Synthesis of cyclic peptides through hydroxyl side-chain anchoring

Article abstract of DOI:10.1016/j.tetlet.2003.11.107

A general method was developed for the synthesis of serine or threonine containing cyclic peptides utilizing the β-hydroxyl side-chain of these residues as an anchor point to Wang resin. The peptide chain was assembled by conventional Fmoc/tBu solid-phase chemistry followed by palladium catalyzed exposure of the allyl protected C-terminus group and on-resin cyclization. The cyclic heptapeptide stylostatin 1 was prepared to demonstrate the utility of this technique.

Full text of DOI:10.1016/j.tetlet.2003.11.107

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 923–925
Synthesis of cyclic peptides through hydroxyl side-chain anchoring
Liang Z. Yan,* Patrick Edwards, David Flora and John P. Mayer*
Lilly Research Laboratories, A Division of Eli Lilly and Company, Lilly Corporate Center, Indianapolis, IN 46285, USA
Received 27 August 2003; revised 23 October 2003; accepted 24 November 2003
Abstract—A general method was developed for the synthesis of serine or threonine containing cyclic peptides utilizing the b-hy-
droxyl side-chain of these residues as an anchor point to Wang resin. The peptide chain was assembled by conventional Fmoc/tBu
solid-phase chemistry followed by palladium catalyzed exposure of the allyl protected C-terminus and on-resin cyclization. The
cyclic heptapeptide stylostatin 1 was prepared to demonstrate the utility of this technique.
ꢀ 2003 Elsevier Ltd. All rights reserved.
The on-resin cyclization of peptides is a particularly
advantageous approach in that it avoids the isolation
and purification of the linear intermediate, minimizes
dimerization side-reactions and results in increased
overall yield of the final product. Practical application of
this method to homodetic Ôhead-to-tailÕ cyclic peptides,
however, requires a specialized solid-phase strategy,
including orthogonal protection for the C-terminus as
well as solid support linkage to either an amino acid
side-chain or peptide backbone.1;2 To date, side-chain
anchoring strategies have been devised for a number of
amino acid side-chain functionalities. Merrifield and
co-workers first reported the synthesis of a hexapeptide
through dinitrophenyl linkage to a histidine imidazole.3
Subsequent application of this concept utilized the side-
chains of Asp/Asn and Glu/Gln in conjunction with
temporary allyl protection for the C-terminal a-carboxyl
group.4;5 This strategy was further extended to other
side-chains using active carbonate linker for hydroxyl
side-chains of Ser, Thr,6 and Lys,7 as well as the recently
reported chroman linkage for Arg.8 Additionally, the
phenol group of Tyr has been successfully linked to solid
support through Mitsunobu chemistry.9
synthesis of C-terminal peptide alcohols.10 We reasoned
that the b-hydroxyl group of serine and threonine would
function equally well as substrates in the BF3 etherate
catalyzed loading of trichloroacetimidate activated
Wang resin. Indeed, Fmoc-Ser-OAllyl11 was loaded with
a substitution level of 0.44 mmol/g, and similarly Fmoc-
Thr-OAllyl achieved a loading of 0.35 mmol/g using
previously reported conditions.10;12 Briefly, commer-
cially available trichloroacetimidate Wang resin
(1.0 mmol, 0.77 mmol/g) was swollen in DCM for 30 min
and then washed several times with dry THF. Fmoc-Ser-
OAllyl (4.0 mmol) was dissolved in dry THF and
transferred to the resin bed. Following the addition of
BF3 etherate (1.0 mmol) via syringe, the mixture was
gently agitated for 1 h at room temperature. Methanol
(2.0 mL) was then added and the reaction was allowed to
proceed for another 5 min. The substitution level was
determined by the method of Meienhofer.13
To demonstrate the utility of this technique, we carried
out the synthesis of stylostatin 1, a cycloheptapeptide
originally isolated from the South Pacific Ocean
sponge14 and synthesized previously by several groups.15
The side-chain immobilized Fmoc-Ser-(Wang resin)-
OAllyl prepared above was used in the Fmoc/tBu-based
synthesis. The primary sequence was assembled using an
ABI 433A synthesizer utilizing a single coupling, DCC/
HOBt activation protocol, a four-fold excess of each
amino acid residue and a conventional protecting group
scheme. The removal of allyl protection was carried out
according to the method by Albericio and co-workers.16
Two 30 min treatments at room temperature with a cat-
alytic amount of Pd(PPh3)4 (0.1 equiv) and an excess of
allyl acceptor phenylsilane (24 equiv) in DCM resulted
We recently reported a procedure to link amino acid
alcohols to activated Wang resin as an initial step in the
Keywords: Hydroxyl side-chain loading; Cyclic peptide synthesis;
Trichloroacetimidate resin.
* Corresponding authors. Tel.: +1-317-433-7283; fax: +1-317-276-1177
(L.Z.Y.); tel.: +1-317-277-9152; fax: +1-317-276-1177 (J.P.M.);
e-mail addresses: lyan@lilly.com; j.mayer@lilly.com
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.107

