Antibody catalysis of peptide bond formation.

Abstract

An antibody generated against a neutral phosphonate diester transition-state (TS*) analog catalyzes the formation of an amide bond between a phenylalanyl amino group and an acyl azide derived from L-alanine. The antibody is selective for L-vs. D-alanine and does not catalyze the hydrolysis of the acyl azide to an appreciable degree. A rate acceleration of 10,000-fold relative to the uncatalyzed reaction is observed. The antibody may achieve its catalytic efficiency both by acting as an entropy trap and by stabilizing the deprotonated form of the amine nucleophile. These experiments constitute a first step toward a general strategy for the generation of sequence-specific peptide ligases. We wished to investigate the requirements for catalysis of peptide bond formation using a catalytic antibody. Previously reported studies in this area focused on condensation reactions involving activated esters and-aryl amines (7,8). We have applied a design strategy that was found to be highly successful for bimolecular ester formation (9). Antibodies were generated against the neutral phosphonate diester 1 that contains elements ofthe acyl donor 5, acceptor 7, and leaving group in a tetrahedral geometry mimicking that of the presumed transition state (Fig. la). One antibody was isolated that accelerated the formation of peptide 8 from the corresponding substrates and was characterized with respect to its substrate specificity and reaction kinetics. MATERIALS Diphenyl 1-(N-carbophenethyloxyamno)ethylphosphonate (10). To a solution ofphosgene in toluene (31 ml, 1.9 M) at 00C was added phenethyl alcohol (6.0 ml, 50 mmol). N,NDimethylaniline (6.4 ml, 50 mmol) was added dropwise over a 10-min interval, and after an additional 15 min, 8 ml of methylene chloride was added, and the reaction was warmed to room temperature. After 4 h residual phosgene was removed under reduced pressure, and the crude chloroformate was treated with 2.9 M aqueous ammonia (70 ml) and stirred rapidly for 30 min. The product was extracted into 50 ml of methylene chloride. The 64.94; H 5.69; N 3.29; P, 7.28. Found: C, 64.88; H, 5.71; N, 3.27; P, 7.28. N-(4-Nitrobenzoyl)-1,3-dlaminopropane (11). To a suspension of 4-nitrobenzoyl chloride (7.2 g, 39 mmol) in 150 ml of methylene chloride at OC was added triethylamine (14 ml, 98 mmol) and 3-bromopropylamine hydrobromide (8.6 g, 39 mmol). The solution was warmed to room temperature, and after 15 min the reaction mixture was diluted with 100 ml of methylene chloride and extracted twice with 100-ml portions of 0.1 M HCO and twice with 100-ml portions of 0.1 M NaOH. The The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

A synthetic peptide ligase

“…3 for definitions). The observed catalytic efficiency of the 33-residue peptide compares favourably to that of the two catalytic antibodies [25][26][27] selected for amino-acid couplings but lags behind that of engineered subtilisin ligases 28 .…”

Catalytic antibodies: prospects for the use in organic synthesis

“…A similar approach was used to obtain CA catalysing the formation of a dipeptide (alanylphenylalanine) derivative 71. 46 N-Benzyloxycarbonyl-L-or -D-alanine azide (72) prepared in situ from the corresponding phenyl ester and sodium azide was used as a peptidyl donor in the condensation reaction. The amino group of phenylalanine the C-terminus of which was protected as the amide with N-(4-nitrobenzoyl)-1,3-diaminopropane has served as an acceptor.…”

A Peptide–Nucleic Acid Replicator Origin for Life

“…The release of the produced peptide could occur stochastically or through periodic environmental changes (e.g., changes in temperature). There is some experimental support for entropy traps [47,48] and indeed they may even play a role in peptide bond formation in the extant ribosome [49].…”