Kipukasins, nucleoside derivatives from Aspergillus versicolor

Article abstract of DOI:10.1021/np070241l

Seven new aroyl uridine derivatives (kipukasins A-G; 1-7) were isolated from solid-substrate fermentation cultures of two different Hawaiian isolates of Aspergillus versicolor. The structures of compounds 1-7 were determined by analysis of NMR and MS data. The nucleoside portion of lead compound 1 was assigned as uracil-1-β-D-ribofuranoside by spectral comparison with an authentic standard. The bioactivity of the original A. versicolor extracts was accounted for mainly by the presence of the known metabolite sterigmatocystin, but kipukasins A and B showed modest activity in assays against Gram-positive bacteria.

Full text of DOI:10.1021/np070241l

1
308
J. Nat. Prod. 2007, 70, 1308-1311
Kipukasins, Nucleoside Derivatives from Aspergillus Wersicolor
†
†
,†
‡
Ping Jiao, Sanjay V. Mudur, 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 May 23, 2007
Seven new aroyl uridine derivatives (kipukasins A-G; 1-7) were isolated from solid-substrate fermentation cultures
of two different Hawaiian isolates of Aspergillus Versicolor. The structures of compounds 1-7 were determined by
analysis of NMR and MS data. The nucleoside portion of lead compound 1 was assigned as uracil-1-â-D-ribofuranoside
by spectral comparison with an authentic standard. The bioactivity of the original A. Versicolor extracts was accounted
for mainly by the presence of the known metabolite sterigmatocystin, but kipukasins A and B showed modest activity
in assays against Gram-positive bacteria.
Our continuing interest in mycoparasitic and fungicolous fungi
as sources of new bioactive secondary metabolites1 prompted us
to investigate the chemistry of an isolate of Aspergillus Versicolor
-3
(
Vuill.) Tiraboschi (MYC-2236 ) NRRL 35600). Although A.
Versicolor is known as a producer of mycotoxins and other
compounds,4 isolation of A. Versicolor as a colonist of other fungi
has not been previously reported to our knowledge. This isolate
was obtained from a basidioma of Gandoderma australe found
growing on a tree in a montane mesic forest in Hawaii and was
cultured by solid-substrate fermentation on rice. The crude extract
of the resulting cultures showed significant antiinsectan activity.
,5
5
Sterigmatocystin was encountered as a major component and was
responsible for the antiinsectan activity of the original crude extract.
However, initial analyses indicated the presence of a set of major
constituents unrelated to sterigmatocystin. Further investigation
afforded five new nucleoside derivatives, which we named kipuka-
sins A-E (1-5). At the same time, analysis of extracts from
cultures of a different fungicolous isolate of A. Versicolor (also
from Hawaii, but from a different location) led to recognition of
the presence of a similar set of compounds. Studies of this second
isolate yielded two additional related compounds (kipukasins F and
G; 6 and 7). Details of the isolation, structure elucidation, and
stereochemical assignment of these metabolites are described here.
Results and Discussion
The crude EtOAc extract from cultures of A. Versicolor NRRL
5600 was subjected to solvent partitioning, chromatography on
3
Sephadex LH-20, and reversed-phase HPLC to afford samples of
sterigmatocystin and kipukasins A-E (1-5). Sterigmatocystin was
identified by comparison of NMR and MS data to those of a
previously isolated sample. The molecular formula of kipukasin A
(
24 2
1) was determined as C21H N O10 (11 unsaturations) on the basis
1
furanose moiety. 13C NMR data were consistent with this conclu-
sion. The base was identified as uracil by comparison of the relevant
NMR signals to literature data. Observation of HMBC correlations
of H-6 with C-2, C-4, and C-5 and of H-5 with C-4 and C-6
confirmed the presence of the uracil moiety. The δ-values for the
of NMR and MS data. The H NMR spectrum revealed the presence
of a 1,2,3,5-tetrasubstituted benzene ring, a 1,2-disubstituted olefin
6
(
J ) 8.1 Hz), four oxymethine protons, one oxymethylene unit,
two methoxy groups, two aryl or acetyl methyl groups, and one
8.65). 13C NMR data were consistent with
exchangeable proton (δ
H
anomeric proton and carbon signals (δ
H
6.11; d, J ) 6.4 Hz; δ
C
these observations and also indicated the presence of four carboxy
or amide carbons. Aside from the data for the substituted benzene
unit, the H and C NMR data were suggestive of a nucleoside
derivative, more specifically, a uridine analogue. Analysis of H
NMR shifts and J-values confirmed that the four oxygenated
87.9) were consistent with the presence of a C-N glycosidic
1
13
linkage. HMBC correlations of H-1′ to C-2 and C-4 confirmed the
connection of the furanose unit to the uracil moiety at the expected
N-1 position.
