The formation of dipeptides from amino acids and the 2′(3′)-glycyl ester of an adenylate

Summary The yields of dipeptide obtained from the reaction of 0.2M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and 0.2M amino acid at pH 8.2 ranged from 0.1% to 35.5% for a group of 15 amino acids. The yields of glyser (35.3%), gly-cys (11.8%) and gly-thr (5.4%) were considerably greater than dipeptide yields obtained from any of the other 12 amino acids ( 1.7%). Aminolysis of 0.05M 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by 0.4M serine ethyl ester yielded 53% glycylserine diketopiperazine, via N-(glycyl)-serine ethyl ester as a transient intermediate. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - serOEt serine ethyl ester - gly-serOEt N-(glycyl)-serine ethyl ester - Boc-gly N-tertbutyloxycarbonylglycine - cyclo-(gly-ser-) glycylserine diketo-piperazine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - gly-ser N-glycylserine     

The formation of peptides from the 2′(3′)-glycyl ester of a nucleotide

Summary We have synthesized 2(3)-O-(glycyl)-adenosine-5-(O-methylphos-phate), an analogue of the 3-terminus of aminoacylated tRNA. A 0.4M solution of this compound maintained at pH 8.2, yields 5.5% of diglycine and 11.5% of diketopiperazine, in addition to the hydrolysis products glycine and adenosine-5-(O-methylphosphate). Under the same conditions, glycine ethyl ester reacts much more slowly, but ultimately gives similar yields of diglycine and diketopiperazine.The aminolysis of 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) by free glycine is relatively inefficient, but serine reacts 20 times more rapidly and yields up to 50% of N-glycylserine. The prebiotic significance of these reactions is discussed.Abbreviations MepA adenosine-5-(O-methylphosphate) - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-gly 2,3-O-(bis-glycyl)-adenosine-5-(O-methylphosphate) - DKP diketopiperazine - gly Et glycine ethyl ester - gly-ser N-glycylserine - O-gly-ser O-glycylserine - O-(gly)-gly-ser O-(glycyl)-glycylserine - Boc-gly N-tert-butyloxycarbonylglycine - MepA-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine-5-(O-methylphosphate) - MepA-bis-Boc-gly 2,3-O-(bis-Boc-glycyl)-adenosine-5(O-methylphosphate) - (gly)₂ diglycine - (gly)₃ triglycine     

Poly(U)-directed peptide-bond formation from the 2′(3′)-glycyl esters of adenosine derivatives

Summary The self-condensation of 2(3)-O-glycyl esters of adenosine, adenosine-5-(O-methylphosphate) and P₁, P₂-diadenosine-5-pyrophosphate in 6.2 mM solutions at pH 8.0 and -5°C in the presence of 12.5 mM poly(U) yields approximately 3 times as much diketopiperazine as reactions without poly(U). As the concentration of 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate is decreased from 6.2 mM to 1.5 mM the yield of diketopiperazine in the presence of poly(U) decreases slightly from 6.6% to 5.2%, whereas, in the absence of poly(U) the yield of diketopiperazine decreases substantially from 2.4% to 0.75%. The enhanced yield of diketopiperazine that is attributed to the template action of poly(U) is temperature dependent and is observed only at temperatures below 10°C (5°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) and below 23°C (15°C to -5°C) for 6.2 mM 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate. The absence of a template effect at high temperatures is attributed to the melting of the organized helices. The hydrolysis half-lives at pH 8.0 and -5°C of 2(3)-O-(glycyl)-adenosine, 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate), 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate, and 5-O-(glycyl)-adenosine in the presence of poly(U) are substantially larger than their half-lives in the absence of poly(U). The condensation of 2(3)-O-(glycyl)-adenosine yields 5% of 5-O-(glycyl)-adenosine in the presence of poly(U) compared to 0.7% in the absence of poly(U).Abbreviations DKP diketopiperazine - (gly)₂ glycylglycine - (gly)₃ glycylglycylglycine - AppA-gly 2(3)-O-(glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - MepA-gly 2(3)-O-(glycyl)-adenosine-5-(O-methylphosphate) - Ado-2(3)-gly 2(3)-O-(glycyl)-adenosine - Ado-5-gly 5-O-(glycyl)-adenosine - Boc-gly N-tert-butyloxycarbonylglycine - AppA P₁, P₂-diadenosine-5-pyrophosphate - MepA adenosine-5-(O-methylphosphate) - AppA-Boc-gly 2(3)-O-(Boc-glycyl)-P₁, P₂-diadenosine-5-pyrophosphate - Ado-5-Boc-gly 5-O-(Boc-glycyl)-adenosine - Ado-2(3)-Boc-gly 2(3)-O-(Boc-glycyl)-adenosine     

2' (or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-triphosphate as a probe for the binding site of heavy meromyosin ATPase.

