Revisiting Boronate/Diol Complexation as a Double Stimulus-Responsive Bioconjugation

Article abstract of DOI:10.1021/acs.bioconjchem.7b00080

This study presents a quantitative assessment of the complexation between boronic acids and diols as a reversible and double-stimulus (oxidation and acidification)-responsive bioconjugation reaction. First, by using a competition assay, we have evaluated the equilibrium constants (water, pH 7.4) of 34 boronate/diol pairs, using diols of both aliphatic and aromatic (catechols) nature; in general, catechols were characterized by constants 3 orders of magnitude higher than those of aliphatic diols. Second, we have demonstrated that successful complexation with diols generated in situ via enzymatic reactions, and the boronate complexation was also employed to calculate the Michaelis-Menten parameters for two catechol-producing reactions: the demethylation of 3-methoxytyramine and the 2-hydroxylation of estradiol, respectively, mediated by P4502D6 and P4501A2. Third, we have prepared phenylboronic acid-functionalized hyaluronic acid (HA) and demonstrated the pH and H₂O₂-responsive character of the adducts that it formed with Alizarin Red S (ARS) used as a model catechol. The versatility and selectivity of the complexation and the mild character of the chemical species involved therefore make the boronate/catechol reaction an interesting candidate for bioconjugation purposes.

Full text of DOI:10.1021/acs.bioconjchem.7b00080

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Article
Revisiting boronate/diol complexation as a
double stimulus-responsive bioconjugation
Arianna Gennari, Chirag Gujral, Erwin Hohn, Enrique Lallana, Francesco Cellesi, and Nicola Tirelli
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00080 • Publication Date (Web): 05 Apr 2017
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Bioconjugate Chemistry
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Revisiting Boronate/Diol Complexation as a Double Stimulusꢀ
Responsive Bioconjugation
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Arianna Gennari1, Chirag Gujral1¶, Erwin Hohn1§§, Enrique Lallana1, Francesco Cellesi2,
Nicola Tirelli1*
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1 NorthWest Centre of Advanced Drug Delivery (NoWCADD), School of Health Sciences,
University of Manchester, Oxford Road, Manchester, M13 9PT, United Kingdom
2 1. Dipartimento di Chimica, Materiali ed Ingegneria Chimica "G. Natta". Politecnico di
Milano, Via Mancinelli 7, 20131 Milan, Italy 2. Fondazione CEN ꢀ European Centre
forNanomedicine, Piazza Leonardo da Vinci 32, 20133 Milan, Italy
¶ Current address: School of Parmacy, Queen’s University Belfast, 97 Lisburn Road, BT7
1NN, Belfast, United Kingdom
§ Current address: Sandoz Biopharmaceuticals and Oncology Injectables, Lehenau 10a, 5325
Plainfeld, Austria
* corresponding author
Nicola.tirelli@manchester.ac.uk
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ABSTRACT
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This study presents a quantitative assessment of the complexation between boronic acids and diols as
a reversible and doubleꢀstimulus (oxidation and acidification)ꢀresponsive bioconjugation reaction.
Firstly, by using a competition assay, we have evaluated the equilibrium constants (water, pH 7.4) of
34 boronate/diol pairs, using diols of both aliphatic and aromatic (catechols) nature; in general
catechols were characterized by constants three order of magnitude higher than those of aliphatic
diols.
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Secondly, we have demonstrated that successful complexation with diols generated in situ via
enzymatic reactions, and the boronate complexation was also employed to calculate the Michaelisꢀ
Menten parameters for two catecholꢀproducing reactions: the demethylation of 3ꢀmethoxytyramine
and the 2ꢀhydroxylation of estradiol, respectively mediated by P4502D6 and P4501A2.
Thirdly, we have prepared phenylboronic acidꢀfunctionalized hyaluronic acid (HA) and demonstrated
the pH and H2O2ꢀresponsive character of the adducts that it formed with Alizarin Red S (ARS) used
as a model catechol.
The versatility and selectivity of the complexation and the mild character of the chemical species
involved make therefore the boronate/catechol reaction an interesting candidate for bioconjugation
purposes.
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INTRODUCTION
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The reaction between boronates and diols in aqueous solution is quantitative, selective and orthogonal
to the reactivity of a majority of biologically relevant functional groups1, 2, thus fulfilling the typical
requirements of a bioconjugation reaction3. Indeed, the boronate/diol complexation has been used to
decorate the surface of living cells (their glycocalyx) with synthetic polymers4, 5, to selectively
recognize saccharides in biological fluids6, and for protein immobilization7, 8. The complexation
between boronateꢀfunctionalized polymers and aliphatic9 or aromatic10 polyols has also been
employed to prepare crossꢀlinked colloids, or hydrogels that acquire selfꢀhealing properties from the
reversibility of the reaction11. A recent review covering the various applications of boronic acidꢀ
containing polymers (and their diol complexes) has been recently published by Sumerlin12 and
provides an upꢀtoꢀdate overview of the field. We are specifically interested in applying this versatile
bioconjugation to hyaluronic acid (HA, Scheme 1). HA is a glycosaminoglycan found in virtually all
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extracellular matrices13ꢀ17, with a broad spectrum of FDAꢀapproved applications as a biomaterial18, 19
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HA conjugation with bioactive molecules is appealing, because it combines the biocompatibility,
degradability and nonꢀimmunogenicity of HA, the improvement in solubility and halfꢀlife of the
coupled (pro)drug20, 21, and the possibility to target HA receptors such as CD44, which is
overexpressed in several pathological conditions22 (e.g. tumor progression23ꢀ26 or inflammatory
disorders27, 28). Following this approach, we have recently used boronic acidꢀbearing HA derivatives
for the reversible bioconjugation and delivery (in a nanoparticle form) of a biocidal polyphenol, tannic
acid29.
Scheme 1. HA structures were prepared via DMTMMꢀmediated amidation with dopamine (DOP), veratrylamine (VER:
negative, non boronateꢀbinding control) and 3ꢀaminophenyl boronic acid (APBA). DOP and APBA derivatives can complex
respectively boronateꢀ and diolꢀ containing molecules, and in both cases HA conjugates presenting cyclic boronic ester
groups as side chains are produced. The latter bear a negative charge at neutral/basic pH due to quaternarization of boron by
OH anions; at acid pH, their neutralization decreases the complex stability and thus determines the release of payloads via
hydrolytic cleavage and production of a diol and a boronate. As a second destabilization mechanism, the lability of the
carbonꢀboron bond in an oxidizing environment releases a phenol and a cyclic borate, the latter eventually releasing a diol.
