Peculiar stability of amino acids and peptides from a radical perspective

Article abstract of DOI:10.1021/ja9027583

(Chemical Equation Presented) Photochemical reactions of free and N-acetyl α-amino acids with chlorine and deuterium labeled hydrogen peroxide have been used to determine both the relative rates of reaction of molecules of these classes and the relative reactivity of their different types of hydrogen toward abstraction by chlorine and oxygen centered radicals. The relative rates of reaction of these species range over more than 3 orders of magnitude; however, where data are available from more than one amino acid for a particular type of group at a specific position on the side chain, the values are remarkably similar. The predictive utility of these results has been demonstrated for the regioselective chlorination of tripeptides. More generally this analysis shows that the backbone and adjacent side chain positions of amino acids and peptides are peculiarly resistant to hydrogen atom transfer, and a similar pattern of reactivity has been noted from earlier studies of reactions of modified substrates catalyzed by isopenicillin-N-synthetase. Such resistance stands out in contrast to the common occurrence of free radical reactions of α-amino acids, peptides, and proteins and their importance in biology. Nevertheless, it provides a reason for the ability of amino acids and their derivatives to avoid degradation in Nature where they are constantly exposed to radicals, and it accounts, at least in part, for the anomalous ability of enzymes to catalyze free radical reactions without being broken down by the radical intermediates.

Full text of DOI:10.1021/ja9027583

Published on Web 07/24/2009
Peculiar Stability of Amino Acids and Peptides from a Radical Perspective
Zachary I. Watts and Christopher J. Easton*
ARC Centre of Excellence for Free Radical Chemistry and Biotechnology, Research School of Chemistry,
Australian National UniVersity, Canberra ACT 0200, Australia
Received April 6, 2009; E-mail: easton@rsc.anu.edu.au
Free radical reactions of R-amino acids and peptides are generally
thought of as ubiquitous. They are involved in many metabolic
processes1,2 and have been associated with a wide variety of
pathological conditions such as aging,3-5 inflammation,6 cancer,7
and neurodegenerative disorders including Alzheimer’s and
Parkinson’s diseases.8-11 Despite these observations, here we
show that the amino acid and peptide structural motifs are in
fact peculiarly resistant to radical reactions. We have used the
photochemical reactions of free and N-acetyl amino acids with
chlorine and deuterium labeled hydrogen peroxide to quantify
both the overall relative reactivity of molecules of these classes
and the relative reactivity of their different types of hydrogen
toward radical abstraction. The results show that the backbone
and adjacent side chain positions of amino acids and their
derivatives are extensively deactivated toward reaction.
of its propensity to undergo reactions with oxygen-centered
radicals,13,14 Leu is the most reactive amino acid, and it is more
than 1000 times more reactive than Asp. The krelH values also cover
wide ranges. Nevertheless, where data are available from more than
one amino acid for a particular type of group at a specific position
on the side chain, the values are remarkably similar (see Supporting
Information). For example, the krelH values of the γ-CH3 groups of
Abu, Val, Tle, and Ile differ by less than a factor of 2. Other trends
in the reactivity data are also readily apparent. At a given distance
from the amino acid backbone, such as the γ-position, CH is more
reactive than CH2, which is in turn more reactive than CH3,
H
consistent with the normal pattern. The krel data also show that
the reactivity of CH, CH2, or CH3 is less the closer it is to the
amino acid backbone. This accounts for the anomalies mentioned
above. Leu is 13 times more reactive than Val, because its CH and
CH3 groups are further out along the side chain, and the Ile δ-CH3
is 7 times more reactive than the γ-CH3 for the same reason. More
generally, the results show that the R- and adjacent side-chain
positions of amino acids are substantially deactivated toward
hydrogen atom transfer. The reactivity of an R-CH and an R-CH2
group is in each case reduced by more than a factor of 50 relative
to that of corresponding remote side chain and hydrocarbon groups.
