Raman Spectroscopy As a Discovery Tool in Carbohydrate Chemistry

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Anal. Chem. 2000, 72, 2093-2098

Raman Spectroscopy As a Discovery Tool in Carbohydrate Chemistry P. H. Arboleda and G. R. Loppnow*

Department of Chemistry, University of Alberta, Edmonton, AB T6G 2G2, Canada

Raman spectra of nine anomerically stable monosaccharides have been obtained in aqueous solution in the 7001700 cm-1 spectral range. Good-quality spectra are obtained of solutions with concentrations as low as 10 mM and volumes as small as 15 µL. Interestingly, the Raman spectra appear to be exquisitely sensitive to the configuration of the carbon centers; unique spectra are obtained of all nine monosaccharides. The unique Raman spectral fingerprint observed for each monosaccharide, and for each anomer of each monosaccharide, suggests that Raman spectroscopy may be a useful technique for the identification and characterization of biologically relevant oligosaccharides. To test this idea, Raman spectra of three unknown disaccharides were obtained in a single-blind study. Identification of the individual monosaccharide components and their anomeric configuration was completely successful. All of these results suggest that development of Raman spectroscopy as a fast, sensitive discovery tool in glycobiology and carbohydrate chemistry is straightforward. Oligosaccharides are increasingly recognized as the molecular agents in tissue and cellular recognition. One reason for this role is thought to be the enormous potential for diversity; oligosaccharides can be made from hundreds of different monomers or their derivatives. The structures of four common monosaccharides are shown in Chart 1. This large library which nature can draw on compares favorably with the relatively limited number of building blocks available for nucleic acids and proteins of 5 and 20, respectively. In addition to this extensive library of building blocks, oligosaccharides frequently develop branched structures, compared with the linear sequences found in naturally occurring proteins and nucleic acids. This branching is possible because of the large number of reactive hydroxyl groups on typical sugars. Biological systems have developed an exquisitely selective recognition system capable of detecting small changes in chemical composition of oligosaccharides. For example, the A and B blood group antigens, tetrasaccharides covalently linked to glycoproteins and glycolipids of mammalian cell surfaces, differ in a single side group on a single monosaccharide.1 Blood group antigen A has an acetamido group at C-2′ of the terminal galactose while blood group antigen B has a normal hydroxy group at this position and * To whom correspondence should be addressed. E-mail: glen.loppnow@ ualberta.ca. or www: http://www.chem.ualberta.ca/∼gloppnow. (1) Davidsohn, I.; Stejskal, R. Haemotologia 1972, 6, 177-184. 10.1021/ac991389f CCC: $19.00 Published on Web 03/25/2000

© 2000 American Chemical Society

Chart 1

yet the physiological effects of such a small mismatch are significant and potentially life-threatening. One of the challenges in glycobiology is finding tools for the structural elucidation of these complex molecules composed of units which are very similar in their chemical and physical properties. Indeed, many monosaccharides only differ in their configuration, having identical molecular masses, chemical composition, and functional groups. These similarities present serious difficulties in identifying and characterizing oligosaccharide structure. Traditionally, NMR has been the probe of choice in determining oligosaccharide composition and structure.2-7 Although one-dimensional NMR spectroscopy is useful at obtaining some information about the composition of oligosaccharides, detailed structural elucidation including the anomeric conformation of each individual monosaccharide and its linkage sites generally requires two-dimensional techniques such as Nuclear Overhauser Effect Spectroscopy (NOESY). This technique can require large amounts of sample (up to milligrams), significant accumulation times (hours to days), and powerful assignment algorithms. These disadvantages drive the search for new techniques in the structural elucidation of novel oligosaccharides, both synthetic and naturally occurring. Raman spectroscopy is a powerful probe of structure and is increasingly being applied to problems in biology.8 In this technique, an incident laser beam, usually at a visible or ultraviolet (2) Bock, K.; Pedersen, C. Adv. Carbohydr. Chem. Biochem. 1983, 41, 27-66. (3) Vliegenthart, J. F. G.; Dorland, L.; van Halbeek, H. Adv. Carbohydr. Chem. Biochem. 1983, 41, 209-374. (4) Pratt, S. L. J. Carbohydr. Chem. 1984, 3, 493-511. (5) Bax, A.; Egan, W.; Kovac, P. J. Carbohydr. Chem. 1984, 3, 593-611. (6) Pfeffer, P. E. J. Carbohydr. Chem. 1984, 3, 613-639. (7) Dill, K.; Berman, E.; Pavia, A. A. Adv. Carbohydr. Chem. Biochem. 1985, 43, 1-49.

