Synthesis of two sequential polypeptides by dispersion in benzene and their circular dichroism spectra in aqueous solution: Poly(L-glu-L-lys-L-ala-gly) and poly(L-ala-D-glu-L-lys-D-ala-gly)

The monomers γ-benzylglutamyl-ε-benzyloxycarbonyl-lysylalanylglycine pentachlorophenyl ester and alanyl-γ-benzyl-D -glutamyl-ε-benzyloxycarbonyllysyl-D -alanyl-glycine pentachlorophenyl ester, were polymerized in dilute solutions of dimethylform-amide (DMF) or as dispersions in the same volume of benzene. After deprotection with hydrogen bromide, the products were either chromatographed on Sephadex G-50 or dialyzed. The polymers derived from the polymerization in benzene were considerably larger than those from DMF. The results in benzene indicated that high monomer to solvent ratios are not necessary for the production of high-molecular-weight sequential polypeptides. Circular dichroism spectra of the polymers and monomers at neutral and acid pH indicated that poly(L -Glu-L -Lys-L -Ala-Gly) exists in a random coil configuration and poly(L -Ala-D -Glu-L -Lys-D -Ala-Gly) exists in a β conformation.     

Synthesis of poly(L-Tyr-L-Glu-L-Ala-Gly) and poly(L-Glu-L-Lys-L-Ala) and their circular dichroism spectra in aqueous solution

Two sequential polypeptides, poly(O-benzyl-L -Tyr-γ-benzyl-L -Glu-L -Ala-Gly) and poly(ε-benzyloxycarbonyl-L -Lys-L -Glu-L -Ala), were synthesized, the former by the pentachlorophenyl ester of the tetrapeptide monomer and the latter by the azide of the tripeptide monomer. After deprotection and dialysis, poly(L -Tyr-L -Glu-L -Ala-Gly) was obtained in 71% yield and had a molecular weight of 53,000. The circular dichroism spectra (CD) of the polymer at pH's 7.2, 10.5, and 11.8 and of oligomers and of the monomer at pH 7.2 indicated that the polymer exists in an α-helical conformation. After deprotection, poly(L -Lys-L -Glu-L -Ala) was obtained in 37% yield and had a molecular weight of 3000. The CD spectra of the polymer at pH 7.2 and 2.8, and of the monomer at pH 7.2, indicated that the polymer is in a randomly coiled configuration.     

Racemization in the synthesis of sequential polypeptides using N-hydroxysuccinimide esters

Racemization in the synthesis of peptide intermediates and their polymers was investigated, using L-amino acid oxidase. The formation of N-hydroxysuccinimide esters from N-protected peptide acids yielded optically pure products in contrast to p-nitrophenyl and pentachlorophenyl active esters. The racemization in the polymerization step was found to be base sensitive. Partially racemized polymer can result from optically homogeneous monomer. Thus, the optical integrity of active monomer species carries no guarantee for that of the polymer.     

Sequential polypeptides containing arginine as histone models: synthesis and conformational studies

The sequential polypeptides (L -Arg-X-Gly)_n, where X represents amino acid residues Ala, Val, and Leu, were prepared as models of arginine-rich histones to be used in studying their structure and their interactions with DNA. The polymerization was carried out on the pentachlorophenyl active esters of the appropriate tripeptides, while the toluene-4-sulfonyl group was used for protecting the arginine guanido group. CD was employed to investigate the conformation of (L -Arg-X-Gly)_n polymers in aqueous solutions, at different pH, as well as in trifluoroenthanol and hexafluoroisopropyl alcohol solutions. In aqueous solutions (at pH 7 and 12) the prepared sequential polymers behaved as a random coil. The CD spectra in various trifluoroethanol–water or hexafluoroisopropyl alcohol–water mixtures indicated that the degree of helical conformation of the studied polytripeptides increased in the order of Ala → Val → Leu. The opposite was true for the β-structure. Characteristics of β-turn are excluded from the poly(L -Arg-L -Leu-Gly), which assumed the most pronounced helical conformation. The poly(L -Arg-L -Val-Gly) exerts a significant preference to the β-turn structure compared to that of poly(L -Arg-L -Ala-Gly). Thus the probability for helical, β-structure or β-turn conformations of the polymers was analyzed in relation to the bulkiness and length, and to the special features of the X-residue side chain (β-branching). We concluded that the prepared sequential arginine-containing polypeptides are plausible models for histone fractions, f₃ and f₂α₁.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

