Amine-promoted β-elimination of a β-aryloxy aldehyde for fluorogenic chemodosimeters

Article abstract of DOI:10.1021/jo200947e

We previously reported a fluorescent chemodosimeter for ozone. The β-elimination step after the ozonolysis of the chemodosimeter was too slow to be practical for real-time monitoring of ozone. We examined primary, secondary, and tertiary amines at various

Full text of DOI:10.1021/jo200947e

NOTE
pubs.acs.org/joc
Amine-Promoted β-Elimination of a β-Aryloxy Aldehyde for
Fluorogenic Chemodosimeters
Amanda K. Leslie, Dandan Li, and Kazunori Koide*
Department of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260, United States
S Supporting Information
b
ABSTRACT: We previously reported a fluorescent chemodo-
simeter for ozone. The β-elimination step after the ozonolysis of
the chemodosimeter was too slow to be practical for real-time
monitoring of ozone. We examined primary, secondary, and
tertiary amines at various pHs. It was found that pyrrolidine in
pH 9 buffer could accelerate the elimination to generate a
fluorescence signal. The elimination step is now sufficiently
rapid to monitor ozone exposure in real time. We also dis-
covered that azetidine was distinctly effective for the same
elimination reaction in a pH 6 buffer.
nstrument-generated ozone is frequently used for the sanitiza-
tion of swimming pools, drinking water,1 food contact sur-
At the outset of this study, it was unclear whether the
β-elimination proceeds by an E2 mechanism or an E1cb mech-
anism13 or both. Considering the ease of fluorescence measure-
ment in a high-throughput manner and the multiple parameters
that can influence the rate of β-elimination near neutral pH,14 we
chose to screen for optimal pH and reagents.
I
faces,2 houses, and surgical rooms.3 In contrast to these bene-
ficial applications of ozone, natural atmospheric ozone negatively
impacts human health. It has been argued that4 ozone at ground
level may induce acute respiratory symptoms among asthmatics5
and might be a cause of asthma.6 Therefore, measurement of
personal exposure to ozone would prove highly beneficial for
workers in high-ozone areas and those who already suffer from
respiratory illness. Ozone badges are commercially available, but
more sensitive, selective, and robust colorimetric or fluorometric
methods need to be developed.
Unlike our previous work,7 in this work we isolated and
characterized aldehyde 3 after exposure of compound 1 to ozone,
although this aldehyde could not be fully purified because of its
instability.15 Using the moderately pure aldehyde 3 as the starting
material, we examined ammonia, cyclohexylamine, pyrrolidine,
and triethylamine as primary, secondary, and tertiary amines
at pH 5, 6, 7, 8, and 9 (Figure 1). Anilines, although poten-
tially catalytically active in this case,16 were excluded from the
screening efforts because of their air sensitivity. Generally, the
β-elimination, as determined by the fluorescence signal from
compound 5, was faster at higher pHs (Figure 1, “no amine”).
We reported a fluorescence-based method that was selec-
tive for ozone (Scheme 1).7 In this method, chemodosimeter
1 reacted with ozone by a 1,3-dipolar cycloaddition mechanism
to form molozonide 2.8 This species underwent retro-[3 + 2]
cyclization to form aldehyde 3 and compound 4. In water, these
compounds presumably did not react with each other to form the
corresponding ozonide.9 In the final step, a β-elimination
occurred to convert the nonfluorescent aldehyde 3 to the
fluorescent compound 5. In tissue culture media, the β-elimina-
tion was found to be facile,7 presumably due to the presence of
albumin.10 However, for environmental samples, the β-elimina-
tion required more than 1.5 h at 37 °C7 and even more hours at
room temperature (see below). This slow step would be detri-
mental to real-time monitoring of ozone exposure.
In order to enable real-time monitoring of ozone, it was
necessary to develop a method to accelerate the β-elimination
step (3 to 5). Such a method would be beneficial not only for this
ozone detection project but also for the development of chemo-
dosimeters that exploit a similar β-elimination.11 Compound
5 displays maximal fluorescence between pH 5 and pH 9.12
Therefore, we set out to develop a method to accelerate the
β-elimination step within this pH range.
