Novel 19F-MRS β-galactosidase reporter molecules incorporated nitrogen mustard analogues

Article abstract of DOI:10.1111/cbdd.12992

In this study, we propose a novel molecular platform-integrated fluorinated antitumor nitrogen mustards for ¹⁹F-MRS assay of β-galactosidase (β-gal) activity. Following this idea, we have designed, synthesized, and characterized 2-fluoro-4-[bis

Full text of DOI:10.1111/cbdd.12992

DR. JIAN-XIN YU (Orcid ID : 0000-0001-5476-1794)
Article type : Research Article
Novel 19F-MRS β-Galactosidase Reporter Molecules
Incorporated Nitrogen Mustard Analogues
Zijun Yu,1† Jianru Zhao,2 Zhiming Hua,2 Xinping Wang,3
Xiaobo Wang,1 Hanqin Wang,1 Jian-Xin Yu 1*
1
2
3
Center of Translational Medicine, 5th School of Medicine/Suizhou Central Hospital, Hubei University
of Medicine, 8 Wenhua Park Road, Suizhou, Hubei 441300, China.
College of Chemistry and Chemical Engineering, Xinjiang University, 14 Victory Road, Urumqi,
Xinjiang 830046, China.
Department of Chemistry and Chemical Engineering, Foshan University School of Sciences, 18
Jiangwan Road, Foshan, Guangdong 528000, China.
† Summer student from the Rice University, 6100 Main St, Houston, TX 77005, USA.
* Correspondence:
Jian-Xin Yu, Ph.D.
Center of Translational Medicine
5th School of Medicine/Suizhou Central Hospital
Hubei University of Medicine
8 Wenhua Park Road, Suizhou, Hubei 441300, China
This article has been accepted for publication and undergone full peer review but has not been
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differences between this version and the Version of Record. Please cite this article as doi:
10.1111/cbdd.12992
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Phone: 18935717422
E-mail: jianxinyu707@gmail.com
Abstract
In this paper, we propose a novel molecular platform integrated fluorinated antitumor nitrogen
mustards for 19F-MRS assay of β-galactosidase (β-gal) activity. Following this idea, we have
designed, synthesized and characterized 2-fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-
galactopyranoside
5,
2-fluoro-4-{bis[2’-O-(β-D-galactopyranosyl)ethyl]amino}phenyl
β-D-
galactopyranoside 8, 2-fluoro-4-{bis[[1’’-(β-D-galactopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl]
amino}phenyl β-D-galactopyranoside 14 and 2-fluoro-4-{bis[[1’’-(β-D-glucopyranosyl)-1’’, 2’’, 3’’-
triazol-4’’-yl]methyl]amino}phenyl β-D-galactopyranoside 15 through glycosylation and click reaction
strategies, and their structures were confirmed by NMR and HRMS or elemental analysis data.
Among of them, 2-fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-galacto-pyranoside 5 was found very
sensitive to β-gal (E801A) in PBS at 37°C with big ΔδF response. Here, we demonstrated the
feasibility of this platform for assessing β-gal activity in solution, and in vitro with lacZ transfected
human MCF7 breast and PC3 prostate tumor cells, by the characterization of β-gal responsive 19F-
chemical shift changes ΔδF and hydrolytic kinetics.
Keywords
β-Galactosidase detection, 19F-MRS lacZ gene reporter, Prodrug, Nitrogen mustard,
Synthesis.
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INTRODUCTION
19F nucleus has a high gyromagnetic ratio (γ=40.05MHz/T), 100% natural isotopic abundance,
large chemical-shift dispersion. Importantly, there are essentially no intrinsic 19F signals detectable in
living tissue and thus no interferences from background signals.[1,2] Therefore, 19F-magnetic
resonance spectroscopy (MRS) has been widely applied in in vivo studies on drug absorption,
distribution, metabolism and excretion by reasons of its favorable MR properties, simplicity and high
sensitivity.[3] We have developed a series of 19F-MRS β-galactosidase (β-gal) reporters with
structures exploiting fluorinated/trifluoromethylated phenols, fluoropyridoxine and fluorinated Fe-
chelator aglycones, and demonstrated the success for assessing β-gal activity in solution, in vitro with
lacZ transfected tumor cells, and in vivo in human tumor xenografts growing in mice.[4-6] In this paper,
we propose a novel molecular platform integrated fluorinated antitumor nitrogen mustards for 19F-
MRS detection of β-gal activity.
RESULTS AND DISCUSSION
Design and Synthesis
Nitrogen mustards are widely used in the treatment of cancers.[7-9]
But, their efficacies for chemotherapy are mainly limited by bone marrow toxicity and genotoxicity,[10]
insufficient drug concentrations in tumors, and lack of selectivity for neoplastic cells.[7] The promising
solution to these problems is to use targeting strategies such as antibody-directed enzyme prodrug
therapy and gene-directed enzyme prodrug therapy. As demonstrated, a number of clinical trials
using suicide gene therapy are ongoing.[11,12]
The lacZ gene encoding β-gal has been recognized as the standard means of assaying clonal
insertion, transcriptional activation, protein expression and interaction in molecular/ cellular biology,
small animal investigations, and clinical trials.[13-16] So when using fluorinated nitrogen mustards[17,18]
as aglycones, their β-D-galactosides would work as prodrugs. Upon delivery and cleavage at the lacZ
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transfected tumor, the fluorinated antitumor nitrogen mustards will be lacZ (or β-gal)-specifically
released, in which the companied 19F chemical shift changes and hydrolytic kinetics will recognize the
presence and progression of lacZ (or β-gal) , offering a potential for simultaneously monitoring of
cancer therapy. Moreover, this probe design is based on the lacZ gene-directed enzyme prodrug
strategy, in which the prodrug proceeds in the β-gal responsive way to selectively convert into a toxic
drug at the tumor, such localized activation will then reduce the normal tissue toxicity associated with
conventional cytotoxic chemotherapy of nitrogen mustards as reported.[10]
The synthesis started with 2-fluoro-4-nitrophenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-
galactopyranoside 1, which was readily obtained in excellent yield (99%) by phase-transfer catalyzed
approach using tetrabutylammonium bromide (TBAB) in CH2Cl2-H2O (pH 8-9) at 50°C.[4a]
Hydrogenation of 1 in ethanolic solution catalyzed by 5% Pd/C converted to the corresponding 2-
fluoro-4-aminephenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-galactopyranoside 2 in quantitative yield.
Treatment of 2 with ethylene oxide in AcOH/benzene (1:5 V/V’) from 0°C to room temperature gave
2-fluoro-4-[bis(2’-hydroxyethyl)amino]phenyl 2’’, 3’’, 4’’, 6’’-tetra-O-acetyl-β-D-galactopyranoside 3 in
85% yield. The direct chlorination of 3 with an excess amount of SOCl2 in CH2Cl2 obtained 2-fluoro-4-
[bis(2’-chloroethyl)amino]phenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-galactopyranoside 4 in 77% yield,
which was then deacetylated with NH3/MeOH from 0°C to room temperature, giving the target
molecule 2-fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-galactopyranoside 5 in excellent yield
(Figure 1). The structures of 1-5 were characterized by NMR and HRMS or elemental analysis data.
4
1
Their anomeric β-D-configuration in the C1 chair conformation was confirmed by the H-NMR
chemical shifts of the anomeric protons at δH-1’/δH-1’’=4.60-5.15ppm, and the coupling constants of
J1’,2’/J1’’,2’’= ~8Hz and J2’,3’/J2’’,3’’=~10Hz, as well as the anomeric carbon resonances appeared at δC-
1’/δC-1’’ =100.20-103.00ppm.[4-6]
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Carbohydrate-associated prodrugs in clinical applications have showed improved water
solubility and permeability, leading to better selectivity and efficacy of chemotherapy.[19] For these
reasons, we sought to introduce additional carbohydrate units to the nitrogen mustard. Condensation
of 3 directly with 1.3 equivalents of 2, 3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide in
anhydrous CH2Cl2/MeCN catalyzed by Hg(CN)2 as a promoter, furnished the 2-fluoro-4-{bis[2’-O-(2’’,
3’’, 4’’, 6’’-tetra-O-acetyl-β-D-galactopyranosyl)ethyl] amino}phenyl 2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-
galactopyranoside 7 in 89% yield. The HRMS displayed the expected quasimolecular ions [M+H]+ at
m/z 1206.3852 (calculated m/z 1206.3889) and [M+Na]+ at m/z 1228.3660 (calculated m/z
1228.3708), corresponding to the fully adorned derivative with additional two 2, 3, 4, 6-tetra-O-acetyl-
β-D-galactopyranosyl units, in which the β-D-configuration linkages were established based on the
anomeric protons H-1’’ of D-galactoses at δH-1’’=4.50ppm with the well resolved doublets J1’’,2’’=8.0Hz
and J2’’,3’’=10.6 Hz, whereas the anomeric carbons C-1’’ at δC-1’’=101.13ppm are in accordance.
