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1.
Angew Chem Int Ed Engl ; 55(17): 5255-8, 2016 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-27008042

RESUMO

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate-limitations can be at the mono-, but also at the di- and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro-approach). In this approach, NTPs are masked by two bioreversible units at the γ-phosphate. Using a procedure involving H-phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme-triggered delivery of NTPs was demonstrated by pig liver esterase, in human T-lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro-compounds of some HIV-inactive nucleoside analogues showed marked anti-HIV activity. For cellular uptake studies, a fluorescent TriPPPro-compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.


Assuntos
Fármacos Anti-HIV/farmacologia , HIV/efeitos dos fármacos , Nucleosídeos/farmacologia , Polifosfatos/farmacologia , Pró-Fármacos/farmacologia , Nucleotídeos de Timina/farmacologia , Animais , Fármacos Anti-HIV/química , Fármacos Anti-HIV/metabolismo , Fármacos Anti-HIV/farmacocinética , Linfócitos T CD4-Positivos/virologia , Linhagem Celular , Permeabilidade da Membrana Celular , Esterases/metabolismo , Infecções por HIV/tratamento farmacológico , Humanos , Nucleosídeos/química , Nucleosídeos/metabolismo , Nucleosídeos/farmacocinética , Polifosfatos/química , Polifosfatos/metabolismo , Polifosfatos/farmacocinética , Pró-Fármacos/química , Pró-Fármacos/metabolismo , Pró-Fármacos/farmacocinética , Suínos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
2.
Org Lett ; 17(11): 2586-9, 2015 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-25970636

RESUMO

Novel α,ß-CH2 and ß,γ-NH (1a) or α,ß-NH and ß,γ-CH2 (1b) "Met-Im" dTTPs were synthesized via monodemethylation of triethyl-dimethyl phosphorimido-bisphosphonate synthons (4a, 4b), formed via a base-induced [1,3]-rearrangement of precursors (3a, 3b) in a reaction with dimethyl or diethyl phosphochloridate. Anomerization during final bromotrimethylsilane (BTMS) deprotection after Mitsunobu conjugation with dT was avoided by microwave conditions. 1a was 9-fold more potent in inhibiting DNA polymerase ß, attributed to an NH-group interaction with R183 in the active site.


Assuntos
Nucleotídeos de Timina/síntese química , Modelos Moleculares , Estrutura Molecular , Nucleotídeos de Timina/química
3.
Bioorg Khim ; 39(6): 718-27, 2013.
Artigo em Russo | MEDLINE | ID: mdl-25696933

RESUMO

The interaction of CDI-activated diethyl phosphonoacetate with methyl 4-aminobenzoat or 3,5-difluoromethylphenylamine followed by treatment with Me3SiBr in DMF led to N-aryl aminocarbonylmethyl phosphonates and their ethyl esters. Their coupling with 3'-acetyl-α-thymidine followed by removal of the acetyl groups gave (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl-, aminocarbonyl- and carboxy)phenyl]-aminocarbonylmethyl phosphonates, (α-D-thymidine-5'-il)-[3,5-bis(trifluoromethyl)phenylaminocarbonyl]methyl phosphonate and their ethyl esters. The phosphonates were stable in different conditions, low cytotoxic (in Vero and K562 cells) and were able to penetrate into K562 cells. The only ethyl ester of (α-D-thymidine-5'-il) N-[4-(methoxycarbonyl)phenyl]-aminocarbonylmethyl phosphonate in high concentration (200 µg/mL) inhibited in vitro the growth of laboratory sensitive strain of Mycobacterium tuberculosis H37Rv.


Assuntos
Inibidores Enzimáticos/síntese química , Mycobacterium tuberculosis/efeitos dos fármacos , Nucleotídeos de Timina/síntese química , Tuberculose/microbiologia , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Humanos , Espectroscopia de Ressonância Magnética , Mycobacterium tuberculosis/enzimologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Tuberculose/tratamento farmacológico
4.
J Med Chem ; 55(16): 7245-52, 2012 Aug 23.
Artigo em Inglês | MEDLINE | ID: mdl-22827702

