Novel Fe3+-based 1H MRI β-galactosidase reporter molecules

Article abstract of DOI:10.1002/cplu.201100072

There is increasing interest in the development of reporter agents to reveal enzyme activity in vivo using imaging in small animals. We have previously demonstrated the feasibility of detecting lacZ gene activity using the commercially available 3,4-cyclohexenoesculetin-β-D-galactopyranoside (S-Gal) as a 1H MRI reporter. Specifically, β-galactosidase (β-gal) releases the aglycone, which forms a magnetic resonance contrast-inducing paramagnetic precipitate in the presence of Fe3+. Contrast was primarily T2-weighted signal loss, but T1 effects were also observed. Since T1-contrast generally provides signal enhancement as opposed to loss, it appeared attractive to explore whether analogues could be generated with enhanced characteristics. We now report the design and synthesis of novel analogues together with characterization of 1H MRI contrast based on both T1 and T2 response to b-gal activity in vitro for the lead agent.

Full text of DOI:10.1002/cplu.201100072

DOI: 10.1002/cplu.201100072
1
Novel Fe3+-Based H MRI b-Galactosidase Reporter
Molecules**
Jian-Xin Yu,[a] Praveen K. Gulaka,[b] Li Liu,[a] Vikram D. Kodibagkar,[a, b] and Ralph P. Mason*[a, b]
There is increasing interest in the development of reporter
agents to reveal enzyme activity in vivo using imaging in small
animals. We have previously demonstrated the feasibility of de-
tecting lacZ gene activity using the commercially available
trast was primarily T2-weighted signal loss, but T1 effects were
also observed. Since T1-contrast generally provides signal en-
hancement as opposed to loss, it appeared attractive to ex-
plore whether analogues could be generated with enhanced
characteristics. We now report the design and synthesis of
3,4-cyclohexenoesculetin-b-d-galactopyranoside (S-Gal) as
a
1H MRI reporter. Specifically, b-galactosidase (b-gal) releases
the aglycone, which forms a magnetic resonance contrast-in-
ducing paramagnetic precipitate in the presence of Fe3+. Con-
novel analogues together with characterization of H MRI con-
1
trast based on both T1 and T2 response to b-gal activity in vitro
for the lead agent.
Introduction
Given the importance of reporter genes in various applications
ranging from molecular biology to clinical trials, the develop-
ment of non-invasive techniques to assay gene expression
in vivo is becoming increasingly significant.[1,2] Traditionally, the
lacZ gene encoding b-galactosidase (b-gal) was the most pop-
ular reporter including assays of clonal insertion, transcriptional
activation, protein expression, and protein interaction.[3,4] The
broad specificity of b-gal activity allows diverse molecular
structures for substrates and successful detection techniques
include colorimetric,[5,6] fluorescence,[7–9] bioluminescence,[10]
chemiluminescence,[11–13] as well as radiotracers for positron
emission tomography (PET)[14] or single-photon emission com-
puted tomography (SPECT)[15] and probes for 1H magnetic reso-
nance imaging (MRI)[16–20] and 19F NMR approaches.[21–26]
ic precipitate in the presence of Fe3+, generating T2*-weighted
1H MRI contrast.[20] This approach was also used to detect ge-
netically engineered b-gal expressing bone marrow cells by
MRI in vivo following labeling in vitro.[30] Both studies exploited
T2*-weighted signal loss to identify b-gal activity. We had no-
ticed that there was additionally T1 relaxivity, but the high
T2 relaxivity (up to 100 s-1 for 15 mm S-Gal) tended to mask
T1 effects. These results prompted us to examine whether mo-
lecular modifications could provide T1 activity without the high
T2 relaxivity.
b-Galactosidase catalyzes the hydrolysis of b-d-galactopyra-
nosides by cleavage of the CꢀO bond between d-galactose
and the aglycone. MRI detection of b-gal based on S-Gal de-
pends on contrast produced by the formation of a complex
between the 3,4-cyclohexenoesculetin aglycone and Fe3+
ions.[20,31] Schwert et al.,[32] Davies et al.,[33] and Raymond
et al.[34] have described the design and evaluation of series of
siderophores that contain catechol-binding groups (catecho-
late ligands) to coordinate Fe3+. Thus, we considered analo-
Many approaches have been demonstrated for in vitro de-
tection, but few have been applied in vivo to date,[8,13,17,18] and
these often required direct injection into the tissue of inter-
est.[24,25,27] While we focus on the detection of transgene activi-
ty in stably transfected human tumor cells, it is important to
note that expression may also arise in normal tissues following
exposure to stress such as radiation or doxorubicin-induced
senescence activated b-galactosidase.[28,29] Moreover, epithelial
exposure of lactase (the human analogue of b-galactosidase)
has been associated with metaplasia in developing esophageal
cancer.[12]
[a] Prof. J.-X. Yu, Dr. L. Liu, Prof. V. D. Kodibagkar,++ Prof. R. P. Mason
Department of Radiology
The University of Texas Southwestern Medical Center
5323 Harry Hines Blvd, Dallas, TX 75390-9058 (USA)
Fax: (+1)214-648-4538
E-mail: Ralph.Mason@UTSouthwestern.edu
The pioneering study of Moats et al. demonstrated
T1-weighted MRI contrast based on b-gal activated unmasking
of a gadolinium ligand,[16] and this was later applied to tracing
the developing cell lineages in frog embryos following direct
intracellular injection of substrate.[17] We recently demonstrated
the feasibility of detecting b-gal activity in vitro in cultured
cancer cells and in vivo in mice with human breast tumor
MCF7 xenografts using 3,4-cyclohexenoesculetin-b-d-galacto-
pyranoside (S-Gal). Specifically, b-gal cleaves S-Gal to release
[b] Dr. P. K. Gulaka,+ Prof. V. D. Kodibagkar,++ Prof. R. P. Mason
Joint Program in Biomedical Engineering
The University of Texas at Arlington and
The University of Texas Southwestern Medical Center
5323 Harry Hines Blvd, Dallas, TX 75390-9058 (USA)
[+] Current address:
Samsung Electronics, Suwon (South Korea)
++] Current address:
[
School of Biological and Health Systems Engineering
Arizona State University, Tempe, AZ (USA)
the cyclohexenoesculetin aglycone, which forms a paramagnet- [**] MRI=magnetic resonance imaging.
370
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ChemPlusChem 2012, 77, 370 – 378

1H MRI b-Galactosidase Reporter Molecules
gous dihydroxycoumarin-based catecholate aglycones. Cou-
marins are reported to have numerous therapeutic applications
including antibacterial, anti-inflammatory, and anti-coagulant
as well as photochemotherapy and anti-HIV therapy.[35] There-
fore, structure–activity relationships and synthetic procedures
have been widely examined. Inspired by these studies, we de-
signed four analogues based on the structure of 3,4-cyclohexe-
noesculetin (1, aglycone of S-Gal): 7,8-dihydroxy-3,4-cyclohexe-
nocoumarin (2), 6,7-dihydroxy-4-methylcoumarin (3), 7,8-dihy-
droxy-4-methylcoumarin (4), and 6-methoxy-7,8-dihydroxycou-
marin (5; see Figure 1).
We now report the design, synthesis, and evaluation of
these novel analogues of S-Gal, and in vitro assessment of
their hydrolytic kinetics. MRI contrast with respect to lacZ-
transfected human MCF7 breast and PC3 prostate cancer cells
is presented for the most promising agent.
Results and Discussion
Aglycone synthesis
Noting the variety of strategies for synthesizing coumarins,[32]
we chose the Pechmann reaction, coupling the two compo-
nents (phenol and b-ketoester) with ZrCl4 (10 mol%) as cata-
lyst.[36] We started the synthesis by subjecting pyrogallol or
1,2,4-benzenetriol to the Pechmann reaction with ethyl cyclo-
hexanone-2-carboxylate or ethyl
Figure 1. Comparison of MRI contrast for aglycone ligands 1–5 with Fe3+
.
1
200 MHz H MRI of vials of ferric ammonium citrate (FAC; 3.0 mm) in PBS/
DMSO (v/v 1:1) alone (leftmost) or mixed with ligands shown above (1–5;
9.0 mm). Upper row of images: T1-weighted 1H MRI with TR=300 ms,
TE=20 ms, 1.5 mm slice with, 128ꢀ128 resolution over 50ꢀ50 mm2. Lower
1
row: Corresponding T2-weighted H MRI with TR=2000 ms, TE=80 ms.
acetoacetate for coumarins 1–4,
while 6-methoxy-7,8-dihydroxy-
coumarin (5) was purchased
commercially. The reactions were
performed at 808C in toluene to
give 1–4 in high yields (92–95%)
within 30 min. After confirming
the structure of coumarins 1–4,
we evaluated the T1-and T2-
weighted MR image contrast of
their Fe3+ complexes. Each
showed substantial T1-weighted
Scheme 1. General reaction scheme: a) pyrogallol (5 mmol), ethyl cyclohexanone-2-carboxylate (5 mmol), ZrCl4
(0.5 mmol), toluene (20 mL), 808C, N2, 20 min, 93% (!2); b) 2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl bromide
(2.5 mmol), 2 (2.5 mmol), TBAB (0.5 mmol), CH2Cl2–H2O (60 mL), pH 8–9, room temperature, N2, 3–4 h, 88% (!6);
c) 0.5m NH3–MeOH, 08C!room temperature, 24 h, quantitative yields.
contrast (Figure 1), suggesting potential as Fe-based 1H MRI
lacZ gene reporters. The greatest T1 response was observed for
6,7-dihydroxy-4-methylcoumarin(3)/Fe3+, which also showed
the greatest T2-weighted MRI contrast.
