Synthesis of extended chromophores derived from 5,5-dimethyloxyluciferin by barton-kellogg methodology

Article abstract of DOI:10.1055/s-0033-1338599

An approach to the synthesis of modified methyl ether-protected dimethyloxyluciferin derivatives is presented, focusing on the enlargement of the π-system. This was achieved by introducing an alkene bridge at the 4-position of the thiazoline moiety to lin

Full text of DOI:10.1055/s-0033-1338599

1114▌
PAPER
paper
Synthesis of Extended Chromophores Derived From 5,5-Dimethyloxyluciferin
by Barton–Kellogg Methodology
Extended Oxyluciferin-Based Chromophores
Hendryk Würfel,a Luiz Francisco Monteiro Leite Ciscato,b Dieter Weiß,a Helmar Görls,c Rainer Beckert*a
a
Institut für Organische und Makromolekulare Chemie, Friedrich-Schiller Universität, Humboldtstr. 10, 07743 Jena, Germany
Fax +49(3641)948212; E-mail: rainer.beckert@uni-jena.de
b
Universidade Federal do ABC, Centro de Ciências Naturais e Humanas, Rua Santa Adélia, 166 Santo André, SP 09210-170, Brazil
c
Institut für Anorganische und Analytische Chemie, Friedrich-Schiller Universität, Humboldtstr. 9, 07743 Jena, Germany
Received: 18.11.2013; Accepted after revision: 29.01.2014
and quinoline to replace the benzothiazole moiety of lucif-
Abstract: An approach to the synthesis of modified methyl ether-
erin.5b All those compounds show light emission with a
protected dimethyloxyluciferin derivatives is presented, focusing
broad range of quantum yields when incubated with fire-
fly luciferase (EC 1.13.12.7). A different approach to
on the enlargement of the π-system. This was achieved by introduc-
ing an alkene bridge at the 4-position of the thiazoline moiety to link
the two chromophoric substructures. The resulting derivatives show modifying similar bioluminescent systems involves the
strong absorbing properties over a wide range of the visible spec-
trum. Also, the fluorescence properties of these novel dimethyl-
oxyluciferin derivatives are unique and might lead to new
mutation of luciferase enzymes from the firefly and relat-
ed organisms.6
One oxyluciferin representative that has been well inves-
tigated is 2-(6-hydroxy-1,3-benzothiazol-2-yl)-5,5-di-
methyl-1,3-thiazol-4-(5H)-one (5a; Scheme 1), which can
act as an inhibitor of the luciferin–luciferase biolumines-
cent system.7,8 More interestingly, this compound shows a
bright greenish-yellow fluorescence in its neutral pheno-
lic form 5a, but when the phenolic moiety is deprotonated
(phenolate 5b), the chromophore emits light in the red re-
gion of the visible spectrum.7 Phenol 5a itself is a potent
chromophore as a result of the partially double-bond char-
acter of the C2–C2′ bond connecting the two heterocyclic
moieties; this leads to an almost planar structure for the
molecule, permitting efficient light absorption.9
photophysical studies and bioanalytical applications.
Key words: chromophores, heterocycles, Barton–Kellogg reac-
tion, fluorophores, luciferins
Bioluminescent molecules, especially those of the class
derived from the firefly Photinus pyralis (firefly lucifer-
ins, known also as just ‘luciferins’), are of great interest in
relation to biochemical research1 and to medical applica-
tions,2 especially in the field of highly sensitive bioanalyt-
ics. A wide variety of well-studied derivatives have been
prepared that contain the parent firefly luciferin moiety as
a core.3 McElroy and co-workers synthesized a series of
several derivatives (Figure 1) with many structural varia-
tions, including D,L-homoluciferin (1), methylluciferin
(2), 6′-aminoluciferin (3), and 5′-chloroluciferin (4)
among others, and they tested them as inhibitors or sub-
strates for firefly luciferase.4
N
S
S
N
N
S
S
N
B
BH+
O
HO
O
O
5b
5a
blue-green fluorescence (λmax~ 500 nm)
red fluorescence (λmax~635 nm)
Scheme 1 Phenol and phenolate form of dimethyloxyluciferin 5
S
N
N
S
N
S
S
N
HO
HO
CO2H
To the best of our knowledge, there are no reports of stud-
ies on structural modifications of dimethyloxyluciferin
that have examined the effects of enlarging the conjugated
π-system at the 4-position of the thiazoline moiety on the
chromophoric and fluorophoric properties of the mole-
cule. One report deals with the olefination of 5,5-dimeth-
ylluciferin but, unfortunately, no analytic data are given
for any of the synthesized compounds.10 We could, there-
fore, draw no correlations between our work and any work
reported in the literature.
