Prebiotic dimer and trimer peptide formation in gas-phase atmospheric nanoclusters of water.

Insight into the origin of prebiotic molecules is key to our understanding of how living systems evolved into the complex network of biological processes on Earth. By modelling diglycine and triglycine peptide formation in the prebiotic atmosphere, we provide a plausible pathway for peptide growth. By examining different transition states (TSs), we conclude that the formation of diglycine and triglycine in atmospheric nanoclusters of water in the prebiotic atmosphere kinetically favors peptide growth by an N-to-C synthesis of glycines through a trans conformation. Addition of water stabilizes the TS structures and lowers the Gibbs free activation energies. At temperatures that model the prebiotic atmosphere, the free energies of activation with a six water nanocluster as part of the TS are predicted to be 16 kcal mol-1 relative to the prereactive complex. Examination of the trans vs. cis six water transition states reveals that a homodromic water network that maximizes the acceptor/donor nature of the six waters is responsible for enhanced kinetic favorability of the trans N-to-C pathway. Compared to the non-hydrated trans TS, the trans six-water TS accelerates the reaction of diglycine and glycine to form triglycine by 13 orders of magnitude at 217 K. Nature uses the trans N-to-C pathway to synthesize proteins in the ribosome, and we note the similarities in hydrogen bond stabilization between the transition state for peptide synthesis in the ribosome and the transition states formed in nanoclusters of water in the same pathway. These results support the hypothesis that small oligomers formed in the prebiotic atmosphere and rained onto earth's surface.

Formation of Fluorescent Maillard Reaction Intermediates of Peptide and Glucose during Thermal Reaction and Its Mechanism.

The dynamic changes in fluorescence intensity of the Maillard reactions of l-alanyl-l-glutamine (Ala-Gln)/Diglycine (Gly-Gly)/glycyl-l-glutamine (Gly-Gln) and glucose were investigated. It was found that the fluorescence intensity would increase with the reaction time; however, it would decrease after longer heating at higher temperatures, which was accompanied by rapid browning. The strongest intensity occurred at 45, 35, and 35 min at 130 °C for Ala-Gln, Gly-Gly, and Gly-Gln systems, respectively. The simple model reactions of Ala-Gln/Gly-Gly and dicarbonyl compounds were selected to reveal the formation and mechanism of fluorescent Maillard compounds. It was confirmed that both GO and MGO could react with peptides to form fluorescent compounds, especially GO, and this reaction was sensitive to temperature. The mechanism was also verified in the complex Maillard reaction of pea protein enzymatic hydrolysates.

Glycine, Diglycine, and Triglycine Exhibit Different Reactivities in the Formation and Degradation of Amadori Compounds.

A series of Amadori compounds of glucose were prepared from glycine (G-ARP), diglycine (DiG-ARP), and triglycine (TriG-ARP), and identified by UPLC-MS/MS and NMR. The formation rate of ARPs was TriG-ARP > DiG-ARP > G-ARP, and their activation energies were 63.48 kJ/mol (TriG-ARP), 72.84 kJ/mol (DiG-ARP), and 84.76 kJ/mol (G-ARP), respectively, suggesting that ARP was formed more easily from small peptides than from amino acid. Although 1-DG was formed much more difficultly than 3-DG, the same order of the formation of 1-DG, 3-DG, and browning was DiGly > TriGly > Gly. It was also confirmed that more methylglyoxal and glyoxal would be formed from small peptides than equimolar amino acids. Compared with free amino acid, ARP, deoxyglycosones, and their secondary degradation products were more easily formed from dipeptide and tripeptide, thereby stronger browning occurred and higher reactivity was exhibited in Maillard reaction of di- or tripeptide.

Hydration of Simple Model Peptides in Aqueous Osmolyte Solutions.

