Caryophyllene sesquiterpenoids from a fungicolous isolate of Pestalotiopsis disseminata

Article abstract of DOI:10.1021/np050460b

Three new caryophyllene-type sesquiterpene alcohols, 6-hydroxypunctaporonin E (1), 6-hydroxypunctaporonin B (2), and 6-hydroxypunctaporonin A (3), have been isolated from cultures of the fungicolous fungus Pestalotiopsis disseminata. The structures of 1-3

Full text of DOI:10.1021/np050460b

608
J. Nat. Prod. 2006, 69, 608-611
Caryophyllene Sesquiterpenoids from a Fungicolous Isolate of Pestalotiopsis disseminata
Stephen T. Deyrup,† Dale C. Swenson,† James B. Gloer,*,† and Donald T. Wicklow‡
Department of Chemistry, UniVersity of Iowa, Iowa City, Iowa 52242, and Mycotoxin Research Unit, Agricultural Research SerVice,
National Center for Agricultural Utilization Research, USDA, Peoria, Illinois 61604
ReceiVed NoVember 8, 2005
Three new caryophyllene-type sesquiterpene alcohols, 6-hydroxypunctaporonin E (1), 6-hydroxypunctaporonin B (2),
and 6-hydroxypunctaporonin A (3), have been isolated from cultures of the fungicolous fungus Pestalotiopsis disseminata.
The structures of 1-3 were determined mainly by analysis of 1D and 2D NMR data. The structure and absolute
configuration of 6-hydroxypunctaporonin E (1) was confirmed through X-ray crystallographic analysis of its mono-
bromobenzoate derivative. Compounds 1 and 2 showed activity against Gram-positive bacteria.
Explorations into the chemistry of fungi have shown them to be
a plentiful source of a diverse array of natural products. Although
fungal chemistry has been widely investigated, many ecological
niche groups remain underexplored. One unique niche is occupied
by fungicolous fungi, those that colonize other fungal species. Our
chemical studies of fungicolous fungi have shown them to be rich
sources of novel metabolites, even among genera that have been
examined previously.1-6
The genus Pestalotiopsis has been the subject of a handful of
prior chemical studies. Chemical investigation of a phytopathogenic
isolate of P. oenotherae yielded both polyketide and terpenoid
constituents.7 Studies of endophytic Pestalotiopsis spp. have yielded
the caryophyllene-derived pestalotiopsins, pestalotiopsolides, and
taedolidols.8-10 Recently, our studies of a fungicolous isolate of
Pestalotiopsis disseminata (Thuem.) Stey. (Amphisphaeriaceae;
MYC-1444 ) NRRL 36915) have afforded three new sesquiterpene
alcohols named 6-hydroxypunctaporonin E (1), 6-hydroxypuncta-
poronin B (2), and 6-hydroxypunctaporonin A (3). Details of the
isolation and structure determination of these compounds are
presented here.
The punctaporonins are a set of six caryophyllene sesquiterpe-
noids that were originally isolated from the coprophilous fungus
Poronia punctata (Linnaeus:Fries).11-14 The structures of puncta-
1
poronins A-F were originally determined via H NMR, MS, and
X-ray crystallography, and the absolute configurations of puncta-
poronins A and D were determined by enantiospecific total
synthesis.15
Results and Discussion
NMR and DEPT data with the molecular formula indicated that
Fractionation of an organic extract from solid-substrate fermenta-
there must be four exchangeable protons, requiring the presence
of four free hydroxy groups. Signals for four oxygenated carbons
tion of P. disseminata using silica gel, followed by purification
employing reversed-phase HPLC, afforded compounds 1-3. These
sesquiterpene alcohols are new 6-hydroxy derivatives of puncta-
(one methylene, two methines, and a quaternary carbon) were
observed, along with nine additional sp3 signals (three quaternary
poronins E, B, and A, respectively.
carbons, one methine, two methylenes, and three methyl groups)
The molecular formula for compound 1 was determined to be
in the 13C NMR spectrum. The 1H NMR spectrum showed that all
C15H24O4 (four degrees of unsaturation) by analysis of 1H and 13
C
three methyl groups are isolated, as is the primary alcohol unit.
