Studies on structure of kinetin riboside and its analogues by variable-temperature NMR

Article abstract of DOI:10.1016/j.molstruc.2019.05.112

Kinetin riboside and its four base-modified analogues were synthesized and their structures in solution were examined by multinuclear 1D and 2D variable-temperature NMR techniques. At lowered temperature kinetin riboside was found to exist as a mixture of two distinct conformers resulting from the restricted rotation around the C6-N⁶ bond. For 8-azakinetin riboside and 2-fluorokinetin riboside the presence of two rotamers was observed at room temperature.

Full text of DOI:10.1016/j.molstruc.2019.05.112

Accepted Manuscript
Studies on structure of kinetin riboside and its analogues by variable-temperature
NMR
Daniel Baranowski, Grzegorz Framski, Eliza Wyszko, Tomasz Ostrowski
PII:
S0022-2860(19)30684-2
DOI:
https://doi.org/10.1016/j.molstruc.2019.05.112
MOLSTR 26619
Reference:
To appear in: Journal of Molecular Structure
Received Date: 1 February 2019
Revised Date: 23 May 2019
Accepted Date: 27 May 2019
Please cite this article as: D. Baranowski, G. Framski, E. Wyszko, T. Ostrowski, Studies on structure of
kinetin riboside and its analogues by variable-temperature NMR, Journal of Molecular Structure (2019),
doi: https://doi.org/10.1016/j.molstruc.2019.05.112.
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ACCEPTED MANUSCRIPT
compd 5,
H10'
H10"
2'OH 3'OH
room temp.
H2'
H10'B
H10"B
10
N
Kinetin riboside (3):
X = CH, Y = N, Z = H
6
HN
ppm
8.6
O
Y
8.8
Analogues:
X
4
5
6
7
X = CH, Y = CH, Z = H
X = N, Y = N, Z = H
X = CH, Y = N, Z = NH2
X = CH, Y = N, Z = F
9.0
H6B-H10'B
H6B-H10"B
H6B
N
N
z
9.2
HO
O
9.4
H6
H6-H10'
H6-H10"
9.6
9.8
HO OH
10.0
10.2
10.4
5.6
5.4
5.2
5.0
4.8
4.6
ppm

ACCEPTED MANUSCRIPT
Studies on structure of kinetin riboside and its analogues by variable-temperature NMR
Daniel Baranowski, Grzegorz Framski, Eliza Wyszko*, Tomasz Ostrowski*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14,
61-704 Poznan, Poland
* Corresponding authors. Tel.: +48 61 8528503; fax: +48 61 8520532.
E-mail addresses: tostr@ibch.poznan.pl (T.Ostrowski), eliza.wyszko@ibch.poznan.pl
(E.Wyszko).
Abstract
Kinetin riboside and its four base-modified analogues were synthesized and their structures in
solution were examined by multinuclear 1D and 2D variable-temperature NMR techniques.
At lowered temperature kinetin riboside was found to exist as a mixture of two distinct
conformers resulting from the restricted rotation around the C6-N6 bond. For 8-azakinetin
riboside and 2-fluorokinetin riboside the presence of two rotamers was observed at room
temperature.
Keywords: Kinetin riboside; Kinetin riboside analogues; Synthesis; Variable-temperature
NMR; 15N NMR; Restricted rotation
1. Introduction
Multinuclear 1D and 2D NMR is a valuable tool for investigation of the dynamic
intramolecular processes in adenine derivatives, among others of amino-imino tautomerism
and rotation around C-N bond. It has been reported that the amino form of adenine is
absolutely dominant in solution, however additional substitution may shift the tautomeric
amino-imino equilibrium [1]. For N6-alkoxy-9-alkyladenines and N6-alkoxyadenosines the
imino form is more favored in all cases where tautomerism has been clearly found to exist by
1H NMR in DMSO-d6 at room temperature [2]. Modification of N6-methoxy-9-methyladenine
by introduction of selected substituents in its 2 position has been shown to shift the ratio of
tautomers toward the amino tautomer as determined by 1H, 13C and 15N NMR in DMSO-d6
[3,4]. The percent amino tautomer is much higher in solvents with increased dipole moment
and with the ability to take part in hydrogen bonding [4].
1