CO2H
2
1
Cl
N
S
S
N
S
S
N
N
H2N
CO2H
CO2H
3
4
Figure 1 Luciferin derivatives synthesized by McElroy and co-
workers4
McCutcheon et al. prepared a series of benzoimidazole-
and imidazole-containing luciferin analogues,5a and fur-
ther research has been conducted by using naphthalene
We have carried out some related research on 2-substitut-
ed oxazoline-4-thiones as precursors in a Barton–Kellogg
olefination.11 These potent fluorophoric materials were
later used to determine the rate constants of the chemi-
excitation step in the chemiluminescence of peroxyoxa-
late.12
SYNTHESIS 2014, 46, 1114–1120
Advanced online publication: 25.02.2014
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DOI: 10.1055/s-0033-1338599; Art ID: ST-2013-T0727-OP
© Georg Thieme Verlag Stuttgart · New York

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Extended Oxyluciferin-Based Chromophores
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The thiazoline ring of 2-(6-methoxy-1,3-benzothiazol-2- These results prompted us to consider a different olefina-
yl)-5,5-dimethyl-1,3-thiazol-4(5H)-one (6), the methyl- tion strategy. The need to transform the carbonyl group of
protected derivative of 5a, was the starting point for our ether 6 into a more reactive derivate led us to the Barton–
investigations on the enlargement of the conjugated π-sys- Kellogg reaction.16 This olefination method (Scheme 4)
tem. Various approaches for transforming the carbonyl employs a thiocarbonyl compound I, that reacts by 1,3-di-
group at the 4-position of the thiazoline ring were investi- polar cycloaddition with a diazo derivate II to form an un-
gated. More-direct approaches using the McMurry, stable 1,3,4-thiadiazole III. After extrusion of nitrogen
Knoevenagel, and Horner–Wadsworth–Emmons method- and the formation of a thiirane IV, the resulting interme-
ologies failed (Scheme 2). When the McMurry reaction diate can be desulfurized by using triphenylphosphine or
was performed by using titanium tetrachloride and zinc copper powder to give the corresponding olefin V in high
powder as a reducing agent13 and adamantan-2-one (7) as yield.17
the ketone coupling partner, the reaction led to the isola-
tion of symmetric adamantylideneadamantane as the only
identifiable product in the reaction mixture. The desired
product 8 (R = adamantylidene) was not obtained.
N
N
N
N
N
N
R1
R1
R2
R2
S
S
R2
R2
R2
R2
R1
R1
III
IIa
IIb
I
When we attempted a Horner–Wadsworth–Emmons
olefination14 with phosphonate 9 and lithium diisopropyl-
amide as the base in tetrahydrofuran as solvent, monitor-
ing by thin-layer chromatography (TLC) showed no
consumption of the starting material; this was confirmed
by the recovery of ether 6 and by NMR spectroscopic
analysis. An attempted Knoevenagel condensation with
diethyl malonate (10) in basic methanol gave similar re-
sults.
– N2
R1, R2 = aliphatic, aromatic
heat
Ph3P or Cu
R1
R1
R2
R2
R1 R2
R1
R2
– S=PPh3 or
– CuS
S
V
IV
Scheme 4 Barton–Kellogg methodology employing a thiocarbonyl
compound I and a diazo compound II to obtain an olefin
The low reactivity of the 4-thiazoline carbonyl group to-
wards nucleophiles prompted us to use stronger nucleo-
philes, such as organomagnesium compounds.15 Ether 6
did indeed react when the Grignard reagent isopropyl-
magnesium bromide (11) was added to a solution of the
compound in tetrahydrofuran, but the nucleophilic
Grignard species did not add at the expected 4-position of
the thiazoline ring, but instead reacted at the 2-position to
give a racemic mixture of thiazolidines 12 (Scheme 3).
The formation of these unexpected structures was con-
firmed by spectroscopy and by x-ray crystallography.15
The reaction of ether 6 with Lawesson’s reagent [2,4-
bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-
disulfide] in refluxing xylene gave 2-(6-methoxy-1,3-
benzothiazol-2-yl)-5,5-dimethyl-1,3-thiazole-4(5H)-thi-
one (13) as a dark-yellow crystalline solid in yields of up
to 65%. This transformation led to a strong downfield
shift of approximately 41 ppm in the 13C NMR signal of
the ring carbon in the 4-position of the thiazoline. No re-
arrangement reaction of the type described by Koltai et. al
for 2,5,5-triaryl-2-thiazolin-4-ones was observed.18
OEt
O
P
OEt
9
THF, LDA
O
7
N
S
S
N
N
S
S
N
THF, TiCl4, Zn
MeO
EtO
OEt
O
MeO
R
6
8
O
O
10
MeOH, KOH
Scheme 2 Various attempted approaches to olefination of methyl ether 6
1. THF, Et2O
2. H+, H2O
N
Br
N
S
S
N
Mg
NH
O
S
MeO
S
MeO
O
12
6
11
Scheme 3 A Grignard reaction of methyl ether 6 leads to the formation of a racemic mixture of thiazolidines 12
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1114–1120

1116
H. Würfel et al.
PAPER
We then treated thione 13 with a series of diazo com-
pounds generated in one of two ways (Scheme 5). The
first method that we used was the oxidation of the corre-
sponding hydrazone 16–18. For this reaction, good results
were obtained by treating the hydrazones with nickel per-
oxide (14) at low to ambient temperatures (–78 °C to r.t.)
