Peptide Bond Research Articles

The Peptide Bond: Resonance Increases Bond Order and Complicates Fragmentation.

The enhancement of the peptide bond order by a resonance in the lone pair of N and the π-bond of CO is analyzed. A decomposition of the bond order in terms of localized molecular orbitals is developed and applied to the peptide bond. A combination of two rotations of hybrid orbitals is proposed to improve the boundary treatment in the fragment molecular orbital method. The developed approach is applied to peptide bonds, and it is found crucial to retain the π orbital in the variational space of both fragments across the boundary. The interaction energies between conventional amino acid residues in Trp-cage (1L2Y) are discussed.

Backbone Modification in a Protein Hydrophobic Core.

Targeted protein backbone modification can recreate tertiary structures reminiscent of folds found in nature on artificial scaffolds with improved biostability. Incorporation of altered monomers in such entities is typically limited to sites distant from the hydrophobic core to avoid potential disruptions to folding. This is limiting, as it is advantageous in some applications to incorporate artificial connectivity at buried sites. Here, we report an examination of protein backbone modification targeted specifically to hydrophobic core positions and its impacts on tertiary folded structure and fold stability. Different artificial monomer types are placed at core, core-flanking, or solvent-exposed positions in a compact three-helix protein. Effects on structure and folding energetics are assessed by NMR spectroscopy and biophysical methods. Results show that artificial residues can be well accommodated in the hydrophobic core of a defined tertiary fold, with effects on stability only modestly larger than identical changes at solvent-exposed sites. Collectively, these results provide new insights into folding behavior of protein-like artificial chains as well as strategies for the design of such molecules.

Tailoring interactions for cisPro peptide bond stabilization

Elucidation of the nature of non-covalent interactions that govern the rate-limiting cis-trans isomerism at Xaa-Pro peptide bonds is fundamental to unravelling the protein folding mechanism, the stereoelectronic control elements of the structure and dynamics of the peptide bond, and the design of novel peptide isosteres. CisPro rotamers are stabilized by very few interactions compared to the transPro rotamer and are hence relatively scarcely populated. Design of novel interactions that can bias cisPro stability, with least mutations to the prolyl peptide bond, is crucial for accessing the cisPro motif in a variety of peptides. This mini review discusses the various interactions tailored for improving cisPro stability.

The conceptual design of pH responsive ZnO-adamantane nanosystems for insulin amyloidosis

Adamantane derivatives are already being explored in amyloidogenic diseases, while ZnO nanoparticles (NPs) have the potential for affecting amyloidosis. Herein, the transformation of ZnO NPs and adamantane-1-carboxylic acid (ADA) to advanced pH responsive nanosystems is reported. Primary ZnO NPs were isolated under a simple solvothermal self-assembly synthetic procedure in the sole presence of octadecylamine (ODA). By adjusting the reaction time (8–24 h), wurtzites of crystallite sizes 19, 21 and 23 nm, respectively, were synthesized with co-crystallized ODA in monoloayer or bilayer conformation. The NPs with the bilayer of ODA have been functionalized with ADA through an amide bond, while those with the ODA monolayer were further PEGylated and ADA was loaded. Loaded ADA-ZnO nanosystems affected insulin amyloidosis in acidic pH (4.8), both in terms of final aggregation status (40 %) and by retarding the lag phase of amyloids formation. The sustained release profile of ADA was confirmed, while interaction with calf thymus DNA (CT-DNA) was evidenced by hyperchromicity. The pH of 4.8 was not enough to cleave the peptide bond, and thus the direct conjugation of ADA was effective only in prolonging the lag phase, without affecting the final amyloid formation, giving rise to the inherent properties of ZnO, foregrounding a prodrug character.

Observation of complex organic molecules containing peptide-like bonds towards hot core G358.93--0.03 MM1

