Fmoc Experimental Procedure

In this section we provide experimental procedures for the synthesis of various 2-nitrophenyl-containing amino acid derivatives glutamic acid derivative 109 230 (Scheme 28), glycine derivative 112 230 and aspartic acid derivative 114 230 (Scheme 29), Fmoc-protected tyrosine derivative 119 234 (Scheme 30), and glycine derivatives 120 235 and 122 232 (Scheme 31). 

As in the case of Boc protection, the Fmoc group is not usually introduced on solid phase, but rather in solution, by the use of an activated Fmoc derivative (e.g. the chloroformate Fmoc-Cl or O-Fmoc-.V-hydroxysuccinimide, Fmoc-OSu) and aqueous base (Experimental Procedure 10.3)., V-/ lkylamino acids bound to cross-linked polystyrene have been Fmoc-protected by treatment with Fmoc-Cl (4 equiv.) and DIPEA (6 equiv.) in DCM for 2 h [132,259], Primary amines on insoluble supports can also be converted into Fmoc derivatives under these conditions [260]. 

Transesterification under strongly basic reaction conditions has been used to acy-late support-bound alcohols with alkyl esters (Entry 10, Table 13.12). For sensitive acids, the Mitsunobu reaction is a particularly mild method of esterification. This reaction gives high yields with support-bound primary aliphatic alcohols and proceeds under essentially neutral reaction conditions (Experimental Procedure 13.4). Mitsunobu esterification of PEG with /V-Fmoc amino acids has also been reported [172]. 

Standard solid-phase peptide synthesis requires the first (C-terminal) amino acid to be esterified with a polymeric alcohol. Partial racemization can occur during the esterification of N-protected amino acids with Wang resin or hydroxymethyl polystyrene [200,201]. /V-Fmoc amino acids are particularly problematic because the bases required to catalyze the acylation of alcohols can also lead to deprotection. A comparative study of various esterification methods for the attachment of Fmoc amino acids to Wang resin [202] showed that the highest loadings with minimal racemization can be achieved under Mitsunobu conditions or by activation with 2,6-dichloroben-zoyl chloride (Experimental Procedure 13.5). iV-Fmoc amino acid fluorides in the presence of DMAP also proved suitable for the racemization-free esterification of Wang resin (Entry 1, Table 13.13). The most extensive racemization was observed when DMF or THF was used as solvent, whereas little or no racemization occurred in toluene or DCM [203]. 

During acylations with Fmoc-protected amino acids, addition of bases should be avoided as these could lead to partial deprotection and thence to multiple incorporation of the amino acid. Small amounts of DIPEA or pyridine, however, do not usually cause major problems (see Experimental Procedure 13.5). 

Examples of off-bead yield estimation that do not require cleavage include reactions where new species are stoichiometrically formed in solution, and their quantification provides an indirect, but accurate, yield estimation of the SPS step. The classical Fmoc deprotection of amines in peptide synthesis is a widely used example. In a typical experimental procedure, the beads are treated with a 20% DMF solution of piperidine at rt for 20 min and the solution is recovered together with the resin washings. The solution is brought to a constant volume (typically 10 mL) by addition of DMF, and the quantitation is carried out by reading the UV absorbance of the piperidine-... 

Experimental procedures for synthesis of peptides on solid supports by the Fmoc/tBu chemistry are reported in Section 4.3.2.2.3, and for solution and solid-phase synthesis of glyco- and phosphopeptides or sulfated peptides in Sections 6.3, 6.5, and 6.6, respectively. 

The following experimental procedures describe the solid-phase synthesis of peptides by means of Fmoc chemistry and TBTU activation (Scheme 8), as well as by HBTU-mediated coupling of Boc amino acid derivatives and in situ neutralization (Scheme 9). Segment condensations on solid phase with C-ternoinal glycine or proline residues can be easily achieved, even on a multigram scale (Scheme 10). Segment condensations involving the activation of other amino acids require the careful optimization of reaction conditions in order to suppress extensive epimerization (see Scheme 11 and Section 3.8.1.2). 

The synthesis and on-resin cyclization of polycyclic peptides of type I is described in the experimental part of Section 6.8.3.2.3. If side-chain-to-side-chain cyclization is needed the couplings of H-Gly-OFm and Fmoc-Gly-OH should be omitted from that procedure. 

Experimenter s notes The same procedure can be used to attach the sugar to Rink amide resin for Fmoc-based synthesis. The Rink amide resin must be deprotected as usual before coupling of the sugar. 

---