Pachymoside A - A novel glycolipid isolated from the marine sponge Pachymatisma johnstonia

Article abstract of DOI:10.1139/v03-183

Crude extracts of the North Sea marine sponge Pachymatisma johnstonia showed promising activity in a new assay for inhibitors of bacterial type III secretion. Bioassay-guided fractionation resulted in the isolation of the pachymosides, a new family of sponge glycolipids. A major part of the structural diversity in this family of glycolipids involves increasing degrees of acetylation and differing positions of acetylation on a common pachymoside glycolipid template. All of the metabolites with these variations in acetylation pattern were converted into the same peracetylpachymoside methyl ester (2) for purification and spectroscopic analysis. Pachymoside A (1) is the component of the mixture that has natural acetylation at the eight galactose hydroxyls and at the C-6 hydroxyls of glucose-B and glucose-D. Chemical degradation and transformation in conjunction with extensive analysis of 800 MHz NMR data was used to elucidate the structure of pachymoside A (1).

Full text of DOI:10.1139/v03-183

1
02
Pachymoside A — A novel glycolipid isolated from
1
the marine sponge Pachymatisma johnstonia
Kaoru Warabi, William T. Zimmerman, Jingkai Shen, Annick Gauthier,
Marilyn Robertson, B. Brett Finlay, Rob van Soest, and Raymond J. Andersen
Abstract: Crude extracts of the North Sea marine sponge Pachymatisma johnstonia showed promising activity in a new
assay for inhibitors of bacterial type III secretion. Bioassay-guided fractionation resulted in the isolation of the pachy-
mosides, a new family of sponge glycolipids. A major part of the structural diversity in this family of glycolipids in-
volves increasing degrees of acetylation and differing positions of acetylation on a common pachymoside glycolipid
template. All of the metabolites with these variations in acetylation pattern were converted into the same peracetyl-
pachymoside methyl ester (2) for purification and spectroscopic analysis. Pachymoside A (1) is the component of the
mixture that has natural acetylation at the eight galactose hydroxyls and at the C-6 hydroxyls of glucose-B and
glucose-D. Chemical degradation and transformation in conjunction with extensive analysis of 800 MHz NMR data
was used to elucidate the structure of pachymoside A (1).
Key words: Pachymatisma johnstonia, marine sponge, pachymoside, glycolipid.
Résumé : Les produits bruts extraits de l’éponge marine de la mer du Nord Pachymatisma Johnstonia présentent une
activité intéressante dans un nouvel essai pour les inhibiteurs de la sécrétion bactérienne de type III. Des fractionne-
ments orientés par des bioessais ont permis d’isoler les pachymosides, une nouvelle famille de glycolipides des épon-
ges. Une partie importante de la diversité structurelle de cette famille de glycolipides implique des degrés croissants
d’acétylation et des différences dans les positions de l’acétylation sur un gabarit commun du glycolipide pachymoside.
Tous les métabolites comportant ces variations dans le patron d’acétylation ont été transformés dans le même ester mé-
thylique du peracétylpachymoside (2) aux fins de purification et d’analyse spectroscopique. Le pachymoside A (1) est
le composant du mélange qui correspond à l’acétylation naturelle au niveau des huit hydroxyles du galactose et aux hy-
droxyles en C-6 du glucose-B et du glucose-D. Afin d’élucider la structure du pachymoside A (1), on a utilisé une dé-
gradation et une transformation chimique de concert avec une analyse extensive des données RMN à 800 MHz.
Mots clés : Pachymatisma johnstonia, éponge marine, pachymoside, glycolipide.
[
Traduit par la Rédaction] Warabi et al. 112
Introduction
fantile diaherria that kills several thousand children world-
wide each year, have served as important models for study-
ing TTSSs and their role in pathogenicity. While many
bacterial pathogens attach themselves to host cell proteins,
these pathogenic E. coli are remarkable because they use a
TTSS to deliver their own receptors into the host cells to
which they attach. The bacterial protein receptor delivered
into the host cell by the E. coli TTSS is called Tir for
“translocated intimin receptor”. Once inside the host cell,
Tir binds to an E. coli protein “intimin” on the surface of the
bacterium to give intimate attachment, a required prelude to
Several important gram-negative pathogenic bacteria de-
liver virulence factors directly into host cells via a “type III
secretion system” (TTSS) (1–4). These include Bordetella
pertussis, the cause of whooping cough, Chlamydia tracho-
matis, the cause of a sexually transmitted disease that leads
to blindness, enterohemorrhagic Escherichia coli (EHEC),
the cause of “hamburger” disease, and Salmonella typhi, the
cause of typhoid fever. EHEC (5) and the closely related
enteropathogenic E. coli (EPEC) (6), the major cause of in-
Received 23 June 2003. Published on the NRC Research Press Web site at http://canjchem.nrc.ca on 15 December 2003.
Dedicated to Professor Edward Piers, a wonderful colleague and friend.
2
3
K. Warabi, W.T. Zimmerman, J. Shen, and R.J. Andersen. Departments of Chemistry, Earth and Ocean Sciences, University
of British Columbia, 2036 Main Mall, Vancouver, BC V6T 1Z1, Canada.
A. Gauthier, M. Robertson, and B.B. Finlay. Departments of Biochemistry and Microbiology, University of British Columbia,
Vancouver, BC V6T 1Z3, Canada.
R. van Soest. Department of Coelenterates and Porifera, Zoologisch Museum, University of Amsterdam, Amsterdam, The
Netherlands.
1
This article is part of a Special Issue dedicated to Professor Ed Piers.
On leave from DuPont Crop Protection, Stine-Haskell Research Center, Newark, DE, U.S.A.
Corresponding author (email: randersn@interchange.ubc.ca).
2
3
Can. J. Chem. 82: 102–112 (2004)
doi: 10.1139/V03-183
© 2003 NRC Canada

Warabi et al.
