Synthesis and characterization of model compounds for the neutral green fluorescent protein chromophore

Article abstract of DOI:10.1055/s-2007-990852

Two new derivatives of the green fluorescent protein (GFP) chromophore each containing a charged substituent (CH₂CH₂NH ₃⁺ and CH₂CH₂NMe₃ ⁺, respectively) on the imidazolone ring have been prepared. These compounds are model systems for the neutral GFP chromophore with an unprotonated phenol moiety. The ammonium groups allow for electrospray ionization and the possibility to study the optical properties of the isolated chromophores in the gas phase. A derivative containing a CH₂CH₂OH substituent arm was also prepared. Georg Thieme Verlag Stuttgart.

Full text of DOI:10.1055/s-2007-990852

PAPER
3635
Synthesis and Characterization of Model Compounds for the Neutral Green
Fluorescent Protein Chromophore
New
D
erivatives of the
i
G
reen
c
Fluorescen
h
t
P
rotein
C
h
a
romophoreel Åxman Petersen,a Peter Riber,a Lars H. Andersen,b Mogens Brøndsted Nielsen*a
a
Department of Chemistry, University of Copenhagen, Universitetsparken 5, 2100 Copenhagen Ø, Denmark
Fax +4535320212; E-mail: mbn@kiku.dk
b
Department of Physics and Astronomy, University of Aarhus, 8000 Aarhus C, Denmark
Received 22 June 2007; revised 22 August 2007
the spectator group may nevertheless perturb the optical
properties of the chromophore. Thus, we have recently in-
vestigated the strong influence which a charged spectator
group exerts on the neutral retinal Schiff base chro-
Abstract: Two new derivatives of the green fluorescent protein
(GFP) chromophore each containing
a charged substituent
+
+
(CH2CH2NH3 and CH2CH2NMe3 , respectively) on the imidazol-
one ring have been prepared. These compounds are model systems
for the neutral GFP chromophore with an unprotonated phenol moi- mophore.4 In compound 1, one of the ammonium hydro-
ety. The ammonium groups allow for electrospray ionization and
gen atoms can in fact form a hydrogen bond to the
the possibility to study the optical properties of the isolated chro-
imidazole carbonyl oxygen, which is likely to disturb the
mophores in the gas phase. A derivative containing a CH2CH2OH
optical properties. For this reason, we have designed a
substituent arm was also prepared.
new and better model system for the neutral chromophore,
Key words: amides, amines, chromophores, heterocycles, phenols
namely compound 2, in which the three ammonium hy-
drogen atoms are substituted by three methyl groups
(Figure 1). Here we present the synthesis of these two
model systems, the chloride salt 1 and the iodide salt 2,
and absorption properties in solution. Moreover, we have
prepared a model system containing CH2CH2OH as R
group.
The green fluorescent protein (GFP) is an important pho-
toactive protein that is widely employed in molecular and
cell biology owing to its fluorescent properties.1 The chro-
mophore moiety sits at the center of a b-barrel that con-
sists of 238 amino acids in a single chain. The absorption
spectrum of GFP shows two maxima, one at 479 nm
which is ascribed to the excitation of a deprotonated (phe-
nolate) chromophore and a main peak at 397 nm which
supposedly correlates to a protonated (neutral phenol)
chromophore.1 Yet, different mutants show spectral be-
havior that cannot be explained by ionization of the phe-
nol group alone suggesting other ionizable sites in the
immediate surroundings of the chromophore.2 In order to
elucidate how the absorption maxima are perturbed by the
protein environment, the gas-phase absorption properties
of the isolated chromophore in its neutral charge state
(i.e., as a phenol) was recently investigated by state-of-
the-art experiments in a heavy-ion storage ring.3 Compar-
ison between gas-phase and protein absorption data pro-
vides directly the extent to which the protein tunes the
chromophore properties, while one would have to correct
for solvent effects when comparing protein data to solu-
tion data. Compound 1 was chosen as a suitable model
system for the neutral chromophore. In order for the chro-
mophore to be transferred to the gas phase by electrospray
ionization, an ammonium group was attached as a so-
called spectator group. Thus, the chromophore moiety is
still neutral while the molecule as a whole is charged. In
the absence of a charged spectator group, the chro-
mophore moiety itself would become protonated or ion-
ized under the conditions of electrospray ionization. Yet,
HO
O
N
N
R
Me
GFP chromophore
Cl
HO
O
NH3
N
N
O
Me
1
I
HO
NMe3
N
N
Me
2
Figure 1 GFP chromophore and model compounds
Our synthesis starts from 4-[(Z)-4-acetoxybenzylidene]-
2-methyloxazol-5(4H)-one (3) (Scheme 1), which was
prepared from N-acetylglycine and 4-hydroxybenzalde-
hyde.5 Conversion of the oxazol-5(4H)-one ring into an
imidazol-5(4H)-one ring has previously been accom-
plished by treatment with an amine in the presence of
K2CO3.6 In a modified procedure, we first treated 3 with
ethylenediamine and isolated the ring-opened diamide 4.
