New strategies for an efficient removal of the 9- fluorenylmethoxycarbonyl (Fmoc) protecting group in the peptide synthesis

Article abstract of DOI:10.1002/(SICI)1099-0690(200002)2000:4<573::AID-EJOC573>3.0.CO;2-I

The aluminium trichloride/toluene system is investigated as a novel, unusual and straightforward reagent for the removal of the 9- fluorenylmethoxycarbonyl (Fmoc) protecting group in the solution peptide synthesis. This procedure avoids any undesired side reactions, such as the frequently observed inversion of the amino acid configuration.

Full text of DOI:10.1002/(SICI)1099-0690(200002)2000:4<573::AID-EJOC573>3.0.CO;2-I

SHORT COMMUNICATION
New Strategies for an Efficient Removal of the 9-Fluorenylmethoxycarbonyl
(
Fmoc) Protecting Group in the Peptide Synthesis
[
a]
[a]
[a]
[a]
Antonella Leggio, Angelo Liguori,* Anna Napoli, Carlo Siciliano, and
Giovanni Sindona[
a]
Keywords: Amino acids / Peptides / Fmoc chemistry / Protecting groups / Aluminium
The aluminium trichloride/toluene system is investigated as
a novel, unusual and straightforward reagent for the removal
of the 9-fluorenylmethoxycarbonyl (Fmoc) protecting group
in the solution peptide synthesis. This procedure avoids any
undesired side reactions, such as the frequently observed in-
version of the amino acid configuration.
One of the most widely used strategies in the solid phase toluene reacting system was thus tested as an alternative
synthesis of peptides is based on the masking of the aminic tool for the selective and highly efficient removal of the
function by the 9-fluorenylmethoxycarbonyl (Fmoc) Fmoc group from both N-protected amino acids and di-
[
1]
group. This approach has found extended applications peptide units.
due to the many advantages presented by the Fmoc pro-
The feasibility of the methodology was previously as-
tecting group, which can be easily removed under very mild sessed using lipophilic amino acids as appropriate model
[
2]
basic conditions. However, some limitations exist when compounds. In a typical experimental protocol, one mmol
this procedure is applied to the preparation of peptide frag- of the N-Fmoc protected derivatives 1a or 1b was treated
[
3]
ments in solution. In this case, in fact, a molar excess of with three mmol of AlCl in dry toluene under an inert
3
nitrogenated base is required either for a complete removal atmosphere (Scheme 1). The reactions went to completion
of the Fmoc group from protected elongating peptide in three hours at room temperature, as detected by TLC.
chains or for the quenching of dibenzofulvene which is gen- After this time, the reaction mixture was acidified to give
erated during the unblocking step. Furthermore, it is neces- the hydrochloride of the corresponding amino acid derivat-
sary to remove the unreacted base from the reaction envir- ive 2a or 2b. A simple solvent extraction with diethyl ether
onment before performing the next coupling.[3,4] Finally, of the aqueous phase containing the unblocked compound
the pH controlled conditions adopted in the workup pro- afforded the substituted dibenzofulvenetoluene adduct 3 as
cedure do not allow an easy separation of the unprotected a mixture of the o- and p-tolyl isomers. From these reac-
peptide from the reaction mixture containing the residual tions, pure -alanine methyl ester (4a) or -isoleucine N-
base and the dibenzofulvene-base adduct.
benzyl amide (4b) were obtained in good to excellent yields
To overcome these problems, the use of alternative (86 and 95%, respectively). Each product was allowed to
methods, such as catalytic hydrogenation,[ has been evalu- react with a molar excess of acetic anhydride in chloroform,
ated to accomplish a successful removal of the Fmoc pro- at room temperature, to give 5a and 5b, whose structures
5]
[6]
tecting group. Tetrabutylammonium fluoride (TBAF) in were confirmed by GC-MS analysis performed in parallel
the presence of thiols[ has been found to be a suitable sys- with analytical samples of each N-derivatised compound.
tem for this purpose. The presence of sulfurated nucleo-
philes can ensure a rapid capture of dibenzofulvene and
also minimises the major drawbacks associated with the
purification of the reaction mixture containing different ni-
trogenated compounds.
