2,7-Di-tert-butyl-Fmoc-P-OSu: a new polymer-supported reagent for the protection of the amino group.

Article abstract of DOI:10.1016/S0960-894X(02)00261-5

The polymer-supported (2,7-di-tert-butyl-9-fluorenyl)methyl succinimidyl carbonate (Dtb-Fmoc-P-OSu), derived from (2,7-di-tert-butyl-9-fluorenyl)methyl chloroformate (Fmoc-Cl) and a polymeric N-hydroxysuccinimide (P-HOSu), has been used for the preparatio

Full text of DOI:10.1016/S0960-894X(02)00261-5

Bioorganic & Medicinal Chemistry Letters 12 (2002) 1817–1820
2,7-Di-tert-butyl-Fmoc-P-OSu: A New Polymer-Supported
Reagent for the Protection of the Amino Group
Rafael Chinchilla, David J. Dodsworth, Carmen Najera* and Jose M. Soriano
´
Departamento de Quımica Organica, Universidad de Alicante, Apartado 99, 03080 Alicante, Spain
´
Received 30 November 2001; accepted 12 February 2002
Abstract—The polymer-supported (2,7-di-tert-butyl-9-fluorenyl)methyl succinimidyl carbonate (Dtb-Fmoc-P-OSu), derived from
(2,7-di-tert-butyl-9-fluorenyl)methyl chloroformate (Fmoc-Cl) and a polymeric N-hydroxysuccinimide (P-HOSu), has been used for
the preparation of Dtb-Fmoc-protected amines and amino acids. After the N-protection reaction, the liberated P-HOSu can be
recovered and reused. This Dtb-Fmoc-protection improves the solubility of the Fmoc-protected analogues. # 2002 Elsevier Science
Ltd. All rights reserved.
The 9-fluorenylmethoxycarbonyl (Fmoc) group has
grown enormously in popularity since its introduction
as a protecting group for primary and secondary amines
and especially for amino acids. It is employed widely in
peptide synthesis due to its stability to acid and lability
to base, the chloroformate ester Fmoc-Cl 1 being tradi-
tionally employed for its incorporation.1,2 However, in
spite of its properties as a protecting group, very few
modifications to the basic structure of the Fmoc group
have been reported in order to increase its versatility.3
Thus, 9-(2-sulfo)fluorenylmethoxycarbonyl chloride (2,
Sulfmoc-Cl) has been prepared by sulfonation of Fmoc-
Cl 1 and used for the incorporation of the Sulfmoc group
on the free amine of a growing peptide chain during a
solid-phase synthesis,4a allowing the easy ion-exchange
chromatographic purification of the final cleaved pep-
tide. In addition, 9-(2,7-dibromo)-fluorenylmethoxy car-
bonyl chloride 3 has been prepared by bromination of 1.
The presence of the two electron-withdrawing bromine
groups favors an easier final removing of the N-protec-
tion using less basic amines.4b However, the use of
reagents 2 and 3 has not been very extensive.
Cl) has been reported and used as a protecting group
reagent, enhancing solubilities of the corresponding
Dtb-Fmoc-protected derivatives by up to 2orders of
magnitude compared to the Fmoc-protected counter-
parts.5 The deprotection can be carried out with 20%
solution of piperidine in DMF, and the piperidine
adduct formed upon deprotection can be easily removed
by hexane extraction.5 In addition, 2,7-bis(trimethyl-
silyl)-9-fluorenylmethoxycarbonyl chloride (5, Bts-
Fmoc-Cl) has been obtained and used as a protecting
reagent showing also a noticeable enhancement in the
solubility of the final systems, its preparation is, however,
rather cumbersome.6
In spite of the wide use of chloroformates, mainly
Fmoc-Cl 1, it is somewhat unstable and toxic and tends to
form ‘Fmoc-dipeptide’ by-products.7 For these reasons,
efforts have been devoted to the preparation of Fmoc-
protecting reagents which presents a different leaving
group instead of the chlorine.8 Amongst the reagents
developed, the 9-fluorenylmethyl succinimidyl carbon-
ate Fmoc-OSu 6 (Su=succinimidyl) is probably the
reagent that has shown more applicability due to its
shelf-stability and high performance.1,7,8d,9 In respect to
this compound, our group has recently reported the
Recently, other 2,7-disubstituted Fmoc-derived reagents
have been prepared in order to overcome the problem of
the poor solubility of many Fmoc-protected amino acids
in organic solvents. Thus, 2,7-di-tert-butyl-9-fluorenyl-
methoxycarbonyl chloride 4 (Fmoc*-Cl or Dtb-Fmoc-
*Corresponding author. Fax: +34-96-590-3549; e-mail: cnajera@
ua.es; URL: www.ua.es/dept.quimorg
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(02)00261-5

