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Phytochemistry: Advances in Research, 2006: 1-22 ISBN: 81-308-0034-9 Editor: Filippo Imperato

1 Mass spectrometry in the structural elucidation of natural products: Glycosides

Gabriela M. Cabrera Departamento de Quimica Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Unìversitaria, Pab. II (1428) Buenos Aires, Argentina

Abstract Mass Spectrometry is an important tool for the identification and structural elucidation of natural products. A review of the last fifteen years of research in this field, applied to natural glycosides, is presented.

Introduction The natural products field, in terms of structural elucidation, emerged around 150 years ago and the identification of the structure of these products was a major focus of organic chemistry research. Methods on natural products isolation required large quantities

Correspondence/Reprint request: Dr. Gabriela M. Cabrera, Departamento de Quimica Orgánica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Ciudad Unìversitaria, Pab. 11, (1428) Buenos Aires Argentina. E-mail: [email protected]

Gabriela M. Cabrera 2

of pure samples to solve the structure determination by chemical transformations. Structure elucidation was a driving force in the development of organic chemistry and analytical techniques during the first half of the nineteenth century. The development of NMR allowed the reduction of the quantities required for structure elucidation and transformed completely this area of research. Most of the structural determinations are nowadays practically done without the use of “wet” chemistry. Mass Spectrometry (MS) was primarily used to obtain molecular weights. Former ionization techniques, as electron ionization, limited this use to non polar, volatiles and thermostable substances. The improvement of softest ionization techniques allowed gradually the analysis of polar and thermolabile compounds, having currently unlimited physical or chemical properties to be analyzed by MS. But the development of tandem MS allowed MS could be applied also to structure elucidation [1-3]. Furthermore, hyphenated MS techniques, such as GC, LC and CE MS, permit the analysis of very low sample amounts and no requirement of purity. Although it enormous capabilities, demonstrated by a large number of published papers, MS is still not fully employed as a normal tool for structural determination. By far the most studied natural products by MS are the flavonoids, although a wide range of other metabolites are being currently explored. It is important to mention the recent excellent reviews about MS of flavonoids [4,5]. The purpose of this review is to show the enormous potential of Mass Spectrometry determining structures, beginning with the case of natural glycosides. Just the case of employing MS to determine molecular weights will not be considered, the same as the application on food stuffs. This review covers the literature published during the last fifteen years. The use of MS for glycosides structural elucidation can be divided according to the information that can be obtained [4]:

1. Type of glycoside 2. Sequence determination of monosaccharide components 3. Glycosidic linkage to the aglycone 4. Interglycosidic linkage 5. Stereochemistry, ring size and anomeric configuration of the

individual sugars.

1. Type of glycoside Mass Spectrometry can be used to easily determine the number and type of monosaccharide unit components of the glycoside. Attained information

Mass spectrometry of natural glycosides 3

indicate if the compound is a mono-, di-, tri-, tetrasaccharide (or more), if the sugar moiety is pentose, hexose or deoxyhexose and if the glycoside is an O-glycoside, N-glycoside or a C-glycoside. Different ionization techniques and a lot of different experiments were employed to gain these valuable findings. The fragment ions nomenclature was designated in this paper according to the nomenclature proposed by Domon and Costello for glycoconjugates [6] (figure 1). Ions containing the aglycone are labelled k,lXj, Yj and Zj, where j is the number of the interglycosidic bond broken, counting from the aglycone, and the superscripts k and l indicate the cleavages within the carbohydrate rings. The glycosidic bond linking to the aglycone is numbered 0. When the charge is retained on the carbohydrate residue, fragments are named k,lAi and Bi, where i represents the number of glycosidic bonds cleaved, counting from the non-reducing end [6]. The fragmentation processes usually are accompanied by a H-transfer, and for this reason, even fragment losses are observed (table 1). Another simpler nomenclature coexists for the major fragment ions (figure 2) [7]. For common abbreviations or acronyms, see the appendix. 1.1. FAB, LSIMS and TSP techniques FAB, LSIMS or CF-LSIMS in the positive ion mode were employed to determine the presence of monosaccharide units attached to aglycones like benzoxazinoids [8], iridoids [8,9], flavones, isoflavones, flavonoids [4,5, 10], pterocarpans [7], anthraquinones [11], triterpenes [12,13], alkaloids [14],

HO

O

HOCH2

OH

OH

O

O

HOCH2

OH

OH

O

OHOCH2

OH

OH

Oaglycone

Z0

Y0

Y1

Y2

B1

B2

B3

Z2

Z1

0,2A2

0,2A1

0,2A3

0,2X2

0,2X1

0,2X0

Figure 1. Fragment ions nomenclature according to Domon and Costello [6].


