Synthesis of the antigenic tetrasaccharide side chain from the major glycoprotein of Bacillus anthracis exosporium

Abstract

A synthesis of the pententyl glycoside of the tetrasaccharide side chain from the major glycoprotein of Bacillus anthracis by a [3+1] approach is described. The construction of the 1,2-trans-glycosidic linkage in the terminal anthrose moiety was achieved through the application of known α-nitrilium ion-mediated β-selective glycosylation methodology. An iterative glycosylation strategy was used for the assembly of the trirhamnan building block. A new route to the anthrose saccharide was developed from D-galactose.

Introduction

Due to the recent uses of Bacillus anthracis, a gram positive bacteria and the etiological agent of anthrax,^1,2 as a biological weapon, inexpensive, effective methods of detection and vaccination against this organism are highly desired. The similarity of the B. anthracis spore-cell-surface antigens with those of the opportunistic human pathogen B. cereus and other bacteria of this group complicates the design of reliable selective antibody-based detection systems. The spores of Bacillus anthracis are enclosed by a prominent loose fitting layer called the exosporium, which is the primary barrier of the spore and the source of the spore antigens,^3,4 and which interacts with the environment, detection devices and with spore-binding cells in the mammalian host and host defenses. The exosporium therefore plays an important role in the spore survival and/or pathogenesis. A highly immunogenic glycoprotein BclA, an important constituent of the exosporium, was found to be substituted with multiple copies of an O-linked oligosaccharide 1,⁵ which contains the previously unknown terminal substituent, 2-O-methyl-4-(3-hydroxy-3-methylbutylamino)-4,6-dideoxy-D-glucose. This sugar, which is also referred to as anthrose, appears to be specific for the spores of this bacteria as it was not found in spores of either Bacillus cereus or Bacillus thuringiensis, the most phylogenetically similar species.⁵ Thus, tetrasacchride 1 is a very attractive tool for the development of species-specific biomarkers, as well as of novel vaccines which target anthrax spores. In view of the fact that pure cell surface oligosaccharides are often difficult to obtain by isolation, effective synthetic approaches to the tetrasaccharide and its analogs are highly desirable.

Since its isolation in 2004,⁵ the preparation of several synthetic tri-⁶ and tetrasaccharide^7–10,11 analogs of 1 has been reported in the literature (Fig. 1). The first total synthesis described, that of the tetrasaccharide analog 2,⁷ featured a terminal pentenyl group, which served as a convenient point of attachment to a carrier protein in vaccine development. In this first synthesis a convergent approach was utilized to facilitate access to analogs and shorter sequences, and assembly of the terminal anthrose was accomplished in a short, straightforward way starting from D-fucose. A different [3+1] tack was taken in the synthesis of 3;^8–10 both a stepwise approach in the direction of the proceeding from the downstream toward the upstream end of the oligosaccharide,^9,10,12 as well as sequential glycosylation with different types of glycosyl donors⁸ were explored in the construction of the trirhamnan. In this second approach the terminal anthrose building block was synthesized by a more lengthy strategy from a 4-azido-4,6-dideoxy-D-mannose derivative.^13–16 A similar, [2+1] approach was taken for the synthesis of trisaccharide analogs 4–7,⁶ but in this case an azido deoxy galactose derivative¹⁷ was used as precursor of the terminal anthrose unit.

Figure 1.

Literature Approaches to the Target

Despite the different overall strategies, these syntheses have many points in common. First, glycosylation with neighboring group (bromoacetyl or levulinoyl) participation was used in the construction of the β-linkage to the terminal anthrose unit,^7–10 thereby necessitating subsequent removal of the protecting group at 2-position and then methylation.¹⁸ Second, α-selectivity in the rhamnosylation reactions was achieved in all but one case by anchimeric assistance.^6,7 In all cases the synthesis of the anthrose building block required extensive protective group manipulations,^13–16 or an expensive starting material.⁷ In planning our synthesis of 2 in a [3+1] manner, we wanted to address these problems through a) application of known α-nitrilium mediated β-selective glycosylation^19,20 methodology for the construction of the 1,2-trans glycosidic linkage in the terminal anthrose moiety; b) the straightforward assembly of the rhamnan building block through the recently developed iterative glycosylation strategy²¹ using thiorhamnoside donors, known to give good α-selectivity without neighboring group participation; and c) developing a new, shorter route to the anthrose saccharide, utilizing inexpensive D-galactose as a precursor.

Results and Discussion

We began our synthesis of the anthrose monosaccharide building block with the galactose derivative 8, which is readily available from D-galactose in 4 steps (Scheme 1).²² Regioselective installation of a 2-naphthylmethyl (Nap)^23–25 group at the 3-position through the intermediacy of a tin acetal,^26,27 followed by methylation, gave 10. Deprotection in acidic media, followed by regioselective substitution of the primary 6-OH in the presence of the secondary 4-OH with a phenylseleno group²⁸ gave 12. Reduction with tributyltin hydride^29,30 gave the 6-deoxygalactose derivative 13. Displacement of the 4-O-triflate leaving group, introduced by the reaction of 13 with triflic anhydride, with azide gave desired donor 14.

Scheme 1.

Synthesis of the Anthrose Donor

With 14 in hand, we proceeded to the synthesis of the trirhamnan building blocks. Benzylation of known n-pentenyl rhamnoside 15,³¹ readily available through Fischer glycosylation of L-rhamnose and further acetonide protection,³¹ gave 16 (Scheme 2). Cleavage of the 2,3-O-isopropylidene group in acidic media, followed by stannyl activated regioselective benzylation, gave 17,⁷ which was converted, using a known procedure,³² to the desired dibromide acceptor 18. Building block 19³³ was synthesized using a known procedure³³ and then converted to the acceptor 20,³³ following the protocol outlined in Scheme 3. A higher yield of 20 was achieved by utilizing a mixture of methanol and dichloromethane as a solvent.

Scheme 2.

Synthesis of Monosaccharide 18

Scheme 3.

Synthesis of Monosaccharide 19

The assembly of the trirhamnan moiety 22 started with preactivation of donor 19, followed by the addition of acceptor 20. Subsequent quenching of the reaction mixture with triethyl phosphite^34,35 provided disaccharide 21, predominantly as the α-anomer. In a similar manner, but omitting the quenching step, 21α was allowed to react with 18, to give the crude trisaccharide, which was further deprotected under oxidative conditions to give the desired trisaccharide building block 22 (Scheme 4).

Scheme 4.

Trisaccharide Synthesis

In order to find an optimal promoter/solvent combination for the synthesis of the tetrasaccharide, the glycosylation of donor 14 was investigated using model acceptor 23³⁶ and the activation systems outlined in Scheme 5. In the case of preactivation of 14 with 1-benzenesulfinyl piperidine (BSP)/Tf₂O³⁷ in the presence of 2,4,6-tri-tert-butylpyrimidine (TTBP) and subsequent addition of acceptor 23, a considerable improvement in the β-selectivity was observed, as expected, when the solvent was changed from dichloromethane to propionitrile.^19,20 Use of analogous promoters (Ph₂SO,³⁵ 1 or 2 eq) did not improve the selectivity. However, activation of the donor 14 in the presence of acceptor 23 by the NIS/TfOH system^38–40 resulted in a significant enhancement of the stereoselectivity, as well as in the overall yield of the reaction.

Scheme 5.

