Fluorophores related to the green fluorescent protein

Article abstract of DOI:10.1016/j.tetlet.2004.06.072

Imidazolin-5-one derivatives and isosteres (oxazolinones, butenolides, and pyrrolinones) of the 4-hydroxybenzylidene-imidazolinone chromophore of the GFP have been synthesized and their photophysical properties have been investigated.

Full text of DOI:10.1016/j.tetlet.2004.06.072

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 6343–6348
Fluorophores related to the green fluorescent protein
Maryline Bourotte,a Martine Schmitt,a Anny Follenius-Wund,b Claire Pigault,b
Jacques Haiechb and Jean-Jacques Bourguignona,*
aLaboratoire de Pharmacochimie de la Communication Cellulaire, 74 route du Rhin, F-67400 Illkirch, France
bLaboratoire de Pharmacologie et Physico-chimie des Interactions Cellulaires et Molꢀeculaires,
74 route du Rhin, F-67400 Illkirch, France
Received 22 March 2004; revised 3 June 2004; accepted 16 June 2004
Abstract—Imidazolin-5-one derivatives and isosteres (oxazolinones, butenolides, and pyrrolinones) of the 4-hydroxybenzylidene-
imidazolinone chromophore of the GFP have been synthesized and their photophysical properties have been investigated.
ꢀ 2004 Elsevier Ltd. All rights reserved.
The green fluorescent protein (GFP) from the jellyfish
Aequorea victoria has found many applications in
molecular biology, because the chromophore is an
integral part of the protein sequence. Consequently,
in situ expression of GFP results in an intrinsically
fluorescent protein that can be fused to another protein
and used to report on protein expression and transport
within cells.1–3 Wild-type GFP autocatalytically gener-
ates the 4-hydroxybenzylidene-imidazolinone chromo-
phore, shown in Figure 1, via a posttranslational
internal cyclization of the Ser65-Tyr66-Gly67 tripeptide
followed by 1,2-dehydrogenation of the tyrosine.4;5 The
absorption spectrum of wild-type GFP contains two
main bands at about 395 and 475 nm, and these are
usually assigned to the neutral and the anionic forms of
the chromophore, respectively. The emission spectrum
shows an intense fluorescence emission band, centered at
508 nm with a high quantum yield (U ¼ 0:79).6 It is now
well known that when it is isolated from the protein, the
chromophore totally loses its fluorescence.4;6 Optimiza-
tion of the fluorescence properties of the chromophore
might lead to efficient fluorescent dyes. In a previous
paper7 we described the photophysical properties of
imidazolinone derivatives with significant fluorescence
quantum yields. The aim of this work is the synthesis
and the study of the spectroscopic properties of some
hydrophobic imidazolin-5-ones (compounds 1, R2 ¼ Ar)
and their isosteres (oxazolinones 2, pyrrolinones 3, and
butenolides 4), when compared with the parent chro-
mophore illustrated in Figure 1.
The oxazolinones 2 were synthesized from aroylglycine8
and aromatic aldehydes in sodium acetate–acetic anhy-
dride mixture, whereas butenolides 4 were synthesized
H
O
O
N
R
N
N
O
Ser-Tyr-Gly
N
N
H
N
R2
HO
HO
HO
O
GFP chromophore
Figure 1. Structural formula of the wild-type GFP chromophore and its derivatives.
Keywords: Green fluorescent protein; Imidazolinones; Oxazolinones; Butenolides; Pyrrolinones; Fluorescence.