O

CH2OH

OH

OH

OH

O

O

CH2OH

OH

OH

O

O

CH2OH

OH

OH

O

O

CH2OH

OH

OH

O aglycone

H

H

H

H

F1H2+

F2H2+

F3H2+

AH2+

Figure 2. Major fragment ions nomenclature [7]. sphingolipids [15,16], gibberellins [17], diacylglycerols [18], macrolactones [19,20], cardenolides [21,22] and steroids [10,23-25]. The most common used matrices are glycerol, magic bullet (dithiothreitol/ditioerythritol) or NBA, and sometimes other mixtures are employed as glycerol-thyoglycerol-TFA [11]. The matrix may have a considerable effect on the relative peak intensities [26]. In most cases, the presence of [M+H-sugar]+ could be observed directly from the spectra of the glycosides without the presence of any additive, indicating the type of monosaccharide according to the table 1. In other cases the same information was obtained with the use of an additive, a cation salt, most commonly an alkali metal salt, to enhance the molecular adduct ion intensity (M+C)+, where C is Li [9], Na, K, Rb [10] or Ag [33].

Table 1. Characteristic fragments by sugar losses.


Neutral losses of water and methanol or acetic acid, if methoxy substituents or acetyl groups were present, were common accompanying fragments from any ion. When these important fragment ions were of low intensity or just to enhance its intensity, application of CID with high energy using daughter scans like B/E or MIKE could be successfully applied. In these experiments the parent ion [M+H]+ or [M+C]+ (or [M-H]- in the negative ion mode) was selected and fragmented by collisions with He gas. The obtained product ions were then determined. Li cationized FAB spectra of iridoid monoglycosides [9] yielded upon CID important fragments [Y0 + Li]+, the litiated aglycone, often the base peak, and [sugar+Li]+. These later ones had characteristic m/z values, m/z 187 for hexose, m/z 169 for anhydrohexose and m/z 186 for aminodeoxyhexose. The CID spectra of adduct ions [M + C]+ (C = Li, Na, K, Rb) of flavonoid monoglycosides showed also [Y0 + C]+ ions, but in the case of β-sitosteryl-β-D-glucoside, only [sugar + C]+ was observed. This fact would indicate that alkali metal cations bind to aglycone moiety with variable strength depending on the aglycone structure, stronger with flavonoids and slightly with sitosterol [10]. The same experiments with flavonoid diglycosides showed a tendency for the relative small cations Li+ and Na+ to bind to the sugar moiety (higher RA fragments [B2 + C]+), whereas the large cations K+ and Rb+ bind to the aglycone moiety (higher RA fragments [Y0 + C]+) [10]. There are fewer examples of the use of thermospray (TSP) than the other techniques in the elucidation of natural products. Flavonol glycosides from Epilobium species were analyzed by TSP in the positive ion mode, showing the spectra the characteristic Y0

+ ions [34]. Other flavonoids were analyzed in the same way [35,36]. FAB or LSIMS in the negative ion mode can also be used to determine the sugar composition, although is more employed to sequence glycosides, since usually the positive ion mode registers better sensitivity [5]. Some examples of glycosides, analyzed in negative mode, include a spirosten lactone [37], triterpenes [38,39], iridoids [40], benzyl alcohols [40], phenylpropanoids [41], cardenolides [22], megastigmanes [40], hydroxy fatty acids [42] and macrolactones [43]. Typical matrices used in the negative ion mode are glycerol [38] and triethanolamine [37]. The characterization of positional synthetic isomers of 2-O- and 3-O-sulfoglycopyranoses was established by LSIMS B/E, comparing the abundances of selected daughter ions [44]. Unsaturated C-glycosides behaved in a different way, compared with saturated, as could be seen for synthetic derivatives, used as models, where the retro-Diels-Alder reaction RDA (figure 3) became a significant step of fragmentation [45].


