# US 2016/0257945 A1 · NON-NATURAL AMINO ACID tRNA SYNTHETASES FOR PYRIDYL TETRAZINE

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2016/0257945 A1
- Document ID: US20160257945A1
- Status: Published application
- Kind: A1
- Application number: 15/028,379
- Filed: 2014-10-10
- Published: 2016-09-08
- Earliest claimed: 2013-10-11
- Assignees: Sutro Biopharma, Inc.
- Applicants: Sutro Biopharma, Inc.
- Inventors: Erik S. Zimmerman; Christopher Thanos
- Web page: https://patentgrep.com/patent/US20160257945A1

## CPC

- C12N 9/93: {Ligases (6)}
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C12Y 601/01: Ligases forming aminoacyl-tRNA and related compounds (6.1.1)
- C12Y 601/01001: Tyrosine-tRNA ligase (6.1.1.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}
- C12Y: ENZYMES

## Abstract

The invention provides methods and compositions for a mutein aminoacyl-tRNA synthetase that preferentially charges a tRNA with a non-natural amino acid. Also provided are methods for incorporating the non-natural amino acids, pyridinyl-amino tetrazine amino acids, into a protein, and further conjugating a biologically active adduct to the pyridinyl-amino tetrazine.

## Claims

### Claim 1

A composition comprising an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridin-2-yl)propanoic acid or a compound having the formula:
compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: L;
b) at position L65 amino acid: V;
c) at position H70 amino acid: A
d) at position F108 amino acid: W;
e) at position Q109 amino acid: S;
f) at position D158 amino acid: V;
g) at position 1159 amino acid: A; and
h) at position L162 amino acid: S or V.

### Claim 2 (depends on 1)

The composition of claim 1, having the amino acid substitution L162S.

### Claim 3 (depends on 1)

The composition of claim 1, having the amino acid substitution L162V.

### Claim 4 (depends on 1)

The composition of claim 1, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 5 (depends on 1)

The composition of claim 1, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 2 and 3.

### Claim 6

A polynucleotide encoding an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridin-2-yl)propanoic acid or a compound having the formula:
compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: L;
b) at position L65 amino acid: V;
c) at position H70 amino acid: A
d) at position F108 amino acid: W;
e) at position Q109 amino acid: S;
f) at position D158 amino acid: V;
g) at position 1159 amino acid: A; and
h) at position L162 amino acid: S or V.

### Claim 7 (depends on 6)

The polynucleotide of claim 6, encoding the RS having the amino acid substitution L162S.

### Claim 8 (depends on 6)

The polynucleotide of claim 6, encoding the RS having the amino acid substitution L162V.

### Claim 9 (depends on 6)

The polynucleotide of claim 6, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 10 (depends on 6)

The polynucleotide of claim 6, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 2 and 3.

### Claim 11

A cell free protein synthesis system for selectively incorporating (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridin-2-yl)propanoic acid or a compound having the formula:
into a protein of interest, the system comprising:
a) a cell free extract of bacteria having biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis;
b) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
c) the compound of Formula I in a concentration sufficient to permit selective incorporation of the compound of Formula I into the protein of interest;
d) a tRNA able to be charged with the compound of formula I and complementary to the suppression codon of the protein of interest; and
e) an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with the compound of Formula I compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: L;
b) at position L65 amino acid: V;
c) at position H70 amino acid: A
d) at position F108 amino acid: W;
e) at position Q109 amino acid: S;
f) at position D158 amino acid: V;
g) at position 1159 amino acid: A; and
h) at position L162 amino acid: S or V.

### Claim 12 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the RS has the amino acid substitution L162S.

### Claim 13 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the RS has the amino acid substitution L162V.

### Claim 14 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 15 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 2 and 3.

### Claim 16 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the cell free extract has an active oxidative phosphorylation system.

### Claim 17 (depends on 11)

The cell free protein synthesis system of claim 11, wherein the protein of interest is an antibody or antibody fragment.

