Family of site-selective molecular optical switches

Article abstract of DOI:10.1021/jo048207o

(Chemical Equation Presented) We describe the design, synthesis, and characterization of a family of thiol-reactive optical switches for labeling proteins and other biomolecules. Site-selective introduction of photochromic probes within biomolecules is be

Full text of DOI:10.1021/jo048207o

Family of Site-Selective Molecular Optical Switches
Tomoyo Sakata, Yuling Yan,† and Gerard Marriott*
Department of Physiology, University of Wisconsin-Madison, 1300 University Avenue,
Madison, Wisconsin 53705, and Department of Mechanical Engineering, University of Hawaii,
2450 Dole Street, Honolulu, Hawaii 96822
gm@physiology.wisc.edu
Received October 11, 2004
We describe the design, synthesis, and characterization of a family of thiol-reactive optical switches
for labeling proteins and other biomolecules. Site-selective introduction of photochromic probes
within biomolecules is being used as part of a new approach for optical control of biomolecular
interactions and activities within cells. The thiol-reactive photochromic probes described in this
report include a spironaphthoxazine and five spirobenzopyrans. The location of the thiol-reactive
group on the spirobenzopyran is different for each probe; this feature can be used to control the
geometry of the optical switch within a bioconjugate. The photochromes undergo rapid and
reversible, optically driven transitions between a colorless spiro (SP) state and a brightly colored
merocyanine (MC) state. The MC absorption of a spironaphthoxazine conjugate is red shifted by
more than 100 nm compared to the equivalent spirobenzopyran, which may be exploited for the
independent control of the MC to SP transition for up to two different spironaphthoxazine and
spirobenzopyran conjugates within the same sample.
Introduction
trophenyl (caged) group, which functions as a one-way,
single-use optical switch. We are improving this tech-
nique through the development of new optical switch
reagents whose interactions and activities within a
bioconjugate can be rapidly, reversibly, and efficiently
modulated with light.
This new approach exploits the properties of photo-
chromic probes that undergo rapid and reversible, light-
directed transitions between a colorless, weakly polarized
spiro (SP) state and a colorful and highly polarized
merocyanine (MC) state8-14 (schematized in Figure 1):
excitation of SP at 365 nm light generates MC, while
excitation of MC at 546 nm generates SP. These optical
switches must be specifically labeled to a biomolecule
The increasing need to understand the molecular basis
of cell behavior and physiology is driving the development
of new optical probes and microscope techniques to study
the interactions and regulation of specific proteins within
a cell.1-3 A necessary requirement for these studies is the
ability to selectively perturb the interactions or activity
of a specific protein in a cell against a background of up
to 30,000 other proteins. To this end, we introduced the
technique of light-directed activation of “caged” proteins,
which has been applied to study protein function within
complex molecular and cellular systems.3-7 However, this
approach is limited by the photochemistry of the 2-ni-
* Address correspondence to this author.
† University of Hawaii.
(8) Inouye, M. Mol. Cryst. Liq. Cryst. A 1994, 246, 169-172.
(9) Giordano, L.; Jovin, T. M.; Irie, M.; Jares-Erijman, E. A. J. Am.
Chem. Soc. 2002, 124, 7481-7489.
(1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. Rev.
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(2) Yan, Y.; Marriott, G. Curr. Opin. Chem. Biol. 2003, 7, 635-640.
(3) Roy, P.; Rajfur, Z.; Jones, D.; Marriott, G.; Loew, L.; Jacobson,
K. J. Cell Biol. 2001, 153, 1035-1048.
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Am. Chem. Soc. 2004, 126, 30-31.
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10.1021/jo048207o CCC: $30.25 © 2005 American Chemical Society
Published on Web 02/10/2005
J. Org. Chem. 2005, 70, 2009-2013
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Sakata et al.
FIGURE 1. Schematic representation of the reversible optical switching between the SP and MC states of a spirobenzopyran
(13) linked to a cysteine residue in a protein.
SCHEME 1. Reaction Scheme Employed for the Synthesis of the Five Thiol-Reactive Spirobenzopyrans (3,
6, 9, 12, and 13) Described in This Papera
a Reagents: (a) 3-chloromethyl-5-nitrosalicylaldehyde, THF; (b) NaI, acetone; (c) 5-nitrosalicylaldehyde, EtOH; (d) PPh3, DIAD, maleimide,
neopentyl alcohol, THF; (e) PPh3, CBr4, THF.
such that the MC state, but not the SP state, competes
against a functional ligand for specific interactions within
the conjugate. Optical switching between the MC to SP
states using cycles of 365 and 546 nm light is then used
to reversibly control the MC-SP states and their inter-
actions within the conjugate and thereby modulate its
function.
lysine residue in proteins;10,12,13 however, the multiple
and randomly labeleld photochromes in these protein
conjugates exhibit complex photochemistry and are ca-
pable of forming dimers that have different optical
properties compared to the monomer and may exhibit
irreversible photochemistry.11,13
In this report we describe a family of photochromic
probes that harbor maleimide, bromomethyl, or iodo-
methyl groups for selective labeling of thiol groups within
biomolecules. Previous studies have described photochro-
mic reagents that react with the amino group of the
Results and Discussion
Syntheses for the six thiol-reactive optical switches (3,
6, 9, 12, 13, 17) are summarized in Schemes 1-3.