Exploratory Couplings to the Anthrose Donor

To complete the synthesis, the precursor to the anthrose unit was coupled to trisaccharide 22 using the optimal NIS/TfOH/propionitrile conditions to afford 25 as an approximately 3:1 β:α-mixture of anomers in 92% overall yield. Thus, the increased efficiency of the direct introduction of the anthrose moiety in this manner is achieved at the expense of some loss of selectivity. Debromination³² of 25β gave 26 from which oxidative cleavage of the 2-naphthylmethyl group afforded tetrasaccharide 27, suitable for further modifications in the terminal anthrose moiety, such as were found to be essential for the constitution of the highly specific antigenic determinant.⁴¹ The moderate yield of 27 is attributable to the previously reported problem of the competing debenzylation reactions in the course of the DDQ promoted cleavage of 2-naphthylmethyl/p-methoxyphenyl ethers.³³ To overcome this difficulty the mixture of debenzylated byproducts of the 2-naphthylmethyl deprotection reaction was benzylated under standard conditions to give 28, suitable for further transformations to the target molecule through the same routes as 27. Final removal of the benzyl protecting groups and transformation of azide moiety to the amine was achieved by application of sodium in liquid ammonia to the mixture of 27 and 28. Subsequent coupling to the 3-hydroxy-3-methylbutyric acid under peptide-coupling conditions⁴² led to the tetrasaccharide 2, whose spectroscopic data was identical to that previously reported (Scheme 6).⁷

Scheme 6.

Completion of the Synthesis

In conclusion, a synthesis of an antigenic tetrasaccharide from Bacillus anthracis has been accomplished in a [3+1] manner. A straightforward route to the anthrose building block from D-galactose was developed in which no extensive protective group manipulations or expensive starting materials were necessary. The assembly of the trirhamnan building block via an iterative glycosylation strategy, and the construction of the 1,2-trans-glycosidic linkage to the terminal anthrose moiety through the α-nitrilium ion-mediated β-selective glycosylation methodology, enables the use of a minimal protecting group strategy and increases the efficiency of the overall synthesis.

Experimental Section

General

Unless otherwise noted, reactions were conducted under an inert atmosphere of argon or nitrogen. All ¹H and ¹³C spectra were recorded in CDCl₃, except for 2, where CD₃OD was used as a solvent.

S-Phenyl 4,6-O-Benzylidene-3-O-(2-naphthylmethyl)-β-D-thiogalactopyranoside (9)

A suspension of 8²² (490 mg, 1.36 mmol) and dibutyltin oxide (610 mg, 2.45 mmol) in toluene (7 mL) was heated to reflux in a Dean-Stark apparatus for 4 h, after which most of the solvent was distilled off. The reaction mixture was cooled to room temperature and the residual solvent was evaporated under reduced pressure. CsF (414 mg, 2.72 mmol), 2-(bromomethyl)naphthalene (602 mg, 2.72 mmol), tetrabutylammonium iodide (1.00 g, 2.72 mmol), and DMF (6 mL) were then added. The reaction mixture was heated to reflux overnight, diluted with ethyl acetate, washed (sat. aq. NaHCO₃, brine), dried (Na₂SO₄), and concentrated. Purification by column chromatography (SiO₂, 1/9 to 1/3 ethyl acetate/hexanes) gave 9 (490 mg, 60%) as a white solid. M.p. 175–176 °C. [α]²¹_D +32.4 (c 0.38, CHCl₃). ¹H NMR (400 MHz) δ: 7.73 – 7.85 (m, 4H), 7.66 – 7.72 (m, 2H), 7.44 – 7.52 (m, 3H), 7.34 – 7.44 (m, 5H), 7.22 – 7.32 (m, 3H), 5.42 (s, 1H), 4.90 (s, 2H), 4.51 (d, J = 9.5 Hz, 1H), 4.34 (dd, J = 12.3, 1.6 Hz, 1H), 4.15 (dd, J = 3.4, 0.7 Hz, 1H), 3.92 – 4.02 (m, 2H), 3.56 (dd, J = 9.3, 3.3 Hz, 1H), 3.41 – 3.43 (m, 1H), 2.52 (d, J = 1.9 Hz, 1H); ¹³C NMR (101 MHz) δ: 137.8, 135.5, 133.8, 133.2, 133.1, 130.6, 129.1 128.9, 128.3, 128.2, 127.9, 127.7, 126.8, 126.6, 126.2, 126.1, 125.8, 101.2, 87.1, 80.2, 73.4, 71.9, 70.1, 69.4, 67.3; ESIHRMS Calcd for C₃₀H₂₈NaO₅S [M+Na]⁺: 523.1555. Found 523.1562.

S-Phenyl 4,6-O-Benzylidene-2-O-methyl-3-O-(2-naphthylmethyl)-β-D-thiogalacto-pyranoside (10)

A solution of 9 (9.34 g, 18.7 mmol) in DMF (300 mL) was cooled to −15 °C, NaH (60% in mineral oil, 1.12 g, 28.0 mmol) was added slowly and the reaction mixture was allowed to stir for 30 min at this temperature. Methyl iodide (1.98 mL, 31.7 mmol) was then added slowly and the reaction mixture was allowed to warm up to r.t. and stirred overnight. The reaction mixture was quenched with MeOH, diluted with CHCl₃, washed (water, brine), dried (Na₂SO₄), and concentrated. Purification by column chromatography (SiO₂, 1/4 ethyl acetate/hexanes) gave 10 (7.7 g, 80%) as a white solid. M.p. 174–176 °C. [α]²¹_D +28.4 (c 0.40, CHCl₃). ¹H NMR (400 MHz) δ: 7.76 – 7.87 (m, 4H), 7.68 – 7.73 (m, 2H), 7.45 – 7.55 (m, 5H), 7.36 – 7.43 (m, 3H), 7.19 – 7.27 (m, 3H), 5.46 (s, 1H), 4.93 (d, J = 12.7 Hz, 1H), 4.86 (d, J = 12.6 Hz, 1H), 4.49 (d, J = 9.4 Hz, 1H), 4.33 (dd, J = 12.3, 1.5 Hz, 1H), 4.12 (dd, J = 3.4, 0.7 Hz, 1H), 3.94 (dd, J = 12.4, 1.6 Hz, 1H), 3.64 (t, J = 9.2 Hz, 1H), 3.59 (s, 3H), 3.57 (dd, J = 9.2, 3.4 Hz, 1H), 3.35 – 3.37 (m, 1H); ¹³C NMR (101 MHz) δ: 137.9, 135.7, 133.2, 133.0, 132.9, 132.6, 129.1, 128.8, 128.2, 127.9, 127.7, 127.5, 126.7, 126.6, 126.2, 126.0, 125.8, 101.4, 86.4, 81.1, 76.9, 74.0, 72.1, 69.8, 69.4, 61.1; ESIHRMS Calcd for C₃₁H₃₀NaO₅S [M+Na]⁺: 537.1712. Found 537.1696.

S-Phenyl 2-O-Methyl-3-O-(2-naphthylmethyl)-β-D-thiogalactopyranoside (11)

A mixture of 10 (510 mg, 1.0 mmol), p-toluenesulfonic acid monohydrate (19 mg, 0.1 mmol) and MeOH (10 mL) was heated to reflux for 45 min, then cooled to r.t., diluted with ethyl acetate, washed (sat. aq. NaHCO₃, brine), dried (Na₂SO₄), and concentrated. Purification by column chromatography (SiO₂, 2/3 to 1/1 ethyl acetate/hexanes) gave 11 (401 mg, 95%) as a white solid. M.p. 144–145 °C. [α]²¹_D −11.7 (c 0.62, CHCl₃). ¹H NMR (400 MHz) δ: 7.78 – 7.87 (m, 4H), 7.46 – 7.57 (m, 5H), 7.22 – 7.32 (m, 3H), 4.88 (s, 2H), 4.54 (d, J = 9.2 Hz, 1H), 4.02 – 4.05 (m, 1H), 3.90 – 3.98 (m, 1H), 3.73 – 3.81 (m, 1H), 3.65 (s, 3H), 3.42 – 3.55 (m, 3H), 2.74 (br. s., 1H), 2.28 – 2.34 (m, 1H); ¹³C NMR (101 MHz) δ: 135.1, 133.6, 133.2, 133.1, 131.7, 129.0, 128.5, 127.9, 127.8, 127.5, 126.8, 126.4, 126.2, 125.7, 87.4, 82.1, 78.8, 78.0, 72.4, 67.6, 62.7, 61.4; ESIHRMS Calcd for C₂₄H₂₆NaO₅S [M+Na]⁺: 449.1399. Found 449.1391.

