Design, synthesis and structure of a frustrated benzoxaborole and its applications in the complexation of amines, amino acids, and protein modification

Article abstract of DOI:10.1039/d0ob00572j

This study describes the design and synthesis of arylboronic acid 2, the first example of a permanently open "frustrated" benzoxaborole, along with an exploration of its application in bioconjugation. An efficient and high yielding seven-step synthesis was optimized. NMR experiments confirmed that compound 2 exists in the open ortho-hydroxyalkyl arylboronic acid structure 2-I, a form that is effectively prevented to undergo a dehydrative cyclization as a result of unfavorable geometry. Compound 2-I conjugates effectively with amines to form stable hemiaminal ether structures, including a highly effective reaction with lysozyme. Complexation with cysteine induces an open structure containing a free hydroxymethyl arm, with the amino and thiol groups reacting preferentially with the formyl group to form a N,S-acetal.

Full text of DOI:10.1039/d0ob00572j

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DOI: 10.1039/D0OB00572J
ARTICLE
Design, Synthesis and Structure of a Frustrated Benzoxaborole
and its Applications in the Complexation of Amines, Amino Acids,
and Protein Modification
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Jasmine Bhangu,a Randy M. Whittal,b Dennis G. Hall,*a
This study describes the design and synthesis of arylboronic acid 2, the first example of a permanently open "frustrated"
benzoxaborole, along with an exploration of its application in bioconjugation. An efficient and high yielding seven-step
synthesis was optimized. NMR experiments confirmed that compound 2 exists in the open ortho-hydroxyalkyl arylboronic
acid structure 2-I, a form that is effectively prevented to undergo a dehydrative cyclization as a result of unfavorable
geometry. Compound 2-I conjugates effectively with amines to form stable hemiaminal structures, including a highly
effective and irreversible reaction with lysozyme. Complexation with cysteine induces an open structure containing a free
hydroxymethyl arm, with the amino and thiol groups reacting preferentially with the formyl group to form a N,S-acetal.
Introduction
Boronic acids and their cyclic hemiboronic acid derivatives
have recently emerged as new and unconventional
chemotypes in drug discovery efforts.1 These compounds offer
desirable properties as mild Lewis acids, with the unique ability
to undergo reversible exchange of their boron-hydroxy bonds
with alcohols and carboxylate groups on target biomolecules.
In particular, benzoxaboroles are a class of stable cyclic 5-
membered hemiboronic acids that has received significant
attention owing to the recent approval of two new drugs,
tavaborole and crisaborole (Figure 1a), which are marketed for
the treatment of onychomycosis and psoriasis, respectively.2,3
Other derivatives have shown potential towards a range of
health issues,4 along with significant success and further
promise in other applications like carbohydrate recognition,
catalysis, and as biomaterials.4,5 To fully exploit the potential of
benzoxaboroles, complementary studies must be conducted to
understand their structural and physicochemical properties. In
this regard, our group recently demonstrated, through NMR
spectroscopic evidence, that benzoxaborole exists largely in its
closed form in aqueous media, yet it can undergo rapid and
reversible hydrolysis to the open form (Figure 1b).6
a.Department of Chemistry, University of Alberta, 4-010 Centennial Centre for
Interdisciplinary Science, Edmonton, Alberta, Canada T6G 2G2.
b.Mass Spectrometry Laboratory, Department of Chemistry, University of Alberta.
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Fig. 1. Benzoxaborole derivatives (a); and various, closed and open hydrated forms (b)-
(d).
This sort of new and important knowledge can help provide a
better understanding of the pharmacokinetic and
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pharmacodynamic behaviour of benzoxaborole drugs. benzoxaborole ring) needs to be raised significantly.
