Allosteric effectors

7/21/2019 Allosteric effectors

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DOI: 10.1002/cmdc.201000366

Polyphosphates and Pyrophosphates of Hexopyranoses asAllosteric Effectors of Human Hemoglobin: Synthesis,Molecular Recognition, and Effect on Oxygen Release

Konstantina C. Fylaktakidou,[a, b] Carolina D. Duarte,[a, c, d] Alexandros E. Koumbis,[a, e]

Claude Nicolau,*[a, c, f] and Jean-Marie Lehn*[a]

Introduction

A most fundamental physiological process in the blood of

aerobic organisms resides in the delivery of oxygen bound to

hemoglobin (Hb) in red blood cells (RBCs) to all tissues.

Oxygen delivery is regulated by allosteric effectors that bind to

Hb and decrease its oxygen binding affinity. As numerous dis-

eases including cardiovascular disease and cancer involve hy-

poxia, achieving increased oxygen release is expected to re-

store normoxia, and to thereby possess significant therapeuticpotential. Therefore, finding allosteric effectors of Hb that in-

crease oxygen release and delivery by RBCs represents a goal

of very special interest.

In humans, allosteric regulation is effected by 2,3-bisphos-

phoglycerate (BPG, I, Figure 1),[1] the binding of which to the

allosteric pocket of the Hb tetramer is well characterized.[2]

Among a variety of polyphosphates that are able to decrease

the affinity of human Hb for oxygen,[1] the natural substance

myo-inositol hexakisphosphate (IHP, II, Figure 1) is the most

powerful allosteric effector identified to date.[1a] It displaces

Hb-bound 2,3-BPG, occupying the allosteric pocket with higher

affinity.[1b,3] Thus, it triggers a decrease in the affinity between

O2 and Hb, and when loaded into circulating RBCs, it subse-

quently leads to increased and regulated release of oxygen

upon tissue demand.[4]

We showed previously that myo-inositol trispyrophosphate

(ITPP, III, Figure 1), the derivative of IHP that contains three

seven-membered cyclic pyrophosphate groups, is a mem-

brane-permeant allosteric effector of Hb, which increasesoxygen release in vitro, both in free Hb and whole blood, in a

concentration-dependent manner.[5] As a result, ITPP was iden-

tified as a novel and highly effective anti-angiogenic and anti-

cancer agent, counteracting the effects of hypoxia and hinder-

ing cancer progression.[6] It suppresses HIF-1a and significantly

Polyphosphorylated and perphosphorylated hexopyranose

monosaccharides and disaccharides were synthesized from

parent or partially protected carbohydrates as potential alloste-

ric effectors of hemoglobin. A study toward the construction

of seven- and eight-membered cyclic pyrophosphates was also

performed on the sugars which had the proper orientation,

protection, and number of phosphates. All final compounds

were tested for their efficiency on oxygen release from human

hemoglobin. Several compounds presented higher potency

than myo-inositol hexakisphosphate, which is the most effi-

cient of the known allosteric effectors of hemoglobin. Struc-

tureactivity relationships were analyzed. The affinity and effi-

ciency depend on the number of phosphates attached to the

carbohydrate skeleton and are related primarily to the number

of negative charges present. Other effects operate, but play a

lesser role.

Figure 1.Structures of allosteric effectors of Hb (IIII). Structures of seven-

and eight-membered cyclic pyrophosphates (IVandV).

[a] Prof. K. C. Fylaktakidou, Dr. C. D. Duarte, Prof. A. E. Koumbis,

Prof. C. Nicolau, Prof. J.-M. Lehn

Institut de Science et dIngnierie Supramolculaires

Universitde Strasbourg

8 Alle Gaspard Monge, 67000 Strasbourg (France)

Fax: (+33)368 855140

E-mail: [email protected]

[email protected]

[b] Prof. K. C. Fylaktakidou

Current address: Department of Molecular Biology and Genetics

Democritus University of Thrace, 68100 Alexandroupolis (Greece)

[c] Dr. C. D. Duarte, Prof. C. Nicolau

NormOxys Inc., 200 Boston Avenue, Medford, MA 02155 (USA)

[d] Dr. C. D. Duarte

Current address: Quintiles Strasbourg, Parc dInnovation

Rue Jean Dominique Cassini, 67400, Illkirch Graffenstaden (France)

