Cyclodextrin retinylidene: A biomimetic kinetic trap model for rhodopsin

Article abstract of DOI:10.1016/j.bmcl.2005.12.052

All trans retinal was attached to both the primary face and the secondary face of β-cyclodextrin via a Schiff base linkage, analogous to that in rhodopsin. The new models were evaluated and compared with n-butylamine retinylidene Schiff base for their rates of hydrolysis, and factors that influence such rates. Competition studies using adamantane carboxylate demonstrated the kinetic trap theory by diminishing the binding of retinal in the cyclodextrin, thereby augmenting the rate of hydrolysis. NMR experiments indicate that the retinylidene is most probably bound in the form of a dimer.

Full text of DOI:10.1016/j.bmcl.2005.12.052

Bioorganic & Medicinal Chemistry Letters 16 (2006) 1523–1526
Cyclodextrin retinylidene: A biomimetic kinetic trap model
for rhodopsin
Kafui Kpegba, Matthew Murtha and Nasri Nesnas*
Department of Chemistry, Florida Institute of Technology, Melbourne, FL 32901, USA
Received 12 November 2005; revised 8 December 2005; accepted 9 December 2005
Available online 27 December 2005
Abstract—All trans retinal was attached to both the primary face and the secondary face of b-cyclodextrin via a Schiff base linkage,
analogous to that in rhodopsin. The new models were evaluated and compared with n-butylamine retinylidene Schiff base for their
rates of hydrolysis, and factors that influence such rates. Competition studies using adamantane carboxylate demonstrated the
kinetic trap theory by diminishing the binding of retinal in the cyclodextrin, thereby augmenting the rate of hydrolysis. NMR
experiments indicate that the retinylidene is most probably bound in the form of a dimer.
ꢀ 2005 Elsevier Ltd. All rights reserved.
Retinal, 1, is the chromophore responsible for triggering
the enzymatic cascade leading to vision in vertebrates. In
its 11-cis geometry, 1b, it is bound by the protein opsin,
covalently linked to Lys296 via a Schiff base generating
a retinylidene, which forms the photoactive photorecep-
tor, rhodopsin.1–3 Upon absorption of light, the 11-cis-
retinylidene undergoes an isomerization to the all trans
retinylidene, which in turn triggers the activation of
the G-protein. The active state of rhodopsin is referred
to as the meta II or MII (see Fig. 1). In the vertebrate
visual cycle, the all trans retinylidene is then hydrolyzed
and is released out of the chromophore binding pocket.
The all trans retinal is then shuttled through a series of
retinol binding proteins (RBPs) that convert it back to
the 11-cis geometry, thereby completing the visual cycle
(Fig. 1).3 The hydrolysis of the Schiff base is a crucial
step and has not been fully explored mechanistically.
Exploring the rate of hydrolysis is pivotal in understand-
ing the abnormalities that affect the visual cycle, such as
Stargardt disease.4 It has been shown that some mutants
of rhodopsin result in varying hydrolysis rates for the all
trans retinal, often leading to adverse visual effects.5,6
For instance, an accumulation of all trans retinal can
lead to severe problems including a common form of
age-related macular degeneration (AMD), in which
two retinal molecules will condense with a phosphatidyl
ethanolamine leading to a highly fluorescent pigment
known as A2E.7–10 The role of hydrogen bonding in
Schiff base hydrolysis in rhodopsin has been carefully
studied via conservative mutations of critical residues
near the binding site.11 We hereby present an alternative
approach to understanding hydrolysis via a simple arti-
ficial biomimetic model using b-cyclodextrin12 as the
recognition site for retinal, for which it is known to bind
in.13,14
b-Cyclodextrin (b-CD), 2, was functionalized from both
the primary and the secondary sides using standard
techniques12,15–20 to afford the primary 6-aminocy-
clodextrin and the secondary 3-aminocyclodextrin (see
Fig. 2). The aminocyclodextrins were subsequently
reacted with 1 equiv of retinal, 1, in the presence of
0.9 equiv of acetic acid in DMSO, to generate 3 and 4
in 98% and 93% yields, respectively. The latter Schiff
bases were isolated via lyophilization. The Schiff base
of retinal with n-butylamine, 5, was also generated and
used as a control in the hydrolysis studies.
