Synthesis and nitric oxide releasing properties of novel fluoro S-nitrosothiols

Article abstract of DOI:10.1039/C8CC08868C

A series of new fluoro S-nitrosothiols is reported as potential nitric oxide (NO) donors. A three-step synthesis and the NO releasing kinetic profiles of these species are presented. The stoichiometric release of NO, with the clean formation of correspond

Full text of DOI:10.1039/C8CC08868C

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Synthesis and nitric oxide releasing properties
of novel fluoro S-nitrosothiols†
Cite this: Chem. Commun., 2019,
55, 401
a
a
Yang Zhou,
Mark E. Meyerhoff
Jinyi Tan,a Yuping Dai,b Yanmin Yu,b Qi Zhang
and
a
Received 7th November 2018,
Accepted 4th December 2018
*
DOI: 10.1039/c8cc08868c
rsc.li/chemcomm
A series of new fluoro S-nitrosothiols is reported as potential nitric desired functionalities is of continuous interest and has
oxide (NO) donors. A three-step synthesis and the NO releasing significance in all branches of chemistry.
kinetic profiles of these species are presented. The stoichiometric
In the recent development of NO-releasing polymers for use
release of NO, with the clean formation of corresponding disulfides, in preparing implanted antimicrobial and thromboresistant
confirms that these new species can facilitate their application as biomedical devices (e.g., intravascular catheters, subcutaneous
NO donors for various applications including creating novel anti- cannula, etc.), chemical leaching of the NO donor and its decom-
microbial and thromboresistant fluoropolymer-based medical position products has become a critical concern, owing to possible
devices.
toxicity issues.22 Very recently, our group reported the incorpora-
tion of a novel fluorinated NO donor (S-nitroso-N-pentafluoro-
Nitric oxide (NO), an endogenous signaling molecule,1–3 has propionyl-penicillamine, C2F5-SNAP) into polyvinylidene fluoride
been extensively studied for its vasodilatory,4 antithrombotic,5 for preparation of fluoropolymer-based biomaterials that exhibit
immune modulatory,6 antimicriobial,7 anticancer8 and wound NO release and significant antimicrobial properties.23 Due to the
healing9 properties. However, due to its high reactivity, the specific fluorous–fluorous interactions present between the fluori-
intravascular lifetime of NO is very short (B2 s).10 Therefore, nated NO donor and the fluoropolymer, leaching of the fluorinated
precursor molecules (NO donors) are commonly utilized to NO donor and the corresponding decomposition products was
generate NO in situ for chemical and biochemical studies. So far, demonstrated to be quite low (o10%, total), compared to that
various NO donors have been reported,11 including S-nitrosothiols, observed in the use of non-fluorinated NO donors. Thus, the
N-diazeniumdiolates and their derivatives, metal nitrosyls, and development of new fluorinated NO donors is of great interest
N-nitrosamines, as well as organic nitrates and nitrites. Current for potentially creating fluoropolymer-based NO release bio-
NO donors are also widely investigated as promising medicinal materials from which chemical leaching will be greatly reduced
and therapeutic agents for disease treatments.12–14
for potential applications of biomedical implants and devices.
Fluorine-containing compounds have significant potential
To the best of our knowledge, fluorinated S-nitrosothiol type
applications in medical and materials chemistry.15,16 In pharma- NO donors have not received much attention in the medical
ceuticals, almost 150 fluorinated drugs have successfully reached and biomaterials research fields. Indeed, to date, only the
the market since the first fluorine-containing drug (fludrocortisone) C2F5-SNAP molecule has been reported as a fluorinated NO
was approved in 1955.16 Currently, 20–30% administered drugs donor.23 However, the C2F5-SNAP NO donor was found to be
contain fluorine atoms or fluoroalkyl groups.16 Fluorocarbon-based quite unstable even upon storage within a freezer, resulting in the
materials also have been widely applied in clinical biomaterials,17 difficulty in handling and storage of this species.23 In contrast, the
affinity chromatography,18 enzyme immobilization,19 crystal non-fluorinated S-nitroso-N-acetylpenicillamine (SNAP, see Fig. 1)
engineering20 and supermolecular chemistry.21 Overall, the has great stability, and has extensively been doped into various
development of fluorinated compounds and materials with polymers for the long-term NO release over several months.22
Therefore, there is a need to develop new and relatively stable
a Department of Chemistry, University of Michigan, Ann Arbor, MI 48109, USA.
fluorinated structural analogs of SNAP as NO donors for
fluoropolymer-based biomedical devices applications.
