New antitumor 6-chloropurine nucleosides inducing apoptosis and G2/M cell cycle arrest

Article abstract of DOI:10.1016/j.ejmech.2014.11.019

Treating cancer has been challenging for decades, following countless approaches and attempts. Nucleosides, alone or as part of nucleotides, are vital elements of living systems and have shown pharmacological effects, e.g. as antibiotic or antiviral agent

Full text of DOI:10.1016/j.ejmech.2014.11.019

Accepted Manuscript
New antitumor 6-chloropurine nucleosides inducing apoptosis and G2/M cell cycle
arrest
Stefan Schwarz , Bianka Siewert , René Csuk , Prof. Dr. Amélia P. Rauter
PII:
S0223-5234(14)01041-1
10.1016/j.ejmech.2014.11.019
EJMECH 7512
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 21 July 2014
Revised Date: 29 October 2014
Accepted Date: 9 November 2014
Please cite this article as: S. Schwarz, B. Siewert, R. Csuk, A.P. Rauter, New antitumor 6-chloropurine
nucleosides inducing apoptosis and G2/M cell cycle arrest, European Journal of Medicinal Chemistry
(2014), doi: 10.1016/j.ejmech.2014.11.019.
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ACCEPTED MANUSCRIPT
Graphical abstract:
New antitumor purine nucleosides inducing apoptosis and G2/M cell cycle arrest
Stefan Schwarz, Bianka Siewert, René Csuk, and Amélia P. Rauter*
NHAc
OBn
OBn
O
N
N
N
BnO
BnO
N
Cl
GI50 (HT-29) = 1.5 ± 0.1 µM
A series of β-configurated 6-chloropurine nucleosides embodying a hexopyranosyl glycon was prepared.
Low micromolar GI50 values were determined on human melanoma, lung and ovarian carcinomas, and
colon adenocarcinoma. The most active compound provided data of the same order of magnitude as that
of the chemotherapeutic cladribine, induced apoptosis and produced a G2/M cell cycle arrest.

ACCEPTED MANUSCRIPT
New antitumor 6-chloropurine nucleosides inducing apoptosis and G2/M cell cycle arrest
Stefan Schwarz,1,2 Bianka Siewert,2 René Csuk,2 and Amélia P. Rauter1*
1Centro de Química e Bioquímica, Faculdade de Ciências, Universidade de Lisboa, Campo Grande, Ed. C8, Piso 5,
1749-016 Lisboa, Portugal; email: aprauter@fc.ul.pt
2 Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-06120 Halle (Saale),
Germany;
Corresponding author:
Prof. Dr. Amélia Pilar Rauter
Faculdade de Ciências, Universidade de Lisboa
Centro de Química e Bioquímica/Departamento de Química e Bioquímica
Campo Grande, Ed. C8, Piso 5
1749-016 Lisboa
Portugal
Tel.: +351 21 7500952
Fax: + 351 21 7500088
e-mail: aprauter@fc.ul.pt
Abstract:
Treating cancer has been challenging for decades, following countless approaches and attempts. Nucleosides, alone or
as part of nucleotides, are vital elements of living systems and have shown pharmacological effects, e.g. as antibiotic or
antiviral agents. We investigated the antitumor potential on human melanoma, lung and ovarian carcinomas, and on
colon adenocarcinoma of a new series of purine nucleosides based on a 6-chloropurine or a 2-acetamido-6-chloropurine
scaffold linked to perbenzylated hexosyl (glucosyl, galactosyl and mannosyl) residues. All compounds were tested in a
sulforhodamine B (SRB) assay for their cytotoxicity and provided micromolar GI50 values with order of magnitude
comparable to structurally similar chemotherapeutics, namely 2-chloro-2′-deoxyadenosine (cladribine). Furthermore,
the induction of apoptosis was established and cell cycle analysis was accomplished demonstrating a G2/M cell cycle
arrest.
Keywords:6-chloropurine; N-glycosylation; antitumor; SRB assay; cell cycle analysis; acridine orange.
1. Introduction
Purine nucleosides and nucleotides have been major targets of anticancer research for several decades. One of the first
investigations was accomplished by the group of Noell in 1962 synthesizing a series of thioguanines and 2-amino-6-
alkylthiopurine derivatives [1]. Studies dealing with compounds containing purine moieties, e.g. in gold (III) complexes
[2] or adenine derivatives [3-5] have also been reported and effective camptothecin purine derivatives possessing GI50
values in micromolar range were synthesized by Li et al. [6]. Caba et al [7] reported on an antiproliferative agent
against the MCF-7 adenocarcinoma with micromolar GI50 value embodying tetrahydrobenzoxazepine N9-linked to a 6-
chloropurine while a coumarin N9-linked to a 2-amino-6-chloropurine showed moderate activity (25 – 35 µM) against

the HeLa, HepG2 and SW620 cell linAesC[9C].EVPolTleEr eDt aMl. AaccNomUpSliCshRedIPsuTbstitutions at different positions of a 6-
aminopurine scaffold and have shown that some of the ribosides exhibited micromolar anticancer activity while their
N7- or N9-linked glucoside analogues were not active [8]. In addition, a 9-norbornyl-6-chloropurine was recently
reported as a novel antileukemic compound [10]. The previously reported anticancer potential of 6-chloropurine derived
compounds encouraged us to investigate, for the first time, a series of purines embodying a 6-chloro substitution (CP)
or both 6-chloro- and 2-acetamido groups (ACP), linked at N7 or N9 to perbenzylated
D
-glucosyl,
D
-mannosyl and -
D
galactosyl residues. The reaction conditions described by Marcelo et al. [11] using a silylated base were optimized for
this type of nucleosides. The anticancer activity was determined using a sulforhodamine B (SRB) assay to yield GI50
values for human melanoma, lung and ovarian carcinoma, and colon adenocarcinoma cancer cell lines. Furthermore, the
substances were tested on murine embryonic fibroblasts (NiH 3T3) to investigate their tumor cell-to-control-specifity.
In addition, acridine orange/propidium iodide assays, DNA laddering experiments and cell cycle analyses were
performed for the most active compound to gain some information about the mode of action of this family of new
anticancer molecular entities.
2. Results
2.1. Chemistry
Nucleoside synthesis can be performed by a two steps procedure, starting with the acetylation [12] or halogenation
[13,14] of suitable precursors, followed by reaction with the heterocyclic base. A direct access to nucleosides can be
gained by Lewis acid activation with tin chloride [15,16] or TMSOTf [17] of methyl glycosides in a reaction employing
a persilylated purine. Marcelo et al. applied TMSOTf in acetonitrile to link regioselectively bicyclic pyranosides to
purine scaffolds at their N7 position with β-stereoselectivity [11]. These conditions were investigated in this study for
this nucleoside series. Therefore, monosaccharidyl donors were prepared according to established procedures [18]. In
the next step, the molarity of TMSOTf was investigated regarding its impact on the reaction yield and N7/N9 ratio. The
proportions of the reaction products formed, determined by 1H NMR, are compiled in Table 1, showing that the use of
eight equivalents of TMSOTf worked best regarding the overall yields, while the N7/N9 ratio did not change
significantly by increasing the concentration of TMSOTf. However, the expected N7 regioselectivity obtained by
Marcelo et al. [11] in the presence of TMSOTf in acetonitrile occurred only for the β-mannosylation and the β-
galactosylation of 2-acetamido-6-chloropurine, while the thermodynamically controlled N9 nucleoside was the major β-
anomer resulting from the N-glucosylation and the N-galactosylation of 6-chloropurine. All four isomers (α/β, N7/N9)
could be detected using these reaction conditions [19]. Nevertheless, only the β-anomers were subjected to biological
testings, in as much as they were the predominant species when introducing the glycosyl or galactosyl moieties and the
α-anomers appeared in very low amount. Therefore, also the minor β-mannosyl nucleosides were isolated and tested in
order to receive comparable biological data to correlate structure with bioactivity in this family of compounds. The
hydroxy groups of the glycosyl moities remained benzylated, since the presence of benzyl groups enhances the
cytotoxicity of glycosylated compounds [20,21]. Moreover, benzyl groups represent a suitable metabolic protection.
The configuration of the anomeric center as well as the purine substitution pattern could be confirmed by the observed
chemical shifts in the 1H and 13C NMR spectra and the 3JH-1,H-2 coupling constant of the anomeric proton. Chemical
shifts of δ = 5.4 ppm to δ = 5.7 ppm and coupling constants of J = 7 to J = 9 Hz were obtained for
galacto configurated derivatives proving an axial position of the anomeric proton, while coupling constants of approx. J
= 1 Hz were determined for
-manno configurated compounds. In the 13C NMR spectra, the chemical shifts of δ = 83 to
D
-gluco and
D
-
D