Subsequent deacetylation of 7 with NH3/MeOH from 0°C to room temperature gave the extra two β-D-
galactopyranosyl-associated molecule 2-fluoro-4-{bis[2’-O-(β-D-galactopyranosyl)ethyl]amino} phenyl
β-D-galactopyranoside 8 in excellent yield (95%). However, similar glycosylation with 2, 3, 4, 6-tetra-
O-acetyl-α-D-glucopyranosyl bromide for the synthesis of 2-fluoro-4-{bis[2’-O-(2’’, 3’’, 4’’, 6’’-tetra-O-
acetyl-β-D-glucopyranosyl)ethyl]amino}phenyl 2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-galactopyranoside 9
then to its corresponding free β-D-galactopyranoside 10 (Figure 2) was not successful, instead by
recovering the starting material 2-fluoro-4-[bis(2’-hydroxyethyl) amino]phenyl 2’, 3’, 4’, 6’-tetra-O-
acetyl-β-D-galactopyranoside 3.
‘Click Chemistry’ using CuI-catalyzed azide-alkyne cycloaddition reaction has been proven as
one of the most powerful tools in drug discovery, chemical biology, and proteomic applications.[20]
Inspired by these successes, we proposed another strategy to associate additional carbohydrate
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units with the nitrogen mustard through the 1, 3, 4-triazole rings, hoping them together to participate
in hydrogen-bonding and dipole interactions, thereby favoring molecular recognition processes and
enzymatic reaction with β-gal.[20-22] Reaction of 2-fluoro-4-aminephenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-
galactopyranoside 2 with propargyl bromide in DMF at
80°C for 3h gave 2-fluoro-4-
[bis(propargyl)amino]phenyl 2’’, 3’’, 4’’, 6’’-tetra-O-acetyl-β-D-galactopyranoside 11 in 88% yield. The
peracetylated β-D-galacto- and glucopyranosyl azides were prepared according to the previously
described method.[23] Click reaction of alkyne 11 with azides catalyzed by CuSO4/NaAsc in DMF-H2O
at room temperature for 12h afforded 2-fluoro-4-{bis[[1’’-(2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-
galactopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl]amino}phenyl 2’’’’, 3’’’’, 4’’’’, 6’’’’-tetra-O-acetyl-β-D-
galactopyranoside 12 (82%) and 2-fluoro-4-{bis[[1’’-(2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-
glucopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl]amino}phenyl 2’’’’, 3’’’’, 4’’’’, 6’’’’-tetra-O-acetyl-β-D-
galactopyranoside 13 (85%), respectively. After workup and deacetylation, the desired molecules 2-
fluoro-4-{bis[[1’’-(β-D-galactopyranosyl)-1’’,
2’’,
3’’-triazol-4’’-yl]methyl]amino}phenyl
β-D-
galactopyranoside 14 and 2-fluoro-4-{bis[[1’’-(β-D-glucopyranosyl)-1’’,
2’’,
3’’-triazol-4’’-
yl]methyl]amino}phenyl β-D-galactopyranoside 15 were obtained in 91-94% yields (Figure 3). The
structures of 11-15 were characterized by NMR and HRMS data. The expected quasimolecular ions
of 12: [M+H]+ at m/z 1280.4073 (calculated m/z 1280.4018), [M+Na]+ at m/z 1302.3614 (calculated
m/z 1302.3838), [M+K]+ at m/z 1318.1919 (calculated m/z 1318.3577); 13: [M-H]+ at m/z 1278.4196
(calculated m/z 1278.3862), [M-H+Na]+ at m/z 1301.3931 (calculated m/z 1301.3759); 14/15: [M+H]+
at m/z 776.2817/776.2834 (calculated m/z 776.2750), [M+Na]+ at m/z 798.3064/798.3345 (calculated
m/z 798.2570), [M+K]+ at m/z 814.2819/814.2741 (calculated m/z 814.2309), are matching to the fully
ornamented derivatives with additional two 2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl (→12 and
14) or 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl (→13 and 15) units by the fused 1, 3, 4-triazole
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linkers, in which the β-D-configuration linkages of N-glycosides were established based on the
anomeric protons H-1’’’ of N-galacosides (→12 and 14) and N-glucosides (→13 and 15) at δH-
1’’’=5.40-5.90ppm with the well resolved doublets J1’’’,2’’’=8.0Hz, whereas the anomeric carbons C-1’’’
at δC-1’’’=85.90-88.20ppm are in agreement.[24] In particular, the regioselectivity of the formation 1,4-
disubstituted 1, 3, 4-triazoles in 12-15 was confirmed by the δC-4’’, δC-5’’ and Δ(δC-4’’-δC-5’’) values [12:
δC-4’’=145.34ppm, δC-5’’=120.81ppm, Δ(δC-4’’-δC-5’’)=24.53ppm; 13: δC-4’’=145.77ppm, δC-5’’= 120.82ppm,
Δ(δC-4’’-δC-5’’)=24.95ppm; 14: δC-4’’=144.27ppm, δC-5’’=122.00ppm, Δ(δC-4’’-δC-5’’)= 22.27ppm; 15: δC-
4’’=144.14ppm, δC-5’’=122.45ppm, Δ(δC-4’’-δC-5’’)=21.69ppm] in the ranges of δC-4’’=132.00-148.00ppm,
δC-5’’=120.00-128.00ppm, Δ(δC-4’’-δC-5’’)=21.00-27.00ppm, which are the typical characteristics for the
identification of the 1,4-disubstituted 1,3,4-triazole structures.[20,25]
β-Gal Activity
The reactivity of 5, 8, 14 and 15 to β-gal was evaluated by kinetic 19F-
MRS.[4-6] 5, 8, 14 and 15 in PBS (0.1M, pH=7.4) at 37°C each gave a narrow 19F-signal at δF=-55.65, -
56.29, -56.85 and -56.78ppm, respectively. Following the reaction with β-gal (E801A) in PBS (0.1M,
pH=7.4) at 37°C, the kinetic 19F-MRS measurements of 19F chemical shift and signal intensity
changes at each time point (0-40min) indicated that only 5 converted in the high hydrolytic rate
ν=738.4μM/min/unit to the fluorinated nitrogen mustard 2-fluoro-4-[bis(2’-chloroethyl)amino]phenol
(2F4ClPh) (δF=-59.44ppm). 8, 14 and 15 however are not reactive to β-gal as expected at the same
condition, keeping their 19F-signals unchanged over the period of 60min. Meanwhile, we found that
the liberated fluorinated nitrogen mustard 2-fluoro-4-[bis(2’-chloroethyl)amino]phenol (2F4ClPh) was
unstable and slowly hydrolyzed with H2O by displacement of the chlorine atoms in the rate
ν=378.0μM/min to the dialcohol derivative 2-fluoro-4-[bis(2’-hydroxyethyl)amino]phenol (2F4OHPh)
(δF=-44.33 ppm) (Figure 4). This observation was further confirmed by monitoring the reaction of 2-
fluoro-4-[bis(2’-hydroxyethyl)amino]phenyl β-D-galactopyranoside 6 (δF=-55.95ppm) with β-gal at the
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same condition, with the direct releasing of 2-fluoro-4-[bis(2’-hydroxyethyl)amino]phenol (2F4OHPh)
at δF=-44.33ppm. Figure 5 showed the kinetic hydrolysis time courses of 2-fluoro-4-[bis(2’-
chloroethyl)amino]phenyl β-D-galactopyranoside 5 with β-gal (E801A) in PBS (0.1M, pH=7.4) at 37°C.
In vitro Assay
Further in vitro evaluation of 5 with the lacZ transfected human MCF7
breast and PC3 prostate cancer cells in PBS (0.1M, pH=7.4) at 37°C under 5% CO2 in air with 95%
humidity, using wild type (WT) MCF7 and PC3 cancer cells at the same conditions as the controls,
19F-MRS analysis of the mixtures at different time points (0-180min) exhibited a single narrow 19F-
signal of 5 (δF=-55.65ppm) at the starting point (0min, or with MCF7-WT and PC3-WT for 3h as
controls), and a new single 19F-peak of the fluorinated nitrogen mustard 2-fluoro-4-[bis(2’-
chloroethyl)amino] phenol (2F4ClPh) at δF=-59.44ppm progressively grown with the time going in the
rates of ν=6.14 (with MCF7-lacZ cells) and 2.30 (with PC3-lacZ cells) μM/min/per million cells,
respectively. However, incubation of 5 with the equal numbers of lysed MCF7-lacZ and PC3-lacZ
cells showed the rates of conversion were 1.5 (with lysed MCF7-lacZ cells) and 11.1 (with lysed PC3-
lacZ cells) times higher than the intact cells, respectively. These differences between intact and lysed
cells suggest that membrane permeation is a bigger obstruction for 5 to reaction of β-gal in PC3-lacZ
cells than in MCF7-lacZ cells, due to the cell membranes of human MCF7-lacZ breast and PC3-lacZ
prostate cancer cells have different selective permeabilities.[26]
Cytotoxicity
The cytotoxicity of prodrug 5 was examined with both wild-type and lacZ
expressing MCF7 and PC3 cells in 24h incubation at 37°C under 5% CO2 in air with 95% humidity.
