Liver esterase (57.six units) as well as the reaction mixture was incubated at 37 with gentle agitation. At numerous time points, aliquots in the reaction mixture (50 … L) had been transferred to an Eppendorf tube containing MeCN (50 … L). Following filtration, the hydrolysis solution was monitored by LC-MS. Data are shown in Fig. 2.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptResults and discussionPreparation of reagent two started with Cbz-Pmab(But2)-OMe (3), which is an intermediate in our previously reported synthesis of 1 (Liu et al. 2009). Benzyl trans-esterification of 3 to 4 was accomplished by initial LiOH-mediated hydrolysis with the methyl ester followed by reaction of the free acid with benzyl bromide (NaHCO3 in DMF, 80 yield for two steps, Scheme 1). Subsequent therapy of four with pivaloyloxymethyl iodide (POMI) (Bandgar et al. 2011) and diisopropylethyl amine (DIPEA) in DMF supplied the corresponding bisPOM-protected intermediate (5). Hydrogenolytic removal of amino and carboxyl defending groups and introduction of N-Fmoc-protection by treatment with 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu) in aqueous THF with NaHCO3, gave the desired reagent two (55 yield from 4) (Scheme 1).Amino Acids. Author manuscript; offered in PMC 2014 November 01.Qian and BurkePageIn order to demonstrate the usefulness of 2 for the incorporation of Pmab(POM)2 into peptides by solid-phase approaches, we chose the sequence, Ac-Pro-Leu-His-Ser-Pmabamide, which has previously shown to bind with excellent affinity for the polo-like kinase 1 (Plk1) polo box domain (PBD) (Yun et al. 2009; Liu et al. 2011; Liu et al. 2012b, a). PBDbinding ligands could potentially serve as anticancer agents by blocking the spatial organization expected for Plk1 to function in oncogenic processes (van de Weerdt et al. 2008). The syntheses of Ac-Pro-Leu-His-Ser-Pmab(POM)2-amide (six) was accomplished on NovaSyn TG Sieber resin working with common Fmoc protocols. Histidine and serine have been employed in their 4-methoxytrityl (Mtt) and trityl (Trt) side chain-protected types, respectively, to enable their cleavage beneath mildly acidic conditions (Scheme two). Following synthesis completion, the resin-bound Ac-Pro-Leu-His(Mtt)-Ser(Trt)-Pmab(POM)2-amide (7) was subjected to remedy with 1 TFA, which resulted in removal of histidine and serine defending groups and cleavage from the peptide from the resin with retention of Pmabbis-POM functionality. Purification by HPLC provided Ac-Pro-Leu-His-Ser-Pmab(POM)2amide (6) as a white solid (Scheme two).Silver(I) trifluoromethanethiolate web So as to examine the bio-reversibility of the Pmab POM protection of 6, we performed in vitro assays using porcine liver estase (PLE) in phosphate buffer at pH 7.Formula of 2653202-15-2 4 (Srivastva and Farquhar 1984).PMID:27017949 We observed that the parent Pmab bis-POM-containing 6 was rapidly converted for the mono-POM-containing product (t1/2 5 minutes) with further deprotection for the free of charge Pmab-containing peptide occurring at a slower price (20 conversion over eight h) (Fig. 2). Reduction in the price of enzymatic cleavage of a second POM group is recognized (Srivastva and Farquhar 1984).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAcConclusionsOur present paper presents the synthesis of a reagent [Fmoc-Pmab(POM)2-OH (two)] that enables the facile synthesis of peptides containing the phosphatase-stable pThr mimetic, Pmab, bearing bio-reversible POM protection. This represents a rare instance of a reagent that permits the solid-phase synthesis o.