N-to-C solid-phase peptide and peptide trifluoromethylketone synthesis using amino acid tert-butyl esters

Solid-phase peptide synthesis in the N-to-C direction, opposite to the classical C-to-N direction of peptide synthesis, provides the synthetically versatile C-terminal carboxyl group for further modification into C-terminally modified peptide mimetics. These are of general interest as potential bioactive agents, particularly as protease inhibitors. Elaboration of peptide mimetics on the solid-phase would facilitate synthesis of peptide mimetic combinatorial libraries. This report describes an effective strategy for solid-phase inverse peptide synthesis based on readily available amino acid tert-butyl esters. The potential of this approach for peptide mimetic synthesis is demonstrated by the solid-phase synthesis of two peptide trifluoromethylketones.     

Reverse proteolysis promoted by in situ generated peptide ester fragments

In this contribution we describe a general synthesis concept for the in situ preparation of protease specific reactants using methyl thioesters as universal precursors. The precursor esters are readily available by standard synthesis procedures and can be used directly as reactants for protease-mediated peptide coupling reactions. Alternatively, they can serve as initial building blocks for the in situ preparation of various types of substrate mimetics. The synthesis of the latter is achieved by a one-pot spontaneous transthioesterification reaction of the parent thioester (Y-(Xaa)(n)-SMe-->Y-(Xaa)(n)-SR; R: CH(2)CH(2)COOH, CH(2)C(6)H(5), C(6)H(4)NHC(:NH)NH(2)), which proceeds efficiently in both a sequential manner and parallel to the subsequent enzymatic reaction. The resulting substrate mimetics act as efficient acyl donor components and show the typical behavior of substrate mimicry enabling irreversible reactions with originally nonspecific acyl moieties. Neither a workup of the substrate mimetic intermediate nor changes of the reaction conditions during the whole synthesis process are required. Model peptide syntheses using trypsin, alpha-chymotrypsin, and V8 protease as the biocatalysts proved the function of the approach and illustrated its synthetic value for protease-mediated reactions and the compatibility of the approach with state-of-the-art solid-phase peptide ester synthesis methods.     

Solid-phase synthesis of "mixed" peptidomimetics using Fmoc-protected aza-beta3-amino acids and alpha-amino acids

[structure: see text] A solid-phase fluorenylmethyloxycarbonyl (Fmoc)-based synthesis strategy is described for "mixed" aza-beta3-peptides as well as a convenient general approach for their required building blocks, the aza-beta3-amino acid residues (aza-beta3-aa). These monomers allow the synthesis of relatively large quantities of pure mixed aza-beta3-peptides. The required Fmoc-substituted aza-beta3-amino acids are accessible by convenient synthesis, and a number of monomers including those containing side chains with functional groups have been synthesized. The method was applied toward the solid-phase synthesis of aza-beta3-peptide mimetics of a biologically active histone H4 sequence.     

Design and synthesis of type-III mimetics of ShK toxin

ShK toxin is a structurally defined, 35-residue polypeptide which blocks the voltage-gated Kv1.3 potassium channel in T-lymphocytes and has been identified as a possible immunosuppressant. Our interest lies in the rational design and synthesis of type-III mimetics of protein and polypeptide structure and function. ShK toxin is a challenging target for mimetic design as its binding epitope consists of relatively weakly binding residues, some of which are discontinuous. We discuss here our investigations into the design and synthesis of 1st generation, small molecule mimetics of ShK toxin and highlight any principles relevant to the generic design of type-III mimetics of continuous and discontinuous binding epitopes. We complement our approach with attempted pharmacophore-based database mining.     

Synthesis of constrained analogues of cholecystokinin/opioid chimeric peptides

In our ongoing research on the synthesis of constrained analogues of CCK/opioid chimeric peptides, a bicyclic dipeptide mimetic for Nle-Asp was designed and synthesized. Starting from β-allyl substituted aspartic acids, the terminal double bond was oxidized resulting in spontaneous cyclization to form racemic hemiaminals. Allylation of the hemiaminals afforded 5-allyl substituted proline analogues, which on oxidation, Horner-Emmons olefination, asymmetric hydrogenation, and bicyclization afforded bicyclic dipeptide mimetics for Nle-Asp. Constrained CCK/opioid peptide analogues containing bicyclic dipeptide mimetics for Nle-Gly, Nle-Asp, and homoPhe-Gly were then synthesized and analyzed at both the CCK and opioid receptors.     

