A backbone linker for BOC-based peptide synthesis and on-resin cyclization: Synthesis of stylostatin 1

Article abstract of DOI:10.1021/jo9818780

We have developed a new 4-alkoxybenzyl-derived linker that anchors the C-terminal amino acid to the resin through the α-nitrogen atom. The linker allows BOC solid-phase peptide assembly and peptide cleavage using standard HF protocols. This linking strategy provides a versatile on-resin route to cyclic peptides and avoids the diketopiperazine formation that is prominent when using FMOC chemistry on backbone linkers. The linker was prepared by forming the aryl ether from 4-hydroxybenzaldehyde and bromovaleric acid. Subsequent reductive amination of the aldehyde with an allyl-protected amino acid ester and acylation of the resulting secondary amine provided the tertiary amide. After linking the amide to the resin, standard BOC SPPS, followed by allyl deprotection, cyclization, and HF cleavage gave cyclic peptides in high purity. To exemplify the strategy, the cytotoxic heptapeptide, stylostatin 1, was synthesized from two linear precursors. For comparison purposes, the yields of the on-resin and solution-phase cyclization were determined and found to be dependent upon the linear precursor. This linker technology provides new solid-phase avenues in accessing libraries of cyclic peptides.

Full text of DOI:10.1021/jo9818780

J . Org. Chem. 1999, 64, 3095-3101
3095
A Ba ck bon e Lin k er for BOC-Ba sed P ep tid e Syn th esis a n d
On -Resin Cycliza tion : Syn th esis of Stylosta tin 1†,§
Gregory T. Bourne, Wim D. F. Meutermans, Paul F. Alewood, Ross P. McGeary,
Martin Scanlon, Andrew A. Watson, and Mark L. Smythe*
Centre for Drug Design and Development, The University of Queensland,
St. Lucia 4072, Brisbane, Australia
Received September 15, 1998
We have developed a new 4-alkoxybenzyl-derived linker that anchors the C-terminal amino acid
to the resin through the R-nitrogen atom. The linker allows BOC solid-phase peptide assembly
and peptide cleavage using standard HF protocols. This linking strategy provides a versatile on-
resin route to cyclic peptides and avoids the diketopiperazine formation that is prominent when
using FMOC chemistry on backbone linkers. The linker was prepared by forming the aryl ether
from 4-hydroxybenzaldehyde and bromovaleric acid. Subsequent reductive amination of the aldehyde
with an allyl-protected amino acid ester and acylation of the resulting secondary amine provided
the tertiary amide. After linking the amide to the resin, standard BOC SPPS, followed by allyl
deprotection, cyclization, and HF cleavage gave cyclic peptides in high purity. To exemplify the
strategy, the cytotoxic heptapeptide, stylostatin 1, was synthesized from two linear precursors.
For comparison purposes, the yields of the on-resin and solution-phase cyclization were determined
and found to be dependent upon the linear precursor. This linker technology provides new solid-
phase avenues in accessing libraries of cyclic peptides.
In tr od u ction
and, provided bioactivity is maintained, serve as models
for the design of bioavailable drugs. Some cyclic peptides
are drugs in their own right; examples include oct-
reotide10 and cyclosporin A.11 To obtain generic access to
arrays of small cyclic peptides, we sought to establish
solid-phase strategies that allowed on-resin cyclization
prior to cleavage/deprotection.
Several approaches to on-resin cyclization have been
established using two types of linking strategies (Scheme
1): (a) through electrophilic linkers that are labile to free
amines (Method A),12-15 where cyclization effects cleavage
from the solid support and (b) through attachment of an
amino acid side chain to the solid support (Method B)15-17
followed by on-resin cyclization and cleavage/deprotec-
tion. With library development in mind, we were seeking
to develop a versatile linker that does not require the
presence of side chain functionality at the C-terminus
and allows on-resin head to tail cyclization. J ensen et
al.18,19 have recently reported a TFA labile backbone
The drug lead discovery process has recently under-
gone dramatic changes through the advent of combina-
torial chemistry and high throughput screening.1-7
Arrays of analogues are accessed simultaneously via
solid-supported synthetic strategies.3-7 Cyclic peptides
are appealing targets for combinatorial library develop-
ment.8,9 They are excellent tools to examine the confor-
mational requirements of peptide or protein recognition
* To whom correspondence should be addressed: Tel: (+61) 7 3365
1271. Fax: (+61) 7 3365 1990. E-mail: m.smythe@mailbox.uq.edu.au.
† Presented in part as a lecture at BBC-2 (Brisbane Biological
Chemistry Symposium) J une 1996 and as a poster at the second
Australian Peptide Symposium, Fraser Island, Oct 1996.
§ Abbreviations used as follows; BOC, tert-butoxycarbonyl; BOP,
benzotriazol-1-yloxy-tris(dimethylamino) phosphonium hexafluoro-
phosphate; Bz, benzyl; DAST, diethylaminosulfur trifluoride; DCC 1,3-
dicyclohexylcarbodiimide; DIEA, diisopropylethylamine; DMF, dime-
thylformamide; DMSO, dimethylsulfoxide; ES-MS, electron-spray mass
spectrometry; EtOAc, ethyl acetate; FMOC, fluorenyl-methyloxycar-
bonyl; HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium
hexafluorophosphate; HF, anhydrous hydrogen fluoride; HOAc, acetic
acid; HPLC, high-perfomance liquid chromatography; MeOH, metha-
nol; NMM, N-methylmorpholine; NMR, nuclear magnetic resonance;
SPPS, solid-phase peptide synthesis; TBAF, tetrabutylammonium
fluoride; TFA, trifluoroacetic acid; THF, tetrahydrofuran.
(1) J ung, G.; Beck-Sickinger, A. G. Angew. Chem., Int. Ed. Engl.
1992, 31, 367-386.
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Marbach, P.; Petchr, T. J .; Pless, J . Life Sci. 1982, 31, 1133-1140.
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Lacy, E.; Crabtree, G. R. Science 1989, 246, 1617-1620.
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Gordon, E. M. J . Med. Chem. 1994, 37, 1233-1251.
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F.; Barany, G. J . Am. Chem. Soc. 1998, 120, 5441-5452.
