O→S Relay Deprotection: A General Approach to Controllable Donors of Reactive Sulfur Species

Article abstract of DOI:10.1002/anie.201802845

Reactive sulfur species (RSS) are biologically important molecules. Among them, H₂S, hydrogen polysulfides (H₂S_n, n>1), persulfides (RSSH), and HSNO are believed to play regulatory roles in sulfur-related redox biology. However, these molecules are unstable and difficult to handle. Having access to their reliable and controllable precursors (or donors) is the prerequisite for the study of these sulfur species. Reported in this work is the preparation and evaluation of a series of O-silyl-mercaptan-based sulfur-containing molecules which undergo pH- or F^?-mediated desilylation to release the corresponding H₂S, H₂S_n, RSSH, and HSNO in a controlled fashion. This O→S relay deprotection serves as a general strategy for the design of pH- or F^?-triggered RSS donors. Moreover, we have demonstrated that the O-silyl groups in the donors could be changed into other protecting groups like esters. This work should allow the development of RSS donors with other activation mechanisms (such as esterase-activated donors).

Full text of DOI:10.1002/anie.201802845

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Accepted Article
Title: O to S Relay Deprotection, A General Approach for the Design of
Controllable Donors of Reactive Sulfur Species
Authors: Ming Xian, Jianming Kang, Shi Xu, Miles N Radford, Wenjia
Zhang, Shane S Kelly, and Jacob J Day
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201802845
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à
O S Relay Deprotection, A General Approach for the Design of
Controllable Donors of Reactive Sulfur Species
Jianming Kang,† Shi Xu,† Miles N. Radford, Wenjia Zhang, Shane S. Kelly, Jacob J. Day, and Ming
Xian*
Dedicated to Prof. Jin-Pei Cheng on the occasion of his 70th birthday
Abstract: Reactive sulfur species (RSS) are biologically important
molecules. Among them, hydrogen sulfide (H2S), hydrogen
polysulfides (H2Sn, n>1), persulfides (RSSH), and S-nitroso hydrogen
sulfide (HSNO) are believed to play regulatory roles in sulfur-related
redox biology. However, these molecules are unstable and difficult to
handle. Their instability is the major roadblock hindering their
biological understanding. Having the access to their reliable and
controllable precursors (or donors) is the prerequisite for the study of
these sulfur species. In this work, we report the preparation and
evaluation of a series of O-silyl mercaptan based sulfur containing
molecules. These compounds can undergo pH- or F- mediated
desilylation to release the corresponding H2S, H2Sn, RSSH, and
HSNO in a controlled fashion. This OàS relay deprotection serves as
a general strategy for the design of pH- or F- triggered RSS donors.
Moreover, we have demonstrated that the O-silyl groups in the donors
could be changed to other protecting groups like esters. This should
allow the development of RSS donors with other activation
mechanisms (such as esterase-activated donors).
species rely on the use of commercially available inorganic salts
(e.g. Na2Sn for H2Sn), or ‘in-situ’ generation of RSSH or HSNO
based on the reactions of H2S (with RSSR to form RSSH; with
RSNO to form HSNO). The unavoidable sulfide impurity in the
salts and the presence of H2S in the reaction mixtures make these
methods less useful as it is impossible to clearly differentiate the
properties of the desired RSS from that of H2S. The ‘ideal’ method
for specific RSS generation should produce the desired RSS
molecules in a clean, controllable, non-H2S or other RSS involved,
and free of redox conditions (because RSS are sensitive to redox
conditions). With these considerations in mind, we herein report a
general strategy for the design of RSS releasing systems that
meet the aforementioned criteria. Most importantly, this modular
design can be easily adopted to release a number of RSS
including H2S, H2Sn, RSSH, HSNO, etc.
In the design of RSS releasing systems, specific sulfur (S-)
protecting groups are often needed to cage the sulfur containing
molecules and also serve as the triggers for the release. However,
sulfur-based protecting groups that can be deprotected under bio-
compatible conditions are rather limited. In contrast, oxygen (O-)
based protecting groups are much more developed. A large
number of O-protection groups are known and have been
employed in O-based prodrug or sensor developments. We
wondered if O-protection/deprotection could be used in S-
cage/decage. That would require OàS relay protection or
deprotection. We envisioned a geminal thiol-hydroxyl moiety
could be useful for this design. As shown in Scheme 1, the
removal of the O-protection group on substrate I should generate
a geminal thiol-hydroxyl derivative II, which should be unstable
and readily degrade to form the SH-containing molecule III. This
achieves the OàS relay deprotection. We also envisioned many
different S-derivatives of I could be prepared. Therefore, a variety
of RSS can be released by this general method.