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R. Chinchilla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1817–1820
chloride 4 was prepared from 2,7-di-tert-butyl-9-fluor-
enylmethanol5 in 95% yield by reaction with tri-
phosgene, instead of using the toxic phosgene.5 The
reaction of a solution of chloroformate 4 in acetone
with polymeric N-hydroxysuccinimide 8 (P-HOSu),12 in
the presence of K2CO3 in water afforded Dtb-Fmoc-P-
OSu 9 as a stable white solid, which could be kept for
days in an open flask at ambient temperature without
appreciable decomposition.
This obtained Dtb-Fmoc-P-OSu polymer 9 showed
identical C=O and C–O stretching bands in the IR
spectrum to those of Fmoc-P-OSu 710 and additional
C–H bending doublet bands at 1382and 1363 cm À1, the
long wavelength band more intense, typical of tert-butyl
groups.
Scheme 1. Synthesis of Dtb-Fmoc-P-OSu 9.
preparation of a reagent that incorporates the succini-
mide moiety into a polymer.10 This polymer-supported
Fmoc-OSu (Fmoc-P-OSu) 7 is partially soluble in ace-
tone and is as efficient as the non-polymeric 6, allowing
the easy separation and recovery of the polymer-sup-
ported N-hydroxysuccinimide (P-HOSu) once the pro-
tection reaction has finished, something especially
valuable when working on a small scale. From these
antecedents we proposed the development of other
polymer-supported Fmoc-OSu-related reagents useful
for the protection of the amino group but affording
more soluble protected compounds. The 2,7-di-tert-
butyl substitution on the fluorenyl system employed in
reagent 4 seemed the appropriate choice due to the easy
direct introduction of the tert-butyl groups to the fluorene
using a Friedel–Crafts alkylation reaction.11
The Dtb-Fmoc-P-OSu 9 prepared was used as a solid-
supported N-Dtb-Fmoc-protecting reagent. Thus, the
amino group of amines (Table 1, entries 1–3) and dif-
ferent free a-amino acids (Table 1, entries 4–8) were
protected after reaction with 9 in the presence of K2CO3
as base in acetone/water as solvent and at room tem-
perature, to give the corresponding Dtb-Fmoc-pro-
tected systems after acidification and filtration. The
filtrate contained pure Dtb-Fmoc-derivatives (1H
NMR), whereas the solid consisted of P-HOSu 8 which
could be easily recovered and re-used for the prepara-
tion of new 9.
Following this protocol, Dtb-Fmoc-protected amines
such as p-methoxybenzylamine were obtained in 82%
yield in 7 h reaction time, a yield comparable to that
reported when the chloroformate 4 was used (88%).5 It
is also interesting to compare the obtained yields of
N-Dtb-Fmoc-systems with the corresponding less-bulky
Fmoc-derivatives. Thus, when the N-protection reaction
was performed on isopropylamine, the obtained final
N-Dtb-Fmoc-protected derivative was obtained in 94%
yield (Table 1, entry 2), which is higher than when the
corresponding Fmoc-protected amine was obtained
using a reported Fmoc-protecting reagent such as poly-
styrene-supported in situ generated Fmoc-OBt
(Bt=benzotriazol-1-yl) (78%),13 and identical to when
using Fmoc-P-OSu 7. Moreover, the reaction of 9 with
2-morpholinoethylamine afforded the corresponding
The synthesis of polymer-supported di-tert-butyl-Fmoc-
OSu 9 (Dtb-Fmoc-P-OSu) was accomplished following
the synthetic methodology outlined in Scheme 1. Thus,
starting 2,7-di-tert-butyl-9-fluorenylmethoxycarbonyl
Table 1. N-Protection of amines and amino acids with reagent 9
Mp (ꢀC)c
[a]2D5,
d
Entry
N-Protected compounda
t (h)
Yield (%)b
1
2
p-MeOC6H4CH2NH-Dtb-Fmoc
i-PrNH-Dtb-Fmoc
7
7
–143
82142
94
143–144
189–190
3
7
96
4
5
6
7
8
Dtb-Fmoc-Gly
Dtb-Fmoc-Ala
Dtb-Fmoc-Val
Dtb-Fmoc-Phe
Dtb-Fmoc-Aib
24
24
24
24
24
87
83
87
83
76
188–189
97–98
102–103
97–98
116–117
À25.7
À26.1
À20.3
aAll compounds gave consistent spectroscopic (IR, 1H NMR, 13C NMR) data.
bIsolated pure crude yield (1H NMR).
cFor the crude products.
dMeasured in DMF (c=1).

