Abstract
Glycosyltransferases (GTs) play a crucial role in mycobacterial cell wall biosynthesis and are necessary for the survival of mycobacteria. Hence, these enzymes are potential new drug targets for the treatment of tuberculosis (TB), especially multiple drug-resistant TB (MDR-TB). Herein, we report the efficient syntheses of Araf(a 1→5)Araf, Galf(β 1→5)Galf and Galf(β 1→6)Galf disaccharides possessing a 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) unit that were prepared as fluorescent disaccharide acceptors for arabinosyl- and galactosyl-transferases respectively. Such analogs may offer advantages relative to radiolabeled acceptors or donors for studying the enzymes and for assay development and compound screening. Additionally, analogs possessing a 5-azidonaphthalene-1-sulfonamidoethyl unit, were prepared as photoaffinity probes for their potential utility in studying active site labeling of the GTs (arabinosyl and galactosyl) in Mycobacterium tuberculosis (MTB). Beyond their preparation, initial biological testing and kinetic analysis of these disaccharides as acceptors towards glycosyltransferases is also presented.
Keywords: arabinofuranose, galactofuranose, fluorescent probe, photoaffinity probe, glycosyltransferases, arabinosyltransferases, galactosyltransferases, tuberculosis
1. Introduction
Tuberculosis continues to be a major public health issue throughout the world.1 Disease incidence remains high, and there are particular concerns relative to the appearance and increase of multi-drug resistant forms of tuberculosis (MDR-TB).2 The emergence of drug-resistance strains of mycobacteria is oftentimes a result of patient non-compliance to treatment protocols, and can be attributed in part to the lengthy treatment times and side effects of current therapies. With advanced HIV disease, immunocompromised patients have a higher risk of developing active tuberculosis - either from new exposure to TB or reactivation of quiescent mycobacteria. Without the aid of an active immune system, treatment can be more difficult and the disease may be more resistant to therapy.3 TB treatment can be exacerbated in AIDS patients undergoing chronic, combined therapies for HIV and attendant opportunistic diseases due to the large number of different agents and an increased likelihood of drug interactions.4 MTB is an obligate intracellular pathogen that persists within macrophages in the human host, and these cells are involved in dissemination of infection.5 Intracellular bacilli are more resistant to treatment due to limited access of drugs to bacteria within macrophages, necessitating chronic treatment with high therapeutic doses of multiple antibiotics lasting six months or longer for effective control and treatment of the disease.6 For these reasons, recent research in tuberculosis has focused on the identification of new drug targets in TB, with a particular emphasis on drugable targets and the development of new, safer agents for the treatment of this disease.
Among the front line drugs for treatment of TB, two drugs isoniazid (INH) and ethambutol (EMB) target the assembly of the mycobacterial cell wall that is essential for the survival of pathogen.7 The structure of the cell wall has now been thoroughly elucidated in terms of its essential complex polysaccharides, the specific chemical linkages therein and the macromolecular structure of the mycolylarabinogalactan complex.8 The cell wall core of members of the Mycobacterium genus consists of an extensively cross-linked peptidoglycan to which is attached the linear D-galactan composed of alternating 5- and 6-linked β-D-Galf units. Chemical analysis of the degradation fragments arising from the reducing end of the arabinogalactan (AG) obtained from MTB, Mycobacterium bovis BCG and Mycobacterium leprae demonstrated the existence of the terminal sequence →5)-D-Galf-(β1→6)-D-Galf-(β1→5)-D-Galf-(β1→4)-L-Rhap-(α1→3)-D-GlcpNAc. This unit is crucial to the cell wall infrastructure and anchors the exterior, waxy mycolate units, known targets of the first line agent INH, to the interior peptidoglycan. The mycobacterial cell wall oligosaccharide is composed of three major sugars, mannopyranose (Manp), galactofuranose (Galf) and arabinofuranose (Araf) in a variety of defined glycosidic linkages. The two major oligosaccharide portions are AG and lipoarabinomannan (LAM). AG is composed of alternate (β1→5) and (β1→6) linked Galf units, linear (α1→5) linked Araf and branched arabinan hexasaccharide at the terminus that consist of (α1→5), (α1→3), (β1→2) linked Araf units (Figure 1). LAM is Manp capped arabinan. The AG complex is critical for the survival of M. tuberculosis, and is a critical part of the cell wall barrier that protects the bacillus within the macrophage and functions as an effective barrier to antibiotics that are commonly used in the treatment of typical Gram positive bacteria.
Figure 1.
Structure representation of M. tuberculosis arabinogalactan (AG) structure
The assembly of the arabinan portions of cell wall polysaccharides in mycobacteria involves a family of arabinosyltransferases (AraTs) that promote the polymerization of decaprenolphosphoarabinose (DPA) as the donor (represented in Figure 2). The galactan portion is prepared through the actions of a bifunctional galactosyltransferase (GalT) enzyme preparing both the (β1→5) and (β1→6) linkages along with another recently characterized enzyme (Rv3782), and both enzymes utilize the donor uridinediphosphate-galactofuranose (UDP-Galf) (represented in Figure 3).8,9 UDP-Galf is produced by the conversion of UDP-galactopyranose (UDP-Galp) by the enzyme UDP-Galp mutase. Mycobacterial viability requires an intact arabinan and galactan, and thus compounds that inhibit these glycosyltransferases are both useful biochemical tools as well as potential lead compounds for new anti-tuberculosis agents.
Figure 2.
Prototypical arabinosyltransferase-catalyzed reaction.
Figure 3.
Prototypical galactosyltransferase-catalyzed reaction.
We have pursued disaccharide analogs designed as probes for mycobacterial cell wall biochemistry and, specifically, as potential glycosyltransferase substrates and inhibitors.10 As an example, photoaffinity probes can play an important role in specific labeling and identification of target biomolecules, and they have been a useful in the identification of receptors and binding sites for various ligands and in locating functional sites of macromolecules.11 Cory et al. utilized the sulfonamide derivatives for N-terminal amino acid labeling of proteins.12 This technique uses photoactivatable, heterobifunctional reagents, which are generally azide derivatives that, upon exposure to ultraviolet light, photo decompose into a reactive nitrene intermediate that subsequently forms a cross-link between the ligand and its binding protein under physiological conditions. These adducts can then be analyzed using gel electrophoresis. This technique is potentially useful as a marker because of its high sensitivity and ease of handling.13
In the present communication we would like to provide a detailed account of the synthesis of fluorescent disaccharide probes (1 – 3) bearing a highly fluorescent 5-N,N-dimethylaminonapthalene-1-sulfonamido14 (dansyl) function (Chart 1). These substrates were designed for the development of non-radiolabelled second generation competitive based acceptor assays that can potentially eliminate the use of radiolabelled donors (DPA and UDP-Galp). Furthermore, this paper describes the preparation of related disaccharides (4 – 6) bearing 1-azido-5-naphthalene sulfonamido13 functionality as potential pro-fluorescent photoaffinity probes to study the nature of AraTs and GalT (Chart 1). Synthetic details and preliminary biological data relating to acceptor activity of these disaccharide probes are discussed.
Chart 1.
Fluorescent (1 – 3) and Photo affinity (4 – 6) probes for glycosyltransferases in MTB.
2. Result and Discussion
2.1 Strategy
We have previously described syntheses of Araf and Galf disaccharides based on linkages present in the cell wall of MTB as acceptors/inhibitors of specific mycobacterial glycosyltransferases (GTs).10 In earlier reports, we briefly communicated the synthesis of fluorescent Araf disaccharides14 and related photoaffinity probes,15 and a Manp photoaffinity probe16 as well as acceptor activity for their respective GTs. The present account, while using a similar synthetic strategy, reports detailed methods with modifications for improving the preparation of final targets in both the photoaffinity and fluorescent disaccharide series.
Several different strategies were adopted for Araf disaccharide preparation to introduce an ethylamino functionality for attachment of naphthylsulfonamido group at the anomeric center (Figure 4). Starting from the readily synthesized Araf thiocresylglycoside 7, a nitroethyl group was introduced at the anomeric center by reaction with commercially available 1-nitroethanol that afforded nitroethyl glycoside 8 in excellent yield. Unfortunately, deacetylation of 8 using several different reagents proved ineffective, and only a poor yield (48%) of deacetylated glycoside was achieved using K2CO3 at −20 °C. Alternatively, glycoside 7 was reacted with commercially available N-(2-hydroxyethyl)phthalimide to afford the desired ethylphthalimido a -glycoside. This product on deacetylation followed by blocking of the hydroxyl groups with tert-butyldimethylsilyl (TBDMS) groups and selective deprotection of the 5-OTBDMS group yielded the acceptor saccharide containing 5-OH group. This acceptor sugar was coupled with thioglycoside donor 7 resulting in the desired a 1→5-linked disaccharide 9. The de-phthaloylation of disaccharide 9 with hydrazine hydrate under several conditions failed to give the desired disaccharide possessing a free ethylamino disaccharide as single product, yielding an inseparable mixture of the blocked amino byproducts as a result of acetyl group migration.
Figure 4.
Finally, a chloroethyl group was introduced at the glycosidic linkage on the reducing end followed by displacement of chloride with azide to synthesize the desired a(1? 5) linked disaccharide. This approach was also utilized to prepare the 1-O-ethylazido glycosides in the Galf disaccharide probes. The fluorescent and photoaffinity disaccharides Araf(a1? 5)Araf, Galf(β1? 5)Galf and Galf(β1? 6)Galf probes (1 – 6) were readily synthesized as described (Scheme 1 – 5).
Scheme 1.
Reagents and Conditions: (a) Cl(CH2)2OH, SnCl4, CH3CN, rt, 85%, (b) NaN3, DMF, 85 °C; 7N NH3 – MeOH, rt, 70% in two steps, (c) BzCl, Py, −78 °C – rt, 58%, (d) TBDMSCl, DMF, imidazole, 85 °C, 81%, (e) 7N NH3 – MeOH, rt, 93%, (f) BF3.Et2O, CH2Cl2, − 20 °C, 66%, (g) Ph3P, benzene, H2O, 50 °C, 87%, (h) Dansyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 95%, (i) 7N NH3 – MeOH, rt, 93%, (j) Et4N+F−, THF, rt, 97%.
Scheme 5.
Reagents & Conditions: (a) 26, NIS, trflic acid, CH2Cl 2, −20 °C, 43%, (b) Ph3P, benzene, H2O, 50 °C, 75%, (c) Dansyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 60%, (d) Azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 68%, (e) SnCl 4, CH2Cl2, thiophenol, −78 to −20 °C, 46: 83%, 48: 81%, (f) 7N NH3 – MeOH, rt, 3: quant., 6: 80%.
2.2 Synthesis of fluorescent probe for AraT’s (Scheme 1)
2.2.1 Synthesis of building blocks
Compound 10, 1,2,3,5-tetra-O-acetyl-D-arabinofuranoside, was obtained from D-arabinose by reported methods.17 Reaction of 10 with chloroethanol in the presence of SnCl 4 gave 11 as the pure α-isomer, anomeric proton as singlet at δ 5.48, after silica gel column chromatography.18 The chloroethyl compound 11 was then heated with NaN3 in dry DMF for 6 hours followed by deacetylation with 7N NH3/MeOH to give 12 in an overall yield of 70% (2 steps) after purification. The 5-position was selectively blocked using 1.0 eq. of benzoyl chloride in dry pyridine at −78 °C followed by warming to room temperature and leaving overnight. Standard workup and chromatography on Silica gel G gave compound 13 in moderate (58%) yield. Both the 2- and 3-positions were blocked with a TBDMS group to give glycoside 14 followed by treatment with 7N NH3/MeOH to obtain the acceptor 15 in high yield. The trichloroacetimidate donor 16 was synthesized as reported.19
2.2.1 Glycosylation and reduction
The trichloroacetimidate donor 16 and the acceptor azidoethyl-2,3-O-di-tert-butyldimethylsilyl-α-D-arabinofuranoside (15) were reacted for 2 hours in the presence of the Lewis acid promoter BF3• Et2O (1.0 eq., dissolved in 2 mL of dry CH2Cl2). Additions were done at 0 °C, and the reaction was carried out under an inert atmosphere in dry CH2Cl2 at room temperature over powdered 4Å molecular sieves. Column chromatography on Silica gel G afforded the pure disaccharide 17 in 66% yield. The azido group was reduced by heating with Ph3P in a benzene/H2O mixture followed by rapid purification via flash chromatography to afford compound 18.
2.2.2 Incorporation of the fluorescent group
The relative instability of 18 necessitated immediate reaction with dansyl chloride in the presence of N-methylimidazole to produce the fluorescently-labeled disaccharide 19 in high yield. Lastly, compound 20 obtained from 19, was de-protected to give the final fluorescent target 1.
2.3 Synthesis of photo affinity probe for AraT’s (Scheme 2)
Scheme 2.
Reagents and conditions: (a) TBDMSCl, DMF, imidazole, 60 °C, 90%, (b) TFA – water (1:1), 0 °C, 68%, (c) 7, NIS, Sn(OTf)2, CH2Cl2, −20 °C, 91%, (d) HCO2NH4, MeOH, 5% Pd/C, rt, 87%, (e) 5-Azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 81%, (f) 7N NH3 – MeOH, rt, 87%, (g) Et4N+F−, THF, rt, 80%.
2.3.1 Synthesis of building blocks
The synthesis of the target probe 4 is represented in Scheme 2. A slightly different approach was adapted that was shorter and more efficient than the synthesis of 22 via selective protection of the 5-position with a benzoyl group as shown in Scheme 1. Compound 19 was first reacted with 3.5 eq. TBDMSCl in the presence of imidazole at 60 °C for two days affording compound 21 in 90% yield, followed by selective deprotection of only the 5-OTBDMS group using TFA – water (1:1) system at 0 °C. The preparation of compound 15 from compound 21 was successfully achieved in one step with a yield of 68% after column purification.
2.3.2 Glycosylation and reduction
The 1-thiocresyl-2,3,5-tri-O-acetyl-a-D-arabinofuranoside donor 720 and the acceptor azidoethyl-2,3-O-di-tert-butyldimethylsilyl-α-D-arabinofuranoside 1522 were reacted for 2 h in the presence of the Lewis acid promoter Sn(OTf)2 and activator N–iodosuccinimide. Addition of the reagents, and the subsequent reaction, were carried out at −20 °C under an argon atmosphere in dry CH2Cl2 over powdered 4 Å molecular sieves. Standard work-up followed by column chromatographic purification on Silica gel G afforded the pure disaccharide 22 in 91% yield. The trans glycosidation (α-linked disaccharide) was confirmed by the appearance of a singlet anomeric proton in 1HNMR spectrum.15 The azido group was reduced by reaction with ammonium formate (H2CO2NH4) in dry methanol using 5% Pd/C as catalyst for 2 h at room temperature followed by rapid purification via flash chromatography to afford compound 23 in 87% yield. Again, the relative instability (probably a result of the acetyl blocking groups) of 23 necessitated immediate reaction of the free amine.
2.3.3 Incorporation of fluorescent group
Disaccharide 23 was reacted immediately with 5-azidonaphthalenesulfonyl chloride in the presence of N–methylimidazole at 0 °C over 4 h. The disaccharide 24 was obtained in good yield (81%). Lastly, compound 24 was deprotected via 25 to give the final target compound (5-azidonaphthalene-1-sulfonamidoethyl)-5-O-(a-D-arabinofuranosyl)-a-D-arabinofuranoside 4.15
2.4 Synthesis of probes for GT
2.4.1 Synthesis of building blocks
Thiocresyl 2,3,5,6-tetra-O-acetyl-β-D-galactofuranoside 26, the building block for disaccharides Galf(β 1? 5)Galf (2) and a Galf(β 1? 5)Galf (3), was readily obtained from D-galactose by reported method.10d,21 In a reaction sequence starting from thioglycoside 26, both acceptors 33 and 36 were prepared possessing either a free 5-OH or 6-OH for coupling with thioglycoside donor 26 as represented in Scheme 3. Compound 26 was reacted with 1-chloroethanol in presence of NIS and Lewis acid promoter Sn(OTf)2 in dichloromethane at −20 °C to produce 27 in 86% yield and α-glycosidation was confirmed by 1H NMR spectra. Compound 27 on further reaction with NaN3 in DMF at 70 °C gave the glycoside 28 possessing the ethylazido functionality. Compound 28 was deacetylated using 7N NH3/MeOH to give a quantitative yield of the deblocked glycoside 29. The 5,6-hydroxyl groups in 29 were first blocked with an isopropylidene group using 2,2′-dimethoxypropane in the presence of a catalytic amount of (1S)-(+)-10-camphorsulphonic acid to yield 30. Treatment of glycoside 30 with p-methoxybenzyl chloride and NaH afforded compound 31 in 98% yield. The ether protecting group was used due to the potential for ester group (benzoyl) migration between hydroxyl groups,10d and the p-methoxybenzyl protecting group was used specifically allowing ready deprotection of the disaccharides without affecting the azido group.
