Absolute configuration of the α,ω-bifunctionalized sphingolipid leucettamol a from leucetta microrhaphis by deconvoluted exciton coupled CD

Article abstract of DOI:10.1021/np800549n

The configuration of leucettamol A (1), a known long-chain "two-headed" sphingolipid (dimeric sphingolipid) from the marine sponge Leucetta microrhaphis, was determined by conversion to an N, N', O, O'-tetrabenzoyl derivative, measurement of the exciton coupled circular dichroism spectrum (ECCD), and quantitative analysis by deconvolution of superposed exciton couplets. Contrary to the earlier assignment that claimed leucettamol A (1) was racemic, the CD approach unambiguously reveals the natural product is chiral and optically active and displays pseudo-C2 symmetry. The configuration of each end of the chain has erythro stereochemistry with an absolute configuration of 2R,3S,28S,29R. We show that deconvolution ECCD reliably predicts erythro versus threo vicinal amino alcohols in all cases with greater sensitivity (<5 nmol) compared to ¹H NMR J-based methods and provides verification of optical purity and unequivocal elucidation of absolute configuration in this difficult class of natural products.

Full text of DOI:10.1021/np800549n

J. Nat. Prod. 2009, 72, 353–359
353
Absolute Configuration of the r,ω-Bifunctionalized Sphingolipid Leucettamol A from Leucetta
microrhaphis by Deconvoluted Exciton Coupled CD
Doralyn S. Dalisay,† Sachiko Tsukamoto,‡ and Tadeusz F. Molinski*,§
Department of Chemistry and Biochemistry, and Skaggs School of Pharmacy and Pharmaceutical Sciences, UniVersity of California,
San Diego, La Jolla, California 92093-0358, and Department of Chemistry, Graduate School of Science, Chiba UniVersity, 1-33 Yayoi-cho,
Inage-ku, Chiba 263-8522, Japan
ReceiVed August 30, 2008
The configuration of leucettamol A (1), a known long-chain “two-headed” sphingolipid (dimeric sphingolipid) from the
marine sponge Leucetta microrhaphis, was determined by conversion to an N,N′,O,O′-tetrabenzoyl derivative, measurement
of the exciton coupled circular dichroism spectrum (ECCD), and quantitative analysis by deconvolution of superposed
exciton couplets. Contrary to the earlier assignment that claimed leucettamol A (1) was racemic, the CD approach
unambiguously reveals the natural product is chiral and optically active and displays pseudo-C2 symmetry. The
configuration of each end of the chain has erythro stereochemistry with an absolute configuration of 2R,3S,28S,29R.
We show that deconvolution ECCD reliably predicts erythro versus threo vicinal amino alcohols in all cases with
1
greater sensitivity (<5 nmol) compared to H NMR J-based methods and provides verification of optical purity and
unequivocal elucidation of absolute configuration in this difficult class of natural products.
Leucettamols A (1) and B (2) are long-chain bifunctionalized
sphingolipids reported by Kong and Faulkner from Leucetta
microrhaphis from Palau.1 Dimeric sphingolipids (DSs) are com-
prised of long-chain C28-C30 vicinal amino alcohols or 2-amino-
1,3-alkanediols that are related to sphingosine (3). Analogues of
D-sphingosine and phytosphingosine, including their ceramides, are
commonly encountered in marine invertebrates, but bifunctionalized
sphingolipids (dimeric bis-R,ω-amino alcohols) are rare and, so far,
have been reported only from marine sponges. In addition to 1 and
2, the known members include rhizochalin (4) from Rhizochalina
incrustata from Madagascar,2 oceanapiside (5)3 and oceanalin A4
from two different species of Oceanapia from Australia, calyxoside
(7) from Calyx sp. from Sulawesi,5 rhizochalins C (8) and D (9),6
and a compound named BSR1 (10) from an unidentified sponge7a
and L. microrhaphis,7b both from the Great Barrier Reef. DSs
display a range of biological activity: 4 exhibits weak antibacterial
activity against Staphylococcus aureus and cytotoxic activity against
mouse Ehrlich carcinoma cells. The glucoside (7) induces DNA
damage in cells and inhibits protein kinase C.3 Significantly, the
aglycone 5a of oceanapiside shows approximately 10-fold greater
antifungal activity against the pathogenic fluconazole-resistant yeast
Candida glabrata2a than monomeric vicinal amino alcohols such
as 3, implying amplification of activity through a multivalent effect.8
Leucettamol A (1) has recently been shown to inhibit the
Ubc13-Uev1A ubiquitin conjugating enzyme complex.9
Analysis of the stereostructure of DSs presents interesting
challenges. The biosynthesis of DSs displays stereochemical
heterogeneity between sponges; each terminus may be elaborated
with the same or different relative and absolute configurations. Since
there are four stereogenic centers in each DS, the maximum number
of possible stereoisomers is 16. If the biosynthesis of DSs follows
the well-established pathways for sphingosine and phytosphin-
gosine, the first step is predicted to be condensation of alanine or
serine with an activated fatty acyl precursor, with concomitant
decarboxylation followed by reduction. The products are 2-ami-
noalkanols or 2-amino-1,3-alkanediols, respectively, with either
Dedicated to Dr. David G. I. Kingston of Virginia Polytechnic Institute
and State University for his pioneering work on bioactive natural products.