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Interestingly, boronic esters are also environmentally responsive groups, being cleavable under acidic
and oxidizing conditions (bottom left in Scheme 1): A) BꢀO bonds have a markedly different
hydrolytic stability when involving triꢀcoordinated boron atoms (at low pH, easily hydrolyzable) or
quaternarized e.g. by hydroxyl groups (at neutral or basic pH, more stable against hydrolysis)30. This
has often been exploited to release drugs at acidic pH10, 31 or to design carriers that would disassemble
under acidic condition allowing their cargo to be released10, 32. B) CꢀB bonds can be easily cleaved by
oxidants such as hydrogen peroxide33, a feature used in boron organic chemistry since the 50s34ꢀ36 and
more recently investigated as a tool to deliver payloads under oxidative conditions (e.g. sites of
inflammation)37. Recent literature offers also examples of bioconjugates sensitive to the combination
of acidic pH and reactive oxygen species (ROS), and used to image apoptotic phenomena38.
In order for the boronate/diol conjugation to have practical applications, the advantages of its stimulus
responsiveness must be accompanied by a good stability in the absence of the stimuli. Therefore, the
quantitative evaluation of boronic ester stability becomes critical; for example, its dependency on the
diol pKa is well known39. Here, we have focused on the comparative evaluation of the binding
strength (pH = 7.4) of a library of diols (Scheme 2) when binding to 3ꢀaminophenylboronic acid
(APBA) as a low molecular weight ligand, as well as a functional side group in a macromolecular
structure (HA). The diols encompass a number of different structures, including sugars, catechols with
high (HAꢀdopamine and PEGꢀdopamine) and low molecular weight and negative controls (1,3 or
methylated catechols). Some substrates were enzymatically generated in situ (dopamine, 2ꢀ
hydroxyestradiol) and/or solubilized thanks to a surfactant or a molecular host (quercetin, 2ꢀ
hydroxyestradiol).
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From the analytical point of view, the most popular assay employed in similar studies is based on the
fluorescence of Alizarin Red S (ARS, a low pKa antraquinonic catechol), whose emission markedly
increases when complexed to a bor(on)ic acid. The affinity of a diol of interest and the bor(on)ic acid
can be easily calculated through its competition with ARS and the correspondingly reduced emitted
intensity30, 40. This approach, however, suffers of two drawbacks; first, the use of the Benesi–
Hildebrand method, which for weak complexes (i.e. when the diol is a strong competitor such as a
catechol) may produce large systematic errors41. Second, fluorescence readings can be seriously
affected by scattering, which means that complex dispersions can be a challenge. In this paper, to
overcome the aforementioned limitations, we present a method based on the ARS hypsochromic
shifts: the absorption spectrum of ARS shifts from red to yellow after binding boronic acids, which
has been often used to qualitatively assess the formation of boronic esters10, 38, 40
.
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Scheme 2. Structures of diols (columns in the left and in the centre) and boronic acids (right column) used in this study.
Dopamine was used in a free aromatic diol form, but was also generated in situ via enzymatic demethylation from 3ꢀ
methoxytyramine; a second aromatic diol was prepared in situ, i.e. ꢀ2ꢀhydroxyestradiol obtained via enzymatic oxidation of
estradiol in cyclodextrin suspension. As negative controls, we have used compounds cannot bind (strongly) to boronic acids
either because of the nonꢀvicinal position (phloroglucinol) or the methylation of OH groups.
RESULTS AND DISCUSSION
Preparation of hyaluronic acid derivatives
In this study we have prepared and employed HA derivatives bearing 16ꢀ19% mol of functional
residues, and with a weight average molecular weight in the range of 200ꢀ300 kDa (from SECꢀ
MALLS measurements; see Supporting Information, Section 1SI, Additional materials and methods).
HA was functionalised with amineꢀcontaining compounds using 4ꢀ(4,6ꢀdimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ
4ꢀmethylmorpholinium chloride (DMTMM) as a water soluble activating agent (experimental
procedure provided in the Supporting Information). DMTMM was preferred to the more popular Nꢀ
(3ꢀdimethylaminopropyl)ꢀN'ꢀethylcarbodiimide (EDC): the reaction product of the latter is both
strongly hydrogen bonding and partially protonated at physiological pH, and therefore difficult to
remove from HA in a quantitative fashion. While the size of HA with low and medium molecular
weight (ꢀꢁ = 51 and 210 kDa, respectively) did not show significant decrease following
functionalization, degradation was observed when the starting material was high molecular weight
HA (see Supporting Information, Table 1SI). The choice of the degree of derivatization was a
compromise between high functionalization and retention of enzymatic degradability. Since
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mammalian hyaluronidases bind to HA utilizing its anionic carboxylic groups, with a binding region
spanning at least a hexasaccharide sequence,42 the introduction of groups bearing no negative charge
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may alter its degradability, as it happens for a number of HA derivatives such as the HYAFF
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family.43,
However, although with a slower kinetics than the pristine HA, all the derivatives
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prepared in this study substantially maintained their enzymatic degradability (see Supporting
Information, Figure 1SI A). The presence of boronic acid groups caused negligible cytotoxicity on
two cellular models with IC50>1% wt. for both L929 fibroblasts, J774.2 macrophages (see
Supporting Information, Figure 1SI B); veratrylamine still caused relatively low toxicity, with IC50
values in the range 5ꢀ10 mg/mL, but the free catechols of HAꢀDOP caused the IC50 to drop in the
proximity to 1ꢀ2 mg/mL in both cell lines.
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Boronic acid/diol equilibrium constants through a spectral shift method
The UVꢀVis spectrum of ARS largely changes upon complexation with boronic acids: in water the
wavelength of the absorption maximum of ARS shifts from 510 nm in the free state to 460 nm in the
complexed form (Figure 1A); this redꢀtoꢀyellow colour change is accompanied by the development of
fluorescence associated the boronic ester and the two phenomena have been extensively reported in
the literature.30 Both fluorescence and spectral shifts are associated to the formation of the complex,
thus either of them can provide the boronateꢀbound / free ARS molar ratio, allowing to calculate the
corresponding equilibrium constants (see experimental section for a detailed description).
