synthesis and conformational analysis of methyl 2-o-(α-d-mannopyranosyl)-α-d-mannopyranoside

T. Peters 135

Synthesis and Conformational Analysis of Methyl 2-O-(a-~-Mannopyranosyl)-a- D-mannop yranoside Thomas Peters

lnstitut fur Biophysikalische Chemie der Universitat Frankfurt, Theodor-Stern-Kai 7 - 15, D-6000 Frankfurt/M. 70

Received August 6, 1990

Key Words: Yeast mannans / Oligosaccharide synthesis 1 Thioglycosides 1 Flexibility of glycosidic linkages

NMR experiments such as steady state NOE experiments and spin lattice ‘H relaxation time measurements were performed on the synthetic disaccharide 10 that constitutes part of the polysaccharide backbone in fungal mannans. The spectroscopic data were compared with a theoretical model that is based upon the HSEA force field and statistical mechanics, taking into account the flexibility around the glycosidic linkage. Potential energy calculations were performed with a modified version of the GESA program in order to derive the global

minimum conformation of disaccharide 10 and to calculate ensemble average NOE and TI values by averaging all ener- getically accessible conformational microstates, using a normalized Boltmann distribution function. The fit between experimental and theoretical results improved significantly when we applied a dynamic model instead of a single state model based solely on the global minimum conformation of 10. Efficient synthesis of 10 was achieved by a recently published protocol for the in situ activation of thioglycosides.

The disaccharide moiety a-D-Man-(l--+2)-a-D-Man is a frequently occurring structural motif in a variety of glyco- conjugates1). It is located at the nonreducing ends of the antennae of mannosidic type N-glycoproteins and can be assumed to play an important role in the interaction of this class of compounds with certain receptor molecules. It also constitutes the major structural moiety of the repeating unit of several fungal polysaccharides that are homopolymers of D-mannose, such as the D-mannan found in Saccharornyces cerevisiae2).

A number of conformational analysis studies have been performed for a-D-Man-(l-+3)-a-D-Man- and a-D-Man- (1+2)-a-~-Man-containing oligosa~charides~) but nothing has yet been published on the dynamic behaviour of the a- 1,Zlinkage. Conformational analysis studies of Brucella A- type di~accharides~), which are closely structurally related to the disaccharide 10 under investigation, have revealed that flexibility, in terms of variability of the dihedral angles 4 and IJI at the glycosidic linkage, plays an important role for this type of glycosidic bond. Thus, it should be expected that these findings will also apply to disaccharide 10. An improvement of the published synthesis5) of 10 could be achieved by using a recently developed in situ glycosylation method6). Synthetic 10 was then subjected to detailed conformational analysis. For the conformational analysis, an approach was adopted that utilized NMR experiments in aqueous solution in combination with theoretical calculations based upon the HSEA force field7). A comparison was performed for a single state conformational model vs. a multi state model that predicts ensemble averaged NMR

parameters such as NOE and TI values in order to assess the importance of flexibility around the glycosidic linkage4-8).

Synthesis

Starting from D-mannose 1, the orthoester 2 was prepared following literature-based procedures’) in good overall yield. Derivative 2 was transformed into glycosyl donor 3 by treatment with HBr in ether, by analogy with the preparation of the corresponding chloride”), and into acceptor alcohol 5 by methanolysis in dry methanol and acetyl chloride’). It is known that storage of orthoester 3 leads to decomposition resulting in a mixture of the corresponding methyl glycoside 4 and small amounts of the 2-OH free methyl glycoside 59). Thus, it was desirable to convert freshly prepared 2 into a more stable glycosyl donor. Thioglycosides perfectly fulfill both these requirements of stability and easy activation for glycosylation reactions ‘ 7 “ ) . Thioglycoside 7 was prepared by treatment of 2 with catalytic amounts of Et,O-BF, in the presence of excess thioethanol as nucleophile. Formation of minor amounts of methyl glycoside 4 was observed as a side reaction, as expected from published work9). Methyl glycoside 4 was converted into acceptor 5 by deacetylation.

Alternatively, heating of 2 in 60% acetic acid’’ and sub- sequent acetylation of the resulting syrup gave 1,2-di-0- acetate 6 which in turn was converted into 7, again with thioethanol and boron trifluoride in dichloromethane”). Yields were comparable for both routes but the first protocol, comprising direct conversion of 2 into 7, was more convenient. Thioglycoside 7 could be converted into bromide 3 under mild conditions utilizing bromine in dichl~romethanel~).