Prodrug 5 showed neither toxicity to MCF7- nor PC3-WT cells, for prodrug 5 viability exceeded 98%
for both MCF7- and PC3-WT cells at all concentrations tested. To MCF7- and PC3-lacZ cells, prodrug
5 exhibited less cytotoxicity but in different behaviors, for MCF7-lacZ cells survival was much more
decreased to 65% at 1mM, whereas for PC3-lacZ cells survival was >92% at the same concentration
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(Figure 6). This observation was in good agreement with the differences of selective permeability
between MCF7 breast and PC3 prostate cancer cells.
CONCLUSION
We present here a novel molecular platform integrated fluorinated antitumor nitrogen mustards
for 19F-MRS detection of β-gal activity. Following this design, we have successfully synthesized
carbohydrate-associated prodrugs 2-fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-galactopyranoside
5, 2-fluoro-4-{bis[2’-O-(β-D-galactopyranosyl)ethyl] amino}phenyl β-D-galactopyranoside 8, 2-fluoro-4-
{bis[[1’’-(β-D-galactopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl] amino}phenyl β-D-galactopyranoside 14
and 2-fluoro-4-{bis[[1’’-(β-D-glucopyranosyl)-1’’, 2’’, 3’’ -triazol-4’’-yl]methyl]amino}phenyl β-D-
galactopyranoside 15 through glycosylation and click reaction strategies. Among of them, 2-fluoro-4-
[bis(2’-chloroethyl)amino]phenyl β-D-galactopyranoside 5 was found very sensitive to β-gal (E801A)
in PBS at 37°C in the rate ν=738.4μM/min/unit and Δδ response (Δδ=~4ppm). Herein we
demonstrated the feasibility of this platform for assessing β-gal activity in solution, and in vitro with
lacZ transfected human MCF7 breast and PC3 prostate tumor cells, by the characterization of β-gal
responsive 19F-chemical shift changes ΔδF and hydrolytic kinetics.
EXPERIMENTAL
General Methods NMR spectra were recorded on a Varian Unity INOVA 400 spectrometer
(400MHz for 1H, 100MHz for 13C, 376 MHz for 19F), 1H and 13C chemical shifts are referenced to TMS
as internal standard with CDCl3, or DMSO-d6 as solvents, 19F to a dilute solution of NaTFA in a
capillary as external standard, chemical shifts are given in ppm. All compounds were characterized by
NMR at 25°C. Microanalyses were performed on a Perkin-Elmer 2400CHN microanalyser. Mass
spectra were obtained by positive and negative ESI-MS using a Micromass Q-TOF hybrid
quadrupole/time-of-flight instrument (Micromass UK Ltd).
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Solutions in organic solvents were dried with anhydrous Na2SO4, and concentrated in vacuo
below 45°C. Hg(CN)2 was dried before use at 50°C for 1h, CH2Cl2 was dried over Drierite, acetonitrile
was dried on CaH2 and kept over molecular sieves under nitrogen. 2, 3, 4, 6-Tetra-O-acetyl-α-D-
galactopyranosyl bromide and 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide were purchased
from the Sigma Chemical Company. β-Gal (E801A) was purchased from Promega (Madison, WI,
USA) and enzymatic reactions were performed at 37°C in PBS solution (0.1M, pH=7.4). Column
chromatography was performed on silica gel (200-300mesh) and silica gel GF254 used for analytical
TLC was purchased from the Aldrich Chemical Company. Detection was effected by spraying the
plates with 5% ethanolic H2SO4 (followed by heating at 110°C for 10mins.) or by direct UV illumination
of the plate. The purity of the final products was determined by HPLC with ≥ 95%.
2-Fluoro-4-nitrophenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-galactopyranoside 1 A solution of 2,
3, 4, 6-tetra-O-acetyl-α-D-galactopyranosyl bromide (820 mg, 2.0 mmol) in CH2Cl2 (15 mL) was added
dropwise to a vigorously stirred solution of 2-fluoro-4-nitrophenol (378 mg, 2.4 mmol, 1.2 equiv.) and
tetrabutylammonium bromide (480 mg, 1.5 mmol) in H2O (25 mL, pH 8-9) and CH2Cl2 (25 mL) at 50
°C in 3-neck round-bottom flask equipped with condenser and thermometer. After TLC showed
complete reaction (~1 h) the organic layer was separated, washed with water, dried (Na2SO4),
evaporated under reduced pressure and purified by column chromatography on silica gel to give 1
(1.0 g, 99%) as white crystals, Rf 0.35 (3:2 cyclohexane-EtOAc), δH (CDCl3): 8.03 (2H, m, Ar-H), 7.32
(1H, m, Ar-H), 5.13 (1H, d, J1’,2’ = 7.8 Hz, H-1’), 5.58 (1H, dd, J2’,3’ = 10.2 Hz, H-2’), 5.15 (1H, dd, J3’,4’
= 3.6 Hz, H-3’), 5.40 (1H, d, J4’,5’ = 3.6 Hz, H-4’), 4.10 (1H, dd, J5’,6a’ = 4.5 Hz, J5’,6b’ = 5.0 Hz, H-5’),
4.26 (1H, dd, J6a’,6b’ = 11.0 Hz, H-6a’), 4.19 (1H, dd, H-6b’), 2.21, 2.11, 2.08, 2.04 (12H, 4s,
4×CH3CO) ppm; δC (CDCl3): 170.45, 170.26, 170.21, 169.44 (4s, 4×CH3CO), 152.23 (d, JF-C = 252.0
Hz, Ar-C), 150.07 (d, JF-C = 10.6 Hz, Ar-C), 120.51 (d, JF-C = 9.2 Hz, Ar-C), 118.53 (d, JF-C = 11.7 Hz,
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Ar-C), 113.11 (d, JF-C = 23.7 Hz, Ar-C), 100.28 (d, JF-C = 3.1 Hz, C-1’), 68.34 (s, C-2’), 70.57 (s, C-3’),
66.81 (s, C-4’), 71.83 (s, C-5’), 61.45 (s, C-6’), 20.80, 20.77, 20.72, 20.33 (4s, 4×CH3CO) ppm;
HRMS: [M+Na]+, C20H22NO12FNa, Calcd: 510.1024, Found: 510.1014; [M+K]+, C20H22NO12FK, Calcd:
526.0763, Found: 526.0751.
2-Fluoro-4-aminophenyl 2’, 3’, 4’, 6’-tetra-O-acetyl-β-D-galactopyranoside 2 Hydrogenation
(H2, 30 psi) of 1 (1.12 g, 2.30 mmol) in anhydrous EtOH (80 mL) catalyzed by Pd/C (5%, 200 mg) at
room temperature overnight furnished the amine derivative 2 (1.05 g, 100%) as white foam solid, Rf
0.30 (1:1 cyclohexane-EtOAc), δH (CDCl3): 7.00 (1H, t, J = 8.8 Hz, Ar-H), 6.41 (1H, dd, J = 2.8, 12.4
Hz, Ar-H), 6.33 (1H, dd, J = 2.8, 8.8 Hz, Ar-H), 4.74 (1H, d, J1’,2’ = 8.0 Hz, H-1’), 5.44 (1H, dd, J2’,3’
=
10.4 Hz, H-2’), 5.06 (1H, dd, J3’,4’ = 3.2 Hz, H-3’), 5.42 (1H, d, J4’,5’ = 3.6 Hz, H-4’), 3.93 (1H, m, H-5’),
4.23 (1H, dd, J5’,6a’ = 6.4 Hz, J6a’,6b’ = 11.2 Hz, H-6a’), 4.14 (1H, dd, J5’,6b’ = 6.8 Hz, H-6b’), 3.60 (2H, br,
NH2), 2.18, 2.11, 2.06, 2.01 (12H, 4s, 4×CH3CO) ppm; δC (CDCl3): 170.55, 170.49, 170.34, 169.86
(4s, 4×CH3CO), 154.57 (d, JF-C = 244.9 Hz, Ar-C), 144.50 (d, JF-C = 9.2 Hz, Ar-C), 136.70 (s, Ar-C),
123.47 (d, JF-C = 1.5 Hz, Ar-C), 110.35 (s, Ar-C), 103.55 (d, JF-C = 3.8 Hz, Ar-C), 102.99 (s, C-1’),
68.80 (s, C-2’), 70.87 (s, C-3’), 67.02 (s, C-4’), 71.07 (s, C-5’), 61.33 (s, C-6’), 20.83 - 20.76 (4s,
4×CH3CO) ppm.
Anal. Calcd. for C20H24NO10F (%): C, 52.52, H, 5.29, N, 3.06; Found: C, 52.47, H, 5.24, N,
3.02.
