Expanded structural and stereospecificity in peptide synthesis with chemically modified mutants of subtilisin

Employing the strategy of combined site directed mutagenesis and chemical modification, we previously generated chemically modified mutant enzymes (CMMs) of subtilisin Bacillus lentus (SBL). We now report the use of these SBL-CMMs for peptide coupling reactions. The SBL-CMMs exhibit dramatically altered substrate specificity, including the acceptance of d-amino acid acyl donors, generating dipeptides containing d-Phe, d-Ala and d-Glu in up to 66% yield, which was not possible using wild-type SBL (WT-SBL). In addition, SBL-CMMs accommodate α-branched amino acids such as l-Ala-NH₂ as acyl acceptors in their S₁′ pockets, which WT-SBL will not.     

Expanding the utility of proteases in synthesis: broadening the substrate acceptance in non-coded amide bond formation using chemically modified mutants of subtilisin

The strategy of combined site directed mutagenesis and chemical modification creates chemically modified mutants (CMMs) with greatly broadened substrate specificities. We have previously reported that the CMMs of subtilisin Bacillus lentus (SBL) are efficient catalysts for the coupling of both l- and d-amino acids. We now report that these powerful catalysts also allow amide bond formation between a variety of non-coded carboxylic acids, including β-alanine and β-amino homologues of phenylalanine, with both l- and d-amino acid nucleophiles. As a guide to enzyme efficiency, a hydrolysis assay indicating pH change has been employed. CMMs selected by this screen furnished higher yields of coupling products compared to the wild-type enzyme (WT). Furthermore, both WT and CMM enzymes allow highly stereoselective aminolysis of a meso diester with an amino acid amine. These results highlight the utility of CMMs in the efficient formation of non-coded amides as potential peptide isosteres.     

One-armed artificial receptors for the binding of polar tetrapeptides in water: probing the substrate selectivity of a combinatorial receptor library

We have recently developed a new class of one-armed artificial receptors 1 for the binding of the polar tetrapeptide N-Ac-D-Glu-L-Lys-D-Ala-D-Ala-OH (EKAA) 2 in water using a combined combinatorial and statistical approach. We have now further probed the substrate selectivity of this receptor library 1 by screening a second tetrapeptide substrate (3) with the inverse sequence N-Ac-D-Ala-D-Ala-L-Lys-D-Glu-OH (AAKE). This "inverse" substrate is also efficiently bound by our receptors, with K(ass) approximately 6000 M(-1) for the best receptors, as determined both by a quantitative on-bead binding assay and by UV and fluorescence titration studies in free solution. Hence, the inverse tetrapeptide 3 is in general bound two to three times less efficiently than the "normal" peptide 2 (K(ass) approximately 17,000 M(-1)), even though the complexation mainly involves long-range electrostatic interactions and both the receptor and substrate are rather flexible. Molecular modeling and ab initio calculations have been used to rationalize the observed substrate selectivity and to analyze the various binding interactions within the complex.     

Reaction mechanisms in peptide synthesis. Part 1. Semiquantitative characteristics of the reactivity of 2-methyl-5(4H)-oxazolone with water and ammonia in the gas phase and weakly polar media

Summary 2,4-Dialkyl-5(4H)-oxazolones are well-recognized intermediates in some aminolysis reactions in peptide synthesis. Using the MOPAC molecular orbital programs, detailed geometric and energetic characteristics of the elementary reaction pathways for the additions of water and ammonia to 2-methyl-5(4H)-oxazolone have been determined at the AM1 level. The results demonstrate that the additions must be parsed into a two-step mechanism involving formation of the -hydroxyimine followed by tautomerization to the parent N-acetylamino acid or amide.     

Convergent solid phase peptide synthesis vi : synthesis by the fmoc procedure with a modified protocol of two protected segments, sequence 5-17 and 18-31 of the neurotoxin ii of the scorpion androctonus australis hector.

