Synthesis of β-amino acids using Boc-Z-amino acid pentafluorophenyl esters

Summary The pentafluorophenyl esters of Boc-/Z-amino acids are used for the preparation of the key intermediates α-aminoacyldiazomethanes during the homologation of α-amino acid to β-amino acid. Thus, all the Boc-/Z-amino acid diazoketones and the corresponding β-amino acids were obtained as crystalline solids in satisfactory yields.     

Rapid and Efficient Synthesis of Peptides Containing α,α-dialkylamino acids employing KOBt

Summary Several Fmoc-α,α-dialkylamino acids and their acid chlorides have been prepared, isolated and characterised. The synthesis of peptides containing sterically hindered dialkylamino acids has been accomplished using acid chloride/KOBt in dichloromethane. The yields as well as the purity of the peptides were satistactory.     

α-Amino acid behaves differently from β- or γ-amino acids as treated by trimetaphosphate

Summary. The condensation reactions of sodium trimetaphosphate with single amino acids, namely glycine, L-alanine, β-alanine and γ-aminobutyric acid or pairs of these amino acids were reinvestigated by electrospray ion-trap mass spectrometry and high performance liquid chromatography. It was found when mixtures were treated by sodium trimetaphosphate only in the presence of α-amino acid dipeptides were formed. Without addition of α-amino acids, the β-amino acid or γ-aminobutyric acid could not form peptide either by themselves or with their mixtures under the same conditions. From the data it is concluded that phosphate might select α-amino acids to produce the peptides being important precursors for the origin of life. Authors’ address: Dr. Pengxiang Xu, The Key Laboratory for Chemical Biology of Fujian Province, Department of Chemistry, Xiamen University, Xiamen 361005, China     

Domino reactions with fluorinated five-membered heterocycles. α-Trifluoromethyl α-amino acids with unsaturated side-chains

Summary. α-Trifluoromethyl α-amino acids with unsaturated side-chains have been prepared from 5-fluoro-4-trifluoromethyloxazole and allyl, propargyl as well as terpene alcohols in a one-pot procedure.     

Preferred structures of constrained peptides from achiral α,α-dialkyiated glycyl residues with acyclic side chains

Conformational energy computations of the monopeptides from three achiral α,α-dialkylated glycyl residues with acyclic side chains (namely α,α-dimethyl-; α,α-diethyl-; and α, α-di-n-propylglycines) are reported as a function of the relevant N-C^α-C′ bond angle. In parallel, experimental studies were performed in the solid state (infrared absorption and X-ray diffraction) and in solution (infrared absorption and proton magnetic resonance) on the corresponding protected homo-peptide series (the former series to the dodecamer, the other two series to the pentamers). The results obtained unequivocally indicate that the preference from a helical to a fully extended conformation increases as side-chain bulkiness increases. The longest homo-peptides from α,α-dimethylglycine form stable 3₁₀-helices. A picture of the mode of self-association of the helical structures has also been determined. The results of the theoretical analyses fit well with the experimentally observed conformational properties in the solid state and in chloroform solution.     

Nitric oxide and polyamine pathway-dependent modulation of neutrophil free amino- and α-keto acid profiles or host defense capability

Summary. We have examined the effects of N_ω-nitro-L-arginine-methylester-hydrochloride [L-NAME; inhibitor of nitric oxide synthase], S-nitroso-N-acetyl-penicillamine [SNAP; nitric oxide donor], α-difluoro-methyl-ornithine [DFMO; inhibitor of ornithine decarboxylase] arginine or ornithine as well as the combination of arginine or ornithine with L-NAME, SNAP or DFMO on intracellular free amino- and α-keto acid profiles and the immune function markers superoxide anion and hydrogen peroxide generation as well as released myeloperoxidase activity in neutrophils (PMN). Although the underlying mechanisms still remain unclear, we believe from our results that nitric oxide as well as polyamine-dependent pathways are involved in the signal transmission of free radical molecule, beneficial nutritional therapy or maleficient pharmacological stress-induced alterations in PMN nutrient composition. Relevant changes in intragranulocyte free amino- and α-keto acid homeostasis and metabolism, especially, may be one of the determinants in PMN nutrition that positively or negatively influences and modulate neutrophil host defence capability and immunocompetence.     