O

OR

RO

CHR1R2

O

OR

RO

CR1R2

RO CR1R2

O

RO

RDA

(M-H)-

Figure 3. Fragmentation pathway of unsaturated C-glycosides. In all cases the interpretation of sugar losses in the mass spectra should be taken carefully, since some common naturally occurring substituents, which can be easily lost in fragmentation pathways, have the same molecular weights of sugars. This is the case of ferulic acid, which can be lost as ketene (176 u) or as acid (194 u) [4,41]. 1.2. API, MALDI techniques Since Atmospheric Pressure Ionization techniques were introduced, the number and diversity of applications has been increased each year. ESI and/or APCI in the positive ion mode has been employed to determine the structure of glycosides of pregnanes [46,47], flavonoids [4,5,48-51], cardenolides [2,52], iridoids [53], naphtopyranones [54], chromones [55], isoflavonoids [56-58], mycosporine-glutamicol [59], phenylpropanoids [60], everninomicins [61], sterols [62] and aminoglycosides [63]. As usual, the information about molecular weight and type of monosaccharide units could be obtained. As these are softer techniques, [M+H]+ and [M+C]+ (and [M-H]- in the negative ion mode) ions are the only ones usually observed in the spectra, called the first order spectra. CID experiments are then needed to obtain structural information. As abundant [M+H]+ or [M+Na]+ are required to run the CID experiments to obtain sugar information, the ESI source declustering potential should be first optimized. For pregnanes it was set at low values (10-30 eV) to obtain [M+H]+ and higher voltages to detect [M+Na]+ [46]. In most cases CID experiments were done with CID energy ranged from 10-20 eV [46]. As mentioned above, structural characterization of glycosides were also achieved through metal complexation followed by tandem mass spectrometry. Complexation could also be obtained with the use of a ligand and a metal.


1,3-Diaminopropane and diethylenetriamine were investigated as ligands in conjunction with different transition metal ions to study the fragmentation of N-acetyl-α-D-neuraminic acid in order to establish patterns to recognize sialic acids [64]. The loss of CO2 from the carboxylic acid functionality was the first step in the fragmentation pathways. 0,4X cross ring cleavage was favored by Co, Ni and Zn and 1,3-diaminopropane, whereas diethylenetriamine with three coordinating nitrogens promoted 0,2X, 0,4X and 0,4X-H2O with any metal [64]. MS/CID/MS analysis (second order spectra), also allowed the differentiation of C-glycoside vs. O-glycoside. Characteristic fragment ions, showing typical losses 0,iX, gave information about not only the presence of a C-glycoside, but also the type of attached monosaccharide (table 2) [4, 65]. These fragments were observed also by LSIMS [66]. Chromone C-glucosides were characterized in a toxic herbal remedy by MS [55]. Characteristic 0,2X fragment of a C-hexosidic moiety was determined. In the same way, fragments Y0, 0,2X and 0,3X allowed the identification of isoflavone O-glycosides [57]. ESI in the negative ion mode has been employed to determine the structure of glycosides of anthraquinones [67,68], coumarins [67], flavonoids [4,31,67,69,70], iridoids [67,71], aromatics [30,67], cardenolides [29], isoflavonoids [57] and triterpenes [67,72]. Usually, simpler fragmentation patterns than in the positive ion mode were observed, being the Yi

- the main CID fragments [57,67,70]. With some compounds, it has been shown a higher sensitivity [5], related to the aglycone part of the molecule. With monoglycosides, the aglycone fragment Y0

-, loss of 162 u in a hexoside, could be accompanied by a radical aglycone ion [Y0-H]-·, loss of 163 u in a hexoside, fragment which dominated at high collision energies [57,70]. Cone voltages ranged about 30-40 eV [70-72] to obtain [M-H]-. Cone voltage fragmentation CVF was applied to induce fragmentation of parent ions [72] with instruments with no capabilities to run MS/MS experiments. In these cases cone voltages up to 75 eV [72] were employed. O- and C-glycosides could be distinguished [30] in the same way as in positive ion mode, as the same sugar losses, shown in table 2, were observed.

Table 2. Characteristic fragment losses in C-glycosides [4].


