A fluorogenic aldehyde bearing a 1,2,3-triazole moiety for monitoring the progress of aldol reactions

Article abstract of DOI:10.1021/jo900013w

We have developed a new type of fluorogenic aldehyde bearing a 1,2,3-triazole moiety that is useful for monitoring the progress of aldol reactions through an increase in fluorescence. Whereas 6-methoxy-2naphthaldehyde was highly fluorescent, the fluorogenic aldehyde, 4-formylbenzene connected to the 6-methoxy-2-naphthyl group through a 1,2,3-triazole moiety, was essentially nonfluorescent in aqueous solutions. We suggest that the 4-formylphenyl group acts as a quencher to suppress the fluorescence of the 6-methoxy-2- naphthyltriazole moiety. The product of the aldol reaction of this aldehyde does not have a quenching moiety and showed more than 800-fold higher fluorescence than the aldehyde. Assay systems using the fluorogenic aldehyde were validated by screening of aldol catalysts, ranking of the activities of the catalysts, and evaluation of reaction conditions.

Full text of DOI:10.1021/jo900013w

A Fluorogenic Aldehyde Bearing a 1,2,3-Triazole Moiety for
Monitoring the Progress of Aldol Reactions
Hai-Ming Guo and Fujie Tanaka*
Department of Molecular Biology, The Scripps Research Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
ftanaka@scripps.edu
ReceiVed January 3, 2009
We have developed a new type of fluorogenic aldehyde bearing a 1,2,3-triazole moiety that is useful for
monitoring the progress of aldol reactions through an increase in fluorescence. Whereas 6-methoxy-2-
naphthaldehyde was highly fluorescent, the fluorogenic aldehyde, 4-formylbenzene connected to the
6-methoxy-2-naphthyl group through a 1,2,3-triazole moiety, was essentially nonfluorescent in aqueous
solutions. We suggest that the 4-formylphenyl group acts as a quencher to suppress the fluorescence of
the 6-methoxy-2-naphthyltriazole moiety. The product of the aldol reaction of this aldehyde does not
have a quenching moiety and showed more than 800-fold higher fluorescence than the aldehyde. Assay
systems using the fluorogenic aldehyde were validated by screening of aldol catalysts, ranking of the
activities of the catalysts, and evaluation of reaction conditions.
Introduction
and rapid characterization of catalysts.1-3 Assay systems using
fluorogenic substrates are critical for measurements and ranking
of catalytic activities in the creation of designer enzymes and
in directed enzyme evolution in vitro.3 Fluorogenic substrates
have also been employed in assessment of the catalytic activities
of catalysts under different conditions and in screening of
catalysts useful for synthetic organic chemistry.4 Detection of
formation of fluorescent products from nonfluorescent fluoro-
genic substrates (i.e., detection of an increase in fluorescence)
is typically more sensitive than detection of formation of
nonfluorescent products from highly fluorescent reactants/
substrates (i.e., detection of a decrease in fluorescence).2 Here
we report the development of a new type of fluorogenic aldehyde
Fluorogenic substrates that afford fluorescent products can
be used for monitoring the progress of chemical reactions by
fluorescence growth and are useful for high-throughput screening
(1) (a) Goddard, J.-P.; Reymond, J.-L. Trends Biotechnol. 2004, 22, 363,
and references cited therein. (b) List, B.; Barbas, F., III.; Lerner, R. A. Proc.
Natl. Acad. Sci. U.S.A. 1998, 95, 15351. (c) Carlson, R. P.; Jourdain, N.;
Reymond, J.-L. Chem.sEur. J. 2000, 6, 4154. (d) Yee, D. J.; Balsanek, V.;
Sames, D. J. Am. Chem. Soc. 2004, 126, 2282. (e) Froemming, M. K.; Sames,
D. Angew. Chem., Int. Ed. 2006, 45, 637. (f) Froemming, M. K.; Sames, D.
J. Am. Chem. Soc. 2007, 129, 14518. (g) Xing, B.; Khanamiryan, A.; Rao, J.
J. Am. Chem. Soc. 2005, 127, 4158. (h) Watzke, A.; Kosec, G.; Kindermann,
M.; Jeske, V.; Nestler, H.-P.; Turk, V.; Turk, B.; Wendt, K. U. Angew. Chem.,
Int. Ed. 2008, 47, 406. (i) Wood, W. J. L.; Patterson, A. W.; Tsuruoka, H.; Jain,
R. K.; Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 15521. (j) Javor, S.; Delort,
E.; Darbre, T.; Reymond, J.-L. J. Am. Chem. Soc. 2007, 129, 13238. (k) Wu,
Q.; Anslyn, E. V. J. Am. Chem. Soc. 2004, 126, 14682. (l) Wang, Q.; Cahill,
S. M.; Blumenstein, M.; Lawrence, D. S. J. Am. Chem. Soc. 2006, 128, 1808.
(m) Konarzycka-Bessler, M.; Bornscheuer, U. T. Angew. Chem., Int. Ed. 2003,
42, 1418. (n) Khersonsky, O.; Tawfik, D. S. ChemBioChem 2006, 7, 49. (o)
Reymond, J.-L.; Fluxa, V. S.; Maillard, N. Chem. Commun. 2009, 34, and
references cited therein. .
(3) (a) Jiang, L.; Althoff, E. A.; Clemente, F. R.; Doyle, L.; Rothlisberger,
D.; Zanghellini, A.; Gallahe, J. L.; Betker, J. L.; Tanaka, F.; Barbas, C. F., III.;
Hilvert, D.; Houk, K. N.; Stoddard, B.; Baker, D. Science 2008, 319, 1387. (b)
Gildersleeve, J.; Varvak, A.; Atwell, S.; Evans, D.; Schultz, P. G. Angew. Chem.,
Int. Ed. 2003, 42, 5971. (c) Tanaka, F.; Lerner, R. A.; Barbas, C. F., III. J. Am.
Chem. Soc. 2000, 122, 4835. (d) Tanaka, F.; Barbas, C. F., III. J. Am. Chem.
Soc. 2002, 124, 3510. (e) Tanaka, F.; Fuller, R.; Barbas, C. F., III. Biochemistry
2005, 44, 7583.
(2) (a) Tanaka, F.; Thayumanavan, R.; Barbas, C. F., III. J. Am. Chem. Soc.