[e] Prof. A. E. Koumbis

Current address: Laboratory of Organic Chemistry

Aristotle University of Thessaloniki, 54124 Thessaloniki (Greece)

[f] Prof. C. Nicolau

Friedman School of Nutrition Science and Policy

Tufts University, Boston, MA 02115 (USA)

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decreases VEGF in cells, thus blocking the route that leads to

angiogenesis.[6] Furthermore, it was found that ITTP is capable

of increasing exercise capacity in both normal and transgenic

mice with severe heart failure.[7] Therefore, it appears that the

biological effects resulting from the enhanced release of

oxygen caused by ITPP may have general utility in the treat-

ment of a variety of disease states in which tissues suffer from

low oxygen tension, such as cardiovascular and oncological ail-

ments, ischemic insult, heart attacks, stroke, and tumor pro-

gression.

These results greatly warrant a more complete exploration

of other molecules that may present related properties. We

have therefore undertaken a wide-range research program to

this end and present herein the synthesis of an initial series of

such compounds, phosphates and cyclic pyrophosphates de-

rived from various carbohydrates, as well as data regarding

their effect on oxygen release from pure human Hb.

Rationale

Research into the action of organophosphates as allosteric ef-

fectors of Hb has not progressed for many years. [8] Indeed, or-

ganophosphates are rarely examined as drug candidates due

to their highly charged structure, which consequently prevents

high concentrations at the targeted site, thus showing poor

oral bioavailability and/or cell penetration. Nevertheless, the re-

markable properties of IHP and ITPP as allosteric effectors [47]

prompted us to extend our investigations to compounds

closely related to inositols, such as carbohydrates. Polyphos-

phorylated carbohydrates (with three or more phosphate

groups) have been reported to possess numerous biological

activities. Specifically, various alkyl glycosides of phosphorylat-ed d-galactose, di-galactose, or lactose derivatives exhibit insu-

linic[9] and anti-inflammatory activity,[10] or have been used as

scaffolds for mechanistic studies into the processes of cell sur-

face receptor recognition.[11] For the glucose series, tris phos-

phorylated analogues were found to exhibit anti-inflammatory

activity[10a] or to prevent restenosis.[12] Other derivatives such as

d-glucose 2,4,6-trisphosphate and its 1,3-deoxy analogues

showed moderate inhibition against acid sphingomyelinase rel-

ative to myo-inositol 1,3,5-triphosphate,[13] whereas 4-nitro-

phenyl 2,3,4,6-tetrakis phosphorylated a- and b-glucopyrano-

sides were used for mechanistic studies on hydrolysis.[14] Inter-

estingly, oligosaccharides composed of repeating glucose

units, such as maltotriose, cellobiose, or heparin-like pentasac-

charide analogues, were found to possess antithrombotic ac-

tivity.[15] Finally, sucrose phosphate ester derivatives were in

use some decades ago for the improvement of films for cine-

matographic and photographic materials owing to their gelling

properties.[16]

The structural similarity of mannose tris phosphorylated de-

rivatives with a-trinositol (d-myo-inositol 1,2,6-trisphosphate

sodium salt), which has anti-inflammatory and analgesic activi-

ties, and with d-myo-inositol 1,4,5-trisphosphate, which is a

known secondary messenger, led to the synthesis of those car-

bohydrate derivatives as their mimics. It was found that

methyl-a-d-mannopyranoside 2,3,4-trisphosphate sodium salt

has less activity than a-trinositol, but is fivefold more resistant

to dephosphorylation.[17] The same 1- and/or 6-alkyl-protected

derivatives (as well as some rhamnose, fructopyranose, and

arabinitol analogues) were also found to prevent restenosis,[12]

and to present growth factor modulating or other activities. [18]

The linear diphosphate (DP, also termed linear pyrophos-

phate) moiety is a quite common unit in nature and is present

in ADP, for example. For the carbohydrate scaffolds, the glu-

cose pentakis linear DP derivative is a metabolite isolated from

bacteria.[19] However, seven-membered cyclic pyrophosphates

(PPs) are generally rather rare in nature, with the exception of

the cyclic 2,3-bisphosphoglycerate (cBPG, IV, Figure 1), which

was found in methanogenic bacteria.[20] Nevertheless, it would

seem that no seven-membered cyclic PP (like those present in

ITPP) has been ever constructed or identified on a carbohy-

drate scaffold.

