Preventing protein oxidation with sugars: Scavenging of hypohalous acids by 5-selenopyranose and 4-selenofuranose derivatives

Article abstract of DOI:10.1021/tx3003593

Heme peroxidases including myeloperoxidase (MPO) are released at sites of inflammation by activated leukocytes. MPO generates hypohalous acids (HOX, X = Cl, Br, SCN) from H₂O₂; these oxidants are bactericidal and are key components o

Full text of DOI:10.1021/tx3003593

Article
pubs.acs.org/crt
Preventing Protein Oxidation with Sugars: Scavenging of
Hypohalous Acids by 5‑Selenopyranose and 4‑Selenofuranose
Derivatives
Corin Storkey,†,∥ David I. Pattison,†,‡,∥ Jonathan M. White,§ Carl H. Schiesser,§,∥
and Michael J. Davies*,†,‡,∥
†The Heart Research Institute, 7 Eliza Street, Newtown, NSW 2042, Australia
‡Faculty of Medicine, University of Sydney, Sydney, NSW 2006, Australia
§School of Chemistry and Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria 3010,
Australia
∥ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, Australia
S
* Supporting Information
ABSTRACT: Heme peroxidases including myeloperoxidase
(MPO) are released at sites of inflammation by activated
leukocytes. MPO generates hypohalous acids (HOX, X = Cl,
Br, SCN) from H2O2; these oxidants are bactericidal and are
key components of the inflammatory response. However,
excessive, misplaced or mistimed production can result in host
tissue damage, with this implicated in multiple inflammatory
diseases. We report here methods for the conversion of simple
monosaccharide sugars into selenium- and sulfur-containing
species that may act as potent water-soluble scavengers of
HOX. Competition kinetic studies show that the seleno
species react with HOCl with rate constants in the range 0.8−
1.0 × 108 M−1 s−1, only marginally slower than those for the most susceptible biological targets including the endogenous
antioxidant, glutathione. The rate constants for the corresponding sulfur-sugars are considerably slower (1.4−1.9 × 106 M−1 s−1).
Rate constants for reaction of the seleno-sugars with HOBr are ∼8 times lower than those for HOCl (1.0−1.5 × 107 M−1 s−1).
These values show little variation with differing sugar structures. Reaction with HOSCN is slower (∼102 M−1 s−1). The seleno-
sugars decreased the extent of HOCl-mediated oxidation of Met, His, Trp, Lys, and Tyr residues, and 3-chlorotyrosine formation,
on both isolated bovine serum albumin and human plasma proteins, at concentrations as low as 50 μM. These studies
demonstrate that novel selenium (and to a lesser extent, sulfur) derivatives of monosaccharides could be potent modulators of
peroxidase-mediated damage at sites of acute and chronic inflammation, and in multiple human pathologies.
phagocytic cells that eliminate parasites and related organisms.
Unlike neutrophils, which phagocytose their target organisms
and subsequently release MPO primarily into the phagolyso-
somal compartment, eosinophils exocytose their granule
contents on to the parasite surface to which they are attached.3
Salivary peroxidase or LPO is found in multiple human
exocrine secretions including tears, milk, saliva, and vaginal
fluid. In each case, the primary role of these peroxidases is as a
first line of defense against invading microorganisms.4
For each peroxidase, the proportions of HOCl/HOBr/
HOSCN produced are determined by the selectivity constants
of the enzymes for each substrate anion, and the physiological
concentrations of Cl−, Br−, and SCN−. At neutral pH and
normal physiological plasma anion concentrations, ∼45% of the
1. INTRODUCTION
There is considerable interest in the roles played by mammalian
heme peroxidase enzymes (e.g., myeloperoxidase (MPO),
eosinophil peroxidase (EPO), and lactoperoxidase (LPO)), in
both the innate immune system, where these species play a
critical role in killing invading pathogens, and in human
pathologies associated with chronic inflammation. MPO is
released at sites of inflammation from intracellular granules by
activated neutrophils, monocytes, and some tissue macro-
phages.1 Activation of these cells results in the generation of
hydrogen peroxide (H2O2) by NADPH oxidase enzymes via a
respiratory burst.2 MPO utilizes this H2O2 to oxidize halide and
pseudohalide ions, predominantly chloride (Cl−), bromide
(Br−), and thiocyanate (SCN−), to generate the potent
oxidants, hypochlorous (HOCl), hypobromous (HOBr), and
hypothiocyanous acids (HOSCN), respectively. EPO is a major
granule protein of eosinophils, which are specialized human
Received: August 17, 2012
Published: October 17, 2012
© 2012 American Chemical Society
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H2O2 consumed by MPO is used to generate HOCl, 50%
HOSCN, and the remaining 5% HOBr.5 In contrast, EPO
generates predominantly HOBr and HOSCN, while LPO
favors HOSCN.6,7 These oxidants are bactericidal or
bacteriostatic and are key components of the inflammatory
response. These properties underlie the widespread use,
particularly of HOCl (a major active component of household
bleach), in the water purification, hygiene, and disinfection
fields.8,9 Excessive or misplaced production of these species in
vivo has, however, been linked to several human pathologies as
a result of collateral damage to host tissues.10
seleno-L-talitol [7 (SeTal)] from glucose and mannose,
respectively, are described, together with a kinetic and product
analysis evaluation of these compounds as water-soluble
protective agents against HOCl-, HOBr-, and HOSCN-
mediated protein damage.
