A two-step, one-pot synthesis of diverse N-pyruvoyl amino acid derivatives using the Ugi reaction

A 100-member combinatorial library of -ketoamides, which was designed to search potent cysteine protease inhibitors, was generated by a parallel solution-phase synthesis. A two-step one-pot synthesis, in which the Ugi reaction followed by pyridinium dichromate (PDC) oxidation was employed in the same reaction vessel, easily afforded the -ketoamides in a short time.     

Transglucosylation of hydroquinone catalysed by α-glucosidase from baker's yeast

Hydroquinone α-isomaltoside and hydroquinone α-glucoside were synthesized by transglucosylation in an aqueous system with baker's yeast α-glucosidase from hydroquinone and maltose as a glucosyl donor. Only one phenolic group was glucosylated, with α-selectivity, and the nature of the reaction products was governed by the concentration of hydroquinone. The optimal conditions for synthesis of glycosides were 9 mM hydroquinone and 1.5 M maltose in a 100 mM sodium citrate/phosphate buffer at pH 5.0 and 30 °C for 20 h. Under these conditions both hydroquinone α-glycosides were obtained in nearly equimolar amounts with a total molar yield of 28% with respect to hydroquinone and a total glycoside concentration of 1 mg/mL in the reaction mixture.     

Selective activation of RNA polymerase I in fat body nuclei of Sarcophaga peregrina larvae by β-ecdysone

RNA synthesis in fat body nuclei of Sarcophaga peregrina larvae was temporarily activated after injection of β-ecdysone: increased synthesis was detectable 2 hr after injecting the hormone and lasted for at least 2 hr. This increased RNA synthesis was insensitive to α-amanitin and was observed in KCl-free reaction mixture, indicating that β-ecdysone activated RNA polymerase I but not RNA polymerase II. No activation was observed when protein synthesis was inhibited by cycloheximide, suggesting that protein synthesis was essential for the activation of the nuclei.     

Availability of tyrosine amide for α-chymotrypsin-catalyzed synthesis of oligo-tyrosine peptides

Oligo-tyrosine peptides such as Tyr-Tyr having angiotensin I-converting enzyme (ACE) inhibitory activity could be synthesized by α-chymotrypsin-catalyzed reaction with l-tyrosine ethyl ester in aqueous media. However, peptide yield in the reaction was below 10%. Since l-tyrosine amide showed highly nucleophilic activity for the deacylation of enzyme through which a new peptide bond was made, its application to the enzymatic peptide synthesis was evaluated in this study. Addition of tyrosine amide into the reaction produced Tyr-Tyr-NH₂, of which yield exceeded 130% on the basis of tyrosine ethyl ester. Although purified Tyr-Tyr-NH₂ did not inhibit ACE activity, α-chymotrypsin could act on the dipeptide amide and convert about 40% of it to Tyr-Tyr. The use of both ester and amide forms of tyrosine is expected to be a potent procedure for α-chymotrypsin-catalyzed synthesis of antihypertensive peptides.     

Synthesis of 2, 3-dinor-PGFα metabolites

Thir report described the preparation of various 2,3-dinor-PGFα prostaglandins. Of particular importance is the synthesis of 2,3-dinor-15(S)-15-methyl PGF₂α, the primary metabolite in the enzymatic degradation of 15(S)-15-methyl-PGF₂α (1). Introduction of the three carbon β,γ-unsaturated carboxyl side chain was achieved in a one-step Wittig reaction. The 2,3-dinor structural assignments were established by carbon magnetic resonance (crm) spectroscopy.     

Catalytic activity of α-chymotrypsin in enzymatic peptide synthesis in ionic liquids

The catalytic activity of α-chymotrypsin in the enzymatic peptide synthesis of N-acetyl-l-tryptophan ethyl ester with glycyl glycinamide was examined in ionic liquids and organic solvents. The water content in 1-ethyl-3-methylimidazolium bis(fluorosulfonyl)imide ([emim][FSI]) affected the initial rates of peptide synthesis and hydrolysis. The activity of α-chymotrypsin was influenced by a kind of anions consisting of the same cation, [emim], when an ionic liquid was used as a solvent. The initial rate of peptide synthesis was improved 16-fold by changing from an organic solvent, acetonitrile, to an ionic liquid, [emim][FSI], at 25 °C. The activity of α-chymotrypsin in the peptide synthesis in [emim][FSI] was 17 times greater than that in acetonitrile at 60 °C, although the activity of α-chymotrypsin in the peptide synthesis gradually decreased with an increase in reaction temperature in [emim][FSI], similar to organic solvents. Moreover, α-chymotrypsin exhibited activity in [emim][FSI] and [emim][PF₆] at 80 °C.     