ACCEPTED MANUSCRIPT
On the other hand variable-temperature NMR technique has been used to demonstrate
the restricted rotation of the C6-N6 bond in N6-substituted adenine derivatives. Martin and
Reese have found that the spectrum of N6,N6-dimethyl-2’,3’-O-isopropylideneadenosine in
CDCl3 exhibits two N6-methyl signals at -60 °C and one signal at 40 °C [5]. They have
interpreted it as an example of hindered internal rotation, with slow exchange occurring
below, and fast exchange occurring above 0 °C. Engel and von Hippel have proposed the
preferential syn conformation for N6,9-dimethyladenine at low temperature in CDCl3 [6].
Dodin et al. have postulated that for 9-alkyl-N6-methyladenines the favored syn conformer is
stabilized by N6H-N7 hydrogen bonding, whereas the minor anti rotamer is stabilized by
N6H-N1 bonding but destabilized by the steric interaction of the methyl group [7]. These
authors have observed a 1:1 ratio of both rotamers for 1-alkyl-4-methylaminopyrazolo[3,4-
d]pyrimidines (9-alkyl-N6-methyl-8-aza-7-deazaadenines) at -40 °C. Hindered rotation of C6-
N6 bond at room temperature has been reported for N6,3-dialkyladenines in DMSO-d6 and
CDCl3 [8]. Geometrical isomerism has been also found by means of 1H and 13C NMR in
liquid ammonia containing potassium amide at -50 °C for the anions of adenine and 2-
aminopurine [9].
Novotna et al. have reported the existence of amino and 1H-imino tautomeric forms
for 2-chloro-N6-furfuryladenine (Fig. 1: 1) and 2-chloro-N6-(5-methylfurfuryl)adenosine (2) at
room temperature based on NMR studies performed in DMSO-d6 and DMF-d7 [10]. At
increased temperature the spectra of these compounds show only a single set of signals
attributed to a time-averaged contribution of both tautomers due to fast chemical exchange.
The C2-non-substituted congener of 2, N6-furfuryladenosine (kinetin riboside, KR; 3), has
been shown to occur at room temperature only as amino tautomer. The abovementioned
results motivated us to extend the described studies to 3 and its selected modified analogues,
7-deazakinetin riboside (7-deazaKR; Fig. 2: 4), 8-azakinetin riboside (8-azaKR; 5), 2-
aminokinetin riboside (2-aminoKR; 6) and 2-fluorokinetin riboside (2-fluoroKR; 7) at various
temperatures in order to have a broader view on their structures in solution.
O
O
CH3
HN
HN
O
N
N
N
N
N
N
HN
N
N
N
N
Cl
N
HO
HO
O
O
N
H
Cl
HO
OH
HO OH
1
2
3
Fig. 1. Compounds discussed in ref. [10].
2

ACCEPTED MANUSCRIPT
O
O
HN
HN
N
N
N
N
N
N
N
N
HO
O
HO
O
HO OH
HO OH
5
4
O
O
HN
N
HN
N
N
N
N
N
N
O
N
O
NH2
F
HO
HO
HO OH
HO OH
7
6
Fig. 2. Synthesized compounds discussed in this paper.
2. Results and discussion
2.1. Syntheses
Kinetin riboside (3) was prepared by N6-alkylation of N6-acetyl-2’,3’,5’-tri-O-
acetyladenosine with furfuryl alcohol under Mitsunobu conditions as described [11]. In a
similar manner the analogous new compounds, 7-deazakinetin riboside (4) and 8-azakinetin
riboside (5) were synthesized starting from 7-deazaadenosine (tubercidin; 8) and 8-
azaadenosine (9), which were converted into their tetraacetates (10 and 11, respectively) and
subsequently subjected to Mitsunobu reaction followed by deacetylation of the product
formed. Compound 9 was obtained by cyclization of 5-amino-1-(2,3,5-tri-O-acetyl-β-D-
ribofuranosyl)-1H-[1,2,3]triazole-4-carbonitrile (12) [12] with diethoxymethyl acetate and
methanolic ammonia as elaborated earlier for the synthesis of related compounds [13].
2-Chloroadenosine (13) was reacted with furfurylamine and diisopropylethylamine
according to the previously reported general procedure for the synthesis of N2-substituted 2-
aminoadenosines [14], but satisfactory yield of the resulting 2-(furfurylamino)adenosine (14)
was achieved using larger excess of furfurylamine and with 2-methoxyethanol as solvent [15].
The synthesis of 2-aminokinetin riboside (6) was performed by the same method, making use
of 2-amino-6-chloropurine riboside (15) as starting material. To obtain the hitherto
undescribed 2-fluorokinetin riboside (7) 6-chloro-2-fluoropurine (16) was first treated with
furfurylamine in the presence of triethylamine to afford 2-fluorokinetin (17) which was
separated from the mixture containing also 6-chloro-2-(furfurylamino)purine. Compound 17
3

ACCEPTED MANUSCRIPT
was ribosylated and the resulting product 18 was afterwards deprotected to give the desired
compound 7. The synthetic route for the discussed compounds is outlined in Scheme 1.
O
NH2
NHAc
HN
N
ii, iii
N
i
N
N
N
N
N
N
N
HO
O
AcO
HO
O
O
10
8
4
HO OH
OAc
HO OH
AcO
O
NH2
N
NHAc
HN
N
CN
N
N
N
N
N
N
iv
ii, iii
i
N
N
N
N
N
N
N
N
N
NH2
N
AcO
O
HO
O
AcO
HO
O
O
12
9
11
5
AcO OAc
HO OH
OAc
HO OH
AcO
NH2
NH2
N
N
N
v
N
N
O
N
O
N
Cl
N
N
H
HO
HO
O
13
14
HO OH
HO OH
O
Cl
N
HN
N
N
N
v
N
N
N
O
N
O
NH2
NH2
HO
HO
15
6
HO OH
HO OH
O
O
HN
N
HN
O
N
HN
Cl
N
N
N
N
N
N
N
vi
vii
viii
N
N
O
F
N
F
HO
AcO
O
N
H
N
H
N
F
N
F
16
17
18
7
HO OH
AcO OAc
Scheme 1. Synthesis of compounds 4-7 and 14. Reagents and conditions: (i) Ac2O, Pyr, rt →
60 °C, then imidazole, EtOH, rt; (ii) furfuryl alcohol, Ph3P, DEAD, THF, rt; (iii) 25%
NH4OH, MeOH, CH2Cl2, rt; (iv) DEMA, 90 °C, then NH3-MeOH, rt; (v) furfurylamine,
DIPEA, 2-methoxyethanol, 125 °C; (vi) furfurylamine, Et3N, MeOH, 60 °C; (vii) 1,2,3,5-
tetra-O-acetyl-β-D-ribofuranose, p-TsOH, chlorobenzene, 145 °C; (viii) 25% NH4OH, MeOH,
CHCl3, rt.
4

