Fast reactions of arylnitrenium ions with amino acids and proteins: A laser flash photolysis study

Article abstract of DOI:10.1002/poc.1031

Laser flash photolysis was used to examine the reaction of N-methyl-N-(4-biphenylyl)nitrenium ion with various amino acids and proteins in aqueous media. This nitrenium ion was found to react rapidly (> 10 ⁸ M^-1 s^-1) with tryptophan, tyrosine, methionine and cysteine, more slowly (10⁷-10⁸M ^-1s^-1) with lysine, histidine, and arginine. Rapid reaction was also seen with several representative proteins including bovine serum albumin, lysozyme, and chymotrypsin. These results suggest that reaction with proteins is likely to be a significant pathway in the reactions of nitrenium ions generated in vivo. Copyright

Full text of DOI:10.1002/poc.1031

JOURNAL OF PHYSICAL ORGANIC CHEMISTRY
J. Phys. Org. Chem. 2006; 19: 291–294
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/poc.1031
Fast reactions of arylnitrenium ions with amino acids
and proteins: a laser flash photolysis study
*
Selina I. Thomas and Daniel E. Falvey
Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland, USA
Received 5 December 2005; revised 20 January 2006; accepted 21 January 2006
ABSTRACT: Laser flash photolysis was used to examine the reaction of N-methyl-N-(4-biphenylyl)nitrenium ion
with various amino acids and proteins in aqueous media. This nitrenium ion was found to react rapidly (>108 MÀ1 sÀ1
)
with tryptophan, tyrosine, methionine and cysteine, more slowly (107–108 MÀ1 sÀ1) with lysine, histidine, and
arginine. Rapid reaction was also seen with several representative proteins including bovine serum albumin, lysozyme,
and chymotrypsin. These results suggest that reaction with proteins is likely to be a significant pathway in the reactions
of nitrenium ions generated in vivo. Copyright # 2006 John Wiley & Sons, Ltd.
Supplementary electronic material for this paper is available in Wiley Interscience at http://www.interscience.
wiley.com/jpages/0894–3230/suppmat/
KEYWORDS: nitrenium ion; N-methyl-N-(4-biphenylyl)nitrenium ion; amino acids, proteins; hydrazine dimer;
methionine; photochemistry; arylnitrenium ion
INTRODUCTION
This caused us to consider the possibility that nitrenium
ions generated in vivo might also be trapped by
nucleophilic and/or readily oxidized amino acids in
proteins. The LFP experiments described below support
this prediction. This nitrenium ion is quenched by
tryptophan, tyrosine, methionine, and cysteine, at or near
the diffusion limit and somewhat more slowly by serine,
histidine, lysine, and arginine. LFP experiments carried
out with: bovine serum albumin (BSA), bovine pancreatic
nuclease, lysozyme (lyz), insulin (Ins), and chymotrypsin
(Chym) show that native proteins are also reactive toward
this nitrenium ion.
Arylnitrenium ions are short-lived, electrophilic inter-
mediates characterized by a positively charged, dicoor-
dinate nitrogen atom which is bound to one or two
aromatic groups. A series of elegant experiments carried
out several decades ago showed that the carcinogenic
effects of aromatic amines could be attributed to
enzymatic oxidation of the amines followed by ester-
ification of the resulting hydroxylamine.1–3 The key DNA
damaging step was inferred to be heterolysis of this ester,
forming an arylnitrenium ion, which in turn reacts with
guanine bases in DNA forming covalent adducts.4–7
Subsequent kinetic studies using both competitive
trapping and laser flash photolysis (LFP) confirmed this
general mechanism.6,8–10 In particular those arylnitre-
nium ions derived from highly carcinogenic amines were
found to react very rapidly with guanine, but to be far less
reactive toward water.
There is far less information regarding the stability of
nitrenium ions toward other biological molecules. Novak
and others have shown that glutathione can rapidly trap
nitrenium ions.11 However, we are unaware of any direct
kinetic data regarding the stability of arylnitrenium ions
toward proteins or their component amino acids. Earlier
studies from this laboratory on N-methyl-N-4-bipheny-
lylnitrenium ion (2) showed that this intermediate was
trapped rapidly by amines, and electron-rich arenes.12,13
RESULTS AND DISCUSSION
The method for the photochemical generation and
detection of 2 has been described elsewhere.14 Briefly
this reactive intermediate is generated by photolysis of
1-(N-methyl-N-4-biphenylyl)-2,4,6-trimethylpyridinium
tetrafluoroborate (1) using the third harmonic (355 nm,
20 mJ, 4 ns) of a Nd:YAG laser. The resulting singlet
nitrenium ion can be detected through its absorption at
460 nm. In aqueous solution, the latter lives for 590 ns. Its
typical decay reactions have been previously shown to
include: (a) addition of nucleophiles to the aromatic ring
carbons; (b) addition of electron rich arenes to the
nitrogen and the ortho carbon on the proximal phenyl
ring: and a net reduction of the nitrenium ion through
what is inferred to be a relatively long-lived complex with
the arene.
*Correspondence to: D. E. Falvey, Department of Chemistry and
Biochemistry, Building 091, University of Maryland, College Park,
MD 20742, USA.
E-mail: falvey@umd.edu
Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 291–294

