Comparison of Reductive Ligation-Based Detection Strategies for Nitroxyl (HNO) and S-Nitrosothiols

Article abstract of DOI:10.1002/open.201500200

Phosphine-based detection strategies for both nitroxyl (HNO) and S-nitrosothiols (RSNO) were investigated and compared. Phosphorus NMR studies show that azaylides derived from HNO or organic RSNO efficiently participate in subsequent reductive ligation required for fluorescence generation in properly substituted substrates. S-Azaylides derived from biological RSNO containing free amine and carboxylic acid groups primarily yield phosphine oxides suggesting these groups facilitate nonligation pathways such as hydrolysis. The fluorescence response of a phosphine-based fluorophore toward the same RSNO confirms these differences and indicates that these probes selectively react with HNO. Flow cytometry experiments in HeLa cells reinforce the reactivity difference and offer a potential fast screening approach for endogenous HNO sources.

Full text of DOI:10.1002/open.201500200

DOI: 10.1002/open.201500200
Comparison of Reductive Ligation-Based Detection
Strategies for Nitroxyl (HNO) and S-Nitrosothiols
[
a]
Zhengrui Miao and S. Bruce King*
Phosphine-based detection strategies for both nitroxyl (HNO)
and S-nitrosothiols (RSNO) were investigated and compared.
Phosphorus NMR studies show that azaylides derived from
HNO or organic RSNO efficiently participate in subsequent re-
ductive ligation required for fluorescence generation in proper-
ly substituted substrates. S-Azaylides derived from biological
RSNO containing free amine and carboxylic acid groups pri-
marily yield phosphine oxides suggesting these groups facili-
tate nonligation pathways such as hydrolysis. The fluorescence
response of a phosphine-based fluorophore toward the same
RSNO confirms these differences and indicates that these
probes selectively react with HNO. Flow cytometry experi-
ments in HeLa cells reinforce the reactivity difference and offer
a potential fast screening approach for endogenous HNO
sources.
phosphine oxide (2) and azaylide (3), which in the presence of
an electrophilic ester, undergoes Staudinger ligation to yield
amide (4) and the corresponding fluorescent alcohol (5,
Scheme 1).
S-Nitrosothiols (RSNO) represent an important type of post-
translational modification that preserves and amplifies NO sig-
[9]
naling and regulates protein activity. Variation and dysregula-
tion of RSNO levels are associated with the etiology of diverse
[10]
diseases. Faster and specific detection and quantification of
RSNO will better elucidate their behavior in vivo and define
a better understanding of their therapeutic potential. Current
RSNO detection depends on indirect assays that limit the over-
[11]
all specificity of the measurements. A recent report on re-
ductive ligation of phosphines with some model RSNO pro-
[12]
vides insight into new RSNO detection approaches.
Like
HNO, phosphines react with RSNO to give phosphine oxide (2)
and an S-azaylide (6), which undergoes ligation to form a sulfe-
[
12]
Nitroxyl (HNO), the one-electron reduced/protonated form of
nitric oxide (NO), shows distinct physiology and pharmacology
namide (7) and an alcohol (5, Scheme 1). The reactivity of
phosphines with RSNO has been exploited to develop fluores-
cent and mass spectrometric-based probes for RNSO detection
[
1]
from NO. Specifically, HNO inhibits the activity of various
thiol-containing enzymes and regulates cardiovascular signal-
ing, making it an intriguing candidate for many physiological
disorders such as alcoholism and congestive heart failure
[13]
that possess similar structures to those for HNO.
While increasing efforts have been made in designing new
phosphine-based fluorescent probes for HNO and RSNO detec-
tion, little attention has been paid to the cross reactivity of
HNO and RSNO with the same phosphine-based detection sys-
tems. Prior experiments consistently demonstrate that HNO in-
duces a greater fluorescence response than S-nitrosogluta-
[
2]
(
CHF). The lack of fast and reliable HNO detection methods
applicable to living cells limits the biological understanding of
HNO and identification of endogenous sources. New detection
approaches have been developed for robust HNO identifica-
[
3]
tion including copper-based fluorescent complexes, a cobalt-
thione (GSNO) or S-nitrosocysteine (CysNO) upon reaction with
[
4]
[6a,b,d,e,13b]
porphyrin electrochemical method, a membrane inlet mass
the same phosphine.
