A novel spin trap for targeting sulfhydryl-containing polypeptides

Article abstract of DOI:10.1039/b509903j

A novel spin trap containing an iodoacetamide group has been synthesized and then used to target polypeptides, i.e. glutathione and bovine serum albumin, by which the resulting covalently bonded bioconjugates exhibit great potential for the application of

Full text of DOI:10.1039/b509903j

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A novel spin trap for targeting sulfhydryl-containing polypeptides{
Yang Ping Liu,a Yi Qiong Ji,a Yu Guang Song,a Ke Jian Liu,*b Bin Liu,a Qiu Tiana and Yang Liu*a
Received (in Cambridge, UK) 14th July 2005, Accepted 27th August 2005
First published as an Advance Article on the web 7th September 2005
DOI: 10.1039/b509903j
2. Finally, the iodination of nitrone 2 afforded a colorless or pale
yellow solid.
A novel spin trap containing an iodoacetamide group has been
synthesized and then used to target polypeptides, i.e. glu-
tathione and bovine serum albumin, by which the resulting
covalently bonded bioconjugates exhibit great potential for the
application of spin trapping of transient radicals in biological
systems.
To evaluate the reactivity of the nitrone 3 with the sulfhydryl
group in polypeptides, first, we probed its targeting attack to
glutathione (GSH) which is the most abundant non-protein
sulfhydryl-containing tripeptide in the cell. The coupling reaction
was carried out overnight at room temperature in darkness. The
water-soluble bioconjugate (GS-PBN) was subsequently obtained
by several extractions and chromatographic purification on
Sephadex LH-20. The covalent link between glutathione and the
Much of the current interest in spin trapping lies in the detection of
biological radicals which are implicated in many physiological and
pathological processes.1 However, these reactive species are
unstable and low in concentration, and may site-specifically
transform to secondary radical species.2 Thus, the issue of how to
selectively make a spin trap accumulate at the defined site of free
radical evolution is very important for the purpose of enhancing
spin trapping efficiency and precisely demonstrating the biological
significance of the trapped radicals. Recently, a few nitrones
derived from a-phenyl-N-tert-butyl nitrone (PBN) were prepared
for targeting mitochondria,3 cell membranes4 and lipid mem-
branes5 via noncovalent interactions. A recent study also reported
a polyclonal antibody that can recognize adduct species as an
antigen which is produced upon spin trapping of suitable protein
radicals by 5,5-dimethylpyrroline N-oxide (DMPO).6 Despite the
availability of a nitrone bioconjugate as an antigen produced by a
covalent conjugation between DMPO derivative and ovalbumin,6d
little attention has been paid to developing site-specific spin traps
for targeting polypeptides via covalent connections. With this aim
in mind, we report herein the synthesis of N-[4-(iodoacetamido)-
benzylidene]-N-tert-butylamine N-oxide. This compound has two
important properties: while the iodoacetamido moiety can undergo
facile reaction with the sulfhydryl group of polypeptides,7 the
nitronyl group can be used to trap free radicals. Thereafter, its
covalent targeting for glutathione and bovine serum albumin
(BSA) (Scheme 1(A)) as well as enzymatic recognition and
proteolysis for the polypeptide-linked spin adducts are also
demonstrated.
1
iodoacetamide moiety of the nitrone 3 was characterized by H,
13C NMR and MS spectroscopies. To confirm that the covalent
connection does not affect the nitronyl group that is the functional
site for trapping free radicals, we performed a few spin-trapping
EPR experiments in which several oxygen-, carbon- and sulfur-
centered radicals were captured by GS-PBN in aqueous or DMSO
solution (Table 1). A typical nitroxide signal is demonstrated in
Fig. 1(A). The resulting EPR spectrum is unambiguously
composed of a triplet of doublets derived from the splitting of
nitronyl-N and a-H nuclei, and exhibits the slight anisotropy
Scheme 1 (A) Coupling of the functional nitrone to sulfhydryl-contain-
ing biomolecules. (B) The synthesis of the functional nitrone 3. Reagents:
(a) chloro acetyl chloride, pyridine, dry ether; (b) N-tert-butyl hydro-
xylamine, a small amount of MgSO4, benzene; (c) sodium iodine, acetone.
Functional nitrone 3 was prepared according to a three-
step synthesis as indicated in Scheme 1(B). First, 4-(chloro-
acetamido)benzaldehyde
1 was obtained by acylation of
4-aminobenzaldehyde with chloroacetyl chloride. Then, the
condensation of N-tert-butylhydroxylamine on compound 1 led
to N-[4-(chloroacetamido)benzylidene]-N-tert-butylamine N-oxide
Table 1 EPR hyperfine splitting constants of GS-PBN spin adducts
Adduct
Resource
Solvent
aN/mT aH/mT
aState Key Laboratory for Structural Chemistry of Unstable Species,
Center for Molecular Science, Institute of Chemistry, Chinese Academy
of Sciences, Beijing, 100080, China. E-mail: yliu@iccas.ac.cn;
Fax: +86 10 62559373; Tel: +86 10 62571074
EtO
t-BuO
Ethanol/Pb(OAc)4
(t-BuO)2/hn
DMSOa 1.37
DMSOa 1.52
0.20
0.32
0.33
0.35
0.43
0.19
?
?
CH3CH OH Fe2+/H2O2/ethanol
Water
1.62
1.65
1.60
1.51
?
CH3
p-ClPh
Fe2+/H2O2/DMSOa Water
?
bCollege of Pharmacy, University of New Mexico, 2502 Marble NE,
Albuquerque, NM 87131, USA. E-mail: jliu@unm.edu;
Fax: (505) 272-6749; Tel: (505) 272-9546
p-CDTb/hn
Na2SO3/K2Cr2O7
Water
Water
?
22
?
SO3
a
b
DMSO, dimethyl sulfoxide. p-CDT, para-chlorophenyl diazonium
tetrafluoraborate.
{ Electronic supplementary information (ESI) available: Experimental
section. See http://dx.doi.org/10.1039/b509903j
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Chem. Commun., 2005, 4943–4945 | 4943

