In order to obtain target compounds 1–5, universal synthetic blocks 13, 21, and 33 were designed. They, on one hand, would ensure the stereoselective building of 1,2-cis-glycosidic bonds between galactopyranose residues and, on the other hand, would allow the regioselective deprotection of hydroxyl groups at C-2, C-3, and C-6 for the efficient synthesis of branched fragments and chain extension. Both goals were achieved by the rational selection of protecting groups in the galactopyranosyl donors. Thus, donor 13, which is the precursor of the branched unit, carries a non-participating allyl (All)-protecting group at O-2 and participating acyl groups at O-3 (levulinoyl, Lev) (Komarova et al., 2014; 2015; 2023), O-4 (pentafluorobenzoyl, PFB) (Komarova et al., 2018a), and O-6 atoms (fluorenylmethyloxycarbonyl, Fmoc), which favored the α-stereocontrol of the glycosylation reaction. According to the literature data, each of the above groups can be selectively removed under orthogonal conditions without affecting the other protecting groups (Prabhu et al., 2003; Ágoston et al., 2016).

Galactosyl donor 13 was synthesized from the well-known monosaccharide precursor 6 (Thijssen et al., 1998) (Scheme 1). Its primary hydroxyl group at C-6 was regioselectively protected by Fmoc to form 3,4-diol 8 (Argunov et al., 2016). The introduction of the levulinoyl group using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) hydrochloride and dimethylaminopyridine (DMAP) at −18^оС proceeded exclusively at O-3 (Hirose et al., 2015), and the subsequent treatment of product 10 with pentafluorobenzoyl chloride in the presence of pyridine (Py) allowed obtaining a fully protected monosaccharide 11. The p-methoxyphenyl-protecting group was removed from the anomeric center by ceric ammonium nitrate (CAN) in a mixture of acetonitrile, benzene, and water to form the corresponding hemiacetal 12, which was then converted to N-phenyltrifluoroacetimidate 13. It is important to note that glycosyl donors of this type are usually purified on silica gel with the addition of triethylamine to the eluent in order to neutralize the silica gel and reduce the cleavage of the acid-labile leaving group. In the case of Fmoc-containing donors, the presence of triethylamine led to a loss of yield as a result of partial Fmoc removal (Oberli et al., 2008). Chromatography of donor 13 on neutral aluminum oxide without triethylamine has made it possible to mitigate side reactions and isolate the product with a sufficiently high yield of 82%.

Monosaccharide blocks 15 and 17 were obtained based on 2-O-benzylated triol 7 (Zhu and Yang, 2012) (Scheme 1). Protecting groups (6-O-Fmoc and two benzoate groups at C-3 and C-4) were introduced sequentially with considerably high yields at each step. The resulting monosaccharide 14 was partially converted to acceptor 15 after Fmoc removal under the action of piperidine in tetrahydrofuran (THF). Hemiacetal 16 was also obtained from monosaccharide 14 and then treated with N-phenyltrifluoroacetimidoyl chloride in acetone. As in the case of donor 13, chromatographic purification of the resulting 6-O-Fmoc-bearing donor was performed on neutral Al₂O₃, yielding compound 17 with a considerably high yield of 84%.

The conditions for the selective removal of the chosen protecting groups (Fmoc, Lev, and All) were optimized using the model monosaccharide 11 (Scheme 2). The use of the standard Fmoc-removal procedure in the piperidine/THF system led to a rapid cleavage of the Fmoc group (Pennington and Dunn, 1994; Werz, 2012). However, under these conditions, a side reaction was observed, which consisted of the substitution of a fluorine atom in 4-O-pentafluorobenzoate by piperidine. This process was confirmed by HRMS data and the emergence of piperidine ring signals in ¹H NMR spectra at 3.31 ppm 1.65 ppm and ¹³C NMR at 52.1 ppm and 23.9 ppm, respectively. Thus, these conditions can be used only for compounds without a pentafluorobenzoyl group or only at the last synthetic steps of the complete deprotection. Nevertheless, the Fmoc group in the presence of pentafluorobenzoate was successfully removed under milder conditions under the action of N-methylmorpholine in methylene chloride for 2 days. The levulinoyl group was efficiently and selectively removed by hydrazine acetate in pyridine, resulting in compound 19 with a 96% yield. The allyl substituent at O-2 was selectively removed using (1,5-cyclooctadiene)bis (methyldiphenylphosphine)iridium(I) hexafluorophosphate ([Ir(COD) (PMePh₂)₂]PF₆), which was pre-reduced with hydrogen (Laroussarie et al., 2015). This resulted in compound 20 with an almost quantitative yield.

