Pyridine Nucleosides Neopetrosides A and B from a Marine Neopetrosia sp. Sponge. Synthesis of Neopetroside A and Its β-Riboside Analogue

Article abstract of DOI:10.1021/acs.jnatprod.5b00256

Neopetrosides A (1) and B (2), new naturally occurring ribosides of nicotinic acid with extremely rare α-N-glycoside linkages and residues of p-hydroxybenzoic and pyrrole-2-carboxylic acids attached to C-5′, were isolated from a marine Neopetrosia sp. spo

Full text of DOI:10.1021/acs.jnatprod.5b00256

Article
pubs.acs.org/jnp
Pyridine Nucleosides Neopetrosides A and B from a Marine
Neopetrosia sp. Sponge. Synthesis of Neopetroside A and Its
β‑Riboside Analogue
†
,†
†
‡
†
‡
Larisa K. Shubina, Tatyana N. Makarieva,* Dmitry V. Yashunsky, Nikolay E. Nifantiev,
†
†
Vladimir A. Denisenko, Pavel S. Dmitrenok, Sergey A. Dyshlovoy, Sergey N. Fedorov,
Vladimir B. Krasokhin, Seung Hun Jeong, Jin Han, and Valentin A. Stonik
†
§
§
†
†G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-Eastern Branch of the Russian Academy of Sciences, Prospect 100-let
Vladivostoku 159, Vladivostok 690022, Russian Federation
‡
N.D. Zelinsky Institute of Organic Chemistry of the Russian Academy of Sciences, Leninsky Prospect, 47, 119991, Moscow, Russian
Federation
§
National Research Laboratory Mitochondrial Signaling, Cardiovascular and Metabolic Disease Center (CMDC), Department of
Physiology, College of Medicine, Inje University, Busan 614-735, South Korea
*
S Supporting Information
ABSTRACT: Neopetrosides A (1) and B (2), new naturally
occurring ribosides of nicotinic acid with extremely rare α-N-
glycoside linkages and residues of p-hydroxybenzoic and pyrrole-
2
-carboxylic acids attached to C-5′, were isolated from a marine
Neopetrosia sp. sponge. Structures 1 and 2 were determined by
NMR and MS methods and confirmed by the synthesis of 1 and
its β-riboside analogue (3). Neopetroside A (1) upregulates
mitochondrial functions in cardiomyocytes.
arine sponges of the genus Neopetrosia have been
age-related disorders, characterized by defective mitochondrial
functions, and as a protector against high-fat-diet-induced
obesity. It was also revealed that pyridine nucleosides and
M
reported to be a rich source of secondary metabolites
1
13
with diverse chemical structures and biological activities. This
2
group consists of tetrahydroisoquinolines, polycyclic alka-
nucleotides play an important role in regulating a wide variety
of functions in cardiovascular tissues. Deregulation of pyridine
nucleotides is immediately involved in the pathogenesis of heart
failure, arrhythmia, and age-associated abnormalities of the
3
−6
7
8
loids,
tricyclic peptides, pentacyclic hydroquinones, and
9
sesquiterpene benzoquinones found in different species of the
genus. The isolated compounds demonstrate a broad spectrum
of biological activities, including potent cytotoxic effects against
1
2
heart. In addition, modified pyridine nucleotides and
nucleosides can provide therapeutic benefits. For instance,
tiazofurin and benzamide riboside, synthetic nucleosides, are+
used clinically as anticancer agents. They form cytotoxic NAD
2
,4,5
cancer cells,
inhibitory activity against pathogenic mi-
4
3
crobes, and inhibition of indoleamine-2,3-dioxygenase and
7
amoeboid invasion. As part of our continuing search for
bioactive secondary metabolites from marine organisms,
1
0,11
14−16
analogues in the metabolism of humans.
As natural products, modified pyridine nucleosides are
exceedingly rare. So far, only three natural modified pyridine
nucleosides have been discovered, namely, clitidine (3-carboxy-
new pyridine α-ribosides (1, 2) from a Neopetrosia sp. sponge
have been isolated. Similar pyridine nucleosides, having both an
α-glycosidic bond and an acyl substituent at C-5′, have never
been isolated from natural sources or synthesized.
4
-imino-l-(β-D-ribofuranosyl)-1,4-dihydropyridine) from the
17
poisonous mushroom Clitocybe acromelalga, 1-α-D-ribofur-
Pyridine nucleosides are well-known biosynthetic precursors
of nicotinamide adenine dinucleotides, NAD+ and NADH,
important regulators of energy production, metabolism, redox
reactions, some ion channels, and processes of cell survival and
anosyl-4-pyridone-3-carboxamide from the urine of normal
18
human individuals and leukemic patients, and α-nicotinamide
19
riboside from the marine sponge Protophlitaspongia aga.