For a comparison of the two techniques, we have used the complexation of ARS to two model
compounds: phenyl boronic acid (PBA) and 3ꢀaminophenyl boronic acid (APBA). PBA allows a
comparison to literature data, whereas APBA is a model for more functional boronic acids. From
fluorescence, we have obtained ꢂꢃꢄꢅꢆꢇꢈ(ꢉꢊꢋ) = 1648 ± 97 Mꢀ1, a value very similar to what obtained
by Wang30 using the same method (1300 Mꢀ1), whereas the spectral shift method provided
ꢂꢃꢌꢍꢎꢄꢏ(ꢉꢊꢋ) = 2357 ± 172 Mꢀ1. For the second equilibrium, we found ꢂꢃꢄꢅꢆꢇꢈ(ꢋꢉꢊꢋ) = 1349 ± 73
Mꢀ1 and ꢂꢃꢌꢍꢎꢄꢏ(ꢋꢉꢊꢋ) = 2465 ± 132 Mꢀ1. The spectral shift method therefore provided higher
equilibrium constants, as a graphical comparison may also suggest (Figure 1B: the empty circles reach
a plateau before the black squares). This difference may stem from the higher sensitivity of
fluorescence to factors such as aggregation and scattering; in fact, in some cases the fluorescence
signal showed a significant decrease in intensity with time (see Supporting Information, Figure 3SI),
which is possibly due to selfꢀquenching caused by the increased proximity of fluorophores in
aggregates. In order to confirm the possibly higher accuracy of the spectral shift method, we have
used a third technique to estimate the equilibrium constants for the two systems above; the values
1
obtained via H NMR (ꢂꢃꢐꢑꢒ(ꢉꢊꢋ) = 3100 Mꢀ1 and ꢂꢃꢐꢑꢒ(ꢋꢉꢊꢋ) = 3600 Mꢀ1; see Supporting
Information, additional material and methods and Figure 4SI) were indeed closer, albeit 30ꢀ40%
higher, to those obtained with the spectral shift method.
Due to its better stability, we have then employed the ARS spectral shifts for all further complexation
studies. In particular, we have used the ARS as a reporter in competitive equilibria with other diols
(Figure 1C, see Scheme 2 for the chemical structures of all diols used), estimating the binding
constants of these diols to APBA and HAꢀAPBA (Table 1). The reliability of the method was
confirmed by using three negative controls: the dimethylated catechol of veratrylamine, and the allꢀ
meta aromatic hydroxy groups of phloroglucinol did not show any appreciable competition with ARS.
In accordance to literature, the affinity for boronates of sugar diols was markedly lower than that of
aromatic diols30; this is due to a combination of lower acidity (hence negligible deprotonation at
physiological pH) and a less favourable conformation of the vicinal OH groups. The latter point
explains why mannose – a cis diol ꢀ binds more strongly than glucose, while both factors rationalize
the higher binding of ascorbic acid.
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Figure 1. A: the Vis spectra of 0.15 mM ARS in PBS show a strong (up to 50 nm) hypsochromic shift with increasing
content of the boronic acid APBA (graph on the right); the clear isosbestic point at 490 nm ensures the presence of only two
species in the equilibrium: ARS and ARSꢀAPBA. The λmax of the curves obtained as linear combinations of the spectra of
ARS and of the ARS/APBA complex (see Supporting Information, Figure 2SI) has a sigmoidal dependence on xA that can be
fitted with a Boltzmann equation, generating a ‘master curve’ that has an excellent agreement with the experimental data
(graph on the left). B: the shift in the absorption maximum and the fluorescence intensity provide comparable results, but in
some cases fluorescence data are affected by additional phenomena. For example, while the two methods provide identical
results for the complexation of ARS with phenyl boronic acid (PBA), the use of APBA (featuring an additional NH2 group)
causes a progressive increase of the fluorescence, possibly related to scattering; additionally, when using HAꢀAPBA, the
fluorescence intensity exhibited a clear time dependency (see Supporting Information, Figure 3SI). C: sketch of the
competitive equilibria used to calculate the affinity of diols to APBA (or HAꢀAPBA); considering that ꢂꢓꢔꢕ ꢖ ꢗ , from the
ꢘꢙꢚꢛ
ꢗꢜꢝꢞ
knowledge of ꢂꢟꢒꢠ, [ARS] and [ARSꢀAPBA] it is possible to obtain ꢂꢡꢎꢇꢅ (Table 1).
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Table 1. Equilibrium constants for the complexation of diols with APBA and HAꢀAPBA at pH 7.4 in
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0.1 M PBS buffer. SD are calculated over n = 3.
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Diol
K [APBA]
K [HA-APBA] a
optimal
pH b
(Mꢀ1)
(Mꢀ1)
Class
Name
ARS
pKa
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Positive
control
2550 ± 150 c
6.5
2465 ± 132
8.545
=
8.7
=
Phloroglucinol
Veratrylamine
HAꢀVER
0
0
0
0
=
0
0
=
8.646
8.947
8.8
8.9
Pyrocatechol
Dopamine
1490 ± 70
1555 ± 66
1844 ± 110
1955 ± 117
Dopamine from
3ꢀmethoxytyramine
8.947
8.9
1550 ± 73
1839 ± 110
8.648
8.649
8.7
8.8
Epinephrine
1445 ± 65
1545 ± 66
1910 ± 114
1969 ± 118
Norepinephrine
2ꢀhydroxy estradiol
from estradiol
9.7 d
9.4
955 ± 38 e
1061 ± 67 e
9.150
7.650
9.0
8.3
Pyrogallol
Quercetin
Mannose
1796 ± 78 f
1240 ± 98 g
6 ± 1
2325 ± 140 f
1312 ± 115 g
6 ± 1
12.151
12.351
4.252
10.5
10.6
6.5
Glucose
4 ± 1
4 ± 1
Ascorbic acid
6 ± 1
7 ± 1
(8.9) h
(8.9)h
HAꢀDOP
1440 ± 61
1717 ± 103
1432 ± 86
(8.9) h
(8.9)h
PEGꢀdopamine
1025 ± 42
a The equilibrium constants were measured by using HAꢀAPBA(1100).
The optimal pH was calculated according to Yan et al.53 as ꢢꢣꢇꢤꢏꢎꢥꢦꢅ ꢖ (ꢢꢂꢦ ꢧꢨꢩꢨꢪꢫꢬ ꢭꢬꢫꢮ + ꢢꢂꢦ ꢮꢫꢨꢯ)/2 . The pKa
b
(
)
(
)
of HAꢀAPBA is assumed to be in the region of 9, similarly to APBA.
