Increasing the dynamic range of metal ion affinity changes in Zn 2+ photocages using multiple nitrobenzyl groups

Article abstract of DOI:10.1021/ic400465g

Two generations of DiCast photocages that exhibit light-induced decreases in metal ion affinity have been prepared and characterized. Expansion of the common Zn²⁺ chelator of N,N-dipicolylaniline (DPA) to include additional aniline ligand provides N,N′-diphenyl-N,N′-bis(pyridin-2- ylmethyl)ethane-1,2-diamine, a tetradentate ligand that was functionalized with two photolabile groups to afford DiCast-1. Uncaging of the nitrobenzhydrol reduces the electron density on two metal-bound aniline ligands, which decreases the Zn²⁺ affinity 190-fold. The analogous MonoCast photocage with a single nitrobenzhydrol group only undergoes a 14-fold reduction in affinity after an identical photochemical transformation. A second series of DiCast photocages based on a N,N′-(pyridine-2,6-diylbis(methylene))dianiline scaffold, which allows the introduction of two additional Zn²⁺- binding ligands into a preorganized chelator, expand on the multi-photolabile group strategy. DiCast-2 includes two pyridine ligands while DiCast-3 adds two carboxylate groups. Addition of bridging pyridine to the second generation photocages leads to more stable Zn²⁺ complexes, and photolysis of two photolabile groups increases the Zn²⁺ affinity changes to 480-fold. The Zn²⁺, Cu²⁺, and Cd²⁺ binding properties were examined in all the DiCast photocages and the corresponding photoproducts using UV-vis spectroscopy. Further insight into the photocage Zn ²⁺-binding motifs was obtained by X-ray analysis of DiCast-2 and DiCast-3 model ligands.

Full text of DOI:10.1021/ic400465g

Article
pubs.acs.org/IC
Increasing the Dynamic Range of Metal Ion Affinity Changes in Zn2+
Photocages Using Multiple Nitrobenzyl Groups
Celina Gwizdala,† Prem N. Basa,‡ John C. MacDonald,‡ and Shawn C. Burdette*,‡
‡Department of Chemistry and Biochemistry, Worcester Polytechnic Institute, 100 Institute Road, Worcester, Massachusetts
01609-2280, United States
†Department of Chemistry, University of Connecticut, 55 North Eagleville Road U-3060, Storrs, Connecticut 06269, United States
S
* Supporting Information
ABSTRACT: Two generations of DiCast photocages that exhibit light-induced decreases in metal ion affinity have been
prepared and characterized. Expansion of the common Zn2+ chelator of N,N-dipicolylaniline (DPA) to include additional aniline
ligand provides N,N′-diphenyl-N,N′-bis(pyridin-2-ylmethyl)ethane-1,2-diamine, a tetradentate ligand that was functionalized
with two photolabile groups to afford DiCast-1. Uncaging of the nitrobenzhydrol reduces the electron density on two metal-
bound aniline ligands, which decreases the Zn2+ affinity 190-fold. The analogous MonoCast photocage with a single
nitrobenzhydrol group only undergoes a 14-fold reduction in affinity after an identical photochemical transformation. A second
series of DiCast photocages based on a N,N′-(pyridine-2,6-diylbis(methylene))dianiline scaffold, which allows the introduction
of two additional Zn2+-binding ligands into a preorganized chelator, expand on the multi-photolabile group strategy. DiCast-2
includes two pyridine ligands while DiCast-3 adds two carboxylate groups. Addition of bridging pyridine to the second
generation photocages leads to more stable Zn2+ complexes, and photolysis of two photolabile groups increases the Zn2+ affinity
changes to 480-fold. The Zn2+, Cu2+, and Cd2+ binding properties were examined in all the DiCast photocages and the
corresponding photoproducts using UV−vis spectroscopy. Further insight into the photocage Zn2+-binding motifs was obtained
by X-ray analysis of DiCast-2 and DiCast-3 model ligands.
disruptions can produce severe consequences leading to
imbalances in intracellular [Zn2+]. Excess Zn2+ can accumulate
within cells and lead to damage and death. We have focused on
designing new Zn2+ photocages to explore potential signaling
functions and disease pathology.11−14
INTRODUCTION
■
Photocages are indispensable tools for studying molecular
biology.1−3 The light-induced release of bioactive species allows
time and delivery location to be controlled. Such photo-
chemical tools permit quantitative manipulation of bioactive
species with greater precision than alternative methods. While
photocaged complexes have facilitated significant insight into
Ca2+ signaling pathways,2,4 other metal ions like Zn2+, Fe2+, and
Cu+ have equal importance in complex cellular processes, but
the development of analogous tools only began recently.5,6
Free or chelatable zinc is present in the mammalian forebrain
where it is stored within the synaptic vesicles of glutamatergic
neurons.7 Neuronal activity releases vesicular zinc into the
synaptic cleft, which modulates the activity of many synaptic
targets. While synaptic zinc functions have not been elucidated
fully, Zn2+ can bind to GABA (γ-aminobutyric acid) and
NMDA (N-methyl-D-aspartate) receptors as well as regulate
activity of many postsynaptic channels.8 Zinc may function as a
neural signaling agent in healthy individuals, but it may also be
involved in many neurological diseases.9,10 Homeostasis
Inspired by the Nitr family of photocages for Ca2+, we
developed ZinCast-1,13 a Zn2+ photocage that integrates a N,N-
dipicolylaniline (DPA) ligand with a light sensitive o-nitro-
benzyl moiety. The metal ion release in [Zn(ZinCast-1)]2+
relies on attenuation of the aniline nitrogen atom−Zn2+ bond
strength upon light-initiated formation of the nitrosobenzo-
phenone photoproduct [Zn(ZinUnc-1)2+] (Scheme 1). The
resonance delocalization of the anilino lone pair onto the newly
formed carbonyl group decreases the Zn2+ affinity and shifts the
binding equilibrium toward solvated metal ion. Change in metal
affinity due to photolysis can be expressed quantitatively by
ΔKd, which is a ratio of photoproduct’s dissociation constant
Received: February 21, 2013
Published: July 10, 2013
© 2013 American Chemical Society
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Article
utilize crown ether ligands.15,16 These results indicate that
increased chelator affinity in the Zn2+ complexes reduces metal
ion release efficiency. Complexes containing more weakly
binding chelators exhibit significantly larger changes in Zn2+
affinity upon photolysis, but the low affinity prohibits use in
biology where proteins maintain Zn2+ homeostasis at minimal
basal levels.17 Addressing these practical problems requires
designing a photocage possessing a high affinity for Zn2+ that
decreases drastically upon uncaging.
Scheme 1. Uncaging process in ZinCast-1
To maximize the Zn2+ offloading capability with concurrent
preservation of pre-photolysis metal ion buffering capacity, we
envisioned introducing a second o-nitrobenzyl photocaging
group. Utilization of two photolabile groups in photocages has
precedent in organic synthesis18 as well as in small molecule
photocages.19 In the first Ca2+ photocage utilizing two
photolabile groups Nitr-8, the affinity of the Ca2+-selective
chelator decreases 1600-fold upon uncaging; however, Nitr-8
was reported in a review that lacked extensive characterization
data.4 In the structurally related photocage Nitr-T that also
contains two nitrobenzyl groups, the Ca2+ affinity decreases
>3000-fold from the pre-photolysis Ca2+ Kd of 520 nM.20 The
photoproducts resulting from photolysis of Nitr-8 and Nitr-T
photocages contain two metal-bound aniline nitrogen atoms
with reduced electron density compared to the corresponding
photocaged complex. While inclusion of a second photocaging
group has been implemented for Ca2+, this strategy has not
(ZinUnc Kd) to the dissociation constant of the photocage
(ZinCast Kd), ΔKd = (ZinUnc Kd)/(ZinCast Kd).
Additional complementary ZinCast photocages differ only in
Zn2+-receptor design, where the methylene linker between
aniline nitrogen and the pyridine moiety of ZinCast-1 was
extended to an ethylene on one (ZinCast-2) or both spacers
(ZinCast-3).14 While all three ZinCast photocages exhibit
reduced Zn2+ affinity following photolysis, the most pro-
nounced changes occur in ZinCast-3, which has the lowest Zn2+
affinity. In contrast, ZinCast-1 forms the highest affinity pre-
photolysis Zn2+ complex, but a reduced ΔKd upon uncaging.