δ = 85 ppm for the anomeric carbon arAe iCn fCulEl aPgTreeEmDentMwAithNdaUtaSgCiveRnIiPn Tthe literature for the anomeric carbon of
other β-hexopyranosyl nucleosides [22-24]. The distinction between N7 or N9 substitution at the purine scaffold can be
achieved by the chemical shift of purine carbon 5. In CP and ACP nucleosides the resonance of C-5 ranges from δ = 131
ppm to δ = 128 ppm indicating N9 substitution while N7 substitution was characterized by C-5 chemical shifts of about
δ = 122 ppm for CP and about δ = 118 ppm for ACP nucleosides. These assignments were also confirmed with NMR
HMBC experiments.
Table 1: Experimental conditions for the reaction of methyl 2,3,4,6-tetra-O-benzyl-α- -glycoside with the purine (CP or
D
ACP). Yields and product ratios were determined by 1H NMR experiments. 1 Equivalent of the monosaccharide and 1.5
equivalents of the silylated purine in dry acetonitrile were used, conventional heating at 65 °C was performed.
Entry
Purine
CP
eq. TMSOTf
Time
24 h
2 h
β-N7/N9
Overall yield
n.d.a
1
2
3
4
5
6
7
8
1
2
4
8
1
2
4
8
CP
1/1.9
1/4.5
1/3.8
43 %
CP
2 h
62 %
CP
2 h
65 %
ACP
ACP
ACP
ACP
24 h
2 h
n.d.
1/2.4
1/1
1 %
2 h
62 %
2 h
1/1.3
63 %
an.d: no product detected
Scheme 1. Synthesis of the nucleosides. CP: 6-chloropurine; ACP: 2-acetamido-6-chloropurine. Reagents and
conditions: (a) TMSOTf, CH3CN, 65 °C, 2 h.

ACCEPTED MANUSCRIPT
2.2. Biology
The cytotoxic activities of all synthesized compounds are represented by their GI50 values given in Table 2. The values
were determined in photometric SRB assays, using four different human tumor cell lines as well as murine embryonic
fibroblasts (NiH 3T3). Some general tendencies could be observed within the bounds of our study. While the purines CP
and ACP did not show any activity below 30 µM (cut-off), their N-glycosylation, increased considerably their cytotoxic
activity. In general, the ACP nucleosides showed higher activities than the corresponding CP analogues. Furthermore,
N7 derivatives showed lower GI50 values when compared to their N9 regioisomers.
Table 2: Cytotoxicity (GI50 in µM) of compounds 1-12 measured in SRB-assays with 4 different human cancer cell
lines and non-malignant murine embryonic fibroblasts (NiH 3T3) in comparison to their parent purines CP and ACPa as
well as to betulinic acid [25,26] and tamoxifen [27].
Purine,
purine
N7/N9
518A2
A2780
(lung
A549
HT-29
(colon
NiH 3T3
(melanoma)
(ovarian
nucleosideb
carcinoma)
carcinoma) adenocarcinoma)
CP
ACP
>30
>30
>30
>30
>30
>30
>30
>30
>30
>30
GlcCP
N7
N9
N7
N9
N7
N9
N7
N9
N7
N9
N7
N9
-
4.7 ± 0.1
>30
6.3 ± 0.1
>30
4.2 ± 0.2
17.9 ± 8.7
8.1 ± 0.7
20.2 ± 16.4
9.4 ± 0.2
29.5 ± 6.4
4.6 ± 0.4
23.2 ± 0.6
4.1 ± 0.4
17.5 ± 6.8
1.6 ± 0.2
13.0 ± 0.1
14.9 ± 0.7
9.7 ± 0.7
6.8 ± 0.4
n.d.
6.9 ± 0.1
29.2 ± 13.9
7.9 ± 2.9
18.6 ± 6.1
9.8 ± 2.4
11.2 ± 5.2
3.6 ± 0.1
8.8 ± 0.3
4.2 ± 0.8
7.3 ± 3.0
1.4 ± 0.1
8.7 ± 0.2
10.0 ± 0.5
7.3 ± 0.5
1
2
GlcCP
GalCP
9.0 ± 0.3
>30
4.0 ±1.0
>30
19.7 ± 1.1
n.d.
3
GalCP
4
ManCP
ManCP
GlcACP
GlcACP
GalACP
GalACP
ManACP
ManACP
Betulinic acid
Tamoxifen
13.5 ± 0.6
13.7 ± 3.4
3.4 ± 0.1
7.6 ± 0.1
3.8 ± 0.1
8.9 ± 4.4
2.0 ± 0.1
11.5 ± 0.3
11.9 ± 0.6
7.6 ± 0.5
10.9 ± 0.1
27.1 ± 1.9
3.5 ± 0.1
15.6 ± 0.8
5.5 ± 0.3
11.0 ± 3.2
2.2 ± 0.1
15.4 ± 0.2
11.0 ± 0.6
7.8 ± 0.5
11.0 ± 1.1
25.3 ± 1.5
3.9 ± 0.1
>30
5
6
7
8
4.1 ± 0.6
15.7 ± 8.4
1.5 ± 0.1
13.2 ± 0.2
16.1 ± 0.8
-
9
10
11
12
-
-
-
aGI50 values represent mean values of 3 independent measurements and were calculated applying the two-parametric
Hill slope equation; bBetulinic acid and tamoxifen are standard anticancer drugs.

ACCEPTED MANUSCRIPT
The influence of the glycosyl moiety was, however, less significant. The introduction of glucosyl and galactosyl groups
showed similiar results, e.g. GI50 values from 18 to above 30 µM were determined for compounds 2 and 4, both N9 CP
regioisomers, exhibiting the lowest activities amongst the CP nucleosides tested. In contrast, those nucleosides bearing a
mannosyl group showed the highest cytotoxic potential. The only exception was compound 5, a N7 CP nucleoside,
possessing GI50 values between 9 and 14 µM. The corresponding ACP derivative 11 exhibited GI50 values of 1.4 to 2.2
µM and is the most potent compound of this investigation. This compound exhibited a higher cytotoxicity than betulinic
acid (GI50 from 11.0 to 14.9 µM [25,26]) or tamoxifen (GI50 from 7.6 to 9.7 µM [27]), two common antitumor drugs.
However, no selectivity was detected when these substances were tested on murine embryonic fibroblasts NiH 3T3, a
non-malignant cell line.
Figure 1. Results of AO/PI assay using HT-29 cells treated with compound 11 (5 µM) for 24 h, floated cells collected
Figure 2. DNA fragmentation scatter of the DNA laddering assay using HT-29 cells tested with compound 11 (5 µM)
for 24 h

ACCEPTED MANUSCRIPT
The ability to trigger apoptosis is a fundamental quality of potent anticancer drugs. Consequently, the most active
compound 11 of this study was subjected to further screening, applying a dye exclusion assay (acridine
orange/propidium iodide, AO/PI) [28,29], DNA laddering assay [25] and cell-cycle analysis [30]. The appearance of
green fluorescent cells with sections of diverse intensity indicated that 11 is able to trigger apoptosis in HT-29 cells
(Figure 1). This circumstance was confirmed by the results of the DNA laddering test (Figure 2) showing the
characteristic DNA fragmentation.
As a result of the AO/PI assay, the induction of a programmed cell death (PCD) [31] by 11 could be demonstrated.
Membrane integrity is a typical hallmark of PCDs, like e.g. apoptosis. For an accidental cell death (necrosis) a
membrane disruption is described, instead. Necrotic cells are stained in this assay red, due to intercalation of the cell
membrane impermeable dye (propidium iodide, PI) into the DNA double strand [32].
Additionally, investigation of the cell morphology suggested a PCD mediated by apoptosis, due to the occurrence of
typical membrane blabbing. Also, the occurrence of many mitotic cells indicates a potential interruption in the last cell
cycle phase (mitosis); some of the cells seem to be arrested in the telophase.
To establish the induction of apoptosis, the DNA enriched fraction of the death-cells was submitted to a gel-
electrophoresis based fragmentation assay (DNA-ladder assay). As depicted in Fig 1 by the occurrence of well-defined
178 kBp DNA fragments and multiples thereof, treatment of the cells with 11 led to DNA fragmentation and thus, to the
activation of the caspase-activated DNAse (CAD) [33]. The latter is another typical hallmark of the caspase mediated
apoptosis.
Triggering of apoptosis is known to be possible via several caspase dependent routes, besides an intrinsic and an
extrinsic way, an inhibition of the cell cycle regulating cyclin-dependend kinases (cdks) is known to effect the cell cycle
distribution and hence to trigger apoptosis [34]. 2,6,9-Trisubstituted purine derivatives are able to inhibit CDKs and
some of them led to a specific G2 phase arrest, promoting apoptosis in MT-2 cells [35], while several other CDK
inhibiting purine analogues were shown to induce a significant G1 arrest or to trigger apoptosis independently from the
trapping of the cell cycle [36]. To gain a deeper insight into the modulation of the cell cycle, cells were treated with 11
and submitted to a cell cycle distribution assay. As shown in fig. 3, a significant G2/M phase arrest was induced by the
compound after an incubation period of 24 h.
Figure 3. Results of the cell cycle analysis in a size-number-diagram using HT-29 cells, left – control, right –
compound 11 (5 µM); incubation time was 24 h

