Amino acid nitrosation products as alkylating agents

Article abstract of DOI:10.1021/ja010348+

Nitrosation reactions of α-, β-, and γ-amino acids whose reaction products can act as alkylating agents of DNA were investigated. To approach in vivo conditions for the two-step mechanism (nitrosation and alkylation), nitrosation reactions were carried ou

Full text of DOI:10.1021/ja010348+

7506
J. Am. Chem. Soc. 2001, 123, 7506-7510
Amino Acid Nitrosation Products as Alkylating Agents
M. del Pilar Garc´ıa-Santos, Emilio Calle, and Julio Casado*
Contribution from the Departamento de Qu´ımica F´ısica, UniVersidad de Salamanca,
E-37008 Salamanca, Spain
ReceiVed February 7, 2001
Abstract: Nitrosation reactions of R-, â-, and γ-amino acids whose reaction products can act as alkylating
agents of DNA were investigated. To approach in vivo conditions for the two-step mechanism (nitrosation
and alkylation), nitrosation reactions were carried out in aqueous acid conditions (mimicking the conditions of
the stomach lumen) while the alkylating potential of the nitrosation products was investigated at neutral pH,
as in the stomach lining cells into which such products can diffuse. These conclusions were drawn: (i) The
alkylating species resulting from the nitrosation of amino acids with an -NH2 group are the corresponding
lactones; (ii) the sequence of alkylating power is: R-lactones > â-lactones > γ-lactones, coming respectively
from the nitrosation of R-, â-, and γ-amino acids; and (iii) the results obtained may be useful in predicting the
mutagenic effectiveness of the nitrosation products of amino acids.
Introduction
damage DNA, in which process the cytotoxic and mutagenic
effects of carcinogenic compounds are believed to reside.16,17
The nitrosation of amino acids is particularly interesting. In
the case of those with a secondary amino group, besides direct
nitrosation by NO+ and N2O3, a nitrosation mechanism through
the initial formation of a nitrosyl carboxylate followed by a slow
intramolecular rearrangement has been reported.18,19
Regarding the nitrosation of amino acids with a -NH2 group,
some results suggested that mutagenic products arose primarily
from nitrosation of the primary amine rather than the amide or
indole group.20,21 It thus became of interest to identify the
alkylating agent as well as its alkylating potential.
This research was carried out in two stages: (i) Kinetic study
of the nitrosation of amino acids; (ii) identification of alkylating
agents, and investigation of their alkylating potential through
their reactions with 4-(p-nitrobenzyl)pyridine, NBP (introduced
to trap alkylating agents22). NBP has nucleophilic characteristics
similar to those of DNA.
Since the report by Magee and Barnes1 that dimethyl-
nitrosamine induces liver cancer when fed to rats this finding
has prompted and expanded study of the series of nitroso
compounds. Biologists were mainly interested in the use of these
compounds as models for producing a broad range of cancers,2-4
whereas chemists were more interested first in the mechanisms
of formation of nitroso compounds5-8 and then in blocking or
inhibiting the mechanisms of formation of these species.9-13
Studies showing that dimethylnitrosamine is converted en-
zymically into a methylating agent14,15 sparked interest in the
mechanism of carcinogenesis through the alkylation of proteins
and nucleic acids by N-nitroso compounds.
Some nitroso compounds decompose or can be metabolized
in vivo to form strongly alkylating electrophiles that may
* To whom correspondence and reprint requests should be addressed.
E-Mail: jucali@gugu.usal.es.
(1) Magee, P. N.; Barnes, J. M. Br. J. Cancer 1956, 10, 114.
(2) Mirvish, S. S. Cancer Lett. 1995, 93, 17.
(3) Lijinsky, W. Chemistry and Biology of N-Nitroso Compounds;
Cambridge University Press: Cambridge, UK, 1992.
(4) Laires, A.; Gaspar, J.; Gomes da Costa, G.; Rueff, J. Toxicologist
1995, 15, 78.
