Peptide synthesis on fluorous support

Article abstract of DOI:10.1016/j.tetlet.2004.03.013

New fluorous supports were synthesized and used to prepare a peptide having a C-terminal COOH based on fluorous chemistry. The hexakisfluorous chain-type support was suitable for the synthesis of a pentapeptide or a peptide derivative on a fluorous support whose fluorine content is over 40 w/w%. A bioactive peptide, Leu-enkephalin, was easily synthesized using an Fmoc-strategy based on fluorous chemistry.

Full text of DOI:10.1016/j.tetlet.2004.03.013

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 3425–3428
Peptide synthesis on fluorous support
Mamoru Mizuno,a,* Kohtaro Goto,a Tsuyoshi Miura,a Takeshi Matsuuraa and
Toshiyuki Inazua,b
aThe Noguchi Institute, School of Engineering, Tokai University, Japan
bDepartment of Applied Chemistry, School of Engineering, Tokai University, Japan
Received 20 January 2004; revised 2 March 2004; accepted 4 March 2004
Abstract—New fluorous supports were synthesized and used to prepare a peptide having a C-terminal COOH based on fluorous
chemistry. The hexakisfluorous chain-type support was suitable for the synthesis of a pentapeptide or a peptide derivative on a
fluorous support whose fluorine content is over 40 w/w%. A bioactive peptide, Leu-enkephalin, was easily synthesized using an
Fmoc-strategy based on fluorous chemistry.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Recently, fluorous chemistry has been studied in several
fields such as catalytic chemistry, combinatorial chem-
istry,1 and oligosaccharide synthesis.2 A fluorous (highly
fluorinated) solvent is immiscible in an organic solution,
and a fluorous compound partitions out of the organic
phase and into the fluorous phase. Therefore, a fluorous
compound is readily separated from nonfluorinated
compounds by a simple ‘fluorous/organic’ extraction.
Similar to a solid-phase synthesis, fluorous synthesis
does not resort to chromatography. Therefore, the
strategy of ‘fluorous synthesis’ is designed to combine
the advantages of solid-phase synthesis (facile purifica-
tion) with those of traditional organic synthesis in
the liquid-phase synthesis (purification of intermediate
and reaction monitoring; large scale reaction).
As a fluorous support, a hexakisfluorous chain-type
amine 1 (Hfa) was used in this study. A 4-hydroxy-
methylphenoxyacetyl (HMPA)-type fluorous support
24;5 was prepared for the synthesis of a C-terminal
COOH-type peptide. The loading of the amino acid on
the fluorous support 2 was investigated using Fmoc-Ala-
OH. Although 1.5 equiv of Fmoc-Ala-OH did not
provide a sufficient esterification yield using either DCC-
DMAP and PyBop-DMAP, 3.0 equiv of Fmoc-Ala-OH
produced a high yield. (Fig. 1, Table 1).
The cleavage of the amino acid from the HMPA-type
fluorous support was examined. Fmoc-Ala-HMPA-Hfa
was treated with 95% aqueous TFA for 2 h at room
temperature. After the partition with an organic solvent
(MeCN) and a fluorous solvent (FC-72),6 the Fmoc-
Ala-OH was obtained in 46% yield from the organic
layer. The HMPA-Hfa and a fluorous compound having
a fluorene moiety were recovered from the FC-72 layer.
It appeared that the fluorene moiety was not the Fmoc
groupbased on the 1H NMR spectra. We postulated
that the Fmoc groupwas damaged under the cleavage
conditions. To study whether or not this side reaction
The C-terminal of almost all natural peptides is an
amide type or a carboxyl type. In a fluorous peptide
synthesis, a suitable linker corresponding to the C-ter-
minal form is introduced to a fluorous support the same
as in a solid-phase method. Previously, we described the
synthesis of the C-terminal amide peptide using a
Rinke-type fluorous support in fluorous chemistry.3 In
this study, we synthesized new fluorous supports for the
synthesis of a C-terminal carboxyl peptide, and a bio-
active peptide, Leu-enkephalin, was easily synthesized
by an Fmoc-strategy based on fluorous chemistry.
C8F17
C8F17
O
O
C8F17
C8F17
N
O
O
N
H
N
H
N
Hfa
C8F17
N
N
C8F17
O
O
N
H
1
Keywords: Fluorous support; Leu-enkephalin; Fluorous chemistry;
Peptide synthesis.
* Corresponding author. Tel./fax: +81-359443214; e-mail: mmizuno@
noguchi.or.jp
Figure 1. Hexakisfluorous chain-type amine (Hfa) 1.
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.03.013

