200ꢀ CHIMIAꢀ2010,ꢀ64,ꢀNo.ꢀ3ꢀ
doi:10.2533/chimia.2010.200ꢀ
Columns
Universities of Applied Sciences
FH
Fachhochschulen – Hautes Ecoles Spécialisées
HES
Fig. 1. The two major
Thermal Cleavage of the Fmoc Protection Group
rotamers of the carbamate
group.
Stefan Höck*a, Roger Martib, Rainer Riedla, and Marina
Simeunovicc
O
O
*Correspondence:ꢀDr.ꢀS.ꢀHöcka,ꢀTel.:ꢀ+41ꢀ58ꢀ934ꢀ5574,ꢀ
E-mail:ꢀstefan.hoeck@zhaw.ch,ꢀ
R
R
O
N
H
O
N
H
aZHAWꢀZurichꢀUniversityꢀofꢀAppliedꢀSciences,ꢀInstituteꢀofꢀChemistryꢀandꢀ
ꢀBiologicalꢀChemistryꢀ(ICBC),ꢀCampusꢀReidbach,ꢀEinsiedlerstrasseꢀ31,ꢀꢀ
CH-8820ꢀWädenswil
bEcoleꢀd’Ingénieursꢀetꢀd’ArchitectesꢀdeꢀFribourg,ꢀDépartementꢀdesꢀTechnologiesꢀ
Industrielles,ꢀFilièreꢀdeꢀChimie,ꢀBdꢀdeꢀPérollesꢀ80,ꢀCH-1705ꢀFribourg
cPartꢀofꢀMS’sꢀBachelorꢀthesis,ꢀCurrentꢀAddress:ꢀEmpaꢀDübendorf,ꢀ
Überlandstrasseꢀ129,ꢀCH-8600ꢀDübendorf
base-free cleavage of the Fmoc group that can easily be tested
for any given substance with a simple high-temperature NMR
experiment. This method is also applicable in the presence of
free carbonic acid groups, where the pH of the reaction mixture
is slightly acidic throughout the whole reaction.
Abstract: TheꢀFmocꢀprotectionꢀgroupꢀisꢀamongꢀtheꢀmostꢀcom-
monlyꢀ usedꢀ protectionꢀ groupsꢀ forꢀ theꢀ aminoꢀ function.ꢀ Aꢀ fastꢀ
methodꢀforꢀtheꢀthermalꢀdeavageꢀofꢀthisꢀprotectionꢀgroupꢀunderꢀ
base-freeꢀconditionsꢀwithoutꢀtheꢀneedꢀforꢀdibenzofulveneꢀsca-
vengersꢀ isꢀ presented.ꢀ Theꢀ advantagesꢀ ofꢀ thisꢀ methodꢀ includeꢀ
straightforwardꢀtestabilityꢀbyꢀmeansꢀofꢀaꢀsimpleꢀhigh-tempera-
tureꢀNMRꢀexperiment,ꢀusuallyꢀhighꢀyields,ꢀandꢀgoodꢀselectivityꢀ
towardsꢀtheꢀBOCꢀprotectionꢀgroupꢀandꢀt-butylꢀethers.
2. Results and Discussion
2.1 Preliminary Testing in High-temperature NMR
Experiments
The applicability of the thermal Fmoc cleavage for a given
molecule was at first tested for each substrate in a high-temper-
ature NMR experiment by dissolving the Fmoc-protected amine
in d6-DMSO and keeping the solution at a sample temperature of
120 °C. The reaction’s progress was monitored by comparison of
the increasing signal of the olefinic hydrogen atoms of dibenzo-
fulvene (s at 6.21 ppm) with the signals of the aromatic hydrogen
atoms (7 – 8 ppm; Fig. 2).
Keywords: Dibenzofulvene · Fmoc protection group ·
High-temperature NMR · Peptide synthesis · Thermal cleavage
1. Introduction
1.1 The Fmoc Protection Group
The Fmoc (fluorenylmethoxy carbonyl) protection group is
among the most commonly used protection groups for the amino
function especially in solid-phase peptide synthesis (for a review,
see ref. [1]). It can be easily introduced by coupling an amine
with an activated 9-fluorenylmethyl carbonate like Fmoc-Cl,[2]
Fmoc-OSu,[3] or Fmoc-OBt.[3a,b] One major advantage of the
Fmoc group is its stability towards acidic conditions, which al-
lows the selective removal of other protection groups like BOC
(t-butyl carbamate) in the presence of strong acids. The Fmoc
group can be easily cleaved by using amine bases such as 20%
piperidine or 50% morpholine in DMF.[1] Since Fmoc cleavage
results in the liberation of a primary or secondary amine, auto-
catalytic decomposition of the Fmoc group in only slightly basic
environments has been observed.[4]
2.2 Solvent Effects and Mechanistic Aspects
In a first set of experiments, the role of the solvent in the
thermal cleavage of the Fmoc group was investigated. At 120
°C quantitative cleavage of the Fmoc group from FmocProOH
(Fmoc-l-proline) was observed after 15 min in DMF, NMP (N-
FmocProPheOEt inDMSO
1H NMR
at 24°C (1)
at 110°C (2)
after 15' at 120°C (3)
3
2
1
1.2 Dynamic Effects of the Carbamate Group in NMR
Experiments
One peculiarity of carbamate and amide groups is their high
rotational barrier for the rotation around the C(O)–N bond. In
NMR spectroscopy this usually leads to a broadening or even
doubling of at least some of the molecule’s signals due to the lon-
gevity of the two major rotamers (Fig. 1). The occurrence of such
dynamic effects can be circumvented by increasing the sample
temperature during the NMR measurement. Usually, tempera-
tures of 100 °C and above are needed to observe a single set of
sharp signals.
8.6
8.2
7.8
7.4
7.0
6.6
6.2
4.6
4.2
3.8
2.2
1.8
1.4
1.0
During such a high-temperature experiment in d6-DMSO
we observed that the Fmoc protection was neatly cleaved within
10 min at 120 °C without any amine base being present in the
mixture. Further investigation revealed a facile method for the
Fig. 2. Selected regions of the 1H-NMR spectrum of FmocProPheOEt
at 1) 24 °C; 2) 110 °C; 3) after 15 min at 120 °C. Note the increase in
intensity of the signal at 6.2 ppm, resulting from the olefinic hydrogen
atoms of dibenzofulvene.