First total synthesis of 5'-O-α-d-glucopyranosyl tubercidin

Article abstract of DOI:10.3390/MD18080398

The first total synthesis of 5⁰-O-α-d-glucopyranosyl tubercidin was successfully developed. It is a structurally unique disaccharide 7-deazapurine nucleoside exhibiting fungicidal activity, and was isolated from blue-green algae. The total synt

Full text of DOI:10.3390/MD18080398

marine drugs
Communication
First Total Synthesis of
50-O-α-d-Glucopyranosyl Tubercidin
Wenliang Ouyang, Haiyang Huang * , Ruchun Yang, Haixin Ding and Qiang Xiao *
Jiangxi Key Laboratory of Organic Chemistry, Institute of Organic Chemistry, Jiangxi Science and Technology
Normal University, Nanchang 330013, China; ouyangwl93@yeah.net (W.O.); ouyangruchun@163.com (R.Y.);
dinghaixin_2010@163.com (H.D.)
* Correspondence: huanghaiyang1209@163.com (H.H.); xiaoqiang@tsinghua.org.cn (Q.X.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 July 2020; Accepted: 28 July 2020; Published: 29 July 2020
Abstract: The first total synthesis of 50-O-
α-d-glucopyranosyl tubercidin was successfully developed.
It is a structurally unique disaccharide 7-deazapurine nucleoside exhibiting fungicidal activity,
and was isolated from blue-green algae. The total synthesis was accomplished in eight steps
with 27% overall yield from commercially available 1-O-acetyl-2,3, 5-tri-O-benzoyl-
The key step involves stereoselective
-O-glycosylation of the corresponding 7-bromo-6-chloro-20,
30-O-isopropylidene-
-d-tubercidin with 2,3,4,6-tetra-O-benzyl-glucopyranosyl trichloroacetimidate.
β-d-ribose.
α
β
All spectra are in accordance with the reported data for natural 50-O-
α-d-glucopyranosyl tubercidin.
Meanwhile, 50-O-β-d-glucopyranosyl tubercidin was also prepared using the same strategy.
Keywords: total synthesis; natural product; 7-deazapurine nucleoside; disaccharide nucleoside;
tubercidin
1. Introduction
As the first isolated naturally occurring 7-deazapurine nucleoside, tubercidin (Figure 1, 1) is
a biogenic analogue of adenosine, in which the N-7 atom is replaced by CH (purine numbering is
used throughout this work) [1–3]. It showed significant cytostatic activity in various cancer cell lines.
Although the application of tubercidin in clinical trials has been attempted, its use was eventually halted
because of significant toxicity [
4
–
7]. Since then, 7-deazapurine has been recognized as a privileged
scaffold in developing new antitumor and antiviral nucleosides [8]. Consequently, many 7-deazapurine
nucleosides have been synthesized and their biological activities have been screened in the past
50 years [9–11]. Among them, several promising lead compounds have been discovered [12–14].
OH
NH2
N
NH2
N
O
7
HO
HO
1
N
N
OH
N
HO
N
3
O
5’
O
O
OH OH
OH OH
1
2
Figure 1. Chemical structures of tubercidin (1) and 50-O-α-d-glucopyranosyl tubercidin (2).
50-O-
in 1988 (Figure 1,
α
-d-Glucopyranosyl tubercidin 1 is a disaccharide nucleoside isolated from blue-green algae
) [15]. It is characterized by a unique 50-O-
-d-glucopyranosyl moiety attached
2
α
to tubercidin. Preliminary biological evaluation indicated that it exhibited moderate cytotoxicity
Mar. Drugs 2020, 18, 398; doi:10.3390/md18080398
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and fungicidal activity. Although many disaccharide nucleosides have been identified as naturally
occurring products [16,17], disaccharide 7-deazapurine nucleosides are rarely reported.
Our group has a long-standing interest in total synthesis and biological activity evaluation of
naturally occurring 7-deazapurine nucleosides and their biological activities [18
–22]. Because of
the unique disaccharide structure and a great need for structure-based biological study, structural
confirmation of 50-O-
α-d-glucopyranosyl tubercidin 2 and synthesis of its analogues in a concise and
modular fashion are preferred. Herein, we report the first total synthesis of 50-O-
tubercidin 1.
α-d-glucopyranosyl
Our retrosynthetic analysis is depicted in Scheme 1. From a synthetic point of view, there are two
possible approaches. The first approach is Vorbrüggen glycosylation of disaccharide with nucleobase
directly (Scheme 1, Path a). Because of the labile -O-glycosylic bond of disaccharide , the isomerization
might occur during Vorbrüggen glycosylation. The second approach is postglycosylation of properly
protected nucleoside with glycosyl donor (Scheme 1, Path b). The postglycosylation approach
has been widely used for synthesizing disaccharide nucleosides [23 25]. Considering that a modular
3
4
α
3
5
6
–
strategy is needed, we employed the postglycosylation approach herein.
OBn
Cl
X
NH
CCl3
O
N
BnO
BnO
O
N
N
OBn
H
4
6
OH
NH2
O
Path b
Path a
HO
HO
OAc
Cl
N
N
X
OH
O
N
N
N
AcO
AcO
O
O
N
OAc
HO
O
O
O
OAc
OH OH
O
O
2
OAc OAc
3
5
Scheme 1. Retrosynthetic analysis of 50-O-α-d-glucopyranosyl tubercidin 2.
2. Results
First, 7-deazpurine nucleoside
and coworkers (Scheme 2) [26 29].
9
was synthesized based on a previous report by Seela
After silylation of 6-chloro-7-bromo-7-deazapurine
–
8
with N,O-bis(trimethylsilyl)acetamide (BSA) in freshly distilled acetonitrile (CH3CN),
1-O-acetyl-2,3,5-tri-O-benzoyl- -d-ribose (2 eq.) was added followed by TMSOTf (3 eq.) in one pot.
Then, the resulting reaction mixture was heated at 80 ◦C for 8 h to afford 7-deazpurine nucleoside
β
7
9
with a 76% yield. The 7-bromo substitute is critical for the above Vorbrüggen glycosylation, otherwise
the reaction cannot proceed. The reason may be that the 7-bromo substituent can reduce the reactivity
of N-9, which results in the deazapurine being more purine-like [20,26–29].