Table 1. The hydroxyl pKa values of coumarins 1–5.
Coumarins
pKa[OH-7] pKa[OH-6] pKa[OH-8]
3,4-cyclohexenoesculetin 1
11.84
11.48
10.28
10.35
10.49
8.74
—
8.52
—
—
7.95
—
8.00
7.22
7,8-dihydroxy-3,4-cyclohexenocoumarin 2
6,7-dihydroxy-4-methylcoumarin 3[a]
7,8-dihydroxy-4-methylcoumarin 4[a]
7,8-dihydroxy-6-methoxycoumarin 5
Mono b-d-galactopyranosides
—
To generate b-gal reporters, a b-d-galactopyranosyl group was
added to the coumarins forming b-d-galactopyranosides
(Scheme 1). Each coumarin 2–5 has two hydroxyl groups locat-
ed at the 6,7- or 7,8-positions, which were expected to show
differences in reactivity and hence an opportunity for regiose-
lective synthesis. Indeed, straightforward regioselective mono-
glycopyranosylation[37] and etherification[38] have been reported
at the 7-hydroxyl group of 3,4-cyclohexenoesculetin (1). 13C
[a] Values from reference [36], while others were calculated by using Ad-
vanced Chemistry Development Software (www.acdlabs.com).
ty: 7-hydroxyl>6-hydroxyl>8-hydroxyl. Hydroxyl pKa values in
coumarins 1–5 corresponding to their positions (Table 1) sug-
gested that phase-transfer catalysis at pH 8–9 could provide
regio- and stereoselective synthesis of b-d-galactopyranosides,
as we also exploited previously for 19F NMR b-gal reporters.[40,41]
Reaction of 2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl bromide
with an equimolar amount of the respective coumarin (2–5) at
room temperature catalyzed by tetrabutylammonium bromide
1
and H NMR chemical shifts of coumarin derivatives (dC-7 >dC-5
[39]
>dC-6 >dC-8
)
also suggested that the relative electron defi-
ciency of C-7, C-6, and C-8 would result in the relative reactivi-
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371

R. P. Mason et al.
Figure 2. The kinetic hydrolysis time courses of mono-b-d-galactopyrano-
sides. Evolution of fluorescence following addition of b-gal (10 units, E801A)
to solutions of S-Gal and 10–13 (10 mm) in PBS (1.0 mL, 0.1 m, pH 7.4) at
20–228C showing release the corresponding aglycones S-Gal!1(*), 10!2
^
(
), 11!3 (o), 12!4 (&), 13!5 (~).
Scheme 2. Mono-b-d-galactopyranosides 6–13.
ing that all the b-d-galactopyranosides were effective sub-
strates though with varying hydrolytic rates in the order: n1
(TBAB) in a dichloromethane–aqueous biphasic system (pH 8–
>
9) under N2 afforded 7-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galacto-
n11 >n13 >n10 >n12 (Figure 2). Given that 11 and 13 were con-
siderably better substrates, these were favored for further eval-
uation. In addition the MRI contrast generated by the agly-
cones 1–5 in the presence of Fe3+ (Figure 1) indicated that
both 11 and 13 would show considerable T1 contrast upon hy-
drolysis by b-gal, and since 13 showed much less T2 sensitivity
it was chosen for further evaluation.
pyranosyl)-8-hydroxy-3,4-cyclohexenocoumarin
(6),
7-O-
(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl)-6-hydroxy-4-
methylcoumarin (7), 7-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galacto-
pyranosyl)-8-hydroxy-4-methylcoumarin (8) and 7-O-(2’, 3’, 4’,
6’-tetra-O-acetyl-b-d-galactopyranosyl)-8-hydroxy-6-methoxy-
coumarin (9) in moderate yields (72–88%; Scheme 2). Nuclear
Overhauser enhancements (NOE)
showed that the mono b-d-gal-
actopyranosylations occurred at
the O-7 positions, as predicted.
Subsequent deacetylation with
NH3/MeOH from 08C to room
temperature gave the free mono
galactopyranosides
10–13
(Scheme 2) in nearly quantitative
yields. The anomeric b-d-config-
uration of compounds 10–13 in
the 4C1 chair conformation was
1
confirmed by the H NMR chemi-
cal shifts (dH =4.75–5.03 ppm) of
the anomeric protons and the
J1,2 (Jꢁ8 Hz), and J2,3 (Jꢁ10 Hz)
coupling constants. The anome-
ric carbon resonances appeared
at dC-1’ 100.85–105.53 ppm in ac-
cordance with the b-d-configura-
tion.[40,42]
The coumarins 1–5 are strong-
ly fluorescent (365/440 nm) in
phosphate buffered saline (PBS;
0.1m, pH 7.4); however, their b-
d-galactopyranosides, S-Gal, and
10–13, are weakly or non-fluo-
Figure 3. T1 and T2 response to b-gal. The T1 (a) and T2 (b) maps of solutions of various concentrations of mono-b-
rescent. The measurement of d-galactopyranoside 13 in PBS (0.1m, pH 7.4) in the presence of ferric ammonium citrate (FAC; 5 mm) together
with bar charts for corresponding R1 and R values. b-gal (E801A, 5 units) was added to D–F. A) 13 (15 mm) alone in
fluorescence intensity increased
PBS; B) FAC (5 mm) alone in H2O; C) 13 (15 mm) plus FAC (5 mm) in PBS; D) 13 (15 mm), FAC (5 mm) and b-gal in
following reaction of the cou-
1
PBS; E) 13 (10 mm), FAC (5 mm) and b-gal in PBS; F) 13 (5 mm), FAC (5 mm) and b-gal in PBS. H MRI at 200 MHz.
marin with b-gal (E801A) in PBS
^
c) Dependence of relaxation rates R1 ( ) and R2 (&) on concentration of 13 for constant b-gal (E801A, 5 units)
(0.1m, pH 7.4) at 20–228C show- and FAC (5 mm) in PBS (0.1m, pH 7.4).
372
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ChemPlusChem 2012, 77, 370 – 378

1H MRI b-Galactosidase Reporter Molecules
MRI in solution
and 10–13, as applied successfully to 19F NMR b-gal reporters
previously.[41]
T1 and T2 maps were measured for vials containing various
combinations of mono b-d-galactopyranoside 13 and Fe3+
ions, with or without five units of b-gal (E801A; Figure 3).
Ferric ions alone enhanced R1 relaxation, but the presence of
13 made no difference. Addition of b-gal to the mixture of 13
+ Fe3+ generated much more rapid relaxation, which depend-
ed on the ratio of the two components: specifically DR1 5.9 sꢀ1
(3:1), 5.1 sꢀ1 (2:1), and 2.3 sꢀ1 (1:1), respectively (Figure 3a). T2-
weighted MR contrast showed a very similar effect, though
Fe3+ alone caused minimal relaxation, as expected: for the
complexes [DR2 9.4 sꢀ1 (3:1), 7.0 sꢀ1 (2:1), and 3.2 sꢀ1 (1:1)] (Fig-
ure 3b). The relaxation rates R1 and R2 varied linearly as a func-
tion of the concentration of 13 at a fixed concentration of Fe3+
(Figure 3c).
Di-b-d-galactopyranosides
Condensation of the coumarins 1–5 directly with 2.2 equiva-
lents of 2,3,4,6-tetra-O-acetyl-a-d-galactopyranosyl bromide in
anhydrous CH2Cl2/MeCN catalyzed by Hg(CN)2 as a promoter,
furnished the fully galactopyranosylated coumarins: 6,7-di-O-
(2’’,3’’,4’’ 6’’-tetra-O-acetyl-b-d-galactopyranosyl)-3,4-cyclohexe-
nocoumarin (14; 90%), 7,8-di-O-(2’’,3’’,4’’,6’’-tetra-O-acetyl-b-d-
galactopyranosyl)-3,4-cyclohexenocoumarin (15; 86%), 6,7-di-
O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl)-4-methylcou-
marin (16; 73%), 7,8-di-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galacto-
pyranosyl)-4-methylcoumarin (17; 77%) and 7,8-di-O-(2’,3’,4’,6’-
tetra-O-acetyl-b-d-galactopyranosyl)-6-methoxycoumarin (18;
87%), respectively (Scheme 3). Deacetylation of 14–18 in NH3/
MeOH from 08C to room temperature gave the free di-b-d-gal-
actopyranosides 19–23 in high yields (Scheme 3). The ESI-MS
MRI in cells
To demonstrate the potential for detecting b-gal activity
in vivo, various cells (human MCF7 breast and PC3 prostate
cancer), as well as stably transfected clones expressing b-gal
(MCF7-lacZ and PC3-lacZ) were incubated with 15 mm 13 and
5 mm Fe3+ in PBS (0.1m, pH 7.4) at 378C under 5% CO2 in air
with 95% humidity for 30 min. A significant difference in T1
and T2 was observed between the lacZ transfected and wild
type (WT) cells. In MCF7-WT cells T1 =(1.32ꢂ0.12) s and T2 =
(45ꢂ6) ms, while for MCF7-lacZ T1 =(0.70ꢂ0.10) s and T2 =
(32ꢂ9) ms (Figure 4). Similarly, in PC3-WT cells T1 =(1.50ꢂ
0.07) s, T2 =(39ꢂ6) ms, while for PC3-lacZ T1 =(1.16ꢂ0.04) s,
T2 =(28ꢂ4) ms, respectively).