2. EXPERIMENTAL PROCEDURES
2.1. Synthesis and Characterization of Novel Compounds.
The strategies used to synthesize compounds 5−7 are outlined in
detail in the Results section. NMR, IR, elemental analysis, mass
spectrometry, optical rotation for all compounds (5−7, 9−12, 14−
15), and X-ray crystal structure data for 7 are given in Supporting
Information. The data obtained for compounds 9, 10, 14, and 15 agree
with literature reports.32−34
The role of MPO in the pathogenesis of disease has been
researched extensively, and considerable evidence links this
enzyme and the oxidants that it generates with the pathogenesis
of cardiovascular disease (atherosclerosis), cystic fibrosis, sepsis,
rheumatoid arthritis, asthma, kidney disease, and some
cancers.11−16 The evidence for a role for MPO in the
pathogenesis of atherosclerosis is compelling,11,17,18 with
clinical studies indicating that elevated plasma and blood
MPO levels are a strong independent risk factor and predictor
of outcomes for cardiovascular disease.12 Furthermore, it has
been reported that MPO mRNA, protein, and peroxidase
activity are present in atherosclerotic lesions, that elevated
levels of chlorinated tyrosine residues (3-chloro-Tyr, an
established biomarker of HOCl damage) are present on
proteins extracted from diseased human arteries compared to
healthy tissue, and that there is a direct correlation between the
extent of HOCl-damaged proteins and disease severity.19−22
HOCl and HOBr are powerful oxidants (two-electron
reduction potentials of 1.28 and 1.13 V, respectively)23 that
react rapidly with biological molecules, particularly with sulfur-
centers and amine functions on proteins, unsaturated lipids,
antioxidants, and DNA.24 In contrast, HOSCN is a less
powerful oxidant (two-electron reduction potential of 0.56 V)23
that reacts almost exclusively with sulfur and seleno-
residues.25−29 Kinetic data are consistent with proteins being
major targets for HOCl within biological systems as a result of
their abundance and high reactivity.30 Exposure of isolated
proteins to HOCl results predominantly in modification at side-
chains (Cys, Met, cystine, His, Lys, Trp, and Tyr), with limited
protein fragmentation and aggregation observed at high oxidant
concentrations.10
1H NMR spectra were recorded on Varian Inova 400 (400 MHz) or
Varian Inova 500 (500 MHz) instruments at room temperature, using
CDCl3 or CD3OD as an internal reference deuterium lock, CDCl3 at δ
7.26 ppm and CD3OD at δ 3.31 ppm. The chemical shift data for each
signal are given as δ in units of parts per million (ppm). The
multiplicity of each signal is indicated by s (singlet), d (doublet), t
(triplet), q (quartet), dd (doublet of doublets), dt (doublet of triplets),
and m (multiplet). The number of protons (n) for a given resonance is
indicated by n H. Coupling constants (J) are quoted in Hz and are
recorded to the nearest 0.1 Hz. 13C NMR spectra were recorded on
Varian Inova 400 (400 MHz) or Varian Inova 500 (500 MHz)
instruments using the central resonance of the triplet of CDCl3 at δ
77.23 ppm or the septet of CD3OD at δ 49.00 as an internal reference.
The chemical shift data for each signal are given as δ in units of parts
per million (ppm). Infrared spectra were recorded on a Perkin-Elmer
Spectrum One FT-IR spectrometer in the region 4000−650 cm−1. The
samples were analyzed as thin films from dichloromethane or
methanol. Mass spectra were recorded at the Bio21 Institute, The
University of Melbourne. Low-resolution spectra were recorded on a
Waters Micromass Quattro II instrument (EI and CI). High-resolution
mass spectrometry experiments were conducted using a hybrid linear
ion trap and Fourier transform ion cyclotron resonance (FT-ICR)
mass spectrometer (Finnigan LTQ-FT San Jose, CA), equipped with
ESI. Ions of interest were mass selected in the LTQ using standard
procedures and analyzed in the FT-ICR MS to generate the high-
resolution tandem mass spectrum. Optical specific rotations were
measured using a Jasco DIP-1000 digital polarimeter, in a cell of 1 cm
path length. The concentration (c) is expressed in g/100 cm3
(equivalent to g/0.1 dm3). Specific rotations are denoted [α] and
given in implied units of °dm2·g−1 (T = temperature in °C). Analytical
thin layer chromatography (TLC) was carried out on precoated 0.25
mm thick Merck 60 F254 silica gel plates. Visualization was by
absorption of UV light or thermal development after dipping in an
ethanolic solution of phosphomolybdic acid (PMA) or sulfuric acid
(H2SO4). Flash chromatography was carried out on silica gel [Merck
Kieselgel 60 (230−400 mesh)] under nitrogen pressure. Hydro-
We have recently demonstrated that the carbohydrate
derivatives 1,5-dideoxy-5-thio/seleno-L-gulitol [1 (SGul), 2
(SeGul)] and 1,5-dideoxy-5-thio/seleno-D-mannitol [3
(SMan), 4 (SeMan)] (Figure 1) afford significant protection
to human plasma proteins against HOCl in vitro.31 Here, the
syntheses of a range of sulfur- and selenium-containing
carbohydrate derivatives, 1,5-dideoxy-5-thio/seleno-L-iditol [5
(SId), 6 (SeId)] and the furanose derivative 1,4-dideoxy-4-
genation was carried out in a Buchi GlasUster “miniclave drive”
̈
stainless steel vessel, 100 mL, with a maximum operation pressure of
60 bar. Teflon inserts were used, and reactions were stirred using
magnetic stirrer bars.
Crystals of compound 7 were mounted in low temperature oil and
flash cooled to 130 K using an Oxford Cryostream low temperature
device. Intensity data were collected at 130 K on an Oxford
SuperNova X-ray diffractometer with a CCD detector using Cu−Kα
radiation (λ = 1.54184 Å). Data were reduced and corrected for
absorption (CrysAlis CCD, Oxford Diffraction Ltd., version 1.171.32.5
(release 08-05-2007 CrysAlis171 .NET)). The structures were solved
by direct methods and difference Fourier synthesis using the SHELX
suite of programs,35 as implemented within the WINGX5 software.36
Thermal ellipsoid plots were generated using the program ORTEP-3.
2.2. Biochemical Reagents. All chemicals were obtained from
Sigma/Aldrich/Fluka and were used as received, with the exception of
sodium hypochlorite (in 0.1 M NaOH, low in bromine; BDH
Chemicals). HOCl was quantified by its absorbance at 292 nm at pH
12 using ε292 = 350 M−1 cm−1.37 All studies were performed in 10 mM
Figure 1. Sulfur- and seleno-sugar derivatives prepared and
investigated in these studies.
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phosphate buffer (pH 7.4). Phosphate buffers were prepared using
Milli-Q water treated with washed Chelex resin (Bio-Rad) to remove
contaminating transition metal ions. The pH values of solutions were
adjusted, where necessary, to pH 7.4 using 100 mM H2SO4 or 100
mM NaOH.
size; Rockland Technologies, Newport, DE) packed with octadecyl
silanized silica, equipped with a Pelliguard guard column (2 cm;
Supelco), at 30 °C (using a column oven, Waters Corp., Milford, MA),
with a solvent flow rate of 1 mL·min−1. The mobile phase comprised a
gradient of solvent A (10 mM phosphoric acid with 100 mM sodium
perchlorate, pH 2.0) and solvent B [80% (v/v) MeOH in nanopure
water], programmed as follows: 20% solvent B and 80% solvent A at 0
min increasing to 80% solvent B over 10 min; over the next 5 min, the
proportion of solvent B was held at 80%, before the proportion of
solvent B was reduced to 20% over 4 min, and the column was allowed
to re-equilibrate for 6 min prior to injection of the next sample.
