Azidophenylselenylation of glycals towards 2-azido-2-deoxy-selenoglycosides and their application in oligosaccharide synthesis

Introduction

2-Amino-2-deoxy-aldohexoses are known to be the principal structural and functional components of glycoproteins, proteoglycans [1], glycolipids, lipopolysaccharides and polysaccharides found in mammals, bacteria [2] and fungi [3]. The most abundant are 2-amino-2-deoxy-D-glucopyranosyl and 2-amino-2-deoxy-D-galactopyranosyl units which can be incorporated into natural glycosides as α- and β-anomers. This structural feature dramatically adds to the complexity of synthetic methods for the stereoselective synthesis of natural or related 2-amino-2-deoxyglycosides. Half-century long efforts were concentrated on elaboration of efficient stereospecific approaches towards 2-amino-2-deoxyglycosides, which indicated the substantial impact of the acyl-protected amino group at C-2 on the stereochemistry of glycosylation facilitating the formation of 1,2-trans-glycosides.To date, preparation of compounds of this type is very well designed [4], [5], [6], [7].

On the contrary, the synthesis of 1,2-cis-glycosides (most often they have α-configuration) still represents a challenge and needs quite long sequences of synthetic steps [8], [9], [10], [11], [12], [13], [14], [15], [16], [17], [18]. Conventional synthetic strategy is based on the use of glycosyl donors with a non-participating azido group at C2 as a synthetic precursor of the amino- or amido-groups [17]. General method for the preparation of 2-azido-2-deoxy glycosyl donors is illustrated in Fig. 1 using the example of D-galactose as a starting compound. It includes first preparation of acetylated galactosyl bromide 1, its conversion into galactal 2 followed by the laborious azidonitration according to Lemieux procedure [19] to give the mixture of 2-azido-2-deoxy glycosyl nitrates 3 and their further transformation into appropriate glycosyl donors. Simplification of this sequence can be achieved via direct transformation of glycal into 2-azido-2-deoxy glycosyl donors which is marked with a bold arrow in Fig. 1.

Fig. 1:

Synthesis of 2-azido-2-deoxy galactosyl donors with the use of azidonitration of tri-O-acetyl-D-galactal 2.

In this review, we describe recent works of our and others laboratories on the synthesis and application of phenyl 2-azido-2-deoxy-1-selenoglycosides as efficient starting materials for preparation of 2-amino-2-deoxy-glycosides. These compounds are directly used as glycosyl donors, which can be activated in different ways to control the formation 1,2-cis and 1,2-trans glycosides. Furthermore, they can be readily transformed into the corresponding 2-azido-2-deoxy-1-halides, imidates, and other types of glycosyl donors [15], [16].

After the description in 1991 by Tingoli et al. [20] of anti-Markovnikov one-step azidophenylselenylation (APS) of olefins with a mixture of NaN₃, PhI(OAc)₂, and Ph₂Se₂ in CH₂Cl₂, this reaction was applied by Czernecki et al. [21], [22], [23], [24], [25] to convert peracetylated and perbenzylated D-glucals and D-galactals into corresponding phenyl 2-azido-2-deoxy-1-seleno-α-D-gluco- and galactopyranosides. For galactals, complete stereocontrol was observed, and APS of glucals resulted in the mixture of gluco- and manno-adducts. Santoyo-Gonzalez et al. [26] confirmed poor stereoselectivity of APS of glucals and showed the inapplicability of this procedure to benzylidenated compounds. In contrast to monosaccharide glucals, APS of peracetylated glycals of lactose, maltose and cellobiose performed by Santoyo-Gonzalez et al. [27] afforded low yields of α,β-isomeric mixtures of phenyl 2-azido-2-deoxy-1-seleno-mannopyranosides.