292
S. I. THOMAS AND D. E. FALVEY
Figure 2. Dependence of the pseudo-first order decay rate
constant (kobs) of nitrenium ion 2 on the concentration of
glycine, determined by laser flash photolysis (355 nm, 6 ns,
20 mJ) of 1
Figure 1. Dependence of the pseudo-first order decay rate
of nitrenium ion 2 on the concentration of cysteine, deter-
mined by laser flash photolysis (355 nm, 6 ns, 20 mJ) of 1
The reactivity of the nitrenium ion toward the various
amino acids was determined by LFP. Specifically the
decay of the nitrenium ion’s absorbance at 460 nm was
measured in the presence of varying concentrations of
each amino acid (typically 0–10 mM). In most cases the
decays could be fit to a first order decay function. The
bimolecular rate constants were derived from the
dependence of these pseudo-first order rate constants
on the concentration of the amino acid. Typical data are
shown in Fig. 1 and all of the second order rate constants
are compiled in Table 1. For the amino acids with
aliphatic side chains, carboxylic side chains, and amide
side chains, along with phenylalanine, threonine, and
proline, no change in the lifetime of the nitrenium ion
could be detected. The short lifetime of the nitrenium ion,
coupled with the limited solubility of most amino acids in
the 10% aqueous acetonitrile medium makes it impos-
sible to characterize trapping rate constants less than
105 MÀ1 sÀ1, thus the latter is assumed to be the upper
limit for these amino acids.
with electron rich arenes, such as 1,3,5-trimethoxyben-
zene, and N,N-dimethylaniline, but showed no measur-
able reactivity toward unactivated or weakly activated
arenes (e.g., toluene).12 The nitrenium ion reacts
measurably, but more slowly with amino acids having
amine (lysine, arginine, and histidine) and hydroxyl
(serine) side chains. The rate constants we observe are
somewhat lower than seen for comparable amines in
aprotic solvents. This is readily explained by H-bonding
of water to the traps, which ought to diminish the
reactivity of these groups. It is interesting to note that
serine is weakly reactive (as seen for simple alcohols), but
threonine shows no measurable quenching. However, the
previous studies have shown that the reactivity of the
alcohols is significantly retarded by increasing steric bulk.
While we suspect that threonine is somewhat reactive, it is
apparently too slow to detect by our method.
It was also observed that glycine quenches the
nitrenium ion. This is surprising, given the lack of a
reactive side chain on this amino acid. However, a careful
examination of the pseudo-first order dependence of this
process (Fig. 2) indicates that it is not a simple
bimolecular reaction, but apparently involves reaction
of two or more glycine molecules. This process was not
investigated in any more detail.
The nitrenium ion reacts rapidly (108–109 MÀ1 sÀ1
)
with the electron-rich aromatic amino acids, tyrosine and
tryptophan, but slowly, if at all, with phenylalanine. The
same nitrenium ion has been shown to react very rapidly
Table 1. Bimolecular trapping rate constants of 2 by amino
acids
The reactivity of the sulfide-bearing amino acids,
methionine and cysteine is interesting. The current LFP
experiments show that these amino acids react with 2 with
rate constants comparable to that for tyrosine and
tryptophan. Examples of arylnitrenium ion trapping by
sulfides seems to be largely confined to the reactions of
glutathione with those nitrenium ions implicated in DNA
damaging mechanisms.6,15,16 For example, Novak and
Lin report addition of the sulfur atom of glutathione to the
ring carbons of N-acetyl-N-biphenylylnitrenium ion.11
These products were also accompanied by formation of
the parent amide, 4-biphenylylacetamide. The latter
product was attributed to initial N–S bond formation
followed by SN2 displacement of the glutathione residue
Amino Acids
kq (MS1 sS1
)
L-Glycine
L-Serine
(a)
(7.93 W 0.7) T 106
(2.79 W 0.2) T 108
(1.29 W 0.7) T 109
(2.95 W 0.7) T 108
(2.91 W 0.1) T 109
(5.39 W 0.5) T 107
(8.04 W 1.6) T 107
(3.04 W 0.5) T 107
L-Cysteine
L-Methionine
L-Tyrosine
L-Tryptophan
L-Histidine
L-Lysine
L-Arginine
The remaining amino acids tested showed no measurable quenching.
(a) A non-linear pseudo-first order plot was obtained (Fig. 2).
Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 291–294

