Mollamides B and C, cyclic hexapeptides from the indonesian tunicate Didemnum molle

Article abstract of DOI:10.1021/np700718p

Two new cyclic hexapeptides, mollamides B (1) and C (2), were isolated from the Indonesian tunicate Didemnum molle along with the known peptide keenamide A (3). The structures were established using 1D and 2D NMR experiments. The relative configuration of mollamide B at the thiazoline moiety was determined using molecular modeling coupled with NMR-derived restraints. Their absolute configuration was determined using Marfey's method. The new peptides have been evaluated for their antimicrobial, antimalarial, anticancer, anti-HIV-1, anti-Mtb, and anti-inflammatory activities. Keenamide A and mollamide B show cytotoxicity against several cancer cell lines.

Full text of DOI:10.1021/np700718p

J. Nat. Prod. 2008, 71, 941–945
941
Mollamides B and C, Cyclic Hexapeptides from the Indonesian Tunicate Didemnum molle
Marwa S. Donia,† Bin Wang,† Daniel C. Dunbar,† Prashant V. Desai,‡ Akshay Patny,‡ Mitchell Avery,‡ and Mark T. Hamann*,†
Department of Pharmacognosy, Medicinal Chemistry and National Center for Natural Products Research, School of Pharmacy, The UniVersity
of Mississippi, UniVersity, Mississippi 38677
ReceiVed December 13, 2007
Two new cyclic hexapeptides, mollamides B (1) and C (2), were isolated from the Indonesian tunicate Didemnum molle
along with the known peptide keenamide A (3). The structures were established using 1D and 2D NMR experiments.
The relative configuration of mollamide B at the thiazoline moiety was determined using molecular modeling coupled
with NMR-derived restraints. Their absolute configuration was determined using Marfey’s method. The new peptides
have been evaluated for their antimicrobial, antimalarial, anticancer, anti-HIV-1, anti-Mtb, and anti-inflammatory activities.
Keenamide A and mollamide B show cytotoxicity against several cancer cell lines.
The ascidians have become one of the most important sources
of marine natural products in the last decades, and a significant
number of compounds with unusual structures and bioactivities have
been isolated from various ascidians. The peptide metabolite
didemnin B is an example of an important drug lead isolated from
Trididemnum solidum, a Caribbean tunicate of the family Didem-
nidae.1 Didemnin B was the first marine natural product to enter
phase I and II clinical trials as an anticancer agent.1 The genus
Didemnum is an extremely productive source of bioactive cyclic
peptides characterized by the presence of modified amino acids.
Enzymatic heterocyclization of serine, threonine, and cysteine side
chains with vicinal carbonyl groups results in the formation of five-
ring heterocyclic oxazole and thiazole moieties.2–4 Peptides with
this functionality exhibit a diverse array of bioactivities. Thiazole-,
oxazole-, and thiazoline-containing peptides have been isolated from
the common Indo-Pacific ascidian Didemnum molle from many
different geographic locations, and they share the peculiar reverse
prenylated ethers of serine and threonine amino acids. They include
the heptapeptides mollamide,5 cyclodidemnamides,6,7 and myota-
mides A and B8 and the hexapeptides comoramides A and B8 and
didmolamides A and B.9 As the chemical composition of this genus
changes from one collection to another, Mu¨ller et al. proposed that
these metabolites are actually biosynthesized by symbiotic prochlo-
rophytes, and they isolated prokaryotic algal symbionts often
associated with the didemnid family.10 Furthermore, Schmidt et
al. reported the full sequence and functional expression of the related
peptides, patellamides, from the obligate symbiont Prochloron
didemni.11 Recently, Donia et al. showed that Prochloron spp.
