Journal of Molecular Structure 1040 (2013) 59 64
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Journal of Molec ular Stru cture
journal homepage: www.elsevier.com/locate/molstruc
Potentiometric and NMR complexation studies of phenylboronic acid PBA
and its aminophosphonate analog with selected catecholamines
a,Ń!
Tomasz Ptak a,b, Piotr MÅ‚ynarz , Agnieszka Dobosz b, Agata Rydzewska a, Monika Prokopowicz a
a
_
Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrzeze Wyspiańskiego 27, 50-370 Wrocław, Poland
b
Wroclaw Medical University, Department of Basic Medical Sciences, Borowska 211 Str., 50-556 Wroclaw, Poland
g r a p h i c a l a b s t r a c t
h i g h l i g h t s
" Binding abilities of PBA and receptor
NH3+
HO OH HO NH3+
O
1 with chosen catechol group
HO
B
containing molecules were H3O+
B
investigated.
O
HO
" Potentiometric and NMR studies
were performed.
" Protonation constants for
catecholamines and two boronic
receptors PBA and 1 were calculated.
" Stability constants of complexes PBA
and 1 with catecholamines were
determined. At high pH values the
complexes breakdown was
observed.
" The stepwise binding constants log
Ktet for formed complexes were
calculated.
a r t i c l e i n f o a b s t r a c t
Article history:
Boronic acids are a class of intensively explored compounds, which according to their specific properties
Received 28 November 2012
have been intensively explored in last decades. Among them phenylboronic acids and their derivatives
Received in revised form 7 February 2013
are most frequently examined as receptors for diverse carbohydrates. In turn, there is a large gap in basic
Accepted 8 February 2013
research concerning complexation of catecholamines by these compounds. Therefore, we decided to
Available online 20 February 2013
undertake studies on interaction of chosen catecholamines, namely: noradrenaline (norephinephrine),
dopamine, L-DOPA, DOPA-P (phosphonic analog of L-DOPA) and catechol, with simple phenyl boronic
Keywords:
acid PBA by means of potentiometry and NMR spectroscopy. For comparison, the binding properties of
Catecholamines
recently synthesized phenylboronic receptor 1 bearing aminophosphonate function in meta-position
Boronic receptors
were investigated and showed promising ability to bind catecholamines. The protonation and stability
Phenylboronic acid
constants of PBA and receptor 1 complexes were examined by potentiometry. The obtained results
NMR
Potentiometry demonstrated that PBA binds the catecholamines with the following affinity order: noradrenaline P
dopamine L-DOPA > catechol > DOPA-P, while its modified analog 1 reveals slightly different
preferences: dopamine > noradrenaline > catechol > L-DOPA > DOPA-P.
Ó 2013 Elsevier B.V. All rights reserved.
1. Introduction (Boron Neutron Capture Therapy) through their medicinally useful
enzyme inhibitory functions (VelcadeÒ) [1], up to determination of
Boronic acids have attracted an intensive interest in last three recognition properties towards organic molecule possessing
s
decades being the subject of many studies starting from BNCT neighboring hydroxyl groups in 1,2- or 1,3 positions. This particu-
lar feature makes them a useful analytical tool feasibly recognizing
carbohydrate molecules, which gives a huge hope for their use in
Ń!
Corresponding author. Tel.: [phone].
medicinal diagnostics, where boronic acids might be applied to
E-mail address: [e-mail] (P. MÅ‚ynarz).
