Synthesis and characterization of 4-ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone and its fluorescent amino acid derivatives

Article abstract of DOI:10.1016/S0040-4020(01)00332-5

4-Ethoxymethylene-2-[1]-naphthyl-5(4H)-oxazolone 1b was synthesized from 1-naphthoyl-glycine and triethyl orthoformate as a fluorescent analogue of the hapten 4-ethoxymethylene-2-phenyl-5(4H)-oxazolone. A simple and general method has been devised to prepare conjugates of type 6 between 1b and amines and amino acids by direct reaction. Compounds 6 are stable in solid form and in aqueous solution at 20°C, but are a mixture of two isomers. ¹H- and ¹³C-NMR experiments on model compounds have identified the more stable isomer. Comparative analysis of fluorescence properties of 1b and of amino acid derivatives has identified the spectral properties of two chromophores. An intense and pH-sensitive emission band centered at λ=460 nm (λ_ex=360 nm) in aqueous solution has been discovered. This feature might be useful for monitoring cellular compartments of different pH.

Full text of DOI:10.1016/S0040-4020(01)00332-5

TETRAHEDRON
Pergamon
Tetrahedron 57 42001) 4589±4598
Synthesis and characterization of 4-ethoxymethylene-2-[1]-
naphthyl-5ꢀ4H)-oxazolone and its ¯uorescent amino acid
derivativesq
a
b
c
b
a
c
 Â
Â
Â
Í
Â
Â
È
Gyorgy Koczan, Gabriella Csõk, Antal Csampai, Erika Balog, Szilvia Bosze, Pal Sohar
and Ferenc Hudecza,p
a
È
È
Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Eotvos L. University, Budapest 112, POB 32, Hungary, H-1518
bInstitute of Biophysics and Radiation Biology, Semmelweis University, Budapest, POB 263, Hungary, H-1444
È È
Department of General and Inorganic Chemistry, Eotvos L. University, Budapest 112, POB 32, Hungary, H-1518
c
Received 22 October 2000; revised 23 February 2001; accepted 15 March2001
AbstractÐ4-Ethoxymethylene-2-[1]-naphthyl-544H)-oxazolone 1b was synthesized from 1-naphthoyl-glycine and triethyl orthoformate as
a ¯uorescent analogue of the hapten 4-ethoxymethylene-2-phenyl-544H)-oxazolone. A simple and general method has been devised to
prepare conjugates of type 6 between 1b and amines and amino acids by direct reaction. Compounds 6 are stable in solid form and in aqueous
1
solution at 208C, but are a mixture of two isomers. H- and 13C-NMR experiments on model compounds have identi®ed the more stable
isomer. Comparative analysis of ¯uorescence properties of 1b and of amino acid derivatives has identi®ed the spectral properties of two
chromophores. An intense and pH-sensitive emission band centered at lˆ460 nm 4lexˆ360 nm) in aqueous solution has been discovered.
This feature might be useful for monitoring cellular compartments of different pH. q 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
4Fig. 1). The aminolysis of 1a occurs by unexpected
displacement of the ethoxy group by the amine with
elimination of ethyl alcohol to give a 4-4substituted-
methylene)-2-phenyl-544H)-oxazolone type 2a product.7±9
Compound 1a was ®rst synthesized during war-time efforts
to produce penicillin.10 The interesting chemical reactivity
of this compound and its application in various ®elds of
biological researchprompted us to syntehsize a new
In basic immunology 4-ethoxymethylene-2-phenyl-544H)-
oxazolone 1a has received a great deal of interest.1,2
Administration of 1a induces a strong T-cell response.3,4
This compound has also been used as a hapten for the
analysis of antibody recognition.5,6 It reacts withteh
amino groups of proteins as well as withprimary amines
Figure 1. The reaction of 4-ethoxymethylene-2-aryl-544H)-oxazolone with amines.
q A preliminary account of this work was read at the 4th International Symposium on Amino Acids, Vienna, Austria, 1995.
Keywords: pH sensitive ¯uorescent chromophore; 4-ethoxymethylene-2-aryl-544H)-oxazolones; amino acid conjugate of 4-ethoxymethylene-2-[1]-naphthyl-
544H)-oxazolones; alkylation of amino acid.
   Â
Corresponding author. Research Group of Peptide Chemistry, Hungarian Academy of Sciences, Pazmany P. setany 1A, Budapest, Hungary, H-1117.
p
Tel.: 136 1 372 2828; fax: 136 1 372 2620; e-mail: hudecz@szerves.chem.elte.hu
0040±4020/01/$ - see front matter q 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020401)00332-5

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4590
ethoxymethylene-aryl-oxazolone derivative possessing
¯uorescent properties. For this we have incorporated the
1-naphthyl group as the aryl substituent of the oxazolone
ring at position 4 to produce 1b. In addition we have aimed
at the synthesis of type 2b amino acid conjugates of the new
¯uorescent compound 1b by utilizing the pronounced
reactivity of the ethoxymethylene moiety towards the
amino group.
Figure 2. The synthesis of 4-ethoxymethylene-2-[1]-naphthyl-544H)-
oxazolone 1b.
