Design, synthesis and evaluation of a boronic acid based artificial receptor for l-DOPA in aqueous media

Article abstract of DOI:10.1039/c3cc45275a

Design and synthesis of a boronic acid based artificial receptor which selectively and effectively bound to a neurotransmitter, l-DOPA (1), in aqueous media. In addition, the synthetic receptor was found to effectively inhibit the DDC (l-DOPA decarboxylase) enzymatic reaction under physiological conditions.

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Design, synthesis and evaluation of a boronic acid based artificial
receptor for -DOPA in aqueous media
L
Haruna Ueno, Takayuki Iwata, Nozomi Koshiba, Daisuke Takahashi* and Kazunobu Toshima*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
A)
A designed and synthesised boronic acid based artificial
OH
OH
OH
receptor selectively and effectively bound to
neurotransmitter, -DOPA (1), in aqueous media. In addition,
the synthetic receptor was found to effectively inhibit the
-DOPA decarboxylase) enzymatic reaction under
physiological conditions.
a
HO
HO
HO
HO
CO2H
NH2
L
NH2
L-DOPA (1)
Dopamine (2)
Catechol (3)
10 DDC (
L
OH
OH
B
NH2
NH2
B
H
N
n
HO
O
H
L
ꢀDOPA (3,4ꢀdihydroxyphenylalanine) (1) is a precursor of one
of the important neurotransmitters, dopamine (2). ꢀDOPA is
converted to 2 by ꢀDOPA decarboxylase (DDC, E.C. 4.1.1.28)
N
H
N
NH2
5a: n=1
5b: n=2
5c: n=3
N
O
n
L
H
4a: n=1
4b: n=2
4c: n=3
NH2
L
HO
OH
15 in the presence of the coꢀenzyme, pyridoxalꢀ5’ꢀphosphate (PLP).
In addition, 1 is generally accepted as the gold standard for drugs
for alleviating the symptoms of Parkinson’s disease (PD).1 PD is
a neurodegenerative disease mainly caused by the progressive
loss of 2 in the central nervous system (CNS).2 Although
20 administration of exogenous 1 to PD patients compensates for
inadequate dopamine synthesis and often dramatically alleviates
the symptoms, only 1% of an orally administered dose of 1
reaches the dopaminergic neurons of the brain. The remainder is
metabolized by the peripheral DDC to 2.3 Therefore, coꢀ
25 administration of 1 with peripheral DDC inhibitors (such as
carbidopa4 and benserazide5), which cannot cross the bloodꢀbrain
barrier (BBB), is the most effective symptomatic treatment for
PD. However, early clinical studies comparing carbidopa/1 with 1
monotherapy showed that more than 75% of patients treated with
30 carbidopa/1 experienced marked dyskinesia as a severe side
effect.6 Therefore, the development of novel therapeutic agents
which can prolong the peripheral halfꢀlife of 1 without causing
serious side effects is urgently needed. Herein we describe the
molecular design, chemical synthesis, and evaluation of artificial
35 receptors which can selectively bind to 1 and reduce the rate of
firstꢀpass metabolism by DDC under physiological conditions. To
the best of our knowledge, this is the first report of an artificial
receptor that shows high affinity and selectivity for 1 over 2
under neutral aqueous conditions.
B
HO
OH
B
O
H
N
n
NH2
6a: n=1
6b: n=2
6c: n=3
N
H
NH2
7
NH2
NH
B
)
H
N
N
H
n
O
H
Boronic ester
formation
Ionic Interaction
O
O
H3N
B
O
HO
O
50 Figure 1. A) Chemical structures of
(3), designed artificial receptors 4ꢀ6, and phenylboronic acid (7). B)
Expected binding mode between designed artificial receptors and
DOPA (1).
