On-and-Off Control of Allosteric Affinity toward Flavin Mononucleotide by the Use of a Pseudocyclophane Formed with Cu(I) as an Effector

Full text of DOI:10.1021/jo9619600

2788
J . Org. Chem. 1998, 63, 2788-2789
Sch em e 1. F or m a tion a n d Decom p osition of th e
Mu ltir ecogn ition System
On -a n d -Off Con tr ol of Alloster ic Affin ity
tow a r d F la vin Mon on u cleotid e by th e Use of
a P seu d ocyclop h a n e F or m ed w ith Cu (I) a s a n
Effector
Tatsuya Nabeshima,*,† Akihiro Hashiguchi,†
Suguru Yazawa,‡ Tsutomu Haruyama,‡ and
Yumihiko Yano‡
Department of Chemistry, University of Tsukuba, Tsukuba,
Ibaraki 305-8571, J apan, and Department of Chemistry,
Gunma University, Kiryu, Gunma 376-8515, J apan
Received October 22, 1996 (Revised Manuscript Received March 23,
1998 )
Molecular functions of biological systems such as the
catalytic activity of enzymes, molecular recognition of recep-
tors, etc., are often controlled by allostery to maintain the
material balance necessary for life.1 The allostery of the
molecular functions is also very important and useful in the
construction of artificial functional systems2 regulated by
molecular information.3,4a
Sch em e 2. Syn th esis of th e Ar tificia l Alloster ic Hosts
(1 a n d 2)
We have designed artificial ionophores that contain two
bipyridine moieties to bind a Cu(I) ion as an effector.5
Quantitative conversion from its less selective form to the
more selective one was performed. Recently, control of
molecular recognition by metal coordination has been re-
ported.6 However, on-off action of the recognition ability
in situ has not been achieved by the systems utilizing a
metal ion. Here, we wish to report the first example of an
allosteric system reversible in situ by the use of host 1 for
binding flavin mononucleotide (FMN), which is an important
cofactor of various redox enzymes.7 Host 1 consists of two
dimethyldiphenylmethane skeletons derived from 4,4′-iso-
propylidenediphenol, two 2,2′-bipyridine moieties, and an
ammonium group (Scheme 1). Upon complexation of 1 with
a Cu(I) ion, the formation of pseudocyclophane bearing a
binding cavity with the ammonium moiety in close proximity
results in a considerable positive allosteric effect on extrac-
tion and transport of FMN.
Hosts 1 and 2 were prepared according to a synthetic
route shown in Scheme 2. A bathochromic shift and a MLCT
band (λmax ) 479 nm in CHCl3-CH3CN (95:5, v/v)) charac-
teristic of a tetrahedral complex of bipyridines with Cu(I)5
were observed in the absorption spectrum of 1‚Cu(I). On
addition of CuCl, resonances of the picolyl methyl and meth-
ylene protons of 1 were shifted upfield (3.01 f 2.71 ppm and
2.69 f 2.48 ppm, respectively, in CDCl3-CD3CN-CD3OD
(47:3:50, v/v)) by an anisotropic effect of the bipyridine
moieties due to the tetrahedral geometry because the two
bipyridine parts are forced into a close and orthogonal
geometry.5 Downfield shifts were also observed in protons
of the bipyridine moieties. This result supports coordination
of the ligands to Cu(I), too. Both UV-vis and 1H NMR
spectroscopic titrations using the spectral changes described
above suggested the exclusive formation of a 1:1 complex
between 1 (or 2) and Cu(I) with a tetrahedral geometry. A
FABMS spectrum of Cu(I) complex of 1 ascertained a 1:1
stoichiometry (m/z 1645, [M - Br]+, M calcd for 1‚CuCl).
We call such a framework pseudocyclophane because the
cyclic structure is maintained by metal coordination instead
of covalent bonding, as seen in the pseudocrown ethers.5
Allosteric control of FMN recognition was achieved in an
extraction experiment using a 1,2-dichloroethane-water
biphasic system. After mixing with an aqueous solution of
FMN sodium salt (10) and a 1,2-dichloroethane solution
containing the host, changes of concentrations of 10 in the
aqueous phase were determined from the decrease in the
* To whom correspondence should be addressed. Tel.: Int. code + 298-
53-4223. Fax: Int. code + 298-53-6503. E-mail: nabesima@staff.chem.
tsukuba.ac.jp.
† University of Tsukuba.
‡ Gunma University.
(1) Perutz, M. F. Mechanism of Cooperativity and Allosteric Regulation
in Proteins; Cambridge University Press: Cambridge, 1990.
