New selenyl linker for solid-phase synthesis of dehydropeptides

Article abstract of DOI:10.1016/S0040-4039(03)01309-1

A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide.

Full text of DOI:10.1016/S0040-4039(03)01309-1

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 44 (2003) 5445–5448
New selenyl linker for solid-phase synthesis of dehydropeptides
Kazuhiko Nakamura,a,* Yuki Ohnishi,a,† Eiji Horikawa,a,b Takeo Konakahara,b Masato Kodakaa
and Hiroaki Okunoa,‡
aNational Institute of Advanced Industrial Science and Technology (AIST), Central 6-9, Higashi 1-1-1, Tsukuba,
Ibaraki 305-8566, Japan
bFaculty of Industrial Science and Technology, Tokyo University of Science 2641 Yamazaki, Noda, Chiba 278-8510, Japan
Received 13 March 2003; revised 12 May 2003; accepted 23 May 2003
Abstract—A novel linker possessing selenocyanate and masked carboxylic acid was developed for the solid-phase synthesis of
dehydropeptides. This linker was used to demonstrate the synthesis of the model compound of RGD-conjugated dehydropeptide.
© 2003 Elsevier Science Ltd. All rights reserved.
Dehydropeptides, containing a,b-unsaturated amino
acid (or dehydroamino acid) moiety, are typical abnor-
mal peptides.1 Their structures are rigid and reactive
because they have a double bond conjugated with a
peptide linkage. In a previous study on the design of
artificial peptides, the introduction of dehydroamino
acid residues into normal peptides induced folded con-
formations based on structural factors.2 A number of
dehydropeptides are bioactive substances isolated from
natural resources. Compounds typical of this class
include AM-toxin,3,4 kahalalide F5 and Sch20561.6
Dehydropeptides are also known as fairly reactive
Michael acceptors that react readily with ‘soft’ nucleo-
philes, such as thiols or amines of biological
molecules. This reactivity is thought to be one of the
molecular mechanisms underlying the biological activi-
ties of dehydropeptides.1 However, the instability of
these chemical properties makes it difficult to synthesize
such molecules, especially in the case of solid-phase
synthesis.
dehydroamino acid. The linker was introduced onto a
starting substrate, instead of onto a polystyrene-based
resin, followed by immobilization on a commercially
available resin through activation of a carboxylic acid.
The construction of an unstable double bond was real-
ized simultaneously with oxidative cleavage from the
solid phase in the final stage of synthesis.9 However, to
derive carboxylic acid from the nitro group requires a
multi-step reaction (reduction to amine, then reaction
with succinic anhydride to give hemi-amide), and a
cyclic imide would be formed by a side reaction during
the activation of the carboxylic acid for
immobilization.10
To avoid these problems, we designed a new selenyl
linker. Our purposes required an aryl selenocyanate
with a direct carboxylic acid precursor.11 Thus, we
selected acid treatment to expose the carboxylic acid,
whose condition is orthogonal to Fmoc (N-terminal)
and allyl (C-terminal) deprotection. The typical synthe-
sis of a seleno linker is depicted in Scheme 1. The
synthesis was started with a nitrobenzoic acid 1, which
Recently, the authors synthesized AM-toxin II using a
solid-phase method.7 That synthesis utilized commer-
cially available nitrophenyl selenocyanate8 as an acti-
vatable linker to immobilize
a
precursor of
Keywords: dehydropeptide; solid-phase synthesis; selenyl linker;
RGD-conjugate.
* Corresponding author. Tel.: +81-29-861-6186; fax: +81-29-861-6123;
e-mail: kazuhiko.nakamura@aist.go.jp
† Present address: Advanced Science Research Center, Japan Atomic
Energy Research Institute, Tokai-mura, Ibaraki 319-1195, Japan.
‡ Present address: Faculty of Pharmaceutical Sciences, Toho Univer-
sity, Funabashi, Chiba 274-8510, Japan.
Scheme 1. Synthesis of seleno linker. Keys: (a) Boc2O,
DMAP/tert-BuOH; (b) H2Pd(C)/EtOH (two steps 90%); (c)
NaNO2, dil. HCl then KSeCN (50%).
0040-4039/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0040-4039(03)01309-1

