Efficient synthesis of 2′-deoxyzebularine and its α-anomer by the silyl method of N-glycosylation. Crystal structures and conformational study in solution

Article abstract of DOI:10.1016/j.carres.2014.03.018

2′-Deoxyzebularine and its α-anomer have been efficiently synthesized with relatively high stereoselectivity by a modified procedure of the silyl method of the N-glycosidic bond formation. An SnCl₄- catalyzed condensation of silylated pyrimidin

Full text of DOI:10.1016/j.carres.2014.03.018

Accepted Manuscript
Efficient synthesis of 2’-deoxyzebularine and its α-anomer by the silyl method
of N-glycosylation. Crystal structures and conformational study in solution
Katarzyna Ebenryter-Olbinska, Janina Karolak-Wojciechowska, Elzbieta
Sochacka
PII:
S0008-6215(14)00122-0
DOI:
http://dx.doi.org/10.1016/j.carres.2014.03.018
CAR 6713
Reference:
Carbohydrate Research
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 February 2014
17 March 2014
18 March 2014
Please cite this article as: Ebenryter-Olbinska, K., Karolak-Wojciechowska, J., Sochacka, E., Efficient synthesis of
2’-deoxyzebularine and its α-anomer by the silyl method of N-glycosylation. Crystal structures and conformational
study in solution, Carbohydrate Research (2014), doi: http://dx.doi.org/10.1016/j.carres.2014.03.018
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Efficient synthesis of 2’-deoxyzebularine and its α-anomer by the silyl method of
N-glycosylation. Crystal structures and conformational study in solution.
Katarzyna Ebenryter - Olbinska1, Janina Karolak-Wojciechowska2 and Elzbieta Sochacka1*
1Institute of Organic Chemistry and 2Institute of General and Ecological Chemistry,
Technical University of Lodz, Zeromskiego 116, 90-924 Lodz, Poland
*Corresponding author: tel. +48 42 6313155, fax +48 42 6365530, e-mail:
elzbieta.sochacka@p.lodz.pl
Abstract:
2’-Deoxyzebularine and its α-anomer have been efficiently synthesized with relatively high
stereoselectivity by a modified procedure of the silyl method of the N-glycosidic bond
formation. An SnCl4-catalyzed condensation of silylated pyrimidin-2-one with 1-α-chloro-
3,5-di-O-p-toluoyl-2-deoxy-D-ribofuranose under kinetic control condition (-33°C, 1,2-
dichloroethane) led to the mixture of β- and α-anomeric nucleosides in 3:1 ratio. Analogous
condensation at +35 °C (thermodynamic control conditions) provided mainly p-toluoyl
protected α-2’-deoxyzebularine (α:β = 4:1), easily separated by crystallization from the
anomeric mixture. The structures of both 2’-deoxyzebularine anomers were confirmed by X-
ray analysis of the crystals and conformational studies in solution performed using an NMR
method.
Keywords: 2’-deoxyzebularine, zebularine, modified nucleosides, 2’-deoxynucleosides
synthesis, stereoselective N-glycosylation, nucleoside conformation
1. Introduction
Modified nucleosides are fundamental bioactive compounds applied as antiviral and
anticancer agents.1 Important representatives of such type of nucleosides are 2’-
deoxyzebularine (1-β-D-deoxyribofuranosyl-1,2-dihydropyrimidin-2-one, β-dZeb, 1a) and
zebularine (1-β-D-ribofuranosyl-1,2-dihydropyrimidin-2-one, Zeb, 2, Fig.1) containing 2-
pyrimidinone as an aglycon residue.2
N
N
O
HO
HO
O
O
O
N
N
OH
R
OH
H
R=H 1a
R=OH 2
1b
α
1- -D-deoxyribofuranosyl-1,2-dihydropyrimidin-2-one
1- -D-deoxyribofuranosyl-1,2-dihydropyrimidin-2-one
β
(2'-deoxyzebularine, dZeb, 1a)
β−
(
dZeb, 1b)
α−
1- -D-ribofuranosyl-1,2-dihydropyrimidin-2-one
β
(zebularine, Zeb, 2)
Figure 1. Structures of pyrimidin-2-one nucleosides.
Formally, these nucleosides can be considered as 2’-deoxycytidine/cytidine analogs deprived
of the exocyclic amino group at the C4 atom of the heterobase. The lack of the NH2 function
1

at the pyrimidine ring increases electrophilicity of the resulting 2-pyrimidinone heterobase in
β-dZeb and Zeb, making them active bacteriostatic agents3 and conferring inhibitory
properties towards cytidine deaminase (CDA) and DNA-cytosine methyltransferases
(DNMT).4,5 The inhibitory property of zebularine towards DNMT makes this compound a
good drug candidate for epigenetic anticancer therapy.2 Zebularine exerts its DNA
methyltransferase inhibitory activity after being transformed (on a complex metabolic route6)
into 2’-deoxyzebularine 5’-triphosphate (dZebTP), further incorporated into a DNA chain at
specific CpG methylation sites, and, eventually, trapping DNMT enzymes by a covalent
binding6b,d. Interestingly, the 2’-deoxynucleoside 1a itself cannot be directly used as an
inhibitor of DNMT since it does not serve as a substrate for cellular phosphorylating enzymes
and is not transformed to the active form (dZebTP). Instead, various prodrugs of 2’-
deoxyzebularine, which can be intracellularly unmasked into 2’-deoxyzebularine 5’-
monophosphate derivative and further transformed to the required 5’-triphosphate are tested
as potential DNMT inhibitors.7 Multiple elaborations of 2’-deoxyzebularine prodrug
strategies, as well as studies on a structure-function relationship of methyltransferases
inhibitors using dZeb-derived model oligodeoxyribonucleotides,8-12 support the need for
efficient methods of synthesis of the pyrimidin-2-one nucleosides.
While zebularine 2 can be efficiently synthesized, especially by the Vorbruggen method,13 2’-
deoxyzebularine is not easily accessible. The methods of synthesis of 2’-deoxyzebularine are
based mainly on transformations of 2’-deoxycytidine or 2’-deoxyuridine (which is not a
natural component of DNA),7a,9,14-17 or on deoxygenation of ribonucleoside 2.18
The first synthesis of 2’-deoxyzebularine was carried out by a direct reduction of the 2’-
deoxyuridine derivative with sodium amalgam and gave the desired product with low yield.14
The next method, similarly inefficient, described by Wightman and Holy, was based on
reductive desulfurization of 4-thiouracil nucleoside with deactivated Raney-Ni.15 Then, 2’-
deoxyzebularine was prepared with relatively better yield (~40-50%) by oxidation of a 4-
hydrazinopyrimidinone nucleoside and subsequent rearrangement of the intermediary diazene
with Ag2O in refluxing aqueous dioxane or EtOH.16 However, all these procedures use
metallic components, which should be avoided in synthesis of compounds to be used in
biological experiments because of possible toxic side effects. Only recently, a metal-free
method was published, in which the homogeneous oxidation of 4-hydrazinopyrimidinone
nucleosides was performed with N-phenyltriazolinedione (PTAD) resulting in formation of 5-
methyl derivatives of 2’-deoxyzebularine and 2’,3’-dideoxy-analog.17 An alternative
approach, often used for the 2’-deoxyzebularine preparation, starts with a ribonucleoside
analog as a substrate. Chemical deoxygenation of the 2’-hydroxyl group of a properly
protected zebularine derivative allows to obtain the 2’-deoxynucleoside in moderate overall
yield, although the analytically pure nucleoside is finally isolated by HPLC.18a
Preparative methods of the N-glycosidic bond formation were not extensively used for the
synthesis of 2’-deoxyzebularine since it was assumed that the product would be formed as a
mixture of two anomers, usually in the 1:1 ratio, and the separation of α- and β-anomers
would reduce the overall yield of the desired nucleoside. Indeed, the first glycosylation
procedure employing condensation of the mercury salt of pyrimidin-2-one nucleobase with
3,5-di-O-(p-toluoyl)-2-deoxy-α-D-ribofuranosyl chloride reported by Wightman and Holy,
yielded only traces of the required nucleoside product.15 Another procedure, based on the
coupling of the pyrimidin-2-one chloromercury salt with 3,5-di-O-(p-toluoyl)-2-deoxy-α-D-
ribofuranosyl chloride in dry acetonitrile, afforded only 14-17% of the β-anomeric nucleoside
and 30-33% of the α-anomeric counterpart.19 Condensation of a silylated nucleobase with a
2

