Patent Application
20070195309
The present invention provides methods and compounds for use as reversible optical switches and probes for studying and manipulating biomolecular interactions. Specifically these optical switches are based on the reversible optical chemistry of colorless spirobenzopyran (SP) and colored merocynanine (MC) states.
Optical probes capable of specifically manipulating protein interactions and activities in complex environments1-3 are useful for understanding cellular processes in terms of the reaction mechanisms and its underlying protein function.
A serious limitation, however of certain optical probes such as 2-nitrophenyl-based caged groups is that the photoisomerization reaction that leads to the activation of the protein is irreversible and they function as self-destructing, one-way, single-use, optical switches.
Further, Willner et al11 have shown that although binding of certain conjugates, which are randomly labeled with multiple photochromes, is possible, however, these conjugates are polydisperse and spectroscopically complex.
Accordingly, the need exists to have stereoscopically simpler approaches to reversible, optical switching of biomolecular interactions. Further, the need exists for seeking activity that employs chemically and spectroscopically defined conjugates harboring a single and specifically labeled photochromic probe.
The present invention generally provides compounds and methods for using reversible photochromic compounds as probes. In one embodiment, the present invention provides a compound or a derivative thereof selected from the group consisting of:
wherein R is independently selected from H, CH3, C2H5 and C3H7.
The invention also provides a reversible optical photochromic probe comprising a compound or a derivative thereof as shown above. The probe is capable of undergoing light directed reversible transition between a first state and a second state. The first state is obtained by shining light of about 365 nm on the compound or derivative thereof, whereas the second state is obtained by shining light of about 545 nm to 620 nm on the compound or derivative thereof.
Another embodiment of the invention also provides a method of determining or controlling biomolecular interactions or activity. The method comprises the step of contacting said biomolecule with an optical photochromic probe of a compound or derivative thereof as shown above. Further, the biomolecular interactions may studied or determined using Foerster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoactivation of fluorescence (PAF) technologies and Speckle microscopy. In this method, the optical photochromic probe is capable of undergoing light directed reversible transition between a first and second state. As discussed above, the first state is obtained by shining light of about 365 nm on the compound or derivative thereof and the second state is obtained by shining light of about 545 nm to 620 nm on the compound or derivative thereof. The biomolecules in this method include proteins, DNA, RNA, sugars, or ligands.
The present invention also provides a method of determining free or bound calcium or controlling calcium binding in a subject. The method comprises the step of contacting the subject with a reversible optical photochromic probe of a compound or a derivative thereof as shown above. The free or bound calcium determination or calcium binding is controlled by light directed reversible transition between a first state and a second state. Quantative calcium estimation and controlling calcium binding interactions may be determined using Foerster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoactivation of fluorescence (PAF) technologies and Speckle microscopy. Further, the present invention also provides an optical photochromic probe which has at least two optical switches. Each optical switch may be independently controlled by light directed reversible transition between the first state and the second state.
The present invention also teaches a method of synthesizing a thiol reactive optical switch, comprising the steps of: (a) coupling an indoline derivative with a salycilaldehyde or nitrosonaphthol derivative to yield a spirobenzopyran or a spironaphthoxazine; and (b) conducting a halogen exchange reaction or bromination of alcohol or modified Mitsunobu reaction on the spirobenzopyran or spironaphthoxazine to yield a thiol reactive spirocompound useful as an optical switch. In this method, the indoline derivative is a compound selected from the group consisting of:
wherein R is independently selected from H, CH3, C2H5 and C3H7.
Further, the spirobenzopyran or the spironaphthoxazine is a compound or a derivative thereof as shown above. The indoline derivative may be synthesized by a coupling reaction of an indole derivative and an alkyl halide.
In sum, the present invention represents new compounds and methods of using these compounds as photochromic probes. These and other objects and advantages of the present invention will become apparent from the detailed description and drawings accompanying the claims.
Although the invention is amenable to various modifications and alternative forms, specifics thereof has been shown by way of examples in the drawings and will be described in detail. It should be understood, however, that the invention is not limited to the particular embodiments described. On the contrary, the invention is to cover all modifications, equivalents, and alternatives falling within the scope of the scope and spirit of the present invention.
General
Before the present methods are described, it is understood that this invention is not limited to the particular methodology, protocols, cell lines, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, reference to “a compound” includes a plurality of such compounds and equivalents thereof known to those skilled in the art, and so forth. As well, the terms “a” (or “an”), “one or more” and “at least one” can be used interchangeably herein. It is also to be noted that the terms “comprising”, “including”, and “having” can be used interchangeably.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing the chemicals, cell lines, vectors, animals, instruments, statistical analysis and methodologies which are reported in the publications which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
As defined herein, the term “isomer” includes, but is not limited to strereoisomers and analogs, structural isomers and analogs, conformational isomers and analogs, positional isomers and analogs and the like. In one embodiment, this invention encompasses the use of different positional isomers of a photochromic compounds as described in this invention. It will be appreciated by those skilled in the art that the photochromic compounds useful in the present invention may contain a chiral center. Accordingly, the compounds used in the methods of the present invention may exist in, and be isolated in, optically-active or racemic forms. Some compounds may also exhibit polymorphism. It is to be understood that the present invention encompasses the use of any racemic, optically-active, polymorphic, or stereroisomeric form, or mixtures thereof, which form possesses properties useful in obtaining reversible photochromic probes as described and claimed herein.
This invention further includes method utilizing derivatives of the photochromic compounds. The term “derivatives” includes but is not limited to ether derivatives, acid derivatives, amide derivatives, ester derivatives and the like. In addition, this invention further includes methods utilizing hydrates of the photochromic compounds. The term “hydrate” includes but is not limited to hemihydrate, monohydrate, dihydrate, trihydrate and the like.
As defined herein, “contacting” means that the photochromic compound used in the present invention is introduced into a sample containing the receptor in a test tube, flask, tissue culture, chip, array, plate, microplate, capillary, or the like, and incubated at a temperature and time sufficient to permit binding of the photochromic compound to a receptor. Methods for contacting the samples with the photochromic compound or other specific binding components are known to those skilled in the art and may be selected depending on the type of assay protocol to be run. Incubation methods are also standard and are known to those skilled in the art.
In another embodiment, the term “contacting” means that the photochromic compound used in the present invention is introduced into a subject, and the compound is allowed to come in contact in vitro, i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example, humans. In certain embodiments, the present invention encompasses contacting the compounds useful in the present invention to a patient or subject.
The Invention:
The present invention generally provides compounds and methods for using reversible photochromic compounds as probes. In one embodiment, the present invention provides a compound or a derivative selected from the group consisting of:
wherein R is independently selected from H, CH3, C2H5 and C3H7.
The invention also provides a reversible optical photochromic probe comprising a compound or a derivative thereof as shown above. The probe is capable of undergoing light directed reversible transition between a first state and a second state. The first state is obtained by shining light of about 365 nm on the compound or derivative thereof, whereas the second state is obtained by shining light of about 545 nm to 620 nm on the compound or derivative thereof.
Another embodiment of the invention also provides a method of determining or controlling biomolecular interactions or activity. The method comprises the step of contacting said biomolecule with an optical photochromic probe of a compound or derivative thereof as shown above. Further, the biomolecular interactions may studied or determined using Foerster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoactivation of fluorescence (PAF) technologies and Speckle microscopy. In this method, the optical photochromic probe is capable of undergoing light directed reversible transition between a first and second state. As discussed above, the first state is obtained by shining light of about 365 nm on the compound or derivative thereof and the second state is obtained by shining light of about 545 nm to 620 nm on the compound or derivative thereof. The biomolecules in this method include proteins, DNA, RNA, sugars, or ligands.
The present invention also provides a method of determining free or bound calcium or controlling calcium binding in a subject. The method comprises the step of contacting the subject with a reversible optical photochromic probe of a compound or a derivative thereof as shown above. The free or bound calcium determination or calcium binding is controlled by light directed reversible transition between a first state and a second state. Quantative calcium estimation and controlling calcium binding interactions may be determined using Foerster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoactivation of fluorescence (PAF) technologies and Speckle microscopy. Further, the present invention also provides an optical photochromic probe which has at least two optical switches. Each optical switch may be independently controlled by light directed reversible transition between the first state and the second state.
The present invention also teaches a method of synthesizing a thiol reactive optical switch, comprising the steps of: (a) coupling an indoline derivative with a salycilaldehyde or nitrosonaphthol derivative to yield a spirobenzopyran or a spironaphthoxazine; and (b) conducting a halogen exchange reaction or bromination of alcohol or modified Mitsunobu reaction on the spirobenzopyran or spironaphthoxazine to yield a thiol reactive spirocompound useful as an optical switch. In this method, the indoline derivative is a compound selected from the group consisting of:
wherein R is independently selected from H, CH3, C2H5 and C3H7.
Further, the spirobenzopyran or the spironaphthoxazine is a compound as shown described above. The indoline derivative may be synthesized by a coupling reaction of an indole derivative and an alkyl halide.
Generally, in the present invention, the inventors have shown that dipolar (protein) interactions and activities can be manipulated with unrivalled temporal and spatial resolution3,6 using light-directed activation of caged proteins. When coupled with FRET-based imaging this technique can provide detailed and quantitative information on thermodynamic parameters that define the regulation of specific protein complexes4. In one preferred embodiment, the underlying basis for the photochromic compounds, such as the spirobenzopyrans, which undergo light-directed, includes reversible transitions between a colorless spiro-(SP) state and a colorful merocyanine (MC) state10 (
As part of a new approach for reversible optical switching of biomolecular interactions and activities the inventors present the design, synthesis and chemical and photochemical characterization of five spirobenzopyrans that harbor thiol reactive groups at different sites on a common spirobenzopyran scaffold. The inventors show that the colored merocyanine (MC) specifically linked to proteins acts as an environmentally sensitive probe of dipolar interactions and that the strength of the MC-protein interaction depends on the linkage geometry. Absorption spectroscopy is used to demonstrate that reversible transitions between the spiro-(SP) and MC states on proteins is achieved over many cycles using alternate irradiation with 365 nm and 546 nm light with the full and specific recovery of the MC-protein interaction. The strong interaction between MC and the protein prevent the thermal transition of MC to SP allowing specific control of the two switch states with light. The inventors believe that the higher dipole moment of the MC probe (20 D) compared to SP (5 D) will result in significant differences in the dipolar interactions of SP and MC within the bioconjugate that may be used to differentially and reversibly perturb functional interactions of the bioconjugate with ligands and other proteins.
Differently linked photochromes will exhibit a spectrum of rate constants for both the photo- and thermally driven transitions between MC and SP states. Furthermore the presence of multiple photochromes will lead to the formation of MC and SP dimers in the conjugate having different spectroscopic and photochemical properties compared to the monomer12.
The rate for thermal conversion of MC to SP is 1000 slower for the SBP compared to the SNZ—this difference can be exploited in the design of optical switches that require a stable MC state (SBP) or unstable MC state (SNZ).
In this invention the inventors introduce a different approach for reversible, optical switching of biomolecular interactions and activity that employs chemically and spectroscopically defined conjugates harboring a single and specifically labeled photochromic probe. The inventors introduce a family of thiol reactive spirobenzopyrans that differ only in the position of the reactive group on the common chromophoric ring (
Fluorescence emission from the MC state: The fluorescence of the MC state serves as an intrinsic probe to monitor the status of the optical switch in complex environments e.g. on a surface or within a cell. The emission is centered at 620 nm for the spirobenzopyrans (SBP) probes upon excitation with 546 nm light. The same emission can be generated by uv irradiation of SBP—uv irradiation serves 2 purposes here: first it pumps the S0—S2 transition of MC, which emits fluorescence after returning to the S1 state, and second it serves to maintain a constant population of MC states. As shown in
Photochromic FRET7,8,13 (pcFRET) uses photochromic probes to modulate the quantum yield of the donor emission. The photochromic probes described herein are more suitable probes for pcFRET based analysis of molecular proximity since they can be specifically labeled to unique cysteine residues in proteins rather than randomly introduced through lysine groups. The inventors present evidence that members of the spirobenzopyran family can be used to vary the orientation of the MC dipole in the conjugate thereby providing an experimental system to test the commonly used assumption that the dipole moments for the donor and acceptor probes are randomly orientated in space (i.e. k2 is ⅔)9.
Certain techniques and methodologies of the present invention are described in the following examples. These examples are for illustrative purposes only and should not be deemed to narrow the scope of the present invention.
Materials and Methods
Instrumentation. 1H NMR spectra were measured on a Brucker Ac 300 MHz; mass spectra were carried out on a Micromass AutoSpec for El, a Micromass LCT for ESI, or a Bruker REFLEX II for MALDI. Absorption spectra were recorded on a Hewlett-Packard 82152 diode array spectrophotometer or a Shimadzu 1601 PC instrument. Fluorescence spectroscopy was performed on an SLM-AB2 instrument (Thermoelectron, Madison, Wis.) or an ISS PC1 (Champaign, Ill.). Light-directed switching of the probes described in this work was achieved by irradiating the sample (120-1000 μL) with the 365 nm or 546 nm lines of a 100 W Hg-arc lamp (Zeiss).
Materials. The starting materials for the following syntheses are all commercially available.
Synthesis.