The deactivation extends to the ꢀ- and γ-positions, where the
reduction in reactivity of CH3 is by factors of more than 70 and
10, respectively.
Our initial aim was to prepare side-chain halogenated derivatives
of proteinogenic amino acids, as precursors of hydroxy and dehydro
amino acids that are commonly found in physiologically active
peptide secondary metabolites.2 Accordingly, amino acids were
photolyzed in trifluoroacetic acid (TFA) saturated with chlorine12
but we were unable to rationalize the different rates of reaction of
various amino acids or the sites of reaction of individual species.
For example, Leu was found to be 13 times more reactive than
Val, yet the only structural difference between these two amino
acids is the extra CH2 group of Leu and no reaction was observed
at that part of the molecule. Further, the γ- and δ-CH3 groups of
Ile were found to differ in reactivity by a factor of 7. We therefore
redirected our efforts toward understanding this anomalous behavior.
Accordingly, the reactions of Gly and a series of other R-amino
acids with either aliphatic side chains or side-chain amino or
carboxyl groups (Figure 1) were investigated. The compounds were
photolyzed in chlorine-saturated TFA, and the reactions were
1
followed using H NMR spectroscopy. For any reaction mixture,
1
the 1D H NMR spectrum showed at least one baseline separated
resonance characteristic of each product and starting material (Table
SI1). The rates of reaction relative to that of Phe (krel) (Figure 2a)
were determined by monitoring the relative rates of consumption
of the compounds from mixtures through integration of key
resonances in NMR spectra. The reactions afforded monochlorinated
amino acid derivatives in high corrected yields at up to ∼50%
conversion and with negligible stereoselectivity in regard to the
formation of diastereomers. For Gly and the aliphatic amino acids,
information from NMR spectra on the distribution of products was
combined with the krel values to calculate the per hydrogen reactivity
(krelH) of the various types of CH, CH2, and CH3 groups (Table
1a). Selected nonproteinogenic amino acids were included in this
study in order to obtain reactivity data for groups of each of these
types at the various positions along the amino acid side chains.
H
The krel and krel values reflect the susceptibility of the amino
acids and their constituent groups to hydrogen abstraction by a
chlorine atom. The former show that, consistent with earlier reports
Figure 1. Amino acids used in this study.
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C O M M U N I C A T I O N S
Table 1. Relative Reactivity Per Hydrogen (krelH) of Various Amino
Acid CH, CH2, and CH3 Groupsa b
,
locationc
entry
group
R
ꢀ
γ
δ
ꢀ
ad
CH3
CH2
CH
CH3
CH2
CH
-
e0.03
0.05
0.54
e0.03
0.35
0.84
0.13
0.91
5.2
0.45
2.7
9.3
0.12
0.13
1.1
3.7
-
1.3
5.0
-
2.3
-
e0.05
e0.10
-
-
be
2.0
-
-
0.34
0.12
e0.20
e0.10
-
cf
CH3
CH3
e0.03
e0.02
0.31
0.17
dg
-
a Average reactivity for groups of the same type in various amino acids.
b See the Supporting Information for details of the procedures used to
calculate these values and for the range of reactivities of the groups with
the various amino acids (Table SI2). c Refers to the backbone (R),
adjacent side chain (ꢀ), and more remote side chain (γ, δ, ꢀ) positions.
d Amino acids in TFA saturated with chlorine, on photolysis with a
300-W broad-spectrum sunlamp. e Amino acids with acetylated R-amino
groups, in TFA saturated with chlorine, on photolysis with a 300-W
broad-spectrum sunlamp. f Amino acids in deuterium labeled water and
hydrogen peroxide, acidified with TFA, on photolysis at 254 nm in a
photochemical reactor. g Amino acids with acetylated R-amino groups,
in deuterium labeled water and hydrogen peroxide, acidified with TFA,
on photolysis at 254 nm in a photochemical reactor.
prevent peptide cleavage by the most logical conceptual pathways.
