Autoinductive exponential signal amplification: A diagnostic probe for direct detection of fluoride

Article abstract of DOI:10.1002/chem.201101796

A new example of an exponential signal amplification strategy for the direct detection of fluoride is demonstrated. The amplification occurred through reaction of fluoride with a responsive chromogenic probe. The probe activity is based on a unique dendri

Full text of DOI:10.1002/chem.201101796

FULL PAPER
DOI: 10.1002/chem.201101796
Autoinductive Exponential Signal Amplification: A Diagnostic Probe for
Direct Detection of Fluoride
Rotem Perry-Feigenbaum, Eran Sella, and Doron Shabat*[a]
Abstract: A new example of an exponential signal amplification strategy for the
direct detection of fluoride is demonstrated. The amplification occurred through
reaction of fluoride with a responsive chromogenic probe. The probe activity is
Keywords: analytes · dendrimers ·
based on a unique dendritic chain reaction that generates a fluoride anion, which
dyes/pigments · fluoride · reaction
is the analyte of interest, during the disassembly pathway of the dendritic probe.
mechanisms
This autoinductive amplification mechanism may be applied for detection of other
analytes by coupling activity of a modified probe with that of the fluoride amplifi-
er.
Introduction
system exponentially amplifies a diagnostic signal generated
by fluoride, the analyte of interest.
Signal amplification techniques are often used to boost sig-
nals in diagnostic assays for the detection of various analytes
and biological markers. One of the most familiar techniques
is the enzyme-linked immunosorbent assay (ELISA), a
method that uses the catalytic activity of an enzyme–anti-
body conjugate to amplify a chromogenic signal.[1] In this
type of assay, the initial signal is amplified through a linear
growth pathway by the catalytic activity of the enzyme. Re-
cently, several groups, including ours, have reported new ap-
proaches for signal amplification on the basis of the expo-
nential progress of chemical reactions of small mole-
cules.[2–10] We have introduced a novel modular technique
for the exponential amplification of a diagnostic signal: am-
plification is achieved through a distinctive dendritic chain
reaction (DCR),[11] which is generated by the disassembly of
self-immolative dendrimers.[12,13] A single activation event
initiated by the analyte of interest leads to a chain reaction
that results in complete disassembly of all the probe mole-
cules through an exponential progress to ultimately release
many copies of dye molecules. By taking advantage of the
modular design of the probe, we have applied the DCR
technique to the detection of the analytes hydrogen perox-
ide[14,15] and ubiquitous sulfhydryls.[16] These two examples
were based on DCR probes designed to act under aqueous
conditions. Here we report a new DCR probe, which under-
goes dendritic chain reaction in organic solvent. The probe
Results and Discussion
Fluoride anions are of interest because fluoride treatment
can prevent dental caries and other medical issues such as
osteoporosis.[17] An excess amount of fluoride, however,
causes fluorosis, a type of fluoride toxicity that is related to
an increase in bone density, urolithiasis, or even cancer.[18]
A
large number of sensors for fluoride anion have been devel-
oped to date.[19,20] The United States Environmental Protec-
tion Agency (EPA) enforceable standard for drinking water
is no more than 4 mgLÀ1 (higher levels may result in osteo-
flurosis), and ideal levels should be no more than 2 mgLÀ1
to protect against dental fluorosis. Therefore, relevant sen-
sors for fluorides should be able to produce a detectable di-
agnostic signal this concentration range.
Our new DCR probe 1 was constructed of an AB3 self-im-
molative dendron[21,22] equipped with silyl ether, the trigger-
ing group, which can be cleaved by a fluoride anion
(Scheme 1). Removal of this protecting group by fluoride
exposes a phenolate, which undergoes rapid disassembly to
release
a dye reporter and two additional fluorides
(Scheme 2). These two anions activate two more molecules
of probe 1 to generate an autoinductive disassembly and ex-
ponential amplification of the diagnostic signal.
