Fluorenylmethyloxycarbonyl protecting group explained

The fluorenylmethoxycarbonyl protecting group (Fmoc) is a base-labile protecting group used in organic synthesis.

Protection & Formation

Fmoc carbamate is frequently used as a protecting group for amines, where the Fmoc group can be introduced by reacting the amine with fluorenylmethyloxycarbonyl chloride (Fmoc-Cl), e.g.:^[1]

The other common method for introducing the Fmoc group is through 9-fluorenylmethylsuccinimidyl carbonate (Fmoc-OSu), which may itself be obtained by the reaction of Fmoc-Cl with the dicyclohexylammonium salt of N-hydroxysuccinimide.^[2]

Reacting with 9-fluorenylmethyloxycarbonyl azide (itself made by reacting Fmoc-Cl with sodium azide) in sodium bicarbonate and aqueous dioxane is also a method to install Fmoc group.^[3]

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]

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.^[5] A typical SPPS Fmoc deprotection is performed with a solution of 20% piperidine in N,N-dimethylformamide (DMF).

Common deprotection cocktails for Fmoc during SPPS:

• 20% piperidine in DMF (Fmoc group has an approximate half life of 6 seconds in this solution)^[6]
• 5% piperazine, 1% DBU and 1% formic acid in DMF. This method avoids the use of strictly controlled piperidine.^[7] No side product was observed for a peptide with 9 residues synthesized with this method.^[8]

Notes and References

1. 10.1016/j.molstruc.2007.11.059 . A solid-state 17O NMR, X-ray, and quantum chemical study of N-α-Fmoc-protected amino acids . 2008 . Yamada . Kazuhiko . Hashizume . Shimizu . Ohki . Yokoyama . Journal of Molecular Structure . 888 . 1–3 . 187–196 . Daisuke . Tadashi . Shinobu . Shigeyuki.
2. Paquet, A. . Introduction of 9-fluorenylmethyloxycarbonyl, trichloroethoxycarbonyl, and benzyloxycarbonyl amine protecting groups into O-unprotected hydroxyamino acids using succinimidyl carbonates . . 60 . 976–980 . 1982 . 10.1139/v82-146 . 8 . free.
3. Carpino. Louis A.. Han. Grace Y.. 9-Fluorenylmethoxycarbonyl amino-protecting group. The Journal of Organic Chemistry. 37. 22. 3404–3409. 10.1021/jo00795a005. 1972.
4. Book: 10.1016/s0076-6879(97)89043-x . [4] Standard Fmoc protocols . Solid-Phase Peptide Synthesis . Methods in Enzymology . 1997 . Wellings . Donald A. . Atherton . Eric . 289 . 44–67 . 9353717 . 9780121821906 .
5. J. Jones, Amino Acid and Peptide Synthesis, 2nd edn., Oxford University Press, 2002
6. Book: Wuts. P. G. M.. Greene, T.W.. 2006. Greene's Protective Groups in Organic Synthesis. 10.1002/0470053488. 9780470053485. J. Wiley. NY.
7. Ralhan . Krittika . KrishnaKumar . V. Guru . Gupta . Sharad . Piperazine and DBU: a safer alternative for rapid and efficient Fmoc deprotection in solid phase peptide synthesis . RSC Advances . 8 December 2015 . 5 . 126 . 104417–104425 . 10.1039/C5RA23441G . 2015RSCAd...5j4417R . en . 2046-2069.
8. Lam . Pak-Lun . Wu . Yue . Wong . Ka-Leung . Incorporation of Fmoc-Dab(Mtt)-OH during solid-phase peptide synthesis: a word of caution . Organic & Biomolecular Chemistry . 30 March 2022 . 20 . 13 . 2601–2604 . 10.1039/D2OB00070A . 35258068 . 247175352 . en . 1477-0539.