Trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific immobilization and fluorescent labeling

Article abstract of DOI:10.1039/c1ob05401e

Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native lipase was not immobilized at pH 7 and 25 °C. After the entire modification procedure, the catalytic activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a selective Diels-Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this labeled-CAL-B was also possible by a Diels-Alder reaction on solid phase in excellent yields.

Full text of DOI:10.1039/c1ob05401e

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PAPER
trans,trans-2,4-Hexadiene incorporation on enzymes for site-specific
immobilization and fluorescent labeling†
Marco Filice, Oscar Romero, Jose M. Guisan and Jose M. Palomo*
Received 14th March 2011, Accepted 26th April 2011
DOI: 10.1039/c1ob05401e
Lipase B from Candida antarctica (CAL-B) has been site-directedly modified by the introduction of a
trans,trans-hexadiene moiety onto lipase molecules, identified by MALDI-TOF. This modification on
CAL-B permitted its immobilization on Q-Sepharose supports in excellent yields (>95%) when native
lipase was not immobilized at pH 7 and 25 ◦C. After the entire modification procedure, the catalytic
activity of the protein on the solid support was surprisingly increased 2-fold. A tailor-made
maleimide-fluorophore derivative was specifically covalently linked to the protein in high yield via a
selective Diels–Alder reaction in aqueous media. Furthermore, the NBD-labeled-CAL-B was also
immobilized on the ionic support, retaining around 80% of the specific activity. The preparation of this
labeled-CAL-B was also possible by a Diels–Alder reaction on solid phase in excellent yields.
is very likely that multiple functional handles are introduced when
Introduction
this residue is targeted for modification.11 The properties of the
bioconjugates thus generated are influenced by the degree and site
of modification, therefore it is highly desirable to introduce new
functional groups in a more defined manner.12
The selective modification of proteins has been a long-standing
challenge of modern chemical biology, with many applica-
tions, ranging from the study of natural post-translational
modifications1 to the development of protein-based materials. Site-
specific modifications have been used in investigations of protein
expression and localization, as tools in structure–function studies,
in pharmacokinetics of protein-based drugs and to improve
bioavailability, and in the development of biosensors.2–4
The incorporation of novel functionalities into proteins has been
extensively studied for these items. Fluorescent labels, polymers,
nucleic acids, polypeptides, and carbohydrates have been coupled
to proteins to study or manipulate their function.5–6 These mod-
ifications have also permitted the site-specific immobilization of
proteins for purification, manipulation or property modulation.7–8
Importantly, these reactions occur at or near physiological
pH and are compatible with the complex array of functional
groups commonly found in biological macromolecules allowing
conjugation reactions to be carried out on unprotected substrates.
In most of these cases, the side chain functionalities of the amino
acids cysteine and lysine were targeted for conjugation reactions
because of the reactivity of the free thiol and amine moieties,
respectively.9–10 However, cysteines are often involved in disulfide
bonds that occur inside proteins. Breakage of these bonds can
easily lead to loss of structural integrity and, hence, function.
Lysine residues are positively charged, predominantly located at
the protein surface, and relatively abundant. As a consequence, it
One approach to achieve this involves the modification of some
protein amino acids of which only some residues are exposed to the
solvent. Tyrosine functionalization by organometallic complexes 13
or chemical modifications of tryptophan14 are two examples.
Actually, different kinds of techniques for protein derivatization,
based on synthetic organic chemistry such as expressed protein
ligation,15 Staudinger ligation16 or “click” ligation,17 have been
developed in the context of biological molecules in aqueous media.
However, due to the multifunctionality of biomacromolecules,
there is a major and continuing demand for development of
new technology providing alternatives to the methods men-
tioned above. The required chemistry must be compatible with
the functional groups found in proteins and proceed chemos-
electively under mild conditions and in aqueous solution,
preferably in the absence of any potentially denaturating co-
solvent.
