A total synthesis of a highly N-methylated cyclodepsipeptide [2S,3S-Hmp]-aureobasidin L using solid-phase methods

Article abstract of DOI:10.1016/j.tet.2014.02.036

[2S,3S-Hmp]-Aureobasidin L 2 has been successfully synthesised through a combination of solution- and solid-phase peptide synthesis. All of the Fmoc-protected residues including a depsidipeptide, Fmoc-MeVal-Hmp-OH, were prepared in solution phase. Chain e

Full text of DOI:10.1016/j.tet.2014.02.036

Tetrahedron 70 (2014) 2351e2358
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Tetrahedron
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A total synthesis of a highly N-methylated cyclodepsipeptide
[2S,3S-Hmp]-aureobasidin L using solid-phase methods
,
,
*
Rani Maharani a b, Robert T.C. Brownlee a, Andrew B. Hughes a, Belinda M. Abbott a
a Department of Chemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria 3086, Australia
b Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jatinangor, West Java 45363, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
[2S,3S-Hmp]-Aureobasidin L 2 has been successfully synthesised through a combination of solution- and
solid-phase peptide synthesis. All of the Fmoc-protected residues including a depsidipeptide, Fmoc-
MeVal-Hmp-OH, were prepared in solution phase. Chain elongation on chlorotrityl resin was un-
dertaken using selected coupling reagents including HBTU/HOBt, HATU/HOAt and BTC/collidine. Cleav-
age of the linear depsinonapeptide was followed by cyclisation to give the desired cyclodepsipeptide.
Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
Received 13 December 2013
Received in revised form 10 February 2014
Accepted 17 February 2014
Available online 20 February 2014
Keywords:
Aureobasidin
N-Methylated cyclodepsipeptide
Depsidipeptide
Fmoc-based solid-phase peptide synthesis
1. Introduction
variety of structural features and biological activities of N-methyl-
ated cyclodepsipeptides make the examination of these com-
Cyclodepsipeptides are cyclic heterodetic peptides, which in-
clude ester (depside) bonds as part of their backbone.1 These
molecules have been isolated from both marine and terrestrial
sources and have been found to have a wide spectrum of biological
activity including antifungal, immunosuppressive and anticancer
properties.1e4 Some of the cyclodepsipeptides found in nature
contain N-methylated amino acids, for example, aureobasidins,5
dolastatins,6e8 didemnins,9 petriellins10 and clavariopsins.11 The
pounds very attractive. This study investigates one class of the
N-methylated cyclodepsipeptides, the aureobasidins.
Aureobasidins, isolated from the black yeast Aureobasidium
pullulans R106, are highly N-methylated cyclodepsipeptides with
antifungal properties. A number of these compounds have been
shown to have excellent in vitro activity against several pathogens
including Candida albicans (MIC<0.05
mg/mL) and Cryptococcus
neoformans (MIC<0.9
m
g/mL).12 Over 30 aureobasidin analogues
have been successfully characterised including the major com-
pound, aureobasidin A (AbA) 1 (Fig. 1). All of the structures consist
of one
a hydroxy acid and eight L-amino acids where either three or
four of the residues are N-methylated.12e14 There are a few reports
describing the total synthesis of aureobasidins, all of which have
been carried out in solution phase with predominantly Boc
protection.15e17
Abbreviations: Boc, tert-butyloxycarbonyl; BTC, bis-(trichloromethyl)-carbon-
ate; 13C NMR, carbon-13 nuclear magnetic resonance; COSY, correlation
spectroscopy; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC, N,N0-dicyclohex-
ylcarbodiimide; Hmp, 2-hydroxy-3-methylpentanoic acid; DIPEA, N,N-diisopropy-
lethylamine; DMAP, 4-dimethylaminopyridine; DMF, N,N-dimethylformamide;
ESI-MS, electrospray ionisation mass spectrometry; HATU, 2-(1H-7-
azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium; HBTU, 2-(1H-benzotriazol-1-
yl)-1,1,3,3-tetramethyluronium; HOAt, 7-aza-1-hydroxybenzotriazole; HOBt, N-hy-
droxybenzotriazole; HMBC, heteronuclear multiple bond correlation; 1H NMR,
proton nuclear magnetic resonance; HPLC, high performance liquid chromatogra-
phy; HRMS, high resolution mass spectrometry; HSQC, heteronuclear single
quantum coherence; NMR, nuclear magnetic resonance; NOESY, nuclear overhauser
effect spectroscopy; RP-HPLC, reverse phase high performance liquid chromatog-
raphy; TFA, trifluoroacetic acid; TLC, thin layer chromatography; TNBS, 2,4,6-
trinitrobenzenesulfonic acid.
Total synthesis of cyclic peptides in solution phase has been
clearly shown to be challenging and time consuming.18e24 One
significant disadvantage of synthesis in solution is the large amount
of starting materials required. Many steps are involved, including
preparation of the residues, synthesis of fragments and the linear
peptide ultimately needs to be followed by cyclisation, which can
all result in low yields. Usually purification between steps is also
required, which also impacts on the yield. Solid-phase peptide
synthesis, pioneered by Merrifield,25 is a useful method to over-
come some of the problems inherent in solution chemistry and has
* Corresponding author. Tel.: þ61 3 9479 2520; fax: þ61 3 9479 1266; e-mail
address: b.abbott@latrobe.edu.au (B.M. Abbott).
0040-4020/$ e see front matter Crown Copyright Ó 2014 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2014.02.036

2352
R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
Fig. 1. Structures of AbA 1, [2S,3S-Hmp]-AbL] 2 and AbE 3.
been applied successfully to the synthesis of several cyclopeptides
and cyclodepsipeptides.26e28
also the ability of proline to resist racemisation. Synthesis of the
linear depsipeptide was initiated by attaching the first amino acid,
However, solid-phase peptide synthesis is not without problems
particularly in regard to N-methylated cyclodepsipeptides, such as
the aureobasidins. Acylation of N-methylated residues on resin
often occurs with a low yield as well as a racemised product.29e31
The presence of many nonpolar residues, a significant number of
which are also N-methylated, results in cyclic molecules, which are
very hydrophobic and so their purification by techniques typically
used in peptide synthesis is also challenging.
Fmoc-L-proline, onto the resin. This step was undertaken in
dichloromethane in the presence of DIPEA as the base. To avoid
truncated products, methanol was then added in order to cap any of
the remaining chloro groups on the resin. Each coupling in the
synthesis of the linear depsipeptide was monitored using Kaiser,
2,4,6-trinitrobenzenesulfonic acid (TNBS) and chloranil tests as
appropriate. A small sample from each step was also cleaved from
the resin and analysed by ESI-MS. Fmoc deprotection of the pep-
tidyl resin generally employed 20% piperidine in DMF for 30 min.
