peptide bond

Processing of the gag and pol gene precursor proteins of retroviruses is essential for infectivity and is directed by a viral protease that is itself included in one of these precursors. We demonstrate here that small synthetic peptides can be used as both model substrates and inhibitors to investigate the specificity and molecular parameters of the reaction. The results indicate that a peptide that extends five amino acids but not three amino acids in both directions from a known cleavage site is accurately hydrolyzed by the protease of avian sarcoma-leukosis virus. Substitutions of the amino acids to either side of the peptide bond to be cleaved affect the ability of the peptide (as well as a larger precursor protein) to serve as a substrate. The specificity is more stringent for the amino acid that will become the carboxyl end after cleavage. Some substitutions produced peptides that were not cleaved but could act as inhibitors of cleavage of a susceptible peptide. Thus, small mo...

Mutating the side‐chains of amino acids in a peptide ligand, with unnatural amino acids, aiming to mitigate its short half‐life is an established approach. However, it is hypothesized that mutating specific backbone peptide bonds with bioisosters can be exploited not only to enhance the proteolytic stability of parent peptides, but also to tune its receptor subtype selectivity. Towards this end, four [Y]6‐Angiotensin II analogues are synthesized where amide bonds have been replaced by 1,4‐disubstituted 1,2,3‐triazole isosteres in four different backbone locations. All the analogues possessed enhanced stability in human plasma in comparison with the parent peptide, whereas only two of them achieved enhanced AT2R/AT1R subtype selectivity. This diversification has been studied through 2D NMR spectroscopy and unveiled a putative more structured microenvironment for the two selective ligands accompanied with increased number of NOE cross‐peaks. The most potent analogue, compound 2, has b...

Mechanochemical methods for assembling carbon-nitrogen bonds play a central role in the ongoing green chemistry-driven renovation of organic synthesis, including the synthesis of active pharmaceutical ingredients (APIs). However, chemoselective issues in these transformations, such as the tolerance of unmasked hydroxyl groups, have not been systematically explored. Screening of various amide coupling conditions revealed that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in combination with ethyl acetate as a liquid-assisted grinding (LAG) solvent was the most selective amide coupler, delivering 76-94% yields of the respective amide products from unprotected hydroxycarboxylic acids. Boc-protected tyrosine and serine with unmasked hydroxyl functionalities were successfully involved in peptide couplings. By incorporating green chemistry principles as a driver for innovation, the established protocol was applied to the synthesis of imatinib, an anticancer drug included in the World Health Organization's List of Essential Medicines. The target API was synthesized with an overall yield of 86% and 99% HPLC purity through a two-step mechanochemical C-N bond assembling reaction sequence starting from 4-(hydroxymethyl)benzoic acid. A comparison of green chemistry metrics between the developed mechanochemical approach and similar solution-based approaches revealed reduced waste generation, enhanced eco-friendliness, and a superior safety profile for the proposed strategy. The safety improvement was primarily attributed to the elimination of toxic solvents and a genotoxic intermediate from the production chain.

Plasmids containing 23S rRNA randomized at positions 2057-2063 and 2502-2507 were introduced into Escherichia coli, affording a library of clones which produced modified ribosomes in addition to the preexisting wild-type ribosomes. These clones were screened with a derivative of puromycin, a natural product which acts as an analogue of the 3′-end of aminoacyl-tRNA and terminates protein synthesis by accepting the growing polypeptide chain, thereby killing bacterial cells. The puromycin derivative in this study contained the dipeptide p-*

For over 20 years, native chemical ligation (NCL) has played a pivotal role in enabling the total synthesis and semisynthesis of increasingly complex peptide and protein targets. Classical NCL proceeds by the chemoselective reaction of two unprotected polypeptide chains in near-neutral pH aqueous solution and is made possible by the presence of a thioester moiety on the C-terminus of the N-terminal peptide fragment and a natural cysteine residue on the N-terminus of the Cterminal peptide fragment. The reaction yields an amide bond adjacent to cysteine at the ligation site, furnishing the native protein backbone in a traceless manner. This protocol will highlight a number of recent and powerful advances to the methodology and will outline their particular uses, facilitating their continued application in the synthesis of challenging protein targets.

Isotopic fractionation of nitrogen and carbon is considered during peptide bond hydrolysis. Theoretical considerations suggest that hydrolysis will enrich residual, unhydroiyzed protein in "N while r3C should be relatively unaffected. Preliminary experimental results support this conclusion, although further studies are reauired to auantifv the magnitude of this effect as a function of protein degradation, in particular under natural environmental conditions.

Quantum mechanical/molecular mechanical (QM/MM) free energy simulations are applied for understanding the mechanism of the acylation reaction catalyzed by sedolisin, a representative serine-carboxyl peptidase, leading to the acyl-enzyme (AE) and first product from the enzymecatalyzed reaction. One of the interesting questions to be addressed in this work is the origin of the substrate specificity of sedolisin that shows a relatively high activity on the substrates with Glu at P 1 site. It is shown that the bond making and breaking events of the acylation reaction involving a peptide substrate (LLE*FL) seem to be accompanied by local conformational changes, proton transfers as well as the formation of alternative hydrogen bonds. The results of the simulations indicate that the conformational change of Glu at P 1 site and its formation of a low barrier hydrogen bond with Asp-170 (along with the transient proton transfer) during the acylation reaction might play a role in the relatively high specificity for the substrate with Glu at P 1 site. The role of some key residues in the catalysis is confirmed through free energy simulations. Glu-80 is found to act as a general base to accept a proton from Ser-287 during the nucleophilic attack and then as a general acid to protonate the leaving group (N-H of P 1 0 -Phe) during the cleavage of the scissile peptide bond. Another acidic residue, Asp-170, acts as a general acid catalyst to protonate the carbonyl of P 1 -Glu during the formation of the tetrahedral intermediate and as a general base for the formation of the acyl-enzyme. The energetic results from the free energy simulations support the importance of proton transfer from Asp-170 to the carbonyl of P 1 -Glu in the stabilization of the tetrahedral intermediate and the formation of a low-barrier hydrogen bond between the carboxyl group of P 1 -Glu and Asp-170 in the lowering of the free energy barrier for the cleavage of the peptide bond. Detailed analyses of the proton transfers during acylation are also given.