(5) Iglesias, E. J. Am. Chem. Soc. 1998, 120, 13057.
(6) Iglesias, E.; Garc´ıa-R´ıo, L.; Leis, J. R.; Casado, J. Recent Res. DeV.
Phys. Chem. 1997, 1, 403.
Experimental Section
Amino acid solutions were made up by weight from Merck 99%
glycine and valine; Aldrich 99% alanine, R-aminobutyric acid, R-amino-
isobutyric acid, norvaline, and â-alanine, 97% â-aminobutyric acid and
γ-aminobutyric acid, and 98% aspartic acid. NBP was from Sigma.
Triethylamine (99%) was obtained from Aldrich. Perchloric acid,
sodium hydroxide used for preparing buffer solutions, and the ionic
strength controller sodium perchlorate monohydrate were obtained from
Merck. Sodium dihydrogen phosphate 2-hydrate was from Panreac as
well as the NBP solvents, 99% acetone and 99% ethylene glycol.
(7) Williams, D. L. H. Nitrosation; Cambridge University Press: Cam-
bridge, UK, 1988.
(8) Casado, J. Nitrosation Reactions; Invited Lecture. In Fast Reactions
in Solution; Royal Society of Chemistry Annual Meeting, 1994.
(9) Archer, M. C. In N-Nitroso Compounds. Occurrence, Biological
Effects and ReleVance to Human Cancer; O’Neill, I. K., von Borstel, R.
C., Miller, C. T., Long, J., Bartsch, H., Eds.; IARC Sci. Pub. No. 57: Lyon,
France, 1984; p 263.
(10) Loeppky, R. N.; Bao, Y. T.; Bae, J.; Yu, L.; Shevlin, G. In
Nitrosamines and Related N-Nitroso Compounds: Chemistry and Biochem-
istry; Loeppky, R. N., Michejda, C. J., Eds.; American Chemical Society:
Washington, DC, 1994; p 52.
(16) Galtress, C. L.; Morrow, P. R.; Nag, S.; Smalley, T. L.; Tschantz,
M. F.; Vaughn, J. S.; Wichems, D. N.; Ziglar, S. K.; Fishbein, J. C. J. Am.
Chem. Soc. 1992, 114, 1406.
(17) Lewis, R. S.; Tannenbaum, S. R.; Deen, W. M. J. Am. Chem. Soc.
1995, 117, 3933.
(11) Gonza´lez-Mancebo, S.; Calle, E.; Garc´ıa-Santos, M. P.; Casado, J.
J. Agric. Food Chem. 1997, 45, 334.
(12) Garc´ıa-Prieto, J. C.; Mateos, R.; Calle, E.; Casado, J. J. Agric. Food
Chem. 1998, 46, 3517.
(18) Casado, J.; Castro, A.; Leis, J. R.; Mosquera, M.; Pen˜a, M. E. J.
Chem. Soc., Perkin Trans. 2 1985, 1859.
(19) Gil, R.; Casado, J.; Izquierdo, C. Int. J. Chem. Kinet. 1994, 26,
1167.
(13) Gonza´lez-Mancebo, S.; Garc´ıa-Santos, M. P.; Herna´ndez-Benito,
J.; Calle, E.; Casado, J. J. Agric. Food Chem. 1999, 47, 2235.
(14) Magee, P. N.; Hultin, T. Biochem. J. 1962, 83, 106.
(15) Magee, P. N.; Farber, E. Biochem. J. 1962, 83, 114.
(20) Meier, I.; Shephard, S. E.; Lutz, W. K. Mutat. Res. 1990, 238, 193.
(21) Shephard, S. E.; Wakabayashi, K.; Nagao, M. Food Chem. Toxicol.
1993, 31, 323.
(22) Epstein, J.; Rosenthal, R. W.; Ess, R. J. Anal. Chem. 1955, 27, 1435.