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wavelength, scatters inelastically off the vibrational states of a molecule. This inelastically scattered light is collected, dispersed in a monochromator, and detected. The resulting vibrational spectrum contains similar information as the infrared (IR) vibrational spectrum, with a few important distinctions. The selection rules in Raman spectroscopy are based on changes in polarizability, rather than in permanent dipole moments, as in IR spectroscopy. Thus, C-C and C-H vibrations are more easily observed in the Raman spectrum and may be more sensitive to changes in configuration at a carbon center than O-H and C-O bonds, which are more easily observed in an IR spectrum. Raman spectroscopy is ideally suited for biological samples, as water is a very weak scatterer and does not interfere with Raman scattering from solutes in aqueous solution. Finally, the resolution of a Raman spectrum can be higher than that of an infrared spectrum because of the better monochromator and laser properties in the UV and visible regions of the spectrum. For all of these advantages, Raman spectroscopy has not been applied to oligosaccharides in any detail. The earliest studies were aimed at determining the feasibility of applying Raman spectroscopy to saccharides and examined monosaccharides labeled with strong Raman chromophores, such as thiol and nitrile groups.9,10 Later studies focused on detailed Raman analyses of crystalline monosaccharides as models for the interpretation of the vibrational spectra and structural elucidation of glucan homopolysaccharides.11-24 These early studies provided detailed experimental force fields17-21 and demonstrated that the vibrational modes of monosaccharides involve complex mixing of internal coordinates. While these results indicated that localized group frequency descriptions (e.g., “C1′-O stretch”, “C1′-C2′ stretch”, etc.) of the modes below 1500 cm-1 are physically inaccurate, they did suggest that monosaccharides may exhibit complex and unique spectral fingerprints which can be used as empirical aids in elucidating oligosaccharide structure and composition. More recently, vibrational Raman optical activity (ROA) of mono- and (8) Nakamoto, K. Infrared and Raman Spectra of Inorganic and Coordination Compounds, vol. A and B; Wiley-Interscience: Toronto, 1997; 800 pp. (9) Tu, A. T.; Lee, J.; Lee, Y. C. Carbohydr. Res. 1978, 67, 295-304. (10) Tu, A. T.; Liddle, W. K.; Lee, Y. C.; Myers, R. W. Carbohydr. Res. 1983, 117, 291-297. (11) Mathlouthi, M.; Koenig, J. L. Adv. Carbohydr. Chem. Biochem. 1986, 44, 7-89. (12) Vasko, P. D.; Blackwell, J.; Koenig, J. L. Carbohydr. Res. 1971, 19, 297310. (13) Vasko, P. D.; Blackwell, J.; Koenig, J. L. Carbohydr. Res. 1972, 23, 407416. (14) Cael, J. J.; Koenig, J. L.; Blackwell, J. Carbohydr. Res. 1974, 32, 79-91. (15) Mathlouthi, M.; Luu, D. V. Carbohydr. Res. 1980, 81, 203-212. (16) Mathlouthi, M.; Luu, C.; Meffroy-Biget, A. M.; Luu, D. V. Carbohydr. Res. 1980, 81, 213-223. (17) Dauchez, M.; Derreumaux, P.; Lagant, P.; Vergoten, G.; Sekkal, M.; Legrand, P. Spectrochim. Acta, Part A 1994, 50A, 87-104. (18) Dauchez, M.; Lagant, P.; Derreumaux, P.; Vergoten, G.; Sekkal, M.; Sombret, B. Spectrochim. Acta, Part A 1994, 50A, 105-118. (19) Dauchez, M.; Derreumaux, P.; Lagant, P.; Vergoten, G. J. Comput. Chem. 1995, 16, 188-199. (20) Durier, V.; Tristram, F.; Vergoten, G. J. Mol. Struct. 1997, 395-396, 8190. (21) Wells, H. A., Jr.; Atalla, R. H. J. Mol. Struct. 1990, 224, 385-424. (22) Sekkal, M.; Dincq, V.; Legrand, P.; Huvenne, J. P. J. Mol. Struct. 1995, 349, 349-352. (23) Kacurakova, M.; Mathlouthi, M. Carbohydr. Res. 1996, 284, 145-157. (24) Zhbankov, R. G.; Prihodchenko, L. K.; Kolosova, T. E.; Andrianov, V. M.; Korolevich, M. V.; Ratajczak, H.; Marchevka, M. J. Mol. Struct. 1998, 450, 29-40.

