Mechanistic Insights into the H2S-Mediated Reduction of Aryl Azides Commonly Used in H2S Detection

Article abstract of DOI:10.1021/jacs.5b10675

Hydrogen sulfide (H₂S) is an important biological mediator and has been at the center of a rapidly expanding field focused on understanding the biogenesis and action of H₂S as well as other sulfur-related species. Concomitant with this expansion has been the development of new chemical tools for H₂S research. The use of H₂S-selective fluorescent probes that function by H₂S-mediated reduction of fluorogenic aryl azides has emerged as one of the most common methods for H₂S detection. Despite this prevalence, the mechanism of this important reaction remains under-scrutinized. Here we present a combined experimental and computational investigation of this mechanism. We establish that HS^-, rather than diprotic H₂S, is the active species required for aryl azide reduction. The hydrosulfide anion functions as a one-electron reductant, resulting in the formation of polysulfide anions, such as HS₂^-, which were confirmed and trapped as organic polysulfides by benzyl chloride. The overall reaction is first-order in both azide and HS^- under the investigated experimental conditions with ΔS^? = -14(2) eu and ΔH^? = 13.8(5) kcal/mol in buffered aqueous solution. By using NBu₄SH as the sulfide source, we were able to observe a reaction intermediate (λ_max = 473 nm), which we attribute to formation of an anionic azidothiol intermediate. Our mechanistic investigations support that this intermediate is attacked by HS^- in the rate-limiting step of the reduction reaction. Complementing our experimental mechanistic investigations, we also performed DFT calculations at the B3LYP/6-31G(d,p), B3LYP/6-311++G(d,p), M06/TZVP, and M06/def2-TZVPD levels of theory applying the IEF-PCM water and MeCN solvation models, all of which support the experimentally determined reaction mechanism and provide cohesive mechanistic insights into H₂S-mediated aryl azide reduction.

Full text of DOI:10.1021/jacs.5b10675

Subscriber access provided by CMU Libraries - http://library.cmich.edu
Article
2
Mechanistic Insights into the HS-Mediated Reduction
2
of Aryl Azides Commonly used in HS Detection
Hillary A. Henthorn, and Michael D Pluth
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.5b10675 • Publication Date (Web): 05 Nov 2015
Downloaded from http://pubs.acs.org on November 5, 2015
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a free service to the research community to expedite the
dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts
appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been
fully peer reviewed, but should not be considered the official version of record. They are accessible to all
readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered
to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published
in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just
Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor
changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers
and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors
or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical
Society. 1155 Sixteenth Street N.W., Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the course
of their duties.

