Vacuum UV Circular Dichroism Spectroscopy of Acetamido Sugars

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Vacuum UV Circular Dichroism Spectroscopy of Acetamido Sugars C. A L L E N BUSH and SURESH RALAPATI Department of Chemistry, Illinois Institute of Technology, Chicago, IL 60616

The carbohydrate chains of glycoproteins and glycolipids have non repeating structures containing from four to approxi mately fifteen sugar residues. Since the chains are branched and feature sugars with such chemical functionality as amides and carboxylic acids, they are known as complex carbohydrates. Although the biological function of these complex oligosaccha rides is not well understood, it is the subject of considerable study in connection with such biochemical phenomena as lectin stimulated mitogenesis, hormone binding to cell surfaces and inter-cellular communication generally. The conformational properties and the physico chemical in teractions of the complex oligosaccharides have not been exten sively investigated. As a modest beginning for such studies we will pose certain questions regarding the influence of a sugar on its neighbors in a complex oligosaccharide chain. We will attempt to show how one can initiate studies of the conformation of the glycosidic linkage between sugars and between the sugar and the protein as well as the interactions among sugar residues and between the carbohydrate and the peptide chain of a protein. The biophysical techniques we have used are mainly spectroscopic, primarily circular dichroism (CD) and to a lesser extent NMR. Other methods which may be brought to bear on this problem in clude conformational energy calculations and x-ray crystallo graphy. Small molecule crystallography has already provided some useful insights but protein crystallography has not yet made the major contributions which we would expect, apparently as a result of some fundamental problems in preparing x-ray quality crystals of glycoproteins. I.

CD of Amide Chromophores in Carbohydrates

Peptide CD studies have revealed the importance of the amide η-π* transition in amide spectra. This transition which contributes an important CD band at 222 nm in the polypeptide α helix spectrum also appears in the CD spectra of N-acetyl 0097-6156/81/0150-0293$05.00/0 © 1981 American Chemical Society Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


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amino sugars near 210 nm (Figure 1). This band, although it oc curs near the short wavelength limit of the range of convention al commercial CD instrumentation, has been studied by several research groups (1_, 2_, 3_, 4). Also apparent in the spectra of Figure 1 is a band at shorter wavelength (185-192 nm). This band is associated with the strong amide π-π* transition which has its uv absorption maximum at 189 nm. Although some useful data on these shorter wavelength bands has been obtained from conventional commercial instruments, in our laboratory we have used a vacuum uv instrument which is optimized for use with samples in solution in the wavelength range 160-300 nm. The in strument features a short focal length monochromator with the cell compartment at atmospheric pressure following a design first introduced by Brahms et al. (5j. The optical layout of the instrument in our lab is illustrated in Figure 2 (15). This machine is operated under computer control for repetitive scan ning, signal averaging, and. Fourier digital filtering of the CD spectra (7). The data of Figure 1 illustrate the similarity of the long er wavelength η-π* bands for different anomeric glycosides of 2-acetamido-2-deoxy-glucose and - galactose (GlcNAc and GalNAc). Variations over a factor of two in magnitude without a change in sign of wavelength of the minimum are observed for this band in a wide wariety of oligosaccharides containing these acetamido sugars in aqueous solution. At shorter wavelength, the $ glyco sides generally show a positive band near 192 nm while the α glycosides have a stronger band at 185 nm. These bands certain ly arise from the amide chromophore as is shown by their ab sence in CD spectra of neutral sugar glycosides which lack the 2-acetamido functionality (8). The fact that the wavelength of the maximum of the CD band departs from the maximum of the absorbance (189 nm) suggests that there is mutual coupling be tween the amide π-π* transition and some other strong transi tion, perhaps an η-σ* band of the acetal chromophore. The CD spectra of reducing sugars differ substantially from those of the corresponding glycosides. The data of Figure 3, taken with the instrument in our laboratory, are in substantial agreement with those of Bush (9) taken with the instrumentation in the laboratory of Dr. J. Brahms (5_). The data reported by Buffington et a l . , (1_0) for GlcNAc differ in the 180-190 nm re gion perhaps as a result of a difference in anomeric composition. Our data represent an equilibrium anomeric mixture of approxi mately equal amounts of α and 3 pyranose. In addition to varia bility in the exact anomeric composition, a second feature con tributes to differences between the CD spectra of reducing sugars and those of their corresponding glycosides. In the former case the amide is perturbed by the hemi-acetal chromo phore while in thelatter case it is the acetal chromophore which influences the π-π* rotational strength, perhaps by a coupling mechanism.