Received: March 24, 2015
12
death under normal and pathological conditions. It is known
that the recently studied vitamin nicotinamide riboside may be
used as a nutritional supplement to ameliorate metabolic and
©
XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.5b00256
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
The occurrence of an oxidized nicotinamide adenine
Table 1. NMR Data of Neopetrosides A (1) and B (2) in
a
dinucleotide containing an α-riboside linkage in nature was
CD OD
3
20
first reported by Kaplan and co-workers. A biological role for
+
+
1
2
α-NAD and α-NADP , however, was not uncovered. The
function of naturally occurring α-NADPH anomers in
mammalian physiology was first demonstrated later, after
identifying the catalytic activity of renalase as an α-NADPH
δH mult (J in
δH mult (J in
b
b
position
δ , type
Hz)
δ , type
Hz)
C
C
2
3
4
144.3, CH
139.5, C
9.28, s
144.3, CH
139.0, C
9.27, s
21
oxidase/anomerase.
147.9, CH
8.94, dd (8.0,
148.0, CH
8.94, m
8.06, m
8.95, m
Herein we describe the structure elucidation of two new
modified pyridine α-ribosides, named as neopetrosides A (1)
and B (2), along with the synthesis and biological activity of
neopetroside A.
1
.4)
5
6
7
127.8, CH
143.7, CH
8.06, dd (8.0,
.2)
127.8, CH
143.7, CH
6
8.96, dd (6.2,
.4)
1
RESULTS AND DISCUSSION
The EtOH extracts of the sponge were concentrated and
167.2, C
98.6, CH
74.4, CH
73.3, CH
168.0, C
98.7, CH
74.4, CH
73.3, CH
■
1′
2′
3′
6.50, d (5.3)
4.79, m
6.48, d (5.3)
4.81, m
partitioned between H O and n-BuOH. The n-BuOH-soluble
2
4.33, dd (3.8,
4.33, dd (3.4,
4.5)
materials, after concentration in vacuo were partitioned
between aqueous EtOH and n-hexane. The EtOH-soluble
materials were subjected to chromatographic separation on a
YMC gel column, and further purification using reversed-phase
HPLC afforded neopetrosides A and B.
4
.5)
4′
88.0, CH
4.96, ddd (3.8,
3.6, 4.6)
88.2, CH
4.93, ddd (3.4,
3.6, 4.6)
5′
65.3, CH2 4.50, dd
64.7, CH2 4.48, dd
(4.6,12.2)
(
4.6,12.2)
4
.59, dd (3.6,
4.57, dd (3.6,
12.2)
Neopetroside A (1) was obtained as a light yellow,
1
2.2)
amorphous solid. The HRESIMS spectrum of 1 showed a
1
2
3
″
″
″
122.3, C
+
protonated molecule at m/z 376.1033 [M + H] and an adduct
133.6, CH
117.0, CH
7.93, d (8.8)
6.86, d (8.8)
123.6, C
+
ion at m/z 398.0854 [M + Na] consistent with a molecular
117.7, CH
6.95, dd (1.7,
3.7)
1
13
formula C H NO . Analysis of H, C, HSQC, and COSY
18
17
8
NMR data (Table 1) of 1 showed the presence of a
4
″
164.5, C
111.6, CH
125.9, CH
163.4, C
6.21, dd (2.5,
3.7)
ribofuranoside ring and aromatic carbons at δ 147.9, 144.3,
C
1
43.7, 139.5, 133.6, 127.8, 122.3, and 117.0; one oxygenated
5″
117.0, CH
6.86, d (8.8)
7.93, d (8.8)
7.01, dd (1.7,
2
.5)
methylene (δ 4.50, 4.59 and δ 65.3), and three downfield
H
C
6
″
″
133.6, CH
168.2, C
nonprotonated carbons at δC 164.5, 167.2, and 168.2. GC
analysis of the acetylated methyl acetal derivative, obtained as a
result of methanolysis of 1 followed by acetylation, confirmed
the presence of a ribose unit in this compound. The chemical
7
a
1
Spectra were recorded at 500 MHz for H NMR and 125 MHz for
C NMR.
HMBC data.
13
b
13
C NMR assignments were supported by HSQC and
shifts at δ 9.28 (s), 8.94 (dd, J = 8.0, 1.4 Hz), 8.06 (dd, J = 8.0,
H
6
.2 Hz), and 8.96 (dd, J = 6.2, 1.4 Hz) and coupling constants
1
values in the H NMR spectrum (Table 1), together with
HMBC and COSY data (Figure 2), strongly indicated the
presence of the 3-substituted pyridinium ring. The HMBC
correlations of the anomeric proton at δ 6.50 (H-1′) with C-2
H
and C-6 aromatic carbons indicated that the pyridinium ring is
linked to the sugar moiety through a nitrogen atom. The
presence of a carboxylate group was inferred by the IR
−1
absorption band at 1645 cm and a singlet at δC 167.2.