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disaccharides has been proposed as a technique for the sequencing of oligosaccharides.25-29 However, high concentrations and long accumulation times are needed for sufficient ROA spectral quality, reducing the feasibility of applying this technique to most biologically derived oligosaccharides. In this paper, we describe the results of Raman spectroscopy of mono- and disaccharide samples. Good-quality spectra can be obtained of monosaccharide samples with concentrations as low as 10 mM and volumes as small as 15 µL in as little as 1/2 h. The spectra are unique for each anomer and each monosaccharide of the nine monosaccharides obtained, suggesting Raman spectroscopy can be used to determine the composition of oligosaccharides. This idea was tested successfully by identifying the composition of three disaccharides in a single-blind study. All of these results suggest that Raman spectroscopy may be a fast, sensitive discovery tool in glycobiology and carbohydrate chemistry. EXPERIMENTAL SECTION All mono- and disaccharides were used as received. 1-O-MethylR-D-glucoside, 1-O-methyl-β-D-glucoside, 1-O-methyl-R-D-galactoside, 1-O-methyl-β-D-galactoside, 1-O-methyl-R-D-mannoside, and 1-O-methyl-β-D-mannoside were purchased commercially (Sigma Chemical Co., St. Louis, MO). 1-O-Methyl-R-D-xyloside, 1-O-methylβ-D-xyloside, 1-O-methyl-R-D-N-acetylgalactosamine, and three disaccharides were generously donated by Professor O. Hindsgaul, University of Alberta. The three disaccharides were received “blindly”, i.e., with no knowledge of their chemical composition before identification by Raman spectroscopy. Room-temperature Raman spectra of the saccharides were obtained by spherically focusing the laser either onto a spinning 5-mm o.d. NMR tube containing 0.3-0.5 mL of the sample solution (0.01-1.0 M, H2O) in a 135° backscattering geometry or onto a 1.5-mm-diameter capillary containing 15 µL of the sample solution (0.03-1.0 M, H2O) in a 90° backscattering geometry. Laser excitation was obtained with 0.5-3 W of 514.5-nm light from an Ar+ laser (Coherent, Santa Clara, CA). Rayleigh scattering was rejected with a 514.5-nm holographic filter (Kaiser Optical Co., Ann Arbor, MI). Multichannel detection was obtained with a liquid nitrogen-cooled CCD detector (Princeton Instruments, Trenton, NJ) coupled to a single monochromator (Spex Industries, Metuchen, NJ). Spectral slit widths were 5-7 cm-1, and typical accumulation times were 30 min. Frequency calibration was done by measuring the Raman scattering of solvents of known frequencies (benzene, acetone, toluene, and ethanol). Reported frequencies are accurate to (2 cm-1. The spectra were analyzed by subtracting a water spectrum taken under identical conditions and leveling the baseline by subtracting multiple joined line segments. RESULTS AND DISCUSSION Figures 1 and 2 show the Raman spectra of nine 1-Omethylmonosaccharides. Methylation of the C-1′ oxygen prevents (25) Barron, L. D.; Gargaro, A. R.; Wen, Z. Q. Carbohydr. Res. 1991, 210, 3949. (26) Wen, Z. Q.; Barron, L. D.; Hecht, L. J. Am. Chem. Soc. 1993, 115, 285292. (27) Bell, A. F.; Hecht, L.; Barron, L. D. J. Am. Chem. Soc. 1994, 116, 51555161. (28) Bell, A. F.; Hecht, L.; Barron, L. D. Spectrochim. Acta, Part A 1995, 51A, 1367-1378. (29) Bell, A. F.; Hecht, L.; Barron, L. D. J. Mol. Struct. 1995, 349, 401-404.