Samples were filtered (0.2 μm) to remove particulate matter before
analysis, with 50 μL injected for each run. The eluent was monitored
using a UV detector (λ 280 nm) and an electrochemical detector
(Antec Leyden Intro). The electrochemical detector was set to +1200
mV to quantify the halogenated N-Ac-Tyr products. Peak areas were
quantified using Class VP 7.4 SP1 software (Shimadzu) and compared
to authentic standards. Under these conditions, N-Ac-Tyr was
detected in the UV and electrochemical channel at 8.3 min, N-Ac-3-
Br-Tyr at 11.4 min, and N-Ac-3,5-diBr-Tyr at 13.4 min.
2.7. Competitive Kinetic Studies for SeTal (7) against
HOSCN Using 5-Thio-2-nitrobenzoic Acid (TNB). HOSCN was
prepared by in situ LPO-catalyzed reaction of H2O2 with SCN−.28,39
Thus, LPO (1.5−2 μM) was incubated with H2O2 (3.75 mM) and
NaSCN (7.5 mM) in 10 mM potassium phosphate buffer (pH 6.6) for
15 min at 22 °C. Catalase (1 mg·mL−1; from bovine liver) was
subsequently added to remove unreacted H2O2, followed by
centrifugation at 11300g for 5 min through a 10 kDa molecular-
mass cutoff filter to remove catalase and LPO. HOSCN concentrations
were determined by oxidation of TNB (5-thio-2-nitrobenzoic acid) at
412 nm using ε = 14150 M−1·cm−1.40,41
2.3. Competitive Kinetic Studies for Thio/Seleno-Sugars
against HOCl Using FMoc-Met. The kinetics of the reactions of
HOCl (1 μM) with the thio/seleno-sugar derivatives (“scavenger”; 1.2
μM−20 μM) were investigated in competition with Fmoc-Met (5 μM)
at 22 °C using an adaptation of a previous method,38 in which the
conversion of Fmoc-Met to the Fmoc-Met sulfoxide is quantified, in
the absence and presence of a putative scavenger. The yields of Fmoc-
Met sulfoxide generated in the presence of increasing concentrations
of the sugar derivative (yieldscavenger) were determined by UPLC and
compared to the maximal yield in the absence of added sugar/
scavenger (yieldmax). Using a competition kinetic analysis, the yields are
related by eq 1, and rearrangement of this equation results in the linear
form (y = mx + c), given in eq 2.
yieldscavenger
k
FmocMet[FmocMet]
=
yieldmax − yieldscavenger
kscavenger[scavenger]
(1)
k
scavenger[scavenger]
yieldmax[FmocMet]
=
+ [FmocMet]
yieldscavenger
kFmocMet
(2)
From a plot of [Fmoc-Met]·yieldmax/yieldscavenger against increasing
concentrations of the scavenger ([scavenger]), the slope of the
corresponding line allows determination of the value of kscavenger (i.e., k
for reaction of HOCl with the sugar derivative) using kFmocMet (1.3 ×
108 M−1 s−1) with a set y-intercept equal to [Fmoc-Met].
2.8. Kinetic Measurements of HOSCN Reactions. Stopped-flow
studies were carried out using an Applied Photophysics SX.18MV
stopped-flow system as described previously.42−44 HOSCN was kept
as the limiting reagent with a minimum of a 4-fold excess of the
substrate. The sample chamber was maintained at 22 °C, with ten runs
averaged to improve the signal-to-noise ratio. The rate constant for the
reaction of HOSCN with SeTal (7) was determined by competition
kinetics against TNB at 412 nm using kTNB = 3.8 × 105 M−1·s−1.25
2.9. HPLC Amino Acid Analysis of HOCl-Oxidized BSA and
Plasma. Amino acid analyses of HOCl oxidized BSA and plasma were
carried out essentially as described previously.45 BSA (0.5 mg mL−1,
∼7.6 μM) or human plasma diluted to give a final plasma protein
concentration of 0.5 mg mL−1 (ca. 7.6 μM, chosen to allow
comparison with the isolated protein data) was mixed with buffer
(labeled 0 mM) or increasing concentrations of the Se-sugars (2, 4, 6,
or 7; 50 μM−1 mM), before reaction with a 100-fold molar excess of
HOCl (760 μM), or buffer (labeled “control”). Samples, were
incubated for 3 h at 37 °C before being delipidated and the proteins
precipitated by the addition of 25 μL 0.3% (w/v) deoxycholic acid and
50 μL of 50% (w/v) TCA. The protein pellets were washed once with
5% (w/v) TCA and twice with ice cold acetone, before being
resuspended in 150 μL of 4 M methanesulfonic acid (MSA)
containing 0.2% w/v tryptamine. The samples were then hydrolyzed
under vacuum at 110 °C for 16−18 h. After neutralization with 150 μL
of 4 M NaOH and filtration (centrifuged at 11300g for 2 min through
a PVDF 0.22 μm membrane; 0.5 mL volume; No. UFC30GVNB,
Millipore), the samples were diluted into water (10-fold) and 40 μL
transferred to HPLC vials for analysis.
2.4. UPLC Instrumentation and Methods. Quantification of
Fmoc-Met and Fmoc-Met sulfoxide were carried out on a Shimadzu
Nexera UPLC system (Shimadzu, South Rydalmere, NSW, Australia),
with samples separated on a Shim-pack XR-ODS (Shimadzu, 100 ×
4.6 mm, 2.2 μm) column at 40 °C with a solvent flow rate of 1.2
mL·min−1. The mobile phase comprised of a gradient of solvent A
[(MeOH (20%), THF (2.5%), 1 M NaOAc, pH 5.3 (5%) and H2O
(72.5%)] and solvent B [MeOH (80%), THF (2.5%), 1 M NaOAc,
pH 5.3 (5%), H2O (12.5%)], programmed as follows: 75% solvent B
and 25% solvent A at 0 min, increasing to 87.5% solvent B over 5 min,
followed by a further increase to 100% solvent B over the next 0.5 min
and a wash with 100% solvent B for 2.5 min, before returning to 75%
solvent B over the next 0.5 min with 3.5 min of re-equilibrating
preceding the next injection. Samples were filtered (0.2 μm) to remove
particulate matter before analysis with 5 μL injected for each run. The
eluent was monitored by fluorescence detection (RF-20AXS; λex 265
nm; λem 310 nm), with peak areas determined using Lab Solutions
5.32 SP1 software (Shimadzu) and compared to authentic standards as
required. Using these conditions, Fmoc-Met sulfoxide was detected at
a retention time of 1.7 min, and Fmoc-Met at 2.8 min.
