Spectral assignments and reference data complete1H and 13C NMR spectral assignment of α- And β-adenosine, 2′-deoxyadenosine and their acetate derivatives

Article abstract of DOI:10.1002/mrc.2036

¹H and ¹³C NMR chemical shifts of α- and β-anomers of adenosine, 2′-deoxyadenosine and their acetate derivatives were completely and definitely assigned using the concerted application of one- and two-dimensional experiments (gCOSY, gNOESY, gHSQC and gHMBC). The influence of the stereochemistry of the purine base on the NMR data of the hydrogen and carbon atoms of the furanose moiety was estimated. Copyright

Full text of DOI:10.1002/mrc.2036

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2007; 45: 781–784
Published online 19 July 2007 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/mrc.2036
Spectral Assignments and Reference Data
Complete 1H and 13C NMR spectral
assignment of a- and b-adenosine,
2ꢀ-deoxyadenosine and their acetate
derivatives
based on combined information from 1D and 2D NMR (gCOSY,
gNOESY, gHSQC and gHMBC) experiments. 1H–1H coupling
constants were directly measured from resolution-enhanced 1D
spectra and confirmed, when necessary, by homodecoupling. NOE
analysis allowed the assignment of the nucleoside configuration,
determination of the H-8 resonance and the assignment of H-20˛
and H-20ˇ of 20-deoxy derivatives. In particular, NOE correlations
between H-10 and H-40 are present only in compounds 1, 2, 3 and
4 identified as ˇ-anomers, in accordance with 1D-NOE difference
spectroscopy studies referred to adenosine (1), ˛-adenosine (5)5 and
20-deoxyadenosine (3).6 H-8 resonances were assigned according to
NOESY correlations with H-10 as well as HMBC correlations between
H-10 and C-8. Adenosine (1), 20-deoxyadenosine (3), ˛-adenosine (5)
and ˛-20-deoxyadenosine (7) have H-8 resonance less shielded than
H-2, in agreement with values reported for compounds 1 and 3.7
On the contrary, H-8 of the acetate derivatives 2, 4, 6 and 8 is more
shielded than H-2 in contrast with literature results for adenosine-
20,30,50-triacetate (2)8 and 20-deoxyadenosine-30,50-diacetate (4).9 In
addition, NOESY correlations allowed the assignment of H-20˛ and
H-20ˇ of 20-deoxyadenosine (3), 20-deoxyadenosine-30,50-diacetate (4),
˛-20-deoxyadenosine (7) and ˛-20-deoxyadenosine-30,50-diacetate (8).
In the ˇ-compounds 3 and 4, NOE-contacts between H-8 and the less
shielded H-20 were observed, whereas in the ˛-anomers 7 and 8 H-8 is
in NOE-contact with the more shielded H-20; these findings assigned
H-20ˇ resonance to high frequency H-20. The H-10 resonances of ˇ-
and ˛-nucleosides could be useful to define anomeric configuration.
Significant differences of chemical shifts were observed between
ˇ anomers adenosine (1), adenosine-20,30,50-triacetate (2), and the
corresponding ˛ anomers 5 and 6. In particular, H-10 resonances of
compounds 1 and 2 are more shielded than H-10 of 5 and 6. On the
contrary, H-10 resonances of ˇ- and ˛-20-deoxyribonucleosides show
very similar chemical shifts (Tables 1 and 2) but different coupling
constants. Natural 20-deoxyadenosine (3) and 20-deoxyadenosine-
30,50-diacetate (4) show the coupling constants between H-10 and
H-20˛ (both 6.0 Hz) to be larger than those of the ˛-anomers 7 and 8
(3.1 and 2.1 Hz respectively).
