Rhizochalins C and D from the sponge Rhizochalina incrustata. A rare threo-sphingolipid and a facile method for determination of the carbonyl position in α,ω-bifunctionalized ketosphingolipids

Article abstract of DOI:10.1021/np0704811

Rhizochalins C and D (1, 2), new representatives of two-headed glycosphingolipids, were isolated from the sponge Rhizochalina incrustata. Rhizochalin D is an unexpected C₂₉ homologue of the canonical C ₂₈ dimeric sphingolipid structu

Full text of DOI:10.1021/np0704811

J. Nat. Prod. 2007, 70, 1991–1998
1991
Rhizochalins C and D from the Sponge Rhizochalina incrustata. A Rare threo-Sphingolipid and
a Facile Method for Determination of the Carbonyl Position in r,ω-Bifunctionalized
Ketosphingolipids
Tatyana N. Makarieva,*,† Pavel S. Dmitrenok,† Alexander M. Zakharenko,† Vladimir A. Denisenko,† Alla G. Guzii,†
Ronghua Li,‡ Colin K. Skepper,§ Tadeusz F. Molinski,§ and Valentin A. Stonik†
Laboratory of MaNaPro Chemistry, Pacific Institute of Bioorganic Chemistry of the Russian Academy of Sciences, 690022 VladiVostok-22,
Russian Federation, 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, UniVersity of California, DaVis, California 95616
ReceiVed September 10, 2007
Rhizochalins C and D (1, 2), new representatives of two-headed glycosphingolipids, were isolated from the sponge
Rhizochalina incrustata. Rhizochalin D is an unexpected C29 homologue of the canonical C28 dimeric sphingolipid
structures. Their structures including absolute configurations were established using spectroscopic data, micromolar-
scale Baeyer–Villiger oxidation, and LCMS interpretation of the products. Application of the latter method leads to a
revision of the structure of oceanapiside and placement of the keto group at C-18 rather than C-11.
3
1
Two-headed sphingolipids represent a group of rare bis-R,ω-
amino alcohols from the marine sponges Rhizochalina incrustata,1,2
Oceanapia phillipensis,3 Oceanapia sp.,4 Calyx sp.,5 Leucetta
leptorhapsis,6 and an unidentified sponge.7 Early findings on
biological properties of two-headed sphingolipids included anti-
bacterial activity against Staphylococcus aureus, cytotoxic activity
against mouse Ehrlich carcinoma cells,1 antifungal activity against
the pathogenic fluconazole-resistant yeast Candida glabrata,3,4
selective DNA-damaging activity,5 and inhibition of protein
kinase C.7
In the course of our continuing studies on marine natural
products,8,9 we have found several additional two-headed sphin-
golipids, including two new compounds we have named rhizocha-
lins C (1) and D (2). In this report we describe the isolation and
structure elucidation of both 1 and 2 including absolute configura-
tions. In contrast to the regular (2S,3R)-configuration found in
normal sphingoid bases, rhizochalin C contains the rare (2R,3R)
threo stereochemistry, while 2 possesses an odd-numbered C29
hydrocarbon chain instead of C28 found in canonical members of
this series.1–5
seen for oceanapiside. The H and 13C NMR data of 1 (Table 1)
revealed signals typical of a pseudo-dimeric amino alcohol glyco-
side, reminiscent of 3 and its known analogues.1–5 These included
signals for one secondary methyl group (δH 1.25, δC 16.0), one
oxymethylene group (O-CH2, δH 3.76, 3.64; δC 59.6), two N-
substituted CH carbons (δH 3.12, 3.05; δC 57.5, 53.3), two
oxymethines (δH 3.86, 3.40; δC 77.0, 73.0), and one ketone carbonyl
group (δC 215.0), flanked by two CH2 groups (2 × CH2, δH 2.43;
2.42; δC 43.4; 43.5). The remainder of the signals were assigned
to (CH2)n chains (δH 1.27-1.29; δC 30.8–31.6) and a sugar moiety.
In contrast with 3, the carbon backbone of sphingolipid 1 contains
two different end-groups, the R-terminus formally derived from
serine and the ω-terminus from alanine. The structures of these
moieties were confirmed by 2D NMR data including HMBC
experiments. Collectively, the NMR data of 1 were found to be
similar to those previously seen in oceanpiside except signals due
to the sugar group.3 A galactose unit was suggested by signals at
δC 104.6 and δH 4.32 (d, J ) 7.2 Hz), δC 73.3 and δH 3.51 (dd,
7.2, 9.8), δC 75.1 and δH 3.47 (dd, 3.4, 9.8), δC 71.1 and δH 3.78
(dd, 3.4), δC 77.6 and δH 3.54 (dd, 4.6, 6.5), and δC 63.6 and δH
3.72 and 3.74 (m). The 3J-coupling constant of the anomeric proton
H-1′ (d, J ) 7.2 Hz) indicated that rhizocalin C is a ꢀ-galactoside.
