Direct NMR detection of the unstable "red product" from the reaction between nitroprusside and 2-mercaptosuccinic acid

Article abstract of DOI:10.1039/c5dt04029a

The reaction between nitroprusside (NP, [Fe^II(CN)₅NO]^2-) and organic thiolates (RS^-) in aqueous solution has long been known to produce an unstable red intermediate thus often being referred to as the "red product" (RP) in the literature. While RP has always been formulated as [Fe^II(CN)₅N(O)SR]^3-, it is rather difficult to study it in aqueous solution because it is not only unstable but also exhibits rapid ligand exchange. All previous studies of RP have relied on UV-vis, IR, kinetics measurements, and analysis of decomposed products. Herein we report the first comprehensive multinuclear (¹H, ¹³C, ¹⁵N, and ¹⁷O) NMR characterization of the RP produced from the reaction between NP and 2-mercaptosuccinic acid (MSA). The NMR chemical shifts obtained for the RP are compared with those from the free ligand (S-nitrosothiol, RS-NO) prepared in situ by the reaction of MSA with NaNO₂. We also showed that useful thermodynamic and kinetic properties of RP formation can be readily obtained from ¹H NMR studies.

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Direct NMR detection of the unstable “red product” from reaction
between nitroprusside and 2-mercaptosuccinic acid†
a
a
a
Yin Gao, Brendan Mossing, and Gang Wu*
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
5
DOI: 10.1039/b000000x
II
2–
–
The reaction between nitroprusside (NP, [Fe (CN)
5
NO] ) and organic thiolates (RS ) in aqueous solution
has long been known to produce an unstable red intermediate thus often being referred to as the “red
II
3–
5
product” (RP) in the literature. While RP has always been formulated as [Fe (CN) N(O)SR] , it is rather
difficult to study it in aqueous solution because it is not only unstable but also exhibits rapid ligand
exchange. All previous studies of RP have relied on UV-vis, IR, kinetics measurement, and analysis of
1
0
1
13
15
17
decomposed products. Herein we report the first comprehensive multinuclear ( H, C, N, O) NMR
characterization of the RP produced from reaction between NP and 2-mercaptosuccinic acid (MSA). The
NMR chemical shifts obtained for RP are compared with those from the free ligand (S-nitrosothiol, RS-
2
N=O) prepared in situ by reaction of MSA with NaNO . We also showed that useful thermodynamic and
1
1
5
kinetic properties of RP formation can be readily obtained from H NMR studies.
.
II
3–
to produce RSSR and [Fe (CN)
5
NO] . The latter can further
O and NH
3
Introduction
undergo complex decomposition to produce NO, N
2
among other species, depending strongly on experimental
conditions, as illustrated in Scheme 1. Because of this
extraordinary instability, no crystal structure has ever been
S-nitrosothiols (RSNOs) have been intensely investigated in the
past 20 years, because of their important roles in protein post-
translational modification, NO-mediated bioactivity, and
50
2
2
3
3
4
4
0
5
0
5
0
5
1
-10
potential therapeutic applications.
RSNO bioregulatory action however remains unknown. Recently,
the reactivity of RSNOs towards H S under physiological
conditions has attracted considerable attention
The general mechanism of
Scheme 1 Formation of RP and its subsequent decomposition in aqueous
solution.
2
1
1-15
and also
This new reaction pathway adds
further complication for possible “cross talks” between the two
major gaseous signaling molecules, NO and H S. While transition
3
!
1
6-20
generated controversies.
S
O
Fe(CN) NO]2! + RS!
R
N
[
5
2
metal catalyzed RSNO formation/decomposition has long been
considered to be a possible pathway for RSNO bioregulatory
function in biological systems, the field of coordination chemistry
of RSNOs remains largely unexplored. For example, it is well
NP
Fe(CN)5
RP
2
+
+
known that metal ions such as Hg and Cu can catalyze the
3!
[
Fe(CN) NO] + " RSSR
5
1
,21
decomposition of RSNOs. But the detailed mechanism has not
2
2
yet been firmly established. Recently, Kozhukh and Lippard
2
+
showed that Zn can also catalyze RSNO decomposition to
release gaseous NO and N O. In this case, however, RSSR was
NO NH3 N O
2
2
not found among the decomposed products. In general, well
characterized transition metal coordination complexes containing
55
reported for any RP. The closet analogs to RPs are a series of
Ir(III)-N(O)RS complexes reported by Doctorovich and co-
2
3,24
RSNO ligands are extremely rare.
