Glycobiology Advance Access originally published online on May 11, 2005
Glycobiology 2005 15(10):1043-1050; doi:10.1093/glycob/cwi074
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Banana lectin is unique in its recognition of the reducing unit of 3-O-ß-glucosyl/mannosyl disaccharides: a calorimetric study
2 Department of Biological Chemistry, The University of Michigan Medical School, Ann Arbor, MI; and 3 Department of Organic Chemistry, University of Stockholm, S-10691 Stockholm, Sweden
1 To whom correspondence should be addressed; e-mail: igoldste{at}umich.edu
Received on March 16, 2005; revised on May 3, 2005; accepted on May 5, 2005
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Abstract |
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The binding of banana lectin (BanLec) to laminaribiose (Glcß1,3Glc) and a series of novel synthetic analogues was measured by titration calorimetry to assess the contribution of the hydroxyl groups of the reducing glycosyl moiety and its 3-O-ß-substituent to binding. Key areas of interaction involved the 1, 2, and 6 positions of the reducing-terminal hexose unit. The
-anomeric configuration of the reducing hexose was strongly favored over the ß-anomer. The 2-hydroxyl in the axial position (mannose) also enhanced binding, whereas the 6-hydroxymethyl group was essential, because xylopyranose in the reducing position was inactive. The 3-O-ß-glucosyl unit of methyl -laminaribioside could be replaced by any of its monodeoxy derivatives. However, the 49-deoxy derivative or axial hydroxy (galactosyl) substitution was somewhat detrimental to binding. 3-O-substitution with the (S)tetrahydropyranyl ring or a benzyl group had similar effect as 49-deoxyglucosyl substitution. Surprisingly, p-nitrobenzyl or ß-xylosyl 3-O-substitution greatly enhanced binding of the reducing glucosyl or mannosyl derivative. Chemical syntheses of a number of novel disaccharides and analogues prepared for this study are described. Key words: calorimetric titration / carbohydrate recognition / glucose-binding lectin / glycosynthesis / laminaribiose
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Introduction |
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TopAbstractIntroductionResults and discussionMaterials and methodsNote added in proofAcknowledgmentsReferences |
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Lectins are carbohydrate-recognizing proteins/glycoproteins that bind in a reversible fashion to the sugar units of glycoconjugates on cell surfaces or in solution. For many years it was believed that lectins bound only to the nonreducing termini of oligosaccharides present in polysaccharides and glycoconjugates. Subsequently it was shown that concanavalin A could also bind to internal 2-O-
-linked glucosyl/mannosyl residues on the basis of the availability of their hydroxyl groups at C-3,4, and 6 of these sugars (Goldstein et al., 1973
).
The banana lectin (BanLec) is a glucosyl/mannosyl-binding lectin that binds to terminal nonreducing -D-glucosyl/mannosyl units of oligo-/polysaccharide chain ends (Mo et al., 2001). BanLec is unique in its recognition of internal glucosyl/mannosyl residues linked 1,3-, but not ß1,3-, and especially in its recognition of the reducing sugar unit of laminaribiose (Glcß1,3Glc) and its higher homologues (Goldstein et al., 2001; Mo et al., 2001).
In this article, we describe the synthetic routes to a number of novel derivatives and analogues of laminaribiose. Measurement of binding parameters of these derivatives to BanLec by titration calorimetry serves to determine the molecular basis of the interaction of the BanLec with laminaribiose and its derivatives, that is, the importance of the various hydroxyl groups in its binding to the protein.
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Results and discussion |
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TopAbstractIntroductionResults and discussionMaterials and methodsNote added in proofAcknowledgmentsReferences |
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Synthesis of laminaribioside and analogues
Phase-transfer catalyzed benzylation (Garegg et al., 1976
) of methyl 4,6-O-benzylidene--D-glucopyranoside yielded mainly the key intermediate 3-OH acceptor 1 together with the 3-OBn derivative 2 (Scheme 1). Removal of the benzylidene acetal from 2 gave 3, whereas methylation of 1 (
4) and subsequent hydrogenolysis gave 5. Treatment of 1 with dihydropyran under acidic conditions gave the two stereoisomers 15 and 16, which were separated by silica gel chromatography and subsequently hydrogenolyzed to yield the 3-O-tetrahydropyranyl acetal derivatives 17 and 18 (Scheme 2). For disaccharide synthesis, 1 was used as acceptor and acetobromo- or chlorosugars as donors in silver triflate-promoted glycosylations, except in one case when a thioglycoside donor was used promoted by DMTST (Schemes 2 and 3). Acetobromoglucose gave an intermediate 7, which was deprotected to give methyl -laminaribioside, 8. Acetobromogalactose and acetobromoxylose gave intermediates 10 and 13, respectively, which after deprotection afforded the galactose and xylose analogues 11 and 14. For the monodeoxy analogues, the known monodeoxyacetochlorosugar donors 19, 22, and 25 were employed to give the 6'-, 4'-, and 3'-monodeoxy analogues 21, 24, and 27, respectively, after deprotection. To obtain the right stereochemistry in the 2'-deoxy analogue, thioglycoside 28 with a participating 2-O-acetyl group was used as donor in a DMTST-promoted coupling to give the ß-linked disaccharide 29. Chemoselective removal of the acetyl group afforded the 2'-OH derivative, which was deoxygenated through tributyltinhydrate reduction of the corresponding 2'-imidazolthioester (Barton and McCombie, 1975) to give the 2'-deoxy analogue 31 after deprotection.