A rapid and selective strategy was developed for the screening and identification of glycosides by ESI- in a triple quadrupole. Many glycosides were screened out with high confidence by an energy gradient neutral loss scan (EGNLS) [67]. This method employs a continuous collision energy gradient, which omits the necessity of energy optimization for each individual glycoside, while performing neutral loss scan (CNL) with MS/MS [67]. Besides [M+nH]n+ ions are the most often obtained by ESI, this is not the case with natural products which are molecules usually below 1500 u. These multiple charged ions, mainly [M+2H]2+, may appear in the spectra, generally with low RA and with the characteristic isotopic pattern, 0.5 u (or 1/n u) between isotopes. MALDI has not been very much explored in the natural products field. Rutin, cationized with different metal cations, was investigated by this technique [73]. The PSD-MALDI mass spectra of the [R + Cat]+ ions showed fragmentations [Bi + Cat]+ and [Yi + Cat]+, where the extent of fragmentation decreased as the atomic number increased [73]. Cerebrosides were ionized by MALDI, showing the first order spectra [M + Na]+ and [M-Gal]+ ions [74]. Saponins also showed by MALDI in the first order spectra, the corresponding Y ions, but in the negative ion mode [75]. The most common matrices for natural products are α-cyano-4-hydroxycinnamic acid and 2, 5-dihydroxybenzoic acid.

2. Sequence determination of monosaccharide components MS have been extensively used to sequence monosaccharide moieties in glycosides that mean to determine the order where sugars are connected one to each other or to establish points of ramification.

2.1. DCI, FAB, LSIMS, TSP techniques Although DCI is not an extended and popular soft technique, it was applied to sequence cardenolides using NH3 as chemical reagent [28]. Typical Y fragment ions were observed. While it is not the most common mode of operation in sequence determination, some examples are presented about the use of FAB or LSIMS in the positive ion mode. These included sequence determination of glycosides of macrolactones [19,20], pregnanes [25], cardenolides [22] and steroids [23]. Some authors indicated similarities between both positive and negative modes [19,20]. For other compounds noticeable differences were observed [22]. Figure 4 shows FAB spectra in both modes of operation. Sequence information was clearly obtained in the negative ion mode whereas more additional structural information was obtained in a more complex positive ion mode spectrum. Sometimes spectra were obtained with high resolution (HR) to have more confidence about the molecular formula of the fragments [25].


OH

OH

O

O

OO

OCH2

HO

OH

OHOH2C

HOHO

OHHO O

H3CO

b

a

Figure 4. FAB MS/CAD/MS spectra, a) negative ion mode, b) positive ion mode. Many other glycosides have been sequenced by FAB- or LSIMS- from first order spectra or by CID MS, like glycosides of cardenolides [21,22], triterpenes [39], iridoids [40], benzyl alcohol [40], megastigmanes [40], hydroxy fatty acids [42] and macrolactones [43]. 2.2. API techniques Most of the glycosides isolated nowadays are sequenced by ESI or APCI, employing CID, in the negative or positive ion modes. Examples are glycosides of flavonoids [4,26,76-79], pregnanes [80], triterpenes [27,81-83], aminocyclitol antibiotics [32], naphtopyranones [84], cardenolides [29,52], xanthones [85] and polar natural oligosaccharides [86]. Different strategies to sequence depend on the accessible instruments. Ion traps, which have the ability to perform MSn, are the most suitable for this purpose. With this instrument, it is possible, in a single experiment, to select a precursor ion (i.e. [M+H]+), to collide it with He gas, to select a product fragment ion (i.e. Yi

+), to collide it, to select a new daughter ion (i.e. Yi-1+), and

to repeat this procedure n times, with n = 11 or 12. Selecting adequately the precursors, a complete sequence can be obtained. This is represented [M+H]+>Yi

+>Yi-1+, where each of these ions is a precursor in the experiment.

For other instruments, one experiment is required for each pair precursor-products ions, although most of the times this is not necessary, as the second order spectrum shows all the Y ions needed to sequence.


