrimidine (41.4 g, 0.28.6 mol) was added and stirring with refluxing
continued for 5 hrs. After cooling, the mixture was neutralized with acetic acid and the volume reduced almost to
dryness. After adding 600 mL of 30% acetic acid solution the temperature was increased to 80°C. Sodium nitrite (24.15
g, 0.35 mol) was dissolved in a minimum amount of water and added dropwise; the formation of a deep purple
coloration was observed. The reaction was maintained under stirring at 80°C for 40 min and at the end the warm mixture
was put into a separatory funnel and the water bottom-layer removed.The oily deep purple upper-layer after 1 night at
-20°C gives a crystalline purple solid which was filtered off, washed with water and stored in a desiccator overnight.
Crystallization from chloroform/n-hexane 1:19. Data code = IT 21; X-ray code IT 21 ; yield 85%; Mass spectrometry (APCI,
positive, low t. low f.) = 226.3, 156.2 m/z; elemental analysis: calculated C-47.99, H-6.71, N-31.09; found C-47.71, H-6.5,
N-30.7; 1H NMR (300MHz , DMSO-d6) δ = 10.09 (d, J = 4.2 Hz, 6 H), 7.97 (d, J = 3.8 Hz, 6 H), 7.76 (d, J = 8.7 Hz, 13 H), 4.48
(t, J = 6.8 Hz, 13 H), 1.79 (quin, J = 6.9 Hz, 14 H), 1.46 - 1.28 (m, 27 H), 0.95 - 0.82 (m, 21 H) ppm; 13C NMR (75MHz ,
DMSO-d6) δ = 170.8, 163.5, 150.8, 139.5, 66.7, 28.0, 27.6, 21.8, 13.9 ppm; 13C NMR, (75 MHz, DEPT-135): δ = 13.9 (CH3),
21.8 (CH2), 27.6 (CH2), 28.0 (CH2), 66.7 (CH2) ppm.
101
2,4,5-Pyrimidinetriamine, 6-(pentyloxy) 7
A procedure as reported in a publication [7]: was followed: a solution of ethanol and water (865 mL /251 mL) was
purged with nitrogen for 3 hrs, sodium hydrosulfide (147 g, 2.6 mol) was added and the temperature increased to 80°C.
To the yellow solution was added portionwise product 6 (under nitrogen flow, 94.35 g, 0.41 mol) until complete
dissolution. After cooling, the ethanol was removed by vacuum and the resulting a suspension. The yellow precipitate
was collected, washed with water, dried under vacuum and stored under nitrogen pressure. Data code = IT 22; yield
82.5%; mass spectrometry (APCI, positive, low t. low f.) = 212.1 m/z; elemental analysis: calculated C-51.17, H-8.11, N-
33.15; found C-45.88, H-7.23, N-29.76; 1H NMR (300MHz, DMSO-d6) δ = 5.62 (s, 2 H), 5.22 (s, 2 H), 4.12 (t, J = 6.6 Hz, 2
H), 3.11 (s, 2 H), 1.64 (quin, J = 6.9 Hz, 2 H), 1.44 - 1.22 (m, 4 H), 1.00 - 0.77 (m, 3 H); 13C NMR (75MHz, DMSO-d6) δ =
157.6, 155.5, 155.4, 100.2, 64.7, 28.5, 27.7, 21.9, 13.9; 13C NMR, (75 MHz, DEPT-135): δ = 13.9 (CH3), 21.9 (CH2), 27.7
(CH2), 28.5 (CH2), 64.7 (CH2) ppm.
2-Pteridinamine, 4-(pentyloxy) 8
A procedure as reported in a publication [93]: was followed: in a two-neck round-bottom flask 600 mL of DMF were
purged with nitrogen for 2 hrs. To this were added first glyoxal-hydrate trimer (3.3 g, 15.7mmol) and then product 7 (10
g, 47.4 mmol) giving a green-blue coloration of the mixture. The reaction was stirred at RT for 3 days under nitrogen.
Some insoluble material was filtered off and the filtrate diluted with 600 mL of water. The solution was extracted with
chloroform (three times 400 mL), then the organic layer was washed twice with water, dried using sodium sulfate,
filtered and evaporated to dryness. Crystallization from 160 mL of methanol/water 1:1 gave a yellowish crystalline
product. Data code = IT 23; yield 69%; mass spectrometry (APCI, positive, low t. low f.) = 233.9-235.0, 163.9 m/z;
elemental analysis: calculated C-56.64, H-6.48, N-30.02; found C-54.46, H-6.38, N-28.76; 1H NMR (300MHz,
CHLOROFORM-d) δ = 8.80 (d, J = 2.3 Hz, 1 H), 8.52 (d, J = 2.3 Hz, 1 H), 4.58 (t, J = 7.0 Hz, 2 H), 1.94 (quin, J = 7.3 Hz, 2 H),
1.53 - 1.33 (m, 4 H), 0.98 - 0.90 (m, 3 H) ppm; 13C NMR (75MHz ,DMSO-d6) δ = 157.6, 155.5, 155.4, 100.2, 64.7, 28.5,
27.7, 21.9, 13.9 ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 167.7, 161.5, 157.0, 150.5, 140.3, 124.4, 68.6, 28.1, 27.9,
22.3, 13.9 ppm; 13C NMR, (75 MHz, DEPT-135): δ = 13.9 (CH3), 22.3 (CH2), 27.9 (CH2), 28.1 (CH2), 68.6 (CH2), 140.3 (CH),
150.5 (CH) ppm.
2-Pteridinamine 4-(pentyloxy)- 8-oxide 9
A procedure ad reported in a publication [93] was followed: a solution of product 8 (5.0 g, 21 mmol) in 85 mL of TFA
(trifluoroacetic acid) was cooled to 6°C, and 5 mL of 30% H2O2 were slowly added with stirring. The mixture was kept at
6°C (refrigerator) and after 60 hrs further 2 mL of 30% H2O2 were added. After 30 hrs in the refrigerator at the same
temperature the mixture was concentrated in vacuum to 1/3 of its volume, diluted with 20 mL of water and the resulting
precipitate was collected. The solid was suspended in 100 mL of water and neutralized by concentrated ammonia. The
precipitate was filtered off and dried to give a yellowish chromatographically pure material. Data code = IT 28; X-ray
102
code IT 27CHF; yield 40%; mass spectrometry (APCI, positive, low t. low f.) = 249.8 m/z; 1H NMR (300MHz, DMSO-d6) δ
= 8.52 (d, J = 3.8 Hz, 1 H), 8.20 (d, J = 3.8 Hz, 1 H), 4.46 (t, J = 6.6 Hz, 2 H), 1.81 (quin, J = 7.0 Hz, 2 H), 1.51 - 1.25 (m, 4
H), 0.97 - 0.83 (m, 3 H) ppm; 13C NMR (75MHz, DMSO-d6) δ = 167.4, 160.4, 152.3, 139.3, 135.0, 125.7, 67.8, 27.7, 27.5,
21.8, 13.9 ppm; 13C NMR, (75 MHz, DEPT-135): δ = 13.9 (CH3), 21.8 (CH2), 27.5 (CH2), 27.7 (CH2), 67.8 (CH2), 135.0 (CH),
139.3 (CH) ppm.
2-Pteridinamine, 6-chloro-4-(pentyloxy) 10
A procedure as reported in a publication [93] was followed exactly: at -40°C product 9 (1 g, 4 mmol) was suspended in
10 ml of freshly distilled acetyl chloride, and with stirring 3 mL of TFA (trifluoroacetic acid) were slowly added while
stirring. The solution was warmed to 0°C, stirred for 3 hrs, and then the reaction was stopped by addition of 30 g of ice.
The mixture was neutralized with concentrated ammonia to pH 4, then extracted with chloroform, (6 x 50 mL). The
organic layer was washed with water, dried with sodium sulfate and the solvent evaporated to a small volume. The
residue was purified by chromatography on a silica-gel column with chloroform. The product fraction was evaporated
to dryness and the residue crystallized from isopropanol. Data code = IT 99; X-ray code IT 99; yield 62%; mass
spectrometry (APCI, positive, low t. low f.) = 267.7-269.7 m/z; elemental analysis: calculated C-49.35, H-5.27, N-26.16;
found C-54.35, H-5.66, N-28.69; 1H NMR (300MHz , CHLOROFORM-d) δ = 8.75 (s, 1 H), 5.98 (br. s., 1 H), 4.57 (t, J = 7.0
Hz, 2 H), 1.92 (quin, J = 7.2 Hz, 3 H), 1.58 - 1.30 (m, 4 H), 1.07 - 0.82 (m, 3 H) ppm; 13C NMR (75MHz , CHLOROFORM-d)
δ = 166.9, 161.6, 155.8, 151.0, 142.5, 122.6, 68.7, 28.1, 27.9, 22.3, 13.9 ppm; 13C NMR (75 MHz, DEPT-135): δ = 13.9
(CH3), 21.8 (CH2), 27.5 (CH2), 27.7 (CH2), 67.8 (CH2), 135.0 (CH), 139.3 (CH) ppm.
Ethanone, 1-[2-amino-4-(pentyloxy)-7-methyl-6-pteridinyl] 11
A procedure as reported in a publication [119] was followed: a mixture of product 6 (4 g, 17.8 mmol) and 60 mL of 2,4-
pentanedione were heated under reflux for 7 hrs. The solvent was dried giving an oily black residue. The crude product
was first purified by silica gel column chromatography (ethyl acetate /n hexane 1:1) and then crystallized from a
water/methanol mixture 1:1 yielding a yellow microcrystalline powder. Data code = IT 29; X-ray code IT 29; yield 80%;
mass spectrometry (APCI, positive, low t. low f.) = 290.1 m/z; elemental analysis: calculated C-58.12, H-6.62, N-24.21;
found C-57.50, H-6.52, N-23.64; 1H NMR (300MHz, CHLOROFORM-d) δ = 6.01 (br. s., 1 H), 4.55 (t, J = 6.8 Hz, 2 H), 2.92
(s, 3 H), 2.76 (s, 3 H), 2.04 - 1.81 (m, 2 H), 1.64 - 1.31 (m, 4 H), 1.06 - 0.86 (m, 3 H) ppm; 13C NMR (75MHz, CHLOROFORM-
d) δ = 199.9, 167.9, 163.0, 161.4, 157.1, 142.7, 120.6, 68.5, 28.1, 27.5, 25.1, 22.3, 14.0 ppm; 13C NMR (75 MHz, DEPT-
135): δ = 14.0 (CH3), 22.3 (CH2), 25.1 (CH3), 27.5 (CH3), 28.1 (CH2, CH2), 68.5 (CH2) ppm.