ACCEPTED MANUSCRIPT
Conclusions
A new family of anticancer nucleosides embodying a 6-chloropurine or a 2-acetamido-6-chloropurine linked to a
hexosyl moiety is here disclosed. The structural features common to the most active compounds are the 2-acetamido
group and the N7 glycosyl linkage, in contrast to the N9-linkage present in the antitumor purine derivatives described in
the literature. The mannosyl group was present in the most active nucleoside, but the analogue galactosyl nucleoside
also showed GI50 values of the same order of magnitude. They were active on human melanoma, lung and ovarian
carcinomas, and colon adenocarcinoma in low micromolar range, and the most active compounds provided data of the
same order of magnitude (e.g. compound 11 with GI50 1.5 µM) as that of the structurally simillar chemotherapeutic 2-
chloro-2′-deoxyadenosine (cladribine, GI50 2.43 µM on U266 leukemia [35]).
A membrane-stability analysis (AO/PI assay), as well as a DNA-fragmentation assay with the most potent compound 11
confirmed the cytocidal effect leading to apoptosis. In addition, cell cycle analyses suggest an induced mode-of-action
via modulation of DNA-synthesis. The process is a known hallmark of several clinical-approved and structurally related
deoxyadenosine analogues (e.g. fludarabine, pentostatin or cladribine [38-40]). Treatment with these drugs offered
several benefits, for example promising overall survival rates at 12 years of 75-87% for the treatment of hairy cell
leukemia with 2-chlorodeoxyadenosine [41]. However, resistance of the tumor cells via a down regulation of drug-
activating enzymes (dCK and dGK [42]) urges the necessity of further improvements [37,43]. Our findings confirm the
potential of hexopyranosyl glycon based purines as a rewarding starting point for further pharmacological
investigations.
Acknowledgements
Fundação para a Ciência e a Tecnologia is gratefully acknowledged for the postdoc research grant of Stefan Schwarz
(FCT, Project SFRH/BPD/81065/2011, Portugal) and for the support of the project Pest-OE/QUI/UI0612/2013. The
European Commission is also gratefully acknowledged for the approval of INOVAFUNAGEING commitment. The
authors also wish to thank the support of the “Gründerwerkstatt – Biowissenschaften” and Dr. Ralph Kluge for running
compounds′ elemental analysis. The cell lines were kindly provided by Dr. T. Müller (Dept. of Haematology/Oncology,
Univ. Halle).
3. Experimental Section
3.1. Synthesis and analysis
Reagents were bought from commercial suppliers without any further purification. Melting points were measured with a
Melting Point Apparatus, SMP3, Stuart Scientific, Bibby and were not corrected. NMR spectra were recorded on
BRUKER Avance 400 spectrometer at 298 K with trimethylsilane as an internal standard, δ are given in ppm and J in
Hz. Mass spectra were taken on a FINNIGAN MAT TSQ 7000 (electrospray, voltage 4.5 kV, sheath gas nitrogen)
instrument. Elemental analyses were measured on a Foss-Heraeus Vario EL unit. Optical rotations were determined on a
Perkin-Elmer 341 polarimeter. TLC was performed on silica gel (Merck 5554). Solvents were dried before use
according to usual procedures. The purity of the compounds was checked by HPLC-DAD and found to be > 95 % for
each compound.
3.2. General procedure for the N-glycosylation

ACCEPTED MANUSCRIPT
N,O-Bis(trimethylsilyl)acetamide (BSA) (1.5 eq. for -chloropurine and 3.0 eq. for 2-acetamido-6-chloropurine) was
added to a mixture of the respective purine (1.5 eq.) in dry acetonitrile (10 mL). The mixture was stirred at room
temperature for 40 min. Then, the methyl glycoside (1 eq.), dissolved in dry acetonitrile (2 mL), and trimethylsilyl
trifluoromethanesulfonate (TMSOTf, 8 eq.) were added. After continous stirring at 65 °C for 2 h, the solution was
poured into dichloromethane (10 mL), washed with a saturated solution of Na2CO3 and extracted with dichloromethane
(3 × 15 mL). The combined organic layers were washed with brine, dried (MgSO4) and concentrated. Compounds were
isolated by column chromatography.
3.2.1. 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-
glucopyranosyl)purine (2)
D-glucopyranosyl)purine (1) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-
The compounds were obtained by the reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-glucopyranoside (280 mg, 0.50
mmol) and 6-chloropurine (122 mg, 0.75 mmol) according to the general procedure. Purification by column
chromatography (ethyl acetate/cyclohexane 1:1) afforded 1 (65 mg, 19 %) and 2 (103 mg, 30 %). Data for 1: colourless
oil; Rf = 0.10 (ethyl acetate/hexane 1:2); [α] = -9° (c 1.01, CHCl3); MS (ESI): m/z (%) = 587.1 ([M-Bn+H]+, 90), 609.3
1
([M-Bn+Na]+, 48), 677.0 ([M+H]+, 100); H NMR (400 MHz, CDCl3): δ = 8.84 (s, 1H, H-2), 8.23 (s, 1H, H-8), 7.40-
7.17 (m, 15H, Bn-arom.), 7.07-6.95 (m, 3H, Bn-arom.), 6.74 (m, 2H, Bn-arom.), 5.72 (br, 1H, H-1′), 5.01 (part A of AB
system, 1H, Bn-CHH', J = 10.9), 4.95 (part B of AB system, 1H, Bn-CHH', J = 10.9), 4.89 (part A of AB system, 1H,
Bn-CHH', J = 10.7), 4.64 (part B of AB system, 1H, Bn-CHH', J = 10.7), 4.63 (part A of AB system, 1H, Bn-CHH', J =
11.5), 4.54 (part A of AB system, 1H, Bn-CHH', J = 12.1), 4.48 (part B of AB system, 1H, Bn-CHH', J = 12.1), 4.23
(part B of AB system, 1H, Bn-CHH', J = 11.5), 3.97-3.86 (br, 3H, H-2′ and H-3′ and H-5′), 3.78-3.70 (br, 3H, H-4′ and
H-6′a and H-6′b) ppm; 13C NMR (100 MHz, CDCl3): δ = 161.4 (C-4), 152.1 (C-2), 147.0 (C-8), 143.1 (C-6), 137.8 (Bn-
Cq), 137.5 (Bn-Cq), 137.5 (Bn-Cq), 135.8 (Bn-Cq), 128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-
arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.2 (Bn-arom.), 128.2 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.),
128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.8
(Bn-arom.), 127.8 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 122.1 (C-5'), 86.0 (C-3′), 85.2 (C-1′), 80.1 (C-2′),
78.1 (C-4′), 77.2 (C-5′), 75.9 (Bn-CH2), 75.3 (Bn-CH2), 74.7 (Bn-CH2), 73.5 (Bn-CH2), 68.2 (C-6′) ppm. Elemental
analysis calculated for C39H37ClN4O5 (677.2): C, 69.17; H, 5.51; N, 8.27; found: C, 68.98; H, 5.42; N, 8.02.
Data for 2: colourless crystals; mp 146-149°C; RF = 0.35 (ethyl acetate/hexane 1:2); [α] = -24° (c 1.03, CHCl3); MS
1
(ESI): m/z (%) = 677.1 ([M+H]+, 100), 699.3 ([M+Na]+, 29), 1354.7 ([2M+H]+, 12); H NMR (400 MHz, CDCl3): δ =
8.68 (s, 1H, H-2), 8.03 (s, 1H, H-8), 7.39-7.27 (m, 13H, Bn-arom.), 7.23-7.18 (m, 2H, Bn-arom.), 7.12 (m, 1H, Bn-
arom.), 6.99 (m, 2H, Bn-arom.), 5.59 (d, 1H, H-1′, J = 9.0), 4.99 (part A of AB system, 1H, Bn-CHH', J = 11.0), 4.94
(part B of AB system, 1H, Bn-CHH', J = 11.0), 4.88 (part A of AB system, 1H, Bn-CHH', J = 10.7), 4.65 (part B of AB
system, 1H, Bn-CHH', J = 10.7), 4.61 (part A of AB system, 1H, Bn-CHH', J = 11.7), 4.55 (part A of AB system, 1H,
Bn-CHH', J = 12.2), 4.48 (part B of AB system, 1H, Bn-CHH', J = 12.2), 4.17 (part B of AB system, 1H, Bn-CHH', J =
11.7), 4.07 (t, 1H, H-2′, J = 9.0), 3.91 (dd, 1H, H-3′, J = 9.0, 8.3), 3.87 (dd, 1H, H-4′, J = 9.6, 9.1), 3.76-3.69 (br, 3H, H-
6′a and H-5′ and H-6′b) ppm; 13C NMR (100 MHz, CDCl3): δ = 152.0 (C-2', 151.4 (C-4), 151.0 (C-6), 143.2 (C-8),
137.9 (Bn-Cq), 137.7 (Bn-Cq), 137.6 (Bn-Cq), 136.2 (Bn-Cq), 131.5 (C-5), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5
(Bn-arom.), 128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-
arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.),

