Difficult sequences

Y Sohma, M Sasaki, Y Hayashi, T Kimura, Y Kiso. Novel and efficient synthesis of difficult sequence-containing peptides through O-N intramolecular acyl migration reaction of O-acyl isopeptides. Chem Commun 124, 2004. 

RC de L Milton, SCF Milton, PA Adams. Prediction of difficult sequences in solid-phase peptide synthesis. J Am Chem Soc 112, 6039, 1990. 

C Hyde, T Johnson, D Owen, M Quibell, RC Sheppard. Some difficult sequences made easy. A study of interchain association in sohd-phase peptide synthesis. Int J Pept Prot Res 43, 431, 1994. 

The insolubility of peptides is not limited to the solution phase and can occur during peptide synthesis on the solid phase where it manifests itself as the difficult sequence problem. On-resin peptide aggregation and poor peptide segment solubility are due to the formation of (3-sheet-like structures. Difficult sequences contain predominantly hydrophobic residues that consistently show incomplete acylation and a characteristic shrinkage of the peptide-resin during synthesis.111 Reversible backbone protection was introduced into Fmoc/ tBu SPPS after the study of synthesis failure during the assembly of difficult sequences1121 and is now routinely used to circumvent synthesis failure in SPPS. 

Although this methodology has been successfully used for a number of peptides, its efficacy for the cyclization of peptides with sterically hindered cyclization sites or difficult sequences of preferred extended conformation has not been investigated. 

We have found that although the use of an automatic synthesizer can save time and can be relatively unproblematic for simple sequences, the synthesis of peptides manually allows for more flexibility and control over the assembly process. This is particularly important for the synthesis of difficult sequences as it permits quick and easy intervention at any point. 

Cross-linked polystyrene (PS)-based resins are most commonly used for routine SPPS. Beads of 200 to 400 mesh size distribution (corresponding to a diameter of about 50 pm) and a loading of 0.5 to 0.8 mmol/g present good characteristics for polymer swelling in solvents such as DMF and DCM, diffusion of reactants into the polymer matrix, and accessibility of linker sites buried into the bead. For larger peptides (more than 25 amino acids) or more difficult sequences, a lower loading is required (0.1-0.2 mmol/g). 

Cross-linked polyamide (PA)-based resins and composite PS-polyethylene glycol (PEG)-based resins are much more hydrophilic supports exhibiting physical properties different from PS resins at microscopic and macroscopic levels (7). These supports, often with a lower loading capacity, may represent an alternative to standard cross-linked PS resins for the synthesis of difficult sequences and large peptides. 

Add HBTU (2.9 eq compared with 3 eq of amino acid), screw the cap, and stir for 30 min. Eor difficult sequences it is recommended to preactivate the A-protected amino acid before coupling to the free A -amino function to limit the guanylation side-reaction. 

Yamashiro, D., Blake, J., and Li, C. H. (1976) The use of trifluoroethanol for improved coupling in solid-phase peptide synthesis. Tetrahedron Lett. 17,1469-1472. Milton, S. C. and Milton, R. C. (1990) An improved solid-phase synthesis of a difficult-sequence peptide using hexafluoro-2-propanol. Int. J. Pept. Protein Res. 36, 193-196. 

Ser, Thr, and Trp were left unprotected for short sequences (i.e., 5 to 6 amino acids in length). Initially we used benzyl-protecting groups for the alcohols, but resulting from incomplete deprotection for some libraries, we left the side-chain unprotected. For Trp, alkylations during activation/ deprotection step was sometimes observed. As such we sometimes include the formyl protecting group on the indole for difficult sequences. 

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