generate a diverse library of patellamides, and they used this
information to engineer the production of a new cyclic peptide in
Escherichia coli.12
Results and Discussion
An investigation of the tunicate D. molle collected from Manado
Bay, Indonesia, yielded two new cyclic hexapeptides, mollamides
B (1) and C (2), along with the known keenamide A (3), which
was previously reported from the notaspidean mollusk Pleurobran-
chus forskalii collected from Manado, Indonesia. This mollusk was
and water extracts were exhaustively extracted separately with
shown to feed on the ascidian D. molle on the basis of the analysis
dichloromethane and then ethanol. The dichloromethane and the
of the gut content.13 All three peptides share the peculiar reverse
1
ethanol extracts were combined on the basis of H NMR analysis
prenylated ethers of serine and threonine amino acids.
and were subjected to silica gel vacuum-liquid chromatography
The freeze-dried invertebrate was extracted with water to remove
followed by reversed-phase and amino normal-phase HPLC to yield
the foaming aqueous-soluble polysaccharides. The dried residue
the two new cyclic hexapeptides mollamides B (1) and C (2) and
the known peptide keenamide A (3).
* To whom correspondence should be addressed. Tel: 662-915-5730.
Mollamide B (1) was obtained as a light yellow powder, and
Fax: 662-915-6975. E-mail: mthamann@olemiss.edu.
HRESIMS gave an [M + H]+ peak at 697.3713 for a molecular
formula of C36H53N6O6S (calc 697.3747, ∆ -3.4 mmu, mmu )
† Department of Pharmacognosy.
‡ Department of Medicinal Chemistry.
10.1021/np700718p CCC: $40.75
2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 06/11/2008

942 Journal of Natural Products, 2008, Vol. 71, No. 6
Donia et al.
resonating at δ 7.00 and belonging to the valine 1 residue correlated
with the thiazoline carbonyl carbon (δ 170.8) in the HMBC
spectrum connecting both residues. The NH proton signal (δ 7.60)
of the phenylalanine residue showed an HMBC correlation to the
valine 1 carbonyl signal (δ 170.5). The connection between the
proline and the phenylalanine amino acid residues was established
by ROESY correlations between the R-proton of phenylalanine (δ
4.50) and the R-proton of proline (δ 3.44) and from the aromatic
protons of phenylalanine to the R-proton of proline.
The absolute configuration of mollamide B (1) was determined
by acid hydrolysis and derivatization with Marfey’s reagent15
followed by comparative HPLC analysis with derivatized standard
D- and L-amino acids. The analysis established the L-configuration
for each amino acid residue (L-valine, L-phenylalanine, L-proline,
and L-threonine). A minor peak was also observed for D-valine,
which resulted from partial racemization of the L-valine from the
L-valinylthiazoline moiety. It has been reported that the thiazoline-
based amino acid units in cyclic peptides tend to epimerize easily
during hydrolysis.16,17 The configuration at the thiazoline amino
acid moiety using traditional degradation and derivatization tech-
niques remains a challenge. During acid hydrolysis, the thiazoline
moiety hydrolyzes to cysteine, which then incompletely derivatizes
by FDAA (N-alkylation, S-arylation, formation of bis-derivative,
and oxidation to cystine), leading to unpredictable results.16 The
synthesis of these compounds has historically been the only
stereochemical solution to these moieties, but it is time-consuming
and costly.
Figure 1. Key ROESY correlations for mollamide B (1).
Due to the inherent limitations of experimental data to assign
the absolute configuration of functional groups such as these,
molecular modeling coupled with NMR-derived restraint informa-
tion has exhibited significant potential for assigning relative and
absolute configuration of many natural products and pharmaceuti-
cally relevant compounds.18–20 A similar approach has been used
to assign the unknown configuration at the R-carbon of the
thiazoline moiety of mollamide B. Molecular modeling was utilized
to provide a reasonable prediction of configuration at the R-carbon
of the thiazoline moiety based on conformational changes to the
molecule associated with D- or L-configuration at this center. Using
the ROESY data as distance restraints (Figure 1), molecular
mechanics calculations involving stepwise minimization followed
by restrained simulated annealing were performed to generate an
ensemble of conformations for the two possible configurations at
the R-carbon (D-Tzn (S): MolB-S; L-Tzn (R): MolB-R). Additionally,
since molecular mechanics energies are empirical in nature, more
accurate semiempirical quantum mechanical calculations were
performed to calculate the heats of formation (∆Hf) for the lowest
energy conformations. MolB-R appeared to be more stable than
MolB-S by 8.5 kcal/mol. Also, MolB-R was found to be consistent
with NMR-derived distance restraints, while MolB-S showed two
significant violations (Figure 2). Thus, the configuration at the
R-carbon of the thiazoline moiety was assigned to be R, corre-
sponding to the L-amino acid configuration.