Coupling of donor 3 and acceptor 5 was achieved using conventional silver triflate promoted glycosylation’) furnish-

Liebigs Ann. Chem. 1991, 135- 141 OVCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 0170-2041/91/0202-0135 $ 3.50+.25/0

136 T. Peters

ing disaccharide 8 in 60- 70% yield. Alternatively, the coupling was performed employing thioglycoside 7 directly as glycosyl donor, taking advantage of a recently published in situ activation procedure6) utilizing bromine as the activat- ing reagent. This latter protocol proved to be more convenient, and gave comparable to better yields than the "clas- sical" procedure. A value of 171 Hz for the heteronuclear coupling constant Jc.l,,i..H in 8 clearly indicated exclusive formation of the a linkage. Deblocking of 8 proceeded without complications following literature-based procedures. Transesterification followed by catalytic hydrogenation yielded the free methyl glycoside disaccharide 10 which was used for conformational analysis.

HO * HO OH

1

R'O R'O % R3

R1 R2 R3

Bzl Ac B r Bzl Ac OMe Bzl H OMe Bzl Ac OAc Bzl Ac SEt

L

R3

I R1 R2 R3

8 Bzl Ac OMe Bzl H OMe

!ll 1 H H OMe

Calculations

Potential energy calculations were performed using a modified version of the GESA program 3b~14) that embodies the rigid body HSEA (Hard Sphere Exo Anomeric) approach of Lemieux') and that is capable of performing the task of motional averaging4.'). Using this method one assumes that the main forces determining the conformation of a saccharide are van der Waals interactions and the exo- anomeric effect Is). Pyranose ring coordinates were obtained from crystallographic X-ray or neutron diffraction data, with hydrogen atoms repositioned with standard geometries and standard C - H bond lengths. Treatment of hydroxyl group protons was deliberately avoided. Thus, different conformations of disaccharide 10 are defined by the dihedral angles c$H(l-H-C-l - 1-0-C-x) and +"(C-l- 1-0-C-x - x-H) at the glycosidic linkages and w(5-0 - C-5 - C-6 -6-0) at the 5,6 linkages. Calculation of the preferred rotamers at the 5,6 bonds requires an additional torsional potential function "I. This point was not of importance here because the glycosidic linkage orientation in 10 is almost independent of the orientation of the hydroxymethyl groups.

The global minimum was found to have dihedral angles of + = -47" and $ = -20" at the cl-1,2 glycosidic linkage, in accordance with values found earlier for similar linkage~~','). A local minimum was found to have 4, $ values of 30" and 22", respectively, the energy being 2.4 kcal/mol higher than for the global minimum. In order to calculate ensemble average values for NOE values, spin lattice relaxation times TI , and distances the molecule was allowed to rotate around the glycosidic linkage by varying the dihedral angles + and $ in 5" steps starting from the global minimum and covering the whole conformational space defined by these two angles. The resulting energy surface is shown in Figure 1, and clearly indicates that the + angle is significantly more restricted than the $ angle, a fact that is due to the em-anomeric effect "I. Averaging of parameters was achieved by assigning a statistical weight to each conformational micro state sampled, according to a normalized Boltzmann distribution function, and subsequently sum- ming over the entire conformational ~pace~. '~ . In order to avoid unnecessary calculations, conformational states more than 10 kcal/mol above the global minimum were not considered for the calculation of motionally averaged parameters.

-179 -'?177 -77 23 123

4 Figure 1. Potential energy surface as a function of 4 and JI for the a-1,2 linkage in disaccharide 10 isoenergy contours are scaled at

1.0 kcal intervals up to 10 kcal above the global minimum

Steady state NOE values were calculated employing the full relaxation matrix approa~h3~~~ '~") . The relaxation matrices were either derived directly from the coordinates of the global minimum to yield NOE values based upon a single conformation model or from motionally averaged r - 6 or rP3 values, in the following denoted as (rP6) and ( F ~ ) , with r being the proton-proton distances, thus leading to motionally averaged NOE values, denoted as (NOE). There is some debate whether one should use (F~)' or ( r P 6 ) values for setting up the relaxation matrix for the calculation of (NOE) values. In the case of proteins there is no doubt that (rP3)' values have to be used, since in most cases internal motions in a protein are much faster than the overall molecular tumbling 18). The situation is different for carbohydrates with the size of disaccharides. Overall mo-

Liebigs Ann. Chem. 1991, 135- 141

Synthesis and Conformation of Methyl 2-0-(a-~-Mannopyranosyl)-a-~-mannopyranoside 137

lecular tumbling is usually in the extreme narrowing limit, which means z, values of the order of lo-‘’ s, values one would expect as well for the magnitude of correlation times z, for internal motions such as vibrations of pyranose rings around glycosidic linkages. Rotations around the 5,6 linkages in hexopyranose-containing oligosaccharides may exhibit even larger zI values. Following the formulas given in reference’8b), one can conclude that where the internal molecular tumbling time zi is equal to the overall molecular tumbling time z, the elements of the relaxation matrix are derived from ( ( F ~ ) * + (rP6))/2. Thus, motionally averaged NOE values were calculated using both, ( v P 6 ) and ( T - ~ ) ’

values, in order to estimate deviations that could originate from either treatment. As a matter of fact, in the present case no deviations were detected that could have been ver- ified experimentally, and thus, here, the question of whether one should use ( r - 3 ) 2 or values has no relevance from an experimentalist’s point of view. Calculation of ( Tl) values was also performed on the basis of ( r p 6 ) and ( r - 3 ) * values (Table 4) and, alternatively, on the basis of the global minimum coordinates, in order to evaluate the importance of conformational flexibility.

