Salvadenosine, a 5β-deoxy-5β-(methylthio) nucleoside from the Bahamian tunicate Didemnum sp.

Article abstract of DOI:10.1021/jo501486p

Salvadenosine, (1) a rare 5β-deoxy-5β-(methylthio) nucleoside, was isolated from the deep-water Bahaman tunicate Didemnum sp. The structure was solved by integrated analysis of MS and 1D and 2D NMR data. We revise the structure of the known natural product, hamiguanosinol, which is a constitutional isomer of 1, to 5 by interpretation of the spectroscopic data and comparison with synthesized nucleosides.

Full text of DOI:10.1021/jo501486p

Article
pubs.acs.org/joc
Salvadenosine, a 5′-Deoxy-5′-(methylthio) Nucleoside from the
Bahamian Tunicate Didemnum sp.
Matthew T. Jamison,† Christopher N. Boddy,‡ and Tadeusz F. Molinski*,†,§
§
†Department of Chemistry and Biochemistry and Skaggs School of Pharmacy and Department of Pharmaceutical Sciences,
University of California, San Diego, La Jolla, California 92093-0358, United States
‡Departments of Chemistry and Biology, University of Ottawa, Ottawa, Ontario, Canada K1N 6N5
S
* Supporting Information
ABSTRACT: Salvadenosine, (1) a rare 5′-deoxy-5′-(methylthio)
nucleoside, was isolated from the deep-water Bahaman tunicate
Didemnum sp. The structure was solved by integrated analysis of MS
and 1D and 2D NMR data. We revise the structure of the known
natural product, hamiguanosinol, which is a constitutional isomer of 1,
to 5 by interpretation of the spectroscopic data and comparison with
synthesized nucleosides.
INTRODUCTION
■
Modified nucleosides are relatively rare from marine organisms,
but they have a long history. Early pioneering work1 by
Bergmann on Caribbean sponges of the genera Tethya and
Cryptotethya resulted in the isolation of spongosine, spongo-
thymidine,2 and spongouridine2b,c (the first arabino-nucleo-
sides) and others. Predating the modern era of marine natural
products chemistry, these seminal discoveries were the
inspiration3 for development of the clinically important
antitumor drugs Ara A4 and Ara C.5 In our investigations of
antifungal and antitumor compounds using “nanomole-scale”
techniques,6 we examined several extracts of rare tunicates that
displayed antifungal activity against a panel of Candida spp. and
Cryptococcus spp. Here, we report salvadenosine (1), an
uncommon 5′-deoxy-5′-(methylthio) nucleoside from an
encrusting deep-water tunicate Didemnum sp. (Figure 1). In
addition, we revise the structure of the known compound,
hamiguanosinol (2), reported by Proksch and co-workers from
the Pacific sponge Hamigera hamigera.7 Salvadenosine (1) joins
the family of rare marine-derived nucleosides that include
Bergmann’s arabino-nucleosides from Cryptotethya sp.2 and the
Figure 1. Structures of marine nucleosides (1−3), the tautomers of
guanosine (4a, 4b), and original and revised structures of
hamiguanisinol (2 and 5).