Scheme 3.
Reagents and Conditions: (a) HOCH2CH2Cl, NIS, Sn(OTf)2, CH2Cl2, −20 °C – rt, 86%, (b) NaN3, DMF, 70 °C, quant., (c) 7N NH3 – MeOH, rt, quant., (d) 2,2-Dimethoxypropane, camphor sulfonic acid, acetone, rt, 82%, (e) p-methoxybenzyl chloride (MPMCl), NaH, DMF, 0 °C – rt, 98%, (f) 60% aq. AcOH, 60 °C, 77%, (g) Bu2SnO, MPMCl, toluene, reflux – rt, 90%, (h) TrCl, DMAP, Py, rt − 50 °C, 80%, (i) MPMCl, NaH, THF, 0 °C – rt, 86%, (j) 5% TFA in CH3Cl, 0 °C, 70%.
Saccharide 32 was regio-selectively benzylated via stannylated intermediates after reaction with dibutyltin oxide in boiling toluene and azeotropic removal of water.10d Evaporation gave the crude 5,6-O-stannylene acetal intermediate which was then selectively p-methoxybenzylated at the 6-position. Glycoside 33, possessing a free 5-OH for coupling, was produced in 90% yield after silica gel flash column chromatography.
Glycoside 32 was selectively blocked using trityl chloride in pyridine at 50 °C to give the 6-tritylated derivative 34 in 72% yield. Glycoside 34 was then p-methoxybenzylated to give 35 in 86% yield. Finally, de-tritylation with 10% CF3COOH in chloroform and further purification using column chromatography gave glycoside 36 possessing free 6-OH group in 70% yield.
2.4.2. Glycosylation and deprotection
Once the synthesis of acceptor glycosides 33 and 36 was achieved, disaccharides 37 and 43 were prepared using thioglycoside 26 as the donor, NIS as the promoter, and triflic acid as the Lewis acid as represented in Schemes 4 and 5 respectively.10d
Scheme 4.
Reagents and Conditions: (a) 26, NIS, triflic acid, CH2Cl2, −20 °C, 66%, (b) Ph3P, benzene, H2O, 50 °C, 85%, (c) Dansyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 77%, (d) Azidonaphthalenesulfonyl chloride, N-methylimidazole, CH2Cl2, 0 °C, 68%, (e) 7N NH3 – MeOH, rt, 40: quant., 42: 92%, (f) SnCl4, CH2Cl2, thiophenol, −78 to −20 °C, 2: 33%, 5: 78%.
2.4.2.1. Synthesis of Galf(β 1? 5)Galf disaccharide
The synthesis of β 1? 5 linked Galf disaccharide was carried out by coupling the acceptor glycoside 33 with thiocresyl glycoside donor 26 in dry dichloromethane at −20 °C in presence of promoter NIS and triflic acid to produce disaccharide 37 in 66% yield after column chromatography. β-Glycosidation was confirmed by the appearance of a singlet anomeric proton.14 The azido group was reduced to the amine to produce disaccharide 38 by reduction with H2CO2NH4 in the presence of 5% Pd/C in dry MeOH. Rapid purification gave pure disaccharide 38 in 92% yield as a colorless oil that was stored in a freezer (−20 °C) for further use to prepare the β 1? 5 linked Galf probes.
2.4.2.2. Synthesis of Galf(β 1? 6)Galf disaccharide
The synthesis of the β 1? 6 linked Galf disaccharide was carried out by coupling glycoside 36 and the thiocresyl glycoside donor 26 in dry dichloromethane at −20 °C in the presence of promoter NIS and trifluoromethanesulfonic acid to produce disaccharide 43 in 65% yield after column chromatography. Again, the azido group in disaccharide 43 was reduced to an amino group using H2CO2NH4 over 5% Pd/C in dry MeOH and purification gave disaccharide 44 in 98% yield. It was stored in the freezer (−20 °C) for later use.
2.5. Synthesis of fluorescent and photo affinity probes for GT
2.5.1. Galf(β 1? 5)Galf fluorescent disaccharide (2) and photo affinity probe (5)
Disaccharide 2 was prepared by reaction of 38 with dansyl chloride at 0 °C in the presence of N-methylimidazole and dry CH2Cl2 to give disaccharide 39 in 77% yield after chromatography as shown in Scheme 4. Disaccharide 39 was de-acetylated using 7N NH3 in methanol to produce 40 in quantitative yield after purification. Treatment of 40 with ceric ammonium nitrate to remove the p-methoxybenzyl group in dichloromethane under standard conditions22 was not effective. Addition of tin(IV) chloride and thiophenol to a cold dichloromethane solution of disaccharide 40 at −78 °C and further reaction at −20 °C overnight provided the deblocked disaccharide 2 in moderate 33% yield after purification. In a similar fashion, photoaffinity probe 5 was prepared by reaction of 38 with 5-azidonaphthalene sulfonyl chloride to produce disaccharide 41. Deacetylation followed by debenzylation under conditions used to prepare 2 gave the target 5 in 78 % yield.
2.5.2. Galf(β 1? 6)Galf fluorescent disaccharide (3) and photoaffinity probe (6)
Synthesis of fluorescently labeled β 1? 6 linked Galf disaccharide 3 and photo affinity probe 6 were carried out in a similar fashion to the β 1? 5 linked Galf disaccharides as represented in Scheme – 5. Disaccharide 44 was reacted with dansyl chloride or 5-azidonaphthalene sulfonyl chloride and N-methylimidazole to produce 45 and 47 respectively. These two disaccharides (45 and 47) were first debenzylated with SnCl4 and thiophenol at sub zero temperatures to produce disaccharides 46 and 48 in 83 and 81% respective yields followed by deacetylation to give 3 (quantitative) and 6 (80%).
2.6. Structural elucidation
All mono- and disaccharides were characterized using NMR, MS and CHN analysis. Whenever necessary, NOE, decoupling and DEPT experiments were performed in order to confirm NMR assignments and stereochemistry at the anomeric center of all sugars. Coupling constant correlations were used to assign the individual protons. In Araf glycosides the anomeric proton is seen as singlet or doublet with the 3J1,2 value = 2.0 Hz, suggesting the formation of the α-stereocenter with the D-arabino configuration.23 In Araf disaccharides the H-1 anomeric protons of the reducing sugar (4.58 – 4.83 ppm) are generally seen at lower ppm as compare to non-reducing sugar H-1′ (4.87 – 5.38 ppm) with coupling constant 3J1,2 and 3J1′,2′ = 2.0 Hz (in most cases the coupling constant was found to be 0.0 Hz), suggesting trans-glycosylation and formation of α-linked disaccharides. Similar trends were seen in 13C NMR spectra of disaccharides with C-1 present at higher ppm value (low field region) as compare to C-1′ in the range 110 – 105 ppm. The β-stereochemistry, trans-glycosylation, of all of the D-Galf glycosides was evident by their appearance as a singlet or doublet with 3J1,2 value = 2.0 Hz in the range of 4.98 – 5.08 ppm.24 It is notable that H-1 appeared as ddd at 4.00 ppm with 3J1,2 = 2.4 Hz in compound 29 (D2O as solvent) with additional long-range coupling with H-3 and H-4 protons (J values less = 1.0 Hz), and this observation may be attributable to conformational change in a polar solvent. 1H NMR spectra of Galf disaccharides showed a singlet or doublet due to anomeric H-1 proton in the range of 4.61 – 5.05 ppm and anomeric H-1′ in the range of 4.90 – 5.42 ppm with coupling constants 3J1,2 = 2.0 Hz. As with the Araf disaccharides, the anomeric proton of the reducing sugar in Galf disaccharides is seen at higher field as compared to a non-reducing sugar. In 13C NMR spectra of Galf glycosides the C-1 signal appeared between 108.6 – 105.4 ppm. However, in Galf disaccharides the C-1 proton is seen between 105.8 – 109.9 ppm and C-1′ at higher field in a range between 105.2 – 109.5 ppm. Detailed NMR data (1H and 13C NMR) on all disaccharides (sugar portion) are presented in Table 1 – 4 and the values confirmed the proposed configuration at the anomeric centers and the structures of synthesized disaccharides.
Table 1.
1H NMR Chemical Shifts for Araf Disaccharides
| Shifts δ (ppm), J (Hz) |
|||||||
|---|---|---|---|---|---|---|---|
| Compd | Unit | H-1 (J1,2) | H-2 (J2,3) | H-3 (J3,4) | H-4 (J4,5a) | H-5a (J5a,5b) | H-5b (J4,5b) |
| 17 | Araf | 4.83 (d) (1.4) | 4.06 (dd) (3.7) | 3.99 (dd) (6.5) | 4.08 (m) (5.2) | 3.89 (dd) (11.2) | 3.75 (dd) (3.7) |
| Araf ′ | 5.38 (s) (0) | 5.59 (d) (1.1) | 5.56 (d) (4.8) | 4.62 (ddd) (3.2) | 4.84 (dd) (11.8) | 4.62 (dd) (4.7) | |
|
| |||||||
| 18 | Araf | 4.83 (d) (1.5) | 4.04 (dd) (3.4) | 3.99 (dd) (5.8) | 4.07 (m) (5.4) | 3.89 (dd) (11.0) | 3.72 (dd) (4.3) |
| Araf ′ | 5.37 (s) (0) | 5.58 (s) (0) | 5.57 (d) (7.0) | 4.62 (ddd) (3.1) | 4.84 (dd) (11.8) | 4.69 (dd) (4.7) | |
|
| |||||||
| 19 | Araf | 4.69 (d) (1.8) | 4.04 (dd) (3.3) | 3.91 (m) (n.d.) | 3.98 (m) (4.6) | 3.84 (dd) (10.9) | 3.61 (dd) (3.5) |
| Araf ′ | 5.32 (s) (0) | 5.57 (s) (0) | 5.56 (d) (4.0) | 4.62 (ddd) (3.1) | 4.83 (dd) (11.6) | 4.68 (dd) (4.8) | |
|
| |||||||
| 20 | Araf | 4.68 (d) (1.5) | 3.92 (dd) (3.4) | 3.77 (m) (n.d.) | 3.99 (m) (n.d.) | 3.77 (m) (10.8) | 3.58 (dd) (3.2) |
| Araf ′ | 5.07 (s) (0) | 4.07 (d) (0.8) | 3.99 (m) (2.3) | 4.17 (dd) (2.4) | 3.86 (dd) (11.8) | 3.77 (m) (2.3) | |
|
| |||||||
| 22 | Araf | 4.83 (d) (1.5) | 4.05 (dd) (3.6) | 3.97 (dd) (6.4) | 4.01 (ddd) (4.6) | 3.81 (dd) (11.2) | 3.63 (dd) (3.8) |
| Araf ′ | 5.11 (s) (0) | 5.15 (d) (1.3) | 4.97 (dd) (4.8) | 4.29 (ddd) (3.0) | 4.42 (dd) (11.3) | 4.22 (dd) (5.6) | |
|
| |||||||
| 23 | Araf | 4.82 (d) (1.4) | 3.99 (m) (n.d.) | 3.99 (m) (n.d.) | 3.99 (m) (4.6) | 3.82 (dd) (10.6) | 3.60 (dd) (4.2) |
| Araf ′ | 5.09 (s) (0) | 5.14 (d) (1.5) | 4.97 (dd) (4.7) | 4.28 (ddd) (2.9) | 4.42 (dd) (11.2) | 4.22 (dd) (5.8) | |
|
| |||||||
| 24 | Araf | 4.66 (d) (1.9) | 3.91 (m) (n.d.) | 3.91 (m) (n.d.) | 3.91 (m) (4.5) | 3.78 (dd) (10.8) | 3.51 (m) (n.d.) |
| Araf ′ | 5.07 (s) (0) | 5.14 (d) (1.5) | 4.99 (dd) (4.9) | 4.29 (ddd) (3.1) | 4.43 (dd) (11.4) | 4.23(5.6) | |
|
| |||||||
| 25 | Araf | 4.58 (d) (1.8) | 3.80 (dd) (4.1) | 3.93 (dd) (6.3) | 3.71 (m) (n.d.) | 3.71 (m) (n.d.) | 3.62 (m) (n.d.) |
| Araf ′ | 4.87 (d) (1.8) | 3.99 (dd) (4.0) | 3.86 (ddd) (6.6) | 3.94 (ddd) (3.2) | 3.74 (dd) (11.9) | 3.63 (dd) (4.9) | |
|
| |||||||
| 1 | Araf | 4.68 (d) (1.5) | 3.92 (dd) (3.4) | 3.77 (m) (n.d.) | 3.99 (m) (n.d.) | 3.77 (m) (10.7) | 3.58 (dd) (3.2) |
| Araf ′ | 5.07 (s) (0) | 4.05 (d) (0.8) | 3.99 (m) (4.5) | 4.17 (dd) (2.3) | 3.86 (dd) (11.8) | 3.77 (m) (2.3) | |
|
| |||||||
| 4 | Araf | 4.63 (d) (1.2) | 3.79 (dd) (2.8) | 3.84 (m) (5.8) | 3.96 (ddd) (3.3) | 3.73 (dd) (11.9) | 3.63 (dd) (5.5) |
| Araf ′ | 4.89 (d) (1.3) | 3.96 (dd) (3.5) | 3.84 (m) (5.8) | 3.84 (m) (4.8) | 3.76 (dd) (10.9) | 3.52 (dd) (3.8) | |
Table 4.
13C NMR Chemical Shifts for Galf Disaccharides.
| Compd | Unit | Shifts δ (ppm) |
|||||
|---|---|---|---|---|---|---|---|
| C-1 | C-2 | C-3 | C-4 | C-5 | C-6 | ||
| 37 | Gal f | 106.2 | 87.9 | 83.2 | 80.5 | 73.9 | 70.5 |
| Gal f ′ | 105.1 | 80.9 | 76.5 | 80.4 | 69.4 | 63.0 | |
|
| |||||||
| 38 | Gal f | 106.0 | 87.8 | 83.2 | 80.5 | 74.0 | 70.5 |
| Gal f ′ | 105.1 | 80.9 | 76.5 | 80.4 | 69.3 | 63.0 | |
|
| |||||||
| 39 | Gal f | 105.8 | 87.4 | 82.9 | 81.1 | 74.1 | 70.3 |
| Gal f ′ | 105.2 | 81.0 | 76.4 | 80.3 | 69.3 | 63.0 | |
|
| |||||||
| 40 | Gal f | 106.6 | 86.2 | 83.5 | 81.9 | 73.4 | 69.3 |
| Gal f ′ | 105.3 | 87.4 | 78.7 | 78.6 | 70.9 | 63.8 | |
|
| |||||||
| 41 | Gal f | 105.9 | 87.4 | 82.9 | 81.3 | 74.3 | 70.2 |
| Gal f ′ | 105.2 | 81.1 | 76.5 | 80.3 | 69.4 | 63.1 | |
|
| |||||||
| 42 | Gal f | 107.8 | 81.2 | 77.6 | 84.7 | 77.1 | 62.7 |
| Gal f ′ | 105.6 | 80.1 | 76.2 | 81.8 | 69.2 | 61.6 | |
|
| |||||||
| 43 | Gal f | 106.5 | 88.1 | 82.1 | 80.5 | 75.1 | 67.8 |
| Gal f ′ | 105.7 | 81.1 | 76.5 | 80.1 | 69.3 | 62.7 | |
|
| |||||||
| 44 | Gal f | 105.9 | 87.4 | 81.9 | 81.1 | 75.5 | 67.7 |
| Gal f ′ | 105.7 | 81.2 | 76.5 | 80.0 | 69.3 | 62.7 | |
|
| |||||||
| 45 | Gal f | 108.3 | 86.1 | 79.2 | 78.5 | 71.0 | 69.3 |
| Gal f ′ | 106.3 | 81.8 | 76.0 | 79.9 | 69.1 | 62.6 | |
|
| |||||||
| 46 | Gal f | 106.0 | 87.4 | 81.9 | 81.2 | 75.5 | 67.6 |
| Gal f ′ | 105.7 | 81.1 | 76.5 | 80.1 | 69.3 | 62.8 | |
|
| |||||||
| 47 | Gal f | 108.2 | 85.7 | 79.4 | 78.1 | 69.7 | 69.1 |
| Gal f ′ | 106.2 | 79.9 | 76.1 | 81.7 | 69.2 | 62.7 | |
|
| |||||||
| 2 | Gal f | 109.4 | 83.1 | 78.5 | 84.3 | 77.2 | 64.3 |
| Gal f ′ | 109.2 | 82.6 | 78.8 | 85.0 | 72.3 | 62.8 | |
|
| |||||||
| 3 | Gal f | 109.9 | 82.9 | 78.9 | 85.0 | 72.6 | 70.6 |
| Gal f ′ | 109.5 | 82.8 | 78.8 | 85.6 | 71.1 | 64.5 | |
|
| |||||||
| 5 | Gal f | 109.3 | 82.6 | 78.5 | 84.2 | 77.1 | 64.2 |
| Gal f ′ | 109.1 | 83.1 | 78.7 | 84.9 | 72.2 | 62.7 | |
|
| |||||||
| 6 | Gal f | 109.9 | 82.9 | 78.9 | 85.5 | 71.0 | 70.5 |
| Gal f ′ | 109.4 | 82.8 | 78.8 | 85.0 | 72.5 | 64.4 | |
2.8. Glycosyltransferase activity of disaccharides
The effectiveness of these compounds as photoaffinity and fluorescent probes to study the mycobacterial glycosyltransferases (AraT’s and GalT) requires that they act as enzyme substrates. As such, a cell-free assay system using the membrane fraction from M. smegmatis was used to evaluate their acceptor activity.10c,d,24
2.8.1. Arabinosyltransferases activity
Based on the previous use of specific arabinose based neoglycolipid acceptors,10 probe disaccharides 1 and 4 were assayed for acceptor activity. As expected, both compounds showed acceptor activity in assays performed in the presence of mycobacterial membrane fractions containing GT activity and resulted in [14C]Araf incorporation from DP-[14C]A as the arabinose donor. TLC/autoradiography for the reaction products from 1 is shown in Figure 5. Data for probe 4 were described earlier.15 Both probes demonstrated the enzymatic conversion of the disaccharides to their corresponding trisaccharide products. Different concentrations of 1 (2 mM maximum) and of 4 (8 mM maximum) were used for arabinosyltransferase activity. Calculation of kinetic constants revealed that probe 1 possessed a Km value 0.68 mM whereas probe 4 possessed a Km value 3.07 mM. Based on these data, both probes are clearly substrates for the arabinosyltransferases. Compound 1 may be useful in the development of competition-based acceptor assay for the discovery of inhibitors of a crucial mycobacterial enzyme. Disaccharide 4, on the other hand, may be useful as a photoaffinity probe for labeling active site residues of the arabinosyltransferase.
