Published on the web February 25, 2012
313
Synthesis of a Bridging Ligand with a Non-denatured Protein Pendant:
Toward Protein Encapsulation in a Coordination Cage
Daishi Fujita,1 Kosuke Suzuki,1 Sota Sato,1 Maho Yagi-Utsumi,3,4 Eiji Kurimoto,4,5
Yoshiki Yamaguchi,4,6 Koichi Kato,*3,4 and Makoto Fujita*1,2
1Department of Applied Chemistry, Graduate School of Engineering, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656
2CREST, Japan Science and Technology Agency (JST), 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656
3Okazaki Institute for Integrative Bioscience, Institute for Molecular Science,
5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787
4Graduate School of Pharmaceutical Sciences, Nagoya City University,
3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603
5Faculty of Pharmacy, Meijo University, 150 Yagotoyama, Tempaku-ku, Nagoya, Aichi 468-8503
6Structural Glycobiology Team, Systems Glycobiology Research Group, Chemical Biology Department, RIKEN,
Advanced Science Institute, 2-1 Hirosawa, Wako, Saitama 351-0198
(Received November 25, 2011; CL-111131; E-mail: kkato@phar.nagoya-cu.ac.jp, mfujita@appchem.t.u-tokyo.ac.jp)
Toward protein encapsulation by a synthetic host, we
synthesized an ubiquitin-dangled ligand, a potential precursor
of a nanoscale coordination cage. The key step is the addition
of a C-terminal Cys76 SH group, which was introduced by
Gly76Cys mutation, to a maleimide acceptor on the ligand. The
C-terminal mutation and the SH addition to the ligand did not
damage the ubiquitin moiety at all, neither structurally nor
conformationally.
spherical cages are comparable to those of proteins (up to
10 nm), we had an ambition to encapsulate proteins in the
discrete cage host. The first step toward the protein encapsula-
tion inside the cage is the development of a mild synthesis of
a protein-functionalized ligand without denaturization of the
protein’s native folding. As our first target, we choose ubiquitin,
a relatively small globular protein (76 residues, 8.6 kDa
approximately 3-4 nm in diameter) which plays an important
role in proteasomal degradation.6 Here we report that terminal
mutation (Gly76 to Cys76) of ubiquitin followed by coupling
with a maleimide-functionalized bent ligand provides a mild and
high-yielding method for attaching a protein on a ligand without
denaturization.
In our strategy for the protein encapsulation, one of the
ligands involved in the cage 2 should be replaced by a protein-
functionalized ligand. The key requirements in the synthesis of
the protein-functionalized ligand are (1) selective coupling at
a specific residue of ubiquitin with the ligand and (2) mild
conditions that do not denature the ubiquitin native structure.
The existing functional groups in ubiquitin (for example, NH2 of
Lys and COOH of Asp or Glu) are unavailable for the coupling
with the ligands because two or more of these residues exist in
the ubiquitin sequence and no selectivity among these residues is
expected. In addition, chemical derivation of internal residues
may lead to denaturization. We therefore designed the introduc-
tion of a Cys residue (SH group) by a mutation technique. Since
the ubiquitin C-terminal sticks out from the folding structure and
is conformationally flexible, mutation at this site is expected to
retain the native structure. Thus we examined the mutation of
C-terminal Gly76 into Cys76 (Gly76Cys mutation).7
Encapsulation of proteins in synthetic hosts may enable the
control of protein functions. With the expectation of enhanced
stability and enzymatic activities, some proteins have been
accommodated in synthetic host materials such as solid
supports,1 polymer matrices,2 and reverse micelles.3 In these
structurally nonuniform hosts, however, the protein functions are
dispersed. Furthermore, functional control and elaboration of the
encapsulated proteins are difficult because they can no longer be
analyzed by common spectroscopic or crystallographic methods.
We envisioned the encapsulation of proteins in a structurally
well-defined host, where the protein functions are not dispersed
but distinctly controlled and can be observed using spectro-
scopic and crystallographic methods. This chemical approach
has a great advantage over biological strategies with natural
cages such as viruses which present difficulties in preparation
and structural modification.4
Recently, rigid and bent ligands 1 have been shown to self-
assemble into nanoscale spherical cages 2 upon complexation
with Pd(II) ions (Figure 1).5 Noting that the diameters of the
The Gly76Cys-mutated ubiquitin was constructed by stand-
ard polymerase chain reaction (PCR) and genetic engineering
techniques. The recombinant ubiquitin mutant was expressed
and purified as described previously.6 Among the number of
methods available to couple a protein with an organic functional
group, we chose thiol-maleimide coupling8,9 since a covalent
bond is irreversibly formed under very mild coupling conditions.
The maleimide part is covalently coupled to the concave of
ligand 1. We established the synthesis of ligand 11 via two
synthetic routes. Initially, ligand 11 was prepared by route a
(Scheme 1a). Commercially available 3 was acetylated (3 ¼ 4;
Figure 1. Schematic representation of self-assembly of a
coordination sphere.
Chem. Lett. 2012, 41, 313-315
© 2012 The Chemical Society of Japan