Moreover, both aromatic signals at δ 9.28 (H-2) and 8.94 (H-
H
4) were coupled to the carboxylate carbon at δC 167.2 by
HMBC. These data located the carboxylate function at C-3. On
1
13
Figure 1. Structures and ESIMS/MS analysis of neopetrosides A (1)
and B (2).
the whole, the H and C NMR data for atoms C-1−C-7
Table 1) were in agreement with those of the betaine alkaloids
(
22
trigonelline, pyridinebetaine A, and agelongine and strongly
indicated the presence of nicotinic acid as the aglycone in 1.
The remaining aromatic signals exhibited an AA′XX′ spin
system with δ 7.93 (d, J = 8.8 Hz, 2H) and 6.86 (d, J = 8.8 Hz,
H
2
H), suggesting the presence of a 1,4-disubstituted aromatic
moiety. This was further corroborated by COSY and HMBC
data (Figure 2), while the HMBC correlations from the H-2″
and H-6″ to a carbonyl carbon with δ 168.2 as well as
C
downfield shifted signals of methylene protons in the ribosyl
moiety (Table 1) together with MS data were consistent with
the presence of a p-hydroxybenzoic acid unit. This was
supported by comparing the spectroscopic data with those
reported in the literature for p-hydroxybenzoyloxy-containing
Figure 2. Key COSY (bold), HMBC (blue arrows), and NOE (red
arrows) correlations for the structural assignment of 1 and 2.
2
3
natural compounds. The HMBC correlations of the
B
DOI: 10.1021/acs.jnatprod.5b00256
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Journal of Natural Products
Article
Scheme 1. Synthesis of 1
Scheme 2. Synthesis of 3
methylene protons H -5′ to the carbonyl C-7″, which in turn
its HRESIMS spectrum The spectroscopic properties of 2 were
similar to those of 1 (Table 1). The difference between the
NMR spectra of 1 and 2 was in the signals belonging to acyl
2
showed correlations with the aromatic protons H-2″/H-6″,
indicated that the p-hydroxybenzoyloxy group was attached to
C-5′ of the sugar moiety. The structure of 1 was further
confirmed by ESIMS/MS analysis, showing fragmentations
with the loss of nicotinic acid or a p-hydroxybenzoyl group
1
substituents. The H NMR spectrum of 2 showed three proton
signals at δ 6.21 (dd, J = 2.5, 3.7 Hz), 6.95 (dd, J = 1.7, 3.7
H
Hz), and 7.01 (dd, J = 1.7, 2.5 Hz) and an exchangeable proton
(
Figure 1). The ribofuranoside ring configuration was
at δ 11.97 (br s, in the spectrum recorded in DMSO) instead
H
established on the basis of NOESY correlations between H-
of two aromatic proton doublets of the p-hydroxybenzoyl
fragment in 1. The corresponding 13C chemical shifts (δC
111.6, 117.7, 125.9) and COSY connectivities from H-3″ to H-
5″ and from NH to H-5″ as well as HMBC data (Figure 2)
1
′/H-3′, H-5′ and H-4′/H-2, H-6 (Figure 2). This assignment
was further supported by the chemical shifts and coupling
constants of the pentose unit that were close to those in α-
24
26
nicotinamide riboside.
were consistent with a substituted pyrrole.
The determination of the absolute configuration of the sugar
unit was carried out after acid hydrolysis of 1 with 2 M TFA
followed by preparation of acetylated (+)-2-octylglycosides. GC
analysis of these compounds and comparison with authentic
samples according to the procedure of Leontein et al.
revealed the D-configuration of the sugar.
Moreover, the base peak at m/z 110.0243 in the HRESIMS/
MS spectrum (negative ion mode) corresponding to a
C H NO fragment was in agreement with the presence of
pyrrole-2-carboxylic acid unit in the structure. Therefore,
compound 2 is a new pyridine nucleoside esterified at C-5′
with a pyrrole-2-carboxylic acid residue.
5
4
2
25
Neopetroside B (2) was obtained as an amorphous solid. Its
5′-O-p-Hydroxybenzoylated and 5′-O-pyrrole-2-carboxylated
ribosides of both nicotinic acid and nicotinamide are
unprecedented among natural products. Until now only 5′-O-
molecular formula was established as C H N O from a
1
6
16
2
7
−
−
prominent [M − H] ion peak at m/z 347.0887 [M − H] in
C
DOI: 10.1021/acs.jnatprod.5b00256
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Journal of Natural Products
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Figure 3. Evaluation of mitochondrial function. (A) Action of 1 on C2C12 cells after 24 h of treatment. (B) ATP levels in C2C12 cells after 1 h of
#
treatment by 1. (C) OCR in C2C12 cells after 1 h of treatment by 1. Four independent in vitro experiments were performed. p < 0.05 vs control
group.
benzoyl-β-ribosides of nicotinic acid and nicotinamide have
EXPERIMENTAL SECTION
■
been synthesized as cofactors for DT-diaphorase and nitro-
General Experimental Procedures. Optical rotations were
measured using a JASCO DIP-360 polarimeter. UV spectra were
recorded on a Shimadzu UV-1601 PC spectrophotometer. CD spectra
were recorded with an Applied Photophysics Chirascan Plus
spectropolarimeter. IR spectra were recorded using a Bruker Vector
2
7,28
reductase.
