Equilibration of alditol anhydrides in acetic acid

Dehydration of pentitols in acetic acid containing an acidic catalyst parallels that in aqueous sulfuric acid; 1,4(2,5)-dehydration occurs with inversion of configuration at C-2 or C-4. Acetylated alditols undergo similar processes via intermediates having free hydroxyl groups. Configurational inversion of 1,4- or 1,5-anhydroalditols is attributed to intermediate acyloxonium ions that are also proposed as intermediates in the structural isomerisation. Drastic treatment of each alditol gives equilibrium mixtures. The equilibrium concentrations are used to calculate free-energy differences.     

Polymerization of amino acid methyl esters via their copper complexes

Polymerization of glycine methyl ester catalyzed by cupric ions in organic solvents yields oligoglycines with a degree of polymerization up to nine. With a trifunctional amino acid, the yeild and degree of polymerization were much lower. Extension of this reaction to an aqueous medium was not successful even when copper ions were complexed with substances like montmorillonite or fatty acids. The prebiotic significance of this reaction is discussed.Contribution to the 4th International Conference on the Origin of Life, Barcelona, June 1973.     

Synthesis of D-allose fatty acid esters via lipase-catalyzed regioselective transesterification

The acylation of the rare sugar, D-allose (the C-3 epimer of D-glucose), with fatty acid vinyl esters was successfully carried out using Candida antarctica lipase in acetonitrile at 45 degrees C to give D-allose 6-alkanoates with high regioselectivity in good yields.     

Synthesis of novel vasodilatory active nicotinate esters with amino acid function

A variety of N-[(ethyl-4,6-diaryl-3-pyridinecarboxylate)-2-yl]amino acid esters 6a–h were synthesized through the reaction of 2-bromonicotinates 4 with a number of primary amino acid ester hydrochlorides 5 in refluxing tetrahydrofuran in the presence of triethylamine as dehydrohalogenating agent. Similarly, reaction of 4 with N-glycylglycine ethyl ester hydrochloride 7 ‘as a representative example of dipeptide derivative’ afforded smoothly the corresponding N-[(ethyl-4,6-diaryl-3-pyridinecarboxylate)-2-yl]-N′-glycylglycine ethyl ester analogues 8. However, reaction of 4 with 5 in refluxing pyridine yielded the unexpected 2-aminonicotinate esters 9. Vasodilation activity screening for the synthesized nicotinate esters was investigated in vitro on the contractile response of vascular thoracic aorta smooth muscle from Wistar rats, where all the tested compounds exhibit considerable vasodilatory properties. In addition, few prepared compounds especially, 6b, 6h and 9b reveal remarkable vasodilation potency (IC₅₀).     

The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site

The structure of L-amino acid oxidase (LAAO) from Calloselasma rhodostoma has been determined to 2.0 A resolution in the presence of two ligands: citrate and o-aminobenzoate (AB). The protomer consists of three domains: an FAD-binding domain, a substrate-binding domain and a helical domain. The interface between the substrate-binding and helical domains forms a 25 A long funnel, which provides access to the active site. Three AB molecules are visible within the funnel of the LAAO-AB complex; their orientations suggest the trajectory of the substrate to the active site. The innermost AB molecule makes hydrogen bond contacts with the active site residues, Arg90 and Gly464, and the aromatic portion of the ligand is situated in a hydrophobic pocket. These contacts are proposed to mimic those of the natural substrate. Comparison of LAAO with the structure of mammalian D-amino acid oxidase reveals significant differences in their modes of substrate entry. Furthermore, a mirror-symmetrical relationship between the two substrate-binding sites is observed which facilitates enantiomeric selectivity while preserving a common arrangement of the atoms involved in catalysis.     

Polymerization of amino acid methyl esters via their copper complexes.

Polymerization of glycine methyl ester catalyzed by cupric ions in organic solvents yields oligoglycines with a degree of polymerization up to none. With a trifunctional amino acid, the yield and degree of polymerization were much lower. Extension of this reaction to an aqueous medium was not successful even when copper ions were complexed with substances like montmorillonite or fatty acids. The prebiotic significance of this reaction is discussed.     

Enzymatic resolution of 2-aryloxy-1-propanols via lipase-catalyzed enantioselective acylation using acid anhydrides as acyl donors

Pseudomonas sp. lipase-catalyzed enantioselective acylation procedure using acid anhydrides as acyl donors was exploited for the resolution of 2-aryloxy-1-propanols carrying different substituents on the benzene ring. These primary alcohols, which belong to primary alcohols with an oxygen atom at the stereocenter, were resolved generally with moderate to good enantioselectivity (E of up to 55) through the acylation with hexanoic anhydride in diisopropyl ether at 25 °C in a short reaction time. With the alcohol substrate, which gave a low enantioselectivity in the acylation at ordinary temperature, the selectivity proved to be enhanced by conducting the reaction at low temperature (−10 °C). By this acylation procedure employing the acid anhydride, enantiomerically pure (R)-2-phenoxy-1-propanol was prepared in a gram-scale reaction.     

