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A bromotyrosine alkaloid family of antimicrobial agents was synthesized using the known structure of a natural inhibitor of the mycobacterial mycothiolS-conjugate amidase (MCA) as a template. This series of compounds represents a novel class of anti-infective agents against Gram-positive pathogens, including mycobacteria and meticillin- and vancomycin-resistantStaphylococcus aureus. The fact that these compounds are active against mycobacterial strains in which the MCA gene is deleted and against Gram-positive bacteria lacking mycothiol suggests the existence of an alternative target for these compounds. One member of this family, EXEG1706, was identified as the lead compound possessing low MICs (2·5–25 μg ml−1) for several clinical isolates, whilst having low toxicity for THP-1 monocytes and macrophages.

Mycothiol (1-D-myo-inosityl 2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, MSH or AcCys-GlcN-inositol (Ins)) is the major reducing agent in actinomycetes, including Mycobacterium tuberculosis. The biosynthesis of MSH involves a deacetylase that removes the acetyl group from the precursor GlcNAc-Ins to yield GlcN-Ins. The deacetylase (MshB) corresponds to Rv1170 of M. tuberculosis with a molecular mass of 33,400 Da. MshB is a Zn2+ metalloprotein, and the deacetylase activity is completely dependent on the presence of a divalent metal cation. We have determined the x-ray crystallographic structure of MshB, which reveals a protein that folds in a manner resembling lactate dehydrogenase in the N-terminal domain and a C-terminal domain consisting of two beta-sheets and two alpha-helices. The zinc binding site is in the N-terminal domain occupying a position equivalent to that of the NAD+ co-factor of lactate dehydrogenase. The Zn2+ is 5 coordinate with 3 residues from MshB (His-13, Asp-16, His-147) and two water molecules. One water would be displaced upon binding of substrate (GlcNAc-Ins); the other is proposed as the nucleophilic water assisted by the general base carboxylate of Asp-15. In addition to the Zn2+ providing electrophilic assistance in the hydrolysis, His-144 imidazole could form a hydrogen bond to the oxyanion of the tetrahedral intermediate. The extensive sequence identity of MshB, the deacetylase, with mycothiol S-conjugate amidase, an amide hydrolase that mediates detoxification of mycothiol S-conjugate xenobiotics, has allowed us to construct a faithful model of the catalytic domain of mycothiol S-conjugate amidase based on the structure of MshB.

Binding sites analogous to those of sp(3) carbon are presented by concanavalin A. This lectin has now been cross-linked with a bismannopyranoside which contains the C(2) spacer required to form the computer-modeled diamondlike three-dimensional protein lattice shown in the picture.

Ab initio calculations show that sulfhydryl anion has a significantly lower potential than the hydroxide anion for stabilizing the products of its attack on carbonyl moieties - the tetrahedral complexes (TC). In this paper we analyze the factors that contribute to this phenomenon. Quantum mechanical MO ab initio calculations were used for studies of two reaction series, one for the attack of hydroxyl and one for the attack of sulfhydryl anion on different carbonyl compounds and their analogs. All of the anionic TCs formed by HS- are characterized by higher charge transfer, but are significantly less stable than the relevant TC of HO-. To explain the phenomenon we used a simple qualitative model based on Density Functional Theory (DFT). The crucial role of the occupied valence MOs is demonstrated in the process of electronegativity equalization between the donor and acceptor fragments in the final TC product. The sulfhydryl anion has significantly lower potential to stabilize TC products in comparison with the hydroxide anion because of the larger extent of electron back-donation from the electrophile′s HOMOA to the nucleophile′s LUMOD. This electron back-donation thus reduces the stability of the anionic TC in the case of HS- and may account for the calculational results. Applications of this work to enzyme reactions help in understanding the differences in mechanisms of serine and cysteine proteases and may be used to guide the design of inhibitors for these enzymes. In perspective, the back-donation phenomenon discussed here may be applied to the study of electron transfer processes involving oxidation-reduction enzymes.