Comparison of negative vs. positive ion modes of operation was achieved with saponins [82,83]. The highest instrument response was obtained using negative-ion ESI and a basic NH4OH solution and also showed that elevated pH values increased the base catalyzed hydrolysis of the sugar substituent followed by decarboxylation and dehydration of the aglycone [82]. In other study, saponins gave strong molecular species both in positive and negative ion modes [83]. Despite this fact, it was established an enormous difference, between both modes, in the CID spectra obtained from (M+Na)+ and (M-H)-. (M+Na)+ was the predominant ion in the first order spectrum, even when no sodium ion was added, due to the inevitable presence of this ion during the process of sample preparation. In the positive ion mode, (M+Na)+ yielded Y, Z, B and X type of ions whereas in the negative ion mode, (M-H)- ions only yielded Y ions and no cross-ring ions were detected. These differences were attributed to an exothermic combination of a saponin molecule with a sodium ion during the formation of positive ions. As a consequence, the energy released was deposited into the internal degree of freedom and could excite more bond cleavages under CID conditions [83]. These differences between positive and negative ion modes and the effects upon metal adduct formation on CID had been also observed with cardenolides ionized by FAB [87]. When APCI was employed, the relative intensities of (M+H)+ and Y+ ions could be manipulated by adjusting the APCI vaporizer temperature. In some cases, full information about molecular weight of the glycoside and sequence was obtained in the first-order spectrum without the need to undertake tandem MS [77]. ESI+ LC-MS and LC-MS/MS, with low energy CID, have shown to be very useful in the structural elucidation of trace level impurities in aminoglycoside antibiotics without prior separation [32]. The product ions derived from the glycosidic cleavages were also the major ions in the CID spectra of the [M+2H]2+ ions of these compounds [88]. Formic acid was used to enhance the ionization efficiency of cardenolides, but [M+HCOO]- ions were obtained [29]. Curiously, the MS3 spectrum (adduct [M+HCOO]- > [M-H]- >) gave Z0, the dehydrated strophanthidin aglycone, instead of Y0. Special cares should be taken when assigning ramified glycosides [83] or when chains of sugar are attached to several points of an aglycone [27], as the number of Y ions increase (figure 5). It was reported that an inner glucose residue was lost at low-energy collision from the [M+H]+ of flavonoids. This result could lead to an incorrect sequence [89-91]. The fragmentation of the corresponding [M+Na]+ or [M-H]-

ions provided the right sequence in this case [89,90].


HO

OCH2OH

OH

OH

O

OOH

OH

O

OOH

OH

O aglycone

Y2α+

Y1α+

Y0+

OHO

OCH2OH

OH

OH

OHO

OCH2OH

OH

OHY2β

+

Y1β+

Figure 5. Y ions in branched glycosides. In situ formation of C-glycosides during ESI MS/MS of cholesteryl polyethoxy neoglycolipids, leading to erroneous conclusions, was also reported [92]. 3. Glycosidic linkage to the aglycone Some strategies have been developed to discern the correct position where a sugar chain is connected to an aglycone, when different locations are possible. Determination of the sugar linkage position in flavonoid C-glycosides was resolved in many ways, using FAB or LSIMS, and tandem MS with high-energy collision [66,93] or ESI and tandem MS with low-energy collision [4,65,66,94-97]. LSI B/E mass spectra, selecting Y1 ions as precursors, showed the radio of ion abundances 0,2X0 and 0,3X0 of approximately 2:1 for C-6 conjugates and 4:1 for C-8 conjugates [66]. Losses of molecules of water were also more pronounced for C-6 conjugates [93], as there are two hydroxyl groups between the C-6 glycosidation site in many flavonoids and only one neighbour in the C-8 site. These hydroxyl groups would participate in the water loss [93]. The same fragmentations were observed in the low-energy [M+H]+ CID spectrum of flavonoids [65,94,95], although Waridel et al. found out that these fragmentations were collision energy dependent and it was difficult to draw rules for an unambiguous distinction between C-6 and C-8 isomers from [M+H]+ [65]. These authors suggested guidelines for identification based on


several spectra in both negative and positive ion modes and MS/MS on fragments 0,2X [65]. The differences on the ability to lose the glycan residue from [M+H]+ or [M+Na]+ was used to distinguish between different O-glycosides of flavonoids, being a glycan substituent located at the position 3 more easily lost compared to that of the position 7 [78,98]. These results were in full accordance with previous work on FD, where the order 5>3>3´=5´>4´>7 was observed [99]. It is obviously difficult to assign the point of attachment only on these bases and without the use of reference compounds. Recently, simple guidelines for the unambiguous information about the glycosidation site in flavonoid mono-O-glycosides using high-energy CID were presented [100]. The presence of a B-ring (+Na+) product ion, containing the sugar residue, in the CID spectrum of the [M+Na]+ ion, indicated 4´-O-glycosidation, while 7-O and 3-O-glycosides could be differentiated by the loss of B-ring and Y0

