Photo-cleavable analog of BAPTA for the fast and efficient release of Ca2+

Article abstract of DOI:10.1039/c7cc02056b

A new photocleavable analog of BAPTA chelating ligand has a high affinity towards Ca²⁺ ions (K = 2.5 × 10⁶ M^-1). The use of photolabile 3-(hydroxymethyl)-2-naphthol core in the design of photo-BAPTA allows for the efficient (Φ = 0. 63) and very fast (τ < 12 μs) release of Ca²⁺ ions upon 300 or 350 nm irradiation.

Full text of DOI:10.1039/c7cc02056b

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Photo-cleavable Analog of BAPTA for the Fast and Efficient
Release of Ca2+
Mariia V. Sutton, Matthew McKinley, Revathy Kulasekharan and Vladimir V. Popika*
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
OH
N
O
O
O
O
Q
O
O
O
O
h
A new photocleavable analog of BAPTA chelating ligand has a high
affinity towards Ca2+ ions (K= 2.5*106 M-1). The use of photolabile
3-(hydroxymethyl)-2-naphthol core in the design of photo-BAPTA
allows for the efficient (ꢀ=0. 63) and very fast (ꢀ ꢁ12 ꢂs) release of
Ca2+ ions upon 300 or 350 nm irradiation.
H
CO2
Ca2+
HO2C
HO2C
N
N
N
Ca
HO2C
Ca2+
O
O
O
+
O
CO2H
N
CO2H
OH
OH
O
N
CO2H
CO2H
O
H2
1
NQMP-BAPTA,
O
Free calcium ions (Ca2+) play an important role as a secondary
messenger for a wide variety of regulatory functions in physiological
and biological processes in cells.1 The fluctuations of intracellular
Ca2+ concentration regulate many cellular functions, such as
neurotransmitter release,2 operation of ion channels in the plasma
membrane,3 hormone secretion,4 muscle contraction,3 and many
others. In recent years, the flash photolysis of photoresponsive
calcium-ion chelators has become a common method in biochemical
studies.5 The light-triggered release of free Ca2+ allows for following
the system response to a spike in calcium ion concentration.
The majority of photoresponsive calcium chelators are developed
around polydentate ligands with high affinity to Ca2+, such as
ethylenediaminetetraacetic acid (EDTA),6,7 ethylene glycol-bis(ꢀ-
aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA),7,8 and 1,2-bis(o-
aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA).9,10 For
biomedical applications, BAPTA-based ligands are preferable since
BAPTA offers the highest selectivity towards calcium ions (versus
magnesium and other divalent cations).
Two general strategies are commonly employed in the design of
photo-responsive calcium chelators. In the first approach, a
photoreactive moiety conjugated to the amino group can change the
availability of the nitrogen atom lone pair, thus modulating the
calcium binding constant upon irradiation.10 For example, the Tsien’s
design of the first photoresponsive calcium chelator was based on
light-driven intramolecular oxidation of p-hydroxymethyl group of
aniline moiety into a ketone.9 Alternatively, a photolabile linker can
be incorporated between two aminodicarboxylic acid fragments. The
2
NQM-APDA,
O
CO2H
N
OH
OH
NQMP-APDA,
CO2H
3
Scheme 1.
calcium affinity of tridentate ligands, produced upon photochemical
cleavage, is many orders of magnitude lower than that of the
octadentate precursor.8 The latter strategy for calcium photorelease
is preferable to the former, as it permits a much higher overall
change in Ca2+ affinity of a ligand. However, photo-cleavable
analogues of calcium-selective BAPTA chelators have not been
reported so far. Additional consideration in the design of photolabile
chelator is the rate of ion release. The photochemical cleavage of o-
nitrobenzyl or o-nitroveratryl ether linkers, that are commonly
employed in photo-cleavable chelators,8 is a relatively slow process.