Combinatorial in design, the synthetic approach involves
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Family of Thiol-Reactive Optical Switches
SCHEME 2. Preparation of Indoline Derivativesa
a Reagents: (a) NaNO2, SnCl2‚H2O, HCl; (b) 3-methyl-2-bu-
tanone, H2SO4, EtOH; (c) 2-iodoethanol, CH3CN; (d) KOH, H2O;
(e) MeI, CH2Cl2.
coupling members of a family of indoline derivatives (1,
4, 7, 10) with specific nitrosalicylaldehydes or nitrosonaph-
thol to yield the four key spirobenzopyrans (2, 5, 8, 11)15,16
or spironaphthoxazin (16).15 Indoline 1 is commercially
available (Aldrich). Indoline 4 is prepared from com-
mercially available 2,3,3-trimethyl-3H-indole in two steps.
Indolines 7 and 10 are synthesized from compounds 14
and 15, which are generated as a mixture and separated
by chromatography, according to Scheme 2. The thiol-
reactive spirobenzopyrans (3, 6, 9, 12, 13) are prepared
from the corresponding spirobenzopyrans via the halogen
exchange reaction, bromination of alcohol, or modified
Mitsunobu reaction for the maleimides.17 The yields for
the conversion to the thiol-reactive probes are 15% (6),
60% (9), 24% (12), and 55% (13). Compound 3 is easily
prepared from commercially available 1 and 3-chloro-
methyl-5-nitrosalicylaldehyde through compound 2 and
is purified only at the final step with a yield of 78%.
FIGURE 2. (A) Normalized absorption spectra for the lowest
energy transition of the MC state of the five thiol-reactive
probes attached to the reactive cysteine residue in BSA (Cys-
34). The letters a-e refer to compounds 13, 9, 12, 6, and 3,
respectively. (B) Absorption spectra of the spironaphthoxazine
(17) labeled G-actin (Cys-374) in the SP-state (a) and MC state
(b). The MC state was generated by irradiating a 50% glycerol
solution of a with 365 nm light for 10 s.
The MC state of the five spirobenzopyrans has a
ground-state dipole moment of 20 D.18,19 To the best of
our knowledge, this is the largest dipole moment of all
commonly used optical probes; we suggest that this
property underlies the strong dipolar interactions be-
tween the MC probe and polar groups on the protein. The
MC absorption spectra of the five spirobenzopyrans BSA
conjugates are shown in Figure 2A and the spironaph-
thoxazine-G-actin conjugate is shown in Figure 2B. The
most red-shifted MC-BSA conjugate is seen for com-
pound 6 (Figure 2A, curve d; 544.5 nm), while the
absorption maximum progresses to higher energy within
the BSA conjugates of 12 (c, 534 nm); 9 (b, 520) nm; 3 (e,
515.5 nm); 13 (a, 503.5 nm). Since the five spirobenzopy-
ran reagents harbor their thiol-reactive group at different
sites on a common photochromic scaffold, the MC dipole
moment should adopt a different geometry, and interac-
tions, within each BSA conjugate, which is reflected in
their absorption maxima. This demonstration satisfies
an important criterion in our approach for optical switch-
ing of biomolecular function in cells; thus, in its most
optimal form, optical switching will be realized when a
specific and essential dipolar interaction between the
conjugate and its binding partner is inhibited by a
competing interaction involving the highly polarized MC
state, but not by the weakly polar SP state.
The thiol-reactive spironaphthoxazine (17) is prepared
via a same method as compound 13 with a yield of 15%
(Scheme 3).
The six optical switch reagents efficiently label cysteine
residues in proteins; in this study we describe spiroben-
zopyran conjugates of BSA and the spironaphthoxazine
conjugate of G-actin. BSA is often used as a model protein
to test new thiol-reactive probes21 (although BSA harbors
35 cysteine residues, all but Cys-34 engage in disulfide
bond formation);22 consequently, most studies show that
BSA can only be labeled with a single thiol-reactive probe
at residue Cys-34.21,23,24
(15) Inouye, M.; Ueno, M.; Tsuchiya, K.; Nakayama, N.; Konishi,
T.; Kitao, T. J. Org. Chem. 1992, 57, 5377-5383.
