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     

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     

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     

Ability of Adenosine-2′(3′)-deoxy-3′(2′)-triphosphates and Related Analogues to Replace ATP as Phosphate Donor for all Human and Drosphila melanogaster Deoxyribonucleoside Kinases

Abstract

Six non-conventional adenosine-2′- and 3′-triphosphate analogues of ATP were tested as potential phosphate donors for all four human, and D. melanogaster, deoxyribonucleoside kinases. With dCK (only dAdo as acceptor), TK1, TK2 and dNK only 3′-deoxyadenosine-2′-triphosphate was an effective donor (5–60% that for ATP). With dCK (dCyd as acceptor) and dGK (dGuo as acceptor), sharing 45% sequence identity, donor activities ranged from 13 to 119% that for ATP. Products were 5′-phosphates. In some instances, kinetics are dependent on the nature of the acceptor, and donor and acceptors properties are mutually interdependent. Results are highly relevant to studies on the modes of interaction with the enzymes, and to interpretations of reported crystal structures of dCK and dNK with bound ligands.     

Inhibition of the peptidyltransferase acceptor site by 2′(3′)-O-cycloleucyl- and α-aminoisobutyryl derivatives of cytidylyl-(3′–5′)adenosine

2′(3′)-O-(N-Benzyloxycarbonylcycloleucyl)adenosine (1a) was prepared by esterification of 5′-O-(4-methoxytrityl)adenosine with N-benzyloxycarbonylcycloleucine in the presence of dicyclohexylcarbodiimide and subsequent deprotection in acidic medium. The compound 1a was separated into pure 2′- and 3′-isomers using HPLC; these isomers were found to undergo an easy interconversion. Compound 1a was coupled with N-dimethylaminomethylene-2′,5′-di-O-tetrahydropyranylcytidine 3′-phosphate in the presence of dicyclohexylcarbodiimide to give, after subsequent deblocking, cytidylyl(3′→5′)2′(3′)-O-cycloleucyladenosine (1c). Compound 1c, as well as the related cytidylyl(3′→5′)2′(3′)-O-(α-aminoisobutyryl)adenosine (1d), inhibited the peptidyltransferase catalyzed transfer of an AcPhe residue to puromycin in the Ac[¹⁴C]Phe-tRNA·poly(U)·70 S E. coli ribosome system. A half of the maximum inhibition of AcPhe-puromycin formation (at 10^?5 M puromycin) was achieved at 9.5·10^?6 M of compound 1c and 9·10^?5 M of compound 1d, respectively. The inhibition of the puromycin reaction by compound 1d shows a mixed-type of inhibition kinetics. Further, none of the compounds 1c and 1d was an acceptor in the peptidyltransferase reaction. Both compounds 1c and 1d inhibited the binding of C-A-C-C-A[¹⁴C]Phe to the A site of peptidyltransferase in a system containing tRNA^Phe·poly(U)·70 S E. coli ribosomes, in which compound 1d was a much stronger inhibitor than 1c. These results indicate that the derivatives such as compounds 1c and 1d which contain an anomalous amino acid with a substituent in lieu of α-hydrogen can interfere with the peptidyltransferase A site; however, they are not acceptors in the peptidyltransferase reaction probably due to a misfit of the α-substituent.     

The elongation factor Tu·GTPase reaction: Effect of 2′(3′)-O-aminoacyl oligoribonucleotides

The activity of synthetic (2′(3′)-O-aminoacyl trinucleotides, C-C-A-Phe, C-C-U-Phe, C-U-A-Phe, U-C-A-Phe and C-A-A-Phe, in promoting the EF-Tu·70 S ribosome-catalyzed GTP hydrolysis was investigated. It was found that the activity decreases in the order C-C-A-Phe > C-U-A-Phe > U-C-A-Phe > C-A-A-Phe ⪢ C-C-U-Phe. Thus, the substitution in ‘natural’ C-C-A sequence with other nucleobases weakens binding of 2′(3′)-O-aminoacyl trinucleotides to EF-Tu, with the substitution at the 3′-position having the most profound effect. Since the 2′(3′)-O-aminoacyl oligonucleotides mimic the effect of the aa-tRNA 3′-terminus on EF-Tu·GTPase, it follows that EF-Tu probably directly recognizes structure of nucleobases in the aa-tRNA 3′-terminus, with the 3′-terminal adenine playing the most important role.     