Specific 12β-Hydroxylation of Cinobufagin by Filamentous Fungi

Biotransformation of natural products has great potential for producing new drugs and could provide in vitro models of mammalian metabolism. Microbial transformation of the cytotoxic steroid cinobufagin was investigated. Cinobufagin could be specifically hydroxylated at the 12β-position by the fungus Alternaria alternata. Six products from a scaled-up fermentation were obtained by silica gel column chromatography and reversed-phase liquid chromatography and were identified as 12β-hydroxyl cinobufagin, 12β-hydroxyl desacetylcinobufagin, 3-oxo-12β-hydroxyl cinobufagin, 3-oxo-12β-hydroxyl desacetylcinobufagin, 12-oxo-cinobufagin, and 3-oxo-12α-hydroxyl cinobufagin. The last five products are new compounds. 12β-Hydroxylation of cinobufagin by A. alternata is a fast catalytic reaction and was complete within 8 h of growth with the substrate. This reaction was followed by dehydrogenation of the 3-hydroxyl group and then deacetylation at C-16. Hydroxylation at C-12β also was the first step in the metabolism of cinobufagin by a variety of fungal strains. In vitro cytotoxicity assays suggest that 12β-hydroxyl cinobufagin and 3-oxo-12α-hydroxyl cinobufagin exhibit somewhat decreased but still significant cytotoxic activities. The 12β-hydroxylated bufadienolides produced by microbial transformation are difficult to obtain by chemical synthesis.     

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

Highly conserved glycoside hydrolase family 70 glucansucrases are able to catalyze the synthesis of α-glucans with different structure from sucrose. The structural determinants of glucansucrase specificity have remained unclear. Residue Leu⁹⁴⁰ in domain B of GTF180, the glucansucrase of the probiotic bacterium Lactobacillus reuteri 180, was shown to vary in different glucansucrases and is close to the +1 glucosyl unit in the crystal structure of GTF180-ΔN in complex with maltose. Herein, we show that mutations in Leu⁹⁴⁰ of wild-type GTF180-ΔN all caused an increased percentage of (α1→6) linkages and a decreased percentage of (α1→3) linkages in the products. α-Glucans with potential different physicochemical properties (containing 67–100% of (α1→6) linkages) were produced by GTF180 and its Leu⁹⁴⁰ mutants. Mutant L940W was unable to form (α1→3) linkages and synthesized a smaller and linear glucan polysaccharide with only (α1→6) linkages. Docking studies revealed that the introduction of the large aromatic amino acid residue tryptophan at position 940 partially blocked the binding groove, preventing the isomalto-oligosaccharide acceptor to bind in an favorable orientation for the formation of (α1→3) linkages. Our data showed that the reaction specificity of GTF180 mutant was shifted either to increased polysaccharide synthesis (L940A, L940S, L940E, and L940F) or increased oligosaccharide synthesis (L940W). The L940W mutant is capable of producing a large amount of isomalto-oligosaccharides using released glucose from sucrose as acceptors. Thus, residue Leu⁹⁴⁰ in domain B is crucial for linkage and reaction specificity of GTF180. This study provides clear and novel insights into the structure-function relationships of glucansucrase enzymes.     