Then, all benzoyl groups of nucleoside 9
were removed in saturated ammonia in methanol at 0 ◦C
to give nucleoside 10 in a 94% yield. It should be noted that the C-6 chloride was retained to avoid
extra manipulation of protecting groups in the following steps. Next, p-toluenesulfonic acid-catalyzed
reaction of nucleoside 10 with 2, 2-dimethoxy propane in acetone afforded isopropylidene-protected
nucleoside 11 with a 98% yield, which left the 50-OH free for further glycosylation.
With nucleoside 11 in hand, we focused on carrying out the key glycosylation reaction. In general,
1,2-trans
donor, which is very reliable and highly stereoselective [29]. In contrast, the construction of our desired
1,2-cis -O-glucoside is much more challenging. It requires glycosyl donors having nonassisting
functionality at C-2 position, and even so, the reaction normally produces a mixture of
isomers [30 32]. In this work, we chose 2,3,4,6-tetra-O-benzyl-glucopyranosyl trichloroacetimidate
β-O-glucoside can be prepared by the neighboring group participation of the 2-O-acyl glucose
α
α
and
β
–
6
as

Mar. Drugs 2020, 18, 398
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the glucosyl donor, which was synthesized from 2,3,4,6-tetra-O-benzyl glucose with trichloroacetonitrile
in dichloromethane (DCM) using anhydrous potassium carbonate as catalysis (98% yield,
β ≈
1:4) [33 34]. After optimization (Supplementary Materials), TMSOTf catalyzed glycosylation of
nucleoside 11 with glycosyl donor at isomer 12 and isomer 13 (4:1) in
30 ◦C gave a mixture of
79% overall yield. Fortunately, they can be separated by careful silica gel chromatography to afford the
α
:
,
6
−
α
β
desired nucleoside 12 with
the J1”-2” coupling constant (
α glycosylic configuration. These two isomers can be clearly identified by
α
isomer 3.5 Hz,
β
isomer 8.0 Hz) and 13C chemical shift of C1” (
α
isomer
96.4 ppm, β isomer 102.9 ppm).
Cl
N
Br
Cl
N
Cl
N
Br
Br
N
N
Cl
N
Br
N
BzO
N
OAc
HO
N
O
BzO
b
HO
a
N
O
c
O
O
N
N
OBz OBz
O
O
H
OBz OBz
OH OH
9
7
8
11
10
Cl
Br
OBn
OBn
Cl
N
N
OBn
Br
O
O
NH
BnO
BnO
N
d
N
BnO
BnO
N
O
O
BnO
BnO
OBn
O
CCl3
O
N
OBn
O
OBn
O
O
O
O
O
6
13
12
e
OBn
OBn
OBn
NH2
N
Cl
N
NH2
N
Br
Br
O
OBn
O
O
BnO
BnO
BnO
BnO
BnO
BnO
g
N
f
OBn
N
OBn
N
N
N
N
O
O
O
O
O
O
OH OH
OH OH
OH OH
16
14
15
OH
O
NH2
N
NH2
N
OH
HO
HO
h
O
N
OH
HO
HO
N
N
N
O
O
O
OH
O
OH OH
OH OH
2
17
Scheme 2. Total synthesis of 50-O-
) bis(trimethylsilyl)acetamide (BSA), TMSOTf, CH3CN, 80 C, 1 h, 75%; (
12 h, 94%; ( ) 2,2-dimethoxy propane, p-toluenesulfonic acid, acetone, rt., 4 h, 98%; (
CH2Cl2, , 4:1); ( ) NH3, MeOH, 130
20 ◦C, 4 h, 79% ( ) 80% aqueous acetic acid, 50 ◦C, 12 h, 85%; (
◦C, 12 h, 95%; (
) H2, 10% Pd/C, Et3N, THF/MeOH, rt. 5 h, 83.5%; ( ) H2, 20% Pd(OH)2/C, THF/MeOH,
40 ◦C, 12 h, 80%.
α-d-glucopyranosyl tubercidin 2. Reagents and conditions:
◦
◦
(
a
b
) NH3, MeOH, 0 C,
c
d
) TMSOTf,
−
α
:
β
e
f
g
h
Next, the isopropylidene-protecting group was removed in 80% aqueous acetic acid to give
nucleoside 14 with an 85% yield. Then, the substitution of C-6 chloride with freshly prepared saturated
ammonium in methanol at 130 ◦C afforded nucleoside 15 with a 95% yield. Then, we tried to remove
the C-7 bromide atom and all benzyl-protecting groups by hydrogenation with 10% Pd/C at the same

Mar. Drugs 2020, 18, 398
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time. However, the presence of triethylamine in the reaction due to the need to neutralize HBr, which is
generated through bromide reduction, prevented the removal of the benzyl group. Only bromide was
reduced, to afford nucleoside 16 in 84% yield. Finally, further hydrogenation with 20% Pd(OH)2/C
gave 50-O-
reported data of the authentic naturally occurring product.
Since the corresponding 50-O-
-d-glucopyranosyl tubercidin has not been previously
reported, its synthesis provides more evidence for structural elucidation and biological activity.
50-O-
-d-Glucopyranosyl tubercidin 17 was also prepared according to a similar procedure for the
α-d-glucopyranosyl tubercidin 2 with 80% yield. All spectra were in accordance with the
β
β
synthesis of nucleoside 2.
3. Materials and Methods
All reagents were purchased from commercial sources and used without purification unless
specified. Acetonitrile, pyridine and CH2Cl2 were refluxed with CaH2 and distilled prior to use.
THF was dried with LiAlH4 and distilled prior to use. Thin-layer chromatography was performed
using silica gel GF-254 plates (Qingdao Chemical Company, Qingdao) detected by UV (254 nm) or
charting with 10% sulfuric acid in ethanol. Column chromatography was performed on silica gel
(200–300 mesh, Qingdao Chemical Company, Qingdao). NMR spectra were recorded on a Bruker
AV400 spectrometer. Chemical shifts (δ) are reported in ppm and coupling constants (J) are reported in
Hz. Then, 1H and 13C NMR spectra were calibrated with TMS as an internal standard. The specific
rotation was measured on a Rudolph autopol IV polarimeter. ESI-MS was acquired with a Bruker
Dalton microTOFQ II spectrometer.
