Methods for Removing the Finoc Group

Abstract

The electron withdrawing fluorene ring system of the 9-fluorenyl-methyloxycarbonyl (Fmoc) group renders the lone hydrogen on the β-carbon very acidic and, therefore, susceptible to removal by weak bases (, ). Following the abstraction of this acidic proton at the 9-position of the fluorene ring system, β-elimination proceeds to give a highly reactive dibenzofulvene intermediate (, , , , ). Dibenzofulvene can be trapped by excess amine cleavage agents to form stable adducts (, ). The stability of the Fmoc group to a variety of bases (, , , , ) is reported in Table 1. The Fmoc group is, in general, rapidly removed by primary (i.e., cyclohexylamine, ethanolamine) and some secondary (i.e., piperidine, piperazine) amines, and slowly removed by tertiary (i.e., triethylamine [Et3N], N, N-diisopropylethylamine [DIEA]) amines. Removal also occurs more rapidly in a relatively polar medium (N, N-dimethyl-formamide [DMF] or N-methylpyrrolidone [NMP]) compared to a relatively nonpolar one (dichloromethane [DCM]). During solid-phase peptide synthesis (SPPS), the Fmoc group is removed typically with piperidine, which in turn scavenges the liberated dibenzofulvene to form a fulvene-piperidine adduct. Standard conditions for removal include 30% piperidine-DMF for 10 min (), 20% piperidine-DMF for 10 min (, ), 55% piperidine-DMF for 20 min (), 30% piperidine in toluene-DMF (1:1) for 11 min (, , , ), 23% piperidine-NMP for 10 min (), and 20% piperidine-NMP for 18 min ().Table 1 Removal of the Fmoc Group Compound Base Solvent Time, min Deprotection, % Reference Fmoc-Gly-PS 10% Morpholine DCM 240 18 a Fmoc-Gly-PS 10% Morpholine DMF 240 75 a Fmoc-Gly-PS 50% Morpholine DCM 240 100 a Fmoc-Val 50% Morpholine DMF 1 50 b Fmoc-Ala-OtBu 50% Morpholine DCM 120 100 c Fmoc-Gly-PS 10% Piperidine DCM 240 100 a Fmoc-Val 20% Pipendine DMF 0.1 50 b Fmoc-Gly-HMP-PS 23% Piperidine NMP 0.25 50 d Fmoc-Ala-OtBu 50% Piperidine DCM <5 100 c Fmoc-Val 5% Piperazine DMF 0.33 50 b Fmoc-Ala-OtBu 50% Piperazine DCM 60 100 c Fmoc-PCA 59% 1,4-bis-(3-aminopropyl)piperazine CDC13 2 100 e Fmoc-Val 50% Dicyclohexylamine DMF 35 50 b Fmoc-Ala-OtBu 50% Dicyclohexylamine DCM >1080 100 c Fmoc-Val 50%DIEA DMF 606 50 b Fmoc-Ala-OtBu 50% DIEA DCM >1080 100 c Fmoc-Val 10% 4-Dimethylaminopyridine DMF 85 50 b Fmoc-Ala-OtBu 50% DBU DCM <5 100 c Fmoc-Ala-OtBu 50% Pyrrolidine DCM <5 100 c Fmoc-Ala-OtBu 50% Cyclohexylamine DCM <5 100 c Fmoc-Ala-OtBu 50% Ethanolamine DCM <5 100 c Fmoc-Ala-OtBu 50% Diethylamine DCM 180 100 c Fmoc-Ala-OtBu 50% Triethylamine DCM >1080 100 c Fmoc-Ala-OtBu 50% Ammonia DCM >1080 100 c Fmoc-Ala-OtBu 50% Tributylamine DCM >1080 100 c Fmoc-Ala-OtBu 1.0 mM triethylenediamine DCM >1080 100 c Fmoc-Ala-OtBu 1 0 mM Hydroxylamine HC1 DCM >1080 100 c Fmoc-Ala-OtBu 0 5 mmol Proton sponge DCM >1080 100 c Fmoc-Ala-OtBu 2 0 mmol NaOH 30% CH3OH-p-dioxane <5 100 c Fmoc-PCA 50% Tris(2-aminoethyl)amme CDCl3 2 100 c Fmoc-PCA 59% 1,3-Cyclohexanebis-(methylamine) CDC13 2 100 c a Deprotection of Fmoc-Gly-PS was quantitated spectrophotometrically at 273 nm (6) b Deprotection of Fmoc-Val was quantitated by amino acid analysis (7) c Deprotecùon of Fmoc-Ala-O-tu was quantitated by thin-layer chromatography (8) d Deprotection of Fmoc-Gly-HMP-PS was quantitated by ninhydrin analysis (9) e Jeprotection of 9-fluorenylmethyl N-p-chlorophenyl carbamate (Fmoc-PCA) was quantitated by 1H-NMR (10). Dibenzofulvene was scavenged in 2 min by tris(2-aminoethyl)amine, 15 min by 1,3-cyclohexanebis-(methylamine), and 50 min by l,4-bis-(3-aminopropyl)piperazine.