Sidues of cyt c, while the imidazole moiety on the acyl chain – protruding into the hydrophobic pocket – appeared to interact with the heme iron to lock the catalytic site and form a high affinity complex [6]. In this complex, H2O2 has no or little access to the heme catalytic site. Specific mitochondrial accumulation of 12-imidazole-substituted stearic acid (ISA) has been achieved by conjugating it with a lipophilic cation, triphenylphosphonium (TPP) [6, 18]. In line with the prediction, TPP-12-ISA suppressed peroxidase activity of cyt c/CL complexes with a prototypical phenolic substrate, Amplex Red, inhibited oxidation of bound TLCL, and, importantly, prevented irradiation-induced injury in vitro and in vivo [6]. However, molecular mechanisms and structural optimization of the inhibitory action of TPP-ISA on peroxidase activity or anti-apoptotic propensities, particularly with regards to the closest positioning of the imidazole functionality to heme-iron, have not been performed. In the current study, we designed and synthesized a group of TPP-ISA homologues with alternating position of the imidazole group at 6, 8, 10, 13 and 14th carbons, and experimentally tested their efficiency of interaction with cyt c heme-iron and anti-peroxidase inhibitory activity in model biochemical systems as well as their anti-apoptotic potential after exposure of mouse embryonic cells (MECs) to -irradiation. The proposed molecular mechanisms of inhibitory action of different TPP-ISA homologues were also studied by employing computational modeling.Author Manuscript Author ManuscriptReagentsMaterials and MethodsHorse heart cyt c (type C-7752, 95 ), diethylenetriaminepentaacetic acid (DTPA), H2O2, and fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St.Cetrorelix Acetate Louis, MO).Niraparib 1,2Dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,1,2,2-tetraoleoyl-cardiolipin (TOCL) were purchased from Avanti Polar Lipids (Alabaster, AL).PMID:24576999 Amplex Red (N-acetyl-3,7dihydroxyphenoxazine) was obtained from Life Technologies (Carlsbad, CA). Preparation of TPP-conjugated Imidazole Fatty Acids The initial syntheses of imidazole fatty acids were performed using the naturally occurring hydroxy-fatty acid, ricinoleic acid, and the reduced product 12-hydroxy-stearic acid [6]. The lack of abundant other naturally occurring hydroxy-acids meant that these materials had to be prepared from short chain precursors. TPP-6-ISA and TPP-8-ISA were synthesized from unsymmetrical ozonolysis of cyclohexene and cyclooctene respectively [19, 20]. TPP-10ISA can, in theory, be synthesized by the unsymmetrical ozonolysis of cyclodecene, but this starting material is very expensive. Rather, we started from 1,10-decanediol which was selectively monobenzylated [21], oxidized to the aldehyde with pyridinium chlorochromate in dichloromethane [22], chain extended with a Grignard reagent from 8-bromooctane, mesylated and substituted with imidazole as previously described [6]. Following debenzylation, oxidation provided the 10-imidazole stearic acid that was then conjugated as an ester with (3-hydroxypropyl)-triphenylphosphonium bromide as previously described. Full synthetic details are found in the supplementary materials. TPP-13-ISA was prepared starting with the monobromination of 1,12-dodecandiol, chain extension to the 17-Author Manuscript Author ManuscriptFree Radic Biol Med. Author manuscript; available in PMC 2015 June 01.Jiang et al.Pagebromoheptadecan-6-ol, and one-carbon homologation with sodium cyan.