1
Analysis of additional HMBC data enabled location of both
methoxy groups and an aryl methyl on the benzene ring. Correla-
tions to the remaining two carbonyl groups enabled extension of
the aromatic ring to a benzoate moiety and also revealed the
presence of an acetate group. Both of these carbonyls were
connected to oxygen atoms of the sugar moiety to form ester
methines and the oxygenated methylene are linked to form a
*
To whom correspondence should be addressed. Tel: 319-335-1361.
Fax: 319-335-1270. E-mail: james-gloer@uiowa.edu.
†
‡
University of Iowa.
National Center for Agricultural Utilization Research.
1
0.1021/np070241l CCC: $37.00
© 2007 American Chemical Society and American Society of Pharmacognosy
Published on Web 07/03/2007

Nucleoside DeriVatiVes from Aspergillus Versicolor
Table 1. 1H NMR Data [δ
Journal of Natural Products, 2007, Vol. 70, No. 8 1309
H
(mult; J
H
3
)] for Kipukasins A-E (1-5; 300 MHz, CDCl )
position
1
2
3
4
5
5
6
5.79 (d, 8.1)
7.76 (d, 8.1)
6.11 (d, 6.4)
5.57 (dd, 6.4, 5.6)
5.67 (dd, 5.6, 2.8)
4.35 (q, 2.8)
5.75 (d, 8.1)
7.73 (d, 8.1)
6.06 (d, 6.7)
5.55 (dd, 6.7, 5.5)
5.63 (dd, 5.5, 2.4)
4.35 (q, 2.3)
5.80 (d, 8.1)
7.71 (d, 8.1)
6.09 (d, 6.8)
5.63 (dd, 6.8, 5.7)
5.75 (dd, 5.7, 2.5)
4.39 (q, 2.5)
5.78 (d, 8.1)
7.67 (d, 8.1)
5.85 (d, 6.4)
4.63 (t, 6.0)
5.62 (dd, 5.5, 2.6)
4.35 (q, 2.5)
a 3.99 (dd, 12, 2.4)
b 3.94 (dd, 12, 2.2)
5.76 (d, 8.1)
7.59 (d, 8.1)
5.95 (d, 5.0)
5.70 (t, 5.3)
1
2
3
4
5
′
′
′
′
′
4.63 (t, 5.2)
4.14 (dt, 4.8, 2.4)
a 4.03 (dd, 12, 2.4)
b 3.88 (dd, 12, 2.5)
3.99 (br m)
3.99 (br m)
4.01 (br d, 2.5)
7
3
5
8
2
2
4
′
′′
′′
′′
2.06 (s)
2.05 (s)
2.07 (s)
6.34 (br s)
6.34 (br s)
2.32 (s)
6.26 (d, 2.1)
6.24 (d, 2.1)
2.28 (s)
6.35 (d, 2.3)
6.34 (d, 2.3)
2.58 (s)
6.37 (d, 2.2)
6.40 (d, 1.5)
2.38 (s)
6.34 (d, 2.2)
6.38 (d, 2.2)
2.34 (s)
′′-OH
′′-OMe
′′-OMe
11.35 (s)
a
3.84 (s)b
3.87 (s)b
8.22 (br s)
3.84 (s)c
3.82 (s)c
8.00 (br s)
3.82 (s)
3.81 (s)
8.65 (br s)
3.79 (s)
a
3.82 (s)
7.98 (br s)
NH
7.89 (br s)
a-c
Assignments with identical superscripts are interchangeable. The appearance and/or presence of signals for most of the exchangeable protons
was variable and sample-dependent.
linkages. The acetate CH singlet at δ
correlations to C-2′ (δ 73.4) and C-6′ (δ
of the acetate group at C-2′. A correlation between H-3′ (δ
and the second ester carbonyl at δ 167.5 established the attachment
of the modified benzoate unit to C-3′. The locations of the
substituents on the aromatic ring (already noted above to be 1,2,3,5-
tetrasubstituted) were assigned by analysis of chemical shifts and
3
H
2.06 showed HMBC
170.2), enabling location
5.67)
Table 2. 13C NMR Data (δ
C
) for Kipukasins A-E (1-5; 100
C
C
MHz, CDCl3)
H
2a
3a
position
1
4
5
C
2
4
5
6
1′
2′
150.8
163.1
103.6
141.1
87.9
150.6
162.9
103.3
140.7
87.0
150.7
162.6
104.0
141.2
88.6
150.8
162.9
103.4
141.4
91.1
150.0
162.5
103.1
141.5
89.9
HMBC correlations. The chemical shifts of H-3′′ (δ
C-3′′ (δ 96.7) were suggestive of oxygen substitution at both ortho
positions. H-3′′ showed HMBC correlations to C-2′′, C-4′′, and
C-5′′. Correlations from aryl CH -8′′ to C-5′′, C-6′′, and C-7′′
H
6.34) and
C
73.4
73.4
73.1
73.8
75.5
3
4
5
6
7
′
′
′
′
′
72.2
84.2
62.5
170.2
20.9
72.3
84.3
62.2
169.8
20.7
72.9
73.8
70.1
84.2
83.5
85.3
3
62.3
62.7
62.2
located the methyl group at C-6′′. A long-range HMBC correlation
between H-5′′ and C-7′′ established the location of the ester carbonyl
at C-1′′. The two methoxy singlets showed correlations to C-2′′
and C-4′′, thereby completing the assignment of structure 1 as
shown.