2003, 125, 8523. (b) Tanaka, F.; Mase, N.; Barbas, C. F., III. J. Am. Chem. Soc.
2004, 126, 3692. (c) Guo, H.-M.; Minakawa, M.; Tanaka, F. J. Org. Chem.
2008, 73, 3964. (d) Tanaka, F. Chem. Rec. 2005, 5, 276.
(4) (a) Tanaka, F.; Thayumanavan, R.; Mase, N.; Barbas, C. F., III.
Tetrahedron Lett. 2004, 45, 325. (b) Mase, N.; Tanaka, F.; Barbas, C. F., III.
Org. Lett. 2003, 5, 4369. (c) Mase, N.; Tanaka, F.; Barbas, C. F., III. Angew.
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10.1021/jo900013w CCC: $40.75
Published on Web 02/17/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 2417–2424 2417

Guo and Tanaka
SCHEME 1. Fluorogenic Aldols, Fluorescent Aldehydes,
useful for monitoring the progress of aldol reactions through
an increase in fluorescence.
and Related Compounds
The aldol reaction is an important C-C bond-forming
reaction in enzyme catalysis and synthetic organic chemistry.2b,3-5
The development of efficient aldol catalysts is the topic of a
number of current research efforts.3-5 Because factors that affect
the catalysis of aldol reactions are not completely understood
and because design of aldol catalysts is often difficult, ap-
proaches that allow rapid screening of libraries are important
for the development of aldol catalysts and for the investigation
of aldol reactions.3-5 That is, there is a high demand for
fluorogenic substrates that can be used for monitoring the
progress of aldol reactions.2b,3-5 Whereas many fluorogenic
substrates for bond-cleaving reactions, including fluorogenic
substrates for retro-aldol reactions, are available,1,3 few fluo-
rogenic substrates for C-C bond-forming reactions have been
reported,2 and only our fluorogenic aldehydes have been reported
for aldol reactions.2b Because of the lack of the fluorogenic
substrates for aldol reactions, catalysts of aldol reactions have
often been identified or evaluated by retro-aldol reactions using
fluorogenic and chromogenic substrates for retro-aldol reactions.3,6
Catalysts of aldol reactions have also been evaluated using a
fluorescence-based assay method involving Michael addition to
a fluorogenic maleimide.2a,4 However, aldol catalysts do not
necessarily accelerate both aldol and retro-aldol reactions and
retro-aldol catalysts or Michael catalysts may not catalyze aldol
reactions. With appropriate fluorogenic aldehydes, aldol catalysts
can be screened and evaluated on the basis of aldol reactions.
For rapid evaluation of aldehyde transformations, including
aldol reactions, we previously developed fluorogenic aldehydes
and demonstrated their use in monitoring the progress of aldol
reactions.2b The design of our previous fluorogenic aldehydes
was a conceptual advance. However, improved fluorogenic
aldehydes are required. For example, the solubility of our
fluorogenic aldehydes was low in aqueous solutions. For the
fluorescence-based evaluation of aldol catalysts (such as designer
protein and peptide catalysts and small organocatalysts) in water,
high water solubility of the fluorogenic aldehydes is required.
Ratios of fluorescence intensities of aldol/aldehyde in the assay
systems using our previous fluorogenic substrates were up to
78-fold in aqueous solutions and 19-fold in organic solvents.2b
To efficiently detect the progress of aldol reactions using a
fluorogenic aldehyde, the fluorogenic aldehyde should show little
or no fluorescence and the aldol product should be highly
fluorescent. The fluorogenic range (or ratio of fluorescence
intensities of product/substrate) should be high enough to allow
monitoring of product formation during the initial stages of the
reactions; higher fluorogenic range provides better detection of
the product formation in assays. Fluorogenic aldehydes and
assay systems using fluorogenic aldehydes that allow efficient
monitoring of the progress of aldol reactions are needed for
creation and discovery of new aldol catalysts and for research
into the chemistry of aldol reactions.
Results and Discussion
(5) (a) Krattiger, P.; Kovasy, R.; Revell, J. D.; Ivan, S.; Wennemers, H. Org.
Lett. 2005, 7, 1101. (b) Dickerson, T. J.; Janda, K. D. J. Am. Chem. Soc. 2002,
124, 3220. (c) Oberhuber, M.; Joyce, G. F. Angew. Chem., Int. Ed. 2005, 44,
7580. (d) Revell, J. D.; Wennemer, H. Curr. Opin. Chem. Biol. 2007, 11, 269.
(e) Kofoed, J.; Reymond, J.-L. J. Comb. Chem. 2007, 9, 1046. (f) Nakadai, M.;
Saito, S.; Yamamoto, H. Tetrahedron 2002, 58, 8167. (g) Mahrwald, R. Modern
Aldol Reactions: Wiley-VCH; New York, 2004.
(6) (a) Kofoed, J.; Nielsen, J.; Reymond, J.-L. Bioorg. Med. Chem. Lett.
2003, 13, 2445. (b) Kofed, J.; Reymond, J.-L. Org. Biomol. Chem. 2006, 4,
3268. (c) Zhong, G.; Shabat, D.; List, B.; Anderson, J.; Sinha, R. A.; Lerner,
R. A.; Barbas, C. F., III. Angew. Chem., Int. Ed. 1998, 37, 2481. (d) Tanaka, F.;
Kerwin, L.; Kubitz, D.; Lerner, R. A.; Barbas, C. F., III. Bioorg. Med. Chem.
Lett. 2001, 11, 2983. (e) Shamis, M.; Barbas, C. F., III.; Shabat, D. Bioorg.
Med. Chem. Lett. 2007, 17, 1172.
Strategy for Design of Fluorogenic Aldehydes. Many
benzene and naphthalene derivatives that bear both electron-
donating and electron-withdrawing groups are fluorescent.1d,f,7
For example, aldehyde 1 (Scheme 1), which is fluorescent in
aqueous buffers,8 has a methoxy group (an electron-donating
group) and an aldehyde group (an electron-withdrawing group)
(7) Rettig, W. Angew. Chem., Int. Ed. Engl. 1986, 25, 971.
(8) Wierzchowski, J.; Wroczynski, P.; Laszuk, K.; Interwicz, E. Anal.
Biochem. 1997, 245, 69.
