Targeting peptides with an iron-based oxidant: Cleavage of the amino acid backbone and oxidation of side chains

Article abstract of DOI:10.1021/ja075075i

The oxidation of protected amino acids using an iron-based oxidant is described. Substrates of the general formula Ac-X-NH^tBu, where X = Gly (1), Ala (2), Val (3), Phe (4), Tyr (5), Trp (6), and Met (7) were constructed to model individual amino acid residues within a polypeptide chain. Oxidation of 1 by the iron catalyst [Fe^II(N4Py)(MeCN)](ClO₄)₂ (8) and KHSO₅ leads to scission of the amino acid backbone and produces N-acetylformamide as the major product. Decomposition of the iron-based oxidant [Fe^IV(O)(N4Py)]²⁺, derived from 8, is slower in the presence of 2,2-d₂-1 (96% D) than with 1, giving a kinetic isotope effect of 4.8, which is consistent with [Fe^IV(O)(N4Py)]²⁺ cleaving an α-CH bond of 1. Aliphatic amino acid substrates 2 and 3 do not react with [Fe^IV(O)(N4Py)]²⁺ under the same conditions used with 1. With substrates 4-7 oxidation of the amino acid side chain is observed. Decomposition of [Fe^IV(O)(N4Py)]²⁺ upon treatment with 10 equiv of 1 and 4-7 revealed that 5 is the most reactive toward the Fe^IVO species. Pseudo-first-order rate constants of 17.0(5) × 10^-3, 3.15(8) × 10^-3 and 5.8(2) × 10^-5 s^-1 were obtained for decomposition of [Fe^IV(O)(N4Py)]²⁺ ([Fe] = 1 mM, 1:1 H₂O/MeCN) by 6, 7, and 1, respectively. Copyright