R. Chinchilla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1817–1820
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Dtb-Fmoc-protected amine in 96% yield (Table 1, entry
3), which is similar to the yield reported when a meta-
thesis-generated polymeric HOSu-derived Fmoc-reagent
(93%)14 or Fmoc-P-OSu 7 (94%)10a were employed.
and evaporated affording pure protected amino acids
(see Table 1).
Acknowledgements
When the protection reaction was carried out on differ-
ent a-amino acids, the corresponding N-Dtb-Fmoc-
protected derivatives were obtained after 1 day (Table 1,
entries 4–8). In almost all cases, yields were higher than
80%, something remarkable considering, for instance,
that polystyrene-supported in situ generated Fmoc-OBt
afforded only 43% of Fmoc-Val,13 and Fmoc-P-OSu 7
gave a 75% yield.10a In addition, the protection reaction
of a sterically hindered amino acid such as the amino-
isobutyric acid (Aib) (Table 1, entry 8) afforded a 76%
of the corresponding protected amino acid. These Dtb-
Fmoc-protected amino acids presented a much higher
solubility than the corresponding Fmoc-derivatives,
being soluble for instance in toluene.
We thank the Direccion General de Ensenanza Superior
e Investigacion Cientıfica (project no. 1FD97–0721) of
the Ministerio de Educacion y Cultura (MEC) and
the Conselleria de Cultura Educacio i Ciencia of the
Generalitat Valenciana (project no. GV99-33-1-02) for
financial support.
References and Notes
1. (a) Albericio, F. Biopolymers (Peptide Science) 2000, 55,
123. (b) Green, T. W.; Wuts, P. G. M. Protective Groups in
Organic Synthesis; J. Wiley & Sons: New York, 1999; p 506.
(c) Kocienski, P. J. Protecting Groups; Thieme: Stuttgart,
1994; p 202. (d) Atherton, E.; Sheppard, R. C. In The Pep-
tides; Uderfriend, S., Meienhofer, J., Eds.; Academic: New
York, 1987; Vol. 9, p 1. (e) Bodanszky, M.; Bodanszky, A.
The Practice of Peptide Synthesis; Springer: Berlin, 1984.
2. (a) Carpino, L. A.; Beyerman, H. G.; Bienert, M. J. Org.
Chem. 1991, 56, 2635. (b) Carpino, L. A. Acc. Chem. Res.
1987, 20, 401.
3. Fmoc-protected amino acids have been nitrated to give (2-
nitrofluoren-9-yl)methoxycarbonyl amino acids, which are
useful for the detection of peptides in HPLC: Henkel, B.;
Bayer, E. Synthesis 2000, 1211.
4. (a) Merrifield, R. B.; Bach, A. E. J. Org. Chem. 1978, 43,
4808. (b) Carpino, L. A. J. Org. Chem. 1980, 45, 4250.
5. Stigers, K. D.; Koutroulis, M. R.; Chung, D. M.; Nowick,
J. S. J. Org. Chem. 2000, 65, 3858.
We also prepared the analogous new non-polymeric
reagent Dtb-Fmoc-OSu 10,15 in 90% yield from 4, using
the same methodology used for the preparation of 9.
This reagent presented identical characteristic IR bands
than 9 and was used for performing some of the pro-
tection reactions carried out using its polymeric coun-
terpart 9, under the same reaction conditions. Yields
obtained using 10 were rather higher than when using 9.
Thus, the reaction of p-methoxybenzylamine with Dtb-
Fmoc-OSu afforded the corresponding N-protected
derivative in 97% yield, whereas the N-protection reac-
tion carried out on alanine gave a 93% yield.
6. Carpino, L. A.; Wu, A.-C. J. Org. Chem. 2000, 65, 9238.
7. Lapatsanis, L.; Milias, G.; Froussios, K.; Kolovos, M.
Synthesis 1983, 671.