Herein, we consider the amine-based nucleophilic addition
chemistry of thiolactone (see Fig. 1), which has been widely
used to incorporate free thiol moieties as the precursors of
S-nitrosothiols.24–27 3-Acetamido-4,4-dimethylthietan-2-one
E-mail: mmeyerho@umich.edu
b Beijing Key Laboratory for Green Catalysis and Separation,
Department of Chemistry and Chemical Engineering,
Beijing University of Technology, Beijing 100124, China
† Electronic supplementary information (ESI) available: Experimental details. See
DOI: 10.1039/c8cc08868c
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remained stable after being stored at À20 1C over several
1
months. For example, H NMR studies of 3l indicated that no
decomposition of 3l was observed after storage for 40 days at
À20 1C under dark conditions (see Fig. S1, ESI†). Hence, the
presence of fluoro/trifluoromethyl groups on the aromatic ring
in targets 3a–3n do not appear to decrease the stability of the
resulting S-nitrosothiols as observed for C2F5-SNAP. This result
is also consistent with the literature finding that tertiary
S-nitrosothiols are known to be thermally stable.29
Fig. 1 Chemical structure of SNAP and the design of fluorinated S-nitroso-
thiols from thiolactone.
To further investigate the NO release profiles from 3a–3n, we
carried out kinetic studies of their photolytic and thermal
decompositions. For example, the steady-state photolysis of
3k (150 mM) was carried out under 350 nm irradiation in a
mixture of PBS and DMSO (50 : 50, v/v) at 23 1C. The UV-Vis
spectra of its photo-decomposition are shown in Fig. 2a, in
which the characteristic UV-Vis band of the nO - p* transition
(342 nm) continuously decreases and eventually flattens. The
kinetic profile was analyzed using the absorbance changes at
342 nm (Fig. 2a inset), and was fitted to a first-order rate equation
giving an observed rate constant kobs = (1.214 Æ 0.052) Â 10À2 sÀ1
(t1/2 B 57 s). The corresponding rate constants and half-lives for
the photo-decomposition of the other NO donors synthesized in
this work are summarized in Table 1. All exhibit first-order
decomposition with half-lives between 50 to 80 s under light
irradiation. Compared to the photostability of SNAP (t1/2 B 46 s),
3a–3n exhibit slower photo-decomposition rates. Targets 3m and
3n have similar decomposition rate constants to 3a–3l, indicating
that substitution groups on the phenyl ring do not influence the
photolytic release rate of NO.
Thermal decomposition studies of 3a–3n at 37 1C were also
performed in a mixture of PBS and DMSO (50 : 50, v/v). The
absorbance data vs. time for the decomposition of 3k at 342 nm
(see Fig. 2b) was monitored and this kinetic data was fitted to a
zero-order rate equation, yielding an observed rate constant
kobs of (1.446 Æ 0.001) Â 10À4 mM minÀ1. All of the thermal
decomposition kinetic data for compounds 1 and 3a–3n are
reported in Table 2. NO donors 3a–3n are less stable in solution
when compared to SNAP. NO donors 3d–3e and 3j–3l decom-
posed via a zero-order reaction model, similar to SNAP. But
the others, 3a–3c, 3f–3i and 3m–3n, exhibit first-order decom-
position kinetics.
(gem-dimethyl thiolactone) was selected as the thiolactone to
imitate the structure of SNAP. To prevent the strong electron
withdrawing effects from fluorine or fluoroalkyl groups on the
stability of S–N bond, we consider fluorinated benzylamine
derivatives as nucleophiles. Specifically, the phenyl ring acts as
a linker to diminish the strong electron withdrawing effects from
fluoro/trifluoromethyl groups. We hypothesize that a nucleophilic
ring opening of the thiolactone in the presence of fluorinated
benzylamines should yield amide-based tertiary free thiols, which
can be subsequently nitrosylated into fluorinated S-nitrosothiols
(see Fig. 1).
In total, 12 fluorinated benzylamines with various degrees of
fluorine substitution and 2 non-fluorinated benzylamines were
utilized in the synthesis of the new amide-based S-nitrosothiols
shown in Scheme 1. The gem-dimethyl thiolactone intermediate
was easily obtained from the reaction between D-penicillamine
and excess acetic anhydride. In the presence of various benzyl-
amines as nucleophiles, a ring opening of the gem-dimethyl
thiolactone yielded amide-based free thiol 2 intermediates.