1. General tert-Butyl hydroperoxide solution (TBHP, packed in FEP bottles, 5.5 M in decane, over molecular sieve 4Å) was purchased from Sigma-Aldrich. N-hydroxyphthalimide (NHPI, 98%) was purchased from Alfa Aesar. Thin-layer chromatography (TLC) was conducted with Merck 60 F254 pre-coated silica gel plate (0.2 mm thickness) and visualized with UV and potassium permanganate staining. Flash chromatography was performed using Merck silica gel 60 with distilled solvents. 1 H NMR spectra were performed on a Bruker AVIII 400 NMR spectrometer and are reported in ppm downfield from SiMe 4 (δ 0.0) and relative to the signal of chloroform-d. Data reported as: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, b = broad; coupling constant(s) in Hz; integration. Proton-decoupled 13 C NMR spectra were recorded on a Bruker AVIII 400 (100 MHz) spectrometer and are reported in ppm using CDCl 3 as an internal standard. IR spectra were recorded as thin films on NaCl plates on a Bio-Rad FTS 165 FTIR spectrometer and are reported in frequency of absorption (cm -1 ). High resolution mass spectral analysis (HRMS) was performed on Waters Q-Tof Permies Mass Spectrometer. 2-Aryloxy-1-acetophenone derivatives 3 were prepared by the literature procedures cited in the references.

Aminopeptidase P (APP), dipeptidyl peptidase II (DP II), dipeptidyl peptidase IV (DP IV) and prolyl oligopeptidase (POP) are proline specific peptidases. Hence, they are able to cleave peptide bonds containing the imino acid proline. Amino acid pyrrolidides (Pyrr) and thiazolidides (Thia) are well-known product analogue inhibitors of DP IV and POP. For the first time we describe the influence of a thioxo amide bond, incorporated into these compounds, on the inhibition of the proline specific peptidases. Taking into account the substrate specificity of these peptidases, we have synthesized Xaa-i[CS-N]-Pyrr and Xaa-i[CS-N]-Thia of the amino acids Ala, Phe, Val and Ile. The inhibition constants were determined for the above mentioned proline specific peptidases isolated from different sources. As a result, the serine proteases DP II, DP IV and POP were inhibited competitively, whereas metal-dependent APP displayed a linear mixed-type inhibition with inhibition constants up to 10 34 M. Thioxylation of Xaa-Pyrr and Xaa-Thia led to a slight decrease of inhibition of DP IV and POP compared to Xaa-Pyrr and Xaa-Thia, though the inhibition constants were still in the range up to 10 37 M. As Xaa-Thia exist as two isomers, we investigated isomer specific inhibition with regard to DP IV. Thus, our studies have revealed that DP IV was only inhibited by the Z isomer of the Xaa-i[CS-N]-Thia. For the first time, Xaa-Pyrr and Xaa-Thia were characterized as inhibitors of DP II with inhibition constants in the micromolar range. In contrast to DP IV inhibition, the Xaa-i[CS-N]-Pyrr and Xaa-i[CS-N]-Thia have proven to be more potent inhibitors of DP II than the corresponding Xaa-Pyrr and Xaa-Thia. Thus, these Xaa-i[CS-N]-Thia are new potent inhibitors especially suitable for DP II with K i values ranging in the upper nanomolar concentration.

Recently, considerable insight has been gained into the modular organization and catalytic properties of nonribosomal peptide synthetases. However, molecular and biochemical aspects of the condensation of two aminoacyl substrates or a peptidyl and an aminoacyl substrate, leading to the formation of a peptide bond, have remained essentially impenetrable. To investigate this crucial part of nonribosomal peptide synthesis, an in vitro assay for a dipeptide formation was developed. Two recombinant holomodules, GrsA (PheATE), providing D-Phe, and a C-terminally truncated TycB, corresponding to the first, L-Pro-incorporating module (Pro-CAT), were investigated. Upon combination of the two aminoacylated modules, a fast reaction is observed, due to the formation of the linear dipeptide D-Phe-L-Pro-S- enzyme on ProCAT, followed by a noncatalyzed release of the dipeptide from the enzyme. The liberated product was identified by TLC, high pressure liquid chromatography-mass spectrometry, 1 H and 13 C NMR, and comparison with a chemically synthesized standard to be the expected D-Phe-L-Pro diketopiperazine. Further minimization of the two modules was not possible without a loss of transfer activity. Likewise, a mutation in a proposed active-site motif (HHXXXDG) of the condensation domain giving ProCAT(H147V), abolished the condensation reaction. These results strongly suggest the condensation domain to be involved in the catalysis of nonribosomal peptide bond formation with the histidine 147 playing a catalytic role.

Background: Nonribosomal peptide synthetases (NRPSs) are large multidomain proteins that catalyze the formation of a wide range of biologically active natural products. These megasynthetases contain condensation (C) domains that catalyze peptide bond formation and chain elongation. The natural substrates for C domains are biosynthetic intermediates that are covalently tethered to thiolation (T) domains within the synthetase by thioester linkages. Characterizing C domain substrate speci¢city is important for the engineered biosynthesis of new compounds. Results: We synthesized a series of aminoacyl-N-acetylcysteamine thioesters (aminoacyl-SNACs) and show that they are small-molecule substrates for NRPS C domains. Comparison of rates of peptide bond formation catalyzed by the C domain from enterobactin synthetase with various aminoacyl-SNACs as downstream (acceptor) substrates revealed high selectivity for the natural substrate analog L-Ser-SNAC. Comparing L-and D-Phe-SNACs as upstream (donor) substrates for the ¢rst C domain from tyrocidine synthetase revealed clear D-versus L-selectivity. Conclusions: Aminoacyl-SNACs are substrates for NRPS C domains and are useful for characterizing the substrate speci¢city of C domain-catalyzed peptide bond formation.