8-(Chloromethyl)spirobenzopyran (2) A THF solution (10 ml) of 3-chloromethyl-5-nitrosalicylaldehyde (50 mg, 0.23 mmol) and 1,3,3-trimethyl-2-methyleneindoline (40 mg, 0.23 mmol) was refluxed for 4 hours. Evaporation of the solvent gave 2 as a crude product, which was used for the subsequent reaction without further purification. MS(EI): 370(M+, 45), 336(72), 159(73); HRMS(EI): M+370.1096 (Calc. 370.1084); 1H NMR (CDCl3) δ1.22 (s, 3H), 1.32 (s, 3H), 2.71, (s, 3H), 4.32 (d, J=11.7 Hz, 1H), 4.38 (d, J=11.7 Hz, 1H), 5.92 (d, J=10.3 Hz, 1H), 6.55 (d, J=7.3 Hz, 1H), 6.89 (dd, J=7.3, 7.3 Hz, 1H), 6.95 (d, J=10.3 Hz, 1H), 7.09(d, J=7.3 Hz, 1H), 7.19(dd, J=7.3, 7.3 Hz, 1H), 8.00 (d, J=2.8, 1H), 8.14 (d, J=2.8 Hz, 1H).
8-(lodomethyl)spirobenzopyran (3). An acetone solution (5 ml) of crude product 2 (56 mg, ca. 0.15 mmol) and Nal (70 mg, 0.47 mmol) was stirred overnight. After evaporation, the residue was purified by column chromatography (silica gel; eluent, CH2Cl2) to afford 3 (55 mg, 78%). MS(EI): 335([M−I]+, 23), 159(13), 71 (100); HRMS: [M−I]+335.1385 (calc. 335.1396); 1H NMR (CDCl3) δ1.24 (s, 3H), 1.38 (s, 3H), 2.77, (s, 3H), 4.13 (d, J=9.3 Hz, 1H), 4.22 (d, J=9.3 Hz, 1H), 5.94 (d, J=10.4 Hz, 1H), 6.59 (d, J=7.5 Hz, 1H), 6.91 (dd, J=7.5, 7.5 Hz, 1H), 6.95 (d, J=10.4 Hz,1H), 7.12 (dd, J=1.0, 7.5 Hz, 1H), 7.21 (ddd, J=1.0, 7.5, 7.5 Hz, 1H), 7.95 (d, J=2.8, 1H), 8.08 (d, J=2.8 Hz, 1H).
1′-(Hydroxyethyl)spirobenzopyran (5). A solution of 2,3,3-trimethyl-3H-indole (1 ml, 6.3 mmol) and 2-iodoethanol (0.56 ml, 8.8 mmol) in MeCN (4 mL) was refluxed for 1 day. After being cooled to r.t., the reaction mixture was suspended in hexane, and the precipitated solid was sonicated and filtered. A part of the obtained purple solid (53 mg out of 1.37 g) was dissolved in 1N KOH (2 mL) and stirred at r.t. for 30 min. After extraction with ether, the organic layer was evaporated to afford 4 as yellow oil, which was used for the next reaction without further purification. A solution of 5-nitrososalicylaldehyde (38 mg, 0.23 mmol) and the obtained 4 in EtOH (5 mL) was refluxed for 4 hours. The mixture was evaporated and purified by column chromatography (silica gel; eluent, hexane:AcOEt=1:1) to afford purple crystal 5 (56 mg, 66% based on 2,3,3-trimethyl-3H-indole). MS(EI): 352(M+, 15), 337(5), 321(9), 83(100); HRMS(EI): M+352.1411 (calc.352.1423); 1H NMR(CDCl3) δ1.20 (s, 3H), 1.30 (s, 3H), 3.34 (ddd, J=5.1, 5.1, 14.7 Hz, 1H), 3.47 (ddd, J=5.5, 7.3, 14.7 Hz, 1H), 3.69-3.82 (m, 2H), 5.89 (d, J=10.5 Hz, 1H), 6.67 (d, J=7.5 Hz, 1H), 6.77 (d, J=8.5 Hz, 1H), 6.90 (dd, J=7.5, 7.5 Hz, 1H), 6.91 (d, J=10.5 Hz, 1H), 7.10 (dd, J=1.1, 7.5 Hz, 1H), 7.50 (ddd, J=1.1, 7.5, 7.5 Hz, 1H), 8.00 (d, J=2.5, 1H), 8.03 (dd, J=2.5, 8.5 Hz, 1H).
1′-(Maleimidoethyl)spirobenzopyran (6). 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, 5 (32 mg, 91 μmol) in dry THF (0.3 ml) was added over 2 min, and the mixture was stirred for 5 minutes. Neopentyl alcohol (4 mg, 4 μmol) and maleimide (9 mg, 9 μmol) were added sequentially to the reaction mixture as solids. After stirred for 5 minutes, the reaction mixture was allowed to warm up to r.t. and stirred for additional 1 hour. The reaction mixture was concentrated and then applied to preparative TLC twice (silica gel; hexane:EtOAc=2:1, then CH2Cl2) to afford 6 (6 mg, 15%). MS(MALDI): 432 [M+H]+; HRMS(ESI): [M+Na+MeOH]+486.1657 (calc.486.1641); 1H NMR (CDCl3) δ1.18 (s, 3H), 1.31 (s, 3H), 3.43 (t, J=6.7 Hz, 1H), 3.77 (t, J=6.7 Hz, 1H), 5.98 (d, J=10.4 Hz, 1H), 6.68 (dd, J=1.0, 7.5 Hz, 1H), 6.72 (s, 2H), 6.79 (d, J=10.1 Hz, 1H), 6.92 (ddd, J=1.0, 7.5, 7.5 Hz, 1H), 6.97 (d, J=10.4 Hz, 1H), 7.12 (dd, J=1.3, 7.5 Hz, 1H), 7.23 (ddd, J=1.3, 7.5, 7.5 Hz, 1H), 8.05 (d, J=2.5, 1H), 8.06 (dd, J=2.5, 10.1 Hz, 1H).
4-(Hydroxymethyl)-2,3,3-trimethyl-3H-indole (14) and 6-(hydroxymethyl)-2,3,3-trimethyl-3H-indole (15). To 3-aminobenzylalcohol (2.0 g, 16 mmol) in conc. HCl (6.4 mL) was added an aqueous solution (5.6 mL) of NaNO2 (1.1 g, 16 mmol) at 0°. After 30 minutes, SnCl2.2H2O (10 g, 44 mmol) in conc. HCl (11 mL) was added to the reaction mixture. The reaction mixture was stirred for an additional 30 minutes, then washed with ether, neutralized with NaOH, and extracted with ether. The ether extract was evaporated to afford 3-hydrazinobenzyl alcohol, which was used for the next reaction without further purification. The obtained 3-hydrazinobenzyl alcohol was dissolved in EtOH (10 mL) and refluxed with 3-methyl-2-butanone (1.7 ml, 16 mmol) and concentrated H2SO4 (1 ml) for 17 hours. After concentration, the reaction mixture was washed with CH2Cl2, basified with Sat. Na2CO3, and extracted with CH2Cl2. The CH2Cl2 extract was subjected to column chromatography (silica gel; eluent, EtOAc) and preparative TLC (silica gel; EtOAc) to give 14 (86 mg, 3% based on 3-aminobenzyl alcohol) and the isomer 15 (131 mg, 4%). 14: MS(EI): 189 (M+, 88), 174 (48), 156 (36), 83 (100); HRMS(EI): M+189.1161 (calc. 189.1154): 1H NMR (CDCl3) δ1.43(s, 6H), 2.29(s, 3H), 4.88(2H, s), 7.26 (d, J=7.5 Hz, 1H), 7.35 (dd, J=7.5 Hz, 1H), 7.50 (d, J=7.5 Hz, 1H). 15: MS(EI): 189(M+, 100), 174 (56), 158(19), 144(18); HRMS(EI): M+189.1155 (calc.189.1154); 1H NMR (CDCl3) δ1.29(s, 6H), 2.27(s, 3H), 4.73(s, 2H). 7.22 (d, J=7.6 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 7.54 (s, 1H).
4′-(Hydroxymethyl)spirobenzopyran (8). A solution of 14 (51 mg, 0.27 mmol) and CH3I (0.15 ml, 2.4 mmol) in CH2Cl2 (1 mL) was refluxed for overnight. The reaction mixture was filtered, and the filtrate was dissolved in 0.5 N KOH (1 ml) and stirred for 15 min. After extraction with CH2Cl2, the extract was evaporated to afford oil 7 as a crude product. With 5-nitrosalicylaldehyde (45 mg, 0.27 mmol), 7 was stirred in EtOH (2 mL) at r.t. over night. The reaction mixture was evaporated and subjected to column chromatography (silica gel; eluent, EtOAc) to afford 8 (34 mg, 36% based on 14). MS(EI): 352 (M+, 22), 337 (4), 189 (10), 83 (100); HRMS(EI): M+352.1408 (calc.352.1423); 1H NMR (CDCl3) 1.25 (s, 3H), 1.28 (s, 3H), 2.74 (s, 3H), 4.74 (d, J=12.5 Hz, 1H), 4.82 (d, J=12.5 Hz, 1H), 5.85 (d, J=10.4 Hz, 1H), 6.55 (d, J=7.7 Hz, 1H), 6.77 (d, J=9.0 Hz, 1H), 6.93 (d, J=7.7 Hz, 1H), 6.96 (d, J=10.4 Hz, 1H), 7.2 3 (dd, J=7.7, 7.7 Hz, 1H), 8.01 (d, J=2.8, 1H), 8.02 (dd, J=2.8, 9.0 Hz, 1H).
4′-(Bromomethyl)spirobenzopyran (9). To a THF solution (1 mL) of 8 (34 mg, 97 μmol) and CBr4, (70 mg, 211 μmol) was dropped Ph3P (50 mg, 191 μmol) at 0°. The reaction mixture was stirred at 0° for 30 minutes and at r.t. overnight. After evaporation of the reaction mixture, the residue was subjected to column chromatography (silica gel; eluent, hexane:EtOAc=5:1) to afford 9 (24 mg, 60%). MS(EI): 416 (76), 414 (M+, 74), 335 (41), 85 (100), 83 (99); HRMS(EI): M+414.0580 (calc.414.0579); 1H NMR(CDCl3) 1.25 (s, 3H), 1.32 (s, 3H), 2.73 (s, 3H), 4.51 (d, J=10.3 Hz, 1H), 4.65 (d, J=10.3 Hz, 1H), 5.84 (d, J=10.1 Hz, 1H), 6.51 (d, J=7.8 Hz, 1H), 6.79 (d, J=8.5 Hz, 1H), 6.87 (d, J=7.8 Hz, 1H), 6.97 (d, J=10.1 Hz, 1H), 7.20 (dd, J=7.8, 7.8 Hz, 1H), 8.01 (s, 1H), 8.03 (dd, J=2.8, 8.5 Hz, 1H).
6′-(Hydroxymethyl)spirobenzopyran (11). A solution of 15 (50 mg, 0.27 mmol) and CH3I (100 ul, 1.6 mmol) in CH2Cl2 (2 mL) was refluxed for 12 hours. 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 hours, and then the reaction mixture 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 (calc.352.1423); 1H NMR (CDCl3) 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 minutes at −78° C., and the reaction mixture was stirred for 5 minutes. To this solution, 6′-(hydroxymethyl)spirobenzopyran 11 (34 mg, 97 μmol) in dry THF (0.3 ml) was added over 2 minutes, and the mixture was stirred for 5 minutes. Neopentyl alcohol (4 mg, 4 μmol) and maleimide (9 mg, 9 μmol) were added sequentially to the reaction mixture as solids. After stirred for 5 minutes, the reaction mixture was allowed to warm up to r.t. and stirred for additional 1 h. The reaction mixture was concentrated and then applied to preparative TLC (silica gel; hexane:EtOAc=1:1) to afford 12 (10 mg, 24%). MS(EI): 431 (M+, 10), 416 (3), 268 (8), 83 (100); HRMS(EI) M+431.1497 (calc.431.1481); 1H NMR (CDCl3) δ1.61 (s, 3H), 1.27 (s, 3H), 2.74 (s, 3H), 5.84 (d, J=10.2 Hz, 1H), 6.54 (s, 1H), 6.73 (s, 2H), 6.78 (d, J=8.6 Hz, 1H), 6.88 (d, J=7.6 Hz, 1H), 6.92 (d, J=10.2 Hz, 1H), 7.02 (d, J=7.6 Hz, 1H), 8.01 (d, J=2.4, 1H), 8.03 (dd, J=2.4, 8.6 Hz, 1H).
6′-(Bromomethyl)spirobenzopyran (13). To a THF solution (1.5 mL) of 11 (28 mg, 78 μmol) and CBr4, (53 mg, 160 μmol) was dropped a THF solution (0.5 mL) of Ph3P (42 mg, 160 μmol) at 0 C. The reaction mixture was stirred at 0° for 30 min and at r.t. 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(EI): 416 (1), 414(M+, 1), 335 (2); HRMS(EI): M+414.0577 (calc.414.0579); 1H NMR(CDCl3) 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).
