US Pat. No. 9,447,450

RED-SHIFTED LUCIFERINS AND METHODS OF USING SAME

PROMEGA CORPORATION, Mad...

1. A compound according to Formula (Ia):

wherein
X is CN or

each Y is independently halo, SO3H, C1-4 alkyl, substituted C1-4 alkyl, OR1 or NR1R2;

each R1 is H, C1-10 alkyl or substituted C1-10 alkyl;

each R2 is H, C1-10 alkyl or substituted C1-10 alkyl; or

R1 and R2 together form a 4 to 8 membered ring; and

n is 1 to 6.

US Pat. No. 9,139,836

IMIDAZO[1,2-A]PYRAZINE DERIVATIVES

PROMEGA CORPORATION, Mad...

1. A compound of formula (Ia) or formula (Ib):

wherein R2 is selected from the group consisting of


 or C2-5 straight chain alkyl;

R6 is selected from the group consisting of —H, —OH, —NH2, —OC(O)R or —OCH2OC(O)R;

R8 is selected from the group consisting of —CH2C6H5, —C(R3)(R4)H, H or lower cycloakyl;

wherein R3 and R4 are both H or both C1-2 alkyl;

W is —NH2, halo, —NHC(O)R, —CO2R;

X is —S—, —O— or —NR22—;

Y is —OR11;

Z is —CH— or —N—;
each R11 is independently —C(O)R? or —CH2OC(O)R?;

R22 is H, CH3 or CH2CH3;

each R is independently C1-7 straight-chain alkyl or C3-7 branched alkyl;

R? is C1-7 straight-chain alkyl or C3-7 branched alkyl;

and the dashed bonds indicate the presence of a ring, which may be saturated or unsaturated.

US Pat. No. 9,416,353

COVALENT TETHERING OF FUNCTIONAL GROUPS TO PROTEINS AND SUBSTRATES THEREFOR

PROMEGA CORPORATION, Mad...

1. A fusion polypeptide of a protein of interest and a mutant haloalkane dehalogenase having at least 85% sequence identity
to the wild type dehalogenase polypeptide of SEQ ID NO:82 and comprising one to three amino acid substitutions at positions
106, 130, and 272 relative to the wild-type dehalogenase.
US Pat. No. 9,290,745

LUCIFERASE BIOSENSOR

PROMEGA CORPORATION, Mad...

1. An isolated polynucleotide encoding a modified beetle luciferase comprising an internal insertion of a heterologous amino
acid sequence which interacts with a molecule of interest, wherein the activity of the modified beetle luciferase is altered
after the molecule of interest interacts with the insertion sequence relative to the activity before the interaction, wherein
the internal insertion is at a residue or region in a beetle luciferase sequence which is tolerant to modification, the isolated
polynucleotide further comprising a polynucleotide encoding a tag of at least one amino acid fused to the N-terminus, C-terminus,
or both, of the modified beetle luciferase.
US Pat. No. 9,551,705

COMPOSITIONS AND METHODS FOR CAPTURE OF CELLULAR TARGETS OF BIOACTIVE AGENTS

Promega Corporation, Mad...

1. A method of capturing a cellular target comprising the steps of:
(a) administering to a cell, a conjugate comprising a small molecule bioactive agent tethered by a carbamate linker to a chloroalkane
capture ligand, the cell comprising a cellular target of the small molecule bioactive agent, under conditions such that the
cellular target non-covalently binds the small molecule bioactive agent;

(b) lysing the cell to produce a cell lysate;
(c) contacting the cell lysate with a solid surface displaying a capture protein comprising a dehalogenase modified to covalently
bind to the chloroalkane capture ligand under conditions in which the capture protein forms a covalent bond with the chloroalkane
capture ligand; and

(d) separating the solid surface with the bound cellular target from the cell lysate.
US Pat. No. 9,487,814

STABILIZED FORMULATION FOR LUMINESCENT DETECTION OF LUCIFERASE AND NUCLEOSIDE PHOSPHATES

PROMEGA CORPORATION, Mad...

1. A luciferase reaction composition for detecting luciferase activity, the composition comprising, a mixture of a concentration
of l-luciferin and a concentration of D-luciferin, wherein the concentration of L-luciferin exceeds the concentration of D-luciferin,
and the ratio of L-luciferin to D-luciferin is less than about 2:1, wherein the composition retains at least about 50% of
the original activity in a luciferase reaction after being stored for at least about 5 days at a temperature from about 0°
C. to about 35° C., and the concentration of L-luciferin in the mixture is at least about 100 ?M.

US Pat. No. 9,139,868

SILICON AND GERMANIUM DYES FOR USE IN GENETIC IDENTITY

PROMEGA CORPORATION, Mad...

1. A method of detecting the presence of a nucleic acid polymer in a sample comprising:
a) contacting a sample suspected of containing a nucleic acid polymer with a composition comprising a conjugate comprising
a compound of Formula (I) and an oligonucleotide; and

b) detecting the presence or amount of the compound in the sample;
wherein the compound of formula (I) is a component in an ET cassette; and
wherein the compound of formula (I) is as shown below:
wherein
Z is Si(R11)(R12) or Ge(R11)(R12);

each R11 and R12 are independently selected from C1-10 linear, branched or cyclic alkyl, C1-10 alkyl interrupted with one or more heteroatoms, C6-10 aryl, heteroaryl or R11 and R12 may together form a ring;

each X is independently selected from OR2 or N(R3)(R4);

each R1 is independently selected from H, C1-4 alkyl, sulfonate or halo;

R2 is H, C1-10 alkyl, L-R or L-CS;

R3 and R4 are independently selected from H, C1-4 alkyl, C1-4 alkyl interrupted with one or more heteroatoms, C6-10 aryl, peptidyl, heteroaryl, L-R or L-CS;

R6-10 are independently H, halo, alkoxy, amino, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;

L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups
or single, double, triple or aromatic carbon-carbon bond;

R is a reactive group; or
CS is a conjugated substance;

one or more of R3 and R4 or R3 and R1 or R4 and R1 may together form a ring;

and
one or more of R6-10 may together form a ring.

US Pat. No. 9,732,373

LUCIFERASE SEQUENCES UTILIZING INFRARED-EMITTING SUBSTRATES TO PRODUCE ENHANCED LUMINESCENCE

Promega Corporation, Mad...

1. An isolated polynucleotide encoding a click beetle red luciferase (CBR) variant polypeptide having at least 80% amino acid
sequence identity to SEQ ID NO: 1 and comprising an amino acid substitution at a position corresponding to positions 389,
444, and 251 of SEQ ID NO: 1, or an amino acid substitution at a position corresponding to positions 334 and 351 of SEQ ID
NO: 1, wherein the variant CBR polypeptide has at least one of enhanced luminescence, altered light emission wavelength, altered
substrate specificity, or a combination thereof, as compared to a CBR polypeptide of SEQ ID NO: 1.

US Pat. No. 9,459,249

PROTEIN LABELING WITH CYANOBENZOTHIAZOLE CONJUGATES

PROMEGA CORPORATION, Mad...

1. A compound of formula I:
wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate,
alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or (C1-C10)alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (?O), nitro, thiol, or halo;

each R1 is independently H, F, Cl, Br, I, CN, (C1-C6)alkyl, (C1-C6)alkoxy, or (C1-C6)alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl,
cycloalkyl, aryl, alkyl sulfonate, or CO2M wherein M is H, an organic cation, or an inorganic cation;

n is 0, 1, or 2;
Y is a linking group comprising (C1-C16)alkyl optionally substituted with one or more halo, oxo (?O), (C1-C6)alkyl, or (C1-C6)alkoxy, and optionally interrupted with one or more N(R1), O, S, or —N—C(?O)— groups; or Y is optionally absent when X is N3; and

X is a fluorescent dye, biotin, HisTag, chitin, a solid support, N3, H, or OH, provided that when X is H or OH, the compound of formula I comprises a radioactive moiety or an isotopic variant
of any atom other than the carbon or nitrogen atom of the 2-nitrile moiety.

US Pat. No. 9,458,499

NUCLEIC ACID BINDING DYES AND USES THEREFOR

PROMEGA CORPORATION, Mad...

1. A method determining cell viability, comprising:
a) contacting a sample comprising a cell with a compound according to Formula 1:

wherein
R1, R2 and R3 are each H

Y is S or O;
W taken together with the atoms to which it is attached is a 6-membered heterocyclic ring;
n is 0;
Q is Q2:

wherein
Y is —CR13?CR14—; m is 1 and p is 0;

R6 is unsubstituted (C1-C8)alkyl, unsubstituted aryl, or unsubstituted (C1-C8)alkaryl;

R9, R11, and R12 are each independently H, amino, or halo;

R10 is H, methoxy, amino or halo;

one of R13 and R14 is H and the other is NR15R16;

R15 is (C1-C6)alkyl or (C1-C6)alkyl-NR17R18 or C3H7—N+(CH3)3 wherein R17 and R18 are each independently H or (C1-C6)alkyl; and

R16 is (C1-C6)alkyl-NR17R18 or C3H7—N+(CH3)3, wherein R17 and R18 are each independently H or (C1-C6)alkyl;

wherein at least one of R15 and R16 is C3H7—N+(CH3)3;

or a salt thereof;
b) detecting a fluorescent signal in the sample; and
c) correlating the amount of fluorescence in the sample with the viability of the cell in the sample;
wherein an increase in the fluorescent signal correlates to a loss in cell membrane integrity.

US Pat. No. 9,206,474

NUCLEIC ACID BINDING DYES AND USES THEREFOR

PROMEGA CORPORATION, Mad...

1. A method to quantify the amount of a nucleic acid in a sample subjected to nucleic acid amplification, comprising:
a) contacting the sample with a compound according to Formula 1:

wherein
R1, R2 and R3 are each H;

Y is S or O;
W taken together with the atoms to which it is attached is a membered heterocyclic ring;
n is 0;Q is Q2:
wherein
Y is —CR13?CR14—; m is 1 and p is 0;

R6 is unsubstituted (C1-C8)alkyl, unsubstituted aryl, or unsubstituted (C1-C8)alkaryl;

R9, R11, and R12 are each independently H, amino, or halo;

R10 is H, methoxy, amino or halo;

one of R13 and R14 is H and the other is NR15R16;

R15 is (C1-C6)alkyl or (C1-C6)alkyl-NR17R18 or C3H7—N+(CH3)3, wherein R17 and R18 are each independently H or (C1-C6)alkyl; and

R16 is (C1-C6)alkyl-NR17R18 or C3H7—N+(CH3)3, wherein R17 and R18 are each independently H or (C1-C6)alkyl;

or a salt thereof;
b) detecting fluorescence in the amplification reaction; and
c) correlating the amount of fluorescence in the amplification reaction with the amount of nucleic acid in the reaction.

US Pat. No. 9,487,520

COELENTERAZINE DERIVATIVES AND METHODS OF USING SAME

PROMEGA CORPORATION, Mad...