In this communication we describe the synthesis of 4-ethoxy-
methylene-2-naphthyl-544H)-oxazolone, 1b. The prepara-
tion and characterization of several amine and amino acid
derivatives produced by N-alkylation are also reported. The
detailed analysis of the spectroscopic properties of 1b and
its type 6 amino acid conjugates suggest that these
compounds are ¯uorescent, possessing bene®cial spectral
characteristics. Based on these observations, we propose
the application of 1b as a new ¯uorophore for labelling
proteins and peptides and also the incorporation of type 6
amino acid conjugates into peptides by chemical synthesis.
as starting material. 1-Naphthoyl-glycine11 was synthesized
by a previously described method using different reaction
conditions 4e.g. benzene has been replaced by dioxane) and
a more detailed description is therefore provided. The
resulting compound possessed a higher and narrower
melting point range when compared to the published data.
The reaction of 1-naphthoyl-glycine with acetic anhydride
and TEOF was performed in EtOAc under re¯ux conditions.
The crystalline product 1b was obtained after 3 hwithsatis-
factory isolated yield 459%) and was found to be stable at
148C for a longer period of time 4.24 months). Similarly,
it does not decompose in cooled non-aqueous solutions
4.1 month).
2. Results and discussion
2.1. Synthesis
Aminolysis of 1b by amino acids yielded type 6 derivatives
4Fig. 3). In order to avoid oxazolone ring opening by
hydrolysis, non-aqueous reaction conditions have been
used.10 The reaction was carried out in a heterogeneous
phase; amino acid 45) was suspended in methanol and
4-Ethoxymethylene-2-[1]-naphthyl-544H)-oxazolone, like
4-ethoxymethylene-2-phenyl-544H)-oxazolone7,10 was
obtained by heating the corresponding acyl-glycine 43)
with acetic anhydride and triethyl orthoformate 4Fig. 2).
For the preparation of 1b, 1-naphthoyl-glycine was used
Figure 3. The synthesis of amino acid derivatives type 6 of 4-ethoxymethylene-2-[1]-naphthyl-544H)-oxazolone 1b.
Table 1. Characteristics of amino acid derivatives of 1b
Compound
Formula/molar mass Yield 4%) Mp 48C)
Elemental analysis 4calcd/found)
Rf 4TLC)a
Rt 4HPLC)b
Code Amino acid
C
H
N
System A System B Isomer 1 Isomer 2
6a
6b
6c
6d
6e
6f
6g
6h
6i
Gly
l-Ala
d-Ala
C16H12N2O4/296.28 81
C17H14N2O4/310.30 87
C17H14N2O4/310.30 85
207 4dec.) 64.86/64.89 4.08/4.08 9.46/9.41
0.24
0.34
0.34
0.51
0.51
0.62
0.24
0.46
0.46
0.79
0.85
0.85
0.86
0.86
0.90
0.74
0.88
0.88
0.52
0.84
0.91
8.3c
11.1pp
15.4pp
16.3pp
27.4pp
27.2pp
27.6pp
148
149
129
130
81
103
186
186
214
109
157
65.80/65.78 4.55/4.59 9.03/9.10
65.80/65.83 4.55/4.60 9.03/9.13
72.01/72.15 8.12/8.23 7.87/7.73
72.01/72.19 8.12/8.01 7.87/7.69
68.17/68.10 5.72/5.62 7.95/8.09
67.85/67.81 4.79/4.71 8.33/8.27
71.49/71.55 4.70/4.70 7.25/7.20
71.49/71.48 4.70/4.64 7.25/7.18
11.1
11.9
23.5
23.3
23.2
13.5d
24.7
24.0
29.9
40.8
37.9
l-Leu£DCHA C32H43N3O4/533.70 95
d-Leu£DCHA C32H43N3O4/533.70 54
l-Ile
l-Pro
C20H20N2O4/352.38 87
C19H16N2O4/336.34 78
C23H18N2O4/386.40 87
C23H18N2O4/386.40 81
C20H16N4O4/376.37 83
C25H19N3O4/425.44 67
C25H29N3O6/467.51 85
l-Phe
d-Phe
l-His
l-Trp
a-Boc-l-Lys
27.0pp
27.0pp
30.7p
6j
6k
6l
63.82/63.60 4.28/4.16 14.89/14.65 0.00
70.58/70.45 4.50/4.41 9.88/9.73
64.23/64.31 6.25/6.28 8.99/8.98
0.34
0.49
42.4ppp
39.5pppp
a
TLC, system A: pyridine/acetic acid/H2O/EtOAc 420:6:11:333, v/v/v/v); system B: n-butanol/acetic acid/H2O 44:1:1, v/v/v).
b
21
Ê
RP-HPLC, column: C18 ODS, 300 A, 10 mm 4150£3.9 mm I.D.); eluent A: 0.1% TFA/H2O, B: 0.1% TFA/MeCN±H2O 480:20, v/v); vˆ1 mL min
gradients: p5±60% B in 30 min; pp40±80% in 30 min; ppp5±60% in 45 min; or pppp10±100% in 40 min.
Rt value of the more stable isomer peak is printed in bold.
;
c
d
Only a single peak is detectable.

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4591
solid 1b was added. In this way amino acid conjugates could
be obtained in fairly good yields 4Table 1). The products
were readily puri®ed. Minor variations of the work-up
procedure were required in a few cases. The purity of the
compounds was checked by TLC using two solvent systems
and by analytical RP-HPLC. Most of the compounds had
sharp melting points 4Table 1). Apart from isomerization
4see below) no decomposition of compounds 6 was detected
in solid form at 2208C.
CH2Cl2. Products were obtained witha narrow melting
point range and acceptable yield 465±92%). In the HPLC
chromatogram of 2a, two peaks could be detected with
Rtˆ13.43 and 19.5 min. Similarly, compound 2c also
showed two components with Rtˆ12.85 and 18.3 min. In
both cases the ®rst peak corresponded to the more stable
isomer. The N,N-dimethylamine derivative, 2b displayed
only a single peak under identical conditions. All three
compounds are insoluble in water, but soluble in CHCl3.