View ArticIletOwnlianse
DOI: 10.1039/D0OB00572J
Remarkably, there appears to be no example of a permanently anticipated that such a frustrated benzoxaborole could be
open benzoxaborole derivative. Lulinski and Serwatowski designed by imposing ring strain into the putative closed form.
studied the ortho-phtalaldehyde derivative
found to exist predominantly in its closed form
organic solvent, with 1-III as a minor open form (Figure 1c).7 as a result of its incorporation into a strained 5-membered
The only reported instance of a temporary open member of hemiacetal. Presumably, the secondary hydroxy group of
this general class of boron heterocycles is an aliphatic -amino would be incapable of undergoing a dehydrative cyclization to
oxaborole bound to the NS3 serine protease (Figure 1d).8 Thus the corresponding benzoxaborole,
II, a tricyclic fused ring
1
-
I, which was In line with this stratagem, the
-hydroxyalkyl unit of
1
-
II in wet arylboronic acid is expected to be geometrically restricted
2-I
2-I

2
-
it remains unknown what unique properties a predominantly system rendered highly unfavorable due to the extreme angle
and permanently open benzoxaborole would possess, and how strain imposed on the fused arene unit. Molecular modeling
these properties can guide the development of new (AM1) of both forms
applications in chemical biology and catalysis. Herein, we this proposal. Even when normalized for bond enthalpy due to
present the design and synthesis of compound the loss or addition of water, compared to benzoxaborole the
geometrically restricted ortho-hydroxyalkyl arylboronic acid closed form
II was found to be significantly higher in energy.§
that is incapable of undergoing a dehydrative cyclization to Indeed, the energy-minimized bowl-shaped structure of II
2-I and 2-II provides strong support for
2
, a
2
-
2
-
afford its closed form (Figure 2). Being prevented from existing shows significant distortion of the arene ring with the central
in the closed form of benzoxaboroles normally preferred, arene carbon laying out of plane with bond angles that deviate
ortho-hydroxyalkyl boronic acid
"frustrated benzoxaborole". Moreover, it was recognized that such an sp2 carbon (Figure 2b). Also presaging of the
, a hemiacetal, may interconvert with its hydroxy-aldehyde unlikelihood of form II is the lack of precedence for most of
form 2-IV, leading to an efficient complexation of biological the analogous C–C isosteres, such as 4a which are
amines and amino alcohols to give adducts
(Figure 2a). presumably too unstable to prepare or even exist.9
Herein, the structure of compound is investigated along with
its potential applications in the complexation of amines and The synthesis of target compound
2 thus can be viewed as a significantly (up to 13°) from the ideal planar configuration of
2
-
I
2-
-f,
3
2
2
from commercial material
involved the preparation of the known dibromide with
small modifications to the published procedures.10 From
formation of aldehyde was achieved in high yield using a
standard lithiation/formylation reaction. A classical Miyaura
amino acids, and protein modification.
5
7
7
,
(a)
8
HO
HO
O
HO
OH
B
B
O
NH
O
B
OH
O
borylation of
intermediate
8
9
installed the requisite boronyl group leading to
in a moderate yield. The formyl group was then
HO
NH2
O
X
reduced to the corresponding alcohol 10 without difficulties.
Attempts to transform this intermediate directly into by a
– H2O
2-I (open)
2-II (closed)
3
2
concomitant acidic hydrolysis of both the acetal and the
boronic ester led to issues of product isolation. Instead, the
conversion of boronate 10 into the corresponding
trifluoroborate salt is effectively achieved using aqueous KHF2,
releasing HF in the reaction medium, which in turn cleaves the
acetal in situ to yield 11 as a readily isolable precipitate. The
final hydrolytic step in this synthesis proved to be tricky as it
– H2O
HO
HO
OH
B
O
O
B
OH
OH
2-III (hydroxy)
H
OH
2-IV (hydroxyaldehyde)
was predicted that the target compound
2 would be highly
water-soluble and therefore difficult to isolate and purify. This
problem was easily overcome by using a strong, solid
fluorophile, in this case silica gel, a procedure developed by
Molander and co-workers to extract the fluorides from boron
without any contamination of the aqueous phase with salts.11
However, since silica gel is partially soluble in water, the clean
product from the aqueous phase was contaminated with a
small amount of silica gel. To fix this issue, the reaction is
instead diluted with acetone, then silica gel is easily separated
by filtration and the filtrate concentrated in vacuo. For
purification, the white crude solid was dissolved in water, and
any intractable organic impurities are separated by filtration.