Such differences suggest a rela-
[
5]
spectrometry (MIMS) approach, and a series of phosphine-
tive specificity for HNO over RSNO in their reaction with organ-
ic phosphines. The overall similarity of these two described re-
action pathways complicates phosphine-based detection strat-
egies of both species and opens the possibility of false positive
results in vivo. Concerns regarding the reliability of these
probes exist, and in vivo screening of HNO with these probes
remains risky without a rationale for the diminished fluores-
cence response from GSNO. Here, we directly investigated the
reactions of two phosphines (1a–b) with HNO and RSNO and
reveal important differences vital for the fluorescence re-
sponse. Unlike the HNO-derived azaylide or the S-azaylide de-
scribed in earlier reports, the S-azaylide formed from the reac-
tion of phosphines with GSNO or CysNO does not efficiently
participate in the reductive ligation needed for fluorophore
generation. This difference was further confirmed by using 1b
[
6]
based fluorescent probes. Organophosphines have been in-
tensively studied due to their fast and selective reaction with
[
7]
HNO compared with other nitrogen oxides. Despite their vari-
ous structures, all of the reported phosphine probes react with
[
8]
HNO in a similar fashion (Scheme 1). Two equivalents of
phosphine (1) react with HNO to produce equal amounts of
[a] Z. Miao, Prof. S. B. King
Department of Chemistry, Wake Forest University
Winston-Salem, NC 27109 (USA)
E-mail: kingsb@wfu.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/open.201500200. Experimental details includ-
ing synthetic procedures, NMR and mass spectra, and flow cytometry
protocols are available in the Supporting Information.
[6e]
ꢀ
2015 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
to detect HNO (vs. RSNO) in HeLa cells using flow cytometry.
This is an open access article under the terms of the Creative Commons
Attribution-NonCommercial-NoDerivs License, which permits use and
distribution in any medium, provided the original work is properly cited,
the use is non-commercial and no modifications or adaptations are
made.
Fluorescence generation described in Scheme 1 relies on
productive ylide formation and reductive ligation with release
of a competent fluorophore. For the reaction of 1 with HNO,
a productive ligation sequence should yield an equivalent of
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Scheme 1. Triarylphosphine reactions with HNO and RSNO.
phosphine oxide amide (4), phosphine oxide ester (2), and the
alcohol (or fluorophore, 5). Given that 2 and 5 could arise from
either oxidative or hydrolytic pathways, 4 represents a distinct
indicator of a productive ligation process. For the RSNO reac-
tion, the sulfenamide (7) represents an analogous ligation
product, which can be reduced to 4 in the presence of excess
[
12]
phosphine (Scheme 1).
We monitored the reactions of phosphine probes with HNO
and GSNO by phosphorus nuclear magnetic resonance (NMR)
spectroscopy and HPLC-MS (Figures S3–S8 in the Supporting
Information). Treatment of phenyl 2-(diphenylphosphino)ben-
zoate (1a) with Angeli’s salt (AS), a common HNO donor, re-
veals the formation of 4 (d=34.8 ppm), the corresponding
phosphine oxide (2a, d=33.7 ppm) as expected, and an addi-
tional peak (d=35.8 ppm), identified by HPLC-MS as 2-(diphe-
nylphosphino)benzoic acid oxide, possibly from the hydrolysis
of 2a (Figure 1A). The amide peak (4, d=34.8 ppm) indicates
productive ligation. In contrast to reported efficient reductive
3
1
Figure 1. P NMR spectra for the reaction of 1a–b (0.01 mmol) with HNO or
GSNO (0.02 mmol) in CD CN/THF/Tris-HCl Buffer (100 mm, pH 7.4, 3:1:2) after
3
60 min. A–B) Resonances correspond to 4 (34.8 ppm) and 2a (33.7 ppm).
