Creation of an artificial metalloprotein with a Hoveyda-Grubbs catalyst moiety through the intrinsic inhibition mechanism of α-chymotrypsin

Article abstract of DOI:10.1039/c2cc16898g

An l-phenylalanyl chloromethylketone-based inhibitor equipped with a Hoveyda-Grubbs catalyst moiety was regioselectively incorporated into the cleft of α-chymotrypsin through the intrinsic inhibition mechanism of the protein to construct an artificial organometallic protein. The Royal Society of Chemistry 2012.

Full text of DOI:10.1039/c2cc16898g

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COMMUNICATION
Creation of an artificial metalloprotein with a Hoveyda–Grubbs catalyst
moiety through the intrinsic inhibition mechanism of a-chymotrypsinw
Takashi Matsuo,*a Chie Imai,a Takefumi Yoshida,a Takashi Saito,b Takashi Hayashib and
Shun Hirotaa
Received 7th November 2011, Accepted 6th December 2011
DOI: 10.1039/c2cc16898g
An L-phenylalanyl chloromethylketone-based inhibitor equipped
with a Hoveyda–Grubbs catalyst moiety was regioselectively
incorporated into the cleft of a-chymotrypsin through the intrinsic
inhibition mechanism of the protein to construct an artificial
organometallic protein.
Designed metalloproteins are expected to give rise to unique
reaction systems mediated by metal ions, because the highly well-
constructed and pre-organized structures of proteins provide a
specific coordination space to suitably control and/or enhance
the reactivities of a metal ion.1 Chiral environments constructed
by L-amino acid residues will be useful for stereo-selective
syntheses.2 Common methods used to construct artificial metallo-
proteins include the incorporation of a metal ion into a protein
through ligation of functional groups in amino acid residues, the
conjugation of a synthetic metal complex to the protein surface at
Cys or Lys residues and the insertion of a metal complex into the
interior space of proteins through supramolecular (non-covalent)
interactions.1,3,4 In most cases, metal complexes associated with
proteins are categorized as inorganic complexes (i.e. metal com-
Fig. 1 Structures of inhibitors and a-chymotrypsin; (a) structures of 1-L
and 1-D; (b) substrate-binding site of a-chymotrypsin (PDB code: 4CHA).
plexes with N, O, or S-coordination by amino acid residues or
cofactors such as heme). In contrast, some organometallic com-
plexes (C-coordination) are also possible candidates for metallo-
of a-chymotrypsin, a serine protease recognizing hydrophobic
amino acid residues.10 This is accomplished by the intrinsic
features of the protein to create an organometallic biocatalyst
with olefin metathesis activity. Furthermore, this approach is
useful for introducing a synthetic molecule site-specifically into
the interior of the protein, thereby affording a biocatalyst with a
non-natural function.11
protein designs, because they show unique reactivities which are
difficult to obtain using inorganic complexes (e.g. C–C bond
formation). Certain research groups have attempted to prepare
organometallic proteins,5–8 although the resulting functions of
these constructed proteins are, in most cases, similar to the types
of reactions seen in naturally occurring systems (oxidations,
reductions and electron transfer reactions etc.).9 In this communi-
cation, we describe the incorporation of compound 1-L (see
Fig. 1(a)) with a Hoveyda–Grubbs catalyst moiety into the cleft
a-Chymotrypsin from bovine pancreas has a cleft between
the two domains, where four subsites (S1 through S4 sites)
interact with the amino acid residue units of a peptidyl
substrate.12 The S1 site is a key site with respect to the
substrate-selectivity of a-chymotrypsin (Fig. 1(b)), and 1-L
was designed with reference to these structural characteristics.
The structure of 1-L is composed of three parts: the Hoveyda–
Grubbs catalyst part, the linker part, and the inhibitor part.
The Hoveyda–Grubbs catalyst moiety was prepared using a
previously reported method.13 The inhibitor part is based on
the inhibition mechanism of N-tosyl L-phenylalanyl chloro-
methylketone (TPCK) toward a-chymotrypsin,14,15 and
synthesized from Boc-L-Phe with reference to the synthesis of
a Graduate School of Materials Science, Nara Institute of Science and
Technology (NAIST), 8916-5 Takayama-cho, Ikoma, Nara 630-0192,
Japan. E-mail: tmatsuo@ms.naist.jp; Fax: +81-743-72-6119;
Tel: +81-743-72-6112
b Department of Applied Chemistry, Graduate School of Engineering,
Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan
w Electronic supplementary information (ESI) available: Experimental
procedures, synthetic scheme, calculated structure of 1-L, FPLC
chromatogram, mass spectra of chymotrypsins, CD spectra in the
high energy region, UV-vis spectra of the ruthenium complexes in
CH2Cl2, 1H NMR spectrum and HPLC chromatogram of the reaction
mixture. See DOI: 10.1039/c2cc16898g
c
1662 Chem. Commun., 2012, 48, 1662–1664
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an HIV-1 protease inhibitor (the total synthetic route is shown