An analysis to elucidate the dependence of calculated (NOE) values on the cutoff energy showed that only certain NOE values were sensitive to motional averaging. The cutoff energy is defined as the maximum allowed energy difference between the energy of a particular conformational state and the energy of the global minimum up to which conformational states are considered for the calculation of (NOE) values. The most striking example is provided by the NOE experiment, where 1’-H was saturated to give enhancements of 2’-H and 2-H. For this analysis, calculation of absolute (NOE) values was performed assuming an overall correlation time z, of 1.6 x s, a value which was found satisfactorily to reproduce the experimental NOE values given in Table 2. The cutoff energy was varied from 0.0 up to 2.0 kcal/mol in 0.2 kcal/mol steps. The curves in Figure 2 clearly indicate that the interglycosidic NOE between 1’- H and 2-H is sensitive to motional averaging, whilst the intraglycosidic NOE between 1’-H and 2’-H is not.

10

9 0 0.5 1.0 1.5 2.0

Cutoff Energy [kcal]

Figure 2. Dependence of the interglycosidic NOE between 1’-H and 2-H (triangles) and the intraglycosidic NOE between 1’-H and 2‘-

H (squares) on the energy cutoff used for motional averaging

The analysis of dynamic behaviour of saccharides must, of course, focus on those experimental values which show such a dependence. This analysis also demonstrates that for the saccharide under investigation conformational states more than ca. 2 kcal/mol above the global minimum do not contribute significantly to motionally averaged (NOE) values and thus the cutoff energy of 10 kcal/mol (see above) chosen for the calculation of (NOE) values in Table 3 is a safe choice.

NMR Spectroscopic Experiments

Complete assignment of all ’H-NMR resonances (Table 1 a) was accomplished by the phase-sensitive H,H-COSY 19)

experiment. The assignment achieved is in accordance with the published values2’) for the chemical shifts of the anomeric protons in 10. A comparison of the chemical shifts in 10 with literature data for methyl a-D-mannopyranoside2’) re- veals downfield shifts greater than 0.1 ppm for 1’-H, 2’-H, 5’-H, 1-H, and 3-H (Table lb). The downfield shift of 0.26 ppm for 1’-H is readily explained by a short 1’-H - 3-0 distance across the glycosidic linkage. For the global minimum this distance was found to be 2.6 A, for the averaged distance a value of 2.9 8, is calculated. Both values are con- sistent with the experimental data. All other protons men- tioned did not exhibit close (< 3.0 A) oxygen contacts across the glycosidic linkage, neither for the global minimum conformation nor for performing motional averaging. More- over, for 2-H a close contact to 5’-0 was found (2.6 8, in the global minimum, 2.8 8, for motional averaging) which obviously did not result in any significant shift (Table lb). It has been proposed earlier*’) that the downfield shift of 1-H is attributable to a close 1-H - 5’-0 contact across the glycosidic linkage. This is not confirmed by the calculations, which give a value of 3.5 8, for this distance in the case of the global minimum and a value of 3.3 A when this distance is motionally averaged. These findings may be explained if one assumes that both the solvation shells and the preferred

Table la. ‘H-NMR chemical shifts in 6 values and couplin constants J in Hz (the latter in parentheses) for a-D-Man-OMeK21 and

10 in D?O at 296 and 303 K, ?$acetone) = 2.225

Compound 1-H 2-H 3-H 4-H 5-H 6-Ha 6-Hb O M

10 5.02 4.06 3.84 3.62 3.76 3.89 3.72 (1.8) (3.4) (9.6) (9.8) (12.0) (6.7)

(2.0)

4.99 3.95 3.86 3.68 3.60 3.90 3.76 3.40 (1.6) (3.4) (9.6) (9.8) (12.2) (6.1)

(2.4)

a-D-Man- 4.76 3.92 3.75 3.63 3.61 3.89 3.75 3.40 OM2 (1.6) (3.5) (10.0) (10.0) (12.0) (5.8)

(1.9)

Table 1 b. Differences in chemical shifts (6 values) between a-ii-Man- OMc and 10

1-H 2-H 3-H 4-H 5-H 6-Ha 6-Hb OM

-0.26 -0.14 -0.09 0.01 -0.15 0.00 0.03 -0.23 -0.03 -0.11 -0.05 -0.01 -0.01 -0.01 0.00

Liebigs Ann. Chem. 1991, 135-141

T. Peters 138

orientations of the hydroxyl functions of the two individual mannose residues, including the oxygen atom now involved in the glycosidic linkage, are significantly changed when the a-1,2-linked disaccharide 10 is formed.

