Large Scale Solid Phase Synthesis of Peptide Drugs: Use of Commercial Anion Exchange Resin as Quenching Agent for Removal of Iodine during Disulphide Bond Formation
The S-acetamidomethyl (Acm) or trityl (Trt) protecting groups are widely used in the chemical synthesis of peptides that contain one or more disulfide bonds. Treatment of peptides containing S-Acm protecting group with iodine results in simultaneous removal of the sulfhydryl protecting group and disulfide formation. However, the excess iodine needs to be quenched or adsorbed as quickly as possible after completion of the disulfide bond formation in order to minimize side reactions that are often associated with the iodination step. We report here a simple method for simultaneous quenching and removal of iodine and isolation of disulphide bridge peptides. The use of excess inexpensive anion exchange resin to the oxidized peptide from the aqueous acetic acid/methanol solution affords quantitative removal of iodine and other color impurities. This improves the resin life time of expensive chromatography media that is used in preparative HPLC column during the purification of peptide using preparative HPLC. Further, it is very useful for the conversion of TFA salt to acetate in situ. It was successfully applied commercially, to the large scale synthesis of various peptides including Desmopressin, Oxytocin, and Octreotide. This new approach offers significant advantages such as more simple utility, minimal side reactions, large scale synthesis of peptide drugs, and greater cost effectiveness. 1. Introduction Many naturally occurring peptides contain intradisulphide bridges, which play an important role in biological activities. Disulfide bond-containing peptides have long presented a particular challenge for their chemical synthesis [1]. There are many ways to form a disulphide bridge in solution, and solid phase synthesis is well known and widely used in peptide community. Solution phase cyclization is most commonly carried out using air oxidation and or mild basic conditions [2]. The cyclization of free thiols by air oxygen usually leads to low yields of target peptides (10–15%) [3]. The application of potassium ferricyanide [4] or dimethyl sulphoxide [5] usually results in homogeneous reaction mixtures, and yields of cyclic peptides are considerably higher. However, a multistage purification of peptide is necessary for the removal of excess of these oxidative reagents [6]. Beginning with the initial discovery by Kamber et al. [7] that in peptides, where thiols are protected by Acm groups, iodine offered the potential to carry out removal and simultaneous oxidative disulphide bond formation in one-single step, several disulphide-bonded peptides have been
References
[1]
I. Annis, B. Hargittai, and G. Barany, “Disulfide bond formation in peptides,” Methods in Enzymology, vol. 289, pp. 198–221, 1997.
[2]
C. Kellenberger, H. Hietter, and B. Luu, “Regioselective formation of the three disulfide bonds of a 35-residue insect peptide,” Peptide Research, vol. 8, no. 6, pp. 321–327, 1995.
[3]
E. V. Kudryavtseva, M. V. Sidorova, and R. P. Evstigneeva, “Some peculiarities of synthesis of cysteine-containing peptides,” Russian Chemical Reviews, vol. 67, no. 7, pp. 545–562, 1998.
[4]
R. Eritja, J. P. Ziehler-Martin, P. A. Walker et al., “On the use of s-t-butylsulphenyl group for protection of cysteine in solid-phase peptide synthesis using fmoc-amino acids,” Tetrahedron, vol. 43, no. 12, pp. 2675–2680, 1987.
[5]
J. P. Tam, C. R. Wu, W. Liu, and J. W. Zhang, “Disulfide bond formation in peptides by dimethyl sulfoxide. Scope and applications,” Journal of the American Chemical Society, vol. 113, no. 17, pp. 6657–6662, 1991.
[6]
D. Andreu, F. Albericio, N. A. Sole, M. C. Munson, M. Ferrer, and G. Barany, “In methods in molecular biology, formation of disulphide bonds in synthetic peptides and proteins,” in Peptide Synthesis Protocols, M. W. Penington and B. M. Dunn, Eds., vol. 35, pp. 91–169, Humana Press, Totowa, NJ, USA, 1994.
