Tandem ligation at X-Cys and Gly-Gly positions via an orthogonally protected auxiliary group.

4,5-dimethoxy-2-mercaptobenzylamine (Dmmb) has been protected by acetamidomethyl (Acm) and incorporated into a peptide thioester for use in tandem native chemical ligation. Upon ligation between the thioester and a Cys-peptide, Acm was removed from Dmmb using silver acetate, and a second ligation reaction was done at the Dmmb position. Dmmb removal using TFMSA-TFA effected overall tandem ligation at X-Cys and Gly-Gly.

Assembly of high-affinity insulin receptor agonists and antagonists from peptide building blocks.

Insulin is thought to elicit its effects by crosslinking the two extracellular alpha-subunits of its receptor, thereby inducing a conformational change in the receptor, which activates the intracellular tyrosine kinase signaling cascade. Previously we identified a series of peptides binding to two discrete hotspots on the insulin receptor. Here we show that covalent linkage of such peptides into homodimers or heterodimers results in insulin agonists or antagonists, depending on how the peptides are linked. An optimized agonist has been shown, both in vitro and in vivo, to have a potency close to that of insulin itself. The ability to construct such peptide derivatives may offer a path for developing agonists or antagonists for treatment of a wide variety of diseases.

Peptides identify the critical hotspots involved in the biological activation of the insulin receptor.

We used phage display to generate surrogate peptides that define the hotspots involved in protein-protein interaction between insulin and the insulin receptor. All of the peptides competed for insulin binding and had affinity constants in the high nanomolar to low micromolar range. Based on competition studies, peptides were grouped into non-overlapping Sites 1, 2, or 3. Some Site 1 peptides were able to activate the tyrosine kinase activity of the insulin receptor and act as agonists in the insulin-dependent fat cell assay, suggesting that Site 1 marks the hotspot involved in insulin-induced activation of the insulin receptor. On the other hand, Site 2 and 3 peptides were found to act as antagonists in the phosphorylation and fat cell assays. These data show that a peptide display can be used to define the molecular architecture of a receptor and to identify the critical regions required for biological activity in a site-directed manner.

Synthesis and application of peptide dendrimers as protein mimetics.

The multiple antigenic peptide (MAP) is a novel approach to preparing peptide immunogens. The MAP consists of an inner core matrix built up of a large layer of Lys residues and a surface of peptide chains attached to the core matrix. Because of its dendrimeric structure, MAP can be very useful as a template for assembling potential peptide surfaces. Two methods for preparing MAP systems are described in this unit. The direct approach for preparing MAP systems is presented in the first two protocols, including the procedure for b-butyloxycarbonyl (Boc) chemistry and the procedure for 9 -fluorenylmethyloxycarbonyl (Fmoc) chemistry. An indirect approach for preparing MAP systems, in which peptide and core matrix are synthesized separately and conjugated by several ligation methods, is then described. The cMAP approach is also executed using either the direct or indirect approach, but requires an additional cyclization step to constrain the peptides after synthesis. The synthesis of cMAP is described, and the preparation of cyclic peptides is illustrated. A support protocol describes the ninhydrin assay to assess the completeness of the coupling reaction. In most cases, MAP systems can be used directly after simple dialysis or desalting. Some immunological studies, however, require purified MAPs. Additional support protocols describe MAP system purification by dialysis and high-performance gel-filtration chromatography.