Under physiological conditions the amino groups of free amino
acids and N-terminal amino acid residues in peptides are protonated,
and those of non- and C-terminal amino acid residues in peptides
are acylated, as they are with the free and acetylated amino acids
studied here. The systems studied here are carboxylic acids, whereas
in biological systems free and peptidic amino acids are generally
either carboxylates or carboxamides. However, carboxylic acids,
carboxylates, and carboxamides have very similar reactivities in
these systems, and for example, the behavior of Asn and Gln is
directly analogous to that of Asp and Glu, respectively. Therefore,
the compounds used in this investigation are relevant models of
the natural amino acids and peptides. This claim is substantiated
by the results of the direct chlorination of several tripeptides. Taken
as representative of N-terminal and other amino acid residues, the
data in Table 1 (entries a and b, respectively) indicate that at least
48% of the reaction of (S)-Phe-(S)-Leu-(S)-Ala should occur at the
γ-position of the Leu residue and another 41% at the Leu δ-position.
In the event, the γ-chloro-Leu derivative was isolated in 44% yield,
after HPLC, and the 1NMR spectrum of the crude product mixture
showed a similar combined yield of the diastereomers of the
δ-chloro-Leu derivative. Even though (S)-Abu-(S)-Nva-(S)-Ala
comprises three CH3, three CH2, and three CH groups, the data in
Table 1 suggest that at least 50% and 36% of the reaction should
occur at γ-CH2 and δ-CH3 of the Nva residue, respectively. In that
Figure 2. Relative rates of reaction (krel) of (a) amino acids in TFA saturated
with chlorine on photolysis with a 300-W broad-spectrum sunlamp [krel(Phe)
) 1.0]; (b) the corresponding amino acids with acetylated R-amino groups
under the same conditions [krel(Phe) ) 1.0]; (c) amino acids in deuterium
labeled water and hydrogen peroxide, acidified with TFA, on photolysis at
254 nm in a photochemical reactor [krel(Tle) ) 1.0]; and (d) the corre-
sponding amino acids with acetylated R-amino groups under the same
conditions [krel(Tle) ) 1.0]. See the Supporting Information for full details
of the methods used.
Under the acidic conditions used in this investigation, the amino
groups of the amino acids are protonated, as illustrated in Figure
1; otherwise there is extensive literature that shows N-chlorination
and consequent processes occur with free amines.1 The correspond-
ing series of compounds with acetylated R-amino groups were also
studied using the same conditions and methods, and the derived
H
krel and krel values are shown in Figure 2b and Table 1b,
respectively. These data show the same trends as observed for the
protonated amino acids: the krel and krelH values cover wide ranges
with Leu being the most reactive of the proteinogenic amino acids;
1
case the H NMR spectrum of the crude product mixture showed
that the corresponding chlorides were produced cleanly and in a
5:4 ratio. They were isolated in corrected yields of 29% and 27%,
respectively, after HPLC.
H
similar krel values were measured for comparable groups of
different amino acids, and for example, those of the γ-CH3 groups
of Abu, Val, Tle, and Ile differ by less than a factor of 1.5. At a
given position on the amino acid side chain the order of reactivity
is CH > CH2 > CH3, and, a CH, CH2, or CH3 group closer to the
amino acid backbone is much less reactive.
The observation of this deactivation of CH, CH2, and CH3 groups
in proximity to the amino acid backbone raises the intriguing
possibility that one of the many factors that may contribute to the
existence of amino acids and peptides in biology is their peculiar
resistance to radical degradation and consequent ability to survive
in the natural environment where radicals are omnipresent. The
deactivation only needs to be effective at the R- and ꢀ-positions to
The deactivation of CH, CH2, and CH3 groups closer to the amino
acid backbone can be attributed to polar inductive and field effects,15
that is, repulsion of the incoming electronegative chlorine atom by
the electron withdrawing carboxyl, protonated amino, and acetamido
groups of the free and acetylated amino acids. Similar, very much
weaker effects are well-known for aliphatic carboxylic acids and
amines,15 but the magnifying effect of the combination of functional
groups is quite striking. The extent of the effect is sufficient to
override the normal thermodynamic preference for formation of
more stable radicals. The R-carbon-centered radicals of the acety-
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C O M M U N I C A T I O N S
lated amino acids are 30-80 kJ mol-1 more stable than the side-
chain aliphatic radicals.16
show that a contributing factor to this stability is possibly the
inherent resistance to radical reactions peculiar to the R-amino acid
and peptide structural frameworks. We do not contend that amino
acids and peptides do not participate in radical reactions, only that
their basic skeletons are deactivated toward radical degradation.
