Facile solid phase synthesis of N-cycloguanidinyl-formyl peptides

Article abstract of DOI:10.1016/j.tetlet.2014.01.100

A novel strategy of solid phase synthesis of N-cycloguanidinyl-formyl peptides has been established and investigated which involved coupling orthogonal protected diaminoacid with resin bound peptide, α-amino group deprotection, guanidinylation of α-amino group by bis-Cbz-1H-pyrazole-1- carboxamidine followed by cleavage and cyclization in solution, and finally removing Cbz by palladium catalyzed hydrogenation. Through this method, cycloguanidine could be introduced to either N-terminus or sidechain of designated peptides. The reaction conditions were facile, straightforward, and totally adaptive to common solid phase peptide synthesis strategy.

Full text of DOI:10.1016/j.tetlet.2014.01.100

Tetrahedron Letters 55 (2014) 1733–1735
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Facile solid phase synthesis of N-cycloguanidinyl-formyl peptides
⇑
⇑
Xiaoxiao Yang , Guoliang Bai, Hao Lin, Dexin Wang
Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing City Key Laboratory of Active Substances Discovery and
Drugability Evaluation, Beijing 10050, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel strategy of solid phase synthesis of N-cycloguanidinyl-formyl peptides has been established and
Received 29 November 2013
Revised 15 January 2014
Accepted 23 January 2014
Available online 31 January 2014
investigated which involved coupling orthogonal protected diaminoacid with resin bound peptide,
a-amino group deprotection, guanidinylation of a-amino group by bis-Cbz-1H-pyrazole-1-carboxami-
dine followed by cleavage and cyclization in solution, and finally removing Cbz by palladium catalyzed
hydrogenation. Through this method, cycloguanidine could be introduced to either N-terminus or side-
chain of designated peptides. The reaction conditions were facile, straightforward, and totally adaptive
to common solid phase peptide synthesis strategy.
Keywords:
Cycloguanidine
Solid-phase peptide synthesis
Intramolecular cyclization
Peptidomimetics
Ó 2014 Elsevier Ltd. All rights reserved.
Microwave-assisted peptide synthesis
Guanidine as a strong basic cation pharmacophore could be
found in various bioactive natural products from microorganism,
plants, marine organism, and vertebrates. Among guanidine com-
pounds, cyclic guanidines with unique structure and bioactivity
such as antimicrobial,1 cytotoxic,2 antitumor activity,2 ion channel
inhibition,3 HIV-1 protease inhibition,4 and HIV-1 Nef inhibition5
are under the spotlight of medicinal chemistry research. Natural
microorganism derived unusual amino acids including enduracid-
idine,6 Tetrahydrolathyrine7 and capreomycidine8 consist of five
or six-membered cycloguanidine.
acid (Dab) was loaded on aminomethyl-polystyrene (AM) resin
and reaction was monitored by ninhydrin test11 (Scheme 1). How-
ever, due to low nucleophilicity of a-NH2 of amino acids, it was not
only incapable to be guanidinylated by 1a but also insufficient to
attack the carbon atom of the side chain guanidine intermediate 2.
Thus, we introduced N,N’-dicarbobenzoxyl-1H-pyrazole-1-car-
boxamidine (1b) which reported12 to be capable of guanidinylating
a-NH2 of tyrosine residue to guanidinylate a-NH2 of resin bound
diaminobutyric acid. After cleavage by TFA, cyclization was carried
out by diluting and neutralizing with sodium hydroxide in water.
Reaction was monitored by HPLC which achieved completion after
1 h. Cbz-group was then removed by hydrogenation to give cyclo-
guanidine carboxylic acid 5.13 Formation of compound 4 was con-
firmed by 2D-NMR HMBC test in which quaternary carbon atom of
guanidine carbon (dC 153.11) showed long range correlations with
We have previously reported9 a feasible method to synthesize
five or six-membered aliphatic cycloguanidine by one-pot guani-
dination of diamine by bis-Cbz-S-methylisothiourea (1a) followed
by intramolecular 1,4-nucleophilic addition–elimination reaction.
