11,12-Difluororhodopsin and related odd-numbered fluororhodopsins. The use of J(F,F) for following a cis-trans isomerization process

Full text of DOI:10.1021/ja990495w

J. Am. Chem. Soc. 1999, 121, 5803-5804
5803
Scheme 1. (a) N-Bromosuccinimide. (b) C15-phenyl sulfone +
BuLi.8 (c) MeN-OMe Li+. (d) MeLi.9 (e) Cyanophosphono-
acetate, base. (f) Dibal-H.
11,12-Difluororhodopsin and Related Odd-Numbered
Fluororhodopsins. The Use of JF,F for Following a
Cis-trans Isomerization Process
Leticia U. Colmenares, Xiang-long Zou, Jin Liu,
Alfred E. Asato and Robert S. H. Liu*
Department of Chemistry, UniVersity of Hawaii
2545 The Mall, Honolulu, Hawaii 96822
Angel R. de Lera and Rosana Alvarez
Departamento de Quimica Fisica y Quimica Organica
Facultad de Ciencias, UniVersidade de Vigo
36200 Vigo, Spain
ReceiVed February 16, 1999
ReVised Manuscript ReceiVed April 8, 1999
Table 1. F NMRa and UV-vis Data for the 11,12-F2-, 9-F-, 11-F-,
and 13-F-retinal and their SB, PSB, and Bovine Rhodopsin
Analogues
Fluorine-19 NMR spectroscopy has become popular in studies
of complex molecules mainly because of the natural abundance
and the high sensitivity of the 19F nucleus and its wide range of
chemical shifts, extending beyond 300 ppm. The sensitivity of
the 19F nucleus to changes in the environment further enhanced
its appeal as a label in complex biochemical systems.1 However,
possible use of the stereochemically sensitive vicinal F,F cou-
plings2 in structural studies have not been fully explored.
As part of an extended study of F-labeled retinals and retinal
binding proteins,3 the possible incorporation of vicinal fluorine
probes into a retinal analogue (13,14-difluoro-13-demethylretinal)4
was demonstrated earlier by following the Middleton procedure
for vicinal difluorination:5 reaction of ethyl acetoacetate (or a
â-diketone) with diethylaminosulfur trifluoride (DAST) in an
aprotic dipolar solvent.4 In the process, we took advantage of the
unusual but well-known stability of the cis isomers of vicinal
difluoroalkenes,6 as revealed in the allylic bromination in the
scheme below, that allowed introduction of the cis-difluoro linkage
into the polyene chain. Now, after employing a modified sequence
of reactions (Scheme 1), we have completed a stereoselective
synthesis of the important 11-cis-11,12-difluororetinal (1) and a
related analogue 11-cis-11,12-F2-13-demethylretinal (2). The
synthesis of odd-numbered F retinals was reported recently.7
The cis isomer of 11,12-F2-retinal is characterized by two sets
of signals in its F NMR spectrum (CD2Cl2): d × d at -118.5
(F-11, with a small JF,F ) 7.5 and JH,F ) 25.6) and d at -138.3
ppm (F-12) while the trans isomer is characterized by d × d at
-135.5 (F-11 with a large JF,F ) 107.5 and JH,F ) 31.9 Hz) and
d at -149.0 ppm (F-12). Detailed F NMR data including that for
other F retinals, their Schiff bases (SB), and protonated Schiff
bases (PSB) are listed in Table 1.
Rh,
F shift, ppm
UV-vis
c
retinal
CHO
SB
PSB
Rh
FOSb λmax
11,12-F2
11-cis 11-F -118.5
-129.0
-103.4
-101.6 1.8
-117.1 19
503.5
J10,11 ) 25.6 J10,11 ) 25.6 J10,11 ) 25.6
12-F -138.3
J11,12 ) 7.5
all-t 11-F -135.5
-136.9
J11,12 ) 9.1
-143.8
-136.1
J11,12 ) 0
-123.7
NA
NA NA
J10,11 ) 32.1 J10,11 ) 29.5 J10,11 ) 29.5
12-F -149.0 -148.7 -147.5
J11,12 ) 107.5 J11,12 ) 108.8 J11,12 ) 103.9
11,12F2-13-deMe
11-cis 11-F -122.1
J10,11 ) 28.1 J10,11 ) 28.4
-130.2
no
pigm.
