Oxidative damage of proline residues by nitrate radicals (NO3): A kinetic and product study

Article abstract of DOI:10.1039/d0ob01337d

Tertiary amides, such as in N-acylated proline or N-methyl glycine residues, react rapidly with nitrate radicals (NO3) with absolute rate coefficients in the range of 4-7 × 108 M-1 s-1 in acetonitrile. The major pathway proceeds through oxidative electron transfer (ET) at nitrogen, whereas hydrogen abstraction is only a minor contributor under these conditions. However, steric hindrance at the amide, for example by alkyl side chains at the α-carbon, lowers the rate coefficient by up to 75%, indicating that NO3-induced oxidation of amide bonds proceeds through initial formation of a charge transfer complex. Furthermore, the rate of oxidative damage of proline and N-methyl glycine is significantly influenced by its position in a peptide. Thus, neighbouring peptide bonds, particularly in the N-direction, reduce the electron density at the tertiary amide, which slows down the rate of ET by up to one order of magnitude. The results from these model studies suggest that the susceptibility of proline residues in peptides to radical-induced oxidative damage should be considerably reduced, compared with the single amino acid.

Full text of DOI:10.1039/d0ob01337d

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DOI: 10.1039/D0OB01337D
ARTICLE
•
Oxidative damage of proline residues by nitrate radicals (NO3 ): A
kinetic and product study
Joses G. Nathanael,a Jonathan M. White,a Annika Richter,a,b Madison R. Nuskea and Uta Wille*a
Received 00th January 20xx,
Accepted 00th January 20xx
•
Tertiary amides, such as in N‐acylated proline or N‐methyl glycine residues, react rapidly with nitrate radicals (NO3 ) with
DOI: 10.1039/x0xx00000x
absolute rate coefficients in the range of 4 ‐ 7  108 M−1 s−1 in acetonitrile. The major pathway proceeds through oxidative
electron transfer (ET) at nitrogen, whereas hydrogen abstraction is only a minor contributor under these conditions.
However, steric hindrance at the amide, for example by alkyl side chains at the ‐carbon, lowers the rate coefficient by up
•
to 75%, indicating that NO3 ‐induced oxidation of amide bonds proceeds through initial formation of a charge transfer
complex. Furthermore, the rate of oxidative damage of proline and N‐methyl glycine is significantly influenced by its position
in a peptide. Thus, neighbouring peptide bonds, particularly in the N‐direction, reduce the electron density at the tertiary
amide, which slows down the rate of ET by up to one order of magnitude. The results from these model studies suggest that
the susceptibility of proline residues in peptides to radical‐induced oxidative damage should be considerably reduced,
compared to the single amino acid.
peptides, proton‐coupled ET (PCET) at the secondary amide
Introduction
Air pollution has become the largest environmental risk for
society, being responsible for the premature death of about
seven million people worldwide every year.1 In particular, the
moiety was identified as an important reaction pathway for
NO3 , in addition to HAT from activated C–H bonds, for example
from the α‐carbon.10,11
Of the proteinogenic amino acids proline stands out due to
its cyclic structure and secondary amine motif, which may lead
to different reactivity towards oxidative damage, compared
with the linear aliphatic amino acids (Scheme 1).
•
•
noxious gaseous pollutants nitrogen dioxide (NO2 ) and ozone
(O3) are recognised as a serious problem not only for human
health2,3 but also for plants by causing damage to cuticles and
photosynthetic pigments, which reduces plant growth and
productivity.4,5 Interestingly, studies on human health effects
using rat models showed that exposure to combined NO2• and
O3 has significant synergistic effects compared to the isolated
pollutants, such as increased lipid peroxidation and protein
nitration.6 These findings suggest that highly reactive nitrate
radicals (NO3 ) could be formed in situ, according to eqn. 1:7
•
Scheme 1. Suggested radical reaction pathways in proline.
NO2• + O3

NO3• + O2 (eqn. 1)
For example, recent computational studies revealed that
hydroxyl radicals (HO•) could react with proline through HAT
NO3• is an O‐centred, electrophilic and strongly oxidising radical
[Eo (NO3 /NO3 ) = 2.3–2.5 V vs NHE],8 which reacts with organic
molecules through electron transfer (ET), addition to π systems
or by hydrogen atom transfer (HAT).7 We have recently shown
through kinetic and product studies that phenylalanine, which
•
−
from the N–H group to form an N‐centred radical
lead to spontaneous decarboxylation of proline, or through HAT
from the δ‐CH2 moiety to give radical , which is predicted to be
1, which could
2
energetically less favourable than HAT from N–H.12 However,
when no N‐H bond is present, HAT occurs from the δ‐CH2
moiety, as identified in kinetic studies of the reaction of N‐Boc
protected proline with cumyloxyl radicals (PhCMe2O•, CumO•)
•
does not react with NO2• or O3 in isolation, is damaged by NO3
through oxidative ET at the aromatic ring.9,10 In reactions with
the less reactive aliphatic acyclic amino acids and short
in acetonitrile.13 On the other hand, HAT from the
proline to form the capto‐datively stabilised
radical is only competitive pathway in some
conformations.14 Formation of a radical cation of type
through
oxidative ET at N has been suggested as the major pathway in
reactions of amino acids with alkylated N‐termini, for example