3.1. H NMR, 13C NMR and HRMS of 2,3,4,6-O-Tetrabenzyl-d-glucopyranose
Rf = 0.32 (PE/EA = 3:1); mp 153–155 ◦C; 1H NMR (400 MHz, CDCl3):
7.18–7.12 (m, 2H, Bn), 5.31–5.19 (m, 1H, H-1), 4.98–4.48 (m, 8H, 4CH2), 4.09–3.94 (m, 2H, H-4,5),
3.74–3.58 (m, 4H, H-2,3,6), 3.06 (d, J = 2.2 Hz, 1H, 1-OH); 13C NMR (101 MHz, CDCl3):
138.7 (C), 138.2
δ 7.36–7.28 (m, 18H, Bn),
δ
(C), 137.8 (2C), 128.5 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.7 (CH), 127.6
(CH), 91.3 (CH-1), 81.7 (CH-5), 80.0 (CH-2), 77.7 (CH-3), 75.7 (CH2), 75.0 (CH2), 73.5 (CH2), 73.3 (CH2),
70.3 (CH-4), 68.6 (CH2-6); HRMS Calcd. For C34H36O6Na+ [M + Na]+: 563.2404, found: 563.2410.
3.2. Synthesis of 2,3,4,6-Tetra-O-benzyl-glucopyranosyl Trichloroacetimidate (6)
To the suspension of anhydrous K2CO3 (10 g, 101 mmol) in dry dichloromethane (100 mL) was
added 2,3,4,6-O-tetrabenzyl-d-glucopyranose (10 g, 18.5 mmol) and trichloroacetonitrile (11.3 mL,
112 mmol) sequentially. The mixture was stirred at room temperature for 4 h until the reaction was
completed (monitored by TLC, PE/EA = 6:1, Rf = 0.21). After evaporation of the solvent under
reduced pressure, the crude product was directly purified by flash column chromatography on silica
gel (Et3N/PE/EA = 0.1:3:1) to provide 12.4 g compound
6
as a white solid (98% yield,
-NH), 8.60 (s, 0.2H, -NH), 7.32 (m, 18H),
-H-1), 5.84 (d, J = 6.1 Hz, 0.8H, -H-1), 5.01–4.46 (m, 9H), 3.81–3.76
(m, 4H), 3.67 (d, J = 4.6 Hz, 1H); 13C-NMR (101 MHz, CDCl3):
161.2 (C), 138.4 (C), 138.1 (C), 138.0 (C),
α
:
β ≈ 1:4); mp:
◦
1
77–79 C; H NMR (400 MHz, CDCl3):
7.20 (m, 2H), 6.55 (d, J = 3.2 Hz, 0.2H,
δ 8.73 (s, 0.8H, β
α
β
δ
138.0 (C), 128.5 (CH), 128.4 (CH), 128.4 (CH), 128.4 (CH), 128.1 (CH), 128.0 (CH), 128.0 (CH), 127.9 (CH),
127.8 (CH), 127.7 (CH), 127.6 (CH), 127.6 (CH), 98.4 (CH), 84.6 (CH), 81.0 (CH), 76.8 (CH), 75.9 (CH2),
75.6 (CH2), 75.0 (CH2), 74.9 (CH2), 73.4 (CH), 68.2 (CH); HRMS Calcd. For C36H36Cl3NO6Na+ [M +
Na]+: 706.1500, found: 706.1509.
3.3. Synthesis of 7-Bromo-6-chloro-pyrrole [2,3-d]pyrimidine (8)
To the stirred suspension of 6-chloro-pyrrole[2,3-d]pyrimidine (12.0 g, 78.2 mmol) in dry
dichloromethane (500 mL) was added NBS (16.1 g, 91.1 mmol) in batches. After the addition
was finished, the mixture was stirred at room temperature for another 10 h until completion (monitored

Mar. Drugs 2020, 18, 398
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by TLC, DCM/EA = 5:1, Rf = 0.41) and then poured into ice water (500 mL). The resulting brown
precipitate was filtered under reduced pressure and the filter cake was washed with water (500
and dried to afford compound
DMSO): δ
12.95 (s, 1H, NH), 8.59 (s, 1H, H-2), 7.91 (s, 1H, H-8); 13C NMR (100 MHz, DMSO):
×
3 mL)
8
as a brown solid (17.2 g, 95% yield); mp: >300 ◦C; 1H NMR (400 MHz,
δ
151.5
(C), 151.4 (CH), 150.7 (C), 129.1 (CH), 114.1 (C), 86.3 (C); HRMS Calcd. For C6H4BrClN3+ [M + H]+:
231.9272, found: 231.9270.
3.4. Synthesis of 7-Bromo-6-chloro-9-(20,30,50-O-tribenzoyl-
β-d-ribofuranose-yl)-pyrrole[2,3-d]pyrimidine (9)
To the stirred suspension of 7-bromo-6-chloro-pyrrole[2,3-d]pyrimidine 8 (5.0 g, 21.5 mmol)
in◦dry acetonitrile (40 mL) was added BSA (N,O-bis(trimethylsiyl)acetamide, 5.4 g, 26.0 mmol) at
0 C under argon. The resulting mixture was stirred at room temperature for 15 min, and then
1-O-ethanoyl-2,3,5-O-tribenzoyl-β-d-ribofuranose (16.0 g, 32.5 mmol) and TMSOTf (8.0 g, 46 mmol)
were added sequentially. After the addition was completed, the mixture was stirred at 80 ◦C for 1 h
(monitored by TLC, CH2Cl2: PE, 5:1, Rf 0.27). After the mixture was cooled to room temperature,
water (200 mL) was added to quench the reaction and the resulting mixture was extracted with ethyl
acetate (200
×
3 mL). The organic layers were separated and washed with saturated NaHCO3 (200 mL)
and saturated brine (200 mL) and dried with anhydrous Na2SO4. After the solvent was removed under
reduced pressure, the crude product was purified by flash column chromatography on silica gel to
provide 10.1 g compound
9
(75.5% yield) as a white solid. mp: 142–143 ◦C; [α]D25
= −110 (c = 0.30,
CHCl3); 1H-NMR (400 MHz, CDCl3): δ (ppm) 8.57 (s, 1H), 8.10 (d, J = 8.0 Hz, 2H), 7.99 (d, J = 8.0 Hz,
2H), 7.91 (d, J = 7.9 Hz, 2H), 7.61–7.32 (m, 10H), 6.67 (d, J = 5.2 Hz, 1H), 6.12 (m, 2H), 4.89 (d, J = 12.1 Hz,
1H), 4.80 (d, J = 3.1 Hz, 1H), 4.67 (dd, J = 12.2, 3.1 Hz, 1H); 13C NMR (101MHz, CDCl3):
δ (ppm) 166.1
(C), 165.4 (C), 165.1 (C), 152.7 (C), 151.6 (CH), 150.7 (C), 133.9 (CH), 133.9 (CH), 133.7 (CH), 129.9 (4CH),
129.7 (2CH), 129.2 (C), 128.8 (2CH), 128.6 (2CH; C), 128.6 (2CH), 128.3 (C), 126.5 (CH), 115.9 (C), 90.2
(C), 86.7 (CH), 80.6 (CH), 74.1 (CH), 71.4 (CH), 63.5 (CH); HRMS Calcd. For C32H23BrClN3O7Na+ [M +
Na]+: 698.0300, found: 698.0305.
