Thermodynamic and structural study of complexation of phenylboronic acid with salicylhydroxamic acid and related ligands

Article abstract of DOI:10.1002/aoc.4405

Stability constants of boronate complexes with a highly efficient bioconjugation ligand salicylhydroxamic acid, its derivatives and some structurally related compounds were determined by potentiometric and spectroscopic titrations at variable pH allowing one to obtain detailed stability – pH profiles and to identify the optimum pH for complexation with each ligand. The N,O-binding of salicylhydroxamic acid via condensation of boronic acid with phenolic OH and hydroxamic NH groups was established by crystal structure determination of isolated complexes with phenylboronic and 4-nitrophenylboronic acids. Although this type of binding is impossible for N-methylated salicylhydroxamic acid it still forms stable boronate complexes supposedly involving unusual 7-membered –O-B-O- cycle supported by ¹H NMR studies. Hydroxamic acids lacking ortho-OH group and salicyloyl hydrazide form less stable boronate complexes, which nevertheless possess stabilities similar to those of catechole complexes and may be useful for conjugation applications. In contrast to other ligands, which form tetrahedral anionic complexes, salicylamidoxime forms tetrahedral, but neutral boronate complex with high stability in weakly acid solutions. The highest affinity in neutral and acid solutions surpassing that of salicylhydroxamic acid is observed with 2,6-dihydroxybenzhydroxamic acid (K_obs?=?5.2?×?10⁴ at pH?7.4). Fairly stable mono- and bisboronate complexes are formed with 2,5-dihydroxy-1,4-benzdihydroxamic acid, which also possesses intense fluorescence and may serve as a boronic acid sensor with detection limit 4?μM. Results presented in this study provide quantitative basis for rational applications of hydroxamic acid derivatives in bioconjugation and sensing.

Full text of DOI:10.1002/aoc.4405

Received: 19 February 2018
DOI: 10.1002/aoc.4405
Revised: 4 April 2018
Accepted: 6 April 2018
F U L L P A P E R
Thermodynamic and structural study of complexation of
phenylboronic acid with salicylhydroxamic acid and related
ligands
Mayte A. Martínez‐Aguirre | Marcos Flores‐Alamo
| Anatoly K. Yatsimirsky
Facultad de Química, Universidad
Stability constants of boronate complexes with
a
highly efficient
Nacional Autónoma de México, 04510
México D.F., Mexico
bioconjugation ligand salicylhydroxamic acid, its derivatives and some struc-
turally related compounds were determined by potentiometric and spectro-
scopic titrations at variable pH allowing one to obtain detailed stability – pH
profiles and to identify the optimum pH for complexation with each ligand.
The N,O‐binding of salicylhydroxamic acid via condensation of boronic acid
with phenolic OH and hydroxamic NH groups was established by crystal
structure determination of isolated complexes with phenylboronic and 4‐
nitrophenylboronic acids. Although this type of binding is impossible for
N‐methylated salicylhydroxamic acid it still forms stable boronate complexes
Correspondence
Anatoly K. Yatsimirsky, Facultad de
Química, Universidad Nacional
Autónoma de México, 04510 México D.F.,
Mexico.
Email: anatoli@unam.mx
Funding information
Consejo Nacional de Ciencia y Tecnología,
Grant/Award Number: RTQS No. 281251;
Facultad de Química de la UNAM;
Programa de Apoyo a la Investigación y el
Posgrado, Grant/Award Number: PAIP
5000‐9042; CONACyT, Grant/Award
Number: 271108
1
supposedly involving unusual 7‐membered –O‐B‐O‐ cycle supported by H
NMR studies. Hydroxamic acids lacking ortho‐OH group and salicyloyl hydra-
zide form less stable boronate complexes, which nevertheless possess stabilities
similar to those of catechole complexes and may be useful for conjugation
applications. In contrast to other ligands, which form tetrahedral anionic
complexes, salicylamidoxime forms tetrahedral, but neutral boronate complex
with high stability in weakly acid solutions. The highest affinity in neutral
and acid solutions surpassing that of salicylhydroxamic acid is observed with
2,6‐dihydroxybenzhydroxamic acid (Kobs = 5.2 × 104 at pH 7.4). Fairly stable
mono‐ and bisboronate complexes are formed with 2,5‐dihydroxy‐1,4‐
benzdihydroxamic acid, which also possesses intense fluorescence and may
serve as a boronic acid sensor with detection limit 4 μM. Results presented in
this study provide quantitative basis for rational applications of hydroxamic
acid derivatives in bioconjugation and sensing.
KEYWORDS
boronic acid, complexation, crystal structure, hydroxamic acid, stability constant
1 | INTRODUCTION
common type of complexation of boronic acids in water is
formation of diol esters which finds applications in
Complex formation with boronic acids represents one of
important types of bioorthogonal chemical reactions
often employed in bioconjugation chemistry.[1] The most
molecular recognition of sugars and other polyols[2] as
well as in self‐assembly of pH and/or sugar responsive
supramolecular structures.[3–6] When fragments of
Appl Organometal Chem. 2018;e4405.
wileyonlinelibrary.com/journal/aoc
Copyright © 2018 John Wiley & Sons, Ltd.
