Uncapped Synthesis, Chemistry Essay Example

The synthesis of pure uncapped (61-95) alpha-syn was performed through the solid-phase peptide synthesis using the microwave-assisted 9-fluorenylmethoxycarbonyl (Fmoc) peptide synthesis. The role of Fmoc is to protect amino acids from the coupling of dissolved amino acids. Base-stable protecting groups of Fmoc protect the free amino acid side chains via the substitution of phenol by t-butyl on the tyrosine side chain and tert-butoxycarbanyl protected amino side chains on lysine. Wang resin with 100-200 mesh beads of polystyrene polymer functionalized at a density of 0.7 mM/g with phydroxybenzyl alcohol groups to which the amino acid carboxyl attaches was used as the solid support. The synthesis of the target peptide was performed from the C-terminus to N-terminus on a scale of 0.35 mM by reacting two molar equivalents of protected amino acid and coupling agents for each coupling step. Overall, all the steps totaled 10 mL of reaction volume.

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The C-terminal residue was coupled to the resin by activating the carboxyl group on the free amino acid with diisopropylcarbodiimide (DIC), using hydroxybenzoyltriazole (HOBt) to suppress racemization in the first step of Fmoc-peptide synthesis. During this first coupling step in which the C-terminal amino acid is coupled to the resin, catalytic acceleration of the activation of the amino acid carboxyl was achieved by adding 0.1 molar equivalents to Wang resin of 4-dimethylaminopyridine (DMAP). The reactants were agitated in dimethylformamide (DMF), which acted as the solvent for the coupling step for 5 minutes. The reactants were removed upon completion of the reaction by washing with dimethylformamide. The solid resin was retained through the glass frit of the vessels. The resin bound residues were deprotected through suspension and agitation of the resin in a 1:4 Piperidine/DMF (v/v) solution for 3 minutes in order to enable it to react with the Fmoc group. The resin-bound amino acid was prepared for coupling to the next residue in the sequence by removing the Fmoc protecting group via the procedure as described above with the coupling time decreased to 3 minutes for the other residues.

The Fmoc protected amino acid coupling and deprotection steps are repeated in the primary structure of the target peptide. Dichloromethane was used to wash the 61-95 alpha syn in order to remove it from the resin upon complete synthesis of the peptide chain. The resin was suspended in 20 mL trifluoroacetic acid (TFA)/DCM/triisopropylsilane/H2O 75:22:1.5:1.5 and the suspension agitated to break the bond between the peptides C-terminal carboxyl and resin, followed by dissolving the peptide into the acid. The purpose of triisopropylsilane and H2O is to scavenge the t-butyl and tert-butoxycarbanyl protecting groups cleaved by the acid.

The Fmoc was cleaved from the resin by adding 20 mL of trifluoroacetic acid (TFA)/DCM/triisopropylsilane/H2O 75:22:1.5:1.5. The resin was allowed to rock in this solution for 1 hour. Triisopropylsilane and H2O were used to scavenge the t-butyl and tert-butoxycarbanyl protecting groups cleaved by the acid. The cleavage cocktail was prepared in a round bottom flask equipped with a stirrer and cooled in an ice-bath during the preparation. The cold mixture was flushed with nitrogen. The reactants were then added slowly under an inert atmosphere and controlled temperature. The cooling bath was removed upon completion of the addition and the reaction was allowed to proceed at ambient temperature for the predetermined time. The cleavage mixture was filtered and the resin rinsed with neat trifluoroacetic acid. The filtrate was slowly added to cool Methyl tert-butyl ether (8-10 mL ether/mL cleavage cocktail) and the precipitate left to settle and filtered off. The residue was resuspended in the ether, stirred and left to settle in the cold and filtered off. The steps were repeated until the TFA and scavengers were eliminated. It is strongly recommended that sample cleavages should be done using different cocktail compositions (to be monitored by analytical HPLC) and to compare yield and quality of the crude resin before choosing the final one. A simultaneous time course evaluation is also done that should be complete in 2-3 hours at room temperature during the development of the cleavage conditions to minimize side reactions arising from long exposure to strong acids. The steps followed to synthesize the target protein are illustrated in Figures 11-13. The synthesis vessel was cleaned with dimethylformamide after cleaving the peptide, and the wash was combined with the trifluoroacetic acid solution. Trifluoroacetic acid and dimethylformamide were then purged with nitrogen gas in order to remove them, while the peptide was precipitated with ethyl ether. In the final steps of the preparation, the peptide was pelleted from the ether via mild centrifugation in a 15 mL polypropylene tube and then dried under vacuum to obtain the synthetic peptide.