7]
The sensitivity of the base-labile Fmoc group to acid con-
ditions has not been recognised before. This remarkable
possibility could increase the potentiality of the protecting
group either in the peptide synthesis or in the preparation
of more complex base-sensitive organic substrates.
It seemed to be very attractive to exploit the Lewis acid
assisted unblocking of the aminic function using new and
3
straightforward strategies which could not employ het- Scheme 1. Removal of the Fmoc group by the AlCl /toluene
reagent system
eroatom-containing reagents. The aluminium trichloride/
It could be supposed that, under the adopted conditions,
[
a]
Dipartimento di Chimica, Universit a` della Calabria,
I-87030 Arcavacata di Rende (CS), Italy
Fax: (internat.) ϩ 39-984/492-044
the interaction between the Lewis acid AlCl and the ful-
3
vene moiety affords a highly reactive electrophilic species
which, in turn, forms a lipophilic adduct by reacting with
E-mail: A.Liguori@unical.it
Eur. J. Org. Chem. 2000, 573Ϫ575

WILEY-VCH Verlag GmbH, D-69451 Weinheim, 2000
1434Ϫ193X/00/0204Ϫ0573 $ 17.50ϩ.50/0
573

A. Leggio, A. Liguori, A. Napoli, C. Siciliano, G. Sindona
In conclusion we have been able to develop a new and
SHORT COMMUNICATION
toluene. The advantages of this new methodology mainly
consist in the use of toluene (the solvent of choice to carry convenient system for the removal of the Fmoc protecting
out the unblocking step) as a suitable trapping agent for the group either from N-protected lipophilic amino acids or
fulvene derivative and the high lipophilicity of the dibenzof- their ester or amide derivatives. We have also shown that
ulvenetoluene adducts (which do not contain nitrogen) an efficient trapping of the dibenzofulvene adduct, formed
which can easily be separated from the other reaction sys- during the AlCl -promoted unblocking step, can be per-
3
tem components. It is worth noting that the removal of the formed by the reaction solvent. The latter aspect is one of
Fmoc group from the N-protected amino acid derivatives the most important features of the proposed procedure.
1
a and 1b occurred without any racemisation of the chiral This strategy can also be applied in the solution phase syn-
α carbon atom. This aspect was extensively investigated us- thesis of dipeptides thus avoiding any unwanted inversion
ing the N-Fmoc--isoleucine methyl ester (6), selected as the of the amino acid stereochemistry. Further applications of
appropriate model to achieve our aim.
the optimised methodology to the removal of the Fmoc
When 6 was treated with AlCl in toluene, only the - group from some side-chain functionalised amino acids,
3
isoleucine methyl ester (7) was recovered in satisfactory and to the preparation of longer peptide chains, is currently
overall yields (Scheme 2); no traces of the -allo-isoleucine under investigation.
methyl ester (9) were detected. The absence of the inverted
amino acid derivative 9 was confirmed by subjecting the
crude material obtained from the treatment of 6 with AlCl3
to acetylation. A GC-MS analysis of the resulting mixture
Experimental Section
did not reveal any N-acetyl--allo-isoleucine methyl ester General: Solvents and reagents were purified and dried by standard
(
10), as established by injecting an authentic sample of com- procedures and distilled prior to use. Melting points were deter-
pound 10.
