Differently Glycosidated 2‐Amino‐2‐deoxy‐d‐glucopyranosiduronic Acids as Building Blocks in Peptide Synthesis

Seven differently glycosidated sugar amino acids (SSAs) derived from glucosamine have been prepared. Following standard solution‐phase peptide‐coupling procedures, the glycosidated 2‐amino‐2‐deoxy‐D ‐glucopyranosiduronic acids were condensed with natural amino acids to furnish useful heterodi‐ and ‐trimeric building blocks to be used in peptide synthesis. Combinations of these building blocks yielded hetero‐oligomeric peptides with two sugar amino acid units in different distances to each other. These were prepared to evaluate the influence of glycosidic side chains on the peptide backbone. Conformations of selected examples were examined by means of ROESY spectroscopy in combination with molecular dynamics (MD) simulations and circular‐dichroism (CD) studies.     

Quaternary β2,2‐Amino Acids: Catalytic Asymmetric Synthesis and Incorporation into Peptides by Fmoc‐Based Solid‐Phase Peptide Synthesis

β‐Amino acid incorporation has emerged as a promising approach to enhance the stability of parent peptides and to improve their biological activity. Owing to the lack of reliable access to β^2,2‐amino acids in a setting suitable for peptide synthesis, most contemporary research efforts focus on the use of β³‐ and certain β^2,3‐amino acids. Herein, we report the catalytic asymmetric synthesis of β^2,2‐amino acids and their incorporation into peptides by Fmoc‐based solid‐phase peptide synthesis (Fmoc‐SPPS). A quaternary carbon center was constructed by the palladium‐catalyzed decarboxylative allylation of 4‐substituted isoxazolidin‐5‐ones. The N?O bond in the products not only acts as a traceless protecting group for β‐amino acids but also undergoes amide formation with α‐ketoacids derived from Fmoc‐protected α‐amino acids, thus providing expeditious access to α‐β^2,2‐dipeptides ready for Fmoc‐SPPS.     

Syntheses and CD‐Spectroscopic Investigations of Longer‐Chain β‐Peptides: Preparation by Solid‐Phase Couplings of Single Amino Acids,Dipeptides, and Tripeptides

The synthesis and CD‐spectroscopic analysis of eleven water‐soluble β‐peptides composed of all‐β³ or alternating β²‐ and β³‐amino acids is described. Different approaches for the efficient syntheses of longer‐chain β‐peptides (>9 residues) were investigated. They were synthesized on solid phase with Fmoc‐protected amino acids or Fmoc‐protected di‐ or tripeptide fragments (assembled using solution‐phase synthesis). The use of preformed fragments significantly increased the purity of the crude peptides and facilitated purification. Especially, the use of Fmoc‐protected β²/β³‐dipeptides for the synthesis of a ‘mixed' β²/β³‐nonapeptide proved to be remarkably effective, yielding the crude peptide in 95% purity and without detectable epimerization of the β²‐amino acid residues. This is a significant improvement over previously reported procedures for the solid‐phase synthesis of β‐peptides, and foreshadows that the field of β‐peptide research will now switch from synthesis to the design and study of complex functional ‘β‐proteins'.     

Heavy‐Atom Labeled Transmembrane β‐Peptides: Synthesis,CD‐Spectroscopy,and X‐ray Diffraction Studies in Model Lipid Multilayer