Figure 11. Peptide synthesis coupling reaction

Figure 12. Peptide synthesis deprotection reaction

Figure 13. Peptide synthesis cleavage reaction

N-Capped and Both Capped Synthesis

The protocol for the synthesis of N-uncapped alpha-synuclein (61-95) was repeated for the synthesis of the N-capped peptide, which began with the Wang residue. For deprotection, the resin-bound residues occurred through the suspension and agitation of the resin in a 1:4 Piperidine/DMF (v/v) solution for 3 minutes to allow the Fmoc group to react. The resin-bound amino acid was prepared for coupling to the next residue in the sequence by removing the Fmoc protecting group via the procedure as described above with the coupling time decreased to 3 minutes for the other residues. The Fmoc protected amino acid coupling and deprotection steps were also repeated in the primary structure of the target peptide. Dichloromethane was used to wash the 61-95 alpha-syn in order to remove it from the resin upon complete synthesis of the peptide chain.

The resin was suspended in 20 mL trifluoroacetic acid (TFA)/DCM/triisopropylsilane/H2O 75:22:1.5:1.5 and the suspension agitated to break the bond between the peptides C-terminal carboxyl and resin, followed by dissolving the peptide into the acid. Triisopropylsilane and H2O were used to scavenge the t-butyl and tert-butoxycarbonyl protecting groups cleaved by the acid. The cleavage cocktail was prepared in a round bottom flask equipped with a stirrer and cooled in an ice-bath during the preparation. The cold mixture was flushed with nitrogen. The reactants were then added slowly under an inert atmosphere and controlled temperature. The cooling bath was removed upon completion of the addition and the reaction was allowed to proceed at ambient temperature for the predetermined time. The cleavage mixture was filtered and the resin rinsed with neat trifluoroacetic acid. The filtrate was slowly added to cool Methyl tert-butyl ether (8-10 mL ether/mL cleavage cocktail) and the precipitate left to settle and filtered off. The residue was resuspended in the ether, stirred and left to settle in the cold and filtered off. The steps were repeated until the TFA and scavengers were eliminated. It is strongly recommended that sample cleavages should be done using different cocktail compositions (to be monitored by analytical HPLC) and to compare yield and quality of the crude resin before choosing the final one. A simultaneous time course evaluation is also done that should be complete in 2-3 hours at room temperature during the development of the cleavage conditions to minimize side reactions arising from long exposure to strong acids.

The capping following the last coupling was performed using acetyl anhydride acetyl amide group (CH3-CO-NH2). The presence of unreacted amino acids after coupling of the target residues was blocked (capped) to avoid the formation of unwanted sequences. Capping will yield a truncated sequence (shortened peptide) that are significantly different from the final target peptide and can be readily separated from the intended peptide. Capping can be achieved by a short treatment of the peptide resin with a large excess of a highly reactive unhindered acid derivative and a base, usually acetic anhydride or benzoyl chloride and pyridine. In this study, acetic anhydride and pyridine were used in capping Rink amide resin. The capping solution was prepared by combining acetic anhydride and pyridine in a 3:2 ratio (acetic anhydride/pyridine). Care was taken to ensure that the capping solution remained fresh each time. A Pasteur pipette was used to stir the reactants (acetic anhydride/pyridine) in a scintillation vial. The capping solution was dumped on the resin and agitated for 30 min at room temperature. After capping is complete, the capping solution was pushed out by washing the resin 4 times with DMF. Before moving to the next step, a color test was performed using the Bromophenol Blue test. The process of capping was also used to reduce the loading of the resin. Consequently, a deficient amount of Fmoc amino acid was coupled to the preloaded resin. At this stage, the loaded resin was ready to go through repeated Fmoc-deprotections and amino acid couplings to build the rest of the peptide.