Anal. Chem. 1998, 70, 3009-3014
Stereochemical Differentiation of Mannose, Glucose, Galactose, and Talose Using Zinc(II) Diethylenetriamine and ESI-Ion Trap Mass Spectrometry Sara P. Gaucher and Julie A. Leary*
Department of Chemistry, University of California, Berkeley, California 94720
Diastereomeric diethylenetriamine N-glycoside zinc(II) complexes are investigated using electrospray ionization followed by tandem mass spectrometry in a quadrupole ion trap. Dissociation ions specific to stereochemical differences at C2 and C4 in hexose complexes are observed in the MS2 and MS3 spectra, thus allowing unambiguous differentiation of glucose, galactose, mannose, and talose. Labeling studies incorporating 2H and 13C are used to probe the mechanisms of dissociation involved with these diastereomers, and MS2 studies on deoxyglucose complexes are implemented to support proposed sites of deprotonation within the complexes. Carbohydrates have been implicated in a wide variety of cellular interactions. In particular, studies are ongoing to determine the mechanism of action of these ubiquitous molecules which are involved in molecular targeting, cell-cell recognition, and cell-cell adhesion.1 The first step in such studies must be structural elucidation, which requires a knowledge of monosaccharide composition and sequence, linkage position, and branch points, as well as anomeric configuration of each glycosidic bond. The ultimate goal of this project is a methods development in carbohydrate sequencing where metals or metal-ligand systems are used to cationize the oligosaccharide. Tandem mass spectrometry (MS/MS) is then employed in order to probe carbohydrate structure. Although mass spectrometry traditionally has not been used in stereochemical determination, some data exists which reveal the potential of such a technique.2-4 Current methodologies for carbohydrate analysis include NMR, permethylation/hydrolysis, periodate oxidation, and enzymatic reduction.5 These techniques require a substantial amount of highly purified material and time-consuming labor. Furthermore, the results often prove difficult to interpret. Tandem mass spectrometry, on the other hand, is a sensitive technique in which picomole quantities or less are consumed during sample analysis. Purity requirements are lowered because the ion of interest is isolated from the initial mass spectrum before analysis. In (1) Stryer, L. Biochemistry; W. H. Freeman: New York, 1988; pp 331-348. (2) Splitter, J. S., Turecek, F., Eds. Applications of Mass Spectrometry to Organic Stereochemistry; VCH: New York, 1994. (3) Smith, G.; Leary, J. A. J. Am. Chem. Soc. 1996, 118, 3293. (4) Smith, G.; Pedersen, S. F.; Leary, J. A. J. Org. Chem. 1997, 62, 2152. (5) Chaplin, M. F., Kennedy, J. F., Eds. Carbohydrate Analysis; Oxford: New York, 1994. S0003-2700(98)00023-7 CCC: $15.00 Published on Web 05/22/1998
© 1998 American Chemical Society
addition, the labor and analysis time are substantially reduced. Recent studies using MS and MS/MS have provided unambiguous linkage type and sequence information for certain branched and straight-chain oligomers,6-17 as well as monosaccharide substitution patterns; i.e., hexose, N-acetylhexosamine, fucose, etc.18 Glucose, galactose, and mannose (Glu, Gal, and Man) are the three most common hexoses present in mammalian physiology19 and, therefore, have been chosen for our studies due to their biological relevance. These isomers differ only in the axial/ equatorial configuration of their hydroxyl groups (see Figure 1). For MS/MS studies presented here, diastereomeric complexes of Glu (1), Gal (2), Man (3), and Talose (Tal, 4) (see Figure 2) were prepared according to procedures similar to those reported by Yano and co-workers, who have previously prepared, isolated, and characterized nickel diaminopropane glucose and mannose complexes.20,21 The reaction mixture