The HMBC spectrum also showed a cross-peak from H-1′ (δH 4.32)
to C-3 (δ 77.0), confirming the attachment of the galactopyranosyl
fragment to O-3. Hydrolysis of 1 gave an aglycone named
rhizochalinin C (1b). The NMR spectra of 1b were congruent with
those of 1. Therefore, rhizochalin C (1) was formulated as a C-1-
hydroxy analogue of 3.
Results and Discussion
The EtOH extract of lyophilized R. incrustata, collected in
Madagascar,1 was concentrated and partitioned between n-hexane
and aqueous EtOH. The aqueous EtOH-soluble materials were
further separated by column chromatography on Polychrome-1
(powdered Teflon) by eluting with EtOH-H2O, 1:1, followed by
Si gel chromatography (CHCl3-EtOH-H2O, 3:2:0.2) to obtain a
crude mixture, which was purified by preparative HPLC (Dynamax
C18 column) to provide pure rhizochalin (3)1 and rhizochalins C
(1) and D (2) (0.12%, 0.008%, and 0.011%, respectively, based on
dry weight of the sponge).
The molecular formula C34H68N2O9 of 1 was obtained from a
high-resolution mass measurement of the [M + H]+ ion peak in
the HRFABMS and interpretation of MALDI-TOF-MS and ESIMS
data, as well as ESIMS data for the corresponding octaacetate (1a).
Interestingly, the base peak at m/z 325 in the ESIMS of 1
corresponded to a doubly charged ion [M + H2]2+ as previously
The molecular formula C35H71N2O8 of rhizochalin D (2) was
obtained from HRFABMS measurement of the [M + H]+ ion,
together with interpretation of MALDI-TOF-MS and ESIMS data,
and ESIMS data of the corresponding peracetate (2a). As for
compound 1, the base peak in the ESIMS of 2 was the doubly
charged ion at m/z 324 corresponding to [M + H2]2+. The 1H and
13C NMR data of 2 (Table 1) showed signals typical of a pseudo-
dimeric amino alcohol glycoside similar to 3,1–5 but the MS data
showed a molecular mass for 2 that was 14 amu higher than those
reported for 3.1 The 13C NMR, COSY, and HMBC data of 2 (Table
1) were essentially the same as those of 3, suggesting that the carbon
chain of 2 is extended by one additional CH2 group with respect to
3. Hydrolysis of 2 gave an aglycone designated as rhizochalinin D
(2b).
* To whom correspondence should be addressed. Tel: 7 (4232) 31-11-
68. Fax: 7 (4232) 31-40-50. E-mail: makarieva@piboc.dvo.ru.
† Pacific Institute of Bioorganic Chemistry.
§ UC San Diego.
The assignment of the D-configuration of the sugar was based
on comparison of NMR data for H-2 and H-3 adjoining the sugar
‡ UC Davis. Present address: Sanofi-Aventis, New Jersey.
10.1021/np0704811 CCC: $37.00
2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 12/06/2007

1992 Journal of Natural Products, 2007, Vol. 70, No. 12
MakarieVa et al.
in 2 and 2a with those of related1,2,4 and model compounds, as it
is found that the latter signals are sensitive to the sugar absolute
configuration.10 The D-galactose is the most common sugar
encountered among the rhizochalinins from the sponge R. incrustata1,2
and is also found here in rhizochalin C (1).
carbonyl in 3. As little as about 0.13 mg of 3a is sufficient for
identification of principal ions at m/z 724 [M + Na]+ in the
MALDI-TOF-MS of 9 and 12 and at m/z 338 [M + Na]+ in the
MS of 14 and 16. Furthermore, preparative isolation of 9, 12, 14,
and 16 obtained from a larger amount of 3a (2.0 mg) using HPLC
1
gave sufficient amounts of target compounds to record H NMR
The foregoing evidence established the structures of rhizochalins
C and D as unusual glycosylated two-headed sphingolipids, having
a ketone group embedded in a hydrocarbon chains. However,
interpretation of spectroscopic data could not establish the position
of the carbonyl group or the configuration of these natural products.
