Synthesis of the cyclic heptapeptide axinellin A

Article abstract of DOI:10.1016/j.tet.2009.11.090

The first synthesis of the naturally occurring cyclic peptide axinellin A has been achieved. Cyclization and subsequent deprotection of linear precursors containing either a t-butyl protected Thr residue or a Thr(Ψ^Me,Mepro) derivative gave a cyclic peptide, identical in all respects to the naturally occurring material, with the exception that the synthetic peptide does not exhibit the cytotoxic activity reported for the natural product.

Full text of DOI:10.1016/j.tet.2009.11.090

Tetrahedron 66 (2010) 935–939
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Synthesis of the cyclic heptapeptide axinellin A
,
Kelly A. Fairweather a, Nima Sayyadi a, Christos Roussakis b, Katrina A. Jolliffe a
*
a School of Chemistry, The University of Sydney, 2006 NSW, Australia
b Faculte´ de Pharmacie, 1 Rue Gaston Veil, F-044035 Nantes Cedex 01, France
a r t i c l e i n f o
a b s t r a c t
Article history:
The first synthesis of the naturally occurring cyclic peptide axinellin A has been achieved. Cyclization and
Received 22 September 2009
Received in revised form
28 October 2009
Accepted 20 November 2009
Available online 27 November 2009
subsequent deprotection of linear precursors containing either a t-butyl protected Thr residue or
a Thr(J
Me,Mepro) derivative gave a cyclic peptide, identical in all respects to the naturally occurring
material, with the exception that the synthetic peptide does not exhibit the cytotoxic activity reported
for the natural product.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic peptide
Pseudoproline
Cyclization
Natural product
1. Introduction
Thr residues.20 After cyclization the turn-inducers were readily re-
moved to reveal free Thr residues. Having successfully utilized this
Naturally occurring cyclic peptides have generated much interest
in recent years owing to their intriguing chemical structures and
potent biological activity. An emerging class of proline-rich cyclic
peptides, isolated mainly from South Pacific marine organisms, are
characterized by the presence of seven or eight amino acids with the
presence of two or three proline residues. Examples of such natural
products include the axinastatins,1 the phakellistatins,2–4 the
hymenamides,5,6 stylostatin 1,7 the rollamides,8 and the axi-
nellins.9,10 Compounds from this class are frequently isolated as
mixtures of geometrical isomers, with the cis-and trans-amide
conformers unable to interconvert due to steric constraints. This
makes these structures demanding targets for synthesis. To date, the
synthesis of a handful of cyclic peptides from this ‘proline-rich’ class
of natural products has been achieved,11–17 however the head-to-tail
cyclizations of the representative linear peptides are generally low
yielding operations. This is not surprising as the cyclization of small
linear peptides (three to eight amino acids) is notoriously difficult,
often resulting in epimerization of the C-terminal amino acid and/or
the formation of linear and cyclic oligomers.18,19 We recently
methodology in the synthesis of the heptapeptide mahafacyclin B21
it was of interest to investigate whether it could be used to improve
the synthesis of the more conformationally constrained proline-rich
peptides. In addition, we wanted to investigate whether the in-
corporation of a cis-amide inducing Thr(J
Me,Mepro) residue would
affect the conformation of the cyclic peptide obtained after the
removal of this protecting group. We herein report on the applica-
tion of this methodology to the total synthesis of the naturally
occurring proline-rich cyclic peptide, axinellin A. Axinellin A (1) is
a cyclic heptapeptide [cyclo(Asn-Pro-Phe-Thr-Ile-Phe-Pro)], which
was isolated together with axinellin B from the marine sponge
Axinella carteri (Axinellidae).9 The structure and conformation of
axinellin A were elucidated by Randazzo et al., however, to date the
structure has not been confirmed by synthesis. The natural product
was reported to have moderate in vitro antitumor activity against
a human bronchopulmonary non-small cell carcinoma cell line
(IC50¼3.0
mg/mL against NSCLC-N6 cells).