103
Acetic acid, 2-[[2,4-diamino-6-(pentyloxy)-5-pyrimidinyl] imino]- ethyl ester 12
A modified procedure similar to one reported in a publication [118] was followed: product 7 (73 g 0.34 mol) was
dissolved in a mixture of methanol and water (5.2 L/390 mL), the reactant (Acetic acid, 2-ethoxy-2-hydroxy-, ethyl ester,
see below) was added and the solution kept under stirring at RT for 1 hr. The yellow precipitate was filtered off and
washed with a minimum amount of diethyl ether. To the filtrate was added circa 500 mL of water and kept in a
refrigerator overnight obtaining a second fraction of product. The recrystallization procedure was repeated till a yield
of 74.3 % was achieved. Data code = IT 77 S1; yield 74,3%; mass spectrometry (APCI, positive, low t. low f.) = 296.1 m/z;
elemental analysis: calculated C-52.87, H- 7.17, N-23.71; found C-52.86, H-7.00, N-23.3; 1H NMR (300MHz,
CHLOROFORM-d) δ = 8.33 (s, 1 H), 5.81 (br. s., 1 H), 4.97 (br. s., 1 H), 4.48 - 4.19 (m, 4 H), 1.78 (quin, J = 7.0 Hz, 2 H),
1.51 - 1.27 (m, 7 H), 1.01 - 0.84 (m, 3 H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 165.4, 164.0, 163.2, 161.2, 141.8,
102.7, 77.2, 66.8, 60.9, 28.5, 28.2, 22.3, 14.3, 13.9 ppm; 13C NMR (75 MHz, DEPT-135): δ = 13.9 (CH3), 14.3 (CH3), 22.3
(CH2), 28.2 (CH2), 28.5 (CH2), 60.9 (CH2), 66.8 (CH2), 141.8 (CH) ppm.
Acetic acid, 2-ethoxy-2-hydroxy-, ethyl ester - reactant
A modified procedure similar to one reported in a publication [120] was followed: a solution of glyoxylic acid
monohydrate in ethanol (9.2 g in 100 mL) was heated for 114 hrs at 80°C. After reduction of volume at rotary evaporator
the crude product was used without further purification taking into account ca. 70% of yield. Data code = IT 66, yield
70%); 1H NMR (300MHz ,CHLOROFORM-d) δ = 4.94 (s, 1 H), 4.37 - 4.20 (m, 2 H), 3.93 - 3.79 (m, 1 H), 3.79 - 3.59 (m, 2
H), 1.37 - 1.23 (m, 6 H) ppm.
7(8H)-Pteridinone, 2-amino-4-(pentyloxy) 13
A modified procedure similar to one reported in a publication [118] was followed: product 12 (20 g, 67.7 mmol) was
dissolved in a mixture of 4 L of ethanol and 540 mL of sodium hydrogen carbonate solution (0.5 N). The mixture was
refluxed for 2 hrs, than another 135 mL of sodium hydrogen carbonate solution were added and the reflux was
continued for 1 hr. To the mixture were added around 10 g of active charcoal as fine powder, and the solution filtered
while still warm. After cooling, the yellow solution was brought to pH 5 with acetic acid giving a white powder which
was filtered off, washed with water in order to remove the excess acetic acid and kept in the desiccator overnight. Data
code = IT 77 S2/ CT-OXO; yield 60% ; mass spectrometry (APCI, positive, low t. low f.) = 250.0 m/z; elemental analysis:
calculated C-53.00, H-6.07, N-28.10; found C-52.53, H-5.697, N-26.97; 1H NMR (300MHz, DMSO-d6) δ = 7.42 (s, 1 H),
6.52 (br. s., 2 H), 5.75 (s, 1 H), 4.30 (t, J = 6.8 Hz, 2 H), 1.73 (t, J = 7.0 Hz, 2 H), 1.50 - 1.19 (m, 4 H), 0.97 - 0.76 (m, 3 H)
104
ppm; 13C NMR (75MHz, DMSO-d6, TFA) δ = 166.4, 160.7, 150.8, 141.4, 110.9, 110.2, 67.5, 28.2, 27.9, 22.1, 13.9 ppm; 13C
NMR (75 MHz, DEPT-135, DMSO-d6, TFA): δ = 13.9 (CH3), 22.1 (CH2), 27.9 (CH2), 28.2 (CH2), 67.5 (CH2), 141.4 (CH) ppm.
2-Pteridinamine, 7-chloro-4-(pentyloxy) 14
A procedure as reported in publications [35, 121] was followed: to a mixture of phosphoryl chloride (615.76 mL, in
excess) and potassium chloride (30.71 g 0.4 mol) was added product 13 (29.2 g 0.12 mol). The flask was put in a hot oil
bath at 140°C. After heating for 12 min with stirring, the mixture was cooled with an ice-bath (the glassware has to be
in good condition in order to avoid leaking of the solvent that can react violently with water). The excess of phosphoryl
chloride was evaporated in vacuum (with two cooling traps in order to protect the vacuum pump). The residue was
treated with 600 g of ice and 500 mL of chloroform. After formation of two layers, the upper part was neutralized using
sodium hydrogen carbonate), shaken and neutralized many times until no more acidity was observed in the aqueous
layer. The organic layer was separated, the aqueous fraction extracted twice with chloroform, and the combined organic
extracts dried using sodium sulfate and evaporated. The crude product was purified by chromatography column short-
path aluminium oxide (neutral) using chloroform as eluent. The collected product precipitated in n-hexane and was
filtered off yielding a pure pale yellow powder (microcrystalline needles). Data code = IT 82; X-ray code CT CL yield 40%;
mass spectrometry (APCI, positive, low t. low f.) = 267.8-269.7 m/z; elemental analysis: calculated C-49.35, H-5.27, N-
26.16; found C-47.4, H-5.87, N-20.20; 1H NMR (300MHz, DICHLOROMETHANE-d2) δ = 8.38 (s, 1 H), 4.51 (t, J = 6.8 Hz, 2
H), 1.88 (quin, J = 7.1 Hz, 2 H), 1.51 - 1.33 (m, 4 H), 0.98 - 0.87 (m, 3 H) ppm; 13C NMR (75MHz, DICHLOROMETHANE-d2)
δ = 167.9, 163.1, 157.1, 153.7, 139.5, 122.7, 68.9, 28.5, 28.3, 22.7, 14.1 ppm; 13C NMR (75 MHz, DEPT-135): δ = 14.1
(CH3), 22.7 (CH2), 28.3 (CH2), 28.5 (CH2), 68.9 (CH2), 139.5 (CH) ppm.
2-Pteridinamine, 4-(pentyloxy)-7-(propylthio) 15
A procedure as reported in publications [35, 118]: was followed: to a mixture of phosphoryl chloride (615.76 mL, in
excess) and potassium chloride (30.71 g 0.4 mol) product 13 was added (29.2 g 0.12 mol). The flask was put in a hot oil
bath at 140°C. After heating for 12 min with stirring, the mixture was cooled with an ice-bath. The excess of phosphoryl
chloride was evaporated in vacuum (with two cooling traps in order to protect the vacuum pump). The residue was
treated with 600 g of ice and 500 mL of chloroform, after separation of the organic phase the aqueous fraction was
extracted twice with chloroform, and the combined organic extract dried using sodium sulfate and then evaporated.
The residue consisting in crude product 14 was treated with a prepared solution of 1-propanethiol (30 mL, use carefully
is extremely smelly, all residue or waste hast to be treated with a sodium hypochlorite solution 13%!) in 500 ml of 0.25
N sodium methoxide. The solution was stirred for 1 hr at RT, then neutralized with acetic acid and evaporated. The
residue was treated with 200 mL of water and the precipitate collected and purified by short-path aluminium oxide
105
(neutral) chromatography eluting with gradient from chloroform to 10% methanol/chloroform. Recrystallization from
chloroform/n-hexane 1:19. Data code = IT 84; X-ray code IT 84, MADXM5; yield 70%; mass spectrometry (APCI, positive,
low t. low f.) = 307.9 m/z; elemental analysis: calculated C-54.70, H-6.89, N-22.78, S-10.43; found C-58.9, H-7.22, N-
24.26, S-10.94; 1H NMR (300MHz, CHLOROFORM-d) δ = 8.27 (s, 1 H), 5.45 (br. s., 2 H), 4.53 (t, J = 7.0 Hz, 2 H), 3.33 (t, J
= 7.2 Hz, 2 H), 1.99 - 1.86 (m, 2 H), 1.86 - 1.73 (m, 2 H), 1.53 - 1.31 (m, 4 H), 1.07 (t, J = 7.4 Hz, 3 H), 1.00 - 0.86 (m, 3 H)
ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 167.8, 164.8, 161.6, 157.0, 140.3, 119.4, 68.2, 31.5, 28.2, 27.9, 22.3, 22.0,
13.9, 13.4 ppm; 13C NMR (75 MHz, DEPT-135): δ = 13.4 (CH3), 13.9 (CH3), 22.0 (CH2), 22.3 (CH2), 27.9 (CH2), 28.2 (CH2),
31.5 (CH2), 68.2 (CH2), 140.3 (CH) ppm.
1-Propanone, 1-[2-amino-4-(pentyloxy)-7-(propylthio)-6-pteridinyl] 16
A procedure as reported in a publication [35] was followed: to a suspension of product 15 (4.5 g, 14.6 mmol) in 360 mL
of acetic acid/water 3:1 and propionaldehyde (17.8 mL, 17 equivalents) were added dropwise simultaneously 25.5 g of
FeSO4.*7H2O (25.5 g, 91.8 mmol) in 15 of mL water and 11.6 mL of tert-butyl hydroperoxide 70% aqueous solution with
vigorous stirring within around 60 seconds. After stirring for 2 minutes, 720 mL of water were added, the precipitate
was collected and washed with water and dried to give a pure pale yellow powder. Data code = IT 90; X-ray code IT 90
COL; yield 78%; mass spectrometry (APCI, positive, low t. low f.) = 364.3 m/z; elemental analysis: calculated C-56.17, H-
6.93, N-19.27, S-8.82; found C-56.22, H-6.73, N-19.22, S-8.676; 1H NMR (300MHz, CHLOROFORM-d) δ = 5.89 (br. s., 1
H), 4.54 (t, J = 6.8 Hz, 2 H), 3.52 - 3.12 (m, 4 H), 1.92 (quin, J = 7.0 Hz, 2 H), 1.86 - 1.71 (m, 2 H), 1.59 - 1.36 (m, 4 H), 1.29
- 1.16 (m, 3 H), 1.10 (d, J = 7.2 Hz, 3 H), 0.96 (t, J = 7.0 Hz, 3 H) ppm; 13C NMR (75MHz ,CHLOROFORM-d) δ = 201.4, 168.2,
165.5, 163.2, 156.5, 139.9, 117.6, 68.4, 32.3, 31.6, 28.1, 28.1, 22.3, 21.3, 14.0, 13.7, 7.9 ppm; 13C NMR (75 MHz, DEPT-
135): δ = 7.9 (CH3), 13.7 (CH3), 14.0 (CH3), 21.3 (CH2), 22.3 (CH2), 28.1 (CH2), 31.6 (CH2), 32.3 (CH2), 68.4 (CH2) ppm.