Reactive sulfur species (RSS) are a group of sulfur containing
molecules existing in biological systems and play regulatory roles.
Representative RSS include thiols (like cysteine and glutathione),
hydrogen sulfide (H2S), persulfides (RSSH), polysulfides
(RSSnSR, n>0), hydrogen polysulfides (H2Sn, n>1), as well as S-
modified cysteine adducts such as nitrosothiols (RSNO) and
sulfenic acids (RSOH).[1] Since the discovery of H2S as a nitric
oxide (NO)-like signaling molecule, understanding chemical
biology of H2S and H2S-derived RSS, especially RSSH, H2Sn, and
HSNO, has become a fast growing research field.[1] On the other
hand, many of these RSS are unstable and highly reactive
molecules, causing significant difficulty in their studies. To solve
this problem, considerable efforts have been put into the
development of unique releasing systems (e.g. donors) or in situ
generation systems for RSS.[2] Meanwhile, the development of
specific and sensitive detection methods for RSS has also
attracted much attention.[3] Progress in chemical tool
development, especially those targeting H2S, has dramatically
advanced the RSS field. However, reliable and controllable
releasing systems for other RSS including RSSH, H2Sn, HSNO,
etc., are still lacking. Current methods for generating these
Scheme 1. The design of a general approach for RSS releasing
Based on this hypothesis, we believed the O-protected geminal
hydroxyl-mercaptans should be the common precursors for all
RSS releasing compounds. As the first proof-of-concept, we
decided to prepare a series of O-silyl mercaptans (1 and 2,
Scheme 2). These compounds were prepared from the
corresponding aldehyde reacting with silyl chloride and H2S gas.[4]
These compounds were found to be quite stable. They could be
[*]
J. Kang, S. Xu, M. N. Radford, W. Zhang, S. S. Kelly, J. J. Day,
Prof. Dr. M. Xian
Department of Chemistry, Washington State University
Pullman, WA 99164 (USA)
E-mail: mxian@wsu.edu
J.Kang and S. Xu contributed equally to this work.
†
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201802845
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purified by column chromatography and fully characterized (see
the supporting information (SI)). Due to the sensitivity of silyl
groups toward acidic conditions, 1 and 2 were expected to be pH-
sensitive H2S donors. As such, H2S release profiles of 1/2 were
measured in buffers using a H2S-selective electrode. Indeed,
TMS-protected donors (1a-d) exhibited pH- and structure-
dependent H2S releasing ability. In general, more and faster H2S
release was observed under more acidic pH. This is shown in
Figure 1 using 1d as the example. Other donors’ profiles are
shown in Fig. S1 in the SI. Their structures also played a role in
controlling H2S release as more hindered donors tended to
release slower and less H2S. TES-protected donors (2a-d)
showed enhanced stability. These donors released H2S in a much
slower fashion. In particular, 2c and 2d released barely detectable
H2S under these conditions. To further confirm their H2S
production in buffers we also carried out H2S gas trapping
experiments. The representative results were shown in Fig. S2,
which matched well with the H2S-electrode results. These results
indicate that O-silyl mercaptans could serve as effective H2S
donors and their H2S release profiles could be controlled by
structural modifications.
reported acyl disulfides as H2Sn donors.[6] It is known that acyl
disulfides are highly reactive and can easily react with
nucleophiles like amines.[7] As such, off-target effects of acyl
disulfide based donors could be a concern. Seeking more stable
and controllable H2Sn donors has become one of our research
goals. We realized that O-silyl mercaptans could be readily
converted to the corresponding disulfides and thus prepared a
series of such compounds (3a-f, Scheme 3). It should be noted
that in theory these compounds should just release H2S2 upon
desilylation, given their structural identity. However, regardless of
what the starting H2Sn species is, in solutions mixed polysulfides
(including H2S2, H2S3, H2S4, etc.) will be formed rapidly. Therefore,
these compounds were expected to be H2Sn donors. 3a-f were
then tested for their pH-dependent degradation to release H2Sn.