To be useful in vivo, reporter molecules must exhibit suffi-
cient water solubility and it appeared that 10–13 were less
soluble than S-Gal. Thus, we sought to enhance solubility by
conjugating an additional b-d-galactopyranosyl unit to S-Gal
Scheme 3. The structures of di-b-d-galactopyranosides 14–23.
of 19–23 showed the expected molecular ions, corresponding
to the fully galactopyranosylated derivatives. Again, the identi-
ties of 19–23 were established from their 1H and 13C NMR spec-
tra. The anomeric protons H-1’, H-1’’ or H-1’’’ of d-galactoses
linked to the 7- and 6- or 8-positions of coumarins 1–5 at d=
5.26–4.82 ppm with the well-resolved doublets (J1,2 =8.0 Hz,
J
2,3 =10 Hz) confirming both d-galactoses in the b-configura-
tion.
As expected, the synthesized di-b-d-galactopyranosides 19–
23 are soluble in PBS (0.1m, pH 7.4) in high concentrations,
unlike 10–13, which required the addition of DMSO. The hy-
drolysis of di-b-d-galactopyranosides 19–23 by b-gal (E801 A)
in PBS (0.1m, pH 7.4) at 20–228C showed that relative hydrolyt-
ic rates were similar though a little slower than for the corre-
sponding mono-galactopyranosides and 23 was considerably
slower than expected (Figure 5).
Figure 4. T1 and T2 effects due to lacZ transfected cells. The T1 and T2 maps
of mono b-d-galactopyranoside 13 (15 mm) and FAC (5 mm) in PBS (0.1m,
pH 7.4, 200 mL) incubated at 378C under 5% CO2 in air with 95% humidity
for 30 min. T1 maps: A) MCF7-lacZ and MCF7-WT: 4ꢁ106 cells each; B) PC3-
lacZ and PC3-WT: 4ꢁ106 cells each; corresponding T2 maps C,D). MRI param-
eters: 200 MHz, matrix=128ꢁ128, FOV=40ꢁ40, 2 mm slice
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373

R. P. Mason et al.
tection was effected by spraying the plates with 5% ethanolic
H2SO4 (followed by heating at 1108C for 10 min) or by direct UV il-
lumination of the plate.
Pechmann condensation for synthesis of coumarins 1–4
General procedure: To an equimolar mixture of the phenol (pyrogal-
lol or 1,2,4-benzenetriol, 10 mmol) and the b-ketoester (ethyl cyclo-
hexanone-2-carboxylate or ethyl acetoacetate, 10 mmol) in toluene
(40 mL) was added ZrCl4 (377.3 mg, 1.0 mmol, 10 mol%), and the
mixture was stirred at 808C under N2 until TLC showed complete
reaction (<30 min). After solvent evaporation under reduced pres-
sure, the mixture was washed with cold water, and recrystallized
from hot EtOH/H2O to give the pure coumarins 1–4.
Figure 5. The kinetic hydrolysis time courses of di-b-d-galactopyranosides.
Evolution of fluorescence (365/440 nm) following addition of b-gal (10 units,
E801 A) to solutions of 19–23 (10 mm) in PBS (1.0 mL, 0.1m, pH 7.4) at 20–
228C showing release of the corresponding aglycones 19!1( ), 20!2(&),
*
^
21!3( ), 22!4(~), 23!5(o).
1
3,4-Cyclohexenoesculetin 1: Yield: 2.14 g, 92%; H NMR ([D6]DMSO,
400 MHz): dH =6.93 (s, 1H; H-5), 9.25 (s, 1H; OH-6), 9.98 (s, 1H;
OH-7), 6.69 (s, 1H; H-8), 2.64 (t, J=4.0 Hz, 2H; H-1’), 1.69 (m, 4H;
H-2’,3’), 2.34 ppm (t, J=4.0 Hz, 2H; H-4’); 13C NMR ([D6]DMSO,
100 MHz): dC =164.02 (-CO), 102.64 (C-3), 148.04 (C-4), 142.74 (C-5),
118.81 (C-6), 146.00 (C-7), 108.40 (C-8), 148.81 (C-9), 111.57 (C-10),
24.78 (CH2-1’), 21.37, 21.05 (CH2-2’,3’), 23.66 ppm (CH2-4’); elemen-
tal analysis calcd (%) for C13H12O4: C 67.23, H 5.21; found: C 67.21,
H 5.19.
Conclusion
In conclusion, we have successfully synthesized nine novel b-
d-galactopyranosides and demonstrated their potential to
detect b-gal activity based on MRI contrast in the presence of
Fe3+ ions. The di-b-d-galactopyranosides react a little slower,
but exhibit much higher water solubility, suggesting greater
potential for use in vivo. MRI clearly revealed WT versus lacZ
expressing cells in culture upon incubation with 13 based on
significant differences in both T1 and T2. Signal gain providing
contrast in T1-weighted images is potentially preferable to T2-
weighted signal loss observed previously with S-Gal in vivo.
However the combination of both T1 and T2 response may be
most promising since the concerted effect will add certainty to
observations in vivo, where tissue heterogeneity may other-
wise be misinterpreted. These mono- and di-b-d-galactopyra-
7,8-Dihydroxy-3,4-cyclohexenocoumarin 2: Yield: 2.16 g, 93%; 1H
NMR ([D6]DMSO, 400 MHz): dH =7.00 (d, J=8.0 Hz, 1H; H-5), 6.75
(d, J=8.0 Hz, 1H; H-6), 9.84 (s, 1H; OH-7), 9.20 (s, 1H; OH-8), 2.68
(t, J=4.0 Hz, 2H; H-1’), 1.69 (m, 4H, H-2’,3’), 2.36 ppm (t, J=4.0 Hz,
2H; H-4’); 13C NMR ([D6]DMSO, 100 MHz): dC =160.08 (-CO), 148.15
(C-9), 141.61 (C-4), 132.00 (C-7), 118.34 (C-5), 113.90 (C-6), 112.82 (C-
10), 112.10 (C-8), 107.23 (C-3), 24.79 (CH2-1’), 21.37, 21.02 (CH2-
2’,3’), 23.61 ppm (CH2-4’); elemental analysis calcd (%) for C13H12O4:
C 67.23, H 5.21; found: C 67.21, H 5.20.
6,7-Dihydroxy-4-methylcoumarin 3: Yield: 1.83 g, 95%; 1H NMR
([D6]DMSO, 400 MHz): dH =6.07 (s, 1H; H-3), 6.98 (s, 1H; H-5), 9.38
(brs, 1H; OH-6), 10.19 (brs, 1H; OH-7), 6.71 (s, 1H; H-8), 2.29 ppm
(s, 3H; CH3-4); 13C NMR ([D6]DMSO, 100 MHz): dC =160.69 (-CO),
102.74 (C-3), 150.20 (C-4), 142.86 (C-5), 111.58 (C-6), 147.80 (C-7),
110.45 (C-8), 153.31 (C-9), 110.49 (C-10), 18.29 ppm (CH3-4); elemen-
tal analysis calcd (%) for C10H8O4 (%): C 62.50, H 4.20; found: C
62.47, H 4.18.
1
nosides show promise as H MRI lacZ gene reporters and we
are currently evaluating them for potential application in
human tumor xenografts in vivo.
Experimental Section
7,8-Dihydroxy-4-methylcoumarin 4: Yield: 1.81 g, 94%; 1H NMR
([D6]DMSO, 400 MHz): dH =6.10 (s, 1H; H-3), 7.06 (d, J=8.2 Hz, 1H;
H-5), 6.80 (d, J=8.2 Hz, 1H; H-6), 10.03 (s, 1H; OH-7), 9.27 (s, 1H;
OH-8), 2.33 ppm (s, 3H; CH3-4); 13C NMR ([D6]DMSO, 100 MHz): dC =
160.29 (-CO), 110.27 (C-3), 149.47 (C-4), 132.24 (C-5), 115.59 (C-6),
143.36 (C-7), 112.19 (C-8), 154.01 (C-9), 112.84 (C-10), 18.33 ppm
(CH3-4); elemental analysis calcd (%) for C10H8O4 (%): C 62.50, H
4.20; found: C 62.48, H 4.19.
General methods
NMR spectra were recorded on a Varian Unity INOVA 400 spec-
trometer (400 MHz for 1H, 100 MHz for 13C) with CDCl3 or
[D6]DMSO as solvents at 258C, and H and 13C chemical shifts are
1
referenced to internal TMS. Microanalyses were performed on a Per-
kinElmer 2400CHN microanalyser. Mass spectra were obtained by
positive-ion and negative-ion ESI-MS using a Micromass Q-TOF
hybrid quadrupole/time-of-flight instrument (Micromass UK Ltd).
Solutions in organic solvents were dried with anhydrous sodium
sulfate, and concentrated in vacuo below 458C. 3,4-Cyclohexenoes-
culetin-b-d-galactopyranoside (S-Gal), 2,3,4,6-tetra-O-acetyl-a-d-gal-
actopyranosyl bromide and 6-methoxy-7,8-dihydroxycoumarin (5)
were purchased from the Sigma Chemical Company. b-Gal (E801A)
was purchased from Promega (Madison, WI), and enzyme reactions
were performed at 20–228C in PBS solution (0.1m, pH 7.4). Fluores-
cence was measured by using a Fluorolog 3 spectrometer (Jobin–
Yvon Horiba, Edison, NJ) with lex at 365 nm and lem at 440 nm.