X
(c)
(b)
O
Y
Y
4e Y = O
4f Y = CH2
4a X = H2, Y = O
4b X = O, Y = O
4c X = H2, Y = CH2
4d X = O, Y = CH2
Fig. 2. (a) Design of a permanently open, "frustrated" benzoxaborole compound, 2-I; (b)
energy-minimized model of the putative closed form 2-II showing severe distortion of
the arene unit; (c) corresponding carbon isosteres of 2-II.
Results and Discussion
Design and Synthesis
Product
2
is obtained in good yield with only minor impurities
1
(<5% according to H NMR) after a simple evaporation of the
aqueous phase.
To favor the open form of an ortho-hydroxyalkyl arylboronic
acid, the intrinsic energy of the closed form (i.e.,
a
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Fig. 3. Diagnostic NMR signals (DMSO-d6, with one drop of H2O) for benzoxaborole,
derivatives 12-13, and target compound 2.
1
The H chemical shift of the protons on the B(OH) moiety also
act as a diagnostic signal. In wet DMSO, these B(OH) protons
do not appear to exchange rapidly, thus providing sharp and
distinct resonances. With benzoxaborole, the B(OH) resonance
1
shows up at 9.15 ppm while all the other B(OH) H shifts on
open boronic acid compounds, 12, 13 and 2, appear at ~8.0
ppm. The AB resonances of the diastereotopic CH2O unit
within the benzofuran unit are also in line with those
assumptions. Compound
approximately 4.9–5.1 ppm that are very similar to that of 13
and about 0.5 ppm higher than the corresponding resonances
2 displays chemical shifts of
,
in 12 and the open derivative 10
.
Scheme 1. Synthesis of target compound 2.
1
The H NMR spectrum of
2 in slightly wet acetone (again to
prevent the dimerization and oligomerization of B(OH) groups)
was also very informative (Figure 4). Under these conditions,
all hydroxy protons were found to exchange with the solvent
at a rate slower than the spectrometer frequency, and are thus
observable as distinct, sharp peaks. The singlet at 7.6 ppm,
with an integration of 2H, was attributed to the boronic acid
resonances, whereas the doublet at 6.6 ppm was tentatively
assigned to the hemiacetal OH group (coupled with the
benzylic methine). These assignments were confirmed by a
deuterium-exchange experiment; not only did these peaks
disappear, the doublet at 6.6 ppm became a singlet, thus
confirming this resonance belongs to the methine of the
benzylic hemiacetal. Altogether, these NMR spectroscopic
studies in wet polar solvent provide overwhelming evidence
Structure Elucidation
With the synthesis of
conclusively address its equilibrium position in aqueous and
non-aqueous media and whether or not it exists under form
(cf. Figure 2). To this end, product was analyzed initially by
2 complete, the next task was to
2
-
I
2
1H NMR spectroscopy using several organic solvents. In very
dry DMSO, the 1H NMR spectrum revealed an extensive
formation of boroxine and other oligomeric anhydrides.§ To
hydrolyze these species, one drop of H2O was added to the
NMR tube, which considerably cleaned the spectrum. The
absence of resonances associated with a diastereotopic
methylene and aldehydic hydrogens ruled out the existence of
forms 2-III and 2-IV
. One way to assess if a benzoxaborole
structure is closed or open is to compare NMR shifts of
relevant derivatives. Chosen for this purpose were
benzoxaborole, along with 2-methoxymethylphenyl boronic
acid (12) and intermediate 13, a methylated thus permanently
that compound
2 exists exclusively in its open form, 2-I, not in
the closed oxaborole form 2-II
.
A key feature of a free boronic acid moiety is the ability to
dehydrate trimolecularly to form a 6-membered boroxine
under dry solvent conditions or in the gas phase. With this
open derivative of
2 (Figure 3). When ran in DMSO-d6 (with
one drop of H2O to suppress boronic anhydrides),
benzoxaborole displays a 11B chemical shift of 32.3 ppm
compared to 29.5 ppm and 28.7 ppm, respectively, for 12 and
13. The newly designed frustrated benzoxaborole
11B chemical shift of 30.3 ppm, which suggests its structure is
similar to the open control compounds 12 13
knowledge in mind, the behavior of compound
examined by high-resolution ESI-MS. A trimeric boroxine was
not observed.§ Instead, compound
displays the loss of only
two molecules of water to form the putative dimer , which
can only originate through the involvement of the alcohol unit
in the open form (Figure 5a). Such a doubly dehydrated
2 was
2
2 displays a
2-V
-
.