To confirm the unusual structure of 1, we have synthesized
this compound and its β-analogue (3) from 2,3-O-benzylidene-
D-ribose (Scheme 1) and 1,2,3-tri-O-acetyl-D-ribose (Scheme
1
13
2
2 spectrophotometer. The H and C NMR spectra were obtained
2
), respectively. Synthesis of 1 included the selective
29
using Bruker Avance III-700, Bruker Avance 600, and Bruker Avance
III HD-500 spectrometers. Chemical shifts were referenced to the
corresponding residual solvent signal (δ 7.26/δ 77.20 for CDCl , δ
H
benzylidene formation (4 → 5) and the introduction of the
p-hydroxybenzoic acid residue at the C-5′ position (5 → 6).
The corresponding ribofuranose derivative (7) as a pure β-
H
C
3
3
.30/δ 49.60 for CD OD, and δ 2.50/δ 39.60 for DMSO-d ). ESI
C 3 H C 6
isomer was obtained by treatment of 6 with the Ph P-CCl4
mass spectra (including HRESIMS) were measured on a MicrOTOF
II (Bruker Daltonics) instrument and an Agilent 6510 Q-TOF LC-MS
spectrometer by direct infusion in MeOH. GC analysis was conducted
on an Agilent 6580 Series apparatus, equipped with a capillary column
3
system in DMF. Nucleophilic displacement of the Cl group
with the ethyl ester of nicotinic acid proceeded with completed
inversion to give the required derivative 8. The synthesis was
completed by the removal of protective groups by aqueous
(
HP-5 MS, 30 m × 0.25 mm) with helium carrier gas (1.7 mL/min)
over the temperature range 100−250 °C at 5 deg/min. The
temperatures of the injector and detector were 150 and 280 °C,
respectively. Low-pressure column chromatography was performed
using YMC gel ODS-A (Japan) and silica gel 60 (40−63 μm, E.
Merck). HPLC was performed using a Shimadzu Instrument equipped
with a UV−vis detector and a Develosil ODS-UG-5 (250 × 4.6 mm)
column. TLC was performed on silica gel 60 F254 plates (E. Merck),
and visualization was accomplished using UV light or by charring at
NH in MeCN (8 → 9) and then with aqueous TFA to give
3
pure 1 in its salt form. Treatment of the TFA salt form of 1
with aqueous NH in MeOH gave 1 as a betaine. All the
3
spectroscopic data of synthetic 1 were identical to those of the
natural product (Supporting Information S19, S20, S21, and
Table S1).
The synthesis of β-riboside analogue 3 was carried out from
30
150 °C with 10% (v/v) H PO in EtOH. All air- or moisture-sensitive
3 4
the D-ribose triacetate derivative 10 by introduction of the p-
acetoxybenzoyl group into the 5′-position (10 → 11) followed
by treatment with the ethyl ester of nicotinic acid (11 → 12)
with TMSOTf as the catalyst. Removal of the acetate groups
and hydrolysis of the ethyl ester by aqueous NH in CH CN
reactions were carried out using dry solvents under dry argon.
Chemicals were purchased from Acros, Fluka, or Aldrich and used
without further purification.
Animal Material. A Neopetrosia sp. sponge was collected by scuba
during the 38th scientific cruise of R/V Academic Oparin, May 2010,
near Con Son Island (08°40,9 N; 106°44,4 E, depth 6−15 m) in
Vietnamese waters. The shape of the sponge varies from a 1−2 cm
thick-lobed encrustation up to 15 cm in the lateral dimension to
massive. The surface is usually even and smooth or slightly velvety,
with oscules scattered up to 5 mm in diameter, flush or on top with a
conical elevation up to 5 mm high. The color is dull blue external and
cream internal, distinguishing this animal from other Neopetrosia spp.
The consistency is hard and brittle. The choanosome is dense. The
ectosome is a tangential isodictial regular reticulation of single spicules.
The skeleton is a rather regular reticulation of ascending and
interconnecting multispicular spicule tracts, up to 150 μm thick,
forming rounded meshes up to 400 μm in diameter. In deeper parts of
the choanosome the skeleton is rather more compact and irregular.
Erect spicule brushes extend outwardly through the surface. The
spicules are oxeas, curved with an acute ending reaching 220 μm long
by 10 μm wide. Until now about 30 species of Neopetrosia have been
described. Definition of these sponges to species level is difficult. The
morphologies of the skeleton and spicules are important taxonomic
characteristics, but clearly insufficient in this case because of their
ecological plasticity. This is a new undescribed species. A very similar
3
3
provided 3. The comparison of the NMR spectra of 1 and 3
revealed substantial differences in chemical shifts and spin-
coupling constants of the ribofuranose fragments (Table S2).
