Study of α-crustacyanin utilizing halogenated canthaxanthins

Article abstract of DOI:10.1021/ol026146f

(Matrix presented) R = OH; Astaxanthin R = F, Cl, Br, I; Halogenated canthaxanthins The preparations and spectroscopic characteristics of five all-trans halogenated canthaxanthins are described in this letter. The air/light-sensitive halogenated canthaxan

Full text of DOI:10.1021/ol026146f

ORGANIC
LETTERS
2002
Vol. 4, No. 15
2521-2524
Study of r-Crustacyanin Utilizing
Halogenated Canthaxanthins
,†
Jin Liu,* Nicole L. Shelton,† and Robert S. H. Liu‡
Department of Chemistry, Murray State UniVersity, Murray, Kentucky 42071, and
Department of Chemistry, UniVersity of Hawaii, Honolulu, Hawaii 96822
jin.liu@murraystate.edu
Received May 7, 2002
ABSTRACT
The preparations and spectroscopic characteristics of five all-trans halogenated canthaxanthins are described in this letter. The air/light-
sensitive halogenated canthaxanthins were used to study r-crustacyanin, a blue astaxanthin−protein complex, which is isolated from the
carapace of the lobster. Steric and electronegative effects of the halogen substituents on the noncovalent interaction between astaxanthin and
the protein in r-crustacyanin were observed.
Bacteriorhodopsin,1 a retinal-protein complex, plays several
important biological roles. In recent years, considerable effort
has been concentrated on the understanding of astaxanthin-
protein complexes. The well-known astaxanthin-protein
complexes, crustacyanins, are isolated from the carapace of
the lobster Homarus gammarus.2
R-Crustacyanin and γ-crustacyanin are two isolated crus-
tacyanins that have a deep blue color. UV-vis absorptions
of R-crustacyanin and γ-crustacyanin are 632 and 625 nm,
respectively. The unusual characteristic of R-crustacyanin
and γ-crustacyanin is the large bathochromic shift (R-
crustacyanin, 5100 cm-1; γ-crustacyanin, 5050 cm-1) caused
by the noncovalent interaction between astaxanthin and the
proteins.3 Because of a lack of the crystal structures of
crustacyanins, some techniques such as solid-state 13C NMR,4
Stark spectroscopy,5 and 19F NMR6 have been used to study
the structure of R-crustacyanin to explain its large batho-
chromic shift. Recombination studies between the colorless
* Corresponding author. Phone: 270-762-6626. Fax: 270-762-6474.
† Murray State University.
‡ University of Hawaii.
(1) Lueke, H.; Schibert, B.; Richter, H. T.; Cartailler, J. P.; Lanyi, J. P.
J. Mol. Biol. 1999, 291, 899-911.
(2) Zagalsky, P. F. Carotenoids 1A: Isolation and Analysis; Britton, G.,
Liaaen-Jensen, S., Pfander, H., Eds.; Birkha¨user Verlag: Basel, Switzerland,
1995; pp 287-294.
(4) Weesie, R. J.; Askin, D.; Jansen, F. J.; de Groot, H. J. M.; Lugtenburg,
J.; Britton, G. FEBS Lett. 1995, 362, 34-38.
(5) Krewczyk, S.; Britton, G. Biochim. Biophys. Acta 2001, 1544, 301-
310.
(3) Britton, G.; Weesie, R. J.; Askin, D.; Warburton, J. D.; Gallardo-
Guerrero, L. Jansen, F. J.; de Groot, H. J. M.; Lugtenburg, J.; Cornard,
J.-P.; Merlin, J.-C. Pure Appl. Chem. 1997, 69, 2075-2084.
(6) Hoischen, D.; Colmenares, L. U.; Liu, J.; Simmons, C. J.; Britton,
G.; Liu, R. S. H. Bioorg. Chem. 1998, 26, 365-374.
10.1021/ol026146f CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/27/2002