It involves several dark steps and might take tens of seconds to
complete.11
Here, we report the synthesis, photochemical properties, and Ca2+
release kinetics of a novel photo-cleavable analogue of BAPTA (1,
Scheme 1). 3-(Hydroxynaphth-2-yl)methyl ether servers as
photolabile linker connecting two aminodicarboxyl “claws”. Upon
excitation, the former ƵŶĚĞƌŐŽĞƐꢀǀĞƌLJꢀĨĂƐƚꢀ;׬ΕϭϮꢀђƐͿꢀƐĐŝƐƐŝŽŶꢀŽĨꢀƚŚĞꢀ
benzylic ether bond to produce o-napthoquinone methide (o-NQM,
structure highlighted in the intermediate 2).12 3-(Hydroxynaphth-2-
yl)methyl derivatives are, therefore, dubbed NQMPs for o-
NaphthoQuinone Methide Precursors. In neutral aqueous solutions,
o-NQMƐꢀ ƵŶĚĞƌŐŽꢀ ƌĂƉŝĚꢀ ;׬Εϳꢀ ŵƐͿꢀ ŚLJĚƌĂƚŝŽŶꢀ ƚŽꢀ ƉƌŽĚƵĐĞꢀ ϯ-
hydroxymethyl-2-haphthols (e.g., 3, Scheme 1). NQMP derivatives
13
show no dark- and photo-cytotoxicity below 100 ђD͘
The synthesis of NQMP-BAPTA starts from commercially available
methyl 3-hydroxy-2-naphthanoate (4, Scheme 2). The protection of
a.Department of Chemistry, University of Georgia, Athens, GA 30677, USA.
Electronic Supplementary Information (ESI) available: Detailed synthetic procedures
and NMR spectra of newly prepared compounds. See DOI: 10.1039/x0xx00000x
This journal is © The Royal Society of Chemistry 20xx
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DOI: 10.1039/C7CC02056B
HO
H
N
O2
O
Me
CO2
e
d
O
f
c
b
O
OH
OH
OEOM
OEOM
OR
O
OEOM
OEOM
8
7
6
NO2
9
4 R= H
,
a
OEOM
5 R= CH OEt
,
10
(EOM)
2
O
O
O
O
H2N
MeO
N
2C
HO
N
O
2C
O
O
j
O
O
k
O
O
h
MeO2C
g
N
N
HO
Ca
2C
O
O
O
O
O
NH2
N
Me
CO2
N
H
CO2
OEOM
11
OR
OH
Me
CO2
OH
12 R=CH OEt
13 R= H
,
,
H
CO2
1
2
i
Scheme 2. Reagents and conditions: a) i) NaH, ii) chloromethyl ethyl ether, 99 %; b) LiAlH4, 72 %; c) PCC, AcONa, molecular sieves, DCM,
89%; d) Trimethylsulfonium iodide, NaH, DMSO, THF, 99%; e) DMF-H2O, 100 °C, 60%; f) 1-Fluoro-2-nitrobenzene , NaH, DMF, 120 °C, 78%;
g) Zn, AcOH, MeOH, THF, 98%; h) Methyl bromoacetate, 1,8-bis(dimethylamino)naphthalene, NaI, 75%; i) Amberlyst-15(H), MeOH, 63%; k)
KOH, MeOH, 1,4-dioxane, quant.; l) CaCO3, HEPES/KCl.
the phenolic hydroxyl with ethoxymethyl chloride (EOMCl), followed (scheme 3).15 The photoproducts 14, 16, and 17 were isolated in the
by LAH reduction of the ester produced 3-(hydroxymethyl)-2- preparative photolysis of 13 and fully characterized.15 The
naphthyl EOM ether (6).14 The PCC oxidation of the latter gave identification of 16 required the independent preparation,15 as
naphthaldehyde 7, which was converted to epoxide 8 under Corey- spectroscopic data corresponding to the structure of the lactone was
Chaykovsky conditions in an excellent yield.
previously assigned to a lactam isomer of 16.16 Phenol 15 was also
Several attempts of the nucleophilic ring opening of epoxide 8 with prepared independently, however, it always contains impurities of
2-nitrophenolate produced only mediocre yields of o-nitrophenyl lactone 16; and on silica gel, it forms lactone 16 quantitatively.
ether.15 We, therefore, employed an alternative approach to the Based our previous experience with NQMP derivatives, we believe
introduction of 2-nitrobenzyl moieties. First, the hydrolysis of 8 that the initial photo-cleavage of 13 occurs through the excited state
produced diol 9, which, in turn, was treated with 2 equivalents of o- scission of the benzylic C-O bond. This results in the formation of
fluoronitrobenzene to give bis(o-nitrophenylether) 10. The nitro phenol 15 and o-aminophenoxymethene-N,N-diaacetic acid
groups of the latter were quantitatively reduced by zinc in acetic acid. derivative of o-naphthoquinone methide (NQM-APDA, 2, Scheme 1).
