improved synthesis of α-l-fuc(1→4)-β-d-glcnac and α-l-fuc(1→6)-β-d-glcnac building blocks: a...

T. Peters, T. Weimar 237

Improved Synthesis of a-L-Fuc( 1-+4)-P-D-GlcNAc and a-L-Fuc( 1-+6)-P-D- GlcNAc Building Blocks: A Convergent Strategy Employing 4-0-6-0 Acetyl Migration; NOE Data of the Protected a-1,4-Linked Disaccharide Thomas Peters" and Thomas Weimar

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

Received October 19. 1990

Key Words: Oligosaccharide synthesis / Thioglycosides / Acetyl migration / Glycosidic linkage conformation 1 Carbohydrates

The synthesis of the two ethylthio disaccharide building blocks 10 and l? was achieved by coupling of fucosyl bromide 9 with the acceptor alcohols 6 and 7 under in situ anomerization conditions. The selectively protected 2-deoxy-2-phthalimidoglucose derivative ? was derived from 6 by utilizing an optimized acetyl migration reaction. The ethylthio function in disaccharides 10 and 17 was activated with bromine, and excess bromine was removed with cyclohexene. The sensitive

l,g-linkage in the disaccharide 10 proved to be stable under these activating conditions. The disaccharide bromides 11 and 18 were treated with methanol to afford after deblocking the methyl glycosides 16 and 23. Homonuclear 'H-NOE data were obtained for the protected 1,4-linked disaccharide 17 suggesting that its preferred solution conformation is rather similar to the solution conformation of the deblocked disaccharide 23 in aqueous solution as known from literature data.

In the course of a project dealing with the conformational analysis of carbohydrate chains containing flexible glycosidic linkages we started to synthesize selected oligosaccharide fragments'). It is of considerable interest how carbohydrate chains behave conformationally when being in- corporated into membranes2), and answers to the question of whether they alter their conformational preferences whilst in the two environments3) would be of importance for a better understanding of biological recognition phenomena such as cell-cell differentiation and adhesion4), antigenicity of bacterial polysaccharides 'I, and malignant transforma- tions of cells6). The two disaccharides ct-~-Fuc(l-+6)-P-~- GlcNAc 16 and ct-~-Fuc(l-+4)-P-~-GlcNAc 23, the latter a dissacharide component of the Lewis blood-group system'), the former a ubiquitosyl occurring structural motif in N- linked glycoproteins *I, were studied for the following rea- sons: the ct- l ,Clinked disaccharide provides a glycosidic

Scheme 1

0 ~ 3 X I

R20 R'O R4 H3C & 0 ~ ~ 1

NPhth

1-7

I dBzI BzlO

8 X=SEt 9 X = B r I R1 R2 R3 R4

Ac Ac Ac OAc Ac Ac Ac SEt H H H SEt H H Trt SEt Ac Ac Trt SEt Ac Ac H SEt Ac H Ac SEt

R'O R'O I OR' I OR'

I

NR' R 2 0 R20 & R4 17 - 23 NR3

I R' R2 R3 R4 10 - 16

10,17 11,18 12,19 13,20 14,21 15,22 16.23

Phth = phthaloyl

Bzl Ac Phth SEt Bzl Ac Phth Br Bzl Ac Phth OMe Bzl H Phth OMe Bzl Ac Ac, H OMe Bzl H A c , H OMe H H Ac,H OMe

R4

linkage with a single energy minimum'), whereas ct-1,6-linkages in general exhibit several energetically accessible local minima"). This opens the possibility that conformational rigidity vs. flexibility may be investigated for two structur- ally related saccharides. In order to allow coupling of the disaccharides to various carrier molecules to give glycocon- jugates that can be used for building artificial membranes or neoglycoproteins we designed the synthesis of suitable disaccharide glycosyl donors. Thioglycoside methods ' I ) of- fered the most attractive route to achieve this goal, and in this paper we report on the synthesis of the two thioglycoside building blocks 10 and 17 and their transformation into the methyl glycosides 16 and 23.

Liebigs Ann. Chem. 1991, 237-242 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1991 0170-2O41/91/0303 -0237 $ 3.50-t .25/0