An analogous trend was observed for Cast type photocages that
Figure 1. (A) Structures of Ca2+ photocages Nitr-8 and Nitr-T that utilize two nitrobenzyl groups. (B) Structures of MonoCast, DiCast-1, DiCast-2,
and DiCast-3. MonoCast and DiCast-1 allow the direct comparison of the effects from using a single versus two nitrobenzyl groups. DiCast-2 and
DiCast-3 are second generation DiCast photocages designed to have higher affinity for Zn2+.
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N-Phenyl-N,N′-bis(pyridin-2-ylmethyl)-N′-(p-tolyl)ethane-1,2-dia-
mine (10). To a solution of 9 (0.464 g, 2.05 mmol) in CH3CN (30
mL) were added 2-(bromomethyl)pyridine hydrobromide (1.55 g,
6.15 mmol), potassium hydrogen phosphate (K2HPO4) (2.14 g, 12.3
mmol), and KI (0.34 g, 2.05 mmol). After the reaction mixture was
refluxed overnight, the solvent was removed, water (30 mL) was
added, and pH was adjusted to approximately 9. The crude product
was extracted with EtOAc (3 × 50 mL) and dried over MgSO4. Flash
chromatography on basic alumina (2:3 EtOAc/hexanes) yielded 10 as
a yellow solid (0.653 g, 75.4%). Mp: 90−91 °C. 1H NMR (400 MHz,
CDCl3): δ 8.55 (s, 2 H), 7.52 (t, J = 4.0 Hz, 2 H), 7.17−7.11 (m, 6 H),
6.97 (d, J = 8.0 Hz, 2 H), 6.68 (m, 3 H), 6.60 (d, J = 8.0 Hz, 2 H), 4.64
(d, J = 8.0 Hz, 4 H), 3.73 (s, 4 H), 2.20 (s, 3 H). 13C NMR (100 MHz,
CDCl3): δ 159.47, 159.26, 149.85, 149.79, 147.96, 145.82, 136.90,
130.19, 129.65, 126.40, 122.18, 122.15, 121.15, 121.04, 117.07, 112.39,
57.54, 57.28, 49.08, 48.98, 20.38. IR (KBr pellet, cm−1): 1616, 1590,
1570, 1520, 1505, 1475, 1459, 1445, 1431, 1386, 1344, 1211, 1167,
994, 941. HRMS (+ESI): calcd for MH+, 409.2392; found, 409.2389.
(4,5-Dimethoxy-2-nitrophenyl)(4-((pyridin-2-ylmethyl)(2-((pyri-
din-2-ylmethyl)(p-tolyl)amino)ethyl)amino)phenyl)methanol
(MonoCast, 3). Trimethylsilyl trifluoromethanesulfonate (TMSOTf)
(0.74 mL, 4.08 mmol) and 2,6-lutidine (0.63 mL, 5.44 mmol) were
added successively to a mixture of 10 (0.575 g, 1.36 mmol) and 6-
nitroveratraldehyde (11, 0.374 g, 1.76 mmol) in CH2Cl2 (40 mL). The
resulting solution was stirred for 6 h, and then tetrabutylammonium
fluoride (TBAF) in THF (1 M, 4.1 mL, 4.08 mmol) was added to the
reaction mixture. After 1 h, the solvent was removed under reduced
pressure. Flash chromatography on basic alumina (1:4 hexanes/
EtOAc) afforded 3 as a yellow solid (0.632 g, 75.0%). Mp: 136−137
°C. 1H NMR (400 MHz, CDCl3): δ 8.52 (s, 2 H), 7.58 (s, 1 H), 7.51
(t, J = 4.0 Hz, 2 H), 7.39 (s, 1 H), 7.11−7.05 (m, 6 H), 6.95 (d, J = 8.0
Hz, 2 H), 6.57 (d, J = 8.0 Hz, 4 H), 6.45 (d, J = 4.0 Hz, 1 H), 4.59 (d, J
= 4.0 Hz, 4 H), 3.94 (s, 3 H), 3.91 (s, 3 H), 3.69 (s, 4 H), 2.86 (d, J =
4.0 Hz, 1 H), 2.19 (s, 3 H). 13C NMR (100 MHz, CDCl3): δ 159.33,
158.91, 153.55, 149.82, 149.72, 147.87, 147.63, 145.67, 140.10, 136.96,
135.02, 130.52, 130.19, 128.68, 126.45, 122.25, 122.19, 121.15, 120.99,
112.67, 112.12, 110.31, 108.30, 71.45, 57.46, 57.13, 56.63, 56.55,
48.97, 20.37. IR (KBr pellet, cm−1): 3105, 1596, 1570, 1520, 1507,
1470, 1433, 1360, 1335, 1276, 1248, 1235, 1211, 1159, 1066, 1027.
HRMS (+ESI): calcd for MH+, 620.2828; found, 620.2819.
(4,5-Dimethoxy-2-nitrosophenyl)(4-((pyridin-2-ylmethyl)(2-((pyri-
din-2-ylmethyl)(p-tolyl)amino)ethyl)amino)phenyl)methanone
(MonoUnc, 12). Photolysis of 3 (57 mg, 0.92 mmol) in 3 mL of
CH3CN for 8 h followed by chromatography on basic alumina (1:1
EtOAc/hexanes) yielded MonoUnc as an orange oil (26 mg, 47%). 1H
NMR (400 MHz, CDCl3): δ 8.53 (t, J = 8.0 Hz, 2 H), 7.66 (d, J = 8.0
Hz, 2 H), 7.53 (q, J = 4.0 Hz, 2 H), 7.13−7.08 (m, 4 H), 7.02−6.96
(m, 3 H), 6.59 (t, J = 8.0 Hz, 4 H), 6.30 (s, 1 H), 4.66 (s, 2 H), 4.58 (s,
2 H), 4.01 (s, 3 H), 3.88 (s, 3 H), 3.79 (d, J = 4.0 Hz, 2 H), 3.72 (d, J
= 4.0 Hz, 2 H), 2.20 (s, 3 H). 13C NMR (100 MHz, CDCl3): δ 193.99,
160.05, 159.05, 157.52, 156.01, 152.23, 150.16, 150.09, 149.78, 145.60,
140.14, 137.12, 136.96, 132.90, 130.28, 127.73, 126.93, 122.57, 122.31,
121.33, 120.81, 112.97, 111.19, 109.82, 91.90, 57.67, 77.55, 77.23,
76.92, 57.67, 57.00, 56.91, 56.42, 49.08, 48.80, 20.39. IR (KBr, cm−1):
2926, 1611, 1590, 1570, 1463, 1434, 1328, 1268, 1207, 1179, 1062,
908, 796. HRMS (+ESI): calcd for MH+, 602.2767; found, 602.2771.
N,N′-Diphenyl-N,N′-bis(pyridin-2-ylmethyl)ethane-1,2-diamine
(14). Synthesis of 14 followed the same procedure as synthesis of 10
using compound 13 (1.01g, 4.76 mmol), 2-(bromomethyl)pyridine
hydrobromide (3.00g, 11.9 mmol), potassium hydrogen phosphate
(K2HPO4) (4.14 g, 23.8 mmol), and KI (0.789 g, 4.76 mmol). Flash
chromotography on basic alumina using a solvent gradient (1:2 to 3:2
EtOAc/hexanes) afforded 14 as a white crystalline solid (1.26 g,
64.9%). Mp: 141−142 °C. 1H NMR (400 MHz, CDCl3): δ 8.56 (d, J
= 8.0 Hz, 2 H), 7.54 (t, J = 8.0 Hz, 2 H), 7.18−7.12 (m, 8 H), 6.70−
6.67 (m, 6 H), 4.66 (s, 4 H), 3.76 (s, 4 H). 13C NMR (100 MHz,
CDCl3): δ 159.20, 149.87, 147.95, 16.93, 129.67, 122.21, 121.07,
117.16, 112.43, 57.29, 48.90. IR (KBr pellet, cm−1): 1597, 1588, 1571,
1506, 1469, 1443, 1432, 1394, 1351, 1341, 1215, 1170, 991, 941, 750.