10.1021/jo9818780 CCC: $18.00 © 1999 American Chemical Society
Published on Web 04/02/1999

3096 J . Org. Chem., Vol. 64, No. 9, 1999
Bourne et al.
Sch em e 1. Meth od s for Solid -P h a se Syn th esis of
Cyclic P ep tid es
Backbone linking allows one to select, at least in
theory, any linear peptide sequence and any position of
cyclization (except proline at the C-terminus). It thus
provides a means to target diverse libraries of cyclic
peptides. Further, it is well known that a different
position of cyclization can drastically affect the yield of
target product. The ability to include several linear
precursors to the same target in a designed library
improves the chances of successful cyclization and thus
the quality of the target library. In addition, we and
others have found that backbone-substituted peptides
cyclize more readily then their nonsubstituted ana-
logues.22 The position of our linker, i.e., on the first
backbone nitrogen of the linear peptide, improves the
flexibility of the linear peptide by presumably decreasing
the cis-trans rotational barrier and therefore increases
the propensity for cyclization (as opposed to oligomer-
ization).
We employed the novel linker strategy in the synthesis
of the cyclic heptapeptide stylostatin 1 (Scheme 2) that
has been isolated from Stylotella aurantium.23 Stylostatin
1 serves as an interesting model to evaluate our linker
in the context of library development of small cyclic
peptides. It has recently been the focus of a small
combinatorial library, which involved the attachment of
Asn of stylostatin and related analogues to resin.24
Sch em e 2. Lin k er Design a n d Stylosta tin 1a
Resu lts a n d Discu ssion
The backbone linker 1 is attached to the amine of the
first residue of the peptide via an alkoxybenzyl group.
The linker should have improved acid stability, accom-
modating standard BOC chemistry protocols, in particu-
lar, TFA stablity but very strong acid or HF lability.
We chose to evaluate this linker at two positions in
the stylostatin 1 (2) backbone. This involved the attach-
ment of the linker to the backbone nitrogens of Phe and
Ile, with (a) Phe and (b) Ile at the C-terminus (Scheme
2). The positions of the linker at these two sites were
selected for the following reasons. Site a: Pro-Phe is a
common motif in a large number of natural cyclic
peptides such as hymenistatin,25 malaysiatin,26 axinas-
tatin,26,27 and stylopeptide.28 Additionally, this dipeptide
unit has been widely used in peptide chemistry to
introduce conformational turns, e.g., analogues of soma-
tostatin.29 Consequently, successful attachment of the
linker and cyclization at this point would presumably
a
Positions a and b indicate the selected sites for linkage to the
resin.
linker (Method C) suitable for FMOC SPPS, where the
C-terminal residue is linked via its R-nitrogen atom. The
strategy is particularly useful for accessing C-terminal
modified peptides such as amides, alcohols, or aldehydes.
In the case of C-terminal carboxylic acids or esters,
however, the strategy suffers from substantial diketopip-
erazine formation.20 N-1 amide-substituted dipeptides,
when left in the unprotonated form (e.g., during FMOC
deprotection), are highly prone to this side reaction.
Clearly, alternative protection strategies need to be
developed. Here we report on a novel backbone linker
based on the original backbone linker of J ensen et al. but
designed for BOC solid-phase peptide synthesis (Scheme
2). The R-amine remains protonated after deprotection
and thus unreactive prior to the in situ neutralization
coupling step,21 thereby minimizing potential diketopip-
erazine formation. Such linkers should enable facile solid-
phase access to cyclic peptides using readily available
BOC-protected amino acids and inexpensive starting
materials.
(22) Ehrlich, A.; Klose, J .; Heyne, H.-U.; Beyermann, M.; Carpino,
L. A.; Bienert, M. In Peptides: Chemistry, Structure and Biology
(Proceedings of the 14th American Peptide Symposium); Kaumaya, P.
T. P., Hodges, R. S., Eds.; Mayflower Scientific Ltd: U.K., 1996; Vol.
14, 75-76.
(23) Pettit, G. R.; Srirangem, J . K.; Herald, D. L.; Erickson, K. L.;
Doubek, D. L.; Schmidt, J . M.; Tackett, L. P.; Bakus, G. L. J . Org.
Chem. 1992, 57, 7217-7220.
(24) Spatola, A. F.; Romanovskis, P. Cyclic Peptide Libraries: Recent
Developments. In Peptides and Non-Peptide Libraries- A Handbook
for the Search of Lead Structures; J ung, G., Ed.; VCH Publishers:
Weinheim, 1996; Chapter 11, 327-347.
(25) Pettit, G. R.; Clewlow, P. J .; Dufresne, C.; Doubek, D. L.; Cerny,
R. L.; Ru¨tzler, K. Can. J . Chem. 1990, 68, 708-711.
(26) Konat, R. K.; Matha B.; Winkler J .; Kessler H. Liebigs Ann.
Chem. 1995, 765-774.
(27) Pettit, G. R.; Herald, D. L.; Boyd, M. R.; Leet, J . E.; Dufresne,
C.; Doubek, D. L.; Schmidt, J . M.; Cerny, R. L.; Hooper, J . N. A.;
Ru¨tzler, K. J . Med. Chem. 1991, 34, 3339-3340.
(28) Pettit, G. R.; Srirangem, J . K.; Herald, D. L.; J un-ping, X.; Boyd,
M. R.; Cichacz, Z.; Kamano, Y.; Schmidt, J . M.; Erickson, K. L. J . Org.
Chem. 1995, 60, 8257-8261.
(20) Fresno, M.; Alsini, J .; Barany, G.; Albercio, F. Tetrahedron Lett.
1998, 39, 2639-2642.
(21) Schno¨lzer, M.; Alewood, P.; J ones, A.; Alewood, D.; Kent, S. B.
H. Int. J . Pept. Protein Res. 1992, 40, 180-193.
(29) Huang, Z.; Probstyl, A.; Spencer, J . R.; Yamazaki, T.; Goodman,
M. Int. J . Pept. Protein Res. 1993, 42, 352-365.

Synthesis of Stylostatin 1
J . Org. Chem., Vol. 64, No. 9, 1999 3097
Sch em e 3. Syn th esis of th e Dip ep tid e-Lin k er
Sch em e 4. HF -In d u ced Clea va ge of th e Dip ep tid e
Ac-P r o-P h e-OH fr om th e Lin k er Un ita
Un its 7a a n d 7ba
a
Reagents: (i) THF, rt, 5 min. (ii) Ac2O, DIEA, DCM, 2 h; (iii)
0.1 M LiOH, THF/H2O 1:1, rt, 1 h; (iv) HF/p-cresol 10:1, 0 °C, 1 h.
removed with TBAF followed by purification of the
dipeptide-linker units 7a and 7b.
