Cell-surface carbohydrates and their interactions

124

Biochimica et Biophysica Acta, 399 (1975) 124--130 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

BBA 27 684

CELL-SURFACE C A R B O H Y D R A T E S AND T H E I R INTERACTIONS I. NMR OF N-ACETYL NEURAMINIC ACID

ELLEN B. BROWN, WALLACE S. BREY, Jr, and WILLIAM WELTNER, Jr

Department of Chemistry, University of Florida, Gainesville, Fla. (U.S.A.) (Received December 2nd, 1974)

Summary 1. The proton nuclear magnetic resonance spectrum of the sialic acid N-acetyl neuraminic acid was measured and completely assigned. The coupling constants for interactions between protons are obtained and the assignments checked by calculating the observed line intensities. 2. It was verified that the pyranose ring exists in the 1C (~C4) conformation as the ~-D anomer and no mutarotation was detectable. Coupling constants for the glycerol side chain were interpreted to yield its most likely conformation.

Introduction The oligosaccharides of the glycoprotein and glycolipids located at the cell surface have been shown to be involved in or responsible for many of the functions of animal cell membrane. These carbohydrate chains have been implicated in the dynamic processes of cellular adhesion [ 1 - 3 ] , contact inhibition [2--5] and transport [6--8] and have been shown to constitute the sites of viral receptors [9--13] and antigens [14,15]. Such functions imply a specific conformation requirement for the sugar chains. Indeed, one mechanism [3] proposed for cell adhesion is a dynamic, multiple enzyme-substrate process in which membrane glycosyltransferases bind to the carbohydrate substrates on neighboring cells. Sialic acids, which are located at the non-reducing end of carbohydrate chains in glycoproteins and gangliosides, have often been shown to be integrally involved in these membrane processes. Removal of sialic acid from the carbohydrate cell surface by neuraminidase markedly alters or prevents many membrane functions, including interactions of erythrocyte cells with influenza virus

125 [9,12,13], adhesion and contact inhibition in certain cells [1--5], and the rate of potassium and protein transport in leukemia cells [7,8]. N-Acetyl neuraminic acid (Ac-Neu) is the most commonly occurring sialic acid and the most relevant carbohydrate with which to begin an investigation of cell surface molecular interactions. Proton nuclear magnetic resonance (PMR) has been chosen as a probe because of its demonstrated success in elucidating detailed information concerning saccharide conformation and the location of binding sites in complexes formed between carbohydrates (specifically, inositols [ 16] ) and metallic cations. The earlier enzymatic work of Comb and Roseman [17], and the preparation and reaction of a lactone of Ac-Neu by Kuhn and Baschang [18] served to establish the stereochemistry of C-4 through C-8 (see Fig. 1). The synthesis of two methyl ketosides, by Yu and Ledeen [19], and the study of their tendency to form lactones has made possible the assignment of their anomeric structures. The reaction of only one of them with neuraminidase established that it had the naturally occurring stereochemistry for the glycosidic bond and that it was an a anomer. The correct name for Ac-Neu is then 5-amino-3,5-dideoxy-aD-glycero-D-galacto-2-nonulopyranosonic acid. This has recently been reviewed by Bentley [20]. Early investigations of Ac-Neu by 60 MHz PMR spectroscopy produced no assignments except for the strong absorption of the N-acetyl methyl group [21,22]. The 100 MHz spectra of derivatives of Ac-Neu in organic solvents by Lutz et al. [23] have been sufficiently assigned to determine that these molecules assume the 1C (1C4) conformation. In the work presented herein, the first complete assignment of Ac-Neu has been made. The measurements were made in aqueous solvent to better reproduce in vivo conditions. The results presented serve as a prelude to further study of structure vs function relationships involving sialic acids and other cell-surface carbohydrates. H

F~ ~CH20H

OXYGEN

OH

CARBON C) HYDROGEN

Fig. 1. T h e sialicacid N-acety! neuraminic acid (Ac-Neu).

126 H4

H5

H8

H7

H9'

I

I

]

I

H3e

Me

H3o

H9

I

I

40

:3.5

2.5

2_0

8 (ppm) Fig. 2. 2 7 0 MHz NMR s p e c t r u m and a s s i g n m e n t of Ac-Neu in 2 H 2 0 .

