Liposomal circular dichroism. assignment of remote stereocenters in plakinic acids K and L from a plakortis-xestospongia sponge association

Article abstract of DOI:10.1021/ol100249v

"Figure Presented" Two new ωtu-phenyl polyketide peroxides, plakinic acids K and L, were isolated from a two-sponge association of Plakortis halichondroides and Xestospongia deweerdtae. The absolute configurations of the remote dimethyl-branched stereocenters in plakinic acid K were assigned by degradation of plakinic acid K to a long-chain naphthamide and analysis by liposomal circular dichroism (L-CD) and comparison with synthetic standards.

Full text of DOI:10.1021/ol100249v

ORGANIC
LETTERS
2010
Vol. 12, No. 7
1524-1527
Liposomal Circular Dichroism.
Assignment of Remote Stereocenters in
Plakinic Acids K and L from a
Plakortis-Xestospongia Sponge
Association
Doralyn S. Dalisay,†,§ Tim Quach,† and Tadeusz F. Molinski*,†,‡
Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and
Pharmaceutical Sciences, UniVersity of California, San Diego, 9500 Gilman DriVe,
La Jolla, California 92093
tmolinski@ucsd.edu
Received January 31, 2010
ABSTRACT
Two new ω-phenyl polyketide peroxides, plakinic acids K and L, were isolated from a two-sponge association of Plakortis halichondroides
and Xestospongia deweerdtae. The absolute configurations of the remote dimethyl-branched stereocenters in plakinic acid K were assigned
by degradation of plakinic acid K to a long-chain naphthamide and analysis by liposomal circular dichroism (L-CD) and comparison with
synthetic standards.
The marine sponges of the genera Plakortis and Plakinas-
trella1 are prolific producers of cyclic polyketide peroxides
that exhibit a broad spectrum of biological properties,
including antifungal,1 antimalarial,2 antiprotozoal,3 and im-
munosuppressant4 activities. Sponge-derived cyclic peroxides
may occur as 1,2-dioxane or 1,2-dioxolane ring systems.5
We recently reported the stereochemical assignments of two
ω-phenyl polyketide peroxides, plakinic acids I (1) and J
† Department of Chemistry and Biochemistry.
(3) Perry, T. L.; Dickerson, A.; Khan, A. A.; Kondru, R. K.; Beratan,
D. N.; Wipf, P.; Kelly, M.; Hamann, M. T. Tetrahedron 2001, 57, 1483–
1487.
‡ Skaggs School of Pharmacy and Pharmaceutical Sciences.
§ Present address: Department of Chemistry, University of British
Columbia, Vancouver, BC, Canada V6T 1Z1.
(4) Costantino, V.; Fattorusso, E.; Mangoni, A.; Di Rosa, M.; Ianaro,
A. J. Am. Chem. Soc. 1997, 119, 12465–12470.
(1) Phillipson, D. W.; Rinehart, K. L., Jr. J. Am. Chem. Soc. 1983, 105,
7735–7736. (b) Davidson, B. S. J. Org. Chem. 1991, 56, 6722–6724. (c)
Qureshi, A.; Salva, J.; Harper, M. K.; Faulkner, D. J. J. Nat. Prod. 1998,
61, 1539–1542. (d) Sandler, J. S.; Colin, P. L.; Hooper, J. N. A.; Faulkner,
D. J. J. Nat. Prod. 2002, 65, 1258–1261.
(5) Different polyketide peroxides have been found in tunicates: (a)
Reyes, F.; Rodr´ıguez-Acebes, R.; Ferna´ndez, R.; Bueno, S.; Francesch, A.;
Cuevas, C. J. Nat. Prod. 2010, 73, 83–85. (b) Fontana, A.; Gonzalez, M. C.;
Gavagnin, M.; Templado, J.; Cimino, G. Tetrahedron Lett. 2000, 41, 429–
432. (c) Dura´n, R.; Zub´ıa, E.; Ortega, M. J.; Naranjo, S.; Salva´, J.
Tetrahedron 2000, 56, 6031–6037.