+
Ammonia (pKa = 9.2 for NH4 ) and Et3N (pKa = 10.75 for
Et3NH+) did not appear to accelerate the β-elimination signifi-
+
cantly. Cyclohexylamine (pKa = 10.66 for c-HexNH3 ) acceler-
ated the β-elimination, particularly in pH 8 and 9 buffers.
Pyrrolidine (pKa = 11.3 for pyrolidinium ion) was found to be
even more effective, especially in the pH 9 buffer. These results
indicate that it is critical to form an iminium ion with a secondary
amine (e.g., 6, Scheme 2) or an imine with a primary amine dur-
ing the elimination reaction.
We also tested other secondary amines, piperidine, morpho-
line, and Et2NH (Figure 1). The most sterically hindered Et2NH
was found to be the least effective promoter for the conversion of
3 to 5. Piperidine (pKa = 11.2 for piperidinium ion) accelerated
the reaction better than less basic but sterically similar morpholine
Received: May 13, 2011
Published: July 26, 2011
r
2011 American Chemical Society
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The Journal of Organic Chemistry
NOTE
Scheme 1. Fluorescence-Based Detection of Ozone Using
Compound 1 as an Indicator
Scheme 2. Plausible Pathway Leading to Compound 5
and that the pH values of the reaction solutions and the pKa values
of amines were related to the rates of the β-eliminations of
β-hydroxy and β-acetoxy ketones.14,18
In order to gain insight into the mechanism of the pyrrolidine-
promoted β-elimination, preliminary kinetic studies were per-
formed in a pH 7 phosphate buffer.19 The reaction rate, as
determined by fluorescence, appeared to show a saturation curve
with regard to the concentrations of pyrrolidine in the 0ꢀ2 mM
range (Figure 2a). A likely scenario is that the rate-determining
step (RDS) in this range is the reversible formation of iminium
ion 6 (Scheme 2). The RDS presumably starts shifting to the
elimination step when the concentration of pyrrolidine is above
2 mM, as the rate becomes linearly proportional to the concen-
tration of pyrrolidine. The rate could not be measured accurately
at higher pyrrolidine concentrations due to the altered pH of the
reaction media. The reaction was too fast to be measured
accurately when the concentrations of phosphate salts were high
(see below).
The reaction rate increased nearly linearly when the concen-
trations of phosphate ions were high (Figure 2b). We used a low
concentration of pyrrolidine (0.25 mM) in this experiment to
slow down the reaction so that we could measure the rate
accurately. Under these conditions, phosphate ions accelerated
the reaction possibly by either enhancing the formation of
iminium ion 6 with high ionic strength or deprotonating the
R-proton of 3 and 6 to facilitate the elimination reaction. This is
the second system from our laboratory that benefited from a
phosphate-accelerated fluorogenic reaction.20
At this point, our findings are that the rate of the β-elimination
(3 to 5) increases as the concentrations of pyrrolidine and
phosphate ions increase and that pH 9 was optimal within the
aforementioned allowed pH range. Therefore, it became appar-
ent that higher concentrations of pyrrolidine and borate21 at pH
9 would further facilitate the β-elimination. In the next experi-
ment, the concentration of pyrrolidine was raised to 47.5 mM,
and a commercially available concentrated pH 9 buffer (0.5 M
borate) was used. As Figure 2c shows, the initial rate of the
β-elimination was 30 times greater in the presence of pyrrolidine
(47.5 mM) under these conditions. The β-elimination reaction
was essentially complete in 3 min at room temperature unlike our
previous work where the β-elimination required >1.5 h.
We proceeded to determine whether chemodosimeter 1 could
be used to detect ozone in a pyrrolidine solution at pH 9
(Figure 2d). In the absence of pyrrolidine, the chemodosimeter
apparently reacted with ozone as indicated by stronger fluorescence
Figure 1. Conversion of the nonfluorescent aldehyde 3 to the fluor-
escent compound 5 in the presence of various amines in pH 5ꢀ9 buffers.
[3] = 1 μM, [amine] = 1 mM, 25 °C, 20 min, 1:9 (v/v) MeOH/buffer.
The fluorescence intensity was normalized. The same graph is shown
from two different angles.