antiproliferative trachycladine A (3) from the sponge
Trachycladus laevispirulifer.8
RESULTS
■
the 5′-deoxy-5′-(methylthio)ribose moiety. Cross peaks arising
from a modified ribose corresponded to the following
contiguous spin system: anomeric proton H-1′ (δH 5.87, d, J
The n-BuOH soluble partition of the methanol extract of
Didemnum sp. was separated by reversed-phase HPLC to give 1
in addition to tryptamine and the known natural product 6-
bromotryptamine.9 The molecular formula of 1, C11H15N5O4S,
established from HRMS (ESI-TOF m/z 312.0777 [M − H]−),
was isomeric with 2 (Table 1). COSY correlations confirmed
Received: July 5, 2014
Published: October 3, 2014
© 2014 American Chemical Society
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1
Table 1. H and 13C NMR Data for 1 (Formate Salt, CD3OD)
a
b
1
atom
1 δ 13C
1 δ H (mult, J, integ)
gHMBC (1H→13C)
COSY (1H→1H)
1
c
c
2
152.3
8.08 (s, 1H)
4, 5
3
4
148.7
105.2
148.7
5
6
7
8
154.0
87.9
72.0
74.5
85.1
37.4
1′
2′
3′
4′
5′a
5′b
6′
5.87 (d, J = 4.9 Hz, 1H)
5.12 (t, J = 4.9 Hz, 1H)
4.42 (t, J = 4.9 Hz, 1H)
2′, 4, 6, 8
2′
1′, 3′
2′, 4′
3′, 5′a, 5′b
4′
1′, 5′
3′
3′, 4′, 6′
4.07 (ddd, J = 4.9, 5.5, 7.2 Hz, 1H)
2.88 (dd, J = −14.0, 5.5 Hz, 1H)
2.81 (dd, J = −14.0, 7.2 Hz, 1H)
2.10 (s, 3H)
16.1
5′a, 5′b
a
b
c
1
125 MHz. 600 MHz. JCH = 203.4 Hz.
= 4.9 Hz) to H-2′ (δH 5.12, t, J = 4.9 Hz) to H-3′ (δH 4.42, t, J
= 4.9 Hz) to H-4′ (δH 4.07, ddd, J = 4.9, 5.5, 7.2 Hz) and the
diastereotopic protons H-5′a and H-5′b of a methylene
attached to S instead of O (δH 2.88, dd, J = −14.0, 5.5 Hz;
2.81, dd, J = −14.0, 7.2 Hz). An HMBC correlation from the
three-proton singlet (δH 2.10, s, δC 16.1, 1JCH = 138.4 Hz) to C-
5′ established the location of the MeS group.
carbon, C-1′, a correlation common to guanosine nucleosides.10
The HSQC (DMSO-d6) showed the aforementioned downfield
proton was attached to a carbon with a 13C signal (δC 151.7, s)
more compatible with an imidazolone CO group than the
imidazole C-8 chemical shift of 2 (δC 135.0, s). A better match
for the NMR data of 1 was obtained by replacement of guanine
with 8-oxoadenine, a nucleobase isomeric with the former. The
downfield 1H NMR signal (δH 8.08, s, 1H) was assigned to H-2,
and all inconsistencies were resolved. For example, the expected
long-range correlation between C-1′ and H-8 for a guanine ring
systembut missing in 2is replaced by 2,4J correlations of H-
2 to C-4 and C-5 in an 8-oxoadenine, respectively. The 13C
chemical shifts (DMSO-d6, Table 2) of the quaternary ring
junction carbons C-4 and C-5 (δC 146.7, s; 103.6, s) of 1 are
more polarized (Δδ [C-4 − C-5] = 43.1 ppm) than the
corresponding signals of 2 (Δδ = 35 ppm), guanosine, or
adenosine but closer in magnitude than the 8-oxoadenosines,
aplidiamine, erinacean, and caissarone.11 Additionally, we
measured the heteronuclear coupling constants of the down-
field sp2 1H NMR singlets in 1 and several purine nucleosides
(Table 3), revealing a better match between 8-oxoadenosine
1
Distinct differences were apparent between the H and 13C
chemical shifts of 1 and those published for hamiguanosinol (2,
Table 2), particularly the sp2 13C NMR signals. An HSQC
a
Table 2. Comparison of 1H and 13C NMR Data for 1 and 2
(DMSO-d6)
1 δ
1 δ
2 δ
2 δ
a
b
c
c
1
atom
1H
13C
1H
13C
Δδ H (1−2) Δδ 13C (1−2)