Abstract In the star formation regions, the complex organic molecules that contain peptide bonds (–NH–C(=O)–) play a major role in the metabolic process because –NH–C(=O)– is connected to amino acids (R-CHNH2–COOH). Over the past few decades, many complex organic molecules containing peptide-like bonds have been detected in hot molecular cores, hot corinos, and cold molecular clouds, however, their prebiotic chemistry is poorly understood. We present the first detection of the rotational emission lines of formamide (NH2CHO) and isocyanic acid (HNCO), which contain peptide-like bonds towards the chemically rich hot molecular core G358.93–0.03 MM1, using high-resolution and high-sensitivity Atacama Large Millimeter/submillimeter Array (ALMA) bands 6 and 7. We estimate that the column densities of NH2CHO and HNCO towards G358.93–0.03 MM1 are (2.80±0.29)×10^{15} cm^{−2} and (1.80±0.42)×10^{16} cm^{−2} with excitation temperatures of 165±21 K and 170±32 K, respectively. The fractional abundances of NH2CHO and HNCO towards G358.93–0.03 MM1 are (9.03±1.44)×10^{−10} and (5.80±2.09)×10^{−9}. We compare the estimated abundances of NH2CHO and HNCO with the existing three-phase warm-up chemical model abundance values and notice that the observed and modelled abundances are very close. We conclude that NH2CHO is produced by the reaction of NH2 and H2CO in the gas phase towards G358.93–0.03 MM1. Likewise, HNCO is produced on the surface of grains by the reaction of NH and CO towards G358.93–0.03 MM1. We also found that NH2CHO and HNCO are chemically linked towards G358.93–0.03 MM1.

Raman Spectra of Amino Acids and Peptides from Machine Learning Polarizabilities.

Raman spectroscopy is an important tool in the study of vibrational properties and composition of molecules, peptides, and even proteins. Raman spectra can be simulated based on the change of the electronic polarizability with vibrations, which can nowadays be efficiently obtained via machine learning models trained on first-principles data. However, the transferability of the models trained on small molecules to larger structures is unclear, and direct training on large structures is prohibitively expensive. In this work, we first train two machine learning models to predict the polarizabilities of all 20 amino acids. Both models are carefully benchmarked and compared to density functional theory (DFT) calculations, with the neural network method being found to offer better transferability. By combination of machine learning models with classical force field molecular dynamics, Raman spectra of all amino acids are also obtained and investigated, showing good agreement with experiments. The models are further extended to small peptides. We find that adding structures containing peptide bonds to the training set greatly improves predictions, even for peptides not included in training sets.

Effects of Phosphorylation on Protein Backbone Dynamics and Conformational Preferences.

Phosphorylations are the most common and extensively studied post-translational modification (PTM) of proteins in eukaryotes. They constitute a major regulatory mechanism, modulating protein function, protein-protein interactions, as well as subcellular localization. Phosphorylation sites are preferably located in intrinsically disordered regions and have been shown to trigger structural rearrangements and order-to-disorder transitions. They can therefore have a significant effect on protein backbone dynamics or conformation, but only sparse experimental data are available. To obtain a more general description of how and when phosphorylations have a significant effect on protein behavior, molecular dynamics (MD) currently provides the only suitable framework to study these effects at a large scale in atomistic detail. This study develops a systematic MD simulation framework to explore the influence of phosphorylations on the local backbone dynamics and conformational propensities of proteins. Through a series of glycine-backbone peptides, we studied the effects of amino acid residues including the three most common phosphorylations (Ser, Thr, and Tyr), on local backbone dynamics and conformational propensities. We further extended our study to investigate the interactions of all such residues between position i to positions i + 1, i + 2, i + 3, and i + 4 in such peptides. The final data set comprises structural ensembles for 3393 sequences with more than 1 μs of sampling for each ensemble. To validate the relevance of the results, the structural and conformational properties extracted from the MD simulations are compared to NMR data from the Biological Magnetic Resonance Data Bank. The systematic nature of this study enables the projection of the gained knowledge onto any phosphorylation site in the proteome and provides a general framework for the study of further PTMs. The full data set is publicly available, as a training and reference set.

From Zero to Hero: The Cyanide-Free Formation of Amino Acids and Amides from Acetylene, Ammonia and Carbon Monoxide in Aqueous Environments in a Simulated Hadean Scenario.

Amino acids are one of the most important building blocks of life. During the biochemical process of translation, cells sequentially connect amino acids via amide bonds to synthesize proteins, using the genetic information in messenger RNA (mRNA) as a template. From a prebiotic perspective (i.e., without enzymatic catalysis), joining amino acids to peptides via amide bonds is difficult due to the highly endergonic nature of the condensation reaction. We show here that amides can be formed in reactions catalyzed by the transition metal sulfides from acetylene, carbon monoxide and ammonia under aqueous conditions. Some α- and β-amino acids were also formed under the same conditions, demonstrating an alternative cyanide-free path for the formation of amino acids in prebiotic environments. Experiments performed with stable isotope labeled precursors, like 15NH4Cl and 13C-acetylene, enabled the accurate mass spectroscopic identification of the products formed from the starting materials and their composition. Reactions catalyzed using the transition metal sulfides seem to offer a promising alternative pathway for the formation of amides and amino acids in prebiotic environments, bypassing the challenges posed by the highly endergonic condensation reaction. These findings shed light on the potential mechanisms by which the building blocks of life could have originated on early Earth.

