| Title: |
NOVEL FLUORINATED RHODAMINES AS PHOTOSTABLE FLUORESCENT DYES FOR LABELLING AND IMAGING TECHNIQUES |
| Document Number: |
20120135459 |
| Publication Date: |
May 31, 2012 |
| Appl. No: |
13/380148 |
| Application Filed: |
June 26, 2009 |
| Abstract: |
The present invention relates to novel fluorinated 3,6-diaminoxanthene compounds derived from the basic structural formula (I) and to their uses as photostable fluorescent dyes, e.g. for immunostainings and spectroscopic and microscopic applications, in particular in conventional microscopy, stimulated emission depletion (STED) reversible saturable optically linear fluorescent transitions (RESOLFT) microscopy, and fluorescence correlation spectroscopy. The claimed compounds are also useful as molecular probes in various spectroscopic applications. |
| Inventors: |
Hell, Stefan W. (Goettingen, DE); Belov, Vladimir N. (Goettingen, DE); Mitronova, Gyuzel (Goettingen, DE); Bossi, Mariano (Buenos Aires, AR); Moneron, Gael (Goettingen, DE); Wurm, Christian A. (Goettingen, DE); Jakobs, Stefan (Goettingen, DE); Eggeling, Christian (Goettingen, DE); Bierwagen, Jakob (Goettingen, DE); Meyer, Lars (Goettingen, DE) |
| Assignees: |
Max-Planck-Gesellschaft zur Foerderung der Wissenschaften e.V. (Muenchen, DE) |
| Claim: |
1. A fluorinated 3,6-diaminoxanthene compound derived from the basic structural formula I which exists in the open form I-OF or in the closed form I-CF [chemical expression included] and which compound has one of the following structural formulae Ia-Ij or of the corresponding closed forms thereof: [chemical expression included] wherein Y1=Y2=F; [chemical expression included] wherein R11=R12=CH2CF3, X1=X2=SO3H, and R21=R22=H; [chemical expression included] wherein R11=R12=CH2CF3, Y1=Y2=F, and X1=X2=H; [chemical expression included] wherein X1=X2=SO3H, Y1=Y2=F, and R11=R12=R=R22=H; [chemical expression included] wherein R11=R12=CH2CF3, X1=X2=SO3H, Y1=Y2=F, and R21=R22=H; [chemical expression included] wherein Y1=F; [chemical expression included] wherein R11=CH2CF3, X1=SO3H, and R21=H; [chemical expression included] wherein R11=CH2CF3, Y1=F and X1=H; [chemical expression included] wherein X1=SO3H; Y1=F and R11=R12=H; [chemical expression included] wherein R11=CH2CF3, X1=SO3H, Y1=F, and R21=H; and wherein in each of formulae Ia-Ij X3=H, 5-COOH or 6-COOH or an ester thereof, and undefined substituents from the set R11, R12, R21, R22, X1, X2, Y1, Y2, alone or two substituents taken together, may be hydrogen atoms or any alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl groups, or any functionally substituted alkyl, cycloalkyl, aryl, heterocyclic aryl groups, or any combination of the said groups; and wherein in each of formulae Ia-Ij Z=OR3 or NR4R5, where R3, R4 and R5 is (are) hydrogen atom(s), any alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl group(s), or any functionally substituted alkyl, cycloalkyl, aryl, heterocyclic aryl group(s), or any combination of the said groups. |
| Claim: |
2. The compound according to claim 1, having the basic formula II [chemical expression included] and being selected from the group of compounds 10-22 with the following substitution patterns: 10: Y=Y2=F; R11=R12=X1=X2=X3=H 11: Y1=Y2=F; R11=R12=X1=X2=H; X3=COOH 12: Y1=Y2=F; R11=R12=X3=H; X1=X2=SO3H 13: Y1=Y2=F; R1=R12=H; X=X2=SO3H; X3=COOH 14: Y=Y2=X1=X2=X3=H; R11=R12=CH2 CF3 15: Y1=Y2=X1=X2=H; R=R12=CH2 CF3; X3=COOH 16: Y1=Y2=X3=H; X1=X2=SO3H; R11=R12=CH2 CF3 17: Y1=Y2=H; X1=X2=SO3H; R11=R12=CH2 CF3; X3=COOH 18: Y1=Y2=F; X1=X2=X3=H; R=R12=CH2CF3 19: Y1=Y2=F; X1=X2=H; R11=R12=CH2 CF3X3=COOH 20: Y1=Y2=F; X1=X2=SO3H; R11=R12=CH2 CF3; X3=H 21: Y1=Y2=F; X1=X2=SO3H; R11=R12=CH2 CF3; X3=COOH 22: Y1=Y2=X1=X2=H; R11=R12=CH2 CF3; X3=COOMe. |
| Claim: |
3. The compound according to claim 1, having the basic structural formula III [chemical expression included] and being selected from the group of compounds 23-28 with the following substitution patterns: 23: R11=R12=X3=H 24: R11=R12=H; X3=COOH 25: R11=R12=CH2CF3; X3=H 26: R11=R12=CH2CF3; X3=COOH 27: R11=R12=Me; X3=H 28: R11=R12=H; X3=COOH. |
| Claim: |
