Fmoc chemistry (revision 15)
Old revision·05:45, 31 Dec 2024·StopperingSteff
| Fmoc chemistryProtecting-group scheme | |
|---|---|
| Temporary group | Fluorenylmethyloxycarbonyl (Fmoc), base-labile |
| Side-chain groups | Acid-labile (tBu, Trt, Pbf) |
| Deprotection reagent | Piperidine in DMF |
| Final cleavage | Trifluoroacetic acid with scavengers |
| Analytical method infobox · conventions | |
Fmoc chemistry is the protecting-group scheme that dominates modern solid-phase peptide synthesis. The temporary N-terminal group, fluorenylmethyloxycarbonyl, is removed by base; the side-chain protecting groups are removed by acid. The two are orthogonal, so each can be removed without disturbing the other.[1]
Its predecessor, Boc chemistry, used acid for both and therefore required a much stronger acid for final cleavage — hydrogen fluoride — with the handling requirements that implies. Boc chemistry was the scheme of the original solid-phase method.[2] Orthogonality is what made peptide synthesis a routine operation.[1]
Deprotection is monitored spectrophotometrically: the dibenzofulvene released when Fmoc is removed absorbs strongly, so the extent of each deprotection can be followed in real time. This is one of the few in-process checks available in stepwise synthesis.[1]
The cycle
[edit]Fmoc is removed with a solution of piperidine in dimethylformamide, which abstracts the fluorenyl proton and triggers elimination. The released dibenzofulvene is trapped by excess piperidine to prevent it alkylating the peptide.[1]
The resin is then washed and the next Fmoc-protected amino acid coupled using an activating reagent. See Peptide coupling reagent. The cycle repeats once per residue.
Side-chain protection uses acid-labile groups: tert-butyl for hydroxyl and carboxyl side chains, trityl for cysteine and asparagine, and sulfonyl-type groups for arginine. All are removed in the final acidic cleavage. See Resin cleavage.[3]
Characteristic side reactions
[edit]Repeated base exposure has costs. Aspartimide formation is the most consequential: an aspartate residue can cyclise with the following backbone nitrogen under basic conditions, and the resulting succinimide reopens to give both the desired product and an isomer. Aspartate followed by glycine is the worst context — the same sequence dependence seen in deamidation.[1]
Racemisation occurs mainly at the activation step rather than at deprotection, and is worst at histidine and cysteine. It is invisible to mass spectrometry, since epimers are isobaric.[1]
Diketopiperazine formation can cleave the first two residues from the resin when the third is being added, particularly with proline or glycine in those positions. Linker choice mitigates it.[3]
See also
References
- ^ a b c d e f Behrendt R, White P, Offer J. "Advances in Fmoc solid-phase peptide synthesis." Journal of Peptide Science 22(1):4–27 (2016). PMID 26785684.
- ^ Merrifield RB. "Solid phase peptide synthesis. I. The synthesis of a tetrapeptide." Journal of the American Chemical Society 85(14):2149–2154 (1963). DOI:10.1021/ja00897a025.
- ^ a b United States Pharmacopeia, General Chapter <1503>, Quality Attributes of Synthetic Peptide Drug Substances.