* To whom correspondence should be addressed. Tel: +1 (858) 551-
1662. Fax: +1 (858) 822-0386. E-mail: tmolinski@ucsd.edu.
† Department of Chemistry, UC San Diego.
‡ Chiba University.
threo and erythro relative configuration and S or R absolute
configuration (with respect to the R-amino acid). Nevertheless, the
biosynthesis is not stereorandom. Each species of sponge appears
to produce a limited number of DS analogues but always with only
§ SSPPS, UC San Diego.
10.1021/np800549n CCC: $40.75
2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 01/21/2009

354 Journal of Natural Products, 2009, Vol. 72, No. 3
Dalisay et al.
Scheme 1
sitivity. The method readily converges upon a unique stereochemical
assignment and was used by one of us (T.F.M.) to assign 5,3b 4,2c
6,12 8, and 95 and by Kingston and co-workers to assign 7.5
Applying this method, we now report that 1 is not racemic as first
suggested, but an optically actiVe compound with pseudo-C2
symmetry.
Results and Discussion
Leucettamol A (1) was isolated from MeOH extracts of L.
microrhaphis by solvent partition and reversed-phase HPLC.13 The
compound showed spectroscopic values identical with literature
values,1 was highly prone to decomposition by autoxidation, and
was kept frozen (-20 °C) until needed. The structures of 1 and 2
are each unsymmetrical due to the offset placement of skipped
polyolefins in the C30 linear carbon long chain; however if one
considers only end groups for stereochemical analysis, the problem
can be reduced in complexity after removal of the double bonds.
In order to achieve this aim, a sample of 1 (ca. 0.5 mg) was
hydrogenated (Scheme 1) to give the constitutionally symmetrical
dimeric C30 bis-2,29-diamino-3,28-diol 11 (m/z 485.36 [M + H]+),
which was immediately converted without purification to the
corresponding N,N′,O,O′-tetrabenzoyl derivative 12 (freshly pre-
pared benzoyl N-imidazolide 13, DBU, dry CH3CN, 60-70 °C).14
The final product was purified by HPLC (6:94 i-PrOH-n-hexane)
Figure 1. Representations of major conformers contributing to
exciton coupling CD11a in threo and erythro isomers of N,O-
arenecarboxy derivatives of 2-amino-3-alkanols (i and ii) and
2-amino-1,3-alkanediols (iii and iW). Blue-shaded spheres represent
N-arenecarboxamide chromophores, and red spheres are O-aren-
ecarboxylate chromophores (e.g., arenecarboxy ) Ph(CO)O, ben-
zoate, λmax ≈ 228 nm). A positive exciton coupling (+ sign) is
defined by a split Cotton effect with a maximum CD signal at longer
wavelengths and minimum at shorter wavelengths. Contributions
to ECCD from the primary C-1 chromophore (e.g., benzoate) in
compounds iii and iW modulate the fine structure and intensity of
the split Cotton effect, but the signs remain the same as their
respective counterparts i and ii. The enantiomers of i-iW would
show mirror image CD spectra.
1
and characterized by MS (m/z 923.43, [M + Na+]) and H NMR
(CDCl3, 600 MHz, 1.7 mm microcryoprobe), which showed half
the number of H signals compared to the formula C58H80N2O6,
consistent with a structure of 12 with either C2 or meso symmetry.
In particular, downfield methine signals were observed due to H2/
H29 CH-NHBz groups (δ 4.46, m, 2H), H3/H28 CH-OBz groups
(δ 5.22, m, 2H), and the benzamide NH (δ 6.99, d, J ) 7.8 Hz,
2H).
Elucidation of the stereostructure assignment of 1 can be reduced
to defining which of the 10 unique stereoisomers 12a-j (Figure 2)
corresponds to 12 formed by hydrogenation of 1. It should be noted
that symmetry considerations reduce the maximum number of
possible stereoisomers from 16 for 1 to only 10 for 12; these consist
of two enantiomeric pairs of C2 isomers, 12a and 12d, two
enantiomeric pairs of C1 isomers, 12b and 12c, belonging to the
group that lacks symmetry, and two achiral compounds, the meso
dimers 12i and 12j.15
one stereostructure, but this configuration may differ in compounds
from different sponges.
Two claimed exceptions to this motif are 1 and 2, which were
each described as “an optically inactive...oil”. On the basis of
analysis of the relative configuration of 1 and a specific rotation of
[R]D ) 0, the authors conclude, “it must be assumed that leucettamol
A...is racemic”.10 Since we were aware that low specific rotations
were characteristic of natural long-chain dimeric amino alcohols,
and enzyme-catalyzed C-C bond formation in sphingosine is highly
enantiospecific, this anomaly may be explained if the leucettamols
were actually chiral and optically pure but of very low rotatory
power. Fortuitous procurement of a sample of 1 by reisolation from
Leucetta aff. microrhaphis (Haekel, 1872), collected in Indonesia,
allowed us to make a complete configurational assignment of 1
and 2, which is the subject of this report.