1H NMR coupling information enabled identification of the C2-
C3, C6-C7, and C9-C10-C11 units. HMBC data were used to
establish the connectivity of these units with the remainder of the
molecule. HMBC correlations from geminal methyls H3-13 and H3-
14 to C-3, C-4, and C-5 extended the C2-C3 fragment through
C-4 and C-5. Correlations were also observed from H3-15 to C-1,
C-7, C-8, and C-9 and from H-6 to C-5, linking the C6-C7 and
C9-C10-C11 fragments via methylated quaternary carbon C-8.
Key correlations between H2-12 and C-1, C-2, C-8, and C-11
located the primary alcohol unit and enabled closure of the five-
and six-membered rings in 1. HMBC correlations from H-2 to C-11
and C-6 required the presence of a cyclobutane ring to complete
NMR data (Table 1) in conjunction with DEPT results, and this
conclusion was confirmed by HRESIMS. The 13C NMR data
showed that compound 1 had only two olefinic carbons and no
carbonyl groups and must therefore be tricyclic in order to account
for the remaining degrees of unsaturation. DEPT and 1H NMR data
indicated that the olefin must be 1,2-disubstituted, and the small
J-value (5.7 Hz) is consistent with the presence of a cis-olefin unit,
1
most likely incorporated into a small ring. Comparison of the H
* To whom correspondence should be addressed. Tel: 319-335-1361.
Fax: 319-335-1270. E-mail: james-gloer@uiowa.edu.
† University of Iowa.
‡ USDA.
10.1021/np050460b CCC: $33.50
© 2006 American Chemical Society and American Society of Pharmacognosy
Published on Web 03/11/2006

Caryophyllene Sesquiterpenoids from Pestalotiopsis
Journal of Natural Products, 2006, Vol. 69, No. 4 609
Table 1. NMR Data for 6-Hydroxypunctaporonin E (1)
a
δΗ
HMBCc
correlations (C#)
NOESYd
correlations (H#)
b
position
(multiplicity, JHH
)
δC
1
2
3a
3b
4
5
6
7a
7b
8
50.3
42.9
35.8
2.00 (dd, 12.8, 7.4)
2.14 (dd, 12.8, 8.3)
1.59 (dd, 8.3, 7.4)
e
e
3b, 6, 11, 13
2, 3a
3b, 12b, 14
1, 2, 4, 5, 12f, 14
42.1
80.3
69.3
44.0
3.58 (dd, 10.5, 5.7)
1.80 (dd, 13.5, 5.7)
1.73 (dd, 13.5, 10.5)
5, 7
13
1, 5, 6, 8, 9, 15
1, 5, 6, 8, 9, 15
15
6, 9
53.3
147.4
129.8
81.6
9
5.83 (d, 5.7)
5.68 (dd, 5.7, 2.7)
4.12 (d, 2.7)
4.20 (d, 11.4)
3.83 (d, 11.4)
1.12 (s)
1, 7, 8, 10, 11, 15
1, 8, 9, 11, 15f
2, 8, 9, 10
7b, 15
11
2, 10
10
11
12a
12b
13
14
15
61.4
1, 2, 11
1, 2, 8, 11
3, 4, 5, 14
3b, 12a
12b, 15
2, 6
24.2
23.3
27.9
1.13 (s)
1.24 (s)
3, 4, 5, 13
3a
1, 6f, 7, 8, 9
7b, 9, 12a
a Recorded in CDCl3 at 300 MHz. b Recorded in acetone-d6 at 75 MHz. c Recorded in acetone-d6 at 600 MHz (1H dimension). d Recorded in
f
CDCl3 at 400 MHz. e Fortuitous overlap of these signals in acetone-d6 prevented unambiguous assignment of their correlations. Four-bond correlation.
Figure 2. Selected NOESY correlations for 6-hydroxypunctaporo-
nin B (2).
system. H-10 correlated to H-2, thus requiring the E-geometry for
the C-1/C-11 olefin, and H3-15 correlated to H-11, requiring the
Figure 1. X-ray model of 12-(4-bromobenzoyl)-6-hydroxy-
Z-configuration for the C-9/C-10 olefin (Figure 2). The H3-15 to
punctaporonin E (4). Thermal ellipsoids are shown at the 30% level.
H-11 correlation also permits the assignment of H3-15 to the top
face of the ring system. This relative configuration matches that of
the ring system. This planar structure is similar to that of
punctaporonin E, differing only in the presence of an additional
hydroxy group at C-6. It was therefore assigned the trivial name
6-hydroxypunctaporonin E (1).
punctaporonin B.12 The absolute configuration is presumed to be
analogous to that of 1 (2R, 5S, 6S, and 8S). The difference in the
R/S designation at position 8 is due to a change in priority
assignments in 2 relative to 1.