Among them the so-called
3
6,37
workers
In addition to their poor stabilities, RPs often exhibit
“red products” (RPs), which are produced from reaction between
II
2–
–
rapid ligand exchange as indicated in Scheme 1, making them
rather difficult to characterize by NMR. As a result, all previous
nitroprusside (NP, [Fe (CN)
5
NO] ) and organic thiolates (RS )
2
5-35
in aqueous solution, are the most extensively studied.
It is
5
N(O)SR] , where
II
3–
60 studies of RPs have relied on UV-vis, IR, kinetics measurement,
and chemical analysis of decomposed products. While RPs
themselves are diamagnetic, many decomposed products are
commonly accepted that the RP is [Fe (CN)
1
RSNO is coordinated to the Fe(II) center in the κ -N binding
mode. However, RPs are generally unstable, decomposing rapidly
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1
paramagnetic and thus can be studied by EPR. Clearly, it is
highly desirable to have a more direct means of probing the
formation and transformation of unstable RPs. Some time ago,
ppm) are consistent with the κ -N binding mode as shown in
1
5
Scheme 1. Similar N coordination shifts were also observed for
1
43,44
C-nitroso metal compelxes in the κ -N mode of binding.
It is
3
3
1
Stasicka and co-workers reported that the RP generated from
interesting to note that, for the κ -O mode of binding, while the
1
5
5
0
5
0
reaction between NP and 2-mercaptosuccinic acid (MSA) is 60 N coordination shifts (200 ppm) are similar to those found for
1
17
relatively stable. We decided to utilize the stability of this
the κ -N complexes, significantly larger O coordination shifts
1
7
15
13
1
42
17
particular RP to obtain its multinuclear ( O, N, C, H) NMR
signatures. The present work was also motivated by our recent
finding that the red-violet and blue transient intermediates in
(600 ppm) were detected. This illustrates the uniqueness of
O
NMR to differentiate between the two distinct modes of metal
1
5
17
binding. Table 1 summarizes the N and O NMR data reported
1
1
2
Gmelin reaction (between nitroprusside and hydrogen sulfide) are 65 for related compounds in the literature. A parallelism between
4
–
4–
38
15
17
in fact [Fe(CN)
5
N(O)S] and [Fe(CN)
5
N(O)SS] , respectively.
N and O chemical shifts is clearly seen for the RSNO-related
compounds. However, it is important to point out that it took only
–
The thionitro ligand in the former complex, [SNO] , is the
deprotonated form of HSNO, which can be considered to be the
1
7
4 min to acquire the O NMR data shown in Figure 2a, whereas
4
–
15
smallest S-nitrosothiol. Thus, [Fe(CN)
5
N(O)S] may be seen as a
the corresponding N NMR spectra shown in Figure 2b were
special type of RP. Indeed, aqueous solution of 70 recorded in 50 min. This drastically short experimental time in
4
–
17
[
Fe(CN)
5
N(O)S] exhibits nearly the same red coloration (λmax
=
O NMR experiments is due to the rapid quadrupolar relaxation,
5
30 nm) as do all RPs. Another objective of this work is to
1
7
continue our effort to explore the use of O NMR as a new
technique for probing highly reactive and unstable
3
9
17
intermediates/products. The advantage of O NMR over the
1
5
more conventional N NMR in studying NO related compounds
1
7
is that the very short O spin-lattice relaxation time allows very
rapid data acquisition, thus permitting detection of very short-
3
8
lived species as we demonstrated recently.
2
5
Results and discussion
1
Figure 1 shows the UV-vis and H NMR spectra of the reaction
solution containing NP and MSA in a 1:1 molar ratio. The
formation of the RP is evident from the brilliant red color of the
solution (λmax = 526 nm). Under the conditions employed in this
3
3
4
4
5
5
0
5
0
5
0
5
½
study, this RP has a half-life (t ) of about 2 hrs at pH 11.