‐position in

‐amino acid
a. School of Chemistry, Bio21 Institute, The University of Melbourne, 30 Flemington
Road, Parkville, Victoria 3010, Australia. Email: uwille@unimelb.edu.au
b. Institute of Chemistry, Humboldt‐University Berlin, Brook‐Taylor‐Str. 2, 12489
Berlin, Germany.
Electronic Supplementary Information (ESI) available: Synthetic procedures and
spectroscopic details for all substrates, details of kinetic studies, X‐ray data,
Gaussian archive entries. See DOI: 10.1039/x0xx00000x
3
a
4
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N‐methyl glycine, which can be regarded as an open‐chain
model for proline.15
Results and discussion
View Article Online
Synthesis of materials and experimental seDt OupI: 1fo0.r10k3in9e/Dti0cOsBtu0d13ie37sD
Free proline is present in various organisms, where it acts as
a key molecule in cell signalling, stress protection and energy
production.16 In particular, it has been found that free proline
accumulates in plant tissues during abiotic stress, where it could
act as a scavenger of HO• to reduce oxidative damage caused by
this radical.17,18,19 However, proline’s antioxidant capacity has
recently been questioned because of its inability to scavenge
All amino acids, di‐ and tripeptides studied in this work were
synthesised as enantiomerically pure materials (where chiral
centres are present). The purity of the compounds was
confirmed by analytical HPLC. To increase the solubility in the
solvent acetonitrile, all amino acids and peptides had the C‐
termini protected as methyl esters and the N‐termini as
acetamides (Ac) or phthalimides (Phth), respectively. Synthetic
details and spectroscopic data for all compounds are given in
the Electronic Supporting Information (ESI).
The nanosecond laser flash photolysis experiments were
carried out at 298 ± 1 K on an Edinburgh Instrument LP920
spectrometer. The 3rd harmonic of a Quantel Brilliant B Nd:YAG
laser was used to generate NO3• in the presence of the amino
acid or short peptide through photoinduced ET from cerium(IV)
−
singlet oxygen (1O2), superoxide (O2 ), peroxynitrite (ONOO−),
• 20
nitric oxide (NO•) and NO2 .
Irrespective of these
controversies, it has been demonstrated that proline supplied
at low concentrations is beneficial for the growth of plants
exposed to salinity stress and decreases peroxidative damage
to the lipid membranes,17 suggesting that such application may
significantly increase plant growth and crop yield under
environmental stress.21
ammonium nitrate (CAN) at

= 355 nm, according to eqn. 2:7
Proline is also an integral part of proteins, with the demand
for proline in protein synthesis being the highest among all
other amino acids (on a per‐gram basis).22 Given the ongoing
discussion over the role of proline during salinity and drought
stress, for example as essential protein building block,
osmotolerant or antioxidant,23 a fundamental understanding of
radical‐induced oxidative damage in proline and proline
residues in peptides is essential, in particular whether and how
the environment in the peptide influences the reactivity of this
(NH4)2Ce(NO3)6 + h  NO3• + (NH4)2Ce(NO3)5
(eqn. 2)
•
Kinetic data for the reaction of NO3 with all substrates were
obtained under pseudo‐first order conditions by measuring the
decay of the NO3• signal at
λ
= 630 nm as a function of excess
•
amino acid, di‐ and tripeptide concentration, according to: NO3
+ substrate

products, as described previously.9e,10,11 Details
•
of the kinetic measurements are provided in the ESI.
amino acid. In previous work, we have demonstrated that NO3 ,
apart from being an important environmental oxidant, is also an
excellent ‘tool’ to investigate irreversible radical‐induced
oxidative damage in biomolecules, since back‐electron transfer
cannot occur, which is a typical complication in studies where
the oxidant is produced through photo‐induced ET in donor‐
acceptor pairs.
Reaction of NO3• with tertiary amides
The obtained absolute second‐order rate coefficients k for the
reaction of NO3• with N‐acetylated amino acids and dipeptides
containing tertiary amide moieties, i.e., proline and its open‐
chain model N‐methyl glycine are in the range 108 – 109 M−1 s−1
(Table 1). These data are two‐three orders of magnitudes higher
than those for aliphatic amino acids, di‐ and tripeptides with
secondary amide motifs,11 suggesting that the reaction should
occur through ET at the tertiary amide group. The experimental
confirmation of ET as the predominant mechanism will be
outlined in the next section.
Here we present the results of our kinetic and product
studies of the reaction of NO3• with proline and proline‐mimics
in short peptides. Through a ‘bottom‐up’ approach the
fundamental reactivity between the biomolecule and the
oxidant NO3• is studied without any interfering interactions with
the environment (such as biological fluids or salts or even a
longer peptide chain itself), which could significantly complicate
data analysis and interpretation. Previous product and kinetic
studies have demonstrated that acetonitrile is a highly suitable
solvent for such purposes due to its low reactivity with both
NO3• and radical intermediates formed in these reactions.9,10,11
While data obtained from such model studies may not
necessarily be transferable to biological conditions in their
entirety,‡ having first a detailed understanding of the
fundamental reactivity in model systems under exclusion of
solvent interactions (the aim of this work) is crucial to minimise
misinterpretation of subsequent experiments performed in
more complex environments, such as in aqueous systems.‡‡
This work reveals that the rate of proline oxidation is
influenced by electron‐withdrawing neighbouring amide bonds
in the peptide backbone. These inductive effects should
considerably reduce the susceptibility of proline residues in
peptides to radical‐induced oxidative damage compared with
the single amino acid.
The rate coefficient for the reaction of diproline (Ac‐Pro‐Pro‐
OMe, entry 2) of 16.7