Figure 1. Raman spectra of the four most common 1-O-methylmonosaccharides, (A) 1-O-methyl-R-D-glucoside, (B) 1-O-methyl-βD-glucoside, (C) 1-O-methyl-R-D-galactoside, and (D) 1-O-methyl-βD-galactoside, excited with 2 W of 514.5-nm light.

racemization of the anomeric carbon and allows the spectrum of a pure anomer in aqueous solution to be obtained. The spectra are all of good quality with the exception of that of 1-O-methylR-D-N-acetylgalactosamine, which exhibited a high level of fluorescence. We suspect the fluorescence arises from some impurity in the sample and is not intrinsic to 1-O-methyl-R-D-N-acetylgalactosamine. No vibrations were observed at frequencies above 1500 cm-1 in any of the samples. At first glance, the spectra appear complex and similar. Indeed, this is to be expected as each compound should exhibit 70-80 vibrations, all of which should be Raman allowed. The spectra of the R and β anomers of glucose, galactose, and mannose are expected to be similar, because the chemical composition and functional groups are all identical in these six monosaccharides. However, a detailed examination of the spectra shows that each one exhibits a unique spectral fingerprint. While space prohibits a global comparison, a simple comparison of the Raman spectra of 1-O-methyl-R-D-glucoside (Figure 1A) and 1-O-methyl-β-Dglucoside (Figure 1B) should be sufficient to highlight the salient points. The spectra of the two glucose derivatives are clearly different. The Raman spectrum of 1-O-methyl-R-D-glucoside exhibits peaks at 761, 846, 1082, and 1130 cm-1 which are absent in the spectrum of 1-O-methyl-β-D-glucoside, while that of 1-O-methylβ-D-glucoside exhibits peaks at 879, 951, and 1307 cm-1 which are absent in that of 1-O-methyl-R-D-glucoside. In addition, for those peaks which occur at similar frequencies, the relative

Figure 2. Raman spectra of five other common 1-O-methylmonosaccharides, (A) 1-O-methyl-R-D-N-acetylgalactosamine, (B) 1-O-methyl-R-D-mannoside, (C) 1-O-methyl-β-D-mannoside, (D) 1-Omethyl-R-D-xyloside, and (E) 1-O-methyl-β-D-xyloside, excited with 2 W of 514.5-nm light.

intensities are quite different. For example, the relative intensities of the 906 and 1013 cm-1 peaks in the two glucosides are quite different. These differences are significant enough that 1-O-methylR-D-glucoside and 1-O-methyl-β-D-glucoside are easily distinguishable by Raman spectroscopy. Indeed, comparison of the spectra for each anomeric pair demonstrates that the same could be said for each monosaccharide. To determine detection limits, Raman spectra were obtained of 1-O-methyl-R-D-glucoside at a number of concentrations. The results are shown in Figures 3 and 4. Even at concentrations as low as 10 mM, the characteristic peaks are easily distinguished from noise. Given the signal-to-noise ratio of the 900 cm-1 peak at 10 mM, we estimate that the minimum concentration detectable under these conditions could be as small as 0.5-1 mM, although it is unclear whether different monosaccharides would be distinguishable at these low concentrations. Figure 4 demonstrates that similar-quality spectra can be obtained with volumes as small as 15-µL drops in a capillary tube. The results in Figure 4 demonstrate that reasonable-quality spectra for identification and characterization can be obtained on amounts as small as 90 µg, amounts comparable or smaller than those needed for NMR, the current technique of choice. Enhancement techniques could potentially make Raman spectroscopy more sensitive than NMR, perhaps even approaching the sensitivity of mass spectrometry. Well-known enhancement Analytical Chemistry, Vol. 72, No. 9, May 1, 2000

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Figure 3. Raman spectra of 1-O-methyl-R-D-glucoside at various concentrations excited with 2 W of 514.5-nm light.

