11B NMR sensing of d-block metal ions in vitro and in cells based on the carbon-boron bond cleavage of phenylboronic acid-pendant cyclen (cyclen = 1,4,7,10-tetraazacyclododecane)

Article abstract of DOI:10.1021/ic201507q

Noninvasive magnetic resonance imaging (MRI) including the "chemical shift imaging (CSI)" technique based on ¹H NMR signals is a powerful method for the in vivo imaging of intracellular molecules and for monitoring various biological events. However, it has the drawback of low resolution because of background signals from intrinsic water protons. On the other hand, it is assumed that the ¹¹B NMR signals which can be applied to a CSI technique have certain advantages, since boron is an ultratrace element in animal cells and tissues. In this manuscript, we report on the sensing of biologically indispensable d-block metal cations such as zinc, copper, iron, cobalt, manganese, and nickel based on ¹¹B NMR signals of simple phenylboronic acid-pendant cyclen (cyclen = 1,4,7,10- tetraazacyclododecane), L⁶ and L⁷, in aqueous solution at physiological pH. The results indicate that the carbon-boron bond of L ⁶ is cleaved upon the addition of Zn²⁺ and the broad ¹¹B NMR signal of L⁶ at 31 ppm is shifted upfield to 19 ppm, which corresponds to the signal of B(OH)₃. ¹H NMR, X-ray single crystal structure analysis, and UV absorption spectra also provide support for the carbon-boron bond cleavage of ZnL⁶. Because the cellular uptake of L⁶ was very small, a more cell-membrane permeable ligand containing the boronic acid ester L⁷ was synthesized and investigated for the sensing of d-block metal ions using ¹¹B NMR. Data on ¹¹B NMR sensing of Zn²⁺ in Jurkat T cells using L⁷ is also presented.

Full text of DOI:10.1021/ic201507q

ARTICLE
pubs.acs.org/IC
11B NMR Sensing of d-Block Metal Ions in Vitro and in Cells Based on
the CarbonꢀBoron Bond Cleavage of Phenylboronic Acid-Pendant
Cyclen (Cyclen = 1,4,7,10-Tetraazacyclododecane)
Masanori Kitamura,†,‡ Toshihiro Suzuki,§,‡ Ryo Abe,§,‡ Takeru Ueno,† and Shin Aoki*,†,‡
†Faculty of Pharmaceutical Sciences, Tokyo University of Science, 2641 Yamazaki, Noda 278-8510, Japan
‡Center for Technologies against Cancer (CTC) and §Research Institute for Biological Sciences, Tokyo University of Science,
2669 Yamazaki, Noda 278-0022, Japan
S Supporting Information
b
ABSTRACT: Noninvasive magnetic resonance imaging (MRI)
including the “chemical shift imaging (CSI)” technique based
on 1H NMR signals is a powerful method for the in vivo imaging
of intracellular molecules and for monitoring various biological
events. However, it has the drawback of low resolution because
of background signals from intrinsic water protons. On the
other hand, it is assumed that the 11B NMR signals which can be
applied to a CSI technique have certain advantages, since boron
is an ultratrace element in animal cells and tissues. In this
manuscript, we report on the sensing of biologically indispen-
sable d-block metal cations such as zinc, copper, iron, cobalt, manganese, and nickel based on 11B NMR signals of simple
phenylboronic acid-pendant cyclen (cyclen = 1,4,7,10-tetraazacyclododecane), L6 and L7, in aqueous solution at physiological pH.
The results indicate that the carbonꢀboron bond of L6 is cleaved upon the addition of Zn2+ and the broad 11B NMR signal of L6 at
31 ppm is shifted upfield to 19 ppm, which corresponds to the signal of B(OH)3. 1H NMR, X-ray single crystal structure analysis, and
UV absorption spectra also provide support for the carbonꢀboron bond cleavage of ZnL6. Because the cellular uptake of L6 was very
small, a more cell-membrane permeable ligand containing the boronic acid ester L7 was synthesized and investigated for the sensing
of d-block metal ions using 11B NMR. Data on 11B NMR sensing of Zn2+ in Jurkat T cells using L7 is also presented.
’ INTRODUCTION
paramagnetic metal ions such as copper, iron, and nickel have
remained underdeveloped11 because these metals typically act as
fluorescence quenchers.12
d-Block metal cations such as zinc, copper, iron, cobalt,
manganese, and nickel are involved in one-third of all human
proteins as catalytic centers and structural cofactors, which
makes them essential for life.1 To maintain the homeostasis of
metal ions both at the cellular and at whole organism levels,
nature uses sophisticated metal complexes with DNA, proteins,
and other biomelecules.2,3 For example, several families of
proteins that are integral transmembrane transporters, metallor-
egulatory sensor proteins, and diffusible cytoplasmic metallocha-
perone proteins deliver the metal ions to target molecules. In
recent years, it has been recognized that a metal imbalance in cells
and tissues causes a number of diseases such as taste disorders,
Alzheimer’s disease, Menkes and Wilson’s diseases, amyotrophic
lateral sclerosism, and cancer.1c,4 For example, it is well estab-
lished that Zn2+ levels is markedly decreased in prostate cancer
and other cancer cells.5 Accordingly, considerable efforts have
been devoted to the development of fluorescent molecular
sensors for the metal cations in living systems.6ꢀ10 However,
this sensing technique sometimes suffers from photobleaching
of the fluorescent molecule and invisibility especially in tissue
more than a few millimeters in depth because of light scattering
and absorption. In addition, fluorescent sensors for cellular
1
In this regard, H magnetic resonance imaging (MRI) is a
powerful method and is a widely used medical imaging technique
for in vivo visualization because it is a noninvasive method
capable of producing three-dimensional images of opaque
organisms.13 However, recent studies dealing with the MRI
detection of metal ions14 such as Zn2+,15 Cu2+/Cu+,16 Fe2+,17
and Ca2+18,19 are mostly limited to Gd3+-based contrast agents
1
that are used to observe changes in H NMR signals. Further-
more, there are only a few reports that describe the MRI
detection of metal cations in cells.14a
Meanwhile, it has been established that 1,4,7,10-tetraazacy-
clododecane (cyclen, L1) forms extremely stable complexes with
metal ions such as Zn2+, Cu2+, Ni2+, Mn4+/Mn3+, and Co3+ in
aqueous solutions at neutral pH (Scheme 1 depicted for Zn2+
complex 1 (ZnL1))20ꢀ24 and Zn2+-selective cyclen-based fluor-
escent molecules (Chart 1) such as 2 (L2),8 3 (L3),9 4a (L4),10a,b
and 4b (L5).10c It should be noted that Zn2+ in 1a possesses
Received: July 15, 2011
Published: October 19, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ic201507q Inorg. Chem. 2011, 50, 11568–11580
|

Inorganic Chemistry
ARTICLE
strong Lewis acidity which facilitates the deprotonation of Zn2+-
bound H2O to form 1b even at neutral pH (pKa = 7.8).10b,c
Moreover, the Zn2+-bound HOꢀ in 1b can also function as a
nucleophile or base.
boron in metal-free 5 and 6 to the sp3 boron in the corresponding
metal complexes 7 and 8 at neutral pH, based on the hypothesis
that the metal-bound H2O (or HOꢀ) would interact with boron,
resulting in a 11B NMR spectral change.
