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Table 1. Synthetic protocol in Liberty Peptide SynthesizerParametersCoupling of AminoacidsCoupling of Fmoc-Arg(Pbf)-OHDeprotection10 mL x 10 min), DMF (3 x 10 mL), i-Pro (2 x 10 mL), n-hexane (2 x 10 mL) and dried invacuo for 24 h at RT. The loading of the amino acid/g of substituted resin was 0.7 mmol/g,calculated by weight and amino acid analysis. Removal of Fmoc group was achieved by therepetitive treatment with piperidine/DMF (1:4, 10 mL) for 10 and 20 min and the resin wasthoroughly washed with DMF (3 x 10 mL), i-Pro (2 x 10 mL), n-hexane (2 x 10 mL) [3,4].3. Esterification of Fmoc-Leu-OH on Wang resin: Wang resin (0.3 g, 0.6 mmol OH-/g ofresin) was left to swell in dry DMF (5 ml) for 10 min. In a separate flask were dissolvedN-Fmoc-Leu-OH (0.795 g, 2.25 mmol), HOBt (0.304 g, 2.25 mmol), DIC (0.35mL, 2.25mmol) in the minimum amount of DMF. The solution was added to the resin. DMAP(0.11g, 0.9mmol) was dissolved in the minimum amount of DMF and was added to theresin. The mixture was stirred for 3 hours at room temperature. The solvent was removedby filtration under reduced pressure and then was added 15 mL solution aceticanhydride/pyridine/DCM (2:3:5) for 30 minutes [5]. The resin was filtered and washed 5times with DMF, 3 times with DCM, and finally 3 times with methanol. After the finalmethanol wash, dry the resin was dried in vacuo. The substitution of the resin wasestimated 0.7 mmol/g. Removal of Fmoc group was achieved by the repetitive treatmentwith piperidine/DMF (1:4, 10 mL) for 10 and 20 min and the resin was thoroughly washedwith DMF (3 x 10 mL), i-Pro (2 x 10 mL), n-hexane (2 x 10 mL) [6].4. Solid phase synthesis using microwave energy: The two different resins was transferredto Liberty Peptide Synthesizer and the two synthetic procedures were performed in thesame conditions, on 0.25 mmol scale, using HOBt/DIC as coupling reagents (Table 1). Thetotal synthesis was completed in 11 hours.5. Cleavage from the resin: Cleavage from both resins were performed by treating the resinwith 95/3/2 TFA/anisole/water for 2 hours in ambient temperature. Crude peptides wereprecipitated with diethyl ether, dissolved in DMSO or MeOH and analyzed in allianceHPLC (Figure 1). Separation was performed with stepped linear gradient from 5% (B) to100% (B) in 30 min with a flow rate of 1 mL/min. [(A):TFA solution in H 2 O 0,08%(v/v),(B):TFA solution in AcN 0,08%(v/v)]. Crude signal peptides were purified in Prep HPLCusing a stepped linear gradient from 5% (B) to 60% (B) over 45 min at a flow rate of 12mL/min. The identity of the peptide was verified using ESI mass spectroscopy.This study was performed to determine the stability of two different resins using thesame conditions and microwave energy. The present comparison of the two syntheticprotocols showed that the results were quite similar. The synthetic protocols were prettysimilar and both final crude products were of high purity. The yields for Cltr-Cl and Wangresin were 74% and 68% respectively. Also, compared to the traditional method the abovesynthetic protocols, using microwave energy, afforded cleaner peptides with higher yields.AcknowledgmentsThis work was supported by Eldrug S.A..References1. Choo, K.H., et al. BioMed Central 6, 249-257 (2005).2. Friligou, I., et al. J. Pept. Sci. 14, 90-93 (2008).3. Androutsou, M.E., et al. Amino Acids 38, 985-990 (2010).4. Matsoukas, J., et al. J. Med. Chem. 48, 1470-1480 (2005).5. Jubilut, G.N., et al. Chem. Pharm. Bull 55, 468-470 (2007).6. Tselios, T., et al. J. Med. Chem. 45, 275-283 (2002).Protocol in Microwave Peptide SynthesizerTime1 x 300sec2 x 300 sec1 x 30 sec1 x 180 secConditionsReagents: HOBt/ DIC30W, 75 o CReagents: HOBt/ DIC25W, 60 o CReagents: Pip/ DMF (1:5)Stage 1: 52W, 60 o CStage 2: 70W, 60 o C101