The oxidation of dicarboxylic acid CoA esters via peroxisomal fatty acyl-CoA oxidase

Evidence supporting a common peroxisomal beta-oxidation pathway for the coenzyme A thioesters of medium-chain-length dicarboxylic acids (DCn-CoA) and monocarboxylic acids (MCn-CoA) has been obtained. Using the mono-CoA esters of dodecanedioic acid (DC12-CoA) and lauroyl-CoA (MC12-CoA) as substrates, parallel inductions of activities and parallel increases in specific activities during purification of peroxisomal fatty acyl-CoA oxidase (EC 1.3.99.3) from rat liver after di(2-ethylhexyl)phthalate treatment were seen. The purified enzyme was used for antiserum production in rabbits; antiserum specificity was verified by immunoblot analysis. Coincident losses of oxidase activities with MC12-CoA and DC12-CoA were found in immunotitration experiments with rat liver homogenates, supporting the hypothesis that peroxisomal fatty acyl-CoA oxidase is solely responsible for the oxidation of medium-chain length dicarboxylic acid substrates. Kinetic studies with purified enzyme using the mono-CoA esters of sebacic (DC10-CoA), suberic (DC8-CoA), and adipic (DC6-CoA) acids along with DC12-CoA revealed substrate inhibition. Although these substrates exhibited similar calculated Vmax values, with decreasing chain length, the combination of increasing Km values and decreasing substrate inhibition constant (Ki) caused the maximum obtainable velocity to decrease. These studies offer an explanation for the previously observed limit of the ability of peroxisomes to chain-shorten dicarboxylates and increased urinary excretion of adipic acid when peroxisomal oxidation of dicarboxylic acids is enhanced.     

Heterologous expression of Rhodococcus opacus L-amino acid oxidase in Streptomyces lividans

L-amino acid oxidase (L-AAO) from Rhodococcus opacus is a highly enantioselective enzyme with a broad substrate specificity that catalyses the oxidation of L-amino acids to keto acids. The lao-gene (AY053450) from R. opacus was cloned into different Escherichia coli and Streptomyces lividans expression vectors. Expression in E. coli resulted in the accumulation of insoluble protein, but S. lividans was a suitable host for the heterologous production of L-AAO. When using the thiostrepton-inducible vector pIlaao, a specific activity of 0.18 Umg(-1) was obtained in the crude extract of S. lividans 1326. For the vector pUlaao, which contains the constitutive ermEp(*) promoter, a specific activity of 0.05 Umg(-1) was reached. Both the wild type and the recombinant L-AAO were purified to homogeneity. The expression systems described here now allow the structural and biochemical analysis of the L-AAO using genetic engineering methods.     

Presence of endogenous inhibitor of aromatic L-amino acid decarboxylase in monkey serum

Summary In the course of our studies on the developmental changes of aromatic L-amino acid decarboxylase (AADC) in the serum of Japanese monkeys (Macaca fuscata fuscata), we found the presence of an endogenous inhibitor of AADC in all stages of monkey life. This inhibitor inhibited the serum enzyme activity completely with L-5-hydroxytryptophan (L-5-HTP) as substrate, while the activity was partially inhibited with L-DOPA as substrate. The inhibitor was non-dialyzable, but it could be removed from the monkey serum by DEAE-Sephacel chromatography. After this treatment AADC activities could be detected in the monkey serum by using both L-DOPA and L-5-HTP as substrates. Moreover, the total activity for L-DOPA was augmented by 3-fold in the serum after the removal of the inhibitor. Serum AADC was partially purified from monkey and compared with that of rat using both L-DOPA and L-5-HTP as substrates, but the ratio of the activities for the two substrates did not change significantly in each fraction during purification from either monkey or rat serum.On leave from the University of Rajshahi, Rajshahi, Bangladesh.     

Cooperative polymerization of photosynthetic pigments in formamide-water solution

