Efficient microwave-assisted synthesis of human islet amyloid polypeptide designed to facilitate the specific incorporation of labeled amino acids

Abstract

A cost-efficient, time-reducing solid-phase synthesis of the amyloidogenic, 37 residue islet amyloid polypeptide (IAPP) is developed using two pseudoprolines (highlighted blue in sequence) in combination with microwave technology. A yield twice that obtained with conventional syntheses is realized. The utility of this protocol is demonstrated by the synthesis of a (13)C(18)O-labeled Ser-20 IAPP variant, a prohibitively expensive and chemically challenging site to label via other protocols. TEM analysis shows the peptide forms normal amyloid (abstract image).

Figure 1

Primary sequence of IAPP, illustrating the coupling scheme used for the microwave solid-phase peptide synthesis. Residues that were double coupled are underlined. The pseudoproline residues are colored blue, and all other double coupled residues are underlined.

Figure 2

(a) Reversed-phase analytical HPLC trace and (b) MALDI spectrum of crude, reduced IAPP. The HPLC was run on a C18 Vydac analytical column at a flow rate of 1 mL/min with a gradient of 0–90% buffer B (80% acetonitrile, 0.045% HCl) over 90 min.

Figure 3

(a) Reversed-phase analytical HPLC trace and (b) MALDI mass spectrum of pure, oxidized IAPP. The HPLC was run on a C18 Vydac analytical column at a gradient of 0–90% buffer-B (80% acetonitrile, 0.045% HCl) over 90 min. The pure peptide elutes at 48 min, corresponding to 48% buffer-B.

Figure 4

Thioflavin-T monitored kinetics of IAPP synthesized by microwave (red) and conventional (black) synthesis.