1
2
8.00
150.8
153.5
−2.7
3
4
146.7
103.6
147.2
153.0
118.0
157.2
−6.3
−14.4
−10.0
5
6
1
and the natural product (1: H-2, JCH = 203.4 Hz; 8-
7
1
1
oxoadenosine: H-2 JCH = 201.5 Hz; guanosine: H-8 JCH
=
8
151.7
85.8
69.9
72.9
83.0
36.1
7.90
5.60
4.50
3.95
4.05
2.80
2.70
135.0
87.0
74.0
71.5
84.0
35.0
16.7
213.5 Hz).
1′
2′
3′
4′
5′a
5′b
6′
5.65
4.96
4.19
3.88
2.80
2.69
2.03
0.05
0.46
−1.2
−4.1
1.4
The natural products salvadenosine (1) and hamiguansinol
are clearly not identical, but isomeric (Figure 1). Naturally, the
0.24
−0.17
0.0
−1.0
1.1
1
1
1
Table 3. H, 13C, and JCH NMR Data for Downfield H
NMR Singlets of Purine Nucleosides
−0.01
−0.02
a
b
c
15.5
2.05
b
16.0
−0.5
1
compd
atom δ H
δ
13C
1JCH
a
c
d
d
d
d
d
d
d
Formate salt, 600 MHz. 125 MHz. Reference 7.
adenosine (8)
8
2
8
8
8
2
2
8.13
8.35
7.94
7.93
8.01
8.03
152.9
140.4
135.6
135.8
150.7
150.9
199.1
213.4
213.5
213.3
201.5
203.5
d
d
d
d
d
guanosine (4b)
correlation from the sole downfield nonexchangeable aromatic
Hamiguanosinal (2)
8-oxoadenosine (10)
1
signal (δH 8.00, δC 150.8, JCH = 203.4 Hz) revealed a 13C
chemical shift that was deshielded (Δδ 15.8 ppm) compared to
that of the H-8 of 2 (δH 7.90, δC 135.0). Another notable
difference was observed for the most shielded sp2 13C signal of
1 (δC 103.6, s) and 2 (δC 118.0, s, C-8; Δδ 14.4 ppm). HMBC
correlations of 1 were observed from the anomeric proton H-1′
signal, and sp2 13C signals were also inconsistent with guanine;
for example, no correlation was observed between the
5′-chloro-5′-deoxy-8-oxoadenosine
(11)
f
e
e
e
e
salvadenosine (1)
2
2
8.08
8.06
152.3
152.1
203.4
202.4
g
1
a
b
c
500 MHz. 125 MHz. Measured from 13C satellites in the 1H NMR
d
e
f
g
spectrum (500 MHz). DMSO-d6. CD3OD. Formate salt. Free
1
downfield H NMR signal (δH 8.08, s, 1H) and the anomeric
base.
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question of validity of the structure of 2 arises. The nucleobase
in hamiguanosinol (incorrectly named “6-hydroxyguanine”)7
was assigned as the enol tautomer (cf. enol form of guanosine
4a, Figure 1) based on a 13C shift for C-6 of δC 157.2 “instead of
a ca.170 ppm for a keto amide function”;7 however, we find this
less convincing for three reasons. Keto groups in purines
(guanine, inosine, etc.) are not electronically equivalent to
simple amides and often exhibit relatively high field chemical
shifts in the range observed for 3; the C-6 13C chemical shift
reported for hamiguanosinol is not incompatible with the 6-
keto tautomer 4b. Numerous studies have shown the keto form
of guanosine (4b) and deoxyguanosine are the naturally stable
tautomers in protic solvents: in fact, Watson−Crick pairing of
G−C in DNA is crucially dependent upon it. Given that
electronic differences between guanosine and 2 are insignif-
icant, there are no compelling reasons to expect that 2 would be
“locked” in the enol form and unable to spontaneously
tautomerize to the keto form in protic solvent. Lastly,
compound 5 has been synthesized by van Tilburg and co-
workers12 who characterized it as the keto tautomer. We
repeated the synthesis of 5 (Scheme 1) from guanosine (4b)
Scheme 2. Synthesis of 5′-Deoxy-5′-(methylthio)-8-
oxoadenosine (1)
Scheme 1. Synthesis of 5′-Deoxy-5′-(methylthio)guanosine
(5)
Figure S1). Therefore, salvadenosine is assigned the structure 1
with high confidence.
