Configuration and conformation of a novel uridine analogue: 1H and 13C NMR spectra of (5′S)-1-[2′ -(2-hydroxyethyl)tetrahydropyran-5′-yl]-1H-pyrimidine-2,4-dione

Article abstract of DOI:10.1002/mrc.1001

A combination of homo- and heteronuclear 1D and 2D NMR techniques provided the assignment of the ¹H and ¹³C resonances of the major component of a reaction product consisting of the two possible diastereomers of (5′S)-1-[2′-(2-hydroxyethyl)tetrahydropyran-5′-yl] -1H-pyrimidine-2,4-dione and showed that the tetrahydropyranyl ring in the major 5′S,2′S-isomer adopts the twist conformation. Copyright

Full text of DOI:10.1002/mrc.1001

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2002; 40: 244–246
Note
Configuration and conformation of a novel uridine
analogue: 1H and 13C NMR spectra
of (5’S)-1-[2’-(2-hydroxyethyl)tetrahydropyran-5’-yl]-
1H-pyrimidine-2,4-dione
George Balayiannis,1 Dionissios Papaioannou1 and Anastassios Troganis2∗
1
Department of Chemistry, University of Patras, GR-265 00 Patras, Greece
Department of Biological Applications and Technologies, University of Ioannina, GR-451 10 Ioannina, Greece
2
Received 25 June 2001; Revised 7 November 2001; Accepted 23 November 2001
A combination of homo- and heteronuclear 1D and 2D NMR techniques provided the assignment of
1
the H and 13C resonances of the major component of a reaction product consisting of the two possible
diastereomers of (5ꢀS)-1-[2ꢀ-(2-hydroxyethyl)tetrahydropyran-5ꢀ-yl]-1H-pyrimidine-2,4-dione and showed
that the tetrahydropyranyl ring in the major 5ꢀS,2ꢀS-isomer adopts the twist conformation. Copyright 
2002 John Wiley & Sons, Ltd.
KEYWORDS: NMR; H NMR; 13C NMR; nucleoside analogues; conformation; configuration
INTRODUCTION
O
N
NH2
N
G
HN
20,30-Dideoxynucleosides (ddN), such as AZT, AZddU and
ddC (Scheme 1), are potent anti-HIV agents targeting the
enzyme reverse transcriptase (RT).1 We have recently
reported a methodology which allows the total syntheses
of novel ddN analogues, incorporating a tetrahydrofuranyl
or tetrahydropyranyl ring as the pseudosugar moiety,
using the lactones of chiral hydroxyamino acids (HAA)
as key intermediates.2 Application of this methodology,
described diagrammatically in Scheme 2, to the HAA
1, readily obtained from L-glutamic acid,3 led to the
preparation of an inseparable (by TLC) mixture of the cis-
(4a) and trans- (4b) uridine analogues (Scheme 2) through
the corresponding intermediates 2 and 3, also obtained
as inseparable mixtures of diastereomers (G. Balayiannis
and D. Papaioannou, unpublished results. The yields of the
reactions in Scheme 2 were not optimized. Abbreviations
used are as follows: DCC, N,N0-dicyclohexylcarbodiimide;
Ts, 4-toluenesulphonyl).
N
O
O
O
HO
HO
O
N3
ddC
AZT ; G = Me
AZddU ; G = H
H
6'
H
H
H
2'
H
3'
H
5'
O
H
H
1
2
3
N
H
O
H
O
1''
4'
N
1'
H
H
H
4
2''
H
6
OH
5
H
4a
Scheme 1. Structures of dideoxynucleoside analogues con-
sidered in this work.
namely the twist conformation, and the configuration of the
major isomer.
The 1H and 13C NMR spectra of the major isomer 4a
(4a:4b ³ 4 : 1) at 300 K were assigned using a combination
of homo- and heteronuclear 1D and 2D techniques. Further-
more, these techniques also allowed the determination of
the conformation adopted by the tetrahydropyranyl ring,
EXPERIMENTAL
An isomeric mixture of uridine analogues 4a and 4b was
prepared from the N-tritylated HAA 1 through a sequence
of 10 steps with a total yield of 3% as described in Scheme 2.
The inseparable (TLC) mixture of compounds 4 was purified
by flash column chromatography and was obtained as an oil.
NMR experiments were performed on a Bruker AMX-
400 instrument at 300 K. A 20 mM solution of 4 in deuturated
ŁCorrespondence to: A. Troganis, Department of Biological
Applications and Technologies, University of Ioannina, GR-451 10
Ioannina, Greece. E-mail: atrogani@cc.uoi.gr
Contract/grant sponsor: General Secretariat for Research and
Technology, Greek Ministry for Development.
DOI: 10.1002/mrc.1001
Copyright  2002 John Wiley & Sons, Ltd.

