Mandelalides A-D, cytotoxic macrolides from a new Lissoclinum species of South African tunicate

Article abstract of DOI:10.1021/jo3008622

Mandelalides A-D are variously glycosylated, unusual polyketide macrolides isolated from a new species of Lissoclinum ascidian collected from South Africa, Algoa Bay near Port Elizabeth and the surrounding Nelson Mandela Metropole. Their planar structures were elucidated on submilligram samples by comprehensive analysis of 1D and 2D NMR data, supported by mass spectrometry. The assignment of relative configuration was accomplished by consideration of homonuclear and heteronuclear coupling constants in tandem with ROESY data. The absolute configuration was assigned for mandelalide A after chiral GC-MS analysis of the hydrolyzed monosaccharide (2-O-methyl-α-l-rhamnose) and consideration of ROESY correlations between the monosaccharide and aglycone in the intact natural product. The resultant absolute configuration of the mandelalide A macrolide was extrapolated to propose the absolute configurations of mandelalides B-D. Remarkably, mandelalide B contained the C-4′ epimeric 2-O-methyl-6- dehydro-α-l-talose. Mandelalides A and B showed potent cytotoxicity to human NCI-H460 lung cancer cells (IC₅₀, 12 and 44 nM, respectively) and mouse Neuro-2A neuroblastoma cells (IC₅₀, 29 and 84 nM, respectively).

Full text of DOI:10.1021/jo3008622

Article
pubs.acs.org/joc
Mandelalides A−D, Cytotoxic Macrolides from a New Lissoclinum
Species of South African Tunicate
Justyna Sikorska,† Andrew M. Hau,† Clemens Anklin,‡ Shirley Parker-Nance,§
Michael T. Davies-Coleman,∥ Jane E. Ishmael,† and Kerry L. McPhail*,†
†Department of Pharmaceutical Sciences, College of Pharmacy, 203 Pharmacy Building, Oregon State University, Corvallis, Oregon
97331, United States
‡Bruker BioSpin, 15 Fortune Drive, Billerica, Massachusetts 01821, United States
§Department of Zoology, Nelson Mandela Metropolitan University, PO Box 77000, Port Elizabeth 6031, South Africa
∥Department of Chemistry, Rhodes University, PO Box 94, Grahamstown 6140, South Africa
S
* Supporting Information
ABSTRACT: Mandelalides A−D are variously glycosylated, unusual
polyketide macrolides isolated from a new species of Lissoclinum
ascidian collected from South Africa, Algoa Bay near Port Elizabeth and
the surrounding Nelson Mandela Metropole. Their planar structures
were elucidated on submilligram samples by comprehensive analysis of
1D and 2D NMR data, supported by mass spectrometry. The
assignment of relative configuration was accomplished by consideration
of homonuclear and heteronuclear coupling constants in tandem with
ROESY data. The absolute configuration was assigned for mandelalide
A after chiral GC-MS analysis of the hydrolyzed monosaccharide (2-O-
methyl-α-L-rhamnose) and consideration of ROESY correlations
between the monosaccharide and aglycone in the intact natural
product. The resultant absolute configuration of the mandelalide A
macrolide was extrapolated to propose the absolute configurations of mandelalides B−D. Remarkably, mandelalide B contained
the C-4′ epimeric 2-O-methyl-6-dehydro-α-L-talose. Mandelalides A and B showed potent cytotoxicity to human NCI-H460 lung
cancer cells (IC50, 12 and 44 nM, respectively) and mouse Neuro-2A neuroblastoma cells (IC50, 29 and 84 nM, respectively).
from the ascidian Aplidium albicans and is biosynthetically
similar to peptides from the marine cyanobacterium Lyngbya
majuscula8 (now Moorea producta).
INTRODUCTION
■
Over the last 50 years, ascidians have been shown to be a
prolific source of natural products with promising biomedical
potential.1 Indeed, ascidian-derived natural products have
yielded promising drug leads, among which ecteinascidin 743
(Yondelis) and dehydrodidemnin B (Aplidin) are in clinical use
for the treatment of specific cancers.2 The diverse chemotypes
reported from Lissoclinum species collected globally have
important biological properties that range from cancer cell
toxicity to antifungal and antibacterial activities,1 and include
peptides, alkaloids, chlorinated diterpenes, polyether amides,
lactones, and macrolides such as haterumalide B3 and
patellazoles B and C.4,5 It has been noted that many secondary
metabolites isolated from Lissoclinum, as well as other ascidians
species, are present in bacteria, sponges, and mollusks.6−8 This
diversity of ascidian metabolites can be explained by the fact
that tunicates are often hosts to cyanobacterial and
heterotrophic bacterial symbionts, as well as being fed upon
by predatory mollusks.7−10 Therefore, identification of the
biogenetic origin of ascidian natural products is very often
challenging.11 This is highlighted by the recent isolation of
didemnin B (an analog of Aplidin) from the α-proteobacterium
Tistrella mobiliz.8,12 This compound was originally identified
In our search for new potential anticancer compounds, we
encountered the highly cytotoxic organic extract (IC50 = 0.7
μg/mL in NCI-H460 lung cancer cells) of a new Lissoclinum
species from Algoa Bay, South Africa. Bioassay-guided
fractionation of this extract, yielded a series of new macrolides,
named mandelalides A−D (1−4), two of which were tested
and show good cytotoxicity to mouse Neuro-2A neuroblastoma
and human NCI-H460 lung cancer cells. While the relative
configuration of the compounds could be assigned from
analysis of ROESY data in tandem with homonuclear (3JHH
)
and heteronuclear (2,3JCH) coupling constants, the assignment
of absolute configuration relied on hydrolysis of the
glycosylated mandelalide A and chiral GC-MS analysis of the
released monosaccharide. Remarkably, mandelalide A contains
2-O-methyl-α-L-rhamnose, while mandelalide B contains the C-
4′ epimer 2-O-methyl-6-dehydro-α-L-talose.
Received: April 29, 2012
Published: June 19, 2012
© 2012 American Chemical Society
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(δ 3.40) and deshielded methyl C-7′ (δ 59.1) indicated
methylation of the C-2′ hydroxyl group. An HMBC correlation
from H-7 (δ 3.82) to C-1′ placed the monosaccharide
(fragment B) at C-7 of fragment A.
RESULTS AND DISCUSSION
■
A new Lissoclinum ascidian species was collected from White
Sands Reef in Algoa Bay, South Africa. The organic extract
(1.45 g) was subjected to bioassay-guided fractionation through
consecutive LH-20 and RP-HPLC chromatography to yield
mandelalides A−D (1−4), of which mandelalide D degraded to
deacylmandelalide D (4b).
The second compound characterized, mandelalide B (2), was
assigned a molecular formula of C37H58O13, based on HR-ESI-
MS data for [M + Na]+ m/z 733.3773, which is consistent with
1
9 degrees of unsaturation. Inspection of the H and 13C NMR
spectra for mandelalide B (2) suggested that it was structurally
related to mandelalide A (1). However, the 1H NMR spectrum
for 2 lacked the coupled olefinic signals at δ 6.01 (H-2) and
6.97 (H-3) and the relatively deshielded diastereotopic H2-24
signals at δ 3.81 and 3.61. Instead an additional oxymethine
double doublet at δ 5.48 was evident, as well as a methyl triplet
(δH 0.95) indicative of an aliphatic chain. Correspondingly,
comparison of the 13C NMR spectra for 1 and 2 revealed the
absence of olefinic 13C resonances at δ 123.1 (C-2) and 147.1
(C-3) from the spectrum for 2, and the presence of two
additional midfield 13C resonances (δ 79.3 and 69.5), as well as
a second carbonyl resonance (δC 173.6). These data accounted
for 8 of the 9 degrees of unsaturation, implying the presence of
an additional cycle in 2. The analysis of HSQC and HMBC
data for 2 permitted assignment of an oxygenated quaternary
C-2 (δ 79.3) and an oxymethine CH-3 (δ 69.5, δ 5.48). While
the same C-1/C-23 macrolactone linkage was apparent in both
compounds, two-and three-bond HMBC correlations from H2-
24 (δ 1.93, 2.39) to C-2 and C-3, respectively, indicated a C-2/
C-24 bond in 2. These data described a γ-butyrolactone ring
around the ester linkage of the mandelalide B macrocycle.
