We have recently demonstrated that neutrophils
oxidize nearly all of the amino acids commonly found in plasma to a
corresponding family of aldehydes in high yield. The reaction is
mediated by hypochlorous acid (HOCl), the major oxidant generated by
the myeloperoxidase-H2O2-Cl
system of phagocytes. We now present evidence for the underlying mechanism of this reaction, including the structural requirements and
reaction intermediates formed. Utilizing mass spectrometry and
isotopically labeled amino acids, we rule out hydrogen atom abstraction
from the
-carbon as the initial event in aldehyde formation during
amino acid oxidation, a pathway known to occur with ionizing radiation.
Aldehyde generation from amino acids required the presence of an
-amino moiety;
- and
-amino acids did not form aldehydes upon
oxidation by either the myeloperoxidase system or HOCl, generating
stable monochloramines instead. UV difference spectroscopy, high
pressure liquid chromatography, and multinuclear
(1H,15N) NMR spectroscopy established that the
conversion of
-amino acids into aldehydes begins with generation of
an unstable
-monochloramine, which subsequently decomposes to yield
an aldehyde. Precursor product relationships between
-amino acid and
-monochloramine, and
-monochloramine and aldehyde were confirmed
by high pressure liquid chromatography purification of the reaction
intermediate and subsequent 1H and 15N NMR
spectroscopy. Collectively, these results detail the chemical mechanism
and reaction intermediates generated during conversion of amino acids
into aldehydes by myeloperoxidase-generated HOCl.
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INTRODUCTION |
Activated phagocytes both secrete the heme enzyme myeloperoxidase
and generate hydrogen peroxide
(H2O2)1
(1, 2). We have recently shown that the enzyme can use H2O2 and chloride ions (Cl
) to
oxidize nearly all of the common amino acids into a family of aldehydes
(3). Because these products react readily with biological constituents
and may be generated in significant quantities at sites of
inflammation, we set out to elucidate the chemical mechanism by which
they are generated.
Our study explored two potential mechanisms
(Scheme I). Pathway A, known to be a
route by which amino acids are oxidized by ionizing radiation, involves
initial hydrogen atom abstraction to generate an
-carbon-centered
radical (4-9). This short-lived intermediate decomposes into carbon
dioxide and an imine, which then may form an aldehyde in the presence
of H2O2 through liberation of ammonia (4-9).
Metal-catalyzed oxidation of amino acids may similarly convert amino
acids into aldehydes (9, 10), as first suggested by Dakin (11-13)
nearly a century ago. Pathway B involves initial chlorination of the
-amino moiety, generating an
-monochloramine. This reaction is
plausible because myeloperoxidase generates hypochlorous acid (HOCl)
from H2O2 and Cl
(14), and
monochloramines form readily when HOCl reacts with amines (15-17).
Zgliczynski and colleagues (18) first suggested that monochloramines of
common amino acids might serve as precursors in aldehyde formation.
These conclusions, however, were drawn from indirect evidence and
confirmed neither the structures of products formed nor the putative
monochloramine intermediates. In fact, monochloramines are generally
thought to be relatively stable compounds under physiological
conditions (15-17, 19-23). The stability of the presumed
monochloramine generated from taurine is exploited as a quantitative
assay for HOCl production (16). Despite the widely held belief that the
myeloperoxidase-H2O2-Cl
system
chlorinates primary amines to form monochloramines, structural characterization of these species has been indirect (e.g. UV
absorbance) rather than direct (e.g. NMR).
In this study, we use a variety of techniques to explore in detail the
chemical mechanism of myeloperoxidase-mediated amino acid oxidation. We
rule out hydrogen atom abstraction (a requirement for Pathway A)
through mass spectrometric analysis of isotopically labeled amino
acids. We then demonstrate that aldehydes can be generated only from
amino acids that have free
-amino and
-carboxylic acid groups. We
show that chlorination of
-amino acids by HOCl yields an unstable
-monochloramine, as in Pathway B, utilizing a variety of techniques
including UV spectroscopy, 1H and 15N NMR, and
HPLC. The monochloramine then loses both
-carboxylic acid and NHCl
groups to yield an aldehyde. Collectively, these results describe
the underlying mechanism for aldehyde formation from
-amino acids by
the phagocytic oxidant HOCl. The relative abundance of
-amino acids
in plasma (24), interstitial fluid, and intracellular compartments (25)
suggests that aldehydes derived from these precursors should be readily
formed at sites of inflammation.
 |
EXPERIMENTAL PROCEDURES |
Materials
HPLC solvents were purchased from Baxter (McGaw Park, IL).
Sodium phosphate, ethyl acetate, H2O2, and
sodium hypochlorite were obtained from Fisher. D2O,
L-[15N]tyrosine, and
L-[d8]phenylalanine were purchased
from Cambridge Isotopes, Inc. (Andover, MA). All other materials were
purchased from Sigma, except where indicated.