T. Peters, T. Weimar 238

Synthesis Syntheses of oligosaccharides containing the sequences

a-~-Fuc(1+6)-~-~-GlcNAc’*) and a-L-Fuc(l-+4)-P-~- GlcNAc”) have been reported in the literat~re’’,’~’. In order to prepare the a-1,4- and a-1,6-linked building blocks 10 and 17 from a common aglycon precursor, we first synthe- sized the glycosyl acceptor 6 with free 6-OH group. Com- pound 6 subsequently could be converted via 4-0+6-0 acetyl migrationI4’ into the acceptor 7 with free 4-OH group. The synthesis of 6 was achieved according to literature procedures: 1,3,4,6-tetra-O-acetyl-2-deoxy-2-phthalimido-~-~- gluc~pyranose’~) (1) was converted into the S-ethyl glycoside 2 in %0/0 yield by using the boron trifluoride catalyzed gen- eration of the acetoxonium ion and in situ reaction with ethanethiolI6). We found that the use of boron trifluoride as Lewis acid is more convenient and furnished higher yields than the use of titanium tetrachloride”). Transesterification of 2 with sodium methoxide in methanol to 3 proceeded with almost quantitative yield. Subsequent tritylation, acetylation, and detritylation furnished the glycosyl acceptor 6 in 50 - 60% overall yield. The detriylation step needs care because fast 4-0+6-0 acetyl migration may occur if traces of pyridine are present 14’. Exposure of alcohol 6 to aqueous acetic acid should be limited to the time required for complete detritylation. NMR control experiments revealed that 75% acetic acid caused slow 4-0+6-0 acetyl migration (Table 1) and thus traces (up to 5 -loo/,) of the acceptor 7 with free 4-OH group were also isolated after the detritylation step. As seen from Table 1, pure pyridine does not induce any acetyl migration at all, which allows the conclusion that the presence of water is essential for the rearrangement reaction to take place. The acceptor 6 with free 6-OH group was either purified on a silica gel column or, in this particular case, where both disaccharides, the a-1,4- and the ct-1,6-linked, were desired, the crude reaction product could be used for the following glycosylation reaction because separation of the resulting a-1,4- and a-1,6-linked disaccharides 10 and 17 posed no problem.

Table 1. Amount of 4-0-6-0 acetyl migration in different solvent systems (in %), the data were obtained from ‘H-NMR experiments; 75% [D,Jacetic acid refers to a 75% solution in D20, solvent A: 75% [D,Jacetic acid/[DJpyridine, 45: 7, v/v, solvent B: 75%

[D,Jacetic acid, solvent C: [DJpyridine

Solvent system Time interval [d] 3 5 8 10 13

A 12 29 44 50 55 B n.d. 9 11 14 14 C 0 0 0 n.d. n.d.

The alcohol 7 was prepared from 6 according to a protocol utilizing pyridine/water mixtures 14‘) to achieve efficient 4-0+6-0 acetyl migration. It was rep~rted’~‘’ that although the reaction is faster in alcohol/alkaline hydroxide mixtures pyridine/water is superior because no transesterification was observed as a side reaction. We monitored the conversion of 6 into 7 utilizing ‘H-NMR spectroscopy in order to find

the optimum reaction conditions. The signals for the methyl groups of the 0-acetyl groups in compounds 6 and 7 served as reporter groups to be integrated after fixed time intervals. Half-live times for the rearrangement of 6 in different pyridine/water mixtures are compiled in Table 2. As the half- life time was not found to be independent of the initial concentration (Table 2) the rearrangement cannot be inter- preted in terms of a first-order kinetics. Nevertheless, it is evident that acetyl migration is significantly accelerated by increasing the amount of water in the solvent system. As a general conclusion we can assume that the best results are achieved with these types of rearrangements when the water content is as high as the solubility allows. Transferring our results to preparative-scale experiments using pyridine/water (1 : 3, v/v), we obtained an equilibrium mixture of 95% aglycon 7 and 5% starting compound 6, which was not purified further prior to subsequent glycosylation.

Table 2. 4-0-6-0 acetyl migration rate on the basis of ‘H-NMR measurements at 300 K (see experimental part); p = [DJpyridine, w = D20. T ~ , ~ and qI4 are the time intervals after which the initial concentration has dropped to a half or a quarter of its initial value, respectively. The initial concentration was 10 mg/0.5 ml. For a discussion of the data in the text the kinetic isotope effect was

neglected

p:w (v/v)

26230 74820 3: 1 1:l 542 1453 1 :3 180 45 1

The glycosylation reactions were performed under in situ anomerization conditions lZJ by using the perbenzylated fu- cosy1 bromide 919) as glycosyl donor, which in turn origi- nated from treatment of the corresponding S-ethyl glycoside 8 with bromine in dichloromethane”). It proved advanta- geous not to isolate the bromide 9 but rather to remove excess bromine with cyclohexene*”, and to use the resultant bromide solution directly for glycosylation. The yields for the coupling reactions to give the c(-1,6- and the a-lA-linked disaccharides 10 and 17 were in the range 60-80%. If a mixture of the two aglycons 6 and 7 was used, both disaccharides 10 and 17 were formed. Separation was easily achieved by chromatography on silica gel.