In contrast, eight-membered cyclic PPs attached mainly to a

ribofuranose core (V, Figure 1), have received attention due to

cyclic ADP-ribose (or adenosine 3,5-PP),[21] a natural product

that was found to control calcium levels.[22] Other 3,5-PP nu-cleotide analogues have shown effects on germinating

B. cereus 569 spores,[23] cytidilate cyclase activity,[24] or antitu-

mor activity.[25] 3,5-PP nucleotide derivatives phosphorylated

at position 2 have been isolated from the red seaweed Por-

phyra umbilicalis, which is a constituent of herbal medicines. [26]

Finally, several 3,5-deoxyribose phosphate and PP nucleotides

were proven to be human P2Y6 receptor ligands, P2Y1 recep-

tor antagonists and partial agonists, and recombinant rat P2X

receptor agonists and antagonists.[27,28]

In the work presented herein, we targeted polyphosphates

(with three or more phosphate groups) and seven- and/or

eight-membered cyclic PPs of hexopyranoses, which are allnovel compounds, except one. Furthermore, their binding to

human Hb and their effect on oxygen release were investigat-

ed in order to gain insight into molecular recognition features

and structureactivity relationships in the allosteric regulation

of Hb.

Results and Discussion

Synthesis of polyphosphate and cyclic pyrophosphate

derivatives of hexopyranoses

Our synthetic plan should provide valuable information on sev-

eral questions regarding molecular recognition in the allosteric

pocket of Hb, such as the effect of the number of phosphates,

the most appropriate conformation for binding (mannose and

galactose with one axial hydroxy group are more closely relat-

ed to IHP), and the role of the anomeric phosphate (a or b),

which is chemically the most labile. To serve these purposes,

double protection of positions 1 and 6 of monosaccharides

could lead to the corresponding tris phosphorylated deriva-

tives. Proper selective unmasking of positions 1 or 6 of hexoses

would allow the synthesis of tetrakis phosphorylated deriva-

tives, and subsequently the possible simultaneous formation

of two cyclic PPs in a row, which is the closest that can be ach-

ieved in mimicking the ITPP structure. In addition, protection

154 www.chemmedchem.org 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ChemMedChem 2011, 6, 153 168

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of position 6 may allow the synthesis of both a- and b-anom-

ers of the tetrakis phosphorylated derivatives. Finally, perphos-

phorylation of naked monosaccharides and disaccharides is ex-

pected to give pentakis- and octakisphosphates, respectively.

For our purpose, we selected the three more common mono-

saccharidesglucose, mannose, and galactosewhich, upon

proper protection and deprotection reactions, would provide

tris- to pentakisphosphates and bispyrophosphates, and two

disaccharides (a reducing one: lactose, and a nonreducing

one: sucrose), expected to give octakisphosphates.

Synthesis of tris phosphorylated monosaccharides

The sodium salts of the tris phosphorylated glucose and man-

nose derivatives 8 and 10 were prepared from the known 6-O-

tert-butyldiphenylsilyl (TBDPS) glucose and mannose methyl

glycosides (1[29] and 2,[30] respectively), as depicted in

Scheme 1. Compounds 1 and 2 were individually subjected to

a phosphorylation reaction using dibenzyl N,N,-diisopropyl-

phosphoramidate and tetrazole in dry acetonitrile under argon

at room temperature for 24 h. The initially formed phosphites

were directly oxidized with meta-chloroperbenzoic acid

(mCPBA) to give compounds 3 and 4 in 76 and 73% yields for

glucose and mannose, respectively. Removal of the TBDPS pro-

tecting group was achieved with a buffered solution of tetra-

butylammonium fluoride (TBAF) at 08C, and yielded com-

pounds 5 and 6 (84% in both cases). This synthetic pathway

allows further substitution at position 6, for instance for the

preparation of more lipophilic derivatives.

The benzyl esters 5 and 6 were deprotected upon catalytic

hydrogenolysis (H2 in the presence of Pd/C and triethylamine)

to give the triethylammonium salts7 and 9 . These were trans-

formed into sodium salts 8 and 10 by using a sequence of

cation exchange columns first in H+ and subsequently in Na+

forms (Scheme 1).