Potent inhibitors of proteases are constantly sought because of their potential as new therapeutic lead compounds. In this paper we report a simple computational methodology for obtaining new ideas for functional groups that may act as effective inhibitors. We relate this study to serine proteases. We have analyzed all of the factors that operate in the enzyme-substrate interactions and govern the free energy for the transformation of the Michaelis complex (MC) to the anionic covalent tetrahedral complex (TC). The free energy of this transformation ( ΔΔG MC-TC ) is the quantitative criterion that differentiates between the catalytic and inhibitory processes in proteases. The catalytic TC is shifted upwards (ΔΔG MC-TC > 0) relative to the MC in the free energy profile of the reaction, whereas the inhibitory tetrahedral species is shifted downward (ΔΔG MC-TC < 0). Therefore, the more stable the TC, the more effective it should be as an inhibitor. We conclude that the dominant contribution to the superstabilization of an anionic TC for transition state analog inhibitors originates from the formation of a σ-covalent bond between the reactive centers of the enzyme and its inhibitor. This energetic effect is a quantitative value obtained in ab initio calculations and provides an estimate as to whether a functional group is feasible as potent inhibitor or not. To support our methodology, we describe several examples where good agreement is shown between modeled ab initio quantum chemical calculations and experimental results extracted from the literature.

We developed an algorithm to analyze the distribution and geometry of simple and complex salt bridges in 94 proteins selected from the Protein Data Bank. In this study, the term "salt bridging" denotes both non-bonded and hydrogen-bonded paired electrostatic interactions between acidic carboxyl groups and basic amino groups in single or adjacent protein chains. We defined complex salt bridges as those joining more than two charged residues, including Asp, Glu, Lys and Arg, and excluding His. The survey related the following special features of complex salt bridges. (1) The abundance of complex salt bridges is high; one-third of all residues participating in salt-bridge formation were part of complex salt bridges. (2) The geometry of the interaction between acidic and basic residues is very similar in simple and complex salt bridges. Adding one residue to a simple interaction represents a minor change in the geometry but provides the molecule with a more complex interaction, a phenomenon that may explain the cooperative effect of salt bridges in proteins. Such moderate changes in salt-bridge networks can be generated stepwise and reversibly without trapping the protein in a local energetic minimum. (3) One important role of complex salt bridges is connecting protein subunits or joining two secondary structures to form quaternary structures, where they can connect as many as five secondary structure units. (4) Arginine serves as a key connector and/or a branching unit because its geometry allows three possible directions of interactions. The information gained from this study of complex salt bridges should enhance the understanding of protein structure.

Three independent strategies are tested toward the synthesis of the protected 1-aminobicyclo[2.2.2]octene ketodiester 1. One of these three is found to be completely regioselective. It proceeds by Diels–Alder addition of dimethyl acetylenedicarboxylate to the silyl enol ether of 3-benzyloxycarbonyl-2-cyclohexenone, followed by a chemoselective Curtius rearrangement.

Picornavirus 3C proteases are prime targets for rational drug design. This viral protease appears in a large number of viruses from the Picornavirus family that cause serious disease syndromes, and it has an important role in the life cycle of the virus, processing the translation product of the Picornavirus genome by progressive co- and posttranslational cleavages. It is, therefore, important to gain structural and mechanistic information about this family of enzymes. We concentrate in this paper on the specific features of the 3C; particularly, we are trying to show that the 3C constitute a new family to enzymes which is neither a serine- nor a cysteine-type protease. General basic theory on the behavior of sulfur nucleophile vs the behavior of oxygen nucleophile regarding the nucleophilic attack on carbonyl compounds and the possible determinants in the structure of 3C viral proteases of rhinovirus 1A are being discussed.