170.3
21.0
1′′
2′′
114.8
162.4
96.7
159.3
107.3
139.4
167.5
113.9
159.4
97.2
159.6
109.6
139.2
167.3
20.1
104.7
166.7
99.5
114.7
162.7
96.9
114.0
168.8
96.7
3
4
5
6
7
′′
′′
′′
′′
′′
165.4
112.3
143.5
159.2
108.1
141.0
166.7
162.5
107.7
140.9
166.4
20.6
Structure elucidation of kipukasins B-E (2-5) was straightfor-
ward due to their close relationships with compound 1. Compound
2
22 2
was assigned the molecular formula C20H N O10, having one
methylene unit less than 1, on the basis of NMR and MS data. The
8′′
2′′-OMe
20.6
25.4
55.7
20.9
1
13
55.8b
55.8c
d
d
H and C NMR spectra were consistent with the absence of one
55.9
56.4
55.7
4′′-OMe
56.3b
56.5c
of the methoxy groups in 1. The structure was independently
assigned as shown by analysis of HMQC and HMBC data. Due to
a
These shifts were assigned on the basis of HMBC and HMQC
b-d
the proximity of the two key carbon signals for C-2′′ and C-4′′ (δ
59.6 and 159.4), some uncertainty remained regarding the position
C
correlations.
changeable.
Assignments with identical superscripts are inter-
1
assignment for the methoxy group. Therefore, a NOESY experiment
was carried out for compound 2. A correlation between the methoxy
signal and the aromatic H-3′′ resonance but not the H-5′′ signal
suggested that the methoxy group was located at C-2′′, rather than
C-4′′. This assignment was verified by isolation of the regioisomer
Kipukasin E (5) possessed the same molecular formula as that
1
13
of 4, as deduced from its NMR and MS data. The H and C NMR
spectra were very similar to those of 4, with only a subtle difference
in the 1H NMR spectrum, consisting of a somewhat different
splitting pattern for some of the ribose oxymethine proton signals.
Homonuclear decoupling experiments were employed to establish
proton signal assignments and revealed that the signal for H-3′ was
shifted upfield, while the H-2′ resonance was shifted downfield,
thereby leading to the location of the acyl group at C-2′ in 5 as
shown. This conclusion was confirmed by observation of an HMBC
correlation from H-2′ to C-7′′.
A second isolate of the same species (MYC-1793 ) NRRL
35641) obtained from a different location showed similar biological
activity and was also investigated. The known A. Versicolor
metabolite sterigmatocystin was again recognized after solvent
partitioning, again accounting for the antiinsectan activity. Most
of the sterigmatocystin was removed by trituration with MeOH,
and the soluble portion was subjected to chromatography on silica
gel, followed by HPLC to afford two additional new benzoylated
nucleosides (kipukasins F and G; 6 and 7).
3
.
1
13
The H and C NMR and MS data for compound 3 revealed
1
that 3 is an isomer of 2. The only significant difference in the H
NMR spectrum was the presence of an exchangeable proton
resonance at δ 11.35, which was identified as a hydrogen-bonded
H
phenolic OH group signal. The presence of this signal in the
spectrum of 3, but not in that of 2, is consistent with location of
the OH group ortho to the ester carbonyl in 3. Moreover, the
methoxy proton signal at δ
H-3′′ and H-5′′, unambiguously locating the methoxy group at C-4′′.
22 2 9
Kipukasin D (4) was assigned the molecular formula C19H N O
H
3.82 showed NOE correlations to both
1
13
on the basis of NMR and MS data. The H and C NMR chemical
shift assignments (Tables 1 and 2) matched well with those of the
corresponding signals for 1 and revealed the same structural features
present in 1 except for the absence of the acetate, which was
consistent with the difference in formula. Correspondingly, the H-2′
resonance was shifted significantly upfield (by 0.94 ppm) relative
to that of 1, leading to assignment of the structure of kipukasin D
The elemental composition of kipukasin F (6) was determined
to be C21H
24
2
N O10 on the basis of NMR and HRESIMS data. The
1
(4) as shown.
H NMR spectrum displayed resonances that were nearly identical

1
310 Journal of Natural Products, 2007, Vol. 70, No. 8
Jiao et al.
to those observed in the spectrum of kipukasin B (2). The presence
of a broad phenolic OH singlet at δ 6.50, rather than an
intramolecularly hydrogen bonded OH signal, supported an analo-
gous regiochemical assignment for the substituted benzene ring.