Both building blocks 10 and 17 were designed to be linked to different carrier molecules. Here, we would like to de- scribe their activation for use as disaccharide building blocks and the formation of the corresponding methyl glycosides 12 and 19. It is well-known that fucosyl 1,6-linkages as found in 10 are acid-sensitive”), especially in the presence of benzyl ether-protecting groups, and thus activation of the ethylthio function had to proceed under mild nonacidic conditions. Isolation of the corresponding bromide 11 after treatment of the thioglycoside 10 with bromine failed and led to cleav- age of the a-1,6-linkage instead, resulting in a 1,2-elimina- tion product of the phthalimidoglucose unit and a fucose derivative with free 1-OH group. It turned out that a stable

Liebigs Ann. Chem. 1991, 237-242

a-~-Fuc( l~4)-P-~-GlcNAc and a-~-Fuc( 1+6)-P-~-GlcNAc Building Blocks 239

disaccharide bromide solution could be obtained by again removing excess bromine with cyclohexene*’). The freshly prepared bromide solution was treated with methanol as acceptor alcohol in the presence of silver triflate as catalyst and powdered 10-A molecular sieves to trap hydrogen bromide **) formed during glycosylation. The corresponding methyl glycoside 12 was obtained in approx. 60% yield. The same experimental protocol was used to prepare the a-1,4- linked methyl glycoside 19 in comparable yields. Deblocking of both saccharides, 12 and 19, proceeded via transesterification with sodium methanolate in methanol, hydrazinolysis with hydrazine, and catalytic hydrogenation with 5% palladium on charcoal according to literature-based procedures in good overall yield.

NOE Experiments

Finally, we would like to report on NOE effects observed with the ~-1P-linked protected disaccharide 17. The conformation of the corresponding deblocked disaccharide 23 has been investigated by Lemieux et al. 9), utilizing NMR spectroscopy and HSEA (Hard Sphere Exo Anomeric) calculations. The obtained data suggested that for this particular disaccharide the flexibility around the glycosidic linkage is rather restricted, and the minimum energy conformation derived from simple HSEA calculations with dihedral angles @ (1-H-C-1-4-0-C-4) = 55” and Y(C-l-l-O-C-4- 4-H) = 25” at the glycosidic bond was found to provide a molecular model in accordance with NMR experimental results on the solution conformation of this dissacharide. These findings are in good aggreement with our experimental data obtained for the protected disaccharide 17 in CDC13 as solvent. NOE experiments showed strong dipolar inter- actions between 1’-H and 4-H (NOESY experiment, data not shown) and between 3-H and 5’-H as seen from the one- dimensional NOE difference spectrum (Figure 1). Since the chemical shifts of 5’-H and 5-H are rather similar, Figure 1 also shows the NOE difference spectra obtained upon irradiation on 1-H and 6’-CH3 which allow both of the pro-

D

C

5‘-H B

5.0 4.0 3.0 2.0 1.0 6

Figure 1. NOE difference spectra for the disaccharide 17 in CDCI, at 270 MHz; trace A showing the normal ‘H-NMR spectrum; trace C shows the NOE enhancement of 5’-H upon irradiation on 3-H; traces B and D represent irradiations on 6’-CH3 and 1-H, respec-

tively

tons in trace A of Figure 1, 5’-H and 5-H to be identified unequivocally. The NOE contact observed between 3-H and 5’-H greatly restricts the conformations possible at the glycosidic linkage. Model building using the programs GESA”) and INSIGHT24) showed that the solution conformation of the protected disaccharide 17 in CDC13 is rather similar to the solution conformation of the corresponding free disaccharide as known from the literature’). As a conclusion, the protective groups present in disaccharide 17 do not significantly alter the glycosidic linkage conformation, thus em- phasizing the importance of the exo-anomeric effect 25) on the conformational behavior of oligosaccharides. This is in accordance with recent ab initio calculations performed for several dialkoxymethane derivatives suggesting that values for the energetic contribution of the exo-anomeric effect have been underestimated in earlier work26).

We wish to thank Prof. Dr. H . Riiterjans, Institute of Biophysical Chemistry, University of Frankfurt, for access to Bruker AM 270 and AM 500 spectrometers and to the computing facilities. T. P. thanks the Fonds der Chemischen Industrie for a stipend. This work was supported by a grant from the Deutsche Forschungsgemein- schaft and is also part of a project partially financed by a NATO research grant CRG 890356. Mrs. U. Bergmann is thanked for skil- full technical assistance.

Experimental Genera[ Procedures: 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. - Melting points are uncorrected. - Preparative scale silica gel separations were performed on silica gel 60 (230 - 400 mesh, Merck) applying flash chromatography. No more than 1 g of compound was loaded per 50 g of adsorbent. - Reactions performed with dried solvents were conducted under dry nitrogen. Glycosylations were performed in the dark. - The ‘H- and I3C- NMR spectra were recorded with Bruker AM 270 and AM 500 spectrometers. When necessary, assignments were made on the basis of routine homo- and heteronuclear shift correlation experiments*”. Solutions in organic solvents were referenced to internal tetrame- thylsilane and solutions in D 2 0 to internal acetone (6 , = 2.225). Chemical shifts and coupling constants are first-order values. 1D NOE experiments were performed in the difference mode2*) under steady-state conditions. Saturation of signals was accomplished by single-line irradiationz9). Phase-sensitive 2D NOE 30) spectra were recorded by application of the TPPI method3’) with 512 increments in t , and 2K data points in t2. The data matrix was multiplied by a x/3-shifted sine-bell function in both dimensions and Fourier- transformed to give a 2K x 2K spectral matrix.