ACCEPTED MANUSCRIPT
2.2. NMR study
Characteristic broadening of signals of the CH2 group attached to the N6 atom of
adenosine has been reported previously by others and ascribed to a chemical exchange
process connected to the restricted conformational change [16-18]. To our knowledge,
however, studies at lower temperature in order to obtain separated signals of particular
conformers have not been carried out as far. In the 1H NMR spectrum of kinetin riboside (3)
in DMSO-d6 recorded at room temperature we observe broad signal of N6-CH2 (specified as
C10-H2 in this paper) at 4.70 ppm and broad signal of N6-H at 8.31 ppm (Table 1). In the 1H
NMR spectrum of 3 in acetone-d6 these protons are reflected by broad signals at 4.87 ppm and
7.42 ppm, respectively (Table 2). Decreasing the temperature causes the appearance of double
signals starting from 0 °C. The spectrum of 3 in acetone-d6 at -20 °C exhibits two sets of
signals corresponding to major and minor components of the mixture (in a ratio of
approximately 9:1, according to integration). We note the presence of two doublets of
doublets of C10-H2 (at 4.87 and 4.82 ppm) corresponding to the predominant conformer and
two minor doublets of doublets (at 5.34 and 5.27 ppm). Besides, there are two triplets of N6-H
(major at 7.90 ppm and minor at 7.82 ppm), two singlets of C8-H (major at 8.23 ppm and
minor at 8.31 ppm) and two singlets of C2-H (major at 8.27 ppm and minor at 8.13 ppm). The
crucial assignment of N6-H protons is supported by 1H-1H COSY spectrum recorded at -20 °C
showing distinct crosspeaks between the triplets at 7.90 ppm and at 7.82 ppm and the
respective signals of C10-H2 for both components of the mixture, but no crosspeaks between
these triplets and the signals of C2-H (Fig. 3) are observed. Proton vicinal coupling constants
(3JHH) between N6-H and C10-H2 are of 6.0 Hz and 6.7-6.9 Hz for major and minor
component, respectively. The 13C spectrum of 3 recorded at -20 °C shows two sets of signals
of C10, C6, C5, C2, C8, C11 and C14 (Table 2). In the 1H-15N gHSQC spectrum of 3 at -20
°C two separated one-bond 1H-15N correlation signals are detected: strong at 85.1 ppm and
weak at 93.6 ppm. The spectrum confirmed the coupling of N6 and N6-H for the major
component, but indicated also the coupling of N6 and N6-H for the minor one (Fig. 4). This
means that both components are in one tautomeric form – the amino form. Additionally, 1H-
15N gHMBC spectrum reveals the long range correlations of N1 with N6-H and C2-H at 231.4
ppm, thus unambiguously supporting for aromatic character of N1 (Supplementary data: Fig.
16). These results in turn clearly show the restricted rotation around the C6-N6 bond in the
molecule of 3 resulting in the existence of syn and anti rotamers at temperature below 0 °C
(Fig. 5). Comparison of the 1H NMR spectra recorded between 35 °C and -30 °C in steps of 5
5

ACCEPTED MANUSCRIPT
°C (Supplementary data: Fig. 67) reveals that hydroxyl protons of the ribose moiety undergo
downfield shift of frequency resonance by ca 0.42-0.68 ppm because of increased hydrogen
bonding with the solvent. However in the case of N6-H the observed broad singlet at 35 °C is
shifted downfield by 0.69 ppm and at -30 °C gives undeniable triplet for both components of
the mixture. This effect results from slowing down intermolecular proton exchange leading to
the split of N6-H signal with observable coupling to nearby protons. Analogously, the same
effect of splitting the signal depending on temperature is observed for C10-H2. It is
noteworthy that C2-H or C8-H signals of minor conformer are shifted upfield or downfield
respectively, compared with C2-H or C8-H of major component due to the anisotropic
shielding/deshielding effects of the aromatic ring of furan.
6