was then ionized by electrospray (ESI) and examined by sequential stages of mass spectrometry (MSn). The dissociation pattern of each complex is unique and, as evidenced here, can be used to differentiate each of the four hexose diastereomers. Although previous MS/MS studies of nickel diaminopropane hexose (Ni-dap-hexose) diastereomers indicated differentiation of mannose from glucose and galactose, distinction between the latter two isomers was not possible.3,4 The implementation of a zinc diethylenetriamine (Zndien) metal-ligand system in place of Ni-dap has circumvented (6) Egge, H.; Peter-Katalinic, J. Mass Spectrom. Rev. 1988, 6, 331. (7) Zhou, Z.; Ogden, S.; Leary, J. A. J. Org. Chem. 1990, 55, 5444. (8) Hofmeister, G. E.; Zhou, Z.; Leary, J. A. J. Am. Chem. Soc. 1991, 113, 5964. (9) Staempfli, A.; Zhou, Z.; Leary, J. A. J. Org. Chem. 1992, 57, 3590. (10) Garozzo, D.; Impallomeni, G.; Montaudo, G.; Spina, E. Rapid Commun. Mass Spectrom. 1992, 6, 550. (11) Garozzo, D.; Giuffrida, M.; Impallomeni, G.; Ballistreri, A.; Montaudo, G. Anal. Chem. 1990, 62, 279. (12) Fura, A.; Leary, J. A. Anal. Chem. 1993, 65, 2805. (13) Hayes, G. R.; Williams, A.; Costello, C. E.; Enns, C. A.; et al. Glycobiology 1995, 5, 227. (14) Reinhold, V.; Reinhold, B. B.; Costello, C. E. Anal. Chem. 1995, 67, 1772. (15) Cancilla, M. T.; Penn, S. G.; Carroll, J. A.; Lebrilla, C. B. J. Am. Chem. Soc. 1996, 118, 6736. (16) Sible, E. M.; Brimmer, S. P.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1997, 8, 32. (17) Asam, M. R.; Glish, G. L. J. Am. Soc. Mass Spectrom. 1998, 8, 987. (18) Gillece-Castro, B. L.; Burlingame, A. L. Methods Enzymol. 1990, 193, 689. (19) Ginsburg, V. In Biological Mass Spectrometry; Burlingame, A. L., and McCloskey, J. A., Eds.; Elsevier: New York, 1990; p 363. (20) Yano, S. Coord. Chem. Rev. 1988, 92, 113. (21) Yano, S.; Kato, M.; Shioi, H.; Takahashi, T.; Tsubomura, T.; Toriumi, K.; Ito, T.; Hidai, M.; Yoshikawa, S. J. Chem. Soc., Dalton Trans. 1993, 1699.
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methods required to deduce monosaccharide identity and stereochemistry, linkage position of larger oligomers, and glycosidic anomericity through MS/MS of metal-ligand derivitized oligosaccharides.3,4,7-9,12,16,23-26 We are currently in the process of developing a combined hydrolysis/online HPLC separation method, after which postcolumn metal derivatization23 and other modifications are implemented prior to online MS/MS analysis of a complex carbohydrate. This project is nearing completion and is the focus of another manuscript.
Figure 1. Isomeric monosaccharides.
Figure 2. Structures of hexose complexes examined in this study.
this deficiency, thus providing unambiguous differentiation of all four hexoses. Overall, the many advantages of MS/MS such as speed, sensitivity, and low purity requirements make it an ideal analytical tool for oligosaccharides. Unfortunately, the biggest disadvantage is that there is currently no single sample introduction system and mass analyzer which can be used for total structural elucidation. In addition, many of the derivatization steps needed either to volatilize the sample or to distinguish linkage/branching are often quite time-consuming and require inert atmosphere conditions.22 The results presented herein are one part of a larger analytical challenge: development of a method for total structural identification of complex carbohydrates using one type of mass analyzer. It is also anticipated that a minimum of hydrolysis/LC and timeconsuming derivatization steps would be required. As of the reporting of these results, we now have all the preliminary (22) (a) Merkle, R. K.; Poppe, I. Methods Enzymol. 1994, 230, 1. (b) Geyer, R.; Geyer, H. Methods Enzymol. 1994, 230, 86. (c) Dell, A.; Reason, A. J.; Khoo, K.-H.; Panico, M.; McDowell, R. A.; Morris, H. R. Methods Enzymol. 1994, 230, 108.