DISCUSSION
■
The nature of the 5′-(methylthio) group in 1 and 5 deserves
some comment. One plausible origin of 1 is from S-adenosyl
methionine (SAM). The two CH2 groups and one CH3 group
bonded to the S in SAM are electrophilic in nature. Common
biological methylation involves SN2 nucleophilic substitution of
the electrophilic Me-S bond through attack by C-, N-, O-, S-
centered nucleophiles, generating S-adenosylhomocysteine: the
latter is cycled back to adenosine and homocysteine. In the
biosynthesis of both the chlorinated antitumor drug salinospor-
amide A19 and rare fluorinated natural products, substantial
biochemical and structural evidence supports participation of
the 5′-CH2−S bond of SAM in SN2 substitutions by halide ions
(Figure 2). For example, Streptomyces cattleya produces 5′-
fluoroadenosine through nucleophilic attack at the 5′-CH2 of
SAM by F− (path a), catalyzed by the enzyme fluorinase;20 the
former in turn is catabolized to fluoroacetate. A homologous
“chlorinase” catalyzes displacement at 5′-CH2 of SAM by Cl−
(path a) in salinosporamide A biosynthesis.19 Phylogenetic and
biochemical studies21 have shown a bifurcation of this
biosynthetic motif. The gene duf62, represented in Nature in
about 100 bacterial and archael genomes, expresses a protein,
DUF62, that has high structural homology to the halogenases.
DUF62, a “protein of unknown function,” lacks halogenase
activity, but carries out hydrolysis of SAM by nucleophilic
attack at the 5′-CH2 group of adenosine group by HO− (path
b) to liberate L-methionine and adenosine. Attack at the 5″-
CH2 of SAM is similarly rare, with the best-characterized
example occurring in the biosynthesis of nocardicin.22 Transfer
of the 3-amino-3-carboxypropyl group from SAM has also been
proposed in the biosynthesis of modified bases for bacterial and
yeast tRNAPhe,23 the natural product discadenine,24 nicotian-
amine (a precursor of plant siderophores),25 and homoserine-
based betaine lipids.26 In addition, transfer of the 3-amino-
propyl group from decarboxylated SAM is involved in
and showed the 13C chemical shifts of the product were
essentially the same as those reported for natural hamiguano-
sinol (Supporting Information, Table S2).13 Therefore, the
structure of hamiguanosinol is the keto tautomer 5,14 not the
enol 2: synthetic 5 and hamiguanosinol are identical.
In order to verify the structure of salvadenosine (1), the
natural product was synthesized by extension of the sequence
of reactions used to prepare 5. N,O-Protected adenosine15 was
subjected to bromination (Scheme 2, saturated Br2−H2O, pH 4
NaOAc buffer), but only the cyclized product 7 was formed
presumably through intramolecular attack by the 5′-OH group
after bromination at C-8.16 The same result was observed under
aprotic conditions (NBS, DMF; 5,5-dimethyl-1,3- dibromohy-
dantoin, DBH, DMF);17 consequently, we turned to a
“protecting-group free” strategy (Scheme 2). Adenosine (8)
was converted to 8-bromoadenosine (9) (saturated Br2−H2O,
pH 4 buffer) followed by an efficient conversion to the 8-
oxoadenosine (10) under Chatgilialoglus conditions.18 Com-
pound 10 was converted to primary chloride 11 with improved
yield (SOCl2, DMPU, 70%); the latter, in turn, was subjected to
nucleophilic substitution with sodium thiomethoxide to yield 1
in four steps and 17% yield from adenosine. Since the sample of
natural 1 was purified as the formate salt, synthetic 1 was also
1
converted to the formate salt for comparison. The H and 13C
NMR spectra (Supporting Information, Table S3) as well as
UV, IR, and CD data of the two samples 1·HCO2H matched in
every way. Co-injection of natural 1 with synthetic 1 by HPLC
resulted in a single peak (see the Supporting Information,
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Figure 2. Hypothetical biosynthetic origin of 1 from S-adenosylmethionine (SAM). See text for discussion.
polyamine biosynthesis.27 Common to these biological
reactions is the release of 5′-deoxy-5′-(methylthio)adenosine.
