微波合成：天冬酰亚胺形成研究

Application Note BIO-0002

Microwave Synthesis: Aspartimide Formation Studies

INTRODUCTION

VIP (vasoactive intestinal peptide), a peptide hormone, has been implicated in a number of biological processes including: increasing digestive system motility, stimulating pepsinogen secretion, controlling the mammalian circadian timekeeping machinery, regulating prolactin secretion, and causing vasodilation. One problem with synthesis of VIP is aspartimide formation during Fmoc deprotection. Aspartimide formation occurs most frequently in peptides containing Asp followed by Asn(Trt), Gly, Thr, or Ser. The use of 5% piperazine with 0.1 M HOBt for the deprotection solution can readily reduce the amount of aspartimide formation during microwave peptide synthesis providing the VIP peptide in high crude purity compared to the conventional method.

Figure 1: Aspartimide Formation

MATERIALS AND METHODS Reagents

All Fmoc amino acids, O -(Benzotriazol-1-yl)-N ,N ,N ’,N ’-tetramethyluronium

hexafluorophosphate (HBTU) and 1-hydroxybenztriazole hydrate (HOBt) were obtained from CEM Corporation. Rink Amide MBHA resin was obtained from Novabiochem (San Diego, CA).

Diisopropylethylamine (DIEA), piperazine,

trifluoroacetic acid (TFA), triisopropylsilane (TIS), and 3,6-dioxa-1,8-octanedithiol (DODT) were obtained from Sigma Aldrich (St. Louis, MO). Dichloromethane (DCM), N ,N -dimethylformamide (DMF), N -methylpyrrolidone (NMP), anhydrous diethyl ether, acetic acid, HPLC grade water and acetonitrile were obtained from VWR (West Chester, PA).

Peptide Synthesis:

HSDAVFTDNYTRLRKQMAVKKYLNSILN-NH 2 The peptide was prepared using the CEM Liberty automated microwave peptide synthesizer on 0.278 g of Rink Amide MBHA resin (0.36 meq/g substitution). Deprotection was performed in two stages with an initial deprotection of 30 s followed by 3 min at 75 °C (5 min followed by 10 min at 25 °C for conventional synthesis). Fresh reagents were used each time with one of the two solutions: (i) 5% piperazine in DMF; or (ii) 5% piperazine with 0.1 M HOBt in DMF. Coupling reactions were performed with a 5 fold excess of Fmoc-AA-OH with 1:0.9:2 AA/HBTU/DIEA for 5 min at 75 °C (30 min at 25 °C for conventional synthesis). Cleavage was performed using 92.5:2.5:2.5:2.5 TFA/H 2O/TIS/DODT for 40 min at 38 °C (4 hours at 25 °C for conventional synthesis). Following cleavage the peptide was precipitated and washed in diethyl ether. Peptide Analysis

The peptide was analyzed on a Waters Atlantis C 18 column (2.1 ×150 mm) at 214

Application Note BIO-0002

nm with a gradient of 5 - 95% MeCN (0.1% formic acid), 0 – 20 min. Mass analysis was performed using an LCQ Advantage ion trap mass spectrometer with electrospray ionization (Thermo Electron, Waltham, MA).

RESULTS

Microwave synthesis of the peptide without HOBt in the deprotection solution has a crude purity of 27% due to the formation of significant amounts of the aspartimide side product (Figure 2). Microwave synthesis with 0.1 M HOBt in the deprotection solution increased the crude purity to 80% (Figure 3). As a comparison, conventional synthesis of the peptide with 0.1 M HOBt in

the deprotection solution has a crude purity of

only 45% (Figure 4). The conventional synthesis was completed in 35 hours while the microwave enhanced synthesis was completed in only 17 hours.

CONCLUSION

The use of 5% piperazine with 0.1 M HOBt for the deprotection solution can readily reduce the amount of aspartimide formation during microwave peptide synthesis. This optimized method for microwave synthesis improves the crude purity of the peptide and the rate of synthesis.