Preparation of S-glycoside surfactants and cysteine thioglycosides using minimally competent Lewis acid catalysis

Abstract

Here we report a method for the preparation of anomerically pure β-S-glycopyranosides (1,2-trans-glycosides) from the corresponding peracetate donors. S-glycosylation was performed in CHCl₃ at reflux in the presence of a catalytic amount of InBr₃. Deacylation of the intermediate peracetates were achieved under Zemplén conditions. Five pyranose examples, monosaccharides D-glucose and D-galactose and disaccharides cellobiose, maltose, and lactose, were used as donors, and five thiols including an α/ω dithiol and Fmoc-L-cysteine were used as acceptors. Melting points, high res MS, [α]_D and NMR data (¹H and ¹³C, including COSY, HSQC and HMBC) are reported for compounds not previously described.

Glycolipids represent a diverse class of compounds typically found in biological membranes¹ and are regarded as environmentally friendly bio-surfactants with broad use in cosmetics, food, environmental and other industrial applications,² as well as potential therapeutic drug delivery systems.^3,4 Despite high surface activity,⁵ high biodegradability, and low toxicity, their use is still hampered by a lack of knowledge regarding physical properties, including phase behavior. Even less is known about the S-linked thioglycosides. Thioglycosides (S-glycosides)⁶ may be regarded as relatively inert moieties in vivo,⁷ or as chemical intermediates that can be oxidatively activated⁸ to generate glycoside donors.^9–11 Thioglycosides were previously prepared from monosaccharide peracetates using a molar excess of BF₃•Et₂O or SnCl₄ as promoters by Kihlberg in the 1990s.¹² Here we disclose the catalytic use of a much weaker Lewis acid, InBr₃,¹³ which we dub a “minimally competent” Lewis acid.¹⁴ It should be noted that InBr₃ and other Lewis acids have been used in conjunction with the O-glycosylation of trichloroacetimidate donors.¹⁵ We have previously reported the catalytic use of InBr₃¹⁴ and other Lewis acids¹⁶ to generate O-glycosides from weakly electrophilic peracetate sugars.

The surfactant β-S-octyl-glucopyranoside and several of its analogues were previously prepared by S-alkylation of 2-(2,3,4,6-tetra-O-acetyl-β-glucopyranosyl) thiopseudourea•HBr by Saito and Tsuchiya.¹⁷ The present approach (Fig. 1) seems to be superior, both in terms of the number of steps and in overall yield. β-D-glucopyranose peracetate 1 was treated with two molar equivalents of 1-octanethiol in CHCl₃ at reflux for 1 hour in the presence of 1% InBr₃, to form the desired β-peracetate, which was purified by short column chromatography (SiO₂). No α anomer was observed. We propose that the formation of the S-glycoside occurs via a similar route to the previously published O-glycoside example, resulting in only the β-S-glycoside.¹⁴ The peracetate product was deprotected using Zemplén deacylation¹⁸ to provide the desired surfactant.¹⁷ The glycosylation produced a similar yield in PhCH₃, but the reaction in CH₃CN decreased the yield.

Fig. 1.

Preparation of β-thioglycosides.

Several mono- and disaccharide peracetates (1–5) and thiols (6–11, Table 1) were subjected to the reaction conditions to provide a variety of S-glycosides. Bolaamphiphile 15a and mono-glycoside 16a were prepared by adjusting the stoichiometry of the glycosyl donor and acceptor to favor either mono- or di-glycosylation of the α/ω dithiol.

Table 1. Results of InBr₃ promoted glycosylations.

Thioglycosides from InBr₃ promoted glycosylations of various thiolipids (6–10) and Fmoc-L-cysteine (11) with five peracetylated mono- and disaccharides (1–5) to produce compounds 12–33. After Zemplén deacylation of the lipids, compounds 12a–28a were isolated and characterized. Compounds 12a,^19,20 13a,²⁰ 14a,²⁰ 17a,^19,21 18a,²² 19a,²² 20a,²³ 22a,²⁴ 23a,²³ 24a,²⁵ 25a,²⁴ 26a,²³ 28a,²⁶ 29,²⁷ and 30^28,29 have been previously reported using alternate reaction conditions. Previously reported deacylated thioglycosides are noted in lit. % indicates combined yields.