2-Fluoro-4-[bis(2’-hydroxyethyl)amino]phenyl
2’’,
3’’,
4’’,
6’’-tetra-O-acetyl-β-D-
galactopyranoside 3 To a solution of the amine 2 (1.0 g, 2.2 mmol) in benzene (50 mL) and acetic
acid (10 mL) at 0°C was added ethylene oxide (2.2 mL, 44.0 mmol, 10 equiv.), and the mixture was
stirred for 48 h at room temperature at the end of time TLC showed reaction complete, then
evaporated under reduced pressure and purified by column chromatography on silica gel to give 3
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(1.0 g, 85%) as syrup, Rf 0.45 (EtOAc), δH (CDCl3): 7.07 (1H, t, J = 9.2 Hz, Ar-H), 6.42 (1H, dd, J =
2.8, 13.6 Hz, Ar-H), 6.33 (1H, dd, J = 2.8, 9.2 Hz, Ar-H), 4.74 (1H, d, J1’’,2’’ = 8.0 Hz, H-1’’), 5.44 (1H,
dd, J2’’,3’’ = 10.4 Hz, H-2’’), 5.06 (1H, dd, J3’’,4’’ = 3.2 Hz, H-3’’), 5.41 (1H, d, J4’’,5’’ = 2.4 Hz, H-4’’), 3.92
(1H, m, H-5’’), 4.23 (1H, dd, J5’’,6a’’ = 6.4 Hz, J6a’’,6b’’ = 11.2 Hz, H-6a’’), 4.15 (1H, dd, J5’’,6b’’ = 7.2 Hz, H-
6b’’), 3.53 (4H, t, J1’,2’ = 4.8 Hz, -NCH2CH2OH), 3.83 (4H, t, J1’,2’ = 4.8 Hz, -NCH2CH2OH), 3.35 (2H,
br, -N(CH2)2OH), 2.19, 2.11, 2.04, 2.01 (12H, 4s, 4×CH3CO) ppm; δC (CDCl3): 170.58, 170.49,
170.37, 169.91 (4s, 4×CH3CO), 154.74 (d, JF-C = 243.4 Hz, Ar-C), 146.09 (d, JF-C = 10.0 Hz, Ar-C),
135.62 (d, JF-C = 12.0 Hz, Ar-C), 123.43 (s, Ar-C), 107.91 (s, Ar-C), 103.10 (s, Ar-C), 101.03 (s, C-1’’),
68.82 (s, C-2’’), 70.88 (s, C-3’’), 66.98 (s, C-4’’), 71.06 (s, C-5’’), 61.27 (s, C-6’’), 55.57 (s, -
NCH2CH2OH), 60.69 (s, -NCH2CH2OH), 21.85 - 20.78 (4s, 4×CH3CO) ppm.
Anal. Calcd. for C24H32NO12F (%): C, 52.84, H, 5.91, N, 2.57; Found: C, 52.78, H, 5.88, N,
2.53.
2-Fluoro-4-[bis(2’-chloroethyl)amino]phenyl
2’’,
3’’,
4’’,
6’’-tetra-O-acetyl-β-D-
galactopyranoside 4 To a well stirred solution of 3 (0.5 g, 1.0 mmol) in anhydrous CH2Cl2 (30 mL)
at 0°C was added 2M SOCl2 in CH2Cl2 (3.0 mL, 6.0 mmol, 3 equiv.), and the mixture was continually
stirred for 2 h at 0°C and 1 h at room temperature until TLC showed reaction complete, then
evaporated under reduced pressure and purified by flash silica gel column chromatography to afford
4 (411 mg, 77%) as syrup, Rf 0.67 (2:3 cyclohexane-EtOAc), δH (CDCl3): 7.10 (1H, t, J = 8.2 Hz, Ar-
H), 6.31 (1H, dd, J = 3.8, 12.2 Hz, Ar-H), 6.23 (1H, dd, J = 3.8, 8.2 Hz, Ar-H), 4.76 (1H, d, J1’’,2’’ = 8.0
Hz, H-1’’), 5.45 (1H, dd, J2’’,3’’ = 10.2 Hz, H-2’’), 5.07 (1H, dd, J3’’,4’’ = 3.8 Hz, H-3’’), 5.41 (1H, d, J4’’,5’’
=
3.4 Hz, H-4’’), 3.91 (1H, m, H-5’’), 4.22 (1H, dd, J5’’,6a’’ = 6.6 Hz, J6a’’,6b’’ = 11.6 Hz, H-6a’’), 4.14 (1H,
dd, J5’’,6b’’ = 7.4 Hz, H-6b’’), 3.58 (4H, t, J1’,2’ = 4.8 Hz, -NCH2CH2Cl), 3.77 (4H, t, J1’,2’ = 4.8 Hz, -
NCH2CH2Cl), 2.18, 2.11, 2.03, 2.00 (12H, 4s, 4×CH3CO) ppm; δC (CDCl3): 170.52, 170.47, 170.34,
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169.83 (4s, 4×CH3CO), 155.08 (d, JF-C = 244.0 Hz, Ar-C), 144.04 (d, JF-C = 5.0 Hz, Ar-C), 135.78 (d,
JF-C = 12.0 Hz, Ar-C), 123.97 (s, Ar-C), 106.90 (s, Ar-C), 103.01 (s, Ar-C), 100.43 (s, C-1’’), 68.86 (s,
C-2’’), 70.93 (s, C-3’’), 68.34 (s, C-4’’), 71.14 (s, C-5’’), 67.03 (s, C-6’’), 59.08 (s, -NCH2CH2Cl), 61.28
(s, -NCH2CH2Cl), 21.24 - 20.80 (4s, 4×CH3CO) ppm.
Anal. Calcd. for C24H30NO10FCl2 (%): C, 49.50, H, 5.19, N, 2.41; Found: C, 49.44, H, 5.13, N,
2.37.
2-Fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-galactopyranoside 5 A solution of 4 (400
mg) in anhydrous MeOH (30 mL) containing 0.5M NH3 was vigorously stirred from 0°C to room
temperature overnight until TLC showed that the reaction was completed, and evaporated to dryness
in vacuo. Chromatography of the crude syrup on silica gel with ethyl acetate-methanol afforded the
corresponding β-D-galactopyranoside 5 (270 mg, 95%) as white foam solid, Rf 0.37 (5:1 MeOH-
EtOAc), δH (DMSO-d6): 7.11 (1H, t, J = 12.0 Hz, Ar-H), 6.63 (1H, dd, J = 4.8, 12.4 Hz, Ar-H), 6.47 (1H,
dd, J = 4.8, 12.0 Hz, Ar-H), 4.66 (1H, d, J1’’,2’’ = 8.0 Hz, H-1’’), 3.80 - 3.24 (14H, m, H-2’’, 3’’, 4’’, 5’’, 6’’,
-NCH2CH2Cl), 5.13 (1H, d, JH-2’’,OH-2’’ = 4.0 Hz, HO-2’’), 4.49 (1H, d, JH-3’’,OH-3’’ = 4.2 Hz, HO-3’’), 4.82
(1H, d, JH-4’’,OH-4’’ = 3.2 Hz, HO-4’’), 4.60 (1H, t, JH-6’’,OH-6’’ = 4.2 Hz, HO-6’’) ppm; δC (DMSO-d6): 153.14
(d, JF-C = 241.0 Hz, Ar-C), 142.56 (d, JF-C = 9.0 Hz, Ar-C), 136.04 (d, JF-C = 11.0 Hz, Ar-C), 120.01 (s,
Ar-C), 107.65 (s, Ar-C), 102.80 (s, Ar-C), 100.81 (s, C-1’’), 70.30 (s, C-2’’), 73.35 (s, C-3’’), 68.05 (s,
C-4’’), 75.47 (s, C-5’’), 60.31 (s, C-6’’), 41.23 (s, -NCH2CH2Cl), 52.32 (s, -NCH2CH2Cl) ppm.
Anal. Calcd. for C16H22NO6FCl2 (%): C, 46.39, H, 5.35, N, 3.38; Found: C, 46.34, H, 5.31, N,
3.34.
