Oceanapiside, an antifungal bis-α,ω-amino alcohol glycoside from the marine sponge Oceanapia phillipensis

Article abstract of DOI:10.1021/np990190v

The structure of oceanapiside, an antifungal α,ω-bis- aminohydroxylipid glycoside from the temperate marine sponge Oceanapia sp., was elucidated by a combination of 2D NMR, chemical degradation/ correlation, and MALDI MS-MS spectrometry. Oceanapiside exhibits antifungal activity against Candida glabrata at 10 μg/mL (MIC).

Full text of DOI:10.1021/np990190v

1678
J . Nat. Prod. 1999, 62, 1678-1681
Ocea n a p isid e, a n An tifu n ga l Bis-r,ω-a m in o Alcoh ol Glycosid e fr om th e Ma r in e
Sp on ge Ocea n a pia ph illipen sis
Gillian M. Nicholas, Tamilyn W. Hong, Tadeusz F. Molinski,* Melissa L. Lerch, Mark T. Cancilla, and
Carlito B. Lebrilla
Department of Chemistry, One Shields Avenue, University of California, Davis, California 95616
Received April 23, 1999
The structure of oceanapiside, an antifungal R,ω-bis-aminohydroxylipid glycoside from the temperate
marine sponge Oceanapia sp., was elucidated by a combination of 2D NMR, chemical degradation/
correlation, and MALDI MS-MS spectrometry. Oceanapiside exhibits antifungal activity against Candida
glabrata at 10 µg/mL (MIC).
The structures of sphingolipids from marine inverte-
brates share features of sphingolipids derived from plants
and animals. In higher animals, the base is sphingosine
(erythro-D-E-2-amino-4-octadecene-1,3-diol), while sphin-
ganine (D-2-amino-octadecan-1,3-diol) is most common in
plant sphingolipids.1 A significant departure from this
motif is found in the structures of dimeric amino alcohol
bases from certain calcareous sponges. Leucettamols A (1)
and B, from Leucetta sp.;2 compound BRS1, from an
unidentified sponge;3 rhizochalin (2), from Rhizochalina
incrustata,4 and related compounds; coriacenins, from the
Mediterranean Clathrina coriacea;5 and rhapsamine, from
the Antarctic Leucetta leptorhapsis,6 are all aminolipids
composed of symmetrical, or almost symmetrical, very long
hydrocarbon chains (C28-C30) functionalized at both ter-
minii as vicinal amino alcohols. We report here a new
glycosidic amino alcohol lipid, oceanapiside (3) from a
temperate-water, non-calcareous sponge, Oceanapia phil-
lipensis Dendy, 1895 (Haplosclerida, Phloeodictyidae) col-
lected in Southern Australia. Oceanapiside is a naturally
occurring “bolaamphiphile”sa term coined7 to describe
amphiphilic lipids with polar functionality at both termini
whose structures suggest a resemblance to the bola, a
weapon fashioned from two leather balls tethered to the
ends of a string. Oceanapiside exhibits significant antifun-
gal activity against the pathogenic, Fluconazole-resistant
yeast, Candida glabrata.
The n-BuOH-soluble portion of a MeOH extract of
Oceanapia phillipensis was fractionated by reversed-phase
(C18) chromatography to afford a highly polar amino lipid,
(-)-oceanapiside (3), C34H68N2O9 (HRFABMS (MH+
649.5003, ∆ mmu ) -1.3), which gave a positive ninhydrin
test. Oceanapiside was insoluble in CHCl3 and EtOAc, but
soluble in MeOH and water. The UV spectrum of 3 showed
no absorbance above λ 210 nm. Interestingly, the base peak
in the ESIMS of 3 was the doubly charged ion at m/z 325
(z ) 2) that corresponds to [M + H2]2+. Taken together with
the positive ninhydrin reaction, this suggested a readily
protonated diamine. The presence of a keto group was
indicated by FT-IR (ν 1712 cm-1, CdO stretch) and 13C
NMR spectra (δ 214.3, s). Although it was later confirmed
that 3 contains two free primary amines, treatment of 3
with t-BOC carbonate (THF-MeOH-Et3N) gave only a
mono-t-BOC derivative 4 (C39H75N2O11, ESIMS, m/z 748,
M+), presumably because the second NH2 group was
hindered by the glycosyl group (see below). Oceanapiside
gave no precipitate with AgNO3 (absence of Cl-), so we
formulated 3 as the free base, but cannot exclude a salt
with other anions.