Ethyl 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-l-thio-~-~-glucopyranoside (2): 20.0 g (42 mmol) of the peracetylated glucopyranose derivative 1 was dissolved in 400 ml of dry dichloromethane. After adding 1.5 g of powdered 4-A molecular sieves, 7.9 ml (105 mmol) of ethanethiol, and 7.9 ml(63 mmol) of Et20 -BF3 the solution was stirred at room temp. until complete conversion of 1 into product 2 (TLC: hexane/ethyl acetate, 3: 1, v/v). Dropwise addition of 9.0 ml (67 mmol) of triethylamine, filtration, and removal of the solvents in vacuo afforded the crude reaction product, which was purified by gel filtration over silica gel (hexane/ethyl acetate, 1 : 1, v/v) to give the crystalline product; yield 19.05 g (95%), m.p. 110-113°C


240 T. Peters, T. Weimar

(ref.") 118-119"C), [a]E = +49.5 (c = 1.7, CHCI3) (ref.17'

SCH2CH3), 1.85, 2.00, 2.09 (3 s, 9H, 3 OAc), 2.67 (m, 2H, [a]? = +44). - 'H NMR (270 MHz, CDC13): 6 = 1.20 (dd, 3H,

SCH,CH3), 3.88 (ddd, J5,6a = 2.2 HZ, J5,6b = 5.0 Hz, J4.5 = 10.2 HZ, 1 H, 5-H), 4.15 (dd, J61,6b = 12.4 HZ, 6-Ha), 4.28 (dd, 1 H, 6-Hb), 4.36 (dd, J1,2 = 10.3 Hz, IH, 2-H), 5.15 (dd, J3,4 = 9.2 Hz, IH, 4-H), 5.46 (d, ZH, I-H), 5.83 (dd, l H , 3-H), 7.88-7.65

10.4 Hz, J2.3 =

(m, 4H, Pht) (ref.") 'H NMR (100 MHz, CDCI3): 6 = 5.48 (d, 1 H, 10 Hz, H-1)).

CzzH25N09S (479.5) Calcd. C 55.11 H 5.26 N 2.92 Found C 55.09 H 5.12 N 2.95

Ethyl 3,4-Di-O-acetyl-2-deoxy-2-phthalimido-i-thio-6-0-trityl-~- o-glucopyranoside (5): 34.0 g (70.1 mmol) of thioglycoside 2 was dissolved in 1 1 of dry methanol. After addition of 15 ml of a solution of 3% sodium methanolate in dry methanol the solution was stirred at room temp. until removal of the acetates was complete (TLC: hexane/ethanol, 1 : 2, v/v). The reaction mixture was neutralized with IR 120 H + ion-exchange resin, filtered, and the solvent was removed in vacuo to give 24.6 g (quant. yield) of alcohol 3 as a syrup; [a]E = + 9.4 (c = 0.4, CHCI3). Dissolution of 3 in 700 ml of dry pyridine, addition of 50 g (186 mmol) of trityl chloride furnished the dialcohol 4 after stirring at 50°C for 10 h (TLC: hexanei ethanol, 1 : 1, v/v). After filtration and removal of the solvents in vacuo the crude reaction product was purified by column chromatography (hexane/ethyl acetate, 1 : 1, v/v; after unreacted trityl chloride was removed: hexane/ethanol, 1 : 1, v/v). The solvents were evaporated and the resulting dialcohol 4 was dissolved in 375 ml of pyridine/acetic anhydride (2: 1, v/v). The solution was kept at room temp. until acetylation of the free hydroxyl functions was complete (TLC: hexane/ethyl acetate, 1 : 1, v/v). 200 ml of water was carefully added and the solution was stirred for 30 min. After removal of the solvents in vacuo the resulting syrup was dissolved in 500 ml of dichloromethane and washed with 15% aqueous KHS04 solution, saturated aqueous NaHC03 solution, and half saturated aqueous NaCl solution (300 ml each). The dichloromethane solution was dried with Na2S04 and, after rotary evaporation, 41.8 g (88%) of compound 5 was isolated as an amorphous product; [a]E = +62.1 (c = 0.8, CHC13). - 'H NMR (270 MHz, CDCI3): 6 = 1.33 (dd, 3H, SCH2CH3), 1.73, 1.85 (2 S, 6H, 2 OAC), 2.63 to 2.90 (m, 2H, SCHZCH~), 3.15 (dd, Jsa,6b = 8.5 HZ, J5,6a = 5 HZ, 1 H, 6-H,), 3.3 (dd, J5,6b = 2.0 Hz, IH, 6-Hb), 3.78 (ddd, J4 ,5 = 10.2 Hz, IH, 5-H), 4.45 (dd, J1,2 = 9.0 Hz, J2.3 = 10.0 Hz, IH, 2-H), 5.23 (dd, J3.4 = 9.2 Hz, 1 H, 4-H), 5.50 (d, 1 H, 1-H), 5.79 (dd, 1 H, 3-H), 7.20-7.60 (m, 15H, 3 Ph), 7.65-7.95 (m, 4H, Phth).