2418 J. Org. Chem. Vol. 74, No. 6, 2009

Fluorogenic Aldehyde Bearing Triazole Moiety
SCHEME 2. Fluorogenic Aldehydes, Fluorescent Aldols, and Related Compounds
on the naphthalene ring. Aldol 2, which does not have an
electron-withdrawing group, is not fluorescent in aqueous buffers
at the wavelengths used for the detection of fluorescence of
aldehyde 1.9 Because of the fluorescence features of aldehyde
1 and aldol 2, aldol 2 has been used as a fluorogenic substrate
for the retro-aldol reaction.1b,3a,c-e On the other hand, arylal-
dehyde moieties can act as intramolecular quenchers to suppress
fluorescence in some molecules, and such arylaldehydes may
be used as fluorogenic substrates for the aldehyde trans-
formations.2b Therefore, arylaldehydes within or connected to
aromatic systems bearing electron-donating groups may be either
fluorescent or nonfluorescent depending on the structure of the
molecule and depending on wavelengths to observe the fluo-
rescence. Electron-donating groups attached to arylaldehydes
often influence the fluorescence.10 Length of π-conjugation
usually correlates with UV absorption and/or fluorescence
emission wavelengths of fluorescent molecules.11 Use of
aromatic systems with electron-donating groups should provide
absorption and fluorescence at longer wavelengths than those
without electron-donating groups. In addition, attachment of
oxygen- or nitrogen-containing groups (such as methoxy group
or dialkylamino group) to aromatic hydrocarbons often increases
the solubility of the compounds in aqueous solutions. We
reasoned that arylaldehydes with fluorescent aromatic systems
bearing electron-donating groups would be fluorogenic when
there was no conjugation between the arylaldehyde moiety and
the electron-donating group; in such a system, the arylaldehyde
moiety would act as a quencher.12 Although many fluorescent
molecules are known, it is often difficult to predict fluorescence
or fluorescence-quenching features of a given molecule in a
particular solvent on the basis of the chemical structure of the
molecule. Therefore, to develop fluorogenic aldehydes that are
useful for assays of aldol reactions in aqueous solutions, we
synthesized a series of aldehydes with aromatic moieties bearing
electron-donating groups, their aldols, and related compounds
(compounds 1-23 in Schemes 1 and 2) and analyzed the
fluorescence of these compounds. As described below, we found
that aldehyde 20 was the most useful fluorogenic aldehyde of
those tested for evaluation of the aldol reaction in aqueous
solutions.
Fluorescence of Aldehydes, Aldols, and Related Com-
pounds. Fluorescence of compounds 1 and 3-21 (Schemes 1
and 2) was analyzed in 5% DMSO/50 mM sodium phosphate,
pH 7.0. The results, including excitation and emission wave-
lengths (λex and λem, respectively), that provided a significant
difference in fluorescence between aldehyde and the corre-
sponding aldol are summarized in Table 1. Selected fluorescence
spectra are shown in Figure 1. All of the aldehydes shown in
Scheme 1 (6-oxygen-substituted-2-naphthaldehydes 1, 4, and
6; 2-naphthaldehydes 7, 9, and 11 bearing 6-amono groups;
aldehyde 15 containing a coumarin moiety with a diethylamino
group; aldehyde 17 containing a coumarin moiety with a
hydroxy group) were fluorescent (entries 1-8 and 10 and Figure
1A and B). For the tested 6-oxygen- or 6-nitrogen-substituted
2-naphthaldehydes and the corresponding aldols shown in
Scheme 1 (aldehyde/aldol pairs 1/3, 4/5, 7/8, 9/10, and 11/12),
wavelengths that provided higher fluorescence of aldol than the
corresponding aldehyde were not found. Aldols 8, 10, and 12
showed fluorescence at some excitation and emission wave-
lengths, but the corresponding aldehydes also showed fluores-
cence at these wavelengths. Thus, like aldol 2, aldols 3, 5, 8,
10, and 12 may be used as fluorogenic substrates for the retro-
aldol reaction to detect the formation of the aldehyde by
fluorescence increase, but the corresponding aldehydes are not
fluorogenic aldehyde substrates for aldol reactions in this buffer.
Of the 6-nitrogen-substituted 2-naphthylaldehydes 7, 9, and 11,
6-pyrrolidyl-2-naphthaldehyde (7) showed the highest fluores-
cence, although 6-methoxy-2-naphthaldehyde (1) showed higher
fluorescence than aldehyde 7. It has been reported that the
nitrogen lone electron pair of the N-phenylpyrrolidine is
conjugated with the phenyl group but there is no such favored
conjugation in N-phenylpiperidine.13 Higher fluorescence of 7
than that of 9 or of 11 in the buffer may be explained by the
(9) Aldol 2 is fluorescent in some organic solvents. For fluorescence of
2-methoxynaphthalene, see: Balomenou, I.; Pistolis, G. J. Am. Chem. Soc. 2007,
129, 13247.
(10) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 2nd ed.;
Kluwer Academic: New York, 1999.
(11) Yamaguchi, Y.; Matsubara, Y.; Ochi, T.; Wakamiya, T.; Yoshida, Z.
J. Am. Chem. Soc. 2008, 130, 13867.
(12) For fluorescence quenching by ketones and enones in 2-methoxynaph-
thalene derivatives, see ref 1f.
(13) Rozeboom, M.; Houk, K. N.; Searles, S.; Seyedrezai, E. J. Am. Chem.
Soc. 1982, 104, 3448.
J. Org. Chem. Vol. 74, No. 6, 2009 2419

Guo and Tanaka
TABLE 1. Fluorescence of Aldehydes and Aldols in Aqueous Buffera
aldehyde
aldol or enone
entry
λex (nm)
λem (nm)
compd
fluorescence intensityb
φc
compd
fluorescence intensityb
φc
1
2
315
315
319
380
450
445
445
450
1
4
6
7
7.6 × 102
1.6 × 102
90
0.56
0.12
0.06
0.22
3
5
<5
<5
0.00
0.00
3
4d
3.0 × 102
8
13
8
13
10
12
16
16
<5
<5
<5
40
<5
<5
50
0.00
0.00
0.00
0.03
0.00
0.00
0.04
0.17
5e
380
535
7
1.0 × 102
0.07
6
7
8
9
10
11
12
13f
14g
15h
365
355
390
390
325
300
260
260
260
260
530
520
485
430
450
365
375
370
385
375
9
11
15
15
17
18
20
20
20
20
80
55
0.06
0.04
0.51
7.0 × 102
11
2.3 × 102
60
<5
<5
<5
<5
<5
0.04
0.00
0.00
0.00
0.00
0.00
19
21
21
21
21
7.2 × 102
8.8 × 102
8.8 × 102
8.8 × 102
4.2 × 103
0.53
0.64
0.64
0.64
a The fluorescence was recorded on a microplate spectrophotometer using 100 µL of a 5 µM solution in 5% DMSO/50 mM sodium phosphate, pH 7.0
in a 96-well plate at 26 °C. b Relative fluorescence intensity after background correction. c Relative to 2-aminopyridine in 0.1 N H2SO4 as a standard
(λex 300 nm, φ ) 0.60). d Wavelengths for detection of formation of 7 from 8. e Wavelengths for detection of formation of 13 from 8. f In 5% DMSO/
50 mM NaH2PO4, pH 5. g In 5% DMSO/50 mM Na2HPO4, pH 9. h Compound concentration 50 µM.