C–D) Resonances correspond to 4 (34.8 ppm) and 2b (33.7 ppm).
[
12]
ligation results,
treatment of this model phosphine with
GSNO only yields 2a (d=33.7 ppm) with trace amounts of 4 or
sulfenamide (7, Figure 1B), revealing inefficient ligation. Incu-
bation of fluorescent probe (1b) with AS gives a similar
[6d]
in the Supporting Information).
These results clearly show
31
P NMR spectrum showing complete conversion of 1b to 4
d=34.8 ppm), the corresponding phosphine oxide (2b, d=
3.7 ppm, Figure 1C) and 2-(diphenylphosphino)benzoic acid
oxide (d=35.8 ppm). The formation of 4 correlates with
different reactivity of phosphine probes with HNO and GSNO
under these conditions. The HNO reaction reliably gives Stau-
dinger ligation products (that yield fluorescence in properly
designed compounds) but the GSNO reaction primarily yields
phosphine oxide with little evidence of ligation, which corre-
sponds to the low fluorescence response observed in previous
(
3
[
6e]
a strong and rapid fluorescence response. Similarly, incuba-
tion of 1b with GSNO yields mostly phosphine oxide (2b, d=
[6a,b,d,e]
3
3.7 ppm) and a trace amount of another phosphorus product
Figure 1D). A previously reported coumarin-derived fluores-
cent HNO probe (P-CM) demonstrates similar results (Figure S1
reports.
While reductive ligation phosphine-based fluorescent detec-
(
[6]
tion strategies for HNO are becoming well established, the
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use of similar compounds for RSNO detection remains to be
fully developed. Early studies show the ability of phosphines to
react with RSNO to form S-azaylides that undergo ligation re-
actions but these discoveries have not been translated to
[
12]
a robust RSNO detection system.
A mass spectrometric
phosphine-based method for GSNO has been reported and
this work also shows that probes with similar structures to 1a–
[
13a]
b do not undergo ligation with GSNO.
The reasons for the
observed differences in the ligation reactivity of phosphines
and HNO (reliable) and RSNO (unreliable) remain to be defined.
Kinetic studies on the reaction of phosphines with HNO or
RSNO show that initial phosphine addition occurs rapidly (with
[
7,13a,14]
the HNO reaction being slightly faster).
Differences in
the reactivity of the azaylide intermediates (3 or 6) likely play
a role in the observed reactivity as the rate determining step
and rate of classical Staudinger ligations vary significantly with
[
15]
31
azaylide stability. Examination of previous work shows that
in general only organic RSNOs or fully protected versions of
biological RSNO derivatives (fully protected CysNO) yield suc-
Figure 2. P NMR for reaction of 1a (0.02 mmol) with CysNO and its deriva-
tives (0.01 mmol): A) N-acetyl-CysNO-methyl ester, B) CysNO-methyl ester,
C) N-acetyl-CysNO, D) CysNO in CD
:1:2) in dark after 30 min.
3
CN/THF/Tris-HCl Buffer (100 mm, pH 7.4,
3
[
6a,d,e,12–13]
cessful and reliable reductive ligation.
We speculate
that the S-azaylides derived from GSNO and CysNO that con-
tain both free carboxylic acid and amine groups may be less
stable than those from the organic RSNO. Both of these RSNO
exist as a zwitterion at physiological pH, and these functional
groups may react with the S-azaylide facilitating other possible
reaction pathways such as hydrolysis to yield the observed
phosphine oxide.
