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1.
Bioconjug Chem ; 27(5): 1276-84, 2016 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-27098672

RESUMO

A disulfide-bridged peptide drug development candidate contained two oligopeptide chains with 11 and 12 natural amino acids joined by a disulfide bond at the N-terminal end. An efficient biotechnology based process for the production of the disulfide-bridged peptide was developed. Initially, the two individual oligopeptide chains were prepared separately by designing different fusion proteins and expressing them in recombinant E. coli. Enzymatic or chemical cleavage of the two fusion proteins provided the two individual oligopeptide chains which could be conjugated via disulfide bond by conventional chemical reaction to the disulfide-bridged peptide. A novel heterodimeric system to bring the two oligopeptide chains closer and induce disulfide bond formation was designed by taking advantage of the self-assembly of a leucine zipper system. The heterodimeric approach involved designing fusion proteins with the acidic and basic components of the leucine zipper, additional amino acids to optimize interaction between the individual chains, specific cleavage sites, specific tag to ensure separation, and two individual oligopeptide chains. Computer modeling was used to identify the nature and number of amino acid residue to be inserted between the leucine zipper and oligopeptides for optimum interaction. Cloning and expression in rec E. coli, fermentation, followed by cell disruption resulted in the formation of heterodimeric protein with the interchain disulfide bond. Separation of the desired heterodimeric protein, followed by specific cleavage at methionine by cyanogen bromide provided the disulfide-bridged peptide.


Assuntos
Biotecnologia , Dissulfetos/química , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Escherichia coli/genética , Modelos Moleculares , Peptídeos/genética , Multimerização Proteica , Estrutura Quaternária de Proteína
3.
J Org Chem ; 80(14): 7019-32, 2015 Jul 17.
Artigo em Inglês | MEDLINE | ID: mdl-26151079

RESUMO

Clopidogrel is a prodrug anticoagulant with active metabolites that irreversibly inhibit the platelet surface GPCR P2Y12 and thus inhibit platelet activation. However, gaining an understanding of patient response has been limited due to imprecise understanding of metabolite activity and stereochemistry, and a lack of acceptable analytes for quantifying in vivo metabolite formation. Methods for the production of all bioactive metabolites of clopidogrel, their stereochemical assignment, and the development of stable analytes via three conceptually orthogonal routes are disclosed.


Assuntos
Microssomos Hepáticos/metabolismo , Piperidinas/síntese química , Inibidores da Agregação de Plaquetas/síntese química , Inibidores da Agregação de Plaquetas/metabolismo , Pró-Fármacos/síntese química , Ticlopidina/análogos & derivados , Fenômenos Biológicos , Clopidogrel , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Piperidinas/química , Inibidores da Agregação de Plaquetas/química , Pró-Fármacos/química , Estereoisomerismo , Ticlopidina/síntese química , Ticlopidina/química , Ticlopidina/metabolismo
4.
Angew Chem Int Ed Engl ; 53(45): 12091-6, 2014 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-25244630

RESUMO

Physicochemical properties constitute a key factor for the success of a drug candidate. Whereas many strategies to improve the physicochemical properties of small heterocycle-type leads exist, complex hydrocarbon skeletons are more challenging to derivatize because of the absence of functional groups. A variety of C-H oxidation methods have been explored on the betulin skeleton to improve the solubility of this very bioactive, yet poorly water-soluble, natural product. Capitalizing on the innate reactivity of the molecule, as well as the few molecular handles present on the core, allowed oxidations at different positions across the pentacyclic structure. Enzymatic oxidations afforded several orthogonal oxidations to chemical methods. Solubility measurements showed an enhancement for many of the synthesized compounds.


Assuntos
Carbono/química , Hidrogênio/química , Produtos Biológicos/química , Oxirredução , Solubilidade
5.
J Ind Microbiol Biotechnol ; 39(12): 1789-99, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22926342

RESUMO

Biotransformation of the highly substituted pyridine derivative 2-amino-4-methyl-3-nitropyridine by Cunninghamella elegans ATCC 26269 yielded three products each with a molecular weight of 169 Da which were identified as 2-amino-5-hydroxy-4-methyl-3-nitropyridine, 2-amino-4-hydroxymethyl-3-nitropyridine, and 2-amino-4-methyl-3-nitropyridine-1-oxide. Biotransformation by Streptomyces antibioticus ATCC 14890 gave two different products each with a molecular weight of 169 Da; one was acid labile and converted to the other stable product under acidic conditions. The structure of the stable product was established as 2-amino-4-methyl-3-nitro-6(1H)-pyridinone, and that of the less stable product was assigned as its tautomer 2-amino-6-hydroxy-4-methyl-3-nitropyridine. Four of the five biotransformation products are new compounds. Several strains of Aspergillus also converted the same substrate to the lactam 2-amino-4-methyl-3-nitro-6(1H)-pyridinone. Microbial hydroxylation by C. elegans was found to be inhibited by sulfate ion. In order to improve the yield and productivity of the 5-hydroxylation reaction by C. elegans, critical process parameters were determined and Design of Experiments (DOE) analyses were performed. Biotransformation by C. elegans was scaled up to 15-l fermentors providing 2-amino-5-hydroxy-4-methyl-3-nitropyridine at ca. 13 % yield in multi-gram levels. A simple isolation process not requiring chromatography was developed to provide purified 2-amino-5-hydroxy-4-methyl-3-nitropyridine of excellent quality.