The biology and chemistry of proteins and peptides are inextricably linked with water as the solvent. The reason for the high stability of some proteins or uncontrolled aggregation of others may be hidden in the properties of their hydration water. In this study, we investigated the effect of stabilizing osmolyte-TMAO (trimethylamine N-oxide) and destabilizing osmolyte-urea on hydration shells of two short peptides, NAGMA (N-acetyl-glycine-methylamide) and diglycine, by means of FTIR spectroscopy and molecular dynamics simulations. We isolated the spectroscopic share of water molecules that are simultaneously under the influence of peptide and osmolyte and determined the structural and energetic properties of these water molecules. Our experimental and computational results revealed that the changes in the structure of water around peptides, caused by the presence of stabilizing or destabilizing osmolyte, are significantly different for both NAGMA and diglycine. The main factor determining the influence of osmolytes on peptides is the structural-energetic similarity of their hydration spheres. We showed that the chosen peptides can serve as models for various fragments of the protein surface: NAGMA for the protein backbone and diglycine for the protein surface with polar side chains.

The Interfacial Interactions of Glycine and Short Glycine Peptides in Model Membrane Systems.

The interactions of amino acids and peptides at model membrane interfaces have considerable implications for biological functions, with the ability to act as chemical messengers, hormones, neurotransmitters, and even as antibiotics and anticancer agents. In this study, glycine and the short glycine peptides diglycine, triglycine, and tetraglycine are studied with regards to their interactions at the model membrane interface of Aerosol-OT (AOT) reverse micelles via 1H NMR spectroscopy, dynamic light scattering (DLS), and Langmuir trough measurements. It was found that with the exception of monomeric glycine, the peptides prefer to associate between the interface and bulk water pool of the reverse micelle. Monomeric glycine, however, resides with the N-terminus in the ordered interstitial water (stern layer) and the C-terminus located in the bulk water pool of the reverse micelle.

Synthesis, Docking, Computational Studies, and Antimicrobial Evaluations of New Dipeptide Derivatives Based on Nicotinoylglycylglycine Hydrazide.

Within a series of dipeptide derivatives (5-11), compound 4 was refluxed with d-glucose, d-xylose, acetylacetone, diethylmalonate, carbon disulfide, ethyl cyanoacetate, and ethyl acetoacetate which yielded 5-11, respectively. The candidates 5-11 were characterized and their biological activities were evaluated where they showed different anti-microbial inhibitory activities based on the type of pathogenic microorganisms. Moreover, to understand modes of binding, molecular docking was used of Nicotinoylglycine derivatives with the active site of the penicillin-binding protein 3 (PBP3) and sterol 14-alpha demethylase's (CYP51), and the results, which were achieved via covalent and non-covalent docking, were harmonized with the biological activity results. Therefore, it was extrapolated that compounds 4, 7, 8, 9, and 10 had good potential to inhibit sterol 14-alpha demethylase and penicillin-binding protein 3; consequently, these compounds are possibly suitable for the development of a novel antibacterial and antifungal therapeutic drug. In addition, in silico properties of absorption, distribution, metabolism, and excretion (ADME) indicated drug likeness with low to very low oral absorption in most compounds, and undefined blood-brain barrier permeability in all compounds. Furthermore, toxicity (TOPKAT) prediction showed probability values for all carcinogenicity models were medium to pretty low for all compounds.

Degronomics: Mapping the Interacting Peptidome of a Ubiquitin Ligase Using an Integrative Mass Spectrometry Strategy.

Human cells make use of hundreds of unique ubiquitin E3 ligases to ensure proteome fidelity and control cellular functions by promoting protein degradation. These processes require exquisite selectivity, but the individual roles of most E3s remain poorly characterized in part due to the challenges associated with identifying, quantifying, and validating substrates for each E3. We report an integrative mass spectrometry (MS) strategy for characterizing protein fragments that interact with KLHDC2, a human E3 that recognizes the extreme C-terminus of substrates. Using a combination of native MS, native top-down MS, MS of destabilized samples, and liquid chromatography MS, we identified and quantified a near complete fraction of the KLHDC2-binding peptidome in E. coli cells. This degronome includes peptides that originate from a variety of proteins. Although all identified protein fragments are terminated by diglycine or glycylalanine, the preceding amino acids are diverse. These results significantly expand our understanding of the sequences that can be recognized by KLHDC2, which provides insight into the potential substrates of this E3 in humans. We anticipate that this integrative MS strategy could be leveraged more broadly to characterize the degronomes of other E3 ligase substrate receptors, including those that adhere to the more common N-end rule for substrate recognition. Therefore, this work advances "degronomics," i.e., identifying, quantifying, and validating functional E3:peptide interactions in order to determine the individual roles of each E3.

