Synthesis Of Long Peptides - Bioscience Technology Online

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Synthesis Of Long Peptides

Introduction

The attraction of peptides as therapeutics or research tools comes from their high specificity to targeted sites, their diversity and their usually low toxicity. As the demand for peptides increases, and the price of raw material for synthesis decreases, research groups and pharmaceutical companies alike are able to purchase peptides at lower cost and with increasing length and complexity. However, the synthesis of long and complex peptides, as one might expect, is not simple and alternative synthesis methods have been devised to meet this challenge.

Background

Peptide synthesis is a very well established field and can be divided into three general methods: solution phase synthesis, biosynthesis, and solid phase synthesis. Solution phase synthesis can be the best strategy to obtain large amounts of short length peptides while keeping costs low. However, the method becomes ill-suited to produce peptides longer than 10 amino acids, as the growing peptide chain will need to be purified between each coupling and deprotection step. The biosynthesis of peptides offers the option to obtain long peptidic chains, even proteins, by recombinant techniques; moreover the method can be scaled up for large production batches. Unfortunately, biosynthesis cannot be adapted to introduce unnatural amino acids or other in-chain modifications. Finally, solid phase synthesis methods developed by B. Merrifield⁽¹⁾ allow the synthesis of peptides that are difficult to obtain by biosynthesis and which contain a wide variety of in-chain modifications and unnatural amino acids. Specific chemistries have been developed that allow the stepwise synthesis of the peptides while minimizing unwanted side reactions via protecting groups attached to side chains and amino groups. The most commonly used synthetic methods are referred as Boc and Fmoc strategies. Peptides are synthesized in a linear, stepwise fashion beginning with their C-terminus bound to an insoluble support. The major advantage of using a solid support is the ability to easily wash away the excess reagents and byproducts after each amino acid coupling step. Nevertheless, the synthesis of peptides longer than ~50 amino acids using solid phase peptide synthesis (SPPS) can be difficult due to aggregation (hydrophobic interaction) or the formation of secondary structures (hydrogen bonding) between the growing peptide chains. The poor coupling efficiencies due to these interactions result in peptides that are produced at low purity, if at all.

The methods available for synthesizing peptides containing "difficult sequences", or long peptides, will be briefly reviewed.

Fragment synthesis

A peptide attached to a solid support is elongated, using protected peptide fragments instead of single amino acids. The protected fragments can be generated using either solution phase or solid phase synthesis methods, according to their desired length. The peptide fragments must have a free Cterminus carboxylic group, and the amino acid side chains and N-terminus must also be appropriately protected for peptide synthesis (Figure 1). This method can be used for the synthesis of peptides that are difficult to obtain using standard chemistries, such as poly-proline containing peptides, collagen peptides or highly hydrophobic peptides. The method is often limited by poor coupling efficiency of the fragments to the growing peptide chain due to steric hindrance or weak solubility in the coupling solvent.

Fragment synthesis of a peptide. Three fragments are successively assembled and the peptide is finally cleaved from the solid support

Native chemical ligation (NCL)

Developed by Kent and coworkers, this strategy permits the chemoselective ligation of unprotected peptide fragments in aqueous buffer.^{(2, 3} Ligation occurs between a peptide containing a cysteine at its N-terminus and another peptide modified with a thioester at its C-terminus. NCL is the most popular method used to obtain long peptide chains and proteins that cannot be synthesized by SPPS.

A recent example illustrating the power of NCL was the full synthesis of a glycoprotein containing an intact complex-type sialyloligosaccaride by P. E. Dawson and coworkers.⁽⁴⁾ The authors used a mixture of Fmoc and modified-Boc strategies to synthesize two peptide fragments and one sialglycopeptide thioester. The sequential ligation of the peptides yielded a 76 amino acid protein with a human type oligosaccharide modification.