Methods
General Procedures--
Aldehydes were synthesized and analyzed
by electron impact gas chromatography/mass spectrometry and reverse
phase HPLC as described (3).
UV Absorbance Spectroscopy--
UV absorbance spectra were
obtained on a Beckman DU7 spectrophotometer equipped with a
thermostatically controlled cuvette holder. UV difference spectra of
amino acid-derived monochloramines were performed at the indicated
temperature with the starting amino acid used as a reference. Kinetic
studies of monochloramines were carried out similarly by monitoring the
time-dependent changes in absorbance at 254 nm.
Glycolaldehyde Production--
Glycolaldehyde production was
measured by reverse phase HPLC following derivatization with
3-methyl-2-benzothiazolinone hydrazone hydrochloride as described
previously (26).
Multinuclear NMR Studies--
Monochloramines for NMR
experiments were synthesized by dropwise addition of NaOCl (1:1;
mol/mol) to the indicated amino acid in 20 mM sodium
phosphate buffer (pH 7.0) in 10% D2O, 90% H2O (v/v) at 0 °C with constant mixing. NMR spectra were acquired on a
Varian Unity-Plus 500 spectrometer (499.843 MHz for 1H). A
Nalorac indirect detection probe was employed for the 1H
and 1H,15N two-dimensional NMR studies. Sample
temperature was maintained (±0.1 °C) with an Oxford Instruments
temperature controller. 1H chemical shifts were referenced
to external sodium
3-(trimethylsilyl)-propianate-2,2,3,3,d4 in
D2O. The intense HOD signal was attenuated by transmitter
pre-irradiation, and digital signal processing was employed to suppress
phase distortions for 1H spectra.
The proton NMR spectrum of L-tyrosine
-monochloramine
was recorded from 32 transients under the following conditions:
pre-acquisition delay = 1 s, acquisition time = 1.89 s (37,760 complex data points), pulse width = 5 µs
(62° flip angle) and spectral width = 10,000 Hz. The free
induction decays were processed with a combination of gaussian and
exponential weighting functions.
15N NMR chemical shifts of
L-[15N]tyrosine and
L-[15N]tyrosine
-monochloramine were
established through heteronuclear multiple bond correlation
spectroscopy experiments collected at 0 °C. Collection conditions
for the t2 domain (1H) include
transmitter presaturation of the intense water signal, a spectral width
of 6,000 Hz, and collection of eight transients containing 2,048 complex data points. The t1 domain
(15N) included 128 increments over a spectral width of
7,000 Hz (evolution time ~18 ms). The 90° pulse duration was 8 and
26 µs for the 1H and 15N channels,
respectively. Data were collected and processed in the hypercomplex
mode employing sine bell and gaussian weighting in
t2 and t1, respectively.
The 15N chemical shifts were established through 3-bond
connectivities to the benzylic protons and were referenced to
L-[15N]tyrosine arbitrarily set to 0 ppm.
 |
RESULTS |
Only Amino Acids with Free Amino and Carboxylic Acid Groups on the
-Carbon Yield Aldehydes When Oxidized by the
Myeloperoxidase-H2O2-Cl
System--
To explore the reaction mechanism by which the
myeloperoxidase system generates aldehydes during oxidation of
-amino acids, we first determined which structural features the
amino acids must possess. Incubation of
-alanine (1-aminopropanoic
acid) with the complete
myeloperoxidase-H2O2-Cl
system
resulted in the near quantitative conversion of the
-amino acid into
its corresponding aldehyde, acetaldehyde (Table
I) (3). In contrast, incubation of
-alanine (2-aminopropanoic acid) with the complete myeloperoxidase
system generated no detectable aldehyde. We next examined aldehyde
yield during regiospecific chlorination of either the
- or
-
amino moieties of lysine. Incubation of the
-amino blocked analog
(N
-acetyllysine) with the
myeloperoxidase-H2O2-Cl
system
resulted in aldehyde formation in high yield (85%; Table I). In
contrast, incubation of the
-amino blocked analog
(N
-acetyllysine) with the complete
myeloperoxidase system failed to generate detectable aldehyde. No
aldehyde was formed when the myeloperoxidase system oxidized taurine
(
-aminosulfonic acid) or ethanolamine (Table I). Thus, only amino
acids that have both the primary amino and carboxylic acid groups in
the
-position are oxidized to aldehydes by the
myeloperoxidase-H2O2-Cl
system.
There were no significant differences in overall aldehyde yield from
D- or L-tyrosine. Finally, aldehyde formation
required that both the
-amino and
-carboxylic acid groups be free
since no detectable p-hydroxyphenylacetaldehyde (pHA) was
formed during myeloperoxidase-catalyzed oxidation of
N
-acetyltyrosine, tyrosinamide,
N
-acetyltyrosine ethyl ester, or
tyrosine methyl ester (Table I).
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Table I
Aldehyde production and monochloramine half-life of amino compounds
oxidized by myeloperoxidase and reagent HOCl
Aldehyde production from non-aromatic compounds was quantified by
reverse phase HPLC of the PFB-oxime derivatives (3). pHA production
from tyrosine and its analogues was determined as described (3).