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M. Mizuno et al. / Tetrahedron Letters 45 (2004) 3425–3428
Table 1. Loading of amino acid on the HMPA-type fluorous support 2
HMPB-Hfa 3 was performed using 4-(4-hydroxymethyl-
3-methoxyphenoxy)-butyric acid via the same method
of preparing 2. Although the esterification yield was
slightly lower than the HMPA-type, the cleavage of
Fmoc-Ala-OH completely occurred (Scheme 1).
Fmoc-Ala-OH
HO
Fmoc-Ala
O
Hfa
Hfa
coupling reagent
O
O
O
O
2
C4F9OEt-CH2Cl2
r. t., 24 h
[ HMPA-Hfa ]
Additionally, we investigated the property of the Hfa-
type support for the peptide elongation. Two peptide
syntheses were demonstrated using Rinke-Hfa (Fig. 3).9
For test peptide 1, although the hexapeptide derivative
(F content ¼ 40 w/w%) was easily prepared, the hepta-
peptide derivative (F content ¼ 39 w/w%) precipitated in
the F-C72 layer. For test peptide 2, although the pen-
tapeptide derivative (F content ¼ 43 w/w%) was
obtained without any trouble, the hexapeptide deriva-
tive (F content ¼ 40 w/w%) produced an organic layer
emulsion in the partition step. Based on these results, a
pentapeptide or a peptide derivative on a fluorous sup-
port whose fluorine content is over 40 w/w% can be
synthesized by the Hfa-type support (Scheme 2).
Fmoc-Ala-OH
Coupling reagenta
PyBOP (1.1 equiv)
DIEA (1.2 equiv)
DMAP (0.1 equiv)
DCC (1.1 equiv)
DMAP (0.1 equiv)
1.5 equiv
3.0 equiv
46%
95%
74%
95%
a Equivalent of a coupling reagent corresponds to Fmoc-Ala-OH.
damaged not only the Fmoc groupbut also the cleavage
of the amino acid from the fluorous support, the
naphtylacetyl group, which is stable under the depro-
tection condition of the Fmoc group, was used for the
amino group protection instead of Fmoc group. The
naphtylacetyl-Ala-HMPA-Hfa was then treated under
the same conditions, and the naphtylacetyl-Ala-OH was
obtained in 94% yield from the MeCN layer. From these
results, it was found that this side reaction had no
influence on the cleavage of the amino acid from
HMPA-Hfa, and the HMPA-Hfa can be used for the
synthesis of a peptide containing no Fmoc group
(Fig. 2).
Based on these results, the synthesis of Leu-enkephalin
(F content of protected Leu-enkephalin 6 is 47 w/w%)
was demonstrated. HMPA-Hfa was used as a fluorous
support to investigate the possibility that the HMPA-
type support can be used for peptide synthesis. Fmoc-
Leu-OH was esterified onto HMPA-Hfa in 94% yield.
The Fmoc groupwas cleaved by 40% Et NH/MeCN–
2
FC-72 (1:1) solution, and the coupling was carried out
using PyBOP as the coupling reagent in the mixed sol-
vent, C4F9OEt8–CH2Cl2–DMF (5:4:1). A 4-fold excess
of the amino acid derivative was used in each coupling
reaction. During these reaction steps, the reaction mix-
ture was partitioned with a fluorous solvent FC-72 and
MeCN. The peptide having a fluorous support 6 was
Furthermore, the properties of the 4-(4-hydroxymethyl-
3-methoxyphenoxy)-butyl (HMPB)-type fluorous sup-
port 37 regarding the loading and the cleavage were
examined using Fmoc-Ala-OH. The synthesis of
Fmoc-Ala-OH
Organic layer
46%
95% aq. TFA
Fmoc-Ala O
Hfa
HO
Hfa
O
r. t., 2 h
O
Fluorous layer
O
O
+
Fluorous compound having
a fluorene moiety
95% aq. TFA
r. t., 2 h
Ala
OH
O
Ala
Hfa
O
O
O
O
94%
Figure 2. Cleavage of amino acid from HMPA-Hfa.
MeO
MeO
HO
Fmoc Ala O
a
b
Hfa
Hfa
Fmoc-Ala-OH
96%
O
O
88%
O
O
3
[HMPB-Hfa]
Scheme 1. Loading and cleavage to HMPB-Hfa. Reagents and conditions: (a) Fmoc-Ala-OH (3.0 equiv), PyBOP (3.3 equiv), DIEA (3.6 equiv),
DMAP (0.3 equiv)/C4F9OEt–CH2Cl2, rt, 24 h; (b) 95% aq TFA, rt, 2 h.