Enzymatic synthesis of salicin glycosides through transglycosylation catalyzed by amylosucrases from Deinococcus geothermalis and Neisseria polysaccharea

Amylosucrase (ASase, EC 2.4.1.4) is a member of family 13 of the glycoside hydrolases that catalyze the synthesis of an α-(1→4)-linked glucan polymer from sucrose instead of an expensive activated sugar, such as ADP- or UDP-glucose. Transglycosylation reactions mediated by the ASases of Deinococcus geothermalis (DGAS) and Neisseria polysaccharea (NPAS) were applied to the synthesis of salicin glycosides with sucrose serving as the glucopyranosyl donor and salicin as the acceptor molecule. Two salicin glycoside transfer products were detected by TLC and HPLC analyses. The synthesis of salicin glycosides was very efficient with NPAS with a yield of over 90%. In contrast, DGAS specifically synthesized only one salicin transglycosylation product. The transglycosylation products were identified as α-d-glucopyranosyl-(1→4)-salicin (glucosyl salicin) and α-d-glucopyranosyl-(1→4)-α-d-glucopyranosyl-(1→4)-salicin (maltosyl salicin) by NMR analysis. The ratio between donor and acceptor had a significant effect on the type of product that resulted from the transglycosylation reaction. With more acceptors present in the reaction, more glucosyl salicin and less maltosyl salicin were synthesized.     

Enantioselective ester hydrolase from Sphingobacterium sp. 238C5 useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis

A novel enzyme, β-phenylalanine ester hydrolase, useful for chiral resolution of β-phenylalanine and for its β-peptide synthesis was characterized. The enzyme purified from the cell free-extract of Sphingobacterium sp. 238C5 well hydrolyzed β-phenylalanine esters (S)-stereospecifically. Besides β-phenylalanine esters, the enzyme catalyzed the hydrolysis of several α-amino acid esters with l-stereospecificity, while the deduced 369 amino acid sequence of the enzyme exhibited homology to alkaline d-stereospecific peptide hydrolases from Bacillus strains. Escherichia coli transformant expressing the β-phenylalanine ester hydrolase gene exhibited an about 8-fold increase in specific (S)-β-phenylalanine ethyl ester hydrolysis as compared with that of Sphingobacterium sp. 238C5. The E. coli transformant showed (S)-enantiomer specific esterase activity in the reaction with a low concentration (30 mM) of β-phenylalanine ethyl ester, while it showed both esterase and transpeptidase activity in the reaction with a high concentration (170 mM) of β-phenylalanine ethyl ester and produced β-phenylalanyl-β-phenylalanine ethyl ester. This transpeptidase activity was useful for β-phenylalanine β-peptide synthesis.     

Levels of collagen mRNA in dedifferentiating chondrocytes

Chondrocytes liberated from chick embryo sterna were maintained in monolayer cultures and allowed to dedifferentiate. mRNA was prepared from these cultures at several intervals over a total period of 6 weeks. Levels of α1(I) and α2(I) procollagen mRNA were assayed by cell-free translation and by Northern blots using cDNA clones specific for the respective procollagen chains. Dedifferentiating chondrocytes first take up synthesis of α1(I) mRNA which is followed after several days by synthesis of α2(I) mRNA. This two-step mechanism for the onset of procollagen type I mRNA synthesis is accompanied by a proceeding loss of α1(II) procollagen gene expression.     

Hyperosmotic Stress as a Stimulant for Proinflammatory Cytokine Production

The synthesis of proinflammatory cytokines involves members of the mitogen-activated protein (MAP) kinase stress pathway, particularly p38 MAP kinase and c-jun NH₂-terminal kinase. In this report we used hyperosmotic stress to study changes in steady-state mRNA levels and synthesis of proinflammatory cytokines in freshly obtained human peripheral blood mononuclear cells (PBMC)in vitro.There was no evidence of interleukin (IL)-8 gene expression in freshly obtained human blood despite 30 cycles of amplification of reverse-transcribed mRNA using the polymerase chain reaction. In contrast, exposure of PBMC to hyperosmotic conditions (330–410 mOsM) by the addition of NaCl to tissue culture medium induced gene expression for IL-1α, IL-1β, and IL-8. Routine tissue culture medium is hyperosmotic (305 mOsM) compared to human plasma (280–295 mOsM), but decreasing the osmolarity to the physiological range resulted in a 50% reduction in baseline IL-8 synthesis (P< 0.001). Although hyperosmotically induced accumulation of steady-state mRNA levels for IL-1α and IL-1β increased 50- and 7-fold over control, respectively, these were poorly translated into each respective cytokine. However, in PBMC stimulated by hyperosmotic stress, the addition of femtomolar concentrations of bacterial lipopolysaccharide, IL-1, or 1% normal human serum resulted in a synergistic synthesis (at least twice that expected) of IL-1α, IL-1β, TNF-α, and IL-8.     

