An efficient peptide ligation using azido-protected peptides via the thioester method

Article abstract of DOI:10.1016/j.tetlet.2008.07.037

Azido-protected Fmoc-Lys-OH (Fmoc-Lys(N₃)-OH) was synthesized from Fmoc-Lys-OH by the copper(II)-catalyzed diazo transfer method, and introduced to a peptide by the ordinary Fmoc-based solid-phase peptide synthesis. This azido peptide could be condensed with a peptide thioester by the Ag⁺-free thioester method without any significant side reactions. The azido group was easily reduced to an amino group by Zn powder after peptide condensation.

Full text of DOI:10.1016/j.tetlet.2008.07.037

Tetrahedron Letters 49 (2008) 5492–5494
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Tetrahedron Letters
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An efficient peptide ligation using azido-protected peptides
via the thioester method
b
a,
Hidekazu Katayama a, Hironobu Hojo a, , Tsuyoshi Ohira , Yoshiaki Nakahara
*
*
a Department of Applied Biochemistry, Institute of Glycotechnology, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
b Department of Biological Sciences, Faculty of Science, Kanagawa University, Hiratsuka, Kanagawa 259-1293, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Azido-protected Fmoc–Lys–OH (Fmoc–Lys(N3)–OH) was synthesized from Fmoc–Lys–OH by the cop-
per(II)-catalyzed diazo transfer method, and introduced to a peptide by the ordinary Fmoc-based
solid-phase peptide synthesis. This azido peptide could be condensed with a peptide thioester by the
Ag+-free thioester method without any significant side reactions. The azido group was easily reduced
to an amino group by Zn powder after peptide condensation.
Received 9 June 2008
Revised 2 July 2008
Accepted 4 July 2008
Available online 9 July 2008
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Azido peptide
N-Alkyl cysteine
Pigment dispersing hormones
Thioester method
The solid-phase peptide synthesis (SPPS) is usually limited to a
length of approximately less than 50 residues long. To overcome
this problem, condensation methods involving two or more
peptide segments are employed for the synthesis of longer peptide
sequences. To date, two methods have been developed: native
chemical ligation1 and the thioester method.2,3 In the native chem-
ical ligation reaction, no protecting group is needed for amino or
thiol groups, although a cysteine residue is required at the ligation
point. On the other hand, although any residue at the ligation point
can be used in the thioester method, protecting groups are re-
quired for amino and thiol groups.
For protection of thiols in the ordinary fluorenylmethoxycar-
bonyl (Fmoc)-based SPPS, the trityl group is usually used. This
group is removed by the trifluoroacetic acid (TFA) treatment com-
monly used for cleavage from the solid support. However, the
released peptide having free thiol(s) cannot be used directly for
the thioester method condensation reaction. To avoid this inconve-
nience, acetamidomethyl group is used for the synthesis of peptide
segment in the thioester method because of its stability under both
basic and acidic conditions.3 For the protection of lysine side
chains, tert-butoxycarbonyl (Boc) groups are usually introduced
to the peptide segments used for this method after the cleavage
from the resins and the purification steps.3 To overcome this incon-
venience, benzyloxycarbonyl (Z) groups have been used for protec-
tion of the amines. Z groups are stable under TFA acidic condition,
and the peptide segments having Z groups can be used directly for
the thioester method without additional protection of amines.
However, the strong hydrophobicity of the Z groups sometimes
gives difficulty in the purification of the peptide segment. In addi-
tion, this protecting group is removed during the deprotection of
the benzyl groups used for the hydroxyl groups of the carbo-
hydrate moiety in our glycopeptide synthesis.4,5 Recently, Chen
et al. reported that the ivDde group could be used for the protec-
tion of the lysine side chains.6 This protecting group is stable under
both acidic and basic conditions, and can be specifically removed
by hydrazine treatment;7 however, it is also hydrophobic and
may cause the problem stated above when many lysine residues
exist in the peptide. Hence, an alternative less-hydrophobic pro-
tecting group for amines stable under both acidic and basic condi-
tions was desired.