Experimentally derived steady state NOE values are sum- marized in Table 2. The quality of the data obtained may be assessed from inspecting Figure 3, which depicts the difference spectra recorded. Additionally, a 2D-ROESY experiment2*) (data not shown) was performed to exclude dipolar interactions that could not be searched for by sep- arately irradiating proton signals and that would qualita- tively violate the theoretical model. In fact, no such ROE values could be detected. The quantitative analysis of NOE data was performed as discussed previously4! A comparison of relative NOE values resulting from the experimental data with theoretically derived results is given in Table 3, and in the following the individual steady state experiments will be discussed.

1'-H cy 21-x 1'-H

2-H

1-H w %r

5 4 6

Figure 3. NOE difference spectra for disaccharide 10

Table 2. NOE values observed at 310 (303) K in DzO; irradiation time was 5.0 s

Proton satlllilted NOE observed (%) (100%)

1'-H 2'-H 2-H 12.8 17.8

(11.9) (15.7)

2'-H 1'-H 3'-H 9.0 12.9

(9.9) (14.0)

1 -H 2-H 5'-H OMe 8.5 8.3 10.7

(7.4) (7.9) (10.4)

2-H 1'-H 1-H 3-H 20.2 8.3 11.4

(20.9) (9.6) (12.5)

Irradiation of 1'-H resulted in one interglycosidic NOE to 2-H and an intraglycosidic NOE to 2'-H. The assumption that the solution conformation of disaccharide 10 is exclu-

Table 3. Comparison of theoretically and experimentally derived relative NOE values; the experimental values are the mean of the

two measurements in Table 2

Proton NOE NOE <NOE> Sat. Obs. Experiment GlobalMin. <r%

1'-H 2'-H 0.42 0.55 0.49 0.50 0.45 0.51 0.50 2-H 0.58

0.43 0.40 0.40 2'-H 1'-H 0.41 0.57 0.60 0.60 3'-H 0.59

1-H 2-H 0.49 0.42 0.40 0.42 5'-H 0.51 0.58 0.60 0.58

2-H 1'-H 0.50 0.42 0.46 0.45 1-H 0.22 0.18 0.16 0.17 3-H 0.28 0.40 0.38 0.38

sively represented by the global minimum would lead to a false prediction of the relative magnitude of these two NOE values. Introduction of flexibility around the glycosidic linkage into the model approach by averaging over the conformational micro states significantly improved the situation, as has also been observed recently in the similar case of a Brucella A-type disaccharide4'. The relative ordering of the two NOE values was now correct. Comparing the NOE values observed upon irradiation of 1-H with the values calculated on the basis of the single state (global minimum) and the multi state conformational models lead to satisfactory correspondence in both cases. Obviously the interglycosidic NOE contact between 1-H and 5'-H was much less sensitive to motional averaging than the one between 1'-H and 2-H, although this first NOE is sensitive to the glycosidic linkage conformation. Of course, the NOE between 1- H and 5'-H is dependent on the orientation of the hydroxymethyl group in the nonreducing mannose unit. From the vicinal coupling constants J5,6 given in Table l a it could be concluded that the gg(o = -60") and g t ( o = 60") con- formers are equally populated in both the reducing and the nonreducing mannose units. The relative NOE values presented in Table 3 were derived from calculations where the hydroxymethyl groups were fixed to the gg orientation. Al- tering the conformation towards the gt orientation gave a less satisfying fit between experimental and theoretical data for both the single state and the multi state approach. A more detailed analysis for this NOE contact would have to apply an additional torsional potential function to the C-5 -C-6 linkages, and, in addition, averaging would have to include rotation of the hydroxymethyl groups around the C-5 -C-6 bonds'6b). Irradiation of 2-H lead to the obser- vation of two intra- and one interglycosidic NOE. Here again, an improvement between experiment and theory was achieved when switching to motional averaging. The relative NOE values to 1'-H and 3-H showed improvement whereas the value for 1-H was slightly too low. The latter effect may be explained by the fact that our model did not take into account fast rotation of 0-methyl groups, necessitating a different mathematical protocol for the calculation of NOE values involving relaxation via such fast rotating protons. Irradiation of 2'-H did not result in any interglycosidic NOE values. Nevertheless, a slight improvement was observed for


Synthesis and Conformation of Methyl 2-O-(ct-~-Mannopyranosyl)-ct-~-mannopyranoside 139

the intraglycosidic NOE values applying the multi state approach, as this is evident from the data given in Table 3. This is readily explained by the fact that relaxation of the observed protons involves dipolar contacts across the glycosidic linkage, which in turn influences the size of these NOE values. In this particular case, the dipolar interaction between 1'-H and 2-H, which has been shown above to be very sensitive to motional averaging, may contribute most to the observed effect. In all cases discussed the NOE values derived from motional averaging were essentially independent of whether ( r P 6 ) or ( rP3) ' values were used for setting up the relaxation matrix (compare Table 3).