HRMS (+ESI): calcd for MH+, 395.2236; found, 395.2221.
been explored for other metal ions with different coordination
chemistry requirements.
By combining the multi-photolabile group strategy utilized in
Ca2+ photocages with Zn2+-binding properties of ZinCast-1,
new photocaged chelators were envisioned with increased Zn2+
stability and increased ΔKd upon uncaging. The DiCast
photocage nomenclature evokes the original Cast ser-
ies,13−16,21−23 where the suffix implies release by “casting off”,
and adds the prefix “Di-” indicating the presence of two
nitrobenzyl groups. To quantify the magnitude of ΔKd changes
induced by two photocaging groups, MonoCast-1 and DiCast-1
were designed to mimic elements of the 3-nitrogen donor-
based DPA ligand of ZinCast-1 and the 4-nitrogen donor
chelator EBAP (ethylene-bis-α,α′-(2-aminomethyl)pyridine). A
modular synthetic strategy provided access to derivatives that
could be functionalized with 1 or 2 nitrobenzyl groups. Since
the receptor in MonoCast-1 and DiCast-1 contains only an
additional aniline ligand when compared to ZinCast-1, only a
modest increase in absolute Zn2+ affinity was anticipated, but
the photocages would allow a direct comparison of ΔKd trends.
To increase photocage metal ion affinity, a second generation
of DiCast photocages was designed with additional chelating
ligands (Figure 1). Replacement of the ethylene linker in
DiCast-1 chelator by a bridging pyridine leads to increase in
ligand denticity and reduces ligand flexibility to further increase
binding strength. DiCast-2 retains the aniline and pyridine
ligands; however, carboxylates were used in DiCast-3 to
enhance water solubility.
EXPERIMENTAL SECTION
■
General Experimental. All materials were purchased from Acros
Organic or TCI America. Solvents were sparged with argon and dried
in Seca Solvent Purification System. N,N′-Diphenylethane-1,2-diamine
(13)24−26 and 2,6-bis-(bromomethyl)pyridine (16)27 was prepared as
previously described. Bulk photolysis was carried out using a 1000 W
lamp with λ = 300−800 nm. All chromatography and TLC were
performed on silica from Silicycle or activated basic alumina from
Acros Organics. 1H and 13C NMR spectra were recorded with a Bruker
400 MHz spectrometer and referenced to CDCl3. IR spectra were
recorded on a Nicolet 205 FT-IR instrument, and samples were
analyzed as KBr pellets. High resolution mass spectra were obtained
on a Waters Micromass Q-Tof-2 mass spectrometer at the University
of Connecticut Biotechnology-Bioservices Center or a Bruker
micrOTOF II at the University of Notre Dame Mass Spectrometry
& Proteomics Facility operating in positive ion mode. The instrument
was calibrated with Glu-fibrinopeptide B 10 pmol/μL by using a 50:50
solution of CH3CN/H2O with 0.1% acetic acid.
N-Phenyl-N′-(p-tolyl)ethane-1,2-diamine (9). Tris-
(dibenzylideneacetone)dipalladium (Pd2(dba)3) (40 mg, 1 mol %),
2,2′-bis(diphenylphosphino)-1,1′-binaphthyl (BINAP, 82 mg, 3 mol
%), NaOtBu (1.27 g, 1.32 mmol), and degassed toluene (15 mL) were
combined in a Schlenk tube. N-Phenylethylenediamine (7, 636 μL,
4.86 mmol) and p-bromotoluene (8, 544 μL, 4.42 mmol) were
successively added, and the reaction mixture was stirred at 80 °C. After
24 h, the reaction mixture was cooled, diluted with H2O (30 mL),
extracted with EtOAc (3 × 30 mL), and dried over MgSO4 and the
solvent was removed. Flash chromatography on silica (1:4 EtOAc/
hexanes) yielded 9 as a yellow solid (0.481 g, 48.1%). Mp: 44−45 °C.
1H NMR (400 MHz, CDCl3): δ 7.18 (t, J = 8.0 Hz, 2 H), 7.00 (d, J =
8.0 Hz, 2 H), 6.72 (t, J = 8.0 Hz, 1 H), 6.64 (d, J = 8.0 Hz, 2 H), 6.57
(d, J = 8.0 Hz, 2 H), 3.78 (s, 2 H), 3.37 (s, 4 H), 2.24 (s, 3 H). 13C
NMR (100 MHz, CDCl3): δ 148.31, 145.99, 130.04, 129.54, 127.33,
117.99, 113.46, 113.24, 43.93, 43.61, 20.59. IR (KBr pellet, cm−1):
3418, 3408, 1601, 1525, 1459, 1341, 1294, 1263, 1209, 1178, 1135,
1090. HRMS (+ESI): calcd for MH+, 227.1548; found, 227.1552.
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((Ethane-1,2-diylbis((pyridin-2-ylmethyl)azanediyl))bis(4,1-
phenylene))bis((4,5-dimethoxy-2-nitrophenyl)methanol) (DiCast-1,
4). Synthesis of DiCast-1 followed the same procedure as synthesis of
MonoCast using 14 (0.310 g, 0.759 mmol), 11 (0.353 g, 1.51 mmol),
2,6-lutidine (0.62 mL, 5.31 mmol), TMSOTf (0.69 mL, 3.80 mmol),
and 1 M TBAF (4.54 mmol). Flash chromatography on basic alumina
(1:99 MeOH/EtOAc) yielded DiCast-1 as an orange solid (0.398 g,
64.2%). Mp: 106−108 °C. 1H NMR (400 MHz, CDCl3): δ 8.50 (d, J
= 4.0 Hz, 2 H), 7.57 (s, 2 H), 7.50 (t, J = 8.0 Hz, 2 H), 7.42 (s, 2 H),
7.11−7.03 (m, 8 H), 6.54 (d, J = 8.0 Hz, 4 H), 6.44 (d, J = 4.0 Hz, 2
H), 4.48 (s, 4 H), 3.94 (s, 6 H), 3.90 (s, 6 H), 3.65 (s, 4 H), 3.16 (d, J
= 4.0 Hz, 2 H). 13C NMR (100 MHz, CDCl3): δ 158.80, 153.58,
149.76, 147.87, 147.50, 140.08, 137.09, 135.05, 130.90, 128.75, 122.33,
121.02, 112.23, 110.28, 108.34, 71.39, 57.11, 56.64, 56.55, 48.94. IR
(KBr pellet, cm−1): 3510, 1611, 1571, 1517, 1462, 1437, 1392, 1330,
1271, 1215, 1179, 1063, 911. HRMS (+ESI): calcd for MH+,
817.3197; found, 817.3338.
((Ethane-1,2-diylbis((pyridin-2-ylmethyl)azanediyl))bis(4,1-
phenylene))bis((4,5-dimethoxy-2-nitrosophenyl)methanone)
(DiUnc-1, 15). Photolysis of 4 (57 mg, 0.070 mmol) for 16 h followed
by flash chromatography on basic alumina (EtOAc) afforded DiUnc-1
as a yellow oil (12 mg, 22%). 1H NMR (400 MHz, CDCl3): δ 8.52 (d,
J = 4.0 Hz, 2 H), 7.67 (d, J = 8.0 Hz, 4 H), 7.56 (t, J = 8.0 Hz, 2 H),
7.14 (t, J = 8.0 Hz, 2 H), 7.08 (s, 2 H), 7.03 (d, J = 8.0 Hz, 2 H), 6.60
(d, J = 8.0 Hz, 4 H), 6.31 (s, 2 H), 4.65 (s, 4 H), 4.01 (s, 6 H), 3.88 (s,
6 H), 3.84 (s, 4 H). 13C NMR (100 MHz, CDCl3): δ 194.10, 160.05,
157.18, 156.08, 151.97, 150.24, 150.17, 139.94, 137.23, 132.95, 128.14,
122.78, 121.02, 111.45, 111.21, 109.81, 107.07, 91.97, 57.03, 56.88,
56.43, 48.84. IR (KBr): 1608, 1587, 1514, 1452, 1381, 1327, 1267,
1217, 1180, 1061, 1023, 1003, 819, 795, 758 cm−1. (+ESI): calcd for
MH+, 781.2986; found, 781.3120.