The relative configuration of 1 was determined via analysis of
NOESY data (Table 1). The structure of 1 was confirmed and the
absolute configuration assigned through single-crystal X-ray dif-
fraction analysis of the mono-bromobenzoate derivative (4; Figure
1). The inclusion of a bromine atom in the molecule allowed
determination of the absolute configuration as shown in 1 (1R, 2R,
5S, 6S, 8R, and 11R). These data allow for the tentative assignment
of the absolute configuration of punctaporonin E by analogy.
Compound 2 is an isomer of 1, but contains two additional sp2
carbons, indicating the presence of an additional double bond and
only two rings in 2. In addition, one of the oxygenated methine
signals observed for 1 was replaced by an additional oxygenated
The NMR data for compound 3 were nearly identical to those
of 1, except that the HMBC data for 3 showed a correlation from
H3-15 to an oxygenated methine carbon (C-9) rather than an olefinic
carbon, and H2-12 correlated with an olefinic carbon (C-11) rather
than an oxymethine carbon, indicating that the hydroxy and olefinic
groups were transposed from their positions in 1.
The NOESY spectrum of 3 closely resembled that of 1 (Table
1). H-2 showed correlations to H-3b, H-6, and H3-13, placing all
of these hydrogens on the same face of the ring system. H2-12
showed correlations to H-3a and H3-15, placing these protons on
the opposite face of the ring system relative to H-2. Correlation of
H-9 with H3-15 indicated their cis relationship. Again, the absolute
configuration is presumed to be analogous to that of 1 and
punctaporonin A15 (1S, 2R, 5S, 6S, 8S, and 9R).
1
quaternary carbon signal in the 13C NMR spectrum of 2. H-1H
coupling and HMBC data revealed the presence of the same C2-
C7 structural unit found in 1, as well as a new conjugated diene
system corresponding to the C1/C9-C11 unit in 2. HMBC
correlations of H3-15 to C-7, C-9, and the new oxygenated
quaternary carbon (C-8) enabled connection of C-15, C-7, and C-9
to C-8. Correlations of H2-12 to C-1, C-2, and C-11 led to
completion of the gross structure as shown in 2.
The relative configuration of 2 was assigned by analysis of
NOESY data. As in 1, H-2 correlated to H-6, requiring a trans
ring fusion, and placing H-2 and H-6 on the same face of the ring
The punctaporonins were originally characterized using 1D NMR
techniques, and their structures were verified by X-ray crystal-
lography. On the basis of the HMQC results obtained for 3, the
1
reported H NMR data11 for punctaporonin A appear to contain a
minor misassignment. In the literature report, the proton at C-2
was assigned as one of the methylene protons of C-3. This
assignment was understandably complicated by the fact that the
geminal coupling constant between the protons on C-3 is only 7.6
Hz, while the vicinal coupling between the downfield C-3 proton

610 Journal of Natural Products, 2006, Vol. 69, No. 4
Deyrup et al.
6-Hydroxypunctaporonin E (1): white powder; [R]25D -20 (c 0.08,
CH3OH); HPLC tR 14.7 min under the above conditions; UV (CH3-
OH) λmax (log ꢀ) 201 (3.38); IR (KBr) νmax 3401, 2918, 1630, 1384,
1095, 1046 cm-1; 1H NMR, 13C NMR, HMBC, and NOESY data, see
Table 1; EIMS (70 eV) m/z 250 (M - H2O; rel int 2), 232 (3), 219 (5),
194 (42), 176 (12), 163 (16), 150 (49), 125 (100), 107 (53), 91 (62),
55 (75); HRESIMS m/z 267.1599 [M - H]-, calcd for C15H23O4,
267.1596.
and the C-2 proton is 12 Hz. Similar HMQC correlations were
observed in 1 and 2, therefore resulting in a similar adjustment in
the corresponding assignments for punctaporonins A, B, and E.