The solid-phase synthesis of the tetrapeptidyl resin 9 proceeded
smoothly, employing HATU/HOAt, HBTU/HOBt or BTC/collidine as
Aureobasidin L (AbL) is a closely related structure differing by
two amino acids from AbA 1. In this work, we describe the first
solid-phase methodology for the total synthesis of [2S,3S-Hmp]-
aureobasidin L ([2S,3S-Hmp]-AbL) 2 (Fig. 1), where the
residue of the natural product is replaced with the more accessible
(2S,3S)-2-hydroxy-3-methylpentanoic acid ( -Hmp). As a conse-
quence, all of the starting materials are readily available and in-
expensive in order to develop the synthetic methodology.
Importantly, the methodology presented here will be transferable
to other members of the aureobasidin class as well as other N-
methylated cyclodepsipeptides.
D
-Hmp
coupling reagents. However, attachment of the fifth residue, L-Hmp,
would result in a free hydroxyl group at the terminus of the pen-
tapeptidyl resin. Esterification on resin characteristically gives
a product in low yield compared to amidation.38 To avoid this po-
tential problem in the next step of the synthesis, it was decided to
attach the hydroxy acid as part of a depsidipeptide, a strategy
employed by Sleebs et al. in the synthesis of petriellin A.28 The
L
synthesis of depsidipeptide Fmoc-
with the conversion of -Ile 14 into its
according to the protocol by Kolasa and Miller,39 as shown in
Scheme 2. Introduction of a tert-butyl protective group to the
L-N-MeVal-L-Hmp-OH 16 began
L
a
-acetyloxy acid analogue
2. Results and discussion
a
-
acetyloxy acid was followed by the removal of the acetyl group
affording the protected (2S,3S)-2-hydroxy-3-methylpentanoic acid,
Preparation of the required of Fmoc-N-methylvaline 4 and
Fmoc-N-methylphenylalanine 5 (Fig. 2) was undertaken using the
appropriate Fmoc-amino acids via an oxazolidinone in-
termediate.32,33 A two-step method was chosen since it gives
a good to excellent yield of product in each step and no race-
misation was found during the reaction compared to other
N-methylation methods.34e37
L-Hmp-t-Bu, 15. The desired depsidipeptide was then formed by the
conjugation of the
L-Hmp-t-Bu 15 to the sixth residue, Fmoc-L-N-
MeVal 4.
Attachment of the Fmoc-depsidipeptide 16 onto the peptidyl
resin was explored using two different coupling conditions. A first
attempt employed the azabenzotriazole-based coupling reagent
HATU with the additive HOAt but required a double coupling pro-
tocol. Sufficient coupling in one cycle was obtained, however, using
BTC/collidine as described by Thern et al.27 BTC/collidine was found
to give the best coupling conditions for not only the attachment of
the depsidipeptide but also the N-methylated residues for which it
was developed.
Unfortunately, incomplete deprotection of the Fmoc-
hexapeptidyl resin 10 was observed but a second cycle of 20% pi-
peridine in DMF resolved the issue, which may have been caused by
the particular peptide sequence present on the resin that made any
efficient reaction, including removal of the Fmoc group by the base,
difficult.40 Later optimisation found that a basic cocktail of piperi-
dine/DBU/DMF (2:2:96) provided a suitable alternative protocol.
Fig. 2. Fmoc-N-methylvaline 4 and Fmoc-N-methylphenylalanine 5.
The addition of the seventh residue, Fmoc-
L-leucine, was also
Solid-phase peptide synthesis of the [2S,3S-Hmp]-AbL
2
problematic despite the use of double coupling protocol.
a
linear precursor was performed on acid-labile chlorotrityl resin
(Scheme 1). A site between proline and valine was chosen for
cyclisation due to the potential role of proline as a turn inducer and
BTC/collidine as the coupling agent in the first cycle followed by
HATU/HOAt was initially trialed. However, unprotected hexapep-
tide was found to be present by ESI-MS when a small amount of

R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
2353
Scheme 1. Solid-phase synthesis of [2S,3S-Hmp]-AbL] 2. Reagents and conditions: (a) (1) Fmoc-L-Pro, CH2Cl2, DIPEA; (2) MeOH; (3) Piperidine in DMF. (b) (1) Fmoc-L-N-MePhe 5,
HBTU/HOBt, HATU/HOAt, or BTC/collidine, DIPEA, CH2Cl2/DMF (1:1); (2) Piperidine in DMF. (c) (1) Fmoc-L-Phe, HATU/HOAt, DIPEA, CH2Cl2/DMF (1:1); (2) Piperidine in DMF. (d) (1)
Fmoc-L-N-MeVal 4, HBTU/HOBt or HATU/HOAt, DIPEA, CH2Cl2/DMF (1:1); (2) Piperidine in DMF. (e) (1) Fmoc-L-N-MeVal-L-Hmp-OH 16, BTC/collidine, DIPEA, THF; (2) 20% piperidine
in DMF 30 min, two cycles or piperidine/DBU/DMF (2:2:96) 20 min with an additional 10 min cycle. (f) (1) Fmoc-L-Leu, BTC/collidine, DIPEA, THF then HATU/HOAt, DIPEA, CH2Cl2/
DMF (1:1); (2) 1. 20% piperidine in DMF 30 min, two cycles or 2. Piperidine/DBU/DMF (2:2:96) 20 min. (g) (1) Fmoc-L-N-MeVal 4, BTC/collidine, DIPEA, THF; (2) Piperidine/DBU/DMF
(2:2:96), 20 min. (h) (1) Fmoc-L-Val, 1. BTC/collidine, DIPEA, THF 2. HATU/HOAt, DIPEA, CH2Cl2/DMF (1:1); (2) Piperidine/DBU/DMF (2:2:96), 20 min. (i) (1) TFA/CH2Cl2 (1:19) over
15 min; (2) HATU, DIPEA, CH2Cl2.
peptide was cleaved from the resin. When a double coupling of
either HATU/HOAt or BTC/collidine was carried out, no improve-
ment in the coupling efficiency was observed. Overall, the first
combination was found to give the best result.
Cleavage of the linear depsinonapeptide from the resin 13 was
undertaken by TFA/dichloromethane (1:19) and was followed by
lyophilisation to give 1.3 g of crude material. Purification of the
crude depsipeptide using semi-preparative RP-HPLC gave the de-
sired linear precursor, albeit in a low yield (7.8%). While some dif-
ficulties were encountered during the solid-phase synthesis, which
in part was detrimental to yield and the highly hydrophobic nature
of the material also proved cumbersome, the synthesis was suc-
cessfully completed.
Cyclisation of the purified linear depsinonapeptide was un-
dertaken using the optimised conditions of HATU in dichloro-
methane for 24 h. Purification of the crude cyclic product was also
difficult due to its small quantity and hydrophobicity, but flash
column chromatography was eventually able to purify the product
to obtain 4 mg of [2S,3S-Hmp]-AbL 2 in 10.2% yield, as determined
from the purified linear depsipeptide.