Moreover, deacetylation had occurred under these condi-
SYNTHESIS 2007, No. 23, pp 3635–3638
x
x.
x
x
.
2
0
0
7
Advanced online publication: 29.10.2007
DOI: 10.1055/s-2007-990852; Art ID: T09707SS
© Georg Thieme Verlag Stuttgart · New York

3636
M. Å. Petersen et al.
PAPER
H2N
tions to afford the unprotected phenol group. The isolated
product contained minor impurities that could not be re-
moved at this stage as purification was limited to repeated
dissolution/precipitation steps. Formation of the imida-
zolone ring was next accomplished by treatment with
K2CO3 in refluxing ethanol. Subsequent acidification with
hydrochloric acid resulted in protonation of both the phe-
nolate and amino groups to provide the product as its chlo-
ride salt 1 (precipitates upon addition of diethyl ether),
together with traces of the E-isomer. Prolonged exposure
to acidic conditions was observed to result in an increase
of E/Z ratio. As the workup involved tedious removal of
the coprecipitated KCl, we designed another route to ob-
tain the salt 1 in pure form. Thus, we found that simply
heating compound 4 in ethanol containing molecular
sieves (4 Å) resulted in ring-closure to the imidazolone 5.
Upon treatment of this compound with HCl, the pure chlo-
ride salt 1 precipitated.
NMe2
HO
HN
NMe2
3
HN
O
O
EtOH, r.t., 15 min
82%
Me
6
K2CO3, EtOH
reflux, 22 h
46%
HO
O
MeI, CH2Cl2
56%
2
NMe2
N
N
Me
7
Scheme 2
H2N
HO
O
H2N
OH
3
OH
N
N
EtOH, MS (4 Å)
reflux, overnight
43%
AcO
O
NH2
Me
EtOH, r.t., 15 min
ca. 90%
N
O
8
3
Me
Scheme 3
NH2
HO
HN
MS (4 Å)
EtOH
Compounds 1 and 2 exhibit similar absorption maxima in
H2O, lmax = 368 and 369 nm, respectively, as well as in
MeOH, lmax = 370 and 372 nm, respectively. These val-
ues are similar to those observed for related model sys-
tems devoid of the ammonium arm,7 yet blue-shifted
relative to the value of 397 nm observed for the protein.
Moreover, we studied the absorption behavior in CH2Cl2
containing 0.4% DMSO (Figure 2). The neutral amine 5
and the ammonium salt 2 absorb at the same maximum
(lmax = 373 nm) in this medium. Addition of 1 equivalent
of HBF4 to the amine causes a slight broadening of the
spectrum. However, addition of 10 equivalents of HBF4 to
the amine causes a significant red-shift in the absorption
maximum to 405 nm as now not only the amine group but
also the imidazolone nitrogen should be protonated. Pre-
vious studies on the protonated 4-hydroxybenzylidene-
imidazolone have revealed an absorption maximum at
395 nm in CH2Cl2 and at 383 nm in DMSO.7d The ob-
served red-shift to lmax 405 nm after protonation of 5 may
reflect the influence exerted by a hydrogen bond between
the carbonyl oxygen and the ammonium group. This in-
tramolecular hydrogen bond formation is expected at least
in the gas phase.3 The protonated GFP chromophore was
previously found to absorb at 406 nm in the gas phase8 and
one would accordingly expect a red-shift from this value
for diprotonated 5 in the gas phase. Compound 2 lacks the
possibility for forming a hydrogen bond to the carbonyl
oxygen, and we expect its gas-phase absorption spectrum
to be blue-shifted relative to that of compound 1 (for
which lmax = 415 nm in the gas phase3). Gas-phase inves-
tigations are currently being undertaken. These nontrivial
experiments will allow us to elucidate spectroscopic
changes induced by forming a hydrogen bond without in-
45%
HN
O
O
reflux, overnight
Me
4
HO
O
1) K2CO3, EtOH
reflux, 22 h
2) HCl (aq)
NH2
N
N
<80%
Me
5
HCl (aq)
100%
1
Scheme 1
Next, compound 3 was treated with N,N-dimethylethyl-
enediamine to provide the ring-opened diamide 6 in which
deacetylation of the phenol has also occurred. Ring-
closure was accomplished by treatment with K2CO3 in re-
fluxing EtOH, affording the imidazolone 7, which was pu-
rified by column chromatographic workup. This
compound was then methylated at room temperature with
methyl iodide in dichloromethane. The iodide salt 2 con-
veniently precipitated in analytically pure form from the
1
solution (Scheme 2). H NMR spectroscopy confirmed
that methylation had only occurred at the amine unit.