108 M−1 s−1 is about 2.5 times higher
than for proline itself (Ac‐Pro‐OMe, entry 1), indicating that
consecutive proline residues are particularly susceptible to
oxidative damage. The reactivity of proline‐phenylalanine (Ac‐
Pro‐Phe‐OMe, entry 3) is with 7.2

108 M−1 s−1 only marginally
higher than that of proline itself, suggesting that the reaction
should take place at the proline residue only, which is supported
by the much lower rate coefficient of 1.1

107 M−1 s−1 for the
•
reaction of NO3 with phenylalanine (in Ac‐Phe‐OMe)
determined previously.10
•
Surprisingly, the rate coefficient for the reaction of NO3
with the dipeptide possessing the inverted sequence, i.e., Ac‐
Phe‐Pro‐OMe, is only 1.7

108 M−1 s−1 (entry 4). A similar
outcome was obtained in glycine‐proline Ac‐Gly‐Pro‐OMe
(entry 5) and leucine‐proline Ac‐Leu‐Pro‐OMe (entry 6). These
findings indicate that amino acid residues in the N‐direction
could lower the electron density at the tertiary amide moiety in
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proline through inductive effects, thereby reducing the compared with their proline counterparts, a drop of the rate
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susceptibility of proline to radical‐induced oxidative damage in coefficient by up to one order of magnituDdOeI:w10a.s10fo39u/nDd0OwBh0e13n37ND‐
peptides. This suggestion is supported by the rate coefficient for methyl glycine is the C‐terminal residue in the dipeptide (entries
•
the reaction of NO3 with the dipeptide

‐alanine‐proline (Ac‐ 11‐14 vs 10). Overall, the data indicate that the N‐terminal

Ala‐Pro‐OMe, entry 7), which is nearly as high as for proline secondary amide pulls the electron density away from the C‐
itself and could be rationalised by the additional methylene terminal tertiary amide, slowing down the oxidation rate at this
group in ‐alanine that dampens the electron‐withdrawing site.
effect of the N‐terminal amino acid. As will be shown in the next
section, HAT from the activated ‐CH2 moiety should not be Mechanism: HAT vs ET
competitive under these conditions. Irrespective of this, the


Kinetic studies were performed to confirm the proposed ET
mechanism as the primary pathway in the reaction of NO3• with
tertiary amides. The results are compiled in Table 2.
kinetic data for Ac‐Ala‐Pro‐OMe suggest that β‐peptides,
which are stable to proteolytic degradation,24 should be
significantly more prone to radical‐induced oxidative damage
Table 2. Absolute second‐order rate coefficients k to determine the mechanism of the
reaction of NO3• with tertiary amide moieties in N‐acylated amino acids.a,b
than
‐peptides, which could be one of the reasons why they
are not common in nature.
•
Table 1. Absolute second‐order rate coefficients k for the reaction of NO3 with N‐
acetylated amino acids and dipeptides containing proline or N‐methyl glycine residues.a,b
Entry
1
2
3
4
5
6
7
8
Substrate
Ac‐Gly‐OMe
Ac‐Ala‐OMe
k /  108 M‐1 s‐1
0.03c
0.3
1.0
3.2
2.0
1.7
3.4
2.5
1.4
1.2
Ac‐Lys(NHAc)‐OMe
Ac‐(Me(d3))Gly‐OMe(d3)
Me‐Glp‐OMe
Me(d3)‐Glp‐OMe(d3)
Ac‐(Me)Ala‐OMe
Ac‐(Me)Val‐OMe
Ac‐(Me)Aib‐OMe
Ac‐(tBu)Gly‐OMe
Entry
Substrate
k /  108 M‐1 s‐1
a) Proline and proline‐containing dipeptides
1
2
3
4
5
6
7
Ac‐Pro‐OMe
6.8
16.7
7.2
1.7
2.2
1.7
5.7
9
10
Ac‐Pro‐Pro‐OMe
Ac‐Pro‐Phe‐OMe
Ac‐Phe‐Pro‐OMe
Ac‐Gly‐Pro‐OMe
Ac‐Leu‐Pro‐OMe
Ac‐Ala‐Pro‐OMe
aIn acetonitrile, at 298  1 K. bExperimental error 10%. cFrom ref. 11.
•
We recently found that NO3 reacts with glycine through HAT
from the
with each pathway contributing 1