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Binding of laminaribiose and derivatives to BanLec
Table I presents the binding data determined by isothermal titration calorimetry of the interaction of the BanLec with oligosaccharides related to laminaribiose. It is of considerable interest that laminaribiose (7) binds to the BanLec for two reasons: the disaccharide has a ß-glucosidic bond and it contains a 1,3-linkage. As noted above, it is the C-3, C-4, and C-6 hydroxyl groups of D-glucose and D-mannose that are essential for binding to lectins that recognize these sugars [sophorose, 2-O-ß-D-glucosyl-D-glucose, binds to glucose/mannose-binding lectins because the 3-, 4-, and 6-hydroxyl groups are available for reaction (Goldstein et al., 1967); see Figure 1 for structures of these disaccharides]. Until demonstrated for BanLec, no oligosaccharide with a glycosyl group at the C-3 position in ß-linkage was shown to react with a lectin.
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The evidence supporting the view that the BanLec recognizes the reducing glucosyl unit of laminaribiose is the following:
- Reduction of laminaribiose and its higher homologues (e.g., laminariheptaose) with NaBH4 abolishes binding to the BanLec (Goldstein et al., 2001);
- The methyl -glycoside of laminaribiose has a higher Ka than does the free sugar, whereas the ß-glycoside is a poorer ligand than the free sugar (Table I, 8 vs. 7, 9); and
- Substitution of mannose, as its methyl -glycoside (methyl 3-O-glucosyl--mannopyranoside) increases binding over that of methyl -laminaribioside, showing that it is indeed the reducing unit which is the primary site of interaction with the BanLec (Table I, 11 vs. 8).
Next we studied the effect of modifying the nonreducing terminal sugar moiety on the binding activity of the resulting disaccharide. Changing the 3-O-glucosyl group to a D-galactosyl group (10 vs. 8) decreased the Ka value 3-fold, which could indicate failure of the C-4' axial hydroxyl group to form a hydrogen bond with the protein, with or without additional steric interference. The four deoxy derivatives in the nonreducing glucosyl moiety of methyl -laminaribioside were prepared and studied. Only the 4'-deoxy derivative differed significantly, being about one-half as effective as the parent disaccharide (14 vs. 8,12,13, and 15), suggesting that interaction with the equatorial 4' hydroxyl group contributes to a modest extent to the binding, whereas steric interference of the axial hydroxyl of galactose is minor. On the other hand, the 3-O-xylosyl analogue (methyl 3-O-ß-D-xylopyranosyl--D-glucopyranoside, 23) of methyl -laminaribioside showed an unexpectedly high Ka value. This effect is most likely attributed to steric hindrance of the C-6 hydroxymethyl group on the nonreducing glucosyl residue of laminaribiose.
Replacement of the nonreducing sugar moiety of laminaribiose with essentially hydrophobic groups also provided interesting insight. 3-O-methyl substitution on glucose or its methyl glycosides (16–18 vs. 1–3) gave only a slight positive or negative effect. The two diastereoisomeric 3-O-tetrahydropyranyl derivatives of methyl -glucopyranoside were prepared and resolved (see experimental section) to give the R and S isomers. The configuration of the tetrahydropyranyl linkage is of importance. The diastereoisomer with the lower molar optical rotation was assigned the S configuration, analogous to the ß-anomer of a pyranose ring [assuming the 4C1 configuration of the tetrahydropyran-2-yl (THP) ring] and was preferred over the R (-anomer) structure by 4-fold (20 vs. 19).
Although the S-THP derivative lacks the 6'-hydroxymethyl group as does the xylosyl derivative, it does not show enhanced binding over the 4'-deoxyglucosyl derivative (20 vs. 14) indicating that at least one of the other hydroxyl groups on the nonreducing moiety also contributes to binding, most likely the 2'-hydroxyl (cf. 12 vs. 8).