Column chromatography was performed on silica gel (200–300
mesh) by elution with cyclohexane–ethyl acetate, and silica gel
GF254 was used for analytical TLC (Aldrich Chemical Company). De-
Regioselective mono b-d-galactopyranosylation of coumar-
ins 2–5 for preparation of acetylated mono b-d-galactopyra-
nosides 6–9
General procedure: A solution of 2,3,4,6-tetra-O-acetyl-a-d-galacto-
pyranosyl bromide (1.04 g, 2.52 mmol) in CH2Cl2 (30 mL) was
added dropwise to a vigorously stirred biphasic mixture (pH 8–9)
of coumarins 2–5 (2.52 mmol) and tetrabutylammonium bromide
(TBAB; 160 mg, 0.5 mmol) in CH2Cl2–H2O (50 mL, 1:1 v/v) over
a period of 1 h at room temperature under N2, and the stirring was
continued until TLC showed that the reaction was complete (ca.
374
www.chempluschem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2012, 77, 370 – 378

1H MRI b-Galactosidase Reporter Molecules
3 h). Extraction with CH2Cl2 (4ꢀ30 mL), washing, drying (Na2SO4),
and evaporation under reduced pressure gave a syrup, which was
purified by column chromatography on silica gel yielding acetylat-
ed mono b-d-galactopyranosides 6–9.
138.34 (C-5), 136.07 (C-6), 139.23 (C-7), 116.27 (C-8), 149.31 (C-9),
116.77 (C-10), 103.57 (C-1’), 68.60 (C-2’), 70.51 (C-3’), 66.74 (C-4’),
71.76 (C-5’), 61.03 (C-6’), 56.41 (CH3O-6), 20.06, 20.88, 20.79,
20.76 ppm (4 ꢁ CH3CO); elemental analysis calcd (%) for C24H26O14:
C 53.53, H 4.87; found: C 53.52, H 4.85.
7-O-(2’’,3’’,4’’,6’’-Tetra-O-acetyl-b-d-galactopyranosyl)-8-hydroxy-3,4-
cyclohexenocoumarin 6: Yield: 1.25 g, 88%; Rf =0.40 (1:1 cyclohex-
1
ane–EtOAc); H NMR (CDCl3, 400 MHz): dH =7.23 (d, J=8.0 Hz, 1H;
Mono b-d-galactopyranosides 10–13
H-5), 6.83 (d, J=8.0 Hz, 1H; H-6), 4.84 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’),
5.53 (dd, J2’’,3’’ =12.0 Hz, 1H; H-2’’), 5.07 (dd, J3’’,4’’ =4.0 Hz, 1H; H-
3’’), 5.40 (d, J4’’,5’’ =3.2 Hz, 1H; H-4’’), 3.95 (m, 1H; H-5’’), 4.19 (dd,
General procedure: A solution of 7-O-(acetylated b-d-galactopyrano-
syl)coumarins 6–9 (900 mg) in anhydrous ammoniacal MeOH (0.5m
100 mL) was vigorously stirred from 08C to room temperature
overnight until TLC showed that the reaction was complete, and
then the mixture was evaporated to dryness in vacuo. Chromatog-
raphy of the crude syrup on silica gel with EtOAc/MeOH afforded
the corresponding free mono b-d-galactopyranosides 10–13 in
nearly quantitative yields.
J
5’’,6a’’ =7.2 Hz, J6a’’,6b’’ =11.8 Hz, 1H; H-6a’’), 4.16 (dd, J5’’,6b’’ =5.6 Hz,
1H; H-6b’’), 2.68 (t, J=4.0 Hz, 2H; H-1’), 1.76 (m, 4H; H-2’,3’), 2.47
(t, J=4.0 Hz, 2H; H-4’), 2.00, 1.99, 1.98, 1.96 ppm (4 s, 12H; 4 ꢁ
CH3CO); 13C NMR (CDCl3, 100 MHz): dC =170.92, 170.56, 170.30,
170.14 (4 ꢁ CH3CO), 161.24 (-CO), 112.83 (C-3), 147.64 (C-4), 131.06
(C-5), 120.88 (C-6), 145.87 (C-7), 114.35 (C-8), 151.96 (C-9), 120.69
(C-10), 104.52 (C-1’’), 68.11 (C-2’’), 70.68 (C-3’’), 66.83 (C-4’’), 71.87
(C-5’’), 61.12 (C-6’’), 25.49 (CH2-1’), 21.80, 21.49 (CH2-2’,3’), 24.00
(CH2-4’), 20.91, 20.82, 20.75, 20.70 ppm (4 ꢁ CH3CO); elemental
analysis calcd (%) for C27H30O13 (%): C 57.65, H 5.38; found: C 57.63,
H 5.35.
7-O-(b-d-Galactopyranosyl)-8-hydroxy-3,4-cyclohexenocoumarin 10:
Yield: 599.43 mg, 95%; Rf =0.41 (1:3 MeOH–EtOAc); 1H NMR
([D6]DMSO, 400 MHz): dH =7.30 (d, J=8.4 Hz, 1H; H-5), 6.82 (d, J=
8.4 Hz, 1H; H-6), 4.75 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 3.67 (dd, J2’’,3’’
10.2 Hz, 1H; H-2’’), 3.56 (dd, J3’’,4’’ =4.0 Hz, 1H; H-3’’), 3.45 (d, J4’’,5’’
=
=
6.3 Hz, 1H; H-4’’), 3.40 (m, 1H; H-5’’), 3.38 (m, 2H; H-6’’), 2.74 (t, J=
4.0 Hz, 2H; H-1’), 1.72 (m, 4H; H-2’,3’), 2.51 ppm (t, J=4.0 Hz, 2H;
H-4’); 13C NMR ([D6]DMSO, 100 MHz): dC =160.81 (-CO), 112.12 (C-
3), 147.99 (C-4), 131.60 (C-5), 119.62 (C-6), 145.89 (C-7), 113.45 (C-8),
153.46 (C-9), 118.15 (C-10), 105.53 (C-1’’), 71.40 (C-2’’), 73.27 (C-3’’),
67.86 (C-4’’), 75.77 (C-5’’), 59.95 (C-6’’), 24.83 (CH2-1’), 21.36, 21.02
(CH2-2’, 3’), 23.60 ppm (CH2-4’); elemental analysis calcd (%) for
C19H22O9: C 57.87, H 5.62; found: C 57.84, H 5.58.
7-O-(2’,3’,4’,6’-Tetra-O-acetyl-b-d-galactopyranosyl)-6-hydroxy-4-meth-
ylcoumarin 7: Yield: 0.95 g, 72%; Rf =0.33 (2:3 cyclohexane–EtOAc);
1H NMR (CDCl3, 400 MHz): dH =6.16 (s, 1H; H-3), 7.07 (s, 1H; H-5),
6.95 (s, 1H; H-8), 5.03 (d, J1’,2’ =8.0 Hz, 1H; H-1’), 5.45 (dd, J2’,3’
11.4 Hz, 1H; H-2’), 5.15 (dd, J3’,4’ =3.4 Hz, 1H; H-3’), 5.42 (d, J4’,5’
7.2 Hz, 1H; H-4’), 4.10 (m, 1H; H-5’), 4.16 (dd, J5’,6a’ =4.0 Hz, J6a’,6b’
=
=
=
7.2 Hz, 1H; H-6a’), 4.13 (dd, J5’,6b’ =5.0 Hz, 1H; H-6b’), 2.32 (s, 3H;
CH3-4), 2.15, 2.08, 2.07, 1.99 ppm (4 s, 12H; 4 ꢁ CH3CO); 13C (CDCl3,
100 MHz): dC =170.95, 170.64, 170.24, 170.04 (4 ꢁ CH3CO), 161.15
(-CO), 104.15 (C-3), 147.88 (C-4), 143.45 (C-5), 116.17 (C-6), 146.77
(C-7), 110.03 (C-8), 152.30 (C-9), 114.03 (C-10), 100.85 (C-1’), 69.18
(C-2’), 70.23 (C-3’), 66.82 (C-4’), 71.91 (C-5’), 61.46 (C-6’), 21.06,
20.84, 20.77, 20.69 (4 ꢁ CH3CO), 18.98 ppm (CH3-4); elemental anal-
ysis calcd (%) for C24H26O13 (%): C 55.17, H 5.02; found: C 55.15, H
5.00.
7-O-(b-d-Galactopyranosyl)-6-hydroxy-4-methylcoumarin 11: Yield:
1
567.62 mg, 93%; Rf =0.40 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
400 MHz): dH =6.16 (s, 1H; H-3), 7.07 (s, 1H; H-5), 6.95 (s, 1H; H-8),
5.03 (d, J1’,2’ =8.0 Hz, 1H; H-1’), 5.45 (dd, J2’,3’ =11.4 Hz, 1H; H-2’),
5.15 (dd, J3’,4’ =3.4 Hz, 1H; H-3’), 5.42 (d, J4’,5’ =7.2 Hz, 1H; H-4’),
4.10 (m, 1H; H-5’), 4.16 (dd, J5’,6a’ =4.0 Hz, J6a’,6b’ =7.2 Hz, 1H; H-6a’),
4.13 (dd, J5’,6b’ =5.0 Hz, 1H; H-6b’), 2.32 ppm (s, 3H; CH3-4); 13C
NMR ([D6]DMSO, 100 MHz): dC =161.15 (-CO), 104.15 (C-3), 147.88
(C-4), 143.45 (C-5), 116.17 (C-6), 146.77 (C-7), 110.03 (C-8), 152.30
(C-9), 114.03 (C-10), 100.85 (C-1’), 69.18 (C-2’), 70.23 (C-3’), 66.82 (C-
4’), 71.91 (C-5’), 61.46 (C-6’), 18.98 ppm (CH3-4); elemental analysis
calcd (%) for C16H18O9: C 54.24, H 5.12; found: C 54.20, H 5.09.