C39H37NOsS (679.8) Calcd. C 68.91 H 5.49 N 2.06 Found C 68.69 H 5.44 N 1.90

Ethyl 3,4-Di-O-acetyl-2-deoxy-2-phthalimido-l-thio-~-~-glucopyranoside (6): 4.8 g (7.1 mmol) of finely ground compound 5 was dispersed in 500 ml of 75% aqueous acetic acid. The mixture was vigorously stirred at 55 "C for 1 h (TLC: hexane/ethyl acetate, 1 : 1, v/v). The solvent was evaporated, and the syrup was codistilled with toluene until acetic acid was completely removed. After purification on silica gel (hexane/ethyl acetate, 8: 1-3: 1, v/v) alcohol 6 was isolated as a crystalline product; yield 2.0 g (64%), m.p. 142-144"C, [ ~ ] 2 = +49.9 (C = 1.4, CHC13). - 'H NMR (270 MHz, CDC13): 6 = 1.22 (dd, Jv,c = 7.4 Hz, 3H, SCH,CH3), 1.85, 2.10 (2 s, 6H, 2 OAc), 2.58-2.79 (m, 2H, SCH2CH3), 3.65 (dd,

= 4.4 HZ, .16a,6b = 12.0 HZ, IH, 6-H,), 3.69-3.85 (m, 3H, 6- Hb, 5-H, 6-OH), 4.38 (dd, J ~ J = 10.1 Hz, J2,3 = 10.1 Hz, l H , 2-H),

5 . 1 2 ( d d , J 3 , ~ ~ 9 . 0 H ~ , J ~ , ~ ~ 9 . 6 H ~ , l H , 4 - H ) , 5 . 5 0 ( d , 1 H , l - H ) , 5.86 (dd, l H , 3-H), 7.70-7.90 (m, 4H, Phth).

C20H23NOsS (437.5) Calcd. C 54.91 H 5.30 N 3.20 Found C 55.10 H 5.35 N 3.34

Ethyl 3,6-Di-O-acetyl-2-deoxy-2-phthalimido-l-thio-~-o-glucopyranoside (7): 1.0 g (2.5 mmol) of the alcohol 6 was dissolved in 10 ml of pyridine. 30 ml of water was added and the solution was stirred at room temp. for 30 min (two-dimensional TLC: hexane/ ethyl acetate, 1 : 3, v/v). The solvent was removed in vacuo and after threefold codestillation with toluene an equilibrium mixture of 7 and 6 was obtained as a syrup. The ratio of the two compounds was determined from the 'H-NMR spectra by integration of the methyl signals of the 0-acetyl groups; yield 1.0 g (95% 7, 5% 6).

1.94, 2.14 (2 s, 6H, 2 OAc), 2.60-2.75 (m, 2H, SCH,CH,), 3.58-3.78 (m, 3H, 5-H, 4-H, 4-OH), 4.25-4.42 (m, 2H, 6-H,, 2-

- 'H NMR (270 MHz, CDC13) Of 7: 6 = 1.21 (dd, 3H, SCHICH3),

H), 4.52 (dd, J5.66 = 4.0 HZ, J6a,6b = 12.2 HZ, 1H, 6-Hb), 5.47 (d, J1,Z = 10.8 Hz, IH, I-H), 5.68 (dd, J2 ,3 = 8.5 Hz, J3 ,4 = 10.2 Hz, IH, 3-H), 7.70-7.90 (m, 4H, Phth).

CZoH23NOsS (437.5) Calcd. C 54.91 H 5.30 N 3.20 Found C 54.91 H 5.42 N 3.29

Ethyl 3,4-Di-O-acetyl-2-deoxy-2-phthalimido-l-thio-6-O-(2,3,4-tri- O-benzyl-a-~-~ucopyranosyl/-P-o-glucopyranoside (10): 1 .O g (2.28 mmol) of compound 6 was dissolved in 60 ml of dichloromethane. 800 mg (2.48 mmol) of tetrabutylammonium bromide, 1.0 ml (5.74 mmol) of ethyldiisopropylamine, and 1.0 g of powdered 4-A molecular sieves were added. The mixture was stirred at room temp. for 1 h. A solution of 1.1 g (2.29 mmol) of thioglycoside 8 in 20 ml of dichloromethane was cooled to O T , and 0.3 ml (5.8 mmol) of bromine was added. After 10 min dry cyclohexene was dropped into the reaction mixture until the bromine color disappeared. The resulting solution of bromide 9 was then added to the solution of acceptor 6 and stirred at room temp. until glycosylation was complete (approx. 5 d; TLC: hexane/ethyl acetate, 1 : 1, v/v). The mixture was filtered over Celite and washed with saturated NaHCO, solution and water. Removal of the solvents in vacuo and purification on a silica gel column (hexane/ethyl acetate, 5 : 1 , viv) afforded pure 10 as a syrup; yield 1.54 g (79%), [a]E = +9.4 (c = 1.1, CHCI3). - 'H NMR (270 MHz, CDC13): 6 = 1.10- 1.20 (m, 6H, SCH2CH3, 6'-H), 1.85, 1.98 (2 s, 6H, 2 OAc), 2.48-2.73 (m, 2H, SCH2CH,),

IH, 6-H,), 3.86-3.98 (m, 3H, 3'-H, 5'-H, 5-H), 4.04 (dd, Jlz =

9.4 Hz, 1H, 2-H), 4.60-5.00 (m, 8H, 6 CH2Ph, 1'-H, 6-Hb), 5.10

(dd, l H , 3-H),7.10-7.50(m, 15H, 3 Ph), 7.68-7.89(m,4H, Phth).