Transmembrane β‐peptides are promising candidates for the design of well‐controlled membrane anchors in lipid membranes. Here, we present the synthesis of transmembrane β‐peptides with and without tryptophan anchors, as well as a novel iodine‐labeled d ‐β³‐amino acid. By using one or more of the heavy‐atom labeled amino acids as markers, the orientation of the helical peptide was inferred based on the electron‐density profile determined by X‐ray reflectivity. The β‐peptides were synthesized through manual Fmoc‐based solid‐phase peptide synthesis (SPPS) and reconstituted in unilamellar vesicles forming a right‐handed 3₁₄‐helix secondary structure, as shown by circular dichroism spectroscopy. We then integrated the β‐peptide into solid‐supported membrane stacks and carried out X‐ray reflectivity and grazing incidence small‐angle X‐ray scattering to determine the β‐peptide orientation and its effect on the membrane bilayers. These β‐peptides adopt a well‐ordered transmembrane motif in the solid‐supported model membrane, maintaining the basic structure of the original bilayer with some distinct alterations. Notably, the helical tilt angle, which accommodates the positive hydrophobic mismatch, induces a tilt of the acyl chains. The tilted chains, in turn, lead to a membrane thinning effect.     

Butelase‐Mediated Macrocyclization of d‐Amino‐Acid‐Containing Peptides

Macrocyclic compounds have received increasing attention in recent years. With their large surface area, they hold promise for inhibiting protein–protein interactions, a chemical space that was thought to be undruggable. Although many chemical methods have been developed for peptide macrocyclization, enzymatic methods have emerged as a promising new economical approach. Thus far, most enzymes have been shown to act on l ‐peptides; their ability to cyclize d ‐amino‐acid‐containing peptides has rarely been documented. Herein we show that macrocycles consisting of d ‐amino acids, except for the Asn residue at the ligating site, were efficiently synthesized by butelase 1, an Asn/Asp‐specific ligase. Furthermore, by using a peptide‐library approach, we show that butelase 1 tolerates most of the d ‐amino acid residues at the P1′′ and P2′′ positions.     

Selection of New Sequence‐Selective Unnatural Peptides Binding to Double‐Stranded Deoxyribonucleic Acids (dsDNA) by Means of a Gel‐Retardation Experiment for Library Analysis

Previously, we developed a methodology for the solid‐phase screening of peptide libraries for interaction with double‐stranded deoxyribonucleic acids (dsDNA). In the search for new and more‐potent DNA ligands, we investigated the strategy of solution‐phase screening of chemical libraries consisting of unnatural oligopeptides. After synthesis of the selected amino acid building blocks, libraries were constructed with the general structure Ac‐Arg‐Ual‐Sar‐X¹‐X²‐X³‐Arg‐NH₂, where X represents each of twelve unnatural or natural amino acids. Optimization of the sequence of binding peptides was performed with an iterative deconvolution procedure. Selection of interacting peptides was carried out in solution by means of gel‐retardation experiments, starting with libraries of 144 compounds. A 14‐base‐pair double‐stranded DNA fragment was chosen as the target. After several cycles of synthesis and screening of libraries and individual peptides, an oligopeptide was selected with an apparent dissociation constant of 9⋅10⁻⁵ M , as determined by gel‐retardation experiments. This peptide was studied by NMR spectroscopy. A certain degree of conformational pre‐organization of the peptides was shown by temperature‐dependent circular‐dichroism experiments. Finally, DNase‐I‐footprinting studies indicated a preferential interaction with a 6‐base‐pair mixed sequence 5′‐CTGCAT‐3′. This study demonstrates that gel‐shift experiments can be used for the solution‐phase screening of library mixtures of peptides against dsDNA. In general, this technique allows the selection of new sequence‐selective dsDNA‐interacting molecules. Furthermore, novel dsDNA‐binding unnatural oligopeptides were developed with affinities in the 0.1 mM range.     

Flow‐Through Synthesis on Teflon‐Patterned Paper To Produce Peptide Arrays for Cell‐Based Assays

A simple method is described for the patterned deposition of Teflon on paper to create an integrated platform for parallel organic synthesis and cell‐based assays. Solvent‐repelling barriers made of Teflon‐impregnated paper confine organic solvents to specific zones of the patterned array and allow for 96 parallel flow‐through syntheses on paper. The confinement and flow‐through mixing significantly improves the peptide yield and simplifies the automation of this synthesis. The synthesis of 100 peptides ranging from 7 to 14 amino acids in length gave over 60 % purity for the majority of the peptides (>95 % yield per coupling/deprotection cycle). The resulting peptide arrays were used in cell‐based screening to identify 14 potent bioactive peptides that support the adhesion or proliferation of breast cancer cells in a 3D environment. In the future, this technology could be used for the screening of more complex phenotypic responses, such as cell migration or differentiation.     