(pKa = 8.46 for morpholinium ion). Pyrrolidine and piperidine are
similarly basic (pKa = 11.3 for pyrrolidinium ion), but pyrrolidine
was reproducibly17 superior as a promoter in this system. Proline
was also tested but was less effective than pyrrolidine (data not
shown). It is noteworthy that a series of Spencer0s seminal studies
showed that pyrrolidine was more effective than other amines14
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NOTE
Figure 2. (a) [3] = 1 μM, [pyrrolidine] = 0, 0.25, 0.5, 1, 2, 4, and 8 mM, 25 °C, 4 min, 5:95 (v/v) MeOH/pH 7 phosphate (50 mM) buffer. (b) [3] = 1
μM, [pyrrolidine] = 0.25 mM, 25 °C, 3 min, 5:95 (v/v) MeOH/pH 7 phosphate (154, 308, and 615 mM) buffer. (c) [3] = 10 nM, [pyrrolidine] = 0 or
47.5 mM, 25 °C, 5:95 (v/v) MeOH/pH 9 borate (0.50 M) buffer. (d) [1] = 1 μM, [pyrrolidine] = 0 or 47.5 mM, 25 °C, 5:95 (v/v) MeOH/pH 9 borate
(0.50 M) buffer.
after pyrrolidine was added. On the other hand, the chemodosi-
meter did not react with ozone in the presence of pyrrolidine. This is
consistent with the reactivity of amines with ozone at higher pHs.22
Therefore, in order to develop an ozone detection kit based on
compound 1, it is recommended that compound 1 be exposed to
ozone in the absence of pyrrolidine and that pyrrolidine be added
before fluorescence measurement (for a represntative example, see
Figure 3 and “Ozone detection with compound 1” in the Experi-
mental Section).
To justify the pyrrolidine-promoted β-elimination of 3, we
hypothesized that ring strain of the pyrrolidine-derived iminium
ions might play a crucial role. More specifically, the iminium ion
A (Scheme 2) with a smaller n value might suffer from ring strain
to a greater extent, thus increasing a degree of contribution from
resonance structure A0. The more carbocation-like character of
the iminium ion would increase the acidity of the adjacent CꢀH,
which would then facilitate the deprotonation step.
To test this hypothesis, additional cyclic secondary amines
were examined in pH 6 and 7 buffers (Figure 4). In both buffers,
azetidine promoted the β-elimination of 3 most efficiently.
Specifically, the initial rate of the azetidine-promoted elimination
in a pH 6 buffer was 33 times and 6 times greater than those of the
background and pyrrolidine-promoted reactions, respectively.
Although aziridine was an effective catalyst in a different iminium
ion system,23 azetidine was more effective in our system. These
data are consistent with our hypothesis, but it would be pre-
mature to attribute the rate enhancement exclusively to strain.
Figure 3. Fluorescence increase after exposure of 1 to ozone.
More rigorous mechanistic studies, as represented by a series of
studies by the Spencer group for a related system,14,18 are needed
to understand the structureꢀreactivity relationship in a quanti-
In conclusion, we have developed a more rapid method to
detect ozone by fluorescence than our previous one. Pyrrolidine
tative manner.
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The Journal of Organic Chemistry
NOTE
Chart 1
control of a Windows-based PC running FluorEssence software. The
samples were excited at 496 nm and the emission intensities were col-
lected between 500 and 600 nm wavelengths. All spectra were cor-
rected for emission intensity using the manufacturer supplied photo-
multiplier curves.
Other fluorescence spectra were recorded in black 96-well polypro-
pylene assay plates on a Modulus II Microplate Multimode Reader with
a blue optical kit (maximum excitation wavelength 490 nm, emission
wavelength 510ꢀ570 nm).
The fluorescence signals of compound 5 in 1% DMSO/50 mM
phosphate pH 7 buffer were measured at various concentrations as
shown in Figure S1 (Supporting Information).
Figure 4. Conversion of the nonfluorescent aldehyde 3 to the fluor-
escent compound5 inthe presenceof secondary cyclic amines in(a) pH 6
or(b) pH7 buffer. Conditions:[3] = 1 μM, [amine] = 200 μM, 25°C, 1:9
(v/v) MeOH/buffer. The experiments were performed in duplicate, and
the average values are shown in the graphs. Error bars were omitted for
clarity. See Figure S5 in the Supporting Information for error bars.