In general, the triethylammonium salts are required for the

preparation of the corresponding PPs, whereas the sodium

salts could also be directly obtained by performing the hydro-

genation reaction in the presence of sodium bicarbonate. The

direct formation of sodium salts was realized for several deriva-

tives; however, it is important to note that the gummy starting

material should be very carefully dried and weighted, because

an exact amount of sodium bicarbonate is required (one equiv-

alent per phosphate) in order to avoid contamination of the

final product. In contrast, the excess base (triethylamine) is

easily removed under vacuum when the corresponding triethyl-

ammonium salts are prepared. Transformation of the triethyl-

ammonium salt into the H+ and then Na+ forms using ion-ex-

change procedures provides an indirect and safe way to

obtain the sodium salts. In some cases this alternative ap-

proach is preferable. Finally, hydrogenations in the absence of

base should be avoided in order to prevent potential compli-

cations from the labile anomeric phosphates in an acidic envi-

ronment.

Compound10 is the only derivative among those studied inthis work that has been previously reported;[17] however, it was

prepared by following a different pathway. Notably, under the

reaction conditions used, no migration of phosphate group(s)

was observed as indicated by NMR spectral data.

Synthesis of tetrakis phosphorylated monosaccharides

We first envisaged construction of the 1,2,3,4-tetrakis phos-

phorylated analogues in order to examine the role of the

anomeric orientation in molecular recognition of Hb. Reaction

of parent sugars with TBDPS chloride was used to selectively

block the primary hydroxy group at position 6. Whereas prepa-ration of the silylated mannose derivative 15 has not been re-

ported, those of glucose and galactose, 14 [31] and16 ,[32] respec-

tively, have been. However, we followed a modified procedure

for the synthesis of all of them (Scheme 2).

Phosphorylation of these 6-O-silylated precursors proceeded

smoothly in acetonitrile and with a similar yield of ~80% in

each case. Both anomers were formed for all three sugars, al-

though in different proportions. Glucose and mannose gave a-

and b-anomers (17/18 and 19/20, respectively) in a ratio of

~5:3, whereas the opposite ratio (~3:5) was observed for the

galactose anomers 21/22. Glucose and mannose anomers

were easily separated by column chromatography. The galac-

tose derivatives were practically inseparable in large scale and

were taken forward as a mixture. Nevertheless, a small amount

of each anomer was isolated for full characterization. Removal

of the TBDPS protecting group was performed under carefully

controlled conditions (0 8C, near neutral pH) to prevent loss of

the sensitive and labile anomeric phosphate (yields 6181 %,

compounds 2328). At this stage we were also able to sepa-

rate the galactose anomers.

This synthetic scheme again allows further substitution at

position 6, in case the synthesis of PPs or compounds with in-

creased lipophilicity are desired. Finally, hydrogenation in the

presence of sodium bicarbonate directly provided the sodium

salts of both anomers of all monosaccharides (2934) in excel-

Scheme 1.Synthesis of the tris phosphorylated derivatives8 and 10 of glu-

cose and mannose respectively, from their silylated methyl glycoside precur-

sors1 and 2 : a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, CH3CN, RT; 2.mCPBA, CH2Cl2,

40 8C!RT; b) TBAF, AcOH, THF, 0 8C; c) H2 (1 atm), Pd/C, Et3N, EtOH/H2O

(1:1), RT; d) Dowex H+ , H 2O then Dowex Na+, H2O. [DBP=P(O)(OBn)2].

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Allosteric Effectors of Human Hemoglobin


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lent yields (>99%). Again, no phosphate migration was ob-

served for all of these derivatives.