Ab initio molecular orbital theory was used to study the following enol-carbonyl pairs: H 2 C=C(OH)RCH 3 C(=O)R, CH 3 CH=C(OH)RCH 3 CH 2 C(=O)R, (CH 3 ) 2 C=C(OH)R(CH 3 ) 2 CHC(=O)R. Geometries were optimized with the 3-21G basis set, and for most molecules single-point 6-31G calculations were also performed

alpha-Neurotoxins from scorpion venoms constitute the most studied group of modifiers of the voltage-sensitive sodium channels, and yet, their toxic site has not been characterized. We used an efficient bacterial expression system for modifying specific amino acid residues of the highly insecticidal alpha-neurotoxin LqhalphaIT from the scorpion Leiurus quinquestriatus hebraeus. Toxin variants modified at tight turns, the C-terminal region, and other structurally related regions were subjected to neuropharmacological and structural analyses. This approach highlighted both aromatic (Tyr10 and Phe17) and positively charged (Lys8, Arg18, Lys62, and Arg64) residues that (i) may interact directly with putative recognition points at the receptor site on the sodium channel; (ii) are important for the spatial arrangement of the toxin polypeptide; and (iii) contribute to the formation of an electrostatic potential that may be involved in biorecognition of the receptor site. The latter was supported by a suppressor mutation (E15A) that restored a detrimental effect caused by a K8D substitution. The feasibility of producing anti-insect scorpion neurotoxins with augmented toxicity was demonstrated by the substitution of the C-terminal arginine with histidine. Altogether, the present study provides for the first time an insight into the putative toxic surface of a scorpion neurotoxin affecting sodium channel gating.

This paper discusses the benefit of mapping paired cysteine mutation patterns as a guide to identifying the positions of protein disulfide bonds. This information can facilitate the computer modeling of protein tertiary structure. First, a simple, paired natural-cysteine-mutation map is presented that identifies the positions of putative disulfide bonds in protein families. The method is based on the observation that if, during the process of evolution, a disulfide-bonded cysteine residue is not conserved, then it is likely that its counterpart will also be mutated. For each target protein, protein databases were searched for the primary amino acid sequences of all known members of distinct protein families. Primary sequence alignment was carried out using PileUp algorithms in the GCG package. To search for correlated mutations, we listed only the positions where cysteine residues were highly conserved and emphasized the mutated residues. In proteins of known three-dimensional structure, a striking pattern of paired cysteine mutations correlated with the positions of known disulfide bridges. For proteins of unknown architecture, the mutation maps showed several positions where disulfide bridging might occur.

The stereochemistry of the Diels-Alder reactions of 3 cyclopropenes was determined unequivocally by X-ray and NOE studies. Tetrachlorocyclopropene yields exo -adducts with ( E )-1-R-1,3-butadiene (ROCH3, OCOCH3, OSiMe3), with 1,4-diphenylbutadiene and with furan. 1,2-bromochlorocyclopropene yields with ( E )-1-R-butadienes (ROCH3, OSiMe3) endo : exo -adducts in a ratio of 9:1. Cyclopropene + 1-methoxybutadiene yield only the endo -adduct.

Differences between SiH + 5 and CH + 5 are more significant than the similarities. The proton affinity of SiH 4 exceeds than of CH 4 by ≈25 kcal/mol. but the heat of hydrogenation of SiH + 3 is smaller than that of CH + 3 by nearly the same amount. Like CH + 5 the C 5 structures of SiH + 5 are preferred, but SiH + 5 is best regarded as a weaker SiH + 3 —H 2 complex. D 3h , C 2v , and C 4v forms are much higher in energy and SiH + 5 should not undergo hydrogen scrambling (pseudorotation) readily, as does CH + 5 The neutral BH 5 is only weakly bound toward loss H 2 , and the D 3h . C 2v , and C 4v forms are also high in energy. The contral-atom electronegativities, C + > B > Si + , control this behavior. The electronegativities also determine the ability to bear positive charges. Thermodynamically. SiH + 5 and SiH + 3 are more stable than CH + 5 and CH + 3 , respectively; hydride transfer occurs from SiH 4 to CH + 3 and proton transfer from CH + 5 to SiH 4 .

A 5-cyano substituent decelerates the solvolysis rate of exo-2-norbornyl brosylate (1-H) by a factor of 1790. A much smaller deceleration (24–90 fold) is observed for the secondary endo and for both exo and endo tertiary 2-norbornyl derivatives. These results support the occurrence of σ-participation in the solvolysis of 1-H.