P. Ciuffreda,1 S. Casati1 and A. Manzocchi2∗
1
Dipartimento di Scienze Precliniche LITA Vialba, Universita` degli Studi
di Milano, Via G.B. Grassi, 74-20157 Milano, Italy
2
Dipartimento di Chimica, Biochimica e Biotecnologie per la Medicina,
Universita` degli Studi di Milano, Via Saldini 50-20133 Milano, Italy
Received 22 March 2007; revised 11 May 2007; accepted 15 May 2007
1H and 13C NMR chemical shifts of a- and b-anomers of
adenosine, 2ꢀ-deoxyadenosine and their acetate deriva-
tives were completely and definitely assigned using the
concertedapplicationof one-and two-dimensionalexper-
iments (gCOSY, gNOESY, gHSQC and gHMBC). The
influence of the stereochemistry of the purine base on
the NMR data of the hydrogen and carbon atoms of the
furanose moiety was estimated. Copyright  2007 John
Wiley & Sons, Ltd.
KEYWORDS: 1H NMR; 13C NMR; gCOSY; gNOESY; gHSQC;
gHMBC; ˇ- and ˛-adenine nucleosides; ˇ- and ˛-adenine
nucleoside acetates
INTRODUCTION
Although ˛-nucleosides are not found in nucleic acids, they are con-
stituents of smaller molecules, e.g. vitamin B12, present in living cells.
Some of them exert biological activities equal to or even exceeding
those of corresponding ˇ anomers.1 Recently it was demonstrated
that ˛-20-deoxyadenosine in DNA is a substrate of E. coli, human
and S. Cerevisiae endonucleases.2 Current projects in our laboratory
involve the synthesis of modified purine nucleosides and derivatives
with both anomeric configurations. 1H and 13C NMR spectroscopy
can be a very useful tool to achieve the identification of the two
anomers which have very similar chromatographic behaviors. The
assignment of anomeric configurations is an important topic in
nucleoside chemistry. Some NMR methods have already been used
to detect spectral differences between natural and ˛ anomers of
purine nucleosides, such as (i) methyl 1H resonances of isopropyli-
dene derivatives;3 (ii) C-10 versus C-40 chemical shifts for adenosine
and base-modified adenosine analogues;4 (iii) comparison of cou-
pling constants of H-10 for 20-deoxyadenosine and derivatives, and
(iv) 1D-nuclear Overhauser effect (NOE) difference spectroscopy.5
To our knowledge, complete 1H NMR data of ˛-adenosine and ˛-
20-deoxyadenosine are not reported in the literature. Moreover, no
data are available for other nucleoside derivatives of our interest,
i.e. acetates of ˛ anomers. Chemical structures of compounds1-8 are
shown in Fig. 1.
13C resonance assignments were in accordance with data
reported for adenosine (1),4,10 ˛-adenosine (5)4,11 and 20-deoxyade-
nosine (3).12 C-10 and C-20 resonances of the ribonucleosides 1,
2, 5 and 6 are significatively affected by the C-10 configuration,
whereas corresponding resonances of 20-deoxyribose moiety in the
compounds 3, 4, 7 and 8 are not (Table 3). C-10 and C-20 of 1 and 2
are less shielded than the corresponding atoms of the ˛-anomers 54
and 6.
Some of the previously reported NMR methods used to
establish anomeric configuration of purine nucleosides are not
straight, requiring chemical transformation to 20, 30-isopropylidene
derivatives or 1H NOE experiments. The finding that C-40 is less
shielded in ˛-anomers than C-10 whereas it is more shielded in ˇ-
ones, is valid neither for the acetates 2 and 6 nor for the 20-deoxy
compounds 3, 4, 7 and 8. The anomeric configuration of nucleosides
1–8 can be easily assigned by the analysis of the 1H NMR spectrum.
