A Series of Analogues to the AT2R Prototype Antagonist C38 Allow Fine Tuning of the Previously Reported Antagonist Binding Mode

We here report on our continued studies of ligands binding to the promising drug target angiotensin II type 2 receptor (AT₂R). Two series of compounds were synthesized and investigated. The first series explored the effects of adding small substituents to the phenyl ring of the known selective nonpeptide AT₂R antagonist C38 , generating small but significant shifts in AT₂R affinity. One compound in the first series was equipotent to C38 and showed similar kinetic solubility, and stability in both human and mouse liver microsomes. The second series was comprised of new bicyclic derivatives, amongst which one ligand exhibited a five-fold improved affinity to AT₂R as compared to C38 . The majority of the compounds in the second series, including the most potent ligand, were inferior to C38 with regard to stability in both human and mouse microsomes. In contrast to our previously reported findings, ligands with shorter carbamate alkyl chains only demonstrated slightly improved stability in microsomes. Based on data presented herein, a more adequate, tentative model of the binding modes of ligand analogues to the prototype AT₂R antagonist C38 is proposed, as deduced from docking redefined by molecular dynamic simulations.     

Synthesis of New Chrial Building Blocks for Novel Peptide Nucleic Acids

N-Boc protected amino acids of analogues of peptide nucleic acid (PNA),which are a class of conformationally constrained building blocks based on 4-aminoproline backbone with chirality at 2-c and 4-c,have been synthesized.Those monomers can be used for the construction of novel peptide nucleic acid analogues.     

Radical-driven dissociation of odd-electron peptide radical ions produced in 157 nm photodissociation

Odd-electron a+1 radical ions generated in the 157 nm photodissociation of peptide ions were investigated in an ion trap mass spectrometer. To localize the radical, peptide backbone amide hydrogens were replaced with deuterium. When the resulting radical ions underwent hydrogen elimination, no H/D scrambling was obvious, suggesting that without collisional activation, the radical resides on the terminal α-carbon. Upon collisional excitation, odd-electron radical ions dissociate through two favored pathways: the production of a-type ions at aromatic amino acids via homolytic cleavage of backbone C_α-C(O) bonds and side-chain losses at nonaromatic amino acids. When aromatic residues are not present, nonaromatic residues can also lead to a-type ions. In addition to a-type ions, serine and threonine yield c _n−1 and a _n−1+1 ions where n denotes the position of the serine or threonine. All of these fragments appear to be directed by the radical and they strongly depend on the amino acid side-chain structure. In addition, thermal fragments are also occasionally observed following cleavage of labile Xxx-Pro bonds and their formation appears to be kinetically competitive with radical migration.     

Intramolecularly lactam stapled oxyntomodulin analogues inhibit cancer cell proliferation in vitro

As a glucagon (GCG) receptor (GCGR) and glucagon-like peptide 1 (GLP-1) receptor (GLP-1R) dual agonist, oxyntomodulin (OXM) has been attracting scientific attentions due to its efficacies of suppressing appetite, increasing energy expenditure, and inducing body weight loss in obese humans. Based on the scaffold of native OXM, specific helix-favoring amino acids substitutions and the consequent salt bridge formations were believed to offer enhanced and balanced GCGR/GLP-1R activations through increasing α-helical conformation. Novel OXM analogues are obtained by intramolecular lactam stapling of positions [Glu16 & Lys20] or [Lys17 & Glu21] to further strengthen conformationally constrained stabilization. Even though the lactam staple does not provide additional dual GCGR/GLP-1R activations in vitro, the stapled OXM analogues are firstly reported to have higher or lower anti-PANC-1 cell proliferation activity, meanwhile which has no obvious inhibitory effect on the proliferation of HeLa cells. Therefore, it is speculated that the stapled analogues may have the potential to inhibit the proliferation of specific cancer cell types. Among the stapled peptides as well as their precursors, analogue 6 has the most prominent anti-PANC-1 proliferation activity with the IC₅₀ value of 115.1 μmol/L. Its mechanism of actions including effective signal pathways should be worth further investigations in future.     

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.     