In this manuscript, we report on our finding about the CꢀB
bond hydrolysis of 5 and 6 upon the formation of complexes with
Zn2+ and other d-block metal ions to give 9 (ML8) and boric acid
(B(OH)3), resulting in a substantial and measurable change in
the 11B NMR signals (Scheme 2). Data on the in-cell 11B NMR
for the sensing of Zn2+ in Jurkat T cells using 6 are also presented.
These data present a basic concept for sensing of Zn2+ and other
d-block metal ions based on shifts in the 11B NMR signal, the
mechanism of which is different from that of Gd3+-based contrast
agents.
The objective of this work was to focus on the chemical and
spectroscopic behaviors of simple Zn2+ꢀcyclen complexes con-
taining a Lewis acidic boronic acid side chain such as 5 (L6) and 6
(L7) (Scheme 2). It is because the boron is regarded as a
nonessential or “ultratrace” element in mammals25 and the 11
B
nucleus has higher NMR sensitivity (16.5% for 11B and 2.0% for
10B relative to 1H NMR) and higher natural abundance than 10
B
(80.4% for 11B vs 19.6% for 10B),26 while both isotopes have
almost similar structures and binding or pharmacokinetic effects
of the agents. We were interested in structural changes of the sp2
’ EXPERIMENTAL SECTION
Scheme 1
General Information. ZnSO4 7H2O and CuSO4 5H2O were
3
3
purchased from Yoneyama Chemical Industry Co. Ltd. Zn(NO3)2
3
6H2O, MgCl2 6H2O, Cd(NO3)2 4H2O, and FeCl3 6H2O were ob-
tained from Kanto Chemical Co. Ltd. FeCl2 4H2O, NiCl2, and CoCl2
were purchased from Wako Pure Chemical Industries, Ltd. Anhydrous
3
3
3
3
CaCl2 was obtained from Nacalai Tesque, Inc. MnSO4 H2O was
3
purchased from Sigma-Aldrich Co. Acetonitrile (CH3CN) and dichlor-
omethane (CH2Cl2) were distilled from calcium hydride. All aqueous
solutions were prepared using deionized and distilled water. The
Good’s buffer reagents (Dojindo) were obtained from commercial
sources: MES (2-morpholinoethanesulfonic acid, pKa = 4.8), HEPES
(N-(2-hydroxyethyl)piperazine-N0-2-ethanesulfonic acid, pKa = 7.5),
EPPS (N-(2-hydroxyethyl)piperazine-N0-3-propanesulfonic acid,
pKa = 8.0), CHES (2-(cyclohexylamino)ethanesulfonic acid, pKa = 9.5),
CAPS (3-(cyclohexylamino)propanesulfonic acid, pKa = 10.4). Buffer
solutions (acetic acid/sodium acetate, pH 4; MES, pH 5, 5.5, 6; HEPES,
pH 7, 7.4; EPPS, pH 8, 8.5; CHES, pH 9; CAPS, pH 10, 11) were used.
All other reagents and solvents were of the highest commercial quality
and were used without further purification unless otherwise noted.
Melting points were measured on a YANACO Micro Melting Point
Apparatus and are uncorrected. IR spectra were recorded on a JASCO
Chart 1
Scheme 2
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Inorganic Chemistry
ARTICLE
FTIR-410 and a PerkinElmer Spectrum100 spectrophotometer at room
temperature (rt). 1H (400 MHz), 13C (100 MHz), and 11B (128 MHz)
NMR spectra were recorded on a JEOL Lambda 400 spectrometer.
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a
JEOL Always 300 spectrometer. Chemical shifts (δ) in CDCl3 were
determined relative to an internal reference of tetaramethylsilane
Boronic Acid Ester (6). A EtOH solution (3 mL) of 5 2TFA
3
(162 mg, 0.3 mmol) and bicyclohexyl-1,10-diol 1329 (59 mg, 0.3 mmol)
was refluxed overnight. After concentration under reduced pressure, the
residue was purified by column chromatography using Fuji Silysia
Chromatorex Chromatography Silica Gel NH (CHCl3/MeOH = 9:1)
1
to afford the 6 as a colorless solid (133 mg, 95% yield): H NMR
1
(TMS) for H NMR and CDCl3 for 13C NMR. The sodium salt of
(300 MHz, CDCl3/TMS): δ = 1.15ꢀ1.33 (6H, m), 1.67ꢀ1.82 (14H,
m), 2.54ꢀ2.67 (15H, m), 2.80 (4H, t, J = 4.9 Hz), 3.96 (2H, s), 7.21
(1H, td, J = 7.4, 1.0 Hz), 7.43 (1H, td, J = 7.5, 1.4 Hz), 7.58 (1H, d, J = 7.3
Hz), 7.81 (1H, dd, J = 7.4, 1.4 Hz) ppm; 13C NMR (75 MHz, CDCl3):
δ = 22.33, 25.62, 32.26, 45.15, 46.27, 47.10, 51.57, 57.14, 84.41,
125.68, 128.12, 128.85, 130.86, 135.69, 146.11 ppm; 11B NMR (128
3-(trimethylsilyl)propionic-2,2,3,3-d4 acid (TSP) was used as an exter-
nal reference for 1H NMR and 1,4-dioxane for 13C NMR measurements
in D2O. 11B NMR spectra were measured in a quartz NMR tube
using boron trifluoride diethyl ether complex in CDCl3 as an external
reference (0 ppm). The pD values in D2O were corrected for a
deuterium isotope effect using pD = (pH-meter reading) + 0.40.
Elemental analyses were performed on a Perkin-Elmer CHN 2400
analyzer. Electrospray ionization (ESI) mass spectra were recorded
on a JEOL JMS-SX102A and Varian 910-MS. Inductively coupled
plasmaꢀatomic emission spectrometry (ICPꢀAES) measurements
were performed on a Shimadzu ICPE-9000. Thin-layer chromatography
(TLC) was performed using a Merck Silica 5554 (silica gel) TLC plate.
Silica gel column chromatographies were performed using Fuji Silysia
Chemical FL-100D or Fuji Silysia Chromatorex Chromatography Silica
Gel NH.
1-[(2-Boronophenyl)methyl]-4,7,10-tris(tert-butyloxycar-
bonyl)-1,4,7,10-tetraazacyclododecane (12). A mixture of
3Boc-cyclen 1027 (1.18 g, 2.50 mmol), 2-(bromomethyl)phenylborane
1128 (806 mg, 3.75 mmol), and K2CO3 (691 mg, 5.00 mmol) in CH3CN
(10 mL) was refluxed for 3 h. After the addition of water, the reaction
mixture was extracted with CH2Cl2. The combined extract was washed
with brine, dried over Na2SO4, and evaporated. The resulting residue
was purified by silica gel column chromatography (hexane/AcOEt = 1:1
to MeOH:CH2Cl2 = 1:10) to afford 12 as a colorless amorphous
solid (1.31 g, 86% yield): mp 74ꢀ77 °C. 1H NMR (400 MHz, CDCl3
with 3 drops of MeOH-d4/TMS) δ = 1.43ꢀ1.50 (27H, m), 2.83
(4H, brs), 3.35 (4H, brs), 3.44 (4H, brs), 3.52 (4H, brs), 3.77 (2H,
brs), 7.16 (1H, brs), 7.29ꢀ7.33 (2H, m), 7.83 (1H, brs) ppm; 13C NMR
(100 MHz, CDCl3 with 3 drops of MeOH-d4) δ = 28.21, 28.24, 28.30,
28.31, 45.95 (br), 48.48 (br), 49.83 (br), 60.95, 80.14, 80.19, 127.23,
129.72, 130.35, 135.07, 135.93, 140.69, 156.21 ppm; 11B NMR (128
MHz, CDCl3 with 3 drops of MeOH-d4): δ = 29.02 (brs) ppm; IR
(neat): ν = 3390, 2976, 2930, 1694, 1463, 1415, 1366, 1250, 1165, 1031,
921, 858, 732, 647, 438 cmꢀ1. Anal. Calcd (%) for C30H51BN4O8: C,
59.40; H, 8.47; N, 9.24. Found: C, 59.11; H, 8.66; N, 8.84. HRMS
(ESI+): calcd for [M (C30H5110BN4O8) + H]+, 606.3897: found,
606.3909.
