Amnon Albeck

Stereocontrolled Synthesis of Erythro N-Protected α-Amino Epoxides and Peptidyl Epoxides.

Abstract N-protected α-amino epoxides of erythro configuration, derived from α-amino acids, were synthesized in a stereoselective manner. The erythro (2S,3S) configu- ration was achieved by the synthetic sequence: amino acid → haloketone → halohydrin → epoxide. A mechanistic explanation for the observed stereoselectivity is presented. This stereoselective synthetic approach was applied to the synthesis of a variety of short peptidyl epoxides, bearing a predefined absolute configuration of the chiral epoxide moiety.

Utilization of L-serine in an oxime olefin cycloaddition route to a functionalized asymmetric pyrrolidine, a selective α-glucosidase inhibitor

Abstract A new route for asymmetric aza-sugar analogs starting with L-serine and utilizing an intramolecular oxime olefin cycloaddition has been successfully developed. A member of this family of branched chain sugar amino di(hydroxymethyl) pyrrolidines ( 1 and 2 ) exhibits selective inhibition of α-glucosidase, while no inhibition of β-glucosidase was detected.

Octa-O-bis-(R,R)-Tartarate Ditellurane (SAS)—a Novel Bioactive Organotellurium(IV) Compound: Synthesis, Characterization, and Protease Inhibitory Activity†

Octa‐O‐bis‐(R,R)‐Tartarate Ditellurane (SAS) is a new TeIV compound, comprised of two tellurium atoms, each liganded by four oxygen atoms from two carboxylates and two alkoxides of two tartaric acids. Unlike many other TeIV compounds, SAS was highly stable in aqueous solution. It interacted with thiols to form an unstable Te(SR)4 product. The product of the interaction of SAS with cysteine was isolated and characterized by mass spectroscopy and elemental analysis. SAS selectively inactivated cysteine proteases, but it did not inactivate other families of proteolytic enzymes. It displayed selectivity towards the cysteine protease cathepsin B, a human enzyme of pharmaceutical interest, with a second order rate constant ki/Ki=5900 M−1 s−1.

Is there a weak H-bond LBHB transition on tetrahedral complex formation in serine proteases?†

The transformation of a weak hydrogen bond in the free enzyme into a low‐barrier hydrogen bond (LBHB) in the tetrahedral intermediate has been suggested as an important factor facilitating catalysis in serine proteases. In this work, we examine the structure of the H‐bond in the Asp102–His57 diad of serine proteases in the free enzyme and in a covalent tetrahedral complex (TC) with a trifluoromethylketone inhibitor. We apply ab initio quantum mechanical calculations to models consisting of a large molecular fragment of the enzyme active site, and the combined effect of the rest of the protein body and the solvation by surrounding bulk water was simulated by a self‐consistent reaction field method in our novel QM/SCRF(VS) approach. Potential profiles of adiabatic proton transfer in the Asp102–His57 diad in these model systems were calculated. We conclude that the hydrogen bond in both the free enzyme and in the enzyme‐inhibitor TC is a strong ionic asymmetric one‐well hydrogen bond, in contrast to a previous suggestion that it is a weak H‐bond in the former and a double‐well LBHB in the latter. Proteins 2004;54:000–000.

Synthesis, biological studies and molecular dynamics of new anticancer RGD-based peptide conjugates for targeted drug delivery.

New cyclic RGD peptide-anticancer agent conjugates, with different chemical functionalities attached to the parent peptide were synthesized in order to evaluate their biological activities and to provide a comparative study of their drug release profiles. The Integrin binding c(RGDfK) penta-peptide was used for the synthesis of Camptothecin (CPT) carbamate and Chlorambucil (CLB) amide conjugates. Substitution of the amino acid Lys with Ser resulted in a modified c(RGDfS) with a new attachment site, which enabled the synthesis of an ester CLB conjugate. Functional versatility of the conjugates was reflected in the variability of their drug release profiles, while the conserved RGD sequence of a selective binding to the αv integrin family, likely preserved their recognition by the Integrin and consequently their favorable toxicity towards targeted cancer cells. This hypothesis was supported by a computational analysis suggesting that all conjugates occupy conformational spaces similar to that of the Integrin bound bio-active parent peptide.