As we reported before [28], the efficiency and selectivity of the procedure by Tingoli et al. [20] depends on the scale of the reaction. Presumably, the lower efficiency is connected with heterogeneity of the reaction mixture and low solubility of NaN₃ in CH₂Cl₂ in combination with high exothermicity of the chemical process. To avoid these obstacles and to make the APS procedure of glycals scalable and reproducible, we elaborated [28] a homogeneous procedure which involves trimethylsilylazide (TMSN₃) as a soluble azide donor to replace NaN₃. Treatment of a solution of triacetylgalactal 2 (1 mmol), Ph₂Se₂ (1 mmol) and diacetoxyiodobenzene (1 mmol) in CH₂Cl₂ (5 ml) with TMSN₃ (2 mmol) at −10°C in 4 h gave a 9:1:1 mixture of the target phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-galactopyranoside (2a), its regioisomer with a talo-configuration 2b, and the corresponding 1,2-di-azide 2c with galacto-configuration in 90% overall yield. For a deeper insight into the mechanism of APS, we employed [29] a spin trap technique with nitrones and nitroso compounds as spin traps to confirm the presence of azido radicals in the reaction mixture, and demonstrated that these radicals are generated by decomposition of azidoiodinane derivatives.

Recently, with a view to an efficient and safe scale up, Guberman et al. [30] applied a continuous flow technology to homogeneous APS of triacetylgalactal 2 and optimized the conditions for a better yield of 2a. The process was adapted to room temperature, and the production was scaled up to 5 mmol of galactal in 3 h, yielding 1.2 mmol/h of the target compound 2a. Depending on the reaction conditions, up to six by-products [30] were detected in the reaction mixtures.

Differently to the Tingoli method [20], homogeneous APS of galactals (Fig. 2) proved to be compatible with a wide variety of protecting groups. Thus, 3,4,6-tri-O-benzyl-galactal 4 in these conditions was transformed [28] into corresponding phenyl 2-azido-2-deoxy-1-seleno-α-D-glycoside 4a in 72% yield. Homogeneous APS of 3,4-di-O-acetyl-D-fucal 5 gave 3,4-di-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-fucopyranoside 5a in good yield, as reported by Bedini et al. [31] and Hagen et al. [32].

Fig. 2:

APS of galactals 2, 4, and 5.

Homogeneous APS was also successfully applied [28] to the transformation of 2,3,4-tri-O-acetyl-D-glucal. In different solvents and at varied temperature levels it afforded corresponding phenyl 2-azido-2-deoxy-1-selenoglycosides. The major products were phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-glucopyranoside (see gluco-isomer in Table 1) and phenyl 3,4,6-tri-O-acetyl-2-azido-2-deoxy-1-seleno-α-D-mannopyranoside (manno-isomer) with excellent yield (91%) and ratio 2.7:1. We examined [33] the influence of the solvent, temperature and amount of azide source on the ratio of gluco- and manno-isomers formed in APS of 7. In non-polar solvents (dichloromethane, hexafluorobenzene, tetrachloromethane, and toluene), the ratio of gluco- and manno-isomers was 2.7–3.2 to 1, in ethyl acetate and tetrahydrofuran it was substantially lower (1.3–1.5 to 1), while in polar solvents no regioselectivity was observed. Meanwhile, variation of the temperature in the range from −10 to −40°C had no effect on regioselectivity of APS.

Table 1:

Examples of APS of variously protected glucals.

Entry	R1	R2	R3	The ratio of gluco (a) and manno (b) products, yield	Ref.
1	Ac	Ac	Ac	2.7:1 (91 %)	28
2	TIPS	–PhC–		Only gluco, 77 %	28
3	Piv	–CMe2–		1:1	33
4	TIPS	–CMe2–		Only gluco, 85 %	33
5	Bz	Bz	Bz	1:1	33
6	Piv	Piv	Piv	1.6:1	33
7	TMS	TMS	TMS	3:1	33
8	TIPS	TIPS	TIPS	Only gluco, 81 %	33
9	Ac		Ac	1.1:1, 89 %	33
10	TES		TES	Only gluco, 81 % [33], 56 % [34]	33, 34
11	Ac		Ac	1:1, 75 %	33
12	TES		TES	Only gluco, 69 %	33