in an inert solvent.19 9-Diazo-9H-fluorene, however,
could only be generated by using a variation of the
Bamford–Stevens method, through treatment of the tosyl-
hydrazone of 9H-fluoren-9-one 15 with aqueous potassi-
um hydroxide in 1,4-dioxane.20 All the diazo compounds
reacted promptly with thione 13 with release of nitrogen.
The resulting thiiranes were immediately desulfurized to
form yellow- to red-colored olefins 19–22 in medium to
excellent yields (Scheme 5).
Figure 2 Molecular structure of olefin derivative 19. The ellipsoids
represent a probability of 40%. Hydrogen atoms are omitted for the
sake of clarity.
In olefin 20, the two phenyl rings connected to the olefinic
moiety are twisted out of the general plane of the mole-
cule, blocking conjugation between these moieties (Fig-
ure 3). One of the phenyl rings is oriented almost parallel
to the C10–C11–C12 plane, preventing closer contact
with the methyl groups on the thiazoline ring. The other
phenyl ring is twisted out of the olefin plane that is gener-
ated by the two sp2 carbon atoms C13 and C14. The tor-
sion angle C13–C14–C21–C26 of –38.9(2)° shows that
the phenyl ring participates only slightly to the π-conjuga-
tion system of the molecule. Although the influence for
the conjugation effects of the phenyl ring is weak, it can-
not, however, be neglected.
1. R=N-NH2 (15–18), NiO2 (14)
CH2Cl2
–78 °C (up to r.t.)
N
S
S
N
N
S
S
N
MeO
R
MeO
S
2. toluene, Cu
reflux, 12 h
19–22
13
16
MeO
OMe
hydrazone: R =
olefin: R =
17
20 (40)
18
21 (40)
15a
22 (48)
19 (99)
(yield in %)
Scheme 5 Synthesis of olefin derivatives 19–22 from thione 13.
a For 15, the tosylhydrazone was treated with aq KOH in 1,4-dioxane.
Olefins 19–22 showed some interesting features; for ex-
ample, the benzothiazole and the thiazoline moieties are
almost coplanar to one another. The connecting carbon at-
oms C1 and C2 are separated by 1.45 Å (Table 1), a value
that is intermediate between that of a C–C single bond and
that of a C=C double bond. The thiazoline ring shows a
small distortion in its planar geometry caused by the sp3-
hybridized carbon and the bulky sulfur atom. The substit-
uents on the newly generated olefin moiety do not show
comparable behavior. The isopropyl groups of olefin 19
are oriented so that the hydrogen at the isopropyl carbon
(C15) points toward the methyl groups of the thiazoline
ring, thereby minimizing the repulsion between these
bulky groups (Figure 2).
Figure 3 Molecular structure of olefin 20. The ellipsoids represent a
probability of 40%. Hydrogen atoms are omitted for the sake of clar-
ity.
Substitution at the 4-position, as in olefin 21, produces
significant changes in the UV/visible absorption and
emission spectra. Unfortunately, the crystal structure of
olefin 21 could not be determined and, therefore, a com-
plete comparison of structures was not possible at this
point.
Table 1 Structure Parameters for Luciferin Derivatives from X-ray
Crystal Structure Analyses
Bond length (Å)
C1–C2
Torsion anglea (°)
Compound
C13–C14
19
20
22
1.454(2)
1.459(2)
1.4465(19)
1.352(2)
1.353(2)
1.372(2)
–1.8(2)
–0.4(2)
–4.5(2)
Olefin 22, however, occupied an exceptional position in
this series. The whole molecule, with its large fluorenyl
moiety, is almost completely planar (Figure 4). The fluo-
renyl moiety shows only a slight twist of –9.9(3)° in rela-
tion to the olefinic plain C10–C13–C14–C15. Despite this
small deviation, the aromatic fluorenyl moiety was in-
cluded in the conjugated π-system of the luciferin, result-
a Torsion angle S2–C1–C2–N1.