(2) Hu, J.-F.; Gao, H.-F.; Kelly, M.; Hamann, M. T. Tetrahedron 2001,
57, 9379–9383.
10.1021/ol100249v  2010 American Chemical Society
Published on Web 03/05/2010

(2) (Figure 1), from the symbiotic two-sponge association
Plakortis halichondroides-Xestospongia deweerdtae Lehnert
& van Soest, 1999,6 which showed differential inhibition
against haplodeficient lag1∆/LAG1 strains of Saccharomyces
cereVisiae.7 The configurations of the remote methyl-
branched C8/C7 stereocenters of 1 and 2 were solved by
the application of liposomal circular dichroism (L-CD),8 a
very sensitive technique (limit of detection ∼16 nmol) that
amplifies CD Cotton effects (CEs) through ordering of the
long-chain lipids in highly uniform, unilamellar liposomes.7
Here, we show a remarkable long-range perturbation of
naphthamide chromophores by a single methyl branch, oVer
eight bonds away, that discriminates between spectroscopi-
cally indistinguishable diastereomers. L-CD was used to
assign configurations of both proximal C7/C8 and distal C12/
C11 methyl-branched centers in two new ω-phenyl polyketide
peroxides, plakinic acids K (3) and L (4).
an additional methyl group (δH 0.82, d, J ) 6.4 Hz; δC 19.9,
CH3). Inspection of the COSY and HMBC spectra estab-
lished the position of the second methyl branch at C12 (see
the Supporting Information). Using a similar analysis,
plakinic acid L (4, 7.5 × 10-3 % yield w/w wet weight, [R]D
-26.2 (c 1.90, CHCl3) C25H40O4; HREIMS [M+] m/z
404.2918) was shown to be a homologue of 2.
The common origin of 1-4, similar specific rotations and
1H NMR suggest that the respective stereocenters at C3, C4,
and C6 have the same configurations as those of 1 and 2.10
The configurations of the proximal C8/C7 stereocenters in
1 and 2 were assigned as R by interpretation of the intense
CEs in L-CD of the derived 6-methoxy-2-naphthamide (in
contrast, CD of the naphthamides in isotropic media, such
as methanol solutions, showed only baseline).7 We had
proposed7 that the strong, bisignate CEs displayed in L-CD
spectra arise from pairwise intramolecular exciton coupling
of naphthamides within the liposomal bilayers. Phospholipid
bilayers, comprised of saturated long chains, promote
hexagonal close packing of extended CH2 groups.
Scheme 1. Degradation of 3 and Conversion to 7
Figure 1. Structures of plakinic acids I (1), J (2), K (3), and L (4).
P. halichondroides-X. deweerdtae, collected from the
Bahamas, was extracted with MeOH/CH2Cl2 (1:1) to yield
a dark-brown residue which was sequentially extracted with
n-hexane, chloroform, n-butanol, and water. Antifungal
bioassay-guided fractionation revealed that the CHCl3-soluble
extract exhibited potent activity against S. cereVisiae lag1∆/
LAG1 strain. This fraction was subjected to flash chroma-
tography (silica, 0-100% MeOH-CHCl3, stepped gradient)
and the active fraction further purified by HPLC (RP-C18
CH3CN/H2O) to yield known compounds 1 and 2,7 new
methyl-branched plakinic acids K (3) and L (4), and plakinic
acid M, a linear homologue of 1.9
The dimensions of the problem expand significantly for
assignment of the remote C12/C11 methyl-branched stereo-
centers in 3 and 4. We surmised the remote methyl branch
in 3 and 4 may alter the chain packing and influence the
magnitude and form of the naphthamide CEs in L-CD. This
would constitute net transmission of stereochemical informa-
tion from the remote C12 center to the naphthamide group;
however, it was not certain the effect would be large enough
to be readily observable in the CD spectrum.
Plakinic acid K (3) was isolated as an optically active
colorless oil (4.74 × 10-3 % yield, w/w, wet weight), [R]D
-137 (c 1.31, CHCl3), of molecular formula C26H44O4 as
determined by HREIMS [M+] m/z 432.3239, with 14 mass
1
units greater than 1. The UV, IR, and H and 13C NMR
spectra of 3 were almost identical to those of 1, except for
(6) While n- and isoalkyl cyclic peroxides are found in free-living P.
halichondroides, we found only the P. halichondroides-X. deweerdtae
association produces ω-phenyl-terminated polyketide peroxides.