mined on a Kofler hot-stage apparatus and are uncorrected. IR
spectra were obtained with a Perkin–Elmer FT Paragon 1000 PC
1
spectrometer. H NMR spectra were recorded at 300 MHz on a
Bruker ACP 300 spectrometer with tetramethylsilane as internal
standard and CDCl or [D ]DMSO as solvents. Chemical shifts (δ)
3 6
are given in ppm. GC/MS analyses were carried out on a Hewlett-
Packard HP 5890 Series II gas chromatograph coupled to an HP
5
972 A Series mass-selective detector with a 30 m HP-5MS capil-
lary column with a 0.25 mm internal diameter and a 0.25 µm film
thickness (Hewlett-Packard). The mass detector was operated in
the electron-impact ionisation mode (EIMS) with an electron en-
ergy of 70 eV. Mass spectra were recorded on a Fisons Vacuum
Generators ZAB-2F spectrometer, from 2 mL of 3-nitrobenzyl al-
Scheme 2. The absence of racemisation was tested on the model
compound N-Fmoc--Ile-OMe
cohol (NBA)/sample mulls, by fast atom bombardment
ϩ
(
FAB MS), with a neutral xenon beam operating at 8 keV and a
total current of 10 µA. Reaction mixtures were monitored by TLC
using Merck Silica Gel 60-F254 precoated glass plates. When re-
quired, the reactions were carried out under inert atmosphere (N ).
2
We further report that any racemisation process was also
suppressed in the unblocking of the N-Fmoc protected di-
peptides (Scheme 3). As an example, N-Fmoc--Ile--Val-
OMe (11) and N-Fmoc--allo-Ile--Val-OMe (13) were Synthesis of N-acetyl-l-Ile-NHCH Ph (5b): AlCl (0.40 g, 3 mmol)
2
3
treated with AlCl under experimental conditions similar to was added to a magnetically stirred solution of N-Fmoc--Ile-
3
NHCH
sulting mixture was maintained at room temperature for 3 h, mon-
itoring the conversion of the protected precursor by TLC (CHCl
CH OH, 90:10). HCl 1 was then added and the acidified solution
pH ϭ 2) was extracted with diethyl ether (3 ϫ 10 mL). The aque-
ous phase was made basic with a saturated NaHCO solution, then
extracted with chloroform (4 ϫ 10 mL) and washed with distilled
water (2 ϫ 10 mL). The organic layer was dried (Na SO ) and
2
Ph (1b) (0.44 g, 1 mmol) in dry toluene (20 mL). The re-
those adopted for the removal of the protecting group from
a, 1b and 6. The resulting mixtures were then acetylated
1
3
/
in chloroform to afford the N-acetyl derivatives 12 and 14,
respectively, in very high yields. All the obtained com-
pounds showed retention of configuration of the chiral car-
bon atom of the precursors, as confirmed by GC-MS and
3
(
3
1
H NMR spectroscopy.
2
4
evaporated to dryness to give pure 4b (0.21 g, 95% yield). A solu-
tion of the latter compound (0.21 g, 0.95 mmol) in chloroform
(
8 mL) was allowed to react with acetic anhydride (0.18 mL,
.9 mmol) at room temperature for 20 min. The mixture was made
basic with a saturated NaHCO solution, then extracted with chlo-
roform (3 ϫ 6 mL). The organic phase was washed with distilled
water (2 ϫ 6 mL), then dried (Na SO ) and evaporated to dryness
1
3
2
4
to afford 5b (0.17 g, 92% yield) as a white solid, m.p. 51–53 °C. –
IR (KBr): nu(tilde) ϭ 3286, 3088, 2964, 1632, 1540, 1380, 730, 696
–
1
1
cm . – H NMR ([D
and CH CH ), 1.10 (m, 1 H, CH
1.72 (m, 1 H, CH CH), 1.89 (s, 3 H, CH
6
]DMSO): δ ϭ 0.75–0.90 (m, 6 H, CH
CH ), 1.42 (m, 1 H, CH CH
CO), 4.22 (m, 1 H,
3
CH
),
Scheme 3. AlCl
of the complete retention of the amino acid stereochemistry
3
/toluene-induced unblocking of dipeptides: a proof
3
2
3
2
3
2
3
3
574
Eur. J. Org. Chem. 2000, 573Ϫ575

Efficient Removal of the 9-Fluorenylmethoxycarbonyl (Fmoc) Protecting Group
SHORT COMMUNICATION
NCHCONHCH
2
Ph), 4.28 (d, J ϭ 5.9 Hz, 2 H, HNCH
.38 (m, 5 H, Ar-H), 7.98 (d, J ϭ 8.7 Hz, 1 H, CH CONHCH), tained by adopting a procedure similar to that previously described
.53 (t, J ϭ 5.9 Hz, 1 H, HNCH ). – GC-MS (EI); m/z (%): 262 for the preparation of 5b.