Interpretation of the 1H and 13C NMR spectra of 3 in
CD3OD (Table 1) revealed one acetal group (δ 4.30, d, J )
7.6 Hz; δC 104.5 d), seven additional methines attached to
oxygen (δ 80.1, 78.1, 77.7, 74.8, 73.1, 71.6), and a lipid chain
(CH2 envelope, δ 1.28, br s). Six of the 13C signals were
consistent with the presence of a sugar, which was identi-
fied as D-glucose as follows. Methanolysis of 3 (2M HCl in
MeOH, 80 °C, 16 h) gave two fractions, separable by Si gel
chromatography. The first fraction was a mixture of
anomeric methyl glycosides (C7H14O6, m/z 180, MH+ - Me),
identified as an R:â mixture of 1-O-methyl-D-glucopyrano-
sides by high-performance TLC and comparison with an
authentic sample (obtained by methylation of D-glucose,
HCl, MeOH, 80 °C, 2 h). Glycoside 3 has the â-configuration
at the anomeric carbon as shown by the coupling constant
for the anomeric proton signal (δ 4.30, d, J ) 7.6 Hz).8 The
second fraction eluted from the column with NH3-MeOH-
CHCl3 and was identified as the novel polar aglycon 5
(FABMS; m/z 487.5, MH+).
A downfield 13C signal (δ 214.3, s) for a keto group and
the glycopyranosyl ring accounted for all of the degrees of
unsaturation required by the formula of 3. The remainder
of the formula was attributed to a linear aminolipid chain.
Interpretation of COSY, gHMQC, and gHMBC data re-
vealed the two terminal sequences of oceanapiside algycon
as -CH(OH)CH(NH2)CH3 and -CH(O-)CH(NH2)CH2OH-
and enabled assignment of the corresponding two sets of
13C NMR signals: CH2-O (δ 58.9, t, C-1), CH-N (57.2, d,
C-2), CH-O (80.1, d, C-3) and CH3 (16.0, q, C-28), CH-N
(53.5, d, C-27), and CH-O groups (73.1, d, C-26). The two
CH2 groups (δ 2.43, t, J ) 6.5 Hz, 4H; δC 43.4, t) R to the
ketone carbonyl were clearly resolved and readily assigned
by comparison with 3-heptanone (δ 2.37, t, J ) 7 Hz; δC
44.9, t).9 The methyl terminus (δΗ ∼1.3, d, δC 16.0, q) was
obscured in the 1H NMR spectrum of 3 by the CH2
envelope, but inferred from observation of the expected
COSY and HMQC cross-peaks together with DEPT data,
and conveniently revealed in the 1H NMR spectrum (CDCl3,
Table 1) of the octaacetyl derivative 6 (δ 1.05, d, J ) 6.7
Hz) or the t-BOC derivative 4 (CDCl3, δ 1.14, d, J ) 6.5
Hz).
Oceanapiside (3) gave an octaacetyl derivative, 6 (Ac2O,
pyridine, 16 h, 25 °C). The COSY spectrum (500 MHz) of 6
1
revealed downfield shifts (∆δ ∼0.5-1 ppm) of carbinol H
* To whom correspondence should be addressed. Tel: (530) 752-6358.
Fax: (530) 752-8995. E-mail: tfmolinski@ucdavis.edu.
signals due to acetylation of the OH groups, including both
10.1021/np990190v CCC: $18.00
© 1999 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/23/1999

Notes
J ournal of Natural Products, 1999, Vol. 62, No. 12 1679
Ta ble 1. 1H, 13C NMR Data for Oceanapiside (3) and the Octaacetyl Derivative 6
Oceanapiside 3 (CD3OD, 500 MHz)
octaacetyl 6 (CDCl3, 500 MHz)
δH (mult, J Hz) COSY
atom no.