CD studies of synthetic polypeptides as histone models: Poly(L-Arg-L-Ile-Gly), poly(L-Arg-L-Nva-Gly), and poly(L-Arg-L-Nle-Gly)

Sequential polypeptides (L -Arg-X-Gly)_n were prepared as synthetic models of arginine-rich histones to study their structure and their stereospecific interactions with DNA. In our previous work the conformational characteristics of poly(L -Arg-L -Ala-Gly), poly(L -Arg-L -Val-Gly), and poly(L -Arg-L -Leu-Gly) have already been analyzed. To obtain further insight into the influence of the X residue side chain on the conformation of the (L -Arg-X-Gly)_n polytripeptides, we now report their synthesis and cd properties when X represents the amino acid residues Ile, Nva, and Nle. The pentachlorophenyl active esters of the appropriate tripeptides were used to perform the polymerization, and the toluene-4-sulfonyl group was used to protect the arginine guanido group. CD spectroscopy showed that, in 100% trifluoroethanol, the degree of helical conformation increased in the order Ile → Nle → Nva. An equilibrium between β-turn, α-helix, and random-coil conformers occurred in 100% hexafluoroisopropyl alcohol, while a rise in the temperature or the addition of water favored the α-helix, the highest percentage of which was observed in a mixture of hexafluoroisopropyl alcohol: water (20 : 80) and in the order Ile → Nle → Nva. In aqueous solutions (at pH 7 and 12) the polymers behaved as a random coil, but they were forced to a less aperiodic structure, over a range of ionic strengths (0–0.5M NaF). A rise in temperature of up to 70°C in 100% trifluoroethanol resulted in a decrease of the α-helix percentage of the polymers, while in aqueous solutions the aperiodic structure decreased with increasing temperature. This study proved the importance of the nature of the X residue (length, C_β branching) in relation to the structural order of the sequential polypeptides. We concluded that the polymers prepared are suitable models for arginine-rich histones.     

Polypeptides with known repeating sequences of amino acids. Comparison of several methods used for the synthesis of poly-gamma-D- and L-glutamylglycine and investigation of its serological reaction with antianthrax immune serum

To make a comparison of some of the various polymerization methods, poly-γ-D -and L -glutamylglycine were synthesized through the mixed anhydride, carbodiimide, and pentachlorophenyl active ester methods. This last method proved to be best, on the basis of weight-average molecular weights (M?_w). A detailed study was also carried out to determine the optimum conditions for this method. The M?_w, values of the polymers were determined by the sedimentation equilibrium method; it was found that the pentachlorophenyl active ester gave M?_w values of up to 11, 500 but the mixed anhydride and carbodiimide methods gave M?_w, values of only about 1000. Tests with rabbit antianthrax immune serum showed that neither D - nor L -isomer precipitated with the antiserum, but that, both were able to inhibit the precipitation of the antiserum by anthrax polypeptide. In addition, the D -polymer gave more inhibition than the L , which would indicate a specificity of the antibody for D -glutamic acid residues.     

Microwave assisted facile synthesis of amino acid benzyl ester p-toluenesulfonate and hydrochloride salts

Summary A simple method for the synthesis of several amino acid benzyl esterp-toluenesulfonate salts from the corresponding amino acid and benzyl alcohol in presence ofp-toluenesulfonic acid accelerated with microwave irradiation is described. Under similar condition, the amino acid benzyl ester hydrochloride salts have also been obtained by using thionyl chloride instead ofp-toluenesulfonic acid in good yield and purity.     