The Power of Molecular Dynamics Simulations and Their Applications to Discover Cysteine Protease Inhibitors.

A large family of enzymes with the function of hydrolyzing peptide bonds, called peptidases or cysteine proteases (CPs), are divided into three categories according to the peptide chain involved. CPs catalyze the hydrolysis of amide, ester, thiol ester, and thioester peptide bonds. They can be divided into several groups, such as papain-like (CA), viral chymotrypsin-like CPs (CB), papain-like endopeptidases of RNA viruses (CC), legumain-type caspases (CD), and showing active residues of His, Glu/Asp, Gln, Cys (CE). The catalytic mechanism of CPs is the essential cysteine residue present in the active site. These mechanisms are often studied through computational methods that provide new information about the catalytic mechanism and identify inhibitors. The role of computational methods during drug design and development stages is increasing. Methods in Computer-Aided Drug Design (CADD) accelerate the discovery process, increase the chances of selecting more promising molecules for experimental studies, and can identify critical mechanisms involved in the pathophysiology and molecular pathways of action. Molecular dynamics (MD) simulations are essential in any drug discovery program due to their high capacity for simulating a physiological environment capable of unveiling significant inhibition mechanisms of new compounds against target proteins, especially CPs. Here, a brief approach will be shown on MD simulations and how the studies were applied to identify inhibitors or critical information against cysteine protease from several microorganisms, such as Trypanosoma cruzi (cruzain), Trypanosoma brucei (rhodesain), Plasmodium spp. (falcipain), and SARS-CoV-2 (Mpro). We hope the readers will gain new insights and use our study as a guide for potential compound identifications using MD simulations.

Development and Challenges of Cyclic Peptides for Immunomodulation.

Cyclic peptides are polypeptide chains formed by cyclic sequences of amide bonds between protein-derived or non-protein-derived amino acids. Compared to linear peptides, cyclic peptides offer several unique advantages, such as increased stability, stronger affinity, improved selectivity, and reduced toxicity. Cyclic peptide has been proved to have a promising application prospect in the medical field. In addition, this paper mainly describes that cyclic peptides play an important role in anti-cancer, anti-inflammatory, anti-virus, treatment of multiple sclerosis and membranous nephropathy through immunomodulation. In order to know more useful information about cyclic peptides in clinical research and drug application, this paper also summarizes cyclic peptides currently in the clinical trial stage and cyclic peptide drugs approved for marketing in the recent five years. Cyclic peptides have many advantages and great potential in treating various diseases, but there are still many challenges to be solved in the development process of cyclic peptides.

Destruxin E backbone modification effects on osteoclast Morphology: Synthesis and SAR study of N-Desmethyl and N-Methyl analogs

The design and synthesis of N-desmethyl and N-methyl destruxin E analogs have been demonstrated. The X-ray single crystal structure of destruxin E (1a) revealed a stable three-dimensional (3D) structure, including a s-cis amide bond at the MeVal–MeAla moiety and two intramolecular hydrogen bonds between NH(β-Ala) and OC(Ile) and between NH(Ile) and OC(β-Ala). N-Desmethyl analogs 2a (MeAla → Ala) and 2b (MeVal → Val) were synthesized through macrolactonization similar to our previously reported synthesis of 1a. Conversely, for the synthesis of N-methyl analogs 2c (Ile → MeIle) and 2d (β-Ala → Meβ-Ala), macrolactonization did not proceed; therefore, cyclization precursors 10c and 10d were designed to maintain the intramolecular hydrogen bonds described above during their cyclization. The macrolactamization proceeded despite the presence of a less reactive N-methylamino group at the N-terminus in both cases. Analog 2a, which exhibits multiple conformers in solutions, was inactive at 50 μM, whereas analog 2b, which exhibits a conformation similar to that of 1a in solutions, exhibited morphological changes against osteoclast-like multinuclear cells at 1.6 μM. The activity of the MeIle analog 2c, which cannot take the intramolecular hydrogen bond (Ile)NH•••OC(β-Ala) in 1a, was markedly diminished compared with that of 1a, and that of the Meβ-Ala analog 2d, which cannot take the intramolecular hydrogen bond (β-Ala)NH•••OC(Ile) in 1a, was further reduced to one-fourth of that of 2c. The overall results indicate that both the s-cis amide bond at the MeVal–MeAla moiety and two intramolecular hydrogen bonds (β-Ala)NH•••OC(Ile) and (Ile)NH•••OC(β-Ala) are important for constraining the conformation of the macrocyclic peptide backbone in destruxin E, thereby exhibiting its potent biological activity.