We compare recent quantum mechanical computations of alternative reaction pathways for carboxypeptidase A, a zinc proteinase, in an "enzyme environment" to similar calculations in the "gas phase" that include the minimal chemical entities that are required for a non-catalytic reaction. The main question that we address is whether anything may be learned from such reduced representations. Two general acid-general base alternative pathways and one nucleophilic pathway are compared. The original calculations were run on a relatively large model (120 atoms) of the active site of carboxypeptidase A which included zinc and its ligands, as well as the residues Arg145, Arg127, Glu270, a water molecule and a model dipeptide. The "gas-phase" pathways include only the dipeptide, water and Glu270 and serve as models for the non-catalytic pathway. The calculations were performed by semiempirical MNDO/H/d that includes modifications for d-orbital representations as well as for intra-and intermolecular multiple H-bond formation. The gas-phase results strengthen our previous conclusion about the preference for general acid-general base pathways for peptide cleavage by carboxypeptidase A rather than a "direct nucleophilic" pathway. The bottleneck of the reaction is proton transfer to the nitrogen in the peptide bond, preceding the peptide cleavage.

Kinetic measurements on a fluorescent peptide analog of the p17/p24 cleavage site of the Gag polyprotein demonstrate the conformational selectivity of human immunodeficiency virus, type 1 protease for the trans conformation of the Tyr-Pro bond. A mean cis/trans ratio of 0.3, and a cis 3 trans isomerization rate constant of 0.022 s ؊1 are determined at T ‫؍‬ 22 °C. This rate is in excellent agreement with that predicted by 19 F NMR studies of structurally analogous peptides containing a fluorine/hydroxyl substitution on the tyrosyl residue. Addition of recombinant human cyclophilin resulted in a significant enhancement of this rate, and it is proposed that this enzyme, which has been shown to be associated with the Gag protein, functions as an auxiliary enzyme for the protease during cleavage in the virion.

Fluorine nuclear magnetic resonance studies of the cleavage of peptides containing a 4-fluorophenylalanine (FPhe)-Pro bond have been performed in order to determine the conformational specificity of FPhe-Pro bond cleavage by pepsin. The peptides selected were substrates of HIV protease or of avian sarcoma virus protease, both of which have been reported to be cleaved specifically at X-Pro by pepsin as well as by the corresponding viral protease enzyme. By working at 0 °C, it was possible to separate kinetically cleavage and cis/trans isomerization. For the case of the protease substrate, Ser-Gln-Asn-FPhe-Pro-Ile-Val-Gln, cleavage was shown to be specific for the trans conformation. A value for the rate constant for hydrolysis of the trans peptide divided by the Michaelis constant, k tH /K M trans ) 0.3 min -1 mM -1 was obtained with this substrate, and the Michaelis constant appears to be considerably higher than the substrate concentration, 3.7 mM, used in the study. On a slower time scale, additional cleavages can readily be detected. For the avian leukemia virus protease substrate, Thr-Phe-Gln-Ala-FPhe-Pro-Leu-Arg-Glu-Ala, the cleavage was both slower and less specific. In addition to the primary cleavage at the FPhe-Pro site, cleavage also occurs at the Ala-FPhe bond on a somewhat slower time scale. In addition to the conformational specificity of the cleavage reaction, these results indicate that pepsin is a better model for HIV protease than for avian leukemia virus protease.

The Ca 2+ -dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca 2+ -dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca 2+ -loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae , resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca 2+ -dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

the layers were separated. The aqueous layer was neutralized formed, 5-10 ml of methanol was added and crystallization with sodium bicarbonate and extracted with three 50-ml portions occurred upon refrigeration of the mixture. Recrystallization of ether. The combined ether extracts were concentrated a t was effected with ethanol or benzene (see Table ). reduced pressure on the steam bath.18 If the residue contained a mixture of solid and an oil, it was washed with 10 ml of cold Registry "3.-3b, 1148-87-4; 3C, 3672-51-3; 3d, (0") methanol and filtered immediately. If a solid was not

Collagen induced arthritis (CIA) is the most studied animal model for rheumatoid arthritis and is associated with the MHC class II molecule A q . T-cell recognition of a peptide from type II collagen, CII256-270, bound to A q is a requirement for development of CIA. Lysine 264 is the major T-cell recognition site of CII256-270 and CIA is in particular associated with recognition of lysine 264 after posttranslational hydroxylation and subsequent attachment of a b-D D-galactopyranosyl moiety. In this paper we have studied the structural requirements of collagenous glycopeptides required for T-cell stimulation, as an extension of earlier studies of the recognition of the galactose moiety. Synthesis and evaluation of alanine substituted glycopeptides revealed that there are T-cells that only recognise the galactosylated hydroxylysine 264, and no other amino acid side chains in the peptide. Other T-cells also require glutamic acid 266 as a T-cell contact point. Introduction of a methylene ether isostere instead of the amide bond between residues 260 and 261 allowed weaker recognition by some, but not all, of the T-cells. Altogether, these results allowed us to propose a model for glycopeptide recognition by the T-cells, where recognition from one or the other side of the galactose moiety could explain the different binding patterns of the T-cells.