To employ a dendritic chain reaction for detection of fluo-
ride, two major chemical reactivities must be in hand: a
“disassembly” chemical pathway that enables the release of
a covalently linked fluoride, and a specific protecting group
that is cleaved by reaction with a fluoride. These two reac-
tions are illustrated in Scheme 3. The release of a covalently
linked fluoride was achieved through the design of mole-
cules like compound 2a. The synthesis of this model com-
pound and probe 3 was achieved as shown in Scheme 4. 4-
[a] Dr. R. Perry-Feigenbaum, E. Sella, Prof. D. Shabat
School of Chemistry
Raymond and Beverly Sackler Faculty of Exact Sciences
Tel Aviv University, Tel Aviv 69978 (Israel)
Fax : (+972)0-3-640-9293
E-mail: chdoron@post.tau.ac.il
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line was monitored by UV/Vis
spectroscopy. As expected, the
presence of triethylamine with
salt 2a resulted in release of 4-
nitroaniline from probe 3,
whereas no release was ob-
served in the absence of the
base (Figure 1). This model
system clearly demonstrated
the proposed disassembly path-
way, which resulted in release
of a covalently linked fluoride
to activate a silyl-ether protect-
ing-group-based probe.
Based on the design present-
ed in Scheme 1, we synthesized
DCR probe 1 with a TBDMS
protecting group as the trigger
and 4-nitroaniline as a reporter
Scheme 1. Autoinductive disassembly of a DCR probe for the detection of fluoride.
Hydroxybenzyl-alcohol was treated with carbamoyl-chloride
2b to give carbamate 2c. Fluorination of the latter with (di-
ethylamino)sulfur trifluoride (DAST) gave benzyl fluoride
2d, which was deprotected under acidic conditions to pro-
duce compound 2a in the form of an ammonium salt. Probe
3 was obtained in quantitative yield by an addition reaction
of silyl-protected 4-hydroxybenzyl alcohol to 4-nitrophenyl
isocyanate.
(Scheme 5). To achieve the triggered release of fluoride, a
fluorine was covalently attached to the benzylic positions of
the AB3 dendron, similar to the synthesis described for com-
pound 2a. The synthesis of the probe was performed accord-
ing to Scheme 6. Phenol derivative 1a was protected with
When triethylamine was incubated with ammonium salt
2a, a rapid cyclization occurred to generate a phenloate that
underwent 1,6-elimination to release a fluoride. The free
fluoride treated with probe molecule 3 removed the tert-bu-
tyldimethylsilyl (TBDMS) protecting group, thereby initiat-
ing another 1,6-elimination and decarboxylation to release
the reporter 4-nitroaniline.
The salt form of amine 2 (ammonium trifluoroacetate 2a)
and probe 3 were incubated together in methanol with or
without of triethylamine, and the release of the 4-nitroani-
Scheme 3. Activation of a silyl ether trigger by release of covalently at-
tached fluoride.
Scheme 2. Disassembly pathway of AB3 self-immolative dendron 1 to release its reporter and the two fluorides.
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Chem. Eur. J. 2011, 17, 12123 – 12128

Exponential Signal Amplification
FULL PAPER
quinone methide elimination to
release 4-nitroaniline and two
fluoride anions. The two fluo-
rides can then activate two ad-
ditional AB3 dendrons. The rate
of the disassembly should in-
crease exponentially until all of
the 4-nitroaniline molecules
have been released. The signal
can be detected by monitoring
the yellow color of the released
4-nitroaniline.
To test whether an exponen-
tial amplification was indeed
produced by the DCR probe,
probe 1 was incubated in aceto-
Scheme 4. Chemical synthesis of compounds 2a and probe 3.
nitrile/dimethylsulfoxide (1:1)
with 5% H2O and various amounts of tetrabutyl-ammo-
niumfluoride (TBAF). The release of 4-nitroaniline was
monitored by visible spectroscopy (Figure 2).
When 1.0 equiv of TBAF was used, the probe disassem-
bled immediately (data not shown) to give a signal indica-
tive of the complete release of the 4-nitroaniline. As expect-
ed, probe 1 reached complete disassembly after a longer
period of time when less fluoride was used. Complete reac-
tion occurred with as little as 0.004 equiv of fluoride (2 mm).
The sigmoidal plots observed for various equivalents of
TBAF are characteristic of an exponential progress of disas-
sembly. The background signal due to some spontaneous hy-
drolysis of probe 1 in the absence of fluoride was also ampli-
fied. Nevertheless, the background amplification com-
menced only after about 10 h. Since the ideal and enforcea-
ble standards for fluoride in drinking water are between 1–
4 mgmLÀ1, the designed colorimetric probe is sufficiently
sensitive for fluoride anion detection.
Figure 1. Release of 4-nitroaniline from probe 3 in the presence of the
salt-form amine 2a and triethylamine (squares). No reaction was ob-
served in the absence of triethylamine (triangles).
