Overview of Solid Phase Peptide Synthesis (SPPS) HEADING_TITLE

General Solid Phase Peptide Synthesis Scheme

General Solid Phase Peptide Synthesis Scheme
The general process for synthesizing peptides on a resin starts by attaching the first amino acid, the C-terminal residue, to the resin.  To prevent the polymerization of the amino acid, the alpha amino group and the reactive side chains are protected with a temporary protecting group.  Once the amino acid is attached to the resin, the resin is filtered and washed to remove byproducts and excess reagents.  Next, the N-alpha protecting group is removed in a deprotection process and the resin is again washed to remove byproducts and excess reagents.  Then the next amino acid is coupled to the attached amino acid.  This is followed by another washing procedure, which leaves the resin-peptide ready for the next coupling cycle.  The cycle is repeated until the peptide sequence is complete.  Then typically, all the protecting groups are removed and the peptide resin is washed, and the peptide is cleaved from the resin.General Solid Phase Peptide Synthesis Scheme

The general process for synthesizing peptides on a resin starts by attaching the first amino acid, the C-terminal residue, to the resin.  To prevent the polymerization of the amino acid, the alpha amino group and the reactive side chains are protected with a temporary protecting group.  Once the amino acid is attached to the resin, the resin is filtered and washed to remove byproducts and excess reagents.  Next, the N-alpha protecting group is removed in a deprotection process and the resin is again washed to remove byproducts and excess reagents.  Then the next amino acid is coupled to the attached amino acid.  This is followed by another washing procedure, which leaves the resin-peptide ready for the next coupling cycle.  The cycle is repeated until the peptide sequence is complete.  Then typically, all the protecting groups are removed and the peptide resin is washed, and the peptide is cleaved from the resin.

SPPS cycle

General Solid Phase Peptide Synthesis Cycle

Selective Protection and Orthogonality

The side chains of many amino acids are reactive and may form side products if left unprotected.  For successful peptide synthesis, these side chains must remain protected despite repeated exposure to N alpha deprotection conditions.  Ideally, the N alpha protecting group and the side chain protecting groups should be removable under completely different conditions, such as basic conditions to remove the N alpha protection and acidic conditions to remove the side chain protection.  Such a protection scheme is called “orthogonal” protection.  

 

Boc/Bzl Protection

In the Boc/Bzl protection scheme, Boc protecting groups are used to temporarily protect the N alpha nitrogen groups of the amino acids and benzyl-based protecting groups provide more permanent protection of sidechains.  Boc and benzyl-based protecting groups are both acid labile, so Boc/Bzl is not a true orthogonal protection scheme.  It is practically utilized though, because the Boc group is removed under moderate conditions ( 50% TFA in DCM) while benzyl-based protection groups require very strong acids, such as HF or TFMSA, to remove them.  AAPPTec offers competitively priced Boc-protected amino acids for peptide synthesis.

 

Boc Deprotection Mechanism

As shown in the mechanism below, tert-butyl carbonium ions are formed during Boc-deprotection.  These cations react further with nucleophiles, forming isoprene or tert-butyl adducts.  Tryptophan (Trp), cysteine (Cys) or methionine (Met) residues within a peptide can react with tert-butyl carbonium ions and produce undesired peptide side products.  Adding 0.5% dithioethane (DTE) to the cleavage solution scavenges the tert-butyl cations and prevents the formation of peptide side products.  

Boc deprotection mechanism

 

Boc Deprotection Mechanism

 

After the Boc group has been removed by treatment with TFA, the deprotected amine is in the form of a TFA salt.  The salt must be converted to the free amine before the next amino acid can be coupled.  Typically this is achieved by treating the resin-peptide with a 50% solution of diisopropylethylamine (DIEA) in dichloromethane (DCM), followed by several washes. 

Castro and coworkers have reported using an in situ neutralization procedure with BOP/DIEA.1   Kent and Alewood have developed in situ neutralization with HATU or HBTU coupling.2  In addition to saving time through eliminating the separate neutralization and washing procedures, in situ neutralization can improve coupling yields when aggregation causes problems.  Since aggregation occurs mainly in the neutral resin-peptide, in situ neutralization presumably minimizes aggregation by minimizing the period of time that the deprotected resin-peptide is in the neutral state.

Fmoc/tBu Protection

In this protection scheme, the alpha nitrogen of the amino acids is protected with the base labile Fmoc group and the side chains are protected with acid labile groups based either on the tert-butyl protecting group or the trityl (triphenylmethyl) group.  This is an orthogonal protection system, since the side chain protecting groups can be removed without displacing the N-terminal protection and visa versa. It is advantageous when sidechains need to be selectively modified, as when the peptide is selectively labeled or cyclized through the side chain.  AAPPTec offers a large selection of Fmoc-protected amino acids.