Donors	Sulfide acceptors
	R′
	R=	Ac	H	Lit	%	Ac	H	Lit	%	Ac	H	Lit	%	Ac	H	Lit	%	Ac	H	Lit	%	Ac	Lit	%
		12	12a	19,20	81	13	13a	20	88	14	14a	20	84	15	15a	–	86	16	16a	–	69	29	27	72
		17	17a	19,21	89	18	18a	22	90	19	19a	22	92									30	28,29	81
		20	20a	23	88	21	21a	–	66	22	22a	24	76									31	–	65
		23	23a	23	85	24	24a	25	87	25	25a	24	88									32		85
		26	26a	23	87	27	27a		82	28	28a	26	80									33		83

Similar reaction conditions were applied to Fmoc-L-cysteine. Glycosylation of neurologically active endogenous peptides has been applied to a number of systems,³⁰ although these modifications have typically been performed by O-glycosylation. O-glycosyl linkages have the drawback of being susceptible to enzymatic degradation and hydrolysis. Therefore, S-glycosylated amino acid building blocks compatible with common solid-phase peptide synthesis conditions are an attractive target. Fmoc-L-Cys(S-D-Gal)-OH has been previously reported by Kamerling et al,²⁹ and Pieters et al,²⁸ although their methodology required the use of excess SnCl₄. We report similar yields using catalytic quantities of the minimally competent Lewis acid activator, and show the broad applicability of this approach through conjugation of five mono- and disaccharide glycosyl donors 29–33 to Fmoc-L-cysteine (Fig. 2).

Fig. 2.

Preparation of L-cysteine β-thio-glycosides.

The importance of this study is twofold. First, only catalytic amounts of the Lewis acid promoter are required. Secondly, weakly electrophilic peracetates, generally regarded as synthetic intermediates rather than reactive donors, are suitable. This reduces the number of steps and greatly expands the application of this methodology. We have not observed the corresponding α-thioglycosides in any of the reactions.

1. Experimental section

1.1. Materials and instrumentation

¹H and ¹³C NMR spectra were recorded on a Bruker 400 (400 MHz) spectrometer using CDCl₃ and D₃COD as solvents with TMS as the internal standard. All NMR spectra were analyzed and interpreted using MestReNova^® software. First-order chemical shifts and coupling constants were obtained from 1D spectra; proton and carbon resonances were assigned from COSY, HSQC and HMBC experiments. Mass spectrometry was achieved on a Thermo Finnigan LCQ Deca with positive and negative detection and a 1:1 CH₃OH:H₂O solvent system. Optical rotation measurements were performed using a JASCO P-1010 polarimeter using the Na D line (589 nm) in MeOH at room temperature, and specific rotation was determined using the JASCO software package. For flash chromatography Silia Flash P60 Silica Gel, 40–63 μm, 60 Å (Silicycle, R1200308) was employed and a Biotage Inc. Horizon HPFC system was utilized.

Preparative HPLC separations were performed on a Gilson HPLC using a Phenomenex C-18 reverse phase column (1000 Å, 50 × 250 mm) with a flow rate of 25 mL/min and detection at 280 nm. Solvent systems A (5% acetonitrile in 0.1% aqueous F₃CCOOH) and B (80% acetonitrile in 0.1% aqueous F₃CCOOH) were used. A Dikma Technologies Inspire C-18 column (4.6 × 250 mm) and a flow rate of 1.0 mL/min were used for analytical HPLC separations. Thin layer chromatography (TLC) was performed on silica gel 60 F254 (Merck) with detection by UV light and charring with H₂SO₄. Solvents were dried and distilled according to literature procedures or purchased as anhydrous solvents from a manufacturer. All chemicals were purchased from Aldrich Chemical Company, Acros Chemicals, Chem-Impex International, or EMD Chemicals.

1.2. Alkyl-β−1-thio-D-glycopyranoside (12a–14a, 16a–28a)

Peracetyl-β-D-glycopyranose (1–5, 10 mmol), InBr₃ (106 mg, 0.3 mmol, 0.03 eq), and alkane-1-thiol (20 mmol, 2 eq) were suspended in CHCl₃ (7 mL). The reaction mixture was heated to reflux with magnetic stirring. The course of the reaction was followed by TLC (hexane:EtOAc 1:1). After 1 hour the peracetate starting material could no longer be detected by TLC. The reaction was cooled to RT, diluted with CH₂Cl₂ (20 mL), and this solution was applied to a short column (4 cm × 12 cm) of SiO₂. The HOAc that was formed from the reaction and the excess alkane-1-thiol were eluted with hexanes. The desired thio-glycoside peracetate was eluted with hexanes:EtOAc 7:3. The fractions containing the desired material were combined, concentrated in vacuo, and the intermediate peracetates (12–14, 16–28) were characterized by 1D, 2D, ¹H-NMR and ¹³CNMR in CDCl₃ (in Tables in Supplementary data).