### Claim 18

A method for selectively incorporating (S)-2-amino-3-(5-((6-methyl-1,2,4,5-tetrazin-3-ylamino)methyl)pyridin-2-yl)propanoic acid or a compound having the formula:
into a protein of interest, the method comprising the steps of:
a) combining a cell free extract of bacteria having containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis with the following reagents:
i) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
ii) the compound of Formula I in a concentration sufficient to permit selective incorporation of the compound of Formula I into the protein of interest;
iii) a tRNA able to be charged with the compound of formula I and complementary to the suppression codon of the protein of interest; and
iv) an aminoacyl-tRNA synthetase (RS) wherein the RS:
preferentially aminoacylates to a degree of greater than 90%, a tRNA with the compound of Formula I compared to the 20 common naturally occurring amino acids;
has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
using SEQ ID NO:1 as a reference sequence, has:
a) at position Y32 amino acid: L;
b) at position L65 amino acid: V;
c) at position H70 amino acid: A
d) at position F108 amino acid: W;
e) at position Q109 amino acid: S;
f) at position D158 amino acid: V;
g) at position 1159 amino acid: A; and
h) at position L162 amino acid: S or V; and
b) incubating the combination of step (a) under conditions permitting selective incorporation of the compound of Formula I into the protein of interest.

### Claim 19 (depends on 18)

The method of claim 18, wherein the RS has the amino acid substitution L162S.

### Claim 20 (depends on 18)

The method of claim 18, wherein the RS has the amino acid substitution L162V.

### Claim 21 (depends on 18)

The method of claim 18, wherein the RS has sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 22 (depends on 18)

The method of claim 18, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 2 and 3.

### Claim 23 (depends on 18)

The method of claim 18, wherein the cell free extract has an active oxidative phosphorylation system.

### Claim 24 (depends on 18)

The method of claim 18, wherein the protein of interest is an antibody or antibody fragment.

### Claim 25 (depends on 18)

The method of claim 18, further comprising the step of conjugating a biologically active adduct to the compound of Formula I within the protein of interest.

### Claim 26 (depends on 25)

The method of claim 25, wherein the conjugation is by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a trans-cyclooctene on a biologically active adduct.

### Claim 27 (depends on 25)

The method of claim 25, wherein the conjugation is by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a methylcyclopropene on a biologically active adduct.

### Claim 28

A composition comprising an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: G;
b) at position L65 amino acid: V or A;
c) at position H70 amino acid: A or H
d) at position F108 amino acid: P, F or W;
e) at position Q109 amino acid: Q, H or S;
f) at position D158 amino acid: G;
g) at position 1159 amino acid: I or A; and
h) at position L162 amino acid: S or G.

### Claim 29 (depends on 28)

The composition of claim 28, having the amino acid substitution L65A.

### Claim 30 (depends on 28)

The composition of claim 28, having the amino acid substitution L65V.

### Claim 31 (depends on 28)

The composition of claim 28, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 32 (depends on 28)

The composition of claim 28, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

### Claim 33

A polynucleotide encoding an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
iii) using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: G;
b) at position L65 amino acid: V or A;
c) at position H70 amino acid: A or H
d) at position F108 amino acid: P, F or W;
e) at position Q109 amino acid: Q, H or S;
f) at position D158 amino acid: G;
g) at position 1159 amino acid: I or A; and
h) at position L162 amino acid: S or G.

### Claim 34 (depends on 33)

The polynucleotide of claim 33, encoding the RS having the amino acid substitution L65A.

### Claim 35 (depends on 33)

The polynucleotide of claim 33, encoding the RS having the amino acid substitution L65V.