Full text of DOI:10.1021/ja075075i

Published on Web 09/26/2007
Targeting Peptides with an Iron-Based Oxidant: Cleavage of the Amino Acid
Backbone and Oxidation of Side Chains
Anil R. Ekkati and Jeremy J. Kodanko*
Wayne State UniVersity, Department of Chemistry, 5101 Cass AVenue, Detroit, Michigan 48202
Received July 9, 2007; E-mail: jkodanko@chem.wayne.edu
Proteins are an integral part of the chemistry of life. In an oxygen-
rich environment, the amino acid residues of proteins are susceptible
to attack by reactive oxygen species (ROS) such as singlet oxygen,
superoxide, or hydroxy radical.1,2 This damage can disable proteins,
Figure 1. Model amino acid substrates for iron-catalyzed oxidation
and peptide oxidation has been implicated in numerous disease
reactions.
states, as well as in the progression of aging. How ROS damage
Scheme 1. Oxidation of the Amino Acid Backbone in 1 by KHSO5
and 1 mol % of 8.
proteins is well understood, and the reaction products of amino
acid degradation by ROS have been characterized.3 In contrast,
much less is understood about how metal-based oxidants react with
amino acid residues.4,5 This is unfortunate because metal-based
oxidants have greater potential than ROS to modify amino acid
residues of proteins with high selectively and specificity, considering
they are typically less reactive, nondiffusible, and could be directed
to sites on a protein. Recent achievements regarding the generation
of high-valent iron complexes,6,7 coupled with the high abundance
of iron in biological systems, make iron-based oxidants attractive
for targeting proteins of interest. Toward this end, herein we
describe the oxidation of amino acids by an iron-based oxidant,
which results in cleavage of the amino acid backbone and modi-
fication of side chains.
To study how iron-based oxidants might react with amino acid
residues in a protein, a series of protected amino acid substrates
(1-7, Figure 1) was constructed that were designed to model
individual residues within a polypeptide chain. Starting from the
parent amino acid, the N-terminus was acetylated. On the C-ter-
minus, a tert-butyl amide was installed to block the weak C-H
bonds that would be equivalent to the R-position of the next residue
in a polypeptide chain.
Next, using substrates 1 and 4, conditions of the oxidation
reaction were optimized for iron-based catalyst and stoichiometric
oxidant. Water was chosen as a solvent because it is most relevant
to biological systems, and MeCN was incorporated to solublize
the amino acid substrates. For these studies, a series of catalysts
were picked that are known to generate iron-based oxidants,
including [FeII(N4Py)(MeCN)](ClO4)2 (8) that can generate a
reactive FeIVdO species which is stable in aqueous media8 and is
powerful enough to oxidize a C-H bond of cyclohexane9,10 as well
as complexes FeII(TPA)(OTf)2 (9)11 and [FeII(BPMEN)](OTf)2 (10)
(Figure S1).12 For optimization, substrates 1 and 4 were screened
against catalysts 8-10 and oxidants H2O2, CH3CO3H, PhIO, and
KHSO5 (Oxone). The combination of 8 and KHSO5 was found to
be most effective so these conditions were adopted for the following
studies.
Results with the glycine substrate 1 prove that an iron-based
oxidant can attack the amino acid backbone and lead to its scission.
Treatment of 1 with 1 mol % of the FeII catalyst 8 and 5 equiv of
KHSO5 in a mixture of H2O/MeCN (5:1) resulted in formation of
aldehyde 11 as the major product (41%), along with recovered 1
(17%), aldehyde 12 (11%), the R-hydroxyglycine derivative 13
(5%), and glyoxamide 14 (5%) as lesser components (Scheme 1).
Importantly, no reaction occurred between 1 and KHSO5 in the
absence of iron catalyst 8, nor when using Fe(ClO4)2 or FeII(EDTA)
as the catalyst, which indicates that the ligand N4Py, which
produces a well-characterized FeIVdO species, is required. Also
important was that similar product mixtures were formed in the
reactions of 1 under aerobic and anaerobic conditions, which
excludes autoxidation by atmospheric O2 acting as the dominant
reaction pathway.
Because products 11-14 are characteristic of a peptide cleavage
pathway that involves alkoxyradical intermediates,2,13 more support
was sought for the hypothesis that an FeIVdO species attacks the
backbone. Therefore, a green solution of the iron-based oxidant
[FeIV(O)(N4Py)]2+ was generated,8,9 then treated with 10 equiv of
substrate 1 ([Fe] ) 1 mM, 1:1 H2O/MeCN) and the reaction was
monitored by UV-vis spectroscopy. Decay of absorbance at 680
nm fit well to a single-exponential equation, furnishing a pseudo-
first-order rate constant of 5.8(2) × 10-5 s-1 (Supporting Informa-
tion). Decay of [FeIV(O)(N4Py)]2+ under the same conditions was
slower with the 2,2-d2 isotopomer of 1 (96% D), giving a kinetic
isotope effect of 4.8, which is consistent with the FeIVdO species
cleaving an R-CH bond of 1. An inverse deuterium kinetic isotope
effect of 0.95 was observed for the reaction of 1 in D2O/CD3CN
solvent, which rules out the possibility that oxidation of 1 is initiated
by NH abstraction. Taken together, these observations are most
consistent with an FeIVdO attacking the glycine backbone and
initiating a radical chain process carried by KHSO5 that leads to
formation of 11-14 in the catalytic reaction of Scheme 1.
The same backbone attack that occurred with 1 was not observed
with alanine and valine substrates 2 and 3. These aliphatic amino
acids do not react under the same catalytic conditions used with 1,
nor do they promote decomposition of the FeIVdO species faster
than the control experiment without substrate added. On the basis
of first principles, 2 and 3 would be expected to react faster because
the R-position is a tertiary carbon. However, these results are
consistent with the observations of others,14 that H-atom abstraction
9
12390
J. AM. CHEM. SOC. 2007, 129, 12390-12391
10.1021/ja075075i CCC: $37.00 © 2007 American Chemical Society