0022-2860/$ - see front matter Ó 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.molstruc.2013.02.013
60 T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59 64
recognize cell carbohydrates and glycoproteins [2 4]. However, interactions with diols [8]. It is generally known that boronic moi-
there are many literature reports describing boronic receptors as ety may be esterified by 1,2 and 1,3- diols with formation of trigo-
host molecules for other types of organic diols [5]. Among hun- nal and tetrahedral adducts. These equilibria are frequently
dreds of papers only few were dedicated to their interaction with presented as a cyclic   square  equation [11]. However, recently
catecholamines, including neurohormones (e.g. dopamine, L-DOPA, many few papers consider trigonal boronic group as a crucial form,
noradrenaline). Thus, boronic acids were found to be carriers for which is directly responsible for a tetrahedral complex formation
catecholamines in selective membrane electrodes [6], synthetic [13]. Recent work of Tomsho and Benkovic demonstrates a scheme
host molecules for dopamine and noradrenaline [7] and receptors of a multi-equilibrium complexation of ARS by phenylboronic acid,
for catechol molecules [8]. Amongst studied catecholic objects where the intermediate complex is initially formed only by reac-
the most popular and the most frequently explored molecule is tion with one catechol hydroxyl unit, which replaces one hydroxyl
Alizarin Red S (ARS), which is generally used for determination of entity of a trigonal boronic group [14].
association constants in competition tests with diols [9,10]. There- PBA potentiometric and NMR studies were performed in order
fore we decided to examine binding properties of phenylboronic to determine the protonation constants. This HA (110) type ligand
11
acid (PBA) and N-benzylamino-3-boronbenzylphosphonic acid 1 showed B NMR signal shift from trigonal (ca. 30 ppm) to tetrahe-
(Scheme 1) towards selected catecholam Presented paper re- dral (3 ppm) form of a boronate entity with fast exchange in
ines.
ports a study of binding ability of PBA and receptor 1 with chosen NMR time scale between these two forms. Introduction of the
catechol group containing molecules. aminophosphonate group to PBA molecule yielded three additional
protonation sites: one basic amine group and two acidic ones
derived from phosphonic unit. Thus, the receptor itself should be
2. Results and discussion
a H4A (410) type molecule, but the last protonation site is too
low to be detected accurately by potentiometry.
2.1. Protonation constants
In this work the protonation and stability constants for com- 2.2. Complexation studies
plexes of PBA and receptor 1 (Scheme 1) with dopamine, noradren-
aline, L-DOPA, catechol and DOPA-P have been determined. Similar The process of complexation of catecholamines (here shown for
detailed research of complexation equilibria were performed to- dopamine) by PBA can be generally described as a set of following
wards carbohydrate binding by PBA and its ortho-substituted ami- equilibria (Scheme 2). This scheme presents the combinations of
no derivatives [11]. possible created complexes including trigonal as well as tetrahe-
All catecholamines contain detectable in the investigated pH dral form of boronic entity but without formation of trigonal
range two invariant protonation sites, namely a catechol moiety complexes.
with the first stepwise acidity constant of about 8.7 and the second The addition of catecholamines to the PBA receptor induced an
one originating from an amine entity with the value of ca. 10 (Ta- appearance of a slow exchange equilibrium visible as two NMR sig-
11
ble 1). Therefore, they can be considered as a H2L (201) type li- nals (1H, B) originating from free receptor and its molar fraction
gands. This stoichiometry is characteristic for two involved in the formed complexes. One of these signals corre-
catecholamines: dopamine and noradrenaline. Due to additional sponding to the unbound PBA appeared at 30 ppm (trigonal sp 2
acidic protonation side L-DOPA and its phosphonic analog DOPA- form), while the second one from the complex arose at 10 ppm
P are H3L (301) type ligands. (tetrahedral sp 3 form). Interestingly, no shift induced by complex-
The phenylboronic acid receptor is considered as a monoproto- ation was observed for the signal deriving from the sp 2 boron atom,
nated molecule HA (110), where electron deficient trigonal boron which rather excluded the formation of trigonal complexes. How-
11
atom reacts with one water molecule simultaneously leading to a ever due to a large half-width of B NMR this particular signal
proton liberation. The pKa value of this reaction is 8.80 and remains might not be observable.
