system is described for solid-phase synthesis of custom peptide synthesis under continuous-flow conditions with liquid chromatographic equipment, conventional polystyrene supports, and well-defined chemistry. The model tetrapeptide Leu-Ala-Gly-Val was assembled in 99.3% purity in about 4 hr on microporous copoly(styrene-1% divinylbenzene).
During coupling, the preformed symmetric anhydrides were conserved by being recycled. Relative yields of the peptide products were determined quantitatively in 20 min by reverse-phase high-pressure liquid chromatography. This rapid assay system was used to examine the influence on product yields of (i) the time and number of couplings per cycle, (ii) microporous versus macroporous polystyrene, and (iii) tert-butoxycarbonyl (Boc) group versus 9-fluorenylmethoxycarbonyl for amine protection. Use of microporous polystyrene and two 30-min couplings of Boc-amino acids per cycle gave the best results. This continuous-flow system provides a rapid and efficient approach to solid-phase custom peptide synthesis. A 17-residue peptide from chicken ovalbumin was obtained in similar purity and yield from a discontinuous synthesis and from a continuous-flow synthesis.
Exemplified on fifty derived from proteins of the (), we summarize our experiences with rapid solid-phase multiple synthesis under low-pressure continuous-flow conditions on standard -based resin in a series of concatenated flow reactors with adjustable volume. Alternatively, multiple batchwise synthesis in plastic syringes may be used as a still simpler but a more elaborate procedure. Completeness of acylation of resin-bound free amino groups was monitored by the bromophenol blue. Efficiency of the coupling reaction was increased by exposure to elevated temperature in an ultrasonic bath. Up to 100 mg of crude were purified on a mass-overloaded Vydac reversed-phase semipreparative HPLC column in one run, this procedure ensuring high recovery of homogeneous . The synthesized were serologically relevant to the detection of anti-antibodies in positive sera.