Exciton coupled circular dichroism (ECCD) between vicinal
dibenzoates is a powerful nonempirical method for assignment of
absolute configuration of glycols11 and also cyclic11b and acyclic
amino alcohols11c including “picomole” scale stereochemical
analyses of sphingosine and phytosphingosines.11d On the basis of
this work, we devised a method for quantitative ECCD to assign
all four stereocenters in DSs simultaneously3b that takes advantage
ofpairwiseexcitoncouplingsbetweenvicinalpairsofbenzamide-benzoate
chromophores (Figure 1). Stereochemical assignment requires a
single CD measurement of the corresponding perbenzoyl derivative,
only requiring samples as low as 5 nmol, but effectively decon-
volutes the superposed exciton couplings between as many as six
vicinal N,O-dibenzoyl pairwise interactions with nanomolar sen-
Assignment of relative configuration and absolute configuration
followed from ECCD of the tetrabenzoyl derivative 12 (see below);
however, independent verification of the erythro/erythro relative
configuration of 1 reported by Kong and Faulkner’s assignment1
was evident in the 1H NMR spectrum of 12. We had earlier shown2b
3
that the vicinal coupling constant JH2-H3 in N,O-dibenzoyl-2,3-
aminoalkanols can be used to assign threo (3J ) 5.2 Hz) versus
erythro (3J ) 2.6-2.7 Hz) relative configurations; however, the
differences between these values diminish in the corresponding
2-amino-1,3-alkanediol derivatives (3J ) 3.8 Hz threo; 3J ) 3.9-4.1
Hz, erythro). The J values also become difficult to measure
accurately in the latter compound due to the strong mutual coupling
of the C-1 oxymethylene protons and magnetically inequivalent
vicinal coupling to H-2. Both factors may lead to erroneous

Leucettamol A Configuration by ECCD
Journal of Natural Products, 2009, Vol. 72, No. 3 355
Figure 2. Representation of all possible stereoisomers of dimeric R,ω-dimeric amino alcohols 12a-j (only end groups depicted) corresponding
to the hydrogenation product of leucettamol A (1). The first four rows depict chiral stereoisomers in the left column and their corresponding
enantiomers in the right column.
assignments of configuration in amino alcohols. Fortunately, a
highly reliable diagnostic indicator for relative configuration of the
vicinal amino alcohols is the chemical shift of the amide NH in
perbenzoyl derivatives in both 2-amino-3-alkanols and 2-amino-
1,3-alkanediols. Differences in the degree of intramolecular hy-
drogen bonding lead to an NH signal in the threo stereoisomer that
occurs at an unusually high field (δ ≈ 6.38-6.63, d, J ) 8.9-9.2
Hz) compared to that of the erythro isomer (δ ≈ 6.97-7.10, d, J
) 7.5-8.7 Hz).2b The leucettamol A derivative 12 showed a
downfield NH signal (δ 6.99, 2H, J ) 7.8 Hz) strongly suggestive
of the erythro configuration at each end of the chain.16 This is
entirely consistent with the relative configuration that Kong and
Faulkner assigned to 1 and 2, as depicted in the reported
structures.17
The specific rotation of 12, with attendant errors of a low rotatory
compound, is not expected to be informative of the configuration
at all four stereocenters. In contrast, CD readily revealed 12 was
chiral and nonracemic even with only 90 µg of sample. Tetrabenzoyl
derivative 12 gave a positive bisignate CD spectrum (split Cotton
effect, MeOH, c 20 µM) (Figure 3) of moderate intensity (λ 222
nm, ∆ꢀ -2.80; λ 238 nm, ∆ꢀ +10.29) due to ECCD between vicinal
N-benzamide-O-benzoate pairs. Two erythro/erythro configurations
can be formulated for 12; one in which the absolute configuration
at each corresponding stereogenic center is the same (overall C2
symmetry, 12a) or one in which the absolute stereostructures of
the opposing termini are inverted with respect to one another by a
mirror plane that bisects C-15-C-16 (overall meso symmetry, 12i).
Clearly, the meso isomerssand for that matter, racemates of C1
and C2 stereoisomerssare optically inactive and can be eliminated
here by observation of a nonzero CD spectrum for 12 (Figure 3).
The final assignment of 1 was readily made by quantitative
comparison of the CD spectrum of its derivative 12 with “hybrid
CD spectra” generated by all possible linear combinations of CD
spectra of two well-characterized model compounds 14 and 15
prepared from (2S)-alanine2b and their enantiomeric “virtual CD
spectra” obtained by inversion of the first two. Figure 3 shows
overlays of the measured CD spectrum of 12 with “hybrid CD
spectra” (note: only nonzero combinations 12a-h are shown here).
An excellent match is seen between the hybrid CD spectrum of
the ent-erythro/ent-erythro pair 12e and the measured CD spectrum
of 12, showing the two share the same absolute configuration. The
same match was found when the spectra were recorded in CH3CN,
even though the nonprotic solvent produces slightly different
amplitudes of maxima and minima for all CD spectra, due to slightly
different blue shifts in λmax benzamide and benzoate chromophores
and other minor changes in fine structure at ∼200 nm. These CD
results provide a second independent verification of the erythro/
erythro relative configuration assigned first by Kong and Faulkner1
and corroborated here by NMR (see above). Therefore, leucettamol
A (1) is not racemic but a C2 diastereomer of the configuration
(2R,3S,28S,29R). Since leucettamol B (2) co-occurs with 1 in L.
microrhaphis,1 it is almost certain both compounds have the same
configuration at C-2, C-3, C-28, and C-29.18
Leucettamol A (1) has erythro end groups, like D-sphingosine
(3), but is of the opposite absolute configuration. The minor anomaly
of reported “zero rotation” can now be explained. Leucettamol A
(1) is clearly chiral and its optical rotation cannot be zero, except
by coincidence. In our hands, the specific rotation of 1 was

356 Journal of Natural Products, 2009, Vol. 72, No. 3
Dalisay et al.