103
virulence. The TTSS, which forms a syringe-like pore that
spans both bacterial and host cell membranes, consists of
more than 20 proteins. Deleting or mutating any part of the
TTSS significantly reduces the virulence of the affected E.
coli. Non-pathogenic intestinal E. coli do not have TTSSs.
The TTSS is considered an attractive target for the devel-
opment of new antibiotics that would selectively target
pathogenic gram negative bacteria but would not harm the
ordinary microflora that do not possess this virulence mech-
anism (1, 7–9). An added benefit of this type of hypothetical
antibacterial agent is that by targeting a virulence mecha-
nism, there would be little or no selective pressure for viabil-
ity, potentially reducing the development of resistance. No
TTSS inhibitors were known at the outset of our research.
Consequently, there was a significant need to discover TTSS
inhibitors that could be used to provide proof principle dem-
onstrations and drug leads for this novel approach to treating
important infectious diseases.
Marine sponges, which are the richest source of bioactive
marine natural products (10, 11), are constantly exposed to
bacteria in seawater and it has frequently been proposed that
they contain chemical agents that have evolved to prevent
pathogenic bacteria from infecting them (12, 13). We have
screened a library of crude extracts from marine sponges for
their ability to inhibit type III secretion of E. coli secreted
proteins (Esps) by EPEC without affecting the growth or
general secretion of the bacterium (1). Two extracts showed
promising activity in the screen. Recently, we reported the
structure of caminoside A, an antimicrobial glycolipid iso-
lated from the Caribbean sponge Caminus sphaeroconia that
ural products into individual components failed. To facilitate
purification of the mixture, it was first methylated with
(trimethylsilyl)diazomethane and then acetylated with
Ac O–pyridine in the presence of DMAP. Reversed-phase
2
HPLC separation of the mixture of derivatized natural prod-
ucts gave a pure peracetylpachymoside A methyl ester (2) as
a colourless glass.
Peracetylpachymoside A methyl ester (2) gave a [M +
+
Na] ion at m/z 2333.9639 in the ESI-HR-MS consistent
with a molecular formula of C106H159NO54 (calcd. for
1
2
13
C105 CH159NO Na, 2333.9658), requiring 28 sites of
54
1
3
unsaturation. The C NMR spectrum of 2 (100 MHz) re-
corded in C D showed the same general features of a glyco-
6
6
lipid observed in the spectrum of the natural product
mixture. Thus, it was possible to attribute clusters of peaks
to ester or amide carbonyls (δ 168–170), monosaccharide
anomeric carbons (δ 100–102), carbinol methines and
methylenes (δ 67–81), methyl groups attached to O or N (δ
4
9–52), acetate methyl carbons (δ 29–31), and a significant
1
saturated aliphatic fragment (δ 14–43). The H NMR spec-
trum of 2 was very congested at 500 MHz so all of the H
1
1
D and 2D data used in the structure elucidation was re-
corded at 800 MHz in C D , which gave excellent disper-
6
6
sion.
The first step in the structure elucidation of 2 involved
chemical degradation of the mixture of natural pachymo-
sides to liberate the individual monosaccharides. Treatment
of the pachymoside mixture with acetyl chloride in dry
MeOH cleaved the glycosidic linkages and converted the
individual monosaccharides into mixtures of α- and β-
methylglycopyranosides. After removal of the reagents in
was the first compound active in the TTSS inhibition screen
4
(
14, 15). The second active extract came from the sponge
vacuo, the resulting residue was acetylated with Ac O in
2
Pachymatisma johnstonia (Bowerbank, 1842) collected
along the coast of the Isle of Mann. Bioassay-guided frac-
tionation of the P. johnstonia extract yielded a complex fam-
ily of glycolipids that were active in the TTSS assay. The
structure elucidation of pachymoside A (1), a novel glyco-
lipid that is a representative member of this family, is de-
scribed below.
pyridine and a catalytic amount of DMAP to give the pera-
cetylated methylglycosides of the individual monosaccha-
rides. Fractionation of the peracetylated methylglycosides
via reversed-phase HPLC gave an inseparable mixture of
methyl 2,3,4,6-tetra-O-acetyl-α-glucopyranoside (3) and
methyl 2,3,4,6-tetra-O-acetyl-α-galactopyranoside (4) in a
ratio of ≈2:1, respectively. The difference in intensities of
the resonances for each of 3 and 4 made it possible to iden-
tify each of the component compounds by analysis of the 1D
and 2D NMR data obtained for the mixture. To confirm the
assigned structures of 3 and 4, authentic samples of D-
glucose and D-galactose were converted into their peractyl-
ated methyl α-glycopyranosides and the NMR chemical
shifts of the authentic compounds were compared with the
degradation products. Chiral GC analysis of the alditol ace-
tates prepared from authentic L- and D-glucose and D-
galactose and the alditol acetates prepared from the mono-
saccharides obtained by aqueous hydrolysis of the pachy-
moside mixture showed that the glucose and galactose
residues in the pachymosides had the D configuration.
Results and discussion
The TTSS inhibitory fraction obtained from an initial
Sephadex LH20 chromatographic separation (eluent: MeOH)
of the crude P. johnstonia extract gave spectroscopic data
that was indicative of a complex mixture of closely related
glycolipids. LR-FAB-MS analysis of the mixture revealed a
series of clusters of molecular ions with centres at m/z 1918,
1
932, 1944, 1961, 1973, 1988, 2003, 2015, and 2046. The
1
13
H and C NMR spectra of the mixture showed evidence for
the presence of a number of monosaccharide anomeric car-
bons, a large number of carbinol methines and methylenes,
extensive acetylation, and a long aliphatic chain. All chro-
matographic attempts to resolve the complex mixture of nat-
A very non-polar fraction, which contained a mixture of
the aglycon portions of the pachymosides, was also obtained
4
While the current study was underway, another group reported screening a chemical library for inhibition of YopE transcription with a luci-
ferase reporter assay, yielding three compounds that inhibited the reporter signal expressed from the yopE promoter and effector protein se-
cretion with little effect on bacterial growth (15). One compound appears to be affecting type III regulation, and this and one other
compound are specific to the virulence-associated TTSS, and do not affect flagellum assembly and motility. Overall, these results are evi-
dence in support of TTSS-specific inhibitors, although the bacterial target(s) and ability to inhibit TTSS in infected host cells awaits further
study.
