Deuterium isotope effect on 13C chemical shifts of tetrabutylammonium salts of Schiff bases amino acids

Article abstract of DOI:10.1002/mrc.1852

Deuterium isotope effects on ¹³C chemical shift of tetrabutylammonium salts of Schiff bases, derivatives of amino acids (glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-methionine) and various ortho-hydroxyaldehydes in CDCl₃ have been measured. The results have shown that the tetrabutylammonium salts of the Schiff bases amino acids, being derivatives of 2-hydroxynaphthaldehyde and 3,5- dibromosalicylaldehyde, exist in the NH-form, while in the derivatives of salicylaldehyde and 5-bromosalicylaldehyde a proton transfer takes place. The interactions between COO^- and NH groups stabilize the proton-transferred form through a bifurcated intramolecular hydrogen bond. Copyright

Full text of DOI:10.1002/mrc.1852

MAGNETIC RESONANCE IN CHEMISTRY
Magn. Reson. Chem. 2006; 44: 881–886
Published online 1 June 2006 in Wiley InterScience
(www.interscience.wiley.com) DOI: 10.1002/mrc.1852
Note
Deuterium isotope effect on 13C chemical shifts of
tetrabutylammonium salts of Schiff bases amino acids
Z. Rozwadowski∗
Institute of Chemistry and Environmental Protection, Szczecin University of Technology, Al. Piasto´ w 42, 70-065 Szczecin, Poland
Received 7 March 2006; Revised 21 April 2006; Accepted 25 April 2006
Deuterium isotope effects on 13C chemical shift of tetrabutylammonium salts of Schiff bases, derivatives
of amino acids (glycine, L-alanine, L-phenylalanine, L-valine, L-leucine, L-isoleucine and L-methionine)
and various ortho-hydroxyaldehydes in CDCl3 have been measured. The results have shown that the
tetrabutylammonium salts of the Schiff bases amino acids, being derivatives of 2-hydroxynaphthaldehyde
and 3,5-dibromosalicylaldehyde, exist in the NH-form, while in the derivatives of salicylaldehyde and
5-bromosalicylaldehyde a proton transfer takes place. The interactions between COO− and NH groups
stabilize the proton-transferred form through a bifurcated intramolecular hydrogen bond. Copyright 
2006 John Wiley & Sons, Ltd.
Supplementary electronic material for this paper is available in Wiley InterScience at http://www.interscience.wiley.com/
jpages/0749-1581/suppmat/
KEYWORDS: Schiff bases; amino acids; tetrabutylammonium salts; 13C NMR; deuterium isotope effects; intramolecular
hydrogen bond
of low temperature measurements of 13C chemical shift and
deuterium isotope effects on 13C chemical shift in CDCl3.
INTRODUCTION
Schiff bases, derivatives of ortho-hydroxyaldehydes and
amino acids, have been widely studied because of their
biological activity and wide applications as ligands of
complexes used as enantioselective catalysts.1–8 In various
biologically important reactions Schiff bases are formed
as intermediate products between pyridoxal phosphate
and lysine residue.9–11 The presence of an intramolecular
hydrogen bond is essential for their enzymatic function
and the proton transfer process from oxygen to nitrogen
atom is the first step of the catalytic cycle.12 In the
studies of hydrogen bond and proton transfer processes,
measurements of deuterium isotope effects on 13C chemical
shift are especially informative.13,14 For some systems this
simple method permits a direct detection of equilibrium and
allows a determination of the mole fractions of tautomers.
