Reprogramming the specificity of sortase enzymes

Staphylococcusaureus sortase A catalyzes the transpeptidation of an LPXTG peptide acceptor and a glycine-linked peptide donor and has proven to be a powerful tool for site-specific protein modification. The substrate specificity of sortase A is stringent, limiting its broader utility. Here we report the laboratory evolution of two orthogonal sortase A variants that recognize each of two altered substrates, LAXTG and LPXSG, with high activity and specificity. Following nine rounds of yeast display screening integrated with negative selection, the evolved sortases exhibit specificity changes of up to 51,000-fold, relative to the starting sortase without substantial loss of catalytic activity, and with up to 24-fold specificity for their target substrates, relative to their next most active peptide substrate. The specificities of these altered sortases are sufficiently orthogonal to enable the simultaneous conjugation of multiple peptide substrates to their respective targets in a single solution. We demonstrated the utility of these evolved sortases by using them to effect the site-specific modification of endogenous fetuin A in human plasma, the synthesis of tandem fluorophore –protein–PEG conjugates for two therapeutically relevant fibroblast growth factor proteins (FGF1 and FGF2), and the orthogonal conjugation of fluorescent peptides onto surfaces.Chemistry and Chemical Biolog

Design, Synthesis, and Evaluation of Irciniastatin Analogues: Simplification of the Tetrahydropyran Core and the C(11) Substituents

The design, synthesis, and biological evaluation of irciniastatin A (<b>1</b>) analogues, achieved by removal of three synthetically challenging structural units, as well as by functional group manipulation of the C(11) substituent of both irciniastatins A and B (<b>1</b> and <b>2</b>), has been achieved. To this end, we first designed a convergent synthetic route toward the diminutive analogue (+)-<i>C</i>(8)-desmethoxy-<i>C</i>(11)-deoxy-<i>C</i>(12)-didesmethylirciniastatin (<b>6</b>). Key transformations include an acid-catalyzed 6-<i>exo</i>-tet pyran cyclization, a chiral Lewis acid mediated aldol reaction, and a facile amide union. The absolute configuration of <b>6</b> was confirmed via spectroscopic analysis (CD spectrum, HSQC, COSY, and ROESY NMR experiments). Structure–activity relationship (SAR) studies of <b>6</b> demonstrate that the absence of the three native structural units permits access to analogues possessing cytotoxic activity in the nanomolar range. Second, manipulation of the C(11) position, employing late-stage synthetic intermediates from our irciniastatin syntheses, provides an additional five analogues (<b>7</b>–<b>11</b>). Biological evaluation of these analogues indicates a high functional group tolerance at position C(11)