NA NA
12-F -147.2
J11,12 ) 8.1
-146.0
J11,12 ) 7.7
J12,13 ) 27.4 J12,13 ) 26.4
9-F
9-cis 9-F -110.8
J8,9 ) 27.6
-115.0
J8,9 ) 28.6
-103.3
J8,9 ) 27.1
-96.4 6.9
463
J9,10 ) 18.7 J9,10 ) 19.3 J9,10 ) 18.1
11-F
9-cis 11-F -95.9
-104.1 -87.1
J10,11 ) 31.0 J10,11 ) 29.6 J10,11 ) 32.2
J11,12 ) 36.9 J11,12 ) 37.6 J11,12 ) 35.7
-81.0 6.1
-75.9 2.1
474
11-cis 11-F -90.0
-97.6
-78.0
488.5
J10,11 ) 28.6 J10,11 ) 28.6 J10,11 ) 29.7
J11,12 ) 22.4 J11,12 ) 23.2 J11,12 ) 23.0
all-t 11-F -97.7
-106.0
-88.0
NA
NA
NA NA
J10,11 ) 29.9 J10,11 ) 29.6 J10,11 ) 32.8
J11,12 ) 36.8 J11,12 ) 37.6 J11,12 ) 36.0
13-F
11-cis 13-F -100.8
NDd
501.5
J12,13 ) 33.9
J13,14 ) 32.4
a For CHO, SB (n-butyl), and PSB (trichloroacetic acid), in CD2Cl2,
while for Rh, in 2% CHAPS. Coupling constants in Hz. b F shift (Rh)
- F shift (PSB) in ppm. c In nm. d ND, not determined due to
overwhelming intensity of -121 ppm peak.
To demonstrate the possible use of three-bond F,F coupling
constants in following a cis to trans isomerization reaction, Figure
1 shows the F NMR spectrum of the configurationally unstable
hydrobromide of the n-butyl Schiff base (PSB) of 11-cis-11,12-
F2-retinal. Immediately after addition of HBr to the Schiff base,
the room-temperature spectrum shows a mixture of the anti
(major) and the syn (minor) isomers (assignments based on J15,16
values: 15.7 Hz for anti and 10.6 Hz for syn).10 Upon standing,
this was displaced by a new set of signals of all-trans-PSB of
the same anti/syn ratio. The changes in peak structure reflect the
increase of the F,F coupling constants from ∼0 Hz for the 11-cis
isomers to 103.6 and 104.6 Hz (anti and syn) for the trans. The
large change in coupling constant has been put to use in following
the cis-to-trans photoisomerization of a visual pigment analogue
where excessive line width is commonly encountered in NMR
studies of detergent-solubilized solutions of proteins of this size.
(1) Gerig, J. T. Prog. NMR Spectrosc. 1994, 293-370.
(2) (a) Emsley, J. W.; Phillips, L.; Wray, V. Prog. Nucl. Magn. Reson.
Spectrosc. 1977, 10, 85-756. (b) Everett, T. S. In Chemistry of Organic
Compounds II: A Critical ReView; Hudlicky, M., Pavlath, A. E., Eds.; 1995,
pp 1037-1086.
(3) See, e.g., (a) Colmenares, L. U.; Liu, R. S. H. Tetrahedron 1996, 52,
109-118. (b) Colmenares, L. U.; Niemczura, W. P.; Asato, A. E.; Liu, R. S.
H. J. Phys. Chem. 1996, 100, 9175-9180.
(4) Asato, A. E.; Liu, R. S. H. Tetrahedron Lett. 1986, 27, 3337-3340.
(5) Unpublished results of W. Middleton, communicated to RSHL, cited
in ref 4.