Page 1 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60



.
Introduction
conditions including hypertension,11 Down syndrome,12
and diabetes.13
Hydrogen sulfide (H2S) has emerged as an important bio-
logical molecule of interest and joins nitric oxide (NO) and
carbon monoxide (CO) as the third recognized gas-
otrasmitter.1 Highlighting the importance of biological H2S,
endogenous sulfide plays critical roles in the cardiovascu-
lar, gastrointestinal, immune, and nervous systems.2 En-
zymatic H2S synthesis occurs primarily from cystathionine-
-synthase (CBS),3 cystathionine--lyase (CSE),4 and 3-
mercaptopyruvate sulfurtransferase (3-MST) working in
concert with cysteine aminotransferase (CAT), although
recent reports have also documented efficient H2S synthe-
sis from D-Cys with D-aminoacid oxidase (DAO) and 3-
MST.5 Because H2S is a weak acid, about 80% of H2S exists
as monoanionic HS– under physiological conditions. These
different protonation states, along with the complex redox
chemistry of sulfur, provide H2S with a more complex reac-
tivity profile than NO or CO, resulting in reaction of H2S
with both organic and inorganic targets. For example, the
oxidative posttranslational S-persulfidation of cysteine
residues can enhance enzyme activity,6 and reaction of H2S
with metalloprotein transition metal centers may also con-
tribute to key signaling pathways.7 Although many of the
molecular mechanisms of action remain to be elucidated,
H2S is now known to be involved in a diverse array of im-
portant biochemical processes including vasorelaxation,4
neuronal long-term-potentiation,3 insulin regulation,8 an-
giogenesis,9 and inflammation.10 Similarly, misregulation of
endogenous H2S concentrations is associated with various
Paralleling the discovery of new biological roles of H2S
has been the rapid expansion of chemical tools to study
H2S in biological contexts. Such advances have included the
development of chemical methods for H2S quantification,14
methods of H2S delivery,15 and reaction-based tools for H2S
imaging. Fluorescent probe development has emerged as
the most common imaging strategy and has relied on three
main detection strategies, which includes metal precipita-
tion,16 nucleophilic attack,17 and reduction of azide or nitro
groups. Of these methods, the H2S-mediated reduction of
azides (Figure 1) has emerged as the most common strate-
gy for H2S detection. In these systems, protection of a fluo-
rogenic amine nitrogen as an azide renders the fluoro-
phores non-emissive until reduction of the azide to the
amine by H2S. Highlighting the prevalence and impact of
this sensing strategy, over 65 papers have been published
reporting azide-based H2S-probes since the initial report of
SF1 and SF2 in 2011.18 Despite this widespread use, the
mechanism by which H2S reduces aryl azides remains un-
derinvestigated, and key questions related to this process
remain. For example, does H2S act as a one- or two-
electron reductant? Is H2S(g) or HS– the active sulfide spe-
cies involved in the reduction? How many equivalents of
H2S are required for azide reduction? In addition to provid-
ing key insights into H2S sensing, a functional understand-
ing of how this reaction occurs would also provide valua-
ble insights into the design of next generation sensors.
ACS Paragon Plus Environment

Journal of the American Chemical Society
Based on this critical need, we report here experimental
and computational investigations that provide new in-
sights into the mechanism of H2S-mediated azide reduc-
tion.
Page 2 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 1
Figure 1. (top) Example of H2S-mediated azide reduction
with a fluorescent turn-on response. (bottom) Model azide
system used here for mechanistic studies.
Kinetic Studies of the H2S-mediated Reduction of C7-Az.
To investigate the mechanism of sulfide-mediated azide
reduction, we chose to use the previously-reported 4-
methyl-7-azidocoumarin (C7-Az) as a model system due to
its ease of synthesis and amenable spectroscopic proper-
ties (Figure 1).20 Treatment of aqueous buffered solutions
of C7-Az with excess sodium hydrosulfide (NaSH) under
pseudo-first order conditions, resulted in a decrease in the
C7-Az absorbance (max = 326 nm) and concomitant for-
mation of C7-NH2 (max = 343 nm) with a well-anchored
isosbestic point at 334 nm (Figure 2a). Under our experi-
mental conditions, conversion to the amine is complete
within 60 min, making the reaction amenable for kinetic
investigations. We also verified that the fluorescence turn-
on profile of C7-Az matched the UV-vis data. Plots of the
absorbances at 326 and 343 nm vs. time (Figure 2b) both
fit the first-order rate equation and such fittings were used
to obtain rate constants (kobs) for subsequent experiments
unless otherwise specified (Figure S1). Although sulfide-
mediated azide reduction is likely a multistep process, no
intermediates were observed by UV-Vis spectroscopy.
Results and Discussion
On the basis of the high nucleophilicity of HS–, as well
as its prevalence under physiological conditions, it is likely
that HS– rather than H2S is the active species required for
azide reduction. Drawing parallels to analogous reactions
of azides with other nucleophilic reductants, such as phos-
phines in the Staudinger reaction, in which the terminal
nitrogen is the site of nucleophilic attack,19 our expectation
is that the sulfide-mediated reduction of aryl azides pro-
ceeds through initial nucleophilic attack on the electro-
philic azide. After this initial attack to form an anionic az-
idothiol intermediate, we envisioned two possible mecha-
nisms to generate the aryl amine product (Scheme 1). The
first mechanism proceeds through an intramolecular reac-
tion, in which proton transfer generates a terminal thio-
late, which subsequently attacks the azide through either a
4-membered triazathiocyclobutyl or 3-membered diaza-
thiocyclopropyl transition state to release N2, S0, and the
aryl amine product (Mechanism I). In this scenario, H2S
would act as a two-electron reductant and a large, negative
S‡ would be expected. By contrast to this intramolecular
mechanism, the anionic azidothiol intermediate could be
attacked by a second equivalent of HS–. In this scenario,
protonation of the anionic azidothiol to generate a neutral
azidothiol intermediate could enhance its electrophilicity,
thus resulting in further activation for attack by a second
–
equivalent of HS– to release N2, HS2 , and the aryl amine
product (Mechanism II). In this mechanism, H2S acts as a
one-electron reductant and a moderately negative S‡,
corresponding to bimolecular reaction, would be expected.
Figure 2. UV-Vis spectra (a) and time course data (b) of
the reaction of C7-Az (black) with NaSH to form C7-NH2
(red) under pseudo first-order conditions (20 M probe,
2.0 mM NaSH, 50 mM PIPES, 100 mM KCl, pH 7.4, 25 C).
ACS Paragon Plus Environment