‐carbon and PCET from the secondary amide moiety,
106 M‐1 s‐1 to the overall rate

b) N‐Methyl glycine and N‐methyl glycine‐containing dipeptides
coefficient (entry 1), whereas HAT from the Me groups at both
8
9
Ac‐(Me)Gly‐OMe
Ac‐(Me)Gly‐(Me)Gly‐OMe
Ac‐(Me)Gly‐Phe‐OMe
Ac‐Phe‐(Me)Gly‐OMe
Ac‐Gly‐(Me)Gly‐OMe
Ac‐Leu‐(Me)Gly‐OMe
Ac‐Val‐(Me)Gly‐OMe
4.1
10.6
6.0
0.8
0.9
0.6
0.5
N‐ and C‐terminus in Ac‐Gly‐OMe is not competitive.10,11 The
•
10
11
12
13
14
comparatively low rate coefficient for NO3 ‐induced HAT from
the
Easton, Radom and coworkers, showing that deactivating polar
effects in the early, reactant‐like transition state of the ‐HAT
‐carbon in ‐amino acids confirms previous work by

disfavour this pathway for highly reactive electrophilic
radicals.25,26 However, the rate coefficient for the reaction of
aIn acetonitrile, at 298  1 K. bExperimental error 5%.
NO3• with Ac‐

Ala‐OMe is one order of magnitude higher than
The rate coefficients obtained for N‐methyl glycine (Ac‐
(Me)Gly‐OMe, entry 8) and N‐methyl glycine‐containing
dipeptides listed in entries 9‐14 show a similar trend. While the
reactivity of these substrates towards NO3• is up to 60% lower
for glycine (entry 2), suggesting that the reaction occurs
primarily via HAT from the activated ‐CH2 moiety.
Because the reaction of proline with HO• and CumO• occurs
through HAT from the δ‐CH2 group (see above),12b,13 kinetic
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•
studies were performed to explore whether NO3 reacts acids decreased with increasing steric demand at tVhieewAr‐tcicalerObnolinne,
similarly with Ac‐Pro‐OMe or with Ac‐(Me)Gly‐OMe, in the dropping from 4.1 x 108 for the unhindDeOreI:d10A.1c0‐3(9M/De0)GOBly0‐1O33M7De
latter case through HAT from the N‐Me group. Firstly, we (Table 1, entry 8), to 3.4 x 108 for alanine (Ac‐(Me)Ala‐OMe,
•
investigated the NO3 ‐induced HAT from an Namide‐CH2 moiety entry 7), 2.5 x 108 for valine (Ac‐(Me)Val‐OMe, entry 8) and to
using the side chain in lysine as a model system for the δ‐CH2 1.4 x 108 M‐1 s‐1 for the most sterically hindered 2‐
group in Ac‐Pro‐OMe. Protection as a secondary amide (i.e., Ac‐ aminoisobutyric acid (Ac‐(Me)Aib‐OMe, entry 9). A similar
Lys(NHAc)‐OMe) was performed to both lower its susceptibility outcome was obtained for Ac‐(tBu)Gly‐OMe (entry 10), where
•
to ET by NO3 and to mimic N‐acetylation in Ac‐Pro‐OMe. The the bulky t‐butyl substituent at the amide led to a drop of the
rate coefficient of 1

108 M‐1 s‐1 obtained for the reaction of rate coefficient to only 25% of that of Ac‐(Me)Gly‐OMe. Because
•
NO3• with this amino acid (entry 3) clearly suggests a fast HAT the t‐butyl group is inert towards NO3 ‐induced HAT,11 it would
process at the Namide‐CH2 motif remote to the electron‐ be expected that its larger inductive effect, compared with that
withdrawing carboxyl group. Assuming that the HAT reaction of a methyl group, should facilitate amide oxidation. The
•
from both secondary and tertiary Namide‐CH2 moieties are opposite outcome clearly shows that the rate of NO3 ‐induced
approximately similar, the nearly seven times higher reactivity oxidation of tertiary amides is influenced by their accessibility
•
of Ac‐Pro‐OMe (Table 1, entry 1) suggests that HAT is only a for approach by NO3 , confirming earlier findings for the PCET
minor contributor to the overall reaction. Therefore, the major reaction with secondary amides, which proceeds through initial
pathway should procced via NO3 ‐induced ET at the tertiary formation of a CT complex.11
•
amide moiety, for which an upper limit of ca. 5.8
can be tentatively given.