N,N′-(Pyridine-2,6-diylbis(methylene))dianiline (17). Synthesis of
17 followed the same procedure as synthesis of 10 using 16 (2.16 g,
8.15 mmol), aniline (2.23 mL, 24.4 mmol), K2HPO4 (5.68 g, 32.6
mmol), and KI (1.35 g, 8.15 mmol). The product was extracted with
CH2Cl2 instead of EtOAc. Flash chromatography on silica using a
solvent gradient (1:4 to 2:3 EtOAc/hexanes) yielded 17 as a yellow oil
(1.08 g, 45.8%). 1H NMR (400 MHz, CDCl3): δ 7.58 (t, J = 8.0 Hz, 1
H), 7.22−7.16 (m, 6 H), 6.74 (t, J = 8.0 Hz, 2 H), 6.68 (d, J = 8.0 Hz,
4 H), 4.73 (s, 2 H), 4.47 (s, 4 H). 13C NMR (100 MHz, CDCl3): δ
158.28, 148.11, 137.42, 129.46, 120.06, 117.83, 113.27, 49.45. IR (KBr,
cm−1): 3414, 3050, 1603, 1575, 1506, 1455, 1429, 1317, 1260, 1179,
749, 692. HRMS (+ESI): calcd for MH+, 290.1657; found, 290.1615.
N,N′-(Pyridine-2,6-diylbis(methylene))bis(N-(pyridin-2-ylmethyl)-
aniline) (18). 2-Pyridinecarboxaldehyde (1.05 mL, 18.4 mmol) and 17
(1.07 g, 3.69 mmol) were combined in CH2Cl2 (40 mL) with sodium
triacetoxyborohydride (2.35 g, 18.4 mmol). After 24 h, the reaction
mixture was washed with saturated NaHCO3 (40 mL) and saturated
NaCl (40 mL). The solvent was removed and recrystallization from
EtOAc yielded 18 as a white solid (0.883 g, 50.7%). Mp: 181−182 °C.
1H NMR (400 MHz, CDCl3): δ 8.58 (d, J = 4.0 Hz, 2 H), 7.61−7.51
(d, J = 4.0 Hz, 2 H), 6.20 (d, J = 8.0 Hz, 2 H), 4.80−4.53 (m, 4 H),
3.98 (s, 3 H), 3.96 (s, 3 H), 3.89 (s, 6 H), 3.54−3.49 (m, 4 H). 13C
NMR (100 MHz, CDCl3): δ 160.41, 160.31, 155.88, 153.57, 149.04,
148.96, 147.42, 139.75, 137.91, 137.91, 137.28, 136.57, 131.99, 131.90,
128.62, 122.15, 120.77, 119.80, 112.94, 109.82, 108.34, 71.22, 57.47,
56.67, 56.62, 56.54, 55.10. IR (KBr pellet, cm−1): 3280, 1612, 1576,
1520, 1436, 1384, 132, 1270, 1217, 1179, 1062, 815, 796, 749. HRMS
(+ESI): calcd for MH+, 894.3463; found, 894.2460.
(((Pyridine-2,6-diylbis(methylene))bis((pyridin-2-ylmethyl)-
azanediyl))bis(4,1-phenylene))bis((4,5-dimethoxy-2-nitrosophenyl)-
methanone) (DiUnc-2, 19). Photolysis of 5 (54 mg, 0.060 mmol) for
16 h followed by chromatography on silica (1:99 CH3OH−CH2Cl2)
1
lead to isolation of DiUnc-2 as a yellow oil (10 mg, 19%). H NMR
(400 MHz, CDCl3): δ 8.54 (d, J = 4.0 Hz, 2 H), 7.65 (d, J = 12.0 Hz, 4
H), 7.60−7.55 (m, 3 H), 7.17−7.04 (m, 8 H), 6.61 (d, J = 8.0 Hz, 4
H), 6.28 (s, 2 H), 4.79 (s, 4 H), 3.99 (s, 3 H), 3.86 (s, 3 H), 3.62−3.57
(m, 10 H). 13C NMR (100 MHz, CDCl3): δ 194.12, 160.07, 157.79,
157. 54, 156.08, 152.86, 150.20, 150.14, 138.12, 137.20, 132.74,
128.21, 122.65, 120.96, 119.76, 111.77, 109.83, 91.88, 60.63, 57.32,
57.04, 56.44. HRMS (+ESI): calcd for MH+, 858.3251; found,
858.4223.
Diethyl 2,2′-((Pyridine-2,6-diylbis(methylene))bis-
(phenylazanediyl))diacetate (20). Synthesis of 20 followed the
same procedure as synthesis of 10 using 17 (0.930 g, 3.21 mmol),
ethyl bromoacetate (0.85 mL, 23.3 mmol), K2HPO4 (3.36 g, 19.3
mmol), and KI (0.533 g, 3.21 mmol). The product was extracted with
CH2Cl2 instead of EtOAc. Flash chromotography on silica with a
solvent gradient (1:4 to 2:3 EtOAc/Hex) yielded 20 as a yellow oil
(1.15 g, 77.4%). 1H NMR (400 MHz, CDCl3): δ 7.52 (t, J = 8.0 Hz, 1
H), 7.24−7.17 (m, 6 H), 6.75 (t, J = 8.0 Hz, 2 H), 6.66 (d, J = 8.0 Hz,
4 H), 4.73 (s, 4 H), 4.24−4.17 (m, 8 H), 1.26 (t, J = 8.0 Hz, 6 H). 13C
NMR (100 MHz, CDCl3): δ 171.22, 159.02, 148.29, 137.79, 129.45,
119.52, 117.93, 112.65, 61.30, 58.43, 53.85, 14.45. IR (KBr pellet,
cm−1): 1744, 1598, 1575, 1504, 1447, 1383, 1346, 1260, 1177, 1023,
990, 958, 746, 690. HRMS (+ESI): calcd for MH+, 462.2393; found,
462.2395.
Diethyl 2,2′-((Pyridine-2,6-diylbis(methylene))bis((4-((4,5-dime-
thoxy-2-nitrophenyl)(hydroxy)methyl)phenyl)azanediyl))diacetate
(21). A solution of 20 (0.139 g, 0.301 mmol) and 11 (0.127 g, 0.602
mmol) in 10 mL of CH2Cl2 was cooled to −10 °C, and 2,6-lutidine
(868 μL, 0.602 mmol) and TMSOTf (135 μL, 0.602 mmol) were
added over 1 h. The resulting solution was stirred for 6 h, and then
TBAF in THF (1 M, 1.8 mL, 1.8 mmol) was added to the reaction
mixture. After 1 h the solvent was removed under reduced pressure.
Recrystallization from CH3OH yielded 21 as a yellow crystalline solid
1
(0.130 g, 48.9%). Mp: 79−81 °C. H NMR (400 MHz, CDCl3): δ
7.58 (s, 2 H), 7.50 (t, J = 8.0 Hz, 1 H), 7.39 (s, 2 H), 7.16−7.09 (m, 6
H), 6.51 (d, J = 8.0 Hz, 4 H), 6.44 (s, 2 H), 4.65 (s, 4 H), 4.17 (q, J =
8.0 Hz, 4 H), 4.03 (d, J = 8.0 Hz, 4 H), 3.95 (s, 6 H), 3.91 (s, 6 H),
2.76 (s, 2 H), 1.23 (t, J = 8.0 Hz, 6 H). 13C NMR (100 MHz, CDCl3):
δ 171.22, 158.55, 153.54, 148.05, 147.87, 140.11, 137.81, 134.91,
131.39, 128.45, 119.65, 112.53, 110.31, 108.30, 71.48, 61.41, 58.07,
56.63, 56.56, 53.90, 14.44. IR (KBr pellet, cm−1): 3513, 1731, 1611,
1575, 1513, 1461, 1385, 1267, 1205, 1178, 1060, 1022, 984, 960.
HRMS (+ESI): calcd for MH+, 884.3354; found, 884.3304.