The chemical incorporation of a diene group is an interesting
modification for site-specific introduction of different molecules
into proteins such as fluorescent label, polymers, peptides, by
chemoselective cycloaddition reaction such as the Diels–Alder
[4p+2p] reaction. The characteristic of the Diels–Alder reaction
makes it as an ideal methodology for bioconjugation18–20 because
it is accelerated in aqueous media at very mild reaction conditions
(pH 6, room temperature). This reaction has been successfully
applied for site-specific modification of streptavidin or Rab
proteins.21
Departamento de Biocata´lisis, Instituto de Cata´lisis (CSIC), c/marie
curie 2, Cantoblanco, Campus UAM, 28049 Madrid, Spain. E-mail:
josempalomo@icp.csic.es; Fax: +34-91-585-47-60
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c1ob05401e
This chemical strategy could be quite interesting when ap-
plied to enzymes, in particular lipases whose properties can be
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modulated to create artificial catalysts with new activities and
high selectives.22–23
500 nm. This experiment clearly showed the differences between
CAL-B and modified-CAL-B (Fig. 2). The CAL-B spectrum
showed three different peaks at 215, 238 and 260 nm. This
broad peak at 260 nm, corresponding to the absorption bands
of aromatic residues in the protein, was extremely decreased in
the diene-modified CAL-B, indicating a loss on the aromaticity
probably of the indole group in Trp by the chemical modification.14
Therefore, in the present manuscript we describe the chemical
modification of Candida antarctica (fraction B) lipase (CAL-
B), one of the most used lipases in chemistry,24 by a trans-
trans hexadiene derivative for selective immobilization on ionic
exchange matrices and for the direct or subsequent chemical
incorporation of fluorescent labels by Diels–Alder reaction.
Results and discussion
Site-directed incorporation of trans,trans-2,4-hexadiene on CAL-B
The heterobifunctional cross-linker 1 (Scheme 1) was prepared
as previously described25 in two steps by esterification of succinic
anhydride and hexadienol followed by transformation of the diene
derivative into the N-hydroxysuccimidyl ester. This linker was
attached to CAL-B molecules by acylation of N activated residues
at pH 7 through the N-hydroxysuccinimidyl (NHS) group (Scheme
1). The molar ratio between the cross-linker and CAL-B was
kept relatively low (3 : 1) to prevent multiple labeling of the lipase
molecules.
Fig. 2 UV-visible absorption spectra of modified-CAL-B. (A) CAL-B,
(B) 2.
Tryptic hydrolysis was performed to determine which amino
acids were modified with diene molecules. A peptide profile from
MALDI was obtained after the hydrolysis for CAL-B and diene
modified CAL-B (2) (Fig. 3, Figures 2SI–3SI) corresponding
approximately to 60% of the protein sequence (Fig. 4). Mainly,
four different peptides were found (Fig. 3). A clear difference in
the peptide sequence was found between these two enzymes.
Scheme 1 Site-specific modification of CAL-B by 1.
However, analysis of the MALDI-TOF spectra of modified
CAL-B (2) revealed that on average each lipase was equipped
with three diene linkers (Fig. 1).
Fig. 3 MALDI spectra after tryptic hydrolysis. A. CAL-B. B. diene–
CAL-B (2).
Fig. 1 MALDI-TOF spectra. A. CAL-B; B. diene–CAL-B (2).
Considering that lysines are not reactive at the pH of the
modification (pKa 10.2), the chemical modification of the protein
was performed on different groups; for instance at the amino
terminal residue and for example at the tryptophan moiety.14
Analyzing the tridimensional structure of CAL-B we observed
that this lipase shows five tryptophans (Trp52, Trp65, Trp104,
Trp113, Trp155) (Figure 1SI).
A peak at 1519.734 [Mmodified CAL-B + Na] was found corresponding
to the peptide sequence LPSGSDPAFSQPK including the termi-
nal amino group modified with the diene molecule (Fig. 3). The
other peptides between both proteins were identical; the sequence
included three tryptophans, Trp104, Trp113 and Trp 155 (Fig. 4),
which were not modified.
