Facile formation of dehydroalanine from S-nitrosocysteines

Article abstract of DOI:10.1021/ja905558w

(Chemical Equation Presented) A reductive elimination of S-nitrosocysteines using phosphine substrates has been developed. This reaction converts unstable S-nitrosocysteines to dehydroalanine derivatives under very mild conditions. It can potentially be applied for the detection of protein S-nitrosation.

Full text of DOI:10.1021/ja905558w

Published on Web 08/28/2009
Facile Formation of Dehydroalanine From S-Nitrosocysteines
Hua Wang, Jiming Zhang, and Ming Xian*
Department of Chemistry, Washington State UniVersity, Pullman, Washington 99164
Received July 6, 2009; E-mail: mxian@wsu.edu
Scheme 1
Nitric oxide (NO) is a signaling molecule which plays important
roles in biological systems. NO exerts its actions by chemical
modifications of targets, preferentially with thiol groups. S-
Nitrosation of Cys residues in proteins is a principal reaction of
NO and NO-derived species. This redox-based post-translational
modification has been implicated in the cGMP-independent control
of a broad spectrum of cellular functions in a variety of cell types.1
S-Nitrosothiols (RSNO) are products of S-nitrosation that are
synthesized, stored, transported, and degraded in cell systems.
However, the detection of RSNO in biological samples remains a
challenge because of the lability of the SNO moiety.2 We believe
if new reactions can be developed to convert unstable SNO to stable/
detectable molecules, such reactions should hold considerable
promise for the detection of RSNO. We recently discovered several
phosphine-based reactions which can selectively convert SNO to
stable conjugates such as sulfenamides and disulfide-iminophos-
phoranes.3 In these reactions, an azaylide intermediate is formed
upon treatment of SNO with the phosphine substrate.3,4 Then, a
cascade intramolecular ligation occurs on an electrophile (such as
an ester) attaching to the phosphine, leading to the formation of
stable conjugates (eq 1, Scheme 1).3,5 In the meantime, we
envisioned that if the electrophile is absent, the nucleophilic N of
the azaylide intermediate could serve as a base in some cases. If
an acidic proton is present, such as the R-proton in Cys derivatives
(eq 2, Scheme 1), an elimination may take place to furnish
dehydroalanine (Dha) derivatives. In this process, pentacoordinated
P intermediates C or D might be the byproducts. Upon hydrolysis,
it should provide corresponding phosphine oxide and HSNH2, which
is an unstable species.6
Dha product (see Supporting Information). Therefore, HMPT is
not a selective reagent for SNOCs. We also examined several
triphenylphosphine derivatives (entries 6-10). As shown in Table
1, when an electron-deficient group was attached to P, such as 4a,
no Dha formed. With electron-donating substituents, such as 4b
and 4c, Dha was obtained in moderate to high yields. In 4c, the
ortho-dimethylamino group may help to stabilize the azaylide
intermediate and facilitate the intramolecular elimination process.
We also considered the steric effect as smaller phosphine substrates
should lead to more efficient deprotonation of the R-proton.
Therefore, smaller substrates than triphenylphosphines, i.e. 4d and
4e, were tested, and they both gave the desired product in excellent
yields. With the best substrate we have found so far, i.e. 4e, we
tested the reaction in other solvents including DMSO, DMF,
dioxane, and MeOH (entries 11-14). In all cases, the formation
of Dha was efficient. In addition, it should be mentioned that this
phosphine mediated reductive elimination of SNOC proved to be
fast (typically completed within 20 min at rt).
Dha is a potential site-specific chemical handle. It could allow
straightforward chemoselective incorporation of various reporter
molecules through a Michael addition. In fact, Dha formation from
phosphorylated proteins has been used to enrich and analyze
phosphoproteome.7 Because S-nitrosocysteine (SNOC) is the basic
adduct for protein S-nitrosation, a mild and selective transformation
from SNOC to Dha would be potentially useful for the detection
of protein S-nitrosation. Herein we report the first phosphine
mediated Dha formation from SNOCs.
With the optimized conditions in hand, we studied the generality
of this reaction using a series of SNOC derivatives (Table 2). As these
Table 1
With the proposed mechanism (Scheme 1) in mind, we first tested
the reaction between a freshly prepared SNOC derivative 2a and
triphenylphosphine (2 equiv) in THF (entry 1, Table 1). As
expected, a Dha product 3a was formed in 48% yield. The only
byproduct observed was the corresponding disulfide derivative from
2a, which is a known decomposition product from unstable
S-nitrosothiols.8 To optimize this reaction, a series of phosphine
compounds were screened (all in 2 equiv, Table 1). With PBu3,
PEt3, and P(OEt)3, 3a was obtained in moderate yields (entries 2-4).
Interestingly, treatment of 2a with HMPT [P(NMe2)3] led to 3a in
83% yield (entry 5). HMPT has been reported by Davis et al. for
a desulfurization of disulfides via a mechanism involving the Dha
formation.9 In a separate experiment, we also found that HMPT
reacted with Cys-based disulfide, and it furnished the corresponding
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13238 J. AM. CHEM. SOC. 2009, 131, 13238–13239
10.1021/ja905558w CCC: $40.75  2009 American Chemical Society