Receptor-independent effects of 2′(3′)-O-(4-benzoylbenzoyl)ATP triethylammonium salt on cytosolic pH

The effect of the relatively potent P2X7 receptor agonist 2′(3′)-O-(4-benzoylbenzoyl)adenosine 5′-triphosphate triethylammonium salt (BzATP-TEA) on cytosolic pH (pH_i) was studied using MC3T3-E1 osteoblast-like cells, which endogenously express P2X7 receptors. pH_i was measured fluorimetrically using the pH-sensitive dye 2′,7′-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. BzATP-TEA (0.3–1.5 mM) elicited fast-onset alkalinization responses. In contrast, adenosine 5′-triphosphate disodium salt (5 mM) failed to reproduce the BzATP-TEA-induced responses, indicating a P2 receptor-independent mechanism. We speculated that triethylamine, which is present in solutions of BzATP-TEA, permeates the plasma membrane, and is protonated intracellularly, leading to an increase in pH_i. Consistent with this hypothesis, triethylammonium (TEA) chloride mimicked the effects of BzATP-TEA on pH_i. Moreover, measurements using a Cytosensor microphysiometer revealed that TEA chloride transiently suppressed proton efflux from cells, whereas washout of TEA transiently enhanced proton efflux. BzATP-TEA also elicited a sustained increase in proton efflux that was blocked specifically by the P2X7 antagonist A-438079. Taken together, we conclude that BzATP-TEA-induced alkalinization is unrelated to P2X7 activation, but is due to the presence of TEA. This effect may confound assessment of the outcomes of P2X7 activation by BzATP-TEA in other systems. Thus, control experiments using TEA chloride are recommended to distinguish between receptor-mediated and nonspecific effects of this widely used agonist. We performed such a control and confirmed that BzATP-TEA, but not TEA chloride, caused the elevation of cytosolic free Ca²⁺ in MC3T3-E1 cells, ruling out the possibility that receptor-independent effects on pH_i underlie BzATP-TEA-induced Ca²⁺ signaling.     

Thermodynamic characterization of RNA 2 × 3 nucleotide internal loops

To better elucidate RNA structure-function relationships and to improve the design of pharmaceutical agents that target specific RNA motifs, an understanding of RNA primary, secondary, and tertiary structure is necessary. The prediction of RNA secondary structure from sequence is an intermediate step in predicting RNA three-dimensional structure. RNA secondary structure is typically predicted using a nearest neighbor model based on free energy parameters. The current free energy parameters for 2 × 3 nucleotide loops are based on a 23-member data set of 2 × 3 loops and internal loops of other sizes. A database of representative RNA secondary structures was searched to identify 2 × 3 nucleotide loops that occur in nature. Seventeen of the most frequent 2 × 3 nucleotide loops in this database were studied by optical melting experiments. Fifteen of these loops melted in a two-state manner, and the associated experimental ΔG°(37,2×3) values are, on average, 0.6 and 0.7 kcal/mol different from the values predicted for these internal loops using the predictive models proposed by Lu, Turner, and Mathews [Lu, Z. J., Turner, D. H., and Mathews, D. H. (2006) Nucleic Acids Res. 34, 4912-4924] and Chen and Turner [Chen, G., and Turner, D. H. (2006) Biochemistry 45, 4025-4043], respectively. These new ΔG°(37,2×3) values can be used to update the current algorithms that predict secondary structure from sequence. To improve free energy calculations for duplexes containing 2 × 3 nucleotide loops that still do not have experimentally determined free energy contributions, an updated predictive model was derived. This new model resulted from a linear regression analysis of the data reported here combined with 31 previously studied 2 × 3 nucleotide internal loops. Most of the values for the parameters in this new predictive model are within experimental error of those of the previous models, suggesting that approximations and assumptions associated with the derivation of the previous nearest neighbor parameters were valid. The updated predictive model predicts free energies of 2 × 3 nucleotide internal loops within 0.4 kcal/mol, on average, of the experimental free energy values. Both the experimental values and the updated predictive model can be used to improve secondary structure prediction from sequence.     

Synthesis of Cytidylyl(3′→5′)cytidylyl(3′→5′)-(2′)3′-O-[Lα-alanyl]adenosine

Abstract

A new synthesis of protected C-C-A-[L^α-Ala] 14 is reported using a new set of complementary groups such as 2-phenylsulfonylethoxycarbonyl (PSEC) for the protection of exocyclic amino functions, o-chlorophenyl (o-CIPh) for the internucleotide phosphotriester, 3-methoxy-1,5-dicarbomethoxypentan-3-yl (MDMP) and the 4-monomethoxytrityl (MMTr) for the protection of the ã-amino fuction of the amino acid. 14 could be deprotected in two steps by treatments with 1,1,3,3-tetramethylguanidinium oximate under a dry condition and then by neat trifluoroacetic acid. Treatment with neat trifluoroacetic acid produced a stable salt: [C-C-A-Ala-N^ãH₃+ CF₃CO₂-] and did not promote any internucleotide phosphate migration or degradation of the oligomeric molecule. This salt was considerably more stable than C-C-A-Ala conjugate with a free ã-amino group, and, therefore, it could be easily purified on a silica gel column and was isolated in 82 % yield. This strategy should be useful for the synthesis of longer oligonucleotide-aminoacyl conjugate.     