* Corresponding author. Tel.: +33-3-90-24-42-20; fax: +33-3-90-24-43-10; e-mail: jjb@pharma.u-strasbg.fr
0040-4039/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2004.06.072

6344
M. Bourotte et al. / Tetrahedron Letters 45 (2004) 6343–6348
(NOESY experiments16). It is noteworthy that in all
cases (compounds 1–4 and 6), only one signal could be
observed for this proton, supporting the existence of
only one single structure for these compounds, (ii) spe-
cific deshielding effects could be clearly identified on the
styryl proton, as the result of the presence of oxygen
(X ¼ O) in both the oxazolone and butenolide series
(compare 2 and 1, 4 and 3 in Fig. 2, D ¼ þ0:31 and
þ0.50 ppm, respectively). Similar deshielding effects
were found by N-substitution at the pyrrolinone ring
(compare 3 and 6, D ¼ þ0:30 ppm). These data highlight
modulation of the oxygen carbonyl electronegativity,
which may account for direct effects on Hs deshielding
(electronic delocalization), or indirect effects (aniso-
tropic field), (iii) within the series of butenolides 4,
introducing a second electron-withdrawing group in Ar2
(compounds 4g and 4h) dramatically enhanced the
deshielding of the styryl proton (d ¼ 7:98 ppm). It sup-
ports a strong electronic delocalization in these systems,
as illustrated by the highest value of the extinction
coefficient at the absorption maximum found for the
butenolide 4g (e ¼ 38,300 Mꢀ1 cmꢀ1).
Table 1. Synthesis of compounds 6 with R ¼ i-Bu
Compds
R1
R2
Method A Method B
yield (%)
yield (%)
6b
6g
6h
6i
CN
H
10
28
23
20
30
a
a
CO2Me
CO2Me
CN
4-NO2
4-CN
4-NHAc
H
a
62
a
6j
6k
NO2
NO2
67
76
4-OMe
a: not performed.
from b-aroylpropionic acids9 with aromatic aldehydes in
sodium acetate–acetic anhydride mixture. Imidazolin-5-
ones derivatives were prepared as described in our pre-
vious papers.7;10 The butenolides 4 were allowed to react
with ammonium acetate in concentrated ammonia to
yield the corresponding pyrrolinones 3 (30–90%).11 N-
substitution, besides specific electronic effects, signifi-
cantly increased their solubility in aprotic solvents
(CHCl3, dioxane). When heating the butenolides with
primary amines (e.g., isobutylamine), the N-substituted
pyrrolinones 6 were obtained in low yields ( 630%,
Method A12, Table 1). However a second step of heating
the intermediate 5 in acetic acid was needed to afford
dehydro compounds 6. Using a microwave apparatus
(Scheme 1, Method B13), the compounds could be ob-
tained with better yields after recrystallization in ethanol
(62–76%).
As all the compounds had a low solubility and were
nearly not fluorescent in water, the photophysical
properties of all the compounds (concentrations of
approximately 3 lM) were determined in 1,4-dioxane at
20 ꢁC. This solvent is an aprotic solvent with a very
small dielectric constant (n ¼ 2:2 at 25 ꢁC), commonly
referred to as a good simulator of the hydrophobic
environment of a chromophore buried inside a protein
matrix. Figure 3 shows the absorbance and emission
spectra of compounds 1–4e, which are representative of
the four series of derivatives. Imidazolin-5-ones 1 and
oxazolinones 2 have structured absorption spectra,
characterized by a main absorption peak with a shoul-
der. The spectra of pyrrolinones 3 and butenolides 4 are
not structured at all, and are largely red shifted. They
also show greatly increased widths at half maximum
(Dk).
Within each class of analogues 1–4, the aryl unsubsti-
tuted derivatives 1a–4a were considered as reference
compounds. Monosubstituted (R1 ¼ H, referred to d or
R2 ¼ H, referred to b, c, j), or disubstituted derivatives
(e, f, g, h, i, k) were studied (Table 2). They bear either
electron-withdrawing groups (CN, CO2Me, NO2) or
donor groups (OMe, NHAc) in R2, mainly in para
position. Taking into account our previous results with
the imidazolone series,7 only electron-withdrawing
groups were introduced on the Ar1 aromatic ring to
increase the fluorescence emission properties.