Further evidence for the importance of motional averaging emerged from the comparison of experimental and theoretical TI values. The values given in Table 4a were derived from a non selective inversion recovery experiment. Relative T, values are compared in Table 4b. It is obvious that in all cases the motional averaging approach improved the fit between experimentally and theoretically derived results. Again, the effect of switching between ( r P 6 ) and ( rP3) ' values was of minor importance for the calculation of ( T , ) values.

Table 4a. Observed Tl values at 300 K in DzO

1'-H 2 ' - H 1-H 2-H

ObservedT1 1.4 2 .1 1.3 1.0 [s 1

Table 4b. Comparison of experimentally derived and theoretically calculated relative Tl values

Relative T1 2'-H/l'-H l-H/l'-H 2-H/1'-H

Experiment 1.5 0.7 0.9 GlobalMin. 1.0 0.6 0.6 <r-6> 1.3 0.7 0.7 <,-3>2 1 .2 0.7 0.7

Understanding the processes of carbohydrate protein-receptor interactions requires profound knowledge of the conformational behaviour of the oligosaccharide chains in aqueous solution"). Analysis of the solution conformation of oligosaccharides mainly focuses on the glycosidic linkage conformation as the pyranose rings can be considered as

The NMR data obtained for disaccharide 10 indicated that the a-1,2 glycosidic linkage exhibits conformational flexibility that was simulated utilizing a dynamic model approach4%*) based on statistical mechanics and the HSEA force field. In particular, it was found that interglycosidic NOE values were well-interpreted within this motional averaging model, which encourages more investigation on similar cases in the future. Upon binding of carbohydrate chains by protein receptors such as monoclonal antibodies the global minimum conformation or ones close to it may be frozen out and thus entropy is lost, as this is known from binding studies23). It is evident that the understanding of the thermodynamics and mechanisms of such

binding processes will improve with a more detailed knowledge of the dynamic behaviour of oligosaccharides in aqueous solution. As a final conclusion from the experiments performed, a model for the tetrasaccharide containing the a-1,2 linkages of the polysaccharide backbone of Saccharo- myces cerevisiae is presented in Figure 4, A representing the global minimum and B modelling conformational states allowed within the 2 kcal/mol limit of Figure 1. The different conformations in B were chosen at random with the reducing unit kept fixed.

A

a-D -Man-(1 +2)-a-D-%-( 1 +2)-a-D-Man-(l +Z)-a-D-Man

Figure 4. A: global minimum of 10, and B conformations of 10 within the 2 kcal/mol boundary of Figure 1 with the reducing man-

nose residue kept fixed

I thank the Deutsche Forschungsgmeinschaft for a grant within the Sonderforschungsbereich 169 at the University of Frankfurt and the Fonds der Chemischen Industrie for a Liebig stipend. Stimulating discussions with Dr. J.-R. Brisson from the National Research Council Canada have been made possible on the basis of a NATO research grant CRG.890356. I wish to thank Prof. Dr. H . Riiterjans, Institute of Biophysical Chemistry, University of Frankfurt, for ac- cess to the 500-MHz spectrometer and the computing facilities, and Mrs. U. Bergmann for valuable technical assistance.

Experimental Computational Methods: All calculations were performed on a

micro-VAX and a CONVEX C1. Energies of conformational states were calculated utilizing a modified version4) of the GESA pro- gram3b,'4) that applies the HSEA force field '). Motional averaging was performed by sampling the conformational space spanned by the dihedral angles 4 and + in 5" steps starting with the global minimum conformation and assigning a statistical weight exp(-AE,/RT), with A& = energy difference between micro state i and the global minimum, R = gas constant, and T = 310 K, to each conformational micro state i. The statistical weights were normalized and both distances r, and as r F 6 and r;3 values were multiplied with these normalized Boltzmann factors to give motionally averaged parameters (I), ( r - 6 ) , and ( r - 3 ) , as this has been described before4). Conformational states more than 10 kcal/mol above the global minimum were not considered for the averaging. NOE and TI values for the global minimum were calculated according to the procedure given by Heatley et al. "I, assuming dipole-


140 T. Peters

dipole relaxation to be the dominant relaxation mechanism. Mo- tionally averaged (NOE) values were also calculated on the basis of this full relaxation matrix approach with the relaxation matrices based either on (r-6) or ( r - 3 ) 2 values, and the same holds for the calculation of (TI) values. Motional correlation times 5, for the calculation of NOE values, (NOE), TI, and ( Tl) values were obtained by fitting the calculated (NOE) between 1'-H and 2'-H to the experimentally observed values in Table 2. A value for T~ of 1.6 x lo-'' s gave satisfactory correspondence. Isotropic reorientation of disaccharide 10 was assumed. For graphical representation of molecular geometry the program SCHAKALZ5) was used.

