Cleavage Of Amide Bond Research Articles

Modeling the Complete Catalytic Cycle of Aspartoacylase.

The complete catalytic cycle of aspartoacylase (ASPA), a zinc-dependent enzyme responsible for cleavage of N-acetyl-l-aspartate, is characterized by the methods of molecular modeling. The reaction energy profile connecting the enzyme-substrate (ES) and the enzyme-product (EP) complexes is constructed by the quantum mechanics/molecular mechanics (QM/MM) method assisted by the molecular dynamics (MD) simulations with the QM/MM potentials. Starting from the crystal structure of ASPA complexed with the intermediate analogue, the minimum-energy geometry configurations and the corresponding transition states are located. The stages of substrate binding to the enzyme active site and release of the products are modeled by MD calculations with the replica-exchange umbrella sampling technique. It is shown that the first reaction steps, nucleophilic attack of a zinc-bound nucleophilic water molecule at the carbonyl carbon and the amide bond cleavage, are consistent with the glutamate-assisted mechanism hypothesized for the zinc-dependent hydrolases. The stages of formation of the products, acetate and l-aspartate, and regeneration of the enzyme are characterized for the first time. The constructed free energy diagram from the reactants to the products suggests that the enzyme regeneration, but not the nucleophilic attack of the catalytic water molecule, corresponds to the rate-determining stage of the full catalytic cycle of ASPA.

Employing vibrational spectroscopy to confirm the catalytic activity of Ce(III) and Cr(III) towards amide bond cleavage

The dependence of the Ce(III) and Cr(III) halide concentration on the spectrum of formamide is investigated for the first time. The downshifts observed for the νCO mode in the IR spectra and the upshifts of the νCN vibration in the Raman spectra are consistent with the stabilization of the HO−CN+H2 form, which is directly involved in the amide bond cleavage reaction. The νCN region was used for the quantitative treatment and reveals that Ce(III) is surrounded by 8 formamide molecules, in a square prismatic arrangement, while 4 formamide molecules are around Cr(III) and the counter-ion occupies two vertices of the octahedron in the cis-configuration. The spectral changes in the region of the solvent are in excellent agreement with those observed for the metal-ligand vibrations.

A novel N-terminal degradation reaction of peptides via N-amidination

The cleavage of amide bonds requires considerable energy. It is difficult to cleave the amide bonds in peptides at room temperature, whereas ester bonds are cleaved easily. If peptide bonds can be selectively cleaved at room temperature, it will become a powerful tool for life science research, peptide prodrug, and tissue-targeting drug delivery systems. To cleave a specific amide bond at room temperature, the decomposition reaction of arginine methyl ester was investigated. Arginine methyl ester forms a dimer; the dimer releases a heterocyclic compound and ornithine methyl ester at room temperature. We designed and synthesized N-amidinopeptides based on the decomposition reaction of arginine methyl ester. Alanyl-alanine anilide was used as the model peptide and could be converted into N-degraded peptide, alanine anilide, via an N-amidination reaction at close to room temperature. Although the cleavage rate in pH 7.4 phosphate buffered saline (PBS) at 37°C was slow (t1/2=35.7h), a rapid cleavage rate was observed in 2% NaOH aq (t1/2=1.5min). To evaluate the versatility of this reaction, a series of peptides with Lys, Glu, Ser, Cys, Tyr, Val, and Pro residue at the N-terminal were synthesized; they showed rapid cleavage rates of t1/2 values from 1min to 10min.

Acylation of β‐Amino Esters and Hydrolysis of β‐Amido Esters: Candida antarctica Lipase A as a Chemoselective Deprotection Catalyst

AbstractN‐Acylation by lipase A from Candida antarctica (CAL‐A) in ethyl butanoate was applied to the kinetic resolution of tert‐butyl esters of 3‐amino‐3‐phenylpropanoic acid (E>100), 3‐amino‐4‐methylpentanoic acid (E>100) and 3‐aminobutanoic acid (E=60) on 1.0–2.0 m scale. With the N‐acylated resolution products, the exceptional ability of CAL‐A to hydrolyse amides and bulky tert‐butyl esters was then studied. In all N‐acylated tert‐butyl esters, chemoselectivity favoured the amide bond cleavage. The tert‐butyl ester bond was left intact with 3‐amino‐3‐phenylpropanoate, whereas with 3‐amino‐4‐methylpentanoate and 3‐aminobutanoate the CAL‐A‐catalysed hydrolysis of tert‐butyl ester followed the amide hydrolysis.