Literature descriptions of the biological activity of the puncta-
poronins have varied from “of little interest”14 to “remarkable”.15
Compounds 1 and 2 exhibited activity in standard agar disk
diffusion assays16,17 at 100 µg/disk against Bacillus subtilis (ATCC
6051), each causing a 12-mm zone of inhibition. Staphylococcus
aureus (ATCC 29213) was inhibited to a lesser extent by 1 and 2,
the zone being 8 mm in both cases. No activity was observed for
3 against B. subtilis or S. aureus. All three compounds were for
inactive in assays against Escherichia coli (ATCC 25922) and
Candida albicans (ATCC 14053) at 100 µg/disk or against
Aspergillus flaVus (NRRL 6541) and Fusarium Verticillioides
(NRRL 25457) at 200 µg/disk.
6-Hydroxypunctaporonin B (2): colorless glass; [R]25 -80 (c
D
0.08, CH3OH); HPLC tR 28.5 min under the above conditions; UV
(CH3OH) λmax (log ꢀ) 198 (3.62) 206 (3.51); IR (KBr) νmax 3414, 2932,
1
1705, 1642, 1384, 1120, 1065 cm-1; H NMR (CDCl3, 300 MHz) δ
5.87 (1H, d, J ) 2.2 Hz, H-11), 5.72 (1H, d, J ) 13 Hz, H-9), 5.63
(1H, ddd, J ) 13, 2.2, 2.1 Hz, H-10), 4.12 (1H, dd, J ) 11, 2.1 Hz,
H-12a), 4.02 (1H, d, J ) 4.8 Hz, H-6), 3.88 (1H, d, J ) 11 Hz, H-12b),
3.35 (1H, dd, J ) 12, 8.2 Hz, H-2), 2.82 (1H, dd, J ) 16, 4.8 Hz,
H-7a), 2.15 (1H, dd, J ) 12, 10 Hz, H-3a), 1.54 (1H, dd, J ) 10, 8.2
Hz, H-3b), 1.41 (1H, d, J ) 16 Hz, H-7b), 1.26 (3H, s, H3-13), 1.24
(3H, s, H3-15), 1.10 (3H, s, H3-14); 13C NMR (CDCl3, 75 MHz) δ
141.2 (d, C-9), 138.2 (s, C-1), 127.8 (d, C-11), 124.4 (d, C-10), 82.7
(s, C-5), 74.2 (s, C-8), 71.8 (d, C-6), 65.1 (t, C-12), 46.9 (t, C-7), 40.4
(s, C-4), 39.2 (d, C-2), 33.7 (t, C-3), 32.5 (q, C-15), 24.6 (q, C-13),
23.8 (q, C-14); HMBC (acetone-d6, 600 MHz) H-2 f C-1, 3, 4, 5, 6,
11, 12; H-3a f C-1, 2, 4, 5, 13, 14; H-3b f C-2, 4, 5, 6, 13; H-6 f
C-8; H-7a f C-5, 6, 8, 9, 15; H-7b f C-5, 6, 8, 9, 15; H-9 f C-1, 7,
11; H-10 f C-1, 8; H-11 f C-1, 2, 5, 9, 10, 12; H-12a f C-1, 2, 11;
H-12b f C-1, 2, 11; H3-13 f C-3, 4, 5, 14; H3-14 f C-3, 4, 5, 13;
H3-15 f C-7, 8, 9; NOESY (acetone-d6, 400 MHz) H-2 f H-3b, 6,
10, 13; H-3a f H-3b, 12a, 12b, 14; H-3b f H-2, 3a, 12a, 12b, 13;
H-6 f H-2, 7a, 7b, 13; H-7a f H-6, 7b, 11, 15; H-7b f H-6, 7a; H-9
f H-15; H-10 f H-2; H-11 f H-7a, 12b, 15; H-12a f H-3a, 12b;
H-12b f H-3a, 11, 12a; H3-13 f H-2, 6; H3-14 f H-3a; H3-15 f
H-7a, 9, 11; EIMS (70 eV) m/z 250 (M - H2O; rel int 1), 232 (4), 217
(4), 204 (18), 175 (9), 161 (16), 147 (17), 131 (48), 107 (60), 91 (83),
55 (100); HRESIMS m/z 267.1594 [M - H]-, calcd for C15H23O4,
267.1596.