4. A method for preparing 4,5-disulfono-3,6-bis[N,N′-(2,2,2-trifluoroethyl)amino]xanthenes Ib,e or 4-sulfono-6-amino-3-[N′-(2,2,2-trifluoroethyl)amino]xanthene derivatives Ig,j by direct sulfonation of a corresponding unsulfonated N[,N′-bis](2,2,2-trifluoroethyl)-substituted 3,6-diaminoxanthene derivative at positions 4 and 5 in the course of a one-step procedure by reacting with a sulfonating agent. |
| Claim: |
5. A method for preparing rhodamine amides bearing two sulfonic acid residues in positions 4 and 5 of a xanthene fragment according to formulae Ib, d, e, g, i, j in claim 1 or formula IV below [chemical expression included] wherein in the formula IV R2≦H or SO3H, pairs of substituents R11 - - - Y1, R12 - - - Y2 independently denote any branched or unbranched substituted or unsubstituted alkyl or alkenyl chain or any combination of the said chains with a cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl group, or any functionally substituted alkyl, cycloalkyl, aryl, heterocyclic aryl group; X3=H, 5-COOH or 6-COOH or an ester thereof, and wherein R4 and R5 is (are) hydrogen atom(s), any alkyl, cycloalkyl, heterocycloalkyl, aryl, heterocyclic aryl group(s), or any functionally substituted alkyl, cycloalkyl, aryl, heterocyclic aryl group(s), or any combination of the said groups, by reacting of corresponding unsulfonated rhodamine amides having hydrogen atoms in the said positions with oleum in the course of one-step procedure under temperature conditions which leave an amide bond intact. |
| Claim: |
6. The method according to claim 5, wherein the reaction is conducted at a temperature in a range from −5 to +20° C. |
| Claim: |
7. A method of using compounds according to claim 1 or any of their stable conjugates with biomolecules or any other chemical substances as fluorescent dyes. |
| Claim: |
8. The method according to claim 7, wherein the compound is used in spectroscopy or microscopy. |
| Claim: |
9. A method of using hydrophilic compounds of claim 1 in a free form or attached to antibodies or other biomolecules for microinjections into cells and for immunostainings. |
| Claim: |
10. A method of using compounds according to claim 1 or any of their conjugates with biomolecules or with any other chemical substances as molecular probes. |
| Claim: |
11. The method according to claim 10 wherein absorption and/or emission spectra and/or fluorescence quantum yields or lifetimes of the molecular probes are changed in response to a change in a polarity of a medium or microenvironment. |
| Claim: |
12. The method according to claim 11, wherein the change of the absorption and/or emission spectra of the molecular probes involves a change in a positions, shape and/or relative intensity of bands of the absorption and/or emission spectra of the molecular probes. |
| Claim: |
13. The method according to claim 11, wherein the medium or microenvironment is a solvent, interior of a cell, cell organelle, protein or protein domain, (lipid) membrane or ion-channel. |
| Claim: |
14. The method according to claim 6, wherein the temperature is from 0 to +4° C. |
| Claim: |
15. The method according to claim 7, wherein the biomolecules are selected from the group consisting of peptides, proteins, lipids, carbohydrates, nucleic acids and toxins. |
| Claim: |
16. The method according to claim 8, wherein the compound is used in reversible saturable optically linear fluorescent transitions (RESOLFT) microscopy, stimulated emission depletion (STED) microscopy, fluorescence correlation spectroscopy (FCS), ground state depletion with individual molecular return (GSDIM) imaging, fluorescence recovery after photobleaching (FRAP) technique, fluorescence life-time imaging (FLIM), resonance energy transfer (RET) studies, or conventional microscopy. |
| Claim: |
17. The method according to claim 10, wherein the biomolecules are selected from the group consisting of peptides, proteins, lipids, carbohydrates, nucleic acids and toxins. |
| Claim: |
18. A conjugate of a compound according to claim 1 and a biomolecule selected from the group consisting of peptides, proteins, lipids, carbohydrates, nucleic acids and toxins. |
| Claim: |
19. The conjugate of claim 18, adapted for use as a fluorescent dye, an immunostain or a molecular probe. |
| Current U.S. Class: |
435/405 |
| Current International Class: |
01; 07; 07; 07 |
| Accession Number: |
edspap.20120135459 |
| Database: |
USPTO Patent Applications |