For the 20-deoxynucleosides 3, 4, 7 and 8, anomers can be identified
assessing the coupling constants between H-10 and H-20˛. In the case
of ribonucleosides 1, 2, 5 and 6, the magnitude of the H-10 chemical
shifts (Tables 1 and 2) or the evaluation of differences between υ-
values of the H-2 and H-10 protons can be used for identifying the
anomer. The magnitude of these differences is larger than 2.15 ppm
in ˇ-derivatives (1, 5) and smaller than 1.85 ppm in the ˛-ones
(2, 6)
Here, we present the complete 1H and 13C NMR spec-
tral assignment of adenosine (1), adenosine-20, 30, 50-triacetate (2),
20-deoxyadenosine (3), 20-deoxyadenosine-30, 50-diacetate (4) and
their ˛-anomers i.e. ˛-adenosine (5), ˛-adenosine-20, 30, 50-triacetate
(6), ˛-20-deoxyadenosine (7) and ˛-20-deoxyadenosine-30, 50-diacetate
(8), comparing signals of relevance to distinguish between ˛ and ˇ
anomers. Chemical structures of compounds 1–8 are shown in Fig. 1.
EXPERIMENTAL
Materials
Adenosine (1), 20-deoxyadenosine (3), ˛-adenosine (5) and ˛-20-
deoxyadenosine (7) are commercial products. All the acetylated
compounds were prepared using acetic anhydride in pyridine. Usual
work-up and purification by chromatographic column on silica gel
afforded pure 2, 4, 6 and 8.
RESULTS AND DISCUSSION
Complete 1H and 13C NMR data of all compounds are shown in
Tables 1–3. 1H and 13C resonances were unequivocally assigned
Adenosine-20, 30, 50-triacetate (2): [˛]25 -30.7 (c 1, CHCl3); m/z
394 [M C 1]; 416 [M C Na], 809 [M C M CDNa]; 20-deoxyadenosine-
ŁCorrespondence to: A. Manzocchi, Dipartimento di Chimica, Biochimica e
Biotecnologie per la Medicina, Universita` di Milano, Via Saldini 50-20133
Milano, Italy. E-mail: ada.manzocchi@unimi.it.
30, 50-diacetate (4): [˛]25 -24.4 (c 1, CHCl3); m/z 394 [M C 1], 809
D
[M C M C Na]; ˛-adenosine-20, 30, 50-triacetate (6): [˛]25D C 25.1 (c 1,
Copyright  2007 John Wiley & Sons, Ltd.

782
Spectral Assignments and Reference Data
Table 1. 1H-NMR data for compounds 1, 3, 5 and 7 (υ, ppm; J, Hz)a,b in DMSO-d6 solutions
H
1
3
5
7
2
8.13 (s)
8.34 (s)
7.33 (bs)
5.87 (d)
6.2
8.13 (s)
8.33 (s)
8.13 (s)
8.31 (s)
7.20 (bs)
–
8.15 (s)
8
8.38 (s)
NH2
1ꢀ˛
7.30 (bs)
6.34 (dd)
6.0; 8.0
7.27 (bs)
–
–
–
1ꢀˇ
–
–
6.32 (d)
5.4
6.33 (dd)
–
–
3.1; 8.0
2ꢀ˛
–
2.25 (ddd)
2.7; 6.0; –13.1
2.72 (ddd)
5.7; 8.0; –13.1
4.41 (dddd)
2.6; 2.7; 4.0; 5.7
3.88 (ddd)
2.6; 4.2; 4.4
3.62 (ddd)
4.4; 5.0; –12.0
3.52 (ddd)
4.2; 6.7; –12.0
–
–
2.33 (ddd)
–
–
2.7; 3.1; –14.2
2ꢀˇ
4.61 (ddd)
5.1; 6.2; 6.3
4.14 (ddd)
3.0; 4.6; 5.1
3.96 (ddd)
3.0; 3.6; 3.7
3.67 (ddd)
3.6; 4.4; –12.1
3.55 (ddd)
3.7; 7.2; –12.1
5.43 (d)
6.3
4.36 (ddd)
5.4; 5.4; 5.6
4.15 (ddd)
4.9; 5.4; 5.6
4.08 (ddd)
3.5; 5.2; –12.0
3.58 (ddd)
3.5; 5.2; –12.0
3.48 (ddd)
4.2; 6.2; –12.0
5.46 (d)
5.6
2.76 (ddd)
6.9; 8.0; –14.2
3ꢀ
4.30 (dddd)
2.7; 2.9; 4.6; 6.9
4ꢀ
4.13 (ddd)
2.9; 4.5; 4.5
5ꢀa
3.40–3.48 (AB part of ABXY system)
–
5ꢀb
3.40–3.48 (AB part of ABXY system)
–
–
2ꢀ-OH
3ꢀ-OH
5ꢀ-OH
–
–
5.17 (d)
4.6
5.30 (d)
5.44 (d)
5.6
5.76 (d)
4.6
4.0
5.41 (dd)
4.4; 7.2
5.23 (dd)
5.0; 6.7
4.86 (dd)
5.2; 6.2
4.84 (dd)
5.7; 5.7
a Multiplicity in parentheses.