Synthesis of two protected peptides thirteen and fourteen residues long, sequence 5-17, i.e. Fmoc-Tyr(cHex)- Ile-Val-Asp(Bzl)-Asp(Bzl)-Val-Asn-Cys(Acm)-Thr(Bzl)-Tyr(cHex)- Phe-Cys(Acm)-Gly-OH, and 18-31, i.e. Fmoc-Arg (Tos)-Asn-Ala- Tyr(cHex)-Cys(Acm )-Asn-Glu(Bzl)-Glu(Bzl)-Cys(Acm)-Thr(Bzl)- Lys(Z)-Leu-Lys(Z)-Gly-OH, of the scorpion neurotoxin II from , was performed by the solid phase method. The hydroxymethylphenoxymethyl copoly(styrene - 1% -divinylbenzene) type resin was used in combination with Fmoc-amino acids for both syntheses. A general protocol minimizing side reactions has been developed for the use of the base labile Fmoc--amino protecting group. The time of reaction with piperidine (50% in N,N'-dimethylformamide) has been shortened to three times one minute and coupling was performed mainly in methylene chloride with just dicyclohexyl or diisopropyl-carbodiimide. The side chain protecting groups of the Fmoc--amino acids were of the hydrogen fluoride labile type, which permitted, after trifluoroacetic acid cleavage of the peptide to resin ester bond, obtainment of protected peptides. The crude segments, precipitated from N,N'-dime- thylacetamide with water, were highly purified by HPLC and chemically characterized for future use in convergent solid phase assembling.     

Advances in ultra-high load polymer-supported peptide synthesis with phenolic supports: 2. Use of ‘hydrazinolysis-hplc’ to check for racemization on c-terminal amino acid loading and to determine peptide homogeneity during synthesis with selectively labile c-terminal anchoring linkages

2, 4-Dinitrophenyl-L-phenylalanine has been coupled to L-, D-, and DL-amino acid phenyl esters pendant upon a polymer matrix. The esters had been prepared by di-isopropylcarbodiimide-mediated condensation, catalyzed by 4-dimethylaminopyridine. Reverse phase high pressure liquid chromatography (HPLC), using elution solvents consisting of 10 vol.-% trifluoroacetic acid in water/acetonitrile mixtures, has been used to investigate the 2,4-dinitrophenyl L-L and L-D dipeptide mixtures obtained on hydrazinolysis of each of the dipeptide-matrix assemblies. ‘Hydrazinolysis-HPLC’ has been used also to determine intermediate peptide homogeneity in ultra-high load solid (gel) phase synthesis with Boc amino acids. Cross-linked poly(N-[2-(4-hydroxyphenyl)ethyl]acrylamide) and two derived polymers incorporating spacer groups have been used as supports. The spacer groups made possible peptide C-terminal attachment by either HF-labile benzyl ester or HF-labile cyclohexyl ester bonds, while still incorporating the phenolic ester linkage susceptible to rapid hydrazinolytic scission.     

Regioselectivity and diasteroselectivity in Pt(II)-mediated "green" catalytic epoxidation of terminal alkenes with hydrogen peroxide: mechanistic insight into a peculiar substrate selectivity

Recently developed electron-poor Pt(II) catalyst 1 with the "green" oxidant 35% hydrogen peroxide displays high activity and complete substrate selectivity in the epoxidation of terminal alkenes because of stringent steric and electronic requirements. In the presence of isolated dienes bearing terminal and internal double bonds, epoxidation is completely regioselective toward the production of terminal epoxides. Insight into the mechanism is gained by means of a reaction progress kinetic analysis approach that underlines the peculiar role of 1 in activating both the alkene and H2O2 in the rate-determining step providing a rare example of nucleophilic oxidation of alkenes by H2O2.     

Electrostatic versus steric effects in peptidomimicry: synthesis and secondary structure analysis of gramicidin S analogues with (E)-alkene peptide isosteres

A concise synthetic strategy was used for the first preparation of GS analogues with trisubstituted (E)-alkene peptide bond replacements. Solution and solid state conformational analysis demonstrated that the bistrifluoromethylated analogue was a superior mimic of the natural product, whereas the incorporation of methyl groups into the alkene peptide isostere led to a far greater perturbation of the secondary structure features of GS. The difference between CF3- and CH3-substitution can be explained by the superior electrostatic carbonyl group mimicry of the former function.     