MHz, D2O/BF3 Et2O): δ = 30.98 (brs) ppm; IR (ATR): ν = 2931,
3
2852, 1600, 1570, 1442, 1387, 1368, 1344, 1311, 1285, 1273, 1254, 1237,
1146, 1133, 1113, 1070, 1055, 1040, 937, 911, 826, 805, 748, 727, 671,
656, 545, 507 cmꢀ1. Anal. Calcd (%) for C27H45BN4O2 0.5H2O: C,
3
67.92; H, 9.71; N, 11.73. Found: C, 68.03; H, 9.71; N, 11.47. HRMS
(ESI+): calcd for [M (C27H4510BN4O2) + H]+, 468.3749: found,
468.3745.
1-[(3-Boronophenyl)methyl]-4,7,10-tris(tert-butyloxycar-
bonyl)-1,4,7,10-tetraazacyclododecane (15). A mixture of 3Boc-
cyclen 1027 (172 mg, 0.363 mmol), 3-(bromomethyl)phenylborane 1430
(78 mg, 0.363 mmol), and Na2CO3 (77 mg, 0.73 mmol) in CH3CN
(16 mL) was refluxed for 22 h. After filtration through a Celite pad, the
filtrate was evaporated and purified by silica gel column chromatography
(hexane/AcOEt = 3:2) to afford 15 as a colorless amorphous solid (192 mg,
87% yield): mp 118ꢀ121 °C. 1H NMR (300 MHz, MeOH-d4/TMS)
δ = 1.43 (18H, brs), 1.49 (9H, s), 2.64 (4H, brs), 3.25ꢀ3.75 (14H, m),
7.25ꢀ7.31 (2H, m), 7.46ꢀ7.72 (2H, m) ppm; 13C NMR (75 MHz,
MeOH-d4) δ = 28.86, 29.09, 48.23, 48.50, 50.76 (br), 56.56 (br),
57.91 (br), 80.73, 128.38, 132.70, 133.29, 133.91, 134.59, 136.70,
137.05, 137.43, 157.00, 157.18, 157.39 ppm; 11B NMR (128 MHz,
D2O/BF3 Et2O): δ = 29.02 (brs) ppm; IR (ATR): ν = 3377, 2977,
3
2934, 1684, 1459, 1415, 1364, 1323, 1270, 1249, 1151, 1108, 1048,
980, 859, 802, 772, 711, 673, 614, 555, 511 cmꢀ1. Anal. Calcd (%) for
C30H51BN4O8: C, 59.40; H, 8.47; N, 9.24. Found: C, 59.24; H, 8.85;
N, 8.99. HRMS (ESI+): calcd for [M (C30H5110BN4O8) + Na]+, 628.3735:
found, 628.3729.
1-[(3-Boronophenyl)methyl]-1,4,7,10-tetraazacyclodode-
cane (16). To a solution of 15 (289 mg, 0.477 mmol) in CH2Cl2
(3 mL) was added trifluoroacetic acid (2 mL) at rt, and the reaction
mixture was allowed to stir overnight. After evaporation, the resulting
residue was purified by column chromatography using Fuji Silysia
Chromatorex Chromatography Silica Gel NH (CHCl3/MeOH = 9:1)
to afford the 16 as a colorless amorphous solid (133 mg, 87% yield): mp
101ꢀ105 °C. 1H NMR (400 MHz, D2O (pD 9.2)/TSP): δ = 2.90ꢀ2.98
(8H, m), 3.05ꢀ3.08 (4H, m), 3.11ꢀ3.15 (4H, m), 3.80 (2H, s), 7.26
(1H, d, J = 7.2 Hz), 7.38 (1H, t, J = 7.2 Hz), 7.57 (1H, s), 7.61 (1H, d, J =
7.2 Hz) ppm; 13C NMR (100 MHz, D2O (pD 9.2)/1,4-dioxane): δ =
41.81ꢀ42.22 (m), 43.95ꢀ44.33 (m), 48.34ꢀ48.64 (m), 57.20ꢀ57.69
(m), 66.73ꢀ67.66 (m), 128.04ꢀ128.21 (m), 128.77ꢀ128.99 (m),
131.94, 133.30, 133.41, 134.52 ppm; 11B NMR (128 MHz, D2O (pD
1-[(2-Boronophenyl)methyl]-1,4,7,10-tetraazacyclodode-
cane Trifluoroacetic Acid Salt (5 2TFA). To a CH2Cl2 solution
3
(2 mL) of 12 (1.18 g, 2.50 mmol) was added trifluoroacetic acid (2 mL)
at rt, and the reaction mixture was stirred for 1 h. After evaporation, the
resulting residue was recrystallized from Et2O/MeOH to give colorless
crystals of 5 (476 mg, 89% yield), which were determined to be the
2TFA salt by elemental analysis and potentiometric pH titration. The
obtained crystals were suitable for an X-ray crystal structure analysis: mp
163ꢀ166 °C (dec.). 1H NMR (400 MHz, D2O/TSP): δ = 2.88 (4H,
brs), 3.02 (4H, brs), 3.14 (4H, t, J = 5.2 Hz), 3.18 (4H, t, J = 4.9 Hz), 3.89
(2H, s), 7.38 (1H, d, J = 7.6 Hz), 7.42ꢀ7.51 (2H, m), 7.64 (1H, d, J = 7.6
Hz) ppm; 13C NMR (100 MHz, D2O/1,4-dioxane): δ = 41.66, 41.75,
43.90, 48.78, 59.31, 116.37 (q, JCꢀF = 291.7 Hz), 127.88, 130.04, 131.25,
133.43, 139.19, 162.87 (q, JCꢀF = 35.3 Hz) ppm; 11B NMR (128 MHz,
9.2)/BF3 Et2O): δ = 12.45 (brs) ppm; IR (ATR): ν = 3278, 2823, 1648,
3
1600, 1581, 1447, 1377, 1352, 1291, 1256, 1198, 1146, 1112, 1080, 1043,
1016, 979, 944, 796, 746, 719, 660, 626, 581, 534, 499 cmꢀ1. Anal. Calcd
(%) for C15H27BN4O2 MeOH: C, 56.81; H, 9.24; N, 16.56. Found:
3
C, 56.80; H, 9.09; N, 16.20. HRMS (ESI+): calcd for [M
(C15H2710BN4O2) + H]+, 306.2340: found, 306.2336.