M. Mizuno et al. / Tetrahedron Letters 45 (2004) 3425–3428
3427
Test Peptide 1
Obtained (F content: 40%)
Fmoc-Pro-Gln(Trt)-Gly-Leu-Phe-Arg(Pmc)-Gly-Rink-Hfa
Precipitated in FC72 (F content: 39%)
Obtained (F content: 43%)
Test Peptide 2
Fmoc-Arg(Pmc)-Trp(Boc)-Gly-Lys(Boc)-Pro-Val-Rink-Hfa
Emulsion (F content: 40%)
NH
MeO
MeO
Figure 3. Ability of fluorous support for peptide synthesis.
Rink :
O
CO
Deprotection
& Coupling
Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-HMPA-Hfa
6
Fmoc-Leu-HMPA-Hfa
1) 40% Et2NH/MeCN-FC72 (1:1)
2) 95% aq. TFA
H-Tyr-Gly-Gly-Phe-Leu-OH
70%
Deprotection: 40% Et2NH/MeCN-FC72 (1:1), r. t., 1 h
Coupling: Fmoc-AA-OH(4.0 eq.), PyBOP(4.4 eq.), DIEA(6.0 eq.)/C4F9OEt-CH2Cl2-DMF (5:4:1), r. t., 30 min
Scheme 2. Synthesis of Leu-enkephalin on the Hfa-type fluorous support. Reagents and conditions: Deprotection: 40% Et2NH/MeCN-FC72 (1:1) rt,
1 h. Coupling: Fmocc-AA-OH (4.0 equiv), PyBOP (4.4 equiv), DIEA (6.0 equiv)/C4F9OEt–CH2Cl2–DMF (5:4:1), rt, 30 min.
treated with TFA containing 5% H2O to cleave
the peptide from the fluorous support and remove the
side-chain protecting group. After partition between
C4F9OEt–EtOAc (2:1) and water, the desired peptide
was extracted into the water layer. The HPLC chart of
the crude peptide is shown in Figure 4. After purifica-
tion of the water layer by RP-HPLC, the Leu-enkeph-
alin was provided in 70% yield in nine reaction steps
with only one purification as the final step.10
In conclusion, the C-terminal COOH-type peptide was
very easily prepared using fluorous chemistry. Each
synthetic intermediate was able to be easily purified by a
simple FC-72/organic solvent extraction and monitored
by NMR, mass spectroscopy and TLC. The HMPA and
HMPB-type fluorous supports are useful to prepare a
peptide having the C-terminal COOH based on the
fluorous chemistry. A hexakisfluorous chain-type sup-
port was suitable for the synthesis of a pentapeptide or a
peptide derivative on a fluorous support whose fluorine
content is over 40 w/w%.
Acknowledgements
This work was partly supported by a Grant-in-Aid
for Scientific Research (C) (no. 13680680) and a Grant-
in-Aid for Encouragement of Young Scientists (no.
13771349) from the Japan Society for the Promotion of
Science. This work was performed through the Noguchi
Fluorous Project at our institute.
0
20
40
60
References and notes
Retention time (min)
1. (a) Luo, Z.; Zhang, Q.; Oderatoshi, Y.; Curran, D. P.
Science 2001, 291, 1766; (b) Barrett, A. G. M.; Braddock,
Figure 4. HPLC profile of crude Leu-enkephalin synthesized by flu-
orous synthesis.11