Octapeptide Analogs of Somatostatin Containing α,α-Dialkylated Amino Acids with Potent Anticancer Activity

We describe the synthesis and anticancer activities of octapeptide analogs of somatostatin incorporating α,α-dialkylated amino acids. The designed analogs of somatostatin are: d-Phe¹-Cys²-Tyr³-d-Trp⁴-Orn⁵-Xxx⁶-Pen⁷-Thr⁸-NH₂ where Xxx=α-Aminoisobutyric acid (Aib), Diethyl glycine (Deg), 1-Aminocyclopentane carboxylic acid (Ac5c), and, d-Phe¹-Cys²-Tyr³-d-Trp⁴-Lys⁵-Ac5c⁶-Pen⁷-Thr⁸-NH₂ (disulphide bond between Cys² and Pen⁷ in all analogs). The conformational studies two of the designed analogs were carried out by NMR techniques and the experimental results suggest a β-turn structure for one of the designed analog. In vivo tumor regression study of two designed analogs on human primary colon tumor xenografts in nude mice demonstrates the anticancer potential of the synthesized analogs.     

Fatty Acid-Induced Modulation of Ouabain Responsiveness of Rat Na,K-ATPase Isoforms

Membrane phospholipids represent a potential influence on the enzymatic properties of the Na,K-ATPase. Little is known concerning the effects of the fatty acid environment surrounding the enzyme on the kinetic properties of the Na,K-ATPase. We used the most obvious difference among the α isoforms of rat, their affinities for digitalis glycosides, to examine the relationship between the lipid environment and the Na,K-ATPase. Specific membrane environments that differ in their fatty acid composition were produced by drug-induced diabetes, as well as variations in diet. The α1 isoforms in various tissues were then characterized by their resistance to ouabain in Na,K-ATPase-enriched membrane microsomal fractions. The Na,K-ATPase activity in nerves and hearts were altered by diabetes and partially restored in nerves after a fish oil diet. Evaluation of enzyme kinetics (dose-response curves for ouabain) in membrane preparations allowed us to correlate the ouabain affinity of α1 isoform with fatty acid composition. The affinity of the α1 isoform for ouabain was significantly increased with accretions in the total amount of fatty acids of the n-6 series (P < 0.0001). Our observations provide a partial explanation for the observed difference in isoform properties among tissues. Moreover, these results underline the interaction between membrane fatty acids and the glycoside binding site of the Na,K-ATPase α1 subunit. Received: 15 June 1998/Revised: 18 November 1998     

The “unexpected role” of vitamin E in free radical-induced hemolysis of human erythrocytes

Vitamin E involves a group of tocopherols and tocotrienols, in which α-tocopherol with the highest biological activity plays a more efficient role in advanced lesions with aged oxidized tissues. However, the results of the present study reveal that a large amount of endogenous α-tocopherol in human low-density lipoprotein (LDL) in the absence of any other antioxidants may initiate additional free radical propagation under low concentration of free radical initiator (i.e., 2,2′-azobis(2-amidinopropane hydrochloride) [AAPH], a water-soluble free radical source) to peroxide polyunsaturated fatty acids in LDL in the manner of α-tocopherol-mediated peroxidation (TMP). Whether the addition of high concentration of exogenous α-tocopherol to human erythrocytes under low concentration of AAPH can also drive TMP is the concern in this research work. Moreover, the hemolysis extent of human erythrocytes peroxidized by AAPH is followed easily by the determination of the hemoglobin outside the erythrocytes. A series of observations on various concentrations of AAPH-induced hemolysis in the presence of various concentrations of exogenous α-tocopherol demonstrates that the high concentration of exogenous α-tocopherol, coupled with low concentration of AAPH, can initiate TMP in the free-radical-induced peroxidation of human erythrocytes system as well. This result provides direct evidence to support TMP theory and expands its application into in vitro erythrocytes system.     