The detailed photophysical properties of all the com-
pounds are listed in Table 2. The extinction coefficient e
of the compounds was determined at the absorption
maximum and the fluorescence quantum yield / was
determined as described in our previous paper.7 Steady-
state absorption spectra were recorded on a Cary IV
spectrophotometer. Steady-state fluorescence spectra
were obtained on an SLM 48000 spectrofluorometer,
1
The H NMR data of the H styryl proton (Hs) pointed
out the following comments: (i) as the corresponding
imidazolones14 and oxazolones15 are proved to be E-
isomers (Ar1 and CO in trans position), we focused
attention on both pyrrolinones and butenolides for
which a similar configuration was ascertained by NMR
O
O
O
Hs
Ha
O
N
N
a
b
OH
R1
R1
R1
5b, 5g-k
4b, 4g-k
6b, 6g-k
R2
R2
R2
Scheme 1. Synthesis of N-substituted pyrrolinones under microwaves. Reagents and conditions: (a) i-BuNH2 1.2 equiv, lx (NORMATRONR 112
reactor), acetonitrile, 10 min; (b) AcOH, lx, 5 min.

M. Bourotte et al. / Tetrahedron Letters 45 (2004) 6343–6348
Table 2. Photophysical data of compounds 1, 2, 3, 4, and 6
6345
Compd
Class
R
R1
R2
Mp (lit.) (ꢁC)a e ꢂ 10ꢀ3
kambasx=kem
(nm)
/
e ꢂ / ꢂ 10ꢀ3
max
(Mꢀ1 cmꢀ1
)
(Mꢀ1 cmꢀ1
)
1a
1b
1c
1d
1e
1f
Imidazolones
H
H
H
H
H
H
H
H
CN
H
265 (274)b
285
266
13.1
384/444
393/464
394/461
390/455
405/482
403/478
405/510
0.001
0. 02
3.92
4.58
0.78
5.01
9.49
6.53
H
24.8
28.8
23.0
26.5
32.2
25.3
0.158
0.159
0.034
0.189
0.295
0.258
CO2Me
H
CN
H
3,4-DiOMe
3,4-DiOMe
3,4-DiOMe
4-NO2
234
285
CO2Me
CO2Me
274
304
1g
2a
2b
2c
2d
2e
2f
Oxazolones
––
––
––
––
––
––
––
H
CN
H
171 (168)b
287b
32.0
34.2
41.6
37.1
38.1
40.0
33.0
363/447
369/426
370/nfc
378/447
393/489
391/474
389/461
0.002
0.028
0
0.06
0.96
0
H
CO2Me
H
CN
H
195b
3,4-DiOMe
3,4-DiOMe
3,4-DiOMe
4-NHAc
165 (168)b
249
0.020
0.421
0.348
0.540
0.74
16.0
13.9
17.8
CO2Me
CN
208
244
2i
3a
3b
6b
3c
3d
3e
3f
Pyrrolinones
H
H
CN
H
222 (226)b
225
140
14.6
18.9
10.6
15.2
18.6
19.1
19.0
14.4
13.0
19.9
13.0
12.7
12.5
422/539
438/569
440/598
437/544
431/542
457/585
454/574
442/594
438/589
456/584
450/606
456/601
465/616
0.012
0.294
0.153
0.100
0.108
0.188
0.363
0.245
0.192
0.260
0.218
0.033
0.018
0.17
5.55
1.62
1.52
2.01
3.59
6.89
3.53
2.49
5.17
2.83
0.42
0.22
H
H
i-Bu
H
CN
H
CO2Me
H
CN
H
229
232
H
3,4-DiOMe
3,4-DiOMe
3,4-DiOMe
4-NO2
4-CN
H
301
271
H
CO2Me
CO2Me
CO2Me
CN
6g
6h
3i
i-Bu
i-Bu
H
155
147
4-NHAc
4-NHAc
H
281
150
6i
i-Bu
i-Bu
i-Bu
CN
6j
6k
NO2
NO2
145
159
4-OMe
4a
4b
4c
4d
4e
4f
Butenolides
––
––
––
––
––
––
––
––
––
––
––
H
CN
H
153 (155)b
274
182
23.4
30.1
24.8
31.0
31.1
31.1
38.3
34.7
31.2
22.3
30.3
442/493
400/494
401/495
406/495
428/531
423/522
411/493
404/501
424/521
416/518
438/550
0.007
0.027
0.007
0.004
0.450
0.355
0.002
0.002
0.437
0.083
0.043
0.16
0.81
0.17
0.12
14.0
11.0
0.08
0.07
13.6
1.86
1.30
H
CO2Me
H
CN
H
3,4-DiOMe
3,4-DiOMe
3,4-DiOMe
4-NO2
4-CN
149 (147)b
233
191
CO2Me
CO2Me
CO2Me
CN
4g
4h
4i
295
277
4-NHAc
H
4-OMe
285
290 (292)b
276
4j
4k
NO2
NO2
a The microanalyses were in satisfactory agreement with calculated values.