We speculate that 1 may arise through displacement of 5′-
deoxy-5′-(methylthio)adenosine from SAM (path b), possibly
via a LuxI-type mechanism with release of homoseryl
lactone.28,29 The product may then be converted to 1 by
electrophilic or free-radical attack at C-8 with reactive oxygen
species (ROS), a reaction known from purine metabolism and
the biology of DNA damage.30 The anomaly is 5; no guanosine
analogue of SAM has been demonstrated yet, but 5 has been
detected in human urine as a byproduct of nucleotide
catabolism.14
In conclusion, we confirmed the structure of a new
nucleoside salvadenosine (1, 5′-deoxy-5′-(methylthio)-8-oxoa-
denosine) from the tunicate Didemnum sp. through integrated
analysis of spectroscopic data and total synthesis. Re-evaluation
of the published structure of hamiguanosinol and its synthesis
from guanosine requires revision of the structure of
hamiguanosinol7 from the enol tautomer 2 to the keto form 5.
mg). Fraction C was further purified by reversed-phase HPLC
(phenylhexyl column, 10 × 250 mm, linear gradient (initial conditions
90:10 H2O (0.1% HCO2H)−CH3CN to 50:50 over 20 min) to yield
5′-deoxy-5′-(methylthio)-8-oxoadenosine (1·HCO2H, 0.8 mg, tR
=
10.2 min), 6-bromotryptamine (2.2 mg, tR = 12.6 min), and
tryptamine (0.43 mg, tR = 9.4 min). Fraction B was purified under
identical conditions to obtain additional 1 (0.46 mg) and 6-
bromotryptamine (0.34 mg). The combined samples of 1 were
repurified by HPLC over the same column (90:10 H2O−MeOH, 0.5%
HCO2H) to yield 1·HCO2H (0.42 mg, calculated through NMR
quantification of 13C satellite peaks31). Extraction of sample 11-14-039
and purification was carried out in a similar manner to provide
additional 1·HCO2H (∼0.3 mg).
−
Salvadenosine (1): pale yellow powder, HCO2 salt; [α]D +11.3
(c 0.3, MeOH); UV (MeOH) λmax 210 nm (ε log10 4.49), 255 (3.95),
270 (4.00); FTIR (ATR, ZnSe plate): ν 3356, 3188, 2920, 2850, 1710,
1
1662, 1633, 1590, 1379, 1350, 1131, 1092, 1030, 1006 cm−1; H and
13C NMR see Table 1 (CD3OD) and Supporting Information, Table
S1 (DMSO-d6); ESI-TOF m/z 312.0777 [M − H]− (calcd for
C11H14N5O4S 312.0767).
1
6-Bromotryptamine: colorless powder; H, 13C, and HRMS data
were consistent with previously published data.9
1
Tryptamine: colorless powder; MS and H NMR spectra were
EXPERIMENTAL SECTION
■
identical to those of an authentic sample.
General Experimental Procedures. Inverse detected 2D NMR
HPLC Comparison of Synthetic and Natural 1. Samples of
natural and synthetic 1 (see below) were prepared in MeOH as
equimolar solutions (0.025 mg/mL), and aliquots of each solution,
along with an admixture of both (equivolume), were analyzed by
HPLC (Polar-RP column, 4 μm, 80 Å, 150 × 4.6 mm, gradient elution;
(H2O + 0.1% HCO2H/CH3CN, 0−5 min hold at 5% CH3CN, 5−18
min ramp to 50% CH3CN, 1 mL/min, 40 °C column oven). The
following retention times were obtained: natural 1, tR = 10.48 min;
synthetic 1 tR = 10.47 min; combined natural and synthetic samples, tR
= 10.48 min, single peak. (Supporting Information, Figure S1).
