Total synthesis of cyclohexadepsipeptides exumolides A and B

Article abstract of DOI:10.1016/j.tet.2021.131987

Exumolides A and B are two cyclodepsipeptides that was previously isolated from marine fungus. Both peptides were synthesized using a combination of solid- and solution-phase method. The linear precursor was synthesized through solid-phase method on 2-chlorotrityl resin with standard Fmoc strategy. The hydroxy acid, (S)-2-hydroxy-4-methylpentanoic acid (Hmp), was prepared from its precursor L-leucine, and attached on the resin with a double-coupling protocol. The depside bond formation that was carried out at the end of the coupling process, was beneficial, particularly, in the blockage of the diketopiperazine formation during Fmoc deprotection. The cyclic product was obtained through HATU-based cyclisation. Exumolides A and B were successfully synthesized with overall yields of 4.12% and 6.39%, respectively.

Full text of DOI:10.1016/j.tet.2021.131987

Tetrahedron 83 (2021) 131987
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Total synthesis of cyclohexadepsipeptides exumolides A and B
,
Agung Rahmadani a b, Muhammad Amir Masruhim b, Laode Rijai c,
,
,
, d, *
Ace Tatang Hidayat a d, Unang Supratman a d, Rani Maharani a
a Department of Chemistry, Faculty of Mathematics and Natural Sciences, Universitas Padjadjaran, Jalan Raya Bandung-Sumedang Km 21 Jatinangor,
45363, West Java, Indonesia
b Department of Chemistry Education, Universitas Mulawarman, Jalan Muara Pahu, Samarinda, 75119, East Kalimantan, Indonesia
c Pharmaceuticals Research and Development Laboratory of Farmaka Tropis, Pharmacy Faculty, Universitas Mulawarman, Jalan Penajam No.1, Samarinda,
75119, East Kalimantan, Indonesia
d Laboratorium Sentral, Universitas Padjadjaran, Jalan Raya Bandung-Sumedang Km 21 Jatinangor, 45363, West Java, Indonesia
a r t i c l e i n f o
a b s t r a c t
Article history:
Exumolides A and B are two cyclodepsipeptides that was previously isolated from marine fungus. Both
peptides were synthesized using a combination of solid- and solution-phase method. The linear pre-
cursor was synthesized through solid-phase method on 2-chlorotrityl resin with standard Fmoc strategy.
Received 14 December 2020
Received in revised form
15 January 2021
Accepted 25 January 2021
Available online 6 February 2021
The hydroxy acid, (S)-2-hydroxy-4-methylpentanoic acid (Hmp), was prepared from its precursor L-
leucine, and attached on the resin with a double-coupling protocol. The depside bond formation that was
carried out at the end of the coupling process, was beneficial, particularly, in the blockage of the dike-
topiperazine formation during Fmoc deprotection. The cyclic product was obtained through HATU-based
cyclisation. Exumolides A and B were successfully synthesized with overall yields of 4.12% and 6.39%,
respectively.
Keywords:
Exumolide A
Exumolide B
Cyclohexadepsipeptide
Solid phase peptide synthesis
© 2021 Elsevier Ltd. All rights reserved.
1. Introduction
explained that the presence of depside bond in the backbone will
easily induce the formation of diketopiperazine (DKP) [4].
Exumolides A and B are cyclohexadepsipeptides, which contain
one ester bond and five amides bonds in their structures. These
peptides were previously isolated from marine fungus, Scytalidium,
obtained from Exuma Bahama islands. Structurally, exumolide A is
an N-methylated version of exumolide B at the leucine residue
(Fig. 1). Both compounds have a hydroxy acid, (S)-2-hydroxy-4-
methylpentanoic acid (Hmp) in their structures and five L-config-
ured amino acid residues. Both compounds were known to have an
inhibition activity against chlorophyte Dunaliela sp. [1].
In order to explore the biological properties of both peptides, we
are interested to provide the access to the peptides chemically.
Synthesis of cyclodepsipeptide is known to be challenging due to
the difficulties in the depside bond formation compared to the
formation of amide bond [2]. On solid phase, the formation of ester
bond (depside) is known to be even more difficult than the for-
mation of amide bond [3]. In addition to that, Coin et al. (2008)
A combination of solid- and solution-phase methods have been
shown to be effective for the synthesis of cyclodepsipeptides [4,5].