Figure 3. Circular dichroism (CD) spectra of leucettamol A derivative 12 (c ) 2.5 × 10-5 M, MeOH, 23 °C) (dashed line) overlaid with
“hybrid CD” spectra (solid) generated by linear combinations of the measured CD spectra of erythro-14 and threo-15 and their enantiomers
(ent-). (c ≈ 2 × 10-4 M, MeOH, 23 °C, see ref 3b) as follows: (a) erythro-14 + erythro-14, (b) erythro-14 + threo-15, (c) ent-erythro-14
+ threo-15, (d) threo-15 + threo-15, (e) ent-erythro-14 + ent-erythro-14, (f) ent-erythro-14 + ent-threo-15, (g) erythro-14 + ent-threo-15,
(h) ent-threo-15 + ent-threo 15.
established as [R]D -3.8 ( 0.1 (c 4.4, MeOH) (averaged measure-
ments N ) 10). Therefore, the [R]D ) 0 reported for 1 by Kong
and Faulkner1 might have been close to the limits of detection of
R under the conditions of measurement.19 Specific rotations [R]D’s
of optically pure natural product amines and amino alcohols are
characteristically low and often masked by very low measured
optical rotations, especially with small samples, contamination with
highly rotatory substances, and differences between free base and
protonated salt forms. Derivatization of the NH2 and OH groups
can lead to more reliable measurements. For example, the magni-
tudes of [R]D increase by 2- to 3-fold upon acetylation or
benzoylation of sphingosine.20 Discrimination of threo from erythro
ochemical assignment by correlation of configuration with the split
Cotton effects that arise from exciton coupled benzoyl chro-
mophores.24 Nakanishi and co-workers showed greatly improved
sensitivity of ECCDs using N,O,O-trinaphthoyl derivatives of
sphinganine (dihydrosphingosine) and phytosphingosine,14,25 and
even more distinct CD fingerprints have been obtained by dif-
ferential acylation of primary and secondary NH2 and OH groups.25
When the dihedral angle in staggered conformations of vicinal
benzoate-benzamide pairs rotates in the positive direction (positive
helicity, see Figure 4), a positive split Cotton effect is observed; a
negative sign is observed for a negative helicity.11a The magnitude
of the ECCD is related to the dihedral angle and is maximum when
the angle subtended by the electronic transition dipole moments θ
) 70° but zero when θ ) 0° or 180°.11a The motion-averaged
direction of the electronic transition dipole moments lies ap-
proximately along the C-N and C-O single bond, respectively.
Unlike vicinal benzamide-benzoate pairs in rigid cyclic systems,
where the dihedral angles are fixed, the net magnitude and sign of
the split Cotton effect in acyclic systems are subject to conforma-
tional dependences. The observed CD spectrum of threo-i or
erythro-ii is expected to be the sum of ECCD contributions for all
the possible conformers of each diastereomer, and the net effect is
largely determined by the Boltzmann-weighted staggered conforma-
tions a-c shown in Figure 4.
1
isomers of sphingosine by H NMR has been demonstrated with
the corresponding Mosher’s acid derivatives, which are also useful
for estimating optical purities of sphingosine samples containing
as little as 1% of the minor enantiomer.21 Nevertheless, errors of
assignment in configuration of even simple monomeric marine
amino alcohols have been made, and sole reliance upon some
methods may be risky. For example, the xestaminols22 first reported
by Gulavita and Scheuer as (2S,3R) compounds were later corrected
to (2R,3S) after total synthesis and [R]D comparisons of their diacetyl
derivatives.23
N,O-Dibenzoyl derivatives of sphingolipids show characteristic
CD spectra useful for characterization of very small amounts of
compound. Munesada and co-workers first applied CD to perben-
zoyl sphingosine obtained from frog brain cerebrosides for stere-
Investigations of dibenzoyl and derivatives of acyclic vicinal
2-amino-1-alkanols11b show that prediction of the sign of the split

Leucettamol A Configuration by ECCD
Journal of Natural Products, 2009, Vol. 72, No. 3 357
by even small amounts of another diastereomer (e.g., an ent-erythro/
erythro-diasteromeric contaminant in an ent-erythro/ent-erythro
sample) would amplify mismatches between hybrid and measured
CD spectra and be quickly revealed. This is not the case with
measurements of optical activity, which are notoriously unreliable
for natural products with weak [R]D’s or cross-contamination by
highly rotatory congeners.
In the five dimeric sphingolipids we have examined so far, the
natural product has been isolated as a homochiral entity; that is,
compounds from a single sponge specimen appear as only one
diastereomeric modification of one enantiomeric form (>95% ee).
Nature appears to show high fidelity in enantiospecific biosynthesis
of each DS, and this may reflect a tightly coupled enzymic process
that unifies construction of the amino alcohol end group with long-
chain dimerization, although the details of the latter process are
still unclear.