These model compounds together with the l-Ala derivative
of 1b 46b) were dissolved in CDCl3 and for the structural
2.2. Analysis of the isomers
1
assignments H- and 13C-NMR spectra were measured.
The RP-HPLC analysis of compounds 6 showed two
components in accordance with the ®nding that compounds
of type 27,12 and also 2-phenyl-4-4thienylmethylene)-544H)-
oxazolones13 can exist as a mixture of geometric isomers.
Although baseline separation of the two isomers has been
achieved 4data not shown) for compounds 6, except for the
Pro derivative, HPLC attempts to isolate the components by
preparative RP-HPLC under conditions applied at RT have
failed. This might be due to fast isomerization in the acidic
aqueous solvents used as eluents.12 During storage in
organic solution the isomeric composition of compounds
of type 6 may change over time. For example in the case
of 6i the relative intensity of the two peaks in the HPLC
chromatogram measured at lˆ214 nm changed from 0.88
to 2.40 in 1 mg mL21 acetone solution at RT during 20 h.
However, no evidence was found for the isomerization of
deep-frozen solid samples.
We have constructed possible structures for the analysis of
molecules withan N-monosubstituted amino group 2a, 2c,
6b 4Fig. 4). On this basis we ®nd that two tautomers could
be considered 4types I and II). Owing to the presence of the
double bond between C-4 and C-6, tautomer type I shows
geometric isomerism 4isomers I1 and I2). Assuming a
C46)yN double bond due to hindered rotation, two
geometric isomers of type II could be drawn 4II1 and II2).
In addition, due to the free rotation about the single bond
between C-4 and C-6, bothisomers of tautomer0 type II
could adopt two rotameric forms 4II1 and II1 for the
0
trans- and, II2 and II2 for the cis-isomer). Among the
structures described in Fig. 4, I1 and II1 can be stabilized
by chelation.
NMR data are summarized in Tables 2 and 3. Assignments
of 1H- and 13C-NMR spectra for N-methyl-substituted
compound 2a indicate the presence of two components of
9:1 ratio in CDCl3 solution. This ratio changes to ca. 2:1 in
DMSO-d6. The chemical shifts of the CH3 and NH
hydrogens in the two components are 3.15 and 6.45 ppm
In order to assign the isomer components of the RP-HPLC
chromatograms, amine derivatives of 1b withsimpli®ed
structures 2a, 2b, 2c were synthesized. Compound 1b was
reacted withmethylamine, N,N-dimethylamine or aniline in
Figure 4. Isomer structures considered for assignment of 2-[1]-naphthyl-4-4alkylaminomethylene)-544H)-oxazolone derivatives.

Table 2. 1H-NMR data of compounds 1b, 2a±c and 6b in CDCl3 solution at 500 MHza
Compound
Component
CH3 d 43H)b
yCH d 41H)c
H-2 ,d 41H)
H-3 ,t 41H)
H-4 ,d 41H)
H-5 ,d 41H)
H-6 ,t 41H)
H-7 ,t 41H)
H-8 ,d 41H)
NH broad
1b
2a
1.45
3.15
3.59
3.25
3.67
±
7.37
7.31
7.32
7.17
8.20
8.18
8.15
8.17
7.47
7.52
7.94
7.94
7.92
7.91
7.84
7.88
7.52
7.54
7.62
7.60
9.32
9.35
9.40
9.35
4.43d
6.45
5.80
±
major
minor
2b
7.51
7.88
7.90
7.53
7.59
7.68
2ce
6bf
major
minor
major
minor
7.25
7.15
7.40
7.36
8.19
8.16
8.17
8.16
,7.55
7.98
7.96
7.95
7.92
,7.55
9.43
9.40
9.41
9.44
9.35
8.15
7.26g
1.64
1.67
7.88
7.89
a
Chemical shifts in ppm 4dTMSˆ0 ppm). Assignments were supported by 2D-HSC 41b, 2a, 2c and 6b) and 2D-COSY measurements 41b, 2a and 6b) and in the case of 2b by DNOE experiments.
Triplet for 1b, J4CH3, CH2): 7.3; two singlets for 2b; doublet for all other compounds J4CH3, NH) or J4CH3, CH) for 2a and 6b, respectively, for bothcomponents: 5.1 4 2a) and 7.2 46b).
J: 14.2 Hz 4major component, 2a and 6b), 8.0 Hz 4minor component, 2a and 6b); singlet for 1b and 2b.
CH2, quartet.
b
c
d
e
f
Further signals 4phenyl): H-20,60: 7.25 ,d 42H), H-30,50: 7.38 ,t 42H), H-40: 7.16 ,t 41H).
Further signals C4sp3)H ,qi 41H): 4.21 4major component), 5.37 4minor component).
Double doublet, J: 14 and 7 Hz.
g

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4593
Table 3. 13C-NMR chemical shifts 4dTMSˆ0 ppm) of compounds 1b, 2a±c and 6b at 125 MHza
Compound Component CH3 yCH C-2
C-4
C-5
C-1
C-2
C-3
C-4
C-4a C-5
C-6
C-7
C-8
C-8a
ÃOxazole ring!
ÃNaphthalene ring!