However, the NMR data for 6 also contained signals characteristic
(ATCC 6051) at the same level, affording a 12-mm zone of
inhibition. Kipukasins C-G were inactive in these assays when
tested at the same level.
Experimental Section
of an N-CH
3
group (δ
H
3.31; δ
C
27.8) that were absent in the
General Experimental Procedures. Optical rotations were deter-
mined with a Rudolph automatic polarimeter, model AP III 589, and
UV data were recorded with a Varian Cary III UV-visible spectro-
photometer. CD data were collected using an Olis Cary-17 spectrometer
spectrum of 2 and lacked the exchangeable NH singlet. These data
suggested that 6 differs from 2 only in the presence of an N-3
methyl group, and this conclusion was independently confirmed
by analysis of HMBC, HMQC, and NOE difference data. The
1
13
(
1-cm cell). H and C NMR spectra were acquired using Bruker DPX-
300 and DRX-400 spectrometers, respectively. HMQC and HMBC data
3
location of the N-CH group was confirmed by HMBC correlations
were obtained using a Bruker Avance-600 instrument. HPLC was
carried out using a Beckman System Gold instrument with a model
of the corresponding proton signal to C-2 and C-4. The location of
the methoxy group on the benzene ring was confirmed by analysis
of NOE difference data. Irradiation of the methoxy proton signal
1
66 UV detector with UV detection at 215 nm. Other general procedures
16
and instrumentation have been described previously.
(δ 3.75) resulted in NOE enhancement of the signal corresponding
Fungal Material. One of the cultures of A. Versicolor (MYC-2236)
was isolated from the surface of a basidioma of Gandoderma australe
found growing on a living tree in a montane mesic forest in Kipuka
Pauula (Bird Park), Volcanoes National Park, Hawaii. A subculture
has been deposited in the Agricultural Research Service (ARS)
collection at the USDA NCAUR with the accession number NRRL
35600. The second A. Versicolor isolate (MYC-1793) was obtained
from a white mycelial growth found on the undersurface of a dead
hardwood branch in a subalpine dry forest, Puulaau near Highway 200
to H-3′′ (δ 6.28), while no enhancement of the signal corresponding
to H-5′′ was observed.
The mass of kipukasin G (7) was found to be 42 Da lower than
that of 6, and the molecular formula was determined to be
1
C
19
H N O
22 2 9
by analysis of NMR and HRESIMS data. The H and
1
3
C NMR spectra of 7 were very similar to those of 6, lacking
only the signals corresponding to the acetyl group (δ 2.04, δ
69.8), thereby suggesting that kipukasin G (7) is the desacetyl
analogue of 6. The upfield chemical shift of the H-2′ signal (δ
.56) in the spectrum of 7 relative to its position in the spectrum
H
C
1
(milepost 43), Hawaii County, Hawaii. A subculture of this isolate has
been deposited with the ARS Culture Collection at the NCAUR with
the accession number NRRL 35641.
4
of 6 (δ 5.59) supported this conclusion. Proton assignments for
the furanose ring were confirmed by decoupling experiments, and
independent analysis of HMBC data verified the structure of
kipukasin G (7).
General fermentation procedures used have been published else-
where.2 Each culture was incubated on rice (2 × 50 g for NRRL
35600 and 3 × 50 g for NRRL 35641) at 25 °C for 30 days, and the
combined fermentation mixtures were extracted with EtOAc (3 × 500
mL in each case). The combined, filtered EtOAc solutions were
evaporated to dryness, yielding 303 mg of crude extract for NRRL
,15
The sugar moiety in 1 was determined to be a â-ribose unit by
1
analysis of coupling constants and comparison with H NMR data
3
5600 and 445 mg for NRRL 35641.
reported for modified ribose units (although ribose unit proton
signals are often reported as broad).6 In order to establish the
absolute configuration of the ribose moiety, a sample of 1 was
subjected to hydrolysis with 0.1 N NaOH, and the resulting free
nucleoside was purified by RP-HPLC. Comparison of the CD curve
with that of uridine confirmed the presence of uracil-1-â-D-
Extraction and Isolation. The crude extract from NRRL 35600 was
partitioned between MeCN and hexanes (1:1). The MeCN fraction (133
mg) was chromatographed on a Sephadex LH-20 column eluting
-8
successfully with 4:1 CH
CH Cl -acetone to give 10 fractions. Fraction 1 (47 mg), eluted with
:1 CH Cl -hexanes, consisted of the known compound sterigmato-
2
Cl
2
-hexanes, 3:2 CH
2 2
Cl -acetone, and 1:4
2
2
4
2
2
9
cystin, which was identified by spectroscopic comparison with an
authentic standard. Fraction 4 (22 mg) was further separated by
reversed-phase HPLC (Alltech Apollo 5-µm C18 column; 10 × 250
ribofuranoside in 1. The configurations of the ribose moieties
contained in 2-7 are presumed to be the same as in 1.