5′-Chloro-5′-deoxyguanosine (6). A protocol with improved
yield was modeled after a literature procedure.12 Guanosine (0.60 g,
2.12 mmol) was suspended in dry DMPU (10.6 mL) and dissolved
upon heating. After the mixture was cooled in an ice bath, thionyl
chloride (770 μL, 10.6 mmol, 5 equiv) was slowly added with stirring
and the mixture was warmed to 23 °C over 2 h. The mixture was
cooled in an ice bath, diluted with cold H2O (10 mL), and absorbed
onto a column of Dowex 50 × 2−400 resin (200−400 mesh, H+
form). The column was washed with water and the compound eluted
with 5% aqueous ammonia. The volatiles were removed from the
eluate under reduced pressure to yield 6 (478 mg, 1.58 mmol, 75%
spectra were measured on a 500 MHz spectrometer equipped with a 5
1
mm H{13C} 5 mm probe or a 600 MHz NMR spectrometer with a
1.7 mm 1H{13C} microcryoprobe. 13C NMR spectra were collected on
a 125 MHz spectrometer equipped with a 5 mm 13C{1H} cryoprobe.
NMR spectra were referenced to residual solvent signals, CD3OD (δH
3.31, δC 49.00), (CD3)2SO (δH 2.50, δC 39.52). High-resolution mass
data were obtained with an ESI-TOF system. Low-resolution MS
measurements were made on a UHPLC coupled to an MSD single-
quadrupole detector. Automated medium-pressure chromatography
was carried out using a 30 g C18 cartridges under specified gradient
elution conditions. Optical rotations were measured at the D-double
emission line of Na. FTIR spectra were collected on a ZnSe plate. CD
spectra were measured in quartz cells (1 or 2 mm path length). DMPU
and triethylamine were distilled from CaH2 under N2. Other solvents
were dried by passage through activate alumina or molecular sieves
under Ar. Reactions were performed under N2.
Animal Material. Two samples of the tunicate Didemnum sp. (11-
25-039 and 11-14-018) were collected in July 2011 off Little San
Salvador Island, Bahamas, at a depths of −28 and −32 m. Voucher
samples of the tunicates (11-25-039 and 11-14-018) are archived at
UC San Diego.
Extraction and Isolation. The MeOH extract (5 mL) of
Didemnum sp. (11−14−018, 1.21 g dry) was separated by progressive
solvent partition. The MeOH extract was adjusted to approximately
1:9 H2O/MeOH and was extracted repeatedly with hexane (5 mL ×
3) to yield fraction A (7.7 mg). The aqueous layer was then adjusted
to 2:3 H2O/MeOH followed by extraction with CH2Cl2 (5 mL × 3) to
yield fraction B (7.2 mg). The aqueous layer was adjusted to
approximately 9:1 H2O/MeOH before the solution was extracted with
n-BuOH (5 mL × 3) to yield fraction C (18.5 mg). Removal of the
volatiles from the remaining aqueous phase provided fraction D (202
1
yield). The H NMR spectrum of 6 was consistent with published
data.7
5′-Deoxy-5′-(methylthio)guanosine (5). 5′-Chloro-5′-deoxy-
guanosine (0.10 g, 0.33 mmol) was dissolved in dry DMF with
sodium thiomethoxide (232 mg, 3.3 mmol, 10 equiv) at 23 °C. To
this, NaOMe in DMF (1.8 mg, 33 μmol, 0.1 equiv) was added and the
mixture stirred for 3 h. The reaction was neutralized with HCl (1 M)
and extracted with ether three times. The aqueous layer was dried, and
the residue was preabsorbed on C18 stationary phase for solid-phase
loading and purified by automated medium-pressure chromatography
(gradient elution: 0.1% HCO2H/MeOH/H2O, 10−50% over 20 min).
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The volatiles were evaporated under reduced pressure, and the
152.1, 148.8, 148.8, 105.9, 87.9, 85.0, 74.5, 72.0, 37.4, 16.1; ESI-TOF
m/z 312.0773 [M − H]− (calcd for C11H14N5O4S 312.0767).
Preparation of Salvadenosine Formate salt (1·HCO2H). Free
base 1 (10.88 mg) was dissolved in CD3OD (500 μL), HCO2H (90%
aq, 1 equiv) was added, and the solution was dried under nitrogen to
give 1·HCO2H: white powder; [α]D +20.6 (c 0.1, MeOH); FTIR
(ATR, ZnSe plate) ν 3343, 3212, 1710, 1658, 1592, 1475, 1429, 1364,
aqueous residue was lyophilized to yield 5 (60 mg, 0.19 mmol, 56%
1
yield) as a fluffy, white powder. H and 13C NMR data: Supporting
Information, Table S2.
