Pyruvoyl, a novel amino protecting group on the solid phase peptide synthesis and the peptide condensation reaction

Article abstract of DOI:10.1016/j.tetlet.2008.12.005

In one of the peptide condensation methods termed thioester method, an amino protecting group is required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an amino protecting group, we synthesized N^α

Full text of DOI:10.1016/j.tetlet.2008.12.005

Tetrahedron Letters 50 (2009) 818–821
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Pyruvoyl, a novel amino protecting group on the solid phase peptide
synthesis and the peptide condensation reaction
*
*
Hidekazu Katayama , Takumi Utsumi, Chinatsu Ozawa, Yuko Nakahara, Hironobu Hojo ,
Yoshiaki Nakahara
Department of Applied Biochemistry, Institute of Glycoscience, Tokai University, 1117 Kitakaname, Hiratsuka, Kanagawa 259-1292, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
In one of the peptide condensation methods termed thioester method, an amino protecting group is
required in the lysine side chain. In this study, to investigate the efficiency of the pyruvoyl group as an
Received 10 October 2008
Revised 27 November 2008
Accepted 2 December 2008
Available online 6 December 2008
a
e
amino protecting group, we synthesized N -fluorenylmethoxycarbonyl (Fmoc)-N -pyruvoyl-lysine and
introduced it into peptides and glycopeptides by the ordinary Fmoc-based solid phase peptide synthesis.
The pyruvoyl peptide could be condensed with a peptide thioester by the thioester method, and this pro-
tecting group was easily removed by o-phenylenediamine treatment without significant side reactions.
Ó 2008 Elsevier Ltd. All rights reserved.
The thioester method is one of the peptide condensation meth-
ods for synthesizing long peptide chains.1 In this method, since a
peptide thioester is activated by a silver ion and is allowed to react
with a free amino group, protecting groups at amino and thiol
groups are required in the peptide side chains. Until now, the
tert-butoxycarbonyl (Boc) group was usually used for protection
of amino groups in this method.2 However, this group is labile
under acidic conditions, and it is difficult to synthesize a Boc-
protected peptide segment directly by the ordinary fluorenylmeth-
oxycarbonyl (Fmoc)-based solid phase peptide synthesis (SPPS). In
our previous report, we demonstrated that azido group could be
used as an alternative amino protecting group, which is stable
under both acidic and basic conditions.3 In this study, we
developed another amino protecting group.
To investigate the feasibility of the pyruvoyl group, a prototype
-ketoacyl group, as an amino protecting group on Fmoc-SPPS
and the peptide condensation reaction by the thioester method,
of
a
a
e
we synthesized N -Fmoc-N -pyruvoyl-lysine (Fmoc-Lys(Pyv)-OH,
1), and used it for synthesizing model peptide and glycopeptide.
At first, we attempted to introduce the pyruvoyl group directly
to the amino group of Fmoc-Lys-OAll (2) using pyruvic acid and
condensation reagents such as dicyclohexylcarbodiimide (DCC)/
1-hydroxybenzotriazole (HOBt) and HBTU, but the desired product
was not obtained due to the decomposition of the pyruvic acid dur-
ing the activation step. To avoid the decomposition, the carbonyl
group of pyruvic acid was protected by 1,1-dimethylhydrazine,
yielding dimethylhydrazonopropionic acid (3).7 Then, the carboxyl
group of 3 was activated by DCC/HOBt and condensed with 2 to ob-
tain Fmoc-Lys(dimethylhydrazonopropanoyl)-OAll (4) (Scheme 1).
Unexpectedly, the hydrazono group was hydrolyzed during sepa-
ration in a separatory funnel and purification by silica gel column
chromatography, and only Fmoc-Lys(Pyv)-OAll (5) was obtained in
78% yield.8 The allyl group of 5 was then deprotected by 5,5-
dimethylcyclohexane-1,3-dione (Dimedone) and tetrakis(triphen-
ylphosphine)Pd(0) in dimethoxyethane, and the desired product
1 was obtained in 87% yield.9
In 1965, Dixon and Moret reported the removal method for an
N-terminal amino acid residue from proteins.4 In the initial step
of this method, the N-terminal amino group was specifically con-
verted into a carbonyl group via a metal ion-mediated transamina-
tion reaction. The residual
a-ketoacyl group was then cleaved by
the o-phenylenediamine treatment. To date, this transamination
reaction has been widely used for an N-terminus-specific modifica-
tion such as fluorescent labeling and dinitrophenyl group attach-
ment through the hydrazido linkage.5 Recently, Kawakami et al.
