Investigations of in vivo methionine racemization in mammalian tissues

The racemization rate of methionine at pH 7.4 and 100 degrees C was determined to be similar to that of aspartic acid. However, analyses of dentin and ocular lens nucleus samples from known age Rhesus monkeys showed that unlike aspartic acid, methionine does not undergo in vivo racemization in mammalian proteins.     

Unexpected racemization of proline or hydroxy-proline phenacyl ester during coupling reactions with Boc-amino acids.

When L-proline or O-benzyl-trans-4-hydroxy-L-proline phenacyl ester was coupled with Boc-amino acids in dimethylformamide using water-soluble carbodiimide (WSCI) in the presence of anhydrous 1-hydroxybenzotriazole (HOBt) as coupling reagents, extensive racemization was observed at the C alpha of the proline or hydroxy-proline residue. The extent of racemization was measured by HPLC after the coupling with Boc-L-Leu-OH in the presence or absence of HOBt. The extent of racemization increased when HOBt was added to the reaction mixture, but greatly decreased when it was not, indicating that HOBt was needed for inducing racemization. Almost no racemization was observed when the coupling reaction was carried out by the mixed anhydride procedure in tetrahydrofuran or by the carbodiimide method in dichloromethane without using HOBt. In the case of coupling reactions with ordinary L-amino acid phenacyl esters, no racemization was observed. Examination of some model systems yielded sufficient evidence to prove that HOBt is an efficient catalyst for racemizing proline or hydroxy-proline phenacyl ester not only in the stage of cyclic intermediate formation but also in the opening of the ring structure. Thus, the racemization reaction was found to be closely related to the formation of the cyclic carbinol-amine derivative.     

Dimethylmaleic anhydride, a specific reagent for protein amino groups

The reagent dimethylmaleic anhydride does not cause a stable modification of thiol compounds under the conditions used for modification of protein amino groups, in contrast to maleic and monomethylmaleic anhydrides, which produce an irreversible modification of sulfhydryl groups. This behavior and the low reactivity toward hydroxyamino acid residues, shown in a previous work, make dimethylmaleic anhydride a specific reagent for protein amino groups.     

Racemization of alpha-melanotropin

Racemization of the amino acid residues of alpha-melanotropin was measured after exposure of the peptide to alkali for various lengths of time. Rates of racemization were then compared to the rate of transformation by alkali of alpha-melanotropin into a hormone with prolonged melanotropic activity. When in vitro prolongation became maximal, serine, methionine, histidine, phenylalanine and arginine were racemized 50-70%, glutamic acid, tyrosine and tryptophan 30-40% and lysine, proline and valine 10% or less. Racemization of a particular amino acid residue in alpha-melanotropin could not be associated with induction of prolongation of activity. Rather, partial racemization at multiple sites in the molecule seems almost as effective as extensive or total racemization of a single residue in producing a hormone with prolonged biological effects.     

Histidine racemization in the synthesis of an analog of the luteinizing hormone releasing factor. Investigation by HPLC and enzymatic methods

To investigate histidine racemization in the synthesis of a LHRH analog, (D-Trp)6-LHRH2-10 was built up by stepwise elongation of the sequence 3-10 using the solid phase technique on a 1% cross-linked chloromethyl polystyrene. For the whole synthesis the tert.-butyloxycarbonyl (BOC) group was used for temporary N-terminal protection. To protect the pi-nitrogen in histidine the benzyl-oxymethyl (BOM) group was utilized. The condensation position in the (D-Trp)6-LHRH analog was chosen so as to be able to investigate the racemization of histidine. We coupled BOC-His(BOM) with the (D-Trp)6-LHRH3-10 fragment using three different activating agents, mixed anhydride, carbonyldiimidazole (1 equiv.)/1-hydroxybenzotriazole (2 equiv.) and dicyclohexylcarbodiimide/1-hydroxybenzotriazole. The racemization was investigated by enzymatic digestion and by HPLC. For HPLC, (D-His(BOM)2-(D-Trp)6-LHRH2-10 was also synthesized. It could be proved that practically no racemization occurs during the actual peptide synthesis. The small amount (1%) of D-histidine found is due to racemization in the synthesis of BOC-His(BOM).     