RESUMO

Methyl-substituted cycloSal-pronucleotides of d4TMP were synthesized with high diastereoselectivities in satisfying chemical yields. The individual diastereomers were tested against HIV-1 and HIV-2 infected wild-type CEM/0 and HIV-2 infected thymidine kinase deficient CEM cells. All diastereomers tested showed significant antiviral activity in CEM/0 and strong activity in CEM/TK(-) cell cultures. The antiviral activities were strongly dependent on the chirality at the phosphate group and the position of the methyl-group(s) in the cycloSal moiety. In CEM/TK(-) cell cultures the difference in antiviral potency was found to be 7- to 20-fold. The stability of each diastereomer was studied in aqueous phosphate buffer and in CEM/0 cell extracts. Large differences in the half-lives were found. A comparison of the relative lipophilicity of the methyl-substituted cycloSal triesters was performed based on the retention times obtained by reversed phase HPLC. The results obtained clearly confirm the importance of a diastereoselective synthesis of cycloSal-pronucleotides.


Assuntos
Fármacos Anti-HIV/síntese química , Didesoxinucleotídeos/síntese química , Estavudina/análogos & derivados , Nucleotídeos de Timina/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Linhagem Celular , Didesoxinucleotídeos/química , Didesoxinucleotídeos/farmacologia , Estabilidade de Medicamentos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Hidrólise , Mutação , Solventes/química , Estavudina/síntese química , Estavudina/química , Estavudina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Timidina Quinase/genética , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia
5.
Curr Protoc Nucleic Acid Chem ; Chapter 13: Unit13.10, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22700336

RESUMO

A simple, straightforward, reliable, and efficient method for the chemical synthesis of sodium salt of 2'-deoxynucleoside-5'-O-triphosphates (dNTPs), starting from the corresponding nucleoside, is described. This improved "one-pot, three-step" synthetic strategy involves the monophosphorylation of nucleoside, followed by reaction with tributylammonium pyrophosphate and hydrolysis of the resulting cyclic intermediate to provide the corresponding dNTP in good yields (65% to 70%). It is noteworthy that the protocol holds good for both the purine deoxynucleotides, such as 2'-deoxyguanosine-5'-O-triphosphate (dGTP) and 2'-deoxyadenosine-5'-O-triphosphate (dATP), and pyrimidine deoxynucleotides, such as 2'-deoxycytidine-5'-O-triphosphate (dCTP), thymidine-5'-O-triphosphate (TTP), and 2'-deoxyuridine-5'-O-triphosphate (dUTP).


Assuntos
Nucleotídeos de Purina/síntese química , Nucleotídeos de Pirimidina/síntese química , Nucleotídeos de Desoxiadenina/síntese química , Nucleotídeos de Desoxicitosina/síntese química , Difosfatos/química , Hidrólise , Nucleosídeos/química , Nucleotídeos de Timina/síntese química
6.
Chembiochem ; 13(11): 1605-12, 2012 Jul 23.
Artigo em Inglês | MEDLINE | ID: mdl-22733592

RESUMO

Borononucleotides are a family of natural nucleotide monophosphate analogues with a 5'-boronic acid function. As B-O-P linkages are known to be unstable in solution, we evaluated the ability of borononucleotides to be recognized by nucleoside monophosphate kinases and eventually foil the phosphorylation process. In this context, and with the idea of probing the influence of their size, shape, and flexibility, a library of borononucleotides were synthetized starting from the borononucleotide analogue of thymidine, which was shown to behave as a slow substrate of human TMP kinase. This study thus constitutes a good starting point for the development of new monophosphate mimics as potential substrates or ligands for NMP kinases.


Assuntos
Ácidos Borônicos/farmacologia , Corantes Fluorescentes/farmacologia , Núcleosídeo-Fosfato Quinase/antagonistas & inibidores , Nucleotídeos de Timina/farmacologia , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Ácidos Borônicos/síntese química , Ácidos Borônicos/química , Dicroísmo Circular , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/química , Humanos , Modelos Moleculares , Estrutura Molecular , Núcleosídeo-Fosfato Quinase/metabolismo , Fosforilação , Relação Estrutura-Atividade , Especificidade por Substrato/efeitos dos fármacos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química
7.
Chem Asian J ; 6(10): 2747-52, 2011 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-21887745