Full and unambiguous assignment of all 1 H-and 13 C-NMR resonances of the isomers due to restricted C-N amide bond rotation of N-formyl-o-toluidine and N,N'-bisformyl-o-tolidine in DMSO-d 6 is reported. The cis-isomer predominates in the equilibrium mixture of both compounds as 1D-NOE difference experiments show.

The title compound, C10H13N3O2S, was prepared by condensation
of 3,4-dihydroxybenzaldehyde with 4-ethyl-3-thiosemicarbazide.
The molecule adopts an E configuration with
respect to the C N bond. One of the OH substituents on the
dihydroxybenzene ring is disordered over the two possible 3-
positions on either side of the ordered 4-hydroxy group. The
occupancy of the major disorder component refined to
0.633 (7). The molecule is essentially planar, with an r.m.s.
deviation through all non-H atoms of 0.0862 A ° . An intramolecular
N—H     N hydrogen bond forms between the outer
amine residue and the imine N atom, generating an S(5) ring
motif and contributing to the planarity of the molecule. In the
crystal structure, an extensive network of classical O—H     O,
O—H     S and N—H     S hydrogen bonds and weak C—
H     O and S     O [3.301 (3) A ° ] interactions link molecules
into sheets running approximately parallel to the ab plane.

CNBr cleavage of unreduced proenzyme Clr yielded fragment CP2b, isolated by gel filtration and highpressure gel permeation chromatography. This fragment (~ M τ 55000) comprised at least 4 disulphidelinked peptides, which were separated by gel filtration after reduction and alkylation. Peptide CP2bRA4, overlapping the A‐ and B‐chain regions in proenzyme Clr was digested by V8 staphylococcal protease, and the digest separated by reversed‐phase HPLC. N‐terminal sequence analysis of peptide CP2bRA4SP9 established that Clr activation involves the cleavage of a single Arg‐Ile bond, located in the sequence:⋯ Gln‐Arg‐Gln‐Arg‐Ile‐Ile‐Gly‐Gly⋯

Significance Elongation factor Tu (EF-Tu) facilitates rapid and accurate selection of aminoacyl-tRNA (aa-tRNA) by the bacterial ribosome during protein synthesis. We show that EF-Tu dissociates from the ribosome as aa-tRNA navigates the accommodation corridor en route to peptide bond formation. We find that EF-Tu’s release from the ribosome during aa-tRNA selection can be reversible. We also demonstrate that new ternary complex formation, accompanied by futile cycles of GTP hydrolysis, can occur on aa-tRNA bound within the ribosome. These findings inform on the decoding mechanism, the contributions of EF-Tu to the fidelity of translation, and the potential consequences of reduced rates of peptide bond formation on cellular physiology.

An efficient method has been developed for the direct amide bond synthesis between carboxylic acids and amines via (2-(thiophen-2-ylmethyl)phenyl)boronic acid as a highly active bench-stable catalyst. This catalyst was found to be very effective at room temperature for a large range of substrates while slightly higher temperatures were required for challenging ones. This methodology can be applied to aliphatic, α-hydroxyl, aromatic and heteroaromatic acids as well as primary, secondary, heterocyclic and even functionalized amines. Notably, N-Boc protected amino acids were successfully coupled in good yields with very little racemization and a first example of catalytic dipeptide synthesis is reported.

The protein kinase A (PKA)-catalyzed phosphorylation of peptide substrate RRASVA analogs, containing N β-Me-aza-β3-amino acid residues in all subsequent positions, was studied. This work follows along the lines of our previous research of the phosphorylation of aza-β3-analogs of RRASVA (the shortest active substrate of PKA) and allows characterizing the influence of Nβ-methylation of aza-β3-amino acid residues on substrate recognition by PKA on substrate binding and phosphorylation steps. It was found that the effect of Nβ-methylation was dependent upon the position of the structure alteration. Moreover, the presence of a single Nβ-methylation site in the substrate changed the recognition pattern of this series of peptidomimetics, strongly affecting the phosphorylation step. Structure modeling of aza-β3- and N β-Me-aza-β3-containing substrates revealed that Nβ-methylation of aza-β3-moieties changed the peptide bond geometry from trans- to cis-configuration in -CO-NMe- fragments, wit...

Small ubiquitin-like modifier (SUMO)-specific protease SENP1 processes SUMO-1, SUMO-2 and SUMO-3 to mature forms and deconjugates them from modified proteins. To establish the proteolytic mechanism, we determined structures of catalytically inactive SENP1 bound to SUMO-1-modified RanGAP1 and to unprocessed SUMO-1. In each case, the scissile peptide bond is kinked at a right angle to the C-terminal tail of SUMO-1 and has the cis configuration of the amide nitrogens. SENP1 preferentially processes SUMO-1 over SUMO-2, but binding thermodynamics of full-length SUMO-1 and SUMO-2 to SENP1 and K m values for processing are very similar. However, k cat values differ by 50-fold. Thus, discrimination between unprocessed SUMO-1 and SUMO-2 by SENP1 is based on a catalytic step rather than substrate binding and is likely to reflect differences in the ability of SENP1 to correctly orientate the scissile bonds in SUMO-1 and SUMO-2. SUMO has diverse roles in many biological processes and is required for normal growth and development in all eukaryotes studied to date. In the yeast Saccharomyces cerevisiae, there is a single version of SUMO known as Smt3 (ref. 1), whereas in vertebrates, three SUMO genes, SUMO-1, SUMO-2 and SUMO-3, are expressed. Whereas SUMO-2 and SUMO-3 are very similar (96% identity) they are less than 50% identical to SUMO-1. Proteomic analysis has indicated that there are distinct substrates for modification with SUMO-1 and SUMO-2/3 (refs. 2,3), and functional analysis has indicated that the different isoforms have distinct roles in transcriptional regulation 4 and during the cell cycle 5 . Like most other ubiquitin-like modifiers (Ubls), the primary translation products of the SUMO paralogs need to be proteolytically processed to expose the C-terminal glycine residue that will be linked to target proteins. SUMO modification is mediated by a SUMO-activating enzyme (E1; in human, SAE1 and SAE2), a SUMO-conjugating enzyme (E2; Ubc9) and, typically, a SUMO protein ligase (E3), and it results in formation of an isopeptide bond between the C-terminal carboxyl group of SUMO and the ε-amino group of lysine in the substrate protein. Lysine residues