Natural structural motifs that suppress peptide ion fragmentation after electron capture

Series of doubly and triply protonated diarginated peptide molecules with different number of glutamic acid (E) and asparagine (N) residues were analyzed under ECD conditions. ECD spectra of doubly-protonated peptides show a strong dependence on the number of E and N residues. Both the backbone cleavages and hydrogen radical (H^•) loss from the charge-reduced precursor ions ([M+2H]^+•) were suppressed as the number of E and N residues increases. A strong inhibition of the backbone cleavages and H^• loss from [M+2H]^+• was found for peptides with 6E residues (or 4E + 2N residues). The results obtained using these model peptides were re-confirmed by analyzing N-arginated Fibrinopeptide-B (i.e., REGVNDNEEGFFSAR). In contrast to the N-arginated peptide, ECD of the doubly-protonated Fibrinopeptide-B and its analogues show extensive backbone cleavages leading to series of c- and z-ions (∼80% sequence coverage). Based on these results, it is believed that peptide ions with all surplus protons sequestered in arginine-residues would show enhanced stability under ECD conditions as the number of acid-residue increases. The suppression of backbone cleavages and H^• loss from [M+2H]^+• are presumably attributed to the low reactivity of the charge-reduced precursor ions. One of the possible hypothesis is that diarginated E-rich peptides may contain hydrogen bonds between carbonyl oxygen of E side chains and backbone amide hydrogen. These hydrogen bonds would provide extra stabilization for [M+2H]^+•. This is the first demonstration of natural structural motifs in peptides that would inhibit the backbone fragmentation of the charge-reduced peptide ions under ECD conditions.     

Synthesis of New Chiral Building Blocks for Novel Peptide Nucleic Acids

IntroductionAbouttenyearsago ,PNA ,astructuralmimicofDNAinwhichthesugar phosphatebackboneisreplacedbyN (2 aminoethyl)glycine (aeg)linkageemergedasapotentialanti sensetherapeuticagent.1PNAhassomeadvantages:(1)itisstabletocellularnucleasesandproteases,(2 )ithybridizeswithcomplementaryDNAorRNA (cDNA/RNA)sequenceswithhighaffinity ,(3)ithaslownon specificinteractionwithcellularcontentsand (4 )itiseasilysynthesizedbyadoptionofsolidphasepeptidesynthesischemistry .However,thema jorlimitationo…     

Ynamide‐Mediated Thiopeptide Synthesis

Exploration of the full potential of thioamide substitution as a tool in the chemical biology of peptides and proteins has been hampered by insufficient synthetic strategies for the site‐specific introduction of a thioamide bond into a peptide backbone. A novel ynamide‐mediated two‐step strategy for thiopeptide bond formation with readily available monothiocarboxylic acids as thioacyl donors is described. The α‐thioacyloxyenamide intermediates formed from the ynamides and monothiocarboxylic acids can be purified, characterized, and stored. The balance between their activity and stability enables them to act as effective thioacylating reagents to afford thiopeptide bonds under mild reaction conditions. Amino acid functional groups such as OH, CONH₂, and indole NH groups need not be protected during thiopeptide synthesis. The modular nature of this strategy enables the site‐specific incorporation of a thioamide bond into peptide backbones in both solution and the solid phase.     

Synthesis of protected ω-mercapto amino acids: precursors for incorporation of elongated cysteines into peptides

A series of protected ω-mercapto amino acids with side-chain lengths ranging from 3–5 methylene units has been synthesized via nucleophilic substitution of ω-bromo-α-azido acids by 4-methoxy-α-toluenethiol followed by reduction of the azido functionality with SnCl₂. These enantiomerically pure protected cysteine analogues can be used to optimize the length of disulfide connections in cyclically constrained peptide pharmacophores.     

N-Terminal Peptide Sequence Repetition Influences the Kinetics of Backbone Fragmentation: A Manifestation of the Jahn-Teller Effect?

Analysis of large (>10,000 entries) databases consisting of high-resolution tandem mass spectra of peptide dications revealed with high statistical significance (P?<?1?10^–3) that peptides with non-identical first two N-terminal amino acids undergo cleavages of the second peptide bond at higher rates than repetitive sequences composed of the same amino acids (i.e., in general AB- and BA- bonds cleave more often than AA- and BB- bonds). This effect seems to depend upon the collisional energy, being stronger at lower energies. The phenomenon is likely to indicate the presence of the diketopiperazine structure for at least some b₂ ⁺ ions. When consisting of two identical amino acids, these species should form through intermediates that have a symmetric geometry and, thus, must be subject to the Jahn-Teller effect that reduces the stability of such systems.