(8) Julia, M.; Arnould, D. Bull. Soc. Chim. Fr. 1973, 746-750.
(9) Groesbeek, M.; Rood, G. A.; Lugtenburg, J. Recl. TraV. Chim. Pays-
Bas 1992, 111, 149-154.
(10) (a) Sharma, G. M.; Roels, O. A. J. Org. Chem. 1973, 38, 3648-
3651. (b) Pattaroni, C.; Lauterwein, J. HelV. Chim Acta 1981, 64, 1969-1984.
(6) See, e.g., Smart, B. Mol. Struct. Energ. 1986, 3, 141-191.
(7) Francesch, A.; Alvarez, R.; Lopez, S.; de Lera, A. R. J. Org. Chem.
1997, 62, 310-319.
10.1021/ja990495w CCC: $18.00 © 1999 American Chemical Society
Published on Web 06/03/1999

5804 J. Am. Chem. Soc., Vol. 121, No. 24, 1999
Communications to the Editor
thus a measure of the effect of the protein host on the F label of
the substrate) of 13.2 ppm. We have now obtained the F-shift
data for 11-F-rhodopsin and the PSB giving a small13 FOS of
2.1 ppm (Table 1). FOS values for the -117.1 and -101.6 ppm
peaks (19 and 1.8, ppm, respectively) clearly suggest that the two
peaks are, respectively, due to F-12 and F-11 of the pigment.
Photoirradiation of this pigment (>520 nm) resulted in the
disappearance of the two broad peaks, replaced by two broad
singlets at -143.3 and -137.2 ppm and an unsymmetrical doublet
(J ) 103 Hz) near -147 ppm, and intensification of the -120.9
ppm peak. Recalling the long-accepted notion that the chro-
mophore freed from the binding site exists as a mixture of all-
trans-retinal and the corresponding random Schiff bases (from
random linkage between the extruded retinal and amino units
outside the binding pocket)14 and by comparison of chemical shifts
of authentic samples of all-trans-11,12-F2-retinal and its butyl
Schiff base (Table 1), we conclude that the three upfield peaks
can be safely assigned to those of the retinal (F-11 at -137.2
and F-12 at -147.6 ppm) and the random Schiff base (F-11 at
-143.3 and F-12 at -147 ppm). It is interesting to note that only
the shifts of F-11 (and not F-12) are sufficiently different to allow
measurements of aldehydes and random Schiff bases in the
product mixture. The observation of a distinct doublet for F-12
in the photoproduct retinal15 (despite the broad line width, W1/2
) ∼300 Hz, for the protein-containing sample) from, initially, a
broad singlet has successfully demonstrated the use of three-bond
F,F coupling constants for following the cis-to-trans photoisomer-
ization process of the visual pigment analogue. The large three-
bond JF,F, in contrast to the smaller JH,H (<18 Hz) or JH,F (<40
Hz), is clearly needed to study of proteins of broad line width.
An unusually narrow signal near -121 ppm was observed in
all samples in aqueous solution. The peak was apparently
associated with the free chromophore because, in the initial
rhodopsin sample, the peak was weak and intensified dramatically
after photobleaching. In addition, a similar peak was present in
the spectrum of an aqueous solution of all-trans-11,12-F2-retinal
solubilized with CHAPS. The sharpness of the line suggests that
it is a free small species. Subsequently we demonstrated that the
signal is due to fluoride ion because addition of NaF to these
solutions increased the intensity of the -121 ppm peak.
Figure 1. F NMR spectra (376.4 MHz) showing isomerization of 11-
cis-n-butyl-11,12-F2-retinal-PSB to the trans isomer (a) 8 min after
addition of HBr to the Schiff base and (b) 20 min after addition. The
minor peaks in the upper spectrum have not been assigned; however, we
suspect they are due to the 9-cis isomers. The inserts show expansions
of the F-11 signals for the 11-cis isomer (lower) and the trans isomer
(upper), clearly revealing the large change in JF,F
.