The azide moiety might be a good candidate for this purpose. In
a few experiments, azido-protected Fmoc–Lys–OH (Fmoc–Lys(N3)–
OH) was introduced to peptides. These azido peptides were used
for synthesizing dendrimers or the activity-based protein profiling
method via ‘click chemistry’ reaction.8,9 However, the methods for
deprotection (reduction) of azide moieties at the peptide side
chains have not yet been developed. We found that the azide-pro-
tected peptides could be reduced by Zn/AcOH. This useful protocol
enabled us to synthesize the physiologically active peptides by uti-
lizing azido-protected Fmoc–Lys–OH (Fmoc–Lys(N3)–OH) into the
ordinary Fmoc-based SPPS and subsequent segment condensation
by the thioester method. In this study, we synthesized pigment
dispersing hormones (PDHs) of the kuruma prawn, Marsupenaeus
japonicus, as model peptides. It was reported that there were two
* Corresponding authors. Tel./fax: +81 463 58 1211 (H.H.).
E-mail addresses: hojo@keyaki.cc.u-tokai.ac.jp (H. Hojo), yonal@keyaki.cc.
u-tokai.ac.jp (Y. Nakahara).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.07.037

H. Katayama et al. / Tetrahedron Letters 49 (2008) 5492–5494
5493
desired peptide thioester. The yield of 4 after purification by RP-
HPLC was 4.8%. In our preliminary experiment, a peptide thioester
containing azide groups could be prepared by this NAC-mediated
method without significant decomposition of the azide groups.
The peptide segments were condensed by the Ag+-free thioester
method (Scheme 2).6,17 Segments 3a and 4 were dissolved in
dimethylsulfoxide (DMSO) containing 3-hydroxy-4-oxo-3,4-dihy-
dro-1,2,3-benzotriazine (HOObt), and the reaction was initiated
by addition of N,N-diisopropylethylamine (DIEA) (Fig. 2a).18 In this
reaction, the azide group did not produce any by-products. The
coupling reaction was completed within 3 h and the desired pep-
tide 5a was obtained (Fig. 2b). After condensation, the Fmoc group
at the amino terminus was removed by piperidine treatment. The
azide group was converted to amino group by Zn/AcOH treatment
Figure 1. Amino acid sequences of pigment dispersing hormones. An arrow
indicates the peptide coupling site.
PDHs in the sinus gland of M. japonicus; PDH-I and -II.10 As shown
in Figure 1, both consist of 18 amino acid residues and an amidated
C-terminus. Since the N-terminal 10 residues of PDHs are identical
and only 3 residues in the C-terminal half are different, we synthe-
sized an N-terminal 10-residue peptide thioester as the common
peptide segment and two C-terminal 8-residue peptides having
an azide moiety at Lys.13
Fmoc–Lys(N3)–OH 1 was synthesized from Fmoc–Lys–OH 2 by
the copper(II)-catalyzed diazo transfer method (Scheme 1).11,12
Although K2CO3 is generally used as a base for the introduction
of the azido group to the amino acid,13–15 Fmoc group is not stable
in the K2CO3 solution. Therefore, to avoid the cleavage of the Fmoc
group, NaHCO3 was used as the base. Azido-protected lysine 1 was
introduced to the peptide segments, 3a and b, by the Fmoc-based
SPPS. As reported previously, treatment of the TFA cocktail con-
taining triisopropylsilane as a scavenger of the trityl group for
1.5 h did not decompose the azide group,15 although about 20%
of azide group was converted to the amino group within 2 h when
1,2-ethanedithiol was used instead of triisopropylsilane due to the
reducing ability of the thiol compound (data not shown). The seg-
ments 3a and b were purified by reversed-phase (RP)-HPLC. The
isolated yields were 56% and 37%, respectively.
The N-terminal peptide thioester 4 was synthesized by the N-
alkylcysteine (NAC)-assisted thioesterification method developed
in our previous study.16,17 Fmoc-N-ethyl-S-trityl-cysteine (Fmoc–
(Et)Cys(Trt)–OH) was introduced to Rink Amide MBHA resin and
the peptide chain was elongated by ordinary Fmoc-based SPPS.
