Dye-conjugated dendrimers

Patent metadata

Field	Value
Publication number	US-9448173-B2
Application number	US-201213385828-A
Country	US
Kind code	B2
Filing date	Mar 8, 2012
Priority date	Mar 8, 2011
Publication date	Sep 20, 2016
Grant date	Sep 20, 2016

Abstract

The invention provides dendrimers, conjugates thereof, and methods of using dendrimer conjugates. In one embodiment, the invention provides novel polymeric dendrimers as a new class of fluorescent labels. The labels can include multiple fluorescent dye molecules conjugated to a single polymeric backbone or core, such as a dendrimer. The dendrimers can have regular or irregular branched polymeric network structures that allow for the chemical attachment of multiple dye molecules, multiple color dyes, and/or multiple functional groups, in a combinatorial fashion. The fluorescent dendritic nanoprobes (FDNs) thus provide a new class of fluorescent reporters for fluorescence microscopy and imaging.

First claim

What is claimed is:

1. A dendrimer of Formula I:

wherein
the circle of Formula I is a G2-G10 dendrimer;
each R is independently a dye moiety covalently bonded to a branch of the dendrimer;
each linker-X is independently the same or different, wherein linker is S, NH, or O, or a chain of 2-40 atoms that connects a branch of the dendrimer to a group X;
each X is independently R, a reactive group, an affinity label, or a targeting moiety;
n is a value representing about 1% to about 50% of terminal dendrimer groups;
m is a value representing 0% to about 50% of terminal dendrimer groups;
s is a value representing 1% to about 60% of terminal dendrimer groups; and
t is a value representing 0% to about 60% of terminal dendrimer groups;
wherein at least two R groups are different from each other; and wherein the dye moieties of at least two different R groups comprise at least two different Cyanine dyes;
the photobleaching lifetime of the dendrimer is more than 2 times longer than the photobleaching lifetime of a single dye separate from the dendrimer;
the fluorescence emission intensity of the dendrimer is at least two times brighter than a corresponding single dye molecule; and
the diameter of the dendrimer is less than about 20 nm;

or a salt thereof.

2. A dendrimer of Formula II:

wherein
n 1 , n 2 , n 3 , n 4 , n 5 , n 6 , n 7 and n 8 are each independently 0-20, and each of n 1 , n 2 , n 3 , n 4 , n 5 , n 6 , n 7 and n 8 represents a value corresponding to 0-60% of the terminal groups on the dendrimer surface;
the circle at the core of Formula II represents a dendrimer core having a diameter of about 2 nm to about 10 nm, wherein the dendrimer core comprises a chemically symmetrical or unsymmetrical polymer backbone or hyperbranched polymer backbone;
each X′ is independently a linking group selected from —O—, —NH—, —CO 2 —, —OCO 2 —, —S—, —P(O)(OR)O—, —NH(CO)—, —NH(CO)O—, —NH(CS)NH—, —SO 2 —, —SO 3 —, —(CH 2 ) n — where n is 1-10, or X′ is a direct bond to a nitrogen atom or oxygen atom of the dendrimer core;
each Y and Z is independently —OH, —NH 2 , —CO 2 H, —COOR′, —SR′, —P(O)(OR′) 2 , —OC(O)OR′, —NH(CO)OR′, —NH(CS)NHR′, —SO 2 R′, or —SO 3 R′, where R′ is an alkali metal, alkaline earth metal, hydrogen, vinyl, (C 6 -C 10 )aryl, (C 1 -C 12 )alkyl, or a chemical functional group for bioconjugation, an active carbonate, an active carbamate, a pentafluorophenoxy carbamate, benzylguanine, or O2-benzylcytosine;
D 1 , D 2 , D 3 , and D 4 are each independently a dye moiety bonded to a group X; and wherein the dye moiety comprises at least two different Cyanine dyes;
—X′—P 1 is —NH—Ac, and at least one —NH—Ac is present;
the photobleaching lifetime of the dendrimer is more than 2 times longer than the photobleaching lifetime of a single dye separate from the dendrimer; and
the fluorescence emission intensity of the dendrimer is at least two times brighter than a corresponding single dye molecule;
or salt thereof.

3. The dendrimer of claim 2 wherein the dendrimer is a poly(amidoamine) (PAMAM) dendrimer.

4. The dendrimer of claim 1 wherein m is not 0% and linker-X comprises one or more ethylene glycol units, one or more (C 1 -C 6 )amide units, or a combination thereof.

