Until recently the total synthesis of insulin, with its characteristic heterodimeric structure crosslinked by two interchain and one intrachain disulfide (SS) bridge, remained largely an unsolved challenge. By optimizing the synthesis and directed disulfide crosslinking of the two chains, and by applying biomimetic monocomponent proinsulin approaches, efficient insulin syntheses have been realized. Here we report the optimization and characterisation of an alternative strategy, oxidative native chain assembly. In this method unprotected A- and B-chains assemble oxidatively under thermodynamic control to afford bovine pancreatic insulin in 39% yield. Folding is found to proceed predominantly via structured 1SS* and 2SS* intermediates with a common interchain Cys A20 ‒Cys B19 disulfide. These results suggest that native chain assembly, long considered inefficient, may represent a reasonable strategy to access insulin variants. This is supported by the synthesis of human insulin and human type-II relaxin in yields of up to 49 and 47%, respectively, although the application to human insulin Val A16 variant is unsuccessful.

Background: Molecular imaging for detection of the atherosclerotic plaque burden has been highlighted as a modality for the diagnosis of atherosclerosis. We recently developed a novel and noninvasive positron emission tomography (PET) that was functionalized with an apolipoprotein (Apo) A-I mimetic peptide [known as Fukuoka University Apo A-I mimetic peptide (FAMP)] radiolabeled with gallium-68 (68Ga) - 1, 4, 7, 10-tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) to specifically image the status of atherosclerotic plaque in myocardial infarctionprone Watanabe heritable hyperlipidemic rabbits (WHHL-MI). Methods and Results: To achieve more sensitive molecular imaging, FAMP was modified with 4, 11 - bis (carboxymethyl) - 1, 4, 8, 11 - tetraazabicyclo (6.6.2) hexadecane (CB-TE2A) and radiolabeled with copper-64 (64Cu) for PET, and the ability of 64Cu-TE2A-FAMP to image plaque was compared with that of 68Ga-DOTA-FAMP. Japanese white normal (JW) and WHHL-MI rabbits were intravenously injected with 64Cu-CB-TE2A-FAMP or 68Ga- DOTA-FAMP, and subjected to continuous PET (25-30 MBq). Interestingly, 64Cu-CB-TE2A-FAMP was not taken up by atherosclerotic lesions in the aorta of WHHL-MI, whereas 68Ga-DOTA-FAMP was dramatically illuminated in the aorta of WHHL-MI. Moreover, 64Cu-CB-TE2A-FAMP was rapidly decomposed and 64Cu was excreted to the intestine, liver or urinary bladder in both JW and WHHL-MI rabbits. Conclusions: These results demonstrated that FAMP may be a target molecule for atherosclerotic molecular imaging with 68Ga-DOTA, but not with 64Cu-CB-TE2A. The selection of a suitable radio-nuclide and chelator might be important for HDL functioning imaging.

BACKGROUND:There have been no previous reports that apolipoprotein (apo) A-I mimetic peptide improves survival rate after myocardial infarction (MI).METHOD AND RESULTS:Male C57Bl/6J mice were subjected to left coronary artery permanent ligation as a model of MI. We synthesized a novel 24-amino acid apoA-I mimetic peptide-type5 (FAMP5), which potently removes cholesterol via specific ATP-binding cassette transporter A1 (ABCA1). FAMP5 was associated with a significantly improved survival rate by protecting against cardiac rupture compared to the control. mRNA levels for eNOS, Gata-4, CTGF and ANP were significantly increased in the hearts of the FAMP5-treated group, while that for MCP-1 decreased.CONCLUSION:This is the first report that high-density lipoprotein (HDL) therapy with FAMP5 improved the survival rate after MI.

Background: High-density lipoprotein (HDL) plays a major role in reverse cholesterol transport. Many researchers have been working to enhance the biochemical function of HDL for use in therapy. Although HDL therapy using injections of apolipoprotein (apo)-A-I mimetics, ape A-I (Milano) or full-length apo A-I is dramatically effective, it is still unclear whether apo A-I or ape A-I mimetics actually enter atherosclerotic plaque and remove cholesterol from the lipid burden. We synthesized a novel 24-amino acid apo A-I mimetic peptide (known as FAMP) that potently removes cholesterol via specific ATP-binding cassette transporter A1. We then investigated the potential of FAMP to image developing plaque lesions in vivo.Methods and Results: FAMP was modified with 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and radiolabeled with gallium-68 (Ga-68) for noninvasive positron emission tomography (PET) in an animal model (familial hypercholesterolemic myocardial infarction-prone rabbits: WHHL-MI) with atherosclerotic lesions. The Ga-68-DOTA-FAMP was dramatically taken up by atherosclerotic tissues in the blood vessels and aorta of WHHL-Ml rabbits, but not the control rabbits.Conclusions: An apo A-I mimetic peptide with Ga-68-DOTA is a promising candidate diagnostic tracer for PET imaging of the atherosclerotic lipid burden and may contribute to the development of a tool for the diagnosis of plaque with PET.