Substrate mimetics in protease catalysis: characteristics,kinetics, and synthetic utility

This article reviews the latest developments in protease-catalyzed peptide synthesis focusing on the use of substrate mimetics. The substrate mimetics approach takes advantage of the characteristic of this novel type of substrates to direct the enzyme to recognize an alternative site on the acyl donor, i.e. the site-specific ester leaving group, mediating the acceptance of originally poorly reactive acyl moieties. At first the kinetics and catalytic mechanism of substrate mimetics-mediated reactions are discussed on the basis of hydrolysis, peptide synthesis, protein-ligand docking, and molecular dynamics studies. By the example of the Glu-specific V8 protease and the aromatic amino acid-specific chymotrypsin both the empirical and computer-aided design of specific substrate mimetics is described. The influence of the leaving group specifically recognized by the enzyme is also considered. The benefits of these artificial substrates over common acyl donor components are illustrated by selected synthesis reactions of small peptides, peptide isosteres, non-peptidic carboxylic acid amides, and the coupling of peptide fragments at non-specific ligation sites resulting in biologically active peptide products. Finally, this review focuses on critical syntheses that uses specific-amino acid-containing peptides as the reactants of ligation. Based on these, the restrictions of the substrate mimetics approach is critically discussed and techniques to their overcoming are presented.     

Resin-bound sulfonyl azides: efficient loading and activation strategy for the preparation of the N-acyl sulfonamide linker

This paper describes an optimized protocol for the efficient loading of resin-bound aminoethane sulfonyl azides by either Boc- or Fmoc-protected amino thioacids. The resulting N-acyl sulfonamide is a convenient linker for use in Boc- or Fmoc-based solid-phase peptide synthesis. Activation of the N-acyl sulfonamide via a microwave-assisted alkylation procedure and subsequent treatment with functionalized nucleophiles yields C-terminally modified peptides that can be applied in chemoselective (bio)conjugation or ligation reactions.     

9-Fluorenylmethoxycarbonyl-based solid-phase synthesis of peptide α-thioesters

Peptide thioesters play a key role in convergent protein synthesis strategies such as native chemical ligation, traceless Staudinger ligation, and Ag(+) -mediated thioester ligation. The Boc-based solid-phase synthesis provides a very reliable access to peptide thioesters. However, the acid lability of many peptide modifications and the requirements of most parallel peptide synthesizers call for the milder Fmoc-based solid-phase synthesis. The Fmoc-based synthesis of peptide thioesters is more cumbersome and typically proceeds with lower yields than the synthesis of peptide acids and peptide amides. The success of native chemical ligation and related technologies has sparked intensive research effort devoted to the development of new methods. The recent progress in this rapidly expanding field is reviewed.     

Fe(III)-binding collagen mimetics

The synthesis and characterization of hydroxamic acid containing single-chain and TRIS-assembled (where TRIS is tris(carboxyethoxymethyl)aminomethane) collagen mimetics are reported. We have engineered an Fe(III)-binding domain by placing a hydroxamic acid group at the C termini of collagen mimetic chains composed of the Gly-Pro-NLeu sequence. The circular dichroism spectra and thermal denaturation studies show an enhancement in triple-helical thermal stability upon the addition of Fe(III) for the TRIS-assembled structure. No triple-helical structure was detected for the single-chain collagen mimetic. From the absorbance shown in the UV-vis spectra, we believe that the thermal stabilization of the triple helix is the direct result of a coordination complex between Fe(III) and the hydroxamate groups tethered to the C termini of the collagen mimetic peptide chains.     