ACCEPTED MANUSCRIPT
Table 1
Selected 1H, 13C and 15N NMR chemical shifts for compounds 3-7 in DMSO-d6 at room temperature; δ
in ppm (J in Hz)
3 (KR)
4 (7-deazaKR)
5 (8-azaKR)
6 (2-aminoKR)
7 (2-fluoroKR)
N6-H
8.31 brs
8.00 t (6.0)
9.52 t (5.8, 6.0)a
9.14 t (6.4, 6.5)b
7.72 brs
8.88 t (5.1, 5.0)a
8.61 brsb
C10-H2
C8-H
4.70 brs
8.38 s
─
4.70 d (6.0)
7.36 d (4.0)
4.76 d (5.9)a
4.63 brs
7.93 s
4.62 d (8.9)a
5.19 d (6.7)b
5.16 mb
─
─
8.38 brsa
8.40 brsb
C7-H
6.66 d (4.0)
6.27 d (3.2)
─
─
C12-H
6.23 d
(3.2)
6.31 d (3.0)a
6.33 d (2.9)b
6.24 d (3.2)
6.26 d (2.8)a
6.28 brsb
C14-H
C2-H
7.53 dd
(2.0, 0.8)
7.57 brs
8.15 s
7.56 d (0.9)a
7.53 dd
(2.0, 0.8)
7.56 brsa
7.58 brsb
7.54 brsb
8.24 s
8.45 sa
8.34 sb
─
─
C10
C6
36.49
36.58
36.70
36.24
37.23
154.31
155.77
154.16a
155.59b
154.63
155.78 (20.7)
C5
119.82
152.25
148.58
140.01
152.86
106.65
141.83
87.90
103.52
151.20
149.48
122.37
152.87
106.80
141.90
87.54
124.63a
113.73
159.92
151.08
136.18
153.31
106.62
141.65
86.97
118.00
123.83b
C2
156.84a
156.49b
158.48 (204.5)
150.02 (20.1)
C4
148.57a
149.98b
C8
─
140.09a
139.75b
C11
C12
C14
C1’
N6
151.65
151.98a
152.44b
107.29a
107.46b
107.11
142.19a
142.09a
142.36b
142.65b
89.84a
89.52b
87.53
86.1
86.6
96.3a
83.2
92.5a
99.5b
103.4b
N7
240.3
170.3
ndc
─
ndc
241.0
166.4
ndc
241.3
171.8
195.3
ndc
N9
154.6
226.4
224.4
─
238.3
230.4
216.5
─
N1
N3
ndc
ndc
C2-F
─
─
50.60a
52.62b
a Major component. b Minor component. c Not detected.
7

ACCEPTED MANUSCRIPT
Table 2
Selected 1H, 13C and 15N NMR chemical shifts for compounds 3, 4 and 6 in acetone-d6 at room and
low temperatures; δ in ppm (J in Hz)
3 (KR)
-20
4 (7-deazaKR)
6 (2-aminoKR)
-20 °C
25
°
C
°
C
25
°
C
-20
°
C
25
°
C
N6-H
7.42 brs
7.90 t (6.0, 6.0)a
7.82 t (6.7, 6.9)b
7.24 brs
7.56 t
(5.4, 5.4)
7.12 brs 7.63 t (6.0, 6.1)a
7.26 t (5.8, 6.7)b
4.87 dd (6.1, 15.7)a
4.82 dd (6.1, 15.8)a
5.34 dd (6.9, 15.9)b
5.27 dd (6.8, 15.9)b
4.83 dd
4.82 dd
4.72 d (6.0)a
5.23 d (6.6)b
C10-H2
4.87 brsc
4.74 brs
(5.7, 16.5) (5.6, 15.6)d
4.79 dd
(5.6, 16.5) (5.5, 15.3)d
4.77 dd
C8-H
C7-H
C2-H
C10
C6
8.17 s
─
8.23 sa
8.31 sb
7.27 d
(3.6)
7.30 d (3.6)
6.60 d (3.7)
8.18 s
7.74 s
─
7.81 sa
7.88 sb
─
6.60 d
(3.6)
─
8.26 s
37.70
155.98
122.14
152.99
141.67
153.83
142.85
83.9
8.27 sa
8.13 sb
8.19 s
37.95
157.45
106.06
151.97
125.64
154.02
142.83
84.6
─
─
37.28a
37.44
37.46
156.30
116.43
160.70
138.70
154.08
142.71
82.3
37.02
40.22b
155.57a
156.31b
157.07
105.95
151.63
126.01
153.57
141.85
85.5
155.84a
156.58b
C5
121.89a
115.97a
120.85b
114.81b
C2
152.74a
152.53b
160.44a
160.04b
C8
141.85a
138.75a
141.54b
nde
C11
C14
N6
153.46a
153.92b
153.73a
154.39b
142.76a
142.61a
143.04b
142.81b
85.1a
93.6b
83.1a
90.4b
N7
238.2
168.5
237.8a
─
─
237.6
165.0
238.0
241.9b
N9
168.6a
167.4b
152.6
152.4
165.7
N1
N3
231.5
221.1
231.4
220.2
226.3
222.1
225.6
220.9
nde
nde
197.6
nde
a Major component. b Minor component. c Signal partially overlapped with signal of C2’-H. d Signal
overlapped with signal of C2’-OH. e Not detected.
8

