Concise assembly of linear α(1→6)-linked octamannan fluorescent probe

Abstract

Synthesis of a fluorescently labelled (dansylated) linear α(1→6)-linked octamannan, using glycosyl fluoride donors and thioglycosyl acceptors is described. A selective and convergent two-stage activation progression was executed to construct di-, tetra and octa-mannosyl thioglycosides in three glycosylation steps with excellent yield. Further a 5-N,N-Dimethylaminonaphthalene-1-sulfonamidoethyl (dansyl) group was coupled to 1-azidoethyl octamannosyl thioglycoside. Global deprotection of the coupled product afforded the desired dansylated homo-linear α(1→6)-linked octamannan.

Mannans are constituents of many bacterial and fungal cell walls.¹ They are functional components of proteins and play important roles in defining the structures of proteins, their stabilization under physiological conditions, tuning of enzymatic activities, cell–cell recognition and in the adhesion of the microorganism to host cells.² The α(1→6)-linked oligomannans are found in the cell wall polysaccharides of yeast.³ Polysaccharides, especially oligomannan, can be used to deliver drugs, genes and antigens through polysaccharide receptors present in cells and macrophages.⁴ Macrophages are known to express high levels of specific polysaccharide receptors, e.g. mannan, glucan, and galactin receptors, on their membranes that generally bind neutral or charged polysaccharides and internalize these ligands.⁵ Receptor mediated delivery of drug–polysaccharide conjugates is an approach that can deliver small and effective amounts of drugs specifically to target organisms, minimizing patient exposure and potential toxic side effects. We have recently demonstrated the selective, receptor–mediated delivery of an antibacterial drug to macrophages infected with Mycobacterium tuberculosis via conjugation of moxifloxacin with 1,3-β-glucan.⁶

Drug loading on commercially available high molecular weight polysaccharides is very low and it is attributed to their poor solubility in solvents during the drug conjugation reactions. Therefore, we describe here a simple and efficient method for the synthesis of a low molecular weight homo-linear α(1→6)-linked octamannan fluorescent probe 1 (figure 1) that may be utilized to study uptake by macrophages through the mannan receptor present on the plasma membrane. Further, in the future, such conjugation can be exploited for selective receptor–mediated delivery of drugs. We have previously synthesized and reported dansylated disaccharide fluorescent probes to study glycosyltransferases in the biosynthesis of M. tuberculosis cell wall polysaccharides.⁷ Herein, the assembly of dansylated oligomannan fluorescent probe 1 was approached from synthetic homo-linear α(1→6) thiooctamanosyl glycoside 2, 1-azidoethanol 3 and commercially available dansyl chloride 4 fragments as represented in Figure 1.

Figure 1.

Target α(1→6)-linked octamannan fluorescent probe 1 and its precursors.

Branched oligomannan syntheses are reported in the literature using several different methodologies.⁸ Specifically, the homo-linear α(1→6) oligomannans were synthesized by glycosylation using a trichloroacetimidate donor glycoside.⁹ The strategy of combining the chemistry of thioglycosides with that of glycosyl fluorides¹⁰ for the synthesis of oligosaccharides known as the two-stage activation procedure (Figure 2) was developed by Nicolaou et. al.¹¹ Briefly, in the activation stage one, the stable thioglycoside is converted to the more reactive glycosyl fluoride donor by treatment with N-bromosuccinimide (NBS) and diethylaminosulfur trifluoride (DAST). In stage two, the glycosyl fluoride is then reacted with a thioglycoside acceptor to produce a thiodisaccahride for further promulgation of the oligosaccharide chain. Reiteration of this process of converting thiooligosaccharide to a glycosyl fluoride and their coupling with acceptor thioglycosides straightforwardly produces chain elongation (figure 2).

Figure 2.

Two stage activation of thioglycoside for glycoside bond formation. Other protected OH's on sugars are omitted for simplification.

We executed the synthesis of the desired p-thiotolyl α(1→6) octamannoside 2 using a convergent two-stage activation and iterative orthogonal glycosylation technique starting from the coupling of thioglycoside acceptor 5 and glycosyl fluoride donor 6 (Figure 3). The synthesis of mannose building blocks 5 and 6 was achieved from thioglycoside 7 as reported earlier by us.¹² However, the synthesis of thioglycoside 7 was accomplished from the known synthon 1,6-di-O-acetyl-2,3,4-tri-O-benzoyl-α-D-mannose (8) starting from commercially available D-mannose in an 88% overall yield.¹³ Selective de-acetylation of 7 to the acceptor glycoside 5 was achieved by the overnight reaction with an AcCl/MeOH/CH₂Cl₂ (0.1:20:20 v/v, 12.0 mL/mmol) mixture.¹²

Figure 3.