1b
2a
2b
15.7 153.2 159.5 118.1 168.7 122.2 130.4 125.2 133.7 134.3 129.6
126.9 128.5 126.6 131.0
35.9 144.7 155.4 109.9 167.8 123.1 129.2b 125.5 132.3 134.3 129.2b 126.7 127.9 126.6 130.7
40.1 143.6 153.7 108.7 170.2 123.4 128.7
47.2
125.5 131.8 134.4 129.1
126.5c 127.8 126.6c 130.7
2c
6b
±
134.5 156.3 113.1 168.2 122.9 129.4
19.6 141.5 155.3 109.8 168.0 123.1 128.7
19.1 139.5 110.8 169.7
125.3 132.6 134.2 129.1
125.3 132.2 134.2 128.9
131.9
126.8c 128.1 126.7c 130.7
126.8 128.0 126.6 130.6
major
minor
126.9
126.5 130.5
a
Assignments were supported by DEPT for 1b, 2a, 2c and 6b by 2D-HSC and in the case of 1b also by COLOC 4HMBC) measurements. Further signals, CH2
41b): 73.0, phenyl 42c), C-10: 139.8, C-20,60: 117.1, C-30,50: 130.2, C-40: 124.8; C4sp3)H 46b): 56.1 4major component), 53.4 4minor component), COOH:
173.7 4major component), 174.6 4minor component).
Overlapping lines.
Interchangeable assignments.
b
c
4major component) and 3.59 and 5.8 ppm 4minor compo-
14.2 Hz, while it is only 8.0 Hz in the minor counterpart
due to the cis-orientation of the interacting hydrogens.
nent). The large shift difference for the methyl group
40.44 ppm) is clear evidence for hindered rotation around
the C46)yN bond. The value of the NH resonance shifts
Based on these observations and on the similar set of data
p
excludes structures withchelation 4I and II1). From these
1
obtained with 2c and 6b one can assume that the II1 0-type
0
0
data one can conclude that I2, II1 , II2 and II2 are the
plausible variants for further consideration.
structure represents the predominant component of type 6
amino acid conjugates of 1b observed during HPLC
analyses.
In the case of compound 2b withteh
N,N-dimethyl
1
substituent, the H and 13C spectra correspond to a stereo-
homogeneous product 4Tables 2 and 3). All hydrogens and
carbons give only one signals. However, the two methyl
1
groups show separated H- and 13C-NMR signals, where
the intensity ratio is 1:1 and the differences in chemical
shifts are rather signi®cant 40.42 and 7.1 ppm). These data
refer to a mesomeric structure of tautomer type I 4however,
involving the predominance of a zwitterionic limiting form
IIp). In order to determine the rotameric form we measured
the NOE values resulting from saturating two methyl-group
signals. In bothexperiments responses of the ole®nic as well
as of the H-8 naphthalene hydrogens were observed, but the
ratio of the two DNOE signals were characteristically
different. Namely, the irradiation of the up®eld methyl
signal resulted in 14.2 and 1.3% intensity enhancements
of the yCHZN and H-8 signals, while saturating the down-
®eld counterpart of the above N-methyl signal led to 5.9%
and 5.2% responses. This means that the rotation around the
C-6ZN bond is hindered, but possible. In the solid phase,
however the oxo-form is predominant as proven by high n
CyO IR frequency 41750 cm21) measured in a KBr disc.
The similar 1H-NMR shift differences for the methyl signals
in the isomers of 2a and 2b might indicate similar structures.
Considering the strong steric interaction between the
N-methyl and hydroxy groups in structure II2 4Fig. 4), the
two methyl signals of compound 2a could be assigned to
p
p
p
0
II1 0 and II2 0 structures, where the major component is II1
.
The predominance of II1 could be explained by steric
p
0
hindrance between the oxazole ring and the N-methyl
p
group in the II2 isomer. In accordance withthis proposal
0
the chemical shift of the N-methyl hydrogens is higher in the
p
minor component 4II2 0): the anisotropic neighbouring effect
of the oxazole ring N results in the down®eld shift of the
N4CH3) signal. Another observation also underlines the
relevance of the above supposition. We found that the
vicinal CH±NH coupling for the major component
involving the trans-arrangement of these hydrogens is
Figure 5. Absorption 4a) and normalized emission and excitation 4b) spectra
of 1b in ethanol 4cˆ6.6£1025 M) recorded at RT. Corresponding emission
and excitation wavelengths are indicated in the ®gure.

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4594
2.3. UV and ¯uorescence spectroscopic characterization
A typical absorption spectrum of 1b is presented in Fig.
54a). The highest molar extinction coef®cient,
3.8£103 dm3 mol21 cm21 can be measured at 338 nm in
ethanol. When this solution of 1b is excited at the absorption
maximum, light emission can be recorded between 350 and
UV and ¯uorescent spectroscopic properties of 1b as well as
compounds of type 6 were studied and the results are
summarized in Figs. 5±7 and Table 4.
Figure 6. 4a) Normalized emission and excitation spectra of compound 6b recorded at RT in 0.14 M phosphate buffer, pHˆ7.4. Corresponding emission and
excitation wavelengths are indicated in the ®gure. 4b) Excitation anisotropy spectrum 4dashed line) and normalized excitation spectrum 4solid line) of
compound 6b 4lemˆ465 nm) at RT in 0.14 M phosphate buffer, pHˆ7.4. Dotted lines represent the components of multiple Gaussian ®tting.
Figure 7. The pH dependence of emission spectra 4lexˆ360 nm) of compound 6b in 0.1 M phosphate buffer.