108 M‐1 s‐1
ET at tertiary amides in peptides – the role of inductive effects
Next, HAT from the tertiary Namide‐CH3 group in Ac‐(Me)Gly‐
OMe was examined through deuterium kinetic isotope studies
for the pairs Ac‐(Me)Gly‐OMe/Ac‐(Me(d3))Gly‐OMe(d3) (Table
1, entry 8 vs Table 2, entry 4), and of N‐methylpyroglutamic
methylester, i.e., Me‐Glp‐OMe/Me(d3)‐Glp‐OMe(d3) (Table 2,
entry 5 vs. 6). A value of kH/kD = 1.2 was obtained for both
substrate pairs, which indicates that HAT contributes only to a
minor extent to the overall reaction.‡‡‡ From the rate
differences between the deuterated and non‐deuterated
To further explore the role of the N‐terminal amino acid on the
ET rate at a tertiary amide, kinetic studies were performed for
the reaction of NO3• with a series of model di‐ and tripeptides
containing a C‐terminal N‐methyl glycine. The N‐terminus was
protected as an electron‐withdrawing phthalimide to exclude
ET at this site, to reduce the susceptibility of the adjacent ‐C–
H bonds to HAT,27 and to provide a strong electron‐pull on the
adjacent bonds. In the dipeptide series, N‐methyl glycine was
separated from the N‐phthalimide terminus through amino
•
substrates, the rate coefficient for the NO3 ‐induced HAT from
acids with 1‐5 methylene units, i.e., Gly (n=1),
aminobutyric acid (Gaba, n=3), ‐aminovaleric acid (Ava, n=4)
and ‐aminocaproic acid (Ahx, n=5). Likewise, in the tripeptide
series the N‐terminal phthaloyl glycine and the C‐terminal N‐
methyl glycine were separated by Gly, ‐Ala, Gaba, Ava and Ahx,
‐Ala (n=2), ‐
N
amide‐CH3 can be estimated as (6

3) x 107 M‐1 s‐1, leading to a

rate coefficient of about 3.5

108 M‐1 s‐1 for the ET reaction at

the tertiary amide moiety in Ac‐(Me)Gly‐OMe.
The three times higher reactivity of Ac‐Pro‐OMe compared
with Me‐Glp‐OMe (Table 1, entry 1 vs Table 2, entry 5) is
noteworthy, since both are cyclic amino acids with similar
functional groups. A possible rationale is provided in Scheme 2,

respectively. The obtained rate coefficients are given in Table 3.
Comparison of the data for the reaction of NO3• with Phth‐
Gly‐(Me)Gly‐OMe (entry 1) with those for Ac‐(Me)Gly‐OMe
(Table 1, entry 8) shows a drop in reactivity by a factor of 20,
clearly revealing the deactivating effect of the N‐terminal
phthaloyl glycine on the tertiary amide in N‐methyl glycine.§
Increasing the distance between the tertiary amide and
phthalimide moieties by just one additional CH2 group leads to
a significant increase of the rate coefficient by one order of
which shows that the radical cation I formed through ET in Ac‐
Pro‐OMe is stabilised through resonance involving the exocyclic
acetyl group (II). On the other hand, resonance stabilisation of
the radical cation of Me‐Glp‐OMe (III) via IV requires
planarisation of the amide moiety within the 5‐membered ring,
which increases ring strain and should therefore decrease the
rate of oxidation.
magnitude to 2.0
these two groups further increases the reactivity but to a much
lesser extent, reaching 5.6
108 M‐1 s‐1 for the longest linker in

108 M‐1 s‐1 (entry 2). Further separation of

Phth‐Ahx‐(Me)Gly‐OMe (entry 5).
A similar behaviour was found for the tripeptide series
shown in entries 6‐10, where the biggest ‘jump’ in reactivity by
a factor of about four occurred in going from Gly to ‐Ala as the
separating amino acid. It should be noted that the three times
•
higher reactivity of Phth‐Gly‐Gly‐(Me)Gly‐OMe towards NO3
(entry 6) compared with Phth‐Gly‐(Me)Gly‐OMe (entry 1) is due
to the extra glycine residue that separates the electron‐
withdrawing phthaloyl group from the tertiary amide in the
former. Interestingly, this difference in reactivity is already
Scheme 2. Resonance structures of the amide radical cations of Ac‐Pro‐OMe (I and II)
and Me‐Glp‐OMe (III and IV).
Interestingly, the data in Table 2 also show that the rate
•
coefficients for the NO3 ‐induced ET of N‐methylated amino
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eradicated by only one additional CH2 group in the carbon linker in going from Gly to
(entry 2 vs 7).