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EXPERIMENTAL SECTION General. Zn(dien)2Cl2 was obtained from Dr. Steven F. Pedersen, Department of Chemistry, University of California, Berkeley. Glucose and mannose were purchased from Aldrich (Milwaukee, WI). Galactose, talose, glucose (13C-2), and all deoxyglucose compounds were purchased from Sigma (St. Louis, MO). Glucose (2H-6/6′) was purchased from Cambridge Isotope Labs (Andover, MA). All other labeled compounds were obtained from Omicron Biochemicals (South Bend, IN). All reagents were used as received. Solutions for MS. Samples were prepared by dissolving 0.5 mg (2.8 µmol) of monosaccharide and 0.5 mg (1.4 µmol) of Zn(dien)2Cl2 in 50 µL of methanol. The resulting solution was heated at 70 °C for 20 min, after which time 1 µL was diluted with 100% methanol to 500 µL for analysis. Labeled complexes and deoxyglucose complexes were prepared in the same manner using 2Hor 13C-labeled glucose or 2-, 3-, or 6-deoxyglucose. Mass Spectrometry Measurements. Mass spectra were obtained by using a quadrupole ion trap mass analyzer fitted with an electrospray ionization source (Finnigan LCQ, Finnigan MAT, San Jose, CA). A three-point calibration was carried out with a standard mixture of the peptide MRFA, caffeine, and Ultramark. The sample was delivered using direct infusion with a syringe pump at a flow rate of 2 µL/min. Sample concentration was approximately 50 pmol/µL, and the mobile phase was 100% methanol. Ions were produced with a spray voltage of 4.6 keV, with the heated capillary set at 175 °C. To avoid space charge effects, the number of ions present in the trap at one time was regulated by the automatic gain control, which was set at 5 × 107 ions for MS and 3 × 107 ions for MS/MS. Helium was used as the damping gas at a pressure of 10-3 Torr. Voltages across the capillary and the octapole lenses were tuned by an automated procedure to maximize signal from the ion of interest in the mass spectrum. Spectra were collected in the positive ion mode. Each spectrum is an average of 10-20 “individual” scans; each individual scan is composed of three “microscans”. Collision-induced Dissociation (CID) was carried out in the mass analyzer on an ion selected from the mass spectrum, by using the He gas present in the ion trap. In CID, a resonance excitation radio frequency voltage is applied to the endcap electrodes. This causes ions in the mass analyzer to gain kinetic energy in the axial direction. These ions then collide with the He gas, and the excess kinetic energy of the ions becomes internal energy, which promotes dissociation. Typical values for the relative collision energy (peak-to-peak amplitude of the resonance excitation) range from 0.4 to 0.8 eV. (23) Kohler, M.; Leary, J. A. Anal. Chem. 1995, 67, 3501.
Table 1. MS/MS Product Ions from Diastereomeric Hexose Complexes: Precursor Ion at m/z 328
Glu (1a) Gal (2a) Man (3a) Tal (4a)
-H2O
-CH2O
310 310 310
298 298 298 298
-2 H2O
-CH2O/-H2O
-C2H4O2
-C3H6O3
280 280 280
268 268 268 268
238 238 238
292
-C4H6O3
-C4H8O4
226 226
208 208 208 208
Table 2. MS3 Studies with Diastereomeric Complexes of Glucose, Galactose, and Mannose genealogy (m/z) 328 f
310a,b
product ions formed f 292
328 f 298 f 328 f 280 f 328 f 268 f 328 f 238 f 328 f 226 f 328 f 208 f
Figure 3. Representative MS1. Zn(dien)2Cl2 + glucose, reaction mixture.
RESULTS AND DISCUSSION MS/MS Studies. Four diastereomeric N-glycoside complexes of the form 1-[1-diethylenetriamine]-1-deoxy-D-hexose dichloride zinc(II) (1-4, see Figure 2), where hexose ) glucose, galactose, mannose, or talose, were examined by tandem mass spectrometry. A typical mass spectrum recorded for the reaction mixture is shown in Figure 3. The ionic counterparts of a complex such as 1 which are detected in the gas phase include [M - Cl-]+ at m/z 364 and [M - 2Cl- - H+]+ at m/z 328. The latter represents the type of gas-phase ions examined in this study which are labeled na to distinguish them from the solution-phase precursor n (see Figure 2). Additional ions at m/z 490 and 428 represent [Zn(C4H9N3)(C6H11O5)2]+ and [(C4H10N3)(C6H11O5)2 + H]+, respectively, which are ligand-bridging monosaccharide complexes. These particular ions are byproducts of the reaction and are the subjects of another study. Complexes 1a, 2a, 3a, and 4a (Figure 2) were selected and allowed to undergo CID. The results of the MS/MS experiments are shown in Table 1. (Spectra are included in the Supporting Information.) The data show a clear distinction in the dissociation ions for each of the complexes of Glu, Gal, Man, and Tal. Glucose is distinguished from the other three hexoses by the lack of any water loss from the precursor ion. In addition, the cross ring cleavage corresponding to loss of C3H6O3 (m/z 238) is missing for this diastereomer. The MS/MS spectrum of the galactose complex shows the loss of one H2O molecule (m/z 310) as well as the loss of C3H6O3, thus distinguishing it from the glucose complex. Since the axial or equatorial configuration at C4 is the distinguishing structural feature of Glu (equatorial) and Gal (axial), it is significant that a differentiating feature in their MS/MS spectra is the presence or absence of the three-carbon neutral loss, which depends on bond cleavage between C4 and C3. Neither the galactose nor the mannose complex exhibits an ion at m/z 292 corresponding to the loss of two water molecules. However, like its epimer talose, the MS/MS spectrum of the
292 280
280
274 264 246 208
280 268 250 238 226 208 262c 250c 208 250 208 210 208 196 178 208 191 180 163 151
a Top series, products of Gal; bottom series, products of Man. b m/z 310 only observed for Gal and Man. c Only observed for Man.