The assignment of the a- and b-anomers for the glucose

and galactose derivatives was rather easy, in view of the differ-

ence in coupling constants between the two vicinal protons at

positions 1 and 2. A doublet of doublets was always present in

the spectra of all a-anomers (17, 23 , 29 and 21 , 27 , 33) with a

small coupling constant indicating an equatorial/axial (eq/ax)

relative conformation of protons at positions 1 and 2 (3JH-1,H-2=

2.63.4 Hz), and a larger constant due to coupling of proton

H-1 with the neighboring phosphorous nucleus (3JH-1,P=5.4

7.1 Hz). In contrast, for the b-anomers (18, 24, 30 and 22, 28,

34), a triplet was observed, due to similar large values for the

coupling of vicinal protons and for the heteronucleus coupling

(3JH-1,H-23JH-1,P=6.57.9 Hz) thus indicating an ax/ax orienta-

tion of the protons. All the above results are in accordance

with published data for severala- and b-1-phosphorylated car-

bohydrates, in which substituents at position 2 are equatorially

oriented.[33]

For the mannose derivatives, however, both eq/eq and ax/

eq couples of protons give small coupling constants. There-

fore, the assignment was based on comparison of the chemical

shifts with published values for a- and b-1-monophosphorylat-

ed mannose derivatives, which show considerable differences

in both 1H and 13C NMR spectra.

According to these data, for hexopyranonses such as man-

nose, which, based on NMR spectra, seems to present the 4C1conformation, the anomeric proton signal of thea-anomer ap-

pears at lower field than that of the b-form.[34] This was the

case, as expected, for all glucose and galactose derivatives as

well. Shifts for H-5 of mannose a-anomers are found downfield

relative to the corresponding shifts of the b-isomers.[33d,35]

Moreover, the 13C NMR data display downfield shifts for C-3

and C-5 for b-phosphates.[33d,35,36] These characteristic features

also apply for our derivatives, both protected phosphorylated

(19, 20, 25 , 26) and sodium salts (31, 32), as shown in Table 1.

The data obtained for the perphosphorylated mannose deriva-

tives44 ,48 , and 49 provide further evidence for the a-orienta-tion of these carbohydrates (see Scheme 4 below). The anome-

ric configuration of the phosphates was, however, unambigu-

ously established on the basis of the values of the heteronu-

clear coupling constant JC-1,H-1, which was 174 and 175 Hz for

compounds31 and 49 respectively, indicating ana-phosphate,

and 160 Hz for compound 32 , indicating ab-anomer.[35]

To gain access to 2,3,4,6-tetrakis phosphorylated glucose

and mannose derivatives, the commercially available glyco-

sides 35 and 36 were used (Scheme 3). The free hydroxy

groups in 35 and 36 were all simultaneously phosphorylated

(compounds 37 and 38) under the standard protocol (in 94

and 79% yields, respectively) to give, via the triethylammoni-

um salts 39 and 40 , the final sodium salts 41 and 42 in excel-

lent overall yields. These glucose and mannose derivatives,

which have four phosphates in a row and the remaining

anomeric hydroxy group protected as a methyl ether, proved

to be suitable substrates to investigate the formation of PPs.

Synthesis of pentakis phosphorylated monosaccharides

Glucose (11), mannose (12), and galactose (13) were independ-

ently subjected to a phosphorylation reaction using dibenzyl

N,N,-diisopropylphosphoramidate and tetrazole in dry DMF/

acetonitrile, under argon at room temperature for 24 h. The in-

itially formed phosphites were directly oxidized with mCPBA to

Scheme 2.Synthesis of the tetrakis phosphorylated derivatives 2934of glu-

cose, mannose and galactose from the silylated precursors 1416 :

a) TBDPSCl, Et3N, DMAP, DMF, 0 8C!RT; b) 1. (BnO)

2PN(iPr)

2, 1H-tetrazole,

CH3CN, RT; 2.mCPBA, CH2Cl2, 40 8C!RT; c) TBAF, AcOH, THF, 0 8C; d) H2(1 atm), Pd/C, NaHCO3, EtOH/H2O (1:1), RT. [DBP=P(O)(OBn)2].

Table 1. 1H and 13C NMR chemical shifts for the comparative assignment

ofa- andb-anomers for 1-O-phosphorylated mannose derivatives.

d[ppm]

Compd H-1 H-5 C-3 C-5

19(a-anomer) 6.08 4.01 73.873.5 73.873.5

20(b-anomer) 5.55 3.70 75.6 76.1

44(a-anomer) 5.96 4.01 73.373.1 72.071.7

25(a-anomer) 5.90 3.76 73.373.1 73.6

26(b-anomer) 5.34 3.38 75.475.3 76.0

31(a-anomer) 5.45 3.81 72.9 72.9

32(b-anomer) 5.15 3.50 75.1 76.0

48(a-anomer) 5.50 4.123.99 73.072.8 71.871.6

49(a-anomer) 5.46 4.043.95 73.072.8 71.671.3




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give compounds 43, 44, and 45 in 55, 62, and 65% yield, re-

spectively, (Scheme 4).