5446
K. Nakamura et al. / Tetrahedron Letters 44 (2003) 5445–5448
was derived into the corresponding tert-butyl ester.
The nitroaryl ester was reduced to give the conse-
quent aniline 2. A subsequent Sandmeyer reaction12
with potassium selenocyanate in aqueous media gave
an aryl selenocyanate, the desired seleno linker 3.
N,C-Protected serine or threonine was reacted with
the selenocyanate (3) and tributyl phosphine in
CH2Cl2 at 0°C to give the corresponding selenoether13
4 (see table in Fig. 1). According to the result, the
m-substituted selenocyanate 3a provided the desired
selenoether 4a in acceptable yield. A byproduct, dehy-
droamino acid, is presumed to form via a Mitsunobu
intermediate [A] by six-membered ring transition
state.14 The tert-butyl esters of selenoethers 4 could
very much be directly converted into the correspond-
ing carboxylic acids in quantitative yield by the
action of 95% TFA, compared to the o-nitrophenyl
selenoether for the synthesis of AM-toxin.7 Therefore,
the selenoether 4a was selected for the precursor of
side-chain immobilized amino acid for solid-phase
synthesis of dehydropeptides.
(0.14 mmol/g). This amount was the basis on which
the molar scale was calculated for the following reac-
tions.19 After the C-terminal deprotection by palla-
dium chemistry (20 mol% Pd(PPh3)4 and excess
dimedone in THF), the C-terminal condensation was
achieved by phenylalanyl alanine methyl ester hydro-
chloride and HATU-DIEA to give a protected tripep-
tide on the solid support 6. This was followed by
N-terminal elongation using Pioneer™ peptide synthe-
sizer (Applied Biosystems Inc.). Automatic synthesis
was performed by successive coupling with Fmoc-
Asp(tBu)-OH, Fmoc-Gly-OH and Fmoc-Arg(Pfb)-OH
by repeating the HATU chemistry using built-in pro-
tocols.20 After the capping reaction, the acetylated
hexapeptide was obtained on solid support 7.
Finally, oxidative cleavage from the resin via selenox-
ide was performed by treatment with hydrogen perox-
ide in THF to give the desired RGD-conjugated
dehydropeptide11 8 with concomitant formation of a
double bond, which was easily purified by simple
chromatography on silica gel. The yield (77%) was
calculated on the basis of the loading amount deter-
mined for the starting immobilized amino acid. The
final reaction was carried out without loss of any
protecting groups for further chemical modification
after appropriate deprotection (Scheme 2).
Solid-phase synthesis using tert-butyl 3-selenocyanato-
benzoate (3a) was demonstrated by the following syn-
thesis of RGD-conjugated dehydropeptide. The RGD
sequence is the common recognition motif for the
integrin family of receptors, which are involved in
cell–cell and cell–matrix adhesion,15 and it was
reported that b-turn conformation was important for
the binding.16 To the seleno ether 4a, deprotection
was achieved by the action of 95% TFA in quantita-
tive yield. The resultant benzoic acid was immobilized
to a commercially available aminoethyl resin (Cal-
biochem-Novabiochem, NovaSyn™ TG amino resin;
amino content 0.29 mmol/g) with HATU and DIEA.
The amount of seleno amino acid to load on solid
support was determined by Fmoc quantitation17,18
In conclusion, we have demonstrated the potentiality
of the newly developed strategy to synthesize dehy-
dropeptides, as well as the potentiality of our new
selenium-containing linker. We successfully synthe-
sized the model dehydropeptide by both manual (C-
terminal) and automatic (N-terminal) procedures. We
consider that this new synthetic methodology will also
be quite useful in the preparation of unsaturated
compounds using solid-phase chemistry.
Figure 1. Synthesis of selenoether.

