The synthesis of active and stable diaminopimelate analogues of the lantibiotic peptide lactocin S

Lantibiotic peptides are potent antimicrobial compounds produced by Gram-positive bacteria. They can be used in food preservation, and some also show potential for clinical applications. Unfortunately, some of these peptides can be susceptible to inactivation by oxidation of the sulfur-containing amino acid lanthionine, limiting their use. Here we describe the synthesis and testing of diaminopimelate analogues of the lantibiotic lactocin S. These analogues were designed to improve the oxidative stability of the peptide by replacing the sulfur in lanthionine with a methylene unit. Lanthionine was systematically replaced with diaminopimelate during solid-phase peptide synthesis to produce several analogues. One analogue, A-DAP lactocin S, was found to retain full biological activity in addition to displaying increased stability. This is the first time a synthetic lanthionine ring analogue of a lantibiotic has retained natural activity levels. This methodology is potentially very promising for use in producing more stable, medically relevant lantibiotics.     

Proteinstrukturen aus dem Chemie‐Baukasten. Definierte Peptidarchitekturen

This review summarizes some of the various efforts to synthesize defined secondary structures with unnatural building blocks. These molecules are intended to mimic the molecular architecture of naturally occurring biopolymers while displaying (or even improving) biological function. After a general introduction into the principles of protein structure including the concepts of hierarchy and cooperativity, several examples of the synthesis of defined secondary structures are given. In particular, β‐peptides have received considerable attention as a class of molecules with defined structural elements, such as helices and sheets. Finally, preliminary studies towards tertiary structure and biological applications of β‐peptides and semisynthetic enzymes are presented and the increased stability of β‐peptides over their α‐analogues is discussed.     

Combinatorial synthesis and directed evolution applied to the production of alpha-helix forming antimicrobial peptides analogues

Antimicrobial peptides (AMPs) are effector molecules of innate immune systems found in different groups of organisms, including microorganisms, plants, insects, amphibians and humans. These peptides exhibit several structural motifs but the most abundant AMPs assume an amphipathic alpha-helical structure. The alpha-helix forming antimicrobial peptides are excellent candidates for protein engineering leading to an optimization of their biological activity and target specificity. Nowadays several approaches are available and this review deals with the use of combinatorial synthesis and directed evolution in order to provide a high-throughput source of antimicrobial peptides analogues with enhanced lytic activity and specificity.     

Sustainable Peptide Synthesis Enabled by a Transient Protecting Group

The growing interest in synthetic peptides has prompted the development of viable methods for their sustainable production. Currently, large amounts of toxic solvents are required for peptide assembly from protected building blocks, and switching to water as a reaction medium remains a major hurdle in peptide chemistry. We report an aqueous solid‐phase peptide synthesis strategy that is based on a water‐compatible 2,7‐disulfo‐9‐fluorenylmethoxycarbonyl (Smoc) protecting group. This approach enables peptide assembly under aqueous conditions, real‐time monitoring of building block coupling, and efficient postsynthetic purification. The procedure for the synthesis of all natural and several non‐natural Smoc‐protected amino acids is described, as well as the assembly of 22 peptide sequences and the fundamental issues of SPPS, including the protecting group strategy, coupling and cleavage efficiency, stability under aqueous conditions, and crucial side reactions.     

Tryptophan in Multicomponent Petasis Reactions for Peptide Stapling and Late-Stage Functionalisation

Peptide stapling is a robust strategy for generating enzymatically stable, macrocyclic peptides. The incorporation of biologically relevant tags (such as cell-penetrating motifs or fluorescent dyes) into peptides, while preserving their binding interactions and enhancing their stability, is highly sought after. Despite the unique opportunities offered by tryptophan‘s indole scaffold for targeted functionalisation, its utilisation in peptide stapling has been limited as compared to other amino acids. Herein, we present an approach for peptide stapling using the tryptophan-mediated Petasis reaction. This method enables the synthesis of both stapled and labelled peptides and is applicable to both solution and solid-phase synthesis. Importantly, the use of the Petasis reaction in combination with tryptophan facilitates the formation of stapled peptides in a straightforward, multicomponent fashion, while circumventing the formation of undesired by-products. Furthermore, this approach allows for efficient and diverse late-stage peptide modifications, thereby enabling rapid production of numerous conjugates for biological and medicinal applications.     