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https://doi.org/10.1002/aoc.4405

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MARTÍNEZ‐AGUIRRE ET AL.
aliphatic polyols such as sugars are employed for com-
plexation the major problem is the necessity of using high
pH values to generate relatively stable tetrahedral ester
hydroxocomplexes, which still possess rather modest sta-
bility. Higher stability already in less basic solutions is
observed with catechols[7,8] often employed for self‐
assembly in water,[5b, 6b, d, 9] but they suffer from rather
fast autoxidation in solution[5b, 9a] and still have insuffi-
cient affinity in neutral solutions for applications such
as bioconjugation.[10] An important step in the develop-
ment of suitable high affinity ligands was the discovery
of strong binding of phenylboronic acid (PBA) to
salicylhydroxamic acid (SHA),[11] for which the observed
stability constant Kobs = 1.8×104 M−1 at pH 7.4 was
reported[12] (for comparison Kobs = 830 M−1 for pyrocate-
chol and 160 M−1 for fructose at the same pH).[7] The N,
O‐chelated structure 1 for the SHA‐PBA complex
(Scheme 1) was suggested on basis of mass spectrometric
and 11B NMR data, although contradictory to this N‐
methylated SHA derivative (SHA‐NMe) also was found
to bind PBA.[11] In fact, several isomeric structures of
SHA‐PBA complex are possible, which agree with
reported data. Importantly SHA is considered as a tightly
bound ligand for PBA in a wide range of pH in particular
well below ‘physiological’ pH 7.4,[11–13] however the pH‐
profile of the complex stability constant was never
reported and actual complex stability as a function of
pH is unknown.
In view of importance of SHA in bioconjugation[1b, 12,
13]
and only fragmentary information reported so far on
the strength and type of its interaction with boronic acids,
we provide here a detailed study of complex formation of
phenylboronic acid with SHA and its methylated deriva-
tives SHA‐NMe and SHA‐OMe as well as the crystal
structures of SHA complexes with two phenylboronic
acids which confirm the postulated binding mode. In
addition, we include here results for complexation of
PBA with several related ligands (see Scheme 1) such as
benzhydroxamic acid (BHA), salicyloyl hydrazide (SH),
salicylamidoxime (SAO), 2,6‐dihydroxybenzhydroxamic
acid (DHBHA) and 2,5‐dihydroxy‐1,4‐benzdihydroxamic
acid (DHBDHA) to get a more comprehensive picture of
boronic acid binding to salicylate and hydroxamate deriv-
atives, which generally possess appropriate structural fea-
tures to serve as anchor ligands for conjugation. Testing
of these compounds allowed us to discover even more
powerful ligand DHBHA which binds PBA with stability
constant 5 × 104 M−1 at pH 7.4.
2 | RESULTS AND DISCUSSION
At the first step the pKa values of ligands and stability
constants of complexes with PBA were determined by
standard potentiometric titrations in water analyzed in
terms of general reaction (1) where M is the neutral
PBA molecule, L is the neutral form of the ligand, H
is proton and stability constants are defined as
βpqr = [MpLqHr]/{[M]p[L]q[H]r}. The respective dissocia-
tion and stability constants are given in Table 1.
pM þ qL þ rH⇄MpLqHr
(1)
Acid dissociation constants for SHA and BHA given
in Table 1 agree well with reported values (7.40; 9.81
and 8.71 at 25 °C and ionic strength 0.2 M).[14] In all cases
it was found that p = q = 1 and for majority of the ligands
r = −1 indicating formation of anionic boronate
hydroxocomplexes with stability constants β1,1,‐1, which
in case of SHA agrees with formation of complex 1
(Scheme 1). In case of SAO a neutral complex with stabil-
ity constant β1,1,0 is detected additionally and in fact this
complex is the predominant one in neutral solution (see
below). The stability constant β1,1,‐1 is not a simple
association constant because it refers specifically to the
reaction between neutral forms of PBA and a hydroxamic
acid with simultaneous deprotonation. The respective
SCHEME
complexes considered in this study
1
Chemical structures of ligands and boronate

MARTÍNEZ‐AGUIRRE ET AL.