In agreement with previous studies on ketosphingolipids, direct
attempts to determine the position of the carbonyl group in the
compounds by EI mass spectrometry were unsuccessful due to
uncertainty in the interpretation of low-intensity ions from McLaf-
ferty-type rearrangements. In our earlier work, the position of the
CdO group in 3 was established by Baeyer–Villiger oxidation
followed by alkaline hydrolysis and full characterization of the
liberated alcohol and carboxylic moieties.1 The CdO position in
calyxoside was assigned by reductive amination to afford a primary
amine that facilitated R-cleavage in EIMS and location of the
carbonyl group.5 However, the applicability of these degradative
methods is limited in the case of minor metabolites that are available
in submilligram amounts.
In order to secure the location of the CdO group, we have
developed a procedure based on in situ MS analysis of Baeyer–
Villiger oxidation products obtained from the ketone. Preliminary
analyses were carried out using an available sample of rhizochalin
peracetate (3a). In order to ascertain limits of detection (LOD),
variable amounts of 3a (0.13, 0.26, 0.5, 1.0, and 2.0 mg) in methanol
solution were subjected to Baeyer–Villiger conditions (TFA-H2O2)
to effect three successive chemical transformations: oxidation,
hydrolysis of the derived esters (4 and 4a), and methylation of the
resultant carboxylic acids. Removal of the volatiles under vacuum
followed by acetylation (Ac2O-pyr) in one pot and subsequent
analysis of the degradation products by MALDI-TOF-MS without
purification permitted confirmation of the C-18 position of the
spectra (Table 2) in addition to MALDI-TOF-MS.
The foregoing methodology was also applied to rhizochalin C
peracetate (1a), rhizochalin D peracetate (2a), and rhizochalinin D
peracetate (2c) (∼0.2 mg each) to determine the CdO location in
1 and 2. Baeyer–Villiger oxidation of rhizochalin C peracetate (1a)
and subsequent MALDI-TOF analysis (Scheme 1) gave ions at m/z
782 [M + Na]+ and m/z 338 [M + Na]+, consistent with the
presence of fragments 8, 11, 14, and 16. Interpretation of these
results placed the CdO group at C-18 in 1 and flanked by (CH2)14
and (CH2)7 chain fragments. Thus, the CdO group in 1 is placed
at the same position as those of previously described R,ω-
bifunctionalized ketosphingolipids1–5 (see reassignment of ocean-
apiside, below). The same analysis applied to rhizochalin D
peracetate (2a) gave ions at m/z 724 [M + Na]+ and m/z 352 [M
+ Na]+, consistent with the presence of fragments 9, 12, 15, and
17. Rhizochalinin D peracetate gave ions at m/z 436 [M + Na]+
and m/z 352 [M + Na]+, consistent with the presence of fragments
10, 13, 15, and 17. These combined results confirmed that the
carbonyl group in 2 is flanked by (CH2)14 and (CH2)8, in contrast
to all known R,ω-bifunctionalized ketosphingolipids from sponges.
Having validated the Baeyer-Villager/MS method, we turned
our attention to oceanapiside (18), in which the keto group was
assigned to C-11 using charge-localized fragmentation of the Li+
adduct of 18 and d3-18,3 which relied upon an assumption of the
site of Li+ coordination in the molecule. Since the interpretation
of these results led to an unorthodox structuresthe one exception
to the C-18 keto-substitution pattern in the family of dimeric
sphingolipidsswe applied a modification of the Baeyer–Villiger
oxidation-MS analysis (Scheme 2)11 in order to resolve the
question of location of the keto group. Oxidation of oceanapiside

Rhizochalins C and D from Rhizochalina incrustata
Journal of Natural Products, 2007, Vol. 70, No. 12 1993

1994 Journal of Natural Products, 2007, Vol. 70, No. 12
MakarieVa et al.
Table 2. 1H NMR Data for Peracetates 9, 12, 14, and 16 (CDCl3, TMS)
14
16
9
12
δH (mult, J Hz)
atom no.