Using this protected amino acid 1, we attempted to synthesize
the model peptide, pigment dispersing hormone (PDH)-I (6), by
the thioester method. PDH-I is a crustacean neuropeptide isolated
from the prawn, Marsupenaeus japonicus, and consists of 18 amino
acid residues in H-Asn-Ser-Glu-Leu-Ile-Asn-Ser-Leu-Leu-Gly-Ile-
Pro-Lys-Val-Met-Thr-Asp-Ala-NH2 sequence.10
reported that the
a-ketoacyl group of the intermediate could be
used as an N-terminus-specific protecting group.6 Since this pro-
tecting group is stable under both acidic and basic conditions, it
might be a good candidate as an alternative protecting group for
lysine side chains in the thioester method.
As described previously, Fmoc-PDH-I(1-10) thioester (7) was
synthesized by the N-alkylcysteine (NAC)-assisted thioesterifica-
tion method.3,11 The C-terminal segment (8), PDH-I(11-18), whose
lysine side chains were protected by the pyruvoyl group, was also
synthesized by ordinary Fmoc-SPPS. When the pyruvoyl peptide
* Corresponding authors. Tel./fax: +81 463 50 2075 (H.H.).
E-mail addresses: katay@hotmail.co.jp (H. Katayama), hojo@keyaki.cc.u-to-
kai.ac.jp (H. Hojo).
0040-4039/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2008.12.005

H. Katayama et al. / Tetrahedron Letters 50 (2009) 818–821
819
completely oxidized to methionine sulfoxide during the deprotect-
ing reaction. To inhibit the oxidation, dimethylsulfide was added to
the deprotecting reagent at the concentration of 10%. By this mod-
ification, the desired product 6 was obtained in 30% yield, and the
oxidized product was also obtained in 11% yield.14
N
N
N
N
OH
3
O
+
NH2
HN
O
Having established the condensation and deprotection protocol,
we then synthesized Contulakin-G (9), which is a toxic glycopep-
tide isolated from Conus geographus venom. It consists of 16 amino
acid residues in pGlu-Ser-Glu-Glu-Gly-Gly-Ser-Asn-Ala-Thr-Lys-
Lys-Pro-Tyr-Ile-Leu sequence and an O-linked glycan on the threo-
nine side chain.15 The N-terminal peptide thioester segment (10),
Contulakin-G(1-6)-SC6H4CH2COOH, was synthesized by the NAC-
assisted thioesterification reaction.11 Fmoc-N-ethyl-S-trityl-cys-
teine (Fmoc-(Et)Cys(Trt)-OH) was introduced to CLEAR-Amide re-
sin and the peptide chain was elongated by ordinary Fmoc-SPPS.
After cleavage from the solid support, the peptide was thioesteri-
fied by the addition of 4-mercaptophenylacetic acid (MPAA) under
the acidic conditions. The reaction was almost complete within
24 h giving the desired peptide thioester 10. The yield of 10 after
purification by RP-HPLC was 5.8%.
DCC/HOBt
Fmoc
OAll
Fmoc
OAll
N
N
H
H
O
O
2
4
O
O
O
O
HN
HN
Dimedone
Pd(0)
OAll
Fmoc
N
Fmoc
OH
N
H
H
O
O
The Contulakin-G(7-16) segment having N-acetylgalactosamine
on the threonine side chain (11) was synthesized by Fmoc-SPPS
using Fmoc-Leu-CLEAR-Acid resin as a starting material. The pep-
tide chain was elongated manually, and the peptide was cleaved
from the resin by the TFA cocktail treatment. Benzyl groups on
the glycan moiety were deprotected by the low-acidity TfOH treat-
ment.2b,16 The yield of 11 after RP-HPLC purification was 6.5%.