+ [100]. Other recent approach to provide information on the glycosidation site was achieved performing low-energy CID on complexes of the form [Mn(II) (L) (L-H)]+, where L is a monoglycosyl flavonoid [101]. The loss of a hexose residue is indicative of O-glycosylation. The 3-O-hexoside complexes yielded no other significant fragmentation products, but the 4´-O-hexoside complexes presented further losses of hexoses, aglycone and intact flavonoid glycoside, and the 7-O-glycosides displayed the loss of a hexose residue and 0,2X0 [101]. Other metal complexes were also investigated [102]. Co, Ni and Cu complexes did not permit complete and unambiguous identification. On the other hand [Mg(II) (L) (L-H)]+ allowed a complete identification of 3-O, 4´-O, 7-O, C-6 and C-8 flavonoid glycosides in the same way as Mn complexes [102]. There is not a systematic approach to distinguished O- and COO-glycosylation sites in triterpenes. Few examples of ESI and ESI MS/MS analysis, in the negative ion mode, indicated that COO-glycosides have a greater ability to lose the glycan residue than O-glycosides. The absence of substitution in the carboxyl functional group was evident by the loss of CO2 from the low-energy CID of (M-H)- [103]. Differentiation of 14-COO from 17-COO glycosides was also reported. The CID spectrum of the former presented a product ion formed by the loss of the whole unit COO-glucose while the other showed the sequential loss of glucose and CO2 [103]. It was also reported that the cleavage of the oligosaccharide residue in saponins occurred first at the C-20 position of the aglycone than at the C-3 position, when both positions were glycosylated [83].

4. Interglycosidic linkage Isomeric glycosides, differing in the interglycosidic linkage, could be distinguished by MS. Using negative ESI, the linkages Rha (1 2) Glc and Rha


(1 6) Glc were differentiated in flavonoid glycosides [98,104]. Low-energy CID for [M-H]- (or M-.) yielded 0,2X-, low intensity Y1

- and Y0- product ions

for Rha (1 6) Glc flavonoids, but 0,2X-, Y1-, Y0

-, Z1-, 1,3X0

-, and 0,2X0Y1-

for Rha (1 2) Glc flavonoids [104]. These extra fragments, which were of considerable intensity, could be used to clearly differentiate both types of compounds. Similar differentiation was achieved in permethylated flavonoid glycosides, employing the linked scan B/E, selecting [M + H]+ ions obtained by FAB ionization [91]. Metal complexation with an auxiliary ligand has been used to differentiate flavonoid isomers by ESI MS2 and MS3. CAD of complexes of the type [Co(II) (flavonoid-H) ligand]+, where ligand is 4, 7-diphenyl-1,10-phenanthroline (dpphen) yielded a unique fragment ion [complex-aglycone-Glc]+ for the (1 2) isomer [105]. Linkages (1 1), (1 2), (1 3), (1 4) and (1 6) of glucopyranosyl disaccharides, were assigned according to an empirical criteria, comparing characteristic fragments and abundances [106]. 5. Stereochemistry, ring size and anomeric configuration of the individual sugars Nowadays MS is recognized for its capacity to differentiate stereoisomers [107,108]. In this sense many strategies were employed to distinguish isomeric monosaccharides [109-111], furanose and pyranose rings [112] and α and β anomers [106]. The 0,2X / 1,5X abundance ratio ([M+ Na-90]+ / [M+Na-104]+) in the FAB high-energy collision CID MS/MS of sodium cationized di, tri and tetrasaccharides, having L-Ara or D-Xyl non-reducing termini, provided information on the ring size, furanose or pyranose ring, of the non-reducing monosaccharide termini [109]. These ratios were 2-3 times lower when the xylopyranose was the terminal residue than those having arabinofuranose as terminal residue. The ratio differences decreased with increasing number of monosaccharide residues [109]. Although not directly applied to natural products, several attempts were made to distinguish isomeric sugars. Complexes of the type Ni(NH2(CH2)3NH(carbohydrate – H2O))2 Cl2 were prepared for mannose, galactose, glucose and talose. Each complex was ionized by FAB and a fragment ion [Ni (N-Glycoside) – H]+ (figure 6), selected with high resolution, was allowed to undergo unimolecular dissociation [110,113]. The KER values were measured for the resulting product ions. D-Gal and D-Glc, with equatorial C-2-OH configuration gave KER values (30 mV) very different from D-Man and D-Tal, with axial C-2-OH configuration (9 mV) [110]. This method can clearly distinguish between these groups of sugars. This distinction was also obtained comparing relative


