Merrifield resin can be loaded with Boc-amino acids as described in Method 11, or can be purchased pre-loaded with the C-terminal amino acid.
Method 1. Attachment of Boc-amino acids to Merrifield resins
One of the simplest methods for esterification to hydroxymethyl-functionalized linkers (Wang, HMPA, and HMBA resins) is to use the symmetrical anhydride of the protected amino acid in the presence of a catalytic amount of p-dimethylaminopyridine (DMAP) (Method 2)2. However, due to the basic character of this material, enantiomerization and dipeptide formation can be expected; the amount depending on the quantity of DMAP used, the length of the reaction and the nature of the amino acid.
The MSNT method3,4 (Method 3) is the method of choice in difficult circumstances, such as loading of HMBA resins or when attaching enantiomerization prone amino acid derivatives5,6.
Cysteine and histidine are particularly prone to enantiomerization and should not be loaded by this method. For these residues, the use of 2-ClTrt resin is recommended; esterification of the C terminal residue is free from enantiomerization and dipeptide formation7 because attachment does not involve activation of the incoming protected amino acid (Method 5). When peptide acids containing Pro as the C-terminal residue are desired, the use of trityl-based resins is recommended.
Once the resin is loaded the substitution of the resin can be easily determined using Method 10.
Note: This method is not suitable for His or Cys.
Attachment of amino acid derivatives and other carboxylic acids to linkers containing primary amino groups can normally be affected using standard methods of amide bond formation (see Method 4). Hydroxylamine, Weinreb amide, and resins functionalized with secondary amines are much more difficult to load; for these the use of HOAt/DIPCDI or HATU/DIPEA activation is required.
In contrast to benzyl alcohol-based supports, attachment of Fmoc-amino acids to trityl-based resins, such as 2-chlorotrityl or NovaSyn® TGT resins, is free from enantiomerization7, making them ideal for the immobilization of sensitive residues such as Cys and His (Method 5). The resin also protects Cys from enantiomerization during chain extension. They are particularly useful in the synthesis of C-terminal prolyl peptides as the bulk of the trityl linker helps to prevent diketopiperazine formation8-10. When loading 2-chlorotrityl chloride resin, it is important to ensure that all amino-acid derivatives, glassware and solvent are thoroughly dried before use.
NovaSyn® TGT alcohol resins must be converted to the chloride form before attachment of the amino acid (Method 6).
NOTE: it is important to dry all solvents and glassware before use.
Attachment of carboxylic acids
NOTE: it is important to dry all solvents and glassware before use.
Success in using the Dbz strategy11 depends on regioselective acylation of only one of the two linker amines with the C-terminal amino acid residue and to avoid acylation of the unprotected amine during chain extension. Incomplete acylation leads to formation of C-terminally truncated peptides as new chains are propagated by acylation of any unreacted amines during subsequent coupling cycles. Whereas, overacylation results to formation of branched peptides with chains growing off both linker amines. Therefore, the selection of acylation method of attachment of the C-terminal residue and subsequent couplings is critical if good results are to be obtained (Method 7).
Particularly problematic is the coupling of glycine residues, especially if they occur close to the C-terminus of the peptide. This reactive and unhindered amino acid can couple to the free Dbz-amino group if uronium or phosphonium activation is used. In our hands best results are obtained if glycine residues are introduced using Fmoc-Gly-OPfp/HOBt. This precaution may be unnecessary once the peptide is extended beyond 10 residues, as hindrance should reduce the reactivity of unprotected Dbz amine. Furthermore, the use of strong activators like HATU or HCTU should be avoided as their use can lead to branching. In our hands, HBTU/HOBt appears to work well for coupling of all residues except Gly, where the use of the pre-formed OPfp in conjunction with HOBt gives minimal branching.
The use of Alloc protection for blocking the second amino group has been advocated to avoid all issues with branching and truncation12. Dbz resins as supplied contain mostly 3-Fmoc-Dbz, with small amounts of 4-Fmoc-Dbz and bis-Fmoc-Dbz. Capping the resin with Alloc-Cl prior to removal of the Fmoc group will thus reduce the maximum potential for branching or truncation to 6%. For hindered amino acids, it has been found necessary to load the resin prior to capping with Alloc. The Alloc group must be cleaved off with Pd(0) before conversion to the Nbz form (Figure 1).
Figure 1: Alloc protection to avoid branching in Dbz resin
Loading of sulfamyl-based resins is best achieved with carboxylic acids activated with PyBOP® and DIPEA in CHCl3 at -20 °C13 or with DIPCDI/N-methylimidazole (Method 8). In the case of PyBOP® activation, the loading efficiencies are reported to vary from >95% for Cys, Met and His to 44% for Pro, the worst case. Extent of racemization for the loading of Fmoc-Phe and Fmoc-Leu by these methods are 0.5% and 0.3%, respectively. However, in practice the loading obtained by these methods can be highly variable, and problems can occur with over acylation of the linker. Furthermore, the substitution of the support must be determined before starting peptide synthesis.
DHP HM resin consists of 3,4-dihydro-2H-pyran-2-yl-methanol linker14 attached to 100-200 mesh chloromethyl polystyrene, and is a useful tool for the synthesis of peptide alcohols.
In contrast to trityl-based supports, where the use of prolonged reaction times and elevated temperatures are often required to achieve satisfactory loadings, derivatization of DHP HM resin is relatively straightforward, with even secondary alcohols being loaded without difficulty. Typically, this process involves treating the resin in DCE with an excess of alcohol in the presence of pyridinium p-toluenesulfonate (PPTS); full experimental details are given in Method 9.
One of the simplest and most effective methods of preparing peptide aldehydes, which involves the solid-phase immobilization of an amino aldehyde by formation of an oxazolidine between a pre-formed Fmoc-amino aldehyde and H-Thr-Gly-NovaSyn® TG resin15.
After loading the resin, the oxazolidine nitrogen should be blocked by treatment with Boc-anhydride. The resultant acyloxazolidine is stable to base and is compatible with Fmoc protocols.
For peptides containing a C-terminal aspartal, argininal, leucinal, phenylalaninal, or valinal residue, pre-loaded resins are available.
For estimating the loading of resins derivatized with Fmoc-amino acids, the simplest approach involves cleaving the Fmoc group with DBU and measuring the solution concentration of the liberated dibenzofluvene by U.V. spectroscopy16.