Synthesis 2014, 46, 1114–1120
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Extended Oxyluciferin-Based Chromophores
1117
ing in a large bathochromic shift in its UV/visible Olefin 20 in tetrahydrofuran showed two absorption
absorption maximum in comparison to that of the starting bands with maxima at 368 and 393 nm (27174 and 25445
luciferin 6.
cm–1) that almost completely overlapped one another; the
logarithmic absorption coefficients again exceeded 4.
Olefin 21 in tetrahydrofuran also showed two absorption
maxima, in this case at 344 and 417 nm (29070 and 23981
cm–1); these bands did not show as strong an overlap as
those of 20. The methoxy groups affect the position of the
absorption bands, even if the aromatic rings do not fully
participate in the conjugated π-system, as shown in the
crystal structure of 20. The absorption maximum of 21 at
344 nm is hypsochromically shifted by 1896 cm–1 and its
maximum at 417 nm is bathochromically shifted by 1464
cm–1 with respect to the maxima of 20.
Olefin 22 shows the most-structured absorption bands of
all the derivatives that we investigated. An absorption pla-
teau reaching from 372 to 409 nm (26882 to 24450 cm–1)
was observed in tetrahydrofuran; furthermore, the absorp-
tion maximum of 22 in tetrahydrofuran occurred at
458 nm (21834 cm–1), so that the compound showed an in-
tense red coloration.
Figure 4 Molecular structure of 22. The ellipsoids represent a prob-
ability of 40%. Hydrogen atoms are omitted for the sake of clarity.
The UV/visible data for the derivatives showed some in-
teresting properties (Figure 5 and Table 2). The UV/visi-
ble spectrum of olefin 19 in tetrahydrofuran contained a
broad absorption plateau from 337 to 388 nm (29674 to
25773 cm–1) with small maxima, possibly due to vibra-
tional changes in the molecular geometry during the exci-
tation process. In the plateau region, the absorption
coefficient was very high (log ε > 4.0).
The fluorophoric properties of the olefins, measured in
tetrahydrofuran, are shown in Figure 6 and Table 2. Olefin
19 showed a fluorescence maximum at 466 nm with a
shoulder at 437 nm. Compared with 19, olefins 20 and 21
showed bathochromically shifted emission maxima at 533
nm (18762 cm–1) and 548 nm (18248 cm–1), respectively.
The fluorescence spectrum of 21 was more distinct than
that of 20 in that it showed two shoulders at 516 nm and
598 nm. Compound 22 had the most-structured fluores-
cence spectrum. A small emission maximum was present
at 555 nm, and the most bathochromically shifted maxi-
mum, in comparison with 19, was found at 595 nm (16807
cm–1). The compound also presented two shoulders at 529
nm and at 645 nm.
Figure 5 Normalized UV/visible spectra of olefin derivatives 19–22
in tetrahydrofuran
Table 2 Excitation and Emission Maxima with Stokes Shifts and
Quantum Yields for Solutions of the Synthesized Olefins
Compd λmax (ex)a (nm) λmax (em)a (nm) Stokes shift Quantum
(cm–1)
yieldb (%)
19
20
21
22
337–388
368–393
344–417
458
466
533
548
595
4314
6683
5733
5027
5.9
3.8
1.0
0.02
Figure 6 Normalized fluorescence spectra of olefin derivatives 19–
22 recorded in tetrahydrofuran
We also investigated the reactions of two other diazo
compounds: diazomethane and ethyl diazoacetoacetate.
These compounds did not show a comparable reactivity
towards olefin formation (Scheme 6). Diazomethane re-
a In THF.
b In EtOH.
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1114–1120

1118
H. Würfel et al.
PAPER
by TLC (0.2 mm Merck silica gel plates 60 F254). 1H and 13C NMR
spectra (in CDCl3 or DMSO-d6) were recorded on Bruker Avance
250 or 400 spectrometers. IR spectra were recorded by using an IR
Affinity-1 (Shimadzu). Mass spectra were recorded on a VG Trio-
2000 quadrupole mass spectrometer. Elemental analyses (CHNOS)
were performed by using Vario EL III equipment. Melting points
(uncorrected) were measured on a Büchi B-545 apparatus. UV/vis
absorption and fluorescence spectra were recorded on a Ther-
mo/UNICAM UV 500 spectrophotometer and on a JASCO FP-
6500 spectrofluorimeter, respectively.
acted with thione 13 in diethyl ether at 0 °C with instanta-
neous visible extrusion of nitrogen, leading to an intensely
red-colored solid that was not soluble in tetrahydrofuran,
dimethyl sulfoxide, acetone, N,N-dimethylformamide, or
chloroform. Investigation of the dried compound by ele-
mental analysis suggested that the thiirane compound 23
was the main product; mass spectrometry did not lead to a
conclusive result, and NMR studies could not be conduct-
ed because of the poor solubility of the compound. Further
treatment of the product with copper powder in refluxing
toluene resulted in complete degradation of the com-
pound; therefore, no methylene derivative of the
dimethyloxyluciferin series could be obtained or probed.
