Energy transfer dye conjugates for use in biological assays

Patent metadata

Field	Value
Publication number	US-2024060117-A1
Application number	US-202118006554-A
Country	US
Kind code	A1
Filing date	Jul 23, 2021
Priority date	Jul 23, 2020
Publication date	Feb 22, 2024
Grant date	—

Abstract

Energy transfer dye pairs including a donor dye covalently attached to an acceptor dye through a linker, uses of the energy transfer dye pairs, for example, in conjugates of an energy transfer dye pair covalently attached to a quencher and an analyte (e.g., an oligonucleotide), for biological applications including, for example, amplification assays such as quantitative polymerase chain reaction (qPCR) and digital PCR (dPCR).

First claim

We claim:

1 . A fluorescent energy transfer dye conjugate comprising:
i. a donor dye capable of absorbing light at a first wavelength and emitting excitation energy in response; ii. an acceptor dye capable of absorbing the excitation energy emitted by the donor dye and emitting light at a second wavelength in response; and iii. a linker covalently attaching the donor dye to the acceptor dye, wherein the linker comprises one or more of an alkyl portion, an amino-alkyl portion, an oxy-alkylene portion, an amino-alkylene-dialkoxy portion, an alkenylene portion, an alkynylene portion, a polyether portion, an arylene portion, an amide portion, or a phosphodiester portion.

2 . The energy transfer dye conjugate of claim 1 , wherein the second wavelength is longer than the first wavelength.

3 . The energy transfer dye conjugate of claim 1 or 2 , wherein the donor dye is selected from the group consisting of a xanthene dye, a cyanine dye, a BODIPY dye, a pyrene dye, a pyronine dye, and a coumarin dye.

4 . The energy transfer dye conjugate of any one of the preceding claims, wherein the donor dye is a fluorescein dye or a rhodamine dye.

5 . The energy transfer dye conjugate of any one of the preceding claims, wherein the acceptor dye is selected from the group consisting of a fluorescein dye, a cyanine dye, a rhodamine dye, a BODIPY dye, a pyrene dye, a pyronine dye, and a coumarin dye.

6 . The energy transfer dye conjugate of any one of the preceding claims, wherein the conjugate is linked to an analyte and having a basic structure selected from one of

wherein L 1 is a first linker, wherein L 1 is attached to D 1 , D 2 and A through a covalent bond or through a spacer comprising one or more intervening atoms;
wherein L 2 is a second linker, wherein L 2 is attached to each of D 2 and D 3 through a covalent bond or through a spacer comprising one or more intervening atoms;
wherein L 3 is a third linker, wherein L 3 is attached to each PO 4 H and D 1 through a covalent bond or through a spacer comprising one or more intervening atoms;
wherein L 4 is a fourth linker, wherein L 4 is attached to PO 4 H and D 2 through a covalent bond or through a spacer comprising one or more intervening atoms;
wherein A is the analyte;
wherein each of D 1 , D 2 , and D 3 is interchangeably a donor dye or an acceptor dye;
wherein the combination of D 1 and D 2 in L I and L III and D 2 and D 3 in L II forms an energy transfer dye pair.

7 . The energy transfer dye conjugate of claim 6 , wherein the L 1 linker comprises an arylene portion of the formula

wherein
each R 1 is independently —C 1 -C 10 alkyl-N(R 3 )—*, —C 2 -C 10 alkenyl-N(R 3 )—*, —C 2 -C 10 alkynyl-N(R 3 )—*, —OC 1 -C 10 alkyl-*, —C 1 -C 10 alkyl-O—*, —N(R 3 )C 1 -C 6 alkyl-*, —N(R 3 )C 1 -C 6 alkyl-O—*, —OC 1 -C 6 alkyl-N(R 3 )—*; or —N(R 3 )—*;
each R 2 is independently —C(O)N(R 4 ), —C 1 -C 10 alkyl-C(O)N(R 4 ), —C 2 -C 10 alkenyl-C(O)N(R 4 ), —C 2 -C 10 alkynyl-R 4 , —C(O)N(R 4 ), —N(R 3 )—C(O)N(R 4 ), C 1 -C 6 alkyl-O—C(O)N(R 4 ), —OC 1 -C 6 alkyl-C(O)N(R 4 ), —N(R 4 ), halogen, —CO 2 − Z + , —SO 3 R 4 , or —SO 3 − Z + ;
each R 3 is independently H or C 1 -C 6 alkyl;
each R 4 is independently H, C 1 -C 6 alkyl, or a point of attachment to A, wherein the attachment to A is through a covalent bond or through a spacer comprising one or more intervening atoms;
each * represents a point of attachment to D 1 or D 2 , wherein the attachment to D 1 or D 2 is through a covalent bond or through a spacer comprising one or more intervening atoms;
Z + is a cation(s) (e.g., Na + , K + , or NH 4 + );
n is 2, 3 or 4; and
m is 0, 1, 2, 3, or 4, provided that n+m=3 to 6.