2-I
dimer does not occur with either benzoxaborole or the
blocked o-hydroxymethyl arylboronic acid 12 (Figure 5b).
Therefore, mass spectrometric analysis corroborates the
above NMR evidence in support of the open, "frustrated"
boroxole form (2-I) of compound 2.
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a) Dry deuterated acetone ampoule + 10 µL H2O
He
Hc,d
Hb Ha
b) Dry deuterated acetone ampoule + 2 drops D2O
He
Hc,d
Ha
Hb
Fig. 4. 1H NMR spectra of compound 2 in acetone-d6 with (a) 10 L H2O; (b) one drop of D2O.
Equilibrium Dissociation in Water
arylboronic acids. For example, the reported pKa of 7.2 for
benzoxaborole is almost two units lower than that of
Having convincingly demonstrated that compound
predominantly in its open "frustrated benzoxaborole" form
2 exists
phenylboronic acid (8.9).12 Here, the pKa of compound
2 was
2-I,
measured using a 11B NMR titration method (Figure 6). To our
surprise, the resulting pKa of 7.5 is closer to that of
benzoxaborole (reproducibly measured at 7.2 in our hands)
compared to a normal boronic acid. Moreover, the titration
we turned towards a preliminary examination of the reactivity
of this unique molecule, starting with its equilibrium
dissociation in water. Boronic acids behave as Brønsted acids
through indirect ionization of water leading to the formation
of a tetrahedral hydroxyborate anion as the conjugate base. It
is well established that benzoxaboroles generally display a pKa
approximately one to two units lower than the corresponding
curve for 2-I is significantly steeper than the broader transition
curve displayed by benzoxaborole,§ with less than one pH unit
difference from the first to the second plateau. This difference
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may afford a tetrahedral sp3 boron, the low pKa of
2-I Vie2w-IArtmicleaOynlbinee
in the transition suggests that benzoxaborole and
feature different a mechanism of capturing and stabilizing explained by a stabilization of the conjuDgOatI:e10t.r1i0h3y9d/Dro0xOyBb0o0r5a7t2eJ
hydroxy ions. Whereas benzoxaborole's low pKa is likely due to conjugate base VI by the hydrogen-bonding effect of the
2
-
favorable release of angle strain upon hydroxy coordination to ortho
hydroxyalkyl
unit
(Fig.
6a).
Fig. 5. Major species observed in the electrospray (neg. mode) mass spectral
analysis of: (a) compound 2; (b) control compounds: benzoxaborole, and 12.
Reactivity with Monoamines
(a)
It is well established that the carbonyl groups of 2-
formylphenylboronic acid (2-FPBA)13 and 2-acetylphenylboronic
acid (2-APBA)14 can condense readily with amines in aqueous
media to form the corresponding imines, which are stabilized by
a dative B–N bond with the adjacent boronic acid (Figure 7a).
These types of structures, termed “iminoboronates”, provide
reversible bioconjugates.15 The potential ability of
undergo reversible exchange with the hydroxy-aldehyde form
IV could provide a method for ligating amines in aqueous
conditions through formation of aminal derivatives (Figure 2).
By analogy with the "water-inserted" ortho-dimethylaminoalkyl
benzeneboronate structure described by Anslyn and co-
workers,16 these complexes may be particularly favorable in
water. The dynamic nature of the hydroxymethyl arm on
compound 2-I, however, could potentially result in a more
stable and possibly irreversible hemiaminal structure, which
could be favored over the imine isomer (Figure 7b).
2-I to
(b)
2
-
35
30
25
20
15
10
5
3
0
0
5
10
15
pH
Fig. 6. (a) Dissociation equilibrium for compound 2 and proposed conjugate base 2-VI;
and (b) Acid-base titration by 11B NMR in NH4OAc buffer.