The values for the complexation of ARS with HA(235)ꢀAPBA or HA(35)ꢀAPBA are respectively 2605 ± 332 and 2525 ±
c
213 Mꢀ1.
d
Predicted via the ChemAxon pKa calculator (http://www.chemaxon.com/products/calculatorꢀplugins/propertyꢀ
predictors/#pka).
e
In the presence of sulfonated βꢀcyclodextrin to solubilise the steroid. The end point of the enzymatic reaction was used to
calculate the value of the equilibrium constant, assuming complete conversion of estradiol.
f In the presence of TCEP to avoid pyrogallol oxidation.
g In the presence of Tween 20 to solubilise quercetin.
h Due to the structural similarity, we estimate the pKa of these catechols to be substantially analogous to that of dopamine.
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On the other hand, all aromatic diols and polyols exhibited a similar affinity to APBA or HAꢀAPBA,
indicating a negligible influence of the nature, size or polarity of the other parts of the molecule. The
equilibrium constants for these compounds were lower than that of ARS, which is a likely
consequence of their higher pKa (= lower chance of quaternarization of the boronate ester).
It is noticeable that the macromolecular nature of HAꢀAPBA did not appear to have any detrimental
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influence: HAꢀAPBA with
ꢀꢁ= 50 and 300 kDa had indistinguishable affinity for ARS (resp. 2605 ±
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332 and 2525 ± 213 Mꢀ1). Further, the binding of any diol was slightly but consistently higher for the
polymeric HAꢀAPBA than for the low molecular weight APBA; this slight increase is likely to stem
from an avidity effect (= local concentration of boronates being higher than the macroscopic one),
which would also explain the higher binding strength recorded for HAꢀDOP vs. PEGꢀdopamine. It is,
however, important to notice that the equilibrium constants recorded on macromolecular compounds
are average values: the binding strength of HAꢀAPBA with free boronates is likely much higher than
that of the same polymer where the same groups are almost all bound to catechols.
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Enzyme-induced bioconjugation
Catechols can be generated by a variety of enzymatic methods and we investigated whether these
processes can be used for the in situ bioconjugation of the products. As examples, we have used two
variants of cytochrome P450, CYP2D6 as a demethylase54 and CYP1A2 as a hydroxylase55, to
respectively produce dopamine from 3ꢀmethoxytyramine56 and 2ꢀhydroxyestradiol from estradiol57
(Figure 2). These two processes can act as general models for the conjugation of in situ generated
catechols, respectively with a hydrophilic and a hydrophobic nature.
The constants for the dopamine ꢀ APBA/HAꢀAPBA equilibria were measured previously and those
obtained for enzymatically generated dopamine were indistinguishable (Table 1, compare the sixth
and seventh entries), which confirmed both the efficiency in the demethylation of 3ꢀmethoxytyramine
and the robustness of the analytical method. The conversion of estradiol into a catechol was
performed in a more complex environment, since estradiol was solubilized in a sulphated
βꢀ
cyclodextrin guestꢀhost system. The constants that we recorded for the 2ꢀhydroxyestradiol ꢀ
APBA/HAꢀAPBA equilibria were significantly lower than those shown by all other catechols; we
ascribe this to both the more difficult accessibility of the catechol in the guestꢀhost complex and
above all to the electrostatic repulsion between the anionic groups on the cyclodextrin and on ARS.
Most interestingly, the spectral shift method allowed also to follow the kinetics of the enzymatic
reactions, by monitoring the ARS absorbance as a function of time (Figure 2A and 2B). By knowing
the equilibrium constant of the catechol and under the assumption that the complexation is
considerably quicker than the enzymatic conversions, it was then possible to estimate the kinetic
parameters for both reactions (Table 2). For the production of dopamine, a direct comparison of our
results with literature data is hardly possible, because this reaction has never been studied
quantitatively; our data are broadly comparable to examples of CYP2D6ꢀmediated demethylation of
oꢀmethylphenols, which however are largely variable (see Supporting information Table 2SI) possibly
as a result of the large polymorphism of this enzyme58. The only noteworthy point is that our KM
values are rather high, suggesting a lower affinity of the enzyme for a substrate with an OH vicinal to
the target methoxy group. A more quantitative comparison would appear to be possible for the 2ꢀ
hydroxylation of estradiol, for which KM and the enzymatic clearance appear to be respectively in the
proximity of 20 µM and in the range 0.018ꢀ0.06 µL/(pmol P450*min) (see Supporting information
Table 2SI; please note that the reactions are strongly NADPHꢀdependent and the KM vary
considerably depending on the presence of the latter, see Supporting Information, Figure 6SI). We
have observed a very comparable clearance value but a dramatically lower KM; this can be attributed
to the charge and steric hindrance of the estradiol/cyclodextrin complex, but literature values often
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refer to oversaturated solutions (up to 200 µM with estradiol water solubility being 20 µM59) where
phase separation may lead to an overestimated affinity of CYP1A2 for estradiol.
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Figure 2. The oneꢀpot conjugation through in situ enzymatic generation of catechols and binding to boronic derivatives can
be monitored using ARS as an efficient reporter system that allows to extract information about enzymatic kinetics. The
MichaelisꢀMenten plots in A and B are respectively obtained through the P450ꢀmediated demethylation of 3ꢀ
methoxytyramine to dopamine (A) and the hydroxylation of estradiol (B).
Table 2. Kinetic parameters for the P4502D6ꢀmediated demethylation of 3ꢀmethoxytyramine and for
the P4501A2ꢀmediated 2ꢀhydroxylation of estradiol. (HAꢀ)APBA complexation used as a reporter.
Hydrolase activity
(CLint) a
vmax
KM
Substrate
3ꢀmethoxytyramine
Estradiol
Reporter
(pmol/(pmol
P450*min))
(ꢀM)
(ꢀL/(pmol P450/min))
APBA
HAꢀAPBA
APBA
281 ± 8
285 ± 6
49 ± 4
54 ± 3
65 ± 13
0.18
0.19
3800 ± 650
0.017
HAꢀAPBA 3570 ± 990
56 ± 11
0.015
a CLint (intrinsic clearance = vmax/KM) is a common measure of the enzymatic activity in a cell.
Release behaviour and double responsiveness of boronate/catechol conjugates
Despite the relatively low values of the equilibrium constant at neutral pH, the complexation strength
of aliphatic diols with boronates is still sufficiently high to allow e.g. the determination of a variety of
saccharidic structures60, 61. As reported above, the complexation of boronate/catechol couples is orders
of magnitude more stable than that of aliphatic diols, therefore in principle amenable to
bioconjugation; nevertheless, the equilibrium constants are not very large (around 103 Mꢀ1), and they
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may not be able to guarantee stability against dilution, which is a common problem in the design of
injectable pharmaceutical formulations. In this part of the work, we have focused on improving the
stability of the structure through the use of a stoichiometric excess of boronates on the HA structure,
i.e. by locally increasing the carrier avidity. Please note that, due to the higher local concentration of
free boronates, the resulting binding of the catechols is considerably stronger than what could be
assumed on the basis of the equilibrium constants presented in Table 1; while the latter were obtained
using variable boronate/catechol ratios and therefore present an average binding strength, an excess of
free boronates is expected to increase is significantly (see e.g. the difference between experimental
data and theoretical values in Figure 3A).