b Compounds described in the literature.
c Nonfluorescent.
with 4 nm excitation and emission bandwidths. The
fluorescence quantum yields were determined at 20 ꢁC
from the area under the fluorescence spectra, taking as
reference a solution of quinine sulfate in 0.1 M H2SO4
(/QS ¼ 0:51 at this pH)17 or fluorescein in 0.1 M NaOH
(/F ¼ 0:95)18 of the same absorbance at the excitation
wavelength.
When considering data for the third class of isosteric
compounds (pyrrolinones 3 and 6), they support the
following comments: (i) a similar but less pronounced
increase in e by aromatic substitution in R2, (ii) additive
abs
max
bathochromic effects on k
induced by each aromatic
substituent (compare 3a with 3e, 3f, 3i, Dk ꢁ þ30 nm, or
with 3b, 3c, 3d, Dk ꢁ þ15 nm), (iii) N-substitution by an
em
i-Bu group caused the strongest bathochromic effect on
A first analysis focused on the imidazolones 1. When
compared with the nonsubstituted imidazolone 1a,
introducing a substituent in para position of one (1b, 1c,
1d), or both aromatic rings (1e, 1f, 1g) significantly in-
creased simultaneously the extinction coefficient e, the
k
(ꢁ600 nm), but significantly decreased the quantum
max
yield (compare 6b and 3b, 6i and 3i, respectively), (iv) all
the substitutions at the phenyl rings dramatically
increased the quantum yield, with the dimethoxy
derivative 3f as the most potent compound within this
series.
kambasx, kem (bathochromic effects), and more dramatically
max
the quantum yield (300 times). The most important
substitution effect was found with the dimethoxy phenyl
derivative 1f bearing an electron-withdrawing group on
the second aromatic ring (R1 ¼ CO2Me).
The oxazolone series (NH fi O isosteric replacement)
showed high values of e (more than 30,000 Mꢀ1 cmꢀ1).
However the compound 2c, with the highest e value, was

6346
M. Bourotte et al. / Tetrahedron Letters 45 (2004) 6343–6348
O
O
NH
NH
O
N
N
R1
R1
1
3
R2
R2
O
O
O
R1
R1
2
4
R2
R2
class I
class II
Hs
O
Hs
O
Ar1
X
Ar1
X
N
Ar2
Ar2
Imidazolones
Oxazolones
Pyrrolinones
Butenolides
1
2
3
6
4
X
NH
O
NH
N-iBu
O
Hs
(ppm)
7.02 0.12
7.33 0.08
7.15 0.05
7.45 0.07
7.65 0.07
Figure 2. Derivatives (imidazolin-5-ones 1) and isosteres (oxazolinones 2, pyrrolinones 3, and butenolides 4) of the 4-hydroxybenzylidene-imid-
azolinone chromophore.
bearing two instead of one electron-withdrawing groups
in both Ar1 and Ar2 (compounds 4g and 4h,
e > 34; 000 Mꢀ1 cmꢀ1), in relation with existing highly
delocalized p-conjugated systems, and illustrated by
highly deshielded styryl protons. However, due to their
very low fluorescence quantum yields (/ ¼ 0:002), this
type of compounds was no more investigated.