S-Phenyl 2-O-Methyl-3-O-(2-naphthylmethyl)-6-deoxy-6-phenylseleno-β-D-thio-galactopyranoside (12)

A mixture of 11 (1.06 g, 2.49 mmol), diphenyl diselenide (3.89 g, 12.47 mmol), tributylphosphine (4.3 mL, 17.43 mmol), and toluene (20 mL) was heated to reflux for 24 h. Concentration of the reaction mixture, followed by purification by column chromatography (SiO₂, hexanes to 2/3 ethyl acetate/hexanes) gave 12 (1.09 g, 77%) as a white solid. M.p. 128–132 °C. [α]²¹_D +16.4 (c 1.01, CHCl₃). ¹H NMR (500 MHz) δ: 7.78 – 7.88 (m, 4H), 7.58 – 7.63 (m, 2H), 7.43 – 7.54 (m, 5H), 7.18 – 7.34 (m, 6H), 4.87 (s, 2H), 4.48 (d, J = 9.5 Hz, 1H), 4.12 (t, J = 2.5 Hz, 1H), 3.65 (s, 3H), 3.41 – 3.52 (m, 3H), 3.30 (dd, J = 12.8, 6.7 Hz, 1H), 3.18 (dd, J = 12.7, 7.2 Hz, 1H), 2.36 (d, J = 1.8 Hz, 1H); ¹³C NMR (126 MHz) δ: 135.1, 133.7, 133.2, 133.1, 132.6, 132.1, 129.7, 129.2, 128.9, 128.5, 127.9, 127.8, 127.5, 127.1, 126.8, 126.3, 126.2, 125.8, 87.7, 82.4, 78.7, 77.6, 72.5, 67.5, 61.3, 27.5; ESIHRMS Calcd for C₃₀H₃₀NaO₄SSe [M+Na]⁺: 589.0928. Found 589.0900.

S-Phenyl 2-O-Methyl-3-O-(2-naphthylmethyl)-β-D-thiofucopyranoside (13)

A solution of 12 (820 mg, 1.45 mmol), AIBN (98 mg, 0.58 mmol), and Bu₃SnH (576 μL, 2.17 mmol) in benzene (145 mL) was heated to reflux for 3h. The solvent was evaporated and the residual syrup was dissolved in acetonitrile, washed twice with hexanes and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, 1/19 to 1/4 ethyl acetate/hexanes) gave 13 (504 mg, 85%) as a white solid. M.p. 108–111 °C. [α]²⁰_D −11.2 (c 0.33, CHCl₃). ¹H NMR (501 MHz) δ: 7.79 – 7.87 (m, 4H), 7.55 – 7.59 (m, 2H), 7.46 – 7.53 (m, 3H), 7.22 – 7.33 (m, 3H), 4.89 (s, 2H), 4.50 (d, J = 9.7 Hz, 1H), 3.80 (d, J = 2.8 Hz, 1H), 3.65 (s, 3H), 3.51 – 3.55 (m, 2H), 3.43 (t, J = 9.4 Hz, 1H), 2.33 (br. s., 1H), 1.35 (d, J = 6.6 Hz, 3H); ¹³C NMR (126 MHz) δ: 135.3, 133.9, 133.2, 133.1, 132.0, 128.9, 128.5, 128.0, 127.8, 127.4, 126.8, 126.3, 126.2, 125.8, 87.4, 82.6, 78.7, 74.2, 72.3, 69.7, 61.4, 16.8; ESIHRMS Calcd for C₂₄H₂₆NaO₄S [M+Na]⁺: 433.1450. Found 433.1445.

S-Phenyl 4-Azido-4-deoxy-2-O-methyl-3-O-(2-naphthylmethyl)-β-D-thioquinovo-pyranoside (14)

To a solution of 13 (313 mg, 0.76 mmol) and pyridine (185 μL, 2.29 mmol) in CH₂Cl₂ (7 mL) Tf₂O (256 μL, 1.52 mmol) was added at 0 °C. The reaction mixture was stirred for 1.5 h at r.t., diluted with CH₂Cl₂, washed (sat. aq. NaHCO₃, brine), dried (Na₂SO₄), and concentrated. The residue was dissolved in DMF (3 mL) and NaN₃ (64 mg, 0.98 mmol) was added. The reaction mixture was allowed to stir for 2.5 h, diluted with ethyl acetate, washed (brine), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent) and purification by radial chromatography (SiO₂, hexanes to 1/19 ethyl acetate/hexanes) gave 14 (288 mg, 87%) as a white solid. M.p. 85–86 °C. [α]²³_D +32.3 (c 0.25, CHCl₃). ¹H NMR (501 MHz) δ: 7.81 – 7.90 (m, 4H), 7.46 – 7.59 (m, 5H), 7.26 – 7.35 (m, 3H), 5.07 (d, J = 10.8 Hz, 1H), 5.02 (d, J = 10.8 Hz, 1H), 4.53 (d, J = 9.9 Hz, 1H), 3.67 (s, 3H), 3.51 (t, J = 9.1 Hz, 1H), 3.20 – 3.28 (m, 2H), 3.18 (t, J = 9.6 Hz, 1H), 1.38 (d, J = 6.1 Hz, 3H); ¹³C NMR (126 MHz) δ: 135.2, 133.42, 133.35, 133.1, 132.1, 129.0, 128.3, 128.1, 127.8, 127.7, 127.2, 126.3, 126.2, 126.1, 87.4, 84.7, 83.0, 75.7, 74.8, 67.6, 61.2, 18.8; ESIHRMS Calcd for C₂₄H₂₅NaN₃O₃S [M+Na]⁺: 458.1514. Found 458.1505.

n-Pentenyl 4-O-Benzyl-2,3-O-isopropylidene-α-L-rhamnopyranoside (16)

To a solution of 15³¹ (1.46 g, 5.36 mmol) in DMF (8 mL) NaH (60% in mineral oil, 321 mg, 8.04 mmol) was added slowly at 0 °C, followed by benzyl bromide (961 μL, 8.04 mmol), and the reaction mixture was allowed to stir for 3h at r.t. The reaction mixture was diluted with ethyl acetate, washed (sat. aq. NH₄Cl, brine), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 1/19 ethyl acetate/hexanes) gave 16 (1.74 g, 90%) as a white solid. M.p. 38–39 °C. [α]¹⁹_D −46.4 (c 0.71, CHCl₃). ¹H NMR (500 MHz) δ: 7.26 – 7.40 (m, 5H), 5.77 – 5.86 (m, 1H), 5.04 (ddd, J = 17.1, 3.1, 1.5 Hz, 1H), 4.99 (dq, J = 10.2, 1.5 Hz, 1H), 4.96 (s, 1H), 4.92 (d, J = 11.6 Hz, 1H), 4.64 (d, J = 11.6 Hz, 1H), 4.27 – 4.31 (m, 1H), 4.15 (d, J = 5.7 Hz, 1H), 3.66 – 3.74 (m, 2H), 3.43 (dt, J = 9.6, 6.4 Hz, 1H), 3.23 (dd, J = 9.8, 7.1 Hz, 1H), 2.09 – 2.17 (m, 2H), 1.64 – 1.74 (m, 2H), 1.53 (s, 3H), 1.39 (s, 3H), 1.29 (d, J = 6.2 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.4, 138.0, 128.3, 128.0, 127.6, 115.0, 109.1, 97.0, 81.2, 78.7, 76.2, 73.0, 66.8, 64.5, 30.3, 28.6, 28.1, 26.4, 17.9; ESIHRMS Calcd for C₂₁H₃₀NaO₅ [M+Na]⁺: 385.1991. Found 385.1980.