LꢀDOPA (1), dopamine (2), catechol
Lꢀ
designed and synthesized as promising candidates. However,
55 none of these receptors display selectivity for ꢀDOPA over
dopamine and other catecholamines under aqueous conditions. In
this context, to develop ꢀDOPAꢀspecific receptors, we chose a
L
L
phenylboronic acid for boronic ester formation with the 1,2ꢀdiol
in 1, and a guanidino group for ionic interaction with the
60 carboxylic acid in 1. Because no other catecholamines have a
carboxylic acid in their structure, this ionic interaction may
contribute to high affinity and selectivity for 1 (Figure 1B). To
investigate our hypothesis, we designed nine compounds (4aꢀc,
5aꢀc, and 6aꢀc) by changing the position of an amide group to a
65 boronyl group in the benzene ring (ortho, para, and meta
isomers) and the length of the spacer between the benzene ring
and the guanidino group (n = 1ꢀ3) (Figure 1A).
40
To date, several boronic acid based catecholamine receptors
and chemosensors,7,8 which can reversibly bind to a 1,2ꢀdiol in
catecholamines in aqueous media at neutral pH, have been
The synthesis of 4ꢀ6 is outlined in Scheme 1. Amidation
reactions of orthoꢀanilineboronic acid (8) with Bocꢀglycine (9a),
Department of Applied Chemistry, Faculty of Science and Technology,
45 Keio University, 3-14-1 Hiyoshi, Kohoku-ku, Yokohama 223-8522
(Japan). E-mail: dtak@applc.keio.ac.jp, toshima@applc.keio.ac.jp; Fax: 70 Bocꢀβꢀalanine (9b), and 9c were performed using DMTꢀMM in
(+81) 45-566-1576.
† Electronic supplementary information (ESI) available: See
DOI: 10.1039/b000000x/
MeOH to afford 10a-c, respectively. Deprotection of the Boc
group of 9a-c and subsequent removal of pinacol ester provided
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DOI: 10.1039/C3CC45275A
Bpin
Bpin
H
N
O
a)
b, c)
NH2
NHBoc
NHBoc
n
HO
O
n
9a: n=1
9b: n=2
9c: n=3
10a: n=1, 40%
10b: n=2, 83%
10c: n=3, 63%
40
45
50
8
HO
OH
B
H
N
d, e)
: n=1, 43%
4b: n=2, 27%
4c: n=3, 47%
4a
N
NHBoc
NH3 TFA
N
n
O
NBoc
12
11a: n=1
11b: n=2
11c: n=3
Bpin
Bpin
5a: n=1, 32%
5b: n=2, 48%
5c: n=3, 60%
a)
b-e)
9a-c
NH2
HN
Figure 2. Association constants (Ka) obtained for 4ꢀ7 with 1ꢀ3. The Ka
values were determined by H NMR titrations in phosphate buffer (D2O,
100 mM, pH 7.4) and are the average of at least two reproducible
measurements; see Electronic Supplementary Information (ESI).
NHBoc
n
1
13
O
14a: n=1, 92%
14b
: n=2, 62%
14c: n=3, 78%
Bpin
Bpin
A)
HO
OH
H
N
HO
HO
HO
a)
b-e)
O
R
O
R
6a: n=1, 44%
6b: n=2, 47%
6c: n=3, 34%
B
B
B
55
60
65
70
O
9a-c
R
N
NH
NHBoc
n
16a: n=1, 69%
16b: n=2, 65%
16c: n=3, 82%
N
H
NH2
O
15
4a-c
4'
4''
B)
Scheme 1. Synthetic scheme for 4ꢀ6. Reagents and conditions: a) DMTꢀ
MM, MeOH, 40 °C; b) TFA, CH2Cl2; c) Acetone/H2O; d) 12, DIEA,
MeCN/H2O; e) 4.0 M HCl in 1,4ꢀdioxane. pin = pinacol, Boc = tꢀ
butoxycarbonyl, TFA
dimethoxyꢀ1,3,5ꢀtriazinꢀ2ꢀyl)ꢀ4ꢀmethylꢀmorpholinium chloride, DIEA =
NH2
NH
NH2
NH
H
N
H
N
H
N
H
N
NH2
N
H
N
H
H
H
O
O
O
H
N
2
Weak
O
W eak
=
trifluoroacetic acid, DMTꢀMM
=
4ꢀ(4,6ꢀ
O
O
O
H3N
H3N
H3N
O
O
B
O
O
B
O
O
B
O
O
5
HO
HO
HO
N,Nꢀdiisopropylethylamine.
