Fluorogenic imines for fluorescent detection of Mannich-type reactions of phenols in water

Article abstract of DOI:10.1021/jo8003293

(Chemical Equation Presented) Fluorogenic imines and their precursor amines that can be used for fluorescent visualization of Mannich-type reactions of phenols in aqueous buffers have been developed. The precursor amines are aniline derivatives that are c

Full text of DOI:10.1021/jo8003293

fluorescent detection of molecules that react with the fluorogenic
molecules.1 For example, fluorogenic maleimides are nonfluo-
rescent or very weakly fluorescent until they react with thiols;
the reaction products are highly fluorescent, allowing fluorescent
detection or fluorescent visualization of thiols and thiol-
containing molecules.1a–c When intrinsically fluorescent mol-
ecules are used for bond-forming reactions, separation of product
molecules from unreacted fluorescent molecules is required
before analysis; an advantage of use of fluorogenic molecules
is that the reaction products can be analyzed without need for
a separation step.1,2 Although there are many types of fluoro-
genic molecules,1,2 fluorogenic molecules for reactions of
phenols have not been reported.4 The phenol functionality is
present in tyrosine, a building block of proteins, and in many
natural products.5 Tyrosine residues on folded proteins have
been used as modification sites.4 Therefore, fluorogenic mol-
ecules that react with phenols would be useful for screening of
catalysts and reaction conditions for the bond-forming reactions
with phenols including phenol-bearing biomolecules and for
fluorescent labeling and detection of molecules possessing
accessible phenols. Here, we report the first examples of
fluorogenic molecules for Mannich-type reactions of phenols
in aqueous buffers.
Fluorogenic Imines for Fluorescent Detection of
Mannich-Type Reactions of Phenols in Water
Hai-Ming Guo, Maki Minakawa, and Fujie Tanaka*
Department of Molecular Biology, The Scripps Research
Institute, 10550 North Torrey Pines Road,
La Jolla, California 92037
ftanaka@scripps.edu
ReceiVed February 8, 2008
Since imines and iminiums react with phenols through
Mannich-type reactions in aqueous buffers or water-containing
solvents under mild conditions,4a,b we chose imines as phenol-
Fluorogenic imines and their precursor amines that can be
used for fluorescent visualization of Mannich-type reactions
of phenols in aqueous buffers have been developed. The
precursor amines are aniline derivatives that are covalently
conjugated to fluorophores. These amines and their imine
derivatives were nonfluorescent or very weakly fluorescent.
On the other hand, addition products of the imines to phenols
showed more than 100-fold higher fluorescence than the
imines and the precursor amines.
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Fluorogenic molecules that afford fluorescent products upon
bond-forming reactions allow monitoring the progress of the
reactions by increase in fluorescence.1–3 Evaluation of chemical
transformations with these fluorogenic molecules accelerates
characterization and screening of catalysts and/or reaction
conditions.1,2 In addition, these fluorogenic molecules enable
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3964 J. Org. Chem. 2008, 73, 3964–3966
10.1021/jo8003293 CCC: $40.75 2008 American Chemical Society
Published on Web 04/23/2008