6′-(Hydroxymethyl)spironaphthoxazin (16). A solution of 1 5 (62 mg, 0.33 mmol) and CH3I (150 μl, 2.4 mmol) in CH2Cl2 (1 mL) was refluxed for 12 hours. 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 (43 mg) as a crude product. With 1-nitroso-2-naphthol (39 mg, 0.23 mmol), 10 was refluxed in EtOH (10 mL) for 3 hours, and then the reaction mixture was evaporated and subjected to column chromatography (silica gel; hexane:EtOAc 3:1) and preparative TLC (silica gel; hexane:EtOAc 2:1) to afford 16 (43 mg, 53% based on 15). MS(EI): 358(M+, 20), 343(15), 189(20), 83 (100); HRMS(EI): M+358.1670 (calc.358.1681); 1H NMR(CDCl3) 1.35 (s, 3H), 1.36 (s, 3H), 2.78 (s, 3H), 4.70 (m, 2H), 6.63 (s, 1H), 6.88 (d, J=7.3 Hz, 1H), 7.01 (d, J=8.9 Hz, 1H), 7.07 (d, J=7.3 Hz, 1H), 7.40 (dd, J=7.9, 7.9 Hz, 1H), 7.58 (dd, J=7.9, 7.9 Hz, 1H), 7.67 (d, J=8.9 Hz, 1H), 7.75 (s, 1H), 7.75 (d, J=7.9 Hz, 1H), 8.56 (d, J=7.9 Hz, 1H)
6′-(Bromomethyl)spironaphthoxazin (17). To a THF solution (1 mL) of 16 (16 mg, 45 μmol) and CBr4, (30 mg, 91 μmol) was dropped a THF solution (0.5 mL) of Ph3P (23 mg, 88 μmol) at 0 C. The reaction mixture was stirred at 0° for 30 min and at r.t. overnight. after evaporation of the reaction mixture, the residue was subjected to preparative TLC (silica gel; hexane:EtOAc=2:1) to afford 17 (3 mg, 15%) with recovered 16 (11 mg, 69%). MS(EI): 422 (6), 420 (M+, 6), 407(4), 405(4), 199(40), 83(100); HRMS(EI): M+420.0839 (calc. 420.0837); 1H NMR (CDCl3) 1.33 (s, 3H), 1.35 (s, 3H), 2.77 (s, 3H), 4.51 (d, J=10.2 Hz, 1H), 4.55 (d, J=10.2 Hz, 1H), 6.59 (d, J=1.6 Hz, 1H), 6.92 (dd, J=1.5, 7.4 Hz 1H), 7.00 (d, J=9.2 Hz, 1H), 7.03 (d, J=7.9 Hz, 1H), 7.40 (ddd, J=1.1, 7.0, 8.2 Hz, 1H), 1H), 7.58 (ddd, J=1.5, 7.0, 8.2 Hz, 1H), 7.67 (d, J=9.2 Hz, 1H), 7.73 (s, 1H), 7.75 (d, J=8.2 Hz, 1H), 8.55 (d, J=8.2 Hz, 1H)
Reagents: (a) 3-chloromethyl-5-nitrosalicylaldehyde, THF; (b) Nal, acetone; (c) 5-nitrosalicylaldehyde, EtOH; (d) PPh3, DIAD, maleimide, neopentyl alcohol, THF; (e) PPh3, CBr4, THF.
Reagents: (a) NaNO2, SnCl2.H2O, HCl (b) 3-methyl-2-butanone, H2SO4, EtOH; (c) 2-iodoethanol, CH3CN; (d) KOH, H2O (e) Mel, CH2Cl2.
Reagents: (a) 1-nitroso-2-naphthol, EtOH; (b) PPh3, CBr4, THF.
Chelate-Spirocompounds
6-(bromomethyl)-2,3,3-trimethyl-3H-indole (1).
To a CH2Cl2 solution (2.0 mL) of 15 (35 mg, 185 μmol) and NBS, (40 mg, 220 μmol) was dropped Ph3P (58 mg, 220 μmol) at 0° C. The reaction mixture was stirred at 0° C. for 1 hr and at r.t. 3 h. After evaporation of the reaction solvent, the residue was subjected to preparative TLC (silica gel, EtOAc) to afford I (42 mg, 89%). MS(EI): 253(7), 251(M+, 7), 172(M+-Br, 62), 83(100); HRMS(EI): M+251.0320 (calc. 251.0310); 1H NMR (CDCl3) δ1.31(s, 6H), 2.30(s, 3H), 4.57(s, 2H), 7.25-7.28(m, 2H), 7.56(s, 1H).
6-[N′,N′-bis(tert-buthyloxycarbonylmethyl)aminomethyl]-2,3,3-trimethyl-3H-indole (II). To a THF (12 ml) suspension of di-tert-butyl iminodiacetate (203 mg, 835 μmol) and ground K2CO3 (126 mg, 913 μmol) was added a THF (2 ml) solution of I (42 mg, 116 μmol) dropwise at refluxing condition, and the reaction mixture was further refluxed for 6 h. After evaporation of the reaction solvent, the residue was subjected to preparative TLC (silica gel, hexane:EtOAc 3:1) to afford I (46 mg, 67%). MS(EI): 31 5(M+—CO2tBu,55), 215(53), 196(80), 172(100); HRMS(EI): [M-tBuO2C]+315.2076 (calc.315.2073); 1H NMR (CDCl3) δ1.29(s, 6H), 1.47(s, 18H), 2.27(s, 3H), 3.44(s, 4H), 3.94(s, 2H), 7.22(d, J=7.6 Hz, 1H), 7.32(dd, J=1.3, 7.6 Hz, 1H), 7.49(d, J=1.3 Hz, 1H).
To a THF (8 ml) solution of di-tert-butyl iminodiacetate (504 mg, 2.1 mmol) and Et3N (0.54 ml, 3.9 mmol) was added a THF (2 ml) solution of 3-chloromethyl-5-nitrosalicylaldehyde (435 mg, 2 mmol) dropwise at refluxing condition, and the reaction mixture was further refluxed for 4 h. After filtration, the reaction solvent was evaporated to afford V as a mixture with Et3N (10:7), which was used for the next reaction without purification. 1H NMR(CDCl3) δ1.50(s, 18H), 3.46(s, 4H), 4.07(s, 2H), 8.22(d, J=3.8 Hz, 1H), 8.65(d, J=3.8 Hz, 1H), 10.44 (s, 1H)
A solution of II (20 mg, 48 μmol) and CH3I (0.1 ml, 1.6 mmol) in CHCl3 (1.5 mL) was heated at 65° C. for 14 h. To the reaction mixture 0.5 N NaOH (1 ml) was added and stirred for 15 min. After extraction with CH2Cl2, the extract was evaporated to afford oil III as a crude product, which was used for the next reaction without purification and characterization. A solution of crude V (25 mg containing Et3N, ca. 51 μmmol) and the obtained III in THF (2 mL) was stirred for over night. The mixture was evaporated and purified by column chromatography (Sephadex; eluent, hexane:MeOH: CH2Cl2=2:1:1) to afford purple crystal VI (6 mg, 15% based on II). HRMS(ESI): [M+Na]+589.4429 (calc.859.4469); 1H NMR (CDCl3) δ1.18 (s, 3H), 1.26 (s, 3H), 1.39 (s, 18H), 1.49 (s, 18H), 2.70 (s, 3H), 3.23 (d, J=17.0 Hz, 2H), 3.29 (d, J=17.0 Hz, 2H), 3.46 (s, 4H), 3.59 (d, J=15.8 Hz, 1H), 3.66 (d, J=15.8 Hz, 1H), 3.87 (s, 2H), 5.84 (d, J=10.4 Hz, 1H), 6.63 (s, 1H), 6.81 (dd, J=1.4, 7.4 Hz, 1H), 6.91 (d, J=10.4 Hz, 1H), 6.97 (d, J=7.4 Hz, 1H), 7.92 (d, J=2.5 Hz, 1H), 8.30 (d, J=2.5, 1H).
To a CH2Cl2 (0.3 ml) of VI (2.5 mg, 3.0 μmol) was added trifluoroacetic acid (TFA) (0.5 ml) and the reaction mixture was stirred at r.t. for 5 h. After evaporation of the solvent and TFA, the residue was subjected to column chromatography (Sephadex; eluent, hexane:MeOH: CH2Cl2=2:1:1) to afford VII (1 mg, 55%). 1H NMR (CD3OD) δ1.24-1.28 (m, 6H), 2.80 (s, 3H), 3.34 (s, 2H), 3.47 (s, 4H), 4.01 (s, 4H), 4.45 (s, 2H), 6.01 (d, J=10.3 Hz, 1H), 6.73 (s, 1H), 6.93 (d, J=7.6 Hz, 1H), 6.93 (d, J=10.3 Hz, 1H), 7.13 (d, J=10.3 Hz, 1H), 7.19 (d, J=7.6 Hz, 1H), 8.09 (d, J=2.7 Hz, 1H), 8.26 (d, J=2.7, 1H).
A MeCN (0.8 mL) solution of 2,3,3-trimethyl-3H-indole (16 mg, 0.1 mmol) and N,N-bis(tert-buthyloxycarbonylmethyl)-2-bromoethylamine (35 mg, 0.1 mmol), which was prepared according to xxx was heated at 75° C. for 1 day. After being cooled to r.t., the reaction mixture was stirred with 1N KOH (0.5 mL) for 30 min and extracted with CH2Cl2. The organic layer was evaporated to afford IV, which was used for the next reaction without further purification. A THF (2 ml) solution of V containing Et3N (45 mg, ca. 91 μmol) and the obtained IV was stirred over night, and the mixture was evaporated and purified by column chromatography (Sephadex; eluent, hexane:MeOH:CH2Cl2=2:1:1) to afford purple crystal VIII (19 mg, 23% based on 2,3,3-trimethyl-3H-indole). HRMS(ESI): [M+Na]+859.4465 (calc.859.4469); 1H NMR (CDCl3) δ1.16(s, 3H), 1.24(s, 3H), 1.39(s, 18H), 1.43(s, 18H), 2.80-2.95(m, 2H), 3.25(s, 4H), 3.30-3.40(m, 2H), 3.42(s, 4H), 3.58(d, J=6.3 Hz, 1H), 3.65(d, J=6.3 Hz, 1H), 5.96 (d, J=10.5 Hz, 1H), 6.61(d, J=7.5 Hz, 1H), 6.82(ddd, J=1.0, 7.5, 7.5 Hz, 1H), 6.88(d, J=10.5 Hz, 1H), 7.04(dd, J=1.0, 7.5 Hz, 1H), 7.13(ddd, J=1.0, 7.5, 7.5 Hz, 1H), 7.90(d, J=3.0 Hz, 1H), 8.26(d, J=3.0 Hz, 1H).
To a CH2Cl2 (0.5 ml) of VIII (3.7 mg, 4.4 umol) was added trifluoroacetic acid (TFA) (2 ml) and the reaction mixture was stirred at r.t. over night. After evaporation of the solvent and TFA, the residue was subjected to column chromatography (Sephadex; eluent, hexane:MeOH: CH2Cl2=2:1:1) to afford IX (1.8 mg, 67%). 1H NMR (CD3OD) δ1.26(s, 3H), 1.30(s, 3H), 3.33(s, 4H), 3.52-3.62(m, 4H), 3.76(s, 2H), 3.82(s, 4H), 6.11 (d, J=10.4 Hz, 1H), 6.74(d, J=7.7 Hz, 1H), 6.90(dd, J=7.7, 7.7 Hz, 1H), 7.12(d, J=10.4 Hz, 1H), 7.13-7.20(m, 2H), 8.10(d, J=2.5 Hz, 1H), 8.20(d, J=2.5 Hz, 1H).
Reagents: (a) PPh3,NMS, CH2Cl2; (b) Di-tert-butyl iminodiacetate, K2CO3, THF, reflux; (c) Mel, CHCl3, reflux; (d) 0.5N NaOH (e) N,N-Bis[(tert-butoxycarbonyl)methyl]-2-bromoethylamine, CH3CN; (f) Di-tert-butyl iminodiacetate; Et3N, THF, reflux; (g) V, THF; (h) CF3CO2H, CH2Cl2.
Combinatorial Synthetic Approach
Indoline
Indo-1 (1) is available from Sigma-Aldrich. All other indolines except for Indo-6, 12 and 13 were synthesized through the combinatorial approach (
General Synthetic Method
A solution (CH2Cl2 or CH3CN) of indolenine and alkylhalide (excess amount) was refluxed for 12 h. After being cooled to r.t., the reaction mixture was stirred with 0.5 N NaOH (1 ml) for 15 min and extracted with CH2Cl2. The extract was evaporated to afford indoline as a crude product.
Additional Characteristic Data of Indolines:
Indo-5
1H NMR (CDCl3) δ1.53(s, 6H), 3.03(s, 3H), 3.85(s, 2H), 4.57(s, 2H), 6.43(d, J=7.8 Hz, 1H), 6.77(dd, J=7.8, 7.8 Hz, 1H), 7.12(d, J=7.8 Hz, 1H)
Indo-7
1H NMR (CDCl3) δ1.34(s, 6H), 3.41(t, 2H, 6.2), 3.71-3.74(m, 4H), 3.76(t, J=6.2 Hz, 2H), 3.86(d, J=1.6 Hz, 1H), 3.92(d, J=1.6 Hz, 1H), 6.65(d, J=7.5 Hz, 1H), 6.78(dd, J=7.5, 7.5 Hz, 1H), 7.09(d, J=7.5 Hz, 1H), 7.13(dd, J=7.5, 7.5 Hz, 1H)
Indo-8 (n=2)
1H NMR(CDCl3) δ1.34(s, 6H), 1.66-1.71(m, 4H), 2.34-2.39(m, 2H), 3.50-3.54(m, 2H), 3.67(s, 3H), 3.84(s, 1H), 3.87(s, 1H), 6.54(d, J=6.1 Hz, 1H), 6.76(dd, J=7.6, 7.6 Hz, 1H), 7.10(d, J=7.6 Hz, 1H), 7.12(d, J=7.6 Hz, 1H)
Indo-10
1H NMR (CDCl3) δ1.33(s, 6H), 2.47(t, J=7.2 Hz, 4H), 2.64(m, 2H), 2.85(t, J=7.2 Hz, 4H), 3.67(s, 6H), 3.58-3.70(m, 4H), 6.58(d, J=7.6 Hz, 1H), 6.77(dd, J=7.6, 7.6 Hz, 1H), 7.08(d, J=7.6 Hz, 1H), 7.12(d, J=7.6 Hz, 1H)
Indo-14
1H NMR (CDCl3) δ1.33(s, 12H), 3.63-3.68(m, 8H), 3.83(s, 1H), 3.87(s, 1H), 6.58(d, J=7.5 Hz, 1H), 6.79(dd, J=7.5, 7.5 Hz, 1H), 7.10(d, J=7.5 Hz, 1H), 7.11 (dd, J=7.5, 7.5 Hz, 1H)
Salicylaldehyde and Nitrosonaphthol
Sal-1, nit-1 and Sal-2 are commercially available from Sigma-Aldrich and TCl-America. The synthesis of sal-3 (V) is found in the section of chelating probes.