Methods and Materials

N-Acetyl neuraminic acid (Sigma t y p e V) was dissolved in 2H20, lyophilized once, and solvated in ~ H 2 0 at 0.18 M concentration. Frequencies were measured relative to TSP on a 270 MHz Brucker spectrometer with internal deuterium lock. 32 sweeps were made for each spectrum at 25°C. Results

Fig. 2 shows the observed 270 MHz spectrum of Ac-Neu in 2 H 2 0 with the splitting patterns and proton assignments indicated. Derived coupling constants are given in Table I, and observed frequencies and 5 values for the center of each p r o t o n pattern are indicated in columns 2 and 3 of Table II. The rationale for these assignments is given below.

TABLE 1 C O U P L I N G C O N S T A N T S F O R SIALIC A C I D IN 2 H 2 0 [ J ( 3 a x , 3 eq) [ [J(3ax, 4)1 [J(3eq, 4)[ [J(4,5)[ [J(5,6) I [J(6,7)1 IJ(7,8) [ IJ(8,9)t [J(8,9')1 [J(9,9')1

13.1 c.p.s. 11.8 4.7 10.4 10.4 0.8 9.2 6.3 2.5 11.7

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TABLE

II

RESONANCE Proton

H 3ax

Methyl H 3eq H7 H9 H8 H9' H5 H6 H4

FREQUENCIES

OF TRANSITION

CENTERS

Observed frequency for transitions b e t w e e n m i x e d spin s t a t e s

C a l c u l a t e d f r e q u e n c y for t r a n s i t i o n s b e t w e e n u n m i x e d spin s t a t e s

Hz*

Hz*

-509.5 -558.2 -629.4 -971.3 -987.0 -1022.9 -1047.5 -1071.3 -1109.2 -1111.7

b** 1.8872 2.0674 2.3311 3.5974 3.6557 3.7885 3.8796 3.9676 4.1080 4.1172

-509.9 -558.2 -629.0 -971.8 -987.9 -1022.2 -1046.8 -1072.4 -1108.6 -1111.2

5"* 1.8885 2.0674 2.3296 3.5993 3.6589 3.7859 4.0689 3.9719 4.1059 4.1156

* R e l a t i v e to TSP. ** p p m d o w n f i e l d f r o m TSP.

TABLE IlI R E S O N A N C E F R E Q U E N C I E S AND I N T E N S I T I E S OF PEAKS Proton H4 H4 H6 H4 H4 H4, H6 H4 H5

H9'

H8

H9

H7

Observed frequency (Hz) -1125.0 -1120.1 -1114.7 -1109.8 -1108.0 -1103.6 -1098.3 -1081.6 -1071.4 -1060.9 -1054.5 -1051.9 -1042.7 -1040.5 ' -1032.1 -1029.4 -1024.9 -1023.0 -1020.1 -1016.3 -1013.7 -996.1 -989.5 -984.0 -978.0 -975.5 -974.7 -966.8 -965.5

Calculated intensity 0.10 0.10 0.37 0.25 0.25 0.15 0.15 0.40 0.45 0.15 0.17 0.23 0.26 0.36 0.15 0.20 0.12 0.20 0.15 0.08 0.09 0.34 0.26 0.26 0.19 0.58

0.41

(two overlapping H4 peaks) (two overlapping H4 peaks) (H4), 0.66 (H6)

(two overlapping peaks)

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Assignment o f the spectrum Splitting patterns and peak frequencies of the methylene protons at C3 are indicated in Fig. 2. The equatorial proton H3e is distinguished from the axial proton H3a by its expected lower field resonance [24] and its lesser degree of coupling [25,26] with proton H4. Integration of the singlet at 5 = 2.0674 ppm (downfield from TSP) reveals that it belongs to the three methyl protons of the amide group. Selective decoupling experiments allowed the seven protons at lower fields (5 = 3.5--4.3 ppm) to be assigned without ambiguity. Peak intensities which were calculated from theory corroborate the assignments by adequately reproducing the "intensity borrowing" observed over the entire spectrum. The transition intensities (Table III) calculated from mixed spin states determined from the nuclear spin Hamiltonian [27], compare favorably with the observed intensities. Discussion