Aldehydes were prepared by incubating the indicated compounds (100 nmol) individually with myeloperoxidase (20 pmol), H2O2
(100 nmol), and NaCl (100 µmol) in 50 mM sodium phosphate
buffer (pH 7.0; final volume 1 ml) within gas-tight reaction vials at
37 °C for 60 min. Identities of amino acid-derived aldehydes and
their oxime derivatives were confirmed by mass spectrometry (3). The
half-life of individual monochloramines was determined spectrophotometrically (A254). Monochloramines were
prepared by drop-wise addition of HOCl (1.5 mM final) to
each amino acid (1.5 mM each) in 50 mM sodium
phosphate buffer (pH 7.0) at 0 °C. Reaction mixtures were briefly
(15 s) warmed to 37 °C in a H2O bath and then transferred to
thermostatically controlled cuvettes (37 °C).
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The Myeloperoxidase System Does Not Abstract a Hydrogen Atom When
It Oxidizes
-Amino Acids--
The finding that amino acids must
contain an
-amino moiety to serve as substrates for aldehyde
generation during amino acid oxidation suggested either the initial
formation of an unstable
-monochloramine (Pathway B) or an
alternative catalytic strategy involving hydrogen atom abstraction from
the tertiary
-carbon (Pathway A). To determine which of these
reaction mechanisms mediated aldehyde formation, we analyzed the
products generated when myeloperoxidase acted on an amino acid that was
isotopically labeled with deuterium at the
-carbon position
(Scheme II). Oxidation of isotopically labeled L-[d8]phenylalanine in an
aqueous solution would be anticipated to generate an aldehyde with a
molecular ion (M
) of mass to charge ratio
(m/z) 127, if hydrogen atom abstraction of the
-carbon occurred. Chlorination of the primary amino group generating
an
-monochloramine intermediate would instead lead to a product with
a M
of m/z 128.
Electron impact gas chromatography/mass spectrometric analysis of
L-phenylalanine and
L-[d8]phenylalanine oxidized by
the myeloperoxidase-H2O2-Cl
system revealed that the aldehyde formed with the
d8 analogue possessed an M
of
m/z 128 (Fig. 1) and was
identical in isotopic composition to the parent amino acid. Examination
of pentafluorobenzyl (PFB)-oxime derivatives of the aldehydes formed
from the precursor deuterated and non-deuterated amino acids revealed
molecular ions of m/z 323 and m/z 315, respectively. The 8 mass unit increase in the derivatized aldehyde
derived from myeloperoxidase-catalyzed oxidation of
L-[d8]phenylalanine confirms that
the deuteron at the
-carbon position was retained, as in Pathway B. In contrast, L-[d8]phenylalanine
oxidized by a hydroxyl radical-generating system (2 mM
H2O2 and 100 µM
CuSO4) produced only trace amounts of aldehyde, as well as
other products that likely reflect HO· addition products to the
ring (27, 28). The low abundance of the aldehyde formed prevented its
mass analysis by electron impact mass spectrometry; however, gas
chromatography-mass spectrometry analysis of the PFB-oxime derivative
confirmed that the aldehyde lost the
-carbon deuteron (M
of
m/z 322; data not shown). Thus, aldehyde generation by
myeloperoxidase does not involve hydrogen (or deuteron) atom
abstraction from the
-carbon of L-phenylalanine, in
contrast to amino acid oxidation with a hydroxyl radical generating system.

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Fig. 1.
Electron impact mass spectra of
L-phenylalanine and
L-[d8]phenylalanine oxidation
products generated by the
myeloperoxidase-H2O2-Cl
system. L-Phenylalanine (200 µM) or
L-[d8]phenylalanine (200 µM) were incubated with myeloperoxidase (6 nM), H2O2 (200 µM), and sodium chloride (100 mM) in buffer A (50 mM
sodium phosphate (pH 7.0)) within gas-tight reaction vials at 37 °C
for 30 min. Oxidation products were extracted into ethyl acetate (1:1,
v/v) and analyzed by gas chromatography-mass spectrometry in the
electron impact mode with an electron ionization energy of 70 eV. Gas
chromatographic separations were carried out utilizing a Restek RTX-200
column (15 m, 0.33 mm inner diameter, 1-µm film thickness) in the
splitless mode with helium as the carrier gas. The column was run with
the following temperature gradient: 70-150 °C at 10 °C/min;
150-280 °C at 20 °C/min. Upper panel,
L-phenylalanine oxidized by the
myeloperoxidase-H2O2-Cl system.
Prominent ions in the mass spectrum include the molecular ion at
m/z 120 (M ), m/z 91 (M -CHO) and
m/z 65 (M -CHO-C2H2). Lower panel,
L-[d8]phenylalanine oxidized by
the myeloperoxidase-H2O2-Cl
system. The molecular ion of m/z 128 (M ) indicates
that the -carbon deuteron is not abstracted during aldehyde
formation. Prominent fragment ions in the mass spectrum at
m/z 98 and m/z 70 represent (M -CDO) and
(M -CDO-C2D2), respectively.
