Potentiometric and NMR complexation studies of phenylboronic acid PBA and its aminophosphonate analog with selected catecholamines

Potentiometric and NMR complexation studies of phenylboronic acid PBA
and its aminophosphonate analog with selected catecholamines

Tomasz Ptak

, Piotr Młynarz

Department of Bioorganic Chemistry, Faculty of Chemistry, Wrocław University of Technology, Wybrze _ze Wyspian

´skiego 27, 50-370 Wrocław, Poland

Wroclaw Medical University, Department of Basic Medical Sciences, Borowska 211 Str., 50-556 Wroclaw, Poland

h i g h l i g h t s

Binding abilities of PBA and receptor
1 with chosen catechol group
containing molecules were
investigated.

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
K

tet

for formed complexes were

calculated.

g r a p h i c a l

a b s t r a c t

a r t i c l e

i n f o

Article history:
Received 28 November 2012
Received in revised form 7 February 2013
Accepted 8 February 2013
Available online 20 February 2013

Keywords:
Catecholamines
Boronic receptors
Phenylboronic acid
NMR
Potentiometry

a b s t r a c t

Boronic acids are a class of intensively explored compounds, which according to their speciﬁc properties
have been intensively explored in last decades. Among them phenylboronic acids and their deriva tives
are most frequent ly examined as receptors for diverse carbohydrates. In turn, there is a large gap in basic
research concerning complexation of catecholamines by these compounds. Therefore, we decided to
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
acid PBA by means of potentiometry and NMR spectro scopy. For comparison, the binding properties of
recently synthesized phenylboronic receptor 1 bearing aminophosphonate function in meta-position
were investigated and showed promising ability to bind catecholami nes. The protonation and stability
constants of PBA and receptor 1 complexes were examined by potentiometr y. The obtained results
demonstrated that PBA binds the catecholamines with the following afﬁnity order: noradrena line P
dopamine L-DOPA > catechol > DOPA-P, while its modiﬁed analog 1 reveals slightly different
preferences: dopamine > noradrenaline > catechol > L-DOPA > DOPA-P.

1. Introduction

Boronic acids have attracted an intensive interest in last three

decades being the subject of many studies starting from BNCT

(Boron Neutron Capture Therapy) through their medicinally useful
enzyme inhibitory functions (Velcade

)

[1]

, up to determination of

recogniti on properties towards organic molecule

s possessing

neighbori ng hydroxyl groups in 1,2- or 1,3 positions. This particu-
lar feature makes them a useful analytical tool feasibly recognizing
carbohyd rate molecules, which gives a huge hope for their use in
medicinal diagnostics, where boronic acids might be applied to

http://dx.doi.org/10.1016/j.molstruc.2013.02.013

⇑

Corresponding author. Tel.: +48 71 320 45 97.
E-mail address:

piotr.mlynarz@pwr.wroc.pl

(P. Młynarz).

Journal of Molecular Structure 1040 (2013) 59–64

Contents lists available at

SciVerse ScienceDi rect

Journal of Molec ular Stru cture

j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / m o l s t r u c

recognize cell carbohydrat

es and glycoproteins

[2–4]

. However,

there are many literature reports describing boronic receptors as
host molecule s for other types of organic diols

[5]

. Among hun-

dreds of papers only few were dedicated to their interactio n with
catecholam ines, including neurohormone s (e.g. dopamine,

-DOPA,

noradrenaline). Thus, boronic acids were found to be carriers for
catecholam ines in selective membrane electrodes

[6]

, synthetic

host molecules for dopamine and noradrenaline

[7]

and receptors

for catechol molecules

[8]

. Amongst studied catecholic objects

the most popular and the most frequently explored molecule is
Alizarin Red S (ARS), which is generally used for determination of
association constants in competition tests with diols

[9,10]

. There-

fore we decided to examine binding properties of phenylboronic
acid (PBA) and N-benzyla mino-3-boronb enzylphosphon ic acid 1
(

Scheme 1

) towards selected catecholam ines. Presented paper re-

ports a study of binding ability of PBA and receptor 1 with chosen
catechol group containing molecule s.