δH (mult, J Hz)
δH (mult, J Hz)
δH (mult, J Hz)
1
2
3
1.165 (d, 6.8)
4.09 (m)
3.49 (dt, 2.7, 6.3)
1.42 (m
1.165 (d, 6.8)
4.09 (m)
3.49 (dt, 2.7, 6.3)
1.42 (m)
4a,b
5–16
17
17-COOCH3
1.27–1.29 (bs)
4.05 (t, 7.0)
1.27–1.29 (bs)
2.30 (t, 7.0)
3.70 (s)
19-COOCH3
3.70 (s)
19
20–25
26
2.29 (t, 7.0)
1.27–1.29 (bs)
4.85 (m)
4.05 (t, 7.0)
1.27–1.29 (bs)
4.85 (m)
27
4.20 (m)
4.20 (m)
28
1.101 (d, 6.8)
1.101 (d, 6.8)
2-NHAc
5.89 (d, 9.0)
5.89 (d, 9.0)
27-NHAc
5.50 (d, 9.0)
5.50 (d, 9.0)
1′
2′
3′
4′
5′
6′
4.48 (d, 8.0)
4.48 (d, 8.0)
5.16 (dd, 8.0, 10,6)
5.04 (dd, 3.3, 10.6)
5.39 (dd, 0.8, 3.3)
3.91 (dt, 0.8, 6.6)
4.10 (dd, 6.6, 11.3) 4.19 (dd, 6.6, 11.3)
5.16 (dd, 8.0, 10,6)
5.04 (dd, 3.3, 10.6)
5.39 (dd, 0.8, 3.3)
3.91 (dt, 0.8, 6.6)
4.10 (dd, 6.6, 11.3) 4.19 (dd, 6.6, 11.3)
Scheme 1. Degradation of Rhizochalin C, Rhizochalin D, Rhizochalinin D, and Rhizochalin Peracetates (1a, 2a,c, and 3a) by Baeyer–
Villiger Oxidation
18 (TFA-H2O2) gave a mixture of esters, which was hydrolyzed
and directly analyzed by ESIMS without separation. The m/z pattern
of carboxylic acid-alcohol pairs (Scheme 2) was consistent only
with a keto group at C-18, not C-11. The reassignment was
confirmed by reductive amination of oceanapiside and analysis of
EIMS fragmentation patterns as previously described,5 and the
results are entirely self-consistent. Thus, the revised structure of
oceanapiside is depicted as shown.
The absolute configurations of rhizochalins C and D were
1
assigned from analysis of the H NMR and CD spectra of the
corresponding perbenzoyl aglycones following a procedure we
developed specifically for rhizochalin (3) and oceanapiside (18)12,13

Rhizochalins C and D from Rhizochalina incrustata
Journal of Natural Products, 2007, Vol. 70, No. 12 1995
Scheme 2. Degradation of Oceanpiside (18) by Baeyer–Villiger Oxidation
1
Table 3. Partial H NMR Data of the Compounds 18a, 19a, 20a,
Rhizochalinin C (1c), and Rhizochalinin D (2d) Perbenzoates
chemical shifts (ppm)
J-values (Hz)
δ1-H JNH-H-2 JH-2-H-3
R-terminus
δC2-NH
δ3-H
δ2-H
erythro-18a
erythro-19aa
threo-20ab
threo-1c
7.08
7.10
6.63
6.63
6.39
5.38
5.38
5.56
5.54
5.22
4.88 4.61, 4.64
4.88 4.62, 4.66
4.89 4.48, 4.57
4.89 4.48, 4.55
4.54 1.29
8.6
8.7
9.2
9.1
9.0
3.8
4.1
4.8
4.9
5.3
threo-2d
a See ref 5. b See ref 12.
1
Table 4. Partial H NMR Data of the Compounds 3b, 18a, 19a,
Rhizochalinin C (1c), and Rhizochalinin D (2d) Perbenzoates
chemical shifts (ppm)
J-values (Hz)
R-terminus
δC27-NH
δ26-H
δ27-H
δ28-H JNH-H-27 JH-26-H-27
erythro-18a
erythro-19a
threo-3b
threo-1c
threo-2d
6.38
6.38
6.38
6.38
6.39
5.21
5.21
5.21
5.21
5.22
4.53
4.53
4.53
4.53
4.54
1.28
1.29
1.29
1.29
1.29
8.9
9.0
8.9
8.9
9.0
5.2
5.3
5.3
5.3
5.3
of all four stereocenters emerges. Perbenzoyl compounds 1c and
2d were prepared using a conventional procedure (benzoic acid,
EDC, CH2Cl2), purified by HPLC, and their 1H NMR spectra were
compared with those of the perbenzoyl oceanin (18a), perbenzoyl
calyxinin (19a), and 1c as shown in Tables 3 and 4. The similarities
of the chemical shifts and coupling constants for the R-terminus
of rhizochalinin C perbenzoate (1c) with those of the N,O,O′-
tribenzoyl model compound (20a)12 indicated that rhizochalinin C
perbenzoate (1c) and its natural product parent 1 share the threo
configuration at C-2,C-3 of the R-terminus (Table 3). Rhizochalin
C (1), with a rare threo configuration, differs in relative configu-
ration from oceanapiside12 and calyxoside,5 which possess an
and applied, later, by Kingston and co-workers to calyxoside (19;
here, for convenience we refer to the corresponding aglycone as
“calyxinin”).5 By comparing CD spectra of the natural product-
derived perbenzoyl aglycones with “hybrid CD spectra” generated
from 16 linear combinations of all possible terminal group
configurations,12 a unique solution to the absolute configurations

1996 Journal of Natural Products, 2007, Vol. 70, No. 12
MakarieVa et al.