To examine the efficiency of cleavage of the linker-
peptide backbone bond, we initially treated linker unit
7a , after BOC deprotection, with acetic anhydride to give
8 (Scheme 4). Allyl deprotection was accomplished using
LiOH, generating acid 9. Ester 8 and the acid 9 were both
subjected to standard HF deprotection/cleavage condi-
tions (HF/p-cresol 9/1, 0 °C, 1 h). After workup, dipeptide
10 was isolated in excellent yield. No starting material
(8 or 9) could be detected in the crude cleavage product,
indicating that the amide linker was cleaved quantita-
tively under the selected conditions. It should also be
noted that the allyl protecting group of 8 was removed
quantitatively under the HF conditions. In addition, the
linker-dipeptide unit 8 remained unchanged after 2 h
in TFA at 25 °C, demonstrating that this backbone linker
is ideally suited for in situ neutralization BOC SPPS.
We attached the dipeptide-linker units 7a and 7b to
aminomethylated resin (Scheme 5). We have noted
several times that peptide assembly on resin is more
effective when the linker is separated from the resin
backbone by a small peptide sequence. We therefore
introduced a tripeptide spacer group (Gly-Leu-Leu) on
the aminomethylated resin prior to attaching the dipep-
tide-linker units. The linear peptides 12a and 12b were
then assembled on the respective resins 11a and 11b in
a stepwise fashion using in situ neutralization protocols
and HBTU activation.21 Following allyl- and BOC-depro-
tection of resin 12a , HF treatment of the resulting resin
13a produced linear peptide 14a in 70% yield (after
HPLC purification). The analytical HPLC of the crude
cleavage product 14a is shown in Figure 1i. Purified
linear peptide 14b was obtained in the same fashion in
39% yield.
a
Reagents: (i) BrCH2CH2CH2CH2CO2CH2CH2Si(CH3)3, K2CO3,
acetone, ∆, 16 h; (ii) H-Phe-OAllyl or H-Ile-O-Allyl, MgSO4,
CH2Cl2, rt, 3 h; (iii) NaBH3CN, MeOH, rt, 2 h; (iv) (BOC-Pro)2-O,
DIEA, EtOAc, rt, 16 h; or BOC-Ala-F, DIEA, THF, rt, 30 min; (v)
TBAF, THF, rt, 2 h.
allow access to a wide range of biologically active pep-
tides. Appendage site b was chosen to examine sterically
hindered cyclization sites (Ile-Pro in this case), and it
allows an investigation of potential difficulties in acylat-
ing N-substituted â-branched amino acids.
We commenced with the synthesis of the two dipep-
tide-linker units 7a and 7b via solution-phase chemistry
as depicted in Scheme 3. This involved reacting the
p-hydroxybenzaldehyde 3 with 5-bromo trimethylsilyl-
ethylvalerate in the presence of K2CO3 and acetone. The
resulting aldehyde 4 was then condensed with an allyl
ester-protected amino acid, (H-Phe-OAllyl or H-Ile-
OAllyl), and the corresponding imines were reduced in
situ with NaBH3CN.30,31 Acylation of the secondary amine
5a with BOC-Pro-OH was accomplished using the sym-
metrical anhydride procedure.32 Acylation of the more
hindered amine 5b with (BOC-Ala)2-O gave low yields
of the desired product. However, acylation with BOC-
Ala-F33 proceeded well, leading to 44% yield of isolated
product 6b . The silyl protection group was then
The resin-bound peptides 13a and 13b were cyclized
with BOP/2,6-lutidine at -10 °C. Following HF cleavage,
the crude products were analyzed by HPLC and ES-MS.
From comparison of the HPLC profiles it is clear that
the two linear precursors, though generated and cyclized
on the same resin using the same linker and activation
conditions, give significantly different product mixtures.
Whereas 13b cyclizes almost exclusively to stylostatin 1
(Figure 1v, peak b), the precursor 13a generates sty-
lostatin 1 and the corresponding cyclic dimer in a ratio
of 1:1 (Figure 1ii, peaks b and c). The synthetic stylostatin
1 coeluted with native stylostatin 1 in two solvent
systems (water/acetonitrile and water/methanol). In
(30) Look, G.; Holmes, C. P.; Chin, J . P.; Gallop, M. J . Org. Chem.
1994, 59, 7588-7590.
(31) Sasaki, Y.; Coy, D. H. Peptides 1987, 8, 119-126.
(32) Merrifield, R. B.; Vizioli, L. D.; Boman, H. G. Biochemistry 1982,
21, 5020-5031.
(33) Kaduk, C.; Wehschuh, H.; Beyermann, M.; Forner, K.; Carpino,
L. A.; Bienert, M. Lett. Pept. Sci. 1995, 2, 285-289.

3098 J . Org. Chem., Vol. 64, No. 9, 1999
Sch em e 5. Syn th esis of Stylosta tin 1 on Solid Su p p or ta
Bourne et al.
DMSO, the 1H NMR data obtained from the synthetic
material were identical to those reported for the native
product.34
so-called pseudodilution effect.35,36 However, it may be
that the pseudodilution effect in on-resin chemistry is
significant but is counterbalanced by the higher “local”
concentrations of linear peptide (i.e., the resin is not
homogeneously activated across the bead).
To evaluate the effect of resin-bound cyclization on
yields and purity of the product, we carried out control
solution-phase cyclization of the corresponding linear
precursors 14a and 14b (see Table 1). For peptide 14b,
cyclization at 10-3 M (in DMF) proceeded as expected to
yield almost exclusively stylostatin 1. Solution-phase
cyclization of the linear peptide 14a was performed at
10-2 and 10-3 M (see Table 1). The analytical HPLC of
the crude mixtures displayed significant differences in
the ratio of cyclic monomer (stylostatin 1) over higher
oligomers (Figure 1iii and 1iv, respectively). The mono-
mer:dimer ratio was approximately 2:5 at 10-2 M con-
centration. At 10-3 M, this was reversed to 5:2 monomer:
dimer.