In contrast to the above observations, no reaction of thione
13 with ethyl diazoacetoacetate in dichloromethane could
be observed. TLC monitoring of the reaction mixture
showed that both starting materials were present through-
out, and no possible product was observed.
Crystal Structure Determination: The intensity data for the com-
pounds were collected on a Nonius Kappa CCD diffractometer by
using graphite-monochromated Mo-Kα radiation. Data were cor-
rected for Lorentz and polarization effects, but not for absorption ef-
fects.22,23 The structures were solved by direct methods (SHELXS
24) and refined by full-matrix least squares techniques against Fo2
(SHELXL-97 24). The hydrogen atoms of 20 and 22 were located by
difference Fourier synthesis and refined isotropically. The hydro-
gen atoms of 19 were included at calculated positions with fixed
thermal parameters. All nonhydrogen atoms were refined anisotrop-
ically.23 XP (Siemens Analytical X-ray Instruments, Inc.) was used
for structure representations.
CH2N2
Lawesson’s reagent, (diphenylmethylene)hydrazine (17), and ethyl
diazoacetoacetate were obtained commercially. Nickel peroxide
(17), diazomethane, 2-(6-methoxy-1,3-benzothiazol-2-yl)-5,5-di-
methyl-1,3-thiazole-4(5H)-one (6), (1-isopropyl-2-methylpropyli-
dene)hydrazine (16), [bis(4-methoxyphenyl)methylene]hydrazine
(18), and 9H-9-diazo-9H-fluorene were synthesized according to
known literature procedures.25–30
Et2O, 0 °C
N
S
S
N
– N2
MeO
S
N
S
S
N
23
N2CHCO2Et
CH2Cl2, r.t.
MeO
S
13
N
S
S
S
2-(6-Methoxy-1,3-benzothiazol-2-yl)-5,5-dimethyl-1,3-thiazole-
4(5H)-thione (13)
N
MeO
A mixture of ketone 6 (10.0 mmol) and Lawesson’s reagent (8.0
mmol) in dry xylene (50 mL) was refluxed under N2 for 4 h. When
the starting material was completely consumed (TLC), the mixture
was cooled to r.t. and filtered through a short column of silica gel (3
cm), which was subsequently washed with excess toluene. The or-
ganic phases were combined and concentrated, and the resulting
solid was crystallized (heptane) to give a golden solid; yield: 2.0 g
(65%); mp 193–194 °C (heptane).
O
OEt
Scheme 6 Attempted reactions of thione 13 with diazomethane or
ethyl diazoacetoacetate
In conclusion, a synthetic methodology based on the
Barton–Kellogg olefination (the reaction of a diazo com-
pound with a thione), capable of forming very sterically
hindered alkenes, allowed the preparation and isolation of
four new luciferin derivatives. These compounds contain
an alkene bridge at the 4-position of the thiazoline moiety
of dimethyloxyluciferin, forming an expanded luciferin-
like π-system that might be useful in color-modulated bio-
luminescence assays. The photophysical parameters of
the four new substances in organic media showed a broad
distribution of UV/visible absorption and emission maxi-
ma, permitting tuning of those characteristics. The fluo-
rescence quantum yields are also reported. Trends in these
parameters follow the electronic behavior of the extended
π-systems, which show unique features that are correlated
to the spatial arrangement of the two chromophoric sys-
tems involved. The applicability of those compounds in
bioanalytical assays will be evaluated in future works.
IR (ATR): 3429, 1603, 1507, 1454, 1214, 918, 867, 827 cm–1.
1H NMR (250 MHz, DMSO-d6): δ = 8.08 (d, 3J = 9.1 Hz, 1 H), 7.40
(d, 4J = 2.4 Hz, 1 H), 7.19 (dd, 3J = 9.1 Hz, 4J = 2.4 Hz, 1 H), 3.93
(s, 3 H), 1.81 (s, 6 H).
13C NMR (63 MHz, DMSO-d6): δ = 236.8, 186.3, 160.2, 157.0,
149.1, 140.2, 126.0, 118.5, 103.3, 73.1, 55.9, 31.7.
MS (EI): m/z (%) = 310 (53), 308 (100), 293 (56), 275 (53), 250
(15), 217 (36), 208 (57), 190 (30), 165 (12), 147 (14).
Anal. Calcd for C13H12N2OS3: C, 50.62; H, 3.92; N, 9.08; S, 31.19.
Found: C, 50.62; H, 3.85; N, 8.98; S, 30.85.
UV/Vis (CHCl3): λmax (log ε) = 281 (3.64), 420 (4.49) nm.