The chromophore isolated by enzymatic digestion of
GFP lost totally its emissive properties.6 In addition
simpler imidazolinones in which tyrosine vicinal a-
aminoacids have been replaced by alkyl chains
(R ¼ R2 ¼ alkyl, see Fig. 1)6;19 were not fluorescent at
room temperature (/ < 0:0001). Only few papers deal
with optimization of luminescence properties of some
oxazolones and imidazolones.20;21 These works showed
some beneficial effects of electron-withdrawing group in
part R1, but nothing has been reported on part R2 of
these compounds.
Figure 3. Room-temperature steady-state absorption spectra (solid)
and fluorescence emission spectra (dashed) of the compounds 1e, 2e,
3e, and 4e (ꢃ3 lM) in dioxane. The absorption and fluorescence
spectra were normalized to approximately the same maximum ampli-
tude.
When considering the most promising compounds
within each series, a common type of substitution of
aromatics was found for 1e–4e, 1f–4f, and 2i–4i high-
lighting the combined beneficial effects of an electron
donor group in Ar2 (R2 ¼ OMe, NHAc) and an elec-
tron-withdrawing group in Ar1 (R1 ¼ CN, CO2Me,
NO2). Among them was selected 4e as a good compro-
mise between high values of e (31,000 Mꢀ1 cmꢀ1) and
emission of fluorescence at 531 nm with a high fluores-
cence quantum yield of 0.45. N-substituted pyrrolinones
6i–j seemed to be promising, because of the important
red shift observed with emission fluorescence at more
not fluorescent. When considering fluorescence quan-
tum yield values within this series, 2e, 2f, and 2i were
selected as the most interesting compounds.
Similar features were found in the butenolide series
highlighting the most promising compounds 4e, 4f, and
4i (increased e and / values). In addition, a profound
increase in the e value was observed with butenolides

M. Bourotte et al. / Tetrahedron Letters 45 (2004) 6343–6348
6347
4.20; found C% 72.46, H% 4.40, N% 4.25. 4f: mp 191 ꢁC.
1H RMN (DMSO, 300 MHz) d 3.87 (s, 3H, OCH3); 3.90
(s, 3H, OCH3); 3.91 (s, 3H, CO2CH3); 7.15 (d, J ¼ 8:8,
1H, ArH); 7.37 (s, 1H, Ha); 7.4–7.55 (m, 2H, ArH); 7.58
(s, 1H, Hs); 8.00 (d, J ¼ 8:6, 2H, ArH); 8.07 (d, J ¼ 8:6,
2H, ArH). Anal. Calcd C% 68.85, H% 4.95, O% 26.20;
than 600 nm, but disappointing concerning the value of e
(about 13,000 Mꢀ1 cmꢀ1).
In conclusion, we synthesized and investigated new
interesting structural analogues of the GFP chromo-
phore with good fluorescence quantum yields in a
1
found C% 68.51, H% 4.96, O% 26.41. 4i: mp 285 ꢁC. H
hydrophobic environment (/ > 0:24), and large range of
RMN (DMSO, 200 MHz) d 2.11 (s, 3H, CH3); 7.39 (s, 1H,
Ha); 7.58 (s, 1H, Hs); 7.76 (d, J ¼ 8:7, 2H, ArH); 7.88 (d,
J ¼ 8:6, 2H, ArH); 7.95 (d, J ¼ 8:7, 2H, ArH); 8.05 (d,
J ¼ 8:6, 2H, ArH); 10.28 (s, 1H, NHAc). Anal. Calcd with
0.9H2O C% 69.31, H% 4.60, N% 8.09; found C% 69.27,
H% 4.58, N% 8.19.
em
max
emission wavelengths (461 < k
< 594 nm). Most
promising compounds were found in both oxazolone
(2e, 2f, 2i) and butenolide (4e, 4f, 4i) series. Some of
them may constitute a good starting point for building
hydrophobic fluorescent markers of biological materi-
als. Several derivatives may allow very efficient fluores-
cence resonance energy transfer (FRET) and will be
useful for a wide panel of biosensor applications.