(m, 3 H), 7.25 (d, J = 8.0 Hz, 2 H), 7.17−7.11 (m, 8 H), 6.72−6.68
(m, 6 H), 4.81 (s, 4 H), 4.78 (s, 4 H). 13C NMR (100 MHz, CDCl3):
δ 159.12, 159.07, 149.91, 148.49, 137.80, 137.00, 129.51, 122.21,
121.01, 119.33, 117.40, 112.69, 57.47, 57.45. IR (KBr pellet, cm−1):
1600, 1593, 1570, 1506, 1461, 1388, 1348, 1245, 1225, 1181, 986, 946,
780, 756, 744. HRMS (+ESI): calcd for MH+, 472.2501; found,
472.2474.
2,2′-((Pyridine-2,6-diylbis(methylene))bis((4-((4,5-dimethoxy-2-
nitrophenyl)(hydroxy)methyl)phenyl)azanediyl))diacetic Acid (Di-
Cast-3, 6). To a solution of 21 (0.080 g, 0.090 mmol) in THF (1
mL) were added 1 M KOH (0.27 mL, 0.27 mmol) and 1 mL of H2O.
The resultant solution was stirred overnight at room temperature, 10
mL of water was added, and product was precipitated by a dropwise
addition of acetic acid until no more solid formed. The resulting
yellow solid was collected by filtration and dried under nitrogen (0.026
(((Pyridine-2,6-diylbis(methylene))bis((pyridin-2-ylmethyl)-
azanediyl))bis(4,1-phenylene))bis((4,5-dimethoxy-2-nitrophenyl)-
methanol) (DiCast-2, 5). A solution of 18 (0.362 g, 0.767 mmol) and
11 (0.356 g, 1.53 mmol) in CH2Cl2 was cooled to 0 °C, and 2,6-
lutidine (0.62 mL, 5.36 mmol) and TMSOTf (0.695 mL, 3.83 mmol)
were added. The reaction mixture was stirred overnight, and TBAF (1
M, 4.6 mL, 4.6 mmol) was added. After 1 h, the solvent was removed
under reduced pressure. Recrystallization from CH3OH yielded
DiCast-2 as a yellow crystalline solid (0.205 g, 30.0%). Mp: 134−
1
g, 65%). Mp: 169−171 °C. H NMR (400 MHz, DMSO) 7.55−7.47
(m, 5 H), 7.27 (d, J = 8 Hz, 2 H), 6.87 (d, J = 8.0 Hz, 4 H), 6.35 (d, J
= 8.0 Hz, 4 H), 4.52 (s, 4 H), 3.92 (s, 6 H), 3.85 (s, 6 H), 3.53 (s, 4
H). 13C NMR (100 MHz, DMSO): δ 171.78, 159.71, 152.84, 148.46,
147.00, 139.31, 135.84, 129.72, 127.56, 118.85, 111.04, 109.94, 107.72,
79.16, 69.33, 58.01, 56.70, 56.05, 55.99. IR (KBr pellet, cm−1): 3382,
1
136 °C. H NMR (400 MHz, CDCl3): δ 8.53 (d, J = 4.0 Hz, 2 H),
7.79 (d, J = 4.0 Hz, 2 H), 7.64−7.57 (m, 5 H), 7.31 (d, J = 8.0 Hz, 2
H), 7.16−7.19 (m, 6 H), 7.04 (t, J = 8.0 Hz, 2 H), 6.86 (s, 2 H), 6.53
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1575, 1513, 1452, 1382, 1326, 1268, 1215, 1179, 1060, 1024, 983, 817,
795, 746. HRMS (+ESI): calcd for MH+, 828.2728; found, 828.2774.
2,2′-((Pyridine-2,6-diylbis(methylene))bis(phenylazanediyl))-
diacetic Acid (23). Deprotection of 20 (0.59 g, 1.3 mmol) followed
the same procedure as deprotection of 6 using 1 M KOH (5.1 mL, 5.1
mmol). After precipitation and drying, 23 was obtained as a white solid
Table 1. Crystallographic Parameters for DiCast Model
Complexes
[Zn(18)][Zn(NO3)4]
[Zn3K(23)2(H2O)Cl3]
formula
formula wt
crystal size
space group
a, Å
C31H29N5Zn·N4O12Zn
850.41
C46H44Cl3KN6O9Zn3
1166.43
1
(0.41 g, 80%). Mp: 199−201 °C. H NMR (400 MHz, DMSO): δ
0.20 × 0.25 × 0.40
monoclinic, P21/c
10.4293(3)
0.06 × 0.20 × 0.40
monoclinic, P21/c
22.191(3)
7.77 (t, J = 8.0 Hz, 1 H), 7.35 (d, J = 8 Hz, 2 H), 7.09 (t, J = 8.0 Hz, 4
H), 6.63 (t, J = 8 Hz, 2 H), 6.48 (d, J = 8 Hz, 4 H), 4.70 (s, 4 H), 4.28
(s, 4 H). 13C NMR (100 MHz, DMSO): δ 172.60, 158.59, 147.38,
138.62, 129.01, 119.99, 116.70, 111.80, 56.91, 54.00. IR (KBr pellet,
cm−1): 1570, 1504, 1450, 1391, 1349, 1322, 1295, 1229, 1179, 988,
958, 898, 784, 770, 745, 690. HRMS (+ESI): calcd for MH+, 406.1767;
found, 406.1770.
b, Å
15.1047(3)
16.5760(19)
c, Å
22.6113(6)
12.8734(16)
α, deg
β, deg
γ, deg
102.094(1)
94.590(4)
[Zn(18)][Zn(NO3)4]. The ligand 18 (20.0 mg, 0.042 mmol) was
combined with Zn(NO3)2·4H2O (22.0 mg, 0.084 mmol) in 1:1
CH3CN/CH2Cl2 and refluxed for 2 h. Colorless blocks where
obtained by vapor diffusion with diethyl ether. IR (KBr pellet,
cm−1): 3060, 2388, 1614, 1587, 1495, 1467, 1440, 1380, 1353, 1321,
1286, 1259, 1186, 1161, 1126, 1100, 1060, 1032, 1015, 982, 934, 898,
838, 812. HRMS (+ESI): calcd for [Zn(18)]H+, 536.1793; found,
536.1787.
[Zn3K(23)2(H2O)Cl3]. The ligand 23 was washed with a
concentrated solution of KOH in EtOH and dried. Stock solutions
of 23 (135 μL, 0.0839 M, 0.011 mmol) and ZnCl2 (75 μL, 0.182 M,
0.014 mmol) in CH3CN were combined with pyridine (3.5 μL, 0.044
mmol). The solution was diluted with 1 mL of CH3CN and placed in a
diffusion chamber with hexanes to provide colorless needles. IR (KBr
pellet, cm−1): 3586, 3066, 2890, 2323, 2166, 2036, 1646, 1625, 1599,
1576, 1497, 1467, 1441, 1410, 1385, 1327, 1289, 1261, 1243, 1207,
1162, 1134, 1096, 1026, 932, 888, 844. HRMS (+ESI): calcd for MH+,
1160.9825; found, 1160.9820.
Binding Constants. Absorption spectra were recorded on a Cary
50 UV−visible spectrophotometer run under Varian’s Cary WinUV
software. All spectra were acquired at 25 °C in quartz cuvettes with
path length of 1 cm and cell volume of 3.0 mL. Stock solutions of
photocages were prepared at mM concentrations in DMSO and
diluted to prepare 25 μM solutions in the solvent of choice. All metal
binding constants were determined by previously described
procedures. Aqueous stock solutions of the metal perchlorate salts
of Cu2+, Zn2+, and Cd2+ were prepared in mM concentrations. A 25
μM solution of the ligand was prepared in 3000 μL of the solvent and
titrated in triplicate with each of the metal salt stock solutions.
Absorption spectra were corrected for dilution, and the conditional
dissociation constants (Kd) were calculated using XLfit.28
Collection and Reduction of X-ray Data. Structural analysis was
carried out in the X-ray Crystallographic Facility at Worcester
Polytechnic Institute. Crystals were covered in PARATONE oil on
100 μm MiTeGen polyimide micromounts or glued on the tip of a
glass fiber and were mounted on a Bruker-AXS APEX CCD
diffractometer equipped with an LT-II low temperature device.