2.5. Competitive Kinetic Studies for Seleno-Sugars Against
HOBr Using N-AcTyr. HOBr was prepared by mixing HOCl (40 mM
in water, pH 13) with NaBr (45 mM in water) in equal volumes. The
reaction was left for 1 min before dilution to the required
concentration of HOBr (typically 0.1 − 0.2 mM). The kinetics of
the reactions of HOBr (10 μM) with the thio/seleno-sugar derivatives
(20−150 μM) were investigated in competition with N-Ac-Tyr (1
μM) at 22 °C by adapting a previous method,38 using the conversion
of N-Ac-Tyr to N-Ac-3-Br-Tyr, in the absence and presence of a
scavenger. The yields of N-Ac-3-Br-Tyr at increasing carbohydrate
derivative concentration (yieldscavenger) were determined by HPLC and
compared to the maximal yield in the absence of added scavenger
(yieldmax). The data were subsequently analyzed in an analogous
OPA reagent (Sigma-Aldrich, P7914) was activated immediately
before use by the addition of 5 μL of 2-mercaptoethanol to 1 mL of
OPA reagent in an HPLC vial. Five micromolar standards were
prepared by the addition of 10 μL of Sigma-Aldrich amino acid
standards (A9781, 500 μM stock) to 990 μL of water. From these
stock solutions, 1, 2, 3, 4, and 5 μM standards were prepared for
comparative analysis.
manner to that described for HOCl/Fmoc-Met above, with kN‑Ac‑Tyr
=
2.6 × 105 M−1 s−1.38
Analysis and quantification of protein reaction products were
carried out on a Shimadzu Nexera UPLC system (Shimadzu, South
Rydalmere, NSW, Australia). The reaction mixtures were separated on
a Shim-pack XR-ODS (Shimadzu, 100 × 4.6 mm, 2.2 μm) column,
2.6. HPLC Instrumentation and Methods. Quantification of N-
Ac-Tyr and N-Ac-3-Br-Tyr were carried out on a Shimadzu LC-10A
HPLC system (Shimadzu, South Rydalmere, NSW, Australia), on a
Zorbax reverse-phase HPLC column (25 cm × 4.6 mm, 5 μm particle
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a
Scheme 1. Reagents and Conditions for the Chemical Synthesis of 1,5-Dideoxy-5-thio/seleno-L-iditol (5 and 6)
a
(i) BnOH, AcCl; (ii) p-TSA, 2-methoxypropene, Me2CO; (iii) H2, Pd/C, EtOH; (iv) NaBH4, EtOH; (v) MsCl, DMAP, CH2Cl2/Py; (vi) Na2S,
DMF (11) or NaBH4, Se, EtOH (12); (vii) TFA, CH2Cl2.
a
Scheme 2. Reagents and Conditions for the Chemical Synthesis of 1,4-Dideoxy-4-seleno-D-talitol (7)
a
(i) p-TSA, 2,2-dimethoxypropane, Me2CO; (ii) NaBH4, MeOH; (iii) MsCl, DMAP, CH2Cl2/Py; (iv) NaBH4, Se, EtOH; (v) TFA, CH2Cl2.
maintained at 40 °C with a flow rate of 1.2 mL·min−1. The mobile
phase comprised a gradient of solvent A [(MeOH (20%), THF
(2.5%), 1 M NaOAc, pH 5.3 (5%), and H2O (72.5%)] and solvent B
[MeOH (80%), THF (2.5%), 1 M NaOAc, pH 5.3 (5%), and H2O
(12.5%)] programmed as follows: 0% solvent B and 100% solvent A at
0 min, increasing to 25% solvent B over 6 min, maintaining 25% for a
further 1 min, followed by an increase to 62% solvent B over the next
0.5 min and holding this concentration for another 2.5 min, followed
by an increase to 100% B over 2 min, holding at 100% B for another 1
min, before returning to 0% solvent B over the next 0.5 min, and re-
equilibrating at 0% for 3.5 min. The autoinjector was programmed to
add 20 μL of activated OPA reagent to the specified sample (40 μL),
followed by 3 mixing cycles, and a 1 min incubation period. After the
incubation step, 15 μL of the final reaction mixture was injected. The
fluorescence detector was set with λex of 340 nm and λem of 440 nm to
detect the OPA tag. The concentration of each amino acid in the
samples was determined from linear plots of the HPLC peak area
versus concentration from the standards. Any variation in the
efficiency of hydrolysis or sample recovery after the precipitation
and washing steps was taken into account by expressing the
concentration of the amino acids of interest as a ratio with isoleucine
(Ile), as it is not modified by HOCl.
2.10. Analysis of 3-Chlorotyrosine (3-Cl-Tyr) Using LCMS.
Sample preparation for 3-Cl-Tyr analysis was identical to that used for
total amino acid analysis (see above), except that hydrolysis was
achieved by the addition of 150 μL of 6 M HCl and 50 μL of
thioglycolic acid into the PicoTag vessel, before being placed under
vacuum in the oven at 110 °C for 16−18 h. The hydrolyzed samples
were then placed in 1.5 mL centrifuge tubes and dried under vacuum.
Each sample was resuspended in 50 μL of water and filtered
(centrifuged at 11300g for 2 min through a PVDF 0.22 μm membrane,
0.5 mL volume, No. UFC30GVNB, Millipore) to remove any
insoluble precipitate. The samples were then transferred to HPLC
vials for LC-MS analysis.
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Standard solutions were prepared in Milli-Q water to give 2.5 mM
Tyr and 100 μM 3-Cl-Tyr. The two stock solutions were combined
and diluted to give 1:1 mixtures of tyrosine: 3-Cl-Tyr with
concentrations of 100−500 pmol in 20 μL. Then, 40 μL of each
standard was transferred to HPLC vials for LC-MS analysis. Tyr and 3-
Cl-Tyr were quantified by LC-MS in the positive ion mode with a
Thermo LCQ Deca XP ion-trap instrument coupled to a Finnigan
Surveyor HPLC system as described previously.46
(pH 7.4). These conditions were chosen to enable direct
comparison with previous kinetic data for other targets
including Met, Cys, and glutathione (GSH).24,30 Reactions
with selenomethionine (SeMet), a well-established oxidant
scavenger,47 were also investigated to assess the influence of the
ring structure on the reactivity of the selenoether moiety.
Reactions of HOCl (1 μM) with the S-/Se-sugar derivatives
(1−7; 1.2 μM−100 μM) or SeMet (1.2 μM−20 μM) were
investigated using an adaptation of previously established
competition kinetics approach, with Fmoc-Met (5 μM) as the
competing target.38 A decrease in Fmoc-Met sulfoxide was
3. RESULTS
3.1. Synthesis. Compounds 1−4 were prepared as reported
previously.31 The synthesis of 1,5-dideoxy-5-thio/seleno-L-iditol
(5 and 6) followed an analogous synthetic route except with
the inclusion of an anomeric benzyl protecting group.
