9-Azido-9-deoxy-2,3-difluorosialic Acid as a Subnanomolar Inhibitor against Bacterial Sialidases

Abstract

A library of 2(e),3(a/e)-difluorosialic acids and their C-5 and/or C-9 derivatives were chemoenzymatically synthesized. Pasteurella multocida sialic acid aldolase (PmAldolase), but not its Escherichia coli homolog (EcAldolase), was found to catalyze the formation of C5-azido analog of 3-fluoro(equatorial)-sialic acid. In comparison, both PmAldolase and EcAldolase could catalyze the synthesis of 3-fluoro (axial or equatorial)-sialic acids and their C-9 analogs although PmAldolase was generally more efficient. The chemoenzymatically synthesized 3-fluoro (axial or equatorial)-sialic acid analogs were purified and chemically derivatized to form desired difluorosialic acids and derivatives. Inhibition studies against several bacterial sialidases and a recombinant human cytosolic sialidase hNEU2 indicated that sialidase inhibition was affected by the C-3 fluorine stereochemistry and derivatization at C-5 and/or C-9 of the inhibitor. Opposite to that observed for influenza A virus sialidases and hNEU2, compounds with an axial fluorine at C-3 were better inhibitors (up to 100-fold) against bacterial sialidases compared to their 3Fequatorial counterparts. While C-5-modified compounds were less efficient anti-bacterial sialidase inhibitors, 9-N 3 -modified 2,3-difluoro-Neu5Ac showed increased inhibitory activity against bacterial sialidases. 9-Azido-9-deoxy-2-equatorial-3-axial-difluoro-N-acetylneuraminic acid (2e3aDFNeu5Ac9N 3 ) was identified as an effective inhibitor with a long effective duration selectively against pathogenic bacterial sialidases from Clostridium perfringens (CpNanI) and V. cholerae.

Polyvalent transition-state analogues of sialyl substrates strongly inhibit bacterial sialidases.

“…The synthesis of mono-azido-EG 3 20, di-azido-EG 11, 27 21, 22 and peracetylated azido-EG 3 -CD 23 (Scheme 3) was described in previous articles. 30,32 The two polyvalent azides 17 and 19 were obtained as depicted in Scheme 2.…”

“…These results also suggested that CAT domains of SA are sensitive to multivalent inhibitory effects as previously shown with multivalent iminosugars and specific glycosidases. [22][23][24][25] In this work, we select the DANA ligand to develop the first class of multi-and polyvalent sialyl cation transition-state inhibitors of bacterial SA (Figure 1B). The DANA-clusters inhibition potentials were assessed towards SA from Bacterioides thetaiotaomicron (BtSA), and Streptococcus pneumoniae (NanA) both belonging to the GH33 family These enzymes were produced both in their full length (CAT+CBM) and as CAT domains alone (for NanA).…”

Sialidase and Sialyltransferase Inhibitors: Targeting Pathogenicity and Disease

“…This included an azido group modification at the C9 or C5 position to increase selectivity for bacterial over human SAs (10) ( Khedri et al, 2012 ). These compounds displayed weak inhibition, but were improved in more recent work combining the aforementioned C2, C3-difluoro additions to produce a covalent inhibitor (11) ( Li et al, 2019 ). Conjugation of a small peptide (1-4 residues) to the C9 position of Neu5Ac2en via a triazole linkage has been attempted and showed high selectivity for V. cholerae SA, with low micromolar IC 50 values (12) ( Slack et al, 2018 ).…”

Polyvalent Transition‐State Analogues of Sialyl Substrates Strongly Inhibit Bacterial Sialidases**

“…There has been less success with bacterial, fungal or parasitic SAs, and there are few reports of inhibitory activities for bacterial SA in the nanomolar range. [2,3] The most promising inhibitors of bacterial and human SA (NEU1-4) [4] are based on 2-deoxy-2,3didehydro-N-acetylneuraminic (DANA), a transition-state analogue of the sialyl cation after SA cleavage ( Figure 1A). DANA modifications with adequate pharmacophores at the C-5 or C-9 positions were shown to improve affinity and selectivity for specific hNEU isoenzymes.…”

“…Inhibitors of bacterial, fungal, or parasitic SAs have been less successful, with few reports of inhibitory activities in the nanomolar range [2, 3] . The most promising inhibitors of bacterial and human SA (NEU1‐4) [4] are based on 2‐deoxy‐2,3‐didehydro‐ N ‐acetylneuraminic (DANA), a transition‐state analogue of the sialyl cation after SA cleavage (Figure 1 A).…”