Published on Web 03/13/2003
Design of Radical-Resistant Amino Acid Residues: A
Combined Theoretical and Experimental Investigation
Anna K. Croft,† Christopher J. Easton,* and Leo Radom
Contribution from the Research School of Chemistry, Australian National UniVersity,
Canberra, ACT 0200, Australia
Received December 10, 2002; E-mail: easton@rsc.anu.edu.au.
Abstract: Ab initio calculations have been used to design radical-resistant amino acid residues. Optimized
structures of free and protected amino acids and their corresponding R-carbon-centered radicals were
determined with B3-LYP/6-31G(d). Single-point RMP2/6-31G(d) calculations on these structures were then
used to obtain radical stabilization energies, to examine the effect of steric repulsion between the side
chains and amide carbonyl groups on the stability of R-carbon-centered peptide radicals. Relative to glycine,
the destabilization for alanine and valine residues was found to be approximately 9 and 18 kJ mol-1
,
respectively, which correlates with the reactivity of analogous amino acid residues in peptides toward
hydrogen atom abstraction in conventional free radical reactions. To design amino acid residues that would
resist radical reactions, strategies by which the steric effects could be magnified were considered. This
resulted in the identification of tert-leucine and 3,3,3-trifluoroalanine as suitable molecules. With these amino
acid residues, the destabilization of the R-carbon-centered radicals relative to that of glycine is increased
substantially to approximately 36 and 41 kJ mol-1, respectively. The theoretical predictions have been
supported by experimental observations: a tert-leucine derivative was shown to be very slow to react with
N-bromosuccinimide, while the corresponding trifluoroalanine derivative was found to be inert.
Introduction
Amino acid and peptide radicals have been implicated in a
wide variety of biochemical processes and physiological
disorders,1-6 including arteriosclerosis4 and aging.5,6 At a
molecular level, they are associated with protein damage7 and
enzyme function.8-18 As a result, studies of their properties are
both important and topical. In peptides and proteins, R-carbon-
Figure 1. R-Carbon-centered radicals formed from peptides, proteins, and
other amino acid derivatives.
centered radicals (Figure 1) form preferentially,19-24 because
they are extensively stabilized through resonance. Knowledge
of other factors affecting the formation of these radicals is
fundamental to understanding the basis of their effects.
Of the R-carbon-centered radicals produced from peptides
and other amino acid derivatives, glycyl radicals are the most
common and form selectively. This has been observed in EPR
and radiolysis studies.3,25,26 It has also been seen and exploited
in the regioselective photoalkylation of peptides and proteins22-24
and in the bromination of peptides and other amino acid
derivatives.19-21,27,28 Glycyl radicals are also intermediates in
enzymic processes. Backbone glycyl radicals are thought to be
involved in the catalysis displayed by pyruvate formate lyase,11,12
* To whom correspondence should be addressed.
† Current address: Department of Chemistry, University of Wales,
Bangor, Gwynedd, LL57 2UW, UK.
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10.1021/ja029674v CCC: $25.00 © 2003 American Chemical Society
J. AM. CHEM. SOC. 2003, 125, 4119-4124
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