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The boronate/diol complexes are well known to reverse by acidification30, although in a fashion
dependent on the diol pKa. Here, we have placed the solution of an HAꢀAPBA/ARS conjugate at pH
7.4, 7.0, 6.0, 5.0 in dialysis bags, measuring the release of ARS in a 20ꢀfold larger volume of fluid
(thereby at least partially evaluating the effect of dilution) as a function of time. The equilibrium is
apparently reached after 24 hours (Figure 3A), showing that at pH 7 about 30% of the diol was
released, therefore showing a relatively good stability of the complex; on the other hand, at acidic pH
the decomplexation was quantitative; importantly, the release kinetics shows an initial burst followed
by a slower release, but this profile is likely much affected by the transfer through the dialysis
membrane, and the actual release may be considerably more rapid. Importantly, the overall trend
observed was in agreement with the data already reported30 underline once more the little effect of the
HA on the pHꢀinduced release phenomenon.
The oxidative cleavability of the boronate esters is based on cleavage of sp2 carbonꢀboron bond37, and
correspondingly increasing amounts of 3ꢀaminophenol were produced when a APBA/ARS adduct was
incubated with increasing amounts of H2O2 (Figure 3B). It is noticeable that, although the oxidation of
free catechols to ortoꢀquinones is also possible, no trace of ARS quinone were found, thus suggesting
that catechols may be released as oxidationꢀprotected boric acid complexes. This is an important point
to ensure the stability of in principle oxidationꢀsensitive drugs in an oxidizing (e.g. inflamed)
biological environment.
Having proven the principle of the oxidative responsiveness, it is also important to note that this was
not perfectly stoichiometric, and a certain excess of oxidant appeared necessary for a quantitative
conversion of the complexes. For example, stoichiometric equivalence of H2O2 only corresponded to a
35% oxidation of the APBA/ARS, and higher excesses of oxidants were necessary for HAꢀ
APBA/ARS conjugates (50% release with 10 equivalents). The apparently lower reactivity of the
macromolecular complexes may actually stem from the presence of an excess of nonꢀcomplexed
boronic acids (to boost the carrier avidity) that are likely easier to oxidize. However, this event
corresponds to the release of boric acid, not of the drug.
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Figure 3. A: An HAꢀAPBA/ARS conjugate was prepared with 8.5 µmol of boronic acid and 1.5 µmol of ARS and was
incubated at 37 °C in buffers at different pH values (7.4, 7.0, 6.0 and 5.0) in a dialysis bag. The release of free ARS in the
dialysate was measured over 24 hours and expressed as relative to the overall amount. On the basis of the equilibrium
constant presented in Table 1, under the experimental conditions used, >50% of ARS should be released at neutral pH; the
fact that only about a half of it was actually released is attributed to the higher stability arising from the avidity of a structure
with a large excess of uncomplexed boronates. B: The APBA/ARS conjugate was incubated for 2 hours at 37 °C in buffers
0.1 M PBS buffer at pH 7.4 with different amount of H2O2 and then Na2SO3 was added to quench the unreacted H2O2 and
the samples were analysed through HPLC to measure the amount of ARS, APBA and 3ꢀaminophenol (3ꢀAP) produced. It is
noteworthy that the ARS complexes dissociate during HPLC analysis; therefore we analysed separately ARS, APBA and 3ꢀ
PA. Further, we did not discriminate between the ARS released in its free form or as ester with boric acid. C: The HAꢀ
APBA/ARS conjugate was incubated for 2 hours at 37 °C in buffers 0.1 M PBS buffer at pH 7.4 with different amount of
H2O2 and then centrifuged in centrifugal filters with nominal molecular weight limit of 3 kDa to recover the unbound ARS.
Again, we did not discriminate between the ARS released in its free form or as ester with boric acid.
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CONCLUSION
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8
Here we have presented a successful proof of principle for the use of boronic/diol complexation for
the purpose of reversible and responsive bioconjugation. In this demonstration, the use of HA as a
model platform offers a plethora of benefits, including targeting.
We have thus compared the performance of a boronic acidꢀcontaining HA derivative to that of the low
molecular weight APBA in the complexation to a library of diols. Instead of detrimentally affecting
the equilibrium constants (e.g. because of steric hindrance or electrostatic repulsion between anionic
boronic esters), the use of HAꢀAPBA slightly increased (5ꢀ15%) the average binding strength, which
we attributed to the avidity of the polymeric structure.
It is noteworthy that we have adopted a modified analytical method for the evaluation of boronic/diols
equilibria, which uses ARS as an absorbance reporter. In comparison to the more commonly
fluorescence tests (based on ARS emission), this method is specifically advantageous to study
concentrated and possibly heterogeneous samples, allowing for example to monitor this “click”
reaction in a selfꢀemulsifying system (Tween 20 + quercetin) and during enzymatic kinetics, also
performed on solubilized active principles (cyclodextrin + estradiol). Using this absorbance assay, we
have also for the first time demonstrated that CYP2D6 is able to demethylate the monomethyl ether of
dopamine (3ꢀmethoxytyramine). This finding could be important considering that one of the major
drawbacks of Parkinson’s disease therapies based on the use of catecholamine drugs or proꢀdrugs (e.g.
LꢀDOPA) is the inactivation of the exogenous catecholamines through methylation (operated by
catechol Oꢀmethyltransferase, COMT)62.
In the conclusive part of the study, we have then demonstrated that the boronic acidꢀbearing HA
derivatives, allowed the responsive release of catechols in acidic or oxidative environment. Indirect
evidence suggests that in the latter case the released catechols are not further oxidized to quinones
possibly because they are release as boric acid complexes.
Since a slightly acidic pH and the presence of ROS are often associated to pathological states, we
believe that this kind of HA bioconjugation could provide the means for a more efficient delivery due
the combination of receptor (CD44) targeting and triggered (pH or H2O2) release.
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EXPERIMENTAL PROCEDURES
The list of all reagents and the description of the synthetic procedures for all HA derivatives are
reported in the Supporting Information, additional materials and methods.