2. Results and discussion
reported on the use of pseudoprolines (J
Me,Mepro) as removable
turn-inducers to facilitate head-to-tail peptide cyclization.20,21 In-
corporation of these Thr-derived azaacetals into linear peptides was
found to substantially increase the head-to-tail cyclization yields
compared to those for the same peptides having O-TBS-protected
AxinellinAcontains asingle threonineresidue, which makesitan
ideal target on which to test our pseudoproline-assisted cyclization
methodology. In order to place the Thr(J
Me,Mepro) residue near the
center of the linear precursor, and to avoid epimerization of the
C-terminal amino acid we chose a point of cyclization between Asn
and Pro to give the linear precursor 2 with Asn as the N-terminal
amino acid and Pro as the C-terminal amino acid (Scheme 1).
* Corresponding author. Tel.: þ61 2 9351 2297; fax: þ61 2 9351 3329.
E-mail address: jolliffe@chem.usyd.edu.au (K.A. Jolliffe).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.11.090

936
K.A. Fairweather et al. / Tetrahedron 66 (2010) 935–939
Scheme 1. Cyclization point and linear precursor for axinellin A (1).
Scheme 2. Cyclization of pseudoproline containing linear precursor 2.
The synthesis of the required linear peptide 2 was achieved by
standard solid-phase peptide synthesis (SPPS), using Fmoc/HBTU
chemistry on a PS3 automated peptide synthesizer, with the ex-
ception of the N-terminal Fmoc-Asn residue, which was coupled as
the pentafluorophenyl ester to avoid side chain dehydration.22 The
2-chlorotritylchloride resin was chosen as the solid support to
enable cleavage of peptides from the solid phase with the side chain
protecting groups intact. As anticipated, the cyclization of 2 was not
straightforward. The reaction was attempted using a variety of
coupling reagents under high dilution (0.001 M) in DMF. Using
pentafluorophenyl diphenylphosphinate (FDPP), and 4-(4,6-dime-
thoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate
(DMTMM) as coupling reagents the corresponding cyclodimer 4
was obtained as the major product (50 and 39%, respectively) along
with the desired cyclic peptide 3 as the minor product (25 and 18%
yields, respectively). The best cyclization results were obtained
using (O-benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium hexa-
fluorophosphate (HBTU) as the coupling reagent, and adding this
together with 2 slowly over 24 h via syringe pumps. In this case
a 61% yield of the desired 3 was obtained, along with a low yield of
cyclodimer 4 (8%). Upon treatment of 3 with a solution of TFA:
thioanisole: triisopropylsilane: H2O (8.5:0.5:0.5:0.5) for four days,
cyclic peptide undergoes conformational changes to give the single
conformer observed.
To determine if the Thr(
adopt a conformation amenable to cyclization, we prepared and
cyclized the linear peptide 5, in which the Thr(
Me,Mepro) residue
J
Me,Mepro) residue was assisting 2 to
J
was replaced with a t-butyl protected Thr residue. Linear hepta-
peptide 5 was synthesized in an analogous manner to 2, and the
head-to-tail cyclization was investigated using the same range of
coupling reagents. We found that, in all cases, the desired cyclic
peptide 6 was obtained as the major product in good yield and as
a single conformer. Furthermore, the best cyclization results were
obtained using FDPP as the coupling reagent. In this case 77% of
cyclic peptide 6 was obtained, along with trace amounts of cyclo-
dimer 7 (2%). The t-butyl protecting group in 6 was removed in an
analogous manner to the N,O-isopropylidene group in 3 to afford
axinellin A (1) in 80% yield (Scheme 3). The material obtained via
this synthetic route was identical in all respects to that obtained
above.
The cytotoxicity of synthetic axinellin A (1) and synthetic in-
termediates 3, 4, 6 and 7 was evaluated against the human bron-
chopulmonary non-small cell lung cancer lines NSCLC-N6 and
A549 employing the same procedures and cell line (NSCLC-N6) as
used previously for the naturally derived axinellin A (1). In all cases,
deprotection of the Thr(J
Me,Mepro) residue ensued to afford the
cyclic peptide 1 in 75% yield (Scheme 2). This material was found to
have physical and spectral properties identical to those reported for
axinellin A, although the absolute optical rotation was found to be
the synthetic peptides were found to be inactive (all IC50s>90 mM).
