Total synthesis of α-conotoxin MII using a soluble-tag-assisted method

Article abstract of DOI:10.1016/j.tet.2013.01.068

Oxidative disulfide bond formations have been applied to soluble-tag-assisted method successfully to realize the total synthesis of α-conotoxin MII (4) that comprises 16 amino acid residues and possesses 2 disulfide bonds. Orthogonal peptide folding using

Full text of DOI:10.1016/j.tet.2013.01.068

Tetrahedron 69 (2013) 2555e2559
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Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of
method
a-conotoxin MII using a soluble-tag-assisted
,
Shingo Kitada a, Shuji Fujita b, Yohei Okada a, Shokaku Kim a, Kazuhiro Chiba a
*
a Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan
b Research and Development Division, JITSUBO Co., Ltd., 2-24-16 Naka-cho, Koganei, Tokyo 184-0012, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxidative disulfide bond formations have been applied to soluble-tag-assisted method successfully to
Received 1 December 2012
Received in revised form 16 January 2013
Accepted 21 January 2013
Available online 1 February 2013
realize the total synthesis of a-conotoxin MII (4) that comprises 16 amino acid residues and possesses 2
disulfide bonds. Orthogonal peptide folding using DEAD and iodine oxidations in combination with Mmt
and Acm groups for the side chain protection of cysteines could be carried out with no peptide
aggregation.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
a
-Conotoxin MII
Soluble tag
Liquid-phase peptide synthesis
Hydrophobic benzyl amine
Disulfide bond
1. Introduction
with various diseases. In addition, a high content of hydrophobic
residues tends to cause peptide aggregation in aqueous media,
Conotoxins are disulfide-rich peptides comprising 10e30 amino
acid residues, originally isolated from the venom of marine snails
belonging to the genus Conus.1 In recent years, based on their
neurotoxic potentials, conotoxins have received much attention as
novel analgesics that modulate the functions of various ion chan-
impeding the use of standard peptide purification procedures. Such
hydrophobicity also hinders peptide folding, which could be a cru-
cial drawback in the chemical synthesis of disulfide-rich peptides,
because their linear precursors are required to adopt folded con-
formations. Therefore, effective chemical synthesis of
remains challenging.
a-conotoxins
nels.2 In particular,
a-conotoxins, which are characterized by potent
and specific inhibition of nicotinicacetylcholine receptors, consti-
tute one of the largest families of conotoxins3 and their structures
and activities have been investigated in detail both practically and
theoretically.4 However, the restricted amount of naturally occur-
One approach to address peptide aggregation is through the use
of soluble tags;6 this method enables significant control of peptide
solubility, with unique product isolation methodologies to ease
reaction work-up. When hydrophobic supports7 are employed in
combination with less polar solvents, peptide aggregation based on
high contents of hydrophobic residues should be inhibited. We
have developed hydrophobic soluble-tag-assisted methods using
benzyl alcohols bearing long alkyl chains as supports,8 which have
led to versatile preparation of bioactive peptides.9 Using this
method, tagged peptides are highly soluble in less polar solvents
such as THF or CH2Cl2. We have also applied our method to an
existing oxidative strategy in which iodine is used in combination
with acetamidomethyl (Acm) groups for side chain protection of
cysteines, leading to the synthesis of somatostatin (1), a growth-
hormone-inhibiting peptide possessing one disulfide bond
(Scheme 1).10 The linear precursor (2) of somatostatin (1) was
prepared using general Fmoc chemistry, followed by the iodine
oxidation to form the tagged cyclic peptide (3) (Scheme 2). All
protective groups could be removed under acidic conditions in one
ring
promising medicinal candidates. In this context, several chemical
syntheses of -conotoxins have been accomplished; some of these
a-conotoxins has hampered further investigations of these
a
have even resulted in the production of artificial variants of the
native forms, realizing improvements in bioavailability and/or en-
hancements in bioactivity.5
In order for chemical synthesis of peptides to be successful, their
aggregation behavior must be taken into account, especially in
liquid-phase approaches. For example, amyloid
b-peptides and
collagens have a well-known tendency to form insoluble fibrils
through intermolecular hydrogen bonding; these are associated
* Corresponding author. Tel./fax: þ81 42 367 5667; e-mail address: chiba@
cc.tuat.ac.jp (K. Chiba).