n-Pentenyl 2,4-Di-O-benzyl-α-L-rhamnopyranoside (17)

A mixture of 16 (1.25 g, 3.45 mmol) and acetic acid (80% in water, 10 mL) was heated for 4h at 90 °C. Solvents were then evaporated and residual syrup was dried by addition of toluene, followed by evaporation (2 times). Bu₂SnO (1.03 g, 4.14 mmol) was then added to this syrup, followed by benzene (25 mL) and mixture was heated to reflux in a Dean-Stark apparatus for 3 h, after which most of the solvent was distilled off. The reaction mixture was cooled to r. t. and the residual solvent was evaporated under reduced pressure. CsF (1.05 g, 6.90 mmol), benzyl bromide (536 μL, 4.49 mmol) and DMF (20 mL) were then added. The reaction mixture was allowed to stir overnight, diluted with ethyl acetate, washed (water, brine), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 17 (1.06 g, 74%). Spectral data matched that reported in literature.⁷

4,5-Dibromopentyl 2,4-Di-O-benzyl-α-L-rhamnopyranoside (18)

A solution of 17 (300 mg, 0.73 mmol) in THF/acetonitrile (3 mL/6 mL) was added to a mixture of CuBr₂ (812 mg, 3.64 mmol) and LiBr (632 mg, 7.28 mmol) in THF/acetonitrile (6 mL/12 mL). The reaction mixture was stirred overnight in the dark at r.t., then diluted with ethyl acetate, washed (water, 2 times), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent) and purification by radial chromatography (SiO₂, hexanes to 1/4 ethyl acetate/hexanes) gave 18 as a mixture of two diastereomers in the dibromopentyl chain (370 mg, 89%). ¹H NMR (500 MHz) δ: 7.28 – 7.41 (m, 10H), 4.91 (d, J = 10.8 Hz, 1H), 4.81 (d, J = 1.5 Hz, 1H), 4.72 (s, 2H), 4.66 (d, J = 10.8 Hz, 1H), 4.15 – 4.22 (m, 1H), 4.01 – 4.04 (m, 1H), 3.84 – 3.89 (m, 2H), 3.69 – 3.77 (m, 2H), 3.63 (t, J = 10.1 Hz, 1H), 3.48 (t, J = 9.4 Hz, 1H), 3.42–3.47 (m, 1H), 2.60 (s, 1H), 2.20 – 2.30 (m, 1H), 1.79 – 1.93 (m, 2H), 1.67 – 1.76 (m, 1H), 1.34 (d, J = 6.4 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.4, 137.9, 128.6, 128.5, 128.1, 128.0, 127.9, 127.8, 99.1, 99.0, 80.0, 75.5, 72.1, 68.6, 67.5, 66.54, 66.51, 52.6, 52.5, 36.2, 33.01, 32.95, 26.9, 18.0; ESIHRMS Calcd for C₂₅H₃₂NaBr₂O₅ [M+Na]⁺: 593.0514. Found 593.0519.

S-Phenyl 2,4-Di-O-benzyl-α-L-thiorhamnopyranoside (20)

To a solution of 19³³ (1.83 g, 3.17 mmol) in MeOH/CH₂Cl₂ (10 mL/30 mL) DDQ (2.16 g, 9.51 mmol) was added at 0 °C. After 30 min, the reaction mixture was warmed to r. t. and stirred for a further 5 h before it was diluted with CH₂Cl₂ and quenched with sat. aq. Na₂CO₃. The organic phase was separated, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 1/9 ethyl acetate/hexanes) gave 20 (1.03 g, 74%), whose spectral data matched that reported in literature.³³

S-Phenyl 2,4-Di-O-benzyl-3-O-(2-naphthylmethyl)-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-thiorhamnopyranoside (21α) and S-Phenyl 2,4-Di-O-benzyl-3-O-(2-naphthylmethyl)-β-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-thiorhamno-pyranoside (21β)

To a solution of 19 (304 mg, 0.53 mmol), TTBP (262 mg, 1.05 mmol), Ph₂SO (107 mg, 0.53 mmol), and activated 3 Å powdered molecular sieves in CH₂Cl₂ (10 mL) Tf₂O (98 μL, 0.58 mmol) was added at −60 °C. The reaction mixture was stirred for 1 h at this temperature and solution of 20 (391 mg, 0.90 mmol) in CH₂Cl₂ (4 mL) was added dropwise. The reaction mixture was stirred for an additional 30 min at −60 °C and then quenched by the addition of P(OEt)₃ (184 μL, 1.05 mmol). The reaction mixture was warmed r.t., then filtered, diluted with CH₂Cl₂, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 1/9 ethyl acetate/hexanes), followed by the HPLC (SiO₂, hexanes to 1/9 ethyl acetate/hexanes) gave 21α (235 mg, 49%), 21β (34 mg, 7%) and 20 (70 mg, 18%) was recovered. 21α. [α]²⁴_D −61.2 (c 0.20, CHCl₃). ¹H NMR (501 MHz) δ: 7.66 – 7.83 (m, 4H), 7.41 – 7.49 (m, 5H), 7.35 – 7.40 (m, 6H), 7.18 – 7.35 (m, 17H), 5.52 (s, 1H), 5.18 (s, 1H), 5.04 (d, J = 11.2 Hz, 1H), 4.78 (d, J = 12.1 Hz, 1H), 4.67 – 4.74 (m, 3H), 4.60 (d, J = 11.9 Hz, 1H), 4.45 – 4.55 (m, 4H), 4.12 – 4.20 (m, 1H), 4.07 (dd, J = 9.5, 2.9 Hz, 1H), 4.01 – 4.04 (m, 1H), 3.99 (dd, J = 9.5, 3.0 Hz, 1H), 3.81 – 3.88 (m, 1H), 3.73 – 3.76 (m, 1H), 3.70 (t, J = 9.4 Hz, 1H), 3.58 (t, J = 9.4 Hz, 1H), 1.35 (d, J = 6.2 Hz, 3H), 1.30 (d, J = 6.2 Hz, 3H); ¹³C NMR (126 MHz) δ: 139.0, 138.4, 137.8, 136.0, 134.6, 133.3, 133.0, 131.5, 129.1, 128.6, 128.5, 128.4, 128.3, 128.1, 128.0, 127.9, 127.84, 127.81, 127.71, 127.66, 127.6, 127.5, 127.4, 127.0, 126.5, 126.1, 125.9, 99.9 (¹J_CH = 168.7 Hz), 85.5 (¹J_CH = 164.9 Hz), 80.8, 80.5, 79.9, 79.6, 76.0, 75.2, 74.7, 72.6, 72.5, 72.2, 69.5, 68.9, 18.2, 17.9; ESIHRMS Calcd for C₅₇H₅₈NaO₈S [M+Na]⁺: 925.3750. Found 925.3741. 21β. [α]²²_D +7.4 (c 0.50, CHCl₃). ¹H NMR (501 MHz) δ: 7.69 – 7.86 (m, 4H), 7.37 – 7.51 (m, 9H), 7.26 – 7.37 (m, 13H), 7.18 – 7.25 (m, 6H), 5.54 (d, J = 2.9 Hz, 1H), 5.05 (d, J = 10.6 Hz, 1H), 5.01 (d, J = 10.8 Hz, 1H), 5.00 (d, J = 12.5 Hz, 1H), 4.92 (d, J = 12.5 Hz, 1H), 4.66 – 4.75 (m, 2H), 4.69 (d, J = 12.1 Hz, 1H), 4.63 (d, J = 12.1 Hz, 1H), 4.55 (d, J = 10.6 Hz, 1H), 4.48 (d, J = 12.3 Hz, 1H), 4.29 (s, 1H), 4.18 (dd, J = 7. 9, 3.1 Hz, 1H), 4.11 – 4.17 (m, 1H), 3.91 (t, J = 3.0 Hz, 1H), 3.82 (d, J = 2.9 Hz, 1H), 3.63 – 3.70 (m, 2H), 3.40 (dd, J = 9.5, 3.0 Hz, 1H), 3.22 – 3.29 (m, 1H), 1.39 (d, J = 6.2 Hz, 3H), 1.37 (d, J = 6.1 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.9, 138.7, 138.6, 137.7, 135.9, 134.8, 133.3, 133.0, 131.6, 129.1, 128.5, 128.44, 128.37, 128.24, 128.18, 128.14, 128.10, 128.0, 127.9, 127.8, 127.6, 127.4, 127.3, 126.2, 125.9, 125.6, 99.0 (¹J_CH = 154.9 Hz), 85.4 (¹J_CH = 166.2 Hz), 82.2, 80.3, 80.2, 76.5, 76.3, 75.5, 74.7, 74.3, 74.1, 72.12, 72.09, 71.6, 69.0, 18.4, 18.2; ESIHRMS Calcd for C₅₇H₅₈NaO₈S [M+Na]⁺: 925.3750. Found 925.3744.

