Formation of 8-S-L-cysteinylguanosine from 8-bromoguanosine and cysteine

Article abstract of DOI:10.1016/j.bmcl.2013.04.084

When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37 C, a previously unidentified product was formed as a major product in addition to guanosine. The product was identified as a cysteine substitution derivative of guanosine at the 8 position, 8-S-l-cysteinylguanosine. The reaction was accelerated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposed with a half-life of 193 h at pH 7.4 and 37 C. Similar results were observed for incubation of 8-bromo-2′-deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides, RNA, and DNA can react with thiols resulting in stable adducts.

Full text of DOI:10.1016/j.bmcl.2013.04.084

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3864–3867
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Formation of 8-S-L-cysteinylguanosine from 8-bromoguanosine
and cysteine q
⇑
Toshinori Suzuki , Aya Kosaka, Michiyo Inukai
School of Pharmacy, Shujitsu University, Okayama 703-8516, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
When 8-bromoguanosine was incubated with cysteine at pH 7.4 and 37 °C, a previously unidentified
product was formed as a major product in addition to guanosine. The product was identified as a cysteine
substitution derivative of guanosine at the 8 position, 8-S-L-cysteinylguanosine. The reaction was accel-
erated under mildly basic conditions. The cysteine adduct of guanosine was fairly stable and decomposed
with a half-life of 193 h at pH 7.4 and 37 °C. Similar results were observed for incubation of 8-bromo-20-
deoxyguanosine with cysteine. The results suggest that 8-bromoguanine in nucleosides, nucleotides,
RNA, and DNA can react with thiols resulting in stable adducts.
Received 9 March 2013
Revised 23 April 2013
Accepted 26 April 2013
Available online 9 May 2013
Keywords:
8-Bromoguanosine
Cysteine
Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
8-S-L-cysteinylguanosine
Guanosine
Eosinophil peroxidase (EPO) plays an important role in defense
one order of magnitude lower than that of 8-oxo-7,8-dihydroxy-
20-deoxyguanosine (8-oxo-dGuo).10 In diabetic patients, urinary
8-Br-dGuo and 8-Cl-dGuo levels were eightfold higher than the
levels in healthy volunteers, whereas there was a small increase
in 8-oxo-dGuo. This implies that 8-Br-dGuo exists in healthy hu-
mans and that inflammatory diseases greatly increase its level.
Thus, the importance of 8-Br-dGuo is becoming clear. However,
there is little information available about the reaction of 8-Br-dGuo
with intracellular molecules. In the present study, we investigated
mechanisms against parasites mediated by the oxidation of Brꢀ.1
EPO generates a reactive species hypobromous acid (HOBr) from
H2O2 and Brꢀ. An EPO/H2O2/Brꢀ system in the presence of a plasma
concentration of Clꢀ can react with nucleosides to form
brominated nucleosides including 8-bromo-20-deoxyguanosine
(8-Br-dGuo).2–4 Meanwhile, myeloperoxidase (MPO), an enzyme
secreted from neutrophil and monocytic cells, generates hypochlo-
rous acid (HOCl) from H2O2 and Clꢀ.5,6 The formed HOCl is also of
central importance in host defense mechanisms. MPO can generate
chlorinated nucleosides including 8-chloro-20-deoxyguanosine (8-
Cl-dGuo).7 It has been reported that an MPO/H2O2/Clꢀ system in
the presence of a plasma concentration of Brꢀ also generates bro-
minated nucleosides.8 HOCl formed in the MPO system would re-
act with Brꢀ, generating a brominating reagent, HOBr. An in vitro
DNA replication study showed that human DNA polymerases
incorporated 20-deoxyguanosine-50-monophosphate (dGMP), 20-
deoxyadenosine-50-monophosphate (dAMP), and 20-deoxythymi-
dine-50-monophosphate (dTMP) in addition to one-base deletion
opposite to an 8-Br-dGuo residue in an oligodeoxynucleotide, sug-
gesting that 8-Br-dGuo in DNA is a mutagenic lesion.9 Recently,
8-Br-dGuo was detected in urine from healthy volunteers with a
similar concentration of 8-Cl-dGuo, while the concentrations are
the reaction of 8-bromoguanosine (8-Br-Guo) with
and report identification of the products.