Spriobenzopyran and Spironaphthoxazine
Spriobenzopyran and spironaphthoxazine are prepared via coupling reaction of indoline with salicylaldehyde or nitrosonaphthol (
General Synthetic Method
A THF or EtOH solution of indoline and salicylaldehyde (1.2 eq) or nitrosonaphthol (1.2 eq) is stirred at r.t. over night or at the refluxing condition for 6 h. After the evaporation of the reaction solvent, the residue was subjected to column chromatography (SiO2 or Sephadex LH-20) to afford spirocompound.
Additional Characteristic Data of Spirocompounds:
Indo-1-sal-3 (R=Me)
MS(EI): 495(M+, 5), 422 (2), 84(100); HRMS(EI): M+495.1998 (calc.495.2006); 1H NMR (CDCl3) δ1.20(s, 3H), 1.27(s, 3H), 2.69(s, 3H), 3.27(s, 4H), 3.60(s, 3H), 3.59(d, J=14.2 Hz, 1H), 3.66(d, J=14.2 Hz, 1H), 5.87 (d, J=10.1 Hz, 1H), 6.54(d, J=7.6 Hz, 1H), 6.86(dd, J=7.6, 7.6 Hz, 1H), 6.92(d, J=10.1 Hz, 1H), 7.07(d, J=7.6 Hz, 1H), 7.17(dd, J=7.6, 7.6 Hz, 1H), 7.94(d, J=2.6 Hz, 1H), 8.12(d, J=2.6 Hz, 1H)
Indo-1-sal-3 (R=tert-Bu)
1H NMR (CDCl3) δ1.19(s, 3H), 1.27(s, 3H), 1.38(s, 18H), 2.70(s, 3H), 3.23(s, 4H), 3.61(s, 2H), 5.85 (d, J=10.1 Hz, 1H), 6.53(d, J=7.4 Hz, 1H), 6.84(ddd, J=1.2, 7.4, 7.4 Hz, 1H), 6.92(d, J=10.1 Hz, 1H), 7.06(dd, J=0.9, 7.4 Hz, 1H), 7.15(ddd, J=1.2, 7.4, 7.4 Hz, 1H), 7.92(d, J=2.6 Hz, 1H), 8.25(d, J=2.6 Hz, 1H)
Indo-3-sal-3 (R=Et)
HRMS(ESI): [M+H]+554.2515 (calc.554.2502); 1H NMR (CDCl3) δ1.21 (t, J=6.9 Hz, 6H), 1.22 (s, 3H), 1.27(s, 3H), 2.11 (t, J=6.0 Hz, 1H), 2.72(s, 3H), 3.27(s, 4H), 3.17 (d, J=17.5 Hz, 2H), 3.28 (d, J=17.5 Hz, 2H), 3.56 (d, J=14.4 Hz, 1H), 3.60 (d, J=14.4 Hz, 1H), 4.06 (q, J=6.9 Hz, 4H), 4.65 (d, J=6.0 Hz, 2H), 5.89 (d, J=10.2 Hz, 1H), 6.63 (d, J=1.1 Hz, 1H), 6.86 (dd, J=1.1, 7.4 Hz, 1H), 6.94 (d, J=10.2 Hz, 1H), 7.05 (d, J=7.4 Hz, 1H), 7.95 (d, J=2.6 Hz, 1H), 8.08 (d, J=2.6 Hz, 1H)
Indo-5-sal-2
1H NMR (CDCl3) δ1.21 (s, 3H), 1.31 (s, 3H), 2.71, (s, 3H), 4.34 (d, J=11.8 Hz, 1H), 4.40 (d, J=11.8 Hz, 1H), 4.48 (d, J=9.7 Hz, 1H), 4.52 (d, J=9.7 Hz, 1H), 5.59 (d, J=10.1 Hz, 1H), 6.54 (d, J=1.6 Hz, 1H), 6.92 (dd, J=1.6, 7.6 Hz, 1H), 6.96 (d, J=10.1 Hz, 1H), 6.99 (d, J=7.6 Hz, 1H), 8.01 (d, J=2.8, 1H), 8.15 (d, J=2.8 Hz, 1H)
Indo-7-sal-3 (R=tert-Bu)
HRMS(ESI): [M+H]+716.2513 (calc.716.2546); 1H NMR (CDCl3) δ1.19(s, 3H), 1.25(s, 3H), 1.39(s, 18H), 3.25(s, 4H), 3.27-3.81 (m, 6H), 3.43(t, J=6.0 Hz, 2H), 3.62(s, 2H), 5.95 (d, J=10.2 Hz, 1H), 6.61(d, J=7.5 Hz, 1H), 6.85(ddd, J=1.0, 7.5, 7.5 Hz, 1H), 6.91(d, J=10.2 Hz, 1H), 7.06(dd, J=1.0, 7.5 Hz, 1H), 7.15(ddd, J=1.0, 7.5, 7.5 Hz, 1H), 7.92(d, J=2.6 Hz, 1H), 8.25(d, J=2.6 Hz, 1H)
Indo-8-sal-3 (n=2, R=Me)
MS(EI): 595(M+,21), 522 (13), 86(100); HRMS(EI): M+595.2546 (calc.595.2530); 1H NMR(CDCl3) δ1.21(s, 3H), 1.27(s, 3H), 1.57-1.73(m, 4H), 2.31(t, J=6.7 Hz, 2H), 3.12(t, J=6.7 Hz, 2H), 3.30(s, 4H), 3.58(d, J=11.4 Hz, 1H), 3.62(s, 6H), 3.65(s, 3H), 3.66(d, J=11.4 Hz, 1H), 5.89 (d, J=10.2 Hz, 1H), 6.56(d, J=7.5 Hz, 1H), 6.86(dd, J=7.5, 7.5 Hz, 1H), 6.92(d, J=10.2 Hz, 1H), 7.08(d, J=7.5 Hz, 1H), 7.16(dd, J=7.5, 7.5 Hz, 1H), 7.94(d, J=2.8 Hz, 1H), 8.15(d, J=2.8 Hz, 1H)
Indo-10-sal-3 (R=Me)
HRMS(ESI): [M+H]+697.3057 (calc.697.3085); NMR (CDCl3) δ1.17(s, 3H), 1.26(s, 3H), 2.37(t, J=7.2 Hz, 4H), 2.50-2.65(m, 2H), 2.74(t, J=7.2 Hz, 4H), 3.14-3.25(m, 2H), 3.31(s, 4H), 3.61(s, 6H), 3.62(s, 6H), 3.61-3.65(m, 2H), 5.88 (d, J=10.6 Hz, 1H), 6.57(d, J=7.6 Hz, 1H), 6.85(dd, J=7.6, 7.6 Hz, 1H), 6.93(d, J=10.6 Hz, 1H), 7.07(d, J=7.6 Hz, 1H), 7.16(dd, J=7.6, 7.6 Hz, 1H), 7.94(d, J=2.7 Hz, 1H), 8.16(d, J=2.7 Hz, 1H)
Indo-11-sal-1 (R=Et)
HRMS(ESI): [M+Na]+546.8217 (calc.546.2216); NMR (CDCl3) δ1.19 (s, 3H), 1.27 (s, 3H), 1.29 (t, J=7.0 Hz, 6H), 2.75 (s, 3H), 3.61 (s, 4H), 3.92 (s, 3H), 4.19 (q, J=7.0 Hz, 4H), 5.86 (d, J=10.6 Hz, 1H), 6.67 (s, 1H), 6.79 (d, J=8.6 Hz, 1H), 6.84 (d, J=7.3 Hz, 1H), 6.93 (d, J=10.6 Hz, 1H), 7.01 (d, J=7.3 Hz, 1H), 8.01-8.05 (m, 2H)
Indo-14-sal-3 (R=tert-Bu)
HRMS(ESI): [M+Na]+1223.5823 (calc.1223.5892); NMR (CDCl3) δ1.13(s, 3H), 1.14(s, 3H), 1.22(s, 3H), 1.23(s, 3H), 1.38(s, 36H), 3.20-3.60(m, 8H), 3.23(s, 8H), 3.58-3.60(s, 4H), 5.79 (d, J=10.3 Hz, 1H), 5.85 (d, J=10.3 Hz, 1H), 6.54(d, J=6.9 Hz, 1H), 6.57(d, J=6.9 Hz, 1H), 6.77(d, J=10.4 Hz, 1H), 6.84(d, J=10.4 Hz, 1H), 6.82-6.88(m, 2H), 7.05(d, J=7.4 Hz, 2H), 7.09(dd, J=7.4 Hz, 2H), 7.12(dd, J=7.4, 7.4 Hz, 1H, 7.88(m, 2H), 8.24(m, 2H)
Rabbit muscle G-actin was purified according to Marriott14 in G-buffer (2 mM Tris, 0.2 mM CaCl2, and 0.2 mM ATP at pH 8.0). The concentration of G-actin was determined by absorption spectroscopy using an extinction coefficient of 3400 M−1 cm−1 at 290 nm14. 1 ml of a 20 μM solution of G-actin was treated with 20 μL of a 10 mM DMF stock solution of each thiol reactive, spirobenzopyran probe [3, 6, 9, 12, 13]. The reaction mixture was left in the dark for 2 hours at room temperature. The protein was centrifuged for 10 min at 2000 g at 4° and applied to a Bio-Rad PD-10 column equilibrated in G-buffer containing 1 mM DTT. If necessary the conjugate was dialyzed against 1 L of G-buffer at 4°. The labeled G-actin solution was clarified by centrifugation (100000 g for 1 hour) before absorption spectrometry. The extinction coefficient for the SP probe is taken as 35,000 M−1cm−1 for the near ultraviolet absorption maximum and 52,000 M−1cm−1 for the maximum visible wavelength for the MC probe15.
RESULTS AND DISCUSSION for Example I and II
The goal of the synthetic work was to prepare a family of optical switches for the specific labeling of biomolecules. The reactive groups (bromo-, iodo- and maleimido), which were introduced to different sites on the spirobenzopyran ring, were used to attach a common photochrome to thiol groups within biomolecules. This family of photochromic reagents allows control of the chromophore dipole geometry within the bioconjugate. This represents a new development in bioconjugate chemistry that will be useful for spectroscopic studies of biomolecular structure and dynamics.
Synthesis of Thiol Reactive Spiropyrans.
The synthetic approach used to prepare the thiol reactive optical switches is summarized in schemes 1 and 2. The syntheses involve coupling the indoline derivatives (1, 4, 7, 10) with the corresponding salycilaldehydes to yield the four key spirobenzopyrans (2, 5, 8, 11)16-18. 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 reaction19.
Optical Spectroscopy of the Spirobenzopyran Chromophore in Solvents
The characterization of the effects of specific solvent interactions on the MC absorption spectrum will be important to interpret the nature of MC interactions within a protein. The average energy of the lowest energy absorption band for MC contains information on the dielectric constant of the solvent and the presence of specific solvent effects, such as H-bonds and dipole-dipole interactions. The ground and excited state dipole moment for polar aromatic probes such as MC are often defined by the nature and location of polar groups within the conjugated ring system20. In the case of MC, the dipole will most likely be defined by the positive nitrogen atom and the negative nitro-group—these two groups may be considered as monopoles since they are well-separated by the aromatic rings and olefin (
Absorption and fluorescence spectroscopy can provide important information on the nature of molecular interactions between the SP and MC states and their molecular environment15,22. For example, the similarity in the MC absorption spectra of the five spirobenzopyrans (
Further information on MC-solvent interactions can be derived from studying the fluorescence properties of MC. From a practical standpoint, fluorescence from 1MC* is best studied by exciting the spirobenzopyran with mid-ultraviolet light (S0—S2 transition)—this condition maintains a uniform population of MC excited state molecules (see Jablonski diagram,
An explanation for the anomalous polar solvent-induced blue-shifts in the MC absorption spectrum and the insensitivity of MC fluorescence to polar solvents is provided by considering an unusual property of the MC dipole moment—Bletz et al have shown that the MC dipole is lower in the excited state (14 D) compared to the ground state (20 D)21. Dipolar interactions between the MC dipole and polar, H-bonding groups (e.g. water and the peptide bond in proteins) should therefore be stronger in the ground state compared to the excited state. Furthermore the difference in the interaction energy for specific dipolar interactions between solvent molecules and the MC dipole will be greater in the ground state compared to the excited state as the inventors show in the absorption and fluorescence studies (e.g.
Although the observed fluorescence of MC is weak (
Effect of Linkage Geometry on the Absorption Spectrum of Spirobenzopyran Conjugates
Having shown that the MC absorption spectrum is sensitive to specific dipolar interactions related studies were performed to characterize the dipolar environment around MC probes specifically attached to biomolecules.