more
stable 1-alkoxysugar
derivative in the presence of trimethylsilyl
trifluoromethanesulfonate as a catalyst in acetonitrile (3h, 20°C) after laborious
chromatographic purification on an Al2O3 column gave the β-anomer in 30% yield.20
The α-anomer of 2’-deoxyzebularine was stereoselectively synthesized upon glycosylation of
a lithium salt of pyrimidin-2-one with 3,5-di-O-(p-toluoyl)-2-deoxy-α-D-ribofuranosyl
chloride (reaction in acetonitrile at 60°C; 64% yield). Under similar conditions, the reaction
carried in the presence of KOH also yielded only the α–D-nucleoside, whereas the Na salt of
the heterobase gave 28% and 35% of β- and α-anomers, respectively.21
The silyl method of glycosylation was applied for synthesis of several 5-substituted
pyrimidin-2-one 2’-deoxynucleosides, e.g. substituted with the -SCH3 group,22 halogen atoms
(Br, I),23 alkynyl (ethynyl, 1-propynyl) residues,24 or ethyl25 and phenyl26 groups. However,
the syntheses occurred to be rather inefficient and in most cases the α-isomer was the main
product.
In this paper we report an efficient procedure for the synthesis of 2’-deoxyzebularine (β-dZeb,
1a) and its α-anomer (α-dZeb, 1b) under improved conditions of the Vorbruggen silyl
methodology, which offers relatively high stereoselectivity of N-glycosylation of the
pyrimidin-2-one base and allows to obtain both isomers 1a and 1b in good yields. The
structure study of both anomers by the X-ray and NMR methods are also presented.
2. Results and discussion
2.1. Synthesis
In the classical procedure for the synthesis of 2’-deoxyribonucleosides, silylated heterocyclic
bases are treated with suitably protected 2-deoxyribose derivatives in the presence of various
Lewis acid catalysts. The yield of nucleoside products and the ratio of isomers strongly
depend on the reagents and reaction conditions.27 In the case of 2’-deoxyzebularine, the
synthesis by the above mentioned methodology delivers one N-regioisomer only since the
structure of the nucleobase is symmetrical in respect to the location of two nitrogen atoms,
which are equally available for nucleophilic substitution with a sugar moiety. Thus, the
synthetic problem concerns mainly the stereoselectivity of N-glycosylation. Several studies of
the N-glycosylation reactions have generally addressed thermodynamic and kinetic factors
and their roles as determinants of anomeric product distribution.23,26,28-30 It was shown, that in
the case of synthesis of 5-substituted 2’-deoxyzebularines, the formation of undesired α-
anomers was thermodynamically favored under the “equilibrating” conditions employed in
the silyl method of N-glycosylation.23,26 For example, a low-temperature (-20°C),
trimethylsilyl triflate catalyzed condensation of silylated 5-phenylpyrimidinon-2-one with
methyl 3,5-di-O-(p-toluoyl)-2-deoxyribofuranoside in acetonitrile gave the nucleoside
products in 58 % yield with a 2:1 ratio of the β:α anomers, while an analogous reaction
performed at 20°C yielded the mixture containing more of the α-anomer than under previous
conditions (β:α anomers in a 3:2 ratio).
Considering the above observations we started our experiments on 2’-deoxyzebularine
synthesis by the Vorbruggen methodology under conditions typical for kinetic control of the
N-glycoside bond formation, which were expected to give preferably the nucleoside product
with β-anomeric configuration (Scheme 1).
3

N
N
(CH3)3SiCl
N
3
OH
N
4
OSi(CH3)3
Et3N / benzene
SnCl4 (3eq, entry 1,2)
conditions for kinetic control
N
N
O
TolO
TolO
TolO
O
O
O
N
+
+
Cl
N
OSi(CH3)3
N
O
TolO
TolO
TolO
5 (1 eq)
4 (2 eq)
N
6a
6b
SnCl4 (2 eq, entry 3,4)
conditions for thermodynamic control
Yield (%) of
isolated
nucleoside
fraction
β:α on the basis
Entry
Temp / °C
Time / h
Solvent
of 1H NMR
1
2
3
4
-33
-40
35
3.5
3.5
3.0
3.0
ClCH2CH2Cl
CH2Cl2
74
60
91
70
3:1
3:1
1:4
1:4
ClCH2CH2Cl
CH2Cl2
35
Scheme 1. Condensation of 1α-chloro-3,5-di O-p-toluoyl-2-deoxy-D-ribofuranose (5) with
silylated pyrimidin-2-one (4) using SnCl4 as a Lewis acid catalyst under kinetic (entry 1, 2)
and thermodynamic control conditions (entry 3, 4).
Selected as the sugar component 1-α-chloro-3,5-di-O-p-toluoyl-2-deoxy-D-ribofuranose (5)
was prepared in three steps from 2-deoxy-D-ribose according to the modified Hoffer’s
procedure.31 For reproducible results of condensation with the silylated base 4, careful pre-
drying of powdered 5 (4-18 h at 0.1 mm Hg) was needed just before the use. The pure
silylated base was obtained in a large scale by the treatment of pyrimidin-2-one 3 with
trimethylsilyl chloride in benzene and distillation of crude 4 under reduced pressure (yield
72%).
The protected chlorosugar derivative 5 was reacted with 2 equivalents of the silylated base 4
in the presence of 3 equivalents of SnCl4 in 1,2-dichloroethane at -33°C. Such molar excess of
a Lewis acid catalyst was previously reported to be crucial for stereoselective synthesis of β-
anomer of 2’-deoxy-2-thiouridine performed in 1,1,2,2-tetrachloroethane at -40°C.29 The
progress of the reaction of 5 with 4 was monitored by TLC (CHCl3/MeOH, 98/2), and after
3.5 h the disappearance of almost all starting sugar substrate was noticed. After a typical
aqueous work up and chromatographic separation, the nucleoside fraction was isolated in 74%
4