RESUMO

DNA molecules are known to be important materials in sensing, aptamer selection, nanocomputing, and construction of unique architectures. The incorporation of modified nucleobases affords unique DNA properties for applications in areas that would otherwise be difficult or not possible. Earlier, we demonstrated that the boronic acid moiety can be introduced into DNA through polymerase-catalyzed reactions. In order to study whether such incorporation by polymerase is a general phenomenon, we designed and synthesized four boronic acid-modified thymidine triphosphate (TTP) analogues. The synthesis of certain analogues was through the use of a single dialkyne tether for both the Sonogashira coupling with thymidine and the later Cu-mediated [3+2] cycloaddition for linking the boronic acid moiety. This approach is much more efficient than the previously described method, and paves the way for the preparation of a large number of boronic acid-modified TTPs with a diverse set of structural features. All analogues showed very good stability under polymerase chain reaction (PCR) conditions and were recognized as a substrate by DNA polymerase, and thus incorporated into DNA.


Assuntos
Biocatálise , Ácidos Borônicos/síntese química , Ácidos Borônicos/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desenho de Fármacos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/metabolismo , Ácidos Borônicos/química , Química Click , Estrutura Molecular , Reação em Cadeia da Polimerase , Estereoisomerismo , Nucleotídeos de Timina/química
8.
Antiviral Res ; 88(2): 176-81, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-20739003

RESUMO

3'-Deoxy-3'-fluorothymidine (FLT, alovudine(®)) belongs to the most potent agents inhibiting HIV-1 replication. Its 5'-triphosphate (FLTTP) is a potent inhibitor of HIV-1 reverse transcriptase (HIV RT). Unfortunately, FLT exerts substantial hematologic toxicity both in vitro and in vivo. It was suggested that this toxicity may be related to inhibition of human DNA polymerases, especially mitochondrial DNA polymerase γ, by nucleoside analogue 5'-triphosphates leading to termination of DNA synthesis and mitochondrial dysfunction. To decrease the toxicity of FLT, its thiated analogues, 4-SFLT and 2-SFLT, were previously synthesized and shown to be potent inhibitors of HIV-1 with low in vitro cytotoxicity. To explain this phenomenon in the present study the synthesis of 5'-triphosphates of thiated FLT analogues was undertaken and their interaction with recombinant HIV-1 RT and human DNA polymerases γ (pol γ) and ß (pol ß) was investigated. It was shown that 3'-deoxy-3'-fluoro-4-thiothymidine 5'-triphosphate (4-SFLTTP) and 3'-deoxy-3'-fluoro-2-thiothymidine 5'-triphosphate (2-SFLTTP) were, similarly to FLTTP, potent competitive inhibitors of HIV-1 RT, with K(i)(app) values of 0.091 and 0.022 µM respectively. It is of interest that 2-SFLTTP, a compound in an unusual syn conformation around the glycosidic bond was an uncompetitive inhibitor of human mitochondrial DNA pol γ with K(i)(app) of 0.174 µM, while 4-SFLTTP in anti conformation inhibited this enzyme similarly to FLTTP, i.e., non-competitively, with K(i)(app) of 0.055 µM. Both 4-SFLTTP and 2-SFLTTP were competitive inhibitors of human DNA pol ß, with K(i)(app) values of 16.84 and 4.04 µM, respectively. The results point to partially selective inhibition of HIV RT by thiated 3'-fluorothymidine 5'-triphosphate analogues. Of special interest is that 2-SFLTTP, showing syn conformation, is a less potent inhibitor of human mitochondrial pol γ than 4-SFLTTP and FLTTP, both in the anti conformation, and has a higher inhibitory activity against HIV-1 RT than 4-SFLTTP. Moreover, the parent nucleoside 2-SFLT possessing the syn conformation shows a more potent anti-HIV-1 activity and a better selectivity index than its 4-thio isomer in the anti conformation (Matthes et al., 1989; Poopeiko et al., 1995), 2-SFLT is a potent and selective anti-HIV-1 agent with the selectivity index 4-fold higher than that of FLT. Findings regarding the mechanisms of antiviral and cytotoxic activities of FLT and its thioanalogues are discussed.