After cleavage from the resin, the peptide was thioesterified by
the addition of 4-mercaptophenylacetic acid under the acidic con-
dition. This reaction was almost completed within 24 h giving the
NH2
N3
Tf-N3
CuSO4
NaHCO3
Fmoc
Fmoc
N
COOH
N
COOH
Figure 2. RP-HPLC elution profiles of peptide condensation and deprotection. (a)
Coupling reaction mixture of 3a and 4 (0 h). (b) Three hours after the coupling
reaction. (c) Reaction mixture after deprotection of the Fmoc group and the
reduction of azide group. Elution condition: column, YMC-Pack PROTEIN-RP (4.6/
 150 mm, YMC, Japan) at a flow rate of 1 ml/min.
H
H
1
2
Scheme 1. Azido transfer to lysine side chain.
H
IPK(N3)VMTDA NH2
3a
3b
or
COOH
NSELINSLLGIPK(N3)VMTDA
or
5a
5b
Fmoc
NH2
H LPK(N3)FMIDA NH2
Fmoc
S
NSELINSLLG
HOObt/DIEA
DMSO
Fmoc NSELINSLLGLPK(N3)FMIDA NH2
4
1) Piperidine
2) Zn/AcOH
NSELINSLLGIPKVMTDA
or
H
H
NH2
6a
6b
NSELINSLLGLPKFMIDA NH2
Scheme 2. Coupling reaction of peptide segments and deprotection.

5494
H. Katayama et al. / Tetrahedron Letters 49 (2008) 5492–5494
within 30 min, and the desired product 6a was obtained without
any significant side reactions (Fig. 2c). The isolated yield calculated
from the amount of the N-terminal segment 4 used for the cou-
pling reaction was 47%. The peptide segments 3b and 4 were cou-
pled and deprotected in the same manner as described above, and
the desired product 6b was obtained with 57% yield.
References and notes
1. Dawson, P. E.; Muir, T. W.; Clark-Lewis, I.; Kent, S. B. Science 1994, 266, 776–
779.
2. Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117.
3. Aimoto, S. Biopolymers 1999, 51, 247–265.
4. Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara, Y.; Nabeshima, K.; Toole, B. P.;
Watanabe, Y. Tetrahedron Lett. 2003, 44, 2961–2964.
5. Hojo, H.; Matsumoto, Y.; Nakahara, Y.; Ito, E.; Suzuki, Y.; Suzuki, M.; Suzuki, A.;
Nakahara, Y. J. Am. Chem. Soc. 2005, 127, 13720–13725.
6. Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan, K.; Danishefski, S.
Angew. Chem., Int. Ed. 2007, 46, 7383–7387.
7. Chhabra, S. R.; Hothi, B.; Evans, D. J.; White, P. D.; Bycroft, B. W.; Chan, W. C.
Tetrahedron Lett. 1998, 39, 1603–1606.
8. Speers, A. E.; Cravatt, B. F. J. Am. Chem. Soc. 2005, 127, 10018–10019.
9. Arnusch, C. J.; Branderhorst, H.; de Kruijff, B.; Liskamp, R. M.; Breukink, E.;
Pieters, R. J. Biochemistry 2007, 46, 13437–13442.