Measurements of deuterium isotope effect can be easily
performed on a standard NMR spectrometer as one-
tube experiments for partially deuterated sample. Recently,
measurements of deuterium isotope effects on chemical
shifts have been applied in the studies of HIV-1 protease
inhibitors,15 protein hydrogen bonds16 and DNA.17
EXPERIMENTAL
The Schiff bases derived from amino acids were prepared
by condensation of 2-hydroxynaphthaldehyde with glycine,
L-phenylalanine, L-leucine, L-isoleucine, L-methionine; sal-
icylaldehyde and 5-bromosalicylaldehyde with L-valine,
L-phenylalanine; 3,5-dibromosalicylaldehyde with L-alanine,
L-valine and L-phenylalanine according to the procedure
described elsewhere.18,19 The tetrabutylammonium salts of
amino acid Schiff bases (Scheme 1) were prepared by reac-
tion of equimolar amounts of Schiff base and 1.0 M methanol
solution of tetrabutylammonium hydroxide in absolute
ethanol. After the reaction the solvents were removed under
reduced pressure. The products in the form of yellow oils
were dissolved in CHCl3, dried over anhydrous Na2SO4
and next solvent was removed under reduced pressure.
N-(2-hydroxynaphthylidene)-L-leucine methyl ester was pre-
pared by a reaction of L-leucine methyl ester with excess
of 2-hydroxynaphthaldehyde according to the procedure
described in Ref. 20.
In this work we have investigated tetrabutylammo-
nium salts of Schiff bases amino acids, derivatives of
2-hydroxynaphthaldehyde and salicylaldehydes, by means
The 1H and 13C NMR spectra in CDCl3 were recorded on
a BRUKER DPX-400 spectrometer operating at 400.13 MHz
(1H) and 100.62 MHz (13C). Typical spectral parameters
were used for 1H NMR: spectral width 8 kHz, number of
data points 65.5 K, 0.488 Hz per point digital resolution,
acquisition time 4.09 s, relaxation delay 1 s, pulse width
ŁCorrespondence to: Z. Rozwadowski, Institute of Chemistry and
Environmental Protection, Szczecin University of Technology, Al.
Piasto´w 42, 70-065 Szczecin, Poland. E-mail: zroz@ps.pl
Copyright  2006 John Wiley & Sons, Ltd.

882
Z. Rozwadowski
+
+
O
N
O
O
N
R
R
1'
1'
1'
O-
2'
O-
2'
2'
OCH3
N
N
α
N
α
α
H
O
H
O
H
O
8
5
1
1
8
5
1
9
9
7
7
6
6
2
3
2
2
3
6
10
3
5
10
R2
R1
4
4
4
(6) R = CH(CH3)2 R1= R2 = H
(13)
(1) R = H
(2) R = CH2Ph
(7) R = CH2Ph
(8) R = CH(CH3)2
(9) R = CH2Ph
(10) R = CH3
R
R =H
=
1 2
R1= H R2= Br
R1= H R2= Br
R1= R2= Br
(3) R = CH(CH3)CH3CH3
(4) R = CHCH2CH(CH3)2
(5) R = CH2CH2SCH3
(11) R = CH(CH3)2
(12) R = CH2Ph
R1= R2 = Br
R1= R2 = Br
Scheme 1. Studied Schiff bases.
signal towards higher frequencies for C-10 carbon only. A
similar effect was observed for lithium salts of Schiff bases in
DMSO23 as well as in D2O.24 The C-2 atoms, most sensitive
to the position of hydrogen in the intramolecular hydrogen
bond, are deshielded and resonate at υ ¾ 180 ppm. These
υ(C-2) values are close to those observed for other salts of
Schiff bases amino acids.23,24 The shielding of the H-8 and
H-˛ signals in the spectrum of the L-phenylalanine derivative
(2) relative to the chemical shifts of these positions for the
other compounds studied (Table 1) is due to the anisotropy of
the aromatic ring of phenylalanine (Scheme 2). The chemical
shifts of the signals assigned to C O carbons (C-20) are close
to those measured for lithium salts in DMSO.23
7.8 µs, number of scans 16; for 13C NMR: spectral width
24 kHz, number of data points 65.5 K, 1.46 Hz per point
digital resolution, acquisition time 1.37s, relaxation delay
1s, pulse width 9.2 µs, number of scans 1000–8000. The
chemical shifts were referred to TMS as internal standard.