δC
δH (mult, J Hz)
COSY
gHMBCa
ROESY
1a
1b
2
3
4a
4b
5
6,7
8
58.9 (t)
3.86 (dd, 3.8, 11.8)
3.69 (dd, 9.2, 11.8)
3.35 (m)
3.92 (m)
1.52 (m)
1.70 (m)
1.34 (m)
∼1.28 (br s)
∼1.28 (br s)
1.51 (m)
C2, C3
C2, C3
4.2 (m)
4.08 (dd, 8, 11.5)
4.2 (m)
3.65 (m)
1.58 (m)
4.2
57.2 (d)
80.1 (d)
33.1 (t)
H4a, H4b
H3
H3
C2, C4, C5, C1′
C3, C5, C6
1.58, 4.2
3.65
4.48, 6.43
26.7 (t)
30.5
30.2 (t)
24.8 (t)
43.4 (t)
214.3 (s)
43.4 (t)
24.8 (t)
30.2 (t)
9
H10
H9
C10
C9, C11
1.55 (m)
2.36 (m)
2.36
1.55
10
11
12
13
14
15-23
24
25a
25b
26
27
28
1′
2′
3′
4′
5′
6′a
6′b
2-NH
27-NH
Ac
2.43 (t, 7)
2.43 (t, 7)
1.51 (m)
∼1.28 (br s)
∼1.28 (br s)
1.37 (m)
1.39 (m)
1.56 (m)
3.45 (m)
H13
H12
C13, C11
C12
2.36 (m)
1.55 (m)
1.55
2.36
26.2
34.5 (t)
H26
H26
C26, C27
C26, C27
73.1 (d)
53.5 (d)
16.0 (q)
H25a, H25b, H27 C24, C25, C27, C28 4.85 (m)
1.55, 4.20 1.05
4.85
3.10 (dq, 6.9, 6.9)
H28, H26
H27
H2′
C25, C26, C28
C26, C27
C3, C2′, C3′/C5′
C1′, C4′, C3′/C5′
4.2 (m)
∼1.25 (d)b
1.05 (d, 6.7)
4.48 (d, 8.0)
4.96 (dd, 8.0, 9.5)
5.20 (dd, 9.5, 9.5)
5.08 (dd, 9.5, 9.5)
3.70 (m)
4.2
4.85
104.5 (d) 4.30 (d, 7.6)
4.96
3.70, 5.20
74.8 (d)
77.7 (d)
71.6 (d)
78.1 (d)
62.6 (t)
3.22 (dd, 3.6, 9.0)
H3′, H1′
4.48, 5.20
3.35 (m)
3.25 (m)
3.35 (m)
3.63 (dd, 6.9, 11.6) H5′, H6′
3.95 (dd, 2.1, 11.6) H5′, H6′
3.70, 4.48
4.48, 5.20
C2′, C3′/C5′, C6′
3.70
4.2, 5.08
C4′, C3′/C5′
C4′, C3′/C5′
4.2
4.2
6.43 (d, 9.0)
5.54 (d, 9.5)
2.09, s; 2.07, s; 2.20, s;
2.01, s (× 2); 1.99, s;
1.97, s; 1.96, s
4.2
4.2
3.65
a
b
gHMBC recorded at 600 MHz. Obscured by the CH2 envelope.
gHMBC). Clear assignments of the carbinol, CH-NH, and
1
glycosidic H signals were now made that allowed place-
ment of the sugar group. The anomeric proton signal H-1′
(δ 4.30, d, J ) 7.6 Hz) showed a correlation to δ 80.1 d,
which, in turn, was assigned to C-3 from a combination of
COSY and gHMBC data. In addition, the ROESY spectrum
of 6 (CDCl3) showed a strong dipolar correlation between
the H-3 (δ 3.65, m) and H-1′ (δ 4.48, d, J ) 8.0 Hz). Taken
together, these data show the location of the D-glucopyra-
nosyl group at C-3.
Initial attempts to assign the position of the CdO group
in 3 by mass spectrometry were unsuccessful, as neither
the ESIMS nor FABMS of 3 showed R or â cleavage
fragment ions, while the EIMS fragmentation of 6 was
dominated by multiple losses of ketene and CH3CO. We
recognized that the solution to this problem lay in soft-
ionization, preferably with unambiguous charge-localiza-
tion at the glucose group of the quasimolecular ion.
Fortuitously, a sample of 3 left standing in CD3OD (ca. 2
months, 4 °C) underwent adventitious deuterium exchange
of the CH2 hydrogens R to the carbonyl group. After
reexchange of the labile OD and ND deuterons in the
sample with CH3OH, the 1H NMR spectrum of 3-d4 showed
primary OH groups (H2-6′ and H2-1), as well NH acetamide
signals (δ 6.43, d, J ) 9.0 Hz; 5.54, d, J ) 9.5 Hz), which
supported the presence of two primary NH2 groups in 3.
COSY and RELAY experiments identified the two spin
systems associated with NH acetamide proton signals at
each chain terminus.
1
a diminished H signal for the R-CH2 protons (δ 2.43, 2 ×
t)10 and a single 2H NMR (CH3OH) signal (δD 2.41, br s)
due the two R-CD2 groups. The deuterium content of 3-d4
(74 atom % by MS - 49% d4, and lesser amounts of other
d isotopomers) was suitable for MS fragmentation studies.
