As mentioned above, the generation and disappearance of Fmoc based chromophors allows the monitoring of the synthesis. Furthermore, samples may be taken to determine the load of Fmoc peptide. The completion of the deprotection reaction may be checked by cleaving samples and analyzing the obtained peptide.
Better results will be obtained by repeating a coupling with fresh reagents (and changing coupling parameters if a low conversion was obtained) rather than by prolonging the reaction. Generally, coupling protocols may be changed in the course of a synthesis, especially when optimizing an SPPS.
Mengchun Ye, Andrew Edmunds, James Morris, David Sale, Yejia Zhang and Jin-Quan Yu
"A Robust Protocol for Pd(II)-catalyzed C-3 Arylation of (1H)Indazoles and Pyrazoles: Total Synthesis of Nigellidine Hydrobromide"
The base catalyzed elimination of the suffhydryl protecting group affords dehydroalanine and the subsequent addition of piperidine yields the C-terminally modified peptide . This side reaction is minimized (but not avoided!) when trityl is used for the protection of the C-terminal Cys.
The other approach is to introduce backbone protecting groups which will prevent the formation of hydrogen bonds. Such protection is made by the introduction of the Hmb group on the αnitrogen . It has been shown that the presence of a Hmb unit every 6-7 residues is sufficient to disrupt the peptide aggregation . The Hmb protected amino acid is introduced under the form of N,O-bis-Fmoc-N-(2hydroxy-4-methoxybenzyl) derivative, the O-Fmoc protection being cleaved during the following piperidine treatment. At the end of the synthesis the Hmb group is cleaved in the final TFA cleavage.
Shengqing Ye, Weibo Yang, Timothy Coon, Dewey Fanning, Tim Neubert, Dean Stamos and Jin-Quan Yu
"N-Heterocyclic Carbene Ligand-Enabled C(sp3)-H Arylation of Piperidine and Tetrahydropyran Derivatives"