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NMR and LC-MS for any signs of conjugation. Shown in Figure
View Article Online
1
8 is the H NMR spectrum of compound 2-I
DOI: 10.10i3n9/dDe0OutBe0r0a5t7e2dJ
acetone, after the addition of one equivalent of benzylamine
(Figure 8). The most important change in the spectrum is the
upfield shift of the hemiacetal proton Ha, from 6.54 ppm in the
unbound compound 2-I (c.f., Fig. 4), to 6.05 ppm after addition
of benzylamine. This observation confirms that the hemiacetal
hydroxy group on compound 2-I has been replaced by
benzylamine to form the aminal 3a. This result provides strong
evidence that structure 2-I is dynamic in water and affords
enough of the ring-opened isomer 2-IV to enable the
formation of an imine with benzylamine. Subsequent ring
closing of the hydroxymethyl group onto the C=N bond (c.f.,
Fig. 7b) provides the 5-membered hemiaminal structure 3a
.
The 11B NMR spectrum of isolated 3a shows a major resonance
with a chemical shift of 28.9 ppm consistent with a neutral
Fig. 7. Amine conjugation with 2-FPBA, 2-APBA (a); and with reagent 2 (b).
trigonal boronic acid. A minor species (
associated with its partial ionization
trihydroxyboronate conjugate base.

~3 ppm) is likely
Initial studies conducted to study the binding of
benzylamine were performed in acetone and monitored via
2 with
into the
Ha
Fig. 8 1H NMR spectra of conjugate of compound 2-I with benzylamine in acetone-d6. (See Fig. 4 for the spectrum of free 2-I)
The outcome of this NMR analysis was corroborated by the LC- described by Gao and Gois, 2-FPBA and 2-APBA are also known
MS chromatogram of the benzylamine addition product, which to bind to N-terminal cysteines and form a thiazolidino
shows mostly the mass of the hemiaminal product 3a after 0.5 boronate (TzB) structure (Figure 9a).17,18 TzB structures, unlike
h, with only a small amount of leftover starting material.§ As iminoboronates, are stable to competing biomolecules present
expected, 2-hydroxybenzylamine also forms an aminal with in solution, although they are also known to be reversible in
reagent
2. According to mass spectrometric analysis, it slightly acidic or basic solutions. It was proposed that
undergoes further dehydration likely to form a hemiboronic compound
2 would form a similar type of structure, but
ester with the phenolic hydroxy group. §
instead have enhanced stability due to the additional adjacent
hydroxymethyl arm that may form an additional ring on the
Complexation with Amino Acids and Esters
conjugate structure. Therefore, binding with compound 2
could potentially form a fused 3-membered ring conjugate
that would be more stable and irreversible within a wide range
of pH (Figure 9b).
The complexation of bifunctional molecules such as amino acid
derivatives was examined next. ‘Click’ chemistry has become a
valuable tool in the area of bioconjugation as it can provide a
quick, easy and stable method for labeling amino acids and
peptides. Relatively few methods exist where a compound can
undergo a chemoselective bioconjugation reaction with a
naturally occurring functionality on a peptide. One of these
methods utilizes 2-FPBA and 2-APBA, which can form relatively
stable imines with N-terminal residues on proteins.13-15 As
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the strained triple-dehydration tricycle 14
dehydration aminal, 15, may also form, along with a ring-open
thiazolidine 16 similar to that observed by Gao and Gois.17,18
,
Vieaw ArtdicoleuObnlliene-
(a) Thiazolidino boronate (TzB) formation with 2-FPBA or 2-APBA
DOI: 10.1039/D0OB00572J
R
N-terminal L-cysteine
R
HS
O
O
NH2
O
R
There are a few notable details in the resulting 1H NMR
spectrum of Figure 10b. Firstly, similar to the benzylamine
conjugation described above, in the presence of cysteine the
hemiacetal proton Ha has once again shifted upfield, from 6.5
S
aqueous
pH 7
HS
NH
+
NH
OH
Me/H
H/Me
OH
O
B
Me/H
OH
OH
OH
B
B
OH
thiazolidino boronate (TzB),
reversible in slightly
acidic/basic conditions
ppm in free 2-I (c.f. Fig. 4b) to around 6.3 ppm. Secondly, the
