Bio-functionalization of metal-organic frameworks by covalent protein conjugation

Article abstract of DOI:10.1039/c0cc03288c

Bioconjugation of functional proteins onto metal-organic frameworks (MOFs) has been achieved using activation of pendent linking groups of the organic linkers on the surface of MOFs. Fluorescent microscopy revealed successful conjugation of an enhanced fluorescent protein onto MOFs. In addition, Candida-antarctica-lipase-B-conjugated MOFs showed no loss of enantioselectivity and activity in transesterification of (±)-1-phenylethanol. The Royal Society of Chemistry.

Full text of DOI:10.1039/c0cc03288c

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COMMUNICATION
Bio-functionalization of metal–organic frameworks by covalent protein
conjugationw
a
b
b
c
c
Suhyun Jung, Youngmee Kim, Sung-Jin Kim, Tae-Hwan Kwon, Seong Huh* and
a
Seongsoon Park*
Received 16th August 2010, Accepted 17th December 2010
DOI: 10.1039/c0cc03288c
2
Bioconjugation of functional proteins onto metal–organic frame-
works (MOFs) has been achieved using activation of pendent
linking groups of the organic linkers on the surface of MOFs.
Fluorescent microscopy revealed successful conjugation of
an enhanced fluorescent protein onto MOFs. In addition,
Candida-antarctica-lipase-B-conjugated MOFs showed no
loss of enantioselectivity and activity in transesterification of
reactions and introduced several organic groups. Since then
many groups have reported modifications of MOFs using similar
approaches, which require a special functional group, such as
3
an amino group, for further modification. Therefore, these
approaches should be limited for the MOFs bearing such func-
tional groups in the original linker molecules.
We have explored to overcome this limitation by using the
linking groups of MOFs. In fact, typical organic linkers of the
MOFs contain carboxylate groups. For example, a series of
(
ꢀ)-1-phenylethanol.
Metal–organic frameworks (MOFs) are garnering more interest
in material science. Thermally and mechanically stable MOFs
with large void volumes have been useful in advanced applica-
tions such as selective catalysis, sensors, and high-efficiency gas
IRMOFs are composed of Zn
4
O nodes and dicarboxylate
1
organic linkers. Either pendent Zn O nodes or carboxylate
f
4
groups should occupy the surface of the frameworks and
thereby are exposed on the surface of IRMOFs. If pendent
carboxylate groups are present on the surface of MOFs, then
they could be activated and modified by simple organic
reactions. We assumed that a certain amount of the carboxylate
groups are exposed and can be activated by 1-ethyl-3-
(3-dimethylaminopropyl) carbodiimide (EDC) or dicyclohexyl
carbodiimide (DCC). Then, the activated carboxylates can be
conjugated with other compounds or even biomaterials, such
as proteins. We exploited this idea of conjugating proteins to
coordination polymers or MOFs with different structural
architectures, such as one-, two- and three-dimensional (3D)
structures. To our knowledge, the incorporation of functional
proteins to MOFs has not yet been achieved. We first chose an
enhanced green fluorescent protein (EGFP) as a model protein
because its presence and the folding status of EGFP can be
easily tracked by fluorescence microscopy.
1
storage. Although diverse MOF materials have been prepared
by solvothermal reactions between metal ions and organic linker
compounds, organic linkers should possess adequate thermal
stability because of their synthetic conditions.
To incorporate thermally unstable compounds into MOFs, the
‘
‘postsynthetic covalent modification’’ was recently explored by
2,3
several research groups. For instance, Cohen and coworkers
used IRMOF-3 (isoreticular metal–organic framework-3) as a
starting MOF material for postsynthetic modification. IRMOF-3
is composed of Zn(II) ions and 2-amino-1,4-benzene dicarboxylic
2 2
acid (NH -BDC). The two carboxylate groups of NH -BDC link
Zn O nodes but the amino group is intact during the IRMOF-3
4
formation. Thus, the amino groups are available for further
modification of the framework. Cohen and Wang have shown
that the amino groups can be modified by simple organic
A new indium(III)-based one-dimensional (1D) coordination
3 3
polymer was prepared from the reaction between In(NO )
a
Department of Chemistry, Center for NanoBio Applied Technology,
and 1,4-phenylenediacetic acid (H pda) in N,N-diethylformamide
2
Institute of Basic Sciences, Sungshin Women’s University,
Seoul 136-742, Korea. E-mail: spark@sungshin.ac.kr;
Fax: +82 2 920 2047; Tel: +82 2 920 7646
Department of Chemistry and Nano Science,
Ewha Womans University, Seoul 120-750, Korea
Department of Chemistry and Protein Research Center for
Bio-Industry, Hankuk University of Foreign Studies,
Yongin 449-791, Korea. E-mail: shuh@hufs.ac.kr
(DEF) (see ESIw). The crystal structure was shown in Fig. S1
and S2 (ESIw). The coordination mode of indium in the
b
1
D-MOF is 8-coordinated by four carboxylates. The overall
c
bonding scheme of the indium ion is very similar to those
observed in several previous examples of 3D In-MOFs. The
indium ion formed a pseudotetrahedral structure with four
bridging pda ligands chelated in a bidentate manner. However,
the bent geometry of methylene groups of the bridging pda
ligands prevented the pseudotetrahedral motifs from expanding
into a 3D network. Instead, it formed a 1D coordination
polymer bearing a relatively large cavity between two
negatively-charged indium ions and two parallel benzene rings
w Electronic supplementary information (ESI) available: Experimental
details, X-ray crystallographic information of 1D-MOF, PXRD patterns
for activated and EGFP-conjugated MOFs, IR analysis of 1D-MOF,
2
emission spectra and CLSM images of EGFP-MOFs, N adsorption/
desorption isotherms for the CAL-B-3D-MOF and measurements of
the specific activities of CAL-B-MOFs. CCDC 789848. For ESI and
crystallographic data in CIF or other electronic format see DOI:
1
0.1039/c0cc03288c
2
904 Chem. Commun., 2011, 47, 2904–2906
This journal is c The Royal Society of Chemistry 2011