Scheme 1a
a Reagents and conditions: (a) (CF3SO2)2O, pyridine, CH2Cl2, then n-Bu4NF in THF, 49% yield; (b) (CF3SO2)2O, pyridine, CH2Cl2, then
n-Bu4NCl in THF, 50% yield; (c) N,N-dimethyl-N-1-chloro-2-methylpropenylamine, CH2Cl2, 95% yield; (d) (CF3SO2)2O, pyridine, CH2Cl2,
then NaBr in acetone, 97% yield; (e) (CF3SO2)2O, pyridine, CH2Cl2, then NaI in acetone, 98% yield; (f) (i) CH3COCl, pyridine, CH2Cl2,
(ii) N,N-dimethyl-N-1-chloro-2-methylpropenylamine, CH2Cl2, (iii) NaOCH3, CH3OH, 61% yield.
apoprotein of crustacyanin and various carotenoids have
revealed that the 4,4′-keto groups are essential for reconsti-
tuting the blue carotenoprotein.3 The unique solid-state 13C
NMR study of R-crustacyanin reported by Lugtenburg and
co-workers supported the idea that the 4,4′-carbonyl groups
of astaxanthin played a crucial role in the noncovalent
interaction between astaxanthin and the protein. Unfortu-
nately, the resonance signals from the 4,4′-13C-labeled
astaxanthin in the reconstituted R-crustacyanin were weaker
than regular 13C resonance signals.7
Halogenated retinal analogues have been demonstrated to
be useful synthetic chromophores in a number of bacterio-
rhodopsin studies.8-10 In an effort to understand the important
role of the 4,4′-carbonyl groups of astaxanthin in crustacya-
nins, we decided to introduce four different halogen atoms
at the 3- and 3′-positions of astaxanthin and to examine the
effects of the size and electronegativity of halogen atoms
on the noncovalent interactions between astaxanthin and the
proteins.
The strategy for introducing halogen substitutes at the 3-
and 3′-positions is shown in Scheme 1. The 3- and 3′-
hydroxy groups of all-trans astaxanthin were reacted with
triflic anhydride and pyridine in CH2Cl2 to afford a bis-
triflate. Upon treatment with 2 equiv of n-Bu4NF in THF,
the bis-triflate was converted into 3,3′-difluorocanthaxanthin.
Also, 3,3′-dichlorocanthaxanthin, 3,3′-dibromocanthaxanthin,
and 3,3′-diiodocanthaxanthin were prepared by treatment of
the bis-triflate with 2 equiv of n-Bu4NCl in THF, 3 equiv of
NaBr, and 3 equiv of NaI in acetone, respectively. The yields
of the bromination and the iodination were almost quantita-
tive. The yields of the chlorination and fluorination were
∼50%. The low yields were due to the formation of an
elimination product.
To improve the yield of the chlorination, astaxanthin was
reacted with N,N-dimethyl-N-1-chloro-2-methylpropenyl-
amine in CH2Cl2 at room temperature to give the desired
product in a good yield (>95%).11 However, treatment of
astaxanthin with N,N-dimethyl-N-1-fluoro-2-methylpropen-
ylamine failed to give 3,3′-difluorocanthaxanthin ascribed
to the highly electronegative fluorine atom. The starting
material, the elimination product, and other side products
were recovered after the reaction. Diethylaminosulfurtri-
fluoride (DAST) has been proved to be a versatile reagent
(7) Weesie, R. J.; Jansen, F. J. H. M.; Merlin, J. C.; Lugtenburg, J.;
Britton, G.; de Groot, H. J. M. Biochemistry 1997, 36, 7288-7296.
(8) Crouch, R. K.; Scott, R.; Ghent, S.; Govindjee, R.; Chang, C.-H.;
Ebrey, T. Photochem. Photobiol. 1986, 43, 297-303.
(9) Tierno, M. E.; Mead, D.; Asato, A. E.; Liu, R. S. H.; Sekiya, N.;
Yoshihara, K.; Chang, C.-W.; Nakanishi, K.; Govindjee, R.; Ebrey, T. G.
Biochemistry 1990, 29, 5948-5953.
(10) Colmenares, L. U.; Zhou, X.-L.; Liu, J.; Asato, A. E.; Liu, R. S. H.
J. Am. Chem. Soc. 1999, 121, 5803-5804.
(11) Munyeman, F.; Frisque-Hesbain, A.-M.; Devos, A.; Ghosez, L.
Tetrahedron Lett. 1989, 30, 3077-3080.
2522
Org. Lett., Vol. 4, No. 15, 2002