Compound 1 was not cytotoxic against the C2C12 mouse
myoblast cell line up to 100 μM concentration. However,
compound 1 in its salt form modulated mitochondrial functions
in C2C12 cells. To evaluate mitochondrial functions,
mitochondrial ATP levels and oxygen consumption rate
(OCR) in C2C12 cells treated with 10 μM 1 were measured,
and these values were increased in comparison with those in
the control experiments (Figure 3). It is known that nicotinic
acid and its derivatives may provide beneficial effects on blood
31
lipid and cholesterol profiles, and we intend to study similar
effects of the obtained compounds in the future.
In summary, neopetrosides A (1) and B (2) are polyfunc-
32
tional biomolecular systems and the first reported represen-
tatives of a novel class of pyridine nucleosides possessing an α-
riboside linkage, acylated by aromatic acids, and containing a
carboxylate group in the aglycone moieties. The noncytotoxic
compound 1 upregulates mitochondrial functions and, there-
fore, may be a useful lead structure for drug development.
33
sponge was noted in Singapore waters. A voucher specimen is
deposited under registration number O38-059 in the marine
invertebrates collection of the Pacific Institute of Bioorganic
Chemistry (Vladivostok, Russia).
D
DOI: 10.1021/acs.jnatprod.5b00256
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Extraction and Isolation. Animal material (dry weight 120 g) was
cut and extracted with EtOH immediately after collection (3 × 3 L).
The EtOH extract after evaporation in vacuo was partitioned between
Ethyl 1-[2,3-O-Benzylidene-5-O-(4-acetoxybenzoyl)-α-D-
ribofuranosyl]pyridinium-3-carboxylate Chloride (8). A solution of
ribosyl chloride 7 (270 mg, 0.645 mmol) and ethyl nicotinate (2.5
H O and n-BuOH. The n-BuOH-soluble materials were partitioned
mL) in CH
concentrated under vacuum, and triturated two times with ether to
give 330 mg (90%) of nicotinoyl riboside 8: amorphous solid; R 0.32
(15% MeOH/CHCl
); HRESIMS m/z 534.1750 [M]+ (calcd for
534.1759).
Synthetic Neopetroside A (TFA Salt). To a stirred solution of
nicotinoyl riboside 8 (100 mg, 0.175 mmol) in CH CN (2.5 mL) was
added 1.4 N aqueous ammonia solution (2.2 mL). The mixture was
stirred at rt for 5 h, diluted with H O, and concentrated to dryness.
The residue was subjected to silica gel column chromatography
(MeOH/CH Cl 15% → 30%) to give 60 mg (74%) of nicotinoyl
riboside 9. The latter was dissolved in 90% aqueous TFA, and the
mixture was kept at rt for 1 h, concentrated in vacuo, dissolved in H O,
and freeze-dried to give 60 mg (94%) of riboside 1 in its salt form:
3
CN (2.5 mL) was kept at room temperature for 10 h,
2
with aqueous EtOH and n-hexane. The EtOH-soluble layer was
fractionated by flash column chromatography on YMC gel ODS-A (75
f
μm), eluting with a step gradient of H O and EtOH (100:0 to 20:80)
3
2
with monitoring by HPLC. The fractions that eluted with 20% EtOH
were further purified by reversed phase HPLC (Develosil ODS-UG-5,
C H28NO
29 9
0
.7 mL/min, 25 min, UV = 280 nm) using a gradient solvent system
3
from 18% to 80% CH CN to afford neopetrosides A (1, 5 mg, 0.004%
3
from dry weight) and B (2, 3 mg 0.002% from dry weight).
Neopetroside A (1): light yellow, amorphous solid; UV (EtOH)
λmax (log ε) 260 (3.54); ECD (EtOH, c 2.66 × 10−4 M) λmax (Δε) 275
2
2
2
−
1 1
(
+0.21) nm; IR (KBr) νmax 3417, 1708, 1642, 1608, 1385 cm ; H,
1
3
+
C NMR, Table 1; HRESIMS m/z 376.1033 [M + H] (calcd for
C H NO , 376.1027) and 398.0854 [M + Na] (calcd for
2
+
1
8
17
8
1
9
solid; R 0.36 (CHCl /MeOH/H O, 10:10:1); [α] +20 (c 1, H O);
C H NNaO , 398.0846).
