Characterization of a phosphorylated peptide and peptoid and peptoid-peptide hybrids by mass spectrometry

Nano-electrospray tandem mass spectrometry (nano-ES-MS/MS) was used to record collision-induced dissociation (CID) spectra of a set of peptoid-peptide hybrids and the complete peptoid derived from the phosphopeptide Ac-pTyr-Glu-Thr-Leu-NH(2) (1). The presence of B and Y'-type fragment ions in the tandem mass spectra of the protonated molecular ions [M + H](+) allowed confirmation of sequence similar to mass spectrometric sequence analysis in peptides. In the isomeric peptoid compounds studied, one or several amino acid residues were replaced by peptoid residues (N-substituted glycine residues), which resulted in characteristic tandem mass spectra with differently increased relative abundances of Y'-and B-type fragment ions. The increment of a particular Y'-ion was directly correlated to the position of a peptoid residue present. In addition to these increased peak intensities, other characteristic peaks were also observed compared with the spectrum of reference peptide 1. When a peptoid phosphotyrosine was incorporated, the presence of this residue was apparent from the occurrence of a relatively intense peak at m/z 187 representing the positively charged side-chain of phosphotyrosine, which was almost absent in the spectrum of the reference peptide 1. Since the threonine side-chain had to be translated into the homo peptoid analog this substitution was apparent from the presence of [M + H](+) and fragment ions 14 mass units higher than observed in the spectrum of the reference phosphopeptide 1. The presence of an NLeu peptoid residue could be confirmed by the specific fragmentation of the immonium ion showing an intense peak in its tandem mass spectrum at m/z 57, which results from the loss of an neutral imine molecule leading to a positively charged [C(4)H(9)](+) ion. By means of these mass spectrometric characteristics, all isomeric peptoid compounds could be distinguished from each other and characterized. The methods used appear to be very useful in future studies of peptoids and peptoid-peptide hybrids.     

Atroposelective synthesis of N-aryl peptoid atropisomers via a palladium(ii)-catalyzed asymmetric C–H alkynylation strategy

The introduction of chirality into peptoids is an important strategy to determine a discrete and robust secondary structure. However, the lack of an efficient strategy for the synthesis of structurally diverse chiral peptoids has hampered the studies. Herein, we report the efficient synthesis of a wide variety of N-aryl peptoid atropisomers in good yields with excellent enantioselectivities (up to 99% yield and 99% ee) by palladium-catalyzed asymmetric C–H alkynylation. The inexpensive and commercially available l-pyroglutamic acid was used as an efficient chiral ligand. The exceptional compatibility of the C–H alkynylation with various peptoid oligomers renders this procedure valuable for peptoid modifications. Computational studies suggested that the amino acid ligand distortion controls the enantioselectivity in the Pd/l-pGlu-catalyzed C–H bond activation step.

The introduction of chirality into peptoids is an important strategy to determine a discrete and robust secondary structure.     

Novel chiral stationary phases based on peptoid combining a quinine/quinidine moiety through a C9‐position carbamate group

By connecting a quinine or quinidine moiety to the peptoid chain through the C9‐position carbamate group, we synthesized two new chiral selectors. After immobilizing them onto 3‐mercaptopropyl‐modified silica gel, two novel chiral stationary phases were prepared. With neutral, acid, and basic chiral compounds as analytes, we evaluated these two stationary phases and compared their chromatographic performance with chiral columns based on quinine tert‐butyl carbamate and the previous peptoid. From the resolution of neutral and basic analytes under normal‐phase mode, it was found that the new stationary phases exhibited much better enantioselectivity than the quinine tert‐butyl carbamate column; the peptoid moiety played an important role in enantiorecognition, which controlled the elution orders of enantiomers; the assisting role of the cinchona alkaloid moieties was observed in some separations. Under acid polar organic phase mode, it was proved that cinchona alkaloid moieties introduced excellent enantiorecognitions for chiral acid compounds; in some separations, the peptoid moiety affected enantioseparations as well. Overall, chiral moieties with specific enantioselectivity were demonstrated to improve the performance of peptoid chiral stationary phase efficiently.     