Placement of the second carbonyl carbon (δC 173.6) in a
butyrate substituent at C-3 was facilitated by HMBC
correlations to δC 173.6 from δH 5.48 (H-3), as well as 2.35
(H2-2″) and 1.67 (H2-3″). Further analysis of NMR data
confirmed the presence of a 2-O-methyl-6-dehydro sugar in 2.
The HR-ESI-MS data for mandelalide A (1) gave a
pseudomolecular ion [M + Na]+ at m/z 647.3394, which is
consistent with a molecular formula of C33H52O11, and implies
8 degrees of unsaturation. The 13C and multiplicity-edited
HSQC NMR spectra for compound 1 (Table 1) indicated the
presence of an ester carbonyl (δC 167.4), fourteen sp3
methines, twelve of which were oxygen-bearing (δC 94.2−
68.1, 37.3 and 34.2), six olefinic methines (δC 147.1, 141.5,
131.3, 126.9, 123.9 and 123.1), eight methylenes (δC 66.1, 43.1,
39.7, 38.8, 37.6, 36.8, 34.1 and 31.1) and four methyl groups
(δC 59.1, 18.3, 17.7 and 14.5). Interpretation of COSY and
TOCSY correlations delineated two spin systems, one
comprising 25 carbons from C-2 to C-26 (Figure 1a, fragment
A), and the other of 6 carbons from C-1′ to C-6′ (Figure 1a,
fragment B). The acquisition of a semiphase sensitive HMBC13
optimized for a 4 Hz coupling constant (Figure S6, Supporting
Information), revealed a correlation between δH 5.23 (H-23)
and δC 167.4 (C-1) that indicated cyclization of fragment A
into a 23-carbon macrolactone. The C-1 carbonyl also showed
HMBC correlations from the olefinic H-2 and H-3 multiplets,
1
However, differences in both H and 13C shifts for CH-3′ to
CH-6′ compared to compound 1 suggested a different relative
configuration for the monosaccharide in 2 (Table 2).
The HR-ESI-MS data ([M + Na]+ m/z 589.2970) for
mandelalide C (3) supported a molecular formula of
C30H46O10, for 8 degrees of unsaturation. The 1H NMR
spectrum for 3 was reminiscent of that for 2. However, a careful
comparison of the two spectra revealed the absence of midfield
glycosidic signals in the 1H spectrum for 3. Similarly, inspection
of the 13C NMR spectrum for 3 showed a lack of midfield
resonances for a methoxy methyl (δ 59.6, C-7′ in 2), anomeric
carbon (δ 94.8, C-1′ in 2), and other glycosidic carbons.
Together, the MS and 1D NMR data for compound 3 indicated
that the structure of 3 is related to the aglycone of mandelalide
B (2, Table 2). Indeed, the only difference between the 13C
NMR signals for the aglycones of compounds 2 and 3 was a
relatively downfield methine resonance at δ 72.2 in the
spectrum for 3, compared to the methylene signal at δ 37.9
(C-24) in the spectrum for 2. This methine was HSQC-
correlated to a 1H doublet at δH 3.98, and could be assigned to
an oxymethine CH-24 on the basis of HMBC correlations from
the latter 1H doublet to C-1, C-2, C-3 and C-23. Thus,
considering its MS and NMR data, mandelalide C (3) could be
assigned as the C-24 hydroxylated aglycone of mandelalide B
(2). Unfortunately, an attempt to crystallize mandelalide C (0.5
mg) was unsuccessful and degradation of the compound over
several months prevented the acquisition of additional
spectroscopic or biological data.
3
for which a JHH of 15.5 Hz indicated an E double bond
geometry. An HMBC correlation between H-9 (δ 3.32) and C-
5 (δ 73.9) delineated a tetrahydropyran ring, while a correlation
between H-17 (δ 3.98) and C-20 (δ 83.2) was consistent with a
tetrahydrofuran ring, both within fragment A (Table 1, Figure
1a). The four contiguous (TOCSY-coupled) olefinic methines
CH-12 to CH-15 (δH 5.45, 6.28, 6.05, and 5.28 ppm) were
consistent with a conjugated diene, situated β to the
tetrahydrofuran and γ to the tetrahydropyran on the basis of
HMBC correlations from H-15 to C-17 and H-12 to C-10,
respectively. From homonuclear coupling constant values (3JHH
= 14.8 and 10.8 Hz, respectively) it was evident that the
geometry of H-12 and H-13 is trans, while H-14 and H-15 are
3
cis. Knowing that JHH values are not sufficiently characteristic
to assign the orientation between H-13 and H-14, ROESY data
were examined.14 ROEs between H-12 and H-10a, as well as
between H-13 and H-16b, suggested an anti orientation for H-
13 and H-14 (Figure 1b). In the case of fragment B, the
downfield shift of C-1′ (δC 94.2), the presence of four midfield
oxymethine 13C resonances, an upfield methyl doublet (CH3-6′,
δC 17.7, δH 1.27) and an HMBC correlation between H-1′ (δ
5.02) and C-5′ (δ 68.1) suggested that fragment B is a 6-
dehydro monosaccharide. An HMBC correlation between H-2′
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Article
Table 1. NMR Data for Mandelalide A (1) in CDCl3
a
no. δC (mult)
δH (J in Hz)
COSY
HMBC
1, 4
TOCSY
3, 4a, 4b, 5, 6ax, 6eq
ROESY
3 , 4a, 4b, 5
1
167.4 (s)
b
2
123.1 (d) 6.01 (dd, 15.5, 1.2)
3, 4a
b
b
b
3
147.1 (d) 6.97 (ddd, 15.2, 10.4, 4.6)
2, 4a, 4b
2, 3
1, 2, 4, 5
2, 3, 5, 6
2, 3, 5, 6
3, 4
2, 4a, 4b, 5, 6ax, 6eq
2, 3, 5, 6ax, 6eq, 7
3 , 4a , 4b , 5
b
b
b
4a
4b
5
38.8 (t)
2.36 (m)
2, 3 , 5 , 6ax , 6eq
b
b
b
2.39 (ddd, 14.1, 10.6, 10.6)
3, 5
2, 3, 5, 6ax, 6eq, 7
2, 3 , 5 , 6ax , 6eq
2, 3, 4a, 4b, 6eq, 7
73.9 (d) 3.36 (dddd, 11.4, 11.4, 2.3,