(16) Tanaka, M.; Nakamura, M.; Salhin, M. A. A.; Ikeda, T.;
Kamada, K.; Ando, H.; Shibutani, Y.; Kimura, K. J. Org. Chem. 2001,
66, 1533-1537.
(17) Walker, M. A. J. Org. Chem. 1995, 60, 5352-5355.
(18) Bletz, M.; Pfeifer-Fukumura, U.; Kolb, U.; Baumann, W. J.
Phys. Chem. A 2002, 106, 2232-2236.
A demonstration of optical switching between the SP
and MC states of 17 in propylene glycol is shown in the
(19) Gorner, H. Phys. Chem. Chem. Phys. 2001, 3, 416-423.
(20) Marriott, G.; Zechel, K.; Jovin, T. M. Biochemistry 1988, 27,
6214-6220.
(23) Marriott, G.; Jovin, T. M.; Yan-Marriott, Y. Anal. Chem. 1994,
66, 1490-94.
(24) Narazaki, R.; Maruyama, T.; Otagiri, M. Biochim. Biophys. Acta
1997, 1338, 275-281.
(21) Pedersen, A. O.; Jacobsen, J. ′ Eur. J. Biochem. 1980, 106, 291-
295.
(22) He, X. M.; Carter, D. C. Nature 1992, 358, 209-215.
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Sakata et al.
SCHEME 3. Reaction Scheme Employed for the Synthesis of the Thiol-Reactive Spironaphthoxazine (17)a
a Reagents: (a) 1-nitroso-2-naphthol, EtOH; (b) PPh3, CBr4, THF.
Experimental Section
General Procedure for the Synthesis of Thiol-Reac-
tive Optical Switches. 6′-(Hydroxymethyl)spirobenzopy-
ran (11). A solution of 6-(hydroxymethyl)-2,3,3-trimethyl-3H-
indole (15) (50 mg, 0.27 mmol) and CH3I (100 ul, 1.6 mmol) in
CH2Cl2 (2 mL) was refluxed for 12 h. The reaction mixture
was filtered, and the filtrate was dissolved in 0.5 N NaOH and
stirred for 15 min. After extraction with CH2Cl2, the extract
was evaporated to afford oil 10 as a crude product. With
5-nitrosalicylaldehyde (50 mg, 0.30 mmol), 10 was refluxed in
EtOH (2 mL) for 2 h, and then the reaction solvent was
evaporated and subjected to column chromatography (silica
gel; eluent, EtOAc) to afford 11 (70 mg, 75% based on 15): MS-
(EI) 352 (M+, 10), 337 (4), 83 (100); HRMS(EI) M+352.1434
1
(calcd 352.1423); H NMR (CDC13) 1.19 (s, 3H), 1.30 (s, 3H),
2.76 (s, 3H), 4.70 (s, 2H), 5.87 (d, J ) 10.5 Hz, 1H), 6.62 (s,
1H), 6.77 (d, J ) 8.7 Hz, 1H), 6.94 (d, J ) 10.5 Hz, 1H), 7.07
(d, J ) 7.2 Hz, 1H), 8.01 (d, J ) 2.4, 1H), 8.02 (dd, J ) 2.4, 8.7
Hz, 1H).
6′-(Maleimidomethyl)spirobenzopyran (12). To a dry
THF (1 mL) solution of PPh3 (25 mg, 95 µmol), was added
DIAD (18 ul, 95 µmol) over 2 min at -78 °C, and the reaction
mixture was stirred for 5 min. To this solution was added 11
(34 mg, 97 µmol) in dry THF (0.3 mL) over 2 min, and the
FIGURE 3. Absorption spectra of compound 13 (A) and 17
mixture was stirred for 5 min. Neopentyl alcohol (4 mg, 4 µmol)
(B) in propylene glycol at 0 °C. (A) The ability to optically
and maleimide (9 mg, 9 µmol) were added sequentially to the
control the MC to SP states of spirobenzopyrans is shown in
reaction mixture as solids. After being stirred for 5 min, the
the spectrum of a 10 µM solution of 13 following a 10 s
reaction mixture was allowed to warm to rt and stirred for an
irradiation using 365 nm light (a), followed by 10 s of 546 nm
additional 1 h. The reaction mixture was concentrated and
light (b), and 10 s of 365 nm light (c). (B) The ability to optically
then applied to preparative TLC (silica gel; hexane/EtOAc )
control the MC to SP states of spironaphthozaxine (17) is
1:1) to afford 12 (10 mg, 24%): MS(EI) 431(M+, 10), 416 (3),
shown in the spectrum of a 10 µM solution of 17 following a
268 (8), 83 (100); HRMS(EI) M+ 431.1497 (calcd 431.1481); 1H
10 s irradiation using 546 nm light (a), followed by 10 s of 365
NMR (CDCl3) δ 1.61 (s, 3H), 1.27 (s, 3H), 2.74 (s, 3H), 5.84 (d,
nm light (b), 10 s of 546 nm light (c) and finally 10 s of 365
J ) 10.2 Hz, 1H), 6.54 (s, 1H), 6.73 (s, 2H), 6.78 (d, J ) 8.6
nm light (d). The difference in the absorption value for the
Hz, 1H), 6.88 (d, J ) 7.6 Hz, 1H), 6.92 (d, J ) 10.2 Hz, 1H),
MC state between switch cycles observed in Figure 3A is
7.02 (d, J ) 7.6 Hz, 1H), 8.01 (d, J ) 2.4, 1H), 8.03 (dd, J )
caused by slight differences in the method of illumination,
2.4, 8.6 Hz, 1H).