The synthesis of amides and dipeptides from unprotected amino acids by a simultaneous protection-activation strategy using boron trifluoride diethyl etherate

The reaction of l-phenylalanine (1) with boron trifluoride diethyl etherate and primary amines leads to the formation of amides via a cyclic boron intermediate. It is also possible to use the amino dicarboxylic acid l-aspartic acid and N-alkylated amino acids (peptoid building blocks, e.g., NPhe-OH 9). The latter can be used in the preparation of dipeptidomimetics.     

A Multiple Multicomponent Approach to Chimeric Peptide–Peptoid Podands

The success of multi‐armed, peptide‐based receptors in supramolecular chemistry traditionally is not only based on the sequence but equally on an appropriate positioning of various peptidic chains to create a multivalent array of binding elements. As a faster, more versatile and alternative access toward (pseudo)peptidic receptors, a new approach based on multiple Ugi four‐component reactions (Ugi‐4CR) is proposed as a means of simultaneously incorporating several binding and catalytic elements into organizing scaffolds. By employing α‐amino acids either as the amino or acid components of the Ugi‐4CRs, this multiple multicomponent process allows for the one‐pot assembly of podands bearing chimeric peptide–peptoid chains as appended arms. Tripodal, bowl‐shaped, and concave polyfunctional skeletons are employed as topologically varied platforms for positioning the multiple peptidic chains formed by Ugi‐4CRs. In a similar approach, steroidal building blocks with several axially‐oriented isocyano groups are synthesized and utilized to align the chimeric chains with conformational constrains, thus providing an alternative to the classical peptido‐steroidal receptors. The branched and hybrid peptide–peptoid appendages allow new possibilities for both rational design and combinatorial production of synthetic receptors. The concept is also expandable to other multicomponent reactions.     

Cyclization of peptoids by formation of boronate esters

Introduction of conformational constraints into peptoids (N-substituted oligoglycines) will enable new applications in molecular recognition and self-assembly. Peptoids that contain both a phenylboronic acid side chain and a vicinal diol cyclize by intramolecular condensation to form boronate esters. A fluorescent indicator of free boronic acid was used to assay esterification. A galactose moiety 2 to 5 monomer units away from a boronic acid side chain in a peptoid reacts with the boronic acid in competition with the indicator. The intramolecular reaction predominates in each case, with 80-90% of the peptoid cyclized. When the diol is a simple 2,3-dihydroxypropyl group, esterification is less favored but still appreciable.     

Synthesis of low grafting density molecular brush from a poly(N‐alkyl urea peptoid) backbone

The synthesis of a molecular brush was accomplished by combining step‐growth polymerization and reversible addition fragmentation chain transfer (RAFT) polymerization in a “grafting from” methodology. A symmetrical N‐alkyl urea peptoid sixmer containing alkyne functional groups was prepared using a divergent strategy, and the structure of the product was confirmed using NMR spectroscopy and mass spectrometry. A step‐growth process was used to prepare a linear poly(N‐alkyl urea peptoid) by reacting the diamine‐functionalized N‐alkyl urea peptoid sixmer with a diisocyanate. RAFT chain transfer agents were coupled to the poly(N‐alkyl urea peptoid) backbone through a copper‐catalyzed azide/alkyne cycloaddition reaction. The afforded macro‐RAFT agent was used to sequentially polymerize styrene and tert‐butyl acrylate block copolymer arms from the poly(N‐alkyl urea peptoid) backbone. The tert‐butyl groups were removed using dilute trifluoroacetic acid affording hydrophilic polyacrylic acid segments. The molecular brushes were observed to generate micelles in aqueous solution. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

Improving SH3 domain ligand selectivity using a non-natural scaffold

BACKGROUND: Src homology 3 (SH3) domains bind sequences bearing the consensus motif PxxP (where P is proline and x is any amino acid), wherein domain specificity is mediated largely by sequences flanking the PxxP core. This specificity is limited, however, as most SH3 domains show high ligand cross-reactivity. We have recently shown that diverse N-substituted residues (peptoids) can replace the prolines in the PxxP motif, yielding a new source of ligand specificity. RESULTS: We have tested the effects of combining multiple peptoid substitutions with specific flanking sequences on ligand affinity and specificity. We show that by varying these different elements, a ligand can be selectively tuned to target a single SH3 domain in a test set. In addition, we show that by making multiple peptoid substitutions, high-affinity ligands can be generated that completely lack the canonical PxxP motif. The resulting ligands can potently disrupt natural SH3-mediated interactions. CONCLUSIONS: Peptide-peptoid hybrid scaffolds yield SH3 ligands with markedly improved domain selectivity, overcoming one of the principal challenges in designing inhibitors against these domains. These compounds represent important leads in the search for orthogonal inhibitors of SH3 domains, and can serve as tools for the dissection of complex signaling pathways.     