Reversing the relative time courses of the peptide bond reaction with oligopeptides of different lengths and charged amino acid distributions in the ribosome exit tunnel

The kinetics of the protein elongation cycle by the ribosome depends on intertwined factors. One of these factors is the electrostatic interaction of the nascent protein with the ribosome exit tunnel. In this computational biology theoretical study, we focus on the rate of the peptide bond formation and its dependence on the ribosome exit tunnel electrostatic potential profile. We quantitatively predict how oligopeptides of variable lengths can affect the peptide bond formation rate. We applied the Michaelis-Menten model as previously extended to incorporate the mechano-biochemical effects of forces on the rate of reaction at the catalytic site of the ribosome. For a given pair of carboxy-terminal amino acid substrate at the P- and an aminoacyl-tRNA at the A-sites, the relative time courses of the peptide bond formation reaction can be reversed depending on the oligopeptide sequence embedded in the tunnel and their variable lengths from the P-site. The reversal is predicted to occur from a shift in positions of charged amino acids upstream in the oligopeptidyl-tRNA at the P-site. The position shift must be adjusted by clever design of the oligopeptide probes using the electrostatic potential profile along the exit tunnel axial path. These predicted quantitative results bring strong evidence of the importance and relative contribution of the electrostatic interaction of the ribosome exit tunnel with the nascent peptide chain during elongation.

Mechanofluorochromic behaviors and latent fingerprint detection of triphenylamine-based compounds with mono-/bis-BF2 fluorophores

To better understand the relationship between molecular structure of the mono-/bis-BF2-core compounds and mechanofluoroboron behaviors, two pyridine-based difluoroboron compounds with triphenylamine group (TPA-ts-BF2 and TPA-ts-2BF2) were designed and successfully synthesized, which TPA-ts-BF2 including a BF2 fluorophore and TPA-ts-2BF2 containing the bisBF2 fluorophores. Based on the photophysical properties measurements results, it was found that TPA-ts-2BF2 had more excellent intramolecular charge transfer characteristics than that of TPA-ts-BF2, and exhibited significant aggregation-induced emission activity, however, TPA-ts-BF2 displayed typical aggregation-caused quenching phenomenon. Meanwhile, the emission spectrum of the solid powders of TPA-ts-2BF2 was red-shifted 52 nm after grinding, that of TPA-ts-BF2 was red-shifted 46 nm, which was resulted from crystalline state switching to amorphous state. According to the theoretical calculations, we conjectured that TPA-ts-BF2 with uncoordinated amide linkage moiety had a tendency to forming a more twisted conformance and higher molecular polarity, which made that mechanofluorochromic behavior was worse than that of TPA-ts-2BF2. Additionally, TPA-ts-2BF2 was applied to latent fingerprint detection due to its prime aggregation-induced emission property.

LC-MS/MS-based peptidomics and metabolomics reveal different metabolic patterns of common spoilage bacteria in grass carp flesh

Grass carp are highly perishable under the action of spoilage bacteria including Aeromonas rivipollensis, Pseudomonas putida, and Shewanella putrefaciens. However, the metabolic characteristics of the three bacteria in degrading fish proteins and small molecular nutrients are unclear. In this study, we used LC-MS/MS-based peptidomics and metabolomics to detect peptides and small molecular metabolites in grass carp flesh inoculated with the three bacteria. The peptidomic results showed that A. rivipollensis and S. putrefaciens induced an overall increase in the molecular weight of peptides in grass carp flesh. S. putrefaciens tended to hydrolyze peptide bonds composed of at least one hydrophobic amino acid whereas A. rivipollensis and P. putida preferred hydrolyzing Xaa-serine bonds. The metabolomic results showed that amino acid metabolism and nucleotide metabolism are among the most enriched pathways of differential metabolites. P. putida and S. putrefaciens were active in decomposing amino acids and nucleosides, respectively. Besides, lipid metabolism also plays an important role in grass carp spoilage, representing as the degradation from phosphatidylcholine to lysophosphatidylcholine by the three bacteria. To conclude, this study revealed distinct metabolic characteristics of the three bacteria in degrading proteins and small molecular metabolites, and thus contribute to the understanding of fish spoilage mechanisms.