2-Fluoro-4-[bis(2’-hydroxyethyl)amino]phenyl β-D-galactopyranoside 6 Deacetylation of 3
(500 mg) in anhydrous MeOH (50 mL) containing 0.5M NH3, according to the procedures as
described for the preparation of free β-D-galactopyranoside 5, yielded 6 (336 mg, 97%) as white foam
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solid, Rf 0.30 (6:1 MeOH-EtOAc), δH (DMSO-d6): 7.06 (1H, t, J = 9.6 Hz, Ar-H), 6.52 (1H, dd, J = 3.8,
14.4 Hz, Ar-H), 6.40 (1H, dd, J = 3.8, 9.6 Hz, Ar-H), 4.61 (1H, d, J1’’,2’’ = 8.0 Hz, H-1’’), 3.60 - 3.30
(14H, m, H-2’’, 3’’, 4’’, 5’’, 6’’, -NCH2CH2OH), 5.13 (1H, d, JH-2’’,OH-2’’ = 5.2 Hz, HO-2’’), 4.50 (1H, d, JH-
3’’,OH-3’’ = 4.8 Hz, HO-3’’), 4.83 (1H, d, JH-4’’,OH-4’’ = 5.6 Hz, HO-4’’), 4.73 (1H, t, JH-6’’,OH-6’’ = 5.2 Hz, HO-
6’’), 3.68 (2H, t, JH-2’, OH-2’ = 4.0 Hz, -NCH2CH2OH) ppm; δC (DMSO-d6): 153.34 (d, JF-C = 239.0 Hz, Ar-
C), 144.53 (d, JF-C = 9.0 Hz, Ar-C), 134.65 (d, JF-C = 11.0 Hz, Ar-C), 120.23 (s, Ar-C), 106.77 (s, Ar-C),
103.18 (s, Ar-C), 99.64 (s, C-1’’), 70.36 (s, C-2’’), 73.37 (s, C-3’’), 68.07 (s, C-4’’), 75.46 (s, C-5’’),
60.32 (s, C-6’’), 53.46 (s, -NCH2CH2OH), 58.17 (s, -NCH2CH2OH) ppm.
Anal. Calcd. for C16H24NO8F (%): C, 50.93, H, 6.41, N, 3.71; Found: C, 50.89, H, 6.38, N, 3.67.
2-Fluoro-4-{bis[2’-O-(2’’,
3’’,
4’’,
6’’-tetra-O-acetyl-β-D-galactopyranosyl)ethyl]
amino}phenyl 2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-galactopyranoside 7 A solution of 2, 3, 4, 6-
tetra-O-acetyl-α-D-galactopyranosyl bromide (784 mg, 1.9 mmol, 1.3 equiv.) in anhydrous CH2Cl2 (10
mL) was added dropwise into the solution of 3 (400 mg, 0.7 mmol) and Hg(CN)2 (556 mg, 2.2 mmol,
1.5 equiv.) as a promoter in dry CH2Cl2 (20 mL) containing powdered molecular sieves (4 Å, 1.5 g)
with vigorous stirring at room temperature under an argon atmosphere in the dark for 12 h. The
mixture was diluted with CH2Cl2 (70 mL), filtered through Celite, washed with water, dried (Na2SO4)
and concentrated in vacuo. The residue was purified on a silica gel column to yield 7 (787 mg, 89%)
as syrup, Rf 0.56 (EtOAc), δH (CDCl3): 7.07 (1H, t, J = 8.2 Hz, Ar-H), 6.48 (1H, dd, J = 3.8, 15.6 Hz,
Ar-H), 6.39 (1H, dd, J = 3.8, 8.2 Hz, Ar-H), 4.50 (2H, d, J1’’,2’’ = 8.0 Hz, H-1’’), 4.77 (1H, d, J1’’’,2’’’ = 8.0
Hz, H-1’’’), 5.23 (2H, dd, J2’’,3’’ = 10.6 Hz, H-2’’), 5.45 (1H, dd, J2’’’,3’’’ = 10.2 Hz, H-2’’’), 5.00 (2H, dd,
J3’’,4’’ = 3.4 Hz, H-3’’), 5.08 (1H, dd, J3’’’,4’’’ = 3.2 Hz, H-3’’’), 5.39 (2H, d, J4’’,5’’ = 2.8 Hz, H-4’’), 5.43 (1H,
d, J4’’’,5’’’ = 2.6 Hz, H-4’’’), 4.03 - 3.88 (3H, m, H-5’’, 5’’’), 4.30 - 4.05 (6H, m, H-6’’, 6’’’), 3.55 (4H, t, J1’,2’
= 4.0 Hz, -NCH2CH2O-), 3.76 (4H, t, J1’,2’ = 4.0 Hz, -NCH2CH2O-), 2.20, 2.19, 2.18, 2.13, 2.12, 2.07,
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2.06, 2.05, 2.04, 2.02, 1.99, 1.98 (36H, 12s, 12×CH3CO) ppm; δC (CDCl3): 170.56, 170.54, 170.53,
170.51, 170.49, 170.46, 170.40, 170.38, 170.33, 170.27, 169.82, 169.70 (12s, 12×CH3CO), 154.62
(d, JF-C = 244.0 Hz, Ar-C), 146.34 (d, JF-C = 9.0 Hz, Ar-C), 135.52 (d, JF-C = 11.0 Hz, Ar-C), 123.28 (s,
Ar-C), 108.04 (s, Ar-C), 103.07 (s, Ar-C), 101.13 (s, C-1’’), 101.26 (s, C-1’’’), 66.93 (s, C-2’’), 67.04 (s,
C-2’’’), 68.76 (s, C-3’’, 3’’’), 61.19 (s, C-4’’), 61.23 (s, C-4’’’), 70.85 (s, C-5’’), 70.94 (s, C-5’’’), 60.09 (s,
C-6’’), 60.55 (s, C-6’’’), 51.70 (s, -NCH2CH2O-), 54.76 (s, -NCH2CH2O-), 21.21 - 20.64 (12s,
12×CH3CO) ppm; HRMS: [M+H]+, C52H69NO30F, Calcd: 1206.3889, Found: 1206.3852; [M+Na]+,
C52H68NO30FNa, Calcd: 1228.3708, Found: 1228.3660.
2-Fluoro-4-{bis[2’-O-(β-D-galactopyranosyl)ethyl]amino}phenyl β-D-galacto-pyranoside 8
Deacetylation of 7 (700 mg) in anhydrous MeOH (60 mL) containing 0.5M NH3 using the procedures
as described for the preparation of free β-D-galactopyranosides 5 and 6 afforded 8 (387 mg, 95%) as
white foam solid, Rf 0.33 (10:1 MeOH-EtOAc), δH (DMSO-d6): 7.08 (1H, t, J = 8.0 Hz, Ar-H), 6.54 (1H,
dd, J = 3.8, 12.2 Hz, Ar-H), 6.42 (1H, dd, J = 3.8, 8.0 Hz, Ar-H), 4.13 (2H, d, J1’’,2’’ = 8.0 Hz, H-1’’),
4.63 (1H, d, J1’’’,2’’’ = 8.0 Hz, H-1’’’), 3.85 - 3.15 (26H, m, H-2’’, 2’’’, 3’’, 3’’’, 4’’, 4’’’, 5’’, 5’’’, 6’’, 6’’’, -
NCH2CH2O-), 4.85 (2H, d, JH-2’’,OH-2’’ = 4.2 Hz, HO-2’’), 5.14 (1H, d, JH-2’’’,OH-2’’’ = 6.2 Hz, HO-2’’’), 4.38
(2H, d, JH-3’’,OH-3’’ = 4.0 Hz, HO-3’’), 4.49 (1H, d, JH-3’’’,OH-3’’’ = 4.2 Hz, HO-3’’’), 4.71 (2H, d, JH-4’’,OH-4’’
=
4.8 Hz, HO-4’’), 4.83 (1H, d, JH-4’’’,OH-4’’’ = 5.8 Hz, HO-4’’’), 4.59 (2H, t, JH-6’’,OH-6’’ = 5.4 Hz, HO-6’’), 4.68
(1H, t, JH-6’’’,OH-6’’’ = 4.4 Hz, HO-6’’’) ppm; δC (DMSO-d6): 153.40 (d, JF-C = 245.0 Hz, Ar-C), 144.47 (d,
JF-C = 9.0 Hz, Ar-C), 134.79 (d, JF-C = 12.0 Hz, Ar-C), 124.28 (s, Ar-C), 106.93 (s, Ar-C), 106.47 (s, Ar-
C), 103.24 (s, C-1’’), 103.95 (s, C-1’’’), 70.44 (s, C-2’’), 70.66 (s, C-2’’’), 73.44 (s, C-3’’), 73.49 (s, C-
3’’’), 68.15 (s, C-4’’), 68.20 (s, C-4’’’), 75.30 (s, C-5’’), 75.52 (s, C-5’’’), 60.39 (s, C-6’’), 60.47 (s, C-
6’’’), 56.13 (s, -NCH2CH2O-), 58.25 (s, -NCH2CH2O-) ppm; HRMS: [M+H]+, C28H45NO18F, Calcd:
702.2621, Found: 702.2667; [M+Na]+, C28H44NO18FNa, Calcd: 724.2440, Found: 724.2505.