Far-infrared spectra of sequential polypeptides with -helical and polyglycine II structures

The sequential copolymers of glycine and L -alanine, L-valine and L -alanine, L-leucine and L -alanine, and L-phenylalanine and L -alanine and those containing the L-proline residues were synthesized. The infrared spectra in the region from 700 to 200 cm^-1 were measured for these polypeptides with the α-helical conformation or the polyglycine II structure and compared with the spectra of the β-form structures. The results showed that several infrared bands observed in the region from 600 to 200 cm^-1 clearly reflect not only the backbone conformations but also the local conformations of component amino acid residues of polypeptides with the α-helical, β-form and polyglycine II structures.     

Occurrence of a unique amino acid racemase in a basidiomycetous mushroom, Lentinus edodes

Free -alanine was detected in a cell extract of the fruit-body of an edible basidiomycetous mushroom, Lentinus edodes (Shiitake), by means of reverse-phase high performance liquid chromatography. We also found an amino acid racemase activity in L. edodes fruit-body, and purified the enzyme. The enzyme has a molecular weight of approximately 86,000, and consists of two subunits of identical molecular weight (44,000). The optimal pH of the enzyme activity is around pH 9.5 for both -to- and -to- alanine racemization. The enzyme requires pyridoxal 5′-phosphate as a cofactor. K_m and V_max values for -alanine were 37.3 mM and 520 nmol/min/mg, respectively; for -alanine, they were 9.21 mM and 141 nmol/min/mg, respectively. The equilibrium constant was calculated to be 1.10, which is consistent with the theoretical value for the racemase reaction. The ability of the enzyme to catalyze the racemization of various -amino acids was investigated. The enzyme catalyzes the racemization of -serine (relative reaction rate, 144% of rate for -alanine), -alanine (100%), -homoserine (17.1%), -2-aminobutyrate (5.6%), -glutamate (4.5%), and -asparagine (3.2%). To the best of our knowledge, this is the first report of an amino acid racemase produced by a basidiomycetous mushroom.     

Racemization in the Coupling Reaction,Pro-Val+Pro,with the Activated Ester Methods

The degree of racemization in the several activated ester methods of the peptide synthesis was measured in using the critical racemization test, Pro-Val+Pro, with help of gas chromatography. The results were compared with that in the coupling reaction, Leu-Phe+Val, in which no racemization had been reported in the corresponding reaction conditions by F. Weygand et al., when the activated dipeptide esters had been prepared from Z-Leu+Phe-activated esters. The significantly higher racemization was observed in the methods of N-hydroxypiperidine ester and thiophenyl ester, even when the activated dipeptide esters were prepared from Z-Pro+Val-activated esters. On the other hand, almost no racemization was observed in the N-hydroxysuccinimide ester and p-nitrophenyl ester methods. A great extent of the racemization was detected when the activated dipeptide esters were prepared directly from Z-Pro-Val-OH.     

Poly(L-histidyl-L-alanyl-α-L-glutamic acid). I. Synthesis

Poly(L -histidyl-L -alanyl-α-L -glutamic acid) has been prepared in order to test the acid–base catalytic ability of a carboxyl-imidazole hydrogen-bonded system. Two different blocked histidyl-alanyl-glutamic acid monomers were used in the polymerization step. The imidazole ring was blocked with either a dinitrophenyl or a t-butyloxycarbonyl group. The γ-carboxyl of glutamic acid was protected as the benzyl ester. Both the coupling reactions and the polymerization step were via the N-hydroxysuccinimide active ester method. Thiolysis removed the dinitrophenyl group, while hydrogen bromide removed the t-butyloxycarbonyl and the benzyl groups. The water-soluble unblocked polymers obtained were fractionated on Sephadex G-50 or Bio-Gel P30. Fractions within a range of average molecular weights of 2,000 to 25,000 were isolated. Enzymatic oxidation of the acid hydrolyzate of the polymers revealed that no detectable racemization had occurred.     