7-O-(2’,3’,4’,6’-Tetra-O-acetyl-b-d-galactopyranosyl)-8-hydroxy-4-meth-
ylcoumarin 8: Yield: 1.07 g, 81%; Rf =0.47 (1:1 cyclohexane–EtOAc);
1H NMR (CDCl3, 400 MHz): dH =6.17 (s, 1H; H-3), 7.01 (d, J=8.0 Hz,
1H; H-5), 6.93 (d, J=8.0 Hz, 1H; H-6), 4.96 (d, J1’,2’ =8.0 Hz, 1H; H-
1’), 5.44 (dd, J2’,3’ =10.7 Hz, 1H; H-2’), 5.09 (dd, J3’,4’ =4.0 Hz, 1H; H-
3’), 5.41 (d, J4’,5’ =3.0 Hz, 1H; H-4’), 4.01 (m, 1H; H-5’), 4.18 (dd,
7-O-(b-d-Galactopyranosyl)-8-hydroxy-4-methylcoumarin 12: Yield:
1
J
5’,6a’ =5.8 Hz, J6a’,6b’ =11.6 Hz, 1H; H-6a’), 4.16 (dd, J5’,6b’ =7.8 Hz, 1H;
585.93 mg, 96%; Rf =0.38 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
H-6b’), 2.34 (s, 3H; CH3-4), 2.14, 2.06, 2.01, 1.96 ppm (4 s, 12H; 4 ꢁ
CH3CO); 13C NMR (CDCl3, 100 MHz): dC =170.58, 170.46, 170.33,
170.24 (4 ꢁ CH3CO), 160.06 (-CO), 113.77 (C-3), 146.35 (C-4), 135.34
(C-5), 117.28 (C-6), 143.65 (C-7), 113.93 (C-8), 152.63 (C-9), 115.25 (C-
10), 101.64 (C-1’), 69.01 (C-2’), 70.42 (C-3’), 66.83 (C-4’), 71.54 (C-5’),
61.37 (C-6’), 20.91, 20.83, 20.81, 20.75 (4 ꢁ CH3CO), 18.99 ppm
(CH3-4); elemental analysis calcd (%) for C24H26O13 (%): C 55.17, H
5.02; found: C 55.16, H 5.01.
400 MHz): dH =6.17 (s, 1H; H-3), 7.01 (d, J=8.0 Hz, 1H; H-5), 6.93
(d, J=8.0 Hz, 1H; H-6), 4.96 (d, J1’,2’ =8.0 Hz, 1H; H-1’), 5.44 (dd,
J
J
J
2’,3’ =10.7 Hz, 1H; H-2’), 5.09 (dd, J3’,4’ =4.0 Hz, 1H; H-3’), 5.41 (d,
4’,5’ =3.0 Hz, 1H; H-4’), 4.01 (m, 1H; H-5’), 4.18 (dd, J5’,6a’ =5.8 Hz,
6a’,6b’ =11.6 Hz, 1H; H-6a’), 4.16 (dd, J5’,6b’ =7.8 Hz, 1H; H-6b’),
2.34 ppm (s, 3H; CH3-4); 13C NMR ([D6]DMSO, 100 MHz): dC =160.06
(-CO), 113.77 (C-3), 146.35 (C-4), 135.34 (C-5), 117.28 (C-6), 143.65
(C-7), 113.93 (C-8), 152.63 (C-9), 115.25 (C-10), 101.64 (C-1’), 69.01
(C-2’), 70.42 (C-3’), 66.83 (C-4’), 71.54 (C-5’), 61.37 (C-6’), 18.99 ppm
(CH3-4); elemental analysis calcd (%) for C16H18O9: C 54.24, H 5.12;
found: C 54.19, H 5.08.
7-O-(2’,3’,4’,6’-Tetra-O-acetyl-b-d-galactopyranosyl)-8-hydroxy-6-me-
thoxycoumarin 9: Yield: 1.06 g, 78%, Rf =0.52 (1:4 cyclohexane–
1
EtOAc); H NMR (CDCl3, 400 MHz): dH =6.29 (d, J=9.8 Hz, 1H; H-3),
7.51 (d, J=9.8 Hz, 1H; H-4), 6.90 (s, 1H; H-5), 4.80 (d, J1’,2’ =8.0 Hz,
1H; H-1’), 5.44 (dd, J2’,3’ =10.2 Hz, 1H; H-2’), 5.04 (dd, J3’,4’ =3.5 Hz,
1H; H-3’), 5.34 (d, J4’,5’ =6.8 Hz, 1H; H-4’), 3.93 (m, 1H; H-5’), 4.12
(dd, J5’,6a’ =4.4 Hz, J6a’,6b’ =7.8 Hz, 1H; H-6a’), 4.04 (dd, J5’,6b’ =5.2 Hz,
1H; H-6b’), 3.79 (s, 3H; CH3O-6), 2.14, 2.08, 1.97, 1.94 (4 s, 12H; 4 ꢁ
CH3CO) ppm; 13C NMR (CDCl3, 100 MHz): dC =170.64, 170.34,
170.20, 169.75 (4 ꢁ CH3CO), 160.24 (-CO), 115.49 (C-3), 143.36 (C-4),
7-O-(b-d-Galactopyranosyl)-8-hydroxy-6-methoxycoumarin 13: Yield:
575.63 mg, 93%; Rf =0.45 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
400 MHz): dH =6.29 (d, J=9.8 Hz, 1H; H-3), 7.51 (d, J=9.8 Hz, 1H;
H-4), 6.90 (s, 1H; H-5), 4.80 (d, J1’,2’ =8.0 Hz, 1H; H-1’), 5.44 (dd,
1
J
J
J
2’,3’ =10.2 Hz, 1H; H-2’), 5.04 (dd, J3’,4’ =3.5 Hz, 1H; H-3’), 5.34 (d,
4’,5’ =6.8 Hz, 1H; H-4’), 3.93 (m, 1H; H-5’), 4.12 (dd, J5’,6a’ =4.4 Hz,
6a’,6b’ =7.8 Hz, 1H; H-6a’), 4.04 (dd,
J5’,6b’ =5.2 Hz, 1H; H-6b’),
ChemPlusChem 2012, 77, 370 – 378
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chempluschem.org
375

R. P. Mason et al.
3.79 ppm (s, 3H; CH3O-6); 13C NMR ([D6]DMSO, 100 MHz): dC =
160.24 (-CO), 115.49 (C-3), 143.36 (C-4), 138.34 (C-5), 136.07 (C-6),
139.23 (C-7), 116.27 (C-8), 149.31 (C-9), 116.77 (C-10), 103.57 (C-1’),
68.60 (C-2’), 70.51 (C-3’), 66.74 (C-4’), 71.76 (C-5’), 61.03 (C-6’),
56.41 ppm (CH3O-6); elemental analysis calcd (%) for C16H18O10: C
51.90, H 4.90; found: C 51.85, H 4.86.
[M+] (29%), 894 [M+1] (15%); elemental analysis calcd (%) for
C41H48O22: C 55.16, H 5.42; found: C 55.13, H 5.38.
6,7-Di-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl)-4-methylcou-
1
marin 16: Yield: 1.56 g, 73%; Rf =0.35 (1:2 cyclohexane–EtOAc); H
NMR (CDCl3, 400 MHz): dH =6.17 (s, 1H; H-3), 7.30 (s, 1H; H-5), 7.04
(s, 1H; H-8), 5.15 (d, J1’,2’ =J1’’,2’’ =8.0 Hz, 2H; H-1’,1’’), 5.41 (dd,
J
2’,3’ =J2’’,3’’ =10.4 Hz, 2H; H-2’,2’’), 5.10 (dd, J3’,4’ =J3’’,4’’ =4.0 Hz, 2H;
H-3’,3’’), 5.37 (d, J4’,5’ =J4’,5’ =3.2 Hz, 2H; H-4’,4’’), 4.07 (m, 1H; H-5’),
4.00 (m, 1H; H-5’’), 4.15 (dd, J5’,6a’ =J5’’,6a’’ =5.4 Hz, J6a’,6b’ =J6a’’,6b’’
Full b-d-galactopyranosylation of coumarins 1–5 for prepa-
ration of acetylated di-b-d-galactopyranosides 14–18
=
11.0 Hz, 2H; H-6a’,6a’’), 4.13 (dd, J5’,6b’ =J5’’,6b’’ =4.2 Hz, 2H; H-
6b’,6b’’), 2.34 (s, 3H; CH3-4), 2.14, 2.12, 2.07, 2.04, 2.03, 1.96, 1.95,
1.94 ppm (8 s, 24H; 8 ꢁ CH3CO); 13C NMR (CDCl3, 100 MHz): dC =
170.52, 170.33, 170.25, 170.16, 170.13, 170.10, 169.27, 169.11 (8 ꢁ
CH3CO), 160.69 (-CO), 105.43 (C-3), 150.41 (C-4), 132.52 (C-5),
115.27 (C-6), 143.05 (C-7), 114.00 (C-8), 151.74 (C-9), 114.38 (C-10),
100.68 (C-1’), 100.74 (C-1’’), 68.53 (C-2’), 68.90 (C-2’’), 70.79 (C-3’),
70.85 (C-3’’), 67.05 (C-4’,4’’), 71.49 (C-5’), 71.87 (C-5’’), 61.41 (C-6’),
61.51 (C-6’’), 21.12, 20.86, 20.75, 20.73, 20.70, 20.68, 20.66, 20.64 (8
ꢁ CH3CO), 18.69 ppm (CH3-4); ESIMS: m/z: 853 [M+] (25%), 854
[M+1] (9%); elemental analysis calcd (%) for C38H44O22: C 53.52, H
5.20; found: C 53.50, H 5.18.