Finally, we obtained compound 8 in a one-pot reaction by
simply refluxing compound 3 overnight with ethanol-
amine in ethanol (Scheme 3). This compound may be in-
teresting to study as phenolate ion in the gas phase in order
to investigate the influence of hydrogen bonding between
the hydroxy group and the carbonyl oxygen.
Synthesis 2007, No. 23, 3635–3638 © Thieme Stuttgart · New York

PAPER
New Derivatives of the Green Fluorescent Protein Chromophore
3637
+
HRMS-FAB: m/z [M + H]+ calcd for C13H18N3O3 : 264.1348;
found: 264.1343.
(Z)-1-(2-Aminoethyl)-4-(4-hydroxybenzylidene)-2-methyl-1H-
imidazol-5(4H)-one (5)
Compound 4 (197 mg, 0.75 mmol) was dissolved in absolute EtOH
(20 mL) and then molecular sieves (4 Å, 4 g) were added. The mix-
ture was refluxed overnight, filtered, and evaporated on Celite. Pu-
rification by dry column chromatography [EtOH in CH2Cl2,
increasing ratio from 0 to 100% in steps of 20%; elution with neat
EtOH (96%) continued until all product had eluted, Rf = 0.06] gave
pure 5 (83 mg, 45%) as a yellow powder; mp 200 °C (dec.).
IR (KBr): 3352 (m), 3287 (w), 2947 (m), 2877 (m), 2745 (m), 2682
(m), 2583 (m), 1686 (s), 1640 (s), 1599 (s), 1515 (s), 1489 (m), 1448
(s), 1405 (s), 1359 (s), 1313 (s), 1287 (s), 1262 (s), 1168 (s), 1136
(s), 1034 (m), 1011 (m), 986 (m), 886 (m), 852 (m), 837 (s), 768
(w), 731 (w), 708 (w), 662 (w), 634 (w), 609 (w), 592 (w), 540 (w),
512 (w), 493 (m), 461 (w), 416 cm–1 (w).
Figure 2 UV-Vis Absorption spectra in CH2Cl2 containing 0.4%
DMSO
1H NMR (300 MHz, DMSO-d6): d = 8.04 (d, J = 8.2 Hz, 2 H, Ar),
6.85 (s, 1 H, CH=), 6.78 (d, J = 8.2 Hz, 2 H, Ar), 3.52 (t, J = 6.5 Hz,
2 H, NCH2), 2.69 (t, J = 6.5 Hz, 2 H, NCH2), 2.37 (s, 3 H, CH3).
13C NMR (75 MHz, DMSO-d6): d = 170.0, 161.9, 160.5, 135.8,
134.1, 125.5, 124.8, 115.9, 43.3, 40.5, 15.5.
terfering solvent effects and to validate calculational
methods.
In conclusion, we have obtained several new model sys-
tems of the GFP chromophore that are suitable for gas-
phase experiments for elucidating the influence of hydro-
gen bonding on the intrinsic optical properties.
HRMS-FAB: m/z [M + H]+ calcd for C13H16N3O2 : 246.1243;
+
found: 246.1241.