significantly higher {[
sign as that reported for the isolated material {[
a
]
ꢀ178.5 (c 0.2 MeOH)}, but of the same
Although difficult to explain, these results were not entirely
unexpected. In fact, there have been many reports of large
disagreements between the bioactivities of synthetic and natural
proline-rich cyclic peptide samples.11–14,16,17 Given that our
synthetic sample of 1 was identical in every way to that of the
natural material, including in the conformations about the amide
bonds, perhaps the lack of activity can be explained by the naturally
derived cyclic peptide retaining a chemically undetectable amount
of strongly active cytotoxic compound, as has previously been
suggested for the phakellistatins.16,17
D
a
]
D ꢀ98.2 (c 0.003
MeOH)}.9 Notably, even though the pseudoproline containing
precursor 3 is a mixture of conformers, as evidenced by doubling
and/or broadening of signals in the 1H and 13C NMR spectra, the
deprotected material has spectral data identical to the naturally
occurring material, for which a single conformer is observed with
cis-geometry about the Phe–Pro amide bond with all other amide
bonds reported to adopt trans-geometries.9 This indicates that,
under the conditions required for pseudoproline deprotection, the

K.A. Fairweather et al. / Tetrahedron 66 (2010) 935–939
937
4. Experimental
4.1. General procedures
Most reagents were commercially available reagent grade
chemicals and were used without further purification. Dime-
thylformamide was dried over 4 Å molecular sieves prior to use.
Dichloromethane was dried over CaH2 and distilled prior to use.
Melting points were determined using a Gallenkamp melting point
apparatus and are reported in degrees Celsius (uncorrected). 1H
Nuclear magnetic resonance spectra were recorded at a frequency
of 300 MHz or at a frequency of 400 MHz and are recorded as parts
per million (ppm) downfield shift with deuteromethanol (dH 3.34),
deuteroacetonitrile (dH 1.95) or deuteropyridine (dH 8.71, 7.55, 7.19)
as internal references, unless otherwise stated. 13C Nuclear mag-
netic resonance spectra were recorded at a frequency of 75.47 MHz
or at a frequency of 100.62 MHz and are reported as parts per
million (ppm) downfield shift with deuteromethanol (dC 49.00),
deuteroacetonitrile (dH 118.2, 1.3) or deuteropyridine (dH 149.9,
135.5, 123.5) as an internal reference, unless otherwise stated.
Optical rotations were measured on a dual wavelength polarimeter
in a 2.5 cm cell at 22 ꢁC using the indicated spectroscopic grade
solvents. Analytical reverse phase high performance liquid
chromatography (RP-HPLC) was performed using a SunfireÔ C18
column (5
m
m, 2.1ꢂ150 mm ID). Flow rate was maintained at
0.2 mL/min over a linear gradient from 0 to 100% solvent B (solvent
A: 100:0.01 v/v Milli-Q water/TFA, solvent B: 100:0.01 v/v aceto-
nitrile/TFA) over 30 min and eluent monitored from 210 to 320 nm.
Preparative RP-HPLC was performed using a SunfireÔ PrepC18
OBDÔ column (5
m
m, 19ꢂ150 mm ID). Flow rate was maintained at
Scheme 3. Cyclization of linear peptide 5.
7.0 mL/min over a linear gradient from 0% to 100% solvent B
(solvent A: 100:0.01 v/v Milli-Q water/TFA, solvent B: 100:0.01 v/v
acetonitrile/TFA) over 45 min and eluent monitored at wavelengths
214 and 280 nm.
3. Conclusions
4.2. General procedure for the synthesis of linear
In summary, we have successfully completed the first total
synthesis of the naturally occurring cyclic peptide, axinellin A from
two linear precursors, and hence confirmed the elucidated struc-
ture. Contrary to our previous work in this area,21 it appears that
the introduction of a single pseudoproline turn-inducer into the
linear precursor 2 does not enhance the head-to-tail cyclization
yield, compared to that for linear precursor 5, in which the Thr
residue is protected by a t-butyl group. In the case of linear peptide
heptapeptides using solid-phase peptide synthesis (SPPS)
4.2.1. Loading of the resin. Under anhydrous conditions, 2-chloro-
trityl chloride resin (1.0 g, loading 1.4 mmol/g) was swollen in CH2Cl2
(10 mL)for10 min, thena solutionofFmoc-Pro-OH(1.42 g, 4.2 mmol)
and N,N-diisopropylethylamine (DIPEA) (2.2 mL, 12.6 mmol) in
CH2Cl2 (10 mL) was added. Dry N2 was bubbled through the mixture
for 1 h, then the mixture was gently agitated overnight. The solvent
was drained and the resin was washed sequentially with
CH2Cl2:MeOH:DIPEA (17:2:1, 3ꢂ10 mL), CH2Cl2 (2ꢂ10 mL), DMF
(2ꢂ10 mL), CH2Cl2 (2ꢂ10 mL), and ether (2ꢂ10 mL). The resin was
dried under vacuum overnight, to give the Fmoc-proline loaded resin
(0.4–0.7 mmol/g). The proline loaded 2-chlorotrityl chloride resin
(1.0 g, 0.4–0.7 mmol/g) was then placed in a PS3 automated peptide
synthesizer, and the resin was washed with DMF (3ꢂ30 s).