Local electronic structure of the peptide bond probed by resonant inelastic soft X-ray scattering.

The local valence orbital structure of solid glycine, diglycine, and triglycine is studied using soft X-ray emission spectroscopy (XES), resonant inelastic soft X-ray scattering (RIXS) maps, and spectra calculations based on density-functional theory. Using a building block approach, the contributions of the different functional groups of the peptides are separated. Cuts through the RIXS maps furthermore allow monitoring selective excitations of the amino and peptide functional units, leading to a modification of the currently established assignment of spectral contributions. The results thus paint a new-and-improved picture of the peptide bond, enhance the understanding of larger molecules with peptide bonds, and simplify the investigation of such molecules in aqueous environment.

Diglycine Enables Rapid Intrabacterial Hydrolysis for Activating Anbiotics against Gram-negative Bacteria.

Antimicrobial drug resistance demands novel approaches for improving the efficacy of antibiotics, especially against Gram-negative bacteria. Herein, we report that conjugating a diglycine (GG) to an antibiotic prodrug drastically accelerates intrabacterial ester-bond hydrolysis required for activating the antibiotic. Specifically, the attachment of GG to chloramphenicol succinate (CLsu) generates CLsuGG, which exhibits about an order of magnitude higher inhibitory efficacy than CLsu against Escherichia coli. Further studies reveal that CLsuGG undergoes rapid hydrolysis, catalyzed by intrabacterial esterases (e.g., BioH and YjfP), to generate chloramphenicol (CL) in E. coli. Importantly, the conjugate exhibits lower cytotoxicity to bone marrow stromal cells than CL. Structural analogues of CLsuGG indicate that the conjugation of GG to an antibiotic prodrug is an effective strategy for accelerating enzymatic prodrug hydrolysis and enhancing the antibacterial efficacy of antibiotics.

Timely Addition of Glutathione for Its Interaction with Deoxypentosone To Inhibit the Aqueous Maillard Reaction and Browning of Glycylglycine-Arabinose System.

The inhibitory effects of glutathione (GSH) and oxiglutathione (GSSG) on Maillard browning were compared, and it was clarified that free sulfhydryl was the key substance for the inhibition. The Amadori rearrangement product (ARP) derived from glycylglycine (Gly-Gly) and arabinose (Ara) was prepared by aqueous Maillard reaction, and LC-MS/MS was used to investigate the reaction products of GSH and purified ARP. Reaction between GSH and deoxypentosone (DP) was found to alter the pathway of aqueous Maillard reaction, which reduced the production of glyoxal, methylglyoxal, and furfural and thereby inhibited the formation of melanoidins. To determine the optimal conditions for browning inhibition, a stepwise increase of temperature was used to prepare Maillard reaction products (MRPs). The results showed that the optimum browning inhibitory effect was obtained by adding GSH after Gly-Gly and Ara heating at 80 °C for 60 min.

Evaluation of New 99mTc(CO)3 + Radiolabeled Glycylglycine In Vivo.

BACKGROUND: Peptide-based agents are used in molecular imaging due to their unique properties, such as rapid clearance from the circulation, high affinity and target selectivity. Many of the radiolabeled peptides have been clinically experienced with diagnostic accuracy. The aim of this study was to investigate in vivo biological behavior of [99mTc(CO)3(H2O)3]+ radiolabeled glycylglycine (GlyGly). METHODS: Glycylglycine was radiolabeled with a high radiolabeling yield of 94.69±2%, and quality control of the radiolabeling process was performed by thin layer radiochromatography (TLRC) and High-Performance Liquid Radiochromatography (HPLRC). Lipophilicity study for radiolabeled complex (99mTc(CO)3-Gly-Gly) was carried out using solvent extraction. The in vivo evaluation was performed by both biodistribution and SPECT imaging. RESULTS: The high radiolabelling yield of 99mTc(CO)3-GlyGly was obtained and verified by TLRC and HPLRC as well. According to the in vivo results, SPECT images and biodistribution data are in good accordance. The excretion route from the body was both hepatobiliary and renal. CONCLUSION: This study shows that 99mTc(CO)3-GlyGly has the potential to be used as a peptide-based imaging agent. Further studies, 99mTc(CO)3-GlyGly can be performed on tumor-bearing animals.