C O M M U N I C A T I O N S
Table 2
Scheme 3
formation of E-isomer in a small amount. Indeed, when a smaller
phosphine, P(OEt)3, was employed in this reaction, only Z-isomer was
obtained, albeit the yield was lower. In addition, the reaction with
P(OEt)3 (finishing in one minute) was much faster than the one with
4e (finishing in ∼25 min).
In summary, a phosphine-mediated Dha formation from SNOCs
was developed. Mechanistic study suggests that this reaction proceeds
mainly via an intramolecular cis-elimination on the azaylide intermedi-
ate. This Dha formation procedure, under very mild conditions, holds
the potential to be applied for the detection of protein S-nitrosation.
Scheme 2
Acknowledgment. Financial support from the Washington State
University and American Heart Association (0930120N).
Supporting Information Available: Spectroscopic and analytical
data and selected experimental procedures. This material is available
free of charge via the Internet at http://pubs.acs.org.
primary RSNO compounds are unstable species, they were freshly
generated from corresponding Cys starting materials and were used
for the elimination without purification. The yields reported were
overall yields in the two steps. As shown in Table 2 (entries 1-7), all
of the SNOC derivatives provided Dha products in good yields. We
have also tested this elimination in aqueous solutions, such as pH 7.4
PBS buffer. Due to a solubility problem for both SNOC compounds
and phosphine 4e, 20% of THF was needed. As shown in entries 8-11,
the desired Dha products were again obtained in decent yields, albeit
lower than in pure THF. In addition, a homocysteine derivative 1h
was also tested in this reaction (entry 12). As expected, no Dha
formation was observed with this substrate. We believe this transfor-
mation of Cys compounds, under very mild conditions, could serve
as a method to distinguish cysteine and homocysteine.
To examine the chemoselectivity of 4e, we tested the reaction of
4e with Cys-based disulfide 5. Unlike HMPT, 4e did not react with 5
to produce any detectable Dha product. In fact, disulfides were not
very sensitive to 4e. Trace amounts of -S-S- reduction products
were observed after 3 h. Extending the reaction time to 48 h did lead
to reduction product 1a in 40-50% yield (Scheme 2).
To prove the intramolecular elimination mechanism proposed in
Scheme 1, compound 1i was prepared. Upon nitrosation and treatment
with phosphine, an azaylide intermediate should form (Scheme 3). An
intramolecular cis-elimination from an eclipsed conformation (path A)
should give a Z-alkene product 6a. In contrast, an intermolecular trans-
elimination from the staggered conformation (path B) should provide
an E-isomer 6b. When 4e was used as the phosphine reagent, the
Z-isomer 6a was obtained as the major product (54%); we also isolated
some E-isomer 6b in 27% yield. Presumably, with 4e, a large
phosphine substrate, the lower-energied staggered conformation over-
whelmed the preferred cis-elimination to some degree, leading to the
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