Biomimetic nanostructures: creating a high-affinity zinc-binding site in a folded nonbiological polymer

One of the long-term goals in developing advanced biomaterials is to generate protein-like nanostructures and functions from a completely nonnatural polymer. Toward that end, we introduced a high-affinity zinc-binding function into a peptoid (N-substituted glycine polymer) two-helix bundle. Borrowing from well-understood zinc-binding motifs in proteins, thiol and imidazole moieties were positioned within the peptoid such that both helices must align in close proximity to form a binding site. We used fluorescence resonance energy transfer (FRET) reporter groups to measure the change of the distance between the two helical segments and to probe the binding of zinc. We systematically varied the position and number of zinc-binding residues, as well as the sequence and size of the loop that connects the two helical segments. We found that certain peptoid two-helix bundles bind zinc with nanomolar affinities and high selectivity compared to other divalent metal ions. Our work is a significant step toward generating biomimetic nanostructures with enzyme-like functions.     

Peptoids bearing tertiary amino residues in the n-alkyl side chains: synthesis of a potent inhibitor of Semaphorin 3A

A study on the preparation of N-alkylglycines (peptoids) that contain tertiary amino residues on the N-alkyl side chains is reported. The appropriate combination of the submonomer strategy with N-alkylglycine monomer couplings depending upon the structure of the N-alkyl side chain that must be incorporated into the peptoid is determinant for the efficiency of the synthetic pathway. The application of this strategy to the preparation of SICHI, an N-alkyglycine trimer containing tertiary amino residues in the three N-alkyl branches, and that has been identified as a potent Semaphorin 3A inhibitor, is presented.     

Rapid generation of macrocycles with natural-product-like side chains by multiple multicomponent macrocyclizations (MiBs)

A small parallel library of peptoid macrocycles with natural-product-derived side chains of biological importance was produced by Ugi-type multiple multicomponent macrocyclizations including bifunctional building blocks (Ugi-MiBs). Diverse exocyclic elements of high relevance in natural recognition processes, i.e., all functional amino acid residues (e.g., Cys, Arg, His, Trp) and even sugar moieties, can be introduced in a one-pot process into different types of peptoid-containing macrocyclic skeletons. This is exemplified by the use of a diamine/diisocyanide combination of Ugi-MiBs and N-protected alpha-amino acids or carboxy-functionalized carbohydrates as source for the natural-product-like exocyclic elements. Employed as the acid components of the multiple Ugi reactions, they appear as N-amide substituents on the macrocyclic cores. The use of different diamines and diisocyanides allows an easy variation of the N- to C-directionality and therefore of the position of the exocyclic elements.     

Tuning peptoid secondary structure with pentafluoroaromatic functionality: a new design paradigm for the construction of discretely folded peptoid structures

