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1.
Nucleic Acids Res ; 26(16): 3813-24, 1998 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-9685501

RESUMO

Oligodeoxynucleotide N3'-->P5' phosphoramidates are promising candidates for antisense therapeutics, as well as for diagnostic applications. We recently reported a new method for the synthesis of these oligonucleotide analogs which makes use of a phosphoramidite amine-exchange reaction in the key coupling step. We report herein an improved set of monomers that utilize a more reactive, hindered phosphoramidite to produce optimal yields in a single coupling step followed by oxidation, thereby eliminating the need for the previously reported couple-oxidize-couple-oxidize approach. On the 10 micromol scale, the synthesis is performed using only 3.6 equivalents (equiv.) of monomer. An improved oxidation reagent consisting of hydrogen peroxide, water, pyridine and THF is also introduced. Reported here for the first time is the use of a reverse-phase purification methodology employing a ribonucleotide purification handle that is removed under non-acidic conditions, in contrast to the conventional dimethoxytrityl group. The synthesis and purification of uniformly modified N3'-->P5' phosphoramidate oligodeoxy-nucleotides, as well as their chimera containing phosphodiester and/or phosphorothioate linkages at predefined positions, using these new methodologies are included herein. The results of31P NMR studies that led to this improved amine-exchange methodology are also described.


Assuntos
Oligonucleotídeos Antissenso/síntese química , Aminas/química , Sequência de Bases , Cromatografia Líquida de Alta Pressão/métodos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/isolamento & purificação , Compostos Organofosforados/síntese química , Compostos Organofosforados/química , Compostos Organofosforados/isolamento & purificação , Oxirredução
2.
Biochemistry ; 33(47): 14003-10, 1994 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-7947809

RESUMO

Tissue factor (TF) is a membrane-bound glycoprotein that functions as a cofactor for coagulation factor VIIa (VIIa) and initiates blood coagulation at sites of vascular injury. On the basis of sequence alignments, TF was predicted to be a member of the cytokine receptor superfamily. Utilizing the structural information available for the cytokine receptor superfamily, we have used site-directed mutagenesis to identify the binding site on TF for VIIa. The predicted loop regions in TF were systematically replaced with the homologous loops from the gamma-interferon receptor (gamma-IFN-R), the protein most related to TF in the superfamily of cytokine receptors. Six discontinuous regions (residues 16-20, 40-46, 60-69, 101-111, 129-151, 193-207) were identified that are required for interaction with VIIa and enhancement of activity. Individual substitution of 68 residues within these loops with alanine revealed eight residues (K20, D44, W45, K46, Q110, R135, F140, V207) that are required for cofactor activity. These residues fall into two groups, those that are required only for interactions with VIIa (K46, Q110, R135, F140, V207) and those that are also required to induce the conformational change in VIIa required for enhanced activity (K20, D44, W45). The discontinuous regions of TF required for interactions with VIIa form a single binding surface for VIIa that is analogous to the interface defined by the crystal structure of the complex between growth hormone and its receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Alanina/genética , Fator VIIa/metabolismo , Mutagênese Sítio-Dirigida , Tromboplastina/química , Sequência de Aminoácidos , Sítios de Ligação , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Interferon gama , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Conformação Proteica , Receptores de Interferon/química , Receptores de Interferon/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Relação Estrutura-Atividade , Tromboplastina/metabolismo
3.
J Biol Chem ; 268(28): 20808-11, 1993 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-8407909

RESUMO

A new class of thrombin inhibitors based on sequence-specific single-stranded DNA oligonucleotides (thrombin aptamer) has recently been identified. The aptamer-binding site on thrombin was examined by a solid-phase plate binding assay and by chemical modification. Binding assay results demonstrated that the thrombin aptamer bound specifically to alpha-thrombin but not to gamma-thrombin and that hirudin competed with aptamer binding, suggesting that thrombin's anion-binding exosite was important for aptamer-thrombin interactions. To identify lysine residues of thrombin that participated in the binding of the thrombin aptamer, thrombin was modified with fluorescein 5'-isothiocyanate in the presence or absence of the thrombin aptamer, reduced, carboxymethylated, and digested with endoproteinase Arg-C. The digestion products were analyzed by reversed-phase high performance liquid chromatography and the peptide maps compared. Four peptides with significantly decreased modification in the presence of the aptamer were identified and subjected to N-terminal sequence analysis. Results indicated that B chain Lys-21 and Lys-65, both located within the anion-binding exosite, are situated within or in close proximity to the aptamer-binding site of human alpha-thrombin. The thrombin aptamer binds to the anion-binding exosite and inhibits thrombin's function by competing with exosite binding substrates fibrinogen and the platelet thrombin receptor.