©
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1
04
Can. J. Chem. Vol. 82, 2004
from the methanolysis reaction on the pachymoside mixture
described above. HPLC fractionation of the mixture of
aglycons yielded a number of closely related compounds.
Methanolysis of pure peracetylpachymoside A methyl ester
correlations into the amide carbonyl resonance at δ 172.0
and into a deshielded carbon resonance at δ 211.0, assigned
to a saturated ketone. These correlations indicated that the
six proton multiplet was comprised of three separate but co-
incidentally chemical shift-equivalent methylene resonances
that were assigned to a methylene α to the amide carbonyl
and two methylenes α to a saturated ketone. Consistent with
these assignments, there was a small triplet at δ 2.20 in the
(
2), followed by analysis of the non-polar product by HPLC,
H NMR, and EI-MS, revealed that the methyl ester 5 ob-
1
tained from the large-scale methanolysis of the mixture of
pachymosides corresponded to the aglycon fragment of 2.
+
1
The ester 5 gave a M ion at m/z 539.4567 in the EI-HR-MS
H NMR spectrum that was assigned to the methylene α to
appropriate for a molecular formula of C H NO (calcd.
the amide carbonyl in the minor rotamer of 5. The methine
resonance at δ 3.55 showed a HMQC correlation to the car-
bon resonance at δ 72.5 consistent with the presence of a
secondary alcohol in 5, and the triplet at δ 0.87 was assigned
to a methyl at the end of a linear aliphatic chain. Since the
methyl ester, N-methylamide, and ketone carbonyls ac-
counted for the three sites of unsaturation in 5, and the only
terminal residues identified in the NMR data were the
methyl ester and aliphatic methyl groups, the aglycon had to
have a 27-carbon linear chain extending from the N-acyl am-
ide carbonyl to the terminal methyl group, and there had to
be saturated ketone and secondary alcohol functionalities sit-
uated along the chain.
3
2
61
5
5
39.4550), requiring three sites of unsaturation.
Three associated pairs of singlets were apparent in the H
1
NMR spectrum of 5, each of which exhibited an intensity ra-
1
tio of ca. 4:1. A singlet at δ 3.71 in the H NMR spectrum
(
associated peak at δ 3.77), which showed a HMQC correla-
tion to a carbon at δ 52.5 and a HMBC correlation to a car-
bon at δ 170.0, was assigned to a methyl ester. A second
singlet at δ 3.05 (associated peak at δ 2.95), which showed
an HMQC correlation to a carbon at δ 36.5 and HMBC cor-
relations to carbons at δ 172.0 and 49.0, was assigned to a N-
methyl amide. The third singlet at δ 4.11 (associated peak at
δ 4.03), integrated for only two protons relative to the other
singlets. It showed a HMQC correlation to a carbon at δ 49.0
and HMBC correlations to the ester carbonyl at δ 170.0, the
amide carbonyl at δ 172.0, and the N-methyl carbon at
δ 36.5, and was assigned to the α carbon of an N-acyl-N-
methylglycine methyl ester fragment. As is typical for ter-
tiary amides, slow rotation about the N-acyl-N-methyl-
glycine amide bond is presumed to be responsible for the
The positions of the ketone and secondary alcohol
functionalities in 5 were determined by analysis of the EI-
HR-MS data obtained for the compound as shown in Fig. 1.
A major peak at m/z 145.0739 (C H NO ) was attributed to
6
11
3
a McLafferty rearrangement involving the C-4 amide car-
bonyl that results in cleavage of the C-5—C-6 bond, con-
firming the presence of the N-acyl-N-methylglycine methyl
ester fragment at one terminus of the aglycon. A pair of
peaks at m/z 284.2232 (C H NO ) and 341.2557 (C H NO )
1
presence of associated pairs of peaks observed in the H
NMR spectrum for the N-methyl, methyl ester, and glycine
16
30
3
19 35
4
α-methylene resonances.
were attributed to McLafferty rearrangements occurring on
both sides of the ketone functionality, which located the
ketone at C-14. Finally, a peak at m/z 468.3697 (C H NO )
1
Additional features in the H NMR spectrum of 5 in-
cluded a six proton multiplet at δ 2.36, a deshielded one pro-
ton multiplet at δ 3.55, and a three proton triplet at δ 0.87.
The six proton multiplet (δ 2.36) showed HMQC correla-
tions into carbon resonances at δ 38.1 and 42.8, and HMBC
2
7
50
5
was assigned to an α cleavage adjacent to the secondary al-
cohol, which had to be at C-23. The base peak in the mass
spectrum of 5 resulted from a cleavage of the amide bond to
©
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Warabi et al.
105
Fig. 1. HR-EI-MS analysis of the aglycon methyl ester 5.
Fig. 2. Expansion of gHSQC spectrum of peracetylpachymoside A methylester (2) acquired in C D at 800 MHz. Anomeric carbon
6
6
correlations are shown.
give a fragment ion at m/z 104.0709 (C H NO ), which had
the molecular formula of protonated N-methylglycine
methyl ester.
aglycon, accounted for all of the unsaturation required by
the molecular formula of 2. Therefore, what remained was to
determine the connectivity among the aglycon, the six
monosaccharide residues, and the 19 acetates, as well as de-
termining the relative stereochemistry of the polysaccharide
portion of the molecule. These features of 2 were elucidated
by detailed analysis of its 800 MHz NMR data (Figs. 3 and
4
10
2
Subtracting the atoms present in the aglycon (C H NO )
3
2
60
5
from the molecular formula of peracetylpachymoside A
methyl ester (2) (C106H159NO ) left C H O and 25 sites
54
74 99 49
of unsaturation unaccounted for. Preliminary analysis of the
NMR data obtained for 2, in conjunction with the degrada-
tion studies, indicated the remaining atoms had to be part of
glucose, galactose, or acetate residues. Six anomeric carbons
4
).
