Biomedical subjects

Paul A Bartlett

Publications and source records attributed to Paul A Bartlett.

18 recordsLinked to original sources

PubMed2006

Beta strand peptidomimetics as potent PDZ domain ligands.

The search for general strategies for inhibiting protein-protein interactions has been stimulated by recognition of the key role they play in virtually every process of living systems. Multiprotein complex assembly and localization by PDZ domain-containing proteins exemplify processes critical to cell physiology and function that are mediated by beta strand association. Here we describe the development of substituted "@-tides," protease-resistant peptidomimetics incorporating conformationally restricted amino acid surrogates that reproduce the hydrogen-bonding pattern and side-chain functionality of a beta strand. The synthetic flexibility and generality of the substituted @-tide design was demonstrated by the synthesis of a panel of ligands for the alpha1-syntrophin PDZ domain. The rational design of a small molecule of unprecedented affinity for the PDZ domain suggests that these peptidomimetics may provide a general method for inhibiting protein-protein interactions involving extended peptide chains.

Biomimetics↗

PubMed2005

Quantifying amino acid conformational preferences and side-chain-side-chain interactions in beta-hairpins.

The intrinsic conformational biases of individual amino acids and their interstrand side-chain-side-chain (SC-SC) interactions both contribute to the stability of beta-sheets. The relative magnitudes of these effects have been difficult to assess in the context of folded proteins, where tertiary contacts complicate the quantitative analysis of local effects. We now report the results of such an analysis in a much simpler system, a short, stabilized beta-hairpin structure where intrastrand (conformational) and interstrand (SC-SC) influences can be distinguished in the absence of competing protein tertiary interactions. A comprehensive comparison of all pairwise combinations of 11 N-terminal and 7 C-terminal amino acids within an 8-residue, @-tide-stabilized [in which @ denotes the 1,2-dihydro-3(6H)-pyridinyl unit] beta-hairpin reveals distinct differences between the various pairings and shows that the intrastrand and interstrand effects are of comparable magnitude in contributing to the stability of the folded forms over the unfolded forms.

Amino Acids↗

PubMed2005

@-tides as reporters for molecular associations.

The 1,6-dihydro-3(2H)-pyridinone unit is an amino acid surrogate that favors the extended beta-strand conformation when incorporated in an oligopeptide ("@-tide") strand. We now report that the circular dichroism (CD) signature of the vinylogous amide in the @-unit is sensitive to conformation in organic and aqueous solvents and, therefore, is useful as a quantitative measure of @-tide association and folding processes that involve this moiety. Moreover, this method can be employed in the micromolar concentration range, which is not readily accessible using other techniques. Measurements of @-tide dimerization and beta-hairpin folding equilibria not only demonstrate the utility and generality of this approach but also provide a way to quantify amino acid side chain-side chain interactions relevant to beta-sheet stability.

Circular Dichroism↗

PubMed2005

Interaction of phosphonate analogues of the tetrahedral reaction intermediate with 5-enolpyruvylshikimate-3-phosphate synthase in atomic detail.

The enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the penultimate step of the shikimate pathway and is the target of the broad-spectrum herbicide glyphosate. Since the functionality of the shikimate pathway is vital not only for plants but also for microorganisms, EPSPS is considered a prospective target for the development of novel antibiotics. We have kinetically analyzed and determined the crystal structures of Escherichia coli EPSPS inhibited by (R)- and (S)-configured phosphonate analogues of the tetrahedral reaction intermediate. Both diastereomers are competitive inhibitors with respect to the substrates of the EPSPS reaction, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). Remarkably, the (S)-phosphonate (K(iS3P) = 750 nM), whose configuration corresponds to that of the genuine tetrahedral intermediate, is a much weaker inhibitor than the (R)-phosphonate analogue (K(iS3P) = 16 nM). The crystal structures of EPSPS liganded with the (S)- and (R)-phosphonates, at 1.5 and 1.9 A resolution, respectively, revealed that binding of the (R)-phosphonate induces conformational changes of the strictly conserved residues Arg124 and Glu341 within the active site. This appears to give rise to substantial structural alterations in the amino-terminal globular domain of the enzyme. By contrast, binding of the (S)-phosphonate renders the enzyme structure unchanged. Thus, EPSPS may facilitate the tight binding of structurally diverse ligands through conformational flexibility. Molecular docking calculations did not explain why the (R)-phosphonate is the better inhibitor. Therefore, we propose that the structural events during the open-closed transition of EPSPS are altered as a result of inhibitor action.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2005

@-Tide-stabilized beta-hairpins.

