Atment of four with pivaloyloxymethyl iodide (POMI) (Bandgar et al. 2011) and diisopropylethyl amine (DIPEA) in DMF offered the corresponding bisPOM-protected intermediate (five). Hydrogenolytic removal of amino and carboxyl protecting groups and introduction of N-Fmoc-protection by treatment with 9-fluorenylmethyl succinimidyl carbonate (Fmoc-OSu) in aqueous THF with NaHCO3, gave the preferred reagent 2 (55 yield from 4) (Scheme 1).Amino Acids. Author manuscript; accessible in PMC 2014 November 01.Qian and BurkePageIn order to demonstrate the usefulness of two for the incorporation of Pmab(POM)two into peptides by solid-phase strategies, we chose the sequence, Ac-Pro-Leu-His-Ser-Pmabamide, which has previously shown to bind with fantastic affinity for the polo-like kinase 1 (Plk1) polo box domain (PBD) (Yun et al.Galiximab 2009; Liu et al. 2011; Liu et al. 2012b, a). PBDbinding ligands could potentially serve as anticancer agents by blocking the spatial organization essential 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 utilizing normal Fmoc protocols. Histidine and serine had been employed in their 4-methoxytrityl (Mtt) and trityl (Trt) side chain-protected forms, respectively, to let their cleavage below mildly acidic conditions (Scheme 2). 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 protecting groups and cleavage in the peptide in the resin with retention of Pmabbis-POM functionality. Purification by HPLC offered Ac-Pro-Leu-His-Ser-Pmab(POM)2amide (six) as a white solid (Scheme two). So that you can examine the bio-reversibility of your Pmab POM protection of six, we performed in vitro assays applying porcine liver estase (PLE) in phosphate buffer at pH 7.4 (Srivastva and Farquhar 1984). We observed that the parent Pmab bis-POM-containing six was swiftly converted to the mono-POM-containing item (t1/2 5 minutes) with additional deprotection towards the absolutely free Pmab-containing peptide occurring at a slower rate (20 conversion more than 8 h) (Fig. 2). Reduction within the rate of enzymatic cleavage of a second POM group is identified (Srivastva and Farquhar 1984).NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptAcConclusionsOur current paper presents the synthesis of a reagent [Fmoc-Pmab(POM)2-OH (two)] that makes it possible for the facile synthesis of peptides containing the phosphatase-stable pThr mimetic, Pmab, bearing bio-reversible POM protection.AUDA This represents a rare example of a reagent that permits the solid-phase synthesis of polypeptides obtaining a pThr mimetic in bioreversible prodrug form.PMID:24120168 Reagent 2 should locate utility inside a assortment of pharmacological applications.Supplementary MaterialRefer to Net version on PubMed Central for supplementary material.AcknowledgmentsThis analysis was supported by the Intramural Research System on the NIH, Center for Cancer Study, NCIFrederick and the National Cancer Institute, National Institutes of Health.AbbreviationsAcetyl Certain rotation Benzyl bromide[]D BnBr But Cbz CH2Cltert-ButylBenzyloxycarbonyl Dimethyl chlorideAmino Acids. Author manuscript; available in PMC 2014 November 01.Qian and BurkePageCH3CNAcetonitrile Diisopropylethylamine Dimethylformamide Electrospray ionization Ethyl acetate Difluorophosphonomethylphenylalanine 9-Fluorenylmethoxycarbonyl 9-Fluorenylmet.