The linear peptide is synthesized through solid phase method and
cyclisation is carried out in solution phase. Coin et al. (2008) suc-
cessfully synthesized a cyclodepsipeptide contrasin, where the
unprotected hydroxy acid was applied in the synthesis and
attached on the resin at the initial stage [4]. The depside bond
formation with the second amino acid was facilitated with DIC
coupling agent and DBU-based Fmoc deprotection was applied to
avoid the diketopiperazine formation. The cyclodepsipeptide was
obtained in 22% overall yield. Rahim et al. (2020) has also suc-
cessfully synthesized cyclodepsipeptide [leu] [6]-aureobasidin K by
employing an Fmoc-protected hydroxy acid [5]. The formation of
depside bond on solid phase was occurred between the hydroxy
acid at fourth position and the fifth residue. In the synthesis, a
diketopiperazine formation during the Fmoc deprotection was
detected, eventhough the DBU-based deprotection was applied for
the Fmoc elimination during the synthesis. To minimize the for-
mation of DKP, the depside bond formation was placed at the last
residue due to linear depsipeptide step [6]. For this purpose, the
cyclisation site was selected in between phenylalanine at C
* Corresponding author. Department of Chemistry, Faculty of Mathematics and
Natural Sciences, Universitas Padjadjaran, Jalan Raya Bandung-Sumedang Km 21
Jatinangor, 45363, West Java, Indonesia.
E-mail address: r.maharani@unpad.ac.id (R. Maharani).
https://doi.org/10.1016/j.tet.2021.131987
0040-4020/© 2021 Elsevier Ltd. All rights reserved.

A. Rahmadani, M.A. Masruhim, L. Rijai et al.
Tetrahedron 83 (2021) 131987
HOAt was known as a good combination of coupling agents that can
facilitate difficult coupling involving N-methyl amino acid [8].
Meanwhile, the attachment of Fmoc-L-Pro-OH onto the tripeptidyl
of exumolide B only required HBTU/HOBt for the amide bond for-
mation. The next step was the addition of hydroxy acid (S)-2-
hydroxy-4-methylpentanoic acid (Hmp) onto the tetrapeptidyl
resin of exumolide A and B to give the pentapeptidyl resin. HATU/
HOAt-based coupling was undertaken twice to make a complete
amide bond formation. Binding of the last amino acid Fmoc-L-Pro-
OH onto the pentapeptidyl resin involved the formation of depside
bond(ester). The protocol applied in the synthesis of aureobasidin
analogue was followed with a slight modification [5]. The penta-
peptidyl resin was coupled to Fmoc-L-Pro-OH by using a Steglich
esterification method (DIC/DMAP) with the addition of basic DIPEA
as the modified protocol. The reaction was performed for 3 ꢀ 5 h at
45 ꢁC to finally result in Fmoc-hexadepsipeptide-resin. Fmoc
deprotection on the peptidyl resin afforded hexadepsipeptide that
was ready to be cleaved from the resin. Moreover, the linear hex-
adepsipeptide was released from the resin using 20% TFA in
dichloromethane. Crude of linear hexadepsipeptides were charac-
terized by using HR-TOF-MS. The HR-TOF-MS spectra of both crude
showed the correct molecular ion peak of linear hexadepsipeptide
exumolides A and B at m/z 748.3941 [MþH]þ and m/z 734.3800
[MþH]þ, respectively.
Fig. 1. Exumolides A (1) and B (2).
terminus and proline at N terminus. Solution-phase cyclisation was
applied following the protocol described in Maharani et al. (2014)
[7].
Since exumolide A consists of N-methyl leucine in its structure,
coupling involving this residue can be problematic. The coupling
reaction involving N-methyl residue took advantage of HATU/HOAt
through a double coupling protocol [5,7]. In this paper we describe
the first total synthesis of two cyclohexadepsipeptides, exumolides
A and B.
