An NMR and DFT investigation on the interconversion of 9-substituented-N 6-hydrazone-8-azaadenine derivatives: proton migration or conformational isomerization?

Article abstract of DOI:10.1007/s11224-017-1061-5

A newly synthesized N⁶-arylhydrazone-8-azaadenine derivatives (1) showed significant differences in NMR spectra with previously synthesized analogues, specifically, the hydrogens of N^1’H and C^3’H in all the titled compounds showed two groups of signals in their ¹H-NMR spectra. In order to investigate whether the duplication of proton signals were related to a mixture of conformational isomers which rotated around C-N^1’ bond or configurational isomers which resulted from proton migration, variable temperature NMR and 2D-NOESY experiments were carried out in conjunction with density function theory (DFT) calculations at the B3LYP/6-311G (d,p)//B3LYP/6-31G (d,p) level. The results indicated that it was the conformational isomerism rather than hydrogen transfer that induced the reproduction of proton signals, which was attributed to lower barrier energy and larger rate constant of the former process.

Full text of DOI:10.1007/s11224-017-1061-5

Structural Chemistry
https://doi.org/10.1007/s11224-017-1061-5
ORIGINAL RESEARCH
An NMR and DFT investigation on the interconversion
of 9-substituented-N6-hydrazone-8-azaadenine derivatives:
proton migration or conformational isomerization?
Qiangwen Fan1 & Yeming Wang2 & Hong Yan1
Received: 11 October 2017 /Accepted: 16 November 2017
#
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
A newly synthesized N6-arylhydrazone-8-azaadenine derivatives (1) showed significant differences in NMR spectra with pre-
viously synthesized analogues, specifically, the hydrogens of N1’H and C3’H in all the titled compounds showed two groups of
signals in their 1H-NMR spectra. In order to investigate whether the duplication of proton signals were related to a mixture of
conformational isomers which rotated around C-N1’ bond or configurational isomers which resulted from proton migration,
variable temperature NMR and 2D-NOESY experiments were carried out in conjunction with density function theory (DFT)
calculations at the B3LYP/6-311G (d,p)//B3LYP/6-31G (d,p) level. The results indicated that it was the conformational isomer-
ism rather than hydrogen transfer that induced the reproduction of proton signals, which was attributed to lower barrier energy
and larger rate constant of the former process.
6
.
.
.
Keywords N -hydrazone-8-azaadenine Variable-temperature NMR Density-functional theory (DFT) Conformational
.
isomerism Proton migration
Introduction
Moreover, many documents have reported that 8-
azaadenines derivatives showed strong fluorescence proper-
ties and can be applied for analytical applications, for instance,
using as fluorescence probe [12–14]. Besides, 8-azaadenines
and their nucleosides have also been intensively studied as
model systems for elucidating the mechanisms of action of
natural nucleosides and their analogs in various enzyme sys-
tems [15–17]. On the other hand, it is well known that differ-
ent geometric isomers of organic molecules may display dis-
tinct physical and chemical properties. In this sense, the study
of conformational or configurational changes in molecules is
essential in biochemistry, pharmacology, and supramolecular
chemistry. Nowadays, the most popular approaches for studies
of conformational interconversions are based on temperature-
dependence 1H NMR spectroscopy coupled with DFT calcu-
lations [18–23].
Contrasted with natural purines, 8-azaadenines are a class of
compounds characterized by the CH group of the imidazolic
ring replacing by a nitrogen atom. The 8-azaadenine scaffold
which served as an essentially biological activities moiety of
many kinase inhibitors has attracted considerable attentions of
chemists for a long time [1–8]. Some pharmaceuticals with
various substituted 8-azaadenines moiety are of potent anti-
thrombotic, anti-viral, and anti-neoplastic activities [9–11].
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s11224-017-1061-5) contains supplementary
material, which is available to authorized users.
* Hong Yan
With these in mind, herein we described our studies on
preparation and structural analysis of series of 8-
azaadenine derivatives (Scheme 1). Interestingly, we
found that the hydrogens of N1’H and C3’H in all the
target compounds (1) showed two groups of signals in
hongyan@bjut.edu.cn
1
College of Life Science and Bioengineering, Beijing University of
Technology, Chaoyang District, Beijing 100124, China
2
Beijing Tide Pharmaceutical Co., Ltd, No.8 East Rongjing Street,
1
their H-NMR spectra although this phenomenon of some
compounds is less obvious at 298 K. In order to
Beijing Econnomi Technological Development Area(BDA),
Beijing 100176, China

Struct Chem
Scheme 1 Synthetical strategy
for preparation of the target
compounds 1
investigate the source of the phenomenon, variable-
temperature NMR and 2D-NOESY experiments combined
with the density function theory (DFT) calculations were
carried out. To best of our knowledge, it is the first de-
scription of prototropic tautomerism and conformational
interconversion for 8-azaadenine derivatives from experi-
mentally and theoretically.
monohydrate and aromatic aldehyde where through nucleo-
philic substitution and dehydration condensation reaction
(Scheme 1).
General procedure for the preparation of compounds 1 A
250-ml round bottom flask was charged with solution of
N,N′-diisopropylethylamine (DIEPA) (2.8 g 0.022 mol) and
hydrazine monohydrate (1.1 g 0.022 mol) in 40 ml THF and
stirred in ice-water bath for 30 min and then solution of 4
(8.57 g 0.022 mol) in 100 ml THF was added dropwise to
the mixture and stirred until the compound 4 was no longer
detectable by TLC, followed by addition of aromatic aldehyde
into the reaction and stirred for 40 min when TLC showed
complete disappearance of starting materials. The reaction
mixture was treated with 200 ml H2O and extracted with
THF (3*20 ml). The organic layers were combined, washed
with brine, dried with anhydrous Na2SO4, filtered, and evap-
orated to afford crude product and then recrystallized from
THF and hexane (V/V = 3:1), resulting in pure target com-
pounds 1.
Experimental methods
Physical measurements
All chemicals were purchased from commercial sources and
used without further purification. Thin layer chromatography
(TLC) was conducted on silica-gel 60 F254 plates (Merck
KGaA). Melting points were determined on a XT-5A digital
melting-point apparatus without correcting.1H NMR spectra
and 13C NMR spectra were recorded on a Bruker Avance 400
spectrometer at 400 and 100 MHz using DMSO-d6 as the
solvent and tetramethylsilane (TMS) as the internal standard.
High-resolution mass spectral (HRMS) analyses were carried
out using a VG 70SE mass spectrometer from Manchester,
UK, which was operated in electron impact or electrospray
ionization mode.
9-[(1′R,2′S,3′S,4′S)-2′,3′-dihydroxyl-4′-hydroxyethoxy-
c y c l o p e n t a n e - 1 ′ - y l ] - 9 H - 2 - p r o p y l t h i o - 6 - ( 3 -
hydroxylbenzaldehyde hydrazone)-8-azapurine(1a) 1H NMR
(400 MHz, DMSO-d6): δ (ppm) 1.01 (3H, t, CH3, J = 7.2 Hz),
1.73 (2H, m, CH2), 2.07 (1H, m, CH2), 2.69 (1H, m, CH2),
3.14 (2H, CH2), 3.51–3.53 (4H, m), 3.78 (1H, s, CH), 3.97
(1H, s, CH), 4.62 (2H, m, CH, OH), 4.99–5.18 (3H, m, CH,
OH), 6.83 (1H, m, Ph-H), 7.24–7.31 (3H, Ph-H), 8.19, 8.43
Chemical synthesis
(1H, =CH), 9.68 (1H, s, Ph-OH), 12.36, 12.54 (1H, NH). 13
C
NMR (100 MHz, DMSO-d6): δ (ppm) 13.72, 22.82, 32.72,
33.57, 60.78, 61.08, 71.26, 74.27, 74.76, 82.25, 113.32,
117.82, 119.25, 122.79, 130.25, 135.98, 146.95, 151.69,
153.54, 158.10, 168.70. HRMS (ESI) for [M + H]+: calcd
490.18671, found 490.18668.
The titled compounds (1) were prepared by using compounds
2 and 3 as raw materials which underwent a nucleophilic
substitution process giving rise to the intermediate 4, followed
by a diazotization and deprotection reaction to afford another
intermediate 5, which subsequently reacted with hydrazine

Struct Chem
9-[(1′R,2′S,3′S,4′S)-2′,3′-dihydroxyl-4′-hydroxyethoxy-
c y c l o p e n t a n e - 1 ′ - y l ] - 9 H - 2 - p r o p y l t h i o - 6 - ( 3 , 4 -
diflorobenzaldehyde hydrazone)-8-azapurine(1b) H NMR
Computational details
1
Density functional theory at B3LYP/6-31G (d,p) [24] theoret-
ical level was used to optimize the geometries of all stationary
points along the reaction paths. Frequency analysis calcula-
tions were carried out at the same level to classify the located
stationary points as minima (no imaginary frequency) and
transition states (only one imaginary frequency) and to obtain
zero point energies (ZPES) and the thermodynamic correc-
tional data. The B3LYP/6-31G (d,p) intrinsic reaction coordi-
nate (IRC) pathways have also been computed of all the tran-
sition states to determine whether these transition states can
connect the reactants, intermediates or products [25]. The sin-
gle point energies of all the optimized structures were calcu-
lated at B3LYP/6-311G (d,p) level. All the energies listed in
this paper were corrected by thermal corrections. The polar-
ized continuum model (PCM) was used in Consistent
Reaction Field (SCRF) with DMSO as solvent [26–28]. All
of the quantum chemical calculations were performed using
the Gaussian 09 program package [29]. All the geometrical
coordination and single point energies of calculated structures
were provided in Supporting Information.
(400 MHz, DMSO-d6): δ (ppm) 1.01 (3H, t, CH3, J =
7.2 Hz), 1.73 (2H, m, CH2), 2.09 (1H, m, 5′-CH2), 2.67 (1H,
m, 5′-CH2), 3.14 (2H, CH2), 3.52 (4H, m, CH2), 3.78 (1H, s,
CH), 3.98 (1H, s, CH), 4.63 (2H, m, CH, OH), 5.03–5.18 (3H,
m, CH, OH), 7.53–7.92 (3H, m, Ph-H), 8.22, 8.46 (1H, =CH),
12.56, 12.71 (1H, NH). 13C NMR (100 MHz, DMSO-d6): δ
(ppm) 13.71, 22.76, 32.73, 33.80, 60.77, 61.08, 71.28, 74.25,
74.81, 82.25, 115.40 (115.52), 118.39 (118.50), 122.86,
124.78, 132.75, 143.83, 149.36 (149.45), 150.99 (151.08),
153.67, 168.78, 170.72. HRMS (ESI) for [M + H]+: calcd
510.17296, found 510.17287.
9-[(1′R,2′S,3′S,4′S)-2′,3′-dihydroxyl-4′-hydroxyethoxy-
cyclopentane-1′-yl]-9H-2-propylthio-6-(4-nitrobenzaldehyde
hydrazone)-8-azapurine(1c) 1H NMR (400 MHz, DMSO-d6):
δ (ppm) 1.02 (3H, t, CH3, J = 7.2 Hz), 1.73 (2H, m, CH2), 2.11
(1H, m, 5′-CH2), 2.69 (1H, m, 5′-CH2), 3.16 (2H, CH2), 3.35–
3.53 (4H, m), 3.79 (1H, s, CH), 3.98 (1H, s, CH), 4.63 (2H, m,
CH, OH), 5.04–5.18 (3H, m, CH, OH), 8.09 (2H, Ph-H), 8.30
(2H, Ph-H), 8.33, 8.60 (1H, =CH), 8.68 (1H, s, Ph-H), 12.76,
12.88 (1H, NH). 13C NMR (100 MHz, DMSO-d6): δ (ppm)
13.73, 22.75, 32.79, 33.62, 60.76, 61.16, 71.27, 74.24, 74.83,
82.24, 124.52, 128.29, 141.12, 148.11. HRMS (ESI) for [M +
H]+: calcd 519.17688, found 519.17689.
Results and discussion
9-[(1′R,2′S,3′S,4′S)-2′,3′-dihydroxyl-4′-hydroxyethoxy-
cyclopentane-1′-yl]-9H-2-propylthio-6-(4-chlorobenzaldehyde
hydrazone)-8-azapurine(1d) 1H NMR (400 MHz, DMSO-d6):
δ (ppm) 1.00 (3H, t, CH3, J = 7.2 Hz), 1.73 (2H, m, CH2),
2.10 (1H, m, 5′-CH2), 2.68 (1H, m, 5′-CH2), 3.14 (2H, CH2),
3.52(4H, m, CH2), 3.79 (1H, s, CH), 3.98 (1H, s, CH), 4.63
(2H, m, CH, OH), 5.03–5.18 (3H, m, CH, OH), 7.51–7.88
(4H, m, Ph-H), 8.32 8.50 (1H, =CH), 12.50, 12.64 (1H,
NH). 13C NMR (100 MHz, DMSO-d6): δ (ppm) 13.73,
22.79, 33.74, 33.60, 60.78, 61.08, 71.27, 74.28, 74.80,
82.26, 122.86, 129.08, 129.32, 133.75, 134.78, 145.08,
151.70, 153.61, 168.75. HRMS (ESI) for [M + H]+: calcd
508.15283, found 508.15289.
NMR characterization
All the structures of synthesized compounds were determined
by 1H NMR, 13C NMR and HRMS (Supporting Information
Fig. S1–6). Among, the compound 1a was selected as the
model for structural characterization and its NMR spectra
were presented in Fig. 1.
According to the 1H NMR spectrum (Fig. 1a), signals at δ
1.01 (t, 3H), 1.73 (m, 2H), and 3.15 (m, 2H) were attributed to
protons in propyl and peaks at δ 3.78 (t, 1H) and 3.97 (t, 1H)
and 5.18 (d, 1H) due to the three hydrogens of hydroxyl, the
assignments of the remained eight saturated protons signals
attached at cyclopentane and ethylidene corresponding to the
rest of peaks at up-field ranging from 2.05 to 5.09 ppm.
Additionally, the four aromatic protons of phenyl were located
at 6.83~7.81 ppm and the presence of the phenolic hydroxyl at
9.68 ppm. Finally, the hydrogen signals at 8.19~8.43 ppm and
12.36~12.54 ppm were regarded as belonging to C3’H and
N1’H, respectively.
To verified rationality of the assignment of the three pro-
tons at the lowest field in Fig. 1a, D2O-exchange NMR exper-
iment was performed and the spectrum was depicted in Fig.
1b, where both the signals at 9.68 ppm and 12.36~12.54 ppm
were disappeared while the peaks at 8.19~8.43 ppm were
remained, which illustrated that peaks at the disappeared lo-
cation were correspondence with those of active hydrogens
9-[(1′R,2′S,3′S,4′S)-2′,3′-dihydroxyl-4′-hydroxyethoxy-
cyclopentane-1′-yl]-9H-2-propylthio-6-(3-nitrobenzaldehyde
hydrazone)-8-azapurine(1e) 1H NMR (400 MHz, DMSO-d6):
δ (ppm) 1.01 (3H, t, CH3, J = 7.2 Hz), 1.73 (2H, m, CH2), 2.14
(1H, m, 5′-CH2), 2.69 (1H, m, 5′-CH2), 3.15 (2H, CH2), 3.53
(4H, m, CH2), 3.79 (1H, s, CH), 4.00 (1H, s, CH), 4.64 (2H, m,
CH, OH), 5.01–5.19 (3H, m, CH, OH), 7.70–7.76 (1H, m, Ph-
H), 8.21–8.24 (2H, m, Ph-H), 8.58, 8.68 (1H, =CH), 8.68 (1H,
s, Ph-H), 12.64, 12.82 (1H, NH). 13C NMR (100 MHz, DMSO-
d6): δ (ppm) 13.72, 22.75, 32.77, 33.62, 60.78, 61.08, 71.30,
74.25, 74.80, 82.26, 121.49, 122.87, 124.33, 130.70, 133.46,
136.67, 143.95, 148.61, 151.71, 153.62, 168.81. HRMS (ESI)
for [M + H]+: calcd 519.17688, found 519.17691.

Struct Chem
Fig. 1 Spectra of compound 1a. a 1H NMR. b D2O-exchange. c 13C NMR. d HRMS
that assigned as phenolic hydroxyl and NH and hence the
remained one was the proton of C3’H.
molecular system. Contreras and colleagues’ theoretically
studies on the prototropic tautomerism of 8-azaadenine have
shown that there are a number of tautomeric forms in the 8-
azaadenine structure and among them, the 9H-azaade, 8H-
azaade and 7H-azaade are more stable for their lower energy
values [30]. Moreover, Wierzchowski’s group speculated that
2-amino-8-azaadenine can exist in many different tautomeric
forms and they predicted that the photo-induced proton trans-
fer process of such compound is particularly useful for its
application in fluorescence probe (Scheme 2a) [30]. On the
other hand, previous studies about N-acylhydrazone (NAH)
have shown that C = N double bond stereoisomers (E/Z) and
From the 13C NMR spectrum (Fig. 1c), 21 carbon signals
were observed. The first 10 peaks from 13.72 to 82.76 ppm
were correlated with those sp3 hybrid carbon atoms in the
compound. The location of the phenylic and the olefinic car-
bon signals was found at 113.2~135.9 ppm and the distribu-
tion of carbon signals of the 8-azaadenine skeleton was locat-
ed at 151.69~168.70 ppm. HRMS (ESI+) of 1a (Fig. 1d)
shown a molecular ion peak at 490.18671 m/z, which was
consistent with the calculated value (i.e., 490.18668 m/z) for
1a [M]+. Hereto, the structure of 1a was established through
these above data without controversy.
Table 1 1H NMR data of N1’H and C3’H protons for compounds 1
From Fig. 1a, one can readily noticed the duplications of
proton signals at 8.19~8.43 ppm and 12.36~12.54 ppm, which
were assigned as C3’H and N1’H, respectively. Particularly, the
same consequence can be observed within all the titled com-
pounds 1 (Fig. S1), of which the proton of C3’H and N1’H
showed a broaden doublet peak. The relative chemical shifts
were depicted in Table 1.
Compound no.
N1’H (ppm)
C3’H (ppm)
1a
1b
1c
1d
1e
12.36, 12.54
12.56, 12.71
12.76, 12.88
12.50,12.64
12.64, 12.82
8.19, 8.43
8.22, 8.46
8.33, 8.60
8.32,8.50
8.50, 8.68
In general, this phenomenon was introduced by the pres-
ence of configurational isomers or conformational isomers in

Struct Chem
a
b
Scheme 2 a Structure and some possible tautomeric forms of 2,6-diamino-8-azapurine. b Stereoisomers and conformers of NAH
conformers (syn/anti) about the amide CO-NH bond may be
observed (Scheme 2b), which depended on the assemblage of
amide and imine functions. Furthermore, both the protons of
benzyl and alkyl NAHs showed duplication signals, which
was later proved to be induced by a mixture of two conformers
of the samples in solution and the energy barrier of this con-
version was calculated as about 17 kcal/mol via dynamic
NMR experiments [31].
Inspiring by studies on the compounds 8-azapurines and
hydrazones, we speculated that the isomers involved in the 1H
NMR spectrum of compounds 1 would incorporate two of the
following structures displayed in Scheme 3. The compounds
1a was also selected as the structural analysis model.
To find out what was the exactly dominated factor resulting in
the occurrence of the doublet proton signals, primarily, the
1
temperature-dependence H NMR experiment was performed
Scheme 3 Conceivable isomers of compound 1a. (Numbering in the scheme was just for simplicity and not obey the IUPAC rules)

Struct Chem
Fig. 2 Variable temperature 1H NMR spectrum (a) and local amplified NOESY spectrum (b) of 1a.
1
using different temperature at 298 K, 303 K and 313 K, the
spectrum as displayed in Fig. 2a. As can be seen from Fig. 2a,
both the two groups of proton peaks of C3’H and N1’H were
gradually coalesce at 303 K, and then merged into a singlet
one with temperature increasing to 313 K, which implying that
the duplication of these proton signals may be attributed to the
presence of two isomers which interconvert slowly at ambient
and the rate will be accelerated with temperature increasing.
With this result in hand, we then traced back to Scheme
3 for analyzing the possible configuration of 1a in solu-
tion. As shown in Scheme 3, a couple of stereoisomers
(E/Z) would be observed with respect to C3’ = N2’ double
bond, which were capable of forming configurational iso-
mers E12~E15 and Z22~Z25 through 1,3-hydrogen trans-
fer. Additionally, it was also readily accepted that the
existence of conformational isomers of 1a in solution,
generated by rotation of C6 -N1 ’ single bond.
Nonetheless, the rotation of N1’-N2’ bond was not taken
into consideration for the steric repulsion effect between
the arylhydrazone groups and the 8-azapurines moiety. In
this case, combining with the result extracted from
variable temperature H NMR spectra, we ruled out the
possibility of simultaneous occurrence of E/Z on account
of unable to interconvert each other of the two stereoiso-
mers. Subsequently, we decided to proceed with 2D-
NOESY experiments to access the relative stable config-
uration of 1a in solution (Fig. 2b).
According to Fig. 2b, besides the expected diagonal signals
were observed, correlation at (8.17, 12.29 ppm), assigned to
proton of C3’H and hydrogen of N1’H, was the straightforward
evidence that the E configuration was adopted by 1a in solu-
tion. Hence, our follow-up theoretical studies were focused on
investigating the E stereoisomers of 1 by using density func-
tional theory.
Potential energy surface (PES) scan studies
In order to describe the conformational flexibility of the titled
molecules, the energy profile as a function of N2’-N1’-C6-N1
torsion angle of compound 1a was shown in Fig. 3. All the
geometrical parameters were simultaneously relaxed during
the calculations while the variable was increased in steps of
10° from 180° to 360°. The torsional potential surface of the
molecule was obtained by using the PM6 method [32]. From
Fig. 3, two stable local minima cis-E (E11) and trans-E (E10)
arising from aromatic hydrazone bond to N1’ atom were ob-
tained at 0° (360°) and 180°. Further geometrical optimization
of these two structures was proceeded at B3LYP/6-31G (d,p)
theoretical level. According to the DFT calculation the two
conformers possessed similar energy, one was − 1973.960
Eh and the other was − 1973.958 Eh, the difference was just
for 0.02 Eh. Moreover, from the calculations, the optimized
structures of the two isomers were both coplanar between the
aromatic hydrazone and the 8-azapurine ring. These resem-
blance illustrated that the two isomers may exist in solution
may exist in solution simultaneously and therefore induce
duplication of proton signal.
Fig. 3 Potential energy surface scan for the selected dihedral angle of 1a
(in DMSO)

Struct Chem
1a
1c
Fig. 4 Energy profile for conformational isomerism and proton migration process of compounds 1a and 1c
Competition of proton migration and conformational
isomerism process
[33], we concluded that the duplications of hydrogen peaks
were attributed to conformational isomerism.
Additionally, variation tendency of the rate constants, k, for
the 1a and 1c varied with temperature ranging from 298 K to
313 K were calculated via the transtion state theory (TST) and
the resultant curves were shown in Fig. 5. Conspicuously, the
rate constants of conformatioanal isomerism were 1.00E +
016 fold than hydrogen migration, which implied that the
latter process can be considered as nonoccurrence in solution.