8. (a) Carpino, L. A.; Han, G. Y. J. Org. Chem. 1972, 37,
3404. (b) Tessier, M.; Albericio, F.; Pedroso, E.; Grandas, A.;
Eritja, R.; Giralt, E.; Granier, C.; Van Rietschoten, J. Int. J.
Pept. Protein Res. 1983, 22, 125. (c) Schon, I.; Kisfalady, L.
Synthesis 1986, 303. (d) Paquet, A. Can. J. Chem. 1982, 60,
976. (e) Henklein, P.; Heyne, H.-U.; Halatsch, W.-R.;
Niedrich, H. Synthesis 1987, 166.
9. Ten Kortenaar, P. B. W.; Van Dijk, B. G.; Peters, J. M.;
Raaben, B. J.; Adams, P. J. H. M.; Tesser, G. I. Int. J. Pept.
Protein Res. 1986, 27, 398.
We conclude that Dtb-Fmoc-P-OSu 9 is a new, safe,
stable, and efficient solid supported reagent for the Dtb-
Fmoc-protection of the amino group. The use of this
reagent affords results comparable to those obtained
from the non-polymeric counterpart Dtb-Fmoc-OSu 10,
but with the advantage of the easy separation and recy-
cling of the P-HOSu 8 liberated after the protection
reaction.
10. (a) Chinchilla, R.; Dodsworth, D. J.; Najera, C.; Soriano,
J. M. Tetrahedron Lett. 2001, 42, 7579. (b) Chinchilla, R.;
Dodsworth, D. J.; Najera, C.; Soriano, J. M.; Yus, M., ES
Patent P200101169, 2001.
In a typical Dtb-Fmoc-protection reaction of amino
acids using Dtb-Fmoc-P-OSu, to a suspension of 9 (270
mg, 0.4 mmol) in acetone (20 mL) was added a solution
of the corresponding amino acid (0.4 mmol) and K2CO3
(39 mg, 0.4 mmol) in water (15 mL). The suspension
was stirred at rt for 1 day and the solvents were evapo-
rated in vacuo (15 Torr). The solid was suspended in a
mixture of AcOEt (15 mL) and water (15 mL) and
acidified with HCl(c) (2mL). The suspension was fil-
tered and the solid, consisting in P-HOSu, was washed
with AcOEt (2Â10 mL) and water (2Â10 mL). The
combined filtrates were decanted and the organics were
washed with 5% NaHCO3 (3Â20 mL), dried (Na2SO4)
11. 2,7-Di-tert-butylfluorene is now commercially available
(Aldrich).
12. Obtained with a loading of 1.5 mmol gÀ1 by reaction of
inexpensive commercially available styrene/maleic anhydride
co-polymer with an 50% aqueous solution of hydroxylamine
(Chinchilla, R.; Dodsworth, D. J.; Najera, C.; Soriano, J. M.
Tetrahedron Lett. 2001, 42, 4487). This copolymer was easy to
handle in different solvents and presented good mechanical
stability, the cross-linking with diamines, as in the described
case of poly(ethylene-co-N-hydroxymaleimide) (Fridkin, M.;
Patchornik A.; Katchalski, E. Biochem. 1972, 11, 466.
Andreev, S. M.; Tsiryapkin, V. A.; Samoilova, N. A.; Mir-
onova, N. V.; Davidovich, Y. A.; Rogozhin, S. V. Synthesis
1977, 303) was not found to be necessary.

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R. Chinchilla et al. / Bioorg. Med. Chem. Lett. 12 (2002) 1817–1820
13. Dendrinos, K. G.; Kalivretenos, A. G. J. Chem. Soc.,
Perkin Trans. 1 1998, 1463.
14. Barrett, A. G. M.; Cramp, S. M.; Roberts, R. S.; Zecri,
F. J. Org. Lett. 2000, 2, 261.
15. 2,7-Di-tert-butyl-9-fluorenylmethyl succinimidyl carbon-
ate 10 (Dtb-Fmoc-OSu): mp 170–171 ꢀC; H NMR IR (KBr) n
1803, 1786, 1736, 1384, 1364, 1262, 1220 cmÀ1 1H NMR
;
(CDCl3, 300 MHz) d 1.38 (s, 18H), 2.81 (s, 4H), 4.30 (t, J=7.3
Hz, 1H), 4.56 (d, J=7.3 Hz, 2H), 7.44 (m, 2H), 7.64 (m, 4H);
13C NMR (CDCl3, 75 MHz) d 25.4, 31.5, 34.9, 46.3, 73.3,
119.3, 122.0, 125.2, 138.7, 142.5, 150.3, 151.6, 168.5.