Figure 1. Measured CD and “hybrid CD” spectra (see ref 12) (MeOH, 25 °C, c ) 0.13-0.016 mM): (a) perbenzoyl rhizochalinin C (1c);
(b) hybrid CD of (2R,3R,26R,27R)- ent-20a + ent-20c; (c) hybrid CD of (2S,3R,26R,27R)-20b + ent-20c; (d) perbenzoyl rhizochalinin D
(2d); (e) hybrid CD of (2R,3R,26R, 27R)- ent-20c + ent-20c.
erythro configuration at the R-termini, as found in “normal”
sphingoid bases.14 The 1H NMR chemical shifts and coupling
constants for R-and ω-termini of rhizochalinin D perbenzoate (2d)
and rhizochalin perbenzoate (3b) and ω-termini of rhizochalinin C
perbenzoate (1c), oceanin perbenzoate (18a), and calyxinin per-
benzoate (19a) are identical (Tables 3 and 4). Rhizochalin D (2)
thus has the same threo relative configuration at both the R-and
ω-termini as rhizochalin (3),13 and rhizochalin C (1) has the threo
relative configuration at the ω-terminus shared by oceanapiside12
Rhizochalin and oceanapiside exhibited antifungal activity against
the fluconazole-resistant yeast Candida glabrata (ED50 ∼40 µM).3,4
However, rhizochalins C and D were not active (ED50 > 150 µM)
under the same conditions.
Rhizochalin D is the first representative of two-headed sphin-
golipids from sponges containing an odd-numbered carbon chain.
Reassignment of oceanapiside reveals that the position of the keto
group is conserved in all known two-headed ketosphingolipids,
including the new compounds 1 and 2. It is possible that a unified
biosynthetic pathway links these natural compounds to a common,
as yet unidentified biosynthetic lipid intermediate; however the
details of two-headed sphingolipid biosynthesis remain to be
elucidated.
1
and calyxoside. The foregoing H NMR analysis does not distin-
guish S,S or R,R stereoisomers from their antipodes at either
terminus; for this purpose we analyzed the CD spectra of the
perbenzoyl compounds.
Measurement of the CD spectra of rhizochlinin C perbenzoate
(1c) and comparison with “hybrid CD” spectra generated from linear
combinations of CD spectra of model compounds 20a-d, with all
possible terminal group configurational combinations (Figure 1),
clearly showed that 1c bears the (2R,3R,26R,27R) configuration.
The amplitude of the split Cotton effect in the CD spectrum of 1c
(Figure 1a) was considerably smaller in magnitude (A ) 4.5) than
the hybrid CD spectrum (Figure 1c), corresponding to perbenzoyl
oceanin (18a, Figure 1c, A ) 18.6), but matched perfectly in sign
and magnitude the split Cotton effects of the (2R,3R,26R,27R)
hybrid CD spectrum (Figure 1b, rhizochalin numbering), including
the presence of a weak positive shoulder at λ 245 nm. This
surprising finding reveals a rare example of a threo sphingolipid
with inverted 2R configuration at C-2 that is ostensibly derived
from C-2 of the amino acid L-serine.
The CD spectra of rhizochalinin D perbenzoate (2d) (Figure 1d)
showed a CD spectrum that is essentially superimposible upon that
of the (2R,3R,26R,27R)-pseudo-C2 “hybrid CD” spectrum (Figure
1e). Thus, the configuration of the C29 carbon chain sphingolipids
2d and 2 is each (2R,3R,27R,28R). It is worth noting that hybrid
CD spectral comparisons,10 based on deconvolution of CD Cotton
effects by comparison of “virtual” CD spectra constructed from
appropriate amino alcohol and amino diol models of known
configuration, are accurate, sensitive (limits of detection ∼nM), and
responsive to changes in configuration at a single stereogenic center
at either terminus of a dimeric sphingolipid.
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured using a Perkin-Elmer 343 polarimeter and a Jasco DIP-370. CD
spectra were recorded on a Jasco J800 spectrometer using quartz cells
(0.2 mm path length) at a scan rate of 50 nm/s. The NMR spectra were
recorded on Bruker DPX-400, DRX-500, and DRX-600 spectrometers
at 400, 500, and 600 for 1H and 100, 125, and 150 MHz for 13C,
respectively, with (CH3)4Si as an internal standard. MALDI-TOF mass
spectra were obtained on a Bruker Biflex III laser desorption spec-
trometer coupled with delayed extraction using N2 laser (λ 337 nm) on
2,5-dihydroxybenzoic acid (DHB) and R-cyano-4-hydroxycinnamic acid
(CCA) as matrix. ESI mass spectra were obtained on an LCMS system
comprising an MSQ Thermo Finnigan quadrupole spectrometer, coupled
to an Agilent 1100 series HPLC, or by direct infusion in MeOH
containing HCOOH (0.1%). FAB and EI mass spectra were obtained
on an AMD-604S spectrometer (AMD-Intectra, Germany). HRFAB
mass spectra were provided by the University of California, Riverside
mass spectrometry facility.