The alkylation of amino groups in resulting bis-aniline 11 with methyl The latter then adds water to form NQMP-APDA (3, Scheme 1). The
bromoacetate, followed by the deprotection of naphthol with isolated ketone 13 (Scheme 3), is an apparent product of the
Amberlyst-15(H), produced NQMP-BAPTA tetra-methyl ester (13). oxidation of the benzylic hydroxy group in 3 by atmospheric oxygen.
Finally, saponification of the latter gave a quantitative yield of the Second primary product, phenol 15, apparently undergoes
target NQMP-BAPTA chelator (1). Tetra-acid 1 in the neat form slowly intramolecular trans-esterification to produce lactone 16. The
decomposes on prolonged storage. Its potassium salt, however, is detailed investigation of the mechanism of NQMP-BAPTA tetra-ester
perfectly stable at room temperature.
photo-transformations is underway in our laboratory.
The photochemical behavior of NQMP-BAPTA was first studied on The Ca2+ affinity of NQMP-BAPTA (1) was determined by
the example of its tetra-ester 13. Above 250 nm, the UV spectrum of spectrophotometric titration at Ɖ,сϳ͘ϰ. The UV spectrum of
13 in 50% aqueous acetonitrile contains two pairs of close-lying ďƵĨĨĞƌĞĚꢀƐŽůƵƚŝŽŶꢀ;,ꢁWꢁ^͕ꢀƉ,сꢀϳ͘ϰͿꢀŽĨꢀƚŚĞꢀƚĞƚƌĂ-potassium salt of 1 is
ďĂŶĚƐꢀŽĨꢀƐŝŵŝůĂƌꢀŝŶƚĞŶƐŝƚLJ͗ꢀĂƚꢀϮϳϴꢀĂŶĚꢀϮϴϴꢀŶŵꢀ;ůŽŐꢀɸꢀсꢀϯ͘ϴϰꢀĂŶĚꢀϯ͘ϴϮͿ͕ꢀ
as well as at ϯϮϬꢀĂŶĚꢀϯϯϮꢀŶŵꢀ;ůŽŐꢀɸꢀс 3.25 and 3.33).15 The irradiation
of a solution of 13 in 50% aqueous acetonitrile with 300 nm
fluorescent lamps resulted in the efficient cleavage of the starting
ŵĂƚĞƌŝĂůꢀ;ɌꢀсꢀϬ͘ϲϯ±0.03). After 5 min of irradiation, no traces of tetra-
ester 13 could be detected in the photolysate. HPLC analysis of the
reaction mixtures at low conversion (90 s of irradiation), allowed us
to identify two initial photoproducts: ketone 14 and o-
dicarbmethoxymethylamino-phenol 15. Phenol 15 is rather unstable
and undergoes rapid acid-catalysed cyclization to lactone 16.
Exhaustive irradiation of the reaction mixture led to the formation of
an additional photoproduct, 3-hydroxy-2-naphthaldehyde (17)
Me
CO2
MeO2C
MeO2C
N
N
N
Me
CO2
+
MeO2C
MeO2C
Q
O
h
O
OH
15
O
O
OHQ
OH
14
N
h
Me
CO2
MeO2C
Me
CO2
N
13
O
OH
O
O
17
16
Scheme 3. Conditions: 300 nm, CH3CN/water, 1:1.
2 | J. Name., 2012, 00, 1-3
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0.062
0.060
0.058
0.056
0.054
0.052
0.050
0.048
View Article Online
DOI: 10.1039/C7CC02056B
1.00
0.75
0.50
0.25
0.00
K= (2.46±0.08)*106 M-1
2.5x10-6
5.0x10-6
7.5x10-6
1.0x10-5
0.0
300
325
350
375
Ca2+ (M)
Wavelength (nm)
Figure 2. Spectrophotometric titration of 2.16 ʅM aqueous
solution of NQMP-BAPTA with 0.25 mM of CaCl2.
Figure 1. UV spectra of 0.2 mM aqueous solution of potassium salt
of 1 ĂƚꢀƉ,ꢀсϳ͘ϰꢀĂƚꢀǀĂƌŝĂďůĞꢀꢂĂ2+ concentrations.
similar to that of ester 13͕ꢀŽŶůLJꢀďůƵĞꢀƐŚŝĨƚĞĚꢀďLJꢀΕϮꢀŶŵ: bands at 276, midpoint of titration, determined from the absorbance values vs.
