Journal of Natural Products
Article
E 124°35.60′). The lavender-colored sponge is thickly encrusting
across a coral fragment. Consistency is firm and crisp and breaks easily.
The sponge was identified as Haliclona (Halichoclona) sp. POR6118
(family Chalinidae, order Haplosclerida). The ectosomal skeleton is a
tangential subisotropic reticulation of single spicules cemented at the
nodes with spongin. The choanosome consists of a subisotropic
paucispicular skeleton with ill-defined primary and secondary lines
with many loose spicules in confusion. The spicules are stout oxeas
that are abruptly pointed with a dimension of 190−210 × 8−10 μm.
At least 53 species belonging to the genus of Haliclona have been
reported in the Philippines and neighboring areas (Central Indo
Pacific), and only two belong to the same subgenus Halichoclona.
Haliclona (Halichoclona) vanderlandi de Weerdt & van Soest, 2002
from Indonesia has similar spicule dimensions (175−218 × 6−10 μm),
but its growth form is tubular. Haliclona (Halichoclona) centragulata
(Sollas, 1902) from Malaysia also has similar spicule dimensions, 220 ×
7 μm, but has sigmoid microscleres in addition. Eight species hve been
reported from the Philippines, and none of these fit the present species.
In conclusion, the present sponge might very well be an undescribed
species. A voucher sample has been deposited at The Netherlands
Centre for Biodiversity Naturalis in Leiden, The Netherlands (voucher
number: RMNH POR. 6118).
Small-Scale Extraction of the Sponge and Isolation of
Halicloic Acids A (1) and B (2). Freshly collected sponge was frozen
on site and transported frozen. Lyophilized sponge material (10 g) was
cut into small pieces and immersed in and subsequently extracted
repeatedly with MeOH (3 × 50 mL) at rt. The combined MeOH
extracts were concentrated in vacuo, and the resultant residue was then
partitioned between EtOAc (3 × 5 mL) and H2O (15 mL). The
combined EtOAc extract was evaporated to dryness, and the resulting
oil was chromatographed on Sephadex LH-20 with 4:1 MeOH/
CH2Cl2 as eluent. Purification of the active fraction via C8 reversed-
phase HPLC using a Phenomenex 5 μm, Luna 25 × 1.0 cm column,
with 3:1 MeCN/(0.05%TFA/H2O) as eluent, gave pure samples of
halicloic acids A (1) (0.5 mg) and B (2). However, the yield of B
provided us with only enough material to obtain a very weak 1H NMR
spectrum.
Aldehyde 3: pale yellow glass; [α]25 −51 (c 1.1, 3:1 CH2Cl2/
D
MeOH); 1H and 13C NMR, see Table 1; (−)-HRESIMS m/z
545.3990 [M − H]− (calcd for C37H53O3, 545.3995).
Aldehyde 4: pale yellow glass; [α]25 +42 (c 0.3, 3:1 CH2Cl2/
D
MeOH); 1H and 13C NMR, see Table 2; (−)-HRESIMS m/z
545.3989 [M − H]− (calcd for C37H53O3, 545.3995).
Treatment of Both Mandelic Acid and 4-Hydroxymandelic
Acid with DMSO-d6, with and without Trace Amounts of TFA.
To dried 10 mg samples of D/L-mandelic acid and 4-hydroxymandelic
acid was added 600 μL of 4:1 MeCN/(0.05%TFA/H2O), and the
samples were immediately dried. Similarly, to dried 10 mg samples of
D/L-mandelic acid and 4-hydroxymandelic acid was added 600 μL
of 4:1 MeCN/H2O, and again the samples were immediately dried.
To each of the four dried samples was added 600 μL of DMSO-d6, and
the samples were left to sit at rt for 7 days. Each sample was then
heated to 50 °C for 7 days, followed by 78 °C for 4 days, and finally
128 °C for 4 days. The resulting reaction mixture obtained for the
sample of 4-hydroxymandelic acid treated with 4:1 MeCN/(0.05%
TFA/H2O) was the only sample to show both appreciable
decomposition and the significant production of an aromatic aldehyde
as determined by the observation of a singlet resonating at δ 9.76 ppm
and the concomitant appearance of an aromatic doublet at δ 7.74 ppm
1
in the H NMR spectrum. These diagnostic resonances, though only
small, were initially seen after heating to 50 °C for 7 days and
increased in size as the temperature increased. The experiments were
terminated when there appeared to be enough of the desired product
to isolate. The sample was purified using Si gel flash chromatography
(step gradient 19:1 hexanes/EtOAC to EtOAc to 1:9 MeOH/EtOAc,
2 g Sep pak). An early eluting fraction (19:1−9:1 hexanes/EtOAc) was
further purified via C8 reversed-phase HPLC, using a CSC-Inertsil
150A/ODS2 5 μm 25 × 0.94 cm column, with 17:3 H2O/MeCN as
eluent to give 4-hydroxybenzaldehyde (0.5 mg), which was identified
by co-injection and comparison with the UV and NMR spectra of a
standard sample.