4,5-Dibromopentyl 2,4-Di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (22)

To a solution of 21α (677 mg, 0.75 mmol), TTBP (373 mg, 1.50 mmol), Ph₂SO (151 mg, 0.75 mmol), and activated 3 Å powdered molecular sieves in CH₂Cl₂ (22 mL) Tf₂O (139 μL, 0.82 mmol) was added at −60 °C. The reaction mixture was stirred for 1h at this temperature, and solution of 18 (644 mg, 1.13 mmol) in CH₂Cl₂ (7 mL) was added dropwise. After stirring for 30 min at −60 °C the reaction mixture was warmed r.t., then filtered, diluted with CH₂Cl₂, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. The crude was filtered through a pad of silica gel (with ethyl acetate as an eluent) and purified by radial chromatography (SiO₂, hexanes to 1/9 ethyl acetate/hexanes). Fractions containing product were combined and concentrated to give clear oil. To a solution of this oil in CH₂Cl₂/MeOH (18 mL/6 mL) DDQ (400 mg, 1.76 mmol) was added at 0 °C. After 30 min, the reaction mixture was warmed to r. t. and further stirred for 9 h before it was diluted with CH₂Cl₂ and quenched with sat. aq. Na₂CO₃. The organic phase was separated, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 22 as a mixture of two diastereomers in the dibromopentyl chain (413 mg, 45%, 2 steps). ¹H NMR (501 MHz) δ: 7.14 – 7.40 (m, 30H), 5.23 (s, 1H), 5.13 (d, J = 1.7 Hz, 1H), 4.90 (d, J = 11.2 Hz, 1H), 4.89 (d, J = 10.9 Hz, 1H), 4.80 (d, J = 11.7 Hz, 1H), 4.70 – 4.75 (m, 2H), 4.57 – 4.69 (m, 4H), 4.48 – 4.55 (m, 2H), 4.36 (d, J = 11.6 Hz, 1H), 4.20 (dd, J = 9.6, 3.0 Hz, 1H), 4.16 – 4.22 (m, 1H), 4.12 (d, J = 11.7 Hz, 1H), 4.01 – 4.05 (m, 1H), 3.97 (td, J = 9.0, 3.5 Hz, 1H), 3.79 – 3.89 (m, 4H), 3.60 – 3.77 (m, 6H), 3.42 – 3.47 (m, 1H), 3.42 (t, J = 9.5 Hz, 1H), 3.31 (t, J = 9.3 Hz, 1H), 2.29 (d, J = 9.5 Hz, 1H), 2.20 – 2.30 (m, 1H), 1.79 – 1.92 (m, 2H), 1.67 – 1.77 (m, 1H), 1.33 (d, J = 6.2 Hz, 3H), 1.30 (d, J = 6.1 Hz, 3H), 1.25 (d, J = 6.2 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.8, 138.6, 138.5, 138.3, 138.1, 137.8, 128.50, 128.47, 128.4, 128.1, 127.84, 127.78, 127.6, 126.9, 99.2 (¹J_CH = 170.0 Hz), 99.1(¹J_CH = 168.7 Hz), 99.0 (¹J_CH = 170.0 Hz), 98.5 (¹J_CH = 170.0 Hz), 82.2, 81.1, 80.6, 79.9, 79.2, 77.9, 76.8, 75.5, 74.9, 74.7, 74.6, 72.6, 72.5, 72.4, 71.7, 68.9, 68.2, 67.7, 66.4, 52.6, 52.5, 36.1, 33.03, 33.00, 26.9, 18.2, 18.1, 18.0; ESIHRMS Calcd for C₆₅H₇₆Br₂NaO₁₃ [M+Na]⁺: 1245.3550. Found 1245.3521.

Methyl 2,4-Di-O-benzyl-3-O-(2-naphthylmethyl)-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranoside (24α) and Methyl 2,4-Di-O-benzyl-3-O-(2-naphthylmethyl)-β-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyrano-side (24β)