1. From NMR, IR and visible absorption studies of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)-adenosine 5'-triphosphate (TNP-ATP), 2'(or 3')-O-(2, 4, 6-trinitrophenyl) adenosine (TNP-Ad(, and 1-(2'-hydroxyethoxy)-2, 4, 6-trinitrobenzene (TNP-EG), it was concluded that there is an intramolecular interaction between the base and 2, 4, 6-trinitrophenyl (TNP) moieties in the TNP-ATP molecule. 2. A broad new absorption band was observed in the 530-630 nm region when excess indole was added to reaction mixtures containing TNP-ATP dissolved in 50% methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, methanol or dimethyl sulfoxide. On addition of aromatic amino acid derivatives, TNP-ATP and TNP-Ad underwent spectral shifts in the 400-550 nm region. The formation of a 1:1 complex apparently occurred between TNP-ATP and aromatic amino acid derivatives, and the complex with N-acetyltryptophan was stable in 50% methanol. The difference spectrum of TNP-EG vs. TNP-ATP closely resembled that induced by the addition of N-acetyltryptophan to the TNP-ATP solution. 3. The binding of 2'(or 3')-O-(2, 4, 6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) to heavy meromyosin (HMM) was studied by the rapid gel equilibrium method using Sephadex G-25. A dissociation constant of 1.4 muM and a maximum binding number of 1.8 were obtained in 0.15 M KCl, 10 mM MgCl2, and 50 mM Tris-HCl (pH 8.0) at 25 degrees. TNP-ADP bound to the enzyme caused a characteristic spectral shift in the visible region. This spectral shift was explained in terms of an interaction between tryptophanyl residues and the adenine base of TNP-ADP bound to the enzyme. TNP-ADP quenched the tryptophanyl fluorescence, but TNP-EG and TNP-Ad did not. In the presence of 6 M guanidine hydrochloride, TNP-ADP scarcely quenched the tryptophanyl fluorescence, its effect being comparable to that of TNP-Ad.     

Synthesis and biological activities of N-acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some of its lipophilic derivatives

N-Acetyl-1-thiomuramoyl-L-alanyl-D-isoglutamine and some lipophilic analogs were synthesized from benzyl 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]- alpha-D-glucopyranoside (1). O-Debenzoylation of 2, derived from 1 by oxidation, gave 2-acetamido-2-deoxy-4,6-O-isopropylidene-3-O-[D-1-(methoxycarbonyl)ethyl ]-D-glucopyranose (3). Condensation of the alkoxy-tris(dimethylamino)phosphonium chloride (4), formed from 3 by the action of carbon tetrachloride and tris(dimethylamino)phosphine, with potassium thioacetate afforded 2-acetamido-1-S-acetyl-2-deoxy-4,6-O-isopropylidene-3-O-[ D-1-(methoxycarbonyl)ethyl]-1-thio-beta-D-glucopyranose (8). Coupling of the acid 9, obtained from 8 by hydrolysis and subsequent S-acetylation, with the methyl ester of L-alanyl-D-isoglutamine gave N-[2-O-(2-acetamido-1-S-acetyl-2,3-dideoxy-4,6-O- isopropylidene-1-thio-beta-D-glucopyranose-3-yl)-D-lactoyl]-L-alan yl-D- isoglutamine methyl ester (10), which was converted, via O-deisopropylidenation, S-deacetylation, and de-esterification, into the N-acetyl-1-thiomuramoyl dipeptide. Condensation of 11 (derived from 10 by S-deacetylation) and of 12 (obtained from 10 by S-deacetylation and de-esterification) with various acyl chlorides yielded the corresponding 1-S-acyl-N-acetylmuramoyl-L-alanyl-D-isoglutamine derivatives, which were converted into the desired, lipophilic 1-thiomuramoyl dipeptides by cleavage of the isopropylidene group. Condensation of 11 with the alkyl bromides yielded the 1-S-alkyl derivatives, which were also converted, via O-deisopropylidenation and de-esterification, into the corresponding 1-S-alkylmuramoyl dipeptides. The biological activities were examined in guinea-pigs and mice.     