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3'OH
2'OH
H2 H8
H4'
H14
H1'
H10'
H10"
H13
H12
H3'
H5'
H5"
5'OH
H6
H10'B
H10"B
H8B H2B
H6B
ppm
7.7
H2'
H6-H10'B
H6-H10"B
H6B
7.8
7.9
8.0
8.1
8.2
8.3
H6-H10'
H6-H10"
H6
H2B
H8
H2
H8B
8.0
7.5
7.0
6.5
6.0
5.5
5.0
4.5
4.0
ppm
Fig 3. Fragment of 1H-1H COSY spectrum of compound 3 in acetone-d6 recorded at -20 °C.
Fig. 4. Fragment of 1H-15N gHSQC spectrum of compound 3 in acetone-d6 recorded at -20 °C
9

ACCEPTED MANUSCRIPT
10
O
O
HN
NH
N
14
11
1
6
3
7
5
4
12 13
N
N
N
N
N
8
2
N
N
9
5'
3'
HO
O
HO
O
1'
4'
2'
HO OH
OH
HO
Syn
Anti
Fig. 5. Rotameric equilibrium of 3 at lowered temperature and numbering of atoms.
The 1H NMR spectra of 8-azakinetin riboside (5) and 2-fluorokinetin riboside (7) in
DMSO-d6 recorded at room temperature (Table 1) exhibit two sets of signals corresponding to
major and minor components of the mixture (approximately 80% and 20%, respectively,
according to integration). The latter compound, bearing the electron-withdrawing substituent
in position 2 resembles the described previously [10] analogue 2. We observe two doublets of
C10-H2 for 5 (major at 4.76 ppm and minor at 5.19 ppm) and two signals of C10-H2 for 7
(major doublet at 4.62 ppm and minor one at 5.16 ppm). Signals of N6-H occur as two triplets
for 5 (major at 9.52 ppm and minor at 9.14 ppm), whereas for 7 as major triplet at 8.88 ppm
and minor broad signal at 8.61 ppm. In the 1H-1H COSY spectrum of 5 and 7 recorded at 25
°C crosspeaks between both signals of N6-H and the respective signals of C10-H2 are present
(Fig. 6). Proton vicinal coupling constants (3JHH) between N6-H and C10-H2 are of 5.8-6.0 Hz
and 6.4-6.5 Hz for major and minor component of 5, respectively. For 5 there are two signals
of C2-H (major at 8.45 ppm and minor at 8.34 ppm), of C12-H (major at 6.31 ppm and minor
at 6.33 ppm) and of C14-H (major at 7.56 ppm and minor at 7.58 ppm), whereas for 7 of C8-
H (major at 8.38 ppm and minor at 8.40 ppm), of C12-H (major at 6.26 ppm and minor at
6.28 ppm) and of C14-H (major at 7.56 ppm and minor at 7.54 ppm). The 13C spectrum of 5
recorded at room temperature shows two sets of signals of C6, C5, C2, C4, C12, C14 and C1’.
The analogous spectrum of 7 shows two sets of signals of C8, C11 and C14. Splitting of
signals is also detected at room temperature in 1H-15N gHSQC spectrum for 5 (N6: major
signal at 96.3 ppm and pronounced minor one at 103.4 ppm) and for 7 (N6: major signal at
92.5 ppm and slightly visible minor one at 99.5 ppm), and in 19F spectrum for 7 (C2-F: major
signal at 50.60 ppm and minor one at 52.62 ppm). All these separated signals coalesce upon
heating to 80 °C (Table 3). As in the case of 3 at -20 °C, the 1H-15N gHSQC spectrum of 5
recorded at room temperature indicated the coupling of N6 and N6-H for both components
(Fig. 7), suggesting the restricted rotation around the C6-N6 bond and the existence of two
rotamers of 5. The coupling of N6 and N6-H for the minor component is also observed,
although less evidently, for compound 7. Analysis of the 1H NMR spectra recorded for 5 and
10

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7 between 20 °C and 80 °C in steps of 5 °C reveals that signals of hydroxyl protons of ribose
moiety undergo upfield shift of frequency resonance by ca 0.31-0.37 ppm due to breaking
hydrogen bonds with the solvent (Supplementary data: Fig. 69 and 70). The observed coupled
nuclei i.e. N6-H and C10-H2 for 5 and 7 are gradually broadened loosing their splitting pattern
and N6-H is shifted upfield by 0.41-0.47 ppm. The changes of frequency resonances of C2-H
and C8-H of minor component for 5 and 7, respectively, are the same as in the case of 3.
Table 3
Selected 1H, 13C and 15N NMR chemical shifts for compounds 5 and 7 in DMSO-d6 at room and
increased temperatures; δ in ppm (J in Hz)
5 (8-azaKR)
C
7 (2-fluoroKR)
80 °C
25
°
80
°
C
25
°
C
N6-H
C10-H2
C2-H
C6
9.52 t (5.8, 6.0)a 9.14 brs
8.88 t (5.1, 5.0)a 8.44 brs
9.14 t (6.4, 6.5)b
8.61 brsb
4.76 d (5.9)a
5.19 d (6.7)b
4.83 brs
8.40 brs
151.40
ndc
4.62 d (8.9)a
5.16 mb
4.77d
8.45 sa
─
─
8.34 sb
154.16a
155.59b
155.78 (20.7)
118.00
155.67
117.65
158.12
ndc
C5
124.63a
123.83b
C2
156.84a
156.49b
156.21
ndc
158.48 (204.5)
150.02 (20.1)
C4
148.57a
149.98b
C8
─
─
140.09a
139.75b
139.52
151.82
141.56
87.55
C11
C14
C1’
151.65
151.40
141.69
89.76
151.98a
152.44b
142.19a
142.65b
142.09a
142.36b
89.84a
87.53
89.52b
a Major component. b Minor component. c Not detected. d Signal overlapped with signal of C5’-OH.
11