Typical concentration of the samples was 0.1 M. The
temperature was maintained and measured with Eurotherm
BV-T 2000 an accuracy of 1 K. The deuterium isotope
effects were measured as differences between the 13C signals
in the spectra of non-deuterated and deuterated species:
nC(D) D υC(H)–υC(D). Deuteration of the compounds was
achieved by dissolving the sample in CH3OD followed by
evaporation under reduced pressure.
The selected deuterium isotope effects nC(D) on 13C
chemical shifts are presented in Table 1 (all observed
deuterium isotope effects are available as supplementary
materials – Table S2). Positive values of deuterium isotope
effects were observed for carbon C-˛ and C-10, smaller for C-
1. Negative values were found for carbon C-2, while smaller
for positions 3, 4 and 9. Temperature lowering had small
influence on the deuterium isotope effects (Table 1). For
the carboxylate carbon atom (C-20) the positive deuterium
isotope effect observed (30–57 ppb) was relative to those
measured for lithium salts in D2O.
RESULTS
The selected 1H and 13C chemical shifts for tetrabutylam-
monium salts of Schiff bases 1–13 in CDCl3 are collected in
Table 1. The 1H and 13C chemical shifts for all positions and
the whole range of temperatures (295, 270, 250 and 230 K)
are available as supplementary materials (Table S1). Assign-
ments of the signals were made on the basis of the substituent
effects21,22 and NMR correlation 2D experiments.
Tetrabutylammonium salts of
Tetrabutylammonium salts of
N-(2-hydroxynaphthylidene)-amino acids 1–5
The proton signals with highest frequencies for compounds
1–5, assigned to the proton donor group are observed
in the range υ 13.71–13.87 ppm (Table 1) and indicate the
presence of a medium strong hydrogen bond. Lowering the
temperature had very small effect on the positions of these
signals. The signals of the proton of the azomethine group
(H-˛) are characterized by a large coupling constant 3JꢀNH,Hꢁ
of 12–13.1 Hz (Table 1).
N-(R-salicylidene)-amino acids 6–12
For the sake of comparison, a few derivatives of salicy-
laldehydes were also studied. The most deshielded signals
in these compounds are broad, of low intensity and in the
range of 14.46–15.00 ppm. Decrease in temperature caused
sharpening of the signals and shifted this position towards
higher frequencies in the spectra of compounds 6, 7, while
towards lower frequencies for compounds 8–11. For all com-
3
pounds at low temperatures the JꢀNH,Hꢁ coupling constants
The type of the amino acids group has little influence
on the positions of the 13C signals of the naphthalene ring.
More bulky substituents were responsible for shifting the
at the H atom of the azomethine group were in the range of
6–12.8 Hz for derivatives of L-alanine and L-valine, while for
the derivatives of L-phenylalanine they took much smaller
Copyright  2006 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 881–886
DOI: 10.1002/mrc

Deuterium isotope effect on 13C chemical shifts
883
Table 1. Selected 1H and 13C chemical shifts (ppm), deuterium isotope effects (ppb) and 3JꢀNH,Hꢁ coupling constants (Hz) of
tetrabutylammonium salt of Schiff bases in CDCl3
Position 2
Position ˛
Position 20
T
υH
υC
nC(D)
υH
υC
nC(D)
υC
nC(D)
3JꢀNH,Hꢁ
Comp.