In order to increase the efficiency of the α-(1→6)-glycoside bond formation, the conditions for the glycosylation of the spacer-containing acceptor 21 by donor 13 were optimized. Originally, the coupling was carried out in the presence of trimethylsilyl trifluoromethane sulfonate (TMSOTf) at −35°C (Table 1, entry 1). The desired disaccharide 23 was obtained with an insufficient yield of 28%; however, full α-stereospecificity was achieved that can be explained by the presence of three α-directing protecting groups in donor 13. The low yield may be attributed to the presence of three electron-withdrawing groups in the donor, which lower its activity; hence, the side processes of its destruction occur before the glycosylation reaction is completed. An increase in the reaction yield to 47% was achieved by replacing the promoter with triflic acid, along with a gradual increase in temperature to −15°C (Table 1, entry 2). One of the reasons for the low yield of disaccharide 23, in this case, is the removal of Fmoc from the O-6 product by triethylamine, which is used to neutralize unreacted acid after the reaction is completed (Oberli et al., 2008). This is confirmed by an increase in the yield of disaccharide 23 up to 64% when the addition of triethylamine was omitted, and the reaction mixture was immediately filtered and washed with a saturated NaHCO₃ solution (Table 1, entry 3).

The disaccharide acceptor 25 was obtained from 23 by the removal of the 2-O-allyl group with the iridium complex [Ir(COD) (PMePh₂)₂]PF₆ pre-reduced with hydrogen (Scheme 3). Glycosylation of acceptor 25 by galactofuranosyl donor 22, previously obtained by us (B. Krylov et al., 2018), in the presence of TMSOTf, resulted in trisaccharide 28 with a high yield as a pure β-isomer. In the next step, the hydroxyl group at the C-3 atom of the non-reducing residue was recovered by hydrazine acetate in pyridine. However, in addition to the expected disaccharide 29, we observed the formation of a migration product of pentafluorobenzoate from O-4 to O-3 (compound 29i). Both regioisomers 29 and 29i were successfully separated by column chromatography and found applications in the synthetic scheme.

The target trisaccharide 1a was synthesized from the pentafluorobenzoyl migration product 29i in three steps. First, the Fmoc-protecting group was removed with piperidine in THF. Then, without intermediate purification, benzoyl substituents were removed in the presence of sodium methylate in methanol. The following reduction of the azide group in the spacer to the amino group and the simultaneous removal of benzyl groups by treatment with sodium in liquid ammonia yielded the unprotected (1→2)-trisaccharide 1a with a high yield of 98%. Tetrasaccharide 30 was obtained by coupling galactofuranosyl donor 22 and trisaccharide acceptor 29 with a fairly high yield of 87% and exclusively as a pure β-isomer. The sequential removal of protecting groups using a scheme similar to that described above for trisaccharide 29i resulted in unprotected tetrasaccharide 3a.

Unlike in trisaccharide 28, the removal of the 3-O-levulinoyl group in disaccharide 23 did not result in the migration of the pentafluorobenzoyl group from O-4 to O-3. The furanosyl residue was introduced by glycosylation with donor 22, resulting in the formation of β-(1→3)-trisaccharide 26 with an 86% yield. The removal of all protecting groups in compound 26 included (1) 2-O-deallylation (→27); (2) removal of 6-O-Fmoc with piperidine in THF; (3) removal of 4-O-pentafluorobenzoyl and benzoyl groups with sodium methylate in methanol; and (4) hydrogenolysis on Pd(OH)₂/C in the presence of a small amount of hydrochloric acid, which prevents the methylation of the amino group of the target (1→3)-trisaccharide 2a.

The α-(1→6)-linked galactopyranosyl chain in the synthesis of target compounds 4a and 5a was elongated with disaccharide donor 33 (Scheme 4). The glycosylation reaction of p-methoxygalactoside 15 by donor 17 in the presence of TfOH proceeded with the exclusive formation of α-isomer 33 due to the concerted action of three α-stereodirecting acyl groups at O-3, O-4, and O-6 (Baek et al., 2015). The absence of the β-isomer among the reaction products was confirmed by the NMR spectra of the untreated reaction mixture. The removal of the p-methoxyphenyl-protecting group of the anomeric center, followed by the addition of the N-phenyltrifluoroacetimidoyl-leaving group to the hemiacetal 32, yielded disaccharide donor 33.

Glycosylation of prespacer-containing monosaccharide 21 by donor 33 in the presence of TfOH (Scheme 5) resulted in a mixture of α- and β-isomeric trisaccharides in the ratio of 20:1. Their ratio was determined by the integration of the ¹H NMR spectrum of the reaction mixture. After the successful separation of the two isomers by column chromatography, the desired α-product 34 was isolated with a yield of 75%. The trisaccharide acceptor 35 was obtained after the removal of 6-O-Fmoc with piperidine in THF. An attempt of a TfOH-assisted glycosylation of acceptor 35 with a disaccharide donor 33 failed. After an optimization of conditions, it was found that in the presence of TMSOTf and with an increase in temperature from −20°C to −5°C, α-pentasaccharide 36 is formed with a sufficient yield of 55% without any β-isomer admixture.