Zemplén’s method was used to deprotect the peracetates.¹⁸ The thio-glycopyranoside peracetates were dissolved in anhydrous CH₃OH (100 mL) and the basicity was adjusted to pH = 9 by addition NaOCH₃. After 16 hours, the deacetylation was deemed complete by TLC. Dowex^® resin (H⁺ form) was added until the pH was slightly acidic, the resin was filtered off, and the solution was concentrated, to provide 12a–14a, 16a–28a, which were characterized.

1.3. 1,10 Dithio-β-D-glucopyranosyl-decane (15a)

Peracetyl-β-D-glucopyranose (25 mmol), InBr₃ (3 mmol, 0.03 eq), and decane-1,10-dithiol (10 mmol, 1 eq) were reacted using the previous method to provide 15a. M.p. 124–132 °C (crystallization from MeOH–Et₂O), [α]_D −36.5° (c 0.13, in MeOH), MS (ESI HRMS) for C₂₂H₄₂O₁₀S₂ (+Na) 553.21116, found 553.21195 [M + Na]⁺.

1.3.1. 1-Thio-β-D-glucopyranosyl-10-thiol-decane (16a)

M.p. 95–97 °C (crystallization from MeOH–Et₂O), [α]_D −17.8° (c 0.20, in MeOH), MS (ESI HRMS) for C₁₆H₃₂O₅S₂ (+Na) 391.15834, found 391.15875 [M + Na]⁺.

1.3.2. Decyl-β-thio-cellobioside (21a)

M.p. 200–214 °C (crystallization from MeOH–Et₂O), [α]_D −22.1° (c 0.26, in MeOH), MS (ESI HRMS) for : C₂₂H₄₂O₁₀S (+Na) 521.23909, found 521.24009 [M + Na]⁺.

1.3.3. Decyl-β-thio-lactoside (27a)

M.p. Decomp. ~ 200 °C (crystallization from MeOH–Et₂O), [α]_D −18.3° (c 0.40, in MeOH), MS (ESI HRMS) for C₂₂H₄₂O₁₀S (+Na) 521.23909, found 521.24000 [M + Na]⁺.

1.4. N^α-Fluoren-9-ylmethoxycarbonyl-S-(peracetyl-β-D-glyco-pyranosyl)-L-cysteine (29–33)

Peracetyl-β-D-glycopyranose (0.6 mmol, 2eq), InBr₃ (21 mg, 0.06 mmol, 0.2 eq), and N^α-fluoren-9-ylmethoxycarbonyl-L-cysteine (11, 103 mg, 0.3 mmol, 1 eq) were suspended in CHCl₃ (1.5 mL). The reaction mixture was heated to reflux with magnetic stirring. The course of the reaction was followed by analytical HPLC. After 1 hour HPLC indicated >80% conversion (see Table 1) of Fmoc-Cys(SH)-OH to the desired product. Additional reaction time yielded no further reaction progress that could be detected by HPLC. The reaction mixture was concentrated in vacuo, and the residue was purified by preparative HPLC on a C₁₈ reverse phase column with H₂O-CH₃CN gradient. The appropriate fractions were freeze-dried and the solid residue was characterized.

1.4.1. N^α-Fluoren-9-ylmethoxycarbonyl-S-(peracetyl-β-D-cellobiosyl)-L-cysteine (31)

Phase transition point 104–105 °C (lyophilization from CH₃CN/H₂O), [α]_D −20.7° (c 0.15, in MeOH), MS (ESI HRMS) for C₄₄H₅₁NO₂₁S (+Na) 984.25665, found 984.25901 [M + Na]⁺.

1.4.2. N^α-Fluoren-9-ylmethoxycarbonyl-S-(peracetyl-β-D-maltobiosyl)-L-cysteine (32)

Phase transition point 103–104 °C (lyophilization from CH₃CN/H₂O), [α]_D +21.5° (c 0.14, in MeOH), MS (ESI HRMS) for C₄₄H₅₁NO₂₁S (+Na) 984.25665, found 984.25866 [M + Na]⁺.

1.4.3. N^α-Fluoren-9-ylmethoxycarbonyl-S-(peracetyl-β-D-lactosyl)-L-cysteine (33)

Phase transition point 109–110 °C (lyophilization from CH₃CN/H₂O), [α]_D −20.6 (c 0.17, in MeOH), MS (ESI HRMS) for C₄₄H₅₁NO₂₁S (+Na) 984.25665, found 984.25817 [M + Na]⁺.