As described in Scheme 1 for phosphine-based fluorescent
probes, successful ligation processes occur accompanied by
the generation of fluorescence. Hence, the fluorescence re-
sponse of 1b toward these four CysNO derivatives should
remain consistent with the data presented in Figure 2. Indeed,
addition of 2 equiv of N-acetyl CysNO methyl ester to 1b indu-
ces a significant (10.1-fold) fluorescence increase compared
with CysNO methyl ester (2.9-fold), which is much greater than
N-acetyl CysNO and CysNO (Figure 3). These fluorescence
To explore this idea, the reaction of various CysNO deriva-
tives with 1a followed by phosphorus NMR and HPLC-MS anal-
ysis (Figures S9–S12 in the Supporting Information) provides
further information regarding RSNO structure in these reac-
tions. Previous work shows N-acetyl CysNO methyl ester, a fully
protected CysNO derivative, reacts with 1a via ligation to give
[
12]
the sulfenamide in 84% yield. In our hands, treatment of 1a
with N-acetyl CysNO methyl ester yields three phosphorus-
containing products, the expected phosphine oxide (2a, d=
33.7 ppm), sulfenamide (7a, d=34.3 ppm) and the amide (4,
d=34.8 ppm), a ligation product that forms from the reduc-
[
12]
tion of 7a by 1a (Figure 2A). Both 7a and 4 result from a re-
[
12]
ductive ligation process and confirm previous work. Howev-
er, treatment of CysNO methyl ester, which contains a free
amine, with 1a only provides small amounts of 7a and 4 with
the phosphine oxide as the major species (Figure 2B). Incuba-
tion of N-acetyl CysNO, which contains a free carboxylic acid,
results in primarily phosphine oxide (2a) with a small amount
of another downfield phosphorus-containing product (d=
Figure 3. Fluorescence responses of 1b (40 mm) in CH CN/PBS at rt for
3
60 min after addition of CysNO derivatives (2 equiv). Data are the
averageÆSD of two independent experiments.
5
3.5 ppm, Figure 2C), presumed to a phosphonium salt as
[
13a,16]
judged by HPLC-MS (Figure S11).
Reaction of CysNO with
1
a yields phosphine oxide (2a) as the only phosphorus-con-
results correlate with the amount of ligation product observed
by phosphorus NMR (more fluorescence with more ligation
product) and support the idea that RSNO structure ultimately
controls the stability of the derived S-azaylide.
taining product (Figure 2D). These results indicate that the
structure of the RSNO influences the final product selectivity
possibly by influencing the stability of the S-azaylide. The reac-
tions with RSNO substrates containing free carboxylic acid and
amine groups predominantly generate phosphine oxide (Fig-
ure 2B–D) suggesting the presence of these groups facilitate
S-azaylide hydrolysis as the most direct mechanism of phos-
phine oxide formation.
Additional phosphorus NMR experiments followed by HPLC-
MS of the reactions of these S-nitroso cysteine derivatives with
triphenylphosphine reinforces these ideas (Figures S2, S13—16
in the Supporting Information). The reaction of N-acetyl CysNO
methyl ester with PPh gives an expected 1:1 ratio of phos-
3
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phine oxide and the proposed S-azaylide (confirmed by MS)
while CysNO derivatives containing either the free carboxylic
acid and amine groups predominantly generate phosphine
oxide.
tion of protein RSNO by adding diethylamine (DEA)/NONOate
(a well-known NO donor) also does not yield a fluorescence re-
sponse. Addition of AS to cells containing 1b immediately re-
sults in an increase in fluorescence that grows over 30 min.
The successful detection of HNO-induced fluorescence in nu-
merous cells and the lack of response from GSNO or protein
RSNO-derived from NO treatment confirm the difference in re-
activity and reveal the possibility for HNO detection in vivo
using these phosphine probes. The combination of such
probes with flow cytometry may facilitate the search for en-
dogenous HNO formation from different primary cells.
These experiments, along with previously reported work, in-
dicate that HNO and RSNO distinctly react with triarylphosphi-
[
6a,b,d,e,13]
nes.