Fmoc Deprotection Mechanism

The Fmoc group is removed when a base abstracts the relatively acidic proton from the fluorenyl ring system, leading to β-elimination and the formation of dibenzofulvene and carbon dioxide.  Dibenzofulvine is a reactive electrophile and would readily attach irreversibly to the deprotected amine unless it was scavenged.  Secondary amines such as piperidine add to dibenzofulvene and prevent deleterious side reactions.  Hence piperidine is typically used to remove the Fmoc group and also scavenge the dibenzofulvene by-product.  
A report on utilizing 5% piperidine solution to remove Fmoc protecting groups from resin bound amino acids has been published.3   In the preparation of a poly-alanine peptide, the time required to remove the Fmoc group from the first five alanine residues was between 20 and 30 minutes.  For the next five alanine residues (Ala6 through Ala10) the deprotection time jumped to 100 to 170 minutes, probably due to aggregation.  Recently reported optimized fast Fmoc protocols utilize piperidine deprotection of three minutes or less.4 Some automated peptide synthesizers, such as the Fmoc XC series, monitor the absorbance of the dibenzofulvine adduct and automatically extend time for the deprotection reaction until no further adduct formation is detected.
1,8-Diazabicyclo[5.4.0]undec-7-ene (DBU) removes the Fmoc protecting group much faster than piperidine.5   When Fmoc deprotection during a peptide synthesis is slow or incomplete, replacing piperidine with DBU can improve the deprotection yield and thus increase the yield of desired peptide.6 Since DBU is non-nucleophilic and will not react with the fulvulene byproduct, piperidine is often added just to react with this byproduct.7   DBU should not be used when aspartic acid (Asp) residues are part of the peptide-resin for DBU catalyzes aspartimide formation with subsequent reaction with piperidine.
Fmoc deprotection mechanism

Fmoc-Deprotection Mechanism

 

 

Ligation and Fragment Condensation

Although solid phase peptide synthesis methodology has improved to the point where preparing peptides of up to 100 amino acids is feasible,8  larger peptides and small proteins, as yet, are not accessible by solid phase peptide synthesis alone.  Much larger products can be assembled by coupling protected peptide segments in solution.  The synthesis of the 238-amino acid precursor of green fluorescent protein is an outstanding example.9   This technique is often hampered by insolubility of the protected peptide segments and racemization of the fragment C-terminal residues when activated for coupling.  Racemization can be avoided if the fragment to be activated has a C-terminal Gly.  Careful selection of the fragment coupling reagent can also minimize racemization.  TDBTU, for instance, has been shown to produce significantly less epimerization than other common coupling reagents in coupling peptide fragments to form SK&F 107647 and has been utilized to prepare SK&F 107647 on a two kilogram scale.10

Native chemical ligation is a method for coupling unprotected peptide segments in aqueous solution.  In this methodology, a peptide with an unprotected N-terminal cysteine reacts with a peptide thioester forming an S-acyl intermediate, which then undergoes S-N acyl shift to form a standard peptide bond.11  Native chemical ligation has proven very useful for preparing large peptides12 or complex peptides such as glycopeptides.13  The requirement for cysteine residues in appropriate positions within the target product currently is a limitation of native chemical ligation, but a number of ways are being studied to extend the utility of the method.14  The most promising is to convert the cysteine residue to alanine by desulfurization following ligation.15  Making use of the desulfurization strategy, a number of thiol-substituted derivatives have been reported to allow ligation at proline,16 phenylalanine,17 lysine18 and glutamine.19 

Staudinger ligation is another promising method for assembling peptide segments.  The Staudinger ligation couples a peptide thioester with an azide via a phosphinothioester intermediate.20   To illustrate the potential of Staudinger ligagation and native chemical ligation, both methods were used together to assemble functional ribonuclease A.21 

 


 

Footnotes

Le-Nguyen, D; Heitz, A; Castro, B, J. Chem Soc., Perkin Trans 1, 1987, 1915-1919.

2 Schnölzer, M; Alewood, P; Jones, A; Alewood, D; Kent, SBH, Int. J. Peptide Protein Res., 1992, 40, 180-193.

3 Zinieris, N.; Leondiadis, L.;  Ferderigos, N. J. Comb. Chem., 2005, 7, 4-6.

4 Hood, C. A.; Fuentes, G.; Patel, H.; Page, K.; Menakuru, M.; Park, J. H. J. Pept. Sci., 2008, 14, 97-101.

5 Villain, M.; Jackson, P. L.; Krishna, N.R.; Blalock, J.E. in  “Frontiers of Peptide Science,  Proceedings of the 15th American Peptide Symposium Nashville, TN,. 1997” (J.P. Tam and P.T.P. Kaumaya, Eds.)., Kluwer Academic Publishers, Dordrecht, 1999, 255-256.