The product profile of the on-resin cyclization of 13a
(Figure 1ii) compares well with the solution-phase ex-
periment on 14a at 10-2 M (Figure 1iii). The on-resin
cyclization was performed on 250 mg of resin-bound
peptide (sv 0.16 mmol/g, swollen volume ∼ 1 mL) and is
in the order of 10-2 M. It appears that the on-resin
cyclization does not confer any significant benefit via the
A remaining question that currently complicates syn-
thesis of cyclic peptides is the selection of the optimal
position for cyclization. Our method allows relatively fast
comparison of cyclization of several linear precursors in
a combinatorial fashion, with no intermediate purifica-
tion required. From a library perspective, this permits
one to include several precursors to the same target,
thereby increasing the probability of obtaining “pure”
cyclic peptide for each target in the library. This is
exemplified by the fact that one would predict the Ile-
Pro cyclization site to be the most combersome, but of
the two selected in this study, it results in the highest
yield and purity of cyclic material.
In summary, we have developed a new linker that
anchors the C-terminal amino acid to the resin through
the R-nitrogen atom and permits standard BOC chem-
istries to be performed for assembly and cleavage. The
linker can be used on all amino acids except proline and
thus provides new solid-phase avenues to access libraries
of cyclic peptides.
(34) The structure of the synthetic material was calculated on the
basis of distance constraints derived from NOE data recorded for the
peptide in DMSO. The DMSO solution structure of synthetic stylostatin
1 is very similar to the previously reported crystal structure of native
stylostatin 1.23 Backbone atoms of synthetic stylostatin 1 superimpose
on the X-ray structure of the native material with an RMSD of 0.51 (
0.01 Å.
(35) Leznoff, C. C. Chem. Soc. Rev. 1974, 3, 65-87.
(36) McMurray, J . S.; Lewis, C. A.; Obeyesekere, N. U. Pept. Res.
1994, 7, 195-206.

Synthesis of Stylostatin 1
J . Org. Chem., Vol. 64, No. 9, 1999 3099
subsequently through an orifice (100-200 µm diameter) at a
potential of 80 V. Full scan mass spectra were acquired over
a range of 200-2000 Da with a scan step size of 0.1 Da. TLC
(thin-layer chromatography) was performed on silica gel 60
F254 plates. The chromatograms were viewed under uv light
and/or developed with iodine vapor. Preparative column chro-
matography was effected under pressure, using normal-phase
Merck Kieselgel 60. Analytical reverse-phase HPLC was run
on a C18 column (0.46 cm × 25 cm); preparative reverse-phase
HPLC was run on a C18 column (2.2 cm × 25 cm). Both
columns were attached to a HPLC setup fitted with a Holo-
chrome UV detector. Measurements were carried out at λ )
214 nm.
Ma ter ia ls. BOC-L-amino acids and, synthesis grade DMF,
TFA, and DIEA were purchased from Auspep (Parkville,
Australia). HBTU and BOP were purchased from Richelieu
Biotechnologies (Montreal, Canada). AR grade EtOAc, HOAc,
MeOH, CH2Cl2, CHCl3, hexane, and acetone and HPLC grade
CH3CN were all obtained from Laboratory Supply (Australia);
HF was purchased from CIG (Australia). Aminomethylpoly-
styrene resins with a substitution value of 0.21 mmol/g or 0.26
mmol/g were purchased from Novabiochem. All other reagents
were AR grade or better and were obtained from Aldrich or
Fluka.
4-[5-Oxy-(t r im et h ylsilylet h ylva ler a t e)]b en za ld eh yd e
(4). 4-Hydroxybenzaldehyde (3) (12.2 g, 100 mmol), 5-bromo-
(trimethylsilylethyl)valerate (13.82 g, 200 mmol), and K2C03
(40.0 g, 290 mmol) were refluxed in acetone (250 mL) for 16
h. Solids were filtered and washed with acetone, and the
volatiles were removed in vacuo. The product 4 was purified
by column chromatography (hexane/EtOAc, 8:1) to yield a
colorless oil (28.2 g, 87%): 1H NMR (300 MHz, CDCl3) δ 9.87
(s, 1H), 7.82 (d, J ) 7.0 Hz, 2H), 6.98 (d, J ) 7.0 Hz, 2H), 4.20
(t, J ) 6.9 Hz, 2H), 4.05 (t, J ) 6.0 Hz, 2H), 2.42 (m, 2H), 1.80
(m, 4H), 0.96 (t, J ) 6.9 Hz, 2H), 0.10 (s, 9H); 13C NMR (75
MHz, CDCl3) δ 190.80, 173.45, 164.026, 131.99, 131.99, 129.87,
114.72, 114.72, 67.82, 62.63, 34.00, 28.49, 21.55, 17.35, -1.49;
ES-MS Mr 322.4 (calcd 322.2).
N-[4-(5-Oxy-(t r im et h ylsilylet h ylva ler a t e))b en zyl]-L-
p h en yla la n in e Allyl Ester (5a ). The aldehyde 4 (16.2 g, 50.2
mmol), phenylalanine allyl ester (20.5 g, 100 mmol), and excess
MgSO4 (∼40 g) were stirred in CH2Cl2 (75 mL) at rt for 16 h.