D2O/BF3 Et2O): δ = 30.76 (brs) ppm; IR (KBr): ν = 3060, 2848, 1667,
Crystallographic Study of 5 2TFA (L6). Crystals of 5 2TFA
3
3
3
1619, 1467, 1444, 1394, 1353, 1276, 1192, 1133, 1035, 1014, 937, 833,
796, 763, 739, 722, 659, 600, 505, 421 cmꢀ1. Anal. Calcd (%) for
C19H29BF6N4O6: C, 42.71; H, 5.47; N, 10.49. Found: C, 42.58; H, 5.29;
N, 10.35. HRMS (ESI+): calcd for [M (C15H2710BN4O2) + H]+,
306.2339: found, 306.2336.
were obtained from Et2O/MeOH at rt. All measurements were made on
a Rigaku Saturn CCD area detector with graphite monochromated
MoꢀKα radiation at 123 K. C19H29B1F6N4O6, Mr = 534.26, a colorless
platelet crystal, crystal size 0.40 ꢁ 0.35 ꢁ 0.20 mm, monoclinic, space
group P21/c (#14), a = 9.392(5), b = 12.179(7), c = 21.872(12) Å,
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Inorganic Chemistry
ARTICLE
β = 94.892(3)°, V = 2493(2) Å3, Z = 4, Dcalc = 1.423 g cmꢀ3, 17564
(1400 rpm ꢁ7 min at 4 °C) and washed with 0.5% calf serumꢀRoswell
Park Memorial Institute (CSꢀRPMI) medium and PBS (phosphate
buffered saline) to remove extracellular 5, 6, or DMSO. The cells were
collected again by centrifugation (2000 rpm ꢁ10 min at 4 °C) and then
lysed in RIPA (Radio-Immunoprecipitation Assay) buffer (500 μL) on
ice for 30 min. After centrifugation (15000 rpm ꢁ10 min at 4 °C), the
resulting supernatant liquids (400 μL) were diluted with 1 N HCl
(5 mL) and water (4.6 mL) to give sample solutions (10 mL). These
sample solutions were prepared in triplicate. The amount of boron in the
sample solutions (Figure 8) was quantitatively determined by ICPꢀAES
(Shimadzu ICPE-9000, emission at 249.773 nm) using a standardized
curve of B(OH)3.
3
measured reflections, 5601 unique reflections, 2θmax = 55.0°, R1 (wR2) =
0.0754 (0.2009), GOF = 1.160. Details of the crystallographic
analysis of 5 2TFA are given in the Supporting Information, Tables
3
S2ꢀS4 and CIF.
Complexation of 5 (L6) with Zn2+ and Crystallographic
Study of the Complex 9a (ZnL8). A solution of Zn(NO3)2 6H2O
3
(44 mg, 0.15 mmol) in water (0.5 mL) was added to an aqueous solution
(1 mL) of 5 (80 mg, 0.15 mmol) at rt, and pH of the reaction mixture was
adjusted to 7 by adding 0.1 M NaOHaq. After evaporation, the resulting
residue was recrystallized from water (0.2 mL) to provide colorless
1
crystals (36 mg, 40% yield). The H NMR spectrum of the obtained
Typical Procedure of in-Cell 11B NMR Using 6 for the
Detection of Zn2+ in Jurkat T Cells. Jurkat T cells (4 ꢁ 108 cells)
were incubated in 10% FCSꢀRPMI medium (400 mL) of 6 (final
concentration 33 μM) at 37 °C in a 5% CO2 environment for 1 h. After
collecting the cells by centrifugation (1400 rpm ꢁ7 min at 4 °C) and
washing with 0.5% CSꢀRPMI to remove extracellular 6, either
Zn2+ꢀpyrithione33 (2.5 or 10 μM in the culture medium) or DMSO
(negative control) in 10% FCSꢀRPMI was added and incubated for
20 min in a 5% CO2 environment. All of the cells were collected by
centrifugation (1400 rpm ꢁ7 min at 4 °C) and washed with 0.5%
CSꢀRPMI and then PBS buffer (prepared in D2O). The cell pellets
obtained by these centrifugations (1400 rpm ꢁ7 min at 4 °C) were
crystals was consistent with that of an authentic sample of 9a.31 The
obtained crystals were suitable for an X-ray crystal structure analysis and
determined as the complex 9a and boric acid (B(OH)3). All measure-
ments were made on a Rigaku Saturn CCD area detector with graphite
monochromated MoꢀKα radiation at 123 K. C38H55B1F12N8O13,
Mr = 1201.45, a colorless block crystal, crystal size 0.30 ꢁ 0.25 ꢁ
0.20 mm, triclinic, space group P1 (#2), a = 9.968(6), b = 16.226(8),
c = 16.718(9) Å, α = 71.955(18)°, β = 89.71(2)°, γ = 77.420(19)°,
V = 2504(2) Å3, Z = 2, Dcalc = 1.594 g cmꢀ3, 16602 measured
3
reflections, 8975 unique reflections, 2θmax = 51.0°, R1 (wR2) =
0.0456 (0.1251), GOF = 0.999. Full details of crystallographic analysis
are given in the Supporting Information, Table S5ꢀS7 and CIF.
Potentiometric pH Titrations. The preparation of the test
solutions and the method used for calibration of the electrode system
(Potentiometric Automatic Titrator AT-400 and Auto Piston Buret
APB-410, Kyoto Electronics Manufacturing, Co. Ltd.) with a Kyoto
Electronics Manufacturing Co. Combination pH Electrode 98100C171
have been described previously.10,27 All of the test solutions (50 mL)
were maintained under an argon (>99.999% purity) atmosphere. The
potentiometric pH titrations were performed with I = 0.1 (NaNO3)
at 25.0 ( 0.1 °C (0.1 M aqueous NaOH was used as the base).
The deprotonation constants were determined using the “BEST” soft-
placed in quartz NMR tubes using 200 μL PBS buffer (in D2O), and 11
B
NMR spectra of the cell pellets were measured using 830000 scans and
the same parameters as described above. Each spectrum was processed
with 4.6 Hz line broadening and baseline correction, and referenced to
external BF3 Et2O in CDCl3 (as δ = 0 ppm).
3
’ RESULTS AND DISCUSSION
Synthesis of 5 (L6), 6 (L7), 16 (L9), and X-ray Single Crystal
Structure Analysis of 5 (L6). We first synthesized 5 (L6) and 6
(L7) as outlined in Scheme 3. The reaction of Boc-protected
cyclen (1027) with bromide (1128) afforded 12, whose three Boc
groups were deprotected by treatment with trifluoroacetic acid
ware program.32 The KW (equivalent to aH+aOHꢀ), KW (equivalent
ꢀ
to [H+][HOꢀ]), and fH+ values used at 25 °C were 10ꢀ14.00, 10ꢀ13.79
,
and 0.825, respectively. The corresponding mixed constants K2
(= [HOꢀ-bound species]aH+/[H2O-bound species]), were derived
using [H+] = aH+/fH+. The percentage species distribution values against
pH (= ꢀlog[H+] + 0.084) were obtained using the “SPE” software
program.32
(TFA) to give 5 as the 2TFA salt (L6 2TFA). The reaction of 5
3
with bicyclohexyl-1,10-diol 1329 gave 6 (L7). For comparison, the
isomeric ligand 16 (L9) was prepared in a similar manner as for 5
via 15, which was synthesized from Boc-protected cyclen (10)
and bromide 1430 (Scheme 3).
General Procedure for Detection of d-Block Metal Ions
by 11B NMR Spectroscopy. A solution (0.6 mL) of boronic acid
5 (L6) or boronic acid ester 6 (L7) ([5] = [6] = 20 mM) prepared
in HEPES buffer (1 M, pD 7.4) was placed into a quartz NMR tube.
After measuring the 11B NMR spectrum of the ligand alone, an
equimolar amount of the given metal ions in D2O was added, and
11B NMR spectra were collected with a sweep width of 38022 Hz,
4096 data points, a 45° pulse width, a 0.16 s recycle time, and 2850 scans.