Racemization and coupling rate constants of several N-carbobenzoxy-?-methyl-L-glutamic acid and N-carbobenzoxy-P-methyl-L-aspartic acid active esters have been determined. The order of magnitude of coupling rate constants for both amino acid esters is comparable to that reported for the corresponding cysteine active esters. However, significant differences are observed for the racemization rates. The importance of the ratio of coupling to racemization rate constants is discussed. It has been previously reported that racemization of N-carbobenzoxy-S-benzyl-L-cysteine active esters in organic solvents and in the presence of triethylamine occurs through a-hydrogen abstraction and proceeds with unusual f a ~i l i t y . ~-~ It was also reported that the racemization for N-carbobenaoxy-S-benzyl-L-C~Steine pentachlorophenyl ester and N-carbobenzoxy-L-phenylalanine pentachlorophenyl ester in a nonpolar solvent proceeds via isoracemization.616 I n addition the rate of coupling of the cysteine active ester derivatives with L-valine methyl ester was investigated.8~~ From these data an important conclusion was drawn, that a fast coupling active ester which racemizes relatively slowly is probably the best choice for the synthesis of peptides and sequential polypeptides. Numerically this can be best expressed by the ratio of the rate constants of coupling (k,) to racemization (krao); the larger this number, the smaller the racemization to be expected during coupling. The fast racemization of the commonly used active esters of N-carbobenzoxy-S-benzylcysteine, as well as the reports of Anderson,' Liberek,8 and others that several amino acid active ester derivatives racemize in the presence of a tertiary base, led us to study the effect of the side chain of amino acids on the rates of racemization and coupling. The results with glutamic and aspartic acids are reported in this paper. Rates of Racemization of N-Carbobenzoxy-r-methyl-L-glutamic and N-Carbobenzoxy-P-methyl-L-aspartic Acid Active Esters.-The racemization of several frequently used active esters of glutamic and aspartic acids was studied in tetrahydrofuran solution in the presence of triethylamine under the conditions described for cysteinc active ester derivative^.^ The results are given in Table ,Q which contains the pseudo-firstorder as well as the second-order racemization rate (1) P a r t 0 of a series on racemization studies of amino acid derivatives.

We have used ribonuclease T1 and its chemically modified derivatives as substrates, and trypsin as proteinase, to investigate the kinetics of proteolysis of a specific pep- tide bond in the folded and unfolded conformations of a protein. Steady-state kinetic studies showed that Km = 0.27mM and Kcat. = 2.45s-s for the tryptic hydrolysis of the Arg(77)-Val(78) peptide bond in unfolded ribonuclease T1. This Km is somewhat lower than, and the kcat. value similar to, values found for the tryptic hydrolysis of comparable bonds in small peptides. Our data for the initial velocity of hydrolysis of the Arg(7-7)-Val(78) bond in a solution of the folded protein indicate that the bond is at least 1700 times less rapidly hydrolysed in the folded than in the unfolded conform- ation of ribonuclease T1, and do not exclude the possibility that the bond is com- pletely resistant to hydrolysis in the folded protein. Peptide-bond cleavage plays an essential role in many important biological processes, including the post-translational processing steps of protein syn- thesis, the generation of polypeptide hormones, assembly of intracellular organelles, blood clotting, intracellular protein degradation, tumour invasion and alimentary digestion. It is therefore desirable that we understand the kinetics of peptide-bond cleavage by proteolytic enzymes. Most of our knowledge of the action of proteinases is based on kineties studies of the hydrolysis of small peptides, so that much is known of the influence of covalent structure on the kinetics of proteolysis, but there is little information on the influence of conformation, since most small peptides do not form stable folded conformations in aqueous solution (Blundell & Wood, 1982). It is well established that most proteins are ren- dered much more susceptible to proteolysis by denaturation. The significance of this has been much debated since the seminal work of Lin- derstr0m-Lang's laboratory in the late 1940s Abbreviations used: the abbreviations used for amino acid derivatives and N-terminal groups are based on the standard conventions [Biochem. J. (1972) 126, 773-780]. Ribonuclease T, derivatives: Ac, N-acetyl; Cm, S- carboxymethyl; Suc, N-2-carboxypropionyl.

The fragmentation of protonated tripeptides under metastable ion conditions and collision-induced conditions are reported. The majority of protonated tripeptides cleave at the C-terminal amide bond to form b 2 and y 1 ions, with the relative amounts depending on the proton affinities of the amino acids derived from the C-terminal amino acid residue. Protonated tripeptides that have a proline residue at the C-terminal position fragment almost exclusively by cleavage of the amide bond adjacent to the proline and give mainly y 1 ions; where there is a proline residue in the central position, fragmentation of the protonated tripeptide occurs at both the N-terminal and the C-terminal amide bonds to form y 2 and b 2 ions; finally, two of the tripeptides with proline at the N-terminal position, Pro-Pro-Pro and Pro-Gly-Gly, fragment largely by cleavage at the N-terminal, the former by cleavage of the amide bond and the latter by direct formation of the a 1 ion. Protonated Pro-Val-Gly fragments to give predominantly b 2 and a 2 ions at low energy, but the a 1 ion becomes the dominant product at higher energies. In the fragmentation of protonated peptides, the lack of b ions formed by cleavage at the amide bond C-terminal to proline (i.e., with the proline residue in the oxazolone ring) has conventionally been attributed to ring strain in a bicyclic oxazolone. Here we show, however, that the bicyclic oxazolone structure containing proline derived from protonated Gly-Pro-Gly is only 2.7 kcal/mol higher in free energy, at the B3LYP/6-31++G(d,p) level of theory, than the isomeric nonbicyclic oxazolone derived from protonated Pro-Gly-Gly; that is, strain appears to be a small or negligible factor. Formation of the y 2 ion in protonated Gly-Pro-Gly has a barrier of 3 kcal/mol higher free energy than that of the b 2 ion. In comparison, the difference increases to 10 kcal/mol in protonated Gly-Phe-Gly. The neutral products that are formed along with the y 2 ion are CO and methanimine, not aziridinone as once proposed.