G-Actin: The reaction between a ten-fold molar excess of thiol reactive, spirobenzopyran reagents and 20 μM G-actin at 20° is complete within 2 hours. The concentration of spirobenzopyran in each conjugate is determined from the value of the absorption maximum of the SP form of G-actin conjugates using an extinction coefficient of 35,000 M−1cm−1 at 350 nm15 (
Given the sensitivity of the MC absorption spectrum to specific solvent interactions, it is not surprising the average energy and shape of the lowest energy MC-G-actin absorption transition is also dependent on the MC-cysteine-374 linkage geometry (
The near ultraviolet SP absorption spectra in the five spirobenzopyran G-actin conjugates differ to a lesser extent than that found for MC, which the inventors believe is a result of the lower SP ground state dipole moment at 5 D compared to that of MC at 20 D21.
BSA: BSA conjugates of the spirobenzopyran reagents were prepared in a similar fashion to that described for G-actin yielding similar labeling ratios (
Optical Switching of Dipolar Interactions within Spirobenzopyran Conjugates:
The inventors propose a model to understand the origin of different interactions of MC within a protein that is based on a qualitative comparison of the different linkage geometries between the five spirobenzopyran probes and the thiol group on the protein. As shown in
The results from the MC-protein absorption studies suggest that dipolar interactions between MC and the protein are specific and site selective—the free energy (difference) measured for MC probes attached to a unique cysteine residue with different linkage geometries is similar to that found in complexes of actin with actin binding proteins and other ligands. However, the interaction between the MC probe and the protein can be significantly reduced by optically converting MC to SP, which will assume a different location and engage in weaker interactions with the protein. The challenge for ongoing studies is to prepare spirobenzopyran protein conjugates in which the MC or the SP state competes effectively on the protein for the binding site of a regulatory ligand, as depicted in
Thermally-Driven Transitions Between MC and SP
The rate for the thermal equilibration of MC to SP in ethanol was measured using the decrease in the maximum MC absorption value as a function of time in the dark (
Highlighting Applications of Photochromic Probes in FRET:
The ability to differentially project an acceptor probe with a well-defined dipole moment to different locations on a protein can be used as part of a new approach to improve the precision of proximity determinations using FRET; First, the ability to shift the absorption maximum of a common MC chromophore using different probe-protein linkage geometries can be used to change the overlap integral between the donor and acceptor probes and to “tune” the Foerster distance between an identical pair of donor and acceptor probes28. To illustrate this potential the inventors used Foerster theory27 to highlight the effects of MC absorption spectral shifts in G-actin conjugates of compounds 9 and 13 (maximum absorption of 500 nm and 551.5 nm respectively; MC ∈max=52,000 M−1cm−1; K2−⅔; n=1.4, φ=0.7) on the overlap integral (J) with a simulated tetramethylrhodamine (TMR) donor spectrum fluorescence maximum at 575 nm)—j increases from 1.10×1015 M−1·cm−1·nm4 in compound 9 to 4.21×1015 M−1·cm−1·nm4 in compound 13, which would lead to a tuning of the Ro in this simulated TMR-MC-acceptor pair from 4.8 nm to 6.0 nm for compounds 9 and 13 respectively. Clearly by choosing other MC-conjugates of G-actin shown in
Summary for Examples I and II
A family of thiol reactive spirobenzopyran reagents is described that can be specifically attached to biomolecules where they undergo efficient and reversible, light-directed structural transitions between two states (SP and MC) that have widely different structural and physical properties. Differences in the properties and interactions between SP and MC within a bioconjugate are being exploited as part of a new approach to achieve reversible, optical switching of biomolecular interactions. The inventors show that the strength and nature of the dipolar interactions between MC and the biomolecule depend on the MC linkage geometry and that these interactions prevent the thermally-driven transition between MC and SP. The difference in the free energy of interactions between MC probes and G-actin is comparable to that found for complexes of G-actin with regulatory proteins (˜6 kcal/mol)—together the studies described in this work demonstrate the feasibility of using site-selectively labeled photochromes as optical switches for the reversible, modulation of biomolecular interactions e.g., protein-protein, protein-Ca2+. The inventors also highlight how the family of spirobenzopyran reagents described in this work can be used to tune the Foerster distance and to provide an experimental system to evaluate the K2 value in FRET based analysis of molecular complexes.
The concentration of intracellular Ca2+ rapidly increases during the activation of membrane-activated cell signaling pathways that leads to motility, muscle contraction and exocytosis. The change in [Ca2+] is modest (2˜5-fold) and is usually only effective close to the plasma membrane unless intracellular Ca2+ stores are released. Ca2+-binding to signaling proteins is an early step in the regulation of cellular processes and usually leads to structural changes in the protein that trigger a cascade of functional protein interactions and activities that ultimately lead to a physiological response. Understanding the mechanisms underlying calcium ion signaling therefore requires techniques capable of mapping and manipulating [Ca2+] within the cell and performing correlative molecular analysis to show how local changes in [Ca2+] are coupled to the activation of Ca2+-binding signaling proteins and pathways and to the global physiological response. The Tsien laboratory revolutionized the field of Ca2+ signaling through the development and application of optical probes for imaging or perturbing Ca2+ (Tsien, 1989)—these probes include fluorescent Ca2+-indicators such as Indo-1 that bind to Ca2+ in a rapid and reversible reaction and are effective in interrogating intracellular [Ca2+] over many excitation cycles at non-buffering levels, and photomodulatable Ca2+-chelators that release or sequester Ca2+ in a light-driven reaction.
Research aimed at understanding the mechanisms that underlie Ca2+-triggered physiological processes such exocytosis and muscle contraction, which occur on a microsecond timescale, can only be realized using a chemical relaxation approach. Temperature, pressure and flow perturbation techniques are either unsuitable or non-specific in changing intracellular [Ca2+] and so considerable effort has been expended in developing photoactivatable Ca2+-chelators, such as Nitr-5 and NP-EGTA, to generate Ca2+ transients at defined sites with cells. However, these probes can usually only be used to generate a single perturbation of Ca2+ because the photoisomerization reaction, which occurs with a rate of 104 s−1 at best, is irreversible and generates photoproducts that are either reactive or maintain an affinity for Ca2+ (Nitr-5). These properties not only limit the usefulness of the caged Ca2+ probe but their effects must be evaluated in separate, control experiments.
The inventors believe that the ideal Ca2+-perturbation probe would incorporate both Ca2+ sequestering and release activities that are controlled through reversible, efficient and rapid, light-driven reactions without the release of secondary products. As part of a new program to develop such probes, the inventors introduce a new approach in the design of optical probes capable of generating specific and reversible, light-directed perturbations of Ca2+ and Ca2+-binding proteins within cells. The approach is based on interesting properties of benzospiropyrans, which undergo rapid and reversible transitions between a colorless spiro-(SP) state and a colorful merocyanine (MC) state (Inouye, 1994). Transitions between the two states of the switch can be controlled by exciting the SP state with 365 nm light (SP—MC) and the MC state with 546 nm light (MC—SP). A family of optical switches was designed and synthesized with the aim of positioning the four carboxyl groups on the optical switch such that they exhibit a high affinity for Ca2+ in either the SP or MC state. Since the two aromatic rings in the benzospiropyran are orthogonal the inventors reasoned that this state would not bind Ca2+ very tightly whereas the positioning of the four carboxyl groups in the planar MC state would lead to strong Ca2+ chelator (
The dissociation constant for the SP—Ca2+ complex of ˜500 μM for compound X shown in
Irradiation of the SP calcium switch (Compound X in
An attractive feature of the Ca2+ switches described in this invention is the very fast rate of optical switching between the strong and weakly binding Ca2+ states. Studies by Horner (2001) suggest that the photoisomerization of benzospiropyrans occurs via triplet state reactions within 11 μs. If Ca2+ is released from the MC state on a similar timescale then this class of probe will lead to an improvement in the rate of Ca2+-perturbations by at least 3 orders of magnitude (Adams and Tsien, 1989).
A complicating factor in the use of benzospiropyrans is that the MC—SP reaction may also occur in the ground state driven by thermal fluctuations (Sakata et al, 2004). This property may lead to the leaching of Ca2+ from MC—however, the inventors note this effect will have limited impact in most applications since the rate for the thermally driven process is slow (0.02 s1−) at 37 c compared to the excited state (105 s−1). Furthermore the ground state reaction provides an additional degree of control of the Ca2+-switch. The inventors also note that a further reduction in the rate of the thermally driven MC—SP reaction can be realized by attaching the optical switch to proteins (Sakata et al, 2004). Also, the rate for the MC to SP transition for SNZ is 1000 times faster than SBP.
The family of Ca2+-optical switch was introduced into living cells as methyl esters. The efficiency of the loading was determined by fluorescence imaging of the MC state using and excitation wavelength of 546 nm and emission of >600 nm. Since excitation of MC leads to the MC—SP transition and loss of fluorescence this analysis should be conducted using a single pulse of 546 nm light. In most cell studied the optical switch entered the cell within a few minutes and while initially it was localized within vesicles within 30 minutes the fluorescence was generally uniformly distributed within the cell. The efficiency of the de-esterification of the carboxyl groups was not determined but assumed, on the basis of functional Ca2+ responses, to be complete within the incubation period.
The family of Ca2+-optical switch described in this invention has built-in design features that can be used to tune the maximum of the action spectrum for the MC—SP transition. The nitro group generates a weakly fluorescent MC state, which can be used to quantify the kinetics of Ca2+-perturbations and to image the distribution of the MC state within living cells. Interestingly the maximum MC absorption spectrum of switches lacking the nitro group is shifted to 620 nm and leads to a non-fluorescent MC state. However, this class of photochrome exhibits a remarkable degree of photostability that allows fully reversible optical switching between the SP and MC states over numerous irradiation cycles.
A second important design feature is the incorporation of reactive functional groups onto the optical switch, which can be used to prepare conjugates of proteins and other biomolecules as well as surfaces containing amino or thiol groups. These conjugates can be used to target the optical switch to specific sites in the cell e.g. membrane, actin cytoskeleton, or to restrict the optical switch to specific sites on a chip or surface for applications in biotechnology.
The inventors have introduced a new class of calcium ion chelating probe that undergo rapid and reversible, light-directed transitions between two structural states that exhibit widely different affinities for calcium ions. The advantages of this approach to perturbing [Ca2+] compared to the caged Ca2+ approach include: 1), a single probe that can be used to sequester or release Ca2+ using light; 2), the transitions are fully reversible and can occur exclusively in the excited state or a combination of an excited state and a thermally driven ground state reaction; 3), the transitions are rapid (11 μs) and proceed with almost perfect quantum efficiency; 4), the transitions do not involve the release of photoproducts and are therefore free of artifacts associated with 2-nitrophenyl based caged groups; 5), the action spectrum for the MC—SP transition can be tuned over a broad wavelength range (500 nm-750 nm) to limit interference from other optical probes in the sample.
Kinetic mapping of Ca2+-mediated signaling pathways also requires analysis of downstream protein targets. Some years ago the inventors introduced a technique to generate spatially and temporally defined perturbations of specific proteins using light-directed of caged proteins. The caged protein approach is also affected, but to a lesser extent, by the limitations outlined for caged Ca2+ chelators. The inventors will use this approach to generate perturbations of Ca2+-binding proteins, troponin C, calmodulin and CapG, which are known to underlie the regulation of muscle contraction and cell motility respectively. The inventors will also show how a new class of optical probe can be used to generate conjugates of cTnC, calmodulin and CapG whose Ca2+-binding properties can be rapidly and reversibly modulated using light-directed.
Combinatorial Synthesis of Calcium Chelates.
Combinatorial libraries are illustrated in FIGS. 14 and B and
Overall, the present invention envisions a new class of chemical library for drug screening. The ability to synthesize>million member library of SBP and SNZ compounds using the approach outlined in this patent represents new opportunities for drug screening and discovery. Specifically the drugs allow for:
Cell protrusion is characterized by fluctuations in the concentrations and activities of, and/or interactions between, membrane receptors, Rac1, Ca2+, PIP2, cofilin and actin filament barbed end capping proteins. These dynamic events are confined to the lamellipodium and are somehow coupled to the rapid and polarized polymerization of actin filaments in the vicinity of the activated membrane receptor (Barkalow et al, 1996;Hartwig et al, 1995; Machesky & Insall, 1999; Cox et al, 1997; Pollard, 2003; Ghosh et al, 2004; Vallotton et al, 2004). A major challenge in cell motility research is to develop innovative approaches to study the temporal and spatial regulation of these molecular events within the ˜6 fL volume of a typical lamellipodium (3 μm×10 μm×0.2 μm; Abraham et al, 1999; Zhang et al, 2002)—these techniques must be capable of detecting, mapping and resolving interactions for fewer than 1000 barbed ends within the few seconds it takes to form a protrusion.
The late Fred Fay and his colleagues and others have shown that the increase in cell Ca2+ shortly after the activation of membrane receptors correlates with a dramatic increase in the rate of actin filament polymerization and contraction of actomyosin at the cortex (Brundage et al, 1991; 1993; Walker et al, 2001; Hendey et al, 1993; Maxfield, 1993). While Ca2+ activates many cytoskeleton proteins, most notably calmodulin (Hahn et al, 1992), the Ca2+-dependent barbed-end capping proteins, such as CapG and Gelsolin are likely to be the primary targets for cell protrusion (Young et al, 1994)—deletion of either or both of genes leads to a dysfunctional regulation of protrusion in macrophage cells (Witke et al, 1995; 2001). PIP2 is also associated with the regulation of actin filament polymerization (Janmey, 1994; Yin & Janmey, 2003) and is believed to dissociate capping proteins from their complexes with the barbed-end (Schafer et al, 1996; Cooper & Shafer, 2000) and trigger the actin filament-mediated comet-like motion of vesicles in cells (Rozelle et al, 2000). The focus of this proposal is to understand how changes in the concentration, interactions and activities of Ca2+ and PIP2 are integrated in the lamellipodium and coupled to the regulation of the barbed end of actin filament, actin polymerization and cell protrusion.