Substitution of an aromatic ring (3-O-benzyl) also enhanced binding 2-fold over methyl -glucoside (21 vs. 2), similar to a galactosyl or S-tetrahydropyranyl group. A p-nitro group further enhanced binding, as methyl 3-O-p-nitrobenzyl--mannoside was enhanced 
5-fold over the unsubstituted mannoside (22 vs. 4). The p-nitro group is in a similar position as the equitorial 4'-hydroxyl group of the nonreducing glucosyl residue. This suggests that it, too, may participate in hydrogen bonding or possibly electrostatic interaction not available to 3-O-substituents lacking the electronegative function in this position, such as galactosyl, 4'-deoxyglucosyl, THP, or benzyl groups.
Effects of the configura tion of the 2-hydroxyl group of the reducing sugar and the lack of the 6'-hydroxymethyl group are additive, as shown by the further increase in binding by methyl 3-O-ß-D-xylopyranosyl--D-mannopyranoside (24), which had the highest binding constant of any structure tested. However, the negligible reactivity of 3-O-ß-xylopyranosyl-D-xylose (27) shows the critical role of the 6-hydroxyl group of the reducing aldohexapyranose. Although in ß-1,3-xylobiose the reducing xylose group exists as the free reducing sugar rather than being constrained as the methyl -glycopyranoside, it nevertheless exists virtually exclusively as a pyranose ring, with a significant percentage in the -anomeric form, because free xylose exists in solution as 37% -pyranose and 63% ß-pyranose, with insignificant amounts of furanose structures (Stoddart, 1971). Lack of reactivity of methyl -xylopyranoside and methyl 6-deoxy--glucopyranoside, in contrast to methyl -pyranosides of glucose and mannose, supports the critical role played by the hydroxymethyl group at the 6-position in binding the reducing glucose /mannose groups. Further effect of the involvement of the 2-hydroxyl group of the reducing sugar is shown by the relative binding of the methyl -glycosides of glucose, mannose, and their 2-deoxy derivative, whose binding affinities are in the order man>2-deoxy=glc, indicating that the axial hydroxyl contributes to binding, whereas the equatorial hydroxyl is virtually without effect.
On the basis of the modeling studies of Peumans et al. (2000), the assumption was made that binding occurred at one binding site per subunit molecular mass of 14.5 kDa. The low affinities involved (generally at or less than 103 M–1) give titration curves that are nearly hyperbolic and thus do not yield reliable data on the stochiometry of the binding (Cliff et al., 2004). Only when concentrations of the lectin on the order of 1 mM (14.5 mg/mL) or greater [c = 1; (Wiseman et al., 1989)] together with correspondingly high concentrations (>50 mM) of ligands are used do titration curves exhibit a distinct inflection point giving reliable stoichiometry. Use of such concentrations is impractical for other reasons, however. In this study, a sufficiently high concentration (0.4 mM) of the lectin was titrated with the best binding-affinity disaccharides to give slightly sigmoidal titration curves which fit with a high degree of certainty to a stochiometry of 1.8 ± 0.1 sites per subunit. Additionally, X-ray data on the lectin crystallized in the presence of ligands suggests the presence of two binding sites per subunit (Meagher et al., 2005). Accordingly, we reanalyzed all previous data and repeated some titrations. All such data fit somewhat better (n without parentheses in Table I), or nearly as well (n in parentheses), to a stochiometry of 2 binding sites per subunit (1.7–2, depending on the best fit observed with all titrations using a particular lectin preparation). Another consequence of the low affinities is that it is impractical to carry titrations out to total saturation to obtain a baseline heat of dilution. Consequently, baseline correction was estimated from blank titrations of selected ligands into an inert protein (bovine serum albumin) at comparable concentration to the lectin. Data were initially corrected to an appropriate observed value, but corrections were refined to a value giving best fit. In all cases, the fit to independent binding sites of this stochiometry led to somewhat higher binding constants. In no case did the data fit satisfactorily to a multiple interacting-site model.
Table I also shows the estimated entropy of binding, expressed as T
S. Poor binding of methyl ß-glycosides or 3-O-methyl hexosides seems due largely to increased unfavorable entropy (negative TS). However, increased binding of the more hydrophobic groups in the 3-hydroxyl position, such as the THP-, benzyl-, or xylosyl-moieties, appears to arise from a favorable entropic contribution (positive TS), further indicating the contribution of hydrophobic interactions in this region of the molecule. For most ligands with nonreducing hexose units, entropic contributions or penalties are quite small, being less than ±1 kcal/mol.