Effect of copper(II) chloride on suppression of racemization in peptide synthesis by the mixed anhydride and related methods.

In segment couplings by the mixed anhydride method using isobutyloxycarbonyl chloride, the use of copper(II) chloride as an additive suppressed racemization completely in the same manner as in the carbodiimide method reported previously. This was confirmed by employing a number of couplings between Z-dipeptides and amino acid esters. The racemization-suppressing effect of other compounds were also evaluated by employing one of these model couplings to be at best only limitedly effective. Copper(II) chloride was effective also in the related method using EEDQ. Thus, in the couplings where a low level of racemization was observed without an additive, the addition of copper(II) chloride eliminated racemization even at ambient temperature where EEDQ is usually used. The effectiveness of copper(II) chloride was confirmed also in the BOP-Cl method. In the presence of HOBt racemization was reduced to a low but still detectable level, while it was suppressed completely by the addition of copper(II) chloride.     

An Integrated Experimental and Computational Approach for Characterizing the Kinetics and Mechanism of Triadimefon Racemization

Enantiomers of chiral molecules commonly exhibit differing pharmacokinetics and toxicities, which can introduce significant uncertainty when evaluating biological and environmental fates and potential risks to humans and the environment. However, racemization (the irreversible transformation of one enantiomer into the racemic mixture) and enantiomerization (the reversible conversion of one enantiomer into the other) are poorly understood. To better understand these processes, we investigated the chiral fungicide, triadimefon, which undergoes racemization in soils, water, and organic solvents. Nuclear magnetic resonance (NMR) and gas chromatography / mass spectrometry (GC/MS) techniques were used to measure the rates of enantiomerization and racemization, deuterium isotope effects, and activation energies for triadimefon in H₂O and D₂O. From these results we were able to determine that: 1) the alpha‐carbonyl carbon of triadimefon is the reaction site; 2) cleavage of the C‐H (C‐D) bond is the rate‐determining step; 3) the reaction is base‐catalyzed; and 4) the reaction likely involves a symmetrical intermediate. The B3LYP/6–311 + G** level of theory was used to compute optimized geometries, harmonic vibrational frequencies, nature population analysis, and intrinsic reaction coordinates for triadimefon in water and three racemization pathways were hypothesized. This work provides an initial step in developing predictive, structure‐based models that are needed to identify compounds of concern that may undergo racemization. Chirality 28:633–641, 2016. © 2016 Wiley Periodicals, Inc.     

Isolation of R-(−)-3-Hydroxy-β-ionone from Burley Tobacco

The tritium-hydrogen exchange method was used to determine the total racemization of amino acid residues of four proteins (ribonuclease A, lysozyme, soybean protein and casein) during their exposure to an alkali. Tritium was incorporated with first order kinetics into these proteins during their incubation in 0.2 n NaOH at 40°C. The tritium-hydrogen exchange increased as the temperature and the alkali concentration increased. In contrast, pepsin digestibility decreased extensively in the initial stage of the treatment. This phenomenon was verified by the subsite theory of protease, that only minor racemization renders the extended range of the peptide chain around the racemized amino acids non-susceptible to pepsin. General precautions against the racemization of food protein treated with alkalis are discussed briefly in terms of the deterioration of nutrients.     

氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

Habituation to alkali and increased u.v.-resistance in DNA repair-proficient and -deficient strains of Escherichia coli grown at pH 9.0

Repair-deficient strains of Escherichia coli carrying polAI or recA mutations were more alkali-sensitive than was their repair-proficient parent but, like strain 1829 ColV, I-K94, they showed habituation to alkali (induction of increased resistance) when grown at pH 9.0. Occurrence of such increased alkali resistance in the recA mutant implies that habituation to alkali does not depend on induction of SOS-related repair mechanisms. Organisms of repair-proficient and repair-deficient strains also became more resistant to u.v.-irradiation after growth at pH 9.0; this increased u.v.-resistance also appeared to be RecA-independent.     

A new peptide coupling reagent—Dialkyl phosphite

Eighteen N-protected dipeptide esters were synthesized using dialkyl phosphite as a coupling reagent. The reaction proceeded probably through a mixed carboxylic-phosphoric anhydride intermediate. The dipeptide esters were prepared in satisfactory yields and low racemization.     