2 it appears that, while the incorporation of the Thr(J
Me,Mepro)
residue results in the adoption of a predominantly cisoid confor-
mation at the preceding amide bond, this does not provide an
overall linear peptide conformation that is more favorable for cy-
clization than that adopted by the linear precursor 5, and that the
introduction of this conformational constraint instead favors the
formation of the cyclodimer 4. Nevertheless, good yields were
obtained for the cyclizations of both the linear heptapeptide con-
taining the pseudoproline residue, 2, and that containing a t-butyl
protected Thr residue, 5, and the material obtained after removal of
the Thr protecting groups from both the pseudoproline containing
3 and t-butyl protected 6 was identical. This indicates that, in the
case of axinellin A, the incorporation of a cis-amide inducing
pseudoproline residue in the linear precursor does not affect the
geometry of the amide bonds in the deprotected cyclic product.
The total synthesis of 1 has confirmed the structure of the
natural product as originally reported9 and provided further
material for biological testing. The lack of cytotoxic activity in our
synthetic material, as previously observed for similar compounds
from this class,11–14,16,17 provides further evidence that the naturally
derived proline-rich cyclic peptides may be masking small quan-
tities of a highly cytotoxic compound.
4.2.2. Fmoc deprotection. The Fmoc group was removed with 20%
piperidine in DMF (2ꢂ5 min), and then the resin was washed with
DMF (6ꢂ30 s).
4.2.3. Peptide coupling. Fmoc-amino acid (2 equiv) and HBTU
(2 equiv) were activated by treatment with a DIPEA/DMF mixture
for 30 s, before addition to the reaction vessel. After 40 min the
resin was washed with DMF (3ꢂ30 s).
4.2.4. Final coupling to form heptapeptide. The hexapeptide loaded
2-chlorotrityl chloride resin was removed from the peptide syn-
thesizer and suspended in DMF. Fmoc-Asn-pentafluorophenyl ester
(2 equiv) was added and dry N2 was bubbled through the reaction
vessel for 1 h. The solution was then drained and the resin washed

938
K.A. Fairweather et al. / Tetrahedron 66 (2010) 935–939
sequentially with DMF (2ꢂ10 mL), CH2Cl2 (2ꢂ10 mL), and ether
(2ꢂ10 mL). A solution of 20% piperidine in DMF (10 mL) was then
added to the resin and dry N2 was bubbled through the mixture for
5 min, then the resin was washed with DMF (3ꢂ10 mL).
2H), 3.89–3.99 (m, 2H), 4.01–4.11 (m, 2H), 4.21–4.26 (m, 2H), 4.27–
4.44 (m, 4H), 4.49–4.55 (m, 2H), 4.57–4.70 (m, 2H), 4.75–4.92 (m,
2H), 4.95–5.10 (m, 2H), 7.08–7.34 (m, 20H), NH and NH2 signals not
observed; MS (ESI) m/z 1713 ([MH]þ, 100), 1735 ([MNa]þ, 10%);
HRMS
C90H120N16O18Na.
m/z¼1735.8838
[MNa]þ,
1735.8859
calcd
for
4.2.5. Cleavage from the resin. A mixture of hexafluoroiso-
propanol:trifluoroethanol:CH2Cl2 (1:2:7, 20 mL) was added to the
resin bound heptapeptide and N2 was bubbled through the vessel
for 30 min. The resin was washed with CH2Cl2 (3ꢂ10 mL) and the
filtrate was collected. Concentration in vacuo gave the crude hep-
tapeptide, which was purified by RP-HPLC.