1
Interestingly, the H NMR spectrum of the reaction solution
displays two sets of peaks suggesting that RP and free MSA are
at equilibrium. This observation also suggests that, for MSA, the
reversible process of the RP formation is slow on the NMR
timescale (vide infra). Because of this rather slow ligand
exchange process, it is possible to fully characterize RP for the
1
17
15
13
first time by multinuclear ( H, O, N, C) NMR. As seen from
1
7
Figure 2, the
O NMR signal obtained for the RP,
II
17
3–
17
38
[
Fe (CN)
5
N( O)SR] , appears at 1036 ppm. This O NMR
which makes it possible to detect very short-lived species.
signal is quite broad with a full width at the half-height (FWHH)
of ca. 4.2 kHz. This is because the relatively large size of the RP
1
7
15
induces a very rapid O nuclear quadrupole relaxation. The
N
75 Fig. 1 (a) UV-vis spectrum (0.1 mm pathlength) of the RP prepared by
reacting 50 mM MSA with 50 mM NP in aqueous solution (pH 11, 50
mM sodium carbonate buffer, 0.1 M NaCl, 0.5 mM EDTA and 12 mM
NMR signal for the RP is at 607 ppm. In both cases, weaker
signals from NP were also observed, in agreement with the
1
KCN). The brilliant red color of the RP is shown in the inset. (b) H NMR
1
conclusion drawn from the H NMR data shown in Figure 1 that
1
spectrum of 10 mM MSA in aqueous solution at pH 11. (c) H NMR
both RP and free ligands are present. To compare these NMR 80 spectrum of the same reaction solution as in (a).
data with those from a free RSNO ligand, we reacted MSA with
NaNO
2
in a 1:0.7 molar ratio and immediately recorded NMR
1
7
15
As this particular RP is relatively stable, we were also able to
spectra. As shown as insets in Figures 2a and 2b, the O and
N
1
3
record a C NMR spectrum at the natural abundance; see Figure
NMR signals from a free RSNO ligand appear at 1200 and 761
ppm, respectively. It is immediately clear that, upon coordination
1
3
2
c. The most interesting observation in the C NMR spectrum is
1
7
15
85 that the equatorial and axial CN groups appear at 167.6 and 162.8
ppm, respectively. These are drastically different from the
corresponding signals found in NP, 134.5 (CNeq) and 132.3
to the Fe(II) center, both O and N chemical shifts of the
RSNO ligand change significantly. Considering that the
1
7
15
42
δ( O)/δ( N) ratio is typically 1.8 for nitroso compounds, the
3
7
13
1
5
17
(
CNax) ppm. Recently, we reported that the experimental
C
observed nearly equal N and O coordination shifts (ca. 160
2
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1
7
15
13
Fig. 2 (a) O, (b) N, and (c) C NMR spectra of the RP prepared by reacting 100 mM MSA with 100 mM NP in aqueous solution (pH
1
7
15
1
1, 50 mM sodium carbonate, 0.1 M NaCl, 0.5 mM EDTA and 12 mM KCN). In (a) and (b), O- and N-labled NPs were used
1
7
15
respectively. The experimental time to acquire the O (recycle delay 40 ms, 5435 transients), N (recycle delay 1.3 s, 2300 transients),
1
3
17
15
and C (recycle delay 1.2 s, 1840 transients) NMR spectra was 4, 50, and 37 min, respectively. In the insets of (a) and (b), O and
N
5
NMR spectra obtained for the free RSNO ligand are shown for comparison. The free RSNO ligand was prepared in situ by reacting MSA
1
5
17
with NaNO
2
(either 60% N and 20% O labeled) in a 1:0.7 molar ratio. In (c) the two signals marked by * are due to the presence of a
4
–
13
very small amount of decomposed products: [Fe(CN)
6
]
and RSSR. The C NMR signals from NP were too weak to be seen because
1
3
they have longer C spin-relaxation times than those from RP and were thus partially saturated under the current experimental condition.
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1
5
17
–1
Table 1 Comparison of N and O chemical shifts (δ in ppm) and vibrational frequencies (ν in cm ) between RP and other related RSNO compounds.
1
5
17
Compound
δ( N)
δ( O)
ν
NO
ν
758
NS
Ref.