2. Results and discussion
Crude linear hexadepsipeptide of exumolides A or B was
cyclized using coupling agent HATU in a basic DIPEA condition
(10ꢂ3 M) for 48 h, following the protocol employed by Maharani
et al. (2014) [7]. The reaction was controlled with thin layer chro-
matography and mass spectrometry. The separation of the cyclo-
hexadepsipeptides was carried out using column chromatography
with eluent of n-hexane:ethyl acetate affording white solid 3.1 mg
(4.12%) exumolide A 1 and 5.3 mg (6.39%) exumolide B 2. The purity
of 1 and 2 was analyzed using analytical RP-HPLC, showing a single
peak at retention time of 31.4 min and 26.3 min, respectively. The
presence of two residual prolines in the peptidic backbone, where
one of them positioned at the N terminal is beneficial to make a
complete cyclisation [2]. Puentes et al. (2017) explained the pres-
ence of proline can increase the cyclisation process, due to the role
of proline as beta turn inducer [9].
The strategy for the synthesis of exumolides A and B was shown
in Scheme 2. The linear precursors were constructed on 2-
chlorotrityl resin and were then cyclized in solution phase to
afford exumolides A and B. At the initial stage, the precursor of
exumolides A and B, (S)-2-hydroxy-4-methylpentanoic acid (Hmp),
was prepared through a diazotisation reaction between L-leucine
and sodium nitrite in acid condition, resulting in (S)-2-hydroxy-4-
methylpentanoic acid in 81% yield (Scheme 1). The purity of in-
termediate 4 has been confirmed by analytical RP-HPLC analysis,
showing a single peak at retention time of 23.2 min.
Once the precursor ready, the next step was to construct the
linear depsipeptide on resin. Since a site between proline as N
terminal and phenylalanine as C terminal was selected for the final
head-to-tail cyclisation, Fmoc-L-Phe-OH became the first amino
acid attached on the 2-chlorotrityl chloride resin. Once the first
residue attached on the resin, the following step was to capped the
unreacted resin using methanol and to deprotect the Fmoc-
protecting group on Fmoc-Phe-resin using 20% piperidine on
DMF to afford NH2-Phe-resin.
HBTU/HOBt was employed to connect the second amino acid
Fmoc-L-Phe-OH to the NH2-Phe-resin to give the dipeptidyl resin.
The next Fmoc-L-MeLeu-OH coupling using HBTU/HOBt onto the
dipeptidyl resin, followed by the Fmoc deprotection step, resulted
in tripeptidyl resin of exumolide A. By using similar steps, Fmoc-L-
Leu-OH was attached on the resin to afford the tripeptidyl resin of
exumolide B.
The fourth residue, Fmoc-L-Pro, was coupled onto the tri-
peptidyl resin of exumolide A by employing HATU/HOAt in a
double-coupling protocol to facilitate a complete amide formation.
This strategy was also applied in the synthesis of highly N-meth-
ylated cyclodepsipeptide, the aureobasidin analogues [5,7]. HATU/
Cyclic peptide 1 was characterized using HR-TOF-MS, 1H NMR
and 13C NMR. HR-TOF-MS spectrum showed correct molecular ion
peak for the desired product with m/z 752.3970 [MþNa]þ (calcu-
lated m/z 752.3999 for C41H55N5O7Na). 1H NMR and 13C NMR in
CDCl3 showed protons and carbons that are consistent with the
structure of exumolide A (Supplementary material). The spectra of
1H NMR exhibiting the presence of two proton signals of NH amides
at 8.21 and 6.98 ppm. The existence of six alpha protons were
shown in the chemical shifts at 5.15, 5.08, 4.78, 4.41, 4.40 and
3.31 ppm. The chemical shift at 2.43 ppm with a singlet multiplicity
is a characteristic of N-methyl of exumolide A. 13C NMR spectra
showed the presence of five carbonyl signals of amides at 172.1,
171.5, 170.3, 169.7, 169.5 and one carbonyl signal of ester at
167.5 ppm. The presence of depside bond in exumolide A was
confirmed through the presence of CeO sp [3] chemical shift at
70.8 ppm. The other five alpha carbons were shown in the chemical
shift at 59.4, 59.0, 56.4, 54.5 and 53.7 ppm. The presence of
chemical shift at 28.9 ppm showed the existence of N-methyl from
exumolide A.