10.1021/ja010348+ CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/13/2001

AA Nitrosation Products as Alkylating Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7507
Table 1. Nitrosation Rate of Amino Acids
amino acid
glycine
alanine
R-aminobutyric acid
R-aminoisobutyric acid
valine
norvaline
â-Alanine
â-aminobutyric acid
γ-aminobutyric acid
pH
k3 exp‚102 (M-2 s-1
)
3.00
3.05
3.05
3.00
3.05
3.03
3.00
3.05
3.14
11.3 ( 0.3
3.91 ( 0.09
4.8 ( 0.1
0.87 ( 0.04
7.6 ( 0.2
5.1 ( 0.4
2.80 ( 0.08
1.34 ( 0.02
0.89 ( 0.03
Table 2. Alkylating Potential of the Amino Acid Nitrosation
Products
λmax
tnitrosation
talkylation
amino acid
glycine
alanine
R-aminobutyric acid
R-aminoisobutyric acid
valine
norvaline
â-alanine
â-aminobutyric acid
aspartic acid
(nm)
(min)b
(min)b
Figure 1. Nitrosation rate as a function of pH. (b) [nor-Val]o ) 0.300
M, (0) [â-Ala]o ) 0.300 M, (2) [γ-Amb]o ) 0.300 M; [Nit]o ) 0.010
M, [NaH2PO4] ) 0.50 M, I ) 1.00 M, T ) 298 K.
562
521
525
550
532
525
587
588
598
585
100
60
60
very long
100
40
150
long
300
long
20
10
7
≈60
Nitrosation reactions were carried out in aqueous acid conditions
(mimicking the conditions of the stomach lumen), while the alkylating
potential of the nitrosation products was investigated at neutral pH, as
in the stomach lining cells into which such products can diffuse.
10
10
125
very long
400
very long
For studying alkylation reactions, equimolar concentrations of amino
acid and sodium nitrite were incubated in NaH2PO4/HClO4 buffer
solution (“nitrosation mixture”). At different times (“nitrosation time”),
aliquots were removed and added to a NBP solution (NBP was
dissolved in acetone, ethylene glycol, and sodium phosphate buffer,
pH 8, at a ratio of 1:4:1).
γ-aminobutyric acid
a Nitrosation reactions (pH ≈ 3.5), [amino acid]o ) 0.060 M,
[NaNO2]o ) 0.060 M, [NaH2PO4] ) 0.50 M, I ) 1.00 M (NaClO4), T
) 298 K. Alkylation reactions (pH ≈ 7), [NBP]o ) 0.116 M, T ) 298
K. b Times for maximum adduct concentration to be reached.
The “alkylation mixture” (neutral pH) was prepared with 1 mL of
nitrosation mixture and 3 mL of NBP solution. The mixture was
incubated at 298 K.
Second-order with respect to the nitrite concentration shows
that the effective nitrosating agent should be dinitrogen tri-
oxide.23
Aliquots of 2.4 mL of the alkylation mixture were removed at
different times (“alkylation time”) and added to the cuvette containing
0.6 mL of the 99% triethylamine reagent (to stop the alkylation reaction
and render the adduct stable), measuring absorbance at the wavelength
of maximum absorption.
Table 1 shows the values of k3exp obtained in the nitrosation
of R-, â-, and γ-amino acids. The observed sequence of k3exp
values is: R-amino acids > â-amino acids > γ-amino acids.
Alkylation Reactions. Formation of the adduct between NBP
and the alkylating agent resulting from the nitrosation was
studied as a function of the nitrosation time at different
alkylation times (see Experimental Section).
Spectrophotometric measurements were carried out on a Shimadzu
UV-2101 PC spectrophotometer equipped with a thermoelectric six-
cell holder temperature-control system ((0.1 °C). IR spectra were
obtained with a Bonem FT MB-100 spectrometer. NMR proton spectra
were obtained with a 200 MHz Bruker, model WP 200 SY spectrometer.