FIGURE 1. Fluorescence emission spectra. Compound (5 µM) in 5% DMSO/50 mM NaH2PO4-Na2HPO4, pH 7.0. Key: open square, aldehyde;
solid circle, aldol. (A) Aldehyde 7, aldol 8, and enone 13; λex 380 nm. (B) Aldehyde 15, aldol 16; λex 390 nm. (C) Aldehyde 18, aldol 19; λex 300
nm. (D) Aldehyde 20, aldol 21; λex 260 nm.
efficient conjugation between the aryl π and the N lone pair in
7. Features of the conjugation of the N lone pair with aryl π
vary depending on solvent14 and thus fluorescence features of
these molecules vary depending on solvent (see below). Enone
13 showed fluorescence (entry 5 and Figure 1A), as did 14.1f
Under some conditions, formation of 13 from aldol 8 may be
detected by fluorescence.
For the aldehyde 15 and aldol 16 pair, aldol 16 showed only
weak fluorescence under the excitation and emission wave-
lengths (λex 390 nm, λem 485 nm) optimal for the fluorescence
of 15 (entry 8 and Figure 1B). On the other hand, aldol 16
showed fluorescence higher than that of aldehyde 15 at λex 390
nm and λem 430 nm (entry 9 and Figure 1B). Therefore, aldehyde
15 and aldol 16 may be used as fluorogenic substrates for aldol
and retro-aldol reactions, respectively, when the reactions are
monitored at optimized emission wavelengths.
In contrast to the aldehydes described above, aldehyde 18
was not fluorescent; aldol 19 showed high fluorescence at λex
300 nm and λem 365 nm (entry 11 and Figure 1C). Fluorescence
of aldol 19 was more than 140-fold higher than that of aldehyde
18. Although fluorescence of 18 was analyzed at a number of
excitation and emission wavelengths, wavelengths where alde-
hyde 18 showed high fluorescence were not found. Like the 18
and 19 pair, aldehyde 20 was not fluorescent and aldol 21
showed high fluorescence (entry 12 and Figure 1D). At λex 260
(14) Wu, L.; Burgess, K. Org. Lett. 2008, 10, 1779.
2420 J. Org. Chem. Vol. 74, No. 6, 2009

Fluorogenic Aldehyde Bearing Triazole Moiety
nm and λem 375 nm, aldol 21 showed more than 170- and 800-
fold higher fluorescence than aldehyde 20 at 5 and 50 µM,
respectively (entries 12 and 15). Because aldehyde 20 was
essentially nonfluorescent in aqueous solution, the fluorogenic
range of >800 for 21/20 was achieved. Note that aldehyde 22
and aldol 23 were both nonfluorescent.
For aldehyde 20, the arylaldehyde moiety may quench the
fluorescence of the methoxynaphthyltriazole moiety. The meth-
oxynaphthyl, 1,2,3-triazole, and formylphenyl groups may not
be on a single plane because of steric interaction between the
aryl groups attached to the triazole ring; that is, there may be
no π-conjugation between the formylphenyl group and meth-
oxynaphthyl group in 20, and this may be why the formylphenyl
moiety quenches the fluorescence. In the reported X-ray crystal
structures of 4,5-diphenyl-1,2,3-triazole derivatives, one phenyl
ring and the triazole group are almost on a single plane but the
other phenyl ring is not on the plane, indicating that there is no
π-conjugation between the two phenyl groups.15 Aldehyde 20
and aldol 21 should have conformations similar to these crystal
structures, supporting that the methoxynaphthyl group does not
have π-conjugation with the formylphenyl moiety in aldehyde
20. In aldol 21, no moiety quenches the fluorescence, and this
aldol is fluorescent. The aldol reaction of aldehyde 20 proceeded
efficiently. The aldol reaction of aldehyde 20 was faster than
that of aldehyde 18 in aqueous buffer under the same conditions
(see below). These results also support that there is no
π-conjugation between the methoxynaphthyl group and the
formylphenyl moiety in aldehyde 20. Note that aldehyde 15, in
which the formylphenyl group is connected to the coumarin
moiety with a single bond, showed high fluorescence. For
aldehydes 15 and 17, π-conjugation between the coumarin
moiety and formylphenyl group is possible, and this is presum-
ably why these compounds are fluorescent.
Use of the 1,2,3-triazole moiety to connect the methoxynaph-
thyl group and the formylphenyl group in aldehyde 20 may be
the key for preventing π-conjugation between the two aryl
groups attached on the 4- and 5-positions of the 1,2,3-triazole.
If cis olefin is used instead of the triazole ring, the cis olefin
may be isomerized to trans olefin under the UV required for
the fluorescence measurements.16 In the resulting trans form,
fluorescence may be different from that of the cis form; thus
the cis-trans isomerization may complicate analysis of the
fluorescence data. With the 1,2,3-triazole group, there is no
opportunity for such isomerization.
FIGURE 2. Fluorescence emission spectra of 21 (5 µM) in 5% DMSO/
50 mM NaH2PO4, pH 5, in 5% DMSO/50 mM NaH2PO4-Na2HPO4,
pH 7.0, and in 5% DMSO/50 mM Na2HPO4, pH 9.
SCHEME 3. Fluorogenic Aldehyde and Its Fluorescent
Aldol Product
disruption of the π-conjugation between the formylphenyl
moiety and the 6-methoxynaphthylalkynyl moiety by rotation
of the single bonds may lead the fluorescence quenching.