The selectivity of the DCR probe was tested by the addi-
tion of other common anions. As shown in Figure 3, only
fluoride anions elicited a strong visual signal after 10 h from
probe 1, whereas iodide, bromide, chloride, and acetate
caused no signal significantly above background under simi-
lar conditions.
The principles of the DCR system are based on autoin-
ductive amplification generated by the release of trigger
molecules. Once these molecules are free, they acquire the
activity of the analyte of interest. Thus the free reagents
have than the ability to trigger the disassembly of additional
probes and thereby initiate new diagnostic cycles. The DCR
approach offers considerable advantages over diagnostic
probes that are based on a stoichiometric reaction between
the analyte and the probe. In the DCR technique, the stoi-
chiometric reaction of the analyte and the probe is exponen-
tially amplified to generate a strong diagnostic signal. The
DCR-based assay presented here can be extended to ampli-
fy diagnostic signals for the detection of other analytes by
simply coupling it with another probe activity. During the
preparation of this manuscript, a closely related example for
signal amplification based on fluoride, was reported by Phil-
Scheme 5. DCR-based probe for detection of fluorides.
3 equiv of TBSCl to give tri(silyl ether) 1b. Reduction of
the ester functional group with diisobutylaluminum hydride
(DIBAL-H) afforded benzyl alcohol 1c, which was treated
with 4-nitrophenyl isocyanate to give carbamate 1d. Selec-
tive deprotection of the benzylic silyl ethers by using p-tolu-
ene sulfonic acid (p-TsOH) gave diol 1e, which then was
fluorinated by DAST to produce probe 1.
The DCR amplification cycle obtained by reaction of
fluoride with probe 1 is illustrated in Scheme 5. Cleavage of
the TBDMS trigger by a fluoride anion generates the unpro-
tected phenol, which is further disassembled through triple
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D. Shabat et al.
Scheme 6. Chemical synthesis of probe 1.
ponent dendritic chain reaction (2CDCR).[15] This approach
is based on disassembly mechanisms of a dendritic amplifier
moiety and a chromogenic probe. Both are equipped with
identical triggering systems designed for activation by a spe-
cific analyte. The amplifier component releases reagent
units with the same chemical reactivity of the analyte, there-
by initiating amplification cycles, and the probe component
generates a chromogenic signal. The synthesis of this two-
component amplification system is rather simple compared
to the synthesis of a DCR probe that includes both the ana-
lyte and the chromogenic probe. In addition, the two-com-
ponent system provides additional flexibility. Once the am-
plifier component is in hand, the diagnostic assay can be
performed in combination with different types of probe mol-
ecules. Furthermore, the produced diagnostic signal could
then be tuned by adjusting the ratio of the amplifier and the
chromogenic probe. The 2CDCR approach could also be ap-
plied in the current example, in which the amplifier compo-
nent releases three equivalents of fluoride and the second
component equipped with the same silyl trigger generates
the chromogenic molecule.
Figure 2. Release of 4-nitroaniline from probe 1 (500 mm) in acetonitrile/
dimethylsulfoxide (1:1) upon addition of the indicated equivalents of
TBAF. The reaction progress was monitored at a wavelength of 400 nm
for the indicated time period.
Conclusion
In summary, we have demonstrated a new DCR probe for
detection of fluoride that exponentially amplifies a visual di-
agnostic signal. The probe is based on a dendritic chain re-
action that generates a fluoride anion, the analyte of inter-
est, during the disassembly pathway of the probe molecule.
Aqueous fluoride solutions can be directly analyzed by di-
luting them into organic solvent mixture of acetonitrile and
dimethylsulfoxide. The sigmoidal curves observed upon re-
action of the DCR probe with substoichiometric quantities
of fluoride provide a clear indication that the autoinductive
amplification mechanism occurs through the designed disas-
sembly pathway.
Figure 3. Absorbance of free 4-nitroaniline [400 nm] released from probe
1 (500 mm) upon addition of various anions (0.1 equiv) in acetonitrile/di-
methylsulfoxide (1:1); 10 h incubation time, 258C.