Stereospecific Synthesis and Anti-HIV Activity of (Z)2′- and (E)3′-Deoxy-2′(3′)-C-(chloromethylene) Pyrimidine Nucleosides

Abstract

Reaction of 1-[2,5(and 3,5)-di-O-trityl-β-D-erythro-pentofuran-3 (and 2)-ulosyl]uracil derivatives 5 and 6 with (chloromethyl)triphenylphosphorane resulted in the stereoselective formation of (E)-3′- and (Z)-2′-chloromethylene derivatives 7 (69%) and 8 (53%), respectively, deprotection of which gave 9 and 10. Transformation of the uracil nucleoside 7 into cytosine one followed by deprotection yielded 12. The latter was converted into the arabinoside 14. The fully deprotected chloromethylene nucleosides were tested for their activity against HIV-1 and HIV-2.     

Interaction of (2′ -5′) and (3′ -5′) Linked 2-Aminoadenylyl-3-aminoadenosines with Polyuridylic Acid

Abstract

2′-5′ and 3′-5′ linked 2-aminoadenylyl-2-aminoadenosines [(2′-5′)n²Apn²A (1) and (3′-5′)n²Apn²A (2)] were synthesized by condensation of 5′-O-monomethoxytrityl-N ² N ⁶-dibenzoyl-2-aminoadenosine and N ²,N ⁶,2′,3′-O-tetrabenzoyl-2-aminoadenosine 5′-phosphate using dicyclohexylcarbodiimide (DCC). The conformational properties of these dimers 1 and 2 were examined by UV, NMR and CD spectroscopy. The results reveal that the 2′-5′-isomer 1 takes a stacked conformation, which contains a larger base-base overlap and is more stable against thermal perturbation with respect to the 3′-5′-isomer 2. Interactions of 1 and 2 with polyuridylic acid (Poly (U)) were also examined by Tm, mixing curves, UV and CD spectra. Both the dinucleoside isomers 1 and 2 formed a complex of 1 : 2 stoichiometry with poly(U), which was much more stable than that of the corresponding ApA isomer     

甲基乙烯基醚和2′(3′)-O-甲氧乙基-5′-核糖核苷二磷酸的合成

本文报导了一种制备甲基乙烯基醚(MVE)的简易方法,其合成反应途径是:(1) CH_3CHO+CH_3OH+HCl -20℃→CH_3OCH(Cl)CH_3+H_2O(2) CH_3OCH(Cl)CH_3+C_5H_5N -20°→[CH_3OCH(Cl)CH_3·C_5H_5N](3) [CH_3OCH(Cl)CH_9·C_5H_5N] -120°→CH_3OCH=CH_2+C_5H_5N·HCl 在三氟乙酸催化作用下,MVE与各种5’-核苷二磷酸(ppN)反应,用纤维素柱层析方法分离反应产物,制备了五种2’(3’)-O-甲氧乙基-5’-核苷二磷酸(ppN-2’(3’)-O-ME):ppA-2’(3’)-O-ME,ppG-2’(3’)-O-ME,ppC-2’(3’)-O-ME,ppU-2’(3’)-O-ME,以及ppΨ-2’(3’)-O-ME,产率一般为50～70%。     

Conformational activation of the EF-Tu·GTPase activity Stimulation of the 2′(3′)-O-l-phenylalanyladenosine-promoted EF-Tu·GTPase upon binding of the tRNAPhe lacking the terminal adenosine residue

The elongation factor Tu (EF-Tu) dependent GTPase (in the presence of aurodox) is stimulated by analogs of the aminoacyl tRNA 3′-terminus in the following order: A-Phe < C-A-Phe < C-C-A-Phe. The GTPase-promoting activity of A-Phe is strongly enhanced by tRNA-C-C (devoid of 3′-terminal adenosine residue) but not by intact tRNA-C-C-A. On the other hand, the activity of C-A-Phe as the EF-Tu·GTPase promoter is only slightly enhanced by tRNA-C-C.     