2.3)
4a, 4b, 6ax, 6eq
2, 3, 4a, 4b, 6ax, 6eq, 7, 8ax, 8eq
b
6ax
6eq
37.6 (t)
1.20 (m)
5, 6eq, 7
4, 5, 8
7, 8
3, 4a, 4b, 5, 6eq, 7
4b , 7
b
2.02 (dddd, 12.6, 4.4, 2.3,
5, 6ax, 7, 8eq
4a, 4b, 5, 6ax, 7, 8ax,
6ax , 4a, 4b, 5, 7, 1′
c
1.6)
b
b
7
73.1 (d) 3.82 (dddd, 11.1, 10.5, 4.4,
6ax, 6eq, 8ax, 8eq 8, 9, 1′
4a, 4b, 5, 6ax, 6eq, 8ax, 8eq, 9
5, 6ax , 6eq, 8ax , 8eq, 9, 1′
c
4.4)
b
b
8ax
8eq
9
39.7 (t)
1.22 (m)
1.87 (m)
7, 8eq, 9
6, 7, 9, 10
6eq, 7, 8eq, 9, 10b
6eq , 7, 8eq
b
6eq, 7, 8ax, 9
6, 7
5, 7
6eq, 7, 8ax, 9, 10a, 10b
7, 8ax , 9, 10b, 5′
b
72.5 (d) 3.32 (dddd, 11.2, 11.2. 2.2,
8ax, 8eq, 10a,
10b
6eq, 7, 8ax, 8eq, 10a, 10b, 11, 12, 13, 25 7, 8eq, 10a, 10b , 25
c
2.2)
c
b
b
10a 43.1 (t)
10b
1.21 (ddd, 15.2, 9.6, 2.2)
9, 10b, 11
9, 10a, 11
11, 12, 25
8eq, 9, 10b, 12, 13, 14, 25
8eq, 9, 10b , 11 , 12, 25
c
b
b
1.51 (ddd, 15.2, 11.2, 3.7)
8, 9, 11, 12, 25 8ax, 8eq, 9, 10a, 11, 12, 13, 25
8ax, 8eq, 9, 10a, 10b, 11, 12, 13, 14, 15
8eq, 9 , 10a , 11, 25
c
b
b
b
11
12
13
14
15
34.2 (d) 2.37 (dqd, 9.6, 6.5, 3.7)
141.5 (d) 5.45 (dd, 14.8, 9.7)
123.9 (d) 6.28 (dd, 14.8, 11.0)
131.3 (d) 6.05 (dd, 10.9, 10.9)
126.9 (d) 5.28 (ddd, 10.8, 10.8, 5.6)
10a, 10b, 12, 25 10, 12, 25
10a , 10b, 12 , 13, 25
b
b
b
11, 13
10, 11, 14, 25 9, 10a, 10b, 11, 13, 14, 15, 16a, 16b, 25
10, 11, 14, 15 10a, 10b, 11,12, 14, 15, 16a, 16b, 17, 25
11 , 10a, 13 , 14 , 25
b
b
b
12, 14
11, 12 , 14 , 15 , 16b, 21
b
b
b
13, 15
12, 13,16, 17
13, 16, 17
14, 15, 17
10a, 11, 12, 13, 15, 16a, 16b, 17, 25
12, 13, 14, 16a, 16b, 17, 18, 26
12, 13, 14, 15, 16b, 17, 18, 19a, 26
12 , 13 , 15 , 16a
b
b
b
b
14, 16a, 16b
14, 15, 16b, 17
15, 16a, 17
16a, 16b, 18
13 , 14 , 16a, 16b , 17
b
16a 31.1 (t)
16b
1.88 (m)
2.28 (ddd, 13.1, 11.4, 11.4)
14, 15, 16b, 17 , 26
b
b
14, 15, 17, 18 12, 13, 14, 15, 16a, 17, 18, 19a, 19b, 26
15, 19, 20 14, 15,16a, 16b, 18, 19a, 19b, 20, 21, 26
13,15 ,16a,17 ,19a,21,26
b
b
17
18
81.0 (d) 3.98 (ddd, 11.1, 8.1, 1.8)
15, 16a , 16b ,18, 26
b
b
37.3 (d) 2.52 (dddq, 12.0, 7.0, 7.0,
17, 19a, 19b, 26 16, 17, 19, 26 15, 16a, 16b,17, 19a, 19b, 20,21, 22a, 26 17, 19a , 19b, 20, 26
c
7.0)
b
19a 36.8 (t)
19b
1.17 (ddd, 11.9, 11.9, 10.3)
18, 19b, 20
18, 19a, 20
19a, 19b, 21
20, 22a, 22b
18, 20, 21, 26 17, 18, 19a, 20, 21, 22a, 22b, 26
16b, 18 , 21, 22b, 26
c
2.01 (ddd, 12.2, 7.0, 5.6)
17, 18
16b, 17, 18, 19a, 20, 21, 22a, 26
17, 18, 19a, 19b, 21, 22a, 22b, 26
18, 19a, 20, 22a, 22b, 26
b
20
21
83.2 (d) 3.63 (m)
21, 22
18, 19b, 21 , 22a, 22b
b
73.0 (d) 3.42 (ddd, 11.1, 8.8, 1.8)
20, 22, 23
17, 18, 19a, 19b, 20, 22a, 22b, 23, 24a, 24b, 13, 16b, 19a, 22a , 22b, 23
26
b
b
22a 34.1 (t)
22b
1.46 (ddd, 14.1, 11.1, 1.9)
1.76 (ddd, 13.9, 11.7, 1.8)
21, 22b, 23
21, 22a, 23
20, 21
23, 24
18, 19a, 19b, 20, 21, 22b, 23, 24a, 24b, 26 19b, 21 , 22b , 23, 24a, 24b
b
b
19a, 20, 21, 22a, 23,24a, 24b
19a, 19b, 21, 22a ,23 , 24a,
24b
b
23
72.3 (d) 5.23 (dddd, 11.7, 4.9, 2.9, 1.9) 22a, 22b, 24a,
24b
1
21, 22a, 22b, 24a, 24b
21, 22a, 22b , 24a, 24b
24a 66.1 (t)
24b
3.61 (m)
23, 24b
23, 24a
11
22, 23
23, 24b
22a, 22b, 23, 24b
22a, 22b, 23, 24a
c
3.81 (dd, 12.2, 2.9)
22, 23
22b, 23, 24a
b
25
26
1′
18.3 (q) 0.85 (d, 6.6)
14.5 (q) 1.03 (d, 6.9)
10, 11, 12
17, 18, 19
7, 2′, 3′, 5′
3′, 4′, 7′
9, 10a, 10b, 11, 12, 13, 14
15, 16a, 16b, 17, 18, 19a, 19b, 20 21
2′, 6′
9, 10a, 10b, 11 , 12
b
18
16a, 16b, 18 , 19a, 19b
94.2 (d) 5.02 (d, 1.1)
80.8 (d) 3.40 (dd, 3.8, 1.4)
71.7 (d) 3.68 (m)
2′
6eq, 7, 8eq, 2′, 7′
1′
2′
1′, 3′
2′, 4′, 8′
3′, 5′
4′, 6′
1′, 3′, 4′, 5′, 6′, 8′, 9′
2′, 4′, 5′, 6′, 8′, 9′
3′, 5′, 6′, 8′, 9′
b
3′
2′, 4′
b
b
4′
74.3 (d) 3.34 (dd, 9.4, 9.4)
68.1 (d) 3.62 (m)
3′, 5′, 6′
3′, 4′, 6′
4′, 5′
3′ , 5′ , 6′
b
5′
4′, 6′, 8′, 9′
8eq, 4′ , 6′
b
6′
17.7 (q) 1.27 (d, 6.3)
59.1 (q) 3.45 (s)
1′, 3′, 4′, 5′, 8′
4′, 5′
7′
2′
1′
OH-3′
OH-4′
2.24 (s)
1.54 (s)
2, 4a, 4b
2, 3
2′, 3′, 4′, 5′, OH-4′
3′, 4′, 5′, OH-3′
a
b
c
3
HMBC correlations are presented from proton to indicated carbon. COSY artifacts observed in ROESY spectrum. JHH values obtained from
DQFCOSY and/or NMRSim.
The molecular formula of mandelalide D (4) was assigned by
HR-ESI-MS ([M + Na]+ m/z 659.3424) as C34H52O11,
implying 9 degrees of unsaturation. Comparison of the similar
1H NMR spectra (Table 2) for mandelalides C (3) and D (4)
indicated a second aglycone structure. A key difference between
the 1H spectra for the two compounds was the further
deshielded H-24 doublet (δH 5.17) for 4 relative to that for 3
(δH 3.98). In the HMBC spectrum for 4, the H-24 doublet was
correlated to a carbonyl 13C resonance (δC 173.1, C-1″), which
also showed correlations from H-2a″, H-2b″ and H-3″,
consistent with an additional butyrate substituent at C-24.
Thus, mandelalide D (4) is 24-butyro mandelalide C (3). Over
1
a period of 12 months, we observed changes in the H NMR
spectrum of 4 that were consistent with the loss of both
butyrate moieties, resulting in the new compound deacylman-
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3
provided very weak, but measurable JHC between H-10a and
C-25 (0.9 Hz), and between H-10b and C-25 (8.9 Hz).