1. A compound of formula (II):

wherein R2 is —(CH2)n-T or C1-5 alkyl;

R6 is selected from the group consisting of —H, —OH, —NH2, —OC(O)R or —OCH2OC(O)R;

R8 is selected from the group consisting of


 H or lower cycloalkyl;
R11 is selected from the group consisting of a peptide containing from 2 to 35 amino acids, an amino acid, —O—RA, —OC(O)O—RA, —N(RB)2, or —NHC(O)ORA;

wherein R3 and R4 are both H or both C1-2 alkyl;

RA is C1-4 alkyl, substituted C1-4 alkyl, —CH2—Rc or —CH2—V—Rc;

each RB is independently —H or —RA;

Rc is aryl, heteroaryl, substituted aryl or substituted heteroaryl;

L? is a direct bond or a linker, wherein the linker is selected from the group consisting of
 wherein each RE is independently H, halogen or NO2, each RD is independently H or Me, and each X is independently NH, NMe, O or S;
n is 0 to 3;
each R is independently a C1-7 alkyl;

T is aryl, heteroaryl, substituted aryl, substituted heteroaryl or cycloalkyl; and
V is —S— or —O—.
US Pat. No. 9,290,794

MUTANT PROTEASE BIOSENSORS WITH ENHANCED DETECTION CHARACTERISTICS

PROMEGA CORPORATION, Mad...

1. A polynucleotide encoding a circularly-permuted thermostable luciferase comprising a peptide linker linking the original
N- and C-termini of the non-permuted thermostable luciferase, wherein the peptide linker comprises a granzyme B recognition
site, wherein the circularly-permuted thermostable luciferase has increased luminescence following cleavage by granzyme B
when compared to an uncleaved circularly-permuted thermostable luciferase, wherein the granzyme B recognition site is selected
from the group consisting of GRIEADSE (SEQ ID NO:80), KSVGPDFG (SEQ ID NO:81), and GIETDSG (SEQ ID NO:82), and wherein the
non-permuted thermostable luciferase has at least 90% identity to the amino acid sequence of SEQ ID NO:2 or at least 90% identity
to the amino acid sequence of SEQ ID NO:4.
US Pat. No. 9,593,316

POLYNUCLEOTIDES ENCODING MUTANT HYDROLASE PROTEINS WITH ENHANCED KINETICS AND FUNCTIONAL EXPRESSION

Promega Corporation, Mad...

1. An isolated polynucleotide comprising a first sequence encoding a mutant dehalogenase that has dehalogenase activity and
forms a covalent bond with a dehalogenase substrate, said mutant dehalogenase having at least 85% amino acid sequence identity
to the polypeptide of SEQ ID NO: 1, wherein said mutant dehalogenase further comprises (a) substitutions corresponding to
substitutions S58T, D78G, A155T, A172T, K175M, C176G, A224E, P291S, and A292T in SEQ ID NO: 1, and (b) substitutions at positions
corresponding to positions 272 and/or 106 of SEQ ID NO: 1.

US Pat. No. 9,540,402

COVALENT TETHERING OF FUNCTIONAL GROUPS TO PROTEINS

PROMEGA CORPORATION, Mad...

1. A composition comprising a dehalogenase substrate of the formula R-linker-A-X, wherein R is an affinity molecule that forms
a covalent bond with an acceptor molecule, A-X is a substrate for said dehalogenase, X is a halogen, and the linker is a group
that separates R and A; wherein R, linker, A, and X are covalently linked.
US Pat. No. 9,371,531

VECTORS FOR DIRECTIONAL CLONING

PROMEGA CORPORATION, Mad...

1. A vector encoding a fusion polypeptide prepared by ligating
a DNA fragment comprising a first open reading frame that begins with start codon but does not end with an in-frame stop codon,
a 5? end generated after cleavage of a first recognition site with a first restriction enzyme which generates an end compatible
with an end generated after cleavage of a SgfI recognition site by SgfI, and a 3? end generated after cleavage of a second
recognition site with a second restriction enzyme that generates a blunt end, and

a DNA segment comprising a second open reading frame which ends with a stop codon, a 3? end generated after cleavage of a
third recognition site with a third restriction enzyme which generates an end compatible with an end generated after cleavage
of a SgfI recognition site by SgfI, a 5? end generated after cleavage of a fourth recognition site for a fourth restriction
enzyme which generates a blunt end, and a promoter located at the 3? end of the DNA segment and between the stop codon and
the third recognition site,

wherein the first recognition site is cleaved by SgfI, the third recognition site is cleaved by SgfI or both the first and
third recognition sites are cleaved by SgfI, wherein ligation of the blunt ends yields a third open reading frame comprising
the first and second open reading frames which encodes the fusion polypeptide, wherein the promoter is 5? to and operably
linked to the first open reading frame following the ligation of the first recognition site and the third recognition site,
wherein a SgfI site is located between the promoter and the first open reading frame following the ligation of the first recognition
site and the third recognition site, and wherein the first and third recognition sites are cleaved by SgfI and yield i) an
exchange site comprising GCGATCGCnATGG (SEQ ID NO: 92), wherein n is C, A, T or G, or ii) an exchange site comprising GCGATCGCnATG
(SEQ ID NO: 93), wherein n is C, A, T or G.

US Pat. No. 9,273,343

COMPOUNDS AND METHODS FOR ASSAYING REDOX STATE OF METABOLICALLY ACTIVE CELLS AND METHODS FOR MEASURING NAD(P)/NAD(P)H

PROMEGA CORPORATION, Mad...

1. A compound is of Formula (IV):

wherein R2 is selected from —CH2-aryl or —CH2-heteroaryl;

R5 is selected from —CH2-aryl or —CH2-heteroaryl;

R7 is selected from aryl or heteroaryl;

R14 is H, C1-4 alkyl, C1-4 alkoxyl, C2-4 hydroxylalkyl, C2-4alkoxyl, C2-4 carboxylic acid, or C2-4 amide;

R9 and R10 are independently selected from C1-4 alkyl;

R11, R12 and R13 are independently selected from H, C1-4 alkyl, C1-4 alkoxyl, bromo, chloro or amino, or R11 and R12 can form a fused phenyl ring;

X is O, NH or a direct bond;
L is a direct bond or —C6(R16)4CH2— or —(CH2)mC(R17)2(CH2)n—Y—C(O)—;

R16 is independently H, halogen, CH3, OCH3, or NO2;

R17 is independently H, C1-4 alkyl; or both R17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR15; and

R15 is H, C1-4 alkyl, C2-4 hydroxylalkyl, C2-4alkoxyl, C2-4 carboxylic acid, or C2-4 amide.

US Pat. No. 9,702,824

PH SENSORS

PROMEGA CORPORATION, Mad...

1. A pH sensor agent comprising:
wherein R1 and R2 are independently alkyl sulfonic acids, and wherein R3-R6 are independently: H, F, Cl, Br, I, OH, an alkoxide
group, an alkyl group, a heteroalkyl group, an aryl group, CO2H, SO3H, L-CO2H, L-SO3H, L-W, or L-CS; wherein L is a linear, branched, and/or cyclic covalent linkage comprising 0-16 non-hydrogen atoms and comprising
any suitable combination of: ester, acid, sulphonic acid, sulfamide, amine, amide, alcohol, ether, thioether, halide, single
bonds, double bonds, triple bonds, and aromatic bonds; wherein W is a reactive group; wherein X is: O, CR17, MR17; wherein R17 is: H, F, Cl, Br, I, OH, an alkoxide group, an alkyl group, an aryl group, CO2H, SO3H, L-CO2H, L-SO3H, L-Y, or L-CS; and wherein M is selected from C, N, O, Si, P, S, Ge, As, Se, Sn, Sb, Te, Pb, Bi, and Po.

US Pat. No. 9,689,868

PROTEIN LABELING WITH CYANOBENZOTHIAZOLE CONJUGATES

PROMEGA CORPORATION, Mad...

1. A method for immobilizing a protein of interest, comprising:
a) contacting a sample comprising an N-terminal cysteine labeled protein of interest with a cyanobenzothiazole derivative
of Formula I


wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate,
alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or C1-C10 alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (?O), nitro, thiol, or halo;

each R1 is independently H, F, Cl, Br, I, CN, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl,
cycloalkyl, aryl, alkyl sulfonate, or CO2M wherein M is H, an organic cation, or an inorganic cation;

n is 0, 1, or 2;
Y is a linking group comprising C1-C16 alkylene optionally substituted with one or more halo, oxo (?O), CrO6alkyl, or C1-C6alkoxy, and optionally interrupted with one or more N(R1), O, S, or —NH—C(?O)— groups; and

X is a solid support;
and
b) immobilizing the protein of interest.

US Pat. No. 9,248,201

MUTANT PROTEASE BIOSENSORS WITH ENHANCED DETECTION CHARACTERISTICS

PROMEGA CORPORATION, Mad...

1. A method of detecting the presence or activity of a target molecule in a sample comprising:
a) contacting the sample with
i) a polynucleotide encoding a biosensor polypeptide comprising a modified circularly-permuted thermostable luciferase of
a parental thermostable luciferase that is modified by a substitution of an amino acid at a position that corresponds to position
507 of SEQ ID NO:2, wherein the encoded amino acid sequence of the parental thermostable luciferase has at least 90% identity
to the amino acid sequence of SEQ ID NO:2, wherein the circularly-permuted thermostable luciferase has a C-terminal portion
of the thermostable luciferase joined to an N-terminal portion of the thermostable luciferase by a peptide linker comprising
a sensor region capable of interacting with a target molecule in a cell, and wherein the modified circularly-permuted thermostable
luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to at least one of:

A) the parental circularly-permuted thermostable luciferase in the presence of the target molecule, or
B) the modified circularly-permuted thermostable luciferase in the absence of the target molecule; and
ii) a substrate for the modified thermostable luciferase; and
b) detecting luminescence in the sample.
US Pat. No. 9,096,889

COMPOSITIONS AND METHODS FOR MONITORING TRANSMEMBRANE TRAFFICKING

PROMEGA CORPORATION, Mad...

1. A method of detecting endocytosis in a living cell comprising:
(a) tethering a reporter element to a cell-surface analyte to create a fusion element, wherein a detectable signal from said
reporter element is altered upon endocytosis of said fusion element; and

(b) detecting said detectable signal from said reporter element outside the living cell;
(c) allowing endocytosis of said fusion element;
(d) detecting an altered detectable signal from said reporter element within the living cell; and
(e) comparing said detectable signal to said altered detectable signal, thereby detecting endocytosis.
US Pat. No. 9,879,306

LUCIFERASE BIOSENSORS FOR CAMP

PROMEGA CORPORATION, Mad...