(Z)-1-(2-Aminoethyl)-4-(4-hydroxybenzylidene)-2-methyl-1H-
imidazol-5(4H)-one Hydrochloride (1)
Chemicals were purchased from Aldrich and used as received. TLC
was carried out using aluminum sheets pre-coated with silica gel
60F (Merck 5554). The plates were inspected under UV light. Melt-
ing points were measured on a Reichert melting point apparatus
equipped with a microscope or on a Büchi melting point apparatus
and are uncorrected. 1H NMR (300 MHz) and 13C NMR (75 MHz)
spectra were recorded at 25 °C on a Varian instrument, using the re-
sidual solvent (DMSO; dH = 2.5; dC = 39.5) as the internal standard.
All chemical shifts are quoted on the d scale (ppm), and all coupling
constants (J) are expressed in Hz. Fast atom bombardment (FAB)
mass spectra were obtained on a Jeol JMS-HX 110 Tandem Mass
spectrometer in the positive ion mode using 3-nitrobenzyl alcohol
(NBA) as matrix or polyethylene glycol (PEG). Microanalyses were
performed at the Microanalytical Laboratory of the Department of
Chemistry, University of Copenhagen. Solution absorption spectra
were recorded on a Cary 50 (Varian Inc.) with pure solvent as base-
line.
Method 1: Compound 4 (135 mg, 0.51 mmol) was dissolved in ab-
solute EtOH (10 mL), and then K2CO3 (260 mg, 1.88 mmol) was
added. After stirring for 16 h at reflux, the mixture was subjected to
centrifugation in order to remove the K2CO3 precipitate and then
acidified by dropwise addition of aq 4 M HCl until acidic. Precipi-
tation by addition of Et2O (100 mL) followed by filtration yielded 1
as an orange solid containing also KCl (116 mg, <80%). Removal
of KCl was attempted by redissolving in absolute EtOH, but this
was unsuccessful. The compound was instead obtained pure by
Method 2.
Method 2: Compound 5 (23 mg, 0.09 mmol) was dissolved in EtOH
(10 mL), whereupon aq 4 M HCl was added until pH ~ 2 was
reached (2 drops), followed by precipitation by addition of Et2O (90
mL). The solvent was decanted off, and the precipitate was washed
with Et2O (50 mL) to give, after drying under vacuum (oil pump), 1
(27 mg, ca. 100%) as a yellow powder with traces of EtOH and E-
isomer; mp 175 °C (dec.).
(Z)-2-(Acetylamino)-N-(2-aminoethyl)-3-(4-hydroxyphe-
nyl)acrylamide (4)
IR (KBr): 3008 (s), 1745 (s), 1645 (s), 1599 (s), 1541 (s), 1514 (s),
1424 (m), 1368 (w), 1322 (w), 1285 (s), 1232 (m), 1177 (s), 1154
(s), 1032 (w), 1009 (w), 928 (w), 881 (w), 841 (m), 799 (w), 750
(w), 711 (w), 591 (w), 536 (w), 515 (w), 482 cm–1 (w).
Compound 3 (526 mg, 2.14 mmol) was dissolved in absolute EtOH
(25 mL) and then ethylenediamine (2.5 mL, 37.4 mmol) was added,
resulting in an immediate red solution. After stirring for 15 min at
r.t., the mixture was evaporated in vacuo at 30 °C. The orange-red
oil was dissolved in hot EtOH (10 mL). A solid was precipitated by
the addition of Et2O (100 mL). This procedure was repeated three
times followed by drying the solid under vacuum for 2 h. Com-
pound 4 was obtained as a yellow powder containing residual EtOH
and minor impurities (520 mg, ca. 90%); mp 130 °C (dec.).
+
1H NMR (300 MHz, DMSO-d6): d = 8.24 (br s, 3 H, NH3 ), 8.07 (d,
J = 8.2 Hz, 2 H, Ar), 7.00 (s, 1 H, CH=), 6.88 (d, J = 8.2 Hz, 2 H,
Ar), 3.89 (m, 2 H, CH2), 3.06 (m, 2 H, CH2), 2.48 (s, 3H, CH3).
13C NMR (75 MHz, DMSO-d6): d = 167.9, 163.3, 160.8, 134.5,
130.5, 127.1, 123.9, 116.1, 38.4, 37.1, 14.8.
IR (KBr): 3266 (s), 2683 (m), 2594 (m), 1656 (s), 1604 (s), 1510 (s),
1371 (s), 1274 (s), 1208 (s), 1172 (s), 1110 (m), 977 (m), 916 (m),
833 (s), 720 (m), 632 (m), 603 (m), 534 cm–1 (s).
