Cyclic peptide formation catalyzed by an antibody ligase

Abstract

Cyclic hexapeptides represent a class of compounds with important, diverse biological activities. We report herein that the antibody 16G3 catalyzes the cyclization of D-Trp-Gly-Pal-Pro-Gly-Phe⅐p-nitrophenyl ester (8a) to give c-(D-Trp-Gly-Pal-Pro-Gly-L-Phe) (11a). The antibody does not, however, catalyze either epimerization or hydrolysis. The resulting rate enhancement of the cyclization by 16G3 (22-fold) was sufficient to form the desired product in greater than 90% yield. In absolute rate terms, the turnover of 16G3 is estimated to be 2 min ؊1 . The background rate of epimerization of 8a was reduced from 10 to 1% and hydrolysis from 50 to 4% in the presence of 16G3. As expected, the catalytic effects of 16G3 were blocked by the addition of an amount of the hapten equal to twice the antibody concentration. We also synthesized three diaste- S everal years ago, we undertook the generation of antibodies to catalyze peptide bond formation in part because manmade catalysts of peptide bond formation had not been described (1). We were also intrigued by the contrast between medicinal chemistry and antibody design. Generally, the former seeks by design or screening to discover small molecules that interact with macromolecules such as enzymes or receptors leading to enzyme inhibitors or to hormone͞neurotransmitter agonists͞antagonists. In contrast, catalytic antibody research involves the synthesis of haptens, designed to generate novel macromolecules (antibodies), which in turn are capable of catalyzing predetermined chemical reactions. The Design of Hapten 1a: Novel Chemistry and the Generation ofAntibody 16G3 for Bimolecular Peptide Bond Formation. We reported the synthesis of hapten 1a (Fig. 1), designed to induce the formation of antibodies capable of catalyzing the formation of dipeptides of the general structure acetyl-XXX-D-Trp⅐NH 2, wherein XXX represents hydrophobic L-amino acids typified by L-Phe (1). This endeavor generated antibody 16G3, which gave rate enhancements on the order of 2 ϫ 10 4 over the background reaction with pleasingly high turnover rates (Ϸ2 min Ϫ1 ). Our study of the kinetics implicated a sequential mechanism with no preferred order of substrate binding and no evidence for acylation of the antibody by the ester before peptide bond formation. Antibody 16G3 did not, however, catalyze either the hydrolysis or racemization of the active ester. The generation of antibodies that catalyze nonsolvolytic bimolecular bond formation is a greater challenge than the production of antibodies designed to catalyze solvolysis because an important requirement for the former is to prevent hydrolysis. The acylating agents employed in these coupling reactions were the p-nitrophenyl esters of acetyl-XXX. Although the hapten had been designed to generate antibodies that can catalyze the formation only of dipeptides, we subsequently found that antibody 16G3 also catalyzes the coupling of the p-nitrophenyl ester of N-acetyl-LPhe with D-Trp-Gly⅐NH 2 and the p-nitrophenyl ester of N-acetylGly-L-Phe with D-Trp-Gly⅐N...

A Catalytic Antibody Produces Fluorescent Tracers of Gap Junction Communication in Living Cells

“…Fluorescein (42 mg, 0.125 mmol) and carbamoyl chloride 6 (73 mg, 0.25 mmol in 500 ml of dry THF) in 1 ml of dry pyridine was treated with a catalytic amount of dimethylaminopyridine and stirred for 5 days at room temperature. After work up with ethyl acetate and saturated aqueous NH 4 Cl, the mixture was purified by column chromatography (hexanes/ethyl acetate 1:2) to give carbamate 7 (13 mg, 0.0154 mmol, 12%). High resolution mass spectroscopy calculated for C 44 H 52 N 4 O 13 -Cs ϩ : 977.2585, observed 977.2602.…”

“…Catalytic antibodies have proven utility for chemical catalysis in vitro (1)(2)(3)(4)(5)(6)(7). In living cells, these protein catalysts could accomplish a variety of reactions to modify cell behavior or generate reporter molecules for biological assays.…”

The cyclization of peptides and depsipeptides

“…Shorter peptides do not cyclize, but for the larger peptides yields of 30%-85% proved possible. The antibody ligase 16G3 has been shown to catalyse the cyclization of D-Trp-Gly-Pal-Pro-Gly-Phe-p-nitrophenyl ester [66] to give cyclo (D-Trp-Gly-Pal-Pro-Gly-Phe) in >90% yield with a rate enhancement of 22-fold.…”

Generation, characterization, and docking studies of DNA‐hydrolyzing recombinant Fab antibodies

“…Investigations of catalytic mechanisms employed by autoantibodies should provide a clearer understanding of their biological significance. One mechanism proposed to be employed by catAbs is antibody‐induced conformational distortion of the substrate resulting in activation of the ground state (Smithrud et al ., 2000).…”