Accordingly, D-glucose (8) was reacted with benzyl alcohol in
the presence of acetyl chloride, followed by further reaction of
the isolated crude product with 2-methoxypropene in the
presence of catalytic p-toluenesulfonic acid under anhydrous
conditions to afford the 1-benzyl-2,3,4,6-di-O-isopropylidene-D-
glucoside (9) as the major product in 80% yield over two steps
(Scheme 1).
detected with increasing concentrations of (1−7) (yieldscavenger
)
with these data compared to the maximal yield in the absence
of added scavenger (yieldmax). The linear graphs (Figure 2a)
obtained by plotting [Fmoc-Met]·(yieldmax/yieldscavenger) against
the sugar concentration ([scavenger]) gave a slope from which
the rate constant for reaction of HOCl with the S-/Se-sugar
derivatives (Table 1) could be calculated, using k(HOCl+Fmoc‑Met)
The benzyl group was then cleaved by hydrogenation before
the crude reaction mixture was reacted with sodium
borohydride in ethanol to yield the diol (10), which was
purified by chromatography in an overall yield of 90% over two
steps. The subsequent step involved conversion of the diol (10)
to the corresponding bis-mesylate intermediate (10a), by
reaction with methanesulfonyl chloride in dichloromethane and
pyridine with catalytic 4-dimethylaminopyridine (DMAP). The
bis-mesylate (10a) was then reacted with either sodium sulfide
in DMF at 100 °C to give the protected S-sugar (11) in a 74%
yield or with selenium metal and sodium borohydride in EtOH
at 70 °C to give the protected Se-sugar (12) in 53% yield.
Deprotection of 11 and 12 was achieved by treatment with
trifluoroacetic acid in dichloromethane, and the resulting
residues were purified by flash chromatography to yield SId
(5) and SeId (6) as colorless oils. Characterization data for
these materials are provided in Supporting Information.
The synthesis of the pyranose 1,4-dideoxy-4-seleno-D-talitol
(7) began from D-mannose (13). This was reacted with 2,2-
dimethoxypropane in the presence of catalytic p-toluenesul-
fonic acid and acetone before reduction of the hemiacetal with
sodium borohydride in methanol to yield the diol (14) as the
major product in 82% over two steps (Scheme 2). The diol
(14) was subsequently converted to the corresponding bis-
methanesulfonate intermediate (14a), by reaction with
methanesulfonyl chloride in dichloromethane and pyridine
with catalytic 4-dimethylaminopyridine (DMAP). Reaction of
this bis-methanesulfonate species (14a) with selenium and
sodium borohydride in EtOH at 70 °C gave the isopropylidene-
protected species (15) in 55% yield. Deprotection of (15) was
achieved by treatment with trifluoroacetic acid in dichloro-
methane, and the resulting residue was purified by flash
chromatography to yield the desired seleno-sugar derivative
SeTal (7) as a colorless oil. Full characterization data for (7)
(NMR, IR, elemental analysis, mass spectrometry, optical
rotation, and X-ray crystal structure) are provided in
Supporting Information.
Figure 2. Kinetic data for the reaction of S-/Se-sugar derivatives 1, 2,
and 7 with (a) HOCl and (b) HOBr. Representative data for three
■
▲
sugar (“scavenger”) derivatives, SGul (1; ), SeGul (2; ), and SeTal
●
(7; ), are presented. (a) HOCl scavenging was measured by
competition against Fmoc-Met using UPLC with fluorescence
detection to quantify Fmoc-Met sulfoxide yields. Slope = k(HOCl +
sugar)/k(HOCl + Fmoc-Met) with k(HOCl + Fmoc-Met) = 1.3 × 108
M−1 s−1.19 The insert shows expanded data for SeGul (2) and SeTal
(7). (b) HOBr scavenging was measured in competition with N-Ac-
Tyr using HPLC with electrochemical detection to quantify N-Ac-
BrTyr formation. Slope = k(HOBr + sugar)/k(HOBr + N-Ac-Tyr)
with k(HOBr + N-Ac-Tyr) = 2.6 × 105 M−1 s−1.18 Errors bars for all
points are expressed as standard error of the mean (n > 4); in some
cases, the error bars are smaller than the symbols.
3.2. Kinetic Studies with HOCl, HOBr, and HOSCN. The
reactivity of the thio- and seleno-containing pyranose
carbohydrate derivatives (1−7) with HOCl, HOBr, and
HOSCN was investigated and the second-order rate constants
for their reactions determined at 22 °C and physiological pH
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the small changes in absorbance, an accurate value for the
second-order rate constant could not be determined, but an
estimate was obtained using eq 3 (with kTNB 3.8 × 105 M−1
s−1)25 for each set of concentrations. It has also been noted
previously that analysis of rate constants of HOSCN using
TNB as a competitive substrate is complicated by rapid
secondary reactions of the TNB sulfenyl thiocyanate (or
sulfenic acid) product with a further TNB molecule to form the
disulfide, DTNB.26 With these limitations in mind, an
approximate second-order rate constant was determined for
reaction of HOSCN with 7 of ∼102 M−1 s−1 (Table 1). As this
rate constant could not be assessed accurately, experiments
with 1−6 were not undertaken.
Table 1. Summary of the Second Order Rate Constants for
Scavenging of HOCl, HOBr, and HOSCN by the S-/Se-
Sugar Derivatives (1−7), SeMet, Met, Cys, and GSH
a
k (HOCl)/107 M−1 k (HOBr)/107 M−1
k (HOSCN)/104
compd
s−1
s−1
M−1 s−1
SeGul 2
SeMan 4
SeId 6
SeTal 7
SeMet
Met
9.4 ( 0.3)
8.0 ( 0.5)
9.0 ( 0.3)
10 ( 2)
1.3 ( 0.2)
1.09 ( 0.03)
1.33 ( 0.03)
1.52 ( 0.06)
1.4 ( 0.1)
∼0.01
b
32 ( 1)
0.28 ( 0.02)
c
d
e
2.95 ( 0.07)
0.36 ( 0.03)
<0.1
SGul 1
SMan 3
SId 5
0.190 ( 0.007)
0.141 ( 0.006)
0.140 ( 0.002)
0.112 ( 0.004)
0.099 ( 0.003)
0.115 ( 0.004)
yieldscavenger
c
f
b
k
TNB[TNB]
Cys
31.2 ( 0.6)
1.2 ( 0.2)
7.8 ( 1.4)
≈
c
b
GSH
10.9 ( 0.1)
2.5 ( 0.4)
yieldmax
kSeTal[SeTal] + kTNB[TNB]
(3)
a
Values were determined by UPLC (for HOCl) or HPLC (for HOBr)
competition kinetic assays using fluorescence (HOCl) or electro-
chemical (HOBr) detection. HOSCN rate constants were determined
by competitive kinetics using stopped-flow methods to measure
absorbance changes at 412 nm. Errors are expressed as 95% confidence
3.3. Protection Against HOCl-Mediated Protein
Oxidation. The rate constants reported above indicate that
2, 4, 6, and 7 are rapid scavengers of HOX, and therefore, the
potential of these compounds to protect against HOCl-
mediated protein oxidation was examined. These studies
focused on HOCl, as HOSCN does not react at an appreciable
rate with the seleno species. Studies were carried out with
isolated bovine serum albumin (BSA, as a model of the major
protein in human plasma, human serum albumin) and human
plasma itself. The latter system contains multiple alternative
targets for HOCl (antioxidants, lipids) allowing a more realistic
examination of scavenging potential. Protein oxidation was
quantified by HPLC analysis of total amino acids following
acid-catalyzed protein hydrolysis.30
b
c
intervals (= standard error × t95). From ref 29. Pattison, D. I.,
d
Storkey, C., and Davies, M. J., unpublished data. Data for NAc-Met-
e
OMe are from ref 38. Upper limit is reported in ref 25; the actual
f
value may be lower. From ref 38.