1
Table 1. Partial H NMR Data and UV-vis Absorption Maxima of the Five Halogenated Canthaxanthins
compounds
H-3,3′a
H-7,7′
H-8,8′
H-10,10′
H-11,11′
H-12,12′
astaxanthin
3,3′-diF
3,3′-diCl
3,3′-diBr
3,3′-diI
4.31
5.09
4.71
4.91
5.27
4.31, 4.71
6.21
6.19
6.18
6.18
6.17
6.18, 6.21
6.43
6.43
6.42
6.42
6.41
6.42
6.30
6.30
6.30
6.30
6.30
6.30
6.66
6.65
6.65
6.65
6.65
6.65
6.45
6.44
6.44
6.45
6.45
6.45
3-Cl-3′-OH
b
compounds
H-14,14′
H-15,15′
5-Me
1,1′-Me,Me
λmax
astaxanthin
3,3′-diF
3,3′-diCl
3,3′-diBr
3,3′-diI
6.30
6.30
6.30
6.30
6.30
6.30
6.68
6.65
6.65
6.65
6.65
6.65
1.95
1.93
1.95
1.95
1.96
1.95
1.21, 1.32
1.25, 1.32
1.23, 1.30
1.23, 1.29
1.20, 1.26
1.21, 1.23, 1.30, 1.32
477
477
477
477
478
477
3-Cl-3′-OH
a In CDCl3, 200 MHz. b Units ) nm, in acetone.
in preparation of bioactive molecules with fluorine atoms.12
Because astaxanthin was decomposed by DAST, it was
unsuitable for the preparation of 3,3′-difluorocanthaxanthin.
The first step in the preparation of 3-chloro-3′-hydroxy-
canthaxanthin was the protection of one hydroxy group of
astaxanthin as an acetoxy group using acetyl chloride and
pyridine. Treatment of 3-acetoxy-3′-hydroxycanthaxanthin
with N,N-dimethyl-N-1-chloro-2-methylpropenylamine, fol-
lowed by deprotection with sodium methoxide, gave 3-chloro-
3′-hydroxycanthaxanthin in 61% overall yield.
All the analogues obtained as deep red solids are air/light-
sensitive. The all-trans isomers of the five halogenated
canthaxanthins were isolated by HPLC (YMC Carotenoid
Column, 5µ, 60:25:15 CH3CN/CH3OH/EtOAc, flow rate )
1 mL/min) in dim red light. The structures of the analogues
were confirmed by their 1H and 13C NMR spectra, HR-MS,
and UV-vis data and by comparison with those of astax-
anthin and canthaxanthin (Table 1).13,14 Because the recon-
stitution of R-crustacyanin with (3S,3′S)-, (3R,3′R)-, and
(3R,3′S)-astaxanthins gave the same reconstituted R-crusta-
cyanin, separation of the stereoisomers of the halogenated
canthaxanthins was considered unnecessary for the study of
R-crustacyanin.15
carbonyl groups of astaxanthin and hydrogen donors from
the protein caused the usual red-shift of the chromophore in
R-crustacyanin.7 The electronegative fluorine atoms at the
3- and 3′-positions should induce weaker hydrogen bonds
between the carbonyl groups and hydrogens in the caroteno-
protein, thus causing the smaller bathochromic shift of the
reconstituted analogue.
Also, 3,3′-dichlorocanthaxanthin, 3,3′-dibromocanthaxan-
thin, and 3,3′-diiodocanthaxanthin were combined with the
apoprotein to form red canthaxanthin-protein complexes
(λmax ≈ 500 nm) within 1 min of mixing. The halogenated
canthaxanthins were completely dissociated from the protein
after 12 h at 4 °C. The results show that if the size of a
substituent is larger than that of a fluorine atom, the
substituents at the 3,3′-positions of astaxanthin affected the
interactions between astaxanthin and the protein. This is
probably due to the rigid portion of the protein host around
the 3,3′-OH groups of astaxanthin. The protein surrounding
for the six-membered rings of astaxanthin in R-crustacyanin
Reconstitutions of the five halogenated canthaxanthins
with the apoprotein isolated from natural R-crustacyanin were
carried out.6 3,3′-Difluorocanthaxanthin was combined with
the apoprotein to give a blue R-crustacyanin analogue (λmax
) 600 nm). The spectra (Figure 1) show that the fluorine
atoms at the 3- and 3′-positions cause the blue-shifted UV-
vis absorption of the R-crustacyanin analogue. It was
suggested that hydrogen-bonding interactions between the
(12) Chen, S.-H.; Huang, S.; Farina, V. Tetrahedron Lett. 1994, 35, 41-
44.
(13) See, for example, in: Carotenoids 1B: Spectroscopy; Britton, G.,
Liaaen-Jensen, S., Pfander, H., Eds.; Birkha¨user Verlag: Basel, Switzerland,
1995.
(14) Liu, J.; Colmenares, L. U.; Liu, R. S. H. Tetrahedron Lett. 1997,
38, 8495-8498.
(15) Berger, H.; Rønneberg, H.; Borch, G.; Liaaen-Jensen, S. Comput.
Biochem. Physiol. 1982, 71B, 253-258.
Figure 1. Absorption spectra of R-crustacyanin (1) and the
R-crustacyanin analogue reconstituted with 3,3′-difluorocanthax-
anthin (2) in 50 mM sodium phosphate buffer.
Org. Lett., Vol. 4, No. 15, 2002
2523