methylene protons, Hc and Hd, have shifted vastly from 5.0
ppm to 4.5 ppm. By analogy with the model compounds of
Figure 3 (and also compound 10), this chemical shift most
likely indicates that the methylenoxy arm is open. Together
this information strongly suggests that the conjugate between
(b) Proposed condensation to a fused 3-membered ring with reagent 2
HO
O
O
NH2
XH
OHC
O
N
H
cysteine with compound
2 has a structure with an open –
N
HN
B
(HO)2B
CH2OH arm, which rules out structures such as 14 and 15. Thus
the proposed thiazolidine adduct 16 is consistent with the
formation of TzB adducts observed with 2-FPBA and 2-APBA
(c.f. Fig. 9a), not with a tricyclic adduct such as 14, which is
presumably too strained to form. Moreover, the 11B NMR
chemical shift of 11.3 ppm§ for isolated 16 is in line with data
obtained for the simpler TzB adducts (~10 ppm) and supports a
neutral, tetrahedral species with N–B coordination promoted
by the strained nature of this acyloxy adduct.17 The HRMS data
of 16 further supports this structure.§ Attempts to observe a
X
3 H2O
2-IV
X = S (N-terminal cysteine)
= O (N-terminal serine)
potentially stable and
irreversible bioconjugate
Fig. 9. N-terminal amino acid conjugation with 2-FPBA, 2-APBA and compound 2.
As an initial test for cysteine and serine conjugation,
compound
2 was dissolved in ammonium acetate buffered
aqueous solution with one equivalent of each amino acid and
left to stir at room temperature. In the case of N-terminal
cysteine, a white solid material crashed out of solution within
5 minutes. This material was collected by filtration and
analyzed by 1H NMR spectroscopy. Some of the possible
condensation adducts are shown in Figure 10a. In addition to
conjugate between serine and compound
2 met with minimal
success, with only starting materials and a few minor peaks
appearing in the 1H NMR spectrum after 24 hours.19
(a)
(b)
Ha
Hb,c
Fig. 10. (a) Possible structures of the equimolar adduct between compound 2 and free cysteine. (b) 1H NMR spectrum of cysteine conjugate of compound 2 in in acetone-d6 with
one drop H2O. (See Fig. 4 for the spectrum of free 2-I)
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To emulate a Cys-terminal peptide, the O-protected L-cysteine reversibility, as indicated by the similar appearance of the
ethyl ester was reacted with reagent under the same mass spectra before and after the addition of fructose.
conditions (Figure 11). Surprisingly, despite the lack of a free Although 2-APBA also displays strong amine binding, it is
carboxylate, again a white solid material crashed out of poorly soluble in water in comparison with reagent . Thus,
2
2
solution within 5 minutes. Signals from the ethyl group of with excellent reactivity and increased aqueous solubility,
1
protected L-cysteine were not found in the H NMR spectra of frustrated benzoxaborole
the resulting white solid. Spectra in both acetone-d6 and bioconjugation reagent.
DMSO-d6 were identical to that of adduct 16 with free
2 presents attractive attributes as a
cysteine.§ The HRMS spectrum of the white solid material
further confirmed the loss of ethoxide. Therefore, during the
reaction, the ethyl ester group was readily hydrolyzed, possibly
as a result of internal activation by the Lewis acidic boron unit.
Fig. 11. Formation of adduct 16 by condensation and hydrolysis of cysteine ethyl ester
with compound 2.
Lysozyme Conjugation
To assess the potential of compound
2 as a bioconjugation
reagent for biologically relevant targets, we utilized as a model
protein, lysozyme (aka, muramidase), an antimicrobial enzyme
previously utilized to evaluate the 2-FPBA reagent in protein
bioconjugation.13 Functionalization of amino groups of up to
the six lysine residues on lysozyme was monitored using ESI-
Fourier transform ion cyclotron resonance (FTICR)-MS. The
conjugation of reagent
2 with lysozyme was compared to that
of 2-FPBA and 2-APBA after stirring with an excess of the
boron reagent for 30 min in 50 mM ammonium acetate buffer
at room temperature. Under these conditions, reagent
2 was
found to modify the model protein multiple times (Figure 12).
