The first successful investigation into a cyclodextrin-based enzyme model as an efficient catalyst for luminol chemiluminescent reaction.

Article abstract of DOI:10.1039/b111018g

The chemiluminescence of the luminol-H2O2 system is found for the first time to be remarkably enhanced by the Ce(IV) complexes of cyclodextrin dimers.

Full text of DOI:10.1039/b111018g

The first successful investigation into a cyclodextrin-based enzyme model
as an efficient catalyst for luminol chemiluminescent reaction
De-Qi Yuan,* Jianzhong Lu, Masato Atsumi, Ayako Izuka, Masaaki Kai and Kahee Fujita
Faculty of Pharmaceutical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan.
E-mail: deqiyuan@net.nagasaki-u.ac.jp; Fax: +81-95-846 5736; Tel: +81-95-846 5736
Received (in Cambridge, UK) 4th December 2001, Accepted 19th February 2002
First published as an Advance Article on the web 6th March 2002
The chemiluminescence of the luminol–H
found for the first time to be remarkably enhanced by the
Ce(IV) complexes of cyclodextrin dimers.
2
O
2
system is
2 2
luminol solution, 40 ml of 0.25 M H O , and then 50 ml of 0.5
mM Ce(IV) complex solution. As soon as the addition was
finished, the resultant solution was mixed on an auto-mixer, and
the measurement of light output was immediately started. For a
typical run, the time interval was about 5s between the mixing
of components and CL measurement.
A characteristic of cyclodextrins (CDs) is their ability to form
inclusion compounds in aqueous solution with a large variety of
chemical species ranging from organic molecules to inorganic
ions. The molecules included in the CD cavity usually
demonstrate chemical and physical properties quite different
from those of their free forms in the bulk aqueous environment.
Therefore, CDs are widely employed as molecular recognition
sites to develop functional systems for diverse purposes, among
Fig. 1 shows the CL decays of luminol. Catalysts are of
paramount importance in aqueous luminol CL. Luminol was
almost chemiluminescently mute in the absence of catalysts.
None of the dual CDs, Ce(IV) ion and EDTA–Ce(IV) complex
demonstrated obvious influence on the CL reaction. However,
as soon as 6–Ce(IV) complex was used, the emission of luminol
was remarkably enhanced. The result is indicative of the
catalytic importance of the 6–Ce(IV) complex. The complex
formation was found to take a few minutes to complete and this
enabled the examination of activity–time dependence of the
mixture of 6 and Ce(IV) freshly prepared from their individual
solutions. The test showed the catalytic ability of the mixture
increased rapidly with the mixing time and approached a
1
which the construction of CD-based biomimetic materials and
2
molecular sensors lies in the center of interest and has been
witnessing great progress. Upon complex formation, molecular
sensors (either themselves or their bound guests) frequently
demonstrate changes in absorption/photoluminescence proper-
2
3
ties, but less frequently a change in chemiluminescence (CL).
As enzyme mimics of oxidases, metalloporphyrins are conju-
gated to CD to effect oxidation of double bonds or saturated C–
H bonds in a controlled manner,1 and selenium is introduced in
,4
5
CDs to scavenge the active oxygen. In this paper, we report the
first example of dual CD-based metal complexes to catalyze the
CL reaction of luminol.
The synthesis and structures of the dual cyclodextrins are
depicted in Scheme 1. b-CD was modified with an amino group6
at one of the C-6 and C-3 positions by literature procedures.
The reactions of EDTA dianhydride with a slight excess of CD
amines 3 ~ 5 were undertaken in dry DMF at room temperature
and subsequent chromatography of the reaction mixtures on a
reverse-phase Lobar column afforded the products 6 ~ 8 in 85,
7
1 and 33% yields, respectively. The structures of the dual CDs
6
~ 8 were confirmed by FAB-MS and NMR spectra.
Cerium complexes† of the dual CDs were obtained by mixing
the aq. solution of dual CDs with freshly prepared aq. solution
of Ce(NH
4
)
2
(NO
3
)
4
, and their catalytic properties were tested
CO
on the CL reactions of luminol. To 100 ml of 0.1 M Na
2
3
M
Fig. 1 The CL intensity decays in the luminol chemiluminescent
reactions.
2
5
solution were successively added 10 ml of 1.0 3 10
3 3 3
Scheme 1 The synthesis of dual cyclodextrins: (a) TsCl–pyridine; (b) NaN –DMF; (c) NaH–CH I–DMF; (d) Ph P–DMF; (e) 2-naphthalenesulfonyl
chloride–30% aq. CH CN; (f) K CO –H O; (g) NaN –H O; (h) EDTA dianhydride–DMF.
3
2
3
2
3
2
7
30
CHEM. COMMUN., 2002, 730–731
This journal is © The Royal Society of Chemistry 2002