For the tetrahydrofuran ring of 1, H-17 and H-20 could be in
a cis or trans configuration. There are two close conformational
minima (envelope and half-chair) for THF rings.15 However, in
the envelope conformation CH3-26, H-18, H-19a and H-19b in
1 would be eclipsed, while these same moieties would be gauche
in the half-chair conformation and there would be no eclipsed
interaction along the C-18/19 bond.15 Therefore we decided to
analyze ROESY correlations in the context of a half-chair THF
ring to assign the relationship of H-17 and H-20. The relatively
3
large JHH couplings between vicinal proton pairs H-16b/H-17
(11.4 Hz) and H-20/H-21 (8.8 Hz) supported their anti
orientation in each case (Figure 3a). Moreover, ROESY
correlations between H-16b and H-19a, H-16b and H-21, and
H-19a and H-21 were consistent with a cis localization of H-17
and H-20 (Figure 3b), despite that no direct ROESY
correlation was observed between H-17 and H-20. Instead,
the proposed conformation, consistent with ROESY correla-
tions between H3-26/H2-16 and H-16b/H-21, brings ROE-
correlated H-18 and H-20 into close proximity (Figure 3b). For
Figure 1. (a) Planar structure of 1 showing TOCSY correlations from
H-2, H-6, H-8, H-10b, H-13, H-17, H-21, H-1′, H-2′ (black circles)
indicated as bolded lines and key HMBC correlations represented by
single-headed arrows. (b) Assignment of geometry around the C-13/
C-14 bond in 1 using key ROESY correlations indicated by double-
headed arrows.
delalide D (4b, m/z 519.3 [M + Na]+, Table S6, Supporting
Information).
3
2
the C-21 chiral center, the large values of JHH and JHC (8.8
and −4.0 Hz, respectively), in parallel with the lack of NOE
contact between H-20 and H-21 (resolved in py-d5), are
consistent with their anti orientation (Figure 3a), as stated
Consideration of the differences in the planar macrocyclic
structures, and available amounts, of mandelalides A−D (1−4)
lead to the conclusion that the assignment of relative
configuration should be performed on mandelalide A and one
3
of mandelalides B−D. Similarities in coupling constant (3JHH
)
above. Analysis of JHH between H-21/H-22a (11.1 Hz), and
H-21/H-22b (1.8 Hz), placed H-22a anti and H-22b gauche
with respect to H-21 (Figure 3c). ROESY correlations between
H-19a and H-22b, H-19b and H-22a, and H-19b and H-22b
suggested that H2-22 are directed away from the center of
macrocycle, with the C-21 hydroxyl pointing toward the center.
Analogous reasoning was used to resolve the relative
values for mandelalides B−D suggested retention of the relative
configuration of the macrocycle between these structures
(Table 2). Therefore besides mandelalide A, mandelalide B
(2) was selected for further analysis because it also possesses a
monosaccharide moiety, although it lacks the C-24 chiral center
present in 3 and 4. For both mandelalides A (1) and B (2), the
relative configuration of the macrocycle could be assigned fully
using a combination of ROESY and J-based configuration
analysis of data acquired in CDCl3 and pyridine-d5, which were
consistent between the two solvents.
3
configuration at C-23. The respective large and small JHH
values between H-22b/H-23 (11.7 Hz) and H-22a/H-23 (1.9
Hz) suggested anti and gauche orientations, respectively (Figure
3d).
Configurational assignment of the mandelalide A mono-
Considering the tetrahydropyran ring of 1, ROESY
correlations between H-5, H-7 and H-9 supported their axial
orientation, consistent with a chair conformation and equatorial
C-7 glycosidic bond (Figure 2a). The relative configuration for
fragment C-9 to C-11 (−CHCH2CH−) was assigned on the
basis of coupling constants obtained from the 1H spectrum and
a DQFCOSY. In CDCl3, 3JHH values of 2.2 and 11.2 Hz for H-
9/H-10a and H-9/H-10b, respectively, localized H-10a gauche
and H-10b anti to H-9 in two possible rotamers (Figure 2b),
the correct one of which should be distinguishable from
ROESY data. 1H signal overlap in the CDCl3 ROESY spectrum
for mandelalide A (1) obscured ROE interactions in the region
of interest. However, acquisition of NMR data for 1 in py-d5
provided sufficient resolution (0.16 ppm, Figure S20,
Supporting Information) between H-8ax and H-10a to reveal
ROESY correlations between H-8ax and H-10b, and H-8eq and
H-10a. This lead to the conclusion that rotamer A is correct,
orienting H-10a to the outside of the macrocycle, (Figure 2).
The assignment of relative configuration around the C-10/C-11
3
saccharide (C-1′ to C-6′) relied on JHH values, obtained from
the 1H NMR and assigned by DQFCOSY, given the overlap for
H-3′ to H-5′ signals in both CDCl3 and py-d5 ROESY spectra.
Although the small value of 3JH‑1′/H‑2′ = 1.1 Hz was inconclusive,
3
H-2′ was assigned as equatorial based on JH‑2′/H‑3′ = 3.8 Hz.
The H-4′ multiplet indicated couplings of 9.4 Hz with both H-
3′ and H-5′, indicative of axial hydrogens, although the direct
analysis of H-3′ and H-5′ multiplets was impeded by their
1
overlap with other H shifts. Overall, these data for CH-2′ to
CH-5′ were consistent with the relative configuration of 2-O-
methylrhamnose. To confirm this and to assign the
monosaccharide as α- or β-2-O-methylrhamnose, we decided
to analyze the magnetization transfer pattern of TOCSY signals
originating from the anomeric center (C-1′), following the
method proposed by Gheysen et al.16 This “TOCSY matching”
3
approach takes advantage of the fact that the size of the JHH
scalar couplings affect the rate of magnetization transfer
through a 1H spin system during the TOCSY spin-lock period.
The acquisition of a TOCSY with a spin-lock of 100 ms (Figure
S9, Supporting Information) affords a differential presence or
absence of the CH3-6′ signal in α- and β-rhamnose, respectively.
In the TOCSY acquired for mandelalide A (1), the intensity of
peaks fit to previously reported data for α-rhamnose (Figure
S9).16 Additional confirmation that the monosaccharide
attached to 1 is 2-O-methyl-α-rhamnose was provided by the
measurement of 1JHC = 167.2 Hz for the anomeric CH-1′ from
3
bond required direct measurement of JHH values from
3
DQFCOSY, again due to significant overlap. In CDCl3, JHH
values of 9.6 Hz for H-10a/H-11 and 3.7 Hz for H-10b/H-11
indicated anti and gauche orientations, respectively (Figure 2c).
Finally, ROESY correlations between H-9 and H3-25, and H-11
and H-13 were consistent with the relative configuration shown
in Figure 2a. This assignment was supported by HETLOC data
in CDCl3 (Figures S11, Supporting Information), which
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1
Table 2. H and 13C NMR Data for Mandelalides B−D (2−4) in CDCl3
Mandelalide B (2)
δH (J in Hz)
Mandelalide C (3)
δH (J in Hz)
Mandelalide D (4)
δH (J in Hz)
no.