Peptide segments 10 and 11 were dissolved in 5% HOObt/DMSO
and then DIEA was added to this solution (Scheme 3, Fig. 1). The
condensation reaction was almost complete within 2 h, and the
crude peptide was purified by gel filtration HPLC. After lyophiliza-
tion, the protecting group was cleaved in the same manner as for
the PDH-I synthesis. However, the yield of 9 after RP-HPLC purifi-
cation was only 17%, and two by-products with molecular masses
larger by 28 and one with molecular mass larger by 56 were ob-
tained. These results suggested that the pyruvoyl groups were par-
tially converted to formyl groups. This side reaction was
suppressed when the pH of the deprotection conditions was chan-
ged from pH 5.0 to pH 4.0, and the yield increased to 32%.17 As
shown in Figure 1c, no formylation was observed in these
conditions.
To test the stability of pyruvoyl group under the conditions of
NAC-assisted thioesterification reaction, peptide 8 was treated
with the solution, 2% MPAA/6 M urea dissolved in 0.1% TFA/50%
CH3CN. However, a part of pyruvoyl group (10–20%) was converted
into thioketal within 24 h, which might be promoted by the acidic
conditions used in the NAC method. If other post-SPPS thioesterifi-
cation methods, which use basic or neutral conditions, such as
safety-catch linker,18 cysteinylproryl ester,19 cysteine derived oxa-
zolidinone,20 and N-acylurea forming linker,21 are used, a pyruvoyl
peptide thioester would be obtained in better yield.
1
5
Scheme 1.
was cleaved from a solid support by Reagent K,12 which is a TFA
cocktail containing ethanedithiol as a scavenger of the trityl group,
the
a-carbonyl group on the pyruvoyl moiety was partially con-
verted to a thioketal structure. On the other hand, triisopropylsi-
lane could not be utilized as a scavenger due to its ability to
reduce the carbonyl group to a hydroxyl group. To avoid the
decomposition of the pyruvoyl group, we decided to add ethanedi-
thiol to the TFA cocktail just before the cleavage reaction is com-
plete to catch the trityl cation. Using this method, thioketal
formation was completely suppressed, and no significant side reac-
tion was observed (see Supplementary figure). In reversed-phase
(RP)-HPLC analysis of the crude peptide, the desired product gave
a broad, sometimes split peak due to the equilibrium between
keto- and hydrate-forms or the intramolecular hemiacetal at the
pyruvoyl group. The yield of 8 after RP-HPLC was 34%.
The peptide segments were condensed by the Ag+-free thioester
method (Scheme 2).13 Segments 7 and 8 were dissolved in dimeth-
ylsulfoxide (DMSO) containing 5% 3-hydroxy-3,4-dihydro-4-oxo-
1,2,3-benzotriazine (HOObt), and the coupling reaction was initi-
ated by the addition of diisopropylethylamine (DIEA) at a concen-
tration of 5%. The reaction was almost complete within 2 h, and the
N-terminal Fmoc group was cleaved by the piperidine treatment.
Then, the crude peptide was precipitated by diethyl ether. When
the pyruvoyl group was deprotected by the method reported by
Kawakami et al.,6 methionine residue in the product was almost
Scheme 2.

820
H. Katayama et al. / Tetrahedron Letters 50 (2009) 818–821
O
O
O
O
GalNAc
NH
GalNAc
NH
2
2
HOObt/DIEA
DMSO
S
H
Contulakin G(1-6)
H
Contulakin G(7-16)
H
Contulakin G
OH
+
OH
COOH
10
11
12
GalNAc
(NH2)2
o
-Phenylenediamine
Acetate buffer
Contulakin G
H
OH
9
Scheme 3.
nology of Japan (No. 20380069). 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).
Supplementary data
Supplementary data (experimental procedures and the HPLC
chromatograms after cleavage of peptide 8 from the resin) associ-
ated with this article can be found, in the online version, at
doi:10.1016/j.tetlet.2008.12.005.
References and notes
1. (a) Hojo, H.; Aimoto, S. Bull. Chem. Soc. Jpn. 1991, 64, 111–117; (b) Aimoto, S.
Biopolymers (Pep. Sci.) 1999, 51, 247–265.