Page 3 of 9
Journal of the American Chemical Society
Reaction Order and Activation Parameters. To deter-
reaction, this highly colored intermediate slowly decayed
to an absorbance corresponding to the C7-NH2 product
(Figure 4a). A plot of the absorbance values for C7-NH2 at
334 nm and the intermediate at 473 nm fit cleanly to a
first-order rate equation with nearly identical kobs values of
0.204 s-1 and 0.197 s-1, respectively (Figure 4b). These rate
data support that rate-limiting decomposition of the ob-
served intermediate leads to C7-NH2 product formation. As
control experiments, we confirmed that 473 nm absorb-
ance does not correspond to other possible products
formed during the course of the reaction including hydro-
1
2
3
4
5
6
7
8
mine the reaction order of [C7-Az] and [NaSH], we meas-
ured the rate of the reaction as a function of reactant con-
centrations at constant pH and temperature. Working un-
der pseudo first-order conditions in sulfide, varying
[NaSH] allowed for determination of the [NaSH] order by
plotting log(kobs) versus log([NaSH]). This plot provided
linear relationship with a slope of 0.99, suggesting a first-
order rate dependence on [NaSH] (Figure 3a, S3). To de-
termine the reaction order in C7-Az, kinetic data were col-
lected at constant pH, [NaSH], and temperature. To aid
experimental measurement of kobs for these experiments,
we monitored initial rates due to the small changes in the
absorbance of C7-Az and C7-NH2 during the course of the
reaction. The resultant plot of log(kobs) versus log([C7-Az])
also provided a linear plot with a slope of 0.94, which is
also consistent with a first-order rate dependence on [C7-
Az] (Figure 3b, S3). Supplementing the reaction order data,
we also measured the rate of the reduction from 288 – 307
K under pseudo first order conditions in [NaSH] and calcu-
lated the 2nd-order rate constants from the resultant kobs
data at different [NaSH]. Eyring analysis of the resultant
second-order rate datarevealed H‡ = 13.8(5) kcal/mol
and S‡ = -14(2) eu (Figure S2).21 The moderately nega-
tive S‡ supports a rate limiting step that proceeds
through a bimolecular transition state rather than the
highly-strained intramolecular transition state associated
with Mechanism I.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
polysulfide anions (HSx ), trisulfide radical ion (S3·–), or
–
deprotonated amine (C7-NH–) (Figure S5, S7). Further
supporting the anionic structure of the intermediate, addi-
tion of an excess of H2O abolished the 473 nm absorbance
and resulted in partial product formation, suggesting that
protonation of the azidothiolate intermediate is required
for subsequent sulfide attack (Figure S6). Using fluores-
cence spectroscopy we also confirmed that the C7-Az fluo-
rescence response is due to C7-NH2 formation rather than
formation of the intermediate.
Figure 3. (a) Plot of log(kobs) vs log([NaSH]) for the reac-
tions of 0.25–2.00 mM NaSH with 20 M C7-Az. (b) Plot of
log(kobs) vs log([C7-Az]) for the reactions of 2.00 mM NaSH
with 5–80 M C7-Az. Standard conditions: 50 mM PIPES,
100 mM KCl, pH 7.4, 25 °C. Each measurement was repeat-
ed at least in triplicate.
Figure 4. Reaction of C7-Az with excess NBu4SH (20 M
probe, 2.0 mM NBu4SH, MeCN). Full spectra overlay (a)
and time course (b) at 334 (blue) and 473 (red) nm.
In addition to providing a solvent in which an inter-
mediate could be observed, use of aprotic MeCN in combi-
nation with NBu4SH allowed for titration of C7-Az to de-
termine the reaction stoichiometry of HS– required to
complete the reduction. These experiments demonstrated
that complete conversion of the azide starting material to
the amine product did not occur until two equivalents of
HS– were added, which not only suggests that HS– func-
tions as a one electron reductant, but also that two equiva-
lents of HS– are required for the reaction (Figure 5). The
requirement of a second equivalents of HS– supports
Mechanism II (Scheme 1), in which one additional equiva-
lent of HS– is required.
Observation of Reaction Intermediates. To better iso-
late and differentiate the impacts of H2S and HS– protona-
tion states, we also investigated the reduction reaction in
organic solution. Additionally, based on the mechanisms