For aldol 21, the λem max of the fluorescence slightly shifted
depending on pH; λem max was 370 nm at pH 5, 375 nm at pH
7, and 385 nm at pH 9 at λex 260 nm (entries 12-14 and Figure
2). This may be related to the dependence of deprotonation of
the triazole moiety on pH. Aldehyde 20 showed no fluorescence
at pH 5, pH 9, or pH 7.0. Note that the λem max of the
fluorescence of aldehyde 1 was unchanged over the pH range
from 5 to 9.3
Fluorogenic aldehyde 20 was soluble at 50 or 100 µM in
aqueous buffers containing small amounts of water-miscible
organic solvents. The triazole group of 20 may contribute to
the water solubility as 20 was more soluble than 18 in aqueous
buffer. Aldehyde 20 was also more soluble than fluorogenic
aldehyde 24, which we previously reported2b (Scheme 3), in
aqueous solutions. The ratio of fluorescence intensities of 21/
20 was more than 170 at 5 µM and was more than 800 at 50
µM as described above, a higher ratio than that of 25/24 (∼80).
Thus, fluorogenic aldehyde 20 is superior to 24 for monitoring
the progress of aldol reactions in aqueous solutions with respect
to the fluorescence increase upon aldol reaction and to the
solubility of the aldehyde.
Fluorescence of the compounds was also analyzed in organic
solvents (Table 2). For aldehydes 7, 9, and 11 and aldols 8, 10,
and 12, either aldehyde or aldol was fluorescent at 5 µM in
organic solvents, such as DMSO or CH3CN, depending on
excitation and emission wavelengths (entries 1-8). However,
the fluorescence of aldols 8, 10, and 12 did not correlate with
concentration; the fluorescence of these aldols at 100 µM was
not higher than that at 5 µM in DMSO at λex 260 nm. Thus,
aldehydes 7, 9, and 11 may be used for the detection of
formation of aldol products only under certain conditions. For
For aldehyde 18, the arylaldehyde group may act as a
quencher of the fluorescence of the 6-methoxynaphthylalkynyl
moiety or 6-methoxynaphthylalkynylphenyl moiety. We initially
expected that aldehyde 18 might emit fluorescence at longer
wavelengths than aldehyde 1 because π-conjugation in 18 is
longer than that in 1. Interestingly, aldehyde 18 differed from
our expectation; aldehyde 18 was not fluorescent and aldol 19
was highly fluorescent, as described above. Although reported
crystal structures of 1,2-diphenylacetylene suggested strong
π-conjugation between the two phenyl groups, some of the
structures were distorted from a single plane, suggesting flexible
rotation of the single bonds.17 For aldehyde 18, frequent
(15) (a) Adelwohrer, C.; Rosenau, T.; Kloser, E.; Mereiter, K.; Netscher, T.
Eur. J. Org. Chem. 2006, 2081. (b) Kokkou, S. C.; Pentzeperis, P. J. Acta
Crystallogr., Sect. B 1975, B31, 2788.
(16) Saltiel, J.; Krishna, T. S. R.; Turek, A. M. J. Am. Chem. Soc. 2005,
127, 6938.
(17) Thomas, R.; Lakshmi, S.; Pati, S.; Kulkarni, G. U. J. Phys. Chem. B
2006, 110, 24674.
J. Org. Chem. Vol. 74, No. 6, 2009 2421

Guo and Tanaka
TABLE 2. Fluorescence of Aldehydes and Aldols in Organic Solventsa
aldehyde
aldol or enone
entry
1d
solvent
DMSO
λex (nm)
λem (nm)
compd
fluorescence intensityb
φc
compd
fluorescence intensityb
φc
375
470
7
8.0 × 102
0.59
8
13
8
13
8
13
8
10
10
12
12
16
16
19
21
21
21
<5
<5
<10
0.00
0.00
<0.01
0.81
2e
3
DMSO
DMSO
375
260
555
420
7
7
<5
0.00
0.00
1.1 × 103
2.0 × 103
<5
<5
0.00
0.01
0.04
4
5
6
7
8
9
10
11
12
13
14
CH3CN
DMSO
DMSO
DMSO
DMSO
DMSO
CH3CN
DMSO
DMSO
CH3CN
EtOAc
375
375
260
360
260
375
375
275
260
260
260
475
470
420
475
415
460
455
370
370
370
370
7
9
9
1.2 × 103
1.2 × 103
1.2 × 102
6.2 × 102
30
0.88
0.88
0.09
0.45
0.02
1.1
15
60
2.2 × 103
60
11
11
15
15
18
20
20
20
0.04
1.3 × 103
1.5 × 103
1.7 × 103
<50
<5
0.00
<0.01
2.0
0.51
0.60
0.55
1.2
<10
<0.04
<0.01
0.01
0.01
2.8 × 103
7.0 × 102
8.3 × 102
7.5 × 102
<15
12
12
a The fluorescence was recorded on a microplate spectrophotometer using 100 µL of a 5 µM solution in DMSO or in 5% DMSO/indicated solvent in
a 96-well polypropylene plate at 26 °C. b Relative fluorescence intensity after background correction. c Relative to 2-aminopyridine in 0.1 N H2SO4 as a
standard (λex 300 nm, φ ) 0.60). d Wavelengths for detection of formation of 7 from 8. e Wavelengths for detection of formation of 13.
FIGURE 3. Fluorescence assay of amine-catalyzed aldol reaction of acetone and aldehyde 20.18 Conditions: [amine catalyst] 12.5 mM, [aldehyde
20] 100 µM for (A) or 50 µM for (B) and (C), [acetone] 5% (v/v) (680 mM) in 5% DMSO-170 mM Na2HPO4, pH 9 in a 96-well plate at 26 °C;
(a) reaction in the presence of pyrrolidine; (b) reaction in the presence of DBU; (c) reaction without catalyst (blank). RFU ) relative fluorescence
intensity. (A) Time course for 12 min at λex 260 nm and λem 370 nm. (B) Time course for 60 min. (C) Emission spectra (λex 260 nm) of reactions
at 60 min.