δC (mult)
δC (mult)
δC (mult)
1
175.6 (s)
79.3 (s)
69.5 (d)
36.3 (t)
174.7 (s)
82.0 (s)
68.3 (d)
36.4 (t)
172.9 (s)
81.3 (s)
68.6 (d)
36.2 (t)
2
3
5.48 (dd, 6.0, 1.7)
1.59 (m)
5.51 (dd, 6.6, 0.9)
5.56 (dd, 6.6, 0.7)
1.68 (m)
4a
1.65 (m)
4b
5
2.14 (ddd, 15.0, 11.7, 1.5)
3.30 (dddd, 11.4, 11.4, 1.5, 1.5)
1.10 (ddd, 11.7, 11.7, 11.7)
1.89 (m)
2.14 (ddd, 15.6, 11.7, 1.0)
3.25 (dddd, 11.4, 11.4, 1.8, 1.8)
1.11 (ddd, 12.5, 11.4, 11.4)
1.86 (m)
2.18 (ddd, 15.4, 11.5, 0.9)
3.21 (dddd, 11.2, 11.2, 1.3, 1.3)
1.12 (ddd, 11.3, 11.3, 11.3)
1.85 (dddd, 12.2, 4.6, 1.5, 1.5)
3.75 (m)
72.7 (d)
37.4 (t)
72.3 (d)
41.2 (t)
72.2 (d)
41.2 (t)
6ax
6eq
7
73.3 (d)
39.6 (t)
3.75 (m)
68.3 (d)
41.8 (t)
3.76 (dddd, 10.9, 10.9, 4.6, 4.1)
1.13 (ddd, 12.5, 11.2, 11.2)
1.83 (m)
68.4 (d)
41.8 (t)
8ax
8eq
9
1.23 (m)
1.12 (ddd, 11.3, 11.3, 11.3)
1.83 (dddd, 12.2, 4.6, 1.5, 1.5)
3.36 (dddd, 11.0, 11.0, 2.2, 2.2)
1.19 (ddd, 13.9, 11.5, 2.8)
1.57 (ddd, 14.0, 11.5, 4.0)
2.48 (m)
1.81 (dddd, 12.4, 4.8, 1.8, 1.8)
3.39 (dddd, 11.0, 11.0, 2.2, 2.2)
1.18 (ddd, 14.1, 12.3, 2.8)
1.57 (m)
72.4 (d)
42.2 (t)
72.3 (d)
42.1 (t)
3.37 (dddd, 10.9, 10.9, 2.1, 2.1)
1.19 (ddd, 14.2, 12.1, 2.8)
1.57 (m)
72.2 (d)
42.1 (t)
10a
10b
11
12
13
14
15
16a
16b
17
18
19a
19b
20
21
22a
22b
23
24a
24b
25
26
33.9 (d)
142.1 (d)
123.3 (d)
131.0 (d)
127.1 (d)
30.7 (t)
2.50 (m)
34.0 (t)
2.49 (m)
34.2 (d)
142.3 (d)
123.1 (d)
131.1 (d)
127.1 (d)
30.6 (t)
5.50 (dd, 15.1, 9.8)
6.40 (dd, 14.7, 11.3)
6.10 (dd, 11.1, 11.1)
5.28 (ddd, 11.1, 11.1, 5.2)
1.90 (m)
142.2 (d)
123.2 (d)
131.1 (d)
127.1 (d)
30.7 (t)
5.50 (dd, 14.6, 9.6)
6.39 (dd, 14.6, 11.4)
6.10 (dd, 11.0, 11.0)
5.28 (ddd, 11.0, 11.0, 5.4)
1.90 (ddd, 13.6, 5.2, 1.1)
2.30 (ddd, 13.8, 11.9, 11.9)
3.95 (ddd, 11.5, 7.5, 1.1)
2.53 (m)
5.50 (dd, 14.8, 9.7)
6.38 (dd, 15.0, 11.3)
6.10 (dd, 11.0, 11.0)
5.28 (ddd, 11.3, 11.3, 5.4)
1.90 (ddd, 13.6, 5.3, 1.0)
2.29 (ddd, 13.1, 11.3, 11.3)
3.94 (ddd, 12.0, 7.5, 1.1)
2.54 (dddq, 12.3, 7.5, 7.02, 6.8)
1.34 (ddd, 12.3, 12.3, 9.2)
2.08 (ddd, 12.2, 7.02, 7.02)
3.79 (ddd, 9.3, 9.3, 7.0)
3.73 (ddd, 10.0, 10.0, 1.6)
1.47 (ddd, 14.2, 10.5, 1.0)
1.65 (m)
2.31 (ddd, 13.6, 11.6, 11.5,)
3.95 (ddd, 11.6, 7.46, 1.6)
2.52 (m)
81.5 (d)
38.3 (d)
35.6 (t)
81.6 (d)
38.3 (d)
35.7 (t)
81.6 (d)
38.3 (d)
35.7 (t)
1.33 (ddd, 12.2, 12.2, 8.2)
2.05 (ddd, 12.8, 6.9, 6.9)
3.77 (m)
1.33 (ddd, 12.2, 12.2, 9.1)
2.10 (ddd, 12.5, 7.0, 6.8)
3.82 (ddd, 9.2, 9.2, 7.0)
3.73 (ddd, 10.8, 9.5, 1.2)
1.59 (m)
82.1 (t)
74.5 (d)
37.9 (t)
82.4 (d)
74.4 (d)
32.1 (t)
82.2 (d)
74.3 (d)
32.5 (t)
3.76 (m)
1.51 (m)
1.64 (m)
1.82 (ddd, 14.4, 11.0, 1.0)
5.01 (ddd, 11.2, 2.0, 2.0)
3.98 (d, 2.8)
74.9 (d)
39.7 (t)
4.84 (dddd, 10.4, 10.4, 4.9, 1.0)
1.93 (dd, 12.7, 10.7)
2.39 (dd, 12.8, 4.9)
1.07 (d, 6.6)
78.9 (d)
72.2 (d)
76.7 (d)
74.0 (d)
5.13 (ddd, 10.8, 3.3, 0.8)
5.17 (d, 3.3)
18.5 (q)
14.2 (q)
OH-21
OH-2
18.4 (q)
14.2 (q)
OH
1.06 (d, 6.2)
1.03 (d, 6.9)
2.80 (br s)
18.3 (q)
14.3 (q)
OH
1.07 (d, 6.6)
1.04 (d, 6.8)
2.71 (br s)
2.78 (br s)
1.03 (d, 6.9)
2.70 (br s)
2.87 (s)
OH
1′
94.8 (d)
80.9 (d)
5.04 (s)
173.4 (s)
36.3 (t)
173.6 (s)
36.3 (t)
2′a
2′b
3′
3.33 (ddd, 3.0, 1.5. 1.3)
2.34 (td, 7.5, 1.2)
2.40 (dt, 15.5, 7.3)
2.43 (dt, 15.5, 7.3)
1.70 (m)
66.6 (d)
73.3 (d)
67.4 (d)
16.6 (q)
59.6 (q)
OH-3′
3.74 (m)
18.7 (t)
13.9 (q)
1.65 (m)
18.6 (t)
13.9 (q)
4′
3.49 (ddt, 11.8, 3.3, 1.2, 1.2)
3.85(br q, 6.6)
1.23 (d, 6.5)
0.94 (t, 7.5)
0.96 (t, 7.4)
5′
6′
7′
3.45 (s)
2.86 (d, 11.6)
2.74 (d, 11.6)
OH-4′
1″
173.6 (s)
36.3 (t)
173.1 (s)
36.0 (t)
2″a
2″b
3″
2.35 (t, 7.5)
2.31 (dt, 12.8, 7.5)
2.33 (dt, 12.8, 7.5)
1.63 (m)
18.8 (t)
14.0 (q)
1.67 (tq, 7.4, 7.4)
0.95 (t, 7.4)
18.6 (t)
13.8 (q)
4″
0.93 (t, 7.3)
a HETLOC experiment (Figure S12, Supporting Information).
This value is in agreement with that reported previously for 2-
O-methyl-α-rhamnose, although is smaller than in the case of
nonmethylated common sugars (∼170 Hz).17,18
As in the case of 1, the coupling constants values and ROESY
correlations measured from both CDCl3 and py-d5 data for
mandelalide B (2) were comparable, indicating similar
conformations in both solvents. The chair conformation of
the THP ring in 2 (C-5 to C-9) was confirmed by the presence
of diaxial ROESY correlations between H-5, H-7 and H-9
(Figure 4a). For the C-9/C-11 segment, coupling constants of
2.2 and 11 Hz, between H-9/H-10a, and H-9/H-10b confirmed
gauche and anti relationships, respectively. Similarly for H-10a/
H-11 and H-10b/H-11, large (12.3 Hz) and small (4.1 Hz in
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Figure 2. (a) Key ROESY correlations indicated on the C-5/C-15 fragment of 1. (b) Two possible rotamers around the C-9/C-10 bond. (c) Most
feasible rotamers around the C-10/C-11 bond of 1, with anticipated large/small heteronuclear coupling constant indicated on each. For b and c, the
JHH and JHC values (CDCl3) are listed below the Newman projections and the rotamer of best fit is indicated by a dashed outline.
Figure 3. (a) Newman projections along the C-16/C-17 and C-20/C-21 bonds in 1. (b) The most feasible cis orientation of the THF ring with
double-headed arrows indicating ROESY correlations. (c) Newman projection along the C-21/C-22 bond with applicable JHH and JHC values
(CDCl3) indicated on the right. (d) Newman projection along the C-22/C-23 bond and relevant coupling constants.
Figure 4. (a) Key ROESY correlations for the C-3/C-5 fragment are indicated by double-headed arrows on the partial structure. (b) The most
probable rotamer along the C-3/C-4 bond highlighted by the dashed outline and alternative equilibrium of two rotamers explaining medium size
coupling constants. But = butyrate.
CDCl3, 3.8 Hz in py-d5) coupling constants assigned from
DQFCOSY supported anti and gauche relationships, respec-
tively, overall leading to the same relative configuration in this
fragment confirmed that a cis conformation of the THF ring is
conserved in mandelalides A (1) and B (2).