If not otherwise stated, all solutions were prepared in 0.1 M PBS obtained by dissolving sodium
dihydrogen orthophosphate dihydrate, disodium hydrogen orthophosphate dihydrate and sodium
chloride, supplied by BDH (U.K.), in concentrations respectively of 2.3 g/L, 11.8 g/L and 9 g/L in
water purified in a MilliꢀQ system (Millipore, U.K.).
Study of boronate-diol equilibria
We have employed a BioTek Synergy 2 multiꢀmode microplate reader to record absorbance spectra
(in the range λ = 400ꢀ800 nm) and fluorescence readings (filters at λexc = 485±20 nm and λem
=
620±40 nm); temperature was kept at 25 °C, except for the spectra of enzymatic reactions which were
recorded at 37 °C. All the spectra were corrected by subtracting the scattering component of the
buffer solution using appropriate blanks.
In this analysis we have not differentiated between the equilibria leading to the formation of the
trigonal or the tetragonal form of the diolꢀboronic esters. The binding constants provided for the
diolꢀboronic equilibria are overall affinities at pH 7.4.
In this discussion we will identify ARS as A, the boronic acid partner as B, any competitive diol as D,
the complex between A and B as AB and the complex between B and D as DB.
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1) Determination of ARS/boronic acids binding constants through fluorescence
3
General conditions. The experimental design developed by Springsteen et al.30 was adapted to be
performed in 96 well plates. Briefly, 125 µL ARS solution (1.8ꢁ10ꢀ5 M) were titrated with an equal
volume a solution of PBA or APBA with concentrations ranging between 3.6ꢁ10ꢀ3 and 1.8ꢁ10ꢀ4 M. The
plates were incubated 30 min in the plate reader at 25 °C and then the fluorescence intensities were
recorded (λexc = 485±20 nm and λem = 620±40 nm).
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8
9
Analytical method used for the calculation of binding constants. The binding of ARS to PBA and
APBA can be studied by recording the increase in fluorescence intensity of an ARS solution upon
addition of the boronic acids, a method pioneered by Wang40. Fluorescence data were fitted according
to the BenesiꢀHildebrand method which can be used to measure the association constant (Keq) of a
reaction when two species are in equilibrium with their oneꢀtoꢀone complex63. The mathematical
approach is based on the assumption that when either one of the reactants is present in molar amount
higher than 10 equivalents over the other reactant, its concentration can be considered constant. Under
these conditions a set of data relating the recorded changes in fluorescence intensity (ꢀIf) at different
initial concentrations of the boronic acid containing compound (CB) can be fitted according to the
following equation:
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ꢃ
ꢃ
ꢖ (∆ꢲꢢꢳꢴꢟꢂꢓꢵ)ꢶꢃ
∙
+ (∆ꢲꢢꢳꢴꢟ)ꢶꢃ
ꢷ
ꢸ
(1)
∆ꢰ
ꢱ
Where CA is the total concentration of ARS, and ꢀkp0 is a constant derived from the quantum yield
and instrumental parameters. Thus by plotting 1/ꢀIf vs 1/CB, it is possible to obtain Keq dividing the
intercept by the slope of the fitted line.
2) Determination of ARS/boronic acids binding constants through hypsochromic shifts
General conditions. For binding experiments, 125 µL of a solution of APBA or PBA at a
concentration ranging from 0.00 to 4.05 mM (at least 23 different concentrations were prepared) were
mixed in each well of a 96 well plate with 125 µL of a solution 0.30 mM of ARS (final ARS
concentration = 0.15 mM, total volume = 250 ꢀL/well). The plate was incubated 30 min in the plate
reader at 25 °C and then the absorbance readings were recorded between 400 and 800 nm (Figure 1A
provides a picture of a typical plate during the assay).
Analytical methods used for the calculation of binding constants. When using boronic acids such as
APBA or PBA (B) that lack a significant absorption in the visible or near UV spectral region, the only
absorbing species are ARS (A) and the ARSꢀboronic ester (AB). The spectra of ARS alone and of its
complex, obtained from ARS in the presence of a large excess of boronic acid containing compound,
can be accurately fitted with Gaussian models (see Supporting Information, Figure 2SIA), it is
possible to recreate the absorption spectrum of any mixture of ARS with its boronic ester, expressing
it as a linear combination of the spectra of the two pure substances (see Supporting Information,
Figure 2SIB):
ꢽꢾ(ꢿꢽꢿ
)
ꢽꢾ(ꢿꢽꢿ
)
ꢾ
ꣀꣁꣂꣃ
ꣀꣁꣂꢾ
꣄
ꢾ
ꢋꢧꢹ ꢖ ꢺꢟꢴꢟꢻꢃꢼ[
ꢾ + (1 − ꢺꢟ)ꢴꢟꢻꣅꢼ[
(2)
]
]
꣄
ꣃ
where the absorbance Abs depends on the molar concentrations of the two products (expressed as the
molar fraction xA times the total concentration of ARS, (CA), and on parameters obtained from the
spectra of the two pure products: λmax1 and λmax2, ε1 and ε2, ω1 and ω2, which are, respectively, the
wavelengths of the maxima of the spectra of ARS and of its boronic ester, the corresponding
extinction coefficients and parameters related to the band width. By plotting the wavelength of the
absorbance maxima of these calculated spectra vs xA one obtains a sigmoidal graph (Figure 2, right).
This graph allows one to relate the location of the maximum absorption for a given ARS/boronated
compound mixture to the fraction of free ARS in that mixture (xA). By knowing xA as a function of the
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constant of the equilibrium between ARS and APBA or PBA (K1) and of the initial concentrations of
the reactants, CA and CB:
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7
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9
[ꢟ꣆]
[ꢟ꣆]
ꢂꢃ ꢖ [ꢟ [꣆] ꢖ (ꢷ ꢶ ꢟ꣆ )(ꢷ ꢶ ꢟ꣆ )
(3)
]
[
]
[
ꢸ
]
ꢜ
ꣃ
ꣃ
ꢾ
ꢷ ꣇ꢷ ꣇
ꢶ (ꢷ ꣇ꢷ ꣇ ) ꢶ꣊ꢷ ꢷ
꣉
ꢜ ꢸ ꢜ ꢸ
꣈
ꢜ
ꢸ
꣈
ꣃ
ꣃ
[ꢋꢊ] ꢖ
(4)
(5)
ꣅ
ꣃ
ꣃ
ꢾ
ꢷ ꣇ꢷ ꣇
ꢶ
(ꢷ ꣇ꢷ ꣇ ) ꢶ꣊ꢷ ꢷ
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꣉
ꢜ
ꢸ
ꢜ
ꢸ
ꢜ
ꢸ
꣈
꣈
ꣃ
ꢺꢟ ꢖ 1 − [ ] ꢖ 1 −
ꢟ꣆
ꣃ
ꢷ
ꢜ
ꣅꢷ
ꢜ
Using a fixed concentration CA (0.15 mM) and varying CB (between 0.00 and 2.03 mM) it is possible
to obtain a set of xA values, where xA = f(CB). Equation 5 can be edited as user defined function in a
data analysis software (Origin 8.5.1), allowing to calculate K1 as a fittable parameter.