2'(3')-O-L-Phenylalanyl derivatives of N2,5'-anhydroformycin and N4,5'-anhydroformycin: new substrates for ribosomal peptidyltransferase with a fixed anti and syn conformation of the base

The 2'(3')-O-L-phenylalanyl-N2,5'-anhydroformycin (1c) and 2'(3')-O-L-phenylalanyl-N4,5'-anhydroformycin (2c), obtained by chemical synthesis, are substrates for ribosomal peptidyltransferase from Escherichia coli. Nucleoside 1c, which mimics an anti conformation of antibiotic formycin, has 80% of the acceptor activity of puromycin at 5 x 10(-4) M determined by the release of N-Ac-Phe residue from the 70 S ribosome-poly(U)-N-Ac-[14C]Phe-tRNA complex. The reaction product, 2'(3')-O-(N-acetyl)-L-phenylalanyl-L-phenylalanyl-N2,5'-anhydroformyc in (1d), was characterized by paper electrophoresis before and after alkaline hydrolysis. By contrast, nucleoside 2c, which resembles a syn conformation of formycin, exhibited only 20% of the acceptor activity of puromycin at 5 x 10(-4) M. The results which are in accord with previous models have shown that a substrate with its base in an anti conformation is preferable for the acceptor site of peptidyltransferase than the corresponding syn counterpart. Nevertheless, it is possible that an intermediate conformation, for example, high anti (amphi-minus), is an optimal arrangement for acceptor site substrates.     

Chemical aminoacylation of transfer ribonucleic acid. The reaction of Escherichia coli tRNA with ethyl N-benzyloxycarbonylorthoglycinate.

The alpha-carbethoxypentadecyltrimethylammonium (Septonex) salt of tRNA (Ib) was condensed with ethyl N-benzyloxycarbonylorthoglycinate (II) in dimethylformamide in vacuo and in the presence of H3PO4 as catalyst. Pancreatic RNAase degradation and phenylalanine acceptor activity showed a 55--60% conversion to the 2',3'-cyclic orthoglycinate derivative of tRNA (IIIb). The orthoester grouping of IIIb was quantitatively hydrolyzed in 80% formic acid at 0 degrees C for 15 min to give 2'(3')-O-(N-benzyloxycarbonyl)glycyl tRNA (IVb). The latter was stripped at pH 8.8 to give tRNA whose behavior on DEAE cellulose column and gel electrophoresis was similar to that of starting tRNA. The phenylalanine acceptor activity amounted to almost 80% of the starting tRNA.     

Lipase catalyzed synthesis of l-alanyl, l-leucyl and l-phenylalanyl esters of d-glucose using unprotected amino acids

Enzymatic synthesis of l-alanyl, l-leucyl and l-phenylalanyl esters of D-glucose was carried out in a non-polar solvent using lipases from Rhizomucor miehei and porcine pancreas. The unprotected amino acids at millimolar concentrations were used in presence of 10 to 50% (w/w) glucose of the lipases to give ester yields up to >99%. The reaction mixture on analysis by 2-D NMR showed that the product is a mixture of 6-O-, 3-O- and 2-O-monoesters and 2,6-di-O- and 3,6- di-O-esters.     

Substrate specificity of ribosomal peptidyltransferase. Effect of modification in the heterocyclic, carbohydrate and amino acid moiety of 2'(3')-O-L-phenylalanyladenosine.