Brant; Solution Properties of Polysaccharides ACS Symposium Series; American Chemical Society: Washington, DC, 1981.


B U S H A N D RALAPATi

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Acetamido

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Wavelength (nm) Dordrecht-Holland Figure 1.

CD spectra of methyl pyranosides of 2-acetamido-2-deoxy hexoses (9)

Figure 2. Schematic of the vacuum UV CD apparatus: (A) 200-W deuterium lamp; (B) CaF collimating lens; (C) McPherson 218 monochromator vacuum chamber; (D, E) focusing mirrors; (F) plane grating; (G) MgF rochon polarizer; (H) modulator; (I) CaF lens; (J) sample chamber at atmospheric pressure; (K) mask for extraordinary beam, (L) photomultiplier (6). 2

2

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Analytical Chemistry


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Circular Dichroism Spectra of Oligosaccharides Containing Acetamido Sugars

The effect on the CD spectrum of linking two GlcNAc resi dues by a 3 1-4 linkage is essentially that of forming a Β gly coside as may be seen from comparison of the spectrum of chitobiose (Figure 4) with that of 3-methyl-GlcNAc (Figure 1). This fact is also clear from a spectrum of chitotriose ( £ ) . It is also true for 3 1-6 linked acetamido sugars as may be seen in the spectrum of GlcNAc(3 l-6)GlcNAc (9). In contrast, sugars linked at the C-3 position, directly adjacent to the amide chromophore, show a strong negative band at 180 nm which is not seen in spectra of simple glycosides. Figure 5 shows the spectrum of Gal(3 l-3)GalNAc which has nega tive CD in the 190 nm region, a feature which is characteristic of three and four linked oligosaccharides having reducing termi nal acetamido sugars. Lacto-N-tetraose (LNT) has a 3 linked GlcNAc residue which is also substituted at the C-3 position. Therefore its spectrum shows a positive band at 190 nm charac teristic of a 3 linked acetamido sugar in addition to the nega tive band at 180 nm which is characteristic of C-3 substitution. Although data presently available for oligosaccharides contain ing acetamido sugars are sufficient to make a few generalizations, it will be necessary to examine a wider variety of linkages in order to recognize details of explicit interactions between residues. III.

Vicinal Diacyl Amino Sugars and Glycopeptides

In many glycoproteins the connection between the carbohy drate and peptide occurs through a glycosyl amide linked to an asparagine side chain in the protein. The linked sugar, in variably a 3 GlcNAc residue, is therefore a vicinal diacyl amino sugar. Such sugars have two amide chromophores in sufficiently close proximity to interact by exciton coupling in a manner analagous to that in polypeptides. The CD spectrum which arises from this interaction is characterized by a pair of strong posi tive and negative bands crossing the axis near the wavelength of the ahsorbance maximum for the coupled bands. The CD spec trum of 4-N-(2-acetamido-2-deoxy-3-D-glucopyranosyl)-asparagine (GlcNAc-Asn) of Figure 6 shows just such a pair of strong CD bands, crossing the axis near the maximum of the amide π-π* absorbance. That these bands, which are much larger than those of the other acetamido sugars discussed above, arise from the interaction of the vicinal di-equatorial amides is shown by the CD spectrum of 2-acetamido-2-deoxy-l-N-Acetyl-3-D-glucopyranosyl amine ( 3 l , 2 DAG). Its similarity to that of GlcNAc-Asn implies that the large bands arise from amide interaction, not from the amine and carboxyl functions of the amino acid. Since the CD due to this exciton coupling is quite sensitive to the geometric



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BUSH A N D RALAPATi

Figure 3.

Acetamido

CD spectra of Ν-acetyl

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glucosamine ( mine ( )

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) and of ^-acetyl galactosa-

Figure 4. CD spectrum of GlcNAc l,4)GlcNAc (chitobiose)


(β-


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Figure 5. CD spectra of Gal (β-1,3) GalNAc ( ; and of Gal(fi-l,3)GlC' Ν A c(fi-l ,3)Gal^-l ,4 )-Glc (Lacto-N-tetraose) ( ).