A framework to understand the roles of Ca2+, PIP2 and key cytoskeleton-associated proteins in the regulation of cell protrusion is shown in
Regulation of molecular interactions at the barbed-end of the actin filament: On the basis of the model shown in
CapG:CapG is a 40 kD, Ca2+ regulated barbed-end capping protein that is found at high levels in macrophage cells where it regulates actin mediated membrane ruffling during phagocytosis and motility. CapG, unlike Gelsolin, does not sever actin filaments. In vitro studies show that CapG binds to the barbed-end of the actin filament in a Ca2+-dependent fashion with a kd of 1 μM (Southwick & DiNubile, 1986). Unlike Gelsolin and CP, the complex between the barbed end and CapG dissociates at low Ca2+. The interaction of CapG with actin is perhaps the simplest and the most tractable experimental model system to study the regulation of actin filament barbed-ends during protrusion since: (1), CapG is a monomeric protein that exhibits a straightforward interaction with G- and F-actin (Yu et al, 1990); (2), CapG binds to actin only in the presence of Ca2+ and does not sever filaments; (3), CapG binds to the same site on G- and F-actin, which facilitates structural and mechanistic investigations on the regulation of this complex (Tanaka et al, 2003); (4), the CapG-actin complex, like other capping proteins, is regulated by PIP2; (5), The regulation of the CapG-actin complex can be studied in macrophage cells derived from CapG and/or Gelsolin-null mice, which are known to exhibit defects in ruffling and protrusion (Witke et al, 2001).
PIP2: PIP2 regulates actin filament dynamics by dissociating Gelsolin, CapG, and CP from the barbed end of the actin filament (Yin, 1987; Yin & Janmey, 2003; Hartwig et al, 1995; 1996; Shafer & Cooper, 2000; Sun et al, 1999). Consistent with this mode of regulation, platelet cell activation is accompanied by a rise in PIP2, which precedes actin polymerization and protrusion (Bartalow et al, 1995). Further support for this model comes Botelho et al (2000), who showed that PIP2 regulates actin polymerization during phagocytosis, and a study involving the PI's laboratory (Rozelle et al, 2000), that equated an increase in cellular PIP2 with actin polymerization and the motility of vesicles in cells.
Ca2+-independentregulation of barbed ends: Ghosh et al (2004) has shown that free barbed-ends are generated in the lamellipodium by the weak severing activity of cofilin. This discovery is important because it provides an explanation for the large increase in the number of free barbed ends during cell protrusion that cannot be accounted for by the one time severing/capping activity of gelsolin (Bartalow et al, 1995; Yin &Janmey, 2003). Cofilin, on the other hand, may engage in multiple, Ca2+-independent actin filament severing events that generate free barbed ends that are capped by CP (Mejillano et al, 2004), or else lead to a Ca2+-independent polymerization of actin filaments. We will investigate the role of Ca2+-independent generation of free barbed ends in cell protrusion using light direct activation of a constitutively active caged cofilin in cells and using new fluorescent KabC probes (Tanaka et al, 2003) to image the distribution of free barbed ends by uncaging cofilin in cells.
Functional redundancy:The mechanisms underlying Ca2+ and PIP2-mediated regulation of molecular interactions at the barbed-end during cell protrusion are not fully understood—these investigations are further complicated because CapG, CP, radaxin and gelsolin cap filament barbed ends using the same site on actin (Schafer et al, 1996; Tsukita et al, 1989; Klenchin et al, 2003; Tanaka et al, 2003; Kim et al, 2004). On the other hand, gene knock out studies show that CapG, gelsolin and CP each play essential roles in regulating cell protrusion and motility (Mejillano et al, 2004; Witke et al, 1995; 2001). We will address the potential problem of functional redundancy by focusing our studies on the Ca2+-dependent capping interactions of CapG in macrophage cells derived from CapG-null, Gelsolin-null, and Gelsolin/CapG-double-null mice (Dr. Walter Witke has agreed to provide the PI with these mice and protocols to isolate and culture primary macrophages. We will use the RNAi approach of Mejillano et al (2004) to suppress CP function in cells. In addition to macrophage cells, we will conduct key experiments within the highly protrusive Neuro-2a neuroblastoma (Rosner et al, 1995). These cells have large and dynamic lamellipodia and are amenable to transfection and microinjection.
A biophotonics approach to understand the molecularregulation of cell protrusion: We will employ a biophotonics approach to correlate changes in the interactions between Ca2+, PIP2 and CapG with events at the barbed end of the actin filament. These studies, which involve collaborations with Prof. Peter So and Yuling Yan, will involve optimizing imaging techniques including Foerster resonance energy transfer (FRET), fluorescence recovery after photobleaching (FRAP), photoactivation of fluorescence (PAF) and speckle (Yan & Marriott, 2003; Lanni & Ware, 1984; Theriot & Mitchison, 1991; Waterman-Storer & Salmon, 1997) for time-correlated optical perturbations of caged compounds and proteins (Adams et al, 1997; Marriott et al, 2003) and our new optical switches.
Rapid and reversible, optical switching of Ca2+: Ca2+-transients can be artificially generated within cells using light-directed activation of caged Ca2+ chelators (Adams et al, 1997; Ellis-Davies, 2003). However, the relatively slow and irreversible photochemistry of the 2-nitrophenyl caging groups and their toxic photoproducts limit the usefulness of this approach. The ideal Ca2+ perturbation probe would be one whose affinity for Ca2+ changes rapidly and reversibly via optical transitions between two switch states. Transitions between these two states and the accompanying change in their Ca2+ binding affinity would more closely mimic the nature of Ca2+ transients that exist in cells. We are designing optical switches based on the spirobenzopyran photochrome that incorporate this desired property (
Rapid and reversible modulation of protein interactions using optical switches: Spirobenzopyran conjugates provide an opportunity to optically control the interactions and activities of specific proteins in complex environments (Willner et al, 1994)—our interest in these probes center on developing optical switches to control interactions between Ca2+ and barbed end capping proteins during cell protrusion. We have found that the action spectrum of these switches can be tuned over a wide wavelength range using spirobenzopyran and spironaphthoxazine based reagents. Since the MC states of spirobenzopyran and spironaphthoxazine protein conjugates can be selectively controlled using different excitation wavelengths, this feature will allow control of multiple optical switch conjugates and their interactions and activities within a cell. We propose to develop a simple approach to prepare optical switch conjugates focusing our initial studies on the simple Ca2+ binding protein parvalbumin.
Specifically we propose to position the MC state of a thiol reactive spirobenzopyrans to a specific site on a engineered parvalbumin where it engages in MC—, but not SP, dependent interactions that block Ca2+ binding to the optical switch conjugate (schematized in
New probes to image molecular events at the barbed-end of actin filaments during cell protrusion: The studies outlined in this proposal require the development of fluorescent probes that selectively report on changes in the distribution of free barbed ends during cell protrusion. Ideally, these probes would act as indicators of uncapping events at the barbed end. We have previously shown that actin-targeted, macrolide drugs typified by KabC (
Design, synthesize and characterize optical switches for Ca2+
The ideal probe to modulate Ca2+ would incorporate both Ca2+ sequestering and release activities that are controlled through rapid and reversible, light-driven reactions without the release of secondary products. As part of a new program to develop such probes, we introduce a family of tetracarboxylic chelating reagents based on the spirobenzopyran photochrome that undergo rapid and reversible, light-directed transitions between two stable isomeric states, a colorless spiro-(SP) state and a colorful merocyanine (MC) state (Inouye, 1994)—these probes are designed to exhibit different affinities for Ca2+ in the SP and MC states. We suppose that this property can be introduced by positioning two pairs of N-linked carboxyl groups on the optical switch with a geometry that favors Ca2+ binding in either the SP or the MC state. In the case of the compound X, the ester precursor of the tetracarboxylic chelator, we reasoned that the close proximity of the two pairs of carboxyl groups in the planar MC state would favor Ca2+ binding over the SP state—we reasoned that the opposite would be true for compound VIII as indicated in
Reagents: (a) PPh3, CBr4, CH2Cl2; (b) diethyl iminodiacetate, K2CO3, THF, reflux; (c) methyl iodide, CHCl3, reflux; (d) 0.5 N NaOH; (e) methyl acrylate, MeOH, 40° C.; (f) 2,3,3-trimethyl-3H-indole, CH3CN, reflux; (g) diethyl iminodiacetate, Et3N, THF, reflux; (h) III, EtOH; (i) dimethyl iminodiacetate hydrochloride, Et3N, THF, reflux; (j) VI, EtOH.
Analytical Data:
6-(bromomethyl)-2,3,3-trimethyl-3H-indole (I): Yield: 17%; MS(EI): 253(7), 251(M+,7), 172(M+—Br,62), 83(100); HRMS(EI): M+251.0320 (calc. 251.0310); 1H NMR (CDCl3) δ1.31 (s, 6H), 2.30(s, 3H), 4.57(s, 2H), 7.25-7.28(m, 2H), 7.56(s, 1H).
6-[N′,N′-bis(ethyloxycarbonylmethyl)aminomethyl]-2,3,3-trimethyl-3H-indole (II): Yield: 54%; MS(EI): 360(M+,5), 287(56), 273(15), 172(100); HRMS(EI): M+360.2046 (calc.360.2049); 1H NMR(CDCl3) δ1.28(t, J=7.2 Hz, 6H), 1.31(s, 6H), 2.30(s, 3H), 3.58(s, 4H), 3.97(s, 2H), 4.18(q, J=7.2 Hz, 4H), 7.24(d, J=7.5 Hz, 1H), 7.30(d, J=7.5 Hz, 1H), 7.51(s, 1H).
6-[N′,N′-bis(ethyloxycarbonylmethyl)aminomethyl]-1,3,3-trimethyl-exo-methyleneindoline (III):Unstable, used for the next reaction without purification and characterization.
2-[N,N-bis(methyloxycarbonylethyl)amino]ethanol (IV): Used for the next reaction without purification. 1H NMR (CDCl3) δ2.48(t, J=6.5 Hz, 4H), 2.60(t, J=6.5 Hz, 4H), 2.80(t, J=7.7 Hz, 2H), 3.60(t, J=7.7 Hz, 2H), 3.69(s, 6H).
N,N-bis(methyloxycarbonylethyl)-2-bromoethylamine (V): Yield: 91%; MS(EI): 297(7), 295(M+,7), 224(88), 222(89), 202(100); HRMS(EI): M+295.0408 (calc. 295.0419); 1H NMR(CDCl3) δ2.47(t, J=6.5 Hz, 4H), 2.84(t, J=6.5 Hz, 4H), 2.85(t, J=7.7 Hz, 2H), 3.35(t, J=7.7 Hz, 2H), 3.69(s, 6H).
N-[N′,N′-bis(methyloxycarbonylethyl)aminoethyl]-3,3-dimethyl-exo-methyleneindoline (VI): Unstable, used for the next reaction without purification and characterization.
3-[N,N-bis(ethoxycarbonylmethyl)aminomethyl]-5-nitrosalicylaldehyde (VII): Used for the next reaction without purification. 1H NMR (CDCl3) δ1.28(t, J=7.0 Hz, 6H), 3.57(s, 4H), 4.05(s, 2H), 4.20(q, J=7.0 Hz, 4H), 8.27(d, J=2.9 Hz, 1H), 8.55(d, J=2.9 Hz, 1H), 10.28 (s, 1H).
Spirobenzopyran 8,6′-tetraester (VIII): Yield: 22% based on II; HRMS(ESI): [M+Na]+747.3202 (calc.747.3217); 1H NMR (CDCl3) δ1.18 (s, 3H), 1.19 (t, J=7.1 Hz, 6H), 1.26 (s, 3H), 1.28 (t, J=7.1 Hz, 6H), 2.70 (s, 3H), 3.26 (d, J=7.6 Hz, 2H), 3.33 (d, J=7.6 Hz, 2H), 3.57 (s, 4H), 3.63 (s, 2H), 3.89 (s, 2H), 4.06 (q, J=7.1 Hz, 4H), 4.18 (q, J=7.1 Hz, 4H), 5.85 (d, J=10.3 Hz, 1H), 6.63 (s, 1H), 6.81 (d, J=7.5 Hz, 1H), 6.91 (d, J=10.3 Hz, 1H), 6.98 (d, J=7.5 Hz, 1H), 7.93 (d, J=2.5 Hz, 1H), 8.18 (d, J=2.5, 1H).
3-[N,N-bis(methoxycarbonylmethyl)aminomethyl]-5-nitrosalicylaldehyde (IX): Used for the next reaction without purification. 1H NMR (CDCl3) δ3.62(s, 4H), 3.78(s, 6H), 4.12(s,2H), 8.35(d, J=2.7 Hz, 1H), 8.61(d, J=2.7 Hz, 1H), 10.36 (s, 1H).
Spirobenzopyran 8,1′-tetraester (X): Yield: 10% based on V; HRMS(ESI): [M+H]+697.3057 (calc.697.3085);
1H NMR (CDCl3) δ1.71(s, 3H), 1.26(s, 3H), 2.37(t, J=7.2 Hz, 4H), 2.50-2.65(m, 2H), 2.74(t, J=7.2 Hz, 4H), 3.14-3.25(m, 2H), 3.31(s, 4H), 3.61(s, 6H), 3.62(s, 6H), 3.61-3.65(m, 2H), 5.88(d, J=10.6 Hz, 1H), 6.57(d, J=7.6 Hz, 1H), 6.85(dd, J=7.6, 7.6 Hz, 1H), 6.93(d, J=10.6 Hz, 1H), 7.07(d, J=7.6 Hz, 1H), 7.16(dd, J=7.6, 7.6 Hz, 1H), 7.94(d, J=2.7Hz, 1H), 8.16(d, J=2.7Hz, 1H).