proposed in Scheme 1, we envisioned that use of an aprotic
solvent could facilitate observation of the proposed anionic
intermediates. Based on these requirements, we chose to
use MeCN as a solvent due to its high dielectric constant
and aprotic nature. Supporting our hypothesis that HS–,
rather than H2S(g), is the active sulfide source in the reduc-
tion reaction, treatment of C7-Az with excess H2S(g) failed
to produce any reaction. By contrast, addition of NBu4SH,
which is a source of HS– that is soluble in organic solvents,
resulted in immediate formation of an orange intermediate
with an absorbance at 473 nm. During the course of the
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 4 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 2
To gain further insights into the identity of the oxi-
–
polysulfides supports HSx formation during the azide re-
duction process.
dized sulfur products formed during azide reduction, we
treated C7-Az with 3 equiv. of NBu4SH in MeCN and
trapped the sulfur species present at the end of the reac-
tion with BnCl. On the basis of the proposed reaction stoi-
chiometry, we expected to observe 1 equiv. of unreacted
HS– and 1 equiv. of oxidized sulfur products. Consistent
with this hypothesis, we observed 1 equiv of Bn2S (3.67
ppm) and Bn2S2 (3.72 ppm), as well as a small peak corre-
sponding to higher order benzylpolysulfides (4.45 ppm)
(Figure 6). Importantly, the formation of Bn2S2 confirmed
Proposed Mechanism. Taken together, the experi-
mental results detailed above support Mechanism II, which
is outlined in greater detail in Scheme 2. The first step of
the reaction proceeds by reversible nucleophilic attack of
HS– on the terminal azide nitrogen of C7-Az (1), which is
the most electrophilic nitrogen on the azide.22 For example,
nucleophilic attack on the terminal nitrogen has precedent
in the Staudinger reaction19 as well as the addition of car-
bon-based nucleophiles to azides.22 This initial nucleo-
philic attack generates the anionic azidothiol intermediate
2, which we assign to the absorbance at 473 nm observed
in MeCN based on the experiments described above as well
as qualitative TD-DFT calculations that support a batho-
chromic shift upon formation of the anionic azidothiol in-
termediate (Figure S9). Although anionic azidothiol 2
could be attacked directly by HS–, it is more plausible that
2 is transiently protonated to generate neutral 3, which
would be significantly more electrophilic. Subsequent at-
tack on 3 by a second equivalent of HS– then generates
deprotonated amine 4, N2, and H2S2, after which proton
transfer, likely mediated by solvent, results in formation of
C7-NH2 (5). We note that anion 4 is a significantly stronger
base than intermediate 2 or HS2– and should undergo facile
proton transfer to generate 5. This proposed mechanism is
consistent with the observed first-order dependence of HS–
and azide, as well as the moderately negative S‡ for the
reaction.
–
that HS2 was formed during the azide the reduction reac-
tion.
Figure 5. (a) UV-Vis titration showing the reduction of C7-
Az (20 M in MeCN) to C7-NH2 (red) by NBu4SH. (b) Moni-
toring the absorbance of the C7-NH2 product as a function
of NBu4SH establishes that 2 equiv. of HS– are required for
azide reduction.
Rate Law Analysis. On the basis of the mechanism out-
lined in Scheme 2, the overall rate law for product for-
mation can be described in (1), in which two equiv. of HS–
and one equiv. of aryl azide are involved in the reaction.
(1)
To simplify this reaction scheme, we note that conver-
sion of 1 to 3 requires the net addition of H2S(g) to 1, thus
allowing for simplification of the rate law to (2).
Figure 6. 1H NMR spectrum (600 MHz) of the products
formed during the reduction of C7-Az (12 mM) with
NBu4SH (36 mM, 3 equiv.) in MeCN. Polysulfide anions
formed during the reaction were trapped by BnCl (12
equiv.) and NEt3 (12 equiv) after azide reduction was com-
plete. The observation of Bn2S2 and higher order organic
(2)
Under this scenario, anionic azidothiol intermediate 3
is formed through a rapid pre-equilibrium before the rate
limiting attack by HS–, thus leading to the simplified steady
state rate equation shown in (3).
ACS Paragon Plus Environment

