Chemoenzymatic Synthesis of Functional Sialyl Lewis^X Mimetics with a Heteroaromatic Core

Abstract

Functional mimetics of the sialyl Lewis^X tetrasaccharide were prepared by the enzymatic sialylation of a 1,3-diglycosylated indole and a glycosyl azide, which was subsequently transformed into a 1,4-diglycosylated 1,2,3-triazole, by using the trans-sialidase of Trypanosoma cruzi. These compounds inhibited the binding of E-, L-, and P-selectin-coated nanoparticles to polyacrylamide-bound sialyl-Lewis^X-containing neighboring sulfated tyrosine residues (sTyr/sLe^X-PAA) at low or sub-millimolar concentrations. Except for E-selectin, the mimetics showed higher activities than the natural tetrasaccharide.

Introduction

Cell–cell interactions are a prerequisite for the functioning of multicellular organisms. Besides protein–protein interactions, the recognition of glycostructures on the cell surface by cognate (glyco)protein receptors—lectins—is one of the two major mechanisms of cell adhesion.[1] In humans and animals, the attractive interactions between leukocytes, which patrol the bloodstream, and the activated endothelium of blood vessels, which indicates tissue inflammation, play a key role in the inflammatory cascade, which is an important part of the immune response against pathogenic microbes. Recognition of the tetrasaccharide sialyl Lewis^X (sLe^X, α-Neup5Ac-(2→3)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-GlcpNAc, 1; Scheme 1) by endothelial (E-), platelet (P-), and lymphocyte (L-)selectin constitutes the initial step in this physiological process.[2]

Scheme 1.

Structures of the sialyl Lewis^X tetrasaccharide and mimetic 2.

The excessive accumulation of leukocytes in an inflamed tissue, initiated by exaggregated leukocyte adhesion to activated vascular endothelia, is thought to be the main cause of secondary tissue destruction in various chronic diseases, such as asthma,[3] psoriasis,[4] and rheumatoid arthritis.[5] Selectin-based cell adhesion is also involved in pathomechanisms, such as tumor metastasis[6] or the reperfusion syndrome,[7] which is observed upon restoring circulation after a period of ischemia in a diverse range of tissues and can contribute to complications in the transplantation of organs. In all of these cases, inhibition of the sLe^X–selectin interactions is desirable. To achieve this inhibition, numerous mimetics of the sLe^X molecule have been synthesized that are capable of competing with their natural antetype for the binding site in the C-type lectin domain of the selectins.7a, [8] These mimetics belong to various structural classes, ranging from all-carbohydrate compounds to peptides and N-heterocycles.[9]

The crystal structures of complexes of sLe^X with E- and P-selectin show that, among all four saccharide units, the GlcNAc residue has the weakest interactions with both receptors.[2] In previously reported mimetics, the GlcNAc residue has been substituted by cyclic and acyclic 1,2-diols to imitate the relative arrangement of the 3-sialylated galactose residue and the fucose moiety in sLe^X.[8] In particular, (1R,2R)-cyclohexanediol was found to be a well-suited GlcNAc surrogate (see mimetic 2; Scheme 1), whereas the use of more flexible or improperly aligning spacer units resulted in a loss of affinity towards E-selectin.[10] We wanted to investigate the possibility of replacing GlcNAc by a rigid heterocycle, which ideally should: 1) simplify the preparation of the respective mimetic; 2) permit the late-stage variation of the fucose moiety; and 3) use N- or C-glycosidic bonds to attach the pending hexose units to prevent enzymatic cleavage, whilst 4) retaining or even improving the affinity of the native tetrasaccharide.

Results and Discussion

Attempts to bring the galactose and fucose moieties into similar spatial proximity as in sLe^X by preparing diglycosylacetylenes and subjecting them to 1,3-dipolar cycloaddition or by transforming them into 2,3-diglycosylquinoxalines through oxidation into 1,2-diones and subsequent reaction with o-phenylenediamine met with little success. In contrast, C,N-diglycosylated o-alkynylbenzamides of type 5 could be readily prepared from glycosylamines by amide formation with 2-iodobenzoyl chloride; subsequent Sonogashira reaction with glycosylacetylenes to provide the starting materials for a Larock iodocyclization has been reported to furnish isoindolinones of type 7 (Scheme 2).[11]

Scheme 2.

Diglycosylated heterocycles as potential core structures.

Although the reaction products exhibited spectroscopic properties that were very similar to literature data, the structural assignment of the known iodocyclization products was found to have been wrong and that isobenzofurans of type 6 were formed instead.[12] These latter products should not only provide a less-suitable spatial arrangement of the glycosyl moieties, but their structure also explains the observed instability towards the conditions for protecting-group removal.

The copper- (CuAAC)[13] or ruthenium-catalyzed (RuAAC)[14] 1,3-dipolar cycloaddition[15] of glycosylazides to glycosylacetylenes provided the expected 1,4- and 1,5-diglycosylated 1,2,3-triazoles, respectively, in appreciable yields.11a In particular, the CuAAC reaction was highly reliable and largely insensitive towards steric hindrance. Moreover, both types of triazoles were stable towards the hydrogenolytic removal of benzyl protecting groups on the glycosylacetylene moiety. Selective enzymatic sialylation of the 3-OH group of the galactopyranose unit was employed to transform β-d-galactopyranosylazide (8) into disaccharide azide 10. This reaction uses the readily available p-nitrophenylsialoside 9 as the sialyl donor and is catalyzed by the trans-sialidase of Trypanosoma cruzi, the causal agent of Chagas disease (Scheme 3).[16] Subsequent CuAAC reaction with fucosylacetylene 11 furnished C-fucoside 12, which was subsequently debenzylated to yield mimetic 13 (Scheme 3).