With pure thiol intermediates 2 in hand, a nitrosylation reac-
tion using NaNO2 in presence of strong acid under dark
conditions yielded the pure target products 3a–3n as greenish
powders. NO donors 3a–3n were obtained with total yields of
between 21–30% in three steps (see Section S2 in ESI†).
Although S-nitrosothiols are known to be relatively unstable
due to the inherently weak S–N bond (25–31 kcal molÀ1),28,29
SNAP is very stable at room temperature. For the previously
prepared C2F5-SNAP NO donor, due to the presence of fluori-
nated groups, C2F5-SNAP was found to be unstable at room
temperature and even slowly decompose at À20 1C.23 All new
targets 3a–3n were found to be stable during the vacuum drying
process at room temperature in the dark. Additionally, they
Fig. 2 (a) UV/Vis spectra for the photolysis of 3k (150 mM) at 23 1C in a
mixture of PBS and DMSO (50 : 50, v/v). Inset: Plot of absorbance at
342 nm vs. time and fitting into a first-order rate equation. (b) Plot of
absorbance at 342 nm vs. time for the thermal decomposition of 3k (100 mM)
at 37 1C in a mixture of PBS and DMSO (50 : 50, v/v).
Scheme 1 Synthesis of NO donors 3a–3n.
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Table 1 Kinetic photo-decomposition data for NO donors 1 and 3a–3n
under irradiation of 350 nm (4 W) in a mixture of PBS and DMSO (50 : 50, v/v)
at 23 1C
NO donors
Rate constant (sÀ1
)
Half-lives (s)
1 (250 mM)
3a (250 mM)
3b (250 mM)
3c (250 mM)
3d (250 mM)
3e (250 mM)
3f (250 mM)
3g (250 mM)
3h (250 mM)
3i (250 mM)
3j (150 mM)
3k (150 mM)
3l (250 mM)
3m (250 mM)
3n (250 mM)
(1.495 Æ 0.027) Â 10À2
(0.945 Æ 0.031) Â 10À2
(1.006 Æ 0.023) Â 10À2
(1.012 Æ 0.063) Â 10À2
(1.384 Æ 0.046) Â 10À2
(1.047 Æ 0.050) Â 10À2
(0.911 Æ 0.022) Â 10À2
(1.009 Æ 0.013) Â 10À2
(0.864 Æ 0.017) Â 10À2
(1.011 Æ 0.015) Â 10À2
(1.191 Æ 0.071) Â 10À2
(1.214 Æ 0.052) Â 10À2
(1.177 Æ 0.075) Â 10À2
(0.972 Æ 0.030) Â 10À2
(1.020 Æ 0.034) Â 10À2
46
73
69
68
50
66
76
69
80
69
58
57
58
71
68
Table 2 Thermal decomposition kinetic data of NO donors 1 and 3a–3n
at 37 1C in a mixture of PBS and DMSO (50 : 50, v/v)
Fig. 3 (a) Stoichiometric NO determination observed for photolysis of 3k
(1.50 Â 10À7 mol) using chemiluminescence NOA detection of emitted NO
in a mixture of PBS (10.0 mM, pH 7.40, with 100 mM EDTA) and DMSO
(50 : 50, v/v) at 23 1C, and (b) NO release reaction for NO donors 3.