in a good accordance with literature data [11]. The same type of The representative potentiometric titration data for of the PBA-
water hydrolysis is adopted for stability constants of Zn 2+ com- dopamine systems (Fig. 1 and Table 1) revealed the following of
plexes calculation [12]. two forms of the predominant complexes: H2AL (2 1 1) and HAL
Although phenylboronic acid is one of the simplest known (1 11). Both of them may be formed either from trigonal or tetrahe-
receptors, it yields a set of equilibria, which appear during mutual dral structure of PBA (Scheme 2a d). However, the most probable
OH
B(OH)2 OH
NH2
NH2
HO
OH HO
OH
OH
Phenylboronic acid, PBA
Catechol 2 Dopamine 3 Norepinephrine 4
PO3H2
COOH PO3H2
N
H
NH2 NH2
HO HO
OH OH
B(OH)2
N-Benzylamino-3-
L-DOPA 5 DOPA-P 6
boronbenzylphosphonic acid, 1
Scheme 1. Structures of studied catecholamines (2 6) and boronic ligands (PBA and 1).
T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59 64 61
Table 1
Potentiometrically calculated protonation constants for catecholamines (2 6) and two boronic receptors (PBA and 1) at 25 °C, I = 0.1 mol dm 3 (KNO3).
PBA 12 3 4 5 6
log b1 8.80 9.91 9.17 10.31 9.82 9.83 10.64
log b2  18.46  19.17 18.48 18.63 19.61
log b3  24.03    20.53 25.15
log Ka1 8.80 (8.8a) 9.91 9.17 (9.17a) 10.31 (10.32b) 9.82 (9.53b) 9.83 (9.89c) 10.64
log Ka2  8.55  8.86 (8.85c) 8.66 (8.58c) 8.80 (8.76c) 8.97
log Ka3  5.57    1.9 5.54
a
Data taken from Ref. [8].
b
Data taken from Ref. [15].
c
Data taken from Ref. [16].
HO
(a)
HO
OH O
+ H3O+
+
B B
HO NH3+ O
OH
NH3+
110 201 211
(b)
HO
HO
HO OH O
+ 2H2O
+
B B
HO NH3+ O
OH
NH3+
010
201 211
(c)
O
HO
HO OH O
+ H2O + OH-
+
B B
HO NH3+ O
OH
NH3+
010 101 -100
111
(d)
O
HO
OH O
+ H2O
+
B B
HO NH3+ O
OH
NH3+
110 101
211
(e)
HO
HO O
O
+ H3O+
B
B
O
O
NH2
NH3+
211
111
Scheme 2. Possible equilibria in the mixture of dopamine and PBA.
11
formation of H2AL species detectab by potentiometry might be This finding is additionally confirmed by a B NMR experiment,
le
created by the trigonal form of boronic moiety with the release where the intensity of a signal at 10 ppm decreases, whereas the
of H3O+. The formation of the next (1 1 1) species H2AL,HAL + H+ one at 3 ppm, which clearly corresponds to the free tetrahedral
may result from deprotonation of the amine group of dopamine boronate ion PhB ðOHÞ 3 , increases.
(Scheme 2e). In general, the formation of the main complex The overlapped species distribution diagram (Fig. 2) obtained
(2 1 1) might be ascribed by the equation shown in Scheme 2a, from the calculated values of the stability constants (Table 2)
but also other pathways cannot be unambiguously excluded. The indicates, that PBA possesses the strongest affinity towards cate-
formation of H3AL species was sought by calculation, but was not cholamines being in the following order: noradrenaline > dopa-
found. This may give a straight evidence for the formation of tetra- mine L-DOPA > catechol > DOPA-P. The same dependence was
11
hedral complexes directly from trigonal boronic group with the found by examina of the boron signal in B NMR spectra,
tion
intermediate stage including complex formation between one hy- where comparison of the obtained points reflects the same trend
droxyl group of a boronic acid and one hydroxyl group of a catechol (Fig. 3). Collected altogether, obtained data confirm the correctness
entity. Raising pH above 10 results in appearance of significant of the chosen model used for calculation of stability constants.
quantities of a free tetrahedral boronate ion PhB ðOHÞ 3 (A ), which The resulting differences in the values of stability constants are
points to the hydrolysis of both complexes H2AL and HAL (Fig. 1). the most probably caused by the overall charge of the formed
62 T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59 64
11
Fig. 3. Second-degree polynomial lines fitted to B NMR titration points of the
Fig. 1. Species distribution curves for the complexes formed in PBA (A)  dopamine
formation of tetrahedral complexes in the pD function, [PBA] = [catechol-
(L) system as a function of pH: sHA, h A , dH2AL, j HAL.
amine] = 0.05 M (4}; 3D; 5s; 6h).