aldehydes 7, 9, and 11, a higher concentration of the compound
provided higher fluorescence in DMSO and in CH3CN at λex
375 nm or at λex 360 nm. Thus, retro-aldol reactions of aldols
8, 10, and 12 that afford aldehydes 7, 9, and 11, respectively,
may be monitored more reliably by fluorescence increase than
aldol reactions using aldehydes 7, 9, and 11. Whereas aldehydes
9 and 11 showed moderate fluorescence compared to aldehyde
1 in aqueous buffer (Table 1, entries 6 and 7 versus entry 1;
approximately 10-fold difference), these compounds showed
similar levels of fluorescence in DMSO at selected excitation
and emission wavelengths (Table 2, entries 1, 5, and 7; within
2-fold difference). These results may be related to the efficient
conjugation between the aryl π and the amine nitrogen lone
pair in organic solvents including DMSO.14 Compound 13 was
also fluorescent in DMSO, and the fluorescence of 13 differed
from the fluorescence of aldehyde 7 and of aldol 8 (Table 2,
entry 2); thus, formation of 13 may be detected by an increase
in fluorescence. For aldehyde 15 and aldol 16, aldehyde 15 was
fluorescent (entries 9 and 10). For aldehyde/aldol pairs 18/19
and 20/21, the aldols were highly fluorescent (entries 11-14).
Aldol 21 showed approximately 50-70-fold higher fluorescence
than aldehyde 20 in DMSO, CH3CN, and EtOAc.
genic aldehyde 20, a time-course of the aldol reaction of this
aldehyde with acetone was monitored by fluorescence. Aldol
reactions (100 µL scale) were performed using 12.5 mM of
amine catalyst, 50 µM or 100 µM of aldehyde 20, and 5% (v/
v) acetone (680 mM) in 5% DMSO-170 mM Na2HPO4, pH 9,
and the fluorescence was recorded at λex 260 nm and λem 370
nm.18 The assay results are shown in Figure 3. Reaction progress
was detected by an increase in fluorescence (Figure 3A, B).
Pyrrolidine, a known catalyst of aldol reactions in water,5b,19
catalyzed the aldol reaction of 20 at pH 9. Reaction in the
presence of DBU20 was slower than the pyrrolidine-catalyzed
reaction; differences in reaction rate were detected by fluores-
cence measurements. By monitoring the fluorescence of the
catalyzed-aldol reactions of fluorogenic aldehyde 20, activities
of catalysts were ranked within 10-60 min.
(18) Reactions were initiated by adding 5 µL of a stock solution of aldehyde
20 (1 or 2 mM in DMSO) to a mixture of 5 µL of acetone, 85 µL of 200 mM
Na2HPO4, pH 9, and 5 µL of a catalyst solution (250 mM in H2O). Final
concentrations: [catalyst] 12.5 mM, [aldehyde 20] 50 µM (from 1 mM stock
solution) or 100 µM (from 2 mM stock solution), [acetone] 5% (v/v) (680 mM)
in 5% DMSO-170 mM Na2HPO4, pH 9. Fluorescence assay was performed in
a 96-well plate on a spectrophotometer at 26 °C.
(19) Chimni, S. S.; Mahajan, D. Tetrahedron 2005, 61, 5019.
(20) Markert, M.; Mulzer, M.; Schetter, B.; Mahrwald, R. J. Am. Chem. Soc.
2007, 129, 7258.
Monitoring of the Progress of Aldol Reactions Using
the Fluorogenic Aldehyde. To demonstrate the use of fluoro-
2422 J. Org. Chem. Vol. 74, No. 6, 2009

Fluorogenic Aldehyde Bearing Triazole Moiety
TABLE 3. Velocities of Aldol Reactions of Acetone and 20
Determined by Fluorescence Assaysa
identification of aldol catalysts, ranking of activities of catalysts,
and evaluation of reaction conditions. Although the fluorescence
assay system using aldehyde 20 does not allow determination
of stereochemistries of the products, superior catalysts based
on the catalytic activity can be rapidly identified using this assay
system.22 Selected catalysts with high activities may be further
analyzed in detail. Methods of the determination of stereose-
lectivities (such as chiral phase HPLC) are generally slower and
generate more waste than the fluorescence assay with 20.23,24
Thus, the assay system using aldehyde 20 will reduce the time
and energy required for the development of aldol catalysts.
Although development of efficient aldol catalysts is a topic
of interest, design of desired aldol catalysts is difficult.3-5
Factors that affect the catalysis of aldol reactions are not
completely understood.3-5 The fluorogenic aldehyde and the
assay system reported here will make discovery of new aldol
catalysts more efficient and will contribute to the study of the
chemistry of aldol reactions.
entry
catalyst
velocity (µM/min)
1
2
3
4
5
6
7
8
pyrrolidine
prolinamide
2,3-diaminopropionic acid
proline
0.10 (0.002b)
0.08
0.04
0.03
0.02
0.01
<0.01
0.004
DBU
2-aminoethanol
each catalyst listed in footnotec
- (blank)
a Conditions: [catalyst] 12.5 mM, [acetone] 5%(v/v) (680 mM),
[aldehyde 20] 50 µM in 5% DMSO-170 mM Na2HPO4, pH 9.
Fluorescence of 100 µL scale reactions was analyzed at λex 260 nm and
λem 370 nm in a 96-well plate.21 Deviations of the velocities were
(20% in duplicated experiments. b Data of the reaction performed in
5% DMSO-170 mM NaH2PO4-Na2HPO4, pH 7.5. c Piperidine,
morpholine, pyridine, imidazole, Et2NH, n-BuNH2, Et3N, i-Pr2NEt, and
DABCO.
Fluorogenic aldehyde 24, which we previously reported, was
useful for fluorescence-based monitoring of the aldol reaction
and both the reduction to form the alcohol and the formation
of the thiazolidine with cysteine.2b,25 Thus, fluorogenic aldehyde
20 may also be useful for monitoring the progress of these
reactions and of other aldehyde transformations24d,26 in aqueous
solutions.
The assay system using aldol reaction of acetone and aldehyde
20 was also employed for the evaluation of a set of amines as
aldol catalysts. Reactions were monitored over 60 min, and
initial velocities were determined based on fluorescence increase
(Table 3).21 When 50 µM of aldehyde 18, instead of 20, was
used for the evaluation of the same set of catalysts under the
same conditions, reactions were slower, and 60 min was not
sufficient time to rank the catalysts or to determine the velocities.