in Scheme S1 of ESIw).16 The hydrophobic side chain of the
L-phenylalanine moiety in the inhibitor part is expected to enter
the S1 site upon binding to a-chymotrypsin, followed by a
nucleophilic attack at the chloromethylketone moiety by the
imidazole of His57, one of the key amino acid residues for the
hydrolase activity of a-chymotrypsin. As a result, 1-L will be
site-specially introduced into the cleft of a-chymotrypsin.17
Based on an MMFF calculation, the distance between the
chlorine atom and the Ru center in 1-L was evaluated to be
ca. 12 A (Fig. S1 of ESIw). Since this distance is close to that
between His57 and the S4 site (ca. 14 A, see Fig. 1(b)), the
Hoveyda–Grubbs catalyst moiety is expected to be situated
near the S4 site, which is located at the edge of the protein cleft.
Therefore, an olefin metathesis substrate can readily bind to the
Ru center and the corresponding product can diffuse smoothly
into the bulk solvent. The programmed positioning of the
metal center will give rise to the following advantages:
(i) site-specific modification, (ii) the location of the metal center
within a chiral environment and (iii) smooth substrate access
and product release. This approach is quite different from
making simple chemical modifications of a protein surface
and/or inserting a synthetic complex through hydrophobic
interactions. In order to confirm the incorporation of 1-L
through the intrinsic inhibition mechanism of the protein, the
D-form inhibitor, 1-D, was also synthesized from Boc-D-Phe.
a-Chymotrypsin was mixed with 2 equivalents of 1-L in 1 mM
HEPES buffer (pH = 7.0) containing 100 mM KCl. After 2 h,
it was found that the hydrolysis activity toward N-succinyl
L-Phe p-nitroanilide (SucPhePNA) was essentially abolished
(Fig. 2(a)). This is because the imidazole of His57 at the active
site was blocked by the chloromethylketone group in 1-L. In
contrast, a-chymotrypsin retains the hydrolase activity in the
presence of the D-form inhibitor 1-D (Fig. 2(b)).18
is shown in Fig. S2 (inset, ESIw). A band is observed around
370 nm, which is characteristic of the Hoveyda–Grubbs
complex.19 This band remains over the period of 24 h at room
temperature, whereas the band shifts to around 325 nm for free
1-L in an aqueous solution because of the ligand exchange at the
Ru center with water molecules.
a-Chymotrypsin modified with 1-L was characterized by
electrospray ionization mass (ES-MS) and MALDI-TOF-MS
spectrometry. In the ES-MS spectra (Fig. S3 in ESIw), both the
modified and the unmodified chymotrypsins show multiple
charged peaks from 9+ to 13+. The appearance of the peaks
in a similar range indicates that no significant structural denatura-
tion occurs with the incorporation of 1-L.20 The deconvolution of
the spectrum yields the molecular weight of 26 366, corresponding
to the molecular weight of a-chymotrypsin + [1-L ꢀ 3Clꢀ]. The
loss of three chloride ions originated from dechlorination with
alkylation of His57, loss of the counter anion and ligand exchange
at the Ru center under the measurement conditions. MALDI-
TOF-MS analysis showed an increased mass number in the
1-L-modified chymotrypsin (Fig. S4 in ESIw). On the basis of
the hydrolase activity measurements and mass analyses, it is
concluded that 1-L is covalently introduced into the active site of
a-chymotrypsin through its intrinsic inhibition mechanism.
Conformational perturbation was evaluated using circular
dichroism (CD) spectroscopy (Fig. S5 and Fig. 3). Slight
changes in the spectra between the modified and unmodified
chymotrypsins were observed in the range from 200 to 280 nm
(Fig. S5 in ESIw). This change may be caused by the occupancy
of the protein cleft by 1-L. In contrast, a distinct difference
between free 1-L and that conjugated to chymotrypsin was
observed in the range from 300 nm to 650 nm of the CD spectra
(Fig. 3(a)). Compound 1-L in a buffer solution (without protein)
has an absorption band around 370 nm without CD signals
The chromatogram obtained using a carbomethoxy (CM)
Sepharose column suggests that no significant conformational
change occurs after the incorporation of 1-L, since the modified
and unmodified a-chymotrypsins are eluted at similar salt
concentrations (Fig. S2 in ESIw). This fact is also supported
by the CD spectra (vide infra). The UV-vis spectrum of the
a-chymotrypsin modified with 1-L after the column purification
Fig. 2 Hydrolase activity of a-chymotrypsin toward SucPhePNA.
(a) In the presence of 1-L. (b) In the presence of 1-D. (c) In the absence
of the inhibitor (control); 1 mM HEPES pH = 7.0 at 25 1C; [protein] =
6.4 mM, [1-L or 1-D] = 12.8 mM, [SucPhePNA] = 600 mM.
Fig. 3 CD spectra of 1-L with/without a-chymotrypsin in 100 mM
KClaq and UV-vis spectrum of a-chymotrypsin with 1-L; (a) CD
spectra; (b) UV-vis spectrum. The dotted lines in spectrum (b) were
drawn based on a wave analysis using Gaussian-type functions.
c
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Chem. Commun., 2012, 48, 1662–1664 1663