0040-4020/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2013.01.068

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S. Kitada et al. / Tetrahedron 69 (2013) 2555e2559
Scheme 1. Structure of somatostatin (1) and hydrophobic tag.
Scheme 2. Synthesis of somatostatin (1).
step to give somatostatin (1), and the couplings and deprotections
were carried out efficiently in the liquid phase. Described herein is
the application of the soluble-tag-assisted method toward the
orthogonal two-step oxidative disulfide bond formation, a mono-
methoxytrityl (Mmt) group was chosen in combination with an
Acm group for side chain protection of cysteines, because the Mmt
group can be removed under weakly acidic conditions. Moreover,
synthesis of disulfide-rich
2. Results and discussion
The present work began with the selection of
a-conotoxin.
the C-terminus of
a-conotoxin MII is the amide form; thus, tag
modification is also required.
Based on the existing linker strategy of solid-phase approaches,
we simply designed a hydrophobic benzyl amine (5) that could be
prepared through reductive amination (Scheme 4). With this tag in
a-conotoxin MII
(4), which comprises 16 amino acid residues and possesses 2
disulfide bonds, as a model (Scheme 3). In order to realize
hand, the linear precursor (6) of a-conotoxin MII (4) was prepared

S. Kitada et al. / Tetrahedron 69 (2013) 2555e2559
2557
then added to the solution. The reaction mixture was stirred at
40 ꢀC until the reaction completed. After completion, MeOH was
added to the reaction mixture to give the product as a precipitate.
The precipitate was dissolved in CH2Cl2, and CH2Cl2 solution was
washed with NaHCO3(aq) and brine. The organic layer was dried
over anhydrous Na2SO4. After filtration and evaporation under re-
duced pressure, the product was obtained in 5.06 g, 93% yield.
4.1.1. Compound (5). Hydrophobic benzyl amine (5), amorphous
solid.
1H NMR (CDCl3, 400 MHz)
d
7.17 (2H, d, J¼7.8 Hz), 7.15 (2H, d,
J¼7.8 Hz), 6.46e6.36 (4H, m), 3.92 (2H, t, J¼6.6 Hz), 3.91 (2H, t,
J¼6.6 Hz), 3.79 (3H, s), 3.78e3.73 (4H, m) 3.77 (3H, s), 1.82e1.68
(4H, m), 1.50e1.17 (76H, m), 0.88 (6H, t, J¼6.9 Hz). 13C NMR (CDCl3,
100 MHz) d 160.2, 159.7, 158.5, 158.1, 130.6, 130.6, 120.0, 119.7, 104.3,
103.7, 99.7, 98.4, 68.1, 67.3, 55.3, 55.2, 48.2, 47.6, 31.9, 29.7, 29.6,
29.6, 29.6, 29.4, 29.4, 29.3, 29.3, 29.2, 26.1, 26.0, 22.7, 14.1. HRMS
[MþH]þ calcd for C60H108NO4906.8278, found 906.8275.
Scheme 3. Structure of a-conotoxin MII (4).
4.2. General method for the introduction of amino acid into
the hydrophobic tag
using general Fmoc chemistry (Scheme 5). In this case, the hydro-
phobic benzyl amine (5) was found to act as an effective soluble tag,
and the resulting (6) was then subjected to weakly acidic conditions
to remove the Mmt groups selectively, followed by DEAD oxidation.
To our satisfaction, formation of the tagged cyclic peptide (7) took
place efficiently, with little deprotection of the side chains. For-
mation of the second disulfide bond was carried out via iodine
oxidation, and all protective groups were removed under acidic
The hydrophobic tag (906 mg, 1.0 mmol) was dissolved in
CH2Cl2 (20 mL). Amino acid (1.5 mol equiv), DIC (189 mg, 1.5 mmol),
and DMAP (12.2 mg, 0.1 mmol) were then added to the solution.