1. A method to detect one or more modulators of a G protein coupled receptor, comprising:
a) contacting a sample with one or more test agents and reagents for a luminescence reaction, wherein the sample comprises
a cell expressing a G-protein coupled receptor and a polynucleotide, wherein the G-protein coupled receptor is a Gs or Gi
coupled receptor; and

b) detecting or determining luminescence in the sample, wherein an increase or decrease in luminescence detected in the sample
compared to a control sample indicates that the test agent is a modulator of the G protein coupled receptor,

wherein the polynucleotide encodes a modified luciferase comprising luciferase sequences and a heterologous amino acid sequence
for a mutant RII?B cAMP binding site inserted at a site or region in the luciferase that is tolerant to modification, wherein
the luciferase sequences in the modified luciferase are circularly permuted relative to a wild-type luciferase and are firefly
luciferase sequences, wherein the permutation is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70
to 88, residue 102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to
313, residue 353 to 408, residue 485 to 495, or residue 535 to 546 of SEQ ID NO: 106, 118, or 120, wherein the mutant RII?B
cAMP binding site has at least 80% amino acid sequence identity to the cAMP binding site of residue 266-414 of SEQ ID NO:
4, wherein the cAMP binding site is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue
102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353
to 408, residue 485 to 495, or residue 535 to 546 of SEQ ID NO: 106, 118, or 120, wherein binding of cAMP to the mutant RII?B
cAMP binding site produces a response from the modified luciferase, wherein the mutant RII?B cAMP binding site has one or
more substitutions that enhance the luminescence signal of the modified luciferase, enhance the response of the modified luciferase
to a change in the amount of cAMP in a cell, reduce affinity of wild-type cAMP binding, or a combination thereof, relative
to a corresponding luciferase with a RII?B cAMP binding site that lacks the one or more substitutions, wherein one or more
of the substitutions are at a position corresponding to residue 266, 282, 284, 286, 296, 316, 333, 338, 382, 389, 404, or
407 of RII?B having SEQ ID NO: 4.

US Pat. No. 9,738,921

COMPOSITIONS AND METHODS FOR MONITORING TRANSMEMBRANE TRAFFICKING

Promega Corporation, Mad...

1. A method of detecting endocytosis comprising:
a) expressing a fusion protein in a cell comprising a cell membrane, wherein said fusion protein comprises a cell-surface
receptor protein and a reporter protein, and said expressing results in the co-expression of the cell surface receptor protein
and the reporter protein as a single fusion protein in the cell, wherein the reporter protein is displayed extracellularly
when the cell-surface receptor is incorporated into the cell membrane of the cell;

b) delivering a reporter substrate to the cell, wherein an association between the reporter protein and reporter substrate
produces a detectable signal when the association occurs extracellularly and an altered signal when the association occurs
intracellularly; and

c) monitoring said detectable signal and said altered signal, wherein a transition from said detectable signal to said altered
signal indicates endocytosis of the fusion protein.

US Pat. No. 9,704,000

MOBILE RFID CONTAINER AND DISTRIBUTION METHOD

PROMEGA CORPORATION, Mad...

1. A method for distributing a plurality of products, each product having an RFID tag, the method comprising:
a. providing a mobile RFID container, the container comprising:
a housing comprising a main body and a door, the main body and the door cooperating to define an interior compartment that
is configured to carry the plurality of products, the door being movable relative to the main body between an open position
to provide access to the interior compartment and a closed position to prevent access to the interior compartment;

a sensor configured to detect opening and closing of the door;
an RFID detector positioned in the housing, the RFID detector being configured to conduct an RFID scan of the interior compartment
and collect scan data concerning the products contained within the compartment;

a controller coupled to the sensor and the at least RFID detector, the controller being configured to trigger an RFID scan
via the RFID detector in response to the sensor detecting that the door has closed and to transmit scan data collected during
the RFID scan to a remote device;

b. placing a plurality of products in the interior compartment of the mobile RFID container;
c. transporting the mobile RFID container to an end user;
d. conducting an RFID scan of the interior compartment of the mobile RFID container in response to sensing the opening and
closing of the door following the transporting step; and

e. transmitting scan data collected via the RFID scan from the mobile RFID container to a remote device;
f. generating invoicing and/or credit data based on the scan data;
wherein the method further comprising transporting the mobile RFID container from the end user to a distribution center and
restocking the container with products; and

wherein the step of restocking is based on scan data transmitted by the mobile RFID container prior to the RFID container
reaching the distribution center.

US Pat. No. 9,469,857

VECTORS FOR DIRECTIONAL CLONING

PROMEGA CORPORATION, Mad...

1. A method of inducing expression of a DNA sequence of interest in a host cell, comprising contacting a recombinant host
cell which is deficient in rhamnose catabolism, and has a recombinant DNA molecule comprising a rhamnose-inducible promoter
operably linked to an open reading frame for a heterologous RNA polymerase, with rhamnose and an expression vector comprising
a promoter for the heterologous RNA polymerase operably linked to a DNA sequence of interest, so as to induce expression of
the DNA sequence of interest, wherein the host cell is an E. coli cell.

US Pat. No. 9,339,561

MUTANT PROTEASE BIOSENSORS WITH ENHANCED DETECTION CHARACTERISTICS

PROMEGA CORPORATION, Mad...

1. A polynucleotide encoding a biosensor polypeptide comprising a modified circularly permuted thermostable luciferase and
a peptide linker linking the C-terminal portion of the circularly-permuted thermostable luciferase to the N-terminal portion
of the same circularly-permuted thermostable luciferase, wherein the modified circularly-permuted thermostable luciferase
is modified relative to an unmodified circularly-permuted thermostable luciferase by one or more amino acid substitutions
or insertions, the peptide linker comprising a sensor region capable of interacting with a target molecule in a cell, wherein
the modified circularly-permuted thermostable luciferase has an enhanced response after interaction of the biosensor with
the target molecule relative to at least one of:
i) the unmodified circularly-permuted thermostable luciferase in the presence of the target molecule, or
ii) the modified circularly-permuted thermostable luciferase in the absence of the target molecule,
wherein the modified circularly-permuted thermostable luciferase comprises a substitution of an amino acid at a position that
corresponds to position 507 of SEQ ID NO:2 and wherein the encoded amino acid sequence of the modified thermostable luciferase
has at least 90% amino acid sequence identity with the amino acid sequence of SEQ ID NO:2.

US Pat. No. 9,056,885

CARBOXY X RHODAMINE ANALOGS

PROMEGA CORPORATION, Mad...

1. A compound according to formula (IIIa), (IIIb) or (IIIc):
wherein
R11 is independently H or C1-4 alkyl, L-R or L-CS;

L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen
atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups
or single, double, triple or aromatic carbon-carbon bond;

R is a reactive group;
CS is a conjugated substance selected from the group consisting of solid supports, resin particles, beads, assay plates, proteins,
nucleotides, polynucleotides, enzyme substrates, antibodies, nanobodies, polypeptides, polypeptide-based toxins, amino acids,
lipids, carbohydrates, haptens, drugs, ion-complexing agents, microparticles, polymers, cells, viruses, fluorophores, chloroalkanes,
and cyanobenzothiazoles;

R2 and R16 can be independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;

R3 and R4 are H, alkyl, L-R, L-CS, L-CO2H, L-SO3H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;

alternatively, R2 and R3 and independently R4 and R16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;

R5, R12, R13, R14 and R15 are independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;

R20, R21, R22 and R23 are independently H or C1-6 alkyl or one or more of R20 and R21, R21 and R22, R22 and R23, together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;

R11 and R12 may together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;

R6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;

X is CHR23, O, S or NR30; and

R30 is H, C1-4 alkyl or —C(O)C1-4 alkyl.

US Pat. No. 9,873,866

MUTANT DEHALOGENASE PROTEINS

Promega Corporation, Mad...

1. An isolated polypeptide comprising a mutant dehalogenase that has dehalogenase activity and forms a covalent bond with
a dehalogenase substrate, said mutant dehalogenase having at least 90% amino acid sequence identity to the polypeptide of
SEQ ID NO: 1, wherein said mutant dehalogenase comprises substitutions corresponding to substitutions at:
(a) one or more positions corresponding to positions in SEQ ID NO:1 selected from the group consisting of positions 106 and
272; and

(b) one or more positions corresponding to positions in SEQ ID NO:1 selected from the group consisting of positions 58, 78,
155, 167, 172, 224, and 291.

US Pat. No. 9,869,670

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. A system comprising
a. a cell expressing a first fusion comprising:
i. a target protein of interest, and
ii. a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO:
2; and

b. a second fusion comprising:
i. an antibody binding moiety, and
ii. a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ
ID NO: 440, wherein a bioluminescent signal produced by the system in the presence of a substrate is substantially increased
when the polypeptide contacts the peptide when compared to a bioluminescent signal produced in the presence of the substrate
but in the absence of contact between the polypeptide and peptide.

US Pat. No. 9,790,537

QUINONE-MASKED PROBES AS LABELING REAGENTS FOR CELL UPTAKE MEASUREMENTS

Promega Corporation, Mad...

1. A compound of formula (I), or a salt thereof,

wherein
A is a reporter moiety;
R14 is H, alkyl, hydroxyalkyl, alkoxy, carboxyalkyl, or amidoalkyl;

R9 and R10 are independently selected from alkyl;

R11, R12 and R13 are independently selected from alkyl;

X is O;
L is —(CH2)mC(R17)2(CH2)n—Y—C(O)—;

R17 is independently H, alkyl or both R17 together can form an alkyl ring having from 3-7 carbons;

m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR15;

R5 is -alkyl-amide, wherein the -alkyl-amide is -alkyl-CON(R32)(R33) or -alkyl-(CO)—NR34—(CRaRb)p—NR35(CO)-T, wherein

R32 and R33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heterocycle,
alkene, polyol, alkenylpolylol, a peptide, a drug, a derivative of a drug, a biologically active moiety, and a dye;

R34 and R35 are each independently selected from hydrogen, and alkyl;

Ra and Rb are each independently selected from hydrogen, alkyl, and carboxy;

p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide,
polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative
of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;

or
R15 is polyalkoxyalkyl, wherein the polyalkoxyalkyl is —(C2-C6-alkoxy)x-alkyl-CON(R32)(R33) or —(C2-C6-alkoxy)x-alkyl-(CO)—NR34-(CRaRb)p—NR35(CO)-T;

x is an integer selected from 1 to 20;
R32 and R33 are each independently selected from hydrogen, alkyl, carboxy, aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl,
heterocycle, hydroxyalkyl, protein, polypeptide, polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar,
carbohydrate, enzyme substrate, drug, derivative of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or
a combination thereof;

R34 and R35 are each independently selected from hydrogen, and alkyl;

Ra and Rb are each independently selected from hydrogen, alkyl, and carboxy;

p is 0 to 6; and
T is selected from aryl, arylalkyl, cycloalkylalkyl, heteroaryl, heteroarylalkyl, heterocycle, hydroxyalkyl, protein, polypeptide,
polypeptide-based toxin, amino acid, nucleotide, polynucleotide, lipid, sugar, carbohydrate, enzyme substrate, drug, derivative
of a drug, polymer-linked nanoparticle, antibody, detergent, and a dye, or a combination thereof;

or
R15 is


wherein
R40 is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, and cycloalkyl,
wherein said aryl, heteroaryl, heterocyclyl, and cycloalkyl are unsubstituted or substituted with one or more suitable substituents;
and

x is an integer selected from 1 to 20.
US Pat. No. 9,631,225

LUCIFERASE-BASED ASSAYS

PROMEGA CORPORATION, Mad...