General procedure: 2,3,4,6-Tetra-O-acetyl-a-d-galactopyranosyl bro-
mide (2.29 g, 5.54 mmol, 2.2 equiv) in CH2Cl2 (50 mL) was added
dropwise to
a vigorously stirred solution of coumarins 1–5
(2.52 mmol) and Hg(CN)2 (2.10 g, 8.31 mmol) in anhydrous MeCN
(100 mL) containing freshly activated 4 ꢂ molecular sieves (5.00 g).
The mixture was stirred in the dark at room temperature under an
N2 atmosphere until TLC indicated complete reaction, then it was
diluted with CH2Cl2 (150 mL), filtered through Celite, washed, dried
(Na2SO4), evaporated under reduced pressure to give a syrup,
which was purified by column chromatography on silica gel to
give the acetylated di-b-d-galactopyranosides 14–18.
6,7-Di-O-(2’’,3’’,4’’,6’’-tetra-O-acetyl-b-d-galactopyranosyl)-3,4-cyclo-
hexenoesculetin 14: Yield: 2.03 g, 90%; Rf =0.41 (2:3 cyclohexane–
EtOAc); 1H NMR (CDCl3, 400 MHz): dH =7.32 (s, 1H; H-5), 7.08 (s,
1H; H-8), 5.14 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 5.20 (d, J1’’’,2’’’ =8.0 Hz,
1H; H-1’’’), 5.48 (dd, J2’’,3’’ =J2’’’,3’’’ =10.0 Hz, 2H; H-2’’,2’’’), 5.16 (dd,
7,8-Di-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl)-4-methylcou-
marin 17: Yield: 1.66 g, 77%; Rf =0.30 (1:2 cyclohexane–EtOAc); H
1
NMR (CDCl3, 400 MHz): dH =6.16 (s, 1H; H-3), 7.25 (d, J=8.0 Hz,
1H; H-5), 7.04 (d, J=8.0 Hz, 1H; H-6), 5.33 (d, J1’,2’ =8.0 Hz, 1H; H-
1’), 5.21 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 5.42 (dd, J2’,3’ =10.8 Hz, 1H; H-
2’), 5.38 (dd, J2’’,3’’ =10.4 Hz, 1H; H-2’’), 5.08 (dd, J3’,4’ =4.0 Hz, 1H; H-
3’), 5.05 (dd, J3’’,4’’ =4.0 Hz, 1H; H-3’’), 5.34 (d, J4’,5’ =2.8 Hz, 1H; H-
4’), 5.32 (d, J4’’,5’’ =3.0 Hz, 1H; H-4’’), 3.95 (m, 1H; H-5’), 3.90 (m, 1H;
H-5’’), 4.16 (dd, J5’,6a’ =J5’’,6a’’ =5.6 Hz, J6a’,6b’ =J6a’’,6b’’ =11.2 Hz, 2H; H-
6a’,6a’’), 4.09 (dd, J5’,6b’ =J5’’,6b’’ =4.8 Hz, 2H; H-6b’,6b’’), 2.35 (s, 3H;
CH3-4), 2.15, 2.14, 2.12, 2.06, 1.97, 1.96, 1.95, 1.94 ppm (8 s, 24H; 8
ꢁ CH3CO); 13C NMR (CDCl3, 100 MHz): dC =170.35, 170.31, 170.29,
170.27, 170.26, 170.10, 169.78, 169.56 (8 ꢁ CH3CO), 159.39 (-CO),
113.93 (C-3), 151.33 (C-4), 134.49 (C-5), 120.02 (C-6), 147.17 (C-7),
116.53 (C-8), 152.25 (C-9), 117.53 (C-10), 101.37 (C-1’), 100.55 (C-1’’),
69.32 (C-2’), 68.75 (C-2’’), 71.04 (C-3’), 70.58 (C-3’’), 67.01 (C-4’),
66.92 (C-4’’), 71.39 (C-5’), 71.30 (C-5’’), 61.23 (C-6’), 61.16 (C-6’’),
21.13, 20.88, 20.77, 20.75, 20.71, 20.69, 20.67, 20.65 (8 ꢁ CH3CO),
18.97 ppm (CH3-4); ESIMS: m/z: 853 [M+] (29%), 854 [M+1] (17%);
elemental analysis calcd (%) for C38H44O22: C 53.52, H 5.20; found: C
53.49, H 5.17.
J
3’’,4’’ =J3’’’,4’’’ =4.0 Hz, 2H; H-3’’,3’’’), 5.43 (d, J4’’,5’’ =J4’’’,5’’’ =3.2 Hz, 2H;
H-4’’,4’’’), 4.06 (m, 2H; H-5’’,5’’’), 4.20 (dd, J5’’,6a’’ =J5’’’,6a’’’ =5.0 Hz,
6a’’,6b’’ =J6a’’’,6b’’’ =11.2 Hz, 2H; H-6a’’,6a’’’), 4.13 (dd, J5’’,6b’’ =J5’’’,6b’’’
J
=
4.0 Hz, 2H; H-6b’’,6b’’’), 2.72 (t, J=4.0 Hz, 2H; H-1’), 1.81 (m, 4H;
H-2’,3’), 2.57 (t, J=4.0 Hz, 2H; H-4’), 2.21, 2.14, 2.10, 2.09, 2.03, 2.02,
2.01, 2.00 ppm (8 s, 24H; 8 ꢁ CH3CO); 13C NMR (CDCl3, 100 MHz):
dC =170.63, 170.43, 170.35, 170.27, 170.24, 170.22, 169.36, 169.20
(8 ꢁ CH3CO), 161.71 (-CO), 105.34 (C-3), 148.84 (C-4), 143.17 (C-5),
122.95 (C-6), 146.53 (C-7), 114.10 (C-8), 149.30 (C-9), 115.68 (C-10),
100.88 (C-1’’), 101.02 (C-1’’’), 68.66 (C-2’’), 69.00 (C-2’’’), 70.87 (C-3’’),
70.96 (C-3’’’), 67.17 (C-4’’), 67.22 (C-4’’’), 71.48 (C-5’’), 71.82 (C-5’’’),
61.57 (C-6’’), 61.64 (C-6’’’), 25.38 (CH2-1’), 21.72, 21.46 (CH2-2’, 3’),
24.13 (CH2-4’), 20.95, 20.90, 20.86, 20.83, 20.80, 20.78, 20.76,
20.74 ppm (8 ꢁ CH3CO); ESIMS: m/z: 893 [M+] (27%), 894 [M+1]
(11%); elemental analysis calcd (%) for C41H48O22: C 55.16, H 5.42;
found: C 55.14, H 5.40.
7,8-Di-O-(2’’,3’’,4’’,6’’-tetra-O-acetyl-b-d-galactopyranosyl)-3,4-cyclo-
hexenocoumarin 15: yield: 1.94 g, 86%; Rf =0.36 (2:3 cyclohexane–
EtOAc); H NMR (CDCl3, 400 MHz): dH =7.27 (d, J=8.0 Hz, 1H; H-5),
7,8-Di-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl)-6-methoxy-
coumarin 18: Yield: 1.91 g, 87%; Rf =0.38 (1:3 cyclohexane–EtOAc);
1H NMR (CDCl3, 400 MHz): dH =6.27 (d, J=8.2 Hz, 1H; H-3), 7.54 (d,
J=8.2 Hz, 1H; H-4), 6.69 (s, 1H; H-5), 4.08 (d, J1’,2’ =8.0 Hz, 1H; H-
1’), 4.01 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 5.42 (dd, J2’,3’ =10.1 Hz, 1H; H-
2’), 5.40 (dd, J2’’,3’’ =10.0 Hz, 1H; H-2’’), 5.04 (dd, J3’,4’ =3.0 Hz, 1H; H-
3’), 5.01 (dd, J3’’,4’’ =3.2 Hz, 1H; H-3’’), 5.36 (d, J4’,5’ =6.4 Hz, 1H; H-
4’), 5.32 (d, J4’’,5’’ =6.2 Hz, 1H; H-4’’), 3.89 (m, 1H; H-5’), 3.83 (m, 1H;
H-5’’), 4.04 (m, 4H; H-6’,6’’), 3.78 (s, 3H; CH3O-6), 2.11, 2.10, 2.05,
1.96, 1.91, 1.90, 1.88, 1.85 ppm (8 s, 24H; 8 ꢁ CH3CO); 13C NMR
(CDCl3, 100 MHz): dC =170.44, 170.36, 170.26, 170.20, 170.15,
170.08, 169.83, 169.42 (8 ꢁ CH3CO), 159.58 (-CO), 105.61 (C-3),
143.15 (C-4), 141.77 (C-5), 136.64 (C-6), 142.44 (C-7), 115.30 (C-8),
149.75 (C-9), 116.11 (C-10), 105.58 (C-1’), 101.45 (C-1’’), 69.48 (C-2’),
69.17 (C-2’’), 70.96 (C-3’,3’’), 66.93 (C-4’), 66.88 (C-4’’), 71.20 (C-5’),
71.10 (C-5’’), 60.93 (C-6’), 60.88 (C-6’’), 56.74 (CH3O-6), 21.07, 20.86,
20.84, 20.80, 20.65, 20.62, 20.60, 20.58 ppm (8 ꢁ CH3CO); ESIMS: m/
z: 869 [M+] (27%), 870 [M+1] (18%); elemental analysis calcd (%)
for C38H44O23: C 52.54, H 5.11; found: C 52.50, H 5.08.