The chemical synthesis of 2'(3')-O-L-phenylalanyl derivatives of nebularine (Ld), 6-methoxynebularine (Ih), N6,N6-dimethyladenosine (Lk), 6-methylthionebularine (Lo), 8-bromoadenosine (Lr), tubercidin (Iu), and 3'-O-L-phenylalanyl derivatives of 1-(beta-D-arabinofuranosyl)cytosine (IIIg) and 9-(beta-D-arabinofuranosyl)adenine (IIIl) is described. 2'(3')-O-(3-Phenyl)propionyladenosine (Iv) was obtained by reaction of adenosine with ethyl 3-phenylorthopropionate and subsequent hydrolysis of the orthoester intermediate IV with formic acid. Compounds Id, Ih, Ik, Io, and Iu were active in the release of Ac-Phe from N-Ac-Phe-tRNA-poly(U)-70S ribosome complex: at 0.01 mM the release of Ac-Phe was 50-100% of that of A-Phe. At 1 mM, compounds Ir and IIIg released 30 and 25% of Ac-Phe relative to A-Phe whereas derivatives Iv and IIIl were virtually inactive. The results indicate the following conclusions regarding ribosomal peptidyltransferase activity of 2'(3')-O-aminoacyl nucleosides: (a) the presence of the 2'-hydroxy group in the ribo configuration is more important for a highly active substrate (A-Phe) than for one of moderate activity (C-Phe); (b) the heterocyclic (purine) residue is in the anti conformation although this requirement is not absolute; (c) the presence of the amino group of the aminoacyl moiety is required; (d) acceptor activity is dependent upon the substituent in the position 6 of the purine moiety.     

Synthesis and reactions of O-acetylated benzyl alpha-glycosides of 6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-N-acetylmuramoyl-L- alanyl-D-isoglutamine esters: the base-catalysed isoglutamine in equilibrium glutamine rearrangement in peptidoglycan-related structures

Condensation of benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2- deoxy-3-O-[(R)-1-carboxyethyl]-alpha-D-glucopyranoside (2) and its 4-acetate (4) with L-alanyl-D-isoglutamine benzyl ester via the mixed anhydride method yielded N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-lacto yl)-L- alanyl-D-isoglutamine benzyl ester (5) and its 4-acetate (6), respectively. Condensation by the dicyclohexylcarbodi-imide-N-hydroxysuccinimide method converted 2 into benzyl 2-acetamido-6-O-(2-acetamido-3,4,6-tri-O-acetyl- 2-deoxy-beta-D-glucopyranosyl)-3-O-[(R)-1-carboxyethyl]-2-deoxy-alpha-D- glucopyranoside 1',4-lactone (7). In the presence of activating agents, 7 underwent aminolysis with the dipeptide ester to give 5. Zemplén O-deacetylation of 5 and 6 led to transesterification and alpha----gamma transamidation of the isoglutaminyl residue to give N-(2-O-[benzyl 2-acetamido-6-O-(2- acetamido-2-deoxy-beta-D-glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyr anosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (8) and -glutamine methyl ester (9). Treatment of 6 with MgO-methanol caused deacetylation at the GlcNAc residue to give a mixture of N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2- deoxy-beta-D-glucopyranosyl)-4-O-acetyl-2,3-dideoxy-alpha-D-glucopyra nosid-3- yl]-(R)-lactoyl)-L-alanyl-D-isoglutamine methyl ester (11) and -glutamine methyl ester (12). Benzyl or methyl ester-protection of peptidoglycan-related structures is not compatible with any of the reactions requiring alkaline media. Condensation of 2 with L-alanyl-D-isoglutamine tert-butyl ester gave N-(2-O-[benzyl 2-acetamido- 6-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-2,3-d ideoxy- alpha-D-glucopyranosid-3-yl]-(R)-lactoyl-L-alanyl-D-isoglutamine tert-butyl ester (16), deacetylation of which, under Zemplén conditions, proceeded without side-reactions to afford N-(2-O-[benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D- glucopyranosyl)-2,3-dideoxy-alpha-D-glucopyranosid-3-yl]-(R)-la cotyl)-L- alanyl-D-isoglutamine tert-butyl ester (17).     