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Oxidation of
-Amino Acids by HOCl Yields Unstable Intermediates
with UV Spectral Features Consistent with
-Monochloramines--
Preliminary studies demonstrated that
oxidation of several common amino acids (glycine, alanine, isoleucine,
phenylalanine, and serine) by the
myeloperoxidase-H2O2-Cl
system
generated a labile intermediate with a UV absorbance band at 252-254
nm, the absorbance maximum of monochloramines (15-17). To explore the
possibility of an unstable
-monochloramine as a reaction
intermediate, amino acids were incubated with HOCl at 0 °C, and
their UV difference spectra were determined. A characteristic absorbance band was observed at 254 nm, suggesting monochloramine formation. Comparisons of the stability of
-,
- and
-monochloramines of amino acids, as assessed by the disappearance of
absorbance at 254 nm at 37 °C, demonstrated that only
-monochloramines were thermally labile (Table I). Parallel
experiments with each of these same
-,
-, and
-amino acids
reacted with purified myeloperoxidase, chloride, and
H2O2 also demonstrated that only
-monochloramines were unstable, as assessed by loss of an absorbance
band at 254 nm (data not shown).
The differing stabilities of
- and
-monochloramines are
illustrated in the experiment depicted in Fig.
2. Either the
-amino or
-amino
moiety of L-lysine was regiospecifically chlorinated with
HOCl using the appropriate N-acetylated parent compound as substrate, and the thermal stability of the resulting
- or
-monochloramine was determined. Chlorination of the
-amino group
of N
-acetyllysine generated a labile
-monochloramine that demonstrated a t1/2 of ~9
min at 37 °C (Fig. 2A). In striking contrast,
chlorination of the
-amino group of
N
-acetyllysine as substrate generated
a
-monochloramine that was apparently stable (Fig.
2B).

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Fig. 2.
Oxidation of
N -acetyllysine and
N -acetyllysine by HOCl. Monochloramines
of N -acetyl lysine (A) or
N -acetyllysine (B) were
generated by dropwise addition of HOCl (1.5 mM) to reaction
mixtures of the amino acids (1.5 mM) in buffer A at
0 °C. Reaction mixtures were rapidly warmed to 37 °C, transferred to thermostatically controlled cuvettes (37 °C), and the initial rate of monochloramine decay monitored spectrophotometrically (A254 nm). Note that at t = 0 min, quantitative chlorination of both the - and -amino moieties
results in near identical UV absorbance profiles. Upon warming to
37 °C, the -monochloramine is unstable and decomposes with
first-order kinetics, whereas the -monochloramine is stable.
Insets in A and B represent the proposed
structures of the monochloramines.
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Examination of L-lysine oxidized with HOCl (1:1, mol/mol)
revealed an initial first-order loss of monochloramine (as monitored by
the change in A254) followed by persistence of
~30% of the original absorbance, despite prolonged incubation (>10
h at 37 °C). The UV spectrum of the reaction mixture demonstrated an
absorbance band at 254 nm, suggesting that a stable
-monochloramine
was still present.
The HOCl-dependent oxidation of acidic
(L-glutamate), alcoholic (L-serine), aliphatic
(L-leucine), amide (L-glutamine), aromatic (L-phenylalanine), and basic (L-lysine) amino
acids all resulted in the formation of a labile intermediate with UV
spectral features consistent with an
-monochloramine (Table
II). Interestingly, chlorination of the
secondary amino group of L-proline also led to formation of
an unstable monochloramine, as assessed by loss of an absorbance band
at 254 nm (Table II). However, this monochloramine did not generate an
aldehyde during its decomposition, since derivatization of the product
yielded neither a PFB-oxime nor a DNPH derivative; moreover, the
reaction products failed to exhibit a positive Tollen's test, a
classic qualitative assay for a reactive carbonyl (29, 30). These
results are consistent with our recent observation that no aldehyde was
formed when proline (an imino acid that contains a secondary amino
group) was oxidized by the
myeloperoxidase-H2O2-Cl
system
(3). Collectively, these results suggest that unstable primary
-monochloramines serve as intermediates when the
myeloperoxidase-H2O2-Cl
system of
phagocytes oxidizes
-amino acids to form aldehydes.
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Table II
Half-life of monochloramines of each class of -amino acids
Monochloramines of amino acids were prepared as described in the legend
to Table I, and the initial rate of monochloramine decay was monitored
spectrophotometrically as the decrease in absorbance at 254 nm.