Although all three compounds show up to six modifications of
the lysozyme protein, 2-FPBA shows the least amount of free
lysozyme, suggesting a superior binding efficacy.§ To test the
reversibility of the boronic acid/lysozyme conjugates, fructose
was added as a biomolecular competitor, and the qualitative
change in the proportion of free lysozyme and modified peaks
was monitored qualitatively.§ In the event, with the addition of
excess fructose, the reversibility of 2-FPBA conjugation was
evident. Not only did a significant increase of the free
lysozyme peak was observed, but most of the other peaks in
the mass spectrum changed from 2-FPBA-lysozyme adducts to
fructose-bound lysozyme adducts.§ In contrast, both 2-APBA
Fig. 12. ESI-FTICR-MS lysozyme binding assays showing the (M+8H)8+ charge state
of lysozyme with excess reagent 2 (top) and competition experiment with excess
fructose (bottom).
Conclusion
This study details the design and synthesis of arylboronic acid
2
, the first example of a permanently open "frustrated"
benzoxaborole, along with an exploration of its application in
bioconjugation. An efficient and high yielding seven-step
and reagent
2 (Figure 12) showed no appearance of
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Organic & Biomolecular Chemistry
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Journal Name
ARTICLE
2014, 22, 4462-4473; (c) A. Adamczyk-Woźniak, K. M. Borys,
A. Sporzyński, Chem. Rev. 2015, 115, 5D2O24I:-1502.14073V.9ie/wD0AOrtiBcl0e0O5n7li2nJe
For recent examples of applications in: Catalysis: (a) S.
Kusano, S. Miyamoto, A. Matsuoka, Y. Yamada, R. Ishikawa,
O. Hayashida, Eur. J. Org. Chem. 2020, Early Access, DOI:
10.1002/ejoc.201901749. Materials science: (b) Y. Chen, W.
D. Wang, D. Wu, H. B. Zeng, D. G. Hall, R. Narain, ACS Appl.
Mater. Interfaces 2019, 11, 44742-44750; (c) D. Wu, W. D.
Wang, D. Diaz-Dussan, Y. Y. Peng, Y. J. Chen, R. Narain, D. G.
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Chen, D. Diaz-Dussan, D. Wu, W. D. Wang, Y. Y. Peng, A. B.
Asha, D. G. Hall, K. Ishihara, R. Narain, ACS Macro Letters
synthesis was optimized, which exploits the hydrophilicity of
the final product with a simple purification that circumvents
the need for chromatography. Through several NMR
5
experiments, compound
hydroxyalkyl arylboronic acid structure
effectively prevented to undergo a dehydrative cyclization as a
result of unfavorable geometry. Boronic acid was found to
2
was found to exist in the open ortho
2-I, a form that is
2-I
have a pKa of 7.5 that is similar to that of benzoxaborole (7.2),
and significantly lower than traditional boronic acids. The
relatively low pKa of compound 2-I is proposed to be the result
of stabilization of the trihydroxyborate conjugate base by the
hydrogen-bond donor ability of the ortho hydroxyalkyl unit.
This low pKa favors solubility in water, which increases the
2018,
7
, 904-908.
6
7
8
S. Vshyvenko, M. L. Clapson, I. Suzuki, D. G. Hall, ACS Med.
Chem. Lett. 2016,
7
, 1097−1101.
S. Luliński, J. Serwatowski, J. Organomet. Chem. 2007, 692
,
potential to use compound
In this regard, compound
2
-
I
in aqueous biological systems.
was found to conjugate
2924.
2-I
X. Li, Y.-K. Zhang, Y. Liu, C. Z. Ding, Y. Zhou, Q. Li, J. J. Plattner,
S. J. Baker, S. Zhang, W. M. Kazmierski, L. L. Wright, G. K.
Smith, R. M. Grimes, R. M. Crosby, K. L. Creech, L. H.