(K)
(ppm)
(ppm)
(ppb)
(ppm)
(ppm)
(ppb)
(ppm)
(ppb)
(Hz)
(1)
Naph-Gly
295
270
250
230
295
270
250
230
295
270
250
230
295
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
295
270
250
230
13.74
13.76
13.76
13.78
13.71
13.71
13.71
13.72
13.84
13.85
13.85
13.87
13.84
13.82
13.82
13.79
13.76
13.73
14.75
14.90Ł
14.89
15.00
14.43
14.54
14.63
14.77
14.87
14.83
14.79
14.80
14.45
14.50
14.62
14.69
14.76
14.73
14.72
14.69
14.61
14.57
14.52
14.47
14.58
14.54
14.50
14.46
14.70
14.76
14.78
14.77
180.18
180.21
180.19
180.10
179.94
179.97
179.95
179.91
179.96
179.90
179.85
179.71
179.88
179.82
179.90
180.00
180.05
180.06
167.22
168.07Ł
168.38
169.66
164.53
164.48
164.55
164.78
167.60
168.33
168.51
169.45
164.74
165.03
165.37
165.74
168.75
169.35
169.51
169.84
169.22
169.50
169.67
169.91
168.81
169.19
169.64
169.91
170.63
171.56
172.55
173.79
−278
−254
−234
−225
−252
−248
−231
−213
−261
−248
−231
−218
−275
−221
n.o.
8.60
8.62
8.62
8.64
7.96
7.93
7.91
7.90
8.56
8.56
8.54
8.52
8.71
8.65
8.69
8.69
8.69
8.69
8.21
8.17Ł
8.16
8.14
7.94
7.92
7.92
7.93
8.06
8.08
8.06
8.04
7.77
7.64
7.71
7.69
8.05
8.05
8.05
8.06
7.89
7.89
7.88
7.88
¾7.16
7.10
7.06
7.03
8.96
8.93
8.90
8.86
157.45
157.57
157.72
158.00
155.64
155.63
155.64
155.73
155.89
155.91
155.97
156.04
156.18
156.23
155.98
156.00
156.05
156.16
163.39
163.34Ł
163.51
163.52
163.87
163.94
164.01
164.02
162.22
162.27
162.39
162.48
162.56
162.58
162.64
162.60
161.99
162.04
162.16
162.31
162.30
162.37
162.44
162.55
161.77
161.72
161.69
161.66
159.71
159.40
159.09
158.70
374
377
364
362
368
358
347
357
345
353
336
340
362
338
n.o.
392
390
374
n.o.
269
271
283
n.o.
n.o.
218
248
n.o.
262
274
279
n.o.
n.o.
n.o.
br.
n.o.
218
218
215
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.o.
420
463
512
170.31
170.47
170.62
170.75
172.22
172.33
172.42
172.55
172.74
172.80
172.95
172.97
173.60
174.07
172.38
172.53
172.69
172.85
173.44
173.28Ł
173.43
173.35
173.54
173.60
173.66
173.70
172.82
172.75
173.03
173.06
173.06
173.60
173.22
173.29
172.12
172.06
172.14
172.23
171.38
171.26
171.33
171.39
170.95
170.92
170.91
170.92
171.75
171.75
171.75
171.75
48
42
31
30
n.o.
42
43
44
n.o.
57
46
n.o.
n.o.
30
n.o.
n.o.
30
12.0
12.6
12.7
12.8
12.4 br.
13.1
13.1
13.1
12.2
12.7
12.8
12.8
11.6
12.8
11.8
12.6
12.7
12.8
n.o.
(2)
Naph-Ph
(3)
Naph-Ile
(4)
Naph-Le
(5)
Naph-Met
−281
−262
−245
n.o.
49
(6)
Sal-Val
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
44
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
n.o.
71
n.o.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.o.
n.o.
n.o.
n.o.
74a
br.
6.1
7.1
n.o.
n.o.
n.o.
−73
br.
(7)
Sal-Ph
n.o.
n.o.
349a
175a
n.o.
br.(¾2.5)
(8)
Br-Val
n.o.
n.o.
n.o.
6.8
7.9
n.o.
n.o.
n.o.
−72
br.
(9)
Br-Ph
n.o.
n.o.
n.o.
br.
br.(¾3.5)
(10)
diBr-Ala
n.o.
n.o.
−276
∼−262
−233
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.m.
n.o.
br.
12.0
12.3
n.o.