The profile of the UV-visible absorption spectrum for the soluble
CAL-B and diene–CAL-B (2) was studied in a range of 199–
Thus, by consideration of the loss in UV signal for modified
CAL-B in Fig. 2 together with these results, we could estimate that
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Scheme 3 Maleimide–NBD derivative 6 incorporation in CAL-B 2
Fig. 4 Amino acid sequence of CAL-B. Peptides identified by tryptic
molecules by Diels–Alder conjugation.
hydrolysis in red.
CAL-B was probably modified in Trp52 and Trp65 in addition to
the terminal amino group.
compatible with reactive amino acids such as lysines, aspartic
acids, glutamic acids or histidines.
Site-directed incorporation of NBD labeled molecules on CAL-B
by Diels–Alder cycloaddition in aqueous media
Immobilization of modified-CAL-B (2, 7)
The modified-CAL-B (2), anchoring a water-stable diene, is an
excellent candidate for further combination with a variety of
maleimide probes to perform a Diels–Alder reaction in aque-
ous media. An interesting application of this classic synthetic
organic reaction on proteins is to label them with fluoro-
phores.26
A 7-nitrobenzofurazan (NBD)-labeled maleimide 6 was pre-
viously synthesized (Scheme 2). NBD-Cl (3) was transformed
on EDA-NBD 4 in 90% yield by a two step synthesis; an
aromatic substitution with the previously synthesized N-Boc-
1,2-diaminoethane27 and a Boc deprotection by TFA treatment.
After that, 4 was modified with 6-(maleimide)hexanoyl chloride
(5) for the incorporation of the maleimide group on the NBD
molecule. Compound 6 was synthesized in 77% overall yield after
purification by flash chromatography.
The stability of the hexadiene function in aqueous solution and
its compatibility with the functional groups present in amino
acids opens up the opportunity to combine the Diels–Alder
ligation method with other techniques, for example the oriented
immobilization of enzymes.
Particularly, the immobilization of proteins by ionic exchange
on cationic supports goes through a multipoint mechanism, where
the enzyme is oriented by the richest area of carboxylic groups
on the protein surface.28 This is an interesting methodology
because represents a reversible, although strong, immobilization
methodology.28
This characteristic permits us to work with the proteins on
the solid-phase, for example for further modifications, and finally
recovery by a simple desorption with salt.
However, commercial CAL-B was not immobilized on DEAE
or Q-Sepharose – supports with a high density ◦of tertiary or
quaternary amines respectively – at pH 7 and 25 C. Increasing
pH up to 9 did not improve the result.
The diene–CAL-B 2 was successfully immobilized by ionic
exchange on these supports at these mild conditions. More than
95% yield of immobilized protein was achieved in one hour even
using 1 mg up to 12 mg pure protein per g support (Fig. 5A). To
demonstrate that the immobilization is exclusively through ionic
interactions, the immobilized enzyme preparation was incubated
at different concentrations of NaCl (Figure 4SI). A full desorption
of the protein from the support was found at 1 M of NaCl,
indicating that the enzyme was strongly immobilized on the
resin.
Surprisingly, the specific activity of the modified enzyme in the
hydrolysis of pNPB was increased 2 fold after the immobilization
(Fig. 5B) which never has been observed for a lipase in an
ionic exchange interaction, a characteristic only observed for
this enzyme on the hydrophobic interfaces.29 Modified CAL-B 2
showed the same specific activity as native CAL-B in solution. The
conformational change of Phe48 (Figure 4SI) – after the chemical
modification of the protein with the diene – could be responsible
for this enzyme immobilization or hyperactivation.
Scheme 2 Synthesis of NBD-maleimide derivative 6. a:1) NH2CH2CH2-
NHBoc, DIPEA, CH2Cl2; 2) TFA, CH2Cl2, r.t.; b: 5, DIPEA, CH2Cl2, r.t.
The protein–hexadiene complex 2 was treated with fluorescent
NBD-labeled maleimide derivative 6 (30-fold excess) in 5 mM
sodium phosphate buffer at pH 6.5 and room temperature for 24
h (Scheme 3). Subsequently unreacted dienophile was removed by
membrane centrifugation affording fluorescent protein complex 7.