Cyclic acetals of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto-sucrose and their conversion into methyl ether derivatives

The acid-catalysed reaction of 4,1',6'-trichloro-4,1',6'-trideoxy-galacto- sucrose (1) with 5.5 equiv. of 2-methoxypropene in N,N-dimethylformamide followed by acetylation gave 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene-6-O-(1-methoxy-1-methylethyl)-galacto-sucrose (2, 2%), 6,3',4'- tri- O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O-isopropylidene-galacto -sucrose (3, 31%), 3',4'-di-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (4, 38%), 3'-O-acetyl-4,1',6'-trichloro-4,1',6'-trideoxy-2,3-O- isopropylidene- galacto-sucrose (5, 13%), and 2,3',4'-tri-O-acetyl-4,1',6'-trichloro- 4,1',6'-trideoxy-galacto-sucrose (6, 13%). Methylation of 4 followed by removal of the protecting groups gave 4,1',6'-trichloro-4,1',6'-trideoxy-6-O-methyl- galacto- sucrose (8). 4,1',6'-Trichloro-4,1',6'-trideoxy-3-O-methyl-galacto-sucrose (11) was synthesised from 6 by preferential tert-butyldiphenylsilylation of HO-6 followed by methylation and removal of the protecting groups. Likewise, 4,1',6'-trichloro- 4,1',6'-trideoxy-4'-O-methyl-galacto-sucrose (14) was synthesised from 5. Treatment of 3 with aqueous acetic acid followed by methylation and removal of the protecting groups afforded 4,1',6'-trichloro-4,1'6'-trideoxy-2,3-di-O-methyl- galacto-sucrose (17).     

Splicing of unnatural amino acids into proteins: a peptide model study

S-Ethyl 2-azidohexanethioate (N3-Hex-SEt), an unnatural amino acid analog of leucine, is coupled with L-cysteine ethyl ester (NH2-Cys-OEt) to obtain N3-Hex-Cys-OEt by native chemical ligation. Coupling of this dipeptide with N-t-butoxycarbonyl-2-diphenylphosphinoethanethioglycinate produces the tripeptide, t-Boc-Gly-Hex-Cys-OEt, in high yield. These reactions suggest an approach for the incorporation of unnatural amino acids into proteins by successive native chemical ligation and Staudinger ligation.     

Affinity chromatography and binding studies on immobilized 5'-monophosphate and adenosine 2',5'-bisphosphate of nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa.

1. Nicotinamide nucleotide transhydrogenase from Pseudomonas aeruginosa was purified to apparent homogeneity with an improved method employing affinity chromatography on N6-(6aminohexyl)-adenosine 2', 5'-bisphosphate-Sepharose 4B. 2. Polyacrylamide gel electrophoresis of the purified transhydrogenase carried out in the presence of sodium dodecyl sulphate, indicated a minimal molecular weight of 55000 +/- 2000. 3. The kinetic and regulatory properties of the purified transhydrogenase resembled those of the crude enzyme, i.e., NADPH, adenosine 2'-monophosphate and Ca2+ were activators whereas NADP+ was inhibitory. 4. Nicotinamide nucleotide-specific release of binding of the transhydrogenase to N6-(6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and N6-(-aminohexyl)-adenosine-5'-monophosphate-Sepharose suggests the presence of at least two separate binding sites for nicotinamide nucleotides, one that is specific for NADP(H) and one that binds both NAD(H) and NADP(H). 5. Binding of transhydrogenase to N6-)6-aminohexyl)-adenosine-2',5'-bisphosphate-Sepharose and activation of the enzyme by adenosine-2',5'-bisphophate showed a marked pH dependence. In contrast, inhibition of the Ca2+-activated enzyme by adenosine 2',5'-bisphosphate was virtually constant at various pH values. This descrepancy was interpreted to indicate the existence of separate nucleotide-binding effector and active sites.     

稻瘟病菌(Magnaporthe grisea)诱导的水稻早期反应基因ER1的5' 端cDNA片段克隆及序列分析

研究高等生物基因表达与调控的一个重要方面是分离基因的编码区及其上游的调控序列（ＤｅＶｅｅｒ等１９９７）,这需要获得一个基因的ｃＤＮＡ全长及从植物基因组获取全基因。在前文（周建明等１９９９）中曾经分离了稻瘟病菌侵染诱导的水稻早期反应基因ＥＲ１的ｃＤＮＡ片段,但是运用ｍＲＮＡ差异显示技术分离的ｃＤＮＡ片段往往只有近ｍＲＮＡ３’端的一部分,难以反映基因的结构及功能特点,因此,必须进一步分离其５’端的部分才有可能比较全面地了解此基因的特点。ＲＡＣＥ（ｒａｐｉｄａｍｐｌｉｆｉｃａｔｉｏｎｏｆｃＤＮＡｅｎ…     

Fast and efficient peptide bond formation using bis-[alpha,alpha-bis(trifluoromethyl)-benzyloxy]diphenylsulfur. Part I.