Figure 5.
Kinetic analysis of acceptor 1 [SRI#20309 - the inset shows the double reciprocal plot for 1 as a substrate for the arabinosyltransferase. The TLC autoradiogram shows the fluorescent trisaccharide reaction product mixture resulting from a(1→5) and β(1→2) arabinofuranosyltransferase activities as reported earlier25 through the inclusion of 1 at the highest concentration (2 mM), mycobacterial membranes and [14C]-DPA. TLC/autoradiography was performed using chloroform:methanol: NH4OH:H2O (65:25:0.5:3.6) and products revealed through exposure to Kodak X-Omat film at −70 °C for 48 h.
2.8.2. Galactosyltransferases activity
All four Galf probes 2, 3, 5 and 6 were screened for acceptor activity.10d Assays were performed in the presence of membranes and the cell wall enzymatic fraction P60 and resulted in excellent [14C]Galf incorporation from UDP-[14C]Galp, following endogenous conversion to UDP-[14C]Galf and transferase activity for all Galf probes. Different concentrations of probes 2 and 3 in the range of 0 to 4 mM were used for galactosyltransferase activity. In the case of probes 5 and 6, a concentration range of 0 to 6 mM was utilized. TLC/autoradiography (Figure 6 and 8) demonstrated acceptor activity and the likely enzymatic conversion of all Galf disaccharides to their corresponding trisaccharide products and incorporation of [14C]Gal to the 5′-OH of 2 and 5 and the 6′-OH of 3 and 6. Galf disaccharides 3, 4 and 6 gave rise to a second, slower migrating band (Figure 6 and 8) which, based on relative migration profiles, would be anticipated to be a tetrasaccharide product resulting from further elongation of the trisaccharide products at the 6′-OH or 5′-OH consistent with the alternating linkage pattern of AG. Calculation of kinetic constants (Figure 7 and 9) revealed that 2 and 3 possessed Km values 7.33 mM and 2.38 mM, respectively whereas 5 and 6 possessed Km values 2.77 mM and 1.98 mM, respectively. The acceptor data clearly suggested that disaccharide probes 2 and 3 can be used for the development of competitive-based assays for inhibitor discovery, and that disaccharides 5 and 6 may be useful for probing the bi-functional galactosyltransferase.
Figure 6.
An autoradiogram of reaction products produced through the inclusion of disaccharides 2 (SRI 20413, upper panel) and 3 (SRI 20414, lower panel), mycobacterial membranes and UDP[14C]Galf. Lane 1, no acceptor; Lane 2, 0.25 mM; Lane 3, 0.5 mM; Lane 4, 0.75 mM; Lane 5, 1.0 mM; Lane 6, 1.5 mM; Lane 7, 2.0 mM; and Lane 8, 4.0 mM. TLC/autoradiography was performed using CHCl3:MeOH:NH4OH:H2O (65:25:0.4:3.6) and products revealed through exposure to Kodak X-Omat film at −70 °C for 3 days.
Figure 8.
An autoradiogram of reaction products produced through the inclusion of disaccharides 5 (SRI 20668) and 6 (SRI 20669), mycobacterial membranes and UDP[14C]Galf. Lane 1, no acceptor; Lane 2, 0.5 mM; Lane 3, 0.75 mM; Lane 4, 1.0 mM; Lane 5, 2.0 mM; Lane 6, 4.0 mM; and Lane 7, 6.0 mM. TLC/autoradiography was performed using CHCl3:MeOH:NH4OH:H2O (65:25:0.4:3.6) and products revealed through exposure to Kodak X-Omat film at −70 °C for 3 days.
Figure 7.
Kinetic analysis of acceptor disaccharides 2 (SRI 20413) and 4 (SRI 20414). The inset illustrates the double reciprocal plot for disaccharides as a substrate for the mycobacterial galactosyltransferase.
Figure 9.
Kinetic analysis of acceptor disaccharides 5 (SRI 20668) and 6 (SRI 20669). The inset illustrates the double reciprocal plot for disaccharides as a substrate for the mycobacterial galactosyltransferase.
2.9. Conclusion
In summary, we reported a detailed account of the syntheses of Araf (a1? 5) Araf, Galf (β1? 5) Galf and Galf (β1? 6) Galf disaccharides as potential fluorescent and photoaffinity probes (1 – 6). All probe disaccharides were found to be very stable at room temperature but were stored in the refrigerator at 4 °C in dark bottles when not in use. These disaccharides have shown acceptor activity in cell free assay systems for the target glycosyltransferases. Extensive developmental work utilizing these compounds is in progress and will be reported elsewhere. Competition-based assays are being developed to substitute a fluorescence-based assay for the reported assays based on radiolabeled decaprenolphosphoarabinose (14C-DPA) for AraT’s and UDP-[14C]Galf (used for in situ preparation of UDP-[14C]Galp) for GalT. Recently, we have reported the use of Galf (β1? 5) Galf and Galf (β1? 6) Galf disaccharides as potential photoaffinity probes to identify the galactosyltransferase (glfT) in MTB.§ Very recently the isolation and purification of glfT in milligram quantities has been reported by expression of the Rv3808c gene in Escherichia coli C41 (DE3) and purification of protein from the culture.26 These probes may also be useful for labeling and identification of active site residues.
3. EXPERIMENTAL SECTION
3.1. Synthesis
3.1.1. General Procedures
The reactions were performed under a dry argon atmosphere and reaction temperatures were measured externally. Anhydrous solvents were directly purchased and used as such in reactions. Whenever necessary, compounds and starting materials were dried by azeotropic removal of water with toluene under reduced pressure. The reactions were monitored by thin-layer chromatography (TLC) on precoated silica gel (60F254) plates (0.25 mm) and visualized using UV light (254 nm) and/or heating after spray with (NH4)2SO4 solution (150 g ammonium sulfate, 30 mL H2SO4, 750 mL H2O). All solvents used for work-up and chromatography were reagent grade. Flash column chromatography was carried out on silica gel 60 (230–400 Mesh). 1H and 13C NMR spectra were recorded at 300 MHz and 75 MHz respectively. Specific 1HNMR spectra were recorded at 600 MHz as required. The coupling constants (J) are reported in Hz and chemical shifts are in ppm (δ) relative to a residual solvent peak or an internal standard. FAB mass spectra were recorded either by adding NBA (3-nitrobenzyl alcohol) or LiCl and in some cases ESI-MS spectra were recorded on a BioTof-2 time-of-flight mass spectrometer. The presence of water, when presented in the analytical results, was confirmed by 1H NMR as per journal requirements.
3.1.2. Chloroethyl 2,3,5-tri-O-acetyl-a-D-arabinofuranoside (11)
To a dry CH3CN (800 mL) solution of 2,3,5,6-tetra-O-acetyl-α-D-arabinofuranoside 10 (26.0 g, 81.69 mmol) was added SnCl 4 dropwise and the mixture was stirred for 30 min at room temperature. 1-Chloroethanol (7.5 mL) was added dropwise to the reaction which was stirred for another 1 h at room temperature. Celite (5.0 gm) was added, the suspension was cooled in an ice-water bath, and a saturated aqueous NaHCO3 solution was added dropwise to precipitate tin salts. After complete precipitation, the mixture was filtered through celite and extracted with chloroform (2×500 mL). The chloroform layer was again extracted with a cold saturated solution of NaHCO3 (2×100 mL) followed by water (100 mL) and brine (100 mL). The organic layer was dried over Na2SO4 and concentrated to an oil. Column chromatography (CHCl3 100%) afforded pure α-isomer 11 (22.1 g, 85%) as an oil. FABMS: m/z 525 [M+H]+. Anal. calcd for C13H19ClO8: C, 46.14; H, 5.65. Found C, 46.09; H, 5.69. 1H NMR (DMSO-d6): δ 5.61 (d, 1H, J 4.4 Hz, H-3), 5.48 (s, 1H, H-1), 5.41 (d, 1H, J 1.2 Hz, H-2), 4.76 (dd, 1H, J 5.0, 13.8 Hz, H-5a), 4.64 (dd, 1H, J 4.7, 13.8 Hz, H-5b), 4.65 (m, 1H, H-4), 3.95 (m, 1H, OCH2), 3.87 (m, 3H, OCH2, CH2Cl).
3.1.3. Azidoethyl a-D-arabinofuranoside (12)
To a dry DMF (500 mL) solution of compound 11 (31.0 g, 91.52 mmol) was added NaN3 (8.9 g, 137.28 mmol). The reaction mixture was heated at 85 °C for 6 h, left at rt for 2 days at which time 300 mL of acetone/ether (2: 1) was added. It was filtered through celite, and the solid was washed with acetone/ether mixture (20 mL). The combined filtrate was concentrated to an oil. 7N NH3/MeOH (200 mL) was added under inert atmosphere and the mixture was stirred overnight at rt. The reaction mixture was concentrated to an oil and purified by column chromatography using CHCl3 – MeOH (95:5) as the eluting solvent to yield compound 12 (14.4 g, 70%). FABMS: m/z 220 [M+H]+. Anal. calcd for C7H13N3O5 • 1.0 H 2O: C, 35.44; H, 6.37; N, 17.71. Found C, 35.39; H, 6.57; N, 17.89. 1H NMR (DMSO-d6): δ 5.36 (d, 1H, J 5.4 Hz, 2-OH), 5.12 (d, 1H, J 5.4 Hz, 3-OH), 4.77 (d, 1H, J1,2 1.9 Hz, H-1), 4.72 (t, 1H, J 5.6 Hz, 5-OH), 3.83 (dd, 1H, J1,2 1.9 Hz, J2,3 4.2 Hz, H-2), 3.78 (m, 2H, H-4, OCH2), 3.68 (dd, 1H, J2,3 4.2, J3,4 7.1 Hz, H-3), 3.60 (dd, 1H, J4,5a 3.0 Hz, J5a,5b 11.9 Hz, H-5a), 3.57 (m, 1H, OCH2), 3.46 (dd, 1H, J4,5b 5.7 Hz, J5a,5b 11.9 Hz, H-5b), 3.44 (m, 2H, CH2N3).
3.1.4. Azidoethyl 5-O-benzoyl-a-D-arabinofuranoside (13)
The compound 12 (14.4 g, 65.69 mmol) was azeotroped with pyridine and re-dissolved in 500 mL of dry pyridine. It was cooled in to −78 °C and BzCl (7.6 mL, 65.69 mmol) was added dropwise. After complete addition, the reaction mixture was stirred overnight at rt. It was poured in ice-water mixture and extracted with CHCl3 (200 mL). The organic layer was washed with water (2×50 mL), dried over Na2SO4 and concentrated to oil. Column chromatography using CHCl3 – MeOH (98:2) as the eluting solvent gave compound 13 (12.4 g, 58%). FABMS (LiCl): m/z 330 [M+Li]+. Anal. calcd for C14H17N3O6 • 1.0 H2O: C, 49.25; H, 5.61; N, 12.31. Found C, 49.29; H, 5.58; N, 12.29. 1H NMR (DMSO-d6): δ 7.98, 7.67, 7.54 (each m, Ph), 5.54 (d, 1H, J 5.1 Hz, 2-OH), 5.43 (d, 1H, J 5.3 Hz, 3-OH), 4.85 (d, 1H, J1,2 2.0 Hz, H-1), 4.51 (dd, 1H, J4,5a 2.9 Hz, J5a,5b 11.9 Hz, H-5a), 4.31 (dd, 1H, J4,5b 5.9 Hz, J5a,5b 11.9 Hz, H-5b), 4.07 (ddd, 1H, J3,4 2.9 Hz, J4,5a 2.9 Hz, J4,5b 5.9 Hz, H-4), 3.87 (ddd, 1H, J1,2 2.0 Hz, J2,3 4.6 Hz, H-2), 3.78 (m, 2H, H-3, OCH2), 3.59 (m, 1H, OCH2), 3.40 (m, 2H, CH2N3).
3.1.5. Azidoethyl 2,3-O-di-tert-butyldimethylsilyl-5-O-benzoyl-a-D-arabinofuranoside (14)
Compound 13 (12.4 g, 38.39 mmol) was dissolved in dry DMF (500 mL) and to it was added TBDMSCl (21.5 g, 141 mmol) and imidazole (19.2 g, 282 mmol). The reaction mixture was heated at 60 °C for one days and concentrated. The residue was dissolved in CHCl3 (40 mL) and washed with water (10 mL). Organic layer was dried over Na2SO4 and concentrated to syrup. Column chromatography (cyclohexane – EtOAc, 9:1) afforded compound 21 (17.1 g, 81%) as an oil. FABMS (NBA): m/z 558 [M+H]+. Anal. calcd for C26H45N3O6Si2: C, 56.59; H, 8.23; N, 7.62. Found C, 56.58; H, 8.19; N, 7.70. 1H NMR (CDCl3): δ 8.07, 7.57, 7.44 (each m, Ph), 4.89 (d, 1H, J1,2 1.1 Hz, H-1), 4.57 (dd, 1H, J4,5a 3.3 Hz, J5a,5b 11.9 Hz, H-5a), 4.36 (dd, 1H, J4,5b 5.7 Hz, J5a,5b 11.9 Hz, H-5b), 4.23 (ddd, 1H, J4,5a 3.3 Hz, J4,5b 5.7 Hz, J3,4 6.2 Hz, H-4), 4.10 (dd, 1H, J1,2 1.3 Hz, J2,3 3.3 Hz, H-2), 4.04 (dd, 1H, J2,3 3.3 Hz, J3,4 6.2 Hz, H-3), 3.91 (m, 1H, OCH2), 3.59 (m, 1H, OCH2), 3.46 (m, 1H, CH2N3), 3.35 (m, 1H, CH2N3).
3.1.6. Azidoethyl 2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (15)
Method A
Compound 14 (17.0 g, 30.52 mmol) was dissolved in 7N NH3/MeOH (200 mL) under inert atmosphere and stirred overnight at rt. The reaction mixture was concentrated to an oil and subjected to column chromatography using cyclohexane – EtOAc (9:1) as the eluting solvent to give compound 15 (12.5 g, 93%) as a colorless oil.