Enthalpic and polar effects in the reactions of perfluoroalkyl radicals: New selective synthetic developments with alkenes and heteroaromatic bases

Perfluoroalkyl radicals, generated by iodine abstraction from perfluoroalkyl iodides by phenyl radical, react with low selectivity with protonated heteroaromatic bases, due to their electrophilic character and the prevalent enthalpic effect on the reaction. In the presence of alkenes, perfluoroalkyl radicals add very rapidly to the double bond and the polar character of the radical adduct is reversed, allowing the selective substitution of protonated heteroaromatic bases. The mechanism of the reaction and the key role of enthalpic and polar effects are discussed.     

Synthesis of a series of ethylene glycol modified water-soluble tetrameric TPE-amphiphiles with pyridinium polar heads: Towards applications as light-up bioprobes in protein and DNA assay,and wash-free imaging of bacteria

Available online Development of water soluble AIE-active “light-up” bioprobes for the detection of biomacromolecules has drawn huge research interests in recent past. In this study, a series of ethylene glycol modified water soluble tetrameric tetraphenylethylene amphiphiles with pyridinium polar heads (TPE-xEG-Py, x = 3, 4, 6 or 1a-c) have been synthesized by varying the ethylene glycol spacer. Their unique structure allows them to form vesicles and other nanoaggregates in aqueous solutions. These amphiphiles were successfully utilized for fluorimetric detection and quantitation of BSA and DNA based on the electrostatic interactions to trigger AIE-emission from the TPE moiety. The electrostatic interaction was also proved very effective in wash-free imaging of both Gram-negative (E. coli) and Gram-positive (S. aureus) bacteria with up to 92 folds increase in fluorescence response within bacterial concentration 0–12 × 10⁸ CFU mL^?1. The strategy is advantageous due to cost-effective and easy synthesis, high water solubility, and fast response.     

The use of combinatorial chemical vapor deposition in the synthesis of Ti(3-delta)O4N with 0.06 < delta < 0.25: a titanium oxynitride phase isostructural to anosovite

We employ, for the first time, a unique combinatorial chemical vapor deposition (CVD) technique to isolate a previously unreported transition-metal mixed-anion phase. The new oxynitride phase, Ti(3-delta)O4N (where 0.06 < delta < 0.25), is the first example of a complex titanium oxynitride and was synthesized within composition graduated films formed from atmospheric pressure CVD of TiCl4, NH3, and ethyl acetate. Characterization was performed by X-ray diffraction, X-ray photoelectron spectroscopy, UV-visible spectra, and SQUID magnetometry. The material crystallizes in the Cmcm space group, with the ordered nitrogen ions stabilizing the orthorhombic analogue of the monoclinic anosovite structure, beta-Ti3O5. The lattice parameters are sensitive to composition, but were determined to be a = 3.8040(1) A, b = 9.6486(6) A, and c = 9.8688(5) A for Ti(2.85(2))O4N. Powder samples were prepared through delamination of the thin films for synchrotron X-ray diffraction and magnetic measurements. It is the first example of a new phase to be synthesized using such a combinatorial CVD approach and clearly demonstrates how such techniques can provide access to new materials. This metastable phase with unusual nitrogen geometry has proved to be elusive to conventional solid-state chemistry techniques and highlights the value of the surface growth mechanism present in CVD. Furthermore, the ease and speed of the synthesis technique, combined with rapid routes to characterization, allow for large areas of phase space to be probed effectively. These results may have major implications in the search for new complex mixed-anion phases in the future.     

Fragmentation with supernucleophiles—VI: β-Haloalkyl groups as functional protection in peptide synthesis. A kinetic study of the reaction of the cobalt(I)phthalocyanine anion with organic halides

The kinetics of the reaction between cobalt(I)phthalocyanine anion and primary alkyl monohalogenides was studied by the use of conductivity measurements. The data agree with an S_N2 mechanism. In the case of geminally polyhalogenated compounds a somewhat different type of mechanism seems to prevail. The alkylation of cobalt(I)phthalocyanine anion by β-haloethyl esters, urethanes, phenolethers, phenolthioethers, amines and amides, followed by reductive fragmentation of the organo-cobalt intermediate, are model experiments related to the extension of an earlier developed protective group technique for peptide synthesis. In the studied cases the rate of the overall reaction is roughly the same as the rate of the initial alkylation step.     