Novel nucleoside derivatives have been reported from a variety
mm; MeCN-H O, 40-60% over 20 min, 60-100% over 5 min at a
2
10-14
of sources such as sponges, red algae, mushrooms, and bacteria,
flow rate of 2 mL/min) to give compounds 1 (7.7 mg) and 3 (0.3 mg).
Fraction 5 (13 mg) was processed by HPLC under the same conditions
to afford compounds 1 (1.1 mg), 3 (0.5 mg), 4 (1.8 mg), and 5 (0.7
and representatives of this class have been reported to show
antitumor, antiviral, antifungal, and antibiotic activity. However,
such reports are relatively uncommon, despite the ubiquitous nature
of nucleosides. Unlike the kipukasins, none of these previously
known metabolites display acylation at positions on the sugar
residue, and to our knowledge, kipukasins A-G (1-7) are the first
naturally occurring aroyl nucleosides to be reported.
mg). Fraction 6 (17 mg), eluted with 3:2 CH
Sephadex column, was separated using the same HPLC column
MeCN-H O, 30-50% over 20 min, 50-100% over 5 min) to give
compounds 2 (0.9 mg), 4 (1.5 mg), and 5 (0.9 mg). Compound 2 (1.8
mg) was also obtained from fraction 7 (15 mg), eluted with 3:2 CH
Cl -acetone from the Sephadex column, using the same HPLC
conditions as above for fraction 6.
The crude NRRL 35641 extract was similarly partitioned between
MeCN and hexanes (1:1). In this instance, the CH CN-soluble portion
365 mg) was evaporated to dryness and then CH OH (5 mL) was
OH-insoluble solid was collected by filtration using
2 2
Cl -acetone from the
(
2
2
-
2
2
′-3′-Transesterification is known to occur in some ribose
15
derivatives, and such a process could be involved in the formation
of some of these compounds (e.g., via interconversion of species
such as 4 and 5); however, warming samples of 4 and 5 in solution
3
(
3
added. The CH
3
(acetone) did not result in observable interconversion. On a related
Whatman #2 filter paper. This process was repeated two more times
with decreasing volumes of CH OH to obtain a sample of the precipitate
47 mg), which was identified as sterigmatocystin. The combined filtrate
was evaporated to dryness (301 mg) and then subjected to silica gel
column chromatography, eluting successively with CH Cl -CH OH
49:1, 200 mL; 97:3, 75 mL; 19:1, 150 mL; 9:1, 200 mL; 87:13, 100
mL; 21:4, 100 mL; 7:3, 75 mL) and CH Cl -CH OH-acetone (6:3:1,
point, compounds 1-3 and 6 each contain acetate groups, but these
units seem unlikely to arise from the ethyl acetate used routinely
as an extraction solvent, since the compounds were not exposed to
this solvent during any of the purification steps, and none of the
presumably more accessible primary alcohol positions were acetyl-
ated. However, re-fermentation and analysis of extracts made with
a different solvent was not carried out.
3
(
2
2
3
(
2
2
3
100 mL; 2:2:1, 100 mL) to afford several fractions. HPLC separation
of the seventh fraction (22 mg; eluted with 19:1 CH Cl -CH OH)
eluting with CH CN and H O (same column as above; 30% CH CN in
O over 15 min, 30% to 86% CH CN in H O over 20 min, and 86%
to 100% CH CN in H O over 1 min) afforded kipukasin G (7; 3 mg).
The sixth fraction (32 mg; eluted with 19:1 CH Cl OH) was
2
2
3
Although the original extract showed modest antifungal activity,
kipukasins A-G showed no activity in disk assays against
Aspergillus flaVus (NRRL 6541), Fusarium Verticillioides (NRRL
3
2
3
H
2
3
2
3
2
2
5457), or Candida albicans (ATCC 14053) at 200 µg/disk. In
2
2
-CH
3
antibacterial assays, compound 1 was active against Staphylococcus
aureus (ATCC 29213), causing an 18-mm zone of inhibition at
subjected to HPLC (Rainin Dynamax C-18 column, 8-µm particles;
40% CH CN in H O over 10 min and 40% to 100% CH CN in H O
3
2
3
2
100 µg/disk. Compound 2 showed activity against Bacillus subtilis
over 5 min at 10.0 mL/min) to obtain kipukasin F (6; 12 mg).