yield. Its 1H NMR spectrum showed the signals characteristic for both anomeric nucleosides.
Clearly separated signals for H-6 heterobase protons indicate a 3:1 ratio of isomers, but the
overlapping signals for the H-1’ sugar protons with the heterobases H-5 protons (Fig. 2, A1;
entire spectrum Fig. S2a, Supplementary Material) do not allow for precise determination of
the anomeric configuration of the major product, usually based on the empirical “triplet-
quartet peak” rule.25,32,33 According to this well experimentally established method, the signal
of the anomeric proton in the β-series of 2’-deoxynucleosides consists of three lines (a
pseudo-triplet) and that of the α-series consists of four lines (quartet), which, at low
resolution, appears often as a pseudo-doublet. In our 2’-deoxyzebularine synthesis, the better
resolution of the anomeric protons was obtained after removal of the p-toluoyl groups from
the sugar residue. Analysis of the 1H NMR spectrum recorded for a mixture of the deprotected
isomers revealed that the main product has the β configuration as the signal of the anomeric
proton of higher intensity appears as the pseudotriplet (Fig. 2, A2; Fig. S2b Supplementary
Material).
Kinetic control
Thermodynamic control
α + β H1’ + H5
βH6 αH6
α+β H5+H1'
β H6
α H6
8.15 8.10
6.40
6.35
6.30
6.25
8.2
ppm
ppm
ppm
6.4
6.3
ppm
A1) a mixture of p-toluoyl nucleosides
B1) a mixture of p-toluoyl nucleosides
β H6
α H6
β H1’ + α H1’
βH6
αH6
βH1' αH1'
8.4
6.1
ppm
ppm
8.30
8.25
8.20
6.0
ppm
ppm
A2) a mixture of the sugar deprotected
nucleosides
B2) a mixture of the sugar
deprotected nucleosides
1
Figure 2. H-6, H-5 heterobase and anomeric protons regions of H NMR spectra of reaction
mixtures: A) under kinetic control conditions (-33°C, in ClCH2CH2Cl); B) under
thermodynamic control conditions (+35°C, in ClCH2CH2Cl).
The desired β-anomer fraction of 3’,5’-O-(p-toluoyl)-2’-deoxyzebularine (6a) containing
1
(according to the H NMR spectrum, Fig. S4) only ~7% of the α-anomer was obtained from
5

the filtrate remaining after precipitation of the α-nucleoside 6b in a CHCl3/hexane solvents
system. Further separation of the anomers using silica gel column chromatography (with n-
butanol as an eluent) afforded the pure β-nucleoside 6a (Fig. S5) in overall 44% yield. It is
worth noting that observed easy crystallization of the α-anomer 6b from the mixture of
isomers, even at its low content, significantly facilitates isolation of the desired β-anomer 6a.
Lowering the temperature of the condensation of the chlorosugar 5 with the silylated base 4 to
-40°C (possible when methylene chloride was used as a solvent), resulted in decreasing of the
overall yield of the nucleoside fraction and did not improve a β:α ratio (Scheme 1, entry 2).
Removal of the p-toluoyl protecting groups from the nucleoside 6a by standard sodium
methoxide/methanol transesterification lead almost quantitatively to the deacylated nucleoside
1a. The β configuration of this product was unambiguously confirmed by its spectral
characteristics (1H and13C NMR, CI-MS, IR, UV, Supplementary Material, Fig. S6-Fig. S10)
and by X-ray analysis (see Structural study part).
An analogous reaction of the chlorosugar derivative 5 with 2 equivalents of the silylated base
4 in the presence of 2 equivalents of SnCl4 in 1,2-dichloroethane performed under
thermodynamic control conditions (+35°C) provided predominately the α-nucleoside 6b. The
1
ratio of α:β as 4:1 was determined by integrating the heterobase H-6 protons in a H NMR
1
spectrum of the isolated nucleoside fraction (Fig. 2, B1 and B2; entire H NMR spectra –
Supplementary Material, Fig. S11b and S11b). The pure α-anomer 6b was obtained by
fractional crystallization. The first crop of crystals from CHCl3/hexane gave the α–nucleoside
1
6b contaminated with ~6% of the β-anomer (according to the H NMR spectrum, Fig. S12).
Recrystalization of this product from the same solvents system afforded the pure α-isomer 6b
(Fig. S13) in 53.5 % overall yield. Also in this case, the use of methylene chloride as a solvent
did not improve the α:β ratio and, unfavorably, decreased the overall yield of the nucleoside
fraction (scheme 1, entry 4).
The p-toluoyl nucleoside 6b was efficiently deprotected by standard MeONa/MeOH
treatment to give α-anomer of 2’-deoxyzebularine 1b. The structure of the nucleoside was
fully confirmed by spectral data (Fig. S14-Fig. S19, Supplementary Material) and by an X-ray
analysis (see Structural study part).
The results of our studies on the synthesis of 1,2-pyrimidin-2-one 2’-deoxynucleosides
strongly indicate that the β-anomer 6a is the kinetic product and the α-nucleoside 6b is the
thermodynamic product. Indeed, when the pure β-nucleoside 6a was treated with the silylated
base 4 and SnCl4 in ClCH2CH2Cl at+35°C it underwent transglycosylation resulting in the
mixture of the α and β isomers at a 4:1 ratio, the same as in a direct glycosylation reaction
carried out at the same temperature (Fig. S20 compare to Fig. S11a).
2.2. Structural study
2.2a X-ray structure analysis
The ORTHEP drawings of 2’-deoxyzebularine (β-dZeb, 1a) and its α-anomer (α-dZeb, 1b)
are presented in Fig. 3 showing the main conformational features of the molecules. Selected
geometrical parameters, indispensable for conformation discussion, and the remaining
crystallographic data are given in Supplementary Material (Table S1-S4). The crystal
structures of β-dZeb and α-dZeb have not been published until now and the complete
crystallographic data have been deposited at Cambridge Crystallographic Data Center
(deposition numbers CCDC 970241-970242).
6