It is interesting to note that rabbit antibodies raised against a laminaribiose azo-bovine-serum albumin conjugate displayed specificity for the nonreducing 3-O-ß-glucosyl moiety (Allen et al., 1970), as did the family 6 carbohydrate-binding module (CBM6) from a bacterial laminarinase (van Bueren et al., 2005). CBM6 is also distinct in its preference for laminarihexaose over smaller laminarin oligosaccharides. These observations highlight the differences in binding specificity among these three classes of Glcß1,3Glc-binding proteins.
At the present time, no xylose-binding lectin has been reported. The fact that 3-O-xylosyl glucose (or mannose) binds with high affinity to the BanLec suggests its potential utility in assays for 3-O-ß-xylosyltransferases, utilizing glucose or mannose as acceptor.
Summarizing, the BanLec is unique in its carbohydrate-binding properties: in common with other glucose/mannose-binding lectins, it binds to -glucosyl and -mannosyl terminal nonreducing units. Unique is both its recognition of internal 3-O--glucosyl/mannosyl residues and the reducing terminal 3-O-ß-glucosyl/mannosyl unit of oligosaccharides. The X-ray crystal structure of the BanLec in its native form and complexed with its various oligosaccharide ligands should clarify its carbohydrate-binding properties.
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Materials and methods |
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TopAbstractIntroductionResults and discussionMaterials and methodsNote added in proofAcknowledgmentsReferences |
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Banana lectin, prepared as previously described (Peumans et al., 2000
), was available from previous studies. Carbohydrate ligands other than those whose synthesis is described below were from various commercial sources or were available from previous studies.
Calorimetric titrations were performed essentially as previously described (Mo et al., 2001). All calculations were performed using the Bindworks software provided installed in the instrument; some earlier data were recalculated using the newer version of Bindworks.
Synthesis of mono- and disaccharide derivatives: general methods
Nuclear magnetic resonance (NMR) spectra were recorded at 25°C and 300 or 400 MHz in CDCl3 with Me4Si as the internal standard (
= 0), unless otherwise stated. Thin layer chromatography (TLC) was performed on silica gel 60 F254 with detection by UV light and charring with 8% sulfuric acid. Silica gel (0.040–0.063 mm) was used for column chromatography.
Methyl 3-O-benzyl--D-glucopyranoside (3)
Compound 2 (0.1 g, 269 µmol) in HOAc (70% aq., 25 mL) was stirred at 70°C for 2 h. The mixture was concentrated and freeze-dried to give 3 (76 mg, 267 µmol, 99%). 13C NMR (CD3OD): , 55.5, 62.6, 71.5, 73.6, 76.1, 83.6, 101,3, 128.3, 128.9, 129.0, and 140.3 ppm.
Methyl 3-O-methyl--D-glucopyranoside (5)
A solution of 1 (0.1 g, 0.269 mmol) and MeI (57 mg, 0.404 mmol) in dimethyl formamide (DMF) (2 mL) was added dropwise to a suspension of NaH (13 mg, 0.538 mmol) in DMF (4 mL). The reaction was monitored on TLC (toluene/EtOAc 2:1). When conversion was complete, the reaction was quenched with MeOH (2 mL) and the mixture co-concentrated with toluene. The residue was purified on a silica gel column (toluene/EtOAc 15:1) to give 88 mg (0.228 mmol, 85% yield) of 4. Compound 4 and Pd/C (catalytic amount) in EtOH/H2O/HOAc (7:1:2) were stirred under H2 (110 psi) overnight, then filtered through celite, and concentrated. Recrystallisation from EtOAc gave 5 (30 mg, 0.144 mmol, 50%). 13C NMR (D2O): , 55.5, 61.2, 62.5, 71.3, 73.4, 73.5, 85.1, and 101.2 ppm.
Methyl ß-D-glucopyranosyl-(13)--D-glucopyranoside (8)
A solution of 1 (0.5 g, 1.34 mmol), 6 (1.0 g, 2.43 mmol), and molecular sieves in CH2Cl2 (60 mL) was cooled to 0°C in an ice bath. The mixture was stirred for 20 min and then silver triflate (0.62 g, 2.4 mmol) was added. The reaction was monitored on TLC (toluene : EtOAc 2 : 1). When complete conversion of the starting material was observed, the reaction mixture was neutralized with Et3N (5 mL), diluted with CH2Cl2 (140 mL), filtered through celite, and concentrated. The residue was purified on a silica gel column (toluene/EtOAc 4:1) to give 7 (0.55 g, 0.78 mmol, 58%). 13C NMR: , 20.87, 20.92, 21.0, 21.1, 55.6, 61.9, 62.2, 68.2, 69.1, 71.9, 72.0, 73.3, 74.2, 78.4, 78.8, 80.6, 98.9, 101.0, 101.5, 126.1–137.9, 169.4, 169. 6, 170.4, and 170.7 ppm. Compound 7 was dissolved in MeOH (25 mL), and NaOMe (1M in MeOH) was added to the mixture until it was basic. The reaction mixture was stirred overnight at room temperature and was then neutralized with Dowex H+ ion exchange resin, filtered, and concentrated. The residue was dissolved in EtOH/H2O/HOAc 7:1:2 and hydrogenolyzed over Pd/C (catalytic amount) at 100 psi overnight. The reaction mixture was filtered (celite) and concentrated, to give 0.24 g of crude 8. Recrystallisation from EtOH/diethyl ether yielded pure 8 (0.14 g, 0.399 mmol, 51%). 13C NMR (D2O): , 55.3, 60.8, 60.9, 68.3, 69.8, 71.0, 71.6, 73.7, 75.8, 76.2, 82.7, 99.3, and 103.0.