(7) Dalisay, D. S.; Quach, T.; Nicholas, G. M.; Molinski, T. F. Angew.
Chem., Int. Ed. 2009, 48, 4367–71.
(10) The absolute configurations at stereocenters around the 1,2-dioxane
ring in 1 were solved conventionally by integrated analysis of ROESY
spectra and the modified Mosher’s ester method. The absolute configurations
of C4 and C5 within the 1,2-dioxolane ring of 2 were assigned by
comparison of the [R]D with those of synthetic “plakinates” of defined
configuration. Dai, P.; Trullinger, T. K.; Liu, X.; Dussault, P. H. J. Org.
Chem. 2006, 71, 2283–92, and ref 7.
(8) (a) MacMillan, J. B.; Molinski, T. F. J. Am. Chem. Soc. 2004, 126,
9944–5. (b) MacMillan, J. B.; Linington, R. G.; Andersen, R. J.; Molinski,
T. F. Angew. Chem., Int. Ed. 2004, 43, 5946–51.
(9) See S1, Supporting Information, for complete characterization.
Org. Lett., Vol. 12, No. 7, 2010
1525

In order to test this hypothesis, 311 was degraded (Scheme
1) by cleavage of the C6-C7 bond using Fe(II)-promoted
reductive fragmentation (FeCl2, aq CH3CN-H2O, N2-
sparged) to give primary alkyl chloride 5,12 which was
subsequently transformed by a three-step sequence7 to give
naphthamide 7.
Remarkably, no signal appeared in the CD spectrum of 7
(Figure 2a) immediately after formulation in liposomes, but
over 24 h intense, complex CEs gradually appeared that were
stable (>30 days) and reproducible [λ 196 nm (∆ε -11.6),
213 (+ 37.4), 226 (-6.5), 255 (+ 5.6)]. Fitting the time-
dependence of the CE at λ ) 226 nm in the L-CD spectrum
of 7 (Figure 2c) to an exponential function gave t1/2 ) 385
min. Evidently, the kinetics of lipid chain reorganization of
liposomal 7 at room temperature are considerably slower than
the corresponding naphthamides derived from 1 and 2 in
which less complex CEs appeared essentially immediately.14
The assignment of configuration in 7 was completed by
comparison with synthetic model compounds (2S,6R)-8 and
(2R,6R)-9, which were prepared as follows.
Scheme 2. Synthesis of an Epimeric Mixture of Acids 17
Tosylation of (S)-(-)-citronellol15 10 gave 1116 in 87%
yield (Scheme 2), which upon treatment with lithium
phenylacetylide (reflux, THF), afforded enyne 12 in 92%
yield.
Two-step Johnson-Lemieux oxidation17 of 12 provided
aldehyde 14 (Scheme 2), which was subjected to Horner-
Wadsworth-Emmons olefination to give R,ꢀ-unsaturated
ester 15 as an inseparable 1:1 mixture of E/Z isomers.
Hydrogenation of enyne 15 (Pd/C) afforded saturated ester
16, which was saponified (LiOH, THF/water) to give acid
17 as a 1:1 mixture of diastereomers, epimeric at C2. Acid
17 was coupled with (S)-phenethylamine (HATU, i-Pr2NEt)
(Scheme 3) to provide a mixture of diastereomeric amides
(2S,6R)-18 and (2R,6R)-19, which were separated by silica
gel chromatography.18 Amides 18 and 19 were individually
treated with BH3·THF to afford secondary amines 20 and
Figure 2. Liposomal circular dichroism (L-CD) spectra (T ) 296
K): (a) 7 from t ) 0 to 48 h after formulation of liposomes (H2O,
distearoyl-sn-glycero-3-phosphocholine, 2 mg/mL; molar ratio of
phospholipid:7 ) 20:1); (b) 7, c ) 2.24 × 10-4 M and (2S,6R)-8,
c ) 2.25 × 10-4 M; (c) time dependence of the Cotton effect of 7
(λ ) 226 nm), postformulation; (d) (2R,6R)-9, c ) 2.47 × 10-4
and ent-9 (calcd).