Table 2 shows the wavelength of maximum absorption for
each adduct. Figure 2, a-c, shows the evolution of the NBP
alkylation process as a function of the nitrosation and alkylation
times.
Results and Discussion
Nitrosation Reactions. We investigated the nitrosation of
six R-amino acids: glycine (Gly), DL-alanine (Ala), DL-R-
aminobutyric acid (R-Amb), R-aminoisobutyric acid (R-Amib),
valine (Val), and norvaline (nor-Val); two â-amino acids:
â-alanine (â-Ala) and DL-â-aminobutyric acid (â-Amb); and one
γ-amino acid: γ-aminobutyric acid (γ-Amb).
Table 2 reveals important differences between the nitrosation
and alkylation times that lead to the maximum concentration
of the adduct. The nitrosation times required for the maximum
adduct concentration to be reached follow the sequence:
R-amino acids < â-amino acids < γ-amino acids, which is
coherent with the k3exp values found in the nitrosation reaction
(Table 1).
Since the alkylation time necessary for the maximum adduct
concentration to be reached is very different, depending on
whether the precursor is R-, â-, or γ-amino acid, a study was
made of the alkylating potential of the nitrosation products of
aspartic acid, which has two acid groups at positions R and â
with respect to the amino group.
Since the HNO2/NO2- system (henceforth nitrite, Nit) shows
a maximum in the absorption spectrum at λ ) 371 nm, nitrite
was used as the control species for the continuous measurement
of the amino acid nitrosation.
Figure 2d shows the results obtained on studying the
alkylation potential of the nitrosation products of aspartic acid.
All reactions studied exhibited experimental rate equations
of the form:
As may be seen from Table 2, the nitrosation and alkylation
times corresponding to aspartic acid are similar to those obtained
with the â-amino acids. This means that the carboxyl group
rate ) k3exp [amino acid] [Nit]2
(1)
(23) Cachaza, J. M.; Casado, J.; Castro, A.; Lo´pez-Quintela, M. A. Z.
Krebsforsch. 1978, 91, 279.
k3exp showing a strong dependence on pH (Figure 1).

7508 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Garc´ıa-Santos et al.
Figure 2. Formation of NBP adduct with the nitrosation time, at different alkylation times. (a) Glycine, [Gly]o ) 0.060 M, [Nit]o ) 0.060 M, pH
(nitrosation) ) 3.55; [NBP]o ) 0.116 M, pH (alkylation) ) 7.0. Alkylation time: (b) 3 min, (0) 5 min, (2) 10 min, (O) 15 min, (9) 20 min. (b)
â-Alanine, [â-Ala]o ) 0.060 M, [Nit]o ) 0.060 M, pH (nitrosation) ) 3.53; [NBP]o ) 0.116 M, pH (alkylation) ) 7,0. Alkylation time: (b) 5 min,
(0) 25 min, (2) 50 min, (O) 75 min, (9) 100 min, ([) 125 min. (c) γ-Aminobutyric acid, [γ-Amb]o ) 0.116 M, [Nit]o ) 0.116 M, pH (nitrosation)
) 2.78; [NBP]o ) 0.116 M, pH (alkylation) ) 7.0. Alkylation time: (b) 60 min, (0) 300 min, (2) 1440 min, (O) 2880 min, (9) 8640 min. (d)
Aspartic acid, [Asp]o ) 0.060 M, [Nit]o ) 0.060 M, pH (nitrosation) ) 3.53; [NBP]o ) 0.116 M, pH (alkylation) ) 7.0. Alkylation time: (b) 25
min, (0) 50 min, (2) 100 min, (O) 150 min, (9) 200 min, ([) 300 min, (4) 400 min.
located at the â position with respect to the amino group exerts
a greater influence than that situated at the R position.
Scheme 1 shows the proposed mechanism. Following nitro-
sation, the corresponding diazo compound is formed, which
rapidly loses N2.
