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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.

In another aspect, the invention features methods of generating light that include providing a luciferase having a binding pocket sized for any luciferin described herein (or an equivalent thereof, e.g., a salt or ester thereof); and combining the luciferin with the luciferase. The luciferase can be a wild-type luciferase, such as a firefly, click beetle, or railroad worm luciferase, or a mutated luciferase.

In another aspect, the invention features methods of imaging living cells or animals, e.g., mammals, or humans, that include providing a cell expressing a luciferase, or an animal having at least one cell expressing a luciferase; administering to the cell or animal any one or more of the luciferins described herein (or an equivalent thereof, e.g., a salt or ester thereof); and detecting emission therefrom.

Aspects and/or embodiments of the invention can have any one of, or combinations of, any of the following advantages. Relative to oxyluciferin, the luciferins described herein emit red-shifted light when combined with a suitable luciferase. For example, the luciferins can emit light having a wavelength of greater than 590 nm, e.g., greater than 600 nm, 610 nm, 615 nm, 620, nm, 625 nm, 630 nm, 635 nm, 640 nm, 645 nm, 650 nm, 665 nm, or even greater than 675 nm. Emission from the luciferins can be insensitive to pH. While generally the novel luciferins emit light that is red-shifted relative to oxyluciferin, the precise wavelength emitted can be tuned by selection of the functional groups attached to the luciferin. It is expected that the luciferins described herein will be well tolerated by animals. The luciferins are likely to be more cell permeable than native luciferin, potentially allowing greater access to luciferase within the mouse. Furthermore, the novel luciferins are likely to have a higher affinity for luciferase, which would allow robust light output under conditions where the luciferin is not present at wild-type luciferin saturating concentrations. Modifications in the N-alkyl group(s) could also allow for modulation of wavelength and light output, leading to luciferin substrates with different rates and durations of light output, to best suit the imaging experiment. When luciferins are covalently bonded in a pocket of a luciferase, fluorescent proteins can be provided. In such instances, the chromophore can be protected from the chemical environment in which the fluorescent protein is placed.

“Luciferase” as used herein is an enzyme that operates on a luciferin to produce light, e.g., near-infrared or visible light. The luciferase can be wild-type, or it can be a mutated luciferase. An example of a luciferase is wild-type firefly luciferase. A number of suitable luciferases are known in the art (e.g., Branchini et al., Anal. Biochem. (2005) 345:140-148; Nakatsu et al., Nature (2006) 440:372-376; Viviani et al., Biochemistry (1999) 38:8271).

“Luciferin” as used herein is a material, such as a pure compound or mixture of compounds, that in the presence of a luciferase produces light. An example of a luciferin is firefly luciferin.

“A near infrared fluorophore” as used herein is one having a maximum emission of greater than 600 nm at physiological pH, e.g., 650 nm or above.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference herein in their entirety for all that they contain. In case of conflict, the present specification, including definitions, will control. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed inventions, or that any publication specifically or implicitly referenced is prior art. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 is a series of structures for firefly luciferin (1) and the corresponding oxyluciferin (represented by (1′A) and (1′B)), that results from enzymatic oxidative decarboxylation with firefly luciferase (LUC).

FIG. 2A is a series of generalized structures for some novel amino luciferins.

FIG. 2B is a series of structures for specific amino luciferins in which the nitrogen atom of the amino group is not a member of a ring.

FIGS. 2C and 2D are two series of structures for specific novel amino luciferins in which the nitrogen atom of the amino group is a member of one or more rings.

FIGS. 3A-3D are representations of synthetic strategies for making various novel amino luciferins.

FIG. 4A is a series of generalized structures for novel amino luciferins that include a NIR acceptor fluorophore at positions 1 and 3.

FIG. 4B is a series of generalized structures for novel amino luciferins, illustrating specific NIR fluorophores and spacers.

FIG. 4C is a generalized structure for some novel NIR fluorophore-substituted luciferins described herein.

FIG. 5 is a schematic representation of a specific novel amino luciferin (1c) having a mustard group reacting with a thiol group in the pocket of (LUC).

DETAILED DESCRIPTION

Generally, luciferins, or salts or derivatives thereof, e.g., acid esters thereof, are provided that are sized to fit a binding pocket of a luciferase, such as a wild-type or a mutated luciferase.

Luciferins

Generally, and by reference to FIG. 2A, compounds of Structure (I), or salts or acid esters thereof are provided.