N-(tert-Butoxycarbonyl)-5′-O,8-cyclo-2′,3′-O-isopropylide-
neadenosine (7). A solution of N-(tert-butoxycarbonyl)-2′,3′-O-
isopropylideneadenosine (1.30 g, 3.19 mmol) in MeOH (25 mL) and
NaOAc buffer (25 mL, 0.5 M, pH 4) was treated by slow addition of
saturated Br2−water (32.5 mL) and the resulting mixture stirred at
room temperature for 48 h. The mixture was decolorized by addition
of NaHSO3 (5 M) and adjusted to pH 7 with NaOH aqueous (2 M)
to give a precipitate which was filtered, washed with water, and dried
under reduced pressure. The residue was preabsorbed onto C18
stationary phase for solid-phase loading and purified by automated
medium-pressure chromatography (gradient elution, 10−80% 0.1%
HCO2H/MeOH/H2O over 20 min). The volatiles were evaporated
under reduced pressure and the aqueous phase was lyophilized to yield
7 (290 mg, 0.72 mmol, 23% yield) as an off-white powder: [α]D −33.9
(c 0.1, MeOH); UV (MeOH) λmax 266 nm (ε, log10 4.24), 210 (4.42);
FTIR (ATR, ZnSe plate) ν 3336, 3187, 2990, 2932, 2363, 2337, 1710,
1
1129, 1096, 1037 cm−1; UV−vis was identical to free base 1; H and
13C NMR, see the Supporting Information, Table S3.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C, and 2D NMR spectra of 1 and 7 as well as H NMR
1
spectra of 6 and 11. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +1 (858) 534-7115. Fax: +1 (858) 822-0386. E-mail:
tmolinski@ucsd.edu.
■
1
1645, 1606, 1462, 1377, 1292, 1084, 849 cm−1; H NMR (CD3OD)
δH 8.51 (s, 1H), 6.33 (s, 1H), 5.18 (d, 1H, J = 5.7 Hz), 4.87 (d, 1H, J =
5.7 Hz), 4.77 (d, 1H, J = 1.0 Hz), 4.70 (dd, 1H, J = 2.1, −13.0 Hz),
4.30 (d, 1H, J = −13.0 Hz), 1.57 (s, 9H), 1.53 (s, 3H), 1.36 (s, 3H);
13C NMR (CD3OD) δc 157.3, 152.6, 152.4, 151.2, 149.4, 120.0, 114.1,
88.3, 87.5, 86.7, 82.6, 82.6, 76.4, 28.5, 26.4, 24.7; ESI-TOF m/z
406.1723 [M + H]+ (calcd for C18H24N5O6 406.1721). Attempted
bromination of N-(tert-butoxycarbonyl)-2′,3′-O-isopropylideneadeno-
sine (18.0 mg, 0.058 mmol) in DMF (300 μL) with 5,5-dimethyl-1,3-
dibromohydantoin (DBH, 25.3 mg, 0.089 mmol, 1.5 equiv)17 or NBS
(16.2 mg, 0.089 mmol, 1.5 equiv) resulted in rapid loss of starting
material (∼30 min, TLC), no detection of starting material, and only 7
as the product (LCMS).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank P. Stout for assistance with collection of Didemnum
sp. and Y. Su and A. Mrse (UCSD) for MS data and help with
NMR measurements, respectively. The 500 MHz NMR
spectrometer and the HPLC TOFMS were purchased with
funding from the NSF (Chemical Research Instrument Fund,
CHE0741968) and the NIH Shared Instrument Grant
(S10RR025636) programs, respectively. This work was
supported by grants from NIH (AI1007786 to TFM) and
NSERC (CNB).
8-Bromoadenosine (9). Kohyama’s protocol was used to yield 9
(1.04 g, 3.01 mmol, 85% yield). 1H NMR, 13C NMR, and MS data of 9
were identical to literature values.32
8-Oxoadenosine (10). The title compound was prepared
according to a published method18 and purified by automated flash
chromatography (0.1% HCO2H/MeOH/H2O, gradient elution 10−
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