3.5. Synthesis of 7-Bromo-6-chloro-9-(β-d-ribofuranose-yl)-pyrrole[2,3-d]pyrimidine (10)
7-Bromo-6-chloro-9-(20,30,50-O-tribenzoyl-β-d-ribofuranose-yl)-pyrrole[2,3-d]pyrimidine 9 (10 g,
14.77 mmol) was dissolved in methanolic ammonia (methanol was saturated with NH3 at 0 ◦C, 300 mL)
and the mixture was stirred at 0 ◦C for 12 h until the reaction was completed (monitored by TLC,
CH2Cl2:CH3OH, 50:1, Rf 0.45). The mixture was concentrated and the crude product was purified
by flash column chromatography on silica gel (CH2Cl2/CH3OH, 70:1) to provide 5.1 g compound 10
(94.0% yield) as a white solid. mp: 179–180 ◦C; [α]D25
DMSO):
20-OH), 5.25 (d, J = 4.9 Hz, 1H, 30-OH), 5.15 (t, J = 5.3 Hz, 1H, 50-OH), 4.40 (dd, J = 11.1, 5.6 Hz, 1H,
H-20), 4.13 (dd, J = 8.3, 4.5 Hz, 1H, H-30), 3.96 (d, J = 3.4 Hz, 1H, H-40), 3.71–3.56 (m, 2H, H-50); 13
=
−
64 (c = 0.1, CH3OH); 1H NMR (400 MHz,
δ
8.72 (s, 1H, H-2), 8.27 (s, 1H, H-8), 6.24 (d, J = 5.8 Hz, 1H, H-10), 5.49 (d, J = 6.1 Hz, 1H,
C
NMR (101 MHz, DMSO): δ 151.6 (CH-2), 151.0 (C-6), 151.0 (C-4), 128.75(CH-8), 114.9 (C-5), 87.7 (C-7;
CH-10), 86.0 (CH-40), 74.9 (CH-20), 70.8 (CH-30), 61.7 (CH2-50); HRMS Calcd. For C11H11BrClN3O4Na+
[M + Na]+: 385.9514, found: 385.9511.
3.6. Synthesis of 7-Bromo-6-chloro-9-(20,30-O-isopropylidene-β-d-ribofuranose-yl)-pyrrole[2,3-d]
pyrimidine (11)
To the stirred suspension of compound 10 (4.0 g, 11 mmol) in dry acetone (200 mL) was added
p-toluenesulfonic acid (200 mg, 1.2 mmol) and 2,2-dimethoxy propane (6.0 g, 55 mmol). The mixture
was stirred at room temperature for 4 h until the reaction was completed (monitored by TLC,
CH2Cl2:CH3OH, 50:1, Rf 0.57). After that, the mixture was neutralized by triethylamine (70.0 mg,
0.7 mmol) and concentrated under reduced pressure. The crude product was purified by flash column
chromatography on silica gel (CH2Cl2/CH3OH, 70:1) to provide 4.4 g compound 11 (98.0% yield) as
a white solid. mp: 73–74 ◦C; [α]D25
= δ 8.73 (s,
63 (c = 0.05, CH3OH); 1H NMR (400 MHz, DMSO):
−

Mar. Drugs 2020, 18, 398
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1H, H-2), 8.24 (s, 1H, H-8), 6.36 (d, J = 2.1 Hz, 1H, H-10), 5.32–5.04 (m, 2H, H-20,50-OH), 5.03–4.83
(m, 1H, H-30), 4.21 (s, 1H, H-40), 3.56 (s, 2H, H-50), 1.54 (s, 3H, Me), 1.31 (s, 3H, Me); 13C NMR (101
MHz, DMSO):
δ
151.8 (CH-2), 151.2 (C-4), 150.5 (C-6), 129.2 (CH-8), 115.0 (C-5), 113.6 (C), 90.0 (CH-10),
87.8 (C-7), 86.9 (CH-40), 84.4 (CH-20), 81.4 (CH-30), 61.9 (CH2-50), 27.5 (Me), 25.6 (Me); HRMS Calcd.
For C14H15BrClN3O4Na+ [M + Na]+: 425.9827, found: 425.9811.
3.7. Synthesis of 7-Bromo-6-chloro-(20,30-O-isopropylidene-50-O-(2”,3”,4”,6”-O-tetrabenzyl-α-d-
glucopyranosyl)tubercidin (12) and 7-bromo-6-chloro-(20,30-O-isopropylidene-50-O-(2”,3”,4”,6”-O-
tetrabenzyl-β-d-glucopyranosyl)tubercidin (13)
Compound
dichloromethane (300 mL) and the resulting mixture was stirred at room temperature for 5 min under
argon. After that, TMSOTf (16.5 mg, 0.07 mmol) was added to the mixture at
20 ◦C. The mixture
6 (6.4 g, 9.3 mmol) and compound 11 (2.5 g, 6.2 mmol) were dissolved in dry
−
was slowly warmed to room temperature and stirred for another 4 h until the reaction was completed
(monitored by TLC, PE: EA, 3:1, Rf 0.5). The mixture was concentrated and the crude product was
purified by flash column chromatography on silica gel (CH2Cl2/CH3OH, 70:1) to provide 8.0 g of
compounds 12 and 13 (79% yield) as a light yellow oil.