Solids were filtered, and volatiles were removed in vacuo to
yield the crude imine as a yellow oil. MeOH (200 mL) and
HOAc (3 mL) were added, and the reaction mixture was cooled
to 10 °C. NaBH3CN (6.1 g, 100 mmol) was added portionwise
to the stirred solution. The reaction mixture was allowed to
warm to room temperature before being stirred for an ad-
ditional 2 h. Volatiles were removed in vacuo, and the resulting
residue was diluted with H2O and extracted with EtOAc. The
combined EtOAc extracts were washed with saturated brine
and water before being dried over MgSO4. Volatiles were
removed in vacuo, and the resulting oil was purified by flash
chromatography (hexane/EtOAc, 1:1) to yield 5a as a clear
colorless oil (20.2 g, 79%): 1H NMR (300 MHz, CDCl3) δ 7.28
(m, 5H), 7.20 (d, J ) 7.0 Hz, 2H), 6.85 (d, J ) 7.0 Hz, 2H),
5.80 (m, 1H), 5.28 (dd, J ) 12.1 Hz, 1.7 Hz, 1H), 5.23 (dd, J )
10.0 Hz, 1.7 Hz, 1H), 4.55 (d, J ) 6.4 Hz, 2H), 4.15 (t, J ) 6.9
Hz, 2H), 3.92 (m, 2H), 3.80 (dd, J ) 12.2 Hz, 1.2 Hz, 2H), 3.65
(dd, J ) 11.7 Hz, 1.2 Hz, 2H), 3.58 (m, 1H), 3.05 (m, 1H), 2.25
(m, 2H), 1.80 (m, 4H), 0.95 (t, J ) 6.9 Hz, 2H), 0.10 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 173.56, 173.00, 158.32, 136.78,
131.96, 130.67, 129.57, 129.57, 129.27, 129.27, 128.39, 128.39,
126.76, 118.77, 114.36, 114.36, 67.33, 66.48, 62.51, 61.60,
51.13, 39.18, 34.08, 28.68, 21.62, 17,32, -1.51; ES-MS Mr 511.1
(calcd 511.3).
F igu r e 1. Analytical HPLC of crude mixtures: (i) HF cleav-
age of 13a ; (ii) on-resin cyclization of 13a followed by HF
cleavage; (iii) solution-phase cyclization of 14a (1 × 10-2 M);
(iv) solution-phase cyclization of 14a (1 × 10-3 M); (v) on-resin
cyclization of 13b followed by HF cleavage. a ) H-Asn-Ser-
Leu-Ala-Ile-Pro-Phe-OH (14a ); b ) stylostatin 1 (2); c ) cyclic
dimer of 14a ; d ) cyclic trimer of 14a .
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined on a
Gallenkamp melting point apparatus and are uncorrected. 1H
NMR spectra were recorded on a 300 MHz spectrometer, and
the chemical shifts are reported in δ parts per million
downfield from tetramethylsilane. Coupling constants (J ) refer
to proton-proton coupling (J H,H). 13C NMR spectra were also
recorded on the spectrometer at 75.5 MHz. TOCSY and
ROESY spectra were obtained on a 500 MHz spectrometer.
Data analysis and structure calculations were undertaken
using standard protocols.37 Mass spectra were acquired on a
triple-quadrupole mass spectrometer equipped with an Ion-
spray atmospheric pressure ionization source. Samples (10 µL)
were injected into a moving solvent (30 µL/min; 50:50 CH3-
CN/0.05% TFA) coupled directly to the ionization source via a
fused silica capillary interface (50 µm i.d. × 50 cm length).
Sample droplets were ionized at a positive potential of 5 kV
and entered the analyzer through an interface plate and
BOC-L-P r o-[N-(4-(5-oxy-(tr im eth ylsilyleth ylva ler a te))-
ben zyl)]-L-p h en yla la n in e Allyl Ester (6a ). BOC-Pro-OH
(8.61 g, 40.0 mmol) was dissolved in EtOAc (30 mL), to which
was added DCC (4.12 g, 20.0 mmol). After activation for 10-
15 min to form the symmetric anhydride, the mixture was
filtered. The filtrate was added to a solution of the amine 5a
(5.11 g, 10.0 mmol) and DIEA (2.67 mL, 15 mmol) in EtOAc
(20 mL). The reaction was stirred at rt for 16 h. EtOAc (100
mL) was added, and the reaction mixture was washed with a
(37) Scanlon, M. J .; Naranjo, D.; Thomas, L.; Alewood, P. F.; Lewis,
R. J .; Craik, D. J . Structure 1997, 5, 1585-1597.

3100 J . Org. Chem., Vol. 64, No. 9, 1999
Bourne et al.
Ta ble 1. Yield s of Isola ted Cyclic Mon om er , Dim er a n d Tr im er fr om On -Resin a n d Solu tion -P h a se Cycliza tion of Tw o
Stylosta tin 1 P r ecu r sor s
yield (%)
cyclization
peptide
conditions
monomer
dimer
trimer
H-Asn-Ser-Leu-Ala-Ile-Pro-Phe-OH
soln (1 × 10-2 M)
soln (1 × 10-3 M)
resina
11
48
10
43
21
25
7
1
5
H-Pro-Phe-Asn-Ser-Leu-Ala-Ile-OH
soln (1 × 10-3 M)
67
25
<1
<1
<1
<1
resina
a
Yields based on weight of isolated material and the original resin substitution value.
10% K2CO3 solution, brine, and H2O before being dried over
MgSO4. Volatiles were removed in vacuo, and the resulting
oil was purified by flash chromatography (hexane: Et2O, 5:1)
to yield 6a as a clear colorless oil (3.55 g, 60%): 1H NMR (300
MHz, CDCl3) δ 7.20 (m, 7H), 6.85 (d, J ) 7.0 Hz, 2H), 5.98 (m,
1H), 5.20 (m, 2H), 4.50 (m, 3H), 4.20 and 4.13 (rotomers, m,
1H), 4.15 (t, J ) 6.9 Hz, 2H), 3.92 (m, 2H), 3.71 (m, 2H), 3.31
(m, 4H), 2.25 (m, 2H), 2.05 (m, 4H), 1.80 (m, 4H), 1.48 (br s,
9H), 0.95 (t, J ) 6.9 Hz, 2H), 0.10 (s, 9H); 13C NMR (75 MHz,
CDCl3) δ rotomers 173.54 and 173.00, 172.42, rotomers 170.08
and 169.47, rotomers 158.68 and 158.50, rotomers 154.31 and
153.98, rotomers 138.35 and 138.05, rotomers 132.45 and
131.96, 129.40, 129.40, 129.10, 128.91, 128.63, 128.63, 127.52,
rotomers 126.75 and 126.62, rotomers 118.26 and 118.06,
114.32, 114.32, rotomers 79.96 and 79.19, 67.34, rotomers
65.96 and 65.80, 62.55, rotomers 60.68 and 60.58, rotomers
57.44 and 56.94, 51.37, rotomers 46.83 and 46.77, rotomers
35.11 and 34.97, 34.07, rotomers 30.84 and 29.78, 28.67, 28.46,
rotomers 24.02 and 22.77, 21.68 17.32, -1.50; ES-MS Mr 708.6
(calcd 708.4).