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Intermediates with UV Spectral Features Consistent with a
Monochloramine Are Precursors for
-Amino Acid-derived
Aldehydes--
The labile intermediates formed during HOCl-mediated
oxidation of
-amino acids are stabilized at low temperatures and
have UV spectral features consistent with an
-monochloramine;
aldehydes were also formed when these reaction mixtures were warmed
(Table I). Direct demonstration of this precursor-product relationship was achieved by isolating the unstable intermediate and demonstrating its conversion into an aldehyde. L-Tyrosine was first
cooled to 0 °C, and aliquots of HOCl were added. Examination of the
reaction products by reverse-phase HPLC at 0 °C demonstrated a
product whose retention time was longer than that for the anticipated end product of the reaction, pHA (Fig.
3A). UV difference
spectroscopy of the intermediate demonstrated an absorbance band at 254 nm, suggesting the formation of an
-monochloramine. A
precursor-product relationship between the presumed
-monochloramine
intermediate and pHA was confirmed by gradually warming the isolated
intermediate and subjecting the solution to reverse phase HPLC
analysis. As the putative
-monochloramine disappeared, the aldehyde,
pHA, was formed (Fig. 3B). A parallel set of experiments
with L-[14C]tyrosine and scintillation
counting demonstrated that production of the presumed
-monochloramine intermediate from L-tyrosine and HOCl
was nearly quantitative at 0 °C (Fig.
4, left panel); loss of the
intermediate upon warming was accounted for by the generation of pHA
(Fig. 4, right panel). Collectively, these results demonstrate that oxidation of
-amino acids by
myeloperoxidase-generated HOCl produces an unstable intermediate with
UV spectral features consistent with an
-monochloramine. This labile
intermediate then serves as a precursor for the ultimate generation of
a reactive aldehyde.

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Fig. 3.
Reverse phase HPLC analysis of
L-tyrosine oxidation products generated by HOCl.
A, 4 aliquots of HOCl (5 µmol) were added dropwise with
constant stirring to L-tyrosine (20 µmol) in 10 ml of
ice-cold buffer A. Following each addition, the reaction mixture was
analyzed by reverse phase HPLC with the column and sample loop packed
in ice. Addition of HOCl resulted in the near quantitative consumption
of L-tyrosine yielding a product whose retention time was
slightly longer than that for p-hydroxyphenylacetaldehyde. B, the ice-cold reaction mixture containing
L-tyrosine -monochloramine was warmed repeatedly to
37 °C for 10 min, cooled to 0 °C to halt monochloramine
decomposition, and then analyzed by reverse phase HPLC as described
above. Note the consumption of the L-tyrosine -monochloramine and production of the aldehyde,
p-hydroxyphenylacetaldehyde, upon sample warming. Identity
of the intermediate as L-tyrosine -monochloramine was
confirmed utilizing multinuclear NMR spectroscopy as described under
"Results."
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Fig. 4.
Progress curve of
L-tyrosine oxidation by HOCl. Aliquots of
L-[14C]tyrosine (1 µmol) in buffer A (1 ml)
were cooled to 0 °C. The indicated amounts of HOCl were then added
(left panel), and the reaction products were analyzed by
reverse phase HPLC as described in the legend to Fig. 3. Fractions
containing L-tyrosine (AA), L-tyrosine -monochloramine (RNHCl), and
p-hydroxyphenylacetaldehyde (RCHO) were
collected, dried, and quantified by scintillation spectrometry.
Right panel, L-tyrosine -monochloramine was
prepared at 0 °C, warmed to 37 °C for the indicated times, and
then rapidly cooled to 0 °C. Products were then isolated by reverse
phase HPLC and quantified by scintillation spectrometry as described in
the legend to Fig. 3. Note that warming results in the loss of
monochloramine and the near quantitative production of
p-hydroxyphenylacetaldehyde.
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NMR Spectroscopy Confirms That
-Monochloramines Are
Intermediates in Aldehyde Formation during HOCl-mediated Oxidation of
-Amino Acids--
Because UV difference spectroscopy cannot
establish unequivocally the structure of a reaction intermediate, we
utilized multinuclear NMR to confirm the structure of the thermally
labile intermediate generated during incubation of an
-amino acid
and HOCl. 15N-Labeled L-tyrosine was employed
since the presumed
-monochloramine of the aromatic amino acid is
readily resolved at 0 °C on reverse phase HPLC (Fig. 3) and since
the 15N resonance position should serve as a non-perturbing
and sensitive probe into the immediate chemical environment at the
-amino nitrogen atom. L-[15N]Tyrosine was
incubated with HOCl (1:1, mol/mol) in 50 mM sodium phosphate buffer (pH 7.0) at 0 °C. The intermediate formed was then
analyzed by 1H NMR and 1H,15N
two-dimensional NMR spectroscopy. The chemical shifts, integrated areas, and coupling constants of resonances in the 1H NMR
spectrum of the compound all were consistent with the formation of
L-tyrosine
-monochloramine (Fig.
5). The chemical environment of the
15N atom of the initial
L-[15N]tyrosine/HOCl oxidation product was
interrogated by heteronuclear multiple bond correlation spectroscopy.