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TABLE 1 Equilibrium constants for complexation of PBA with hydroxamic acids determined by potentiometric titrations (25 °C, 0.05 M
NaCl)a
Kobs, M−1
b
Ligand
pKa1
pKa2
logβ1,1,‐1
optimum pH
pH 7.4
UV–Vis titration (pH)
1
SHA
7.55
10.03
−2.95
−3.17
−2.58
−4.63
−4.17
−4.14
−4.95
−4.17
−4.22
−5.35 3.12
−2.29
2.6 × 104 (8.2)
1.1 × 104 (8.1)
8.1 × 103 (7.7)
4.1 × 103 (8.85)
1.6 × 104
7.8 × 103
7.9 × 103
5.5 × 102
1.4 × 103
1.4 × 103
270
1.1 × 104 (7.4)
6.5 × 103 (7.4)
6.6 × 103 (7.4)
3.7 × 103 (8.9)
2
SHA‐OMe
SHA‐NMe
BHA
7.35
3
6.55
11.05
4
8.85
5
3‐NO2‐BHA
3‐NMe3‐BHA
8.21
6
8.06
c
7
BHA
8.85
d
8
BHA
8.85
1.4 × 103
1.0 × 103
1.3 × 103
3.6 × 104
9
SH
7.82
11.44
2.4 × 103 (8.3)
1.3 × 103 (6.6)
4.0 × 104 (7.9)
2.2 × 103 (8.3)
1.4 × 103 (6.5)
5.2 × 104 (7.4)
e
10
11
SAO
4.30
8.85
f
f
DHBHA
7.00 6.86
9.73 9.94
aStandard errors in logK are less than 0.05; pKa of PBA was determined to be 8.84.
bOptimum pH is given in parentheses.
cwith 4‐MeO‐PBA.
dwith 4‐Cl‐PBA.
elogβ110
.
fSpectrophotometric titration.
L
L
observed association constant defined in terms of total
concentrations of components (Kobs), which is the stabil-
ity constant experimentally measured e.g. by spectro-
scopic titration at a given pH, depends on pH in
Usually Ka2 < < Ka1 and pKa2 is above 10, which
makes it possible to exclude the Ka2L/[H+] term from
the equation (2) for analysis in practically important pH
range around 7. With these assumptions the equation (2)
can be substituted with more familiar simplified equa-
tion (3).
accordance with theoretical equation (2),[8] where Ka1
L
L
and Ka2 are the acid dissociation constants of the ligand
B
and Ka is the dissociation constant of PBA (Scheme 2).
À
Á ÈÀ
ÁÀ
ÁÉ
À
Á
Kobs ¼ β1;1;−1=½Hþꢀ = 1 þ ½Hþꢀ=Ka1 1 þ KaB=½Hþꢀ
L
Kobs ¼ β1;1;−1=½Hþꢀ =
(2)
(3)
ÈÀ
ÁÀ
ÁÉ
1 þ ½Hþꢀ=Ka1 þ Ka2L=½Hþꢀ 1 þ KaB=½Hþꢀ
L
The equation (3) predicts a bell‐shaped form of the pH
profile of Kobs with a maximum Kobs value at optimum
L
B
pH = (pKa1 + pKa )/2. The profiles of logKobs vs. pH
calculated with equation (2) are shown in Figure 1 and
Table 1 contains Kobs values at optimum pH and at
“physiological” pH 7.4 for each ligand. Also for compari-
son Table 1 contains Kobs determined spectrophotometri-
cally (Supplementary Material) either at pH 7.4 or at
optimum pH.
Inspection of Table 1 shows that the value of
Kobs = 1.6×104 M−1 calculated for SHA at pH 7.4
from potentiometric data practically coincides with
reported value at this pH determined by 11B NMR (see
Introduction),[12] but the spectrophotometrically deter-
mined Kobs is somewhat smaller. The discrepancy can
be attributed to a difference in reaction conditions most
probably to previously noticed buffer inhibitory effect
SCHEME 2 Acid–base and complexation equilibria involved in
the interaction between SHA and PBA. The first dissociation
constant of SHA is attributed to deprotonation of phenolic OH
[15]
[15]
group in accordance with Ref
(potentiometric titrations were performed in

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MARTÍNEZ‐AGUIRRE ET AL.
FIGURE
2
Hammett plots for Kobs determined by
FIGURE 1 Profiles of logKobs vs. pH calculated for complexation
of hydroxamic acids with PBA in accordance with equilibrium
constants given in Table 1 and equation (2). The black dashed line
is the profile for catechol as a ligand shown for comparison
spectrophotometric titrations for substituted phenylboronic acids
with SHA at pH 7.4 (squares) and for respective β11‐1 values
(circles)
unbuffered solutions). Indeed, lower spectrophotometri-
cally determined Kobs values as compared to those calcu-
lated from potentiometric data are observed for majority
of ligands in Table 1.