1-Acetone, 1-[2-amino-4-(pentyloxy)-7-(propylthio)-6-pteridinyl] 17
A procedure as reported in a publication [35]: was followed: to a suspension of product 15 (650 mg, 2.1 mmol ) and
acetaldehyde (2 mL, 17 equivalent) in 50 mL of acetic acid/water 3:1 were added dropwise simultaneously 3.7 g of
FeSO4.7H2O in 17 of mL water and 1.7 mL of tert-butyl hydroperoxide with vigorous stirring within around 60 sec. After
stirring for 2 minutes, 100 mL of water were added, the precipitate was collected and washed with water and dried to
give a pure pale yellow powder. Data code = CT 39 yield 85%; X-ray code CT 39; mass spectrometry (APCI, positive, low
t. low f.) = 350.0 m/z; elemental analysis: calculated C-54.99, H-6.63, N-20.04, S-9.18; found C-53.56, H-6.06, N-19.31,
S-9.192; 1H NMR (300MHz, CHLOROFORM-d) δ = 5.69 (br. s., 1 H), 4.49 (t, J = 6.8 Hz, 2 H), 3.29 - 3.10 (m, 2 H), 2.69 (s, 3
H), 1.87 (quin, J = 7.0 Hz, 2 H), 1.74 (sxt, J = 7.3 Hz, 2 H), 1.63 (s, 1 H), 1.53 - 1.30 (m, 4 H), 1.04 (t, J = 7.4 Hz, 3 H), 0.96 -
0.85 (m, 3 H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 198.8, 168.2, 165.5, 163.3, 156.5, 140.1, 117.7, 68.5, 32.3,
106
28.1, 28.1, 26.4, 22.3, 21.3, 14.0, 13.8 ppm; 13C NMR (75 MHz, DEPT-135): δ = 13.8 (CH3), 14.0 (CH3), 21.3 (CH2), 22.3
(CH2), 26.4 (CH3), 28.1 (CH2), 28.1 (CH2), 32.3 (CH2), 68.5 (CH2) ppm.
1- isopropyloxy(thiocarbonyl)thio]propanyl, 1-[2-amino-4-(pentyloxy)-
-7-(propylthio)-6-pteridinyl] 18
To a stirred solution of product 16 (3 g, 8.2 mmol) and 2-pyrrolidone (3.5 equivalents, 2.2 mL) in dry THF at 60°C was
added slowly portion-wise PHT (pyrrolidone hydrotribromide, 14.23 g, 3.5 equivalents) within around 5 hrs. After 3 days
of heating the volume was reduced almost to dryness. The product was solubilized in 80 mL of ethyl acetate, washed
three times with water and once with brine. The crude product was suspended in 60 mL of acetone and potassium
isopropylxanthate (1.43 g, 1 equivalent) was added and the solution was kept stirring overnight. The mixture was filtered
and the mother liquor dried on a rotary evaporator and the residue dissolved in ethyl acetate. The organic solution was
washed three times with water and once with brine, and dried with sodium sulfate.
The pure product was obtained after short-path aluminium oxide (neutral) column chromatography using chloroform
as eluent. The compound was stored in a dark place in order to avoid possible photochemical side reactions.
Data code = IT 103; X-ray code IT 103 FIN; yield 65%; mass spectrometry (APCI, positive, low t. low f.) = 497.7, 363.8
m/z; elemental analysis: calculated C-50.68, H-6.28, N-14.07, S-19.33; found C-50.68, H-6.17, N-13.09, S-18.85; 1H NMR
(300MHz, CHLOROFORM-d) δ = 5.84 (q, J = 7.2 Hz, 1 H), 5.65 (quind, J = 6.2, 12.4 Hz, 1 H), 4.53 (dt, J = 1.9, 6.7 Hz, 2 H),
3.26 (t, J = 7.2 Hz, 2 H), 1.98 - 1.85 (m, 2 H), 1.85 - 1.72 (m, 2 H), 1.65 (d, J = 7.2 Hz, 4 H), 1.57 - 1.37 (m, 4 H), 1.30 (d, J =
6.2 Hz, 6 H), 1.08 (t, J = 7.4 Hz, 3 H), 1.01 - 0.91 (m, 3 H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 211.8, 196.3,
168.2, 166.2, 163.5, 156.6, 138.3, 117.8, 78.2, 68.4, 49.2, 32.3, 28.2, 28.1, 22.3, 21.3, 21.2, 21.1, 16.6, 14.0, 13.7 ppm; 13C NMR (75 MHz): δ = 13.8 (CH3), 14.0 (CH3), 16.6 (CH3), 21.1 (CH3), 21.2 (CH3), 21.3 (CH2), 22.4 (CH2), 28.1 (CH2), 28.2
(CH2), 32.3 (CH2), 49.2 (CH), 68.5 (CH2), 78.2 (CH) ppm.
1- diethylamino(thiocarbonyl)thio]propanyl, 1-[2-amino-4-(pentyloxy)-
-7-(propylthio)-6-pteridinyl] 19
To a stirred solution of product 16 (3 g, 8.2 mmol) and 2-pyrrolidone (3.5 equivalents, 2.18 mL) in dry 300 mL of dry THF
at 60°C was added slowly portion-wise 14.23 g (3.5 equivalents) of PHT within around 5 hrs. After 3 days the volume
was reduced almost to dryness. The product was solubilized in 80 mL of ethyl acetate and washed three times with
water and once with brine. The crude product was suspended in 60 mL of acetone, sodium diethyldithiocarbamate (1.4
g, 1 equivalents) was added and the solution kept stirring overnight. The mixture was filtered, the mother liquor dried
on a rotary evaporator and the residue dissolved in ethyl acetate. The organic solution washed three times with water
and once with brine, then dried with sodium sulfate. The pure product was obtained by short-path aluminium oxide
107
(neutral) column chromatography with chloroform and crystallized from a chloroform/n-hexane mixture. The
compound was stored in a dark place in order to avoid possible photochemical side reactions.
Data code = IT 108/122; X-ray code IT 122 FIN; yield 62%; mass spectrometry IT 108 (APCI, positive, low t. low f.) = 511.5
m/z.
1-diethylamino(thiocarbonyl)thio]propanyl, 1-[2-amino-7-(propylthio)-6-pteridinyl] 20
The deprotection was achieved dissolving 0.5 g (0.97 mmol) of product 19 in 100 ml dichloromethane and diethyl ether
1:1 mixture with 10 equivalents of concentrated sulfuric acid at RT overnight under stirring. The volume of the reaction
mixture was reduced almost to dryness, 10 mL of acetone were added and the mixture kept under stirring until a
homogeneous suspension was obtained. Then 40 g of ice in 10 mL of water were added slowly until complete
precipitation of the yellowish powder. The solution was neutralized with sodium hydrogen carbonate and the product
filtered out. The precipitate was washed with water and acetone and kept in a dessicator under vacuum overnight. The
compound was stored in a dark place in order to avoid possible photochemical side reactions. Data code = IT 108 CARB.,
IT 124; 73% of 20, mass spectrometry (APCI, positive, low t. low f.) = 19-511.6; 20-364 m/z; elemental analysis: 20
calculated C-46.34, H-5.49, N-19.07, S-21.83; found C-45.45, H-5.20, N-17.88; S-21.43; 1H NMR 20 (300MHz, DMSO-d6)
δ = 5.95 - 5.77 (m, 1 H), 3.90 (d, J = 6.2 Hz, 1 H), 3.86 - 3.71 (m, 3 H), 3.06 (t, J = 7.2 Hz, 2 H), 1.66 (sxt, J = 7.3 Hz, 3 H),
1.51 (d, J = 7.3 Hz, 3 H), 1.25 (t, J = 7.0 Hz, 3 H), 1.12 (t, J = 6.9 Hz, 3 H), 0.98 (t, J = 7.3 Hz, 3 H) ppm; 13C NMR 20 (75MHz,
DMSO-d6) δ = 195.9, 192.5, 163.4, 160.1, 156.8, 155.9, 136.7, 122.8, 50.6, 48.8, 47.1, 31.2, 21.5, 16.5, 13.5, 12.4, 11.3
ppm; 13C NMR 20 (75 MHz, DEPT-135): δ = 11.3 (CH3), 12.4 (CH3), 13.5 (CH3), 16.5 (CH3), 21.5 (CH2), 31.2 (CH2), 47.1
(CH2), 48.8 (CH2), 50.6 (CH) ppm.
1,3-dithiol-2-one-4-methyl-5-(2-pteridinamine, 7-(propylthio) 21
A small flask containing product 18 (3 g, 6 mmol) was kept in an ice bath with stirring. After 10 min 3.2 mL of
concentrated sulfuric acid (10 equivalents) were added slowly and dropwise until the formation of a homogeneous
mixture was observed. The flask was kept in the bath under stirring for 3 hrs, upon which RT was reached. The viscous
mixture was poured into ice for the precipitation of a yellow powder. The suspension was neutralized with sodium
hydrogen carbonate, taking care that the strong evolution of gas did not cause a loss of product. After filtration the
product was washed with water and acetone and stored under vacuum overnight. The compound was stored in a dark
place in order to avoid possible photochemical side reactions. Data code = IT 111 DEP FIN; yield 60%; mass spectrometry
(APCI, positive, low t. low f.) = 367.7 m/z; elemental analysis: calculated C-42.49, H-3.57, N-19.06, S-26.18; found C-
38.55, H-3.59, N-17, S-26.15; 1H NMR (300MHz, DMSO-d6) δ = 7.39 (br. s., 1 H), 3.17 (t, J = 7.2 Hz, 3 H), 2.12 (s, 3 H),
1.69 (sxt, J = 7.3 Hz, 2 H), 0.97 (t, J = 7.3 Hz, 3 H) ppm; 13C NMR (75MHz, DMSO-d6) δ = 190.5, 161.9, 160.1, 154.5, 136.1,
108
131.6, 124.2, 122.2, 31.4, 21.8, 15.3, 13.3 ppm; 13C NMR (75 MHz, DEPT-135): δ = 13.3 (CH3), 15.3 (CH3), 21.8 (CH2), 31.4
(CH2) ppm.
