Communications
DOI: 10.1002/anie.200900595
Enzyme Mimics
Downsizing of Enzymes by Chemical Methods: Arginine Mimics with
Low pKa Values Increase the Rates of Hydrolysis of RNA Model
Compounds
N. Johan V. Lindgren, Lars Geiger, Jesus Razkin, Carsten Schmuck,* and Lars Baltzer*
The downsizing of enzymes is an attractive goal.[1] It increases
our understanding of enzyme function and enables many
applications in biotechnology. The fine-tuning of amino acid
pKa values to match reaction mechanisms, which are con-
trolled in proteins by the active-site molecular environment,
is a critically important aspect of enzyme catalysis that is
difficult to mimic in model peptides. The catalytic residues are
exposed to solvent, and effects on pKa values by neighboring
groups are limited to an increase or decrease of approx-
imately one unit.[2] In contrast, the chemical synthesis of
amino acids with non-natural side chains can be useful for the
control of dissociation constants with high accuracy and offers
a fast and robust route to improved catalysts. Herein we
report on the use of a synthetic arginine mimic, a guanidi-
niocarbonyl pyrrole derivative named Gcp, with a pKa value
of 6–7 in aqueous solution. This value is several units lower
than that of Arg.[3] Gcp was introduced in a polypeptide
scaffold to provide transition-state stabilization and general-
acid/general-base catalysis in reactions that mimic RNA
hydrolysis.
catalytic machinery, but full ribonuclease activity remains an
elusive goal, most likely because of the difficulties encoun-
tered in combining all of the necessary catalytic components
in the correct orientation in a small catalyst.
We previously reported a helix–loop–helix motif (HNI,
Figure 1) based on commonly occurring amino acids and
capable of catalyzing the hydrolysis of RNA models with rate
enhancements of two orders of magnitude or more.[9] HNI
forms a helix–loop–helix motif with two His residues in
opposite corners of an approximately square-shaped active
site and two Arg residues in the remaining two corners. The
imidazole side chains most likely act as general-acid and/or
general-base catalysts. The flanking arginine residues were
introduced to bind the negatively charged phosphate ester
and the even more negatively charged transition state by
electrostatic interactions as well as by hydrogen bonding.
Since the transition state is more negatively charged than the
substrate, we expect it to be bound more strongly than the
substrate and expect transition-state binding to contribute
significantly to catalysis.
Phosphodiesters are extremely stable towards hydrolysis,
and the mechanism for the catalysis of RNA hydrolysis by
enzymes and ribozymes has been studied thoroughly.[4]
Enzymes that have evolved to catalyze phosphodiester
hydrolysis are among the most efficient known; they show
rate enhancements of eighteen orders of magnitude or
more.[5] Ribonucleases provide general-acid and general-
base catalysis as well as substrate binding and transition-
state stabilization. Numerous model systems ranging from
metalloenzyme mimics[6] and metallocomplexes[7] to poly-
amines[8] have been designed to mimic all or part of the
Although arginine residues are considered to be good
binding groups for phosphates, the pKa values of the guani-
dino group and phosphate groups are not well-matched. The
arginine residue has a pKa value of more than 12 in short
peptides,[10] whereas the pKa value of the phosphate group in a
phosphodiester is approximately 1.3.[11] It seemed very likely
that an arginine mimic with a pKa value as low as the
pH value of the solvent for the reaction would offer consid-
erable improvements in catalytic efficiency in phosphoryl-
transfer reactions through improved hydrogen bonding. The
concept has previously been demonstrated elegantly in
nonpeptidic catalysts.[12] Furthermore, general-acid and gen-
eral-base catalysis by His residues is hampered by the short
side chain and short reach relative to the distances between
residues on the surface of a helix–loop–helix motif. We
introduced the Gcp residue in HNI to replace both Arg
residues (JL1), both His residues (JL2), and both Arg as well
as both His residues (JL3; Figure 1). The pKa value of the
guanidinium residue of this non-natural amino acid in
aqueous solvents is approximately 6–7, close to that of
His.[13] The guanidiniocarbonyl pyrrole cation has been shown
to be an excellent receptor for carboxylates with dissociation
constants in the millimolar range, even in aqueous solvents.[14]
The peptides were synthesized by solid-phase peptide
synthesis as reported previously for HNI, but by attaching the
guanidiniocarbonyl pyrrole group to selectively deprotected
ornithine residues by using amide-coupling chemistry.[15,16]
After deprotection and cleavage from the solid support, the
[*] Dr. L. Geiger, Prof. C. Schmuck
Institut fꢀr Organische Chemie
Fakultꢁt fꢀr Chemie, Universitꢁt Duisburg-Essen
Universitꢁtstrasse 7, 45141 Essen (Germany)
Fax : (+49)201-183-4259
E-mail: carsten.schmuck@uni-due.de
N. J. V. Lindgren, Prof. L. Baltzer
Department of Biochemistry and Organic Chemistry
Uppsala University, Box 576, 75123 Uppsala (Sweden)
Fax : (+46)18-471-3818
E-mail: lars.baltzer@biorg.uu.se
Dr. J. Razkin
Departamento de Quꢂmica Aplicada, Universidad Pfflblica de Navarra
31006 Pamplona, Navarra (Spain)
Fax : (+34)948-169-606
Supporting information for this article is available on the WWW
6722
ꢀ 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2009, 48, 6722 –6725