Perfluoroaryl Bicyclic Cell‐Penetrating Peptides for Delivery of Antisense Oligonucleotides

Exon‐skipping antisense oligonucleotides are effective treatments for genetic diseases, yet exon‐skipping activity requires that these macromolecules reach the nucleus. While cell‐penetrating peptides can improve delivery, proteolytic instability often limits efficacy. It is hypothesized that the bicyclization of arginine‐rich peptides would improve their stability and their ability to deliver oligonucleotides into the nucleus. Two methods were introduced for the synthesis of arginine‐rich bicyclic peptides using cysteine perfluoroarylation chemistry. Then, the bicyclic peptides were covalently linked to a phosphorodiamidate morpholino oligonucleotide (PMO) and assayed for exon skipping activity. The perfluoroaryl cyclic and bicyclic peptides improved PMO activity roughly 14‐fold over the unconjugated PMO. The bicyclic peptides exhibited increased proteolytic stability relative to the monocycle, demonstrating that perfluoroaryl bicyclic peptides are potent and stable delivery agents.     

Beta-amino acid-containing hybrid peptides--new opportunities in peptidomimetics

Hybrid peptides consisting of alpha-amino acids with judiciously placed beta-amino acids show great promise as peptidomimetics in an increasing range of therapeutic applications. This reflects a combination of increased stability, high specificity and relative ease of synthesis.     

Synthesis of functionalized rab GTPases by a combination of solution- or solid-phase lipopeptide synthesis with expressed protein ligation

Prenylated proteins with non-native functionalities are generally very difficult to obtain by recombinant or enzymatic means. The semisynthesis of preparative amounts of prenylated Rab guanosine triphosphatases (GTPases) from recombinant proteins and synthetic prenylated peptides depends largely on the availability of functionalised prenylated peptides corresponding to the proteins' native structure or modifications thereof. Here, we describe and compare solution-phase and solid-phase strategies for the generation of peptides corresponding to the prenylated C terminus of Rab7 GTPase. The solid-phase with utilisation of a hydrazide linker emerges as the more favourable approach. It allows a fast and practical synthesis of pure peptides and gives a high degree of flexibility in their modification. To facilitate the analysis of semisynthetic proteins, the synthesised peptides were equipped with a fluorescent group. Using the described approach, we introduced fluorophores at several different positions of the Rab7 C terminus. The position of the incorporated fluorescent groups in the peptides did not influence the protein-ligation reaction, as the generated peptides could be ligated onto thioester-tagged Rab7. However, it was found that the positioning of the fluorescent group had an influence on the functionality of the Rab7 proteins; analysis of the interaction of the semisynthetic Rab7 proteins with REP (Rab escort protein) and GDI (guanosine diphosphate dissociation inhibitor) molecules revealed that modification of the peptide side chains or of the C-terminal isoprenoid did not significantly interfere with complex formation. However, functionalisation of the C terminus was found to have an adverse effect on complex formation and stability, possibly reflecting low structural flexibility of the Rab GDI/REP molecules in the vicinity of the lipid-binding site.     

Fmoc-based synthesis of peptide alpha-thioesters using an aryl hydrazine support

C-Terminal peptide thioesters are key intermediates in the synthesis/semisynthesis of proteins and of cyclic peptides by native chemical ligation. They are prepared by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. Until recently, the chemical synthesis of C-terminal alpha-thioester peptides by SPPS was largely restricted to the use of Boc/Benzyl chemistry due to the poor stability of the thioester bond to the basic conditions required for the deprotection of the N(alpha)-Fmoc group. In the present work, we describe a new method for the SPPS of C-terminal thioesters using Fmoc/t-Bu chemistry. This method is based on the use of an aryl hydrazine linker, which is totally stable to conditions required for Fmoc-SPPS. When the peptide synthesis has been completed, activation of the linker is achieved by mild oxidation. This step converts the acyl hydrazine group into a highly reactive acyl diazene intermediate which reacts with an alpha-amino acid alkyl thioester (H-AA-SR) to yield the corresponding peptide alpha-thioester in good yield. This method has been successfully used to prepare a variety of peptide thioesters, cyclic peptides, and a fully functional Src homology 3 (SH3) protein domain.     