Ring conformation The large J(3a, 4) coupling constant is consistent with an axial position for H4, which can only occur if sialic acid is in the 1C conformation. This conformation has previously been established for derivatives of sialic acid in [2 H] chloroform. We have also observed in water that the 1C conformation is very stable over a 24-h period at room temperature. In addition, no mutarotation was observed over that same time period. Conversion from the a to the/3 conformer would require the axial OH group at C2 to become equatorial, which would in turn greatly shift the resonance of H3a. Lemieux and Stevens [28] have provided a set of empirical rules describing the effect of the orientation of neighboring h y d r o x y groups on proton chemical shifts in pyranoses. According to these rules, the change from axial OH to equatorial OH incurred at C2 would cause the H3a resonance to shift 0.30 ppm downfield. No such shift was observed. The lack of mutarotation is in agreement with the literature [15].

Conformation o f glycerol side chain The Karplus relationship [25,26] predicts the coupling constant to be large when the dihedral angle between two vicinal protons is around 0 °, with a m a x i m u m value around 180 ° , and a minimum around 90 ° . Rotation about the C-C bonds of the glycerol side chain is sufficiently facile that the frequencies and coupling constants observed for each proton are averages over rotamer states. Since the chemical shift and the H8 coupling constant of each proton on C9 are determined by the average chemical environment observed on each proton, the differences in chemical shifts and coupling constants indicate that unequal amounts of time are spent in the contributing rotamer conformations. The large differences in values of J(8,9) and J(8,9') are interpreted to mean t h a t one proton is more likely to be found trans to H8 than the other. There are two configurations possible wherein one proton on G9 is trans to H8. The configuration which places the OH groups trans to one another introduces an unfavorable steric interaction of the h y d r o x y l groups on C7 and C9. The con-

129

H9' 07-~H8

OH 09"~H8

OH C5"~0

H9~ ~'~OH OH

H7~'C6 OH

HT~~[~'~C8 H6

A

B

C

Fig. 3. C o n f o r m a t i o n o f g l y c e r o l side c h a i n .

figuration with the more thermodynamically stable arrangement of 7--9 OH groups is shown in Fig. 3A. In this configuration the proton trans to H8 will be deshielded by the vicinal OH group on C8. The proton gauche to H8 will be strongly deshielded by the OH group on C7. (Examples which illustrate this last effect are found in ref. 29, p. 138). Generally the former effect is larger than the latter, which would lead us to the conclusion that the configuration with the unstable arrangement of 7--9 OH groups is the more prevalent rotamer. However, there is very convincing evidence from X-ray crystallographic studies [30] of glycerol and PMR studies [29] of solvated m e t h o x y hexitols that the rotamer illustrated in Fig. 3A is more likely. And since predictions of relative deshielding from neighboring h y d r o x y groups are very useful, but are n o t correct 100% of the time, we believe the rotamer in Fig. 3A is the more energetically favored and abundant one. A strong preference for the configuration in which H7 and H8 are trans (Fig. 3B) is evident from the relatively large value of the J(8,7) coupling constant. The coupling constant J(6,7) is very small, indicating a stronger preference for a gauche conformer with a dihedral angle of 60--90 ° between H6 and H7. In one of the two possible gauche rotamers the glycerol side chain is wellremoved from the ring and N-acetyl group, thus relieving much of the steric interaction of these masses. This preferred rotamer is shown in Fig. 3C. The lowest energy conformation of the entire D-glycerol side chain is presented in Fig. 1.

Hydrogen bonding The proposed lowest energy conformation for the glycerol side chain offers t w o possible sites for intramolecular hydrogen bonding. The hydroxyl group on C7 can easily hydrogen bond with the glycosidic oxygen of the ring. Hydrogen bond formation is further facilitated if the dihedral angle between H6 and H7 is closer to 90 ° than 60 °, which may account for the very low coupling constant observed for H6, H7. The second possible intramolecular hydrogen-bonding scheme is between the OH group of C8 and the oxygen of the C9-OH moiety. Addendum

Work in progress shows that Ca 2÷ is selectively b o u n d to sialic acid, in contrast to other cations, at a site produced b y a change in the conformation

130

of the glycerol side chain. 13C NMR spectra are also being measured and analyzed.

Acknowledgement The authors would like to thank R. Rosanski for his excellent technical assistance in obtaining the 270 MHz NMR spectra analyzed here.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

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