Fmoc-SPPS chemistry compatible approach for the generation of (glyco)peptide aryl thioesters

A new approach is described for the general Fmoc-based solid-phase synthesis of (glyco)peptide aryl thioesters. A peptide alkyl oxoester obtained by standard Fmoc-based chain elongation undergoes an O-to-S acyl shift, and is followed by alkyl thioester exchanges with a large excess of aryl thiol, affording the corresponding peptide aryl thioester. The newly developed methodology is useful for the chemical synthesis of post-translationally modified proteins because of its compatibility with standard Fmoc-SPPS conditions. In addition, the peptide aryl thioesters are essential intermediates for chemical synthesis of proteins by kinetically controlled convergent strategy.     

New developments in the synthesis of pyrrolizidinone-based dipeptide isosteres

1,3-Dipolar cycloaddition of acrylamide with the cyclic nitrone derived from proline tert-butyl ester has been employed in the synthesis of bicyclic Gly-(s-cis)Pro isosteres suitably protected for the Fmoc-based solid phase peptide synthesis. (R)-1-Phenylethylamine was introduced as chiral auxiliary to resolve racemic intermediates and obtain enantiopure compounds. Using methacrylamide as dipolarophile, the analogous Ala-Pro mimetics have been prepared in racemic form, whereas the same strategy applied to methyl itaconate failed to give the corresponding Asp-Pro mimetic.     

Total synthesis of cyclic heptapeptide euryjanicin B

The first synthesis of the naturally occurring cyclic peptide euryjanicin B has been achieved.A general method was described to synthesize the cyclic peptide by a two-step solid-phase/solution synthesis strategy.All the amino acids in this study are L-configuration. The linear heptapeptide was assembled by standard Fmoc chemistry on solid-phase and subsequently cyclization was carried out by solution method.     

Solid-phase synthesis of N-nosyl- and N-Fmoc-N-methyl-alpha-amino acids

We report here a convenient and simple solid-phase synthesis of N-nosyl-N-methyl-alpha-amino acids and N-Fmoc-N-methyl-alpha-amino acids, important building blocks for the synthesis of conformationally restricted and protease-resistant natural peptides and peptide analogues. The methodology involves the use of 2-chlorotrityl chloride resin to temporarily protect the carboxylic group of alpha-amino acids and of diazomethane as the reagent to methylate the sulfonamidic function. The approach developed is particularly efficient also with alpha-amino acids bearing appropriately protected functionalized side chains.     

Semi-Synthesis and Analysis of Chemically Modified Zif268 Zinc-Finger Domains

Total synthesis of proteins can be challenging despite assembling techniques, such as native chemical ligation (NCL) and expressed protein ligation (EPL). Especially, the combination of recombinant protein expression and chemically addressable solid-phase peptide synthesis (SPPS) is well suited for the redesign of native protein structures. Incorporation of analytical probes and artificial amino acids into full-length natural protein domains, such as the sequence-specific DNA binding zinc-finger motifs, are of interest combining selective DNA recognition and artificial function. The semi-synthesis of the natural 90 amino acid long sequence of the zinc-finger domain of Zif268 is described including various chemically modified constructs. Our approach offers the possibility to exchange any amino acid within the third zinc finger. The realized modifications of the natural sequence include point mutations, attachment of a fluorophore, and the exchange of amino acids at different positions in the zinc finger by artificial amino acids to create additional metal binding sites. The individual constructs were analyzed by circular dichroism (CD) spectroscopy with respect to the integrity of the zinc-finger fold and DNA binding.     

Stereoselective synthesis of alpha-monofluorinated phosphonate mimetics of naturally occurring phosphoserine and phosphothreonine,via electrophilic fluorination of lithiated bis-lactim ethers

Electrophilic fluorinations of lithiated bis-lactim ethers derived from cyclo-[L-AP4-D-Val] allow a direct access to alpha-monofluorinated phosphonate mimetics of naturally occurring phosphoserine and phosphothreonine, in enantiomerically pure form and suitably protected for solid-phase peptide synthesis.     