The time required for the maximum concentration of adduct
to be reached (Table 2) rules out the hypothesis of a carbocation
as the alkylating agent. We thought in terms of a product
resulting from its cyclization, such as lactones.
On the basis of the tricentric, tetracentric, and pentacentric
structures of the lactones corresponding to the R-, â-, and
γ-amino acids (Scheme 2), it is to be expected that the reactivity
of such lactones would decrease when their size increases. That
is, the alkylation rate would decrease in the same order, and
their alkylation time would increase, as was, in fact, observed
(Table 2).
These results are consistent with those reported in biomedical
research: â-propiolactone and four-membered-ring lactones are
generally, but not always, active carcinogens. â-Propiolactone
(in our case resulting from the nitrosation of â-alanine) has been
extensively investigated and has been found to be a potent,
although slow-acting, carcinogen.24 Unlike the â-lactones,
γ-lactones are not inherently reactive because of their lack of
ring strain. This lack of reactivity of γ-butyrolactone is therefore
in accordance with its inactivity as a carcinogen.24
stability. All of the lactones derived from R-amino acids have
similar alkylating times, with the exception of the lactone
deriving from the nitrosation of R-aminoisobutyric acid, with a
slightly longer alkylation time. The cause of this must lie in
the fact that in this case there are two methyl groups bound
directly to the electrophilic carbon atom that provide charge
density.
For the reasons mentioned above, the â-amino acids have
longer alkylation times than the R-amino acids. Upon comparing
the alkylation due to the â-lactones coming from â-alanine and
â-aminobutyric acid, it may be seen that it is much slower in
the case of the latter; the reason for this may lie in the presence
of the methyl group adjacent to the electrophilic carbon, thus
providing charge density.
In the case of aspartic acid, the alkylation time observeds
similar to that of â-amino acidssis coherent with the formation
of a lactone with the intervention of the carboxyl group located
at â-position with respect to the amino group, less strained than
the lactone formed with the R-carboxyl group.
Following its nitrosation, the γ-amino acid studied leads to
a highly stable γ-lactone and, as a result, is only sparingly
reactive in both chemical and biological terms.
With a view to checking experimentally that the lactones were
indeed responsible for the alkylation, they were identified.
Owing to their greater stability the â- and γ-lactones were
chosen.
The times of alkylation by R-lactones are shorter than those
corresponding to â- and γ-lactones (Scheme 2) owing to ring
Amino acids and NaNO2 were made to react in acid medium.
After a sufficient nitrosation time for a significant concentration
of alkylation reagent to be reached, extraction was carried out
(24) Lawley, P. D. In Chemical Carcinogens, 2nd ed.; Searle, C. E.,
Ed.; ACS Monograph 182; American Chemical Society: Washington, DC,
1984; Vol. 1, p 428.

AA Nitrosation Products as Alkylating Agents
J. Am. Chem. Soc., Vol. 123, No. 31, 2001 7509
Scheme 1. Alkylation Mechanism
Scheme 2. Lactones Resulting from Nitrosation of R-, â-,
and γ-Amino Acids; Arrow V Shows Electrophilic Center;
talkylation Corresponds to the Maximum Adduct Concentration
Identification of γ-Butyrolactone. The IR spectrum of the
nitrosation products of γ-aminobutyric acid was obtained; this
featured a band centered at 1771 cm-1
.
The lactones with a pentagonal ring show a band correspond-
ing to the CdO group at 1760-1780 cm-1. In particular,
γ-butyrolactone shows a band at 1770 cm-1 25,28 which can be
identified with the one observed by us.