N M R Experiments: Disaccharide 10 was dissolved twice in 99.96% D20 (Aldrich) and the solvent evaporated, before dissolving it in 99.996% D20 (Aldrich). The sample was degassed by repeated freeze-pump-thaw cycles and was sealed under argon. The concen- tration of the sample was 15 mg/0.5 ml (84 mM). All experiments were performed on a Bruker AM 500 MHz spectrometer. The coupling constants and chemical shifts reported are first order values. Acetone (l%, v/v) was used as internal reference (6, = 2.225).

NOE Difference Spectroscopy and ' H Spin Lattice Relaxation Time Measurements: NOE experiments were performed in the difference mode26J under steady state conditions. Saturation of signals was accomplished by single line irradiation2". 'H spin lattice relaxation times TI were measured by the nonselective inversion recovery techniquez8' utilizing a composite 180" pulse29J. Calculation of TI values was achieved by applying standard Bruker software.

Two Dimensional Spectroscopy: Phase-sensitive homonuclear COSY spectra were obtained with the TPPI methodi9) with 512 increments in tl and 2K data points in t2. The spectral width was 3 ppm in both dimensions. The data matrix was multiplied by sine bell window functions and Fourier transformed to give a 2K x 2K spectral matrix. 2D-ROESY experiments **) were performed in the phase-sensitive mode, also applying the TPPI procedure. A pulsed spin lock field utilizing equally spaced 30" pulses3') was applied, resulting in a total spin lock time of 150 ms and a field strength Bl of 2500 kHz. The time domain consisted of 512 data points in t l and 2K data points in t2. Prior to Fourier transformation a n/3 shifted squared sine bell window function was applied in both dimensions to finally yield a 2K x 2K spectral matrix.

Synthesis: All reactions were monitored by TLC on silica gel- coated aluminium foil (Merck, silica gel 60 F254). Detection was achieved by UV absorption and/or charring with 5% H2S04 in ethanol. Optical rotations were determined on a Perkin-Elmer 243 polarimeter. Preparative scale silica gel separations were performed on silica gel 60 (230-400 mesh, Merck) applying flash chromatography. Reactions performed with dried solvents were conducted under dry nitrogen. Glycosylations were performed in the dark. The 'H- and ',C-NMR spectra were recorded with Bruker AM 270 and AM 500 spectrometers. Tetrametbylsilane was used as internal reference for solutions in organic solvents. Chemical shifts and coupling constants are first order values.

1,2-Di-O-acetyl-3,4,6-tri-O-benzyl-cc-~-mannopyranose (6): 2.0 g (4.0 mmol) orthoester 2 was dissolved in 40 ml of 60% acetic acid and kept at 70°C until no more 2 could be detected (TLC: hexane/ ethyl acetate, 3: 1 , v/v). The mixture was concentrated to a syrup and taken up in 40 ml of pyridine/acetic anhydride (2: 1, v/v). The solution was kept at 40°C until conversion into 6 was complete (TLC: hexane/ethyl acetate, 3: 1, v/v). 10 ml of water were added, and stirring was continued for another 0.5 h. Solvents were removed in vacuo and the resulting syrup was purified on a silica gel column (hexane/ethyl acetate, 6: 1, v/v) to give 6 as a syrup; yield 890 mg (42%), 26 = f37.0 (C = 1.28, CHC13). - 'H NMR (270 MHz,

CDCI,): 6 = 2.07, 2.18 (2 S, 6H, 2 OAc), 3.69 (dd, J5,6a = 1.8 Hz, J6a,6b = 11.0 Hz, l H , 6-H,), 3.77-3.92 (m, 2H, 5-H, 6-H& 3.94-4.04 (m, 2H, 3-H, 4-H), 4.47-4.90 (m, 6H, 3 CH,Ph), 5.36

7.10-7.40 (m, 15H, 3 Ph). (dd, J1,2 = 1.8 Hz, Jl,, = 3.2 Hz, IH, 2-H), 6.12 (d, IH, 1-H),

C31H3408 (534.6) Calcd. C 69.65 H 6.41 Found C 69.56 H 6.30

Methyl glycoside 4 was isolated as a byproduct; yield 680 mg (32%), [a]g = +27.5 (c = 1.3, CHCl,) (ref.9) [a]: = +27.9 (c = 2.24,