As minimalist versions of beta-structure, two-stranded beta-hairpins are commonly employed as platforms for assessing the interactions that stabilize beta-sheets in proteins. We have found that the presence of a 1,6-dihydro-3(2H)-pyridinone moiety (the "@-unit") as an amino acid replacement at the i - 1 or i + 4 positions relative to a beta-turn strongly stabilizes the hairpin conformation. Hybrids of this type bridge the gap between natural beta-hairpins and unnatural beta-sheets because the @-unit only replaces one residue in a peptide while stabilizing the hairpin conformation to a greater extent than a normal amino acid. In this report, we describe the synthesis of a variety of @-tide-templated hairpins and the NMR and CD characterization of their conformations in both polar and nonpolar solvents.

Hydrogen Bonding↗

PubMed2004

Facile synthesis of @-tide beta-strand peptidomimetics: improved assembly in solution and on solid phase.

The synthesis of @-tide beta-strand peptidomimetics has been improved such that oligomers now can be obtained from solution- and solid-phase synthesis protocols approaching the efficiency and flexibility of peptide chemistry. These methods enable the synthesis of @-tide oligomers with a variety of amino acids and with lengths up to 13 units. [reaction: see text]

Combinatorial Chemistry Techniques↗

PubMed2004

Enzymatic synthesis of a ring-contracted analogue of 5-enolpyruvylshikimate-3-phosphate.

[reaction: see text] Three ring-contracted mimics of shikimate-3-phosphate, formed from the triols by shikimate kinase, were evaluated as substrates of the next enzyme in the pathway, EPSP synthase. The cyclopentylidene analogue (+)-2P was converted enzymatically to the enolpyruvyl derivative, thus demonstrating the second step of an artificial biosynthetic sequence.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2004

Conservation of mechanism in three chorismate-utilizing enzymes.

Chorismate is the end-product of the shikimate pathway for biosynthesis of carbocyclic aromatic compounds in plants, bacteria, fungi, and some parasites. Anthranilate synthase (AS), 4-amino-4-deoxychorismate synthase (ADCS), and isochorismate synthase (IS) are homologous enzymes that carry out the initial transformations on chorismate in the biosynthesis of tryptophan, p-aminobenzoate, and enterobactin, respectively, and are expected to share a common mechanism. Poor binding to ADCS of two potential transition state analogues for addition of a nucleophile to C6 of chorismate implies that it, like AS and IS, initiates reaction by addition of a nucleophile to C2. Molecular modeling based on the X-ray structures of AS and ADCS suggests that the active site residue K274 is the nucleophile employed by ADCS to initiate the reaction, forming a covalent intermediate. The K274A and K274R mutants were shown to have 265- and 640-fold reduced k(cat) values when PabA (the cognate amidotransferase) + glutamine are used as the nitrogen source. Under conditions of saturating chorismate and NH(4)(+), ADCS and the K274A mutant have identical k(cat) values, suggesting the participation of NH(4)(+) as a rescue agent. Such participation was confirmed by the buildup of 2-amino-2-deoxyisochorismate in the reactions of the K274A mutant but not ADCS, when either NH(4)(+) or PabA + glutamine is used as the nitrogen source. Additionally, the inclusion of ethylamine in the reactions of K274A yields the N-ethyl derivative of 2-amino-2-deoxyisochorismate. A unifying mechanism for AS, ADCS, and IS entailing nucleophile addition to C2 of chorismate in an S(N)2' ' process is proposed.

Anthranilate Synthase↗

PubMed2003

Identification of novel macrocyclic peptidase substrates via on-bead enzymatic cyclization.