Peptide‐Stabilized,Fluorescent Silver Nanoclusters: Solid‐Phase Synthesis and Screening

Few‐atom silver nanoclusters (AgNCs) can exhibit strong fluorescence; however, they require ligands to prevent aggregation into larger nanoparticles. Fluorescent AgNCs in biopolymer scaffolds have so far mainly been synthesized in solution, and peptides have only found limited use compared to DNA. Herein, we demonstrate how solid‐phase methods can increase throughput dramatically in peptide ligand screening and in initial evaluation of fluorescence intensity and chemical stability of peptide‐stabilized AgNCs (P‐AgNCs). 9‐Fluorenylmethyloxycarbonyl (Fmoc) solid‐phase peptide synthesis on a hydroxymethyl‐benzoic acid (HMBA) polyethylene glycol polyacrylamide copolymer (PEGA) resin enabled on‐resin screening and evaluation of a peptide library, leading to identification of novel peptide‐stabilized, fluorescent AgNCs. Using systematic amino acid substitutions, we synthesized and screened a 144‐member library. This allowed us to evaluate the effect of length, charge, and Cys content in peptides used as ligands for AgNC stabilization. The results of this study will enable future spectroscopic studies of these peptide‐stabilized AgNCs for bioimaging and other applications.     

Synthesis of Histidine‐Containing Oligopeptides via Histidine‐Promoted Peptide Ligation

Histidine‐containing peptides are valuable therapeutic agents for a treatment of neurodegenerative diseases. However, the synthesis of histidine‐containing peptides is not trivial due to the potential of imidazole sidechain of histidine to act as a nucleophile if unprotected. A peptide ligation method utilizing the imidazole sidechain of histidine has been developed. The key imidazolate intermediate that acts as an internal acyl transfer catalyst during ligation is generated by deprotonation. Transesterification with amino acids or peptides tethered with C‐terminal thioester followed by N→N acyl shifts led to the final ligated products. A range of histidine‐containing dipeptides could be synthesized in moderate to good yields via this method without protecting the imidazole sidechain. The protocol was further extended to tripeptide synthesis via a long‐range N→N acyl transfer, and tetrapeptide synthesis.     

Radical Stability Directs Electron Capture and Transfer Dissociation of β‐Amino Acids in Peptides

We report on the characteristics of the radical‐ion‐driven dissociation of a diverse array of β‐amino acids incorporated into α‐peptides, as probed by tandem electron‐capture and electron‐transfer dissociation (ECD/ETD) mass spectrometry. The reported results demonstrate a stronger ECD/ETD dependence on the nature of the amino acid side chain for β‐amino acids than for their α‐form counterparts. In particular, only aromatic (e.g., β‐Phe), and to a substantially lower extent, carbonyl‐containing (e.g., β‐Glu and β‐Gln) amino acid side chains, lead to N? C_β bond cleavage in the corresponding β‐amino acids. We conclude that radical stabilization must be provided by the side chain to enable the radical‐driven fragmentation from the nearby backbone carbonyl carbon to proceed. In contrast with the cleavage of backbones derived from α‐amino acids, ECD of peptides composed mainly of β‐amino acids reveals a shift in cleavage priority from the N? C_β to the C_α? C bond. The incorporation of CH₂ groups into the peptide backbone may thus drastically influence the backbone charge solvation preference. The characteristics of radical‐driven β‐amino acid dissociation described herein are of particular importance to methods development, applications in peptide sequencing, and peptide and protein modification (e.g., deamidation and isomerization) analysis in life science research.     