Scheme 3.

Chemoenzymatic synthesis of mimetic 13.

The specific role of the α-l-fucopyranosyl unit within the sLe^X tetrasaccharide is the complexation of a selectin-bound Ca²⁺ ion by the hydroxy groups at the 2-, 3-, and 4-positions.[2, 17] α-d-Mannopyranosides present the same spatial arrangement of their three secondary alcohol functions with the axial 2-OH-group of mannose being equivalent to the 4-OH-group of fucose. Based on this analogy, various functional mannose-derived sLe^X mimetics were prepared.[18] To test whether the activity of mimetic 13 could be retained or even improved in a similar fashion, the mannose analogue of compound 13, C-mannoside 16, was prepared accordingly from mannosylacetylene 14 (Scheme 4).

Scheme 4.

Synthesis of C-mannoside 16.

1,3-Diglycosylindoles are another class of potential trisaccharide mimetics and present the same relative arrangement of the two pendant glycosyl moieties as in 1,4-diglycosyl-1,2,3-triazoles. Although the installation of 1,3- and 2,3-diglycosylation patterns on the indole core has already been established,[19] the former compound class is particularly attractive, owing to the possibility of introducing both carbohydrate substituents in a straightforward sequential manner. The result of scaffold hopping from 1,2,3-triazole to indole on mimetic 16 is C-mannoside 21, which could be prepared by the reaction of d-galactose with indoline and subsequent dehydrogenation of the resulting β-N-galactoside with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ),[20] followed by O-acetylation to afford protected 1-glycosylindole 17. α-Selective C-glycosylation at the 3-position with peracetylated mannosyl trichloroacetimidate 18 produced 1,3-diglycosylindole 19, which was deacetylated under Zemplén conditions to give trisaccharide mimetic 20. The enzymatic sialylation reaction proceeded with complete regioselectivity and furnished sLe^X mimetic 21 as the sole product in 13 % yield (24 % based on recovered starting materials; Scheme 5).

Scheme 5.

Chemoenzymatic synthesis of mimetic 21.

Evaluation of Selectin Inhibition

The binding of the prepared sLe^X mimetics to the selectins was studied by using a competitive inhibition assay. In this assay, potential inhibitors compete with the binding of L-, P-, or E-selectin to the synthetic sulfo-tyrosine/sLe^X biligand under flow conditions.[21] Mimetics 13, 16, and 21 were compared to the performance of the sLe^X tetrasaccharide in this assay (Figure 1). Binding of L- and P-selectin is not inhibited by the natural sLe^X ligand within the tested concentration range since additional binding sites on these selectins need to be addressed. However, E-selectin is inhibited, with an IC₅₀ value of about 0.7 mm (Table 1). In contrast, mimetic 13 shows inhibition of L- and P-selectin, with IC₅₀ values of 2.2–2.3 mm, but no significant inhibition of E-selectin binding within the tested concentration range. Mimetic 16 only shows inhibition of L-selectin, with IC₅₀ values within the same concentration range as for mimetics 13 and 21. Indole-based mimetic 21 can inhibit all three selectins, with IC₅₀ values from 0.7 mm (P-selectin) to 7.4 mm (E-selectin), and is as effective for L-selectin inhibition as the other two mimetics.

Figure 1.

Performance of mimetics 13, 16, 21, and sLe^X in a competitive selectin-inhibition assay, expressed as a plot of relative binding versus concentration.

Table 1.

IC₅₀ values [mM] of the sLe^X mimetics, as calculated from the dose-response curves

Compound	IC50 value
	L-selectin	P-selectin	E-selectin
13	2.3	2.2	–
16	1.8	–	–
21	1.8	0.7	7.4
sLeX	–	–	0.7

Thus, the mimetics tested show selectivity towards the three selectins: Whereas sLe^X is the best inhibitor of E-selectin and does not affect the other selectins within the tested concentration range, compound 21 is the best inhibitor of P-selectin and shows lower potency towards L- and E-selectin. Compound 13 inhibits P- and L-selectin in a similar manner and compound 16 shows a clear preference for L-selectin.

Conclusions

In summary, three heteroarene-based mimetics of sialyl Lewis^X were synthesized by using chemoenzymatic methods. All three compounds were found to be better inhibitors than the native tetrasaccharide for at least one of the three selectins, with E-selectin the weakest binding partner in all cases. Whereas triazole-based C-mannoside 16 only showed appreciable binding to L-selectin, the analogous C-fucoside (13) inhibited P- and L-selectin binding with similar potency. The most-active compound was indole-based mimetic 21, which affected all three selectins. Mimetic 21 showed a clear preference for P- over L-selectin and had less effect on E-selectin binding, thereby resulting in an IC₅₀ value about one order of magnitude higher than that of sLe^X. However, the binding of L- and P-selectin was even more effectively inhibited than by the natural tetrasaccharide.