Low-pressure column liquid chromatography was performed using
Si gel L (50/160 µm, Sorbpolymer, Russia). Si gel plates (4.5 × 6.0
cm, 5–17 µm, Sorbfil, Russia) were used for thin-layer chromatography.
Preparative HPLC for isolation and separation of sphingolipids was
carried out using a Rainin binary HPLC system (Dynamax C18 column
10 × 250 mm, 5 µm, 3 mL/min) in 80:20:0.1% MeOH-H2O-TFA
coupled to a refractive index detector (Waters R401). Preparative HPLC
separation of Baeyer–Villiger oxidation products was performed on a
YMC Pack-ODS-A column (10 × 250 mm, 5 µm, 1.0 mL/min) in

Rhizochalins C and D from Rhizochalina incrustata
Journal of Natural Products, 2007, Vol. 70, No. 12 1997
80:20 EtOH-H2O using an Agilent Series 1100 instrument equipped
with the differential refractometer RID-DE14901810. Preparative HPLC
separation of perbenzoate products was performed on the same
instrument and column with slightly different solvent conditions (1.4
mL/min in 93% EtOH-H2O).
Animal Material. The sponge Rhizochalina incrustata (phylum
Porifera, class Demospongiae, subclass Ceratinomorpha, order Hap-
losclerida, family Phloeodictyidiae) was collected using scuba (depth
3–5 m) during the 3-day scientific cruise on board R/V “Akademik
Oparin” (November 1986, Madagascar) and identified by Prof. V. M.
Koltun (Zoological Institute, St-Petersburg, Russia). A voucher speci-
men is kept under registration number PIBOC #03-98 in the marine
invertebrate collection of Pacific Institute of Bioorganic Chemistry
(Vladivostok, Russia).
Extraction and Isolation. The fresh collection of the sponge R.
incrustasta was immediately lyophilized and kept at -20 °C. The
lyophilized material (18 g) was extracted with EtOH (200 mL × 3).
The combined EtOH extract was concentrated under reduced pressure
and redissolved in EtOH-H2O (9:1). This solution was partitioned three
times with equal volumes of n-hexane. The aqueous EtOH layer was
evaporated in Vacuo at 50 °C to give a brown oil, which was separated
over Polichrome I (powder Teflon, Biolar, Latvia) by elution with
gradient of H2O f 1:1 H2O-EtOH f EtOH. The two-headed
sphingolipid fraction (ninhydrin positive) was eluted with 50% EtOH.
The latter was further separated on a Si gel column (3:2:0.2,
CHCl3-EtOH-H2O) to give a mixture of the known rhizochalin (3)1
and new compounds 1 and 2.
Preparative HPLC of the mixture (Dynamax C18 80:20:0.1%
MeOH-H2O-TFA) gave 3 (20.0 mg, 0.12% based on dry weight of
sponge) and rhizochalins C (1, 1.4 mg, 0.008% based on dry weight
of sponge) and D (2, 1.9 mg, 0.011% based on dry weight of sponge).
Rhizochalin C (1): colorless solid; [R]25D -14.0 (c 0.093 MeOH);
MALDI-TOF-MS m/z 671 [M + Na]+; ESIMS m/z [M + H]+ 649
(25%), [M + H2]2+ 325 (100%); HRFABMS m/z 649.5000 [M + H]+
calcd, C34H68N2O9 649.5003; NMR (CD3OD, see Table 1).
Rhizochalin C Peracetate (1a). A sample of 1 (0.2 mg) was
dissolved in a mixture of pyridine (50 µL) and Ac2O (50 µL) and
allowed to stand at 25 °C for 18 h. Removal of the volatile materials
gave a residue (0.2 mg) of 1a: MALDI-TOF-MS m/z 1007 [M + Na]+;
ESIMS m/z 1007 [M + Na]+.