Figure 4. Raman spectra of microsamples of 1-O-methyl-R-Dglucoside at various concentrations excited with 0.5 W of 514.5-nm light.

techniques in Raman spectroscopy include resonance enhancement,30 by exciting within an absorption band of the analyte molecule, and surface enhancement,31 in which a colloidal solution of silver or other metal is added to the analyte. Carbohydrates do not have significant absorption bands above 200 nm, thus (30) Pelletier, M. J. Analytical Applications of Raman Spectroscopy; Blackwell Science: Malden, MA, 1999; 478 pp. (31) Neddersen, J.; Chumanov, G.; Cotton, T. M. Appl. Spectrosc. 1993, 47, 1959-1964.

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Figure 5. Raman spectra of unknown disaccharide A and 1-Omethyl-R-D-glucoside excited with 3 W of 514.5-nm light.

resonance enhancement is not possible. Surface enhancement of the Raman signal from saccharides has not been reported and our attempts at surface enhanced Raman spectroscopy of the saccharides here were unsuccessful, even though we did observe enhancement of benzene and pyridine vibrational modes, as expected. Recently, more practical techniques, such as the use of a Raman waveguide,32 have been suggested to increase the effective Raman signal. We are considering such possibilities for increased sensitivity and spectral quality. These results all suggest that Raman spectroscopy could be a potentially powerful probe of oligosaccharide composition and structure. Figures 5-7 show the Raman spectra obtained of three unknown disaccharides, provided as part of a single-blind study, to test this idea. The spectra are all of good quality with the exception of that of unknown C, which exhibited a high level of fluorescence. Again, we believe the fluorescence arises from an impurity. However, all of the spectra have enough characteristic peaks that the composition and anomeric configuration of each unknown can be determined. Identification of the monosaccharide composition and anomeric configuration was accomplished simply by visual comparison of the mono- and disaccharide Raman spectra and will be discussed in detail separately for each unknown disaccharide. Specifically, peaks unique to a single monosaccharide were first identified in the disaccharide spectra. Spectral signatures were then compared peak by peak to confirm the presence of the candidate monosaccharides. Initial attempts to quantitatively analyze the disaccharide spectra by linear fitting to the monosaccharide component spectra were unsuccessful due to the noise and variable baselines in some of the monosaccharide spectra (e.g., Figure 2 parts E and A, respectively). Improvements in the quality of the monosaccharide spectra are ongoing and (32) Marquardt, B. J.; Vahey, P. G.; Synovec, R. E.; Burgess, L. W. Anal. Chem. 1999, 71, 4808-4814.

Figure 6. Raman spectra of unknown disaccharide B, 1-O-methylβ-D-glucoside, and 1-O-methyl-R-D-glucoside excited with 3 W of 514.5-nm light.

Figure 7. Raman spectra of unknown disaccharide C, 1-O-methylβ-D-glucoside, and 1-O-methyl-β-D-galactoside excited with 3 W of 514.5-nm light.

should facilitate a more rigorous quantitative identification of unknown oligosaccharides. Figure 5 shows the Raman spectrum for unknown disaccharide A. The strong band at ∼910 cm-1 in the disaccharide spectrum is found with such an intensity only in the 1-o-methyl-R-D-glucose spectrum, indicating that this monosaccharide is one of the components. Indeed, careful comparison of the disaccharide and