924
L. Z. Yan et al. / Tetrahedron Letters 45 (2004) 923–925
BF3·Et2O,
dry THF
NH
peptide chain assembly
using Fmoc chemistry
FmocHN
Allyl-O
O
Cl3C
O
FmocHN
OH
O
O
Allyl-O
O
O
O
O
O
H
H
N
N
O
N
Allyl
Fmoc
N
H
N
H
N
H
N
H
NH
O
O
O
Trt
O
a,b,d
a,b,c,d
NH2
OH
HN
O
O
OH
OH
O
O
O
O
O
H
N
H
N
N
N
H
O
H2N
N
H
N
H
N
c
NH
NH
H
O
O
O
O
O
NH2
HN
HN
O
N
O
Stylostatin 1
O
Scheme 1. Synthesis of stylostatin 1 (all reactions were carried out at room temperature unless otherwise specified): (a) 20% piperidine in DMF;
(b) Pd(Ph3P)4/PhSiH3 in DCM, 30 min twice; (c) PyBOP/HOBt/DIEA in DMF; (d) TFA.
in clean removal of the allyl protecting group. The resin-
bound peptide was then used in the on-resin cyclization,
while a portion was subjected to acidolytic cleavage to
obtain the linear precursor (Scheme 1).
PyBOP/HOBt and 10 equiv of DIEA) at highly diluted
conditions17 was completed within 4 h at room temper-
ature and afforded the reference compound stylostatin 1.
The product was purified to homogeneity on reverse
phase HPLC (Fig. 1B) and showed the expected mass
(theoretical mass 742.8; mass observed MHþ 743.7,
(M+Na)þ 765.8) with a yield of 59.9%.
The crude linear peptide showed more than 70% purity
on reverse-phase HPLC (Fig. 1A) and the desired mass
(theoretical mass 760.9; found MHþ 761.6, (M+Na)þ
783.4). This further demonstrated that the loading of
Fmoc-Ser-OAllyl was successful and the peptide chain
assembly on this preloaded resin was efficient. Cycliza-
tion of the crude linear peptide in solution (4 equiv
On-resin cyclization was accomplished with 4 equiv of
PyBOP/HOBt and 10 equiv of DIEA in DMF overnight
at room temperature. The cyclic product was then
deprotected and cleaved from the resin support using a
mixture of TFA/Tis/H2O/thioanisole (92.5/2.5/2.5/2.5,
v/v) for 2 h at room temperature. The final product
stylostatin 1 was purified to homogeneity (Fig. 1C) and
showed the expected mass (theoretical mass 742.8;
observed MHþ 743.7, (M+Na)þ 765.4). Based on the
initial loading, overall yield of the on-resin stylostatin
synthesis was calculated at 13.9%.
A
B
0
15
Minutes
30
0
15
Minutes
30
A mixture (1:1) of stylostatin 1 obtained by on-resin
cyclization and by the solution method was homogenous
and showed a single peak on reverse phase HPLC (Fig.
1D), which suggests that the two methods afford the
same product.
C
D
In summary, we have demonstrated that the b-hydroxyl
function of serine and threonine could be conveniently
used as a tether point for solid-phase linkage. With
appropriate temporary, orthogonal protection for the
C-terminal a-carboxyl group this resin construct can
then function in an on-resin protocol for cyclic peptide
synthesis. This technique effectively expands the scope of
on-resin Fmoc/tBu-based cyclic peptide synthesis to
include serine and threonine containing peptides as
synthetic targets.
0
15
Minutes
30
0
15
30
Minutes
Figure 1. HPLC traces of specific compounds: (A) Crude linear sty-
lostatin 1; (B) purified stylostatin 1 cyclized in solution; (C) purified
stylostatin 1 cyclized on-resin; (D) mixture (1:1) of stylostatin 1 from
cyclizations on-resin and in solution. HPLC conditions: Solvent A:
water with 0.1% TFA; solvent B: acetonitrile with 0.1% TFA; C18
ꢀ
Vydac reverse-phase, 4.6 · 250 mm, 5 lM, 300 A pore size; gradient:
linear from 0% B to 65% B over 20 min, then 90% B for 5 min, and 0%
B for another 5 min. Wavelength used for detection: 214 nm.

L. Z. Yan et al. / Tetrahedron Letters 45 (2004) 923–925
925
10. Yan, L. Z.; Mayer, J. M. J. Org. Chem. 2003, 68, 1161–
1162.
Acknowledgements
11. Prepared according to a literature method: Vorherr, T.;
Bannwarth, W. Bioorg. Med. Chem. Lett. 1995, 5, 2661–
2664.
We thank Drs. Lianshan Zhang and Wayne Kohn for
helpful discussions.
12. Hanessian, S.; Xie, F. Tetrahedron Lett. 1998, 39, 733–736.
13. Meienhofer, J.; Waki, M.; Heimer, E. P.; Lambros, T. J.;
Makofske, R. C.; Chang, C. D. Int. J. Pept. Res. 1979, 13,
35–42.
14. Pettit, G. R.; Srirangam, J. K.; Herald, D. L.; Erickson, K.
L.; Doubek, D. L.; Schmidt, J. M.; Tackett, L. P.; Bakus,
G. J. J. Org. Chem. 1992, 57, 7217–7220.
15. (a) Bourne, G. T.; Meutermans, W. D. F.; Alewood, P. F.;
McGeary, R. P.; Scanlon, M.; Watson, A. A.; Smythe, M.
L. J. Org. Chem. 1999, 64, 3095–3101; (b) Hashimoto, C.;
Atuzawa, Y.; Kudo, M.; Kodomari, M. Peptide Science
1999, 35, 473–476; (c) Rosenbaum, C.; Waldmann, H.
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17. Linear stylostatin 1 (15.3 mg, 0.02 mmol) in 3 mL of dry
DMF was added continuously to a solution of PyBOP/
HOBt/DIEA in DMF (3 mL)/DCM (7 mL) over a period
of 2 h through a syringe pump. The reaction was allowed
to proceed for another 2 h at RT and LCMS confirmed
that there was no detectable linear starting material.
Solvent removal was followed by RP-HPLC purification
to give the final product stylostatin 1 (8.9 mg, a yield of
59.9%).
ꢁ
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