Figure 2. Energy-minimized conformations of MolB-R (1) and
MolB-S (2).
millimass unit), requiring 14 degrees of unsaturation. The peptidic
1
nature of the compound was suggested by the IR, and H and 13C
NMR spectra; four amide protons were observed at δ 7.00, 7.61,
7.86, and 9.14 ppm, and six 13C NMR signals resonated between
δ 168 and 175 ppm, corresponding to amide carbonyls and
indicating the presence of six amino acids. In addition, the IR
spectrum showed bands at 3256, 3356, and 1683 cm-1, confirming
the presence of an amide functionality. The compound was negative
to ninhydrin, suggesting a blocked N-terminus or a cyclic peptide.
The structure of mollamide B (1) was determined using both
one- and two-dimensional NMR spectral measurements including
COSY, HMQC, HMBC, and ROESY experiments. Six partial
structures were constructed that were assigned as one phenylalanine,
two valine units, one threonine carrying a dimethylallyl ether group,
one proline unit, and one thiazoline unit. The ether linkage between
the isoprene moiety and the threonine amino acid was assigned on
the basis of the HMBC correlation between the ꢀ-proton of
threonine resonating at δ 3.57 ppm and the quaternary carbon of
the isoprene group resonating at δ 78.0 ppm.
The assignment of the thiazoline residue was accomplished using
HMQC, HMBC, COSY, and HRESIMS spectrometric techniques.
The methylene protons (δ 4.03, 3.46) showed HMBC correlations
to the carbonyl signal at δ 174.5, which was assigned as part of
the valine 2 residue, and to the carbonyl signal at δ 170.8. The
same protons also showed COSY correlations to a methine proton
at δ 5.13, which provides a strong HMBC correlation to the valine
2 carbonyl (δ 174.5). The cis/trans configuration of Pro was
determined to be cis on the basis of the ∆δꢀγ (differential value of
13C chemical shifts of Cꢀ and Cγ in Pro)14 value of 8 ppm.
The amino acid sequence and the connection of amino acid
residues of 1 were accomplished by long-range 1H-13C correlation
experiments (HMBC) and ROESY. The NH proton (δ 9.10) and
R-proton (δ 4.80) signals of threonine provide HMBC correlations
to the carbonyl (δ 171.1) signal of proline. The NH proton (δ 7.80)
and R-proton (δ 4.37) signals of valine 2 showed HMBC correla-
tions to the threonine carbonyl resonance (δ 168.9). The NH
The minor component mollamide C (2) was obtained as a white
solid. The HRESIMS showed an [M + H]+ 619.3259 (calc
619.3277, ∆ -1.8 mmu) peak, indicating a molecular formula of
C30H46N6O6S, which accounts for 15 degrees of unsaturation and
one more additional unsaturation unit than the known peptide
1
keenamide A (3). The H NMR spectrum showed a comparable
spectrum to keenamide A (3). Three 1H NMR resonances are
lacking (δ 5.0 (d), 3.60, and 3.70 (m)) corresponding to the
thiazoline unit, and instead an additional singlet peak at δ 7.9 ppm
appears. Two additional aromatic carbon resonances (δ 122.4, 149.2
ppm) were observed, indicating the presence of an aromatic ring
system. Analysis of HMBC data revealed that the aromatic singlet
proton resonating at δ 7.9 ppm correlates to the leucine carbonyl
carbon (δ 170.7 ppm) and to a quaternary aromatic carbon (δ 149.2
ppm). The same proton showed a strong correlation to the carbonyl

Cyclic Hexapeptides from Didemnum molle
Journal of Natural Products, 2008, Vol. 71, No. 6 943
Table 1. NMR Spectroscopic Data for Mollamide B (1)a,b
amino acid
proline
position
δC, mult.