Interestingly, scaffold hopping from 1,2,3-triazole to indole on mimetic 16 generated high affinity for P-selectin whilst retaining the same activity for L-selectin. These results demonstrate that functional sLe^X mimetics can be obtained without any particular relative preorganization of the 3-sialylgalactose unit with the fucose moiety or a suitable substitute through an exo-anomeric effect. Substitution of the GlcNAc portion of sLe^X, the impact of which on the overall conformation of the tetrasaccharide has been thoroughly investigated,[22] by two simple heteroarenes even leads to compounds with improved affinity for P- and/or L-selectin without the need to address distal binding pockets.[23] Although the metabolic stability of the as-prepared mimetics has not yet been tested, complete inertness of the two modified glycosidic bonds against degrading glycosidases is to be expected because the essential structural features for the action of these enzymes are missing. It is likely that heteroarene-based glycomimetics can also be used to target further carbohydrate-binding proteins, which may constitute an interesting opportunity for drug development.

Experimental Section

For materials and methods, see the Supporting Information.

Expression and Purification of Trans-sialidase

T. cruzi trans-sialidase,16c, [25] which contained a hexahistidine tag, was expressed in E. coli M15 pRep4 cells (Qiagen) in terrific broth medium at 18 °C for 16 h and 120 rpm. The cells were harvested, re-suspended in 50 mm sodium phosphate buffer (pH 8.0) that contained 0.3 m NaCl and 0.05 % Lubrol, and sonicated. After centrifugation, the supernatant was applied onto a His-Trap matrix (GE Healthcare) and washed with 50 mm sodium phosphate buffer (pH 8.0) that contained 0.3 m NaCl. Trans-sialidase was eluted with 50 mm sodium phosphate buffer (pH 8.0) that contained 250 mm imidazole with a purity of 95 % and dialyzed against 20 mm Tris buffer (pH 7.6) that contained 30 mm NaCl.

Competitive Selectin-Inhibition Assay

Sialyl Lewis^X mimetics were tested for their ability to inhibit selectin-mediated binding by using a competitive SPR assay, as described by Enders et al.[21] Briefly, recombinant selectin Fc-chimeras (R&D Systems) were coupled to gold nanoparticles that were coated with protein A (15 nm average, Aurion) in running buffer (20 mm HEPES pH 7,4; 150 mm NaCl, 1 mm CaCl₂) and passed over the surface of a sensor chip of a BIAcore X device (GE Healthcare). The sensor-chip surface was divided into two flow cells: a measurement cell, which was functionalized with sLe^X and sulfated tyrosine coupled to a polyacrylamide backbone, and a reference cell, which displays the non-binding LacNAc polymer in a similar manner. Both conjugates were immobilized onto the streptavidin sensor chip by using biotin. The unspecific binding signal from the reference cell was subtracted during the measurements. An aliquot (35 μL) of the selectin-coupled gold particles with the buffer or inhibitors was injected at a flow rate of 20 μL min⁻¹; the dissociation phase was 120 s. The data points were collected by using the difference of the binding signal immediately before injection (baseline) and at the end of the dissociation phase. All of the measurements were performed at least three times and averaged. The inhibitors were incubated for 18 min with the selectin gold particles prior to injection. The resulting reduced binding signal was divided by the binding values without the inhibitor (100 %) at each corresponding point, which was calculated by linear regression of the 100 % values taken before and after each series of measurements. The data were plotted as the percentage relative binding versus the concentration of inhibitor. The IC₅₀ values were calculated by using a dose-response curve fit (log inhibitor versus normalized response-variable slope) in GraphPad Prism 5.

3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galactonon-2-ulopyranosyl)-β-d-galactopyranosyl azide (10)

The title compound was prepared according to a method reported by Neubacher et al.16b Azide 8[26] (400 mg, 1.95 mmol) and 4-nitrophenyl-α-sialoside 9[27] (492 mg, 1.10 mmol) were dissolved in Tris-HCl-buffer (8 mL, 100 mm, pH 7.9). The mixture was cooled to 13 °C by using a cryostat and an aqueous solution of recombinant trans-sialidase (4 mL, c=0.93 mg mL⁻¹) was added. The mixture was kept at that temperature for 3 days. The enzyme was denatured by the addition of EtOH (10 mL) and the mixture was centrifuged. The supernatant was removed and the solvent was evaporated by lyophilization. The lyophilisate was purified by flash chromatography on silica gel (cyclohexane/EtOAc, 3:1→EtOAc/MeOH, 10:1→MeOH). The product contained remaining TRIS-buffer (13 mol % as judged by NMR spectroscopy), which could neither be removed by the weakly acidic cation-exchange resin Amberlite IRC-86 nor by repeated chromatography. Yield: 257 mg (0.518 mmol, 47 %); colorless solid; R_f=0.12 (BuOH/AcOH/H₂O, 5:2:2); m.p. 175.0–176.0 °C; [α]=±0.0 (c=0.40, CD₃OD); ¹H NMR (COSY, 400 MHz, CD₃OD): δ=4.49 (d, ³J_1,2=8.5 Hz, 1 H; H-1^gal), 4.04 (dd, ³J_2,3=9.6 Hz, ³J_3,4=2.7 Hz, 1 H; H-3^gal), 3.95 (d, ³J_3,4=2.7 Hz, 1 H; H-4^gal), 3.86–3.59 (m, 9 H; H-5^gal, H-6a/b^gal, H-4^sia, H-5^sia, H-6^sia, H-8^sia, H-9a/b^sia), 3.55 (pseudo-t, ³J_1,2=³J_2,3=9.2 Hz, 1 H; H-2^gal), 3.50 (m, 1 H; H-7^sia), 2.84 (dd, ²J=11.8 Hz, ³J_3,4=3.3 Hz, 1 H; H-3a^sia), 2.01 (s, 3 H; CH₃^NHAc), 1.76–1.70 ppm (m, 1 H; H-3b^sia); ¹³C NMR, HSQC (100.6 MHz, CD₃OD): δ=175.5 (2 C; C=O^sia, C=O^NHAc), 101.1 (C-2^sia), 92.3 (C-1^gal), 77.7 (C-3^gal), 70.3 (C-2^gal), 70.0 (C-7^sia), 78.9, 74.9, 72.9, 69.3 (C-5^gal, C-4^sia, C-6^sia, C-8^sia), 68.9 (C-4^gal), 64.4 (C-6^gal), 62.7 (C-9^sia), 53.9 (C-5^sia), 42.0 (C-3^sia), 22.6 ppm (CH₃^NHAc); MS (ESI): m/z (%): 519.1 [M+Na]⁺ (100); HRMS (ESI): m/z calcd for [C₁₇H₂₈N₄O₁₃+Na]⁺: 519.1551 [M+Na]⁺; found: 519.1546.