286, and ϯϯϭꢀŶŵꢀ;Ŷŵꢀ;ůŽŐꢀɸꢀсꢀϯ͘ϴϲ, 3.77, and 3.26) are observed.15 [Ca2+]/[1] ratio plot, indicates 1:1 binding stoichiometry (Insert in
dŚĞꢀďĂŶĚꢀǁŝƚŚꢀʄmaxсꢀϯϭϴꢀŶŵꢀďůĞŶĚƐꢀǁŝƚŚꢀďƌŽĂĚꢀabsorbance around Figure 2).
300 nm. Upon the increase in Ca2+ ion concentration, absorbance The release of calcium ions from NQMP-BAPTA:Ca2+ complex under
around 300 nm is progressively reduced, and 331 nm band suffers irradiation was explored with the help of Calcein indicator. The
red shift (Figure 1). These changes saturate at higher Ca2+ binding constant of the latter is 3-4 orders of magnitude lower than
concentrations. The final UV spectrum Ca2+:NQMP-BAPTA complex that of NQMP-BAPTA,17 thus calcein only forms the complex with
contains three absorbance bands ĂďŽǀĞꢀϮϱϬꢀŶŵ͗ꢀϮϲϵꢀ;ůŽŐꢀɸꢀсꢀϯ͘ϴϬͿ͕ꢀ free Ca2+ ions in solution. Since the response of this indicator is
ϮϳϳꢀŶŵꢀ;ůŽŐꢀɸꢀсꢀϯ͘ϴϰͿ͕ꢀĂŶĚ 333 nm (ůŽŐꢀɸꢀсꢀϯ͘ϯϵ͕ꢀ&ŝŐƵƌĞꢀϭ͘Ϳ15
known to depend on the media, we have built the calibration curve
Due to the high calcium affinity of NQMP-BAPTA, the titration of 1 under the conditions of irradiation.15 An aqueous solution (HEPES
ǁĂƐꢀ ĐŽŶĚƵĐƚĞĚꢀ Ăƚꢀ Ϯ͘ϭϲꢀ ʅDꢀ ĐŽŶĐĞŶƚƌĂƚŝŽŶꢀ ŽĨꢀ ƚŚĞꢀ ƐƵďƐƚƌĂƚĞ͘ꢀ dŚĞꢀ ďƵĨĨĞƌ͕ꢀƉ,сꢀϳ͘ϰ͕ꢀ/^ꢀсꢀϬ͘ϭꢀDͿꢀcontaining 1.13 mM of NQMP-BAPTA and
buffered solution of potassium salt of 1 ;,ꢁWꢁ^͕ꢀƉ,сꢀϳ͘ϰͿꢀwas titrated 1.25 mM of CaCl2 were irradiated with 300 nm fluorescent lamps.
with 0.25 mM of CaCl2 following the changes in absorbance at 300 The 3.5 mL samples were exposed to UV light for various duration of
nm (Fig. 2).
time, from 30 s to 8 min, and a solution of Calcein was added to each
The absorbance of the NQMP-BAPTA solution at 300 nm kept sample to achieve 0.7 nm concentration of the indicator. The
decreasing with successive addition of CaCl2 until it levels off above concentration of free calcium in solution was calculated from the
ϭϬꢀђDꢀꢂĂ2+ concertation (Figure 2). The calcium ion binding constant observed fluorescence intensity at 513 nm. As Figure 3 illustrates, the
of NQMP-BAPTA was calculated using eq. 1:
photorelease of calcium from NQMP-BAPTA occurs in a dose-
Eq. 1 dependent manner and is essentially complete after 6 min of
irradiation.
The
increase
of
calcium
concentration
1
ꢃ
1
ꢃ
ꢅ
([ꢀ]ꢂ + [ꢁ]ꢂ +
2
)
([ꢀ]ꢂ + [ꢁ]ꢂ +
4
)
ꢄ
[
]
ꢀꢁ =
+
ꢆ [ꢀ]ꢂ[ꢁ]ꢂ
Where [LM] is the equilibrium concentration of NQMP-BAPTA (1) –
Ca2+ complex, [L]0 and [M]0 are stoichiometric concentrations of
ligand 1 and Ca2+, K is a binding constant. We assumed that the
difference in absorbance of the solution of 1 at [Ca2+΁ꢀсꢀϬ (A0) and
at [Ca2+] > 10 ђDꢀ ;Aꢁ) is mostly due to the differences in the
extinction coefficients between the free and fully complexed ligand.