IDO Inhibition Assays. Enzyme activity assays were performed
with human recombinant IDO (rh-IDO) expressed in E. coli as
previously described.23 The activity of rh-IDO in the absence and
presence of inhibitory compounds was determined using an end-point
assay as previously described24 with the following changes: The assay
was performed in 100 mM potassium phosphate buffer (pH 6.5)
containing 10 mM sodium ascorbate (Sigma), 1.25 μM methylene
blue (Sigma), 10 μg/mL catalase (Sigma), and 400 μM L-tryptophan
(Sigma). The reaction was started by the addition of rh-IDO (100 nM)
and allowed to progress at 37 °C for 60 min before termination with 30%
(w/v) trichloroacetic acid. The samples were further incubated for 15 min
at 60 °C prior to addition of 2% (w/v) 4-(dimethylamino)-
benzaldehyde (Sigma). After 5 min at rt, the absorbance at 480 nm
was measured with a Tecan infinite M200 plate reader, and the
kynurenine concentration was determined from the extinction
coefficient (15 820 M−1 cm−1) for kynurenine.25 Nonlinear regression
of the enzyme activity assays and calculations of IC50 values were
performed using GraphPad Prism 4.
Decomposition of Halicloic Acid A (1) to Aldehyde 3. After
lyophilizing halicloic acid A (1) overnight in an NMR tube and adding
600 μL of DMSO-d6, 1 was observed to decompose cleanly to aldehyde 3
within 24 h at 4 °C.
Larger Scale Extraction of the Sponge and Isolation of
Halicloic Acids A (1) and B (2). The larger scale workup started
with 32 g of lyophilized sponge material and proceeded as described
above to yield 15.8 and 3.7 mg of halicloic acids A (1) and B (2),
respectively.
Controlled Decomposition of Halicloic Acids A (1) and B (2)
to Aldehydes 3 and 4, Respectively. Halicloic acids A (1) and B
(2) appeared stable when the two samples were dried down directly in
NMR tubes and spectra acquired in MeOH-d4 or in mixtures of
MeOH-d4/CD2Cl2. The spectra obtained in these NMR solvents were,
however, poorly resolved, with broad peaks and of no use for structural
elucidation. With this in mind, after both 1 and 2 had first been dried
overnight in separate NMR tubes on a freeze drier, 600 μL of DMSO-d6
was added to an additional 2.1 mg of halicloic acid A (1) and to 0.6 mg
of halicloic acid B (2). Immediately after the addition of the DMSO-d6,
1D and 2D NMR spectra were run for both samples. Fortunately, this
time, for both samples there was only partial conversion after 16 h of
collecting NMR data (5% and 20%, for 1 and 2, respectively).
Then over a period of several weeks initially at 4 °C and later at rt both
1 and 2 were observed to cleanly convert to aldehydes 3 and 4,
respectively.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 2D NMR spectra of compounds 1, 2, 3, and 4.
This material is available free of charge via the Internet at
Halicloic acid A (1): pale yellow glass; [α]25 −66 (c 6.1, MeOH);
D
AUTHOR INFORMATION
1
■
UV (3:1 MeCN/(0.05%TFA/H2O)) λmax 208, 224, 272 nm; H and
13C NMR, see Table 1; (−)-HRESIMS m/z 591.4054 [M − H]−
(calcd for C38H55O5, 591.4050).
Corresponding Author
*Tel: 604 822 4511. Fax: 604 822 6091. E-mail: raymond.
Halicloic acid B (2): pale yellow glass; [α]25D −5.0 (c 2.1, MeOH);
1
UV (3:1 MeCN/(0.05%TFA/H2O)) λmax 208, 224, 272 nm; H and
13C NMR, see Table 2; (−)-HRESIMS m/z 591.4039 [M − H]−
Notes
(calcd for C38H55O5, 591.4050).
The authors declare no competing financial interest.
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dx.doi.org/10.1021/np300345j | J. Nat. Prod. 2012, 75, 1451−1458