6 Wade, J. D.; Bedford, J.; Sheppard, R. C.; Tregear, G. W., Pept. Res., 1991, 4, 194-199; Dettin, M.; Pegoraro, S.; Rovero, P.; Bicciato, S.; Bagno, A.; Di Bello, C., J. Pept. Res., 1997, 49, 103-111.

7 Fields, C. J.; Mickelson, D. J.; Drake, S. L.; McCarthy, J. B.; Fields, G. B., J. Biol. Chem., 1993, 268, 14153-14160.

8 White, P.; Keyte, J. W.; Bailey, K.; Bloomberg, G. J. Pept. Sci., 2003, 10, 18-26;  Kakizawa, T.; Koide-Yoshida, S.; Kimura, T.; Uchimura, H.; Hayashi, Y.; Saito, K.; Kiso, Y. J. Pept. Sci., 2008, 14, 261-266.

9 Nishiuchi, Y.; Inui, T.; Nishio, H.; Bódi, J.; Kimura, T.; Tsuji, F. I.; Sakakibara, S. Proceed. Natl. Acad. Sci. U.S.A., 1998, 95, 13549-13554.

10 J. Hiebl, H. Baumgartner, I. Bernwieser, M. Blanka, M. Bodenteich, K. Leitner, A. Rio, F. Rovenszky, D.P. Alberts, P.K. Bhatnagar, A.F. Banyard, K. Baresch, P.M. Esch, H. Kollmann, G. Mayrhofer, H. Weihtrager, W. Welz, K. Winkler, T. Chen, R. Patel, I. Lantos, D. Stevenson, K.D. Tubman, K. Undheim, J. Peptide Res., 1999, 54, 54-65.

11 Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science, 1994; 266, 776 - 779.

12 Li, X.; de Leeuw, E.; Lu, W. Biochemistry, 2005, 44, 14688-14694; Durek, T.; Torbeev, V.Y.; Kent, S. B. H. Proceed. Natl. Acad. Sci. U.S.A., 2007, 104, 4846-4851.

13 Shin, Y.; Winans, K. A.; Backes, B. J.; Kent, S. B. H.; Ellman, J. A.; R. Bertozzi, C. R. J. Am. Chem. Soc., 1999, 121, 11684-11689; Dudkin, V. Y.; Miller, J. S.; Danishefsky, S. J., J. Am. Chem. Soc., 2004, 126, 736–738; Yang, Y.-Y.; Ficht, S.; Brik, A.; Wong, C.-H., J. Am. Chem. Soc., 2007, 129, 7690-7701.

14 Crich, D.; Banerjee, A. J. Am. Chem. Soc., 2007, 129, 10064–10065; Pentelute, B. L.; Kent, S. B. H., Org. Lett., 2007, 9, 687–690;  Chen,  G.; Warren, J. D.; Chen, J.; Wu, B.; Wan, Q.; Danishefsky, S. J., J. Am. Chem. Soc., 2006, 128, 7460–7462; Tchertchian, S.; Hartley, O.; Botti, P., J. Org. Chem., 2004, 69, 9208–9214; Macmillan, D.; Anderson, D. W.; Org. Lett., 2004, 6, 4659–4662; Yan, L. Z.; Dawson, P. E., J. Am. Chem. Soc., 2001, 123, 526–533; Canne, L. E.; Bark, S. J.; Kent, S. B. H., J. Am. Chem. Soc., 1996, 118, 5891 –5896.

15 Yan, L. Z.; Dawson, P. E., J. Am. Chem. Soc., 2001, 123, 526 –533; Pentelute, B. L.; Kent, S. B. H., Org. Lett., 2007, 9, 687–690; Kan, C.; Trzupek, J. D.; Wu, B.; Wan, Q.; Chen, G.; Tan, Z.; Yuan, Y.; Danishefsky, S. J., J. Am. Chem. Soc., 2009, 131, 5438–5443.

16 Shang, S.; Tan, Z.; Dong, S.; Danishefsky, S. J., J. Am. Chem. Soc., 2011, 133, 10784–10786.

17 Critch, D.; Banerjee, A., J. Am. Chem. Soc., 2007, 129, 10064–10065.

18 Yang, R.; Pasunooti, K. K.; Li, F.; Liu, X.-W.; Liu, C.-F., J. Am. Chem. Soc., 2009, 131, 13592–13593.

19 Siman, P.; Karthikeyan, S. V.; Brik, A., Org. Lett., 2012, 14, 1520–1523.

20 Nilsson, B. L.; Kiessling, L. L.; Raines, R. T., Org. Lett., 2000, 2, 1939–1941; Nilsson, B. L.; Kiessling, L. L.; Raines, R. T., Org. Lett., 2001, 3, 9–12; Soellner, M. B.; Tam, A.; Raines, R. T. J., Org. Chem., 2006, 71, 9824–9830.

21 Nilsson, B. L.; Hondal, R. J.; Soellner, M. B.; Raines, R. T., J. Am. Chem. Soc., 2003, 125,  5268–5269.