12: [α]2D5 = −7 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSO): δ 8.71 (s, 1H), 8.23 (s, 1H), 7.34–7.22 (m,
18H), 7.13–7.04 (m, 2H), 6.41 (d, J = 2.6 Hz, 1H), 5.20 (dd, J = 6.1, 2.7 Hz, 1H), 4.97 (dd, J = 6.1, 3.2 Hz, 1H),
4.90 (m, 2H), 4.72–4.61 (m, 4H), 4.40 (m, 4H), 3.78–3.67 (m, 2H), 3.61 (dd, J = 10.9, 4.1 Hz, 1H), 3.56–3.37
(m, 5H), 1.54 (s, 3H), 1.29 (s, 3H); 13C NMR (101 MHz, DMSO):
δ 151.8 (CH), 151.3 (C), 150.4 (C),
139.3 (C), 138.9 (C), 138.7 (C), 138.6 (CH), 129.2 (CH), 128.7 (CH), 128.6 (CH), 128.2 (CH), 128.0 (CH),
128.0 (CH), 127.9 (CH), 127.8 (CH), 115.1 (C), 114.0 (C), 96.4 (CH), 89.6 (CH), 88.3 (C), 84.5 (CH), 84.3
(CH), 81.5 (CH), 81.4 (CH), 79.8 (CH), 77.7 (CH), 75.0 (CH2), 74.4 (CH2), 72.8 (CH2), 71.8 (CH2), 70.4
(CH), 68.8 (CH2), 68.0 (CH2), 27.5 (Me), 25.8 (Me); HRMS Calcd. For C48H49BrClN3O9Na+ [M + Na]+:
948.2233, found: 948.2236.
13: [α]2D5
= δ 8.67 (s, 1H), 8.17 (s, 1H), 7.24 (m,
19 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSO):
−
14H), 7.14 (m, 6H), 6.36 (d, J = 2.5 Hz, 1H), 5.19 (dd, J = 6.1, 2.6 Hz, 1H), 4.92 (dd, J = 6.1, 2.8 Hz,
1H), 4.78–4.64 (m, 4H), 4.56–4.30 (m, 6H), 4.04–3.98 (m, 1H), 3.76–3.38 (m, 6H), 3.24 (m, 1H), 1.50 (s,
3H), 1.23 (s, 3H); 13C NMR (101 MHz, DMSO):
δ 151.7 (CH), 151.2 (C), 150.3 (C), 139.0 (C), 138.9 (C),
138.7 (C), 138.6 (C), 129.3 (CH), 128.7 (CH), 128.6 (CH), 128.4 (CH), 128.2 (CH), 128.0 (CH), 128.0 (CH),
127.9 (CH), 127.9 (CH), 127.8 (CH), 115.1 (C), 113.8 (C), 102.9 (CH), 90.4 (CH), 87.9 (C), 85.0 (CH),
84.4 (CH), 84.1 (CH), 82.1 (CH), 81.4 (CH), 78.1 (CH), 74.9 (CH2), 74.4 (CH2), 74.4 (CH2), 74.0 (CH2), 72.7
(CH), 69.7 (CH2), 69.2 (CH2), 27.4 (Me), 25.6 (Me); MS (ESI): HRMS Calcd. For C48H49BrClN3O9Na+
[M + Na]+: 948.2233, found: 948.2235.
3.8. Synthesis of 7-Bromo-6-chloro-50-O-(2”,3”,4”,6”-O-tetrabenzyl-α-d-glucopyranosyl) Tubercidin (14)
Compound 12 (1.0 g, 1.08 mmol) was added to 80% acetic acid aqueous solution (50 mL) and
the solution was stirred at room temperature for 30 min. After that, the mixture was transferred to
preheated oil and stirred at 50 ◦C for another 12 h until the reaction was completed (monitored by TLC,
CH2Cl2:CH3OH,20:1, Rf 0.35). The mixture was concentrated and the crude product was purified by
flash column chromatography on silica gel (CH2Cl2/CH3OH, 50:1) to provide 0.9 g compound 14 (85.0%
yield) as a white solid. mp: 49–50 ◦C; [α]D25
= δ 8.71
26◦ (c = 0.3, CH3OH); 1H NMR (400 MHz, DMSO):
−
(s, 1H, H-2), 8.37 (s, 1H, H-8), 7.42–7.21 (m, 18H, H-Ph), 7.16–7.09 (m, 2H, H-Ph), 6.35 (d, J = 6.4 Hz, 1H,
H-10), 5.60 (d, 1H, 20-OH), 5.37 (s, 1H, 30-OH), 5.01 (d, J = 11.1 Hz, 1H, H-1”), 4.84 (m, 2H, H-CH2),
4.77–4.65 (m, 3H, H-CH2; H-20), 4.45 (m, 4H, H-CH2), 4.19 (d, J = 2.3 Hz, 1H, H-30), 4.12 (s, 1H, H-50),
3.82–3.62 (m, 3H, H-50; H-40), 3.58–3.56 (m, 3H, H-3”; H-6”), 3.52–3.40 (m, 2H, H-2”; H-4”); 13C NMR
(101 MHz, DMSO): δ 151.7 (CH-2), 151.3 (C-6), 151.1 (C-4), 139.3 (C), 138.8 (C), 138.6 (2C), 129.6 (CH),
128.7 (CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH), 127.9 (CH), 127.89 (CH), 114.9
(C-5), 96.4 (CH-1”), 88.4 (C-7), 87.3 (CH-10), 83.8 (CH-5”), 82.0 (CH-40), 79.5 (CH-4”), 77.8 (CH-3”),

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75.3 (CH2), 75.1 (CH-20), 74.6 (CH2), 72.8 (CH2), 71.9 (CH2), 71.6 (CH-30), 70.6 (CH-2”), 69.0 (CH2-6”),
67.9 (CH2-50); HRMS Calcd. For C45H46BrClN3O9+ [M + H]+: 886.2100, found: 886.2098.