Ethanaminium, N-(1,3-dithiol-2-ylidene-4-methyl-5-(2-pteridinamine, 7-(propylthio))-
-N-ethyl, sulfate 22
A small flask containing product 20 (3 g, 6.8 mmol) was kept in an ice bath with stirring. After 10 min 3.6 mL of
concentrated sulfuric acid (10 equivalents) was added slowly and dropwise until the formation of a homogeneous
mixture was observed. The flask was kept in the bath under stirring for 3 hrs, upon which the RT was reached. The
viscous mixture was poured into ice and neutralized with sodium hydrogen carbonate. The product was partly extracted
four times with chloroform. The organic phases were collected and dried on a rotary evaporator obtaining a brownish
oil, which was kept in a dessicator overnight. The analytical data were obtained after the attempt of recrystallization in
methanol at low temperature. The compound was stored in a dark place in order to avoid possible photochemical side
reactions. Data code = IT 124, IT 125; yield estimated around 40%; mass spectrometry (APCI, positive, low t. low f.) =
423.3 m/z; 1H NMR (300MHz, DMSO-d6) δ = 8.33 (br. s., 2 H), 5.96 - 5.68 (m, 1 H), 4.05 - 3.65 (m, 4 H), 3.12 (t, J = 7.0 Hz,
2 H), ), 2.55-2.45 (s, 3 H) overlap with DMSO signal, 1.68 (sxt, J = 7.3 Hz, 2 H), 1.52 (d, J = 7.2 Hz, 3 H), 1.25 (t, J = 6.8 Hz,
3 H), 1.11 (t, J = 6.8 Hz, 3 H), 1.05 - 0.93 (m, 3 H); 13C NMR (75MHz, DMSO-d6) δ = 188.4, 180.4, 164.9, 159.0, 157.1,
156.5, 131.6, 122.5, 55.0, 47.7, 31.1, 28.3, 21.5, 13.4, 11.7 ppm; 13C NMR (75 MHz, DEPT-135): δ = 11.4 (CH3, CH3), 13.2
(CH3), 21.3 (CH2), 28.0 (CH3), 30.8 (CH2), 47.4 (CH2, CH2) ppm.
1-[[(1,1-dimethylethyl)dimethylsilyl]oxy], 1-[2-amino-4-(pentyloxy)-7-(propylthio)-6-pteridinyl] 23
To a suspension of product 15 (0.5 g, 1.6 mmol) and the aldehyde H (5.2 g, 17 equivalents) in 40 mL of acetic acid/water
3:1 were added dropwise simultaneously 2.5 g of FeSO4.7H2O (2.5 g. 9.2 mmol) in 13 mL of water and 1.3 mL of tert-
butyl hydroperoxide 70% aqueous solution with vigorous stirring within around 60 seconds. After stirring for 2 minute,
80 mL of water were added, the precipitate was collected and washed with water and dried to give a pure pale yellow
powder. Data code = CT 38; yield 70%; mass spectrometry (APCI, positive, low t. low f.) = 494.2 [(without AcO-)+H], 362
m/z; elemental analysis (with a molecule of acetic acid): calculated C-54.22, H-7.83, N-12.65, S-5.79; found C-55.868, H-
7.922, N-12.778, S-7.12;1H NMR (300MHz, CHLOROFORM-d) δ = 4.54 (t, J = 6.6 Hz, 2 H), 4.08 (t, J = 6.8 Hz, 2 H), 3.43 (t,
J = 6.6 Hz, 2 H), 3.25 - 3.16 (m, 2 H), 2.11 (s, 3 H), 1.91 (quin, J = 7.0 Hz, 2 H), 1.77 (sxt, J = 7.4 Hz, 2 H), 1.56 - 1.37 (m, 4
H), 1.08 (t, J = 7.4 Hz, 3 H), 0.99 - 0.92 (m, 3 H), 0.85 (s, 9 H), 0.05 (s, 6 H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ =
199.1, 176.3, 168.2, 165.7, 163.2, 155.8, 140.0, 117.4, 68.6, 59.3, 41.5, 32.3, 28.1, 28.1, 25.8, 22.3, 21.7, 21.1, 18.2, 14.0,
13.7, -5.4 ppm;13C NMR (75 MHz, DEPT-135): δ = -5.4 (CH3, CH3), 13.7 (CH3), 14.0 (CH3), 21.1 (CH3), 21.7 (CH2), 22.3
(CH2), 25.8 (CH3, CH3, CH3), 28.1 (CH2), 28.1 (CH3), 32.3 (CH3), 41.5 (CH3), 59.3 (CH3), 68.6 (CH3) ppm;29Si NMR (60MHz,
CHLOROFORM-d) δ = 19.88 ppm.
109
1-2-[(4RS)-2,2-Dimethyl-1,3-dioxolan-4-yl]-1-,1-[2-acetamido-4-(pentyloxy)-
-7-(propylthio)-6-pteridinyl] 24
To a suspension of product 15 (0.5 g, 1.6 mmol) and the aldehyde I (3.9 g, 17 equivalents) in 40 mL of acetic acid/water
3:1 were added dropwise simultaneously 2.5 g of FeSO4.7H2O (2.5 g. 9.2 mmol) in 13 mL of water and 1.3 mL of tert-
butyl hydroperoxide 70% aqueous solution with vigorous stirring within around 60 seconds. After stirring for 1 minute,
80 mL of water were added, the precipitate was collected and washed with water and dried to give a pure pale yellow
powder. Data code = ACT 20; yield 82.5 %; X-ray MAD205F; mass spectrometry (APCI, positive, low t. low f.) = 450.0
m/z; 1H NMR (300MHz, CHLOROFORM-d) δ = 4.75 - 4.61 (m, 1 H), 4.54 (t, J = 6.0 Hz, 2 H), 4.30 (dd, J = 6.2, 8.0 Hz, 1 H),
3.81 (dd, J = 5.6, 17.4 Hz, 1 H), 3.71 (t, J = 7.5 Hz, 1 H), 3.32 (dd, J = 7.6, 17.3 Hz, 1 H), 3.20 (t, J = 7.4 Hz, 2 H), 2.11 (s, 3
H), 1.97 - 1.85 (m, 2 H), 1.83 - 1.70 (m, 2 H), 1.34 – 1.54 (m, 10 H), 1.09 (t, J = 7.3 Hz, 3 H), 0.96 (t, J = 6.8 Hz, 3 H) ppm.
1-[2-Amino-4-(pentyloxy)-6-pteridinyl]-1-propanone 26
Method B in a procedure as reported in a publication [122] was followed: a pre-dried 100 mL Schlenk with a
PTFE/silicone septum was charged with product 16 (6.48 g, 17.8) and palladium on charcoal 10% (380 mg, 0.356 mmol
). After careful (the fine powder charcoal can be lost) evacuation of the flask and installation of an nitrogen gas
atmosphere around 15 mL of THF (dry and oxygen free) were added and kept in an ice bath. Using a syringe triethylsilan
(8.5 mL, 3 equivalents) was added and the mixture kept at ice-bath temperature for 40 min (evolution of gas was
observed) and at RT for 5 hrs. The suspension was filtered through a patch of celite to remove the charcoal and the
volume was reduced. The crude mixture was dissolved in 50 mL of methanol and stirred overnight at RT in order to
remove the silane reacted with the amino function (see chapter results and discussion: product 25). On a rotary
evaporator the methanol was removed and the residue purified through aluminium oxide (neutral) short-path column
chromatography. The first fraction (eluent chloroform) was the unreacted starting material and the second (10%
methanol/chloroform eluent) was product 26. Data code = IT 127; yield 40%; mass spectrometry (APCI, positive, low t.
low f.) = 289.9 m/z; elemental analysis: calculated C-58.12, H-6.62, N-24.21; found C-57.18, H-6.324, N-23.6; 1H NMR
(300MHz, CHLOROFORM-d) δ = 9.45 (s, 1 H), 5.74 (br. s., 1 H), 4.58 (t, J = 6.8 Hz, 2 H), 3.27 (q, J = 7.3 Hz, 2 H), 1.95 (quin,
110
J = 7.0 Hz, 2 H), 1.57 - 1.38 (m, 5 H), 1.25 (t, J = 7.3 Hz, 4 H), 1.03 - 0.90 (m, 3 H); 13C NMR (75MHz, CHLOROFORM-d) δ =
200.9, 168.2, 162.7, 158.5, 149.8, 142.9, 132.1, 122.2, 68.8, 64.2, 30.8, 28.1, 22.4, 14.0, 7.7, 0.0 ppm; 13C NMR (75 MHz,
DEPT-135): δ = 7.7 (CH3), 14.0 (CH3), 22.4 (CH2), 28.1 (CH2), 28.1 (CH2), 30.8 (CH2), 68.8 (CH2), 149.8 (CH) ppm.
1- isopropyloxy(thiocarbonyl)thio]propanyl, 1-[2-amino-4-(pentyloxy)-6-pteridinyl] 27
To a stirred solution of 3 g product 26 (10.3 mmol) and 2-pyrrolidone (3.5 equivalents, 2.7 mL) in dry THF at 60°C was
added slowly portion-wise PHT (pyrrolidone hydrotribromide, 17.9 g, 3.5 equivalents) within around 3 hrs and the
mixture was kept under stirring overnight. The volume was reduced almost to dryness, the product was solubilized in
80 mL of ethyl acetate and washed three times with water and once with brine. The crude product was suspended in
60 mL of acetone and 1.8 g of potassium isopropylxanthate (1 equivalent) was added. The solution was kept stirring
overnight. The mixture was filtered and the mother liquor dried on a rotary evaporator. The residue was dissolved in
ethyl acetate, the organic solution washed three times with water and once with brine, then dried with sodium sulfate.