Potency-Enhanced Peptidomimetic VHL Ligands with Improved Oral Bioavailability.

The von Hippel-Lindau (VHL) protein plays a pivotal role in regulating the hypoxic stress response and has been extensively studied and utilized in the targeted protein degradation field, particularly in the context of bivalent degraders. In this study, we present a comprehensive peptidomimetic structure-activity relationship (SAR) approach, combined with cellular NanoBRET target engagement assays to enhance the existing VHL ligands. Through systematic modifications of the molecule, we identified the 1,2,3-triazole group as an optimal substitute of the left-hand side amide bond that yields 10-fold higher binding activity. Moreover, incorporating conformationally constrained alterations on the methylthiazole benzylamine moiety led to the development of highly potent VHL ligands with picomolar binding affinity and significantly improved oral bioavailability. We anticipate that our optimized VHL ligand, GNE7599, will serve as a valuable tool compound for investigating the VHL pathway and advancing the field of targeted protein degradation.

Titin mechanical unloading disrupts sarcomere tensional homeostasis triggering fast, non-canonical myocardial fibrosis

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): We acknowledge funding from Ministerio de Ciencia e Innovación (Madrid, Spain) through grant FJC2021 047055-I and European Research Council (ERC) through grant H2020-ERC-2020-COG (ProtMechanics-Live). Background/Introduction Stresses exerted during myocardial contraction and relaxation cycles are mainly braced by mechanical proteins located in the extracellular matrix (ECM) and cardiomyocytes (CMs). Titin (TTN) is a gigantic protein that scaffolds the sarcomere (i.e. the basic unit of contraction). Since most of the CM cytoskeleton is occupied by sarcomeres, titin is an essential structural hub for the maintenance of a homeostatic level of mechanical tension. Purpose It has been shown that mechanical disruption of the ECM affects the equilibrium forces between CMs and the ECM, leading to morphological changes in sarcomeres. However, potential alteration of tensional homeostasis resulting from defective mechanics of CM proteins remains unknown due to the lack of tools for in vivo studies. Therefore, the aim of this study is to explore the in vivo response of the myocardium to the abrupt cessation of the mechanical properties of titin. Methods For this purpose, we have experimentally challenged CMs to a tensional unloading by means of a titin cleavage model (TEVs-TTN) [1], both in vitro, using neonatal CM (NMCM), and in vivo. In this model, a cassette with a tobacco etch virus protease (TEVp) recognition sequence has been included in the I-band of titin. Transducing TEVp with adeno-associated virus (myoAAV), we were able to cleave titin in living CMs, ceasing only its mechanical properties. Samples were analyzed using an array of techniques such as histology, immunofluorescence and bulk and single-nucleus RNAseq. Results Our results show that while in NMCM we obtain a complete titin cleavage, in vivo we observe mosaic expression of TEVp and no more than 30% cleavage of the protein. In both cases, TEVp expression in TEVs-TTN does not affect cell viability. However, mild myocardial titin cleavage leads to a fibrotic response in less than six days, characterized by an increase in interstitial collagen and an expansion of cardiac fibroblasts population. Transcriptional and phospo-SMAD2/3 analysis results suggest that this fibrotic response occurs without activation of the canonical TGFβ pathway. Conclusion Taken together, our results suggest that the loss of titin mechanical-structural function in cardiomyocytes derived from the cleavage of a single peptide bond elicits a fast fibrotic compensatory response in the myocardium through intercellular communication with fibroblasts.