Removal of the Fmoc protecting group from the Fmoc-
heptapeptidyl resin 11 using either 20% piperidine in DMF or the
piperidine/DBU/DMF solution resulted in some diketopiperazine
formation and production of a Fmoc-depsipentapeptide. An at-
tempt to suppress diketopiperazine formation during deprotection
using 10% DBU in DMF with shorter reaction times was made but
was unsuccessful. Fortunately, as already described, the hydroxyl
group of this by-product depsipeptide was unlikely to react with
the eighth residue. Chain elongation of the heptapeptidyl resin 11
with Fmoc-MeVal 4 was straightforward using BTC/collidine. A
combination of a coupling cycle of BTC/collidine followed by a cycle
of HATU/HOAt then attached Fmoc- -valine to 12, after double
L
couplings of either BTC/collidine or HATU/HOAt was found to be
ineffective.
[2S,3S-Hmp]-AbL 2 was characterised by ESI-MS and NMR.
LRMS (ESI) determined the [MþH]þ mass to be 1071.7 and

2354
R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
Table 1 (continued)
Assignment Conformer A
1H
Conformer B
1H
13C
13C
d
in ppm
(ppm)
d
in ppm
(ppm)
(SH, m, J in Hz)
(SH, m, J in Hz)
MePh
CO
171.8
76.9
36.7
168.8
71.0
36.8
a
b
-CH
-CH2
2.95e3.03 (1H, m)
3.05e3.16 (1H, m)
2.72e2.77 (1H, m)
3.62 (1H, dd, 11.9; 2.7)
2.95e3.03 (1H, m)
3.38e3.50 (1H, m)
Cg (ar.)
Cd (ar.)
C 3 (ar.)
Cz (ar.)
NeCH3
Pro
135.3
129.4
137.9
129.0
128.7
127.2
40.6
7.14 (2H, d, 7.0)
6.22 (2H, d, 7.7)
7.05 (2H, t, 7.0)
7.26e7.33 (1H, m)
2.40 (3H, s)
7.26e7.33 (2H, m) 129.0
7.26e7.33 (1H, m) 128.0
3.04 (3H, s)
32.6
CO
170.9
59.3
30.6
172.3
63.7
29.7
a
b
-CH
-CH2
4.20 (1H, t, 8.7)
3.00e3.05 (2H, m)
4.69 (1H, d, 7.7)
2.27e2.33 (1H, m)
0.79e0.89 (1H, m)
2.06e2.13 (1H, m)
1.50e1.70 (1H, m)
4.34 (1H, t, 8.4)
g-CH2
2.06e2.13 (1H, m)
1.50e1.70 (1H, m)
3.30e3.38 (1H, m)
3.39e3.50 (1H, m)
24.3
46.5
24.1
47.7
d-CH2
Scheme 2. Synthesis of depsidipeptide 16. Reagents and conditions: (a) (1) acetic acid,
NaNO2; (2) tert-butanol, (Boc)2O, DMAP; (3) K2CO3 in MeOH. (b) (1) THF, DMAP, DCC,
0
3.65e3.72 (1H, m)
Val
CO
ꢀC; (2) TFA/CH2Cl2 (1:1).
170.3
56.4
30.8
20.7
22.6
170.0
56.3
30.8
20.9
22.9
a
b
g
g
-CH
-CH
-CH3
-CH3
4.40 (1H, t, 9.2)
4.45 (1H, t, 10.7)
2.46e2.55 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
7.78 (1H, d, 8.6)
2.17e2.26 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
9.18 (1H, d, 8.8)
[MþNa]þ 1093.7 while HRMS (ESI) was [MþNa]þ 1093.6661. This
data was strongly supported by fragmentation of the molecular
mass of the cyclic product with a [MþH]þ of 471.3 representing the
MePhe-Pro-Val-MeVal fragment and a [MþH]þ of 649.4 repre-
senting fragment of MeVal-Hmp-MeVal-Phe-MePhe. Molecular
ions at m/z 470þH and 648þH were also observed by Ikai et al.
following the fragmentation of the natural product.41
A structural assignment of [2S,3S-Hmp]-AbL 2 was achieved on
the basis of 1D and 2D NMR spectra including 1H, 13C NMR, COSY,
HSQC, DEPT-135 and also HMBC in combination with the spectral
data comparison with AbA 1 (conformers A and B) and aur-
eobasidin E (AbE) 3 (Fig. 1). The complete 1H and 13C NMR reso-
nance assignment of [2S,3S-Hmp]-AbL 2 is presented in Table 1.
NH
MeVal2
CO
169.6
61.5
26.4
19.1
19.5
31.4
169.5
61.5
26.5
18.9
18.7
30.8
a
b
g
g
-CH
-CH
-CH3
-CH3
5.25 (1H, t, 11.4)
2.27e2.33 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
3.27 (3H, s)
5.25 (1H, t, 11.4)
2.35e2.39 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
3.34 (3H, s)
NeCH3
Leu
CO
171.3
50.1
41.5
170.0
49.6
41.8
a
b
-CH
-CH2
4.78 (1H, q, 5.8)
4.85 (1H, q, 7.8)
1.72e1.85 (1H, m)
1.09e1.39 (1H, m)
1.50e1.70 (1H, m)
0.90e1.00 (3H, m)
0.90e1.00 (3H, m)
7.75 (1H, d, 7.8)
1.72e1.85 (1H, m)
1.09e1.39 (1H, m)
1.50e1.70 (1H, m)
0.90e1.00 (3H, m)
0.90e1.00 (3H, m)
7.98 (1H, d, 8.6)
g-CH
25.2
23.3
22.3
25.0
23.1
22.3
d
d
-CH3
-CH3
Table 1
NH
MeVal3
CO
1H and 13C NMR assignment in CDCl3 for conformer A and B of [2S,3S-Hmp]-AbL 2
170.6
62.5
26.6
18.4
18.2
39.8
170.7
61.1
26.5
19.2
19.6
39.1
Assignment Conformer A
1H
Conformer B
1H
a
b
g
g
-CH
-CH
-CH3
-CH3
4.97 (1H, d, 11.2)
2.27e2.33 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
2.88 (3H, s)
3.36e3.40 (1H, m)
2.27e2.33 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
2.82 (3H, s)
13C
13C
d
in ppm
(ppm)
d
in ppm
(ppm)
(SH, m, J in Hz)
(SH, m, J in Hz)
NeCH3
Hmp
CO
2-CH
3-CH
4-CH2
172.87
74.9
36.1
172.92
74.5
36.2
5.05 (1H, d, 4.4)
5.15e5.20 (1H, m)
1.72e1.85 (1H, m)
1.45e1.55 (1H, m)
1.28e1.32 (1H, m)
0.90e1.00 (3H, m)
0.79e0.89 (3H, m)
The 1H and 13C NMR spectra clearly showed the presence of two
conformers in a ratio of 61:39, which greatly aided the assignment
in an otherwise complex task. Rotational isomerism was also ob-
served for AbA in a similar ratio (57:43) but not for AbE 3, where the
1.72e1.85 (1H, m)
1.45e1.55 (1H, m)
1.61e1.71 (1H, m)
0.90e1.00 (3H, m)
0.79e0.89 (3H, m)
22.1
23.6
4-CH3
5-CH3
MeVal1
CO
15.6
11.8
15.4
11.9
hydroxyl group of the
b-HOMePhe residue next to the Pro was
found to result in only a cis-geometry of the amide bond (Fig. 3).42
The conformations of the amide bonds of [2S,3S-Hmp]-AbL 2
were determined on the basis of NOESY experiments and a com-
parative study of the chemical shifts and coupling constants with
168.8
61.6
26.4
168.8
61.4
26.5
14.35
19.8
30.6
a
b
g
g
-CH
-CH
-CH3
-CH3
5.15e5.20 (1H, m)
2.17e2.26 (1H, m)
0.79e0.89 (3H, m)
0.90e1.00 (3H, m)
2.76 (3H, s)
3.28e3.34 (1H, m)
2.27e2.32 (1H, m)
14.42 0.79e0.89 (3H, m)
19.9
39.5
0.90e1.00 (3H, m)
3.20 (3H, s)
NeCH3
Phe
CO
170.1
51.8
39.2
169.9
51.8
39.4
a
b
-CH
-CH2
5.34 (1H, q, 7.8)
3.20e3.29 (1H, m)
2.72e2.77 (1H, m)
5.10e5.15 (1H, m)
3.30e3.40 (1H, m)
2.59e2.62 (1H, m)
Cg (ar.)