Figure 3. ORTHEP drawings of 2’-deoxyzebularine (β-dZeb, 1a) and its α-anomer (α-dZeb,
1b); Conformational parameters of β-dZeb - C3’-endo/C2’-exo; anti, gauche (+) and α-dZeb -
C2’-endo/C3’-exo, anti, trans.
In both studied nucleosides the pyrimidin-2-one ring is essentially planar and its geometry is
similar to those of natural pyrimidine heterobases.34 However, the absence of the acceptor
carbonyl function at the C-4 position and the lack of the donor hydrogen atom at N-3 in the
heterobase of 1a and 1b significantly influence the conformational features of these
nucleosides, mainly due to different possibilities in the hydrogen bonding interactions in
comparison to the natural pyrimidine nucleosides.34,35 Comprehensive descriptions of the
observed hydrogen bonds pattern for both structures are summarized in Table 1. The
corresponding illustrations are placed in Supplementary Material (Fig. S21 and S22 for β-
dZeb and α-dZeb, respectively).
Table 1. The geometry of hydrogen bonds in the crystal of β-dZeb (1a) and α-dZeb (1b).
D-H..A
(sym. code)
D…A (Å)
D-H..A (◦)
O3'-H3'O…N3
O5'-H5'O…O2
O5'-H5'O…O4'
O5'-H5'O…N3
O3'-H3'O…O5'
1/2+x,3/2-y,-z
1+x,y,z
2.8623(16)
2.7716(15)
2.8952(12)
2.8087(14)
2.8004(13)
167
168
104
152
155
β-dZeb
α-dZeb
intermolecular
-x,-1/2+y,1/2-z
1-x,1/2+y,1/2-z
The single-crystal X-ray structures of 1a and 1b reveal that both nucleosides adopt the
expected anti conformation around the N-glycoside bond, with χ= -166.3° and χ=167.9°,
respectively. The sugar ring of β-dZeb has a nearly ideal twist conformation with C3’-
endo/C2’-exo puckering (P=5.6° and ψm=35.8°, an N-type conformation), which is not often
found in 2’-deoxynucleosides in the solid state.34a As expected,34b the opposite conformation
to that of β-nucleoside is observed in the molecules of the α-anomer 1b, with C2’-endo/C3’-
exo (P=174.3°, ψm=33.8°, an S-type conformation) sugar pucker. For the arrangement around
the C4’-C5’ sugar bond, the most populated gauche (+) conformation is found in the structure
of the β-anomeric nucleoside 1a, while trans orientation is preferred for the α-nucleoside 1b,
due to different hydrogen bonds system in both crystals.
2.2b. NMR data
Various NMR techniques were used to determine the conformation of the pyrimidin-2-one 2’-
deoxynucleosides 1a and 1b in an aqueous solution. Generally, nucleoside conformation in
solution is characterized by a dominant sugar pucker, a preferred glycosidic bond arrangement
and conformation around the C4’-C5’ bond.36-38 The chemical shifts for hydrogen and carbon
7

atoms of 1a and 1b were assigned by analysis of the 1H and 13C NMR spectra and the selected
data are given in Table 2 and in Supplementary Material (Figs. S6, S7 and S14-S16).
Additionally, a NOE experiment was used to confirm the assignments of the chemical shifts
for hydrogen atoms H-2’ and H-2’’ located at the same or the opposite side of the sugar ring as
the 4’-CH2OH function (Table 3). In a β-dZeb molecule, a weaker NOE effect (1.4%) from
H-1’ to the proton resonating at 2.35 ppm indicates its trans location (H-2’), because a
stronger NOE interaction (5.7%) between H-1’ and the hydrogen atom resonating at 2.66 ppm
confirms a shorter distance expected for H-2’’. In the case of α-dZeb, the opposite orientation
of H-2’ and H-2’’ in respect to H-1’ results in a stronger NOE between H-1’ and H-2’ (4.0%)
and a weaker one between H-1’ and H-2’’(1.2%). This confirms the assignment of the signal
at 2.79 ppm to H-2’ and that at 2.27 ppm to H-2”. The values of proton-proton coupling
constants essential for conformational analysis of a nucleoside sugar moiety are presented in
1
Table 2b. Analyzing a H NMR spectrum for α-dZeb we encountered a problem (typical for
the α–anomers of 2’-deoxynucleosides38) with determination of H2’-H3’, H2’’-H3’ and H3’-
H4’ vicinal coupling constants. It was resolved numerically using a DAISY (Bruker Biospin)
deconvolution procedure for the overlapping sugar proton resonances (the real and simulated
1H NMR spectra for 1b are shown on Fig.S15, Supplementary Material).
Table 2. NMR data of β-dZeb and α-dZeb; a) 1H chemical shifts, b) proton-proton coupling
constants, c) 13C chemical shifts
Chemical shifts
a)
1’
2’
2’’
3’
4’
5’
5’’
4
5
6
protons
β-dZeb
α-dZeb
α-dZeb*
6.23
6.18
6.18
2.35
2.79
2.79
2.66
2.27
2.27
4.42
4.44
4.41
4.19
4.58
4.58
3.90
3.75
3.75
3.79
3.67
3.67
8.63
8.63
8.63
6.78
6.78
6.78
8.52
8.44
8.44
Coupling constants
1’-2’ 1’-2’’ 2’-2’’ 2’-3’ 2’’-3’ 3’-4’ 4’-5’ 4’-5’’ 5’-5’’ 4-5
b)
5-6
4-6
6.1
7.0
7.2
6.6
1.4
1.5
14.3
15.0
15.0
6.5
nd
4.7
nd
4.3
nd
3.5
4.2
4.1
5.4
5.6
5.3
12.6 4.4 6.7 2.6
12.3 4.4 6.6 2.6
12.4 4.5 6.6 2.7
β-dZeb
α-dZeb
α-dZeb*
5.6
1.8
1.5
*values from simulated 1H spectrum
c) 13C
Chemical shifts
C-1’
88.2
90.3
C-2’
40.0
40.1
C-3’
70.0
71.0
C-4’
87.6
90.0
C-5’
C-2
C-4
C-5
C-6
60.9
61.7
156.7
156.7
166.5
166.2
106.5
106.1
145.2
145.7
β-dZeb
α-dZeb
Conformation of a nucleoside sugar ring in solution is usually described in terms of the
equilibrium between two more stable puckered forms – C2’-endo (an S type conformer) and
C3’-endo (an N type conformer). Their relative populations can be calculated on the basis of
proton-proton vicinal coupling constants from the following equation: conformer Smolar fraction
=
JN - Jexp / JN – JS, where Jexp is the experimentally determined coupling constant J3’-4’, JN =8.4
Hz and JS= 1.1 Hz are the limiting values of J3’-4’ for 2’-deoxynucleosides.37-39 The obtained
data for β-dZeb (S=0.57) and for α-dZeb (S=0.95) reveal that in both isomers the S type
conformation is preferred, with a greater extent in the α-anomer. It is worth to notice that the
dominant S conformation of β-dZeb in solution is different from the N-type sugar pucker
8