3428
M. Mizuno et al. / Tetrahedron Letters 45 (2004) 3425–3428
D. C.; Catterick, D.; Chadwick, D. P.; Henschke, J.;
gel chromatography [CHCl3–MeOH–H2O, 90:10:0.6] to
give compound 2 in 97% from 1.
McKinnell, R. M. Synlett 2000, 847; (c) Curran, D. P.
Pure Appl. Chem. 2000, 72, 1649; (d) Curran, D. P. Angew.
Chem., Int. Ed. 1998, 37, 1174; (e) Studer, A.; Hadida, S.;
Ferritto, R.; Kim, S. Y.; Jeger, P.; Wipf, P.; Curran, D. P.
Science 1997, 275, 823; (f) Pozzi, G.; Cavazzini, M.; Quici,
S. Tetrahedron Lett. 1997, 38, 7605.
5. Compound 2: amorphous solid, 1H NMR (600 MHz,
CDCl3) d ¼ 1:72–1.95 (m, 8H), 1.98–2.16 (m, 8H), 2.29–
2.77 (m, 16H), 3.31–3.69 (m, 24H), 4.55–4.72 (s, 2H), 4.61
(m, 4H), 6.76–6.90 (m, 2H), 7.27–7.30 (m, 2H). MALDI-
TOF MASS: Calcd for C91H67F102N7NaO9[MþNa]þ:
3363.37, found: 3363.63.
2. (a) Miura, T.; Hirose, Y.; Ohmae, M.; Inazu, T. Org. Lett.
2001, 3, 3947; (b) Miura, T.; Goto, K.; Hosaka, D.; Inazu,
T. Angew. Chem., Int. Ed. 2003, 42, 2047; (c) Miura, T.;
Inazu, T. Tetrahedron Lett. 2003, 44, 1819.
6. FC-72 is a commercially available fluorocarbon solvent
(3M, Tokyo), which consists of perfluorohexane (C6F14)
isomers and is called Fluorinerte FC-72.
3. Mizuno, M.; Goto, K.; Miura, T.; Hosaka, D.; Inazu, T.
Chem. Commun. 2003, 972.
7. Compound 3: amorphous solid, 1H NMR (600 MHz,
CDCl3) d ¼ 1:77–1.93 (m, 8H), 1.99–2.15 (m, 10H), 2.35–
2.80 (m, 18H), 3.30–3.72 (m, 24H), 3.79–3.85 (m, 3H),
3.95–4.04 (m, 2H), 4.55–4.62 (m, 2H), 6.34–6.49 (m, 2H),
7.10–7.16 (m, 2H). MALDI-TOF MASS: Calcd for
C94H73F102N7NaO10[MþNa]þ: 3421.45, found: 3420.66.
8. C4F9OEt is a commercially available fluorocarbon solvent
(3M, Tokyo), which is called Novece HFE-7200.
9. To avoid a diketopiperadine formation, the C-terminal
amide type linker (Rinke) was used in Figure 3.
4. Synthesis of 2. A hexakisfluorous chain-type amine 1
(1.62 g, 0.50 mmol) and 4-hydroxymethylphenoxyacetic
acid (0.14 g, 0.75 mmol) were dissolved in a mixed solution
of EtOC4F9 (15 mL), CH2Cl2 (15 mL) and DMF (7 mL).
To the solution, PyBOP (0.39 g, 0.75 mmol) and DIEA
(0.26 mL, 1.50 mmol) were added and stirred at room
temperature for 1 h. The reaction mixture was concen-
trated to approximately 1/3 volume, and MeCN (50 mL)
was then added. The mixture was extracted with FC-72
(3 · 50 mL) and concentrated. The residue was dissolved in
a mixed solution of EtOC4F9 (15 mL) and MeOH (15 mL).
To the solution, a catalytic amount of NaOMe was added
and stirred at room temperature for 1 h. After concentra-
tion, the MeCN (50 mL) was added to the residue and
extracted with FC-72 (3 · 50 mL). The FC-72 layer was
concentrated in vacuo. The residue was purified by silica-
10. Amino acid analysis of Leu-enkephalin: Gly 2.00 (2), Leu
1.08 (1), Tyr 0.87 (1), Phe 0.68 (1). MALDI-TOF MASS:
Calcd for C28H38N5O7[MþH]þ:
556.28, found:
556.31.
11. Elution conditions of crude peptide: column, GL Sciences
Inertsil ODS-3 (4.6 · 250 mm); eluent, 10–80% MeCN/
H2O–0.1%TFA (v/v/v), 60 min; flow rate, 1.0
mL/min.