δH (J in Hz)
3.44, m
2.1, 0.75, dd (6.0, 12.0)
1.6, m
HMBCc
1, 3, 4, 5
1, 2, 4, 5
2, 3
1
2
3
4
5
6
7
8
171.1, qC
61.2, CH
29.8, CH2
21.8, CH2
46.2, CH2
170.1, qC
54.7, CH
37.6, CH2
135.1, qC
129.5, CH
129.0, CH
127.6, CH
129.0, CH
129.5, CH
3.3, 3.48, t (10.0)
3, 4
phenylalanine
4.5, t (7.2)
3.0, m
8
6, 7, 9, 10
9
10
11
12
13
14
NH
15
16
17
18
19
NH
20
21
22
23
24
25
26
27
NH
28
29
30
31
NH
32
33
34
35
36
7.1, dd (1.2, 7.6)
7.2, m
7.2, m
7.2, m
7.2, m
8, 12, 14
9, 13
9, 11
8, 10, 12
15
7.6, d
valine 1
170.5, qC
57.9, CH
32.4, CH
17.8, CH3
18.7, CH3
4.18, t (7.6)
2.0, m
0.97, m
0.97, m
7.0, d (7.6)
17, 18, 15
18, 19, 16, 15
16, 17
16, 17
16, 20
thiazoline
valine 2
170.8, qC
78.0, CH
34.0, CH2
174.5,qC
58.0, CH
30.6, CH
16.1, CH3
20.2, CH3
5.13, m
4.03, 3.46, dd (4.4, 11.2)
20, 23
20, 21, 23
4.37, dd (3.2, 6.0)
2.27, m
0.93, d (7.2)
1.0, d (6.8)
7.8, d (6.4)
23, 25, 26, 27, 28,
26, 27
24, 25, 27
24, 25, 26
24, 25, 28
threonine
isoprene
168.9, qC
56.1, CH
70.1, CH
17.8, CH3
4.8, dd (4.0, 9.6)
3.57, m
1.43, d (6.8)
9.1, d (9.6)
31, 30, 28
31, 29, 32, 28
29, 30
1
78.0, qC
27.0, CH3
25.9, CH3
142.4, CH
115.3, CH2
1.32, s
1.37, s
5.8, dd (10.8, 17.6)
5.1, m
34, 32, 35
33, 32, 35
34, 33, 32
32, 35
a
1H and 13C NMR data were measured at 400 and 100 MHz, respectively. b All spectra were measured in CDCl3 and referenced to the residual
solvent signal at δH 7.26 and δC 77.4. c HMBC correlations are from the proton(s) stated to the indicated carbons.
carbon forming the amide bond with the isoleucine amino acid,
supporting the presence of the thiazole-modified amino acid. Careful
analysis of COSY, HMQC, and HMBC NMR data indicated that
the rest of the molecule is identical to keenamide A. ∆δꢀγ of the
proline in mollamide C was calculated to be 2.8 ppm, showing
that the proline amide bond is trans, which is the same as in
keenamide A (3) (∆δꢀγ 3.1 ppm).
The absolute configuration of the thiazole-containing peptide
mollamide C (2) was determined by the procedure described by
McDonald and Ireland.21 This procedure uses ozone to destroy the
aromaticity of the thiazole in order to facilitate hydrolysis and
prevent racemization. The analysis established the L-configuration
of each amino acid residue (L-isoleucine, L-leucine, L-serine, and
L-proline).
Mollamide B (1) and keenamide were examined for antimicrobial
activity against MRSA, Mycobacterium intracellulaire, Candida
albicans, C. glabrata, C. krusei, and Cryptococcus neoformans and
exhibited no activity.
Mollamides B and C as well as keenamide have shown no anti-
inflammatory activity in rat neonatal microglia, and neither of them
inhibited cyclooxygenase enzyme (COX-2) activity in a cell-based
assay.