BOC-L-P r o-[N-(4-(5-oxyva ler ic a cid )ben zyl)]-L-p h en yl-
a la n in e Allyl Ester (7a ). The ester 6a (2.0 g, 2.82 mmol) was
stirred in THF (20 mL) at rt TBAF (3 mL, 1 M in THF) was
added dropwise, and stirring proceeded for an addition 3 h.
H2O (100 mL) and HOAc (3 mL) were added to the reaction
mixture. The acid 7a was extracted into EtOAc and washed
with a 10% solution of citric acid followed by H2O before being
dried over MgSO4. Volatiles were removed in vacuo, and the
resulting oil was purified by flash chromatography (CH2Cl2/
MeOH, 19:1) to yield a white solid (2.54 g, 90%): mp 28-30
°C; 1H NMR (300 MHz, CDCl3) δ 8.89 (br s, 1H), 7.20 (m, 7H),
6.75 (dd, J ) 7.1 Hz, J ) 1.9 Hz, 2H), 5.88 (m, 1H), 5.25 (m,
2H), 4.50 (m, 3H), 4.20 and 4.13 (rotomers, dd, J ) 6.9 Hz,
1.9 Hz, 1H), 3.88 (m, 2H), 3.71 (m, 2H), 3.41 (m, 4H), 2.25 (m,
2H), 2.05-1.85 (m, 8H), 1.48 (br s, 9H); 13C NMR (75 MHz,
CDCl3) δ rotomers 179.09 and 177.04, 173.05, rotomers 170.08
and 169.48, rotomers 158.64 and 158.44, rotomers 154.28 and
153.96, rotomers 138.31 and 138.02, rotomers 132.43 and
131.94, 129.41, 129.41, 128.99, 128.69, 128.48, 128.48, 127.50,
rotomers 126.78 and 126.65, rotomers 118.30 and 118.10,
114.37, 114.37 rotomers 80.17 and 79.38, 67.30, rotomers 65.99
and 65.84, rotomers 60.72 and 60.54, rotomers 57.49 and 57.00,
51.40, rotomers 46.86, rotomers 35.09 and 34.95, 33.56,
rotomers 30.83 and 29.78, rotomers 28.46 and 20.76, rotomers
24.00 and 22.78, 21.39; ES-MS Mr 608.3 (calcd 608.3).
(d, J ) 8.0 Hz, 2H), 6.85 (d, J ) 8.0 Hz, 2H), 5.98 (m, 1H),
5.31 (d, J ) 27.2 Hz, 1H), 5.27 (dd, J ) 13.2 Hz, 1.7 Hz, 1H),
5.10 (dd, J ) 11.2 Hz, 1.7 Hz, 1H), 4.65 (m, 2H), 4.15 (t, J )
6.9 Hz, 2H), 3.92 (m, 2H), 3.81 (d, J ) 13 Hz, 1H), 3.60 (d, J
) 13 Hz, 1H), 3.17 (m, 1H), 2.90 (m, 1H), 2.35 (m, 2H), 1.80
(m, 2H), 1.52 (m, 1H), 1.20 (m, 1H), 0.95 (t, J ) 6.9 Hz, 2H),
0.92 (d, J ) 7.6 Hz, 3H), 0.90 (t, J ) 7.0 Hz, 3H), 0.10 (s, 9H);
13C NMR (75 MHz, CDCl3) δ 174.55, 174.25, 158.96 132.66,
131.22, 130.48, 130.48, 119.45, 115.02, 115.02, 68.05 65.92,
65.52, 63.20, 52.36, 38.74, 34.78, 29.39, 29.39, 26.35, 22.34,
18.02, 16.23, 12.13, -0.81; ES-MS Mr 477.3 (calcd 477.3).
BOC-L-Ala -[N-(4-(5-oxy-(tr im eth ylsilyleth ylva ler a te)-
ben zyl)]-L-isoleu cin e Allyl Ester (6b). BOC-Ala-OH (2.89
g, 15.0 mmol) was dissolved in CH2Cl2 (30 mL), to which was
added DAST (4.12 g, 20.0 mmol). After activation for 10-15
min to form the acid fluoride, the mixture was washed with
cold H2O and dried over MgSO4, and the volatiles were
removed in vacuo. The acid fluoride was then added im-
mediately to a solution of the amine 5b (4.78 g, 10.0 mmol)
and DIEA (2.67 mL, 15 mmol) in THF (20 mL). The reaction
was stirred at rt for 16 h. EtOAc (100 mL) was added, and
the reaction mixture was washed with 10% K2CO3 solution,
brine, and H2O before being dried over MgSO4. Volatiles were
removed in vacuo, and the resulting oil was purified by flash
chromatography (hexane/diethyl ether, 1:5) to yield 6b as a
clear colorless oil (2.86 g, 44%): 1H NMR (300 MHz, CDCl3) δ
7.24 (d, J ) 8.0 Hz, 2H), 6.85 (d, J ) 8.0 Hz, 2H), 5.98 (m,
1H), 5.31 (d, J ) 14.2 Hz, 1H), 5.23 (d, J ) 12.0 Hz, 1H) 4.65
(m, 3H), 4.15 (t, J ) 6.9 Hz, 2H), 3.92 (m, 2H), 3.81 (d, J ) 13
Hz, 1H), 3.60 (d, J ) 13 Hz, 1H), 3.17 (m, 1H), 2.90 (m, 1H),
2.35 (m, 2H), 1.80 (m, 2H), 1.52 (m, 1H), 1.45 (s, 9H), 1.20 (m,
1H), 0.95 (t, J ) 6.9 Hz, 2H), 0.97 (s, 3H), 0.92 (d, J ) 7.6 Hz,
3H), 0.90 (t, 3H, J ) 7.0 Hz), 0.10 (s, 9H); ES-MS Mr 648.5
(calcd 648.4).