The formation of this new fluorescent protein was verified by SDS-
PAGE via UV-detection (Scheme 3). Incubation of unmodified
CAL-B and dienophile molecule 6 resulted in no detectable specific
product (Scheme 3), confirming that the observed conjugation of
maleimide probes proceeded via Diels–Alder ligation.
Therefore these results revealed that the Diels–Alder reaction is
an interesting methodology for chemical modification of lipases,
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Fig. 5 Immobilization of CAL-B on Q-Sepharose supports. A) Immo-
bilization scheme. B) Relative specific activity of unmodified CAL-B and
diene CAL-B 2. Specific activity was measured on the hydrolysis of pNPB
(see experimental part).
Fig. 7 Fluorescent labeling of immobilized CAL-B 2 by Diels–Alder.
SDS-PAGE: Lane 1: CAL-B 2, lane 2: protein supernatant from the boiled
preparation 8.
The oriented immobilization of the labeled-CAL-B (7) on
Q-Sepharose support is shown in Fig. 6. The adduct 7 was
immobilized on this support at pH 7 and 25 ◦C.
unreacted dienophile was removed by simple filtration and several
washing steps using distilled water.
Finally, 100 mg of CAL-B- Q-Sepharose derivative 8 (Fig. 7)
were treated with 100 mL of rupture buffer and boiled for 5 min.
Then 12 mL of the solution were rapidly injected on the SDS-PAGE
gel and the Diels–Alder ligation was analyzed via UV-detection
(Fig. 7). Therefore, the site-directed incorporation of NBD was
possible on the lipase on the solid-phase.
Conclusions
A convenient method to modify CAL-B in a site-specific way
has been developed. A trans,trans-hexadiene moiety was in-
troduced by modification of the amino terminal residue and
tryptophans on the lipase molecules to act as a functional handle.
Subsequently, a tailor-made fluorophore was covalently linked
via the highly selective Diels–Alder reaction in aqueous media.
This modification procedure on CAL-B permitted its immo-
bilization on Q-Sepharose supports in excellent yields (>95%)
when native lipase was not immobilized at pH 7 and 25 ◦C.
After the entire modification procedure, catalytic activity was
surprisingly increased 2-fold once immobilized on the solid ionic
support.
Finally, the labeled-CAL-B also was immobilized on this
support although retaining the same initial activity and the
incorporation of the fluorophore was also successfully performed
directly on the immobilized modified enzyme. This solid phase
method presents many advantages for an easy coupling and
purification with high final yields.
Fig. 6 Immobilization of labeled-CAL-B 7 on Q-Sepharose supports. A)
Immobilization scheme. B) Relative specific activity of unmodified CAL-B
and diene CAL-B 7. Specific activity was measured on the hydrolysis of
pNPB (see experimental section).
In this case, 80% of the offered protein (3 mg g-1 support) was
immobilized after 1 h although no hyperactivation of the modified
enzyme was observed after immobilization, the specific activity of
the immobilized 7 was similar to that in solution and even similar
to native CAL-B.
In particular, the site-specific incorporation of fluorophores in
lipases could be useful for studying the catalytic mechanism by
controlling the opening and closing of the lid for example using
a FRET system,30 the enzyme dynamics at different interfaces,31
to quantify surface diffusion by fluorescence recovery after
Labeling of diene–CAL-B (2) on the solid-phase
The site-specific incorporation of the fluorophore on the CAL-
B was performed on the solid phase too. The hyperactivated
and immobilized CAL-B diene was treated with NBD-labeled
maleimide 6 (60-fold excess) in 5 mM sodium phosphate buffer
at pH 6.5 and room temperature for 48 h (Fig. 7). Then, the
31
photobleaching (FRAP) or for binding and characterization
on surfaces, such as magnetic nanoparticles.32
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(t, 2H, J = 7.35, CH2 next to carbonyl), 1.74–1.64 (m, 4H,
maleimide-2CH2), 1.45–1.37 (m, 2H, maleimide-CH2). HRMS
(ESI+): m/z calcd for C18H20N6O6 [M + H]+ 417.39, found 417.15.