Recognition of the Diglycine C-End Degron by CRL2KLHDC2 Ubiquitin Ligase.

Aberrant proteins can be deleterious to cells and are cleared by the ubiquitin-proteasome system. A group of C-end degrons that are recognized by specific cullin-RING ubiquitin E3 ligases (CRLs) has recently been identified in some of these abnormal polypeptides. Here, we report three crystal structures of a CRL2 substrate receptor, KLHDC2, in complex with the diglycine-ending C-end degrons of two early-terminated selenoproteins and the N-terminal proteolytic fragment of USP1. The E3 recognizes the degron peptides in a similarly coiled conformation and cradles their C-terminal diglycine with a deep surface pocket. By hydrogen bonding with multiple backbone carbonyls of the peptides, KLHDC2 further locks in the otherwise degenerate degrons with a compact interface and unexpected high affinities. Our results reveal the structural mechanism by which KLHDC2 recognizes the simplest C-end degron and suggest a functional necessity of the E3 to tightly maintain the low abundance of its select substrates.

Peptide bond conformation in peptides and proteins probed by dipolar coupling-chemical shift tensor correlation solid-state NMR.

Multidimensional magic-angle spinning solid-state NMR experiments are described that permit cis and trans peptide bonds in uniformly 13C,15N-labeled peptides and proteins to be unambiguously distinguished in residue-specific manner by determining the relative orientations of the amide 13C' CSA and 1H-15N dipolar coupling tensors. The experiments are demonstrated for model peptides glycylglycine and 2,5-diketopiperazine containing trans and cis peptide bonds, respectively. Subsequently, the measurements are extended to two representative proteins that contain exclusively trans peptide bonds, microcrystalline B3 immunoglobulin domain of protein G and Y145Stop human prion protein amyloid fibrils, to illustrate their applicability to a wide range of protein systems.

Density Functional Studies on Secondary Amides: Role of Steric Factors in Cis/Trans Isomerization.

Cis/trans isomerization of amide bonds is a key step in a wide range of biological and synthetic processes. Occurring through C-N amide bond rotation, it also coincides with the activation of amides in enzymatic hydrolysis. In recently described QM studies of cis/trans isomerization in secondary amides using density functional methods, we highlighted that a peptidic prototype, such as glycylglycine methyl ester, can suitably represent the isomerization and complexities arising out of a larger molecular backbone, and can serve as the primary scaffold for model structures with different substitution patterns in order to assess and compare the steric effect of the substitution patterns. Here, we describe our theoretical assessment of such steric effects using tert-butyl as a representative bulky substitution. We analyze the geometries and relative stabilities of both trans and cis isomers, and effects on the cis/trans isomerization barrier. We also use the additivity principle to calculate absolute steric effects with a gradual increase in bulk. The study establishes that bulky substitutions significantly destabilize cis isomers and also increases the isomerization barrier, thereby synergistically hindering the cis/trans isomerization of secondary amides. These results provide a basis for the rationalization of kinetic and thermodynamic properties of peptides with potential applications in synthetic and medicinal chemistry.

Self-assembled nanomicelles of amphiphilic clotrimazole glycyl-glycine analogue augmented drug delivery, apoptosis and restrained melanoma tumour progression.