Method B
To a THF (8.86 mL) solution of Compound 21 (1.8 g, 3.20 mmol) was added TFA – H2O (1:1, 17.5 mL) at 0 °C and the solution was stirred for 4 h. A saturated aqueous solution of NaHCO3 (20 mL) was added and the mixture was diluted with CHCl3 (50 mL). The organic layer was washed with 15 mL of deionized water and concentrated. Purification by column chromatography (cyclohexane – EtOAc, 3:1) afforded 15 (980 mg, 68%) as colorless oil. Spectral and analytical data reported earlier.13
3.1.7. Azidoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (17)
A dry CH2Cl2 (20 mL) solution of 15 (1.00 g, 2.24 mmol) and 16 (1.63 g, 2.69 mmol) was cooled to −20 °C and stirred for 15 min. To it BF3·Et2O (0.34 mL, 2.69 mmol) was added dropwise and the reaction was stirred for another 2 h at −20 °C. The reaction mixture was poured into a cold, saturated solution of NaHCO3 (20 mL). The organic layer was washed with water (2×25 mL), brine (20 mL) and dried over Na2SO4. After concentration and column chromatography (cyclohexane – EtOAc, 5:1), pure disaccharide 17 (1.2 g, 60%) was obtained as an oil. Spectral and analytical data have been reported.14
3.1.8. Aminoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (18)
Compound 17 (1.0 g, 1.16 mmol) was dissolved in 50 mL of benzene, Ph3P (608 mg, 2.32 mmol) was added, and the reaction was heated to 50 °C. After 1 hr, water (2.8 mL) was added and heating was continued for 5 h. The reaction mixture was concentrated and dissolved in CHCl3, washed with water (2×10 mL) and brine (10 mL). The organic layer was dried over Na2SO4, concentrated and chromatographed (CHCl3 – MeOH, 95:5) to give 18 (845 mg, 87%) as a colorless oil. Spectral and analytical data have been reported.13
3.1.9. 5-N,N-Dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(2,3,5-tri-O-benzoyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (19)
To a dry CH2Cl 2 (25 mL) solution of 18 (805 mg, 0.93 mmol) was added N-methylimidazole and the mixture was cooled to 0 °C. To it 5-N,N-dimethylaminonaphthalene-sulfonyl chloride (378 mg, 1.40 mmol) was added and the reaction was stirred at 0 °C for 3 h. The mixture was washed with water and brine, and the organic layer was dried over Na2SO4. Concentration followed by column chromatography (cyclohexane – EtOAc, 5:1) gave 19 (970 mg, 95%) as a light yellow oil. Spectral and analytical data have been reported.13
3.1.10. 5-N,N-Dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (20)
Compound 19 (900 mg, 0.82 mmol) was debenzoylated with 7N NH3/MeOH (30 mL) as described for the preparation of 15. Purification by column chromatography (CHCl3 – MeOH, 96:4) afforded 20 (567 mg, 88%) as a light yellow oil. FABMS (LiCl): m/z 793.5 [M+Li]+. Anal. calcd for C36H62N2O11SSi2: C, 54.94; H, 7.95; N, 3.56. Found: C, 54.98; H, 7.92; N, 3.50. 1H NMR (CDCl3): non-sugar signals, δ 8.53 (d, 1H, J 8.6 Hz, Ph), 8.29 (d, 1H, J 8.7 Hz, Ph), 8.22 (dd, 1H, J 1.2, 7.4 Hz, Ph), 7.53 (m, Ph), 7.18 (d, 1H, J 7.6 Hz, Ph), 5.30 (m, 1H, NH, D2O exchangeable), 3.50 (m, 2H, OCH2), 3.25 (d, 1H, J 11.4 Hz, 3′-OH), 3.08 (m, 2H, CH2NH), 2.89 (s, 2H, 2xNCH3), 0.87, 0.86 (s, 9H each 6xCH3), 0.08 (s, 3H, CH3), 0.06 (s, 6H, CH3), 0.05 (s, 3H, CH3). 13C NMR (CDCl3): non-sugar signals, δ 151.9, 134.7, 130.4, 129.9, 129.6, 129.5, 128.3, 123.1, 118.9, 115.2 (Ph), 66.8 (OCH2), 45.4 (NCH3), 43.3 (CH2NH), 25.7, 25.7 (CH3), 17.83, 17.82 (C), −4.3, −4.61, −4.67, −4.8 (CH3).
3.1.11. Azidoethyl 2,3,5-tri-O-tert-butyldimethylsilyl-a-D-arabinofuranoside (21)
Compound 12 (780 mg, 3.56 mmol) was silylated by reaction with TBDMSCl (1.84 mg, 12.5 mmol) and imidazole (1.5 mg, 21.36 mmol) in dry DMF (40 mL) as described for the preparation of 14. Column chromatography (cyclohexane – EtOAc, 5:1) afforded 21 (1.80 g, 90%) as an oil. Spectral and analytical data have been reported.14
3.1.12. Azidoethyl 5-O-(2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (22)
Alcohol 15 (980 g, 2.05 mmol) and thioglycoside donor 1-Deoxy-1-thiocresyl-2,3,5-tri-O-acetyl-a-D-arabinofuranoside19 7 (942 mg, 2.46 mmol) and activated, powdered 4Å molecular sieves (100 mg) in dry CH2Cl2 (30 mL) were cooled at 0 °C under argon atmosphere. The mixture was stirred for 15 min, and NIS (553 mg, 2.46 mmol) followed by Sn(OTf)2 (86 mg, 0.21 mmol) were added to initiate coupling. The reaction mixture was stirred for 30 min at rt, and the reaction was quenched by addition of Et3N (5 mL), diluted with CH2Cl2 (60 mL) and filtered through a celite pad. The filtrate was washed with 10% Na2S2O3 (20 mL), followed by washing with saturated aqueous NaHCO3 (20 mL). The organic layer was dried over Na2SO4, the solvent was removed under vacuum, and the residue was purified by column chromatography (cyclohexane – EtOAc 3:1) to give pure disaccharide 22 (1.33 g, 91%) as an oil. ESI-MS: m/z 728.3233 [M+Na]+, calcd 728.3216 [M+Na]+ for C30H55N3O12Si2. 1H NMR (CDCl3): non-sugar signals, d 3.88 (m, 1H, OCH2), 3.57 (m, 1H, OCH2), 3.44 (m, 1H, CH2N3), 3.34 (m, 1H, CH2N3), 2.10 (s, 6H, 2xCOCH3), 2.09 (s, 3H, CH3), 0.99 (s, 9H, 3xCH3), 0.88 (s, 9H, 3xCH3), 0.102 (9H, s, 3xCH3), 0.099 (s, 6H, 2xCH3), 0.081 (s, 3H, CH3). 13C NMR (CDCl3): non-sugar signals, d 170.6, 170.1, 169.4 (COCH3), 66.4 (OCH2), 50.8 (CH2N3), 25.8, 25.7 (C), 20.75, 20.72, 20.7 (COCH3), 17.83, 17.79 (CH3), −4.3, −4.7, −4.8, −4.9 (CH3).
3.1.13. Aminoethyl 5-O-(2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (23)
Disaccharide 22 (500 mg, 0.71 mmol) was dissolved in dry MeOH (10 mL) and 5% Pd/C was added (500 mg) under argon atmosphere. H2CO2NH4 (179 mg, 2.83 mmol) was added and the reaction mixture was stirred at rt for 2 h. TLC showed complete conversion, and the reaction was filtered through a short celite pad and concentrated to syrup. The concentrate was dissolved in CHCl3 (20 mL), washed with deionized water (10 mL) and dried over Na2SO4. Evaporation of the organic layer afforded the crude disaccharide 23 (420 mg) that was used as such to prepare 24. 1H NMR (CDCl3): major non-sugar signals in crude sample, δ 3.71 (m, 1H, OCH2), 3.43 (m, 1H, OCH2), 2.86 (m, 2H, CH2NH), 2.10 (s, 6H, CH3), 2.09 (s, 3H, CH3), 0.90 (s, 12H, CH3), 0.88 (s, 12H, CH3), 0.10, 0.09, 0.08 (CH3’s).
3.1.14. 5-azidonaphthalene-1-sulfonamidoethyl 5-O-(2,3,5-tri-O-acetyl-a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (24)
Crude 24 (420 mg, 0.62 mmol) was coupled with 5-azidonaphthalenesulfonyl chloride (248 mg, 0.93 mmol) in the presence of N-methylimidazole (102 mg, 1.24 mmol) using dry CH2Cl2 (50 mL) as solvent as described for the preparation of 19. Purification by column chromatography (cyclohexane – EtOAc, 3:1) afforded 24 (453 mg, 81%) as a light yellow oil (light sensitive). Spectral and analytical data have been reported.14
3.1.15. 5-azidonaphthalene-1-sulfonamidoethyl 5-O-(a-D-arabinofuranosyl)-2,3-O-di-tert-butyldimethylsilyl-a-D-arabinofuranoside (25)
Compound 24 (430 mg, 0.47 mmol) was deacetylated by 7N NH3/MeOH (6 mL) as described for the preparation of 20. Column chromatography (CHCl3 – MeOH, 95:5) afforded pure 25 (322 mg, 87%) as a light sensitive, yellow oil. Spectral and analytical data have been reported.14
3.1.16. Chloroethyl 2,3,5,6-tetra-O-acetyl-β-D-galactofuranoside (27)
β-D-Galactofuranose pentaacetate (26)21 (20.0 g, 51.28 mmol) was treated with SnCl 4 (6.0 mL, 51.28 mmol) and 1-chloroethanol (4.12 mL, 61.52 mmol) in dry CH3CN (150 mL) as described for the preparation of 11. Purification by column chromatography (cyclohexane – EtOAc, 2:1) yielded 27 (13.44 g, 64%) as an oil. FABMS (LiCl): m/z 417 [M+Li]+. Anal. calcd for C16H23ClO10: C, 46.78; H, 5.64. Found: C, 46.69; H, 5.60. 1H NMR (300 MHz, CDCl3) δ: 5.39 (ddd, 1H, J4,5 3.7 Hz, J5,6a 4.7 Hz, J5,6b 7.0 Hz, H-5), 5.09 (dd, 1H, J1,2 0.7 Hz, J2,3 2.0 Hz, H-2), 5.08 (s, 1H, H-1), 5.01 (ddd, 1H, J1,3 0.7 Hz, J2,3 2.0 Hz, J3,4 6.0 Hz, H-3), 4.34 (dd, 1H, J5,6a 4.7 Hz, J6a,6b 11.8 Hz, H-6a), 4.33 (ddd, 1H, J1,4 0.3 Hz, J4,5 3.5 Hz, J3,4 6.0 Hz, H-4), 4.21 (dd, 1H, J5,6b 7.0 Hz, J6a,6b 11.8 Hz, H-6b), 3.90 (m, 1H, OCH2), 3.78 (m, 1H, OCH2), 3.66 (m, 2H, CH2Cl), 2.14, 2.12, 2.10, 2.07 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): δ 170.5, 170.00, 169.95, 169.6 (COCH3), 105.5 (C-1), 81.3 (C-2), 80.1 (C-4), 76.3 (C-3), 70.0 (C-5), 67.7 (OCH2), 62.4 (C-6), 42.6 (CH2Cl), 20.8, 20.72, 20.68, 20.63 (4xCOCH3).
3.1.17. Azidoethyl 2,3,5,6-tetra-O-acetyl-β-D-galactofuranoside (28)
To a dry DMF (70 mL) solution of compound 27 (4.80 g, 11.66 mmol) was added NaN3 (1.89 g, 29.2 mmol), and the reaction mixture was stirred at 70 °C overnight. The mixture was concentrated to near dryness and 100 mL of acetone – ether (2:1) was added and the mixture was stirred for 20 min at rt. It was filtered and concentrated to dryness. The resulting oil was re-dissolved in CHCl3 (200 mL), washed with water (2×40 mL), dried over Na2SO4 and concentrated. Column chromatography (cyclohexane – EtOAc, 2:1) gave compound 28 as pale yellow oil (4.88 g, quant.). FABMS (LiCl): m/z 424 [M+Li]+. Anal. calcd for C16H23N3O10: C, 46.04; H, 5.56; N, 10.07. Found: C, 46.06; H, 5.62; N, 9.99. 1H NMR (300 MHz, CDCl3): δ 5.41 (ddd, 1H, J4,5 3.7 Hz, J5,6a 4.7 Hz, J5,6b 7.0 Hz, H-5), 5.08 (d, 1H, J2,3 1.9 Hz, H-2), 5.07 (s, 1H, H-1), 5.02 (ddd, 1H, J1,3 0.4 Hz, J2,3 1.9 Hz, J3,4 5.7 Hz, H-3), 4.34 (dd, 1H, J5,6a 4.7 Hz, J6a,6b 11.8 Hz, H-6a), 4.32 (ddd, 1H, J4,5 3.7 Hz, J3,4 5.7 Hz, H-4), 4.23 (dd, 1H, J5,6b 7.0 Hz, J6a,6b 11.8 Hz, H-6b), 3.89 (ddd, 1H, J 3.6, 6.9, 10.6 Hz, OCH2), 3.66 (ddd, 1H, J 3.4, 6.1, 10.6 Hz, OCH2), 3.45 (ddd, 1H, J 3.4, 6.9, 13.3 Hz, CH2N3), 3.36 (ddd, 1H, J 3.6, 6.1, 13.3 Hz, CH2N3), 2.14, 2.12, 2.09, 2.07 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): δ 170.3, 169.9, 169.8, 169.4 (COCH3), 105.4 (C-1), 81.1 (C-2), 80.2 (C-4), 76.2 (C-3), 69.1 (C-5), 66.5 (OCH2), 62.3 (C-6), 50.3 (CH2N3), 20.6, 20.54, 20.48, 20.43 (4xCOCH3).
3.1.18. Azidoethyl β-D-galactofuranoside (29)
Compound 28 (4.48 g, 10.7 mmol) was debenzoylated with 7N NH3/MeOH (10 mL) as described for the preparation of 20. Purification by column chromatography (CHCl3 – MeOH, 5:1) afforded 29 (2.67 g, 98%) as an oil. FABMS (NBA): m/z 250 [M+H]+. Anal. calcd for C8H15N3O6 •1.5 H2O: C, 34.78; H, 6.57; N, 15.21. Found: C, 34.77; H, 6.59; N, 15.20. 1H NMR (300 MHz, D 2O): δ 4.00 (ddd, 1H, J1,2 2.4 Hz, J1,3 0.6 Hz, J1,4 0.7 Hz, H-1), 3.98 (m, 2H, H-2, H-3), 3.90 (ddd, 1H, J1,4 0.7 Hz, J3,4 6.0 Hz, J4,5 4.0 Hz, H-4), 3.80 (m, 1H, OCH2), 3.73 (ddd, 1H, J4,5 4.0 Hz, J5,6a 4.5 Hz, J5,6b 7.4 Hz, H-5), 3.64 (m, 1H, OCH2), 3.60 (dd, 1H, J5,6a 4.5 Hz, J6a,6b 11.7 Hz, H-6a), 3.55 (dd, 1H, J5,6b 7.4 Hz, J6a,6b 11.7 Hz, H-6b), 3.42 (m, 2H, CH2N3). 13C NMR (75 MHz, D2O): δ 107.6 (C-1), 83.0 (C-4), 81.3 (C-2), 76.9 (C-3), 70.9 (C-5), 66.9 (OCH2), 63.0 (C-6), 50.6 (CH2N3).
3.1.19. Azidoethyl 5,6-isopropyledine-β-D-galactofuranoside (30)
To a solution of compound 29 (2.60 g, 10.4 mmol) in dry acetone (20 mL) was added 2,2′-dimethoxypropane (2.57 mL, 20.9 mmol) and (1S)-(+)-10-camphorsulfonic acid (242 mg, 1.04 mmol) at rt. After 1 h of stirring, the pH was adjusted to 7 by adding Et3N. Concentration under vacuum gave the product which was dissolved in CHCl3 (250 mL), washed with saturated NaHCO3 (50 mL) and water (50 mL), dried over Na2SO4, and concentrated to an oil. Purification by column chromatography (CHCl3 – MeOH, 99:1) yielded 30 (2.46 g, 82%) as an oil. FABMS (NBA): m/z 290 [M+H]+. Anal. Calcd for C11H19N3O6•0.5 H2O: C, 44.29; H, 6.76; N, 14.09. Found: C, 44.29; H, 6.79; N, 14.01. 1H NMR (300 MHz, CDCl3): δ 5.08 (s, 1H, H-1), 4.37 (ddd, 1H, J4,5 1.4 Hz, J5,6a 6.5 Hz, J5,6b 7.2 Hz, H-5), 4.14 (dd, 1H, J3,4 1.8 Hz, J4,5 1.4 Hz, H-4), 4.12 (dd, 1H, J5,6a 6.5 Hz, J6a,6b 8.3 Hz, H-6a), 4.05 (m, 3H, H-2, H-3, 3-OH), 4.02 (dd, 1H, J5,6b 7.2 Hz, J6a,6b 8.3 Hz, H-6b), 3.93 (m, 1H, OCH2), 3.65 (m, 1H, OCH2), 1.46 (m, 2H, CH2N3), 2.88 (d, 1H, J 11.6 Hz, 2-OH). 13C NMR (75 MHz, CDCl3): δ 110.0 (C), 108.5 (C-1), 85.6 (C-4), 78.6 (C-3), 78.4 (C-2), 75.5 (C-5), 65.9 (C-6), 65.5 (OCH2), 50.6 (CH2N3), 25.5, 25.9 (2xCH3).