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2-Fluoro-4-[bis(propargyl)amino]phenyl
2’’,
3’’,
4’’,
6’’-tetra-O-acetyl-β-D-
galactopyranoside 11 To a well stirred dry DMF (50 mL) solution of the amine 2 (1.0 g, 2.2 mmol)
and K2CO3 (1.2 g, 8.8 mmol) at 80°C was added propargyl bromide solution (1.0 g, 6.6 mmol, 80%
wt. in toluene) dropwise, and the stirring continued for 3 h until completion of the reaction (TLC). The
mixture was diluted with CH2Cl2 (100 mL), filtered, washed (H2O), dried (Na2SO4) and concentrated in
vacuo. The residue was purified on a silica gel column to yield 11 (1.0 g, 88%) as syrup, Rf 0.51 (1:1
cyclohexane-EtOAc), δH (CDCl3): 7.13 (1H, t, J = 8.8 Hz, Ar-H), 6.70 (1H, dd, J = 3.8, 12.1 Hz, Ar-H),
6.60 (1H, dd, J = 3.8, 8.8 Hz, Ar-H), 4.80 (1H, d, J1’’,2’’ = 8.0 Hz, H-1’’), 5.46 (1H, dd, J2’’,3’’ = 10.0 Hz,
H-2’’), 5.07 (1H, dd, J3’’,4’’ = 3.8 Hz, H-3’’), 5.41 (1H, d, J4’’,5’’ = 2.8 Hz, H-4’’), 3.94 (1H, m, H-5’’), 4.23
(1H, dd, J5’’,6a’’ = 6.6 Hz, J6a’’,6b’’ = 11.8 Hz, H-6a’’), 4.15 (1H, dd, J5’’,6b’’ = 7.0 Hz, H-6b’’), 4.05 (4H, d, J
= 4.0 Hz, -NCH2C≡CH), 2.27 (2H, t, J = 4.0 Hz, -NCH2C≡CH), 2.18, 2.11, 2.04, 2.01 (12H, 4s,
4×CH3CO) ppm; δC (CDCl3): 170.47, 170.41, 170.27, 169.74 (4s, 4×CH3CO), 154.29 (d, JF-C = 245.0
Hz, Ar-C), 145.54 (d, JF-C = 8.0 Hz, Ar-C), 137.70 (d, JF-C = 12.0 Hz, Ar-C), 122.75 (s, Ar-C), 111.17
(d, JF-C = 3.0 Hz, Ar-C), 104.50 (d, JF-C = 22.0 Hz, Ar-C), 102.63 (s, C-1’’), 68.77 (s, C-2’’), 70.86 (s, C-
3’’), 67.00 (s, C-4’’), 71.14 (s, C-5’’), 61.33 (s, C-6’’), 40.95 (s, -NCH2C≡CH), 78.77 (s, -NCH2C≡CH),
73.24 (s, -NCH2C≡CH), 21.81 - 20.74 (4s, 4×CH3CO) ppm.
Anal. Calcd. for C26H28NO10F (%): C, 58.53, H, 5.29, N, 2.63; Found: C, 58.50, H, 5.25, N,
2.60.
2-Fluoro-4-{bis[[1’’-(2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-galacto- or glucopyranosyl) -1’’,
2’’,
3’’-triazol-4’’-yl]methyl]amino}phenyl
2’’’’,
3’’’’,
4’’’’,
6’’’’-tetra-O-acetyl-β-D-
galactopyranosides 12, 13 A freshly prepared solution of (+)-sodium L-ascorbate (40 mg, 0.2
mmol) in H2O (5 mL) was dropwise added into the solution of 11 (530 mg, 1.0 mmol), 2, 3, 4, 6-tetra-
O-acetyl-β-D-galactopyranosyl azide or 2, 3, 4, 6-tetra-O-acetyl-β-D-glucopyranosyl azide (900 mg,
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2.4 mmol), and CuSO4⋅5H2O (25 mg, 0.1 mmol) in DMF (30 mL)-H2O (5 mL) with vigorous stirring at
room temperature under an argon atmosphere, and the reaction mixture was continually stirred for 12
h until completion as monitored by TLC (cyclohexane-EtOAc 1:5). EtOAc (200 mL) was added to
dilute the mixture. The organic phase was washed by saturated brine, dried (Na2SO4), concentrated
and purified by column chromatography.
2-Fluoro-4-{bis[[1’’-(2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-galactopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-
yl]methyl]amino}phenyl 2’’’’, 3’’’’, 4’’’’, 6’’’’-tetra-O-acetyl-β-D-galactopyranoside 12 (1.05 g, 82%) as
syrup, Rf 0.47 (1:5 cyclohexane-EtOAc), δH (CDCl3): 7.73 (1H, s, H-5’’), 7.64 (1H, dd, J = 4.0, 8.0 Hz,
Ar-H), 7.48 (1H, dd, J = 4.0, 12.0 Hz, Ar-H), 6.56 (1H, t, J = 12.0 Hz, Ar-H), 5.80 (2H, d, J1’’’,2’’’ = 8.0
Hz, H-1’’’), 4.73 (1H, d, J1’’’’,2’’’’ = 8.0 Hz, H-1’’’’), 5.46 (2H, dd, J2’’’,3’’’ = 10.2 Hz, H-2’’’), 5.39 (1H, dd,
J2’’’’,3’’’’ = 10.0 Hz, H-2’’’’), 5.21 (2H, dd, J3’’’,4’’’ = 3.8 Hz, H-3’’’), 5.02 (1H, dd, J3’’’’,4’’’’ = 4.0 Hz, H-3’’’’),
5.50 (2H, d, J4’’’,5’’’ = 3.0 Hz, H-4’’’), 5.36 (1H, d, J4’’’’,5’’’’ = 2.8 Hz, H-4’’’’), 3.92 - 3.85 (3H, m, H-5’’’, 5’’’’),
4.25 - 4.04 (6H, m, H-6’’’, 6’’’’), 4.56 (4H, s, -NCH2-), 2.19 - 1.80 (36H, 7s, 12×CH3CO) ppm; δC
(CDCl3): 170.40 - 169.70 (7s, 12×CH3CO), 154.33 (d, JF-C = 244.0 Hz, Ar-C), 146.12 (d, JF-C = 9.0 Hz,
Ar-C), 145.34 (s, C-4’’), 132.49 (s, Ar-C), 130.95 (s, Ar-C), 128.84 (s, Ar-C), 120.81 (s, C-5’’), 109.73
(s, Ar-C), 86.30 (s, C-1’’’), 103.00 (s, C-1’’’’), 68.18 (s, C-2’’’), 67.94 (s, C-2’’’’), 70.76 (s, C-3’’’), 68.70
(s, C-3’’’’), 66.95 (s, C-4’’’), 66.90 (s, C-4’’’’), 74.12 (s, C-5’’’), 71.00 (s, C-5’’’’), 61.22 (s, C-6’’’), 61.16
(s, C-6’’’’), 46.59 (s, -NCH2-), 20.75 - 20.19 (7s, 12×CH3CO) ppm; HRMS: [M+H]+, C54H67N7O28F,
Calcd: 1280.4018, Found: 1280.4073; [M+Na]+, C54H66N7O28FNa, Calcd: 1302.3838, Found:
1302.3614; [M+K]+, C54H66N7O28FK, Calcd: 1318.3577, Found: 1318.1919.
2-Fluoro-4-{bis[[1’’-(2’’’, 3’’’, 4’’’, 6’’’-tetra-O-acetyl-β-D-glucopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-
yl]methyl]amino}phenyl 2’’’’, 3’’’’, 4’’’’, 6’’’’-tetra-O-acetyl-β-D-galactopyranoside 13 (1.10 g, 85%) as
syrup, Rf 0.41 (1:5 cyclohexane-EtOAc), δH (CDCl3): 7.73 (1H, s, H-5’’), 7.70 (1H, dd, J = 3.0, 8.0 Hz,
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Ar-H), 7.52 (1H, dd, J = 3.0, 12.0 Hz, Ar-H), 6.54 (1H, t, J = 12.0 Hz, Ar-H), 5.85 (2H, d, J1’’’,2’’’ = 8.0
Hz, H-1’’’), 4.78 (1H, d, J1’’’’,2’’’’ = 8.0 Hz, H-1’’’’), 5.45 (2H, dd, J2’’’,3’’’ = 10.6 Hz, H-2’’’), 4.61 (1H, dd,
J2’’’’,3’’’’ = 10.8 Hz, H-2’’’’), 5.20 (2H, dd, J3’’’,4’’’ = 4.0 Hz, H-3’’’), 5.05 (1H, dd, J3’’’’,4’’’’ = 4.0 Hz, H-3’’’’),
5.40 (3H, d, J4’’’,5’’’ = J4’’’’,5’’’’ = 3.4 Hz, H-4’’’, 4’’’’), 4.03 - 3.91 (3H, m, H-5’’’, 5’’’’), 4.29 - 4.08 (6H, m, H-
6’’’, 6’’’’), 4.58 (4H, s, -NCH2-), 2.20 - 2.03 (36H, 8s, 12×CH3CO) ppm; δC (CDCl3): 170.59 - 169.18
(8s, 12×CH3CO), 154.44 (d, JF-C = 244.0 Hz, Ar-C), 146.05 (d, JF-C = 9.0 Hz, Ar-C), 145.77 (s, C-4’’),
132.62 (s, Ar-C), 131.04 (s, Ar-C), 128.96 (s, Ar-C), 120.82 (s, C-5’’), 109.71 (s, Ar-C), 85.94 (s, C-
1’’’), 102.81 (s, C-1’’’’), 70.36 (s, C-2’’’), 68.80 (s, C-2’’’’), 72.69 (s, C-3’’’), 70.89 (s, C-3’’’’), 68.31 (s,
C-4’’’), 67.89 (s, C-4’’’’), 75.37 (s, C-5’’’), 71.11 (s, C-5’’’’), 61.76 (s, C-6’’’), 61.26 (s, C-6’’’’), 47.52 (s,
-NCH2-), 20.83 - 20.24 (8s, 12×CH3CO) ppm; HRMS: [M]+, C54H66N7O28F, Calcd: 1279.3940, Found:
1279.4218; [M-H]+, C54H65N7O28F, Calcd: 1278.3862, Found: 1278.4196; [M-H+Na]+,
C54H65N7O28FNa, Calcd: 1301.3759, Found: 1301.3931.