HRMS-FAB: m/z [M – Cl]+ calcd for C13H16N3O2 : 246.1243;
found: 246.1243.
+
1H NMR (300 MHz, DMSO-d6): d = 9.27 (br s, 1 H, ArOH), 7.80
(t, J = 5.3 Hz, 1 H, NH), 7.39 (d, J = 8.4 Hz, 2 H, Ar), 6.97 (s, 1 H,
CH=), 6.76 (d, J = 8.4 Hz, 2 H, Ar), 3.51 (br s, NH2, NH, H2O), 3.12
(m, 2 H, NCH2), 2.59 (m, 2 H, NCH2), 2.00 (s, 3 H, CH3).
13C NMR (75 MHz, DMSO-d6): d = 169.6, 165.5, 158.7, 131.3,
128.2, 127.0, 124.7, 115.6, 42.3, 41.0, 23.0.
(Z)-2-(Acetylamino)-N-[2-(dimethylamino)ethyl]-3-
(4-hydroxyphenyl)acrylamide (6)
Compound 3 (2.21 g, 9.01 mmol) was dissolved in absolute EtOH
(100 mL) and then N,N-dimethylethylenediamine (16 mL, 146
mmol) was added. After stirring for 15 min at r.t., the red mixture
was evaporated in vacuo. The red oil was dissolved in EtOH (20
mL). A solid precipitated by the addition of Et2O (100 mL). The sol-
Synthesis 2007, No. 23, 3635–3638 © Thieme Stuttgart · New York

3638
M. Å. Petersen et al.
PAPER
vent was decanted off, and this procedure was performed three
times, whereupon 6 was isolated as a yellow solid (2.16 g, 82%).
This product was used in the next step without further purification;
mp 90 °C.
1H NMR (300 MHz, DMSO-d6): d = 9.26 (s, 1 H, ArOH), 7.38 (d,
J = 8.5 Hz, 2 H, Ar), 7.01 (s, 1 H, CH=), 6.77 (d, J = 8.5 Hz, 2 H,
Ar), 3.20 (q, J = 6.8 Hz, 2 H, NCH2), 2.30 (t, J = 6.8 Hz, 2 H,
NCH2), 2.15 [s, 6 H, N(CH3)2], 1.99 (s, 3 H, COCH3).
lecular sieves (10 g) were added. The mixture was refluxed over-
night, filtered, and evaporated on Celite. Purification by dry column
chromatography (MeOH in CH2Cl2, starting from 0% with 1% rise)
gave 8 as a yellow powder contaminated with N-(2-hydroxyeth-
yl)acetamide. Repeated washings with hot Et2O (2 × 100 mL) fol-
lowed by drying under vacuum (oil pump) gave pure 8 (318 mg,
43%); mp 181.5–183 °C.
IR (KBr): 3155 (s), 2969 (m), 2814 (m), 1708 (s), 1640 (s), 1599 (s),
1556 (m), 1515 (m), 1454 (s), 1415 (s), 1365 (m), 1312 (w), 1302
(m), 1285 (s), 1255 (m), 1235 (m), 1172 (s), 1144 (s), 1105 (w),
1066 (m), 1035 (w), 1019 (w), 974 (w), 922 (w), 852 (w), 834 (m),
765 (w), 723 (w), 663 (w), 633 (w), 600 (w), 535 (w), 513 (w), 486
(m), 442 cm–1 (w).
1H NMR (300 MHz, DMSO-d6): d = 10.07 (br s, 1 H, ArOH), 8.08
(d, J = 8.6 Hz, 2 H, Ar), 6.9 (s, 1 H, CH=), 6.83 (d, J = 8.6 Hz, 2 H,
Ar), 4.94 (t, J = 5.6 Hz, 1 H, OH), 3.61 (t, J = 5.3 Hz, 2 H, CH2),
3.51 (m, 2 H, CH2), 1.79 (s, 3 H, CH3).
+
HRMS-FAB: m/z [M + H]+ calcd for C15H22N3O3 : 292.1661;
found: 292.1663.
(Z)-1-[2-(Dimethylamino)ethyl]-4-(4-hydroxybenzylidene)-2-
methyl-4H-imidazol-5-one (7)
Compound 6 (2.835 g, 9.73 mmol) was dissolved in absolute EtOH
(150 mL), and then K2CO3 (4.5 g, 33 mmol) was added. After stir-
ring for 22 h at reflux, the mixture was filtered and concentrated in
vacuo to give a red oil. The residue was subjected to column chro-
matography (SiO2, 10% MeOH in CH2Cl2) to give the product as a
yellow solid (1.23 g, 46%); mp 218 °C (dec.).
