Enzamide analogues as prospective high-affinity CD33 ligands applying iterative rounds of focused library synthesis coupled with glycan array screening to simultaneously address affinity and selectivity for this siglec. It was reasoned that an optimal C9 substituent combined with the 4-cyclohexyl-1,two,3-triazole in the C5 position could work synergistically to attain higher affinity and selectivity for hCD33. As a 1st step towards this goal, an initial series of 9-benzamide substituents have been synthesized and analysed by glycan array (Fig. 1, compounds 3-6). It was noted that replacing the biphenyl substituent PARP1 Activator list having a single benzamido group (3) absolutely abolished MAO-A Inhibitor Source binding to hCD33 (Fig. 1). Interestingly, on the other hand, addition of an acetylene moiety to the meta- (five) but not para- (six) position of the benzamide ring re-established this affinity acquire and enhanced selectivity. Notably, click chemistry-derived solutions of (five) having a variety of azides totally abolished binding to hCD33 and recommended a potential steric clash of huge moieties at this position (data not shown). Hence, we initially sought to explore if other substituents in the meta position of your benzamide ring, specifically small ones, could yield further improvements over 5. Accordingly, a tiny library of C9-analogues with meta-substituted benzamide rings had been generated within the 2-6 linked scaffold (Fig. 1, compounds 7-12). This was achieved by way of a very simple synthetic technique involving enzymatic transfer of a 9-amino sialic acid to an azide or Cbz-protected lactosyl–O-ethylamine scaffold (Scheme 1, A and B), followed by N-acylation in the C9 position of sialic acid, and deprotection on the linker to the absolutely free amine needed for microcontact printing (Scheme 1).42 On a five?0 mg scale, this procedure reproducibly provided compounds in great yield and purity. Using this method, analogues with both small (7-11) and large (12) substituents at the meta position from the benzamide ring have been designed. Upon glycan array evaluation, compound 7, having a 3methylbenzamido substituent, yielded the most promising enhance in affinity and selectivity over 5 (Fig. 1b-c and Fig. S1, ESI). It ought to be noted that we routinely confirm that allChem Sci. Author manuscript; available in PMC 2015 June 01.Rillahan et al.Pagecompounds are equally printed employing the 2-6-linkage specific plant lectin SNA, that is not affected by the presence of 9-substituents (Fig. S2, ESI).33, 43,NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptWith a objective to improve upon compound 7, yet another library containing C9-appended, 3methylbenzamide substituents, was developed with additional perturbations towards the benzamide ring (Fig. 1, Compounds 13-16). From this library, 13, containing a three,5-dimethylbenzamide substituent, gave a further improvement in affinity and selectivity for hCD33 (Fig. 1b and Fig. S1, ESI), whilst the two,3-dimethyl isomer 14 abolished binding. Because the methyl group from the 3-methylbenzamide is vital for binding to hCD33 (examine 3 and 7), the additional increase in avidity for the three,5-dimethylsubstituent may very well be an entropic effect due to the symmetry from the resulting ring. It was notable that all substitutions at the 2 and 5-position in the benzamide ring abrogated binding to hCD33 (14 and 15), when modifications at the 4-positon have been from time to time tolerated (four and 16). To extend these observations, we constructed a panel of C9-substituted three,5-dimethylbenzamide analogues with varying alterat.