The mis-assignment of 1 and 2 as “racemic”1 is understandable
from one point of view. Low molar rotations are often observed
for acyclic and alicyclic aminoalkanes and amino alcohols and can
be further lowered when functional groups contribute molar
rotations of opposing signs. Nevertheless, the biosynthesis of
sphingolipids can be expected to occur under stringent enzyme
control with high enantioselectivity, although the stereospecificity
may vary between different species of sponge and produce
stereodiads at termini with different configurations. Although
racemic and partially racemic chiral long-chain lipids28 and
terpenoids29 have been observed in Nature, these seem to be
exceptions. It seems unlikely that leucettamols would be biosyn-
thesized with complete racemization. Even if they were, it seems
even less likely that only one diastereomer of 1 instead of two or
more would be present in the same sponge.
Finally, it is of interest to note that L. microrhaphis, the sponge
that produces 1 and 2, belongs to the less common group of
calcareous sponges; all other DSs come from Demospongia of the
family Oceanapiidae. The outstanding compound in the series, the
polyene dimeric sphingolipid BRS1 (10), is clearly a leucettamol
analogue and has been isolated from two sponges from the Great
Barrier Reef: another specimen of L. microrhaphis7b,30 and the
earlier finding7a from a sponge of uncertain identity.
Figure 4. Staggered conformers contributing to exciton coupling
CD in threo (ia-ic) and erythro (iia-iic) isomers of acyclic
dibenzoyl 2-amino-3-alkanols. Blue- and red-shaded spheres rep-
resent N-Bz and O-Bz chromophore groups, respectively (λmax
≈
228 nm). The net signs of the observed split Cotton effects are
consistent with predominant contributions from conformers ia and
iia, respectively. Conformers ib and iic have antiparallel transition
dipole moments and give zero contributions to the ECCDs. See
also Figure 1.
Cotton effect is upheld by both CD measurements and calculations
of conformer populations. Our own empirical CD and NMR
investigations of 3-amino-2-alkanols3b also support this model; for
example, the larger H2-H3 vicinal coupling constant in the threo
diastereomer (Figure 4, R ) n-Pr, J ) 5.2 Hz) shows the dominant
conformer is ia with the antiperiplanar arrangement of the
H2-C2-C3-H3 bonds, but in the erythro isomer (J ) 2.6 Hz) it
is gauche iia. It is of interest to note that erythro and threo
diastereomers of vicinal benzamide-benzoates are opposite in sign,
with magnitudes that are similar but not equal (Figure 1). This is
in contrast to vicinal dichromophoric derivatives of 1,2-glycols in
which the magnitude of the ECCD in the threo isomer is almost
zero due to a dihedral angle θ ) 180° between the C-O bonds in
the predominant anti-periplanar conformer.26 The reason for the
difference is likely related to hydrogen bonding of the NH in the
amide group to the vicinal benzoate oxygen in both ia and iia that
stabilize these gauche conformations with consequently larger
contributions to the net ECCDs.
We have exploited N,O-benzoyl derivatives by extension to
dimeric sphingolipids using a simple one-step perbenzoylation and
interpretation of the resultant unique CD pattern. The results are
easily read; simple superposition of the exciton coupled Cotton
effects from expected linear combinations of threo and erythro
isomeric end groups in 2-amino-3-alkanols can be deconvoluted
by permutations of the constituent short-chain perbenzoyl
derivatives.3b
The unambiguous assignment of 1 now allows some summary
comparative notes on this interesting class of natural products and
outcomes from application of the deconvolution of ECCD. The ∆ꢀ
values measured in the ECCD of benzoate-benzamide pairs in these
studies are recorded using highly dilute solutions (10-4-10-5 M)
underconditionswhere,likeUV-visspectroscopy,theBeer-Lambert
law is obeyed.27 The method is applicable even to natural products
that are obtained in submilligram amounts, since accurate concen-
trations of perbenzoyl derivatives are reliably measured from UV
absorbance values together with the precisely recorded molar
absorbtivities, ꢀ, that we reported in the original study.3b In turn,
this allows confidence in estimating stereochemical purity and
possible inhomogeneity or “contamination” of natural product long-
chain DSs. Homochiral perbenzoyl derivatives of homologues and
some analogues of DSs exhibit CD spectra that are matched both
by sign and by magnitude of the Cotton effects. Cross-contamination
In conclusion, we have completed the stereochemical assignment
of leucettamol A (1) by CD and report the complete configuration.
The deconvolution ECCD method for assignment of dimeric
sphingolipids,2c,3 as well as simple monomeric 2-amino-3-alkanols
(e.g., 6),12 is well suited to distinguish enantiomers of both threo
and erythro stereoisomers of amino alcohols at nanomole sensitivity.
This is particularly useful for those natural products with [R]D’s
that, through happenstance, have magnitudes near zero and
deceptively present themselves as racemic or meso.
Experimental Section
General Experimental Procedures. Optical rotations were measured
using a Jasco P1020 polarimeter. 1H NMR spectra were recorded on a
Bruker DRX-600 (600 MHz, 1H, 1.7 mm CPTCI microcryoprobe) and
Varian Mercury 400 (400 MHz) spectrometers in CDCl3 using residual
CHCl3 (δ 7.26 ppm) as an internal standard. CD spectra were measured
on a Jasco J810 spectropolarimeter using spectroscopic grade solvents
(Fisher EM) and a 2 mm quartz cell with 50 nm/min scan rate and 1
nm slit. FTIR was measured using a Jasco FT-4100 spectrometer
equipped with a Pike MIRacle ZnSe ATR plate. LR ESI mass spectra
were obtained on a ThermoElectron MSQ single quad mass spectrom-
eter coupled to an Accela UPLC. HRESI mass spectra were provided
by the University of California, Riverside, mass spectrometry facility.