1. A method of determining the effect of a compound on an ATP-generating enzyme activity in a sample not containing living
cells, comprising:
(a) contacting a compound, ADP, a sample containing or suspected of containing an ATP-generating enzyme, and a substrate for
the ATP-generating enzyme, so as to produce a first reaction mixture;

(b) contacting the first reaction mixture with a reagent composition comprising luciferase, luciferin, and a tolerance enhancement
agent so as to produce a second reaction mixture, wherein the tolerance enhancement agent is present in an amount effective
to substantially protect the activity of the luciferase from interference from the compound, thereby reducing the likelihood
of a false positive resulting from the interfering effect of the compound;

(c) detecting luminescence in the second reaction mixture; and
(d) determining the effect of the compound, if any, on the ATP-generating enzyme activity by comparing the luminescence of
the second reaction mixture to a control reaction mixture.

US Pat. No. 9,359,635

PERMUTED AND NONPERMUTED LUCIFERASE BIOSENSORS

PROMEGA CORPORATION, Mad...

1. An isolated polynucleotide comprising a nucleic acid sequence encoding a circularly permuted luciferase, the circularly
permuted luciferase comprising a cyclic nucleotide binding site, which circularly permuted luciferase being permuted at a
site or in a region which in a corresponding nonpermuted luciferase is tolerant to modification, and wherein luciferase activity
of the circularly permuted luciferase is detectable, wherein the cyclic nucleotide binding site comprises at least a portion
of a protein selected from the group consisting of exchange protein directly activated by cAMP (Epac), cyclic nucleotide gated
ion channels, neuropathy target esterase, PKA regulatory type II? subunit, PKA regulatory type I? subunit, catabolite activating
protein, cGMP dependent protein kinase (GK), GAF regulatory region in phosphodiesterase, adenyl cyclases and FnlA, wherein
the permutation is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue 102 to 126, residue
139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353 to 408, residue 485
to 495, or residue 535 to 546 of a firefly luciferase of SEQ ID NO: 210, a region corresponding to residue 15 to 30, residue
112 to 122, residue 352 to 362, residue 371 to 384, residue 393 to 414, or residue 485 to 495 of a click beetle luciferase
of SEQ ID NO: 3, or a region corresponding to residue 2 to 12, residue 26 to 47, residue 64 to 74, residue 85 to 116, residue
147 to 157, residue 223 to 234, or residue 301 to 311 of a Renilla luciferase of SEQ ID NO: 308.
US Pat. No. 9,045,730

LUCIFERASE BIOSENSORS FOR CAMP

PROMEGA CORPORATION, Mad...

1. A polynucleotide encoding a modified luciferase comprising luciferase sequences and a heterologous amino acid sequence
for a mutant RII?B cAMP binding site inserted at a site or region in the luciferase that is tolerant to modification, wherein
the luciferase sequences in the modified luciferase are circularly permuted relative to a wild-type luciferase and are firefly
luciferase sequences, wherein the permutation is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70
to 88, residue 102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to
313, residue 353 to 408, residue 485 to 495, or residue 535 to 546 of SEQ ID NO: 106, 118, or 120, wherein the mutant RII?B
cAMP binding site has at least 80% amino acid sequence identity to the cAMP binding site of residue 266-414 of SEQ ID NO:
4, wherein the cAMP binding site is in a region corresponding to residue 2 to 12, residue 32 to 53, residue 70 to 88, residue
102 to 126, residue 139 to 165, residue 183 to 203, residue 220 to 247, residue 262 to 273, residue 303 to 313, residue 353
to 408, residue 485 to 495, or residue 535 to 546 of SEQ ID NO: 106, 118, or 120, wherein binding of cAMP to the mutant RII?B
cAMP binding site produces a response from the modified luciferase, wherein the mutant RII?B cAMP binding site has one or
more substitutions that enhance the luminescence signal of the modified luciferase, enhance the response of the modified luciferase
to a change in the amount of cAMP in a cell, reduce affinity of wild-type cAMP binding, or a combination thereof, relative
to a corresponding luciferase with a RII?B cAMP binding site that lacks the one or more substitutions, wherein one or more
of the substitutions are at a position corresponding to residue 266, 282, 284, 286, 296, 316, 333, 338, 382, 389, 404, or
407 of RII?B having SEQ ID NO: 4.

US Pat. No. 9,938,564

SUBSTITUTED IMIDAZO[1,2-A]PYRAZINES FOR USE IN BIOLUMINOGENIC METHODS

PROMEGA CORPORATION, Mad...

1. A compound selected from the group consisting of:
US Pat. No. 9,840,730

OPLOPHORUS-DERIVED LUCIFERASES, NOVEL COELENTERAZINE SUBSTRATES, AND METHODS OF USE

PROMEGA CORPORATION, Mad...

1. An isolated polynucleotide encoding a polypeptide comprising an amino acid sequence having at least 95% sequence identity
with the amino acid sequence of SEQ ID NO: 1 with the exception of one or more amino acid substitutions at a position corresponding
to positions 18, 27, 43, 38, 109, 15, 19, 21, 22, 36, 40, 56, 58, 70, 93, 95, 102, 110, 112, 119, 127, 128, 149, 152, 155,
158, 159, 1, 6, 10, 14, 16, 24, 25, 31, 32, 39, 42, 46, 47, 48, 49, 50, 55, 59, 60, 66, 67, 69, 71, 74, 86, 87, 94, 96, 97,
98, 100, 106, 111, 113, 117, 125, 126, 129, 130, 136, 142, 145, 146, 147, 148, 150, 154, 163, or 168 of the amino acid sequence
SEQ ID NO: 1, and optionally one or more amino acid substitutions at a position corresponding to positions 4, 33, 54, 68,
72, 75, 90, and 166 of the amino acid sequence of SEQ ID NO: 1; and wherein said polypeptide has enhanced luminescence activity
relative to the amino acid sequence of SEQ ID NO: 3.
US Pat. No. 9,816,127

OXIDIZED GLUTATHIONE ASSAY

PROMEGA CORPORATION, Mad...

1. A method for detecting GSSG comprising:
a. contacting a sample with a sulfhydryl alkylating agent;
b. contacting the sample with an excess of a reducing agent, glutathione-S-transferase, and a substrate for glutathione-S-transferase
which is converted to a signal-generating compound in the presence of GSH and glutathione-S-transferase, wherein the reducing
agent inactivates the sulfhydryl alkylating agent and reduces any GSSG in the sample to GSH; and

c. detecting the signal generated from step (b), thereby confirming the presence of GSSG in the sample, wherein neither the
inactivated sulfhydryl alkylating agent nor the reducing agent is removed from the sample.

US Pat. No. 10,000,792

METHODS FOR CYCLIC NUCLEOTIDE DETERMINATION

PROMEGA CORPORATION, Mad...

1. A method for determining the presence or amount of endogenous cyclic nucleotide in a sample comprising a cell lysate, the method comprising:a) contacting a sample comprising a cell lysate which may contain endogenous cyclic nucleotide, with (I) a cyclic nucleotide-dependent protein kinase capable of being activated by the endogenous cyclic nucleotide, wherein said protein kinase is Protein Kinase A and (II) a detection system, the detection system comprising:
i) a substrate capable of being phosphorylated by the cyclic nucleotide-dependent protein kinase;
ii) a luciferase enzyme capable of utilizing ATP to generate a bioluminescent signal; and
(iii) ATP; and
b) detecting or measuring the bioluminescent signal thereby determining the presence or amount of the endogenous cyclic nucleotide present in the sample, wherein the cell lysate comprises the cellular debris and fluid that is released from a cell when the cell membrane is broken apart or lysed,
wherein the endogenous cyclic nucleotide is cAMP.

US Pat. No. 9,977,928

RADIO FREQUENCY IDENTIFICATION TRAY SYSTEMS AND METHODS

PROMEGA CORPORATION, Mad...

1. A radio frequency identification (RFID) tray system, comprising.an RFID tray including an embedded agent, an RFID reader, and a communication component operatively coupled to the embedded agent; and
a cloud network operatively coupled to the embedded agent through the communication component in the RFID tray,
wherein the RFID tray is configured to:
perform, with at least the RFID reader, an RFID scan of one or more RFID-tagged items placed on or in the RFID tray; and
send, by the embedded agent via the communication component, tag information to an inventory management application via the cloud network, wherein the inventory management application tracks and manages an inventory of the RFID-tagged items.

US Pat. No. 9,979,095

RADIO FREQUENCY IDENTIFICATION TECHNIQUES IN AN ULTRA-LOW TEMPERATURE ENVIRONMENT

PROMEGA CORPORATION, Mad...

12. A system comprising:at least one RFID antenna assembly for use in a cold-storage apparatus, wherein:
the RFID antenna assembly is configured to scan a plurality of RFID tags on a corresponding plurality of items; and
the RFID antenna assembly includes a plurality of individual RFID antenna elements, wherein the plurality of individual RFID antenna elements are formed on two printed circuit boards,
wherein each of the plurality of individual RFID antenna elements comprises at least one patterned conductive layer, at least one patch, and at least one dielectric region interposed between the patterned conductive layer and the patch, and
wherein the dielectric region comprises a low dielectric constant foam, a first layer of glass-reinforced epoxy laminate interposed between the patterned conductive layer and the low dielectric constant foam, and a second layer of glass-reinforced epoxy laminate interposed between the patch and the low dielectric constant foam; and
at least one controller in communication with the RFID antenna assembly, wherein the controller is configured to:
individually and separately control each of the plurality of individual RFID antenna elements; and
activate only one of the plurality of individual RFID antenna elements at a time.
US Pat. No. 9,969,991

INTERNAL PROTEIN TAGS

Promega Corporation, Mad...

1. A polypeptide comprising:an N-terminal segment of a protein of interest, a C-terminal segment of a protein of interest, and an internal tag between the N-terminal and C-terminal segments, wherein the internal tag comprises an amino acid sequence having less than 100% and greater than 45% sequence identity with SEQ ID NO: 2 inserted within a protein of interest; wherein a detectable bioluminescent signal is produced in the presence of a coelenterazine substrate when the internal tag contacts a polypeptide consisting of SEQ ID NO: 440 and wherein the polypeptide is not a naturally occurring polypeptide.

US Pat. No. 9,932,365

PROTEIN LABELING WITH CYANOBENZOTHIAZOLE CONJUGATES

PROMEGA CORPORATION, Mad...