Peptoids, or oligomers of N-substituted glycine, are an important class of non-native polymers whose close structural similarity to natural alpha-peptides and ease of synthesis offer significant advantages for the study of biomolecular interactions and the development of biomimetics. Peptoids that are N-substituted with alpha-chiral aromatic side chains have been shown to adopt either helical or "threaded loop" conformations, depending upon solvent and oligomer length. Elucidation of the factors that impact peptoid conformation is essential for the development of general rules for the design of peptoids with discrete and novel structures. Here, we report the first study of the effects of pentafluoroaromatic functionality on the conformational profiles of peptoids. This work was enabled by the synthesis of a new, alpha-chiral amine building block, (S)-1-(pentafluorophenyl)ethylamine (S-2), which was found to be highly compatible with peptoid synthesis (delivering (S)-N-(1-(pentafluorophenyl)ethyl)glycine oligomers). The incorporation of this fluorinated monomer unit allowed us to probe both the potential for pi-stacking interactions along the faces of peptoid helices and the role of side chain electrostatics in peptoid folding. A series of homo- and heteropeptoids derived from S-2 and non-fluorinated, alpha-chiral aromatic amide side chains were synthesized and characterized by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. Enhancement of pi-stacking by quadrupolar interactions did not appear to play a significant role in stabilizing the conformations of heteropeptoids with alternating fluorinated and non-fluorinated side chains. However, incorporation of (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomers enforced helicity in peptoids that typically exhibit threaded loop conformations. Moreover, we found that the incorporation of a single (S)-N-(1-(pentafluorophenyl)ethyl)glycine monomer could be used to selectively promote looped or helical structure in this important peptoid class by tuning the electronics of nearby heteroatoms. The strategic installation of this monomer unit represents a new approach for the manipulation of canonical peptoid structure and the construction of novel peptoid architectures.     

A threaded loop conformation adopted by a family of peptoid nonamers

Non-natural polymers with well-defined three-dimensional folds offer considerable potential for engineering novel functions that are outside the scope of biological polymers. Here we describe a family of N-substituted glycine or "peptoid" nonamers that folds into an unusual "threaded loop" structure of exceptional thermal stability and conformational homogeneity in acetonitrile. The structure is chain-length-specific and relies on bulky, chiral side chains and chain-terminating functional groups for stability. Notable elements of the structure include the engagement of the positively charged amino terminus by carbonyl groups of the backbone through hydrogen bonding interactions and shielding of polar groups from and near-complete exposure of hydrophobic groups to solvent, in a manner resembling a folded polypeptide globular domain turned inside-out. The structure is stable in a variety of organic solvents but is readily denatured in any solvent/cosolvent milieu with hydrogen bonding potential. The structure could serve as a scaffold for the elaboration of novel functions and could be used to test methodologies for predicting solvent-dependent polymer folding.     

Cyclic peptoids

Foldamers are an intriguing family of biomimetic oligomers that exhibit a propensity to adopt stable secondary structures. N-Substituted glycine oligomers, or "peptoids", are a prototypical example of these foldamer systems and are known to form a helix resembling that of polyproline type I. Ongoing studies seek to improve the stability of peptoid folding and to discover new secondary structure motifs. Here, we report that peptoids undergo highly efficient head-to-tail macrocyclization reactions. A diverse array of peptoid sequences from pentamers to 20mers were converted to macrocyclic products within 5 min at room temperature. The introduction of the covalent constraint enhances conformational ordering, allowing for the crystallization of a cyclic peptoid hexamer and octamer. We present the first X-ray crystallographic structures of peptoid hetero-oligomers, revealing that peptoid macrocycles can form a reverse-turn conformation.     

Pep-Calc.com: a set of web utilities for the calculation of peptide and peptoid properties and automatic mass spectral peak assignment

The ability to calculate molecular properties such as molecular weights, isoelectric points, and extinction coefficients is vital for scientists using and/or synthesizing peptides and peptoids for research. A suite of two web utilities: Peptide Calculator and Peptoid Calculator, available free at http://www.pep-calc.com, are presented. Both tools allow the calculation of peptide/peptoid chemical formulae and molecular weight, ChemDraw structure file export and automatic assignment of mass spectral peaks to deletion sequences and metal/protecting group adducts. Peptide Calculator also provides a calculated isoelectric point, molar extinction coefficient, graphical peptide charge summary and β-strand contiguity profile (for aggregation-prone sequences), indicating potential regions of synthesis difficulty. In addition to the unique automatic spectral assignment features offered across both utilities, Peptoid Calculator represents a first-of-a-kind resource for researchers in the field of peptoid science. With a constantly expanding database of over 120 amino acids, non-natural peptide building blocks and peptoid building blocks, it is anticipated that Pep-Calc.com will act as a valuable asset to those working on the synthesis and/or application of peptides and peptoids in the biophysical and life sciences fields.     