Mollamide B (1) has been tested at 100 µM against four cancer
cell lines: the non-small cell lung cancer cell line H460, the breast
cancer cell line MCF7, and the CNS cancer cell line SF-268.
Mollamide B showed significant percentage growth inhibition. The
percentage growth was H460 (29), MCF7 (44), and SF-268 (42),
which allowed the compound to be evaluated by the National
Cancer Institute (NCI) in the 60-cell-line panel. No cell lines showed
any sensitivity to mollamide B that exceeded the mean.
Mollamide C was tested in an in Vitro disk diffusion assay that
defines the differential cell killing among nine cell types examined.
These are two leukemias (murine L1210 and human CCRF-CEM),
five solid tumors (murine colon 38, human colon HCT-116, human
lung H125, human breast MCF-7, and human prostate LNCaP),
and a murine and human normal cell (hematopoietic progenitor cell,
CFU-GM). The assay is designed to determine large differences in
the relative sensitivity of solid tumors, normal cells, and leukemias.
The greater the zone differential in the solid tumor colonies and
only minor toxicity for both the leukemia and normal cells, the
higher the selectivity of the compound to solid tumors. Mollamide
C showed a unit zone differential value of 100 against L1210,
human colon HCT-116, and human lung H125 and a value of 250
against murine colon 38 and was not considered to be solid tumor
selective.
Mollamide B (1) exhibited moderate antimalarial activity against
Plasmodium falciparum (D6 clone and W2 clone), with IC50 values
of 2.0 and 2.1 µg/mL, respectively. Mollamide B also exhibited
marginal activity against Leishmania donoVani, with IC50 and IC90
values of 18 and 35 µg/mL, respectively, and against HIV-1 in
human PBM cells with an EC50 value of 48.7 µM in Vitro.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a JASCO DIP-310 digital polarimeter. IR and UV spectra were
obtained using an AATI Mattson Genesis Series FTIR and a Perkin-

944 Journal of Natural Products, 2008, Vol. 71, No. 6
Table 2. NMR Spectroscopic Data for Mollamide C (2)a,b
Donia et al.
amino acid
glycine
position
δC, mult.
δH (J in Hz)
HMBCc
1, 3
1
2
NH
3
4
5
6
7
8
169.0, qC
43.2, CH2
3.5, d (3.6), 4.6, d (10.2)
6.7, d (9.6)
proline
170.8, qC
63.4, CH
28.9, CH2
26.1, CH2
47.4, CH2
161.8, qC
57.0, CH
36.9, CH
23.1, CH2
11.9, CH3
16.7, CH3
4.0, dd (6.0,10.2)
2.1, dt (6.6), 2.0, m
1.8, m
5
3, 4, 6, 7
3.6 (9.0, 11.4), 3.7 (9.6)
4, 5
isoleucine
9
4.5 (3.0, 10.2)
1.9, m
1.3, m
0.8, t (7.2)
1.0, d (7.2)
8.9, d (8.4)
8, 10, 11, 13
11
10
11
12
13
NH
14
15
16
17
18
19
20
21
22
NH
23
24
25
NH
26
27
28
29
30
10, 11
9, 10, 11
14
thiazole
leucine
171.6, qC
149.2, qC
122.4, CH
170.7, qC
50.6, CH
44.5, CH2
26.4, CH
21.4, CH3
23.6, CH3
7.9, s
14, 15, 17
5.2, ddd, (4.2, 8.4, 15.0)
1.8, dd (4.8, 9.0)
1.9, m
19, 23
17, 18, 21
19, 21, 22
19, 20, 22
19, 20
1.0, d
1.0, d (6.6)
8.1, d (7.8)
18, 23
serine
171.7, qC
51.1, CH
62.9, CH2
4.9, ddd (3.6, 7.2, 9.3)
3.4, 3.5 dd, (4.2, 9.0)
8.5, d (9.6)
1, 23
23, 26
1
isoprene
77.4
25.6
25.3
142.4
115.2
1.2, s
1.3, s
28, 26, 29
26, 27, 29
26
5.7 dd, (10.8, 17.4)
5.07 d, (10.8), 5.1 (17.4)
26, 29
a
1H and 13C NMR were recorded at 600 and 150 MHz, respectively. b All spectra were measured in CDCl3 and referenced to the residual solvent
signal at δH 7.26 and δC 77.4. c HMBC correlations are from the proton(s) stated to the indicated carbons.