(C3’-endo/C2’-exo) adopted by this nucleoside in the solid state due to hydrogen bond
network in the crystals.
A considerable preference of the S conformation over the N one in the β-anomer in solution
and a very strong preference in the α-anomer is a characteristic property of 2’-
deoxynucleosides with the anti arrangement around the N-glycosidic bond.38 Indeed, the
predominating anti conformation around the C1’-N1 bond was confirmed for both anomers
using 1D NOE experiments.36,40 Irradiation of H-6 of β-dZeb revealed the combined NOEs of
6.3% to H-2’ and H-3’ and only 1.8% to the H-1’, while irradiation of H-6 in α-dZeb led to
relatively strong NOE to H-4’ (7.5%) in comparison to weaker interaction with H-1’(2.1%)
(Table 3, Fig.S24 and S26; Supplementary Material), what is typical for anti 2’-
deoxynucleosides in β or α anomeric configuration, respectively.40
Table 3.Results of 1D NOE experiments for β-dZeb and α-dZeb, (25°C in D2O at 700 MHz)
Irradiated
proton
Irradiated
proton
Compound
NOE (%)
Compound
NOE (%)
β-dZeb
H-6
H-1’ (1.8)
α-dZeb
H-6
H-1’ (2.1)
H-2’ (5.2)
H-2” (2.0)
H-3’ (1.1)
H-2’+H-3’ (6.3)
H-4’
H-2’ (1.1)
H-2” (2.0)
H-3’ (1.1)
H-4’ (7.5)
H-1’
H-2’ (1.4)
H-2” (5.6)
H-1’
H-2’ (4.0)
H-2” (1.2)
Table 4. Comparison of β-dZeb and α-dZeb structure in crystals and in solution.
β-dZeb
α-dZeb
a) Crystal structure
Sugar moiety C3’-endo C2’-exo C2’-endo C3’-exo
conformation
N-type
S-type
C4’-C5’
C1’-N1
gauche (+)
anti
trans
anti
b) conformation in solution
Sugar moiety
conformation
56% C2’-endo
S-type
94% C2’-endo
S-type
46% gauche (+)
42% trans
12% gauche (-)
42% gauche (+)
38% trans
20% gauche (-)
C4’-C5’
C1’-N1
anti
anti
9

3. Conclusions
2’-Deoxyzebularine and its α-anomer were synthesized in good overall yields by a modified
Vorbruggen silyl procedure of N-glycosylation using 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-
ribofuranosyl chloride as a sugar component and SnCl4 as a Lewis acid catalyst. Low-
temperature condensation under kinetic control condition (-33°C, 1,2-dichloroethane) led
predominantly to the β-anomeric nucleoside (β:α in a 3:1 ratio), while analogous condensation
at +35 °C (thermodynamic control conditions) provided mainly p-toluoyl protected α-2’-
deoxyzebularine (α:β =4:1). Isolation of anomeric pure nucleosides is strongly facilitated by
the susceptibility of p-toluoylated α-2'-deoxyzebularine to easy crystallization from the
mixture of isomers (even at its low content) using a CHCl3/hexane solvents system.
The specific, symmetric structure of the pyrimidin-2-one heterobase in respect to nitrogen
atom location, that resulted in only one N-regioisomer formation, and the obtained relatively
high anomeric stereoselectivity under our modified conditions of N-glycosylation offers the
presented procedure as an alternative, effective route to pyrimidin-2-one 2’-deoxynucleosides
as compared to the methods consisting of multi-step transformation of 2’-deoxynucleoside
substrates (2’-deoxyuridine or 2’-deoxycytidine).
The anomeric configuration of both obtained 2’-deoxyzebularine isomers (1a and 1b) was
unambiguously confirmed by X-ray analysis and 1H NMR data. Additionally, the
conformational analysis of 2’-deoxyzebularine and its α-anomer in aqueous solution allowed
to compare the conformational parameters of both nucleosides to that observed in the crystals
(Table 4).
4.Experimental
General
Reagents: 2-Hydroxypyrimidine was purchased from Alfa Aesar and predried (2 days, under
reduced pressure) before silylation; 2-deoxy-3,5-di-O-(p-toluoyl)-α-D-ribofuranosyl chloride
was prepared in three steps from 2-deoxy-D-ribose,31 carefully powdered and dried for 4-18
hours in a vacuum desiccator over P2O5. SnCl4 was distilled from P2O5 under reduced
pressure.
Solvents: ClCH2CH2Cl and CH2Cl2 were predried with K2CO3 and distilled from
K2CO3/P2O5; pyridine was stirred with NaOH, refluxed over ninhydrin for several hours,
distilled from ninhydrin and then from CaH2; MeOH was distilled from Mg; toluene was
distilled from Na.
Chromatography: TLC was performed on analytical silica plates (Kieselgel 60 F254/ 0.2 mm
thickness), silica gel 60 (230-400 mesh, Merck) was used for column chromatography.
Spectral analysis: NMR spectra were obtained on a Bruker DPX 250 MHz and a Bruker
1
Avance II Plus 700 MHz instrument. H and 13C NMR chemical shifts are in ppm, and J
values are in Hz. Mass spectra were obtained on a Finnigan MAT 95 spectrometer. UV-data
were obtained on a HITACHI UV Vis U-2800A instrument.
Synthesis of modified nucleosides
2-Trimethylsilyloxypyrimidine (4)
Trimethylsilyl chloride (8.7 mL, 68.8 mmol) was added in one portion to a suspension of 2-
hydroxypyrimidine (7.3 g, 55 mmol) in anhydrous benzene (550 mL) and then triethylamine
(17.8 mL, 126.5 mmol) dissolved in 25 mL of anhydrous benzene was slowly dropped in. The
reaction mixture was stirred at room temperature for 7 days. After this time, the precipitated
Et3NxHCl was filtered off with exclusion of moisture. The filtrate was evaporated to a small
10