The new structures 3 and 4 were characterized by UV,
1H NMR,21 and FAB mass spectrometry. Their UV
absorption in DMSO was at a kmax of 370 and
372 nm, with molar absorptivities of 112,800 and
98,400 molꢀ1 cmꢀ1, respectively. The 1H NMR in
DMSO-d6 revealed a distinctive peak for the aldimine
proton (the former aldehyde proton), which was at d
8.36 and at 8.45 ppm in structures 3 and 4, respective-
ly. FAB mass spectrometry revealed a mass of 1401.37
for the two structures corresponding to the [M+H]
ion.
Keywords: Cyclodextrin; Retinal; Cyclodextrin retinylidene; Schiff base
hydrolysis; Kinetic trap; Rhodopsin mimic; Dimer.
*
Corresponding author. Tel.: +1 321 674 8902; fax: +1 321 674
8951; e-mail: nesnas@fit.edu
0960-894X/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.12.052

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K. Kpegba et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1523–1526
phosphatidyl
ethanolamine
several RBPs
and enzymes
dehydrogenase
O
A2E
OH
all trans retinal
vitamin A
11-cis-retinal
1
O
Schiff base
hydrolysis
1b
Schiff base
hydrolysis
OPSIN
OPSIN
OPSIN
light
β
-ionone
H
binding
pocket
N
lys296
11-cis-retinylidene
PSB (Protonated
Schiff Base)
N
retinal is no longer
bound by the opsin
lys296
N
H
meta II
lys296
meta III
Rhodopsin
all trans retinylidene
leading to vision
Figure 1. A simplified outline of the visual cycle.
ed Schiff bases at their corresponding kmax, which was
442 nm for 3 and 447 nm for 4, in pH 4 buffer. Since
it is expected that retinal will bind into cyclodextrin13,14
in water, analogous to retinal’s binding into opsin, we
sought to evaluate the effect of the binding of the hydro-
phobic retinoid on the rate of Schiff base hydrolysis. It is
established that in rhodopsin, the binding that 11-cis-
retinal has for opsin ensures its stability in the dark
state, such that if hydrolysis of the 11-cis conformation
occurs, the bound chromophore ligand simply reforms
the Schiff base, hence the term kinetic trap.5 However
after isomerization of the 11-cis-retinal to the all trans,
the b-ionone moiety of the all trans retinal no longer
binds into the opsin binding pocket, thereby exposing
the Schiff base to water molecules that will lead to its
hydrolysis. Alternatively, the conformational change
imposed by the isomerization of retinal could reorient
the amino acid residues within the Schiff base binding
site in such a manner as to orchestrate a hydrogen bond-
ing network that will catalyze the hydrolysis of the Schiff
base.11 In our model studies, we envision compound 4
forming a dimer in water as depicted in Figure 3. The
NMR data presented below support our reasoning be-
hind it. Introduction of a strong hydrophobic b-cyclo-
OH
6
O
O
2
3
HO
OH
2
β-cyclodextrin
ref. 12
ref. 15-20
OH
6
NH2
6
O
H2N
O
O
O
2
2
3
3
H2N
OH
HO
OH
6−amino-β-cyclodextrin
3−amino-β-cyclodextrin
all trans
HOAc
retinal
DMSO
1
all trans
HOAc
retinal
DMSO
1
all trans
HOAc
DMSO
retinal
1
dextrin ligand, such as adamantane carboxylate which
has a binding constant in b-cyclodextrin of 104 Mꢀ1
,
22
will undoubtedly break up the dimer ‘releasing’ the ret-
inal from the hydrophobic cyclodextrin cavity. It is
therefore expected that the rate of hydrolysis will be en-
hanced in the presence of adamantane carboxylate. In
other words, in our model, adamantane carboxylate
N
N
N
β
β
5
4
3
N
Figure 2. The synthesis of the three retinylidene Schiff bases.
Hydrolysis studies were carried out at 25 ꢁC and in pH
4.0 buffer. Although the hydrolysis of the three Schiff
N
1
bases was followed by H NMR to prove the identity
of the products, the rates were measured more precisely
by following the decrease in absorption of the protonat-
Figure 3. Proposed dimer of 4 formed in aqueous solutions.