1
7.06 (d, J=8.0 Hz, 1H; H-6), 5.37 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 5.25
(d, J1’’’,2’’’ =8.0 Hz, 1H; H-1’’’), 5.48 (dd, J2’’,3’’ =10.4 Hz, 1H; H-2’’),
5.45 (dd, J2’’’,3’’’ =10.2 Hz, 1H; H-2’’’), 5.13 (dd, J3’’,4’’ =4.0 Hz, 1H; H-
3’’), 5.10 (dd, J3’’’,4’’’ =4.0 Hz, 1H; H-3’’’), 5.44 (d, J4’’,5’’ =3.6 Hz, 1H; H-
4’’), 5.42 (d, J4’’’,5’’’ =3.8 Hz, 1H; H-4’’’), 3.99 (m, 1H; H-5’’), 3.93 (m,
1H; H-5’’’), 4.21 (dd, J5’’,6a’’ =J5’’’,6a’’’ =7.6 Hz, J6a’’,6b’’ =J6a’’’,6b’’’ =6.4 Hz,
2H; H-6a’’,6a’’’), 4.16 (dd, J5’’,6b’’ =J5’’’,6b’’’ =5.8 Hz, 2H; H-6b’’,6b’’’),
2.73 (t, J=4.0 Hz, 2H; H-1’), 1.84 (m, 4H; H-2’,3’), 2.54 (t, J=4.0 Hz,
2H; H-4’), 2.19, 2.18, 2.17, 2.16, 2.12, 2.00, 1.99, 1.94 ppm (8 s, 24H;
8 ꢁ CH3CO); 13C NMR (CDCl3, 100 MHz): dC =170.36, 170.33, 170.31,
170.27, 170.13, 170.10, 169.87, 169.65 (8 ꢁ CH3CO), 160.44 (-CO),
116.79 (C-3), 148.91 (C-4), 134.35 (C-5), 122.81 (C-6), 145.51 (C-7),
117.87 (C-8), 150.07 (C-9), 118.62 (C-10), 101.45 (C-1’’), 100.72 (C-
1’’’), 69.29 (C-2’’), 68.80 (C-2’’’), 71.07 (C-3’’), 70.62 (C-3’’’), 67.01 (C-
4’’,4’’’), 71.31 (C-5’’), 71.24 (C-5’’’), 61.25 (C-6’’), 61.20 (C-6’’’), 25.47
(CH2-1’), 21.58, 21.36 (CH2-2’,3’), 24.06 (CH2-4’), 20.91, 20.90, 20.76,
20.74, 20.71, 20.69, 20.67, 20.66 ppm (8 ꢁ CH3CO); ESIMS: m/z: 893
376
www.chempluschem.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemPlusChem 2012, 77, 370 – 378

1H MRI b-Galactosidase Reporter Molecules
Free di-b-d-galactopyranosides 19–23
70.40 (C-2’’), 73.11 (C-3’), 73.33 (C-3’’), 68.18 (C-4’), 68.35 (C-4’’),
75.78 (C-5’), 75.92 (C-5’’), 60.49 (C-6’), 60.65 (C-6’’), 18.13 ppm (CH3-
4); ESIMS: m/z 517 [M+] (5%), 518 [M+1] (9%); elemental analysis
calcd (%) for C22H28O14: C 51.16, H 5.47; found: C 51.12, H 5.44.
Di-O-(2’,3’,4’,6’-tetra-O-acetyl-b-d-galactopyranosyl) coumarins 14–
18 (1.00 g) were deacetylated as described above for free mono b-
d-galactopyranosides 10–13 to afford the corresponding free di-b-
d-galactopyranosides 19–23 in high yields.
7,8-Di-O-(b-d-galactopyranosyl)-4-methylcoumarin
563.24 mg, 93%; Rf =0.26 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
400 MHz): dH =6.27 (s, 1H; H-3), 7.48 (d, J=8.0 Hz, 1H; H-5), 7.26
22:
Yield:
1
6,7-Di-O-(b-d-galactopyranosyl)-3,4-cyclohexenoesculetin 19: Yield:
585.93 mg, 94%; Rf 0.29 (1:3 MeOH–EtOAc); 1H NMR ([D6]DMSO,
400 MHz): dH =7.40 (s, 1H; H-5), 7.14 (s, 1H; H-8), 5.29 (br, 1H; HO-
2’’,2’’’, exchangeable with D2O), 4.67 (br, 2H; HO-3’’,3’’’, exchangea-
ble with D2O), 5.00 (br, 2H; HO-4’’,4’’’, exchangeable with D2O),
(d, J=8.0 Hz, 1H; H-6), 5.36 (d,
JH-2’,OH-2’ =4.0 Hz, 1H; HO-2’,
exchangeable with D2O), 4.94 (d, JH-2’’,OH-2’’ =4.0 Hz, 1H; HO-2’’,
exchangeable with D2O), 4.60 (d, JH-3’,OH-3’ =4.0 Hz, 1H; HO-3’,
exchangeable with D2O), 4.58 (d, JH-3’’,OH-3’’ =4.0 Hz, 1H; HO-3’’,
exchangeable with D2O), 4.91 (d, JH-4’,OH-4’ =4.0 Hz, 1H; HO-4’,
exchangeable with D2O), 4.89 (d, JH-4’’,OH-4’’ =4.0 Hz, 1H; HO-4’’,
4.70 (br, 2H; HO-6’’,6’’’, exchangeable with D2O), 4.82 (d, J1’’,2’’
8.0 Hz, 1H; H-1’’), 4.91 (d, J1’’’,2’’’ =8.0 Hz, 1H; H-1’’’), 3.68 (dd, J2’’,3’’
=
=
exchangeable with D2O), 4.73 (d,
J
H-6a’,OH-6’ =4.0 Hz, JH-6b’,OH-6’
=
J
2’’’,3’’’ =10.0 Hz, 2H; H-2’’,2’’’), 3.65 (dd, J3’’,4’’ =J3’’’,4’’’ =4.0 Hz, 2H; H-
6.2 Hz, 1H; HO-6’, exchangeable with D2O), 4.43 (d, JH-6a’’,OH-6’’
4.0 Hz, JH-6b’’,OH-6’’ =6.6 Hz, 1H; HO-6’’, exchangeable with D2O), 5.07
(d, J1’,2’ =8.0 Hz, 1H; H-1’), 4.83 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 3.70 (dd,
=
3’’,3’’’), 3.47 (d, J4’’,5’’ =J4’’’,5’’’ =3.4 Hz, 2H; H-4’’,4’’’), 3.52 (m, 2H; H-
5’’,5’’’), 3.72 (m, 2H; H-6’’,6’’’), 2.74 (t, J=4.0 Hz, 2H; H-1’), 1.75 (m,
4H; H-2’,3’), 2.39 ppm (t, J=4.0 Hz, 2H; H-4’); 13C NMR ([D6]DMSO,
100 MHz): dC =161.30 (-CO), 103.82 (C-3), 147.74 (C-4), 143.49 (C-5),
120.55 (C-6), 147.23 (C-7), 103.92 (C-8), 149.28 (C-9), 113.66 (C-10),
101.23 (C-1’’), 102.05 (C-1’’’), 70.05 (C-2’’), 70.26 (C-2’’’), 72.76 (C-3’’),
72.94 (C-3’’’), 67.99 (C-4’’), 68.17 (C-4’’’), 75.61 (C-5’’), 75.70 (C-5’’’),
60.34 (C-6’’), 60.54 (C-6’’’), 24.60 (CH2-1’), 21.14, 20.84 (CH2-2’,3’),
23.58 ppm (CH2-4’); ESIMS: m/z: 557 [M+] (7%), 558 [M+1] (10%);
elemental analysis calcd (%) for C25H32O14: C 53.96, H 5.80; found: C
53.94, H 5.76.
J
2’,3’ =J2’’,3’’ =10.0 Hz, 2H; H-2’,2’’), 3.44 (dd, J3’,4’ =J3’’,4’’ =4.2 Hz, 2H,
H-3’,3’’), 3.76 (d, J4’,5’ =J4’’,5’’ =3.8 Hz, 2H; H-4’,4’’), 3.36 (m, 2H; H-
5’,5’’), 3.51 (m, 4H; H-6’,6’’), 2.40 ppm (s, 3H; CH3-4); 13C NMR
([D6]DMSO, 100 MHz): dC =159.72 (-CO), 112.29 (C-3), 152.63 (C-4),
133.08 (C-5), 120.48 (C-6), 147.12 (C-7), 112.98 (C-8), 153.32 (C-9),
115.39 (C-10), 103.82 (C-1’), 102.72 (C-1’’), 71.38 (C-2’), 70.64 (C-2’’),
73.26 (C-3’), 72.61 (C-3’’), 68.16 (C-4’), 68.02 (C-4’’), 75.97 (C-5’),
75.76 (C-5’’), 60.45 (C-6’), 60.19 (C-6’’), 18.23 ppm (CH3-4); ESIMS:
m/z: 517 [M+] (7%), 518 [M+1] (15%); elemental analysis calcd
(%) for C22H28O14: C 51.16, H 5.47; found: C 51.13, H 5.43.