3
–
–
–
[
Fe(CN)
5
N(O)SR] (R = -CH(COO )(CH
2
)-COO )
607
1035
1390
1380
1505
This work
3
–
[
Fe(CN)
5
N(O)SEt]
⎯
761
⎯
⎯
35
–
–
R-S-N=O (R = -CH(COO )(CH
2
)-COO )
1200
⎯
This work
a
R-S-N=O (R = a variety of groups)
765-830
700
⎯
⎯
⎯
40
38
38
39
39
36
41
4
–
[
[
[
[
[
Fe(CN)
Fe(CN)
Fe(CN)
Fe(CN)
5
5
N(O)S]
1028
938
1254
1358
1352
1935
1431
2008
805
⎯
4
–
N(O)SS]
632
3
–
5
5
N(O)H]
640
1099
419
⎯
2
–
(NO)] (NP)
373
⎯
–
IrCl
IrCl
4
5
(MeCN)N(O)SR] (R = -CH
2
Ph)
⎯
⎯
⎯
⎯
778
⎯
–
[
(NO)]
a
15
The listed values are obtained by adding 39 ppm to those reported in ref. 39. This is because the N chemical shifts in ref. 39 were referenced by setting
1
5
15
2
the N NMR signal of Na NO to 570 ppm, but in our work the same signal was determined to be at 609 ppm.
4
–
5
0
5
0
5
0
5
0
5
signal from the CNeq groups in [Fe(CN) N(O)S] is 174.7 ppm
and this signal is predicted by quantum chemical computation
to shift to 159.3 ppm, if the RSNO ligand is neutral, i.e.,
(a)
14
15
!NO"
N- N
3
–
[
5
Fe(CN) N(O)SH] . We then hypothesized that the CNeq
signal should be a sensitive probe to the protonation state of the
HSNO ligand. Indeed, our observation that the RP exhibits
1
1
2
2
3
3
4
1
3
13
δ( Ceq) = 167.6 ppm confirms this hypothesis. A full list of
C
chemical shifts are provided in the ESI. The small signal at 177
ppm (marked with * in Figure 2c) is due to the presence of a
4
–
trace amount of [Fe(CN)
6
]
from RP decomposition. Over a
-0.10
0
1
3
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400
period of about 3.5 hrs, all the C NMR signals from RP
disappear, producing a set of new signals attributable to RSSR;
see ESI.
!
/cm#1"
(b)
RP
After having obtained a complete set of multinuclear NMR
data for the RP, we decided to further characterize this
particular stable RP with FTIR. Figure 3 shows the FTIR
spectra obtained for both free RSNO ligand and RP. The
relevant IR data from the literature are also given in Table 1.
The νNO stretching for the free RNSO ligand was observed at
!NS"
14
15
!NO"
! 4
NP
N- N
0.00
-0.05
-0.10
0.4
0.3
0.2
0.1
0
!NO"
!CN"
–
1
!
CN"
1
505 cm , and it is shifted to a lower wavenumber in the RP,
–
1
1390 cm . This is consistent with that reported by Schwane
and Ashby. A similar trend was also noted by Doctorovich
3
4
3
6,37
2400 2200 2000 1800 1600 1400 1200 1000 800 600 400
and co-workers
in the Ir-RSNO complexes. As seen from
!
/cm#1"
Figure 3b, we were also able to detect a small peak attributable
–
1
to νNS at 758 cm . This appears to be the only reported νNS for
RPs. However, we should point out that similar νNS values
Fig. 3 ATR-FTIR spectra of (a) free RSNO ligand prepared in situ
from mixing MSA with NaNO (1:0.7 molar ratio) and (b) RP prepared
under the same conditions as described in Fig.2b.
3
6,37
were reported for Ir-RSNO complexes.
It is also interesting
4
5
2
to note that the νNO stretch of RP is higher than that found in
4
–
[
Fe(CN)
5
N(O)S] , but the νNS stretch is lower. This
observation further confirms the deprotonated state of the
4
–
As mentioned earlier, the observation of both RP and MSA
HSNO ligand in [Fe(CN)
b that both NP and RP are present in solution, which is in
agreement with the NMR data discussed earlier. In RP, two νCN
5
N(O)S] . It is also clear from Figure
1
signals in the H NMR spectra suggests that the ligand
3
50
exchange is slow on the NMR timescale. This provides us with
a rare opportunity to actually determine the thermodynamic and
kinetic properties of the reaction between NP and MSA. To this
–
1
signals were observed, 2084 and 2054 cm , both being
considerably shifted to lower wavenumbers than that found in
1
–
1
end, we recorded H NMR spectra of the NP/MSA reaction
NP, 2145 cm . This was also noted by Stasicka and co-
3
3
workers.