K. Kpegba et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1523–1526
1525
competitively displaces the b-ionone from its binding
pocket (the cyclodextrin cavity), which is a mimic for
the light induced isomerization of the retinal, which
releases the b-ionone from its binding pocket in
rhodopsin.
the bound retinal exposing it to the solvent. The rate
enhancement was nearly twofold in the case of the sec-
ondary face attached retinylidene 4 and about 50% in
the case of the primary face attached retinylidene 3. It
is likely that the secondary side of b-cyclodextrin, pos-
sessing the larger opening, will bind the retinal (in par-
ticular the b-ionone portion) better than the primary
side. Thus, the retinal bound to the secondary side of
b-cyclodextrin, being bound stronger in a dimeric form
(Fig. 3) than its primary counterpart, experiences a
greater rate enhancement upon release of the retinal
via competition with excess adamantane carboxylate.
Additional evidence for the binding of the retinoid into
the b-cyclodextrin (b-CD) cavity comes from NMR
anisotropy experiments carried out in D2O, using
CH3CN as an internal standard for accurate calibration
of chemical shifts. The binding of adamantane carboxyl-
ate into b-cyclodextrin resulted in an upfield shift of the
b-CD H-2, H-4, H-3, and H-5 by ca. 0.056 ppm and a
downfield shift of 0.101 ppm for the 3ꢁ protons of
AdCOꢀ2 . The well-resolved anomeric proton (H-1) of
b-CD is quite informative to follow as it exhibited an
upfield shift of 0.049 ppm. The anomeric proton of
structure 4 was also upfield shifted, albeit to a lesser ex-
tent, by ca. 0.041 ppm. This could be a result of the
smaller bulk of the b-ionone unit relative to the adaman-
tane structure. Introduction of the AdCOꢀ2 resulted in a
further upfield shift of the anomeric protons by about
0.035 ppm, indicating displacement of the retinoid from
the b-CD cavity. The upfield shift observed upon addi-
tion of AdCOꢀ2 to 4 is considerably smaller than the
0.056 ppm upfield shift observed when an equal concen-
tration of the AdCOꢀ2 was introduced in b-CD. This
indicated that the b-cyclodextrin cavity in 4 was already
occupied by a hydrophobic moiety, and that the smaller
0.035 ppm upfield shift is a result of the adamantane
being a stronger recognition element thereby displacing
the retinoid out of the cavity. The adamantane 3ꢁ pro-
tons experienced nearly the same exact 0.100 ppm down-
field shift upon binding into the b-CD cavity of 4 as they
have done so in b-cyclodextrin. Therefore, these studies
point to the direction that the b-CD cavity had a hydro-
phobic moiety bound in it, namely the retinoid, that was
displaced by the stronger ligand AdCOꢀ2 , which resulted
in the release of the retinoid. Unfortunately, the
b-ionone proton peaks were not clearly resolved and
we were unable to accurately monitor their shifts. We
were challenged by the rapid hydrolysis of 4, which
made the NMR measurements somewhat cumbersome.
The faster rate observed for the hydrolysis of the Schiff
base of the 1ꢁ face versus 2ꢁ face of cyclodextrin is a
combination of the binding effect of the different sides
of cyclodextrin as well as the difference in pKa of imines
(Schiff bases). The imine attached to the secondary face
of cyclodextrin is situated closer to the anomeric carbon
of the cyclodextrin rendering the electron density more
withdrawn than in the case of the imine of the primary
1
face. This is evidenced by the difference in H NMR
chemical shifts of the aldimine protons, which is
0.09 ppm downfield in structure 4 relative to 3, a result
of a more electron-withdrawing environment.21 There-
fore, according to the mechanism23 presented in Figure
4, a greater percentage of Schiff base 3 will be protonat-
ed shifting the equilibrium in favor of the attack of the
water molecule on the protonated Schiff base.