Assuntos
Fármacos Anti-HIV/farmacologia , DNA Polimerase beta/antagonistas & inibidores , Didesoxinucleotídeos/farmacologia , Transcriptase Reversa do HIV/antagonistas & inibidores , HIV-1/efeitos dos fármacos , Inibidores da Síntese de Ácido Nucleico , Inibidores da Transcriptase Reversa/farmacologia , Nucleotídeos de Timina/farmacologia , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Polimerase do DNA Mitocondrial , DNA Polimerase Dirigida por DNA , Didesoxinucleosídeos/farmacologia , Didesoxinucleotídeos/síntese química , Didesoxinucleotídeos/química , HIV-1/enzimologia , Humanos , Imagem por Ressonância Magnética , Conformação Molecular , Polirribonucleotídeos/metabolismo , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química
9.
J Med Chem ; 52(11): 3464-73, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19438207

RESUMO

Recently, we reported on 3,3'-bis-(cycloSaligenyl-2',3'-dideoxy-2',3'-didehydrothymidine monophosphates) (3,3'-bis-(cycloSal-d4TMPs) 4) as the first pronucleotides with a mask-to-drug ratio of 1:2 that is still a novelty in the field of pronucleotides. Here, we report on a new set of compounds of these unique type of cycloSaligenyl prodrugs 5 that bear a biaryl axis at the 5-position of the cycloSal residue. All compounds 5 showed pronounced in vitro activity against HIV-1 and HIV-2 in wild-type CEM cell cultures and better retained their antiviral activities in thymidine kinase-deficient CEM cells than the compound 4 series. Moreover, compound 5b is the first bis-(cycloSal-d4TMP) that even showed complete retention of antiviral activity in TK-deficient CEM cells. The complex hydrolysis behavior of 5 was investigated, and the proposed hydrolysis mechanism was proven by means of (31)P NMR spectroscopy and HPLC analysis.


Assuntos
Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/farmacologia , Didesoxinucleotídeos/síntese química , Didesoxinucleotídeos/farmacologia , Estavudina/análogos & derivados , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/farmacologia , Linhagem Celular Tumoral , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Hidrólise , Ressonância Magnética Nuclear Biomolecular , Estavudina/síntese química , Estavudina/farmacologia , Relação Estrutura-Atividade
10.
J Med Chem ; 51(24): 8115-23, 2008 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-19053827

RESUMO

In our attempt to further develop the cycloSal pronucleotide concept, we report on 5-(1-acetoxyvinyl)-cycloSal-d4TMPs as a new type of enzyme-activated pronucleotides. These compounds were converted into 5-acetyl-cycloSal-d4TMPs by (carboxy)esterase cleavage inside the cells. The enzymatic reaction led to the formation of a strong electron-withdrawing substituent that strongly accelerates the chemical hydrolysis of the cycloSal nucleotide to give d4TMP. For some cycloSal-d4TMPs a separation into the diastereomers was achieved. The absolute configuration was assigned by correlation of circular dichroism spectra with similar compounds. Most of the compounds showed complete retention of antiviral activity in TK-deficient CEM/TK(-) cells, which proves the TK-bypass potential of this approach. Interestingly, (S(P))-isomers of cycloSal phosphate triesters showed better antiviral activity in HIV-2-infected thymidine-kinase deficient CEM/TK(-) cells than their (R(P))-counterparts.


Assuntos
Química Farmacêutica/métodos , Didesoxinucleotídeos/química , Didesoxinucleotídeos/síntese química , Nucleotídeos/química , Estavudina/análogos & derivados , Nucleotídeos de Timina/síntese química , Fármacos Anti-HIV/farmacologia , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Desenho de Fármacos , Elétrons , HIV-1/metabolismo , HIV-2/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Químicos , Estavudina/síntese química , Estavudina/química , Nucleotídeos de Timina/química
12.
Nucleic Acids Symp Ser (Oxf) ; (52): 81-2, 2008.
Artigo em Inglês | MEDLINE | ID: mdl-18776263

RESUMO

The alpha-P-borano modification, where one of the alpha-phosphate oxygens is replaced by borane, of chain terminating nucleoside triphosphates are currently being tested in cell culture and are showing promise as effective viral polymerase inhibitors. The goal of this project is to combine the alpha-P-borano and Nanogel drug delivery technology to increase the antiviral potency of chain terminating sugar and base modified purine nucleosides versus the Hepatitis C Viral RNA dependent RNA polymerase (HCV RdRp). Here we show the synthesis of Cordycepin and 2'-O-methyl alpha-P-borano triphosphate via a one-pot phosphorochloridite synthesis under mild conditions. These analogues will be used for future structure-activity relationship (SAR) studies.