Elmer Lambda 3B UV/vis spectrometer. The 1H and 13C NMR spectra
were recorded in CDCl3 using Bruker spectrometers operating at 400
or 600 MHz for 1H and 100 or 150 MHz for 13C NMR. Chemical shifts
are reported in parts per million (ppm), and coupling constants (J) in
Hz. The ESI-FTMS was acquired on a Bruker-Magnex BioAPEX 30es
ion cyclotron Fourier transform mass spectrometer by direct injection
into an electrospray interface. Silica gel (200-400 mesh) and alumina
(63-200 µm) were obtained from Natland International Corporation
and Scientific Adsorbents Incorporation, respectively. TLC analyses
were carried out using precoated silica gel G254 or aluminum oxide
Alox-100 UV254 500 µM. HPLC were performed on a Waters 510
model system and Agilent 1100-Bruker microTOF system.
Animal Collection and Taxonomy. The tunicate was collected at
10-20 m depth from Manado Bay, Indonesia, on September 28, 2003,
where it was extraordinarily abundant. The tunicate is massively
encrusting with a tough and elastic texture. In life, the external color
of the tunicate is grayish green. The animal was easily identified as
Didemnum molle (Herdman, 1886), the most commonly encountered
sea squirt in much of the Indo-Pacific.
Extraction and Isolation. Six kilograms of D. molle was collected
in Manado Bay, Indonesia, in 2003. The 450 g dry wt of tunicate was
extracted with H2O. The H2O extract was exhaustively partitioned
between H2O and CH2Cl2. The animal residue was extracted with EtOH.
Both the EtOH and CH2Cl2 extracts were combined on the basis of
TLC, yielding 19 g of dried extract. The extract was subjected to Si
gel VLC using a step gradient elution system of hexanes/EtOAc/MeOH
in 25% increments to yield 10 fractions. Fractions 6 and 7 eluted with
EtOAc/MeOH (9:1) and EtOAc/MeOH (7.5:2.5), respectively, were
enriched with peptides based on 1H NMR analysis. Both fractions were
purified separately with reversed-phase HPLC (Phenomenex Prodigy
10 µm, ODS 250 × 50 mm; λ 230 nm) eluting with a gradient system
from 20% CH3CN in H2O to 100% CH3CN. Mollamide B (1) (40 mg,
8.8 × 10-3 % dry wt) was eluted with 2% H2O in CH3CN. A mixture
of keenamide A and a minor peptide was eluted with 14% H2O in
CH3CN. The mixture was further purified using a NH2 HPLC column
(Phenomenex Luna 5 µm, 250 × 21.2 mm) and an isocratic elution
system of 20% CH2Cl2/hexanes, yielding mollamide C (2) (2.0 mg,
4.4 × 10-4 % dry wt) and keenamide A (3) (40 mg, 8.8 × 10-3 % dry
wt).
Mollamide B (1): yellow solid; [R]25D -119.9 (c 0.25, CHCl3); UV
(MeOH) λmax (log ꢀ) 202 (4.53) nm; IR (KBr film) νmax 3356, 3256,
1
2966, 2931, 1683, 1650, 1530, 1447 cm-1; H NMR and 13C NMR,
see Table 1; LRESIMS m/z 697 [M + H]+; HRESIMS m/z [M + H]+
697.3713 (calcd for C36H53N6O6S, 697.3747, ∆ -3.4 mmu).
Mollamide C (2): white solid; [R]25D 0 (c 0.1, CHCl3); UV (MeOH)
λmax (log ꢀ) 200 (5.15); IR (KBr film) νmax 3318, 2959, 2926, 2873,
1
1729, 1685, 1628, 1541, 1456, 1271, 1140, 1070 cm-1; H NMR and
13C NMR, see Table 2; HRESIMS m/z [M + H]+ 619.3259 (calcd for
C30H46N6O6S 619.3277, ∆ 1.8 mmu).