Convenient and simple homologation of Nα-urethane protected α-amino acids to their β-homologues with concomitant pentafluorophenyl ester formation

Summary The Wolff rearrangement of α-aminodiazoketones derived fromN ^α-urethane protected α-amino acids in the presence of pentafluorophenol catalyzed by silver acetate at low temperature results in the homologation of Fmoc-/Boc-/Z-α-amino acids to β-amino acids with concomitant formation of the corresponding pentafluorophenyl esters in good yield and purity.     

Microwave assisted Wolff rearrangement: A facile method for the synthesis of Fmoc-β-amino acids

Summary The Wolff rearrangement of α-diazoketones, derived from Fmoc-α-amino acids, under no base conditions on exposure to microwave irradiation for 40 to 60 sec to Fmoc-β-amino acids with retention of configuration in good yield (91–95%) is described.     

Alterations in neutrophil (PMN) free intracellular alpha-keto acid profiles and immune functions induced by L-alanyl-L-glutamine, arginine or taurine

Summary. The objective of this study was to determine the dose as well as duration of exposure-dependent effects of L-alanyl-L-glutamine, arginine or taurine on polymorphonuclear neutrophil (PMN) free α-keto acid profiles and, in a parallel study, on PMN immune functions. Exogenous L-alanyl-L-glutamine significantly increased PMN α-ketoglutarate, pyruvate PMN superoxide anion (O₂⁻) generation, hydrogen peroxide (H₂O₂) formation and released myeloperoxidase (MPO) activity. Arginine also led to significant increases in α-ketoglutarate, pyruvate, MPO release and H₂O₂ generation. Formation of O₂⁻ on the other hand was decreased by arginine. Incubation with taurine resulted in lower intracellular pyruvate and α-ketobutyrate levels, decreased O₂⁻ and H₂O₂ formation and a concomitant significantly increased MPO activity. We therefore believe that considerable changes in PMN free-α-keto-acid profiles, induced for example by L-alanyl-L-glutamine, arginine or taurine, may be one of the determinants in cell nutrition that considerably modulates the immunological competence of PMN.     

Synthesis of conformationally constrained hydroxy-α-amino acids by intramolecular conjugate addition

Summary. An efficient and easily applicable method for the synthesis of a variety of hydroxy-α-amino acids analogues of serine and phenylalanine has been established. The method involves the stereoselective intramolecular conjugate addition of the benzamide group to cyclohexenone promoted by Lewis acid. Subsequent transformations of functional groups pro-vide the conformationally constrained 2-hydroxy- and 2,4-dihydroxy-6-phenylcyclohexane-α-amino acids. Received February 5, 1999, Accepted May 16, 1999     

Inhibition of collagen synthesis by α, α′-dipyridyl in human skin fibroblasts in culture

Summary In human diploid skin fibroblasts in culture we have shown that nonhydroxylated collagen precursors remain in the cell when proline hydroxylation is inhibited by α, α′-dipyridyl, a chelator of ferrous ions. The inhibition of proline hydroxylation is reversed by addition of fresh medium containing 50 μg per ml of sodium ascorbate, whereupon nonhydroxylated collagen precursors are hydroxylated within the cell and extruded into the medium. Extrusion of collagen already formed within the cell is not appreciably affected by α, α′-dipyridyl inhibition. Under normal conditions collagen is released from the monolayer into the medium within 3 hr of a pulse ofL[¹⁴C]proline. In the presence of α, α′-dipyridyl, about 35% of theL[¹⁴C]proline incorporated into protein is released into the medium within 8 hr as a proline-rich, hydroxyproline-deficient protein; at the same time, approximately 15% of the protein-boundl-[¹⁴C]proline remains in the cell for as long as 12 hr. When proline hydroxylation is restored after 2 and 12 hr of α, α′-dipyridyl inhibition, approximately the same amount of hydroxyproline is formed after each time interval in the monolayer. Therefore, nonhydroxylated collagen precursors retained in the cell are not appreciably degraded during at least 12 hr of inhibition by α, α′-dipyridyl and are extruded into the medium only upon restoration of hydroxylation. This work was supported in part by a grant from the Easter Seal Research Foundation, and by Project 236, Health Services and Mental Health Administration, Department of Health, Education and Welfare, Grant HD-03110 from the National Institute of Child Health and Human Development, an American Cancer Society Institutional Grant (1N 15-J), a General Research Support Award (5-S01-FR-05406) from the National Institutes of Health, a University Research Council Grant, a National Science Foundation Equipment Grant (GB-4577), and a Research Career Development Award (5-K3-AM-5058) from the National Institute of Arthritis and Metabolic Disease (G.K.S.).     