6a and 1
6b and 1
6c and 1
orthoꢀsubstituted phenylboronic acid derivatives 11a-c.
Treatment of 11a-c with 12, followed by deprotection of the Boc
groups, provided orthoꢀsubstituted artificial receptors 4a-c,
10 respectively. Similarly, paraꢀ and metaꢀsubstituted artificial
receptors 5a-c and 6a-c were also synthesized in moderate yields.
After chemical synthesis of the designed compounds, the
NH2
NH2
H
N
H
N
H
N
H
N
NH2
N
H
NH
H
N
H
NH
H
O
O
O
H
N
2
Weak
Weak
H3N
H3N
H3N
B
O
O
B
O
O
B
O
O
HO
HO
HO
6a and 2
Boronic ester formation
6b and 2
6c and 2
Ionic interaction
1
binding abilities of 4ꢀ7 with 1ꢀ3 were examined using H NMR
Hydrogen bonding
titration binding assays9 in phosphate buffer (D2O, 100 mM, pH
15 7.4). The association constants (Ka) were calculated based on the
integration of the selected peak area corresponding to the free
receptor (boronic acid) and the guest complex (see Figures S1a
and S1b in ESI). The results are summarized in Figure 2. It was
found that 7, which does not possess a guanidino group,
20 moderately bound to the 1,2ꢀdiol in 1ꢀ3; the Ka values obtained
for 7 with respect to 1ꢀ3 were 770, 800 and 560 Mꢀ1, respectively.
In contrast, the orthoꢀsubstituted compounds 4aꢀc did not bind to
1ꢀ3 (Ka <10). These results suggested that the orthoꢀsubstituents
in 4aꢀc are close to the boron atom, and 4aꢀc exist mainly in
25 equilibrium between their stable cyclic form 4’ and 4’’, owing to
the partial aromatic character of the boron in heterocycles, as
shown in Figure 3A.10 The 11B NMR chemical shift of 4a also
supports the existence of the cyclic form (see Table S1 in ESI).
The Ka values for the paraꢀsubstituted compounds 5aꢀc bound
30 to 1ꢀ3 showed a similar tendency to the values obtained for 7 with
respect to 1ꢀ3. Differences in the length of the spacer in 5aꢀc did
not affect binding. These results clearly indicated that the
positively charged guanidino side chain of 5aꢀc could not reach
the negatively charged carboxyl group in 1, and thus the
35 guanidino and carboxyl groups could not ionically interact. In
contrast, when the metaꢀsubstituted compounds 6aꢀc were used
Figure 3. A) Boron containing heterocycles 4’ and 4’’ and the formation
of 4a-c. B) Proposed binding mode between 6aꢀc and 1 or 2.