Cd (ar.)
C 3 (ar.)
137.3
130.6
137.9
129.8
129.7
126.9
7.23 (2H, d, 4.4)
7.43 (2H, d, 7.4)
7.26e7.33 (2H, m)
7.26e7.33 (1H, m)
9.10 (1H, d, 9.70)
7.26e7.33 (2H, m) 129.4
7.26e7.33 (2H, m) 126.9
8.28 (1H, d, 8.95)
C
z (ar.)
Fig. 3. Conformers of the MePhe residue of AbA 1 and [2S,3S-Hmp]-AbL 2 (left, centre)
and the b-HOMePhe residue of AbE 3 (right).
NH

R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
2355
AbA 1 and AbE 3. NOESY experiments showed that conformer A of
[2S,3S-Hmp]-AbL 2 has the same structural form as conformer A of
AbA 1 where all of the amide bonds have trans geometries. Con-
former B of [2S,3S-Hmp]-AbL 2 has the same configuration as AbE 3
and conformer B of AbA 1 where all of the amide bonds are trans-
configured except for the amide bond between Pro and MePhe.
weights of all compounds were determined based on calculations
performed by ChemDraw Ultra 12.0.
Analytical RP-HPLC was performed on a Shimadzu HPLC in-
strument using an LC-20AD Prominence Liquid Chromatograph,
SIL-20A HT Prominence Autosampler and SPD-M20A Prominence
Diode Array Detector or a Shimadzu HPLC instrument using an LC-
20AD Prominence Liquid Chromatography, SIL-20A HT Prominence
Autosampler, SPD-M20A Prominence Diode Array Detector, CTO-
20A Prominence Column Oven and CBM-20A Prominence Com-
munications Bus Module. Semi-preparative RP-HPLC was carried
out on a Beckman HPLC instrument using a System Gold 125 Sol-
vent Module and System Gold 166 Detector. Either Phenomenex
3. Conclusion
[2S,3S-Hmp]-Aureobasidin L 2 was successfully synthesised
using a combination of solution-phase and solid-phase methods. In
particular, the solid-phase peptide synthesis protocols developed
for [2S,3S-Hmp]-aureobasidin L 2 will be an important solution for
the synthesis of highly N-methylated cyclodepsipeptides, in par-
ticular the aureobasidins and their analogues.
Jupiter or Apollo
5
m,
C18 column (250 mmꢁ4.6 mm or
250 mmꢁ10 mm) were used with HPLC grade acetonitrile and
ultra-pure water with the addition of 0.1% TFA. Flash column
chromatography used silica gel 60 (230e400 mesh ASTM) pur-
chased from Merck Chemicals Co Ltd. Distilled organic solvents
were employed as the mobile phase. TLC was carried out on Merck
Kieselgel 60F254 plates and visualised by a UV lamp together with
molybdenum polyphosphoric acid staining reagent, which was
made from 10% molybdenum polyphosphoric acid in ethanol.
4. Experimental section
4.1. General procedures
Commercial-grade reagents and solvents were used without
further purification unless specified. Solvents were purified or
dried as described by Perrin and Armarego.43 All Fmoc-amino acids
were purchased from Peptide Solutions Ltd (Melbourne).
All melting points were recorded on a Reichert ‘Thermopan’
microscope hot-stage apparatus and are uncorrected. Optical ro-
tation was measured with a PerkineElmer 141 Polarimeter or Atago
POLAX-2L polarimeter at the D line of sodium (589 nm) in a 1 dm
tube at the temperature and in the solvent indicated. Infrared
spectra were recorded on a PerkineElmer 1720-X Fourier Transfer-
Infra Red spectrometer by using a diffuse reflectance accessory or
KBr discs for solid samples and by using NaCl plates for liquid
samples.
4.2. Synthesis of [2S,3S-Hmp]-AbL 2
4.2.1. (2S)-N-Fluorenylmethyloxycarbonyl-2-aminomethyl-3-
methylbutanoic acid (Fmoc-N-methylvaline) 4. Fmoc-L-valine
(4.10 g, 12.0 mmol) was dissolved in toluene (144 mL). To this so-
lution were added p-toluenesulfonic acid (252 mg, 1.32 mmol) and
dry paraformaldehyde (360 mg, 12.0 mmol). The mixture was
refluxed until the solution turned clear. Excess paraformaldehyde
(2.88 g, 96.0 mmol) was added in small portions waiting for the
solution to become clear after each addition. The reaction mixture
was then refluxed until it remained clear. The reaction mixture was
concentrated to yield a residue, which was dissolved in ethyl ace-
tate (240 mL). Subsequently, the solution was partitioned by the
addition of 5% sodium bicarbonate solution (3ꢁ120 mL). The ethyl
acetate phase was dried (MgSO4) and evaporated. The residue was
purified by flash chromatography (10e60% ethyl acetate in hexane)
to give the desired Fmoc-valinyloxazolidinone as a yellow solid
(3.16 g, 85%): mp 85e87 ꢀC (lit.33 mp 70e74 ꢀC); IR (NaCl) nmax
(cmꢂ1) 3065, 3041, 3018 (aromatic CH), 2963, 2932, 2912, 2876
(aliphatic CH), 1799, 1712 (C]O), 1503, 1478, 1464, 1460, 1419
(C]C); 1H NMR (300 MHz, CDCl3) dH (ppm) 7.75 (2H, d, J¼7.5 Hz,
ArH), 7.53 (2H, d, J¼7.2 Hz, ArH), 7.40 (2H, t, J¼7.5 Hz, ArH), 7.31 (2H,
td, J¼1.2, 7.5 Hz, ArH), 5.4 (1H, br s, NCHHO), 5.00 (1H, dd, J¼0.6,
4.5 Hz, NCHHO), 4.67 (2H, br s, CHCH2O), 4.21 (1H, t, J¼5.1 Hz,
(Ar)2CHCH2), 3.61 (1H, br s, NCHCO), 1.72e1.24 (1H, m, CH3CHCH3),
0.86e0.68 (6H, m, CH3CHCH3) (IR and 1H NMR data were in
agreement with that previously described);33 13C NMR (75 MHz,
CDCl3) dC (ppm) 170.8, 153.0, 142.9, 141.1, 127.6, 127.5, 126.8, 124.1,
119.7, 77.9, 67.1, 59.5, 46.8, 30.6, 17.4.
