Mixed anhydrides in peptide synthesis. factors afffectlng urethane formation and racemization

A comparative study on the effectiveness of two mixed anhydrides procedures in the synthesis of a series of hydrophobic tripeptides by using three solvents and four different hydrophobic nucleophiles has been carried out. Dicyclohexylcarbodiimide (DCC) method has also been used as reference for comparative purposes. The results show that despite the system Dpp/DMF provides the best chemical yields with these specific couplings, when retention of configuration is concerned the coupling method of choice should be DCC/HOBt using either THF or DMF as solvent system.     

A new method for the detection of racemization during peptide synthesis: stereoselective hydrolysis of diastereomeric peptides by leucine aminopeptidase

A suitably protected dipeptide of configuration L -D , e.g. Z-L -Ala-D -Ala is coupled with an all L alanine peptide, e.g. L -Ala-L -Ala-ONb

1 Abbreviations according to the IUPAC-IUB rules, ‘Symbols for Amino-Acid Derivatives and Peptides, Recommendations (1971)’'. see e.g. J. biol. Chemistry 247, 977 (1972). In particular the following abbreviations have been used: Z = benzyloxycarbonyl-, -ONb = p-nitrobenzyloxy-, -ONSu = succinimido-oxy-. Additional abbreviations are LAP = leucine aminopeptidase, DCCI = N,N′-dicyclohexylcarbodiimide, DMF = dimethylformamide.

. The blocking groups are removed and the free peptide hydrolyzed by leucine amino peptidase (E.C. 3.4.1.1). This enzyme shows absolute L -specificity for the penultimate peptide bond from the amino end and therefore cleaves only the all L peptide formed through racemization. The amount of free alanine determined by amino acid analysis gives a multiple of the degree of racemization. The sensitivity of the test allows 0.1% of (L -Ala)₄ to be detected in the synthesis of L -Ala-D -Ala-L -Ala-L Ala. Coupling of Z-L -Ala-D -Ala and Z-L -Ala-D -Phe with di- and trialanine peptides has been studied using DCCI and DCCI + 1-hydroxybenzotriazole as coupling reagents. The degree of racemization was around 80% for the coupling by DCCI in DMF but was reduced to 0.2–0.4% in the presence of 2 equivalents of 1-hydroxybenzotriazole. Coupling using the succinimide esters Z-L -Ala-D -Ala-ONSu and Z-L -Ala-D -Phe-ONSu resulted in 0.8 to 10% racemization, depending on the solvent and base used.     

A simplified method for the determination of fenpropathrin residues in fruits

Summary A simple and efficient method is described for the determination of fenpropathrin in oranges, pears, apples and strawberries. The procedure is based on the extraction of each homogenized fruit sample with hexane:acetone (1∶1, v/v) mixture, followed by a cleanup technique on a column packed with florisil, using a hexane:ethyl ether (7∶3, v/v) mixture, and gas chromatographic analysis with electron capture detection (ECD). The fortification levels (0.5;1.0;2.0 mg kg⁻¹) were selected according to the maximum residue limits (MRLs) established for fenpropathrin by Brazilian legislation. Mean recoveries from five replicates of fortified fruit samples ranged from 83 % to 98%, with coefficients of variation from 1.4 to 13.5 and detection limits varying from 0.1 to 0.2 mg kg⁻¹.     

The relative susceptibility to racemization of L- and D-residues in peptide synthesis

An activated D-residue of a protected dipeptide when coupled racemizes twice as much as an activated L-residue, which is contrary to the case for couplings of acylamino acids.     

Segment coupling in peptide synthesis—II : A simple predictive equation correlating racemization and primary structure

A predictive equation based on extrathermodynamic assumptions is proposed, that allows the prediction of the degree of epimerization in tripeptide model reactions of condensation of peptide segments as a function of the primary structure. The experimental validation of this equation is presented in the field of the 125 tripeptides containing alanine, leucine, phenylalanine, valine and isoleucine, the prediction requires 13 epimerization measurements on reference reactions. Sixteen statistically chosen predictions are checked experimentally showing a good fit in spite of the drastic basic assumptions needed to set up the equation.     

Copper(II) chloride as a new,efficient additive suppressing racemization in peptide synthesis by the carbodiimide method

Copper(II) chloride was found to be an extremely efficient additive suppressing racemization in the carbodiimide mediated couplings.     

A new reagent for the mediation of amide bond formation in peptide synthesis.

The potential application of 1-oxo-1-chlorophospholane (5) as a novel reagent for the in situ activation of Nα -protected amino acids for use in peptide bond forming reactions has been examined. Wherever possible, 32.4Mhz ³¹P nuclear magnetic resonance (n.m.r.) spectroscopy was employed to follow both the formation of the intermediate phospholanic-carboxylic mixed anhydride and the subsequent aminolysis reaction.     