volume (15 mL) and the residue was distilled under reduced pressure (10 mmHg, 92˚C) to
afford silylated 2-hydroxypyrimidine 4 (6.6 g, 39.6 mmol; 72% yield).
Reaction under kinetic control conditions - synthesis of the β-anomer of pyrimidin-2-one
2’-deoxyriboside (2’-deoxyzebularine, 1a)
2-Deoxy-3,5-di-O-(p-toluoyl)-α-D-ribofuranosyl chloride (3.1 g, 8 mmol) was dissolved in 65
mL of anhydrous ClCH2CH2Cl. The sugar component was added immediately in one portion
to the previously prepared solution of 2-trimethylsilyloxypyrimidine (2.7 g, 16 mmol) and
SnCl4 (2.8 mL, 24 mmol) in 130 mL of 1,2-dichloroethane and the reaction mixture was
cooled to -33°C. After 3.5 hours, the reaction was judged to be complete by TLC (CHCl3:
MeOH 98:2, the disappearance of the sugar fraction with Rf=0.26 and the presence of a
nucleoside fraction with Rf=0.17) and the mixture was warmed to -20°C. Pyridine was added
(15 mL), followed by H2O (65 mL) and after 10 min stirring the mixture was filtered through
a Celite layer. Then the Celite was carefully rinsed with CH2Cl2, the filtrate was washed with
H2O (2x60 mL) and dried (MgSO4) prior to concentration under reduced pressure. The oily
residue was coevaporated with toluene (2x10 mL) and subjected to chromatographic
purification. Elution of a silica gel column with CHCl3/MeOH (a gradient from 100% to 97%
v/v) gave a nucleoside fraction as a mixture of anomers in a 3:1 ratio (2.8 g, 6.2 mmol, yield
74%).
Crystalization of the anomeric mixture from CHCl3/hexane gave crystals of 3`,5`-di-O-(p-
toluoyl)-α-2`-deoxyzebularine (0.7 g, 1.4 mmol) containing ~10% of β anomer according 1H
NMR; m.p. 188-190°C (lit. m.p. 190 °C21;191-193°C19).
1
The oily β-anomer (containing ~7% of the α anomer according to the H NMR spectrum) was
obtained from the filtrate (2.0 g, 4.4 mmol). Further separation using silica gel column
chromatography with n-butanol as an eluent (TLC, Rf= 0.46 for the α-anomer and Rf= 0.38
for the β-anomer) gave small amount of the pure α-anomer (0.12 g) and a fraction containing
the desired pure β-nucleoside 6a (1.6 g, 3.6 mmol, overall yield 44%).
1H NMR (250 MHz, CDCl3): β anomer (6a) 2.46, 2.49 (2s, 6H, 2CH3), 2.26-2.37 (m, 1H, H-
3
3
2
2’), 3.20-3.29 (ddd, JH1’-H2"=5.8 Hz, JH2"-H3’=1.7 Hz, JH2’-H2"=14.5 Hz, 1H, H-2"), 4.71 (d,
2H, H-4’+H-5"), 4.83 (dd, 1H, 3JH4’-H5’=4.0 Hz , 2JH5’-H5"=13.2 Hz, H-5’), 5.65 (m, 1H, H-3’),
6.29 (dd, 1H, JH5-H4=4.0 Hz, JH5-H6=6.4 Hz, H-5), 6.38 (dd, 1H, JH1’-H2’=7.7 Hz , JH1’-H2’’
3
3
3
3
=
3
3
3
5.8 Hz, H-1’), 8.19 (dd, 1H, JH6-H4=2.8 Hz, JH6-H5=6.4 Hz, H-6), 8.60 (dd, 1H, JH4-H5=4.0
Hz, 3JH4-H6=2.8 Hz, H-4), 7.27, 7.32, 7.87, 8.00 (4d, 8H, aromatic protons).
Removal of p-toluoyl protecting groups
The per-toluoyl derivative (6a) (1g, 2.2 mmol) was suspended in a 0.1M solution of MeONa
in MeOH (45 mL) and the mixture was stirred at room temperature. After 1 h, when TLC
checking showed complete removal of the p-toluoyl groups the mixture was worked up with
Dowex (pyridinium salt form). After the resin was filtered off, anhydrous pyridine and
toluene were added. The solution was evaporated in vacuo and coevaporated with toluene.
The oily residue was dissolved in water and washed with diethyl ether. The aqueous layer was
frozen and lyophilized. Pure 2’-deoxyzebularine (1a) was obtained as a white foam (0.44g,
yield 95%). A small sample of 1a was crystallized from ethanol to give nucleoside crystals
with m.p. 138-140°C (lit. m.p. 136-138°C11).
3
3
2
1H NMR (700 MHz, D2O): 2.35 (dt, 1H, JH1’-H2’=6.1 Hz, JH3’-H2’=6.5 Hz, JH2’-H2"=14.3 Hz,
H-2’), 2.66 (ddd, 1H, 3JH1’-H2"=6.6 Hz, 3JH3’-H2"=4.7 Hz, 2JH2’-H2"=14.3 Hz, H-2"), 3.79 (dd, 1H,
3JH4’-H5"=5.4 Hz, JH53’-H5"=12.6 Hz H-5"), 3.90 (dd, 1H, JH4’-H5’=3.5 Hz, JH5’-H5"=12.6 Hz, H-
2
3
2
3
3
5’), 4.19 (ddd, 1H, JH3’-H4’=4.3 Hz, JH4’-H5’=3.5 Hz, JH4’-H5"=5.4 Hz H-4’), 4.42 (ddd, 1H,
11

3
3
3
3
3JH3’-H2’=6.5 Hz, JH3’-H2"=4.7 Hz, JH3’-H4’=4.3 Hz, H-3’), 6.23 (t, 1H, JH1’-H2’=6.1 Hz, JH1’-
3
3
3
H2"=6.6 Hz, H-1’), 6.78 (dd, 1H, JH5-H4=4.4 Hz, JH5-H6=6.7 Hz, H-5), 8.52 (dd, 1H, JH6-
H4=2.6 Hz, 3JH6-H5=6.7 Hz, H-6), 8.63 (dd, 1H, 3JH4-H5=4.4 Hz, 3JH4-H6=2.6 Hz, H-4).
13C NMR (700 MHz, D2O): 40.0 (C-2’), 61.0 (C-5’), 70.0 (C-3’), 87.6 (C-4’), 88.1 (C-1’),
106.5 (C-5), 145.2 (C-6), 156.7 (C-2), 166.5 (C-4).
MS-FAB, m/z (%): 213.0 (100) [M+H]+; calculated for C9H12O4N2: 212. UV-Spectrum
(H2O): λmax 213 nm (ε=6610), 304 nm (ε=4266).
Condensation in CH2Cl2 as a solvent
The reaction was carried out analogously as in ClCH2CH2Cl, with the exception for lower
temperature applied (-40°C).
Components: 1.16 g (3 mmol) of chlorosugar in 25 mL of CH2Cl2; 1.0 g (6 mmol) of 2-
trimethylsilyloxypyrimidine and 1.05 mL (9 mmol) of SnCl4 in 50 mL of CH2Cl2; 3.5 h at
-40°C. The nucleoside fraction was isolated in 60% yield with the same anomeric ratio as in
ClCH2CH2Cl.
Reaction under thermodynamic control conditions - synthesis of α-anomer of pyrimidin-
2-one 2’-deoxyriboside 1b
2-Deoxy-3,5-di-O-(p-toluoyl)-α-D-ribofuranosyl chloride (2.7 g, 7 mmol) was dissolved in 60
mL of anhydrous ClCH2CH2Cl. The sugar component was added immediately in one portion
to the previously prepared solution of 2-trimethylsilyloxypyrimidine (2.35 g, 14 mmol) and
freshly distilled SnCl4 (1.5 mL, 14 mmol) in ClCH2CH2Cl (120 mL) warmed to +35°C. After
3.0 hours, the reaction was judged to be complete by TLC (chloroform:methanol 98:2, the
absence of the sugar substrate in the reaction mixture) and the mixture was cooled to 10°C.
Pyridine was added (10 mL), followed by H2O (50 mL) and after 10 min stirring the mixture
was filtered through a Celite layer and rinsed with CH2Cl2. The filtrate was washed with H2O
(2x20 mL) and dried (MgSO4) prior to concentration under reduced pressure. The residue was
coevaporated with toluene. Purification by silica gel column chromatography (a
CHCl3/MeOH gradient from 100% to 95% v/v) afforded a nucleoside product in 91% (2.9 g,
6.5 mmol) consisting of both anomers with significant majority of the α-isomer (TLC, BuOH,
1
Rf= 0.46 for the α-anomer and Rf= 0.38 for the β-anomer; 4:1 α: β according to H NMR).
Crystalization of the nucleosides mixture from CHCl3/hexane gave crystals of 3’,5’-p-toluoyl-
1
α-2’-deoxyzebularine containing ~ 6% of β-anomer, as it was determined by H NMR
analysis (2.0 g,4.7 mmol). Recrystalization of this fraction from the same solvents gave pure
α-isomer (1.7g, yield 53.5 %).
1H NMR (250MHz, CDCl3): α-anomer (6b) 2.32, 2.37 (2s, 6H, 2CH3), 2.63-2.73 (m, 1H, H-
2’), 2.9-3.01 (m, 1H, H-2"), 4.53 (d, 2H, H-5’+H-5"), 4.86 (d, 1H, H-4’), 5.51 (d, 1H, H-3’),
3
3
3
6.36 (d, 1H, H-1’), 6.41 (dd, 1H, JH5-H4=4.1 Hz, JH5-H6=6.7 Hz, H-5), 8.09 (dd, 1H, JH6-
3
3
3
H4=2.8 Hz, JH6-H5=6.7 Hz, H-6), 8.69 (dd, 1H, JH4-H5=4.1 Hz, JH4-H6=2.8 Hz, H-4), 7.09,
7.21, 7.54, 7.87 (4d, 8H, aromatic protons).
Removal of p-toluoyl protecting groups
The pertoluoyl derivative 6b (1g, 2.2 mmol) was suspended in a 0.1M solution of MeONa in
MeOH (45 mL) and the mixture was stirred at room temperature. After 1 h, when TLC
checking showed complete removal of p-toluoyl groups, the mixture was worked up with
Dowex (pyridinium salt form). After the resin was filtered off, anhydrous pyridine and
toluene were added. The solution was evaporated in vacuo and coevaporated with toluene.
The oily residue was dissolved in water and washed with diethyl ether. The aqueous layer was
frozen and lyophilized. Pure compound 1b was obtained as a white foam (0.44g, 2 mmol,
12