In such compounds of 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.

The first moiety including up to 12 carbon atoms can also include, e.g., one or more of N, O, P, S, F, Cl, Br, or I. For example, N can be part of an amino group, an amide group or an imine group. For example, O can be part of hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group or an ether group. For example, S can be part of a thio-ester group, a thiol group or a thio-ether group. For example, P can be part of a phosphate group, a phosphonate group, a phosphine group, or a phosphoramide group.

For example, the first moiety including up to 12 carbon atoms can be or can include a hydrocarbon fragment, e.g., an alkyl group, an alkenyl group, an alkynyl or an aryl group, or a hydrocarbon fragment that is substituted with one or more of N, O, P, S, F, Cl, Br, or I.

The first moiety can also include a near infrared fluorophore, as described herein.

In compounds of Structure (I), R3 is H, OH, a second moiety including up to 12 carbon atoms, or a second moiety including a near infrared fluorophore.

The second moiety including up to 12 carbon atoms can be any of those moieties described above in reference to R1 and R2. The second moiety can also include a near infrared fluorophore, as described herein.

In compounds of Structure (I), R4 and R5 are each independently H; OH; or a moiety including up to 6 carbon atoms. The moiety including up to 6 carbon atoms can also include, e.g., one or more of N, O, P, S, F, Cl, Br, or I. For example, N can be part of an amino group, an amide group, or an imine group. For example, O can be part of hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group or a ether group. For example, S can be part of a thio-ester group, a thiol group or a thio-ether group. For example, P can be part of a phosphate group, a phosphonate group, a phosphine group, or a phosphoramide group. For example, the moiety including up to 6 carbon atoms can be or can include a hydrocarbon fragment, e.g., an alkyl group, an alkenyl group, an alkynyl or an aryl group, or a hydrocarbon fragment that is substituted with one or more of N, O, P, S, F, Cl, Br, or I.

In compounds of Structure (I), R6 and R7 are each independently H, or a moiety including up to 8 carbon atoms. The moiety including up to 8 carbon atoms can also include, e.g., one or more of N, O, P, S, F, Cl, Br, or I. For example, N can be part of an amino group, an amide group, or an imine group. For example, O can be part of hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group, or a ether group. For example, S can be part of a thio-ester group, a thiol group, or a thio-ether group. For example, P can be part of a phosphate group, a phosphonate group, a phosphine group, or a phosphoramide group. For example, the moiety including up to 8 carbon atoms can be or can include a hydrocarbon fragment, e.g., an alkyl group, an alkenyl group, an alkynyl or an aryl group, or a hydrocarbon fragment that is substituted with one or more of N, O, P, S, F, Cl, Br, or I.

Referring now also to FIG. 2B, compounds of Structure (I) in which the amino nitrogen is not a member of a ring are exemplified by Structures (1a), (1b), (1c), (1d), (1e) and (1f). Structure (1c) represents a reactive N-mustard, which can allow for the luciferin to be functionalized with a nucleophile-containing moiety, such as compound bearing an amino or a thiol group. As will be discussed below, such a mustard can allow for, e.g., the covalent bonding of the luciferin in a binding pocket, and can enable, e.g., the preparation of fluorescent proteins.

When the amino nitrogen is not a member of a ring, one or more of R1-R7 can include one or more chiral, or pro-chiral centers.

In compounds of Structure (I), 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. For example, the one or more rings can further include in a ring or substituted on the ring, e.g., one or more of N, O, P, S, F, Cl, Br, or I. For example, the balance of the 14 carbons atoms not in a ring can substitute a ring, e.g., in the form of hydrocarbon fragments, e.g., an alkyl group, an alkenyl group, an alkynyl or an aryl group, or a hydrocarbon fragment that is substituted with one or more of N, O, P, S, F, Cl, Br, or I. For example, N can be part of an amino group, an amide group or an imine group. For example, O can be part of hydroxyl group, a carboxylic acid group, an ester group, an anhydride group, an aldehyde group, a ketone group or a ether group. For example, S can be part of a thio-ester group, a thiol group or a thio-ether group.

In preferred embodiments, the one or more ring systems define one or more 5, 6, and/or 7-membered rings.



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stats Patent Info
Application #
US 20120276564 A1
Publish Date
11/01/2012
Document #
File Date
12/21/2014
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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