As shown in Table 1, the time needed to reach complete
degradation of these donors varies based on their structures.
Again, more hindered donors showed slower degradation and
more acidic conditions could promote faster degradation. It should
be noted that H+-promoted H2Sn production from these donors
could be detected by DSP-3,[8] a specific H2Sn fluorescent senor
(Figure S3 in SI). We also used DSP-3 to measure fluoride-
triggered H2Sn release from the donors. Figure 2 showed the time-
dependent release profiles of these donors. Structural effects
were obvious. With these results we believe O-silyl disulfides are
effective H2Sn donors.
Scheme 2. The structures of O-silyl mercaptans
Scheme 3. Structures of H2Sn donors (3a-f)
Table 1. Time needed to release H2Sn from the donorsa
Donors
3a
pH=5.0
2.5 h
4 h
pH=6.0
6 h
pH=7.4
10 h
3b
12 h
14 h
3c
4.8 h
7 h
>24 h
14 h
>24 h
20 h
3d
3e
12 h
13 h
16 h
23 h
3f
>24 h
>24 h
adonor concentration: 20 mM, in DMF/PBS buffer (4/1)
Figure 1. Time-dependent H2S release curves of 1d (100 µM) in different pH
buffers.
H2Sn are another group of sulfur-based signaling molecules that
have received increasing attention recently.[1,5] Due to their
instability in aqueous environments, slow and controllable H2Sn
donors are expected to be useful research tools. However, this
has not been well studied. Only a recent work by Wang et al.
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Figure 2. Time-dependent H2Sn release from 3a-3f (indicated by fluorescence
changes). Donors (0.5 mM) were treated with TBAF (2.0 mM) in ethanol and
then sensed by DSP-3.
Table 3. Trapping persulfides with iodoacetamide
Persulfides (RSSH) are another group of biologically important
RSS.[1,9] The formation of persulfides on proteins has been
recognized as a critical posttranslational modification. It provides
a possible mechanism by which RSS alter the functions of a wide
range of cellular proteins and enzymes. Methods that allow easy
and reliable access to persulfides are the prerequisite for the
study of persulfides. Previously, acyl disulfides (RSSAc)[10] and 9-
fluorenylmethyl disulfides (RSSFm)[11] have been developed as
acid- or base-triggered persulfide donors, respectively. We
envisioned that O-silyl mercaptan derived unsymmetric disulfides
could serve as a new category of persulfide donors and would
benefit the study of persulfides. With this idea in mind, a series of
such disulfides (4a-f, Scheme 4) were prepared (see SI for
syntheses and characterization).
HSNO is the simplest S-nitrosothiols and has been proposed to
be the crucial intermediate in the ‘crosstalk’ between NO and
H2S.[12] However, the biochemistry (formation, transport,
decomposition) of HSNO remains poorly understood. Recent
results show that HSNO could be generated from the treatment of
RSNO with H2S,[12] or from NO and H2S gases in high
concentration.[13] These methods are not ideal for HSNO studies
as other reactive species (like NO, H2S) could also present.
Therefore, it is impossible to attribute the observed activities only
to HSNO. Methods that allow ‘clean’ generation of HSNO (without
the interference of other RSS) are critical for better understanding
HSNO chemical biology. To this end, we found O-silyl mercaptans
(1, 2) could be readily converted to the corresponding SNO
adducts (as shown in Scheme 5). These compounds showed the
characteristic red color of S-nitrosothiols (due to n®p* transition
of the SNO motif).[14] Their representative UV-vis spectra are
shown in Figure S4 in SI. Their absorbance peaks around 565 nm
were observed (with e=11~15 L/mol/cm). Like typical alkyl S-
nitrosothiols, 6a-f were found to be unstable. But their identity
could be confirmed by NMR and MS analysis (see SI).