Proposed structure of SHA complex 1 seems reason-
able because of known lability of NH bond in BHA,
which dissociates simultaneously with OH bond,[16] how-
ever alternative structures involving binding to oxygen
atoms of phenolic and hydroxamic acid groups cannot
be excluded on basis of given results, in particular taking
into account the observation of PBA binding to N‐
methylated SHA[11] for which structure 1 is obviously
impossible. Previously the methylated derivatives SHA‐
OMe and SHA‐NMe were tested as ligands for PBA by
11B NMR and mass spectrometry and were found to form
boronate complexes, but their stabilities even relative
were not determined.[11] Also unknown are pKa values
for these ligands which are needed to characterize the
complex stability at variable pH. As one can see from
Table 1 pKa of SHA‐OMe is very close to pKa1 of SHA
in agreement with assignment of first deprotonation of
SHA to phenolic OH[15] and pKa1 of SHA‐NMe is signifi-
cantly lower than that of SHA as is typical for N‐
substituted hydroxamic acids.[17] Both methylated deriva-
tives form anionic deprotonated complexes with PBA
with β1,1,‐1 values slightly smaller in case of SHA‐OMe
and even larger in case of SHA‐NMe than that for SHA.
The Kobs values for both derivatives in pH range 7–9 are
smaller than Kobs for SHA only by a factor of 2
(Figure 1). Thus, methylation does not affect significantly
the affinity of SHA to PBA. This is an expected result for
O‐methylated derivative because complex 1 involves
undissociated hydroxamic OH group and therefore the
structure 2 (Scheme 1) for a complex of similar stability
can be logically proposed for SHA‐OMe. However if the
Figure 1 shows a complete pH‐profile of Kobs for SHA
from which one can see that this ligand retains a signif-
icant affinity to PBA even in weakly acid solutions, e.g.
at pH 5.8 Kobs for SHA equals 800 M−1 similar to Kobs
for catechol at pH 7.4. In comparison with catechol
SHA is more than one order of magnitude stronger
ligand at pH 7.4 and lower pH values, although Kobs
values for these ligands differ only by a factor of 2 when
compared at optimum pH for each ligand, 8.2 for SHA
and 9.1 for catechol (Figure 1). Thus, an important
advantage of SHA is its relatively low pKa value, which
shifts the pH‐profile to lower pH in accordance with
equation (3).
To get a deeper insight into the nature of boronic acid
– SHA interaction the binding constants Kobs were deter-
mined spectrophotometrically at pH 7.4 with substituted
phenylboronic acids. The results are shown in Figure 2
in coordinates of the Hammett equation. The plot is
non‐linear, but the respective β1,1,‐1 values calculated
from these results show good linear correlation (solid cir-
cles in Figure 2) with the slope ρ = 1.35 0.09. The pos-
itive slope agrees with formation of the anionic complex
and its value is close to ρ = 1.5 0.1 reported for com-
plexation of substituted phenylboronic acids with tiron,
a diol with pKa 8.16 close to that for SHA.8 It is worth
to note that Kobs for 4‐nitrophenylboronic acid reaches a
very large value of 5x104 M−1 at pH 7.4.

MARTÍNEZ‐AGUIRRE ET AL.
5 of 10
structure 1 is valid the N‐methylation of SHA should
block completely the binding to PBA. An alternative
structure 3 for SHA‐NMe complex (Scheme 1) involving
O,O‐chelation of doubly deprotonated SHA without
direct involvement of phenolic OH group was sug-
gested.[11] Although structures of this type were reported
for some isolated diphenylborinic acid complexes of N‐
substituted hydroxamic acids,[18] less electrophilic boric
acid does not interact with N‐substituted hydroxamic
acids.[19] One may speculate however, that intramolecu-
lar ion pairing with deprotonated ortho‐hydroxyl group
can stabilize electrostatically the ammonium ion making
structure 3 in principle possible. The question in this case
is how stable are PBA complexes with hydroxamic acids
without involvement of ortho‐hydroxyl group in boronic
acid binding.
Previously only interaction of BHA with boric acid
was reported with logβ1,1,‐1 = −5.70,[20] significantly
smaller than that for PBA (Table 1), as was observed for
SHA (vide supra) and is typical also for diol boronate
complexes.[20] Structure 4 (Scheme 1) agrees with the
reaction stoichiometry as well as with reported structure
of bis(benzohydroximato)borate complexes.[21] In com-
parison with SHA the affinity of BHA to PBA is one to
two orders of magnitude lower both in terms of β1,1,‐1
and Kobs values; see Table 1 and Figure 1. Titrations
of BHA by substituted phenylboronic acids allowed
us to calculate ρ = 1.6 0.2 for logβ1,1,‐1 values in agree-
ment with formation of anionic complex 4. Using
benzhydroxamic acids with electron acceptor groups
including a cationic Me3N‐ group improve very little the
affinity to PBA (Table 1). Thus on basis of these results
the structure 3 is inconsistent with high affinity of SHA‐
NMe to PBA.