The fragmentation of protonated tripeptides under metastable ion conditions and collision-induced conditions are reported. The majority of protonated tripeptides cleave at the C-terminal amide bond to form b 2 and y 1 ions, with the relative amounts depending on the proton affinities of the amino acids derived from the C-terminal amino acid residue. Protonated tripeptides that have a proline residue at the C-terminal position fragment almost exclusively by cleavage of the amide bond adjacent to the proline and give mainly y 1 ions; where there is a proline residue in the central position, fragmentation of the protonated tripeptide occurs at both the N-terminal and the C-terminal amide bonds to form y 2 and b 2 ions; finally, two of the tripeptides with proline at the N-terminal position, Pro-Pro-Pro and Pro-Gly-Gly, fragment largely by cleavage at the N-terminal, the former by cleavage of the amide bond and the latter by direct formation of the a 1 ion. Protonated Pro-Val-Gly fragments to give predominantly b 2 and a 2 ions at low energy, but the a 1 ion becomes the dominant product at higher energies. In the fragmentation of protonated peptides, the lack of b ions formed by cleavage at the amide bond C-terminal to proline (i.e., with the proline residue in the oxazolone ring) has conventionally been attributed to ring strain in a bicyclic oxazolone. Here we show, however, that the bicyclic oxazolone structure containing proline derived from protonated Gly-Pro-Gly is only 2.7 kcal/mol higher in free energy, at the B3LYP/6-31++G(d,p) level of theory, than the isomeric nonbicyclic oxazolone derived from protonated Pro-Gly-Gly; that is, strain appears to be a small or negligible factor. Formation of the y 2 ion in protonated Gly-Pro-Gly has a barrier of 3 kcal/mol higher free energy than that of the b 2 ion. In comparison, the difference increases to 10 kcal/mol in protonated Gly-Phe-Gly. The neutral products that are formed along with the y 2 ion are CO and methanimine, not aziridinone as once proposed.

The in vitro metabolism of transglutaminase-synthesized substance P analogs has been characterized comparing their stability to that of the parent peptide. The major metabolites have been purified and their structures elucidated by mass spectrometry. Our results 5 demonstrated that gln spermidine and spermine analogs of substance P possess an enhanced resistance to the action of proteases. 7 8 Moreover spermine, a large size hydrophilic compound, specifically prevented the hydrolysis at Phe -Phe bond.

Experimental Near-Edge X-ray Absorption Fine-Structure (NEXAFS) spectra of Nmethyltrifluoroacetamide (FNMA), which is a peptide model system, measured at the C, N, O and F K-edges are reported. The features in the spectra have been assigned by Static-Exchange (STEX) calculations. Using the same method, we have also assigned previously measured NEXAFS spectra of another peptide model system, N-methylacetamide (NMA). To facilitate the NEXAFS feature assignments, X-ray Photoelectron Spectroscopy (XPS) measurements for NMA and FNMA have been carried out with the aim to obtain the 1s electron ionization potentials, which are compared with the values predicted by our Hartree-Fock (∆HF) and Multi Configuration Self Consistent Field (∆MCSCF) calculations. We also demonstrate an approach to compensate for screening effects that are neglected in the STEX method. Ion yield measurements of FNMA associated with excitation of several C, N, O, and F K-shell pre-edge resonances have revealed site-specific fragmentation in some cases which we interpret with the aid of our theoretical calculations.

The use of proper computational methods and models has allowed answering the controversial question of whether zwitterionic species exist in the mechanism of peptide bond synthesis in aqueous solution. In fact, the different conformations of zwitterionic species open the door to different mechanistic paths.

In bacteria, ribosome stalling during translation of ErmBL leader peptide occurs in the presence of the antibiotic erythromycin and leads to induction of expression of the downstream macrolide resistance methyltransferase ErmB. The lack of structures of drug-dependent stalled ribosome complexes (SRCs) has limited our mechanistic understanding of this regulatory process. Here we present a cryo-electron microscopy structure of the erythromycin-dependent ErmBL-SRC. The structure reveals that the antibiotic does not interact directly with ErmBL, but rather redirects the path of the peptide within the tunnel. Furthermore, we identify a key peptide-ribosome interaction that defines an important relay pathway from the ribosomal tunnel to the peptidyltransferase centre (PTC). The PTC of the ErmBL-SRC appears to adopt an uninduced state that prevents accommodation of Lys-tRNA at the A-site, thus providing structural basis for understanding how the drug and the nascent peptide cooperate to inhibit peptide bond formation and induce translation arrest.