The pure product was obtained by short-path aluminium oxide column chromatography using chloroform as eluent.
The compound was stored in a dark place in order to avoid possible photochemical side reactions. Data code = IT 128,
X-ray data IT 128f, yield 67%,Mass spectrometry (APCI, positive, low t. low f.) = 423.7, 289.9 m/z; elemental analysis:
calculated C-51.04, H-5.95, N-16.53, S-15.14; found C-46.45, H-5.084, N-23.64 , S-13.6.
1H NMR (300MHz,CHLOROFORM-d) δ = 9.41 (s, 1 H), 5.86 (q, J = 7.2 Hz, 1 H), 5.67 (td, J = 6.2, 12.5 Hz, 1 H), 4.64 - 4.51
(m, 2 H), 1.94 (quin, J = 7.0 Hz, 2 H), 1.66 (d, J = 7.2 Hz, 3 H), 1.58 - 1.39 (m, 5 H), 1.34 - 1.26 (m, 6 H), 1.00 - 0.91 (m, 3
H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 211.5, 196.1, 168.1, 163.1, 158.4, 150.2, 141.1, 122.2, 78.3, 68.7, 48.2,
28.1, 22.3, 21.2, 21.1, 16.2, 14.0 ppm; 13C NMR (75 MHz, DEPT-135): δ = 14.0 (CH3), 16.2 (CH3), 21.1 (CH3), 21.2 (CH3),
22.3 (CH2), 28.1 (CH2, CH2), 48.3 (CH3), 68.8 (CH2), 78.3 (CH), 150.2 (CH) ppm.
Ethanone, 1-[2-amino-4-(pentyloxy)-7-methyl(o-isopropyl dithiocarbonate)-6-pteridinyl] B’
To a stirred solution of product 11 (3 g; 8.2 mmol) and 2-pyrrolidone (3.5 equivalents, 2.2 mL) in dry THF at 55°C was
added slowly portion-wise 14.23 g (3.5 equivalents) of PHT (pyrrolidone hydrotribromide) within around 7 hrs and the
mixture was kept under stirring overnight. The volume was reduced almost to dryness. The product was solubilized in
80 mL of ethyl acetate and washed three times with water and once with brine. The crude product was suspended in
60 mL of acetone and potassium isopropylxanthate (1.43 g, 1 equivalents) was added. The solution was kept stirring
overnight. The mixture was filtered and the mother liquor dried on a rotary evaporator. The residue was dissolved in
ethyl acetate, the organic solution washed three times with water and once with brine, then dried with sodium sulfate.
The pure product was obtained by short-path aluminium oxide (neutral) column chromatography using chloroform as
eluent. The compound was stored in a dark place in order to avoid possible photochemical side reactions. Data code =
IT 32 PU, yield 72%; elemental analysis: calculated C-51.04, H-5.95, N-16.53, S-15.14; found C-50.70, H-5.77, N-16.32, S-
111
15.17; 1H NMR (300MHz, DMSO-d6) δ = 7.80 (br. s., 1 H), 7.68 (br. s., 1 H), 5.60 (spt, J = 6.2 Hz, 1 H), 4.90 (s, 2 H), 4.51
(t, J = 6.6 Hz, 2 H), 2.64 (s, 3 H), 1.84 (quin, J = 6.9 Hz, 2 H), 1.53 - 1.33 (m, 4 H), 1.30 (d, J = 6.4 Hz, 6 H), 0.96 - 0.85 (m, 3
H); 13C NMR (75MHz, DMSO-d6) δ = 212.5, 198.9, 166.8, 163.4, 157.2, 156.9, 139.9, 120.4, 78.3, 67.6, 27.6, 26.8, 21.8,
20.9, 13.9; 13C NMR (75 MHz): δ = 13.9 (CH3), 20.9 (CH3, CH3), 21.8 (CH2), 26.8 (CH3), 27.6 (CH2, CH2), 40.2 (CH2), 67.6
(CH2), 78.3 (CH) ppm.
Ethanone, 1-[2-acetamido-4-(pentyloxy)-7-methyl(o-isopropyl dithiocarbonate)-6-pteridinyl]
White solid
Method as reported in a publication [92] was followed: a suspension of B’ (8 g, 18.8 mmol) in a mixture of 250 mL acetic
acid and 500 mL of acetic anhydride was refluxed for 3 hrs. Then the volume of the solution was reduced almost to
dryness. The residue dissolved in chloroform and washed with water three times, then dried with sodium sulfate and
the solvent was removed on a rotary evaporator The crude product (a dark brown solid) was purified by short-path
aluminium oxide (neutral) column chromatography with ethyl acetate/n-hexane as eluent mixture 1:1), the second
fraction is collected and crystallized in mixture of chloroform and n-hexane. The purified substance was a bright white
powder.
Data code = IT 59, yield 25%; 1H NMR (300MHz, DMSO-d6) δ = 10.92 (s, 1 H), 5.60 (quin, J = 6.2 Hz, 1 H), 4.98 (s, 2 H),
4.63 (t, J = 6.6 Hz, 2 H), 2.71 (s, 3 H), 2.35 (s, 3 H), 1.88 (quin, J = 6.9 Hz, 2 H), 1.53 - 1.35 (m, 4 H), 1.31 (d, J = 6.4 Hz, 6
H), 1.00 - 0.81 (m, 3 H) ppm.
Ethanone,1-[2-amino-4-(pentyloxy)-7-methyl(diethyldithiocarbamate)-6-pteridinyl]
Carbamate derivative
To a stirred solution of product 11 (3 g; 8.2 mmol) and 2-pyrrolidone (3.5 equivalents, 2.2 mL) in dry THF at 55°C was
added slowly portion-wise 14.23 g (3.5 equivalents) of PHT (pyrrolidone hydrotribromide) within around 7 hrs and the
mixture was kept under stirring overnight. The volume was reduced almost to dryness. The product was solubilized in
80 mL of ethyl acetate and washed three times with water and once with brine. The crude product was suspended in
60 mL of acetone and Sodium diethyldithiocarbamate (1.4 g, 1 equivalents) was added. The solution was kept stirring
overnight. The mixture was dried on a rotary evaporator and the residue was dissolved in ethyl acetate, the organic
solution washed three times with water and once with brine, then dried with sodium sulfate. The pure product was
obtained by short-path aluminium oxide (neutral) column chromatography using chloroform as eluent. The compound
was stored in a dark place in order to avoid possible photochemical side reactions.
112
Data code = IT 37, yield 68%; 1H NMR (300MHz, DMSO-d6) δ = 7.91 - 7.50 (m, 2 H), 4.99 (s, 2 H), 4.50 (t, J = 6.6 Hz, 2 H),
3.91 (d, J = 6.8 Hz, 2 H), 3.76 (d, J = 7.2 Hz, 2 H), 2.64 (s, 3 H), 1.84 (t, J = 6.8 Hz, 2 H), 1.55 - 1.30 (m, 4 H), 1.28 - 1.09 (m,
7 H), 0.90 (t, J = 7.0 Hz, 3 H) ppm.
Ethanone,1-[2-amino-4-(pentyloxy)-7-methyl(diethyldithiocarbamate)-6-pteridinyl]- sulfate salt
Carbamate derivative protonated
The product was exposed to the nitrogen flow containing hydrochloric acid for around 5 minutes, as described in the
chapter “Result and Discussion”. The product was characterized without further purification. Data code = IT 39, yield
26%; 1H NMR (300MHz, CHLOROFORM-d) δ = 9.82 (br. s., 1 H), 7.72 (br. s., 1 H), 4.90 (s, 2 H), 4.72 (t, J = 6.6 Hz, 2 H),
3.86 (q, J = 6.9 Hz, 2 H), 3.73 (q, J = 6.7 Hz, 2 H), 2.82 (s, 3 H), 1.55 - 1.38 (m, 4 H), 1.32 (t, J = 7.0 Hz, 3 H), 1.18 (t, J = 7.2
Hz, 3 H), 1.01 - 0.91 (m, 3 H) ppm.
Ethanone, 1-[2-amino-4-(pentyloxy)-7,8-thiazolone)-6-pteridinyl]- C’
Method as reported in a publication [92] was followed: a suspension of B’ (8 g, 18.8 mmol) in a mixture of 250 mL acetic
acid and 500 mL of acetic anhydride was refluxed for 6 hrs. Then the volume of the solution was reduced almost to
dryness, the residue dissolved in chloroform and washed with water three times, then dried with sodium sulfate and
the solvent was removed on a rotary evaporator. The crude product (a dark brown solid) was purified by silica gel column
chromatography with ethyl acetate/n-hexane as eluent mixture 1:1 and crystallized in mixture of chloroform and n-
hexane. The purified substance was a bright red powder.
The collected powder is structure C’ but with the protected amino function (red solid, see below). The product was
dissolved in 500 mL of methanol and kept under stirring overnight, in this time the hydrolysis of the protection is
achieved. The volume of the solution reduced and the crude product recrystallized from a mixture of chloroform and n-
hexane.
Data code = IT 70, overall yield 35% (referring to the moles of product B’, comprehensive the hydrolysis of the protection
of the amino function), elemental analysis: calculated C-51.86, H-4.93, N-20.16, S-9.23; found C-51.57, H-4.68, N-19.54,
S-9.19; mass spectrometry (APCI, positive - negative, low t. low f.) = 348.3, 278.2 - 346.2 m/z; 1H NMR (300MHz,
CHLOROFORM-d) δ = 7.70 (s, 1 H), 5.69 (br. s., 2 H), 4.43 (t, J = 6.8 Hz, 2 H), 2.62 (s, 3 H), 2.04 (s, 1 H), 1.95 - 1.73 (m, 2
H), 1.56 - 1.33 (m, 4 H), 1.02 - 0.86 (m, 3 H); 13C NMR (75MHz, CHLOROFORM-d) δ = 198.0, 167.0, 166.2, 162.0, 149.4,
113
141.5, 125.0, 108.3, 103.3, 67.9, 28.3, 28.1, 25.7, 22.3, 14.0; 13C NMR (75 MHz): δ = 14.0 (CH3), 22.3 (CH2), 25.7 (CH3),
28.1 (CH2), 28.2 (CH2), 67.9 (CH2), 103.3 (CH) ppm.