This study towards the development of sulfurane-based coupling agents shows that bis-[alpha,alpha-bis(trifluoromethyl)-benzyloxy]diphenylsulfur (BTBDS) can facilitate rapid amide bond formation between Nalpha-urethane-protected l-amino acids and l-phenylalanine ethyl ester in the absence of an external base. The corresponding dipeptide esters were obtained in excellent yields and with no detectable racemization, as judged by analysis of the formed dipeptides by chiral-phase HPLC. In addition, BTBDS-mediated condensation of benzoyl-l-phenylalanine with l-phenylalanine ethyl ester was also investigated. The results indicate that sulfuranes can be useful for application in racemization-sensitive systems, such as segment condensation.     

Mutations at alternative 5' splice sites of M1 mRNA negatively affect influenza A virus viability and growth rate

Different amino acid sequences of influenza virus proteins contribute to different viral phenotypes. However, the diversity of the sequences and its impact on noncoding regions or splice sites have not been intensively studied. This study focuses on the sequences at alternative 5' splice sites on M1 mRNA. Six different mutations at the splice sites were introduced, and viral growth characteristics for those mutants generated by reverse genetics with 12 plasmids were examined, for which G12C (the G-to-C mutation at the first nucleotide of the intron for the mRNA3 5' splice site), C51G (at the 3' end of the exon of the M2 mRNA 5' splice site), and G146C (for the first nucleotide of the intron for mRNA4) are lethal mutations. On the other hand, mutants with the mutation G11C (at the 3' end of exon of the mRNA3 5' splice site), G52C (for the first nucleotide of the intron for M2 mRNA), or G145A (at the 3' end of the exon of mRNA4) were rescued, although they had significantly attenuated growth rates. Notably, these mutations did not change any amino acids in M1 or M2 proteins. The levels of precursor (M1 mRNA) and spliced products (M2 mRNA, mRNA3, and mRNA4) from the recombinant mutant virus-infected cells were further analyzed. The production levels of mRNA3 in cells infected with G11C, G52C, and G145A mutant viruses were reduced in comparison with that in wild-type recombinant virus-infected ones. More M2 mRNA was produced in G11C mutant virus-infected cells than in wild-type-virus-infected cells, and there was little M2 mRNA and none at all in G145A and G52C mutant virus-infected ones, respectively. Results obtained here suggest that introducing these mutations into the alternative 5' splice sites disturbed M1 mRNA splicing, which may attenuate viral growth rates.     

Synthesis of 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses

Five 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine)-d-glucopyranoses (lipophilic, muramoyl dipeptide analogs) were synthesized from benzyl 2-(benzyloxycarbonylamino)-3-O-(d-1-carboxyethyl)-2-deoxy-5,6-O-isopropylidene-β-dglucopyranoside (1). Methanesulfonylation of 3, derived from the methyl ester of 1 by O-deisopropylidenation, gave the 6-methanesulfonate (4). (Tetrahydropyran-2-yl)ation of 4 gave benzyl 2-(benzyloxycarbonylamino)-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-6-O-(methylsulfonyl)-5-O-(tetrahydropyran-2-yl)-β-d- glucofuranoside, which was treated with sodium azide to give the corresponding 6-azido derivative (6). Condensation of benzyl 6-amino-2-(benzyloxycarbonyl-amino)-2,6-dideoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-5-O-(tetrahydropyran-2-yl)-β-d-glucofuranoside, derived from 6 by reduction, with the activated esters of octanoic, hexadecanoic, and eicosanoic acid gave the corresponding 6-N-fatty acyl derivatives (8–10). Coupling of the 2-amino derivatives, obtained from compounds 8, 9, and 10 by catalytic reduction, with the activated esters of the fatty acids, gave the 2,6-(diacylamino)-2,6-dideoxy derivatives (11–15). Condensation of the acids, formed from 11–15 by de-esterification, with the benzyl ester of l-alanyl-d-isoglutamine, and subsequent hydrolysis, afforded benzyl 2,6-di(acylamino)-2,6-dideoxy-3-O-(d-2-propanoyl-l-alanyl-d-isoglutamine benzyl ester)-β-d-glucofuranosides. Hydrogenation of the dipeptide derivatives thus obtained gave the five lipophilic analogs of 6-amino-6-deoxymuramoyl dipeptide, respectively, in good yields.     