An increased degree of utilization of the potential N-glycosylation site in the fourth repeat unit of the human~protein may be involved in the inability of~to bind to the corresponding tubulin sequence(s) and in the subsequent development of the paired helical filaments of Alzheimer's disease. To model these processes, we synthesized the octadecapeptide spanning this region without sugar, and with the addition of an N-acetyl-glucosamine moiety. The carbohydrate-protected, glycosylated asparagine was incorporated as a building block during conventional Fmoc-solid phase peptide synthesis. While the crude non-glycosylated analog was obtained as a single peptide, two peptides with the identical, expected masses, in approximately equal amounts, were detected after the cleavage of the peracetylated glycopeptide. Surprisingly, the two glycopeptides switched positions on the reversed-phase high performance liquid chromatogram after removal of the sugar-protecting acetyl groups. Nuclear magnetic resonance spectroscopy and peptide sequencing identified the more hydrophobic deprotected peak as the target peptide, and the more hydrophilic deprotected peak as a peptide analog in which the aspartic acid-bond just preceding the glycosylated asparagine residue was isomerized resulting in the formation of a i-peptide. The anomalous chromatographic behavior of the acetylated i-isomer could be explained on the basis of the generation of an extended hydrophobic surface which is not present in any of the other three glycopeptide variants. Repetition of the syntheses, with altered conditions and reagents, revealed reproducibly high levels of aspartic acid-bond isomerization of the glycopeptide as well as lack of isomerization for the non-glycosylated parent analog. If similar increased aspartic acid-bond isomerization occurs in vivo, a protein modification well known to take place for both the amyloid deposits and the neurofibrillary tangles in Alzheimer's disease, this process may explain the aggregation of glycosylated~into the paired helical filaments in the affected brains.

Backbone cyclization has become an important method for generating or stabilizing the bioactive conformation of peptides without affecting the amino acid side-chains. Up to now, backbone cyclic peptides were mostly synthesized with bridges between N-amino-and N-carboxy-functionalized peptide bonds. To study the influence of a more flexible backbone on the biological activity, we have developed a new type of backbone cyclization which is achieved via the N-functionalized moieties of acylated reduced peptide bonds. As described in our previous publications, the formation of N-functionalized dipeptide units facilitates the peptide assembly compared with the incorporation of N-alkyl amino acids. Besides the racemization-free synthesis of Fmoc-protected pseudodipeptide esters with reduced peptide bonds, the new type of backbone modification allows the use of a great variety of-aminoand h,-dicarboxylic acids differing in chain length and chemical properties. Best results for the coupling of the-aminoand h,-dicarboxylic acids to the reduced peptide bond were obtained by the formation of mixed anhydrides with alkyl chloroformates. Whereas the protecting group combination of Z/OBzl in the dipeptide unit and Boc/OtBu for the N-functionalized moiety leads to the formation of 2-ketopiperazine during hydrogenation, the combination of Fmoc/ OtBu and Alloc/OAll is very suitable for the synthesis of backbone cyclic peptides on solid support.

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with Elongation Factor-Tu (EF-Tu) and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and consequently the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of EF-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer (smFRET). The time resolution of these measurements was extended to 2.5 and 10ms, a 10-50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

Aminoacyl-tRNA (aa-tRNA), in a ternary complex with Elongation Factor-Tu (EF-Tu) and GTP, enters the aminoacyl (A) site of the ribosome via a multi-step, mRNA codon-dependent mechanism. This process gives rise to the preferential selection of cognate aa-tRNAs for each mRNA codon and consequently the fidelity of gene expression. The ribosome actively facilitates this process by recognizing structural features of the correct substrate, initiated in its decoding site, to accelerate the rates of EF-Tu-catalyzed GTP hydrolysis and ribosome-catalyzed peptide bond formation. Here, the order and timing of conformational events underpinning the aa-tRNA selection process were investigated from multiple structural perspectives using single-molecule fluorescence resonance energy transfer (smFRET). The time resolution of these measurements was extended to 2.5 and 10ms, a 10-50-fold improvement over previous studies. The data obtained reveal that aa-tRNA undergoes fast conformational sampling within the A site, both before and after GTP hydrolysis. This suggests that the alignment of aa-tRNA with respect to structural elements required for irreversible GTP hydrolysis and peptide bond formation plays a key role in the fidelity mechanism. These observations provide direct evidence that the selection process is governed by motions of aa-tRNA within the A site, adding new insights into the physical framework that helps explain how the rates of GTP hydrolysis and peptide bond formation are controlled by the mRNA codon and other fidelity determinants within the system.

Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by ≈6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance...

Hydrogen bonds (H-bonds) are ubiquitous in peptides and proteins and are central to the stabilization of their structures. Inter-residue H-bonds between non-adjacent backbone amide NH and C=O motifs lead to the well-known secondary structures of helices, turns and sheets, but it is recognized that other H-bonding modes may be significant, including the weak intra-residue H-bond (called a C5 H-bond) that implicates the NH and C=O motifs of the same amino acid residue. Peptide model compounds that adopt stable C5 H-bonds are not readily available and the so-called 2.05-helix, formed by successive C5 H-bonds, is an elusive secondary structure. Using a combination of theoretical chemistry and spectroscopic studies in both the gas phase and solution phase, we have demonstrated that derivatives of 3-amino-1-methylazetidine-3-carboxylic acid, Aatc(Me) can form sidechain–backbone N–H···N C6γ H-bonds that accompany—and thereby stabilize—C5 H-bonds. In the capped trimer of Aatc(Me), extended ...

Previously only one of the six disulphide bonds within the β-subunit of bovine thyrotropin (bTSHβ) has been unequivocally assigned. In the present investigation, the fluorescent alkylating reagent 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulphonic acid has been employed as part of a double-alkylation strategy to allow the relative reactivities and the location of the six disulphide bonds of bTSHβ, after selective reduction, to be assigned by using reversed-phase HPLC peptide mapping techniques and associated methods of structural analysis. The most reactive disulphide bond was Cys88–Cys95; the second most reactive group of disulphide bonds involved the half-cystine residues Cys16, Cys19, Cys67 and Cys105 with the experimental results consistent with the assignment of disulphide bonds to Cys16–Cys67 and Cys19–Cys105. The least reactive group of half-cystine residues consisted of Cys2, Cys27, Cys31, Cys52, Cys83 and Cys85. The isolation, by high-performance ion-exchange chromatogr...

