Because the fluorenyl group is highly fluorescent, certain UV-inactive compounds may be reacted to give the Fmoc derivatives, suitable for analysis by
reversed phase HPLC. Analytical uses of Fmoc-Cl that do not use chromatography may be limited by the requirement that excess Fmoc-Cl be removed before an analysis of
fluorescence.
Cleavage & Deprotection
The Fmoc group is rapidly removed by base. Piperidine is usually preferred for Fmoc group removal as it forms a stable adduct with the dibenzofulvene byproduct, preventing it from reacting with the substrate.[4][5]
Roles in SPPS
The use of Fmoc as a temporary protecting group for amine at the
N-terminus in SPPS is very widespread for Fmoc/tBu approach, because its removal with piperidine solution does not disturb the acid-labile linker between the peptide and the resin.[6] A typical SPPS Fmoc deprotection is performed with a solution of 20%
piperidine in
N,N-dimethylformamide (DMF).[7]
Common deprotection cocktails for Fmoc during SPPS:
20% piperidine in DMF (Fmoc group has an approximate half life of 6 seconds in this solution)[7]
5%
piperazine, 1%
DBU and 1%
formic acid in DMF. This method avoids the use of strictly controlled piperidine.[8] No side product was observed for a peptide with 9 residues synthesized with this method.[9]
References
^Yamada, Kazuhiko; Hashizume, Daisuke; Shimizu, Tadashi; Ohki, Shinobu; Yokoyama, Shigeyuki (2008). "A solid-state 17O NMR, X-ray, and quantum chemical study of N-α-Fmoc-protected amino acids". Journal of Molecular Structure. 888 (1–3): 187–196.
doi:
10.1016/j.molstruc.2007.11.059.
^Carpino, Louis A.; Han, Grace Y. (1972). "9-Fluorenylmethoxycarbonyl amino-protecting group". The Journal of Organic Chemistry. 37 (22): 3404–3409.
doi:
10.1021/jo00795a005.