Ethanone, 1-[2-acetamido-4-(pentyloxy)-7,8-thiazolone)-6-pteridinyl]
Red solid
Data code = IT 59C1; 1H NMR (300MHz ,CHLOROFORM-d) δ = 8.16 (s, 1 H), 7.78 (s, 1 H), 4.50 (t, J = 6.8 Hz, 2 H), 2.68 (s,
3 H), 2.66 (s, 3 H), 1.88 (quin, J = 7.0 Hz, 2 H), 1.56 - 1.34 (m, 4 H), 1.04 - 0.86 (m, 3 H) ppm.
Ethanone, 1-[2-amino-7,8-thiazolone)-6-pteridinyl]
Product C’ without pentyloxy chain
The removal of the pentyloxy chain was achieved following the method reported in publication [93]: the product was
dissolved in a minimum amount of 0.1 N solution of hydrochloric acid and refluxed for 1 hr. After cooling, the precipitate
was collected, washed with H20 and EtOH, and then dried.
IT 83 (deprotected); yield 42% C-43.32, H-2.54, N-25.26, S-11.57; found C-41.27, H-3.22, N-21.9, S-10.06.
Complexation product (G’, IT 114)
The reaction was conducted under inert gas atmosphere (nitrogen). The product C’ (100 mg, 0.28 mmol) were added in
200 mL of methanol (oxygen free). Five mL of aqueous solution containing 0.5 equivalents of K3Na[MoO2(CN)4]*6H2O
and 10 equivalent of potassium hydroxide were added slowly (the water was first purged with nitrogen for around 30
minutes). The mixture was kept under reflux for 1 hr. The deep red colour solution was evaporated and the residue
washed with diethyl ether and acetonitrile (both solvents oxygen free). The final filtrate was washed with a small
amount of methanol (around 7 mL) and the resulting product dried under vacuum. Data code = IT 114 LAV; elemental
analysis: found C-32.11, H-2.73, N-18.91, S-4.78.
114
3-(t-Butyldimethylsilyloxy)propan-1-ol G-first step
A procedure as reported in a publication [112] was followed: sodium hydride (80% suspension in oil, 7.5 g, 249 mmol)
was suspended in THF (500 mL) after being washed with n-hexane. 1,3-propanediol (15 mL, 15.8 g. 207.5 mmol) was
added to the mixture at RT and stirred for 45 min, after which time an opaque white precipitate had formed. Tert-
butyldimethylsilyl chloride (31.3 g, 207.5 mmol) was then added and vigorous stirring was continued for 45 min. The
mixture was poured into diethyl ether (500 mL), washed with 10% potassium carbonate (150 mL) and brine (150 mL),
dried with sodium sulfate and concentrated in vacuo. The resulting oil was purified by flash column chromatography
using ethyl acetate/petrol (1:4) as eluent. Data code = CT 1, yield 80%, mass spectrometry (APCI, positive, low t. low f.)
= 191.1. m/z.
3-(t-Butyldimethylsilyloxy)butanal G-second step
Method A as reported in a publication [112] was followed: a solution of oxalyl chloride (0.9 mL, 10 mmol) in
dichloromethane (100 mL) was cooled to -78°C (for cooling an ethyl acetate /N2 bath was used; -84°C) to which was
added dropwise dried dimethyl sulfoxide (1.5 mL, 20.8 mmol, dimethyl sulfoxide anhydrous ≥99.9%, sigma Aldrich).
After 15 min at -78°C a solution of product G-first step (1.75 g 8.58 mmol) in dichloromethane (20 mL) was added
dropwise and the reaction mixture stirred at -78°C for further 25-30 min. The reaction was quenched by the addition of
triethylamine (6 mL) and allowed to warm to RT. After 1 hr the reaction was poured into saturated aqueous sodium
bicarbonate solution (50 mL). The layers were separated and the aqueous layers extracted with dichloromethane. The
combined organic layers were washed with brine (50 mL), dried with sodium sulfate and concentrated in vacuo. Flash
column chromatography of the residue using 1:19 ethyl acetate: petrol as eluent, gave the desired product. Data code
= CT 4, yield 75%; 1H NMR (300MHz, CHLOROFORM-d) δ = 9.77 (t, J = 2.1 Hz, 1 H), 3.96 (t, J = 6.0 Hz, 2 H), 2.57 (dt, J =
2.1, 5.9 Hz, 2 H), 0.86 (s, 10 H) ppm; 13C NMR (75MHz, CHLOROFORM-d) δ = 201.9, 57.3, 46.5, 25.7, 18.1, -5.5 ppm; 13C
NMR (75 MHz): δ = -5.5 (CH3, CH3), 25.7 (CH3, CH3, CH3), 46.5 (CH2), 57.3 (CH2), 201.9 (CH) ppm.
2,2-Dimethyl-1,3-dioxolane-4-acetaldehyde (RS) H
The product was synthesized with the collaboration with the project member Mohsen Ahmadi and following the
procedures reported in the publications [123, 124]. The synthetic description will be reported in detail in the PhD thesis
of Mohsen Ahmadi.
115
IR and UV-VIS spectra
6
7
8
116
10
11
117
13
14
15
118
16
17
119
18
20
120
21
121
23
26
122
27
B’
C’
123
Product C’ without pentyloxy chain
124
Molecular structures and X-ray data
6
9
Identification code IT27CHF Empirical formula C12 H15 D Cl3 N5 O2 Formula weight 369.65 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/c Unit cell dimensions a = 16.080(3) A alpha = 90 deg. b = 9.5697(19) A beta = 97.70(3) deg.
c = 10.649(2) A gamma = 90 deg. Volume 1624.0(6) A^3 Z, Calculated density 4, 1.512 Mg/m^3 Absorption coefficient 0.578 mm^-1 F(000) 760 Crystal size 0.458 x 0.228 x 0.207 mm Theta range for data collection 3.249 to 27.176 deg. Limiting indices -20<=h<=20, -12<=k<=12, -11<=l<=13 Reflections collected / unique 13736 / 3523 [R(int) = 0.0384] Completeness to theta = 25.242 99.7 % Absorption correction None Max. and min. transmission 0.9818 and 0.9447 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 3523 / 0 / 212 Goodness-of-fit on F^2 1.061 Final R indices [I>2sigma(I)] R1 = 0.0364, wR2 = 0.0864 R indices (all data) R1 = 0.0524, wR2 = 0.0921 Extinction coefficient n/a Largest diff. peak and hole 0.440 and -0.390 e.A^-3
Identification code Shelx it 21f Empirical formula C9 H15 N5 O2 Formula weight 225.26 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, C 2/c Unit cell dimensions a = 13.610(3) A alpha = 90 deg. b = 9.884(2) A beta = 111.06(3) deg.
c = 17.359(4) A gamma = 90 deg. Volume 2179.1(9) A^3 Z, Calculated density 8,1.373 Mg/m^3 Absorption coefficient 0.101 mm^-1 F(000) 960 Crystal size 0.380 x 0.279 x 0.230 mm Theta range for data collection 3.201 to 29.245 deg. Limiting indices -18<=h<=15, -13<=k<=13, -23<=l<=23 Reflections collected / unique 11888 / 2954 [R(int) = 0.0399] Completeness to theta = 25.242 99.7 % Absorption correction None Max. and min. transmission 0.9818 and 0.9447 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 2954 / 0 / 207 Goodness-of-fit on F^2 0.524 Final R indices [I>2sigma(I)] R1 = 0.0393, wR2 = 0.1049 R indices (all data) R1 = 0.0500, wR2 = 0.1223 Extinction coefficient n/a Largest diff. peak and hole 0.326 and -0.184 e.A^-3
125
11
14
Identification code CTCl Empirical formula C11 H14 Cl N5 O Formula weight 267.7 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/c Unit cell dimensions a = 10.538(2) A alpha = 90 deg. b = 4.6688(9) A beta = 97.42(3) deg.
c = 26.226(5) A gamma = 90 deg. Volume 1279.5(4) A^3 Z, Calculated density 4, 1.390 Mg/m^3 Absorption coefficient 0.295 mm^-1 F(000) 560 Crystal size 0.493 x 0.452 x 0.123 mm Theta range for data collection 1.949 to 26.371 deg. Limiting indices -13<=h<=13, -5<=k<=5, -32<=l<=29 Reflections collected / unique 10316 / 2608 [R(int) = 0.0833] Completeness to theta = 25.242 99.9 % Absorption correction Numerical Max. and min. transmission 0.8180 and 0.4728 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 2608 / 0 / 173 Goodness-of-fit on F^2 0.954 Final R indices [I>2sigma(I)] R1 = 0.0520, wR2 = 0.1181 R indices (all data) R1 = 0.1079, wR2 = 0.1476 Extinction coefficient 0.014(3) Largest diff. peak and hole 0.252 and -0.285 e.A^-3
Identification code Shelx IT 29 Empirical formula C14 H19 N5 O2 Formula weight 289.34 Temperature 293(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/n linic, P -1 Unit cell dimensions a = 13.746(3) A alpha = 90 deg. b = 7.0835(14) A beta = 104.97(3) deg.
c = 15.970(3) A gamma = 90 deg. Volume 1502.2(6) A^3 Z, Calculated density 4, 1.279 Mg/m^3 Absorption coefficient 0.089 mm^-1 F(000) 616 Crystal size 0.440 x 0.132 x 0.076 mm Theta range for data collection 3.165 to 29.270 deg. Limiting indices -17<=h<=18, -9<=k<=9, -21<=l<=21 Reflections collected / unique 15884 / 4059 [R(int) = 0.1730] Completeness to theta = 25.242 99.8 % Absorption correction None Max. and min. transmission 0.9818 and 0.9447 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 4059 / 1 / 206 Goodness-of-fit on F^2 0.910 Final R indices [I>2sigma(I)] R1 = 0.0580, wR2 = 0.1285 R indices (all data) R1 = 0.1545, wR2 = 0.2512 Extinction coefficient 0.040(6) Largest diff. peak and hole 0.353 and -0.377 e.A^-3
126
15
16
Identification code MADXM5a Empirical formula C15 H25 N5 O2 S Formula weight 339.46 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Triclinic, P -1 Unit cell dimensions a = 9.765(2) A alpha = 71.03(3) deg. b = 12.019(2) A beta = 80.36(3) deg.
