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Proceedings of the 31 st European Peptide SymposiumMichal Lebl, Morten Meldal, Knud J. Jensen, Thomas Hoeg-Jensen (Editors)European Peptide Society, 2010Racemization in Automated Solid Phase SynthesisKrzysztof Darlak, Miroslawa Darlak, and Thomas E. HopkinsCreosalus Inc., Louisville, KY, 40228, U.S.A.IntroductionAutomated peptide synthesis allows for preparation of a large number of peptide sequencesin a fairly straightforward manner. Currently, the most widely applied methodologiesutilize Fmoc-protection in combination with uronium/phosphonium activating agents forsynthesis of peptides. The racemization process has been extensively studied with theseactivating agents on solid support [1,2]. Most experiments were carried out via manualprotocols. Automated peptide synthesis adds another limitation affecting racemizationprocess. Our recently presented studies covering the two most racemization-prone aminoacids, cysteine and histidine, pointed out optimal conditions for their incorporation to apeptide chain using an automated peptide synthesis [3]. We have expanded the currentwork to the following commonly used amino acids: Asp, Leu, Ser and Tyr. In order toobtain a baseline resolution of the studied isomers we have applied in our experiments thepreviously published model peptide H-Gly-Xxx-Phe-NH 2 [4]. Herein we reevaluate thedegree of racemization of Asp, Leu, Ser and Tyr using several popular activating agentsapplied to a typical automated peptide synthesis using an in-situ activation method.ExperimentalModel peptides H-Gly-Xxx-Phe-NH 2 , Xxx=Asp, Leu, Ser and Tyr, were prepared onFmoc-Phe-Rink-MBHA resin using the Tetras (Thuramed) multiple peptide synthesizerwith in situ activation. Reagents used in the studies were: 0.6 M solution in NMP (BOP,PyBOP ® , HCTU, HATU, COMU), 0.4 M solution in NMP (HBTU, PyClock ® ), 0.9 MDIEA solution in NMP, 1.0 M solution in NMP (DIC, HOBt, 6-Cl-HOBt, Oxyma Pure).Coupling conditions for uronium/phosphonium reagents (order of addition to the 150 mg ofresin, 0.6 meq/g, no preactivation): Fmoc-amino acid 0.5 mmole, amine 0.9 mmole, reagent0.5 mmole, mixing for 120 min; with DIC/additive: additive 0.5 mmole, Fmoc-amino acid0.5 mmole, DIC 0.5 mmole, mixing for 120 min. Target peptides were cleaved from theresin with the mixture of TFA : water : phenol : TIPS (87.5:5:5:2.5) for 2 hours at RT. Theresin was filtered off and TFA was evaporated under reduced pressure. The product wasprecipitated by addition of cold diethyl ether, centrifuged and dried. Chromatographicanalysis of the obtained products was performed on Waters Alliance HPLC system using aVydac C 18 column (4.6x250 mm, 218TP54) with the linear gradients: 1-21% B in 30 minfor H-Gly-Asp-Phe-NH 2 and H-Gly-Ser-Phe-NH 2 , 10-30 % B in 30 min for H-Gly-Leu-Phe-NH 2 , 5-35% B in 30 min for H-Gly-Tyr-Phe-NH 2 . Buffers used for the analysis whereA: 0.1% TFA in Water: MeCN 98:2 (v/v) and B: 0.1% TFA in MeCN: Water 98:2 (v/v)with flow of 1ml/min and detection at 220 nm. The content of LDL isomer was calculatedas relative peak areas (Absorbance) from HPLC as: A (LDL isomer)/[A (LDL isomer + A(LLL isomer)] x 100. Results of the analysis are presented in Table 1.Results and DiscussionWe have used listed above peptides as targets to evaluate the degree of racemization duringincorporation of Fmoc-Asp(OBut)-OH, Fmoc-Leu-OH, Fmoc-Ser(But)-OH and Fmoc-Tyr(But)-OH to the solid support utilizing a Tetras (Thuramed) automated peptidesynthesizer with in situ activation. Selected reagents are presented in Table 1, includingrecently introduced reagents: COMU [3,4], PyClock [5] and Oxyma Pure [3,4,6,7]. Duringroutine peptide synthesis the same activating method is used to prepare an entire peptidesequence. This approach is necessary due to the limitation of instrumentation (lack ofsufficient number of reagents precisely delivered on board of synthesizer) or the user’schoice. Our previous studies indicated that use of DIC with various additives was found tobe a method of choice for introduction of cysteine and histidine residues using anautomated peptide synthesis. Presented results in Table 1 show clearly, that in all studiedcases (Asp, Leu, Ser, and Tyr) the level of racemization was found below 1%. The majorityof the examples implementing DIC mediated activation with various additives gave lowerracemization than uronium/phosphonium reagents. Changing the amount of base to one192