De-esterification: At the time of submission we have not established conditions for the quantitative removal of all four methyl or ethyl ester groups in compounds VIII and X. This will be necessary to determine the Ca2+-bimding constants for the SP and MC states. We have established however, that the spirobenzopyran group is sensitive to the basic condition that is usually employed for ester hydrolysis. We are now employing acid hydrolysis as an alternative since the SP and MC states are stable in acid medium (Raymo et al, 2004). Even more recently, we have achieved the synthesis of the t-butyl ester compound closely related to compound X, which should yield the de-esterified form in the presence of trifluoroacetic acid.
Cell loading: The carboxylic esters of the putative Ca2+ chelators facilitate the entry of the optical switch into cells. In the case of the ethyl ester (VIII), the fact that the probe remains within the cell after washing with medium suggests that one or more of the ester groups are removed by intracellular esterases. Loaded cells do not exhibit a red fluorescence until they are irradiated with a short pulse of 365 nm light (
Local Increases in Ca2+ are Coupled to the Activation of Actin Filament Polymerization and the Global Response of Cell Protrusion
Design, Synthesis and Application of Fluorescent Barbed-End Probes:
7-(4-aminomethyl)-1H-1,2,3-triazol-1-yl analogue of kabiramide C (AMT-KabC) KabC was converted into 7-azido KabC via Mitsunobu reaction (Ko, 2002) by using hydrazoic acid as nucleophile in the presence of PPh3 and DIAD under nitrogen atmosphere. Then 7-azido KabC was reacted with 3-(fluoren-9-yl-methoxycarbonyl)aminopropyne in the presence of catalytic amount of copper (I) iodide and Et3N (Horne et al, 2003, Tornoe et al, 2002, and Rostovsev et al, 2002) to afford amino protected KabC. This compound was confirmed by the presence of aromatic proton signals of Fmoc in the 1H NMR spectrum. Deprotection of Fmoc with 20% piperidine in dry CH2Cl2 gave AMT-KabC (
Fluorescent KabC: New probes for the barbed end of actin filaments The KabC derivatives of tetramethylrhodamine (TMR), fluorescein diester (FDE) and IC5 (
FDE-KabC readily permeates the plasma membrane of living cells and fluoresces only after one or both acetate groups are hydrolyzed by intracellular esterases—a robust fluorescein fluorescence is visible within a few minutes of cell loading. The TMR—KabC traverses the plasma membrane and is retained floowoing washing with fresh medium. Confocal imaging of the FDE-KabC (
Preparation and characterization of caged CapG: Caged CapG was prepared using a modification of our published method for caging actin and profilin (Marriott, 1994; Marriott et al, 2003). In brief a purified solution of CapG (1 mg/ml) was dialyzed against 20 mM borate buffer, pH 8.5 overnight. The protein was centrifuged and the OD280 nm measured to determine the protein concentration (
Light Directed Activation of Constitutively Active Caged Cofilin
Caged cofilin (constitutively active S3A) was prepared independently of Ghosh et al (2004) using our standard NVOC—Cl approach (Marriott, 1994) that is described above for caged CapG. Constitutively active cofilin increases the rate of F-actin depolymerization as seen in the fluorescence emission ratio (465/502 nm) of Prodan actin following dilution below the critical concentration (
Methodology:
Relationships between Ca2+-mediated capping activities, actin filament dynamics and cell protrusion are studied by locally triggering: (a), cell Ca2+ using caged and optical switch Ca2+ probes (Adams et al, 1997; Ellis-Davies, 2003); (b), caged and optical switch conjugates of CapG; (c), Caged Rac1; (d), caged cofilin; (e), caged PIP2. Fluorescent tags attached to KabC, CapG and actin in CapG/Geloslin double null macrophage cells, NIH 3T3 and the highly protrusive, Neuro-2a cell line, will be used to map events at the barbed-end of the filament and the rate of actin polymerization in response to a rise in cell Ca2+ and PIP2. These studies also integrate phase contrast images that collectively allows correlation of local changes in cell-Ca2+ are coupled to the regulation of actin filament polymerization and integrated to achieve the global response of cell protrusion.
Methodologies for Designing, Synthesis and Characterization of Optical Switches for Reversibly Modulating Ca2+
Preliminary results described the synthesis of potential Ca2+-optical switches—these probes were designed to place two different N-linked pairs of carboxylic esters at different sites the same spirobenzopyran scaffold. We envision using these optical switches to rapidly and reversibly modulate Ca2+ in cells according to
These measurements are made using a competition binding assay with fluorescent divalent metal ion indicators (Fluo-3, X-Rhod-1 and X-Rhod-2; Molecular Probes). Recently established nitrospirobenzopyran probe has been listed as sensitive to the basic conditions required to remove saponification of the esters. Other reagents may also be used to hydrolyze these esters. These studies may be limited to using acid hydrolysis (the probe is stable in acid medium; Raymo et al, 2004) and, since we know at least some esters are cleaved in vivo by intracellular esterases (
A family of optically switches for Ca2+ that exhibit the following properties may be generated, wherein:
Optimization of the Ca2+-binding constants for the SP and MC states, discrimination between Ca2+ and Mg2+ and rate constants for optical switching can be realized by varying the coordination geometry of the two pairs of carboxylic acids. For compound Vil these groups are close in the SP state and far apart in the MC state and we envision that the SP state would bind more tightly to Ca2+ than the MC state. On the other hand the very short distance between the two pairs of carboxylic acids in the SP of compound X, and the more optimal distance observed in the MC state, coupled with the employment of longer and more flexible linker groups is designed to improve the Ca2+ affinity of MC over SP. The MC state of compound X positions the pairs of carboxyl groups at the same molecular distance on the planar aromatic ring as those in Fluo-3.
The combinatorial approach to the synthesis of these optical switches allows is to mix and match different functional groups within libraries of indolines and salicylaldehydes derivatives. Thus by selecting appropriately di-carboxyl indoline and salicylaldehyde reagents, we can rapidly and systematically control the location and geometry of carboxyl groups on the spirobenzopyran as well as the length and flexibility of the linker between the nitrogen atom and the pair of carboxyl groups (indicated as* in
The absorption properties of the MC state of the Ca2+ switch can be tuned using different 1-nitroso-2-naphthol. For example the MC state of spironaphthoxazine (Compound 17) exhibits an absorption maximum of about 620 nm. Furthermore, the probe undergoes a far more rapid thermal reversion to the SP state (within seconds) compared to the nitrospirobenzopyran group (time constant of 370 seconds). We will investigate the photophysics of optimized Ca2+ switches based on spironaphthoxazine. We anticipate, based on our observations of the thermally-induced MC to SP transition that the rate of photochemistry for the spironaphthoxazine will be faster that the 11 μs reported for the spirobenzopyran group (Gorner, 2001).
Design and development of a multi-photon, pulse-probe imaging microscope for optical switches:
Concentration defined perturbations of Ca2+, PIP2 and CapG will be generated by irradiating these loaded cells at defined sites with rapid pulses of 355 nm light. In addition a separately controlled port will be incorporated to deliver pulses of 532 nm light delivered by a 50 mW, frequency-doubled cw-Nd—YAG laser (Laser 2000). Separate timing controls for each laser is used to alternate the 355 nm and 532 nm pulses for optical switching of Ca2+ and CapG light for switches probes and conjugates for Ca2+, PIP2 and CapG. These pulse-probe imaging techniques may be used for multi-photon excitation. Optical switches provide new opportunities and potential improvements over the caged approach that include reversible optical control of the levels, interactions and activities of Ca2+ or protein, faster perturbation kinetics, optical readout of one state of the switch, ability to control multiple switches within a single cells using the nitro and non-nitro forms of the optical switch. In addition to the studies described here, Ca2+ optical switches will also be used within the optical switch microscope to study the effects of generating spatially and temporally-defined Ca2+ transients on barbed end capping reactions in studies detailed above.
Design, Synthesis and Characterization of Thiol Reactive, Optical Switches and their Conjugates
Optical switching to modulate the interactions and activities of cytoskeleton proteins: Having demonstrated the principle and practice of optically switching specific dipolar interactions between MC and G-actin, this property may be used for optical switching of cytoskeleton-associated Ca2+-binding proteins as illustrated in
The difference in average energy of the MC absorption in the five G-actin conjugates (1,868 cm−1) greatly exceeds that measured for the SP state in the same conjugates (504 cm−1). This energy difference for the MC-G-actin interaction is comparable to that found for the interaction of G-actin with ligands and actin binding proteins (Pollard, 2003). Accordingly, the strong interaction between the MC group and specific polar groups within a protein conjugate is used to compete with the interactions underlying the binding of the MC-conjugate with a functional ligand or protein. Proteins may be engineered such that a functional interaction is perturbed for MC but not SP—therefore, optical switching between the SP and MC states would serve to reversibly modulate interactions of the spirobenzopyran conjugate.
A family of spirobenzopyran reagents may be used to project the MC dipole moment to different sites from a common attachment site where they engage in specific dipolar interactions with the protein. The origin of the remarkable differences in the dipolar interactions between these MC probes and the protein is illustrated in a qualitative study of the relative orientations of the MC and SP probes within a hypothetical protein (
Optical perturbation of CapG function: CapG is known to bind Ca2+ in a similar manner to gelsolin—the Ca2+ ion is actually located at the interface of actin with gelsolin (McLoughlin et al, 1993). Existing cysteine residues in CapG will be replaced with Serine. 3-4 different residues in the vicinity of the Ca2+ binding site in CapG to cysteine may be mutated and tested to study whether the Ca2+- and barbed end binding activities of these mutants, or not, before proceeding to the labeling with spirobenzopyran. CapG spirobenzopyran conjugates that exhibit Ca2+ and barbed end binding properties in the SP but not the MC state will be identified. In a second approach to preparing a CapG optical switch, we will generate CapG mutants harboring single cysteine residues in the long α-helix of domain 1. This helix is known to interact with actin in the cleft that forms between subdomains 1 and 3. The spirobenzopyran conjugates of these CapG mutants will be tested for their ability to bind to actin in the SP but not the MC state. CapG conjugates whose functional interactions can be optically switched will be used to rapidly and reversibly perturb the barbed end capping activity of the conjugate in vitro and within CapG-null and gelsolin-null macrophage cells (Witke et al, 1995; 2001). The barbed end capping activity of the CapG optical switch will be quantified by imaging the distribution of TMR—KabC or FDE-KabC as described earlier.
Optical Switching of MC Fluorescence in Spirobenzopyran Protein Conjugates
The MC fluorescence of spirobenzopyran conjugates can be rapidly and reversibly modulated using alternate cycles of 365 nm and 546 nm light (
The G-actin conjugate of compound 9 (
Parallel FRAP-PAF Microscopy Using Fluorescence Optical Switches
The optical switching of MC fluorescence in spirobenzopyran protein conjugates may be used in dynamic optical imaging techniques that combine measurements of FRAP and PAF on the same actin photochromic probe in the cell same. Conducting independent measurements of the diffusion of an identical spirobenzopyran conjugate will overcome several limitations of the PAF and FRAP techniques including the issue of toxicity caused by the release of toxic photoproducts, triplet oxygen in the case of FRAP (Stavreva & McNally, 2004) and 2-nitrosobenzophenone in the case of caged fluorophores (Theriot & Mitchison, 1991). The ability to optically switch a spirobenzopyran conjugate or labeled ligand between the fluorescing MC and non-fluorescing SP state via high quantum yield photo-isomerization reactions, without the generation of toxic photoproducts, provides an internal control that is simply not possible using PAF or FRAP alone. The spirobenzopyran conjugates of G-actin will be injected into Neuro-2a, macrophage and NIH 3T3 cells to show the properties and performance of a FRAP/PAF technique and measure the diffusion rate for F-actin retrograde flow during protrusion. These measurements will be made in cells subject to optical perturbations of Ca2+, PIP2, CapG perturbations will be compared to other studies (Lanni & Ware. 1984; Wang, 1985; Theriot & Mitchison, 1991). The application of multiphoton microscopy to achieve more rapid, high energy pulses of 535 nm and 355 nm light will be used to improve the temporal resolution of optical switching.
Speckle microscopic imaging of fluorescence optical switches: The fluorescence emission from a pixel element containing one or a few TMR-actin or spirobenzopyran-actin molecules will be imaged using speckle microscopy according to Waterman-Storer and Salmon et al (1997). An important factor for successful applications of the speckle microscopy technique is a requirement to show that the fluorescence emission emanating from a region of interest originates from the few to single fluorescent protein conjugates rather than an endogenous or spurious signal. A simple solution to address this issue that exploits the ability to control the fluorescence of MC in a protein conjugate using irradiation of the image field with 365 nm and 546 nm light (
Image processing: Image analysis and quantitative fluorescence microscopy (FRET and FP; Marriott et al, 1994; Yan & Marriott, 2003b) may be used for the analysis of time-series image data obtained after specific perturbations of cell Ca2+, PIP2 and cytoskeleton proteins to calculate kinetic rate constants for the ensuing reactions on a pixel-by pixel basis—this allows the process or reaction to be represented and analyzed in terms of an absolute physical parameter i.e. where each pixel represents an independently determined rate constant for the reaction (Marriott et al, 1994; Yan & Marriott, 2003).