Experimental
R
Qꢀ-Sepharose 4BCL was from GE Healthcare (Uppsala, Sweden).
trans,trans-2,4-Hexadienol, p-nitrophenyl butyrate (pNPB),
succinic anhydride, N-hydroxysuccinimide (NHS), 1-ethyl-
3-(3-dimethylaminopropyl)-carbodiimide
Preparation of the CAL-B–diene conjugate (2)
(EDC),
4-chloro-
To a solution of commercial CAL-B (50 mg, 1.43 nmol) in 5%
dioxane in 5 mM NaH2PO4 pH 6.5 buffer solution, the diene
cross-linker 1 (4.3 nmol in 500 ml of dioxane) was added and kept
stirring overnight at 25 ◦C. The reaction mixture solution was
dialyzed with four changes of distilled water. After the samples
were concentrated and characterized by MALDI-TOF.
7-nitrobenzofurazan (NBD-Cl), N,N-diisopropylethylamine
ethylenediamine (DIPEA), ethylenediamine (EDA) and 6-
maleimidohexanoic acid were purchased from Sigma Chem Co.
Candida antarctica lipase (fraction B) (CAL-B) was purchased
from Novozymes. MALDI MS spectra were recorded by
using 2,5-dihydroxybenzoic acid (DHB) or a-cyano-4-hydroxy
cinnamic acid (CHCA) as matrix. The tryptic hydrolysis and
the protein identification by LC-MS/MS were carried out in the
‘CBMSO PROTEIN CHEMISTRY FACILITY’, a member of
the ProteoRed network.
Enzymatic activity assay
The activities of the soluble lipase, supernatant and enzyme
suspension were analyzed spectrophotometrically measuring the
increment in absorbance at 348 nm produced by the release of
p-nitrophenol (pNP) (e = 5.150 M-1 cm-1) in the hydrolysis◦of 0.4
mM pNPB in 25 mM sodium phosphate at pH 7 and 25 C. To
initialize the reaction, 0.05–0.2 mL of lipase solution or suspension
was added to 2.5 mL of substrate solution in magnetic stirring.
Enzymatic activity is given as mmol of hydrolyzed pNPB per
minute per mg of enzyme (IU) under the conditions described
above.
6-(Maleimido)-N-(2-(7-nitrobenzo[c][1,2,5]oxadiazol-4-
ylamino)ethyl)hexanamide (6)
NBD-Cl (3) (0.25 g, 1.245 mmol) was added at room temperature
to a solution of N-Boc-1,2-diaminoethane (0.2 g, 1.245 mmol)
and N,N-diisopropylethylamine (0.434 ml, 2.49 mmol) in CH2Cl2
(10 mL). The resulting dark brown solution was stirred until
disappearance of the starting products monitored by TLC (ethyl
acetate : n-hexane, 6 : 4). After 3 h the solution was diluted with
10 mL of CH2Cl2 and the organic phase was washed with 3 ¥
10 mL of NaHCO3 5% in distilled water and 2 ¥ 10 mL with
water. The recollected organic phase was dried over anhydrous
Na2SO4, filtered and concentrated in vacuo. Subsequently, the
obtained crude was dissolved in 5 mL of CH2Cl2 and then
5 mL of trifluoroacetic acid were slowly added dropwise at room
temperature. After 1 h, diethyl ether (60 ml) was added. The
precipitated product was collected by vacuum filtration, washed
with cold diethyl ether and dried, yielding 4 (90%).
6-(Maleimido)hexanoic acid chloride (5) was prepared by
adding dropwiseoxalylchloride (4.73mmol, 0.4mL)to an ice-bath
cooled solution of maleimidocaproic acid (0.946 mmol, 0.2 g) and
10 mL of anhydrous DMF in CH2Cl2 (5 mL). After a few minutes,
the mixture is allowed to reach room temperature and left under
stirring over night. Volatiles were removed under reduced pressure
and the residue was azeotroped three times with 5 ml of CH2Cl2
each time. The resulting yellow crude residue was used without
any further purification.