L-cysteine (Cys)
A solution of 100 lM 8-Br-Guo (Santa Cruz Biotechnology, TX,
USA) and 50 mM Cys (Sigma, MO, USA) was incubated in
100 mM potassium phosphate buffer at pH 7.4 and 37 °C for 48 h
in the dark. When the reaction mixture was analyzed by reversed
phase (RP) HPLC, two product peaks appeared in the chromato-
gram (Fig. 1). The products were collected and subjected to spec-
trometric measurements. The product eluted at the retention
time of 6.5 min showed a UV spectrum with kmax = 252 nm and
an ESI-TOF/MS spectrum with m/z = 282 in the negative mode.
The product was identified as guanosine (Guo) by coincidence of
the RP-HPLC retention time and UV and MS spectra of an authentic
Guo. The product eluted at the retention time of 7.7 min showed a
UV spectrum with kmax = 272 nm (Fig. 1, inset). An ESI-TOF/MS
spectrum showed m/z = 401 and 314 in the negative mode
(Fig. 2). High-resolution ESI-TOF/MS (negative) of the molecular
ion showed m/z = 401.088576, which agreed with the theoretical
molecular mass for C13H17N6O7S composition within 1 ppm. 1H-
NMR showed no aromatic proton signal and three aliphatic proton
signals in addition to six aliphatic ribose proton signals. 13C-NMR
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
⇑
Corresponding author. Tel.: +81 86 271 8346; fax: +81 86 271 8320.
E-mail address: tsuzuki@shujitsu.ac.jp (T. Suzuki).
0960-894X/$ - see front matter Ó 2013 The Authors. Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.084

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3864–3867
3865
B
A
Cys
100
80
60
40
20
0
100
80
200
300
400
8-Br-Guo
Wavelength (nm)
Cys-Guo
Guo
60
40
20
0
10
20
30
0
Time (min)
0
25
50
75
100
0
24
48
72
96
Time (h)
Cys (mM)
Figure 1. RP-HPLC chromatogram of a reaction mixture of 8-Br-Guo with Cys
detected at 260 nm. The inset is the on-line UV spectrum of Cys-Guo. A solution of
100 lM 8-Br-Guo and 50 mM Cys was incubated in 100 mM potassium phosphate
buffer at pH 7.4 and 37 °C for 48 h in the dark. The HPLC system consisted of
Shimadzu LC-10ADvp pumps and an SCL-10Avp system controller. On-line UV
spectra were obtained with a Shimadzu SPD-M10Avp UV–vis photodiode-array
detector. For the RP-HPLC, an Inertsil ODS-3 octadecylsilane column of
Figure 4. (A) The time course of the concentration changes in 8-Br-Guo (circle),
Cys-Guo (square), and Guo (triangle) when a solution of 100 M 8-Br-Guo and
50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 7.4 and
37 °C for 0–96 h in the dark. (B) The Cys dose dependence of the concentration
changes in 8-Br-Guo (circle), Cys-Guo (square), and Guo (triangle) when a solution
of 100 lM 8-Br-Guo and 0–100 mM Cys was incubated in 100 mM potassium
phosphate buffer at pH 7.4 and 37 °C for 48 h in the dark. The concentration was
determined by RP-HPLC. Means S.D. (n = 3) are presented.
l
4.6 ꢁ 250 mm and particle size 5
lm (GL Science, Tokyo) was used. The eluent
was 20 mM ammonium acetate (pH 7.0) containing 10% methanol. The column
temperature was 40 °C and the flow rate was 1 mL/min.
48 h. The concentrations of the products increased with increasing
Cys dose.
401
100
Figure 5 shows the pH dependence of the concentrations of the
products and 8-Br-Guo. Consumption of 8-Br-Guo increased with
increasing pH value of the solution from pH 7 to pH 8. The concen-
tration of Cys-Guo increased with increasing pH from 7 to 8. How-
ever, the concentration of Guo gradually decreased as the pH
increased.