Tandem Mass Spectrometric Characterization of Thiol Peptides Modified by the Chemoselective Cationic Sulfhydryl Reagent (4-Iodobutyl)Triphenylphosphonium—

Fixed charge chemical modifications on peptides and proteins can impact fragmentation behaviors in tandem mass spectrometry (MS/MS). In this study, we employed a thiol-specific cationic alkylation reagent, (4-iodobutyl)triphenylphosphonium (IBTP), to selectively modify cysteine thiol groups in mitochondrial proteome samples. Tandem mass spectrometric characteristics of butyltriphenylphosphonium (BTP)-modified peptides were evaluated by comparison to their carbamidomethylated (CAM) analogues using a quadrupole time-of-flight (Q-TOF) instrument under low energy collision-induced dissociation (CID) conditions. Introduction of the fixed charge modification resulted in the observation of peptide and fragment (b_n and y_n) ions with higher charge states than those observed for CAM-modified analogues. The charged BTP moiety had a significant effect on the neighboring amide bond fragmentation products. A decrease in relative abundances of the product ions at the corresponding cleavage sites was observed compared with those from the CAM-modified derivatives. This effect was particularly noticeable when an Xxx-Pro bond was in the vicinity of a BTP group. We hypothesized that the presence of a phosphonium moiety will reduce the tendency for protonation of the proximal amide bonds in the peptide backbone. Indeed, calculations indicated that proton affinities of backbone amide bonds close to the modified cysteine residues were generally 20–50 kcal/mol lower for BTP-modified peptides than for the unmodified or CAM-modified analogues with the sequence motif -Ala-Cys-Ala_n-Ala₂-, -Ala-Cys-Ala_n-Pro-Ala-, and -Ala-Pro-Ala_n-Cys-Ala-, n = 0–3.     

Total synthesis of biseokeaniamides A–C and late-stage electrochemically-enabled peptide analogue synthesis

The first total synthesis of cytotoxic cyanobacterial peptide natural products biseokeaniamides A–C is reported employing a robust solid-phase approach to peptide backbone construction followed by coupling of a key thiazole building block. To rapidly access natural product analogues, we have optimized an operationally simple electrochemical oxidative decarboxylation–nucleophilic addition pathway which exploits the reactivity of native C-terminal peptide carboxylates and abrogates the need for building block syntheses. Electrochemically-generated N,O-acetal intermediates are engaged with electron-rich aromatics and organometallic reagents to forge modified amino acids and peptides. The value of this late-stage modification method is highlighted by the expedient and divergent production of bioactive peptide analogues, including compounds which exhibit enhanced cytotoxicity relative to the biseokeaniamide natural products.

A late-stage electrochemical decarboxylation enables rapid access to structural analogues of biseokeaniamides A–C, cytotoxic lipopeptide natural products.     

A Three‐Site Mechanism for Agonist/Antagonist Selective Binding to Vasopressin Receptors

Molecular‐dynamics simulations with metadynamics enhanced sampling reveal three distinct binding sites for arginine vasopressin (AVP) within its V₂‐receptor (V₂R). Two of these, the vestibule and intermediate sites, block (antagonize) the receptor, and the third is the orthosteric activation (agonist) site. The contacts found for the orthosteric site satisfy all the requirements deduced from mutagenesis experiments. Metadynamics simulations for V₂R and its V_1aR‐analog give an excellent correlation with experimental binding free energies by assuming that the most stable binding site in the simulations corresponds to the experimental binding free energy in each case. The resulting three‐site mechanism separates agonists from antagonists and explains subtype selectivity.     

Synthesis and conformational properties of model dipeptides containing novel axially chiral α,β-didehydroamino acids at the (i+1) position of a β-turn conformation

A series of model dipeptides containing some novel axially chiral α,β-didehydroamino acids at the (i+1) position has been synthesised by reaction of the corresponding 4-(4-alkylcyclohexylidene)-2-phenyl-1,3-oxazol-5(4H)-one with (S)-phenylalanine cyclohexylamide. The conformations of two dipeptides in the crystal state have been studied by X-ray diffraction crystallographic analysis. The backbone torsion angles suggest that both peptides adopt similar type-II′ β-turn conformations. NMR spectroscopy has revealed that relatively rigid β-turn structures also persist in solution and that the absolute configurations of the axially chiral α,β-didehydroamino acids do not significantly influence the conformation of the peptide chain. Both heterochiral and homochiral dipeptides are found to accommodate the same βII′-turn conformation. Axially chiral α,β-didehydroamino acids (R_a)- and (S_a)-4-methyl-, 4-phenyl- and (4-tert-butylcyclohexylidene)glycine can be considered as elongated structural analogues of alanine, phenylglycine and tert-leucine of R and S configuration since, in these chiral α,β-didehydroamino acids, the methyl, phenyl and tert-butyl groups are located about 4.3 Å away from the peptide backbone in which they are incorporated.     