Page 5 of 9
Journal of the American Chemical Society
the electrophilic sulfhydryl group of azidothiol intermedi-
1
2
′
ate 3 and is also consistent with the high homophilicity of
sulfur. The calculated PES indicates that the second nucle-
ophilic attack is the rate-determining step with an overall
activation enthalpy of 16.2 kcal/mol, which is in good
agreement with the experimentally-measured H‡ of
13.8(5) kcal/mol. No significant difference in the relative
energies was observed when comparing the results be-
tween the DFT functionals and basis sets, highlighting the
robust energy landscape derived from these computation
studies. Although one would expect diffuse functions and
dispersion forces to play an important role in this trans-
formation, they have little impact on the thermochemistry
of this PES (Figure S8). Significant efforts to locate transi-
tion state structures corresponding to the 3- or 4-
membered transitions states required in Mechanism I
were unsuccessful.
[
][
]
푘
ArN H S
3
2
′
1
−
[
]
(
푘
rate = 푘3 HS
)
(3)
−
]
[
HS +푘
3
−1
3
4
5
6
7
8
9
In the limiting case in which k3[HS–] << k’–1, which
would result at low sulfide concentrations or if the uni-
molecular disassociation of azidothiol intermediate 3 is
very fast, the rate law would condense to (4). However,
under the conditions in which k3[HS–] >> k’–1, which
would occur at higher [HS–] the rate law condenses to (5).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
′
푘
′
1
[
][ −][
]
rate = 푘 푘3 ArN3 HS H2S
(4)
(5)
−1
′
[
][
]
rate = 푘1 ArN3 H2S
Mechanistic Insights. The experimental and computa-
tional investigations outlined above provide a cohesive
mechanism for sulfide-mediated reduction of aryl azides. A
number of key points are highlighted below. First, the
mechanistic studies establish that HS–, rather than H2S, is
the active species responsible for sulfide-mediated azide
reduction. Because HS– is the dominant species under
physiological conditions, these results highlight that the
azide-reduction method reports on the most prevalent
form of available sulfide. Additionally, the initial addition
of HS– to the azide is reversible, suggesting that generation
of the important azidothiol intermediate may be difficult
under conditions in which sulfide concentrations are very
low. Additionally, it is possible that other sulfhydryl-
containing biomolecules, such as GSH, may participate in
the reaction after the initial attack by the more-
nucleophilic hydrosulfide anion. Furthermore, the mecha-
nistic insights suggest that protonation of the azidothiolate
intermediate is an important step for the reaction. One
would expect that addition of functional groups that could
make this intermediate more basic and favor protonation
would help to drive the reaction, thus facilitating nucleo-
philic attack in the subsequent and rate-limiting step. Ad-
ditionally, these mechanistic studies show that sulfide, and
likely one equivalent of thiol, are consumed during each
reduction of an aryl azide, highlighting that such sensing
methods perturb sulfide levels and the redox state of the
system. Based on the growing importance of the reduction-
labile sulfane sulfur pool, such reaction-based detection
methods may inadvertently disturb the distribution of sul-
fide between the bound and free pool, thus opening new
questions related to how H2S sensor impact redox homeo-
stasis. With the absence of reversible H2S sensing mecha-
nisms that operate under physiological conditions, such
perturbations remain a reality of reaction-based sensors,
and need to be considered for biological inquiries in which
irreversible, reaction-based reporters are used.
Under the experimental reaction conditions, we ob-
serve a 1st-order dependence in both [ArN3] and in [HS–],
which would be expected when [HS–] in in excess and is
consistent with (5). Our attempts to shift the reaction to
the other regime in which a 2nd-order dependence on sul-
fide is observed, including using C7-Az as the flooding sub-
strate, have maintained a 1st-order sulfide dependence,
suggesting that we are experimentally unable to access the
other limiting condition. However, when taken in combina-
tion with the reaction stoichiometry data and trapping of
–
the HS2 reaction product (vide supra), and DFT reaction
coordinate calculations (vide infra) these kinetic data sup-
port the proposed mechanisms for sulfide-mediate aryl
azide reduction.
DFT Calculations. To provide added insights into the
feasibility and energetics of the mechanism proposed in
Scheme 2, we also conducted a full conformational search
of each reactant, product, and intermediate and located