10. Oumouch, S.; Bourotte, M.; Schmitt, M.; Bourguignon, J.
J. Synthesis, submitted for publication.
11. General procedure for compounds 3: Compound 4e
(150 mg, 0.6 mmol), dry ammonium acetate (56 mg,
0.72 mmol), and 30% ammonia solution (94 lL,
0.72 mmol) were suspended in MeOH (3 mL). The mixture
was placed in a sealed tube, stirred, and heated to 90 ꢁC
for 5 h. The solution was evaporated to dryness and
CH2Cl2 (5 mL) was added. The product 3e was filtered and
washed with water. Yield 73%. Mp 301 ꢁC. 1H NMR
(DMSO, 200 MHz) d 3.85 (s, 3H, OCH3); 3.88 (s, 3H,
OCH3); 6.85 (s, 1H, Ha); 7.0–7.2 (m, 2H, Hs and ArH);
7.4–7.7 (m, 2H, ArH); 7.90 (d, J ¼ 8:4, 2H, ArH); 7.99 (d,
J ¼ 8:5, 2H, ArH); 10.65 (s, 1H, NH ech./D2O). Anal.
Calcd with 0.2H2O C% 71.50, H% 4.92, N% 8.34; found
C% 71.48, H% 4.74, N% 8.43. 3f: mp 271 ꢁC. 1H NMR
(DMSO, 200 MHz) d 3.81 (s, 3H, OCH3); 3.84 (s, 3H,
OCH3); 3.87 (s, 3H, CO2CH3); 6.82 (s, 1H, Ha); 7.0–7.2
(m, 2H, Hs and ArH); 7.4–7.6 (m, 2H, ArH); 7.91 (d,
J ¼ 8:3, 2H, ArH); 8.00 (d, J ¼ 8:3, 2H, ArH); 10.59 (s,
1H, NH ech./D2O). Anal. Calcd with 0.4H2O C% 67.70,
H% 5.36, N% 3.76; found C% 67.56, H% 5.28, N% 3.90.
3i: mp 281 ꢁC. 1H NMR (DMSO, 200 MHz) d 2.07 (s, 3H,
CH3); 6.83 (s, 1H, Ha); 7.10 (s, 1H, Hs); 7.67 (d, J ¼ 8:8,
2H, ArH); 7.83 (d, J ¼ 8:8, 2H, ArH); 7.86 (d, J ¼ 8:4,
2H, ArH); 7.96 (d, J ¼ 8:4, 2H, ArH); 10.17 (s, 1H, NH
ech./D2O); 10.59 (s, 1H, NH ech./D2O). Anal. Calcd with
0.6H2O C% 70.62, H% 4.80, N% 12.36; found C% 70.66,
H% 4.64, N% 12.44.
Acknowledgement
We thank Cyril Antheaume for carrying out 2D NMR
experiments.
References and notes
1. Tsien, R. Y. Annu. Rev. Biochem. 1998, 67, 509–544.
2. Zimmer, M. Chem. Rev. 2002, 102, 759–781.
3. Chalfie, M.; Tu, Y.; Euskirchen, G.; Ward, W. W.;
Prasher, D. C. Science 1994, 263, 802–805.
4. Shimomura, O. FEBS Lett. 1979, 104, 220–222.
5. Heim, R.; Prasher, D. C.; Tsien, R. Y. Proc. Natl. Acad.
Sci. 1994, 91, 12501–12504.
6. Niwa, H.; Inouye, S.; Hirano, T.; Matsuno, T.; Kojima,
S.; Kubota, M.; Ohashi, M.; Tsuji, F. I. Proc. Natl. Acad.
Sci. U.S.A. 1996, 93, 13617–13622.
7. Follenius-Wund, A.; Bourotte, M.; Schmitt, M.; Iyice, F.;
Lami, H.; Bourguignon, J. J.; Haiech, J.; Pigault, C.
Biophys. J. 2003, 85, 1839–1850.