2. (a) Kawakami, T.; Yoshimura, S.; Aimoto, S. Tetrahedron Lett. 1998, 39, 7901–
7904; (b) Hojo, H.; Haginoya, E.; Matsumoto, Y.; Nakahara, Y.; Nabeshima, K.;
Toole, B. P.; Watanabe, Y. Tetrahedron Lett. 2003, 44, 2961–2964; (c) Ozawa, C.;
Hojo, H.; Nakahara, Y.; Katayama, H.; Nabeshima, K.; Akahane, T.; Nakahara, Y.
Tetrahedron 2007, 63, 9685–9690.
3. Katayama, H.; Hojo, H.; Ohira, T.; Nakahara, Y. Tetrahedron Lett. 2008, 49, 5492–
5494.
4. Dixon, H. B. F.; Moret, V. Biochem. J. 1965, 94, 463–469.
5. (a) Dixon, H. B. F.; Fields, R. Methods Enzymol. 1972, 25, 409–419; (b) Wu, P.;
Brand, L. Methods Enzymol. 1997, 278, 321–330; (c) Duan, X.; Zhao, Z.; Ye, J.; Ma,
H.; Xia, A.; Yang, G.; Wang, C.-C. Angew. Chem., Int. Ed. 2004, 43, 4216–4219; (d)
Dong, S.-Y.; Ma, H.-M.; Duan, X.-J.; Chen, X.-Q.; Li, J. J. Proteome Res. 2005, 4,
161–166.
6. (a) Kawakami, T.; Hasegawa, K.; Teruya, K.; Akaji, K.; Horiuchi, M.; Inagaki, F.;
Kurihara, Y.; Uesugi, S.; Aimoto, S. Tetrahedron Lett. 2000, 41, 2625–2628; (b)
Kawakami, T.; Hasegawa, K.; Teruya, K.; Akaji, K.; Horiuchi, M.; Inagaki, F.;
Kurihara, Y.; Uesugi, S.; Aimoto, S. J. Peptide Sci. 2001, 7, 474–487.
7. Tapia, I.; Alcazar, V.; Moran, J. R.; Caballero, C.; Grande, M. Chem. Lett. 1990, 19,
697–700.
Figure 1. RP-HPLC elution profiles. (a) Coupling reaction mixture of 10 and 11 (0 h).
*
(b) Two hours after the reaction. (c) Reaction mixture after deprotection.
,
**
pyruvoyl-deprotected compound of the remaining C-terminal segment 11. , non-
peptidic components. Elution conditions: column, Mightysil RP-18 GP
(4.6 Â 150 mm, Kanto, Japan) at a flow rate of 1 ml/min; eluent, A, 0.1% TFA, B,
acetonitrile containing 0.1% TFA.
8. Analytical data of compound 5: [
a
]
À1.4 (c, 1.0). Rf 0.25 (75:25 toluene/
D
EtOAc). 1H NMR (CDCl3): d 7.76 (d, 2H, J = 7.6 Hz, Ar), d 7.60 (d, 2H, J = 7.2, Ar), d
e
7.39 (t, 2H, J = 7.6, Ar), d 7.31 (t, 2H, J = 7.6, Ar), d 7.03 (br, 1H, N H), d 5.90 (m,
1H, –CH@CH2), d 5.45 (d, 1H, J = 8.4, N H), d 5.33 (d, 1H, J = 16.8, –CH@CH2), d
In our preliminary experiments, pyruvoyl group could be specif-
ically removed from a peptide containing pyruvoyl, Fmoc and azi-
do groups without undesirable side reaction (data not shown).
These results indicated that a site-specific amino group modifica-
tion after peptide condensation reaction came to be possible by
this protecting group.
In conclusion, we have synthesized Fmoc-Lys(Pyv)-OH and have
introduced it into the peptide and the glycopeptide by Fmoc-SPPS.
The pyruvoyl peptide could be used for the peptide condensation
reaction by the thioester method, and the pyruvoyl groups were
easily cleaved without significant side reactions. This protecting
group was stable under both acidic and basic conditions, and was
compatible with acid-labile protecting groups such as benzyl
groups used for the protection of carbohydrate moieties as shown
in this study.
a
5.26 (d, 1H, J = 10.4, –CH@CH2), d 4.64 (d, 2H, J = 5.6, –OCH2–), d 4.41 (m, 3H,
a
e
C H, CH2(Fmoc)), d 4.22 (t, 1H, J = 6.8, CH(Fmoc)), d 3.28 (m, 2H, C H2), d 2.45 (s,
3H, Pyv), d 1.87 (m, 1H, CbH2), d 1.70 (m, 1H, CbH2), d 1.56 (m, 2H, CdH2), d 1.39
c
(m, 2H, C H2).