Carrier design: Conformational studies of amino acid (X) and oligopeptide (X-DL-Alam) substituted poly(L-lysine)

The present study was undertaken to examine the influence of the reversal of the sidechain sequential order on the conformation of branched polypeptides. At the same time, the influence of the optically active amino acid joined directly to the poly (L -Lys) backbone and the DL -Ala oligomer grafted as chain-terminating fragment were separately analyzed. Therefore two sets of polypeptides were synthesized corresponding to the general formula poly [Lys-(X_i,)] (XK) and poly[Lys-(DL -Ala_m-X_i)] (AXK) when X = Ala, D -Ala, Leu, D -Leu, Phe, D -Phe, Ile, Pro, Glu.,D -Glu, or His. For coupling amino acid X to polylysine, three types of active ester methods were compared: the use of pentafluorophenyl or pentachlorophenyl ester, and the effect of the addition of an equimolar amount of 1-hydroxybenzotriazole. After cleavage of protecting groups, AXK polypeptides were synthesized by grafting short oligo (DL -Ala) chains to XK by using N-carboxy-DL -Ala anhydride. The CD measurements performed in water solutions of various pH values and ionic strengths were used for classification of the polypeptide conformations as either ordered (helical) or unordered. Different from what was observed with the unsubstituted poly (L -Lys), poly[Lys-(X_i)] type polypeptides can adopt ordered structure even under nearly physiological conditions (pH 7.3, 0.2M NaCl). These data suggest that the introduction of amino acid residue with either (ar) alkyl side chain (Ala, Leu, Phe) or negatively charged side chain (Glu) promotes markedly the formation of ordered structure. Comparison of chiroptical properties of poly [Lys- (DL -Ala_m-X_i)] and of poly [Lys- (X_i)] reveals that side-chain interactions play an important role in the stabilization of ordered solution conformation of AXK type branched polypeptides. The results give rather conclusive evidence that not only hydrophobic interactions, but also ionic attraction, can be involved in the formation and stabilization of helical conformation of branched polypeptides. © 1993 John Wiley & Sons, Inc.     

Racemization in the synthesis of polytripeptide models of a collagen.

Racemization in the synthesis of tripeptide intermediates and their polymers was investigated, using L -amino acid oxidase. Stepwise investigation of peptide intermediates showed no racemization during peptide coupling steps or deprotection of benzyl esters by hydrogenolysis. Saponification of one of the methyl esters produced some racemization. Preparation of active esters from N-protected tripeptide acids containing optically active C-terminal amino acid, with one exception, produced racemization. The fractionated polymers were found to contain less racemized amino acids than the crude products or starting monomeric tripeptides, indicating that the racemized sequences gave rise to lower molecular-weight oligomers. The sequences investigated were -Pro-Pro-Ala-, -Ala-Pro-Pro-, -Val-Pro-Pro-, -Pro-Pro-Leu-, -Pro-Gly-Leu-, -Pro-Gly-Phe-, -Pro-Gly-Val-, -Gly-Val-Pro-, -Phe-Pro-Gly-, -Leu-Pro-Gly-, and Ile-Pro-Gly-.     

Poly(L-alanine): specific heat measurements at low temperatures (1.8-20 degrees K) and one-dimensionality

Low-temperature specific heat measurements have been made on random poly(L -alanine) (Pilot). The effective one-dimensional (intra-molecular polymeric) Debye temperature is 323°K, and the corresponding three-dimensional temperature is 54°K. An anomaly, similar to that seen in polyethylene and other high polymers, is observed. The method will be a useful tool for the study of low-frequency vibrations in polypeptides.     

Synthesis and characterization of four sequential glycyl/glutamate polypeptides

Poly(Glu(OBzl)-Gly)_n, poly(Glu-Gly)_n, poly(Gly)-(Glu(OBzl)-Gly), and poly(Gly-Glu-Gly) were synthesized from the pentachlorophenyl esters of the sequential monomer. Both of the polymers containing free glumatic-acid residues are soluble in water, as is the lower molecular weight fraction of the polytripeptides with the benzyl ester in place. Circular dichroism studies and infrared dichroism studies suggest that the 2₁ helix is favored for the polydipeptide with removal of the benzyl ester reducing the conformational integrity. The polytripeptide showed evidence of 3₁ helix in addition to the 2₁ form, depending on solvent. A rationale for the conformations observed is developed based on the bulkiness of the side-chain residues and conformational stabilization, in certain cases, by hydrophobic interactions between the benzyl ester groups.     