Rhizochalinin C Perbenzoatate (1c). A mixture of rhizochalinin
C (0.5 mg, 1.0 µmol), DMAP (1 mg), benzoic acid (9.2 mg, 0.075
mmol), and EDC (1-ethyl-3-[3-dimethylaminopropyl] carbodiimide
hydrochloride, 12.2 mg, 0.067 mmol) in CH2Cl2 (1 mL) was stirred at
room temperature for 6 days. The product was purified by Si gel column
chromatography (20:1 CHCl3-EtOAc) followed by preparative HPLC
(93:7 EtOH-H2O) to obtain rhizochalin C perbenzoate (1d, 0.3 mg):
MALDI-TOF-MS m/z 1029 [M + Na]+; 1H NMR (see Tables 3
and 4).
Rhizochalin D (2): colorless solid; [R]25 -12.0 (c 0.10 MeOH);
D
MALDI-TOF-MS m/z 669 [M + Na]; ESIMS m/z [M + Na]+ 647
(17%), [M + H2]2+ 323 (100%); HRMSFAB m/z 647.5234 [M + H]+
calcd, C35H71N2O8; 647.5210; NMR (CD3OD, see Table 1).
Rhizochalin D Peracetate (2a). A sample of 2 (0.2 mg) was
dissolved in a mixture of pyridine (50 µL) and Ac2O (50 µL) and
allowed to stand at 25 °C for 18 h. Removal of the volatile material
under reduced pressure gave a residue of 2a (0.2 mg): 1H NMR
(CDCl3): 1.18 (3 H, d, J ) 6.7, 3-H); 4.09 (1 H, m, 2-H); 3.49 (1 H,
td, J ) 2.2, 6.6, 3-H); 2.37 (2 H, t, J ) 7.5, 17-H); 2.38 (2 H, t, J )
7.5, 19-H); 4.85 (1 H, m, 26-H); 4.20 (1 H, m, 27-H); 1.10 (3 H, d, J
) 6.5, 28-H); 4.48 (1 H, d, J ) 8.0, 1′-H); 5.16 (1 H, dd, J ) 8.0,
10.6, 2′-H); 5.04 (1 H, dd, J ) 3.3, 10.6, 3′-H); 5.39 (1 H, J ) dd, 0.8,
3.3, 4′-H); 3.91 (1 H, J ) dt, 0.8, 6.6, 5′-H); 4.10 (1-H, J ) dd, 6.6,
11.3, 6′-H); 4.19 (1-H, J ) dd, 6.6, 11.3, 6′-H); MALDI-TOF-MS m/z
963 [M + Na]+.
Baeyer–Villiger Oxidation of Rhizochalin D Peracetate (2a).
Compound 2a (0.2 mg) was subjected to Baeyer–Villiger oxidation
followed by acetylation under essentially the same conditions used for
1a, and the mixture of peracetates was analyzed by MALDI-TOF-MS:
m/z 724 [M + Na]+; m/z 352 [M + Na]+.
Rhizochalinin D (2b). Rhizochalin D (0.5 mg) was hydrolyzed in
2 N HCl in MeOH and separated, as described above for rhizochalin
C, to provide the ninhydrin-positive product rhizochalinin D (2b, 0.5
mg): MALDI-TOF-MS m/z 485 [M + H]+.
Rhizochalinin D Peracetate (2c). A sample of 2b (0.2 mg) was
acetylated and purified, as described above for rhizochalinin C
peracetate, to give a residue of rhizochalinin D peracetate (2c, 0.2 mg):
MALDI-TOF-MS m/z 675 [M + H]+.
Baeyer–Villiger Oxidation of Rhizochalinin D Peracetate (2c).
Compound 2c (0.2 mg) was subjected to Baeyer–Villiger oxidation,
followed by acetylation, as described above for 1c to provide a mixture
of peracetates that was analyzed by MALDI-TOF-MS: m/z 436, m/z
352.
Baeyer–Villiger Oxidation of Rhizochalin C Peracetate (1a). A
mixture of TFA (100%) and H2O2 (30%) [TFA-H2O2, 3:1 (60 µL)]
was added to a solution of 1a (0.2 mg) in MeOH (50 µL). The mixture
was heated at 60 °C for 2 h, then left for 200 h at room temperature.
The volatiles were removed under reduced pressure and the residue
was acetylated with Ac2O-pyridine (1:1, 100 µL). The resulting mixture
of peracetates was analyzed by MALDI-TOF-MS: m/z 782 [M + Na]+,
m/z 338 [M + Na]+.
Rhizochalinin D Perbenzoate (2d). Rhizochalinin D (2, 0.3 mg)
was perbenzoylated according to the procedure described above for 1
and purified by preparative HPLC to give rhizochalin C perbenzoate
1
(1d, 0.1 mg): MALDI-TOF-MS m/z 923 [M + Na]+; H NMR (see
Tables 3 and 4).