Calculs ab initio sur les composes MCH 2 X avec M=Li ou Na et X=CH 3 , NH 2 , OH, F, SiH 3 , PH 2 , SH ou Cl, avec corrections de correlation electronique. Comparaison avec especes anioniques correspondantes

The worldwide rapid increase in bacterial resistance to numerous antibiotics requires on-going development of new drugs to enter the market. As the development of new antibiotics is lengthy and costly, early monitoring of compound's toxicity is essential in the development of novel agents. Our interest is in a rapid, simple, high throughput screening method to assess cytotoxicity induced by potential agents. Some intracellular pathogens, such as Mycobacterium tuberculosis primary site of infection is human alveolar macrophages. Thus, evaluation of candidate drugs for macrophage toxicity is crucial. Protocols for high throughput drug toxicity screening of macrophages using flow cytometry are lacking in the literature. For this application we modified a preexisting technique, propidium iodide (PI) exclusion staining and utilized it for rapid toxicity tests. Samples were prepared in 96 well plates and analyzed by flow cytometry, which allowed for rapid, inexpensive and precise assessment of compound's toxicity associated with cell death.

The title compounds have been isolated and their structures determined by X-ray crystallography. Their relative stability is discussed in terms of theory and experiment. The endo-adduct is the thermodynamically more stable one.

The title compounds have been isolated and their structures determined by X-ray crystallography. Their relative stability is discussed in terms of theory and experiment. The endo-adduct is the thermodynamically more stable one.

The stereochemistry of the Diels-Alder reactions of 3 cyclopropenes was determined unequivocally by X-ray and NOE studies. Tetrachlorocyclopropene yields exo -adducts with ( E )-1-R-1,3-butadiene (ROCH3, OCOCH3, OSiMe3), with 1,4-diphenylbutadiene and with furan. 1,2-bromochlorocyclopropene yields with ( E )-1-R-butadienes (ROCH3, OSiMe3) endo : exo -adducts in a ratio of 9:1. Cyclopropene + 1-methoxybutadiene yield only the endo -adduct.

Ab initio calculations, mostly with the 3-21G basis-set were carried out for cyclopropabenzene (1), the linear- and angularcyclobutacyclopropabenzenes (2 and 3 respectively), the linear- and angular dicyclopropabenzenes 4 and 5 respectively, tricyclopropabenzene (6), alkylidenecyclopropabenzene (7) and two calecene homologues, i.e., 8 - containing an exocyclic cyclopropene moiety and 9 - containing an exocyclic cyclopentadiene moiety. The geometries and the total and relative energies of compounds l-9 are reported. The calculated geometries of 1 and 2 are in excellent agreement with recent low-temperature X-ray structures. In all molecules, except 6, the fused bonds are shorter than the other aromatic bonds, in conflict with the Mills-Nixon “bond fixation” concept. The following strain energies were calculated for compounds 1-6 (in kcal/mol): (70, also measured experimentally), 2 (102); 3 (103); 4 (133); 5 (140); 6 (217). The surprising theoretical prediction that the strain of the cyclopropane ring in 2 is slightly lower than that in has been confirmed by recent experiments. Compounds 7-9 exhibit considerable polarization of the u-electrons, so that the cyclopropabenzene skeleton is positively charged in 7 and 9 and negatively charged in 8. Consequently, the direction of the dipole moment in 8 is opossite to that in 7 and 9.

Appearance energy measurements for the loss of Br˙ from ionized 2-bromocyclopropabenzene (5c), in conjunction with thermochemical data, suggest that the 2-cyclopropaphenyl cation (2) is stabilized, relative to the phenyl cation (1a), by at least 27.6 kcal/mol, in very good agreement with a previous prediction based on ab initio MO calculations ( 23 kcal/mol at 3-21G*); the heat of formation of (2) is estimated to be 311 kcal/mol (1 cal = 4.184 J).