Useful information on the type of complexes was
1
obtained by comparison of H NMR spectra of free and
bound to PBA ligands, Figure 3. As follows from these
results, the character of changes in the pattern of signals
of aromatic protons upon complexation is essentially the
same for all three ligands, which means that phenolic
oxygen is always involved thus excluding the structure 3
for the complex with SHA‐NMe. Complexation with
SHA‐OMe induces strong up‐field shift of the signal of
OMe group in agreement with binding of boronic acid
to hydroxamic acid nitrogen (see spectra ‘c’ and ‘d’) in
the structure 2, but the signal of NMe group remains
practically not shifted (see spectra ‘e’ and ‘f’) that again
contradicts the structure 3 needing formation of a positive
charge on nitrogen atom. A plausible structure of the
complex with SHA‐NMe consistent with observed
stoichiometry and the small shift of the signal of NMe
group is 5 (Scheme 1). It is worth to note that formation
of similar 7‐membered cycle is preferred over possible
formation of 5‐ and 6‐membered cycles in the structures
of diphenylborinic acid complexes of N‐methylated
salicylaldoxime and some related ligands.[22] Since
stability of SHA‐NMe complex is close to that of SHA
complex (see Table 1), formation of 5 points to a possibil-
ity that in fact the structure 1 may be wrong and the real
structure of the SHA complex is 6. However, as will be
FIGURE 3 1H NMR spectra for free SHA, SHA‐OMe and SHA‐NMe (spectra “a”, “c” and “e”) and their PBA complexes 1, 2 and 5 (spectra
“b”, “d” and “f”) obtained in situ by adding 1 equivalent of phenyl‐d5‐boronic acid and NaOH to respective ligands in D2O

6 of 10
MARTÍNEZ‐AGUIRRE ET AL.
shown below the crystal structure of SHA complex
with phenylboronic acids confirms the validity of the
structure 1.
salicylaldoxime.[23] Bond distances and bond angles
around B atom are collected in Table 2. The tetrahedral
character (THC) of boron atom calculated from these
results in accordance with Höpfl's formula[24] is 89.1 for
4‐nitrophenylboronic acid and 84.1% for PBA. Such high
THC values are indicative of covalent B‐N bonds found in
e.g. amine boranes while boronate adducts typically have
THC about 70%.[25] In accordance with this B‐N distances
are about 1.55 Å (the average B‐N distance in boronate
complexes is 1.69 Å).[25]
Salicyloyl hydrazide (SH), which differs from SHA
just by the presence of NH2 group instead of OH in Ph–
C(O)NH‐ fragment could be equally good ligand for
PBA, but in fact it binds with 10 times smaller association
constant (Table 1). Still the affinity of SH to PBA is simi-
lar to that of catechol at pH 7.4 and below (Figure 1). The
first pKa values of SH and SHA are very close to each
other so binding to phenolic oxygen should be similar,
but the second pKa is 1.4 units higher for SH making
more difficult substitution at NH group.
Crystals of SHA complexes with PBA and 4‐
nitrophenylboronic acid suitable for single crystal X‐Ray
analysis were grown from DMSO solutions. The struc-
tures shown in Figure 4a,b confirm the postulated O,N‐
binding of boronic acid to SHA with NH rather than
OH deprotonation of the hydroxamic acid group. Dimeric
structures apparently result from condensation of two
respective monomeric complexes with elimination of
two water molecules in the solid state. Similar dimeric
structure was reported for PBA complex with
We tested also salicylamidoxime (SAO) as possible
ligand and found that in this case the predominant
boronate complex is neutral with fairly high stability con-
stant 1.3 × 103 M−1 at optimum pH 6.6 (Table 1), which
remains unchanged in a wide pH interval from 5.5 to
8.5 providing tighter binding than SHA at pH below 6
and slightly tighter binding than catechol at pH 7.4
(Figure 1). This ligand can be useful therefore for boronic
acid assembly at weakly acidic media. Figure 5 shows the
11B NMR spectrum of a 1:1 mixture of SAO and PBA in
D2O at pD 7. The peak at 28.91 ppm belongs to free
PBA and the peak at 3.18 ppm can be attributed to the
complex. The position of the signal indicates tetrahedral
coordination of boron in the complex[25] to which
TABLE 2 Bond distances and bond angles around B atom in
structures of complexes
Bond distances (Å)
Bond angles (°)
NPBA+SHA
B—N
B—O
B—O
B—C
1.544
1.467
1.487
1.624
C—B—N
C—B—O
C—B—O
O—B—O
N—B—O
N—B—O
C—B—N
C—B—O
C—B—O
O—B—O
N—B—O
N—B—O
109.9
111.5
111.6
104.6
109.3
109.8
PBA + SHA
B—N
B—O
B—O
B—C
1.553
1.472
1.48
11184
111.54
111.95
103.41
108.65
109.07
FIGURE 4 Perspective views of the molecular structures of SHA
dianionic complexes with 4 nitrophenylboronic acid (a) and PBA (b)
(counterions and DMSO molecules are omitted for clarity).
Ellipsoids are shown at the 50% probability. Color scheme: C, black;
O, red; N, blue; B, pink; H, white
1.619

MARTÍNEZ‐AGUIRRE ET AL.