The magnitude of the effect is likely to depend on the electro-
philic nature of the incoming radical. Radical bromination of amino
acid derivatives typically occurs at the R-position,2,17 whereas there
is already good evidence that side chain reactions predominate with
oxygen-centered radicals more commonly encountered in biological
systems.13,14,18 To clarify trends for the hydroxyl radical, the free
amino acids shown in Figure 1 and the corresponding series of
compounds with acetylated R-amino groups were also photolyzed
in deuterium labeled water and hydrogen peroxide acidified with
TFA. The use of deuterium labeled reagents allowed for the
reactions to be followed using 1H NMR spectroscopy as described
for the chlorinations. The krel values were calculated as before
(Figure 2c and d) but relative to that of Tle instead of Phe. Analysis
of the primary products was complicated by secondary processes,
Acknowledgment. This work was carried out under the
Australian Research Council Centres of Excellence Program. We
thank S. Nicoll for analysis of the reactivity of Asn and Gln relative
to Asp and Glu, respectively.
Supporting Information Available: Procedures used to calculate
the data shown in Figure 2 and Table 1; spectroscopic data and yields
for chlorination products (Table SI1); 1H NMR spectra of chlorination
reaction mixtures; reactivity data for individual amino acids used to
calculate the average values shown in Table 1 (Table SI2); illustration
of reactions of isopenicillin-N-synthetase substrates (Scheme SI1). This
material is available free of charge via the Internet at http://pubs.acs.org.
H
so it was only feasible to determine krel values for the different
types of CH3 groups (Table 1, entries c and d). Nevertheless the
patterns of reactivity are similar to those observed for the chlorina-
tions. The most reactive amino acids have relatively long aliphatic
side chains, as reported previously,13,14,18 and groups closer to the
backbone of the amino acids are substantially deactivated. The latter
point can be derived from the krel data for CH and CH2 groups,
while for CH3 groups it is shown directly by the krelH values. Thus,
the tendency of amino acids and peptides to resist hydrogen transfer
radical reactions is seen in processes involving oxygen-centered
radicals as well as chlorine.
Many biological reactions of peptides are enzyme-catalyzed, in
which cases the enzymes are likely to control the regioselectivity.
Even so, in the context of the current work, it is interesting to note
that Baldwin’s studies of the reactions of modified substrates of
isopenicillin-N-synthetase also show a trend toward radical func-
tionalization more remote from the peptide backbone.19 As il-
lustrated in the Supporting Information, whereas the natural
substrate, δ-((S)-R-aminoadipoyl)-(R)-Cys-(R)-Val, undergoes re-
giospecific reaction at the ꢀ-position of the C-terminal Val residue,
the analogous Ile and Abu derivatives also react at the γ-position.
With the Nva analogue, the change in regioselectivity is complete
within the limits of detection, which the investigators conclude
represents at least a 10-fold preference for reaction of the γ-CH2
over the ꢀ-CH2. That is, the much more reactive CH2 of the Nva
derivative is the more remote from the peptide backbone and not
the closer CH2 corresponding to the preferred site of reaction of
the natural substrate.
References
(1) Davies, M. J.; Dean, R. T. Radical-Mediated Protein Oxidation: From
Chemistry to Medicine; Oxford University Press: Oxford, 1997.