4.5. NH2-Asn-Pro-Phe-Thr(tBu)-Ile-Phe-Pro-OH (5)
Preparation was in accordance to that described in the general
procedure for the SPPS of heptapeptides. Heptapeptide 5 (700 mg)
was obtained as a colorless solid: mp 135–136 ꢁC, [
a
]
D ꢀ57.2 (c 0.1
4.3. NH2-Asn-Pro-Phe-Thr(
j
Me,Mepro)-Ile-Phe-Pro-OH (2)
in MeOH); 1H NMR (400 MHz, CD3OD)
d 0.85–0.94 (m, 9H), 1.03 (d, J
6.2 Hz, 3H), 1.21 (s, 9H), 1.38–1.58 (m, 2H), 1.61–1.91 (m, 4H), 1.92–
2.02 (m, 2H), 2.06–2.26 (m, 2H), 2.70–2.80 (m, 1H), 2.84–2.94 (m,
1H), 2.96–3.09 (m, 2H), 3.10–3.25 (m, 2H), 3.44–3.50 (m, 1H), 3.59–
3.67 (m, 1H), 3.70–3.80 (m, 1H), 4.00–4.10 (m, 1H), 4.22–4.29 (m,
1H), 4.35–4.43 (m, 2H), 4.44–4.51 (m, 2H), 4.63–4.70 (m, 1H), 7.16–
7.37 (m,10H), 7.74 (d, J 9.0 Hz,1H), 7.83 (m, 1H), 8.18 (d, J 7.9 Hz,1H),
one NH and two NH2 (Asn) not observed; 13C NMR (100 MHz,
Preparation was in accordance to that described in the general
procedure for the SPPS of heptapeptides. Heptapeptide 2 (265 mg)
was obtained as a colorless solid: mp 145–146 ꢁC, [
a]
D ꢀ88.5 (c 0.1
in MeOH); 1H NMR (400 MHz, CD3OD)
d 0.89 (d, J 6.2 Hz, 3H), 0.95
(d, J 6.8 Hz, 3H), 1.03–1.12 (m, 1H), 1.13–1.23 (m, 2H), 1.28 (s, 3H),
1.39–1.49 (m, 3H), 1.53 (s, 3H), 1.50–1.73 (m, 3H), 1.74–1.87 (m, 1H),
1.88–2.09 (m, 5H), 2.10–2.32 (m, 2H), 2.61–2.72 (m, 1H), 2.79–3.21
(m, 5H), 3.35–3.55 (m, 1H), 3.57–3.71 (m, 2H), 3.72–3.88 (m, 2H),
4.10–4.24 (m, 1H), 4.34–4.61 (m, 3H), 7.13–7.36 (m, 12H), 8.05 (d, J
7.0 Hz, 1H), 8.13 (m, 1H), 8.20 (d, J 7.0 Hz, 1H), 8.26 (m, 1H), NH2
CD3OD)
d
11.7, 16.0, 19.0, 25.4, 25.8, 25.9, 28.70 (tBu), 28.74 (2ꢂCH2),
30.1, 30.5, 38.2, 38.5, 38.8, 53.9, 56.1, 59.0, 59.1, 60.6, 61.9, 68.3, 68.4,
76.2, 76.3, 127.8, 127.9, 129.45 (2ꢂCH), 129.54 (2ꢂCH), 130.45
(2ꢂCH), 130.52 (2ꢂCH), 138.0, 138.6, 168.7, 171.1, 171.7, 172.8, 173.0,
173.1, 173.8, 175.2; MS (ESI) m/z 891 ([MH]þ, 10), 913 ([MNa]þ,
100%); HRMS m/z¼891.4990 [MH]þ, 891.4980 calcd for
(Asn) not observed; 13C NMR (100 MHz, CD3OD)
d 11.7, 15.9, 19.6,
23.1, 24.3, 25.8, 25.9, 26.5, 30.1, 30.8, 35.4, 37.7, 38.0, 38.5, 40.0, 40.1,
41.2, 50.1, 53.8, 59.9, 60.5, 61.8, 67.7, 77.1, 98.1, 127.8, 128.2, 129.4,
129.6, 130.5, 130.8, 138.2 (2ꢂArC), 168.4, 170.0, 170.7, 171.1, 171.9,
172.4, 172.9, 175.2; MS (ESI) m/z 875 ([MH]þ, 100), 897 ([MNa]þ,
20%); HRMS m/z¼875.4539 [MH]þ, 875.4400 calcd for
C46H67N8O10
.