3.60-3.70 (m, IH, 4'-H), 3.75 (dd, J5,ea = 5.8 HZ, J6a,6b = 12.0 HZ,

3.4 Hz, JZ.3, = 10.0 Hz, IH, 2'-H), 4.39 (dd, Jj,2 = 10.6 Hz, J2.3 =

(dd, J4,5 = 9.2 Hz, J3,4 = 10.1 Hz, lH, 4-H), 5.43 (d, l H , 1-H), 5.82

- ''C NMR (69.9 MHz, CDCI3): 6 = 80.3 (C-I), 98.0 (C-1').

C47H51N012S (854.0) Calcd. C 66.10 H 6.02 N 1.64 Found C 66.30 H 6.20 N 1.45

Ethyl 3,6-Di-O-acetyl-2-deoxy-2-phthalimido-l-thio-4-0-~2,3,4- tri-O-benzy~-a-L-fucopyranosy/)-~-D-g~uco~yranoside (17): 500 mg (1.14 mmol) of 7 was dissolved in 30 ml of dichloromethane. 400 mg (1.25 mmol) of tetrabutylammonium bromide, 0.5 ml (2.87 mmol) of ethyldiisopropylamine, and 500 mg of powdered 4-A molecular sieves were added. The mixture was stirred at room temp. for 1 h. A solution of 500 mg (1.13 mmol) of thioglycoside 8 in 10 ml of dichloromethane was cooled to 0°C and 0.2 ml (3.9 mmol) of bro-

Liebigs Ann. Chem. 1991, 237 - 242

a-L-Fuc(l-+4)-P-~-GlcNAc and a-~-Fuc(1+6)-f3-~-GlcNAc Building Blocks 241

mine was added. After 10 min dry cyclohexene was dropped into the reaction mixture until the bromine color disappeared. The subsequent procedure was the same used for the preparation of 10 and afforded pure 17 as a syrup; yield 319 mg (33%) [a]E = t23.7 (C = 1.7, CHCI3). - ‘H NMR (270 MHz, CDC13): 6 = 0.97 (d, Js,,c = 6.8 Hz, 3H, 6’-H), 1.22 (dd, JVic = 7.4 Hz, 3H, SCHZCH,), 1.82, 2.02 (2 s, 6H, 2 OAc), 2.54-2.75 (m, 2H, SCH2CH3), 3.59 (m, 1H,4’-H), 3.65-4.00(m, 5H,2’-H, 3’-H, 5’-H,4-H, 5-H),4.17-4.32 (m, 2H, 2-H, 6-Ha), 4.55-4.95 (m, 8H, 6 CH,Ph, 1’-H, 6-Hb), 5.51

lH, 3-H), 7.10-7.50 (m, 15H, 3 Ph), 7.60-7.90 (m, 4H, Phth). - (d, J1.2 = 10.6 Hz, 1 H, 1-H), 5.62 (dd, J2.3 = 10.5 Hz, J3,4 = 8.0 Hz,

13C NMR (69.9 MHz, CDC13): 6 = 80.8 (C-l), 100.9 (C-1’). C47HSiN012S (854.0) Calcd. C 66.10 H 6.02 N 1.64 Found C 66.22 H 6.11 N 1.71

Methyl 3,4-Di-0-acetyl-2-deoxy-2-phthalimido-6-0-(2,3,4-tri-0- benzyl-cc-L-fucopyranosyl)-p-D-glucopyranoside (12): 400 mg (0.47 mmol) of thioglycoside 10 was dissolved in 20 ml of dichloromethane. After cooling the solution to 0°C 0.2 ml (3.9 mmol) of bromine was added. The formation of the disaccharide bromide 11 was complete within 10 min (TLC: hexane/ethyl acetate, 1 : 1, v/v), and excess bromine was removed with an appropriate amount of dry cyclohexene. 600 mg of powdered 4-A molecular sieves, 300 mg of powdered lo-A molecular sieves, and 360 mg (1.4 mmol) of silver triflate were dissolved in 70 ml of dichloromethane/methanol (5: 2, v/v). The mixture was kept in the dark and stirred under nitrogen at room temp. for 1 h and was then cooled to -78°C. The solution of bromide 11 was added slowly with stirring, and the reaction mixture was allowed to warm up to room temp. within ca. 12 h (TLC: hexane/ethyl acetate, 1 : 1, v/v). 5 ml of pyridine was added and the mixture filtered over a Celite layer. Solvents were removed in vacuo, and the reaction product was dissolved in 50 ml of dichloromethane and washed with saturated NaHC03 solution and water. Rotary evaporation of dichloromethane furnished crude 12, which was purified on a silica gel column (hexane/ethyl acetate, 5: 1 , v/v) to give pure 12 as a syrup; yield 272 mg (54%), [a12 = -7.6 (C = 1.1, CHCl3). - ‘H NMR (270 MHz, CDC13): 6 = 1.14 (d, J5,,6, = 6.4 Hz, 3H, 6‘-CHJ, 1.84, 1.97 (2 S, 6H, 2 OAC), 3.3 (s, 3H, OMe), 3.77 (dd, Jsba = 3.7 HZ, J(ja,6b = 12.0 Hz, IH, 6-H,), 4.06 (dd, Jl,,z. = 3.4 Hz, J T , ~ = 10.0 Hz, l H , 2’-H), 4.28 (dd, J1,z =