each transition state using Gaussian 09 at the B3LYP/6-
31G(d,p), B3LYP/6-311++G(d,p), M06/TZVP, and
M06/def2-TZVPD levels of theory applying the IEF-PCM
water and MeCN solvation models. The calculated poten-
tial energy surface (PES) for the B3LYP/6-311++G(d,p)
and M06/def2-TZVPD levels of theory with the water solv-
ation model is shown in Figure 6. Starting from C7-Az (1),
the transition state of the first nucleophilic attack TS12 was
located with a calculated energy barrier of 8.8 kcal/mol
and proceeds to generate anionic azidothiol intermediate
2, with an overall endothermicity of 4.1 kcal/mol. Attempts
to locate transition state structures derived from the attack
of HS– on anionic 2 were unsuccessful, whereas protona-
tion of 2 to form neutral azidothiol intermediate 3 pro-
ceeds with a modest enthalpic penalty of 3.0 kcal/mol.23
Subsequent nucleophilic attack by HS– on 3 proceeds
through transition state TS34, which has a local energy bar-
rier of 8.6 kcal/mol to generate the deprotonated amine
which is then protonated to generate the highly thermody-
namically-favored C7-NH2 product (-58.9 kcal/mol). This
second nucleophilic attack proceeds with the HS– attacking
ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 6 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 7. Calculated potential energy surface for proposed mechanism. (B3LYP/6-311++G(d,p) (black) and M06/def2-
TZVPD (red) PCM: MeCN)
Conclusions. Our mechanistic investigations into the
sulfide-mediate reduction of aryl azides revealed that this
reaction is first-order C7-Az and NaSH. This reaction pro-
ceeds with activation parameters consistent with a bio-
molecular mechanism (S‡ = -14(2) eu) with a moderate
enthalpic barrier (H‡ = 13.8(5) kcal/mol). Reactions of
C7-Az with NBu4SH in MeCN allowed for observation of an
intermediate which we attribute to the anionic azidothiol
intermediate, and HS– titrations in this system establish
that two equivalents of HS– are required for complete re-
duction, which is consistent with sulfide acting as a one-
electron reductant. Computational studies supported the
experimental observations, and indicate that the second
attack of sulfide is the rate-determining-step of the reduc-
tion and the calculated activation energy (16.2 kcal/mol) is
in good agreement with the experimental enthalpy of acti-
vation (13.8(5) kcal/mol). Taken together, these mecha-
nistic studies provide new insights into the individual
steps required for H2S-mediated azide reduction and may
be useful in the refinement of this important sensing strat-
egy for H2S.
deoxygenated by three freeze-pump-thaw cycles. Tetrabu-
tylammonium sulfide (NBu4SH) and C7-Az were prepared
as described in the literature.24 Anhydrous sodium hydro-
gen sulfide (NaSH) was purchased from Strem Chemicals
and handled under nitrogen. Piperazine-N,N′-bis(2-
ethansulfonic acid) (PIPES, Aldrich) and potassium chlo-
ride (99.999%, Aldrich) were used to make buffered solu-
tions (50 mM PIPES, 100 mM KCl, pH 7.4) with Millipore
water. Buffered solutions were degassed by vigorous
sparging with N2 and stored in an inert atmosphere glove-
box. Stock solutions of C7-Az were prepared in MeCN or
DMSO. Gas-tight Hamilton syringes were used for solution
transfers outside of the glove box.
Spectroscopic Methods. UV-Vis measurements were col-
lected on an Agilent Cary 100 UV-Vis spectrophotometer
equipped with a QNW dual cuvette temperature controller
at 25.00 ± 0.05 C. All cuvette-based spectroscopic meas-
urements were made under anaerobic conditions with
solutions prepared under an inert atmosphere in 1.0 cm
path length septum-sealed cuvettes obtained from Starna
Scientific and were repeated at least in duplicate. NMR
spectra were acquired on a Bruker Avance-III-HD 600 with
a Prodigy multinuclear broadband CryoProbe at 25.0 ºC.
Experimental Details
Materials and Methods. All manipulations were per-
formed under an inert atmosphere using standard Schlenk
techniques or an Innovative Atmospheres N2-filled glove
box with O2 levels less than 1.0 ppm. Anhydrous and air-
free MeCN was obtained from a Pure Process Technologies
solvent purification system. Deuterated solvents were
dried by distillation over the appropriate drying agent and
Computational Details. Calculations were performed
using the Gaussian 09 software package.25 Chemcraft was
used to analyze results and generate graphical representa-
tions. Conformational searches were conducted at the
B3LYP/6-311++G(d,p), M06/TZVP, and M06/def2-TZVPD
levels of theory applying the IEF-PCM water and MeCN
ACS Paragon Plus Environment

