Journal of Natural Products
NOTE
Figure 1. Key HMBC and selected COSY correlations for bathymo-
diolamide A (1).
Lucky Strike location with a latitude of 37ꢀ17.630 N, longitude of
32ꢀ16.530 W, and depth of 1733 m.3 Once aboard the research
vessel, the mussels were dissected, and samples of adductor
muscle, gill, and mantle tissues were frozen at -70 ꢀC for
subsequent analyses. One hundred grams wet weight of the
tissues from approximately 20 gills was extracted in MeOH. One
part of the MeOH-soluble extract was dissolved in DMSO and
was tested for apoptosis induction as assessed by the ApopScreen
protocol.4-6 Screening was expected to identify compounds that
possess proapoptotic, and potentially anticancer, activity.
The extract induced apoptosis and therefore was fractionated,
with subsequent purification by analytical reversed-phase HPLC.
Using this strategy, two compounds were isolated. The chemical
structures of these two compounds (1 and 2) were ascertained by
standard spectroscopic techniques, as described below.
Figure 2. Compounds 3-6 resulting from chemical degradation of 1.
The molecular formula of 1 was established as C49H89NO7 on
the basis of HRMALDITOFMS [m/z 804.6712 (M þ H)þ].
The 13C NMR spectrum for 1 clearly indicated resonances for
three ester or amide carbonyl carbons (δC 173.2, 173.4, 173.8),
as well as four oxygen-bearing carbons (δC 59.9, 62.5, 63.7, 66.1)
and one nitrogen-bearing carbon (δC 53.5, δH 4.29). Analysis of
the multiplicity-edited HSQC spectrum for 1 revealed that these
heteroatom-substituted carbons comprise two methylenes (C-
H2-1, δC 63.7, δH 3.65; CH2-5, δC 59.9, δH5a 4.20, δH5b 4.45)
and three methines (CH-2, δC 53.5, δH 4.29; CH-3, δC 62.5, δH
4.01; CH-4, δC 66.1, δH 5.25). The HMBC correlations between
H-2 and the carbonyl at δC 173.2, H-4 and the carbonyl at δC
173.4, and H-5a and H-5b and the carbonyl at δC 173.8 suggested
that 1 has an amide moiety attached to C-2 and two ester
moieties attached to C-4 and C-5. Considering the molecular
formula and all carbon signals for 1, the remaining oxygen-
bearing carbons C-1 and C-3 are substituted with hydroxy
groups, resulting in primary and secondary alcohols at C-1 and
C-3, respectively. The connectivity of C-1 through C-5 of 1 was
established from COSY data as shown in Figure 1, confirming the
presence of the amino alcohol moiety in 1.
The remaining partial structures for 1 could be assigned as
three acyl moieties. Given the six degrees of unsaturation of 1
based on its molecular formula, three double bonds must be
present in one or more of the three acyl moieties. The HMBC
correlations from δH 5.35 to δC 26.5 and from δH 2.81 to
δC 127.5, taken together with the 4H integral of the signal at δH
2.81 in the 1H NMR spectrum of 1, indicated the occurrence of
four bis-allylic protons and two bis-allylic methylene carbons,
suggesting that all three double bonds are located in one branch
of the aliphatic side chain. Since bis-allylic carbon signals for Z
and E isomers are observed at ca. δC 27 and 32, respectively,7,8
the 26.5 ppm shift suggested that all double bonds have a cis
geometry (Z).
Figure 3. Compounds 50 and 60 resulting from chemical degradation
of 2.
routine workup of the reaction, the nonpolar organic extract
was analyzed by GC-MS and ESIMS, and three ion peaks [M þ
H]þ at m/z 215.2010, 265.2166, and 271.2636 were observed,
corresponding to dodecanoic acid methyl ester (4), hexadeca-
trienoic acid methyl ester (5), and hexadecanoic acid methyl
ester (6), respectively (Figure 2).
The regiochemical distribution of these three acyl chains was
established using different ionization mass spectrometry anal-
yses. The amide bond was most readily recognized21 by MAL-
DITOFMS/MS [M þ H]þ at m/z 184.1, corresponding to the
cleavage of the dodecanoyl moiety. ESIMS9 was used to differ-
entiate the acyl attached at C-4 and C-5. Fragmentation by
positive-mode ESIMS generated a more stable fragment ion from
cleavage of the acyl chain attached at C-5 than cleavage of the acyl
chain attached to C-4. In contrast, the negative-mode ESIMS
fragment ion resulting from cleavage of the acyl chain attached at
C-4 is more stable. Successive losses of dodecanoyl and hexade-
catrienoyl moieties were observed in the negative-mode ESIMS
(m/z 584.2 and 367.1, respectively). From these analyses it was
concluded that the dodecanoyl moiety is attached to C-2, while
the hexadecatrienoyl and hexadecanoyl moieties are attached
to C-4 and C-5, respectively. To verify this conclusion, a selective
enzyme reaction was performed. Upon regioselective enzymic
hydrolysis of 1 with lipase enzyme type III in dioxane/H2O (1:1)
at 37 ꢀC for 4 h,10 only hexadecanoic acid was obtained, as evidenced
by ESIMS and comparison with an authentic sample. Thus, it was
concluded that the hexadecanoyl residue is attached to C-5 of 1.
The aqueous phase from the hydrolysis product of 1 yielded
an aminotetraol (3), which was identified as (2S,3S,4R)-2-amino-
1,3,4,5-pentane tetraol. The relative configuration of 3 was
To deduce the identity of the fatty acids, 1 was subjected to a
transesterification reaction in MeOH/NaOMe (2 h). After
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dx.doi.org/10.1021/np100601w |J. Nat. Prod. 2011, 74, 842–846