8.4 Hz, J2,3 = 10.7 Hz, 1 H, 2-H), 4.94 (d, 1 H, 1’-H), 5.26 (d, 1 H, I-H), 5.78 (dd, J3.4 = 9.1 Hz, lH, 3-H).

C d b ~ N 0 1 3 (823.9) Calcd. C 67.06 H 5.99 N 1.70 Found C 66.28 H 6.04 N 1.66

Methyl 3,6-Di-O-acetyl-2-deoxy-2-phthalimido-4-O-(2,3,4-tri-0- benzyl-a-L-fucopyranosyl)-~-D-glucopyranoside (19): 280 mg (0.33 mmol) of thioglycoside 17 was dissolved in 15 ml of dichloromethane. After cooling the solution to 0°C 0.15 ml(2.9 mmol) of bromine was added. The formation of the disaccharide bromide 11 was complete within 10 min (TLC: hexane/ethyl acetate, 1 : 1, v/v), and excess bromine was removed with an appropriate amount of dry cyclohexene. The bromide solution was then treated as described above for the preparation of the methyl glycoside 12 with methanol. The corresponding amounts of reagents were: 400 mg of powdered 4-A molecular sieves, 200 mg of powdered lo-A molecular sieves, 260 mg (1.01 mmol) of silver triflate, 45 ml of dichloromethane/ methanol (7: 2, v/v). Workup and isolation were performed according to the procedure used for disaccharide 12; yield 144 mg (54%), [a13 = -8.26 (c = 1.3, CHCI3). - ‘H NMR (270 MHz, CDCI3):

3.41 (s, 3H, OMe), 3.96 (dd, Jl.,2. = 3.4 Hz, J2.,3, = 10.4 Hz, 1 H, 6 = 0.98 (d, Jy.6. = 6.4 Hz, 3H, 6’-CH3), 1.83,2.01 (2 S , 6H, 2 OAC),

2’-H),4.12(dd,J1,2 =8.5Hz,Jz,3 = 10.7Hz,lH,2-H),4,86(d,lH, 1’-H), 5.29 (d, 1 H, I-H).

C46H49N013 (823.9) Calcd. C 67.06 H 5.99 N 1.70 Found C 66.37 H 6.08 N 1.67

Methyl 2-Acetamido-2-deoxy-6-0-(2,3,4-tri-0-benzyl-cc-~-fuco- pyranosylj-8-o-glucopyrunoside (14): 165 mg (0.2 mmol) of methyl glycoside 12 was dissolved in 10 ml of methanol. After addition of 1 ml of a 3% solution of sodium methanolate in methanol the solution was kept at room temp. until complete removal of the acetyl groups (TLC: hexane/ethyl acetate, 1 : 1, v/v). It was neutralized with IR 120 H + ion-exchange resin, filtered, and the solvent removed in vacuo to give the dialcohol 13 which was dissolved in 50 ml of ethanol. 12 ml(247 mmol) of a 98% solution of hydrazine hydrate was added and the solution was refluxed at 90- 110°C for 2 h (TLC: chloroform/methanol, l O : l , v/v). After removal of the solvents in vacuo the crude reaction product was codistilled twice with toluene and then taken up in 30 ml of pyridine/acetic anhydride (2: 1, v/v). After completion of the acetlyation (TLC: chloroform/methanol, 10: 1, v/v) 20 ml of water was added. The solvents were removed in vacuo, and after repeated codistillation with toluene the resulting oil was purified on a silica gel column (hexane/ ethyl acetate, 1: 1, v/v) to give pure 14 as an amorphous product; yield 104.7 mg(71%), [a]g = -24.0(~ = 3.8, CHCI,). - ‘H NMR

1.96 (2 s, 6H, 2 OAc), 2.01 (s, 3H, NAc), 3.38 (s, 3H, OMe), 4.47 (270 MHz, CDC13): 6 = 1.11 (d, J5.,6 = 6.4 Hz, 3H, 6’-H), 1.93,

(d, J1,2 = 8.3 Hz, l H , I-H), 5.73 (d, J Z , N H = 9.0 Hz, l H , NH).