‐Ala as the spacer amino acid (see Table
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DOI: 10.1039/D0OB01337D
3), the rate coefficient increased with increments of only ca (3
1) 
106 M‐1 s‐1 for each additional CH2 moiety that separates

•
Table 3. Absolute second‐order rate coefficients k for the reaction of NO3 with N‐
phthaloylated di‐ and tripeptides possessing N‐methyl glycine residues.a,b
the amide from the electron‐withdrawing C‐terminus.
Although it is not possible to exclude that HAT from the
N
amide‐CH2 group might also occur to some extent, this data
nevertheless shows that reactions at the amide moiety itself are
slowed down by negative inductive effects caused by the C‐
terminus, however to a much lesser extent than those caused
by electron‐withdrawing groups in the N‐direction.
Product studies
We were interested to explore whether the position‐dependent
oxidation rate of proline is also reflected by different reaction
outcomes and analysed the products formed in the reaction of
NO3• with the isomeric dipeptides Ac‐Pro‐Phe‐OMe and Ac‐Phe‐
Pro‐OMe. For these experiments NO3• was generated in situ in
acetonitrile through the reaction of NO2• with O3 (see eqn. 1) in
the presence of the substrate, using experimental conditions
developed previously by us.§§9c,d Details are given in the ESI.
Entry
Substrate
k /  108 M‐1 s‐1
1c
2
Phth‐Gly‐(Me)Gly‐OMe
Phth‐Ala‐(Me)Gly‐OMe
Phth‐Gaba‐(Me)Gly‐OMe
Phth‐Ava‐(Me)Gly‐OMe
Phth‐Ahx‐(Me)Gly‐OMe
Phth‐Gly‐Gly‐(Me)Gly‐OMe
Phth‐Gly‐Ala‐(Me)Gly‐OMe
Phth‐Gly‐Gaba‐(Me)Gly‐OMe
Phth‐Gly‐Ava‐(Me)Gly‐OMe
Phth‐Gly‐Ahx‐(Me)Gly‐OMe
0.2
2.0
3.7
4.8
5.6
0.6
2.2
3.6
4.6
5.6
3
4
5
6
7
8
9
10c
aIn acetonitrile, at 298  1 K. bExperimental error 5%, unless stated otherwise.
cExperimental error 10%.
To explore the role of the C‐direction amino acid on the rate of
•
amide oxidation by NO3 , we used dipeptides with a central
secondary amide moiety as the site for PCET and an N‐terminal
glycine, which was deactivated for radical attack through N‐
phthalimide protection. The data in Table 4 reveal a gradual
increase of the rate coefficient with increasing distance
between the C‐terminal ester and the amide motif.
•
Table 4. Absolute second‐order rate coefficients k for the reaction of NO3 with N‐
phthaloylated dipeptides possessing secondary amide moieties.a,b
Entry
Substrate
k /  106 M‐1 s‐1
1
2
3
4
5
Phth‐Gly‐Gly‐OMe
Phth‐Gly‐Ala‐OMe
Phth‐Gly‐Gaba‐OMe
Phth‐Gly‐Ava‐OMe
Phth‐Gly‐Ahx‐OMe
1.5
3.7
7.8
10.3
13.1
Scheme 3. Reaction of Ac‐Pro‐Phe‐OMe and Ac‐Phe‐Pro‐OMe with NO3• generated by
reaction of NO2• with O3. Free energies in kJ mol‐1, calculated with M062X/6‐31+G* in
acetonitrile.
aIn acetonitrile, at 298  1 K. bExperimental error 10%.
However, compared with an electron‐withdrawing N‐terminus,
where the rate coefficient increased by one order of magnitude
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The reaction of Ac‐Pro‐Phe‐OMe led to formation of the di‐ is flanked by aliphatic amino acids and such
Journal Name

‐complexes are not
View Article Online
DOI: 10.1039/D0OB01337D
nitro derivative NO2‐Pro‐Phe(NO2)‐OMe in which the N‐acetyl formed, deactivation of the tertiary amide by neighbouring N‐
group was replaced by NO2 and the Phe residue converted into terminal amide bonds is the dominant factor that lowers the
4‐nitro phenylalanine, as confirmed by the X‐ray structure susceptibility of Pro residues to oxidation.
•
(Scheme 3). When a lower amount of NO2 was used, only
mononitration of Ac‐Pro‐Phe‐OMe was observed. Analysis by
ESI‐HRMS indicated that the major product was the N‐nitro
Conclusions
proline dipeptide NO2‐Pro‐Phe‐OMe (calcd. for [C15H19N3O5]+:
322.1398 [M + H]+, found 322.1395) in which the Phe residue
remained unchanged (not shown). The result is consistent with
the kinetic studies where the Pro residue is found to be more
reactive than Phe in acetonitrile. Control experiments showed
that no reaction occurred with Ac‐Pro‐Phe‐OMe in the absence
of O3 (see ESI).
This model study revealed that NO3• reacts in acetonitrile with
tertiary amides, such as proline residues in peptides,
predominantly by oxidative ET at the nitrogen, whereas HAT
from the