c = 16.561(3) A gamma = 79.22(3) deg. Volume 1793.9(7) A^3 Z, Calculated density 4, 1.257 Mg/m^3 Absorption coefficient 0.197 mm^-1 F(000) 728 Crystal size 0.402 x 0.181 x 0.099 mm Theta range for data collection 3.247 to 26.372 deg. Limiting indices -12<=h<=12, -14<=k<=15, -20<=l<=20 Reflections collected / unique 15005 / 7288 [R(int) = 0.0901] Completeness to theta = 25.000 99.5 % Absorption correction None Max. and min. transmission 0.9818 and 0.9447 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 7288 / 41 / 44296 / 323 / 444 Goodness-of-fit on F^2 0.984 Final R indices [I>2sigma(I)] R1 = 0.0706, wR2 = 0.1659 R indices (all data) R1 = 0.1418, wR2 = 0.2079 Extinction coefficient n/a Largest diff. peak and hole 0.243 and -0.347 e.A^-3
Identification code Shelx, IT 90 Empirical formula C17 H25 N5 O2 S Formula weight 363.48 Temperature 293(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/c Unit cell dimensions a = 4.5429(9) A alpha = 90 deg. b = 17.490(4) A beta = 92.92(3) deg.
c = 24.684(5) A gamma = 90 deg. Volume 1958.7(7) A^3 Z, Calculated density 4, 1.233 Mg/m^3 Absorption coefficient 0.185 mm^-1 F(000) 776 Crystal size 0.445 x 0.066 x 0.045 mm Theta range for data collection 1.428 to 24.730 deg. Limiting indices -5<=h<=5, -20<=k<=20, 0<=l<=28 Reflections collected / unique 6452 / 3304 [R(int) = 0.3422] Completeness to theta = 25.000 95.5 % Absorption correction Numerical Max. and min. transmission 0.9236 and 0.5309 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 3304 / 0 / 226 Goodness-of-fit on F^2 0.817 Final R indices [I>2sigma(I)] R1 = 0.1125, wR2 = 0.2073 R indices (all data) R1 = 0.4148, wR2 = 0.3529 Extinction coefficient n/a Largest diff. peak and hole 0.249 and -0.240 e.A^-3
127
17
Identification code CT39 Empirical formula C16 H23 N5 O2 S Formula weight 349.45 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/n Unit cell dimensions a = 4.2777(9) A alpha = 90 deg. b = 23.560(5) A beta = 94.44(3) deg.
c = 17.673(3) A gamma = 90 deg. Volume 1775.8(6) A^3 Z, Calculated density 4, 1.307 Mg/m^3 Absorption coefficient 0.201 mm^-1 F(000) 744 Crystal size 3.459 x 0.065 x 0.063 mm Theta range for data collection 3.459 to 25.689 deg. Limiting indices -5<=h<=5, -28<=k<=28, -21<=l<=21 Reflections collected / unique 12607 / 3359 [R(int) = 0.1173] Completeness to theta = 25.242 99.4 % Absorption correction Numerical Max. and min. transmission 0.9972 and 0.8172 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 3359 / 1 / 228 Goodness-of-fit on F^2 0.989 Final R indices [I>2sigma(I)] R1 = 0.0569, wR2 = 0.1083 R indices (all data) R1 = 0.1410, wR2 = 0.1402 Extinction coefficient n/a Largest diff. peak and hole 0.311 and -0.345 e.A^-3
18
Identification code IT103FIN Empirical formula C21 H31 N5 O3 S3 Formula weight 497.69 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Triclinic, P -1 Unit cell dimensions a = 7.8816(12) A alpha = 92.603(12) deg. b = 10.5953(17) A beta = 95.277(11)
deg. c = 15.836(2) A gamma = 106.688(12) deg. Volume 1257.8(3) A^3 Z, Calculated density 2, 1.314 Mg/m^3 Absorption coefficient 0.326 mm^-1 F(000) 528 Crystal size 0.481 x 0.078 x 0.050 mm Theta range for data collection 3.399 to 24.710 deg. Limiting indices -9<=h<=8, -12<=k<=12, -18<=l<=18 Reflections collected / unique 9015 / 4263 [R(int) = 0.1592] Completeness to theta = 24.710 99.5 % Absorption correction Numerical Max. and min. transmission 0.9869 and 0.8403 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 4263 / 70 / 328 Goodness-of-fit on F^2 0.966 Final R indices [I>2sigma(I)] R1 = 0.0862, wR2 = 0.1520 R indices (all data) R1 = 0.2370, wR2 = 0.2070 Extinction coefficient n/a Largest diff. peak and hole 0.319 and -0.308 e.A^-3
128
19
24
Identification code IT122 Empirical formula C22 H34 N6 O2 S3 Formula weight 510.73 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Triclinic, P -1 Unit cell dimensions a = 6.9442(14) A alpha = 86.74(3) deg. b = 13.200(3) A beta = 89.22(3) deg.
c = 14.319(3) A gamma = 89.28(3) deg. Volume 1310.2(5) A^3 Z, Calculated density 2, 1.295 Mg/m^3 Absorption coefficient 0.313 mm^-1 F(000) 544 Crystal size 0.138 x 0.104 x 0.080 mm Theta range for data collection 3.164 to 23.256 deg. Limiting indices -6<=h<=7, -14<=k<=14, -15<=l<=15 Reflections collected / unique 7747 / 3733 [R(int) = 0.0611] Completeness to theta = 23.256 99.0 % Absorption correction None Max. and min. transmission 0.9869 and 0.8403 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 3733 / 101 / 357 Goodness-of-fit on F^2 0.840 Final R indices [I>2sigma(I)] R1 = 0.0439, wR2 = 0.0781 R indices (all data) R1 = 0.1134, wR2 = 0.0960 Extinction coefficient n/a Largest diff. peak and hole 0.170 and -0.213 e.A^-3
Identification code MAD205F, ACT 20 Empirical formula C23 H35 N5 O6 S Formula weight 509.62 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Triclinic, P -1 Unit cell dimensions a = 8.8777(18) A alpha = 96.70(3) deg. b = 9.2872(19) A beta = 93.09(3) deg.
c = 16.197(3) A gamma = 93.58(3) deg. Volume 1321.2(5) A^3 Z, Calculated density 2, 1.281 Mg/m^3 Absorption coefficient 0.168 mm^-1 F(000) 544 Crystal size 0.232 x 0.108 x 0.071 mm Theta range for data collection 3.160 to 25.679 deg. Limiting indices -10<=h<=10, -11<=k<=11, -19<=l<=19 Reflections collected / unique 10478 / 4996 [R(int) = 0.0606] Completeness to theta = 25.242 99.7 % Absorption correction Numerical Max. and min. transmission 0.9818 and 0.9447 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 4996 / 323 / 444 Goodness-of-fit on F^2 0.907 Final R indices [I>2sigma(I)] R1 = 0.0580, wR2 = 0.1285 R indices (all data) R1 = 0.1392, wR2 = 0.1622 Extinction coefficient n/a Largest diff. peak and hole 0.365 and -0.240 e.A^-3
129
27
E’
Identification code IT128F Empirical formula C18 H25 N5 O3 S2 Formula weight 423.55 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Triclinic, P -1 Unit cell dimensions a = 10.845(2) A alpha = 113.34(3) deg. b = 14.928(3) A beta = 107.35(3) deg.
c = 15.236(3) A gamma = 94.38(3) deg. Volume 2107.8(9) A^3 Z, Calculated density 4, 1.335 Mg/m^3 Absorption coefficient 0.281 mm^-1 F(000) 896 Crystal size 0.239 x 0.204 x 0.092 mm Theta range for data collection 3.261 to 23.257 deg. Limiting indices -12<=h<=12, -16<=k<=16, -16<=l<=16 Reflections collected / unique 13507 / 6003 [R(int) = 0.0782] Completeness to theta = 23.257 99.3 % Absorption correction Numerical Max. and min. transmission 0.9946 and 0.9083 Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 6003 / 149 / 543 Goodness-of-fit on F^2 1.004 Final R indices [I>2sigma(I)] R1 = 0.0902, wR2 = 0.2475 R indices (all data) R1 = 0.2353, wR2 = 0.3209 Extinction coefficient n/a Largest diff. peak and hole 0.345 and -0.445 e.A^-3
Identification code IT70c-19 Empirical formula C15 H18 N5 O4 S Formula weight 364.40 Temperature 100(2) K Wavelength 0.6199 A Crystal system, space group Orthorhombic, P c a 21 Unit cell dimensions a = 30.990(6) A alpha = 90 deg. b = 3.9700(8) A beta = 90 deg.
c = 25.430(5) A gamma = 90 deg. Volume 3128.7(11) A^3 Z, Calculated density 8, 1.547 Mg/m^3 Absorption coefficient 0.241 mm^-1 F(000) 1528 Crystal size 150 x 20 x 20 um Theta range for data collection 1.146 to 18.057 deg. Limiting indices -30<=h<=30, -3<=k<=3, -25<=l<=25 Reflections collected / unique 10970 / 3190 [R(int) = 0.0725] Completeness to theta = 18.057 98.9 % Absorption correction None Absolute structure parameter 0.6(10) Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 3190 / 2 / 161 Goodness-of-fit on F^2 2.783 Final R indices [I>2sigma(I)] R1 = 0.2388, wR2 = 0.5420 R indices (all data) R1 = 0.2643, wR2 = 0.5796 Extinction coefficient n/a Largest diff. peak and hole 2.810 and -2.814 e.A^-3
130
F’
Identification code shelx Empirical formula C9 H17 N5 Na2 O6 S Formula weight 369.32 Temperature 170(2) K Wavelength 0.71073 A Crystal system, space group Monoclinic, P 21/c Unit cell dimensions a = 15.111(3) A alpha = 90 deg. b = 12.922(3) A beta = 90.51(3) deg.
c = 9.0809(18) A gamma = 90 deg. Volume 1773.1(6) A^3 Z, Calculated density 4, 1.383 Mg/m^3 Absorption coefficient 0.264 mm^-1 F(000) 768 Crystal size 0.171 x 0.147 x 0.085 mm Theta range for data collection 3.046 to 24.619 deg. Limiting indices -17<=h<=15, -15<=k<=15, -10<=l<=10 Reflections collected / unique 11683 / 2967 [R(int) = 0.0747] Completeness to theta = 25.242 92.6 % Absorption correction None Absolute structure parameter 0.6(10) Refinement method Full-matrix least-squares on F^2 Data / restraints / parameters 2967 / 13 / 250 Goodness-of-fit on F^2 0.932 Final R indices [I>2sigma(I)] R1 = 0.0559, wR2 = 0.1428 R indices (all data) R1 = 0.0961, wR2 = 0.1590 Extinction coefficient n/a Largest diff. peak and hole 0.348 and -0.394 e.A^-3
131
References
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Eigenständigkeitserklärung
Hiermit erkläre ich, dass diese Arbeit bisher von mir weder an der Mathematisch Naturwissenschaftlichen
Fakultät der Ernst-Moritz-Arndt-Universität Greifswald noch einer anderen wissenschaftlichen Einrichtung
zum Zwecke der Promotion eingereicht wurde.