b Coupling constants (J) in italics.
CHCl3); m/z 336 [M C 1], 358 [M C Na], 693 [M C M C Na]; ˛-20-
deoxyadenosine-30, 50-diacetate (8): [˛]25 C 33.0 (c 1, CHCl3); m/z
336 [M C 1], 358 [M C Na], 693 [M C M CDNa].
NMR spectroscopy
NMR spectra were recorded on a Bruker AVANCE 500 spectrometer
equipped with a 5-mm broadband reverse probe with field z-
gradient, operating at 500.13 and 125.76 MHz for 1H and 13C,
respectively. DMSO-d6 (isotopic enrichment 99.95%) and CDCl3
(isotopic enrichment 99.95%) were used as solvents for nucleosides
(1, 3, 5 and 7) and for acetates (2, 4, 6 and 8), respectively. The
samples contained 20 mmol of product dissolved in 0.5 ml of solvent
(40 mM). The central peak of DMSO-d6 signals (2.50 ppm for 1H
and 39.50 ppm for 13C) and of CDCl3 signals (7.26 ppm for 1H
and 77.7 ppm for 13C) were used as internal reference standard.
The sample temperature was maintained constant at 298 K. For
1D acquisition, the parameters were as follows: 1H spectral width
of 5000 Hz and 32 K data points providing a digital resolution
of ca 0.305 Hz per point, relaxation delay 2 s; 13C spectral width
of 29 412 Hz and 64 K data points providing a digital resolution
of ca 0.898 Hz per point, relaxation delay 2.5 s. The experimental
error in the measured 1H–1H coupling constants is š0.5 Hz. The
experiments were performed through standard pulse sequences.
gCOSY-45 and phase sensitive gNOESY experiments were acquired
with 512 t1 increments; 2048 t2 points; spectral/spectrum width
10.0 ppm. The g-NOESY experiments were performed on degassed
samples and with a mixing time of 800 ms. gHSQC and gHMBC
experiments were acquired with 512 t1 increments; 2048 t2 points;
spectral/spectrum width 10.0 ppm for 1H and 180 ppm for 13C.
Figure 1. Chemical structure and numbering for compounds 1–8.