Solid-phase synthesis of bicyclic dipeptide mimetics by intramolecular cyclization of alcohols, thiols, amines, and amides with N-acyliminium intermediates

A general strategy for the solid-phase synthesis of structurally diverse bicyclic dipeptide mimetics is presented. Depending on the amino acid side-chain (R(1)), peptide-derived N-acyliminium intermediates may undergo nucleophilic attack from either side-chain functional groups (-OH, -NH(2), -SH, and -CONH(2)) or the amide backbone (-CONH-) of the peptide, thus affording a range of aza-, thia-, and oxabicycloalkanes in excellent purity and diastereoselectivity. [reaction: see text]     

Synthesis and Solution Conformation of β‐Hairpin Mimetics Utilizing a Template Derived from (2S,3R,4R)‐Diaminoproline

A novel template was synthesized for stabilizing β‐hairpin conformations in cyclic peptide mimetics. The template is a diketopiperazine derived formally from L ‐aspartic acid and (2S,3R,4R)‐diaminoproline, the latter being available by an efficient synthetic route from vitamin C. The template was incorporated by solid‐phase peptide synthesis into a cyclic loop mimetic containing the sequence (‐Ala‐Asn‐Pro‐Asn‐Ala‐Ala‐template‐). This mimetic was shown by NMR to adopt a stable β‐hairpin conformation in (D₆)DMSO solution. The template may prove to be generally useful for creating small‐molecule mimetics of hairpin loops on proteins of diverse function.     

Convergent strategies for the attachment of fluorescing reporter groups to peptide nucleic acids in solution and on solid phase

The site-selective conjugation of peptide nucleic acids (PNA) with fluorescent reporter groups is essential for the construction of hybridisation probes that can report the presence of a particular DNA sequence. This paper describes convergent methods for the solution- and solid-phase synthesis of multiply labelled PNA oligomers. The solid-phase synthesis of protected PNA enabled the selective attachment of fluorescent labels at the C-terminal end (3' in DNA) which demonstrated that further manipulations on protected PNA fragments are feasible. For the conjugation to internal sites, a method is introduced that allows for the on-resin assembly of modified monomers thereby omitting the need to synthesise an entire monomer in solution. Furthermore, it is shown that the application of a highly orthogonal protecting group strategy in combination with chemoselective conjugation reactions provides access to a rapid and automatable solid-phase synthesis of dual labelled PNA probes. Real-time measurements of nucleic acid hybridisation were possible by taking advantage of the fluorescence resonance energy transfer (FRET) between suitably appended fluorophoric groups. Analogously to DNA-based molecular beacons, the dual labelled PNA probes were only weakly fluorescing in the single-stranded state. Hybridisation to a complementary oligonucleotide, however, induced a structural reorganisation and conferred a vivid fluorescence enhancement.     

A highly practical RCM approach towards a molecular building kit of spirocyclic reverse turn mimics

The development of privileged molecular scaffolds efficiently mimicking reverse turn motifs and thus increasing both binding and selectivity and enabling the elucidation of the bioactive conformation of a natural peptide has attracted remarkable interest. The frequent occurrence of proline in various turn patterns initiated the design of proline-based reverse turn mimetics. As a structural hybridization of a highly potent type VI beta-turn inducer 1 with saturated spirocyclic lactams 3 efficiently mimicking type II beta turns, we developed a versatile synthetic route towards unsaturated spirocyclic lactams of type 2, when Seebach's self-reproduction of chirality methodology was combined with a peptide coupling reaction and Grubbs' ring-closing metathesis. By this means, a variety of model peptides with six- up to nine-membered lactam rings were accessible following a uniform pathway. Introduction of suitably protected templates into solid-phase peptide synthesis gave rise to unsaturated spirocyclic analogues of the naturally occurring neuropeptide neurotensin. Spectroscopic investigations as well as DFT calculations on a high level of theory revealed a remarkable dependence of the reverse-turn inducing potency on the ring size. While the secondary structure of the unsaturated spirocyclic epsilon-lactam 12 closely agrees with the reference gamma-lactam 3 a, the unsaturated delta-lactam 11 serves as an extraordinarily potent beta-turn inducer which is even superior to beta-lactams of type 3 b. The eight-membered unsaturated spirocyclic lactam 13 adopts a conformation almost ideally matching the prerequisites for a canonical type II beta turn with the highest stability of the whole series. In contrast, the nine-membered spirolactam 14 represents a scaffold with a high conformational flexibility.