Synthesis of γ-amino acid analogues from natural α-amino acids by a radical pathway

Summary. New γ-amino esters and amides were prepared by a radical 1,4-addition of carbon radicals to acrylic derivatives. α-Amino acids derivatives holding chiral auxiliaries as radical precursors and different chiral olefins were used and chiral induction on the C-γ center was discussed.     

An Approach to Prebiotic Synthesis of α-Oligoribonucleotides and Description of Their Properties: Selective Advantage of β-RNA over α-RNA

Oligomerization of α-adenosine 5′-phosphorimidazolide (α-ImpA) has been done in an aqueous solution using a uranyl-ion catalyst or a poly(U) template as a model process of prebiotic synthesis of RNA with α-glycosidic linkage. α-Oligoriboadenylates up to hexamer were formed from α-ImpA by the uranyl-ion catalyst. 3′-5′ Linkage was mainly formed in the oligomerization. The poly(U) template mediated the oligomerization of α-ImpA, but to a very low extent. The yield and chain length of the resulting α-oligomers were far lower than those of the corresponding β-oligomer formation under the same conditions. Physico-chemical properties of α-oligoriboadenylates are presented along with those of the corresponding β-oligoriboadenylates. The results indicate that β-RNA is more advantageous than α-RNA from the points of their synthesis and properties. Received: 10 February 1997 / Accepted: 31 March 1997     

Control of carbon partitioning and photosynthesis by the triose phosphate/phosphate translocator in transgenic tobacco plants (Nicotiana tabacum L.). I. Comparative physiological analysis of tobacco plants with antisense repression and overexpression of the triose phosphate/phosphate translocator

 The physiological properties of transgenic tobacco plants (Nicotiana tabacum L.) with decreased or increased transport capacities of the chloroplast triose phosphate/phosphate translocator (TPT) were compared in order to investigate the extent to which the TPT controls metabolic fluxes in wild-type tobacco. For this purpose, tobacco lines with an antisense repression of the endogenous TPT (αTPT) and tobacco lines overexpressing the TPT gene isolated from the C₄ plant Flaveria trinervia (FtTPT) were used. The F. trinervia TPT expressed in yeast cells exhibited transport characteristics identical to the TPT from C₃ plants. Neither antisense TPT plants nor FtTPT overexpressors showed a phenotype when grown in a greenhouse in air. Contents of starch and soluble sugars in upper source leaves were similar in TPT underexpressors and FtTPT overexpressors compared to the wild type at the end of the photoperiod. The FtTPT overexpressors incorporated more ¹⁴CO₂ in sucrose than the wild type, indicating that the TPT limits sucrose biosynthesis in the wild type. There were only small effects on labelling of amino acids and organic acids. The mobilisation of starch was enhanced in αTPT lines but decreased in FtTPT overexpressors compared to the wild type. Enzymes involved in starch mobilisation or utilisation, such as α-amylase or hexokinase were increased in αTPT plants and, in the case of amylases, decreased in FtTPT overexpressors. Moreover, α-amylase activity exhibited a pronounced diurnal variation in αTPT lines with a maximum activity after 8 h in the light. These changes in starch hydrolytic activities were confirmed by activity staining of native gels. Activities of glucan phosphorylases were unaffected by either a decrease or an increase in TPT activity. There were also effects of TPT activities on steady-state levels of phosphorylated intermediates as well as total amino acids and malate. In air, there was no or little effect of altered TPT transport activity on either rates of photosynthetic electron transport and/or CO₂ assimilation. However, in elevated CO₂ (1500 μl · l⁻¹) and low O₂ (2%) the rate of CO₂ assimilation was decreased in the αTPT lines and was slightly higher in FtTPT lines. This shows that the TPT limits maximum rates of photosynthesis in the wild type. Received: 26 March 1999 / Accepted: 21 August 1999     

