Klaus Schneider

Aspartimide formation in base-driven 9-fluorenylmethoxycarbonyl chemistry

Abstract Aspartimide and its piperidine adduct formed between -Asp-Asn- has been found during an Fmoc-based synthesis of an EGF-like domain in a blood coagulation factor. Model studies with seven susceptible -Asp-X- pentapeptides show that -Asp(Ot-Bu)-Asn(Trt)- and -Asp(Ot-Bu)-Gly- are the most problematic sequences.

Evidence That Formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) Complex Mediates Mast Cell Activation following FcϵRI/CC Chemokine Receptor 1 Cross-talk

Background: CC chemokine ligand 2 (CCL2) recruits leukocytes in inflammatory tissues. Results: Vimentin, a cytoskeletal protein, interacted with phosphorylated MAPKs, was critical for CCL2 production in mast cells activated via FϵcRI and a CC chemokine receptor. Conclusion: Vimentin was involved in optimal CCL2 production in mast cells. Significance: This work contributes to understanding of mechanisms for chemokine production in mast cells, which are therapeutic targets for allergic inflammation. Accumulating evidence points to cross-talk between FcϵRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcϵRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β′-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcϵRI and CCR1 activation.

Structure of chain d of the gigantic hemoglobin of the earthworm

The extracellular hemoglobin of the earthworm has four major O2-binding chains, a, b, c and d, together with additional non-heme structural chains that are required for assembly. Although the abc trimer self-associates extensively at least to (abc)10, addition of chain d results in the formation of a discrete 280 kDa complex corresponding to (abcd)4. Thus a primary function of chain d is to cap the abc association and convert an abc trimer that binds O2 with weak cooperativity to a highly cooperative (abcd)4 complex. Amino-acid sequences of the major globin chains a, b, c have been determined previously by peptide and cDNA analysis. However, the peptide sequence reported for the major chain d (Shishikura, F., Snow, J.W., Gotoh, T., Vinogradov, S.N. and Walz, D.A. (1987) J. Biol. Chem., 262. 3123-3131), has a calculated molecular mass 134-167 Da higher than masses for components of chain d determined by mass spectrometry (Owrby, D.W., Zhu, H., Schneider, K., Beavis, R.C., Chait, B.T. and Riggs, A.F. (1993) J. Biol. Chem. 268, 13539-13547). Reverse-phase HPLC confirms the presence of two distinct polypeptides, d1 and d2, together with d1, a variant of d1, cDNA derived amino acid sequences have been determined for chains d1 and d2 by application of the polymerase chain reaction with primers based on the NH2-terminal sequences and oligo-dT. Each of the two cDNA-derived sequences has 140 residues and they differ by 28 substitutions. The data show that the sequence originally reported had been derived from peptides generated from both polypeptides.

Characterization of a side reaction using stepwise detection in peptide synthesis with Fmoc chemistry

Publisher Summary Solid phase peptide synthesis almost always employs either Boc or Fmoc chemistry; Boc chemistry requires acidic conditions for deblocking, and the potential side reactions have been extensively studied; however, in Fmoc chemistry repetitive basic conditions are required for deblocking, and only a few of the base-catalyzed side reactions have been characterized. This chapter describes a method used to demonstrate that a side reaction well known in Boc chemistry, but thought not to occur under the conditions of Fmoc. Stepwise analysis is an efficient and direct method to monitor the progress of side reactions in Fmoc chemistry. The analysis involves stepwise micro-scale TFA cleavage in conjunction with HPLC and MS. Micro-scale TFA cleavage can be conveniently carried out on a small amount of sample. MS analysis of peptide ladders provides a rapid method for monitoring and identification of side reactions in peptide synthesis.