Immobilization of proteases to porous chitosan beads and their catalysis for ester and peptide synthesis in organic solvents.

alpha-Chymotrypsin (CT), subtilisin BPN' (STB), and subtilisin Carlsberg (STC) were immobilized by adsorption to porous chitosan beads (Chitopearl, CP). The immobilized enzymes showed higher catalytic activities than free enzymes for amino acid esterification in many hydrophilic organic solvents except for methanol and DMF. In ethanol, the initial rate of the esterification increased with water content, whereas in ethyl acetate, the maximum rate was obtained at 2%-3% water. CP-immobilized CT also catalysed transesterification of Ac-Tyr-OMe in ethanol and peptide synthesis in acetonitrile from Ac-Tyr-OH or its ethyl ester and amino acid amides. The immobilized enzymes are highly stable in organic solutions, and can easily be separated from the reaction solutions. Repeated esterifications of Ac-Tyr-OH in acetonitrile by a CP-immobilized CT gave almost constant yields of the ester for more than 3 weeks.     

Cyclic 3',5'-AMP phosphodiesterase in Physarum polycephalum. I. Chemotaxis toward inhibitors and cyclic nucleotides.

The effect of several inhibitors of the enzyme cyclic 3',5'-AMP phosphodiesterase as chemoattractants in Physarum polycephalum was examined. Of the compounds tested, 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Roche 20-1724/001) and 1-ethyl-4-(isopropylidinehydrazino)-1H-pyrazolo-(3,4-b)-pyridine-5-carboxylic acid ethyl ester, hydrochloride (Squibb 20009) were the most potent attractants. 3-Isobutyl-1-methyl xanthine, theophylline, and morin (a flavanoid) were moderate attractants and sometimes gave negative chemotaxis at high concentrations. Cyclic 3',5'-AMP was an effective, but not potent attractant. A repellent effect following the positive chemotactic action was sometimes observed with cyclic 3',5'-AMP at concentrations as high as 1 . 10(-2) M. Dibutyryl cyclic AMP appeared to be a somewhat more potent attractant than cyclic 3',5'-AMP. The 8-thiomethyl and 8-bromoderivatives of cyclic AMP, which are poorly hydrolyzed by the phosphodiesterase, were not attractants in Physarum. Possible participation of cyclic 3',5'-AMP in the directional movement in P. polycephalum is discussed.     

Synthetic resin-bound truncated Candida antarctica lipase B for production of fatty acid alkyl esters by transesterification of corn and soybean oils with ethanol or butanol

A gene encoding a synthetic truncated Candida antarctica lipase B (CALB) was generated via automated PCR and expressed in Saccharomyces cerevisiae. Western blot analysis detected five truncated CALB variants, suggesting multiple translation starts from the six in-frame ATG codons. The longest open reading frame, which corresponds to amino acids 35-317 of the mature lipase, appeared to be expressed in the greatest amount. The truncated CALB was immobilized on Sepabeads? EC-EP resin and used to produce ethyl and butyl esters from crude corn oil and refined soybean oil. The yield of ethyl esters was 4-fold greater from corn oil than from soybean oil and was 36% and 50% higher, respectively, when compared to a commercially available lipase resin (Novozym 435) using the same substrates. A 5:1 (v/v) ratio of ethanol to corn oil produced 3.7-fold and 8.4-fold greater yields than ratios of 15:1 and 30:1, respectively. With corn oil, butyl ester production was 56% higher than ethyl ester production. Addition of an ionic catalytic resin step prior to the CALB resin increased yields of ethyl esters from corn oil by 53% compared to CALB resin followed by ionic resin. The results suggest resin-bound truncated CALB has potential application in biodiesel production using biocatalysts.