When calculating the stability constants of the complexes
formed between receptor 1 and catecholamines two possible mod-
els were obtained. The best fitting of titration curve was achieved
using four species model, which contains succeeding complexes of
stoichiometry: H4AL, H3AL, H2AL and HAL (Table 2, Fig. 4  dopa-
mine-3 and 1 system as an example).
The exceptional binding mode was found for complexes with
DOPA-P, where additional form of H5AL stoichiomet was de-
ry
tected. The presumed explanation of the presence of the H5AL
and H4AL forms might be the interaction of deprotonated phos-
phonic moiety with one hydroxyl group of catecholam which
ine,
could be deduced by experiencing downfield chemical shift of a
phosphorus signal (Fig. 5) at pD around 6.
The appearance of an interaction between the catechol and
phosphonic group was postulated earlier in the literature [17,18].
Another explanation may be the formation of trigonal complexes,
similar to those found in the potentiometric studies by Bosh and
1
Fig. 2. The distribution of a sum of free species of PBA and its complexes with: 2 (h, coworkers [11]. The H NMR studies for the mixture of receptor
j), 3 (s, d), 4 (r, .), 5 (D,N), and 6 (}, ) in the pH function.
1 and 2 at pH 5 revealed the differences in the chemical shifts of
the protons when compared these spectra with those registered
complexes and the deprotonation reaction of hydroxyl catechol en-
for individua compounds. The greatest changes were detected
l
tity. Noradrenaline possesses the lowest log Ka followed by L-DOPA
for aromatic protons of 2 as well as protons originating from a-car-
and then by dopamine with the highest value found for DOPA-P,
bon atom and methylene group of benzylamine fragment of 1.
which is partly in accordance with the stabilities of the formed
Further deprotonation appearing as an equilibrium between
complexes. The neurotransmiters L-DOPA and DOPA-P include
two complexes H4AL,H3AL + H+ corresponds to the formation
additional negatively charged carboxylic and double negative
of tetrahedral boron complexes with catecholamines. Next two
phosphonic groups respectively but no interactions during pre-
,
steps, namely H3AL,H2AL + H+ and H2AL,HAL + H+ reflect to
sented studies were detected between these units and the boronic
two ammonium group deprotonations one of the receptor 1 and
,
acid.
the second one from the guest molecule. At high pH values the
Table 2
Stability constants calculated for complexes PBA and 1 with catecholamines.
PBA 1
234 6523465
log bH5 AL 46.82 Ä… 0.07
log bH4 AL 41.43 Ä… 0.06 40.28 Ä… 0.06 41.14 Ä… 0.05 40.19 Ä… 0.07
log bH3 AL 31.12 Ä… 0.07 34.51 Ä… 0.04 33.22 Ä… 0.06 33.82 Ä… 0.07 33.06 Ä… 0.06
log bH2 AL 23.73 Ä… 0.05 23.29 Ä… 0.01 23.84 Ä… 0.03 23.16 Ä… 0.02 23.78 Ä… 0.04 25.25 Ä… 0.07 23.69 Ä… 0.05 24.29 Ä… 0.05 23.62 Ä… 0.06
log bHAL 13.61 Ä… 0.02 13.73 Ä… 0.07 13.52 Ä… 0.002 14.09 Ä… 0.17 13.45 Ä… 0.04 14.04 Ä… 0.06 14.89 Ä… 0.05 13.62 Ä… 0.06 14.36 Ä… 0.04 13.57 Ä… 0.05
log KH5AL 5.68
log KH4AL 6.92 7.06 7.32 7.13
log KH3AL 7.34 9.26 9.53 9.53 9.44
log KH2AL 10.00 9.77 9.75 9.71 9.74 10.36 10.07 9.93 10.05
Charges for the species are omitted for clarity.