10. Yang, W.-J.; Aida, K.; Nagasawa, H. Gen. Comp. Endocrinol. 1999, 114, 415–424.
11. Speers, A. E.; Cravatt, B. F. Chem. Biol. 2004, 11, 535–546.
12. Sodium azide (1.45 g, 22.3 mmol) was dissolved in distilled H2O (4.3 ml) with
CH2Cl2 (7.2 ml). Triflic anhydride (0.73 ml, 4.46 mmol) was added slowly, and
stirred for 2 h. The CH2Cl2 phase was removed and aqueous phase was
extracted with CH2Cl2 (3.6 ml  2). The organic fractions containing triflyl
azide were combined, washed with 5% aq NaHCO3, and used without further
purification. Fmoc–Lys–OH hydrochloride (900 mg, 2.23 mmol), NaHCO3
To examine the biological activity of the synthetic PDHs, 6a and
b (1 lg each/individual) were injected into the eyestalk-ablated
prawns. The melanophore index was used to assess the pigment
dispersing activity.19 After injection of 6a and b, the melanophores
indices were changed from 1 or 2 to 5 within 15 min, and the dis-
persion was maintained for 60 min. On the other hand, the change
of indices was not observed in negative control. These results indi-
cated that the synthetic peptides were fully active.
In conclusion, we have synthesized Fmoc–Lys(N3)–OH and
introduced it to the peptide by the Fmoc-based SPPS. The azido
peptides could be used for the peptide condensation by the Ag+-
free thioester method, and the azide groups were easily converted
to amino groups without any undesirable reactions. Azide groups
were compatible with acid-labile protecting groups such as the
benzyl groups used for the protection of carbohydrate and phos-
phate moieties. It is, therefore, likely that this method is a good
tool for synthesizing glyco- and/or phosphoproteins. Application
of this method for the synthesis of a large glycoprotein is now in
progress.
(1.87 g, 22.3 mmol), and CuSO4 pentahydrate (5.5 mg, 22.3 lmol) were
dissolved in distilled H2O (8 ml) and methanol (16 ml). Triflyl azide in CH2Cl2
(14.4 ml) was added with stirring at room temperature and the reaction
continued overnight. The organic solvents were removed under high vacuum,
and the remained solution was acidified at pH 2 by the addition of aq HCl. After
extraction with ethyl acetate (EtOAc, 2 Â 20 ml), the organic fractions were
combined, washed with saturated NaCl aq solution, and dried over Na2SO4.
After concentration, the crude product was chromatographed on silica gel with
toluene/EtOAc (3:1) containing 1% AcOH to give Fmoc–Lys(N3)–OH 1 as white
powder quantitatively.
Acknowledgments
13. Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2001, 3, 781–783.
14. Lundquist, J. T.; Pelletier, J. C. Org. Lett. 2002, 4, 3219–3221.
15. Rijkers, D. T.; van Vugt, H. R.; Jacobs, H. J.; Liskamp, R. M. Tetrahedron Lett. 2002,
43, 3657–3660.
16. Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett.
2007, 48, 25–28.
This work was partly supported by Grant-in-Aid for Scientific
Research from the Ministry of Education, Sport, Science and Tech-
nology of Japan (No. 18580107). We thank Tokai University for a
Grant-in-Aid for high-technology research and the Japan Society
for the Promotion of Science for a Grant-in-Aid for Creative Scien-
tific Research (No. 17GS0420).
17. Hojo, H.; Murasawa, Y.; Katayama, H.; Ohira, T.; Nakahara, Y.; Nakahara, Y. Org.
Biomol. Chem. 2008, 6, 1808–1813.
18. Peptide segments 3a (450
dissolved in DMSO (50 l) containing 10% 3-hydroxy-4-oxo-3,4-dihydro-1,2,3-
benzotriazine (HOObt) and 5% DIEA, and incubated at room temperature for
3 h. Piperidine (12.5 l) was then added to this solution, and kept at room
lg, 0.50 lmol) and 4 (390 lg, 0.27 lmol) were
l
Supplementary data
l
temperature for 20 min. The crude peptide was precipitated by the addition of
10 times volume of diethyl ether, washed twice with ether, and dried under
vacuum. The precipitant was dissolved in 50% aq AcOH (300 ll) containing
The photographs of melanophores before and after the syn-
thetic peptide injections are available free of charge via the inter-
net. Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.tetlet.2008.07.037.
excess amount of Zn powder, and vortexed for 30 min. Zn powder was
removed by filtration, and the desired product 6a was purified by RP-HPLC.
19. Hogben, L.; Slome, D. Proc. R. Soc. London B 1931, 108, 10–53.