A seminaphthofluorescein-based fluorescent chemodosimeter for the highly selective detection of cysteine

Article abstract of DOI:10.1039/c2ob25178g

A fluorescent chemodosimeter for cysteine detection was developed based on a tandem conjugate addition and intramolecular cyclization reaction. The method exhibited an excellent selectivity for cysteine over other biothiols such as homocysteine and glutathione.

Full text of DOI:10.1039/c2ob25178g

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A seminaphthofluorescein-based fluorescent chemodosimeter for the highly
selective detection of cysteine†
Xiaofeng Yang,*a,b Yixing Guoa and Robert M. Strongin*a
Received 24th January 2012, Accepted 22nd February 2012
DOI: 10.1039/c2ob25178g
It has been known for several years that the conjugate addition
of Cys to acrylates (1) will generate the corresponding thioether
(2), which can further undergo an intramolecular cyclization to
yield 3-carboxy-5-oxoperhydro-1,4-thiazepine (3a) (Scheme 1).6
Recently, a new design principle for a probe capable of dis-
tinguishing Cys and Hcy was developed in our labs. The (hydro-
xymethoxyphenyl)benzothiazole (HMBT)-based fluorescent
probe functioned based on a combined PET and ESIPT mech-
anism.7 Since HMBT has a relatively low quantum yield8 and
short excitation wavelength (304 nm) which may limit its appli-
cation in biological samples, herein we report a long wavelength
fluorescent chemodosimeter for Cys. It couples a conjugate
addition/cyclization mechanism to a xanthene dye spirolactone-
opening reaction. Seminaphthofluorescein (SNF) bis-acrylate 4
was synthesized by the condensation of SNF with acryloyl chlor-
ide (ESI, Scheme S1†). SNF was selected as a fluorescent repor-
ter because of its long wavelength absorption and emission.9
Interestingly, upon mixing Cys in colorless solution of 4
in 1.0 mM cetyltrimethylammonium bromide media (CTAB)
buffered at pH 7.4, both a pink color and strong fluorescence
appeared gradually, while solutions other amino acids and
biothiols, such as Hcy and GSH afforded no obvious changes in
the same conditions. This interesting feature indicates that 4 can
serve as a selective “off-to-on” dosimeter for Cys (Fig. 1).
Fluorescence spectra of solutions containing 4 and increasing
concentrations of Cys are shown in Fig. 2. The fluorescence
intensity at 621 nm increases with increasing Cys concentration.
The observed fluorescence intensity is nearly proportional to the
Cys concentration up to 10 μM. Moreover, as low as 0.2 μM Cys
can be readily detected by using the proposed dosimeter (ESI,
Fig. S3†). The time course of the fluorescence assay is shown in
Fig. 3. It can be seen that the fluorescence of the reaction with
Cys is increasing with time and reaches a plateau at 20 min,
whereas for Hcy and GSH, no significant fluorescence increase
is observed during this period.
A fluorescent chemodosimeter for cysteine detection was
developed based on a tandem conjugate addition and
intramolecular cyclization reaction. The method exhibited an
excellent selectivity for cysteine over other biothiols such as
homocysteine and glutathione.
Cysteine (Cys) is an essential amino acid. It plays a key role in a
variety of important cellular functions, such as protein synthesis,
detoxification and metabolism, etc. Elevated levels of Cys have
been associated with neurotoxicity.1 Cys deficiency is involved
in a number of other disorders.2
In recent years, several fluorescent sensors and probes for
biological thiols have been developed.3 Most are based on the
inherent nucleophilicity of the sulfhydril group.3a Although
these probes show high sensitivity towards mercapto biomole-
cules, the direct detection of Cys is highly challenging due to
interference from other biothiols. In our previous work,4 we
introduced fluorescein aldehydes as fluorescent probes for Cys
and Hcy based on the cyclization of N-terminal Cys (or Hcy)
residues to form the corresponding thiazolidines (or thiazinanes).
Because both sulfhydryl and amino groups are responsible for
the sensing mechanism, the selectivity of these types of probes
is higher than traditional probes based mainly on non-specific
thiol nucleophilicity.