Preparation of 3. Ozone was bubbled into a stirred solution of
alkene 1 (49.0 mg, 0.11 mmol) in CH2Cl2 (2 mL) at ꢀ78 °C under an
air atmosphere for 5 min, upon which the reaction solution turned from
yellow to orange. The solution was then purged with nitrogen at ꢀ78 °C
and then treated with dimethyl sulfide (17 μL, 0.22 mmol). The
resulting solution was gradually warmed to 25 °C. After 12 h at the
same temperature, the solvents were removed in vacuo. The resulting
residue was purified by preparative TLC (50% EtOAc in hexanes) to
afford aldehyde 3 (27.0 mg, 55% yield) as a pale yellow solid. This
material could not be purified further due to its instability.
presumably reacts with aldehyde 3 to form the putative iminium
ion 6, rendering the R-protons more acidic. One of the R-protons
is abstracted by a base to trigger the elimination process. The
azetidine-promoted elimination reaction suggests that azetidine
derivatives may serve as organocatalysts.
Data for 3 (because of the equilibrium between 3 and its cyclic ether
form, NMR spectra showed more peaks than the structure of 3 indi-
cates): Rf = 0.29 (50% EtOAc in hexanes); IR (KBr pellet) 3401 (br),
2925, 1715, 1631, 1591, 1488, 1433, 1412, 1268, 1177 cmꢀ1; 1H NMR
(300 MHz, 295K, 1% CD3OD/CDCl3) δ 9.90 (t, 1H, J = 1.8 Hz),
7.42ꢀ7.29 (m, 3H), 6.91ꢀ6.78 (m, 5H), 5.32 (s, 2H), 4.40 (dt, 2H, J =
6.0, 2.4 Hz), 3.00 (t, 2H, J=6.0Hz);13C NMR (75 MHz, 293K, DMSO-d6)
δ 201.3, 154.0, 153.8, 153.7, 149.0, 148.8, 148.7, 144.6, 138.3, 138.2, 129.0,
128.8, 128.6, 128.44, 128.36, 123.0, 121.5, 118.0, 116.8, 116.7, 115.7, 115.5,
103.2, 101.7, 82.0, 72.2, 72.0, 63.3, 48.6, 42.3 (more than 23 peaks due the
tautomerization); HRMS (EI+) m/z calcd. for C23H16Cl2O5 [M]+
442.0375, found 442.0370.
Conversion of the Nonfluorescent Aldehyde 3 to the Fluo-
rescent Compound 5 in the Presence of Various Amines in
pH 5ꢀ9 Buffers (Figure 1). (a). Preparation of Stock Solutions.
The concentrated pH 7 phosphate buffer was diluted with ultrapure
water to a 50 mM phosphate solution. 100 mM solutions of NH4Cl,
cyclohexylamine, pyrrolidine, piperidine, morpholine, Et2NH, and Et3N
were prepared in the 50 mM phosphate pH 7 buffer. These solutions
(0.40 mL of each) were diluted with ultrapure water (3.6 mL) to prepare
the 10 mM amine solutions with 5 mM phosphate. Aldehyde 3 was
dissolved in MeOH to prepare a 10 μM solution.
’ EXPERIMENTAL SECTION
General Techniques. Methylene chloride (CH2Cl2) was distilled
from calcium hydride. The conversion of 1 to 3 was monitored by thin-
layer chromatography (TLC) carried out on 0.25-mm silica gel plates
(60F-254) using heat or UV light (254 or 365 nm) for visualization.
Silica gel (230ꢀ400 mesh) was used for flash chromatography. Ultra-
pure water was prepared from a Barnstead Nanopure Diamond Ultra-
pure Water System.
NMR spectra were recorded on an AM300 instrument and calibrated
using a solvent peak as an internal reference. The following abbreviations
are used to indicate the multiplicities: s = singlet, d = doublet, t = triplet,
q = quartet, m = multiplet, br = broad, app = apparent. High-resolution
mass spectra were obtained using either a VG Autospec having EBE
geometry and electron impact ionization (EI) or a Q-TOF API-US with
electrospray ionization (ESI) in the positive ion modes.