This result inspired us to develop a simple and convenient method
to synthesize novel cycloguanidine containing peptides. Though
early research reported the synthesis of six-membered cycloguani-
the signal of a-H (dH 4.653, 1H, t, J = 6.0 Hz) and the c-H (dH 3.729,
2H, m) as well (Scheme 2).14 Free cycloguanidinoacid 5 showed
identical analytical property with the reported compound,10
including specific rotary power, melting point, and 1H NMR
spectrum data.
dine carboxylic acid by prolonged intramolecular reaction of
a-
amino-
c
-guanidinobutyricacid in aqueous solution,10 it would be
more convenient and feasible to directly introduce cycloguanidine
to the resin bound peptide on solid-phase rather than synthesizing
N-protected cycloguanidine carboxylic acid by solution-phase syn-
thesis separately. To verify if we could apply the same synthesis
strategy on diamino acid with the same guanidinylation reagent
1a used in previous study, orthogonal protected diaminobutyric
By this method, we managed to introduce five-, six-, or seven-
membered cycloguanidine to the literature reported bioactive pep-
tides to substitute their arginine, pyro-glutamine, or cyclourea at
either N-terminus or side chain to mimic their structures
(Scheme 3, Table 1).
The typical synthesis of compound 11a–h involved conven-
tional peptide synthesis on Rink amide resin or 2-Cl-Trt resin
(for 11f) under microwave-assistance, followed by coupling
⇑
Corresponding authors. Tel.: +86 10 63165251.
E-mail addresses: yangxx@imm.ac.cn (X. Yang), wangdx@imm.ac.cn (D. Wang).
http://dx.doi.org/10.1016/j.tetlet.2014.01.100
0040-4039/Ó 2014 Elsevier Ltd. All rights reserved.

1734
X. Yang et al. / Tetrahedron Letters 55 (2014) 1733–1735
O
H
O
N
HN Cbz
H2N
HN Cbz
N
Fmoc
Boc
N
H
N
H
S
a
N
b
d
NR
NR
N Cbz
1a
N
1b
Cbz
NH
NH
Boc
H2N
O
O
H
O
N
H
N
N
H
Boc
N
H
c
Boc
N
H
a
H
N
NH
Cbz
NH
NH2
N
2
Fmoc
Cbz
Scheme 1. Reagents and conditions (AM resin as solid support): (a) 20% piperidine/DMF; (b) 1a (3.0 equiv), NMM, pyridine, rt; (c) 1a (3.0 equiv), NMM, pyridine, rt, then 90%
TFA/DCM; (d) NMM (2.0 equiv), pyridine (NR: no reaction).
the synthesis of 11h, Boc-Lys(Fmoc)-OH was coupled with N-ter-
Fmoc
HN
O
O
minus Leu followed by coupling Fmoc-Dap(Boc)-OH. Fmoc was
removed followed by guanidinylating by 1b. Boc group of Lys
and Dap was removed by TFA cleavage simultaneously before
H2N
b
a
O
O
Cl
H
H
N
N
Boc
Boc
cyclization. Since a-amino group of Lys was insufficient to attack
Cbz Cbz
Cbz
Cbz
HN
HN
guanidinyl group, 10h should be the only proper product. Reac-
tion was smoothly completed overnight except for the seven-
membered product 10b, which took 15 days in pyridine solution
to achieve merely 50% conversion (by HPLC). Attempt had also
been made to synthesize eight-membered ring product by
guanidinylated lysine at N-terminus (n = 4 in compound 11b),
however, probably due to increasing ring strain, no evidence of
cyclization was observed even after 15 days. Furthermore, by
refluxing its pyridine reaction solution, though new peaks were
monitored by HPLC, neither was proved to be target product by
LC–MS. The phenomenon indicated that the cyclization reaction
was more favorable for the synthesis of five- and six-membered
cycloguanidine.
HN
N
O
N
O
d
f
c
HN
O
OH
3
H
N
H2N
TFA
Boc
Cbz
O
O
H
N
H
N
N
HN
HN
ONa
e
OH
HN
4
5
Scheme 2. Reagents and conditions (2-Cl-Trt resin as solid support): (a) Fmoc-
Dab(Boc)-OH, DIEA (4.0 equiv); (b) 20% piperidine/DMF; (c) 1b, TEA, pyridine; (d)
TFA/TESi/H2O (95:2.5:2.5, v:v); (e) NaOH (2.0 equiv) in H2O (C = 10 lmol/ml); (f)
10% Pd/C, H2, HOAc/H2O, rt.