Stepwise pH-responsive nanoparticles containing charge-reversible pullulan-based shells and poly(β-amino ester)/poly(lactic-co-glycolic acid) cores as carriers of anticancer drugs for combination therapy on hepatocellular carcinoma

Stepwise pH-responsive nanoparticle system containing charge reversible pullulan-based (CAPL) shell and poly(β-amino ester) (PBAE)/poly(lactic-co-glycolic acid) (PLAG) core is designed to be used as carriers of paclitaxel (PTX) and combretastatin A4 (CA4) for combining antiangiogenesis and chemotherapy to treat hepatocellular carcinoma (HCC). CAPL-coated PBAE/PLGA (CAPL/PBAE/PLGA) nanoparticles displayed step-by-step responses to weakly acidic tumor microenvironment (pH ≈6.5) and endo/lysosome (pH ≈5.5) respectively through the cleavage of β-carboxylic amide bond in CAPL and the “proton-sponge” effect of PBAE, thus realized the efficient and orderly releases of CA4 and PTX. In human HCC HepG2 cells and human umbilical vein endothelial cells, CAPL/PBAE/PLGA nanoparticles significantly enhanced synergistic effects of PTX and CA4 on cell proliferation and cell migration. In HepG2 tumor-bearing mice, CAPL/PBAE/PLGA nanoparticles showed excellent tumor-targeting capability and remarkably increased inhibitory effects of PTX and CA4 on tumor growth and angiogenesis. In conclusion, this novel nanoparticle system is a promising candidate as carrier for drugs against HCC.

Exploitation of the Ornithine Effect Enhances Characterization of Stapled and Cyclic Peptides.

A method to facilitate the characterization of stapled or cyclic peptides is reported via an arginine-selective derivatization strategy coupled with MS/MS analysis. Arginine residues are converted to ornithine residues through a deguanidination reaction that installs a highly selectively cleavable site in peptides. Upon activation by CID or UVPD, the ornithine residue cyclizes to promote cleavage of the adjacent amide bond. This Arg-specific process offers a unique strategy for site-selective ring opening of stapled and cyclic peptides. Upon activation of each derivatized peptide, site-specific backbone cleavage at the ornithine residue results in two complementary products: the lactam ring-containing portion of the peptide and the amine-containing portion. The deguanidination process not only provides a specific marker site that initiates fragmentation of the peptide but also offers a means to unlock the staple and differentiate isobaric stapled peptides.

ChemInform Abstract: Manganese(II)‐Catalyzed Esterification of N‐β‐Hydroxyethylamides.

AbstractThis protocol enables selective cleavage of amide bonds through a mechanism involving N,O—acyl rearrangement and transesterification.

Amide bond cleavage initiated by coordination with transition metal ions and tuned by an auxiliary ligand.

The reaction of ligand , N,N-bis(pyridin-2-ylmethyl)acetamide, with five transition metal salts, FeCl3·6H2O, CuCl2·2H2O, Cu(ClO4)2·6H2O, ZnCl2 and K2PtCl4/KI, produced five metal complexes, [(μ-O)(FeClL')(FeCl3)] (), [CuLCl2] (), [CuBPA(ClO4)(CHCN)] ClO4 (), [ZnLCl2] () and [PtLI2] (), where = 1-(2,4,5-tri(pyridin-2-yl)-3-(pyridin-2-ylmethyl)imidazolidin-1-yl)ethanone which formed in situ, and BPA = bis(pyridin-2-ylmethyl)amine. The ligand and complexes were characterized by a variety of spectroscopic techniques including X-ray single crystal diffraction where applicable. Depending on the metal ion and auxiliary ligand of the complex, the acetyl group of the ligand could be either intact or cleaved. When ferric chloride hexahydrate was used, the deacetylation proceeded even further and a novel heterocyclic compound () was formed in situ. A possible mechanism was proposed for the formation of the heterocyclic compound found in complex . Our results indicate that to cleave effectively an amide bond, it is essential for a metal centre to bind to the amide bond and the metal centre is of sufficient Lewis acidity.

Discovery of highly potent and selective Bruton's tyrosine kinase inhibitors: Pyridazinone analogs with improved metabolic stability.

BTK inhibitor GDC-0834 (1) was found to be rapidly metabolized in human studies, resulting in a suspension of clinical trials. The primary route of metabolism was through cleavage of the acyclic amide bond connecting the terminal tetrahydrobenzothiophene with the central linker aryl ring. SAR studies were focused on reducing metabolic cleavage of this amide, and resulted in the identification of several central aryl linker substituents that conferred improved stability. The most promising substituted aryl linkers were then incorporated into an optimized pyridazinone scaffold, resulting in the identification of lead analog 23, possessing improved potency, metabolic stability and preclinical properties.