以L-天冬氨酸为原料制备D-天冬氨酸的新方法

以L-天冬氨酸为原料经过酯化、消旋、拆分和水解制备D-天冬氨酸。使L-2,3-二苯甲酰酒石酸(L-DBTA)与DL-天冬氨酸-β-甲酯在水溶液中于65~70℃反应形成非对映体盐,冷却到室温,D-天冬氨酸.L-DBTA盐析出,过滤后再经水解得到D-天冬氨酸,收率78.2%,旋光纯度达到99%以上。     

Metabolic engineering of E. coli for β-alanine production using a multi-biosensor enabled approach

β-alanine is an important biomolecule used in nutraceuticals, pharmaceuticals, and chemical synthesis. The relatively eco-friendly bioproduction of β-alanine has recently attracted more interest than petroleum-based chemical synthesis. In this work, we developed two types of in vivo high-throughput screening platforms, wherein one was utilized to identify a novel target ribonuclease E (encoded by rne) as well as a redox-cofactor balancing module that can enhance de novo β-alanine biosynthesis from glucose, and the other was employed for screening fermentation conditions. When combining these approaches with rational upstream and downstream module engineering, an engineered E. coli producer was developed that exhibited 3.4- and 6.6-fold improvement in β-alanine yield (0.85 mol β-alanine/mole glucose) and specific β-alanine production (0.74 g/L/OD₆₀₀), respectively, compared to the parental strain in a minimal medium. Across all of the strains constructed, the best yielding strain exhibited 1.08 mol β-alanine/mole glucose (equivalent to 81.2% of theoretic yield). The final engineered strain produced 6.98 g/L β-alanine in a batch-mode bioreactor and 34.8 g/L through a whole-cell catalysis. This approach demonstrates the utility of biosensor-enabled high-throughput screening for the production of β-alanine.     

Preparative resolutions of an α-methyl carboxylic acid through selective transformations of readily epimerizable intermediates

Two preparations of the enantiomers of 2 are described. The first makes use of the chromatographic separation of the diastereomeric amides 6a and 6b. Standard hydrolysis of these amides caused racemization, so a milder sequence was developed which utilized carbonyldiimidazole and 1 equivalent of 1 N LiOH. The second preparation involved classical resolution of 9 with (?)-cinchonidine. Subsequent transformations of this substrate involved ester formation, Friedel–Crafts acylation, and ester hydrolysis, all without racemization. The most notable of these reactions was the use of EtAlCl₂ in the Friedel–Crafts step, which provided a mild acylation of 10. This second preparation affords a high yield, mild process for the potential preparation of kilogram quantities of (?)-(R)-2b.     

Synthesis of sequential polypeptides containing L-β-3,4-dihydroxyphenyl-α-alanine (DOPA) and L-lysine

Six different sequential polypeptides with the repeating units L -lysyl-L -DOPA, L -DOPA-L -lysine, L -lysyl-L -lysyl-L -DOPA, L -DOPA-L -DOPA-L -lysine, L -lysyl-L -lysyl-L -lysyl-L -DOPA, and L -DOPA-L -DOPA-L -DOPA-L -lysine have been synthesized by solution polymerization of the p-nitrophyenyl esters of the corresponding di-, tri-, and tetrapeptides. The O,O′-dimethyl and N-ε-2-chlorobenzyloxycarbonyl groups were used to protect side chains of L -DOPA and L -lysine. The monomers for the polytripeptides and polytetrapeptides were prepared by stepwise elongation, using the dicyclohexylcarbodiimide coupling method. Moderately high molecular weight sequential polypeptides were obtained. The addition of 1-hydroxybenzotriazole increased their molecular weights, but not so effectively. The protected groups of the side chains were removed simultaneously by use of boron tribromide in chloroform. Water-soluble sequential polypeptides containing L -DOPA were obtained.