1. A method for labeling a peptide or protein, comprising:contacting a sample comprising an N-terminal cysteine labeled peptide or protein with a cyanobenzothiazole derivative of Formula I

wherein
Z is H, F, Cl, Br, I, CN, amino, alkylamino, dialkylamino, alkyl ester, carboxy, carboxylic acid salt, alkyl amide, phosphate, alkyl phosphonate, sulfate, alkyl sulfonate, nitro, or C1-C10 alkyl optionally unsaturated and optionally substituted with amino, hydroxy, oxo (?O), nitro, thiol, or halo;
each R1 is independently H, F, Cl, Br, I, CN, C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 alkylthio, wherein each alkyl, alkoxy, or alkylthio is optionally substituted with F, Cl, Br, I, amino, alkenyl, alkynyl, cycloalkyl, aryl, alkyl sulfonate, or CO2M wherein M is H, an organic cation, or an inorganic cation;
n is 0, 1, or 2;
Y is absent or is a linking group comprising C1-C16 alkyl optionally substituted with one or more halo, oxo (?O), C1-C6alkyl, or C1-C6alkoxy, and optionally interrupted with one or more N(R1), O, S, or —NH—C(?O)— groups; and
X is a fluorescent dye;
so as to yield a fluorescently-labeled peptide or protein.

US Pat. No. 9,868,977

RED-SHIFTED LUCIFERINS AND METHODS OF USING SAME

PROMEGA CORPORATION, Mad...

1. A compound according to Formula (XI):

wherein
X is CN or

Y is OR; and
R is

US Pat. No. 9,816,054

CLEAVABLE SURFACTANTS

PROMEGA CORPORATION, Mad...

1. A compound of formula I:

wherein:
Q is (C1-C6)alkyl, (C6-C10)aryl, (C5-C10)heteroaryl, or (C6-C10)aryl-NH(C1-C6)alkyl;

Y is —O—C(?Z)—X—;
Z is O or S;
A is aryl, aryl(C1-C6)alkyl, heteroaryl, or a direct bond;

X is O, NH, or S;
V is C;
M is H, an alkali metal, or tetra(C1-C20)alkylammonium;

L is —X—C(?Z)—X— or a direct bond;
R1 is (C6-C16)aryl, or (C5-C10)heteroaryl;

R2 and R3 are each independently H or (C10-C12)alkyl;

wherein any alkyl, alkenyl, aryl, or heteroaryl, carbocyclic ring, or heterocyclic ring, is optionally substituted with one
or more (e.g., 1, 2, 3, 4, or 5) (C1-C20)alkyl, (C2-C20)alkenyl, (C2-C10)alkynyl, (C3-C10)cycloalkyl, (C1-C20)alkoxy, (C1-C20)alkylcarbonyl, (C1-C20)alkylcarboxyl, halo, hydroxyl, —CO2Rx, —SO2Rx, —SO3Rx, nitro, amino, N(Rx)2, mercapto, (C1-C20)alkylthio, (C6-C16)aryl, (C6-C30)arylthio, trifluoromethyl, ?O, heteroaryl, or heterocycle groups; provided that Q is not substituted with CO2H; and

each Rx is independently H, (C1C6)alkyl, (C6-C16)aryl, or (C1C6)alkyl-(C6-C16)aryl; or a salt thereof; and

wherein R2 and R3 are not both H.

US Pat. No. 10,000,750

METHOD OF ISOLATING NUCLEIC ACID

PROMEGA CORPORATION, Mad...

1. A method for isolating nucleic acids from a sample, the method comprising:(a) contacting the sample with a boron carbide composition under conditions sufficient to form a boron carbide-nucleic acid complex;
(b) separating the complex from the sample; and
(c) eluting the nucleic acids from the complex, thereby isolating the nucleic acids.

US Pat. No. 10,000,500

COELENTERAZINE ANALOGUES

PROMEGA CORPORATION, Mad...

1. A compound of formula (I)
or a tautomer, or a salt thereof, wherein
R5 is hydrogen, halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl;
R6 is alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl; or
R5 and R6 together with the atoms to which they are attached, form a 5- or 6-membered partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteratoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ?O, ?S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl; and
R8 is alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, or cycloalkenyl;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, bicyclic aryl, tricyclic aryl, heteroaryl, bicyclic heteroaryl, tricyclic heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ?O, ?S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl;
provided that the following compounds are excluded from formula (I):
8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; and
8-benzyl-2-(furan-2-ylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one.

US Pat. No. 9,927,430

PRO-SUBSTRATES FOR LIVE CELL APPLICATIONS

PROMEGA CORPORATION, Mad...

1. A compound of formula (I), or a salt thereof,
wherein,
RA is selected from the group consisting of —(CR1R2)m—N(R3)C(O)R4, —(CR5R6)m—SO3R7, and —(CR10R11)m—CO2R12, wherein
m at each occurrence is independently 2, 3, or 4;
R1, R2, R3, R5, R6, R7, R10, R11, and R12 at each occurrence are independently hydrogen or C1-C4 alkyl;
R4 is C1-C4 alkyl, C1-C4 alkoxy, carboxy-C1-C4 alkoxy-C1-C4 alkyl, phenyl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halogen, C1-C4 alkoxy, and C1-C4 haloakyl,

 wherein
R41, R42, and R44 at each occurrence are each independently carboxy-C1-C4 alkyl;
R43 and R45 are each independently selected from the group consisting of hydrogen, —C(O)alkyl, and

x and y are each independently an integer selected from 1 to 20;
RB is —(CR46R47)t—NR48R49, wherein
t is 1, 2, 3, 4, 5, 6, 7, or 8
R46 and R47 at each occurrence are independently hydrogen or C1-C4 alkyl;
R48 and R49 at each occurrence are independently hydrogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, alkylaminoalkyl, di(alkyl)aminoalkyl, cyanoalkyl, —(CR50R51)z—OC(O)R52; or R48 and R49 together with the nitrogen atom to which they are attached form a heterocyclyl, wherein said heterocyclyl is unsubstituted or substituted with 1, 2, 3, 4, 5 or 6 substituents independently selected from the group consisting of C1-C4 alkyl, C1-C4 alkenyl, C1-C4 alkynyl, —NO2, —CN, halogen, oxo, —OR96, —OC(O)R97, and —C(O)R116, wherein
R50 and R51 at each occurrence are independently hydrogen or C1-C4 alkyl;
R52, R96, R97, and R116 at each occurrence are independently hydrogen, C1-C6 alkyl, or aryl;
z is 1, 2, 3, or 4;
RC is —(CH)0-3-T or C1-5alkyl; wherein T is aryl, heteroaryl, or cycloalkyl, wherein said aryl, heteroaryl, and cycloalkyl are each independently unsubstituted or substituted with 1, 2, or 3 substituents independently selected from the group consisting of halogen, alkyl, haloalkyl, hydroxyalkyl, alkoxyalkyl, haloalkoxyalkyl, aminoalkyl, alkylaminoalkyl, di(alkylaminoalkyl), cyanoalkyl, alkoxy, haloalkoxy, cyano, hydroxy, amino, alkylamino, and di(alkyl)amino;
RD is hydrogen, C1-C4 alkyl, benzyl, or C3-C6 cycloalkyl;
RE is hydrogen, hydroxy, alkoxy, amino, alkylamino, di(alkyl)amino, —OC(O)alkyl, or —OCH2OC(O)alkyl;
wherein the dash bonds together indicate the presence of an optional 6-membered ring in the compound of formula (I), wherein the optional ring is saturated or unsaturated; and
wherein the optional 6-membered ring is absent.
US Pat. No. 9,777,311

SYNTHETIC OPLOPHORUS LUCIFERASES WITH ENHANCED LIGHT OUTPUT

PROMEGA CORPORATION, Mad...

1. A method comprising:
(a) expressing a luminescent polypeptide in a cell, wherein the luminescent polypeptide comprises a modified luciferase with
at least 70% sequence identity with SEQ NO: 1 and having one or more amino acid substitutions at positions 4, 11, 43, 44,
54, 72, 75, 90, 115, 124, 138, and 166 relative to SEQ ID NO: 1, wherein the modified luciferase has at least one of enhanced
luminescence, enhanced signal stability; and enhanced protein stability relative to a luciferase of SEQ ID NO; 1;

(b) exposing said cell to a substrate for said modified luciferase; and
(c) detecting luminescence.
US Pat. No. 9,757,478

MUTANT PROTEASE BIOSENSORS WITH ENHANCED DETECTION CHARACTERISTICS

Promega Corporation, Mad...

1. A method of detecting the presence or activity of a target molecule in a sample comprising:
a) contacting the sample with
i) a polynucleotide encoding a biosensor polypeptide comprising a modified circularly-permuted thermostable luciferase of
a parental thermostable luciferase that is modified by a substitution of an amino acid at a position that corresponds to position
503 of SEQ ID NO:2, wherein the encoded amino acid sequence of the parental thermostable luciferase has at least 90% identity
to the amino acid sequence of SEQ ID NO:2, wherein the circularly-permuted thermostable luciferase has a C-terminal portion
of the thermostable luciferase joined to an N-terminal portion of the thermostable luciferase by a peptide linker comprising
a sensor region capable of interacting with a target molecule in a cell, and wherein the modified circularly permuted thermostable
luciferase has an enhanced response after interaction of the biosensor with the target molecule relative to at least one of:

A) the parental circularly-permuted thermostable luciferase in the presence of the target molecule, or
B) the modified circularly-permuted thermostable luciferase in the absence of the target molecule; and
ii) a substrate for the modified thermostable luciferase; and
b) detecting luminescence in the sample.

US Pat. No. 9,574,223

LUMINESCENCE-BASED METHODS AND PROBES FOR MEASURING CYTOCHROME P450 ACTIVITY

PROMEGA CORPORATION, Mad...

1. A method for determining the effect of a compound on cytochrome P450 enzyme activity in an animal comprising:
(a) administering a test compound to an animal;
(b) administering a luminogenic molecule to the animal, wherein the luminogenic molecule is a cytochrome P450 substrate and
a pro-substrate of bioluminescent enzyme;

(c) obtaining a biological sample from said animal;
(d) contacting the biological sample with a reaction mixture comprising a bioluminescent enzyme; and
(e) determining cytochrome P450 enzyme activity of said animal after exposure of said animal to the test compound by measuring
and comparing luminescence from said biological sample taken from a second biological sample taken from a second animal not
exposed to said test compound;

and wherein the luminogenic molecule is a compound of formula:
wherein
R1 represents hydrogen, hydroxyl, amino, C1-20 alkoxy, substituted C1-20 alkoxy, C2-20 alkenyloxy, substituted C2-20 alkenyloxy, halogenated C2-20 alkoxy, substituted halogenated C2-20 alkoxy, C3-20 alkynyloxy, substituted C3-20 alkynyloxy, C3-20 cycloalkoxy, substituted C3-20 cycloalkoxy, C3-20 cycloalkylamino, substituted C3-20 cycloalkylamino, C1-20 alkylamino, substituted C1-20alkylamino, di C1-20alkylamino, substituted diC1-20alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl, C1-20alkylamino, substituted C2-20alkenyl C1-20alkylamino C3-20alkynylamino, substituted C3-20alkynylamino, di C3-20alkynylamino, substituted di alkylamino, C3-20alkynyl C2-20alkenylamino, or substituted C3-20alkynyl C2-20 alkenylamino;

R2 and R3 independently represents C or N;

R4 and R5 independently represents S, O, NR8, wherein R8 represents hydrogen or C1-20 alkyl, CR9R10 wherein R9 and R10 independently represent H, C1-20 alkyl, or fluorine;

R6 represents CH2OH; COR11 wherein R11 represents H, OH, C1-20 alkoxide, C2-20 alkenyl, or NR12R13 wherein R12 and R13 are independently H, or C1-20 alkyl; or —OM+ wherein M+ is an alkali metal or a pharmaceutically acceptable salt; and

R7 represent H, C1-6 alkyl, C1-20 alkenyl, halogen, or C1-6 alkoxide,
with the proviso that R1 is not OH or NH2, R7 is not H, R6 is not COR11 R11 is not OH, R3 and R2 are not both carbon, and R4 and R5 are not both S at the same time (luciferin and aminoluciferin).