Since the byproduct benzyl carbamate generated with com-
pounds 10 could be successively decomposed by final hydrogena-
tion to toluene and ammonia, direct hydrogenation of crude
compound 10 followed by evaporation of the filtrate thoroughly
would give target peptides in excellent purity. All final products
were confirmed by HRMS and representative oligopeptides 11a,
11b, and 11e were also verified by 1H NMR, 13C NMR, COSY, HSQC,
and HMBC.
In conclusion, a novel strategy of solid phase synthesis of cyclo-
guanidinyl-formyl peptide has been established and investigated.
The reaction conditions are facile, straightforward, and totally
adaptive to common solid phase peptide synthesis strategy. In
the present letter, we carried out cyclization post-cleavage in solu-
tion-phase, however, it could also be accomplished on solid-phase
before cleavage with proper linker viz safety-catch linker22 which
was stable to the N-deprotection condition of the side chain amino
group. Since compounds 11 were modified from bioactive
peptides, their bioactivities are going to be further evaluated.
commercially available orthogonal protected L-2,3-diaminoprop-
anoic acid (Dap, 6a), L-2,4-diaminobutanoic acid (Dab, 6b), or
L-2,5-diaminopentanoic acid (Orn, 6c), typically in
protected and side chain Boc protected form. After removal of
Fmoc group by piperidine, -amino group was successively
a-Fmoc
a
guanidinylated by 1b21 (2 equiv) with triethylamine (2 equiv) in
pyridine as solvent which gave intermediate 8. Excessive 1b could
be recycled by evaporating the drained reaction solution or di-
rectly applying to another solid-phase guanidinylation reaction.
Peptide cleavage and Boc-deprotection was achieved simulta-
neously by TFA based cleavage cocktail treatment to yield com-
pound 9. Intramolecular cyclization was attained by diluting
compound 9 in acetonitrile or methanol to 10 lmol/ml concen-
tration and neutralizing by 1.0 equiv of N-methylmorpholine
(NMM). The reaction was then monitored by RP-HPLC. As for
Cbz
H
O
H
N
O
N
N
H2N
O
Cbz
OH
Fmoc
Boc
Peptide
a
b
Peptide
H
HN
Boc
H2N
+
Peptide
Boc
n
n
N
H
N
H
6a~c
7a~h
n
N
H
8a~h
Cbz
N
N
O
O
H
O
Cbz
H
N
Cbz
e
c
d
N
HN
Peptide
R
Peptide
Peptide
R
HN
R
N
n
HN
n
n
HN
TFA H2N
9a~h
10a~h
11a~h
(n=1~3; R=-NH2 or -COOH)
Scheme 3. Reagents and conditions (Rink amide resin or 2-Cl-Trt resin as solid support): (a) HBTU, Cl–HOBt, DIEA; (b) 1b, TEA, pyridine; (c) TFA/TESi/H2O (95:2.5:2.5, v:v);
(d) NMM/MeCN; (e) 10% Pd/C in MeOH.

X. Yang et al. / Tetrahedron Letters 55 (2014) 1733–1735
1735
Table 1
Data of cycloguanidine peptides in Scheme 3
Entry
No.
Structure
Refs.
15
HRMS (m/z) [M+H]+
Yield (%)
Calculated
Found
O
H
HN
HN
N
1
2
3
4
5
11a
11b
11c
11d
11e
387.2139
401.2296
786.4621
394.2197
363.1888
387.2139
82
38
70
81
83
Phe-Pro-NH2
O
H
N
HN
HN
15
16
17
18
401.2298
786.4603
394.2199
363.1898
Phe-Pro-NH2
O
O
O
H
N
HN
Gly-Dab-Pro-Tyr-Ile-Leu-NH2
Gly-Pro-Pro-NH2
HN
H
N
HN
HN
H
N
His-Pro-NH2
HN
HN
O
O
H
N
6
7
11f
Gly-Ile-OH
19
19
300.1666
313.1983
300.1676
313.1979
87
86
HN
HN
HN
H
N
11g
Gly-Ile-NH2
HN
H2N-Lys-Leu-Arg-Pro-Ala-NH2
HN
HN
NH
N
8
11h
20
694.4471
694.4501
74
O
H
5. Olszewski, A.; Sato, K.; Aron, Z. D.; Cohen, F.; Harris, A.; McDougall, B. R.;
Robinson, W. E., Jr.; Overman, L. E.; Weiss, G. A. Proc. Natl. Acad. Sci. U.S.A. 2004,
101, 14079–14084.