The reaction mixture was stirred at room temperature until the
reaction completed. After the completion, MeCN was added to the
reaction mixture to give the product as a precipitate.
4.3. General method for the deprotection of Fmoc group of
the tagged peptide
conditions in one step to give
a-conotoxin MII (4). The tagged
peptide was highly soluble in less polar solvents, and aggregation
posed little problem throughout the overall procedure, enabling
efficient orthogonal two-step oxidative disulfide bond formation.
The tagged peptide was dissolved in 1% DBU and 1% piperidine
in THF (20 mL). The reaction mixture was stirred at room temper-
ature until the reaction completed. After the completion, 12 M HCl
was added to the reaction mixture to neutralize, and then MeCN
was added to give the deprotected product as a precipitate.
4.4. General method for the coupling of amino acid to the
tagged peptide
The tagged peptide was dissolved in THF (20 mL). Amino acid
(1.2 mol equiv), HBTU (1.2 mol equiv), HOBt (1.2 mol equiv), and
DIPEA (2.4 mol equiv) were then added to the solution. The reaction
mixture was stirred at room temperature until the reaction com-
pleted. After the completion, MeCN was added to the reaction
mixture to give the coupled product as a precipitate.
Boc-Gly-Cys(Acm)-Cys(Mmt)-Ser(tBu)-Asn(Trt)-Pro-Val-Cys-
(Acm)-His(Trt)-Leu-Glu(tBu)-His(Trt)-Ser(tBu)-Asn(Trt)-Leu-Cys-
(Mmt)-N(Dmb)-TAG (6), amorphous solid.
Scheme 4. Preparation of hydrophobic benzyl amine (5).
3. Conclusion
HRMS [Mþ2Na]2þ calcd for C266H341N25O32S4Na22287.2293,
found 2287.2295.
In conclusion, we successfully applied the soluble-tag-assisted
method to oxidative disulfide bond formation, leading to the total
synthesis of a-conotoxin MII (4). DEAD and iodine oxidations were
4.5. Acidic deprotection of Mmt groups
employed in combination with the use of Mmt and Acm groups for
side chain protection of cysteines to achieve orthogonal peptide
folding with no peptide aggregation. This strategy is expected to be
a considerable aid to further investigation of the therapeutic pos-
Tagged peptide (6) (35.0 mg, 0.010 mmol) was dissolved in 1%
TFA and 5% triisopropylsilane (TIS) in CH2Cl2 (5 mL). The reaction
mixture was stirred at room temperature until the reaction com-
pleted. After the completion, DIPEA was added to the reaction
mixture to neutralize, which was then poured into CH2Cl2, and the
CH2Cl2 solution was washed with brine. The deprotected product
was obtained through evaporation under reduced pressure.
sibilities of a-conotoxins.
4. Experimental section
4.1. Preparation of the hydrophobic benzyl amine (5)
4.6. Oxidative disulfide bond formation using DEAD
2,4-Bis(docosyloxy)benzaldehyde (4.53 g, 6.0 mmol) was dis-
solved in toluene (60 ml). 2,4-Dimethoxybenzylamine (3.01 g,
18.0 mmol), NaBH(OAc)3 (3.81 g,18.0 mmol), and DMF (60 ml) were
Tagged peptide was dissolved in THF (10 mL). DEAD
(10 mol equiv) was then added to the solution. The reaction

2558
S. Kitada et al. / Tetrahedron 69 (2013) 2555e2559
Scheme 5. Synthesis of a-conotoxin MII (4).
mixture was stirred at room temperature until the reaction com-
pleted. After the completion, the cyclized product was obtained
through evaporation under reduced pressure.
was filtrated by hydrophilic PTFE filter, and then diisopropyl ether
(DIPE) was added to the filtrate to give the product as a precipitate
in 12.1 mg, 71% yield over four steps.
Boc-Gly-Cys(Acm)-c(Cys-Ser(tBu)-Asn(Trt)-Pro-Val-Cys(Acm)-
His(Trt)-Leu-Glu(tBu)-His(Trt)-Ser(tBu)-Asn(Trt)-Leu-Cys)-N(Dmb)-
TAG (7), amorphous solid.