75 with 3, Ka values in the range of 1500ꢀ1690 Mꢀ1 were obtained,
which is almost three times greater than the value obtained for 7
with 3. Although we have yet to obtain direct evidence, these
results may suggest that NH/π interaction between the benzene
ring of 3 and the hydrogen atom(s) in the guanidino group in 6aꢀc
80 contribute to the increase in Ka values. In addition, it was found
that 6a showed the highest affinity and selectivity for 1 over 2
and 3. Furthermore, the order of the Ka values obtained for 6aꢀc
with 1 was 6a > 6c ≈ 6b, whereas the order of the Ka values
obtained for 6aꢀc with 2 was 6c > 6a ≈ 6b. A comparison of the
85 Ka values for 6aꢀc with 1 suggests that the expected ionic
interaction between the guanidino group in 6a and the carboxyl
group in 1 is sufficient for selective recognition. In addition, from
a comparison of the Ka values for 6a-c with 2, it appears that
hydrogen bonding between the amide group in 6a-c and the
90 amino group in 1 or 2, except in case of 6a and 1, is also an
important factor in binding. However, we have not obtained
direct evidence for the hydrogen bonding yet. Although the exact
binding mode remains unclear, taking these results together
indicates that 6a can selectively and effectively bind to 1 by a
2
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DOI: 10.1039/C3CC45275A
0.4
0.3
0.2
0.1
0
twoꢀpoint interaction as follows: 1) boronic ester formation
60
L-DOPA
No compound
L-DOPA+BM1
between 6a and 1,2ꢀdiol in 111; and 2) ionic interaction between
the guanidino group in 6a and the carboxyl group in 112 (Figure
3B).
6a
L-4DaOPA+BO1
5
Next, to confirm the selective binding ability of 6a, binding
assays were conducted under identical conditions using other
biologically important potential guests in humans that may show
affinity with 6a. These compounds included sugars 17 and 18
possessing 1,2ꢀdiols, sialic acid 19 possessing both 1,2ꢀdiols and
65
0
30
60
Time (min)
10 a carboxylic acid, amino acids 20 and 21 possessing both an
aromatic ring and an αꢀamino acid structure, and neurotransmitter
22 which possesses a phenol, an amino group and an aromatic
ring. These assays showed that 17ꢀ22 all have low affinity for 6a
(Table 1), and indicate that our designed molecule 6a selectively
15 binds to 1 with high affinity under neutral aqueous conditions.
70 Figure 4. Time courses of DDC enzymatic reaction in the absence (black)
or presence of 4a (blue) and 6a (red). DDC (3.5 ꢀg/mL) was incubated
with 1 (1.0 mM) and PLP (0.1 mM) in the absence or presence of each
receptor molecule (1.0 mM) in 50 mM HEPES buffer (containing 100
mM NaCl, pH 7.2) at 37 °C for 30 or 60 min. To the reaction mixture was
75 added TNBS (5% in H2O), and the mixture was incubated at 37 °C for 20
min. Then, 2,4,6ꢀtrinitrophenyl (TNP)ꢀdopamine, which was formed by
reaction between 2 and TNBS, was extracted with benzene. The progress
of the DDC enzymatic reaction was monitored by UV analysis (340 nm)
of the extracted TNPꢀdopamine.
Table 1 Association constants (Ka) obtained for 6a with several guests.
80 novel therapeutic agents that can regulate the concentration of 2
in the CNS without causing serious side effects.14 The
development of more specific and higher affinity binding
receptors for 1 is now under investigation in our laboratory.
The authors wish to thank Prof. Dr. S. Shimizu, Faculty of
85 Science and Technology, Keio University, for valuable
discussion on cell viability assay.
20
25
30
Entry
Guest
Ka [Mꢀ1]a
Notes and references
1
2
3
4
5
6
D
D
ꢀGlucose (17)
ꢀMannose (18)
<10
<10
<10
<10
<10
<10
1. A. H. V. Schapira, M. Emre, P. Jenner and W. Poewe, Eur. J. Neurol.,
2009, 16, 982.
90 2. (a) H. Bernheimer, W. Birkmayer, O. Hornykiewicz, K. Jellinger and
F. Seitelberger, J. Neurol. Sci., 1973, 20, 415; (b) J. A. Gingrich and
M. G. Caron, Annu. Rev. Neurosci., 1993, 16, 299.
3. B. Zorc, M. Ljubić, S. Antolić, J. FilipovićꢀGrčić, D. Maysinger, T.
AlebićꢀKolbah and I. Jalšenjak, Int. J. Pharm., 1993, 99, 135.