The Fmoc-valinyloxazolidinone (2.80 g, 8.00 mmol) was dis-
solved in chloroform (40 mL). To this solution, trifluoroacetic acid
(1.85 mL, 24.0 equiv) and triethylsilane (3.83 mL, 24.0 equiv) were
added. The mixture was stirred at room temperature and left until
the reaction was complete (24e72 h). The solution was concen-
trated and partitioned between ether and 5% sodium bicarbonate
solution. The combined aqueous phases were acidified with 5 M
hydrochloric acid until reaching pH 2 and then it was extracted
with ethyl acetate. The desired organic phase was dried (MgSO4)
and concentrated to give Fmoc-N-methylvaline 4 as a white solid
(2.35 g, 83%). Recrystallisation of Fmoc-N-methylvaline using ethyl
acetate gave white needles: mp 186e187 ꢀC (lit.33 mp 185e187 ꢀC);
LRMS (ESI) m/z [MþH]þ 354.1 (100%) [MþNa]þ 376.1 (61%); IR
(NaCl) nmax (cmꢂ1) 3420 (broad) (OH), 2964 (aliphatic CH), 1700
(C]O), 1445, 1305 (C]C); 1H NMR (300 MHz, CDCl3) dH (ppm) 7.63
NMR spectra were performed on
a Bruker Avance 300
(300.13 MHz) instrument or a Bruker Avance 500 (500.19 MHz)
instrument. 1H NMR was performed at 300.13 MHz or 500.19 MHz
and 13C was obtained at 75 MHz or 125 MHz (La Trobe University)
or 200 MHz (Bio21 Institute, The University of Melbourne). Deu-
terochloroform (Cambridge Isotope Laboratories Inc.) was used as
a solvent and as an internal standard unless otherwise stated.
Chemical shifts were reported as d values in parts per million (ppm)
and coupling constants (J) in hertz. The DEPT, COSY, HSQC and
HMBC spectra were obtained on Bruker Avance 300 (300 MHz) and
Bruker Avance 500 (500.19 MHz) spectrometers. The NOESY spec-
tra were obtained on
spectrometer.
a Bruker Avance 500 (500.19 MHz)
Low Resolution-Electrospray Ionisation Mass Spectrometry (LR-
ESI) was carried out using a Bruker Daltonics (Germany) Esquire
6000 ion trap mass spectrometer at 300 ꢀC with a scan rate 5500 m/
z/s that was carried out at the Mass Spectrometry & Proteomics
Facility, La Trobe University. High Resolution-Electrospray Ionisa-
tion Mass Spectrometry (HR-ESI) was performed on Agilent 6224
TOF LC/MS Mass Spectrometer coupled to an Agilent 1290 Infinity
(Agilent, Palo Alto, CA) and was undertaken at the Monash Institute
of Pharmaceutical Sciences, Monash University. All data were ac-
quired and reference mass corrected via a dual-spray electrospray
ionisation (ESI) source. Each scan or data point on the Total Ion
Chromatogram (TIC) is an average of 13,700 transients, producing
a spectrum every second. Mass spectra were created by averaging
the scans across each peak and background subtracted against the
first 10 s of the TIC. Acquisition was performed using the Agilent
Mass Hunter Data Acquisition software version B.05.00 Build
5.0.5042.2 and analysis was performed using Mass Hunter Quali-
tative Analysis version B.05.00 Build 5.0.519.13. Methanol, aceto-
nitrile and water were used as the mobile phase. The molecular

2356
R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
(2H, d, J¼7.5 Hz, ArH), 7.46 (2H, d, J¼6.9 Hz, ArH), 7.26 (2H, t,
J¼7.2 Hz, ArH), 7.17 (2H, t, J¼7.5 Hz, ArH), 4.28 (2H, m, CHCH2O),
4.22 (1H, m, (Ar)2CHCH2), 4.13 (1H, m, NCHCO), 2.79 (3H, d,
J¼4.2 Hz, NCH3), 2.02 (1H, m, CH3CHCH3), 0.88 (3H, dd, J¼6.6,
19.2 Hz, CH3CHCH3), 0.72 (3H, dd, J¼6.6, 13.8 Hz, CH3CHCH3) (IR
and 1H NMR data were in agreement with that previously de-
scribed);33 13C NMR (75 MHz, CDCl3) dC (ppm) 175.5, 159.6, 146.6,
143.9, 130.4, 129.7, 127.7, 122.6, 70.3, 66.9, 49.9, 33.1, 30.2, 22.5, 21.7.