Olefinations of 2-(6-Methoxy-1,3-benzothiazol-2-yl)-5,5-di-
methyl-1,3-thiazole-4(5H)-thione (13); General Procedure
A mixture of the appropriate hydrazone (4.0 mmol or 8.0 mmol for
the aliphatic hydrazone) and NiO2 (14; 33.0 mmol) in anhyd CH2Cl2
(50 mL) was stirred at 0 °C for 2 h under N2 (–78 °C for 1 h for the
aliphatic hydrazone). The mixture was filtered under inert condi-
tions by using standard Schlenk techniques to give an intensely col-
ored solution. A soln of thione 13 (1.0 mmol) in anhyd CH2Cl2 (20
mL) was added at 0 °C. The stirred mixture was allowed to warm to
r.t. and kept at that temperature for additional 3 h. The solvent was
then removed in a rotary evaporator and the remaining solid was
mixed with copper powder, taken up in dry toluene (20 mL), and re-
fluxed for 12 h. The mixture was then cooled to r.t., filtered, and
concentrated to give a crude product that was purified by crystalli-
zation or by column chromatography.
All reagents were purchased from commercial sources. Nickel per-
oxide (14), 2-(6-methoxy-1,3-benzothiazol-2-yl)-5,5-dimethyl-1,3-
thiazole-4(5H)-one (6), (1-isopropyl-2-methylpropylidene)hydra-
zine (17), [bis(4-methoxyphenyl)methylene]hydrazine (18), and 9-
diazo-9H-fluorene were synthesized according to known literature
procedures. Solvents were purified according to standard meth-
ods.21 Spectroscopic-grade solvents were used in UV/vis and fluo-
rescence measurements. The progress of reactions was monitored
Synthesis 2014, 46, 1114–1120
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Extended Oxyluciferin-Based Chromophores
1119
2-[4-(1-Isopropyl-2-methylpropylidene)-5,5-dimethyl-4,5-di-
hydro-1,3-thiazol-2-yl]-6-methoxy-1,3-benzothiazole (19)
Yellow crystals; yield: 0.37 g (99%); mp 202–203 °C (heptane–
EtOAc).
IR (ATR): 3051, 2964, 2930, 1602, 1584, 1488, 1440, 1264, 1220,
1139, 822, 808, 724 cm–1.
1H NMR (250 MHz, CDCl3): δ = 8.83 (d, 3J = 7.2 Hz, 1 H), 8.02 (d,
3
3J = 7.9 Hz, 1 H), 7.90 (d, J = 9.0 Hz, 1 H), 7.54–7.70 (m, 2 H),
7.10–7.40 (m, 5 H), 7.03 (dd, 3J = 9.0 Hz, 4J = 2.4 Hz, 1 H), 3.80 (s,
IR (ATR): 3447, 2959, 2925, 2866, 1601, 1496, 1266, 1219, 864
cm–1.
3 H), 2.26 (s, 6 H).
1H NMR (250 MHz, DMSO-d6): δ = 7.97 (d, 3J = 9.0 Hz, 1 H), 7.31
(d, 4J = 2.5 Hz, 1 H), 7.08 (dd, 3J = 9.0 Hz, 4J = 2.5 Hz, 1 H), 3.88
(s, 3 H), 3.04 (sept, 3J = 6.8 Hz, 1 H), 2.40–2.60 (m, 1 H), 1.83 (s, 6
H), 1.36 (d, 3J = 6.9 Hz, 6 H), 1.12 (d, 3J = 7.0 Hz, 6 H).
13C NMR (63 MHz, DMSO-d6): δ = 160.6, 158.7, 155.1, 154.3,
150.4, 148.4, 137.8, 124.8, 116.0, 103.9, 62.2, 55.8, 31.0, 23.2,
22.0, 20.8, 19.0.
13C NMR (63 MHz, CDCl3): δ = 165.2, 164.7, 159.5, 159.1, 148.7,
142.0, 139.8, 138.8, 138.7, 135.4, 131.1, 129.6, 128.0, 127.7, 126.4,
126.3, 125.4, 120.1, 118.9, 117.2, 103.8, 65.1, 56.0, 28.3.
MS (EI): m/z (%) = 440 (52), 425 (76), 234 (62), 208 (56), 202
(100), 191 (60), 165 (38).
Anal. Calcd for C26H20N2OS2: C, 70.88; H, 4.58; N, 6.36; S, 14.56.
Found: C, 70.65; H, 4.57; N, 6.32; S, 14.76.
MS (EI): m/z (%) = 376 (38), 375 (84), 374 (100), 359 (72), 341
(26), 331 (34), 208 (20), 43 (14).
UV/vis (THF): λmax (log ε) = 336 (4.11, sh), 372 (4.26), 391 (4.25),
435 (4.40), 458 (4.48), 484 (4.40), 522 (4.04, sh) nm.
