# US 2021/0340593 A1 · Substrates for covalent tethering of proteins to functional groups or solid surfaces

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- Number: US 2021/0340593 A1
- Document ID: US20210340593A1
- Status: Published application
- Kind: A1
- Application number: 17/374,460
- Filed: 2021-07-13
- Published: 2021-11-04
- Earliest claimed: 2013-03-15
- Assignees: Promega Corporation
- Applicants: Promega Corporation
- Inventors: Carolyn W. Hitko; Thomas Kirkland; Sergiy Levin; Poncho Meisenheimer; Rachel Friedman Ohana; Harry Tetsuo Uyeda; Ji Zhu
- Web page: https://patentgrep.com/patent/US20210340593A1

## CPC

- C12Q 1/34: involving hydrolase
- C12N 9/14: Hydrolases (3)
- G01N 33/54353: {with ligand attached to the carrier via a chemical coupling agent (coatings G01N33/54393)}
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The present invention provides haloalkane substrates, and linkers for connecting such substrates to functional elements (e.g., tags, labels, surfaces, etc.). Substrates and linkers described herein find use, for example, in labeling, detection, and immobilization of proteins, cells, and molecules. In particular, the linkers provided herein find use within substrates for dehalogenase variants that form covalent bonds with their haloalkane substrates.

## Claims

### Claim 1

A compound of formula R—L1-M-L2-A—X;
wherein R is a functional group, L1 is a first linker portion, M is a carbamate group, L2 is a second linker portion, A is an alkyl group, and X is a halogen;
wherein L2-A separates M and X by 6-18 linearly connected atoms; and
wherein L1 separates R and M by 2 or more linearly connected atoms.

### Claim 2 (depends on 1)

The compound of claim 1, wherein A is (CH2)6.

### Claim 3 (depends on 1, 2)

The compound of one of claims 1-2, wherein L2-A separates M and X by 12 linearly connected atoms.

### Claim 4 (depends on 1, 2, 3)

The compound of one of claims 1-3, wherein the functional group comprises a affinity tag, fluorophore, or solid surface.

### Claim 4 (depends on 1, 2, 3)

The compound of one of claims 1-3, wherein the functional group is a drug, drug compound, biomolecule or small molecule.

### Claim 5 (depends on 1, 2, 3, 4)

The compound of one of claims 1-4, wherein L2 does not comprise a carbamate group.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The compound of one or claims 1-5, wherein L2 comprises linearly connected CH2 and O groups.

### Claim 7 (depends on 6)

The compound of claim 6, wherein L2 consists of linearly connected CH2 and O groups.

### Claim 8 (depends on 6, 7)

The compound of one of claim 6 or 7, wherein L2 comprises ((CH2)2O)x, wherein x=0-5.

### Claim 9 (depends on 8)

The compound of claim 8, wherein L2 comprises ((CH2)2O)2.

### Claim 10 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9)

The compound of one of claims 1-9, wherein L1 comprises linearly connected CH2 and O groups.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The compound of one of claims 1-10, wherein L1 comprises a carbamate.

### Claim 12 (depends on 11)

The compound of claim 11, wherein L1 is 2-8 linearly connected atoms in length.

### Claim 13 (depends on 11)

The compound of claim 11, wherein L1 comprises NHCOO—((CH2)2O)x, wherein x=1-8.

### Claim 14 (depends on 13)

The compound of claim 13, wherein L1 comprises NHCOO—((CH2)2O)3.

### Claim 15 (depends on 1)

The compound of claim 1, wherein L1 comprises NHCOO—((CH2)2O)3, wherein L2 comprises ((CH2)2O)2, and wherein A is (CH2)6.

### Claim 16 (depends on 15)

The compound of claim 15, wherein the functional group comprises an affinity tag, fluorophore, or solid surface.

### Claim 17 (depends on 15)

The compound of claim 15, wherein the functional group is a drug, drug compound, biomolecule or small molecule.

### Claim 18

A composition comprising a protein linked to a functional group by a linker comprising L1-M-L2-A;
wherein L1 is a first linker portion, M is a carbamate group, L2 is a second linker portion, and A is an alkyl group;
wherein L2-A separates M from the protein by 8-16 linearly connected atoms; and
wherein L1 separates the functional group and M by 2 or more linearly connected atoms.

### Claim 19 (depends on 18)

The composition of claim 18, wherein said protein comprises a mutant dehalogenase.

### Claim 20 (depends on 18, 19)

The composition of one of claims 18-19, wherein said protein is part of a fusion protein.

### Claim 21 (depends on 18, 19, 20)

The composition of one of claims 18-20, wherein A is (CH2)6.

### Claim 22 (depends on 18, 19, 20, 21)

The composition of one of claims 18-21, wherein L2-A separates M from the protein by 12 linearly connected atoms.

### Claim 23 (depends on 18, 19, 20, 21, 22)

The composition of one of claims 18-22, wherein the functional group comprises an affinity tag, fluorophore, or solid surface.

### Claim 24 (depends on 18, 19, 20, 21, 22)

The compound of one of claims 18-22, wherein the functional group is a drug, drug compound, biomolecule or small molecule.

### Claim 25 (depends on 18, 19, 20, 21, 22, 23, 24)

The composition of one of claims 18-24, wherein L2 does not comprise a carbamate groups.

