Chemoenzymatic Posttranslational Modification Reactions for the Synthesis of Ψ[CH2 NH]-Containing Peptides.

The Ψ[CH2 NH] reduced amide bond is a peptide isostere widely used in the development of bioactive pseudopeptides. Reported here is a method of chemoenzymatic posttranslational modification for the synthesis of Ψ[CH2 NH]-containing peptides converted from ribosomally expressed peptides. The posttranslational conversion composed of an enzymatic cyclodehydration and facile two-step chemical reduction achieves deoxygenation of a specific amide bond present in a nonprotected peptide in water. This method generates the Ψ[CH2 NH] bond in peptides and is applicable to various peptide sequences, potentially enabling the preparation of a library of Ψ[CH2 NH]-containing peptides.

One-pot synthesis of azoline-containing peptides in a cell-free translation system integrated with a posttranslational cyclodehydratase.

Azoline moieties in the backbones of peptidic natural products are important structural motifs that contribute to diverse bioactivities. Some azoline-containing peptides (Az-peptides) are produced from ribosomally synthesized precursor peptides, in which cysteine, serine, and threonine residues are converted to their corresponding azolines by posttranslational modification through a cyclodehydratase. We have devised an in vitro biosynthesis system of Az-peptides, referred to as the FIT-PatD (flexible in vitro translation) system, by the integration of a cell-free translation system with the posttranslational cyclodehydratase PatD. This system enabled the "one-pot" synthesis of a wide variety of Az-peptide derivatives expressed from synthetic DNA templates. The FIT-PatD system also facilitated mutagenesis studies on a wide array of precursor peptide sequences, unveiling unique in vitro substrate tolerance of PatD.

Systemic delivery systems of angiogenic gene by novel bubble liposomes containing cationic lipid and ultrasound exposure.

Recently, we developed polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping ultrasound (US) gas and reported that the combination of BL and US exposure was an effective tool for the delivery of pDNA directly into skeletal muscles of an ischemic hindlimb model with local injection. To achieve gene delivery to deeper tissues, we attempted to prepare novel Bubble liposomes which were able to be loaded with pDNA and useful for systemic injection. We prepared BLs using cationic lipid and analyzed the interaction with the BLs and pDNA using flow cytometry. The solution of pDNA-loaded BLs (p-BLs) was further injected into the tail vein of hindlimb ischemia model mice, and transdermal US exposure was applied to ischemic hindlimb. The effects of transfection on angiogenic factors were investigated by real-time PCR. Blood flow was determined using a laser Doppler blood flow meter. The interaction with BLs and pDNA increased in the presence of DOTAP and short PEG chains and resulted in increased stability of pDNA in the serum. Transfection with pDNA encoding the bFGF gene using p-BLs and US induced various angiogenic factors and improved the blood flow. The gene delivery system into the ischemic hindlimb using the combination of p-BLs and US exposure could be an effective tool for angiogenic gene therapy via systemic injection.

Efficient siRNA delivery using novel siRNA-loaded Bubble liposomes and ultrasound.

Recently, we developed novel polyethyleneglycol (PEG)-modified liposomes (Bubble liposomes; BLs) entrapping an ultrasound (US) imaging gas and reported that the combination of BLs and US was useful for the delivery of siRNA directly into the cytoplasm. However, the results were obtained using a mixture of BLs and naked siRNA. With systemic injections, it is important to control the biodistribution of both BLs and siRNA. In addition, the delivery of siRNA is affected by nuclease degradation after intravenous administration. In this study, we prepared novel siRNA-loaded BLs (si-BLs) using a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). We demonstrated that siRNA could be loaded onto BLs containing DOTAP and that siRNA-loaded BLs were stable in serum. A specific gene-silencing effect was also achieved by transfection with si-BLs. Thus, the combination of si-BLs with US exposure can be used for delivery of siRNA to a specific tissue via systemic injection.

Design and evaluation of new antipsoriatic antedrug candidates having 16-en-22-oxa-vitamin D3 structures.

Design, synthesis, and in vitro and in vivo evaluation of a series of antipsoriatic antedrugs having 16-en-22-oxa-vitamin D3 are described. Among the seven compounds examined, two are promising: ester 5c and amide 5f, both of which exhibit greater potent antiproliferation activity with lessened calcemic activity than the presently prescribed maxacalcitol (2).

Novel vitamin D3 antipsoriatic antedrugs: 16-En-22-oxa-1alpha,25-(OH)2D3 analogs.

A series of 16-en-22-oxa-derivatives of vitamin D3 based on the structure of maxacalcitol (2) were prepared. Maxacalcitol is currently used topically for the treatment of psoriasis and is recognized as the most successful antedrug of natural vitamin D(3) because it retains the original antiproliferative activity of calcitriol without increased calcemic activity. We introduced 16-olefinic functionality to accelerate the oxidative metabolism of the drug in liver, presumed to be essential for the reduction of calcemic activity, and modified the side-chain moiety by placing the 22-oxygen on the more labile allylic carbon center. Novel 22-oxa analogs (7a-i), carrying either the 24-alkynyl bond or 24-hydroxy functionality in addition to the 16-double bond were synthesized and their pharmacokinetics were evaluated.

Design and synthesis of 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols as a novel series of antiatherogenic antioxidants.

Antioxidants have been considered as potential antiatherogenic agents by inhibiting oxidation of low-density lipoprotein (LDL), albeit vitamin E, a natural antioxidant, has failed to show reduction on atherosclerosis in clinical trials. We have rationally designed and synthesized a novel series of antioxidants, 4,6-di-tert-butyl-2,3-dihydro-5-benzofuranols, to overcome the clinical limitation of vitamin E. In vitro, the compounds showed a potent inhibitory effect on lipid peroxidation detected as 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1,2-a]pyrazin-3-one (MCLA)-dependent chemiluminescence in linoleic acid autoxidation. They also inhibited the LDL oxidation induced by Cu(2+), and the inhibition is more potent than that of vitamin E and probucol. In vivo, 4,6-di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653, 1f), an optimal compound, showed the highest concentration in plasma and LDL fraction in Watanabe heritable hyperlipidemic rabbits, due to its high affinity to LDL. The isolated LDL samples from the 1f-treated rabbits showed potent resistibility to LDL oxidation. Compound 1f has been taken into clinical trials.