A reductive ligation based fluorescent probe for S-nitrosothiols

Article abstract of DOI:10.1039/c4cc01288g

A reductive ligation based fluorescent probe (SNOP1) for the detection of S-nitrosothiols (SNO) was developed. The probe showed good selectivity and sensitivity for SNO. The Royal Society of Chemistry 2014.

Full text of DOI:10.1039/c4cc01288g

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A reductive ligation based fluorescent probe for
S-nitrosothiols†
Cite this: Chem. Commun., 2014,
50, 4806
Di Zhang,‡ab Wei Chen,‡a Zhengrui Miao,c Yong Ye,*b Yufen Zhao,b S. Bruce Kingc
and Ming Xian*a
Received 19th February 2014,
Accepted 10th March 2014
DOI: 10.1039/c4cc01288g
www.rsc.org/chemcomm
A reductive ligation based fluorescent probe (SNOP1) for the detec- However this has not been well studied. In 2009, we reported our
tion of S-nitrosothiols (SNO) was developed. The probe showed first generation of fluorescent probes for SNO, which was based on a
good selectivity and sensitivity for SNO.
SNO-mediated oxidation of phosphine substrates.5 Although the
probes showed good sensitivity for SNO vs. other reactive sulfur
S-Nitrosation is an important post-translational modification that not species (RSS), potential oxidation by other oxidative species such as
only modulates biological activity of nitric oxides (NO), but also H2O2 could be a problem. To solve this problem we envisioned that
regulates protein functions.1 The levels of S-nitrosation are finely the reductive ligation of SNO could be useful in the development of
regulated, and dysregulation is associated with the etiology of several specific SNO fluorescent probes. Here we report the design, synthesis,
pathologies. Currently the detection of S-nitrosation in biological and evaluation of a reductive ligation-based probe for SNO.
systems is still a challenge due to the labiality of the products of
The mechanism of reductive ligation is described in Scheme 1.4a
S-nitrosation, i.e. S-nitrosothiols (SNO).2 Since SNO are unstable SNO can react with triaryl phosphine 1 to form the azaylide inter-
species, most current methods (such as chemiluminescence based mediate 2, which in turn undergoes a rapid intramolecular acyl
assays, colorimetry based assays, and biotin-switch based assays) are transfer and hydrolysis to furnish sulfenamide 3 and R0OH. This
indirect methods, which in fact detect the decomposition products of reaction provides a unique and specific way to remove the acylated
SNO (either the S part or the NO part).3 Careful control experiments group on hydroxyl groups. It is known that acylation on many
are needed if these methods are used, otherwise false positive results fluorophores can quench the fluorescence and de-acylation can
could be generated. In this regard, direct methods which target the reform the fluorescent species.6 This strategy has been widely used
entire SNO moiety would have advantages. In the past several years in the design of many reaction based fluorescent probes.6 Therefore
our laboratory has developed a series of phosphine-based bio- we expected that if an –OH sensitive fluorophore is introduced into
orthogonal reactions of SNO.4 These reactions specifically target the triarylphosphine acylate, the resultant compound 5 would be a
SNO groups and can directly convert unstable SNO to stable and specific probe for SNO as it will selectively react with SNO to release
detectable species. While we are continuing to work on these the fluorophore.
reactions (our goal is to utilize these reactions to develop novel
reagents for directly enriching or labeling protein SNO), we realized
that fluorescent probes may be developed based on these reactions.
Fluorescence methods are known to have both high sensitivity and
high spatiotemporal resolution for visualizing biomolecules in vitro
and in vivo. Experimental operations are easy to perform. Therefore,
fluorescence methods for SNO detection should be attractive.
a Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
E-mail: mxian@wsu.edu; Fax: +1 509-335-8867
b College of Chemistry and Molecular Engineering, Zhengzhou University,
Zhengzhou 450052, China. E-mail: yeyong03@tsinghua.org.cn
c Department of Chemistry, Wake Forest University, Winston-Salem, NC 27109, USA
† Electronic supplementary information (ESI) available: Experimental proce-
dures, characterization data, and additional spectra. See DOI: 10.1039/
c4cc01288g
Scheme 1 The design of reductive ligation-based probes for SNO.
‡ Two authors contributed equally to this work.
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To test this hypothesis, we proposed a SNO probe SNOP1, as
shown in Scheme 2. A fluorescein (6) was selected as the fluorophore
as it is known that bis-OH acylation of fluoresceins quenches their
fluorescence.6d–f In addition, according to our previous experience,
acylation on both OH groups usually leads to a higher level of
fluorescence.7 With two phosphine moieties in the structure, the
probe could react with SNO molecules to release either the free
fluorescein or the mono-acylated fluorescein, both are strong fluor-
escent species with the same emission wavelengths. SNOP1 was
easily prepared in one step from fluorescein and 2-(diphenylphos-
phino)benzoic acid (7). The compound was fully characterized by 1H,
13C, 31P NMR and MS (see ESI†).
