DOI: 10.1039/C5CC02967H
Page 3 of 4
Journal Name
ChemComm
COMMUNICATION
M tris(2-carboxyethyl)phosphine (TCEP)·HCl aq., pH 7.0, with
reduced glutathione as a hydrogen atom source and VA-044 as
a radical initiator (final concentrations: 6 M, 0.2 M, 40 mM,
1
6
and 20 mM, respectively). The desulfurization went to
completion within 18 hours, yielding the Acm-protected
SEAlide peptide
Acm groups on the resulting peptide
action of AgOTf-anisole in TFA followed by incubation in the
5
in 53% isolated yield over two steps. The
5
were removed by the
1
7
presence of dithiothreitol. After HPLC purification, the native
N-half 74-residue glycopeptide fragment was obtained in
6
54% isolated yield.
Convergent assembly of
6 and the 88-residue C-half
6
fragment
7
was accomplished by SEAlide-mediated NCL in
the presence of phosphate salts (Fig. 3D). The NCL went
almost to completion within 30 hours to afford the
monoglycosylated 162-residue peptide 15 in 40% isolated yield,
also affording the epimerized product 15’ at ligation site Ser
18
(
13%). After the main product 15 was folded (Fig. 3E) and
purified by HPLC, a product with molecular weight identical to
that of the folded monoglycosylated native GM2AP (calcd:
17,784.3; found: 17,783.8) was obtained in 52% isolated yield.
CD spectrum of the folded product was similar to that of
1
9
previously synthesized GM2AP analog (see ESI†). Assay of
GM2-degradation using the synthesized native GM2AP in the
presence of HexA clearly exhibited the conversion of GM2 to Fig. 3 HPLC analysis of reactions for 6: (A) ligation of 2 and 3 (t < 5 min);
GM3 (Fig. 3F), indicating that the synthetic protein should be (B) ligation of 2 and 3 (t = 8 h); (C) desulfurization of 4 (t = 18 h). HPLC
correctly folded and work similarly to intrinsic GM2AP.
In conclusion, we developed
analysis of reactions for monoglycosylated native GM2AP: (D) NCL of 6 and
novel 7 (t = 30 h); (E) folding of 15 (t = 24 h). (F) TLC monitoring of degradation
a
ligation/desulfurization procedure enabling N-glycosylated Asn of GM2 with HexA and synthesized GM2AP. HPLC and TLC conditions: see
*
site ligations. This ligation procedure allowed the total ESI.† MPAA.
synthesis of the monoglycosylated native GM2AP. Combining
8
9
(a) S. Tsuda, A. Shigenaga, K. Bando and A. Otaka, Org. Lett., 2009,
, 823; (b) K. Sato, A. Shigenaga, K. Tsuji, S. Tsuda, Y. Sumikawa,
this newly developed ligation method with the chemoenzymatic
strategy should open new avenues for the preparation of
1
1
6
K. Sakamoto and A. Otaka, ChemBioChem, 2011, 12, 1840.
(a) C. T. T. Wong, C. L. Tung and X. Li, Mol. BioSyst. 2013, 9, 826;
homogeneous glycoproteins. Chemistry-based derivatizations
of GM2AP for protein therapeutics and protein probes are
ongoing in our laboratory.
(b) L. R. Malins and R. J. Payne, Aust. J. Chem., in press (DOI:
0.1071/CH14568).
This research was supported in part by a Grant-in-Aid for
Scientific Research (KAKENHI) and research grants from the
Takeda Science and the Uehara Memorial Foundations. K.S. is
grateful for a scholarship from the Yoshida Scholarship
Foundation.
1
1
1
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,
5
Notes and references
Institute of Biomedical Sciences and Graduate School of Pharmaceutical
Sciences, Tokushima University, Tokushima 770-8505, Japan. E-mail:
aotaka@tokushima-u.ac.jp; Fax:+81-88-633-9505; Tel:+81-88-633-7283
1
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1
1
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1
2 R. E. Thompson, B. Chan, L. Radom, K. A. Jolliffe and R. J. Payne,
Angew. Chem. Int. Ed., 2013, 52, 9723.
3 Y. Asahina, M. Kanda, A. Suzuki, H. Katayama, Y. Nakahara and H.
Hojo, Org. Biomol. Chem., 2013, 11, 7199.
†
Electronic Supplementary Information (ESI) available: Experimental
details of syntheses including HPLC charts and CD spectrum. See DOI:
4 K. C. Nicolaou, A. A. Estrada, M. Zak, S. H. Lee and B. S. Safina,
Angew. Chem. Int. Ed., 2005, 44, 1378.
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0.1039/b000000x/
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Jolliffe and R. J. Payne, J. Am. Chem. Soc., 2014, 136, 8161.
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1
1
8 Partial epimerization at Ser site using SEAlide-mediated ligation was
previously observed. See: ref. 6.
9 D. Ravasi, M. Masserini, G. Vecchio, Y.-T. Li and S. C. Li,
Neurochem. Res. 2002, 27, 785.
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(a) S. B. H. Kent, Chem. Soc. Rev., 2009, 38, 338; (b) L. R. Malins
and R. J. Payne, Curr. Opin. Chem. Biol., 2014, 22, 70.
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