Semipreparative HPLC was carried out using a Rainin binary HPLC
system (Dynamax C18 column, 10 × 250 mm, 5 µm, 3 mL/min or
Dynamax silica 4.6 × 250 mm, 3 µm in specified solvent systems)
coupled to a refractive index detection (Waters R401) and Isco UV-6
detector equipped with a filter for 250 nm. Flash column chromatog-
raphy was carried out on silica (43-60 µm, EM 360) using a low-
pressure solvent delivery system, and TLC was performed on silica

358 Journal of Natural Products, 2009, Vol. 72, No. 3
Dalisay et al.
gel coated 0.2 mm aluminum backed plates with visualization by
vanillin-H2SO4-EtOH or ceric ammonium nitrate(aq).
Supporting Information Available: Measured CD spectra of 12
and “hybrid spectra” in CH3CN, MS and 1H NMR spectra of 12.
This material is available free of charge via the Internet at http://
pubs.acs.org.
Animal Material. The sponge Leucetta aff. microrhaphis (Haekel,
1872) was collected by hand using scuba at a depth of 10 m in North
Sulawesi, Indonesia (September 2006), and immediately soaked in
EtOH. The sponge was identified by Rob van Soest, and a voucher
sample (ZMAPOR20126) was deposited in the Institute for Systematics
and Ecology, University of Amsterdam, The Netherlands.
References and Notes
(1) Kong, F. H.; Faulkner, D. J. J. Org. Chem. 1993, 58, 970–971.
Extraction and Isolation. The EtOH extract of the sponge was
evaporated, and the aqueous residue extracted with EtOAc followed
by n-BuOH. The n-BuOH fraction (0.61 g) was subjected to reversed-
phase column chromatography (2:3 CH3CN-H2O) to afford leucettamol
A (1, 202 mg, 0.25% w/w) with spectroscopic properties (MS, 1H NMR,
13C NMR) identical with literature values.1 [R]D -3.8 ( 0.1 (c 4.4,
MeOH, 10 measurements), lit.1 [R]D 0 (c 1.26, MeOH).
(2) (a) Makarieva, T. N.; Denisenko, V. A.; Stonik, V. A.; Milgrom, Yu.
N.; Rashkes, Ya. W. Tetrahedron Lett. 1989, 30, 6581–6584. (b)
Makarieva, T. N.; Guzii, A. G.; Denisenko, V. A.; Dmitrenok, P. S.;
Santalova, E. A; Pokanevich, E. V.; Molinski, T. F.; Stonik, V. A. J.
Nat. Prod. 2005, 68, 255–257. (c) Molinski, T. F.; Makarieva, T. N.;
Stonik, V. A. Angew. Chem., Int. Ed. 2000, 39, 4076–4079.
(3) (a) Nicholas, G. M.; Hong, T. W.; Molinski, T. F.; Lerch, M. L.;
Cancilla, M. T.; Lebrilla, C. B. J. Nat. Prod. 1999, 62, 1678–1681.
(b) Nicholas, G. M.; Molinski, T. F. J. Am. Chem. Soc. 2000, 122,
4011–4019.
No leucettamol B (2) was detected in this sample of L. aff.
microrhaphis.
Hydrogenation of Leucettamol A (1). Hexahydroleucettamol A
(11). A mixture of 1 (0.5 mg) in MeOH (2.0 mL) and 10% Pd-C (0.2
mg) was stirred under an atmosphere of hydrogen for 18 h. The mixture
was filtered through Celite and concentrated to give crude 11 (ca. 0.1
mg). LR ESIMS m/z 485.4 [M + H]+; calcd 485.5 for C30H65N2O2.
This material was used immediately in the next step.
(4) Makarieva, T. N.; Denisenko, V. A.; Dmitrenok, P. S.; Guzii, A. G.;
Santalova, E. A.; Stonik, V. A.; MacMillan, J. B.; Molinski, T. F.
Org. Lett. 2005, 7, 2897–2900.
(5) Zhou, B. N.; Mattern, M. P.; Johnson, R. K.; Kingston, D. G. I.
Tetrahedron 2001, 57, 9549–9554.
(6) Makarieva, T. N.; Dmitrenok, P. S.; Zakarenko, A. M.; Denisenko,
V. A.; Guzzi, A. G.; Li, R.; Skepper, C. K.; Molinski, T. F.; Stonik,
V. A. J. Nat. Prod. 2007, 70, 1991–1998.
(7) (a) Willis, R. H.; De Vries, D. J. Toxicon 1997, 35, 1125–1129. (b)
Kehraus, S.; Konig, G. M.; Wright, A. D.; Woerheide, G. J. Org.
Chem. 2002, 67, 4989–4992.
(8) Nicholas, G. N.; Li, R.; MacMillan, J. B.; Molinski, T. F. Bioorg.
Med. Chem. Lett. 2002, 12, 2159–2162.
(9) Tsukamoto, S.; Takeuchi, T.; Rotinsulu, H.; Mangindaan, R. E. P.;
van Soest, R. W. M.; Ukai, K.; Kobayashi, H.; Namikoshi, M.; Ohta,
T.; Yokosawa, H. Bioorg. Med. Chem. Lett. 2008, 18, 6319–6320.