Anal. Calcd for C20H26N2OS2: C, 64.13; H, 7.00; N, 7.48; S, 17.12.
Found: C, 64.27; H, 7.11; N, 7.50; S, 17.09.
Crystal data:31 C26H20N2OS2, Mr = 440.56 g·mol–1, red prism, size
0.06 × 0.05 × 0.04 mm3, monoclinic, space group Cc,
a = 11.0168(3), b = 16.3502(4), c = 11.6237(2) Å, β = 92.584(1)°,
V = 2091.61(8) Å3, T = –140 °C, Z = 4, ρcalcd = 1.399 g·cm–3, μ
(Mo-Kα) = 2.77 cm–1, F(000) = 920, 6369 reflections in h(–13/14),
k(–19/21), l(–14/15), measured in the range 2.89° ≤ θ ≤ 27.47°,
UV/vis (CHCl3): λmax (log ε) = 337 (4.24), 351 (4.26), 368 (4.26),
388 (4.26) and 412 (4.00, sh) nm.
Crystal data:31 C20H26N2OS2, Mr = 374.55 g·mol–1, colorless prism,
size 0.05 × 0.05 × 0.04 mm3, triclinic, space group P1;
a = 8.1086(3), b = 8.1910(3), c = 17.0767(7) Å, α = 98.671(2),
β = 90.205(2), γ = 119.001(2)°, V = 976.78(6) Å3, T = –140 °C,
Z = 2, ρcalcd = 1.273 g·cm–3, μ (Mo-Kα) = 2.83 cm–1, F(000) = 400,
6336 reflections in h(–10/10), k(–10/10), l(–22/21), measured in the
range 2.91° ≤ θ ≤ 27.53°, completeness θmax = 96.9%, 4370 inde-
pendent reflections, Rint = 0.0201, 3854 reflections with Fo > 4σ(Fo),
233 parameters, 0 restraints, R1obs = 0.0405, wR2obs = 0.0894,
R1all = 0.0491, wR2all = 0.0974, GOOF = 1.027, largest difference
peak and hole: 0.364/–0.269 e Å–3.
completeness
θmax = 99.5%, 4326 independent reflections,
Rint = 0.0192, 4276 reflections with Fo > 4σ(Fo), 361 parameters, 2
restraints, R1obs = 0.0254, wR2obs = 0.0644, R1all = 0.0259,
wR2all = 0.0651, GOOF = 1.057, Flack parameter 0.03(4), largest
difference peak and hole: 0.191/–0.206 e Å–3.
2-{4-[Bis(4-methoxyphenyl)methylene]-5,5-dimethyl-4,5-di-
hydro-1,3-thiazol-2-yl}-6-methoxy-1,3-benzothiazole (21)
Bright yellow crystals; yield: 0.20 g (40%); mp 184–185 °C (hep-
tane).
2-[4-(Diphenylmethylene)-5,5-dimethyl-4,5-dihydro-1,3-thia-
zol-2-yl]-6-methoxy-1,3-benzothiazole (20)
IR (ATR): 3046, 2960, 2834, 1602, 1509, 1490, 1448, 1266, 1243,
1224, 1180, 1115, 1026, 918, 828, 804 cm–1.
Yellow crystals; yield: 0.18 g (40%); mp 182–183 °C (heptane).
1H NMR (250 MHz, CDCl3): δ = 8.00 (d, 3J = 9.0 Hz, 1 H), 7.31–
3
4
IR (ATR): 3057, 3004, 2966, 1595, 1491, 1266, 1221, 1122, 1020,
923, 760, 699 cm–1.
1H NMR (250 MHz, CDCl3): δ = 8.01 (d, 3J = 9.0 Hz, 1 H), 7.20–
7.46 (m, 11 H), 7.12 (dd, 3J = 9.0 Hz, 4J = 2.5 Hz, 1 H), 3.89 (s, 3
H), 1.56 (s, 6 H).
13C NMR (63 MHz, CDCl3): δ = 161.2, 159.8, 159.0, 157.7, 148.3,
141.7, 139.2, 138.2, 137.0, 131.0, 130.8, 127.7, 127.6, 127.2, 127.0,
125.0, 116.5, 103.7, 63.2, 55.8, 30.9.
7.40 (m, 3 H), 7.22–7.28 (m, 2 H), 7.11 (dd, J = 9.0 Hz, J = 2.4
Hz, 1 H), 6.91 (d, 3J = 8.5 Hz, 2 H), 6.84 (d, 3J = 8.8 Hz, 2 H), 3.89
(s, 3 H), 3.85 (s, 3 H), 3.82 (s, 3 H), 1.55 (s, 6 H).