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Fig. 2 UV-VIS spectra (H2O) of (a) bovine serum albumin (BSA)
(dashed line), (b) the BSA linked PBN (solid line) and (c) the nitrone 3
(dotted line).
Fig. 1 EPR spectra of free and bound GS-PBN adducts. (A) UV
irradiation of the mixture of GS-PBN (3 mM) and para-chlorophenyl
diazonium tetrafluoraborate (8 mg ml21). (B) The same as (A), except
glutathione s-transferase (10 mM) was added. (C) The same as (A), except
glutathione s-transferase (50 mM) was added.
such as rotational-hindrance or binding to immobilized environ-
ments. Assignment of the broad signal can be performed by
analysing a well-resolved spectrum after proteolysis of the protein-
linked spin adduct. As shown in Fig. 3(B), the broad signal became
well-resolved after the sample was incubated with pronase, a
nonspecific protease. Comparing the hyperfine splitting constants
which is characteristic of the incorporation of a bulky glutathionyl
group into the nitroxide.
Being an intracellular antioxidant and antitoxicant, GSH takes
part in many of the redox and detoxication processes mediated
by enzymes such as glutathione peroxidase and glutathione
s-transferases (GSTs).8 Indeed, GSH, and even GSH derivatives
may be recognized by these enzymes. In a previous study, a spin-
labeled derivative of GSH was recognized by GST and used to
characterize the kinetic and binding properties of GST.9 Thus, it
is conceivable that GS-PBN and its spin adducts, as GSH
derivatives, may be the substrates for GST. As shown in Fig. 1(B),
two EPR spectral components arising from free and bound
nitroxides were observed when 10 mM of GST were added to the
sample from Fig. 1(A). Increasing the GST concentration to 50 mM
resulted in the larger proportion of the bound component
(Fig. 1(C)). These results demonstrate that GS-PBN spin adducts
can be recognized by GST and probably employed to report the
biological environment around the adducts.
(hfsc) for the EPR signal from Fig. 3(B) (aN 5 1.60 mT; aH
5
0.42 mT) and that from spin adduct of GS-PBN and para-
chlorophenyl radicals (aN 5 1.60 mT; aH 5 0.43 mT), we find that
both are dramatically concordant, by which the trapped radical
can be confidently determined to be the para-chlorophenyl radical.
In summary, the newly synthesized nitrone 3 can efficiently
target some sulfhydryl-containing biomolecules, such as glu-
tathione and BSA. The resulting bioconjugates, GS-PBN and
BSA-PBN, not only can efficiently trap reactive radicals, but more
interestingly, their spin adducts further exhibit the characteristics
of immobilization, like spin labelling (either directly by a protein-
linked spin adduct or indirectly through an enzyme-recognized
immobile nitroxide). The present study demonstrates that with the
help of the covalent link or molecular recognition, the targeting for
polypeptides makes possible the transportation of a spin trap to
the area(s) of interest in which the monitored radical species is site-
specifically generated. In addition, characterization of EPR spectra
for immobile adducts can be achieved through proteolysis.
An in vitro targeting attack to the sulfhydryl-containing protein
was performed with a mixture of the nitrone 3 and BSA. The
mixture was then incubated overnight at 30 uC in darkness. Excess
nitrone 3 was subsequently removed by Sephadex G-25. UV-VIS
spectra indicated that besides the protein-specific absorbance at
280 nm the BSA linked PBN (BSA-PBN) exhibited another
absorbance shoulder at 316 nm (Fig. 2). The appearance of the
new absorbance implies the existence of a chromophoric group
(PBN moiety) in the modified protein. To estimate the spin
trapping properties of the nitrone-labeled BSA, para-chlorophenyl
radicals were generated by UV photolysis of para-chlorophenyl
diazonium tetrafluoraborate in the presence of BSA-PBN. After
the irradiation, a spectrum characteristic of an immobilized
nitroxide was observable, as illustrated in Fig. 3(A). Under the
same experimental conditions, UV-irradiation of BSA-PBN or
diazonium alone did not produce any detectable EPR signal (data
not shown). Although the broad line in protein-linked spin adduct
hampers the expedient recognition of the adduct, we can obtain
some additional information concerning protein dynamic effects,
Fig. 3 (A) UV irradiation of the aqueous solution of a small amount of
para-chlorophenyl diazonium tetrafluoraborate and the attached protein
(0.5 mM). (B) The sample from spectrum (A) was treated with pronase
(20 mg ml21) for 24 h at room temperature.
4944 | Chem. Commun., 2005, 4943–4945
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This work was supported by the National Natural Science
Foundation of China (No. 30128003, No. 20175032 and No.
20473098 ) and the Outstanding Overseas Chinese Scholars Fund
of Chinese Academy of Sciences.
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