On the other hand, the purified cyclic peptide 2 was character-
ized using HR-TOF-MS, 1H NMR and 13C NMR. HR-TOF-MS spectra
showed a correct molecular ion peak for the desired product with
m/z 716.4019 [MþH]þ, that suit to the calculated m/z 716.4023 for
C
40H54N5O7. 1H NMR and 13C NMR in CDCl3 showed protons and
carbons that are consistent with the structure of exumolide B
(Supplementary material). The presence of three signals of NH
Scheme 1. Synthesis of (S)-2-hydroxy-4-methylpentanoic acid. Reagent and condi-
tions (a) NaNO2 in water, H2SO4 1 M, 0 ꢁC for 2 h and then rt for overnight (81% yield).
2

A. Rahmadani, M.A. Masruhim, L. Rijai et al.
Tetrahedron 83 (2021) 131987
Scheme 2. Synthesis route of exumolides A and B (a) (1) Fmoc-L-Phe-OH, DIPEA, DCM, 24 h, rt; (2) MeOH/dichloromethane/DIPEA (3:7:1), (3) 20% piperidine in DMF, (b) Fmoc-L-
Phe-OH, HBTU, HOBt, DIPEA, DMF, 4 h, rt (2) 20% piperidine in DMF, (c) (1)Fmoc-L-N-MeLeu-OH(7)/Fmoc-L-Leu-OH(8), HBTU, HOBt, DIPEA, DMF, 4 h, rt, (2) 20% piperidine in DMF,
(d) (1) Fmoc-L-Pro-OH, HATU, HOAt, DIPEA, DMF, 2 ꢀ 4 h, rt, (2) 20% piperidine in DMF, (e) (S)-2-hydroxy-4-methylpentanoic acid, HATU, HOAt, DIPEA, DMF, 2 ꢀ 4 h, rt, (f) Fmoc-L-
Pro-OH, DIC, DMAP, DIPEA, DCM: DMF (95:5), 3 ꢀ 5 h, 45 ꢁC, (2) 20% piperidine in DMF (g) 20% TFA in dichloromethane, 2 ꢀ 10 min, rt, (h) HATU, DIPEA, DCM, 48 h, rt.
amide protons at 7.35, 7.02 and 6.47 ppm were shown in 1H NMR
spectra. Meanwhile, the presence of six alpha protons were per-
formed in the chemical shifts at 4.84, 4.44e4.84, 4.38, 4.24, 3.85
and 3.78 ppm. 13C NMR spectra showed the existence of five
carbonyl signals of amides at 172.7, 171.8, 171.8, 170.4, and
170.0 ppm and one carbonyl signal of ester at 169.5 ppm. The
presence of depside bond in exumolide B was confirmed through
the presence of CeO sp [3] chemical shift at 72.9 ppm. The other
five alpha carbons were shown in the chemical shift at 60.8, 59.9,
58.8, 54.0 and 51.0 ppm. 1H NMR and 13C NMR exumolides A and B
synthesized were in a good agreement with those of the natural
products (Table 1) [1].
analogues, can be potentially explored for their biological proper-
ties as antimicrobial agent since many cyclodepsipeptides are
known to have antimicrobial properties.
4. Experimental part
4.1. Materials
1H and 13C NMR spectra were recorded on Agilent NMR
500 MHz (1H) and 125 MHz (13C) using CD3OD and CDCl3 solvent.
Mass spectrometry spectra were recorded on Waters HR-TOF-MS
Lockspray, Analytical RP-HPLC was performed on Waters Alliance
2998 using photo diode array (PDA) detector with Acquity BEH C18
1H NMR data of both synthetic exumolides have chemical shifts
differences in the range of 0.01e0.08 ppm that are similar to the
chemical shift differences between the natural and synthetic des-
truxin E, reported by Yoshida et al. (2010) that are also in the range
of 0.01e0.08 ppm [11]. The 13C NMR data of exumolide A and B also
show chemical shift differences that are not more than 1 ppm
(0.1e0.7 ppm) between the experimental and the reported data.