2-Fluoro-4-{bis[[1’’-(β-D-galacto-
or
glucopyranosyl)-1’’,
2’’,
3’’-triazol-4’’-yl]
methyl]amino}phenyl β-D-galactopyranosides 14, 15 Deacetylation of 12 and 13 (900 mg) in
anhydrous MeOH (80 mL) containing 0.5M NH3, as described for the preparation of free β-D-
galactopyranosides 5, 6 and 8, afforded 14 and 15, respectively.
2-Fluoro-4-{bis[[1’’-(β-D-galactopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl]amino}phenyl β-D-
galactopyranoside 14 (513 mg, 94%) as syrup, Rf 0.36 (10:1 MeOH-EtOAc), δH (DMSO-d6): 8.20 (1H,
s, H-5’’), 7.08 (1H, t, J = 8.0 Hz, Ar-H), 6.81 (1H, dd, J = 4.0, 12.0 Hz, Ar-H), 6.66 (1H, dd, J = 4.0, 8.0
Hz, Ar-H), 5.48 (2H, d, J1’’’,2’’’ = 8.0 Hz, H-1’’’), 4.13 (1H, d, J1’’’’,2’’’’ = 8.0 Hz, H-1’’’’), 4.05 - 3.33 (18H,
m, H-2’’’, 2’’’’, 3’’’, 3’’’’, 4’’’, 4’’’’, 5’’’, 5’’’’, 6’’’, 6’’’’), 5.24 (2H, d, JH-2’’’,OH-2’’’ = 4.4 Hz, HO-2’’’), 5.13 (1H,
d, JH-2’’’’,OH-2’’’’ = 4.0 Hz, HO-2’’’’), 4.49 (2H, d, JH-3’’’,OH-3’’’ = 4.0 Hz, HO-3’’), 4.28 (1H, d, JH-3’’’’,OH-3’’’’ = 4.0
Hz, HO-3’’’’), 5.03 (2H, d, JH-4’’’,OH-4’’’ = 4.0 Hz, HO-4’’’), 4.82 (1H, d, JH-4’’’’,OH-4’’’’ = 5.2 Hz, HO-4’’’’), 4.73
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(2H, t, JH-6’’’,OH-6’’’ = 5.6 Hz, HO-6’’’), 4.68 (1H, t, JH-6’’’’,OH-6’’’’ = 4.6 Hz, HO-6’’’’), 4.63 (4H, s, -NCH2-)
ppm; δC (DMSO-d6): 152.91 (d, JF-C = 241.0 Hz, Ar-C), 144.27 (s, C-4’’), 144.08 (d, JF-C = 9.0 Hz, Ar-
C), 135.86 (d, JF-C = 12.0 Hz, Ar-C), 131.65 (d, JF-C = 20.0 Hz, Ar-C), 128.65 (s, Ar-C), 122.00 (s, C-
5’’), 108.29 (s, Ar-C), 88.12 (s, C-1’’’), 102.87 (s, C-1’’’’), 69.39 (s, C-2’’’), 70.35 (s, C-2’’’’), 73.73 (s,
C-3’’’), 73.39 (s, C-3’’’’), 67.40 (s, C-4’’’), 68.52 (s, C-4’’’’), 74.48 (s, C-5’’’), 75.47 (s, C-5’’’’), 62.82 (s,
C-6’’’), 60.50 (s, C-6’’’’), 48.64 (s, -NCH2-) ppm; HRMS: [M+H]+, C30H43N7O16F, Calcd: 776.2750,
Found: 776.2817; [M+Na]+, C30H42N7O16FNa, Calcd: 798.2570, Found: 798.3064; [M+K]+,
C30H42N7O16FK, Calcd: 814.2309, Found: 814.2819.
2-Fluoro-4-{bis[[1’’-(β-D-glucopyranosyl)-1’’, 2’’, 3’’-triazol-4’’-yl]methyl]amino}phenyl β-D-
galactopyranoside 15 (496 mg, 91%) as syrup, Rf 0.32 (10:1 MeOH-EtOAc), δH (DMSO-d6): 8.26 (1H,
s, H-5’’), 7.71 (1H, dd, J = 4.0, 8.0 Hz, Ar-H), 7.68 (1H, dd, J = 4.0, 12.0 Hz, Ar-H), 7.09 (1H, t, J =
12.0 Hz, Ar-H), 5.53 (2H, d, J1’’’,2’’’ = 8.0 Hz, H-1’’’), 4.13 (1H, d, J1’’’’,2’’’’ = 8.0 Hz, H-1’’’’), 3.82 - 3.22
(18H, m, H-2’’’, 2’’’’, 3’’’, 3’’’’, 4’’’, 4’’’’, 5’’’, 5’’’’, 6’’’, 6’’’’), 5.39 (2H, d, JH-2’’’,OH-2’’’ = 4.0 Hz, HO-2’’’), 5.28
(1H, d, JH-2’’’’,OH-2’’’’ = 4.0 Hz, HO-2’’’’), 4.50 (2H, d, JH-3’’’,OH-3’’’ = 3.8 Hz, HO-3’’), 4.83 (1H, d, JH-3’’’’,OH-3’’’’
= 4.0 Hz, HO-3’’’’), 5.17 (2H, d, JH-4’’’,OH-4’’’ = 4.0 Hz, HO-4’’’), 5.13 (1H, d, JH-4’’’’,OH-4’’’’ = 5.0 Hz, HO-
4’’’’), 4.66 (2H, t, JH-6’’’,OH-6’’’ = 5.8 Hz, HO-6’’’), 4.60 (1H, t, JH-6’’’’,OH-6’’’’ = 5.2 Hz, HO-6’’’’), 4.62 (4H, s, -
NCH2-) ppm; δC (DMSO-d6): 152.90 (d, JF-C = 241.0 Hz, Ar-C), 144.14 (s, C-4’’), 144.03 (d, JF-C = 9.0
Hz, Ar-C), 135.89 (d, JF-C = 12.0 Hz, Ar-C), 131.66 (d, JF-C = 21.0 Hz, Ar-C), 128.66 (s, Ar-C), 122.45
(s, C-5’’), 108.33 (s, Ar-C), 87.51 (s, C-1’’’), 102.88 (s, C-1’’’’), 72.11 (s, C-2’’’), 70.34 (s, C-2’’’’), 77.02
(s, C-3’’’), 73.40 (s, C-3’’’’), 69.61 (s, C-4’’’), 68.11 (s, C-4’’’’), 79.98 (s, C-5’’’), 75.49 (s, C-5’’’’), 62.83
(s, C-6’’’), 60.73 (s, C-6’’’’), 48.63 (s, -NCH2-) ppm; HRMS: [M+H]+, C30H43N7O16F, Calcd: 776.2750,
Found: 776.2834; [M+Na]+, C30H42N7O16FNa, Calcd: 798.2570, Found: 798.3345; [M+K]+,
C30H42N7O16FK, Calcd: 814.2309, Found: 814.2741.
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Stable lacZ Transfected Cancer Cells. E.coli lacZ gene (from pSV-β-gal vector, Promega,
Madison, WI, USA) was inserted into high expression human cytomegalovirus (CMV) immediate-early
enhancer/promoter vector phCMV (Gene Therapy Systems, San Diego, CA, USA) 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, USA) using GenePORTER2 (Gene
Therapy Systems, Genlantis, Inc., San Diego, CA, USA), as described in detail previously.[4-6] The
highest β-gal expressing colony was selected using the antibiotic G418 disulfate (800μg/mL,
Research Products International Corp, Mt Prospect, IL, USA) and G418 (200μg/mL) was also
included for routine culture. The cells were maintained in Dulbecco’s modified Eagle’s medium
(DMEM, Mediatech Inc., Herndon, VA, USA) containing 10% fetal bovine serum (FBS, 0.1M, pH=7.4,
Atlanta Biologicals, Inc., Lawrenceville, GA, USA) with 100units/mL of penicillin, 100units/mL
streptomycin, and cultured in a humidified 5% CO2 incubator at 37°C. The β-gal activity of lacZ-
transfected tumor cells was measured using a β-gal assay kit with o-nitrophenyl-β-D-
galactopyranoside (Promega, Madison, WI, USA), and confirmed by X-gal staining.[4-6] Cell lysis was
achieved by a freeze/thaw method: equal numbers of MCF7-lacZ or PC3-lacZ cells were suspended
in PBS (0.1M, pH=7.4) and then frozen at -80°C for 10 mins before thawing at room temperature over
3 cycles.