O

HO

HO

N

NiN

HO

O

H

H

H

Figure 6. [Ni (N-Galactoside) – H]+ fragment ion, selected to measure KER. ion intensities of fragments obtained by CID of complexes of the same sugars with Zn, ionized by ESI [114,115]. Similarly, differentiation of diasteromeric N-acetylhexosamine monosaccharides was achieved using complexation with Co (III) and diaminopropane (DAP) as ligand [116]. In this case, a complex ion [CoIII(DAP)2(HexNAc)-2H]+ obtained by ESI, afforded by MS2 and MS3 [complex - DAP]+, [complex - C4H8O4]+ and [complex - C5H9NO3]+ fragment ions, of relative ion intensities that allowed the desired differentiation [116]. This method was employed even to quantify diasteromeric hexosamine monosaccharides [117]. Glucose and galactose, and the pentoses xylose, arabinose and apiose, were differentiated in peracetylated flavonoids. The method was applied successfully to identify the sugar components of an unknown isorhamnetin glycoside [111]. The intensity ratio [m/z 127] / [m/z 109] from the low-energy CID spectrum obtained for the [B1 -2 CH3COOH - 2CH2CO]+, generated by FAB or ESI, allowed the distinction between Glc and Gal (figure 7). The ion m/z 127 had a higher relative abundance than m/z 109 for Gal whereas the opposite held for Glc [111]. In a similar way, the intensity ratio [m/z 139] / [m/z 199] allowed the identification of the pentoses, being the ratio order apiose > arabinose > xylose. Apiose also showed a characteristic fragment [B1 – CH3COOH – CH2CO - CO]+ at m/z 129 [111]. Similar results were previously obtained with peracetylated methyl pentosides and methyl 6-deoxyhexosides, although comparing the intensity ratio [B1 - 2CH3COOH]+ / [B1 -CH3COOH]+ [112,118]. The CID spectra of B1 ions of pyranose and furanose isomers, produced by CI (CH4), showed fragment ions, of particular intensity ratios that could differentiate furanoses from pyranoses. As examples, peak intensity ratio m/z 142 / m/z 205 ([B1-2AcOH]+/ [B1-AcOH]+) was 104.1 for ribofuranose and 46.3 for ribopyranose and the peak intensity ratio m/z 156 / m/z 219 ( [B1-2AcOH]+/ [B1-AcOH]+) was 36.7 for deoxyglucofuranose and 7.2 for deoxyglucopyranose [112].


O

H3COCO

H3COCO

H3COCO OCOCH3

- CH3CO2H

- CH2COm/z 229

m/z 331

- CH3CO2H

- CH2CO m/z 127

- 2 CH3CO2H

m/z 109

O

H3COCO

H3COCO OCOCH3

- CH3CO2H

m/z 259

m/z 199 m/z 139- CH3CO2H

Figure 7. Fragmentation pathways of the B1

+ ions of peracetylated saccharides of flavonoid glycosides. These results implied that the pyranoses show single CH3COOH loss far more efficiently than double loss, whereas for the furanosides the opposite is true. The method described in previous sections, performing low-energy CID of Mn (II) complexes also allowed the distinction of Gal vs. Glc and Ara vs. Xyl in flavonoids [101]. Despite being non-natural, α and β anomers of synthetic C-glycosides were differentiated in the FAB CAD-MIKE spectra of [M+H]+ ions, and in the self-ionization CID spectra [119], as both anomers exhibited different fragmentation behavior. This result could be applied to natural products [120,121]. Conclusion The presented data show the potential of Mass Spectrometry in the structural elucidation of natural products, particularly glycosides. With easy experiments and in short times, it is possible to know the number, sequence and characteristics as ring size, stereochemistry, and linkages of all of the monosaccharide components. Despite it is not fully applied today, Mass Spectrometry will be undoubtedly employed to identify sugar components and full structures in the near future.