Further, we extended [33] the reaction conditions to a group of mono-, di-and trisaccharide glucals (Table 1) and studied the effect of the nature of protecting group on efficiency and regioselectivity of APS of these compounds. Variation of protecting groups showed that a combination of a non-polar protecting group (triisopropylsilyl, tributylsilyl, trimethylsilyl) at O3 and a bulky substituent at O4 (TBS, TIPS, mono- or disaccharide residue) facilitates the formation of a gluco-isomer (Table 1, entries 2, 4, 8, 10 and 12). High regioselectivity and efficiency of APS of lactal and sialyllactals provides an easy way towards derivatives of lactosamine, as in the synthesis of inhibitors of galectin-3 [34] and sialyllactosamine which are common glycoprotein motifs in bacteria and mammals.

Phenyl 2-azido-2deoxy-1-selenoglycosides as glycosyl donors

Phenyl selenoglycosides were introduced as versatile glycosyl donors by Mehta and Pinto [35], [36], [37], and now are commonly used [38] in carbohydrate synthesis as glycosyl donors and glycosyl acceptors. In 2001, Tseng et al. [39] performed glycosylation of a blocked derivative of threonine with phenyl 2-azido-4,6-benzylidene-3-O-chloroacetyl-2-deoxy-1-seleno-α-D-galactopyranoside promoted by AgOTf in the presence of K₂CO₃ or tetramethylurea. Notably, the α/β ratio substantially varied in these reactions from 1/1 to 7.5/1 depending on the type of the base and the temperature.

Per-O-acetylated 2-azido-2-deoxy-1-selenogalactoside 2a was used as a glycosyl donor by Kärkkäinen et al. [40] in the presence of I₂ and DDQ or I₂ alone for glycosylation of simple aliphatic alcohols, L-serine and L-threonine. In these reactions, glycosides were produced with low to moderate yields, and the α/β ratio depended on the solvent and promoting compounds. On average, the mixture toluene – dioxane 1:3 facilitated α-glycosylation, but the yields were lower. However, under promotion with NIS/TfOH in CH₂Cl₂, the reaction of glycosyl donor 2a with bis(2-chloroethoxy)ethanol showed no stereocontrol [41].

In 2016 we published [42] the results of a comprehensive study of glycosylation by per-O-acetylated (2a) and per-O-benzoylated (6) phenyl 2-azido-2-deoxy-1-seleno-α-D-galactopyranosides as glycosyl donors. It was found, that the stereoselectivity of glycosylation with these compounds depends on the reactivity of glycosyl acceptors and the reaction conditions. In acetonitrile, glycosylation of 3-trifluoroacetamidopropanol with 2a and 6 was β-stereospecific (Table 2, entries 3 and 6). Glycosylation of 3-trifluoroacetamidopropanol with donors 2a and 6 in the presence of NIS and TfOH at -35 °C in CH₂Cl₂ (Table 2, entries 1 and 4) and in diethyl ether (Table 2, entries 2 and 5) showed, that benzoyl protecting groups favored the formation of α-glycoside as compared to acetylated donor 2a. The use of PhSeCl/AgOTf for promotion of glycosylation with 6 substantially increased the proportion of α-glycoside (Table 2, entries 7 and 8).

Table 2:

Glycosylation of 3-trifluoroacetamidopropanol with donors 2a and 6 [42].