Method A

To a solution of 14 (17 mg, 0.039 mmol), TTBP (19 mg, 0.078 mmol), BSP (8 mg, 0.039 mmol), and activated 3 Å powdered molecular sieves in CH₂Cl₂ (1.5 mL) Tf₂O (7.2 μL, 0.043 mmol) was added at −60 °C. The reaction mixture was stirred for 5 min at this temperature, and solution of 23 (17 mg, 0.048 mmol) in CH₂Cl₂ (0.6 mL) was added dropwise. The reaction mixture was stirred for an additional 2 min at −60 °C, then slowly warmed to r.t., filtered, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent) and purification by means of radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 24α (19 mg, 72%) and 24β (3 mg, 10%). 24α. [α]²³_D +252.3 (c 0.13, CHCl₃). ¹H NMR (500 MHz) δ: 7.79 – 7.87 (m, 4H), 7.54 (d, J = 8.4 Hz, 1H), 7.41 – 7.48 (m, 4H), 7.28 – 7.40 (m, 8H), 5.10 (d, J = 3.3 Hz, 1H), 5.06 (d, J = 10.8 Hz, 1H), 4.96 (d, J = 11.0 Hz, 1H), 4.82 – 4.87 (m, 2H), 4.74 (d, J = 12.1 Hz, 1H), 4.69 (s, 1H), 4.59 (d, J = 10.6 Hz, 1H), 4.04 (dd, J = 8.9, 2.7 Hz, 1H), 3.92 (t, J = 9.5 Hz, 1H), 3.81 – 3.88 (m, 2H), 3.62 – 3.71 (m, 2H), 3.55 (s, 3H), 3.39 (dd, J = 9.4, 2.9 Hz, 1H), 3.31 (s, 3H), 3.13 (t, J = 9.8 Hz, 1H), 1.35 (d, J = 5.5 Hz, 3H), 1.15 (d, J = 6.1 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.4, 138.0, 135.6, 133.3, 133.0, 128.4, 128.1, 128.0, 127.8, 127.73, 127.71, 127.67, 127.0, 126.2, 126.0, 125.9, 98.8 (¹J_CH = 168.7 Hz), 93.5 (¹J_CH = 167.4 Hz), 82.6, 79.8, 75.64, 75.56, 75.4, 74.3, 72.8, 68.3, 68.0, 66.4, 59.5, 54.7, 18.5, 18.1; FABHRMS Calcd for C₃₉H₄₅N₃NaO₈ [M+Na]⁺: 706.3104. Found 706.3097. 24β. [α]²³_D +12.2 (c 0.18, CHCl₃). ¹H NMR (400 MHz) δ: 7.81 – 7.89 (m, 4H), 7.56 (dd, J = 8.3, 1.5 Hz, 1H), 7.44 – 7.51 (m, 2H), 7.39–7.43 (m, 4H), 7.27 – 7.37 (m, 6H), 5.1 (d, J = 11.0 Hz, 1H), 4.96 – 5.02 (m, 2H), 4.84 (d, J = 12.3 Hz, 1H), 4.74 (d, J = 12.3 Hz, 1H), 4.55 – 4.65 (m, 3H), 4.03 (dd, J = 8.9, 3.2 Hz, 1H), 3.82 (dd, J = 3.1, 1.8 Hz, 1H), 3.69 (s, 3H), 3.59 – 3.68 (m, 2H), 3.45 (t, J = 9.0 Hz, 1H), 3.29 (s, 3H), 3.11 – 3.22 (m, 3 H), 1.36 (d, J = 5.9 Hz, 3H), 1.29 (d, J = 5.4 Hz, 3H); ¹³C NMR (101 MHz) δ: 138.8, 138.5, 135.5, 133.3, 133.1, 128.4, 128.24, 128.18, 128.0, 127.9, 127.8, 127.7, 127.5, 127.1, 126.3, 126.1, 126.0, 103.7 (¹J_CH = 160.9 Hz), 99.7 (¹J_CH = 168.3 Hz), 84.9, 82.8, 80.8, 79.1, 78.4, 75.5, 74.8, 73.5, 70.3, 67.9, 67.7, 60.8, 54.5, 18.6, 18.0; FABHRMS Calcd for C₃₉H₄₅N₃NaO₈ [M+Na]⁺: 706.3104. Found 706.3080.

Method B

To a solution of 14 (18 mg, 0.042 mmol), TTBP (21 mg, 0.085 mmol), BSP (9 mg, 0.042 mmol), and activated 3 Å powdered molecular sieves in propionitrile (1.5 mL) Tf₂O (7.9 μL, 0.047 mmol) was added at −60 °C. The reaction mixture was stirred for 5 min at this temperature, and solution of 23 (19 mg, 0.052 mmol) in propionitrile (0.6 mL) was added dropwise. The reaction mixture was stirred for an additional 2 min at −60 °C, then warmed to r.t., filtered, diluted with CH₂Cl₂, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent) and purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 24α (10.3 mg, 36%) and 24β (9.7 mg, 34%).

Method C

A solution of 14 (27 mg, 0.062 mmol), 23 (18 mg, 0.050 mmol) and activated 3 Å powdered molecular sieves in propionitrile (1 mL) was stirred for 10 min at r.t., then cooled to −60 °C and N-iodosuccinimide (14 mg, 0.062 mmol), followed by TfOH (1.1 μL, 0.012 mmol) were added. The reaction mixture was stirred for 8h at −60 to −65 °C, then quenched with Et₃N (9 μL, 0.062 mmol), filtered, diluted with CH₂Cl₂, washed (sat. aq. Na₂S₂O₃, sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent) and purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 24α (8 mg, 23%) and 24β (25 mg, 70%).

4,5-Dibromopentyl 4-Azido-4-deoxy-2-O-methyl-3-O-(2-naphthylmethyl)-α-D-quinovopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (25α) and 4,5-Dibromopentyl 4-Azido-4-deoxy-2-O-methyl-3-O-(2-naphthylmethyl)-β-D-quinovopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (25β)

A solution of 22 (118 mg, 0.096 mmol), 14 (50 mg, 0.115 mmol), and activated 3 Å powdered molecular sieves in propionitrile (2.4 mL) was stirred for 10 min at r.t., then cooled to −60 °C and N-iodosuccinimide (26 mg, 0.116 mmol), followed by TfOH (1.7 μL, 0.019 mmol) were added. The reaction mixture was stirred for 9h at −60 to −65 °C, then quenched with Et₃N (16 μL, 0.115 mmol), filtered, diluted with CH₂Cl₂, washed (sat. aq. Na₂S₂O₃, sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 25α as a mixture of two diastereomers in the dibromopentyl chain (32 mg, 21%) and 25β of two diastereomers in the dibromopentyl chain (105 mg, 71%). 25α. ¹H NMR (500 MHz): 7.74 – 7.87 (m, 4H), 7.51 (dd, J = 8.4, 1.7 Hz, 1H), 7.42 – 7.47 (m, 2H), 7.12 – 7.35 (m, 30H), 5.21 (s, 1H), 5.10 (d, J = 1.5 Hz, 1H), 5.00 – 5.04 (m, 2H), 4.91 (d, J = 11.0 Hz, 1H), 4.85 (d, J = 10.8 Hz, 1H), 4.82 (d, J = 10.8 Hz, 1H), 4.75 (d, J = 11.7 Hz, 1H), 4.70 (d, J = 1.5 Hz, 1H), 4.62 – 4.67 (m, 2H), 4.55 – 4.61 (m, 3H), 4.51 – 4.55 (m, 2H), 4.49 (s, 1H), 4.40 (d, J = 11.6 Hz, 1H), 4.11 – 4.21 (m, 3H), 3.99 – 4.01 (m, 1H), 3.76 – 3.90 (m, 8H), 3.55 – 3.71 (m, 5H), 3.37 – 3.46 (m, 5H), 3.34 (dd, J = 9.4, 3.5 Hz, 1H), 3.10 (t, J = 9.8 Hz, 1H), 2.18 – 2.27 (m, 1H), 1.77 – 1.89 (m, 2H), 1.63 – 1.74 (m, 1H), 1.282 (d, J = 6.2 Hz, 3H), 1.278 (d, J = 6.1 Hz, 3H), 1.24 (d, J = 6.2 Hz, 3H), 1.06 (d, J = 6.2 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.6, 138.4, 138.3, 138.0, 135.6, 133.3, 133.0, 128.47, 128.42, 128.34, 128.26. 128.12, 128.09, 128.0, 127.8, 127.68, 127.65, 127.60, 127.56, 127.5, 127.0, 126.2, 126.0, 125.9, 99.5 (¹J_CH = 169.9 Hz), 99.1 (¹J_CH = 169.2 Hz), 99.0 (¹J_CH = 169.2 Hz), 98.9 (¹J_CH = 168.5 Hz), 93.1 (¹J_CH = 165.3 Hz), 82.6, 80.8, 80.5, 79.9, 79.7, 77.6, 75.52, 75.49, 75.4, 75.2, 74.7, 74.5, 72.6, 72.4, 72.3, 68.8, 68.2, 68.1, 66.3, 59.1, 52.6, 52.5, 36.1, 33.01, 32.97, 26.8, 18.5, 18.10, 18.05; ESIHRMS Calcd for C₈₃H₉₅N₃NaO₁₆Br₂ [M+Na]⁺: 1570.4977. Found 1570.4920. 25β. ¹H NMR (501 MHz) δ: 7.82 – 7.87 (m, 4H), 7.53 – 7.57 (m, 1H), 7.44 – 7.50 (m, 2H), 7.38 – 7.42 (m, 2H), 7.16 – 7.37 (m, 28H), 5.12 (s, 1H), 5.09 (s, 1H), 5.06 (d, J = 10.8 Hz, 1H), 4.95 – 5.00 (m, 2H), 4.87 (d, J = 10.8 Hz, 1H), 4.66 – 4.72 (m, 2H), 4.54 – 4.66 (m, 7H), 4.44 – 4.50 (m, 3H), 4.10 – 4.21 (m, 3H), 3.99 – 4.01 (m, 1H), 3.88 – 3.91 (m, 1H), 3.81 – 3.87 (m, 4H), 3.75 – 3.80 (m, 1H), 3.66 – 3.69 (m, 4H), 3.59 – 3.71 (m, 3H), 3.55 (t, J = 9.1 Hz, 1H), 3.35 – 3.45 (m, 3H), 3.16 (t, J = 8.4 Hz, 1H), 3.08 (t, J = 9.4 Hz, 1H), 3.00 – 3.08 (m, 1H), 2.17 – 2.28 (m, 1H), 1.77 – 1.89 (m, 2H), 1.64 – 1.74 (m, 1H), 1.283 (d, J = 6.1 Hz, 3H), 1.279 (d, J = 6.1 Hz, 3H), 1.26 (d, J = 6.2 Hz, 3H), 1.12 (d, J = 5.5 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.8, 138.5, 138.3, 138.2, 135.5, 133.4, 133.1, 128.5, 128.33, 128.28, 128.2, 128.13, 128.07, 128.0, 127.73, 127.69, 127.66, 127.60, 127.58, 127.42, 127.39, 127.1, 126.3, 126.1, 126.0, 103.6 (¹J_CH = 161.2 Hz), 100.3 (¹J_CH = 168.7 Hz), 99.0 (¹J_CH = 170.0 Hz), 98.9 (¹J_CH = 169.2 Hz), 84.9, 82.8, 81.0, 80.61, 80.55, 80.0, 79.4, 78.6, 78.1, 75.49, 75.46, 74.84, 74.76, 74.7, 73.4, 72.5, 72.2, 70.3, 68.7, 68.4, 68.1, 67.8, 66.3, 60.8, 52.6, 52.5, 36.1, 33.02, 32.99, 26.8, 18.5, 18.1, 18.0; ESIHRMS Calcd for C₈₃H₉₅N₃NaO₁₆Br₂ [M+Na]⁺: 1570.4977. Found 1570.4930.