(10) Also implicit in the logic of their deduction is that leucettamols must
be chiralsand racemicsbecause both ends of each molecule have the
same configuration and that the positions of the skipped olefins in the
long chains make them constitutionally unsymmetrical, but the
molecules would still be chiral even if they were constitutionally
symmetrical (with the exception of the meso form, in which each end
has the same relative configuration but with mirror image absolute
configurations; see text and Figure 1 for further discussion). Com-
parison of the rotation of C28 rhizochalin aglycone (4a, [R]D +11, ref
2a) with the zero specific rotation observed for 1 and 2 is also invoked
to further support the conclusion that “both leucettamols. .are racemic”;
however as we have shown elsewhere (ref 2c), the former molecule
represents a pseudo-C2 molecule that is fully expected to be chiral,
regardless of the presence of the near mid-chain keto group.
(11) (a) Harada, N.; Nakanishi, K., Circular Dichroic Spectroscopy: Exciton
Coupling in Organic Stereochemistry; University Science Books: Mill
Valley, CA, 1983; p 460. (b) Kawai, M.; Nagai, U.; Katsumi, M.
Tetrahedron Lett. 1975, 16, 3165–3166. (c) Searle, P. A.; Molinski,
T. F. J. Org. Chem. 1993, 58, 7578–7580. (d) Kawamura, A.; Berova,
N.; Dirsch, V.; Mangoni, A.; Nakanishi, K.; Schwartz, G.; Bielawska,
A.; Hannun, Y.; Kitagawa, I. Bioorg. Med. Chem. 1996, 4, 1035–
1043.
(12) Kossuga, M. H.; MacMillan, J. B.; Rogers, E. W.; Molinski, T. F.;
Nascimento, G. S. F.; Rocha, R. M.; Berlinck, R. G. S. J. Nat. Prod.
2004, 67, 1879–1881.
(13) No leucettamol B (2) was detected in these extracts.
(14) Ikemoto, N.; Lo, L.-C.; Nakanishi, K. Angew. Chem., Int. Ed. 1992,
31, 890–891.
(15) Although the hydrogenation product 11 of leucettamol A (1) was
revealed to be a constitutional C2 dimer, the assignment of configu-
ration in 1 would have been made more complicated if it were C1
because of the need to place each inequivalent end group at correct
ends of the unsymmetrical skipped polyene chain. Herein lies a
limitation to the CD method. However the correct assignment could
still be made by 2D NMR spin correlation (e.g., TOCSY, HMBC) of
the compound prepared by per-benzoylation of 1 without hydrogena-
tion and using the empirical NH chemical shift method described in
the text to identify threo and erythro end groups.
N-Benzoylimidazolide (13).31 A suspension of imidazole (4.54 g,
0.0668 mol) in dry benzene (200 mL) was treated with a solution of
benzoyl chloride (4.69 g, 0.0333 mol) in benzene (20 mL) at 8 °C in
a flask fitted with a drying tube. The mixture was allowed to warm to
room temperature and stirred for 16 h. The precipitated imidazole
hydrochloride was removed by filtration through a fritted funnel, and
the clear filtrate concentrated under reduced pressure to give N-benzoyl
imidazolide (13) as a viscous, hygroscopic oil (5.78 g, quant.), which
1
was stored at 0 °C in a tightly stoppered vessel. H NMR (CDCl3)
8.05 (s, 1H), 7.78 (bd, 2H, J ) 8.2 Hz), 7.67 (tt, 2H, J ) 8.2, 1.5 Hz),
7.58 (bt, 1H, J ) 7.4 Hz), 7.53 (bs, 1H), 7.16 (bs, 1H).
N,N′,O,O′-Tetrabenzoylhexahydroleucettamol A (12). A solution
of 11 (0.1 mg, 0.2 µmol) in dry CH3CN (0.5 mL) was treated with a
solution of freshly prepared N-benzoylimidazole 13 (0.36 mg, 2.0 µmol)
in dry CH3CN and DBU (0.28 mg, 1.8 µmol) at room temperature,
then heated (70 °C) with stirring under an atmosphere of nitrogen for
18 h. The volatiles were removed under a stream of N2, and the residue
was dissolved in 0.3 mL of CHCl3, loaded in a pipet column (silica),
and eluted with 3:7 EtOAc-hexanes. After elution of nonpolar UV-
active byproducts, the more polar product 12 was eluted and further
purified by HPLC (6:94 i-PrOH-n-hexane, Alltech 5 µm silica, 4.6 ×
250 mm, tR ) 7.25 min, 1.5 mL/min). UV (MeOH) λ 227 nm (ꢀ
39 900). CD (MeOH) λ 222 nm (∆ꢀ -2.80), 229 (∆ꢀ 0), 238 (∆ꢀ
+10.30). CD (CH3CN) λ 221 nm (∆ꢀ -4.59), 229 (∆ꢀ 0), 237 (∆ꢀ
+10.03). See Figure 3 for CD spectra in MeOH and Supporting
Information for CD spectra in CH3CN. FTIR (ATR) ν 3600bs, 2264,
2362, 1623 cm-1; 1H NMR (CDCl3) δ 8.09 (d, 4H, J ) 8.4 Hz; ortho-
PhCOO), 7.76 (d, 4H, J ) 8.4 Hz; ortho-PhCONH), 7.41-7.60 (m,
12H; aryl H), 6.99 (d, 2 × 1H, J ) 7.8 Hz, C2/C29-NH), 5.22 (m, 2
× 1H, H3/H28), 4.46 (m, 2 × 1H, H2/H29), 1.20-1.30 (m, 48H),
1.29 (d, 2 × 3H, J ) 6.6 Hz, H1/H30); LR ESIMS m/z 923.5908 [M
+ Na]+, calcd for C58H80N2NaO6 923.5914.