Entry	Donor	Promoter	Solvent	Temperature (°C)	The α/β ratio of glycosides, yield
1	2a	NIS, TfOH	CH2Cl2	−35	1:2 (88 %)
2	2a	NIS, TfOH	Et2O	−35	1:3 (95 %)
3	2a	NIS, TfOH	MeCN	−35	Only β (88 %)
4	6	NIS, TfOH	CH2Cl2	−35	1:1 (92 %)
5	6	NIS, TfOH	Et2O	−35	1:1.3 (78 %)
6	6	NIS, TfOH	MeCN	−35	Only β (75 %)
7	6	PhSeCl, AgOTf	CH2Cl2	0	2.4:1 (95 %)
8	6	PhSeCl, AgOTf	Et2O	0	3.3:1 (95 %)

Glycosylation of methyl 2,3-O-isopropylidene-α-L-rhamnopyranoside by donors 2a and 6 demonstrated the α-directing effect of diethyl ether in comparison to CH₂Cl₂ (Table 3, entries 1 and 2 for donor 2a; entries 4 and 5 for donor 6), and confirmed the preferential formation of β-glycosides in MeCN (Table 3, entries 3 and 6). The highest α-selectivity was observed when PhSeCl/AgOTf system was used to promote glycosylation of methyl 2,3-O-isopropylidene-α-L-rhamnopyranoside with benzoylated donor 6 in diethyl ether (Table 3, entry 8). The results shown in Tables 2 and 3 provide the data needed for the choice of optimal solvents for stereocontrolled α- or β-glycosylation with phenyl 2-azido-2-deoxy-1-seleno-α-D-glycopyranosides, as summarized in Fig. 3.

Table 3:

Glycosylation of methyl 2,3-O-isopropylidene-α-L-rhamnopyranoside with donors 2a and 6.

Entry	Donor	Promoter	Solvent	Temperature (°C)	The α/β ratio of glycosides, yield
1	2a	NIS, TfOH	CH2Cl2	−35	1:1.7 (80 %)
2	2a	NIS, TfOH	Et2O	−35	3.5:1 (87 %)
3	2a	NIS, TfOH	MeCN	−35	1:6 (87 %)
4	6	NIS, TfOH	CH2Cl2	−35	2.5:1 (80 %)
5	6	NIS, TfOH	Et2O	−35	3.6:1 (88 %)
6	6	NIS, TfOH	MeCN	−35	1:3.8 (80 %)
7	6	PhSeCl, AgOTf	CH2Cl2	0	3:1 (72 %)
8	6	PhSeCl, AgOTf	Et2O	0	4.7:1 (99 %)

Fig. 3:

Possible mechanisms of Et₂O and MeCN effects on stereoselectivity of glycosylation.

Stereoselectivity of 2-deoxy-2-azido-fucosylation was studied by Hagen et al. [32] on a series of phenyl 2-azido-2-deoxyselenofucosides as glycosyl donors in the presence of a diphenyl sulfoxide (Ph₂SO)/triflic anhydride (Tf₂O) reagent couple (Fig. 4). ¹H NMR study of reactive intermediates generated from di-O-benzyl-fucosazide 7 upon treatment with Ph₂SO and Tf₂O in CD₂Cl₂ at −80°C revealed the presence of corresponding covalent intermediate 15 as α-triflate (δ 6.06 ppm, J=3.2 Hz) and α-oxosulfonium triflate (δ 6.10 ppm, J=3.2 Hz), which are in equilibrium with the corresponding ionic intermediate 16. Glycosylation of ethanol, 2-fluoroethanol, 2,2-difluoroethanol and 2,2,2-trifluoroethanol with donors 7−12 revealed the increase of α-stereoselectivity with the decrease of nucleophilicity of glycosyl acceptors. The authors [32] explained it with inability of weak nucleophiles to displace a covalent leaving group in covalent intermediates thus inhibiting the formation of β-glycoside and facilitating α-glycosylation of the ionic equilibrium partner. It is important to note, that intermediates generated from 7 started to decompose at −20°C, and decomposition of similar intermediates generated from 8 began around 0°C.