Ferner erkläre ich, dass ich diese Arbeit selbstständig verfasst und keine anderen als die darin angegebenen
Hilfsmittel und Hilfen benutzt und keine Textabschnitte eines Dritten ohne Kennzeichnung übernommen habe.
Unterschrift des Promovenden
136
Lebenslauf - Ivan Trentin
Schulbildung und Studium seit 10/2012 Promotion – Universität Greifswald, Institut für Biochemie MOCOMODELS‐European Research Council „Modelling the Molybdenum cofactor, synthesis of pterin-dithiolene ligand system “ Kooperation: Institut für Molekulare Enzymologie – Universität Potsdam Betreuerin: Prof. Dr. rer. nat. Carola Schulzke 03/2012‐ 10/2003 Studium der Chemie – Università degli Studi dell’ Insubria – Como – Italien 03/2012‐ 09/2007 Chemie Master of Science Masterarbeit im Fach anorganische und metallorganische Chemie „Synthese und Charakterisierung von polynukleare pyrazol-komplexen mit Kupfer(II).“Betreuer: Prof. Dr. G. Attilio Ardizzoia, Dr. Stefano Brenna 10/2010‐ 09/2010 Auslandsaufenthalt an der Sprachschule „Bridge Mills“ Galway – Irland Intensivkurs mit Zertifikat C1 10/2009‐ 10/2008 Auslandsstudium – Universität Bremen SOKRATES Stipendium ‐ ERASMUS Programm Besuch von Polymerchemie Vorlesungen und Erlernen von physikalischen Methoden 10/2007‐ 10/2003 Chemie Bachelor of Science Bachelorarbeit im Fach anorganische und metallorganische Chemie „Synthese und Charakterisierung von Koordinationspolymeren der Gruppe11 Betreuer: Prof. Dr. G. Attilio Ardizzoia, Dr. Fulvio Castelli. 10/2005‐ 08/2005 Auslandsaufenthalt in Proitzener Mühle – Uelzen – Deutschland Mühlenmitarbeiter 09/2003‐ 08/2003 Auslandsaufenthalt in Ballytoughey – Clare Island – Irland Farmmitarbeiter auf der „Macalla Farm“ 07/2003 Abitur I.T.I.S. Setificio “Paolo Carcano” – Technikum für Textilien – Como – Italien 08/2002 Auslandsaufenthalt an der Sprachschule „Regent School” Margate Pre‐intermediate – Kent – England Arbeitserfahrung im Rahmen der Chemie seit 10/2018 Wissenschaftlicher Mitarbeiter – CATALIGHT‐ CRC/Transregio Project and der Universität Ulm: Light-driven Molecular Catalysts in Hierarchically Structured Materials, Synthesis and Mechanistic Studies. 10/2018‐ 10/2012 Aktive Mitarbeit am MOCOMODELS – European Research Council Project “Synthesis of mono‐dithiolene molybdenum complexes and their evaluation as potential drugs for the treatment of human isolated sulphite oxidase deficiency.” Betreuerin: Prof. Dr. rer. nat. Carola Schulzke 07/2012‐ 03/2012 Wissenschaftlicher Mitarbeiter Synthese und Manipulation von luftempfindlichen Verbindungen bei inerter
Atmosphäre mittels Schlenktechnik Fakultät für Naturwissenschaften und Hochtechnologie – Como – Italien Laborleitung: Prof. Dr. G. Attilio Ardizzoia
137
Wissenschaftliche Beiträge 13‐12/03/2018 14th Koordinationschemie‐Treffen (KTC) Heidelberg – Deutschland Posterpräsentation: “Synthesis of ligand System that mimic different features of Molybdopterin.” 23‐18/06/2018 10th Molybdenum and Tungsten Enzymes Conference (MoTEC) Santa Fe – New Mexico – USA Vortrag: “Molybdenum and Tungsten enzyme’s Active Site Models – Blueprints for mimicking Molybdopterin.” 07‐05/03/2017 13th Koordinationschemie‐Treffen (KTC) Potsdam – Deutschland Vortrag: “Mimicking the Molybdopterin System by Synthesis of Molybdenum-Pterin Complexes.” 24‐23/02/2017 26th Industrial Inorganic Chemistry‐Material and Processes (ATC) DECHEMA‐ House, Frankfurt am Main – Deutschland Posterpräsentation: “Synthesis of ligand System that mimic different features of Molybdopterin.” 01/09‐28/08/2016 13th European Biological Inorganic Chemistry Conference (EuroBIC) Budapest – Ungarn Posterpräsentation: “Mimicking the Molybdenum Cofactor by synthesis of Molybdenum-Pterin Complexes” 28‐23/08/2015 25th International Society of Heterocyclic Chemistry Congress Santa Barbara – California – USA Posterpräsentation: “Synthesis of ligand System that mimic different features of Molybdopterin.” 10‐06/09/2015 9th Molybdenum and Tungsten Enzymes Conference (MoTEC) Balaton – Ungarn Posterpräsentation: “Mimicking the Molybdopterin System by Synthesis of Molybdenum-Pterin Complexes.” 28‐24/08/2014 12th European Biological Inorganic Chemistry Conference (EuroBIC) Zürich – Schweiz Posterpräsentation: “Mimicking the Molybdopterin System by Synthesis of Molybdenum-Pterin Complexes.” 12‐11/09/2014 17th Norddeutsches Doktorandenkolloquium (NDDK) LIKAT Rostock – Deutschland Posterpräsentation: “Mimicking the Molybdopterin System by Synthesis of Molybdenum-Pterin Complexes.” 20‐19/09/2013 16th Norddeutsches Doktorandenkolloquium (NDDK) Bremen – Deutschland Posterpräsentation: “Synthesis of ligand System that mimic different features of Molybdopterin.” 20‐16/07/2013 8th Molybdenum and Tungsten Enzymes Conference (MoTEC) Sintra – Portugal Posterpräsentation: “Synthesis of ligand System that mimic different features of Molybdopterin.” Zusatzqualifikationen Wissenschaftliche Methoden Spektroskopie: NMR, GC‐MS, MS, IR, GC, UV / VIS Schlenktechnik, Chromatographie, Lösungsmittel Destillation (Wasser/Sauerstoff‐frei) Kurse 24/06/2016 „Intercultural Communication“ One‐day training program mit Zertifikat 10‐04/06/2015 Summer school on Spectroscopy
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“COST action CM1305 Spectroscopy of Spin in Catalysis, Bioinorganic and Materials Chemistry : spinning a web of theory and practice.” University of Groningen – Niederlande 17‐12/06/2016 Summer school on Organic Synthesis 41th Edition of the “A. Corbella” Gargnano – Italien 16/052017 Arbeitssicherheit und Gesundheitsschutz in Laboratorien Fortbildungsveranstaltung – Greifswald – Deutschland EDV Kenntnisse MS Office Excel, Word, PowerPoint Chemoffice ChemDraw Database Scifinder, Reaxys, Beilstein, Pubmed Wissenschaftliche Software EndNote, Mercury Sprachen Italienisch Muttersprache Deutsch B2 – fliesend Englisch C1 – fliesend, verhandlungssicher Veröffentlichungen
„Modelling the Molybdenum cofactor, synthesis of pterin-dithiolene ligand system .“
Ivan Trentin, Nicolas Chrysochos, Mohsen Ahmadi, Carola Schulzke. In Progress
im Rahmen des Projektes ERC‐ MOCOMODELS
https://cordis.europa.eu/result/rcn/179750_en.html
EU Research ‐ Journal
https://issuu.com/euresearcher/docs/eur10_digital_mag/14
„Crystal Structures of Bis[3-Methyl-1,3-Ene-Dithiol-2-One] Disulfide and Bis[3-Methyl-1,3-Ene-Dithiol-2-One] Diselenide“
Ivan Trentin, Claudia Schindler and Carola Schulzke, From Research Communications, Acta Crystallographica Section E, (2018) 74, 840‐845.
„Pd/PTABS: Catalyst for Room Temperature Amination of Heteroarenes“
Murthy Bandaru, Siva Sankar; Bhilare, Shatrughn; Chrysochos, Nicolas; Gayakhe, Vijay; Trentin, Ivan; Schulzke, Carola; Kapdi, Anant R.From Organic Letters (2018), 20(2), 473‐476.
„Engineering the Active Site of the Amine Transaminase from Vibrio fluvialis for the Asymmetric Synthesis of Aryl–Alkyl Amines and Amino Alcohols“
Nobili, Alberto; Steffen‐Munsberg, Fabian; Kohls, Hannes; Trentin, Ivan; Schulzke, Carola; Hoehne, Matthias; Bornscheuer, Uwe T.From ChemCatChem (2015), 7(5), 757‐760.
„The Goldilocks principle in action: Synthesis and structural characterization of a novel [125] cubane stabilized by monodentate ligands“
G. Attilio Ardizzoia, Stefano Brenna, Sara Durini, Bruno Therrien and Ivan Trentin, From Dalton Transactions (2013), 42(34), 12265‐12273.
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Acknowledgement
Firstly, I would like to express my sincere gratitude to my supervisor Prof. Dr. rer. nat. Carola Schulzke for
supporting me during my PhD study and related research, for her motivation, patience and immense
knowledge. Her guidance helped me in all though out my research and this thesis. She has been a great
example to me as both researcher and person.
I thank my colleagues for the stimulating discussions, for the camaraderie, and for all the fun we have had in
these years. In particular for the mutual support during the long working periods in the laboratory.
I could not have finished my research work without the help from technical and non technical staff from our
institute. Indeed I would like to thank Dr. Gottfried Palm for the measurement of my compound at the
Synchrotron.
Also I thank my friends in Como for the support gave me in these years and for visiting me in Greifswald,
despite the distance I never felt that I was away from my hometown.
I am grateful to Katharina and all the people I met in Greifswald for the help in all aspect of my life and for
making me feel at home.
Last but not the least, I would like to thank my family: my father Silvano, my mother Ornella, my sister Isabella
and Marco for supporting me throughout my studies.
