Design and synthesis of Fmoc-Thr[PO(OH)(OPOM)] for the preparation of peptide prodrugs containing phosphothreonine in fully protected form.

The design and efficient synthesis of N-Fmoc-phosphothreonine protected by a mono-(pivaloyloxy)methyl (POM) moiety at its phosphoryl group (Fmoc-Thr[PO(OH)(OPOM)]-OH, 1, is reported. This reagent is suitable for solid-phase syntheses employing acid-labile resins and Fmoc-based protocols. It allows the preparation of phosphothreonine (pThr)-containing peptides bearing bis-POM-phosphoryl protection. The methodology allows the first reported synthesis of pThr-containing polypeptides having bioreversible prodrug protection, and as such it should be useful in a variety of biological applications.

Investigation of unanticipated alkylation at the N(π) position of a histidyl residue under Mitsunobu conditions and synthesis of orthogonally protected histidine analogues.

We had previously reported that Mitsunobu-based introduction of alkyl substituents onto the imidazole N(π)-position of a key histidine residue in phosphothreonine-containing peptides can impart high binding affinity against the polo-box domain of polo-like kinase 1. Our current paper investigates the mechanism leading to this N(π)-alkylation and provides synthetic methodologies that permit the facile synthesis of histidine N(π)-modified peptides. These agents represent new and potentially important tools for biological studies.

General method for the synthesis of caged phosphopeptides: tools for the exploration of signal transduction pathways.

[reaction: see text] An interassembly approach for the synthesis of peptides containing 1-(2-nitrophenyl)ethyl-caged phosphoserine, -threonine, and -tyrosine has been developed. Photochemical uncaging of these peptides releases the 2-nitrophenylethyl protecting group to afford the corresponding phosphopeptide. The peptides described herein are based on phosphorylation sites of kinases involved in cell movement or cell cycle regulation and demonstrate the versatility of the method and compatibility with the synthesis of polypeptides, including a variety of encoded amino acids.

A new synthetic ether aminophosphoglyceride exhibits partial modulator activity towards the glucocorticoid receptor.

Modulator is an endogenous low-molecular weight regulator of both glucocorticoid and mineralocorticoid receptors, as well as protein kinase C. Analogs of the putative modulator structure have been synthesized. These compounds include 1-O-(3'-carboxypropyl) or (5'-carboxypentyl)-L-glycero-3-phospho-L-serine or L-threonine, and the D-glycerol stereoisomers. These compounds were tested for in vitro modulator activity using the glucocorticoid-receptor complex activation inhibition and steroid-binding stabilization assays. One of the ether phosphoglycerides, 1-O-(5'-carboxypentyl)-L-glycero-3-phospho-L-threonine (H-GPT-1), partially inhibited steroid-receptor complex activation in a dose-dependent manner. However, none of the other compounds exhibited any modulator activity towards the glucocorticoid-receptor complex. Like modulator, H-GPT-1 did not inhibit activated glucocorticoid-receptor complex binding to DNA-cellulose. Surprisingly, in contrast to modulator, H-GPT-1 partially inhibited unoccupied receptor steroid-binding in a dose-dependent manner. These results suggest that although modulator is not exactly mimicked by this compound, H-GPT-1 is the first synthetic organic molecule to exhibit some modulator activity towards the glucocorticoid receptor.

Synthesis of 7-mercaptoheptanoylthreonine phosphate and its activity in the methylcoenzyme M methylreductase system.

The structure of component B of the methylcoenzyme M methylreductase of Methanobacterium thermoautotrophicum was recently assigned as 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (Noll, K. M., Rinehart, K. L., Jr., Tanner, R.S., and Wolfe, R.S. (1986) (Proc. Natl. Acad. Sci. U.S.A. 83, 4238-4242). We report here the chemical synthesis and biochemical activity of this compound. Thiourea and 7-bromoheptanoic acid were used to to synthesize 7,7'-dithiodiheptanoic acid. This disulfide was then condensed with DL-threonine phosphate using N-hydroxysuccinimide and dicyclohexylcarbodiimide. The product was reduced with dithiothreitol to give HS-HTP. It could be oxidized in air in the presence of 2-mercaptoethanol to give the compound as it was isolated from cell extracts. The resulting product was identical to the authentic compound by 1H NMR spectroscopy, mass spectrometry, and coelution using high performance liquid chromatography. The synthetic compound is active in the in vitro methanogenic assay at concentrations comparable to the authentic compound. This confirms the structure of component B as HS-HTP and provides a means to synthesize quantities sufficient for studies of the methylreductase system.