Methyl ß-D-galactopyranosyl-(13)--D-glucopyranoside (11)
Compounds 1 (0.1 g, 269 µmol) and 9 (266 mg, 404 µmol), sym-collidine (30 µl, 241µmol), and molecular sieves were dissolved in CH2Cl2 (12 mL). The mixture was cooled to 0°C. After 30 min, silver triflate (0.12 g, 462 µmol) was added to the reaction mixture. After an additional 4 h more silver triflate (60 mg) was added. The reaction mixture was stirred and allowed to attain room temperature overnight, then neutralized with Et3N (1.7 mL), diluted with CH2Cl2 (30 mL), filtered (celite), and concentrated. The residue was purified on a silica gel column (toluene/EtOAc 3:1), to afford 124 mg (176 µmol, 66% yield) of 10. Compound 10 was dissolved in MeOH and sodium methoxide was added until the mixture was basic. The reaction mixture was stirred at room temperature for 2 days, then neutralized with Dowex H+ ion exchange resin, filtered, and concentrated. The residue was dissolved in EtOH/H2O/HOAc (7:1:2) and hydrogenolyzed over Pd/C (catalytic amount) at 110 psi overnight. The mixture was filtered (celite) and concentrated. The residue was purified on a Biogel P2-column (water containing 1% n-butanol) to give 11 (32 mg, 90 µmol, 59%). 13C NMR (D2O): , 55.3, 61.3, 68.4, 68.8, 70.9, 71.47, 71.53, 72.8, 75.5, 99.3, and 103.5 ppm.
Methyl ß-D-xylopyranosyl-(13)--D-glucopyranoside (14)
Compounds 1 (100 mg, 269 µmol) and 12 (137 mg, 403 µmol) were dissolved in CH2Cl2 (15 mL). The mixture was cooled to 0°C with an ice bath for 45 min. Silver triflate (115 mg, 451 µmol) was added to the reaction mixture. After 2 h at 0°C the reaction mixture was neutralized with Et3N (0.5 mL), diluted with CH2Cl2 (20 mL), filtered (celite), and concentrated. The residue was purified on a silica gel column (toluene/EtOAc 5:1) to give 75 mg (119 µmol, 44% yield) of 13. 13C NMR (CDCl3): , 20.86, 20.88, 20.9, 55.5, 61.6, 62.3, 69.0, 69.1, 71.0, 71.3, 73.9, 76.2, 79.7, 80.6, 98.9, 100.3, 101.6, 126–138.0, 169.6, 170.0, and 170.1 ppm. Compound 13 was deprotected as described for 10 above to give 45 mg (138 µmol, 51% total yield) of 14. 13C NMR (D2O): , 55.1, 60.7, 65.3, 68.1, 69.4, 71.0, 71.7, 73.6, 75.8, 82.3, 99.3, and 103.8 ppm.
Methyl 3-O-(S)tetrahydropyranyl--D-glucopyranoside (17) and methyl 3-O-(R)tetrahydropyranyl--D-glucopyranoside (18)
Compound 1 (1.3 g, 3.51 mmol) was dissolved in chloroform (10 mL) to which 3,4-dihydro-2H-pyran(2 mL) and p-toluenesulfonic acid (8 mg) were added. After 5 min, Et3N (0.3 mL) was added and the mixture was purified on a silica gel column (toluene-EtOAc 6:1) to give 15/16 (1.25 g,79%).13C NMR data (CDCl3): , 19.8, 21.3, 25.1, 30.8 (C-2'-4'), 55.4 (OMe), 60.5, 62.6, 62.9, 65.4, 69.2, 80.5 (C-2–6, C-5',CH2Ph), 100.3, 101.5, 102.1 (C-1, C-1', CHPh), and 126–137.6 (Ph). The mixture (0.5 g, 1.1 mmol) was purified on a silica gel column (toluene-EtOAc 12:1), which gave two products 15 (127 mg, 0.28 mmol) and 16 (140 mg, 0.31 mmol). Compound 15 (46 mg, 0.10 mmol) was dissolved in MeOH–EtOAc (5:1, 8 mL). Amberlite IR-45(OH–) resin (46 mg) and palladium on activated carbon were added. The mixture was hydrogenolyzed at 110 psi overnight. The mixture was diluted (MeOH), centrifuged, and concentrated. Silica gel chromatography of the residue (CHCl3/MeOH 5:1) gave 17 (25 mg, 0.09 mmol, 90%). Following the same procedure 16 (46 mg, 0.10 mmol) was deprotected to give 18 (23 mg, 0.083 mmol, 83%).