Each spectrum was processed with 4.6 Hz line broadening and refer-
A single-crystal X-ray structure analysis of 5 2TFA obtained
3
from Et2O/MeOH shows the presence of a carbonꢀboron bond
and hydrogen bonding between hydroxyl groups of the boronic
acid and the carboxylate group of the TFAꢀ (O(1)ꢀO(4) =
2.74 Å and O(2)ꢀC(3) = 2.67 Å in Figure 1 (See also the
structure 5 2TFA in Scheme 3).
3
Deprotonation Constants of 5 (L6) Determined by Poten-
tiometric pH Titration. A typical potentiometric pH titration
curve for 1.0 mM 5 2TFA (L6 2TFA) + 2.0 mM HNO3
enced to external BF3 Et2O in CDCl3 as δ = 0 ppm (Figure 5 and
3
3
3
Table 2).
against aqueous 0.1 M NaOH with I = 0.1 (NaNO3) at 25 °C
(Figure 2) was analyzed for acidꢀbase equilibrium (1ꢀ3). The
deprotonation constants pKai (i = 1ꢀ5) of 5 were determined to
be <3 (pKa1 and pKa2), 8.4 ( 0.1 (pKa3), 10.5 ( 0.1 (pKa4), and
>12 (pKa5) using the “BEST” software program32 (Table 1).
For comparison, the pKa values for cyclen, phenylboronic acid
(1734 in Chart 2), 2-(dimethylaminomethyl)phenylboronic acid
(1835 in Chart 2), and meta isomer 16 (L9) are also listed in the
Table 1.
Typical Procedure for the Uptake of 5 (L6) and 6 (L7) in
Jurkat T Cells and Its Quantitative Analysis by ICPꢀAES. A
culture medium of 10% fetal calf serumꢀRoswell Park Memorial
Institute (FCSꢀRPMI) medium with 5 or 6 (final concentration
33 μM) was prepared using a stock solution of 5 (100 mM) in water or 6
(10 mM) in dimethylsulfoxide (DMSO). For 5, DMSO was added
to adjust the concentration of DMSO in the medium ([DMSO] = 0.33%
v/v). 10% FCSꢀRPMI including DMSO (0.33% v/v in the medium)
was also prepared for use as a negative control. Jurkat T cells (2 ꢁ 107
cells) were incubated with the medium (20 mL) containing either 5, 6,
or DMSO (negative control) at 37 °C in a 5% CO2 environment. After a
1 h period of incubation, the cells were collected by centrifugation
For the assignment of the pKa value of the B(OH)2 portions of
5 (L6) and 16 (L9), 11B NMR spectra were measured at pH
3ꢀ13. As shown in Figure 3, the pKa values of the B(OH)2
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Scheme 3
Figure 1. ORTEP drawing of 5 2TFA salt (50% probability ellipsoids).
3
Selected bond lengths [Å]: C(15)ꢀB(1) 1.579(4), B(1)ꢀO(1) 1.362(4),
B(1)ꢀO(2) 1.364(4), O(1)ꢀO(4) 2.738, O(2)ꢀO(3) 2.670. One
external trifluoroacetate anion and hydrogen atoms have been omitted
for clarity.
portion of 5 and 16 are about 8.5ꢀ9, which are in fairly good
agreement with the pKa3 values determined by potentiometric
pH titration. It has been reported that the pKa value of 17
is 8.9, and 11B NMR signals of sp2 and sp3 boron appear at
25ꢀ35 ppm and 5ꢀ15 ppm, respectively.36ꢀ38 Accordingly, we
assigned the pKa3 of 8.4 and 8.2 to the B(OH)2 parts of 5 and 16
and that the pKa1, pKa2, pKa4, and pKa5 values of 5 and 16
correspond to the pKa values of four nitrogens of the cyclen ring,
as summarized in Scheme 4 and Scheme S1 in the Supporting
Information (pKa1 and pKa2 are for 5 4H+ (H4L6) a 5 3H+
Figure 2. Typical potentiometric pH titration curve for 1.0 mM
5 2TFA (L6 2TFA) + 2.0 mM HNO3 with I = 0.1 (NaNO3) at 25 °C.
3
3
Eq (HOꢀ) is the number of equivalents of base (NaOH) added.
Table 1. Deprotonation Constants (pKai) of Cyclen (L1),
Phenylboronic acid (17), 18, 5 (L6), and 16 (L9) in Aqueous
Solution
cyclen (L1)a
17b
18c
5 (L6)d
16 (L9)d
3
3
(H3L6) a5 2H+ (H2L6), pKa4 for 19 a 20, and pKa5 for 20 a
3
21 in Scheme 4).
pKa1
pKa2
pKa3
pKa4
pKa5
<2
8.9
5.2
<3
<3
Careful experiments revealed that a small shoulder appears at
pD 9ꢀ12 in the pH-dependent 11B NMR of 5 (Figure 3). In
addition, the pKa4 value (10.5) is somehow greater than that of
the regioisomer 16 (10.0). These data allowed us to consider
some interactions between the cyclen ring and the B(OH)2
portion in 5. In general, the pKa value of an ammonium ion is
<2
11.8
<3
3.2
9.9
11.0
8.4
10.5
>12
8.2
10.0
>12
a From ref 10c. b From ref 34. c From ref 35. d The pKa values determined
by potentiometric pH titration with I = 0.1 (NaNO3) at 25 °C.
9ꢀ10, but the pKa1 value from the ammonium portion of 18 H+
3
is reported to be 5.2, which is much smaller than that of typical
ammonium cations.35 On the other hand, the pKa2 value of
18 was determined to be 11.8, which is greater than that of 17
(8.9). Anslyn et al. explained these phenomena as displayed
in Scheme 5.36 Namely, the assumption is that there is an
equilibrium between 18 and the (18 H+ HOꢀ) form (middle
3
3
in Scheme 5), in which an NꢀB dative bond interaction and an
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Chart 2
Scheme 4
Figure 3. 11B NMR spectral change of 5 (L6) and 16 (L9) with
increasing pD at 25 °C and I = 0.5 (NaNO3). [5] = [16] = 10 mM.
N+H HOꢀBꢀ interaction occur. From these data, we assume
3 3 3
that some weak interactions occur between the nitrogen of
the cyclen ring and the B(OH)2 group of 5, as displayed in
19a and/or 19b (Scheme 4).
ðH
L6Þð5 ꢀ iÞþ a ðH
KaiðL6Þ ¼ ½ðH
L6Þð4 ꢀ iÞþ þ Hþ
:
ð5ꢀiÞ
ð4ꢀiÞ
L6Þð4 ꢀ iÞþꢂ aHþ=½ðH
L6Þð5 ꢀ iÞþꢂ
ð4ꢀiÞ
ð5ꢀiÞ
3
ði ¼ 1, 2Þ
ð1Þ
2þ
ðH2L6Þ þ H2O a ðH2L6 HOꢀÞþ þ Hþ
:
3
Ka3ðL6Þ ¼ ½ðH2L6 HOꢀÞþꢂ aHþ=½ðH2L6Þ2þꢂ
3
3
ð2Þ
ðH
L6 HOꢀÞð5 ꢀ iÞþ a ðH
L6 HOꢀÞði ꢀ 4Þꢀ þ Hþ
:
ð6ꢀiÞ
ð5ꢀiÞ
3
3
spectrum of the obtained complex was consistent with that of
an authentic sample of 9a31 (data not shown), supporting the
aforementioned phenomena.