A simplified method for the determination of total homocysteine in plasma by electrospray tandem mass spectrometry

Hyperhomocysteinemia is a risk factor for different diseases. Several methods have been developed to analyze homocysteine and the immunometric ones, although expensive, they are in widespread use. A rapid LC‐MS/MS method for homocysteine assay has been developed for the application of large clinical chemistry routines. Selected reaction monitoring was performed through the transitions m/z 136.0→90.1 for homocysteine and m/z 140.0→94.0 for the internal standard. ESI was used to generate [H+] adduct ions. Chromatographic isocratic separation was achieved using a strong cation exchange column. The mobile phase was methanol/water (20:80 v/v, containing 0.1% formic acid and 1.5 mmol/L ammonium formate in the water phase) at a flow rate of 0.250 mL/min (35°C). Samples treatment consisted in the reduction with DTT and deproteinization with methanol. Recovery, linearity, LOD, LOQ and total imprecision were evaluated to validate the method. Homocysteine values on 100 serum samples were compared with those obtained by HPLC and immunometric methods. The method is robust, selective and precise in the whole range of values studied. Moreover, low reagent cost and easiness of sample treatment make this method useful, not only for research, but also for routine work.     

A modified HPLC method for the determination of aspartic acid racemization in collagen from human dentin and its comparison with GC

The D/L ratio of aspartic acid enantiomers in proteins of low turnover is generally accepted as a reliable procedure for age determination. In our study, twelve samples of eyetooth dentin were analyzed for age determination. The pure insoluble collagen isolated from eyetooth dentin was obtained by an EDTA demineralization process. Free amino acids obtained after collagen hydrolysis were converted into o-phthaldialdehyde-N-acetyl-L-cysteine (OPA-NAC) derivatives for HPLC analysis under modified conditions and into trifluoroacetic acid isopropyl esters for GC analysis, respectively. The modified HPLC procedure used phosphate buffer and acidified sample matrix prior to injection which resulted in suppression of peak tailing of both diastereomers, thus allowing achievement of both good selectivity and good resolution. To ensure the high accuracy of the developed method the other parameters, i.e. specificity, precision, linearity, LOD, and LOQ, were also determined. Nine collagen samples covering the age range of 18 to 84 years were used for the determination of coefficient of racemization (KR) and calculation of parameters for age estimation. The regression equations for the data set analyzed were as follows: KR= 0.0005 x age + 0.0262 (R2 = 0.9639) for HPLC, and KR= 0.0006 x age + 0.0319 (R2 = 0.9374) for GC, respectively.     

多肽合成中几种形成二硫键方法的介绍

综述了多肽合成中几种形成二硫键的方法。介绍了经典的空气氧化法、铁氰化钾氧化法和碘氧化法及近年来发展起来的二甲亚砜氧化法、三氟乙酸铊氧化法和氯硅烷-亚砜氧化法等方法的特点,同时还介绍了形成两对或多对二硫键的策略。     

A simplified calorimeter for the precise determination of purity

A calorimetric apparatus has been constructed for the determination of the mole fraction of impurity in a highly purified sample of a chemical compound by measurement of the temperature of solid-liquid equilibrium as a function of the fraction of the sample melted. An experimental value of the heat of fusion of the compound is also obtained, thus yielding the cryoscopic constant A. The apparatus, though relatively simple to Operate, has given results of good precision. The method has been tested over a wide temperature range using methylcyclopentane, n-heptane, n-decane, cyclohexylcyclohexane, and n-hexadecane, which have freezing points ranging from —142°C to +18°C.     

A simplified UV spectrometric method for determination of peroxide value in thermally oxidized canola oil

The aim of present study was to develop a simple method on UV spectrometer for the determination of peroxide value (PV) of the frying oil. The basis of the PV determination was the stoichiometric reaction of triphenylphosphine (TPP) with the hydroperoxides present in frying oil to produce triphenylphosphine oxide (TPPO), which exhibits a readily measurable absorption band at 240 nm by ultraviolet region. The PV ranged between 0.15 and 11.66 meq. of active oxygen per kilogram of oil as the canola oil was heated from 0 to 12 h in the fryer at 180 °C. The proposed method was correlated with official AOCS titration method and best correlation coefficient (R² = 0.99525) was achieved, proving that there is no significant difference in the results. Therefore, developed method could serve as an alternative to the titration method, for the determination of PV in frying oils.     

A simplified strategy for the synthesis of dideoxyribonucleotide blocks

The rapid synthesis of dideoxyribonucleotide blocks in 60–85% isolated yields has been achieved by combining the phosphorylation and condensation steps in a sequential reaction series which also allows the recovery of unreacted nucleotides.