Functionalized cyanine dyes (peg)
AssigneePacific Biosciences of California, Inc.
InventorsGene Shen, Stephen Yue
Life of this patent
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Abstract
The invention provides a novel class of cyanine dyes that are functionalized with a linker moiety that facilitates their conjugation to other species and substituent groups which increase the water-solubility, and optimize the optical properties of the dyes. Also provided are conjugates of the dyes, methods of using the dyes and their conjugates and kits including the dyes and their conjugates.
Claims26
A compound having the formula:
- wherein
- Q is:
- Rm is a member selected from substituted or unsubstituted aryl and substituted or unsubstituted heteroaryl;
- Rc, and Rd are independently selected from alkyl and heteroalkyl, substituted with a member selected from sulfonic acid, carboxylic acid, phosphonic acid, and phosphoric acid;
- each Ra and each Re, is independently selected from C(O)R9, OR12, NR12R13, CR12C(O)R13, NR12C(O)2R13, SO3H, and C(O)NR12R13, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl, and a ring structure formed by joining a member selected from two Ra moieties together with the atoms to which they are attached, two Re moieties together with the atoms to which they are attached, and a combination thereof, to form said ring structure, which is a member selected from a substituted or unsubstituted aryl and a substituted or unsubstituted heteroaryl, said ring substituted with at least one SO3H,
- wherein
- R9 is a member selected from OR10, and NH(CH2)tOR11
- wherein
- R10 is a member selected from H and substituted or unsubstituted alkyl;
- t is selected from the integers from 1 to 12;
- R11 is a member selected from H, and
- wherein
- u is selected from the integers from 1 to 8;
- and
- Y is a nucleobase;
- and
- R12 and R13 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalky;
- a and e are integers independently selected from 0, 1, 2, 3 and 4;
- Rf, Rg, Rh, and Ri are independently selected from unsubstituted alkyl, alkylsulfonic acid, and
- wherein
- s is selected from the integers from 1 to 12;
- and
- R9 is a member selected from OH, and
- —NH(CH2)t—O—R10
- wherein
- t is selected from the integers from 1 to 12;
- and
- R10 is a member selected from H, a moiety comprising poly(ethylene glycol), and
- wherein
- Y is a nucleobase;
- and
- u is selected from the integers from 1 to 8.
The compound according to any preceding claim wherein when two Ra moieties are not joined to form said ring substituted with said at least one SO3H moiety, at least one Ra is SO3H.
The compound according to any preceding claim wherein when two Re moieties are not joined to form said aryl ring substituted with said at least one SO3H moiety, at least one Re is SO3H.
The compound according to any preceding claim wherein two Ra moieties are joined, providing said compound with the substructure:
The compound according to any preceding claim wherein two Re moieties are joined, providing said compound with the substructure:
The compound according to any preceding claim having the formula:
- wherein
- each Rf and each Rj are members independently selected from C(O)R29, OR32, NR32R33, CR32C(O)R33, NR32C(O)2R33, SO3H, and C(O)NR32R33, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycloalkyl
- wherein
- R29 is a member selected from OR31, and NH(CH2)t′OR30
- wherein
- R31 is a member selected from H and substituted or unsubstituted alkyl;
- t′ is selected from the integers from 1 to 12;
- R30 is a member selected from H, and
- wherein
- u′ is selected from the integers from 1 to 8;
- and
- Y is a nucleobase;
- R32 and R33 are members independently selected from H, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl and substituted or unsubstituted heterocycloalkyl.
The compound according to any preceding claim having the formula:
The compound according to any preceding claim having the formula:
The compound according to any preceding claim having the formula:
The compound according to any preceding claim having the formula:
The compound according to any preceding claim wherein Ra, Re and Rf and Rj are independently selected from:
The compound according to any preceding claim wherein Rm has a formula which is a member selected from:
- wherein
- Z is selected from N and C;
- R12 is a member selected from OH, and
- —NH(CH2)t′—O—R10′
- wherein
- t′ is selected from the integers from 1 to 12;
- R10′ is a member selected from H, a moiety comprising poly(ethylene glycol), and
- wherein
- u′ is selected from the integers from 1 to 8;
- and
- Y′ is a nucleobase.
The compound according to any preceding claim wherein Rm has the formula:
The compound according to any preceding claim wherein Rm is a member selected from:
- in which w is an integer selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or greater.
The compound according to any preceding claim wherein 1, 2, 3 or 4 of R2, R3, R4, R5 R6 is:
The compound according any preceding claim wherein both Rc and Rd are:
The compound according to any preceding claim in which two, three, four, five or six of Ra, Re, Rj, Rc, Rd, Rf, Rg, Rh, Ri, and Rm in any combination, include an independently selected R11 moiety which is:
The compound according to any preceding claim in which two, three, four, five or six of Ra, Re, Rj, Rc, Rd, Rf, Rg, Rh, Ri, and Rm in any combination, include an independently selected dye moiety or dye linker moiety.
The compound according to any preceding claim in which two, three, four, five or six of Ra, Re, Rj, Rc, Rd, Rf, Rg, Rh, Ri, and Rm in any combination, include an independently selected polyvalent moiety.
A method of monitoring an enzyme reaction, said method comprising:
- (a) forming a reaction mixture by contacting said enzyme with a fluorescent dye according to any preceding claim 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.
Dependencies are inferred from the claim text.