Scheme 4. Structures of persulfide donors (4a-f)
With 4a-f in hand, we measured their pH- and fluoride-
dependent decomposition to release persulfides. As shown in
Table 2, the times needed to complete the decomposition were
used to assess their release profiles. Clearly, TMS-based
substrates (4a, 4b, 4d, 4e) could be triggered to release
persulfides in pH 5.0 and 6.0 solutions. They could also release
persulfides in pH 7.4, but more slowly. The corresponding
products were found to be a mixture of disulfide/trisulfides, which
are the typical end products of persulfides. TES-based donors (4c,
4f), in contrast, appeared to be quite stable and no decomposition
in those buffer systems was observed. When these donors were
treated with KF, they all underwent decomposition via varing rates.
The effects of structure on the rates were obvious.
Table 2. Time needed to release persulfides from the donors
Donors pH=5.0a
pH=6.0a
pH=7.4a
>12 h
>12 h
n.a.
w/ KFb
10 min
15 min
>12 h
5 min
5 min
8 h
4a
4b
4c
4d
4e
4f
3.5 h
4 h
n.a.c
3.8 h
4 h
4 h
Scheme 5. Structures of HSNO donors (6a-f)
4.3 h
n.a.
We next studied H+ and F- triggered HSNO release from these
compounds. Both conditions led to the formation of the
corresponding aldehydes, indicating the release of HSNO. Most
importantly, the formation of HSNO could be observed by direct
MS analysis (Figure 3), showing the desired HSNO peak. The
ultimate degradation product from HSNO was found to be
elemental sulfur and we did not observe the formation of H2S.
Overall, these results indicate O-silyl SNO compounds like 6a-f
are clean and H2S-independent HSNO donors.
4 h
>12 h
>12 h
n.a.
4.5 h
n.a.
n.a.
adonor concentration: 20 mM, in DMF/PBS buffer (4/1); bdonor
concentration: 20 mM, in MeOH. cno reaction detected.
To further prove persulfides were generated from these donors,
we used iodoacetamide to capture the persulfides. As shown in
Table 3, in all the cases the desired trapping products (5a or 5b)
were obtained in moderate to high yields. These results indicate
compounds 4a-f are efficient persulfide donors.
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Acknowledgements
This work is supported by NIH (R01GM125968 to M.X. and
1F31HL137233 to J.J.D.).
Keywords: relay deprotection • sulfide • persulfide • polysulfide •
nitrosothiol• prodrug
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Scheme 6. O-Silyl to O-Acetyl protecting group switch
In summary, we report herein the preparation and evaluation of
a series of O-silyl mercaptan based sulfur containing derivatives.
These molecules can undergo pH and F- mediated desilylation to
release the corresponding reactive sulfur species (RSS) such as
H2S, H2Sn, RSSH, and HSNO. This OàS relay deprotection
serves as a general strategy for the design of controllable RSS
donors. It should be noted that some of the donors (such as 3a-f,
4d-f) were obtained as the mixtures of diastereomers, due to two
chiral centers in their structures. However, the desired RSS were
produced as single compounds upon deprotection. While we only
validated donors for H2S, H2Sn, RSSH, and HSNO in this study,
we expect donors for other RSS like RSOH, RSO2H, RSeSH, SO2,
etc., could also be developed using this concept. Given the
significance of RSS in redox biology and the lack of appropriate
generation methods for many of them, our strategy should be very
useful in further exploration of these sulfur species. Moreover, we
have demonstrated that O-silyl groups of the donors could be
changed to other protecting groups like esters. This should allow
the development of RSS donors with other activation mechanisms
(such as esterase-activated donors). More studies using this
strategy for the design of RSS based donors, pro-drugs, and
specific imaging sensors are currently being developed in our
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A General Structural Template for
Sulfur Precursors/Prodrugs: O-Silyl
mercaptan derived compounds can be
developed as controllable donors for
reactive sulfur species such as H2S,
H2Sn, RSSH, and HSNO. This OàS
relay deprotection can serve as a
general strategy for the design of
sulfur-based chemical tools such as
prodrugs and imaging sensors.
Jianming Kang, Shi Xu, Miles N.
Radford, Wenjia Zhang, Shane S. Kelly,
Jacob J. Day, and Ming Xian *
Page No. – Page No.
O
à
S Relay Deprotection, A General
Approach for the Design of
Controllable Donors of Reactive
Sulfur Species
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