(2) Easton, C. J. Chem. ReV. 1997, 97, 53–82.
(3) Stadtman, E. R. Science 1992, 257, 1220–1224.
(4) Beckman, K. B.; Ames, B. N. Physiol. ReV. 1998, 78, 547–581.
(5) Kirkwood, T. B. L. Nature 2008, 451, 644–647.
(6) Eiserich, J. P.; Hristova, M.; Cross, C. E.; Jones, A. D.; Freeman, B. A.;
Halliwell, B.; van der Vliet, A. Nature 1998, 391, 393–397.
(7) Hussain, S. P.; Hofseth, L. J.; Harris, C. C. Nat. ReV. Cancer 2003, 3,
276–285.
(8) Hensley, K.; Carney, J. M.; Mattson, M. P.; Aksenova, M.; Harris, M.;
Wu, J. F.; Floyd, R. A.; Butterfield, D. A. Proc. Natl. Acad. Sci. U.S.A.
1994, 91, 3270–3274.
(9) Dunnett, S. B.; Bjo¨rklund, A. Nature 1999, 399, A32–A39.
(10) Mattson, M. P. Nature 2004, 430, 631–639.
(11) Mattson, M. P.; Magnus, T. Nat. ReV. Neurosci. 2006, 7, 278–294.
(12) Kollonitsch, J.; Scott, A. N.; Doldouras, G. A. J. Am. Chem. Soc. 1966,
88, 3624–3626.
(13) Goshe, M. B.; Chen, Y. H.; Anderson, V. E. Biochemistry 2000, 39, 1761–
1770.
(14) Nukuna, B. N.; Goshe, M. B.; Anderson, V. E. J. Am. Chem. Soc. 2001,
123, 1208–1214.
(15) Russell, G. A. In Free Radicals; Kochi, J. K., Ed.; Wiley: New York, 1973;
Vol. 1, Chapter 7, pp 275-331.
(16) Croft, A. K.; Easton, C. J.; Radom, L. J. Am. Chem. Soc. 2003, 125, 4119–
4124.
(17) (a) Burgess, V. A.; Easton, C. J.; Hay, M. P. J. Am. Chem. Soc. 1989, 111,
1047–1052. (b) Croft, A. K.; Easton, C. J.; Kociuba, K.; Radom, L.
Tetrahedron: Asymmetry 2003, 14, 2919–2926.
(18) (a) Buxton, G. V.; Greenstock, C. L.; Helman, P. W.; Ross, A. B. J. Phys.
Chem. Ref. Data 1988, 17, 513–886. (b) Takamoto, K.; Chance, M. R.
Annu. ReV. Biophys. Biomol. Struct. 2006, 35, 251–276.
(19) (a) Baldwin, J. E.; Abraham, E. P.; Adlington, R. M.; Chakravarti, B.;
Derome, A. E.; Murphy, J. A.; Field, L. D.; Green, N. B.; Ting, H.-H.;
Usher, J. J. J. Chem. Soc., Chem. Commun. 1983, 1317–1319. (b) Baldwin,
J. E.; Adlington, R. M.; Turner, N. J.; Domayne-Hayman, B. P.; Ting,
H.-H.; Derome, A. E.; Murphy, J. A. J. Chem. Soc., Chem. Commun. 1984,
1167–1170.
Free radical reactions involving peptides and proteins are of
fundamental importance to life.20,21 Indeed, life depends on enzyme-
catalyzed activation of oxygen via free radical intermediates. It has
been difficult to understand why the associated enzymes and other
peptides and proteins are not broken down when surrounded by
and processing reactive radical intermediates,22 but our results now
(20) Stubbe, J.; van der Donk, W. A. Chem. ReV. 1998, 98, 705–762.
(21) Frey, P. A.; Hegeman, A. D.; Reed, G. H. Chem. ReV. 2006, 106, 3302–
3316.
(22) Klinman, J. P. Acc. Chem. Res. 2007, 40, 325–333.
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