K. Nakamura et al. / Tetrahedron Letters 44 (2003) 5445–5448
5447
Scheme 2. Synthesis of model dehydropeptide. Reagents and conditions: (a) TFA; (b) H2N-resin, HATU, DIEA; (c) Pd(PPh3)4,
dimedone/THF; (d) HCl Phe-Ala-OMe, HATU, DIEA/DMF; (e) Fmoc-Asp(tBu), Fmoc-Gly, Fmoc-Arg(Pbf)/HATU-DIEA by
Pioneer™ Peptide Synthesizer; (f) Ac2O–DIEA/DMF; (g) H2O2 aq./THF overnight.
also: Nicolaou, K. C.; Pfefferkorn, J. A.; Cao, G.-Q.
Angew. Chem., Int. Ed. 2000, 39, 734–739; Nicolaou, K.
C.; Cao, G.-Q.; Pfefferkorn, J. A. Angew. Chem., Int. Ed.
2000, 39, 739–743.
Acknowledgements
The authors thank Professor Shigeru Nishiyama of
Keio University for the HRMS measurements of our
products.
10. Unpublished results.
11. An arylselenocyanate linker to modify polystylene-based
supports has been reported: Fujita, K.; Watanabe, K.;
Oishi, A.; Ikeda, Y.; Taguchi, Y. Synlett 1999, 1760–
1762.
12. Sharpless, K. B.; Young, M. W. J. Org. Chem. 1975, 40,
947–949.
References
1. Humphrey, J. M.; Chamberlin, R. Chem. Rev. 1997, 97,
2243–2266.
2. Vijayaraghavam, R.; Kumar, P.; Dey, S.; Singh, T. P. J.
Peptide Res. 1998, 52, 89–94.
3. Okuno, T.; Ishita, Y.; Sawai, K.; Matsumoto, T. Chem.
Lett. 1974, 635–638.
4. Ueno, T.; Nakashima, T.; Hayashi, Y.; Fukami, H.
Agric. Biol. Chem. 1975, 39, 1115–1122.
13. Selected data of the key compounds. Compound 4a: mp
37–38°C; HRMS (EI) calcd for C12H14NO280Se m/z
284.0188, found m/z 284.0187; IR (film): 1714, 1569,
1
1369, 1302, 1163, 1127, 1012, 848, 748 cm−1; H NMR l
(CDCl3): 1.61 (9H, s) 7.48 (1H, t, J=8.0 Hz), 7.82 (1H, d,
J=8.0 Hz), 8.05 (1H, d, J=8.0 Hz), 8.23 (1H, s). Com-
pound 5a: [h]2D3 25.2° (c 1.00, CHCl3); HRMS (EI) calcd
for C32H34NO678Se m/z 606.1558, found m/z 606.1584;
IR (film): 3342, 1713br, 1507, 1450, 1413, 1368, 1300,
`
5. Lo´pez-Macia`, A.; Jime´nez, J. C.; Royo, M.; Giralt, E.;
Albericio, F. J. Am. Chem. Soc. 2001, 123, 11398–11401.
6. Afonso, A.; Hon, F.; Brambilla, R. J. Antibiot. 1999, 52,
383–397.
7. Horikawa, E.; Kodaka, M.; Nakahara, Y.; Okuno, H.;
Nakamura, K. Tetrahedron Lett. 2001, 42, 8337–8339.
8. Grieco, P. A.; Gilman, S.; Nishizawa, M. J. Org. Chem.
1976, 41, 1485–1486.
9. Synthesis of unsaturated compounds by specially pre-
pared selenium reagents on polymer support has been
reported by: Nicolaou, K. C.; Pastor, J.; Barluenga, S.;
Winssinger, N. Chem. Commun. 1998, 1947–1948. See
1
1126, 989, 936, 848, 743 cm−1; H NMR (CDCl3) l 1.57
(9H, s), 3.40 (1H, dd, J=4.9, 13.6 Hz), 3.47 (1H, dd,
J=4.6, 13.6 Hz), 4.20 (1H, t, J=7.0 Hz), 4.33 (3H,
complex), 4.53 (1H, dd, J=5.5, 12.8 Hz), 4.77 (1H, dist.
dt, J=4.6, 7.9 Hz), 5.22 (1H, br. d, J=10.3 Hz), 5.27
(1H, br. d, J=19.0 Hz), 5.63 (1H, br. d, J=7.9 Hz), 5.80
(1H, complex), 7.25–7.35 (3H, arom.), 7.40 (2H, arom.),
7.56 (2H, arom.), 7.70 (1H. arom.), 7.70 (2H, arom.), 7.86
(1H, arom.), 8.17 (1H, s, arom.). Compound 9: MALDI
TOF-MS calcd for C47H67N9O13S·Na (M+Na)+ m/z
1020.45, found m/z 1020.32; [h]2D2 −14.3° (c 1.00, CHCl3);

5448
K. Nakamura et al. / Tetrahedron Letters 44 (2003) 5445–5448
1H NMR (CDCl3, selected) l 1.40 (3H, s), 1.41 (3H, d,
overlapped), 1.41 (3H, s), 1.42 (3H, s), 1.45 (9H, s), 2.08
(3H, s), 2.49 (3H, s), 2.56 (3H, s), 2.94 (3H, s), 3.69 (3H,
s), 5.24 (1H, br.s), 6.33 (1H, br.s), 7.15–7.25 (5H,
arom.). This compound was unstable during SiO2 chro-
matography (MeOH–CHCl3 1:9) by formation of an
MeOH adduct.
London, 1998; pp. 213–235.
18. Loading yield (52%) was calculated based on the
amount of Fmoc-Ser-OAll, and it was in harmony with
weight-based yield (51%). However, Keiser ninhydrin
test17 indicated very low concentration of remaining
reactive amino groups (2.8 mmol/g). Thus, the authors
did not carry out any capping reaction, and the follow-
ing reactions were carried out using this loading
amount.
14. The detailed reaction mechanism and selectivity will be
discussed elsewhere.
19. The peptide elongation was carried out at 0.03 mmol
scale.
15. Giannis, A.; Ru¨bsam, F. Angew. Chem., Int. Ed. 1997,
36, 588–590.
20. Using ‘extended cycle’, Fmoc deprotection 5 min, cou-
pling 60 min.
16. Yamada, T.; Kidera, A. FEBS Lett. 1996, 387, 11–15.
17. Bunin, B. A. The Combinatorial Index; Academic Press: