Observation by NMR of the tautomerism of an intramolecular OHOHN-charge relay chain in a model Schiff base

Article abstract of DOI:10.1016/j.molstruc.2007.04.015

As a model system for the internal and external aldimines of the coenzyme pyridoxal phosphate (PLP) in PLP dependent enzymes we have studied the ¹H and ¹⁵N NMR spectra of the ¹⁵N labeled Schiff base 3-carboxy-5-methyl-salicylidenaniline (1) dissolved in CD₂Cl₂. 1 contains a charge relay system with two strongly coupled intramolecular hydrogen bonds of the OHOHN type. One-bond ¹⁵N{single bond}¹H scalar spin-spin coupling constants and chemical shifts of partially deuterated 1 were measured in the temperature range between 243 and 183 K and analyzed assuming an exchange between three tautomeric states exhibiting well defined hydrogen bond geometries. The analysis shows that the dominant structure 1b corresponds to the zwitterion O{single bond}H?O^-?H{single bond}N⁺, where deuteration of one bond leads to a shortening of the other. This anti-cooperative effect is revealed by the vicinal isotope effects on the proton chemical shifts. By contrast, forms 1a and 1c are characterized by the structures O{single bond}H?O{single bond}H?N and O^-?H{single bond}O?H{single bond}N⁺, correspondingly, whose hydrogen bonds exhibit a cooperative coupling. We predict that 1a will dominate at high temperatures and low dielectric constants, whereas 1c will dominate at low temperatures and large dielectric constants. The comparison with model systems which do not contain the additional COOH-group indicates that the latter is responsible for the dominance of the zwitterionic structure of the OHN hydrogen bond. The implications of these findings for the function of the coenzyme pyridoxal phosphate in its natural environment are discussed.

Full text of DOI:10.1016/j.molstruc.2007.04.015

Available online at www.sciencedirect.com
Journal of Molecular Structure 844–845 (2007) 319–327
www.elsevier.com/locate/molstruc
Observation by NMR of the tautomerism of an intramolecular
OHOHN-charge relay chain in a model Schiff base
a
a
a,b,
b
Nikolai S. Golubev , Sergei N. Smirnov , Peter M. Tolstoy
*, Shasad Sharif ,
c
a
b
Michael D. Toney , Gleb S. Denisov , Hans Heinrich Limbach
a
V.A. Fock Institute of Physics, St. Petersburg State University, Russia
b
Institute of Chemistry, Free University of Berlin, Germany
Department of Chemistry, University of California, Davis, USA
c
Received 23 March 2007; received in revised form 12 April 2007; accepted 13 April 2007
Available online 20 April 2007
Dedicated to Prof. Lucjan Sobczyk on the occasion of his 80th birthday.
Abstract
As a model system for the internal and external aldimines of the coenzyme pyridoxal phosphate (PLP) in PLP dependent enzymes we
have studied the 1H and 15N NMR spectra of the 15N labeled Schiff base 3-carboxy-5-methyl-salicylidenaniline (1) dissolved in CD2Cl2. 1
contains a charge relay system with two strongly coupled intramolecular hydrogen bonds of the OHOHN type. One-bond 15NA1H scalar
spin–spin coupling constants and chemical shifts of partially deuterated 1 were measured in the temperature range between 243 and
183 K and analyzed assuming an exchange between three tautomeric states exhibiting well defined hydrogen bond geometries. The anal-
ysis shows that the dominant structure 1b corresponds to the zwitterion OAHꢀ ꢀ ꢀOꢁꢀ ꢀ ꢀHAN+, where deuteration of one bond leads to a
shortening of the other. This anti-cooperative effect is revealed by the vicinal isotope effects on the proton chemical shifts. By contrast,
forms 1a and 1c are characterized by the structures OAHꢀ ꢀ ꢀOAHꢀ ꢀ ꢀN and Oꢁꢀ ꢀ ꢀHAOꢀ ꢀ ꢀHAN+, correspondingly, whose hydrogen
bonds exhibit a cooperative coupling. We predict that 1a will dominate at high temperatures and low dielectric constants, whereas 1c
will dominate at low temperatures and large dielectric constants. The comparison with model systems which do not contain the addi-
tional COOH-group indicates that the latter is responsible for the dominance of the zwitterionic structure of the OHN hydrogen bond.
The implications of these findings for the function of the coenzyme pyridoxal phosphate in its natural environment are discussed.
ꢀ 2007 Published by Elsevier B.V.
Keywords: Charge relay chain; Hydrogen bond; Schiff base; Tautomerism; Low temperature NMR; Isotope effect
1. Introduction
between an aspartate residue and the pyridine ring which
has been modeled by NMR using pyridine–carboxylic acid
complexes [6–9]. Essential is also an intramolecular OHN
hydrogen bond between the phenolic OH group and the
imino nitrogen. For model Schiff bases tautomeric equilibria
between an OAHꢀ ꢀ ꢀN and an Oꢁꢀ ꢀ ꢀHAN+ forms (see
Fig. 2) have been established for liquid solutions [10,11]. It
has been argued that these fast equilibria are pre-requisite
for the enzymatic activity [12]. The equilibria were studied
by UV–Vis [13] and NMR [14–17] spectroscopy. Many fac-
tors, such a temperature, solvent polarity and chemical sub-
stitution in the molecule influence the equilibrium. The
Pyridoxal phosphate (PLP, vitamin B6) is found in the
active sites of many enzymes where it forms a Schiff base
to a lysine side chain (Fig. 1) or to a free amino acid which
is going to be transformed. The first is called ‘‘internal’’ and
the second ‘‘external’’ aldimine [1–5]. The aldimines exhibit
an intermolecular low-barrier OHN hydrogen bond
*
Corresponding author. Address: Institute of Chemistry, Free Univer-
sity of Berlin, Germany. Tel.: +49 30 8385 3615; fax: +49 30 8385 5310.
E-mail address: tolstoy@chemie.fu-berlin.de (P.M. Tolstoy).
0022-2860/$ - see front matter ꢀ 2007 Published by Elsevier B.V.
doi:10.1016/j.molstruc.2007.04.015

320
N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
A towards B, the AB distance decreases to a minimum for
Lys
the quasi-symmetric complex and then increases again.
Often, NMR parameters of the nuclei in hydrogen
bridges are also correlated with the hydrogen bond geom-
etries [26]. Thus, it is possible to obtain information about
hydrogen bond geometries in liquids by NMR. Unfortu-
nately, in order to exploit very high resolution of NMR
for hydrogen bonded systems one needs to perform difficult
low temperature experiments in order to observe individual
hydrogen bonds in the slow hydrogen bond exchange
regime [21]. Such experiments often fail because of a low
solubility of the target compound. Therefore, inspired by
PLP, we have focussed on 3-carboxy-5-methyl-salicylide-
naniline (1) (Fig. 3a) which represents an intramolecular
charge relay chain. Here, the geometries of both hydrogen
bonds can potentially be studied in a larger temperature
interval using liquid state NMR techniques. 1 exhibits
potentially three different tautomeric states 1a–1c as
depicted in Fig. 3a (Fig. 3b will be discussed later). Our
goal was to characterize these tautomers and their equilib-
ria in more detail by measuring different NMR parameters.
The most important parameter we have measured was
O
O
Tyr
N
P
H
O
O
O
H
O
N
H
CH3
O
O
C
Asp
Fig. 1. Fragment of the active site of aspartate aminotransferase; PLP
cofactor with additional hydrogen bonding to the side chain of
neighbouring amino acid is shown.
+
N
N
H
H
O
−
O
1
the one-bond J(15N,1H) scalar coupling constant which
can give direct information about the tautomerism of
OHN hydrogen bond [27–29]. For that purpose we have
Fig. 2. Common structural element of the PLP-like Schiff bases.
Tautomerism between a molecular OAHꢀ ꢀ ꢀN form and a zwitterionic
Oꢁꢀ ꢀ ꢀHAN+ form is shown.
1
labeled 1 with the 15N isotope. Unfortunately, J(15N,1H)
scalar couplings are rather insensitive to the interconver-
sion between 1b and 1c, because in this process the NH dis-
tance does not change significantly. Therefore, besides
measurements of the chemical shifts of hydrogen bonded
typical range of differences in enthalpy DH and entropy DS
between OH- and NH-forms for alkyl-substituted Schiff
bases are from ꢁ7 to ꢁ13 kJ molꢁ1 and from ꢁ30 to
ꢁ50 J molꢁ1 Kꢁ1, respectively, and the equilibrium shifts
significantly with the temperature [15,18]. For phenyl-
substituted Schiff bases, the OH-form predominates and
the equilibrium shifts only slightly with temperature
[19,20]. Additional intermolecular OHO hydrogen bonding
to the phenolic oxygen has been studied previously by NMR
spectroscopy [9]. It has been demonstrated that additional
bonding increases the acidity of the hydroxyl and shifts
the equilibrium to the NH-form of the Schiff base.
Both the inter- and the intramolecular hydrogen bonds
are coupled in the sense that a proton shift towards the ring
nitrogen also favours the zwitterionic form of the intramo-
lecular OHN hydrogen bond [9,21]. Coupled hydrogen
bonds exhibiting the property of rapidly moving electrical
charges via a coupled proton transfers have also been
called ‘‘charge relay chains’’ [22].
1
protons in 1, we have measured the effect on the H chem-
ical shift of a given H-bond arising from substitution of H
for D in the neighbouring hydrogen bond. Such ‘‘vicinal’’
or ‘‘long-range’’ isotope effects can be used to establish
the mutual coupling scheme of two interacting hydrogen
bonds [30,31]. If the neighbouring hydrogen bonds
are cooperatively arranged as in AHꢀ ꢀ ꢀAHꢀ ꢀ ꢀB or
Aꢁꢀ ꢀ ꢀHAꢀ ꢀ ꢀHB+, deuteration of one of them will lead to
the lengthening of the other one and corresponding shield-
ing of the neighbouring bridging proton. In contrast, if
hydrogen bonds are anti-cooperatively arranged as in
AHꢀ ꢀ ꢀAꢁꢀ ꢀ ꢀHB+, deuteration will lead to shortening of
the other hydrogen bond and corresponding deshielding
of its bridging proton.
2. Experimental
In previous papers it has been shown for carboxylic
acid–pyridine complexes that intrinsic changes of NMR
parameters with temperature may occur which arise from
temperature dependent solute–solvent interactions and
hence temperature dependent hydrogen bond geometries
averaged over various solvent configurations [23] called
‘‘solvatomers’’ by Perrin and Lau [24]. Such geometric
changes have been analyzed in terms of hydrogen bond
correlation techniques [25]. According to the latter, the
Aꢀ ꢀ ꢀH and the Hꢀ ꢀ ꢀB distances in AHB hydrogen bond
are correlated in the sense that when H is transferred from
3-Carboxy-5-methyl-salicylidenaniline was synthesised
from 3-carboxy-5-methyl-salicylaldehyde and aniline-15N
(96% of 15N isotope) using standard techniques for Schiff
base synthesis [32]. The aldehyde–acid precursor was
obtained via the formylation of 5-methylsalicylic acid by
chloroform in alkali media, according to the Reimer–Tie-
mann reaction [33]. Deuteration of the Schiff base was
accomplished by repeated re-crystallization from metha-
nol-OD. The total deuterium fraction was determined by
comparison of the integrated signal intensity of the residual

N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
321
_
O
H
H
H
H
H
H
Ph
Ph
Ph
+
+
O
N
O
O
N
O
O
N
_
O
O
O
CH
CH
CH
3
3
3
1a
1b
1c
O H
O D
O H
N
O H
O D
O H
N
O
H
N
O H
O H
O D
O
H
O
O
O
H
N
O H
N
O
D
H
H
N
N
O
D
N
O
D
N
1a
1b
1c
Fig. 3. (a) Possible tautomeric structures of 3-carboxy-5-methyl-salicylidenaniline (1). (b) Vicinal H/D isotope effects on the hydrogen bond geometry;
cooperatively coupled hydrogen bonds in 1a and 1c; anti-cooperatively coupled hydrogen bonds in 1b.
1
mobile proton NMR signal with those of the immobile
CH-protons. The relatively low deuteration ratios obtained
in this work are due to the H/D exchange between 1 and
the moisture during the handling of the substance, as well
as due to proton exchange between 1 and traces of water
in the NMR sample. NMR spectra were obtained on a
Bruker DPX-300 spectrometer in the temperature range
243–183 K using CD2Cl2 as solvent. The chemical shifts
of 15N nuclei were measured with respect to nitromethane
as an internal standard and then re-calibrated to the
NH4Cl scale. The absolute concentration of the samples
was approximately 0.01 M for 1H NMR and approxi-
mately 0.05 M for 15N NMR measurement.
the spectra. The H and 15N NMR parameters obtained
from Fig. 4 are listed in Table 1. They will be discussed
later.
Temperature dependent 1H NMR spectra were recorded
in the temperature range from 183 to 243 K and are shown
in Fig. 5. At temperatures higher than 243 K intermolecular
proton exchange significantly broadens the lines. The assign-
ment of the peaks follows that of Fig. 4. The H NMR
parameters for all temperatures are collected in Table 2.
1
OHOHN
3. Results
ODOHN
The NMR signals of the hydrogen bond protons and of
the Schiff base nitrogen of 1 dissolved in CD2Cl2, recorded
at 233 K are depicted in Fig. 4. The nitrogen signal was
OHODN
15N
1
obtained in the absence of H decoupling. The molecules
of 1 were partially deuterated in the mobile proton sites;
the deuterium fraction xD was approximately 0.2. The
assignment of the signals to the individual isotopologs
OHOHN, ODOHN and OHODN was accomplished by
comparison of the experimental line intensities with those
predicted for a statistical distribution of the isotopologs.
The d(OHOHN) and d(OHODN) signals are singlets.
The d(OHOHN) and d(ODOHN) signals exhibit a doublet
of doublets due to scalar spin–spin coupling with 15N and
the nearest CH proton. The 15N NMR signal consists of
several subsignals. The d(OHOHN) and d(ODOHN)
signals are split into doublets and the d(OHODN) signal
into a triplet. At xD = 0.2 the concentration of ODODN
is too small to give rise to an observable nitrogen signal.
By the line shape analysis the values of the coupling con-
160 159 158 157 156 155 154 153 152 151 150 ppm
OHOHN
ODOHN
_
O
J
J
(OHOHN) = 85.4 Hz
(HNCH) = 12.5 Hz
H
H
+
Ph
O
N
OHOHN
OHODN
O
CH
3
1H
1
1
stants J(OHO1H15N) ” J(OHOHN) and J(ODO1H15N) ”
J(ODOHN) are obtained, which are negative because of
the negative gyromagnetic ratio of 15N. For convenience,
we omit the sign as it cannot be directly determined from
16.2 16.1 16.0 15.9 15.8 15.7
15.6 15.5 15.4 ppm
Fig. 4. 15N (a) and 1H (b) NMR spectra of the solution, containing
0.05 M of 1 (96% 15N, xD = 0.2) at 233 K in CD2Cl2. Isotopolog ODODN
is not seen in 15N spectrum due to the low deuteration ratio xD.

322
N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
Table 1
4.1. General discussion of NMR parameters
NMR spectroscopic parameters of intramolecular hydrogen bonds for
solution of 1 in CD2Cl2 at 233 K
The analysis of the NMR parameters strongly depends
on whether they represent averages over the different tau-
tomeric forms of Fig. 3a or whether single structures are
present exhibiting temperature dependent hydrogen bond
geometries. In order to answer this question we have plot-
ted in Fig. 6a correlation established previously between
the two natural hydrogen bond coordinates q1 = 1/2
(r1 ꢁ r2) and q2 = r1 + r2 of OHN hydrogen bonds where
r1 and r2 are distances from the proton to the oxygen
and nitrogen atoms, correspondingly [7,25,34]. The dashed
curve refers to equilibrium geometries whereas the dotted
line includes an empirical correction for zero-point vibra-
tions [7]. Similar correlations have been observed for
OHO and NHN hydrogen bonds [35–37]. The geometries
of low-barrier or no-barrier hydrogen bonds correspond
to the points on the correlation curve. Generally, it has
been observed that lowering the temperature and increas-
ing of the dielectric constants compresses an OAHꢀ ꢀ ꢀN
hydrogen bridge and widens a zwitterionic Oꢁꢀ ꢀ ꢀHAN+
bridge [23], corresponding to intersection points 1–4 on
the left and on the right side of the correlation curve in
Fig. 6a. The lines connecting these intersection points rep-
resent then the average geometries in the presence of a
tautomerism between a molecular OAHꢀ ꢀ ꢀN form and a
zwitterionic Oꢁꢀ ꢀ ꢀHAN+ form. At the left and right inter-
section points the equilibrium constants of the tautomer-
ism are K = 0 and K fi 1, correspondingly. The
parameters used to calculate these lines were typical for
six-membered intramolecular OHN hydrogen bonded
cycle in Schiff bases [21]. Line 1 refers to higher tempera-
tures and lower dielectric constants whereas line 4 refers
to lower temperatures and larger dielectric constants.
d(OHOHN) 0.005 (ppm)
d(OHODN) 0.005 (ppm)
15.774
15.911
+0.137
15.714
15.826
+0.112
155.09
151.57
ꢁ3.52
d(OHODN) ꢁ d(OHOHN) 0.007 (ppm)
d(OHOHN) 0.005 (ppm)
d(ODOHN) 0.005 (ppm)
d(ODOHN) ꢁ d(OHOHN) 0.007 (ppm)
d(OHOHN) 0.05 (ppm)
d(OHODN) 0.05 (ppm)
d(OHODN) ꢁ d(OHOHN) 0.07 (ppm)
d(ODOHN) 0.05 (ppm)
155.55
+0.46
d(ODOHN) ꢁ d(OHOHN) 0.07 (ppm)
J(OHOHN) 0.5 (Hz)
85.4
84.6
ꢁ0.8
12.5
J(ODOHN) 0.5 (Hz)
J(ODOHN) ꢁ J(OHOHN) 0.7 (Hz)
J(HCNH) 0.5 (Hz)
15N NMR chemical shifts are referenced to NH4Cl.
243 K
233 K
223 K
213 K
203 K
193 K
1
These lines were estimated from the H vs. 15N chemical
shift correlation for intramolecular OHN hydrogen bonds
of Schiff bases [9] depicted in Fig. 6b. The dotted curve
was estimated for low-barrier or no-barrier hydrogen
bonds exhibiting a sequence of protonation states where
H is shifted from oxygen to nitrogen. The solid straight
183 K
15
1
lines now refer to the average H/15N chemical shifts in
16
ppm
the presence of a tautomerism between a OAHꢀ ꢀ ꢀN form
OHOHN
OHOHN
and a Oꢁꢀ ꢀ ꢀHAN+ form.
ODOHN
The data point symbolized by the filled circle in
Fig. 6b was obtained in this study for 1 in CD2Cl2 at
233 K. Clearly, the data point is not located on the cor-
relation curve but on line 2. This finding is a strong indi-
cation of the presence of a OHN tautomerism in 1. For
similar temperatures and the same solvent, however, we
would have expected for OHN hydrogen bonds of phe-
nyl-substituted Schiff bases chemical shifts and geome-
tries as represented by the open circles in Fig. 6 [9].
We associate this change to the presence of the carbox-
ylic group in 1, which increases the acidity of the hydro-
xyl and strongly shifts the equilibrium between molecular
OHꢀ ꢀ ꢀN and zwitterionic Oꢁꢀ ꢀ ꢀHN+ forms towards the
latter.
OHODN
Fig. 5. 1H NMR spectra of the solution, containing 0.01 M of 1 (96% 15N,
xD = 0.2) in CD2Cl2 in the temperature range 183–243 K.
4. Discussion
In the previous section, we have described the 1H
and 15N NMR spectra of 15N enriched 1 dissolved in
CD2Cl2 in the temperature range between 243 and
183 K. In this section we will analyze these parameters
in order to derive information about the hydrogen bond
geometries and proton transfer mechanisms in this
compound.

N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
323
Table 2
1H NMR spectroscopic parameters of intramolecular hydrogen bonds for solution of 1 in CD2Cl2 at various temperatures
T (K)
d(OHOHN) (ppm)
d(OHODN) (ppm)
d(OHOHN) (ppm)
d(ODOHN) (ppm)
J(OHOHN) (Hz)
243
233
223
213
203
193
183
15.705
15.812
15.910
16.002
16.084
16.152
16.224
15.870
15.965
16.063
16.138
16.211
16.268
16.322
15.820
15.720
15.649
15.575
15.501
15.457
15.409
15.940
15.849
15.780
15.709
15.643
15.596
15.551
84.3
85.0
85.8
86.3
86.9
87.3
87.8
the dependence of the NMR parameters assembled in
Table 2 on temperature. For a given parameter P such as
a chemical shift or a coupling constant it follows that
3.1
3.0
2.9
2.8
2.7
2.6
2.5
2.4
r1 r2
O-H····N
r1 r2
O····H-N
Pobs ¼ xaPa þ xbPb þ xcPc;
ð1Þ
r1 r2
O···H···N
r
where xi represents the mole fraction of the tautomeric
state i and Pi represents its intrinsic but temperature depen-
dent chemical shift or coupling constant; i = a, b, c. The
mole fractions xi depend in the usual way on the equilib-
rium constants Kab and Kbc of the two tautomeric equilib-
ria as illustrated in Fig. 7. These constants can be different
for the different isotopologs of 1. Thus, each NMR param-
eter measured depends on the three intrinsic parameters of
the three forms and on two equilibrium constants. The
number of parameters represents a problem for the fitting
as measurements could be done only in a limited tempera-
ture range because of the low solubility of 1.
Nevertheless, we have plotted in Fig. 8 the average
NMR parameters of Table 2 as a function of temperature
and added the solid curves calculated using Eq. (1) and a
parameter set which was obtained as follows. Firstly, we
have neglected any isotope effect on the equilibrium con-
stants, i.e. set
4
3
r
1
2
K
→ ∞
K
= 0
2
3
q
1
4
-0.6
-0.4
-0.2
0.0
0.2
r2) / Å
0.4
0.6
q1 = ½ ( r1
–
24
22
20
18
16
14
12
10
8
b
O····H····N
O-H····N
O····H-N
1
2
3
4
3
2
4
1
δ
HH
ab
HD
ab
DH
ab
K
K
¼ K ¼ K ¼ expðDSab=RÞ expðꢁDHab=RTÞ and ð2Þ
HH
bc
HD
bc
DH
bc
¼ K ¼ K ¼ expðDSbc=RÞ expðꢁDHbc=RTÞ;
ð3Þ
6
4
where DHij and DSij represent the reaction enthalpy and en-
tropy of the interconversion between tautomeric states i
and j in Fig. 7. Though the isotope effects on equilibrium
constant can often be well pronounced [34b], this simplifi-
cation is in agreement with previous findings for a model
Schiff base [21]. The vicinal H/D isotope effects on the
chemical shift of a given hydrogen bonded proton observed
were then associated with changes of the intrinsic values
after deuteration in the neighbouring site rather than with
changes of equilibrium constant. This type of isotope effect
has been called ‘‘equilibrium averaged intrinsic isotope ef-
fect’’ [21]. Secondly, we have assumed a preliminary set of
NMR and thermodynamic parameters for the structures
1a–1c. Using these parameters we have calculated the ana-
lytical curves for the temperature dependencies of the mole
fractions and of the averaged NMR parameters. Finally,
we have adjusted the parameters in order to fit analytical
curves to the experimental points (see solid lines in
Fig. 8). As a result, we got a ‘‘reasonable’’ but somewhat
0
-40
-80
(OHN) / ppm
-120
-160
-200
δ
Fig. 6. Hydrogen bond correlation of OHN hydrogen bonds. (a)
Geometric OHN hydrogen bond correlation of q2 = r1 + r2 as a function
of q1 = 1/2(r1 ꢁ r2), where r1 and r2 are distances from the bridging proton
to the heavy atoms, according to Ref. [7]. (b) 1H vs. 15N chemical shift
correlation of Schiff bases exhibiting an intramolecular OHN hydrogen
bond and a tautomerism between a molecular OAHꢀ ꢀ ꢀN and a zwitter-
ionic Oꢁꢀ ꢀ ꢀHAN+ forms. Adapted from Ref. [9]. The full circle refers to 1
dissolved in CD2Cl2 at 233 K. The open circle refers to the typical
hydrogen bond properties of phenyl-substituted Schiff bases dissolved in
CD2Cl2. For further description, see text.
4.2. Tautomeric equilibrium model
Whereas Schiff bases studied previously exhibited only a
single OHN hydrogen bond, we need to take the OHO
hydrogen bond of 1 into account, i.e. three tautomeric
states according to Fig. 3a are needed in order to describe

324
N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
1a
1b
1c
JNH = 89
JNH = 0
JNH = 88
16.2 16.2
14.2 16.2
16.0 10.0
_
HH
HH
H
H
H
H
Ph
Ph
H
H
Ph
+
+
Kab
Kab
Kab
Kbc
Kbc
Kbc
O
O
N
O
O
N
O
O
N
_
O
O
O
CH
CH
O
CH
3
3
3
16.46
14.1
H
O
15.9
_
O
H
D
Ph
H
D
D
HD
HD
Ph
Ph
+
+
O
O
N
O
N
N
_
O
O
O
CH
CH
CH
3
3
3
16.46
16.1
H
9.6
H
O
_
O
D
O
Ph
D
H
D
Ph
Ph
+
DH
+
DH
N
O
N
O
O
N
_
O
O
O
CH
3
CH
CH
3
3
HH
HD
DH
HH
HD
DH
Kab = Kab = Kab
Kbc = Kbc = Kbc
ΔH = −5kJ ⋅mol−1
ΔS = −31J ⋅ K −1 ⋅mol−1
ΔH = −17kJ ⋅mol−1
ΔS = −50 J ⋅ K −1 ⋅mol−1
Fig. 7. Equilibrium constants of tautomeric reactions of proton transfer for three isotopologs of 1. Numbers above the structures represent the fitted
intrinsic NMR parameters of the tautomers. Chemical shifts are given in ppm and coupling constants are given in Hz. For more details, see text.
a 1.0
b
d
f
90
60
30
0
xa
xb
xc
0.5
0.0
J
(OHOHN) /Hz
c
16
14
12
10
16
14
12
10
δ
δ
(OHOHN)
δ (OHOHN)
e
0.2
0.0
0.2
0.0
-0.2
-0.2
δ
(ODOHN)–
δ (OHOHN)
(OHODN)–
δ
(OHOHN)
0
100 200 300 400 500
/K
0 100 200 300 400 500
T
T /K
Fig. 8. Temperature dependencies of the observed NMR parameters and of the mole fractions of tautomers of 1. (a) Mole fractions of 1a, 1b and 1c. (b)
Coupling constants J(OHOHN). (c) Hydrogen bonding proton chemical shifts d(OHOHN). (d) Hydrogen bonding proton chemical shifts d(OHOHN). (e)
Vicinal isotope effects d(OHODN) ꢁ d(OHOHN). (f) Vicinal isotope effects d(ODOHN) ꢁ d(OHOHN). Solid lines are calculated using the three-state
tautomeric equilibrium model as described in the text.

N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
325
arbitrary set of intrinsic coupling constants and chemical
shifts, which had to be assumed to be independent of tem-
perature. This set can describe well the experimental data,
but which is not a unique set and also not a set exhibiting
the best agreement with the experiment. All fitted parame-
ters used to calculate the solid curves in Fig. 8 are included
in Fig. 7.
Let us now discuss Fig. 8 in more detail. In Fig. 8a, we
have plotted the calculated mole fractions of the tautomers
1a, 1b and 1c as a function of temperature. Our choice of
reaction enthalpies and entropies leads to a preference of
tautomer 1c at lower temperatures. This choice is justified
because 1c exhibits the largest dipole moment of the tauto-
meric states in Fig. 7 and structures with large dipole
moments are preferred in polar solvents at low tempera-
tures [23,27,38–40]. When temperature is increased, tauto-
mer 1b and eventually tautomer 1a are formed.
In Fig. 8b, we have plotted the coupling constants
J(OHOHN) of 1 as a function of temperature. They are
close to 90 Hz at lowest temperatures and decrease slightly
with increasing temperature because of the formation of 1a
which exhibits a much smaller coupling constant as com-
pared to the zwitterionic structures 1b and 1c.
Figs. 8c and d refer to the chemical shifts d(OHOHN)
and d(OHOHN). Their temperature dependence can be
explained as follows. The OHO hydrogen bridges in 1a
and 1b exhibit similar proton chemical shifts, while the
OHO bridge in 1c is somewhat longer and bridging proton
is more shielded. In contrast, the OHN bridges in 1b and 1c
are shorter than in 1a; therefore, the observed chemical
shift d(OHOHN) decreases when temperature increases.
The hydrogen bond geometries for OHOHN isotopologs
of structures 1a–1c are qualitatively depicted in Fig. 3b.
Fig. 8e shows that the observed vicinal isotope effect
d(OHODN) ꢁ d(OHOHN) increases with increasing tem-
perature. This behaviour can be explained as follows. In
forms 1a and 1c OHO and OHN hydrogen bonds are coop-
eratively coupled. This means that lengthening of a given
bond by its deuteration leads to the lengthening of the
other, as illustrated in Fig. 3b. The latter, in turn, is
observed in a proton NMR as a negative value of the vic-
inal isotope effect on bridging proton chemical shift. In
contrast, in tautomer 1b hydrogen bonds are anti-coopera-
tively coupled (they compete for the central phenolic oxy-
gen). Lengthening of a given hydrogen bond by its
deuteration leads to the shortening of the neighbouring
one and, consequently, to the deshielding of the bridging
proton. Thus, the vicinal isotope effect d(OHODN) ꢁ
d(OHOHN) in form 1b is positive. In the temperature
range between 183 and 243 K the mole fraction of 1b grows
and so does the value of the d(OHODN) ꢁ d(OHOHN)
isotope effect.
In Fig. 8f, we have plotted the values of
d(ODOHN) ꢁ d(OHOHN) isotope effect. Similar reason-
ing as in case of (OHODN) ꢁ d(OHOHN) isotope effect
is valid. The geometric isotope effects are illustrated in
Fig. 3b as well. However, in order to reproduce the
decrease of d(ODOHN) ꢁ d(OHOHN) with temperature
increase we had to use a relatively large negative intrinsic
value for 1a.
In summary, it was possible to explain the experimental
data in terms of a tautomerism between the three states in
a
b
c
R
NH2
LYS
LYS
LYS
R
R
H2N
H
H
H
H
N
HN
H
N
H
N
H
O
O
O
H
H
H
A
A
A
f
e
d
LYS
NH2
LYS
LYS
H
R
R
R
H
H
H
N
HN
HN
N
N
H
H
O
H
O
O
H
H
H
A
A
A
Fig. 9. Potential mechanism of the transimination of the internal aldimine of PLP to the external aldimine, involving geminal diamines as intermediate
steps. For further explanation, see text.

326
N.S. Golubev et al. / Journal of Molecular Structure 844–845 (2007) 319–327
Fig. 7. The data are mainly determined by the intrinsic
NMR parameters for the dominating tautomer 1b, but in
order to describe the dependence of the data on tempera-
ture tautomers 1a and 1c cannot be neglected. The set of
thermodynamic data and of the intrinsic NMR parameters
is only semi-quantitative for which measurements in a lar-
ger temperature interval would be required.
all thermodynamic and NMR parameters of the three-state
tautomerism of 1, depicted in Fig. 3a. It would be worth to
replace the phenyl ring by other groups which increase the
solubility and hence allow one to perform measurements at
low temperatures using deuterated freons as solvents. The
third conclusion is that we could successfully describe the
temperature dependence of the NMR parameters neglect-
ing equilibrium isotope effects on the tautomerism. In other
words, the temperature dependence of the vicinal isotope
effects on the chemical shifts arises mainly from ‘‘equilib-
rium averaged intrinsic isotope effects’’ as proposed previ-
ously [21]. Finally, already now one may understand why
nature did not convert the methyl group of PLP in 2-posi-
tion into a carboxylic group: the additional hydrogen bond
would be too strong and not flexible enough to allow the
reaction sequence depicted in Fig. 9. This sequence might
be a good starting point for future experimental and theo-
retical studies.
4.3. Biological role of additional hydrogen bonds to PLP
The question arises whether the above hydrogen bond
assisted intramolecular tautomerism of 1 has some implica-
tions for the biological function of the cofactor PLP. Let us
remind that PLP dependent enzyme mechanisms uniformly
entail transimination to generate the obligatory external
aldimine intermediate from the internal aldimine [41,42].
This process could either proceed with or without the assis-
tance of water molecules or additional bases, a problem
which is still under debate. A mechanism involving no
water molecule is depicted in Fig. 9. The enzyme has pre-
pared the transimination by deprotonation of the amino
group of the amino acid RNH2, by protonation of the pyr-
idine ring and hence of the Schiff base nitrogen (Fig. 9a)
[9,21,43]. Here, additional hydrogen bonds to other amino
acid residues AH assist this double proton shift as indi-
cated in model studies [21]. This leads to a positive charge
on the a-carbon which allows the amino acid to perform
the nucleophilic attack on the internal aldimine (Fig. 9a)
leading to a geminal diamine. The structures of Figs. 9b
and c have been discussed [44] which interconvert by
CAC bond rotation and hydrogen bond reorganization.
We propose here a subsequent proton transfer to oxygen
leading to the structures of Fig. 9d. This could be associ-
ated with a weakening or opening of the additional hydro-
gen bond to the donor AH. Now reverse processes easily
lead to the external aldimine shown in Fig. 9f.
Acknowledgements
This study has been supported by the Russian Founda-
tion of Basic Research, Grant 05-03-33235, the Deutsche
Forschungsgemeinschaft, Bonn and the Fonds der Chemis-
chen Industrie, Frankfurt.
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