Copyright  2007 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2007; 45: 781–784
DOI: 10.1002/mrc

783
Spectral Assignments and Reference Data
Table 2. 1H data for compounds 2, 4, 6 and 8 (υ, ppm; J, Hz)a,b in CDCl3 solutions
H
2
4
6
8
2
8.36 (s)
7.96 (s)
5.82 (bs)
6.18 (d)
5.3
8.35 (s)
7.98 (s)
8.35 (s)
8.10 (s)
5.64 (bs)
–
8.36 (s)
8.08 (s)
8
NH2
1ꢀ˛
5.78 (bs)
6.43 (dd)
6.0; 8.1
5.82 (bs)
–
–
–
1ꢀˇ
2ꢀ˛
2ꢀˇ
3ꢀ
–
–
6.68 (d)
5.6
6.53 (dd)
2.1; 7.4
–
–
–
2.62 (ddd)
2.5; 6.0; –16.1
2.95 (ddd)
6.3; 8.1; –14.1
5.43 (ddd)
2.4; 2.5; 6.3
4.35
overlaps50b
4.42 (dd)
5.4; –13.0
4.36 (dd)
4.4; –13.0
–
–
2.77 (ddd)
2.1; 2.1; –15.3
2.93 (ddd)
7.2; 7.4; –15.3
5.33 (ddd)
2.1; 2.1; 7.2
4.63 (ddd)
2.1; 4.1; 4.4
4.30 (dd)
4.1; –12.1
4.24 (dd)
4.4; –12.1
–
–
–
5.92 (dd)
5.3; 5.4
5.67 (dd)
4.3; 5.4
4.44
overlaps50a
4.45 (dd)
3.2; –13.1
4.37 (dd)
5.5; –13.1
2.08 (s)
2.14 (s)
2.12 (s)
5.76 (dd)
5.6; 5.7
5.52 (dd)
4.3; 5.7
4.65 (ddd)
3.5; 4.0; 4.3
4.38 (dd)
3.5; –12.5
4.24 (dd)
5.5; –12.5
1.88 (s)
2.11 (s)
2.16 (s)
4ꢀ
5ꢀa
5ꢀb
2ꢀ-CH3.CO/c
3ꢀ-CH3.CO/c
5ꢀ-CH3.CO/c
2.13 (s)
2.12 (s)
2.09 (s)
2.00 (s)
a Multiplicity in parentheses.
b Coupling costants (J) in italics.
c Assigned based on gHMBC spectra.
Table 3. 13C-NMR data (υ, ppm) for compounds 1, 3, 5 and 7 in DMSO-d6 solutions; for 2, 4, 6 and 8 in CDCl3 solutions
C
1
2
3
4
5
6
7
8
2
4a
152.4
149.0
119.3
156.2
139.9
87.9
73.4
70.6
85.9
61.6
–
153.9
150.4
120.8
156.1
139.5
86.9
152.4
148.9
119.3
156.1
139.5
83.9
39.4
71.0
88.0
61.9
–
153.7
150.3
120.8
156.1
139.3
85.3
38.3
75.2
83.2
64.5
–
152.2
149.5
118.0
155.8
141.1
83.3
70.6
70.6
84.9
61.4
–
154.0
150.8
119.7
156.0
140.1
83.3
152.4
148.9
118.9
156.0
139.7
83.5
39.9
70.8
88.5
61.6
–
153.6
150.2
120.6
156.0
139.2
86.0
38.8
75.2
84.6
64.4
–
5
6
8
1ꢀ
2ꢀ
3ꢀ
4ꢀ
5ꢀ
73.8
71.0
71.3
71.9
81.0
81.1
63.8
64.0
2ꢀ-COa
3ꢀ-COa
5ꢀ-COa
2ꢀ-CH3.CO/
3ꢀ-CH3.CO/
5ꢀ-CH3.CO/
170.1
170.3
171.1
21.1
169.3
170.0
171.0
20.8
–
–
171.0
171.1
–
–
–
170.8
171.1
–
–
–
–
–
–
–
–
–
–
21.2
–
21.6
21.5
–
21.2
–
21.5
21.5
–
21.5
–
–
21.5
–
a Assigned based on gHMBC spectra.
1
n
Mass spectroscopy and optical rotations
Delay values were optimized for JC,H 140.0 Hz and JC,H 3.0
Hz. Zero filling in F1 to 1 K, ꢀ/2 shifted sine-bell squared (for
gHSQC) or sine-bell (for gHMBC) apodization functions were used
for processing.
Mass spectra were recorded on Finnigan LCQ-Deca (Termoquest)
°
in ESI positive-ion mode, KV 5.00, 225 C, 15 V. Optical rotations
were measured on a Perkin-Elmer 241 polarimeter (sodium D line at
°
25 C).
Copyright  2007 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2007; 45: 781–784
DOI: 10.1002/mrc

784
Spectral Assignments and Reference Data
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This work was financially supported by Universita` degli Studi di
Milano (Fondi FIRST).
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Magn. Reson. Chem. 2007; 45: 781–784
DOI: 10.1002/mrc