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4595
Table 4. Characteristic spectroscopic properties of amino acid derivatives of 1b
Compound
Code
Absorption spectrum
max 4nm)
Fluorescence spectrum
a
Amino acid
l
1 4dm3 mol21 cm21
)
l
ex 4nm)
lem 4nm)a,c
6a
6b
6c
6d
6e
6f
6g
6h
6i
Gly
l-Ala
d-Ala
l-Leu£DCHA
d-Leu£DCHA
l-Ile
l-Pro
l-Phe
d-Phe
l-His
363.5
364.6
363.1
368.9
369.1
364.8
372.4
366.7
366.8
366.1b
370.9
365.0
17700
16700
12400
19200
19800
13200
18000
18400
17700
n.d.d
380
352
395
364
372
352
376
356
356
368
356
357
472
472
490
469
466
472
463
472
472
478b
466
447
6j
6k
6l
l-Trp
a-Boc-l-Lys
21470
17300
a
Solvent: methanol, 0.01 mg mL21
.
c
b
d
Exciting at lex
.
Solvent: acetonitrile, 0.01 mg mL21
No data.
.
600 nm [Fig. 54b)]. The maxima of these broad emission
bands are at 398 and 420 nm; furthermore two shoulders
can be recognized around 380 and 460 nm. When the
sample is excited at 338 nm, the relative quantum yield of
¯uorescence emission is 0.1. The lifetime of the 1b excited
state can be estimated as 2.1^0.3 ns when lexˆ338 nm and
lemˆ400 nm.
The absorption maxima and corresponding extinction
coef®cients of Na-amino acid derivatives 46a±l) are listed
in Table 4. The lifetime of the 6 excited state can be
estimated as shorter than 1 ns when lexˆ338 nm and
lemˆ400 nm. The emission and excitation maxima of
compounds of type 6 are presented in Table 4. Fig. 64a)
shows the ¯uorescence spectra of derivative 6b in 0.14 M
phosphate buffer, pH 7.4. Here and also in the other Na -
amino acid derivatives the two emission and excitation
bands can be clearly recognized. The more intense emission
band is centred at lˆ460 nm 4lexˆ360 nm) and a weaker
band can be detected at lˆ400 nm 4lexˆ285 nm). In order
to distinguishteh excitation spectra of two different
electronic transitions the excitation spectra of compound
6b was deconvoluted by multiple Gaussian ®tting. The
result is presented in Fig. 64b). The best ®t can be received
as a sum of two Gaussian components, the maxima of these
components show a very good agreement with the two
excitation maxima of 6b [see Fig. 64a)].
The structure of the emission spectrum of 1b obtained at
lexˆ338 nm could be indicative for more than one
electronic transition. In order to elucidate this problem, a
series of emission and excitation spectra were recorded at
different excitation and emission wavelengths, respectively.
When the wavelength of the observation was varied
between 350 and 430 nm, excitation spectra of similar
structure were recorded witha maximum centred at
330 nm. As an example the excitation spectrum belonging
to lemˆ420 nm is presented in Fig. 54b). This spectrum is
slightly blue-shifted as compared to the absorption spectrum
of the same compound. The obvious difference between the
excitation and absorption spectra indicates the presence of
two different electronic transitions 4namely two chromo-
phores). If the observation wavelength is shifted towards
wavelengths longer than lˆ430 nm, the excitation
spectrum changes gradually with a redshift in its maximum
from lˆ330 nm to 360 nm. In Fig. 54b) the excitation
spectrum belonging to lemˆ520 nm is presented as an
example. When the sample is excited at lˆ360 nm or
longer wavelengths, the maximum of the emission spectrum
appears at lˆ465 nm.
The short ¯uorescence lifetime of compound 6b makes
possible the measurement of its excitation anisotropy
spectrum even in diluted solution 40.14 M, pH 7.4). In the
anisotropy spectrum [Fig. 64b)] two wavelengthranges can
be distinguished. Between lˆ250 nm and 320 nm negative
anisotropy 420.05 at 285 nm) was observed, while above
lˆ320 nm positive anisotropy 40.12 at 350 nm) could be
detected. These two wavelength regions ®t well to the
recorded [Fig. 64a)] and calculated excitation bands
[Fig. 64b)]. The result of mathematical spectral analysis
and the excitation anisotropy spectrum con®rms that the
two electron transitions belong to two different chromo-
phores.
These data show that, both in the emission and excitation
spectra of 1b, two overlapping bands can be clearly
distinguished. The maxima of the excitation spectra are
around 330 and 360 nm and the corresponding emission
maxima are at lˆ400 and 465 nm. From the separation of
the excitation and emission bands observed we can conclude
that the sample emits light as a consequence of at least two
electronic transitions. However, the amplitudes of the two
emission bands are different 4data not shown). Under similar
conditions the emitted intensity is higher at lˆ400 nm
4lexˆ330 nm) than at lˆ465 nm 4lexˆ360 nm).
Based on the observations described above it cannot be
excluded that the two electron transitions belong to the
two isomers. To investigate this possibility further spectro-
scopic studies were performed at different temperatures
between 158C and 508C. As mentioned before we found
that the ratio of the isomer composition changed with
temperature during the HPLC analysis. If the two electron
transitions belong to two geometric isomers, the relative

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4596
probability and consequently the relative amplitude of the
emission and excitation maxima have to be dependent on
the temperature. The structures of the emission and
excitation spectra of compound 1b and compound 6b
were always similar to the spectra presented in Figs. 54b)
and 64a), regardless of the temperature. From these results
we can conclude that the isomers of molecule 1b bothhave
two independent electronic transitions 4chromophores).