Submonomer synthesis of peptoids containing trans-inducing N-imino- and N-alkylamino-glycines

The use of hydrazones as a new type of submonomer in peptoid synthesis is described, giving access to peptoid monomers that are structure-inducing. A wide range of hydrazones were found to readily react with α-bromoamides in routine solid phase peptoid submonomer synthesis. Conditions to promote a one-pot cleavage of the peptoid from the resin and reduction to the corresponding N-alkylamino side chains were also identified, and both the N-imino- and N-alkylamino glycine residues were found to favor the trans-amide bond geometry by NMR, X-ray crystallography, and computational analyses.

The use of hydrazones as a new type of submonomer in peptoid synthesis is described, giving access to peptoid monomers that are structure-inducing.     

A CGenFF-based force field for simulations of peptoids with both cis and trans peptide bonds

Peptoids, or poly-n-substituted glycines, are peptide-like polymers composed of a flexible backbone decorated with diverse chemical side chains. Peptoids can form a variety of self-assembling structures based on the type and sequence of the side chains attached to their backbones. All-atom molecular dynamics simulations have been useful in predicting the conformational structures of proteins and will be valuable tools for identifying combinations of peptoid side chains that may form interesting folded structures. However, peptoid models must address a major degree of freedom not common in proteins – the cis/trans isomerization of the peptide bond. This work presents CHARMM general force field (CGenFF) parameters developed to accurately represent peptoid conformational behavior, with an emphasis on a correct representation of both the cis and trans isomers of the peptoid backbone. These parameters are validated against experimental and quantum mechanics data and used to simulate three peptoid side chains in explicitly solvated systems. © 2019 Wiley Periodicals, Inc.     

Drug-Like Properties in Macrocycles above MW 1000: Backbone Rigidity versus Side-Chain Lipophilicity

Large macrocyclic peptides can achieve surprisingly high membrane permeability, although the properties that govern permeability in this chemical space are only beginning to come into focus. We generated two libraries of cyclic decapeptides with stable cross-β conformations, and found that peptoid substitutions within the β-turns of the macrocycle preserved the rigidity of the parent scaffold, whereas peptoid substitutions in the opposing β-strands led to “chameleonic” species that were rigid in nonpolar media but highly flexible in water. Both rigid and chameleonic compounds showed high permeability over a wide lipophilicity range, with peak permeabilities differing significantly depending on scaffold rigidity. Our findings indicate that modulating lipophilicity can be used to engineer favorable ADME properties into both rigid and flexible macrocyclic peptides, and that scaffold rigidity can be used to tune optimal lipophilicity.     

Development and use of an atomistic CHARMM‐based forcefield for peptoid simulation

Peptoids are positional isomers of peptides: peptoid sidechains are attached to backbone nitrogens rather than α‐carbons. Peptoids constitute a class of sequence‐specific polymers resistant to biological degradation and potentially as diverse, structurally and functionally, as proteins. While molecular simulation of proteins is commonplace, relatively few tools are available for peptoid simulation. Here, we present a first‐generation atomistic forcefield for peptoids. Our forcefield is based on the peptide forcefield CHARMM22, with key parameters tuned to match both experimental data and quantum mechanical calculations for two model peptoids (dimethylacetamide and a sarcosine dipeptoid). We used this forcefield to demonstrate that solvation of a dipeptoid substantially modifies the conformations it can access. We also simulated a crystal structure of a peptoid homotrimer, H‐(N‐2‐phenylethyl glycine)₃‐OH, and we show that experimentally observed structural and dynamical features of the crystal are accurately described by our forcefield. The forcefield presented here provides a starting point for future development of peptoid‐specific simulation methods within CHARMM. © 2013 Wiley Periodicals, Inc.     

Synthesis of Peptoid Nucleic Acid with Thymine as Nucleic Base

The sequence specific bonding of oligonucleotide to RNA or double stranded DNA hasattTacted wide attention as the antisense and antigene strategies for treatment of diseasesat the level of gene expression in medicinal chemistry'. Peptide nucleic acids designedas a chemira of nucleobases and polyamidic backbone bind with high affinity andsequence specifity to both comPlementary RNA and DNA and a number of templatefunctions are inhibited on forming PNAasA and PNA/DNA complex'-'. In the …