Ethyl 2-0-Acetyl-3,4,6-tri-O-benzyl-l-thio-~-~-mannopyra~~~side (7). - a) From Orthoester 2 3.0 g (5.9 mmol) of orthoester 2 was dissolved in 50 ml of dry dichloromethane. 0.3 g of powdered 4-A molecular sieves, 30 ml(l35.0 mmol) of ethanethiol, and 0.3 ml(2.4 mmol) of Et,O-BF, were added, and the solution was stirred at room temperature until conversion into 7 was complete (TLC: hexane/ethyl acetate, 3 : 1, v/v). Excess boron trifluoride was neutralized by adding 1 ml (7.1 mmol) of triethylamine. The reaction mixture was filtered, the solvents removed in vacuo, and the crude reaction product was purified by column chromatography (hexane/ethyl acetate, 6: 1, v/v) to yield 2.1 g (66%) of amorphous 7. - Methyl glycoside 4 was isolated as a byproduct; yield 0.6 g (23%), = +27.5 (c = 1.30, CHC1,) (ref.9) [a]:= -1-27.9 (c = 2.24,CHC13)).

b) From Diacetate 6: 2.0 g (3.7 mmol) of 6 was dissolved in 40 ml of dry dichloromethane. 0.2 g of powdered 4-A molecular sieves, 1 ml (13.5 mmol) of ethanethiol, and 1 ml (8.1 mmol) of Et,O-BF, were added, and the solution was stirred at room temperature until complete conversion into 7 (TLC: hexane/ethyl acetate, 3: 1, v/v). Excess boron trifluoride was neutralized by adding 2 ml (14.3 mmol) of triethylamine. Purification as described above furnished amorphous 7; yield 1.8 g (89%), [ c L ] ~ = +73.55 (c = 1.0, CHCI,). - 'H NMR (270 MHz, CDC13): 6 = 1.27 (q, 'JVic = 8.4 Hz, 3H, SCH,CH,), 2.13 (s, 3H, OAc), 2.61 (m, 2H, SCHzCH,), 3.67 (dd,

1 H, 6-Hb), 3.84-3.97 (m, 2H, 3-H, 4-H), 4.15 (ddd, 1 H, 5-H),

4.84 (d, 'Jpem = 12.0 Hz, IH, CHHPh), 5.30 (d, J1.2 = 1.6 Hz, 1 H, 1-H), 5.42 (dd, J,,, = 3.2 Hz, 1 H, 2-H), 7.08-7.38 (m, 15H, 3 Ph).

CHCI?)).

J5,6a = 2.4 HZ, J6a,6b = 12.6 HZ, 1 H, 6-H,), 3.82 (dd, J5,6b = 4.8 HZ,

4.43-4.54 (3 d, 3H, 3 CHHPh), 4.63-4.70 (2 d, 2H, 2 CHHPh),

C31H3606S (536.7) Calcd. C 69.38 H 6.76 Found C 69.45 H 6.41

Methyl 2-~-(2-0-Acety/-3,4,6-tri-O-benzy~-cc-~-mannopyran~- sy/)-3,4,6-tri-O-benzy/-a-o-mannopyranoside (8). - a) Reaction of Bromide 3 with Acceptor 5: 1.0 g (2.0 mmol) of freshly prepared 2 was dissolved in 10 ml of a saturated solution of HBr in ether. The solution was kept at room temperature until no more 2 could be detected (TLC: hexane/ethyl acetate, 1 : 1, v/v). The solvent was removed in vacuo, and the syrup was codistilled twice with dry toluene. The resulting bromide was dissolved in 20 ml of dry dichloromethane and was used for glycosylation without further purification. Alternatively, bromide 3 was prepared by dissolving 1.0 g (1.9 mmol) of thioglycoside 7 in 10 ml of dry dichloromethane and adding 100 pI(1.9 mmol) of bromine. The mixture was kept at room temperature until conversion into bromide 3 was complete (TLC: hexane/ethyl acetate, 3: 1, v/v). After removal of dichloromethane in vacuo the syrup was codistilled twice with dry toluene and was dissolved in 20 ml of dry dichloromethane. A solution of 700 mg (1.5 mmol) of acceptor 5,600 mg of powdered 4-A molecular sieves and 600 mg (2.4 mmol) of silver triflate in 20 ml of dry dichloromethane under nitrogen was stirred at -78°C for 0.5 h. The bromide solution was slowly dropped into the mixture and the temperature was kept at -78°C for 1 h. After warming to room temperature within ca. 12 h the mixture was filtered through Celite,