1-o-methyl-R-D-glucose spectra indicates that they are very similar. Thus, this disaccharide was identified as R-glucose + R-glucose. Interestingly, the appearance of slight differences between the monosaccharide and disaccharide spectra, such as the small peak at ∼780 cm-1 and the shoulder at ∼890 cm-1, indicates that the Raman spectrum may also identify the linkage site. Differences between the disaccharide spectrum and constituent monosaccharide spectra were observed for each unknown. Figure 6 shows the Raman spectrum for unknown disaccharide B. Comparison of the disaccharide spectrum and that of 1-Omethyl-β-D-glucoside shows a number of similarities. In particular, the appearance of two strong bands at ∼1060 and ∼1120 cm-1 indicates either β-glucose or β-xylose is present. The strong ∼1360 cm-1 peak in the disaccharide and an overall comparison of spectral signatures suggests 1-O-methyl-β-D-glucoside is the better candidate. Again, the appearance of a band near 900 cm-1, which is more intense than any other band in the 700-1000 cm-1 region, in the disaccharide spectrum indicates the presence of 1-O-methylR-D-glucoside as the other component. Thus, this disaccharide was identified as R-glucose + β-glucose. Figure 7 shows the Raman spectrum for unknown disaccharide C. This spectrum exhibited a high level of fluorescence, so the spectral quality is not as good as for the other two unknowns. However, successful identification of the monosaccharide constituents should still be possible. As in unknown C, this spectrum has two strong peaks at ∼1080 and ∼1120 cm-1, indicating either β-glucose or β-xylose is present. Only β-glucose has a broad band at ∼1360 cm-1, as is seen in the disaccharide; therefore, 1-Omethyl-β-D-glucoside must be one of the constituents of the disaccharide. Confirmation of this assignment comes from the single peak observed in the disaccharide spectrum at ∼950 cm-1. The spectrum of 1-O-methyl-β-D-glucoside has a single peak in this region, while the spectrum of 1-O-methyl-β-D-xyloside exhibits two peaks of similar intensity here. The spectrum of the disaccharide is still quite different from that of 1-O-methyl-β-D-glucoside, particularly in the 1200-1420 cm-1 region. In this region, the disaccharide spectrum has a broad, unresolved band of moderate intensity with a relatively strong peak at ∼1250 cm-1. Only the spectrum of β-galactose exhibits such a broad, unresolved band with a single strong peak at 1260 cm-1. A further confirmation of this assignment is the additional intensity between the two strong β-glucose peaks at 1080 and 1120 cm-1 in the spectrum of the disaccharide. The spectrum of 1-O-methyl-β-D-galactoside exhibits a strong peak exactly centered between the two β-glucose bands. Thus, this disaccharide was identified as β-glucose + β-galactose. A comparison of the Raman-derived identification of the monosaccharide components of the unknown disaccharides and their anomeric configuration, with the actual molecular species, demonstrates the potential of this approach. Unknown A is R-Dglucose(1f4)-1-O-methyl-R-D-glucose, unknown B is R-D-glucose(1f4)-1-O-methyl-β-D-glucose, and unknown C is β-D-galactose(1f4)-1-O-methyl-β-D-glucose, all in agreement with the Ramanderived assignments. The appearance of slight frequency shifts and relative intensity changes in the disaccharide spectra do not preclude the assignment of composition and configuration. Rather, these slight changes may be spectral clues which can be used to assign linkage sites. We are currently attempting to extract these linkage sites from the Raman spectra. Analytical Chemistry, Vol. 72, No. 9, May 1, 2000

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CONCLUSIONS The application of Raman spectroscopy to the identification and characterization of the monosaccharide components of disaccharides is described in this paper. Good-quality spectra are obtained of solutions with concentrations as low as 10 mM, volumes as small as 10 µL, and required amounts as small as 90 µg, comparable or smaller than those needed for NMR, the current method of choice. Successful identification of the monosaccharide components in three unknown disaccharides illustrates the ease and precision with which this technique can be developed as a discovery tool for glycobiology.

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ACKNOWLEDGMENT The authors wish to thank M. Palcic of the University of Alberta for helpful discussions and suggestions, O. Hindsgaul for providing the disaccharides and some monosaccharides used in this work, and E. Fraga for preparing solutions. G.R.L. acknowledges NSERC for providing financial support for this work.

Received for review December 7, 1999. Accepted February 8, 2000. AC991389F