The chemical shift of the 15N resonance of the thermally
labile intermediate was consistent with a monochloramine (data not
shown); the electron withdrawing halide deshielded the 15N
atom, shifting the resonance to a characteristic downfield position. Thus, multinuclear NMR studies directly confirm that the
-monochloramine is a reaction intermediate in aldehyde
formation.

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Fig. 5.
1H NMR spectrum of
L-tyrosine -monochloramine. An ice-cold solution of
L-[15N]tyrosine (1.8 mM) in a 20 mM sodium phosphate buffer (pH 7.0), supplemented with
D2O (10%, v/v) was converted to its monochloramine at
0 °C as described in the legend to Fig. 3. The reaction mixture was
then analyzed by 1H NMR as described under "Experimental
Procedures." To avoid temperature-dependent shifts in
proton resonances, the spectrum was obtained immediately after warming
the sample to 25 °C. Peak assignments are depicted (inset). The benzylic protons (Hb) are not
degenerate due to the distinct chemical environments generated by the
adjacent chiral -carbon. Protons not observed due to rapid exchange
with solvent are designated (L). The spectral feature at
~4.7 ppm is an artifact resulting from suppression of the intense
water signal.
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To confirm a precursor-product relationship between
L-tyrosine
-monochloramine and pHA, we performed an NMR
experiment similar to the HPLC study described in Figs. 3 and 4.
L-Tyrosine
-monochloramine was warmed from 0 to 25 °C
in the NMR spectrometer with continuous 1H spectra
acquisition. A time- and temperature-dependent
disappearance of the
-monochloramine was observed (Fig.
6). Disappearance of the
-monochloramine occurred in concert with the appearance of pHA and a
second compound that exhibited distinct spectral features (Fig. 6).

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Fig. 6.
1H NMR spectra of
L-tyrosine and its HOCl oxidation products. The
1H NMR spectra of L-tyrosine,
L-tyrosine -monochloramine, and the thermal
decomposition products of L-tyrosine -monochloramine are
illustrated over the spectral region where benzylic protons (Hb; Scheme III) and protons to the benzylic protons
(Ha; Scheme III) are observed. L-Tyrosine (2 mM; 25 °C) is depicted in the bottom spectrum.
L-Tyrosine -monochloramine was formed as described in
the legend to Fig. 5, and its 1H NMR spectrum obtained
immediately following brief warming to 25 °C (to avoid
temperature-dependent shifts observed in proton resonances). Prolonged warming (45 min) at 25 °C results in
significant disappearance of the -monochloramine and formation of
both the aldehyde and another species (putative structures
(I) illustrated in Scheme III). The 1H spectra
obtained after incubation of the -monochloramine at 25 °C for
7 h is also shown. Note that the aldehydic proton (9.58 ppm) of
pHA is not observed in this spectral region but was observed to grow as
the monochloramine disappeared (data not shown). Resonances in the full
1H spectra for L-tyrosine (T),
tyrosine -monochloramine (M), pHA (A), and the
compound(s) in equilibrium with pHA (I) are described in
Table III. Resonance assignments are illustrated in Scheme III.
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Aldehydes Generated during Amino Acid Oxidation by HOCl Exist in a
Complex Equilibrium between Monomeric Aldehyde and Other Species Such
as the Gem Diol, Aldehydic Condensation Products, and Schiff Base
Adducts--
Aldehydes react with amines to form Schiff bases; they
also produce gem diols and condensation products in aqueous solution (31, 32). The 1H NMR spectrum obtained following the
thermal decomposition of tyrosine
-monochloramine (Fig. 6) suggested
that the monomeric pHA product was in equilibrium with one or more of
these species (termed I in
Scheme III). To test this hypothesis, we
used 1H NMR to analyze the distribution of monomeric pHA
and its equilibrium product(s) as a function of temperature. Warming
the reaction mixture greatly increased the proportion of monomeric pHA,
and cooling it increased the proportion of its equilibrium product(s), confirming the reversible nature of the equilibrium (Fig.
7). In another experiment similar to that
shown in Fig. 3B, we monitored the 1H NMR peaks
that appeared as reagent HOCl oxidized L-tyrosine. A peak
for
-monochloramine was clearly present, and it was replaced by a
pHA peak as the reaction proceeded. Resonances in the full 1H spectra for L-tyrosine (T), tyrosine
-monochloramine (M), pHA (A), and the compound(s) in equilibrium
with pHA (I) are summarized in Table III.
Collectively, these results demonstrate that the conversion of
L-tyrosine to pHA involves a monochloramine intermediate. The resulting monomeric aldehyde then exists in equilibrium with a
second compound or compounds.

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Scheme III.
Potential species in equilibrium with
pHA. Oxidation of tyrosine with HOCl generates an unstable
-monochloramine, which subsequently decomposes into the aldehyde
pHA. Species in equilibrium with monomeric aldehyde in aqueous solution
might include the hydrated form (gem diol), the Schiff base adduct
(imine), or a polymerized form such as a trimer (31, 32). Benzylic protons (Hb) and -carbon protons (Ha) are
labeled to facilitate assignment of resonances in the 1H
NMR spectra shown in Fig. 6 and are summarized in Table III.