n-Pentenyl 4-Azido-4-deoxy-2-O-methyl-3-O-(2-naphthylmethyl)-β-D-quinovo-pyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (26)

A mixture of 25β (98 mg, 0.063 mmol), NaI (190 mg, 1.268 mmol), and 2-butanone (6 mL) was heated to reflux for 5 h. The reaction mixture was cooled to r.t., diluted with ethyl acetate, washed (sat. aq. Na₂S₂O₃,), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 1/9 ethyl acetate/hexanes) gave 26 (82 mg, 94 %). [α]¹⁵_D +10.0 (c 0.23, CHCl₃). ¹H NMR (501 MHz) δ: 7.83 – 7.91 (m, 4H), 7.56 (dd, J = 8.5, 1.4 Hz, 1H), 7.45 – 7.51 (m, 2H), 7.15 – 7.43 (m, 30H), 5.76 – 5.85 (m, 1H), 5.13 (br. s., 1H), 5.11 (d, J = 1.5 Hz, 1H), 5.08 (d, J = 11.0 Hz, 1H), 5.01 – 5.06 (m, 1H), 4.97 – 5.01 (m, 3H), 4.88 (d, J = 10.8 Hz, 1H), 4.59 – 4.73 (m, 8H), 4.57 (d, J = 11.2 Hz, 1H), 4.44 – 4.51 (m, 3H), 4.11 – 4.17 (m, 2H), 4.02 – 4.04 (m, 1H), 3.90 (dd, J = 2.6, 1.7 Hz, 1H), 3.82 – 3.87 (m, 3H), 3.77 – 3.82 (m, 1H), 3.62 – 3.71 (m, 6H), 3.56 (t, J = 9.4 Hz, 1H), 3.35 – 3.45 (m, 3H), 3.17 (dd, J = 8.9, 8.0 Hz, 1H), 3.09 (t, J = 9.5 Hz, 1H), 3.01 – 3.09 (m, 1H), 2.06 – 2.14 (m, 2H), 1.62 – 1.69 (m, 2H), 1.30 (d, J = 6.2 Hz, 3H), 1.29 (d, J = 6.1 Hz, 3H), 1.27 (d, J = 6.2 Hz, 3H), 1.12 (d, J = 5.9 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.7, 138.4, 138.33, 138.31, 138.2, 138.0, 135.5, 133.3, 133.1, 128.4, 128.3, 128.2, 128.14, 128.09, 128.05, 128.03, 127.98, 127.7, 127.64, 127.57, 127.53, 127.50, 127.4, 127.1, 126.2, 126.1, 125.9, 114.9, 103.5 (¹J_CH = 161.2 Hz), 100.3 (¹J_CH = 168.7 Hz), 98.9 (¹J_CH = 168.7 Hz), 84.8, 82.7, 80.9, 80.58, 80.55, 80.1, 79.3, 78.5, 78.0, 75.5, 75.4, 74.7, 74.6, 73.3, 72.4, 72.1, 70.2, 68.6, 68.4, 67.9, 67.7, 66.7, 60.8, 30.3, 28.6, 18.4, 18.02, 17.98, 17.95; ESIHRMS Calcd for C₈₃H₉₅N₃NaO₁₆ [M+Na]⁺: 1412.6610. Found 1412.6560.

n-Pentenyl 4-Azido-4-deoxy-2-O-methyl-β-D-quinovopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (27) and n-Pentenyl 4-Azido-3-O-benzyl-4-deoxy-2-O-methyl-β-D-quinovopyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamno-pyranosyl-(1→3)-2,4-di-O-benzyl-α-L-rhamnopyranosyl-(1→2)-3,4-di-O-benzyl-α-L-rhamnopyranoside (28)