Although aldehyde 18 may be useful for special cases, aldehyde
20 was more suitable than 18 for evaluation of catalysts. When
the same catalysts were evaluated in the reaction of 20 in 5%
DMSO-170 mM NaH2PO4-Na2HPO4, pH 7.0 or in 5%
DMSO-170 mM NaH2PO4, pH 5, no significant formation of
21 was detected for any of the amines listed in Table 3. When
the pyrrolidine-catalyzed reaction was performed in 5% DM-
SO-170 mM NaH2PO4-Na2HPO4, pH 7.5, the velocity was
approximately 50-fold lower than that of the reaction at pH 9.
For pyrrolidine to efficiently catalyze the aldol reaction in water,
alkaline conditions were required, consistent with reported
observations.5b,19 These results indicate that the fluorescence
assay system using fluorogenic aldehyde 20 is useful for
Conclusions
We have developed a new fluorogenic aldehyde bearing 1,2,3-
triazole moiety and have demonstrated that the aldehyde is useful
for monitoring the progress of aldol reactions in aqueous
solutions through an increase in fluorescence. Fluorogenic
aldehyde 20 was essentially nonfluorescent, and the aldol
product, 21, showed more than 800-fold higher fluorescence
(23) For bond-cleaving reactions, diastereo- and enantiomerically pure
fluorogenic substrates bearing chiral centers have been employed to assess
diastereo- and enantioselective reactions.1a,c Similarly, diastereo- and enantio-
merically pure fluorogenic substrates bearing chiral centers may also be used to
evaluate progress of stereoselective bond-forming reactions. A pair of enantiomers
with different reporter moieties have also been used to identify enantioselective
catalysts for bond-cleaving reactions: (a) Becker, S.; Hobenreich, H.; Vogel,
A.; Knorr, J.; Wilhelm, S.; Rosenau, F.; Jaeger, K.-E.; Reetz, M. T.; Kolmar, H.
Angew. Chem., Int. Ed. 2008, 47, 5085. Fluorogenic substrates that allow both
the evaluation of the progress of bond forming chemical reactions and the
determination of enantiomeric ratios of the products generated from achiral
substrates have not been reported.
(24) To our knowledge, there are no fluorescence- or color-based assay
methods to detect enantiomers of simple ꢀ-hydroxycarbonyl compounds.
Although methods for the determination of enantiomeric ratios of some
compounds by fluorescence- or color-based assays using small molecule probes
have been developed, these methods often require relatively high concentrations
of compounds of interest (i.e., products) compared to our assay method in which
reaction progress is directly observed as an increase in fluorescence. The
fluorescence-based assay methods for the determination of enantiomeric ratios
based on selective interactions with enantiomers are not usually suited for use
during the course of the chemical reaction while concentrations of the products
vary. For fluorescence- or color-based assays of enantiomeric compounds, see:
(a) Leung, D.; Folmer-Andersen, J. F.; Lynch, V. M.; Anslyn, E. V. J. Am.
Chem. Soc. 2008, 130, 12318. (b) Leung, D.; Anslyn, E. V. J. Am. Chem. Soc.
2008, 130, 12328. (c) Reetz, M. T.; Sostmann, S. Tetrahedron 2001, 57, 2515.
A high-throughput method for the sequential determination of enantioselectivity,
yield, and conversion of starting material in reactions to form acetylated
cyanohydrins has been developed; in this method, three enzymes were employed,
including one enantioselective enzyme used to analyze an enantiomer of the
product: (d) Hamberg, A.; Lundgren, S.; Penhoat, M.; Moberg, C.; Hult, K.
J. Am. Chem. Soc. 2006, 128, 2234.
(21) Whereas higher concentration of aldol 21 correlated with higher
fluorescence in the range of 0.2-50 µM under identical conditions, a solution
of a lower concentration of aldol 21 showed higher fluorescence per µM
compared to that of a higher concentration of 21. This observation is consistent
with data for other highly fluorescent compounds.2b Over an approximately 20-
fold concentration range (such as 0.1-2.0 µM or 0.5-5.0 µM), the relationship
between concentration of aldol 21 and relative fluorescence intensity was
essentially linear. The linear correlation was used for the conversion of the
fluorescent intensity to the concentration of aldol 21 for the determination of
the velocities.
(22) Methods for high-throughput screening of catalysts using other than
fluorogenic substrates: (a) Crabtree, R. H. Chem. Commun. 1999, 1611. (b) Kuntz,
K. W.; Snapper, M. L.; Hoveyda, A. H. Curr. Opin. Chem. Biol. 1999, 3, 313.
(c) Reetz, M. T. Angew. Chem., Int. Ed. 2001, 40, 284. (d) Reetz, M. T. Angew.
Chem., Int. Ed. 2002, 41, 1335. (e) Wahler, D.; Reymond, J.-L. Curr. Opin.
Biotechnol. 2001, 12, 535. (f) Goddard, J.-P.; Reymond, J.-L. Curr. Opin.
Biotechnol. 2004, 15, 314. (g) Revell, J. D.; Wennemers, H. Curr. Opin. Chem.
Biol. 2007, 11, 269. (h) Cooper, A. C.; McAlexander, L. H.; Lee, D.-H.; Torres,
M. T.; Crabtree, R. H. J. Am. Chem. Soc. 1998, 120, 9971. (i) Taylor, S. J.;
Morken, J. P. Science 1998, 280, 267. (j) Reetz, M. T.; Becker, M. H.; Kuhling,
K. M.; Holzwarth, A. Angew. Chem., Int. Ed. 1998, 37, 2647. (k) Reetz, M. T.;
Kuhling, K. M.; Deege, A.; Hinrichs, H.; Belder, D. Angew. Chem., Int. Ed.
2000, 39, 3891. (l) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc. 1999, 121,
4306. (m) Harris, R. F.; Nation, A. J.; Copland, G. T.; Miller, S. J. J. Am. Chem.
Soc. 2000, 122, 11270. (n) Copeland, G. T.; Miller, S. J. J. Am. Chem. Soc.
2001, 123, 6496. (o) Francis, M. B.; Jacobsen, E. N. Angew. Chem., Int. Ed.
1999, 38, 937. (p) Yeung, E. S.; Su, H. J. Am. Chem. Soc. 2000, 122, 7422. (q)
Das, G.; Talukdar, P.; Matile, S. Science 2002, 298, 1600. (r) Shaughnessy,
K. H.; Kim, P.; Hartwig, J. F. J. Am. Chem. Soc. 1999, 121, 2123. (s) Stauffer,
S. R.; Beare, N. A.; Stambuli, J. P.; Hartwig, J. F. J. Am. Chem. Soc. 2001, 123,
4641. (t) Stauffer, S. R.; Hartwig, J. F. J. Am. Chem. Soc. 2003, 125, 6977.