Configuration and conformation of a uridine analogue
245
HN Trt
H
H-2’’H-2’
H-6’e H-6’a
H-5’
H-1’’
HN Trt
H
R1
H-4’a
H-4’e
H-3’e
v, vi,
vii
42%
i, ii,
iii, iv
30%
HO
HO
H-3’a
O
O
R2
a : R1=CH2CO2Me, R2=H
1
2
b : R1=H, R2=CH2CO2Me
O
HN
O
2
NH2
R1
N
R1
viii, ix, x
25%
4
O
O
H
R2
H
R2
3
3
a : R1=CH2CH2OH, R2=H
b : R1=H, R2=CH2CH2OH
a : R1=CH2CH2OBn, R2=H
b : R1=H, R2=CH2CH2OBn
Scheme 2. Outline of the synthesis of diastereomers 4a
and 4b. Reagents: (i) DCC; (ii) iBu2AlH; (iii) Ph3 PCHCO2Me;
(iv) Bu4NF; (v) LiAlH4; (vi) PhCH2Br/NaH; (vii) TsOHÐH2O/iPrOH;
(viii) MeOCH CHCONCO/Et3N; (ix) 2 N H2SO4/dioxane; (x) H2/
Pd–C.
4
ppm
ppm
4
3
2
Figure 1. Aliphatic region of 2D homonuclear proton
DQF-COSY experiment on diastereomer 4a. The cross peaks,
denoted by arrows, are due to long-range interaction between
protons 60 and 40, suggesting that the two protons are
equatorial.
methanol was used in all experiments. Standard programs
were used for 1H and 13C experiments and a Gaussian
apodization function was used, in order to increase the
resolution.
2D homonuclear proton experiments (DQF-COSY,
ROESY) were collected using 2 K ð 512 points, with 4 and 72
scans per increment, respectively. Spectra were transformed
RESULTS AND DISCUSSION
1H and 13C chemical shift values for the major isomer 4a are
provided in Table 1. The 1H and 13C resonance arising from
the minor isomer 4b were assigned and are shown in Table 1
in italics. The possibility of 4b being a conformer of 4a was
°
°
in 1 K ð 1 K real points, using 0 sine and 45 square
sine apodization functions. 2D H,13C-HMQC, HMBC and
1
HSQC-TOCSY experiments were performed using 2 K ð 384
points; 112, 144 and 136 scans were collected per increment,
respectively. Spectra were zero-filled in the F1 dimension to
1
excluded by performing a H NMR experiment at different
temperatures. The numbering of carbons for 4a is shown in
Scheme 1. The assignment of the uracil ring was based on 1D
1H NMR and 2D 1H,13C-HMBC and HMQC experiments. H-
6 resonates at υ 8.34 owing to the neighboring nitrogen atom.
°
give 1 K ð 1 K real points. A 45 square sine apodization
function was used in all experiments. Z-gradients were used
in all 2D experiments for coherence pathway selection.
Table 1. 1H and 13C chemical shifts (υ, ppm, reference TMS) of uridine
analogues 4a and 4b
Protona
Mυ(1H)
mυ(1H)
Carbon
Mυ(13C)
mυ (13C)
C-2
C-4
C-5
C-6
C-20
C-30
166.44
152.88
101.51
145.84
75.95
166.44
152.88
101.51
145.84
75.20
H-5
H-6
H-20
5.68
8.34
3.66
1.62
1.47
2.09
2.03
4.36
4.21
3.98
1.76
3.70
5.68
8.34
3.61
1.70
1.48
2.73
2.52
4.10
4.08
3.78
1.77
3.58
H-30e
H-30a
H-40e
H-40a
H-50
H-60e
H-60a
H-100
H-200
27.46
28.86
C-40
28.04
32.29
C-50
C-60
51.04
69.46
49.92
68.97
C-100
C-200
39.80
59.32
41.21
59.50
a a, Axial; e, Equatorial.
Copyright  2002 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2002; 40: 244–246

246
G. Balayiannis, D. Papaioannou and A. Troganis
1
not show H,13C coupling with C-60 and C-40 in the HMBC
Using this resonance as the starting point, the assignments of
all protons and carbons are obtained easily from HMQC and
HMBC experiments. H-6 gives a cross peak with the carbon at
51.04 ppm in a 1H,13C-HMBC experiment, showing that this
carbon is the 50 position of the tetrahydropyranyl ring. The
experiment.4 H-100 shows an NOE with both H-30, but H-200
NOE only with H-30 a.
These results have as a consequence that both the uracil
ring and the hydroxyl ethyl (Het) side-chain take the cor-
responding equatorial positions and thus the major isomer
has the cis configuration (50S,20S). Also, it is established that
the central ring adopts the energetically preferred twist con-
formation. Furthermore, data from a NOESY experiment
allow for the determination of the conformation of the whole
molecule, including the heterocyclic side-chain, which is the
one shown in Scheme 1.
1
complete H and 13C assignment of the tetrahydropyranyl
ring was obtained by sequential analysis based on the DQF-
1
COSY spectrum in Fig. 1 and the H,13C-HMBC spectrum.
The results were confirmed by 1H,13C-HMBC (30 and 70 ms)
and 1H,13C-HSQC-TOCSY (15 and 40ms) experiments.
The existence of the cross peak between H-60 and H-40,
which resonate at 4.21 and 2.09 ppm, respectively, due to
a long-range coupling between these two protons, demon-
strates that these protons are equatorial. The resonances at
3.98 and 2.03 ppm are due to 60a and 40a, respectively. H-50
shows strong 3J coupling with H-60a and H-40a and a strong
NOE effect with H-60a and H-40e. These results confirm that
H-50 is axial and that H-60a and H-40a are in an antiparallel
conformation. This is strong evidence that the central ring
adopts the energetically preferred twist conformation. H-30
at 1.62 ppm is in an equatorial position since it gives an
NOE with both H-40e and H-40a, whereas H-30 at 1.47 ppm
yields an NOE only with H-40e, so it is axial. Finally, H-20
shows 3J coupling with H-30a but not with H-30e, and it
gives a stronger NOE with H-30e than with H-30a. Hence
H-20 is axial. This is also confirmed by the fact that H-20 does
Acknowledgements
The authors thank the General Secretariat for Research and Tech-
nology of the Greek Ministry for Development for financial support
of this research project. Instrumentation used in these studies was
funded in part by EEC Equipment Grant Stride-Hellas-33.
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Copyright  2002 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2002; 40: 244–246