Methyl (6-deoxy-ß-D-glucopyranosyl)-(13)--D-glucopyranoside (21)
Silver triflate (262 mg, 1.02 mmol) dissolved in dry toluene (5 mL) was added at 0°C to a stirred solution of 19 (210 mg, 0.68 mmol) and 1 (378 mg, 1.02 mmol) in CH2Cl2 (15 mL) containing crushed molecular sieves (4Å). After 2h, Et3N (0.3 mL) was added and the mixture was put on top of a silica gel column and eluted (CHCl3/MeOH 40:1) to give methyl (2,3,4-tri-O-acetyl-6-deoxy-ß-D-glucopyranosyl)-(13)-2-O-benzyl-4,6-O-benzylidene--D-glucopyranoside (20, 150 mg, 34%). 13C NMR (CDCl3): , 17.2 (C-6'), 20.7, 20.6, 20.7 (MeCO), 20.6 (PhCH2), 55.2 (MeO), 62.0, 64.8, 68.9, 69.8, 70.1, 70.2, 71.2, 72.4, 73.7, 81.2 (C-2–6, C-2'-5', PhCH2), 98.6, 101.3, 101.9 (C-1,1', PhCH), 126.0–138.0 (Ph), 169.6, 169.6, and 170.3 (COMe). Compound 20 (140 mg, 0.22 mmol) was dissolved in MeOH and a catalytic amount of NaOMe was added. When TLC showed complete deacylation, the mixture was neutralized with Dowex 50(H+) ion exchange resin, filtered, and concentrated. The residue in 60% HOAc was stirred at 70°C for 2h, then concentrated, co-concentrated twice with toluene, and purified on a silica gel column (CHCl3–MeOH 10:1) to give methyl (6-deoxy-ß-D-glucopyranosyl)-(13)-2-O-benzyl--D-glucopyranoside (80 mg, 86%). 13C NMR data (CDCl3): , 17.7 (C-6'), 55.4 (MeO), 58.4, 62.0, 69.0, 71.3, 72.4, 73.5, 74.1, 75.1, 76.6, 83.0(C-2–6, C-2'-5', PhCH2), 98.2, 103.3, (C-1,1'), and 128.3–138.1 (Ph). This compound (70 mg, 0.16 mmol) was dissolved in MeOH (5 mL) containing Et3N (20 µL). Pd/C was added and the solution was hydrogenolyzed for 2h at 100 psi. The mixture was then filtered, concentrated, and purified on a silica gel column (CHCl3/MeOH 6:1) to give 21 (38 mg, 69%). 13C NMR (CD3OD): , 17.9 (C-6'), 55.5 (MeO), 62.5, 70.0, 72.3, 73.5, 73.6, 75.6, 76.6, 77.5, 86.1 (C-2–6, C-2'-5'), 100.9, and 105.1 (C-1,1'); 1H-NMR (CD3OD) selected data: , 4.7 (H-1); 4.4 (H-1'), 1.3 (H-6').