Synthetic plan for the synthesis of α(1→6)-linked octamannosyl thioglycoside 2

Following the synthesis of starting mannose synthons 5 and 6, the synthesis of 2 was carried out as illustrated in Scheme 1. In the first step of the glycosylation sequence, the p-thiotolyl disaccharide 9 was synthesized in high yield by selective anomeric activation of glycosyl fluoride 6 by AgClO₄ and SnCl₂ in CH₂Cl₂ and its coupling with the acceptor 5.¹² The exclusive formation of α(1→6)-linked disaccharide 6 was supported by its analytical and spectral analysis.¹²

Scheme 1.

Reactions and Reagents. (a) AgClO₄, SnCl₂, dry CH₂Cl₂, 4Ǻ Mol sieves, rt, overnight, 9: 97%, 12: 80%. (c) AcCl/MeOH/CH₂Cl₂ (1:20:20 v/v), rt, overnight, 10: quantitative yield, 13: 86%. (b) DAST, NBS, dry CH₂Cl₂, −20 °C, 4 h, 11: 91%, 14: 84%. (d) Cp₂HfCl₂, AgOTf, dry CH₂Cl₂, 4A Mol sieves, rt, overnight, 61%.

Next, for the assembly of α(1→6)-linked tetramannosyl thioglycoside 12, the disaccharide 9 was converted to acceptor thiodisaccharide 10 and donor 1-fluorodisaccharide 11. Deacetylation of 9 by the overnight reaction with an AcCl/MeOH/CH₂Cl₂ mixture produced the desired acceptor disaccharide 10 in quantitative yield. In the ¹H and ¹³C NMR spectra of compound 10, the absence of signals from the acetate group as seen in compound 9, confirmed the selective deacetylation. The disaccharide 9 was also converted to the donor fluoride 11 in 91% yield using DAST and NBS at −20 °C. The structure of 11 was supported by the ¹H NMR spectra in which two characteristic peaks in thiotolyl glycosides [doublet at δ 7.20 ppm from two protons of tolyl ring and a singlet at δ 2.25 ppm from methyl] were not observed. The α-stereochemistry at the anomeric carbon was supported by a C-1 signal at δ 105.21 ppm (d, J_C-1,F = 222.1 Hz) in the ¹³C NMR spectrum of 11, whereas, the signal at δ 98.11 ppm was attributable to the other anomeric carbon. In the ¹H NMR spectrum of 11, the H-1 signal was found obscured with other protons in the multiplet at δ 5.92 ppm, however, the anomeric proton H-1′ was observed δ 5.15 ppm (J = 1.4 Hz).

With the acceptor disaccharide 10 and fluoride donor disaccharide 11 in hand, we next performed the [2+2] glycosylation reaction to access α(1→6)-linked tetramannosyl thioglycoside 12. The coupling reaction between glycosides 10 and 11 was carried out at room temperature with promotors AgClO₄ and SnCl₂ in CH₂Cl₂ over 4Ǻ molecular sieves and resulted in the desired glycoside 12 in 80% yield after purification by SiO₂ column chromatography. The ¹H NMR spectrum of 12 in CDCl₃ showed four anomeric protons at δ 5.77, 5.25, 5.02 and 4.85 ppm as singlets suggesting α-glycosylation. In the ¹³C NMR spectrum of 12, the four anomeric carbons were observed at δ 98.37, 97.99, 97.79 and 86.91 ppm. The final structural confirmation was obtained by FABMS analysis of 12, which showed a peak at 2085.0 [M+Na]⁺, corresponding to the molecular formula C₁₁₇H₉₈O₃₃SNa.

The α(1→6)-linked tetramannosyl thioglycoside 12 was further converted to thioglycosyl acceptor 13 and glycosyl fluoride donor 14 in 96% and 84% yields by similar reactions performed for the syntheses of glycosides 10 and 11 respectively. The characteristic acetate and thiotolyl groups were found absent in the ¹H NMR and ¹³C NMR spectra of compounds 13 and 14 respectively. In the ¹³C NMR spectra of glycosyl fluoride 14, the four anomeric carbon signals were observed at δ 105.21 (d, J_C-1,F = 223.0 Hz), 98.38, 97.92 and 97.56 ppm. Finally, the FABMS analyses of glycosides 13 and 14 confirmed the formation of these products.