(11)
diBr-Val
br.
12.2
12.4
n.o.
9.2 br.
10.8 br.
12.8
n.o.
n.o.
5.4
7.0
(12)
diBr-Ph
(13)
Naph-
Leu Me
∼0
−88
−245
n.o. not observed
br. broad signals
n.m. not measured
Ł Data from partly deuterated compound
a Uncertain value
Copyright  2006 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 881–886
DOI: 10.1002/mrc

884
Z. Rozwadowski
+
+
N
N
O
O
O
O
N
N+
H
O
H
O-
keto form
zwitterionic form
Scheme 2. Influence of aromatic ring of the L-phenylalanine.
values (¾2.5–3.5 Hz) except 12. For L-phenylalanine deriva-
tives 7, 9, 12 a shielding effect of the phenyl ring was observed
for H-˛ (Table 1). This effect is particularly large for com-
pound 12 (0.8 ppm). Also the signal assigned to H-6 in this
compound is shifted towards lower frequencies by 0.4 ppm
relative to its position in the spectra of the other derivatives
of 3,5-dibromosalicylaldehyde. The C-2 chemical shift value
varies from υ164.5 for 7 up to υ169.9 ppm for 12 and increases
with decreasing temperature.
The selected deuterium isotope effects nC(D) on 13C
chemical shifts for compounds 6–10 are presented in
Table 1 (all observed deuterium isotope effects are available
as supplementary materials – Table S2). Unfortunately, for
compounds 11 and 12 it was not possible to measure the
exact values of deuterium isotope effect because of the very
broad and low intensity signals in spite of high yields of
deuteration. Positive values of deuterium isotope effects
were observed for C-˛, smaller for C-1. Negative values were
found for C-6. Positive and negative values were observed
for other carbons. The nC-2(D) values, most sensitive to
the position of the proton in the hydrogen bridge vary from
positive values for 6 to negative values for 10.
Scheme 3. Plot of nC-2(D) versus ꢂ (mole fraction of NH-form).
Tetrabutylammonium salts of
N-(2-hydroxynaphthylidene)-amino acids 1–5
The values of nC(D) measured for tetrabutylammonium
salts observed for C-2 carbon, most sensitive to the position
of hydrogen in the intramolecular hydrogen bond, are in the
range from ꢀ213 to ꢀ281 ppb and close to those measured
for lithium salts.24 Insignificant temperature-dependence of
nC-2(D), values or the lack of the long-range deuterium
isotope effects of naphthalene ring as well as values of
DISCUSSION
In previous papers we have reported on the proton transfer
equilibrium of lithium salts of amino acids Schiff bases,
derivatives of 2-hydroxynaphthaldehyde in DMSO23 and
water.24 In this work we have extended this study to
tetrabutylammonium salts of these Schiff bases in chloroform
solution and for the sake of comparison we have also
performed NMR measurements for tetrabutylammonium
salts of Schiff bases being derivatives of salicylaldehydes.
In this study we have taken advantage of the measure-
ments of deuterium isotope effects on 13C chemical shifts.
It is known that a deuterium isotope effect on carbon atom
linked to a phenolic group (C-2(D)) has two contribu-
tions: an intrinsic effect (C(D)i) and an equilibrium effect
(C(D)e).14 The dependence of the observed deuterium iso-
tope effect C-2(D) is a non-monotonic function (S-shape) of
the mole fraction of the NH-form (ꢂ) and on the basis of this
relationship a position of the equilibrium can be estimated.14
For the compounds studied the position of the equilibrium
has been evaluated from the above-mentioned plot of nC(D)
(vs) ꢂ (NH mole fraction) presented in Scheme 3.