1-(3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galactonon-2-ulopyranosyl))-β-d-galactopyranosyl-4-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-1 H-1,2,3-triazole (12)

The title compound was prepared according to a method reported by Dondoni and Marra.[28] Fucosylacetylene 11[29] (82 mg, 0.19 mmol) and azide 10 (91 mg, 0.18 mmol) were dissolved in dry DMF (4 mL). N,N-Diisopropylethylamine (DIPEA, 64 μL) and CuI (7 mg, 0.04 mmol, 20 mol %) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 °C. The solvent was removed in vacuo and the residue was co-evaporated three times with toluene (5 mL each). The crude product was purified by flash chromatography on silica gel (CH₂Cl₂/MeOH, 4:1→2:1) to yield the title compound (94 mg, 0.10 mmol, 55 % yield) as a light-yellow oil. [α]=−51.6 (c=0.6, MeOH); ¹H NMR (COSY, 600 MHz, CD₃OD): δ=8.20 (s, 1 H; H-5), 7.41–7.40 (m, 2 H; Ph), 7.37–7.22 (m, 13 H; Ph), 5.65 (d, ³J_1,2=8.9 Hz, 1 H; H-1^gal), 5.24 (d, ³J_1,2=5.4 Hz, 1 H; H-1^fuc), 4.85 (d, ²J=11.2 Hz, 1 H; CH₂–Ph), 4.79 (d, ²J=11.5 Hz, 1 H; CH₂–Ph), 4.77 (d, ²J=11.5 Hz, 1 H; CH₂–Ph), 4.64–4.58 (m, 3 H; CH₂–Ph), 4.27 (dd, ³J_2,3=9.6 Hz, ³J_3,4=2.0 Hz, 1 H; H-3^fuc), 4.21–4.18 (m, 2 H; H-2^gal, H-2^fuc), 4.09 (d, ³J_3,4=2.9 Hz, 1 H; H-4^gal), 4.06 (dd, ³J_2,3=8.9 Hz, ³J_3,4=2.9 Hz, 1 H; H-3^gal), 3.89–3.70 (m, 9 H; H-5^gal, H-6a^gal, H-4^fuc, H-5^fuc, H-5^sia, H-6^sia, H-8^sia, H-9a/b^sia), 3.63–3.60 (m, 2 H; H-6b^gal, H-4^sia), 3.51 (d, ³J=8.9 Hz, 1 H; H-7^sia), 2.90 (dd, ²J=13.1 Hz, ³J_3,4=3.5 Hz, 1 H; H-3a^sia), 2.01 (s, 3 H; CH₃^NHAc), 1.80–1.79 (m, 1 H; H-3b^sia), 1.19 ppm (d, ³J_5,CH3=6.4 Hz, 3 H; CH₃^fuc); ¹³C NMR (HSQC, HMBC, 150.9 MHz, CD₃OD): δ=175.5 (2 C; C=O^sia, C=O^NHAc), 140.1 (3 C; C-4, 2×Cq–Ph), 139.6 (Cq–Ph), 129.4 (4 C), 129.34 (2 C), 129.27 (2 C), 129.2 (2 C), 129.0 (2 C), 128.79, 128.77, 128.67 (Ph), 124.4 (2 C; C-5, C-2^sia), 90.0 (C-1^gal), 79.9, 78.3, 72.9, 70.8, 69.4 (C-5^gal, C-4^fuc, C-5^fuc, C-6^sia, C-8^sia), 79.3 (C-3^gal), 78.0 (C-3^fuc), 76.9 (C-2^fuc), 75.5, 74.2, 73.2 (CH₂–Ph), 75.0 (C-4^sia), 70.0 (C-7^sia), 69.8 (C-1^fuc), 69.6 (C-2^gal), 69.2 (C-4^gal), 64.5 (C-6^gal), 62.7 (C-9^sia), 54.0 (C-5^sia), 42.0 (C-3^sia), 22.6 (CH₃^NHAc), 16.5 ppm (CH₃^fuc); MS (ESI): m/z (%): 939.5 [M+H]⁺ (100); HRMS (ESI): m/z calcd for [C₄₆H₅₈N₄O₁₇+Na]⁺: 961.3695 [M+Na]⁺; found: 961.3692.