water several times and then extracted with saturated sodium bi-
carbonate solution. The combined aqueous solution was acidified
with 2 N hydrochloric acid and then extracted with ether. The or-
ganic layer was dried and evaporated to give acetyloxy acid (14.3 g,
20
51.2%) as a colourless oil: [
a]
ꢂ35.0, c 1 in dichloromethane; 1H
D
NMR (300 MHz, CDCl3) dH (ppm) 4.91 (1H, d, J¼4.5 Hz,
CH3COOCHCOOH), 2.11 (3H, s, CH3COO), 2.02e1.89 (1H, m,
CH3CHCH2), 1.56e1.46 (1H, m, CH3CHHCH), 1.35e1.23 (1H, m,
CH3CHHCH), 0.97 (3H, d, J¼6.9 Hz, CH3CH), 0.90 (3H, t, J¼7.5 Hz,
CH3CH2); 13C NMR (75 MHz, CDCl3) dC (ppm) 174.9, 170.5, 73.9, 36.1,
24.1, 20.1, 14.9, 11.1 (1H NMR and 13C NMR data were in agreement
with that previously described).44
4.2.2. (2S)-N-Fluorenylmethyloxycarbonyl-2-aminomethyl-3-
phenylpropanoic acid (Fmoc-N-methylphenylalanine) 5. To a solu-
tion of Fmoc-L-phenylalanine (4.60 g, 12.0 mmol) in toluene
(144 mL) were added p-toluenesulfonic acid (252 mg, 1.32 mmol)
and paraformaldehyde (360 mg, 12.0 mmol). The mixture was then
refluxed until the solution became clear. Excess paraformaldehyde
(2.88 g, 96.0 mmol) was added to the refluxing solution in small
portions once the solution became clear after each addition. The
reaction was refluxed until the reaction mixture turned clear. The
reaction mixture was further separated between ethyl acetate
and 5% sodium bicarbonate solution. The organic phase was
To a solution of acetyloxy acid (14.3 g, 81.9 mmol) in tert-butanol
(52 mL) were added di-tert-butyldicarbonate (21.3 g, 97.6 mmol)
and 4-dimethylaminopyridine (1.26 g,10.3 mmol). The solution was
stirred under nitrogen at room temperature for 4 h with the
progress of reaction monitored by TLC. The reaction mixture was
evaporated and the resulting crude product was dissolved in ethyl
acetate. The ethyl acetate solution was washed with saturated so-
dium bicarbonate solution, dried over MgSO4 and evaporated. The
crude product was purified by silica gel column chromatography
(hexane/ethyl acetate¼9.5:0.5) to give t-Bu-protected product
dried
(MgSO4)
and
evaporated
to
give
Fmoc-
phenylalaninyloxazolidinone as an oil (4.10 g, 85%): LRMS (ESI) m/
z [MþH]þ 400.1 (28%) [MþNa]þ 422.2 (100%); IR (NaCl) nmax (cmꢂ1
)
(15.5 g, 82%) as a colourless oil: [
a
]
D
20 ꢂ30.0, c 1 in dichloromethane;
3065, 3030 (aromatic CH), 2954, 2915 (aliphatic CH), 1801, 1717
(C]O), 1496, 1477, 1451, 1423 (C]C); 1H NMR (300 MHz, CDCl3) dH
(ppm) 7.76 (2H, d, J¼6.6 Hz, ArH), 7.62e7.48 (2H, m, ArH), 7.41 (2H,
t, J¼7.2 Hz, ArH), 7.35 (2H, t, J¼6.6 Hz, ArH), 7.25e7.13 (4H, m, ArH),
6.71 (1H, br s, ArH), 5.07 (2H, s, NCH2O), 4.69 (1H, dd, J¼5.1, 10.5 Hz,
CHCHHO), 4.51 (1H, br s, NCHCO), 4.25 (1H, t, J¼5.1 Hz, CHCHHO),
4.12e3.98 (1H, m, (Ar)2CHCH2), 3.39e2.42 (2H, m, CHCH2Ar) (IR
and 1H NMR data were in agreement with that previously de-
scribed);33 13C NMR (75 MHz, CDCl3) dC (ppm) 171.4, 152.0, 143.0,
141.1, 134.0, 129.2, 128.4, 127.7, 127.6, 127.1, 126.9, 124.1, 119.85,
119.77, 77.4, 66.0, 67.0, 55.9, 46.9, 35.0.
The Fmoc-phenylalaninyloxazolidinone (4.60 g, 11.6 mmol) was
dissolved in chloroform (58 mL). Trifluoroacetic acid (2.68 mL,
34.8 equiv) and triethylsilane (5.56 mL, 34.8 equiv) were added to
the solution. The reaction mixture was stirred at room temperature
until the reaction was complete (24e72 h). The solution was con-
centrated and partitioned between ether and saturated sodium
bicarbonate solution. The combined aqueous phase was acidified
with 5 M hydrochloric acid to pH 2 and extracted with ethyl acetate.
The desired organic phase was then dried (MgSO4) and concen-
trated to give the product as a dark yellow oil. The crude product
was dissolved in dichloromethane and evaporated repeatedly to
give Fmoc-N-methylphenylalanine as a bþrown solid (3.73 g, 80%):
mp 106e107 ꢀC; LRMS (ESI) m/z [MþH] 402.1 (100%); 1H NMR
(300 MHz, CDCl3) dH (ppm) (rotamers) 7.74 (2H, d, J¼7.5 Hz, ArH),
7.49 (1H, d, J¼7.8 Hz, ArH), 7.43 (1H, t, J¼6.9 Hz, ArH), 7.37 (2H, t,
J¼7.5 Hz, ArH), 7.25e7.21 (6H, m, ArH), 6.95 (1H, d, J¼6.3 Hz, ArH),
4.89 (1H, dd, J¼5.1, 11.1 Hz, NCHCOOH), 4.59e4.53 (1H, m,
(Ar)2CHCH2), 4.38 (2H, d, J¼14.4 Hz, CHCH2O), 3.41e3.35 (1H, m,
CHCHHAr), 3.16e3.08 (1H, m, CHCHHAr), 2.77 (3H, s, NCH3), 2.75
(3H, s, NCH3) (1H NMR data was in agreement with that previously
described);33 13C NMR (75 MHz, CDCl3) dC (ppm) 175.5, 156.5, 143.4,
140.9, 136.4, 128.4, 128.3, 127.3, 126.7, 126.5, 124.6, 124.3, 119.6, 67.6,
60.7, 46.7, 34.3, 32.3.
1H NMR (300 MHz, CDCl3) dH (ppm) 4.73e4.71 (1H, m,
CH3COOCHCOO), 2.07 (3H, s, CH3COO), 1.89e1.87 (1H, m,
CH3CHCH2), 1.41 (9H, s, (CH3)3C), 1.50e1.37 (1H, m, CH3CHHCH),
1.29e1.20 (1H, m, CH3CHHCH), 0.94e0.85 (6H, m, CH3CH,CH3CH2);
13C NMR (75 MHz, CDCl3) dC (ppm) 170.3, 168.3, 81.4, 76.2, 36.2,
27.6, 24.3, 20.2, 14.9, 11.2 (1H NMR and 13C NMR data were in
agreement with that previously described for the 2R,3S isomer).45
tert-Butyl (2S)-acetyloxy-(3S)-methylpentanoate (15.6 g,
67.5 mmol) was dissolved in methanol (250 mL). To this solution
was added potassium carbonate (1.36 g, 9.84 mmol) and the solu-
tion was stirred for 2 h with monitoring by TLC. The reaction
mixture was evaporated and the resulting crude product was
evaporated and partitioned between water and ether. The organic
layer was dried and evaporated to give 15 (6.10 g, 48%) as a col-
ourless oil: [
a]
20 ꢂ15.0, c 1 in dichloromethane; 1H NMR (300 MHz,
D
CDCl3) dH (ppm) 3.93 (1H, d, J¼3.6 Hz, HOCHCOO), 1.79e1.65 (1H,
m, CH3CHCH2), 1.47 (9H, s, (CH3)3C), 1.40e1.25 (1H, m, CH3CHHCH),
1.25e1.15 (1H, m, CH3CHHCH), 0.95 (3H, d, J¼6.9 Hz, CH3CH), 0.88
(3H, t, J¼7.2 Hz, CH3CH2); 13C NMR (75 MHz, CDCl3) dC (ppm) 173.8,
81.9, 74.4, 38.8, 27.7, 23.5,14.9,11.5 (1H NMR and 13C NMR data were
in agreement with that previously described for the 2R,3S
isomer).45
4.2.4. (2S,3S)-2-{(S)-2-[(N-Fluorenylmethyloxycarbonyl)-2-
aminomethyl-3-methylbutanoyl]oxy}-3-methylpentanoic acid 16. To
a solution of Fmoc-N-methylvaline 4 (18.0 g, 51.3 mmol) in dry
tetrahydrofuran (100 mL) were added alcohol 15 (10.6 g,
56.3 mmol) and 4-dimethylaminopyridine (1.90 g, 15.6 mmol).