lipsꢁ group.[23] That report elegantly demonstrates how a sim-
ilar probe can amplify a diagnostic signal for the detection
of threshold levels of palladium(II) when coupled with an
additional component. We have previously demonstrated
how DCR amplification can also be achieved by a two-com-
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Exponential Signal Amplification
FULL PAPER
Compound 1b: Compound 1a[21] (500 mg, 2.25 mmol) was dissolved in
DMF (2 mL) and cooled to 08C. Imidazole (766 mg, 11.25 mmol) and
TBSCl (1.69 gr, 11.25 mmol) were added. The reaction was allowed to
warm to room temperature and was stirred for an additional 2 h. The re-
action was monitored by TLC (EtOAc/hexane 5:95). Upon completion,
the reaction was diluted with diethyl ether and washed with saturated
NH4Cl solution followed by brine. The organic layer was dried over mag-
nesium sulfate and the solvent was removed under reduced pressure. The
crude product was purified by column chromatography on silica gel
(EtOH/hexane 5:95) to give compound 1b (1.07 g, 85%) as a colorless
oil. 1H NMR (400 MHz, CDCl3): d=8.06 (s, 2H), 4.67 (s, 4H), 4.31 (q,
J=7.2 Hz, 2H), 1.33 (t, J=7.2 Hz, 3H), 0.98 (s, 9H), 0.93 (s, 18H), 0.16
(s, 6H), 0.07 ppm (s, 12H); 13C NMR (100 MHz, CDCl3): d=170.01,
156.19, 132.63, 128.57, 124.26, 61.19, 60.03, 26.72, 26.64, 19.57, 19.11,
14.98, À2.53, À4.54 ppm; MS (CI+): m/z calcd for C29H56O5Si3: 568.3;
found: 569.4 [M+H]+.
Experimental Section
General methods: All reactions requiring anhydrous conditions were per-
formed under an argon or N2 atmosphere. All reactions were carried out
at room temperature unless stated otherwise. Chemicals and solvents
were either analytical reagent grade or purified by standard techniques.
Thin-layer chromatography (TLC) was carried out on silica gel plates
Merck 60 F254. The compounds were visualized by irradiation with UV
light. Flash chromatography (FC) was carried out using silica gel Merck
60 (particle size 0.040–0.063 mm); the eluent is given in parentheses.
1H NMR spectra were measured using a Bruker Avance instrument oper-
ated at 400 MHz as indicated. 13C NMR spectra were measured using a
Bruker Avance instrument operated at 100 MHz as indicated. 19F NMR
spectra were measured using a Bruker Avance instrument operated at
188 MHz as indicated. The chemical shifts (d) are expressed relative to
TMS (0 ppm), and coupling constants (J) are in Hz. The spectra were re-
corded in CDCl3 as solvent at room temperature unless stated otherwise.
All general reagents, including salts and solvents, were purchased from
Sigma–Aldrich.
Compound 1c: Compound 1b (983 mg, 1.72 mmol) was dissolved in dry
THF (10 mL) under an Ar atmosphere and cooled to À788C. DIBAL-H
(1m in toluene, 6.91 mL, 6.91 mmol) was added dropwise. The reaction
was stirred under these conditions for 30 min and was monitored by TLC
(EtOAc/hexane 10:90). Upon completion, the reaction was quenched
with saturated NH4Cl solution (5 mL) and diluted with diethyl ether.
Celite was added, and the reaction mixture was stirred at room temp for
30 min. After filtration, the organic layer was dried over magnesium sul-
fate, and the solvent was removed under reduced pressure. The crude
product was purified by column chromatography on silica gel (EtOAc/
hexane 10:90) to give compound 1c (750 mg, 83%) as a colorless oil.
1H NMR (400 MHz, CDCl3): d=7.37 (s, 2H), 4.71 (s, 4H), 4.63 (d, J=
5.8 Hz, 2H), 1.02 (s, 9H), 0.94 (s, 18H), 0.18 (s, 6H), 0.15 ppm (s, 12H);
13C NMR (100 MHz, CDCl3): d=165.87, 132.58, 128.56, 125.79, 66.49,
61.33, 26.71, 26.66, 19.57, 19.12, À2.61, À4.52 ppm; MS (CI+): m/z calcd
for C27H54O4Si3: 526.3; found: 526.2 [M]+.