Four peptide fragments are successively ligated in solution phase. The cysteines are then de-sulfurized into alanines. Adapted from reference 5

A second example, reported by Kent and coworkers, describes the synthesis of HIV-1 protease, a homodimeric protein of 99 amino acids.⁽⁵⁾ The innovation of this synthesis was the use of a modified thioester to increase solubility of the fragments in aqueous buffer. The successive ligation of 4 peptide fragments and the successive de-sulfuration of the cysteines residues yielded a fully active protein in good yield (Figure 2).

The major limitation of NCLC comes from the synthesis of the thioester fragment. The methods currently available to synthesize C-terminus modified thioesters are not easily mastered. The chemical synthesis of Cterminal thioester peptides has been largely restricted to the use of Boc chemistry⁽⁶⁾ or to the use of intein-based bacterial expression systems.⁽⁷⁾ Recently, the development of "safety catch" linkers has allowed the synthesis of peptide thioesters with conventional Fmoc chemistry.⁽⁸⁾

Synthesis enhancement strategies

The synthesis of difficult sequences has been the focus of much research. Several purely synthetic methods have been developed to overcome the aggregation and folding of peptide chains during SPPS.

Alanine-threonine pseudoproline dipeptide is deprotected by trifluoroacetic acid to yield a "natural" alanine-threonine

The development of the pseudoproline dipeptide by Mutter and coworkers⁽⁹⁾ has greatly improved the synthetic quality of peptides by minimizing aggregation of peptide chains during Fmoc-based SPPS. Pseudoprolines consist of serine or threonine-derived oxazolidines with proline-like ring structure. Virtually any amino acid can be associated with the serine or threonine derivatives (Figure 3). The particular structure of the dipeptide causes a kink in a growing peptide backbone and delays, or prevents, aggregation. Currently, the use of pseudoproline has enabled the synthesis of peptides previously inaccessible by classical SPPS, such as peptide sequences responsible for amyloid formation.^{(10, 11)}

Polystyrene-based resins are the most commonly used support for SPPS, but are limited by their own hydrophobicity. The development of more hydrophilic supports led to the commercialization of polyethylene glycol (PEG) based resins. The amphiphilic properties of PEG-based resins allow them to swell in polar and non-polar solvents alike in a manner superior to traditional polystyrene supports. A remarkable example of long and difficult peptide synthesis using PEG-based resins was recently reported with the synthesis of the RANTES chemokine protein.⁽¹²⁾

Conclusion

We have reviewed a few strategies for the synthesis of long or difficult peptides. Unfortunately the degree of difficulty for the synthesis of a peptide can only be assessed empirically, as every peptide sequence possesses its own unique physical properties. Aggregation of the peptide chain due to hydrophobic interactions or hydrogen bonding within the peptide backbone is the main reason for synthesis failure. The methods reviewed in this short article enumerate a few powerful tools, currently widely available, to increase peptide synthesis success rates. More elaborate methods, such as microwave-assisted synthesis, have been shown to improve the efficiency of the synthesis of long peptides.

References

1. Merrifield, R. B., J. Am. Chem. Soc. 85:2149 (1963).

2. Dawson, P. E. et al., Science 266:776-779 (1994).

3. Macmillan, D., Angew. Chem. Int. Ed. Engl. 45:7668-7672 (2006).

4. Yamamoto, N. et al., J. Am. Chem. Soc. 130:501-510 (2008).

5. Johnson, E. C. et al., J. Am. Chem. Soc. 129:11480-11490 (2007).

6. Camarero, J. A. et al., J. Pept. Res. 51:303-316 (1998).

7. Muir, T. W., Annu Rev Biochem. 72:249-289 (2003).

8. Backes, B. J. et al., J. Am. Chem. Soc., 118:3055-3056 (1996).

9. Mutter, M. et al., Pept. Res., 8:145-153 (1995).

10. White, P. et al., J. Pept. Sci. 10:18-26 (2004).

11. Abedini, A. and Raleigh, D. P., Org. Lett. 7:693-696 (2005).