Methyl 2-Acetamido-2-deoxy-4-0-(2,3,4-tri-0-benzyl-cc-r~-fucopyranosyl)-p-D-glucopyranoside (21). - Hydrazinolysis was carried out as described before for the preparation of compound 14. The corresponding amounts of the starting compounds and reagents were: 200 mg (0.243 mmol) of methyl glycoside 19, 17 ml of a 3% solution of sodium methanolate in methanol for the preparation of 20; 70 ml of ethanol, 18 ml (371 mmol) of 98% hydrazine hydrate for the hydrazinolysis; 45 ml of pyridine/acetic anhydride (2: 1, v/v) for the preparation of the N-acetylated disaccharide 21; yield 73 mg (40%), [a]: = -51.1 (C = 3.1, CHC13). - ‘H NMR (270 MHz, CDC13): 6 = 1.04 (d, Jy,c = 6.4 Hz, 3H, 6’-CH3), 1.89, 1.99 (2 S,

6H, 2 OAc), 2.02 (s, 3H, NAc), 3.44 (s, 3H, OMe), 4.37 (d, J1,* = 8.0 Hz, 1 H, I-H), 5.70 (d, JZyH = 9.3 Hz, 1 H, NH).


Methyl 2-Acetamido-2-deoxy-6-0-(a-~-fucopyranosyl)-~-o-glucopyranoside (16): 104 mg (0.14 mmol) of compound 14 was dissolved in 11 ml of a 3% solution of sodium methanolate in methanol. After complete removal of the acetyl groups (TLC: hexane/ ethyl acetate, 1:3, v/v) it was neutralized with IR 120 H + ion- exchange resin, filtered, and the solvents removed in vacuo. The obtained dialcohol 15 was then dissolved in 15 ml of dry methanol and hydrogenated over 200 mg of 5% palladium on charcoal at atmospheric pressure and room temp. until complete conversion into the product (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 16 as a syrup; yield 51 mg (96%), [a]:: = -92.2 (c = 1.2, HzO). - ‘H

2.03 (s, 3H, NAc), 3.49 (s, 3H, OMe), 3.50-3.61 (m, 3H, 3-H, 4-H, NMR (270 MHz, DzO): 6 = 1.22 (d, Jy,C = 6.5 Hz, 3H, 6‘-CH3),

Liebigs Ann. Chem. 1991, 237 - 242

242 T. Peters, T. Weimar

5-H), 3.69 (dd, J ~ J = 8.5 Hz, J2.3 = 8.0 Hz, I H , 2-H), 3.76-3.82 (m, 3H, 6-H,, 2‘-H, 4’-H), 3.90 (dd, J2.,3. = 10.4 Hz, JY,& = 3.3 Hz,

(m, 1 H, 5’-H), 4.43 (d, 1 H, I-H), 4.93 (d, Jl,,2. = 3.8 Hz, 1 H, 1’-H). C I ~ H ~ ~ N O I O (381.4) Calcd. C 47.24 H 7.13 N 3.67 Found C 46.99 H 7.37 N 3.70

1 H, 3’-H), 3.99 (dd, J5,6b = 1.6 HZ, J6a,6b = 12.0 HZ, 1 H, 6-Hb), 4.13

Methyl 2-Acetamido-2-deoxy-4-0- (E-~-fucopyranosyl)-~-~-glucopyranoside (23): 64 mg (0.09 mmol) of disaccharide 21 was sub- jected to the same treatment as described above for the preparation of 16. The amounts of solvents and reagents used were identical. 30 mg (98%) of pure deblocked 23 was obtained as an amorphous product; [a126 = -107.7 (c = 0.6, H20). - ‘H NMR (270 MHz,

3.51 (s, 3H, OMe), 3.54-3.58 (m, 2H, 4-H, 5-H), 3.65 (dd, J2.3 =

3.78-3.88(m,4H,6-Ha,2’-H, 3’-H,4-H),4.00(dd,J6a,6b = 12.3 Hz,

D20): 6 = 1.16 (d, J5.,6. = 6.4 Hz, 3H, 6’-CH3), 2.04 (s, 3H, NAc),

10.0 Hz, J3,4 = 7.2 Hz, I H , 3-H), 3.71 (dd, Jl,2 = 8.7 Hz, l H , 2-H),

l H , 6-Hb), 4.34 (m, IH, 5’-H), 4.44 (d, I H , I-H), 4.95 (d, Jj.,? = 3.0 Hz, 1 H, 1’-H).

Ci5H27N010 (381.4) Caicd. C 47.24 H 7.13 N 3.67 Found C 47.11 H 7.23 N 3.71

CAS Registry Numbers

1: 10022-13-6 ,I 2: 99409-32-2 / 3: 130539-43-4 / 5: 131544-99-5 6: 131545-00-1 / 7: 131545-01-2 ,I 8: 131545-02-3 / 10: 131545- 03-4 1 12: 131566-40-0 ,I 14: 131545-04-5 / 16: 97242-89-2 ,I 17: 131545-05-6 ,I 19: 131545-06-7 ,I 21: 131545-07-8 123: 131545-08-9

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improved synthesis of α-l-fuc(1→4)-β-d-glcnac and α-l-fuc(1→6)-β-d-glcnac building blocks: a...

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