ACCEPTED MANUSCRIPT
127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 85.9 (C-3′), 83.3 (C-1′),
79.9 (C-2′), 78.3 (C-5′), 77.2 (C-4′), 75.9 (Bn-CH2), 75.3 (Bn-CH2), 74.9 (Bn-CH2), 73.5 (Bn-CH2), 68.2 (C-6′) ppm.
Elemental analysis calculated for C39H37ClN4O5 (677.2): C, 69.17; H, 5.51; N, 8.27; found: C, 69.02; H, 5.38; N, 8.11.
3.2.2. 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-
galactopyranosyl)purine (4)
D-galactopyranosyl)purine (3) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-
The compounds were obtained by the reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-galactopyranoside (280 mg, 0.50
mmol) and 6-chloropurine (122 mg, 0.75 mmol) according to the general procedure. Purification by column
chromatography (ethyl acetate/cyclohexane 1:1) afforded 3 (51 mg, 15 %) and 4 (151 mg, 44 %). Data for 3: colourless
oil; Rf = 0.30 (ethyl acetate/hexane 1:1); [α] = -22° (c 1.00, CHCl3); MS (ESI): m/z (%) = 587.0 ([M-Bn+H]+, 100),
1
609.3 ([M-Bn+Na]+, 70), 676.9 ([M+H]+, 7); H NMR (400 MHz, CDCl3): δ = 8.83 (s, 1H, H-2), 8.26 (s, 1H, H-8),
7.44-7.23 (m, 15H, Bn-arom.), 7.11-6.96 (m, 3H, Bn-arom.), 6.75 (m, 2H, Bn-arom.), 5.78 (d, 1H, H-1′, J = 8.1), 5.01
(part A of AB system, 1H, Bn-CHH', J = 11.2), 4.85 (part A of AB system, 1H, Bn-CHH', J = 11.6), 4.78 (part B of AB
system, 1H, Bn-CHH', J = 11.6), 4.70 (part A of AB system, 1H, Bn-CHH', J = 11.4), 4.36 (part B of AB system, 1H,
Bn-CHH', J = 11.2), 4.48 (part A of AB system, 1H, Bn-CHH', J = 11.8), 4.43 (part B of AB system, 1H, Bn-CHH', J =
11.8), 4.28 (part B of AB system, 1H, Bn-CHH', J = 11.4), 4.29 (dd, 1H, H-2), 4.10 (t, 1H, H-4, J = 1.9), 3.86 (t, 1H, H-
5, J = 6.3), 3.81 (dd, 1H, H-3, J = 9.4, 2.5), 3.66-3.58 (m, 2H, H-6′a and H-6′b) ppm; 13C NMR (100 MHz, CDCl3): δ =
161.6 (C-4), 152.1 (C-2), 147.2 (C-8), 143.2 (C-6), 138.2 (Bn-Cq), 137.5 (Bn-Cq), 137.4 (Bn-Cq), 136.2 (Bn-Cq),
128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.2
(Bn-arom.), 128.2 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-
arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.6 (Bn-arom.),
127.6 (Bn-arom.), 122.3 (C-5), 85.2 (C-1′), 83.4 (C-3′), 77.2 (C-2′), 76.6 (C-5′), 75.0 (Bn-CH2), 74.9 (Bn-CH2), 73.6
(Bn-CH2), 73.1 (C-4′), 72.6 (Bn-CH2), 68.2 (C-6′) ppm. Elemental analysis calculated for C39H37ClN4O5 (677.2): C,
69.17; H, 5.51; N, 8.27; found: C, 68.97; H, 5.69; N, 7.99.
Data for 4: colourless oil; Rf = 0.30 (ethyl acetate/hexane 1:2); [α] = -16° (c 0.92, CHCl3); MS (ESI): m/z (%) = 497.1
([M-2Bn+H]+, 8), 519.3 ([M-2Bn+Na]+, 12), 587.1 ([M-Bn+H]+, 23), 609.4 ([M-Bn+Na]+, 24), 677.1 ([M+H]+, 100),
1
699.3 ([M+Na]+, 49); H NMR (400 MHz, CDCl3): δ = 8.72 (s, 1H, H-2), 8.07 (s, 1H, H-8), 7.44-7.24 (m, 15H, Bn-
arom.), 7.16 (m, 1H, Bn-arom.), 7.03 (m, 2H, Bn-arom.), 6.64 (m, 2H, Bn-arom.), 5.68 (d, 1H, H-1′, J = 9.0), 5.05 (part
A of AB system, 1H, Bn-CHH', J = 11.5), 4.84 (part A of AB system, 1H, Bn-CHH', J = 11.7), 4.79 (part B of AB
system, 1H, Bn-CHH', J = 11.7), 4.70 (part B of AB system, 1H, Bn-CHH', J = 11.5), 4.69 (part A of AB system, 1H,
Bn-CHH', J = 11.6), 4.48 (part A of AB system, 1H, Bn-CHH', J = 11.9), 4.43 (part B of AB system, 1H, Bn-CHH', J =
11.9), 4.32 (dd, 1H, H-2′, J = 9.2, 9.0), 4.24 (part B of AB system, 1H, Bn-CHH', J = 11.6), 4.09 (br, 1H, H-4′), 3.87-
3.84 (br, 2H, H-5′ and H-3′), 3.61-3.59 (br, 2H, H-6′a and H-6′b) ppm; 13C NMR (100 MHz, CDCl3): δ = 152.0 (C-2),
151.6 (C-4), 150.8 (C-6), 143.0 (C-8), 138.3 (Bn-Cq), 137.7 (Bn-Cq), 137.4 (Bn-Cq), 136.4 (Bn-Cq), 131.1 (C-5),
128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.3 (Bn-arom.), 128.3 (Bn-arom.), 128.1
(Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-
arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.6 (Bn-arom.),
127.6 (Bn-arom.), 83.4 (C-3′), 82.8 (C-1′), 77.7 (C-2′), 76.6 (C-5′), 75.1 (Bn-CH2), 74.7 (Bn-CH2), 73.6 (Bn-CH2), 73.2
(C-4′), 72.8 (Bn-CH2), 68.2 (C-6′) ppm. Elemental analysis calculated for C39H37ClN4O5 (677.2): C, 69.17; H, 5.51; N,
8.27; found: C, 69.00; H, 5.77; N, 8.17.