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H10'B
2'OH 3'OH H10"B
H10'
H10"
H10'
H10"
3'OH
2'OH
H2'
5'OH
H2'
5'OH
H10'B
H10"B
ppm
8.6
ppm
8.5
8.6
8.7
8.8
8.9
9.0
9.1
9.2
9.3
8.8
H6B-H10'B
H6B-H10"B
H6B
9.0
H6B-H10'B
H6B-H10"B
H6B
9.2
H6
H6-H10'
H6-H10"
9.4
H6
H6-H10'
H6-H10"
9.6
9.8
10.0
10.2
10.4
ppm
5.6
5.4
5.2
5.0
4.8
4.6
5.6
5.4
5.2
5.0
4.8
4.6
4.4
ppm
Fig. 6. Fragments of 1H-1H COSY spectra of compounds 5 (left) and 7 (right) in DMSO-d6
recorded at room temperature
Fig. 7. Fragment of 1H-15N gHSQC spectrum of compound 5 in DMSO-d6 recorded at room
temperature
12

ACCEPTED MANUSCRIPT
There is only one set of signals in 1H, 13C and 1H-15N gHSQC NMR spectra of 7-
deazakinetin riboside (4) and 2-aminokinetin riboside (6) in DMSO-d6 at room temperature
(Table 1). The 1H spectra show a doublet of C10-H2 at 4.70 ppm for 4 and a broad signal of
C10-H2 at 4.63 ppm for 6. The signal of N6-H occurs at 8.00 ppm (triplet) for 4 and at 7.72
ppm (broad singlet) for 6. In acetone-d6 the signal of C10-H2 for 4 appears at 4.83 ppm as two
doublets of doublets, whereas for 6 at 4.74 ppm as a broad singlet (Table 2). The signal of N6-
H is detected for 4 at 7.24 ppm (broad singlet) and for 6 at 7.12 ppm (broad singlet). The
splitting of signals is observed upon lowering the temperature in the case of 6. At -20 °C for 6
there are two doublets of C10-H2 (major at 4.72 ppm and minor at 5.23 ppm), two triplets of
N6-H (major at 7.63 ppm and minor at 7.26 ppm) and two singlets of C8-H (major at 7.81
ppm and minor at 7.88 ppm). The 1H NMR spectrum of 2-(furfurylamino)adenosine (14) in
DMSO-d6 at room temperature is significantly different from that of its regioisomer 6. The
signal of N2-CH2 for 14 occurs at 4.45 ppm and, contrary to N6-CH2 of 6, is a partially
ovelapping doublet of doublets. The signal of N2-H (triplet at 6.59 ppm) is shifted upfield in
comparison with N6-H of 6. These sharp signals observed for 14 indicate that internal rotation
around the exocyclic C2-N2 bond in the case of this compound is not restricted.
In literature there has appeared a brief remark postulating that the presence of two
rotamers around the C-N bond in purine derivatives might be misinterpreted as a tautomeric
equilibrium [1]. Our results seem to reveal the existence of the restricted rotation in kinetin
riboside (3), which is manifested by broad signals of N6-H and N6-CH2 in the 1H NMR
spectrum at room temperature and by separated signals in the 1H, 13C and 1H-15N gHSQC
NMR spectra when recorded at lowered temperature. Significantly less visible double set of
signals at -20 °C is also observed for compound 6. Two sets of signals corresponding to both
rotamers occur at room temperature for nitrogen-enriched compound 5, and for compound 7
possessing the electron-withdrawing substituent in position 2. On the other hand for
compound 4 with nitrogen deficiency in position 7 we do not observe double set of signals
that could indicate decisive effect of nitrogen N7 on restricted rotation along C6-N6 bond as in
the case of other discussed compounds. Our suggestions are in some contrast to the
conclusions formulated by Novotna et al. [10], the latter concerning however other
compounds, namely 1 and 2. In turn, our hypothesis has been confirmed by others who have
reported the restricted rotation around the exocyclic C-N bond in 8-azaadenine derivatives i.e.
the compounds related to 5, 9-(3-chlorophenyl)-N6-cyclopentyl-2-methylthio-8-azaadenine
[19] and 9-substituted N6-arylhydrazone-2-propylthio-8-azaadenines [20].
13