314
50
Figure 6 shows the stability of Cys-Guo. 100 lM Cys-Guo was
incubated in 100 mM potassium phosphate buffer of pH 3.0, 7.4,
0
200 300 400 500 600
Figure 2. Negative ion electrospray ionization time of flight mass spectrometry
(ESI-TOF/MS, MicroTOF, Bruker) spectrum of 8-S-L-cysteinylguanosine (Cys-Guo).
100
80
60
40
20
0
showed five aromatic carbon signals and seven aliphatic carbon
signals with a carboxyl carbon signal (171.0 ppm). Combining
these data, the product was identified as a Cys-substituted deriva-
tive of Guo at the 8 position, (Cys-Guo).11 The structures of the
products are shown in Figure 3. Concentrations of the products
were 15.6 0.2
74.3 0.6
M of unreacted 8-Br-Guo.12 As a control, a similar
experiment was conducted for glycine (Gly). Incubation of a solu-
tion of 100 M 8-Br-Guo with 50 mM Gly at pH 7.4 and 37 °C for
lM for Cys-Guo and 8.9 0.2 lM for Guo with
3
4
5
6
7
8 9 10 11
l
pH
Figure 5. The pH dependence of the concentration changes in 8-Br-Guo (circle),
Cys-Guo (square), and Guo (triangle) when a solution of 100 M 8-Br-Guo and
50 mM Cys was incubated in 100 mM potassium phosphate buffer at pH 3–11 and
37 °C for 48 h in the dark. The concentration was determined by RP-HPLC.
Means S.D. (n = 3) are presented.
l
l
up to 120 h showed no product and no consumption of 8-Br-Guo.
Figure 4A shows the time-dependent changes in concentrations
of Cys-Guo, Guo, and 8-Br-Guo, when 100 lM 8-Br-Guo and
50 mM Cys were incubated in 100 mM potassium phosphate buffer
at pH 7.4 and 37 °C for 0–96 h. The concentrations of Cys-Guo and
Guo increased with increasing incubation time. Cys-Guo formed
with a higher concentration than Guo. Figure 4B shows the Cys
dose-dependence of concentrations of the products and 8-Br-
100
80
60
40
20
0
Guo, when 100
lM 8-Br-Guo and 0–100 mM Cys were incubated
in 100 mM potassium phosphate buffer at pH 7.4 and 37 °C for
O
N
O
N
O
N
0
24
48
72
96
H N
2
N
N
N
Cys
Time (h)
NH
NH2
NH
NH2
NH
NH2
S
Br
+
HO
O
N
N
N
Figure 6. The time course of the concentration changes in Cys-Guo at pH 3.0
(circle), pH 7.4 (triangle), and pH 8.5 (square) when a solution of isolated 100
Cys-Guo was incubated in 100 mM potassium phosphate buffer at pH 3.0, 7.4, or 8.5
and 37 °C for up to 96 h in the dark. The concentration was determined by RP-HPLC.
Means S.D. (n = 3) are presented.
R
R
R
lM
8-Br-Guo
Cys-Guo
Guo
Figure 3. Reaction of 8-Br-Guo with Cys. R denotes ribose.

3866
T. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3864–3867
O
N
O
N
O
H
Cys
N
N
N
−
+
+Cys-SH
NH
NH2
NH
NH2
NH
NH2
−Br , −H
S
Br
Cys
S
Br
N
N
N
N
R
R
R
8-Br-Guo
Cys-SH
Cys-Guo
O
N
NH
NH2
N
N
R
Guo
Scheme 1. Proposed reaction pathways for the reaction of 8-Br-Guo with Cys.
and 8.5 at 37 °C for up to 96 h. At pH 3.0, the concentration of Cys-
Guo did not change. At pH 7.4, Cys-Guo decreased time-depen-
dently with a half-life of 193 h. Although several products were de-
tected in the RP-HPLC chromatogram, Guo did not form. At pH 8.5,
the half-life was 244 h. As a control, 8-Br-Guo was incubated under
the same conditions, but no decomposition was observed.