mannose complex is devoid of an ion at m/z 226 which is present in both the glucose and galactose spectra. This ion was first thought to arise from the loss of dien ligand (102 Da); however, labeling studies (discussed below) clearly indicated that a rather peculiar cross ring cleavage of the hexose moiety was responsible for this ion. This is discussed in detail in the following sections. Finally, talose can be identified from the other isomers by the fact that both one and two water molecules are lost from the precursor ion, and no loss of formaldehyde plus water is observed at m/z 280. The ion at m/z 208, which represents a C4H8O4 cross ring cleavage, is a common ion (>50% relative abundance) for all four diastereomers, suggesting that its ease of formation is related to the stability of this particular cross ring cleavage process. Therefore, among these four complexes, the presence of product ion m/z 226 is indicative of an equatorial C2 hydroxyl group, whereas an axial hydroxyl group blocks this dissociation pathway. Similarly, complexes possessing an axial C4 hydroxyl (Gal and Tal) dissociate more readily by loss of C3H6O3 than do their counterparts possessing an equatorial C4 hydroxyl group (the intensity of the m/z 238 ion, although present in the Man diastereomer, was less than 10%). In addition, the number of H2O losses from the precursor ion in the MS/MS spectra corresponds to the number of axial hydroxyl groups in the complex. MS3 Studies. Although MS2 studies were sufficient to distinguish the four hexoses, MS3 experiments were undertaken for the three biologically relevant hexose complexes (Glu, Gal, Man). It was anticipated that further MSn studies would help ascertain the reasoning behind the observed stereochemical differentiation as well as provide a second check on this distinction. Each product ion from Table 1 was examined by MS3 for the Glu, Gal, and Man complexes; i.e., the genealogies m/z 328 f m/z 310 f and m/z 328 f m/z 298 f, etc. were each investigated. These data are summarized in Table 2. Dissociation patterns for any ion representing a cross ring cleavage in the MS3 experiments (i.e., m/z 328 f 298, 268, and 208 f) were identical for Glu, Gal, and Man; i.e., the same product Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
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Figure 4. Proposed mechanisms of dissociation for Zn-dien-glucose complexes.
ions were observed for each complex. This suggests that there is a loss of stereochemistry during the cross ring cleavage process, possibly through ring opening and subsequent bond rotation. These ions involve losses of CnH2nOn from the various precursor ions. Ions at m/z 310 and 280, representing a loss of water from m/z 328 or 298, respectively, do, however, show differences in their MS3 spectra. As shown in Table 2, products of m/z 328 f 310 f include an initial water loss (m/z 292) for both Gal and Man. The only other product for Man is a formaldehyde loss. Since Glu shows no initial loss of water at m/z 310, it is immediately differentiated from the remaining two isomers. In contrast, products for Gal include another water loss from m/z 292 (m/z 274) and a loss of C4H6O3 to give the familiar ion at m/z 3012 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
208. An ion at m/z 208 is not present for Man, and, in fact, this is the only MS3 experiment in which this specific product ion was not formed. Clearly, there is a fundamental difference between the m/z 310 precursor ions for Gal and Man. Additional ions obtained for the galactose complex appear to include losses of CO from m/z 274 and 292, respectively. Therefore, ions at a particular mass-to-charge ratio which represent water losses may, in fact, involve different gas-phase structures for the Glu, Gal, or Man complexes. This would arise if the loss of water were occurring at a specific, but different, site in each complex, depending on the relative axial and equatorial configuration of the hydroxyl groups. 13C, 2H Labeling Studies. To ascertain the mechanisms involved with these diastereomers and understand why each
Figure 5. MS2 results of deoxyglucose complexes. Table 3. Results of Glucose Labeling Studies: Precursor at m/z 329a type and product ions formed location of label -CH2O -CH2O/-H2O -C2H4O2 -C4H6O3 -C4H8O4 2H-1 2H-2 2H-6,6′ 13C-2 13C-3 13C-4 a
299 299 298 299 299 299
281 281 280b 281 281 281
269 269 268b 269 269 269
227 227 226b 227 226b 226b
209 209 208b 209 208b 208b
Precursor for 2H-6,6′-labeled glucose at m/z 330. b Loss of label.