Matrix-assisted laser desorption ionization (MALDI) MS-
MS of oligosaccharides in the presence of Li+ salts has been
shown to produce well-behaved fragmentation with defined
charge-localization on sugar groups.11 MALDI MS-MS of
The location of the glucopyranosyl group in 3 was
established by gHMBC. Severe overlap in the δ 3.0-4.0
1
region in the H NMR spectrum of 3 (300 or 500 MHz) was
relieved when spectra were recorded at 600 MHz (gHMQC,

1680 J ournal of Natural Products, 1999, Vol. 62, No. 12
Notes
mL, C. glabrata) than the glycoside 3, possibly due to better
cell penetration.
Exp er im en ta l Section
Gen er a l Exp er im en ta l P r oced u r es.These procedures are
described elsewhere.12 FABMS measurements were performed
at the University of California, Riverside Mass Spectrometry
facility. MALDI FT-MS measurements were carried out on a
custom-built instrument at the University of California,
Davis.13
An im a l Ma ter ia l. Oceanapia phillipensis Dendy, 1895 (90-
12-095) was collected by hand using scuba at a depth of 30 m
off Point Lonsdale in Port Phillip Bay, Victoria, Australia, in
1990, and stored at -20 °C until needed. The sponge sample
has an encrusting flat base, mounted by fistula (40-70 mm
in height), and terminating with apical oscula (ca. 10 mm
diameter). The texture is very fibrous and spiculose. The
ectoderm shows red pigmentation (3 mm penetration), while
the interior is reddish brown. In life, the animal emits a
pungent odor. The authoritative descriptive work on Oceana-
pia spp. from Port Phillip Bay is credited to Arthur Dendy.14
Of the four Oceanapia species described by Dendy from the
same location as our specimen, O. mollis and O. imperfecta
are excluded by the absence of microsclera. The characteristic
oxeas of 90-12-095 were slightly curved with a size range of
0.080-0.10 mm, entirely consistent with only one of the
candidates, Oceanapia phillipensis (Dendy, 1895).14,15 Type
samples and spicule mounts are available from the corre-
sponding author.
Extr a ction a n d Isola tion . The lyophilized sponge (40.8
g) was repeatedly extracted with MeOH (× 3) and the
combined MeOH extracts concentrated (∼500 mL). The water
content (% v/v) of the MeOH extract was adjusted before
sequentially partitioning against n-hexane (10% H2O), CHCl3
(40%), and n-BuOH (100%). The n-BuOH extract was concen-
trated to give an orange solid (1.3 g), which was positive to
ninhydrin. Further purification by reversed-phase flash chro-
matography on a solid-phase extraction cartridge (Varian, 20
g, C18 bonded-phase) with stepwise gradient elution using
aqueous MeOH (40-100% MeOH: H2O) afforded pure ocean-
apiside 3 (43.3 mg, 0.11% of dry wt). Early fractions from the
column-cartridge gave a white-yellow precipitate upon addition
of 1 drop of 0.1 M AgNO3, (sea salt), but fractions containing
pure 3 gave no precipitate. Oceanapiside (3) produced a
distinct pink stain on TLC upon treatment with with ninhy-
drin reagent spray.
F igu r e 1. MALDI FT-MS spectra of 3. (a ) MALDI FT-MS spectra of
3 in the presence of Li+. (b) MALDI FT-MS of 3-d4. (cd. 70 atom%) in
the presence of Li+. Laser pulse power was decreased with respect to
(a ) to retain parent ion [M + Li+] intensity. (c) Expansion of M + Li+
of 3-d4 parent ion showing d0-d4 isotopomer cluster.
Ocea n a p isid e (3): colorless solid; C34H68N2O9; [R]D -5.5°
3-d4, (λ 347 nm laser irradiation) revealed the lithiated
parent ion (m/z 659, [M + 7Li]+, d4) and the fragment
arising from charge-remote γ-cleavage two bonds from the
(c 1.2, MeOH); no UV absorbance above 210 nm; IR (ZnSe,
1
film), ν 3400-2900 br s, 1712, cm-1; H and 13C NMR, Table
1; ESIMS m/z 649 (30%) [M + H]+, 325 (100%) [M + H2]2+
;
7
CdO group (m/z 417, [M + Li - C15H31NO]+, d3) only on
HRFABMS m/z [M + H+], 649.5016 (calcd for C34H69N2O9,
649.5003).
the side of the carbonyl containing the glucopyranoside
group.11 Both peaks were clearly identified from observa-
tion of the expected deuterium and Li isotope patterns
(Figure 1). Thus, the ketone carbonyl was located at C-11,
closer to the more oxidized terminus, and the same relative
position was found in 2.