Regioselective synthesis of mannobiose and mannotriose by reverse hydrolysis using a novel 1,6-α- -mannosidase from Aspergillus phoenicis

A novel 1,6-α- -mannosidase was produced by Aspergillus phoenicis grown on a commercial manno-oligosaccharide preparation in liquid culture. The enzyme hydrolysed only α- -Manp-(1→6)- -Manp and did not act on α- -Manp-(1→2)- -Manp, or α- -Manp-(1→3)- -Manp. The 1,6-α- -mannosidase was used for synthesis of manno-oligosaccharides by reverse hydrolysis reaction. The highest yields, expressed as percentages (w/w) of total sugar, were 21% mannobiose and 5% mannotriose, and they were obtained with 45% (w/w) initial mannose concentration at pH 4.5 after 12 days incubation at 55 °C. The disaccharide and trisaccharide products were separated and their structures determined by methylation analysis. Only 1–6 linkages were found in both of them.     

Facile synthesis of glucose-1-phosphate from starch by Thermus caldophilus GK24 α-glucan phosphorylase

The glgP gene encoding α-glucan phosphorylase (α-GP) from the thermopile Thermus caldophilus GK24 has been identified, cloned, and overexpressed in Escherichia coli and used to synthesize d-glucose-1-phospate (G1P) from an inexpensive starch. The enzyme, purified 6.5-fold, was isolated in 31% yield from the transformed E. coli, and gave a single band. The purified enzyme may exist as a homohexamer with an apparent molecular mass of a 550 kDa molecule, consisting of 90 kDa per subunit. The optimal pH and temperature were 7.0 and 70 °C in the α-GP reaction with starch producing G1P. Soluble starch (amylopectin, amylose) turned out to be a better substrate giving a higher yield of G1P than α-1,6-branched α-1,4-glucans (glycogen, potato starch, etc.). As a result, G1P was obtained in a good yield (47%, w/w) from the reaction containing 5% (w/v) soluble starch in 0.7 M potassium phosphate at pH 7.0. T. caldophilus α-GP shows a high tolerance (up to 0.7 M) of potassium phosphate and plays a critical role in shifting the reaction equilibrium in favor of G1P synthesis. The G1P product can be purified simply by ethanol precipitation, after removing the unreacted starch and inorganic phosphate by activated charcoal and magnesium acetate precipitation. It is concluded that T. caldophilus α-GP readily utilized in large scale synthesis of G1P.     

Haloperoxidases: Enzymatic Synthesis of α,β-Halohydrins from Gaseous Alkenes

The enzymatic synthesis of α,β-halohydrins from gaseous alkenes is described. The enzymatic reaction required an alkene, a halide ion, dilute hydrogen peroxide, and a haloperoxidase enzyme. A wide range of gaseous alkenes were suitable for this reaction, including those containing isolated, conjugated, and cumulative carbon-carbon double bonds. Chlorohydrins, bromohydrins, and iodohydrins could be formed. The combining of this enzymatic synthesis with a previously described enzymatic synthesis of epoxides from α,β-halohydrins provides an alternate pathway, other than the well-known enzymatic direct epoxidation pathway, from alkene to an epoxide.     

A Function for Phosphatidylinositol 3-Kinase β (p85α-p110β) in Fibroblasts during Mitogenesis: Requirement for Insulin- and Lysophosphatidic Acid-Mediated Signal Transduction