In present investigation, self-assembled nanomicelles of amphiphilic clotrimazole glycyl-glycine (CLT-GG-SANMs) analogue were customized for augmenting drug delivery, permeability and apoptosis in B16F1 mouse melanoma cancer cells both in vitro and in vivo following intratumoral (i.t.) route of administration. The mean particle size of CLT-GG-SANMs was measured to be 35.9 ±â€¯3.4 nm in addition to zeta-potential of -17.1 ±â€¯3.5 mV. The shape of CLT-GG-SANMs was visualized to be smooth and spherical as like nanoparticles. The critical micellar concentration (CMC) of CLT-GG-SANMs was estimated to be 17 µg/ml using DPH (1,6-diphenyl-1,3,5-hexatriene) as a UV probe. Modification of CLT to CLT-GG-SANMs induced the amorphization in therapeutic moiety. Next, CLT suspension released only 9.7% of the drug within 1 h under dissolution testing and further analysis up to 48 h did not display any remarkable effect on the drug release. On the other hand, CLT-GG-SANMs released 46.2% of the drug significantly (P < 0.01) higher than CLT suspension at 4 h. The IC50 of CLT-GG-SANMs was measured to be 15.1-µM significantly (P < 0.05) lower than CLT suspension (IC50 > 20 µM) in B16F1 cells. Western blotting and histopathological analysis also supported the superior therapeutic efficacy of CLT-GG-SANMs in terms of higher extent of apoptosis, tumour regression and exhibition of strong antioxidant potential against B16F1 cells induced tumour in C57BL6J mice. In conclusion, in vitro and in vivo therapeutic efficacy analysis indicated that CLT-GG-SANMs may be a potential candidate for translating in to a clinically viable product.

Effective combination therapy of percutaneous ethanol injection and chemotherapy based on injectable low molecular weight gels.

Percutaneous ethanol injection (PEI) therapy was used in liver cancer treatment, however, the therapeutic ethanol in PEI easily flew away from injected solid tumours and hinder the treatment effect. In this paper, injectable supramolecular gels formed by self-assembly of low molecular weight gelators (LMWGs) based on glycylglycine modified phenylboronic acid were prepared to localize ethanol and load chemotherapeutic drug for in situ synergistic therapy. The mechanism, morphology and rheological property of supramolecular gels were characterized by NMR, UV, SEM, etc. The rheological study revealed that the gels were formed in situ rapidly and recovered promptly once damaged. The gels were non-toxicity to both normal 3T3 fibroblasts cells and 4T1 breast cancer cells. Doxorubicin (DOX) hydrochloride and ethanol were encapsulated in the gels for the combination of chemotherapy and PEI therapy. The in vivo anticancer activity of the DOX-loaded gels was carried out in tumour bearing mice. The injected gels were coated around tumour tissues to lock ethanol, and DOX was released sustainingly from the gels to maintain effective concentration to induce the apoptosis of tumour cells. DOX-loaded gels and the ethanol exhibited excellent therapeutic efficacy and low side effects in local cancer therapy.

A novel melphalan polymeric prodrug: preparation and property study.

The clinical application of melphalan (Me), an anticancer drug for the treatment of hematologic malignancies, has been limited due to its poor water solubility, rapid elimination and lack of target specificity. To solve these problems, O,N-carboxymethyl chitosan-peptide-melphalan conjugates were synthesized and characterized. All polymeric prodrugs showed satisfactory water solubility. It was found that the molecular weight of O,N-carboxymethyl chitosan (O,N-CMCS) and the peptide spacer played a crucial role in controlling the drug content, diameter and drug release properties of O,N-carboxymethyl chitosan-peptide-melphalan conjugates. The studies of in vitro drug release and cell cytotoxicity by MTT assay revealed that, employing the polymeric conjugation strategy and using the peptides glycylglycine (Gly-Gly) as a spacer, the conjugates have good cathepsin X-sensitivity and lower toxicity and the drug release behavior improved remarkably. In conclusion, O,N-carboxymethyl chitosan-peptide-melphalan conjugates could be promising prodrugs for anticancer application.

Global ubiquitination analysis by SILAC in mammalian cells.