Creation of Single Cell Protein-Producing Mutants of Phaffia Rhodozyma

Recently, there has been an increasing demand for sustainable protein sources for aquaculture. In aquaculture, sustainable protein includes sources that can be produced with a minimal environmental footprint, such as single-cell proteins, proteins from algae, insects, and by-products from the agricultural industry. Single-cell proteins that are derived from microorganisms show a sustainable alternative to traditional protein sources due to their high protein content and rapid growth. This research focuses on creating mutants of the yeast Phaffia rhodozyma to increase single-cell protein production. This yeast has significant industrial potential and biological features. The yeast Phaffia rhodozyma is already known for its unique ability to synthesize astaxanthin, so this case will focus on its ability to produce protein. In the study, we used wild strain yeast and previously obtained white mutants on inorganic nitrogen media for further random mutagenesis methods to introduce specific mutations into the genome aimed at improving their ability to biosynthesize protein. Amino acid inhibitor was used to select potential protein-producing mutants. The protein content of the biomass obtained from the experiments was analyzed in detail using BCA (Protein Assay Reagent) analysis, which confirmed their potential as a source of nutrients for aquaculture. This method is based on the reaction between bicinchoninic acid and the peptide bonds of proteins, which results in the formation of a color whose intensity is proportional to the amount of protein in the sample. The results obtained allow further research into the development of cost-effective and environmentally friendly alternatives for feed in aquaculture.

NMR study of the dynamic equilibria among isomeric species in quinoxalin-2-one derivatives

The understanding of the prototropic lactam/lactim/enol equilibrium and the E/Z isomerism around the N-C(O) bond in amides is important for the determination of different physicochemical properties in compounds, moreover in compounds with more than one equilibrium. In this work, these equilibria were studied for N-acyl and N-carbamoyl-3,4-dihydroquinoxalin-2(1H)-ones using mono- and bi-dimensional NMR experiments, at different temperatures. For both types of derivatives, the lactam form was the only tautomer detected. The results obtained from molecular modelling experiments agree with the experimental results. For the N-acyl derivative, the E isomer is the predominant, while for the N-carbamoyl derivative both geometric isomers are present in a similar ratio. The theoretical calculations allowed the signal assignment for each isomer from the chemical shift values estimated for both geometric isomers corresponding to the studied N-acyl and N-carbamoyl derivatives.

Micropore engineering of polyamide loose nanofiltration membrane for efficient dye/salt separation

Loose nanofiltration membranes (LNF) with efficient sieving and excellent permeance are desirable for dye/salt separation, and engineering the micropore of the LNF membranes is a promising approach to maximize membrane performance. Herein, we present a highly permeable and sieving polyamide LNF membrane with high porosity and suitable pore size that is prepared by interfacial polymerization using twisted aqueous phase monomer and subsequent alkali-treatment. The large-size, rigid and twisted structure of the adamantane-1,3-diamine effectively inhibits the dense-packing of polymer chain segments, resulting in a polyamide membrane with enhanced porosity and enlarged pore size. The alkali treatment hydrolyzes the partial amide linkage to further enlarge the membrane pore size, increasing the flux while maintaining high dye rejection. The optimum membrane exhibits a high salt/dye selective factor of 735 for Na2SO4/Evans blue and an excellent water permeance of 87.72 L m−2 h−1 bar−1. This study is expected to provide a valuable guidance for the targeted design and fabrication of LNF membranes.

Synthesis of N-Hydroxysuccinimide Esters, N-Acylsaccharins, and Activated Esters from Carboxylic Acids Using I2/PPh3.

A method for the syntheses of isolable, active esters is described in which carboxylic acids are treated with triphenylphosphine, iodine, and triethylamine. Active esters accessible in this way include N-hydroxysuccinimide esters, N-hydroxyphthalimide esters (N-(acyloxy)phthalimides), N-acylsaccharins, pentafluorophenol esters, pentachlorophenol esters, N-hydroxybenzotriazole esters, and hexafluoro-2-propanol esters. The approach can be similarly applied toward the formation of N-acylsaccharins and N-acylimidazoles. The method is suitable for the formation of isolable active esters of aromatic and aliphatic activated acids as well as α-amino acid derivatives. These products are widely used reagents in organic synthesis, peptide synthesis, medicinal chemistry, and chemical biology (e.g., for bioconjugations). The method has broad substrate scope, uses simple and inexpensive reagents, avoids the use of carbodiimides or other coupling agents, and occurs at room temperature. Additionally, the diastereomers of compound Boc-Ala-NHCHPh are demonstrated to be distinguishable by 1H NMR (in DMSO-d6), allowing for a straightforward NMR method to establish the degree of racemization of activated esters of Boc-Ala or amide bond formations using Boc-Ala.