12. Method A: Compound 4g (500 mg, 1.83 mmol) was
suspended in CH3CN (5 mL) and 0.184 mL of isobutyl-
amine was added. The mixture was placed in a sealed tube
and heated to 90–100 ꢁC during 4 h. The solution was
evaporated to dryness under reduced pressure and the
residue was washed with Et2O. The intermediate 5g was
8. Rao, Y. S.; Filler, R. Synthesis 1975, 749–764, Analytical
data for representative compounds of oxazolinones class:
2e: mp 249 ꢁC. 1H RMN (DMSO, 300 MHz) d 3.89 (s, 6H,
2OCH3); 7.22 (d, J ¼ 8:4, 1H, ArH); 7.30 (s, 1H, Hs); 7.58
(s, 1H, ArH); 7.79 (d, J ¼ 8:4, 1H, ArH); 7.96 (d, J ¼ 8:1,
2H, ArH); 8.43 (d, J ¼ 8:4, 2H, ArH). Anal. Calcd C%
68.26, H% 4.22, N% 8.38; found C% 68.30, H% 4.22, N%
1
obtained as a white powder. H NMR (CDCl3, 300 MHz)
d 0.79 (d, J ¼ 6:5, 3H, CH2CH(CH3)2); 0.82 (d, J ¼ 6:5,
3H, CH2CHC(CH3)2); 1.71 (m, 1H, CH2CHC(CH3)2);
2.74 (dd, JHAHc ¼ 8:1 and JHAHB ¼ 13:7, 1H, CHAHB-
CHC(CH3)2); 3.29 (dd, JHBHC ¼ 7:5 and JHAHB ¼ 13:7, 1H,
CHAHBCHC(CH3)2); 3.48 (d, J ¼ 2:2, 2H, CH2 pyrrole);
3.55 (s, 1H, OH); 3.92 (s, 3H, CO2CH3); 7.43 (d, J ¼ 8:4,
2H, ArH); 7.52 (m, 1H, Hs); 7.43 (d, J ¼ 9, 2H, ArH);
7.99 (d, J ¼ 8:4, 2H, Ar2); 8.25 (d, J ¼ 9, 2H, ArH). MS
(ES): 447 (MþNa).
1
8.48. 2f: mp 208 ꢁC. H RMN (DMSO, 200 MHz) d 3.96
(s, 3H, CH3); 4.00 (s, 3H, OCH3); 4.01 (s, 3H, OCH3); 7.01
(d, Jo ¼ 8:6, 1H, ArH); 7.18 (s, 1H, Hs); 7.65 (d, Jm ¼ 2,
1H, ArH); 7.86 (dd, Jo ¼ 8:6 and Jm ¼ 2, 1H, ArH); 8.13
(d, J ¼ 8:6, 2H, ArH); 8.25 (d, J ¼ 8:6, 2H, ArH). Anal.
Calcd C% 65.39, H% 4.66, N% 3.81; found C% 65.30, H%
4.76, N% 3.79. 2i: mp 244 ꢁC. 1H RMN (DMSO,
200 MHz) d 2.10 (s, 3H, NHCOCH3); 7.29 (s, 1H, CH–
Ph); 7.82 (d, J ¼ 9, 2H, ArH); 7.93 (d, J ¼ 8:5, 2H, ArH);
8.07 (d, J ¼ 8:8, 2H, ArH); 8.42 (d, J ¼ 8:3, 2H, ArH);
10.43 (s, 1H, NH). Anal. Calcd with 0.3H2O C% 67.77,
H% 4.07, N% 12.48; found C% 67.73, H% 4.04, N% 12.50.