Assuntos
Cunninghamella/metabolismo , Piridinas/química , Piridinas/metabolismo , Streptomyces antibioticus/metabolismo , Aspergillus/isolamento & purificação , Aspergillus/metabolismo , Reatores Biológicos , Biotransformação , Cunninghamella/isolamento & purificação , Fermentação , Concentração de Íons de Hidrogênio , Hidroxilação , Peso Molecular , Óxidos/metabolismo , Piridinas/isolamento & purificação , Projetos de Pesquisa , Streptomyces antibioticus/isolamento & purificação
6.
J Nat Prod ; 71(3): 438-44, 2008 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-18327911

RESUMO

Bioassay-guided fractionation of extracts of various plants, marine organisms, and microorganisms has led to the discovery of new natural sources of a number of known compounds that have significant biological activity. The isolation of interesting and valuable cancer cell growth inhibitors including majusculamide C ( 1), axinastatin 5 ( 5), bengazoles A ( 6), B ( 7), and E ( 8), manzamine A ( 10), jaspamide ( 11), and neoechinulin A ( 19) has been summarized.


Assuntos
Antineoplásicos/farmacologia , Carbazóis/farmacologia , Depsipeptídeos/farmacologia , Alcaloides Indólicos/farmacologia , Oxazóis/farmacologia , Peptídeos Cíclicos/farmacologia , Piperazinas/farmacologia , Antineoplásicos/química , Antineoplásicos/isolamento & purificação , Carbazóis/química , Carbazóis/isolamento & purificação , Depsipeptídeos/química , Depsipeptídeos/isolamento & purificação , Ensaios de Seleção de Medicamentos Antitumorais , Alcaloides Indólicos/química , Alcaloides Indólicos/isolamento & purificação , Biologia Marinha , Estrutura Molecular , Oxazóis/química , Oxazóis/isolamento & purificação , Peptídeos Cíclicos/química , Peptídeos Cíclicos/isolamento & purificação , Piperazinas/química , Piperazinas/isolamento & purificação
7.
J Ind Microbiol Biotechnol ; 30(5): 252-9, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12687489

RESUMO

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates are in high demand by pharmaceutical industries for the preparation bulk drug substances. In this review article, microbial/enzymatic processes for the synthesis of chiral intermediates for antihypertensive drugs, melatonin receptor agonists, and beta3-receptor receptor agonists are described.


Assuntos
Agonistas de Receptores Adrenérgicos beta 3 , Agonistas Adrenérgicos beta/metabolismo , Anti-Hipertensivos/metabolismo , Enzimas/metabolismo , Receptores de Superfície Celular/agonistas , Receptores de Superfície Celular/metabolismo , Receptores Citoplasmáticos e Nucleares/agonistas , Receptores Citoplasmáticos e Nucleares/metabolismo , Biotransformação , Química Farmacêutica , Receptores de Melatonina , Estereoisomerismo
8.
J Nat Prod ; 65(12): 1886-91, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12502333

RESUMO

The bark of the giant neem tree Melia dubia was found to contain 11 euphane-type triterpenes. Five new compounds, meliastatins 1-5 (1-5), proved to inhibit growth of the P388 lymphocytic leukemia cell line (ED(50) 1.7-5.6 microg/mL). Four of the others, the previously known methyl kulonate (8), kulinone (9), 16-hydroxybutyrospermol (10), and kulactone (11), were also found to inhibit (ED(50) 2.5-6.2 microg/mL) the P388 cancer cell line. In addition, two new euphane triterpenes were isolated and named dubione A (6) and dubione B (7). Structures for each of the 11 euphane triterpenes were established by spectral techniques that included HRMS and 2D NMR.


Assuntos
Antineoplásicos Fitogênicos/isolamento & purificação , Melia/química , Plantas Medicinais/química , Triterpenos/isolamento & purificação , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Ensaios de Seleção de Medicamentos Antitumorais , Leucemia P388 , Camundongos , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Filipinas , Espectroscopia de Infravermelho com Transformada de Fourier , Estereoisomerismo , Triterpenos/química , Triterpenos/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacos
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