Buffer solutions used in this study are as follows: pH 5.00 buffer solu-
tion (50 mM potassium hydrogen phthalate, <50 mM sodium hydro-
xide), pH 6.00 buffer solution (50 mM potassium hydrogen phthalate,
<50 mM sodium hydroxide), pH 7.00 buffer concentrate (potassium
phosphate monobasic/sodium hydroxide, 1.23 M), pH 8.00 buffer
(50 mM sodium phosphate, dibasic/potassium phosphate), pH 9.00
buffer (50 mM borate), pH 9.00 buffer concentrate (0.5 M borate). The
pH 7.00 buffer concentrate was diluted with ultrapure water to prepare
the corresponding diluted pH 7 buffer solutions.
(b). Conversion of 3 to 5. A black 96-well plate was used (Chart 1). All
of the following steps were performed at 25 °C.
Step 1: The 50 mM pH 5, 6, 7, 8, or 9 buffer solution (160 μL) was
transferred to each well in rows A, B, C, D, or E, columns 1ꢀ8,
respectively.
Fluorescence spectra for Figure 2c were recorded in a 1-mL cuvette
(340ꢀ800 nm) on a Jobin Yvon FluoroMax-3 spectrometer under the
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NOTE
Step 2: A 5 mM pH 7 phosphate buffer (20 μL) was added to each
well in column 1, rows AꢀE. The 10 mM NH4Cl solution (20 μL)
was added to each well in column 2, rows AꢀE. The 10 mM
cyclohexylamine solution (20 μL) was added to each well in column
3, rows AꢀE. The 10 mM pyrrolidine solution (20 μL) was added to
each well in column 4, rows AꢀE. The 10 mM piperidine solution
(20 μL) was added to each well in column 5, rows AꢀE. The 10 mM
morpholine solution (20 μL) was added to each well in column 6,
rows AꢀE. The 10 mM Et2NH solution (20 μL) was added to each
well in column 7, rows AꢀE. The 10 mM Et3N solution (20 μL) was
added to each well in column 8, rows AꢀE.
Kinetics of the β-Elimination of Aldehyde 3 with High
[Pyrrolidine] and High [Borate] (Figure 2c). (a). Preparation of
Stock Solutions. Pyrrolidine (14.3 mg, 0.20 mmol) was dissolved in 0.5
M borate pH 9 buffer (4.0 mL) to prepare a 50 mM pyrrolidine solution
in the borate buffer.
(b). Conversion of 3 to 5. All of the following steps were performed at
25 °C.
Step 1: A 0.5 M borate pH 9 buffer solution (0.95 mL) was transferred
to a 1-mL cuvette. The 50 mM pyrrolidine solution in 0.5 M borate
pH 9 buffer (0.95 mL) was transferred to another 1-mL cuvette.
Step 2: A 0.2 μM solution of aldehyde 3 in MeOH (50 μL) was added
to each of these two cuvettes.
Step 3: Fluorescence spectra were recorded at the indicated times
(Figure 2c) in these cuvettes. The raw data are shown in Figure S4
(Supporting Information).
Step 3: The MeOH solution of aldehyde 3 (10 μM, 20 μL) was added
to each of the 40 wells (columns 1ꢀ8, rows AꢀE). Final conditions:
[3] = 1 μM, [amine] = 0 or 1 mM, 1:9 (v/v) MeOH:buffer, [buffer
salt] = 45 mM.
Fluorometric Detection of Ozone Using Compound 1 in
the Presence and Absence of Pyrrolidine (Figure 2d; for
Flowchart, See Chart S1 in the Supporting Information). (a).
Preparation of Stock Solutions. Pyrrolidine (286 mg, 4.0 mmol) was
dissolved in a 0.5 M borate pH 9 buffer (40 mL) to prepare a 100 mM
pyrrolidine solution. This solution (20 mL) was diluted with a 0.5 M
borate pH 9 buffer (20 mL) to prepare a 50 mM pyrrolidine solution. A
saturated solution of ozone in MeOH was prepared by continuously
bubbling ozone into MeOH at ꢀ78 °C.