To a solution of 26 (122 mg, 0.088 mmol) in MeOH/CH₂Cl₂ (0.7 mL/2.1 mL) DDQ (60 mg, 0.26 mmol) was added at 0 °C. After 30 min, the reaction mixture was warmed to r. t. and further stirred for 5h before it was diluted with CH₂Cl₂ and quenched with sat. aq. Na₂CO₃. The organic phase was separated, washed (sat. aq. Na₂CO₃), dried (Na₂SO₄), and concentrated. Filtration of the crude through a pad of silica gel (with ethyl acetate as an eluent), purification by radial chromatography (SiO₂, hexanes to 1/9 ethyl acetate/hexanes) gave 27 (74 mg, 67%). [α]¹⁵_D −11.6 (c 0.76, CHCl₃). ¹H NMR (501 MHz) δ: 7.13 – 7.39 (m, 30H), 5.74 – 5.85 (m, 1H), 5.11 (br. s., 1H), 5.10 (d, J = 1.7 Hz, 1H), 5.02 (dq, J = 17.2, 1.5 Hz, 1H), 4.95 – 4.99 (m, 1H), 4.88 (t, J = 10.5 Hz, 2H), 4.70 (d, J = 11.4 Hz, 1H), 4.65 – 4.69 (m, 2H), 4.54 – 4.63 (m, 6H), 4.48 (d, J = 11.9 Hz, 1H), 4.39 – 4.44 (m, 2H), 4.12 (ddd, J = 12.2, 9.4, 3.0 Hz, 1H), 4.00 – 4.02 (m, 1H), 3.87 (dd, J = 2.8, 1.7 Hz, 1H), 3.81 – 3.86 (m, 3H), 3.76 – 3.82 (m, 1H), 3.60 – 3.69 (m, 6H), 3.54 (t, J = 9.0 Hz, 1H), 3.47 (td, J = 9.1, 2.1 Hz, 1H), 3.41 (t, J = 9.5 Hz, 1H), 3.34 – 3.39 (m, 1H), 3.01 – 3.09 (m, 1H), 3.01 (t, J = 9.5 Hz, 1H), 2.97 (dd, J = 9.2, 7. 9 Hz, 1H), 2.66 (d, J = 2.6 Hz, 1H), 2.06 – 2.13 (m, 2H), 1.60 – 1.69 (m, 2H), 1.27 – 1.29 (m, 6H), 1.26 (d, J = 6.4 Hz, 3H), 1.11 (d, J = 5.7 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.7, 138.54, 138.47, 138.4, 138.2, 138.1, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.74, 127.68, 127.62, 127.56, 127.41, 127.38, 115.0, 103.4 (¹J_CH = 161.2 Hz), 100.2 (¹J_CH = 166.2 Hz), 99.0 (¹J_CH = 168.7 Hz), 84.0, 81.1, 80.62, 80.59, 80.1, 79.4, 78.2, 78.1, 75.5, 75.2, 74.8, 74.7, 73.4, 72.5, 72.1, 70.5, 68.6, 68.4, 67.9, 67.4, 66.8, 61.0, 30.3, 28.7, 18.3, 18.1, 18.02, 17.99; ESIHRMS Calcd for C₇₂H₈₇N₃NaO₁₆ [M+Na]⁺: 1272.5984. Found 1272.5943. Further elution of with ethyl acetate gave a mixture of the debenzylated byproducts, which was dissolved in DMF (2 mL). Benzyl bromide (60 μL, 0.50 mmol) was then added, followed by NaH (60% in mineral oil, 20 mg, 0.50 mmol) and the reaction mixture was stirred overnight, then diluted with ethyl acetate, washed (brine, water), dried (Na₂SO₄), and concentrated. Purification by radial chromatography (SiO₂, hexanes to 3/17 ethyl acetate/hexanes) gave 28 (26 mg, 22%, 2 steps). [α]¹⁴_D +2.4 (c 1.08, CHCl₃). ¹H NMR (501 MHz): 7.12 – 7.45 (m, 35H), 5.75 – 5.85 (m, 1H), 5.08 – 5.14 (m, 2H), 5.02 (dq, J = 17.2, 1.7 Hz, 1H), 4.96 – 4.99 (m, 2H), 4.90 (d, J = 10.7 Hz, 1H), 4.87 (d, J = 11.0 Hz, 1H), 4.81 (d, J = 10.6 Hz, 1H), 4.65 – 4.72 (m, 3H), 4.54 – 4.64 (m, 6H), 4.44 – 4.50 (m, 3H), 4.13 (td, J = 9.5, 3.0 Hz, 2H), 4.01 – 4.02 (m, 1H), 3.89 (dd, J = 2.8, 1.7 Hz, 1H), 3.76 – 3.86 (m, 4H), 3.61 – 3.70 (m, 6H), 3.55 (t, J = 9.5 Hz, 1H), 3.42 (t, J = 9.5 Hz, 1H), 3.31 – 3.39 (m, 2H), 3.13 (dd, J = 9.1, 8.0 Hz, 1H), 2.95 – 3.07 (m, 2H), 2.06 – 2.13 (m, 2H), 1.61 – 1.69 (m, 2H), 1.284 (d, J = 6.2 Hz, 3H), 1.279 (d, J = 6.2 Hz, 3H), 1.26 (d, J = 6.2 Hz, 3H), 1.11 (d, J = 5.7 Hz, 3H); ¹³C NMR (126 MHz) δ: 138.80, 138.78, 138.5, 138.40, 138.38, 138.2, 138.1, 138.0, 128.44, 128.40, 128.32, 128.26, 128.12, 128.08, 127.9, 127.71, 127.68, 127.60, 127.56, 127.5, 127.40, 127.37, 114.9, 103.6 (¹J_CH = 161.2 Hz), 100.3 (¹J_CH = 173.7 Hz), 99.1 (¹J_CH = 167.4 Hz), 99.0 (¹J_CH = 167.4 Hz), 84.8, 82.7, 81.0, 80.64, 80.63, 80.1, 79.4, 78.6, 78.1, 75.49, 75.45, 74.8, 74.6, 73.4, 72.5, 72.2, 70.3, 68.7, 68.4, 68.0, 67.7, 66.8, 60.8, 30.3, 28.7, 18.5, 18.1, 18.02, 17.99; ESIHRMS Calcd for C₇₉H₉₃N₃NaO₁₆ [M+Na]⁺: 1362.6454. Found 1362.6404.

n-Pentenyl 4-deoxy-4-(3-hydroxy-3-methylbutanamido)-2-O-methyl-β-D-quinovo-pyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→3)-α-L-rhamnopyranosyl-(1→2)-α-L-rhamnopyranoside (2)

Ammonia was condensed at −78 °C in a reaction vessel, containing a solution of 27 (26 mg, 0.021 mmol) and 28 (9 mg, 0.007 mmol) in THF (6 mL), until a total volume of about 20 mL was reached. Sodium (45 mg, 1.96 mmol) was then added in small pieces, affording a solution of dark blue color. The reaction mixture was stirred for 1h at this temperature and then quenched by the addition of the methanol. The reaction mixture was warmed to r.t., in order to evaporate the ammonia, and then concentrated. Purification by column chromatography (eluting first with CHCl₃ to remove most of the byproducts, and then with 1/4 MeOH/CHCl₃, and finally MeOH) gave a crude product, which was dissolved in (i-Pr)₂EtN and concentrated (2 times). DMF (2 mL) was then added, followed by 3-hydroxy-3-methylbutyric acid (5 mg, 0.042 mmol), HATU (16 mg, 0.042 mmol), and (i-Pr)₂EtN (28 μL, 0.16 mmol). The reaction mixture was stirred overnight and then concentrated. Purification by column chromatography (SiO₂, CHCl₃ to 9/31 MeOH/CHCl₃), followed by purification by preparative TLC (9/31 MeOH/CHCl₃) and further column chromatography (SiO₂, CHCl₃ to 9/31 MeOH/CHCl₃), gave 2 (12 mg, 54%, 2 steps), whose spectral data matched that reported in the literature.⁷ [α]²⁰_D −63.5 (c 0.2, MeOH); Lit.⁷ [α]_D −7.9 (c 0.18, CHCl₃).⁴³ IR (thin film): 3377, 2972, 2931, 2879, 1658, 1649, 1641, 1381, 1122, 1061 cm⁻¹.

Supplementary Material

1si20070410_04. Supporting Information Available.

Full experimental details for substrate preparation and copies of spectra of all compounds. This material is available free of charge via the Internet at http://pubs.acs.org.