Preparative Isolation of Baeyer–Villiger Oxidation Products of
Rhizochalin Peracetate (3a). A mixture of TFA (100%) and H2O2
(30%) [TFA-H2O2, 3:1 (180 µL)] was added to a solution of
rhizochalin peracetate (2.0 mg) in methanol (200 µL). The mixture
was heated at 60 °C for 2 h and then left for about 200 h at room
temperature. The volatiles were removed under reduced pressure, and
the residue was acetylated with Ac2O-pyr (1:1, 200 µL) to obtain a
mixture of peracetates. Preparative separation of the mixture by HPLC
(YMC-Pack ODS-A, 80:20 EtOH-H2O) gave peracetates 9, 12, 14,
and 16.
Baeyer–Villiger Oxidation of Oceanapiside (18). A modification
of the Bayer-Villager oxidation was applied to oceanapiside as follows.
H2O2 (0.0965 g, 2.8 mmol, 30% aqueous solution) was added dropwise
into TFA (0.294 g, 2.6 mmol) at 0 °C. The cold bath was removed and
the solution of oceanapiside (3.0 mg) in MeOH (0.5 mL) was added
dropwise. After addition, the mixture was refluxed for 2 h and then
left stirring overnight at room temperature. Na2S2O4 solution (aq) was
added until the mixture turned basic (pH ∼8). The aqueous mixture
was concentrated and extracted with MeOH to remove most of the
salts. The crude product was redissolved in 2 M HCl in aqueous MeOH
(obtained by adding 1.64 mL of 37% aqueous HCl into 8.36 mL of
MeOH) and heated at 80 °C for 24 h. The reaction mixture was
concentrated and redissolved in MeOH and analyzed by ESIMS. See
Scheme 2.
1
Peracetate 9: MALDI-TOF-MS m/z 724 (M + Na+); H NMR
(CDCl3, see Table 2).
1
Peracetate 12: MALDI-TOF-MS m/z 724 (M + Na+); H NMR
(CDCl3, see Table 2).
Peracetate 14: MALDI-TOF-MS m/z 338 (M + Na+); H NMR
1
Rhizochalinin C (1b). Rhizochalin C (0.5 mg) in 2 N HCl in MeOH
(100 µL) was heated at 80 °C for 24 h in a sealed vial. Then the solution
was cooled and concentrated under a stream of N2. The residue was
subjected to microcolumn Si gel chromatography with successive
elution by (a) 1:4 MeOH-CHCl3 and (b) 9:4:1 CHCl3-MeOH-
(CDCl3, see Table 2).
Peracetate 16: MALDI-TOF-MS m/z 338 (M + Na+); H NMR
1
(CDCl3, see Table 2).
1
Acknowledgment. This investigation was supported by the Fogarty
International Center and NIH (TWOO6301-01) and the National
Institutes of Allergies and Infectious Diseases (AI039987, to T.F.M.).
The research described here was made possible in part by Program of
Presidium of RAS “Molecular and Cell Biology”, Grant No. SS-
6491.2006.4 from the President of RF. The authors kindly acknowledge
R. Kondrat (UC Riverside) for HRFABMS measurements.
NH4OH to provide the ninhydrin-positive product (1b, 0.5 mg): H
NMR (CD3OD) 3.76 (1 H, dd, J ) 4.0, 11.6, 1-H); 3.64 (1 H, dd, J )
6.7, 11.6, 1-H); 3.04 (1 H, m, 2-H); 3.66 (1 H, m, 3-H); 2.435 (2 H, t,
J ) 7.5, 17-H); 2.427 (2 H, t, J ) 7.5, 19-H); 3.43 (1 H, m, 26-H);
3.08 (1 H, m, 27-H); 1.26 (3 H, d, J ) 6.5, 28-H); 13C NMR (CD3OD)
59.7 (C-1); 61.1 (C-2); 69.8 (C-3); 44.1 (C-17); 44.0 (C-18); 73.7 (C-
26); 54.1 (C-27); 16.6 (C-28); MALDI-TOF-MS m/z 487 [M + H]+.

1998 Journal of Natural Products, 2007, Vol. 70, No. 12
MakarieVa et al.
(7) Willis, R. H.; De Vries, D. J. Toxicon 1997, 35, 1125–1129.
Supporting Information Available: This material is available free
of charge via the Internet at http://pubs.acs.org.
(8) Makarieva, T. N.; Guzii, A. G.; Dmitrenok, A. S.; Dmitrenok, P. S.;
Krasokhin, V. B.; Stonik, V. A. Nat. Prod. Commun. 2006, 1, 711–
714.
References and Notes
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(10) To be published in a separate communication.
(11) Li, R. Progress Towards the Total Synthesis of Oceanapiside. MSc.
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