Diels–Alder ligation of the soluble CALB–diene conjugate 2 with
fluorescently labeled dienophile probe (6)
Hexadiene-conjugate 2 at 0.1 mg mL-1 concentration in 5 mM
NaH2PO4 pH 6.5 buffer was incubated with 30-fold excess of
maleimide probe 6 (dissolved in 500 mL of acetonitrile). The
temperature was kept at 25 ◦C for 24 h while shaking. After this
time, the excess dienophile was removed by passing the reaction
mixture through an Amicon Ultra 10kDa (DyeEx columns from
Qiagen) and several times washed with 5% (v/v) acetonitrile in
distilled water. The ligated protein 7 was analyzed by SDS-PAGE
on 10% (w/v) polyacrylamide gels by Coomassie staining. To
detect coupling of fluorescent probe 6 to hexadiene–CAL-B, prior
of the Coomassie staining, the gel was analyzed using a Molecular
Imager Gel Doc XR System - Bio-Rad Laboratories.
Immobilization of CALB–diene (2) and CAL-B–NBD (7) on Q
Sepharose support
One gram of Q-Sepharose was added to 30 mL of modified protein
solution (3 mg CALB–diene 2 or CALB–NBD 7 dissolved in 5mM
NaH2PO4 buffer pH 7). The suspension was then stirred for 1 h at
25 ◦C. Periodically, samples of the supernatants and suspensions
were withdrawn, and the enzyme activity was measured as
described above. After that, the immobilization was stopped, the
supernatant removed by filtration and the supported modified
lipase washed several times with distilled water. Immobilization
yield for CAL-B–diene was >95% and for CAL-B–NBD was 70%.
Aminated-NBD
4
(0.41 mmol, 0.138 g) and N,N-
diisopropylethylamine (1.61 mmol, 0.285 mL) were suspended in
anhydrous CH2Cl2 (4 mL) and the mixture was cooled on an ice
bath. After 10 min, to this mixture, the acyl chloride 5 (0.451
mmol, 0.094 g dissolved in 1 mL of CH2Cl2) was added dropwise.
After 15 min on ice, the reaction mixture was allowed to warm up
to room temperature and stirring over night.
The solution was concentrated under reduced pressure and the
residue was purified by flash column chromatography on silica
gel (CH2Cl2/acetone: 7/3) to give a bright orange solid 6 (0.4 g,
overall yield of entire synthetic pathway 77%). 1H-NMR (CDCl3,
400 MHz): d = 8.48 (d, 1H, J = 8.62, NBD), 7.7 (dd, 1H, J =
8.98, J = 3.27, NH), 7.53 (dd, 1H, J = 9.0, J = 3.3, NH), 6.68 (s,
2H, maleimide), 6.16 (d, 1H, J = 8.63, NBD), 3.73–3.59 (2 m, 4H,
2CH2-EDA), 3.48 (t, 2H, J = 7.10, CH2 next to maleimide), 2.25
Solid-phase Diels–Alder ligation of the supported CAL-B–diene
conjugate with fluorescently labeled dienophile probe 6
CALB-diene immobilized in Sepharose
Q
(3 mgprotein g-1,
0.08571 nmol) was suspended in 20 ml of 5 mM NaH2PO4 pH
6.5 buffer. 60-fold excess of NBD-maleimide probe 6 (2.14 mg,
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5.1425 nmol dissolved in 500 mL of acetonitrile) were added. The
temperature was kept at 25 ◦C for 48 h while shaking. After this
time, the reaction was stopped and the derivative (supported lipase
modified with fluorophore) recovered by filtration and washing
several times with distilled water.
11 Y. Wang, S. Youngster, M. Grace, J. Bausch, R. Bordens and D. F.
Wyss, Adv. Drug Delivery Rev., 2002, 54, 547.