Oceanapiside (3) differs from rhizochalin (2) by the
replacement of the 3-O-â-D-galactopyranosyl group in 2
with a 3-O-â-D-glucopyranoside. Presently, the stereochem-
istry of 3 is undefined due to the difficulty of independent
assignment of the four stereogenic centers at dissimilar
termini in this long-chain aminolipid.
Oceanapiside (3) showed antifungal activity against the
Fluconazole-resistant yeast Candida glabrata with a mini-
mum inhibitory concentration (MIC) of 10 µg/mL in broth
dilution experiments. Under similar conditions, amphot-
ericin B gave an MIC of ca. 1 µg/mL against C. glabrata,
but 3 was essentially inactive against C. krusei; C. albicans
ATCC 14503; and two Fluconazole-resistant strains, C.
albicans 96-489 and C. albicans UCD-FR1. Interestingly,
the aglycon 5 showed higher antifungal activity (MIC 3 µg/
N-t-BOC Ocea n a p isid e (4). A solution of 3 in THF and
MeOH (ca. 2 mL) was treated with freshly distilled Et3N (50
µL) and t-BOC carbonate (ca. 50 mg) and allowed to stir for 2
h. Evaporation of the solvent and Si gel chromatography of
the residue (0.7 × 6 cm, gradient, MeOH-CHCl3) gave a mono-
t-BOC derivative, 4: 1H NMR (CDCl3) 1.14 (d, 3H, J ) 6.5
Hz), 1.22 (br s), 1.41 (s, 9H), 2.35 (2 × t, 4H, J ) 7 Hz), 3.3-
4.3, (br m); C39H75N2O11, ESIMS m/z 748 [M]+.
Meth a n olysis of Ocea n a p isid e (3). A solution of 3 (11.7
mg) in 2M HCl in MeOH (1 mL) was heated in a sealed vial
at 80 °C for 16 h. The mixture was cooled, concentrated, and
subjected to chromatography on Si gel (0.7 × 6 cm) to provide
two fractions.
For Fraction 1, elution of the column with 1:4 MeOH-CHCl3
gave anomeric (+)-1-O-methyl glucopyranosides (2.7 mg); [R]D
+62.7° (c 0.15, MeOH), EIMS m/z 180 [M - MeOH]+, CIMS
1
m/z 212 (100%) [M + NH4]+, identical by H NMR (300 MHz)
and [R]D with authentic (+)-1-O-methyl R/â-D-glucopyranosides
prepared from D-glucose (see below). TLC: A 5 × 20 cm plate
coated with silica GF, was sprayed with an aqueous solution
of 0.1M NaH2PO4 and activated at 120 °C for 3 h prior to use.
Adjacent lanes were spotted with either 1-O-methyl d-glu-

Notes
J ournal of Natural Products, 1999, Vol. 62, No. 12 1681
copyranosides (R:â mixture), or 1-O-methyl R-D-galactopyra-
noside (prepared from the d-glucose and D-galactose, respec-
tively, 1M HCl in MeOH, 80 °C, 2 h) or each co-spotted
together with the methanolysate of 3. The plate was developed
with 40:50:10 n-BuOH-Me2CO-0.1 M NaH2PO4 aqueous and
visualized with vanillin-H2SO4 (heat). The O-Me glycoside
obtained from 3 coeluted with 1-O-methyl D-glucopyranoside
(Rf, R-anomer, 0.55), ahead of 1-O-methyl d-galactopyranosides
(Rf, R-anomer, 0.47).
pulse energy was adjusted until appearance of daughter ions,
m/z 414, [M + 7Li-d4]+ (100%), was observed. See Figure 1 for
the fragmentation patterns.
Ack n ow led gm en t. We are very grateful to Mary Kay
Harper, Scripps Institution of Oceanography, for assistance
with identification of the sponge; Roger Mulder for assistance
with HMQC and HMBC experiments; Karl Bailey for the
ESIMS spectrum of 4; and Paulus Bruins for help with the
2H NMR measurement. This work was partially supported by
the National Institutes of Health (AI 31660, AI 39987). The
500 MHz NMR spectrometer was partially funded through
NIH ISIO-RR04795 and NSF BBS88-04739, and the 600 MHz
instrument was partially funded by NIH RR11973.