Experimental Section
General Experimental Procedures. Optical rotations were mea-
sured with a Rudolph Research Autopol III automatic polarimeter. UV
measurements were performed with a Varian Cary 100 Bio UV-vis
spectrophotometer. IR measurements employed a Perkin-Elmer Spec-
1
trum BX FT-IR instrument. H and 13C NMR spectra were recorded
on either a Bruker AC-300 or a DPX-300 spectrometer. NOESY data
were recorded on a Bruker DRX-400. HMBC and HMQC experiments
were performed on a Bruker AMX-600 spectrometer. Chemical shift
values were referenced to residual solvent signals as follows (δH/δC):
acetone-d6 (2.05/29.8), CDCl3 (7.24/77.0), CD3OD (3.30/49.0). ESIMS
and HRESIMS data were recorded on a Micromass Autospec instru-
ment, and EIMS (70 eV) data were obtained using a Finnigan Voyager
instrument.
Fungal Material. Stromata of an unidentified pyrenomycete growing
on a dead hardwood branch in a hardwood swamp within Reed
Bingham State Park, Adel, GA, were collected by B. W. Horn on April
29, 2000. The stromata, along with portions of the woody substrate on
which the stromata had formed, were placed in plastic bags and stored
at 5 °C. To isolate microfungal colonists, stromatal surfaces were gently
abraded with a surface-sterilized fingernail file to remove portions of
the blackened tissues. Direct plating of stromatal filings was ac-
complished by sprinkling a small portion (100-200 mg) of the filings
over the surface of each of two plates of dextrose-peptone-yeast extract
agar (DPYA) containing streptomycin (25 mg/L) and tetracycline (1.25
mg/L).18 Plates were incubated in the dark at 25 °C for 5 days, and
representative cultures were isolated from each colony type showing a
distinctive morphology on DPYA. After 7-12 days incubation, the
tube cultures isolated from the stromatal filings were segregated into
groups of presumptive species and maintained for identification and
rice fermentation (25 °C).
One of these cultures, MYC-1444, was determined by D.T.W. to
be an isolate of Pestalotiopsis disseminata (Thuemen) Steyaert. A
subculture of this organism was deposited in the ARS collection at the
USDA National Center for Agricultural Utilization Research in Peoria,
IL, with the accession number NRRL 36915.
Fermentation was carried out in a single 500-mL Erlenmeyer flask
containing 50 g of rice. Distilled H2O (50 mL) was added to the flask,
and the contents were soaked overnight before autoclaving at 15 lb/
in.2 for 30 min. The flask was cooled to room temperature, inoculated
with 3.0 mL of spore inoculum, and incubated for 30 days at 25 °C.
After incubation, the fermented rice substrate was mechanically
fragmented and then extracted repeatedly with EtOAc (3 × 100 mL).
The combined EtOAc extracts were filtered and evaporated to give a
yellow oil (419 mg).
6-Hydroxypunctaporonin A (3): white powder; [R]25D -11 (c 0.07,
CH3OH); HPLC tR 18.1 min under the above conditions; UV (CH3-
OH) λmax (log ꢀ) 202 (3.38); IR (KBr) νmax 3394, 2926, 1632, 1384,
1088, 1020 cm-1; 1H NMR (CD3OD, 300 MHz) δ 5.70 (2H, br s, H-10
and H-11), 4.18 (1H, dd, J ) 9.7, 5.2 Hz, H-6), 4.07 (1H, s, H-9),
3.69 (1H, d, J ) 10.5 Hz, H-12a), 3.46 (1H, d, J ) 10.5 Hz, H-12b),
2.38 (1H, dd, J ) 12, 7.5 Hz, H-2), 2.06 (1H, dd, J ) 12, 7.6 Hz,
H-3a), 1.91 (1H, dd, J ) 13, 5.2 Hz, H-7a), 1.63 (1H, dd, J ) 13, 9.7
Hz, H-7b), 1.53 (1H, dd, J ) 7.6, 7.5 Hz, H-3b), 1.19 (3H, s, H3-13),