2. Results and discussion

2.1. Protonation constants

In this work the protonati on and stability constants for com-

plexes of PBA and receptor 1 (

Scheme 1

) with dopamine, noradren-

aline,

-DOPA, catechol and DOPA-P have been determined. Similar

detailed research of complexation equilibria were performed to-
wards carbohydrat e binding by PBA and its ortho-substituted ami-
no derivatives

[11]

All catecholamine s contain detectable in the investigated pH

range two invariant protonati on sites, namely a catechol moiety
with the ﬁrst stepwise acidity constant of about 8.7 and the second
one originating from an amine entity with the value of ca. 10 (

Ta-

ble 1

). Therefore, they can be considered as a H

L (201) type li-

gands.

This stoichiomet

ry is characteristic for two

catecholam ines: dopamine and noradrenaline. Due to additional
acidic protonati on side

-DOPA and its phospho nic analog DOPA-

P are H

L (301) type ligands.

The phenylboronic acid receptor is considered as a monoproto-

nated molecule HA (110), where electron deﬁcient trigonal boron
atom reacts with one water molecule simultaneou sly leading to a
proton liberation. The pK

value of this reaction is 8.80 and remains

in a good accordance with literature data

[11]

. The same type of

water hydrolysis is adopted for stability constants of Zn

com-

plexes calculation

[12]

Although phenylboronic acid is one of the simplest known

receptors, it yields a set of equilibria, which appear during mutual

interactio ns with diols

[8]

. It is generally known that boronic moi-

ety may be esteriﬁed by 1,2 and 1,3- diols with formation of trigo-
nal and tetrahedral adducts. These equilibria are frequently
presente d as a cyclic ‘‘square’’ equation

[11]

. However, recently

many few papers consider trigonal boronic group as a crucial form,
which is directly responsible for a tetrahedral complex formation

[13]

. Recent work of Tomsho and Benkovic demonstrat es a scheme

of a multi-equilibri um complexati on of ARS by phenylboronic acid,
where the intermediate complex is initially formed only by reac-
tion with one catechol hydroxyl unit, which replaces one hydroxyl
entity of a trigonal boronic group

[14]

PBA potentiometric and NMR studies were performed in order

to determine the protonati on constant s. This HA (110) type ligand
showed

B NMR signal shift from trigonal (ca. 30 ppm) to tetrahe-

dral (3 ppm) form of a boronate entity with fast exchange in
NMR time scale between these two forms. Introduction of the
aminoph osphonate group to PBA molecule yielded three additional
protonati on sites: one basic amine group and two acidic ones
derived from phospho nic unit. Thus, the receptor itself should be
a H

A (410) type molecule, but the last protonation site is too

low to be detected accurately by potentiometr y.

2.2. Complexati on studies

The process of complexation of catecholam ines (here shown for

dopamine) by PBA can be generally described as a set of following
equilibria (

Scheme 2

). This scheme presents the combinations of

possible created complexes including trigonal as well as tetrahe-
dral form of boronic entity but without formation of trigonal
complexes .

The addition of catecholam ines to the PBA receptor induced an

appearan ce of a slow exchange equilibrium visible as two NMR sig-
nals (

B) originating from free receptor and its molar fraction

involved in the formed complexes. One of these signals corre-
sponding to the unbound PBA appeared at 30 ppm (trigonal sp

form), while the second one from the complex arose at 10 ppm
(tetrahedral sp

form). Interestingly, no shift induced by complex-

ation was observed for the signal deriving from the sp

boron atom,

which rather excluded the formation of trigonal complexes . How-
ever due to a large half-width of

B NMR this particular signal

might not be observabl e.