(2) Rebek, J ., J r. Acc. Chem. Res. 1984, 17, 258-264.
(3) Lehn, J .-M. Angew. Chem., Int. Ed. Engl. 1990, 29, 1304-1319.
(4) (a) Nabeshima, T. Coord. Chem. Rev. 1996, 148, 151-169. (b) Beer,
P. D.; Smith, D. K. Prog. Inorg. Chem. 1997, 46, 1-96. (c) Linton, B.;
Hamilton, A. D. Chem. Rev. 1997, 97, 1669-1680.
(5) (a) Nabeshima, T.; Inaba, T.; Furukawa, N. Tetrahedron Lett. 1987,
28, 6211-6214. (b) Nabeshima, T.; Inaba, T.; Furukawa, N.; Hosoya, T.;
Yano, Y. Inorg. Chem. 1993, 32, 1407-1416.
(6) (a) Maverick, A. W.; Buckingham, S. C.; Yao, Q.; Bradbury, J . R.;
Stanley, G. G. J . Am. Chem. Soc. 1986, 108, 7430-7431. (b) Schneider, H.-
J .; Ruf, D. Angew. Chem., Int. Ed. Engl. 1990, 29, 1159-1160. (c)
Schwabacher, A. W.; Lee, J .; Lei, H. J . Am. Chem. Soc. 1992, 114, 7597-
7598. (d) Goodman, M. S.; J ubian, V.; Linton, B.; Hamilton, A. D. J . Am.
Chem. Soc. 1995, 117, 11610-11611. (e) Pre´vot-Halter, I.; Smith, T. J .;
Weiss, J . Tetrahedron Lett. 1996, 37, 1201-1204.
(7) Chemistry and Biochemistry of Flavoenzymes; Mu¨ller, F., Ed.; CRC
Press: Boston, 1991; Vol. 2.
S0022-3263(96)01960-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 04/17/1998

Communications
J . Org. Chem., Vol. 63, No. 9, 1998 2789
Ta ble 1. Solven t Extr a ction of F la vin by th e
Ta ble 2. Tr a n sp or t of F la vin s by 1 th r ou gh a Liqu id
Mem br a n ea
Mu ltir ecogn ition System a
decrease of
[flavin]aq (%)
decrease of
[flavin]aq (%)
optical density of flavins in the
receiving phase after 4 d
b
b
host
[10]aq
[11]aq
host
[10]aq
[11]aq
host
FMN Na (10)
riboflavin (11)
1
20 ( 2
56 ( 5
5 ( 1
3 ( 1
2 + Cu+
7 + Cu+
none
3 ( 1
2 ( 1
1
<1
1
0
1
0.05
0.22
0.04
0.07
0.07
0.03
0.02-0.03
0.03
0.02
0.02
0.02
1 + Cu+
1
1 + Cu+
2
2
<1
2 + Cu+
7 + Cu+
none
a
Organic phase; CH2ClCH2Cl/CH3CN ) 10:0.1 (v/v), [1] ) [2]
) 6.0 × 10-5 M, [7] ) 1.2 × 10-4 M, [Cu+] ) 6.6 × 10-5 M; aqueous
phase; [flavin]0: the initial concentration of flavins in the aqueous
0.01-0.02
b
a
phase, 6 × 10-5 M, monitored at 445 nm. Decrease of [flavin]aq
Organic phase (1% CH3CN-CH2ClCH2Cl) 50 mL: [host] )
(%) ) ([flavin]0 - [flavin]aq)/[flavin]0(100), where [flavin]aq is the
1.2 × 10-4 M, [7] ) 2.4 × 10-4 M, [Cu+] ) 1.3 × 10-4 M. Source
phase (dist H2O) 4 mL: [10] ) 1.2 × 10-3 M, [11] ) 3 × 10-4 M.
Receiving phase (dist H2O) 40 mL monitored at 445 nm.
concentration of flavins in the aqueous phase after mixing.
Cu(I) and off with 12 and prohibited by Et3NH+Cl-. As far
as we know, there is no example of an artificial allosteric
recognition system switchable in situ, to date.
Transport ability of 1 toward 10 through a 1,2-dichloro-
ethane layer as a liquid membrane using a dual cylindrical
cell5 is regulated allosterically with Cu(I). Amounts of 10
(after 4 days) in the receiving phase determined by absorp-
tion spectroscopy are summarized in Table 2. A small
amount of 10 was carried in the absence of 1. The amount
of the guest transported by 1 was about twice as much as
that of the control. In the presence of Cu(I), the rate of the
transport of 10 is increased by ca. 4-fold compared to 1 alone.