That the chemical processes led to the formation of the fluoride
ion is suggested by the observation that in previous studies of
even-number-substituted F rhodopsins,3b the -121 ppm peak was
not detected, whereas in the current study of the odd-numbered
F rhodopsins, the ease of formation of the fluoride ion parallels
proximity of the substituent to the electron-withdrawing end group
(i.e., extensive loss of 13-F-retinal and minimal loss of 9-F-
retinal). Hence, we suspect a process involving nucleophilic
addition and fluoride elimination must have taken place, in
competition with pigment formation. The generality of this
reaction in studies with F-labeled retinoid binding proteins will
be examined in a separate study. It will also be of interest to
devise conditions for low-temperature F NMR studies so that the
coupling constant values can be applied to examine structural
details of the photobleaching intermediates.16
Figure 2. F NMR spectra (376.4 MHz) of 11,12-F2-rhodopsin and photo-
bleaching products. Lower: spectrum of a concentrated sample of pigment
in a @Shigemi sample tube (0.5 mL, ∼10-4 M) before photoirradiation
(spectrometer parameters: 23,216 scans; LB, 200 Hz; pulse delay, 2 s).
Upper: after photobleaching. Upfield signals are expanded (insert).
11,12-Difluoro bovine rhodopsin analogues, solubilized in 2%
CHAPS, were prepared11 in good yield (80%).12 For comparison,
we have also prepared rhodopsin analogues from three other odd-
numbered F retinal analogues (9F, 11F, and 13F). The absorption
maxima are listed in Table 1. Concentrated NMR samples were
also prepared in the same manner as described before.
The F NMR spectrum for 11,12-F2-rhodopsin, shown in Figure
2a, consists of two broad peaks at -101.6 and -117.1 ppm and
a sharp peak at -120.9 ppm. Assignment of these signals was
made by comparison with related F rhodopsins. The F shifts for
12-F-rhodopsin and the corresponding PSB were reported earlier,3b
giving a large FOS value13 (F-pigment shift minus F-PSB shift,
Acknowledgment. The work was supported by Grants from U. S.
Public Health Services (DK-17806 to R.S.H.L.), Xunta de Galicia
(XUGA30106B97), and MEC (SAF98-0143 to A.R.L.). We thank Drs.
S. Lopez and A. Francesch for their preliminary synthetic efforts.
JA990495W
(13) The large FOS of F-12 was attributed to its proximity to the
counterion.3b The small FOS for F-11 must, therefore, be due to the orientation
of the 11-F substituent, directed away from the counterion toward a more
open space of the binding pocket. This interpretation is consistent with the
currently accepted model for rhodopsin, see, e.g., Shieh, T.; Han, M.; Sakmar,
T. P.; Smith, S. O. J. Mol. Biol. 1997, 269, 373-384.
(14) See, e.g., (a) Wald, G.; Brown, P. K. J. Gen. Physiol. 1953, 37, 189-
200. (b) Rao, V. R.; Cohen, G. B.; Oprian, D. D. Nature 1994, 367, 639-642.
(15) F-12 signals for the random Schiff bases appear as an unresolved broad
band slightly downfield from the retinal doublet.
(11) Following published procedures for pigment formation and sample
concentration.3
(12) It is interesting to note that while the extra 11-F substituent does not
affect formation of the 11,12-F2-pigment, the slight increase in steric interaction
between 9-Me and F-11 is reflected in the shifts of λmax of 11-cis-11-F-retinal
(364.5 nm) and 11-cis-11,12-F2-retinal (374.5 nm) from those of 11-cis-retinal
(380) and 11-cis-12-F-retinal (382 nm, all in ethanol) and the failure of pigment
formation for 11-cis-11,12-F2-13-demethylretinal (relative to the positive
binding result of 13-demethylretinal).
(16) See, e.g., Smith, S. O.; Courtin, J.; De Groot, H. J. M.; Gebhard, R.;
Lugtenburg, J. Biochemistry 1991, 30, 7409-7415.