Synthesis of mannosidase-stable Man₃ and Man₄ glycans containing S-linked Manα1→2Man termini

Abstract

Oligomannose glycans are of interest as HIV vaccine components but are subject to mannosidase degradation in vivo. Herein, we report the synthesis of oligosaccharides containing a thio linkage at the non-reducing end. A thio-linked dimannose donor participates in highly stereoselective glycosylations to afford tri- and tetramannose fragments. STD NMR studies show that these glycans are recognized by HIV antibody 2G12, and we confirm that the reducing terminal S-linkage confers complete stability against x. manihotis mannosidase.

Carbohydrate or glycoconjugate vaccines¹ are in use or development for prevention of bacterial infections,^{2, 3} cancer⁴ and HIV.^5–7 In HIV vaccine development, there is significant interest in elicitation of antibodies that can bind to the Manα1→2Man moieties of high mannose (Man₉GlcNAc₂) glycans;^8–11 however, we and others have shown that, for glycoconjugate vaccines, mannosidase trimming degrades this motif so that the antibody response is directed against the glycan core or other structures in the glycoconjugate.^{12, 13} A possible solution to this problem is chemical stabilization of the Manα1→2Man linkage against enzymatic hydrolysis, in particular using sulfur^14–20 in the glycosidic linkage. Indeed, antibodies raised against some S-linked glycan analogs exhibit cross-reactivity with the natural oxygen-linked sugars^21–25 but such analogs have not been tested in the case of oligomannose vaccines.

Inspired by a report of anomeric alkylation to produce sulfur-linked Manα1→2Man disaccharide,¹⁹ we wondered whether a disaccharide donor containing this S linkage (see 1, Scheme 1) would participate in stereospecific glycosylation with anchimeric assistance from the thioether linkage. Glycosyl donors containing simple 2-thio substituents are known,^26–33 but only one thio-linked disaccharide donor has been reported, with a gluco- configuration.³⁴ Glycosylation with dimannose derivative 1 would offer an efficient route to serum-stabilized fragments of Man₉GlcNAc₂, or potentially the whole oligosaccharide.

Scheme 1.

Thioether-linkage-assisted stereospecific glycosylation

To prepare the requisite thio-disaccharide donor, we began from known trityl thioglycoside 4a (Scheme 2).^35,18 Exchange to benzyl protecting groups proceeded in 68 % overall yield to afford building block 4b. We wondered whether 5-derived thiolate could displace a 2-triflate derivative with a relatively inert leaving group such as a fluoride already present at C1.

Scheme 2.

Synthesis of S-linked disaccharide donor

Thus, we prepared 1-fluoro glucose derivative 6 by a known protocol including epoxidation of tribenzyl glucal,³⁶ followed by TBAF treatment.³⁷ Following triflation of 6 and triethylsilane/trifluoroacetic acid deprotection of 4b, 5 and 7 were combined and allowed to react in the presence of sodium tert-butoxide to afford the desired disaccharide 8 in 63 % yield.

With dimannose donor 8 in hand, we prepared a suitable monomannose acceptor to produce Man₃. Starting from mannose building block 9,¹⁹ installation of an azidoethyl linker and deprotection at the 2-position efficiently afforded acceptor 12. Glycosylation of 12 with 1.5 equivalents of 8, in the presence of hafnium trifluoromethanesulfonate³⁸ afforded the desired trisaccharide 13 in 64 % yield as a single stereoisomer. After global deprotection with sodium in liquid ammonia the desired S-Man₃ 14 was isolated in 62 % yield (Scheme 3). The stability of the thio linkage under dissolving metal conditions has been observed previously,^{19, 20} but is nevertheless noteworthy. The α configuration of all mannose units was confirmed by carbon-coupled HSQC, which showed all ¹J_CH to be in the range of 171–178 Hz (see Supporting Information).^{39, 40}

Scheme 3.

Synthesis of S-linked Man₃

Similarly, we set about preparation of an S-Man₄ containing a reducing-terminal β-mannose analogous to the core mannose in the natural Man₉GlcNAc₂. We prepared dimannose acceptor 19 by coupling our previously-described β-mannose core 17⁴¹ to known building block 16,⁴² followed by Lev deprotection. 19 coupled smoothly to Man₂ fluoride donor 8 (see Scheme 4) in 77 % yield, again as single stereoisomer. This tetrasaccharide was globally deprotected and converted to azide 21 in three steps with an overall yield of 40 %. 21 exhibited three anomeric ¹J_CH values from 169–174 Hertz for the α linkages, and, as expected, a value of 158 Hz for β linkage (see Supporting Information).

Scheme 4.

Synthesis of S-linked Man₄

With these S-Man₃ and S-Man₄ derivatives in hand, we proceeded to study their recognition by HIV broadly neutralizing antibody 2G12, which binds primarily to the linear trimannose (D1) arm of Man₉GlcNAc₂. STD-NMR (Saturation Transfer Difference NMR) spectroscopy with 25 μM 2G12 IgG and a 200:1 ratio of sugar:antibody showed that, as expected, the greatest saturation transfer is seen for the non-reducing mannose unit in either Man₃ or Man₄ (SI Figure S1 and Figure 1a). In the case of the Man₄ derivative, negligible STD is observed for the reducing-terminal mannose unit. These data are closely analogous to STD NMR data previously acquired for oxygen-linked oligomannose fragments,^{43, 44} and are consistent with crystal structure data for Man₄ bound to 2G12, in which little if any interaction is evident between the antibody and residue D (Figure 1b).

Figure 1.

Binding analysis of S-Man₄ to HIV broadly neutralizing antibody 2G12. a) STD-NMR spectrum of S-Man₄ (21) with 2G12 IgG. Bottom spectrum (blue) shows the reference 800 MHz ¹H NMR whereas the top (red) shows corresponding STD spectrum. See supporting information for details. Numbers indicate selected assignments by carbon number and ring letter. b) Crystal structure for all O-linked Man₄ (22) bound to 2G12 (PDB ID 6MSY).

Lastly, we tested natural and sulfur-substituted Man₄ derivatives against the action of xanthomonas manihotis mannosidase, which cleaves oligomannose Manα1→2Man and Manα1→3Man linkages. S-Man₄ derivative 21 and its oxygen analog 22 were labeled by strain-promoted azide/alkyne cycloaddition with DBCO amine linker 23, in order to facilitate separation and detection of degradation products by LC/MS. After incubation with mannosidase, LC/MS analysis showed no degradation of sulfur-substituted derivative 24 after 48 hours, but nearly complete digestion of natural Man₄ derivative 25 to Man₁ 27 (Scheme 5).

Scheme 5.

Stability of S-Linked Man4 Against Mannosidase Cleavage.

In conclusion, we have demonstrated a facile synthetic route to Man₃ and Man₄ derivatives with a non-reducing-terminal sulfur linkage that is highly resistant to enzymatic degradation. These derivatives are recognized by an HIV antibody, 2G12, through contacts that are similar to those it makes with the natural oligomannose structure. This synthetic strategy should be readily amenable to preparation of higher branched stabilized oligomannose analogs, suitable for immunogenicity studies in the near future.