1.3 × 108 M−1 s−1 (Pattison, D.I., Storkey, C., and Davies, M.J.,
unpublished data). The latter value was determined by
comparable competition kinetic experiments utilizing SCN−
or 3,3′-dithiodipropionic acid as the HOCl scavengers in
competition with Fmoc-Met. Accurate second-order rate
constants of 2.34 × 107 and 1.6 × 105 M−1 s−1 (pH 7.4, 20−
22 °C) for the reactions of HOCl with SCN− and 3,3′-
dithiodipropionic acid, respectively, have been determined
previously using stopped flow methodology,42,48,49 allowing
BSA (0.5 mg·mL−1, ∼7.6 μM) was mixed with buffer
(labeled 0 mM) or increasing concentrations of each Se-sugar
derivative (2, 4, 6, or 7; 50 μM−1 mM), before reaction with a
100-fold molar excess of HOCl (760 μM), or buffer (labeled
“control”). The samples were incubated for 3 h at 37 °C, before
the protein was precipitated, subjected to acid hydrolysis and
quantified, following derivitization with the fluorescent tag
OPA by HPLC. In the maximally oxidized samples (0 mM Se-
sugar), five amino acids (Met, Trp, Tyr, His, and Lys) were
depleted in a significant manner. This depletion was decreased
in a dose-dependent manner by increasing concentrations of 2,
4, 6, and 7 (Figure 3), with each species providing a similar
level of protection. Analogous studies on the oxidation of the
Cys-34 residue of BSA were not carried out (though such
residues are known to be kinetically important targets for both
HOCl and HOBr38,42) as thiol consumption may be
complicated by secondary reactions of the Se-sugar oxidation
products (cf. data for the reaction of low molecular mass thiols
with the selenoxide of SeMet47,50).
k
(HOCl+Fmoc‑Met) to be independently determined from the slopes
of two separate competition plots (where k(HOCl+Fmoc‑Met)
(HOCl+scavenger)/slope). The y intercept for all of these
=
k
competition plots was equal to the Fmoc-Met concentration
used.
The kinetics of the reactions of HOBr (10 μM) with 1−7 or
SeMet (20 μM−300 μM) were investigated in a similar manner
using N-Ac-Tyr (N-Ac-Tyr; 1 mM, 22 °C) as the competing
target.38 The conversion of N-Ac-Tyr to N-Ac-3-bromoTyr
(NAc-BrTyr) in the absence and presence of the S-/Se-sugar
derivatives was monitored by HPLC. The data were analyzed in
a manner analogous to those for the HOCl reactions, and the
second-order rate constants (Table 1) for the reaction of 1−7
with HOBr were obtained from a plot of [N-Ac-Tyr]·(yieldmax
/
yieldscavenger) versus sugar concentration (Figure 2b) using
kHOBr+N‑Ac‑Tyr = 2.6 × 105 M−1 s−1.38
Human plasma was diluted to a final plasma protein
concentration of 0.5 mg mL−1 (∼7.6 μM, to allow easy
comparison with the isolated protein data) and mixed with 50
μM−1 mM of 2, 4, 6, or 7, before reaction with HOCl (760
μM). Samples were processed and analyzed as described for
BSA. Treatment with HOCl resulted in significant loss of Met,
Trp, Tyr, His, and Lys residues, with this decreased in a dose-
dependent manner by increasing levels of the Se-sugars. Data
obtained with 7 are shown in Figure 4 and are representative of
all the species. The concentrations at which 7 elicited complete
protection of the susceptible amino acids were similar in plasma
to those required with isolated BSA.
The kinetics for the reaction of HOSCN with 7 were
investigated in competition with 5-thio-(2-nitrobenzoic acid)
(TNB) at 22 °C, with the consumption of TNB monitored at
412 nm over 2−20 s.25 The corresponding sulfur species was
not examined due to the slow reaction of HOSCN with
thioethers.25 With HOSCN (2 μM) and TNB (8 μM) with or
without 7 (4 mM) the yieldscavenger/yieldmax ratio was ∼0.88,
while with HOSCN (0.5 μM), TNB (2 μM), and SeTal (7)
(4.75 mM), this ratio was decreased to 0.64, indicating that
∼35% of the HOSCN reacted with 7 at these concentrations.
Experiments with higher TNB concentrations did not yield
sufficiently great absorbance differences for analysis. Because of
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Figure 3. Bar graphs depicting the protection of protein side chain residues, (a) His, (b) Lys, (c) Met, (d) Trp, and (e) Tyr, from HOCl-mediated
oxidation in bovine serum albumin (BSA) by SeGul (2) (white), SeMan (4) (light gray), SeId (6) (dark gray), and SeTal (7) (black). Error bars
represent standard error of the mean (n = 4). Statistical significance (p < 0.05) was assessed by 2-tailed one-way ANOVA (with Tukey’s posthoc
test). * Indicates significance relative to the oxidant-free control (demonstrating significant damage by HOCl), and # indicates significance relative to
the oxidized sample in the absence of any HOCl scavengers (demonstrating significant protection by the Se-sugar derivatives).
3.4. Inhibition of 3-ChloroTyr Formation. 3-Chloro-Tyr
(3-Cl-Tyr) is a well-established biomarker of HOCl-mediated
protein oxidation.51 3-Cl-Tyr levels were quantified following
the reaction of isolated BSA or plasma (0.5 mg mL−1 protein,
∼7.6 μM) with HOCl (760 μM, or buffer for controls), in the
absence or presence of increasing concentrations of 2 or 7 (50
μM−1 mM), before processing and analysis of (parent) Tyr
and 3-Cl-Tyr concentrations by LCMS. The 3-Cl-Tyr/Tyr
ratios for each sample were normalized relative to the maximal
value achieved in the absence of the Se-sugar derivative (0 mM
SeSugar bar). Our previous studies have ascertained that in the
absence of Se-sugar, the 3-Cl-Tyr levels generated under these
conditions were 1 0.16 nmol (10.5 nmol per mg protein) for
isolated BSA and 0.3 0.1 nmol (7.75 nmol per mg protein)
for diluted human plasma, with negligible 3-Cl-Tyr levels
measured in the absence of HOCl.46 Data for diluted human
plasma are shown in Figure 5.
