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A Novel Approach to Resin-based Cysteine Alkylation Bin Yang and Richard DiMarchi

S. H. O. O. H. R. X. T. F. A. F. m. o. c. /. t. B. u. c. h. e. m. i. s. t. r. y. N. N. H. N. N. 2. M. e. O. H. /. D. M. F. /. H. O. /. T. r. i. s. (. p. H. 8. 5. ). H. H. S. P. P. S. 2. O. M. B. H. A. R. R. S. S. H. F. c. l.

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A Novel Approach to Resin-based Cysteine Alkylation Bin Yang and Richard DiMarchi

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  1. S H O O H R X T F A F m o c / t B u c h e m i s t r y N N H N N 2 M e O H / D M F / H O / T r i s ( p H 8 . 5 ) H H S P P S 2 O M B H A R R S S H F c l e a v a g e O O H O O H N H S Q G T F T S D Y S K Y L D S R R A Q D F V Q W L M N T N N N G l u c a g o n N N H H H 2 S H H O O H N 0 1 9 [ C y s , A h x , G l u ] G l u c a g o n H N S Q G T F T S E Y S K Y L D S R R A Q D F V Q W L M N T 2 O O O H T M m - d P E G - M A L N 1 2 N O O O O O O O O O O O O O O S S O O H O H S N - H O N O H N N N N H 2 - H O 2 S N H 2 S O S O H N N 2 H F H O M - 1 8 M - 1 8 A Novel Approach to Resin-based Cysteine Alkylation Bin Yang and Richard DiMarchi Department of Chemistry, Indiana University, Bloomington, Indiana, 47405 U.S.A. Abstract A novel method in the synthesis of S-alkylated cysteine peptides is described. The S-alkylation reaction was achieved “on resin” with unprotected cysteine containing peptides, in aqueous methanol/DMF buffered in Tris base (pH, 8.5). Alkylating agents such such as bromoacetic acid, iodoacetic acid, 3-iodopropionic acid and 3-maleimidopropionic acid(MPPA), were used in a 2-5 molar excess to selectively S-alkylate cysteine peptides in yields that range between 50-90%. A model peptide (GCSWARKHT) and a specific glucagon analog that each contained a thiol group and a set of additional nucleophilic functional groups (amino, hydroxy, indole, imidazolyl and guanidine) were utilized as alkylation substrates. Other aklylating agents that introduce chemical substitutions of known biological importance, such as farnesyl bromide were also investigated to further test the generality of this methodology. Introduction Cysteine alkylation is a powerful and versatile chemical approach in peptide and protein structure-function analysis. Modification with structurally diverse alkylating agents has been successfully employed in protein identification, assessment of protein folding, and localization of ligand binding sites. S-alkylation of cysteine also provides a rapid means to optimize a specific side chain structure starting from a single peptide. This minimizes the requirement for total synthesis of individual peptides with a unique unnatural amino acid. With pH control the thiol group (RSH) can be selectively alkylated in the presence of other nucleophilic natural amino acid aide chains, such as amino and hydroxy. Here we report a novel “on-resin” cysteine alkylation method for unprotected peptides in aqueous methanol/DMF, buffered with Tris base. Chemistry Reaction Scheme 1 describes the general details of this on-resin peptide cysteine alkylation procedure. Peptides were typically assembled on 4-methylbenzhyrylamine resin (MBHA) resin using traditional Fmoc/tBu chemistry. All the side chain protection groups of the peptides were removed by anhydrous TFA treatment to yield an unprotected peptidyl-resin. The unprotected peptide still covalently attached to the resin was alkylated in 50% methanol or DMF aqueous that was buffered with Tris at pH 8.5, with 2-5 equiv excess of alkylating agent to susceptible thiol group. Each alkylated peptide was cleaved from the resin with anhydrous HF and the extent and selectivity in cysteine modification was assessed by MS and HPLC. Table 1. “On-resin” Alkylation Condition and Yields with Various Agents and Model Peptides Rxn 1 Rxn 2 Rxn 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. Rxn 7 Rxn 9 12. 13. 14. 15. Fig 1. RP-HPLC analysis of representative alkylated peptides Rxn 1 Rxn 7 Rxn 3 Rxn 9 Rxn 2 Rxn 10 Scheme 1 Synthesis of cysteine S-alkylated peptide by on-resin alkylation • 2-Bromo-1-(5-phenyl-2-thienyl)-ethanone is a selective and strong alkylating agent. We observed it to alkylate the thiol to high degree (rxn 15) and resulted in a dehydration product when alkylating an N-terminal cysteine thiol (rxn 11). The dehydrated S-alkylation product provided additional evidence that the alkylation reaction was selective to the thiol group. Our results demonstrate that 2-bromo-1-(5-phenyl-2-thienyl)-ethanone can function as a new and highly effective cysteine • S-alkylating agent. Rxn 6 • Result and Discussions • BrCH2COOH & ICH2CH2COOH & ICH2CH2CH2COOH BrCH2COOH was highly effective in alkylation of glucagon analogs (rxn 1, 2) and the model peptide (rxn 12). Alkylation with ICH2CH2COOH (rxn 3, 4, 13) was slightly less reactive than BrCH2COOH but still provided high yield and selectivity. ICH2CH2CH2COOH was not reactive in this on-resin reaction (rxn 5). • PEG-MAL Polyethylene glycol (PEG) maleimidopropionyimide was highly reactive and selective using similar reaction conditions to the previously • described halo-acids (rxns 7-9). • Farnesyl Bromide Modification of cysteine by addition of a farnesyl motif is involved in many membrane signal transduction proteins, so alkylation of cysteine by farnesyl bromide is a very desirable modification. The alkylation with the farnesyl bromide was slow and thus required additional time (16hr) • but eventually yielded ~ 50% of the desired products (rxn 10, 14). Rxn 11 Fig 2. ESI-MS analysis of representative cysteine S-alkylated peptides after HF cleavage Acknowledgements We would like to thank David L. Smiley, Jay L. Levy and Jonathan A. Karty for their kind help in peptide synthesis and mass spectrometry analysis. References 1. Or, Y. S., Clark, R. F. and Luly J. R. J. Org. Chem. 56, 3146-3149(1991). 2. Perrey, D. A. and Uckun, F. M. Tetrahedron letters, 42, 1859-1861(2001). 3. Yang, C. C., Marlowe, C. K. and Kania, R. J. Am. Chem. Soc.113, 3176-3177(1991). Scheme 2. Mechanism of formation of dehydration product when 2-bromo-1-(5-phenyl-2-thienyl)-ethanone to alkylate the N-terminal cysteine

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