1-(3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galactonon-2-ulopyranosyl))-β-d-galactopyranosyl-4-(α-l-fucopyranosyl)-1 H-1,2,3-triazole (13)

Triazole 12 (100 mg, 110 μmol) was dissolved in MeOH (5 mL). The solution was degassed by ultrasonication under an argon atmosphere before Pd(OH)₂ on charcoal (45 mg, 20 wt %) was added. After a second degassing cycle, the argon atmosphere was replaced with hydrogen (balloon). After stirring for 3 days at RT, another portion of the catalyst (45 mg) was added, the hydrogen pressure was increased to 3.5 bar (Parr bomb), and the suspension was stirred for a further 5 days at RT. The catalyst was removed by filtration over Celite and thoroughly washed with MeOH. The solvent was removed in vacuo and the crude product was purified by preparative HPLC (ACE-C₁₈-PFP) to yield the title compound (24 mg, 36 μmol, 33 % yield) as a colorless oil. =−39.1 (c=0.5, D₂O); ¹H NMR (COSY, NOESY, ROESY, 600 MHz, D₂O): δ=8.36 (s, 1 H; H-5), 5.78 (d, ³J_1,2=8.9 Hz, 1 H; H-1^gal), 5.30 (d, ³J_1,2=6.6 Hz, 1 H; H-1^fuc), 4.32 (dd, ³J_2,3=9.7 Hz, ³J_3,4=3.0 Hz, 1 H; H-3^gal), 4.27 (pseudo-t, ³J_1,2=8.9 Hz, ³J_2,3=9.7 Hz, 1 H; H-2^gal), 4.19 (dd, ³J_1,2=6.6 Hz, ³J_2,3=10.4 Hz, 1 H; H-2^fuc), 4.08–4.05 (m, 2 H; H-4^gal, H-3^fuc), 3.98 (t, ³J_5,6=6.1 Hz, 1 H; H-5^gal), 3.89–3.80 (m, 5 H; H-4^fuc, H-5^fuc, H-5 ^sia, H-6^sia, H-9a^sia), 3.73 (d, ³J_5,6=6.1 Hz, 2 H; H-6^gal), 3.68–3.66 (m, 1 H; H-4^sia), 3.61–3.58 (m, 2 H, H-8^sia; H-9b^sia), 3.56 (dd, ³J=9.1 Hz, ³J=1.8 Hz, 1 H; H-7^sia), 2.73 (dd, ²J=12.4 Hz, ³J_3,4=4.8 Hz, 1 H; H-3a^sia), 1.99 (s, 3 H; CH₃^NHAc), 1.80 (pseudo-t, ²J=12.4 Hz, ³J_3,4=12.1 Hz, 1 H; H-3b^sia), 1.14 ppm (d, ³J_5,CH3=6.5 Hz, 3 H; CH₃^fuc); ¹³C NMR (HSQC, HMBC, 150.9 MHz, D₂O): δ=174.9 (C=O^NHAc), 173.8 (C=O^sia), 143.3 (C-4) 124.8 (C-5), 99.9 (C-2^sia), 87.6 (C-1^gal), 78.0 (C-5^gal), 75.7 (C-3^gal), 72.8 (C-8^sia), 71.7 (C-6^sia), 71.6 (C-4^fuc), 70.5 (C-1^fuc), 70.1 (C-3^fuc), 69.3 (C-5^fuc), 68.3 (C-4^sia), 68.0 (C-7^sia), 67.9 (C-2^gal), 67.6 (C-4^gal), 67.2 (C-2^fuc), 62.5 (C-9^sia), 60.8 (C-6^gal), 51.6 (C-5^sia), 39.4 (C-3^sia), 22.0 (CH₃^NHAc), 15.6 ppm (CH₃^fuc); MS (ESI): m/z (%): 691.3 [M+Na]⁺ (100), 669.3 [M+H]⁺ (89); HRMS (ESI): m/z calcd for [C₂₅H₄₀N₄O₁₇+Na]⁺: 691.2286 [M+Na]⁺; found: 691.2283; t_r=2.1 min (H₂O/MeCN, 98:2→98:2 (5 min)→80:20 (15 min); flow rate=38.00 mL min⁻¹).

1-(3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galactonon-2-ulopyranosyl))-β-d-galactopyranosyl-4-(2,3,4,6-tetra-O-benzyl-α-d-mannopyranosyl)-1 H-1,2,3-triazole (15)