Compound 2c: Commercially available 4-hydroxybenzyl alcohol (200 mg,
1.61 mmol) was dissolved in pyridine (2 mL), and compound 2b (483 mg,
1.93 mmol) and catalytic amount of DMAP (5 mg) were added. The reac-
tion was stirred in room temperature overnight and was monitored by
TLC (EtOAc/hexane 50:50). Upon completion, the reaction was diluted
with EtOAc and washed twice with saturated NH4Cl solution. The organ-
ic layer was dried over magnesium sulfate and the solvent was removed
under reduced pressure. The crude product was purified by column chro-
matography on silica gel (EtOAc/hexane 50:50) to give compound 2c
(250 mg, 45%) as a colorless oil. 1H NMR (400 MHz, CDCl3): d=7.25
(d, J=6.4 Hz, 2H), 7.01 (d, J=6.4 Hz, 2H), 4.53 (s, 2H), 3.51–3.42 (m,
4H), 3.07–2.85 (m, 6H), 1.44 ppm (s, 9H); 13C NMR (100 MHz, CDCl3):
d=156.30, 155.51, 151.12, 139.18, 128.48, 122.44, 80.69, 64.93, 47.45,
35.91, 35.43, 29.11 ppm; MS (ESI+): m/z calcd for C17H26N2O5: 338.1;
found: 361.2 [M+Na]+.
Compound 1d: Compound 1c (228 mg, 0.43 mmol) was dissolved in dry
THF (5 mL). 4-Nitrophenyl isocyanate (85 mg, 0.52 mmol) was added,
followed by catalytic amount of DBTL. The reaction mixture was heated
to 508C, stirred for 45 min under an Ar atmosphere, and monitored by
TLC (EtOAc/hexane 10:90). After completion, the solvent was removed
under reduced pressure. The crude product was purified by column chro-
matography on silica gel (EtOAc/hexane 10:90) to give compound 1d
(298 mg, 100%) as a yellowish solid. 1H NMR (400 MHz, CDCl3): d=
8.19 (d, J=9.2 Hz, 2H), 7.55 (d, J=9.2 Hz, 2H), 7.43 (s, 2H), 6.96 (brs,
1H), 5.22 (s, 2H), 4.72 (s, 4H), 1.04 (s, 9H), 0.94 (s, 18H), 0.19 (s, 6H),
0.10 ppm (s, 12H); 13C NMR (100 MHz, CDCl3): d=153.54, 149.09,
144.77, 143.64, 132.81, 129.21, 127.03, 125.88, 118.39, 68.75, 61.25, 26.72,
19.50, 19.15, À2.56, À4.55 ppm; MS (ESI+): m/z calcd for C34H58N2O7Si3:
690.3; found: 713.6 [M+Na]+.
Compound 2d: Compound 2c (122 mg, 0.36 mmol) was dissolved in
CH2Cl2 (5 mL) and cooled to À788C. DAST (71 mL, 0.54 mmol) was
added and the reaction was stirred for 35 min and monitored by TLC
(EtOAc/hexane 30:70). Upon completion, the reaction was quenched
with water (2 mL), diluted with EtOAc, and washed with brine. The or-
ganic layer was dried over magnesium sulfate, and the solvent was re-
moved under reduced pressure. The crude product was purified by
column chromatography on silica gel (EtOAc/hexane 20:80) to give com-
pound 2d (61 mg, 50%) as a colorless oil. 1H NMR (400 MHz, CDCl3):
d=7.35 (d, J=8.4 Hz, 2H), 7.10 (d, J=8.4 Hz, 2H), 5.31 (d, J=47.8 Hz,
2H), 3.55–3.46 (m, 4H), 3.09–2.87 (m, 6H), 1.43 ppm (s, 9H); 13C NMR
(100 MHz, CDCl3): d=155.47, 152.47, 133.87, 129.41, 122.80, 85.59, 83.94,
47.67, 35.97, 35.28, 28.40 ppm; 19F NMR (188 MHz, CDCl3):
À205.95 ppm (t, J=47.8 Hz); MS (ESI+): m/z calcd for C17H25FN2O4:
340.1; found: 363.2 [M+Na]+.
Compound 1e: Compound 1d (250 mg, 0.36 mmol) was dissolved in
MeOH (2 mL). A catalytic amount of p-TsOH was added to the suspen-
sion. The reaction mixture was stirred at room temperature for 20 min
and monitored by TLC (EtOAc/hexane 40:60). Upon completion, the re-
action was diluted with EtOAc and washed with saturated NaHCO3 solu-
tion followed by brine. The organic layer was dried over magnesium sul-
fate and the solvent was removed under reduced pressure. The crude
product was purified by using column chromatography on silica gel
(EtOAc/hexane 30:70) to give compound 1e (157 mg, 95%) as a yellow
solid. 1H NMR (400 MHz, MeOD): d=8.10 (d, J=9.0 Hz, 2H), 7.51 (d,
J=9.0 Hz, 2H), 7.30 (s, 2H), 5.07 (s, 2H), 4.57 (s, 2H), 0.94 (s, 9H),
0.10 ppm (s, 6H); 13C NMR (100 MHz, MeOD): d=153.55, 150.32,
145.44, 143.29, 132.84, 130.01, 128.20, 125.75, 118.38, 67.71, 60.66, 26.57,
19.33, À2.97 ppm; MS (ESI+): m/z calcd for C22H30N2O7Si: 462.1; found:
485.2 [M+Na]+.