Ubiquitination is a versatile and dynamic posttranslational modification in cells, regulating almost all cellular events. With rapid developments of affinity capture reagents and high-resolution mass spectrometry, it is now feasible to globally analyze the ubiquitinated proteome (ubiquitome) using quantitative strategies, such as stable isotope labeling with amino acids in cell culture (SILAC). Here we describe in detail a SILAC protocol to profile the ubiquitome in mammalian cells including protein labeling, antibody-based enrichment, and analysis by mass spectrometry.

Peptide bond formation via glycine condensation in the gas phase.

Four unique gas phase mechanisms for peptide bond formation between two glycine molecules have been mapped out with quantum mechanical electronic structure methods. Both concerted and stepwise mechanisms, each leading to a cis and trans glycylglycine product (four mechanisms total), were examined with the B3LYP and MP2 methods and Gaussian atomic orbital basis sets as large as aug-cc-pVTZ. Electronic energies of the stationary points along the reaction pathways were also computed with explicitly correlated MP2-F12 and CCSD(T)-F12 methods. The CCSD(T)-F12 computations indicate that the electronic barriers to peptide bond formation are similar for all four mechanisms (ca. 32-39 kcal mol(-1) relative to two isolated glycine fragments). The smallest barrier (32 kcal mol(-1)) is associated with the lone transition state for the concerted mechanism leading to the formation of a trans peptide bond, whereas the largest barrier (39 kcal mol(-1)) was encountered along the concerted pathway leading to the cis configuration of the glycylglycine dipeptide. Two significant barriers are encountered for the stepwise mechanisms. For both the cis and trans pathways, the early electronic barrier is 36 kcal mol(-1) and the subsequent barrier is approximately 1 kcal mol(-1) lower. A host of intermediates and transition states lie between these two barriers, but they all have very small relative electronic energies (ca. ± 4 kcal mol(-1)). The isolated cis products (glycylglycine + H2O) are virtually isoenergetic with the isolated reactants (within -1 kcal mol(-1)), whereas the trans products are about 5 kcal mol(-1) lower in energy. In both products, however, the water can hydrogen bond to the dipeptide and lower the energy by roughly 5-9 kcal mol(-1). This study indicates that the concerted process leading to a trans configuration about the peptide bond is marginally favored both thermodynamically (exothermic by ca. 5 kcal mol(-1)) and kinetically (barrier height ≈ 32 kcal mol(-1)) according to the CCSD(T)-F12/haTZ electronic energies. The other pathways have slightly larger barrier heights (by 4-8 kcal mol(-1)).

Synthesis of glycylglycine-imprinted silica microspheres through different water-in-oil emulsion techniques.

Sol-gel molecularly imprinted materials (MIMs) are traditionally obtained by grinding and sieving of a monolith formed by bulk polymerization. However, this process has several drawbacks that can be overcome if these materials are synthesized directly in the spherical format. This work aimed at the development of two efficient methods to prepare spherical glycylglycine-templated silica ("whole-imprinted" and surface-imprinted) through a combination of sol-gel and emulsion techniques. The synthesis of the microspheres was optimized regarding emulsion and sol-gel parameters. Imprinting efficiency of the prepared materials was studied by solid phase extraction and flow microcalorimetry. The particles prepared with glycylglycine and functional monomer, in basic medium (using cyclohexane as non-polar continuous medium) presented the highest imprinting factor - 2.5 - and the respective surface-imprinted material presented an imprinting factor of 1.5. The results of flow microcalorimetry confirmed the action of different mechanisms of glycylglycine adsorption: entropically-controlled interactions were present for the "whole-imprinted" material, indicating adsorption inside small imprinted pores; enthalpically-controlled interactions were observed for the surface-imprinted material, a behaviour more compatible with a template/surface-only interaction. Globally, the two approaches allowed for a successful imprinting effect which was more extensive for the "whole-imprinted" material, whereas the surface-imprinting feature confers to the surface-imprinted xerogel advantages regarding mass transfer kinetics. Overall, the spherical particles obtained by both approaches presented characteristics, such as sphericity, mesoporosity, easy/fast accessibility to imprinted sites, important indicators that these materials may be candidates for stationary phases for efficient, selective chromatographic separation.