The title compound was prepared according to a procedure reported by Dondoni and Marra.[28] Mannosylacetylene 14[29] (55.0 mg, 0.1 mmol) and azide 10 (50.0 mg, 0.10 mmol) were dissolved in dry DMF (2 mL). DIPEA (35 μL) and CuI (4.00 mg, 0.01 mmol, 10 mol %) were added in a countercurrent of argon gas and the mixture was stirred for 15 h at 70 °C. The solvent was removed in vacuo and the residue was co-evaporated three times with toluene (5 mL each). The crude product was purified by flash chromatography on silica gel (CH₂Cl₂/MeOH, 2:0.2+0.1 % AcOH→CH₂Cl₂/MeOH 1:1+0.1 % AcOH) to yield the title compound (82.0 mg, 78.0 μmol, 78 % yield) as a light-yellow oil. R_f=0.57 (CH₂Cl₂/MeOH 1:1+0.1 % AcOH); [α]=+2.5 (c=1.0, MeOH); ¹H NMR (COSY, 400 MHz, CD₃OD): δ=8.16 (s, 1 H; H-5), 7.39–7.37 (m, 2 H; Ph), 7.33–7.22 (m, 16 H; Ph), 7.18–7.15 (m, 2 H; Ph), 5.66 (d, ³J_1,2=9.2 Hz, 1 H; H-1^gal), 5.30 (d, ³J_1,2=3.3 Hz, 1 H; H-1^man), 4.75 (d, ²J=11.0 Hz, 1 H; CH₂–Ph), 4.70 (d, ²J=12.3 Hz, 1 H; CH₂–Ph), 4.69 (d, ²J=11.6 Hz, 1 H; CH₂–Ph), 4.61 (d, ²J=11.6 Hz, 1 H; CH₂–Ph), 4.59–4.52 (m, 2 H; CH₂–Ph), 4.51–4.49 (m, 2 H; CH₂–Ph), 4.43 (pseudo-t, ³J_1,2=³J_2,3=3.3 Hz, 1 H; H-2^man), 4.28 (dd, ³J_2,3=9.3 Hz, ³J_3,4=2.6 Hz, 1 H; H-3^gal), 4.22 (pseudo-t, ³J_1,2=³J_2,3=9.2 Hz, 1 H; H-2^gal), 4.09 (d, ³J_3,4=2.6 Hz, 1 H; H-4^gal), 3.99 (pseudo-t, ³J_3,4=³J_4,5=8.0 Hz, 1 H; H-4^man), 3.95 (dd, ³J_2,3=3.3 Hz, ³J_3,4=8.0 Hz, 1 H; H-3^man), 3.86–3.59 (m, 12 H; H-5^gal, H-6a/b^gal, H-5^man, H-6a/b^man, H-4^sia, H-5^sia, H-6^sia, H-8^sia, H-9a/b^sia), 3.52 (dd, ³J=8.9 Hz, ³J=1.5 Hz, 1 H; H-7^sia), 2.89 (dd, ²J=12.7 Hz, ³J_3,4=4.2 Hz, 1 H; H-3a^sia), 2.03 (s, 3 H; CH₃^NHAc), 1.82–1.76 ppm (m, 1 H; H-3b^sia); ¹³C NMR (HSQC, 100.6 MHz, CD₃OD): δ=175.5 (2 C; C=O^sia, C=O^NHAc), 146.2 (C-4), 139.8, 139.7, 139.6, 139.5 (4×Cq–Ph), 129.43 (2 C), 129.41 (4 C), 129.33 (2 C), 129.25 (2 C), 129.2 (2 C), 129.1 (2 C), 129.0 (2 C), 128.8 (2 C), 128.7, 128.6 (Ph), 123.6 (2 C; C-5, C-2^sia), 90.0 (C-1^gal), 79.9 (C-3^man), 77.9 (C-3^gal), 76.6 (C-2^man), 76.0 (C-4^man), 75.3, 74.3, 73.3, 73.1 (CH₂–Ph), 70.8 (C-1^man), 70.1 (C-6^man), 70.0 (C-7^sia), 69.6 (C-2^gal), 69.3 (C-4^gal), 79.8, 75.7, 74.9, 72.9, 69.2 (C-5^gal, C-5^man, C-4^sia, C-6^sia, C-8^sia), 64.4 (C-6^gal), 62.6 (C-9^sia), 54.0 (C-5^sia), 41.8 (C-3^sia), 22.6 ppm (CH₃^NHAc); MS (ESI): m/z (%): 1045.5 [M+H]⁺ (70); HRMS (ESI): m/z calcd for [C₅₃H₆₅N₄O₁₈+H]⁺: 1045.4294 [M+H]⁺; found: 1045.4319.

1-(3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galactonon-2-ulopyranosyl))-β-d-galactopyranosyl-4-(α-d-mannopyranosyl)-1 H-1,2,3-triazole (16)