Compound 2a: Compound 2a was obtained in quantitative yield as a
white solid after compound 2d was dissolved in a mixture of CH2Cl2/tri-
fluoroacetic acid (TFA) (1:1), stirred for 5 min, and the solvents were re-
moved under reduced pressure.
Compound 3: Compound 3a[24] (203 mg, 0.85 mmol) was dissolved in dry
THF (5 mL). 4-Nitrophenyl isocyanate (168 mg, 1.02 mmol) was added,
followed by a catalytic amount of dibutyltin dilaurate (DBTL). The reac-
tion mixture was heated to 508C, stirred for 1 h under an Ar atmosphere,
and monitored by TLC (EtOAc/hexane 30:70). After completion, the sol-
vent was removed under reduced pressure. The crude product was puri-
fied by column chromatography on silica gel (EtOAc/hexane 20:80) to
give compound
3 (342 mg, 100%) as a
yellowish solid. 1H NMR
Compound 1: Compound 1e (150 mg, 0.32 mmol) was dissolved in
CH2Cl2 (5 mL) and cooled to À788C. DAST (206 mg, 1.3 mmol) was
added and the reaction was stirred for 5 min and monitored by TLC
(EtOAc/hexane 20:80). Upon completion the reaction was quenched
with 2 mL water, diluted with EtOAc and washed with brine. The organic
layer was dried over magnesium sulfate and the solvent was removed
under reduced pressure. The crude product was purified by column chro-
(400 MHz, CDCl3): d=8.21 (d, J=9.2 Hz, 2H), 7.53 (d, J=9.2 Hz, 2H),
7.28 (d, J=8.6 Hz, 2H), 7.05 (brs, 1H), 6.84 (d, J=8.6 Hz, 2H), 5.15 (s,
2H), 0.98 (s, 9H), 0.20 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=
156.07, 152.75, 143.87, 142.86, 130.15, 127.91, 125.08, 120.18, 117.68, 67.50,
25.61, 25.45, 18.09, À4.61 ppm; MS (ESI+): m/z calcd for C20H26N2O5Si:
402.1; found: 425.1 [M+Na]+.
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D. Shabat et al.
matography on silica gel (EtOAc/hexane 10:90) to give compound 1
(112 mg, 75%) as a yellow powder. H NMR (400 MHz, CDCl3): d=8.03
[7] R. Weinstain, A. Sagi, N. Karton, D. Shabat, Chem. Eur. J. 2008, 14,
6857–6861.
1
(d, J=9.2 Hz, 2H), 7.54 (d, J=9.2 Hz, 2H), 7.47 (s, 2H), 7.00 (brs, 1H),
5.54 (s, 2H), 5.27 (d, J=47.6 Hz, 4H), 1.04 (s, 9H), 0.09 ppm (s, 6H);
13C NMR (100 MHz, CDCl3): d=153.62, 151.68, 145.39, 143.31, 131.33,
130.34, 126.17, 118.41, 117.37, 81.28, 79.64, 66.94, 26.43, 19.27,
À2.96 ppm; 19F NMR (200 MHz, CDCl3): À209.74 ppm (t, J=47.6 Hz);
MS (ESI+): m/z calcd for C22H28F2N2O5Si: 466.1; found: 489.1 [M+Na]+.
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Assay conditions for detection of fluoride by spectrophotometer: A stock
solution of compound 1 was prepared in DMSO, and a stock solution of
TBAF was prepared in THF. Stock solutions of all other reagents were
prepared in H2O. In a typical assay, compound 1 (500 mm) was incubated
in MeCN/DMSO (1:1). Various amounts of TBAF or a different reagent
were added, and the release of 4-nitroaniline was monitored using a 96-
well microplate reader (l=400 nm).
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132, 3945–3952.
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[17] M. Kleerekoper, Endocrinol. Metab. Clin. North Am. 1998, 27, 441–
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Acknowledgements
D.S. thanks the Israel Science Foundation (ISF), the Binational Science
Foundation (BSF), and the German–Israeli Foundation (GIF) for finan-
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Received: June 12, 2011
Published online: September 9, 2011
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