Starting with the six well-resolved anomeric proton reso-
nances, it was possible by careful analysis of the COSY, 1D-
TOCSY, 2D-TOCSY, HSQC, and HMBC data to assign res-
onances to all of the hydrogen and carbon atoms in each of
the six monosaccharide residues as listed in Table 1. Degra-
dation had shown that the pachymosides contained only D-
1
3
1
and their attached protons (δ
01.4:4.21; 101.4:4.56; 101.4:4.78; 101.5:4.61; 102.1:4.31)
could be readily identified in the HSQC spectrum of 2
Fig. 2). Subtracting the 36 carbons of six hexose
C: H; 100.7:4.67;
1
(
1
monosaccharides (glucose and galactose) from the 74 car-
bons remaining to be accounted for, left 38 carbons, which
corresponded to 19 acetate residues. The six sites of
unsaturation accounted for by the putative monosaccharide
rings, along with the 19 sites of unsaturation associated with
the acetate residues and the three sites of unsaturation in the
glucose and D-galactose monomers. Analysis of the H vici-
nal NMR coupling constants for each of the monosaccharide
residues A to F confirmed that there were four glucose units
(A–D) and two galactose units (F and E) in 2. In addition,
all six of the anomeric proton resonances appeared as dou-
blets with coupling constants between 7.0 and 8.2 Hz, indi-
©
2003 NRC Canada

1
06
Can. J. Chem. Vol. 82, 2004
Fig. 3. Expansion of gHMBC of peracetylpachymoside A methyl ester (2), (natural abundance) acquired at 800 MHz in C D . Assign-
6
6
ments for carbinol methine and methylene HMBC correlations to acetate carbonyls are indicated. No coupling for C-4 to its attached
acetate carbonyl is observed for the unlabeled sample.
cating that all of the glycosidic linkages were β.
Examination of the proton chemical shifts (Table 1) in each
of the monosaccharide residues for acyl shifts predicted that
glucose-A was acetylated at C-3 and C-6, glucose-B was ac-
etylated at C-3 and C-6, glucose-C was acetylated at C-2, C-
while the corresponding ESI-LR-MS of the 13C-acetate la-
+
belled compound gave two [M + Na] major peaks at m/z
1
3
2343/2344, indicating the addition of eight and nine 1 C-
3
acetyl groups. An HMBC spectrum collected on the C-
labelled sample of 2 showed intense peaks for the
4
, and C-6, glucose-D was acetylated at C-2, C-3, C-4, and
monosaccharide proton to acetyl carbonyl correlations in-
1
3
C-6, galactose-E was acetylated at C-2, C-3, C-4, and C-6,
and galactose-F was also acetylated at C-2, C-3, C-4, and C-
volving C-labelled acetate residues and essentially no cor-
relations for the natural abundance acetates (Fig. 5). Using
this intensity difference as a marker of introduced acetate, it
6
. This acetylation pattern, which was confirmed by HMBC
1
3
correlations from the corresponding monosaccharide protons
to the acetate carbonyls (Fig. 3), was consistent with the
presence of 19 acetate residues in 2. Finally, HMBC and
ROESY correlations (Table 1 and Fig. 4) provided the infor-
mation required to identify the linkages between the six
monosaccharide residues A–F and the aglycon as shown in
Fig. 4.
was possible to show that C-acetate had been introduced at
10 sites in the molecule (A-3, A-6, B-3, B-6, C-2, C-6, D-2,
D-3, D-4, D-6; see Fig. 5). Evidence for labelled acetate in-
troduction at C-4 came from the observation of additional
scalar coupling in the glucose-C H-4 resonance observed at
1
13
400 MHz, which was attributed to three bond H/ C cou-
pling. The absence of a corresponding HMBC cross peak in
the 800 MHz spectrum is an anomaly produced by the line
broadening of the C residue H-4 resonance at high field
(vide infra). There was no evidence for introduction of 13C-
labelled acetate at any of the hydroxyl positions of the
galactose-E and galactose-F residues, indicating that they are
fully acetylated in all of the natural pachymosides. Since
there is evidence for introduction of labelled acetate at 11
hydroxyls in 2 and the MS data indicates that only eight or
nine labelled acetates have been introduced in individual
molecules, the natural pachymoside mixture must consist in
part of a major group of compounds that have the same
aglycon 5, are peracetylated on the galactose residues E and
F, and have either two or three acetates distributed among
NMR analysis of the crude mixture of pachymosides
showed that the natural products are partially acetylated. In-
formation about the natural acetylation pattern was lost dur-
ing the peracetylation step required for isolation of pure
peracetylpachymoside A methyl ester (2). To locate the sites
of introduced acetylation in 2, a second portion of the crude
pachymoside mixture was methylated with (trimethyl-
1
3
silyl)diazomethane and then acetylated with (CH3 CO) O,
2
pyridine, and DMAP as before. The crude reaction mixture
was fractionated to give peracetylpachymoside A methyl es-
ter (2) labelled at the sites of synthetic acetylation with
1
3
CH3 CO. The ESI-LR-MS of the unlabeled peracetylpachy-
+
moside A methyl ester (2) gave a [M + Na] at m/z 2335,
©
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Warabi et al.