Once the reaction mixture was cooled to 0 ꢀC, N,N0-dicyclohex-
ylcarbodiimide (6.00 g, 56.6 mmol) was added to the reaction
mixture. The reaction was stirred for 1 h at 0 ꢀC and then 24 h at
room temperature. The reaction mixture was evaporated and the
residue was dissolved in ethyl acetate. Insoluble material was re-
moved from the organic solution by filtration. The ethyl acetate
solution was then washed successively with 10% citric acid solution,
saturated sodium chloride solution, saturated sodium bicarbonate
solution and saturated sodium chloride solution. The solution was
then dried (MgSO4), filtered and evaporated to obtain a residue.
This residue was then purified by silica gel column chromatography
4.2.3. tert-Butyl(2S)-acetyloxy-(3S)-methylpentanoate
15. L-Iso-
leucine 14 (21.0 g, 160 mmol) was dissolved in glacial acetic acid
(240 mL). The stirred solution was cooled occasionally to keep the
solution at room temperature. To this solution was added sodium
nitrite (22.1 g, 320 mmol) in several portions over 1 h. Once the
addition was complete, the solution was stirred for 24 h. The re-
action mixture was then evaporated and the crude product was
dissolved in diethyl ether. The ethereal solution was washed with
(hexane/ethyl acetate¼9.5:0.5) to give the tert-butyl depsidipep-
20
tide (11.8 g, 44%) as a colourless oil: [
a
]
D
ꢂ60.0, c 1 in dichloro-
methane; HRMS (ESI) m/z calcd for C31H41NO6 requires [MþNa]
546.2832 Found 546.2827; 1H NMR (300 MHz, CDCl3) dH (ppm)

R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
2357
(rotamers) 7.75 (2H, d, J¼7.2 Hz, ArH), 7.58 (2H, d, J¼7.2 Hz, ArH),
7.38 (2H, t, J¼7.2 Hz, ArH), 7.29 (2H, t, J¼7.2 Hz, ArH), 4.87e4.82 (1H,
m, OCHCOOH), 4.74e4.55 (1H, m, NCHCO), 4.46e4.40 (2H, m,
CHCH2O), 4.26e4.20 (1H, m, ArCHAr), 2.91 (3H, t, J¼9.0 Hz, NCH3),
2.30e2.12 (1H, m, CH3CHCH3), 1.98e1.88 (1H, m, CH3CHCH2), 1.44
(9H, s, C(CH3)3), 1.35e1.18 (2H, m, CHCH2CH3), 1.09e0.85 (12H, m,
3ꢁ CH3CH; CH3CH2); 13C NMR (75 MHz, CDCl3) dC (ppm) (rotamers)
170.9,170.6,168.9,168.7,157.4, 156.7, 144.5,144.3,141.7,128.0,127.4,
125.5, 125.4, 125.3, 119.1, 120.3, 82.3, 82.2, 77.7, 75.9, 68.1, 64.6, 64.2,
47.7, 36.8, 30.9, 30.7, 28.4, 27.9, 26.4, 25.0, 20.2, 20.1, 19.3, 19.2, 15.7,
14.6, 12.0, 11.9.
To a solution of the tert-butyl depsidipeptide (11.9 g, 22.6 mmol)
in dichloromethane (27 mL) was added trifluoroacetic acid (27 mL).
The reaction mixture was stirred for 5 h resulting in 16 (10.0 g,
95%); LRMS (ESI) m/z [MþH]þ 468.2 (44%) [MþNa] 490.2 (100%);
HRMS (ESI) m/z calcd for C27H33NO6 requires [MþH]þ 468.2386
Found 468.2384; 1H NMR (300 MHz, CDCl3) dH (ppm) (rotamers)
7.75 (2H, d, J¼7.5 Hz, ArH), 7.58 (2H, d, J¼6.9 Hz, ArH), 7.38 (2H, t,
J¼7.2 Hz, ArH), 7.29 (2H, t, J¼6.9 Hz, ArH), 4.89 (1H, dd, J¼21.9,
4.2 Hz, OCHCOOH), 4.56e4.51 (1H, m, NCHCO), 4.51e4.39 (2H, m,
CHCH2O), 4.27e4.20 (1H, m, ArCHAr), 4.16e4.12 (1H, d, J¼10.8 Hz,
NCHCO), 2.91 (3H, s, NCH3), 2.87 (3H, s, NCH3) 2.30e2.15 (1H, m,
CH3CHCH3), 2.11e1.92 (1H, m, CH3CHCH2), 1.55e1.40 (1H, m,
CHCHHCH3), 1.36e1.15 (1H, m, CHCHHCH3), 1.07e0.86 (12H, m, 3ꢁ
CH3CH; CH3CH2); 13C NMR (75 MHz, CDCl3) dC (ppm) (rotamers)
174.0, 170.4, 169.8, 156.6, 156.0, 143.6, 143.5, 143.4, 141.0, 127.9,
127.3, 126.7, 124.6, 124.5, 119.6, 75.9, 67.5, 67.4, 63.9, 63.7, 46.9, 36.0,
35.9, 30.4, 30.2, 27.4, 27.1, 24.0, 19.2, 18.6, 18.3, 15.0, 11.1.
3e24 h. The resin was then filtered and washed with dichloro-
methane, dimethylformamide and dichloromethane successively.
To a small portion of dry resin beads was added a TNBS test solution
for a non-N-methylated amino acid confirmation or a chloranil test
solution for a N-methylated amino acid confirmation.
4.2.9. BTC-mediated coupling. The dry depsipeptidyl resin was
swollen with dry N,N-diisopropylethylamine/tetrahydrofuran (1:1,
5.3 mL:5.3 mL). In a separate vessel, Fmoc-amino acid (5 equiv), bis-
(trichloromethyl)carbonate (1.65 equiv) and dry tetrahydrofuran
(20 mL) were mixed to give a clear solution. Later, sym-collidine
(14.0 equiv) was added to that solution to give a white suspension.
The suspension was stirred for 1 min before it was added to the
swollen resin. The resin was shaken for 3e24 h. The resin was then
filtered and washed with dichloromethane, dimethylformamide
and dichloromethane successively. To a small portion of dry resin
beads was added a TNBS test solution for a non-N-methylated
amino acid confirmation or a chloranil test solution for a N-meth-
ylated amino acid confirmation.
4.2.10. HATU/HOAt-mediated coupling. Tothe dry depsipeptidyl resin
(1.50 mmol) having been swollen by dry dichloromethane/dime-
thylformamide (1:1, 10 mL) was added Fmoc-amino acid (5.00 mmol)
that was previously treated with 2-(1H-7-azabenzotriazol-1-yl)-
1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium
(1.68 g, 5.00 mmol), 1-hydroxy-7-azabenzotriazole (681 mg,
5.00 mmol) and N,N-diisopropylethylamine (5.3 mL,15.0 mmol) in dry
dichloromethane/dimethylformamide (1:1, 10 mL). The resin was
shaken for 3e24 h. The resin was then filtered and washed
with dichloromethane, dimethylformamide and dichloromethane
successively. To a small portion of dry resin beads was added a
4.2.5. Loading the resin. 2-Chlorotrityl resin (1.80 g, 2.00 mmol)
was added to dry dichloromethane (10 mL) and a solution of Fmoc-
L-proline (843 mg, 2.50 mmol) that was previously treated with dry
TNBS test solution for a non-N-methylated amino acid con-
dichloromethane (10 mL) and N,N-diisopropylethylamine (1.71 mL,
10.0 mmol). The mixture was shaken for 3e4 h. Methanol (3 mL)
was then added and the mixture was shaken for another 10 min.