Given extensively overlapped CDCl3 1H chemical shifts for
the C-20/C-23 fragment in 2, analysis of 1H NMR and
DQFCOSY in py-d5 was used to assign the relative
3
region as for 1. For the C-3/C-5 fragment of 2, JHH values of
1.5 and 11.7 Hz between H-4a/H-5 and H-4b/H-5 supported
gauche and anti orientations, respectively. A small coupling of
1.6 Hz between H-3 and H-4b indicated their gauche
relationship (Figure 4b), and was in agreement with large H-
4a/C-3 (−5.6 Hz) and small H-4b/C-3 (0 Hz) heteronuclear
couplings. However, medium couplings for H-3/H-4a (6.0 Hz)
and H-5/C-3 (4.3 Hz) suggested that the staggered orientation
along C-3/4 may be distorted or that more than one
conformation is present along this bond. Clear ROESY
correlations between H-3 and each of H-4b, H-5, H-9, H-11,
H-23 and H-24b, but not H-4a (apparent COSY artifact only),
support a distorted rotamer in which 138° and −102° angles
are present between H-3/H-4a and H-3/H-4b, as calculated
using MestReJ for 3JHH 6.0 and 1.6 Hz respectively (Figure 4b).
The presence of a ROESY correlation between H-18 and H-
20 on the THF ring in mandelalide B (2) placed CH3-26 quasi-
equatorial, consistent with ROESY correlations from the latter
methyl to H2-16, (as for 1, Figure 3). ROESY correlations
(Table S2, Supporting Information) and comparative analysis
of coupling constant values (Table 2) around the C-16/C-21
3
configuration of this region. The JHH values for 2 were similar
to those for mandelalide A (1), despite the presence of the γ-
lactone ring in 2. However, in contrast to 1, in which the more
shielded H-22a (δ 1.46) is anti to H-21 and gauche to H-23
(Figure 3), for 2 the more shielded H-22a (δ 1.65) is gauche to
H-21 (3JHH < 1 Hz) and H-22b (δ 1.77) is anti to H-21 (3JHH
=
9.4 Hz), while H-20 and H-21 remain antiperiplanar (3JHH = 9.4
Hz) (Figure 5a). Coupling constant values obtained directly
from the oxymethine H-23 multiplet, and assigned by
DQFCOSY, indicate anti relationships with H-22a (JHH
=
Figure 5. (a) Assigned rotamers along the C-21/C-22 and C-22/C-23
bonds in 2. (b) ROESY correlations around the γ-butyrolactone of 2.
But = butyrate.
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Figure 6. (a) Key ROE correlations (CDCl3) between the monosaccharide and the macrolide THP moiety in mandelalide A. (b) Key ROE
correlations between the monosaccharide and the macrolide, as well as through ring correlations for mandelalide B.
11.2 Hz), and a gauche relationship with H-22b (JHH = 1.0 Hz).
The orientation of the ester bond in the butyrolactone ring
could be assigned knowing the localization of H-23 anti to H-
22a. The ester oxygen must then be positioned gauche or anti to
H-22b. The ROEs observed between H-24a, H-24b and H-22b
in py-d5, in parallel with the absence of ROEs between H2-24
and H-21 suggested that the ester bond is anti to H-22b (Figure
5a). Finally, knowing the relative configuration of the C-3/C-11
and C-20/C-23 fragments, the chirality of the quaternary C-2 in
the butyrolactone ring was considered. ROESY correlations
between H-24b and both H-3 and H-23 were consistent with
an α-oriented H-24b, while H-24a, showing ROESY
correlations to H2-22, is oriented above the butyrolactone
ring in a β configuration. Similarly, the C-2 hydroxyl could be
oriented to the outside of the macrocycle due to the presence
of weak ROESY signals between this OH-2 and H-4b and H-
24a, suggesting a 2R configuration (Figure 5b).
synthetic standard, confirming that the sugar substituent of 1 is
2-O-methyl-α-L-rhamnose. The absolute configuration of this
sugar could then be extrapolated to the aglycone of 1 based on
the ROESY correlations between H-1′ and H-7, H-1′ and H-6eq,
and H-5′ and H-8eq to provide an assignment of the
mandelalide A (1) aglycone as 2E, 5S, 7S, 9R, 11R, 12E, 14Z,
17R, 18R, 20R, 21R, 23R. Given retention of the macrocycle
configuration (C-5 to C-21) between 1 and 2, the absolute
configuration of 2 could be assigned similarly by considering
ROESY correlations between the monosaccharide and THP
moiety, and key correlations across this more rigid macrocycle
(Figure 6b). Thus the aglycone of mandelalide B (2) could be
assigned as 2R, 3R, 5R, 7S, 9R, 11R, 12E, 14Z, 17R, 18R, 20R,
21R, 23R.
Computational modeling of mandelalide B was used to
examine possible mandelalide B conformations (Figure 7). An
The relative configuration of the 2′-O-methyl, 6′-deoxy
monosaccharide moiety (C1′−C7′) in 2 was established
analogously to that in 1. A very weak ROESY correlation
(py-d5) between H-1′ and H-5′ together with 1JHC = 167 Hz for
anomeric CH-1′ suggested the presence of an α-sugar. A
ROESY correlation between H-3′ and H-5′ localized these
3
protons axial, while the small JHH between H-2′ and H-3′′ (2.8
Hz) localized H-2′ equatorial (Figure 6). The presence of a W
coupling (4JHH = 0.98 Hz) between H-2′ and H-4′, as well as
3JHH′ = 3.2 Hz between H-3′ and H-4′ localized H-4′ equatorial,
overall leading to the conclusion that mandelalide B (2)
contains 2-O-methyl-6-dehydro-α-L-talose, the C-4′ epimer of
the monosaccharide in mandelalide A.
Given the assignment of relative configuration of the
macrocycle and monosaccharide units of mandelalides A (1)
and B (2), it remained to establish the absolute configuration of
these glycosidic macrolides. Despite our desire to conserve the
limited sample quantities available for further biological
investigations, a chemical degradation/derivatization approach
to the absolute configuration would be most rigorous and was
facilitated by the contiguous nature of the stereogenic
fragments throughout the molecular framework. Importantly,
inspection of the CDCl3 and py-d5 ROESY spectra for 1 and 2
revealed clear correlations between protons of the mono-
saccharide and THP of the macrocycle (Figure 6a, b). Given
the well-defined solution structures of monosaccharides and
glycosidic bond conformations,19 assignment of the rhamnose
absolute configuration would permit subsequent relay of
configurational assignments around the macrocycle, as shown
for mandelalide B (Figure 6b). Therefore, a portion (100 μg) of
mandelalide A (1) was sacrificed for hydrolysis of the glycosidic
bond and chiral GC-MS comparison of the liberated derivatized
monosaccharide with permethylated and silylated D- and L-
rhamnose standards. During coinjection of each standard with
the derivatized natural product hydrolysate, the natural product
sugar coeluted with the L (97.4 min) and not the D (96.4 min)
Figure 7. Computational model of the lowest energy conformer for
mandelalide B (2).
attempt to investigate the fit of calculated conformations to
experimental data was based on measurements of ROE
distances across the macrocycle in py-d5.20 However, as could
be expected for a flexible macrolide, for the ten lowest energy
conformations of mandelalide B (2), the differences between
the calculated and experimental average distances between all
analyzed protons except H-21/H-11 and H-21/H-13 were
significantly different (0.2 Å or more). Nevertheless, the lowest
energy computational model assists in visualizing the through
ring ROE contacts that facilitated assignment of the absolute
configuration (Figure 7).
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8.74, CD3OD, 3.31; 13C NMR: CDCl3, 77.23, py-d5, 150.35, CD3OD,
49.15). High-resolution MS data were acquired using an orthogonal
acceleration time-of-flight (oa-TOF) mass analyzer and electrospray
ionization (ESI).
Extraction and Isolation. The ascidian, Lissoclinum sp.
(Ascidiacea, Aplousobranchia, Didemnidae) was collected by hand
using SCUBA at a depth of 18 m (July 20, 2004) from White Sands
Reef in Algoa Bay, Eastern Cape Province, South Africa (33:59.916S,
25:42.573W). The type specimen (SAF2004-55) for this new ascidian
species is housed at the South African Institute for Aquatic Biodiversity
(SAIAB), Grahamstown, South Africa.