ACCEPTED MANUSCRIPT
3.2.3. 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-
mannopyranosyl)purine (6)
D-mannopyranosyl)purine (5) and 6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-
The compounds were obtained by the reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-mannopyranoside (250 mg, 0.45
mmol) and 6-chloropurine (104 mg, 0.67 mmol) according to the general procedure. Purification by column
chromatography (ethyl acetate/cyclohexane 1:1) afforded 5 (49 mg, 16 %) and 6 (40 mg, 13 %). Data for 5: colourless
oil; Rf = 0.31 (ethyl acetate/hexane 1:1); [α] = +62° (c 1.02, CHCl3); MS (ESI): m/z (%) = 587.1 ([M-Bn+H]+, 100),
1
609.3 ([M-Bn+Na]+, 29), 677.0 ([M+H]+, 4); H NMR (400 MHz, CDCl3): δ = 8.77 (s, 1H, H-2), 8.51 (s, 1H, H-8),
7.46-7.23 (m, 15H, Bn-arom.), 6.92-6.86 (m, 3H, Bn-arom.), 6.73 (m, 2H, Bn-arom.), 6.90 (d, 1H, H-1′, J = 1.0), 4.98
(part A of AB system, 1H, Bn-CHH', J = 10.8), 4.90 (part A of AB system, 1H, Bn-CHH', J = 11.7), 4.80 (part B of AB
system, 1H, Bn-CHH', J = 11.7), 4.68 (part A of AB system, 1H, Bn-CHH', J = 11.8), 4.66 (part B of AB system, 1H,
Bn-CHH', J = 10.8), 4.64 (part A of AB system, 1H, Bn-CHH', J = 12.4), 4.56 (part B of AB system, 1H, Bn-CHH', J =
12.4), 4.07 (t, 1H, H-4′, J = 9.5), 4.02 (dd, 1H, H-2′, J = 2.5, 1.0), 3.84 (dd, 1H, H-3′, J = 9.5, 2.5), 3.79-3.77 (br, 2H, H-
6′a and H-6′b), 3.71 (dt, 1H, H-5′, J = 9.5, 3.6), 4.34 (part B of AB system, 1H, Bn-CHH', J = 11.8) ppm; 13C NMR
(100 MHz, CDCl3): δ = 161.8 (C-4), 151.8 (C-2), 147.9 (C-8), 141.1 (C-6), 137.8 (Bn-Cq), 137.7 (Bn-Cq), 137.4 (Bn-
Cq), 135.6 (Bn-Cq), 128.8 (Bn-arom.), 128.8 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5
(Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.3 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-
arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.7 (Bn-arom.),
127.7 (Bn-arom.), 127.7 (Bn-arom.), 120.4 (C-5), 83.9 (C-1′), 82.3 (C-3′), 75.4 (Bn-CH2), 74.1 (Bn-CH2), 74.0 (Bn-
CH2), 73.6 (C-5′), 73.5 (Bn-CH2), 72.8 (C-2′), 73.5 (C-4′), 67.8 (C-6′) ppm. Elemental analysis calculated for
C39H37ClN4O5 (677.2): C, 69.17; H, 5.51; N, 8.27; found: C, 69.04; H, 5.62; N, 8.17.
Data for 6: colourless oil; Rf = 0.24 (ethyl acetate/hexane 1:2); [α] = +56° (c 0.91, CHCl3); MS (ESI): m/z (%) = 677.1
1
([M+H]+, 100), 699.3 ([M+Na]+, 38); H NMR (400 MHz, CDCl3): δ = 8.55 (s, 1H, H-2), 8.38 (s, 1H, H-8), 7.43-7.20
(m, 15H, Bn-arom.), 7.06-6.98 (m, 3H, Bn-arom.), 6.86 (m, 2H, Bn-arom.), 5.81 (d, 1H, H-1′, J = 1.1), 4.95 (part A of
AB system, 1H, Bn-CHH', J = 10.8), 4.84 (m, 2H, Bn-CH2), 4.76 (part A of AB system, 1H, Bn-CHH', J = 11.6), 4.65
(part B of AB system, 1H, Bn-CHH', J = 10.8), 4.62 (part A of AB system, 1H, Bn-CHH', J = 12.3), 4.56 (part B of AB
system, 1H, Bn-CHH', J = 12.3), 4.33 (part B of AB system, 1H, Bn-CHH', J = 11.6), 4.08-4.10 (br, 2H, H-2′ and H-4′),
3.91 (dd, 1H, H-3′, J = 9.4, 2.6), 3.77-3.71 (br, 3H, H-6′a and H-6′b and H-5′) ppm; 13C NMR (100 MHz, CDCl3): δ =
151.3 (C-2), 150.6 (C-6), 149.8 (C-4), 144.6 (C-8), 137.8 (Bn-Cq), 137.8 (Bn-Cq), 137.6 (Bn-Cq), 136.0 (Bn-Cq),
130.7 (C-5), 128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-
arom.), 128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.),
128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.6
(Bn-arom.), 127.6 (Bn-arom.), 83.1 (C-3′), 82.1 (C-1′), 78.8 (C-5′), 75.5 (Bn-CH2), 74.1 (C-4′), 74.4 (Bn-CH2), 73.5
(Bn-CH2), 73.2 (Bn-CH2), 72.6 (C-2′), 68.8 (C-6′) ppm. Elemental analysis calculated for C39H37ClN4O5 (677.2): C,
69.17; H, 5.51; N, 8.27; found: C, 69.04; H, 5.76; N, 8.04.
3.2.4. 2-Acetamido-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-
(2,3,4,6-tetra-O-benzyl-β- -glucopyranosyl)purine (8)
D-glucopyranosyl)purine (7) and 2-Acetamido-6-chloro-9-
D
The compounds were obtained by reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-glucopyranoside (280 mg, 0.50 mmol)