With the probe in hand, we first tested its fluorescence
properties in aqueous buffers. The Tris-HCl buffer system was
found to give the best results, so this system was used in all the
experiments described here. As expected, the probe itself
showed low fluorescence intensity. When a model SNO sub-
strate, 8 (50 mM), was added into the solution of the probe
(10 mM), significant increase of fluorescence intensity (B90 fold) was
observed (Fig. 1A). The fluorescence turn-on response was found to
be fast. The maximum intensity was reached in about 25 min. We
also tested the response of SNOP1 to S-nitrosoglutathione (GSNO),
which is an endogenous SNO (Fig. 1B). GSNO also led to very obvious
fluorescence increase, although at a smaller level (B18 fold) and
slower rate (reaching the maximum at B40 min) compared to
compound 8. GSNO is much more stable than other small molecule
SNO compounds. This is probably due to its structural character
which somehow protects SNO from decomposition. These properties
may also cause low or slow reactivity of GSNO toward reagents like
SNOP1, therefore it gave lower and slower fluorescence response. It
should be noted that the model reaction between SNOP1 and SNO
indeed led to both free fluorescein and mono-acylated fluorescein as
products (Fig. S1, ESI†), confirming the reaction mechanism
proposed in Scheme 2.
Fig. 1 Time-dependent fluorescence enhancement (F/F0) of SNOP1 (10 mM)
to 8 (50 mM) (A) and GSNO (50 mM) (B). The reactions were carried out at 37 1C
in Tris-HCl buffer (50 mM, pH = 7.4) with 1% DMSO (lex/em = 490/518 nm).
intensity was recorded after 45 min. We found that the intensities
increased almost linearly in the range of 0–30 mM for GSNO (Fig. 2).
The detection limits8 were calculated to be 90 nM for GSNO,
suggesting that the probe is reasonably sensitive. We also studied
the effects of pH values on the reaction. The probe worked nicely at
neutral pH values (6.5–7.5) (Fig. S2, ESI†). These results suggest that
the probe may be suitable for detecting SNO in biological samples.
We next examined the selectivity of the probe for other reactive
sulfur species and some common oxidative species (Fig. 3).
To demonstrate the efficiency of the probe in determining SNO
concentrations, a series of different concentrations of GSNO were
treated with SNOP1 (10 mM). For consistency, the fluorescence
Fig. 2 Fluorescence emission spectra of SNOP1 (10 mM) with varied concen-
trations of GSNO (0, 0.3, 0.5, 1, 3, 5, 7, 10, 15, 20, 30, 40, 50 mM for curves 1–13,
respectively). The reactions were carried out for 45 min at 37 1C in Tris-HCl
buffer (50 mM, pH = 7.4) with 1% DMSO (lex/em = 490/518 nm).
Scheme 2 The structure and preparation of SNOP1.
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the activity of SNOP1 toward HNO, we treated the probe (10 mM)
with a solution of Angeli’s salt (10 mM), a well known HNO donor.
The fluorescence signals were recorded in 5 min in Tris-HCl buffer
(50 mM, pH = 7.4, with 1% DMSO) at 37 1C. Indeed the probe
showed a dramatic fluorescence increase (about 235 fold, Fig. 4).
This reactivity with HNO may be a problem for using SNOP1 for
selective detection of SNO. However, it is known that HNO is a
highly reactive species which quickly undergoes dimerization to
hyponitrous acid followed by dehydration to give nitrous oxide
(N2O) and water.11 We suspected that if HNO co-exists with SNO in
the solution, HNO will decompose quickly and not be detected.
Therefore we studied the time-dependent stability of HNO using
SNOP1. In this study, a solution of Angeli’s salt (10 mM) was
prepared. At different time intervals (0–60 min) the probe was used
to monitor HNO. As shown in Fig. 4, the fluorescence intensities
decreased quickly. After about 10 min, there was almost no
fluorescence detected, suggesting a complete decomposition of
HNO. These results suggest that although SNOP1 gives a strong
fluorescence response to HNO, its signal can be observed only
when it is continuously formed and is present at the time of the
measurement. Because of this property, we believe SNOP1 will be
useful for selective detection of SNO, at least in some situations
and might be able to distinguish SNO from HNO based on its
time-dependent response. In biological systems, RSNOs exist
mainly as GSNO or S-nitrosated proteins. These RSNOs are
relatively stable. In contrast, HNO decomposes quickly if its
generation is terminated (the biosynthesis of HNO is still unclear).