Scheme 2 Possible pre-organization of luminol and oxidant by the
catalyst.
constant value about 5 min after the mixing of the individual
Ce(IV) and 6 solutions, which further evidenced the catalytic
importance of complex formation.
Many metal cations are well-known to catalyze this reaction,
increasing the light emission or at least speeding up the
oxidation to produce the emitter and therefore the onset of light
production.7 It has also been reported that the binding of
luminol to hydrophobic regions could strongly enhance the
Fig. 2 CL emission versus catalyst concentrations.
of the primary side near the Ce(IV) center. Indeed the
permethylated dual CD 7–Ce(IV) showed a catalytic activity
only one fourth that of 6–Ce(IV).
We also examined the influence of catalyst concentration on
the CL intensity. Upon varying [6–Ce(IV)] from 0 to 0.4 mM,
the CL intensity increased proportionally at first and then
showed increasing downward deviation (Fig. 2).
8
luminol CL. However, the present system is different from
those cases in that it combines both a hydrophobic binding site
and the catalytic metal complex center in one single molecule
just as most metalloenzymes do. It is therefore the covalent
conjugation and cooperation of b-CD and EDTA-Ce(IV
)
In conclusion, the present research demonstrated the first
enzyme model that has both substrate-binding and catalytic sites
to catalyze the luminol chemiluminescent reaction. The coop-
components that are essential for the catalytic ability since
neither the component individuals nor their mechanical combi-
nation resulted in obvious CL changes.
eration of the hydrophobic binding site and the catalytic Ce(IV
)
Based on the above results we tentatively propose a pre-
organization of the two reactants by the catalyst (Scheme 2) for
center was proved to be of pivotal importance for getting high
efficacy in amplifying the luminol CL. The idea of pre-
organizing the chemiluminescent ‘fuels’ and oxidants by
enzyme models may offer many opportunities to improve the
CL as well as a promising approach to develop high efficacy but
low cost CL systems.
the 6–Ce(IV) catalyzed chemiluminescent reaction: the Ce(IV
)
–
center binds the HOO while the CD hydrophobic cavity brings
2
luminol close to the bound HOO , thus the local concentrations
of both luminol and oxidant are greatly increased. The CL is
switched on only when the metal center and the hydrophobic
cavity can efficiently cooperate, which means the geometry of
the dual cyclodextrin would be very important, and this was
proved to be the case. When the dual CD 8 was used instead of
Notes and references
† Freshly prepared Ce solution should be used to prepare the Ce complexes,
otherwise the activity will be greatly lost. The Ce complexes were stable,
and did not show obvious loss of activity even after being stored for a few
weeks. Even so, the complex solutions were all freshly prepared and used
within one hour.
6
, no enhancement of luminol CL was observed. It is worthy of
note that compounds 8 and 6 differ only in the positions at
which the EDTA linkage is attached to CD moieties. Dual CD
8
has CD cavities of the same shape as, but opposite
arrangement to those of compound 6. CL measurements
indicated that this difference in cavity arrangement in 8 resulted
a dramatic loss of catalytic ability. The remarkable difference in
catalytic ability of the two dual CDs can be rationalized as
follows. The secondary side of CD is less hydrophobic but more
acidic. Under the experimental condition (pH ≈ 11.5), it will
ionize and become even worse for accommodating luminol. On
the other hand, the primary side is more hydrophobic and will
remain un-ionized at the experimental pH and, therefore, is
preferential for binding luminol. Based on this consideration,
dual CD 6 is expected to locate luminol towards the center of the
molecule where the oxidant is bound, whereas 8 directs luminol
outwards to either terminals of the molecule. In addition, the
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ionized secondary OH groups of 8 may compete with HOO in
coordinating to the Ce(IV) on the same side. Breslow demon-
strated that in heptakis(6-methylamino)–CD, the methyl groups
7
8
9
were inserted inwards to the CD cavity forming a floor. It is
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