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WAVELENGTH , nm Biochemistry Figure 6. CD spectra of 4-N-(2-deoxy-2-acetamido glycosyl) asparagine ( ) and of 2-acetamido-2-deoxy-l-^-acetyl-fi-O-glucopyranosyl amine ( ) (11)


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relationship of the two amides, we have been able by means of theoretical models to relate the CD spectrum to the geometric relationship of the amides. A model has been proposed for the conformation of GlcNAc-Asn based on these calculations (VjJ.


IV.

Conformation of the Amide in Acetamido Sugars

In acetamido sugars the amide is in the planar trans conformation as it is in most peptide bonds. This conformation, which is generally lower in energy than the planar ci s amide, has been found in all x-ray crystallographic studies of 2acetamido-2-deoxy sugars (1_2, 1_3, For a planar trans amide there remains the question of rotation about the bond between the amide nitrogen and the pyranose ring carbon atom. There are two conformations allowed by steric contact between the amide and the adjacent groups. In one conformation the amide proton and the pyranose C-2 proton are cis to one another while in the second these protons are trans. Conformational energy calculations show these two conformations to be similar in energy with the cis conformation lying approximately 1 k cal. below the trans conformer (15). Experimental methods capable of identifying these two conformations points firmly in favor of the trans conformer. In the proton nmr spectra of acetamido sugars, the amide proton resonance is located well down field and is easily assigned. The dihedral angle about the C-N bond is related to the amide proton coupling constant by a Karplus relation which has been extensively investigated because of its importance in peptide conformational studies (1_6). These studies show that coupling constants as large as 8 Hz are found only for conformations with very nearly trans related protons. The amide proton coupling constants of acetamido sugars have been investigated in a wide variety of solvents. In all cases studied the coupling constants were high (8 to 9 Hz) implying a trans relationship between the protons for both the amide at C-2 as well as at the anomeric center for glucopyranosyl amides (17.). X-ray crystallography also points to the trans conformation in the solid state for GlcNAc (12_), for chitobiose (T4) and for both the amides of GlcNAc-Asn (T3). Moreover the trans conformer has been assumed in successful CD calculations for 2-acetamido-2deoxy sugars (18) as well as for vicinal diacyl amino sugars (11). ~~ Since there is as yet no clear cut evidence for the existence of 2-acetamido sugars in which the amide proton is in a cis conformation with respect to the pyranose proton one must consider the possibility of some error in the conformational energy calculations. Such an error could arise from the neglect of the effects of solvation. This seems unlikely since the proton coupling constants are found to be similar in solvents of widely differing polarities (17). There remains the interesting


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possibility that the cis conformer occurs under conditions which have not yet been experimentally investigated. The exis tence of these two conformational isomers could be of consi derable biological significance.


V.

Theoretical Interpretation of the CD Spectra of Acetamido Sugars

Theoretical interpretations of the CD of acetamido sugars draw heavily on the extensive studies of the CD spectra of poly peptides whose chromophoric properties are essentially identical to those of acetamido sugars. As in the case of polypeptides, it has been shown that the amide η-π* band is made optically active by the asymmetric electrostatic field of the groups surrounding the amide. Specifically it is the electric dipole moment of the hydroxyl function at C-3 which most influences the CD spectrum in the 210 nm region for 2-acetamido-2-deoxy sugars (18). Since the anomeric configuration of the sugar or its glycosides does not have a strong influence on the electro static field gradient, the CD in the 210 nm region is not very sensitive to anomeric configuration in oligosaccharides. On the other hand, the perturbation of the C-3 hydroxyl group is found to be quite sensitive to the hydrogen bonding properties of the solvent. In contrast to water which is both a hydrogen bond donor and acceptor hexafluoro-2-propanol (HFIP) acts only as a hydrogen bond donor. Dickinson et al. (19) have shown that the CD spectra of GlcNAc and Gal ΝAc in HFIP have positively signed CD bands at 210 nm in contrast to the nega tive bands seen in aqueous solutions (Figure 3). Both the absorbance and CD curve shapes are similar for samples in these two solvents implying that HFIP does not exert a profound ef fect on the chromophoric properties of the amide. Clear experi mental support for the involvment for the C-3 hydroxyl in this effect has been given (19). The amide π-π* transition in glycosides of GlcNAc and GalNAc gains its rotational strength not from electrostatic perturbation but rather from coupling with strong transitions of nearby chromophores, perhaps the η-σ* transitions of the acetal chromophore. Therefore it is reasonable to expect that the position and magnitude of the π-π* bands should depend on anomeric configuration as is observed experimentally (Figure 1). These effects have not yet been successfully treated theoreti cally since the exact electronic nature of the bands with which the π-π* transition is coupled is unknown. The improved under standing of the chromophoric properties of the acetal group which is emerging from CD studies on neutral sugar glycosides may make such a treatment possible. (See contribution to this volume by E.S. Stevens.) For vicinal diacyl amino sugars, i t is the two amide chromo phores which interact. Therefore in this case coupling theories