M
Compound 7 was formulated into a highly uniform,
unilamellar liposomes of distearoyl-sn-glycero-3-phospho-
choline (DSPC) as previously described,13 (phospholipid:7
ratio ) 20:1) prior to measurement of the CD spectrum.
(14) The higher order complexity of the L-CD spectrum of 7 (which is
the subject of our ongoing investigations) is indicative of delocalized
intermolecular excitons, or J-arrays: Fidder, H.; Knoester, J.; Wiersma, D. A.
J. Chem. Phys. 1993, 98, 6564–66.
(11) Plakinic acid L (4) also gave 5 under the same conditions.
(12) Compound 5 arises from C6-C7 bond scission initiated by a
“chloro-Fenton” reaction that captures Cl from the Fe ligand sphere by
radical rebound. See ref 7 and: Sawyer, D. T.; Hage, J. P.; Sobkowiak, A.
J. Am. Chem. Soc. 1995, 117, 106–9.
(15) (-)-Citronellol (10) obtained from TCI-EP (lot GM01) was of low
optical purity (72% ee, [R]20 -3.42 (neat) [lit. [R]20 -4.76 (neat)]:
D
D
Riena¨cker, R.; Ohloff, G. Angew. Chem. 1961, 73, 240). Consequently,
pure (-)-10 (98% ee, [R]20 -5.18 (neat)) was obtained by reduction
D
(13) The crude liposome suspension, prepared by shell evaporation of
a solution of naphthamides and DSPC in CHCl3 (2 mg/mL), followed by
sonication in water and thermal annealing, was repeatedly extruded under
pressure through a 100 nm pore polycarbonate membrane to give uniform
diameter, unilamellar liposomes.8
(NaBH4, MeOH) of (-)-citronellal (Takasago, 98% ee).
(16) Mori, K.; Masuda, S.; Suguro, T. Tetrahedron 1981, 37, 1329–
1340.
(17) Pappo, R.; Allen Jr, D. S.; Lemieux, R. U.; Johnson, W. S. J. Org.
Chem. 1956, 21, 478–9.
1526
Org. Lett., Vol. 12, No. 7, 2010

Scheme 3
.
Synthesis of Standards, Naphthamides 8 and 9
Table 1. Antifungal Activities of Plakinic Acids I-L (1-4)
(Minimum Inhibitory Concentration, MIC (µg/mL))a
1
2
3
4
C. albicans, ATCC 14053
C. albicans 96-489b
C. albicans UCD-FR1b
C. glabrata
0.25 0.25
0.06 <0.03 0.12 0.12
0.25 0.12
0.12 0.06
0.06 0.12
0.50 0.50
0.50 0.50
0.12 0.12
0.12 0.12
0.12 0.50
0.12 0.50
C. krusei
Cryptococcus neoformans var. gattii 0.12 0.25
C. neoformans var. grubii 0.12 0.25
a The in vitro susceptibilities were determined by the microbroth dilution
method according to the guidelines of the National Committee for Clinical
Laboratory Standards (NCCLS). b Fluconazole-resistant (MIC > 64 µg/mL).
active as plakinic acids K (3) and L (4). Compound 2 showed
the most potent activity (MIC < 0.12 µg/mL) against C.
albicans 96-489, C. albicans UCD-FRI, and C. glabrata
suggesting that the 1,2-dioxolane ring and a monomethyl-
branch in the ω-phenylalkyl side chain are key determinants
of antifungal activity.
21. Hydrogenolysis of 20 and 21 (Pd/C, CF3CH2OH)
provided primary amines 22 and 23 in 92% and 72% yield,
respectively. Separate acylation of 22 and 23 (6-methoxy-
2-naphthoyl chloride, Et3N, DMAP) gave naphthamides
(2S,6R)-8 and (2R,6R)-9.