12 K. L. Heredia, D. Bontempo, T. Ly, T. J. Byers, S. Halstenberg and H.
D. Maynard, J. Am. Chem. Soc., 2005, 127, 16955.
13 S. D. Tilley and M. B. Francis, J. Am. Chem. Soc., 2006, 128, 1080.
14 (a) J. M. Antos, J. M. McFarland, A. T. Iavarone and M. B. Francis, J.
Am. Chem. Soc., 2009, 131, 6301; (b) J. M. Antos and M. B. Francis,
J. Am. Chem. Soc., 2004, 126, 10256; (c) H. Chiba, M. Hisatake, M.
Hirose and E. Sugimoto, Biochim. Biophys. Acta (BBA)-Enzym., 1973,
327, 380.
Acknowledgements
15 (a) T. W. Muir, Annu. Rev. Biochem., 2003, 72, 249; (b) V. Muralidharan
and T. W. Muir, Nat. Methods, 2006, 3, 429.
This work has been sponsored by the Spanish Ministry of Science
and Innovation (Project CTQ2009-07568) and CSIC (Intramural
Project 200980I133). We also thank to CONICYT and Programa
Bicentenario Becas-Chile for financial support for Mr. Romero.
We thank Dr Valeria Grazu (Institute of Nanotechnology, Uni-
versity of Zaragoza) for MALDI analytical support.
16 J. Kalia, N. L. Abbott and R. T. Raines, Bioconjugate Chem., 2007, 18,
1064.
17 (a) B. Wieczorek, B. Lemcke, H. P. Dijkstra, M. R. Egmond, R. J. M.
Klein Gebbink and G. Van Koten, Eur. J. Inorg. Chem., 2010, 13, 1929;
N. D. Weikart and H. D. Mootz, ChemBioChem, 2010, 11, 774; K. A.
Kalesh, H. Shi, J. Ge and S. Q. Yao, Org. Biomol. Chem., 2010, 8, 1749;
S. K. Mamidyala and M. G. Finn, Chem. Soc. Rev., 2010, 39, 1252.
18 M. Wiessler, W. Waldeck, C. Kliem, R. Pipkorn and K. Braun, Int. J.
Med. Sci., 2010, 7, 19.
19 J. M. Langenhan and J. S. Thorson, Curr. Org. Synth., 2005, 2, 59.
20 T. K. Tiefenbrunn and P. E. Dawson, Biopolymers, 2010, 94, 95.
21 J. M. Palomo, Eur. J. Org. Chem., 2010, 33, 6303.
Notes and references
1 (a) D. P. Gamblin, S. I. van Kasteren, J. M. Chalker and B. G. Davis,
FEBS J., 2008, 275, 1949; (b) G. Walsh and R. Jefferis, Nat. Biotechnol.,
2006, 24, 1241.
2 L. Wang, L. Harrington, E. Trebicka, H. N. Shi, J. C. Kagan, C. C.
Hong, H. Y. Lin, J. L. Babitt and B. J. Cherayil, J. Clin. Invest., 2009,
119, 3322.
3 N.-N. Aye-Han, Q. Ni and J. Zhang, Curr. Opin. Chem. Biol., 2009, 13,
392–397.
4 L. Tang, A. M. Persky, G. Hochhaus and B. Meibohm, J. Pharm. Sci.,
2004, 93, 2184.
22 J. M. Palomo, Curr. Org. Synth., 2009, 6, 1–14.
23 (a) C. Godoy, B. de las Rivas, M. Filice, G. Fernandez-Lorente, J. M.
Guisan and J. M. Palomo, Process Biochem., 2010, 45, 534–541; (b) J.
´
M Palomo, G. Fernandez-Lorente, R. Fernandez-Lafuente and J. M.
´
´
Guisan, Adv. Synth. Catal., 2007, 349, 1119–1127.