Peptidase-catalyzed formation of macrocyclic lactams on solid phase identifies ring systems that are favorably bound in the enzyme active site. We evaluated several cyclic peptide motifs linked by ester bonds between the P2 and P1' or the P1 and P2' side chains. The depsipeptide represented by structure 5 was readily generated by a variety of peptidases from precursor omega-amino acids or omega-amino esters. This strategy for identifying ring systems for potential macrocyclic transition state analogues was demonstrated with the serine peptidases trypsin and chymotrypsin, with the aspartic peptidase pepsin, and with the zinc peptidase thermolysin.

Amides↗

PubMed2003

Synthesis and evaluation of macrocyclic transition state analogue inhibitors for alpha-chymotrypsin.

Lactams 1 and 2 are readily formed from acyclic precursors in the presence of trypsin and chymotrypsin, respectively, identifying the macrocyclic ring system as a potential motif for constrained transition state analogue inhibitors of the serine peptidases. Ketone 3 was synthesized and shown to be a modest inhibitor of chymotrypsin (Ki = 220 microM), albeit 4-fold more potent than the acyclic hydroxy acid 25 (Ki = 1.5 mM as a mixture of epimers). A precursor (31) to the amino boronic acid 4 was also prepared; although this derivative was a potent inhibitor of chymotrypsin (Ki = 130 nM) by virtue of the boronic acid moiety, it showed no advantage over the des-amino analogue 32 (Ki = 120 nM), which is not capable of cyclizing.

Acids, Acyclic↗

PubMed2003

5-enolpyruvylshikimate 3-phosphate synthase: chemical synthesis of the tetrahedral intermediate and assignment of the stereochemical course of the enzymatic reaction.

A chemical synthesis of both diastereomers of the tetrahedral intermediate involved in 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS) catalysis has been accomplished. Combination of methyl dibromopyruvate with a protected shikimic acid derivative, phosphorylation, and lactonization afforded the intermediates (S)-15 and (R)-15, whose configurations were assigned by NMR. After introduction of the 3-phosphate group and deprotection, photoinitiated radical debromination of the dibromo analogues (S)-5 and (R)-5 was accomplished with tributyltin hydride in mixed aqueous solvents in the presence of surfactant to give the pyruvate ketal phosphates (R)-TI and (S)-TI, respectively. These compounds are stable at high pH, but decompose at pH 7 with a half-life of ca. 10 min. (R)-TI proved to be inert to EPSPS, while (S)-TI was converted by the enzyme to a mixture of 5-enolpyruvylshikimate 3-phosphate, shikimate 3-phosphate, and phosphoenolpyruvate. The demonstration that the enzymatic intermediate possesses the S-configuration at the ketal center confirms the mechanism as an anti addition followed by a syn elimination. Furthermore, it appears that the syn stereochemistry of the second step requires the phosphate leaving group to serve as the base in catalyzing its own elimination.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗

PubMed2002

Amino acid derived heterocycles: lewis acid catalyzed and radical cyclizations from peptide acetals.

Bicyclization of peptide acetals via nucleophilic attack of a phenyl group on an endocyclic acyliminium ion 4 was explored as a route to novel amino acid derived heterocycles and peptidomimetic scaffolds. In the presence of protic acid, bridged structures such as 6 are formed readily from phenylalanine derivatives, but the fused-ring analogues 5 could not be obtained in good yield. In contrast, radical cyclization of the bromophenyl dihydropyrazinone 7 provides an effective alternative for the synthesis of 5 (n = 0, 1, 2). Additional versatility in this process was demonstrated by efficient synthesis of a different fused ring system, represented by the antihelmintic praziquantel, 8.

Acetals↗

PubMed2002

Antihydrophobic solvent effects: an experimental probe for the hydrophobic contribution to enzyme-inhibitor binding.