Reaction of diacetylmonoxime with morpholine N-thiohydrazide in the absence and in presence of a metal ion: Facile synthesis of a thiadiazole derivative with non-bonded SS interaction

The condensation of diacetylmonoxime (damnx) with morpholine N-thiohydrazide (mth) in 1:1 molar ratio in ethanol (16 h) afforded a nitrogen–sulfur zwitterionic heterocyclic compound, N-(3,4-dimethyl-1,2,5-thiadiazole-2-ium-2-yl)morpholine-4-carbothioate (dtmc). However, the same reaction in presence of [Zn(OAc)₂]·2H₂O in ethanol under gentle reflux on (3 h) yielded the zinc complex, [Zn(Hdammthiol)(OAc)(H₂O)]·H₂O, where H₂dammthiol (H₂L²) is the thiol form of tridentate NNS donor thiohydrazone ligand, diacetylmonoxime morpholine N-thiohydrazone (Hdammth). Both the nitrogen–sulfur heterocyclic compound and the zinc complex have been characterized by elemental analyses, spectroscopy (IR, UV–Vis, ¹H NMR and ¹³C NMR) and single crystal X-ray crystallography. It is noteworthy that the heterocyclic compound shows SS interaction with distance 2.738 Å in its planar conformation. The heterocyclic compound forms two dimensional supramolecular sheets through C–HO and ππ interactions while the zinc complex, with distorted square pyramidal geometry, forms 1D supramolecular chain. A mechanism has been proposed for the formation of nitrogen–sulfur heterocyclic compound.     

Particle size and concentration of poly(-caprolactone) and adipate modified starch blend on mineralization in soils with differing textures

The objective of this study was to evaluate the effect of particle size and concentration of poly(-caprolactone) and adipate modified starch blend on mineralization in soils with differing textures, comparing it with polyethylene under the same experimental conditions. Two soil types were used: a Kandiudalfic Eutrudox with a clayey texture and an Arenic Hapludult with a sandy texture. The two different plastic specimens were incorporated in the form of plastic films with three increasing particle sizes and six doses, from 0 to 2.5 mg C g⁻¹ soil. Each plastic dose was incorporated into 200 g of soil placed in a hermetically closed jar at 28 °C, and incubated for a 120-day period to determine CO₂ evolution. Once again it was confirmed that polyethylene is almost non-biodegradable, in contrast to PCL/S, which can be defined as a biodegradable material. Soil texture affected the mineralization kinetics of the plastic specimens, with higher values for the clayey soil. No changes in soil microbial biomass-C or -N were observed by adding polyethylene and PCL/S to the soil. Also, no significant differences were observed on seed emergence and development of rice seedlings (Oryza sativa L.) in plastic modified soil.     

Inclusion compounds of the copper(<Emphasis Type="SmallCaps">ii</Emphasis>) and zinc(<Emphasis Type="SmallCaps">ii</Emphasis>) complexes with cyclam in cucurbit[8]uril: synthesis and structure

Inclusion compounds of the macrocyclic cavitand cucurbit[8]uril (CB[8], C₄₈H₄₈N₃₂O₁₆) with the copper(ii) and zinc(ii) complexes with the tetraazamacrocyclic ligand cyclam, {[Cu(cyclam)(H₂O)₂]@CB[8]}Cl₂·18H₂O (1) and {[Zn(cyclam)]@CB[8]}Cl₂·13H₂O (2), were synthesized. The compounds were characterized by X-ray diffraction analysis, electrospray mass spectrometry, IR spectroscopy, and elemental analysis. The ¹H and ¹³C NMR method revealed only one trans-isomer of the zinc(ii) complex with cyclam in an aqueous solution of inclusion compound 2.