3) Determination of diol/boronic acid binding constants through competitive binding with ARS
General conditions. For competitive binding experiments in each well of a 96 well plate 50
ꢀL of a
0.75 mM ARS solution and 50 L of a 0.75 mM APBA solution were mixed; the colour of the
ꢀ
solution changed from red to orange. Separately, diol solutions with concentrations ranging between
0.13 and 250.00 mM were prepared (corresponding to ABPA/diol molar ratios ranging from 1:0.5 to
1:1000). 150 µL of each diol solution (final ARS and APBA concentration = 0.15 mM, total volume =
250 ꢀL/well) were added, producing a clear orange to red chromatic change at high ABPA/diol molar
ratios. The absorbance readings were then recorded as previously described.
The general procedure was modified for the following cases:
ꢀ Reactions involving veratrylamine and phloroglucinol: due to their limited solubility in water, the
highest APBA/diol molar ratio was 1:100.
ꢀ Reactions involving pyrogallol: owing to the quick oxidation of this electronꢀrich polyol, TCEP
was added to the diol stock solution in a molar ratio pyrogallol/TCEP 1:1.5. Due to acidity of
TCEP, a few drops of 1 M NaOH were added to bring the pH back to 7.4.
ꢀ Reaction involving ascorbic acid: due to the acidic nature of this diol, the pH of the final solution
in 0.1 M PBS was raised to 7.4 by adding few drops of 1 M NaOH.
ꢀ Reaction involving quercetin: owing to the very poor solubility of quercetin in water environment,
Tween 20 (a surfactant widely employed in bioassays and pharmaceutical formulations) was used.
For quercetin concentrations up to 1 mM a 1:1 Tween 20/quercetin molar ratio was sufficient to
completely solubilise it in 0.1 M PBS buffer, in fact quercetin UVꢀVis absorbance reached a
plateau and did not increase with larger amounts of surfactant. The highest concentration of Tween
20 showed to be limited by its ability to hamper the binding reaction: a maximum of 8% w/w of
Tween 20 can be used without affecting the reaction kinetics (data not shown).
ꢀ Reactions involving polymeric species: The amount of polymer was calculated on the basis of its
number average degree of functionalization. HA derivatives were dissolved and kept in solution
for 12 h prior to the addition of any reagent. Finally, TCEP was added to the solutions when
polymers functionalised with dopamine were involved (dopamine/TCEP molar ratio = 1:1.5) to
prevent oxidation.
Calculation of binding constants. The gradual addition of a diol (D) to an ARS boronic ester
determines an increasing batochromic shift due to the production of free ARS. The corresponding
competitive equilibrium AB + D
DB + A is governed by a constant K which can be expressed as
the ratio of the two equilibrium constants for the formation of individual boronic esters, AB and DB,
but can also be expressed as a function of the concentration of free ARS as the only variable (see
equations 6 and 7).
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]
[
[
]
[
]
꣋
]
ꢂꢓꢔꢕ ꢖ [[
ꢂꢓꢔꢕ ꢖ [[
ꢖ
ꢖ
∙
] ꢖ ꢗ
(6)
(7)
ꢟ [꣋꣆]
ꢟ [꣆]
꣋꣆
ꢘꢙꢚꢛ
]
[
][
ꢟ꣆ [꣋]
[ꢟ꣆]
]
꣆
[
ꢗ
ꢜꢝꢞ
]
[ ]
ꢟ (ꢷ ꢶꢷ ꣇[ꢟ])
ꢟ [꣋꣆]
ꢟ (ꢷ ꢶ ꢟ꣆ ) ꢖ (
ꢸ
ꢸ
ꢜ
]
(
[
])
[
])
[ ]
ꢜ
ꢟ꣆ [꣋]
ꢷ ꢶ ꢟ [꣋]
ꢷ ꢶ ꢟ (ꢷ ꢶꢷ ꣇(ꢷ ꢶ ꢟ )
ꢜ
ꢜ
꣌
ꢸ
For each given amount of diol, one obtains a λmax value from which it is then possible to calculate the
fraction of free ARS ([A] = xAꢁCA), as shown in the previous section. From each CD value is possible
9
to obtain ꢂꢓꢔꢕ and finally, its value multiplied by ꢂꢟꢒꢠ is the equilibrium constant of interest, ꢂꢡꢎꢇꢅ.
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The values of ꢂꢡꢎꢇꢅ were averaged through a range of diol concentrations corresponding to
ABPA/diol molar ratio from 1:0.5 to 1:1000.
4) Enzymatic Reactions
°
General conditions. Typical experiments were performed in 96 well plates at 37 C sealed with the
help of PCR plate adhesive seals to prevent evaporation. The plates were prepared by mixing ARS
and APBA as previously (50
ꢀ
L of a 0.75 mM ARS solution and 50
ꢀL of a 0.75 mM APBA
solution). Then 100 L of diol were added followed by 50
ꢀ
ꢀ
L of the enzyme working solution. The
diol and the enzyme working solutions were prepared as follows:
ꢂ Demethylation of 3ꢂmethoxytyramine: the cytochrome P450 variant 2D6 can remove the methyl
group from 3ꢀmethoxytyramine producing dopamine. 3ꢀmethoxytyramine was solubilised in PBS at
concentrations ranging from 0.19 mM to 1.22 mM (final APBA/3ꢀmethoxytyramine molar ratio of
1:0.5 to 1:3.2). The solution of CYP2D6 was prepared by diluting 1:50 a 500 pmol/mL solution of
enzyme with a 8.3 mg/mL (10 mM on anhydrous basis) solution of NADPH in 0.1 M PBS.
- Hydroxylation of estradiol: estradiol can bind to boronic acid only upon the introduction of a second
alcoholic function; this reaction can be carried out by cytochrome P450 variant 1A2. Estradiol (0.9
mg/mL, 3.38 mM) was solubilised in PBS by the addition of βꢀcyclodextrin sulfobutylether (7.3
mg/mL, 1:1 molar ratio). This solution was diluted to obtain diol concentrations ranging from 0.08 to
3.38 mM (final APBA/estradiol molar ratio of 1:0.1 to 1:4.5). The CYP1A2 solution was prepared by
diluting 1:25 a 1000 pmol/mL solution of enzyme with a 10.0 mg/mL (12 mM on anhydrous basis)
solution of NADPH in 0.1 M PBS.