Background Apolipoprotein (apo) A‐I is a major high‐density lipoprotein (HDL) protein that causes cholesterol efflux from peripheral cells through the ATP‐binding cassette transporter A1 (ABCA1), thus generating HDL and reversing the macrophage foam cell phenotype. Pre‐β1 HDL is the smallest subfraction of HDL, which is believed to represent newly formed HDL, and it is the most active acceptor of free cholesterol. Furthermore it has a possible protective function against cardiovascular disease (CVD). We developed a novel apoA‐I mimetic peptide without phospholipids (Fukuoka University ApoA‐I Mimetic Peptide, FAMP). Methods and Results FAMP type 5 (FAMP5) had a high capacity for cholesterol efflux from A172 cells and mouse and human macrophages in vitro, and the efflux was mainly dependent on ABCA1 transporter. Incubation of FAMP5 with human HDL or whole plasma generated small HDL particles, and charged apoA‐I‐rich particles migrated as pre‐β HDL on agarose gel electrophoresis. Sixteen weeks of treatment with FAMP5 significantly suppressed aortic plaque formation (scrambled FAMP, 31.3±8.9% versus high‐dose FAMP5, 16.2±5.0%; P<0.01) and plasma C‐reactive protein and monocyte chemoattractant protein‐1 in apoE‐deficient mice fed a high‐fat diet. In addition, it significantly enhanced HDL‐mediated cholesterol efflux capacity from the mice. Conclusions A newly developed apoA‐I mimetic peptide, FAMP, has an antiatherosclerotic effect through the enhancement of the biological function of HDL. FAMP may have significant atheroprotective potential and prove to be a new therapeutic tool for CVD.

A water-soluble selenoxide (DHS(ox)) having a five-membered ring structure enables rapid and selective conversion of cysteinyl SH groups in a polypeptide chain into SS bonds in a wide pH and temperature range. It was previously demonstrated that the second-order rate constants for the SS formation with DHS(ox) would be proportional to the number of the free SH groups present in the substrate if there is no steric congestion around the SH groups. In the present study, kinetics of the SS formation with DHS(ox) was extensively studied at pH 4-10 and 25 °C by using reduced ribonuclease A, recombinant hirudin variant (CX-397), insulin A- and B-chains, and relaxin A-chain, which have two to eight cysteine residues, as polythiol substrates. The obtained rate constants showed stochastic SS formation behaviors under most conditions. However, the rate constants for CX-397 at pH 8.0 and 10.0 were not proportional to the number of the free SH groups, suggesting that the SS intermediate ensembles possess densely packed structures under weakly basic conditions. The high two-electron redox potential of DHS(ox) (375 mV at 25 °C) compared to l-cystine supported the high ability of DHS(ox) for SS formation in a polypeptide chain. Interestingly, the rate constants of the SS formation jumped up at a pH around the pK a value of the cysteinyl SH groups. The SS formation velocity was slightly decreased by addition of a denaturant due probably to the interaction between the denaturant and the peptide. The stochastic behaviors as well as the absolute values of the second-order rate constants in comparison to dithiothreitol (DTT(red)) are useful to probe the chemical reactivity and conformation, hence the folding, of polypeptide chains.

A series of cationic cyclic heptapeptides based on polymyxin B have been synthesized for use as permeabilizers of the outer membrane of Gram-negative bacteria. Only analogs with the Dab(2)-d-Phe(3)-Leu(4)-Xxx(5) sequence (Xxx = Dab or Orn) showed a synergistic bactericidal effect when combined with conventional antibiotics, indicating that the Dab(2) residue plays a critical role in permeation of the outer membrane of Gram-negative bacteria.

A series of Trp and Arg analogs of antibacterial indolicidin (Ind) was synthesized and the antimicrobial and hemolytic activities were investigated. [L(9)]Ind, [L(11)]Ind, [K(8),L(9)]Ind and [K(6, 8),L(9)]Ind showed desirable characteristics, exhibiting negligible hemolytic activity while keeping strong antibacterial activity. The results indicated that the Trp residue at position 11 essentially contributes to both activities and one can not be exchanged for the other, whereas the Trp residues at positions 4 and 9 play important roles in antimicrobial and hemolytic activities, respectively. The Trp residues at positions 6 and 8 play no important roles in biological activities. We then found that the retro analog of Ind showed higher antibacterial activity than Ind against both Gram-positive and Gram-negative bacteria but remarkably lower hemolytic activity than that of Ind.

Radical polymerization of styrene derivatives having a series of amino acid, alanine, glycine, leucine, valine, Bocleucine, and Boc-valine, in the side chain bound at the C-terminal was conducted to regulate the stereoinduction system in the propagation step. Isotacticity increased in the polymer main chain, especially in the polymerization of monomers bearing N-free L-leucyl and L-valyl esters in THF or DMF at 50 degrees C, by the synergic stereoregulation with chirality control and hydrogen bonding between the radical polymer terminal and the monomer. (C) 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 5593-5602, 2010