2
), 7.22– Synthesis of Dipeptides 12 and 14: The title compounds were ob-
7
8
3
2
ϩ
(1) [M ], 206 (6), 156 (11), 128 (57), 106 (25), 91 (51), 86 (100),
ϩ
ϩ
12: White solid, m.p. 158–161 °C. – IR (KBr): nu(tilde) ϭ 3289,
43 (32). – FAB -MS (ϩ, NBA); m/z (%): 263 (86) [(M ϩ H) ],
Ϫ1
1
2
963, 1733, 1634, 1557, 1262, 1104, 800 cm . – H NMR (CDCl ):
3
128 (100).
δ ϭ 0.80–0.90 [m, 12 H, (CH
m, 1 H, CH CH ), 1.49 (m, 1 H, CH
CH CH), 1.94 (s, 3 H, CH CO), 2.10 [m, 1 H, (CH
H, OCH ), 4.33–4.43 (m, 2 H NCHCO and NCHCOOCH
d, J ϭ 8.5 Hz, 1 H, NH Ile), 6.78 (d, J ϭ 8.3 Hz, 1 H, NH Val). –
GC MS (EI), m/z (%): 230 (11), 128 (100), 100 (18), 86 (90), 72 (43),
3
)
2
CH, CH
3
CH and CH
CH ), 1.73 (m, 1 H,
CH], 3.67 (s,
), 6.50
3 2
CH ], 1.09
(
3
2
3
2
Synthesis of Dipeptides 11 and 13: -Val-OMe hydrochloride
3
3
3 2
)
(
(
0.32 g, 1.91 mmol) dissolved in a 5% aqueous Na
13 mL) was added dropwise to the appropriate N-Fmoc amino
2 3
CO solution
3
(
3
3
acid chloride (1.91 mmol) in ethanol-free chloroform (13 mL). The
resulting mixture was maintained under vigorous magnetic stirring
at room temperature for 40 min, until complete conversion of the
ϩ
ϩ
4
3 (33). – FAB -MS (ϩ, NBA); m/z (%): 287 (100) [(M ϩ H) ].
3 3
methyl ester (TLC: CHCl /CH OH 90:10). The aqueous phase was
1
2
4: White solid, m.p. 167–170 °C. – IR (KBr): nu(tilde) ϭ 3289,
separated, then extracted with chloroform (3 ϫ 10 mL). The col-
lected organic layers were washed with HCl 0.1 (2 ϫ 5 mL), dis-
tilled water (1 ϫ 5 mL) and evaporated to dryness to afford the
corresponding dipeptide (88–96% overall yields).
Ϫ1
1
963, 1733, 1634, 1557, 1262, 1021, 800 cm . – H NMR (CDCl
CH, CH CH and CH CH ], 1.10
), 1.43 (m, 1 H, CH CH ), 1.83 (m, 1 H,
CH), 1.95 (s, 3 H, CH CO), 2.10 [m, 1 H, (CH CH], 3.65 (s,
H, OCH ), 4.40–4.55 (m, 2 H, NCHCO and NCHCOOCH ),
3
):
δ ϭ 0.83–0.88 [m, 12 H, (CH
m, 1 H, CH CH
CH
3
)
2
3
3
2
(
3
2
3
2
3
3
3 2
)
3
3
3
1
2
1: White solid, m.p. 163–165 °C. – IR (KBr): nu(tilde) ϭ 3293,
964, 1736, 1691, 1650, 1533, 1242, 1125, 730, 668 cmϪ1. –
1
6.30 (d, J ϭ 8.8 Hz, 1 H, NH Allo-Ile), 6.65 (d, J ϭ 8.5 Hz, 1 H,
NH Val). – GC MS (EI); m/z (%): 230 (14), 128 (100), 100 (15), 86
H
NMR (CDCl
CH CH ], 1.17 (m, 1 H, CH
m, 1 H, CH CH), 2.15 [m, 1 H, (CH
.11 (m, 1 H, NCHCO), ), 4.21 (m, 1 H, CHCH
O), 4.54 (dd, 5.0 Hz,
NCHCOOCH ), 5.56 (d, J ϭ 8.7 Hz, 1 H, NH Ile), 6.51 (d, J ϭ
.6 Hz, 1 H, NH Val), 7.25–7.78 (m, 8 H, Ar-H). – FAB -MS (ϩ,
3
): δ ϭ 0.80–0.97 [m, 12 H, (CH
CH ), 1.53 (m, 1 H, CH
CH], 3.71 (s, 3 H, OCH
O), 4.39 (m, 2 H,
8.6 Hz, H,
3
)
2
CH, CH
3
CH and
), 1.84
),
ϩ
(
80), 72 (33), 43 (40). – FAB -MS (ϩ, NBA); m/z (%): 287 (100)
3
2
3
2
3
CH
2
ϩ
[(M ϩ H) ].