Step 4: Green fluorescence emission was monitored every minute for
20 min. The raw data at 20 min are shown in Table S1 (Supporting
Information).
Pyrrolidine-Accelerated β-elimination of Aldehyde 3
(Figure 2a). (a). Preparation of Stock Solutions. The 100 mM
pyrrolidine solution in 50 mM pH 7 phosphate buffer (192 μL) was
diluted with a 50 mM pH 7 phosphate buffer (108 μL) to prepare a
64 mM pyrrolidine solution in a 50 mM phosphate pH 7 buffer. This
solution was serially diluted (2ꢁ) with a 50 mM pH 7 phosphate
buffer to prepare 32, 16, 8, 4, 2, and 1 mM pyrrolidine solutions.
A 2.5 μM solution of 3 was prepared in 1:4 (v/v) MeOH/water.
(b). Conversion of 3 to 5. A black 96-well plate was used. All of the
following steps were performed at 25 °C. This experiment was performed
in duplicate. One of the two sets of data is reported in this paper for clarity.
Step 1: The 50 mM pH 7 phosphate buffer (100 μL) was transferred
to each of 7 wells.
Step 2: The 0, 1, 2, 4, 8, 16, or 32 mM pyrrolidine solution in 50 mM
phosphate pH 7 buffer (50 μL) was added to each of these wells.
Step 3: The 2.5 μM solution of 3 (50 μL) was added to each of
these wells.
Step 4: Green fluorescence emission was monitored every 2 min for
10 min. The data at 4 min were analyzed. For each pyrrolidine
concentration, Ft=4 min (fluorescence intensity at 4 min) ꢀ Ft=0 min
(fluorescence intensity at 0 min) was calculated. The raw data are
shown in Table S2 (Supporting Information).
(b). Conversion of 1 to 5. All of the following steps were performed at
25 °C.
Step 1: The 50 mM pyrrolidine solution in a 0.5 M borate pH 9 buffer
(1.8 mL) was transferred to each of vials 1ꢀ6. The 0.5 M borate pH 9
buffer (1.8 mL) was transferred to each of vials 7ꢀ9.
Step 2: A 20 μM solution of chemodosimeter 1 in MeOH (0.20 mL)
was added to each of vials 1ꢀ9.
Step 3: MeOH (20 μL) was added to each of vials 1ꢀ3. The saturated
solution of ozone in MeOH (20 μL) was added to each of vials 4ꢀ9.
Step 4: Approximately 1 min after step 3, the 50 mM pyrrolidine
solution in 0.5 M borate pH 9 buffer (1.8 mL) was added to each of
vials 1ꢀ6. The 100 mM pyrrolidine solution in 0.5 M borate pH 9
buffer (1.8 mL) was added to each of vials 7ꢀ9. These solutions were
incubated for 5 min.
Step 5: Each reaction solution (150 μL) from the nine vials was
transferred to a black 96-well plate. The fluorescence was measured
using the plate reader. The average fluorescence intensity and stan-
dard deviations are reported in Figure 2d after normalization. The raw
data are shown in Table S4 (Supporting Information).
Phosphate-Accelerated β-Elimination of Aldehyde 3
(Figures 2b and S3 (Supporting Information)). (a). Pre-
paration of Stock Solutions. A 50 mM pyrrolidine solution in
50 mM phosphate pH 7 buffer (100 μL) was diluted with ultrapure
water (9.9 mL) to prepare a 1 mM pyrrolidine solution in 0.5 mM
phosphate pH 7 buffer. A 1.23 M phosphate pH 7 buffer concentrate
was serially diluted to prepare 615, 307.5, 153.8, and 76.9 mM
phosphate pH 7 buffer solutions.
(b). Conversion of 3 to 5. A black 96-well plate was used. All of the
following steps were performed at 25 °C. This experiment was per-
formed in duplicate. One of the two sets of data is reported in this paper
for clarity.
Ozone Detection with Compound 1 (Figure 3). A 3 mM
solution of compound 1 in EtOH (50 μL; 150 nmol) was applied to each
of two pieces of 5 ꢁ 5 cm adsorbent paper. The organic solvent was then
evaporated in an open atmosphere. These two pieces of paper (samples
A and B) were placed for 15 min in a fume hood containing an operating
ozone generator. Subsequently, samples A and B were placed in vials.
