RESUMO
Chlorinated analogues of Leu and Ile are incorporated during cell-free expression of peptides fused to protein, by exploiting the promiscuity of the natural biosynthetic machinery. They then act as sites for clean and efficient release of the peptides simply by brief heat treatment. Dehydro analogues of Leu and Ile are similarly incorporated as latent sites for peptide release through treatment with iodine under cold conditions. These protocols complement enzyme-catalyzed methods and have been used to prepare calcitonin, gastrin-releasing peptide, cholecystokinin-7, and prolactin-releasing peptide prohormones, as well as analogues substituted with unusual amino acids, thus illustrating their practical utility as alternatives to more traditional chemical peptide synthesis.
Assuntos
Aminoácidos/metabolismo , Técnicas de Química Sintética/métodos , Peptídeos/metabolismo , Sequência de Aminoácidos , Calcitonina/química , Calcitonina/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Sistema Livre de Células , Peptídeo Liberador de Gastrina/química , Peptídeo Liberador de Gastrina/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Hormônio Liberador de Prolactina/química , Hormônio Liberador de Prolactina/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genéticaRESUMO
The S30 extract from E. coli BL21 Star (DE3) used for cell-free protein synthesis removes a wide range of α-amino acid protecting groups by cleaving α-carboxyl hydrazides; methyl, benzyl, tert-butyl, and adamantyl esters; tert-butyl and adamantyl carboxamides; α-amino form-, acet-, trifluoroacet-, and benzamides; and side-chain hydrazides and esters. The free amino acids are produced and incorporated into a protein under standard conditions. This approach allows the deprotection of amino acids to be carried out in situ to avoid separate processing steps. The advantages of this approach are demonstrated by the efficient incorporation of the chemically intractable (S)-4-fluoroleucine, (S)-4,5-dehydroleucine, and (2S,3R)-4-chlorovaline into a protein through the direct use of their respective precursors, namely, (S)-4-fluoroleucine hydrazide, (S)-4,5-dehydroleucine hydrazide, and (2S,3R)-4-chlorovaline methyl ester. These results also show that the fluoro- and dehydroleucine and the chlorovaline are incorporated into a protein by the normal biosynthetic machinery as substitutes for leucine and isoleucine, respectively.
Assuntos
Aminoácidos/química , Escherichia coli/metabolismo , Aminoácidos/metabolismo , Catálise , Ésteres/química , Estrutura Molecular , Biossíntese de ProteínasRESUMO
A new class of cross-bridged cyclam-based macrocycles featuring phosphonate pendant groups has been developed. 1,4,8,11-tetraazacyclotetradecane-1,8-di(methanephosphonic acid) (CB-TE2P, 1) and 1,4,8,11-tetraazacyclotetradecane-1-(methanephosphonic acid)-8-(methanecarboxylic acid) (CB-TE1A1P, 2) have been synthesized and have been shown to readily form neutral copper(II) complexes at room temperature as the corresponding dianions. Both complexes showed high kinetic inertness to demetallation and crystal structures confirmed complete encapsulation of copper(II) ion within each macrocycle's cleft-like structure. Unprecedented for cross-bridged cyclam derivatives, both CB-TE2P (1) and CB-TE1A1P (2) can be radiolabeled with (64)Cu at room temperature in less than 1 h with specific activities >1 mCi µg(-1). The in vivo behavior of both (64)Cu-CB-TE2P and (64)Cu-CB-TE1A1P were investigated through biodistribution studies using healthy male Lewis rats. Both new compounds showed rapid clearance with similar or lower accumulation in non-target organs/tissues when compared to other copper chelators including CB-TE2A, NOTA and Diamsar.
Assuntos
Quelantes/química , Radioisótopos de Cobre/química , Cobre/química , Compostos Heterocíclicos/química , Compostos Organometálicos/síntese química , Compostos Organometálicos/farmacocinética , Organofosfonatos/química , Animais , Ácidos Carboxílicos/química , Cristalografia por Raios X , Marcação por Isótopo , Ligantes , Masculino , Modelos Moleculares , Conformação Molecular , Compostos Organometálicos/química , Teoria Quântica , RatosRESUMO
3-Chloro-Abu and 4-chloro-Nva are biosynthetically incorporated into E. coli peptidyl-Pro cis-trans isomerase B, as substitutes for Val and Leu, respectively. The extent of incorporation is up to ~90%, and substituted protein is catalytically active. By contrast, 4-chloro-Val is not an effective replacement for Ile.
Assuntos
Aminoácidos/química , Cloro/química , Escherichia coli/enzimologia , Ácidos Graxos/química , Peptidilprolil Isomerase/biossíntese , Sequência de Aminoácidos , Biocatálise , Domínio Catalítico , Sistema Livre de Células , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel de Poliacrilamida , Leucina/química , Leucina/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Peptidilprolil Isomerase/química , Valina/química , Valina/metabolismoRESUMO
A phosphonate pendant-armed cross-bridged cyclam chelator has been synthesized, complexed to Cu(ii), radiolabeled with (64)Cu under mild conditions, and its biodistribution studied.
Assuntos
Aminas/química , Quelantes/farmacocinética , Cobre/química , Organofosfonatos/química , Compostos Radiofarmacêuticos/farmacocinética , Sítios de Ligação , Quelantes/síntese química , Quelantes/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Molecular , Radioisótopos/química , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/química , EstereoisomerismoRESUMO
Biodegradable polyesters represent an important class of materials, and one subset of these polymers are the bacterially produced poly(3-hydroxyalkanoate)s (PHA), a bacterially produced material. These polymers are very hydrophobic, and chemical methods to increase their hydrophilicity will ultimately lead to new applications. Many copolymers of PHA are known that contain simple, nonpolar functionality in the side chain, and we explored the conversion of side-chain olefins to carboxylic acids under conditions that minimize molecular weight degradation. With the use of osmium tetraoxide and oxone, the conversion proceeded to completion with little backbone degradation, which was confirmed with NMR, IR, and gel permeation chromatography (GPC). The solubility character of the polymer before and after reaction is very different, and several solvents were explored including acetone, tetrahydrofuran (THF), and water.