Synthesis and physical properties of protein core mimetics.

A series of mimetic cores composed of a synthetic scaffold and amino acids have been constructed and their properties investigated in chloroform. A relative measure of H-bond strength was obtained by comparing temperature coefficients derived from variable-temperature (1)H NMR experiments. Although most templates had a strong H-bond, only a single template composed of D- and L-phenylalanines was able to form two strong H-bonds. Templates containing D- and L-leucines formed only a single H-bond. The results of these studies suggest that aromatic edge-to-face interactions provide greater stabilization energy than aliphatic-aromatic interactions in the tightly packed hydrophobic cores of proteins. Partial structures of the templates were derived by analyzing a series of two-dimensional (1)H NMR spectra and performing molecular mechanics calculations using AMBER and MMFF94 force fields.

Facile synthesis of rotaxanes through condensation reactions of DCC-[2]rotaxanes.

[reaction: see text] In this Letter, we present an easy method for the synthesis of rotaxanes using a novel DCC-[2]rotaxane. The DCC-[2]rotaxane is composed of dibenzo-24-crown-8 ether, an amino acid tether, and di-tert-butyl phenyl rings as blocking groups. It is relatively stable and can be purified by column chromatography. A series of model rotaxanes were obtained in good yields by condensing the DCC-[2]rotaxanes with N-(2-aminoethyl)-3,5-di-tert-butylbenzylamide in acetonitrile and chloroform.

Cyclic peptide formation catalyzed by an antibody ligase.

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-Phe small middle dotp-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 diastereomers of 8a: the d-Trp(1)-d-Phe(6) (8b), l-Trp(1)-l-Phe(6) (8c), and l-Trp(1)-d-Phe(6) (8d) hexapeptides as well as d-Trp'-l-Trp(6) (12) and d-Phe'-l-Phe(6) (13). As expected, the rate enhancement by 16G3 was greatest for 8a, because the stereochemistry of Trp(1) and Phe(6) matches that of the corresponding residues on the hapten used to induce the biosynthesis of 16G3. A model of the variable domain of 16G3 was generated from the primary sequence using the antibody structural database to guide the model construction. The resulting model provided support for some previously proposed interpretations of the kinetic data, while providing valuable new insights for others.

The state of antibody catalysis.

One of the fascinations of catalytic antibodies is the possibility of harnessing the mechanisms available to enzymes for chemical transformation and applying them to the broad realm of chemistry encountered in organic synthesis. Recently, the catalytic repertoire of antibodies has been extended to include mechanistically more complex bimolecular reactions and the immunological response to the hapten can be more thoroughly examined as a result of the advent of new screening technology using bacterial phages or auxotrophic cell lines.

Dissection of an antibody-catalyzed reaction.

Antibody 43C9 accelerates the hydrolysis of a p-nitroanilide by a factor of 2.5 x 10(5) over the background rate in addition to catalyzing the hydrolysis of a series of aromatic esters. Since this represents one of the largest rate accelerations achieved with an antibody, we have undertaken a series of studies aimed at uncovering the catalytic mechanism of 43C9. The immunogen, a phosphonamidate, was designed to mimic the geometric and electronic characteristics of the tetrahedral intermediate that forms upon nucleophilic attack by hydroxide on the amide substrate. Further studies, however, revealed that the catalytic mechanism is more complex and involves the fortuitous formation of a covalent acyl-antibody intermediate as a consequence of complementary side chain residues at the antibody-binding site. Several lines of evidence indicate that the catalytic mechanism involves two key residues: His-L91, which acts as a nucleophile to form the acyl-antibody intermediate, and Arg-L96, which stabilizes the anionic tetrahedral moieties. Support for this mechanism derives from the results of site-directed mutagenesis experiments and solvent deuterium isotope effects as well as direct detection of the acyl-antibody by electrospray mass spectrometry. Despite its partial recapitulation of the course of action of enzymic counterparts, the reactivity of 43C9, like other antibodies, is apparently limited by its affinity for the inducing immunogen. To go beyond this level, one must introduce additional catalytic functionality, particularly general acid-base catalysis, through either improvements in transition-state analog design or site-specific mutagenesis.