Triazole 15 (155 mg, 148 μmol) was dissolved in MeOH (7 mL) and the solution was degassed by ultrasonication under an argon atmosphere. Then, Pd on charcoal (50 mg, 10 wt %) was added and, after a second degassing cycle, the argon atmosphere was replaced by hydrogen (balloon). After stirring for 1 day at RT, no conversion was observed by TLC. Pd(OH)₂ on charcoal (40 mg, 20 wt %) was added and the mixture was stirred for 4 days at RT under a hydrogen atmosphere; then, the catalyst was removed by filtration through Celite and thoroughly washed with MeOH. The solvent was removed in vacuo and the crude product was purified by preparative HPLC (ACE-C₁₈-PFP) to yield the title compound (63.0 mg, 92 μmol, 62 % yield) as a colorless oil. [α]=+5.4 (c=1.25, D₂O); ¹H NMR (COSY, 400 MHz, D₂O): δ=8.33 (s, 1 H; H-5), 5.80 (d, ³J_1,2=9.0 Hz, 1 H; H-1^gal), 5.24 (d, ³J_1,2=1.9 Hz, 1 H; H-1^man), 4.55–4.54 (m, 1 H; H-2^man), 4.33 (dd, ³J_2,3=9.6 Hz, ³J_3,4=2.9 Hz, 1 H; H-3^gal), 4.28 (pseudo-t, ³J_1,2=9.0 Hz, ³J_2,3=9.6 Hz, 1 H; H-2 ^gal), 4.09 (d, ³J_3,4=2.9 Hz, 1 H; H-4^gal), 4.00 (t, ³J_5,6=6.1 Hz, 1 H; H-5^gal), 3.88–3.81 (m, 5 H; H-6a/b^gal, H-3^man, H-5^sia, H9a^sia), 3.78–3.73 (m, 4 H; H-4^man, H-6a/b^man, H-8^sia), 3.70–3.66 (m, 1 H; H-4^sia), 3.63–3.57 (m, 3 H; H-5^man, H-6^sia, H-9b^sia), 3.44–3.41 (m, 1 H; H-7^sia), 2.75 (dd, ²J=12.5 Hz, ³J_3,4=4.7 Hz, 1 H; H-3a^sia), 2.01 (s, 3 H; CH₃^NHAc), 1.83–1.79 ppm (pseudo-t, ²J=12.5 Hz, ³J_3,4=12.1 Hz, 1 H; H-3b^sia); ¹³C NMR (HSQC, 100.6 MHz, D₂O): δ=175.0 (C=O^NHAc), 173.8 (C=O^sia), 144.1 (C-4), 123.6 (C-5), 100.0 (C-2^sia), 87.7 (C-1^gal), 78.0 (C-5^gal), 75.7 (C-3^gal), 75.4 (C-7^sia), 72.8 (2 C; C-1^man, C-5^man), 71.7 (C-3^man), 70.7 (C-4^man), 69.9 (C-2^man), 68.3 (C-4^sia), 68.0 (C-6^sia), 67.9 (C-2^gal), 67.6 (C-4^gal), 67.1 (C-8^sia), 62.5 (C-9^sia), 60.84 (C-6^man), 60.76 (C-6^gal), 51.6 (C-5^sia), 39.4 (C-3^sia), 22.0 ppm (CH₃^NHAc); MS (ESI): m/z (%): 707.3 [M+Na]⁺ (100), 685.2 [M+H]⁺ (51); HRMS (ESI): m/z calcd for [C₂₅H₄₀N₄O₁₈+Na]⁺: 707.2235 [M+Na]⁺; found: 707.2232; t_r=2.0 min (H₂O/MeCN, 98:2→98:2 (5 min)→80:20 (15 min); flow rate=38.00 mL min⁻¹).

1-(β-d-Galactopyranosyl)-3-(α-d-mannopyranosyl)-indole (20)

To a solution of the peracetylated diglycosylindole 1911a, [19] (26 mg, 33 μmol) in MeOH (5 mL) was added NaOMe until the pH value reached 8.5–9.0. The mixture was stirred at RT for 18 h, neutralized with AcOH, and the solvent was removed in vacuo. The residue was purified by flash chromatography on silica gel (EtOAc/MeOH, 20:1→3:1→2:1) to yield the title compound (14 mg, 32 μmol, 95 % yield) as a colorless oil. R_f=0.45 (1-butanol/H₂O/AcOH, 5:2:2); [α]=+61.0 (c=1.00, MeOH); ¹H NMR (COSY, HSQC, HMBC, 300 MHz, CD₃OD): δ=7.84 (dd, ⁴J_4,6=1.1 Hz, ³J_4,5=7.9 Hz, 1 H; H-4^Indol), 7.58 (dt, ⁴J_7,5=0.8 Hz, ³J_7,6=8.4, 1 H; H-7^Indol), 7.48 (d, ⁴J_2,1−Man=1.3 Hz, 1 H; H-2^Indol), 7.18 (ddd, ⁴J_6,4=1.3 Hz, ³J_6,5=7.1 Hz, ³J_6,7=8.4 Hz, 1 H; H-6^Indol), 7.08 (ddd, ⁴J_5,7=0.8 Hz, ³J_5,6=7.1 Hz, ³J_5,4=7.9 Hz, 1 H; H-5^Indol), 5.41 (d, ³J_1,2=9.0 Hz, 1 H; H-1^Gal), 5.34 (pseudo-t, J_{app, 1,2-Indol}=1.3 Hz, 1 H; H-1^Man), 4.51 (dd, ³J_2,3=3.3 Hz, ³J_2,1=2.0 Hz, 1 H; H-2^Man), 4.22 (pseudo-t, J_app=9.3 Hz, 1 H; H-2^Gal), 4.03 (dd, J=3.2, 0.9 Hz, 1 H; H-4^Gal), 3.98 (dd, ³J_3,2=3.3 Hz, ³J_3,4=9.1 Hz, 1 H; H-3^Man), 3.69–3.86 (m, 7 H; H-3^Gal, H-5^Gal, H-6^Gal, H-4^Man, H-6^Man), 3.26 ppm (ddd, ³J_5,6=2.9 Hz, ³J_5,6=4.0 Hz, ³J_5,4=9.4 Hz, 1 H; H-5 ^Man); ¹³C NMR (DEPT, HSQC, HMBC, 75 MHz, CD₃OD): δ=138.4 (C-7a^Indol), 129.1 (C-3a^Indol), 125.0 (C-2^Indol), 123.3 (C-6^Indol), 121.5 (C-4^Indol), 121.2 (C-5^Indol), 113.4 (C-3^Indol), 111.6 (C-7^Indol), 87.2 (C-1^Gal), 79.2 (C-5^Gal), 76.3 (C-1^Man), 75.9, 75.8 (C-3^Gal, C-5^Man), 73.5 (C-3^Man), 72.1 (C-2^Man), 71.1 (C-2^Gal), 70.5 (C-4^Gal), 69.3 (C-4^Man), 62.8, 62.6 ppm (C-6^Gal, C-6^Man); MS (ESI): m/z (%): 464.0 [M+Na]⁺ (100); HRMS (ESI): m/z calcd for [C₂₀H₂₇NO₁₀+Na]⁺: 464.1533; found: 464.1543; m/z calcd for [M+Na+H]⁺: 464.1611; found: 464.1615; IR (film): =3271, 2929, 1705, 1558, 1461, 1407, 1272, 1221, 1064, 1016, 918, 880, 794, 743, 653 cm⁻¹.