EtOAc 420:6:11:333, v/v/v/v) 4system A), n-butanol/acetic
acid/H2O 44:1:1, v/v/v) 4system B), chloroform 4system C)
and n-hexane/EtOAc 43:1, v/v) 4system D). Spots were
detected by I2, Cl2/toluidine or by UV and/or ¯uorescence
light. RP-HPLC analyses were carried out on a Waters
4Milford, MA, USA) HPLC system using a C18 ODS
column 43.9£150 mm; packed withspherical 10 mm silica
Ê
with300 A pore size). The gradient elution was developed
using 0.1% TFA in water as eluent A and 0.1% TFA in
acetonitrile/waterˆ80/20 v/v as eluent B. During the
analysis the content of eluent B was between 5% and
60%, in 30 min 4system I) or 40±80% in 30 min 4system
II). The ¯ow rate was 1 mL min21 at RT. The samples were
dissolved in eluent B and 20±50 mL of solution was
injected. Peaks were detected l1ˆ214 nm and
l2ˆ360 nm. FAB-MS spectra were obtained on a VG-
ZA-2SEQ spectrometer 4Fisons, UK), and elemental
analyses 4C, H, N) were performed on a Hereaus CHN-O
Rapid 4Hereaus Gmbh, Germany) apparatus. IR spectra
were recorded on a Specord IR Spectrophotometer 4Zeiss,
Germany). The 1H- and 13C-NMR spectra were recorded in
CDCl3 or DMSO-d6 solution in 5 mm tubes at RT, on a
Bruker DRX 500 spectrometer at 500.13 41H) and 125. 76
413C) MHz, using the deuterium signal of the solvent as the
lock and TMS as internal standard. The standard Bruker
microprogram NOEMULT.AU was used to generate
NOE. DEPT spectra were run in a standard manner, using
only the Qˆ1358 pulse to separate CH/CH3 and CH2 lines
phased `up' and `down', respectively. The 2D-HSC spectra
were obtained by using the standard Bruker pulse program
HXCO.AU. UV±Vis and ¯uorescence spectra were
routinely registered on a Perkin±Elmer Lambda 2S spectro-
photometer and on a Hitachi F-4500 spectro¯uorimeter,
respectively.
The emission spectra of compound 6b were recorded also in
0.1 M phosphate buffer solutions of various pH values. As
presented in Fig. 7, the amplitude of the emission band
centred at lˆ465 nm varies according to the pH value of
the solution. The pH-sensitivity was especially marked
above pHˆ10. These changes in the emission intensity
might be correlated with the ionization of the carboxy
group of the alanine moiety. At pH 2.2 it could be fully
protonated, while at higher pH values this group is anionic.
However, pronounced changes observed above pH 10 are
indicative of deprotonation of the oxazole ring nitrogen at
high pH or of a more complex set of interactions occuring
on elevation of the pH. In contrast, the light intensity
detected at 400 nm 4lexˆ285 nm) was independent of the
solvent pH 4data not shown). The pH dependence of the
main electron transition of the Na -amino acid conjugate
indicates that this molecule or other amino acid derivatives
of 4-ethoxymethylene-2-[1]-naphthyl-544H) oxazolone can
be useful indicators of the pH of their environment.
3. Conclusion
A simple and general method has been devised to prepare
conjugates between the oxazolone 1b and amine and amino
acid by direct reaction withteh corresponding amino
component. An ef®cient synthesis of 1b is also reported.
Physico-chemical as well as spectroscopic properties of
1b and its amino acid derivatives indicate that these
compounds could be useful for labelling proteins,
glycoproteins or peptides witha new ¯uorophore possessing
intense and pH-sensitive emission characteristics. The
pH-sensitive feature of the chromophore should be
emphasized and could be advantageous to monitor intra-
cellular movement of compounds in cellular compartments
withdifferent pH values. 1b could be introduced directly to
biomolecules possessing a free amino group, while the
amino acid derivatives±according to preliminary
experiments±could be utilized as building blocks for
solid-phase peptide synthesis.14 In addition, the new,
hapten-analogue amino acids might be investigated in
down-regulation of contact sensitivity.15
4.1.1. Preparation of 1-naphthoyl-glycine ꢀ3). Seventy-
®ve grams 41.0 mol) glycine was dissolved in 1000 mL of
1.0 M NaOH solution 41.0 mol), and 500 mL dioxane was
added. From separate dropping funnels 151 mL 4191 g,
1.0 mol) 1-naphthoyl-chloride and 500 mL 2.0 M NaOH
solution 41.0 mol) were added in 3 hat the same rate using
ef®cient mechanical stirring. The stirring was continued at
RT for a further 4 h. Dioxane was removed in vacuo, and the
resulting solution was extracted 5 times with100 mL of
EtOAc. The aqueous phase was acidi®ed with 5.0 M HCl
to pH 2. The precipitated product was ®ltered off,
washed with water and dried. This can be used for the synth-
esis of 1b without further puri®cation, or can be recrystal-
lized from water. Mp: 1528C [148±1498C11]. Yield: 195g
485%).11
4.1.2. Synthesis of 4-ethoxymethylene-2-[1]-naphthyl-
5ꢀ4H)-oxazolone ꢀ1b). A solution of 1-naphthoyl-glycine
416.4 g, 71.6 mmol), acetic anhydride 467.3 mL,
716 mmol) and triethyl orthoformate 435.7 mL, 215 mmol)
in EtOAc 4100 mL) was re¯uxed vigorously for 3 h. The
product was precipitated with n-hexane 4200 mL) and kept
at 48C in a refrigerator overnight. The crystalline crude
product 1b was ®ltered off, dried in vacuo and recrystallized
twice from MeOH to give 11.3 g 442.3 mmol, 59%) of pale
yellow crystals. Mp: 131±1328C; TLC: Rfˆ0.80 4system C).
