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.

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.

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]

Formation of the 7-Oxa-1,4,10-triazatricyclo[8.2.2(5,12)]tetradecane-2,14-dione Ring System: Misrouted Synthesis of a Peptidomimetic.

An attempted synthesis of the tricyclic peptidomimetic 1, designed to imitate a beta-turn tripeptide in tendamistat, afforded instead the 6,6,8-ring system of 2. The key step in the synthesis entailed acylation of the hindered alpha,alpha'-disubstituted morpholine 4.2, which was approached by acylative ring opening of the 3,6-oxazabicyclo[4.2.0]octane 4.3. However, transannular rather than exocyclic cleavage occurred, giving the 1,6-oxazacyclooctane isomer 4.5. Subsequent ring closures to form the bi- and tricyclic intermediates 7.3 and 8.5 were difficult because of the strain being built into the ring systems. After completion of the synthesis, the structures of the intermediates and final product were elucidated by NMR, with three-bond, heteronuclear multiple-bond correlation experiments providing unambiguous evidence for the ring connectivity, and by molecular modeling, which allowed assignment of the stereochemistry. Compound 2 is a modest inhibitor of the target enzyme alpha-amylase (K(i) = 170 &mgr;M in 5% DMSO/water), binding with similar affinity to the tripeptide Ac-Trp-Arg-Tyr-OMe. Although the side-chain attachment points in the ring system of 2 correspond closely to the relative Calpha-positions in tendamistat (rmsd = 0.24 Å), the alignment of the Calpha-Cbeta bonds is poor, illustrating the importance of side-chain orientation in a peptidomimetic.

Design, Synthesis, and Evaluation of a Depsipeptide Mimic of Tendamistat.

The cyclic hexadepsipeptide framework of enniatin B was identified as a template matching the beta-turn tripeptide of tendamistat. The modified analog 1 was synthesized as a tendamistat mimic and compared to the acyclic derivative 2 and the tripeptide Ac-Try-Arg-Tyr-OMe. These compounds were assembled from the dimeric esters 3-5. As an inhibitor of alpha-amylase, 1 is twice as potent as 2 and comparable to the tripeptide. NMR studies of 1 reveal four conformers in equilibrium in a 50:25:15:10 ratio; the ring conformation of the major component is similar to that of the enniatin B template, with the cis geometry of the alpha-hydroxyisovaleryl-N-methylvaline amide linkage; the other conformers differ in the position or presence of the cis amide linkage.

Potentially Macrocyclic Peptidyl Boronic Acids as Chymotrypsin Inhibitors.

The possibility of forming a peptide boronate adduct in a serine protease active site that mimics the first tetrahedral intermediate in the peptide hydrolysis mechanism was explored with the complex boronic acid analogs 7, 8-OH, and 8-NH(2)(). In these structures, the P(1) and P(2) residues and the P(1)'-P(3)' residues are connected through the P(2) and P(1)' side chains, to encourage formation of the diester or amide-ester adducts via macrocyclization. These inhibitors were assembled from suitably protected derivatives of 2,4-diaminobutanoic acid or 2,4-diaminopentanoic acid (11), borophenylalanine (12), aspartic acid, malic acid or the substituted malic acid analog 13, and Leu-Arg dipeptide. Stereoselective syntheses were developed for the (S,S)-2,4-diaminopentanoate 11 and for the (S,S)-beta-isobutylmalate 13 derivatives. The complex peptidyl boronates 7 (K(i) = 26 nM) and 8-OH (68 nM) are potent inhibitors of alpha-chymotrypsin; however, the affinity of 7 is neither time- nor pH-dependent, and it is only moderately greater than that found for comparison compounds like 8-H (114 nM), 9 (356 nM), and 10 (219 nM) that cannot cyclize or form a diester adduct.

Preparation of diazabicyclo[4.3.0]nonene-based peptidomimetics.

Several functionalized diazabicyclo[4.3.0]nonenes and other heterocycles have been prepared as potential peptidomimetic scaffolds. A novel and efficient method has been developed for the preparation of N-substituted gamma-lactams 13. Preparation of amidine-containing 1,5-diazabicyclo[4.3.0]nonenes 43 and 44 has been achieved through Hg-mediated cyclization of the precursor N-aminopropyl-gamma-thiolactams and subsequent functional group manipulation. Bicycle 43 represents a novel scaffold for potential peptide turn mimetics, whereas 44 could potentially be employed as an alpha-helix template attached to the C-terminus of peptides. These compounds are novel additions to the current range of small-molecule constrained peptidomimetics.

Synthesis of amino acid-derived cyclic acyl amidines for use in beta-strand peptidomimetics.

The acyl amidine represented by the 4,5-dihydro-2(3H)-pyrazinone ring system 2 is isosteric to the vinylogous amide of the 1,2-dihydro-3(6H)-pyridinone 1, but its assembly from separate amine and amide components enables ready incorporation of an amino acid side chain with correct regio- and stereochemistry. beta-Strand peptidomimetics incorporating amino acid analogues based on 2 have recently been shown to be potent, protease-resistant ligands to a PDZ protein-interaction domain. Two routes to the protected dipeptide analogue 3 are described.

Differential inhibition of class I and class II 5-enolpyruvylshikimate-3-phosphate synthases by tetrahedral reaction intermediate analogues.

The shikimate pathway enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) is best known as the target of the herbicide glyphosate. EPSPS is also considered an attractive target for the development of novel antibiotics since the pathogenicity of many microorganisms depends on the functionality of the shikimate pathway. Here, we have investigated the inhibitory potency of stable fluorinated or phosphonate-based analogues of the tetrahedral reaction intermediate (TI) in a parallel study utilizing class I (glyphosate-sensitive) and class II (glyphosate-tolerant) EPSPS. The (R)-difluoromethyl and (R)-phosphonate analogues of the TI are the most potent inhibitors of EPSPS described to date. However, we found that class II EPSPS are up to 400 times less sensitive to inhibition by these TI analogues. X-ray crystallographic data revealed that the conformational changes of active site residues observed upon inhibitor binding to the representative class I EPSPS from Escherichia coli do not occur in the prototypical class II enzyme from Agrobacterium sp. strain CP4. It appears that because the active sites of class II EPSPS do not possess the flexibility to accommodate these TI analogues, the analogues themselves undergo conformational changes, resulting in less favorable inhibitory properties. Since pathogenic microorganisms such as Staphylococcus aureus utilize class II EPSPS, we conclude that the rational design of novel EPSPS inhibitors with potential as broad-spectrum antibiotics should be based on the active site structures of class II EPSP synthases.

@-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.

@-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.

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.

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.

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.

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.

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.

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.