Diffraction data were collected at room temperature or at 100(2) K
using graphite monochromated Mo Kα radiation (λ = 0.71073 Å)
using the omega scan technique. Empirical absorption corrections
were applied using the SADABS program.29 The unit cells and space
groups were determined using the SAINT+ program.29 The structures
were solved by direct methods and refined by full matrix least-squares
using the SHELXTL program.29 Refinement was based on F2 using all
reflections. All non-hydrogen atoms were refined anisotropically.
Hydrogen atoms on carbon atoms were all located in the difference
maps and subsequently placed at idealized positions and given
isotropic U values 1.2 times that of the carbon atom to which they
were bonded. Hydrogen atoms bonded to oxygen atoms were located
and refined with isotropic thermal parameters. Mercury 1.4.2 software
was used to examine the molecular structure.30 Relevant crystallo-
graphic information is summarized in Table 1, selected bond distances
and angles are provided in Tables 2 and 3, and the 50% thermal
ellipsoid plots are shown in Figures 2 and 3.
V, Å3
3482.93(15)
4
4720.1(10)
4
Z
ρ
(g cm−3
)
1.622
1.45
1.641
1.83
calcd
abs coeff (cm−1
)
temp, K
100
296
total no. of data
20511
6133
4869
0.067
0.171
487
37638
8294
7234
0.047
0.115
621
no. of unique data
a
obsd data
b
R, %
c
wR2, %
no. of params
́
max/min peaks, e/Å
0.56/−0.43
0.64/−0.52
a
b
c
Observation criterion: I > 2σ(1). R = ∑||Fo| − |Fc||/∑|Fo|. wR2 =
2
2
[∑(w(Fo − Fc2)2)/∑w(Fo )2]1/2
.
Table 2. Selected Interatomic Distances (Å) and Angles
(deg) for [Zn(18)][Zn(NO3)4]
bond lengths
Zn1−N1
Zn1−N2
Zn1−N3
Zn1−N4
Zn1−N5
bond angles
N1−Zn1−N2
2.286(5)
2.271(5)
2.006(5)
2.002(4)
2.018(4)
157.7(2)
113.8(2)
79.0(2)
80.6(2)
81.6(2)
78.7(2)
112.2(2)
128.9(2)
104.5(2)
126.6(2)
N1−Zn1−N3
N1−Zn1−N4
N1−Zn1−N5
N2−Zn1−N3
N2−Zn1−N4
N2−Zn1−N5
N3−Zn1−N4
N3−Zn1−N5
N4−Zn1−N5
a
Numbers in parentheses are estimated standard deviations in the last
digit(s). Atom labels are provided in Figure 2.
RESULTS AND DISCUSSION
■
Synthesis. The first generation DiCast compounds borrow
design elements of the DPA ligand in ZinCast-1, which
exhibited the highest stability for Zn2+ in the monofunctional
nitrobenzhydrol photocages.13 The similarity between these
DiCast compounds and ZinCast-1 also allows for better
comparison of metal binding and releasing properties resulting
from the use of multiple rather than a single nitrobenzyl group.
Dialkylation of N,N-diphenylethylenediamine with picolylbro-
mide hydrobromide in the presence of potassium hydrogen
phosphate and potassium iodide yields the four-nitrogen donor
receptor for DiCast-1 (Scheme 2). The DiCast-1 receptor
retains the structural features 5−5 chelate rings formed by
ZinCast-1 when considering the second aniline nitrogen atom
as a surrogate for one pyridine donor of DPA. The DiCast-1
receptor adds an additional pyridine ring that forms another 5-
membered chelate ring when bound to the guest metal ion like
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multiple nitrobenzyl groups, installing a methyl group onto one
of the positions para to an aniline group in 13 was envisioned.
A methyl group would block the electrophilic substitution
reaction and afford a photocage with a solitary nitrobenzyl
group. Coupling N-phenylethylenediamine and p-bromoto-
luene with Pd provides 9, and subsequent alkylation with
picolylbromide hydrobromide yields the receptor 10. An
identical TMSOTf promoted coupling reaction used to prepare
DiCast-1 affords MonoCast in an overall 27% yield.
Neither MonoCast nor DiCast-1 was predicted to exhibit an
exceptionally high affinity for Zn2+. While adding another metal
binding ligand may increase the Zn2+ affinity by enhancing the
chelate effect, preorganized binding sites often enhance metal
ion complexation.32 A difunctional pyridine group was
envisioned to act as an ideal ligand scaffold to bring together
two DPA-like ligands into a symmetric receptor designed to
encapsulate a single Zn2+ ion. Reacting 16 with excess aniline
forms the backbone of the second generation DiCast
photocages (Scheme 3). Excess aniline prevents overalkylation
to unwanted higher order products. The common intermediate
17 provides the ability to access a variety of photocages with
different aniline-pendant ligands.
Reductive amination of 17 with 2-pyridinecarboxaldehyde
provides receptor 18. Subsequent treatment of 18 with the
standard reaction sequence affords DiCast-2 containing five
nitrogen donor groups. Alternatively, alkylation of 17 with ethyl
bromoacetate affords 20, which was converted to 21 using a
synthesis analogous to DiCast-2. Saponification of the ester
groups with KOH provides access to DiCast-3. The two
carboxylate groups in DiCast-3 provide a charge neutral
photocaged complex, which could allow passive loading into
cells whereas charged complexes like DiCast-2 may require
more invasive techniques to facilitate transport across lipophilic
membranes.
Table 3. Selected Interatomic Distances (Å) and Angles
(deg) for [Zn3K(23)2(H2O)Cl3]
bond lengths
Zn1−N1
Zn1−N2
Zn1−O2
Zn1−O3
Zn1−O5
bond angles
N1−Zn1−N2
2.373(3)
2.085(4)
2.100(3)
2.000(3)
2.007(3)
76.1(1)
159.1(1)
75.9(1)
N1−Zn1−O2
N1−Zn1−O3
N1−Zn1−O5
N2−Zn1−O2
N2−Zn1−O3
N2−Zn1−O5
O2−Zn1−O3
O2−Zn1−O5
O3−Zn1−O5
N4−Zn2−N5
N4−Zn2−N6
N4−Zn2−O6
N4−Zn2−O7
N4−Zn2−Cl2
N5−Zn2−N6
N5−Zn2−O6
N5−Zn2−O7
N5−Zn2−Cl2
N6−Zn2−O6
N6−Zn2−O7
N6−Zn2−Cl2
O6−Zn2−O7
O6−Zn2−Cl2
O7−Zn2−Cl2
91.4(1)
103.9(1)
116.6(1)
130.1(1)
85.9(1)
103.6(1)
106.2(1)
74.1(1)
Zn2−N4
Zn2−N5
Zn2−N6
Zn2−O6
Zn2−O7
Zn2−Cl2
2.503(3)
2.065(4)
2.444(4)
2.089(3)
2.092(3)
2.264(1)
130.4(1)
72.1(1)
154.6(1)
89.91(9)
73.7(1)
96.3(1)
108.8(1)
148.6(1)
147.9(1)
72.7(1)
98.64(9)
82.5(1)
104.48(9)
97.26(8)
a
Numbers in parentheses are estimated standard deviations in the last
digit(s). Atom labels are provided in Figure 3.
Bulk photolysis of MonoCast and DiCast-1 provides the
corresponding ligand photoproducts, MonoUnc and DiUnc-1,
for use in metal ion binding assays. While the photoproduct of
DiCast-2 could be prepared, the photolysis yields an
incomplete reaction with the presence of some byproducts
that prevent obtaining analytically pure DiUnc-2; however, the
purity was deemed sufficient for metal binding assays. In
contrast, the apparent decomposition of the DiCast-3 photo-
product made isolating DiUnc-3 impossible.
Metal-Binding Properties of MonoCast and DiCast-1.