MS 4FAB): m/zˆ268.0 4MH1, 62), 254.0414), 171.0454),
155.0480), 127.0443), 93.14100). Anal. calculated for
4. Experimental
4.1. General
Amino acids and solvents were obtained from Reanal,
Budapest, Hungary, while other reagents were from Aldrich
and were used without further puri®cation. TLC analyses
were performed on Merck 5553 Kiesgel 60, 0.2 mm silica
plates 4Darmstadt, Germany). For the development the
following eluents were used: pyridine/acetic acid/H2O/

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4597
1
C16H13NO3: C 71.90, H, 4.90, N 5.24. Found C 71.89, H,
4.92, N 5.24. NMR data are presented in Tables 2 and 3.
II). For H- and 13C-NMR data see Tables 2 and 3. Anal.
calculated C20H14N2O2: C 76.42, H, 4.49, N 8.91. Found C
76.36, H, 4.44, N 8.85.
4.1.3. Synthesis and puri®cation of amino acid
derivatives of 1b ꢀ6a±6k). General procedure. 4-Ethoxy-
methylene-2-[1]-naphthyl-544H)-oxazolone 1b 40.5 g,
1.87 mmol) and the corresponding amino acid 45)
41.87 mmol) were suspended in MeOH 420 mL) with
vigorous stirring. The reaction proceeded at RT for 24 h.
Work-up procedures 4A±D) started withremoval of
MeOH in vacuo and continued as outlined below.
Compounds 6d and 6e were isolated as dicyclohexylamine
salts. Procedure A: product was triturated and washed with
4.2. Singlet state studies
Ground-state absorption spectra were recorded by use of a
Cary 4E spectrophotometer. Molar absorption coef®cients
41) were obtained from the slope of the linear part of Beer±
Lambert plots of absorbance versus concentration.
Corrected steady-state emission and excitation spectra
were obtained using an FS900CD spectro¯uorimeter
4EdinburghAnalytical Instruments, UK) withXe lamp
excitation and Hammamatsu photomultiplier 4R955)
detection. Emitted ¯uorescence intensity was measured at
an angle of 908 relative to the exciting light. Resolution of
monochromators was 0.5 nm. The ¯uorescence anisotropy
was de®ned as
water 420 mL) 4l-Ile, l-Trp), withEt O 4l-Pro) or with
2
Et2O and water 4Gly, l-Phe, d-Phe, His), ®ltered off and
dried in vacuo. Procedure B: product was dissolved in
Et2O 420 mL), precipitated with n-hexane, ®ltered off,
dried, suspended in water, ®ltered off, washed with water
4l-Ala, d-Ala). Procedure C: product was dissolved in
Et2O 420 mL), cooled withice, DCHA 4375 mL,
1.87 mmol) was added, the precipitated product was ®ltered
off, washed with Et2O 4l-Leu, d-Leu). Procedure D:
product was recrystallized from 70% methanol±water.
After isolation products were dried over P2O5 in vacuo.
All products are yellowishand are crystalline. Yields,
melting point and Rf values are summarized in Table 1.
Chemical characteristics of compounds 6a±l are presented
in Table 1. For NMR data of compound 6b see Tables 2
and 3.
IVV 2 GIVH
A ˆ
ꢀ1†
IVV 1 2GIVH
where IVV and IVH are observed intensities measured with
emission polarizers parallel and perpendicular to the
vertically polarized exciting beam, respectively. G is a
wavelength-dependent factor used to correct for the
inability of the instrument to transmit differently polarized
light equally. For the ¯uorescence lifetime 4tF) measure-
ments the same instrument was used with a hydrogen-®lled
¯ash-lamp excitation with 1.5 ns pulse width and emission
decays were determined by a single-photon counting
method. Air-saturated samples were excited at the
corresponding excitation maxima at RT. During the
absorption and ¯uorescence measurements the samples
were kept at RT. The sample concentration was 1025 M.
If the absorbance of the sample exceeded 0.05 the observed
¯uorescence intensity was corrected for the inner ®lter effect
of the solution. The ¯uorescence quantum yields of
4.1.4. Synthesis of 4-ꢀmethylaminomethylene)-2-[1]-
naphthyl-5ꢀ4H)-oxazolone ꢀ2a) and 4-ꢀdi-methylamino-
methylene)-2-[1]-naphthyl-5ꢀ4H)-oxazolone ꢀ2b). 1b
40.5g, 1.87 mmol) was dissolved in CH2Cl2 410 mL) and
0.3 M methylamine in CH2Cl2 solution 42a, 31 mL) or
0.3 M dimethylamine in CH2Cl2 solution 42b, 31 mL) was
added. The mixture was stirred at RT for 4 h. Volatile
components were removed in vacuo and the product was
precipitated withwater 420 mL). After ®ltration and
washing with water 43£5 mL) and MeOH 41£5 mL) the
product was dried over P2O5 in vacuo.