KaiðL6Þ ¼ ½ðH
L6 HOꢀÞði ꢀ 4Þꢀꢂ aHþ=½ðH
L6 HOꢀÞð5 ꢀ iÞþꢂ
ð5ꢀiÞ
ð6ꢀiÞ
3
3
3
ði ¼ 4, 5Þ
11B NMR Spectral Change of 5 (L6) upon Complexation
with Zn2+ and d-Block Metal Ions. The results of the X-ray
crystal structure analysis and 1H NMR of 9a (ZnL8) prompted us
to measure the 11B NMR spectral change of 5 upon complexation
with Zn2+. Figure 5a displays a 11B NMR signal of 5 (20 mM) in
the absence of Zn2+ in D2O at pD 7.4 (1 M HEPES buffer)
and 25 °C, and Figure 5bꢀf were collected after the addition of
Zn2+ (20 mM) for 8 min of each measurement time. Upon the
addition of Zn2+, the broad signal of 5 at 31.1 ppm was shifted
to 19.4 ppm, which coincides with the signal for B(OH)3
(Figure 5g).39 This carbonꢀboron cleavage reaction did not
take place in the presence of Zn2+ at pD > 12 (Supporting
Information, Figure S1).
ð3Þ
Detection of the CꢀB Bond Cleavage Reaction of 5 (L6)
upon Complexation with Zn2+, As Evidenced by an X-ray
Crystal Structure Analysis and 1H NMR Spectra. Fine colorless
crystals appeared after incubating a mixture of 5 (L6) and Zn2+ in
water at neutral pH and rt. Surprisingly, a single-crystal X-ray
structure analysis showed no carbonꢀboron bond in the Zn2+
complex structure, and boric acid (B(OH)3) was found as an
external molecule in the crystal (Figure 4). This finding suggests
that a carbonꢀboron bond is cleaved upon the complexation
with Zn2+ to yield 9a (Scheme 2). In addition, the H NMR
1
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Scheme 5
Figure 5. 11B NMR (128 MHz) spectral change of 5 (L6) (20 mM)
in the presence of ZnSO4 (20 mM) in D2O at pD 7.4 (1 M HEPES
buffer) and 25 °C. (a) Before the addition of Zn2+. (b) 0ꢀ8 min,
(c) 8ꢀ16 min, (d) 16ꢀ24 min, (e) 32ꢀ40 min, (f) 64ꢀ72 min
after the addition of Zn2+. (g) B(OH)3 in D2O at pD 7.4 (1 M HEPES
buffer) and 25 °C. All data were referenced to external BF3 Et2O in
3
CDCl3 (δ = 0 ppm).
Table 2. 11B NMR Spectral Change of 5 (L6) (20 mM) upon
Addition of d-Block Metal Ions (20 mM) in 1 M HEPES
Buffer at pD 7.4 and 25 °Ca
δ
Δδ
time
(h)c
δ
Δδ
time
(ppm) (ppm)b
(ppm) (ppm)b (h)c
5 (L6) alone 31.1
Mn2+ 20.6
Ni2+
19.8
Cd2+ 19.2
Ca2+
31.7
Mg2+ 31.9
ꢀ10.5 48
Zn2+
Cu2+
Fe2+
Fe3+
Co2+
19.4
19.5
19.7
30.8
19.6
ꢀ11.7
ꢀ11.6
ꢀ11.6
ꢀ0.3
0.5
1.5
0.5
ꢀ11.3
2
ꢀ11.9 0.1
0.6
0.8
ꢀ11.5
1
a All data were referenced to external BF3 Et2O in CDCl3 (δ = 0 ppm).
3
b Δδ = δ (5 (L6) with metal ions) ꢀ δ (5 (L6)). c Approximate reaction
time for completion of CꢀB bond cleavage.
Figure S8 and Table 2). The results of potentiometric pH
titrations of cyclen (L1) with Fe2+ and Fe3+ (data not shown),
provide proof for complexations with Fe2+ and Fe3+. These
results are consistent with our previous results, which indicated that
complexation with Fe3+ did not facilitate the hydrolysis of
sulfonate group on the side chain of PhSO2-caged 8-quinolinol-
pendant cyclen 23 (L11) (See Scheme 6).10c Hydrolysis of the CꢀB
bond with Mn2+ (Supporting Information, Figure S9 and Table 2)
was slow, possibly because of the complexation of binuclear Mn3+
and Mn4+ produced by air oxidation.24 The carbonꢀboron cleavage
of 5 with Cd2+ was faster than that with Zn2+ (Supporting
Information, Figure S10 and Table 2). It is likely that Cd2+-
bound HOꢀ functions as stronger nucleophile than Zn2+-bound
HOꢀ, as we previously observed.10b,c
For comparison, the same reaction was carried out using the
meta isomer 16 (L9), which resulted in negligible CꢀB bond
cleavage (data not shown). Moreover, the CꢀB bond cleavage
of phenylboronic acid (17) was negligible in the presence of 1
after 3 h (<5% after 1 day). These results allowed us to conclude
that CꢀB bond cleavage of ZnL6 complex (7 in Scheme 2)
proceeds in an intramolecular manner rather than an inter-
molecular manner.
Figure 4. ORTEP drawing of 9a and B(OH)3 obtained after the
reaction of 5 (L6) with Zn2+ (50% probability ellipsoids). Selected
bond lengths [Å]: Zn(1)ꢀN(1) 2.130(3), Zn(1)ꢀN(2) 2.138(3),
Zn(1)ꢀN(3) 2.100(3), Zn(1)ꢀN(4) 2.214(3), B(1)ꢀO(9) 1.376(5),
B(1)ꢀO(10) 1.366(5), B(1)ꢀO(11) 1.370(5). One molecule of 9a,
four trifluoroacetate anions, two waters, and hydrogen atoms have been
omitted for clarity.
Similar spectral changes were observed for Cu2+, Fe2+, Co2+,
and Ni2+ but not for Ca2+ and Mg2+ (Supporting Information,
Figures S2ꢀS7 and Table 2). Interestingly, the 11B signal change
was not observed in the case of Fe3+, while the signal change
was observed when Fe2+ was present (Supporting Information,
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Scheme 6
Figure 6. UV spectral change of 5 (L6) in the presence of Zn2+ at pH
7.4 (100 mM HEPES) and 35 °C. (a) 1 mM 5 (L6) in the absence of
Zn2+. (b) The UV spectrum was measured immediately after the
addition of Zn2+ (1 mM) to the solution (a). Spectra after (c) 9 min,
(d) 30 min, and (e) 50 min of the spectrum (b). (f) The UV spectrum
of an authentic sample 9a (1 mM) at pH 7.4 (100 mM HEPES) and
35 °C. The inset figure shows the time course for the absorption at
268 nm.