If the control experiment is taken into account, the overall
enhancement by 1 and Cu(I) together is 10-fold. In contrast,
host 2 does not transport 10 efficiently, even when the
complexation with Cu(I) occurs. The interaction between 1
and 10 was not examined in the same solvent system due
to the very low solubility of 10. However, the complexation
of 1 with Cu(I) does not enhance transport ability toward
11, although a half amount of the guest is transported
without 1. Thus, these results also indicate the importance
of multirecognition by the pseudocyclophane cavity and the
ammonium moiety in close proximity for a high performance
of the FMN binding. However, all data obtained here are
consistent with the fact that the multiple recognition of the
1‚Cu(I) complex toward 10 is caused by the ammonium
group and the cavity of the pseudocyclophane produced upon
the complexation with Cu(I). In addition, ESIMS spectros-
copy strongly suggests the complexation of 1‚Cu(I)‚10 be-
cause the molecular ion, [1‚Cu‚10 - Na - Br]+, was observed
and the isotope patterns are in good accordance with the
theoretical values.
F igu r e 1. Plausible structure of 1‚Cu(I)‚10.
absorbance of 10 at 445 nm in the aqueous phase (Table 1).
As reflected by such a decrease for 1, a considerable allosteric
effect was observed using CuCl as an effector. In the
absence of Cu(I), the degree of decrease is small (20 ( 2%).
However, the concentration of 10 in the aqueous phase is
decreased drastically (56 ( 5%) in the presence of Cu(I).
Furthermore, a mixture of 7 and Cu(I) did not affect the
amount of 10 in the aqueous phase significantly (2 ( 1%).
Thus, the large decrease observed in the mixture of 1 and
Cu(I) results from the interaction between the pseudocyclo-
phane and 10. In contrast, 2 bearing a t-BuMe2Si group
instead of an ammonium moiety does not exhibit an effective
allostery (5 ( 1% without Cu(I), 3 ( 1% with Cu(I)).
Absorption spectra of the organic phases suggested that
under these conditions both 1 and 2 are converted to the
corresponding 1:1 complexes with Cu(I). A large enhanced
decrease of guest concentrations did not occur in the case of
riboflavin (11), since it does not contain a phosphate group.
These results clearly indicate that presence of the cavity and
the ammonium group is necessary to recognize 10. Namely,
the pseudocyclophane with the ammonium moiety in close
proximity to the cavity exhibits a very good affinity toward
10. This affinity may be explained by cooperative binding
with the two sites in a fashion as illustrated in Figure 1,
which is also suggested by CPK model inspection.
Moreover, reversal of the recognition of 10 by 1‚CuCl was
easily achieved. The absorbance of 10 in the aqueous phase
is highly recovered (77 ( 2% of the initial value of 10, i.e.,
23 ( 2% decrease) by the addition of bathocuproine (12, 4
equiv to 1) into the biphasic system. This recovery is caused
by formation of the very stable tetrahedral 12‚Cu(I) complex.
From 1H NMR spectroscopy, the Cu(I) ion bound in 1 is
completely removed by 4 equiv of the highly Cu(I) selective
reagent 128 in CDCl3-CD3CN-CD3OD (47:3:50, v/v). In-
stead of 12, Et3NH+Cl- was also employed as an additive
to recover 10 into the aqueous phase (94 ( 2% of the initial
value of 10, i.e., 6 ( 2% decrease). This inhibition suggests
the importance of electrostatic interaction between the
ammonium moiety of 1‚Cu(I) and the phosphate anion of
10. Hence, the allosteric recognition is switched on with
Accurate structural analysis of the interactions between
1‚Cu(I) and 10 was not carried so far. Further study is
necessary to elucidate the binding ability in detail. None-
theless, switchable allostery for the solvent extraction and
transport was observed in the present system. The concept
described here is essential for the construction of molecular
systems with sophisticated functions whose activities are
controlled by information at the molecular level. Further
extension using the present framework for allosteric regula-
tion of molecular recognition for other biologically important
molecules and of catalytic activities is in progress.
Ack n ow led gm en t. This work was partially supported
by a Grant-in-Aid for Scientific Research from the Ministry
of Education, Science, Sports, and Culture, J apan (No.
09640622). We thank Associate Professor Dr. Ernst Horn
of the University of Tsukuba for reviewing the English text.
Su p p or tin g In for m a tion Ava ila ble: Details of the synthetic
procedures, 1H and 13C NMR data for all new compounds, ESIMS
data of 1‚Cu‚10, and spectroscopic titrations for complexation with
Cu(I) (14 pages).
(8) For a review, see: Borchardt, L. G.; Butler, J . P. Anal. Chem. 1957,
29, 414-419.
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