f
3
2
D
2
18
17
8
1
Neopetroside B (2): light yellow, amorphous solid; UV (EtOH) λ
H NMR (600 MHz, CD
3
OD) δ
H
4.37 (dd, 1H, J = 3.8, 4.6 Hz, H-3′),
max
−4
(
(
log ε) 266 (3.33); ECD (EtOH c 2.04 × 10 M) λ (Δε) 275
4.51 (dd, 1H, J = 4.5, 12.3 Hz, H-5′a), 4.59 (dd, 1H, J = 3.5, 12.3 Hz,
H-5′b), 4.80 (m, 1H, H-2′), 4.98 (m, 1H, H-4′), 6.54 (d, 1H, J = 5.2
Hz, H-1′), 6.86 (d, 2H, J = 8.5 Hz, H-3″, H-5″), 7.93 (d, 2H, J = 8.5
Hz, H-2″, H-6″), 8.17 (dd, 1H, J = 6.8, 7.9 Hz, H-5), 9.02 (dd, 1H, J =
1.3, 8.0 Hz, H-4), 9.12 (dd, 1H, J = 1.4 and 6.2 Hz, H-6), 9.38 (s, 1H,
max
1
13
1
+0.18) nm; H, C NMR (CD OD), Table 1; H NMR (500 MHz,
3
DMSO-d ) δ 11.97 (1H, s, H-1), 9.14 (1H, s, H-2), 8.80 (1H, m, H-
6
H
4), 8.02 (1H, m, H-5), 8.89 (1H, m, H-6), 6.56 (1H, d, J = 5.3 Hz, H-
1
′), 4.63 (1H, m, H-2′), 4.23 (1H, dd, J = 3.4, 4.5 Hz, H-3′), 4.77 (1H,
H-2); 13C NMR (150 MHz, CD
OD) δ 64.8 (C-5′), 72.8 (C-3′),
m, H-4′), 4.38 (1H, dd, J = 4.6, 12.2 Hz, H-5′) 4.49 (1H, dd, J = 3.6,
3 C
1
2
2.2 Hz, H-5′), 6.89 (1H, dd, J = 1.7, 3.7 Hz, H-3″), 6.21 (1H, dd, J =
74.0 (C-2′), 88.0 (C-4′), 98.3 (C-1′), 116.5 (C-3″, C-5″), 127.8 (C-5),
133.1 (C-2″, C-6″), 139.9 (C-3), 144.2 (C-6), 145.0 (C-2), 147.8 (C-
.5, 3.7 Hz, H-4″), 7.08 (1H, dd, J = 1.7, 2.5 Hz, H-5″); HRESIMS m/
−
z 347.0887 [M − H] (calcd for C H N O , 347.0885); HRESIMS/
MS m/z 110.0243 [C H NO ] (calcd for C H NO , 110.0248).
4), 164.0 (C-4″), 165.3 (C-7), 167.7 (C-7″); HRESIMS m/z 398.0849
16
16
2
7
−
+
[M + Na] (calcd for C18
H17NNaO
, 398.0846).
5
4
2
5
4
2
8
Monosaccharide Analysis. Acetyl chloride in MeOH (1:10 v/v,
00 μL) was added to 1 (0.8 mg), and the solution was heated during
h at 100 °C. The solvent was removed by a stream of argon, and a
mixture of pyridine/acetic anhydride (1:1, v/v, 0.4 mL) was added.
After stirring overnight at room temperature (rt) the mixture was
concentrated in vacuo. GC analysis of the obtained residue was carried
out by comparison with authentic samples of standard sugars prepared
by the same procedure. The retention times for the sample (7.59, 7.95,
Synthetic Neopetroside A (1). To a solution of 2 mg of
neopetroside A (TFA salt) in MeOH (0.5 mL) was added a 1.4 N
aqueous ammonia solution (0.1 mL). The mixture was kept at rt for 20
1
3
1
min and concentrated in vacuo. H NMR, Supporting Information
S19; 13C NMR, Supporting Information S20.
1,2,3-Tri-O-acetyl-5-O-(4-acetoxybenzoyl)-D-ribofuranose (11).
3
0
To a stirred solution of riboside 10 (117 mg, 0.424 mmol) and
triethylamine (0.2 mL) in CH Cl (2 mL) were added p-
acetoxybenzoyl chloride (100 mg, 0.51 mmol, 1.2 equiv) and DMAP
(5 mg). The mixture was stirred at rt for 30 min, diluted with CH Cl
2
2
8
.30, 8.34 min) matched those for ribose (7.58, 7.97, 8.34, 8.37 min).
Acid Hydrolysis and Determination of the Absolute
,
2
2
Configuration of the Monosaccharide. A solution of 1 (0.8 mg)
in 1 M TFA (0.5 mL) in a sealed vial was heated at 100 °C for 2 h.