Proton Mobility in b₂ Ion Formation and Fragmentation Reactions of Histidine-Containing Peptides.

A detailed energy-resolved study of the fragmentation reactions of protonated histidine-containing peptides and their b2 ions has been undertaken. Density functional theory calculations were utilized to predict how the fragmentation reactions occur so that we might discern why the mass spectra demonstrated particular energy dependencies. We compare our results to the current literature and to synthetic b2 ion standards. We show that the position of the His residue does affect the identity of the subsequent b2 ion (diketopiperazine versus oxazolone versus lactam) and that energy-resolved CID can distinguish these isomeric products based on their fragmentation energetics. The histidine side chain facilitates every major transformation except trans-cis isomerization of the first amide bond, a necessary prerequisite to diketopiperazine b2 ion formation. Despite this lack of catalyzation, trans-cis isomerization is predicted to be facile. Concomitantly, the subsequent amide bond cleavage reaction is rate-limiting.

Effect of gamma irradiation at intermediate doses on the performance of reverse osmosis membranes

The goal of this study is to explain the degradation of Polyamide (PA) composite reverse osmosis membrane (RO) in function of the irradiation dose. Irradiations were performed with a gamma 60Co source in wet conditions and under oxygen atmosphere. For different doses of 0.2 and 0.5MGy with a constant dose rate of 0.5kGyh−1, RO membranes performances (NaCl retention, permeability) were studied before and after irradiation. ATR-FTIR, ion chromatography and gas chromatography were used to characterize structural modification. Results showed that the permeability of RO membranes irradiated at 0.2MGy exhibited a small decrease, related to scissions of the PVA coating. However, retention did not change at this dose. At 0.5MGy, permeability showed a large increase of a factor around 2 and retention began to decrease from 99% to 95%. Chromatography measurements revealed a strong link between permselectivity properties variation, ion leakage and oxygen consumption. Add to ATR-FTIR observations, these results emphasized that the cleavages of amide and ester bonds were observed at 0.5MGy, more precisely the loss of hydrogen bonds between polyamide chains. By different analysis, modifications of the polysulfone layer occur until a dose of 0.2MGy.

Online Investigation of Aqueous-Phase Electrochemical Reactions by Desorption Electrospray Ionization Mass Spectrometry.

Electrochemistry (EC) combined with mass spectrometry (MS) is a powerful tool for elucidation of electrochemical reaction mechanisms. However, direct online analysis of electrochemical reaction in aqueous phase was rarely explored. This paper presents the online investigation of several electrochemical reactions with biological relevance in the aqueous phase, such as nitrosothiol reduction, carbohydrate oxidation, and carbamazepine oxidation using desorption electrospray ionization mass spectrometry (DESI-MS). It was found that electroreduction of nitrosothiols [e.g., nitrosylated insulin B (13-23)] leads to free thiols by loss of NO, as confirmed by online MS analysis for the first time. The characteristic mass shift of 29 Da and the reduced intensity provide a quick way to identify nitrosylated species. Equally importantly, upon collision-induced dissociation (CID), the reduced peptide ion produces more fragment ions than its nitrosylated precursor ion (presumably the backbone fragmentation cannot compete with the facile NO loss for the precursor ion), thus facilitating peptide sequencing. In the case of saccharide oxidation, it was found that glucose undergoes electro-oxidation to produce gluconic acid at alkaline pH, but not at neutral and acidic pHs. Such a pH-dependent electrochemical behavior was also observed for disaccharides such as maltose and cellobiose. Upon electrochemical oxidation, carbamazepine was found to undergo ring contraction and amide bond cleavage, which parallels the oxidative metabolism observed for this drug in leucocytes. The mechanistic information of these redox reactions revealed by EC/DESI-MS would be of value in nitroso-proteome research and carbohydrate/drug metabolic studies.