The 1H NMR spectrum of the nitrosation products of
γ-aminobutyric showed the following signals: (200 MHz;
CDCl3) δ (ppm): 4.34 (2H, t, J ) 7 Hz), 2.49 (2H, t, J ) 7.7
Hz), 2.26 (2H, q, J ) 7, J ) 7.7 Hz), in agreement with those
found in the literature.25,28
Further proof in favor of the hypothesis that the lactones are
indeed the alkylating agents was obtained when we reacted
commercial â-butyrolactone with NBP, affording a spectrum
(λmax ) 587 nm) analogous to that observed in the alkylating
reaction of NBP with the nitrosation products of â-Amb (it
should also be mentioned that a â-lactone was obtained by
reaction of aspartame, L-aspartyl-L-phenylalanine methyl ester,
with NaNO2 in a biphasic reaction solution of aqueous HCl and
dichloromethane29).
with CHCl3 (â-lactones) or CH2Cl2 (γ-butyrolactone), separating
the organic phase, which was then washed with a saturated so-
lution of NaCl and dried with anhydrous Na2SO4. This was then
filtered and the solvent evaporated off under reduced pressure.
1
Conclusions
Identification of â-Propiolactone. The H NMR spectrum
of the nitrosation products of â-alanine revealed the following
signals: (200 MHz; CDCl3) δ (ppm): 4.28 (2H, t, J ) 5.8 Hz),
3.55 (2H, t, J ) 5.8 Hz), attributed to â-propiolactone, owing
to their agreement with those reported in the literature.25
(i) The alkylating species resulting from the nitrosation of
amino acids with an -NH2 group are the corresponding lactones.
(ii) The sequence of alkylating potential is: R-lactones >
â-lactones > γ-lactones coming, respectively, from the nitro-
sation of R-, â-, and γ-amino acids, that is the most common
natural amino acids are the precursors of the most powerful
alkylating agents.
1
Identification of â-Butyrolactone. The H NMR spectrum
of the nitrosation products of â-aminobutyric acid revealed the
following signals: 1H NMR (200 MHz; CDCl3) δ (ppm): 4.70
(1H, m, H-4), 3.57 (1H, dd, J ) 16.5 Hz, J ) 6.0 Hz, H-3),
3.07 (1H, dd, J ) 16.5 Hz, J ) 3.6 Hz, H-3), 1.57 (3H, d, J )
6.0 Hz, CH3), which are in agreement with the values found in
the literature for â-butyrolactone.26,27
(26) Griesbeck, A.; Seebach, D. HelV. Chim. Acta 1987, 70, 1320.
(27) Seebach, D. Personal communication, 1999.
(28) Williams, D. H.; Fleming, I. Spectroscopy Methods in Organic
Chemistry, 5th ed.; McGraw-Hill: Berkshire, UK, 1997; pp 46, 156.
(29) Challis, B. C.; Carman, N.; Fernandes, M. H. R.; Glover, B. R.;
Latif, F.; Patel, P.; Sandhu, J. S.; Shuja, S. In Nitrosamines and Related
N-Nitroso Compounds. Chemistry and Biochemistry; Loeppky, R. N.,
Michejda C. J., Eds.; American Chemical Society: Washington, DC, 1994;
pp 74-92.
(25) Pretsch, E.; Clerc, T.; Seibl, J.; Simon, W. Tables of Spectral Data
for Structure Determination of Organic Compounds, 2nd ed.; Springer-
Verlag: Berlin, 1989.

7510 J. Am. Chem. Soc., Vol. 123, No. 31, 2001
Garc´ıa-Santos et al.
(iii) In view of the existence of a correlation between the
mutagenic index of nitrosation products and the alkylating
potential index,30 the results obtained here could be useful in
predicting the mutagenic effectiveness of the nitrosation products
of amino acids since NBP has nucleophilic characteristics similar
to those of DNA.
Acknowledgment. This work is supported in part by the
European Commission (Contract CVVF1-610-0). M.P.G.-S.
also thanks the Universidad de Salamanca for a Ph.D. Grant.
Thanks are also given to the valuable comments made by the
reviewers.
(30) Shephard, S. E; Lutz, W. K. Cancer SurVeys 1989, 8, 401.
JA010348+