The n-butyl Schiff base, 5, is nearly an order of magni-
tude faster due to its exposure to solvent. The addition
of adamantane carboxylate resulted in lowering the rate
of hydrolysis, possibility due to the potentially intimate
ion-pair that renders the protonated Schiff more stable,
and hence more resistant to hydrolysis. It is known that
Glu113 in rhodopsin aids in the stabilization of the pro-
tonated Schiff base.5 In the case of 3 and 4, the binding
of the hydrophobic adamantane ring into the b-CD cav-
ity is more favorable than the formation of an ion-pair.
The rates of hydrolysis of three Schiff bases, 3–5 were
studied in both the presence and the absence of the
hydrophobic moiety adamantane carboxylate. The dis-
ruption of the retinal binding in the hydrophobic cavity
of b-cyclodextrin, most probably in the form of a dimer,
resulted in increased rates of hydrolysis. This mimics the
The results of hydrolysis in the absence and presence of
adamantane carboxylate at pH 4.0 are summarized in
Table 1. The rate constants were obtained by measuring
the initial rates of the various substrates at identical con-
centrations. As expected, the presence of adamantane
carboxylate enhanced the hydrolysis rate by displacing
k1
H
H
O
CD
H2O
CD
CD
Ret
N
H
Ret
N
Ret
N
H
Table 1. Observed rate constantsa for hydrolysis of the Schiff bases at
pH 4 (sꢀ1 · 10ꢀ3
)
Compoundb
In the absence of AdCO2ꢀ
In the presencec
of AdCOꢀ2
H
H
O
k2
O
O
rpt
rpt
3
4
5
8.58
2.02
68.6
12.7
3.84
28.7
CD
H
CD
H
Ret
N
H
CD
H
Ret
N
Ret
N
H
H
H
a The results were reproducible within a 5% error.
rpt = rapid proton transfer
b The concentrations of Schiff bases were 1 lM.
c AdCOꢀ2 is adamantane carboxylate. The concentration used was 100-
fold that of compounds 3–5.
Figure 4. Mechanism of the Schiff base hydrolysis, (CD = b-
cyclodextrin).

1526
K. Kpegba et al. / Bioorg. Med. Chem. Lett. 16 (2006) 1523–1526
12. Breslow, R.; Dong, S. D. Chem. Rev. 1998, 98, 1997.
13. Munoz-Botella, S.; Martin, M. A.; del Castillo, B.; Lerner,
D. A.; Menendez, J. C. Anal. Chim. Acta 2002, 468,
161.
14. Munoz Botella, S.; Martin, M. A.; del Castillo, B.;
Menendez, J. C.; Vazquez, L.; Lerner, D. A. J. Pharm.
Biomed. Anal. 1996, 14, 909.
biological rhodopsin model in which the rate of hydro-
lysis of the retinal is enhanced after its light induced
isomerization from 11-cis to all trans, which releases
the b-ionone ring from its hydrophobic binding pocket.
This indicates that our biomimetic model is in accor-
dance with the kinetic trap theory.
15. Yuan, D.-Q.; Tahara, T.; Chen, W.-H.; Okabe, Y.; Yang,
C.; Yagi, Y.; Nogami, Y.; Fukudome, M.; Fujita, K.
J. Org. Chem. 2003, 68, 9456.
Acknowledgments
16. Yuan, D.-Q.; Ohta, K.; Fujita, K. Chem. Commun. 1996,
821.
17. Yuan, D.-Q.; Dong, S. D.; Breslow, R. Tetrahedron Lett.
1998, 39, 7673.
18. Yuan, D.-Q.; Lu, J.; Atsumi, M.; Izuka, A.; Kai, M.;
Fujita, K. Chem. Commun. 2002, 730.
19. Fujita, K.; Tahara, T.; Imoto, T.; Koga, T. J. Am. Chem.
Soc. 1986, 108, 2030.
This work was supported by the Florida Institute of
Technology startup funds to the principal investigator
and the lab members. The National Science Foundation
(CHE 9013145) support for the AMX-360 MHz NMR
is gratefully acknowledged. We also thank Dr. Yasuhiro
Itagaki (Columbia University) for mass spec analysis.