The hydrophobic component to the binding affinities of one acyclic phosphinate (4) and three macrocyclic phosphonamidate inhibitors (1-3) to the zinc peptidase thermolysin was probed by varying the solvent composition. Increasing the percentage of ethanol in the buffer solution over the range 0-9% increases the inhibition constants, K(i), by up to an order of magnitude. This approach represents an experimental method for distinguishing solvation from conformational or other effects on protein-ligand binding. The size of the "antihydrophobic effect" is correlated with the amount of hydrophobic surface area sequestered from solvent on association of the inhibitor and enzyme, although it is attenuated from that calculated from the surface tension of ethanol-water mixtures. The results are consistent with the Lum-Chandler-Weeks explanation for the size dependence of the hydrophobic effect.

Amides↗

PubMed2002

"@-Tides": the 1,2-dihydro-3(6H)-pyridinone unit as a beta-strand mimic.

The cyclic amino acid surrogate 1 was designed to mimic the extended conformation of a peptide unit and to provide hydrogen bond donor and acceptor functions conducive to beta-sheet formation. A convenient synthesis of this unit and solution and solid-phase methods for its incorporation into an oligomer alternating with peptide units have been devised. The resulting "@-tides", as these oligomers have been designated, show a high propensity for self-association in comparison to oligopeptides; insights into the structure and dynamical properties of their antiparallel dimers have been obtained by NMR.

Kinetics↗

PubMedDate not supplied

Combinatorial synthetic design. Solution and polymer-supported synthesis of heterocycles via intramolecular aza Diels-Alder and imino alcohol cyclizations.

A flexible synthetic strategy for combinatorial chemical applications has been developed on the basis of an aldehyde-bridge-alkene motif as the key component in several intramolecular cycloaddition reactions. This strategy was explored most extensively with the formal aza Diels-Alder cyclization, which affords a series of configurationally and functionally diverse heterocyclic compounds. The substrates included substituted salicylaldehydes, glyoxylic esters and amides, and N-acyl-alpha-aminoaldehydes; all reacted with a variety of anilines to yield different tetrahydroquinoline products. The cyclization of the aminoaldehydes was also translated from solution and optimized for solid phase. The stepwise mechanism of this cycloaddition suggested that the cationic intermediate from initial ring closure could be trapped by a variety of nucleophiles. This suggestion was confirmed by cyclization of amino alcohols and related compounds.

Alcohols↗

PubMedDate not supplied

Amino acid-derived heterocycles as combinatorial library targets: spirocyclic ketal lactones.

The spirocyclic ketal-lactone frameworks of 3 and 4 were designed as novel structures amenable to combinatorial synthesis. The synthesis of representative analogues was developed in solution and on solid support, the scope of effective input materials was determined, and the stability and stereochemistry of the products was evaluated. The spirocycles are obtained in modest overall yields (5-36%) and excellent purities (>72%) and offer a promising motif for combinatorial prospecting libraries.

Acetic Acid↗

PubMedDate not supplied

Amino acid-derived heterocycles as combinatorial library targets: bicyclic aminal lactones.

The incorporation of alpha-amino acids into heterocyclic structures is an effective strategy for generating peptidomimetics and combinatorial library scaffolds. This report describes the synthesis of novel bicyclic aminal lactones 3 by base-catalyzed cyclization of N-(2-oxoalkyl)-dipeptide esters 8. Assembly of the acyclic precursor 8 can be carried out on solid phase, with variation at four positions; cyclative release ensures high product purity in the final step. Cyclization affords the exo isomer stereospecifically when one chiral center is present in the precursor, or when both amino acids have the same configuration.

Alkylation↗

PubMedDate not supplied

Virtual hydrocarbon and combinatorial databases for use with CAVEAT.

Three new virtual databases have been developed for use with the bond-orientation-based database searching program CAVEAT. These consist of a database of trisubstituted monocyclic hydrocarbons having ethyl, vinyl, and phenyl substituents; a database of unsubstituted bicyclic hydrocarbons; and a database of core structures from established combinatorial synthetic methods having hydrogen, ethyl, vinyl, and phenyl substituents at the readily varied positions. Each collection of molecules was subjected to a batch conformational search, minimization, and conversion to a vector database for use with CAVEAT.

Bridged Bicyclo Compounds↗