Dissociation of small-intestinal sucrase - isomaltase complex into enzymatically active subunits.

1. The sucrase - isomaltase complex from rabbit small intestine dissociated into its subunits upon reaction with citraconic anhydride. They can recombine after deacylation under mild acidic conditions. 2. When citraconylated, the subunits could be separated and isolated in a catalytically active form. 3. The previously reported procedure for separation of the subunits by alkaline treatment at pH 9.6 is apparently not due to contaminating degradative enzymes (possibly still present at undetectable levels in the isolated sucrase - isomaltase complex) but to the action of alkali.     

Racemization in the Coupling Reaction with the Several Methods beside the Activated Ester Methods

The degree of racemization in the coupling reaction, Pro-Val + Pro, with the several other methods than the activated ester methods was measured and the results were compared with that in the coupling reaction, Leu-Phe + Val, as well as in the previous paper.¹⁾ In the azide and the mixed anhydride methods, no or almost no racemization was detected, whereas in the other tested methods of peptide synthesis (Pachornik-, DCCD- and phosphazo-methods) the significantly large racemization was observed. It can be attributed to the strong nucleo- philic N-atom in the penultimate amino acids (Pro) and the steric hindrance of C-terminal amino acid (Val), which are favourable to the formation of the oxazolone ring.

This assumption was further systematically confirmed in the synthesis of the other several tripeptides with the DCCD method. The separation of the diastereoisomers (LLL and LDL) of the resulting tripeptides by gas chromatography with a packed column was also here reported.     

Enantioselective autocatalysis. II. Racemization studies on a 1,4-benzodiazepinooxazole derivative

Since the bromofluoro-1,4-benzodiazepinooxazole derivativeI may undergo spontaneous resolution on crystallization from methanol solution, it provides an ideal substrate on which to study the phenomenon of enantioselective autocatalysis involving spontaneous resolution under racemizing conditions (srurc). in order to augment the understanding of factors which might be important in such a process we have now undertaken to study the racemization ofI in more detail by observing its rate under a wide variety of experimental conditions.     

季节、环境温度与黄鼠冬眠的关系

观察了达乌尔黄鼠在实验室内冬眠的一般情况。常温黄鼠的体温有着规律性的年周期,与环境温度的年周期变动不完全呈依从关系。出眠初期(4月下旬),动物体温高而稳定。4月至6月常温黄鼠的平均体温(皮温)为35.6℃,波动菹围32—37.5℃。随着体重达到顶峰,体温逐渐降低。8月份部分黄鼠出现低于32℃的低常体温,表明部分黄鼠自8月盛夏开始冬眠。但就整个种群而言,北京地区实验室内黄鼠冬眠季自9月下半月开始。3月底止,共6.5个月。秋季室温下降,动物入眠趋势增长。浅低体温(31.9—15℃)的比数逐渐升高。9月至12月,低体温(低于31.9℃)的百分比从47％增至84.8％,反映了动物从浅冬眠向深冬眠过渡。1月至2月份,低体温占85％以上,深低体温(低于15℃)占绝对优势。标志着动物种群的深眠月份。秋季动物从常温期向冬眠期转化的界线是不清的,而春季从冬眠期向常温相转化的界限却比较明显。     

Studies on the actomyosin ATPase and the role of the alkali light chains

Myosin isoenzymes, highly enriched in either alkali 1 or alkali 2 light chains have been prepared by light chain exchange in 4.7 M ammonium chloride, under conditions where there is minimal loss of ATPase activity. While the actin-activated ATPase measurements were complicated by a biphasic dependence on actin concentration, the two myosin isoenzymes behaved in a similar manner; at a variety of ionic strength conditions their maximum rates of ATP hydrolysis were nearly identical. Furthermore, under conditions where their Km values could be reliably determined, their apparent affinities for actin in the presence of ATP did not differ greatly. These results suggest that the presence of a particular alkali light chain does not influence the maximum rate of ATP turnover by actomyosin under ionic strength conditions approximating physiological.     

