Novel Super-Resolution Imaging Compositions and Methods Using Same

Patent metadata

Field	Value
Publication number	US-2016115180-A1
Application number	US-201514919216-A
Country	US
Kind code	A1
Filing date	Oct 21, 2015
Priority date	Oct 22, 2014
Publication date	Apr 28, 2016
Grant date	—

Abstract

The invention provides compositions that may be used for imaging intracellular structures. The invention further provides methods of imaging intracellular structures. In certain embodiments, the compositions of the invention include trans-cyclooctene-containing ceramide lipids and tetrazine-containing rhodamine-related dyes.

First claim

What is claimed:

1 . A trans-cyclooctene-containing lipid, or a salt, solvate, stereoisomer, or any mixtures thereof, wherein the lipid comprises TCO-LINK-LIPID, wherein TCO is a trans-cyclooctene group, LINK is a bivalent linker, and LIPID is a lipid.

2 . The trans-cyclooctene-containing lipid of claim 1 , wherein TCO is selected from the group consisting of:

wherein R′ is H or optionally substituted C 1 -C 6 alkyl, and wherein the heteroatom is not attached to the ring double bond.

3 . The trans-cyclooctene-containing lipid of claim 2 , wherein TCO is selected from the group consisting of:

wherein R′ is H or optionally substituted C 1 -C 6 alkyl.

4 . The trans-cyclooctene-containing lipid of claim 1 , wherein LIPID comprises at least one selected from the group consisting of a sphingolipid, phospholipid, sterol lipid and fatty acid.

5 . The trans-cyclooctene-containing lipid of claim 4 , wherein LIPID comprises at least one selected from the group consisting of sphingosine, 1-phosphocholine-sphingosine, 1-phosphoethanolamine-sphingosine, and a 1-glycosyl-sphingosine.

6 . The trans-cyclooctene-containing lipid of claim 5 , wherein LIPID comprises sphingosine.

7 . The trans-cyclooctene-containing lipid of claim 6 , wherein LIPID comprises sphingosine and is covalently conjugated to LINK through the 2-amino group of sphingosine.

8 . The trans-cyclooctene-containing lipid of claim 1 , wherein LINK comprises:
—X(CR′R″) n C(═O)—, wherein
X is covalently conjugated to TCO and is selected from the group consisting of a covalent bond, —C(═O)—, —C(═O)O— and —C(═O)NR′—; each occurrence of R′ and R″ is independently selected from the group consisting of H and optionally substituted C 1 -C 6 alkyl; and n is an integer ranging from 1 to 20.

9 . The trans-cyclooctene-containing lipid of claim 8 , wherein n is 5.

10 . The trans-cyclooctene-containing lipid of claim 1 , which is Cer-TCO or a salt, solvate, stereoisomer, or any mixtures thereof:

11 . A tetrazine-containing dye, or a salt or solvate thereof, which is:

wherein
R 1 is —(CH 2 ) n — or —(CH 2 CH 2 O) n (CH 2 ) m —;
n is an integer ranging from 1 to 6; and
m is 0, 1 or 2.

12 . The tetrazine-containing dye of claim 11 , wherein R 1 is —(CH 2 )—.

13 . A tetrazine-containing dye of claim 11 , which is SiR-Tz, or a salt, solvate, stereoisomer, or any mixtures thereof:

14 . A compound, or a salt, solvate, stereoisomer, or any mixtures thereof, of formula:

wherein in (I):
R 1 is —(CH 2 ) n — or —(CH 2 CH 2 O) n (CH 2 ) m —;
m is 0, 1 or 2;
each occurrence of n is independently an integer ranging from 1 to 6;
R 2 is S, O or NR′;
LINK comprises —X(CR′R″) 1-20 C(═O)—, wherein X is covalently conjugated to R 2 and is selected from the group consisting of a covalent bond, —C(═O)—, —C(═O)O— and —C(═O)NR′—;
LIPID comprises a sphingolipid; and,
each occurrence of R′ and R″ is independently selected from the group consisting of H and optionally substituted C 1 -C 6 alkyl.