To obtain information about the reaction of an 8-bromoguanine
moiety in deoxyribonucleoside with Cys, similar experiments were
conducted for 8-bromo-20-deoxyguanosine (8-Br-dGuo). When a
Cys-Guo was relatively stable (Fig. 5). If Cys adducts at guanine
C8 were formed in RNA and DNA, they would remain for a long
period. Although the serum concentration of Cys is low (34 lM),
the intracellular level of glutathione (GSH, pKa = 8.8) in mamma-
lian cells is in the millimolar range (0.5–10 mM).17,18 GSH may also
react with 8-bromoguanine, generating stable products as well as
free Cys at guanine C8 in RNA and DNA. These thiol adducts at
C8 of guanine can exert an influence on RNA and DNA functions
different from that of 8-bromoguanine. Recently, a new signal
transduction mechanism termed protein S-guanylation was re-
vealed.19 8-Nitroguanosine-30,50-cyclic monophosphate (8-NO2-
cGMP) reacts with cysteine thiols of a protein Keap1, generating
cGMP adduct of the protein. The formation of S-guanylation regu-
lated the redox-sensor signaling protein. The results of the present
study suggest that 8-bromoguanosine-30,50-cyclic monophosphate
(8-Br-cGMP), either generated by endogenously-formed HOBr
from guanosine-30,50-cyclic monophosphate (cGMP) or added
exogenously as a reagent, may also react partially with cysteine
thiols of proteins resulting in cGMP adducts as well as 8-NO2-
cGMP. The reaction of 8-bromoguanine nucleosides with Cys may
be slower than that of 8-nitroguanine nucleosides with Cys. How-
ever, 8-bromoguanine nucleosides are relatively stable, whereas 8-
nitroguanine nucleosides are labile for N-glycosidic bond, resulting
in depurination, with half-lives of 3 min for 8-nitro-20-deoxygua-
nosine and 5 h for 8-nitroguanosine at pH 7 and 37 °C.20,21 Thus,
8-bromoguanine would stay in nucleosides and nucleic acids for
a long period and can react with thiols resulting in adducts.
The present results show that relatively stable Cys adducts can
form from 8-bromoguanine nucleosides under neutral conditions.
It suggests that these adducts may have some importance in eluci-
dating the influence of 8-bromoguanine in nucleosides, nucleo-
tides, RNA and DNA in genotoxicity and signal transduction.
solution of 100 lM 8-Br-dGuo (Santa Cruz Biotechnology) and
50 mM Cys was incubated in 100 mM potassium phosphate buffer
at pH 7.4 and 37 °C in the dark, 8-S-L-cysteinyl-20-deoxyguanosine
(Cys-dGuo)13 and dGuo were generated. At an incubation time of
48 h, the concentrations of the products were 20.1 1.3
l
M for
Cys-dGuo and 9.5 0.7
lM for dGuo with 66.2 2.0 lM of unre-
acted 8-Br-dGuo.14
To obtain information about the reaction mechanism generat-
ing Guo, two experiments were conducted. 100 M Cys-Guo was
l
incubated with 50 mM Cys in 100 mM potassium phosphate buffer
(pH 7.4) at 37 °C for up to 72 h and the reaction mixture was ana-
lyzed by RP-HPLC. The concentration of Cys-Guo decreased, but
Guo did not form. It suggests that Cys does not catalyze the forma-
tion of Guo from Cys-Guo. 100 lM 8-Br-Guo was incubated with
50 mM NaBH4, a reducing agent, in 100 mM potassium phosphate
buffer (pH 7.4) at room temperature for 1 h. However, the concen-
tration of 8-Br-Guo did not decrease and Guo was not detected in
the RP-HPLC chromatogram.
It has been reported that 5-bromo-20-deoxyuridine (5-Br-dUrd)
reacts with Cys generating a Cys adduct, 5-S-L
-cysteinyl-20-deoxy-
uridine (Cys-dUrd), and a debromination product, 20-deoxyuridine
(dUrd).15 The dUrd formation in the 5-Br-dUrd/Cys reaction was
maximal at pH 8, and the Cys-dUrd formation was most favorable
at pH 9. The reaction was very slow below pH 5 and above pH 10.