Next, we pursued the [4+4] glycosylation reaction to access α(1→6)-linked octamannosyl p-tolylthioglycoside 2. The glycosyl donor 14 was treated with glycosyl acceptor 13 in the presence of promoters AgClO₄ and SnCl₂ in CH₂Cl₂. Even after 48 hrs at room temperature, only 20% yield of the desired octasaccharide 10 was achieved along with the recovery of unreacted acceptor glycoside 13. The glycosylation reaction between 13 and 14 was next performed with coupling reagents Cp₂HfCl₂ and AgOTf in CH₂Cl₂ overnight at room temperature. Octamannoside 2 was produced in 61% yield after purification by SiO₂ column chromatography. The ¹H NMR spectrum of 2 in CDCl₃ showed the anomeric protons at δ 5.78, 5.24, 5.06, 5.01, 5.00, 4.99, 4.82 ppm as singlets and at δ 4.96 ppm (J_1,2 = 1.4 Hz) as a doublet supporting the 1,2-trans glycosylation. In the ¹³C NMR spectrum of 2, the anomeric carbons were not well resolved and appeared as three signals at δ 98.41, 97.99 and 86.89 ppm. Lastly, the final structural confirmation was obtained by MALDI-TOF mass analysis of 2, which showed a peak at 3983.9 [M+Na]⁺ corresponding to the molecular formula C₂₂₅H₁₈₆O₆₅SNa.

After the successful synthesis of 2, dansylated octamannan fluorescent probe 1 was synthesized in four steps (Scheme-2). In step one, α(1→6)-linked octamannosyl thioglycoside 2 was reacted with 1-azidoethanol 3¹⁴ in presence of activator NIS and Lewis acid AgOTf at −4 °C for 45 minutes. Usual workup of the coupling reaction followed by purification of the reaction product by column chromatography gave the desired 1-azidoethyl α(1→6)-linked octamannosyl glycoside 15 in 89% yield. In the ¹H NMR and ¹³C NMR spectra of 15, the signals from p-thiotolyl group were found absent. The ¹³C NMR spectrum of 15 clearly indicated the attributable to CH₂N₃ and OCH₂ carbons respectively. The MALDI-TOF mass analysis of 15 showed a peak at 3947.1 [M+Na]⁺ corresponding to the molecular formula C₂₂₀H₁₈₃N₃O₆₆Na.

Scheme 2.

Reactions and Reagents. (a) HOCH₂CH₂N₃, NIS, AgOTf, dry CH₂Cl₂, 4A Mol sieves, −4 °C, 45 min, 89%. (b) Pd/C, HCO₂NH₄, dry MeOH–CH₂Cl₂ (9:1), rt, 4 h, 82%. (c) Dansyl chloride, N-methylimidazole, dry CH₂Cl₂, 0 °C, overnight, 69%. (d) satd. NH₃ in MeOH, dry CH₂Cl₂, rt, overnight, 71%.

In step two, the azido functionality in 15 was reduced using ammonium formate (HCO₂NH₄) over Pd/C in CH₂Cl₂-MeOH (9:1) and was monitored by TLC. Upon completion of the reaction, concentration and the usual workup produced 1-aminoethyl α(1→6)-linked octamannosyl glycoside 16. Without further purification, the crude 16 was reacted overnight at 0 °C in the dark with dansyl chloride 3 and N-methylimidazole. TLC showed a major fluorescent spot in long wave (365 nm) UV lamp and purification by column chromatography gave the desired blocked dansylated α(1→6)-linked octamannosyl glycoside 17 in 69% yield. The ¹H NMR spectrum of 17 indicated formation of the desired product as it showed signals due to a dansyl group in the aromatic region as well as ppm [N(CH₃)₂]. In the APT ¹³C NMR spectrum of 17 in CDCl₃, the carbons of CH₂NH and N(CH₃)₂ were observed at δ 42.67 and 45.39 ppm respectively. In the MALDI-TOF mass analysis of 17 gave a peak at 4155.8 [M+Na]⁺ corresponding to the molecular formula C₂₃₂H₁₉₆N₂O₆₈SNa.

Finally, global de-protection of 17 was accomplished by overnight reaction with satd. NH₃ solution in MeOH at room temperature. TLC in CHCl₃:MeOH:H₂O (65:35:10, lower layer) showed one fluorescent spot under a long wave UV lamp. The reaction mixture was concentrated to dryness and the syrup was washed several times with CH₂Cl₂ and finally with EtOAc. The syrup was purified by column chromatography on Sephadex LH-20 using MeOH as the mobile phase to furnish the desired fluorescent probe α(1→6)-linked octamannosyl glycoside 1 in 71% yield. The signals in the ¹H NMR spectrum of 1 in D₂O were not well resolved at 300 MHz, but showed protons in aromatic and sugar regions along with a singlet at δ 3.11 PPM (CH₂NH) and δ 2.29 [N(CH₃)₂]. Similarly, in the 75 MHz ¹³C NMR spectrum of 1 in D₂O, the presence of signals at δ 46.82 ppm [N(CH₃)₂] and δ 43.56 ppm (CH₂NH) supported the structure. The final structural confirmation was obtained by FABMS analysis of 1, which showed a peak at 1613.1 [M+Na]⁺, corresponding to the molecular formula C₆₂H₉₈N₂O₄₃SNa.

In conclusion, we have reported a successful and efficient synthesis of a dansylated homolinear α(1→6)-linked octamannan fluorescent probe for its future biochemical potential use as a delivery vehicle for drugs via mannan receptors present in the infected macrophages.