Multimeric protected fluorescent reagents

Patent metadata

Field	Value
Publication number	US-2016237279-A1
Application number	US-201615015602-A
Country	US
Kind code	A1
Filing date	Feb 4, 2016
Priority date	Feb 4, 2015
Publication date	Aug 18, 2016
Grant date	—

Abstract

Multimeric protected fluorescent reagents and their methods of synthesis are provided. The reagents are useful in various fluorescence-based analytical methods, including the analysis of highly multiplexed optical reactions in large numbers at high densities, such as single molecule real time nucleic acid sequencing reactions. The reagents contain fluorescent dye elements, that allow the compounds to be detected with high sensitivity at desirable wavelengths, binding elements, that allow the compounds to be recognized specifically by target biomolecules, and protective shield elements, that decrease undesirable contacts between the fluorescent dye elements and the bound target biomolecules and that therefore decrease photodamage of the bound target biomolecules by the fluorescent dye elements. The reagents also contain coupling elements connect monomeric compounds into multimeric forms, thereby increasing brightness.

First claim

1 . A reagent of structural formula (IV):
J-[-B″—S″—Z—[—S′—B′] m′ ] q   (IV); wherein
each Z is independently a multivalent central core element comprising a fluorescent dye element; each S′ is independently an intermediate chemical group, wherein at least one S′ comprises a shield element; each S″ is independently an intermediate chemical group, wherein S″ optionally comprises a shield element; each B′ is independently a terminal chemical group, wherein at least one B′ comprises a binding element; each B″ is independently a terminal chemical group or a bond; J is a joining element; each m′ is independently an integer from 1 to 23; and q is an integer from 2 to 10.

2 . The reagent of claim 1 , wherein the shield element decreases photodamage of the reagent or of a biomolecule associated with the binding element.

3 . The reagent of claim 1 , wherein the shield element decreases contact between the fluorescent dye and the binding element.

4 . The reagent of claim 1 , wherein the shield element comprises a plurality of side chains.

5 - 16 . (canceled)

17 . The reagent of claim 1 , wherein the shield element comprises the structure:

wherein each y is independently an integer from 1 to 6.

18 . The reagent of claim 1 , wherein the shield element comprises an inner layer and an outer layer.

19 . The reagent of claim 1 , wherein at least one S′—B′ group comprises the structure:

wherein Ri and R2 is each independently a side chain;
L is an alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl linker; and
Y is

20 . The reagent of claim 1 , wherein the binding element comprises a nucleotide.

21 . The reagent of claim 1 , wherein the binding element comprises biotin.

22 . The reagent of claim 1 , wherein the binding element comprises a polyphosphate.

23 . The reagent of claim 1 , wherein Z comprises a branching element.

24 - 25 . (canceled)

26 . The reagent of claim 1 , wherein each m′ is independently an integer from 1 to 11.

27 - 38 . (canceled)

39 . The reagent of claim 1 , wherein each B″ is a terminal group, and J and each B″ are connected non-covalently.

40 - 43 . (canceled)

44 . The reagent of claim 1 , wherein each B″ is a bond.

45 - 48 . (canceled)

49 . The reagent of claim 1 , wherein each S′ comprises a shield element.

50 . The reagent of claim 1 , wherein q is an integer from 2 to 4.

51 - 96 . (canceled)

97 . The reagent of claim 1 having structural formula (VI):

wherein
X is a non-fluorescent multivalent central core element;
at least one D is a fluorescent dye element;
each W is independently a branching element;
each W′ is independently a branching element;
each n′ is independently an integer from 1 to 5;
each p′ is independently an integer from 1 to 4; and
each p″ is independently an integer from 1 to 4.

98 . The reagent of claim 97 , wherein X comprises a polyamine.

99 . (canceled)

100 . The reagent of claim 97 , wherein X comprises a substituted cyclohexane.

101 . (canceled)

102 . The reagent of claim 97 , wherein X comprises a substituted 1,3,5-triazine.

103 . The reagent of claim 97 , wherein X comprises a substituted benzene.

104 . (canceled)

105 . The reagent of claim 97 , comprising at least one donor fluorophore and at least one acceptor fluorophore.

106 . The reagent of claim 97 , wherein the shield element comprises a plurality of side chains.

107 - 118 . (canceled)

119 . The reagent of claim 97 , wherein the shield element comprises the structure:

wherein each y is independently an integer from 1 to 6.

120 . The reagent of claim 97 , wherein the shield element comprises an inner layer and an outer layer.

121 . The reagent of claim 97 , wherein at least one S′—B′ group comprises the structure:

wherein R 1 and R 2 is each independently a side chain;
L is an alkyl, heteroalkyl, cycloalkyl, cycloheteroalkyl, aryl, or heteroaryl linker; and
Y is

122 . The reagent of claim 97 , wherein the binding element comprises a nucleotide.

123 . The reagent of claim 97 , wherein the binding element comprises biotin.

124 . The reagent of claim 97 , wherein the binding element comprises a polyphosphate.

125 . The reagent of claim 97 , wherein the branching element comprises a substituted phenyl group.

126 - 130 . (canceled)

131 . The reagent of claim 97 , wherein each B″ is a terminal group, and J and B″ are connected non-covalently.

132 - 135 . (canceled)

136 . The reagent of claim 97 , wherein each B″ is a bond.

137 - 142 . (canceled)

Assignees

• Pacific Biosciences California Inc

Inventors

• Zheng Frank
• Hanes Jeremiah
• Shen Gene
• Brogley Louis
• Yue Stephen
• Lapin Yuri
• Lyle John
• Osuna Honey
• Fedorov Andrei
• Sebo Lubomir

Classifications

• C09B69/00

  Dyes not provided for by a single group of this subclass · CPC title

• C09B23/06 Primary

  three >CH- groups, e.g. carbocyanines · CPC title

• C09B69/105

  containing a methine or polymethine dye · CPC title

• G01N33/533

  with fluorescent label · CPC title

• C09B23/105 Primary

  two >CH- groups · CPC title