‐CH2 group is only a minor pathway. This mechanism
•
highlights the strong oxidising capacity of NO3 and contrasts
that of other O‐centred radicals, such as HO•, which react with
proline through HAT. The rate of NO3 ‐induced ET is
•
As mentioned in the Introduction, reaction at the aromatic
considerably influenced by steric hindrance at the tertiary
amide, where bulky substituents, for example a t‐butyl group on
•
ring in Phe is initiated through ET by NO3 , which under these
conditions is followed by recombination of the arene radical
the amide nitrogen or alkyl side chains at the
‐carbon in a
cation with residual NO2 and deprotonation.§§§9,10 A similar
•
peptide, diminish the oxidation rate by up to 75%. These results
•
process likely occurs also at the Pro residue in Ac‐Pro‐Phe‐OMe,
support earlier findings that NO3 ‐induced oxidation of amide
•
where oxidation of the tertiary amide by NO3 leads to the
bonds in peptides proceeds through initial formation of a CT
complex,11 which becomes more difficult when steric bulk at the
radical cation
intermediate
5
, which is trapped by NO2• to form the cationic
•
6. Subsequent heterolytic cleavage of the N‐acetyl
amide hinders approach by NO3 .
bond leads to the N‐nitro proline residue
7. This suggestion was
N‐Acylated proline is more than two orders of magnitude
•
supported by density functional theory (DFT) studies (see
Experimental section), which showed a modest barrier for the
more reactive towards NO3 than linear N‐acylated aliphatic
amino acids, highlighting the role of the tertiary amide moiety
that results from its cyclic structure. While removal of the N‐
protecting group would be expected to increase the electron
density at nitrogen, thereby rendering free or N‐terminal
loss of Ac+ in
6
of about 7 kJ mol‐1 and a considerable
exothermicity of this step by ca. 36 kJ mol‐1. For comparison,
+
heterolytic loss of NO2 in
6, which would restore the
•
undamaged Ac‐Pro moiety, is associated with a barrier of 62.6
kJ mol‐1 and is endothermic by 9.9 kJ mol‐1 (not shown).
proline in peptides even more susceptible to oxidation by NO3 ,
further experiments are clearly required to test this hypothesis
(see below). On the other hand, nearby electron‐withdrawing
groups, such as neighbouring amide bonds in peptides, reduce
the electron density of the tertiary amide in proline and N‐
methyl glycine residues and slow down the rate of ET. Although
this rate retardation is caused by amide bonds in both N‐ and C‐
direction, the deactivating effect of N‐terminal peptide bonds is
considerably stronger. The kinetic data are supported by
On the other hand, the reaction of Ac‐Phe‐Pro‐OMe with
NO3• led to several products, of which the major product could
be tentatively assigned by ESI‐HRMS as the nitro derivative Ac‐
Phe(NO2)‐Pro‐OMe (calcd. for [C17H22N3O6]+: 364.1504 [M + H]+,
found 364.1503). Reaction through oxidation of the Pro residue
would be expected to lead to formation of the N‐nitro proline
derivative
8 through cleavage of the peptide bond (similar to
•
the reaction with Ac‐Pro‐Phe‐OMe). However, no product of
this mass (either deprotonated or protonated) was found in the
mass spectrum of the reaction mixture (see ESI), indicating that
oxidation of the Pro residue did not occur to a measurable
extent.
products studies, which showed that NO3 ‐induced oxidation of
proline occurs in Ac‐Pro‐Phe‐OMe but not in the dipeptide with
the inverted sequence Ac‐Phe‐Pro‐OMe. The diminished
reactivity of the tertiary amide of Pro in the latter dipeptide is
due to the deactivating effect caused by the N‐terminal amide
The selectivity for a reaction at the N‐terminal Phe in Ac‐
Phe‐Pro‐OMe is in line with our previous findings that oxidation
of the aromatic ring in Phe is significantly facilitated by
neighbouring amide groups, which stabilise the developing
positive charge on the aromatic ring through formation of a ‐
complex.9e Phe oxidation in Ac‐Phe‐Pro‐OMe should even be
easier, since the nucleophilic tertiary amide of the C‐terminal
moiety, in addition to formation of a ‐complex that facilitates
oxidation of the Phe residue through neighbouring amide group
effects.9e
While the data in this work clearly suggest that neighbouring
amide bonds could protect proline residues in peptides against
radical‐induced oxidative damage (at least to some extent), we
wish to re‐iterate the model character of this study. Interactions
with the environment, including hydrogen bonding between
the peptide and solvent, were minimised on purpose to enable
insight into the fundamental reactivity of proline towards
radical oxidation. These data provide the required foundation
for the next phase of our studies, where the role of an aqueous
environment, in particular hydrogen bonding, on radical‐
induced oxidative damage of proline as both single amino acid
(without and with N‐protection) and in short peptides will be
Pro residue should provide more stabilisation in the
(Scheme 3) than a secondary amide.
‐complex
9
Overall, the data suggest that the reduced susceptibility of
•
the Pro residue in Ac‐Phe‐Pro‐OMe for NO3 ‐induced oxidation
is due to two effects that remove the electron density from the
tertiary amide: (i) the electron‐withdrawing N‐terminal amide
moiety, and (ii) participation in a ‐complex that facilitates Phe
oxidation. On the other hand, in peptide sequences where Pro
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Page 7 of 8
Organic & Biomolecular Chemistry
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ARTICLE
explored. This work is currently ongoing in our laboratory, and
we will report on the findings in due course.
Notes and references
View Article Online
‡ A linear correlation between rate coeffiDcOieI:n1t0s.1f0o3r9t/Dh0eOrBe0a1c3t3io7Dn
of NO3• with various organic compounds in water and acetonitrile
has been found, with reactions in acetonitrile generally being
slightly faster, see: S. P. Mezyk, T. D. Cullen, K. A. Rickman and B.
J. Mincher, Int. J. Chem. Kinet., 2017, 49, 635. While these data
show that NO3 is highly reactive in both solvents the possibility
that reaction mechanisms may be different, for example because
of the different ability of these solvents to stabilise intermediates,
cannot be excluded.
Conflicts of interest
There are no conflicts to declare.
Experimental
Synthesis of the starting materials. All compounds used in the
kinetic and products studies were prepared according to
established procedures. Synthetic details and spectroscopic
data are given in the ESI.
‡‡ This caution is justified by own previous work on the oxidation of