SCHEME 1. Amines, Imines, and Mannich-Type Reaction
Products with Phenols Used for the Fluorescence Analyses
reacting moieties for the design of the fluorogenic molecules.
We reasoned that combination of an imine moiety and a
fluorophore in one molecule, in which the imine group acts as
a reaction moiety and also acts a quencher that suppresses the
fluorescence of the fluorophore,1,2a,b should provide a fluoro-
genic molecule for reactions with phenol derivatives. In the
presence of water, imines are often hydrolyzed into amines and
carbonyl compounds; therefore, consideration of fluorescence
of the hydrolyzed products (i.e., amines and carbonyl com-
pounds) was also required for the design of the imine-based
fluorogenic molecules. It has been reported that an aniline amino
group intramolecularly quenches fluorophore’s fluorescence;3g,6
therefore, we reasoned that molecules possessing a fluorophore
and an aniline moiety should be useful precursors of fluorogenic
imines. It has been suggested that presence of a free-rotating
amine, which has a lone pair, of an aniline moiety is key for
fluorescence quenching.6 We hypothesized that the quenching
feature of the aminophenyl moiety should differ between the
amine/imine and the reaction product of the imine with phenol
derivatives.
To test our hypothesis, amines, imines, addition products of
phenol derivatives to the imines, and control molecules shown
in Scheme 1 were designed and synthesized and the fluorescence
of these compounds were analyzed in aqueous buffer (Table
1). Amines 1a-e possessed fluorophores, whereas amine 1f did
not and was a control. Imines 2 and 3 were prepared by mixing
amine 1 with formaldehyde or ethyl glyoxylate, respectively,
in DMSO. The resulting imine was added to the buffer before the
fluorescence measurements. Under the conditions of the fluores-
cence analysis for these imines, the corresponding amine coexisted
with the imine because of partial hydrolysis of the imine.
Addition products 4 and 5 containing fluorophores were
highly fluorescent (entries 3, 9, 13, 15, 18, 22, and 24).7 On
the other hand, amines and imines were nonfluorescent or were
very weakly fluorescent (entries 1, 2, 8, 12, 14, 17, 21, and
23). The fluorescent Mannich-type products 4 and 5 showed
more than 100-fold higher fluorescence than the corresponding
amine and imine under the conditions shown in Table 1.
Fluorescence emission spectra of 1c, 3c, and 5c are shown in
Figure 1A. Control products 4f and 5f (entries 10 and 11), which
do not include fluorophores, and phenol derivative 6 (entries 4
and 20) were not fluorescent at the wavelengths used,8 indicating
that the fluorescence of 4a-e, 5a, and 5c originates from the
fluorophore. Compounds 7a and 7c, which are also possible
addition products4a that can be formed with 4a and 4c,
respectively, were also fluorescent (entries 5 and 16).
On the other hand, compound 8 (entry 6) was not fluorescent;
the substituent on the nitrogen of the aniline moiety significantly
affected the fluorescence. A mixture of amine 1a and phenol
derivative 6 (entry 7) was not fluorescent. These results indicate
that the phenolic hydroxy group in products 4 and 5 is important
for the fluorescence. Intramolecular interaction between the
phenolic hydroxyl group and the amine (O---HN or OH---N)
or π-π stacking interactions between the aryl moieties of the
phenol group and the fluorophore2g may be required for the
fluorescence of the addition products, although other mecha-
nisms are also possible. Addition product 9, which was prepared
by the reaction between p-cresol and imine 3c, also showed
fluorescence similar to that of 5c (entry 19 versus entry 18),
suggesting that addition products of other phenol derivatives
to the fluorogenic imines should be fluorescent if another
quenching moiety is not present in the phenol derivative.
Piperidine-derived amine 10 and its addition product 11 were
both highly fluorescent (entries 25 and 26); the piperidine moiety
did not quench the fluorophore’s fluorescence. Thus, covalent
combination of an aniline moiety and a fluorophore was required
for an amine to be a fluorogenic imine precursor.
In order to demonstrate the use of the fluorogenic molecules
for the reaction with phenols, the reaction between 3c and 6
was performed in aqueous buffer and the fluorescence was
analyzed after 70 min (Figure 1B). The reaction mixture
containing imine 3c and phenol 6 showed significantly higher
fluorescence than the solution of imine 3c alone or of phenol 6
alone in the same buffer.9 These results indicate that the
(6) (a) Munkholm, C.; Parkinson, D.-R.; Walt, D. R. J. Am. Chem. Soc. 1990,
112, 2608. (b) Tanaka, K.; Miura, T.; Umezawa, N.; Urano, Y.; Kikuchi, K.;
Higuchi, T.; Nagano, T. J. Am. Chem. Soc. 2001, 123, 2530.
(7) Addition products 4a, 4c, 5a, and 5c showed fluorescence in 5% DMSO/
50 mM sodium phosphate, pH 6.0 or 8.0 and in 5% DMSO/50 mM Tris, pH 8.0
similar to the fluorescence observed in 5% DMSO/50 mM sodium phosphate,
pH 7.0.
(9) Formation of 5f was also observed in the preparative-scale reaction of
3f with 6 in the buffer.
(8) Compounds 4-6 showed fluorescence characteristic of phenols.
J. Org. Chem. Vol. 73, No. 10, 2008 3965