Amide bonds are among the most interesting and abundant molecules of life and products of the chemical pharmaceutical industry. In this work, we describe a method of the direct synthesis of amides from carboxylic acids and amines under solvent-free conditions using minute quantities of ceric ammonium nitrate (CAN) as a catalyst. The reactions are carried out in an open microwave reactor and allow the corresponding amides to be obtained in a fast and effective manner when compared to other procedures of the direct synthesis of amides from acids and amines reported so far in the literature. The amide product isolation procedure is simple, environmentally friendly, and is performed with no need for chromatographic purification of secondary amides due to high yields. In this report, primary amines were used in most examples. However, the developed procedure seems to be applicable for secondary amines as well. The methodology produces a limited amount of wastes, and a catalyst can be eas...

Selective nitrate-to-ammonia electrochemical conversion is an efficient pathway to solve the pollution of nitrate and an attractive strategy for lowtemperature ammonia synthesis. However, current studies for nitrate electroreduction (NO 3 RR) mainly focus on metal-based catalysts, which remains challenging because of the poor understanding of the catalytic mechanism. Herein, taking single transition metal atom supported on graphitic carbon nitrides (g-CN) as an example, the NO 3 RR feasibility of single-atom catalysts (SACs) is first demonstrated by using density functional theory calculations. The results reveal that highly efficient NO 3 RR toward NH 3 can be achieved on Ti/g-CN and Zr/g-CN with low limiting potentials of −0.39 and −0.41 V, respectively. Furthermore, the considerable energy barriers are observed during the formation of byproducts NO 2 , NO, N 2 O, and N 2 on Ti/g-CN and Zr/g-CN, guaranteeing their high selectivity. This work provides a new route for the application of SACs and paves the way to the development of NO 3 RR.

The interaction between several L‐amino acids and dipeptides and the ZnII complex with ligand L, a macrocycle containing a triamine chain linking the 2,9 positions of a phenanthroline moiety, has been studied by means of potentiometric and 1H NMR spectroscopic measurements in aqueous solutions. In the [ZnL]2+ complex, the metal ion displays an unsaturated coordination environment and the metal can act as a binding site for these substrates. Amino acids and dipeptides in their neutral (zwitterionic) form bind to ZnII through the carboxylate function and, when in their anionic form, through the amine group. In this case the carbonyl of the amide function is probably also involved in metal coordination. Amino acids containing aromatic pendants form the most stable complexes, due to hydrophobic and/or π‐stacking interactions between the aromatic subunits of the substrates and the phenanthroline moiety of the metal receptor. The hydrolytic properties of the ZnII complex with L, involving...

Factor IX (Christmas factor) is a single-chain plasma glycoprotein (mol wt 57,000) that participates in the middle phase of the intrinsic pathway of blood coagulation. It is present in plasma as a zymogen and is converted to a serine protease, Factor IXao, by Factor XIa (activated plasma thromboplastin antecedent) in the presence of calcium ions. In the activation reaction, two internal peptide bonds are hydrolyzed in Factor IX. These cleavages occur at a specific arginyl-alanine peptide bond and a specific arginyl-valine peptide bond. This results in the release of an activation peptide (mol wt-11,000) from the internal region of the precursor molecule and the generation of Factor IXaO (mol wt-46,000). Factor IXa, is composed ofa light chain (mol wt-18,000) and a heavy chain (mol wt-28,000), and these chains are held together by a disulfide bond(s). The light chain originates from the amino terminal portion of the precursor molecule and has an amino terminal sequence of Tyr-Asn-Ser-Gly-Lys. The heavy chain originates from the carboxyl terminal region of the precursor molecule and contains an amino terminal sequence of Val-Val-Gly-Gly-Glu. The heavy chain of Factor IXa, also contains the active site sequence of Phe-Cys-Ala-Gly-Phe-His-Glu-Gly-Gly-Arg-Asp-Ser-Cys-Gln-Gly-Asp-SER-Gly-Gly-Pro. The active site serine residue is shown in capital letters. Factor IX is also converted to Factor IXa,,: by a protease from Russell's viper venom. This activation reaction, however, occurs in a single step and involves only the cleavage of the internal arginyl-valine peptide bond. Human Factor IX,,3 was inhibited by human antithrombin III by the formation of a one-to-one complex of enzyme and inhibitor. In this reaction, the inhibitor was tightly bound to the heavy chain of the enzyme. These data indicate that the mechanism of activation of human Factor IX and its inhibition by antithrombin III is essentially identical to that previously shown for bovine Factor IX.

In addition, to further verify the applicability of the B3LYP/6-311+G* method, the MP2/6-311+G* method was also used to explore the isomers of Gly-Gly •+. Four isomers for Gly-Gly •+ obtained by the MP2/6-311+G* optimizations are similar to those obtained by B3LYP/6-311+G* calculations, as shown in Figure S1 with main structural parameters and relative energies. These results indicate that an additional method only causes the changes in the energy gaps among the same dipeptide isomers, but does not change their relative stability order. Therefore, the DFT optimizations and energy calculations were done utilizing the standard 6-311+G(d) basis set offering the proper description of the conformational isomers of a series of dipeptide cation radicals at a modest computational cost. Besides, for the isomers of Gly-Gly •+ , solvent effects were examined at the same level of theory as that in the gas phase by performing optimizations and single-point calculations on the optimized structures using the conductor polarized continuum model (CPCM) method. S1 In this model, the surrounding was represented by a constant dielectric medium outside a cavity containing the solute.