13C NMR (75 MHz, DMSO-d6): d = 169.9, 162.5, 159.5, 136.2,
134.0, 125.4, 125.3, 115.7, 58.8, 42.8, 15.5.
IR (KBr): 3069 (w), 2984 (m), 2959 (m), 2873 (m), 2768 (m), 2687
(m), 2566 (m), 2482 (m), 1701 (s), 1646 (s), 1600 (s), 1516 (s), 1471
(s), 1445 (s), 1406 (s), 1359 (s), 1329 (m), 1293 (s), 1269 (s), 1189
(m), 1171 (s), 1134 (s), 1102 (m), 1060 (w), 1032 (m), 1009 (m),
946 (m), 900 (w), 855 (m), 831 (m), 799 (m), 780 (w), 768 (m), 709
(w), 657 (w), 635 (w), 611 (m), 536 (m), 509 (m), 490 (m), 470 (m),
427 cm–1 (w).
1H NMR (300 MHz, DMSO-d6): d = 10.10 (s, 1 H, ArOH), 8.07 (d,
J = 8.8 Hz, 2 H, Ar), 6.87 (s, 1 H, CH=), 6.83 (d, J = 8.8 Hz, 2 H,
Ar), 3.63 (t, J = 6.2 Hz, 2 H, NCH2), 2.39 (t, J = 6.2 Hz, 2 H,
NCH2), 2.36 (s, 3 H, =CCH3), 2.17 [s, 6 H, N(CH3)2].
+
HRMS-FAB: m/z [M + H]+ calcd for C13H14N2O3 : 247.1083;
found: 247.1095.
Acknowledgment
The Danish Research Agency (grant # 2111-04-0018) is acknow-
ledged for financial support.
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+
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found: 274.1548.
(Z)-N,N,N-Trimethyl-2-[4-(4-hydroxybenzylidene)-2-methyl-
1H-imidazol-5(4H)-one-1-yl]ethylammonium Iodide (2)
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IR (KBr): 3094 (s), 3012 (s), 1690 (s), 1651 (s), 1603 (s), 1580 (s),
1568 (m), 1513 (s), 1485 (m), 1476 (m), 1438 (m), 1401 (s), 1365
(m), 1344 (m), 1329 (m), 1311 (w), 1274 (s), 1213 (s), 1173 (s),
1159 (w), 1138 (s), 1105 (w), 1089 (w), 1032 (w), 1009 (w), 958
(m), 918 (m), 851 (w), 838 (m), 804 (w), 787 (w), 766 (w), 708 (w),
655 (w), 633 (w), 609 (m), 535 (w), 512 (w), 487 cm–1 (w).
1H NMR (300 MHz, DMSO-d6): d = 10.18 (s, 1 H, ArOH), 8.10 (d,
J = 7.5 Hz, 2 H, Ar), 6.96 (s, 1 H, CH=), 6.85 (d, J = 7.5 Hz, 2 H,
Ar), 4.06 (t, J = 7.1 Hz, 2 H, NCH2), 3.59 (t, J = 7.1 Hz, 2 H, NCH2)
+
3.18 [s, 9 H, N(CH3)3 ], 2.44 (s, 3 H, =CCH3).
13C NMR (75 MHz, DMSO-d6): d = 169.5, 160.6, 159.9, 135.2,
134.3, 126.8, 125.0, 115.8, 61.9, 52.5, 33.7, 15.4.
MS-FAB: m/z = 288 [M – I]+.
Anal. Calcd for C16H22IN3O2: C, 46.28; H, 5.34; N, 10.12. Found:
C, 46.33; H, 5.39; N, 9.97.
(Z)-1-(2-Hydroxyethyl)-4-(4-hydroxybenzylidene)-2-methyl-
1H-imidazol-5(4H)-one (8)
Compound 3 (693 mg, 2.8 mmol) was dissolved in absolute EtOH
(50 mL), whereupon ethanolamine (350 mg, 5.8 mmol) and 4 Å mo-
Synthesis 2007, No. 23, 3635–3638 © Thieme Stuttgart · New York