Early on, during the development of selective aminothiol
probes4b we observed that certain α,β-unsaturated aldehydes
with decreasing electrophilicity exhibited predictable enhanced
selectivity towards Cys. Research in this field has since been
greatly extended and many probes containing aldehyde moieties
have been reported,4a,5 with some exhibiting high selectivity for
either Cys5c,d or Hcy.5b Although some have been developed for
assaying either Cys or Hcy, there is still room for improvement
in terms of selectivity, sensitivity, and performance with alterna-
tive reaction mechanisms.
aDepartment of Chemistry, Portland State University, Portland, OR
97201, USA. E-mail: Strongin@pdx.edu; Fax: +1 503-725-9525;
Tel: +1 503-725-9724
bKey Laboratory of Synthetic and Natural Functional Molecule
Chemistry of Ministry of Education, Institute of Analytical Sciences,
College of Chemistry & Materials Science, Northwest University,
Xi’an710069, China. E-mail: xfyang@nwu.edu.cn
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c2ob25178g
Scheme 1 Formation of 3-carboxy-5-oxoperhydro-1,4-thiazepine (3a)
from the reaction of acrylates (R = alkyl, aryl) and Cys.
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Fig. 1 Color changes of the solution of 4 (10 μM) in the presence of
different biothiols (2 equiv.) in 1.0 mM CTAB media buffered at pH 7.4
(Hepes buffer, 20 mM) after 25 min.
Fig. 4 Fluorescence spectra (λex = 550 nm) of 4 (10 μM) with the
addition of 1 equiv. of Cys, Hcy, GSH, leucine, proline, arginine, histi-
dine, valine, methionine, threonine, glutamine, alanine, aspartic acid,
norleucine, isoleucine, lysine, tryptophan, tyrosine, phenylalanine,
cystine and homocystine for 25 min in 1 mM CTAB buffered at pH 7.4
(Hepes buffer, 20 mM).
Fig. 2 Fluorescence spectra (λex = 550 nm) of 4 (10 μM) with the
addition of increasing concentrations of Cys in 1.0 mM CTAB media
buffered at pH 7.4 (Hepes buffer, 20 mM), 25 min. The inset figure
shows the changes in fluorescence intensity at 621 nm as a function of
Cys concentration.
Scheme 2 Proposed mechanism of discrimination of Cys from Hcy
when using 4.
The selectivity of 4 towards Cys over other biothiols was
examined. Upon mixing Cys with 4, the conjugate addition
product 5a is formed, which undergoes a rapid cyclization reac-
tion to produce 3a while releasing the free SNF, thus strong flu-
orescent emission is recorded (Scheme 2). In the case of GSH,
1,4-addition of thiols to the α,β-unsaturated carbonyl moieties of
4 can occur. However, the ensuing intramolecular cyclization
similar to that of Cys cannot proceed because the free amine
does not attack the ester moiety in the 4–GSH adduct apparently
due to entropic considerations involved in large ring formation.
Thus, the dosimeter still exists in its colorless, non-fluorescent
spirolactone form, even after the initial conjugate addition reac-
tion. Mass spectrometric analysis of the product generated from
the mixture of 4 with GSH in CH3OH–H2O (8 : 1, v/v) confirms
the formation of the 4–GSH adduct, and a prominent peak at m/z
558.1390 corresponding to [4–GSH–2H]2− (calc. 558.1365 for
C51H52N6O19S2) is clearly observed in the HRMS data (ESI,
Fig. S13†). In the case of Hcy, the corresponding conjugate
addition adduct 5b is produced, but the rate for the subsequent
intramolecular cyclization is apparently too slow for 3b to be
observed even after an extended incubation time (3 h) (the
addition adduct 5b was confirmed by HRMS data, Fig. S14, see
Fig. 3 Time course study of as solution of 4 (10 μM) and 1 equiv.
biothiols in 1 mM CTAB media buffered at pH 7.4. λex/λem = 562/
621 nm.