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Fig. 7.
Temperature dependence of equilibrium between
monomeric pHA and other species formed upon -monochloramine
decomposition. L-Tyrosine -monochloramine was
prepared as described in the legend to Fig. 5 and incubated for 7 h at 25 °C. Under these conditions, pHA and a second compound(s)
(termed "equilibrium compound") were apparent by 1H NMR
spectroscopy with monitoring of their benzylic proton resonances (Hb; see Scheme III and Fig. 6). The temperature of the
reaction mixture varied between 5 and 90 °C with continuous
monitoring of the relative mole fraction of each species. Quantitation
of the monomeric aldehyde and the equilibrium species was performed by
integration of their benzylic proton resonances (Hb; Scheme
III).
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Table III
1H NMR spectral features of L-tyrosine and its HOCl
oxidation products
The 1H NMR spectra of L-tyrosine and its HOCl
oxidation products (Figs. 6 and 7) are described. Samples were prepared
as described in the legend to Fig. 7. All spectra were performed at
25 °C as described under "Methods." d, doublet; t, triplet; and
dd, doublet-doublet.
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We used a variety of approaches to determine whether the Schiff base,
the gem diol, or the trimer accounted for the compound that is in
equilibrium with pHA (Scheme III). These species cannot be
distinguished by 1H NMR because the proton resonances at
the benzylic and
-carbon positions of each compound possess nearly
identical chemical shifts. However, even 1H,15N
two-dimensional NMR analysis failed to detect a 15N
correlation with the 1H resonances attributed to the
equilibrium compound(s). This suggests that either the Schiff base is
not a component of the reaction mixture or that the heteronuclear
coupling constants were too small for detection. We also examined the
reaction products by electrospray ionization mass spectrometry, finding
a major ion of m/z 136, the mass of monomeric pHA. Tandem
mass spectrometry with collisional activated dissociation confirmed
that the ion was the M
of pHA (data not shown). There was no
evidence for ions derived from the three equilibrium products suggested
in Scheme III, the Schiff base (gem diol or trimer) suggesting that these compounds, if produced, are thermally labile, even under the
gentle ionization conditions of electrospray.
When we analyzed an aqueous solution of reagent phenylacetaldehyde, the
aldehyde that forms when the myeloperoxidase system oxidizes
phenylalanine (3), we detected 1H NMR peaks corresponding
to both the monomeric form of the aldehyde and a second compound. The
latter had distinctive spectral features that resembled those of the
benzylic and
-carbon proton resonances assigned to the compound in
equilibrium with pHA (labeled I in Fig. 6 and Scheme III).
Because phenylacetaldehyde has no amino groups available for Schiff
base formation under these conditions, the equilibrium compound is
likely to be a gem diol and/or trimer. 1H NMR analysis of
reagent phenylacetaldehyde or pHA extracted into an aprotic solvent
(CDCl3) also demonstrated spectral features consistent with
the monomeric form of each aldehyde (data not shown). These
observations again suggest that a gem diol and/or trimer are the
equilibrium products.
To determine whether Schiff base formation may at least partly account
for the resonances observed in the 1H NMR spectrum of the
compound(s) in equilibrium with pHA, we examined the effect on the
1H NMR spectrum of adding NH4OH to
phenylacetaldehyde in aqueous solution at neutral pH. Our rationale was
that ammonia is present as an end product after HOCl oxidizes an amino
acid to an aldehyde (18). We found that adding NH4OH
decreased the resonances attributed to monomeric aldehyde and increased
the relative intensity of the non-monomeric 1H resonances
(data not shown). These results strongly suggest that the
1H spectra of the compound(s) in equilibrium with monomeric
aldehyde are nearly identical to that of an imine. It therefore appears that a complex equilibrium between a monomeric aldehyde, its gem diol,
aldehydic condensation products, and Schiff base adducts exists in
aqueous solution.
Halide Dependence of Glycolaldehyde Production from Serine by the
Myeloperoxidase-H2O2-Halide
System--
Myeloperoxidase and eosinophil peroxidase use
Cl
, Br
, and I
as substrates
to generate a variety of hypohalous acids (1, 14, 33). Previous studies
have suggested that Cl
is the only halide with which
myeloperoxidase converts L-tyrosine into pHA, however (34).
To explore this halide specificity in greater detail, we studied the
oxidation of L-serine by the enzyme. HOCl generated by
myeloperoxidase converts L-serine to glycolaldehyde, an
-hydroxy aldehyde lacking functional groups that can scavenge reactive chlorinating species (26). We quantified this aldehyde by HPLC
following derivatization with 3-methyl-2-benzothiazolinone hydrazone
hydrochloride (26). Exposing L-serine to either the myeloperoxidase-H2O2-Br
system or
hypobromous acid, the primary oxidant of eosinophil peroxidase (33, 35,
36), generated glycolaldehyde (data not shown). In contrast to HOCl
(26, 34), hypobromous acid generated aldehyde from L-serine
only after prolonged incubation (>1 h at 37 °C). Other halides
(I
, F
) or the pseudohalide
SCN
failed to generate glycolaldehyde in this system,
despite incubations of up to 24 h at 37 °C (data not shown).