107
Table 1. 1D- and 2D-NMR data for pachymoside A peracetate methyl ester (2).a
δ
COSY
HMBC
TOCSY
ROESY
Position
Aglyconb
13C NMR 1H NMR
Multiplicity J (Hz)
(H → H)
(H → C)
(H → H)
(H → H)
1
2
3
4
172.8
—
33.0/32.8 2.04/1.98
25.3
29.7
t
7.5
1
2, 4
3
3,4
2,4
2,3
1.72/1.74
1.30
2, 4
3
5
1
1
1
1
1
1
–10
1
2
3
4
1.26–1.32
1.23
1.58
2.09
—
29.7
24.2
42.7
208.9
42.7
24.2
12
11,13
12
12, 13
13, 14
12, 14
12,13
11,13
11,12
qn
t
7.5
7.5
5
6
2.12
1.64
t
qn
7.5
7.5
16
15, 17
14, 16
14, 15
16,17,18,19,20,21
15,17,18,19,20,20′,
2
1
1
1
1
2
2
2
2
2
2
7
8
9
0
0′
1
2
2′
3
30
30
30
30
1.33
1.44
1.32
1.44
1.38
1.55
1.70
1.57
3.71
16
15, 16
17/19(vw)
16,17,20,21 18,20
18/19(vw)
18/19
20(w)
21,23
21,23
22, 24, A1
15,16,18
15,16,17
20′,21
20
20
22′
22
20′,21,21′
19,20,21,22,23,24
20,22
20,20′,21,22′,23
20,22
—
25.3
34.5
—
80.2
22,22′, 24,24′
20-22/20′-22′,24-
A1,22,22′,24,24′
2
6/24′26′
2
2
2
2
2
2
2
2
4
4′
5
5′
6
6′
7
35.3
—
25.3
—
32.4
—
23.1
14.4
169.9
1.70
1.63
1.52
1.54
1.39
1.34
1.40
0.97
—
24′
24
23,25
25(w)
23,25,25′,26
24,25,26
24,24′(w),26,26′
24,24′,26
23,24,24′,25,25′
25,25′,26,27
28,26,25,24,23
27,26,25,24,23
27(vw)
25, 27
27
26, 28
26, 27
28
28
27
8
t
7.4
1
′
2
′
49.2/51.0 3.91/3.42
35.7/34.5 2.44/2.79
51.3/*
s
s
s
1, 1′
1, 2′
1′
3
′
4
′
3.27/3.24
c
Glu-A
A-1
A-2
A-3
A-4
A-5
A-6
A-6′
Glu-B
B-1
100.7
78.7
75.1
78.8
72.4
62.2
—
4.67
3.79
5.50
3.73
3.82
4.76
4.95
d
dd
t
dd
ddd
dd
dd
7.2
7.2, 9.0
9.0
9.0, 9.6
2.6,4.5,9.6
4.3, 12.0
2.8, 12.0
A2
23, A5
A1, A3, E1
A2, A4
A3, A6, B1
A4
23
E1
A1, A3
A2, A4
A3, A5
A4, A6, A6′
A5, A6′
A5, A6′
B1
A4
A4, A5
102.1
78.1
74.0
76.6
72.2
62.8
—
4.31
3.62
5.29
3.50
3.08
4.10
4.35
d
7.0
B2
A4
A4
F1
B-2
B-3
B-4
B-5
dd
dd
dd
ddd
dd
dd
7.0, 8.6
8.6, 9.6
9.6, 11.2
1.6,5.3,11.3 B4, B6, B6′
5.2, 11.9
1.9, 11.9
B1, B3
B2, B4
B3, B5
B1, B3, F1
B2, B4
B3, B5, C1
B1
B5
B4, B5
B1
B-6
B5,B6′
B5, B6
B-6′
Glu-C
C-1
C-2
C-3
101.4
73.2
79.1
68.4
4.21
5.18
3.89
5.00
d
dd
8.2
8.2, 9.4
C2
B4
C(2,3,4,5)d
C(1,3,4,5)
C1, C2
C1
B4, C3, C6(w)e
C4(vw)
D1, C1(w)
C6′(w)
C1, C3
C2, C4
C3, C5
C1, C3
(nco)
(nco)
f
nco
f
C-4
t
9.6
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1
08
Can. J. Chem. Vol. 82, 2004
Table 1 (concluded).
δ
COSY
HMBC
TOCSY
ROESY
Position
13C NMR 1H NMR
Multiplicity J (Hz)
ncof
(H → H)
(H → C)
(H → H)
(H → H)
g
C-5
C-6
C-6′
Glu-D
D-1
D-2
D-3
D-4
D-5
D-6
D-6′
Gal-E
E-1
72.7
61.9
—
3.51
4.11
4.44
C4, C6, C6′
C5, C6′
C5, C6
C1
C6, C6′
C5, C6′
C5, C6
C2, C6
C5, C6′
C6
dd
dd
2.5, 12.7
4.2, 12.3
C4, C5
C5(w)
101.4
71.6
73.3
68.1
72.2
61.5
—
4.56
5.15
5.32
5.18
3.05
4.32
3.88
d
8.2
D2
C3
C3
A2
B2
dd
dd
dd
ddd
dd
dd
8.2, 9.8
9.4, 9.8
9.4, 10.4
2.1,3.9,10.2 D4, D6, D6′
3.9, 12.5
2.1, 12.5
D1, D3
D2, D4
D3, D5
D1, D3
D2, D4
D3, D5
D1(w)
(nco)
D5, D6′
D5, D6
D4, D5
101.4
69.7
71.6
67.3
70.9
60.9
—
4.78
5.56
5.20
5.54
3.49
4.25
4.27
d
8.0
E2
A2
E-2
E-3
E-4
E-5
dd
dd
dd
ddd
dd
dd
8.0, 10.5
3.4, 10.5
0.8, 3.4
0.8,6.2,7.8
6.2, 11.0
7.7, 11.0
E1, E3
E2, E4
E3, E5(w)
E4(w),E(6/6′)
E5
E1, E3
E2, E4
E2, E3
E4, E6
E4, E5
E4, E5
h
E-6
h
E-6′
Gal-F
F-1
F-2
F-3
F-4
F-5
F-6
F-6′
E5
101.5
68.9
71.4
67.5
71.2
61.3
—
4.61
5.54
5.11
5.42
3.33
4.50
3.80
d
8.0
F2
B2
dd
dd
dd
ddd
dd
dd
8.0, 10.6
3.4, 10.6
0.8, 3.4
0.8,5.3,8.1
7.7, 11.3
5.3, 11.3
F1, F3
F2, F4
F3, F5(w)
F4(w), F6, F6′ F4, F6
F5, F6′
F5, F6
F1, F3
F2, F4(w)
F2, F3
F4, F5
F4, F5
Note: (w) denotes a weak but detectable correlation, (vw) very weak; singlet (s), doublet (d), triplet (t), quartet (q), quintet (qn), unresolved multiplet
m); no coupling observed (nco).