Determination of Absolute Configuration of the Amino Acids in
Mollamide B (1). Hydrolysis of 1 (348 µg; 0.5 µmol) was achieved in
1 mL of 6 N HCl in a sealed vial at 100 °C for 20 h. Traces of HCl
were removed under nitrogen. The resulting hydrolysate was resus-
pended in 100 µL of H2O and derivatized with 20 µL of 10% NR-
(2,4-dinitro-5-fluorophenyl)-L-alaninamide (FDAA) in acetone and 10
µL of 1 N NaHCO3. The mixture was heated at 40 °C for 1 h.15 The
solutions were cooled to room temperature, neutralized with 5 µL of 2
N HCl, and evaporated to dryness.13 HPLC analyses of the FDAA-
derivatized hydrolysate of 1 and 2 and standard FDAA-derivatized
amino acids were carried out using a Waters NOVAPAK C18 (3.9 ×
150 mm column) with a linear gradient of triethylammonium phosphate
(50 mM, pH 3.0)/MeCN (90:10 to 60:40) in 60 min at 1 mL/min (UV
detection at λ 340 nm). Because L-threonine and L-allo-threonine could
not be resolved clearly using this method, they were separated using a
Luna C5 column (4.6 × 150 mm, 5 µm) with a gradient of 40 mM
ammonium acetate, 70% CH3CN, and 30% MeOH from 98:2 to 66:34
in 85 min at 0.8 mL/min, detected by microTOF, and then analyzed
for the extracted ion at m/z 372 for the Marfey’s derivative of threonine.
Ozonolysis of Mollamide C (2). A slow stream of O3 was bubbled
into a 7 mL CH2Cl2 solution of mollamide C (0.3 mg; 0.5 µmol) in a
threaded bomb at 25 °C for approximately 8 min. After removal of the

Cyclic Hexapeptides from Didemnum molle
Journal of Natural Products, 2008, Vol. 71, No. 6 945
solvent under a stream of N2, the residue was subjected to hydrolysis
and derivatization as described above with mollamide B. The D/L-
configuration for serine, proline, and leucine were assigned using the
two important NMR-derived distance restraints in case of MolB-S
(Figure 2). The distances between the R-proton of valine-1 and valine-2
were 5.95 and 6.86 Å, while those between the R-proton of valine-2
and the side chain methyl of threonine were 5.16 and 5.45 Å for MolB-R
and MolB-S, respectively. The former restraint violation appears to be
critical since the two atoms involved in the restraint flank the thiazoline
ring, and thus their positions (coordinates) are expected to be affected
by the chirality at the R-carbon of the ring. In other words, the optimized
conformation of MolB-R appeared to match the experimental distances
derived from the NMR data, while MolB-S showed two violations and
was also energetically less stable than the former. On the basis of these
observations, the chirality at the R-carbon of the thiazoline ring was
assigned to be R, corresponding to the L-amino acid configuration for
the thiazoline moiety.
C18 column, and L-isoleucine and L-allo-isoleucine were assigned using
the C5 column as described above.Then the ion at m/z 384 was extracted
for the four isoleucine isomers.
The analysis of mollamide B (1) using the C18 column established
the presence of L-Pro (34.47) [D-Pro (40.52)], L-Val (43.47), [D-Val
(54.10)], and L-Phe (52.41) [D-Phe (59.86)]. Amino acid standards using
LCTOF revealed retention times as follows: L-Thr (36.42 min) [L-allo-
Thr (34.94), D-Thr (45.46), D-allo-Thr (41.37)].