The latter step was undertaken in two cycles. The resin was then
filtered and washed successively with dichloromethane, dime-
thylformamide and dichloromethane. The resin was dried in vacuo
for 30 min by a stream of air to obtain dry Fmoc-propyl resin (57%).
A 1.5 mmol portion of dry Fmoc-propyl resin was employed in the
synthesis of [2S,3S-Hmp]-aureobasidin L 2.
firmation or a chloranil test solution for a N-methylated amino acid
confirmation.
4.2.11. Resin cleavage. To the depsinonapeptidyl-trichlorotrityl
resin 13 (1.50 mmol) was added a cleavage cocktail of trifluoro-
acetic acid/dry dichloromethane (1:19, 10 mL). The yellow resin
turned bright red. The resin was then shaken for 1 h and then fil-
tered. The resin was washed subsequently with further cleavage
cocktail (10 mLꢁ2) and dry dichloromethane (10 mLꢁ2). The
combined solution was evaporated and the resulting residue was
dissolved in acetonitrile/water (1:1, 10 mL). The solution was then
freeze-dried to obtain a brown solid (1.30 g). The crude solution
4.2.6. Fmoc deprotection 1. Fmoc-peptidyl resin was shaken with
20% dry piperidine in dry dimethylformamide (20 mL) for 30 min.
The resin was then filtered and washed with dichloromethane,
dimethylformamide and dichloromethane successively. The ex-
posed primary amine was tested by the addition of a TNBS test
solution while N-methylated secondary amine was tested by add-
ing chloranil test solution to the resin beads.
(20
mL, 1 mg/1 mL in 50% acetonitrile in water) was subjected to
analytical RP-HPLC on Apollo 5
m
, C18 column (250 mmꢁ4.6 mm),
20%e90% acetonitrile in water over 20 min, flow rate 1 mL/min,
40 ꢀC and monitored at 240 nm. There were three major peaks in
the crude product, which was further purified by semi-preparative
RP-HPLC on an Apollo 5
m, C18 column (250 mmꢁ10 mm) using
4.2.7. Fmoc deprotection 2. Fmoc-depsipeptidyl resin was shaken
with dry piperidine/DBU/dimethylformamide (2:2:96, 20 mL) for
10e20 min. The resin was then filtered and washed with
dichloromethane, dimethylformamide and dichloromethane suc-
cessively. The exposed primary amine was tested by the addition of
a TNBS test solution while N-methylated secondary amine was
tested for by adding chloranil test solution to the resin beads.
30%e70% acetonitrile in water over 60 min, flow rate 3 mL/min and
monitored at 240 nm. The desired depsinonapeptide (98.8 mg) was
isolated and determined to be ready for cyclisation. The purity of
the product was checked by analytical RP-HPLC (20%e90% aceto-
nitrile in water over 20 min, flow rate 1 mL/min, 40 ꢀC, 240 nm).
Yellow solid: mp 132e134 ꢀC; LRMS (ESI) m/z [MþH]þ 1089.7
[MþNa]þ 1111.7; HRMS (ESI) m/z calcd for C59H92N8O11 requires
[MþH]þ 1089.6964 Found 1089.6969.
4.2.8. HBTU/HOBt-mediated coupling. To the dry depsipeptidyl
resin (1.50 mmol) having been swollen by dry dichloromethane/
dimethylformamide (1:1, 10 mL) was added Fmoc-amino acid
(5.00 mmol) that was previously treated with O-benzotriazole-
N,N,N0,N0-tetramethyl-uronium-hexafluoro-phosphate (1.88 g,
5.00 mmol), N-hydroxybenzotriazole (671 mg, 5.00 mmol) and dry
N,N-diisopropylethylamine (5.30 mL, 15.0 mmol) in dry dichloro-
methane/dimethylformamide (1:1,10 mL). The resin was shaken for
4.2.12. Peptide cyclisation in solution. The purified linear dep-
sinonapeptide precursor (40 mg, 37
dichloromethane (135 mL, 270 M). To the clear solution were
added 2-(1H-7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium-
hexafluorophosphate methanaminium (71.5 mg, 190 mol) and
N,N-diisopropylethylamine (65.4 L, 39.0 mol). The solution was
stirred under nitrogen for 2 days and then was concentrated under
mmol) was dissolved in dry
m
m
m
m

2358
R. Maharani et al. / Tetrahedron 70 (2014) 2351e2358
13. Awazu, N.; Ikai, K.; Yamamoto, J.; Nishimura, K.; Mizutani, S.; Takesako, K.; Kato,
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Lett. 1993, 22, 1873.
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ijavallabhan, V. M.; Ganguly, A. K. Tetrahedron Lett. 1996, 37, 5661.
17. Schmidt, U.; Schumacher, A.; Mittendorf, J.; Riedl, B. J. Pept. Res. 1998, 52, 143.
18. Pettit, G. R.; Holzapfel, C. W. J. Org. Chem. 1986, 51, 4580.
19. Rinehart, K. L.; Kishore, V.; Nagarajan, S.; Lake, R. J.; Gloer, J. B.; Bozich, F. A.; Li,
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reduced pressure at room temperature. The resulting crude product
was purified by silica gel column chromatography (hexane/2-
propanol¼9.5:0.5) to obtain the cyclic product 2 (4.00 mg, 10%) as
a white solid: mp 62e64 ꢀC; LRMS (ESI) found [MþH]þ 1071.7 and
[MþNa] 1093.7; HRMS (ESI) m/z calcd for C59H90N8O10 requires
[MþNa]þ 1093.6678 Found 1093.6661.
Acknowledgements
We would like to thank Associate Professor Philip Thompson for
his assistance during this work and Dr. Jason Dang for HRMS
analysis (Monash Institute of Pharmaceutical Science, Monash
University). We also greatly appreciate the access to the 800 MHz
NMR spectrometer provided by Dr. Shenggen Yao (Bio21 Institute,
The University of Melbourne). R.M. is grateful to the Indonesian
government for the award of Directorate General of Higher Edu-
cation Scholarship, Department of National Education.
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Supplementary data
29. Wischnat, R.; Rudolph, J.; Hanke, R.; Kaese, R.; May, A.; Theis, H.; Zuther, U.
The 1H and 13C NMR spectra of [2S,3S-Hmp]-AbL 2. Supple-
mentary data related to this article can be found at http://
dx.doi.org/10.1016/j.tet.2014.02.036.
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