CD Measurements and Generation of “Hybrid CD” Spectra.3
CD spectra were measured on a Jasco J-810 spectropolarimeter using
solutions prepared in HPLC grade MeOH or CH3CN. All measurements
were carried out with N ) 20 scans in dilute solutions (20-200 µM)
in cells of CD grade quartz (2 mm path); scan speed 50 nm/min, slit
width 1 nm. No smoothing or noise reduction was applied to CD data.
Digitized spectral files (1 nm/data point) were exported as ASCII files
and parsed into Excel spreadsheets (MS Office 2004) prior to column
additions-subtractions to yield “hybrid CD” spectra. Tabulated CD
data of 14 and 15 were obtained under similar conditions as reported
earlier3b and plotted against “hybrid spectra” using Kaleidagraph 4.0
(Synergy Software) running on an iMac computer (Apple, Inc.).
(16) In our opinion, the (CO)NH 1H NMR chemical shift in CDCl3 only is
more reliable for assignment of relative configuration, in most cases,
than measurements of vicinal couplings or NOEs in the corresponding
cyclic oxazolidinone1 or thiazolidinone derivatives.
(17) In the text of the paper (ref 1), the authors state, apparently in
contradiction to their depicted structure, that 1 has, “threo stereo-
chemistry at both ends of the molecule”. This appears to be a
typographical error since the 1H NMR data support the all-erythro
configuration for leucettamol A as shown.
Acknowledgment. We thank R. van Soest (University of Amster-
dam) for identification of the sponge. This work was supported by grants
from NIH (AI039987 and CA122256, to T.F.M), grants-in-aid for
Scientific Research (No. 18032033 and 19310140, to S.T.) from the
Ministry of Education, Culture, Sports, Science, and Technology of
Japan, and grants from the Naito Foundation and the Keimeikai
Foundation (S.T.).

Leucettamol A Configuration by ECCD
Journal of Natural Products, 2009, Vol. 72, No. 3 359
(18) Leucettamol B (2) is also an expected product of oxidation of 1 by a
singlet oxygen ene-reaction followed by disproportionation of the
resultant hydroperoxide i and may possibly be an artifact.
(21) Li, S.; Wilson, W. K.; Schroepfer, G. J. J. Lipid Res. 1999, 40, 764–
772.
(22) Gulavita, N. K.; Scheuer, P. J. J. Org. Chem. 1989, 54, 366–369.
(23) Mori, K.; Matsuda, H. Liebigs Ann. Chem. 1992, 131–137.
(24) Munesada, K.; Yuasa, M.; Suga, T. J. Chem. Soc., Perkin Trans. 1
1991, 189–194.
(25) Wiesler, W. T.; Nakanishi, K. J. Am. Chem. Soc. 1989, 111, 3446–
3447.
(26) Zhao, N.; Zhou, P.; Berova, N.; Nakanishi, K. Chirality 1995, 7, 636–
651.
(27) We have not been able to detect second-order effects of the CD spectra
of perbenzoyl DSs due to intramolecular π-π stacking under the
conditions of measurement. Such effects are either too weak to observe
or averaged out by dynamical behavior under the conditions of the
measurements (c ≈ 10-4-10-5 M, 23 °C, MeOH or CH3CN).
(28) Skepper, C. K.; Molinski, T. F. J. Org. Chem. 2008, 73, 2592–2597.
(29) (a) Horton, P.; Inman, W. D.; Crews, P. J. Nat. Prod. 1990, 53, 143–
151. (b) Searle, P. A.; Molinski, T. F. Tetrahedron 1994, 50, 9893–
9908.
(19) The different [R]D we observed may partly be due to instrumental
limitations. In our work, a standard 1 cm “microcell” (volume ) 200
µL) and solution of 44 mg/mL of 1 (MeOH) was used for rotation
measurements (Jasco P2000 digital polarimeter), giving a measured
rotation of-0.017 millidegrees for the solution of 1. Back-calculating
the measured R from the specific rotation reported by Kong and
Faulkner, [R]D 0 (c 1.26, MeOH), gives R )-0.0478 in a 10 cm cell
or R )-0.00478 in a 1 cm cell. The latter value is close to the “limit
of detection” (twice the S/N) of older model polarimeters.
(20) For example, D-erythro-sphingosine, [R]D-7 (c 0.8, CHCl3). Solladie-
Cavallo, A.; Koessler, J. L. J. Org. Chem. 1994, 59, 3240-3242;
N,O,O′-triacetyl sphingosine, [R]D19-24.1 (c 13.7, MeOH), [R]D-13
(c 0.5, CHCl3); N,O,O′-tribenzoyl sphingosine, [R]D-11.2 (c 10,
pyridine), Combined Chemical Dictionary; Taylor and Francis, 2008,
http://www.chemnetbase.com/.
(30) A sample of BSR1 was kindly provided to us in 2003 by Professor
G. Ko¨nig, but had already decomposed before we could investigate
the configuration.
(31) Gerngross, O. Ber. 1913, 46, 1908–1913.
NP800549N