(
a
Spectra recorded in C D at 800 MHz.
Major and (or) minor rotomeric forms indicated where observed.
6
6
b
c
Acetate resonances not indicated.
d
1
D-TOCSY shows correlations: C1-C2, C3, C4, C5 and C6-C2, C4, C5, C6′.
e
Possible overlap with B-4 → C1 in HMBC.
f
Peaks broadened at 800 MHz, C4 appears as a triplet at 400 MHz; (nco)
g
h
1
D-NOE (400 MHz) shows C1-C3-C5 (negative enhancement).
5
Non-first-order coupling (see J resolved in Supplementary information ), COSY cross peaks coincident.
the 11 hydroxyl functionalities of the four glucose residues
A–D.
The mass of a pachymoside having the aglycon 5 and
acetylation at 10 sites is 1918, a mass found in the FAB-MS
of the crude mixture of natural pachymosides prior to
derivatization. Adding an additional acetate to the sugar al-
cohols generates a natural pachymoside with a mass of
D-6, since the HMBC correlation intensities in the 13C-
labelled compound indicate that these latter two positions
have the greatest degree of natural acetylation.
It was clear from the NMR data obtained for the mixture
of natural pachymosides and the aglycons liberated by
methanolysis that there are also natural variations in the
aglycon. There was evidence for aliphatic methyl doublets in
1
1
960, which was also represented in the FAB-MS of the
the H NMR spectrum of both the mixture of aglycon
crude mixture. Therefore, it appears that a major part of the
structural diversity in this family of glycolipids involves in-
creasing degrees of acetylation and differing positions of
acetylation on the pachymoside template. All of these varia-
tions in acetylation pattern would have been converted into
the same peracetylpachymoside A methyl ester (2) upon
derivatization for purification. We have arbitrarily chosen to
define pachymoside A (1) as the component that has natural
acetylation at the eight galactose hydroxyls and at B-6 and
methyl esters and the natural pachymoside mixture indicat-
ing the presence of internal methyl branches on the aglycon
fatty acyl chain. The detailed structures of the various agly-
cons other than 5 have not been determined due to the diffi-
culties of locating branching methyls in a long aliphatic
chain.
During the course of analyzing the NMR data for
peracetylpachymoside A methyl ester (2), an unexpected dif-
ference in the data obtained at 400, 500, and 800 MHz in
5
Supplementary data may be purchased from the Directory of Unpublished Data, Document Delivery, CISTI, National Research Council
Canada, Ottawa, ON K1A 0S2, Canada (http://www.nrc.ca/cisti/irm/unpub_e.shtml for information on ordering electronically).
©
2003 NRC Canada

Warabi et al.
109
Fig. 4. A minimal set of unambiguous ROESY and HMBC correlations linking the substructures of 2.
1
3
Fig. 5. Expansion of gHMBC spectrum of peracetylpachymoside A methyl ester (2) (CH CO-labeled) acquired at 800 MHz in C D .
3
6
6
Assignments of methines and methylenes coupled to 1 C-labeled acetates are indicated. The methine at C-4 exhibits weak coupling at
00 MHz (2D data), while none is observed at 800 MHz. 1D proton data (400 MHz) for this methine exhibits additional splitting
unlabeled appears as triplet, 13C-labeled appears as triplet of doublets). Cross peaks for the methylene to carbonyl coupling are of
generally weaker intensity than the methines and vary in order of decreasing intensity as follows: C6 > A6 > B6 > D6.
3
5
(
C D was encountered. As shown in Fig. 6, the resonance
nal overlap. Therefore, the deterioration of the multiplet
structure on going from 400 to 800 MHz was contrary to our
usual experience with 800 MHz data. One possible explana-
tion for the observation is restricted conformational mobility
at C-4 of glucose-C that is fast on the NMR timescale at
400 MHz leading to a single sharp resonance, but slow on
6
6
assigned to the glucose-C H-4 resonance appeared as a clear
dd (J = 9.9, 9.6 Hz) at 400 and 500 MHz as expected, but
appeared as a broad unresolved multiplet at 800 MHz.
Normally the increased dispersion at 800 MHz simplifies
multiplets by removing many second-order effects and sig-
©
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1
10
Can. J. Chem. Vol. 82, 2004
Fig. 6. NMR spectra (400 and 800 MHz) (C D ) of the glucose-C residue H-4 resonance.
6
6
the NMR timescale at 800 MHz leading to a broadening of
the resonance. Since the H-1, H-2, and H-6/H-6′ resonances
in residue C are sharp at 800 MHz, the conformational equi-
librium probably does not involve a change in the pyranose
conformation. The existence of at least two conformers for
rotation about the C-4 oxygen to acetate carbonyl bond that
interconvert slowly on the 800 MHz NMR timescale, is a
probable explanation for the observed field-dependent NMR
behaviour of H-4 on glucose-C. We are not aware of any lit-
erature reports of similar observations of peak shape change
as a function of increasing field strength.