The benzyl esters were deprotected upon catalytic hydroge-

nolysis (H2 in the presence of Pd/C) to give the triethylammo-

nium salts 46, 48, and 50 in very good yields (>94%). The

latter derivatives were then transformed into the sodium salts

47, 49, and 51 by applying a sequential ion exchange with

Dowex H+ and subsequently Dowex Na+ resins in quantitative

yields (Scheme 4).

Notably, in the cases of the perphosphorylated monosac-

charides there was a dramatic change regarding the selectivity

of the anomeric positions. In contrast to the 1,2,3,4-tetrakisphosphorylated derivatives, only one anomer was formed for

the pentakis phosphorylated analogues. The bulkiness of the

TBDPS protecting group in conjunction with the effect of sol-

vent used (acetonitrile instead of DMF/acetonitrile), could be

considered the main factors that alter the anomeric effect and

which lead to the formation of both anomers.

The a-orientation for glucose and galactose derivatives was

indicated by the 1H NMR spectra, in which the signals of the

anomeric protons appear as a doublet of doublets with a cou-

pling constant corresponding to an eq/ax relative conforma-

tion of protons at positions 1 and 2 (3JH-1,H-2) from 3.0 to 3.4 Hz.

The coupling constant of the anomeric proton with the neigh-

boring phosphorous nucleus (3JH-1,P) was in the range of 6.0 to

7.4 Hz. The coupling constants of compounds 43, 46, 47 and

45, 50 , 51 were clearly quite similar to those of the a-anomers

of the tetrakis phosphorylated derivatives (17, 23, 29 and 21,

27, 33), shown in Scheme 2, and in accordance with published

data for a-1-phosphorylated carbohydrates, in which substitu-

ents at position 2 are equatorially oriented. [33]

For the mannose derivatives 44, 48, and 49, comparison of

their 1H and 13C NMR spectra with those of the 1,2,3,4-tetrakis

phosphorylated derivatives and the value of the heteronuclear

coupling constant (JC-1,H-1=175 Hz) for compound 49 indicate

ana-orientation (Table 1).

Synthesis of octakis phosphorylated disaccharides

Lactose (52), a reducing disaccharide, was subjected to the

same sequence of reactions (phosphorylation, hydrogenation,

and ion exchange) as the monosaccharides above to give the

perphosphorylated lactose derivatives 53 (in a 1:4 ratio ofa-

and b-anomers, Scheme 5). We managed to obtain, by column

chromatography, a small quantity of pure b-53, whereas the

rest remained as a mixture with the a-isomer.

The anomeric ratio of lactose derivatives was determined

based on their 1H NMR spectra. Although it was relatively easy

to make the assignment of the anomeric proton of the minor

isomer of 53, it was practically impossible to observe the

anomeric proton for the major isomer (obscured by the meth-

ylene protons of benzyl groups). Therefore, it proved much

easier to assign the a- and b-anomer in the proton NMR spec-

tra of sodium salts 55. The minor anomer gives a signal at

5.57 ppm in the form of a doublet of doublets, 3JH-1,H-2=3.5 Hz

and 3JH-1,P=7.1 Hz. For the major lactose derivative 55 a triplet

appears at 5.02 ppm for the anomeric proton (3JH-1,H-2=3JH1,P=

8.0 Hz), thus indicating an ax/ax orientation of the protons.

The same distribution of anomers could be easily assigned for

Scheme 3.Synthesis of the tetrakis phosphorylated derivatives of glucose

41and mannose 42 from the corresponding methyl glycosides 35 and 36 :

a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, DMF, CH3CN, RT; 2.mCPBA, CH2Cl2,

40 8C!RT; b) H2(1 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex H+, H2O

then Dowex Na+ , H2O. [DBP=P(O)(OBn)2].

