Peptide Synthesis through Cell-Free Expression of Fusion Proteins Incorporating Modified Amino Acids as Latent Cleavage Sites for Peptide Release.

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.

In situ deprotection and incorporation of unnatural amino acids during cell-free protein synthesis.

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.

Synthesis, Cu(II) complexation, 64Cu-labeling and biological evaluation of cross-bridged cyclam chelators with phosphonate pendant arms.

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.

Incorporation of chlorinated analogues of aliphatic amino acids during cell-free protein synthesis.

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.

A new phosphonate pendant-armed cross-bridged tetraamine chelator accelerates copper(ii) binding for radiopharmaceutical applications.

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.

Poly(3-hydroxyalkanoate)s functionalized with carboxylic acid groups in the side chain.

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.