Methyl (4-deoxy-ß-D-glucopyranosyl)-(13)--D-glucopyranoside (24)
Silver triflate (208 mg) dissolved in dry toluene (5 mL) was added at 0°C to a stirred solution of 22 (226 mg, 0.46 mmol) and 1 (256 mg, 0.69 mmol) in CH2Cl2 (15 mL) containing crushed molecular sieves (4Å). After overnight, Et3N (0.3 mL) was added and the mixture was purified on a silica gel column (CHCl3-MeOH 9:1) to give methyl (2,3,4-tri-O-benzoyl-4-deoxy-ß-D-glucopyranosyl)-(13)-2-O-benzyl-4,6-O-benzylidene--D-glucopyranoside (23, 345 mg, 91%). 13C NMR data (CDCl3): , 33.4 (C-4'), 55.5 (MeO), 62.5, 66.1, 69.2, 69.6, 72.1, 73.5, 74.2, 77.7, 79.6, 80.2 (C-2–6, C-2',3',5',6', PhCH2), 99.2, 101.4, 101.5 (C-1,1', PhCH), 125.5–138.5 (Ph), 165.9, 166.2, and 166.4 (PhCO). Compound 23 (338 mg, 0.41 mmol) was dissolved in MeOH, and a catalytic amount of NaOMe was added. When TLC showed complete deacylation, the mixture was neutralized with Dowex 50 (H+) resin, then filtered, concentrated, and purified on a silica gel column. This product (188 mg, 0.36 mmol, 88%) was dissolved in MeOH (5 mL) containing triethylamine (20 µL), Pd/C was added, and the solution was hydrogenolyzed at 100 psi for 24 h. The mixture was then filtered, concentrated, and purified on a silica gel column (CHCl3/MeOH 8:1) to give 24 (120mg, 0.35 mmol, 98%). NMR data (D2O): 13C, , 36.7 (C-4'), 57.8 (MeO), 63.3, 66.2, 70.9, 72.9, 73.4, 74.1, 75.6, 78.0, 85.8 (C-2–6, C-2',3',5',6'), 101.9, and 106.1 (C-1,1'). 1H-NMR (D2O, 25°C) selected data: , 4.8 (H-1); 4.6 (H-1'), 2.0 (H-4'), 1.4 (H-4'ax)
Methyl (3-deoxy-ß-D-glucopyranosyl)-(13)--D-glucopyranoside (27)
Silver triflate (261 mg, 1.02 mmol) dissolved in dry toluene (5 mL) was added at 0°C to a stirred solution of 25 (140 mg, 0.45 mmol) and 1 (379.8 mg, 1.02 mmol), in CH2Cl2 (10 mL) containing crushed molecular sieves (4Å). After 2h, Et3N (0.3 mL) was added and the mixture was applied to a silica gel column and eluted (6:1 toluene-EtOAc) to give methyl (2,4,6-tri-O-acetyl-3-deoxy-ß-D-glucopyranosyl)-(13)-2-O-benzyl-4,6-O-benzylidene--D-glucopyranoside (26, 263 mg, 90 %). 13C NMR (CDCl3): , 20.9, 21.1, 21.2 (MeCO), 33.3 (C-3'), 55.6 (MeO), 62.4, 63.0, 65.9, 68.0, 69.6, 74.2, 75.2, 77.7, 79.4, 80.6(C-2–6, C-2',4',5',6', PhCH2), 99.1, 101.5, 102.4 (C-1,1', PhCH), 125.5–138.3 (Ph), 169.7, 166.8, and 171.0 (COMe). Compound 26 (260 mg, 0.40 mmol) was deprotected as described for compound 20 to give 27 (86mg, 66%). 13C NMR (CD3OD): , 39.2 (C-3'), 54.4 (MeO), 61.4, 61.4, 64.8, 68.5, 68.9, 71.2, 72.2, 80.8, 84.3(C-2–6, C-2',4',5',6'), 99.7, and 106.0 (C-1,1'). 1H-NMR (CD3OD) selected data: , 4.7 (H-1); 4.4 (H-1'), 2.3 (H-3'), and 1.5 (H-3'ax).
Methyl 2-deoxy-ß-D-glucopyranosyl-(13)--D-glucopyranoside (31)
Compound 28 (290 mg, 0.54 mmol), 1 (134 mg, 0.36 mmol), and 2,6-di-tert-butyl-4-methylpyridine (97.5 mg, 0.5 mmol) were dissolved in CH2Cl2 (10 mL) containing crushed molecular sieves (4Å) and stirred for 30 min before dimethyl(methylthio)sulfonium triflate (DMTST) (185 mg, 0.72 mmol) was added. After 2 h Et3N (2 mL) was added and the mixture was concentrated and purified on a silica gel column (toluene-EtOAc 6:1) to give methyl (3,4,6-O-benzyl-ß-D-glucopyranosyl)-(13)-2-O-benzyl-4,6-O-benzylidene--D-glucopyranoside 29 (318 mg, 70%). 13C NMR (CDCl3): , 21.3(MeCO), 55.6(MeO), 62.3, 68.5, 69.3, 73.6, 74.0, 74.3, 75.1, 75.2, 75.2, 77.8, 78.2, 79.1, 81.1, 83.3 (C-2–6, C-2'-6', PhCH), 99.1, 101.0, 101.8 (C-1,1', PhCH), 126.3–138.5 (Ph), and 169.9 (COMe). Compound 29 (298 mg, 0.35 mmol) was dissolved in MeOH, and a catalytic amount of NaOMe was added. When TLC showed complete deacylation, the mixture was neutralized with Dowex 50(H+) resin, then filtered, concentrated, and purified on a silica gel column (toluene/EtOAc 5:1) to give a deacetylated intermediate (256mg, 90 %). 