20. Breslow, R.; Nesnas, N. Tetrahedron Lett. 1999, 40, 3335.
21. NMR data. Schiff base 3: 1H NMR (360 MHz, DMSO-d6)
d = 8.36 (d, J = 9.5 Hz, 1H, aldimine: 60-CD–CH2–N@C–
H), 6.87 (dd, J = 15.1, 11.3 Hz, 1H, ret-H-11), 6.46 (d,
J = 15.3 Hz, 1H, ret-H-12), 6.02–6.37 (m, 4H, ret-H-7, 8,
10, 14), 5.6–6.0 (m, 14H, 2ꢁ-CD-OH), 4.7–5.0 (m, 7H, CD
anomeric), 4.35–4.65 (m, 6H, 1ꢁ-CD-OH), 3.5–4.0 (m,
28H, CD), 3.1–3.5 (m, 14+, CD + DMSO), 2.05 (s, 3H,
ret-H-130), 1.9–2.0 (m, 2H, ret-H-4), 1.95 (s, 3H, ret-H-90),
1.71 (s, 3H, ret-H-50), 1.55 (m, 2H, ret-H-3), 1.42 (m, 2H,
ret-H-2), 1.00 (2s, 6H, ret-H-10,10). Schiff base 4: 1H NMR
(360 MHz, DMSO-d6) d = 8.45 (d, J = 9.8 Hz, 1H, aldim-
ine: 30–CD–N@C–H), 6.97 (dd, J = 15.1, 11.3 Hz, 1H, ret-
H-11), 6.50 (d, J = 15.3 Hz, 1H, ret-H-12), 6.16–6.35 (m,
3H, ret-H-7, 8, 10), 6.10 (d, J = 9.9 Hz, 1H, ret-H-14), 5.6–
6.0 (m, 13H, 2ꢁ-CD-OH), 4.7–5.0 (m, 7H, CD anomeric),
4.35–4.65 (m, 7H, 1ꢁ-CD-OH), 3.5–4.0 (m, 28H, CD), 3.1–
3.5 (m, 14+, CD + DMSO), 2.14 (s, 3H, ret-H-130), 1.95-
2.10 (m, 2H, ret-H-4), 2.00 (s, 3H, ret-H-90), 1.71 (s, 3H,
ret-H-50), 1.59 (m, 2H, ret-H-3), 1.45 (m, 2H, ret-H-2),
1.02 (2s, 6H, ret-H-10,10).
References and notes
1. Borhan, B.; Souto, M. L.; Imai, H.; Shichida, Y.;
Nakanishi, K. Science 2000, 288, 2209.
2. Nakanishi, K. Chem. Pharm. Bull. 2000, 48, 1399.
3. Rando, R. R. Chem. Rev. 2001, 101, 1881.
4. Sun, H.; Molday, R. S.; Nathans, J. J. Biol. Chem. 1999,
274, 8269.
5. Janz, J. M.; Farrens, D. L. Vision Res. 2003, 43, 2991.
6. Janz, J. M.; Fay, J. F.; Farrens, D. L. J. Biol. Chem. 2003,
278, 16982.
7. Sparrow, J. R. Proc. Natl. Acad. Sci. U.S.A. 2003, 100,
4353.
8. Sparrow, J. R.; Vollmer-Snarr, H. R.; Zhou, J.; Jang, Y.
P.; Jockusch, S.; Itagaki, Y.; Nakanishi, K. J. Biol. Chem.
2003, 278, 18207.
9. Sparrow, J. R.; Fishkin, N.; Zhou, J.; Cai, B.; Jang, Y. P.;
Krane, S.; Itagaki, Y.; Nakanishi, K. Vision Res. 2003, 43,
2983.
22. Carrazana, J.; Jover, A.; Meijide, F.; Soto, V. H.; Tato, J.
V. J. Phys. Chem. B 2005, 109, 9719.
23. Cooper, A.; Dixon, S. F.; Nutley, M. A.; Robb, J. L.
J. Am. Chem. Soc. 1987, 109, 7254.
10. Sparrow, J. R.; Cai, B.; Fishkin, N.; Jang, Y. P.; Krane,
S.; Vollmer, H. R.; Zhou, J.; Nakanishi, K. Adv. Exp.
Med. Biol. 2003, 533, 205.
11. Janz, J. M.; Farrens, D. L. J. Biol. Chem. 2004, 279, 55886.