diastereomer dissociates differently, extensive labeling studies were undertaken. Compound 1 was synthesized using the following labeled glucose: 13C at carbon number 2, 3, or 4; 2H at H1, H2, or H6/H6′ (the nonexchangeable proton(s)). Data from the resulting MS/MS experiments are provided in Table 3. By observing which ions shown in Table 3 retained the labels after dissociation of the precursor 1a, it was deduced that a CH2O neutral loss (formaldehyde) includes C6 and both nonexchangeable protons but not the exchangeable proton on the C6 hydroxyl. A C2H4O2 neutral loss does not include C2, C3, or C4 and, therefore, must include C5 and C6, precluding substantial rearrangement. The C4H8O4 neutral loss must include C3-C6, because the carbon at position 2 and the protons H1 and H2 are retained in the product ion. One of the more interesting results from these labeling studies involves the ion at m/z 226, which is shown to be formed from the loss of C4H6O3 and incorporates carbons 3, 4, 5, and 6 in the neutral fragment. A mechanism for this loss was not readily apparent, since it first seemed that a rather elaborate rearrange-
ment from the C4H8O4 loss with subsequent transfer of H2O to the charged ion would need to take place. In fact, earlier investigators noticed a similar ion, but without labeling experiments they were unable to describe an appropriate mechanism.27 In light of the information gleaned from the labeling studies, a proposed mechanism for the generation of m/z 226 (m/z 227 for labeled complexes) is shown in Figure 4. Also shown in Figure 4 are the proposed mechanisms for the generation of ions resulting from losses of CH2O, C2H4O2, and C4H8O4. These are discussed more extensively below in the context of the MS/MS experiments of the deoxy complexes. MS/MS of Deoxyglucose Complexes and Mechanisms of Dissociation. As can be seen from Figure 4, several different sites of deprotonation must be possible in order to propose realistic mechanisms for the generation of the various product ions. In this regard, MS/MS of several deoxyglucose complexes were critical to assigning various sites of deprotonation and for determining which channels of dissociation would be obviated by the absence of a particular hydroxyl functionality. Therefore, each of the possible deprotonation sites was “removed” in turn by synthesizing Zn-dien-(2-deoxy-Glu), Zn-dien-(3-deoxy-Glu), and Zndien-(6-deoxy-Glu) and recording their MS/MS spectra. In the case where the C6 hydroxyl cannot be deprotonated (6-deoxy-Glu complex), losses of formaldehyde (m/z 298) and the subsequent water loss (m/z 280) are no longer present (Figure (24) Smith, G.; Leary, J. A. J. Am. Soc. Mass Spectrom. 1996, 7, 953. (25) Kohler, M.; Leary, J. A. Int. J. Mass Spectrom. Ion Processes. 1997, 389, 233. (26) (a) Gaucher, S. P.; Leary, J. A. Manuscript in preparation. (b) Desaire, H.; Leary, J. A. Mauscript in preparation. (27) Dallinga, J. W.; Heerma, W. Biomed. Environ. Mass Spectrom. 1989, 18, 363.