In conclusion, liposomal CD differentiates long-chain
methyl-branched naphthamides, with epimeric configurations
at a stereocenter eight bonds remoVed from the chromophore.
Comparison of the L-CD spectrum of the naphthamide,
obtained by degradation of plakinic acid K (3), with those
of diastereomeric synthetic models, allowed assignment of
C8/C7 and C12/C11 of plakinic acids K (3) and L (4),
respectively. This remarkable long-range propagation of
stereochemical information is made possible by amplification
of the Cotton effects through ordering of lipid chains within
the liposomal bilayer. The current work also provides CD
references for interrogation of remote double methyl-
branched polyketides by L-CD. Additional applications of
L-CD are the subject of ongoing investigations in our
laboratories.
Comparisons of the L-CD spectra of three stereoisomers
are shown in Figure 2: 7 derived from 3 or 4 and synthetic
compounds (2S,6R)-8 and (2R,6R)-9. The signs and magni-
tudes of the CEs are primarily dominated by perturbation of
the naphthamide chromophore by the proximal stereocenter
C2 within the first sphere of asymmetry. It is also evident
that longer range perturbation from the remote methyl branch
alters the forms of L-CD spectra of (2S,6R)-8 and (2R,6R)-
9) as a function of the C6 configuration (Figures 2b and d).
Whereas the diastereomers (2S,6R)-8 and (2R,6R)-9 were
indistinguishable by NMR,19 they were clearly differentiated
by the CEs and fine structure of their L-CD spectra. In order
to evaluate the effect of the distal C6 stereocenter, the L-CD
of (2S,6R)-8 was compared with the inverted L-CD of
(2R,6R)-9 (Figure 2d) which corresponds to (2S,6S)-9. The
CEs in 8 [λmax 196 (∆ε + 11.0), 213 (-34.0), 226 (+ 27.3),
258 (-4.9)] were changed in 9, particularly λ 217 nm (∆ꢀ
+17.5) and λ 256 nm (∆ꢀ -6.2) (see Table S4 (Supporting
Information) for complete data).
The L-CD of 7 did not match either enantiomer of 9: it
was equal in magnitude and form but opposite in sign to
that of (2S,6R)-8 (Figure 2b). Therefore, 7 and (2S,6R)-8
are enantiomers, and the complete configurations of 3 and 4
are 3S,4S,6R,8R,12S and 3R,5R,7R,11S, respectively.
Compounds 1-4 were assayed for antifungal activity
against strains of Candida albicans, C. glabrata, C. krusei,
and Cryptococcus neoformans (Table 1). All compounds
were exceedingly potent antifungal agents (MICs e 0.5 µg/
mL) against all seven strains. The monomethyl-branched
ω-phenyl polyketide peroxides 1 and 2 were about twice as
Acknowledgment. We thank A. Marcus (University of
Oregon) for helpful discussions, S. Zea (Universidad Na-
cional de Colombia) for identification of the sponges, and
C. Green (Takasago International Corp.) for a generous gift
of (S)-(-)-citronellal. We are grateful to B. Morinaka (UC
San Diego) and A. Jansma (UC San Diego) for assistance
with NMR experiments and J. Pawlik (University of North
Carolina, Wilmington) and the crew of the RV Seward
Johnson for logistics of sponge collection. EI HRMS data
were provided by Y. Su (UC San Diego). The 500 MHz
NMR spectrometers were purchased with funds from the
NSF (CRIF, CHE0741968). Financial support of this work
was provided by the National Institutes of Health (CA1225601
and AI039987).
Supporting Information Available: Full isolation and
characterization of 3, 4, and plakinic acid M (S1), degradation
1
of 3 and 4, synthetic procedures, H and 13C NMR spectra
of 3-23, and CD spectra of 7-9. This material is available
free of charge via the Internet at http://pubs.acs.org.
(18) The assignment of the C2 configurations in 18 and 19 followed
from 1H NMR comparisons with analogues of known configuration:
Nicholas, G. M.; Molinski, T. F. Tetrahedron 2000, 56, 2921–27.
(19) 13C NMR (CDCl3, 125 MHz): Σ [∆δ (8-9)]2 < 0.07 ppm.
OL100249V
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