The analysis of mollamide C (2) using the C18 column established
the presence of L-Ser (21.56 min) [D-Ser (26.13)], L-Pro (34.07) [D-Pro
(40.25)], and L-Leu (51.82) [D-Leu (60.85)]. Amino acid standards using
LCTOF revealed retention times as follows: L-Ile (52.63) [L-allo-Ile
(52.93), D-Ile (70.42), D-allo-Ile (71.37)]. Due to the close retention
time of L-Ile and L-allo-Ile, mollamide C was co-injected with L-Ile
and L-allo-Ile standards, which clearly revealed that Ile has the
L-configuration. Thus, the analysis of mollamides B (1) and C (2)
established the presence of all L-amino acids.
Molecular Modeling. In order to investigate the chirality at the
R-carbon of the thiazoline moiety, two structures of molecule 1 were
drawn using the BUILDER module in Insight II (Accelrys Inc., San
Diego, CA). All regular amino acids were drawn as S, while the
R-carbon of the thiazoline moiety was drawn with both S and R
configurations to provide two initial configurations of molecule 1:
MolB-R and MolB-S (denoting the chirality at the R-carbon of the
thiazoline moiety to be L-Tzn and D-Tzn, respectively).
The structures were first optimized using a series of concurrent
minimizations employing the CVFF forcefield to a gradient of 0.001.
The optimized structures were then subjected to simulated annealing
using the DISCOVER (v 2.98) module of Insight II. All the simulations
were performed using a distance-dependent dielectric. A forcing
potential of 30 kcal/mol/rad2 was applied to the omega dihedrals for
all amino acids, except for the cis-proline, to maintain the trans
geometry. Also, NMR-derived distance restraints based on ROESY
correlations (Figure 1) were introduced during the MD simulations with
a force constant of 20 kcal/mol/rad2. The molecules were minimized
beginning with 300 steps of steepest descents and 1000 steps of
conjugate gradients to relieve any strain in the starting structures. The
structures were then “heated” gradually from 100 to 1000 K in steps
of 100 K with 1500 fs of simulation at each step. At 1000 K, the
simulation was further continued for 20 000 fs, during which 20
conformations were sampled at an interval of 1000 fs. The conforma-
tions were then gradually “cooled” to 300 K in steps of 100 K with
2500 fs simulation at each step. The structures were finally minimized
using 500 steps of steepest descents, 1500 steps of conjugate gradients,
and 500 steps of VA09A. At this stage, the NMR-derived distance
restraints were removed, and all 20 conformations were again subjected
to the same minimization protocol as given above.
The structures were analyzed, and the lowest energy conformation
of MolB-R obtained without any restraints appeared to maintain
important interproton distances in accordance with the NMR data. The
structures were then further optimized by semiempirical quantum
mechanical calculations. The AM1 Hamiltonian was used to calculate
the heat of formation (∆Hf) using the MOPAC suite of programs (v
6.0). The semiempirical calculations were carried out without any
restraints, and all degrees of freedom in the molecule were optimized.
Minimization was run with the EF algorithm to a GNORM of 0.01.
Since it is well known that most semiempirical methods underestimate
the barrier to rotation in peptides, the key word MMOK was used to
allow for a molecular mechanics correction. Stable points on the
potential energy surface (PES) of the molecule were identified by
running a FORCE calculation and were confirmed by the absence of
any imaginary (negative) frequencies.
Acknowledgment. We appreciate the assistance of F. T. Wiggers
and A. G. Shilabin in acquiring NMR and LC-TOF data. We are grateful
to the National Cancer Institute (NCI) and F. Valeriote at Wayne State
for performing cytotoxicity assays; The National Center for Natural
Products Research for performing the antimicrobial and antimalarial
assays; R. F. Schinazi’s group at Emory for HIV-1; S. G. Franzblau’s
group at UIC for Mtb; and A. M. S. Mayer for anti-inflammatory assays.
Financial support for this work was provided by grant numbers
1R01A136596 and P20 RR021929 from the NIAID and NCRR
components of the National Institutes of Health (NIH), and the contents
are solely the responsibility of the authors and do not necessarily
represent the official view of the NIH. The Egyptian Government and
the CDC are also very gratefully acknowledged for financial support.
This investigation was conducted in a facility constructed with support
from Research Facilities Improvements Program (C06 RR-14503-01)
from the National Center for Research Resources, NIH.
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