Synthesis and NMR elucidation of novel pentacycloundecane‐based peptides

The synthesis and NMR elucidation of two novel pentacycloundecane (PCU)‐based peptides are reported. The PCU cage amino acids were synthesised as racemates and the incorporation of the cage amino acid with (S)‐natural amino acids produced diastereomeric peptides. The diastereomeric ‘cage’ peptides were separated using preparative HPLC and the NMR elucidation of these PCU containing peptides are reported for the first time. The ¹H and ¹³C NMR spectra showed series of overlapping signals of the cage skeleton and that of the peptide, making it extremely difficult to resolve the structure using one‐dimensional NMR techniques only. The use of two‐dimensional NMR techniques proved to be a highly effective tool in overcoming this problem. Copyright © 2010 John Wiley & Sons, Ltd.     

Novel N‐(2,2‐Dimethyl‐2H‐azirin‐3‐yl)‐L‐prolinates as Aib‐Pro Synthons

The syntheses of phenacyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate and allyl N‐(2,2‐dimethyl‐2H‐azirin‐3‐yl)‐L ‐prolinate are reported. Reactions of these 2H‐azirin‐3‐amine derivatives with Z‐protected amino acids have shown them to be suitable synthons for the Aib‐Pro unit in peptide synthesis. After incorporation into the peptide by means of the ‘azirine/oxazolone method’, the C‐termini of the resulting peptides were deprotected selectively with Zn in AcOH or by a mild Pd⁰‐promoted procedure, respectively.     

One‐Pot/Sequential Native Chemical Ligation Using N‐Sulfanylethylanilide Peptide

N‐Sulfanylethylanilide (SEAlide) peptides were developed with the aim of achieving facile synthesis of peptide thioesters by 9‐fluorenylmethyloxycarbonyl (Fmoc)‐based solid‐phase peptide synthesis (Fmoc SPPS). Initially, SEAlide peptides were found to be converted to the corresponding peptide thioesters under acidic conditions. However, the SEAlide moiety was proved to function as a thioester in the presence of phosphate salts and to participate in native chemical ligation (NCL) with N‐terminal cysteinyl peptides, and this has served as a powerful protein synthesis methodology. The reactivity of a SEAlide peptide (anilide vs. thioester) can be easily tuned with or without the use of phosphate salts. This interesting property of SEAlide peptides allows sequential three‐fragment or unprecedented four‐fragment ligation for efficient one‐pot peptide/protein synthesis. Furthermore, dual‐kinetically controlled ligation, which enables three peptide fragments simultaneously present in the reaction to be ligated in the correct order, was first achieved using a SEAlide peptide. Beyond our initial expectations, SEAlide peptides have served in protein chemistry fields as very useful crypto‐peptide thioesters. DOI 10.1002/tcr.201200007     

Incorporation of β‐Silicon‐β3‐Amino Acids in the Antimicrobial Peptide Alamethicin Provides a 20‐Fold Increase in Membrane Permeabilization

Incorporation of silicon‐containing amino acids in peptides is known to endow the peptide with desirable properties such as improved proteolytic stability and increased lipophilicity. In the presented study, we demonstrate that incorporation of β‐silicon‐β3‐amino acids into the antimicrobial peptide alamethicin provides the peptide with improved membrane permeabilizing properties. A robust synthetic procedure for the construction of β‐silicon‐β3‐amino acids was developed and the amino acid analogues were incorporated into alamethicin at different positions of the hydrophobic face of the amphipathic helix by using SPPS. The incorporation was shown to provide up to 20‐fold increase in calcein release as compared with wild‐type alamethicin.     