For fraction 2, further elution of the column (12:18:1
MeOH-CHCl3-NH4OH aqueous) gave amino lipid (+)-5,
C28H58N2O4, as a clear glass (4.8 mg): [R]D +14.7°(c 0.3,
MeOH); 1H NMR (CD3OD) 1.10 (d, 3H, J ) 6.6 Hz), 1.28 (br s,
CH2 envelope), 1.53 (m, 8H), 2.43 (m, 4H), 2.74 (m, 1H), 2.80
(m, 1H), 3.48 (dd, 1H, J ) 7.8, 10.5 Hz), 3.53 (m, 1H), 3.73
(dd, 1H, J ) 4.2, 11.1 Hz) FABMS m/z 487.5 [M + H]+.
Oct a a cet yl d er iva t ive, 6. A sample of 3 (11.7 mg) was
dissolved in pyridine (0.5 mL) and acetic anhydride (0.5 mL)
and allowed to stand at 25 °C for 18 h. Removal of the volatile
material gave a residue (7.6 mg) that was purified by Si gel
chromatography (0.7 × 5 cm, 2:98 MeOH-CHCl3) to provide
6 (2.8 mg) as a clear glass; 1H NMR (CDCl3), see Table 1; EIMS
m/z 925.5 (8%) [M - OAc]+, 866.6 (43) [M - 2OAc]+, 842.5
(28) [M + H - 2CH2dCdO]+, 782.5 (28), 578.5 (55), 518.4
(100).
Refer en ces a n d Notes
(1) Shapiro, D. The Chemistry of Sphingolipids; Hermann: Paris, 1969;
Vol. 9, p 112.
(2) Kong, F. H.; Faulkner, D. J . J . Org. Chem. 1993, 58, 970-971.
(3) Willis, R. H.; De Vries, D. J . Toxicon 1997, 35, 1125-1129.
(4) Makarieva, T. N.; Denisenko, V. A.; Stonik, V. A. Tetrahedron Lett.
1989, 30, 6581-6584.
(5) Casapullo, A.; Fontana, A.; Cimino, G. J . Org. Chem. 1996, 61, 7415-
7419.
(6) J ayatilake, G. S.; Baker, B. J .; McClintock, J . B. Tetrahedron Lett.
1997, 38, 7507-7510.
(7) Fuhrhop, J .-H.; Mathieu, J . Angew. Chem. 1984, 96, 124-37.
(8) Collins, P. M.; Ferrier, R. J . Monosaccharides. Their Chemistry and
Their Roles in Natural Products; J ohn Wiley: Chichester, 1995.
(9) Pouchert, C.; Behnke, J . The Aldrich Library of 13C and 1H FT NMR
Spectra, 1st ed., 1993; Vol. 1.
(10) We surmise that the adventitious exchange of the R-CH2 protons was
promoted, in the case of 3, by the basic amino groups, either by a
general base mechanism or through reversible formation of Schiff-
base intermediates.
(11) Cancilla, M. T.; Penn, S. G.; Carroll, J . A.; Lebrilla, C. B. J . Am. Chem.
Soc. 1996, 118, 6736-6745.
Deu ter iu m Con ten t, 2H NMR, a n d MALDI F T-MS of
3-d 4. A sample of 3 (ca. 25 mg) was stored in CD3OD for ca. 2
months. The volatiles were removed under vacuum and the
residue re-evaporated from CH3OH (× 2) to obtain a sample
of 3-d4. The presence of deuterium label in 3-d4 was detected
2
by both H NMR and MS. The 2H NMR spectrum was recorded
using a solution of 3-d4 in CH3OH on a General Electric QE
300 using the lock channel in observe mode and referenced to
2
the natural abundance of the H solvent signal (CDH2OH, δD
3.30), 2H NMR (CH3OH) δ 2.41 (br s). The isotopomer distribu-
tion of 3-d4 was measured from the MALDI FT-MS quasimo-
lecular ion isotope-cluster in the presence of Na+ [M + Na]+
by Biemann’s method16 (74 atom %; d; 49% d4, 25% d3, 11%
d2, 4% d1, 11% d0). In separate experiments, samples of 3 or
3-d4 (ca 0.1 µg) were doped with LiCl (0.01 µmol) and
dihydroxybenzoic acid (ca. 140 µg) and subjected to MALDI
ionization on a custom-built FT-MS instrument.13 The laser
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