The representative potentiom etric titration data for of the PBA-

dopamine systems (

Fig. 1

and

Table 1

) revealed the following of

two forms of the predomin ant complexes: H

AL (2 1 1) and HAL

(1 1 1). Both of them may be formed either from trigonal or tetrahe-
dral structure of PBA (

Scheme 2

a–d). However, the most probable

COOH

Catechol 2

L-DOPA 5

Dopamine 3

Norepinephrine 4

DOPA-P 6

B(OH)

Phenylboronic acid, PBA

B(OH)

-Benzylamino-3-

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

formation of H

AL species detectab le by potentiometr y might be

created by the trigonal form of boronic moiety with the release
of H

. The formatio n of the next (1 1 1) species H

AL , HAL + H

may result from deprotonati on of the amine group of dopamine
(

Scheme 2

e). In general, the formation of the main complex

(2 1 1) might be ascribed by the equation shown in

Scheme 2

but also other pathways cannot be unambiguou sly excluded. The
formation of H

AL species was sought by calculation, but was not

found. This may give a straight evidence for the formation of tetra-
hedral complexes directly from trigonal boronic group with the
intermediate stage including complex formation between one hy-
droxyl group of a boronic acid and one hydroxyl group of a catechol
entity. Raising pH above 10 results in appearan ce of signiﬁcant
quantities of a free tetrahedral boronate ion PhB ðOHÞ

), which

points to the hydrolysis of both complexes H

AL and HAL (

Fig. 1

This ﬁnding is additionally conﬁrmed by a

B NMR experiment,

where the intensity of a signal at 10 ppm decreases, whereas the
one at 3 ppm, which clearly corresponds to the free tetrahedr al
boronate ion PhB ðOHÞ

, increases.

The overlapped species distribution diagram (

Fig. 2

) obtained

from the calculated values of the stability constant

s (

Table 2

)

indicates, that PBA possesse s the strongest afﬁnity towards cate-
cholamin es being in the following order: noradrenaline > dopa-
mine

-DOPA > catechol > DOPA-P. The same dependence was

found by examina

tion of the boron signal in

B NMR spectra,

where comparison of the obtained points reﬂects the same trend
(

Fig. 3

). Collected altogether, obtained data conﬁrm the correctness

of the chosen model used for calculation of stability constants.

The resulting differences in the values of stability constant s are

the most probably caused by the overall charge of the formed

Table 1
Potentiometrically calculated protonation constants for catechola mines (2–6) and two boronic receptors (PBA and 1) at 25 °C, I = 0.1 mol dm

(KNO

PBA

log b

8.80 9.91

9.17 10.31 9.82 9.83 10.64

log b

–

18.46 –

19.17

18.48

18.63

19.61

log b

–

24.03 –

–

20.53

25.15

log K

)

10.64

log K

–

8.55 –

8.97

log K

–

5.57 –

–

1.9

5.54

Data taken from Ref.

[8]

Data taken from Ref.

[15]

Data taken from Ref.

[16]

(a)

(b)

+ 2H

(c)

+ OH

(d)

(e)

110

201

211

010

201

211

010

101

111

-100

110

101

211

111

Scheme 2. Possible equilibria in the mixture of dopamine and PBA.

T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59–64

complexes and the deprotonati on reaction of hydroxyl catechol en-
tity. Noradrenal ine possesses the lowest log K

followed by

-DOPA

and then by dopamine with the highest value found for DOPA-P,
which is partly in accordance with the stabilities of the formed
complexes. The neurotrans

miters

-DOPA and DOPA-P include

additional negatively charged carboxylic and double negative
phosphonic groups respectively , but no interactio ns during pre-
sented studies were detected between these units and the boronic
acid.

When calculating the stability constant

s of the complexes

formed between receptor 1 and catecholam ines two possible mod-
els were obtained. The best ﬁtting of titration curve was achieved
using four species model, which contains succeeding complexes of
stoichiomet ry: H

AL, H

AL 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 H

AL stoichiomet

ry was de-

tected. The presumed explanation of the presence of the H

and H

AL forms might be the interaction of deprotonated phos-

phonic moiety with one hydroxyl group of catecholam ine, which
could be deduced by experienci ng downﬁeld chemical shift of a
phospho rus signal (

Fig. 5

) at pD around 6.