3
the JꢀNH,Hꢁ coupling constant and the frequencies of the
υC-2 signals of 180 ppm (Table 1) indicate that the proton
transfer equilibrium is strongly shifted towards the NH-form
at room temperature. At low temperatures all compounds
studied (1–5) exist exclusively in the NH-form. A small
decrease in nC-2(D) values with temperature can be
related to slight changes of the interactions in the hydrogen
bridge. The coupling constant values as well as deuterium
isotope effects at room temperature can be affected by the
exchange processes. The values of the deuterium isotope
effect observed for C-2 carbons are comparable to those of
the intrinsic isotope effects for the NH-form (C(D)i) of the
Schiff bases. Similar values of the isotope effect (ꢀ190 ppb)
were observed for the Schiff base being a derivative of
gossypol and benzylamine,25 occurring exclusively in the
tautomeric form NH.26,27
Deuterium isotope effects were also observed on the
carboxylic C-20 carbon (¾50 ppb). This effect can be related to
the long-range deuterium isotope effect or engagement of the
Copyright  2006 John Wiley & Sons, Ltd.
Magn. Reson. Chem. 2006; 44: 881–886
DOI: 10.1002/mrc

Deuterium isotope effect on 13C chemical shifts
885
3JꢀNH,Hꢁ, while small amounts of the OH-form are present for
the derivative of L-phenylalanine at 270 and 250 K.
Comparison of the 13C chemical shifts (Table 1), nC-2(D)
3
and JꢀNH,Hꢁ for the salts of Schiff bases 6–12, suggest
+
that the L-phenylalanine group moves the proton transfer
equilibrium backwards except for compound 12 at 230 K.
This shift of the equilibrium suggests that the conformation
forced by the position of the phenyl ring (Scheme 2) weakens
the interaction between COOꢀ and NH groups stabilizing
the proton-transferred form and hence changes the position
of the equilibrium.
N
O
O
N+
H
O-
CONCLUSIONS
Results presented here shows unambiguously that all tetra-
butylammonium salts of amino acids Schiff base derivatives
of 2-hydroxynaphthaldehyde (1–5) exist in the proton-
transferred NH-form and neither the counter ion nor solvent
influence on the position of the equilibrium. The proton-
transferred form is stabilized by bifurcated intramolecular
hydrogen bond (Scheme 4). For the derivatives of salicylalde-
hydes (6–12) the position of the proton transfer equilibrium is
sensitive to the substituent at the salicylic ring, the amino acid
group and temperature (6–9). An influence of the phenyl ring
in phenylalanine on the conformation of the salicylaldehydes
has been suggested.
Scheme 4. Interactions between COOꢀ and NH stabilizing the
proton-transferred form.
COOꢀ group in hydrogen bond. An isotope effect of 50 ppb
(sign corrected to the author’s definition of the deuterium
isotope effect) was observed on the carboxylate carbon
involved in the intermolecular hydrogen bond in HIV-1
protease inhibitor.15 For Schiff base derivative of pyridoxal
and L-valine the existence of a bifurcated hydrogen bond has
been suggested.8 To verify this hypotheses, the derivative
of 2-hydroxynaphthaldehyde and L-leucine methyl ester
has been studied (13). For this compound, no deuterium
isotope effect on C-20 carbon of the carboxylic group was
observed. The values of the nC-2(D), their variation with
Supplementary material
Supplementary electronic material for this paper is available
in Wiley InterScience at: http://www.interscience.wiley.
com/jpages/0749-1581/suppmat/
3
temperature, and values of the coupling constant JꢀNH,Hꢁ
(Table 1) indicated that the proton transfer equilibrium
was shifted towards the OH-s form at room temperature.
These results show the presence of the bifurcated hydrogen
bond in tetrabutylammonium salt and that interactions
between COOꢀ and NH stabilize the proton-transferred form
(Scheme 4).
The chemical shift values of C-20 (carboxylic group) for
compounds 1–5 (υ170–174 ppm) are closer to those observed
for lithium salts in DMSO (υ168–172 ppm) than that for
lithium salts in D2O (υ178–180 ppm), which indicates that in
the compounds studied the counter ion is in the proximity
of the carboxylic group.
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