yield 91%). A small sample of 1b was crystallized from methanol to give nucleoside crystals
with m.p. 155-157°C (lit. m.p. 156-157°C19).
3
3
2
1H NMR (700 MHz, D2O): 2.27 (m, 1H, JH1’-H2"=1.5 Hz, JH3’-H2"=1.8 Hz, JH2’-H2"=15.0 Hz,
3
3
2
H-2"), 2.79 (m, 1H, JH1’-H2’=7.2 Hz, JH3’-H2’=5.6 Hz, JH2’-H2"=15.0 Hz, H-2’), 3.67 (dd, 1H,
3JH4’-H5"=5.3 Hz, JH5’-H5"=12.4 Hz, H-5"), 3.75 (dd, 1H, 3JH4’-H5’=4.1 Hz, 2JH5’-H5"=12.4 Hz, H-
2
5’), 4.41 (m, 1H, 3JH3’-H2’=5.6 Hz, JH3’-H2"=1.8 Hz, 3JH3’-H4’=1.5 Hz, H-3’), 4.58 (m, 1H, JH3’-
3
3
3
3
3
3
H4’=1.5 Hz, JH4’-H5’=4.1 Hz, JH4’-H5"=5.3 Hz, H-4’), 6.18 (dd, 1H, JH1’-H2’=7.2 Hz, JH1’-
3
3
3
H2"=1.5 Hz, H-1’), 6.78 (dd, 1H, JH5-H4=4.5 Hz, JH5-H6=6.6 Hz, H-5), 8.44 (dd, 1H, JH6-
H4=2.7 Hz, 3JH6-H5=6.6 Hz, H-6), 8.63 (dd, 1H, 3JH4-H5=4.5 Hz, 3JH4-H6=2.7 Hz, H-4).
13C NMR (700 MHz, D2O): 40.1 (C-2’), 61.7 (C-5’), 71.0 (C-3’), 90.0 (C-4’), 90.2 (C-1’),
106.1 (C-5), 145.7 (C-6), 156.7 (C-2), 166.2 (C-4).
MS-FAB, m/z (%): 213.1 (100) [M+H]+; calculated for C9H12O4N2: 212. UV-Spectrum
(H2O): λmax 213 nm (ε=14404), 303 nm (ε=8577).
Condensation in CH2Cl2 as a solvent
The reaction was carried out analogously as in ClCH2CH2Cl.
Components: 0.39 g (1 mmol) of chlorosugar 5 in 8 mL of CH2Cl2; 0.34 g (2 mmol) of 2-
trimethylsilyloxypyrimidine and 0.35 mL (2 mmol) of SnCl4 in 16 mL of CH2Cl2; 3.0 h at
+35°C. The nucleoside fraction was isolated in 70% yield with the same anomeric ratio as in
ClCH2CH2Cl.
Isomerisation of 3`,5`-di-O-(p-toluoyl)-2`-deoxyzebularine (β-anomer) into 3`,5`-di-O-(p-
toluoyl)- 2`-deoxyzebularine (α-anomer)
To 3`,5`-di-O-(p-toluoyl)-β-2`-deoxyzebularine (0.22g, 0.5 mmol) dissolved in anhydrous
ClCH2CH2Cl (5 mL) 2-trimethylsilyloxopyrimidine (0.17g, 1 mmol) in ClCH2CH2Cl (5 mL)
was added and warmed to +35°C. Freshly distilled SnCl4 (0.09 ml, 0.75 mmol) was dissolved
in ClCH2CH2Cl (5 mL) and added dropwise with stirring. After 3 h at +35°C the reaction was
judged to be complete by TLC (n-BuOH, the appearance of the α-anomer with Rf=0.46 ) and
the mixture was cooled to 10°C. Pyridine (1 mL) was added, followed immediately by H2O (5
mL) and after 10 min stirring the mixture was filtered through a Celite layer and rinsed with
CH2Cl2. The filtrate was washed with H2O (2x10 mL) and dried (MgSO4) prior to
concentration under reduced pressure. The residue was coevaporated (2x5 mL) with toluene.
The reaction afforded the nucleoside products (0.18 g, 0.4 mmol, yield=80%). Purification by
silica gel column chromatography (CHCl3/MeOH gradient from 100% to 98% v/v) afforded
nucleoside products in 82% (0.15 g, 0.33 mmol). Deprotection of p-toluoyl groups from the
sugar moiety according to a standard procedure gave the mixture of α- and β-anomers in 4:1
ratio of α:β (as it was evaluated on an 1H NMR spectra).
4.2. X-ray structure analysis
Crystal data for β-anomer (1a) C9H12N2O4, M = 212.21, orthorombic, space group P212121, a
= 7.4740(1) Å, b = 9.2857(1) Å, c = 13.5774(5) Å, V = 942.29(2) Ǻ3, Z = 4, Dx = 1.496 g cm-
3, T = 296 K, µ = 1.010 mm-1, λ = 0.71073 Ǻ, data/parameters = 1711/152; Flack x =
0.00(18), final R1 = 0.0243.
Crystal data for α-anomer (1b) C9H12N2O4, M = 212.21, orthorombic, space group P212121, a
= 9.09800 Å, b = 9.4766 (1) Å, c = 11.1722(1) Å, V = 963.25 (1) Ǻ3, Z = 4, Dx = 1.463 g cm-
3, T = 296 K, µ = 0.988 mm-1, λ = 0.71073 Ǻ, data/parameters = 1801/137; Flack x =
0.11(17), final R1 = 0.0239.
13