Role of surface-exposed charged basic amino acids (Lys,Arg) and guanidination in insulin on the interaction and stability of insulin–insulin receptor complex

Naturally occurring proteins are emerging as novel therapeutics in the protein-based biopharmaceutical industry for the treatment of diabetes and obesity. However, proteins are not suitable for oral delivery due to short half-life, reduced physical and chemical stability and low permeability across the membrane. Chemical modification has been identified as a formulation strategy to enhance the stability and bioavailability of protein drugs. The present study aims to study the effect of charge-specific modification of basic amino acids (Lys, Arg) and guanidination on the interaction of insulin with its receptor using molecular modelling. Our investigation revealed that the guanidination of insulin (Lys-NHC = NHNH₂) enhanced and exerted stronger binding of the protein to its receptor through electrostatic interaction than native insulin (Lys-NH₃⁺). Point mutations of Lys and Arg (R22, K29; R22K, K29; R22, K29R; R22K, K29R) were attempted and the effects on the interaction and stability between insulin/modified insulins and insulin receptor were also analyzed in this study. The findings from the study are expected to provide a better understanding of the possible mechanism of action of the modified protein at a molecular level before advancing to real experiments.     

Microcin J25 has a threaded sidechain-to-backbone ring structure and not a head-to-tail cyclized backbone

Microcin J25 is a 21 amino acid bacterial peptide that has potent antibacterial activity against Gram-negative bacteria, resulting from its interaction with RNA polymerase. The peptide was previously proposed to have a head-to-tail cyclized peptide backbone and a tight globular structure (Blond, A., Péduzzi, J., Goulard, C., Chiuchiolo, M. J., Barthélémy, M., Prigent, Y., Salomón, R. A., Farías, R. N., Moreno, F. & Rebuffat, S. Eur. J. Biochem. 1999, 259, 747-755). It exhibits remarkable thermal stability for a peptide of its size lacking disulfide bonds and in part this was previously proposed to derive from its macrocyclic structure. We show here that in fact the peptide does not have a head-to-tail cyclic structure but rather a side chain to backbone cyclization between Glu8 and the N-terminus. This creates an embedded ring that is threaded by the C-terminal tail of the molecule, forming a noose-like feature. The three-dimensional structure deduced from NMR data suggests that slippage of the noose is prevented by two aromatic residues flanking the embedded ring. Unthreading does not occur even when the molecule is enzymatically digested with thermolysin. The new structural interpretation fully accounts for previously reported NMR and biophysical data and is consistent with the remarkable stability of this potent antimicrobial peptide.     

Molecular design and structural optimization of potent peptide hydroxamate inhibitors to selectively target human ADAM metallopeptidase domain 17

Human ADAMs (a disintegrin and metalloproteinases) have been established as an attractive therapeutic target of inflammatory disorders such as inflammatory bowel disease (IBD). The ADAM metallopeptidase domain 17 (ADAM17 or TACE) and its close relative ADAM10 are two of the most important ADAM members that share high conservation in sequence, structure and function, but exhibit subtle difference in regulation of downstream cell signaling events. Here, we described a systematic protocol that combined computational modeling and experimental assay to discover novel peptide hydroxamate derivatives as potent and selective inhibitors for ADAM17 over ADAM10. In the procedure, a virtual combinatorial library of peptide hydroxamate compounds was generated by exploiting intermolecular interactions involved in crystal and modeled structures. The library was examined in detail to identify few promising candidates with both high affinity to ADAM17 and low affinity to ADAM10, which were then tested in vitro with enzyme inhibition assay. Consequently, two peptide hydroxamates Hxm-Phe-Ser-Asn and Hxm-Phe-Arg-Gln were found to exhibit potent inhibition against ADAM17 (K_i = 92 and 47 nM, respectively) and strong selectivity for ADAM17 over ADAM10 (∼7-fold and ∼5-fold, S = 0.86 and 0.71, respectively). The structural basis and energetic property of ADAM17 and ADAM10 interactions with the designed inhibitors were also investigated systematically. It is found that the exquisite network of nonbonded interactions involving the side chains of peptide hydroxamates is primarily responsible for inhibitor selectivity, while the coordination interactions and hydrogen bonds formed by the hydroxamate moiety and backbone of peptide hydroxamates confer high affinity to inhibitor binding.     