5. The dendrimer claim 4 wherein X comprises an alkyne, an azide, an N-hydroxysuccinimide (NHS)-ester, a pentafluorophenyl ester, a tetrafluorophenyl ester, a pentafluoroethyl ester, a vinylsulfonyl group, a 2-pyridyldisulfide, a methansufinyldisulfide, a 4-nitrophenylcarbonate group, a 4-nitrophenylcarbamate group, a thiol, an imide group, benzylguanine, or O2-benzylcytosine.

6. The dendrimer claim 5 wherein X comprises biotin or a Ni/Co-NTA group.

7. The dendrimer of claim 6 wherein R comprises a fluorescent dye.

8. The dendrimer of claim 7 wherein R comprises Acridine Orange, Acridine Yellow, an Alexa Fluor dye, an Atto dye, a BODIPY dye, a CF dye antibody conjugate, Cascade Blue, coelenterazine, coumarin, a cyanine dye, dansyl dye, 4′,6-diamidino-2-phenylindole (DAPI), erythrosin, FLUO 3, fluorescein, FURA 2, 5-hydroxytryptamine (HAT), a Hoechst dye, INDO 1, JC-1 dye, Lucifer Yellow, Nile Red, Oregon Green dye, propidium iodide, QUIN 2, a rhodamine dye, R-phycoerythrin, R-phycoerythrin-Texas Red, SNARF, or Texas Red.

9. The dendrimer of claim 7 wherein one or more R groups include one or more of Cy2, Cy3, Cy3.5, Cy5, Cy5.5, or Cy7.

10. The dendrimer of claim 1 wherein the molecular diameter of the dendrimer is about 2 nm to about 10 nm.

11. The dendrimer of claim 1 wherein the dendrimer exhibits multiple emission wavelength colors upon excitation to provide a plurality of distinct fluorescent signatures.

12. The dendrimer of claim 1 wherein each of the dye moieties is located within a distance of about 8 nm on the surface of the dendrimer.

13. The dendrimer of claim 12 wherein about 4% to about 15% of the terminal groups of the dendrimer comprise surface bound linker-X groups wherein one or more of the linker-X groups comprises biotin or a Ni/Co-NTA group.

14. A fluorescent probe comprising two or more different dyes conjugated to a dendrimer, and wherein the dyes comprise at least two different Cyanine dyes; wherein at least two of the dyes emit light at different wavelengths upon irradiation, the photobleaching lifetime of the dendrimer is more than 2 times longer than the photobleaching lifetime of a single dye separate from the dendrimer, the fluorescence emission intensity of the probe is at least two times brighter than a corresponding single dye molecule, the dendrimer comprises chains that terminate in N-acetyl groups, and the molecular diameter of the fluorescent probe is less than about 20 nm.

15. A method to provide a distinct spectral fingerprint to a single molecule comprising conjugating a fluorescent probe to a molecule, wherein the fluorescent probe is a dendrimer of claim 1 , the fluorescent probe comprises two or more different color dyes conjugated to the dendrimer core moiety of the probe, thereby providing a single molecule with a multiple wavelength emission when irradiated by two or more excitation light sources that correspond to the absorption spectra of the dyes of the probe.

16. A method for labeling a biomolecule comprising:
contacting a sample that includes one or more biomolecules with a plurality of dendrimers of claim 1 , wherein a reactive group of the dendrimer conjugates the dendrimer to the biomolecule, thereby allowing detection of one or more of the biomolecules by microscopy.

17. The method of claim 16 wherein the biomolecule is DNA, RNA, a protein, a membrane, or an intracellular component.

18. A method for in vivo targeting and labeling an intracellular component comprising:
contacting one or more intracellular components with a plurality of dendrimers of claim 1 , wherein a reactive group of the dendrimer conjugates the dendrimer to one or more of the intracellular components, thereby allowing detection of one or more intracellular component by microscopy.

19. A method of imaging a sample comprising photoswitchable fluorescent probes, the method comprising:
(a) providing a sample labeled with a plurality of photoswitchable fluorescent probes, wherein the photoswitchable fluorescent probes comprise one or more dendrimers of claim 1 , at least some of the dye moieties o

Assignees

• Kim Younghoon
• Kim Sung Hoon
• Tanyeri Melikhan
• Katzenellenbogen John A
• Schroeder Iii Charles M
• Univ Illinois

Inventors

• Kim Younghoon
• Kim Sung Hoon
• Tanyeri Melikhan
• Katzenellenbogen John A
• Schroeder Iii Charles M

Classifications

• G01N21/6456

  Spatial resolved fluorescence measurements; Imaging · CPC title

• G01N21/6428 Primary

  Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53) · CPC title

• G01N33/582

  with fluorescent label · CPC title

• C08G73/14

  Polyamide-imides · CPC title

• C08G83/003

  Dendrimers · CPC title