13C NMR (CDCl3): , 55.6 (MeO), 62.7, 68.8, 69.1, 73.6, 73.7, 75.2, 75.3, 75.8, 76.4, 77.0, 78.7, 79.1, 80.9, 84.5, 83.3 (C-2–6, C-2'-6',PhCH), 98.6,101.5, 104.9 (C-1,1', PhCH), and 126.3–139.1(Ph). A mixture of this compound (246 mg, 0.31 mmol) and 1,1'-thiocarbonyldiimidazole (42 mg, 0.24 mmol) in dry 1,2-dichloroethane (5 mL) was stirred under N2. After 6h, the solution was concentrated and the residue was purified on silica gel (toluene/EtOAc 6:1) to give the 2'-thicarbonylimidazole derivative (175 mg, 63%). 13C NMR (CDCl3): , 54.7 (OMe), 61.5, 67.5, 68.5, 72.9, 73.0, 74.5, 74.5, 74.6, 76.2, 76.5, 78.5, 80.0, 81.1, 81.7 (C-2–6, C-2'-6', PhCH2), 98.1, 100.0, 101.1 (C-1,PhCH), 117.5–137.6 (Im, Ph), 183.0 (CS). A mixture of this compound (152 mg, 0.19 mmol) in dry toluene (5 mL) was added dropwise over 30 min to a stirred solution of refluxing toluene (5 mL) and tri-n-butyltinhydride (1 mL) under N2. After 2 h, the solution was cooled and then concentrated. The residue was extracted with acetonitrile (3 x 10 mL), and the combined extracts were washed with hexane (4 x 10mL). The acetonitrile layer was concentrated and the residue purified on a silica gel column (toluene/EtOAc 6:1) to give 30 (78 mg, 81%). 13C NMR (CDCl3): , 37.2(C-2'), 55.6 (MeO), 62.8, 69.2, 71.7, 73.8, 74.0, 75.3, 75.7, 76.9, 76.9, 78.2, 79.8, 80.4, 81.7 (C-2–6, C-3'-6', PhCH2), 99.0, 101.4, 101.6 (C-1,PhCH), and 126.4–138.9 (Ph). Compound 30 (108mg, 0.14 mmol) in 60% HOAc was stirred at 70°C for 2 h, then toluene was added and the mixture concentrated and purified on a silica gel column to give the debenzylidenated intermediate (78 mg, 81%). 13C NMR (CDCl3): , 36.8 (C-2'), 55.4 (MeO), 63.2, 69.3, 70.4, 71.2, 72.0, 73.7, 73.8, 74.8, 75.3, 78.0, 78.1, 79.4, 84.7 (C-2–6, C-3'-6', PhCH2), 98.3, 101.4 (C-1), and 127.9–138.4 (Ph). This compound (76 mg, 0.11 mmol) was dissolved in MeOH (5 mL) containing Et3N (20µL), Pd/C was added, and the solution was hydrogenolyzed at 100 psi for 24 h. The mixture was then filtered, concentrated, and purified on a silica gel column (CHCl3/MeOH 5:1) to give 31 (33mg, 89%). 13C NMR (CD3OD): , 38.9 (C-2'), 54.4 (MeO), 61.4, 61.5, 68.7, 71.3, 71.7, 71.7, 72.3, 77.0, 82.9 (C-2–6, C-3'–6'), 100.0, 101.0 (C-1); 1H-NMR (CD3OD) selected data: , 4.8 (H-1'); 4.7 (H-1), 2.3 (H-2'), 1.55 (H-2'ax).
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TopAbstractIntroductionResults and discussionMaterials and methodsNote added in proofAcknowledgmentsReferences |
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The crystal structures of banana lectin from two species are published elsewhere in this issue [Meagher, J.L., Winter, H.C., Ezell, P., Goldstein, I.J., and Stuckey, J. (2005) Crystal structure of banana lectin reveals a novel second sugar binding site. Glycobiology, 15, 1033–1042; Singh, D.D., Saikrishnan, K., Kumar, P., Surolia, A., Sekar, K., Vijayan, M. (2005) Unusual sugar specificity of banana lectin from Musa paradisiaca and its probable evolutionary origin. Crystallographic and modelling studies. Glycobiology, 15, 1025–1032].
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This research was supported by grant GM29477 from the National Institutes of Health.
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Abbreviations |
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BanLec, mannose/glucose-binding lectin from banana (Musa acuminata); DMF, dimethyl formamide; DMTST, dimethyl(methylthio)sulfonium triflate; NMR, nuclear magnetic resonance; THP, tetrahydropyran-2-yl; TLC, thin layer chromatography
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