while in Figure 3B, the different MC absorption values result
6′-(Bromomethyl)spirobenzopyran (13). To a THF solu-
from the rapid, thermally driven MC to SP transition (time
tion (1.5 mL) of 11 (28 mg, 78 µmol) and CBr4, (53 mg, 160
constant of 6 s) and simply reflect a difference in the period
µmol) was added dropwise a THF solution (0.5 mL) of Ph3P
between the UV illumination of the SP state and the absorp-
(42 mg, 160 µmol) at 0 °C. The reaction mixture was stirred
tion measurement.
at 0 °C for 30 min and at rt overnight. After evaporation of
the reaction mixture, the residue was subjected to column
chromatography (silica gel; eluent, hexane/EtOAc ) 5:1) to
afford 13 (18 mg, 55%) with recovered 11 (10 mg, 36%): MS-
absorption spectra in Figures 3B. While the SP states
for 17 and the equivalent spirobenzopyran (13) exhibit
similar absorption maxima (Figure 3A,B), the MC state
of 17 is red-shifted by almost 100 nm at 614 nm compared
to compound 13. A similar shift is found in the BSA
conjugates of 13 (Figure 2A) and 17 (Figure 2B). The red
shift is most likely due to the additional phenyl ring of
17. The demonstration that MC absorption spectra of
spirobenzopyran and spironaphthoxazine conjugates ex-
hibit large spectral shifts is significant because it pro-
vides an opportunity to independently control the MC
states of spirobenzopyran and naphthoxazine conjugates
within the same sample.
(EI) 416 (1), 414 (M+, 1), 335 (2); HRMS(EI): M+ 414.0577
1
(calcd 414.0579); H NMR (CDC13) 1.19 (s, 3H), 1.29 (s, 3H),
2.76 (s, 3H), 4.52 (d, J ) 10.5 Hz, 1H), 4.56 (d, J ) 10.5 Hz,
1H), 5.86 (d, J ) 10.2 Hz, 1H), 6.58 (d, J ) 1.3 Hz, 1H), 6.79
(d, J ) 8.4 Hz, 1H), 6.92 (dd, J ) 1.3, 7.3 Hz, 1H), 6.94 (d, J
) 10.3 Hz, 1H), 7.04 (d, J ) 7.3 Hz, 1H), 8.01 (d, J ) 3.2, 1H),
8.04 (dd, J ) 3.2, 8.4 Hz, 1H).
Preparation of Protein Conjugates Using Thiol-Reac-
tive Photochromes. A 20 µM solution of BSA (1 mL; Sigma)
was reacted with 100 µM each thiol-reactive photochrome (3,
6, 9, 12, 13, and 17) for 1 h at room temperature in the dark.
The protein was centrifuged for 10 min at 2000g at 4 °C and
applied to a Bio-Rad PD-10 column equilibrated in G-buffer
containing 1 mM DTT. The concentration of the SP state
within each BSA conjugate was determined from the value of
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Family of Thiol-Reactive Optical Switches
the absorption maximum using an extinction coefficient of
35 000 M-1 cm-1 at 350 nm.15 The G-actin conjugate of 17 was
prepared using a standard protocol.20 Transitions between the
SP and MC states for each photochrome were triggered in 1
mL absorption cuvettes using 365 and 546 nm light from a
100 W Hg-arc lamp attached to a Zeiss Axiovert 35 fluores-
cence microscope.
RR0 8389-01) are greatly acknowledged for NMR spec-
trometry instrumentation.
Supporting Information Available: Experimental pro-
cedures and characterization data for compounds described in
this manuscript and copies of 1H NMR spectra for purified
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
Acknowledgment. This work was supported in part
by a grant awarded to G.M. (NIH R01 HL069970-01).
NSF (CHE-9208463 and CHE-9629688) and NIH (1 S10
JO048207O
J. Org. Chem, Vol. 70, No. 6, 2005 2013