p-cresol as a reversible acylium ion scavenger in solid-phase peptide synthesis

In this work we have defined the nature of the p-cresol and p-thiocresol adducts generated from acylium ions during HF cleavage, following contemporary Boc/benzyl solid-phase peptide synthesis. Contrary to the results in previous reports, we found that both p-cresol and p-thiocresol predominantly form. aryl esters under typical cleavage conditions. Initially we investigated a number of small peptides containing either a single glutamate residue or a C-terminal long-chain amino acid which allowed us to unambiguously characterize the scavenged side products. Whereas, the p-cresol esters are stable at 0 degrees C they rearrange irreversibly at higher temperatures (5-20 degrees C) to form aryl ketones. By contrast, p-thiocresol esters do not undergo a Fries rearrangement but readily undergo further additions of p-thiocresol to form ketenebisthioacetals and trithio ortho esters, even at low temperatures. Importantly, we found by LC/MS and FT-ICR MS analysis that peptides containing p-cresol esters at glutamyl side chains are susceptible to amidation and fragmentation reactions at these sites during standard mild base workup procedures. The significance of these side reactions was further demonstrated in the synthesis of neutrophil immobilization factor, a 26-residue peptide, containing four glutamic acid residues. The side reactions were largely avoided by mild hydrogen peroxide-catalyzed hydrolysis which converted the p-cresol adducts to the free carboxylic acids in near quantitative yield. The choice of p-cresol as a reversible acylium ion scavenger when coupled with the simple workup conditions described is broadly applicable to Boc/benzyl peptide synthesis and will significantly enhance the quality of peptides produced.

The chemistry of novolac resins .3. C-13 and N-15 nmr studies of curing with hexamethylenetetramine

The reactions between novolac resins and hexamethylenetetramine (HMTA) which occur on curing have been studied by C-13 and N-15 high-resolution n.m.r. in both solution and the solid state. Strong evidence for the existence of many curing intermediates is obtained. New curing intermediates are reported along with experimental data to support previously postulated intermediates. The initial curing reactions between novolac and HMTA produce various substituted benzoxazines and benzylamines. Thermal decomposition/oxidation and further reactions of these initial intermediates generate methylene linkages between phenolic rings for chain extension and cross-linking. Among the three kinds of methylene linkages, the para-para methylene linkages are formed at relatively lower temperatures. Various imine, amide and imide side-products also concurrently appear during the process. The initial amount of HMTA plays a critical role in the curing reactivity and chemical structures of the cured resins. The lower the amount of HMTA, the lower the temperature at which curing occurs, and the lower the amount of the nitrogen-containing side-products in the finally cured resins. The ortho-linked intermediates are relatively stable, and can remain in the cured resins up to higher temperatures. The study provides an extensive description of the curing reactions of novolac resins. (C) 1997 Elsevier Science Ltd.

Duck hepatitis B virus expresses a regulatory HBx-like protein from a hidden open reading frame

Duck hepatitis B viruses (DHBV), unlike mammalian hepadnaviruses, are thought to lack X genes, which encode transcription-regulatory proteins believed to contribute to the development of hepatocellular carcinoma. A lack of association of chronic DHBV infection with hepatocellular carcinoma development supports this belief. Here, we demonstrate that DHBV genomes have a hidden open reading frame from which a transcription-regulatory protein, designated DHBx, is expressed both in vitro and in vivo. We show that DHBx enhances neither viral protein expression, intracellular DNA synthesis, nor virion production when assayed in the full-length genome context in LMH cells. However, similar to mammalian hepadnavirus X proteins, DHBx activates cellular and viral promoters via the Raf-mitogen-activated protein kinase signaling pathway and localizes primarily in the cytoplasm. The functional similarities as,well as the weak sequence homologies of DHBx and the X proteins of mammalian hepadnaviruses strongly suggest a common ancestry of ortho- and avihepadnavirus X genes. In addition, our data disclose similar intracellular localization and transcription regulatory functions of the corresponding proteins, raise new questions as to their presumed role in hepatocarcinogenesis, and imply unique opportunities for deciphering of their still-enigmatic in vivo functions.

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis of peptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin (TCP-resin) with 1,4-phenylenediamine, subsequent coupling with desired amino acids by the standard Fmoc protocol, and oxidation of the intermediate para-aminoanilides (pAA) with Oxone(R). This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.