Titration of DiCast-1 and MonoCast with Cu2+, Zn2+, and Cd2+
permits the metal ion binding properties to be evaluated
spectrophotometrically. Although ZinCast-1 measurements
were also obtained in mixed buffer, the limited water solubility
of both new photocages limits the evaluation of metal binding
properties to EtOH and CH3CN. Since Cu2+ oxidizes aniline
groups in CH3CN,22,33 no measurements were made for those
conditions. The apo forms of MonoCast and DiCast-1 both
possess characteristic, aniline-derived absorption bands with
λmax at 260 and 270 nm respectively. Formation of metal
complexes of [M(MonoCast)]2+ and [M(DiCast-1)]2+ leads to
erosion of aniline absorption bands with the simultaneous
formation of a new band at ca. 235 nm (Figure 4A). The
absorption profiles for both MonoUnc and DiUnc-1 include
two bands with λmax at 230 and 350 nm and one band with λmin
at ca. 285 nm (Figure 4B). Upon the addition of metal ions, the
band at 285 increases in intensity while the other two
characteristic features erode. Fitting the absorption changes
Figure 2. Perspective view of DiCast-2 model complex [Zn(18)][Zn-
(NO3)4] showing 50% thermal ellipsoids and selected atom labels.
Hydrogen atoms and [Zn(NO3)4]2− are omitted for clarity.
the aliphatic chelator EBAP. Using the TMSOTf methodology,
which was originally developed for Ca2+ photocages and used
extensively to prepare related compounds,31 the receptor 14
was coupled to 11 (Scheme 2). Deprotection of the crude TMS
ether product with TBAF affords DiCast-1 in 41% overall yield.
To provide a direct comparison between photocages that
possess the same receptor but contain either a single or
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Figure 3. Perspective view of DiCast-3 model complex [Zn3K(23)2(H2O)Cl3] showing 50% thermal ellipsoids and selected atom labels. Hydrogen
atoms are omitted for clarity.
Scheme 2. Synthesis of (A) MonoCast (3) and (B) DiCast-1 (4)
provides apparent binding constants for the respective
photocages and photoproducts.
consistent with replacement of the aliphatic amines in EBAP
with aniline groups. The stability of complexes with Cu2+ and
Cd2+ follows a similar trend. In CH3CN, the affinity of
[Zn(ZinCast-1)]2+ (9.7 μM) also exceeds the afinity of
[Zn(MonoCast)]2+ (12 μM) and [Zn(DiCast-1)]2+ (12 μM).
Unlike ZinCast photocages, MonoCast and DiCast-1 exhibit no
complex stability dependence on solvent polarity or the metal
ion. Stability constants for the [M(MonoCast)]2+ and [M-
The expansion of the DPA chelator of ZinCast-1 with a
second aniline group unexpectedly has a destabilizing effect on
the Zn2+ complex despite the addition of a fourth pendant
ligand. The Kd decreases from 13 μM for [Zn(ZinCast-1)]2+ to
14 μM and 20 μM for [Zn(MonoCast)]2+ and [Zn(DiCast-
1)]2+ respectively in EtOH (Table 4). The low Zn2+ affinity is
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Scheme 3. Synthesis of (A) DiCast-2 (5) and (B) DiCast-3 (6)
(DiCast-1)]2+ complexes in both solvent systems remain within
14−22 μM, indicating that the second nitrobenzhydrol group
has a negligible effect on metal ion affinity of DiCast-1
compared to MonoCast.
(DiUnc-1)]2+ decrease in a pattern that conforms to the
Irving−Williams series: Cu2+ > Zn2+ > Cd2+.
The changes in metal affinity after photolysis are more
pronounced than the absolute affinity comparisons of the
parent photocages. Since uncaging of DiCast-1 modulates the
electron density on two metal-bound nitrogen atoms, stability
constants for the [Zn(DiUnc-1)]2+ and [Cd(DiUnc-1)]2+
decrease to millimolar values, which leads to significantly larger
values for ΔKd compared to complexes of ZinUnc-1. The 190-
fold ΔKd with Zn2+ for DiCast-1 significantly exceeds the 3-fold
change determined for ZinCast-1 in EtOH. The 14-fold ΔKd
for MonoCast reflects the trend that weaker binding Cast
photocages tend to possess larger ΔKd values,14,16 but remains
more comparable to the magnitude of change measured for
ZinCast-1 than for DiCast-1. DiCast-1 also exhibits a
measurable ΔKd in CH3CN whereas none was observed for
ZinCast-1 or MonoCast. While ΔKd for DiCast-1 is
considerably smaller than the ones measured for Nitr-8 and
Nitr-T,20,31 the measurements demonstrate that larger ΔKd
values can be achieved by introducing a second nitrobenzhydrol
group in Cast photocages. The larger ΔKd values for both
The minimal variation in complex stabilities for Cu2+ and
Zn2+ complexes and the ability to accommodate a larger metal
ion like Cd2+ with comparable affinity suggest increased
flexibility in the metal ion receptor. The chelator appears to
adopt different conformations easily, which allow metal ions
with different coordination requirements to be accommodated
readily. The chelator flexibility appears to limit both the
selectivity and the affinity, which limits these photocages’
biological applications; however, these features are ideal for
evaluating properties related to the number of nitrobenzyl
groups.
As expected, the resonance interaction between the nitrogen
lone pair and the carbonyl oxygen atom introduced upon
uncaging decreases the ligand basicity and lowers the affinities
measured for the photoproducts. The binding properties of the
photoproducts appear to be affected by solvent and metal ion
involved (Table 4). Affinities for Zn2+ and Cd2+ ions are
significantly reduced in EtOH compared to CH3CN, suggesting
increased ligand solvation. The stability constants for all
photoproduct complexes of [M(MonoUnc)]2+ and [M-
[Cd(MonoCast)] and [Cd(DiCast-1)] suggest weaker Naniline
−
M2+ bonds. The lack of affinity changes between [Cu-
(MonoUnc)]2+ and [Cu(DiCast-1)]2+ indicates the presence
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Figure 4. Titration of 25 μM DiCast-1 (top, A) and 15 μM DiUnc-1
(bottom, B) with Zn(ClO4)2 in EtOH. The absorbance was fitted to a
1:1 binding isotherm (inset). The error bars represent the variance in
the measurements over three trials.
of a strong Naniline−M2+ interaction imposed by the
coordination chemistry of Cu2+, which allows the metal ion
to compete more effectively with the resonance interaction for
electron density. The trends with Cu2+ and Cd2+ are consistent
with those observed in ZinCast complexes, which were also
attributed to differences in aniline−metal ion interactions.14
Structural Properties of DiCast-2 and DiCast-3
Receptors. Unlike previous Cast photocages, DiCast-2 and
DiCast-3 do not utilize a binding motif directly inspired by a
well-known receptor. Analysis of the receptor coordination
chemistry can provide important insight into the properties of
the corresponding photocage. The modular synthetic strategy
allows the receptors to be accessed simultaneously during the
photocage synthesis for evaluation. The tripyridine receptor 18
forms a mononuclear distorted trigonal bipyramidal Zn2+
complex with a [Zn(NO3)4]2− counterion. The three aromatic
pyridyl nitrogen atoms define the equatorial plane with the
anilino nitrogen atoms in the two axial positions. The Naniline
−
Zn−Naniline bond angle of the axial groups 157.7(2)° shows
significant deviation from the 180° of an ideal trigonal
bipyramid. The three equatorial Zn−NPy bond distances
occupy a narrow range between 2.002(4) and 2.018(4) Å.
The axial nitrogen atoms show modest asymmetry and weaker
interactions with Zn−Naniline bond lengths of 2.286(5) Å and
2.271(5) Å. Additional bond angles are consistent with Zn2+
coordinating chemistry that allows significant flexibility in the
ligand orientations (Table 2).
Receptor 23 exhibits more complicated coordination
chemistry in the solid state. Several different structures can
be identified crystallographically, but only one refined
sufficiently for publication. The reported complex forms a
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heterobimetallic cluster of two ligands, three Zn2+ centers, and
one K+ ion. The cluster forms from the aggregation of two
mononuclear Zn2+−receptor complexes that are connected by
the K+ and the third Zn2+ site. The K+ and third Zn2+ interact
with the two [Zn(23)] units through carboxylate ligands that
bridge between the internal receptor bound Zn2+ centers and
these external metal ions. The external Zn2+ site is tetrahedral
with 2 carboxylate ligands and two Cl− groups, one terminal
and one that bridges to the K+. The K+ is formally 7-coordinate,
with four carboxylate groups, a water ligand, and two Cl−
groups. The 2 Cl− ligands bond at approximately 3.1 Å, a
distance near the ionic radii sum with K+, so the interactions are
highly electrostatic in nature. The other structures observed
contain two [Zn(23)] units, and only the external groups and
solvent content differ from the reported complex.