washed with aqueous 1 M HCl, H O, and saturated aqueous
2
NaHCO , dried, and concentrated. The residue was subjected to
3
The reaction mixture was then washed with CHCl (3 × 0.5 mL) and
silica gel column chromatography in EtOAc/toluene (1:4) to provide
3
evaporated to dryness. One drop of concentrated TFA and 0.3 mL of
147 mg (79%) of riboside 11: syrup; R 0.76 (10% Me CO/CHCl );
f
2
3
[α]19 +55 (c 1, CHCl ); H NMR (600 MHz, CDCl ) δ 2.10 (s,
1
(
+)-2-octanol (Aldrich) were added to the residue, and the reaction
D 3 3 H
mixture was heated in a glycerol bath at 130 °C for 6 h. The solution
was evaporated and treated with a mixture of pyridine/acetic
anhydride (1:1, 0.4 mL) for 20 h at rt. The acetylated (+)-2-
octylglycosides were analyzed by GC using the corresponding
authentic samples prepared by the same procedure. The peaks of
four tautomeric forms of ribose were detected in the hydrolysate at
3H, Ac), 2.13 (s, 6H, 2Ac), 2.32 (s, 3H, Ac), 4.46 (dd, 1H, J = 3.8, 12.0
Hz, H-5′a), 4.57 (m, 1H, H′-4), 4.60 (dd, 1H, J=3.2, 12.0 Hz, H-5′b),
5.33 (dd, 1H, J = 4.8, 6.4 Hz, H′-2), 5.38 (dd, 1H, J = 3.0, 6.7 Hz, H′-
3), 6.46 (d, 1H, J = 4.5 Hz, H′-1), 7.20 (d, 2H, J = 8.5 Hz, C-3″, C-5″),
13
8.07 (d, 2H, J = 8.5 Hz, C-2″, C-6″); C NMR (150 MHz, CDCl ) δ
3
C
20.3 (Ac), 20.6 (Ac), 21.0 (Ac), 21.1 (Ac), 63.9 (C-5′), 69.9 (C-
2′),70.1 (C-3′), 81.7 (C-4′), 94.1 (C-1′), 121.8 (C-3″, C-5″), 131.3
(C-2″, C-6″), 126.9 (C-1″), 154.6 (C-4″), 165.2 (C-7″), 168.7 (Ac),
169.3 (Ac), 169.6 (Ac), 170.1 (Ac); HRESIMS m/z 461.1053 [M +
2
4.04, 24.64, 24.93, and 24.94 min. The retention times for authentic
samples were 24.04, 24.64, 24.93, and 24.95 min (D-Rib) and 24.52,
4.75, 24.83, and 25.06 min (L-Rib). The retention times of the L-
isomer derivatives were determined in accordance with the procedure
2
+
Na] (calcd for C H NaO , 461.1054).
20
22
11
25
of Leontein et al.
Ethyl 1-[2,3-Di-O-acetyl-5-O-(4-acetoxybenzoyl)-β-D-
ribofuranosyl]pyridinium-3-carboxylate Trifluoromethanesulfonate
(12). To a stirred solution of riboside 11 (117 mg, 0.267 mmol) and
ethyl nicotinate (0.056 mL, 0.4 mmol, 1.5 equiv) in CH Cl (1.5 mL)
Syntheses of Neopetroside A (1) and β-Riboside 3. 2,3-O-
Benzylidene-5-O-(4-acetoxybenzoyl)-β-D-ribofuranosyl Chloride (7).
29
To a stirred solution of 2,3-O-benzylidene-D-ribose 5 (200 mg, 0.840
mmol) in pyridine (3 mL) were added p-acetoxybenzoyl chloride (200
mg, 1.02 mmol, 1.2 equiv) and DMAP (5 mg). The mixture was
stirred at rt for 30 min, diluted with CH Cl , washed with aqueous 1 M
2
2
was added TMSOTf (0.084 mL, 0.267 mmol). The mixture was stirred
under reflux for 1 h, diluted with toluene, and subjected to silica gel
column chromatography (CH Cl → 97:3 CH Cl /MeOH) to give
2
2
2
2
2
2
HCl, H O, and saturated aqueous NaHCO , dried, and concentrated
to give riboside 6 (310 mg) as a mixture with a 1,5-diacylated
114 mg (63%) of nicotinoyl riboside 12: colorless, amorphous solid; R
0.52 (15% MeOH/CHCl ); [α] +1 (c 1, MeOH); H NMR (600
3 D
2
3
f
18
1
derivative. The above mixture was treated with triphenylphosphine
MHz, CD OD) δH 1.36 (t, 3H, J = 7.1 Hz, COOCH CH ), 2.02 (s,
3
2
3
(
406 mg, 1.55 mmol, 2 equiv) and CCl (0.15 mL) in DMF (3 mL) at
3H, Ac), 2.16 (s, 3H, Ac), 2.22 (s, 3H, Ac), 2.32 (s, 3H, Ac); 4.49 (q,
4
rt for 2 h, diluted with EtOAc, washed with H O and brine, dried,
concentrated, and subjected to silica gel column chromatography in
2H, J = 7.1 Hz, COOCH CH ), 4.76 (dd, 1H, J = 3.9, 12.9 Hz, H-5′a),
2
2
3
4.88 (br d, H-5′b), 4.97 (m, 1H, H-4′), 5.61 (t, 1H, J = 5.6 Hz, H-3′),
5.71 (dd, 1H, J = 4.7, 5.6 Hz, H-2′), 6.65 (d, 1H, H-1′), 7.20 (d, 2H, J
= 8.5 Hz, H-3″, H-5″), 8.02 (d, 2 H, J = 8.5 Hz, H-2″, H-6″), 8.24 (m,
1H, H-5), 9.10 (d, 1H, J = 8.0 Hz, H-4), 9.34 (d, 1H, J = 6.2 Hz, H-6),
CH Cl to provide 276 mg (78%) of riboside 7: colorless syrup; R
2
2
f
+
0
.56 (10% EtOAc/PhCH ); HRESIMS m/z 441.0706 [M + Na]
3
(
calcd for C H ClNaO , 441.0712).