ACCEPTED MANUSCRIPT
and 2-acetamido-6-chloropurine (156 mg, 0.75 mmol) according to the general procedure. Purification by column
chromatography (ethyl acetate/cyclohexane 1:1) afforded 7 (91 mg, 25 %) and 8 (127 mg, 35 %). Data for 7: yellow
crystals; mp 71-74°C; Rf = 0.09 (ethyl acetate/hexane 1:1); [α] = -13° (c 0.99, CHCl3); MS (ESI): m/z (%) = 734.1
1
([M+H]+, 100), 756.2 ([M+Na]+, 78); H NMR (400 MHz, CDCl3): δ = 8.15 (s, 1H, H-8), 8.06 (s, 1H, NH), 7.39-7.17
(m, 15H, Bn-arom.), 7.10-6.99 (m, 3H, Bn-arom.), 6.80 (m, 2H, Bn-arom.), 5.61 (br, 1H, H-1′), 5.00 (part A of AB
system, 1H, Bn-CHH', J = 10.9), 4.94 (part B of AB system, 1H, Bn-CHH', J = 10.9), 4.88 (part A of AB system, 1H,
Bn-CHH', J = 10.7), 4.64 (part A of AB system, 1H, Bn-CHH', J = 11.5), 4.63 (part B of AB system, 1H, Bn-CHH', J =
10.7), 4.54 (part A of AB system, 1H, Bn-CHH', J = 12.1), 4.48 (part B of AB system, 1H, Bn-CHH', J = 12.1), 4.24
(part B of AB system, 1H, Bn-CHH', J = 11.5), 3.94-3.84 (br, 3H, H-2′ and H-3′ and H-5′), 3.77-3.67 (br, 3H, H-4′ and
H-6′ and H-6′b), 2.64 (s, 3H, Ac-Me) ppm; 13C NMR (100 MHz, CDCl3): δ = 167.6 (Ac-COO), 162.8 (C-4), 152.1 (C-
2), 149.3 (C-8), 143.6 (C-6), 137.7 (Bn-Cq), 137.5 (Bn-Cq), 136.6 (Bn-Cq), 136.0 (Bn-Cq), 128.6 (Bn-arom.), 128.6
(Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.3 (Bn-arom.), 128.3 (Bn-
arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.),
127.9 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 117.9
(C-5), 85.9 (C-1′), 85.9 (C-3′), 78.1 (C-4′), 77.2 (C-2′), 77.2 (C-5′), 75.9 (Bn-CH2), 75.3 (Bn-CH2), 74.7 (Bn-xCH2),
73.5 (Bn-CH2), 68.3 (C-6′), 25.2 (Ac-Me) ppm. Elemental analysis calculated for C41H40ClN5O6 (734.2): C, 67.07; H,
5.49; N, 9.54; found: C, 66.81; H, 5.52; N, 9.33.
Data for 8: colourless oil; Rf = 0.53 (ethyl acetate/hexane 1:1); [α] = -24° (c 1.01, CHCl3); MS (ESI): m/z (%) = 734.2
1
([M+H]+, 84), 756.3 ([M+Na]+, 100), 1468.9 ([2M+H]+, 22), 1489.7 ([2M+Na]+, 8); H NMR (400 MHz, CDCl3): δ =
8.05 (s, 1H, NH), 8.92 (s, 1H, H-8), 7.39-7.18 (m, 15H, Bn-arom.), 7.12-6.98 (m, 3H, Bn-arom.), 6.71 (m, 2H, Bn-
arom.), 5.38 (d, 1H, H-1′, J = 9.1), 5.00 (part A of AB system, 1H, Bn-CHH', J = 11.0), 4.94 (part B of AB system, 1H,
Bn-CHH', J = 11.0), 4.88 (part A of AB system, 1H, Bn-CHH', J = 10.7), 4.64 (part B of AB system, 1H, Bn-CHH', J =
10.7), 4.61 (part A of AB system, 1H, Bn-CHH', J = 11.6), 4.52 (part A of AB system, 1H, Bn-CHH', J = 12.1), 4.45
(part B of AB system, 1H, Bn-CHH', J = 12.1), 4.24 (part B of AB system, 1H, Bn-CHH', J = 11.6), 4.11 (t, 1H, H-2′, J
= 9.0), 3.87 (t, 1H, H-3′, J = 9.0), 3.80 (t, 1H, H-4′, J = 9.0), 3.75-3.68 (br, 3H, H-6′a and H-6′b and H-5′), 2.50 (s, 3H,
Ac-Me) ppm; 13C NMR (100 MHz, CDCl3): δ = 171.1 (Ac-COO), 151.9 (C-2), 151.8 (C-4), 151.2 (C-6), 143.0 (C-8),
137.9 (Bn-Cq), 137.5 (Bn-Cq), 137.5 (Bn-Cq), 136.3 (Bn-Cq), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.),
128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0
(Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-
arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.8 (C-5), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 85.9 (C-3′), 83.8 (C-1′),
75.1 (C-2′), 77.9 (C-5′), 77.2 (C-4′), 75.8 (Bn-CH2), 75.2 (Bn-CH2), 74.5 (Bn-CH2), 73.5 (Bn-CH2), 68.2 (C-6′), 25.2
(Ac-Me) ppm. Elemental analysis calculated for C41H40ClN5O6 (734.2): C, 67.07; H, 5.49; N, 9.54; found: C, 66.81; H,
5.70; N, 9.42.
3.2.5. 2-Acetamido-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-
(2,3,4,6-tetra-O-benzyl-β- -galactopyranosyl)purine (10)
Dgalactopyranosyl)purine (9) and 2-acetamido-6-chloro-9-
D
The compounds were obtained by reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-galactopyranoside (280 mg, 0.50
mmol) and ACP (156 mg, 0.75 mmol) according to the general procedure. Purification by column chromatography
(ethyl acetate/cyclohexane, 1:1) gave 9 (172 mg, 47 %) and 10 (66 mg, 18 %). Data for 9: yellow oil; Rf = 0.26 (ethyl
acetate/cyclohexane 1:1); [α] = -4° (c 1.26, CHCl3); MS (ESI): m/z (%) = 734.2 ([M+H]+, 82), 756.4 ([M+Na]+, 22),

ACCEPTED MANUSCRIPT
1468.9 ([2M+H]+, 100), 1491.2 ([2M+Na]+, 72); 1H NMR (400 MHz, CDCl3): δ = 8.16 (s, 1H, H-8), 8.00 (s, 1H, NH),
7.43-7.23 (m, 15H, Bn-arom.), 7.12-7.01 (m, 3H, Bn-arom.), 6.81 (m, 2H, Bn-arom.), 5.65 (d, 1H, H-1′, J = 7.3), 5.00
(part A of AB system, 1H, Bn-CHH', J = 11.2), 4.84 (part A of AB system, 1H, Bn-CHH', J = 11.6), 4.77 (part B of AB
system, 1H, Bn-CHH', J = 11.6), 4.71 (part A of AB system, 1H, Bn-CHH', J = 11.4), 4.62 (part B of AB system, 1H,
Bn-CHH', J = 11.2), 4.48 (part A of AB system, 1H, Bn-CHH', J = 11.8), 4.43 (part B of AB system, 1H, Bn-CHH', J =
11.8), 4.29 (part B of AB system, 1H, Bn-CHH', J = 11.4), 4.28 (br, 1H, H-2′), 4.09 (t, 1H, H-4′, J = 2.5), 3.84 (br, 1H,
H-5′), 3.79 (dd, 1H, H-3′, J = 9.3, 2.5), 3.66-3.57 (br, 2H, H-6′a and H-6′b), 2.63 (s, 3H, Ac-Me) ppm; 13C NMR (100
MHz, CDCl3): δ = 171.4 (Ac-COO), 162.9 (C-4), 152.0 (C-2), 147.9 (C-8), 143.7 (C-6), 138.1 (Bn-Cq), 137.5 (Bn-Cq),
137.3 (Bn-Cq), 136.0 (Bn-Cq), 128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.4 (Bn-
arom.), 128.4 (Bn-arom.), 128.2 (Bn-arom.), 128.2 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.),
128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9
(Bn-arom.), 127.6 (Bn-arom.), 127.6 (Bn-arom.), 118.7 (C-5), 85.2 (C-1′), 83.6 (C-3′), 77.2 (C-2′), 76.5 (C-5′), 75.1
(Bn-CH2), 75.0 (Bn-CH2), 73.6 (Bn-CH2), 73.2 (C-4′), 72.6 (Bn-CH2), 68.2 (C-6′), 25.2 (Ac-Me) ppm; analysis
calculated for C41H40ClN5O6 (734.2): C, 67.07; H, 5.49; N, 9.54; found: C, 67.00; H, 5.67; N, 9.40.
Data for 10: a colourless oil; Rf = 0.57 (ethyl acetate/hexane 1:1); [α] = -14° (c 1.02, CHCl3); MS (ESI): m/z (%) =
734.3 ([M+H]+, 100), 756.5 ([M+Na]+, 56), 1467.9 ([2M+H]+, 12); 1H NMR (400 MHz, CDCl3): δ = 7.93 (s, 1H, NH),
7.92 (s, 1H, H-8), 7.43-7.22 (m, 15H, Bn-arom.), 7.10 (m, 1H, Bn-arom.), 7.01 (m, 2H, Bn-arom.), 6.73 (m, 2H, Bn-
arom.), 5.35 (d, 1H, H-1′, J = 9.0), 4.96 (part A of AB system, 1H, Bn-CHH', J = 11.4), 4.81 (part A of AB system, 1H,
Bn-CHH', J = 11.7), 4.75 (part B of AB system, 1H, Bn-CHH', J = 11.7), 4.68 (part A of AB system, 1H, Bn-CHH', J =
11.8), 4.64 (part B of AB system, 1H, Bn-CHH', J = 11.8), 4.45 (part A of AB system, 1H, Bn-CHH', J = 11.9), 4.43 (m,
1H, H-2), 4.40 (part B of AB system, 1H, Bn-CHH', J = 11.9), 4.29 (part B of AB system, 1H, Bn-CHH', J = 11.4), 4.06
(d, 1H, H-4′, J = 1.9), 3.79 (t, 1H, H-5′, J = 6.3), 3.76 (dd, 1H, H-3′, J = 9.6, 2.6), 3.54-3.56 (br, 2H, H-6′a and H-6′b),
2.42 (s, 3H, Ac-Me) ppm; 13C NMR (100 MHz, CDCl3): δ = 171.3 (Ac-COO), 152.1 (C-2), 151.9 (C-4), 151.1 (C-6),
143.1 (C-8), 138.2 (Bn-Cq), 137.6 (Bn-Cq), 137.4 (Bn-Cq), 136.6 (Bn-Cq), 128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.4
(Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-
arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.),
128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.8 (C-5), 127.6 (Bn-arom.), 127.6 (Bn-arom.), 83.9 (C-1′),
83.6 (C-3′), 76.5 (C-5′), 75.7 (C-2′), 74.8 (Bn-CH2), 74.8 (Bn-CH2), 73.6 (Bn-CH2), 73.0 (C-4′), 72.6 (Bn-CH2), 68.2
(C-6′), 25.2 (Ac-Me) ppm; analysis calculated for C41H40ClN5O6 (734.2): C, 67.07; H, 5.49; N, 9.54; found: C, 66.92;
H, 5.61; N, 9.47.
3.2.6. 2-Acetamido-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-
(2,3,4,6-tetra-O-benzyl-β- -mannopyranosyl)purine (12)
D-mannopyranosyl)purine (11) and 2-acetamido-6-chloro-9-
D
The compounds were obtained by the reaction of methyl 2,3,4,6-tetra-O-benzyl α-D-mannopyranoside (280 mg, 0.50
mmol) and ACP (156 mg, 0.75 mmol) according to the general procedure. Purification by column chromatography
(ethyl acetate/cyclohexane 1:1) yielded 11 (95 mg, 26 %) and 12 (11 mg, 3 %). Data for 11: yellow crystals; mp 80-
82°C; Rf = 0.36 (ethyl acetate/hexane 4:1); [α] = +74° (c 0.98, CHCl3); MS (ESI): m/z (%) = 734.5 ([M+H]+, 66), 756.5
1
([M+Na]+, 24), 1469.2 ([2M+H]+, 100), 1491.3 ([2M+Na]+, 40); H NMR (400 MHz, CDCl3): δ = 8.34 (s, 1H, H-8),
8.09 (s, 1H, NH), 7.38-7.16 (m, 15H, Bn-arom.), 6.89 (m, 3H, Bn-arom.), 6.71 (m, 2H, Bn-arom.), 5.75 (d, 1H, H-1′, J
= 1.0), 4.90 (part A of AB system, 1H, Bn-CHH', J = 10.9), 4.83 (part A of AB system, 1H, Bn-CHH', J = 11.8), 4.72