Scheme 4.Synthesis of the pentakisphosphorylated derivatives 4651of

glucose (11), mannose (12), and galactose (13): a) 1. (BnO)2PN(iPr)2, 1H-tetra-

zole, DMF, CH3CN, RT; 2.mCPBA, CH2Cl2, 40 8C!RT; b) H2(1 atm), Pd/C,

Et3N, EtOH/H2O (1:1), RT; c) Dowex H+ , H2O then Dowex Na

+, H2O.

[DBP=P(O)(OBn)2].

Scheme 5.Synthesis of the perphosphorylated derivatives 54 and 55 of lac-

tose (52): a) 1. (BnO)2PN(iPr)2, 1H-tetrazole, DMF, CH3CN, RT; 2.mCPBA,

CH2Cl2, 40 8C!RT; b) H2(3 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex

H+, H2O then Dowex Na+ , H 2O.

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Allosteric Effectors of Human Hemoglobin


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the lactose derivatives 53 and 54. Because all perphosphoryla-

tion reactions were performed in the same solvent system, we

speculate that lactose gave a mixture of anomers, with the b-

anomer predominating, due solely to steric factors, whereas

the preference for the formation of a-anomers for glucose,

mannose, and galactose derivatives (43, 44, 45, respectively)

appears to result from the anomeric effect.

Sucrose (56), a nonreducing disaccharide, was subjected to

a phosphorylation reaction to give compound57 in 77% yield

(Scheme 6). This benzyl ester was deprotected upon catalytic

hydrogenolysis to afford the triethylammonium salt 58 in very

good yield. The latter was then converted into the sodium salt

59 via ion exchange on resin columns Dowex H + and subse-

quently Dowex Na+ in excellent yields. In this case, of course,

only one product was obtained, as this disaccharide lacks a

free anomeric hydroxy group.

Synthesis of cyclic pyrophosphates

For the synthesis of PPs, two vicinal phosphates, or an even

number of phosphates, all in pairs, are required. Condensation

reactions of phosphates, particularly in the conversion of IHP

into ITPP, were usually performed in pyridine by using the IHP

pyridinium salt.[5,37] The rather large amount of an unpleasant

and toxic solvent, especially for synthesis on the multigram

scale, prompted us to develop a modified coupling reaction

for the synthesis of ITPP[5,7] via its triethylammonium salt. The

method involves dissolution of the IHP triethylammonium salt

in a mixture of acetonitrile/water (ratio of 2:1) and heating the

solution at reflux in the presence of excess N,N-dicyclohexyl-

carbodiimide (DCC). The combination of these solvents was

used because the two solvents are miscible, and both polar

triethylammonium salt and lipophilic DCC are soluble in the

mixture. For other solvent ratios, either DCC or the salt may

remain partially undissolved. In line with these results, the for-

mation of the triethylammonium salts was also implemented

for the present synthesis of cyclic PP derivatives.

Tris phosphorylated derivatives (Scheme 1) are not good

candidates for the formation of cyclic PPs, as they lack an even

number of phosphates. However, we were interested in check-

ing the reactivity of the unprotected hydroxymethyl group

under the neutral reaction conditions described, and not in

pyridine, where formation of 4,6-cyclic phosphates of carbohy-

drates from their 6-monophosphorylated precursors has been

described.[38] Preliminary results showed mainly the formation

of two products, one that possibly contains the 3,4-pyrophos-

phate, and another that contains the 4,6-cyclic phosphate

along with the 2,3-pyrophosphate. We checked the formation

of cyclic phosphate by the heteronuclear coupling constant

observed for C-6 in 13C NMR spectra. However, because the for-

mation of cyclic phosphates is outside the scope of this work,

we did not pursue further experiments with these derivatives,

nor with the 1,2,3,4-tetrakis phosphorylated derivatives

(Scheme 2), which both have the hydroxymethyl group unpro-

tected.

Complete and clean transformation into cyclic PPs could be

achieved from tetrakis phosphorylated hexopyranoses with the

remaining hydroxy group masked, that is, the anomeric group

in the case of the 2,3,4,6-tetrakis phosphorylated derivatives.

Although substrates 39 and 40 are structurally similar to IHP,

we failed to obtain products with two PPs in a row in a regio-

chemically controlled manner when the reactions were per-formed under the same conditions as applied for ITPP.