Protecting groups in pentasaccharide 36 were removed according to a standardized algorithm: first, Fmoc was removed with piperidine; then, benzoate groups were removed in the presence of sodium methylate in methanol; and, in the last step, the azide group was reduced and benzyl groups removed in the course of catalytic hydrogenolysis. Unprotected pentasaccharide 4a was isolated by gel permeation chromatography with a 70% yield after all stages of deprotection. The ¹H NMR spectrum of the product contains five anomeric proton signals. For each of the monosaccharide residues, α-configuration of the C-1 atom is confirmed both by spin–spin coupling constants (less than 4 Hz) and chemical shifts of the related carbon atoms (signals at 99.4, 98.9, and 98.8 ppm and two more signals at 98.7 ppm in ¹³C NMR).

The synthesis of hexasaccharide 5a began with the removal of 6-O-Fmoc in disaccharide 23 by N-methylmorpholine in a mixture of dichloromethane and THF (Scheme 6). The ¹⁹F NMR spectrum, as well as the absence of piperidine signals in the ¹H spectrum, confirmed that the pentafluorobenzoyl group was not affected in this transformation. The TfOH-promoted glycosylation of the resulting acceptor 38 by disaccharide donor 33 was very slow and required a gradual increase in temperature from −20°C to −8°C. The low reaction rate and, accordingly, the accumulation of a large number of by-products of the destruction of the donor may be attributed to the presence of a strong electron-withdrawing PFB group in the immediate vicinity of the nucleophilic center in the acceptor. Attempts to vary the temperature regime of this reaction, as well as to change the promoter from TfOH to TMSOTf, tert-butyldimethylsilyl trifluoromethane sulfonate (TBDMSOTf), and C₄F₉SO₃H, were not successful. After the separation of α- and β-isomers, obtained in a ratio of 13:1 in the reaction promoted with TfOH, tetrasaccharide 39a was isolated by HPLC with a yield of 51%.

For the synthesis of the branched hexasaccharide, we first obtained acceptor 40, which was then reacted with donor 22. Monofuranosylated pentasaccharide 41 was isolated with a fairly high yield of 84% and only as a β-isomer. The introduction of a second galactofuranosyl residue after the removal of the levulinoyl-protecting group from O-3 resulted in a protected hexasaccharide 43 with an 87% yield and absolute β-stereoselectivity. The target hexasaccharide 5a was obtained after the sequential removal of Fmoc and benzoate groups and hydrogenolysis, with a total yield of 46% for the three steps. Two singlets (δ 5.19 and 5.17 ppm) corresponding to H-1 of furanose rings are observed in the ¹H NMR spectrum. Further notable are four doublets (δ 5.07–4.96 ppm) with J in the range 2.6–3.3 Hz, attributable to H-1 in pyranose residues of the main chain. Chemical shifts of the corresponding carbon atoms in the ¹H–¹³C HSQC spectra also confirm the configurations of the anomeric centers of all six carbohydrate residues of compound 5a (for ¹³C-NMR data of 1a–5a, see Table 2).

Biotinylated oligosaccharides 1b–5b were obtained by the treatment of aminopropyl glycosides 1a–5a with an activated biotin ester and triethylamine in DMF according to the described procedure (Tsvetkov et al., 2012). Following purification on gel TSK-40 afforded 1b–5b in good to excellent yields. The attachment of the biotin entity was confirmed by the presence of characteristic signals in the ¹H NMR spectra (for NMR data of corresponding biotin conjugates, see the Supplementary Material) and by HRMS data.

The NMR spectra of oligosaccharides 1a–5a were totally assigned by applying 2D NMR experiments (Table 2. For ¹H NMR shifts, see Supplementary Table S1 in Supplementary Material). The effects of glycosylation (Table 3, units are labeled A–D as in Figure 2) were calculated as the difference in the ¹³C chemical shifts between two structures, one with and one without a particular type of glycosylation, as described before (Dorokhova et al., 2021). Upon the introduction of a glycosylating residue, the most pronounced spectral effect was observed on the glycosylated carbons, which underwent a down-field shift by 5–8 ppm (α-effect), while the resonances of the adjacent carbon atoms moved up-field to a smaller extent (β-effect) (Lipkind et al., 1988; Shashkov et al., 1988; Kochetkov et al., 1991; Gerbst et al., 2015). The C-1 of the glycosylating residue also underwent a significant down-field shift. Other carbon resonances were much less affected and were excluded from consideration. The α- and β-glycosylation effects for β-(1→2)- and β-(1→3)-galactofuranosylation measured using trisaccharides 1a and 2a agreed well with previously reported data (Dorokhova et al., 2021). The deviations from additivity in the vicinally branched fragment of hexasaccharide 5a (Table 4) were calculated as the difference between the experimental (δ_exp) and calculated (δ_calc) ¹³C chemical shifts, where δ_calc was calculated by the summation of all glycosylation effects.

In spite of the presence of 2,3-vicinal branching and 1,2-cis-pseudobranching in hexasaccharide 5a, the good agreement between theoretical and experimental ¹³C chemical shifts was determined (deviation from additivity did not exceed 0.56 ppm; Table 4). It suggests the independence of conformational flexibility around corresponding interunit linkages connected with the branched Galp-residue of 5a. These suggest that the spatial similarity of 5a to the corresponding fragment within the chain of GXMGal makes 5a a the reliable model for future immunological studies of C. neoformans.