Selective intracellular drug delivery from pH-responsive polyion complex micelle for enhanced malignancy suppression in vivo

The pH-triggered intracellular drug delivery platforms have attracted great interest in malignancy therapy. Herein, a pH-responsive polyion complex (PIC) micelle from anionic acid-sensitive methoxy poly(ethylene glycol)-block-poly(N(ϵ)-((1-carboxy-cis-cyclohexene)-2-carbonyl)-L-lysine) (mPEG-b-PCLL) and cationic doxorubicin (DOX), a model anthracycline antitumor drug, was constructed by electrostatic interaction for directional intracellular drug delivery in malignancy chemotherapy. The PIC micelle kept constant diameter at physiological condition (i.e., pH 7.4), while gradually swelled and finally disassembled at mimicking intratumoral pH (i.e., 6.8) and especially intracellular endo/lysosomal pH (i.e., 5.5). The DOX release from the PIC micelle at pH 7.4 was slow, whereas obviously accelerated at the intracellular acidic condition of pH 5.5. These results should be related to the rapid cleavage of the side amide bond of mPEG-b-PCLL in an acidic environment. The PIC micelle exhibited satisfactory tumor suppression toward the H22 hepatoma-bearing BALB/c mouse model compared with free DOX, which was demonstrated by the upregulated tumor inhibition rate, and the increased necrotic and apoptosis areas in tumor tissue. Furthermore, the enhanced security was also observed in the PIC micelle group in relation to that of free DOX. The above results strongly supported that the acid-sensitive PIC micelle was promising for selective intracellular drug delivery along with upregulated malignancy inhibition.

PHe-induced charge-reversible NIR fluorescence nanoprobe for tumor-specific imaging.

Inspired by the specificity of acid tumor microenvironment, we constructed a flexible charge-reversible near-infrared (NIR) fluorescence nanoprobe in response to tumor extracellular pH (pHe) for effective tumor-specific imaging. The nanoprobe consists of an NIR-emitted CuInS2/ZnS quantum dot (CIS/ZS QDs) core and a tailored lauric acid and 2,3-dimethylmaleic anhydride modified ε-polylysine (ε-PL-g-LA/DMA) shell, which provides not only a dense protective layer for the QDs but also the ability of pHe-induced positive charge-mediated endocytosis into tumor cells. The results showed that the QDs@ε-PL-g-LA/DMA nanoprobe with a uniform size of 40 nm had high chemical stability at pH 7.4 and excellent optical properties. Especially, it swiftly reversed its surface charge to positive in 20 min when exposed to pHe due to the cleavage of the β-carboxyl amide bond of ε-PL-g-LA/DMA. Moreover, the cell uptake of the pHe-sensitive QDs nanoprobe exposed at pH 6.8 into HeLa cells is much more significant than that at pH 7.4, which further verified the availability of the electrostatic adsorptive endocytosis facilitated targeting ability. The pHe-induced targeting imparted the QDs nanoprobe a broad targeting ability in a variety of solid tumors. Furthermore, as an effective alternative mechanism for tumor targeting, responsive charge reversion is also universally applicable to other cancer theranostics agent.

PH-Responsive Reversible PEGylation Improves Performance of Antineoplastic Agent.

The reversible PEGylation endows antitumor drugs with various fascinating advantages, including prolonged circulation time in blood, enhanced accumulation in tumor tissue, increased cellular uptake, and promoted intracellular drug release, to improve the therapeutic efficacy and security. Here, the obtained succinic anhydride (SA)-functionalized DOX (SAD) (i.e., insensitive succinic anhydride-functionalized doxorubicin (DOX)) and aconitic anhydride (CA)-modified DOX (CAD) (i.e., acid-sensitive cis-aconitic anhydride-modified DOX) are conjugated to the terminal of poly(ethylene glycol) (PEG) yielding the unresponsive SAD-PEG-SAD and pH-responsive CAD-PEG-CAD prodrugs, respectively. The prepared prodrugs can self-assemble into micelles in aqueous solution. Both micelles are sufficiently stable at normal physiological pH (i.e., 7.4), while CAD-PEG-CAD micelle gradually swells and finally disassembles at intratumoral (i.e., 6.8) and especially endosomal pHs (i.e., 5.5). DOX release from CAD-PEG-CAD at pH 7.4 is efficiently inhibited, whereas it is significantly accelerated by the rapid cleavage of amide bond at pH 5.5. In addition, CAD-PEG-CAD exhibits more efficient cellular uptake and potent cytotoxicity in vitro, as well as improved tissue distribution and superior tumor suppression in vivo than free DOX and SAD-PEG-SAD. More importantly, the PEGylated DOX exhibits favorable security in vivo. In brief, the smart CAD-PEG-CAD with enhanced antitumor efficacy and decreased side effects shows as a promising powerful platform for the clinical chemotherapy of malignancy.

Changes in physicochemical and transport properties of a reverse osmosis membrane exposed to chloraminated seawater.