9. Schueler, F. W.; Hanna, C. J. Am. Chem. Soc. 1951, 73,
3528–3530, Analytical data for representative compounds
of butenolide class: 4e: mp 233 ꢁC. 1H RMN (DMSO,
300 MHz) d 3.84 (s, 3H, OCH3); 3.87 (s, 3H, OCH3); 7.11
(d, J ¼ 8:2, 1H, ArH); 7.33 (s, 1H, Ha); 7.4–7.5 (m, 2H,
ArH); 7.56 (s, 1H, Hs); 7.94 (d, J ¼ 8:3, 2H, ArH); 7.94 (d,
J ¼ 8:3, 2H, ArH). Anal. Calcd C% 72.06, H% 4.54, N%
The intermediate 5g was placed in a sealed tube and
AcOH (3 mL) was added. The mixture was heated at 90–
100 ꢁC for 4 h. After evaporation, the red powder 6g was
washed with EtOH. Yield 28%. Mp 155 ꢁC. 1H
NMR (CDCl3, 200 MHz) d 0.75 (d, J ¼ 6:6, 6H, CH2-
CH(CH3)2); 1.62 (m, 1H, CH2CH(CH3)2); 3.57 (d,
J ¼ 7:6, 1H, CH2CH(CH3)2); 3.94 (s, 3H, CO2CH3);
6.31 (s, 1H, Ha); 7.56 (s, 1H, Hs); 7.6–7.8 (m, 4H, ArH);
8.09 (d, J ¼ 8:4, 2H, ArH); 8.25 (m, 2H, ArH). Anal.
Calcd with 0.3H2O C% 67.08, H% 5.53, N% 6.80; found
C% 67.03, H% 5.55, N% 6.74.

6348
M. Bourotte et al. / Tetrahedron Letters 45 (2004) 6343–6348
13. Method B: Compound 4j (500 mg, 1.83 mmol) was sus-
pended in CH3CN (5 mL) and 0.184 mL of isobutylamine
was added. The mixture was placed in a sealed tube,
stirred, and submitted to microwave irradiation (ꢁ300 W)
until dissolution (ꢁ10 min). The solution was evaporated
to dryness under reduced pressure and AcOH (3 mL) was
added. Then the solution was submitted to microwave
irradiation (ꢁ300 W) for 5 min. After concentration,
EtOH was added and the red powder 6j was filtered and
between Hs and Ar1 whereas no correlation has been
observed between Ha and Hs.
O
Hs
X
Ar1
R1
Ha
Ar2
1
recrystallized in EtOH. Yield 67%. Mp 145 ꢁC. H NMR
R2
(CDCl3, 200 MHz)
d
0.75 (d, J ¼ 6:6, 6H,
17. Brannon, J. H.; Magde, D. J. Phys. Chem. 1978, 82, 705–
709.
CH2CH(CH3)2); 1.64 (m, 1H, CH2CH(CH3)2); 3.56 (d,
J ¼ 7:6, 1H, CH2CH(CH3)2); 6.15 (s, 1H, Ha); 7.43 (s, 1H,
Hs); 7.4–7.6 (m, 4H, ArH); 7.77 (d, J ¼ 9, 2H, ArH); 8.27
(d, J ¼ 9, 2H, ArH). Anal. Calcd C% 72.40, H% 5.79, N%
8.04; found C% 72.52, H% 5.95, N% 8.13.
18. Velapoldi, R. A.; Mielenz, K. D. NBS Special Publication:
Standard Reference Materials; 1980; pp 260–264.
19. Kojima, S.; Ohkawa, H.; Hirano, T.; Maki, S.; Niwa, H.;
Ohashi, M.; Inouye, S.; Tsuji, F. I. Tetrahedron Lett. 1998,
39, 5239–5242.
14. Dalla Croce, P.; La Rosa, C. J. Chem. Res. 1985, 360–
361
20. Krasovitskii, B. M.; Afanasiadi, L. Sh.; Lysova, I. V.;
Stryukov, M. B.; Lyubarskaya, A. E. Russ. J. Phys. Chem.
1982, 56, 1519–1522.
21. You, Y.; He, Y.; Burrows, P. E.; Forrest, S. R.; Petasis, N.
A.; Thompson, M. E. Adv. Mater. 2000, 12, 1678–1681.
15. Prokof’ev, E. P.; Karpeiskaya, E. I. Tetrahedron Lett.
1979, 8, 737–740.
16. Structures of compounds 3, 4, and 6 have been unambig-
uously assigned by NOESY experiments: strong correla-
tions have been observed between Ha and Ar1 and