15 . The compound of claim 14 , wherein in (I) R 1 is —CH 2 —.

16 . The compound of claim 14 , wherein in (I) R 2 is S, O or NH.

17 . The compound of claim 14 , wherein in (I) LINK is —X(CH 2 ) 1-20 C(═O)—.

18 . The compound of claim 14 , wherein in (I) LINK is —C(═O)NH(CH 2 ) 5 C(═O)—.

19 . The compound of claim 14 , which is:

20 . The compound of claim 14 , which is Cer-SiR, or a salt, solvate, stereoisomer, or any mixtures thereof:

21 . A method of labeling an intracellular structure in a cell, comprising the steps of:
contacting the cell with at least one trans-cyclooctene-containing lipid of claim 1 , wherein the lipid associates with the intracellular structure in the cell; contacting the cell with at least one tetrazine-containing dye of claim 11 , wherein the tetrazine-containing dye undergoes a cycloaddition reaction with the at least one trans-cyclooctene-containing lipid in the intracellular structure, whereby the cycloaddition product labels the intracellular structure.

22 . The method of claim 21 , wherein the cell is in at least one state selected from the group consisting of in vitro, ex vivo and in vivo.

23 . The method of claim 21 , wherein the intracellular structure is visualized using at least one method selected from the group consisting of 3D confocal microscopy, STED super-resolution microscopy, and single molecule switching (SMS) super-resolution imaging.

24 . The method of claim 21 , wherein the cycloaddition product is photostable.

25 . The method of claim 21 , wherein the intracellular structure comprises at least one selected from the group consisting of the Golgi and trans-Golgi network.

26 . The method of claim 21 , wherein the tetrazine-containing dye, trans-cyclooctene-containing lipid and cycloaddition product do not have a significant effect on cell morphology.

27 . The method of claim 21 , wherein the tetrazine-containing dye, trans-cyclooctene-containing lipid and cycloaddition product do not have a significant effect on mobility of a protein within the Golgi, or in cargo traffic from the ER through the Golgi and to the plasma membrane.

28 . The method of claim 21 , wherein the tetrazine-containing dye is SirR-Tz, or a salt or solvate thereof.

29 . The method of claim 21 , wherein the trans-cyclooctene-containing lipid is Cer-TCO, or a salt, diastereoisomer, geometric isomer, solvate, or any mixtures thereof.

30 . The method of claim 21 , wherein the cycloaddition product is Cer-SiR, or a salt, diastereoisomer, geometric isomer, solvate, or any mixtures thereof.

31 . The method of claim 21 , wherein the cell is human.

32 . A kit comprising a tetrazine-containing dye, trans-cyclooctene-containing lipid, applicator, and instructional material for use thereof, wherein the instructional material comprises instructions for labeling an intracellular structure in a cell using the dye and the lipid.

33 . The kit of claim 32 , wherein the tetrazine-containing dye is SirR-Tz, or a salt, solvate, stereoisomer, or any mixtures thereof.

34 . The kit of claim 32 , wherein the trans-cyclooctene-containing lipid is Cer-TCO, or a salt, solvate, stereoisomer, or any mixtures thereof.

Assignees

• Univ Yale

Inventors

• Erdmann Roman
• Shrader Alanna Schepartz
• Toomre Derek

Classifications

• A61K49/0021

  the fluorescent group being a small organic molecule · CPC title

• C07F7/0807 Primary

  comprising Si as a ring atom · CPC title

• C07C271/34 Primary

  with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms · CPC title

• A61K49/0052

  Small organic molecules (oligomers, polymers, dendrimers A61K49/0054) · CPC title

• C09B11/28

  Pyronines {; Xanthon, thioxanthon, selenoxanthan, telluroxanthon dyes} · CPC title