Pd(II) Binding Strength of a Novel Ambidentate Dipeptide-Hydroxypyridinonate Ligand: A Solution Equilibrium Study

A novel ambidentate dipeptide conjugate (H(L1)) containing N-donor atoms of the peptide part and an (O,O) chelate at the hydroxypyridinone (HP) ring is synthesized and characterized. It is hoped that this chelating ligand can be useful to obtain multitargeted Co(III)/Pt(II) dinuclear complexes with anticancer potential. The Pd(II) (as a Pt(II) model but with faster ligand exchange reactions) binding strength of the ligand was studied in an aqueous solution with the combined use of pH-potentiometry and NMR. In an equimolar solution, (L1)⁻ was found to bind Pd(II) via the terminal amino and increasing number of peptide nitrogens of the peptide backbone over a wide pH range. At a 2:1 Pd(II) to ligand ratio, the presence of [Pd₂H_–x(L1)] (x = 1–4) species, with high stability and with the coordination of the (O,O) chelating set of the ligand, was detected. The reaction of H(L1) with [Co(tren)]³⁺ (tren = tris(2-aminoethyl)amine) indicated the exclusive binding of (L1)⁻ via its (O,O) donor atoms to the metal unit, while treatment of the resulting Co-complex with Pd(II) afforded the formation of a Co/Pd heterobimetallic complex in solution with an (NH₂, N_amide) coordination of Pd(II). Shortening the peptide backbone in H(L1) by one peptide unit compared to the structurally similar ambidentate chelator consisting of three peptide bonds resulted in the slightly more favorable formation of the N-coordinated Pd(II) species, allowing the tailoring of the coordination properties.     

Synthesis of Amino Acid Derived Cyclic Phosphorus Ylides

Abstract

We recently reported the thermal elimination of Ph₃PO from suitably protected aminoacyl ylides 1 as a route to acetylenic amino acid analogues 2. Pyrolysis of ylides such as 3 with a free amino group takes a different course. Ethanol is eliminated to give the c h i d cyclic ylides 4 which can be viewed as 3-triphenylphosphoranylidene tetramic acids. Specific examples prepared include 4 (R = Me, Pri), the parent compound 5 (from glycine) and the six-membered ring compound 6 (from p-alanine). Using a similar approach, bicyclic ylides such as 7 (from proline) can be prepared. In the case of the glutamate derived ylide 8, thermolysis initially gives a mixturc of 9 and 10 but these both cyclise to the bicyclic product 11 with time. The structure and reactivity of these interesting cyclic ylides are now being examined.     

Stereospecific control of peptide gas‐phase ion chemistry with cis and trans cyclo ornithine residues

We report non‐chiral amino acid residues cis‐ and trans‐1,4‐diaminocyclohexane‐1‐carboxylic acid (cyclo‐ornithine, cO) that exhibit unprecedented stereospecific control of backbone dissociations of singly charged peptide cations and hydrogen‐rich cation radicals produced by electron‐transfer dissociation. Upon collision‐induced dissociation (CID) in the slow heating regime, peptide cations containing trans‐cO residues undergo facile backbone cleavages of amide bonds C‐terminal to trans‐cO. By contrast, peptides with cis‐cO residues undergo dissociations at several amide bonds along the peptide ion backbone. Diastereoisomeric cO‐containing peptides thus provide remarkably distinct tandem mass spectra. The stereospecific effect in CID of the trans‐cO residue is explained by syn‐facially directed proton transfer from the 4‐ammonium group at cO to the C‐terminal amide followed by neighboring group participation in the cleavage of the CO―NH bond, analogous to the aspartic acid and ornithine effects. Backbone dissociations of diastereoisomeric cO‐containing peptide ions generate distinct [b_n]⁺‐type fragment ions that were characterized by CID‐MS³ spectra. Stereospecific control is also reported for electron‐transfer dissociation of cis‐ and trans‐cO containing doubly charged peptide ions. The stereospecific effect upon electron transfer is related to the different conformations of doubly charged peptide ions that affect the electron attachment sites and ensuing N―C_α bond dissociations.