### Claim 36 (depends on 33)

The polynucleotide of claim 33, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 37 (depends on 33)

The polynucleotide of claim 33, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

### Claim 38

A cell free protein synthesis system for selectively incorporating (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
into a protein of interest, the system comprising:
a) a cell free extract of bacteria having biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis;
b) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
c) the compound of Formula II in a concentration sufficient to permit selective incorporation of the compound of Formula II into the protein of interest;
d) a tRNA able to be charged with the compound of formula II and complementary to the suppression codon of the protein of interest; and
e) an aminoacyl-tRNA synthetase (RS) wherein the RS:
i) preferentially aminoacylates to a degree of greater than 90% a tRNA with the compound of Formula II compared to the 20 common naturally occurring amino acids;
ii) has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO:1; and
iii) using SEQ ID NO:1 as a reference sequence, has:
a) at position Y32 amino acid: G;
b) at position L65 amino acid: V or A;
c) at position H70 amino acid: A or H
d) at position F108 amino acid: P, F or W;
e) at position Q109 amino acid: Q, H or S;
f) at position D158 amino acid: G;
g) at position 1159 amino acid: I or A; and
h) at position L162 amino acid: S or G.

### Claim 39 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the RS has the amino acid substitution L65A.

### Claim 40 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the RS has the amino acid substitution L65V.

### Claim 41 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the RS has a sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 42 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

### Claim 43 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the cell free extract has an active oxidative phosphorylation system.

### Claim 44 (depends on 38)

The cell free protein synthesis system of claim 38, wherein the protein of interest is an antibody or antibody fragment.

### Claim 45

A method for selectively incorporating (S)-2-amino-3-(5-(6-methyl-1,2,4,5-tetrazin-3-ylamino)pyridin-3-yl)propanoic acid or a compound having the formula:
into a protein of interest, the method comprising the steps of:
a) combining a cell free extract of bacteria having containing biologically functioning tRNA, amino acids and ribosomes necessary for cell free protein synthesis with the following reagents:
i) a polynucleotide having a coding region encoding the protein of interest and including a suppression codon selectively positioned within its coding region;
ii) the compound of Formula II in a concentration sufficient to permit selective incorporation of the compound of Formula II into the protein of interest;
iii) a tRNA able to be charged with the compound of formula II and complementary to the suppression codon of the protein of interest; and
iv) an aminoacyl-tRNA synthetase (RS) wherein the RS:
preferentially aminoacylates to a degree of greater than 90% a tRNA with the compound of Formula II compared to the 20 common naturally occurring amino acids;
has a sequence identity of over 80% to Methanococcus jannaschii tyrosyl tRNA synthetase (TyrRS) having SEQ ID NO: 1; and
using SEQ ID NO: 1 as a reference sequence, has:
a) at position Y32 amino acid: G;
b) at position L65 amino acid: V or A;
c) at position H70 amino acid: A or H
d) at position F108 amino acid: P, F or W;
e) at position Q109 amino acid: Q, H or S;
f) at position D158 amino acid: G;
g) at position 1159 amino acid: I or A; and
h) at position L162 amino acid: S or G; and
b) incubating the combination of step (a) under conditions permitting selective incorporation of the compound of Formula II into the protein of interest.

### Claim 46 (depends on 45)

The method of claim 45, wherein the RS has the amino acid substitution L65A.

### Claim 47 (depends on 45)

The method of claim 45, wherein the RS has the amino acid substitution L65V.

### Claim 48 (depends on 45)

The method of claim 45, wherein the RS has sequence identity of at least 90% of SEQ ID NO: 1.

### Claim 49 (depends on 45)

The method of claim 45, wherein the RS is selected from the group of amino acid sequences consisting of SEQ ID NOS: 4 and 5.

### Claim 50 (depends on 45)

The method of claim 45, wherein the cell free extract has an active oxidative phosphorylation system.

### Claim 51 (depends on 45)

The method of claim 45, wherein the protein of interest is an antibody or antibody fragment.

### Claim 52 (depends on 45)

The method of claim 45, further comprising the step of conjugating a biologically active adduct to the compound of Formula II within the protein of interest.

### Claim 53 (depends on 52)

The method of claim 52, wherein the conjugation is by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a trans-cyclooctene on a biologically active adduct.

### Claim 54 (depends on 52)

The method of claim 52, wherein the conjugation is by an inverse electron demand Diels-Alder reaction between the tetrazine of the protein of interest and a methylcyclopropene on a biologically active adduct.