3.9. Synthesis of 7-Bromo-6-chloro-50-O-(2”,3”,4”,6”-O-tetrabenzyl-β-d-glucopyranosyl) Tubercidin (140)
Compound 13 was converted into 140 (79.0% yield) as described for the synthesis of 14: Rf 0.34
(CH2Cl2/CH3OH, 20:1); mp: 49–50 ◦ C; [α]D25
= δ 8.67
12 (c = 0.3, CH3OH); 1H NMR (400 MHz, DMSO):
−
(s, 1H), 8.22 (s, 1H), 7.24 (s, 14H), 7.17–7.05 (m, 6H), 6.23 (d, J = 5.4 Hz, 1H), 5.54 (d, J = 6.1 Hz, 1H), 5.34
(s, 1H), 4.78 (d, J = 11.1 Hz, 2H), 4.69 (d, J = 8.8 Hz, 2H), 4.60–4.37 (m, 7H), 4.12 (m, 3H), 3.86–3.72
(m, 1H), 3.72–3.50 (m, 4H), 3.45 (m, 1H), 3.29–3.23 (m, 1H); 13C NMR (101 MHz, DMSO)
δ 151.6
(CH-2), 151.0 (C-6), 150.9 (C-4), 139.1 (C), 138.8 (C), 138.7 (C), 138.6 (C), 128.8 (CH), 128.7 (CH), 128.6
(CH), 128.6 (CH), 128.5 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.9 (CH),
127.8 (CH), 127.8 (CH), 115.0 (C-5), 103.0 (CH-1”), 88.2 (C-7), 87.9 (CH-10), 84.1 (CH-40), 83.7 (CH-5”),
82.4 (CH-4”), 78.1 (CH-3”), 74.9 (CH-20), 74.7 (CH-30), 74.5 (CH2), 74.4 (CH2), 74.1 (CH2), 72.7 (CH2),
71.0 (CH-2”), 70.0 (CH2-50), 69.2 (CH2-6”); HRMS Calcd. For C45H46BrClN3O9+ [M + H]+: 886.2100,
found: 886.2099.
3.10. Synthesis of 7-Bromo-50-O-(2”,3”,4”,6”-O-tetrabenzyl-α-d-glucopyranosyl)tubercidin (15)
Compound 14 (100 mg, 0.11 mmol) was dissolved in methanolic ammonia (methanol was
saturated with NH3 at 0 ◦C, 50 mL) and the mixture was stirred at 130 ◦C for 12 h (monitored by TLC,
CH2Cl2:CH3OH, 25:1, Rf 0.36). After cooling to room temperature, the mixture was concentrated
and the crude product was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH,
30:1) to provide 92.9 mg compound 15 (94.9% yield) as a white solid. mp: 68–69 ◦C, [α]D25
=
−
33 (c
= 0.1, CH3OH); 1H NMR (400 MHz, DMSO):
δ 8.10 (s, 1H, H-2), 7.87 (s, 1H, H-8), 7.40–7.18 (m, 18H,
H-Bn), 7.18–7.07 (m, 2H, H-Bn), 6.76 (br s, 2H, H-NH2), 6.19 (d, J = 6.6 Hz, 1H, H-10), 5.46 (d, J = 6.3
Hz, 1H, 20-OH), 5.26 (d, J = 4.1 Hz, 1H, 30-OH), 4.98 (d, J = 11.1 Hz, 1H, H-1”), 4.81–4.66 (m, 5H, H-20;
2CH2), 4.49–4.32 (m, 4H, 2CH2), 4.10–4.09 (m, 1H, H-30), 4.06–4.04 (m, 1H, H-5”), 3.78–3.50 (m, 6H,
H-40,50,3”,6”), 3.48–3.39 (m, 2H, H-2”,4”); 13C NMR (101 MHz, DMSO):
δ 157.4 (C-4), 153.0 (C-6), 150.6
(CH-2), 139.3 (C), 138.8 (C), 138.7 (2C), 128.7 (CH), 128.7 (CH), 128.4 (CH), 128.2 (CH), 128.1 (CH),
128.0 (CH), 127.9 (CH), 121.7 (C-5), 96.3 (CH-1”), 87.9 (C-7), 86.5 (CH-10), 83.3 (CH-5”), 82.0 (CH-40),
79.5 (CH-4”), 77.8 (CH-3”), 75.3 (CH2), 74.9 (CH-20), 74.6 (CH2), 72.8 (CH2), 71.8 (CH2), 71.7 (CH-30),
+
70.6 (CH-2”), 69.0 (CH2-60), 68.1 (CH2-50); HRMS Calcd. For C45H48BrN4O9 [M + H]+: 867.2599,
found: 867.2598.
3.11. Synthesis of 7-Bromo-50-O-(2”,3”,4”,6”-O-tetrabenzyl-β-d-glucopyranosyl) Tubercidin (150)
Compound 140 was converted into 150 (79.0% yield) as described for the synthesis of 15; Rf 0.40
(CH2Cl2/CH3OH, 20:1); mp: 145–147 ◦C; [α]D25
= δ
15 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSO)
−
8.11 (s, 1H, H-2), 7.68 (s, 1H, H-8), 7.40–7.19 (m, 14H, H-Bn), 7.19–7.10 (m, 6H, H-Bn), 6.77 (br s, 2H,
NH2), 6.13 (d, J = 5.4 Hz, 1H, H-10), 5.44 (d, J = 6.1 Hz, 1H, 20-OH), 5.26 (d, J = 5.0 Hz, 1H, 30-OH), 4.81
(m, 2H, H-20,1”), 4.71 (d, J = 11.0 Hz, 2H, CH2), 4.60–4.08 (m, 6H, 3CH2), 4.15–4.04 (m, 3H, H-30,50,5”),
3.78–3.42 (m, 6H, H-40,50,2”,3”,6”), 3.29 (s, 1H, H-4”); 13C NMR (101 MHz, DMSO)
δ 157.4 (C-6), 153.0
(CH-2), 150.2 (C-4), 139.1 (CH), 138.8 (CH), 138.7 (CH), 138.6 (CH), 128.7 (CH), 128.7 (CH), 128.6 (CH),
128.5 (CH), 128.5 (CH), 128.2 (CH), 128.1 (CH), 128.0 (CH), 127.8 (CH), 122.0 (C-5), 103.0 (C-7), 101.5
(CH-1”), 87.5 (CH-10), 87.4 (CH-40), 84.1 (CH-5”), 83.0 (CH-4”), 82.3 (CH-3”), 78.1 (CH-20), 74.9 (CH-30),
74.4 (CH2), 74.4 (CH2), 74.2 (CH2), 72.7 (CH2), 71.0 (CH-2”), 70.3 (CH2-50), 69.2 (CH2-6”); HRMS Calcd.