Nucleoside DeriVatiVes from Aspergillus Versicolor
Kipukasin A (1): colorless glass; [R]25
Journal of Natural Products, 2007, Vol. 70, No. 8 1311
D
-26 (c 0.12, MeOH); UV
5.87 (d, J ) 6.3 Hz, H-1′), 5.80 (d, J ) 8.1 Hz, H-5), 5.53 (dd, J )
5.4, 2.9 Hz, H-3′), 4.56 (dd, J ) 6.3, 5.4 Hz, H-2′), 4.26 (m, H-4′),
3.91 (dd, J ) 12, 2.2 Hz, H-5′a), 3.87 (dd, J ) 12, 2.2 Hz, H-5′b),
1
13
(
MeOH) λmax (log ꢀ) 213 (4.5), 258 (4.1) nm; H and C NMR data,
see Tables 1 and 2; HMBC data H-5 f C-4, 6; H-6 f C-2, 4, 5, 1′;
H-1′ f C-2, 6, 2′, 4′; H-2′ f C-1′, 4′, 6′; H-3′ f C-1′, 5′, 7′′; H -7′
f C-2′, 6′; H-3′′ f C-2′′, 4′′, 5′′; H-5′′ f C-1′′, 3′′, 7′′, 8′′; H -8′′ f
C-1′′, 5′′, 6′′; 2′′-OMe f C-2′′, 3′′; 4′′-OMe f C-4′′, 5′′; EIMS m/z
3
3.81 (s, 2′′-OCH
+ one drop CD OD, 100 MHz) δ 166.7 (s, C-7′′), 162.9 (s, C-4), 159.6
3 3 3
), 3.30 (s, 3-CH
), 2.29 (s, H-8′′); 13C NMR (CDCl
3
3
(s, C-4′′), 159.1 (s, C-2′′), 151.5 (s, C-2), 140.6 (s, C-6′′), 138.9 (d,
C-6), 112.7 (s, C-1′′), 110.1 (d, C-5′′), 102.2 (d, C-5), 97.0 (d, C-3′′),
91.1 (d, C-1′), 83.2 (d, C-4′), 73.5 (d, C-2′), 73.3 (d, C-3′), 61.9 (t,
+
+
4
[
4
64 (M ; 34), 353 (44), 179 (100); ESIMS m/z 487 [M + Na] , 951
+
24 2
2M + Na] ; HRESIMS m/z 487.1320 (calcd for C21H N O10Na,
87.1329).
C-5′), 56.0 (q, 2′′-OCH
(CDCl + one drop CD
C-4, C-6; H-6 f C-2, C-4, C-5, C-1′; 3-CH
C-6, C-4′; H-2′ f C-1′, C-3′, C-4′; H-3′ f C-1′, C-4′, C-5′, C-7′′;
H-4′ f C-5′; H -5′ f C-3′, C-4′; H-3′′ f C-1′′, C-5′′, C-7′′; H-5′′ f
C-3′′, C-7′′, C-8′′; H -8′′ f C-1′′, C-5′′, C-6′′; 2′′-OCH f C-2′′;
3
), 27.7 (q, 3-CH
OD, 600 MHz) 3-CH
f C-2, C-4; H-1′ f C-2,
3
), 20.2 (q, C-8′′); HMBC data
Kipukasin B (2): colorless glass; [R]25
-14 (c 0.12, MeOH); UV
f C-2, C-4; H-5 f
D
3
3
3
1
13
(
MeOH) λmax (log ꢀ) 215 (4.4), 256 (4.0) nm; H and C NMR data,
3
see Tables 1 and 2; HMBC data H-5 f C-6; H-6 f C-2, 4, 5, 1′; H-1′
f C-2, 6, 2′; H-2′ f C-1′, 4′, 6′; H-3′ f C-1′, 7′′; H-4′ f C-3′; H-5′
2
f C-3′; H
3
-7′ f C-6′; H-3′′ f C-1′′, 2′′, 5′′; H-5′′ f C-1′′, 3′′, 8′′;
3
3
+
+
H
3
-8′′ f C-1′′, 5′′, 6′′; 2′′-OMe f C-2′′; ESIMS m/z 473 [M + Na] ,
23 2 9
HRESIMS obsd m/z 423.1399 (M + H) , calcd for C19H N O ,
+
9
23 [2M + Na] ; HREIMS m/z 450.1271 (calcd for C20
H
22
N O
2 10
,
423.1404.
4
50.1274).
Alkaline Hydrolysis of Kipukasin A (1). Compound 1 (2.4 mg)
was treated with 0.1 N NaOH (0.5 mL) at room temperature for 48 h.