The appearance of an interactio

n between the catechol and

phospho nic group was postulated earlier in the literature

[17,18]

Another explanat ion may be the formation of trigonal complexes,
similar to those found in the potentiometric studies by Bosh and
coworker s

[11]

. The

H NMR studies for the mixture of receptor

1 and 2 at pH 5 revealed the differences in the chemical shifts of
the protons when compare d these spectra with those registered
for individua

l compounds. The greatest changes were detected

for aromatic protons of 2 as well as protons originating from

-car-

bon atom and methylen e group of benzylamin e fragment of 1.

Further deprotonati

on appearing as an equilibrium between

two complexes H

AL , H

AL + H

corresponds to the formation

of tetrahedr

al boron complexes with catecholamine

s. Next two

steps, namely H

AL , H

AL + H

and H

AL , HAL + H

reﬂect to

two ammonium group deprotonations , one of the receptor 1 and
the second one from the guest molecule. At high pH values the

Table 2
Stability constants calculated for complexes PBA and 1 with catechola mines.

PBA

log b

46.82 ± 0.07

log b

41.43

± 0.06 40.28

± 0.06 41.14

± 0.05 40.19

± 0.07

log b

31.12 ± 0.07

34.51 ± 0.04

33.22 ± 0.06

33.82 ± 0.07

33.06 ± 0.06

log b

23.73 ± 0.05

23.29 ± 0.01

23.84 ± 0.03

23.16 ± 0.02

23.78 ± 0.04

25.25 ± 0.07

23.69 ± 0.05

24.29 ± 0.05

23.62 ± 0.06

log b

HAL

13.61 ± 0.02

13.73 ± 0.07

13.52 ± 0.002

14.09 ± 0.17

13.45 ± 0.04

14.04 ± 0.06

14.89 ± 0.05

13.62 ± 0.06

14.36 ± 0.04

13.57 ± 0.05

log K

5.68

log K

6.92 7.06 7.32 7.13

log K

7.34 9.26 9.53 9.53 9.44

log K

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.

Fig. 3. Second-degree polynomial lines ﬁtted to

B NMR titration points of the

formation of tetrahedral complexes in the pD function, [PBA]

= [catechol-

amine] = 0.05 M (4}; 3D; 5s; 6h).

Fig. 1. Species distribution curves for the complexes formed in PBA (A) – dopamine
(L) system as a function of pH: sHA, h A

, dH

AL, j HAL.

Fig. 2. The distribution of a sum of free species of PBA and its complexes with: 2 (h,
j

), 3 (s, d), 4 (

, .), 5 (D, N), and 6 (}, ) in the pH function.

T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59–64

complexes breakdown was observed, although with much less
yield, than for correspondi ng catechola mine complexes with PBA.
In general, the species distribution diagrams reproduce almost
the same trend with tetrahedr al complex constant s being in the
following order: dopamine > noradrenaline > catechol > L-DOPA >
DOPA-P (

Fig. 6

If considering the second type of calculated model, which

excluded the supramolecular interaction from studied systems
together with lack of the H

AL and H

AL species, the ﬁrst spe-

cies that appears in the pH scale is a tetrahedral complex of
H

AL stoichiometry. Because these ﬁtting paramete

rs were

inferior in comparis

on to the ones resulting from the model

described above, the second model models was not further
considered.

From the obtained stability and protonation constants the step-

wise binding constants log K

tet

for formed complexes can be calcu-

lated (

Table 3

). Determination of these paramete

rs led to the

independen t tetrahedral complex formation constant

s (

Table 2

)

from both ligand and receptor protonati on constants (

Table 1

). In

case of receptor 1 the pK

tet

was calculated by additional subtrac-

tion of pK

value of amino group, which has direct contribution

to the stability constant of b

form Ref.