The two receptor-bound Zn2+ ions adopt different geo-
metries. One Zn2+ is octahedral containing the five donor
groups from one 23 unit and a Cl− group in the coordination
sphere. The central pyridine and chloride adopt trans axial sites
with the equatorial sites occupied by the aniline and carboxylate
groups that wrap around the metal ion. The Cl−Zn−NPy bond
angle of 148.6(1)° illustrates the degree of distortion in the
octahedral geometry at the Zn2+ site. The aniline nitrogen
atoms are cis to each other forming a fac-like arrangement when
including the pyridyl group. The bond lengths and angles are
consistent with expected values, but the two Zn−Naniline bond
lengths of 2.444(4) Å and 2.503(3) Å are significantly longer
that the ones measured in [Zn(18)]2+ and exhibit more
asymmetry. By comparison the Zn−NPy bonds in the two
complexes are nearly identical (Table 3).
Figure 5. Titration of 25 μM DiCast-2 (top, A) and DiUnc-2 (bottom,
B) with Zn(ClO4)2 in EtOH. The absorbance was fitted to a 1:1
binding isotherm (inset). The error bars represent the variance in the
measurements over three trials.
The second Zn2+ adopts a distorted trigonal bipyramid;
however, the fifth ligand comes from a carboxylate ligand that
bridges from the Zn2+ in the octahedral site. Only one of the
two aniline nitrogen ligands in the 5-coordinate site resides
within bonding distance to the metal ion. The unbound aniline
nitrogen sits at approximately 2.7 Å away from the Zn2+ site,
whereas the other Zn−Naniline bond length of 2.373(3) Å is the
Limited access to the uncaged photoproducts limits
quantitative analysis of binding affinity and ΔKd to DiCast-2.
The Kd for [Zn(DiUnc-2)]2+ (4.2 mM) and [Zn(DiUnc-1)]2+
(3.8 mM) are nearly identical, but the ΔKd more than doubles
because of the increased affinity of the DiCast-2 photocage.
The 480-fold ΔKd exceeds all the Cast derivatives investigated
to date. Based on the solvent-dependent affinity trends
observed in Cast photocages,14 the ΔKd would be larger if
DiCast-2 could be evaluated in aqueous solution. Lack of access
to DiUnc-3 prevented quantification of metal ion binding, but
qualitative results suggest that the photoproduct binds more
weakly than the parent photocage. Based on the analysis of the
Zn2+ model complex, however, we hypothesize that the ΔKd for
DiCast-3 would be smaller than for DiCast-2. The distinct
asymmetry of the Naniline−Zn bond lengths suggests an unequal
contribution of these ligands to the overall complex stability.
The DiCast-2 affinity measurements reinforce the con-
clusions from the ZinCast and CrownCast series. The use of
aniline ligands inherently limits the magnitude of the absolute
binding affinity. Even using a well-defined binding pocket and
increasing denticity fails to overcome the use of weakly
coordinating aromatic nitrogen ligands. When stronger binding
ligands are incorporated into an aniline-based Cast receptor,
these ligands dominate metal ion binding and attenuating the
electron density on the anilino nitrogen atom has minimal
effects on complex stability.
shortest found in the complex of ligand 23. The Ocarboxylate
−
Zn−Naniline bond angle (159.1(1)°) that defines the axial sites
of the trigonal bipyramid is similar to the geometry found in
[Zn(18)]2+; however, different ligands define the sites (Table
3).
Metal-Binding Properties of DiCast-2 and DiCast-3. In
addition to evaluating the binding properties of DiCast-2 and
DiCast-3 under the same conditions as Dicast-1, DiCast-3
possesses sufficient solubility to conduct measurements under
simulated physiological conditions (50 mM HEPES, 100 mM
KCl, pH 7). The spectroscopic profiles of DiCast-2 and DiCast-
3 exhibit the same features as DiCast-1 with an aniline-derived
charge transfer band with λmax at 260 nm that decreases in
intensity upon the addition of metal ions (Figure 5A). The
analogous blue-shifted λmax absorption band that forms upon
metal ion binding appears at shorter wavelengths than that of
DiCast-1 and outside of the instrument’s detection limit
(Figure 5B). The inclusion of the third pyridine increases the
Zn2+ affinity of DiCast-2 to 8.7 μM as measured in EtOH
(Table 5). The replacement of pyridine ligands with
carboxylate group further enhances the Zn2+ Kd of DiCast-3
to 5.0 μM. Similar modest increases in binding affinity occur for
both photocages in CH3CN. The trends in increased affinity
between the DiCast-1 and the second generation photocages
also persist in the measurements with Cu2+ and Cd2+.
CONCLUSION
■
The inclusion of two nitrobenzyl groups in a Zn2+ photocage
increases the ΔKd by at least an order of magnitude compared
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a
Table 5. Metal Binding Properties of DiCast-2 and DiCast-3
Kd
Kd
M2+
solvent
[M(DiCast-2)]2+
[M(DiUnc-2)]2+
ΔKd
[M(DiCast-3)]2+
[M(DiUnc-3)]2+
Zn2+
EtOH
(8.7 0.3) × 10−6
(4.2 0.4) × 10−3
480
330
(5.0 0.3) × 10−6
(7.8 0.8) × 10−6
(7.8 0.7) × 10−6
(6.9 0.3) × 10−6
(7.3 0.3) × 10−6
(8.2 0.7) × 10−6
(9.3 0.3) × 10−6
b
b
b
b
b
b
b
b
CH3CN
(1.0 0.5) × 10−5
(3.3 0.2) × 10−3
d
buffer
c
c
Cu2+
Cd2+
EtOH
buffer
(8.6 0.3) × 10−6
(1.1 0.8) × 10−6
2
c
c
EtOH
CH3CN
(9.5 0.5) × 10−6
(∼1.0 0.2) × 10−3
110
110
(8.5 0.5) × 10−6
(∼9.3 0.1) × 10−4
d
buffer
c
c
(1.2 0.1) × 10−5
a
b
c
The 1:1 binding constants were calculated with XLfit. DiUnc-3 of sufficient purity could not be isolated. Metal affinity was not determined due to
d
low solubility of the complex. 50 mM HEPES, 100 mM KCl, pH 7.
Notes
to the analogous monofunctionalized compounds. The results
obtained for all three DiCast Zn2+ photocages are consistent
with trends observed in similar Ca2+ systems. Both difunctional
Ca2+ photocages utilize an identical metal ion receptor, whereas
we have screened several different, yet related, binding motifs in
this investigation. While the DiCast photocages exhibit
significant improvement over the ZinCast precursors, several
aspects related to the complex design will probably limit the
biological application of these photochemical tools.
The dependence on Cast photocages on aniline-based
ligands minimizes the absolute Zn2+ affinity attainable unless
tightly binding ligands are added to the chelator, which
significantly decreases the ΔKd. The DiCast photocages would
be useful for introducing Zn2+ into biological systems with μM
basal Zn2+ level; however, the complexes would perturb
homeostasis mechanisms prior to photolysis in most cells
where [Zn2+] are tightly regulated. The general strategy utilized
by DiCast photocages may be more applicable to investigations
of other biologically relevant metal ions that are present at
higher concentrations.
In addition to metal ion buffering considerations, the
nitrobenzylhydrol−aniline receptor strategy has problems
associated with the efficiency of the nitrobenzyl photo-
chemistry. The Cast photocages exhibit low quantum
yields,14,23 which are exacerbated in the DiCast compounds
as evidenced by the bulk synthesis of the photoproducts.
Incomplete photoreactions and instability of some photo-
products make accurately calculating changes in metal ion
concentrations difficult. Finding a suitable replacement for the
nitrobenzyl photocaging group is a high priority of our current
research. Despite the possible limitations for studying Zn2+, the
findings significantly increase the understanding of multifunc-
tional photocaged complexes and will help facilitate the design
of future tools for studying metal ion homeostasis.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The work was funded by Worcester Polytechnic Institute and
by NSF Grant CHE-0955361.
■
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