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IR spectra of EDC- and EGFP-decorated 1D-MOF showed
different patterns from that of 1D-MOF (Fig. S5 in ESIw).
Thus, it can be concluded that EGFP was successfully
introduced on the surface of the MOFs and the anchored
EGFP is still functional.
Solid-state luminescence measurements also provided the
characteristic spectrum of EGFP (Fig. S6 in ESIw). The surface
modification of the MOFs was further confirmed by using a
confocal laser scanning microscope (CLSM). Most green
emissions from the EGFP-decorated MOFs were observed
from the surface of crystals (Fig. S7 in ESIw). The amounts of
Fig.
D-polymer, [(Et
scopic images of EGFP coated MOFs. (a) 1D + EGFP. (b) 2D + EGFP.
c) 3D + EGFP. An Olympus WIB filter set (lex = 460–490 nm;
em 4 515 nm) was used for recording the fluorescence.
1
Schematic representation of the bioconjugation of the
1
2
NH )(In(pda) )] , with EGFP. Fluorescence micro-
2
2 n
EGFP coated on the MOFs were determined as 0.048, 0.052,
ꢂ1
(
0
.064 mg g of the 1D-, 2D-, and 3D-MOFs, respectively. To
l
our knowledge, this is the first direct bioconjugation of
coordination polymers or MOFs.
from the pda ligands. In fact, the counter cation, diethylammonium
ion, is perfectly captured in this cavity. The diethylammonium
ions were generated from the decomposition of solvent molecules,
We also applied this strategy to conjugate MOFs with a
functional protein, such as an enzyme. We chose CAL-B
(Candida antarctica lipase B) that catalyzes hydrolysis or
formation of an ester. CAL-B is one of the most widely used
DEF, during the reaction. The 2D-MOF, [Zn(bpydc)(H O)ꢁ
2
0
0
8
(
H
2
O)]
n
, is composed of zinc ions and 2,2 -bipyridine 5,5 -
5
enzymes because of its high activity and selectivity. CAL-B
4
dicarboxylate (bpydc). IRMOF-3 was used as the 3D-MOF.
was conjugated to the three MOFs through the same coupling
methods. The amounts of proteins decorated on MOFs were
All organic linkers of the MOFs contain carboxylate groups.
We attempted to conjugate MOFs with EGFP by treating
EDC to activate the dangling carboxylate groups of MOFs
ꢂ1
determined as 0.12, 0.17, and 0.18 mg g of the 1D-, 2D-, and
3D-MOFs, respectively (see Table 1). PXRD patterns showed
that the structural integrity of frameworks was intact after
CAL-B conjugation (Fig. 2). It is noteworthy to realize that
even the hydrolytically unstable 3D IRMOF-3 maintained its
framework structure during the conjugation. Presumably,
hydrophobic CAL-B–protein conjugation enhances water-
(
Fig. 1). We noticed, however, the 1D-polymer was only
activated and conjugated with EGFP. Presumably, pda was
rather easily activated in aqueous media because the aliphatic
6
carboxylate is more reactive than the aromatic one. In addi-
tion, this observation implies that no physical adsorption of
EGFP to the MOFs occurs. For 2D- and 3D-MOFs, we
activated the carboxylates by DCC in dichloromethane instead
of EDC in an aqueous buffer. This activation step did not alter
the crystallinity of the MOFs (Fig. S3 in ESIw).
7
resistance of MOFs. In addition, N sorption measurement
2
at 77 K for the CAL-B–3D-MOF exhibited very low
2
ꢂ1
Brunauer–Emmett–Teller (BET) surface area, 15.0 m
g
,
compared with the activated native 3D-MOF which showed
2
ꢂ1
For the following protein-conjugation step, we need to use a
buffer (PBS, pH 7.3) as a reaction media in order to conjugate
proteins because proteins are not soluble in most organic
solvents. The use of a buffer could be problematic because
MOFs, especially the 3D-MOF (IRMOF-3), may be not stable
the BET surface area of 1605.1 m g (Fig. S8 in ESIw). These
data demonstrate the efficient outer surface functionalization
of 3D-MOF by CAL-B instead of structural disintegration
of 3D-MOF.
7
against water. Therefore, we evaluated the stability of the
Table 1 The catalytic activity and enantioselectivity of CAL-B–MOF
conjugates
3D-MOF in water and PBS buffer by comparing PXRD patterns
before and after soaking the 3D-MOF in water or PBS buffer.
To our surprise, 1-h incubation of the 3D-MOF in a PBS
buffer did not alter the PXRD pattern of the original phase
while longer incubation (for 6 h) in PBS buffer or incubation in
water changed the patterns (Fig. S4 in ESIw). We envision that
this finding will broaden the scope of IRMOF-3 functionaliza-
tion in an aqueous buffer solution. After activation of the
MOFs, we successfully conjugated EGFP with the MOFs.
However, the longer reaction period (4 1 h) for binding
EGFP to 1D- and 3D-MOFs altered the structural integrity
while the 2D-MOF remained unchanged (Fig. S3 in ESIw).
The six-histidine tag attached to the N-terminal of EGFP may
coordinate to the metal ions of the 1D- and 3D-MOFs and
result in the structural alteration but not for the 2D-MOF.
The amount of
CAL-B protein
decorated/mg g
Specific activity/
mmol min mg
ꢂ1
ꢂ1
ꢂ1
a
Enzyme
E
b
Free CAL-B
n.a.c
n.a.
0.12
n.a.
0.17
n.a.
0.18
0.037 ꢀ 0.013
4200
n.a.
4200
n.a.
4200
n.a.
4200
d
1D-MOF
1D + CAL-B
2D-MOF
n.d.
15 ꢀ 3.1
n.d.
22 ꢀ 6.6
n.d.
2D + CAL-B
3
3
D-MOF
D + CAL-B
39 ꢀ 1.3
2 4
The different coordination environment (N O ) of 2D-MOF is
a
E = enantiomeric ratio as defined by C. S. Chen, Y. Fujimoto, G.
b
Girdaukas and C. J. Sih, J. Am. Chem. Soc. 1982, 104, 7294. Errors
presumably more stable than those of the other MOFs. The
presence of EGFP on the surface of the MOFs was confirmed
by a fluorescence microscope. The EGFP-decorated crystals
emitted a uniform green fluorescence (Fig. 1). In addition, the
are standard deviations for three measurements obtained from recycling
d
experiments. n.a. = not available. n.d. = not detected.
c
This journal is c The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 2904–2906 2905