These results suggest that hypobromous acid reacts with amino acids to
form an
-monobromamine intermediate that is more stable to
deamination and decarboxylation than its
-monochloramine
analogue.
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DISCUSSION |
We have recently demonstrated that human neutrophils use the
myeloperoxidase-H2O2-Cl
system to
convert common
-amino acids to an array of reactive aldehydes
(3). Here, we provide evidence for the mechanism of aldehyde
production. Free primary amino and carboxylic acid groups on the
-carbon of an amino acid were required for aldehyde generation. Mass
spectrometric studies with isotopically labeled amino acids
demonstrated that hydrogen atom abstraction from the
-carbon (Scheme
I, Pathway A), a mechanism described for aldehyde formation
by ionizing radiation (4-9) and Fenton systems (9-13), did not occur.
Rather, several independent analytical methods (UV spectroscopy and
1H and 1H,15N two-dimensional NMR
spectroscopies) indicate that chlorination of the
-amino group by
HOCl (a product of the myeloperoxidase pathway) appears to be the
initial event. This generates a labile
-monochloramine which rapidly
decomposes to an aldehyde (Scheme I, Pathway B). Examination
of HOCl oxidation products of multiple
-,
-, and
-amino acids
confirmed that
-monochloramines, but not
- or
-monochloramines, are labile.
We were unable to confirm the structural identity of a stable
intermediate between the
-monochloramine and aldehyde for any of the
amino acids examined. A likely explanation is that the half-lives of
the intermediates are too short for detection under the methods
employed. For example, a short-lived imine might form during
monochloramine decomposition directly in a single step by the concerted
loss of CO2 and Cl
; subsequent loss of
ammonia would then yield an aldehyde. A plausible mechanism for this
rearrangement is illustrated in Scheme
IV. Indeed, a short-lived imine has been proposed as a reaction
intermediate in the oxidation of amino acids by ionizing radiation
(Scheme I; Ref. 9). Our identification of
-monochloramines as
reaction intermediates in myeloperoxidase-mediated oxidation of
-amino acids suggests that these labile intermediates could also
have biological functions at sites of inflammation. By analogy, this may also be true for
-monobromamines formed by either
myeloperoxidase or eosinophil peroxidase, particularly at sites of an
allergic inflammatory response where an eosinophilic infiltrate is a
characteristic finding (37).

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Scheme IV.
Proposed reaction mechanism for conversion
of an -monochloramine into an aldehyde via an imine.
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The 1H NMR studies clearly suggest that aldehydes formed by
the myeloperoxidase system in vivo are likely to exist in
equilibrium with hydrated and condensed forms (31, 32). Aldehydes are also likely to interact with thiols and primary amino groups, generating an array of Michael adducts (32) and Schiff bases (9, 31,
32). We recently have identified the reduced Schiff base adduct between
pHA and the
-amino groups of protein lysine residues in inflamed
human tissues (34). It is likely that adducts between other amino
acid-derived aldehydes and proteins, lipids, and nucleic acids will
also be found. Autoantibodies that recognize proteins modified by
acrolein, an
-
unsaturated aldehyde formed when myeloperoxidase
oxidizes L-threonine (26), are present in animals and
humans with atherosclerosis (38, 39). Anti-acrolein antibodies also
recognize epitopes in atherosclerotic lesions and in lipoproteins
recovered from human aorta (38, 40), suggesting that the
myeloperoxidase system generates reactive aldehydes in the artery wall.
Indeed, the enzyme is abundant and catalytically active in human
atherosclerotic lesions (41, 42). The selective reactivity of
aldehydes, and their ability to form covalent adducts, suggests that
amino acid-derived aldehydes generated at sites of inflammation may
damage cellular targets. The reversible nature of many of these adducts
also may serve to prolong their half-lives. Because reversible covalent
modification has served as a paradigm for biological signaling
processes, labile
-monochloramines and amino acid-derived aldehydes
have the potential to serve as signaling molecules at sites of
inflammation, perhaps to coordinate the oxidative capacity of
phagocytes with the response of other immune cells of the host's
defense system.
Collectively, these observations identify the reaction mechanism for
aldehyde formation by HOCl and strongly suggest that free
-amino
acids, which are present at a concentration of 4-5 mM in
plasma and even higher intracellularly (24, 25), may be major
substrates for oxidation by activated phagocytes. The ability of
aldehydes to react with nucleophilic moieties on proteins, lipids, and
DNA suggests that the generation of such species may represent an
important mechanism for damage of biological targets and the
transduction of biological signals at sites of inflammation.
We thank S. Scotino and D. Mueller for expert
technical assistance. Mass spectrometry experiments were performed at
the Washington University School of Medicine Mass Spectrometry
Resource.