7,8-Di-O-(b-d-galactopyranosyl)-3,4-cyclohexenocoumarin 20: Yield:
1
592.16 mg, 95%; Rf =0.31 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
7,8-Di-O-(b-d-galactopyranosyl)-6-methoxycoumarin
23:
Yield:
400 MHz): dH =7.43 (d, J=8.0 Hz, 1H; H-5), 7.26 (d, J=8.0 Hz, 1H;
H-6), 5.43 (br, 2H; HO-2’’,2’’’, exchangeable with D2O), 4.47 (br, 2H;
HO-3’’,3’’’, exchangeable with D2O), 4.95 (br, 2H; HO-4’’,4’’’, ex-
changeable with D2O), 4.63 (br, 2H; HO-6’’,6’’’, exchangeable with
D2O), 5.08 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 4.82 (d, J1’’’,2’’’ =8.0 Hz, 1H; H-
1
706.17 mg, 97%; Rf =0.35 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
400 MHz): dH =6.41 (d, J=8.0 Hz, 1H; H-3), 7.96 (d, J=8.0 Hz, 1H;
H-4), 7.15 (s, 1H; H-5), 5.36 (br, 1H; HO-2’, exchangeable with D2O),
5.32 (br, 1H; HO-2’’, exchangeable with D2O), 4.46 (br, 2H; HO-
3’,3’’, exchangeable with D2O), 4.88 (br, 2H; HO-4’,4’’, exchangeable
with D2O), 4.57 (br, 2H; HO-6’,6’’, exchangeable with D2O), 5.22 (d,
1’’’), 3.72 (dd, J2’’,3’’ =J2’’’,3’’’ =10.2 Hz, 2H; H-2’’,2’’’), 3.45 (dd, J3’’,4’’
=
J
3’’’,4’’’ =4.0 Hz, 2H; H-3’’,3’’’), 3.39 (d, J4’’,5’’ =J4’’’,5’’’ =3.6 Hz, 2H; H-
J
J
1’,2’ =8.0 Hz, 1H; H-1’), 5.26 (d, J1’’,2’’ =8.0 Hz, 1H; H-1’’), 3.72 (dd,
2’,3’ =J2’’,3’’ =10.0 Hz, 2H; H-2’,2’’), 3.57 (dd, J3’,4’ =J3’’,4’’ =4.0 Hz, 2H;
4’’,4’’’), 3.35 (m, 2H; H-5’’,5’’’), 3.53 (m, 4H; H-6’’,6’’’), 2.77 (t, J=
4.0 Hz, 2H; H-1’), 1.78 (m, 4H; H-2’,3’), 2.43 ppm (t, J=4.0 Hz, 2H;
H-4’); 13C NMR ([D6]DMSO, 100 MHz): dC =160.51 (-CO), 112.98 (C-
3), 147.40 (C-4), 132.95 (C-5), 120.66 (C-6), 145.51 (C-7), 115.42 (C-8),
151.56 (C-9), 118.94 (C-10), 103.85 (C-1’’), 102.95 (C-1’’’), 71.37 (C-
2’’), 70.66 (C-2’’’), 73.28 (C-3’’), 72.60 (C-3’’’), 68.15 (C-4’’), 68.05 (C-
4’’’), 75.94 (C-5’’), 75.77 (C-5’’’), 60.46 (C-6’’), 60.20 (C-6’’’), 24.76
(CH2-1’), 21.13, 20.83 (CH2-2’,3’), 23.61 ppm (CH2-4’); ESIMS: m/z:
557 [M+] (9%), 558 [M+1] (13%); elemental analysis calcd (%) for
C25H32O14: C 53.96, H 5.80; found: C 53.92, H 5.77.
H-3’,3’’), 3.40 (d, J4’,5’ =J4’’,5’’ =3.8 Hz, 2H; H-4’,4’’), 3.46 (m, 2H; H-
5’,5’’), 3.75 (m, 4H; H-6’,6’’), 3.83 ppm (s, 3H; CH3O-6); 13C NMR
([D6]DMSO, 100 MHz): dC =159.94 (-CO), 105.99 (C-3), 144.23 (C-4),
141.58 (C-5), 136.52 (C-6), 142.56 (C-7), 114.38 (C-8), 149.89 (C-9),
114.83 (C-10), 103.33 (C-1’), 103.23 (C-1’’), 71.31 (C-2’), 71.26 (C-2’’),
73.26 (C-3’), 73.18 (C-3’’), 68.06 (C-4’,4’’), 75.88 (C-5’), 75.82 (C-5’’),
60.09 (C-6’,6’’), 56.65 ppm (CH3O-6); ESIMS: m/z 533 [M+] (8%), 534
[M+1] (16%); elemental analysis calcd (%) for C22H28O15: C 49.63, H
5.30; found: C 49.59, H 5.26.
6,7-Di-O-(b-d-galactopyranosyl)-4-methylcoumarin
21:
Yield:
1
545.07 mg, 90%; Rf =0.31 (1:3 MeOH–EtOAc); H NMR ([D6]DMSO,
400 MHz): dH =6.24 (s, 1H; H-3), 7.45 (s, 1H; H-5), 7.16 (s, 1H; H-8),
5.10 (d, JH-2’,OH-2’ =4.0 Hz, 1H; HO-2’, exchangeable with D2O), 5.12
(d, JH-2’’,OH-2’’ =4.0 Hz, 1H; HO-2’’, exchangeable with D2O), 4.54 (d,
Preparation of stable lacZ transfected MCF7 and PC3 cell
lines
J
H-3’,OH-3’ =4.0 Hz, 1H; HO-3’, exchangeable with D2O), 4.58 (d, JH-
3’’,OH-3’’ =4.0 Hz, 1H; HO-3’’, exchangeable with D2O), 4.88 (d, JH-4’,OH-
E.coli lacZ gene (from pSV-b-gal vector, Promega, Madison, WI) was
inserted into high expression human cytomegalovirus (CMV) imme-
diate-early enhancer/promoter vector phCMV (Gene Therapy Sys-
tems, San Diego, CA) giving a recombinant vector phCMV/lacZ.
This was used to transfect wild type MCF7 (human breast cancer)
and PC3 (human prostate cancer) cells (ATCC, Manassas, VA) using
GenePORTER2 (Gene Therapy Systems, Genlantis, Inc., San Diego,
CA), as described in detail previously.[24,25] The highest b-gal ex-
pressing colony was selected by using the antibiotic G418 disulfate
(Research Products International Corp, Mt Prospect, IL, USA);
800 mgmL-1) and G418 (200 mgmL-1) was also included for routine
culture. The cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM, Mediatech Inc., Herndon, VA, USA) containing
4’ =4.0 Hz, 1H; HO-4’, exchangeable with D2O), 4.90 (d, JH-4’’,OH-4’’
4.0 Hz, 1H; HO-4’’, exchangeable with D2O), 4.69 (d, JH-6a’,OH-6’
4.0 Hz, JH-6b’,OH-6’ =6.1 Hz, 1H; HO-6’, exchangeable with D2O), 4.71
(d, JH-6a’’,OH-6’’ =4.0 Hz, JH-6b’’,OH-6’’ =6.0 Hz, 1H; HO-6’’, exchangeable
with D2O), 4.85 (d, J1’,2’ =8.0 Hz, 1H; H-1’), 4.97 (d, J1’’,2’’ =8.0 Hz, 1H;
H-1’’), 3.68 (dd, J2’,3’ =J2’’,3’’ =10.1 Hz, 2H; H-2’,2’’), 3.71 (dd, J3’,4’
J
3.46 (m, 2H; H-5’,5’’), 3.58 (m, 4H; H-6’,6’’), 2.37 ppm (s, 3H; CH3-
4); 13C NMR ([D6]DMSO, 100 MHz): dC =160.24 (-CO), 104.25 (C-3),
150.70 (C-4), 143.65 (C-5), 113.51 (C-6), 149.09 (C-7), 112.04 (C-8),
153.25 (C-9), 113.04 (C-10), 101.30 (C-1’), 102.24 (C-1’’), 70.24 (C-2’),
=
=
=
3’’,4’’ =4.0 Hz, 2H; H-3’,3’’), 3.53 (d, J4’,5’ =J4’’,5’’ =3.6 Hz, 2H; H-4’,4’’),
ChemPlusChem 2012, 77, 370 – 378
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chempluschem.org
377

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10% fetal bovine serum (FBS, Hyclone, Logan, UT, USA) with
100 unitsmL-1 of penicillin, 100 unitsmL-1 streptomycin, and cul-
tured in a humidified 5% CO2 incubator at 378C. The b-gal activity
of tumor cells was measured using a b-gal assay kit with o-nitro-
phenyl-b-d-galactopyranoside (Promega, Madison, WI).
MRI
MRI studies were performed by using a 4.7 T horizontal bore
magnet equipped with a Varian INOVA Unity system (Palo Alto, CA,
USA). T1 and T2 maps were acquired by using a spin-echo sequence
with varying repetition times (TR) and echo times (TE), respectively.
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Acknowledgements
This research was supported in part by the NIH National Cancer
Institute (R21 CA120774), and the Southwestern Small Animal
Imaging Research Program (SW-SAIRP), which is supported in
part by U24 CA126608 and P30 CA142543. NMR experiments
were performed at the Advanced Imaging Research Center, an
NIH BTRP facility (P41RR02584). We are also grateful to Dr. Jian
Zhou and Mindy Zhou for technical assistance.
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Keywords: enzymes
relaxivity · reporter molecules
· hydrolases · NMR spectroscopy ·
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Received: December 5, 2011
Published online on March 13, 2012
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