The solution was evaporated to dryness and purified by RP-HPLC using
Kipukasin C (3): colorless glass; UV (MeOH) λmax (log ꢀ) 214 (4.3),
1
13
2
65 (4.0) nm; H and C NMR data, see Tables 1 and 2; HMBC data
H-5 f C-6; H-6 f H-2, 4, 5, 1′; H-1′ f C-2, 6, 2′; H-2′ f C-1′, 4′,
′; H-3′ f C-1′; H-4′ f C-3′; H -7′ f C-6′; H-3′′ f C-1′′, 4′′, 5′′;
H-5′′ f C-1′′, 3′′, 8′′; H -8′′ f C-1′′, 5′′, 6′′; OH-2′′ f C-1′′, 2′′, 3′′,
′′-OMe f C-4′′; EIMS m/z 450 (M ; 14), 338 (15), 175 (30), 165
0.1% formic acid in H O-MeCN (20/80) with UV detection at 250
2
6
3
nm to afford uridine (1.2 mg); CD (H O) ∆ꢀ 267 (9.2), 240 (-4.0),
2
1
3
215 (-5.0) nm; H NMR (300 MHz, CD OD) δ 8.44 (1H, s), 8.01
3
+
4
(1H, d, J ) 8.1 Hz), 5.90 (1H, d, J ) 4.4 Hz), 5.70 (1H, d, J ) 8.1
Hz), 4.18 (1H, t, J ) 5.3 Hz), 4.15 (1H, t, J ) 5.3 Hz), 4.01 (1H, m),
3.84 (1H, dd, J ) 12, 2.7 Hz), 3.73 (1H, dd, J ) 12, 3.2 Hz).
+
(
100); ESIMS m/z 473 [M + Na] . Optical rotation and HRMS data
were not recorded for 3 due to sample limitations.
Kipukasin D (4): colorless glass; [R]2
5
-27 (c 0.19, MeOH); UV
1 13
D
(
MeOH) λmax (log ꢀ) 210 (4.4), 257 (3.8) nm; H and C NMR data,
Acknowledgment. Financial support from the National Science
Foundation (CHE-0315591) and the National Institutes of Health (GM
60600) is gratefully acknowledged.
see Tables 1 and 2; HMBC data H-5 f C-4, 6; H-6 f C-2, 4, 5, 1′;
H-1′ f C-2, 6, 2′; H-2′ f C-4′; H-3′ f C-1′, 5′, 7′′; H-4′ f C-3′;
H-5′ f C-3′, 4′; H-3′′ f C-1′′, 2′′, 4′′, 5′′, 7′′; H-5′′ f C-1′′, 3′′, 8′′;
Supporting Information Available: 1H NMR spectra for 1-7 and
3
H -8′′ f C-1′′, 5′′, 6′′; 2′′-OMe f C-2′′; 4′′-OMe f C-4′′; EIMS m/z
+
4
22 (M ; 27), 310 (49), 195 (42), 148 (100); HREIMS m/z 422.1321
calcd for C19 , 422.1325).
Kipukasin E (5): colorless glass; UV (MeOH) λmax (log ꢀ) 216 (4.4),
13
C NMR spectra for compounds 1 and 4-7. This material is available
(
22 2 9
H N O
free of charge on the Internet at http://pubs.acs.org.
1
13
2
58 (3.9) nm; H and C NMR data, see Tables 1 and 2; HMBC data
References and Notes
H-5 f H-6; H-6 f C-2, 4, 5, 1′; H-1′ f C-2, 6, 2′; H-2′ f C-1′, 4′,
(
(
(
1) Deyrup, S. T.; Gloer, J. B.; O’Donnell, K.; Wicklow, D. T. J. Nat.
Prod. 2007, 70, 378-382.
2) Mudur, S. V.; Gloer, J. B.; Wicklow, D. T. J. Antibiot. 2006, 59,
7
4
′′; H-3′ f C-1′, 5′; H-4′ f C-3′; H-5′ f C-3′, 4′; H-3′′ f C-1′′, 2′′,
′′, 5′′, 7′′; H-5′′ f C-1′′, 3′′, 4′′, 8′′; H -8′′ f C-1′′, 5′′, 6′′; 2′′-OMe
3
+
f C-2′′; 4′′-OMe f H-4′′; EIMS m/z 422 (M ; 16), 311 (49), 179
198), 68 (100). Optical rotation and HRMS data were not recorded
500-506.
(
3) Jiao, P.; Swenson, D. C.; Gloer, J. B.; Wicklow, D. T. J. Nat. Prod.
for 5 due to sample limitations.
Kipukasin F (6): off-white semisolid; [R]
2006, 69, 636-639.
2
5
D
- 32 (c 0.6, acetone);
(4) Dictionary of Natural Products, Web Version 15.2; Chapman and
Hall/CRC Press, 2007.
UV (CH
3
CN) λmax (log ꢀ) 254 (4.0) nm; IR (film on NaCl plate) νmax
-
1
1
(5) Cole, R. J.; Schweikert, M. A. Handbook of Secondary Fungal
Metabolites; Academic Press: New York, Vol. 1, 2003; pp 597-
3
369, 3018, 2933, 1710, 1606 cm ; H NMR (CDCl
3
, 400 MHz) δ
7
.71 (d, J ) 8.1 Hz, H-6), 6.50 (br s, 4′′-OH), 6.28 (br d, J ) 2 Hz,
598.