[7]
B. Kamber, A. Hartmann, K. Eisler et al., “Synthesis of cystiene peptides by iodine oxidation of S-trityl-cystiene,” Helvetica Chimica Acta, vol. 63, no. 4, pp. 899–915, 1980.
[8]
S. Zhang, F. Lin, M. A. Hossain, F. Shabanpoor, G. W. Tregear, and J. D. Wade, “Simultaneous post-cysteine(S-Acm) group removal quenching of iodine and isolation of peptide by one step ether precipitation,” International Journal of Peptide Research and Therapeutics, vol. 14, no. 4, pp. 301–305, 2008.
[9]
M. Engebretsen, E. Agner, J. Sandosham, and P. M. Fischer, “Unexpected lability of cysteine acetamidomethyl thiol protecting group: tyrosine ring alkylation and disulfide bond formation upon acidolysis,” Journal of Peptide Research, vol. 49, no. 4, pp. 341–346, 1997.
[10]
W. L. Mendelson, A. M. Tickner, M. M. Holmes, and I. Lantos, “Efficient solution phase synthesis of {1-(β-mercapto-β,β-cyclopentamethylenepropionic acid}-2-(O-ethyl-D-tyrosine)-4-valine-9-desglycine]arginine vasopressin,” International Journal of Peptide and Protein Research, vol. 35, no. 3, pp. 249–257, 1990.
[11]
M. Pohl, D. Ambrosius, J. Grotzinger et al., “Cyclic disulfide analogues of the complement component C3a. Synthesis and conformational investigations,” International Journal of Peptide and Protein Research, vol. 41, no. 4, pp. 362–375, 1993.
[12]
E. V. Kudryavtseva, M. V. Sidorova, M. V. Ovchinnikov, Z. D. Bespalova, and V. N. Bushuev, “Comparative evaluation of different methods for disulfide bond formation in synthesis of the HIV-2 antigenic determinant,” Journal of Peptide Research, vol. 49, no. 1, pp. 52–58, 1997.
[13]
D. Sahal, “Removal of iodine by solid phase adsorption to charcoal following iodine oxidation of acetamidomethyl-protected peptide precursors to their disulfide bonded products: oxytocin and a Pre-S1 peptide of hepatitis B virus illustrate the method,” Journal of Peptide Research, vol. 53, no. 1, pp. 91–97, 1999.
[14]
L. Chen, H. Bauerová, J. Slaninová, and G. Barany, “Syntheses and biological activities of parallel and antiparallel homo and hetero bis-cystine dimers of oxytocin and deamino-oxytocin,” Peptide Research, vol. 9, no. 3, pp. 114–121, 1996.
[15]
K. Kunin, In Amber-Hi-Lites. Fifty Years of Ion-Exchange Technology, Rohm and Hass, Philadephia, Pa, USA, 1996.
[16]
D. P. Elder, “Pharmaceutical applications of ion-exchange resins,” Journal of Chemical Education, vol. 82, no. 4, pp. 575–587, 2005.
[17]
L. M. Gayo and M. J. Suto, “Ion-exchange resins for solution phase parallel synthesis of chemical libraries,” Tetrahedron Letters, vol. 38, no. 4, pp. 513–516, 1997.
[18]
G. L. Hatch, J. L. Lambert, and L. R. Fina, “Some properties of the quaternary ammonium anion-exchange resin-triiodide disinfectant for water,” Industrial & Engineering Chemistry Product Research and Development, vol. 19, no. 2, pp. 259–264, 1980.
[19]
H. A. Ezzeldin, A. Apblett, and G. L. Foutch, “Synthesis and properties of anion exchangers derived from chloromethyl styrene codivinylbenzene and their use in water treatment,” International Journal of Polymer Science, vol. 2010, Article ID 684051, 9 pages, 2010.
[20]
Ion exchange India pvt limited.
[21]
V. C. Gerald, “Process for removing iodine/iodide from aqueous solution,” US Patent 5624567, 1997.