T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59 64 63
Fig. 6. Distribution of a sum of free species of compound 1 and its complexes with:
Fig. 4. Species distribution curves for the formed complexes in 1 (A)  3 (L) system
2 (h, j), 3 (s, d), 4 (r, .), 5 (D,N), 6 (}, ) in the pH function.
as a function of pH (dotted line indicates free receptor species:rH3A, D H2A, s HA,
hA; solid line formed complexes: h H4AL, D H3AL,}H2AL, s HAL. The overlapped
31
P NMR titration (d) showing the deprotonation of amine group originating from
compound 1.
Table 3
Logarithm of stepwise binding constants (log Ktet) for complexes of PBA and 1.
Catecholamine PBAa 1b
log Ktet
2 4.44 4.70
3 4.56 5.43
4 4.81 4.83
5 4.53 4.52
6 4.23 4.30
a
log Ktet = log b211  log b102ðNH2þArOHÞ or log b111  log b110ðNH2Þ for catechol.
b
log Ktet = log b311  log b102ðNH2þArOHÞ log b110ðNH2Þor log b211  log b110ðNH2þArOHÞ
 log b110ðNH2Þ for catechol.
from both ligand and receptor protonation constants (Table 1). In
case of receptor 1 the pKtet was calculated by additional subtrac-
tion of pKa value of amino group, which has direct contribution
to the stability constant of bH AL form Ref. [11].
3
31
Fig. 5. P NMR chemical shift for free and complexed species in pD function.
3. Conclusions
Concentration of each component: 0.004 M (N1 + 3 free fraction of ligand; d 1 + 3
complex; h 1 free receptor).
The performed studies allowed for the determination of the
protonation constants of four catecholamines, and catechol, as well
as two receptors: PBA and 1. The stability constants of the formed
complexes breakdown was observed, although with much less
complexes were calculated and showed that the simplest boronic
yield, than for corresponding catecholamine complexes with PBA.
acid PBA, which is used frequently as receptor for 1,2 and 1,3 diols,
In general, the species distribution diagrams reproduce almost
binds the catecholamines in a following order: noradrena-
the same trend with tetrahedral complex constants being in the
line P dopamine L-DOPA > catechol > DOPA-P, while its modi-
following order: dopamine > noradrenaline > catechol > L-DOPA >
fied analog 1 reveals different preference in binding the
DOPA-P (Fig. 6).
catecholamines in order which is as follows: dopamine > noradren-
If considering the second type of calculated model, which
aline > catechol > L-DOPA > DOPA-P.
excluded the supramolecular interaction from studied systems
together with lack of the H5AL and H4AL species, the first spe-
cies that appears in the pH scale is a tetrahedral complex of 4. Experimental
H3AL stoichiometry. Because these fitting parameters were
inferior in comparison to the ones resulting from the model 4.1. Materials
described above, the second model models was not further
considered. PBA, catecholamines and catechol were purchased from Sigma
From the obtained stability and protonation constants the step- Aldrich. The receptor 1 was synthesized according to the previ-
wise binding constants log Ktet for formed complexes can be calcu- ously described procedure [19]. The racemic mixture of a phos-
lated (Table 3). Determination of these paramete led to the phonic analog of L-DOPA (DOPA-P) was obtained according to
rs
independent tetrahedral complex formation constants (Table 2) literature procedure [20].
64 T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59 64
4.2. Potentiometric studies
Acknowledgements
The protonation and stability constants for catecholamines, PBA
The Project was financially supported by Polish Ministry of Sci-
and 1 were calculated from titration curves obtained at 25 °C and
ence and Higher Education (Grant Nr N N 204 134837) and partly
using total volume of 1.8 cm 3. The NaOH solution was added from
by Wroclaw Medical University (Grant ST-697).
3
0.250 cm syringe, which was previously calibrated. All solutions
were prepared using degassed, deionized and distilled water. All
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