3
The JHH values acquired in CDCl3 for mandelalides B−D
(2−4) were all similar, suggesting that the relative configuration
of the macrocycle incorporating a γ-butyrolactone is retained.
The configuration at the remaining C-24 stereocenter present
in mandelalides C (3) and D (4) was assigned from ROESY
data. In each case, the H-24 doublet (δH 3.98 and 5.17 in 3 and
4, respectively) is correlated to the H-3 double doublet,
localizing the C-24 hydroxyl/butyrate moiety antiperiplanar to
H-3 and H-23, in an S configuration.
Cytotoxicity of the organic extract from Lissoclinum sp. was
examined against mouse Neuro-2A neuroblastoma, and human
MDA-MB-231 breast and NCI-H460 lung cancer cell lines
following 48 h exposure. In all cases low μg/mL IC50 values
were obtained (Table 3). The Neuro-2A cell line was chosen to
The freeze-dried organism (15.1 g) was extracted with 2:1 CH2Cl2-
MeOH yielding 1.45 g of organic extract. This organic extract was
fractionated on Sephadex LH-20 (CH2Cl2−MeOH, 1:3) to give eight
fractions, of which fractions six and seven were subjected to reversed
phase C18 solid phase extraction (RP-SPE) using a stepped gradient of
50−100% MeOH in H2O. The 75% MeOH-H2O and 100% MeOH
fractions were further separated by RP-HPLC (C18 column, 250 mm ×
10 mm, 7:3 MeOH-0.1% FA in H2O) to yield mandelalides A (1, 0.8
mg), B (2, 0.5 mg), C (3, 0.8 mg) and D (4, 0.6 mg).
Table 3. Cytotoxicity of Lissoclinum sp. Organic Extract and
Mandelalides A (1) and B (2) to Mouse Neuro-2A
Neuroblastoma and Human NCI-H460 Lung and MDA-MB-
231 Breast Cancer Cells
Mandelalide A (1). Amorphous solid; [α]23D −9 (c = 0.25, MeOH);
UV (MeOH) λmax (log ε) 279 (2.7), 217 (4.1); LR-ESI-MS m/z [M +
Na]+ 647.4; HR-ESI-MS m/z [M+Na]+ 647.3394, (calcd for
C33H52O11Na, 647.3407); 1H and 13C NMR, COSY, HMBC,
TOCSY, ROESY (Tables 1, S1, Supporting Information).
a
IC50 (at 48 h) in Cancer Cell Lines
NCI-H460
Neuro-2A
MDA-MB-231
organic extract
0.7 μg/mL
12 nM
5.6 μg/mL
44 nM
22.1 μg/mL
Mandelalide B (2). Amorphous solid; [α]24D −13 (c = 0.5, MeOH);
UV (MeOH) λmax (log ε) 279 (3.0), 229 (4.1); LR-ESI-MS m/z [M +
Na]+ 733.5; HR-ESI-MS m/z [M + Na]+ 733.3773, (calcd for
mandelalide A (1)
mandelalide B (2)
29 nM
84 nM
a
C37H58O13Na, 733.3775); H and 13C NMR, COSY, HMBC, ROESY
Cell viability was assessed by MTT assay and IC50 values were
derived using nonlinear regression analysis.
1
(Tables 2, S2, S3, Supporting Information).
Mandelalide C (3). Amorphous solid; UV (MeOH) λmax (log ε)
280 (2.7), 229 (4.0); LR-ESI-MS m/z [M + Na]+ 589.5; HR-ESI-MS
m/z [M + Na]+ 589.2970, (calcd for C30H46O10Na, 589.2989); 1H and
13C NMR, COSY, HMBC, TOCSY, ROESY (Tables 2, S4, Supporting
Information).
perform activity-guided fractionation leading to the purification
of mandelalides A−D (1−4). The pure compounds man-
delalides A and B yielded nanomolar IC50 values against Neuro-
2A (44.0 and 83.8, respectively) and NCI-H460 (12.0 and 29.4,
respectively) cell lines (Table 3, Figure S57, Supporting
Information). The potent cytotoxicities of these mandelalides
are somewhat surprising given the reported minimal cytotox-
icity of the related glycosylated polyketides madeirolides A and
B.21 The latter metabolites caused less than 50% inhibition of
AsPC-1 and PANC-1 pancreatic cancer cells at 10 μg/mL, but
showed potent fungicidal activity against Candida albicans.
Madeirolides A and B share essentially the same western
hemisphere of the mandelalides (C-5 to C-22), including a
THP (alternatively substituted), diene, THF and neighboring
hydroxymethine. The two series of compounds vary only in the
closure of the macrocycle: the position of the lactone and
additional cycle. Given that the madeirolides were isolated from
a deep-water lithistid Leiodermatium sponge, their structural
relatedness is consistent with a microbial biogenetic origin.
Side-by-side evaluation of the biological properties of all four
mandelalides and the two madeirolides would provide insight
into the structure−activity relationships that result in potent
cytotoxicity and fungicidal activity, respectively. However, the
inaccessible supply of both source organisms means that further
investigation of these metabolites will likely await their total
syntheses.
Mandelalide D (4). Amorphous solid; [α]25 −50 (c = 0.2,
D
MeOH); UV (MeOH) λmax (log ε) 280 (2.7), 229 (4.0); LR-ESI-MS
m/z [M + Na]+ 659.5; HR-ESI-MS m/z [M + Na]+ 659.3424, (calcd
for C34H52O11Na, 659.3407); 1H and 13C NMR, COSY, HMBC,
TOCSY, ROESY (Tables 2, S5, Supporting Information).
Deacylmandelalide D (4b). Amorphous solid; [α]27D −10 (c = 0.2,
MeOH); UV (MeOH) λmax (log ε) 280 (2.8), 227 (3.8); LR-ESI-MS
m/z [M + Na]+ 519.3; HR-ESI-MS m/z [M + H]+ 497.2752, (calcd
1
for C26H41O9, 497.2751); H and 13C NMR, COSY, HMBC (Table
S6, Supporting Information).
Measurement of 1,2JHC Coupling Constants. The sensitivity and
gradient-enhanced HETLOC (ω1-hetero half-filtered TOCSY) experi-
ment was employed to measure JHC coupling constants, with DIPSI-2
spin-lock set to 60 ms.23 Spectral widths of 6229 and 5597 Hz, with a
data matrix of 4K (F2) × 128 (F1), and 146 or 96 scans were
employed in py-d5 and CDCl3, respectively for mandelalide A. In the
case of mandelalide B, a spectral width of 5597 Hz with a data matrix
of 4K (F2) × 128 (F1) and 136 scans were implemented in CDCl3.
The 1D spectra that were obtained after extraction of F2/F1 slices
were subjected to inverse Fourier transform. The resulting FIDs were
multiplied by the exponential window function prior to linear
prediction processing.24
TOCSY Data Acquisition. To determine the magnetization
transfer pattern in 2-O-methyl-L-rhamnose a 2D TOCSY was acquired
with a DIPSI-2 spin-lock sequence in CDCl3. The spectra were
recorded with a spin-lock mixing time of 100 ms, 5597 Hz spectral
width at 2K (F2) × 512 (F1) data matrix for 40 scans. Data were
processed with a sine-bell squared function with 1.5 Hz (F2) and 0.3
Hz (F1) line broadening before measurement of the absolute volumes
of peaks. The following designation was applied to depict intensity of
peaks; gray ovals indicate intensity or signal greater than 1.5% of
intensity measured for the signal of the anomeric proton, white ovals
indicate intensity of at least 0.5% but no more than 1.5% intensity
EXPERIMENTAL SECTION
■
General Experimental Methods. As described previously.22
Additionally NMR data in CDCl3 were acquired at 700 (1H) and 175
(13C) MHz on a 5 mm inverse cryogenic probe. The NMR data in py-
d5, TOCSY (mixing time 100 ms), HETLOCs and gated-decoupled
13C NMR experiments were obtained at 700 (1H) and 175 (13C) MHz
on a 5 mm 13C cryogenic probe. The spectra were referenced to
internal residual solvent signals in ppm (1H NMR: CDCl3, 7.24, py-d5,
16
1
measured for the anomeric H signal.