95 4. C. Diederich, L. Milakofsky, T. A. Hare, J. M. Hofford, M. Dadmarz
and W. H. Vogel, Pharmacology, 1997, 55, 109.
Neu5AcαꢀOMe (19)
LꢀTyrosine (20)
LꢀTryptophan (21)
Serotonin (22)
aThe Ka values were determined by 1H NMR titrations in phosphate buffer
(D2O, 100 mM, pH 7.4), and are the average of at least two reproducible
35 measurements; see Electronic Supplementary Information (ESI).
5. C. S. Myers, M. Witten, Y.ꢀL. Yu and G. C. Wagner, Mol. Chem.
Neuropathol., 1998, 33, 81.
6. A. Lieberman, A. Goodgold, S. Jonas and M. Leibowitz, Neurology,
We next examined the inhibition activity of the DDC
enzymatic reaction by 4a and 6a, which exhibit low and high
affinity, respectively, towards 1. The progress of the DDC
40 enzymatic reaction was monitored by measuring the production
100
1975, 25, 911.
7. J. K. W. Chui and T. M. Fyles, Supramol. Chem., 2008, 20, 397 and
references cited therein.
8. Although Akkaya’s group have reported a fluorescent sensor for Lꢀ
of dopamine (2) from LꢀDOPA (1) with a spectrophotometric
assay using 2,4,6ꢀtrinitrobenzeneꢀ1ꢀsulfonic acid (TNBS).13 The
results are summarized in Figure 4. It was found that, due to its
low affinity for 1, 4a did not inhibit the DDC enzymatic reaction.
45 In sharp contrast, when equimolar amounts of 6a to 1 were used,
the DDC enzymatic reaction was significantly inhibited. These
results clearly indicate that our designed and synthesized artificial
receptor 6a effectively binds to 1 and inhibits the DDC enzymatic
reaction under physiological conditions.
DOPA consisting of a phenyl boronic acid and a Lucifer yellow dye
bound to 1 effectively (Ka = 1600 Mꢀ1) in aqueous media [A. Coskun
and E. U. Akkaya, Org. Lett., 2004, 6, 3107.], the Ka values obtained
for the sensor with other catecholamines were not provided.
9. M. Maue and T. Schrader, Angew. Chem. Int. Ed., 2005, 44, 2265.
10. M. P. Groziak, A. D. Ganguly and P. D. Robinson, J. Am. Chem. Soc.,
1994, 116, 7597.
105
110
115
120
1
11. (a) The H and 11B NMR studies showed prominent shifts of peaks
for the protons corresponding to both the phenylboronic acid moiety
in 6a and catechol moiety in 1, and a prominent shift of a peak for the
boron atom after bindng (see Figures S1a and S2 in ESI). (b) H.
Otsuka, E. Uchimura, H. Koshino, T. Okano and K. Kataoka, J. Am.
Chem. Soc., 2003, 125, 3493.
50
In conclusion, our designed and synthesized boronic acid based
artificial receptor 6a was found to selectively and effectively bind
to 1 through boronic ester formation and ionic interaction
between the guanidino group in 6a and the carboxyl group in 1.
In addition, we demonstrated that 6a effectively binds to 1 and
12. (a) The 1H NMR studies showed a relatively small shift of a peak for
the protons corresponding to the methylene protons on the side chain
in 6a after binding (see Figure S1b in ESI); (b) S. Jin, M. Li, C. Zhu,
V. Tran and B. Wang, ChemBioChem, 2008, 9, 1431.
55 inhibits the DDC enzymatic reaction under physiological
conditions. This approach should provide a new strategy for
inhibiting the peripheral DDC catalysis of 1. Although the
binding selectivity of 6a toward 1 is moderate, we hope the
results presented here will contribute to the molecular design of
13. A. Charteris and R. John, Anal. Biochem., 1975, 66, 365.
14. It was confirmed that 6a did not exhibit cytotoxicity against normal
human lung fibroblast WIꢀ38 cells (see Figure S3 in ESI).
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