Molecular adaptors for dye conjugates

Patent metadata

Field	Value
Publication number	US-9777320-B2
Application number	US-201113218439-A
Country	US
Kind code	B2
Filing date	Aug 25, 2011
Priority date	Aug 25, 2010
Publication date	Oct 3, 2017
Grant date	Oct 3, 2017

Abstract

In various embodiments, the present invention provides fluorescent dyes that are linked to another species through an adaptor moiety. In an exemplary embodiment, the dye is linked to a polyphosphate nucleic acid through an adaptor. An adaptor can be a component of a linker. These conjugates find use in single molecule DNA sequencing and other applications. In various embodiments, the dye moiety is a cyanine dye. Cyanine dyes that are highly charged, such as those including multiple sulfonate, alkylsulfonate, carboxylate and/or alkylcarboxylate moieties are examples of cyanine dyes of use in the compounds of the invention.

First claim

What is claimed is:

1. A fluorescent cyanine dye of Formula I:
{R 1 -(L 1 ) a -A} y -X  (I)

wherein
R 1 is a fluorescent cyanine dye moiety, of Formula Ia:

in which
B is an indolinium moiety, and B′ is an indole moiety;
R x , R y , R w and R z are members independently selected from
—OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O) 2 R′, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, SO 3 R′, —S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2 , —R′, —N 3 , —CH(Ph) 2 , fluoro(C 1 -C 4 )alkoxy, fluoro(C 1 -C 4 )alkyl, alkylsulfonic acid and heteroalkylsulfonic acid,

wherein
R′, R″, and R′″ are independently selected from hydrogen, (C 1 -C 8 )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 -C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl;

c is an integer selected from 1, 2, 3, 4, or 5;
w and z are integers independently selected from 1, 2, 3, 4, 5, or 6;
1, 2, 3, 4, 5, or 6 of R x , R y , R w and R z are independently selected from alkylsulfonic acid or heteroalkylsulfonic acid, and
at least one of R x , R y , R w and R z is A or is L 1 -A;
A is an adaptor covalently bound to X and, optionally, to L 1 , wherein A comprises a moiety with a formula selected from: piperidinyl;

wherein q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10;
L 1 is a linker independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
a is 0 or 1;
X is a member selected from:
(i) a polyvalent moiety selected from triazine moiety, perylene moiety, and tetra-aminoadamantane moiety, having bonded thereto a moiety of the formula:

wherein
Y′ is a naturally occurring nucleobase; and
u′ is selected from the integers 1, 2, 3, 4, 5, 6, 7 and 8; and

(ii) a moiety according to Formula II:

wherein
Z is a member selected from O, S and NH;
Y is a naturally occurring nucleobase;
u is selected from the integers 3, 4, 5, 6, 7 and 8; and

y is selected from the integers 1, 2, 3, 4, 5, 6, 7 and 8, such that when y is 2 or greater X is said polyvalent moiety.

2. The fluorescent cyanine dye of claim 1 , wherein A is selected from:

3. The fluorescent cyanine dye of claim 1 , wherein A is selected from:

4. A method of monitoring an enzyme reaction, said method comprising:
(a) forming a reaction mixture by contacting said enzyme with the fluorescent cyanine dye according to claim 1 , wherein said dye is a substrate for said enzyme under conditions sufficient for said enzyme and said dye to react; and
(b) monitoring fluorescence of said reaction mixture.

5. The method according to claim 4 , wherein said enzyme is a DNA polymerase and said fluorescent cyanine dye comprises a nucleic acid moiety which is said substrate for said enzyme.

6. The method according to claim 5 , wherein said reaction mixture further comprises a template nucleic acid that complexes with said DNA polymerase, and wherein said enzyme reaction is template directed DNA synthesis.

7. The method according to claim 6 , wherein said enzyme reaction is a component of a single molecule DNA sequencing analysis.

8. The fluorescent cyanine dye according to claim 1 , wherein R 1 is a fluorescent cyanine dye moiety selected from:

9. The fluorescent cyanine dye according to claim 1 , wherein R 1 is a fluorescent cyanine dye moiety selected from:

10. A fluorescent cyanine dye of a formula selected from:

11. A fluorescent cyanine dye of Formula I:
{R 1 -(L 1 ) a -A} y -X  (I)

wherein
R 1 is a fluorescent cyanine dye moiety, of Formula Ia:

in which
B is an indolinium moiety, and B′ is an indole moiety;
R x , R y , R w and R z are members independently selected from —OR′, —NR′R″, —SR′, -halogen, —SiR′R″R′″, —OC(O)R′, —C(O)R′, —CO 2 R′, —CONR′R″, —OC(O)NR′R″, —NR″C(O)R′, —NR′—C(O)NR″R′″, —NR″C(O) 2 R′, —NR—C(NR′R″)═NR′″, —S(O)R′, —S(O) 2 R′, SO 3 R′, —S(O) 2 NR′R″, —NRSO 2 R′, —CN and —NO 2 , —R′, —N 3 , —CH(Ph) 2 , fluoro(C 1 -C 4 )alkoxy, fluoro(C 1 -C 4 )alkyl, alkylsulfonic acid and heteroalkylsulfonic acid,
wherein
R′, R″, and R′″ are independently selected from hydrogen, (C 1 -C 8 )alkyl and heteroalkyl, unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(C 1 -C 4 )alkyl, and (unsubstituted aryl)oxy-(C 1 -C 4 )alkyl;

c is an integer selected from 1, 2, 3, 4, or 5;
w and z are integers independently selected from 1, 2, 3, 4, 5, or 6;
1, 2, 3, 4, 5, or 6 of R x , R y , R w and R z are independently selected from alkylsulfonic acid or heteroalkylsulfonic acid, and
at least one of R x , R y , R w and R z is A or is L 1 -A;
L 1 is a linker independently selected from substituted or unsubstituted alkyl and substituted or unsubstituted heteroalkyl;
a is 1;
A is an adaptor of a formula selected from: piperidinyl, C 1 -C 10 aminoalkyl, —NH(CH 2 ) j C(O)NH(CH 2 ) k ;

wherein
j and k are independently selected from the integers from 1 to 30;
q is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10;

y is 2, 3, 4, 5, 6, 7 or 8;
X is a member selected from a polyvalent moiety selected from a triazine, perylene, and tetra-aminoadamantane moiety, wherein said polyvalent moiety has bonded thereto a moiety having the formula:

wherein
Y′ is a naturally occurring nucleobase; and
u′ is selected from the integers 1, 2, 3, 4, 5, 6, 7 and 8.

Assignees

• Yue Stephen
• Shen Gene
• Brogley Louis
• Sebo Lubomir
• Wegener Jeffrey
• Pacific Biosciences California Inc

Inventors

• Yue Stephen
• Shen Gene
• Brogley Louis
• Sebo Lubomir
• Wegener Jeffrey

Classifications

• C09B23/083

  five >CH- groups · CPC title

• C09B23/164

  containing one nitrogen atom · CPC title

• C07H19/10

  with the saccharide radical esterified by phosphoric or polyphosphoric acids · CPC title

• C09B23/06

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

• C09B23/0025

  the substituent being bound through an oxygen atom · CPC title