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Article
Calculation of Dihedral Bond Angles in MestReJ.25 The
calculation of angles between H-3/H-4a and H-3/H-4b was performed
using the Altona equation with substituents at C-3 defined as CH2OR
and OCOR, and substituents at C-4 as H and CH2OR. Resulting 3JHH
values for H-3/H-4a (138.7°) and H-3/H-4b (−102.2°) were 6.0 and
1.6 Hz, respectively.
the L-glycoside in mandelalide A (Figure S56, Supporting
Information).
Cell Viability Assays. Cytotoxicity of the organic extract and
crude fractions was evaluated in mouse Neuro-2A neuroblastoma, and
human MDA-MB-231 breast and NCI-H460 lung cancer cells (ATCC,
Manassas, VA) using a previously described protocol subjected to
slight modifications.22 Cells were seeded into 96-well plates (20000
cells per well for MDA-MB-231 and NCI-H460, 25000 cells per well
for Neuro-2A) in 50 μL of medium 12 hours before treatment. Each
test sample was added in a 25 μL aliquot generated by serial dilution in
serum-free medium on the day of the experiment, after prior removal
of 25 μL of media from the treated well. Aliquots were generated from
stock solutions of 6 mg/mL compound in 100% DMSO. Pure
compounds 1 and 2 were evaluated in Neuro-2A and NCI-H460 cells
after 48 h treatment, as described above. Each compound was tested at
final concentrations ranging from 0.001 to 10 μg/mL. In all cases, cell
viability was determined after 48 h treatment using a standard 3-(4,5-
dimethylthiazol-2-yl)-2,5,diphenyl tetrazolium bromide (MTT)
assay.22 The cytotoxicity of each pure compound was assessed in at
least three independent cultures with the viability of vehicle-treated
control cells defined as 100% in all experiments. Dose response curves
(Figure S57, Supporting Information) were plotted using contempo-
rary biostatistics and curve fitting software.
Computational Modeling of Mandelalide B (2). Computa-
tional modeling was performed using the 2009 version of a
contemporary software package. Minimization using the Amber*
force field with PRCG algorithm, in pyridine (ε = 12.9, 10000 steps,
maximum derivative less than 0.05 kcal/mol) and constrained torsion
angles H-3/H-4a (138°), H-3/H-4b (−102.2°), H-4b/H-5, H-9/H-
10b, H-20/H-21, H-21/H-22b, H-22a/H-23 and distance H-4b/H-
24b = 5 Å was first performed. All torsions were restrained based on
3
the JHH coupling constants values. The minimized structure was
subjected to conformational search using Amber* force field via the
low mode sampling method with an energy cut off of 21 kJ/mol and
1000 steps (100 steps per rotatable bond).26,27 Torsion restraints H-3/
H-4a (138°), H-3/H-4b (−102.2°), H-4b/H-5, H-9/H-10b, H-20/H-
21, H-21/H-22b, H-22a/H-23 were applied to obtain ground state
conformations. Optimization of the ten lowest energy conformations
using DFT with the B3LYP functional and 6-31G** basis set in the
gas phase resulted in the structure presented in Figure 7.
Methanolysis of Mandelalide A (1). Compound 1 (0.1 mg) was
treated with 1 N methanolic HCl (1.0 mL) and heated for 24 h at 70
°C with stirring. The mixture was concentrated in vacuo, redissolved in
0.75 mL of 1-(TMS)-imidazole/py (1:4) and the reaction was
continued for 40 min at 70 °C with stirring.28 The solution was
concentrated, the final residue partitioned between CH2Cl2 and H2O
(1:1), and the organic fraction used for GC-MS analysis.
ASSOCIATED CONTENT
* Supporting Information
■
S
Tables of 1D and 2D NMR data and NMR spectra for
compounds 1−3 in CDCl3 and py-d5, for compound 4 in
CDCl3, for compound 4b in CD3OD. Computational data for
mandelalide B (2). GC-MS chromatograms for derivatized α-L-
and α-D-rhamnose standards and mandelalide A hydrolysate.
Dose response curves for mandelalides A and B (1 and 2) in
Neuro-2A and NCI-H460 cell lines. This material is available
free of charge via the Internet at http://pubs.acs.org.
Preparation of 1,2-Di-O-methyl-3,4-di-O-TMS-α-L-rhamnose
and 1,2-Di-O-methyl-3,4-di-O-TMS-α-D-rhamnose Standards. L-
rhamnose (50 mg) was dissolved in 1N methanolic HCl, heated for 24
h at 70 °C with stirring, and concentrated in vacuo. The residue was
redissolved in dioxane (0.6 mL) and 2 M TMS-diazomethane in
diethyl ether (1.5 mL) was added, with H3BO3 as catalyst. The
solution was maintained at room temperature with stirring for 5 h and
then evaporated to dryness in vacuo.29 Column chromatography on Si
gel GF (15 μm) with 5% MeOH in CH2Cl2 as mobile phase afforded a
1:3 mixture of 1,2-di-O-methyl-α-L-rhamnose and 1,3-di-O-methyl-α-L-
rhamnose in ∼65% yield from this rate limiting reaction: equivalent to
∼16% yield of the desired 1,2-di-O-methyl-α-L-rhamnose to be carried
forward for silylation. This 1:3 mixture of methylation products was
treated with 1-(TMS)-imidazole/py (1:4) for 40 min at 60 °C and
concentrated in vacuo.28 Finally, the residue was dissolved in CH2Cl2,
loaded on a Si gel column, washed with 100% hexanes, and eluted with
3% EtOAc in hexanes to obtain pure 1,2-di-O-methyl-3,4-di-O-TMS-α-
L-rhamnose in a final yield of 5.4%. The same procedure was applied to
produce 1,2-di-O-methyl-3,4-di-O-TMS-α-D-rhamnose in 6.4% yield.
1H NMR (700 MHz, CDCl3): δ 0.12 (s, 9H), 0.15 (s, 9 H), 3.31 (s,
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 541 737 5808. Fax: +1 541 737 3999. E-mail: kerry.
mcphail@oregonstate.edu
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Professor Bill Fenical of Scripps Institution of
Oceanography, San Diego, CA for the generous donation of
funding which enabled the first large scale SCUBA collection of
marine ascidians in Algoa Bay, South Africa. We are also
grateful to Rodger Kohnert for NMR technical assistance, and
Brian Arbogast and Jeff Morre of the Environmental Health
Sciences Center at OSU for mass spectrometric data acquisition
(NIEHS P30 ES00210). The National Science Foundation
(CHE-0722319) and the Murdock Charitable Trust (2005265)
are acknowledged for their support of the OSU Natural
Products and Small Molecule Nuclear Magnetic Resonance.
Additional funding was provided by the OSU College of
Pharmacy.
3H), 3.32 (dd, J = 1.7, 3.2 Hz, 1H), 3.48 (s, 3H), 3.52 (m, 1H), 3.53
(m, 1H), 3.78 (dd, J = 3.6, 8.4 Hz, 1H), 4.61 (d, J = 1.3, 1H); 13C
NMR (175 MHz, CDCl3): δ 0.7, 1.2, 18.5, 54.8, 60.0, 69.1, 73.1, 74.3,
81.6, 98.8, ppm; CI-LR-MS: m/z 305 calculated for oxonium ion
C13H29O4Si2.
Absolute Configuration of 2-O-Methyl-α-rhamnose from
Mandelalide A (1). Analyses of the synthetic standards and
permethylated and silylated mandelalide A hydrolysate were
performed by GC-MS using a 30 m × 0.25 mm i.d. Cyclosil-B
column, and electron impact (EI) ionization. The injector and detector
were operated at 250 °C, while the temperature gradient was set to
75−175 °C at 0.5°/min. The retention times for 1,2-di-O-methyl-3,4-
di-O-TMS-α-D-rhamnose and 1,2-di-O-methyl-3,4-di-O-TMS-α-L-
rhamnose injected separately were 96.5 and 98.3 min, respectively.
The retention time for the monosaccharide in the derivatized
mandelalide A hydrolysate injected separately was 97.5 min. (Figure
S56, Supporting Information). Therefore coinjection of the natural
product hydrolysate with each standard was performed, yielding two
peaks with retention times of 96.4 and 97.4 min, which is indicative of
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