More than 65% of 13C NMR data of exumolide A are identical with
the data of the natural product. Meanwhile, the larger chemical
shift differences are found in the 13C NMR data of exumolide B even
though they are still in the range of 0.1e0.7 ppm. This can be
explained by the different deuterated solvents used in the NMR
column 1.7
using UVeVis Detector 2489 with LichroCart RP 18e column 5
(4.6 ꢀ 250 mm). All of the amino acid residues, Fmoc-L-Pro-OH,
Fmoc-L-Leu-OH, Fmoc-L-N-MeLeu-OH, Fmoc-L-Phe-OH,
m
m (2.1 ꢀ 50 mm), and also on Waters Alliance e2695
mm
L-
Leucine, 1[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]
pyridinium3-oxide hexafluorophosphate (HATU), 1-hydroxy-7-
azabenzotriazole
(HOAt),
(2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU), N-hydox-
ybenzotriazole (HOBt) and 2-chlorotrityl chloride (2-CTC) resin
were purchased from GL-Biochem Ltd., China. Dichloromethane
(DCM), N,N-dimethylformamide (DMF), N,N-diisopropyletilamine
measurement. In the present experiment, exumolide
B was
(DIPEA),
N,N0-diisopropylcarbodiimide
(DIC),
4-
measured in the deuterated chloroform, while the natural product
was measured in the deuterated dichloromethane.
dimethylaminopyridine (DMAP), sodium nitrite, sodium sulphate,
piperidine, methanol, propanol, n-hexane, ethyl acetate, acetic acid,
sulphuric acid and trifluoroacetic acid (TFA) used in the synthesis
were in proanalyst grade.
3. Conclusions
Exumolides A and B were successfully synthesized using a
combination solid- and solution-phase methods with overall yields
of 4.12% and 6.39%, respectively. The utilization of Hmp without
protecting group and the strategy to put ester bond formation at
the last step on solid-phase have successfully produced the linear
depsipeptides. The synthetic products, together with their
4.2. Synthesis of (S)-2-hydroxy-4-methylpentanoic acid (4)
Amino acid
L-leucine (2 g, 15,2 mmol) was dissolved in 40 mL of
sulphuric acid
1
M. Then, sodium nitrite solution (6.78 g,
98.2 mmol) was added into the solution slowly at 0 ꢁC for 2 h. The
reaction mixture was stirred for 17 h at room temperature. The
3

A. Rahmadani, M.A. Masruhim, L. Rijai et al.
Tetrahedron 83 (2021) 131987
Table 1
1H and13C NMR spectral data of exumolides A and B in CDCl3 and comparison with previously reported spectral data.[1]
.
Residue No
atom
dH (ppm), mult, J (Hz)b dC (ppm)a dH (ppm), mult, J dC (ppm)e dH (ppm), mult, J (Hz)d
dC (ppm)c dH (ppm), mult, J (Hz)b
dC (ppm)e
(Hz)b
Exumolide A [1]