A Diaminodiacid (DADA) Strategy for the Development of Disulfide Surrogate Peptides

Disulfide bond‐containing peptides are useful molecular scaffolds with diagnostic and therapeutic applications due to their good biological activity and good target selectivity, but their utility is sometimes limited by the lability of the disulfide moiety under reducing conditions and in the presence of disulfide bond isomerase. The development of disulfide surrogates with improved redox stability has been an area of ongoing research; and one possible strategy is based on a diaminodiacid (DADA) moiety, which can be used to synthesize the disulfide bond replacement peptides with precise structures and enhanced stability through automated solid‐phase peptide synthesis (SPPS). This review summarizes recent developments in the DADA‐based SPPS of peptide disulfide surrogates. Some representative applications and structural studies on the DADA‐based disulfide surrogates are described.     

Solid-Phase Peptide Modification via Deaminative Photochemical Csp3-Csp3 Bond Formation Using Katritzky Salts

Introduction of unnatural amino acids can significantly improve the binding affinity and stability of peptides. Commercial availability of such amino acids is limited, and their synthesis is a long and tedious process. We here describe a method that allows the functionalization of peptides directly on solid-support by converting lysine residues to Katritzky salts, and subjecting them to a photochemical Giese reaction under mild reaction conditions. The method avoids the need for amino acid synthesis and instead offers a late-stage modification route for rapid peptide diversification. While numerous modification approaches at the lysine amine have been described, this work provides the first example of deaminative functionalization of peptides at lysine. The two-step protocol is compatible with various substrates, lysine analogues, resins, and all proteinogenic amino acids. Finally, by leveraging solid-phase modification, this protocol facilitates the functionalization of longer peptides as was demonstrated using biologically relevant peptides of up to 15 amino acids.     

Fluorous (trimethylsilyl)ethanol: a new reagent for carboxylic acid tagging and protection in peptide synthesis

Starting with a fluorous analogue of 2-(trimethylsilyl)ethanol, we have designed an easy method for preparing a new fluorous tag ((F)TMSE) for the protection of carboxylic acids. Because mild conditions are employed in the tag cleavage (TBAF in the presence of 4 A molecular sieves, which prevent racemization), this tag can be advantageously used in the synthesis of peptides and modified peptides, as we have demonstrated with several examples, including the fluorous synthesis of short alpha- and beta-peptides as well as of modified fluorinated retropeptides.     

Self-assembled heterotrimeric collagen triple helices directed through electrostatic interactions

Collagen, a fibrous protein, is an essential structural component of all connective tissues such as cartilage, bones, ligaments, and skin. Type I collagen, the most abundant form, is a heterotrimer assembled from two identical alpha1 chains and one alpha2 chain. However, most synthetic systems have addressed homotrimeric triple helices. In this paper we examine the stability of several heterotrimeric collagen-like triple helices with an emphasis on electrostatic interactions between peptides. We synthesize seven 30 amino acid peptides with net charges ranging from -10 to +10. These peptides were mixed, and their ability to form heterotrimers was assessed. We successfully show the assembly of five different AAB heterotrimers and one ABC heterotrimer. The results from this study indicate that intermolecular electrostatic interactions can be utilized to direct heterotrimer formation. Furthermore, amino acids with poor stability in collagen triple helices can be "rescued" in heterotrimers containing amino acids with known high triple helical stability. This mechanism allows collagen triple helices to have greater chemical diversity than would otherwise be allowed.     

Peptide macrocyclization through amide-to-amide transpeptidation

Cyclic cysteine peptides are peptide macrocycles endowed with enhanced metabolic stability and potentially, with membrane permeability. They have attracted attention in drug design and interest in their synthesis. The chemical approach for macrocyclization through transpeptidation bears striking similarity to the biological approach using an intein. Both use a similar design of thioester precursors and an amide-to-amide transpeptidation scheme, employing a series of acyl shifts to break and make amide bonds. Here we describe the synthesis of two cyclic cysteine peptides, hedyotide B1 and sunflower trypsin inhibitor-1, highlighting the similarities between the intein-based and chemical amide-to-amide schemes. In our intein-based and chemical schemes, we employed an intein Mxe or a thioethylbutylamido linkage at the C-terminus of their linear precursors, respectively. Our results demonstrated that the chemical approach provides a useful alternative to the intein approach with high efficiency.     