(25) Tanaka, F.; Mase, N.; Barbas, C. F., III. Chem. Commun. 2004, 1762.
(26) (a) Lee, K.-S.; Kim, H.-J.; Kim, G.-H.; Shin, I.; Hong, J.-I. Org. Lett.
2008, 10, 49. (b) Lee, K.-S.; Kim, T.-K.; Lee, J. H.; Kim, H.-J.; Hong, J.-I.
Chem. Commun. 2008, 6173. (c) Rusin, O.; Luce, N. N. S.; Agbaria, R. A.;
Escobedo, J. O.; Jiang, S.; Warner, I. M.; Dawan, F. B.; Lian, K.; Strongin,
R. M. J. Am. Chem. Soc. 2004, 126, 438.
J. Org. Chem. Vol. 74, No. 6, 2009 2423

Guo and Tanaka
Aldehyde 20. A mixture of 18 (56 mg, 0.2 mmol), NaN3 (52
mg, 0.8 mmol), and DMF (10 mL) was stirred at 100 °C for 6 h.
The mixture was concentrated in vacuo, added to water, and
extracted with EtOAc. The organic layers were combined, dried
over MgSO4, filtered, concentrated, and purified by flash column
chromatography to afford 20 (44 mg, 67%) as a pale yellow solid:
1H NMR (400 MHz, CD3OD) δ 9.97 (s, 1H), 7.91-7.88 (m, 3H),
7.82-7.69 (m, 4H), 7.49-7.47 (m, 1H), 7.27 (d, J ) 2.4 Hz, 1H),
7.15 (dd, J ) 8.8 Hz, 2.4 Hz, 1H), 3.92 (s, 3H); 13C NMR (100
MHz, CD3OD) δ 193.5, 160.0, 137.4, 136.2, 131.0, 130.6, 130.1,
129.5, 128.7, 128.6, 127.2, 120.6, 106.7, 55.8; HRMS calcd for
C20H16N3O2 (MH+) 330.1237, found 330.1239.
Aldol 21. Aldol 21 was synthesized using pyrrolidine as a
catalyst.19 The reaction was performed using acetone (0.5 mL),
aldehydes 20 (40 mg, 0.12 mmol), and pyrrolidine (3.0 µL, 0.04
mmol) in H2O (0.5 mL) to afford 21 (26 mg, 55%): 1H NMR (400
MHz, CD3OD) δ 7.84 (s, 1H), 7.70 (d, J ) 8.8 Hz, 1H), 7.61 (d,
J ) 8.8 Hz, 1H), 7.46-7.44 (m, 3H), 7.35-7.33 (m, 2H), 7.19 (d,
J ) 2.0 Hz, 1H), 7.08 (dd, J ) 8.8 Hz, 2.0 Hz, 1H), 5.10 (dd, J )
8.8 Hz, 4.4 Hz, 1H), 3.85 (s, 3H), 2.86 (dd, J ) 16.0 Hz, 8.8 Hz,
1H), 2.73 (dd, J ) 16.0 Hz, 4.4 Hz, 1H), 2.12 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 209.1, 158.1, 142.9, 134.3, 129.7, 128.6,
128.2, 127.2, 127.1, 126.3, 125.9, 119.2, 105.6, 69.5, 55.3, 51.7,
30.7; HRMS calcd for C23H22N3O3 (MH+) 388.1656, found
388.1660.
than the aldehyde in aqueous solutions. The fluorogenic alde-
hyde was useful for evaluation of aldol catalysts and reaction
conditions. Fluorescence assays of aldol reactions using the
fluorogenic aldehyde allowed ranking of a set of aldol catalysts
within a short time. The fluorescence assay system using the
aldol reaction of the fluorogenic aldehyde will be useful for
the development of aldol catalysts.
For fluorogenic aldehyde 20, the methoxynaphthyl, triazole,
and formylphenyl groups may not be on a single plane and the
formylphenyl group efficiently quenches the fluorescence of the
methoxynaphthyltriazole moiety. This type of system for
preparation of a fluorogenic reactant should be useful for the
creation of fluorogenic molecules for monitoring other chemical
transformations.
Experimental Section
Aldehyde 18. A mixture of 2-bromo-6-methoxynaphthalene (1.02
g, 4.30 mmol), 3-ethynylbenzaldehyde (22) (560 mg, 4.30 mmol),
Ph3P (56 mg, 0.22 mmol), PdCl2 (9 mg, 0.05 mmol), and Cu(OAc)2
(10 mg, 0.05 mmol) in anhydrous triethylamine (30 mL) was
refluxed for 6 h.27 After being cooled to room temperature, the
precipitate was filtered off, and the filtrate was concentrated in
vacuo. The crude mixture was purified by flash column chroma-
tography to give aldehyde 18 (380 mg, 31%) as a yellow solid: 1H
NMR (300 MHz, CDCl3) δ 10.0 (s, 1H), 8.01 (s, 1H), 7.87 (d, J )
8.1 Hz, 2H), 7.75-7.73 (m, 2H), 7.70 (d, J ) 8.1 Hz, 2 H), 7.55
(dd, J ) 8.7 Hz, 1.7 Hz, 1H), 7.18 (dd, J ) 8.7 Hz, 2.1 Hz, 1H),
7.12 (d, J ) 2.1 Hz, 1H), 3.93 (s, 3H); 13C NMR (100 MHz, CDCl3)
δ 191.3, 158.5, 135.1, 134.4, 131.9, 131.7. 129.7, 129.5, 129.3,
128.7, 128.3, 126.9, 119.5, 117.2, 105.7, 94.2, 88.2, 55.2; HRMS
calcd for C20H15O2 (MH+) 287.1072, found 287.1076.
Acknowledgment. This study was supported by NIH R21
GM078447 and the Department of Molecular Biology, The
Scripps Research Institute.
Supporting Information Available: Fluorescence spectra,
synthesis and characterization of compounds, and NMR spectra.
This material is available free of charge via the Internet at
http://pubs.acs.org.
(27) Wautelet, P.; Le Moigne, J.; Videva, V.; Turek, P. J. Org. Chem. 2003,
68, 8025.
JO900013W
2424 J. Org. Chem. Vol. 74, No. 6, 2009