US Pat. No. 9,924,073

SUBSTITUTED IMIDAZO[1,2-A]PYRAZINES AS LUCIFERASE SUBSTRATES AND PREPARATION METHOD THEREOF

PROMEGA CORPORATION, Mad...

1. A method for preparing a compound of formula (I):

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl; and

q is 1;
the method comprising:
(i) reacting a compound of formula A:

wherein:
R5 is methyl;

with a compound of formula v:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

in the presence of 1,1,3,3-tetramethylguanidine and a solvent, to provide a compound of formula vi:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl; and

(ii) reducing the compound of formula vi above with sodium borohydride, to provide a compound of formula (I); or
(i) reacting a compound of formula B:

wherein:
R5 is benzyl;

with a compound of formula v:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

in the presence of 1,1,3,3-tetramethylguanidine and a solvent, to provide a compound of formula vi:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl; and

(ii) reducing the compound of formula vi above with sodium borohydride, to provide a compound of formula (I); or
(i) reacting a compound of formula C:

wherein:
R5 is tert-butyl;

with a compound of formula v:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

in the presence of 1,1,3,3-tetramethylguanidine and a solvent, to provide a compound of formula vii:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

(ii) reacting the compound of formula vii above with trifluoroacetic acid, to provide a compound of formula ix:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl; and

(iii) reacting the compound of formula ix above with acetic anhydride followed by sodium borohydride, to provide a compound
of formula (I); or

(i) reacting a compound of formula C:

wherein:
R5 is tert-butyl;

with a compound of formula v:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

in the presence of 1,1,3,3-tetramethylguanidine and a solvent, to provide a compound of formula vii:

wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl;

(ii) hydrogenating the compound of formula vii above in the presence of Rh(PPh3)3Cl, to provide a compound of formula viii:


wherein:
R1 is alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl or phosphonate, wherein, at each
occurrence, the alkyl, alkenyl, alkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, heteroarylcarbonyl and phosphonate are
independently and optionally substituted with 1, 2, 3, 4, 5, 6 or 7 functional groups independently selected from the group
consisting of halogen, oxo, thioxo, cyano, carbamate, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl,
alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl,
heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, alkoxyalkyl, aryloxy, phenoxy, benzyloxy, amino, alkylamino,
acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arvlsulfonyl, aminosulfonyl, alkylsulfinyl,
—COOH, amido, silyl, silyloxy, alkylsulfanyl, arylsulfanyl, thiotriazolyl and acyl; and

(iii) reacting the compound of formula viii above with trifluoroacetic acid followed by carbonyldiimidazole, to provide a
compound of formula (I).

US Pat. No. 9,797,889

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. A polypeptide comprising an amino acid sequence having greater than 40% but not greater than 95% sequence identity with
SEQ ID NO: 440, wherein a bioluminescent signal produced in the presence of a furimazine substrate is substantially increased
when the polypeptide contacts a peptide consisting of SEQ ID NO: 2 when compared to a bioluminescent signal produced by the
polypeptide and furimazine substrate alone, and wherein the amino acid sequence is not a naturally occurring protein or a
fragment thereof.
US Pat. No. 9,797,890

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. A peptide comprising an amino acid sequence having greater than 45% but not greater than 90% sequence identity with SEQ
ID NO: 2, wherein a bioluminescent signal produced in the presence of a furimazine substrate is substantially increased when
the peptide contacts a polypeptide consisting of SEQ ID NO: 440 when compared to a bioluminescent signal produced by the peptide
and furimazine substrate alone, and wherein the amino acid sequence is not a naturally occurring protein or a fragment thereof.
US Pat. No. 10,184,936

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. An isolated composition comprising:(a) a polypeptide comprising SEQ ID NO: 1270;
(b) a first peptide comprising SEQ ID NO: 2587; and
(c) a second peptide comprising SEQ ID NO: 374.
US Pat. No. 10,067,149

RECOGNITION OF CELLULAR TARGET BINDING BY A BIOACTIVE AGENT USING INTRACELLULAR BIOLUMINESCENCE RESONANCE ENERGY TRANSFER

Promega Corporation, Mad...

1. A system comprising:(a) a bioactive small molecule conjugated to a small molecule fluorophore;
(b) a cell that expresses a protein fusion of: (i) a protein of interest and (ii) a luciferase, wherein the bioactive small molecule is capable of binding non-covalently to the protein of interest upon interaction therewith, wherein the emission spectrum of the luciferase overlaps with the excitation spectrum of the small molecule fluorophore; and
(c) a substrate for the luciferase.
US Pat. No. 10,024,862

RECOGNITION OF CELLULAR TARGET BINDING BY A BIOACTIVE AGENT USING INTRACELLULAR BIOLUMINESCENCE RESONANCE ENERGY TRANSFER

Promega Corporation, Mad...

1. A method comprising:(a) expressing a fusion of a cellular protein target and a luciferase within a cell, said luciferase having a first emission spectrum with a first peak emission, and
(b) contacting the cell extracellularly with a conjugate of a bioactive agent and a small molecule fluorophore, said small molecule fluorophore having an excitation spectrum that overlaps said first emission spectrum, said small molecule fluorophore having second emission spectrum with a second peak emission, wherein the conjugate is cell permeable, and wherein the bioactive agent is capable of binding non-covalently to the cellular protein target;
(c) contacting said luciferase with a substrate for said luciferase and
(d) detecting fluorescence within said second emission spectrum, wherein the
intensity of said fluorescence within said second emission spectrum correlates with non-covalent binding between said bioactive agent and said cellular protein target.

US Pat. No. 9,951,372

COMPOUNDS AND METHODS FOR ASSAYING REDOX STATE OF METABOLICALLY ACTIVE CELLS AND METHODS FOR MEASURING NAD(P)/NAD(P)H

PROMEGA CORPORATION, Mad...

1. A method for detecting cellular metabolites in a sample comprising,a. contacting the sample with a compound of formula (II), (III), or (IV), a dehydrogenase amplification enzyme system, NAD or NADP, a diaphorase that utilizes NADH or NADPH as a co-factor and the compound of formula (II), (III), or (IV) as a diaphorase substrate, and a luciferase reaction mixture; and
b. detecting bioluminescence;
wherein the compound of formula (II) is

wherein R1 is H, C1-4 alkyl, C1-4 hydroxylalkyl, C3-7 cyclic ring, aryl, benzyl or substituted benzyl ring, heterocycle, heteroaryl or —(CH2)n?—P(Ph)3; R4 and R5 are independently selected from H, halogen, methyl, and trifluoromethyl; and n? is an integer from 2-7;
the compound of formula (III) is

wherein R4 and R5 are independently selected from H, halogen, methyl, and trifluoromethyl;
the compound of formula (IV) is

wherein R2 is —CH2-aryl or —CH2-heteroaryl; R5 is —CH2-aryl or —CH2-heteroaryl; and R7 is aryl or heteroaryl;
wherein R14 is H, C1-4 alkyl, C1-4 alkoxyl, C2-4 hydroxylalkyl, C2-4alkoxyl, C2-4 carboxylic acid, or C2-4 amide;
R9 and R10 are independently selected from C1-4 alkyl;
R11, R12 and R13 are independently selected from H, C1-4 alkyl, C1-4 alkoxyl, bromo, chloro or amino, or R11 and R12 can form a fused phenyl ring;
X is O, NH or a direct bond;
L is a direct bond or —C6(R16)4CH2— or —(CH2)mC(R17)2(CH2)n—Y—C(O)—;
wherein at least one of X or L is not a direct bond when the compound has formula (II);
R16 is independently H, halogen, CH3, OCH3, or NO2;
R17 is independently H, C1-4 alkyl or both R17 together can form an alkyl ring having from 3-7 carbons;
m is an integer from 0-2;
n is an integer from 0-2;
Y is O or NR15; and
R15 is H, C1-4 alkyl, C2-4 hydroxylalkyl, C2-4alkoxyl, C2-4 carboxylic acid, or C2-4 amide.
US Pat. No. 9,951,373

OPLOPHORUS-DERIVED LUCIFERASES, NOVEL COELENTERAZINE SUBSTRATES, AND METHODS OF USE

Promega Corporation, Mad...

1. An isolated polynucleotide encoding a polypeptide having at least 80% sequence identity to SEQ ID NO: 1 and comprising one or more amino acid substitutions relative to SEQ ID NO: 3, wherein at least one amino acid substitution is at a position corresponding to position 27 of SEQ ID NO: 1, and wherein the polypeptide has enhanced luminescence relative to SEQ ID NO: 3.

US Pat. No. 9,953,197

PROCESSES FOR DISTRIBUTION AND USE OF A MOBILE RFID CONTAINER

PROMEGA CORPORATION, Mad...

1. A method for distributing a plurality of RFID-tagged items carried in a mobile RFID container, the method comprising:providing the mobile RFID container comprising:
an RFID detector configured to conduct RFID scans and generate scan data concerning the RFID-tagged items carried in the mobile RFID container; and
location determining circuitry;
conducting one or more RFID scans, by the RFID detector to generate scan data concerning the plurality of RFID-tagged items in the mobile RFID container;
generating, using the location determining circuitry, location data concerning a location of the mobile RFID container; and
processing the scan data and the location data to determine the location of the mobile RFID container at times when the plurality of RFID-tagged items in the mobile RFID container have changed,
wherein the one or more RFID scans are conducted in response to one or more of the following: the location of the mobile RFID container relative to a defined geographical location, determining that the plurality of RFID-tagged items in the mobile RFID container have been accessed, and receiving a request from a remote device.
US Pat. No. 10,288,605

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. A system comprising:(a) a first fusion comprising:
(i) a target molecule, and
(ii) a peptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 2; and
(b) a second fusion comprising:
(i) an antibody binding moiety to the molecule of interest, and
(ii) a polypeptide comprising an amino acid sequence having less than 100% and greater than 40% sequence identity with SEQ ID NO: 440:
wherein a bioluminescent signal produced by the system in the presence of a substrate is increased when the polypeptide contacts the peptide when compared to a bioluminescent signal produced in the presence of the substrate by the polypeptide or peptide alone.
US Pat. No. 10,233,485

SYNTHETIC OPLOPHORUS LUCIFERASES WITH ENHANCED LIGHT OUTPUT

PROMEGA CORPORATION, Mad...