We performed some model experiments to prove this hypothesis
(Fig. 5). Three solutions (Angeli’s salt only, GSNO only, Angeli’s salt +
GSNO) were prepared separately in Tris-HCl buffer (50 mM,
pH = 7.4, with 1% DMSO). The solutions were incubated for
20 min at 37 1C and then tested with SNOP1. The solution with
only Angeli’s salt gave negligible fluorescence signals while the
solutions with only GSNO gave strong fluorescence, at same levels
of directly treating GSNO with the probe. The solutions of Angeli’s
salt and GSNO together produced fluorescence similar to GSNO
only. These results further suggested that at the time of measure-
ment HNO was completely gone and the fluorescence could be
attributed to GSNO. Overall under these conditions HNO had no
influence on the fluorescence measurement of SNO.
Fig. 3 Fluorescence enhancement (F/F0) of SNOP1 (10 mM) to various RSS
or common oxides. The reactions were carried out for 45 min in Tris-HCl
buffer (50 mM, pH = 7.4) with 1% DMSO at 37 1C. (1) 10 mM SNOP1 only; (2)
1 mM GSH; (3) 100 mM Cys; (4) 100 mM Hcy; (5) 100 mM GSSG; (6) 100 mM
Na2S; (7) 100 mM Na2S2O3; (8) 100 mM Na2SO3; (9) 100 mM H2O2; (10)
100 mM NaClO; (11) 100 mM NaNO2; (12) 50 mM GSNO.
Sulfur containing compounds including glutathione (GSH), cysteine
(Cys), homocysteine (Hcy), oxidized glutathione (GSSG), hydrogen
sulfide (H2S), thiosulfates (S2O32ꢀ), and sulfites (SO32ꢀ) were tested
and did not give any significant fluorescence enhancement. Oxi-
dants such as hydrogen peroxide (H2O2), hypochlorites (ClOꢀ), and
nitrites (NO2ꢀ) were found to be non-reactive either. In comparison,
GSNO showed very significant fluorescent signals. To further inves-
tigate the specificity of the probe for SNO, two tertiary SNO
substrates based on S-nitrosopenicillamine were also tested. These
un-natural SNO showed excellent fluorescence responses to the
probe (Fig. S3, ESI†). These results demonstrated good selectivity
of the probe SNOP1 for SNO.
It should be noted that the reductive ligation was also used by
King et al. in trapping nitroxyl (HNO), although the phosphine-
derived product is slightly different.9 Recently Nakagawa and
coworkers have reported a structurally similar probe which showed
excellent activity for HNO, but with low response to SNO.10 To test
Fig. 4 Time-dependent fluorescence changes of HNO in Tris-HCl buffer
(50 mM, pH = 7.4) with 1% DMSO. Angeli’s salt (10 mM) was used as HNO
equivalent. The solutions of Angeli’s salt (10 mM) were kept at 37 1C for 0, 5,
10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 min, and then monitored using
SNOP1 (10 mM), respectively.
Fig. 5 Fluorescence enhancements of SNOP1 (10 mM) to HNO only (10 mM
AS), GSNO (50 mM) only, and HNO (10 mM AS) + GSNO (50 mM), respectively.
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N. Hogg, J. Joseph and B. Kalyanaraman, J. Biol. Chem., 1996,
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To ascertain the practical applicability of SNOP1 for bio-
logical samples, the detection of GSNO in diluted deproteinized
bovine plasma12 was performed successfully (Fig. S4, ESI†).
Upon gradual addition of GSNO (1 to 50 mM), we observed that
a remarkable emission increase appeared at 518 nm in diluted
deproteinized bovine plasma. An excellent linear correlation
between the added GSNO concentrations and the increased
fluorescence emission at 518 nm can still be obtained. These
results indicated that SNOP1 was suitable for sensitive analysis
of GSNO in biological samples.
In summary, we report in this study a new fluorescent probe for
the detection of SNO compounds. The design was based on SNO-
mediated reductive ligation using triaryl-phosphine substrates. This
probe showed good sensitivity and selectivity for some representa-
tive SNO substrates, including the endogenous GSNO.
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Financial support from the NIH (R01GM088226) and NSF
CAREER to M.X., NIH (R01HL62198) to S.B.K, the National
Science Foundation of China (No. 21375113) and the Program
for New Century Excellent Talents in University (NCET-11-0950)
to Y.Y., and CSC for Graduate Student Overseas Study Program
(grant number 201307040059) is greatly acknowledged.
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