Increasing concentrations of both 2 and 7 decreased the 3-
Cl-Tyr/Tyr ratio (i.e., protected against HOCl-mediated
damage) with significant protection (p < 0.05) obtained with
the lowest concentrations examined (50 μM). Complete
inhibition of 3-Cl-Tyr formation was achieved at 500 μM. No
significant differences were observed between 2 and 7 or
between BSA (data not shown) and diluted human plasma
(Figure 5).
DISCUSSION
■
Elevated levels of MPO and other heme peroxidases are
strongly associated with the development of inflammatory
diseases.10 These enzymes produce the oxidants HOCl, HOBr,
and HOSCN, in varying yields, with the formation of these
oxidants implicated in the pathogenesis of multiple diseases
associated with acute or chronic inflammation.11−16 As a result,
considerable effort has been invested in determining the
kinetics and targets of these oxidants. Sulfur and selenium
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Figure 4. Bar graphs depicting the protection of protein side chain residues, (a) His, (b) Lys, (c) Met, (d) Trp, and (e) Tyr, from HOCl-mediated
oxidation in diluted human plasma by SeTal (7). Error bars represent standard error of the mean (n = 4). Statistical significance (p < 0.05) was
assessed by 2-tailed one-way ANOVA (with Tukey’s posthoc test). * Indicates significance relative to the oxidant-free control (demonstrating
significant damage by HOCl), and # indicates significance relative to the oxidized sample in the absence of any HOCl scavengers (demonstrating
significant protection by the SeTal (7) derivative).
The rate constants for the reactions of these oxidants with
putative scavengers, therefore, provides a potential guide to
their possible efficacy.
Competitive kinetic studies have shown that each of the S-/
Se-sugar derivatives (1−7) examined in this study reacts rapidly
with HOCl and HOBr; the rate constants determined are some
of the largest known values for these oxidants, demonstrating
their potential as HOCl and HOBr scavengers. The data for 1−
7 are collected in Table 1 and compared to the corresponding
data for SeMet, GSH, Cys, and Met.25,29,38,42 The Se-species 2,
4, 6, and 7 react with HOCl with k 0.8−1.0 × 108 M−1 s−1,
Figure 5. Bar graph depicting the ability of SeGul (2) (white) and
SeTal (7) (light gray) to prevent 3-Cl-Tyr formation by HOCl in
human plasma. Error bars represent standard error of the mean (n =
3). # Indicates significance (p < 0.05) relative to the 0 mM Se-sugar
control as assessed by 2-tailed one-way ANOVA (demonstrating
significant protection by SeGul (2) or SeTal (7)).
values that are marginally lower than those of the most reactive
biological targets, SeMet (3.2 × 108 M−1 s−1) and Cys (3.1 ×
108 M−1 s−1) (Pattison, D. I., Storkey, C., and Davies, M. J.,
unpublished data). Notably, these values are comparable to
those for the important endogenous antioxidant GSH (1.1 ×
108 M−1 s−1) and faster than that for Met (3.0 × 107 M−1 s−1,52
Pattison, D. I., Storkey, C., and Davies, M. J., unpublished
data). The rate constants for the corresponding sulfur
derivatives (1, 3, and 5) are 50−70 times lower (k 1.4−1.9 ×
106 M−1 s−1) than their Se analogues, though this difference
decreases to ∼10-fold for SeMet versus Met. This is consistent
species are important targets, particularly the thiol and selenol
moieties present in the side-chains of Cys and selenocysteine
(Sec) residues on proteins and some antioxidants (e.g.,
GSH).24,30,52 Met and selenomethionine (SeMet) residues are
also major targets for HOCl and HOBr, but not HOSCN.29,52
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with the reduced nucleophilicity of the sulfur compared to the
seleno species and possibly some stereoelectronic differences
that make Se more reactive than S in the sugar structures.53,54
The reactions of these modified sugars with HOBr are slower
than that for HOCl, consistent with previous kinetic data for
the reactions of HOBr/HOCl with Cys and Met.38,52 Thus, the
values for the reaction of 4, 6, and 7 with HOBr are ∼8 times
lower than that with HOCl, and the values showed little
variation with sugar structure (1.0−1.5 × 107 M−1 s−1). These
values are similar to that for SeMet, (1.4 × 107 M−1 s−1) and
Cys (1.2 × 107 M−1 s−1) but significantly greater than that for
Met (3.6 × 106 M−1 s−1). The second-order rate constants for
the corresponding sulfur-sugars (1, 3, and 5) with HOBr were
smaller than for the selenium analogues, but this difference was
modest (∼1.5-fold). It should be noted that preparation of
HOBr via oxidation of excess Br− with HOCl results in low
concentrations of BrCl in equilibrium with HOBr;55 thus, it is
possible that the reactive BrCl molecule could contribute to the
reactions under our conditions. However, these effects are likely
to be very small in the current studies as appreciable BrCl
concentrations are present only under highly acidic conditions
or in organic solvents where the rapid hydrolysis of BrCl to
HOBr is prevented.56 Overall, these kinetic data indicate that
the structure of the sugar derivatives do not markedly affect
their reactivity with HOCl or HOBr.
The approximate second-order rate constant estimated for
the reaction of 7 with HOSCN (∼102 M−1 s−1) is 6 orders of
magnitude lower than that for HOCl and HOBr. HOSCN is a
much milder oxidant than HOCl or HOBr and reacts relatively
selectively with thiol and selenol residues with much lower rate
constants (Sec, 1.2 × 106 M−1 s−1; Cys, 7.8 × 104 M−1 s−1;
GSH, 2.5 × 104 M−1 s−1).25,26,29 Met residues do not show
significant reactivity, but SeMet shows some reactivity with
HOSCN (k, 2.8 × 103 M−1 s−1).29 These data indicate that
scavenging of HOSCN is unlikely to be a significant process
with these seleno-sugars in cells, where the GSH levels are high
(∼5 mM),57 but could play a role in plasma, where thiol levels
are much lower (∼500 μM).30
required to show significant protection were similar for both
plasma and BSA, with ≥50 μM being effective. It is unclear as
yet whether such concentrations are achievable in mammals,
but preliminary studies indicate that these compounds do not
exhibit significant toxicity at these levels (Jani, N., Storkey, C.,
Ziogas, J., Wright, C. E., Schiesser, C., and Davies, M. J.,
unpublished data). It should be noted that the flux of HOCl
formed in vivo is also likely to be lower than the bolus additions
used here, and it is possible that lower concentrations of the Se-
sugar derivatives may show efficacy.
