DOI: 10.1002/open.201200044
Metal-Conjugated Affinity Labels: A New Concept to
Create Enantioselective Artificial Metalloenzymes
Thomas Reiner,[b] Dominik Jantke,[a] Alexander N. Marziale,[a] Andreas Raba,[b] and Jçrg Eppinger*[a]
Dedicated to Professor Wolfgang A. Herrmann on the occasion of his 65th birthday
Incorporation of artificial metal centers into proteins and pep-
tides has emerged as an important tool in chemical and bio-
logical research.[1] Current applications include pharmaceuti-
cals,[2] probes for molecular imaging[3] and contrast agents,[4]
tools for biophysical studies targeting metalloprotein func-
tions,[5] metal-directed protein assembly,[6] electrochemical bio-
sensors,[7] and altered electrochemical potential of electron
transporting proteins,[8] as well as the synthesis of functional
metalloenzymes with non-natural catalytic activity.[9] Particular-
ly artificial metalloenzymes received great interest, since they
hold the promise to greatly expand the range of reactions ac-
cessible by biocatalysis. A variety of methods to generate artifi-
cial metal sites were developed including domain-based direct-
ed evolution strategies,[10] engineering of transition-metal bind-
ing sites through introduction of coordinating amino acids at
geometrically appropriate positions[11] or site-directed in vivo
incorporation of artificial metal-chelating amino acids.[12] How-
ever, the site-directed anchoring of artificial cofactors repre-
senting appropriate ligands or metal complexes has so far
been the most successful strategy to achieve good catalytic ac-
tivities and enantioselectivities. Inspired by the pioneering
work of Wilson and Whitesides,[13] Ward revealed the potential
of the supramolecular biotin–(strept)avidin technology, which
in combination with directed or rationally guided evolution[14]
can deliver highly enantioselective organometallic enzyme
hybrid (OMEH) catalysts.[15] Such biotin–(strept)avidin–metal
conjugates were subsequently tested in a variety of catalytic
transformations.[16] In contrast to this supramolecular approach,
covalent anchoring of artificial cofactors on proteins can utilize
a variety of protein hosts and hence is not limited by the sta-
bility range of the biotin–(strept)avidin complex. Originally in-
troduced by Kaiser,[18] covalent attachment of artificial cofactors
has been applied to convert proteases,[19] lipases[20] or other
non-metal proteins[21] into organometallic enzyme hybrids.
However, none of the covalent approaches could so far ach-
ieve the enantioselectivities reached by biotin–(strept)avidin
conjugates.[9d,21e] Although metals are introduced site-specifi-
cally, most systems generated through a covalent approach
possess a flexible linker and hence lack a well-defined localiza-
tion of the metal center on the surface of the host protein,
which is a prerequisite to achieve chiral induction.
We reasoned that a well-defined orientation of the metal
center in the binding pocket of a protease could be achieved,
if the specific binding-affinity pattern of the pocket is utilized
to position a suitable artificial cofactor. Correspondingly, we
decided to synthesize and test metal-conjugated affinity labels
(m-ALs), which consist of 1) an achiral, catalytically active metal
complex linked to 2) a protease-specific reactive group able to
form a covalent bond with the active center and 3) a peptidic
tail to direct the binding orientation of the m-AL (Figure 1). To
test the hydrogenation of ketones, we chose a system based
on cysteine proteases of the papain family as host proteins.
This class of proteases is efficiently inhibited by epoxysuccinyl
ester derivatives of the known calpain inhibitor E64c through
S-alkylation of the reactive epoxide group by the active site
22]
cysteine.[17,
Hence, these well-suited, family-wide affinity
labels[23] were chosen to incorporate catalytically active rhodi-
um- or ruthenium-half-sandwich complexes into the enzymatic
host. M-ALs were synthesized starting from E64c derivatives
(1–3), which were first converted to the corresponding penta-
fluorophenyl esters (1-OPf–3-OPf) and subsequently coupled
to monomeric amino-functionalized half-sandwich complexes
of ruthenium and rhodium[25] (1Ru, 2Ru and 1Rh–3Rh, respectively;
see Scheme 1).
Cysteine proteases of the papain family are readily convert-
ed into organometallic enzyme hybrids through conjugation
with m-ALs 1Rh, 1Ru or 2Rh (Figure 1C). Because this system is
modular, combination of different affinity labels, metal com-
plexes and proteases allows a straightforward generation of
OMEH catalyst libraries, from which promising candidates can
be selected based on their performance in asymmetric cataly-
sis. In this study, we generated a variety of OMEH catalysts
originating from three affinity labels (1, 2 and 3), two metal
complexes (Rh and Ru) and three cysteine proteases (papain,
bromelain and cathepsin L). MALDI-TOF MS was used to con-
firm selective formation of the desired enzyme hybrids. Upon
addition of m-AL 1Rh to papain a new signal at 23.976 kDa ap-
peared, indicating formation of the desired organometallic
enzyme hybrid (1Rh@papain, Figure 2). After incubating papain
with one equivalent of m-AL 1Rh for two hours the signal of
[a] D. Jantke, Dr. A. N. Marziale, Prof. J. Eppinger
KAUST Catalysis Center, KCC
King Abdullah University of Science and Technology, KAUST
Thuwal 23955-6900 (Saudi Arabia)
[b] Dr. T. Reiner, A. Raba
Chemistry Department, Technische Universitꢀt Mꢁnchen
Lichtenbergstr. 4, 85748 Garching (Germany)
Supporting information for this article is available on the WWW under
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.
This is an open access article under the terms of the Creative Commons
Attribution Non-Commercial License, which permits use, distribution and
reproduction in any medium, provided the original work is properly
cited and is not used for commercial purposes.
ꢀ 2013 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemistryOpen 2013, 2, 50 – 54 50