Assuntos
Trifosfato de Adenosina/análogos & derivados , Antivirais/síntese química , Compostos de Boro/síntese química , Trifosfato de Adenosina/síntese química , Trifosfato de Adenosina/química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Antivirais/química , Compostos de Boro/química , Hepacivirus/enzimologia , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/química
13.
Carbohydr Res ; 342(11): 1412-8, 2007 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-17532307

RESUMO

Over-expressed GerB (dTDP-4-keto-6-deoxy-d-glucose aminotransferase) of Streptomyces sp. GERI-155 was used in the enzymatic synthesis of dTDP-4-amino-4,6-dideoxy-D-glucose (2) from dTDP-4-keto-6-deoxy-D-glucose (1). [Carbohydrate structure: see text]. Five enzymes including dTMP kinase (TMK), acetate kinase (ACK), dTDP-glucose synthase (TGS), dTDP-glucose 4,6-dehydratase (DH), and dTDP-4-keto-6-deoxy-d-glucose aminotransferase (GerB) were used to synthesize 2 on a large scale from glucose-1-phosphate and TMP. A conversion yield of up to 57% was obtained by HPLC peak integration given a reaction time of 270min. After purification by two successive preparative HPLC systems, the final product was identified by HPLC and then analyzed by (1)H, (13)C, (1)H-(1)H COSY NMR spectrometry.


Assuntos
Desoxiaçúcares/biossíntese , Nucleotídeos de Timina/biossíntese , Transaminases/química , Sequência de Aminoácidos , Desoxiaçúcares/síntese química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Esporos Bacterianos/química , Esporos Bacterianos/enzimologia , Esporos Bacterianos/genética , Streptomyces/química , Streptomyces/enzimologia , Streptomyces/genética , Nucleotídeos de Timina/síntese química , Transaminases/genética
14.
J Med Chem ; 50(6): 1335-46, 2007 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-17328534

RESUMO

Bis-cycloSal-d4T-monophosphates have been synthesized as potentially anti-HIV active "dimeric" prodrugs of 2',3'-dideoxy-2',3'-didehydrothymidine monophosphate (d4TMP). These pronucleotides display a mask-drug ratio of 1:2, a novelty in the field of pronucleotides. Both bis-cycloSal-d4TMP 6 and bis-5-methyl-cycloSal-d4TMP 7 showed increased hydrolytic stability as compared to their "monomeric" counterparts and a completely selective hydrolytic release of d4TMP. The hydrolysis pathway was investigated via 31P NMR spectroscopy. Moreover, due to the steric bulkiness, compound 6 already displayed strongly reduced inhibitor potency toward human butyrylcholinesterase (BChE), while compound 7 turned out to be devoid of any inhibitory activity against BChE. Partial separation of the diastereomeric mixture of 6 revealed strong dependence of the pronucleotides' properties on the stereochemistry at the phosphorus centers. Both 6 and 7 showed good activity against HIV-1 and HIV-2 in wild-type CEM cells in vitro. These compounds were significantly more potent than the parent nucleoside d4T 1 in HIV-2-infected TK-deficient CEM cells, indicating an efficient TK-bypass.


Assuntos
Fármacos Anti-HIV/síntese química , Pró-Fármacos/síntese química , Estavudina/análogos & derivados , Timidina Monofosfato/análogos & derivados , Nucleotídeos de Timina/síntese química , Fármacos Anti-HIV/química , Fármacos Anti-HIV/farmacologia , Butirilcolinesterase/química , Linhagem Celular Tumoral , Inibidores da Colinesterase/síntese química , Inibidores da Colinesterase/química , Inibidores da Colinesterase/farmacologia , Didesoxinucleotídeos , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Hidrólise , Pró-Fármacos/química , Pró-Fármacos/farmacologia , Estavudina/síntese química , Estavudina/química , Estavudina/farmacologia , Estereoisomerismo , Relação Estrutura-Atividade , Timidina Monofosfato/síntese química , Timidina Monofosfato/química , Timidina Monofosfato/farmacologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia
15.
Nucleic Acids Res ; 35(4): 1222-9, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-17267413