4.6. cyclo[-Asn-Pro-Phe-Thr(tBu)-Ile-Phe-Pro] (6)
C45H62N8O10
.
Linear peptide 5 (0.10 g, 0.11 mmol) and FDPP (0.13 g,
0.33 mmol) were each dissolved in DMF (10 mL) and added drop-
wise via syringe pumps (0.5 mL/h) to a vigorously stirred solution
of DIPEA (0.10 mL, 0.56 mmol) and DMF (110 mL, 1 mM). The re-
action mixture was stirred for three days, after which time the
solvent was removed under reduced pressure and the crude resi-
due was purified by RP-HPLC to afford two fractions, A (tR 29.5 min)
and B (tR 40.4 min). Concentration of fraction A gave cyclic hepta-
4.4. cyclo[-Asn-Pro-Phe-Thr(
j
Me,Mepro)-Ile-Phe-Pro] (3)
Linear peptide NH2-Asn-Pro-Phe-Thr(
j
Me,Mepro)-Ile-Phe-Pro-
OH (2) (0.10 g, 0.11 mmol) and HBTU (0.13 g, 0.33 mmol) were each
dissolved in DMF (10 mL) and added dropwise via syringe pumps
(0.5 mL/h) to a vigorously stirred solution of DIPEA (0.10 mL,
0.56 mmol) and DMF (114 mL, 1 mM). The reaction mixture was
stirred for two days, after which time the solvent was removed
under reduced pressure and the crude residue was purified by RP-
HPLC to afford two fractions, A (tR 26.4 min) and B (tR 31.6 min).
Concentration of fraction A gave cyclic heptapeptide 3 (0.060 g,
peptide 6 (0.076 g, 77%) as a colorless solid: mp 168–170 ꢁC, [
a]
D
ꢀ126.9 (c 0.1 in MeOH); 1H NMR (400 MHz, CD3OD)
d 0.85 (m, 1H),
0.98 (t, J 7.5 Hz, 3H), 1.18 (d, J 6.4 Hz, 3H), 1.23 (d, J 6.4 Hz, 3H), 1.32
(s, 9H), 1.37–1.49 (m, 2H), 1.59–1.69 (m, 2H), 1.69–1.89 (m, 4H),
1.90–2.01 (m, 2H), 2.58 (dd, J 14.6, 5.9 Hz, 1H), 2.81–2.96 (m, 2H),
3.05 (t, J 12.7 Hz, 1H), 3.17 (dd, J 12.7, 4.7 Hz, 1H), 3.26–3.40 (m, 4H),
3.58–3.67 (m, 1H), 3.92 (q, J 8.4 Hz, 1H), 4.04 (dd, J 7.5, 4.6 Hz, 1H),
4.17 (dd, J 8.3, 5.8 Hz, 1H), 4.27–4.39 (m, 2H), 4.40–4.50 (m, 2H),
4.74 (q, J 7.0 Hz,1H), 7.16–7.36 (m,10H), 7.74 (d, J 6.3 Hz,1H), 7.85 (d,
J 4.5 Hz, 1H), 8.53 (d, J 8.0 Hz, 1H), 8.59 (s, 1H), 8.88 (d, J 8.0 Hz, 1H),
61%) as a colorless solid: mp 179–182 ꢁC, [
a
]
ꢀ94.7 (c 0.1 in
D
MeOH); 1H NMR (400 MHz, CD3CN)
d
0.78 (d, J 7.9 Hz, 3H), 0.82 (t, J
7.6 Hz, 3H), 1.08 (d, J 6.9 Hz, 3H), 1.59 (s, 3H), 1.62 (s, 3H), 1.66–1.86
(m, 2H), 2.03–2.14 (m, 1H), 2.50 (dd, J 14.2, 11.2 Hz, 1H), 2.66 (dd, J
14.2, 3.4 Hz, 1H), 2.86–3.00 (m, 4H), 3.01–3.14 (m, 4H), 3.29–3.38
(m, 2H), 3.39–3.56 (m, 4H), 3.61–3.72 (m, 1H), 3.82 (t, J 9.0 Hz, 1H),
4.08–4.21 (m, 2H), 4.24–4.32 (m, 1H), 4.38 (dd, J 8.3, 4.4 Hz, 1H),
4.50 (d, J 7.6 Hz, 1H), 4.54–4.62 (m, 1H), 4.63–4.70 (m, 1H), 4.74–
4.84 (m, 1H), 7.15–7.41 (m, 10H), NH and NH2 signals not observed;
NH2 (Asn) not observed; 13C NMR (100 MHz, CD3OD)
d 11.2, 15.9,
18.3, 22.7, 25.5, 26.5, 28.7, 30.5, 31.4, 37.4, 38.1, 38.9, 39.1, 47.5, 50.4,
55.8, 57.1, 58.9, 59.9, 62.1, 62.5, 67.1, 77.0, 86.8, 127.7, 128.7, 129.4,
130.1, 130.4, 130.8, 137.0, 139.6, 170.1, 172.6, 172.7, 173.0, 173.2, 173.6,
174.3, 174.8; MS (ESI) m/z 873 ([MH]þ, 50%), 895 ([MNa]þ, 100%);
HRMS m/z¼873.4875 [MH]þ, 873.4869 calcd for C46H65N8O9.