Analytical methods used for the calculation of binding constants. All enzymatic tests were based on
the in situ production of a diol in the presence of the ARS/APBA complex. With increasing
production of the catechol, the ARS UVꢀVis band undergoes a timeꢀ and concentrationꢀdependent
batochromic shift. The dependence on time provides information about the kinetics of the enzymatic
conversion; the dependence on the concentration of the precursor provides information about the
binding constant of the diol. In this part of the study we have always assumed that the enzymatic
conversion is the rateꢀdetermining step of the test, i.e. the competitive equilibrium is established in an
‘instantaneous’ fashion as soon as new diol is produced. This assumption is reasonable, because the
diolꢀboronic equilibria are established in a matter of seconds, while tenths of minutes are required to
obtain quantitative yields in diols. We have also assumed that complete substrate conversion was
obtained when the batochromic shifts reached their asymptotic values; in this way it is possible to
replace CD in equation 7 with the initial substrate concentration ([S0]), therefore allowing the
calculation of the diolꢀboronic equilibrium constant ꢂꢡꢎꢇꢅ
It is then possible (see Supporting Information, Section 3SI Additional information about enzymatic
reaction experiments) to use ꢡꢎꢇꢅto express the substrate concentration ([S]) as a function of the
.
ꢂ
molar fraction of free ARS (xA), which can be calculated from the batochromic shift; from the time
dependence of [S] it is possible to calculate the initial rate of the enzymatic reaction. In order to
reduce the experimental error, this was done by fitting the timeꢀdependent data as an exponential
decay ( ꣍ ꢖ [꣍ꢳ] ∙ ꢼꢶ꣎ꢏ).The initial reaction rate v was then plotted vs [S0] and fitted using the usual
[ ]
MichaelisꢀMenten relationship ꣏ ꢖ ꣐
[ꢠ]
ꣀꣁꣂ
ꢗ
꣑
꣇[ꢠ]
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pH-induced release
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Four 1 mL Spectra/Por® FloatꢀAꢀLyzer® (MWCO = 10 kDa) were filled with 1 mL of a solution
containing 20 mg of HAꢀAPBA (corresponding to 8.5 µmol of boronic acid) and 0.5 mg of ARS (1.5
µmol) in 0.1 M phosphate buffer at pH 6.0, 7.0, 7.4 and 0.1 M acetate buffer at pH 5.0 respectively.
The FloatꢀAꢀLyzer® were then immersed in 19 mL of the same buffers and incubated at 37 °C. 150
µL aliquots were collected at different time points (n = 3 for each sample) and the fluorescence
intensities were recorded in a 96 well plate (filters at λexc = 485±20 nm and λem = 620±40 nm) and
compared with a calibration curve of ARS in the same buffer to measure the amount of ARS released.
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H2O2-induced release
A) Oxidation of APBA/ARS. 11 wells of a 96 well plate were filled each with 50 µL of a 2.9 mM
solution of ARS followed by 50 µL of a 2.9 mM solution of APBA; after 1 min 50 µL of hydrogen
peroxide which concentrations ranging from 30 µM to 150 mM were added and the plate was
incubated at 37 °C for 2 hours. Finally, 50 µL of a 0.2 M Na2SO3 were added to quench the unreacted
H2O2 and the samples were analysed through HPLC. All the solutions were prepared in 0.1 M
phosphate buffer at pH 7.4. Samples were analyzed via reverse phase HPLC on a Agilent 1100 series
HPLC equipped with analytical (5 ꢂm particle size, 4.6 x 150 mm) Zorbax Eclipse XDBꢀC18 column
and a Laserchrom S3210 UV/VIS detector set at 254 nm. The analysis was performed at 1 mg/mL in a
mixed eluent composed of 10 mM phosphate buffer at pH 7.4 and acetonitrile with the following
gradients: from 10% to 30% acetonitrile, min 0ꢀ6 min; from 30% to 80% acetonitrile, min 6ꢀ12; from
80% to 10% acetonitrile, min 12 to 15.
B) Oxidation of HAꢀAPBA/ARS 300 µL of a 5.0 mM ARS solution (1.5 µmol) were added to 1.7 mL
of a 10 mg/mL solution of HAꢀAPBA (corresponding to 7.2 µmol of boronic acid), both solutions in
PBS at pH 7.4. 200 µL aliquots of the mixture were placed in Amicon Ultraꢀ0.5 Centrifugal Filters
with 3 kDa MWCO (Millipore) followed by 200 µL of 0.0073, 0.073, 0.73 and 7.3 mM H2O2 (0.01,
0.1, 1 and 10 equivalents respect to ARS or 0.0025, 0.25, 0.25 and 2.5 equivalents respect to
boronates) in PBS. The samples were incubated for 2 hours at 37 °C, then free ARS was recovered via
centrifugation (13,000 rpm for 30 min) and analysed via the above described HPLC method.
ASSOCIATED CONTENT
Supporting Information is provided as a separate document
AUTHOR INFORMATION
Corresponding Author
*For N.T: Eꢀmail: Nicola.tirelli@manchester.ac.uk
ACKNOWLEDGMENTS
The authors acknowledge financial support from the EU FP7 project UniVax (grant number 601738)
and from the Marie Curie IAPP Replixcel (grant number 251420), and from the
AstraZeneca/University of Manchester coꢀfunding of the NorthWest Centre of Advanced Drug
Delivery (NoWCADD). The authors are indebted to Novozymes for the donation of all HA samples
used in this study and want also to thank Dr. Elita Montanari for the helpful discussions.
ABBREVIATIONS
ARS, alizarin red S; APBA, 3ꢀaminophenylboronic acid hydrochloride; PBA, phenylboronic acid;
DOP, dopamine hydrochloride; NADPH, βꢀnicotinamide adenine dinucleotide 2′ꢀphosphate reduced
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tetrasodium salt hydrate; CYP2D6, human cytochrome P450 2D6 yeast reductase; CYP1A2,
recombinant human cytochrome P450 1A2; HA, hyaluronic acid; PEG, poly(ethylene glycol); PEGꢀ
DOP, monomethoxy poly(ethylene glycol); RHAMM, hyaluronanꢀmediated motility receptor,
GlcNAc, Nꢀacetyl glucosamine; GlcA, glucuronic acid; ROS, reactive oxygen species.
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