RESUMO

The boronic acid moiety is a versatile functional group useful in carbohydrate recognition, glycoprotein pull-down, inhibition of hydrolytic enzymes and boron neutron capture therapy. The incorporation of the boronic-acid group into DNA could lead to molecules of various biological functions. We have successfully synthesized a boronic acid-labeled thymidine triphosphate (B-TTP) linked through a 14-atom tether and effectively incorporated it into DNA by enzymatic polymerization. The synthesis was achieved using the Huisgen cycloaddition as the key reaction. We have demonstrated that DNA polymerase can effectively recognize the boronic acid-labeled DNA as the template for DNA polymerization, that allows PCR amplification of boronic acid-labeled DNA. DNA polymerase recognitions of the B-TTP as a substrate and the boronic acid-labeled DNA as a template are critical issues for the development of DNA-based lectin mimics via in vitro selection.


Assuntos
Compostos de Boro/síntese química , DNA/biossíntese , Nucleotídeos de Timina/síntese química , Aptâmeros de Nucleotídeos/química , Compostos de Boro/química , Compostos de Boro/metabolismo , DNA/química , Primers do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Reação em Cadeia da Polimerase , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Moldes Genéticos , Nucleotídeos de Timina/química , Nucleotídeos de Timina/metabolismo
16.
Nucleic Acids Symp Ser (Oxf) ; (50): 145-6, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17150859

RESUMO

Triphosphate of a thymidine analogue bearing acridone was prepared from a modified thymidine nucleotide with a terminal amino group at C5 position and an acridone derivative as a new fluorecsent-tagged nucleotide. The acridone-tagged nucleotide was incorporated into DNA enzymatically during PCR using KOD Dash DNA polymerase. Further, we introduced the acridone derivative into an amino-modified DNA chemically by post-synthetic modification, and investigated their fluorescence properties and hybridization ability. The new fluorescent-labeled DNA bearing acridone will be useful as a DNA probe.


Assuntos
Acridinas/química , Sondas de DNA/química , Corantes Fluorescentes/química , Nucleotídeos de Timina/química , Acridinas/síntese química , Reação em Cadeia da Polimerase , Temperatura , Nucleotídeos de Timina/síntese química
17.
Antivir Chem Chemother ; 17(4): 193-213, 2006.
Artigo em Inglês | MEDLINE | ID: mdl-17066898

RESUMO

With the view to deliver anti-HIV nucleoside and nucleoside-monophosphate (MP) analogues specifically into HIV-infected cells, we synthesized a series of ester and phosphoramidate peptide conjugates of zidovudine (AZT) and of AZT-MP, respectively, wherein the peptide sequences derive from a HIV-protease (PR) hydrolysable substrate. Their in vitro stability with respect to hydrolysis, anti-HIV activity and cytotoxicity, and ability to inhibit the HIV-PR activity were investigated. Concerning the ester AZT-peptide conjugates, their antiviral activity level in thymidine kinase-expressing (TK+) CEM-SS and MT-4 cells was in most cases closely correlated to their hydrolysis rate: the faster the hydrolysis, the closer the anti-HIV activity to that of AZT. None of them was a HIV-PR substrate, indicating that their antiviral activity was not related to their intracellular hydrolysis by this enzyme. None of them inhibited HIV in TK-deficient (TK-) CEM cells, demonstrating that they probably act as prodrugs of AZT. Most of the phosphoramidate peptide conjugates of AZT-MP were rapidly degraded in a physiological buffer into several metabolites including AZT. Their anti-HIV activity in TK+ CEM-SS and MT-4 cells was much lower than that of AZT, indicating that only low amounts of AZT or AZT-MP were released into cells during incubation. Antiviral activities measured on TK- CEM cells for some phosphoramidates suggest that low amounts of AZT-MP could be released intracellularly. However, this AZT-MP release was not initiated by a HIV-PR hydrolysis, as no evidence for peptide cleavage was obtained by HPLC analysis of one representative compound after incubation with HIV-PR.