The pH profile for the product yields is quite different from that of
the present 8-Br-Guo/Cys reaction, suggesting that a different reac-
tion mechanism exists. In the reaction of 8-Br-Guo with Cys, the
yield of Cys-Guo increased with increasing pH with a sigmoidal
profile (Fig. 4). The pKa of Cys is 8.3 for the thiol group.16 At mildly
acidic pH, a simple nucleophilic attack of the thiol group (–SH) of
Cys to C8 of 8-Br-Guo and subsequent elimination of Brꢀ and H+
would generate Cys-Guo. The thiolate (–Sꢀ) of Cys formed at mildly
basic pH attacks the C8 atom more efficiently. In contrast, the yield
of Guo gradually decreased with increasing pH. For the formation
of Guo, the pathway via Cys-Guo was ruled out. Meanwhile, Guo
was not generated from 8-Br-Guo by NaBH4. The reaction mecha-
nism for the formation of Guo from 8-Br-Guo by Cys is unclear.
The possible reaction pathways are summarized in Scheme 1.
References and notes
1. Weiss, S. J.; Test, S. T.; Eckmann, C. M.; Roos, D.; Regiani, S. Science 1986, 234,
200.
2. Henderson, J. P.; Byun, J.; Williams, M. V.; McCormick, M. L.; Parks, W. C.;
Ridnour, L. A.; Heinecke, J. W. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 1631.
3. Shen, Z.; Mitra, S. N.; Wu, W.; Chen, Y.; Yang, Y.; Qin, J.; Hazen, S. L. Biochemistry
2001, 40, 2041.
4. Henderson, J. P.; Byun, J.; Mueller, D. M.; Heinecke, J. W. Biochemistry 2001, 40,
2052.
5. Schultz, J.; Kaminker, K. Arch. Biochem. Biophys. 1962, 96, 465.
6. Kettle, A. J.; Winterbourn, C. C. Redox Rep. 1997, 3, 3.
7. Masuda, M.; Suzuki, T.; Friesen, M. D.; Ravanat, J.-L.; Cadet, J.; Pignatelli, B.;
Nishino, H.; Ohshima, H. J. Biol. Chem. 2001, 276, 40486.
8. Henderson, J. P.; Byun, J.; Williams, M. V.; Mueller, D. M.; McCormick, M. L.;
Heinecke, J. W. J. Biol. Chem. 2001, 276, 7867.
9. Sassa, A.; Ohta, T.; Nohmi, T.; Honma, M.; Yasui, M. J. Mol. Biol. 2011, 406, 679.

T. Suzuki et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3864–3867
3867
10. Asahi, T.; Kondo, H.; Masuda, M.; Nishino, H.; Aratani, Y.; Naito, Y.; Yoshikawa,
13C NMR (125 MHz, DMSO-d6): d (ppm/TMS) 170.0 (COOH), 155.8 (C-6), 153.3
T.; Hisaka, S.; Kato, Y.; Osawa, T. J. Biol. Chem. 2010, 285, 9282.
(C-2), 153.3 (C-4), 142.8 (C-8), 116.9 (C-5), 87.6 (C-40), 83.5 (C-10), 71.0 (C-30),
11. Spectrometric data of 8-S-
L
-cysteinylguanosine (Cys-Guo). 1H NMR (500 MHz,
62.0 (C-50), 53.9 (C- ), 36.7 (C-20), 35.3 (C-b). ESI-TOF/MS (negative ion): m/z
a
DMSO-d6): d (ppm/TMS) 6.92 (s, 2H, NH2), 5.73 (d, J = 6.3 Hz, 1H, H-10), 4.89
385 [MꢀH]ꢀ. HR-ESI-TOF/MS (negative ion): m/z 385.092824 [MꢀH]ꢀ
(dd, 1H, H-20), 4.13 (dd, 1H, H-30), 3.85 (ddd, J = 4.6 Hz, 1H, H-40), 3.71 (ddd, 1H,
(calculated for C13H17N6O6S 385.093577). UV: kmax 272 nm (pH 7.0).