1-[3-O-(5-Acetamido-3,5-didesoxy-d-glycero-α-d-galacto-non-2-ulopyranosyl)-β-d-galactopyranosyl)]-3-(β-d-mannopyranosyl)-indole (21)

4-Nitrophenyl-α-sialoside 9 (25 mg, 56 μmol) and diglycosylindol 20 (50 mg, 113 μmol) were dissolved in degassed Tris-HCl buffer (2 mL, 100 mm, pH 7.5). After the addition of an aqueous solution of recombinant trans-sialidase (400 μL, c=0.5 mg mL⁻¹), the mixture was incubated at RT for 24 h. Another portion (25 mg) of compound 9 was added and the mixture was stirred for a further 48 h. The reaction was stopped by the addition of EtOH (1.5 mL). The solution was centrifuged for 30 min and the supernatant was removed and lyophilized. The crude product was purified by preparative HPLC (Luna-C18; t_r=20–25 min; H₂O/MeCN, 95:5 (60 min)→80:20; flow rate=50 mL min⁻¹) to furnish the title compound (11 mg, 15 μmol, 13 % yield) as a yellowish solid. The HPLC separation also yielded unreacted acceptor 20 (22 mg, 50 μmol), thus indicating that the yield of the enzymatic reaction was 24 % (brsm). [α]=+81.6 (c=1.00, D₂O); ¹H NMR (COSY, TOCSY, 600 MHz, D₂O): δ=7.83 (d, ³J_4,5=8.0 Hz, 1 H; H-4^Indol), 7.58 (d, ³J_7,6=8.4 Hz, 1 H; H-7^Indol), 7.51 (s, 1 H; H-2^Indol), 7.29 (pseudo-t, ³J_6,5/7=7.7 Hz, 1 H; H-6^Indol), 7.18 (pseudo-t, ³J_5,4/6=7.6 Hz, 1 H; H-5^Indol), 5.62 (d, ³J_1,2=8.4 Hz, 1 H; H-1^Gal), 5.34 (s, 1 H; H-1^Man), 4.55 (dd, ³J_2,1=2.3 Hz, ³J_2,3=3.4 Hz, 1 H; H-2^Man), 4.27–4.32 (m, 2 H; H-2^Gal, H-3^Gal), 4.08 (d, ³J_4,3=2.6 Hz, 1 H; H-4^Gal), 4.02 (dd, ³J_3,2=3.4 Hz, ³J_3,4=9.2 Hz, 1 H; H-3^Man), 3.94–3.97 (m, 1 H; H-5^Gal), 3.84 (ddd, ³J_8,9a=2.5 Hz, ³J_8,9b=6.4 Hz, ³J_8,7=9.1 Hz, 1 H; H-8^Sia), 3.78–3.81 (m, 2 H; H-5^Sia, H-9a^Sia), 3.63–3.72 (m, 6 H; H-4^Man, H-6^Man, H-4^Sia, H-6^Gal), 3.61 (dd, ³J_6,7=1.6 Hz, ³J_6,5=10.5 Hz, 1 H; H-6^Sia), 3.56 (dd, ³J_9b,8=6.4 Hz, ³J_9b,9a=12.0 Hz, 1 H; H-9b^Sia), 3.52 (dd, ³J_7,6=1.6 Hz, ³J_7,8=9.1 Hz, 1 H; H-7^Sia), 3.23 (ddd, ³J_5,6=2.8 Hz, ³J_5,6=5.6 Hz, ³J_5,4=9.4 Hz, 1 H; H-5^Man), 2.72 (dd, ³J_3äqu,2=4.6 Hz, ²J_3äqu,3ax=12.3 Hz, 1 H; H-3_äqu^Sia), 1.96 (s, 3 H; COCH₃), 1.80 ppm (t, ²J_3ax,3äqu=12.3 Hz, 1 H; H-3_ax^Sia); ¹³C NMR (HSQC, HMBC, 151 MHz, D₂O): δ=174.9 (C=O), 136.6 (C-7a^Indol), 127.0 (C-3a^Indol), 123.7 (C-2^Indol), 123.1 (C-6^Indol), 120.8 (C-5^Indol), 120.5 (C-4^Indol), 112.1 (C-3^Indol), 110.2 (C-7^Indol), 99.8 (C-1^Gal), 77.0 (C-5^Gal), 76.4 (C-3^Gal), 74.4 (C-1^Man), 74.3 (C-5^Man), 72.8 (C-6^Sia), 71.5 (C-8^Sia), 71.3 (C-3^Man), 70.0 (C-2^Man), 68.1 (C-7^Sia), 68.0, 67.7, 67.6 (C-4^Gal, C-4^Man, C-4^Sia), 67.4 (C-2^Gal), 62.5 (C-9^Sia), 60.8, 60.6 (C-6^Gal, C-6^Man), 51.5 (C-5^Sia), 39.3 (C-3^Sia), 21.9 ppm (COCH₃); MS (ESI): m/z (%): 755.3 [M+Na]⁺ (100); HRMS (ESI): m/z calcd for [C₃₁H₄₄N₂O₁₈+Na]⁺: 755.2487; found: 755.2514.

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