Exumolide A
Exumolide B [1]
Exumolide B
170.4
2S-
Hmp
1
167.4
167.5
170.0
2
3
5.11 (dd) 2, 12.5
1.80 (m);
1.42 (m)
70.8
38.9
5.15 (dd) 3, 12.5 70.9
1.83e1.85 (m); 38.9
1.48e1.51 (m)
4.78 (dd) 1.8, 10.8
1.33 (ddd) 1.8, 9.6, 15.0; 1.80 (m) 39.7
73.4
4.84 (d) 9.5
1.24e1.30 (m)
72.9
39.4
4
5
6
7
8
9
1.58 (m)
0.91 (d) 6.5
0.83 (d) 7
24.5
23.3
21.3
172.1
56.4
29.0
1.61e1.63 (m) 24.5
2.00 (m)
1.04 (d) 6.6
1.05 (d) 6.6
25.4
21.8
23.7
173.1
59.4
31.1
1.90e1.95 (m)
1.01 (d) 6.5
1.03 (d) 6
25.0
21.6
23.5
172.7
58.8
30.7
0.97 (d) 7
0.89 (d) 7
23.4
21.3
172.1
56.4
L-Pro
4.72 (dd) 7.5
1.82 (m);
2.12 (m)
4.78 (t) 6
3.96 (dd) 3.6, 9.0
1.97 (m);
2.18 (m)
3.85 (dt) 6, 6, 10
1.98 (dd) 3.75, 12.25; 2.17
(dd) 3.25, 13.25
1.86e1.91 (m); 29.7
2.15e2.20 (m)
10
11
2.16 (m);
1.96 (m)
3.54 (m);
3.78 (ddd) 14.0, 8.5, 7.0
25.4
47.2
2.20e2.24 (m); 25.5
1.99e2.03 (m)
3.57e3.61 (m); 47.3
3.83 (dd) 6.5,
1.75 (m)
22.8
1.81e1.89 (m)
22.5
46.2
3.32 (ddd) 7.8, 7.8, 12.0; 3.59 (ddd) 46.6
4.8, 8.4, 12.0
3.29 (dd) 7.25, 11.75; 3.58
e3.63 (m)
14.75
L-Leu
12
13
14
169.6
59.0
36.5
169.5
172.6
51.4
38.7
171.8
51.0
38.4
4.35 (m)
1.40 (m);
1.97 (m)
1.37 (m)
0.85 (d) 7.5
0.86 (d) 7.0
2.38 (s)
4.40 (dd) 6, 10.5 59.0
1.45e1.47 (m); 36.5
2.05e2.09 (m)
4.42 (ddd) 6, 9.6, 9.6
1.62 (m);
1.69 (m)
4.38 (ddd) 6, 9, 9
1.61e1.65 (m);
1.68 (dd) 3, 9.5
1.54e1.59 (m)
0.85 (d) 6
15
16
17
18
24.5
21.8
23.3
28.8
1.42e1.45 (m) 24.6
1.60 (m)
25.5
21.7
23.4
NH
25.1
21.5
23.2
NH
0.91 (d) 7
0.92 (d) 7
2.43 (s)
21.8
23.4
28.9
0.91 (d) 6.6
0.98 (d) 6.6
6.95 (d) 9.0
0.93 (d) 6.5
7.02 (d) 11
(NMe)
169.6
53.7
L-Phe 19
20
169.7
53.7
170.5
60.8
170.4
59.9
5.03 (dd) 9.0, 16.5
5.08 (dd) 9,
16.25
3.82 (ddd) 6.6, 6.6, 9.6
3.78 (dd) 3, 9
21
3.16 (dd) 13.5; 2.97 (dd) 37.2
9.0, 13.5
136.2
3.19e3.22 (m); 37.2
3.00e3.05 (m)
136.6
3.21 (dd) 6.6, 13.8; 3.38 (dd) 9.6,
13.8
36.2
3.19 (dd) 6, 14;
3.33 (dd) 10, 14
35.6
22
137.8
137.0
23
24
25
26
7.14 (m)
7.24 (m)
7.26 (m)
8.17 (d) 9.0
129.1 ꢀ 2 7.20e7.23 (m) 129.1 ꢀ 2 7.35 (m)
129.6 ꢀ 2 7.28e7.33 (m)
128.9 ꢀ 2
128.5 ꢀ 2
126.9
128.6 ꢀ 2 7.28e7,32 (m) 128.6 ꢀ 2 7.35 (m)
129.2 ꢀ 2 7.28e7.33 (m)
126.8
NH
7.20e7.23 (m) 126.8
7.27 (m)
6.64 (d) 6.6
127.4
NH
7.16e7.25 (m)
6.47 (d) 6
8.21 (d) 9
NH
NH
L-Phe 27
28
170.3
54.5
170.3
54.5
172.3
54.1
171.8
54.0
4.37 (m)
4.41 (dd) 4.5,
10.5
4.52 (ddd) 4.8, 6.6, 9.6
4.44e4.84 (m)
29
2.73 (dd) 12.0; 3.17 (dd) 40.3
12.5
2.79 (t) 12;
3.24 (dd) 4.25,
8,25
40.3
3.02 (dd) 9.6, 12.6;
3.07 (dd) 9.6, 13.8
40.2
3.00 (dd) 10.25, 12.25; 3.10 39.8
(dd) 4.5, 12,5
30