cholesterol by NO2 , which revealed rapid transformation of the
primary products in the presence of water. Without the experiment
performed in an inert solvent an incorrect radical mechanism instead
of an ionic process would have been proposed, see: A. Zalewski, J. G.
Nathanael, J. M. White and U. Wille, Chem. Commun., 2016, 52,
4060.
Kinetic studies. The kinetic experiments were performed at 298
± 1 K on an Edinburgh Instrument LP920 spectrometer
described previously,9e,10,11 using the third harmonic of a
Quantel Brilliant B Nd:YAG laser (6 ns pulse, 10–20 mJ, λ = 355
nm) to generate the reaction transient. The detection system
employed a Hamamatsu R2856 photomultiplier tube (PMT)
interfaced with a Tektronix TDS 3012C Digital Phosphor
oscilloscope for transient absorption spectra. Kinetic
measurements were carried out under pseudo‐first order
conditions following the established procedure described in
ref.10 Measurements for each substrate were done in triplicate
and the results are reported as the average of these three runs.
Experimental details of the kinetic measurements and data
evaluation are given in the ESI (Figures S1–S8).
‡‡‡ It should be noted that low kH/kD values could also indicate a
highly non‐symmetrical (very early or very late) transition state.
•
However, previous computational studies of reactions of NO3
with aliphatic amino acids (see ref. 11) suggest that such
unsymmetrical transition states are not likely in the reactions
studied in this work.
•
§ The rate coefficients for the reactions of NO3 with the N‐
phthaloylated amino acids Gly, Ala, Gaba, Ava and Ahx, which likely
proceed through HAT from the various CH2 moieties, are lower and
in the range 0.6–1.6  106 M‐1 s‐1 (data are given in the ESI, Table S1).
•
§§ Reaction of these dipeptides with NO3 generated from CAN by
irradiation with 350 nm light in a Rayonet photoreactor gave a large
number of products, which presumably resulted from subsequent
photochemical decomposition and were not identified.
Computational studies. The computations were carried out
with the Gaussian 09 program28 using the M062X method29 in
combination with the 6‐31+G* basis set, which has been
employed previously to investigate radical induced damage in
amino acids.10,11 Calculations in acetonitrile were performed
using the Conductor‐like Polarizable Continuum Model (CPCM)
for acetonitrile.30 The ground and transition structures were
verified by vibrational frequency analysis at the same level of
theory, and all identified transition structures showed only one
imaginary frequency. The Gaussian archive entries, including
free energy data and imaginary frequency of the transition
structure, are given in the ESI.
•
§§§ The NO2 /O3‐mediated nitration of Phe leads to nitroaromatic
products with o/m/p ratios similar to the electrophilic aromatic
nitration (see refs. 9, 10). Regioisomeric products were also formed
in the reaction of Ac‐Pro‐Phe‐OMe, but only the major 4‐nitro
product could be separated by column chromatography, followed by
recrystallisation.
1
2
3
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max = 74.73°, 4959 reflections measured, 2723 independent
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 = 1.54184 Å, Monoclinic, space group

=

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Acknowledgements
This work was supported by the Australian Research Council
through the Linkage, Infrastructure, Equipment and Facilities
(LIEF) Grant Scheme and The University of Melbourne through
the High‐Performance Computing facility.31
8
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