This study contributed to improving our understanding of how disinfectants, applied to control biofouling of reverse osmosis (RO) membranes, result in membrane performance degradation. We investigated changes in physicochemical properties and permeation performance of a RO membrane with fully aromatic polyamide (PA) active layer. Membrane samples were exposed to varying concentrations of monochloramine, bromide, and iodide in both synthetic and natural seawater. Elemental analysis of the membrane active layer by Rutherford backscattering spectrometry (RBS) revealed the incorporation of bromine and iodine into the polyamide. The kinetics of polyamide bromination were first order with respect to the concentration of the secondary oxidizing agent Br2 for the conditions investigated. Halogenated membranes were characterized after treatment with a reducing agent and heavy ion probes to reveal the occurrence of irreversible ring halogenation and an increase in carboxylic groups, the latter produced as a result of amide bond cleavage. Finally, permeation experiments revealed increases in both water permeability and salt passage as a result of oxidative damage.

Metal-Free Csp3–N Bond Cleavage of Amides Using tert-Butyl Hydroperoxide as Oxidant

A mild and efficient protocol for the metal-free C–N bond-cleavage of amides has been developed. The methodology employs iodine as a catalyst to cleave the C(Me)–N bond of dimethylformamide or dimethylacetamide, providing novel access to methylene-bridged bis-1,3-dicarbonyl compounds instead of enol carbamates in the presence of tert-butyl hydroperoxide.

The role of basic residues in the fragmentation process of the lysine rich cell-penetrating peptide TP10.

Selective cleavage effect of basic residues in the fragmentation of short peptides has been studied intensively. In contrast, the role of basic residues in the degradation of large peptides, such as cell-penetrating peptides, is largely unknown. In this work, the fragmentation of a 21 residues cell-penetrating peptide TP10 containing four lysine residues was studied by collision-induced dissociation mass spectrometry and computation methods. The influence of lysine residues on amide bond cleavage and fragmentation products was investigated. The results revealed that the selective cleavage effect of lysine residue did not present when the adjacent lysine residues in TP10 were both protonated. The localized high positive charge density might be the reason of preventing the mobile proton from migrating to the amide bonds in this part of the peptide. In contrast, the mobile proton preferred to reside in the N-terminal part of TP10 which had less positive charge. This preference gave more information of the peptide sequence in the mass spectrometry study and was helpful for stabilizing the C-terminal part of TP10, in which the basic lysine residues were preserved and crucial to the cell-penetrating process.

Biodegradation of propyzamide by Comamonas testosteroni W1 and cloning of the propyzamide hydrolase gene camH

Propyzamide is a widely used benzamide herbicide for controlling weeds in lettuce, soybeans, cotton and other crops. An efficient propyzamide-degrading strain W1 was firstly isolated from activated sludge and identified as Comamonas testosteroni. A metabolite of propyzamide by strain W1 was firstly identified. The novel gene camH encoding a hydrolase that catalyzed the amide bond cleavage of propyzamide was cloned from strain W1. The gene contained an open reading frame of 1452bp, the deduced amino acid sequence showed low identity with other amidases. The recombinant enzyme CamH was expressed in Escherichia coli BL21 and purified. CamH displayed the highest activity at 30°C and pH 8.0 with propyzamide as the substrate. These results provide important knowledge on the fate of propyzamide in the biodegradation, and elucidate the biodegradation mechanism of propyzamide by the strain W1.

Cloning, expression, purification, crystallization and preliminary X-ray characterization of allantoinase from Bacillus licheniformis ATCC 14580.

Allantoinase, a member of the amidohydrolase superfamily, exists in a wide variety of organisms, including bacteria, fungi, plants and a few animals, such as fishes and amphibians. Allantoinase catalyzes the reversible hydrolysis of allantoin into allantoate by hydrolytic cleavage of the N1-C2 amide bond of the five-membered hydantoin ring. Allantoinase from Bacillus licheniformis (AllBali) presents an inverted enantioselectivity towards allantoin (R-enantioselective), which is a distinguishable feature that is not observed for other allantoinases. In this work, B. licheniformis ATCC 14580 allantoinase (AllBali) containing a C-terminal His6 tag was overproduced in Escherichia coli and purified to homogeneity. Crystals of AllBali were obtained by the vapour-diffusion method using 0.1 M potassium thiocyanate, 20%(w/v) polyethylene glycol 3350 as a crystallization solution. X-ray diffraction data were collected to a resolution of 3.5 Å with an Rmerge of 29.2% from a crystal belonging to space group P12₁1, with unit-cell parameters a=54.93, b=164.74, c=106.89 Å, β=98.49°. There are four molecules in the asymmetric unit with a solvent content of 47% as estimated from the Matthews coefficient (VM=2.34 Å3 Da(-1)).