4.8.1.
a
-Conotoxin MII (4). HRMS [MþNa]þ calcd for C67H103
-
N23O22S4Na 1732.6429, found 1732.6432.
HRMS [Mþ1þ2Na]2þ calcd for C226H307N25O30S4Na22014.1017,
found 2014.1017.
Acknowledgements
This work was partially supported by a Grant-in-Aid for Scien-
tific Research from the Ministry of Education, Culture, Sports, Sci-
ence and Technology of Japan.
4.7. Oxidative disulfide bond formation using iodine
The tagged peptide (7) was dissolved in THF (9 mL). I2
(10 mol equiv) in THF (1 mL) was then added to the solution. The
reaction mixture was stirred at room temperature until the reaction
completed. After the completion, 1 M ascorbic acid (1 mL) was
added and product was extracted by CH2Cl2. MeCN was then added
to the reaction mixture to give the cyclized product as a precipitate.
Boc-Gly-c(Cys-c(Cys-Ser(tBu)-Asn(Trt)-Pro-Val-Cys)-His(Trt)-
Leu-Glu(tBu)-His(Trt)-Ser(tBu)-Asn(Trt)-Leu-Cys)-N(Dmb)-TAG (8),
amorphous solid.
Supplementary data
Additional schemes, general information, spectra information,
and copies of 1H and 13C NMR spectra. Supplementary data asso-
ciated with this article can be found in the online version, at http://
dx.doi.org/10.1016/j.tet.2013.01.068.
HRMS [Mþ2Na]2þ calcd for C220H295N23O28S4Na21941.5551,
found 1941.5553.
References and notes
1. Terlau, H.; Olivera, B. M. Physiol. Rev. 2004, 84, 41e68.
2. (a) Carstens, B. B.; Clark, R. J.; Daly, N. L.; Harvey, P. J.; Kaas, Q.; Craik, D. J. Curr.
Pharm. Des. 2011, 17, 4242e4253; (b) Halai, R.; Craik, D. J. Nat. Prod. Rep. 2009,
26, 526e536; (c) Craik, D. J.; Adams, D. J. ACS Chem. Biol. 2007, 2, 457e468; (d)
Nicke, A.; Loughnan, M. L.; Millard, E. L.; Alewood, P. F.; Adams, D. J.; Daly, N. L.;
Craik, D. J.; Lewis, R. J. J. Biol. Chem. 2003, 278, 3137e3144.
4.8. Acidic deprotection of all protective groups
Tagged peptide was dissolved in 2.5% TIS and 2.5% H2O in TFA
(5 mL). The reaction mixture was stirred at room temperature until
the reaction completed. After the completion, the reaction mixture
3. (a) Azam, L.; McIntosh, J. M. Acta Pharmacol. Sin. 2009, 30, 771e783; (b) Ellison,
M.; Gao, F.; Wang, H.-L.; Sine, S. M.; McIntosh, J. M.; Olivera, B. M. Biochemistry

S. Kitada et al. / Tetrahedron 69 (2013) 2555e2559
2559
2004, 43, 16019e16026; (c) Dutton, J. L.; Craik, D. J. Curr. Med. Chem. 2001, 8,
327e344; (d) Harvey, S. C.; McIntosh, J. M.; Cartier, G. E.; Maddox, F. N.; Luetje,
C. W. Mol. Pharmacol. 1997, 51, 336e342; (e) Cartier, G. E.; Yoshikami, D.; Gray,
W. R.; Luo, S.; Olivera, B. M.; McIntosh, J. M. J. Biol. Chem. 1996, 271, 7522e7528;
(f) Johnson, D. S.; Martinez, J.; Elgoyhen, A. B.; Heinemann, S. F.; McIntosh, J. M.
Mol. Pharmacol. 1995, 48, 194e199; (g) McIntosh, J. M.; Yoshikami, D.; Mahe, E.;
Nielsen, D. B.; Rivier, J. E.; Gray, W. R.; Olivera, B. M. J. Biol. Chem. 1994, 269,
16733e16739.