3.1.20. Azidoethyl 5,6-isopropyledine-2,3-di-O-p-methoxybenzyl-β-D-galactofuranoside (31)
To a dry DMF solution (50 mL) of compound 30 (1.50 g, 5.19 mmol) was added NaH (60% suspension in mineral oil - 498 mg, 20.76 mmol) followed by p-methoxybenzyl chloride (2.81 mL, 20.76 mmol) dropwise with cooling at 0 °C. The reaction mixture was stirred at rt overnight. The reaction mixture was cooled to 0 °C and MeOH (23 mL) was added to the reaction. The mixture was evaporated to near dryness, and re-dissolved in CHCl3 (200 mL). It was washed with water (50 mL), dried over Na2SO4, and purified by column chromatography (cyclohexane – EtOAc, 5:1) to give 31 (2.69 g, 98%) as an oil. FABMS (LiCl): m/z 536 [M+Li]+. Anal. calcd for C27H35N3O8: C, 61.24; H, 6.66; N, 7.93. Found: C, 61.11; H, 6.49; N, 7.77. 1H NMR (300 MHz, CDCl3): δ 7.29, 7.18 (m, each 2H, Ph), 6.89 (m, 4H, Ph), 5.07 (s, 1H, H-1), 4.44 (m, 4H, 2xCH2Ph), 4.14 (ddd, 1H, J4,5 6.8 Hz, J5,6a 6.9 Hz, J5,6b 7.3 Hz, H-5), 4.04 (dd, 1H, J1,2 1.0 Hz, J2,3 2.9 Hz, H-2), 4.00 (dd, 1H, J3,4 7.0 Hz, J4,5 6.8 Hz, H-4), 3.94 (m, 1H, OCH2), 3.83 (dd, 1H, J5,6a 6.9 Hz, J6a,6b 6.4 Hz, H-6a), 3.81, 3.80 (s, each 3H, 2xOCH3), 3.77 (dd, 1H, J5,6b 7.3 Hz, J6a,6b 6.4 Hz, H-6a), 3.72 (dd, 1H, J2,3 2.9 Hz, J3,4 3.4 Hz, H-3), 3.61 (m, 1H, OCH2), 3.47 (m, 1H, CH2N3), 3.32 (m, 1H, CH2N3), 1.40, 1.35 (s, each 3H, 2xCH3). 13C NMR (75 MHz, CDCl3) δ: 159.4, 159.3 (2xC), 129.7 (CH), 129.3 (C), 113.8, 113.7 (2xCH), 109.6 (C), 106.4 (C-1), 87.5 (C-2), 83.5 (C-3), 82.0 (C-4), 76.5 (C-6), 71.8, 71.6 (2xCH2Ph), 66.3 (OCH2), 65.3 (C-6), 55.2, 55.2 (2xOCH3), 50.5 (CH2N3), 26.4, 25.4 (2xCH3).
3.1.21. Azidoethyl 2,3-di-O-p-methoxybenzyl-β-D-galactofuranoside (32)
A solution of compound 31 (2.50 g, 4.72 mmol) in 60% aqueous acetic acid (75 mL) was stirred for 4 h at 60 °C. The solution was concentrated in vacuum, coevaporated with diluted with CHCl3 (20 mL) and neutralized with saturated NaHCO3 solution. The organic phase was washed with water (2×50 mL), dried over Na2SO4, and purified by column chromatography (CHCl3 – MeOH, 95:5) to give 32 (1.78 g, 77%) as an oil. FABMS: m/z 507 [M+NH4]+. Anal. calcd for C24H31N3O8 • 0.5 H2O: C, 57.82; H, 6.47; N, 8.43. Found: C, 57.85; H, 6.47; N, 8.39. 1H NMR (300 MHz, CDCl3): δ 7.27, 7.20 (each m, each 2H, Ph), 6.88 (m, 4H, Ph), 5.04 (s, 1H, H-1), 4.46 (m, 4H, 2xCH2Ph), 4.08 (dd, 1H, J3,4 6.8 Hz, J4,5 3.9 Hz, H-4), 4.03 (dd, 1H, J1,2 1.1 Hz, J2,3 2.9 Hz, H-2), 4.00 (dd, 1H, J2,3 2.9 Hz, J3,4 6.8 Hz, H-3), 3.85 (m, 1H, OCH2), 3.81, 3.79 (s, each 3H, 2xOCH3), 3.73 (m, 1H, H-5), 3.65 (m, 2H, H2-6), 3.59 (m, 1H, OCH2), 3.46 (m, 1H, CH2N3), 3.33 (m, 1H, CH2N3), 2.51 (d, 1H, J 7.0 Hz, 5-OH), 2.23 (dd, 1H, J 5.1, 7.3 Hz, 6-OH). 13C NMR (75 MHz, CDCl3) δ: 159.4, 159.32 (2xC), 129.7, 129.5 (3xCH), 129.4, 129.1 (2xC), 113.8, 113.8 (2xCH), 106.6 (C-1), 87.1 (C-2), 83.0 (C-3), 82.2 (C-4), 71.9, 71.8 (2xCH2Ph), 71.4 (C-5), 66.3 (OCH2), 64.3 (C-6), 55.2, 55.2 (2xOCH3), 50.5 (CH2N3).
3.1.22. Azidoethyl 2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (33)
A toluene (150 mL) solution of compound 32 (749 mg, 1.53 mmol) and dibutyltin oxide (380 mg, 1.53 mmol) was refluxed overnight with azeotropic removal of water. The reaction mixture was cooled and evaporated to dryness. The residue was dissolved in dry DMF (10 mL) and anhydrous CsF (465 mg, 3.06 mmol) and p-methoxybenzyl chloride (0.45 mL, 3.06 mmol) were added. The resulting mixture was stirred overnight at rt under argon, concentrated under vacuum, redissolved in chloroform (150 mL), and washed with 10% aqueous KF solution and water (10 mL). The combined organic phase was dried, concentrated and chromatographed (cyclohexane – EtOAc, 3:1) to afford pure 33 (840 mg, 90%) as an oil. FABMS (LiCl): m/z 616 [M+Li]+. Anal. calcd for C32H39N3O9 • 0.5 H2O: C, 62.12; H, 6.52; N, 6.79. Found: C, 62.15; H, 6.53; N, 6.75. 1H NMR (300 MHz, CDCl3): δ 7.23 (m, 6H, Ph), 6.87 (m, 6H, Ph), 5.04 (s, 1H, H-1), 4.44 (m, 6H, 3xCH2Ph), 4.08 (ddd, 1H, J1,4 0.8 Hz, J3,4 6.9 Hz, J4,5 3.5 Hz, H-4), 4.01 (d, 1H, J2,3 2.6 Hz, H-2), 4.00 (dd, 1H, J2,3 2.6 Hz, J3,4 6.9 Hz, H-3), 3.86 (m, 1H, H-5), 3.84-3.76 (m, 1H, OCH2), 3.80, 3.795, 3.790 (each s, each 3H, 3xOCH3), 3.57 (m, 1H, OCH2), 3.48 (d, 2H, J 6.0 Hz, H2-6), 3.43 (m, 1H, CH2N3), 3.31 (m, 1H, CH2N3), 2.40 (d, 1H, J 5.9 Hz, 5-OH). 13C NMR (75 MHz, CDCl3) δ: 159.3, 159.2, 159. 1 (3xC), 129.9, 129.6 (2xC), 129.6, 129.4, 129.2 (3xCH), 128.4 (C), 113.7, 113.67, 113.64 (3xCH), 106.4 (C-1), 87.2 (C-2), 82.8 (C-3), 81.5 (C-4), 72.9 (C-6), 71.7, 71.6, 71.1 (3xCH2Ph), 69.9 (C-5), 66.2 (OCH2), 55.15, 55.13, 55.12 (3xOCH3), 50.4 (CH2N3).
3.1.23. Azidoethyl 2,3-di-O-p-methoxybenzyl-6-O-trityl-β-D-galactofuranoside (34)
Compound 32 (1.29 g, 2.64 mmol) was dissolved in dry pyridine (45 mL) and trityl chloride (1.47 g, 5.28 mmol) was added followed by DMAP (32 mg, 0.26 mmol). The reaction mixture was stirred overnight at 50 °C. The solution was concentrated to dryness and re-dissolved in CHCl3 (200 mL). The CHCl3 layer was washed with water (2×50 mL), brine (2×50 mL) and dried over Na2SO4. Concentration of the combined organic layers followed by column chromatography (cyclohexane – EtOAc, 2:1) gave pure 34 (1.54 g, 80%) as an oil. FABMS (LiCl): m/z 737 [M+Li]+. Anal. calcd for C43H45N3O8: C, 70.57; H, 6.20; N, 5.74. Found: C, 70.67; H, 6.23; N, 5.79. 1H NMR (300 MHz, CDCl3): δ 7.42 (m, 6H, Ph), 7.25 (m, 11H, Ph), 7.16 (m, 2H, Ph), 6.85 (m, 4H, Ph), 5.01 (s, 1H, H-1), 4.42 (m, 4H, CH2Ph), 4.17 (m, 1H, H-4), 4.00 (d, 1H, J2,3 2.4 Hz, H-2), 3.98 (dd, 1H, J2,3 2.4 Hz, J3,4 5.2 Hz, H-3), 3.84-3.76 (m, 2H, H-5, OCH2), 3.80, 3.79 (s, each 3H, 2xOCH3), 3.54 (m, 1H, OCH2), 3.40 (m, 1H, CH2N3), 3.26 (m, 2H, H-6a, CH2N3), 3.17 (dd, 1H, J5,6b 5.9 Hz, J6a,6b 9.4 Hz, H-6b), 2.32 (d, 1H, J 7.1 Hz, 5-OH). 13C NMR (75 MHz, CDCl3) δ: 159.4, 159.2 (2xC), 143.8, 129.7 (2xC), 129.6, 129.4 (2xCH), 129.2 (C), 128.6, 127.7, 126.9 (3xCH), 113.8, 113.7 (2xCH), 106.5 (C-1), 87.2 (C-2), 86.6 (C), 83.0 (C-3), 81.7 (C-4), 71.9, 71.6 (2xCH2Ph), 70.0 (C-5), 66.2 (OCH2), 64.8 (C-6), 55.2 (OCH3), 50.5 (CH2N3).
3.1.24. Azidoethyl 2,3,5-tri-O-p-methoxybenzyl-6-O-trityl-β-D-galactofuranoside (35)
To a dry THF solution (50 mL) of compound 34 (1.54 g, 2.11 mmol) was added NaH (60% suspension in mineral oil - 152 mg, 6.32 mmol) followed by p-methoxybenzyl chloride (0.85 mL, 6.32 mmol) dropwise with cooling at 0 °C. The reaction mixture was stirred at rt overnight. MeOH (20 mL) was added to the reaction, the mixture was evaporated to near dryness, and redissolved in CHCl3 (250 mL). It was washed with water (2×50 mL), dried over Na2SO4, and purified by column chromatography (cyclohexane – EtOAc, 6:1) to give 35 (1.53 g, 86%) as an oil. FABMS (LiCl): m/z 857 [M+Li]+. Anal. calcd for C51H53N3O9 • 0.15 H2O: C, 72.28; H, 6.18; N, 4.86. Found: C, 72.25; H, 6.22; N, 4.89. 1H NMR (300 MHz, CDCl3): δ 7.41 (m, 6H, Ph), 7.21 (m, 13H, Ph), 7.06 (m, 2H, Ph), 6.82 (m, 6H, Ph), 4.98 (d, 1H, J1,2 1.4 Hz, H-1), 4.45 (m, 6H, 3xCH2Ph), 4.10 (dd, 1H, J3,4 7.6 Hz, J4,5 3.1 Hz, H-4), 3.96 (dd, 1H, J1,2 1.4 Hz, J2,3 3.8 Hz, H-2), 3.89 (dd, 1H, J2,3 3.8 Hz, J3,4 7.6 Hz, H-3), 3.80, 3.76, 3.75 (s, each 3H, OCH3), 3.72 (m, 1H, OCH2), 3.63 (ddd, 1H, J4,5 3.1 Hz, J5,6a = J5,6b 5.9 Hz, H-5), 3.50 (m, 1H, OCH2), 3.35 (m, 2H, H-6a, CH2N3), 3.22 (m, 2H, H-6b, CH2N3). 13C NMR (75 MHz, CDCl3) δ: 159.3, 159.19, 159.17 (3xC), 143.9, 130.3, 129.93 (3xC), 129.89 (CH), 129.7 (C), 129.6, 129.5, 128.7, 127.7, 126.9, 113.7, 113.7, 113.6 (CH), 106.1 (C-1), 88.3 (C-2), 86.9 (C), 82.3 (C-3), 80.6 (C-4), 75.7 (C-5), 72.9, 71.7, 71.7 (3xCH2Ph), 66.1 (OCH2), 63.6 (C-6), 55.23, 55.20, 55.1 (3xOCH3), 50.5 (CH2N3).
3.1.25. Azidoethyl 2,3,5-tri-O-p-methoxybenzyl-β-D-galactofuranoside (36)
Compound 35 (1.40 g, 1.64 mmol) in CHCl3 (25 mL) was chilled to 0 °C. 5% TFA/CHCl3 (11 mL) was added dropwise over a period of 1.5 h at 0 °C. The reaction mixture was then stirred for another 5 h at 0 °C, neutralized with satd. NaHCO3 and extracted with CHCl3 (200 mL). The organic layer was washed with water (2×50 mL), dried over Na2SO4 and concentrated. Purification by column chromatography (cyclohexane – EtOAc, 3:1) afforded 36 (700 mg, 70%) as an oil. FABMS (LiCl): m/z 616 [M+Li]+. Anal. calcd for C32H39N3O9: C, 63.04; H, 6.45; N, 6.89. Found: C, 63.00; H, 6.42; N, 6.85. 1H NMR (300 MHz, CDCl3): δ 7.23 (m, 6H, Ph), 6.86 (m, 6H, Ph), 5.06 (s, 1H, H-1), 4.44 (m, 6H, 3xCH2Ph), 4.16 (dd, 1H, J3,4 7.4 Hz, J4,5 4.8 Hz, H-4), 4.02 (dd, 1H, J1,2 1.1 Hz, J2,3 3.3 Hz, H-2), 3.95 (dd, 1H, J2,3 3.3 Hz, J3,4 7.4 Hz, H-3), 3.87 (m, 1H, OCH2), 3.81, 3.78, 3.77 (each s, each 3H, 3xOCH3), 3.64 (m, 4H, H-5, H2-6, OCH2), 3.47 (m, 1H, CH2N3), 3.33 (m, 1H, CH2N3), 2.19 (dd, 1H, J = 4.8, 7.8 Hz, 6-OH). 13C NMR (75 MHz, CDCl3) δ: 159.3, 159.2, 159.1 (C), 130.2 (C), 129.6, 129.5 (2xCH), 129.3, 129.2 (2xC), 113.8, 113.7 (2xCH), 106.2 (C-1), 87.7 (C-2), 82.6 (C-3), 81.6 (C-4), 77.7 (C-5), 72.3, 71.8, 71.6 (3xCH2Ph), 66.2 (OCH2), 62.0 (C-6), 55.2, 55.1 (3xOCH3), 50.5 (CH2N3).