8 . The energy transfer fluorescent dye conjugate of claim 6 , wherein the L 1 linker comprises an arylene portion and one or more of a bis-alkylamino portion or a bis-carboxyamidyl portion, wherein the L 1 linker further comprises a point of attachment to A, wherein the attachment to A is through a covalent bond or through a spacer comprising one or more intervening atoms.

9 . The energy transfer dye conjugate of claim 6 , wherein the L 2 linker comprises an arylene portion of the formula

wherein
each R 1 is independently —C 1 -C 10 alkyl-N(R 3 )—*, —C 2 -C 10 alkenyl-N(R 3 )—*, —C 2 -C 10 alkynyl-N(R 3 )—*, —OC 1 -C 10 alkyl-*, —C 1 -C 10 alkyl-O—*, —N(R 3 )C 1 -C 6 alkyl*-, —N(R 3 )C 1 -C 6 alkyl-O—*, —OC 1 -C 6 alkyl-N(R 3 )—*; or —N(R 3 )—*;
each R 2 is independently —C(O)N(R 3 )—*, —C 1 -C 10 alkyl-C(O)N(R 3 )—*, —C 2 -C 10 alkenyl-C(O)N(R 3 )—*, —C 2 -C 10 alkynyl-(R 3 )—*, —C(O)N(R 3 )—*, —N(R 3 )—C(O)N(R 3 )—*, C 1 -C 6 alkyl-O—C(O)N(R 3 )—*, —OC 1 -C 6 alkyl-C(O)N(R 3 )—*, —N(R 3 )—*, halogen, —CO 2 − Z + or —SO 3 − Z + ;
each R 3 is independently H or C 1 -C 6 alkyl;
each * represents a point of attachment to D 2 or D 3 , wherein the attachment to D 2 or D 3 is through a covalent bond or through a spacer comprising one or more intervening atoms.
Z + is a cation(s) (e.g., Na + , K + , or NH 4 + );
n is 2, 3 or 4; and
m is 0, 1, 2, 3, or 4, provided that n+m=2 to 6.

10 . The energy transfer dye conjugate of any one of the preceding claims, wherein the linker comprises a fragment of the formula

wherein each R 2 , m and * is as defined above.

11 . The energy transfer dye conjugate of claim 6 , wherein the L 3 linker comprises a fragment of the formula.

wherein
R 5 is H or C 1 -C 6 alkyl;
n is 2, 3 or 4;
X is O or CH 2 ;
L 4 is an attachment to D 2 , wherein L 4 is a covalent bond or a spacer comprising one or more intervening atoms;
R 7 is a point of attachment to PO 3 H-A, wherein the attachment to PO 3 H-A is through a covalent bond or through a spacer comprising one or more intervening atoms; and
wherein * represents a point of attachment to D 1 , wherein the attachment to D 1 is through a covalent bond or through a spacer comprising one or more intervening atoms.

12 . The energy transfer dye conjugate of claim 11 , wherein the L 4 linker comprises a phosphodiester portion of the formula

wherein
Y comprises one or more of an alkoxy portion, an alkyl portion, an arylene portion, or an oligonucleotide portion;
p is an integer from 0 to 10;
D 2 or A comprises an oxygen atom, wherein each * represents a point of attachment of the phosphodiester portion to the oxygen atom in D 2 or A, wherein the attachment of the phosphodiester to the oxygen atom in D 2 or A is through a covalent bond or through a spacer comprising one or more intervening atoms.

13 . The energy transfer dye conjugate of claim 12 , wherein Y is C 1 -C 10 alkyl or poly(alkylene glycol).

14 . The energy transfer dye conjugate of any one of claims 6 - 13 , wherein a combination of the L 3 and L 4 linker comprises the formula

Assignees

• Life Technologies Corp

Inventors

• Benson Scott
• Menchen Steven
• Chang Chu-An
• Lee Linda
• Mullah Khairuzzaman

Classifications

• C12Q1/6818 Primary

  involving interaction of two or more labels, e.g. resonant energy transfer · CPC title

• C09K11/06

  containing organic luminescent materials · CPC title

• C09B62/3435

  Specific dyes not provided for in groups C09B62/345 - C09B62/357 · CPC title

• C09K2211/1022

  bridged by heteroatoms, e.g. N, P, Si or B · CPC title

• C09K2211/1007

  Non-condensed systems · CPC title