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(part B of AB system, 1H, Bn-CHH', J = 11.8), 4.61 (part A of AB system, 1H, Bn-CHH', J = 11.8), 4.59 (part B of AB
system, 1H, Bn-CHH', J = 10.9), 4.55 (part A of AB system, 1H, Bn-CHH', J = 12.4), 4.49 (part B of AB system, 1H,
Bn-CHH', J = 12.4), 4.26 (part B of AB system, 1H, Bn-CHH', J = 11.8), 3.98 (t, 1H, H-4′, J = 9.4), 3.91 (br, 1H, H-2′),
3.75 (dd, 1H, H-3′, J = 9.4, 2.5), 3.70 (m, 2H, H-6′a and H-6′b), 3.63 (m, 1H, H-5′), 2.56 (s, 3H, Ac-Me) ppm; 13C
NMR (100 MHz, CDCl3): δ = 172.2 (Ac-COO), 162.9 (C-4), 152.9 (C-2), 148.5 (C-8), 141.5 (C-6), 137.7 (Bn-Cq),
137.7 (Bn-Cq), 137.4 (Bn-Cq), 135.8 (Bn-Cq), 128.8 (Bn-arom.), 128.8 (Bn-arom.), 128.6 (Bn-arom.), 128.6 (Bn-
arom.), 128.5 (Bn-arom.), 128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.2 (Bn-arom.),
128.2 (Bn-arom.), 128.1 (Bn-arom.), 128.1 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.8
(Bn-arom.), 127.8 (Bn-arom.), 127.8 (Bn-arom.), 127.6 (Bn-arom.), 116.9 (C-5), 83.9 (C-1′), 82.3 (C-3′), 78.9 (C-5′),
74.0 (C-4′), 75.2 (Bn-CH2), 74.2 (Bn-CH2), 73.7 (Bn-CH2), 73.4 (Bn-CH2), 72.9 (C-2′), 68.7 (C-6′), 25.2 (Ac-Me) ppm;
analysis calculated for C41H40ClN5O6 (734.2): C, 67.07; H, 5.49; N, 9.54; found: C, 66.87; H, 5.39; N, 9.47.
Data for 12: yellow oil; Rf = 0.61 (ethyl acetate/hexane 1:1); [α] = +77° (c 1.00, CHCl3); MS (ESI): m/z (%) = 734.5
1
([M+H]+, 100), 756.6 ([M+Na]+, 50), 1256.5 ([2M+H]+, 12); H NMR (400 MHz, CDCl3): δ = 8.21 (s, 1H, H-8), 8.19
(s, 1H, NH), 7.38-7.16 (m, 15H, Bn-arom.), 7.03-6.96 (m, 3H, Bn-arom.), 6.83 (m, 2H, Bn-arom.), 5.62 (br, 1H, H-1′),
4.89 (part A of AB system, 1H, Bn-CHH', J = 10.8), 4.79 (m, 2H, Bn-CH2), 4.72 (part A of AB system, 1H, Bn-CHH', J
= 11.6), 4.59 (part B of AB system, 1H, Bn-CHH', J = 10.8), 4.56 (part A of AB system, 1H, Bn-CHH', J = 12.2), 4.49
(part B of AB system, 1H, Bn-CHH', J = 12.2), 4.32 (part B of AB system, 1H, Bn-CHH', J = 11.6), 4.05 (br, 1H, H-2′),
4.02 (t, 1H, H-4, J = 9.5), 3.84 (dd, 1H, H-3′, J = 9.4, 2.5), 3.71-3.65 (br, 3H, H-6′a and H-6′b and H-5′), 2.38 (s, 3H,
Ac-Me) ppm; 13C NMR (100 MHz, CDCl3): δ = 170.4 (Ac-COO), 151.3 (C-4), 150.8 (C-2), 150.6 (C-6), 143.9 (C-8),
137.8 (Bn-Cq), 137.8 (Bn-Cq), 137.6 (Bn-Cq), 136.2 (Bn-Cq), 128.6 (Bn-arom.), 128.6 (Bn-arom.), 128.5 (Bn-arom.),
128.5 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.4 (Bn-arom.), 128.3 (C-5), 128.0 (Bn-
arom.), 128.0 (Bn-arom.), 128.0 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.9 (Bn-arom.), 127.8 (Bn-arom.),
127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 127.7 (Bn-arom.), 82.8 (C-3′), 82.1 (C-1′),
78.7 (C-5′), 75.3 (Bn-CH2), 74.3 (Bn-CH2), 74.1 (C-4), 73.4 (Bn-CH2), 73.1 (Bn-CH2), 72.5 (C-2′), 68.7 (C-6′), 25.0
(Ac-Me) ppm; analysis calculated for C41H40ClN5O6 (734.2): C, 67.07; H, 5.49; N, 9.54; found: C, 66.98; H, 5.54; N,
9.37.
3.3. Cell lines and Culture Conditions
The human cell lines 518A2 (melanoma), A549 (lung carcinoma), A2780 (ovarian carcinoma), HT-29 (colon
adenocarcinoma) and the NiH 3T3 mouse fibroblast cell line were included in this study. Cultures were maintained as
monolayer in RPMI 1640 (PAA Laboratories, Pasching, Germany) supplemented with 10% heat inactivated fetal bovine
serum (Biochrom AG, Berlin, Germany) and penicillin / streptomycin (PAA Laboratories) at 37 °C in a humidified
atmosphere of 5% CO2/ 95% air.
3.4. Cytotoxicity Assay [44]
The cytotoxicity of the compounds was evaluated using the sulforhodamine-B (SRB) (Sigma Aldrich) microculture
colorimetric assay. In short, exponentially growing cells were seeded into 96-well plates on day 0 at the appropriate cell
densities to prevent confluence of the cells during the period of experiment. After 24 h, the cells were treated with serial
dilutions of the compounds (0-100 µM) for 96 h. The final concentration of DMSO or DMF solvent never exceeded
0.5%, which was non-toxic to the cells. The percentages of surviving cells relative to untreated controls were