Fig. 4:

(a) Structures of 2-azido-2-deoxy-L-fucosyl donors 7–12 and model acceptors 13 and 14; (b) glycosylation intermediates.

Glycosylation of cyclohexanol and two selectively protected model mannosides 13 and 14 unambiguously demonstrated that the benzoylated 2-azido-2-deoxy-fucosyl donors 8, 9 and 10 were poor α-donors. On the contrary, selenoglycosides 7, 11 and 12 favored the formation of 1,2-cis-glycosides. On the basis of this study, a straightforward synthesis of trisaccharide TS1 (Fig. 5a) related to the repeating unit of the capsular polysaccharide (CPS) of Staphylococcus aureus type 5, which comprises both β-D-FucNAc and α-L-FucNAc residues, was fulfilled. Thus, phenyl selenoglycoside 17, which is an enantiomer of the aforementioned compound 10, was used as a glycosyl donor for stereoselective preparation of β-glycoside 19 (α/β ratio 1:7), which was 3-O-debenzoylated to give glycosyl donor 20 with a free 3-OH. Subsequent glycosylation of 20 with 11 was α-stereospecific and gave disaccharide 21 in 76% yield.

Fig. 5:

(a) Synthesis of trisaccharide TS1 related to the repeating unit of S. aureus type 5 CPS; (b) synthesis of disaccharides 24 and 27 related to CPS of S. aureus strain M and type 8 [32].

Phenyl selenoglycosides 12 and 22, which were prepared using the APS procedure, were the key synthetic blocks in the synthesis of protected disaccharides 24 and 27 related to repeating units of CPS of S. aureus strain M and type 8. Monosaccharide 23 was glycosylated with selenoglycoside 22 to give α-linked disaccharide 24 as a sole product in 65% yield (Fig. 5b). Disaccharide 27 was obtained in three steps, including two glycosylation steps. Glycosylation of 18 with selenoglycoside 22 yielded a mixture of glycosides 25 with α/β ratio 7:1 (85%) (Fig. 5b). After deblocking, glycosylation of 26 with selenoglycoside 12 was conducted to afford disaccharides 27 with α/β ratio 7:1 (73%).

Preparation of a spacered trisaccharide TS2 (see Fig. 6) related to the repeating unit of S. aureus strain M CPS demanded the introduction of an additional α-galactosaminuronic acid residue. At first, phenyl selenoglycoside 28 was considered as a candidate glycosyl donor. However, model glycosylations by phenyl selenoglycoside 28 of a series of substituted ethanol derivatives in conditions described above [43] showed the prevalence of β-glycoside products even in the case of weak nucleophile – 2,2,2-trifluoroethanol. Alternatively, donor 29 [43] carrying an α-directing 4,6-O-bis-tert-butylsilylene (DTBS) group, which was suggested by Kiso et al. [44], showed excellent α-selectivity (α/β 19:1) in model experiments. Subsequently, donor 29 was successfully applied [43] in the synthesis of a spacered trisaccharide TS2 (Fig. 6) related to the repeating unit of S. aureus strain M CPS. The glycosylation of N-benzyloxycarbonyl-N-benzyl-5-aminopentanol with donor 29 was α-stereospecific and afforded glycoside 30 in 82% yield. Straightforward removal of DTBS protecting group, 6-O-regioselective oxidation, and methylation furnished glycosyl acceptor 23 in 85% overall yield, which was glycosylated with 29 to give 88% of α-glycoside 31 as a sole product. Its subsequent de-O-silylation, C-6-oxidation and methylation gave the acceptor 32, which was glycosylated with 22. Similarly to 30, glycosylation of 32 was α-stereospecific and afforded trisaccharide 33 (79%) as the precursor of TS2 [43].

Fig. 6:

Synthesis of a spacered trisaccharide related to the repeating unit of CPS of S. aureus strain M [43].