1
1.12 (3H, s, H3-14), 1.02 (3H, s, H3-15); H NMR data for 3 were
recorded in both acetone-d6 and CD3OD because the olefinic signals
were fortuitously overlapping in CD3OD, but acetone-d6 obscured the
signal at δ 2.06; 13C NMR (CD3OD, 75 MHz) δ 142.3 (d, C-11), 130.6
(d, C-10), 88.4 (d, C-9), 81.1 (s, C-5), 70.1 (d, C-6), 62.6 (t, C-12),
55.6 (s, C-1), 50.9 (s, C-8), 48.1 (d, C-2), 43.1 (s, C-4), 41.0 (t, C-7),
35.9 (t, C-3), 24.7 (q, C-15), 24.3 (q, C-14), 23.5 (q, C-13); HMBC
(acetone-d6, 600 MHz) H-2 f C-1, 3, 4, 5, 6, 8, 11, 12; H-3a f C-1,
2, 4, 5, 13, 14; H-3b f C-2, 4, 5, 6, 13, 14; H-6 f C-5, 7; H-7a f
C-1, 4, 5, 6, 8, 9, 15; H-7b f C-5, 6, 8, 9, 15; H-9 f C-1, 10, 11, 15;
H-10 f C-1, 8, 9, 11; H-11 f C-1, 8, 9, 10, 12; H-12a f C-1, 2, 8,
11; H-12b f C-1, 2, 8, 11; H3-13 f C-3, 4, 5, 14; H3-14 f C-3, 4, 5,
13; H3-15 f C-1, 7, 8, 9; NOESY (CDCl3, 400 MHz) H-2 f H-3b, 6,
13; H-3a f H-3b, 12a, 14; H-3b f H-3a; H-6 f H-2, 7a, 13; H-7a f
H-6, 7b, 15; H-7b f H-7a, 15; H-9 f H-7a, 15; H-12a f H-3a, 12b;
H-12b f H-12a, 15; H3-13 f H-6; H3-14 f H-3a; H3-15 f H-7a, 7b,
9, 12b; overlap of H-10 and H-11 prevented unambiguous assignment
of NOESY correlations for those signals; EIMS (70 eV) m/z 250 (M
- H2O; rel int 2), 237 (23), 219 (18), 194 (37), 181 (90), 163 (100),
145 (49), 135 (78), 107 (57), 91 (92), 55 (98); HRESIMS m/z 267.1587
[M - H]-, calcd for C15H23O4, 267.1596.
12-(4-Bromobenzoyl)-6-hydroxypunctaporonin E (4): A sample
of 1 (6.0 mg) was dissolved in dry THF and added to a 10-mL round-
bottom flask. To the flask were added 2.7 mg of DMAP and 5.4 mg of
4-bromobenzoyl chloride. Three milliliters of dry CH2Cl2 was added
to aid in the dissolution of the reagents. The reaction vessel was sealed
under an Ar atmosphere and stirred at room temperature for 168 h.
Purification was carried out by reversed-phase HPLC (Beckman
Ultrasphere, semipreparative C-18 5-µm column, 1.0 × 25 cm, 2 mL/
min) with a linear gradient from 20 to 100% CH3CN in water over 40
Isolation of 1-3. The EtOAc extract (419 mg) was partitioned
between hexanes and CH3CN. The CH3CN phase (384 mg) was then
fractionated on a silica gel column, eluting successively with 100%
CH2Cl2, 98:2 CH2Cl2-CH3OH, 95:5 CH2Cl2-CH3OH, 90:10 CH2Cl2-
CH3OH, and 80:20 CH2Cl2-CH3OH, and 20-mL fractions were
collected. Compounds 1, 2, and 3 were present in the 15th fraction (74
mg), eluting at 90:10 CH2Cl2-CH3OH. They were separated by
preparative reversed-phase HPLC (Rainin Dynamax, C-18 column, 2.0
× 30 cm, 10 mL/min) using 20% CH3CN in water for 25 min, followed
by a linear gradient from 20 to 60% CH3CN over 15 min to afford 1
(20 mg), 2 (9 mg), and 3 (12 mg).

Caryophyllene Sesquiterpenoids from Pestalotiopsis
Journal of Natural Products, 2006, Vol. 69, No. 4 611
min. The desired monoacylation product eluted at 27.4 min: 1H NMR
(CDCl3, 300 MHz) δ 7.88 (2H, br d, J ) 8.7 Hz), 7.58 (2H, br d, J )
8.7 Hz), 5.83 (1H, d, J ) 5.6 Hz, H-9), 5.79 (1H, dd, J ) 5.6, 2.6 Hz,
H-10), 5.22 (1H, d, J ) 10.6 Hz, H-12a), 4.77 (1H, dd, J ) 10.6, 1.3
Hz, H-12b), 4.32 (1H, br s, H-11), 3.65 (1H, dd, J ) 9.8, 6.4 Hz,
H-6), 2.24 (1H, dd, J ) 12, 9.0 Hz, H-3a), 1.92 (1H, dd, J ) 12, 7.9
Hz, H-2), 1.86 (1H, dd, J ) 14, 9.8 Hz, H-7a), 1.80 (1H, dd, J ) 14,
6.4 Hz, H-7b), 1.69 (1H, dd, J ) 9.0, 7.9 Hz, H-3b), 1.14 (3H, s, H3-
15), 1.10 (3H, s, H3-14), 1.05 (3H, s, H3-13); ESIMS m/z 473 [M +
Na]+.