We have previously shown that phosphatidylinositol 3-kinase α (PI 3-Kα) (p85α-p110α) is required for DNA synthesis induced by various growth factors (S. Roche, M. Koegl, and S. A. Courtneidge, Proc. Natl. Acad. Sci. USA 91:9185–9189, 1994) in fibroblasts. In the present study, we have investigated the function of PI 3-Kβ (p85α-p110β) during mitogenesis. By using antibodies specific to p110β we showed that PI 3-Kβ is expressed in NIH 3T3 cells. PI 3-Kβ and PI 3-Kα have common features: PI 3-Kβ is tightly associated with a protein serine kinase that phosphorylates p85α, it interacts with the Src-middle T antigen complex and the activated platelet-derived growth factor (PDGF) receptor in fibroblasts in vivo, and it becomes tyrosine phosphorylated after PDGF stimulation. PI 3-Kβ was also activated in Swiss 3T3 and Cos7 cells stimulated with lysophosphatidic acid (LPA), a mitogen that interacts with a heterotrimeric G protein-coupled receptor. In contrast PI 3-Kα was activated to a lesser extent in these cells. Microinjection of neutralizing antibodies specific for p110β into quiescent fibroblasts inhibited DNA synthesis induced by both insulin and LPA but poorly affected PDGF receptor signaling. Therefore, PI 3-Kβ plays an important role in transmitting the mitogenic response induced by some, but not all, growth factors. Finally, we show that while oncogenic V12Ras interacts with type I PI 3-Ks, it could induce DNA synthesis in the absence of active PI 3-Kα and PI 3-Kβ, suggesting that Ras uses other effectors for DNA synthesis.     

Prostaglandin synthesis by rheumatoid synovium and its stimulation by colchicine

The synthesis of prostaglandins by rheumatoid synovial tissue in organ culture was studied utilizing radioimmunoassay, with antisera to PGB₁, PGF_1α and PGF_2α. It was established that PGE₂ and PGF_2α were the major prostaglandins formed by analyses of culture media with the two antisera to PGF, before and after alkali treatment. Indomethacin at 5 μg/ml suppressed prostaglandin synthesis, usually to <1% of control cultures. Colchicine, 0.1 μg/ml resulted in marked stimulation of prostaglandin synthesis, in some cases over 10 fold. It is suggested, because of the colchicine effect, that the state of the microtubules may regulate the rate of prostaglandin biosynthesis. It is possible that prostaglandin E₂ produced by rheumatoid synovia may contribute to the pathogenesis of the inflammatory reaction and lead to destruction of juxta-articular bone in rheumatoid arthritis.     

Synthesis and Secretion of Hydroxyproline-containing Macromolecules in Carrots: II. In vivo Conversion of Peptidyl Proline to Peptidyl Hydroxyproline

The chelator α,α′-dipyridyl prevents the formation of protein-bound hydroxyproline without affecting protein synthesis as measured by the incorporation of ¹⁴C-proline. The inhibitory effect can be overcome by exogenous ferrous ions. Neither α,α′-dipyridyl nor Fe²⁺ interferes in any other known way with the synthesis and secretion of the hydroxyproline-rich proteins normally found in the cell wall. This reversal by Fe²⁺ of the inhibition of proline hydroxylation by α,α′-dipyridyl is used for a study in vivo of the hydroxylation reaction. This reaction has a temperature coefficient of 2.2, suggesting that it is an enzymatic process. Reversal by Fe²⁺ of the α,α′-dipyridyl inhibition can occur after protein synthesis has been arrested with cycloheximide, indicating that peptidyl proline is the substrate of the hydroxylation reaction. Hydroxylation occurs in the cytoplasm, and not in the cell wall, and only for a limited time after the incorporation of proline into the polypeptide chain. This suggests a spatial separation of the enzyme and the substrate.     

Highly Functionalized 1,2–Diamino Compounds through Reductive Amination of Amino Acid-Derived β–Keto Esters

1,2-Diamine derivatives are valuable building blocks to heterocyclic compounds and important precursors of biologically relevant compounds. In this respect, amino acid-derived β–keto esters are a suitable starting point for the synthesis of β,γ–diamino ester derivatives through a two-step reductive amination procedure with either simple amines or α–amino esters. AcOH and NaBH₃CN are the additive and reducing agents of choice. The stereoselectivity of the reaction is still an issue, due to the slow imine-enamine equilibria through which the reaction occurs, affording mixtures of diastereoisomers that can be chromatographically separated. Transformation of the β,γ–diamino esters into pyrrolidinone derivatives allows the configuration assignment of the linear compounds, and constitutes an example of their potential application in the generation of molecular diversity.