31
32
33
34
136.6
137.4
137.8
136.9
7.14 (m)
7.24 (m)
7.26 (m)
6.92 (d) 6.0
129.4 ꢀ 2 7.20e7.23 (m) 129.4 ꢀ 2 7.24 (m)
129.8 ꢀ 2 7.16e7.25 (m)
129.3 ꢀ 2
129.1 ꢀ 2
127.2
NH
128.6 ꢀ 2 7.28e7,32 (m) 128.7 ꢀ 2 7.30 (m)
128.9 ꢀ 2 7.16e7.25 (m)
127.1
NH
7.28e7,32 (m) 127.1
7.27
7.25
127.2
NH
7.16e7.25 (m)
7.35 (d) 6.5
6.98 (d) 6.5
NH
L-Pro
35
36
37
171.3
59.4
31.2
171.5
59.4
1.40e1.42 (m); 31.2
1.74e1.78 (m)
1.74e1.78 (m) 22.3
3.40e3.45 (m); 46.0
3.61e3.66 (m)
169.6
61.3
32.6
169.5
60.8
32.1
3.28 (d) 7.5
1.36 (m);
1.72 (m)
3.33 (d) 7
4.24 (br d) 7.8
2.20 (m);
2.37 (br dd) 6.0; 12.6
1.70 (m)
4.24 (d) 8.5
2.12 (dd) 7.25, 14.75; 2.42
(dd) 6.5, 11.7
1.73e1.80 (m)
3.49e3.56 (m)
38
39
1.66 (m)
22.2
22.5
47.4
22.0
46.9
3.37 (ddd) 8.5, 11.5, 8.5; 46.0
3.55 (m)
3.52 (m)
a
Recorded at 100 MHz in CDCl3.
Recorded at 500 MHz in CDCl3.
Recorded at 100 MHz in CD2Cl2.
Recorded at 600 MHz in CD2Cl2.
Recorded at 125 MHz in CDCl3.
b
c
d
e
reaction process was monitored by using thin layer chromatog-
raphy (propanol:methanol:acetic acid (7:2:1)). The reaction
mixture was then added by sodium bicarbonate until the reaction
mixture reached pH 2. Furthermore, the reaction mixture was
saturated with sodium chloride and extracted with ethyl acetate
(3 ꢀ 50 mL). Ethyl acetate fraction was dried with sodium sulphate,
filtered, and concentrated using rotary evaporation. The purity of
the product was analyzed using analytical RP-HPLC. The product
was then characterized using spectrometry methods (FTIR, HR-
TOF-MS, 1H NMR, and 13C NMR) [10]. White solid; 81% yield; IR
(KBr) nmax (cmꢂ1) 3427 (OH hydroxyl), 2978 (CH aliphatic), 1714
(C]O carboxylic group), 1391 (gem dimethyl), 1076 (CeO); HR-
TOF-MS m/z [M ꢂ H]-131.0703 (calcd. C6H11O3 131.0708). 1H NMR
(500 MHz, CD3OD) dH (ppm) 4.95 (brs, 2H, OH and COOH), 4.14 (d,
J ¼ 5.25; 8.25 Hz,1H, H-2), 1.79e1.90 (m, 1H, H-4),1.54e1.57 (m, 2H,
H-3), 0.95 (d, J ¼ 3,05 Hz, 3H, 4-CH3) 0,94 (d, J ¼ 2,95 Hz, 3H, H-5).
4

A. Rahmadani, M.A. Masruhim, L. Rijai et al.
Tetrahedron 83 (2021) 131987
13C NMR (125 MHz, CDCl3) dc (ppm) 180.1 (C-1), 68.9 (C-2), 43.1 (C-
3), 24.4 (C-4), 23.2(C-5), 21.4(4-CH3).
reaction mixture was concentrated to give crude cyclic product as
yellow solid. Cyclic product was then separated by column chro-
matography using eluent n-hexane:ethyl acetate (1:1). The purity
of the cyclic product was analyzed using analytical RP-HPLC. The
product was then characterized using spectrometry methods (HR-
TOF-MS, 1H NMR, and 13C NMR).