+
For C45H48BrN4O9 [M + H]+: 867.2599, found: 867.2599.
3.12. Synthesis of 50-O-(2”,3”,4”,6”-O-Tetrabenzyl-α-d-glucopyranosyl) Tubercidin (16)
To the stirred suspension of compound 15 (200 mg, 0.23 mmol) in THF (15 mL) and methanol
(15 mL) was added triethylamine (0.3 mL) and 20% Pd(OH)2/C (50 mg, 0.04 mmol), and the mixture was
stirred at room temperature for 5 h under a continuous hydrogen environment until the reaction was

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completed (monitored by TLC, CH2Cl2:CH3OH,20:1, Rf 0.24). The mixture was filtered, and the filtrate
was concentrated to provide the crude product, which was purified by flash column chromatography
on silica gel◦(CH2Cl2/CH3OH, 20:1) to provide 151.8 mg compound 16 (83.5% yield) as a white solid.
mp: 74–75 C; [α]2D5 = 1.8 (c = 0.1, CH3OH); H NMR (400 MHz, DMSO)
δ 8.06 (s, 1H, H-2), 7.63
1
(d, J = 3.6 Hz, 1H, H-8), 7.41–7.21 (m, 18H, H-Bn), 7.17–7.10 (m, 2H, H-Bn), 7.00 (s, 2H, NH2), 6.53
(d, J = 3.5 Hz, 1H, H-7), 6.16 (d, J = 5.9 Hz, 1H, H-10), 5.41 (d, J = 6.2 Hz, 1H, 20-OH), 5.22 (s, 1H,
30-OH), 4.95–4.41 (m, 9H, H-1”,Bn), 4.36–4.32 (m, 1H, H-20), 4.12–4.08 (m, 2H, H-30,5”), 3.83–3.77 (m,
2H, H-40,50), 3.70–3.41 (m, 6H, H-2”,3”,4”,50,6”); 13C NMR (101 MHz, DMSO):
δ 157.5 (C-6), 151.7
(CH-2), 150.8 (C-4), 139.2 (CH), 138.9 (CH), 138.7 (CH), 128.7 (CH), 128.7 (CH), 128.2 (CH), 128.1 (CH),
128.1 (CH), 128.0 (CH), 127.9 (CH), 122.1 (CH-8), 103.1 (C-5), 100.4 (CH-7), 96.3 (CH-1”), 87.0 (CH-10),
82.7 (CH-5”), 82.0 (CH-40), 79.9 (CH-4”), 77.8 (CH-3”), 75.2 (CH2), 74.9 (CH-20), 74.6 (CH2), 72.8 (CH2),
+
72.0 (CH2), 71.4 (CH-30), 70.5 (CH-2”), 69.0 (CH2-6”), 68.0 (CH2-50); HRMS Calcd. For C45H49N4O9
[M + H]+: 789.3494, found: 789.3499.
3.13. Synthesis of 50-O-(2”,3”,4”,6”-O-Tetrabenzyl-β-d-glucopyranosyl) Tubercidin (160)
Compound 150 was converted into 160 (79.0% yield) as described for the synthesis of 16; Rf 0.23
(CH2Cl2/CH3OH, 20:1); mp: 166–167 ◦C; [α]D25
=
21 (c = 0.1, CH3OH); 1H NMR (400 MHz, DMSO):
−
δ
8.03 (s, 1H, H-2), 7.38–7.18 (m, 15H, H-8, Bn), 7.19–7.05 (m, 6H, H-Bn), 6.98 (s, 2H, NH2), 6.59 (d,
J = 3.6 Hz, 1H, H-7), 6.09 (d, J = 5.2 Hz, 1H, H-10), 5.36 (d, J = 6.3 Hz, 1H, 20-OH), 5.22 (d, J = 5.3 Hz, 1H,
OH-30), 4.94–4.26 (m, 10H, H-1”,20,Bn), 4.11–4.05 (m, 3H, H-30,50,5”), 3.70–3.40 (m, 6H, H-2”,3”,40,50,6”),
3.26 (d, J = 8.0 Hz, 1H, H-4”); 13C NMR (101 MHz, DMSO):
δ 157.9 (C-6), 152.3 (CH-2), 150.8 (C-4), 139.1
(CH), 138.8 (CH), 138.7 (CH), 138.6 (CH), 128.7 (CH), 128.7 (CH), 128.6 (C-Bn), 128.6 (CH), 128.5 (CH),
128.2 (CH), 128.1 (CH), 128.1 (CH), 128.1 (CH), 128.0 (CH), 127.9 (CH), 127.8 (CH), 127.8 (CH), 122.0
(CH-8), 103.3 (CH-1”), 103.2 (C-5), 100.5 (CH-7), 87.6 (CH-10), 84.1 (CH-40), 82.6 (CH-5”), 82.1 (CH-4”),
78.1 (CH-3”), 74.9 (CH-20), 74.4 (CH2), 74.3 (CH2), 74.2 (CH2), 74.1 (CH-30), 72.8 (CH2), 71.1 (CH-2”),
70.6 (CH2-50), 69.1 (CH2-6”); HRMS Calcd. For C45H49N4O9+ [M + H]+: 789.3494, found: 789.3499.