Poly(styrene‐co‐glycerol dimethacrylate): Synthesis,characterization, and application as a resin for gel‐phase peptide synthesis

An efficient cross‐linked polymer support for solid‐phase synthesis was prepared by introducing glycerol dimethacrylate cross‐linker to polystyrene network using free radical aqueous suspension polymerization. The support was characterized by various spectroscopic methods. Morphological feature of the resin was analyzed by microscopy. The polymerization reaction was investigated with respect to the effect of amount of cross‐linking agent, which in turn vary the swelling, loading, and the mechanical stability of the resin. The solvent uptake of the polymer was studied in relation to cross‐linking and compared with Merrifield resin. The stability of the resin was tested in different synthetic conditions used for solid‐phase peptide synthesis. Hydroxy group of the support was derivatized to chloro and then amino groups using different reagents and reaction conditions. Efficiency of the support was tested and compared with TentaGel? resin by following different steps involved in the synthesis of the 65–74 fragment of acyl carrier protein. The results showed that the poly(styrene‐co‐glycerol dimethacrylate) (GDMA‐PS) is equally efficient as TentaGel resin in peptide synthesis. The purity of the peptides was analyzed by HPLC and identities were determined by mass spectroscopy and amino acid analysis. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 4382–4392, 2005     

Bioorthogonal Diversification of Peptides through Selective Ruthenium(II)‐Catalyzed C–H Activation

Methods for the chemoselective modification of amino acids and peptides are powerful techniques in biomolecular chemistry. Among other applications, they enable the total synthesis of artificial peptides. In recent years, significant momentum has been gained by exploiting palladium‐catalyzed cross‐coupling for peptide modification. Despite major advances, the prefunctionalization elements on the coupling partners translate into undesired byproduct formation and lengthy synthetic operations. In sharp contrast, we herein illustrate the unprecedented use of versatile ruthenium(II)carboxylate catalysis for the step‐economical late‐stage diversification of α‐ and β‐amino acids, as well as peptides, through chemo‐selective C−H arylation under racemization‐free reaction conditions. The ligand‐accelerated C−H activation strategy proved water‐tolerant and set the stage for direct fluorescence labelling as well as various modes of peptide ligation with excellent levels of positional selectivity in a bioorthogonal fashion. The synthetic utility of our approach is further demonstrated by twofold C−H arylations for the complexity‐increasing assembly of artificial peptides within a multicatalytic C−H activation manifold.     

Solid‐phase Total Synthesis of (−)‐Apratoxin A and Its Analogues and Their Biological Evaluation

Two approaches for the solid‐phase total synthesis of apratoxin A and its derivatives were accomplished. In synthetic route A, the peptide was prepared by the sequential coupling of the corresponding amino acids on trityl chloride SynPhase Lanterns. After cleavage from the polymer‐support, macrolactamization of 10 , followed by thiazoline formation, provided apratoxin A. This approach, however, resulted in low yield because the chemoselectivity was not sufficient for the formation of the thiazoline ring though its analogue 33 was obtained. However, in synthetic route B, a cyclization precursor was prepared by solid‐phase peptide synthesis by using amino acids 13 – 15 and 18 . The final macrolactamization was performed in solution to provide apratoxin A in high overall yield. This method was then successfully applied to the synthesis of apratoxin analogues. The cytotoxic activity of the synthetic derivatives was then evaluated. The epimer 34 was as potent as apratoxin A, and O‐methyl tyrosine can be replaced by 7‐azidoheptyl tyrosine without loss of activity. The 1,3‐dipolar cycloaddition of 38 with phenylacetylene was performed in the presence of a copper catalyst without affecting the thiazoline ring.     