To evaluate the selectivity of 4 for Cys, changes in the fluor-
escence intensity of 4 caused by other analytes were also
measured. Fig. 4 shows the fluorescence spectra of the solution
of 4 after the addition of these analytes after 25 min. It was
observed that other amino acids and biothiols promote almost no
fluorescence intensity changes under the same conditions,
demonstrating the high selectivity of 4 for Cys over other ana-
lytes. To further test the Cys specificity of 4, competition ex-
periments were conducted with added excess amounts of other
amino acids. No significant variation in fluorescence intensity
was found in comparison to solutions containing Cys only (ESI,
Fig. S7†).
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ESI†). This can be explained by the fact that for Cys, a 7-
membered ring intermediate is formed, whereas for Hcy, because
it has an additional methylene group in its side chain, a kineti-
cally less favored 8-membered ring would form.10
proposed chemodosimeter can be used to detect Cys at physio-
logical levels.
Furthermore, experiments were carried out to prove the above
sensing mechanism. Firstly, the product mixture of the reaction
of cysteamine (cysteamine was selected instead of Cys because
of its good solubility in organic solvents) with 4 in CH3OH was
separated and 1,4-thiazepan-5-one (6) and the parent SNF were
Acknowledgements
This work was support by the National Institutes of Health (RO1
EB002044). XF Yang acknowledges financial support from the
China Scholarship Council.
1
obtained, respectively. The structure of 6 was identified by H
NMR, 13C NMR and HRMS (ESI, Fig. S15–S17†). The for-
mation of SNF was confirmed by a major peak at m/z 395.0929,
corresponding to [SNF–H]− (calc. 395.0919 for C25H15O5) was
shown in the HRMS data (ESI, Fig. S18†). Secondly, cysteamine
and 3-mercaptopropanoic acid (MPA) were introduced to the
solution of 4, respectively, and it was observed that the former
gives a prominent increase in fluorescence emission but the latter
produces weak fluorescence increase at the same conditions.
Lastly, N-acetyl-L-cysteine (NAC) was added to a solution of 4
and almost no fluorescence increase was observed under the
same conditions (ESI, Fig. S8†). The above experiments serve as
strong evidence that both sulfhydril and amino groups (the NAC
amine is blocked) of Cys are responsible for the signal.
Compound 4 was used for the quantitative measurement of
Cys content in a human plasma sample. 0.5 mL human plasma
was reduced using triphenylphosphine (0.1 M, 80 μL) in the
presence of HCl (0.2 M, 40 μL) for 15 min at rt.11 Proteins
present in the sample after reduction were precipitated by the
addition of acetonitrile (0.5 mL), followed by centrifugation
(4000 rpm) of the sample for 20 min. The supernatant liquid was
then added to a solution of 4 (10 μM) in pH 7.4 Hepes buffer
solution (0.1 M, 5 mL) in the presence of 1.0 mM CTAB.7 As
shown in Fig. S9,† the fluorescence emission shows a significant
increase with the addition of reduced plasma. However, the
fluorescence emission of the solution of 4 showed no obvious
increase when controls of triphenylphosphine alone or deprotei-
nized plasma (without reducing agent) were added, respectively,
proving that the fluorescent increment of the solution of reduced
plasma and 4 is indeed directly correlated to the presence of
Cys. The amount of Cys in the plasma sample was determined
by the standard addition method to be 172.8 8.7 μM (n = 3),
which is well within the reported Cys concentration range
(135.8–266.5 μM) for human plasma samples from healthy indi-
viduals.12 These results prove that the proposed dosimeter may
be useful for the quantitative detection of Cys in biological
media.
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In conclusion, we have developed a highly selective chemodo-
simeter for Cys over other biothiols including Hcy and GSH in
aqueous solution. The extremely high selectivity of 4 towards
Cys can be explained by the reaction of Cys with 4 to give the
corresponding conjugate 5a, which undergoes intramolecular
cyclization releasing free SNF, resulting in a dual chromo- and
fluorogenic response. Due to its simplicity and selectivity, the
This journal is © The Royal Society of Chemistry 2012
Org. Biomol. Chem., 2012, 10, 2739–2741 | 2741