In the case of conformatioanal isomerism process, the constant
of 1c increased from 15 s−1 to 50 s−1 with temperature chang-
ing from 298 K to 313 K. What is more, the rate of 1c at 298 K
was 50 s−1greater than that of 1a at the same temperature and
15 s−1 at 313 K; Furthermore, the differentials expanded to
150 s−1 when temperature of 1c increased to 313 K.
Undoubtedly, these results supported perfectly the conslusion
that the conformatioanal isomerism was superior to proton
migration and in completely agreement with experimental re-
sults that the duplications of hydrogen peaks for N1’H and
C3’H in 1a occurred at 298 K and then coalesce into a broaden
Aiming at ascertaining the predominant factor between hydro-
gen transfer and interconversion causing reproduction of the
proton peaks and interpreting the signals of N1’H and C3’H
protons of 1c at 298 K (Supporting Information Fig. S3) is
similar to those of the other compounds in Scheme 1 at 313 K,
we calculated the energy barriers for the two isomerization
processes of compounds 1a and 1c, for instance. As shown
in Fig. 4, the barriers for conformational isomerism of 1a and
1c were found be very closely, 15.2 and 14.8 kcal/mol, respec-
tively, and far less than those for proton migration where the
barriers were 39.6 and 39.3 kcal/mol, which indicated that the
conformational isomerization was more favorable. Moreover,
the energy barriers of 1c were 0.4 kcal/mol smaller than 1a,
hinting the conformational isomerization of the former more
accessible, which in good agreement with the 1H NMR exper-
iment result. Having known that reactions with a barrier of
21 kcal/mol or less will proceed readily at room temperature
Fig. 5 The curves of rate constants varied with temperature for the conformatioanal isomerism (a) and proton migration (b) of 1a and 1c

Struct Chem
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Conclusion
In this paper, we described the synthesis of 9-substituented-
N6-hydrazone-8-azapurines derivatives and characterized
their structures via 1H NMR, 13C NMR and HRMS. The ste-
reo structures of compounds 1 and the reason resulting in
duplications of proton signals in 1H NMR spectra of the syn-
thesized compounds were analyzed through 2D-NOESY and
variable temperature NMR techniques as well as DFT calcu-
lations at the level of B3LYP/6-311G (d,p)//B3LYP/6-31G
(d,p). The 2D-NOESY spectrum showed that the E configu-
ration of 1 was adopted in solution. The variable temperature
NMR showed that the reproduction of hydrogen signals was
induced by the presence of either tautomers resulting from
proton transfer process or rotational isomers about the C6-
N1’ bond. The DFT calculations showed that the resultant
energy barriers of proton migration and conformational isom-
erism were about 40 kcal/mol and 15 kcal/mol, respectively.
The rate constant for the conformational isomerism process
was far less than that of proton transfer, 1.00E + 01 s−1 vs
1.00E-015 s−1. Combinding the experimental and theoretical
calculating results, we thought that it was the conformers that
introduced the duplication of proton signals of C3’H and N1’H.
Acknowledgments We are grateful for the financial support from 2015
Beijing Natural Science Foundation (No. KZ201510005007).
15. Da Costa CP, Fedor MJ, Scott LG (2007) 8-Azaguanine reporter of
purine ionization states in structured RNAs. J Am Chem Soc
129(11):3426–3432
Compliance with ethical standards
16. Liu L, Cottrell JW, Scott LG, Fedor MJ (2009) Direct measurement
of the ionization state of an essential guanine in the hairpin ribo-
zyme. Nat Chem Biol 5(5):351
Conflict of interest The authors declare that they have no conflicts of
interest.
17. Viladoms J, Scott LG, Fedor MJ (2011) An active-site guanine
participates in glmS ribozyme catalysis in its protonated state. J
Am Chem Soc 133(45):18388
18. Mohamed TA, Hassan AE, Shaaban IA, Abuelela AM, Zoghaib
WM (2017) Conformational stability, spectral analysis (infrared,
Raman and NMR) and DFT calculations of 2-amino-5-(ethylthio)-
1, 3, 4-thiadiazole. J Mol Struct 1130:434–441
19. Mitchell RH (2002) Conformational changes of 2, 11-dithia [3.3]
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