6. Sanière, L.; Leman, L. C.; Bourguignon, J.-J.; Dauban, P.; Dodd, R. H. Tetrahedron
2004, 60, 5889–5897.
Acknowledgment
The authors would like to thank project 81001366 by NSFC and
basic research program 2012CHX12 of Institute of Materia Medica
for funding this research work.
7. Benohoud, M.; Leman, L.; Cardoso, S. H.; Retailleau, P.; Dauban, P.; Thierry, J.;
Dodd, R. H. J. Org. Chem. 2009, 74, 5331–5336.
8. DeMong, D. E.; Williams, R. M. J. Am. Chem. Soc. 2003, 125, 8561–8565.
9. Yuan, J. H.; Yang, X. X.; Lin, H.; Wang, D. X. Chin. Chem. Lett. 2011, 22, 1399–
1402.
10. Marconi, G. G.; Bodanszky, M. J. Antibiot. 1970, 23, 120–124.
11. Sarin, V. K.; Kent, S. B.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 1981, 117, 147–
157.
12. Ogawa, T.; Miyamae, T.; Murayama, K.; Okuyama, K.; Okayama, T.; Hagiwara,
M.; Sakurada, S.; Morikawa, T. J. Med. Chem. 2002, 45, 5081–5089.
13. Bis-Cbz-1H-pyrazole-1-carboxamidine was synthesized from 1H-pyrazole-1-
carboxamidine according to the reported method: Bernatowics, M. S.; Wu, Y.;
Matsueda, G. R. Tetrahedron Lett. 1993, 34, 3389–3392.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.tetlet.2014.01.
100. These data include MOL files and InChiKeys of the most
important compounds described in this article.
14. Al-Shuhaib, Z.; Bockemeier, H.; Coghlan, L.; Dorksen, E.; Jones, I. V.; Murphy, P.
J.; Nash, R.; Page, J. M. Tetrahedron Lett. 2013, 54, 6716–6718.
15. Gkonos, P. J.; Kwok, C. K.; Block, N. L.; Roos, B. A. Peptides. 1994, 15, 1281–1283.
16. Cusack, B.; McCormick, D. J.; Pang, Y. P.; Souder, T.; Garcia, R.; Fauq, A.;
Richelson, E. J. Biol. Chem. 1995, 270, 18359–18366.
17. Mentlein, R.; Struckhoff, G. J. Neurochem. 1989, 52, 1284–1293.
18. Schwertner, E.; Herrling, S. U.S. Patent 4,045,556 A, 1997.
19. Capecchi, J. T.; Loudon, G. M. J. Med. Chem. 1985, 28, 140–143.
20. Braun, K.; Kuhl, P.; Bernd, M.; Kustscher, B. Pharmazie 2001, 56, 45–49.
21. Bernatowicz, M. S.; Wu, Y.; Matsueda, G. R. Tetrahedron Lett. 1993, 34, 3389–
3392.
References and notes
1. Asaoka, M.; Sakurai, M.; Takei, H. Tetrahedron Lett. 1990, 31, 4759–4760.
2. Regasini, L. O.; Castro-Gamboa, I.; Silva, D. H. S.; Furlan, M.; Barreiro, E. J.;
Ferreira, P. M. P.; Pessoa, C.; Lotufo, L. V. C.; de Moraes, M. O.; Young, M. C. M.;
Bolzani, V. d. S. J. Nat. Prod. 2009, 72, 473–476.
3. Hisamoto, M.; Inaoka, Y.; Sakaida, Y.; Kagazaki, T.; Enokida, R.; Okazaki, T.;
Haruyama, H.; Kinoshita, T.; Matsuda, K. J. Antibiot. (Tokyo) 1998, 51, 607–
617.
4. Jadhav, P. K.; Woerner, F. J.; Lam, P. Y.; Hodge, C. N.; Eyermann, C. J.; Man, H.
W.; Daneker, W. F.; Bacheler, L. T.; Rayner, M. M.; Meek, J. L.; Erickson-Viitanen,
S.; Jackson, D. A.; Calabrese, J. C.; Schadt, M.; Chang, C. H. J. Med. Chem. 1998, 41,
1446–1455.
22. Berst, F.; Holmes, A. B.; Ladlow, M.; Murray, P. J. Tetrahedron Lett. 2000, 41,
6649–6653.