We assumed then that water might be primarily responsible

for the failure of the preparation of these PPs. The reactions

were also conducted in neat acetonitrile, with the hope that

the triethylammonium salt, which is insoluble at room temper-

ature, would be slowly and totally solubilized in the solvent at

reflux. Indeed, when glucose salt derivative 39 was dissolved

in acetonitrile at reflux in the presence of excess DCC, the bis-

PP 60 was formed in 95% yield (Scheme 7). The same result

was obtained in the case of mannose salt 40 . The reaction was

again successful, and after 24 h at reflux, the 31P NMR spectrum

of the crude reaction mixture showed complete consumptionof the starting phosphate and the exclusive formation of 61.

Two pairs of doublets with coupling constants of 25.5 and

22.0 Hz appeared, indicating two AB systems that correspond

respectively to the eight- and seven-membered cyclic PPs. The

same pattern was observed in the spectra of glucose derivative

60, with coupling constants of 24.9 and 17.9 Hz, respectively.

The latter was easily purified by filtration to remove the

formed dicyclohexylurea (DCU) from the resulting aqueous so-

lution. It was then transformed into the corresponding sodium

salt 62 by ion exchange (Scheme 7). In contrast, mannose bis-

PP 61 was found to decompose during the aqueous workup,

possibly due to instability of the cis seven-membered pyro-

phosphate of this compound.[39]

Scheme 6.Synthesis of the perphosphorylated derivatives 58 and 59 of su-

crose (56): a) 1. (BnO)2PN(iPr)2, 1 H-tetrazole, DMF, CH3CN, RT; 2.mCPBA,

CH2Cl2, 40 8C!RT; b) H2 (3 atm), Pd/C, Et3N, EtOH/H2O (1:1), RT; c) Dowex

H+, H2O then Dowex Na+, H2O.

Scheme 7.Synthesis of the pyrophosphates 60-62of the tetrakis phosphory-

lated methyl glycosides of glucose 39 and mannose 40 derivatives: a) DCC,

CH3CN, 828

C; b) Dowex H+

, H2O then Dowex Na+

, H2O.




7/16

Glucose, mannose, and galactose pentakis phosphorylated

analogues lack an even number of phosphate groups, and

they are not suitable substrates for the formation of PPs.

Indeed, triethylammonium salts (46, 48, and 50) failed to form

pyrophosphates in a regiochemically controlled manner. The

same was expected for the lactose derivative 54 (Scheme 6),

because the glucose subunit lacks two pairs of vicinal phos-

phates. Interestingly, the sucrose-derived salt 58, with all its

phosphates properly positioned for the formation of four PPs,

also failed to give a clean reaction. A total of 80% of phos-

phates were transformed into PPs (based on 31P NMR investiga-

tions of the crude mixture) only after prolonged heating. This

reluctance toward PP formation might be due to improper

spatial orientation of the phosphate groups.

Binding affinity of the compounds for and enhancement of

oxygen release from hemoglobin

As indicated above, the phosphorylated compounds BPG and

IHP as well as the trispyrophosphate ITPP (Figure 1) bind to Hband significantly enhance its oxygen release capacity. After

having successfully synthesized the tris, tetrakis, pentakis and

octakis phosphorylated carbohydrate derivatives and a bispyr-

ophosphate described herein, all final compounds were evalu-

ated for their effect on stripped human Hb. To determine their

ability to shift the oxygen saturation curve to higher pO2values, we assessed the partial pressure of oxygen for half-sat-

uration (P50) of Hb in the presence of the compounds and their

corresponding dissociation constants (Kd) to Hb. The results are

depicted below in Table 2 and in Figures 27.

All compounds were able to shift the Hb oxygenation

curves up from 58 to 550%, and the relationship betweenbinding to Hb and oxygen release is illustrated in Figure 2. In

general, the ability of the compounds to lower the Hb affinity

for oxygen is directly related to the number of negative charg-

es present; that is, a greater number of phosphates gives a

higherP50 value. For instance, octakisphosphate carbohydrates

55 (550%) and 59 (550%) were more effective than trisphos-

phate compounds 8 (113%) and 10 (144%). The same trend

was observed for the lead compounds BPG (I) < ITPP (III)

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