3.14. Synthesis of 50-O-α-d-Glucopyranosyl tubercidin (2)
To the stirred suspension of compound 16 (100 mg, 0.13 mmol) in THF (10 ml) and methanol
(10 mL) was added 20% Pd(OH)2/C (50 mg, 0.04 mmol), and the mixture was stirred at 40 ◦C for 12 h
under a continuous hydrogen environment. The mixture was filtered and the filtrate was concentrated
to provide the crude product, which was purified by isocratic HPLC (H2O/CH3OH, 4:1) to provide 43.5
mg compound
(400 MHz, DMSO):
2
(80.1% yield) as a white solid; mp: 120–122 ◦C; [α]D25 = 10.5 (c = 0.08, H2O); 1H NMR
δ
8.09 (s, 1H, H-2), 7.76 (d, J = 3.2 Hz, 1H, H-8), 7.20 (s, 2H, NH2), 6.61 (d, J = 3.4 Hz,
1H, H-7), 6.14 (d, J = 7.0 Hz, 1H, H-10), 5.21 (d, J = 4.6 Hz, 2H, 2”, 30-OH), 5.10 (d, J = 4.3 Hz, 1H, 20-OH),
4.92 (d, J = 4.9 Hz, 1H, 3”-OH), 4.84 (d, J = 3.6 Hz, 1H, 4”-OH), 4.72 (d, J = 3.1 Hz, 1H, H-1”), 4.49 (s, 1H,
6”-OH), 4.43 (m, 1H, H-20), 4.10 (s, 1H, H-40), 4.08 (d, J = 1.7 Hz, 1H, H-30), 3.78 (dd, J = 10.9, 2.8 Hz,
1H, H-50), 3.66–3.63 (m, 1H, H-60), 3.47–3.42 (m, 3H, H-50,4”,6”), 3.40–3.34 (m, 2H, H-2”,5”), 3.13–3.07
(m, 1H, H-3”); 13C NMR (101 MHz, DMSO):
δ 157.3 (C-6), 151.5 (CH-2), 150.6 (C-4), 122.4 (CH-8), 102.8
(C-5), 100.0 (CH-7), 98.5 (CH-1”), 85.9 (CH-10), 83.2 (CH-40), 74.2 (CH-20), 73.3 (CH-4”), 72.7 (CH-5”),
71.5 (CH-2”), 70.9 (CH-30), 70.0 (CH-3”), 67.0 (CH2-50), 60.9 (CH2-6”); HRMS Calcd. For C17H25N4O9
[M + H]+: 429.1616, found: 429.1620.
+
3.15. Synthesis of 50-O-β-d-Glucopyranosyl Tubercidin (17)
Compound 160 was converted into 17 (79.0% yield) as described for the synthesis of
◦C; [α]2D5 37 (c = 0.08, H2O); 1H NMR (400 MHz, DMSO):
8.07 (s, 1H, H-2), 7.38 (d, J = 3.7 Hz, 1H,
2; mp: 156–157
=
−
δ
H-8), 7.09 (s, 2H, 6-NH2), 6.61 (d, J = 3.6 Hz, 1H, H-7), 6.08 (d, J = 5.6 Hz, 1H, H-10), 5.26 (d, J = 5.5 Hz,
1H, 20-OH), 5.17 (d, J = 4.6 Hz, 1H, 30-OH), 5.03 (d, J = 3.7 Hz, 1H, 2”-OH), 4.95 (s, 1H, 3”-OH), 4.95 (s,
1H, 4”-OH), 4.51 (s, 1H, 6”-OH), 4.36 (d, J = 5.0 Hz, 1H, H-20), 4.21 (d, J = 7.8 Hz, 1H, H-1”), 4.14 (d, J =
4.4 Hz, 1H, H-30), 4.02–3.95 (m, 2H, H-40,50), 3.68 (d, J = 9.2 Hz, 1H, H-6”), 3.59 (dd, J = 11.9, 5.7 Hz, 1H,

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H-50), 3.44 (d, J = 6.7 Hz, 1H, H-6”), 3.15 (d, J = 9.9 Hz, 1H, H-3”), 3.12–3.04 (m, 2H, H-4”,5”), 3.00
(d, J = 7.8 Hz, 1H, H-2”); 13C NMR (101 MHz, DMSO):
δ 157.5 (C-6), 151.7 (CH-2), 150.7 (C-4), 122.2
(CH-8), 103.5 (CH-1”), 103.1 (C-5), 100.5 (CH-7), 87.1 (CH-10), 83.1 (CH-40), 77.4 (CH-2”), 77.2 (CH-20),
74.2 (CH-4”), 74.1 (C-5”), 71.1 (CH-30), 70.5 (CH-3”), 69.5 (CH2-50), 61.5 (CH2-6”); HRMS Calcd. For
C17H25N4O9+ [M + H]+: 429.1616, found: 429.1619.
4. Conclusions
In summary, we developed
tubercidin in eight steps with
a α-d-glucopyranosyl
concise total synthesis of 50-O-
2
a
28% overall yield from commercially available
1-O-acetyl-2,3,5-tri-O-benzoyl-β-d-ribose 7. This is the first report on the synthesis of disaccharide
7-deazapurine nucleosides.
This synthetic route features two key steps: (1) a one-pot
with 6-chloro-7-bromo-7-deazapurine , and (2) stereoselective
-O-glycosylation of 7-deazapurine nucleoside 11 with 2,3,4,6-tetra-O-benzyl-glucopyranosyl
Vorbrüggen glycosylation of ribose
α
. Additionally, 50-O-
trichloroacetimidate 6 β-d-glucopyranosyl tubercidin 17 was also prepared.
7
8
A comparison with the natural product’s spectra further confirmed the reported structure. Applications
of this newly developed modular synthetic approach to prepare other glycosylated tubercidin analogs
are ongoing in our laboratory.
Supplementary Materials: The following are available online at http://www.mdpi.com/1660-3397/18/8/398/s1,
Table S1: 13C NMR chemical shifts of naturally occurring nucleoside
2
and synthetic nucleosides
2
and 17
;
,
Table S2: 1H NMR chemical shifts and coupling constants of naturally occurring nucleoside
and 1H-and 13C-NMR charts of all compounds.
2, synthetic 2 and 17
Author Contributions: H.H. and Q.X. conceived and designed this research and analyzed the experimental data;
W.O. prepared compounds and collected their spectral data; R.Y. and H.D. checked the experimental data; W.O.,
H.H. and Q.X. wrote the paper; all authors reviewed and approved the manuscript. All authors have read and
agreed to the published version of the manuscript.
Funding: This work is financially supported by the National Natural Science Foundation of China (No. 21676131
and No. 21462019), the Science Foundation of Jiangxi Province (20161BAB213085), Education Department of
Jiangxi Province (No. GJJ180625), and Jiangxi Science & Technology Normal University (No. 2018BSQD025).
Conflicts of Interest: The authors declare no conflict of interest.
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