Acknowledgements The author thanks UMYMFOR, CONICET, ANPCYT and Universidad de Buenos Aires for partial financial support. Appendix Acronyms [122], nomenclature [123] and abbreviations APCI: Atmospheric Pressure Chemical ionization. Ionization method in which ions are formed by ion-molecule reactions between the analyte and a reactant gas at atmospheric pressure. API: Atmospheric Pressure Ionization, generic name to appoint all the ionization techniques that occur at atmospheric pressure. Ara: arabinose B/E: linked scan, based on scanning simultaneously the magnetic field and the electric sector voltage in a constant rate as B/E, holding the accelerating voltage constant. Spectrum of daughter ions is obtained. CID may be applied in the first field-free region [123]. B2/E: linked scan, based on scanning simultaneously the magnetic field and the electric sector field in a constant rate as B2/E, holding the accelerating voltage constant. Spectrum of precursor ions is obtained [123]. CI: Chemical ionization, method in which ions are formed by ion-molecule reactions between the analyte, in the gas phase, and a reactant gas. CF-LSIMS: Continuous flow LSIMS. CAD: Collisionally activated dissociation, process wherein excitation of a projectile ion of high translational energy is dissociated as a result of interaction with a target neutral species [123]. CID: Collision-induced dissociation, process wherein the projectile ion is dissociated as a result of interaction with a target neutral species [123]. CVF: Cone-voltage fragmentation or up-front CID or in-source CID or nozzle-skimmer fragmentation; fragmentation induced in one of the higher pressure regions of the ion passageway from the source into the mass analyser [124]. CNL: Constant neutral loss, MS/MS scan in which two mass analyzers are fixed just to allow the path of parent ions, which decompose loosing a desired constant neutral fragment, and the corresponding daughter ions. CID (constant energy) is applied between the analyzers. Declustering potential: Electric field applied in the region of expansion of ions into the vacuum (between the API source and the mass analyzer). Collisions, produced by the field, lead to the breaking of hydrogen bonds in cluster ions [7]. DCI: Direct Chemical Ionization. EGNLS: Energy gradient neutral loss scan.


ESI: Electrospray ionization, process in which a liquid stream of dissolved analyte is broken up into small droplets by the action of a high potential. Analyte ions are produced in the process [124,125]. ESI-: Electrospray ionization in the negative ion mode. ESI+: Electrospray ionization in the positive ion mode. FAB: Fast Atom Bombardment ionization. The ionization is produced by interaction of the sample with a beam of neutral atoms having high translational energy [123]. FD: Field desorption, process in which ions in the gas phase are formed from a material deposited in a solid surface in the presence of a high electrical field [123]. Gal: galactose Glc: glucose HR: high resolution, measure of m/z values with high accuracy to enable the atomic composition of an ion. KER: Kinetic energy release, fraction of internal energy in a dissociation process (in excess of that of the ground-state products), partitioned into the kinetic energy of the fragments [126]. LC: Liquid chromatography. LSIMS: Liquid Secondary Ions Mass Spectrometry, ionization produced by interaction of the sample dissolved in a liquid matrix, with a beam of ions having high translational energy. LSI: Liquid secondary ionization. MALDI: Matrix assisted laser desorption ionization, process in which the ionization occurs when a sample, dissolved/co-crystallized/adsorbed in a matrix, is irradiated with a laser beam of a frequency absorbed by the matrix material. Man: mannose MIKE: Mass-analyzed ion kinetic energy scan. MS/MS scan in which a magnetic sector is set to pass only a precursor ion, which decompose in a field free region by unimolecular decomposition or by CID. The product ions of reduced kinetic energy are then analyzed in an electrostatic sector. MS: Mass Spectrometry MS/CID/MS: Process in which a precursor ion is selected by a mass analyzer, collisioned and the daughter ions analyzed. MS/MS: Tandem MS. Process in which a precursor ion is selected by a mass analyzer, and the fragment ions, produced by natural or induced decomposition, are then analyzed by a second analyzer (tandem in space) or by the same analyzer (tandem in time). Only ion traps or Fourier transform-ion cyclotron resonance instruments are capable to perform tandem in time. MSn: Extension of MS/MS with more than two stages of MS. m/z: Mass-to-charge ratio. NBA: Nitrobenzyl alcohol.


Precursor or parent ion: Electrically charged molecular moiety which may dissociate to form fragments [123]. Product or daughter ion: Electrically charged product of reaction of a particular parent ion [123]. PSD: Post-source decay, process of production of product-ion spectra in a time-of-flight mass analyzer. RA: Relative abundance to the intensity of the base peak. Rha: rhamnose Tal: talose TSP: Thermospray ionization, process in which a liquid stream of dissolved analyte and a conductive buffer is broken up into small droplets and ions by the action of a high temperature. u: unified atom mass (1/12 of the mass of an atom of 12C) [123]. Xyl: xylose References 1. Wolfender, J. L., Maillard, M., Marston, A., Hostettmann, K., 1992, Phytochem.

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