Glycolipids are emerging as an important class of struc-
turally diverse sponge metabolites that frequently have inter-
esting biological activities (14, 16–21). The pachymosides
represent a novel family of sponge glycolipids that have un-
precedented glycon and aglycon fragments. The fatty acyl
component of their aglycon has the same number of carbons
and identical positioning of ketone and glycon attachments
as the fatty acyl component of erylusamine E (6), one of a
family of IL-6 receptor antagonists isolated from sponges in
the genus Erylus (19, 20). However, the sugar residues in 1
Experimental
General experimental procedures
1
H, COSY-gr, HMBC-gr (optimized for 2,3J = 8 Hz), and
HMQC-gr for (peracetyl pachymoside A methyl ester) were
initially recorded on a Bruker AMX500 NMR spectrometer.
These experiments plus 1D- and 2D-TOCSY, ROESY,
HSQC, eCOSY, and J-resolved experiments were repeated
on a Varian INOVA 800 MHz spectrometer at the NANUC
facility at the University of Alberta. C NMR data were re-
corded on a Bruker AM400 NMR spectrometer. Chemical
shifts were referenced to solvent peaks (δH 7.15 ppm, δC
128.0 ppm for C6D6). Low-resolution electrospray (ES)
mass spectra were recorded on a Bruker Esquire LC mass
spectrometer, operated by direct infusion. High-resolution
ES mass spectra were recorded on a Micromass LCT mass
spectrometer. Both low- and high-resolution EI mass spectra
were recorded on an AEI MS-50 mass spectrometer. Flash
silica gel column chromatography was performed using
1
3
2
30–400 mesh silica gel 60 (Silicycle). Preparative HPLC
separations were performed using a Waters 515 pump and a
Waters 2487 dual tunable absorbance detector. Solvents
were all HPLC grade (Fisher) and filtered prior to use. Thin
layer chromatography (TLC) was performed with Merck sil-
ica gel 60 F254 aluminum sheets, and visualized with vanillin –
sulfuric acid spray reagent. Pyridine, methanol, benzene,
acetic anhydride, 4-(dimethylamino)pyridine (DMAP), and
(
D-glucose and D-galactose) and 6 (D-arabinose and L-xylose)
are different, the number and connectivity of the sugars are
different, and the amino components of the aglycon amides
are different. The pachymosides have an acidic aglycon
while the erylusamines all have basic aglycons. One interest-
ing structural feature of the pachymosides is the complete
acetylation of the galactose residues and only partial
acetylation of the glucose residues. Further investigation of
the biological activity of the pachymosides revealed that al-
though they showed promising activity in the TTSS inhibi-
tion assay, they are not true TTSS inhibitors. Rather, their
observed activity appears to stem from their ability to effec-
tively activate extracellular bacterial proteases that rapidly
degrade the excreted Esps, resulting in a false indication of
TTSS inhibition in the assay (1).
(
trimethylsilyl)diazomethane (solution in hexanes) used in
derivatization reactions were anhydrous and (or) reagent
grade (Aldrich) and used without further purification. Acetic
anhydride (1,1′- C , 99%) was purchased from Cambridge
Isotope Laboratories, Inc.
1
3
2
Biological material and isolation of pachymosides
Specimens of P. johnstonia (650 g wet weight) were col-
lected by hand using SCUBA from rocky subtidal marine
©
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Warabi et al.
111
habitats along the coast of the Isle of Mann. A voucher sam-
ple (ZMA 17065) has been deposited at the Zoologisch Mu-
seum, University of Amsterdam. Freshly collected sponge
material was frozen immediately and stored at –15 °C until
workup. A portion of the frozen sponge (wet wt 111 g) was
cut into pieces and extracted twice with MeOH over a six
day period. The combined MeOH extracts were filtered and
concentrated by rotary evaporation to an amber semisolid.
This residue was partitioned between water (50 mL) and
ethyl acetate (ea) (100 mL), and the aqueous layer extracted
twice more with ethyl acetate (100 mL ea). The yellow-
colored ethyl acetate solution was evaporated to dryness in
vacuo to afford 1.60 g of a greenish amber amorphous solid.
A portion of this material (0.16 g) was purified by
Sephadex™ LH-20 size exclusion chromatography (28 mm
ID × 100 cm bed length) eluting with 100% MeOH. Early
eluting like fractions (as detected by TLC developed with
scribed above to give pure samples of methyl 2,3,4,6-tetra-
O-acetyl-α-D-glucopyranoside (3) and methyl 2,3,4,6-tetra-
O-acetyl-α-D-galactopyranoside (4).
Chiral GC analysis of alditol acetates
The pachymoside mixture was hydrolyzed for 1 h at re-
flux in H O (20 mL) and 0.25 mL of 6 N HCl. The reaction
2
was worked up by evaporating the H O to dryness under
2
high vacuum and the residue was partitioned between H O
2
and Et O. The water soluble materials were concentrated to
2
dryness via lyophilization and reduced with NaBH4 in
MeOH at rt. After removing the MeOH in vacuo, the residue
was treated with acetic anhydride and pyridine and a cata-
lytic amount of DMAP to give a mixture of alditol acetates.
Normal-phase flash chromatography separated the mixture
into glucitol peracetate and galactitol peracetate. Commer-
cial samples of D-glucose, L-glucose, and D-galactose were
also converted to their alditol peracetates using the same re-
action conditions. The alditol peracetates were analyzed by
GC on a Chrial Select 1000 β-Dex-390 column using flame
ionization detection. The GC analysis showed that the glu-
cose and galactose obtained from hydrolysis of the pachy-
moside mixture both had the D configuration.
1
5% MeOH–CH Cl ) were combined and concentrated in
2 2
vacuo to give a mixture of pachymosides as a white amor-
phous solid (0.106 g). This material exhibited a distribution
of molecular ion clusters centred at m/z 1973/1974 in the
LR-FAB-MS.
Methanolysis of the pachymoside mixture
Pachymosides (60 mg) were dissolved in 10 mL of anhy-
Aglycon (5)
drous methanol and 0.2 mL of CH COCl was slowly added.