4
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(a)
(b)
ppm
3
03 K
NOE
2
3
3
.5
.0
.5
2
2
93 K
83 K
Exchange
RP
MSA
3.4
4.0
2
73 K
4
.4
4.2
4.0
3.8
(1H)/ppm
3.6
3.2
4
.0
3.5
3.0
(1H)/ppm
2.5
ppm
!
!
1
1
Fig. 4 (a) The H
1
region of the 1D H NMR spectra recorded at different temperatures. (b) The entire 2D H NOESY (EXSY) spectrum (298 K, mixing
time of 2 s) of the reaction solution containing 50 mM MSA and 50 mM NP. In (b), the H
1
region is indicated with dotted lines. Note that cross peaks due
5
to chemical exchange (red) and NOE (black) display different phases as compared with the diagonal peaks.
35
particularly suited in the study generally unstable S-nitrosotiol
1
7
solution at different temperatures. As seen from Figure 4a, as
the temperature of the solution decreases, both sets of signals
sharpen, suggesting a slowing down of the ligand exchange,
and the RP concentration increases, indicating a shift of the
equilibrium towards RP production. The ligand exchange
process was further confirmed by recording 2D NOESY
compounds. One can envisgae future studies in which
O
NMR can be used to monitor formation of RSNOs and to
follow chemical transformation of the S-N=O group in
biological systems. It is also possible to use this new approach
1
1
2
0
5
0
40 to study chemical reactions between RSNOs and H
along this line is underway in our laboratory.
2
S. Research
(
EXSY) spectra at different temperatures; Figure 4b. From
1
these VT
H
NMR data, we obtained the following
Acknowledgements
–
1
thermodynamic and kinetic data: ∆H° = –88.1 kJ mol , ∆S° =
–
1
–1
‡
–1
‡
–1
G. W. thanks the Natural Sciences and Engineering Research
Council (NSERC) of Canada for financial support.
–
260.1 J mol K ; ΔH = 25.9 kJ mol , ΔS = –118.2 J mol
–
1
K . These are in reasonable agreement with the results from
2
8
earlier temperature-jump/stop-flow kinetic measurements.
45
Notes and references
This is the first time that this kind of information are directly
1
a
obtained for RPs from H NMR experiments. Our results show
Department of Chemistry, Queen’s University, 90 Bader Lane,
that, as long as the ligand exchange is slow on the NMR
Kingston, Ontario, Canada K7L 3N6. E-
mail:gang.wu@chem.queensu.ca.
1
timescale, RP formation can be readily monitored by H NMR.
†
Electronic supplementary information (ESI) available: synthetic
50
details and additional spectroscopic data.
Conclusions
1
2
.
.
D. L. H. Williams, Acc. Chem. Res., 1999, 32, 868-876.
J. S. Stamler and E. J. Toone, Curr. Opin. Chem. Biol., 2002, 6,
779-785.
1
13
15
We have obtained a complete set of multinuclear ( H, C, N,
1
7
2
5
and O) NMR signatures for the RP prepared from reaction
between MSA and NP. We have also prepared in situ the
55 3. N. Hogg, Annu. Rev. Pharmacol. Toxicol., 2002, 42, 585–600.
4
.
P. G. Wang, M. Xian, X. Tang, X. Wu, Z. Wen, T. Cai and A. J.
Janczuk, Chem. Rev., 2002, 102, 1091–1134.
2
corresponding RSNO by reacting RSH with NaNO . This is the
first time that S-nitrosothiols in both free ligand and metal-
bound states are fully characterized by multinuclear NMR.
When the ligand exchange is slow on the NMR timescale,
useful thermodynamic and kinetic information about the
5
6
.
.
J. S. Stamler, Circ. Res., 2004, 94, 414–417.
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0
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7. D. T. Hess, A. Matsumoto, S.-O. Kim, H. E. Marshall and J. S.
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1
7
study also demonstrates the utility of O NMR as a new
technique for detecting short-lived species, which is
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DOI: 10.1039/C5DT04029A
TOC entry
First NMR characterization of the unstable “Red Product” produced from reaction
between nitroprusside and organic thiolates.
3
!
S
O
1H NMR
15N NMR
17O NMR
R
N
Fe(CN)5
13C NMR
Red Product