Synthesis and Conformation of Methyl 2-0-(a-D-Mannopyranosy~)-a-~-mannopyranoside 141

washed with saturated sodium hydrogen carbonate solution. and ’) J. Montreuil. Adv. Carbohvdr. Chem. Biochem. 37 (1980) 157. - - concentrated to a syrup. Column chromatography (hexaneJethyl acetate, 3: 1, v/v) gave 1.0 g (70%) of pure 8 as a syrup.

b) In situ Glycosylation: A solution of 1.0 g (1.9 mmol) of thioglycoside 7, 700 mg (1.5 mmol) of acceptor 5, 600 mg of powdered 4- A molecular sieves, and 600 mg (2.4 mmol) of silver triflate were stirred in 30 ml of dry dichloromethane at -78°C for 1 h. 93 p1 (1.8 mmol) of bromine was added to the reaction mixture, stirring at this temperature was continued for 1 h, and then the mixture was allowed to warm up to room temperature within ca. 12 h. Neutralization was achieved by addition of 1 ml (7.2 mmol) of triethylamine. Workup as described above gave pure 8 as a syrup; yield

MHz,CDC13):6 = 2.12(s,3H,0Ac),3.27(s,3H,0CH3),3.66-3.76 (m, 4H, 6’-H,, 5-H, 6-Ha, 6-H& 3.78 (dd, JY.6.b = 4.7 Hz, J6.a,6,b =

1.04 g (73%), [a]:: = f21.1 (C = 2.25, CHC13). - ‘H NMR (500

11.4 Hz, 1 H, 6-H,), 3.83 (dd, J3,4 = 10.0 Hz, J4 ,5 = 10.0 Hz, 1 H, 4-H), 3.84 (dd, J3,,4, = 10.0 Hz, J4.,5. = 10.0 Hz, 1 H, 4’-H), 3.90 (dd, J2,3 = 3.3 Hz, l H , 3-H), 3.96 (ddd, J5.,6a, = 2.0 Hz, l H , 5’-H), 3.99 (dd, Jy,3. = 3.3 Hz, l H , 3-H’), 4.01 (dd, Jl,2 = 1.8 Hz, IH, 2-H), 4.38-4.69 (m, IOH, 5 CHHPh), 4.65 (d, IH, 1-H), 4.78 (d, J1,,z = 1.9 Hz, 1 H, 3’-H), 4.84,4.86 (2 d, ’Jgem = 11.0 Hz, ’J&,, = 11.0 Hz, lH, 2 CHHPh), 5.54 (dd, lH, 2’-H), 7.12-7.43 (m, 30H, 6 Ph) (ref.5) 2.08 (s, 3H, OAc), 3.23 (OCH3), 5.08 (d, J = 1.5 Hz, 1 H, lb- H), 5.55 (br. s, 1 H, 2b-H)).

C57H62012 (939.1) Calcd. c 72.90 H 6.65 Found C 73.23 H 6.49

Methyl 2-0-(a-D-Mannopyranosy~)-a-D-mannopyranoside (10): 500 mg (0.5 mmol) of disaccharide 8 was dissolved in 5 ml of dry methanol and 0.5 ml of a 3% solution of sodium methoxide in methanol. The solution was kept at room temperature until deacetylation was complete (TLC: hexaneJethy1 acetate, 2: 1, v/v). It was neutralized with ion-exchange resin IR 120 H+, filtered and evaporated to yield 9 as a syrup {[a1202 = +31.7 (c = 1.42, CHC13) (ref.’) [WID = +31.3 (c = 0.52, CHCI,)), the ‘H-NMR spectrum was in accordance with the assumed structure}. Alcohol 9 was dissolved in 10 ml of dry methanol and hydrogenated over 300 mg of 5% palladium on charcoal at atmospheric pressure and room temperature. Conversion into the product was complete within 5 -6 h (TLC: chloroform/methanol/water, 5 : 4: 1, v/v/v). The reaction mixture was filtered and concentrated, and the crude product was purified on a Biogel P2 column to give pure 10 as a syrup; yield 172 mg

methanol) (ref.5) [a]D = +72.0 (c = 0.49, methanol)). - ‘H NMR: see Table 2 (ref.5) 3.36 (s, 3H, OCH,), 4.94 (s, IH, la-H),

(94%), [a]:: = +67.6 (C = 2.40, H,O), [a]g = +74.4 (C = 1.40,

4.97 (d, J = 1 Hz, I H , lb-H). C13H24011 (346.2) Calcd. C 45.10 H 4.08

Found C 44.78 H 4.08

CAS Registry Numbers

2: 16697-49-7 6: 68567-54-4 / 7: 130525-72-3 1 8 : 80596-05-0 J 9: 80585-62-2 J 10: 59571-75-4

3: 93381-69-2 J 4: 20672-69-9 / 5: 20672-61-7

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[ 1661901

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synthesis and conformational analysis of methyl 2-o-(α-d-mannopyranosyl)-α-d-mannopyranoside

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