Assuntos
Sistemas de Liberação de Medicamentos , Infecções por HIV/tratamento farmacológico , Protease de HIV/química , Pró-Fármacos , Nucleotídeos de Timina/síntese química , Nucleotídeos de Timina/uso terapêutico , Zidovudina/análogos & derivados , Zidovudina/síntese química , Zidovudina/uso terapêutico , Amidas/síntese química , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/uso terapêutico , Células Cultivadas , Didesoxinucleotídeos , Avaliação de Medicamentos , Estabilidade de Medicamentos , Ésteres/síntese química , Protease de HIV/efeitos dos fármacos , HIV-1/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Modelos Biológicos , Ácidos Fosfóricos/síntese química , Pró-Fármacos/síntese química , Pró-Fármacos/uso terapêutico , Inibidores de Proteases/farmacologia , Timidina Quinase/genética
18.
Biotechnol Bioeng ; 93(1): 21-7, 2006 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-16276532

RESUMO

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.


Assuntos
Proteínas de Bactérias/química , Enzimas/química , Açúcares de Nucleosídeo Difosfato/síntese química , Rhizobiaceae/enzimologia , Nucleotídeos de Timina/síntese química , Proteínas de Bactérias/genética , Enzimas/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Rhizobiaceae/genética
19.
Artigo em Inglês | MEDLINE | ID: mdl-15892257

RESUMO

3'-Azido-3',5-dideoxythymidine 5'-phosphonate and 3',5'-dideoxy-5'-difluoromethylenethymidine 5'-phosphonate were prepared by multistep syntheses. The nucleoside 5'-phosphonates were converted to their triphosphates and triphosphate mimics (P3Ms) containing beta,gamma-difluoromethylene, beta,gamma-dichloromethylene, or beta,gamma-imodo by condensation with pyrophosphate or pyrophosphate mimics, respectively. Inhibition of HIV-1 reverse transcriptase by the nucleoside P3Ms is briefly discussed.


Assuntos
Transcriptase Reversa do HIV/antagonistas & inibidores , Mimetismo Molecular , Inibidores da Transcriptase Reversa/síntese química , Inibidores da Transcriptase Reversa/farmacologia , Timidina/análogos & derivados , Nucleotídeos de Timina/síntese química , Zidovudina/análogos & derivados , Didesoxinucleotídeos , Estrutura Molecular , Inibidores da Transcriptase Reversa/química , Relação Estrutura-Atividade , Timidina/síntese química , Timidina/química , Timidina/farmacologia , Nucleotídeos de Timina/química , Nucleotídeos de Timina/farmacologia , Zidovudina/síntese química , Zidovudina/química , Zidovudina/farmacologia
20.
Biochemistry ; 43(15): 4548-58, 2004 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-15078101

RESUMO

The antitumor drug aclacinomycin A is a representative member of the anthracycline subgroup that contains a C(7)-O-trisaccharide chain composed of L-2-deoxysugars. The sugar portion of the molecule, which greatly affects its biological activity, is assembled by dedicated glycosyltransferases; however, these enzymes have not been well-studied. Here we report the heterologous expression and purification of one of these enzymes, AknK, as well as the preparation of dTDP-L-2-deoxysugar donors, dTDP-L-2-deoxyfucose and dTDP-L-daunosamine, and the monoglycosyl aglycone, rhodosaminyl aklavinone. Our experiments show that AknK catalyzes the addition of the second sugar to the chain, using dTDP-L-2-deoxyfucose and rhodosaminyl aklavinone, to create the L-2-deoxyfucosyl-L-rhodosaminyl aklavinone. AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones. Remarkably, AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. These results demonstrate that AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants.


Assuntos
Aclarubicina/biossíntese , Fucosiltransferases/isolamento & purificação , Streptomyces/enzimologia , Aclarubicina/análogos & derivados , Catálise , Clonagem Molecular , Fucose/análogos & derivados , Fucose/biossíntese , Fucose/química , Fucosiltransferases/biossíntese , Fucosiltransferases/genética , Glicosilação , Hexosaminas/biossíntese , Hexosaminas/química , Naftacenos/síntese química , Streptomyces/genética , Especificidade por Substrato , Nucleotídeos de Timina/síntese química , Trissacarídeos/química
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