17, 1612e1617; (j) Clark, R. J.; Fischer, H.; Nevin, S. T.; Adams, D. J.; Craik, D. J. J.
Biol. Chem. 2006, 281, 23254e23263.
6. (a) Shemyakin, M. N.; Ovchinnikov, Y. A.; Kiryushkin, A. A.; Kozhevnikovd, I.
V. Tetrahedron Lett. 1965, 6, 2323e2327; (b) Geckeler, K.; Pillai, V. N. R.;
Mutter, M. Adv. Polym. Sci. 1981, 39, 65e94; (c) Narita, M.; Hirata, M.; Ku-
sano, K.; Itsuno, S.; Ue, M.; Okawara, M. Pept. Chem. 1980, 17, 107e112; (d)
Mutter, M.; Hagenmaier, H.; Bayer, E. Angew. Chem., Int. Ed. Engl. 1971, 10,
811e812; (e) Bayer, E.; Mutter, M. Nature 1972, 237, 512e513; (f) Bayer, E.
Angew. Chem., Int. Ed. Engl. 1991, 30, 113e129; (g) Gravert, D. J.; Janda, K. D.
Chem. Rev. 1997, 97, 489e510; (h) Dickerson, T. J.; Reed, N. N.; Janda, K. D.
Chem. Rev. 2002, 102, 3325e3344; (i) Bergbreiter, D. E. Chem. Rev. 2002, 102,
3345e3384; (j) Bergbreiter, D. E.; Tian, J.; Hongfa, C. Chem. Rev. 2009, 109,
530e582.
7. (a) Palcic, M. M.; Heerze, L. D.; Pierce, M.; Hindsgaul, O. Glycoconjugate J. 1988, 5,
49e63; (b) Stangier, P.; Palcic, M. M.; Bundle, D. R. Carbohydr. Res. 1995, 267,
153e159; (c) Kanie, O.; Barresi, F.; Ding, Y. L.; Labbe, J.; Otter, A.; Forsberg, L. S.;
Ernst, B.; Hindsgaul, O. Angew. Chem., Int. Ed. 1996, 34, 2720e2722; (d) Ding, Y.;
Labbe, J.; Kanie, O.; Hindsgaul, O. Bioorg. Med. Chem. 1996, 4, 683e692; (e)
Nilsson, U. J. J. Chromatogr., A 2000, 885, 305e319; (f) Nilsson, U. J.; Fournier, E.
J. L.; Hindsgaul, O. Bioorg. Med. Chem. 1998, 6, 1563e1575; (g) Sato, R.; Toma, K.;
Nomura, K.; Takagi, M.; Yoshida, T.; Azefu, Y.; Tamiaki, H. J. Carbohydr. Chem.
2004, 23, 375e388; (h) Kanie, O.; Ohtsuka, I.; Ako, T.; Daikoku, S.; Kame, Y.;
Kato, R. Angew. Chem., Int. Ed. 2006, 45, 3851e3854; (i) Pozsgay, V. Org. Lett.
1999, 1, 477e479; (j) Pozsgay, V. Tetrahedron: Asymmetry 2000, 11, 151e172; (k)
Bauer, J.; Rademann, J. J. Am. Chem. Soc. 2005, 127, 7296e7297; (l) Encinas, L.;
Chiara, J. L. Eur. J. Org. Chem. 2009, 2163e2173; (m) Li, J.; Sung, S.; Tian, J.;
Bergbreiter, D. E. Tetrahedron 2005, 61, 12081e12092; (n) Guo, J.; Lu, Y.-J.;
Zhang, L.; Ye, X.-S. Synlett 2012, 1696e1700.
4. (a) Yu, R.; Kaas, Q.; David, J.; Craik, D. J. J. Phys. Chem.