The changes of the absorbance at 300 nm in the course of the
titration is, therefore, represented by the equation 2.
ꢇ = ꢇꢂ ꢆ [ ] (ꢇꢋ ꢆ ꢇꢂ)
Eq. 2
ꢈꢉ
[ꢈ]ꢊ
Fitting of the experimental data to the equations 1 and 2 allowed us
to calculate Kbndсꢀ(2.46±0.08)x106 M-1 ;ĂƚꢀƉ,сꢀϳ͘ϰꢀĂŶĚꢀŝŽŶŝĐꢀƐƚƌĞŶŐƚŚꢀ
of 0.1 M). This value is similar to the parent BAPTA ligand.10a The
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9
S. R. Adams, J. P. Y. Kao, G. Grynkiewicz, A. Minta and R. Y. Tsien,
Figure 3. Photo-release of Ca2+ from the NQMP-BAPTA (1) - Ca2+
complex under 300 nm irradiation.
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DOI: 10.1039/C7CC02056B
J. Am. Chem. Soc., 1988, 110, 3212.
10 (a) R. Y. Tsien, Biochemistry, 1980, 19, 2396; (b) R. Pethig, M.
Kuhn, R. Payne, E. Adler, T.-H. Chen and L. Jaffe, Cell Calcium, 1989,
10, 491; (c) L. Wu, Y. Dai and G. Marriott, Organic Lett., 2011, 13,
2018; (d) S.R. Adams, V. Lev-Ram, and R. Y. Tsien, Chem. Biol., 1997,
4, 867; (e) J. Cui, R. A. Gropeanu, D. R. Stevens, J. Rettig and A. d.
Campo, J. Am. Chem. Soc., 2012, 134, 7733.
11 Y. V. Il'ichev, M. A. Schwörer, J. Wirz, J. Am. Chem. Soc. 2004, 126,
4581.
12 S. Arumuqam and V. V. Popik, J. Am. Chem. Soc., 2012, 134, 8408.
13 S. Arumugam and V. V. Popik J. Am. Chem. Soc., 2011, 133, 5573.
14 E. E. Nekongo and V. V. Popik, J. Org. Chem., 2014, 79, 7665.
15 Supporting Information
under given conditions can be approximated by a first-order rate law
to give the apparent rate constant of (7.4±0.3)*10-3 s-1. It is important
to note that Ca2+ is released quantitatively. This observation also
indicates that the fragmentation of NQMP-BAPTA results in at least
five orders of magnitude loss of calcium ion affinity (well below Kbnd
of calcein).
In summary, we have developed the first photo-cleavable analog of
BAPTA Ca2+chelator, NQMP-BAPTA (1), by incorporating photo-labile
3-(hydroxymethyl)-2-naphthol linker in the structure. NQMP-BAPTA
possess very high calcium affinity (Kbndсꢀ Ϯ͘ϰϲΎϭϬ6 M-1), but upon
irradiation, 1 looses one of its tridentate chelating arms resulting in
the efficient calcium ion release. dŚĞꢀŚŝŐŚꢀƋƵĂŶƚƵŵꢀLJŝĞůĚꢀ;ˇꢀсꢀϬ͘ϲϯͿꢀ
16 E. Cielen, A. Stobiecka, A. Tahri, G. J. Hoornaert, F. C. De
Schryver, J. Gallay, M. Vincent and N. Boens, Perkin Trans. 2, 2002,
The NQMP linker photo- 6, 1197.
of the NQMP-BAPTA cleavage makes it one of the most efficient of
photosensitive calcium-ion chelators.
7,18
cleavage occurs on the microsecond time scale making NQMP-BAPTA
suitable for time-resolved experiments.
17 V.C. Chiu and D.H. Haynes, Biophys. J., 1977, 18, 3.
18 S. R. Adams, V. Lev-Ram, and R. Y. Tsien, Chem. Biol. 1997, 4, 867.
Notes and reference
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J. H. Kaplan and G. C. R. Ellis-Davies, Proc. Natl. Acad. Sci. USA,
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A. Barth, S. R. Martin and J. E. T. Corrie, Photochem. Photobiol.
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G. C. R. Ellis-Davies and R. J. Barsotti, Cell Calcium, 2006, 39, 75;
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4 | J. Name., 2012, 00, 1-3
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