Combined solid‐phase and solution approach for the synthesis of large peptides or proteins
In the synthesis of large peptides or proteins, highly homogeneous segments are indispensable for a convergent strategy either on a solid‐phase resin or in solution. Employing Boc/Bzl chemistry to prepare fully protected segments with a free α‐carboxyl group from the solid support, base‐labile linkers are profitable for practical peptide synthesis since they require no special equipment. For this purpose, an N‐[9‐(hydroxymethyl)‐2‐fluorenyl]succinamic acid (HMFS) linker was adopted. Consequently, there must be high compatibility between the protecting groups of the segment and the anchoring group which is cleavable by treatment with morpholine or piperidine in DMF. Instead of using the 2‐bromobenzyloxycarbonyl (BrZ) group for the Tyr residue and the formyl (For) group for the Trp residue, both of which are the most susceptible protecting groups under these base‐catalysed conditions, the base‐resistant 3‐pentyl (Pen) and cyclohexyloxycarbonyl (Hoc) groups were introduced to the respective side‐chain functional groups. By applying the present strategy, the authors were able to rapidly synthesize homogeneous protected segments for use in the subsequent segment coupling in solution. In the present paper, the utility of the combined solid‐phase and solution approach is demonstrated by synthesizing muscarinic toxin 1 (MTX1) which binds to the muscarinic acetylcholine receptors. Copyright © 2000 European Peptide Society and John Wiley & Sons, Ltd.
Chemical synthesis of kurtoxin, a T-type calcium channel blocker
Kurtoxin isolated from the venom of scorpion,Parabuthus transvaalicus, is a 63-residue peptide with four intramolecular disulfide bonds which inhibits low-threshold T-type Ca2+ channels. Kurtoxin was synthesized by native chemical ligation involving the coupling of (1–26)-thioester peptide and Cys27-(28–63)-peptide. The former was synthesized by standard solid-phase peptide synthesis (SPPS) with Boc chemistry, while the latter was sequentially assembled from three protected segments onto a resin-bound C-terminal segment in a chloroform-phenol mixed solvent followed by deprotection reaction using HF. Each protected segment used for the coupling on a solid support was prepared on anN-[9-(hydroxymethyl)-2-fluorenyl] succinamic acid (HMFS) resin and detached from the resin by treatment with 20% Et3N in DMF to produce it in the form of an α-carboxylic acid. Synthetic kurtoxin obtained after the oxidative folding reaction was found to be identical with the natural product by means of several analytical procedures, and its disulfide structure was determined for the first time to be Cys12-Cys61, Cys16-Cys37, Cys23-Cys44 and Cys27-Cys46 by peptide mapping, sequence analysis and mass measurements.
Monitoring and Optimization of Internal Grinding Process
Summary The grinding process is influenced by many factors such as grinding wheel characteristics, dressing conditions and, course, grinding conditions. Therefore, it is difficult to perform the grinding operation in an optimum state. In this study, in-process monitoring methods using power and acoustics emission sensors are proposed to detect malfunctions in the internal grinding process. In addition, a new internal grinding cycle featuring rapid indeed is proposed to minimize the grinding cycle time.