BOC-L-Ala -[N-(4-(5-oxyva ler ic a cid )ben zyl)]-L-isoleu -
cin e Allyl Ester (7b). The ester 6b (2.0 g, 2.82 mmol) was
stirred in THF (20 mL) at rt TBAF (3 mL, 1 M in THF) was
added dropwise, and saponification proceeded for 3 h. H2O (100
mL) and HOAc (3 mL) was added to the reaction mixture. The
acid was extracted into EtOAc. The combined EtOAc extrac-
tions were washed with saturated brine and water before being
dried over MgSO4. Volatiles were removed in vacuo, and the
resulting oil purified by semipreparative HPLC (0-60% B over
60 min) to yield the tertiary amide 7b as a colorless oil (2.54
g, 44%): 1H NMR (300 MHz, CDCl3) δ 7.22 (d, 2H, J ) 8.0
Hz), 6.80 (d, J ) 8.0 Hz, 2H), 5.91 (m, 1H), 5.21 (d, J ) 14.2
Hz, 1H), 5.22 (d, J ) 11.0 Hz, 1H), 4.65 (m, 3H), 3.92 (m, 2H),
3.81 (d, J ) 13 Hz, 1H), 3.60 (d, J ) 13 Hz, 1H), 3.17 (m, 1H),
2.90 (m, 1H), 2.35 (m, 2H), 1.80 (m, 2H), 1.52 (m, 1H), 1.45 (s,
9H), 1.20 (m, 1H), 0.97 (s, 3H), 0.92 (d, 3H, J ) 7.6 Hz), 0.90
(t, 3H, J ) 7.0 Hz); ES-MS [M + H]+ ) 549.1 (expected 549.3).
Ac-(L-P r o)-[N-(4-(5-oxyva ler ic a cid )ben zyl)]-L-p h en yl-
a la n in e Allyl Ester (8). The BOC-protected tertiary amide
7a (61 mg, 0.1 mmol) was treated with TFA (2 mL) for 10 min
at rt. TFA was removed under reduced pressure, and the
resulting oil was dissolved in DMF (20 mL). Triethylamine (4
mL) was added followed by acetic anhydride (4 mL). The
reaction was stirred at rt for 2 h. Volatiles were removed in
vacuo, and the resulting oil was dissolved in CH3CN/H2O (1:
1, 10 mL). The solution was stirred for an additional 2 h. The
mixture was then passed through a semipreparative HPLC
N-[4-(5-Oxy-(t r im et h ylsilylet h ylva ler a t e))b en zyl]-L-
isoleu cin e Allyl Ester (5b). The aldehyde 4 (16.2 g, 50.2
mmol), isoleucine allyl ester (20.5, 100 mmol), and excess
MgSO4 (∼40 g) were stirred in CH2Cl2 (75 mL) at rt for 3 h.
Solids were filtered, and volatiles were removed in vacuo to
yield the crude imine as a yellow oil. MeOH (200 mL) and
HOAc (3 mL) were added, and the reaction mixture was cooled
to 10 °C. NaBH3CN (6.1 g, 100 mmol) was added portionwise
to the stirred solution. The reaction mixture was allowed to
warm to room temperature before being stirred for an ad-
ditional 2 h. Volatiles were removed in vacuo, and the resulting
residue was diluted with H2O and extracted with EtOAc. The
combined EtOAc extractions were washed with saturated brine
and water before being dried over MgSO4. Volatiles were
removed in vacuo, and the resulting oil was purified by flash
chromatography (1:1 hexane/EtOAc) to yield 5b as a clear
colorless oil (19.6 g, 82%): 1H NMR (300 MHz, CDCl3) δ 7.24

Synthesis of Stylostatin 1
J . Org. Chem., Vol. 64, No. 9, 1999 3101
column (40-100% B over 60 min) to yield 8 as a white powder
(47 mg, 85%): ES-MS Mr 550.7 (calcd 550.3).
to the solution. After the reaction was left to stir for an
additional 2 h at this temperature, all volatiles were removed
in vacuo. The peptide cyclo-(Pro-Phe-Asn-Ser-Leu-Ala-Ile) 2
was isolated by preparative HPLC (30-90% B over 60 min)
to yield a white powder (7.0 mg, 48%): ES-MS Mr 742.2 (calcd
Ac-(L-P r o)-[N-(4-(5-oxyva ler ic a cid )ben zyl)]-L-p h en yl-
a la n in e (9). The allyl ester 8 (25 mg, 45.5 µmol) was dissolved
in THF/H2O (1:1, 10 mL), and LiOH‚H2O (3.64 mg, 91 µmol)
was added portionwise to the solution at rt The solution was
stirred for 2 h before HCl (0.01 M) was added until the solution
was at pH 5.0. The mixture was then passed through a
semipreparative HPLC column (30-90% B over 60 min) to
yield the diacid 9 (20 mg, 86%): ES-MS Mr 510.8 (calcd 510.3).
Ac-P r o-P h e-OH (10). Meth od 1. The peptide 8 (20 mg,
36.2 µmol) was treated with HF/p-cresol, (5 mL, 10:1) for 1 h
at -5 °C. After removal of the HF under reduced pressure,
the crude peptide was dissolved in the HPLC buffer and
lyophilized. The acetylated dipeptide Ac-Pro-Phe-OH 10 was
purified by semipreparative HPLC (30-90% B over 60 min)
to yield a white powder (8.6 mg 78%): ES-MS Mr 304.2 (calcd
304.1).
1
742.4). The H and 13C NMR were identical to reported data.22
Also isolated was the dimer, cyclo-(Asn-Ser-Leu-Ala-Ile-Pro-
Phe-Asn-Ser-Leu-Ala-Ile-Pro-Phe) (3 mg, 21%): ES-MS Mr
1485.2 (calcd 1485.8), and the trimer, cyclo-(Asn-Ser-Leu-Ala-
Ile-Pro-Phe-Asn-Ser-Leu-Ala-Ile-Pro-Phe-Asn-Ser-Leu-Ala-Ile-
Pro-Phe) (0.7 mg, 5%): ES-MS Mr 2229.2 (calcd 2229.2).
Meth od 2. Cyclo-(P r o-P h e-Asn -Ser -Leu -Ala -Ile) (2). A
procedure similar to that above was followed, using precursor
14b (100 mg, 0.131 mmol), BOP (174 mg, 0.393 mmol), and
DIEA (228 µL, 1.31 mmol). The peptide cyclo-(Pro-Phe-Asn-
Ser-Leu-Ala-Ile) 2 was isolated by preparative HPLC (10-70%
B over 60 min) to yield a white powder (10.5 mg, 67%): ES-
MS Mr 742.2 (calcd 742.4).