The efficacy of 2 and 7 in preventing damage was also
assessed by quantifying the generation of the HOCl-specific
biomarker 3-Cl-Tyr on isolated BSA and plasma proteins. The
levels of this stable product were decreased by increasing levels
of the Se-sugars. However, the concentration required to inhibit
3-Cl-Tyr formation in plasma (Figure 5) does not correlate
exactly with that required to diminish the loss of parent Tyr
(Figure 4E; see, for example, the data for 0.2 mM seleno-
sugars). The reason for this discrepancy is not known but may
be linked to the nonstoichiometric conversion of Tyr to 3-Cl-
Tyr and the generation of alternative materials.
Previous studies have demonstrated that the major oxidation
product formed on oxidation of Met by HOCl and HOBr is the
corresponding sulfoxide (reviewed in ref 61), and recent studies
indicate that oxidation of SeMet by a number of different
species results in selenoxide formation.47,62−66 The selenoxide
can be subsequently reduced back to SeMet by a range of
reductants, including biologically relevant species such as
ascorbate and GSH.47,50 Preliminary studies with the seleno-
sugars indicate that these materials are also converted
stoichiometrically to the corresponding selenoxides; this
process may proceed via a transient halogenation of the seleno
center (to yield species such as RR′Se+-X, where X = Cl, Br)
and subsequent rapid hydrolysis of this species (Rahmanto, A.
S., Carroll, L., Fu, S., Hawkins, C. L., Storkey, C., Pattison, D. I.,
and Davies M. J., unpublished data). Furthermore, initial
studies indicate that these selenoxides can be reduced by thiols
(Rahmanto, A. S., Carroll, L., Fu, S., Hawkins, C. L., Storkey,
C., Pattison, D. I., and Davies M. J., unpublished data). These
data rationalize the almost complete protection of Met against
oxidation by 7. Whether recycling of the selenoxide of 7 occurs
at a rate sufficient to induce catalytic removal of the oxidants
remains to be determined.
In summary, we have demonstrated a simple, effective
method for the conversion of monosaccharide sugars into
potent water-soluble oxidant scavengers and a proof of
principle with regard to the potential modulation of oxidative
damage associated with inflammation. Kinetic data indicate that
the reactions of these Se-sugars with HOCl and HOBr are
some of the fastest reactions of these oxidants. In contrast, the
rate constant determined for the weaker, thiol-selective, oxidant
HOSCN are lower, and such reactions may not be relevant in
vivo despite the greater selectivity of HOSCN. All of the Se-
sugar derivatives provided dose-dependent protection against
HOCl-mediated oxidation of BSA and diluted human plasma
samples, and prevented 3-Cl-Tyr formation. Further studies are,
however, required to determine whether these materials afford
protection in more complex situations such as intact plasma
and in animal models of inflammation, where a number of
other factors including bioavailability, metabolism, and
achievable plasma/cellular concentrations are likely to be
critically important. As the activity of these Se sugars appears
to be unaffected by their stereochemistry, and there are a large
In light of these kinetic data, further investigations were
carried out with regard to potential protection of isolated BSA
and human plasma proteins (key targets in vivo30,58,59) from
HOCl-mediated oxidation. In both plasma and isolated BSA,
protein-bound Met, Trp, Lys, His, and Tyr were observed to be
significant targets, consistent with previous data.24,30,42,46 Each
of the Se-sugar derivatives afforded dose-dependent protection
against such damage, with little dependence on the sugar
structure. Whether this reflects a direct removal of the initial
oxidant or repair of reversible intermediates formed on the
proteins by the oxidants (e.g., chloramines and bromamines,
RNHCl and RNHBr, generated by reaction of HOCl and
HOBr with amines such as those on Lys side-chains40,60
)
remains to be determined, though the overall effect would be
expected to be the same. Preliminary studies indicate that a
variety of chloramines are also scavenged rapidly by these
seleno sugars (Carroll, L., Fu, S., Hawkins, C. L., Storkey, C.,
Pattison, D. I., and Davies M. J., unpublished data).
The overall extent of side-chain oxidation detected with
isolated BSA, in the absence of added Se-sugar, was greater than
that detected in diluted human plasma (cf. data for Tyr, His,
and Lys residues) consistent with partial scavenging of the
added HOCl by other materials present in human plasma (e.g.,
ascorbic acid, urate, free amino acids, and SCN−).30 Despite
these differences, the concentrations of Se-sugar derivatives
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number of potential carbohydrate scaffolds, these modified
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numerous potential applications in the treatment of diseases
associated with oxidative damage.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures and spectroscopic data for the
1
compounds, and copies of H NMR, 13C NMR spectra, and
CIF tables. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Tel: +61-2-8208-8900. Fax: +61-2-9565-5584. E-mail:
daviesm@hri.org.au.
Funding
This work was supported by the Australian Research Council
(through the Centres of Excellence Scheme, CE0561607, and
Discovery Programs DP0988311), and the National Heart
Foundation (Grant in Aid G09S4313).
Notes
The authors declare no competing financial interest.
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Physiol. 279, L537−546.
(16) Malle, E., Buch, T., and Grone, H. J. (2003) Myeloperoxidase in
kidney disease. Kidney Int. 64, 1956−1967.
ACKNOWLEDGMENTS
■
We thank Drs. Phillip Morgan and Jihan Talib for development
of the UPLC amino acid analysis method and 3-Cl-Tyr LC-MS
method, respectively.
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Modification of low-density lipoprotein by myeloperoxidase-derived
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ABBREVIATIONS
■
AcCl, acetyl chloride; BnOH, benzyl alcohol; BSA, bovine
serum albumin; 3-Cl-Tyr, 3-chloro-Tyr; DMAP, 4-dimethyla-
minopyridine; DMF, dimethylformamide; EtOH, ethanol;
EPO, eosinophil peroxidase; GSH, glutathione; HOX,
hypohalous acids; HPLC, high performance liquid chromatog-
raphy; HSA, human serum albumin; LCMS, liquid chromatog-
raphy coupled with mass spectrometry; LPO, lactoperoxidase;
MPO, myeloperoxidase; MsCl, methanesulfonyl chloride; N-
Ac-Tyr, N-acetyl-Tyr; N-Ac-BrTyr, N-acetyl-3-bromo-Tyr;
NADPH, nicotinamide adenine dinucleotide phosphate; Py,
pyridine; Sec, selenocysteine; SeMet, selenomethionine; TFA,
trifluoroacetic acid; TNB, 5-thio-(2-nitrobenzoic acid); p-TSA,
p-toluenesulfonic acid; UPLC, ultrahigh performance liquid
chromatography
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