(
4
3
3
)
2
3
2
CHCH
2
J
1
ϭ
J
2
ϭ
1
Acknowledgments
This work was supported by a MURST grant.
3
ϩ
8
ϩ
NBA); m/z (%): 467 (100) [(M ϩ H) ], 435 (2), 336 (2.3), 271 (7),
[
1]
245 (15), 165 (43).
L. A. Carpino, H. G. Beyerman, M. Bienert, J. Org. Chem.
1
991, 56, 2635–2642.
[
[
2]
3]
1
2
3: White solid, m.p. 184–186 °C. – IR (KBr): nu(tilde) ϭ 3294,
L. A. Carpino, Acc. Chem. Res. 1987, 20, 401–407.
L. A. Carpino, B. J. Cohen, K. E. Stephens Jr., S. Y. Sadat-
Aalace, J.-H. Tien, D. C. Langridge, J. Org. Chem. 1986, 51,
926, 1734, 1687, 1648, 1541, 1262, 1094, 756, 738 cmϪ1. –
1
H
NMR (CDCl
CH CH ], 1.14 (m, 1 H, CH
m, 1 H, CH CH), 2.09 [m, 1 H, (CH
.12–4.23 (m, 2 H, NCHCO and CHCH
H, CHCH O), 4.49 (dd, J ϭ 4.8 Hz, J
), 5.33 (d, J ϭ 8.0 Hz, 1 H, NH Allo-Ile), 6.39 (d,
3
): δ ϭ 0.78–0.89 [m, 12 H, (CH
CH ), 1.36 (m, 1 H, CH
CH], 3.64 (s, 3 H, OCH
O), 4.34 (d, J ϭ 6.8 Hz,
ϭ 8.3 Hz, 1 H,
3
)
2
CH, CH
3
CH and
), 1.88
),
3
734–3736.
3
2
3
2
3
CH
2
[4]
M. Beyermann, M. Bienert, H. Niedrich, L. A. Carpino, D.
Sadat-Aalace, J. Org. Chem. 1990, 55, 721-728.
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ron Lett. 1979, 32, 3041–3042.
(
4
2
3
3
)
2
3
[
5]
2
2
1
2
[
[
6]
7]
M. Ueki, M. Amemiya, Tetrahedron Lett. 1987, 28, 6617–6621.
M. Ueki, N. Nishigaki, H. Aoki, T. Tsurusaki, T. Katoh,
Chem. Lett. 1993, 721–725.
NCHCOOCH
J ϭ 8.3 Hz, 1 H, NH Val), 7.18–7.75 (m, 8 H, Ar-H). – FAB -MS
3
ϩ
ϩ
(
(
ϩ, NBA); m/z (%): 467 (100) [(M ϩ H) ], 435 (1.6), 336 (2.4), 271
14), 245 (34), 165 (68).
Received October 2, 1999
[O99548]
Eur. J. Org. Chem. 2000, 573Ϫ575
575