S
derivatives 4FF ) in different media were estimated as
ꢀ
ꢁ2
nS
nR
FS AR
´
FSF ˆ
´
´FRF
ꢀ2†
2a: yieldˆ65%, mp 152±1538C, Rfˆ0.88 4system B),
Rtˆ13.4 min 4isomer 1) and 19.5 min 4isomer 2) 4system
AS FR
1
II). For H- and 13C-NMR data see Tables 2 and 3. Anal.
where FFR is the quantum yield of rhodamine B in ethylene
glycol as reference compound,16,17 A is the absorbance at the
wavelengthof the excitation, F is the integrated area of
emission spectra, n is the refractive index of the solution,
the 4S) superscript refers to the sample and 4R) to the
reference. The integrated ¯uorescence of the sample was
compared to that of the standard after adjusting concen-
trations so that OD values of the sample and reference at
the excitation wavelength were equal and smaller than 0.05.
calculated for C15H12N2O2: C 71.42, H, 4.79, N 11.10.
Found C 71.46, H, 4.76, N 11.18.
2b: yieldˆ92%, mp 173±1748C, Rfˆ0.82 4system B),
1
Rtˆ19.4 min 4system II). For H- and 13C-NMR data see
Tables 2 and 3. Anal. calculated for C16H14N2O2: C 72.16,
H, 5,30, N 10.52. Found C 72.21, H, 5.22, N 10.55.
4.1.5. Synthesis of 2-[1]-naphthyl-4-ꢀphenylaminomethy-
lene)-5ꢀ4H)-oxazolone ꢀ2c). 1b 473 mg, 0.27 mmol) was
dissolved in a mixture of MeOH 41 mL) and CH2Cl2
41 mL). Freshly vacuum-distilled aniline 4100 mL,
1.1 mmol) was added to the solution. The mixture was
stirred at RT for 18 h. Volatile compounds were removed
in vacuo and the product was dried over P2O5.
Acknowledgements
These studies were supported by grants from the Hungarian
Ministry of Education 4FKFP 1191/1997 [GCs, EB]) and
from the Hungarian Research Fund 4OTKA) No.
T-021120, No. T-03838.
Yieldˆ90%, mp 172±1738C, Rfˆ0.51 4system D),
Rtˆ12.9 min 4isomer 1) and 18.3 min 4isomer 2) 4system

 Â
G. Koczan et al. / Tetrahedron 57 42001) 4589±4598
4598
10. Clarke, H. T.; Johnson, J. R.; Robinson, R. In The Chemistry of
Penicillin; Princeton University Press: Princeton, NJ, 1949; pp
803±817.
References
1. Gell, P. G. H.; Harington, C. R.; Rivers, R. P. Br. J. Exp.
Pathol. 1946, 27, 267±286.
11. Krasovitskii, B. M.; Lysova, I. V.; Afanasiadi, L. Sh. Chem.
Heterocycl. Comp. 4Engl. Transl.) 1980, 16, 701±703.
12. Singh, K. K.; Singh, M. K.; Singh, R. M. Ind. J. Chem. 1996,
35B, 920±922.
È
È
2. Makela, O.; Kaartinen, M.; Pelkonen, J. L. T.; Karjalainen, K.
J. Exp. Med. 1978, 148, 1644±1660.
3. Phillips, J.; Artsaenko, O.; Fiedler, U.; Horstmann, C.; Mock,
H. P.; MuÈntz, K.; Conrad, U. EMBO J. 1997, 16, 4489±4496.
4. Haworth, D.; Rees, A.; Alcock, P. J.; Wood, L. J.; Dutta, A. S.;
Gormley, J. J.; Jones, H. B.; Jamieson, A.; Reilly, C. F. Br. J.
Pharmacol. 1999, 126, 1751±1760.
13. Cativiela, C.; Diaz De Villegas, M. D.; Mayoral, J. A.;
Melendez, E. Synthesis 1983, 899±902.
Í
Â
Â
14. Bosze, Sz.; Koczan, Gy.; Csik, G.; Falus, A.; Hudecz, F. A
new ¯uorophore for peptide labelling: synthesis and analysis
of its interaction withinterleukin-6 receptor. In Innovation
and Perspectives in Solid Phase Synthesis and Combinatorial
Libraries, 1998; Epton, R., Ed.; May¯ower: Birmingham,
1999; pp 189±192.
5. Mullink, H.; Boorsma, D. M.; Klein, J. C.; Oostendorp, R.;
Henzen-Logmans, S. C.; Scheper, R. J. J. Immunol. Methods
1987, 99, 199±204.
6. Holm, L.; Laaksonen, L.; Kaartinen, M.; Teeri, T. T.;
Knowles, J. K. C. Prot. Eng. 1990, 3, 403±409.
15. Satoh, T.; Yokozeki, H.; Katayama, I.; Nishioka, K. Int. Arch.
Allergy Immunol. 1993, 102, 94±100.
7. Benoiton, N. L.; Hudecz, F.; Chen, F. F. M. F. Int. J. Peptide
Protein Res. 1995, 45, 266±271.
Â
8. Hudecz, F.; Kajtar, J.; Szekerke, M. Biophys. Chem. 1988, 31,
16. Cantor, Ch. R.; Schimmel, P. R. Biophysical Chemistry, II;
Freeman: San Francisco, 1980.
17. Demas, J. N.; Crosby, G. A. J. Phys. Chem. 1971, 75, 991±
1024.
53±61.
Â
Â
9. Rajnavolgyi, E.; Lanyi, A.; Hudecz, F.; Kurucz, I.; Kiss, K.;
Â
È
Laszlo, G.; Szekerke, M.; Gergely, J. J. Mol. Immunol. 1989,
Â
26, 949±958.