Supporting Information Available: 1H NMR and 13C NMR spectra
of 1-3 and H NMR data for 4 are available free of charge via the
1
Internet at http://pubs.acs.org.
References and Notes
(1) Li, C.; Gloer, J. B.; Wicklow, D. T.; Dowd, P. F. J. Nat. Prod. 2005,
68, 319-322.
(2) Angawi, R. F.; Swenson, D. C.; Gloer, J. B.; Wicklow, D. T. J. Nat.
Prod. 2005, 68, 212-216.
X-ray Crystallographic Analysis of 12-(4-Bromobenzoyl)-6-
hydroxypunctaporonin E (4).19 A colorless needle (0.32 × 0.03 ×
0.03 mm) obtained upon crystallization from 7:3 CH3CN-H2O was
separated from the sample, mounted with grease on the tip of a glass
capillary epoxied to a brass pin, and placed on the diffractometer with
the long crystal dimension (unit cell a-axis) approximately parallel to
the diffractometer phi axis. Data were collected on a Nonius KappaCCD
diffractometer (Mo KR radiation, graphite monochromator) at 190(2)
K (cold N2 gas stream) using standard CCD techniques, yielding 29 073
data. Lorentz and polarization corrections were applied. Equivalent data
were averaged, yielding 3964 unique data (Rint ) 0.069, 3134 F >
4σ(F)). Cell dimensions were determined to be a ) 6.7816(7) Å, b )
16.7520(17) Å, c ) 19.939(2) Å, R ) 90°, â ) 90°, γ ) 90°. On the
basis of a preliminary examination of the crystal, the space group
P212121 was assigned (no significant exceptions to the systematic
absences h00, h ) odd; 0k0, k ) odd; 00l, l ) odd were noted). The
computer programs from the HKL package were used for data reduction.
The preliminary model of the structure was obtained using XS, a direct
methods program. Least-squares refining of the model versus the data
was performed with the XL computer program. Illustrations were made
with the XP program, and tables were made with the XCIF program.
All are in the SHELXTL v6.1 package. Thermal ellipsoids shown in
the illustration are at the 30% level. All non-hydrogen atoms were
refined with anisotropic thermal parameters, and the final refinement
gave R1 ) 0.0400, wR2 ) 0.0736 (refinement on F2). All hydrogen
atoms were included with the riding model using the XL program
default values. A CH3CN molecule of solvation was included in the
crystal structure. It was refined as a rigid group (C-N ) 1.1 Å, C-C
) 1.5 Å, C-H ) 0.99 Å, N-C-C ) linear, C-C-H ) tetrahedral)
with an isotropic thermal parameter and occupancy ) 0.415(7). No
further restraints or constraints were imposed on the refinement model.
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(8) Pulici, M.; Sugawara, F.; Koshino, H.; Uzawa, J.; Yoshida, S.;
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(12) Anderson, J. R.; Briant, C. E.; Edwards, R. L.; Mabelis, R. P.; Poyser,
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Walker, N. P.; Sheldrick, G. M.; Whalley, A. J. J. Antibiot. 1986,
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(19) Crystallographic data for compound 4 have been deposited with the
Cambridge Crystallographic Data Centre (deposition number CCDC
284689). Copies of the data can be obtained, free of charge, on
application to the Director, CCDC, 12 Union Road, Cambridge
CB2 1EZ, UK (fax: +44-(0)1223-336033; or e-mail: deposit@
ccdc.cam.ac.uk).
Acknowledgment. The authors thank the staff of the University of
Iowa High Field NMR and Mass Spectrometry facilities for their
assistance. We are also grateful to Dr. B. W. Horn (USDA National
Peanut Research Lab) for collecting the pyrenomycete stromata from
which the culture of P. disseminata was obtained. Financial assistance
for this research was provided by the National Science Foundation
(#CHE-0315591) and a University of Iowa Presidential Fellowship (to
S.T.D.).
NP050460B