H-b), 3.67 (ddd, 1H, H-50 or 500), 3.59 (ddd, 1H, H-
a
), 3.53 (ddd, 1H, H-50 or 500),
(ppm/TMS) 171.0
(COOH), 156.1 (C-6), 153.5 (C-2), 152.5 (C-4), 142.8 (C-8), 116.8 (C-5), 88.2 (C-
14. The concentrations of the products were evaluated from integrated peak areas
on RP-HPLC chromatograms detected at 260 nm and the molecular extinction
3.29 (ddd, 1H, H-b). 13C NMR (125 MHz, DMSO-d6):
d
coefficients at 260 nm (e260 nm). The e260
values of the authentic samples
nm
10), 85.6 (C-40), 70.6 (C-20), 70.5 (C-30), 62.0 (C-50), 53.9 (C-
a
), 35.3 (C-b). ESI-
were determined from integration of the H30 proton signal of NMR and the
TOF/MS (negative ion): m/z 401 [MꢀH]ꢀ, 314 [MꢀHꢀ Cys]ꢀ. HR-ESI-TOF/MS
(negative ion): m/z 401.088576 [MꢀH]ꢀ (calculated for C13H17N6O7S,
401.088491). UV: kmax 272 nm (pH 7.0).
HPLC peak area detected at 260 nm relative to those of dGuo (e260
nm = 11,800 Mꢀ1 cmꢀ1) in the mixed solution. The estimated e260
values
nm
were 16,100 Mꢀ1 cmꢀ1 for 8-Br-dGuo and 15,400 Mꢀ1 cmꢀ1 for Cys-dGuo.
15. Wataya, Y.; Negishi, K.; Hayatsu, H. Biochemistry 1973, 12, 3992.
16. Wang, K.; Wen, Z.; Zhang, W.; Xian, M.; Cheng, J. P.; Wang, P. G. Bioorg. Med.
Chem. Lett. 2001, 11, 433.
12. The concentrations of the products were evaluated from integrated peak areas
on RP-HPLC chromatograms detected at 260 nm and the molecular extinction
coefficients at 260 nm (e260 nm). The e260
values of the authentic samples
nm
were determined from integration of the H30 proton signal of NMR and the
17. Psychogios, N.; Hau, D. D.; Peng, J.; Guo, A. C.; Mandal, R., et al PLoS One 2011, 6,
e16957.
18. Meister, A.; Anderson, M. E. Ann. Rev. Biochem. 1983, 52, 711.
19. Sawa, T.; Zaki, M. H.; Okamoto, T.; Akuta, T.; Tokutomi, Y.; Kim-Mitsuyama, S.;
Ihara, H.; Kobayashi, A.; Yamamoto, M.; Fujii, S.; Arimoto, H.; Akaike, T. Nat.
Chem. Biol. 2007, 3, 727.
20. Yermilov, V.; Rubio, J.; Becchi, M.; Friesen, M. D.; Pignatelli, B.; Ohshima, H.
Carcinogenesis 1995, 16, 2045.
21. Sodom, R. S.; Fiala, E. S. Chem. Res. Toxicol. 2001, 14, 438.
HPLC peak area detected at 260 nm relative to those of Guo
(e260
values
nm = 11,500 Mꢀ1 cmꢀ1) in the mixed solution. The estimated e260
nm
were 16,000 Mꢀ1 cmꢀ1 for 8-Br-Guo and 15,000 Mꢀ1 cmꢀ1 for Cys-Guo.
13. Spectrometric data of 8-S-L
-cysteinyl-20-deoxyguanosine (Cys-dGuo). 1H NMR
(500 MHz, DMSO-d6): d (ppm/TMS) 6.78 (s, 2H, NH2), 6.16 (dd, J = 6.9 and
8.0 Hz, 1H, H-10), 4.36 (dd, J = 2.9 Hz, 1H, H-30), 3.78 (ddd, 1H, H-40), 3.68 (ddd,
1H, H-b), 3.62 (ddd, 1H, H-50 or 500), 3.53 (dd, 1H, H- ), 3.50 (ddd, 1H, H-50 or
a
500), 3.26 (ddd, 1H, H-b), 3.26 (ddd, 1H, H-20 or 200), 2.04 (ddd, 1H, H-20 or 200).