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developed for polypeptides are directly applicable. Complete calculations involving a consistent treatment of both the η-π* and π-π* amide transitions have qiven an adequate explanation of the large CD bands of GlcNAc-Asn (11_).


Abstract The t i t l e compounds have been studied over the wavelength range 170-220 nm using a new ultraviolet CD instrument whose design features a sealed 200 watt deuterium lamp, a wide aper ture vacuum monochromator and a small sample compartment purged with N2 at atmospheric pressure. The asymmetric electrostatic f i e l d causing the n-π* band at 210 nm to be optically active arises mainly from the hydroxyl group at C-3 in the case of the 2-acetamido-2-deoxy hexoses. As a result of free rotation of the C-3 hydroxyl group, the CD band at 210 nm is quite sensitive to solvent, the sign of the CD of 2-deoxy 2-acetamido hexoses in fluorinated alcohols being opposite to that found for aqueous solutions of amido sugars not substituted or hydrogen bonded at the C-3 oxygen. In vicinal diacyl amino sugars, strong exciton CD bands are seen at 178 and 200 nm due to coupling of the two amide π - π * transitions. Calculations following theories pre viously used in polypeptide CD correlate the observed CD bands with the amide orientation. In a conformational model of the glycopeptide linkage compound, 2-acetamido-2-deoxy-l-L-aspartamido-β-D-glucopyranosylamine, the amides at C-1 and C-2 are both oriented such that the amide protons are trans to their respective ring protons. This conformation has been confirmed by nmr measurements of the amide proton coupling constants. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

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Kabat, E . A . ; Lloyd, K.O.; Beychok, S. Biochemistry, 1969, 8, 747. Stone, A . L . Biopolymers, 1971, 10, 739. Park, J.W.; Chakrabarti, B. Biopolymers, 1978, 17, 1323. Coduti, P . L . ; Gordon, E . C . ; Bush, C.A. Anal. Biochem., 1977, 78, 9. Brahms, S.; Brahms, J.; Spach, G . ; Brack, A. Proc. Natl. Acad. Sci. U . S . A . , 1977, 74, 3208. Duben, Α . ; Bush, C.A. Anal. Chem., 1980, 52, 635. Bush, C.A. Anal. Chem., 1974, 46, 890. Nelson, R . G . ; Johnson, W.C. J. Am. Chem. Soc., 1976, 98, 4296. Pullman, B . ; Goldblum, Ν . , Eds. "Excited States in Organic Chemistry and Biochemistry"; D. Reidel: Dordrecht-Holland., 1977; p. 209. Buffington, L . A . ; Pysh, E . S . ; Chakrabarti, B . ; Balazs, E.A. J . Am. Chem. Soc., 1977, 99, 1730.


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11. 12. 13. 14. 15. 16.


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Bush, C . A . ; Duben, A. J. Am. Chem. Soc., 1978, 100, 4987. Johnson, L.N. Acta. Crystallogr., 1966, 21, 885. Delbaere, L . T . J . Biochem. J., 1974, 143, 197. Mo, F.; Jensen, L.H. Acta. Crystal1ogr., 1978, 1334, 1562. Pincus, M.R.; Burgess, A.W.; Scheraga, H.A. Biopolymers, 1976, 15, 2485. Ramachandran, G.N.; Chandrasekaran, R.; Kopple, K.D. Biopolymers, 1971, 10., 2113. Bush, C . A . ; Duben, Α . ; Ralapati, S. Biochemistry, 1980, 1 9 , 501. Yeh, C . - Y . ; Bush, C.A. J. Phys. Chem., 1974, 78, 1829. Dickinson, H.R.; Coduti, P . L . ; Bush, C.A. Carbohydr. Res., 1977, 56, 249.

Research supported by NSF Grant CHE 76-16783. R E C E I V E D September 11, 1980.


Vacuum UV Circular Dichroism Spectroscopy of Acetamido Sugars

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