seems to be very different from the relatively flexible protein
pocket for the six-membered ring of retinal in bacterio-
rhodopsin.16
Moreover, 3-chloro-3′-hydroxycanthaxanthin was recon-
stituted with the apoprotein to give a similar canthaxanthin-
protein complex initially. The synthetic chromophore was
released from the protein complex after 12 h (Figure 2) due
510 and 562 nm) was more red-shifted than those of the
other red complexes. This was attributed to the hydroxy
group of 3-chloro-3′-hydroxycanthaxanthin, which made it
possible for a half of the synthetic chromophore to be fitted
correctly to the apoprotein. On the basis of our results and
the previous reconstitution studies of R-crustacyanin,3 we
conclude that the large bathochromic shift of R-crustacyanin
can only be achieved when the astaxanthin chromophore
snugly fits into the apoprotein of R-crustacyanin.
In this letter, we demonstrate that the air/light-sensitive
halogenated canthaxanthin analogues can be prepared from
all-trans astaxanthin by convenient methods. These analogues
are useful for the study of R-crustacyanin. Recently, we found
an ideal method for the reconstitution of γ-crustacyanin.
Detailed studies of γ-crustacyanin using these halogenated
canthaxanthins and 19F NMR study of the reconstituted
crustacyanin analogues are in progress.
Acknowledgment. This work was supported by grants
from Kentucky NSF EPSCoR and Howard Hughes Medical
Institution.
Supporting Information Available: Experimental pro-
cedures for preparations of the five halogenated canthaxan-
thins and spectroscopic data. This material is available free
of charge via the Internet at http://pubs.acs.org.
Figure 2. Absorption spectra of the 3-chloro-3′-hydroxycantha-
xanthin-protein complex (1) in 50 mM sodium phosphate buffer
within 1 min of mixing and the dissociated complex (2) in the same
buffer after 12 h at 4 °C.
OL026146F
to the steric limitation. However, UV-vis absorption of the
(16) Asato, A. E.; Li, X.-Y.; Mead, D.; Patterson, G. M.; Liu, R. S. H.
J. Am. Chem. Soc. 1990, 112, 7398-7399.
3-chloro-3′-hydroxycanthaxanthin-protein complex (λmax
)
2524
Org. Lett., Vol. 4, No. 15, 2002