Assuntos
DNA de Cadeia Simples/metabolismo , Trombina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA de Cadeia Simples/química , Fluoresceína-5-Isotiocianato , Hirudinas/metabolismo , Humanos , Lisina/análise , Dados de Sequência Molecular
4.
Biochemistry ; 31(6): 1603-9, 1992 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-1737017

RESUMO

Triple-helix formation by oligodeoxynucleotides in a sequence-specific manner is limited to polypurine tracts of duplex DNA. To increase the number of biologically relevant targets for triple-helix formation, we have utilized oligodeoxynucleotides containing a 3'-3' internucleotide junction to allow for binding to opposite strands of duplex DNA. Molecular modeling was used to aid in the design of the xylose dinucleoside linker 1 that is rigid and minimizes the number of conformers to minimize the entropy of binding. Thermal denaturation studies show that a 3'-3'-linked oligodeoxynucleotide, bearing nine nucleotides on each side of the linker, has a higher Tm (47.6 degrees C) than that of a 21-mer binding to a single polypurine tract (45.3 degrees C). Binding domain minimization studies and sequence-specific alkylation of a target duplex demonstrate a high degree of cooperativity between the two triple-helix binding domains, thus allowing for an increase in the number of biologically relevant targets for triple-helix formation.


Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Composição de Bases , Sequência de Bases , Sítios de Ligação , Temperatura Alta , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/metabolismo , Purinas/metabolismo , Xilose/química
5.
Int J Pept Protein Res ; 36(4): 356-61, 1990 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-1964154

RESUMO

Insulin-like growth factor I (IGF-I), a protein of 70 amino acid residues and 3 cystine bridges, has been synthesized by two solid phase Boc methods. The first method used N-methylpyrrolidinone as the solvent with single coupling cycles while the second synthesis used dimethylformamide and dichloromethane as the solvents with a double-coupling protocol. In both cases, trifluoroacetic acid/trifluoromethanesulphonic acid cleavage of the peptide from the resin was employed. Purification of the cleavage products followed by removal of the S-acetamidomethyl protecting groups gave reduced peptides which were then oxidized under conditions favouring the formation of the correct disulphide bonds. The purified synthetic IGF-I peptides were full agonists of natural IGF-I in a radioimmunoassay, in an IGF-I radioreceptor assay, in a bioassay which measures the stimulation of protein synthesis in rat L6 myoblasts and in an IGF-binding protein competitive binding assay. Moreover, in each of these assays, the synthetic IGF peptides were found to be at least 70% as potent as natural IGF-I.


Assuntos
Fator de Crescimento Insulin-Like I/síntese química , Animais , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Indicadores e Reagentes , Fator de Crescimento Insulin-Like I/isolamento & purificação , Fator de Crescimento Insulin-Like I/metabolismo , Fator de Crescimento Insulin-Like I/farmacologia , Mesilatos , Pirrolidinonas , Receptores de Superfície Celular/metabolismo , Receptores de Somatomedina , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Resinas Vegetais , Solventes
6.
Pept Res ; 2(1): 147-52, 1989.
Artigo em Inglês | MEDLINE | ID: mdl-2577698

RESUMO

Fmoc-Cys(t-Bu)-OH, Fmoc-Cys(Acm)-OH, and Fmoc-Cys(Trt)-OH exhibit excellent synthesis characteristics when used in Fmoc solid phase peptide synthesis on the Applied Biosystems Model 431A peptide synthesizer. The actual 5% scavenger mixture will vary according to the particular amino acid residues present. As was previously mentioned, an anisole/ethanedithiol/ethylmethylsulfide mixture (3:1:1) works well as a general scavenger solution for TFA cleavage of Fmoc synthesized peptide resins. It also may be possible to use lower acid (TFA) concentrations. The syntheses and workups of the peptide Somatostatin utilizing these derivatives demonstrate the ease of using these cysteine derivatives with the Fmoc chemistry approach. The use of either the t-Bu or the Acm moiety produces a peptide containing protected thiol groups after cleavage with 95% TFA. The Fmoc-Cys(Trt)-OH derivative is efficiently deprotected using 95% TFA. This investigation should provide further insight into synthesis options and cleavage protocols when working with cysteine-containing peptides.


Assuntos
Cisteína/análogos & derivados , Fluorenos , Peptídeos/síntese química , Somatostatina/síntese química , Sequência de Aminoácidos , Indicadores e Reagentes , Dados de Sequência Molecular , Ninidrina
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