Establish the Molecular Mechanism Underlying the Regulation of Cell Protrusion
The biophotonics technologies described in above of this invention will be used to test whether the increased levels of cell Ca2+ and PIP2 arising from the activation of membrane receptors regulate molecular interactions at the barbed end of the filament, actin filament dynamics and ultimately protrusion. The studies, summarized pictorially in
The dynamic, quantitative imaging studies using novel optical probes are designed to generate a spatial and temporal resolved analysis of the distributions, interactions and activities of specific signaling molecules and proteins during protrusion. Furthermore by incorporating light-directed perturbation techniques (caged and optical switches) into these imaging studies we can locally control the levels of signaling molecules and show how, or if, these signals are integrated in the lamellipodium and coupled to the global response of cell protrusion. These investigations may be performed using model macrophage cells that will cover molecular events beginning with IgG activation of membrane receptors (Serrander et al 2000) through subsequent interactions and activities of Rac1, Ca2+ and PIP2, CapG, actin filament dynamics and culminating in cell protrusion.
Actin polymerization is likely to be regulated by time dependent variations in Ca2+ and PIP2 levels within the lamellipodium that either promote CapG barbed end capping or dissociate the CapG complex respectively. Thus according to the model (
These studies may be performed within CapG-null and CapG-Gelsolin double null macrophage cells from the Witke lab (Witke et al, 2001) and Neuro-2a neuroblastoma (ATTC). Many of the transients invoked in our model will be generated optically through light directed activation of caged precursors and/or optical switches of Ca2+, PIP2, cofilin and CapG previously loaded into cells. Our microscope workstation is set up for simultaneous uncaging and fluorescence imaging (Marriott & Heidecker, 1996) and will be used to generate rapid and localized perturbations within Neuro-2a and macrophage cells. Analysis of accompanying molecular events at the barbed end will be quantified by time-resolved FRET imaging of appropriately labeled probes (see below; actin, TMR-KabC and CapG; Heidecker et al, 1995) using speckle and/or 2-photon confocal microscopies with Peter So.
Fluorescent Probes to Quantify Binding at the Barbed-End of the Actin Filament:
Fluorescent KabC probes provide unrivalled contrast for imaging molecular events at the barbed end as shown by their actin binding specificity in Tanaka et al (2003) and Klenchin et al (2003). Quantitative fluorescence imaging of the membrane permeable fluorescein-KabC, shown in
The Role of CapG in Regulating Actin Filament Dynamics During Cell Protrusion.
The approach involves imaging the distributions and interactions between CapG and the barbed end using microinjected or genetically encoded fluorescent conjugates of CapG, actin and KabC. Specifically, we will use: (1), GFP-fusions of actin (Choidas et al, 1998) and CapG; (2), functional fluorescent CapG conjugates (fluorescein, TMR and IC5-CapG microinjected into macrophage cells; (3), FDE- and TMR-labeled KabC (preliminary achievements). Capping activity in macrophage cells will be mapped using either the fluorescence emission from: (a), FDE- or TMR—KabC; (b), microinjected TMR—CapG; (c), GFP—CapG. Changes in the distribution of actin filaments will be imaged using either GFP-actin in the case of (a) and (c) or microinjected IC3-actin in the case of (b). This combination of probes will also serve to image interactions between the barbed end and CapG using sensitized FRET emission between pairs of appropriately labeled proteins. Simultaneous acquisition of the phase contrast image will allow us to correlate molecular events associated with the uncapping of actin filaments to the explosive polymerization of F-actin and the associated protrusion of the lamellipodium. The role of CapG in regulating actin polymerization in cell protrusion will be further advanced in studies using light-directed, in vivo perturbation of Ca2+ , PIP2 and CapG from their caged precursors, or optical switches in the case of Ca2+ and CapG (described above), that are loaded into cells by microinjection or as membrane permeable probes. These goals will be met through the following studies:
The criteria for using GFP-actin as a probe of actin structure and dynamics (Choidas et al, 1998) have been discussed. The purified CapG and actin conjugates will also be tested for their binding activity using in vitro assays based on FRET between the donor and acceptor conjugates and the effect of the CapG conjugate on the fluorescence of Prodan-actin (
Role of Cell Ca2+ in Regulating Actin Filament Dynamics During Cell Protrusion
Localized, light directed generation of concentration jumps of Ca2+ from commercially available caged NP-EGTA and DM-Nitrophen and optical switching of Ca2+ using the probes developed as shown above and from the optical switch conjugate of parvalbumin may be used to mimic the effect of receptor-mediated rises in cell Ca2+ within macrophage cells undergoing cell protrusion. This approach will allow us to test whether an increase in level of Ca2+ at the plasma membrane at the barbed end by CapG (
Ca2+-independent generation of free barbed ends: Caged cofilin conjugates may be used together with the specific fluorescent KabC probes to image and quantify free barbed ends that result from the activation of cofilin (
Specifically these experiments involve: Mapping changes in the distributions of IC3-CapG and GFP-actin (Choidas et al, 1998) during light-directed activation of caged cofilin. Image based measurements of FRET between (a) GFP-actin and IC3-CapG and (b), IC3-actin and FDE-KabC or TMR—KabC or IC5-KabC will serve to map the distributions of actin filaments and free barbed-ends before and after activation of cofilin. These experiments will be performed in wild type and gelsolin-null macrophage cells in at low and elevated levels of Ca2+ and PIP2 by uncaging DM-Nitrophen or NP-EGTA and caged PIP2, or else using spirobenzopyran optical switches for chelating Ca2+ and parvalbumin (Aims 1 and 2) will be quantified using Fluo-3 and X—Rhod-1 probes loaded into cells by microinjection or as AM-esters.
Establish the Role of PIP2 in Uncapping Barbed Ends During Cell Protrusion
Light directed concentration jumps of PIP2 from caged PIP2 may be used to mimic the effect of receptor-mediated signaling pathways in macrophage cells. This approach studies whether an increase in PIP2 close to the plasma membrane of the leading edge dissociates CapG from the barbed end of the actin filament triggering polymerization and membrane protrusion (
Caged PIP2: Surprisingly caged PIP2 has not been described in the literature yet this would be a most interesting and useful probe to study PIP2 regulation of essential processes including motility and synaptic signaling. For this, a caged PIP2 derivative will be synthesized through the reaction of 1-(2-nitrophenyl)diazoethane phosphate with PIP2 (Calbiochem) in chloroform/water mixture as described and routinely practiced (Walker et al, 1989).
Caged Rac1: Light directed activation of caged Rac1 will be used to generate localized jumps in PIP2 mediated and to correlate this event with barbed-end uncapping. Constitutively active Racl (G12V) and dominant negative mutants of human Racl have been prepared in >30 mg quantities as described in Faix et al (2001). Caged Rac1 will be prepared using three different methods. (a), Cysteine-189 of Rac1 will be modified using our thiol reactive caging group (Marriott & Heidecker, 1996)—membrane anchoring through this cysteine residue is absolutely required for Rac1 function. Cysteine 189 and a limited number (3-4) other cysteine residues will be labeled with bromomethyl-3,4-dimethoxynitrobenzene (Marriott & Heidecker, 1996). The activity of the constitutively active Rac1 (control) and caged Rac1 conjugate will be measured using our in vitro DGAP1 binding assay (Faix et al, 2001) or by membrane ruffling activity when microinjected into serum deprived cells (Ridley, 1995; Ridley & Hall, 1992) before and after irradiation with a pulse of uv light (Roy et al, 2001); (b), Constitutively active Rac1 will be modified at one or two lysine residues with the photo-cleavable reagent BNBA-NHS (Marriott et al, 1992). The Rac1 conjugate will then be crosslinked to a TMR-labeled, thiolated dextran (Otti et al, 1998) in order to physically block binding sites on the Rac1 molecule. The activity of the unmodified, caged and uncaged Rac1-dextran complex will be determined as described above. Uncaging experiments in cells will be subject to the same controls described in Roy et al (2001). PIP2 may also be quantified using a fluorescent peptide indicator of PIP2 described by Tuominen et al (1999).
The Signaling Pathway Leading to Actin Mediated Cell Protrusion
The signaling pathway leading to cell protrusion by correlating changes may be dissected in the spatial and temporal distributions of these molecules and ions and their interactions to the generation of free barbed ends, the polymerization of actin filaments and cell protrusion as outlined in
The sequence of events in receptor mediated signaling of cell motility is likely to be in the following order: 1. IgG Receptor activation; 2. Rac1 activation; 3. Activation of cofilin; 4. Increase in Ca2+; 5. Increase in PIP2; 6. Uncapping actin filament barbed-ends; 7. Polymerization of actin filaments in the lamellipodium; 8. Cell protrusion. Furthermore, these events may be confined to the plasma membrane.
Specific Methods
Instrumentation. 1H NMR spectra were measured on a Brucker Ac 300 MHz; mass spectra were carried out on a Micromass AutoSpec for El, a Micromass LCT for ESI, or a Bruker REFLEX II for MALDI. Absorption spectra were recorded on a Hewlett-Packard 82152 diode array spectrophotometer or a Shimadzu 1601PC instrument. Fluorescence spectroscopy was performed on an SLM-AB2 instrument (Thermoelectron, Madison, Wis.). Light-directed optical switching is achieved by irradiating the sample (120-1000 μL) with the 365 nm or 546 nm lines of a 100 W Hg-arc lamp (Zeiss).
Live cell microscopy: The multi-model microscope workstation for imaging transmission and fluorescence images of living cells (Choidas et al, 1998) incorporates a 100 W Hg-arc lamp allowing for simultaneous fluorescence and flash photolysis of caged compounds. A double-view dichroic mirror assembly is used that separately projects the GFP and TMR images onto a single camera (Heidecker et al, 1995). This technique is particularly useful for recording molecular interactions between GFP-actin and IC3-protein conjugates by real-time imaging of GFP-fluorescence and TMR-sensitized emission. Cells are maintained at 37° in a perfusion chamber (Choidas et al, 1998).
Cloning. All molecular biology methods used in this proposal are routinely used in the PI's laboratory (see papers by Prassler et al, 1998; Stocker et al, 1999; Westphal et al, 1997; Faix et al, 2001). Cloning and gene expression: Genes encoding CapG, Gelsolin, Rac1 and cofilin are cloned from a mouse brain cDNA library (Stratagene) and the clone for mouse β-parvalbumin obtained from ATCC. The genes are cloned into expression vectors and expressed following induction with IPTG. Gelsolin, cofilin and CapG genes are amplified by PCR using a mouse brain cDNA library (Invitrogen) as a template and gene specific primers and cloned in the HindIII, BamHI site of the pQE30 vector that has an N-terminal His-tag. The M15 bacterial strain is used to express the genes.
Protein purification: A 1.6 L culture is induced with 1 mM IPTG at 30° or 37° for 5 to 6 h. The soluble proteins are purified using Ni—NTA (Qiagen manual). For example the gene encoding CapG is cloned from a mouse cDNA library (Invitrogen) and expressed and purified as a soluble His-tagged protein in E. coli using an NTA-sepharose column. About 50 mg of pure CapG is purified from a 600 ml culture. Rabbit muscle G-actin is purified according to Marriott (1994). The concentration of G-actin was determined by absorption using an extinction coefficient of 3400 M−1 cm−1 at 290 nm14. The purity and activity of actin is determined by SDS-PAGE and polymerization assays.
Antibodies: New polyclonal antibodies have been developed against Gelsolin, cofilin, actin, Rac1 and CapG that work well in Western blots. A new polyclonal antibody has also been developed against the NVOC group and should prove to be useful to quantify uncaging reactions.
Protein labeling: Actin, CapG, Gelsolin and cofilin are labeled with the thiol and amino reactive donor dyes: Acrylodan, 5′-TMR-maleimide (Molecular Probes) or IC3-maleimide, IC5-maleimide and IC3-NHS (Dojindo) and thiol reactive spirobenzopyrans using standard protocols in the inventors' laboratory (Marriott et al, 1988). All fluorescent conjugates are analyzed for labeling ratio (<1:1) and binding to F-actin. The activity of the conjugates is assessed using Prodan-actin assays. The extinction coefficient for SP is taken as 35,000 M−1cm−1 at 350 nm and 52,000 M−1cm−1 at 530 nm for MC.
Cells: The molecular basis of actin-based protrusion will be studied using several model cell lines including macrophage cells. These will be isolated from wild type mice, CapG-null mice, Gelsolin-null mice and CapG/Gelsolin double null mice may be used. Macrophage cells exhibit a dramatic IgG-mediated ruffling that is suppressed in CapG-null mice. This activity is restored after microinjecting CapG. Neuro-2a cells (Rosner et al, 1995) are obtained from ATTC.
Furthermore, the compounds and a method of using the photochromic probes of the present invention may have other applications aside from use calcium ion chelating probes. Additionally, it would be apparent to one of ordinary skill in the art to alter the methods and compositions which have been described herein in the preferred embodiment. Such alterations include altering the starting compound and making substitutions, without departing from the spirit of the invention, or altering the positional chemistry, stereochemistry and conformations of the compounds. Further alterations include creating salts of these compounds by techniques and methods known to one of ordinary skill in the art. Thus, although the invention has been herein shown and described in what is perceived to be the most practical and preferred embodiments, it is to be understood that the invention is not intended to be limited to the specific embodiments set forth above. Rather, it is recognized that modifications may be made by one of skill in the art of the invention without departing from the spirit or intent of the invention and, therefore, the invention is to be taken as including all reasonable equivalents to the subject matter of the appended claims.
The present application seeks priority from U.S. Provisional Patent Application No. 60/522,904, which is herein incorporated by reference for all purposes.
This invention was made with United States government support awarded by the following agency: NIH HL069970. The United States government has certain rights in this invention.
| Number | Date | Country | |
|---|---|---|---|
| 60522904 | Nov 2004 | US |
