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Luciferins

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20120276564 patent thumbnailZoom

Luciferins


Novel luciferins, methods of making luciferins, and uses of the same are disclosed.

Browse recent University Of Massachusetts patents - Shrewsbury, MA, US
Inventor: Stephen C. Miller
USPTO Applicaton #: #20120276564 - Class: 435 8 (USPTO) - 11/01/12 - Class 435 
Chemistry: Molecular Biology And Microbiology > Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip >Involving Luciferase

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The Patent Description & Claims data below is from USPTO Patent Application 20120276564, Luciferins.

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CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No. 13/027,233, filed on Feb. 14, 2011, which is a divisional of U.S. patent application Ser. No. 12/040,812, filed on Feb. 29, 2008, and issued as U.S. Pat. No. 7,910,087 on Mar. 22, 2011, which claim the benefit of U.S. Patent Application Serial No. 60/904,731, filed on Mar. 2, 2007. The entire contents of the foregoing documents are hereby incorporated by reference.

TECHNICAL FIELD

This invention relates to luciferins, methods of making luciferins, and to uses of the same.

BACKGROUND

Firefly luciferase is widely used for bioluminescent imaging in mice. However, when combined with firefly luciferin, the emitted yellow-green light (560 nm) penetrates poorly through tissue due to absorption by hemoglobin and Rayleigh scattering. For optimal bioluminescent imaging, longer wavelengths (>650 nm) would be desirable.

Some mutants of firefly and click beetle luciferases maximally emit light as high as 615 nm (Anal. Biochem., 2005, 345(1):140), and railroad worm luciferase naturally emits light at 623 nm (Biochemistry, 1999, 38(26):8271). Most of these red-shifted luciferases, however, have not been well characterized, and for those examples that have, the bathochromatic shift in emission is concomitant with a substantial loss in light output, and often a significant loss of affinity for both luciferin and ATP.

Referring to FIG. 1, enzymatic oxidation of firefly luciferin (1) with firefly luciferase (LUC), and subsequent decarboxylation, generates oxyluciferin (described by (1′A) and (1′B)) in an electronically-excited state (FIG. 1). This molecule returns to the electronic ground state by emitting a photon with very high quantum yield (0.9) (see, e.g., Arch. Biochem. Biophys., 88 (1960) 136-141). The wavelength of the emitted photon is determined by the structure and electronic properties of the oxyluciferin chromophore within the luciferase binding pocket. At physiological pH, the emission wavelength of wild-type firefly luciferase is 560 nm. At low pH (˜6), this emission is red-shifted to as high as 617 nm, but with a decreased quantum yield.

SUMMARY

Generally, luciferins, e.g., N-substituted amino luciferins, such as N-alkylamino luciferins, or salts or derivatives thereof are disclosed, as well as methods of use thereof. These new luciferins are substrates for luciferases, i.e., they emit light when combined with a luciferase.

In one aspect, the invention features compounds of Structure (I), or salts or acid esters thereof.

In Structure (I), R1 and R2 are each independently H (provided that R1 and R2 are not both H), a first moiety including up to 12 carbon atoms, or a first moiety including a near infrared fluorophore. R3 is H, OH, a second moiety including up to 12 carbon atoms, or a second moiety that includes a near infrared fluorophore. R4 and R5 are each independently H, OH, or a moiety that includes up to 6 carbon atoms. R6 and R7 are each independently H, or a moiety including up to 8 carbon atoms. R1, R2, R3, or R5 may together with one or more of its immediate neighbors define one or more ring systems, each including up to 14 carbon atoms.

In some embodiments, the first and/or second moiety including up to 12 carbon atoms also includes one or more N, O, P, S, F, Cl, Br, or I.

The moieties that include up to 6 carbon atoms and/or the moieties that include up to 8 carbon atoms can also include one or more N, O, P, S, F, Cl, Br, or I.

The first and/or second moieties that include the near infrared fluorophore can also include a spacer including up to 24 carbon atoms, or a polymer fragment, e.g., a polymer fragment of a water-soluble polymer such as a polyethylene glycol or a copolymer thereof. The spacer can also include one or more N, O, P, S, F, Cl, Br, or I.

For example, the one or more defined ring systems can further include one or more N, O, P, S, F, Cl, Br, or I.

In particular embodiments, R3, R4, R5, R6, and R7 are each hydrogen, or an alkyl group, e.g., one having fewer than 6 carbon atoms, or having fewer than 4 carbon atoms.

In some embodiments, R1 and R2 together define a ring, the compounds being represented by Structure (II), which is shown below.

In some embodiments, R1 and R5 together define a ring, the compounds being represented by Structure (III). Such compounds are characterized as having hindered rotation about the Ar—N(R1R2) bond of the of Structure (III). Rotation can be further hindered, e.g., by having a carbon-carbon double bond in the ring. A double bond may also provide additional conjugation with the it-system of the chromophore.

In other embodiments, R2 and R3 together define a ring, the compounds being represented by Structure (IV). Such compounds are characterized as having hindered rotation about the Ar—N(R1R2) bond of the of Structure (IV).

In certain embodiments, R1 and R5 and R2 and R3 together define a ring, the compounds being represented by Structure (V). Such compounds are characterized as having extremely hindered rotation about the Ar—N(R1R2) bond of the of Structure (V). Rotation can be further hindered, e.g., by having one or more carbon-carbon double bonds in one or more rings.

In some instances, the one or more ring systems described above can define a 5, 6, and/or 7-membered rings. In some implementations, R1 and/or R2 and/or R3 comprise a near infrared fluorophore.

In specific implementations, the compounds of Structure (I) are represented by Structure (VII).

In such instances, R1 and/or R2 can be, e.g., alkyl groups, such as methyl groups.

The salts of any of the luciferins described herein can be, e.g., lithium, sodium, potassium, calcium, magnesium or ammonium salts (e.g., trialkylammonium salts). The esters can be, e.g., NHS esters, alkyl esters (e.g., C1-C3 alkyl esters), phenyl esters, benzyl esters or adenosine monophosphate (AMP) esters.

In another aspect, the invention features N-alkyl luciferins, or salts or acid ester thereof. For example, the N-alkyl luciferin can be a mono-alkyl luciferin or a di-N-alkyl luciferin.

In a specific embodiment, the N-alkyl luciferin has Structure (la), which is shown below.

In some embodiments, when R1 is not H, R2 is a substrate for a protease (e.g., an amino acid residue or polypeptide). In some embodiments, the carboxyl group is covalently bound to a protecting group. See, e.g., U.S. Pat. No. 5,035,999 and U.S. Pat. No. 7,148,030.



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stats Patent Info
Application #
US 20120276564 A1
Publish Date
11/01/2012
Document #
File Date
10/25/2014
USPTO Class
Other USPTO Classes
International Class
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