NO donors
Rate constant
Half-lives (h)
1 (100 mM)
3a (250 mM)
3b (250 mM)
3c (250 mM)
3d (100 mM)
3e (100 mM)
3f (250 mM)
3g (250 mM)
3h (150 mM)
3i (250 mM)
3j (100 mM)
3k (100 mM)
3l (100 mM)
3m (250 mM)
3n (250 mM)
(1.586 Æ 0.012) Â 10À5 mM minÀ1
(2.070 Æ 0.027) Â 10À3 minÀ1
(2.720 Æ 0.021) Â 10À3 minÀ1
(2.480 Æ 0.036) Â 10À3 minÀ1
(8.395 Æ 0.028) Â 10À5 mM minÀ1
(4.685 Æ 0.001) Â 10À5 mM minÀ1
(2.290 Æ 0.104) Â 10À3 minÀ1
(2.180 Æ 0.082) Â 10À3 minÀ1
(1.970 Æ 0.039) Â 10À3 minÀ1
(2.130 Æ 0.114) Â 10À3 minÀ1
(1.366 Æ 0.011) Â 10À4 mM minÀ1
(1.446 Æ 0.001) Â 10À4 mM minÀ1
(8.089 Æ 0.062) Â 10À5 mM minÀ1
(2.010 Æ 0.090) Â 10À3 minÀ1
(2.520 Æ 0.075) Â 10À3 minÀ1
56.22
5.58
4.25
4.66
6.45
15.47
5.04
5.30
5.86
5.42
4.70
5.24
6.49
5.75
4.58
S-nitrosothiols.30 To further examine the disulfide formation,
products derived from both the photo and thermal decomposi-
tion of 3k (2.98 mM) in DMSO-d6 were analyzed by 1H NMR
spectroscopy (see Fig. 4). Before the decomposition, methide
and gem-dimethyl groups in 3k had chemical shifts at B5.30,
1.98 and 1.96 ppm. After photolysis for 2 h, 3k was completely
decomposed, to cleanly yield the disulfide, with the methide
resonance at B4.65 ppm and gem-dimethyl groups at B1.33
and 1.25 ppm. This disulfide was further confirmed by HRMS
results (m/z (ESI): calculated for [M + H]+ = 731.1966; found =
731.1965). Interestingly, a partial thermal decomposition of 3k
was observed after 4 d at 37 1C in the dark (see Fig. 4), yielding a
mixture of 3k and its corresponding disulfide. These data
provide evidence that 3k can be employed as a pure NO donor,
to stoichiometrically release NO and the corresponding disulfide.
The mechanism of NO-release from S-nitrosothiols has been
To understand these different kinetic behaviors, we also
performed computational calculations (see Section S5 in ESI†).
Interestingly, the transition state for the homolysis of the S–N
bond resulting in NO release does not exist. We found experi-
mentally that the NO release was mainly determined by the
calculated binding dissociation enthalpy (BDE) of S–N bond.
Compared to SNAP, 3e, 3l and 3m have slightly lower BDE
(see Table S3, ESI†) for the S–N bond. Thus, 3e, 3l and 3m are
less stable than SNAP, consistent with our kinetic results under
the thermal decomposition.
Furthermore, the emitted NO gas released by each new donor
was accurately quantitated by an NOA chemiluminescence
method. For instance, the photolysis of 3k (1.50 Â 10À7 mol)
within a NOA reaction cell was conducted in a mixture of PBS and
DMSO (50 : 50, v/v) under 350 nm lamp irradiation, in which the
corresponding NO gas signal was monitored by the NOA analyzer
(see Fig. 3a). Three independent photolysis reactions gave the
average NO generation yield of 98 Æ 2%. These results indicate
that NO donor 3k can stoichiometrically release NO.
Fig. 4 1H NMR spectra for 3k (2.98 mM) before and after decomposition
in DMSO-d6 (1) before decomposition, (2) after photolysis at 23 1C for 2 h,
(3) after thermal decomposition at 37 1C in the dark for 4 d.
In addition to NO gas, the corresponding disulfide is
reported to be the product derived from the decomposition of
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well-studied under heat, irradiation, and Cu+ mediated reduction
conditions.28,30 A homolysis of the weak S–N bond could yield
equal moles of NO and a thiyl radical. Thiyl radical intermediates
are typically reactive, subsequently resulting in a rapid dimeriza-
tion to yield the corresponding disulfide.28,30 Finally, a possible
NO generation pathway for NO donors 3 is summarized in Fig. 3b,
based on our NO measurement and disulfide characterization.
To summarize, a novel family of fluorinated S-nitrosothiols
has been reported herein. The presence of fluorine or fluoro-
alkyl groups on the benzylamide does not influence the
stability of the S–N bond. Kinetic studies of their photolytic
and thermal decomposition reactions were conducted. The
stoichiometric release of NO and the corresponding disulfide
formation from decomposition was monitored by NO chemi-
luminescence measurements and 1H NMR spectroscopy,
respectively. Detailed theoretical calculations on the relation-
ship between structure and stability of these new fluorinated
NO donors are currently underway. We anticipate that these
new species will have applications as useful NO release agents,
potentially for incorporation into fluorinated biopolymers for
preventing infection and/or thrombosis on surfaces of various
medical devices. Such studies are now ongoing in our laboratory.
The authors acknowledge the Juvenile Diabetes Research
Foundation (JDRF: 2-SRA-2016-230-Q-R) and the National
Institutes of Health (HL-128337) for supporting this research.
We also are grateful to Dr. Paul Sampson at Kent State Uni-
versity for helpful discussion on fluorine chemistry.
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404 | Chem. Commun., 2019, 55, 401--404
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