### Claim 26 (depends on 18, 19, 20, 21, 22, 23, 24, 25)

The composition of one or claims 18-25, wherein L2 comprises linearly connected CH2 and O groups.

### Claim 27 (depends on 26)

The composition of claim 26, wherein L2 consists of linearly connected CH2 and O groups.

### Claim 28 (depends on 26, 27)

The composition of one of claim 26 or 27, wherein L2 comprises ((CH2)2O)x, wherein x=0-5.

### Claim 29 (depends on 28)

The composition of claim 28, wherein L2 comprises ((CH2)2O)2.

### Claim 30 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29)

The composition of one of claims 18-29, wherein L1 comprises linearly connected CH2 and O groups.

### Claim 31 (depends on 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The composition of one of claims 18-30, wherein L1 comprises a carbamate.

### Claim 32 (depends on 31)

The composition of claim 31, wherein L1 comprises NHCOO—((CH2)2O)x, wherein x=1-8.

### Claim 33 (depends on 32)

The composition of claim 32, wherein L1 comprises NHCOO—((CH2)2O)3.

### Claim 33 (depends on 31)

The compound of one of claim 31, wherein the functional group is a drug, drug compound, biomolecule or small molecule.

### Claim 34 (depends on 32)

The composition of claim 32, wherein L1 is 2-8 linearly connected atoms in length.

### Claim 34

A method to identify a target molecule of interest comprising: a) contacting a sample with a substrate of any one of the previous claims, wherein the functional group is a compound of interest; b) contacting the sample and substrate with a solid support comprising a mutant dehalogenases; and c) identifying the molecule of interest bound to the substrate.

### Claim 35 (depends on 18)

The composition of claim 18, wherein L1 comprises NHCOO—((CH2)2O)3, wherein L2 comprises ((CH2)2O)2, and wherein A is (CH2)6.

### Claim 35 (depends on 34)

The method of claim 34, wherein the compound of interest is a drug, drug compound, biomolecule or small molecule.

### Claim 36 (depends on 35)

The composition of claim 35, wherein the functional group comprises an affinity tag, fluorophore, or solid surface.

### Claim 36 (depends on 34)

The method of claim 34, wherein the sample is a cell or cell lysate.

### Claim 37 (depends on 34)

The method of claim 34, wherein the molecule of interest is a fusion protein.

### Claim 38 (depends on 37)

The method of claim 37, wherein the fusion protein comprises a reporter protein.

### Claim 39 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method to detect or determine the presence or amount of a mutant dehalogenase, comprising: a) contacting a mutant dehalogenase with a substrate comprising a compound of one of claims 1-15, wherein the mutant dehalogenase comprises at least one amino acid substitution relative to a corresponding wild-type dehalogenase; and b) detecting or determining the presence or amount of R, thereby detecting or determining the presence or amount of the mutant dehalogenase.

### Claim 40 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method to label a cell, comprising: contacting a cell comprising a mutant dehalogenase with a dehalogenase substrate comprising a compound of one of claims 1-15, wherein the mutant dehalogenase comprises at least one amino acid substitution relative to a corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate, thereby labeling the cell with R.

### Claim 41 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method for detecting the presence or amount of a molecule in a cell, comprising: a) contacting a cell comprising a mutant dehalogenase with a substrate comprising a compound of one or claims 1-15, wherein the mutant dehalogenase comprises at least one amino acid substitution relative to a corresponding wild-type dehalogenase, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate; and b) detecting or determining the presence or amount of R in the cell.

### Claim 42 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method to immobilize isolate a molecule of interest, comprising contacting (a) a solid support displaying a substrate comprising a compound of one of claims 1-15 with (b) a fusion protein which comprises (i) a mutant dehalogenase that forms a covalent bond with said substrate upon interaction with said substrate and (ii) a protein which is bound to the molecule of interest.

### Claim 43 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method to immobilize a protein of interest, comprising: contacting (a) a solid support comprising a substrate of one of claims 1-15 with (b) a fusion of (i) a mutant dehalogenase that forms a covalent bond with said substrate upon interaction with said substrate and (ii) a protein of interest.

### Claim 44 (depends on 34, 35, 36, 37, 38, 39, 40, 41, 42, 43)

A method of one or claims 34-43, wherein the at least one amino acid substitution results in the mutant dehalogenase forming a bond with the substrate which is more stable than the bond formed between the corresponding wild-type dehalogenase and the substrate, wherein the at least one amino acid substitution in the mutant dehalogenase is a substitution at an amino acid residue in the corresponding wild-type dehalogenase that is associated with activating a water molecule which cleaves the bond formed between the corresponding wild-type dehalogenase and the substrate or at an amino acid residue in the corresponding wild-type dehalogenase that forms an ester intermediate with the substrate.

### Claim 45 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A method for preparing a compound one of claims 1-15 comprising coupling a compound of formula R—Y with a compound of formula Z—L1-M-L2-A—X, wherein Y and Z are groups that can react to link R— to -Linker-L1-M-L2-A—X.

### Claim 46 (depends on 45)

The method of claim 45, R—Y is an activated ester of a compound of formula R and wherein Z is an amine suitable to react with the activated ester to form an amide bond.

### Claim 47 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15)

A cell comprising a substrate of one of claims 1-15.