B 2012, 116,
6097e6105; (b) Yu, R.; Craik, D. J.; Kaas, Q. PLoS Comput. Biol. 2011, 7,
e1002011; (c) Armishaw, C.; Jensen, A. A.; Balle, T.; Clark, R. J.; Harpsøe, K.;
Skonberg, C.; Liljefors, T.; Strømgaard, K. J. Biol. Chem. 2009, 284,
9498e9512; (d) Maslennikov, I. V.; Shenkarev, Z. O.; Zhmak, M. N.; Ivanov,
V. T.; Methfessel, C.; Tsetlin, V. I.; Arseniev, A. S. FEBS Lett. 1999, 444,
275e280; (e) Gehrmann, J.; Daly, N. L.; Alewood, P. F.; Craik, D. J. J. Med.
€
Chem. 1999, 42, 2364e2372; (f) Rogers, J. P.; Luginbuhl, P.; Shen, G. S.;
McCabe, R. T.; Stevens, R. C.; Wemmer, D. E. Biochemistry 1999, 38,
3874e3882; (g) Quiram, P. A.; Jones, J. J.; Sine, S. M. J. Biol. Chem. 1999, 274,
ꢀ
19517e19524; (h) Lamthanh, H.; Jegou-Matheron, C.; Servent, D.; Menez, A.;
Lancelin, J. M. FEBS Lett. 1999, 454, 293e298; (i) Quiram, P. A.; Sine, S. M. J.
Biol. Chem. 1998, 273, 11007e11011.
5. (a) de Araujo, A. D.; Callaghan, B.; Nevin, S. T.; Daly, N. L.; Craik, D. J.; Moretta,
M.; Hopping, G.; Christie, M. J.; Adams, D. J.; Alewood, P. F. Angew. Chem., Int. Ed.
2011, 50, 6527e6529; (b) Muttenthaler, M.; Nevin, S. T.; Grishin, A. A.; Ngo, S. T.;
Choy, P. T.; Daly, N. L.; Hu, S. H.; Armishaw, C. J.; Wang, C. I.; Lewis, R. J.; Martin,
J. L.; Noakes, P. G.; Craik, D. J.; Adams, D. J.; Alewood, P. F. J. Am. Chem. Soc. 2010,
132, 3514e3522; (c) Armishaw, C. J.; Dutton, J. L.; Craik, D. J.; Alewood, P. F.
Biopolymers 2010, 94, 307e313; (d) MacRaild, C. A.; Illesinghe, J.; van Lierop, B.
J.; Townsend, A. L.; Chebib, M.; Livett, B. G.; Robinson, A. J.; Norton, R. S. J. Med.
Chem. 2009, 52, 755e762; (e) Walewska, A.; Zhang, M.; Skalicky, J. J.; Yoshi-
kami, D.; Olivera, B. M.; Bulaj, G. Angew.Chem., Int. Ed. 2009, 48, 2221e2224; (f)
Galanis, A. S.; Albericio, F.; Grotli, M. Biopolymers 2009, 92, 23e34; (g)
Whiteaker, P.; Marks, M. J.; Christensen, S.; Dowell, C.; Collins, A. C.; McIntosh,
J. M. J. Pharmacol. Exp. Ther. 2008, 325, 910e919; (h) Armishaw, C. J.; Daly, N. L.;
Nevin, S. T.; Adams, D. J.; Craik, D. J.; Alewood, P. F. J. Biol. Chem. 2006, 281,
14136e14143; (i) Vishwanath, V. A.; McIntosh, J. M. Bioconjugate Chem. 2006,
8. (a) Chiba, K.; Kono, Y.; Kim, S.; Nishimoto, K.; Kitano, Y.; Tada, M. Chem. Com-
mun. 2002, 1766e1767; (b) Kim, S.; Tsuruyama, A.; Ohmori, A.; Chiba, K. Chem.
Commun. 2008, 1816e1818; (c) Iijima, M.; Mikami, Y.; Yoshioka, T.; Kim, S.;
Kamiya, H.; Chiba, K. Langmuir 2009, 25, 11043e11047.
9. Tana, G.; Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Chem. Commun. 2010,
8219e8221.
10. Kitada, S.; Fujita, S.; Okada, Y.; Kim, S.; Chiba, K. Bioorg. Med. Chem. Lett. 2011, 21,
4476e4479.