7 of 10
FIGURE 6 Fluorescence titration of 0.02 mM DHBDHA with
PBA at pH 7.4 (excitation wavelength 355 nm). Inset shows the
titration profile at 450 nm; the solid line is the theoretical fitting
curve generated by HypSpec program
FIGURE 5 11B NMR spectrum of a mixture of 0.01 M SAO and
0.01 M PBA in D2O at pD 7
therefore the zwitterionic structure 7 (Scheme 1) can be
assigned.
In a hope to achieve even stronger boronate binding
than with SHA we have prepared two dihydroxy deriva-
tives DHBHA and DHBDHA (Scheme 1). The presence
of the second hydroxyl group in DHBHA should at least
duplicate the binding constant due to statistical factor.
Results of potentiometric and spectrophotometric titra-
tions (Table 1) show that β11–1 increases even larger by
a factor of 4.6 and similar effect is observed in Kobs mea-
sured spectrophotometrically at pH 7.4. Smaller effect in
Kobs by a factor of 2.3 at pH 7.4 is predicted from potenti-
ometric results, but in any case we have a very powerful
ligand with Kobs about 5 × 104 M−1 at pH 7.4 and with
the pH‐profile of stability constant shifted to lower pH
(see Figure 1).
The ability to bind two boronic acid molecules makes
DHBDHA a potentially useful ligand for cross‐linking of
boronic acid polymeric materials.[26] Strong fluorescence
response to a boronic acid makes it also suitable for appli-
cation as a boronic acid sensor. There are a growing num-
ber of boronic acid drugs both in development stage and
recently approved creating a demand in sensitive sensors
for their monitoring.[27] The green luminescence of
DHBDHA at 475 nm linearly depends on concentration
of PBA up to 80 μM with detection limit 4 μM (Supple-
mentary Material) significantly lower than for sensors
reported so far.[28]
Interaction with DHBDHA was studied only spectro-
photometrically by fitting results to the HypSpec program
(Supplementary Material). The pH profile of the absorp-
tion spectrum allowed us to calculate expected four pKa
values 7.06, 8.64, 10.09 and 10.09 (the last pKa with large
uncertainty) and titration by PBA at pH 7.4 gives two
3 | CONCLUSIONS
The observed stability constant for binding of SHA to
phenylboronic acid reaches its maximum value of
2.6 × 104 M−1 at optimum pH 8.2 and remains larger than
103 M−1 in a pH range from 6 to 10. Both O‐ and N‐
methylation of hydroxamic moiety of SHA induces only
a two‐fold decrease in Kobs at pH 7.4 and a small variation
in optimum pH value. Small effect of O‐methylation
agrees with proposed structure 1 of SHA – phenylboronic
acid complex, but high affinity of N‐methylated derivative
points to either different mode of complexation with SHA
or an unusual type of bonding of SHA‐NMe. Single crys-
tal X‐Ray structures of SHA complexes confirm N,O‐
coordination of this ligand and also indicate unusually
strong for boronate adducts covalent B‐N bonding, which
agrees with high stability of solution complexes. Analysis
of 1H NMR results for SHA‐NMe binding suggests
consecutive binding constants (1.6
0.1) × 104 and
(5.6 0.1) × 103 M−1 for interaction with first and second
PBA molecules respectively. This ligand is fluorescent
with emission maximum at 490 nm. Complexation with
PBA strongly increases the fluorescence intensity and
shifts the maximum to 450 nm, Figure 6. The effect is
reminiscent of previously reported strong fluorescence
enhancement of alizarin red S on PBA binding attributed
to ligand deprotonation.7 The fitting of fluorescence titra-
tion data confirms formation of both 1:1 and 1:2 com-
plexes with slightly larger constants (2.5
and (1.0 0.1) × 104 M−1, respectively.
0.3) × 104

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MARTÍNEZ‐AGUIRRE ET AL.
formation of 7‐membered cycle as the most probable
structure of the complex. Hydroxamic acids lacking
ortho‐OH group form less stable complexes with boronic
acids, but they still possess stability similar to that of cat-
echol complexes and therefore may be employed for con-
jugation processes. Incorporation of additional hydroxyl
groups in SHA leads to increased affinity to PBA and in
a case when OH groups are in para‐position to each other
makes the ligand fluorescent. The fluorescence is strongly
enhanced by complexation with PBA allowing highly
sensitive detection of boronic acids.
(s, 1H), 9.86 (s, 1H), 7.38–7.16 (m, 2H), 6.82 (dd, 2H),
3.18 (s, 3H). MS (EI, m/z) 167 [M]+ for C8H9NO3
(167.058).