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5). Ions representing these losses are present in the MS/MS spectra of 2- and 3-deoxyglucose (m/z 282, 264; see Figure 5), suggesting that deprotonation of the C6 hydroxyl is mandatory in order to initiate these losses. The proposed mechanism for the formaldehyde loss shown in Figure 4A involves the formation of a carbanion-type intermediate, which subsequently undergoes a Wittig rearrangement to generate the deprotonated aminal complex. The rearrangement, which is well known, is intramolecular and calls for a 1,2 H shift of the aminal hydrogen to C1. It is interesting to note that, since this loss initiates ring opening, all further losses are nonstereospecific; i.e., all MS3 studies of ions involving ring opening indicate no differentiation of the four diastereomers (see above section). Furthermore, since the labeling studies indicated specifically that only H’s 6,6′ are involved in this loss, the mechanism for m/z 328 f m/z 298 shown in Figure 4 is supported by all of the available information. Similarly, in the case where the C2 hydroxyl cannot be deprotonated (2-deoxy-Glu complex), the loss of C4H8O4 is absent, while the ion representing this loss (m/z 208) is present in the spectra of 3- and 6-deoxyglucose. This suggests that deprotonation of the C2 hydroxyl is necessary for this loss, known through labeling studies to contain C3-C6. As shown for the C4H8O4 loss in Figure 4B, the site of deprotonation on the C2 hydroxyl group is believed to begin the shift of electrons, resulting in two neutral losses and thus regenerating the alkoxide on the C2 oxygen. Other MS studies involving metal-coordinated oligosaccharides have also shown dissociation mechanisms driven from the lone pair electrons of an alkoxide.9,12 Once again, ring-opening obviates any stereochemical differentiation, and thus this ion is observed for all the diastereomers. The loss of C2H4O2 is present in all of the deoxyglucose complex MS/MS spectra. This suggests that the loss, which contains carbons 5 and 6, does not require deprotonation at the C2, C3, or C6 hydroxyl. Figure 4C,D illustrates our proposed mechanisms with initial deprotonation at the aminal nitrogen. Note that the subsequent ring opening destroys the stereochemistry of the product ion. This is in concert with the fact that MS3 studies of m/z 328 f 268 f produce identical spectra for Glu, Gal, and Man complexes. Two possible pathways are proposed for the formation of the m/z 268 ion, one involving formation of a cyclobutane ring, the other from generation of the carbanion at C4 with subsequent Wittig rearrangement to produce the alkoxide. It is difficult to know for certain which of these pathways, or both, is responsible for formation of m/z 268. Production of a cyclobutane complex is not unusual and has been reported previously for other saccharide complexes which were also subjected to extensive labeling studies.9
3014 Analytical Chemistry, Vol. 70, No. 14, July 15, 1998
Finally, the 102 Da loss from the precursor is present only in the 2-deoxy-Glu complex (m/z 222) but not in the 6-deoxy or 3-deoxy complexes, suggesting that both the C6 and C3 hydroxyl groups play a role in this loss of C4H6O3. Labeling studies also indicated that carbons 3-6 are contained in the neutral loss, as in the loss of C4H8O4 (m/z 208) described above. However, one oxygen and two hydrogens are retained in the product ion at m/z 226 relative to the C4H8O4 loss. In fact, the product at m/z 226 dissociates (m/z 328 f 226 f, Table 2) only by a loss of H2O to form m/z 208. In the proposed mechanism (Figure 4E), after formation of the epoxide and subsequent ring opening, a sixmembered transition state can be formed which involves a proton transfer from the C3 hydroxyl and the loss of the neutral fragment in accordance with the 13C and 2H labeling studies. The fact that different sites of deprotonation lead to specific pathways of dissociation appears to play an important role in our observations of the presence/absence of specific product ions for each of the hexose diastereomers. If different populations of these deprotonated species exist for each of the complexes, certain pathways would be favored, leading to unique MS2 and MS3 spectra for Zn-dien complexes of these four hexoses. CONCLUSIONS We have shown that diastereomeric Zn-dien complexes of glucose, galactose, mannose, and talose can be distinguished using ESI tandem mass spectrometry on an ion trap instrument. Studies of deoxyglucose analogues along with 2H and 13C labeling studies have been utilized to suggest possible mechanisms of dissociation. The data suggest that stereochemical differences are retained in the gas phase, providing the pyranose ring is not opened and remains coordinated to the metal-ligand. Once ring opening occurs, bond rotation precludes any further stereochemical differentiation upon CID. These studies further indicate that metal-ligand systems coordinated to various monosaccharides can be used to distinguish stereochemical features of the saccharides when analyzed by sequential tandem mass spectrometry. ACKNOWLEDGMENT The authors thank Professor Steven Pedersen for helpful discussions and also gratefully acknowledge Omicron Biochemicals for supplying some of the labeled glucose. J.A.L. acknowledges the NIH GM 47356 for financial support.
Received for review January 6, 1998. Accepted April 15, 1998. AC980023K