9. Analytical data of compound 1: [a] +10.7 (c, 0.5). Rf 0.11 (50:50:1 toluene/
D
EtOAc/AcOH). 1H NMR (DMSO-d6): d 7.93 (d, 2H, J = 7.2 Hz, Ar), d 7.77 (d, 2H,
e
J = 7.2, Ar), d 7.59 (s, 1H, N H), d 7.46 (t, 2H, J = 7.6, Ar), d 7.37 (t, 2H, J = 7.6, Ar),
a
d 4.33 (d, 2H, J = 6.4, CH2(Fmoc)), d 4.27 (m, 1H, CH(Fmoc)), d 3.97 (m, 1H, C H),
e
d 3.15 (m, 2H, C H2), d 2.37 (s, 3H, Pyv), d 1.74 (m, 1H, CbH2), d 1.66 (m, 1H,
c
CbH2), d 1.50 (m, 2H, CdH2), d 1.36 (m, 2H, C H2).
10. Yang, W.-J.; Aida, K.; Nagasawa, H. Gen. Comp. Endocrinol. 1999, 114, 415–424.
11. Hojo, H.; Onuma, Y.; Akimoto, Y.; Nakahara, Y.; Nakahara, Y. Tetrahedron Lett.
2007, 48, 25–28.
12. King, D.; Fields, C. G.; Fields, G. B. Int. J. Peptide Protein Res. 1990, 36, 255–266.
13. (a) Chen, G.; Wan, Q.; Tan, Z.; Kan, C.; Hua, Z.; Ranganathan, K.; Danishefsky, S.
Angew. Chem., Int. Ed. 2007, 46, 7383–7387; (b) Hojo, H.; Murasawa, Y.;
Katayama, H.; Ohira, T.; Nakahara, Y.; Nakahara, Y. Org. Biomol. Chem. 2008, 6,
1808–1813.
14. Analytical data of peptide 6: MALDI-TOF mass, found: m/z 1914.3,
calcd: 1914.0 (M+H)+. Amino acid analysis: Asp2.92Thr0.96Ser1.61Glu0.99
Acknowledgements
Pro0.99Gly1Ala0.98Val0.98Met0.79 Ile1.95Leu2.95Lys0.99
.
15. Craig, A. G.; Norberg, T.; Griffin, D.; Hoeger, C.; Akhtar, M.; Schmidt, K.; Low,
W.; Dykert, J.; Richelson, E.; Navarro, V.; Mazella, J.; Watkins, M.; Hillyard, D.;
Imperial, J.; Cruz, L. J.; Olivera, B. M. J. Biol. Chem. 1999, 274, 13752–13759.
This work was partly supported by a Grant-in-Aid for Scientific
Research from the Ministry of Education, Sport, Science and Tech-

H. Katayama et al. / Tetrahedron Letters 50 (2009) 818–821
821
16. Tam, J. P.; Heath, W. F.; Merrifield, R. B. J. Am. Chem. Soc. 1986, 108, 5242–5251.
17. Analytical data of peptide 9: MALDI-TOF mass, found: m/z 1908.3,
calcd: 1907.9 (M+H)+. Amino acid analysis: Asp0.99Thr0.91Ser1.69Glu2.97Pro1.19
19. Kawakami, T.; Aimoto, S. Chem. Lett. 2007, 36, 76–77.
20. Ohta, Y.; Itoh, S.; Shigenaga, A.; Shintaku, S.; Fujii, N.; Otaka, A. Org. Lett. 2006,
8, 467–470.
Gly2Ala1.02Ile0.94Leu1.00 Tyr0.96Lys2.00
.
21. Blanco-Canosa, J. B.; Dawson, P. E. Angew. Chem., Int. Ed. 2008, 47,
18. Ingenito, R.; Bianchi, E.; Fattori, D.; Pessi, A. J. Am. Chem. Soc. 1999, 121, 11369–
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11374.