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decrease of pH caused by formation of a by-product, such as
8
a
acetic acid which can be generated by hydrolysis of vinyl acetate.
After CAL-B was incorporated onto 3D-MOF, the
CAL-decorated 3D-MOF is still bearing unmodified amino
groups. These amino groups may be served as further modifi-
cation sites. Thus, the CAL-B-decorated 3D-MOF can be
conjugated with another protein. We introduced EGFP
ꢂ1
(
0.063 mg g ), which was activated by EDC, to the
CAL-B-decorated 3D-MOF (Fig. S11 in ESIw) to form dual
protein-conjugated MOF. The dual protein-conjugated
3
D-MOF showed multi-functionality, i.e. fluorescence and
ꢂ1
ꢂ1
transesterification activity (38 mmol min mg ).
These results clearly indicate that functional proteins can
be decorated on MOFs without losing their functions.
This approach is, therefore, an important step towards the
functional modification of MOFs.
Fig. 2 PXRD patterns of the 1D-, 2D- and 3D-MOFs, and
CAL-B-conjugated 1D-, 2D- and 3D-MOFs.
This work was supported by Basic Science Research
Program through the National Research Foundation of Korea
To evaluate the activity of the immobilized CAL-B, we tested
the catalytic activity in the transesterification of (ꢀ)-1-phenyl-
ethanol as a model reaction (Table 1). In addition, CAL-B
(
NRF) funded by the Ministry of Education, Science and
Technology (MEST) (NRF-2007-331-C00170).
has high enantioselectivity toward (R)-(+)-1-phenylethanol
9
Notes and references
(
E = 4200). We also measured the enantioselectivity of
the immobilized CAL-B and the free CAL-B.
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MOFs without the activation step or after treatment of the
activated MOFs by n-butylamine to block binding of CAL-B.
The amount of physically adsorbed CAL-B was estimated by
difference of the absorbances of the supernatant at 280 nm, before
and after incubation. The amount of physically bound CAL-B
was negligible because the difference was near the detection limit
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4
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3
D-MOF showed about a 10 -fold higher activity than free
3
4
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the moment, we speculate that the MOFs might provide
confined spaces nearby the surface resided enzymes for substrates
to contact enzymes more efficiently. The enhanced rate
acceleration of 3D-MOF compared with 1D- and 2D-MOFs
could be explained by the same speculation. In addition, the
amino groups of IRMOF-3 presumably help to maintain the
optimum pH for the enzymatic reaction because an addition of
a weak base to an enzymatic reaction media can prevent a
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906 Chem. Commun., 2011, 47, 2904–2906
This journal is c The Royal Society of Chemistry 2011