13C NMR (63 MHz, CDCl3): δ = 160.1, 160.0, 159.0, 158.9, 158.8,
156.8, 148.4, 138.1, 136.4, 134.7, 132.5, 132.0, 131.7, 124.9, 116.5,
113.1, 112.6, 103.8, 63.5, 55.8, 55.2, 31.0.
MS (EI): m/z (%) = 502 (36), 487 (66), 469 (36), 304 (28), 294 (84),
280 (72), 236 (48), 208 (100), 193 (32), 165 (32), 152 (36), 59 (48).
MS (EI): m/z (%) = 444 (9), 443 (18), 442 (63), 409 (100), 408 (78),
244 (40), 234 (56), 208 (48), 165 (59), 121 (20), 59 (48).
Anal. Calcd for C28H26N2O3S2: C, 66.91; H, 5.21; N, 5.57; S, 12.76.
Found: C, 67.15; H, 5.60; N, 5.65; S, 12.69.
Anal. Calcd for C26H22N2OS2: C, 70.56; H, 5.01; N, 6.33; S, 14.49.
Found: C, 70.86; H, 5.02; N, 6.48; S, 14.38.
UV/vis (THF): λmax (log ε) = 343 (4.21) and 418 (4.29) nm.
2-Isopropyl-2-(6-methoxy-1,3-benzothiazol-2-yl)-5,5-dimethyl-
1,3-thiazolidin-4-one (12)
UV/vis (THF): λmax (log ε) = 367 (4.29) and 393 (4.31) nm.
Crystal data:31 C26H22N2OS2, Mr = 442.58 g·mol–1, light-yellow
prism, size 0.06 × 0.05 × 0.04 mm3, monoclinic, space group P21/c,
a = 16.3282(3), b = 8.1573(1), c = 17.2187(3) Å, β = 104.615(1)°,
V = 2219.22(6) Å3, T = –130 °C, Z = 4, ρcalcd = 1.325 g·cm–3, μ
(Mo-Kα) = 2.61 cm–1, F(000) = 928, 12857 reflections in h(–21/21),
k(–10/10), l(–22/17), measured in the range 2.78° ≤ θ ≤ 27.44°,
For preparation and additional analytical data, see Würfel et al.15
Pale yellow crystals; yield: 1.43 g (70%); mp 130–132 °C (heptane–
EtOAc).
1H NMR (250 MHz, DMSO-d6): δ = 9.57 (s, 1 H), 7.84 (d, 3J = 9.0
Hz, 1 H), 7.66 (d, 4J = 2.5 Hz, 1 H), 7.08 (dd, 3J = 9.0 Hz, 4J = 2.5
Hz, 1 H), 3.82 (s, 3 H), 2.71 (m, 3J = 6.7 Hz, 1 H), 1.48 (s, 6 H), 0.98
(m, 6 H).
13C NMR (63 MHz, DMSO-d6): δ = 178.1, 178.0, 176.0, 157.8,
148.4, 136.5, 123.8, 116.2, 105.3, 70.8, 70.7, 56.2, 51.4, 37.0, 30.4,
28.8, 17.9, 17.8.
completeness
θmax = 99.5%, 5052 independent reflections,
Rint = 0.0260, 4488 reflections with Fo > 4σ(Fo), 368 parameters, 0
restraints, R1obs = 0.0389, wR2obs = 0.0958, R1all = 0.0455,
wR2all = 0.1010, GOOF = 0.987, largest difference peak and hole:
0.411/–0.437 e Å–3.
MS (EI): m/z (%) = 337 (38) [M + 1], 336 (8), 293 (100), 191 (40),
43 (20).
2-[4-(9H-Fluoren-9-ylidene)-5,5-dimethyl-4,5-dihydro-1,3-thia-
zol-2-yl]-6-methoxy-1,3-benzothiazole (22)
Red crystals; yield: 0.21 g (48%); mp 220–221 °C (heptane).
© Georg Thieme Verlag Stuttgart · New York
Synthesis 2014, 46, 1114–1120

1120
H. Würfel et al.
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Acknowledgment
The authors thank Roche Diagnostics GmbH, Penzberg, for finan-
cial support and Fundação de Amparo à Pesquisa do Estado de São
Paulo (FAPESP, grant 2012/02428-5).
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(31) Crystallographic data for compounds 19, 20, and 22
(excluding structure factors) have been deposited with the
accession numbers CCDC 967132, 967133, and 967134,
respectively, and can be obtained free of charge from the
Cambridge Crystallographic Data Centre, 12 Union Road,
Cambridge CB2 1EZ, UK; Fax: +44(1223)336033; E-mail:
deposit@ccdc.cam.ac.uk; Web site:
www.ccdc.cam.ac.uk/conts/retrieving.html.
Synthesis 2014, 46, 1114–1120
© Georg Thieme Verlag Stuttgart · New York