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Table 1 Catalytic activity of ring closing metathesis (RCM)a
The authors thank Ms Yuriko Nishiyama and Ms Mika
Yamamura for ES-MS technical support. This work was
supported by a Grant-in-Aid for Science Research on Innovative
Areas (Molecular Activation Directed toward Straightforward
Synthesis, MEXT Japan), Adaptable Seamless Technology
Transfer Program through Target-driven R & D (JST, Japan)
and ‘‘The Green Photonics Project’’ (NAIST, Japan).
Entry
Catalyst
Substrate
Solubilityb
TN (2 h)c
1d
1-L in protein
1-L only
1-L in protein
1-L only
1-L in protein
1-L only
GlcDAA
GlcDAA
DAA
DAA
TDA
TDA
WS, N
WS, N
WS, P
WS, P
IS
20
14
2d,e
3f
N.D.g
o2
4
4f,h
5e,i
6e,i
IS
12
a
b
100 mM KCl; 37 1C. Solubility in water and charge state: WS =
water-soluble, IS = water-insoluble (co-solvent is required to solubilize),
Notes and references
c
N = neutrally charged, P = positively charged. Turnover number at
1 (a) Y. Lu, Curr. Opin. Chem. Biol., 2005, 9, 118–126; (b) Y. Lu,
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751–766; (b) C. M. Thomas and T. R. Ward, Appl. Organomet.
Chem., 2005, 19, 35–39; (c) C. M. Thomas and T. R. Ward, Chem.
d
2 h. [GlcDAA] = 8.0 mM, [catalyst] = 50 mM (0.63 mol%).
Containing 10% DMSO (v/v). [DAA] = 8.0 mM, [catalyst] =
e
f
h
0.2 mM (2.5 mol%). g Product not determined. Containing 10% MeOH
i
(v/v). [TDA] = 1.0 mM, [catalyst] = 25 mM (2.5 mol%).
Soc. Rev., 2005, 34, 337–346; (d) V. Kohler, Y. M. Wilson, C. Lo,
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(Fig. 3(a), blue line), whereas the CD signals clearly appeared
when 1-L was conjugated to a-chymotrypsin (Fig. 3(a), red line).
This finding allows us to separate the UV-vis absorption band as
indicated in Fig. 3(b). The absorption band of 1-L around 370 nm
is assigned as MLCT (metal to NHC and to phenyl ligands) or
hybridization of the metal d orbitals and the ligand s orbitals,
based on the extinction coefficient (B6000 Mꢀ1 cmꢀ1, see the
spectra of the Ru complexes in CH2Cl2 in Fig. S6 of ESIw). The
induction of the CD signals in this region suggests the conforma-
tional perturbations to the NHC ligand and the Hoveyda ligand
upon binding to chymotrypsin. This finding supports our view
that the Ru complex is positioned inside of the protein cleft, not
simply bound to the protein surface.
Table 1 summarizes the activities of ring closing metathesis
(RCM) for two water-soluble diolefin compounds, N,N0-diallyl-
ammonium hydrochloride (DAA) and N,N0-diallyl-3-(1-D-
glucopyranosyl)oxypropanamide (GlcDAA) as well as N-tosyl-
diallylamine (TDA), a popular hydrophobic substrate in RCM
studies. GlcDAA produced the corresponding RCM product in
the presence of 1-L with and without a-chymotrypsin in similar
efficiencies (entries 1 and 2; see also Fig. S7, ESIw). In contrast,
an RCM product from DAA was not detected in the reaction
mediated by 1-L with the protein (entry 3). This is due to the
significant electronic repulsion between DAA and the protein
surface. Both the components are positively charged under the
reaction conditions (pI E 8.1 for a-chymotrypsin). The RCM
activity toward TDA decreased when 1-L was incorporated into
the protein (entries 5 and 6; see also Fig. S8, ESIw), similar to
previous research.11a The decrease in efficiency has resulted from
the restricted substrate access to the Ru reaction site in the
presence of protein. No decrease in the RCM efficiencies for
GlcDAA may originate from the employment of relatively high
substrate concentrations. Another possible reason is that the
microscopic hydration on the positively charged protein surface
would partially inhibit the access of the hydrophobic TDA to
the catalytic site included in the protein.
9 In ref. 7 polymerization of olefins is attained as a non-natural
function.
10 H. Neurath and G. W. Schwert, Chem. Rev., 1950, 46, 69–153.
11 The introduction of a Hoveyda–Grubbs catalyst into proteins by
covalent conjugation at a Cys residue on the protein surface or by
biotin–avidin non-covalent interactions. (a) C. Lo, M. R.
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In conclusion, a Hoveyda–Grubbs catalyst was introduced
into the cleft of a-chymotrypsin by the intrinsic inhibition
mechanism. The prepared protein maintains the ordered-
structure without significant denaturation and retains olefin
metathesis activity. Structural investigation of the modified
chymotrypsin and optimization of olefin metathesis for
development of stereo-selective syntheses are in progress.
18 (a) C. H. Johnson and J. R. Knowles, Biochem. J., 1966, 101,
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c
1664 Chem. Commun., 2012, 48, 1662–1664
This journal is The Royal Society of Chemistry 2012