1. A polypeptide comprising an amino acid sequence with at least 70% sequence identity with SEQ ID NO:1 and comprising at least one amino acid substitution relative to SEQ ID NO: 1 at a position corresponding to position 2, 4, 11, 20, 23, 28, 33, 34, 44, 45, 51, 54, 68, 72, 75, 76, 77, 89, 90, 92, 99, 104, 115, 124, 135, 138, 139, 143, 144, 166, 167, or 169 of SEQ ID NO: 1, wherein the polypeptide is a luciferase that is capable of utilizing coelenterazine as a substrate to generate luminescence, and wherein the polypeptide has at least one of enhanced luminescence, enhanced signal stability, and enhanced protein stability relative to a luciferase comprising the amino acid sequence of SEQ ID NO: 1, wherein the modified luciferase polypeptide has luciferase activity.

US Pat. No. 10,139,400

CARBOXY X RHODAMINE ANALOGS

Promega Corporation, Mad...

7. A method of labeling a biomolecule comprising:a) contacting a sample suspected of containing the biomolecule with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc) so as to yield a mixture:
whereinR11 is independently H or C1-4 alkyl, or L-Cs;L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups or single, double, triple or aromatic carbon-carbon bond;Cs is a conjugated substance selected from the group consisting of solid supports, resin particles, beads, assay plates, proteins, nucleotides, polynucleotides, enzyme substrates, nanobodies, polypeptides, amino acids, lipids, carbohydrates, haptens, drugs, ion-complexing agents, microparticles, polymers, cells, viruses, fluorophores, chloroalkanes, and cyanobenzothiazoles;R2 and R16 can be independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, or L-Cs;R3 and R4 are H, alkyl, L-Cs, L-CO2H, L-SO3H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;alternatively, R2 and R3 and independently R4 and R16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;R5, R12, R13, R14 and R15 are independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, or L-Cs;R20, R21, R22 and R23 are independently H or C1-6 alkyl or one or more of R20 and R21, R21 and R22, R22 and R23, together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;alternatively R11 and R12 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;R6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, or L-Cs;X is CHR23, O, S or NR30; andR30 is H, C1-4 alkyl or —C(O)C1-4 alkyl;b) detecting the presence or amount of the dye conjugate, thereby detecting the presence or amount of the labeled biomolecule in the mixture.
US Pat. No. 10,107,800

ACTIVATION OF BIOLUMINESCENCE BY STRUCTURAL COMPLEMENTATION

Promega Corporation, Mad...

1. A kit comprising:(a) a peptide comprising a peptide amino acid sequence having greater than 45% but not greater than 90% sequence identity with SEQ ID NO: 2, wherein the peptide amino acid sequence is not a naturally occurring protein or a fragment thereof, and wherein the peptide is conjugated to a first binding moiety;
(b) a polypeptide comprising a polypeptide amino acid sequence having greater than 40% but not greater than 95% sequence identity with SEQ ID NO: 440, wherein the polypeptide amino acid sequence is not a naturally occurring protein or a fragment thereof, and wherein the polypeptide is conjugated to a second binding moiety;
wherein a bioluminescent signal produced in the presence of a furimazine substrate is substantially increased when the peptide contacts the polypeptide when compared to a bioluminescent signal produced by either: (i) the peptide and furimazine substrate alone and (ii) the polypeptide and furimazine substrate alone.

US Pat. No. 10,308,975

SUBSTITUTED IMIDAZO[1,2-A]PYRAZINES AS LUCIFERASE SUBSTRATES

Promega Corporation, Mad...

1. A compound of formula (I):
or a tautomer or pharmaceutically acceptable salt thereof,
wherein:
R1 is phenyl; and
q is 1;
wherein said phenyl is substituted with 1, 2, 3, 4, or 5 functional groups independently selected from the group consisting of cyano, carbamate, nitro, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, heterocycloalkyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, allyloxy, aryloxy, benzyloxy, amino, aminoalkyl, sulfonylamino, sulfinylamino, sulfonyl, sulfinyl, —COOH, amide, carbamate, silyl, silyloxy, sulfanyl, and acyl;
provided that R1 is not
wherein Rc is OH, OC(O)C1-C7-alkyl, or OCH2OC(O)C1-C7-alkyl.
US Pat. No. 10,247,711

QUALITY CONTROL REAGENTS AND METHODS

Promega Corporation, Mad...

1. A peptide mixture comprising three or more distinct-mass versions of each of two or more distinct-sequence peptides, wherein two or more of the distinct-sequence peptides are selected from SEQ ID NOS: 1-6, and wherein two or more of the distinct-mass versions of each distinct-sequence peptide comprises one or more amino acids with above natural-abundance levels of one or more heavy isotopes.
US Pat. No. 10,246,690

MUTANT HYDROLASE PROTEINS WITH ENHANCED KINETICS AND FUNCTIONAL EXPRESSION

Promega Corporation, Mad...

1. A polypeptide comprising a mutant dehalogenase that is capable of catalyzing hydrolytic dehalogenation of an alkylhalide substrate and forming a covalent ester intermediate with the substrate, said mutant dehalogenase having at least 85% sequence identity with the polypeptide of SEQ ID NO: 1; wherein said mutant dehalogenase comprises substitutions at:(a) one or more positions corresponding to positions in SEQ ID NO: 1 selected from the group consisting of positions 106 and 272; and
(b) positions corresponding to positions 58, 78, 155, 167, 172, 224, and 291 of SEQ ID NO: 1.

US Pat. No. 10,215,751

CARBOXY X RHODAMINE ANALOGS

Promega Corporation, Mad...

1. A method to detect an interaction between a biomolecule and a protein of interest comprising:a) contacting a sample suspected of containing the protein of interest with a composition comprising a dye conjugate according to formula (IIIa), (IIIb) or (IIIc); and
b) detecting the presence or amount of the dye conjugate, thereby detecting the interaction between the protein of interest and the biomolecule;
whereinR11 is independently H, C1-4 alkyl, L-R or L-CS;
L is a covalent linkage that is linear or branched, cyclic or heterocyclic saturated or unsaturated, having 1-16 non hydrogen atoms such that the linkage contains any combination of ester, acid, amine, amide, alcohol, ether, thioether or halide groups or single, double, triple or aromatic carbon-carbon bond;
R is a reactive group;
CS is the biomolecule, the biomolecule being selected from the group consisting of proteins, nucleotides, polynucleotides, enzyme substrates, nanobodies, polypeptides, amino acids, lipids, carbohydrates, haptens, drugs, cells, and viruses;
R2 and R16 are independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;
R3 and R4 are H, alkyl, L-R, L-CS, L-CO2H, L-SO3H or together form a carbocyclic, aryl, heteroaryl, or heterocyclic ring;
alternatively, R2 and R3 and independently R4 and R16 together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R5, R12, R13, R14 and R15 are independently H, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;
R20, R21, R22 and R23 are independently H or C1-6 alkyl or one or more of R20 and R21, R21 and R22, R22 and R23, together form an aryl, heteroaryl, carbocyclic or heterocyclic ring;
R11 and R12 may together form a carbocyclic, heterocyclic, aryl or heteroaryl ring;
R6-10 are independently H, F, Cl, Br, I, OH, alkyl, aryl, heteroaryl, CO2H, SO3H, L-CO2H, L-SO3H, L-R or L-CS;
X is CHR23, O, S or NR30; and
R30 is H, C1-4 alkyl or C(O)C1-4 alkyl;
wherein at least one of R2-16 is L-CS.
US Pat. No. 10,168,323

COMPOSITIONS AND METHODS FOR CAPTURE OF CELLULAR TARGETS OF BIOACTIVE AGENTS

Promega Corporation, Mad...

1. A kit comprising:(a) a cell that expresses a fusion of a bioluminescent reporter and a cellular target of a small molecule bioactive agent, wherein the cellular target is located within a cell;
(b) the small molecule bioactive agent tethered by a carbamate linker to a chloroalkane capture ligand, wherein the small molecule bioactive agent tethered to a chloroalkane capture ligand is cell permeable, and wherein the small molecule bioactive agent is capable of non-covalently binding to the cellular target upon interaction thereof under intracellular conditions; and
(c) a solid surface displaying a capture protein, wherein the capture protein comprises a modified dehalogenase configured to covalently bind to the chloroalkane capture ligand upon interaction thereof.

US Pat. No. 10,093,806

METHODS FOR SYNTHESIZING RHODAMINE DYES

PROMEGA CORPORATION, Mad...

1. A method of synthesizing a rhodamine dye, the method comprising:reacting a compound of formula (I):

wherein
R1 and R2 are each independently hydrogen, alkyl, or R1a—CO—, wherein R1a is hydrogen, C1-C4 alkyl, C1-C4 haloalkyl, or C1-C4 alkoxy; or R1 and R2, together with the atoms to which they are attached, form a 3-8 membered ring;
Rc is selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl and halogen; and
Ra and Rb are each independently selected from the group consisting of hydrogen, alkyl, alkoxy, haloalkyl and halogen; or
R1 and Ra, together with the atoms to which they are attached, form a 5-8 membered ring, and
R2 and Rb, together with the atoms to which they are attached, form a 5-8 membered ring;
with a compound of formula (II),

wherein
R is selected from the group consisting of halogen, alkyl, haloalkyl, cyano, carboxy, alkoxy, haloalkoxy, alkoxycarbonyl, (carboxyl)heteroalkyl, alkylcarbonyl, alkoxyalkyl, cycloalkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, sulfonate, sulfonamide and amide;
n is 0, 1, 2, 3 or 4;
X is halogen or OR3; and R3 is selected from the group consisting of hydrogen, alkyl, aryl, alkylcarbonyl, alkoxycarbonyl, haloalkyl, haloalkylcarbonyl, haloalkoxycarbonyl, alkylsulfonyl, arylsulfonyl and haloalkylsulfonyl;
to form the rhodamine dye.

US Pat. No. 10,428,075

COELENTERAZINE ANALOGUES

PROMEGA CORPORATION, Mad...

1. A method for in vivo bioluminescent imaging, the method comprising:contacting a live cell with a compound of Formula I or Formula II:

wherein R5 and R5a are hydrogen;
R6 and R6a are aryl optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group consisting of halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, acyl amino, aminoalkyl, alkyl, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, alkoxy, sulfonylamino, sulfonyl, alkylsulfonyl, amide, carbamate, silyl, substituted silyl, acyl, or aminosulfonyl;
R8 and R8a are optionally substituted aryl, heteroaryl, alkyl substituted with aryl, or alkyl substituted with heteroaryl;
Rx is halogen, hydroxy, cyano, nitro, amino, alkylamino, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, or heteroalkyl; and
p is 0, 1, 2, 3, 4, or 5;
provided that the following compounds are excluded from formula (I): 8-benzyl-2-(furan-2-ylmethyl)-6-phenylimidazo[1,2-a]pyrazin-3(7H)-one; and
8-benzyl-2-(furan-2-ylmethyl)-6-(4-hydroxyphenyl)imidazo[1,2-a]pyrazin-3(7H)-one; and
Rx is not 4-hydroxy when p is 1, R5a is not hydrogen, or R8a is not benzyl, or any combination thereof;
and
detecting luminescence; wherein the live cell expresses a coelenterazine-utilizing luciferase.