3
The Et O soluble fraction (25mg) from methanolysis of
The reaction mixture was stirred at 70 °C with refluxing for
2
the pachymoside mixture was loaded on a normal phase sil-
ica gel flash column and eluted with a gradient of hexanes–
EtOAc (5% EtOAc:95% hexanes – 100%EtOAc). The frac-
tions eluting with 20% EtOAc:80% hexanes contained one
major component and one minor component by TLC analy-
sis. The more polar major component was fractionated fur-
ther by isocratic reversed-phase HPLC (eluent: 88%
2
h. MeOH was removed from the reaction mixture in vacuo
and the resulting residue was partitioned between H O and
Et O. The aglycon methyl esters (25 mg) went into the or-
ganic phase and the methyl glycosides (30mg) went into the
aqueous phase.
2
2
Acetylation of monosaccharide methylglycosides
MeOH:12% H O (monitored at 206.0 nm)) to give pure
2
The mixture of monosaccharide methyl glycosides
methyl ester 5 (see Results and discussion for spectroscopic
data).
(
20 mg) obtained from methanolysis of the pachymosides
was added to 9 mL of anhydrous pyridine, 3 mL of acetic
anhydride, and ~1 mg of DMAP as catalyst. The reaction
mixture was stirred overnight at room temperature. After re-
moval of the pyridine and acetic anhydride under high vac-
Esterification of the pachymoside mixture
A 45 mg (ca. 0.024 mmol) sample of the pachymoside
mixture was dissolved in a mixture of 0.5 mL each of
MeOH and benzene and treated with 0.080 mL of
uum, the crude product was partitioned between H O and
Et O. The organic extract soluble materials were purified by
2
2
–
1
isocratic normal phase HPLC (eluent: 31% EtOAc:69%
hexanes) monitored with a RI detector to give one fraction
containing peracetylated glucose/galactose α-methyl glyco-
pyranosides and a second fraction containing peracetylated
glucose/galactose β-methyl glycopyranosides.
(trimethylsilyl)diazomethane (2.0 mol L solution in hex-
anes) and stirred at ambient temperature for 24 min. The re-
action mixture was diluted with 5 mL EtOAc, evaporated to
dryness (aspirator), then redissolved in EtOAc (6 mL),
washed with sat. NaCl solution, dried (Na SO ) and again
2
4
evaporated in vacuo to a film (38 mg). A short reaction time
was found to be critical in this step. If left to react overnight,
most of the acetate groups appeared to be lost, as determined
by NMR. Mass spectroscopy (ES) on this sample again ex-
hibited a distribution of molecular ion clusters centered at
Preparation of monosaccharide standards
Commercial D-glucose or D-galactose (20 mg, 0.11 mmol)
were dissolved in 10 mL MeOH with 0.2 mL of CH COCl
3
(
2 mmol) and stirred with refluxing at 70 °C for 2 h. The
+
solvent was then removed in vacuo to give a residue that was
partitioned between H O and Et O. The aqueous fraction
2012 (Na adducts) with a nearly identical pattern to that
found for the underivatized material. This represents a shift
in molecular weight of plus 14 amu, which is correct for the
methyl ester derivative, and confirms that the acetate groups
of the pachymosides remained fully intact in this step.
2
2
was dried and added to 9 mL (100 mmol) of anhydrous
pyridine and 3 mL of acetic anhydride (30 mmol) with
~
1 mg of DMAP as a catalyst. The acetylation mixture was
a
+
stirred at room temperature overnight. After removal of the
pyridine and acetic anhydride under high vacuum, the crude
product was partitioned between H O and Et O and the or-
LR-ESI-MS data : (Na adducts) 1956 (55), 1969 (75),
1998 (70), 2012 (100), 2040 (65), 2053 (80); exchanged
with CD OD: 1965 (50), 1979 (75), 2006 (55), 2018 (100),
2
2
3
ganic extract was purified by normal-phase HPLC as de-
2046 (50), 2060 (70). (Italic indicates homolog ions that
©
2003 NRC Canada

1
12
Can. J. Chem. Vol. 82, 2004
would afford pachymoside
acetylation).
A
peracetate upon full
tance and use of the facilities. NANUC is funded by CIHR,
NSERC, and the University of Alberta.
Acetylation of the pachymoside methyl ester mixture
The sample of mixed pachymoside methyl esters (38 mg)
was dissolved in anhydrous pyridine (3.0 mL) under argon
and treated with 1.3 mg DMAP, then with 1 g of acetic an-
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(
2
UV detection at 202 and 280 nm, up to 3 mg per injection)
to give pachymoside A methyl ester (2), peracetate (4.0 mg)
1
3
1
1
(
or its corresponding 1- C-acetylated analog).
Data for unlabeled analog
HR-ESI-MS:
m/z
2333.9639
(calcd.
for
12C
13
1
13
CH159NO Na 2333.9658); H and C NMR (800
1
05
54
and 100 MHz, respectively) see Table 1.
1
LR-ESI-MS: Unlabeled acetate derivative: 2335 (100,
+
+
13
Na ), 1178 (2Na ). C-acetate derivative: 2343/2344 (100,
Na ), 1183 (10, 2Na ), indicating the presence of eight to
+
+
1
3
nine C-acetyl groups.
Note: LR-ESI-MS data m/z absolute values estimated at
1 amu at these values; relative values of the weighted clus-
1
1
(
Elofsson. Chem. Biol. 10, 241 (2003)
±
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shows three or more sequential peaks owing to naturally
1
3
abundant C).
1
1
7. V. Costantino, E. Fattorusso, and A. Mangoni. Tetrahedron,
5
6, 5953 (2000).
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(
RJA), Canadian Institutes for Health Research (CIHR) (AG,
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Williams for collecting P. johnstonia and the Canadian Na-
tional High Field NMR Centre (NANUC) for their assis-
2
1. R. Goobes, A. Rudi, Y. Kashman, M. Ilan, and Y. Loya. Tetra-
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©
2003 NRC Canada