Kinetic 19F-MRS Experiments. Relative substrate efficacy of 2-fluoro-4-[bis(2’-
chloroethyl)amino]phenyl β-D-galactopyranoside 5 and 2-fluoro-4-[bis(2’-hydroxyethyl)amino] phenyl
β-D-galactopyranoside 6 was evaluated using 19F-MRS. Enzyme reactions were conducted at 37°C in
PBS (0.1M, pH=7.4) using β-gal (E801A, Promega, Madison, WI, USA). The prodrugs 5 and 6
(13.8μmol) were dissolved in PBS (595μL, pH=7.4) and β-gal (E801A, 5 μL, 1unit/μL) added, followed
by immediate 19F-MRS data acquisition at 37°C with subsequent spectra every 102s providing a
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kinetic curve over 40min. The β-gal hydrolysis rates were calculated according to the formula: ν =
[concentration change of 5 or 6 (Ct1 - Ct2) × 1000] / [reaction time (min) × unites of β-gal], and
expressed in μM/min/unit.
In vitro evaluation of 5 with the wild type (WT), lacZ transfected human MCF7 breast and PC3
prostate cancer cells (5×106) in PBS (0.1M, pH=7.4) at 37°C under 5% CO2 in air with 95% humidity
by using 19F-MRS analysis was conducted at different time points (0-180min) at 37°C with
subsequent spectra every 261s. The hydrolysis rates by MCF7- or PC3-lacZ cells were measured
according to the formula: ν = [concentration change of 5 (Ct1 - Ct2) × 1000] / [reaction time (min) ×
numbers of million MCF7- or PC3-lacZ cells], and expressed in μM/min/per million cells.
Cytotoxicity. The cytotoxicity was assessed for both prodrug 5 and the released fluorinated
nitrogen mustard 2-fluoro-4-[bis(2’-chloroethyl)amino]phenol (2F4ClPh) in both wild-type and lacZ
expressing MCF7 and PC3 cells using a colorimetric CellTiter 96 Aqueous Nonradioactive MTS Cell
Proliferation Assay (Promega). Assays were performed in triplicate using 24-well plates seeded with
103 cells per well in 500 μL of RPMI-1640 without phenol red and supplemented with 10% FCS and 2
mM glutamine.[5,6]
ASSOCIATED CONTENT
Supporting Information
Figure S1. The numbering of structures 3-6; Figure S2. The numbering of structures 7-8; Figure S3.
The numbering of structures 12-15.
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AUTHOR INFORMATION
Corresponding Author
*
Center of Translational Medicine, 5th School of Medicine/Suizhou Central Hospital, Hubei University of
Medicine, 8 Wenhua Park Road, Suizhou, Hubei 441300, China. Phone: 18935717422. E-mail:
jianxinyu707@gmail.com.
Present Address
† Summer student from the Rice University, 6100 Main St, Houston, TX 77005, USA.
ACKNOWLEDGEMENTS
This research was partly supported by the Research/Development Grants, and the Key Discipline
Project from Hubei University of Medicine/Suizhou Central Hospital. Partial NMR experiments were performed
at the Advanced Imaging Research Center of UT Southwestern Medical Center at Dallas. We are grateful to
Jennifer Magnusson and Jennifer McAnally for expert technical assistances.
ABBREVIATIONS
MRS: Magnetic resonance spectroscopy; NMR: Nuclear magnetic resonance; HRMS: High resolution
mass spectrometry; β-gal: β-Galactosidase; TBAB: Tetrabutylammonium bromide; CH2Cl2: Dichloromethane;
AcOH: Acetic acid; SOCl2: Thionyl chloride; PBS: Phosphate buffered saline; DMF: Dimethylformamide;
NaASc: (+)-Sodium L-ascorbate; DMSO: Dimethyl sulfoxide; TLC: Thin layer chromatography.
CONFLICT OF INTEREST STATEMENT
The authors declare no conflict of interest in this work.
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Associates, Inc., 2013, ISBN-13: 978-0878939640; (b) Lodish, H., Berk, A., Kaiser, C. A., Krieger, M.,
Bretscher A. Molecular Cell Biology, 7th ed. Freeman, W. H. & Company, 2012, ISBN-13: 978-1429234139;
(c) Yang, N. J., Hinner, M. J. Getting across the cell membrane: An overview for small molecules, peptides,
and proteins, Methods Mol. Biol., 2015, 1266, 29-53.
FIGURE LEGENDS
Graphic Abstract
Figure 1. The synthesis and the structures of 1-6. Reaction conditions: (a) H2 (30psi), EtOH, Pd/C (5%
W/W’), r.t., overnight, 100%; (b) ethylene oxide (10 equiv.), AcOH-benzene (1:5 V/V’), 0°C→r.t., 48h, 85%
yield; (c) SOCl2 (3.0 equiv.), CH2Cl2, stirring at 0°C for 2h then r.t. for 1h, 77% yield; (d) 0.5M NH3-MeOH,
0°C→r.t., 24h, 95%(→5) and 97%(→6), respectively.
Figure 2. The synthetic route and the structures of 7-10. Reaction conditions: (a) 2, 3, 4, 6-tetra-O-acetyl-α-
D-galactopyranosyl bromide or 2, 3, 4, 6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1.3 equiv.), Hg(CN)2 (1.5
equiv.), 4Å M.S., CH2Cl2, r.t., 12h, 89%(→7); (b) 0.5M NH3-MeOH, 0°C→r.t., 24h, 95%(→8).
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Figure 3. The synthetic route and the structures of 11-15. Reaction conditions: (a) CH≡CCH2Br (1.5 equiv.),
K2CO3 (2.0 equiv.), DMF, 80°C, 3h, 88%; (b) 2, 3, 4, 6-tetra-O-acetyl-β-D-galactopyranosyl azide or 2, 3, 4, 6-
tetra-O-acetyl-β-D-glucopyranosyl azide (1.2 equiv.), CuSO4⋅5H2O (10% mol), (+)-sodium L-ascorbate (20%
mol), DMF-H2O (3:1 V/V’), r.t., 12h, Ar, 82%(→12) and 85%(→13), respectively; (c) 0.5M NH3-MeOH,
0°C→r.t., 24h, 91-94% yields.
Figure 4. 19F-MRS detection of β-gal (E801A) in PBS at 37°C. (1) Control: 2-Fluoro-4-[bis(2’-
chloroethyl)amino]phenyl β-D-galactopyranoside 5 (13.8 µmol), without β-gal (5 units, E801A) in PBS (0.1 M,
pH=7.4, 600 µL) at 37°C; (2) 2-Fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-galactopyranoside 5 (13.8 µmol),
with β-gal (5 units, E801A) in PBS (0.1 M, pH=7.4, 600 µL) at 37°C at the time point, t = 2 minutes. 19F-MRS:
parameter sets: relaxation delay: 1 s, pulse width (90°): 16.6 µs, number of points acquired: 59966, filter
bandwidth: 28000 Hz, each spectrum acquisition time: 0.6 s, number of acquisitions: 64, and enhanced with an
exponential line broadening of 40 Hz. 2-Fluoro-4-[bis(2’-chloroethyl)amino] phenyl β-D-galactopyranoside 5 at
δF=-55.65 ppm, 2-fluoro-4-[bis(2’-chloroethyl) amino]phenol (2F4ClPh) at δF=-59.44 ppm, and 2-fluoro-4-
[bis(2’-hydroxyethyl)amino]phenol (2F4OHPh) at δF=-44.33 ppm.
Figure 5. The kinetic hydrolysis time courses of 2-fluoro-4-[bis(2’-chloroethyl)amino]phenyl β-D-
galactopyranoside 5 (16.8 mM) (•) → 2-fluoro-4-[bis(2’-chloroethyl)amino]phenol (2F4ClPh) (■) → 2-fluoro-4-
[bis(2’-hydroxyethyl)amino]phenol (2F4OHPh) (▲) with β-gal (5 units, E801A) in PBS (0.1 M, pH=7.4, 600 µL)
at 37°C.
Figure 6. The cytotoxicity of prodrug 5 to wild-type (WT) and lacZ expressing MCF7 and PC3 cells, incubation
at 37°C under 5% CO2 in air with 95% humidity for 24h; Histology: β-gal activity validation in MCF7- and
PC3-lacZ cells with deep blue (bottom) and no activity in MCF7- and PC3-WT cells (top), X-gal staining (×200).
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This article is protected by copyright. All rights reserved.

This article is protected by copyright. All rights reserved.