The aggregation of bacteriochlorophyll a and bacteriopheophytin a into large oligomers with maximum optical absorption at 860 nm was studied in a 3:1 (vol/vol) formamide/water solution, using optical absorption spectroscopy and electron microscopy. The aggregation is cooperative and proceeds according to two equilibrium constants. Initially, two pigment molecules form a “seed” that absorbs at ≈860 nm. The equilibrium constant, K_a, governing this reaction equals 1.3 × 10³ M^-1 in the case of bacteriochlorophyll a (due to experimental limitations, K_a for bacteriopheophytin a could not be determined). The addition of monomers to aggregates consisting of two or more units is governed by an equilibrium constant, K_b, equal to 2.2 × 10⁶ M^-1 for bacteriochlorophyll a and ≈ 10⁹ M^-1 for bacteriopheophytin a. The enthalpy and entropy changes that drive the bacteriochlorophyll oligomer formation are -9.25 and ≈0.0 kcal/mol, respectively. Above a threshold concentration, the amount of oligomers remains constant but their length continues to increase. Each oligomer appears to consist of dimers that are associated by hydrophobic interactions among their alcohol residues, forming long strands. Single strands presumably coil into helices that are seen as cylinders. The bacteriochlorophyll a oligomers form cylinders with a constant diameter of 150 Å and an average length of 2,000 Å (at 1.5 × 10^-5 M bacteriochlorophyll a). These cylinders contain 200-250 bacteriochlorophyll a dimers. The bacteriopheophytin oligomers coil into wider cylinders (≈400 Å in diameter) which contain ≈600-700 bacteriopheophytin a dimers. In both cases, the separation between the dimers is ≈20 Å. At such distances, the dipolar interactions among adjacent dimers are negligible and do not affect the optical absorption of each individual pair. Therefore, the optical absorption of these pairs can be a tool for investigating the absorption pattern of photosynthetic pigments in vivo.     

Conformational restrictions of biologically active peptides via amino acid side chain groups

Determining the relationships between conformation and biological activity in peptide hormones and neurotransmitters is an important goal of contemporary biology. A major difficulty in these studies is the conformational flexibility of most peptides and the high dependence of the conformations on environment. The question arises whether conformations determined in solution are relevant to those important to the peptide at the membrane receptor(s). One recent approach to overcome these difficulties has been the use of conformational constraints by covalent bonding of side chain groups of residues in the peptide. In this manner linear peptides are rendered cyclic, and cyclic peptides are further conformationally constrained either by ring contractions or by other conformational constraints. Biologically active peptides specifically designed by this approach have been found to possess several useful properties including: 1) greater conformational integrity; 2) increased agonist or antagonist potency; 3) prolonged biological activity; 4) increased enzymatic stability; and 5) increased specificity for a particular receptor. Careful applications of this approach have provided important new designs features for peptide structure-function studies, and new insights into peptide conformation-activity relationships for oxytocin, somatostatin, enkephalin, bradykinin, vasopressin, and other biologically active peptides.     

Expression of cloned aromatic L-amino acid decarboxylase in Xenopus laevis oocytes.

Sense mRNA coding for bovine adrenal medulla aromatic L-amino acid decarboxylase (AADC) was expressed following microinjection into Xenopus laevis oocytes. The expressed enzyme activity was stereoselective for L-5-hydroxytryptophan and L-DOPA and blocked by NSD-1015 an inhibitor of AADC. Heating the expressed enzyme at 55 degrees C resulted in a parallel loss of activity towards both substrates. Our findings are consistent with the prevailing notion that a single enzyme is able to decarboxylate both substrates in vivo.     

Three-dimensional arrangement of sugar residues along helical polypeptide backbone. 2. Synthesis of periodic N-glycosylated peptides by polymerization of tripeptide active esters containing alpha,alpha-disubstituted amino acid

New type of N-glycosylated peptides having periodic sequence of -[X-Gln(beta-D-GlcNAc)-Aib]- [X = L-Glu(OMe), L-Lys(Ac), L-Ala; Aib = alpha-aminoisobutyric acid] were synthesized by polymerization of glycosylated tripeptides with an active ester methods using Cl(-+)H(3)N-L-Glu(OMe)-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (Np=p-nitrophenyl) (13a), Cl(-+)H(3)N-L-Lys(Ac)-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (13b), and Cl(-+)H(3)N-L-Ala-Gln[beta-D-GlcNAc(Ac)(3)]-Aib-ONp (13c) as the monomers. Polymerizable glycosylated tripeptides were prepared by stepwise N,N-dicyclohexylcarbodiimide (DCC)/1-hydroxybenzotriazole (HOBt) method. Polymerizations of 13a-c were initiated by triethylamine and proceeded in DMSO at 50 degrees C for 5 days in the presence of 1-hydroxy-7-azabenzotriazole (HOAt) as the activator (conversions were 25-75%). The glycopeptides were deacetylated by hydrazine monohydrate in methanol to afford periodic glycopeptides 14 (12-27 residues) without racemization (yield, 35-89%). CD spectra in methanol, trifluoroethanol, and water of deacetylated glycopolymers 14a, 14b, and 14c showed double minima (206 and 222 nm) of negative Cotton effect indicating that N-glycoside (N-acetyl-d-glucosamine) was arranged three-dimensionally along the alpha-helical peptides in water as well as in organic protic solvents. The helix content depends on the solvent, peptide sequence, and spacer between peptide backbone and sugar. Interaction of the glycopeptides with wheat germ agglutinin (WGA) lectin was investigated by fluorescence measurement.