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3. Conclusions
Four base-modified analogues of kinetin riboside were prepared making use of various
synthetic methodologies. Their structures as well as the structure of kinetin riboside were
examined in solution using 1D and 2D multinuclear NMR techniques. Separation of signals as
a result of restricted rotation around the C6-N6 bond was demonstrated at lowered temperature
for kinetin riboside and 2-aminokinetin riboside, whereas at room temperature for 8-
azakinetin riboside and 2-fluorokinetin riboside. The key experimental evidence for the
existence of two rotamers was provided by: i/ 1H-1H COSY spectra showing for both of them
crosspeaks between the triplets of N6-H and the respective signals of C10-H2; ii/ 1H-15N
gHSQC spectra confirming for both the couplings of N6 and N6-H. For 7-deazakinetin
riboside the double sets of signals were not observed due to the lack of nitrogen in the 7
position thus suggesting its crucial role for rotation restriction along C6-N6 bond.
4. Experimental
4.1. General methods
1D and 2D 1H, 13C and 15N NMR spectra were recorded on a Bruker Avance III 500 MHz
spectrometer, equipped with 5 mm broad-band multinuclear (PABBO) probe in DMSO-d6 at
298 K. 1D 19F NMR spectra were recorded on a Bruker Avance II 400 MHz spectrometer.
Chemical shifts (δ) for 1H and 13C NMR were reported in ppm relative to the
tetramethylsilane (TMS) peak. For 31F and 15N NMR δ were reported in ppm, relative to
trichlorofluoromethane and liquid NH3, respectively. Mass spectra were recorded using
Thermo Scientific QExactive mass spectrometer. Thin-layer chromatography (TLC) was
carried out on Merck precoated 60 F254 silica gel plates, while column chromatography on
Merck silica gel 60H (40-63 ꢀm). Anhydrous pyridine was prepared by drying with KOH and
distillation with P2O5. 7-Deazaadenosine, 6-chloro-2-fluoropurine, 2-chloroadenosine, 2-
amino-6-chloropurine riboside and 1,2,3,5-tetra-O-acetyl-β-D-ribofuranose were purchased
from Carbosynth. Other reagents were purchased from Sigma-Aldrich, Merck or Fluorochem.
4.2. 7-Amino-3-(β-
5-Amino-1-(2,3,5-tri-O-acetyl-β-
801 mg, 2.18 mmol) was heated in diethoxymethyl acetate (4 ml) at 90 °C for 7h. The
D
-ribofuranosyl)-3H-[1,2,3]triazole[4,5-d]pyrimidine (8-azaadenosine, 9)
D
-ribofuranosyl)-1H-[1,2,3]triazole-4-carbonitrile [12] (12;
14

ACCEPTED MANUSCRIPT
solution was evaporated and the resulting syrup was dissolved in methanolic ammonia (4 ml).
The mixture was allowed to react at room temperature overnight and the obtained suspension
was evaporated to afford crude material which was then dried under vacuum at 50 °C to
remove acetamide (415 mg, 71% yield). 1H NMR spectrum of 9 as in lit. [21].
4.3. General procedure for acetylation reactions of compounds 8 and 9 (based on lit. [11])
To a suspension of 8 (266 mg, 1.0 mmol) or 9 (268 mg, 1.0 mmol) in anhydrous pyridine (2
ml) was added acetic anhydride (1.02 g, 10 mmol). The mixture was stirred at room
temperature overnight, then at 60 °C overnight. The reaction was cooled down and quenched
by addition of EtOH. The volatiles were evaporated, the residue was co-evaporated with
toluene and finally dissolved in EtOH (2.5 ml). Imidazole (53 mg, 0.78 mmol) was added and
the solution was stirred at room temperature for 8 h. The solution was diluted with EtOAc (5
ml) and washed with brine (2 x 5 ml). The organic layer was dried with Na2SO4 and
evaporated. The residue was subjected to chromatography on silica gel column using CH2Cl2-
MeOH (98:2→95:5) to give solid foam of 10 (369 mg, 85% yield) or 11 (240 mg, 55% yield).
1H NMR data of 4-acetylamino-7-(2,3,5-tri-O-acetyl-β-
d]pyrimidine (10) and of 7-acetylamino-3-(2,3,5-tri-O-acetyl-β-
D
-ribofuranosyl)-7H-pyrrolo[2,3-
-ribofuranosyl)-3H-
D
[1,2,3]triazole[4,5-d]pyrimidine (11) are given in Supplementary data section.
4.4. General procedure for converting tetraacetates 10 and 11 into 4 and 5 (based on lit.
[11])
To a solution of 10 (347 mg, 0.8 mmol) or 11 (349 mg, 0.8 mmol) in anhydrous THF (5 ml)
were added Ph3P (316 mg, 1.2 mmol), furfuryl alcohol (118 mg, 1.2 mmol) and DEAD (as
40% solution in toluene; 209 mg, 1.2 mmol). The mixture was stirred at room temperature
overnight. This was sufficient to achieve complete conversion of 11, but in the case of 10 the
addition one more of the same portion of all reagents and reacting for 7 days was necessary.
The mixture was evaporated and the residue was chromatographed on silica gel column with
CH2Cl2-MeOH (99:1). The resulting partly purified material was next treated with CH2Cl2 (6
ml), MeOH (12 ml) and NH4OH (6 ml), and stirred at room temperature overnight. It was
then evaporated and co-evaporated twice with toluene. The residue was subjected to column
chromatography with CH2Cl2-MeOH (97:3→9:1) to give the desired product as colorless oil.
4.5. 4-Furfurylamino-7-β-D-ribofuranosyl-7H-pyrrolo[2,3-d]pyrimidine (7-deazakinetin
riboside, 4)
15