The mechanism of papain inhibition by peptidyl aldehydes

Various mechanisms for the reversible formation of a covalent tetrahedral complex (TC) between papain and peptidyl aldehyde inhibitors were simulated by DFT calculations, applying the quantum mechanical/self consistent reaction field (virtual solvent) [QM/SCRF(VS)] approach. Only one mechanism correlates with the experimental kinetic data. The His–Cys catalytic diad is in an N/SH protonation state in the noncovalent papain–aldehyde Michaelis complex. His159 functions as a general base catalyst, abstracting a proton from the Cys25, whereas the activated thiolate synchronously attacks the inhibitors carbonyl group. The final product of papain inhibition is the protonated neutral form of the hemithioacetal TC(OH), in agreement with experimental data. The predicted activation barrier g  enz≠ = 5.2 kcal mol−1 is close to the experimental value of 6.9 kcal mol−1. An interpretation of the experimentally observed slow binding effect for peptidyl aldehyde inhibitors is presented. The calculated g  cat≠ is much lower than the rate determining activation barrier of hemithioacetal formation in water, g  w≠ , in agreement with the concept that the preorganized electrostatic environment in the enzyme active site is the driving force of enzyme catalysis. We have rationalized the origin of the acidic and basic pKas on the k2/KS versus pH bell‐shaped profile of papain inhibition by peptidyl aldehydes. Proteins 2011.

Factors determining the relative stability of anionic tetrahedral complexes in serine protease catalysis and inhibition

Quantum mechanical ab initio (RHF/6‐31+G*//RHF/3‐21G) calculations were used to simulate the formation of the tetrahedral complex intermediate (TC) in serine protease active site by substrates and transition‐state analog inhibitors. The enzyme active site was simulated by an assembly of the amino acids participating in catalysis, whereas the substrates and inhibitors were simulated by small ligands, acetamide (1) and trifluoroacetone (2), respectively. For the first time, the principal factors determining the relative stability of the TC in serine proteases are arranged according to their energy contributions. These include (a) formation of the new covalent bond between Ser195 Oγ and the electrophilic center of a ligand; (b) stabilization of the oxyanion in the oxyanion hole; (c) basic catalysis by His57; and (d) hydrogen bond between Asp102 carboxylate and Nδ of the protonated His57. We have directly calculated the gas‐phase relative free energy of formation of TCAS(2) and TCAS(1), the value of ΔΔGg[TCAS(2,1)]. It is ΔEcov, the relative energy of the new covalent bond between the enzyme and the ligand formed in a TC that determines the experimentally observed large difference in the stability of TCs formed by substrates and TS‐analog inhibitors of serine proteases. We demonstrated that the relative stability of TCs formed by a series of mono‐ and dipeptide amides and TFKs, derived from experimental kinetic data, can be rather well approximated by the sum of the theoretically calculated value of ΔΔGg[TCAS(2,1)] and the difference in hydration free energies of isolated ligands. Proteins 2000;40:154–167.

Challenging a paradigm: theoretical calculations of the protonation state of the Cys25-His159 catalytic diad in free papain.

A central mechanistic paradigm of cysteine proteases is that the His–Cys catalytic diad forms an ion‐pair NH(+)/S(−) already in the catalytically active free enzyme. Most molecular modeling studies of cysteine proteases refer to this paradigm as their starting point. Nevertheless, several recent kinetics and X‐ray crystallography studies of viral and bacterial cysteine proteases depart from the ion‐pair mechanism, suggesting general base catalysis. We challenge the postulate of the ion‐pair formation in free papain. Applying our QM/SCRF(VS) molecular modeling approach, we analyzed all protonation states of the catalytic diad in free papain and its SMe derivative, comparing the predicted and experimental pKa data. We conclude that the His–Cys catalytic diad in free papain is fully protonated, NH(+)/SH. The experimental pKa = 8.62 of His159 imidazole in free papain, obtained by NMR‐controlled titration and originally interpreted as the NH(+)/S(−) ⇌ N/S(−)

Epoxidation of Peptidyl Olefin Isosteres. Stereochemical Induction Effect of Chiral Centers at Four Adjacent Cα Positions

{\rm NH}(+)/{\rm S}(-)\rightleftharpoons {\rm N/S}(-)

Efficient and Stereospecific Synthesis of (z)-Hex-3-Enedioic Acid, a Key Intermediate for Gly-Gly cis Olefin Isostere

equilibrium, is now assigned to the NH(+)/SH ⇌ N/SH