Meth od 2. The peptide 9 (20 mg, 39.2 µmol) was treated
with HF/p-cresol, (5 mL, 10:1) for 1 h at -5 °C. After removal
of the HF under reduced pressure, the crude peptide was
dissolved in the HPLC buffer and lyophilized. The acetylated
dipeptide Ac-Pro-Phe-OH 10 was purified by semipreparative
HPLC (30-90% B over 60 min) to yield a white powder (9.7
mg, 82%): ES-MS Mr 304.2 (calcd 304.14).
On -Resin Cycliza tion . Meth od 1. Cyclo-(P r o-P h e-Asn -
Ser -Leu -Ala -Ile) (2). After chain assembly of the linear
peptide 13a (250 mg, 0.167 mmol/g), the allyl protecting group
and the NR-BOC group were removed with [Pd(PPh3)4] (580
mg, 0.5 µmol) and TFA (2 × 1 min treatment), respectively,
as described before. The resin in DMF (20 mL) was then cooled
to -10 °C, and BOP (221 mg, 0.5 mmol) was added. Next, 2,6-
Lutidine (194 µL, 1.66 mmol) was added dropwise, and the
reaction was monitored by ninhydrin test. After completion
of the reaction (amine <1% by ninhydrin), the resin was
filtered, and the cyclic peptide was cleaved from the resin using
HF/p-cresol (11 mL, 10:1) for 1 h at 0 °C. After removal of the
HF under reduced pressure, the crude peptide was precipitated
in anhydrous ether before being dissolved in the HPLC buffer
and lyophilized. The peptide cyclo-(Pro-Phe-Asn-Ser-Leu-Ala-
Ile) 2 was isolated by preparative HPLC (30-90% B over 60
min) to yield a white powder (3.1 mg, 10%): ES-MS Mr
742.2 (calcd 742.4). Also isolated was the dimer, cyclo-(Asn-
Ser-Leu-Ala-Ile-Pro-Phe-Asn-Ser-Leu-Ala-Ile-Pro-Phe) (7.6 mg,
24.5%): ES-MS Mr 1485.2 (calcd 1485.8), and the trimer, cyclo-
(Asn-Ser-Leu-Ala-Ile-Pro-Phe-Asn-Ser-Leu-Ala-Ile-Pro-Phe-
Asn-Ser-Leu-Ala-Ile-Pro-Phe) (0.4 mg, 1%): ES-MS Mr 2229.4
(calcd 2229.2).
Solid -P h a se P ep tid e Syn th esis. H-Asn -Ser -Leu -Ala -Ile-
P r o-P h e-OH (14a ). The peptide was synthesized in a stepwise
fashion by established methods using in situ neutralization/
HBTU activation protocols for BOC chemistry. The xanthyl
protecting group was used for the Asn residue and the benzyl
ether for the Ser residue. Coupling reactions were monitored
by quantitative ninhydrin assay and were typically >99.9%
complete. After chain assembly was complete, the removal of
the allyl protecting group was achieved by the addition of
tetrakis(triphenylphosphine) palladium [Pd(PPh3)4] (580 mg,
0.5 µmol, 3 molar equiv) to the resin in a solution of CHCl3/
HOAc/NMM (37:2:1), and the solution was stirred for 14 h at
rt The resin was washed with a 10% solution of diethyldithio-
carbamic acid (sodium salt trihydrate, (C2H5)N2CS2Na‚3H2O)
in DMF, DMF, MeOH/CH2Cl2 1: 1, and finally CH2Cl2. The
NR-BOC group was removed with neat TFA (2 × 1 min
treatment), and the peptide was cleaved from the resin (200
mg, 0.166 mmol/g) using HF/p-cresol (11 mL, 10:1) for 1 h at
-5 °C. After removal of the HF under reduced pressure, the
crude peptide was precipitated in anhydrous ether, filtered,
dissolved in the HPLC buffer, and lyophilized. The peptide
H-Asn-Ser-Leu-Ala-Ile-Pro-Phe-OH 14a was purified by semi-
preparative HPLC (30-90% B over 60 min) to yield a white
powder (25 mg 78%): ES-MS Mr 760.21 (calcd 760.42).
H-P r o-P h e-Asn -Ser -Leu -Ala -Ile-OH (14b). A procedure
similar to that above was followed, using precursor 7b (200
mg, 0.180 mmol/g). The peptide H-Pro-Phe-Asn-Ser-Leu-Ala-
Ile 14b was purified by semipreparative HPLC (30-90% B
over 60 min) to yield a white powder (10.5 mg, 39%): ES-MS
Mr 760.2 (calcd 760.4).
Meth od 2. Cyclo-(P r o-P h e-Asn -Ser -Leu -Ala -Ile) (2). A
procedure similar to that above was followed, using precursor
13b (200 mg, 0.203 mmol/g), BOP (60 mg, 0.136 mmol), and
2,6-lutidene (237 µL, 2.03 mmol). The peptide cyclo-(Pro-Phe-
Asn-Ser-Leu-Ala-Ile) 2 was isolated by preparative HPLC (30-
90% B over 60 min) to yield a white powder (8.2 mg, 25%):
ES-MS Mr 742.2 (calcd 742.4).
Ack n ow led gm en t. We would like to thank Glaxo
Wellcome, U.K., and Glaxo Wellcome, Australia, for
their financial support. We are also grateful to Dr. Mike
Hann, Mr. Peter Seale, Dr. J ohn Murray, and Dr. Paul
Doyle for helpful discussions. We acknowledge Prof. G.
R. Pettit for a generous gift of native stylostatin 1.
Special thanks to Ms. Trudy Bond for performing the
amino acid analyses.
Solu tion P h a se Cycliza tion . Meth od 1. Cyclo-(P r o-
P h e-Asn -Ser -Leu -Ala -Ile) (2). The linear peptide H-Asn-Ser-
Leu-Ala-Ile-Pro-Phe-OH 14a (15.0 mg, 0.020 mmol) and BOP
(26.1 mg, 0.060 mmol) were stirred in DMF (19.7 mL, 1 × 10-3
M) at -10 °C. DIEA (35 µL, 0.197 mmol) was added dropwise
J O9818780