11B NMR spectra of 5 (L6) and boric acid (B(OH)3) in the
presence of biorelevant molecules such as sugars were investi-
gated because it is known to interact with boronic acids and boric
acid to form esters.40 As shown in Figure S11 and S12 in the
Supporting Information, D-glucose (10 mM), D-fructose (10 mM),
and D-galactose (10 mM) scarcely affected the 11B NMR
spectra of 5 (1 mM) and boric acid (1 mM) at these concen-
trations. We further studied an influence of hydrogen peroxide
(H2O2) on the 11B NMR spectra of 5 (L6). Hydrogen peroxide
is one of reactive oxygen species and known to react with
boronic acids and boronic acid esters especially under basic
conditions.41 As shown in Figure S13 in the Supporting Infor-
mation, the broad signal of 5 (20 mM) at 31 ppm shifted to a
broad signal at 5 ppm upon the addition of H2O2 (20 mM),
implying possible interaction between the boron and H2O2
(Supporting Information, Figure S13b). After 12 and 24 h,
partial formation of boric acid (B(OH)3) was observed (∼20%),
indicating that careful consideration is required in the presence of
H2O2 at this level of concentration (Supporting Information,
Figures S13c and S13d). The further addition of Zn2+ ion to the
mixture of 5 and H2O2 (to the solution of Supporting Informa-
tion, Figure S13d) induced the hydrolysis of the CꢀB bond
within 40 min (Supporting Information, Figure S13e).
Figure 7. pHꢀrate constant profile for the first-order rate constants of
the carbonꢀboron bond cleavage of 1 mM 5 (L6) in the presence of
1 mM Zn2+ at 35 °C.
an authentic sample 9a (curve (f)). Figure S14 in the Supporting
Information displays the time course for the CꢀB bond cleavage
of 5 (1.0 mM), as determined by UV absorption spectral changes
upon the addition of Zn2+ (1.0 mM) at the pH range of 4.0ꢀ11.0
(100 mM buffer solution) and 35 °C, from which the first-order
rate constants, k1 (secꢀ1), at pH 4.0ꢀ11.0 were determined and
plotted in Figure 7. The pHꢀk1 profiles indicate that the reaction
rate of 5 with Zn2+ at neutral pH (k1 = 8 ꢁ 10ꢀ4 sꢀ1 at pH 7.4)
was much greater than the corresponding reation at acidic and
basic pH. It has been reported that the rate constant for
the complex formation of [Zn(OAc)]+ and protonated cyclen
UV Spectral Change of 5 (L6) with Zn2+ and the Kinetic
Study of the CarbonꢀBoron Bond Cleavage. To determine
the kinetic parameters for the Zn2+-promoted CꢀB bond
cleavage of 5, UV absorption measurements were carried out.
The curve (a) in Figure 6 shows a UV absorption spectrum of
1 mM 5 (L6) at pH 7.4 (100 mM HEPES) and 35 °C, which
(HL1) was 1.3 ꢁ 105 Mꢀ1 secꢀ1 in 0.1 M acetate buffer at
3
25 °C and the reaction was too fast to be followed with the polaro-
graphic method at pH values higher than 6.20 Therefore, it can be
assumed that the rateꢀdetermining step involves a carbonꢀboron
bond cleavage step, as discussed in the next paragraph.
shows an absorption maximum at 268 nm (ε = 371 Mꢀ1 cmꢀ1).
3
Curve (b) is the UV spectrum of 5 immediately after the addi-
tion of Zn2+, suggesting the formation of a Zn2+ complex of 5
(7a in Scheme 2 and Scheme 8). After that, the UV spectra
changed to curve (cꢀe) in Figure 6, reaching the UV spectrum of
Reaction Mechanism of the CꢀB Bond Cleavage Reac-
tion. The pHꢀk1 profiles in Figure 7 implies two kinetic
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Scheme 7
Scheme 8
In other work, it was reported that the pKa value of the Zn2+-
bound H2O of 29a (H ZnL12(H2O)), which is a Zn2+ complex
3
of the guanidine-pendant cyclen 28 (L12) shown in Scheme 7,
was significantly lowered as the result of the influence of the
guanidinium cation (pKa = 5.9 for 29a a 29b).42 The deproto-
nation of the guanidinium cation by Zn2+-bound HOꢀ (29b)
resulted in the formation of an unusual Zn2+ꢀguanidine co-
ordination bond (in 30). We thus assume that the first kinetic
pKa of 7 (ca. 5) found in Figure 7 corresponds to the pKa value
for Zn2+-bound H2O in the presence of the adjacent Lewis acidic
boron (for 7a a 7b + H+ in Scheme 2).
From these data, a reaction mechanism for CꢀB bond cleavage
in Zn2+ complex of 5is proposed in Scheme 8, showing two possible
scenarios depending on the pH of the solution. At pH > 5, cyclen
part of L6 binds to Zn2+ to form 7a (ZnL6(H2O)),8ꢀ10 in which the
Zn2+-bound H2O is deprotonated to afford 7b (ZnL6(HOꢀ)),
resulting in carbonꢀboron bond cleavage to yield the complex 9a
(ZnL8). At basic condition (at pH >11), electronic repulsion
between the boronate anion and Zn2+-bound HOꢀ in 7b0, which
would be formed via 7a0, may hamper the cleavage of the CꢀB
bond (The formation of Zn2+ complex of 5 at pD 11 was confirmed
by 1H NMR experiment). Therefore, it is presumed that the second
kinetic pKa of Zn2+ꢀbound H2O in 7 (10ꢀ11) found in Figure 7
may correspond to the equilibrium between 7a0 a 7b0.
Uptake of 5 (L6) and 6 (L7) in Jurkat T Cells, and in-Cell 11
B
NMR for Detection of Zn2+. These results allowed us to conduct
in-cell NMR experiments13a,43 for the detection of intracellular
Zn2+ using 5. Lippard et al. reported a MRI sensor (100 μM in
culture medium) for the detection of Zn2+ (200 μM in culture
medium) introduced by using a Zn2+ complex of pyrithione
(Zn2+ ionophore) into HEK-293 cells.15d To the best of our
knowledge, this is the only report on the MRI sensor used for
metal ions in cells and tissues.
Prior to the NMR measurements, the intracellular uptake
of 5 (L6) was examined utilizing Jurkat T cells (Scheme 9).
The Jurkat T cells (2 ꢁ 107 cells) were incubated in 10%
FCSꢀRPMI medium containing 33 μM 5 (L6) and a 5% CO2
environment at 37 °C for 1 h. After washing the cells with
0.5% CSꢀRPMI medium and PBS, they were lysed in RIPA
buffer on ice for 30 min and analyzed by ICPꢀAES. Unfortu-
nately, the amount of boron was very low in Jurkat T cells
(Figure 8).
Therefore, a more cell-membrane permeable boronic acid
ester 6 (L7) was synthesized (Schemes 2 and 3). These types of
boronic acid esters are known to be stable in aqueous media.44
Indeed, the change in the 1H NMR spectra of the 6 (L7) in D2O
(pD 7.4) without Zn2+ for 12 h at 37 °C was negligible
pKa values (ca. 5 and 10ꢀ11) for 7. For reference, Zn2+-
promoted hydrolysis of the sulfonate group of PhSO2-caged
8-quinolinol-pendant cyclen 22 (L10) and 23 (L11) was previously
reported.10b,c It was concluded that the hydrolysis is promoted by
the Zn2+-bound HOꢀ (24b and 25b in Scheme 6) to give fluores-
cent Zn2+ complexes (26 and 27) and the kinetic pKa values for
Zn2+-bound H2O in 24 and 25 were estimated to be about 7.7.
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Scheme 9
Figure 8. Concentration of 5 (L6) and 6 (L7) in Jurkat T cells (2 ꢁ 107
cells), determined by ICPꢀAES (249.733 nm) after incubation with
33 μM 5 (L6) or 33 μM 6 (L7) for 1 h at 37 °C and lysis using RIPA
buffer. DMSO alone was used for the negative control. The error bars
display standard deviation from three independent experiments.
