B. Wang et al.
these binding constants is the extraordinarily high affinity of
these isoquinolinylboronic acids for the monosaccharide
studied. For example, the binding constants with d-fructose
for all isoquinolinylboronic acids studied were in the range
of 1353–2170mꢀ1. In contrast, the binding constant for 8-
QBA and d-fructose was 108mꢀ1.[31] The difference is over
13-fold. Second, it is also interesting to note that these iso-
quinolinylboronic acids showed much higher affinity for glu-
cose than phenylboronic acid.[21,22] For example, the appar-
ent binding constants of 8-IQBA and 5-IQBA with glucose
were 46 and 42mꢀ1, respectively. In contrast, the binding
constant of phenylboronic acid and glucose was about 5mꢀ1.
Third, the apparent association-constant trend for 4-IQBA
followed the order d-fructose > d-sorbitol > d-glucose.
This is different from the trend observed for other arylbor-
onic acids,[10,32,33] which has the order d-sorbitol > d-fruc-
tose > d-glucose. Even in the case of 8-IQBA, its binding
constants with sorbitol and fructose were essentially the
same, which was unexpected. Fourth, the binding constants
are not directly correlated with the intensity of fluorescent
changes. This is also different from most of the fluorescent
boronic acids that we have been reported, which show
higher magnitude changes for tight binders.
Encouraged by the high affinity of these IQBAs, especial-
ly with glucose, we were interested in probing their ability
to bind the pyranose form of a sugar. This interest stems
from the fact that cell-surface carbohydrates only contain
sugars in the pyranose form and one of the important goals
in our carbohydrate sensor effort is the design and synthesis
of probes for cell-surface carbohydrate biomarkers. Howev-
er, the “general consensus” seems to be that arylboronic
acids do not bind to vicinal diols on six-membered ring, and
thus, application of boronic acids in recognizing glycans in
mammalian systems would be difficult. To address this fun-
damental question, we also tested the binding affinities of
the isoquinolinylboronic acids with methyl a-d-glucopyra-
nose and cis-cyclohexanediol. The selection of methyl a-d-
glucopyranose is to ensure that the sugar is in its cyclic
form. However, the disadvantage is that the hydroxyl group
at the 1 position is no longer available for binding. cis-Cy-
clohexanediol was selected as a representative six-mem-
bered cis-diol. The summary of the binding results is shown
in Table 2, and Figures 5 and 6. Several special binding
properties were observed. First, weak but encouraging bind-
ing with cis-cyclohexanediol was observed for 8-IQBA, 5-
IQBA, and 4-IQBA, with the apparent association constants
(Ka) being 0.4 (15-fold fluorescence intensity change), 1.1
(66% fluorescence intensity change), and 0.8mꢀ1 (6-fold
fluorescence intensity change), respectively. In control ex-
periments, cyclohexanol did not show appreciable binding
or fluorescence changes when added to the same boronic
acids. Second, these isoquinolinylboronic acids showed bind-
ing with a model glycoside, methyl a-d-glucopyranose,
under physiologically relevant conditions. The apparent
binding constants (Ka) for 4-IQBA and 6-IQBA were 3.3
and 2.1mꢀ1, respectively. It should be noted that Hall et al.
reported ortho-hydroxymethyl phenylboronic acid as binder
for methyl a-d-glucopyranose.[20,30] However, this boronic
acid does not change fluorescence upon binding. To the best
of our knowledge, 4-IQBA and 6-IQBA are the very first
examples of boronic acid derivatives that can bind to methyl
a-d-glucopyranose with fluorescence changes.
As a control, we also studied the binding of these isoqui-
nolinylboronic acids with cyclohexanol to see whether their
apparent binding was due to interactions with a single hy-
droxyl group (boronic acid interactions with single hydroxyl
groups do have precedents).[3] As can be seen from Table 2,
6-IQBA was found to bind cis-cyclohexanediol (Ka =1mꢀ1)
and cyclohexanol (Ka =4mꢀ1) and the other IQBAs did not
show any binding with cyclohexanol. Such results indicate
that single-hydroxyl-group binding might play an important
role in the binding of 6-IQBA with methyl a-d-glucopyra-
nose and cyclohexanediol. At this time, it is not clear in
which exactly way 6-IQBA binds to these six-membered-
ring diols.
With the weak but encouraging binding with cis-cyclohex-
anediol for all the isoquinolinylboronic acids discussed
above, it becomes important to address the question of
whether the ability to bind cis-diols on a six-membered ring
is unique to isoquinolinylboronic acids. As a control, phenyl-
boronic acid should be considered first. Binding between
phenylboronic acid and cyclohexanediol had been studied
before and no binding was observed.[34] Such results indicate
that the ability to bind to cis-diols on a six-membered ring is
not universal to all boronic acids. Next, we studied the bind-
ing of 8-QBA and 6-MDDCQ (Scheme 2) with cis-cyclohex-
anediol. 8-QBA was selected because it is a quinolinylboron-
ic acid, 6-MDDCQ was chosen because the boronic acid is
attached to a phenyl ring but the compound also contains a
quinoline moiety. Both boronic acids also showed binding
affinities towards cis-cyclohexa-
nediol (Figure 7). For 8-QBA, a
Table 2. Apparent association constants (Ka) of isoquinolinylboronic acids with representative carbo-
23-fold fluorescence intensity
change was found upon the ad-
dition of 1.5m cis-cylcohexane-
diol at physiological pH in
phosphate buffer. These results
indicated that binding of cis-cy-
clohexanediol is not a unique
property of isoquinolinylboron-
ic acids.
hydrates.[a]
Isoquinolinyl
Methyl a-d-glucopyranose
cis-Cyclohexanediol
Cyclohexanol
boronic acids[a]
Ka [mꢀ1
]
DImax/I0
Ka [mꢀ1
]
DImax/I0
Ka ([mꢀ1
]
DImax/I0
8-IQBA
5-IQBA
4-IQBA
6-IQBA
8-QBA
not observed
not observed
3.3ꢁ0.9
–
–
1
0.4ꢁ0.0
1.1ꢁ0.8
0.8ꢁ0.2
1.0ꢁ0.2
1.2ꢁ0.7
2.0ꢁ0.2
15
not observed
not observed
not observed
4ꢁ1
–
–
–
3
–
–
ꢀ66%
6
2.1ꢁ1.4
ꢀ30%
ꢀ71%
23
ꢀ75%
not observed
not observed
–
–
not observed
not observed
6-MDDCQ
[a] Binding studies were conducted in phosphate buffer (0.1m) at pH 7.4 (all experiments were duplicated).
13532
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 2010, 16, 13528 – 13538