3.1.26. Azidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (37)
Compound 33 (2.92 g, 4.78 mmol) was dissolved in dry CH2Cl 2 (50 mL) and powdered 4Å molecular sieves (~500 mg) were added under argon. The mixture was then cooled to −20 °C. The glycosylation donor 26 (3.02 g, 7.18 mmol) in 15 mL dry CH2Cl2 was added dropwise under argon. The mixture was stirred for 15 min, and NIS (2.16 g, 9.56 mmol) followed by triflic acid (4.0 μL, 0.48 mmol) were added. The reaction mixture was allowed to stir for 30 min at −20 °C, quenched by addition of Et3N (5 mL) and filtered through a celite pad. The filtrate was washed with 10% Na2S2O3 (30 mL), followed by washing with saturated aqueous NaHCO3 (20 mL). The organic layer was dried over Na2SO4, the solvent was removed under vacuum, and the residue was purified by column chromatography (cyclohexane – EtOAc, 2:1) to give 37 (3.72 g, 83%) as an oil. FABMS (LiCl): m/z 946.4 [M+Li]+. Anal. calcd for C46H57N3O18 • 0.5 H2O: C, 58.22; H, 6.16; N, 4.23. Found C, 58.19; H, 6.19; N, 4.27. 1H NMR (600 MHz, CDCl3): non-sugar signals, δ 7.22 (m, 6H, Ph), 6.86 (m, 6H, Ph), 4.50, 4.46, 4.42, 4.41, 4.39 (each d, 6H, J = 11.4 Hz, 3xCH2Ph), 3.83 (m, 1H, OCH2), 3.80, 3.794, 3.793 (each s, each 3H, 3xOCH3), 3.58 (m, 1H, OCH2), 3.44 (m, 1H, CH2N3), 3.32 (m, 1H, CH2N3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.4, 170.03, 170.02, 169.3 (4xCOCH3), 159.4, 159.2, 159.1 (3xC), 129.8, 129.3, 129.1, 113.8, 113.7, 113.6 (12xCH), 72.9, 71.9, 71.7 (3xCH2Ph), 66.3 (OCH2), 55.2, 55.2 (3xOCH3), 50.5 (CH2N3), 20.8, 20.7, 20.6, 20.5 (4xCOCH3).
3.1.27. Aminoethyl 5-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (38)
Disaccharide 37 (3.00 g, 3.19 mmol) was dissolved in dry MeOH (50 mL) and 5% Pd/C was added (1.30 g) under argon atmosphere. H2CO2NH4 (807 mg, 12.8 mmol) was added and the reaction mixture was stirred at rt for 4 h. TLC showed complete conversion, and the mixture was filtered through a short celite pad and concentrated to a syrup. It was dissolved in CHCl 3 (100 mL), washed with deionized water (20 mL) and dried over Na2SO4. Concentration afforded the crude disaccharide and chromatography (CHCl3 – MeOH, 9:1) yielded 38 (2.69 g, 92%) as an oil. FABMS (NBA): m/z 914 [M+H]+. Anal. calcd for C46H59NO18 • 0.5 H 2O: C, 59.86; H, 6.55; N, 1.52. Found C, 59.90; H, 6.49; N, 1.48. 1H NMR (600 MHz, CDCl3): non-sugar signals, δ 7.21 (m, 12H, Ph), 6.86 (m, 6H, Ph), 4.48, 4.40 (m, each 6H, 3xCH2Ph), 3.81 (s, 3H, OCH3), 3.79 (s, 6H, 2xOCH3), 3.69 (m, 1H, OCH2), 3.46 (m, 1H, OCH2), 2.87 (m, 2H, CH2NH2), 2.08, 2.06, 1.98, 1.97 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals δ 170.4, 169.9, 169.9, 169.2 (4xCOCH3), 159.3, 159.2, 159.0 (4xC), 130.0, 129.7, 129.3 (3xC), 129.6, 129.3, 129.0 (3xCH), 113.7 (2xCH), 113.6 (CH), 72.9 (CH2Ph), 71.7 (2xCH2Ph), 69.2 (OCH2), 63.0 (C-6′), 55.2 (OCH3), 55.1 (2xOCH3), 41.9 (CH2NH2), 20.7, 20.6, 20.5, 20.4 (4xCOCH3).
3.1.28. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (39)
Compound 38 (350 mg, 0.38 mmol) was mixed with N-methylimidazole (63 mg, 0.76 mmol) and dansyl chloride (155 mg, 0.57 mmol) in dry CH2Cl2 (40 mL) at 0 °C as described for the preparation of 18. Purification by column chromatography (cyclohexane – EtOAc, 1:1) afforded the 39 (340 mg, 77 %) as an oil. FABMS (NBA): m/z 1148 [M+H]+. Anal. calcd for C58H70N2O20S • 0.5 H2O: C, 60.25; H, 6.19; N, 2.42. Found C, 60.19; H, 6.18; N, 2.41. 1H NMR (600 MHz, CDCl3): non-sugar signals δ 8.53 (d, 1H, J 8.4 Hz, Ph), 8.27 (d, 1H, J 8.4 Hz, Ph), 8.23 (dd, 1H, J 0.6, 7.8 Hz, Ph), 7.50 (dd, 1H, J 7.8, 8.4 Hz, Ph), 7.48 (dd, 1H, J 7.2, 8.4 Hz, Ph), 7.19 (m, 6H, Ph), 7.12 (d, 1H, J 7.2 Hz, Ph), 6.87 (d, 4H, J 8.4 Hz, Ph), 6.83 (d, 2H, J 8.4 Hz, Ph), 5.33 (m, 1H, NH), 4.39 (m, 6H, 3xCH2Ph), 3.799, 3.795, 3.791 (each s, each 3H, 3xOCH3), 3.59 (m, 1H, OCH2), 3.40 (m, 1H, OCH2), 3.07 (m, 2H, CH2NH), 2.86 (s, 6H, N(CH3)2), 2.08, 2.06, 1.98, 1.94 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals δ 170.5, 169.9, 169.2 (4xCOCH3), 159.4, 159.3, 159.0 (C), 151.9 (C) 134.8 (C), 130.3 (CH), 130.1 129.8, 129.7 (C), 129.6, 129.5, 129.4 (CH), 129.2 (C), 129.1, 128.3, 123.1, 118.7, 115.1, 113.8, 113.6 (CH), 72.9 (CH2Ph), 71.7 (2xCH2Ph), 65.7 (OCH2), 55.2, 55.2, 55.1 (3xOCH3), 45.3 (N(CH3)2), 43.0 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4xCOCH3).
3.1.29. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(β-D-galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (40)
Disaccharide 39 (300 mg, 0.26 mmol) in dry methanol (5 mL) was treated with 7 N NH3/MeOH (10 mL) overnight at rt. Concentration under vacuum followed by column chromatography (CHCl3 – MeOH, 9:1) yielded 40 (285 mg, 98%) as an oil. FABMS (NBA): m/z 980 [M+H]+. Anal. calcd for C50H62N2O16S • 1.0 H2O: C, 60.23; H, 6.47; N, 2.81. Found C, 60.19; H, 6.43; N, 2.84. 1H NMR (600 MHz, CDCl3): non-sugar signals δ 8.51 (d, 1H, J 8.4 Hz, Ph), 8.25 (d, 1H, J 9.0 Hz, Ph), 8.19 (dd, 1H, J 1.2, 8.4 Hz, Ph), 7.48 (m, 1H, Ph), 7.46 (m, 1H, Ph), 7.18 (m, 4H, Ph), 7.13 (dd, 2H, J 2.4, 10.8 Hz, Ph), 7.11 (d, 1H, J 7.2 Hz, Ph), 6.85 (m, 6H, Ph), 4.35 (m, 6H, 3xCH2Ph), 3.79, 3.78, 3.76 (s, each 3H, 3xOCH3), 3.56 (m, 2H, H-6′b, OCH2), 3.36 (m, 1H, OCH2), 3.03 (m, 2H, CH2NH), 2.85 (s, 6H, N(CH3)2). 13C NMR (75 MHz, CDCl3): non-sugar signals δ 159.5, 159.3, 159.2 (3xC), 151.8 (C), 134.7 (C), 130.3, 129.8 (CH), 129.7 (C), 129.6 (CH), 129.5 (C), 129.3 (CH), 129.2, 128.9 (C), 128.3, 123.1, 118.8, 115.1, 113.9, 113.8, 113.7 (10xCH), 73.0, 71.7, 71.6 (3xCH2Ph), 65.3 (OCH2), 55.3, 55.22, 55.20 (3xOCH3), 45.3 (N(CH3)2), 42.8 (CH2NH).
3.1.30. 5-azidonaphthalene-1-sulfonamidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,6-tri-O-p-methoxybenzyl-β-D-galactofuranoside (41)
Compound 38 (1.70 g, 1.86 mmol) was mixed with N-methylimidazole (0.3 mL, 3.72 mmol) and 1-azido-5-naphthalenesulfonyl chloride (747 mg, 2.79 mmol) in dry CH2Cl2 (50 mL) at 0 °C as described for the preparation of 18. Purification by column chromatography (cyclohexane – EtOAc, 1:1) afforded the 41 (1.22 g, 68%) as an oil. ESI-MS: m/z 1167.37 [M+Na]+. Anal. Calcd for C56H64N4O20S • 0.5 H2O: C, 58.28; H, 5.68; N, 4.85. Found C, 58.27; H, 5.59; N, 4.80. 1H NMR (300 MHz, CDCl3): non-sugar signals, δ 8.38 (m, 2H, Ph), 8.27 (dd, 1H, J 1.2, 8.4 Hz, Ph), 7.55 (dd, 1H, J 3.7, 7.6 Hz, Ph), 7.52 (dd, 1H, J 3.4, 7.4 Hz, Ph), 7.26 (dd, 1H, J 0.7, 15.5 Hz, Ph), 7.19 (m, 6H, Ph), 6.85 (m, 6H, Ph), 5.39 (t, 1H, J 5.2 Hz, NH), 4.38 (m, 3xCH2Ph), 3.79 (m, 3xOCH3), 3.59 (m, 1H, OCH2), 3.40 (m, 1H, OCH2), 3.07 (m, 2H, CH2NH), 2.08, 2.06, 1.98, 1.94 (s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals δ 170.6, 170.0, 169.4 (4xCOCH3), 159.5, 159.4, 159.1 (C), 137.6 (C) 134.9 (C), 130.3 (CH), 130.1 129.6 (C), 129.6, 129.4 (CH), 129.2 (C), 129.2, 128.5, 128.2 (CH), 127.2 (C), 124.3, 121.0, 114.8, 113.9, 113.7 (CH), 73.0, 71.8, 71.7 (3xCH2Ph), 65.7 (OCH2), 55.3, 55.2 (3xOCH3), 43.2 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4xCOCH3).
3.1.31. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-β-D-galactofuranoside (42)
Disaccharide 41 (1.17 g, 1.02 mmol) was dissolved in 110 mL of CH2Cl2 – H2O (10:1) and DDQ (1.16 g, 5.1 mmol) was added at rt. The reaction mixture was stirred for 4 h, dried over Na2SO4 and concentrated to syrup. Purification over silica gel using CHCl3 – MeOH (95:5) afforded pure disaccharide 42 (734 mg, 92%). ESI-MS: m/z 785.20 [M+H]+. Anal. calcd for C32H40N4O17S • 1.5 H 2O: C, 47.35; H, 5.34; N, 6.90. Found C, 47.29; H, 5.33; N, 6.87. 1H NMR (300 MHz, CDCl3): non-sugar signals δ 8.41 (ddd, 1H, J 0.7, 0.8, 8.8 Hz, Ph), 8.39 (ddd, 1H, J 0.8, 1.2, 8.6 Hz, Ph), 8.27 (dd, 1H, J 1.2, 7.4 Hz, Ph), 7.64 (dd, 1H, J 7.6, 8.8 Hz, Ph), 7.54 (dd, 1H, J 7.4, 8.6 Hz, Ph), 7.35 (dd, 1H, J 0.7, 7.6 Hz, Ph), 5.91 (t, 1H, J 5.9 Hz, NH), 3.69 (m, 1H, OCH2), 3.44 (m, 1H, OCH2), 3.28 (d, 1H, J 6.5 Hz, 3-OH), 3.18 (d, 1H, J 9.3 Hz, 2-OH), 3.09 (dd, 2H, J 5.1, 10.4 Hz, CH2NH), 2.87 (br s, 1H, 6-OH), 2.14, 2.13 (each s, each 3H, OCH3), 2.08 (s, 6H, 2xOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 171.2, 170.4, 170.2 (4xCOCH3), 137.6, 134.7 (C), 130.2 (CH), 129.1 (C), 128.5, 128.1 (CH), 127.1 (C), 124.3, 121.0, 114.8 (CH), 66.0 (OCH2), 42.9 (CH2NH), 20.8, 20.7, 20.5 (OCH3).
3.1.32. Azidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-β-D-galactofuranoside (43)
Glycosylation was carried out by the reaction of acceptor 36 (2.20 g, 3.61 mmol) and donor 26 (2.30 g, 5.41 mmol) in the presence of NIS (1.60 g, 7.22 mmol) and triflic acid (30 μL, 0.36 mmol) in dry CH2Cl2 (60 mL) −20 °C as described for 37. Purification by column chromatography (cyclohexane – EtOAc, 2:1) yielded disaccharide 43 (2.35 g, 69%) as an oil. FABMS (LiCl): m/z 946 [M+Li]+. Anal. calcd for C46H57N3O18: C, 58.78; H, 6.11; N, 4.47. Found C, 58.76; H, 6.08; N, 4.43. 1H NMR (600 MHz, CDCl3): non-sugar signals, δ 7.27 (dd, 2H, J 0.3, 9.0 Hz, Ph), 7.22 (dd, 2H, J 0.3 Hz, 9.0 Hz, Ph), 7.13 (dd, 2H, J 0.3, 9.0 Hz, Ph), 6.89 (dd, 2H, J 0.3, 8.4 Hz, Ph), 6.82 (dd, 2H, J 0.3, 9.0 Hz, Ph), 6.81 (dd, 2H, J 0.3, 9.0 Hz, Ph), 4.60, 4.53, 4.44, 4.42, 4.38, 4.21 (each d, each 1H, J 11.4 Hz, 3xCH2Ph), 3.85 (m, 1H, OCH2), 3.81, 3.764, 3.757 (s, each 3H, 3xOCH3), 3.58 (m, 1H, OCH2), 3.44 (m, 1H, CH2N3), 3.34 (m, 1H, CH2N3), 2.12, 2.10, 2.05, 2.03 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.5, 169.9, 169.5 (4xCOCH3), 159.3, 159.22, 159.19 (3xC), 130.2 (C), 129.8 (CH), 129.7 (C), 129.6 (CH), 129.5 (C), 113.7, 113.67, 113.61 (16xCH), 73.0, 71.7, 71.6 (3xCH2Ph), 66.5 (OCH2), 55.2, 55.20, 55.17 (3xOCH3), 50.5 (CH2N3), 20.8, 20.7, 20.6, 20.6 (4xCOCH3).
3.1.23. Aminoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-β-D-galactofuranoside (44)
Disaccharide 43 (2.35 g, 2.50 mmol) was dissolved in dry MeOH (50 mL) and 5% Pd/C was added (2.0 g) under argon atmosphere. H2CO2NH4 (630 mg, 10.0 mmol) was added and the reaction mixture was stirred at rt for 4 h. TLC showed complete conversion, and the mixture was filtered through a short celite pad and concentrated to syrup. It was dissolved in CHCl3 (100 mL), washed with deionized water (20 mL) and dried over Na2SO4. Concentration afforded the crude disaccharide. Column chromatography (CHCl3 – MeOH, 9:1) gave 44 (2.20 g, 98%) as an oil. FABMS (NBA): m/z 914 [M+H]+. Anal. calcd for C46H59NO18: C, 60.45; H, 6.51; N, 1.53. Found C, 60.48; H, 6.47; N, 1.54. 1H NMR (600 MHz, CDCl3): non-sugar signals δ 7.26, 7.23, 7.13 (m, each 2H, Ph), 6.88 (m, 2H, Ph), 6.82 (m, 4H, Ph), 4.61 (d, 1H, J 11.5 Hz, CH2Ph), 4.51 (d, 1H, J 11.4 Hz, CH2Ph), 4.43, 4.42 (each d, 1H, J 11.5 Hz, CH2Ph), 4.38 (d, 1H, J 11.4 Hz, CH2Ph), 4.22 (d, 1H, J 11.5 Hz, CH2Ph), 3.81, 3.77, 3.76 (s, each 3H, 3xOCH3), 3.81-3.70 (m, 3H, OCH2), 2.87 (t, 2H, J 5.3 Hz, CH2NH2), 2.12, 2.10, 2.05, 2.03 (s, each 3H, 4xCOCH3).