Site specific interaction of protons liberated from Photosystem II oxidation with a hydrophobic membrane component of the chloroplast membrane

Chloroplast membranes have been shown previously to undergo a change in radioactive labeling by chemical modification reagents that is dependent on electron transport and protolytic events in Photosystem II. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis has been used to show that a low molecular weight chloroplast polypeptide (7.2 Kilodaltons) undergoes the most change in acetic anhydride labeling upon Photosystem II electron transport. A similar polypeptide has been identified by other workers as a component of the hydrophobic trans-membrane proton channel in chloroplasts. Photosystem I electron transport does not give the change in level of incorporation of acetic anhydride into this membrane protein. These results suggest that protons liberated from Photosystem II interact with a hydrophobic portion of the chloroplast membrane, perhaps with the trans-membrane proton channel.     

Substitution and racemization of 3-hydroxy- and 3-alkoxy-1,4-benzodiazepines in acidic aqueous solutions

Oxazepam (OX), 3-O-methyloxazepam, 3-O-ethyloxazepam, temazepam (TMZ), 3-O-methyltemazepam, and 3-O-ethyltemazepam underwent acid-catalyzed nucleophilic substitution reaction (hydrolysis) in an acetonitrile–oxygen-18 water mixture to form either OX or TMZ in which the 3-hydroxyl group was either partially or fully labeled with an oxygen-18 atom. The dependence of the hydrolysis rates on solvent composition, temperature, ionic strength, and in deuterated solvent was studied by reversed-phase high-performance liquid chromatography (HPLC). The rates of racemization of enantiomeric compounds in acidic aqueous solutions were studied by both spectropolarimetry and chiral stationary phase HPLC. In acetonitrile: 2.5 M H₂SO₄ (4:1, v/v) at 50°C, enantiomers of OX and TMZ underwent racemization at rates ≥40-fold faster than the rates of hydrolysis. Enantiomeric 3-O-alkyl derivatives of OX and TMZ in acidic aqueous solutions did not themselves undergo racemization and it was their hydrolysis products (either OX or TMZ) that underwent racemization. © 1995 Wiley-Liss, Inc.     

Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP

ATR, a critical regulator of DNA replication and damage checkpoint responses, possesses a binding partner called ATRIP. We have studied the functional properties of Xenopus ATR and ATRIP in incubations with purified components and in frog egg extracts. In purified systems, ATRIP associates with DNA in both RPA-dependent and RPA-independent manners, depending on the composition of the template. However, in egg extracts, only the RPA-dependent mode of binding to DNA can be detected. ATRIP adopts an oligomeric state in egg extracts that depends upon binding to ATR. In addition, ATR and ATRIP are mutually dependent on one another for stable binding to DNA in egg extracts. The ATR-dependent oligomerization of ATRIP does not require an intact coiled-coil domain in ATRIP and does not change in the presence of checkpoint-inducing DNA templates. Egg extracts containing a mutant of ATRIP that cannot bind to ATR are defective in the phosphorylation of Chk1. However, extracts containing mutants of ATRIP lacking stable DNA-binding and coiled-coil domains show no reduction in the phosphorylation of Chk1 in response to defined DNA templates. Furthermore, activation of Chk1 does not depend upon RPA under these conditions. These results suggest that ATRIP must associate with ATR in order for ATR to carry out the phosphorylation of Chk1 effectively. However, this function of ATRIP does not involve its ability to mediate the stable binding of ATR to defined checkpoint-inducing DNA templates in egg extracts, does not require an intact coiled-coil domain, and does not depend on RPA.     

Base-catalyzed racemization of 3-O-acyloxazepam

Enantiomers of 3-O-acyloxazepam (oxazepam 3-acetate; OXA) underwent base-catalyzed hydrolysis and racemization. Kinetics of reaction products formed from an OXA enantiomer in buffered and unbuffered alkaline solutions were analyzed by chiral stationary phase high-performance liquid chromatography. Racemization occurred with varying rates in aqueous solutions with pH ranging from 7.5 to 14. Racemization mechanism was studied by the dependence of rates of hydrolysis and racemization on temperature and pH. Mass spectral analysis of racemization products derived from an OXA enantiomer in a deuterated solvent indicated that racemization was accompanied by a proton exchange with the solvent. The results indicated that a base-catalyzed keto-enol tautomerism between the C2-carbonyl group and the C3 carbon was responsible for the observed racemization. © 1994 Wiley-Liss, Inc.

1 This article is a US Goverment work and, as such, is in the public domain in the United States of America.