´
24 T. Hudlicky and J. W. Reed, Chem. Soc. Rev., 2009, 38, 3117.
25 A. D. de Araujo, J. M. Palomo, J. Cramer, O. Seitz, K. Alexandrov and
H. Waldmann, Chem.–Eur. J., 2006, 12, 6095.
5 (a) W. P. Heal and E. W. Tate, Org. Biomol. Chem., 2010, 8, 731; (b) M.
Sunbul and J. Yin, Org. Biomol. Chem., 2009, 7, 3361; (c) K. N. Allen
and B. Imperiali, Curr. Opin. Chem. Biol., 2010, 14, 247.
6 (a) H. Y. Song, M. H. Ngai, Z. Y. Song, P. A. MacAry, J. Hobley and
M. J. Lear, Org. Biomol. Chem., 2009, 7, 3400; (b) M. A. Gauthier and
H.-A. Klok, Chem. Commun., 2008, 2591; (c) E. Basle´, N. Joubert and
M. Pucheault, Chem. Biol., 2010, 17, 213.
7 (a) F. Rusmini, Z. Zhong and J. Feijen, Biomacromolecules, 2007, 8,
1775; (b) P. Jonkheijm, D. Weinrich, H. Schroder, C. M. Niemeyer and
H. Waldmann, Angew. Chem., Int. Ed., 2008, 47, 9618; (c) D. Weinrich,
P.-C. Lin, P. Jonkheijm, U. T. T. Nguyen, H. Schro¨der, C. M. Niemeyer,
K. Alexandrov, R. Goody and H. Waldmann, Angew. Chem., Int. Ed.,
2010, 49, 1252.
26 (a) A. D. de Araujo, J. M. Palomo, J. Cramer, M. Kohn, H. Schroder,
R. Wacker, C. Niemeyer, K. Alexandrov and H. Waldmann, Angew.
Chem., Int. Ed., 2006, 45, 296; (b) A. Doerr, Nat. Methods, 2006, 3, 77.
27 A. Eisenfuhr, P. S. Arora, G. Sengle, L. R. Takaoka, J. S. Nowick and
¨
M. Famulok, Bioorg. Med. Chem., 2003, 11, 235.
28 R. Torres, C. Mateo, M. Fuentes, J. M. Palomo, C. Ortiz, R. Fernandez-
Lafuente, J. M. Guisan, A. Tam and M. Daminati, Biotechnol. Prog.,
2002, 18, 1221.
´
29 U. Derewenda, A. M. Brzozowski, D. M. Lawson and Z. S. Derewenda,
Biochemistry, 1992, 31, 1532.
30 (a) K. Kikuchi, Chem. Soc. Rev., 2010, 39, 2048; (b) G. R. Szilvay, M.
A. Blenner, O. Shur, D. M. Cropek and S. Banta, Biochemistry, 2009,
48, 11273.
8 (a) L. S. Wong, F. Khan and J. Micklefield, Chem. Rev., 2009, 109,
4025; (b) M. Shi, J. H. Wosnick, K. Ho, A. Keating and M. S. Shoichet,
Angew. Chem., Int. Ed., 2007, 46, 6126.
9 K. Blank, J. Morfill and H. E. Gaub, ChemBioChem, 2006, 7, 1349.
10 F. M. Veronese, B. Sacca, P. P. de Laureto, M. Sergi, P. Caliceti, O.
Schiavon and P. Orsolini, Bioconjugate Chem., 2001, 12, 62.
31 (a) A. W. Sonesson, H. Brismar, T. H. Callisen and U. M. Elofsson,
Langmuir, 2007, 23, 2706; (b) L. J. Cruz, P. J. Tacken, F. Bonetto, S. I.
Buschow, H. J. Croes, M. Wijers, I. J. de Vries and C. G. Figdor, Mol.
Pharmaceutics, 2011, 8, 520.
32 B. E. Feller, J. T. Kellis, L. G. Cascao-Pereira, C. R. Robertson and C.
W. Frank, Langmuir, 2010, 26, 18916.
5540 | Org. Biomol. Chem., 2011, 9, 5535–5540
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