Phenyl 2-azido-2-deoxy-1-selenogalactosides were also successfully applied in our syntheses [45] of a series of biotinylated oligo-α-(1→4)-D-galactosaminides and their N-acetylated derivatives (Fig. 7) which are structurally related to galactosaminogalactan – the cell wall polysaccharide of fungal pathogen Aspergillus fumigatus, which is the most important airborne human fungal pathogen in industrialized countries. Thus, twelve α-(1→4)-D-galactosaminides composed of 1–6 α-GalN-units were readily obtained in a sequence of elongation steps using a versatile donor selenogalactoside 34 (Fig. 7) which was selected from a number of donor candidates. The pattern of O-substituents was tuned to manage α-stereoselectivity of glycosylation. Thus bulky DTBS protecting group at O4 and O6 was used to prevent the nucleophile attack from the β-site [44], [46] while 3-O-benzoyl group was introduced to provide the remote anchimeric participation which favors α-stereoselectivity of glycosylation [17], [47], [48], [49]. This surprising but useful property of 3-O-benzoyl protecting group was already used by us in the synthesis of 1,2-cis-glycoside bond containing oligosaccharides related to α-xylosylated Epidermal Growth Factor repeats of Notch [50], [51], [52], fucoidans [53], [54], [55], α-(1→3)-D-glucan of A. fumigatus [56], [57] and its α-(1→6)-linked isomer [58].

Fig. 7:

Synthesis of neoglycoconjugates related to fragments of galactosaminogalactan from A. fumigatus [45].

Thus, the glycosylation of 3-trifluoroacetamidopropanol 35 by donor 34 under the promotion with Me₂S₂-MeOTf as the optimal activation system afforded 81% of α-product 36. Easy removal of DTBS group by the action of aqueous HF in pyridine followed by regioselective 6-O-benzoylation furnished glycosyl acceptor 37. In a similar way, di-, tri- and tetrameric glycosyl acceptors 38–40 were obtained in 71–72% yields, and the yields of acceptors 41 and 42 in the last two chain elongation step were 60 and 55%, respectively. Thus obtained biotinyl-containing neoglycoconjugates were arrayed on streptavidin-coated plates and used to assess the epitopes of anti-galactosaminogalactan murine monoclonal antibodies and screen the human antibodies in the sera of patients with allergic bronchopulmonary and chronic pulmonary aspergillosis. The obtained data showed that the oligo-α-(1→4)-D-galactosamines and their N-acetylated derivatives allowed the first precise analysis of the specificity of the antibody responses to this extremely complex fungal polysaccharide [45].

Conclusion and prospects

In conclusion, the efficient two-step approach for the preparation of 1,2-cis-glycosides of 2-amino-2-deoxy-D-glycopyranosides, which was recently proposed in our laboratory, is a handy tool for introduction of corresponding monosaccharide residues into complex synthetic oligosaccharides. Straightforward homogeneous regioselective APS of protected galactals opens the way to variously protected versatile glycosyl donors. β-Stereocontrol in glycosylation reactions with phenyl 2-azido-2-deoxyselenogalactosides is readily achieved by the use of acetonitrile as a reaction solvent. To attain α-stereocontrol, diethyl ether was shown to be effective as a reaction solvent. Furthermore, α-stereoselectivity of glycosylations with phenyl 2-azido-2-deoxy-1-selenoglycosides can be increased by taking advantage of stereocontrolling action of appropriate O-blocking groups. Thus, low reactivity and nucleophilicity of the acceptor and the presence of a bulky protecting group, which hinders β-glycosylation, favor the formation of 1,2-cis-glycosides. So far, homogenous APS of glycals and glycosylation with phenyl 2-azido-2-deoxy-1-seleno-α-D-glycopyranosides have been successfully used in preparation of complex oligosaccharides and neoglycoconjugates related to fragments of antigenic polysaccharides of S. aureus type 5, strain M and type 8 [32], [43], as well as to galactosaminogalactan of A. fumigatus [45].