ACCEPTED MANUSCRIPT
Because the attempts to crystallize compound 4 were unsuccessful, its analytical sample was
prepared by heating at 55 °C under vacuum for 15 h to remove acetamide. 1H, 13C and 15N
NMR data of 4 are given in Table 1, Table 2 and in Supplementary data section. HRMS
[M+H]+ calcd for C16H19N4O5: 347.1355; found 347.1344.
4.6. 7-Furfurylamino-3-β-D-ribofuranosyl-3H-[1,2,3]triazole[4,5-d]pyrimidine (8-azakinetin
riboside, 5)
Rechromatography on silica gel column with EtOAc-MeOH (95:5) afforded solid 5, which
was then heated at 55 °C under vacuum for 15 h to remove acetamide. 1H, 13C and 15N NMR
data of 5 are given in Table 1, Table 3 and in Supplementary data section. HRMS [M+H]+
calcd for C14H17N6O5: 349.1260; found 349.1249.
4.7. 2-(Furfurylamino)adenosine (14)
To a solution of 13 (241 mg, 0.8 mmol) in 2-methoxyethanol (3 ml) was added furfurylamine
(699 mg, 7.2 mmol) and DIPEA (931 mg, 7.2 mmol), and the whole was heated at 125 °C for
72 h, then evaporated. The residue was subjected to chromatography on silica gel column
with CH2Cl2-MeOH (92:8→9:1) which gave 14 as the yellowish oily material. It was next
rechromatographed with EtOAc-MeOH (9:1) to obtain white solid (194 mg, 67% yield). 1H
NMR spectrum of 14 as in lit. [15]. 13C NMR data are given in Supplementary data section.
4.8. 2-Amino-N6-furfuryladenosine (6)
To a solution of 15 (241 mg, 0.8 mmol) in 2-methoxyethanol (3 ml) was added furfurylamine
(699 mg, 7.2 mmol) and DIPEA (931 mg, 7.2 mmol) and the whole was heated at 125 °C for
48 h, then evaporated. The residue was chromatographed on silica gel column with CH2Cl2-
MeOH (95:5→92:8) and rechromatographed with EtOAc-MeOH (9:1) to give 6 as white
solid (278 mg, 96% yield). It was next crystallized from EtOAc. 1H, 13C and 15N NMR data of
6 are given in Table 1, Table 2 and in Supplementary data section. HRMS [M+H]+ calcd for
C15H19N6O5: 363.1417; found 363.1401.
4.9. 2-Fluoro-N6-furfuryladenosine (7)
To a solution of 16 (400 mg, 2.32 mmol) in anhydrous MeOH (17 ml) was added
furfurylamine (225 mg, 2.32 mmol) and Et3N (293 mg, 2.9 mmol). The mixture was stirred at
60 °C for 48 h, then concentrated under vacuum. The precipitated product 17 was separated,
washed with cooled MeOH and dried to afford 234 mg of solid which was not further
16

ACCEPTED MANUSCRIPT
purified. The mixture of crude 17 (200 mg, 0.86 mmol), 1,2,3,5-tetra-O-acetyl-β-D-
ribofuranose (280 mg, 0.88 mmol) and p-toluenesulfonic acid monohydrate (17.5 mg, 0.09
mmol) in chlorobenzene (5 ml) was heated at 145 °C for 3 h. It was evaporated and the oily
residue was next chromatographed on silica gel column with hexane-EtOAc (2:1→1:3) to
isolate 119 mg of 18 as solid foam. This material was treated with CHCl3-MeOH-25%
NH4OH (1:2:2, 5 ml), the resulting solution was kept at room temperature overnight and
evaporated. The deacetylated product 7 was purified by column chromatography with CHCl3-
MeOH (9:1), then the obtained foam was dried under vacuum at 55 °C for 8 h to remove the
remaining acetamide (66 mg of 7, 9% yield from 16). The solid sample of 7 precipitated from
its solution in MeOH-chloroform-toluene (1:2:2, 5 ml), then it was dried under vacuum. 1H,
13C, 15N and 19F NMR data of 7 are given in Table 1, Table 3 and in Supplementary data
section. HRMS [M+H]+ calcd for C15H17N5O5F: 366.1214; found 366.1202.
Acknowledgments
This work was supported by the National Science Centre, Poland (grant number
2014/13/B/NZ7/02291 to EW) and by the Polish Ministry of Science and Higher Education
(statutory financing and under the KNOW program). We wish to thank Dr. Lukasz Marczak
for MS spectra measurements.
Supplementary data
Supplementary data associated with this article can be found in the online version at
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19

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Four analogues of kinetin riboside (KR) were synthesized.
Multinuclear NMR was used to study the structure of KR and its analogues in solution.
At lowered temperature KR exists as the mixture of two conformers.
The existence of two forms was found at room temperature for 8-azaKR and 2-fluoroKR.
Conformations of studied compounds result from restricted rotation around C6-N6 bond.