REACTIONS OF NITRENIUM IONS WITH PROTEINS
293
by another glutathione molecule, forming the disul-
fide and the amide. It seems reasonable to assume
that cysteine is reacting via similar pathways. With
methionine, however, the reaction pathway is less clear as
this lacks the acidic S–H bond.
In order to understand the reaction pathway followed
with methionine, we carried out a larger scale reaction by
generating the nitrenium ion in the presence of a protected
analog of methionine (3). The protecting groups were
required to circumvent solubility limits of this amino
acid. LFP experiments wherein the nitrenium ion was
generated in the presence of the protected methionine in
CH3CN showed that the trapping reaction occurs with a
rate constant (1.6 Â 109 MÀ1 sÀ1) comparable to that
observed in aqueous solution.
Three products were isolated from preparative runs: the
parent amine (8), a dimeric hydrazine (7), and an
oxidative dimeric coupling product from the amino acid.
The parent amine has been previously characterized. The
hydrazine dimer (7) is a new compound, not previously
detected in the trapping reactions of the nitrenium ion and
was characterized by NMR and MS.
Scheme 1
The proposed mechanism for the reaction of the
nitrenium ion with the protected methionine is shown in
Scheme 1. It is assumed that the initial reaction is N–S
coupling, forming a relatively unstable amino-sulfonium
ion (4). Homolysis of the N–S bond in this adduct would
produce the aminyl radical (5) and the cation radical of
methionine (6). The parent amine forms when 5 abstracts
H atoms from the a-position of unreacted methionine
molecules. The same parent amine has also been observed
in the reactions of the nitrenium ion with arenes, it is
similarly considered to result from a slow dissociation of
a pi-complex followed by a subsequent H atom transfer to
the resulting aminyl radical.
Because 7 was not observed in the arene experiments, it
seems likely that it is generated from a different pathway.
In particular, we suggest that the intermediate sulfonium
ion reacts with a neutral amine molecule producing the
hydrazine (following deprotonation) and regenerates the
methionine.
1
Aside from the aforementioned products, H-NMR of
the reaction mixture showed a complex mixture of several
minor products. It is assumed that these include various
products of methionine oxidation and perhaps minor
adducts of the nitrenium ion. Because of the low yields,
these were not characterized.
To determine if the reactivity toward these amino acids
was manifest in native proteins, LFP experiments were
carried out with several readily available proteins known
to contain various amounts of the reactive amino acids.
Table 2 shows rate constants observed for the reactions of 2
with BSA, bovine pancreatic ribonuclease (BPR), lyz, Ins,
and Chym. The observed rate constants ranged from 5 to
70Â 108 MÀ1 sÀ1. These are comparable to the rates for
the trapping of 2-fluorenylnitrenium ion by single-stranded
DNA and significantly faster than the reactions of the same
nitrenium ion with double-stranded DNA.8 Also shown in
Table 2. Bimolecular trapping rate constants of 2 measured for each proteins and the molecular weights, number of reactive
amino acids and the percentage of reactive amino acids for each proteins (based on kq of amino acids ‡108 MS1 sS1
)
Molecular weight Number of Number of Number of Number of
Trp
Percentage of
reactive amino acids
Proteins
kq (MS1 sS1
)
of protein (Da)
Met
Cys
Tyr
BSA
BPR
Lysozyme
(8.22 W 0.9) T 108
(4.59 W 0.4) T 108
(5.79 W 0.6) T 108
(8.96 W 0.8) T 108
66 465.8b
13 686c
14 388e
5800f
4
4
2
0
2
35
8
8
6
10
20
6
3
4
3
2
0
6
0
8
10.5b
14.5d
14.7e
19.6f
9.9g
Insulina
Chymotrypsina (7.71 W 0.8) T 109
25 000g
a NH4OH was added to aid the solubility of the proteins in buffer.
b Brown JR. Fed. Proc. Fed. Am. Soc. Exp. Biol. 1975; 34: 591; Abstract 2105.
c Raines RT. Chem. Rev. 1998; 98: 1045-1065.
d Smyth DG, Stein WH, Moore S. J. Biol. Chem. 1963; 238: 227-234.
e Canfield RE. J. Biol. Chem. 1963; 238: 2698-2707.
f Ryle AP, Sanger F, Smith LF, Katai R. Biochem. J. 1955; 60: 541.
g Hartley BS. Nature. 1964; 201, 1284-1287.
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294
S. I. THOMAS AND D. E. FALVEY
Table 2 are the molecular weight, number of reactive
amino acids (methionine, cysteine, tyrosine, and trypto-
phan), and the percentage of reactive amino acids for each
protein. There is no obvious correlation of these rate
constants with any of these properties. It is likely that the
reactivity of a given protein depends on more subtle
structural factors, such as the number of reactive amino
acids that are readily accessible to the nitrenium ion, and
perhaps the ability of the proteins tertiary structure to assist
in any oxidation or addition reactions by the nitrenium ion.
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and isolation and characterization of 7 are available in
Wiley Interscience.
Copyright # 2006 John Wiley & Sons, Ltd.
J. Phys. Org. Chem. 2006; 19: 291–294