“Double‐Click” Protocol for Synthesis of Heterobifunctional Multivalent Ligands: Toward a Focused Library of Specific Norovirus Inhibitors

Abstract

Click, click, hooray! A convenient “double-click” protocol for the conjugation of two different ligands to multivalent scaffolds was developed. The protocol involves activation of the second attachment site as an azide for the second click reaction (see scheme). This method was used to synthesize potent inhibitors for norovirus-like particles. The binding of noroviruses to histo-blood group antigens is an essential part of the viral life cycle and the inhibition of attachment to host cells may provide an opportunity to counter infection. Norovirus belongs to the family of Caliciviridae that comprises animal and human pathogens. It causes a broad spectrum of acute or persistent infections, including respiratory diseases, vesicular lesions, necrotizing hepatitis, and diarrhea.1, 2 The virus is responsible for epidemic outbreaks of non-bacterial gastroenteritis most commonly referred to as “gastric flu”.3, 4 The virus has an icosahedral shell constructed from a 60 kDa viral capsid protein, which can self-assemble in the absence of RNA into virus-like particles (VLP) that have similar morphology and binding specificity to the cell-surface antigens as the whole virus.5–7 Hemagglutination assays, enzyme-linked immuno sorbent assay (ELISA)-based binding assays with saliva and synthetic oligosaccharides as well as crystallography revealed that L-fucose is the smallest fragment of the blood group ABH and Lewis antigens that binds to the viral coat.8–10 More recently, saturation transfer difference (STD) NMR spectroscopy has been employed to investigate the fucose binding site and adjacent sites. By using the commercial Maybridge compound library, twenty-six ligands were found to compete with L-fucose binding and four aromatic ligands were identified that bound to VLPs at sites close to the primary fucose binding site (Scheme 1).11 Previously, we have demonstrated that one-million-fold increases in binding avidity can be achieved for bacterial toxin ligands when association is driven by a hetero-bifunctional ligand in which the second head group is specific for a multivalent templating protein.12 An extension of this concept envisions targeting the second head group of a hetero-bifunctional ligand to a second, adjacent site on the same protein. Here we report the synthesis of ligands designed to achieve this objective with norovirus-like particles. Structures of the non-carbohydrate ligands. A-OH competes with L-fucose binding whereas compounds B-OH to E-NH2 bind to an adjacent binding site as described in the accompanying paper. The compounds A-OH to E-NH2 correspond to compounds 160, 151, 231, 191, and 473 in the accompanying paper.11 In this study, we synthesized hetero-bifunctional ligands either presented on a polymeric scaffold (to study the effect of multivalency on binding avidity) or as univalent monomers for STD NMR studies. All derivatives contain an invariant moiety, the α-L-fucose, and differ in the second variable ligand, a non-carbohydrate molecule selected by STD NMR screening of the Maybridge compound library (Scheme 1). The detailed account of this screening, which directed the design of the hetero-bifunctional ligands, together with NMR studies of the univalent monomers is reported in the accompanying manuscript.11 Compound 1 was obtained by Fisher glycosidation of L-fucose with propargyl alcohol, by using sulfuric acid immobilized on silica as a catalyst.13 This method provides the desired glycoside in a 4:1 (α/β) ratio. After peracetylation, the α anomer was easily purified from an α/β mixture of triacetates. Trans-esterification afforded compound 2 in 50 % overall yield (Scheme 2). Synthesis of compounds 1–7. Reaction conditions: a) i) propargyl alcohol, H2SO4–silica; ii) Ac2O/pyridine; b) MeONa; c) i) 4-nitrophenyl chloroformate, pyridine, CH2Cl2, 30 min; ii) propargylamine, N,N-diisopropylethylamine (DIPEA), overnight; d) propargyl chloroformate, NaHCO3, CH2Cl2, 30 min. The carbamates 3–5 of the aromatic ligands were prepared from the corresponding alcohols by in situ activation with 4-nitrophenyl chloroformate, and then treated with propargylamine. This one-pot protocol was developed because, in the case of ligand A, the intermediate (activated alcohol) was highly unstable and could not be isolated. The carbamates 6 and 7 were obtained from the corresponding aniline derivatives D-NH2 and E-NH2 as well as propargyl chloroformate (Scheme 2). Polymer 8 (≈80 kDa, 3 % payload of NH2 groups) synthesized by trans-amidation of polyacrylamide, was modified by conjugation with the activated trifunctional linker 9 to obtain the polymer 10 with two differently protected amine functionalities. The product was first coupled to the propargyl fucopyranoside 2 by a CuI-mediated cycloaddition reaction, by using the method developed by Finn and co-workers for a multivalent scaffold.14 Treatment of 11 with trifluoroacetic acid removed the tert-butoxycarbonyl (Boc) group but did not affect the fucopyranoside moiety as evident from the presence of H-6 signals in the NMR spectrum of 12 (see the Supporting Information). Conversion of the liberated amine in 12 to the azide by using a diazotransfer reagent, imidazole-1-sulfonyl azide,15 resulted in polymer 13, which was coupled with the non-carbohydrate ligands 3–7 to provide compounds 14–18 by using the click procedure again (Scheme 3). Synthesis of polymeric hetero-bifunctional