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Long Peptide Sequences

Most proteinogenic-based peptides, with the exception of some hydrophobic sequences, can be synthesized in a linear fashion by solid-phase peptide synthesis (SPPS) methodologies. Longer sequences, however, particularly sequences that exceed 70 amino acids in length, often require alternative techniques. Poor solvation of the protected peptide during synthesis and the formation of intermolecular hydrogen bonds (i.e., β-sheets) among fragments can result in inefficient coupling and deprotection.

CPC Scientific has experience in the synthesis of long peptide sequences. We employ a variety of strategies to overcome poor solvation and aggregation; some of these methods include:

  • Polar solvent mixtures (e.g., "Magic Mixture",[1,2] chaotropic salt additives)
  • Increased temperatures and microwave irradiation
  • Protected fragment condensation
  • Native Chemical Ligation (i.e., unprotected fragment condensation)
  • Low resin substitution and high-swelling resins

To help mitigate aggregation in SPPS, polar solvent cocktails have been developed to increase reaction mixture polarity and introduce hydrogen bond acceptors to compete with β-sheets that form between peptide fragments. The "Magic Mixture", introduced in 1992 by Kent and co-workers, contains DMF/DCM/NMP (1:1:1), 1% Triton X-100, and 2 M ethylenecarbonate (a strong hydrogen-bond donor) has lead to improved coupling efficiencies in linear peptides and on-resin cyclizations.[3] Other additives such as chaotropic salts (LiCl, KSCN, guanidine HCl) have be shown to also reduce aggregation caused by peptide secondary structure.

While chaotropic additives, polar cocktails, and high-swelling resins have improved coupling efficiencies in SPPS for longer peptides, their benefits diminish with length and the resultant crude peptides can be exceptionally challenging to purify. Condensation of orthogonally protected peptide fragments have met some of these challenges. In 1973, Wang introduced p-alkoxybenzyl alcohol and p-alkoxybenzyloxycarbonylhydrazide resins suitable for the synthesis of protected peptide fragments bearing a free carboxylic acid or hydrazide group.[4] The preparation of protected fragment is relatively easy for short peptide fragments; however, longer fragments (>10 AAs) can suffer from poor solubility. In addition, coupling of fragments without a C-terminal glycine residue, can result in peptide diastereomers.

Native Chemical Ligation

native chemical ligation mechanism

In 1994, native chemical ligation (NCL) was introduced by Kent and Dawson that revolutionized the coupling of peptide fragments, enabling, for the first time, the preparation of full length proteins.[5] The key benefits of NCL are that the fragments do not require side chain protection and that the couplings can be carried out in aqueous conditions. In NCL, the thiol group of an N-terminal cysteine residue in fragment 1 reacts with a C-terminal thioester group of fragment 2. Following transthioesterification, the thioester intermediate rearranges by an intermolecular S,N-acyl shift to provide a native amide bond. NCL has been proven to be an efficient method for preparing proteins the exceed 150 amino acids that include a 166 amino acid analog of erythropoiesis protein and a 203 amino acid HIV-1 protease.

Methodology References

1. Kent SB, et al. In: Innovations and Perspectives in Solid Phase Synthesis. Epton R, editor. Andover: Intercept Ltd.; 1992. p. 1.

2. L. Zhang, C. Goldammer, B. Henkel, F. Zühl, G. Panhaus, G.Jung, E. Bayer, in “3rd lnternational Symposium on Innovation and Perspectives in SPPS, Oxford UK,” R Epton Ed., Mayflower Scientific Ltd, Birmingham 1994, 711.

3. Thakkar, Amit, Thi Ba Trinh, and Dehua Pei. "Global analysis of peptide cyclization efficiency." ACS Combinatorial Science 15, no. 2 (2013): 120-129.

4. Wang, Su-Sun. "p-Alkoxybenzyl alcohol resin and p-alkoxybenzyloxycarbonylhydrazide resin for solid phase synthesis of protected peptide fragments." Journal of the American Chemical Society 95, no. 4 (1973): 1328-1333.

5. Dawson, Philip E., Tom W. Muir, Ian Clark-Lewis, and S. B. Kent. "Synthesis of proteins by native chemical ligation." Science 266, no. 5186 (1994): 776-779.