US Pat. No. 10,408,819

LUMINESCENCE-BASED METHODS AND PROBES FOR MEASURING CYTOCHROME P450 ACTIVITY

PROMEGA CORPORATION, Mad...

1. A method for measuring cytochrome P450 enzyme activity in animal tissue comprising:(a) contacting an animal tissue with the luminogenic molecule and a bioluminescent enzyme, wherein the molecule is a cytochrome P450 substrate and a pro-substrate of bioluminescent enzyme;
(b) determining cytochrome P450 activity of the tissue by measuring luminescence of the mixture; and
wherein the luminogenic molecule is a compound of formula:
whereinR1 represents hydrogen, hydroxyl, amino, C1-20 alkoxy, substituted C1-20 alkoxy, C2-20 alkenyloxy, substituted C2-20 alkenyloxy, halogenated C2-20 alkoxy, substituted halogenated C2-20 alkoxy, C3-20 alkynyloxy, substituted C3-20 alkynyloxy, C3-20 cycloalkoxy, substituted C3-20 cycloalkoxy, C3-20 cycloalkylamino, substituted C3-20 cycloalkylamino, C1-20 alkylamino, substituted C1-20 alkylamino, di C1-20 alkylamino, substituted di C1-20 alkylamino, C2-20 alkenylamino, substituted C2-20 alkenylamino, di C2-20 alkenylamino, substituted di C2-20 alkenylamino, C2-20 alkenyl C1-20 alkylamino, substituted C2-20 alkenyl C1-20 alkylamino, C3-20 alkynylamino, substituted C3-20 alkynylamino, di C3-20 alkynylamino, substituted di alkylamino, C3-20 alkynyl C2-20 alkenylamino, or substituted C3-20 alkynyl C2-20 alkenylamino;
R2 and R3 independently represent C or N;
R4 and R5 independently represent S, O, NR8, wherein R8 represents hydrogen or C1-20 alkyl, CR9R10 wherein R9 and R10 independently represent H, C1-20 alkyl, or fluorine;
R6 represents CH2OH; COR11 wherein R11, represents H, OH, C1-2 alkoxide, C2-20 alkenyl, or NR12R13 wherein R12 and R13 are independently H, or C1-20 alkyl; or —OM+ wherein M+ is an alkali metal or a pharmaceutically acceptable salt; and
R7 represents H, C1-6 alkyl, C1-20 alkenyl, halogen, or C1-6alkoxide, with the proviso that R1 is not OH or NH2, R7 is not H, R6 is not COR11, R11 is not OH, R3 and R2 are not both carbon, and R4 and R5 are not both S at the same time (luciferin and aminoluciferin).

US Pat. No. 10,400,264

5,5-DISUBSTITUTED LUCIFERINS AND THEIR USE IN LUCIFERASE-BASED ASSAYS

PROMEGA CORPORATION, Mad...

1. A compound of formula (I?),
or a tautomer or a salt thereof, wherein
R1 is hydrogen, halogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR1a, —NR1bR1c, —OG1, —NR1xG1, or —NR1xG10;
R2 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR2a, —NR2bR2c, —SR2d, —SO2R2c, —S(O)R2f, —P(O)OR2gR2h, —OG1, or —NR2xG1;
R3 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR3a, —NR3bR3c, —SR3d, —SO2R3e, S(O)R3f, —P(O)OR3gR3h, —OG1, or —NR3xG1;
R4 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR4a, —NR4bR4c, —SR4d, —SO2R4e, —S(O)R4f, —P(O)OR4gR4h, —OG1, or —NR4xG1;
R5 is hydrogen, halogen, cyano, nitro, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, cycloalkenyl, —OR5a, —NR5bR5c, —SR5d, —SO2R5e, —S(O)R5f, —P(O)OR5gR5h, —OG1, or —NR5xG1;
or R2 and R3 together with the atoms to which they are attached, R3 and R4 together with the atoms to which they are attached, or R4 and R5 together with the atoms to which they are attached, or any combination thereof, form a 5- or 6-membered saturated, partially unsaturated or fully unsaturated ring, the 5- or 6-membered ring optionally containing 1, 2 or 3 heteroatoms or heteroatom groups each independently selected from the group consisting of O, N, S, NO, SO and SO2 as ring members, the 5- or 6-membered ring optionally fused to an aryl, heteroaryl, heterocycle, or cycloalkyl, the 5- or 6-membered ring substituted with 0, 1, 2, 3, or 4 substituents, each independently selected from the group consisting of halogen, ?O, ?S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, acyl, —OG1, —NHG1, and —N(C1-C10alkyl)G1;
R1a, R1b, R1c, R2a, R2b, R2c, R2d, R2e, R2f, R2g, R2h, R3a, R3b, R3c, R3d, R3e, R3f, R3g, R3h, R4a, R4b, R4c, R4d, R4e, R4f, R4g, R4h, R5a, R5b, R5c, R5d, R5e, R5f, R5g, and R5h, are each independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl;
R1x, R2x, R3x, R4x, and R5x are each independently hydrogen or C1-C12alkyl;
G1 comprises a substrate of a first enzyme, wherein biotransformation of the substrate by the first enzyme converts G1 to H;
—NR1xG10 is a group that is cleavable by a second enzyme to convert the —NR1xG10 group to —OH; and
W1 and W2 together with the carbon to which they are attached form a cycloalkyl, cycloalkenyl, or heterocycle;
wherein said alkyl, alkenyl, alkynyl, alkoxy, heteroalkyl, aryl, heteroaryl, heterocycle, cycloalkyl, and cycloalkenyl, at each occurrence, are independently substituted with 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, ?O, ?S, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocycle, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, alkylene, aryloxy, phenoxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, sulfonyl, alkylsulfonyl, arylsulfonyl, aminosulfonyl, sulfinyl, —COOH, ketone, amide, carbamate, silyl, substituted silyl, t-butyldimethylsilyl, alkylsulfanyl, sulfanyl, and acyl.

US Pat. No. 10,395,076

PROCESSES FOR DISTRIBUTION AND USE OF A MOBILE RFID CONTAINER

PROMEGA CORPORATION, Mad...

1. A system comprising:a mobile container configured to carry a plurality of items with a corresponding plurality of RFID tags inside the mobile container, wherein the mobile container includes:
an RFID detector configured to conduct at least one RFID scan and responsively generate scan data concerning the plurality of RFID tags;
location determining circuitry configured to determine location data corresponding to a current location of the mobile container;
a communications interface configured to wirelessly transmit the scan data and the location data; and
a door providing access to an interior region of the mobile container where the plurality of items with the corresponding plurality of RFID tags reside; and
an enterprise resource planning (“ERP”) system remote from the mobile container, wherein the ERP system is configured to:
communicate with the mobile container to receive the scan data and the location data;
determine whether the mobile container is within a geofence boundary of an end user based on the location data, at a distribution center, or in transit between the geofence boundary of the end user and the distribution center;
determine an identity of items removed from the mobile container while in the geofence boundary of the end user based on the scan data and the location data; and
determine restocking information for the mobile container based on the scan data before the mobile container arrives at the distribution center.

US Pat. No. 10,316,070

DUAL PROTECTED PRO-COELENTERAZINE SUBSTRATES

Promega Corporation, Mad...

1. A compound of formula (I)
or a tautomer, or a salt thereof, wherein
Ar1, Ar2, and Ar3 are each independently selected from the group consisting of aryl and heteroaryl, wherein Ar1, Ar2, and Ar3 are each optionally substituted, provided that one of Ar1, Ar2, or Ar3 is substituted by —X-L2-R6;
X is O, S, or NRx, wherein Rx is hydrogen or C1-C6-alkyl;
L1 and L2 are each independently selected from the group consisting of a bond and a linker of 1 to 50 atoms, wherein L1 and L2 are each optionally substituted; and
R3 and R6 are each independently selected from the group consisting of a peptide, an amino acid, a saccharide, and a phosphate.
US Pat. No. 10,101,332

COVALENT TETHERING OF FUNCTIONAL GROUPS TO PROTEINS AND SUBSTRATES THEREFOR

Promega Corporation, Mad...

1. A composition comprising a dehalogenase substrate of the formula R-linker-A-X, wherein R is peptide or polypeptide, A-X is a substrate for said dehalogenase, X is a halogen, and the linker is a group that separates R and A; wherein R, linker, A, and X are covalently linked.

US Pat. No. 10,280,447

SUBSTITUTED IMIDAZO[1,2-A]PYRAZINES TETHERED TO ENERGY ACCEPTORS

Promega Corporation, Mad...

1. A compound of formula (I)
or a tautomer, or a pharmaceutically acceptable salt thereof, wherein
R1 is;

R2 is absent or a substituent selected from the group consisting of alkyl, haloalkyl, halogen, —OH, and —NH2;
A1 is aryl, heteroaryl, heterocyclyl, or cycloalkyl, wherein the aryl, heteroaryl, heterocyclyl, and cycloalkyl are unsubstituted or substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, oxo, thioxo, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, aryloxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arylsulfonyl, aminosulfonyl, —COOH, amide, carbamate, silyl, t-butyldimethylsilyl, alkylsulfanyl, and acyl;
T1 is alkyl, alkenyl, alkynyl, or heteroalkyl, wherein the alkyl, alkenyl, alkynyl, and heteroalkyl are unsubstituted or substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substituents, each independently selected from the group consisting of halogen, oxo, thioxo, cyano, nitro, fluoroalkyl, alkoxyfluoroalkyl, fluoroalkoxy, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, heteroalkyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, cycloalkylalkyl, heteroarylalkyl, arylalkyl, hydroxy, hydroxyalkyl, alkoxy, alkoxyalkyl, aryloxy, benzyloxy, amino, alkylamino, dialkylamino, acylamino, aminoalkyl, arylamino, sulfonylamino, sulfinylamino, alkylsulfonyl, arylsulfonyl, aminosulfonyl, —COOH, ketone, amide, carbamate, silyl, t-butyldimethylsilyl, alkylsulfanyl, and acyl;
E1 is an energy acceptor, wherein the energy acceptor is a fluorescent dye, a quencher, a fluorescent particle, a luminescent metal complex, or a combination of any of the foregoing; and
q is 0, 1, or 2.

US Pat. No. 10,240,184

COVALENT TETHERING OF FUNCTIONAL GROUPS TO PROTEINS

Promega Corporation, Mad...

1. A composition comprising a dehalogenase substrate of the formula R-linker-A-X, wherein R is a fluorogenic or luminogenic molecule, A-X is a substrate for said dehalogenase, X is a halogen, and the linker is a group that separates R and A; wherein R, linker, A, and X are covalently linked.