4.3. Solid-phase peptide synthesis
400 mg of 2-chlorotrityl chloride resin (capacity 0.99 mmol/g)
was swollen in DCM for 10 min. Then, the swollen resin was added
by Fmoc-L-Phe-OH (0.6 mmol) and DIPEA (1.2 mmol) in DCM
(4 mL). The reaction mixture was then shaken for 24 h. Resin was
then washed with DCM and dried. The dry resin was then calcu-
lated for its loading resin resulting in the loading resin value of
0.42 mmol/g. Furthermore, resin was then added by 4 mL solution
of MeOH:DIPEA:DCM (1.5:0.5:8) and was shaken for 2 ꢀ 10 min.
Resin was then filtered, washed using DMF and DCM, and dried.
Fmoc-peptidyl resin was then added by 20% piperidine in DMF
with volume of 4 mL. This deprotection process was carried out by
stirring the mixture for 2 ꢀ 5 min. Resin was then filtered and
washed with DMF and DCM. Then, chloranil test was carried out to
determine the presence of free amine groups.
4.4.1. Exumolide A (1)
White solid; 3.1 mg, 4.12% yield; 1H NMR and 13C NMR data can
be found in Supplementary material. HR-TOF-MS m/z [MþNa]
752.3970 (calcd. C41H55N5O7Na 752.3999).
4.4.2. Exumolide B (2)
White solid; 5.3 mg, 6.39% yield; 1H NMR and 13C NMR data can
be found in supplementary material. HR-TOF-MS m/z [MþH]þ
716.4019 (calcd. C40H54N5O7 716.4023).
Declaration of competing interest
Peptidyl-NH2 resin was developed with DMF (4 mL) and then
added by Fmoc-amino acid (0.51 mmol), HBTU (0.51 mmol), HOBt
(0.51 mmol) and DIPEA (1.04 mmol). The mixture was stirred for
4 h, and then the resin was filtered and washed with DMF and DCM.
Next, the Fmoc deprotection was carried out using 20% piperidine
in DMF. Furthermore, the chloranil test was undertaken to confirm
the presence of free amine groups.
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgement
Peptidyl-NHMe resin was developed with DMF 4 mL and then
added by Fmoc-amino acid (0.51 mmol), HATU (0.51 mol), HOAt
(0.51 mmol) and DIPEA (1.04 mmol). The mixture was stirred for
2 ꢀ 4 h, and then the resin was filtered and washed with DMF and
DCM. Next, the Fmoc deprotection was carried out using 20%
piperidine in DMF. Furthermore, the chloranil test was undertaken
to confirm the presence of free amine groups.
We would like to express our gratitude for the research grant of
The Ministry of Research, Technology, and Higher Education-
Indonesia (Doctoral Research Grant Scheme 1827/UN6.3.1/LT/
2020), which provided vital financial support.
Appendix A. Supplementary data
Pentapeptidyl-OH resin was placed in
a reaction tube,
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2021.131987.
completed with magnetic stirrer. Fmoc-L-Pro-OH (1.18 mmol) was
then added by DCM (4 mL) and transferred into the reaction tube.
Then, a mixture of DIC (1.69 mmol), DMAP (0.27 mmol), and DIPEA
(0.24 mmol) was added into the tube. The mixture was reacted for
3 ꢀ 5 h at 45 ꢁC. After the reaction finished, resin was then filtered
and washed with DMF and DCM. Next, the Fmoc deprotection was
carried out using 20% piperidine in DMF. Furthermore, the chloranil
test was undertaken to confirm the presence of free amine groups.
Hexadepsipeptidyl resin was added by 5 mL of 20% TFA in DCM.
Then, the solution was shaken for 2 ꢀ 10 min and the combined
filtrate was collected. Filtrate was then evaporated to get crude
linear hexadepsipeptide, as pale yellow solid. The product was then
characterized with HR-TOF-MS method.
References
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ꢀ
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Each crude linear hexadepsipeptide of exumolides A or B
(10ꢂ3 M) was dissolved with 100 mL of DCM. Into this solution was
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then added HATU (0.4 mmol) in DMF 400
mL and DIPEA (1.6 mmol).
The mixture was reacted at room temperature for 48 h. Then, the
5