Carballo, M. J. Slater, R. L. Jarvest, P. Thommes, J. A.
Hubbard, M. A. Convery, P. M. Nassau, W. McDowell, T. J.
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Zou, J. Wright, Bioorganic & Medicinal Chemistry Letters
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According to our survey of the literature, none of derivatives
4a-f were isolated and characterized. Only fluoradene
compounds similar to 4f were reported: B. McDowell, H.
Rapoport, J. Org. Chem. 1972, 37, 3261.
effectively with amines to form stable hemiaminal structures,
including highly effective and irreversible reaction with
lysozyme. Complexation of reagent
2 with cysteine was also
demonstrated. Surprisingly, cysteine induced an open
structure containing a free hydroxymethyl arm, as the amino
and thiol group bind preferentially onto the formyl group to
form a N,S-acetal. As a first-of-its-kind permanently open
9
“frustrated benzoxaborole”, compound
2-I demonstrates
unique properties that can be further explored to meet the
ever-growing needs and applications of novel boron containing
drugs and bioconjugates.
10 S. Luliński, I. Madura, J. Serwatowski, H. Szatyłowicz, J.
Zachara, New J. Chem. 2007, 31, 144–154.
11 G. A. Molander, S. L. J. Trice, S. M. Kennedy, S. D. Dreher, M.
T. Tudge, J. Am. Chem. Soc. 2012, 134, 11667.
12 M. Dowlut, D. G. Hall, J. Am. Chem. Soc. 2006, 128, 4226-
4227.
Conflicts of interest
13 P. M. S. D. Cal, J. B. Vicente, E. Pires, A. V. Coelho, L. F.
Veiros, C. Cordeiro, P. M. P. Gois, J. Am. Chem. Soc. 2012,
134, 10299–10305.
There are no conflicts to declare.
14 A. Bandyopadhyay, J. Gao, Chem. Eur. J. 2015, 21, 14748–
14752.
Acknowledgements
15 For Reviews: (a) B. Akgun, D. G. Hall, Angew. Chem. Int. Ed.
2018, 57,13028–13044; (b) S. Cambray, J. Gao, Acc. Chem.
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Carvalho, P. M. S. D. Cal, P. M. P. Gois, Chem. Soc. Rev. 2019,
48, 3513-3536.
This work was funded by the Natural Sciences and Engineering
Research Council (NSERC) of Canada (Discovery Grant to DGH)
and the University of Alberta. The authors thank Dr. Timothy
Morgan and Jonah Curl for help in the early stage of
16 L. Zhu, S. H. Shabbir, M. Gray, V. M. Lynch, S. Sorey, E. V.
Anslyn, J. Am. Chem. Soc. 2006, 128, 1222.
developing a synthesis of compound 2, Mr. Ed Fu (HPLC), and
Mr. Mark Miskolzie (NMR Facility) for their help and advice.
17 A. Bandyopadhyay, S. Cambray, J. Gao, Chem. Sci. 2016,
7
4589.
18 H. Faustino, M. J. S. A. Silva, L. F. Veiros, G. J. L. Bernardes, P.
Notes and references
§ See Supporting Information for details.
M. P. Gois, Chem. Sci. 2016, 7, 5052.
19 For a successful example of 1,2-aminoalcohol complexation
using tris(hydroxymethyl)aminomethane, see: K. Li, M. A.
Kelly, J. Gao, Org. Biomol. Chem. 2019, 17, 5908-5912.
1
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2
3
4
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 9
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Organic & Biomolecular Chemistry
Page 10 of 10
View Article Online
DOI: 10.1039/D0OB00572J
TOC Abstract
HO
HO
OH
HO
O
B
O
B
B
OH
NH
HO NH2
O
O
O
X
or
– H2O
protein
2-I (open)
2-II (closed)
3
NMR and MS evidence
amine, cysteine, protein
Design and synthesis of arylboronic acid 2, the first example of a permanently open "frustrated"
benzoxaborole, is described along with an exploration of its application in the complexation of
amines and amino acids, and protein modification.