Crystals of β-anomer (1a) and α-anomer (1b) were obtained by slow evaporation from
aqueous ethanolic solutions. The measurements were performed on a SMART diffractometer
with graphite-monochromated MoKα radiation (λ= 0.71073 Ǻ) at room temperature. The
structures were solved by a direct method and refine with SHELXTL.41 E-maps provided
positions for all non-H-atoms. The full-matrix least-squares refinement was carried out on
F2’s using anisotropic temperature factors for all non-H-atoms. All C-bound H atoms were
−H = 0.93 Å and U
placed in idealized locations and refined using a riding model, with C iso(H)
= 1.2Ueq(C).
Crystallographic data (excluding structural factors) for the structure reported in this paper
have been deposited at Cambridge Crystallographic Data Centre and allocated with the
deposition number: CCDC 970241-970242 for compounds 1a, 1b. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road, Cambridge CB2 1EW, UK
(Fax: Int code + (1223)336-033; E-mail: deposit@ccdc.cam.ac.uk).
4.3. NMR of modified nucleosides
The one- and two-dimensional NMR experiments were recorded on 700.2 MHz spectrometer
at 25 °C in CDCl3 or D2O with DSS (4,4-dimethyl-4-silapentane-1-sulfonic acid) as the
internal standard. The samples for the NMR measurements were prepared by dissolving 4-6
mg of the nucleosides in 0.7 mL of D2O. Spectra were processed using TopSpin 2.1 software
(Bruker BioSpin). In the case of overlapping signals in 1H NMR spectra, the DAISY (Bruker
BioSpin) deconvolution procedure was applied.
Acknowledgements: This work was supported by the grant from Funds for Young Scientists
at the Faculty of Chemistry, Technical University of Lodz to K.E. and by grant DzS
I-18/15/2013 (Technical University of Lodz)
Supplementary Material
Supplementary data contain 1H NMR spectra recorded for reaction mixtures after
condensation performed at kinetic or thermodynamic control conditions, 1H NMR data for β -
3`,5`-di-O-(p-toluoyl)-2`-deoxyzebularine (6a) isomerization and spectral characteristic (1H,
13C, NMR, MS, IR, UV) as well as crystallographic data for compounds 1a and 1b.
References and notes
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D-H..A
(sym. code)
D…A (Å)
D-H..A (◦)
O3'-H3'O…N3
O5'-H5'O…O2
O5'-H5'O…O4'
O5'-H5'O…N3
O3'-H3'O…O5'
1/2+x,3/2-y,-z
1+x,y,z
2.8623(16)
2.7716(15)
2.8952(12)
2.8087(14)
2.8004(13)
167
168
104
152
155
β-dZeb
α-dZeb
intermolecular
-x,-1/2+y,1/2-z
1-x,1/2+y,1/2-z
Table 1. The geometry of hydrogen bonds in the crystal of β-dZeb (1a) and α-dZeb (1b).
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Table 2. NMR data of β-dZeb and α-dZeb; a) 1H chemical shifts, b) proton-proton coupling
constants, c) 13C chemical shifts
Chemical shifts
a)
1’
2’
2’’
3’
4’
5’
5’’
4
5
6
protons
β-dZeb
α-dZeb
α-dZeb*
6.23
6.18
6.18
2.35
2.79
2.79
2.66
2.27
2.27
4.42
4.44
4.41
4.19
4.58
4.58
3.90
3.75
3.75
3.79
3.67
3.67
8.63
8.63
8.63
6.78
6.78
6.78
8.52
8.44
8.44
Coupling constants
1’-2’ 1’-2’’ 2’-2’’ 2’-3’ 2’’-3’ 3’-4’ 4’-5’ 4’-5’’ 5’-5’’ 4-5
b)
5-6
4-6
6.1
7.0
7.2
6.6
1.4
1.5
14.3
15.0
15.0
6.5
nd
4.7
nd
4.3
nd
3.5
4.2
4.1
5.4
5.6
5.3
12.6 4.4 6.7 2.6
12.3 4.4 6.6 2.6
12.4 4.5 6.6 2.7
β-dZeb
α-dZeb
α-dZeb*
5.6
1.8
1.5
*values from simulated 1H spectrum
c) 13C
Chemical shifts
C1’
88.2
90.3
C2’
40.0
40.1
C3’
C4’
C5’
C2
C4
C5
C6
70.0
71.0
87.6
90.0
60.9
61.7
156.7
156.7
166.5
166.2
106.5
106.1
145.2
145.7
β-dZeb
α-dZeb
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Irradiated
proton
Irradiated
proton
Compound
NOE (%)
H1’ (1.8)
Compound
NOE (%)
H1’ (2.1)
H6
H6
β-dZeb
α-dZeb
H2’ (5.2)
H2” (2.0)
H3’ (1.1)
H2’+H3’ (6.3)
H4’
H2’ (1.1)
H2” (2.0)
H3’ (1.1)
H4’ (7.5)
H1’
H2’ (1.4)
H2” (5.6)
H1’
H2’ (4.0)
H2” (1.2)
Table 3.Results of 1D NOE experiments for β-dZeb and α-dZeb, (25°C in D2O at 700 MHz)
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β-dZeb
α-dZeb
a) Crystal structure
Sugar moiety C3’-endo C2’-exo C2’-endo C3’-exo
conformation
N-type
S-type
C4’-C5’
C1’-N1
gauche (+)
anti
trans
anti
b) conformation in solution
Sugar moiety
conformation
56% C2’-endo
S-type
94% C2’-endo
S-type
46% gauche (+)
42% trans
12% gauche (-)
42% gauche (+)
38% trans
20% gauche (-)
C4’-C5’
C1’-N1
anti
anti
Table 4. Comparison of β-dZeb and α-dZeb structure in crystals and in solution.
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Efficient synthesis of 2’-deoxyzebularine and its α-anomer by the silyl method of
N-glycosylation. Crystal structures and conformational study in solution.
Katarzyna Ebenryter - Olbinska1, Janina Karolak-Wojciechowska2 and Elzbieta Sochacka1
Highlights
Synthesis of α- and β-deoxyzebularine with high stereoselectivity by N-glycosylation
Full spectral characterization of α-and β-2’-deoxyzebularine
Crystal structure and conformation analysis in solution of α-and β-2’-deoxyzebularine
25

Graphical abstract
26