Use of intein-directed peptide biosynthesis to improve serum stability and bioactivity of a gelatinase inhibitory peptide

Screening of phage display libraries allows rapid identification of peptides binding to a target. However, functional analysis of the phage sequences and their reproduction as soluble and stable peptides are often the most time-consuming part in the screening. We have used here intein-based peptide biosynthesis to produce a phage-display derived gelatinase inhibitory peptide CTTHWGFTLC and to identify the critical residues for gelatinase inhibitory activity by performing alanine-scanning mutagenesis. By biosynthetic incorporation of 5-fluorotryptophan, we obtained an inhibitor of MMP-2 and MMP-9 gelatinases that showed a 6-fold enhancement in serum stability in comparison to the wild-type peptide. The new peptide also had an improved ability to inhibit tumor cell migration. These studies indicate the utility of intein methodology for synthesis and design of peptides obtained by phage display.     

Synthesis of cyclic endiamino peptides

Several building blocks for endiamino peptides, as well as several cyclic endiamino peptides themselves, and pyrazin-6-one, which embodies the endiamino group, were prepared. The variety of synthesized compounds shows the potential of this synthesis in the preparation of many different groups of compounds.     

Femtomolar Ln(III) affinity in peptide-based ligands containing unnatural chelating amino acids

The incorporation of unnatural chelating amino acids in short peptide sequences leads to lanthanide-binding peptides with a higher stability than sequences built exclusively from natural residues. In particular, the hexadentate peptide P(22), which incorporates two unnatural amino acids Ada(2) with aminodiacetate chelating arms, showed picomolar affinity for Tb(3+). To design peptides with higher denticity, expected to show higher affinity for Ln(3+), we synthesized the novel unnatural amino acid Ed3a(2) which carries an ethylenediamine triacetate side-chain and affords a pentadentate coordination site. The synthesis of the derivative Fmoc-Ed3a(2)(tBu)(3)-OH, with appropriate protecting groups for direct use in the solid phase peptide synthesis (Fmoc strategy), is described. The two high denticity peptides P(HD2) (Ac-Trp-Ed3a(2)-Pro-Gly-Ada(2)-Gly-NH(2)) and P(HD5) (Ac-Trp-Ada(2)-Pro-Gly-Ed3a(2)-Gly-NH(2)) led to octadentate Tb(3+) complexes with femtomolar stability in water. The position of the high denticity amino acid Ed3a(2) in the hexapeptide sequence appears to be critical for the control of the metal complex speciation. Whereas P(HD5) promotes the formation of polymetallic species in excess of Ln(3+), P(HD2) forms exclusively the mononuclear complex. The octadentate coordination of Tb(3+) by both P(HD) leads to total dehydration of the metal ion in the mononuclear complexes with long luminescence lifetimes (>2 ms). Hence, we demonstrated that unnatural amino acids carrying polyaminocarboxylate side-chains are interesting building blocks to design high affinity Ln-binding peptides. In particular the novel peptide P(HD2) forms a unique octadentate Tb(3+) complex with femtomolar stability in water and an improvement of the luminescence properties with respect to the trisaquo TbP(22) complex by a factor of 4.     

The Facile Synthesis of Dialkyl 1-Aminoalkylphosphonates

During past several years we have been engaged in the synthesis of phosphono peptides, peptide analogues with phosphonic acid replacing C-terminal carboxylate moiety. They became increasingly important since they appeared useful as carriers of toxic aminoalkylphosphonic acids through bacterial cell wall^1–4 or into plant tissues.^1,5 The most succesful method for the synthesis of these peptides is the condensation of N-blocked amino acids with dialkyl 6,7 or diphenyl 8.9 esters of aminoalkylphosphonic acids followed by removal of protecting groups.     

End-stapled homo and hetero collagen triple helices: a click chemistry approach

A CuAAC reaction was established for modular synthesis of end-stapled homo- and hetero-triple helical peptides, generating "clicked" macro-assemblies with enhanced thermal stability.     

Substituted diaryldiazomethanes and diazofluorenes: structure, reactivity and stability

The synthesis of several substituted diaryldiazomethanes and diazofluorenes, and an assessment of their structure, reactivity and stability, is reported.