SYNTHESIS of 2′-DEOXY-2′-FLUOROGUANYL-(3′,5′)-GUANOSINE

ABSTRACT

The protected analogue of 2-amnio-6-chloropurine arabinoside (3b) was subjected to reaction with diethylaminosulfur trifluoride (DAST) and subsequently treated with NaOAc in Ac₂O/AcOH to give N ²,O ^3′,O ^5′-triacetyl-2′-deoxy-2′-fluoroguanosine (5a). After deacetylation of the sugar moiety and protection of 5′-OH by a 4,4′-dimethoxytrityl group, this nucleoside component was converted to 2′-deoxy-2′-fluoroguanyl-(3′,5′)-guanosine (6c, GfpG).     

Absolute configuration of 2′(R)-acetylmontrifoline and 2′(R)-montrifoline,furoquinolines from the fruits of Teclea nobilis

A phytochemical study on the alkaloids from Teclea nobilis yielded five furoquinolines, including the new acetylmontrifoline. The structures were elucidated from extensive 2D-NMR and MS experiments. The absolute configuration of the chiral center of montrifoline and of acetylmontrifoline was established as R by analysing the Mosher's derivatives and chemical modifications, respectively. All the five furoquinolines showed weak in vitro antiplasmodial activity against Plasmodium falciparum, montrifoline being moderately active (56 μg/ml) and not cytotoxic.     

Stereoselective synthesis of β-N-glycosides through 2-deoxy-2-nitroglycal

An efficient, base-free protocol has been developed for the β-stereoselective synthesis of N-glycosides from 2-nitroglycal and secondary amines. Simple protection and deprotection manipulations on the N-glycosides pave a way for the synthesis of biologically significant1,2-diaminosugars and glycopeptides.     

Synthesis of novel isoxazoline-containing podophyllotoxin/2′(2′,6′)-(di)halogenopodophyllotoxin derivatives and their insecticidal/acaricidal activities

In continuation of our program aimed at the development of natural product-based pesticidal agents, a series of isoxazoline-containing podophyllotoxin/2′(2′,6′)-(di)halogenopodophyllotoxin derivatives were prepared, and their structures were well characterized by ¹H NMR, IR, optical rotation, HRMS and mp. Especially the structure of compound Ia was further confirmed by ¹H–¹H COSY and NOESY spectrum. Among them, two compounds showed good insecticidal and acaricidal activities against Mythimna separata and Tetranychus cinnabarinus. Their structure–activity relationships were also observed.     

The nucleotide sequence of a wheat γ-gliadin genomic clone

γ-Gliadins are a subclass of the storage proteins from wheat. We have isolated from a wheat genomic library a clone encoding a γ-gliadin gene. The amino acid sequence derived from the gene sequence reveals a mature protein (308 amino acids, excluding a 19 amino acid signal sequence) with a characteristic repeated structure of the consensus heptapeptide PQQPFPQ corresponding to the high glutamine (35.4%) and proline (17.5%) content. The gene does not contain an intron, and possesses a typical eukaryotic promoter.     

甲基乙烯基醚和2′(3′)-0-甲氧乙基-5′-核糖核苷二磷酸的合成

本文报导了一种制备甲基乙烯基醚(MVE)的简易方法,其合成反应途径是: 在三氟乙酸催化作用下,MVE与各种5′-核苷二磷酸(ppN)反应,用纤维素柱层析方法分离反应产物,制备了五种2′(3′)-O-甲氧乙基-5′核苷二磷酸(ppN-2′(3′)-O-ME):ppA-2′(3′)-O-ME,ppG-2′(3′)-O-ME,ppC-2′(3′)-O-ME,ppU-2′(3′)-O-ME,以及ppΨ-2′(3′)-O-ME,产率一般为50～70%。     

Proteases as catalysts of enantioselective α-amino ester hydrolysis: 2. Pronase-catalysed hydrolysis of tryptophan methyl ester

The influence of pH, temperature, EDTA and metal ions on the rate of hydrolysis of methyl esters of d- and l-tryptophan by pronase from Streptomyces griseus has been studied. pH, Ca²⁺ and Co²⁺ ions have different effects on the hydrolysis of d- and l-substrates, which permits the enantioselectivity of the reaction to be regulated. Maximum enantioselectivity was observed at pH 7 in the presence of 10^?3–10^?2m Co²⁺. The data obtained are compared with the literature data for the hydrolysis of other low molecular weight substrates by pronase. It is suggested that leucine aminopeptidase makes a considerable contribution to the observed esterase activity of pronase.