3.1.34. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-β-D-galactofuranoside (45)
Coupling of 44 (240 mg, 0.26 mmol) with dansyl chloride (106 mg, 0.40 mmol) was carried out in the presence of N-methylimidazole (43 mg, 0.52 mmol) in dry CH2Cl2 (30 mL) at 0 °C as described for 39. Purification by column chromatography (cyclohexane – EtOAc, 1:1) afforded 45 (180 mg, 60 %) as an oil. FABMS (NBA): m/z 1148 [M+H]+. Anal. calcd for C58H70N2O20S: C, 60.72; H, 6.15; N, 2.44. Found C, 60.69; H, 6.13; N, 2.39. 1H NMR (600 MHz, CDCl3): non-sugar signals, δ 8.52 (d, 1H, J 8.4 Hz, Ph), 8.27 (d, 1H, J 8.4 Hz, Ph), 8.22 (dd, 1H, J 1.2, 7.2 Hz, Ph), 7.31 (m, 2H, Ph), 7.21 (m, 5H, Ph), 7.14 (d, 1H, J 8.4 Hz, Ph), 7.13 (d, 1H, J 7.2 Hz, Ph), 6.88 (m, 3H, Ph), 6.84 (m, 2H, Ph), 6.81 (m, 1H, Ph), 5.40 (t, 1H, J 6.0 Hz, NH), 4.60, 4.43, 4.39, 4.38, 4.34, 4.26 (each d, each 1H, J 11.4 Hz, CH2Ph), 3.81 (s, 3H, OCH3), 3.77, 3.75 (each s, each 3H, 2xOCH3), 3.61 (m, 1H, OCH2), 3.35 (m, 1H, OCH2), 3.04 (m, 2H, CH2NH), 2.86 (s, 6H, N(CH3)2), 2.11, 2.09, 2.01, 2.01 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.5, 170.0, 169.9, 169.5 (4x COCH3), 159.3, 159.2, 159.1 (C), 148.1, 134.8 (C), 130.3 (CH), 130.2 (C), 129.8 (CH), 129.7 (C), 129.6, 129.5, 128.2, 123.2, 118.9, 115.2, 113.8, 113.7, 113.6 (CH), 72.9, 71.6, 71.5 (3xCH2Ph), 65.7 (OCH2), 55.2, 55.2, 55.1 (3xOCH3), 45.4 (NCH3), 42.9 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4xCOCH3).
3.1.35. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-β-D-galactofuranoside (46)
A solution of 45 (150 mg, 0.13 mmol) and thiophenol (0.05 mL, 0.46 mmol) in dry CH2Cl2 (10 mL) was cooled to −78 °C under argon. SnCl4 (0.05 mL, 0.40 mmol) was added and the mixture was stirred 1h at −78 °C and further overnight at −20 °C. A saturated NaHCO3 solution (5 mL) was added and the reaction was further diluted with CHCl3 (30 mL). The organic layer was washed with water (2×10 mL), dried over Na2SO4 and concentrated to an oil. Chromatography (CHCl3 – MeOH, 95:5) yielded 46 (85 mg, 83%) as an oil. FABMS (NBA): m/z 787 [M+H]+. Anal. calcd for C34H46N2O17S • 1.0 H2O: C, 50.74; H, 6.01; N, 3.48. Found: C, 50.77; H, 6.00; N, 3.44. 1H NMR (300 MHz, CDCl3, D2O exchanged): non-sugar signals, δ 8.54 (d, 1H, J 8.6 Hz, Ph), 8.26 (d, 1H, J 8.6 Hz, Ph), 8.54 (dd, 1H, J 1.2, 7.4 Hz, Ph), 7.54 (m, 2H, Ph), 7.19 (d, 1H, J 7.4 Hz, Ph), 5.65 (br s, 1H, NH), 3.68 (m, 1H, OCH2), 3.40 (m, 1H, OCH2), 3.08 (dd, 2H, J 4.6, 5.4 Hz, CH2NH), 2.89 (s, 6H, N(CH3)2), 2.14, 2.12, 2.07, 2.06 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.7, 170.3, 170.1, 170.0 (4xCOCH3), 151.9, 134.7 (C), 130.5 (CH), 129.9, 129.5 (C), 129.4, 128.4, 123.2, 118.8, 115.2 (CH), 65.8 (OCH2), 43.00 (CH2NH), 31.8, 29.4 (N(CH3)2), 20.8, 20.76, 20.72, 20.6 (4xCOCH3).
3.1.36. 5-Azidonaphthalene-1-sulfonamidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-2,3,5-tri-O-p-methoxybenzyl-β-D-galactofuranoside (47)
Compound 44 (2.2 g, 2.41 mmol) was reacted with N-methylimidazole (0.38 mL, 4.82 mmol) and 1-azido-5-naphthaenesulfonyl chloride (967 mg, 0.40 mmol) in dry CH2Cl2 (30 mL) at 0 °C for 4 h as described for 41. Purification by column chromatography (cyclohexane – EtOAc, 1:1) gave disaccharide 47 (1.64 g, 68%) as an oil. ESI-MS: m/z 807.40 [M+Na]+. Anal. calcd for C56H64N4O20S: C, 58.73; H, 5.63; N, 4.89. Found C, 58.76; H, 5.69; N, 4.87. 1H NMR (300 MHz, CDCl3): non-sugar signals, δ 8.40 (d, 1H, J 8.7 Hz, Ph), 8.37 (d, 1H, J 8.3 Hz, Ph), 8.27 (dd, 1H, J 1.0, 7.2 Hz, Ph), 7.53 (m, 2H, Ph), 7.27 (m, 1H, Ph), 7.17 (m, 6H, Ph), 6.84 (m, 6H, Ph), 5.48 (t, 1H, J 5.8 Hz, NH), 4.60, 4.42, 4.37, 4.31, 4.26, 4.22 (each d, each 1H, J 11.4 Hz, CH2Ph), 3.82, 3.78, 3.76 (each s, each 3H, 3xOCH3), 3.56 (m, 1H, OCH2), 3.33 (m, 1H, OCH2), 3.08 (m, 2H, CH2NH), 2.12, 2.09, 2.02, 2.01 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.6, 170.1, 170.00, 169.5 (COCH3), 159.3, 159.3, 159.2 (C), 137.5, 134.8 (C), 130.3 (CH), 130.2 (C), 129.8 (CH), 129.6 (C), 129.5, 129.4 (CH), 129.3, 129.1 (C), 128.4, 128.1 (CH), 127.1 (C), 124.3, 121.0, 114.8, 113.8, 113.7, 113.6 (CH), 72.9, 71.7, 71.5 (3xCH2Ph), 65.6 (OCH2), 55.2, 55.2, 55.1 (3xOCH3), 43.0 (CH2NH), 20.8, 20.7, 20.6, 20.5 (4xCOCH3).
3.1.37. 5-Azidonaphthalene-1-sulfonamidoethyl 6-O-(2,3,5,6-tetra-O-acetyl-β-D-galactofuranosyl)-β-D-galactofuranoside (48)
Disaccharide 47 (1.30 g, 1.14 mmol) was dissolved in 110 mL of CH2Cl 2 – H2O (10:1) and DDQ (1.30 g, 5.7 mmol) was added at room temperature. The reaction mixture was stirred for 4 h, dried over Na2SO4 and concentrated to syrup. Column chromatography (CHCl3 – MeOH, 95:5) afforded 48 (720 mg, 81%) as oil. FABMS (NBA): m/z 639.14 [M+Na]+. Anal. calcd for C32H40N4O17S • 1.0 H2O: C, 48.42; H, 5.21; N, 7.06. Found: C, 48.46; H, 5.27; N, 7.01. 1H NMR (600 MHz, CDCl3): non-sugar signals δ 8.41 (d, 1H, J 8.6 Hz, Ph), 8.40 (d, 1H, J 8.3 Hz, Ph), 8.28 (dd, 1H, J 1.0, 7.3 Hz, Ph), 7.65 (dd, 1H, J 7.6, 8.6 Hz, Ph), 7.55 (dd, 1H, J 7.3, 8.3 Hz, Ph), 7.37 (d, 1H, J 7.6 Hz, Ph), 5.87 (t, 1H, J 6.0 Hz, NH), 4.23 (br s, 1H, OH), 3.68 (m, 1H, OCH2), 3.61 (br s, 1H, OH), 3.41 (m, 1H, OCH2), 3.21 (d, 1H, J 8.3 Hz, OH), 3.09 (dd, 2H, J 5.3, 10.4 Hz, CH2NH), 2.14, 2.12, 2.07, 2.06 (each s, each 3H, 4xCOCH3). 13C NMR (75 MHz, CDCl3): non-sugar signals, δ 170.8, 170.4, 170.1, 17.1 (COCH3), 137.6, 134.7 (C), 130.2 (CH), 129.1 (C), 128.5, 128.2 (CH), 127.1 (C), 124.3, 122.0, 114.9 (CH), 65.8 (OCH2), 42.8 (CH2NH), 20.8, 20.77, 20.72, 20.6 (4xCOCH3).
3.1.38. 5-N,N-Dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(α-D-arabinofuranosyl)-α-D-arabinofuranoside (1)
Compound 20 (540 mg, 0.69 mmol) was dissolved in dry THF (20 mL) and tetraethylammonium fluoride (3.9 mg, 2.07 mmol) was added. The reaction mixture was stirred overnight at rt and concentrated to oil. Column chromatography (CHCl3 – MeOH, 5:1) gave compound 1 as an oil that was dissolved in deionized water (5 mL) frozen and lyophilized to yield a light yellow, hygroscopic solid (372 mg, 97%). Spectral and analytical data have been reported.13
3.1.39. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 5-O-(β-D-galactofuranosyl)-β-D-galactofuranoside (2)
Compound 40 (125 mg, 0.13 mmol) was reacted with thiophenol (0.05 mL, 0.46 mmol) and SnCl4 (0.05 mL, 0.40 mmol) in dry CH2Cl2 (10 mL) as described for 46. Column chromatography (CHCl3/MeOH 3:1) gave 2 as a light sensitive oil. An aqueous solution (5 mL) of the oil was passed through a small column packed with Bio-Beads™ SM-4 (20–50 mesh), and the solution was frozen and lyophilized to afford 2 as a hygroscopic solid (27 mg, 33%). FABMS (NBA): m/z 619 [M+H]+. Anal. calcd for C26H38N2O13S • 1.0 H2O: C, 49.05; H, 6.33; N, 4.40. Found C, 49.00; H, 6.36; N, 4.38. 1H NMR (600 MHz, CD3OD): non-sugar signals, δ 8.56 (dt, 1H, J 1.2, 8.4 Hz, Ph), 8.34 (dt, 1H, J 0.6, 9.0 Hz, Ph), 8.20 (dd, 1H, J 1.2, 7.2 Hz, Ph), 7.59 (dd, 1H, J 8.4, 9.0 Hz, Ph), 7.58 (dd, 1H, J 8.4, 9.0 Hz, Ph), 7.28 (dd, 1H, J 0.6, 7.2 Hz, Ph), 3.55 (m, 1H, OCH2), 3.31 (m, 1H, OCH2), 3.05 (dd, 2H, J 5.4, 7.2 Hz, CH2NH), 2.88 (s, 6H, N(CH3)2). 13C NMR (75 MHz, CD3OD): non-sugar signals, δ 153.2, 137.1 (2xC), 131.2 (CH), 131.2, 130.9 (2xC), 130.0, 129.1, 124.3, 120.6, 116.5 (5xCH), 67.6 (OCH2), 45.8 (N(CH3)2), 43.9 (CH2NH).
3.1.40. 5-N,N-dimethylaminonaphthalene-1-sulfonamidoethyl 6-O-(β-D-galactofuranosyl)-β-D-galactofuranoside (3)
Disaccharide 46 (80 mg, 0.10 mmol) was deacetylated by 7N NH3/MeOH (5 mL) as described for 40. Column chromatography (CHCl 3 – MeOH, 3:1) followed by a short column of Bio-Beads™ SM-4 (20–50 mesh), freezing and lyophilization afforded 3 as yellow fluffy solid (44 mg, 70%). FABMS (NBA): m/z 619 [M+H]+. Anal. calcd for C26H38N2O13S • 1.0 H2O: C, 49.05; H, 6.33; N, 4.40. Found C, 48.99; H, 6.32; N, 4.37. 1H NMR (600 MHz, CD3OD): non-sugar signals, δ 8.56 (1H, dt, J 1.2, 8.4 Hz, Ph), 8.34 (1H, dt, J 1.2, 8.4 Hz, Ph), 8.20 (1H, dd, J 1.2, 7.2 Hz, Ph), 7.59 (1H, dd, J 7.2, 8.4 Hz, Ph), 7.58 (1H, dd, J 7.2, 8.4 Hz, Ph), 7.28 (1H, dd, J 1.2, 8.4 Hz, Ph), 3.59 (1H, m, OCH2), 3.31 (1H, m, OCH2), 3.06 (2H, t, J 5.4 Hz, CH2NH) 2.88 (6H, s, N(CH3)2). 13C NMR (75 MHz, CD3OD): non-sugar signals, δ 153.2, 137.1 (2xC), 131.2 (CH), 131.2, 130.9 (2xC), 130.0, 129.1, 124.3, 120.6, 116.5 (5xCH), 67.5 (OCH2), 45.8 (N(CH3)2), 43.9 (CH2NH).
3.1.41. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-(α-D-arabinofuranosyl)-α-D-arabinofuranoside (4)
25 (300 mg, 0.38 mmol) was dissolved in dry THF (20 mL) and Et4N+F− (171 mg, 1.15 mmol) was added. The reaction mixture was stirred overnight and concentrated to oil. Column chromatography (CHCl3 – MeOH, 5:1) gave compound 4 as an oil that was dissolved in deionized water (5 mL), frozen and lyophilized to get a light sensitive, hygroscopic solid (128 mg, 60%). Spectral and analytical have been reported.14
3.1.42. 5-Azidonaphthalene-1-sulfonamidoethyl 5-O-(β-D-galactofuranosyl)-β-D-galactofuranoside (5)
A solution of compound 42 (610 mg, 0.78 mmol) in dry MeOH (15 mL) was cooled to 0 °C under argon and treated with 7N NH3/MeOH. The reaction mixture was stirred overnight at room temperature and concentrated to dryness. An aqueous solution (5 mL) was passed through a small column of Bio-Beads™ SM-4 (25 g, 20–50 mesh) and product 5 was eluted with MeOH – H2O (0 – 100%). The aqueous solution that remained after brief evaporation was frozen and lyophilized to yield a light sensitive solid (375 mg, 78%). ESI-MS: m/z 639.17 [M+Na]+. Anal. calcd for C24H32N4O13S • 1.0 H2O: C, 45.42; H, 5.40; N, 8.53. Found C, 45.49; H, 5.39; N, 8.61. 1H NMR (600 MHz, CD3OD): non-sugar signals δ 8.49 (dt, 1H, J 0.8, 0.9, 8.7 Hz, Ph), 8.40 (dt, 1H, J 0.9, 1.2, 8.5 Hz, Ph), 8.26 (dd, 1H, J 1.2, 7.3 Hz, Ph), 7.71 (dd, 1H, J 7.6, 8.7 Hz, Ph), 7.60 (dd, 1H, J 7.4, 8.5 Hz, Ph), 7.48 (dd, 1H, J 0.8, 7.6 Hz, Ph), 3.51 (m, 1H, OCH2), 3.28 (m, 1H, OCH2), 3.07 (t, 2H, J 5.6 Hz, CH2NH). 13C NMR (75 MHz, CD3OD): 138.8, 137.1 (C), 130.9 (CH), 130.4 (C), 129.1 (CH), 128.4 (C), 125.5, 122.6, 116.1 (CH), 67.5 (OCH2), 43.9 (CH2NH).
3.1.43.5-Azidonaphthalene -1-sulfonamidoethyl 6-O-(β-D-galactofuranosyl)-β-D-galactofuranoside (6)
Disaccharide 48 (700 mg, 0.89 mmol) was deacetylated in 7N NH3/MeOH (20 mL) as described for the preparation of 40. An aqueous solution (5 mL) crude disaccharide was passed through a small column of Bio-Beads™ SM-4 (25 g, 20–50 mesh) and eluted with MeOH – H2O (0 – 100%). The aqueous solution that remained after brief evaporation was frozen and lyophilized to a solid (439 mg, 80%). ESI-MS: m/z 619 [M+H]+. Anal. calcd for C24H32N4O13S • 1.0 H2O: C, 45.42; H, 5.40; N, 8.53. Found C, 45.49; H, 5.41; N, 8.57. 1H NMR (600 MHz, CD3OD): non-sugar signals, δ 8.50 (d, 1H, J 8.5 Hz, Ph), 8.41 (d, 1H, J 8.5 Hz, Ph), 8.27 (dd, 1H, J 1.2, 7.4 Hz, Ph), 7.72 (dd, 1H, J 7.6, 8.7 Hz, Ph), 7.62 (dd, 1H, J 7.4, 8.5 Hz, Ph), 7.50 (d, 1H, J 7.6 Hz, Ph), 3.56 (m, 1H, OCH2), 3.29 (m, 1H, OCH2), 3.08 (t, 2H, J 5.5 Hz, CH2NH). 13C NMR (75 MHz, CD3OD): non-sugar signals, δ 138.8, 137.2 (C), 130.9 (CH), 130.5 (C), 129.1 (CH), 128.4 (C), 125.5, 122.6, 116.1 (CH), 67.4 (OCH2), 43.9 (CH2NH).