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determined 96 h after the beginning of drug exposure. After a 96 h treatment, the supernatant medium from the 96 well
plates was discarded and the cells were fixed with 10% TCA. For a thorough fixation, the plates were allowed to rest at
4 °C. After fixation, the cells were washed in a strip washer. The washing was done four times with water using
alternate dispensing and aspiration procedures. The plates were then dyed with 100 µl of 0.4% SRB (sulforhodamine B)
for about 20 min. After dying, the plates were washed with 1% acetic acid to remove the excess of the dye and allowed
to air dry overnight. 100 µl of 10 mM Tris base solution were added to each well, and absorbance was measured at λ =
570 nm (using a 96 well plate reader, Tecan Spectra, Crailsheim, Germany). The GI50 value was determined from three
independent measurements applying the two-parametric Hill slope equation.
3.5. Acridine orange/propidium iodide dye exclusion assay (AO/PI)
Apoptotic cell death was analysed by acridine orange/ethidium bromide dye using fluorescence microscopy on A549
cells. Therefore approx. 500.000 cells were seeded in cell culture flasks and were allowed to grow for 24 hours. The
medium was removed, and the substance loaded medium was added. After 24-48 h, the supernatant medium was
collected and centrifuged; the pellet was suspended in phosphate-buffer saline (PBS) and centrifuged again. The liquid
was removed and the pellet was suspended in PBS. After mixing the suspension with a solution of AO/PI, analysis was
performed under a fluorescence microscope. While a green fluorescence showed apoptosis, a red coloured nucleus
indicated necrotic cells [28,29].
3.6. DNA laddering
The DNA fragmentation assay was performed as described previously [25].
3.7. Cell cycle analysis
Approximately 1*106 cells (HT29, A2780 or NiH 3T3) were seeded in cell culture flasks (25 cm2), and the cells were
allowed to grow for 24 h. After removing of the used medium, the substance loaded medium was reloaded (or a blank
fresh medium as a control). After 24 or 48 h, the living cells were harvested, washed with PBS (with Mg2+ and Ca2+)
twice and ethanol fixed (70%, 4 °C, 1 h). After removing of the fixation and permeabilization agent, the cells were
washed with PBS buffer (with Mg2+ and Ca2+, containing 1% BSA and 0.1% NaN3, 3x1 ml, 1000 rpm) and adjusted to
1*105 million cells. The pellet was gently suspended in staining buffer (PBS buffer containing BSA, RNAase, NaN3 and
PI analog Darzynkiewicz et al. [30]) and incubated for 30 min at 37 °C. Analyses were performed using the Attune®
FACS machine; collecting data from the BL-3A channel. Doublet cells were excluded from the measurements by
plotting BL-3A against BL-3H. For each cell cycle distribution 20.000 events were collected. Distribution was
calculated by the method of Dean et al. [45].
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•
•
•
•
Synthesis of cytotoxic 6-chloropurine nucleosides with antitumor potential
6-Chloropurine nucleosides that induce apoptosis and G2/M cell cycle arrest
GI50 values of the same order of magnitude as that of chemotherapeutics cladribine
More cytotoxic than the antitumor drugs betulinic acid and tamoxifen
Mannosyl N7-linked to 2-acetamido-6-chloropurine required for the highest activity

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Supporting Information
New antitumor 6-chloropurine nucleosides inducing apoptosis
and G2/M cell cycle arrest
Stefan Schwarz,1,2 Bianka Siewert,2 René Csuk,2 Amélia P. Rauter1,*
1Centro de Química e Bioquímica/Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, Campo Grande, Ed. C8, Piso 5, 1749-016 Lisboa, Portugal; email:
aprauter@fc.ul.pt
2Bereich Organische Chemie, Martin-Luther-Universität Halle-Wittenberg, Kurt-Mothes-Str. 2, D-
06120 Halle (Saale), Germany
Corresponding author. E-mail:aprauter@fc.ul.pt
Centro de Química e Bioquímica/Departamento de Química e Bioquímica, Faculdade de Ciências,
Universidade de Lisboa, Campo Grande, Ed. C8, Piso 5, 1749-016 Lisboa, Portugal. Telephone:
+351 21 7500952. Fax: +351 21 7500088.
S1

ACCEPTED MANUSCRIPT
Table of Contents
Figure S1. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (1)..........................S3
Figure S2. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (1).........................S3
Figure S3. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (2)..........................S4
Figure S4. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (2).........................S4
Figure S5. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (3)........................S5
Figure S6. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (3).......................S5
Figure S7. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (4)........................S6
Figure S8. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (4).......................S6
Figure S9. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (5)........................S7
Figure S10. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (5).....................S7
Figure S11. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (6).......................S8
Figure S12. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (6).....................S8
Figure S13. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (7)
..........................................................................................................................................................................................S9
Figure S14. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (7)
..........................................................................................................................................................................................S9
Figure S15. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (8)
........................................................................................................................................................................................S10
Figure S16. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (8)
........................................................................................................................................................................................S10
Figure S17. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (9)
.........................................................................................................................................................................................S11
Figure S18. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (9)
.........................................................................................................................................................................................S11
Figure S19. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine
(10) .................................................................................................................................................................................S12
Figure S20. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine
(10)..................................................................................................................................................................................S12
Figure S21. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine
(11) ................................................................................................................................................................................S13
Figure S22. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine
(11)..................................................................................................................................................................................S13
Figure S23. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine
(12)..................................................................................................................................................................................S14
Figure S24. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine
(12) .................................................................................................................................................................................S14
S2

ACCEPTED MANUSCRIPT
H
N
OBn
H
N
N
O
BnO
BnO
N
H
OBn
Cl
H
H
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
Figure S1. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (1)
H
N
OBn
H
N
N
O
BnO
BnO
N
H
OBn
Cl
H
H
160 155 150 145 140 135 130 125 120 115 110 105 100
95
f1 (ppm)
90
85
80
75
70
65
60
55
50
45
40
35
30
Figure S2. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (1)
S3

ACCEPTED MANUSCRIPT
Cl
N
H
OBn
H
N
N
N
O
BnO
BnO
H
OBn
H
H
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S3. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (2)
Cl
H
OBn
N
N
H
O
BnO
BnO
N
N
H
OBn
H
H
165
160
155
150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
f1 (ppm)
Figure S4. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (2)
S4

ACCEPTED MANUSCRIPT
OBn
N
OBn
H
N
N
O
H
N
BnO
H
OBn
Cl
H
H
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S5. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (3)
OBn
N
OBn
H
N
N
O
H
N
BnO
H
OBn
Cl
H
H
165 160 155 150 145 140 135 130 125 120 115 110 105 100
95
90
85
80
75
70
65
60
55
50
45
40
35
f1 (ppm)
Figure S6. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (3)
S5

ACCEPTED MANUSCRIPT
Cl
OBn
OBn
N
N
H
O
H
N
BnO
N
H
OBn
H
H
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S7. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (4)
Cl
OBn
OBn
N
N
H
O
H
BnO
N
N
H
OBn
H
H
155 150 145 140 135 130 125 120 115 110 105 100 95
90
85
f1 (ppm)
80
75
70
65
60
55
50
45
40
35
30
25
20
15
Figure S8. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-galactopyranosyl)purine (4)
S6

ACCEPTED MANUSCRIPT
H
N
OBn
OBn
N
N
O
BnO
BnO
N
H
H
Cl
H
H
9.0
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S9. 1H NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (5)
H
N
OBn
OBn
N
N
O
BnO
BnO
N
H
H
Cl
H
H
165 160 155 150 145 140 135 130 125 120 115 110 105 100
f1 (ppm)
95
90
85
80
75
70
65
60
55
50
45
40
35
Figure S10. 13C NMR spectrum of 6-Chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (5)
S7

ACCEPTED MANUSCRIPT
Cl
N
H
OBn
OBn
N
N
N
O
BnO
BnO
H
H
H
H
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S11. 1H NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (6)
Cl
H
OBn
N
N
OBn
O
BnO
BnO
N
N
H
H
H
H
155
150
145
140
135
130
125
120
115
110
105
100
95
90
85
80
75
70
65
60
55
50
45
40
f1 (ppm)
Figure S12. 13C NMR spectrum of 6-Chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-mannopyranosyl)purine (6)
S8

ACCEPTED MANUSCRIPT
H
N
NHAc
OBn
H
N
N
O
BnO
N
BnO
H
OBn
Cl
H
H
8.5
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S13. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (7)
H
N
NHAc
OBn
H
N
N
O
BnO
BnO
N
H
OBn
Cl
H
H
170 165 160 155 150 145 140 135 130 125 120 115 110 105 100 95
f1 (ppm)
90
85
80
75
70
65
60
55
50
45
40
35
30
25
Figure S14. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-7-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (7)
S9

ACCEPTED MANUSCRIPT
Cl
N
H
OBn
H
N
N
N
O
BnO
BnO
NHAc
H
OBn
H
H
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
f1 (ppm)
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
Figure S15. 1H NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (8)
Cl
H
OBn
N
N
H
O
BnO
BnO
N
N
NHAc
H
OBn
H
H
165
155
145
135
125
115
105
95
f1 (ppm)
90
85
80
75
70
65
60
55
50 45
40
35
30
25
20
Figure S16. 13C NMR spectrum of 2-(Acetylamino)-6-chloro-9-(2,3,4,6-tetra-O-benzyl-β-D-glucopyranosyl)purine (8)
S10