C O M M U N I C A T I O N S
Table 1. Pseudo-First-Order and Relative Rate Constants for the
Decomposition of [FeIV(O)(N4Py)]2+ ([Fe] ) 1 mM in 1:1 H2O/
MeCN) by Amino Acid Substrates 1 and 4-7 (10 equiv) at 25 °C
1
substrate
k
obs (s-
)
krel
5
6
7
1
4
n.d.a
>293
293
54
1
<1
17.0(5) × 10-3
3.15(8) × 10-3
5.8(2) × 10-5
n.d.a
Figure 2. Major oxidation products from the reactions of substrates 4, 6,
and 7 with Fe catalyst 8 and KHSO5.
a n.d. ) not determined.
is slower with substituted amino acids because their carbon-centered
radicals, which should enjoy greater stablization, are actually
destabilized relative to the glycine radical because of nonbonding
interactions that disrupt captodative stablization. It is worth noting
that the reactivity of the FeIVdO species is also quite different from
organic oxidants like dioxiranes that are known to hydroxylate the
â-position of valine residues.15
With amino acid substrate 4, side-chain oxidation was observed
(Figure 2). Treatment of 4 with 10 mol % of 8 and 3 equiv of
KHSO5 produced orthoquinone 15 as the major oxidation product
(17%). Formation of 15 from 4 involves three consecutive oxida-
tions, and a logical progression would involve formation of a
tyrosine derivative 5, then a catechol DOPA derivative, then
oxidation to form 15. However, treatment of 5 under the same
conditions as with 4 does not lead to formation of 15, but instead
leads to decomposition. Therefore, formation of 15 from 4 may
involve a metastable iron-bound phenolate as an intermediate.16
Analogous to our observations in the oxidation of 1, control
experiments showed that oxidation of neither 4 nor 5 occurred with
KHSO5 in the absence of catalyst 8, nor when using Fe(ClO4)2 or
FeII(EDTA) as the catalyst.
constant measured for 7 agrees well with the previous observations
in reactions of the [FeIV(O)(N4Py)]2+ with aromatic sulfides.8
In conclusion, we have established that an iron-based oxidant
can facilitate oxidative cleavage of the amino acid backbone and
modification of side chains. Studies are now underway in our
laboratory to characterize the chemoselectivty and order of reactivity
of iron-based oxidants with all twenty natural amino acids, in
addition to studies that will define the mechanism of these reactions.
Furthermore, we anticipate that iron-based oxidants, like that derived
from 8, can be applied to the site-specific modification and/or
cleavage of peptides and proteins, akin to extensive investigations
regarding the cleavage of nucleic acids by bleomycin and its model
complexes.
Acknowledgment. We thank Wayne State University for their
generous financial support of this research and Melody Kelley for
assistance with preparing substrates for these studies.
Supporting Information Available: Experimental procedures for
preparation of 1-7, 11-17, including characterization data, UV-vis
spectra and kinetic fits. This material is available free of charge via
the Internet at http://pubs.acs.org.
Oxidation of the amino acid side chains was also observed with
substrates 6 and 7. However, control experiments concluded that
these substrates react with KHSO5 in the absence of catalyst 8.
With tryptophan substrate 6, oxidation with 1 mol % of 8 and 3
equiv of KHSO5 led to a complex mixture of products. The major
product formed, which was also unique to the iron-catalyzed
reaction, was determined to the acyl lactam derivative 16 (18%)
where oxidation of the aniline to a nitrobenzene had occurred.
Treating the methionine substrate 7 with 1 mol % of catalyst 8 and
2 equiv of KHSO5 led to formation of the sulfone 17 (97%), which
is the same product obtained without 8. Seeing that 6 and 7 react
with KHSO5 alone, evidence was gathered later on that these
substrates will react with the iron-based oxidant [FeIV(O)(N4Py)]2+
as well.
After identifying the major products from the oxidation reactions,
the relative rates of reaction for substrates 1-7 were established.
Reactions were performed under pseudo-first-order conditions in
a 1:1 mixture of H2O/MeCN to obtain full solubility of all
substrates. These experiments established that the tyrosine substrate
5 causes decomposition of the FeIVdO species most rapidly out of
all of the substrates, followed by 6 and then 7. The glycine
derivative 1 was of intermediate reactivity. Phenylalanine substrate
4 was significantly less reactive than 1.17 From reactions with 6
and 7, the decrease of absorbance at 680 nm was fit satisfactorily
to furnish pseudo-first-order rate constants (Table 1). The rate
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