3.2. Biological Activity
3.2.1 Arabinosyltransferase assay
Compounds 1 at a range of concentrations from 0.25 to 2.0 mM and 2 from 0.5 to 8.0 mM were stored as 100 mM ethanol stocks and DP[14C]A (20,000 cpm, 9 mM, 10 μL [stored in chloroform/methanol, 2:1]), were dried under a stream of argon in a microcentrifuge tube (1.5 mL) and placed in a vacuum desiccator for 15 min to remove any residual solvent. The dried constituents of the assay were then resuspended in 8 μl of a 1% aqueous solution of Igepal. The remaining constituents of the arabinosyltransferase assay containing 50 mM MOPS (adjusted to pH 8.0 with KOH), 5 mM β-mercaptoethanol, 10 mM MgCl2, 1 mM ATP, M. smegmatis membranes (250 μg) were added to a final reaction volume of 80 μL. The reaction mixtures were then incubated at 37 °C for 1 h. A CHCl3:CH3OH (1:1, 533 μL) solution was then added to the incubation tubes and the entire contents centrifuged at 18,000 × g. The supernatant was recovered and dried under a stream of argon and re-suspended in C 2H5OH:H2O (1:1, 1 mL) and loaded onto a pre-equilibrated (C2H5OH:H2O [1:1]) 1 mL Whatman strong anion exchange (SAX) cartridge which was washed with 3 mL of ethanol. The eluate was dried and the resulting products partitioned between the two phases arising from a mixture of n-butanol (3 mL) and H20 (3 mL). The resulting organic phase was recovered following centrifugation at 3, 500 × g and the aqueous phase was again extracted twice with 3 mL of n-butanol saturated water, the pooled extracts were back-washed twice with water saturated with n-butanol (3 mL). The n-butanol-saturated water fraction was dried and re-suspended in 200 μL of n-butanol. The total cpm of radiolabeled material extractable into the n-butanol phase was measured by scintillation counting using 10 % of the labeled material and 10 mL of EcoScintA (National Diagnostics, Atlanta). The incorporation of [14C]Araf was determined by subtracting counts present in control assays (incubation of the reaction components in the absence of the compounds). Another 10 % of the labeled material was subjected to thin-layer chromatography (TLC) in CHCl3:CH3OH:NH4OH:H2O (65:25:0.5:3.6) on aluminum backed Silica Gel 60 F254 plates (E. Merck, Darmstadt, Germany). Autoradiograms were obtained by exposing TLC’s to X-ray film (Kodak X-Omat) for 3 days.
3.2.2 Galactosyltrasferase assay
All four disaccharides at a range of concentrations from 0.5 to 6.0 mM were incubated with 0.5 μCi of UDP-[14C]Galp (Amersham, 257 mCi/mmol) in a buffer containing 50 mM MOPS, pH 7.9, 5 mM 2-mercaptoethanol, 10 mM MgCl2, 62.5 μM ATP and 50 μL of a membrane preparation from M. smegmatis (0.5 mg of protein) in a total volume of 160 μL for 1 hour at 37 °C. A chloroform/methanol (1:1, 1.07 mL) solution was then added to the incubation tubes and the entire contents centrifuged at 14,000 rpm. The supernatant was recovered, dried under a stream of argon, resuspended in ethanol:water (1:1, 1 mL) and loaded onto a 1 mL Whatman strong anion exchange (SAX) cartridge. The cartridge was then washed with ethanol (3 mL). The eluants were combined, dried, and the resulting products partitioned between the two phases arising from n-butanol (3 mL) and water (3 mL). The organic phase was recovered and back washed with water saturated with n-butanol twice (3 mL each). The n-butanol fraction was dried, re-suspended in 200 μL of n-butanol saturated with water. Fifty microliters of this solution were subjected to scintillation counting, and another 50 μL were applied to an analytical thin-layer chromatogram developed in chloroform/methanol/ammonium hydroxide/water (65:25:0.5:3.6) and subjected to autoradiography. The incorporation of [14C]Gal was determined by subtracting counts present in control assays (incubation of the reaction components in the absence of the acceptors).
Table 2.
13C NMR Chemical Shifts for Araf Disaccharides.
| Compd | Unit | Shifts δ (ppm) |
||||
|---|---|---|---|---|---|---|
| C-1 | C-2 | C-3 | C-4 | C-5 | ||
| 17 | Araf | 108.5 | 82.1 | 79.1 | 84.1 | 66.5 |
| Araf ′ | 105.9 | 82.1 | 78.1 | 81.2 | 63.7 | |
|
| ||||||
| 18 | Araf | 108.3 | 87.4 | 79.2 | 83.8 | 66.8 |
| Araf ′ | 105.9 | 82.1 | 78.0 | 81.2 | 63.7 | |
|
| ||||||
| 19 | Araf | 108.3 | 82.6 | 78.6 | 83.5 | 66.6 |
| Araf ′ | 106.1 | 82.1 | 77.9 | 81.1 | 63.7 | |
|
| ||||||
| 20 | Araf | 108.5 | 83.5 | 79.1 | 82.5 | 65.8 |
| Araf ′ | 107.5 | 79.2 | 78.0 | 86.9 | 61.9 | |
|
| ||||||
| 22 | Araf | 108.5 | 84.1 | 78.8 | 82.0 | 66.4 |
| Araf ′ | 105.9 | 81.0 | 77.1 | 80.5 | 63.3 | |
|
| ||||||
| 24 | Araf | 108.4 | 82.6 | 78.6 | 83.4 | 66.5 |
| Araf ′ | 106.0 | 81.2 | 77.1 | 80.5 | 63.3 | |
|
| ||||||
| 25 | Araf | 109.6 | 85.3 | 79.9 | 83.2 | 67.4 |
| Araf ′ | 109.8 | 83.4 | 78.4 | 85.3 | 62.7 | |
|
| ||||||
| 1 | Araf | 107.7 | 82.5 | 76.9 | 81.2 | 61.5 |
| Araf ′ | 107.6 | 81.0 | 76.8 | 84.2 | 66.7 | |
|
| ||||||
| 4 | Araf | 109.6 | 83.1 | 79.0 | 85.9 | 67.5 |
| Araf ′ | 109.6 | 83.1 | 78.7 | 84.3 | 63.1 | |
Table 3.
1H NMR Chemical Shifts for Galf Disaccharides.
| Shifts δ (ppm), J (Hz) |
||||||||
|---|---|---|---|---|---|---|---|---|
| Compd | Unit | H-1 (J1,2) | H-2 (J2,3) | H-3 (J3,4) | H-4 (J4,5) | H-5 (J5,6a) | H-6a (J6a,6b) | H-6b (J5,6b) |
| 37 | Galf | 5.05 (s) (0) | 4.01 (s) (0) | 4.07 (m) (5.9) | 4.00 (dd) (3.5) | 4.07 (m) (3.6) | 3.62 (dd) (10.2) | 3.59 (dd) (3.6) |
| Galf ′ | 5.24 (s) (0) | 5.17 (d) (1.8) | 4.96 (dd) (5.4) | 4.35 (dd) (4.2) | 5.32 (ddd) (3.6) | 4.24 (dd) (12.0) | 4.13 (dd) (7.8) | |
|
| ||||||||
| 38 | Galf | 5.04 (s) (1.2)* | 3.98*(dd) (3.0) | 3.99 (m) (n.d.) | 4.06 (m) (n.d.) | 4.06 (m) (3.6) | 3.62 (dd) (10.4) | 3.59 (dd) (3.6) |
| Galf ′ | 5.42 (s) (0) | 5.17 (d) (1.8) | 4.95 (dd) (4.8) | 4.36 (dd) (4.2) | 5.32 (ddd) (3.6) | 4.24 (dd) (12.0) | 4.14 (dd) (7.8) | |
|
| ||||||||
| 39 | Galf | 4.87 (s) (1.2)* | 3.83*(dd) (2.4) | 3.97 (m) (n.d.) | 3.97 (m) (n.d.) | 3.97 (m) (7.2) | 3.55 (dd) (10.4) | 3.50 (dd) (3.6) |
| Galf ′ | 5.39 (s) (0) | 5.15 (d) (1.8) | 4.95 (dd) (5.4) | 4.35 (dd) (3.6) | 5.31 (ddd) (3.6) | 4.24 (dd) (12.0) | 4.12 (dd) (7.8) | |
|
| ||||||||
| 40 | Galf | 4.88 (s) (0) | 3.81 (d) (1.8) | 4.06 (dd) (2.4) | 4.01 (dd) (6.0) | 3.96 (ddd) (4.2) | 3.44 (dd) (n.d.) | 3.44 (dd) (6.0) |
| Galf ′ | 5.26 (s) (0) | 4.01 (s) (0) | 3.79 (m) (5.4) | 3.98 (dd) (5.4) | 3.79 (m) (6.6) | 3.62 (dd) (11.4) | 3.56 (dd) (n.d.) | |
|
| ||||||||
| 41 | Galf | 4.87 (d) (1.2) | 3.79 (m) (n.d) | 3.98 (m) (n.d.) | 3.98 (m) (n.d.) | 3.98 (m) (6.1) | 3.54 (dd) (10.7) | 3.49 (dd) (4.5) |
| Galf ′ | 5.37 (s) (0) | 5.14 (d) (1.6) | 4.95 (dd) (5.6) | 4.34 (m) (3.6) | 5.31 (ddd) (3.6) | 4.25 (dd) (12.2) | 4.12 (dd) (7.5) | |
|
| ||||||||
| 42 | Galf | 4.81 (s) (0) | 3.93† (dd) (1.5) | 4.09 (dd) (3.8) | 4.17 (dd) (3.2) | 3.97 (ddd) (4.9) | 3.86 (dd) (12.2) | 3.78 (dd) (5.8) |
| Galf ′ | 5.05 (s) (0) | 5.34 (d) (1.6) | 4.06 (dd) (5.1) | 4.45 (dd) (3.4) | 5.39 (ddd) (4.4) | 4.39 (dd) (11.9) | 4.22 (dd) (6.9) | |
|
| ||||||||
| 43 | Galf | 5.04 (s) (1.8) | 4.00# (dd) (3.6) | 3.96 (dd) (7.2) | 4.06 (dd) (3.6) | 3.71 (ddd) (4.8) | 3.82 (dd) (10.2) | 3.61 (dd) (7.2) |
| Galf ′ | 5.06 (d) (1.2) | 5.07(dd) (2.4) | 4.95 (dd) (5.4) | 4.23 (dd) (3.6) | 5.38 (ddd) (4.2) | 4.32 (dd) (12.0) | 4.17 (dd) (7.8) | |
|
| ||||||||
| 44 | Galf | 5.04 (s) (0) | 3.95 (m) (n.d.) | 3.95 (m) (6.3) | 4.04 (dd) (3.0) | 3.61 (m) (n.d.) | 3.76 (m) (10.4) | 3.45 (dd) (5.0) |
| Galf ′ | 5.04 (s) (0.5) | 5.07†(dd) (1.8) | 4.98 (dd) (5.6) | 4.24 (dd) (4.0) | 5.38 (ddd) (4.0) | 4.32 (dd) (11.9) | 4.17 (dd) (7.4) | |
|
| ||||||||
| 45 | Galf | 4.86 (s) (0) | 3.81 (d) (3.0) | 3.91 (dd) (6.6) | 4.01 (dd) (3.0) | 3.67 (ddd) (4.8) | 3.79 (dd) (10.2) | 3.56 (dd) (7.2) |
| Galf ′ | 5.01 (s) (0) | 5.07 (d) (1.8) | 4.98 (dd) (5.4) | 4.22 (dd) (3.6) | 5.36 (ddd) (4.2) | 4.30 (dd) (12.0) | 4.17 (dd) (7.2) | |
|
| ||||||||
| 46 | Galf | 4.80 (s) (0) | 3.88 (s) (0) | 4.10 (d) (2.1) | 4.01 (t) (2.1) | 4.03 (ddd) (3.8) | 3.81 (dd) (10.5) | 3.58 (dd) (8.6) |
| Galf ′ | 5.06 (s) (0) | 5.04 (m) (n.d.) | 5.04 (m) (5.8) | 4.31 (dd) (3.6) | 5.36 (ddd) (4.2) | 4.37 (dd) (12.0) | 4.20 (dd) (7.8) | |
|
| ||||||||
| 47 | Galf | 4.83 (s) (0) | 3.77 (m) (3.0) | 3.90 (dd) (6.6) | 3.98 (dd) (3.1) | 3.66 (ddd) (3.1) | 3.77 (m) (n.d.) | 3.56 (m) (6.6) |
| Galf ′ | 5.01 (s) (0) | 5.07 (d) (2.1) | 4.97 (dd) (5.8) | 4.30 (dd) (3.7) | 5.37 (ddd) (3.7) | 4.27 (dd) (11.8) | 4.16 (dd) (7.4) | |
|
| ||||||||
| 48 | Galf | 4.83 (s) (0) | 3.92 (u) (n.d.) | 4.14 (u) (n.d.) | 4.07 (u) (n.d.) | 4.03 (u) (4.0) | 3.80 (dd) (10.5) | 3.58 (dd) (8.5) |
| Galf ′ | 5.06 (s) (0) | 5.04 (d) (2.4) | 5.06 (dd) (5.9) | 4.31 (dd) (3.7) | 5.37 (ddd) (4.2) | 4.37 (dd) (11.9) | 4.20 (dd) (7.1) | |
|
| ||||||||
| 2 | Galf | 4.66 (d) (1.5) | 3.82 (dd) (3.3) | 4.04 (dd) (6.1) | 3.91 (dd) (3.7) | 3.86 (ddd) (6.6) | 3.67 (dd) (11.8) | 3.64 (dd) (5.2) |
| Galf ′ | 5.16 (d) (0.6) | 3.99 (m) (n.d.) | 3.99 (m) (n.d.) | 4.10 (u) (3.1) | 3.72 (ddd) (6.5) | 3.64 (dd) (11.2) | 3.61 (dd) (6.6) | |
|
| ||||||||
| 3 | Galf | 4.67 (d) (1.5) | 3.81 (dd) (3.1) | 3.96$(ddd) (5.4) | 3.82 (m) (n.d.) | 3.82 (m) (4.6) | 3.74 (dd) (10.2) | 3.48 (dd) (7.0) |
| Galf ′ | 4.91 (d) (1.6) | 3.98 (dd) (3.3) | 4.02$(ddd) (5.8) | 3.99 (dd) (3.2) | 3.72 (ddd) (5.7) | 3.64 (dd) (11.2) | 3.61 (dd) (6.9) | |
|
| ||||||||
| 5 | Galf | 4.61 (d) (1.6) | 3.77 (dd) (3.3) | 4.02 (dd) (5.9) | 3.86 (dd) (n.d.) | 3.84 (m) (6.4) | 3.66 (dd) (11.8) | 3.62 (m) (n.d.) |
| Galf ′ | 5.15 (d) (1.5) | 3.99 (m) (n.d.) | 3.99 (m) (n.d.) | 4.09 (m) (5.9) | 3.71 (ddd) (3.1) | 3.62 (m) (11.1) | 3.60 (dd) (6.6) | |
|
| ||||||||
| 6 | Galf | 4.63 (d) (1.4) | 3.76 (dd) (3.1) | 3.95 (dd) (5.7) | 3.77 (dd) (3.8) | 3.81 (ddd) (5.0) | 3.72 (dd) (10.4) | 3.46 (dd) (7.2) |
| Galf ′ | 4.90 (d) (1.4) | 3.98 (dd) (3.3) | 4.02 (dd) (5.9) | 3.99 (dd) (3.2) | 3.72 (dd) (5.6) | 3.63 (dd) (11.2) | 3.61 (dd) (6.9) | |
showed J1,2 = 1.2 Hz;
showed J1,2 = 1.8 Hz;
showed J1,2 = 0.5 Hz;
showed J1,3 = 0.5 Hz;
n.d. = not determined due to complexity of signal, u = unresolved signal
Acknowledgments
This work was supported by NIH/NIAID grant R01AI45317. The authors are thankful to Mr. M. D. Richardson for FABMS, ESI-MS and Ms. Joan Bearden for CHN analysis. The 600 MHz NMR work was supported by NCI grant CA-13148 (the University of Alabama at Birmingham shared NMR Core Facility). GSB acknowledges support from Mr. James Bardrick in the form of a Personal Chair, the Lister Institute as a former Jenner Research Fellow, the Medical Research Council (UK) and the Wellcome Trust.
Footnotes
Alderwick, L. J.; Dover, L. G.; Veerapen, N.; Gurcha, S. S.; Kremer, L.; Roper, D. L.; Pathak, A. K.; Reynolds, R. C.; Besra, G. S. (communicated)
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