21 19 7
E
DOI: 10.1021/acs.jnatprod.5b00256
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
1
3
9
.58 (s, 1H, H-2); C NMR (150 MHz, CD OD) δC 14.8
3
ASSOCIATED CONTENT
Supporting Information
Tables and NMR spectra for synthetic and natural compounds,
D and 2D NMR spectra, HRESIMS spectra for compounds 1
and 2. The Supporting Information is available free of charge
on the ACS Publications website at DOI: 10.1021/
acs.jnatprod.5b00256.
■
(
(
1
3
COOCH CH ), 20.8 (2 Ac) and 21.4 (Ac), 64.8 (C-5′), 64.9
2
3
*
S
COOCH CH ), 71.3 (C-3′), 78.1 (C-2′), 85.1 (C-4′), 99.7 (C-1′),
2
3
23.8 (C-3″,C-5″), 128.6 (C-1″), 130.5 (C-5), 132.8 (C-2″,C-6″, C-
), 143.4 (C-6), 145.5 (C-2) 149.4 (C-4), 157.0 (C-4″), 162.8
1
(
COOEt), 167.1 (C-7″), 171.0 (Ac), 171.7 (Ac), 172.2 (Ac);
+
HRESIMS m/z 530.1641 [M] (calcd for C H NO , 530.1657).
26
28
11
1
-[5-O-(4-Hydroxybenzoyl)-β-D-ribofuranosyl]pyridinium-3-car-
boxylate (3). To a stirred solution of nicotinoyl riboside 12 (55 mg,
.081 mmol) in CH CN (1.15 mL) was added a 1.4 N aqueous
ammonia solution (1.6 mL). The mixture was stirred at rt for 4 h,
diluted with H O, and concentrated to dryness. The residue was
AUTHOR INFORMATION
Corresponding Author
Tel: 7 (4232) 31-11-68. Fax: 7 (4232) 31-40-50. E-mail:
makarieva@piboc.dvo.ru.
0
■
*
3
2
subjected to silica gel column chromatography (90% MeOH/H O−
CH Cl 25% → 50%] to give 21 mg (69%) of nicotinoyl β-riboside 3:
2
2
2
Notes
19
colorless solid; R 0.36 (CHCl /MeOH/H O, 10:10:1); [α] −21 (c
f
3
2
D
The authors declare no competing financial interest.
1
13
1
1
, H O); H, C NMR (500 MHz,CD OD), Table S1; H NMR (600
2
3
MHz, D O) δ 4.45 (dd, 1H, J = 2.7, 12.7 Hz, H-5′a), 4.48 (dd, 1H, J
ACKNOWLEDGMENTS
2
H
■
=
3.5, 5.0 Hz, H-3′), 4.60 (t, 1H, J = 4.9 Hz, H-2′), 4.78 (q, 1H, J = 2.8
The research described in this publication was supported by
Grant 148.2014.4 from the President of RF, Grant 13-03-00986
from RFBR, Grant 14-50-00126 from RSF, and the National
Research Foundation Joint Research Program
2014K2A1A2048550) from the Republic of Korea. The
authors are thankful to Dr. N. Yu. Kim for providing UV and
CD spectroscopic data.
Hz, H-4′), 4.83 (dd, 1H, J = 2.8, 12.7 Hz, H-5′b), 6.65 (d, 1H, J = 4.3
Hz, H-1′), 6.72 (d, 2H, J = 9.7 Hz, H-3″, H-5″), 7.52 (d, 2H, J = 9.7
Hz, H-2″,6″), 7.92 (dd, 1H, J = 6.3, 7.8 Hz, H-5), 8.67 (dd, 1H, J =
13
1.3, 7.9 Hz, H-4), 8.94 (d, 1H, J = 6.2 Hz, H-6), 9.20 (s, 1H, H-2);
C
(
NMR (150 MHz, D O) δ 66.3 (C-5′), 73.9 (C-3′), 80.6 (C-2′), 88.9
2
C
(
1
4
C-4′), 101.9 (C-1′), 118.3 (C-3″, C-5″), 123.0 (C-1″), 130.6 (C-5),
34.5 (C-2″ C-6″), 139.9 (C-3), 142.9 (C-6), 143.3 (C-2), 149.2 (C-
), 163.9 (C-4″), 169.5 (C-7), 170.1 (C-7″); HRESIMS m/z 376.1030
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