H-3′′), 6.26 (br d, J ) 2 Hz, H-5′′), 6.05 (d, J ) 5.8 Hz, H-1′), 5.82
d, J ) 8.1 Hz, H-5), 5.65 (dd, J ) 5.8, 3.4 Hz, H-3′), 5.59 (t, J ) 5.8
Hz, H-2′), 4.32 (m, H-4′), 3.98 (br d, J ) 12 Hz, H-5′a), 3.94 (br d, J
12 Hz, H-5′b), 3.75 (s, 2′′-OCH ), 3.31 (s, 3-CH ), 2.24 (s, H -8′′);
, 100 MHz) δ 169.8 (s, C-6′), 167.3
s, C-7′′), 162.9 (s, C-4), 158.7 (s, C-4′′), 159.2 (s, C-2′′), 151.3 (s,
C-2), 139.2 (s, C-6′′), 138.7 (d, C-6), 113.9 (s, C-1′′), 109.4 (d, C-5′′),
02.5 (d, C-5), 96.9 (d, C-3′′), 89.0 (d, C-1′), 83.6 (d, C-4′), 73.1 (d,
C-2′), 71.5 (d, C-3′), 61.9 (t, C-5′), 55.8 (q, 2′′-OCH ), 27.8 (q, 3-CH ),
0.6 (q, C-7′), 20.0 (q, C-8′′); HMBC data (CDCl , 600 MHz) 3-CH
f C-2, C-4; H-5 f C-4, C-6; H-6 f C-2, C-4, C-5, C-1′; 3-CH
(
6) Pretsch, E.; Buhlmann, P.; Affolter, C. Structure Determination of
Organic Compounds: Tables of Spectral Data; Springer-Verlag:
Berlin, 2000; 421 pp.
(
)
3
3
3
(
7) Ostroswki, T.; Maurizot, J.; Adeline, M.; Fourrey, J.; Clivio, P. J.
Org. Chem. 2003, 6502-6510.
1
3
2
3 3
.04 (s, H -7′); C NMR (CDCl
(
(8) Ohigashi, H.; Kaji, M.; Sakaki, M.; Koshimizu, K. Phytochemistry
1989, 28, 1365-1368.
(9) Miles, D. W.; Inskeep, W. H.; Robins, M. J.; Winkley, M. W.; Robins,
1
R. K.; Eyring, H. J. Am. Chem. Soc. 1970, 92, 3872-3881.
10) Zhao, J.; Ma, M.; Wang, S.; Li, S.; Cao, P.; Yang, Y.; Lu, Y.; Shi,
3
3
(
2
3
3
J.; Xu, N.; Fan, X.; He, L. J. Nat. Prod. 2005, 68, 691-694.
11) Capon, R. J.; Trotter, N. S. J. Nat. Prod. 2005, 68, 1689-1691.
12) Kubo, I.; Kim, M.; Wood, W. F.; Naoki, H. Tetrahedron Lett. 1986,
3
f
(
(
C-2, C-4; H-1′ f C-2, C-6, C-2′, C-3′, C-4′; H-2′ f C-1′, C-4′, C-6′;
H-3′ f C-1′, C-2′, C-4′, C-5′, C-7′′; H-4′ f C-2′, C-3′, C-5′; H -5′ f
C-3′, C-4′; H -7′ f C-6′; H-3′′ f C-1′′, C-4′′, C-5′′, C-7′′; H-5′′ f
C-3′′, C-8′′; H -8′′ f C-1′′, C-5′′, C-6′′; 2′′-OCH f C-2′′; NOE
difference data 2-OCH f H-3′′; H -8′′ f H-5′′; HRESIMS obsd m/z
65.1500 (M + H) , calcd for C21 10, 465.1509.
Kipukasin G (7): colorless glass; [R]
CH CN) λmax (log ꢀ) 256 (4.1) nm; IR (film on NaCl plate) νmax 3384,
2
27, 4277-4280.
3
(13) Sakata, K.; Sakurai, A.; Tamura, S. Tetrahedron Lett. 1974, 15,
4327-4330.
3
3
(14) Sakata, K.; Sakurai, A.; Tamura, S. Tetrahedron Lett. 1975, 16,
3191-3194.
3
3
+
4
25 2
H N O
2
5
(15) Jackson, M. D.; Denu, J. M. J. Biol. Chem. 2002, 277, 18535-18544.
D
-24 (c 0.15, acetone); UV
(
16) H o¨ ller, U.; Gloer, J. B.; Wicklow, D. T. J. Nat. Prod. 2002, 65,
(
1
3
876-882.
-
1
1
710, 1659, 1603 cm ; H NMR (CDCl
3
3
+ one drop CD OD, 400
MHz) δ 7.71 (d, J ) 8.1 Hz, H-6), 6.29 (br s, H-3′′), 6.28 (m, H-5′′),
NP070241L