Concentration of fraction B gave the cyclic dimer 7 (0.002 g, 2%
13C NMR (75 MHz, CDCl3)
d 4.7, 10.3, 15.2, 18.6, 24.9, 25.9, 29.47,
29.52, 33.1, 36.41, 36.44, 37.0, 38.3, 47.7, 49.4, 53.1, 53.9, 56.1, 57.1,
61.2, 68.6, 77.2, 77.8, 104.3, 126.8, 127.0, 128.3, 128.5, 129.0, 129.6,
136.2, 137.3, 169.4, 170.3, 170.6, 171.2, 173.0, 173.2, 173.4 (1 signal
obscured or overlapping); MS (ESI) m/z 857 ([MH]þ, 100), 879
([MNa]þ, 10%); HRMS m/z¼879.4219 [MNa]þ, 879.4300 calcd for
C45H60N8O9Na.
yield) as a colorless solid: mp 236–237 ꢁC, [
a]
ꢀ100.5 (c 0.2 in
D
MeOH); 1H NMR (400 MHz, CD3OD)
d
0.81 (d, J 6.9 Hz, 6H), 0.82 (t, J
6.9 Hz, 6H), 0.89–0.97 (m, 2H), 0.97–1.08 (m, 4H), 1.12 (d, J 6.3 Hz,
6H), 1.15–1.27 (m, 6H), 1.32 (s, 18H), 1.35–1.41 (m, 2H), 1.63–1.77 (m,
4H), 1.78–2.04 (m, 10H), 2.08–2.21 (m, 2H), 2.69–2.79 (m, 2H),
2.82–2.99 (m, 2H), 3.01–3.15 (m, 2H), 3.17–3.28 (m, 2H), 3.38–3.51
(m, 2H), 3.62–3.75 (m, 4H), 3.81–3.91 (m, 2H), 4.10–4.21 (m, 2H),
4.24–4.31 (m, 2H), 4.31–4.41 (m, 4H), 4.42–4.50 (m, 2H), 4.62–4.75
(m, 2H), 4.97–5.09 (m, 2H), 7.14–7.56 (m, 20H), 8.36 (d, J 9.2 Hz, 2H),
six NH and two NH2 (Asn) not observed; 13C NMR (75 MHz, CD3OD)
Concentration of fraction B gave the cyclic dimer 4 (0.008 g, 8%
yield) as a colorless solid: mp 150–158 ꢁC, [
a
]
ꢀ105.5 (c 0.1 in
D
MeOH); 1H NMR (400 MHz, CD3CN)
d 0.61 (d, J 6.8 Hz, 2H),
0.81–0.99 (m, 18H), 1.14–1.22 (m, 6H), 1.37–1.45 (m, 6H), 1.48–1.63
(m, 8H), 1.64–1.98 (m, 16H), 1.20–1.25 (m, 2H), 2.41 (dd, J 14.5,
4.1 Hz, 2H), 2.53 (dd, J 12.9, 10.6 Hz, 2H), 2.80–3.19 (m, 4H), 3.20–
3.29 (m, 2H), 3.29–3.54 (m, 4H), 3.54–3.78 (m, 6H), 3.79–3.85 (m,
d
12.0, 16.2, 18.3, 24.8, 25.7, 26.0, 28.6, 28.7, 28.8, 37.7, 38.7, 53.6,

K.A. Fairweather et al. / Tetrahedron 66 (2010) 935–939
939
56.3, 59.0, 59.8, 61.7, 62.1, 67.4, 77.0, 127.8, 127.9, 129.5, 129.6, 130.4,
Acknowledgements
130.5, 138.2, 139.2, 171.3, 172.3, 172.9, 173.5, 173.9, 174.3, 174.4,
175.4; MS (ESI) m/z 1745 ([MH]þ, 100), 1767 ([MNa]þ, 60%); HRMS
m/z¼1767.9485 [MNa]þ, 1767.9485 calcd for C92H128N16O18Na.