Triarylphosphine probes react rapidly with both
HNO and RSNO to generate a 1:1 mixture of the corresponding
[
8,12]
azaylide and the phosphine oxide (Scheme 1).
In the case
of HNO, these simple unsubstituted azaylides efficiently under-
go Staudinger ligation with properly positioned esters to gen-
erate the fluorophore and the amide phosphine oxide (4, Sche-
In summary, we compared the phosphine detection strat-
egies for HNO and RSNO. The reactions of phosphines with
HNO and organic or biological RSNO demonstrate clear differ-
ences in the ability of the intermediate ylides to undergo liga-
tion leading to fluorophore release. Ylides derived from HNO
or organic RSNO tend to participate in the ligation process,
while ylides from biological RSNO that contain free carboxylate
and amino groups do not readily undergo ligation and prefer-
entially react (perhaps through hydrolysis) to form the phos-
phine oxide. This reactivity difference was confirmed by moni-
toring the fluorescence response in HeLa cells. Successful de-
tection of HNO in cells using 1b by flow cytometry without
biological RSNO interference illustrates the reliability of phos-
phine probes for HNO and may serve as a fast robust screen-
ing approach for endogenous HNO sources.
[
2c,6,13b]
me 1).
While RSNO species also quickly generate an
S-azaylide, these appear to only undergo efficient ligation reac-
tions (required for fluorophore release) with simple organic
RSNO or with fully-protected peptide or amino acid RSNO de-
[
12,13b]
rivatives.
Under predominantly aqueous conditions in the
presence of free carboxylic acid and amine groups, the non-
fluorescent phosphine oxide represents the major phosphorus-
containing product indicating that other nonligation reaction
pathways of the S-azaylide dominate. Considering the physio-
logical environment and the presence of amine and carboxylic
acid groups in many proteins or in biological constituents, pro-
tein RSNO will mostly likely react with phosphine probes to
yield phosphine oxides. Such reactivity suggests that phos-
phine-based fluorescent probes demonstrate selectivity for
HNO and will not to be interfered with by RSNO.
To further confirm the reactivity difference, we measured the
intracellular fluorescence increase in HeLa cells treated with 1b Acknowledgements
by adding an HNO donor and RSNO. Intracellular fluorescence
was determined by flow cytometry, which generates more stat-
istically reliable data by simultaneous measurement of millions
of cells and focuses on normal cells, avoiding faulty data from
abnormal and/or dead cells. Figure 4 and Figures S17–18 in the
Supporting Information show the mean fluorescence intensity
value difference between 1b-treated HeLa cells with different
substrates. Direct addition of GSNO to cells does not yield an
increase in fluorescence (Figure 4). Possible intracellular forma-
This work was supported by Wake Forest University and the
Center for Molecular Communication and Signaling of Wake
Forest University. Flow Cytometry services were supported by the
Comprehensive Cancer Center of Wake Forest University NCI
CCSG P30CA012197 grant.
Keywords: fluorescence · nitroxyl · phosphine · reductive
ligation · S-nitrosothiol
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Received: October 23, 2015
sekera, S. Boncompagni, D. L. Galvan, C. P. Gilman, M. R. Baker, N. Shiro-
Published online on && &&, 0000
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COMMUNICATIONS
Z. Miao, S. B. King*
HNO… it’s me! Call me on my sulfone.
Phosphine-based detection strategies
for both nitroxyl (HNO) and S-nitroso-
thiols (RSNO) were investigated and
compared. Phosphorus NMR and HPLC-
MS studies show that azaylides derived
from HNO or organic RSNO efficiently
participate in subsequent reductive liga-
tion required for fluorescence genera-
tion in properly substituted substrates.
S-Azaylides derived from biological
RSNO containing free amine and car-
boxylic acid groups primarily yield phos-
phine oxides and cannot generate fluo-
rescence effectively.
&& – &&
Comparison of Reductive Ligation-
Based Detection Strategies for
Nitroxyl (HNO) and S-Nitrosothiols
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ÝÝ These are not the final page numbers!