TABLE 1. Fluorescence of Amines, Imines, and Addition
Productsa
In summary, we have developed fluorogenic imines and their
precursor amines that can be used for fluorescent detection of
Mannich-type reactions of phenols in aqueous buffers. These
fluorogenic imines should be useful for detection of phenol-
bearing molecules and for screening of catalysts and conditions
for labeling reactions of phenol-bearing molecules. Our design
strategy for the creation of fluorogenic imines and their precursor
amines should be useful for the development of other fluorogenic
molecules for bond-forming reactions.
entry
compound
λex (nm)
λem (nm)
fluorescence intensityb
1
2
3
4
5
6
7
8
1a
2a
4a
6
7a
8
350
380
<10
<10
1.7 × 103
<10
1.7 × 103
<10
1a + 6c
3a
5a
4f
<10
<10
9
2.8 × 103
Experimental Section
10
11
12
13
<10
5f
1b
4b
<10
General Procedure for the Synthesis of 1. A mixture of (4-
tert-butoxycarbonylaminophenyl)acetic acid (5 mmol), fluorophore
amine (5 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (1.44 g, 7.5 mmol), and DMAP (5.0 mg, 0.04 mmol)
in CH2Cl2 (20 mL) was stirred at room temperature for 24 h. Usual
workup followed by purification by silica gel flash column
chromatography afforded the amide derivative. The Boc group of
this compound (3 mmol) was deprotected in trifluoroacetic acid
(TFA) (10 mL)-anhydrous CH2Cl2 (10 mL) at room temperature,
and then the solvents were removed in vacuo. Usual workup
followed by purification by silica gel flash column chromatography
afforded 1.
<10
1.8 × 103
14
15
16
17
18
19
20
1c
4c
7c
3c
5c
9
415
445
5
1.7 × 103
1.6 × 103
50d
8.0 × 103
7.0 × 103
<10
6
21
22
1d
4d
350
350
325
380
380
395
<10
2.5 × 103
23
24
1e
4e
<10
Compound 1a. 1H NMR (400 MHz, DMSO-d6) δ 10.45 (s, 1H),
7.75 (d, J ) 1.6 Hz, 1H), 7.70 (d, J ) 8.4 Hz, 1H), 7.49 (dd, J )
8.4 Hz, 1.2 Hz, 1H), 6.98 (d, J ) 8.0 Hz, 2H), 6.51 (d, J ) 8.0 Hz,
2H), 6.25 (s, 1H), 4.94 (s, 2H), 3.47 (s, 2H), 2.39 (s, 3H); 13C
NMR (100 MHz, DMSO-d6) δ 170.6, 159.9, 153.5, 152.9, 147.2,
142.5, 129.4, 125.7, 122.1, 114.9, 114.7, 113.7, 112.0, 105.3, 42.6,
17.8; HRMS calcd for C18H17N2O3 (MH+) 309.1234, found
309.1233.
1.5 × 103
25
26
10
11
1.6 × 103
1.1 × 103
a Fluorescence was recorded on a microplate spectrophotometer using
100 µL of 5 µM solution in 5% DMSO/50 mM sodium phosphate, pH
7.0. For the imine, see the text. b Relative fluorescence intensity. c 1a (5
µM) + 6 (5 µM). d The data without background correction.
General Procedure for the Synthesis of 4. Amine 1 (0.1 mmol)
was dissolved in TFA (1 mL)-anhydrous CH2Cl2 (1 mL). To this
solution were added (4-hydroxyphenyl)acetic acid methyl ester (19.9
mg, 0.12 mmol) and 37% formaldehyde solution in water (14.9
µL, 0.2 mmol), and the mixture was stirred for 24 h at room
temperature. Usual workup followed by purification by silica gel
flash column chromatography afforded 4.
Compound 4a. 1H NMR (400 MHz, CD3OD-CDCl3) δ
7.60-7.51 (m, 3H), 7.16-7.14 (m, 2H), 7.11 (d, J ) 2.4 Hz, 1H),
7.05-7.03 (m, 1H), 6.79 (d, J ) 8.4 Hz, 1H), 6.76-6.73 (m, 2H),
6.18 (d, J ) 1.2 Hz, 1H), 4.32 (s, 2H), 3.67 (s, 3H), 3.61 (s, 2H),
3.53 (s, 2H), 2.41 (d, J ) 1.2 Hz, 3H); 13C NMR (100 MHz,
CD3OD-CDCl3) δ 172.9, 171.1, 161.8, 154.7, 153.8, 152.9, 141.7,
129.9, 129.6, 129.1, 124.9, 124.8, 124.2, 124.1, 115.7, 115.6, 114.7,
112.7, 112.6, 106.8, 104.7, 51.8, 45.6, 43.3, 40.0, 18.2; HRMS calcd
for C28H27N2O6 (MH+) 487.1864, found 487.1868.
FIGURE 1. (A) Fluorescence emission spectra (λex 415 nm) of amine 1c
(circle), imine 3c (triangle), and addition product 5c (square) at 5 µM in
5% DMSO/50 mM sodium phosphate, pH 7.0. (B) Fluorescence increase
(λex 415 nm, λem 445 nm) after 70 min in 5% DMSO/50 mM sodium
phosphate buffer, pH 7.0: column 1, phenol derivative 6, 2.5 mM; column
2, imine 3c, 2.5 µM; column 3, 6, 2.5 mM and 3c, 2.5 µM.
Acknowledgment. This study was supported by NIH R21
GM078447.
Mannich product formation of the phenol with the imine was
detected by increase in fluorescence. Note that imines prepared
from formaldehyde and aniline derivatives have been used for
labeling of proteins at tyrosine.4a Therefore, fluorogenic imines
described here may be used for reactions with tyrosine residues
of peptides and proteins.
Supporting Information Available: Additional fluorescence
data, fluorescence spectra, and synthesis and characterization
of compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
JO8003293
3966 J. Org. Chem. Vol. 73, No. 10, 2008