Protection and deprotection of Fmoc
The Fmoc protecting group is a popular choice for solid-phase peptide synthesis. It was the first major viable alternative to the Boc group to be proposed for SPPS. Because its removal entails gentler and less hazardous reagents than Boc, it has become mainstream. All of the common amino acids are widely available as their Fmoc derivatives.
Installation
The Fmoc group is generally installed using Schotten-Baumann conditions. Fmoc-OSu (N-(9-Fluorenylmethyloxycarbonyloxy)succinimide) or Fmoc-Cl (9-Fluorenylmethyl chloroformate) may be used. Fmoc-Cl may lead to activation of carboxylate groups and subsequent amide coupling if the reaction becomes too acidic. Fmoc-OSu may decompose to Fmoc-beta-alanine if more than one equivalent is used (and if excess base is used). In both of these situations, purification may be difficult. Use slightly less than one equivalent of Fmoc-OSu or Fmoc-Cl to get selective reaction at the amine. Work up the reaction the next morning to avoid Fmoc deprotection by any unreacted amine. The amine will remain in the aqueous phase under acidic conditions.
Stability
Fmoc is removed rapidly by secondary amines like piperidine. Fmoc is removed less rapidly by primary amines, and very slowly by tertiary amines like DIPEA. Fmoc is reasonably stable to resin-bound amino acids, but could theoretically be removed by the N-terminus during extremely difficult couplings. Fmoc amino acid solutions in DMF are stable in the refrigerator for at least a week but probably not more than two. Fmoc is extremely stable in strong acids. Fmoc is incompatible with catalytic hydrogenation. Fmoc is stable to the reductive deprotection of pNZ and cleavage of allyl esters and alloc using palladium complexes.
Removal
The mechanism of Fmoc removal has two steps. First deprotonation of the fluorenyl system induces elimination yielding DBF (dibenzofulvene) and the carbamic acid, which decomposes spontaneously to release carbon dioxide along with the free amine. The DBF must be scavenged by a nucleophile otherwise it will react with the liberated amine. Piperidine is an effective scavenger.
Solid Phase
The Fmoc protecting group is efficiently removed from solid-phase synthesis resins by various mixtures of nucleophilic amines in high dielectric solvents. For example:
2% piperidine and 2% DBU in DMF
This is the cocktail used in our lab. DBU is a relatively strong amine base, while the piperidine is nucleophilic enough to scavenge the DBF byproduct. This cocktail uses less piperidine than most Fmoc removal cocktails. Everyone treats for different amounts of time. I like to do a 10-15 second treatment followed by a fresh 10-15 minute treatment. Fmoc is removed quite rapidly but should be removed completely to avoid truncated peptide sequences. 2 X 10 minutes is common. You may choose to agitate the tubes in some way during deprotection, but I usually just let them stand.
20% piperidine in DMF
The classic removal cocktail. Piperidine is toxic and malodorous. Also, piperidine is a DEA List I chemical, which complicates purchase of large quantities.
20% piperidine in DMF containing 0.1M HOBt
Inclusion of HOBt helps prevent DKP and aspartimide formation. This cocktail may also be less likely to cleave esters.
2% HOBt, 2% hexamethyleimine (azepane), 25% N-methylpyrrolidine in 1:1 DMSO/NMP.
Reported to leave thioesters intact.
50-100% morpholine in DMF
Morpholine is less basic than piperidine and less likely to harm delicate functionality.
Solution phase Fmoc deprotection
Removal of Fmoc in solution is ideally avoided. If a peptide is mistakenly cleaved while it still contains an Fmoc group, the Fmoc group may be removed using a volatile amine like diethylamine in acetonitrile. The solvent is evaporated on the rotavap and then the peptide precipitated with ether or an ether/hexanes mixture. Another option is a polystyrene-bound amine (e.g. PS-bound pyrazine). In this case, the amine both deprotonates and scavenges, and filtration removes everything except product.