We thank the Australian Research Council for financial support
and for the award of a Queen Elizabeth II research fellowship to K.A.J.
4.7. Axinellin A (1)
Supplementary data
A solution of TFA:thioanisole:triisopropylsilane:H2O (8.5:0.5:
0.5:0.5, 10 mL) was added to the cyclic heptapetide (3, 75 mg or 6,
85 mg) at rt and the resulting reaction mixture was stirred for 4 d.
Concentration in vacuo gave the crude residue, which was purified
by RP-HPLC to give cyclo[Asn-Pro-Phe-Thr-Ile-Phe-Pro], Axinellin A
(1) (75% from 3 and 80% from 6) as a colorless solid: mp 115–116 ꢁC,
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.tet.2009.11.090.
References and notes
[
a]
ꢀ178.5 (c 0.2 in MeOH) [lit.9
[
a]
ꢀ98.2 (c 0.003 MeOH)]; 1H
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D
NMR (400 MHz, pyridine-d5):
d 0.81 (t, J 7.63 Hz, 3H), 1.14–1.27
(m, 2H), 1.29 (d, J 6.8 Hz, 3H), 1.36–1.54 (m, 5H), 1.46 (d, J 6.42 Hz,
3H), 1.55–1.66 (m, 1H), 1.78–1.90 (m, 1H), 2.05–2.19 (m, 1H), 2.23–
2.30 (m,1H), 3.04 (dd, J 13.6, 3.2 Hz,1H), 3.20 (dd, J 12.0, 4.4 Hz, 2H),
3.41 (t, J 14.4 Hz, 1H), 3.46 (d, J 8.4 Hz, 1H), 3.50–3.62 (m, 2H), 3.66
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1H), 4.28 (dd, J 15.2, 6.4 Hz, 1H), 4.49 (dd, J 9.6, 4.0 Hz, 1H), 4.56 (br
q, J 6.42 Hz, 1H), 4.62 (dd, J 10.0, 4.0 Hz, 1H), 4.98 (t, J 8.4 Hz, 1H),
5.19 (ddd, J 14.4, 8.8, 3.2 Hz, 1H), 5.22 (d, J 9.6 Hz, 1H), 5.35 (ddd, J
13.6, 8.4, 3.2 Hz, 1H), 7.11–7.46 (m, 10H), 7.94 (d, J 9.6 Hz, 1H), 8.53
(br s,1H), 8.54 (br s,1H), 9.81 (br s, 1H), 8.60 (d, J 8.8 Hz,1H), 9.16 (d,
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15.4, 21.6, 22.5, 24.5, 25.7, 29.8, 31.4, 38.5, 39.0, 39.2, 48.0, 47.1, 49.1,
55.2, 56.7, 56.9, 58.4, 61.1, 62.0, 67.7, 127.7, 128.9, 128.6, 129.8, 129.8,
126.7, 136.3, 139.4, 169.8, 170.4, 171.7, 172.1, 172.2, 173.2; MS (ESI) m/
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4.8. Cytotoxicity assays
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Experiments were performed in 96-well microtiter plates
(105 cells/mL for NSCLC-N6 and 2ꢂ104 cells/mL for A549). Cell
growth was estimated by a colorimetric assay based on the con-
version of tetrazolium dye (MTT) to a blue formazan product by live
mitochondria.23 Eight repeats were performed for each concen-
tration. Control growth was estimated from eight determinations.
Optical density at 570 nm corresponding to solubilized formazan
was read for each well on Titertek Multiskan MKII.