Page 7 of 9
Journal of the American Chemical Society
solvation models by exploring reasonable input dihedral
angles. Frequency calculations were performed on located
stationary points to confirm that they were either a local
minima or saddle point. Relative energies were calculated
using the zero-point corrected electronic energies ob-
tained in the frequency calculations.
1
2
3
4
5
6
7
8
9
General Conditions for UV-Vis Experiments. In a sep-
tum-sealed cuvette, 2.970 mL of a C7-Az stock solution (20
mM), in either MeCN or buffered water, was treated with
30 L of 200 mM HS– stock solution. The UV-vis spectra
were monitored as a function of time, and the C7-NH2 ab-
sorbance (343 nm) was used to measure the observed rate
constants.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
General Conditions for NMR Experiments. A septum
sealed NMR tube was charged with C7-Az (12 mM in 0.5
mM of MeCN-d3) under an N2-atmosphere, and 6.6 L of
TEA was added. A 50 L aliquot of a MeCN-d3 solution con-
taining 240 mM NBu4SH was added. The azide is com-
pletely reduced upon sulfide addition and BnCl (5.5 L)
was added to trap the generated HS2-. Bn2S2 was subse-
quently added to confirm the identity of the singlet at 3.72
ppm. All additions were performed using Hamilton syring-
es.
Supporting Information. UV-Vis spectra, Eyring plot,
computational data. The Supporting Information is availa-
ble free of charge on the ACS Publication website at DOI:
10.1021/XXX.


ACS Paragon Plus Environment

Journal of the American Chemical Society
Page 8 of 9
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
ACS Paragon Plus Environment

Page 9 of 9
Journal of the American Chemical Society
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
9
ACS Paragon Plus Environment