Sample A was treated with 50 mM borate pH 9 buffer (5 mL), and
sample B was treated with 500 mM borate pH 9 buffer containing
50 mM pyrrolidine (5 mL). Each solution (200 μL) was transferred to a
96-well plate, and the fluorescence signal was monitored. All of the
operations were conducted at 25 °C. As Figure 3 shows, the conversion
of 3 to 5 was complete in less than 1 min for sample B and in ∼2 h for
sample A.
Step 1: The 1230, 615, 307.5, 153.8, or 76.9 mM phosphate pH 7
buffer solution (100 μL) was transferred to each of five wells.
Step 2: The 1 mM pyrrolidine solution in 0.5 mM phosphate pH 7
buffer (50 μL) was added to each of the five wells.
Step 3: The 2.5 μM solution of 3 (50 μL) was added to each of the
five wells.
Secondary Cyclic Amine-Accelerated β-Elimination of
Aldehyde 3 (Figure 4). Aldehyde 3 was prepared by the ozonolysis
of 1 1 day prior to this experiment and stored at ꢀ80 °C.
Step 4: Green fluorescence emission was monitored every minute for 4
min. The data at 3 min were analyzed. For each phosphate concentration,
Ft=3 min (fluorescence intensity at 3 min) ꢀ Ft=0 min (fluorescence intensity
at 0 min) was calculated. The raw data are shown in Table S3 (Supporting
Information).
Stock Solutions for This Experiment. Solutions: 100 mM
piperidine in 50 mM phosphate pH 7 buffer; 50 mM pyrrolidine in
50 mM phosphate pH 7 buffer; 100 mM azetidine hydrochloride in
50 mM HCl; 10 mM aziridine hydrochloride in 10 mM HCl. Each amine
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NOTE
Chart 2
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(19) The choice of the pH is based on the commercial availability of
a higher concentration solution for pH 7 buffer (up to 1.25 M
phosphate) compared to pH 9 buffer (up to 0.5 M borate).
(20) Song, F.; Carder, E. J.; Kohler, C. C.; Koide, K. Chem.—Eur.
J. 2010, 16, 13500–13508.
solution was diluted with ultrapure water to prepare a solution of 1.6 mM
amine. A 2.5 μM solution of 3 was prepared in 2:3 v/v MeOH/H2O.
Conversion of 3 to 5. A black 96-well plate was used (Chart 2). All
of the following steps were performed at 25 °C. The experiment was
performed in duplicate.
Step 1: The 50 mM phosphate pH 6 or 7 buffer (125 μL) was
transferred to a black 96-well plate according to the diagram.
Step 2: The 1.6 mM amine solution or water (25 μL) was transferred
to the plate according to the diagram.
Step 3: The 2.5 μM solution of 3 (50 μL) was added to all of the wells.
Final reaction conditions: [3] = 1 μM, [amine] = 200 μM, [pH 6 or 7
buffer salt] = 31 mM, 1:9 MeOH/buffer.
Step 4: Green fluorescence emission was monitored for 80 min.
’ ASSOCIATED CONTENT
S
Supporting Information. Spectroscopic data and Chart
b
S1. This material is available free of charge via the Internet at
http://pubs.acs.org.
(21) Concentrated pH 9 phosphate buffers are not commercially
available.
’ AUTHOR INFORMATION
(22) Qiang, Z.; Adams, C.; Surampalli, R. Ozone: Sci. Eng. 2004, 26,
525–537.
(23) Gerasyuto, A. I.; Hsung, R. P.; Sydorenko, N.; Slafer, B. J. Org.
Corresponding Author
*E-mail: koide@pitt.edu.
Chem. 2005, 70, 4248–4256.
’ ACKNOWLEDGMENT
A.K.L. was part of the First Experiences in Research program
at the University of Pittsburgh. D.L. was on leave from the East
China University of Science and Technology and a recipient of
the China Scholarship Council Postgraduate Scholarship Pro-
gram. This work was supported by the US National Science
Foundation (CHE-0616577).
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