Isoxazole‐Derived Amino Acids are Bromodomain‐Binding Acetyl‐Lysine Mimics: Incorporation into Histone H4 Peptides and Histone H3

A range of isoxazole‐containing amino acids was synthesized that displaced acetyl‐lysine‐containing peptides from the BAZ2A, BRD4(1), and BRD9 bromodomains. Three of these amino acids were incorporated into a histone H4‐mimicking peptide and their affinity for BRD4(1) was assessed. Affinities of the isoxazole‐containing peptides are comparable to those of a hyperacetylated histone H4‐mimicking cognate peptide, and demonstrated a dependence on the position at which the unnatural residue was incorporated. An isoxazole‐based alkylating agent was developed to selectively alkylate cysteine residues in situ. Selective monoalkylation of a histone H4‐mimicking peptide, containing a lysine to cysteine residue substitution (K12C), resulted in acetyl‐lysine mimic incorporation, with high affinity for the BRD4 bromodomain. The same technology was used to alkylate a K18C mutant of histone H3.     

Efficient Access to Enantiopure γ4‐Amino Acids with Proteinogenic Side‐Chains and Structural Investigation of γ4‐Asn and γ4‐Ser in Hybrid Peptide Helices

Hybrid peptides composed of α‐ and β‐amino acids have recently emerged as new class of peptide foldamers. Comparatively, γ‐ and hybrid γ‐peptides composed of γ⁴‐amino acids are less studied than their β‐counterparts. However, recent investigations reveal that γ⁴‐amino acids have a higher propensity to fold into ordered helical structures. As amino acid side‐chain functional groups play a crucial role in the biological context, the objective of this study was to investigate efficient synthesis of γ⁴‐residues with functional proteinogenic side‐chains and their structural analysis in hybrid‐peptide sequences. Here, the efficient and enantiopure synthesis of various N‐ and C‐terminal free‐γ⁴‐residues, starting from the benzyl esters (COOBzl) of N‐Cbz‐protected (E)‐α,β‐unsaturated γ‐amino acids through multiple hydrogenolysis and double‐bond reduction in a single‐pot catalytic hydrogenation is reported. The crystal conformations of eight unprotected γ⁴‐amino acids (γ⁴‐Val, γ⁴‐Leu, γ⁴‐Ile, γ⁴‐Thr(OtBu), γ⁴‐Tyr, γ⁴‐Asp(OtBu), γ⁴‐Glu(OtBu), and γ‐Aib) reveals that these amino acids adopted a helix favoring gauche conformations along the central C^γ? C^β bond. To study the behavior of γ⁴‐residues with functional side chains in peptide sequences, two short hybrid γ‐peptides P1 (Ac‐Aib‐γ⁴‐Asn‐Aib‐γ⁴‐Leu‐Aib‐γ⁴‐Leu‐CONH₂) and P2 (Ac‐Aib‐γ⁴‐Ser‐Aib‐γ⁴‐Val‐Aib‐γ⁴‐Val‐CONH₂) were designed, synthesized on solid phase, and their 12‐helical conformation in single crystals were studied. Remarkably, the γ⁴‐Asn residue in P1 facilitates the tetrameric helical aggregations through interhelical H bonding between the side‐chain amide groups. Furthermore, the hydroxyl side‐chain of γ⁴‐Ser in P2 is involved in the interhelical H bonding with the backbone amide group. In addition, the analysis of 87 γ⁴‐residues in peptide single‐crystals reveal that the γ⁴‐residues in 12‐helices are more ordered as compared with the 10/12‐ and 12/14‐helices.     

Design and Synthesis of Nucleoproline Amino Acids for the Straightforward Preparation of Chiral and Conformationally Constrained Nucleopeptides

A straightforward synthesis of orthogonally protected nucleoproline (Nup) amino acids and their coupling to oligomers are described. A key step is the attachment of alkynylated nucleobases to Fmoc‐protected 4‐azidoproline (Fmoc‐Azp‐OH) by a Cu‐catalyzed 1,3‐dipolar cycloaddition (‘click reaction’). The developed protocol allows preparation of the nucleoprolines in scales of >30 g. Solid‐phase peptide synthesis proved to be straightforward with these Nup amino acids. The resulting oligonucleoproline peptides adopt defined helices, are very well H₂O soluble, and show comparable cell‐penetrating properties as recently reported α‐nucleoalanine peptides.