2,6‐Dihydroxybenzhydroxamic acid (DHBHA)
was synthesized following similar procedure. A solution
of hydroxylamine hydrochloride (214.1 mg, 3.1 mmol)
in 5 ml of methanol was cooled in an ice‐water bath
and then potassium hydroxide (347.9 mg, 6.2 mmol)
was added. After stirring for 20 min, 2‐
(methoxycarbonyl)‐1,3‐phenylene diacetate (388 mg,
1.54 mmol, prepared by reaction of methyl 2,6‐
dihydroxybenzoate with acetic anhydride) was added
and the mixture was stirred at room temperature for
3 days. The solution was acidified with acetic acid
(0.3 ml) and the solvent was evaporated in vacuo. The
residue was taken into water (10 ml) and extracted with
ethyl acetate. The organic layer was dried over anhydrous
MgSO4 and evaporated under reduced pressure. The
product was washed with chloroform. 1H NMR
(400 MHz, DMSO‐d6) δ 12.27 (s, 2H), 10.86 (s, 1H), 9.46
(s, 1H), 7.13 (t, J = 8.2 Hz, 1H), 6.34 (d, J = 8.2 Hz,
4 | EXPERIMENTAL
4.1 | General Experimental Methods
Commercially
available
PBA
and
substituted
phenylboronic acids, SHA, BHA, SH, SAO and compo-
nents of buffer solutions (CHES, MOPS, MES) were used
as supplied. All titration experiments were performed at
25 °C and ionic strength 0.05 M created either by buffer
or NaCl.
2H). HRMS (ESI, m/z) 170.0452 [M
[C7H8NO4]+ (calculated 170.0453).
+
H]+ for
SHA‐OMe was synthesized according to literature
method.[11] A solution of methoxylamine hydrochloride
(626.1 mg, 7.5 mmol) in 10 ml of methanol was cooled
in an ice‐water bath and then potassium hydroxide
(634 mg, 11.3 mmol) was added. After stirring for
20 min, 2‐(chlorocarbonyl)phenyl acetate (500 mg,
2.5 mmol) was added and the mixture was stirred at room
temperature for 30 h. The solution was acidified with
acetic acid (0.22 ml) and the solvent was evaporated in
vacuo. The residue was taken into water (10 ml) and
extracted with ethyl acetate. The organic layer was dried
over anhydrous MgSO4 and evaporated under reduced
pressure. The product was purified by silica gel chroma-
tography with 1:1 ethyl acetate:hexane. 1H NMR
(300 MHz, DMSO‐d6) δ 11.80 (s, 1H), 11.74 (s, 1H), 7.67
(d, 1H), 7.42 (t, 1H), 6.93 (dd, 2H), 3.75 (s, 3H). MS (EI,
m/z) 167 [M]+ for C8H9NO3 (167.058).
2,5‐Dihydroxy‐1,4‐benzdihydroxamic
acid
(DHBDHA) was synthesized following similar proce-
dure. solution of hydroxylamine hydrochloride
A
(333.6 mg, 4.8 mmol) in 10 ml of methanol was cooled
in an ice‐water bath and then potassium hydroxide
(505 mg, 9 mmol) was added. After stirring for 20 min,
diethyl 2,5‐dihidroxyterephthalate (300 mg, 1.2 mmol)
was added and the mixture was stirred at room tempera-
ture for 1 day. The solution was acidified with acetic acid
(0.15 ml) and the solvent was evaporated in vacuo. The
residue was taken into water (5 ml) and extracted with
ethyl acetate. The organic layer was dried over anhydrous
1
MgSO4 and evaporated under reduced pressure. H NMR
(400 MHz, DMSO‐d6) δ 11.30 (s, 2H), 9.44 (s, 1H), 7.24 (s,
1H). HRMS (ESI, m/z) 229.0461 [M
+
H]+ for
[C8H9N2O6]+ (calculated 229.0461).
SHA‐NMe was synthesized according to literature
method.[11] A solution of N‐methylhydroxylamine hydro-
chloride (626.1 mg, 7.5 mmol) in 10 ml of methanol was
cooled in an ice‐water bath and then potassium hydrox-
ide (634 mg, 11.3 mmol) was added. After stirring for
20 min, 2‐(chlorocarbonyl)phenyl acetate (500 mg,
2.5 mmol) was added and the mixture was stirred at room
temperature for 30 h. The solution was acidified with
acetic acid (0.22 ml) and the solvent was evaporated in
vacuo. The residue was taken into water (10 ml) and
extracted with ethyl acetate. The organic layer was dried
over anhydrous MgSO4 and evaporated under reduced
pressure. The product was recrystallized with 1:1 ethyl
4.2 | Potentiometry
Potentiometric titrations were performed in a 25 ml
thermostated cell kept under nitrogen at 25 0.1 °C with
0.05 M NaCl as background electrolyte. Depending on
solubility 2–5 mM solutions of ligands in the presence
of equimolar amount of PBA were employed. The pKa
values of all components were determined independently
by titrations in the same conditions and were used as
fixed parameters in the fitting of results for the
PBA − ligand mixtures. Experimental details and proce-
dure for the electrode calibration were the same as in
1
acetate:hexane. H NMR (300 MHz, DMSO‐d6) δ 10.13

MARTÍNEZ‐AGUIRRE ET AL.
