189
Table 2.16
ITL labeled amino
material, luminol, was mainly recovered. This could be due to
the low reactivity of TCI where imidazole is not a very good
leaving group (pKa µ 6.99) compared to the halide substitu-
ent.11,12 N,N-Dimethylformamide (DMF) was found to be the
optimized solvent offering a high solubility of luminol. To
improve the reactivity of reagent, highly reactive thiophosgene
was used instead of TCI. After the reaction proceeded for 24 h,
a yellow product was obtained in moderate yield (Table 1). Its
molecular mass was identified by high-resolution mass spec-
trometry and found to correspond to the molecular weight of ITL
[m/z 220 (M + H+)].
It is known that chemical manipulation of (iso)luminol
structures deteriorates their CL efficiency.9 Therefore, CL
emission of ITL was checked against the starting material. The
CL emission obtained from ITL was comparable to luminol
indicating that the introduction of isothiocyanate group into
luminol did not compromise CL efficiency.
Expected
Found
(m/z)
Entry
acid
Mr
1
2
3
4
5
6
7
8
9
Ala-ITL
Arg-ITL
Asn-ITL
Cys-ITL
Glu-ITL
Leu-ITL
Lys-ITL
Met-ITL
Phe-ITL
Pro-ITL
308.06
393.12
337.05
340.03
366.06
350.10
365.12
368.02
384.09
334.07
309.08
394.13
360.14
341.08
367.07
351.11
366.12
369.07
385.10
335.08
10
In order to ensure that these synthetic conditions could be
used for the introduction of isothiocyanate group into other
amine compounds, the synthetic method was employed for the
preparation of ITIL and phenyl isothiocyanate. After comple-
tion, ITIL and phenyl isothiocyanate were obtained in moderate
62 and 44% yields, respectively. This demonstrated the
efficiency of the protocol for the installation of isothiocyanate
groups into primary amines.
In order to employ ITL as a labeling reagent for various
proteins in the supersensitive CL assay, it is necessary to ensure
that ITL could be used as a labeling reagent for amino acids.
Since all proteins possess various amino acids which are
abundant with -NH2 terminus on their surfaces, the -NH2
terminus is expected to serve as a labeling site for ITL. To test
this hypothesis, 10 representative amino acids were used for CL
labeling with ITL. The labeling of amino acids with ITL was
simply achieved via the incubation of the mixture between a
corresponding amino acid and ITL at 60 °C for 2 h before
analyzing the product by mass spectrometry. It was found that all
amino acids were successfully labeled with ITL (Table 2). The
labeling mechanism is achieved via the nucleophillic addition of
the -NH2 group of the amino acid to the isothiocyanate group of
ITL.
Since ITL could be successfully employed as a CL labeling
reagent for amino acids, we targeted the ITL-labeled protein.
Bovine serum albumin (BSA) was chosen as a model protein
since it is widely used in various immunoassays due to its low
cost and stability.13,14 The labeling conditions (solvent) were
then optimized. A mixture of DMSO and water in a ratio of 1:9
(v/v) was chosen as the solvent since both BSA and ITL were
well dissolved. The optimum temperature and incubation time
were found to be 60 °C for 24 h. To confirm the presence of ITL-
labeled BSA, Gel-filtration liquid chromatography (GFLC) was
employed for identification. After incubating BSA and ITL for
1 h, the labeled product was analyzed by GFLC. Results showed
the presence of a new peak at the retention time of 15 min which
was later identified as ITL-labeled BSA (Figure 3). The intensity
of this peak increased with the progression of reaction time. To
further confirm the identity of ITL-labeled BSA, it was spotted
on a poly(vinylidene fluoride) (PVDF) membrane prior to CL
detection employing a reported protocol.15
Figure 3. GFLC of ITL-labeled BSA. Conditions: injection
volume, 5 ¯L; column, TSK gel T2000SW; eluent, 0.1% (v/v)
¹1
aqueous solution of trifluoroacetic acid; flow rate, 1.0 mL min
;
UV detection, lamp: 275 nm; fluorescence detection, excitation
(mercury lamp)/emission (wavelength cutoff filter): 254/
>360 nm.
membrane. The dextran probe is highly sensitive and able to
detect CYP3A4 protein at the femtomole level.15 The signal
amplication of the dextran probe originated from the multiple
incorporation of luminols onto a macromolecular dextran
template. In order to compare the CL efficiency of ITL-labeled
BSA, Dex-Lu which was prepared according to Kai’s procedure
was used as a reference. The molar concentration of Dex-Lu
(0.15 ¯M) was estimated to be the same as ITL-labeled BSA.
It was found that the CL intensity of ITL-labeled BSA increased
as a function of reaction time. After the reaction was left for
24 h, the CL emission of ITL-labeled BSA on a solid-phase
membrane was at its highest and comparable to that of Dex-Lu
(Figure 4). Comparing to the Mr of dextran (7 © 105) and BSA
(6.6 © 104), the result from a solid-phase CL detection demon-
strated a good efficiency of ITL incorporated into the smaller
BSA template. The obtained CL results on a solid-phase
membrane were in accordance to GFLC data. From preliminary
results, ITL was successfully synthesized and employed as an
effective CL labeling reagent for amino acids and proteins.
In conclusion, ITL was synthesized under optimized
conditions. The introduction of an isothiocyanate group main-
tained its CL efficiency. The conditions were further employed
for the preparation of other isothiocyanate-containing com-
pounds, e.g., ITIL and phenyl isothiocyanate. ITL was success-
Kai et al. synthesized luminol-labeled dextran probe (Dex-
Lu) for the CL detection of CYP3A4 protein on a solid-phase
Chem. Lett. 2011, 40, 188-190
© 2011 The Chemical Society of Japan