Suppression of myofibrillar proteolysis in chick skeletal muscles by α-ketoisocaproate

Summary. We previously reported that L-leucine suppresses myofibrillar proteolysis in chick skeletal muscles. In the current study, we compared the effects of L- and D-enantiomers of leucine on myofibrillar proteolysis in skeletal muscle of chicks. We also assessed whether leucine itself or its metabolite, α-ketoisocaproate (α-KIC), mediates the effects of leucine. Food-deprived (24 h) chicks were orally administered 225 mg/100 g body weight L-leucine, D-leucine or α-KIC and were sacrificed after 2 h. L-Leucine administration had an obvious inhibitory effect on myofibrillar proteolysis (plasma N^τ-methylhistidine concentration) in chicks while D-leucine and α-KIC were much more effective. We also examined the expression of the proteolytic-related genes (ubiquitin, proteasome, m-calpain and cathepsin B) by real-time PCR of cDNA in chick skeletal muscles. Ubiquitin mRNA expression was decreased by D-leucine and α-KIC but not L-leucine. Proteasome and m-calpain mRNA expressions as well as cathepsin B mRNA expression were likewise decreased by L-leucine, D-leucine and α-KIC. These results indicate that D-leucine and α-KIC suppress proteolytic-related genes, resulting in an decrease in myofibrillar proteolysis while L-leucine is much less effective in skeletal muscle of chicks, may be explain by conversion of D-leucine to α-KIC.     

Self-association of α-chymotrypsin: Effect of amino acids

The concentration-dependent self-association of α-chymotrypsin is known to be influenced by various factors including the presence of small molecules and autolysis products. In this connection the effect of various amino acids on the self-association of α-chymotrypsin has been studied, as a point of interest, by measuring the sedimentation coefficient of α-chymotrypsin. The influence of an amino acid is seen to be governed by the nature of its side chain. Some amino acids do not affect the self-association of α-chymotrypsin at all while some affect it moderately and some others considerably. Functional groups such as the - OH group of Ser or the phenolic ring of Tyr do not seem to influence self-association behaviour. Based on these effects, amino acids could be categorized into 3 groups. Activity studies in the presence of amino acids indicate that the site of self-association and the active-site are probably mutually exclusive.     

Domain Evolution in the α-Amylase Family

The available amino acid sequences of the α-amylase family (glycosyl hydrolase family 13) were searched to identify their domain B, a distinct domain that protrudes from the regular catalytic (β/α)₈-barrel between the strand β3 and the helix α3. The isolated domain B sequences were inspected visually and also analyzed by Hydrophobic Cluster Analysis (HCA) to find common features. Sequence analyses and inspection of the few available three-dimensional structures suggest that the secondary structure of domain B varies with the enzyme specificity. Domain B in these different forms, however, may still have evolved from a common ancestor. The largest number of different specificities was found in the group with structural similarity to domain B from Bacillus cereus oligo-1,6-glucosidase that contains an α-helix succeeded by a three-stranded antiparallel β-sheet. These enzymes are α-glucosidase, cyclomaltodextrinase, dextran glucosidase, trehalose-6-phosphate hydrolase, neopullulanase, and a few α-amylases. Domain B of this type was observed also in some mammalian proteins involved in the transport of amino acids. These proteins show remarkable similarity with (β/α)₈-barrel elements throughout the entire sequence of enzymes from the oligo-1,6-glucosidase group. The transport proteins, in turn, resemble the animal 4F2 heavy-chain cell surface antigens, for which the sequences either lack domain B or contain only parts thereof. The similarities are compiled to indicate a possible route of domain evolution in the α-amylase family. Received: 4 December 1996 / Accepted: 13 March 1997