9 of 10
ref.[28] The program Hyperquad 2008[29] was used to
calculate all equilibrium constants.
CrysAlisPro and CrysAlis RED software packages[30]
were used for data collection and data integration.
Analysis of the integrated data did not reveal any decay.
Final cell constants were determined by a global refine-
ment of 8564 and 6510 reflections (θ < 30.089°) for
PBA‐SHA and NPBA‐SHA respectively. Collected data
were corrected for absorbance by using Analytical
numeric absorption correction[31] using a multifaceted
crystal model based on expressions upon the Laue sym-
metry using equivalent reflections.
Structure solution and refinement were carried out
with the SHELXS‐2014[32] and SHELXL‐2014[33] pack-
ages; WinGX v2014.1 software was used to prepare mate-
rial for publication.[34]
Full‐matrix least‐squares refinement was carried out
by minimising (Fo2 – Fc2)2. All non‐hydrogen atoms were
refined anisotropically. H atoms attached to C atoms
were placed in geometrically idealized positions and
refined as riding on their parent atoms, with C–
H = 0.98–1.00 Å and with Uiso(H) = 1.2Ueq(C) for
methyne and methylene groups, and Uiso(H) = 1.5Ueq(C)
for methyl groups. Details of the X‐ray studies for the
compounds are summarized in Table S1.
4.3 | Spectrophotometric and
Fluorometric Titrations
To a 30–100 μM solution of a ligand (4–10 μM for fluo-
rometric titrations) in an appropriate 0.05 M buffer
(MES, MOPS, or CHES in order of increasing pH from
5.5 to 9.5) portions of concentrated solution of
phenylboronic acid in the same buffer were added, and
the mixture was incubated for 3 min after each addition
before recording the spectrum. In an independent exper-
iment, it was established that the system equilibrates
completely during this incubation period. The observed
equilibrium constants of the ester formation (Kobs) for
simple 1:1 equilibria were calculated from the absorbance
or fluorescence intensity (A) vs concentration of boronic
acid (B) profiles at several wavelengths by nonlinear least
squares fitting to the equation (4), and the results were
averaged. In equation (5), subscript T stands for total con-
centration, A0 is the initial absorbance or fluorescence
intensity of the ligand measured in the absence of B,
and Δε is the difference in molar absorptivities or respec-
tive proportionality coefficients for fluorescence between
the ester and free ligand.
The crystallographic data for PBA‐SHA and NPBA‐
SHA compounds reported in this paper has been depos-
ited with the Cambridge Crystallographic Data Centre
as supplementary publication no. CCDC 1819799 and
1505470. Copies of the data can be obtained free of charge
on application to CCDC, 12 Union Road, Cambridge, CB2
1EZ, UK, e‐mail: deposit@ccdc.cam.ac.uk).
È
A ¼ A0 þ 0:5 Δ ε ½LꢀT þ ½BꢀT þ 1=Kobs
ꢀ
ꢁ
À
Á
−
½Lꢀ þ ½Bꢀ þ 1=Kobs 2–4½LꢀT½BꢀT 0:5g
(4)
T
T
ACKNOWLEDGEMENTS
Titration results at variable pH and for more complex
stoichiometry were fitted to HypSpec program.[28]
Mayte A. Martínez‐Aguirre thanks CONACyT for
the Doctoral fellowship (271108). The support from
Programa de Apoyo a la Investigación y el Posgrado (PAIP
5000‐9042), Facultad de Química de la UNAM and RTQS
CONACYT Proyecto No. 281251 is gratefully acknowledged.
4.4 | X‐ray Crystallography
Crystals of the boronate esters suitable for X‐ray diffrac-
tion were grown by slow evaporation from DMSO.
Crystals of SHA complexes with PBA and 4‐
nitrophenylboronic acid mounted on a glass fiber were
studied with Oxford Diffraction Gemini ‘A' diffractometer
with a CCD area detector (λMoKα = 0.71073 Å, monochro-
mator: graphite) source equipped with a sealed tube X‐
ray source at 130 K. Unit cell constants were determined
with a set of 15/3 narrow frame/runs (1° in ω) scans. A
data sets consisted of 208 and 669 frames of intensity data
collected for PBA‐SHA, and NPBA‐SHA respectively
with a frame width of 1° in ω, and a crystal‐to‐detector
distance of 55.00 mm. The double pass method of scan-
ning was used to exclude any noise. The collected frames
were integrated by using an orientation matrix deter-
mined from the narrow frame scans.
ORCID
Marcos Flores‐Alamo
7616
Anatoly K. Yatsimirsky
http://orcid.org/0000-0002-2996-
http://orcid.org/0000-0002-4616-
4831
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