(Supporting Information, Figure S15). The addition of Zn2+
to 6 (20 mM) induced CꢀB bond cleavage to give B(OH)3
and slightly water-soluble diol 13, which are formed by rapid
hydrolysis of the corresponding boronic acid ester (Supporting
Information, Figures S16 and S17). Moreover, a precipitate
formed in this aqueous reaction mixture was isolated and
Figure 9. In-cell 11B NMR spectra of 6 (L7) in the absence of Zn2+ꢀ
pyrithion (ionophore), and in the presence of Zn2+ꢀpyrithion
(4 ꢁ 108 cells) were incubated with 33 μM 6 (L7) in culture medium at
37 °C for 1 h, and then (a) DMSO (as negative control), (b) 2.5 μM
Zn2+ꢀpyrithione, and (c) 10 μM Zn2+ꢀpyrithione at 37 °C for 20 min.
(BF3 Et2O was used as an external references). The Jurkat T cells
3
1
identified as diol 13 by H NMR in CDCl3 (data not shown).
A similar 11B NMR signal change of 6 (L7) was observed for other
metal ions such as Cu2+, Fe2+, Co2+, Ni2+, and Mn2+ (Supporting
Information, Table S1). The intracellular uptake of 6 into Jurkat T
cells was improved (0.63 ( 0.22 fmol/cell), as shown in Figure 8.
We next investigated the Zn2+-induced CꢀB bond cleavage of
6 (L7) in Jurkat T cells (bottom half of Scheme 9). Jurkat T cells
(4 ꢁ 108 cells) were incubated with 6 (final concentration 33 μM)
under the same condition as those with 5, washed with 0.5%
CSꢀRPMI, and incubated with either 2.5 or 10 μM Zn2+ꢀ
pyrithione, or DMSO (as a negative control) at 37 °C for 20 min.
The cells were sequentially washed with 0.5% CSꢀRPMI and
PBS, and transferred to a quartz NMR tube containing external
BF3 Et2O standard using 200 μL of PBS prepared in D2O
3
(estimated volume of whole cells = ca. 1.25 cm3). 11B NMR
spectra were recorded and compared for cell pellets with and
without exogenously introduced Zn2+.
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As shown in Figure 9a, the signal of 6 at about 31 ppm in the
absence of Zn2+ is too broad to be observed at this concentration
in the 11B NMR. In contrast, the signal at 19 ppm that corre-
sponds to B(OH)3 was observed in response to the concentra-
tion of Zn2+ (Figure 9b and 9c).45,46
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’ CONCLUSION
We describe the sensing of biologically essential d-block
metal cations such as Zn2+, Cu2+, Fe2+, Co2+, Ni2+, and Mn2+
based on the 11B NMR signals of 5 (L6) and 6 (L7) in aqueous
solution at physiological pH. The carbonꢀboron bonds of
simple 11B NMR probes, 5 and 6, were cleaved upon the
addition of d-block metal ions and the broad 11B NMR signal
at 31 ppm was shifted to a sharp signal at 19 ppm, which corre-
1
sponds to B(OH)3, as confirmed by H NMR, X-ray single
crystal structure analysis, and UV absorption spectra. In addition,
more cell-membrane permeable boronic acid ester 6 was used
for the 11B NMR sensing of Zn2+ in Jurkat T cells. The signal
corresponding to B(OH)3 was observed in response to the
concentration of Zn2+ in Jurkat T cells, while such a signal
was not observed in the absence of Zn2+.47 These results
suggest that the 11B NMR sensing of Zn2+ and other d-block
metal ions by 6 (L7) and its derivatives may afford a potential
“chemical shift imaging (CSI)”13a,48 technique in the biome-
dical sciences.
It has been suggested that decrease of the Zn2+ and other
metal ions are potent candidates as biomarkers for diagnosis of
prostate and other cancers.5 Moreover, it is suggested that 11
B
and 10B NMR is useful for the detection of 10B-containing
agents such as carborane and L-4-boronophenylalanine in the
boron neutron capture therapy (BNCT).26c,d,f Our results
may provide a basic concept to suggest that 11B NMR sensing
of Zn2+ and other d-block metal ions based on chemical reac-
tion of boron is useful for a diagnosis and treatment of cancers
and other diseases in combination with BNCT and other medical
technologies.
’ ASSOCIATED CONTENT
Supporting Information. 1H NMR, 13C NMR, and 11
B
S
b
NMR of 5 (L6) and 6 (L7). Deprotonation behavior of 16 (L9) in
Scheme S1. Figures S1ꢀS14. Table S1 for the CꢀB bond
cleavage of 6 (L7) with d-block metal ions. Tables and CIF for
the X-ray crystal structure analysis of 5 (H2L6) and 9a. This
material is available free of charge via the Internet at http://pubs.
acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: shinaoki@rs.noda.tus.ac.jp.
’ ACKNOWLEDGMENT
This work was supported by grants-in-aid from the Ministry
of Education, Culture, Sports, Science and Technology (MEXT)
of Japan (Nos. 19659026, 22390005, and 22659055 for S.A.
and No. 20750081 for M.K.) and “Academic Frontier” project
for private universities: matching fund subsidy from MEXT,
2009ꢀ2013. M.K. is thankful for Sasakawa Grants for Science
Fellows (SGSF) from the Japan Science Society.
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(44) Bernardini, R.; Oliva, A.; Paganelli, A.; Menta, E.; Grugni, M.;
Munari, S. D.; Goldoni, L. Chem. Lett. 2009, 38, 750–751.
(45) The intracellular amounts of the boron in Jurkat T cells used
for in-cell NMR experiments were reconfirmed by ICPꢀAES to be
0.64 ( 0.12 fmol/cell, which coincides with the amount of the boron
reported in Figure 8. From these data, the concentration of the boron in
the whole collected cell volume (ca. 1.25 cm3) was calculated to be
0.20 ( 0.04 mM. These results also indicate that intracellular uptake of 6
is negligibly affected by the Zn2+ concentrations (0, 2.5, and 10 μM) in
the culture medium.
(46) The working curve for the determination of the concentration
of B(OH)3 in the cells after the addition of Zn2+ was prepared using an
external BF3 Et2O in CDCl3. Using their curve, the concentrations of
3
B(OH)3 in Jurkat T cells were determined to be 0.11 mM and 0.27 mM
for Figure 9b and 9c, which are slightly lower than the Zn2+ contents in
cells estimated by ICPꢀAES (0.62 fmol/cell (0.20 mM) for Figure 9b
and 3.3 fmol/cell (1.0 mM) for Figure 9c). We assume that the Zn2+
added from outside of cells does not fully react with L7, possibly because
of some interactions of free Zn2+ with intracellular organelles or cell
membranes.
(47) In our preliminary experiments, phenylboronic acid having
iminodiacetate side chain instead of cyclen also undergoes CꢀB bond
hydrolysis upon complexation with Zn2+, Fe2+, and Cu2+. Details will be
reported somewhere else.
(48) (a) Nelson, S. J.; Vigneron, D. B.; Star-Lack, J.; Kurhanewicz, J.
NMR Biomed. 1997, 10, 411–422. (b) Lee, H.; Tikunov, A.; Stoskopf,
€
M. K.; Macdonald, J. M. Mar. Drugs 2010, 8, 2369–2383. (c) Ostlund,
Å.; Bernin, D.; Nordstierna, L.; Nydꢀen, M. J. Colloid Interface Sci. 2010,
344, 238–240.
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dx.doi.org/10.1021/ic201507q |Inorg. Chem. 2011, 50, 11568–11580