[11]

3. Conclusion s

The performed studies allowed for the determinati

on of the

protonati on constants of four catechola mines, and catechol, as well
as two receptors: PBA and 1. The stability constant s of the formed
complexes were calculated and showed that the simplest boronic
acid PBA, which is used frequently as receptor for 1,2 and 1,3 diols,
binds the catechola

mines in a following order: noradren

line P dopamine

-DOPA > catechol > DOPA-P, while its modi-

ﬁed analog 1 reveals different preference in binding the
catechola mines in order which is as follows: dopamine > noradren-
aline > catechol > L-DOPA > DOPA-P.

4. Experimen tal

4.1. Materials

PBA, catecholam ines and catechol were purchased from Sigma –

Aldrich. The receptor 1 was synthesized according to the previ-
ously described procedure

[19]

. The racemic mixture of a phos-

phonic analog of

-DOPA (DOPA-P) was obtained according to

literature procedure

[20]

Fig. 6. Distribution of a sum of free species of compound 1 and its complexes with:
2 (h, j), 3 (s, d), 4 (

, .), 5 (D, N), 6 (}, ) in the pH function.

Table 3
Logarithm of stepwise binding constants (log K

tet

) for complexes of PBA and 1.

Catecholamine PBA

log K

tet

4.44 4.70

4.56 5.43

4.81 4.83

4.53 4.52

4.23 4.30

log K

tet

= log b

211

– log b

102ðNH

þArOHÞ

or log b

111

– log b

110ðNH

for catechol.

log K

tet

= log b

311

– log b

102ðNH

þArOHÞ

– log b

110ðNH

or log b

211

– log b

110ðNH

þArOHÞ

– log b

110ðNH

for catechol.

Fig. 4. Species distribution curves for the formed complexes in 1 (A) – 3 (L) system
as a function of pH (dotted line indicates free receptor species:

A, D H

A, s HA,

A; solid line—formed complexes: h H

AL, D H

AL, } H

AL, s HAL. The overlapped

P NMR titration (d) showing the deprotonation of amine group originating from

compound 1.

Fig. 5.

P NMR chemical shift for free and complexed species in pD function.

Concentration of each component: 0.004 M (N 1 + 3 free fraction of ligand; d 1 + 3
complex; h 1 free receptor).

T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59–64

4.2. Potentiometri c studies

The protonation and stability constant s for catecholamine s, PBA

and 1 were calculated from titration curves obtained at 25 °C and
using total volume of 1.8 cm

. The NaOH solution was added from

0.250 cm

syringe, which was previously calibrate d. All solutions

were prepared using degassed, deionized and distilled water. All
solvents were prepared using 0.1 mol dm

KNO

. The concentra-

tions of individual reagents were in the range of 0.5–2 mM.

The pH-metric titration was performed on Molspin 1000 pH

meter with automatic titration system. Each titration was per-
formed at least three times for 1:1 M ratio. The exact concentr a-
tions of the boronic compounds were determined pH-metri cally
using GRAN method

[21]

. Electrode was calibrated daily with

determination of E

and slope parameters. The SUPERQUAD com-

puter program was used to calculate the stability constant

[22,23]

. The cumulative stability constants are expressed by the

general equation (pH + rA + qL , H

; b

prq

= [H

]/[H]

[A]

) were b

prq

is deﬁned in the terms of concentratio n at the pres-

ence of constant ionic strength 0.1 mol dm

KNO

4.3. Spectroscopic studies

The

P NMR and

B NMR spectra were recorded at

300 MHz DRX Bruker and 600 MHz Bruker Avance instruments at
298 K in D

O using coaxial tube ﬁlled with TSP [trimethylsilyl

propanoic acid], solution of phosphorou

s acid in D

O or BF

ether as the external standards. In the NMR titration experiment
the pH-meter (SevenEasy, Mettler Toledo) and combined micro-
electrode (InLab@micro

, Mettler Toledo) were used. The concen-

trations of PBA and 1 were 1 mM and 0.4–0.5 mM respectivel y.

Acknowled gements

The Project was ﬁnancially supported by Polish Ministry of Sci-

ence and Higher Education (Grant Nr N N 204 134837) and partly
by Wroclaw Medical Universit y (Grant ST-697).

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T. Ptak et al. / Journal of Molecular Structure 1040 (2013) 59–64

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