ligands. Reaction conditions: a) NEt3, H2O; b) compound 2, CuI, pH 8; c) trifluoroacetic acid (TFA); d) imidazole-1-sulfonyl azide, CuII; e) compounds 3–7, CuI, pH 8. After these aromatic ligands were linked, the polymers were rendered much less soluble in water, but remained sufficiently soluble for testing their inhibitory activities against VLPs by using surface plasmon resonance (SPR) (Table 1). The details of the SPR assay are given in the accompanying paper11 and in the Supporting Information. Despite significant precipitation, for several compounds inhibitory activities could be obtained by competitive titration by using STD NMR spectroscopy16 (Table 1, Table 2S in the Supporting Information). During titration of the polymer the decrease of the STD signal for methyl α-L-fucopyranoside in the presence of VLPs was observed. These data correlate well with the values obtained in the SPR assay. Activities of all polymers containing the second binding moiety, which was identified by STD NMR screening, are higher than that of progenitor 13. Thus, the STD NMR-facilitated fragment-based design appears a valid approach to discovery of novel inhibitors for noroviruses. Compound IC50(SPR)[a] [μM] IC50(STD)[a] [μM] 13 80±30 14 0.61±0.03 15 40±4.9 220±80 16 40±10 17 20±6.5 20±4 18 5.8±1.6 8±3 To further investigate the binding by STD NMR spectroscopy, the heterodimer 24 was synthesized as a unimeric analogue of the pendant ligand of the polymer 14 (Scheme 4). Because the initial attempt to sequentially click the fucoside and the aromatic ligand to 1,3-diazidopropan-2-ol failed, resulting only in the corresponding di-fucoside and the starting material, we resolved to follow the “double-click” protocol as already established for multivalent analogues. Thus, 1,3-diazidopropan-2-ol was first converted to the Boc-protected azidoamine 19.17 Then, it was coupled to the propargyl fucoside 1 following the regular click protocol, by using CuSO4 and sodium ascorbate. The Boc protection group was removed to obtain 20 and the azido derivative 21 was formed by the same method as described for compound 13. Finally, a second cycloaddition reaction with compound 3 and deprotection gave the desired bifunctional ligand 24. Synthesis of the monomeric hetero-bifunctional ligand 24. Reaction conditions: a) i) PPh3, 5 % HCl, Et2O; ii) Boc2O; b) compound 1, CuSO4, sodium ascorbate, H2O/THF; c) TFA; d) imidazole-1-sulfonyl azide, CuII; e) compound 3, CuSO4, sodium ascorbate, H2O/THF; f) MeONa, MeOH. Unexpectedly, in the course of NMR experiments, we observed that compound 24 slowly decomposed in D2O at neutral pH (see the Supporting Information). However, when stored as solid or in solution in methanol or dichloromethane, no decomposition of 24 was observed. By using mass spectrometry and NMR spectroscopy we found that the carbamate linkage in 24 was unstable, with a half-life of about eleven days. Hydrolysis of compound 24 with a loss of carbon dioxide gave the initial alcohol A-OH and the corresponding amine 25 (Scheme 5). Decomposition of compound 24. Conjugation of ligands to proteins, polymers, dendrimers, and other scaffolds through a triazole unit is becoming increasingly common and often propargyl carbamates of the ligands are used as a convenient way to activate hydroxyl functionalities for click reaction.18, 19 The discovered instability of compound 24 presents a concern that the shelf life of such conjugates can be affected. To further investigate this phenomenon, we hypothesized that the basicity of the adjacent triazole ring is responsible for intramolecular acceleration of carbamate hydrolysis. As a model, compound 28 was synthesized from 2-azidoethyl lactoside 26 and alkyne 2720 by using a cycloaddition reaction (Scheme 6). To study the stability of this compound, it was incubated at 37 °C in D2O/phosphate buffer at pH 6, 7, or 8 and the decomposition behavior was periodically monitored by NMR spectroscopy. After three months incubation, compound 28 remained intact, thus, demonstrating that the instability of compound 24 is not general and cannot be attributed to the linker comprising triazole and carbamate, but is rather a unique property of the aromatic moiety in this ligand. Despite its shorter life in aqueous solutions, compound 24 was sufficiently stable to perform STD NMR experiments with VLPs. The results are published in the accompanying paper.11 Synthesis of compound 28 for the decomposition study. Reaction conditions: CuSO4, sodium ascorbate, H2O/THF. In conclusion, a convenient “double-click” protocol for conjugation of two different ligands to multivalent scaffolds was developed that involves activation of the second attachment site as an azide for a subsequent click reaction. This protocol supplements the previously reported sequential click reaction strategy, which relies on masking one of the two alkyne moieties in a bifunctional scaffold.21 Financial support for J.G., P.I.K., and D.R.B. was provided by the Alberta Innovates Centre for Carbohydrate Science and the Natural Science and Engineering Research Council of Canada. T.P. thanks the Deutsche Forschungsgemeinschaft (DFG) for grant Pe494/8-1 and for grants HBFG 101/192-1 and ME 1830/1. B.F. thanks the Studienstiftung des deutschen Volkes for a stipend. Detailed facts of importance to specialist readers are published as ”Supporting Information”. Such documents are peer-reviewed, but not copy-edited or typeset. They are made available as submitted by the authors. 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