# US 9,085,514 B2 · Unnatural amino acids comprising a cyclooctynyl or trans-cyclooctenyl analog group and uses thereof

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- Number: US 9,085,514 B2
- Document ID: US9085514B2
- Status: Granted
- Kind: B2
- Application number: 13/982,446
- Filed: 2012-02-03
- Published: 2014-03-13
- Granted: 2015-07-21
- Earliest claimed: 2011-02-03
- Assignees: Embl
- Applicants: Edward Lemke; Carsten Schultz; Tilman Plass; Sigrid Milles; Christine Koehler
- Inventors: Edward Lemke; Carsten Schultz; Tilman Plass; Sigrid Milles; Christine Koehler
- Web page: https://patentgrep.com/patent/US9085514B2

## CPC

- C07C 271/22: to carbon atoms of hydrocarbon radicals substituted by carboxyl groups
- C07C 271/34: with the nitrogen atoms of the carbamate groups bound to hydrogen atoms or to acyclic carbon atoms
- C07K 1/13: Labelling of peptides
- C07K 2/00: Peptides of undefined number of amino acids; Derivatives thereof
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12P 21/02: having a known sequence of two or more amino acids, e.g. glutathione
- C07B 2200/09: Geometrical isomers
- C07C 2601/18: with a ring being at least seven-membered
- C07C 2602/24: the ring system containing nine carbon atoms, e.g. perhydroindane
- C07C 2602/42: the bicyclo ring system containing seven carbon atoms
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- 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)}
- C07B: GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR (preparation of carboxylic acid esters by telomerisation C07C67/47; telomerisation C08F)

## Abstract

The present invention relates to unnatural amino acids comprising a cyclooctynyl or trans-cyclooctenyl analog group and having formula (I) or an acid or base addition salt thereof. The invention also relates to the use of said unnatural amino acids, kits and processes for preparation of polypeptides that comprise one or more than one cyclooctynyl or trans-cyclooctenyl analog group. These polypeptides can be covalently modified by in vitro or in vivo reaction with compounds comprising an azide, nitrile oxide, nitrone, diazocarbonyl or 1,2,4,5-tetrazine group.

## Claims

### Claim 1

A compound of formula I
wherein:
X1 has formula
wherein:
Y1, Y2, Y3, Y5, Y6 independently are —CH2—, —NH—, —S— or —O— provided that at least 4 of Y1, Y2, Y3, Y4, Y5, Y6 are —CH2—;
R2 is hydrogen, halogen, C1-C4-alkyl, CF3, CN, C1-C4-alkoxy, —O—CF3, C2-C5-alkanoyloxy, C1-C4-alkylaminocarbonyloxy or C1-C4-alkylthio;
X2 is —CH2—, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH—, or
X2 is >CH— or >N— wherein the carbon or the nitrogen atom together with two adjacent ring atoms of X1 forms a 3-membered ring, or
X2 is —CH2—CH<, —NH—CH< or —CH2—N< wherein the two carbon atoms or the carbon and the nitrogen atom together with two adjacent ring atoms of X1 form a 4-membered ring, or
X2 is —CH2—CH2—CH<, —NH—CH2—CH<, —CH2—NH—CH<, —CH2—CH2—N<,
wherein the three carbon atoms or the two carbon atoms and the nitrogen atom together with two adjacent ring atoms of X1 form a 5-membered ring;
X3 is C1-C6-alkylene, —(CH2—CH2—O)m—, —(CH2—O)p— or a single bond;
X4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH2)—, —CH(NH2)—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH2)—, —C(O)—NH—C(NH)—NH—, NH—CH(NH2)—C(O)— or —NH—C(NH)—NH—C(O)—;
X5 is hydrogen, C1-C6-alkyl, C1-C6-alkoxy-C1-C2-alkyl, C2-C7-alkanoyloxy-C1-C2-alkyl or C2-C7-alkanoylsulfanyl-C1-C2-alkyl;
R1 is —OH or —NH2;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6,
or an acid or base addition salt thereof.

### Claim 2 (depends on 1)

The compound or salt of claim 1, wherein R2 is hydrogen or halogen.

### Claim 3 (depends on 2)

The compound or salt of claim 2, wherein R2 is fluorine.

### Claim 4 (depends on 3)

The compound or salt of claim 3, wherein R2 is two fluorine bound to one carbon ring atom.

### Claim 5 (depends on 1)

The compound or salt of claim 1, wherein X1 has the formula
wherein R2 is as defined in claim 1.

### Claim 6 (depends on 1)

The compound or salt of claim 1, wherein one of Y1, Y2, Y3, Y4, Y5, Y6 is —NH— while the remaining five of Y1, Y2, Y3, Y4, Y5, Y6 are —CH2—, and R2 is as defined in claim 1.

### Claim 7 (depends on 1)

The compound or salt of claim 1, wherein X1 has a formula selected from
and R2 is as defined in claim 1.

### Claim 8 (depends on 1)

The compound or salt of claim 1, wherein X1 is unsubstituted cyclooctynyl or cyclooctynyl substituted with one or two halogen atoms.

### Claim 9 (depends on 8)

The compound or salt of claim 8, wherein the halogen atoms are fluorine atoms.

### Claim 10 (depends on 1)

The compound or salt of claim 1, wherein X1 is unsubstituted trans-cyclooctenyl.

### Claim 11 (depends on 1)

The compound or salt of claim 1, wherein X2 is —O—.

### Claim 12 (depends on 1)

The compound or salt of claim 1, wherein X3 is —CH2—CH2—O— or a single bond.

### Claim 13 (depends on 1)

The compound or salt of claim 1, wherein the structural element —X2-X3— comprises from 1 to 6 atoms in the main chain.

### Claim 14 (depends on 1)

The compound or salt of claim 1, wherein X4 is —NH—, —C(O)—NH—, —NH—CH(NH2)—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH2)— or —C(O)—NH—C(NH)—NH—.

### Claim 15 (depends on 1)

The compound or salt of claim 1, wherein n is 3 or 4.

### Claim 16 (depends on 1)

The compound or salt of claim 1, wherein the structural element —X2-X3-X4—(CH2)n— comprises from 5 to 12 atoms in the main chain, such as 6, 7, 8, 9, 10 or 11 atoms in the main chain.

### Claim 17 (depends on 1)

The compound or salt of claim 1, wherein X5 is hydrogen, C1-C6-alkoxymethyl, C1-C6-alkoxyeth-1-yl, C2-C7-alkanoyloxymethyl or C2-C7-alkanoylsulfanylethyl.

### Claim 18 (depends on 1)

The compound or salt of claim 1, having S-configuration with regard to the asymmetric carbon atom carrying R1.

### Claim 19 (depends on 1)

The compound or salt of claim 1, wherein —(CH2)n—CHR1—C(O)O—X5 has formula
wherein R1 and X5 are as defined in claim 1.

### Claim 20 (depends on 1)

The compound or salt of claim 1 that is a compound of formula Ia, Ib, Ic or Id
wherein R1, R2, X5, and Y1 to Y6 are as defined in claim 1,
or an acid or base addition salt thereof.

### Claim 21 (depends on 1)

The compound or salt of claim 1 that is a compound of formula
or an acid or base addition salt thereof.

### Claim 22 (depends on 1)

The compound or salt of claim 1 that is a compound of formula Ie
wherein R1, R2, X5, and Y1 to Y6 are as defined herein and X2 is >CH— or >N— wherein the carbon or the nitrogen atom together with two adjacent ring atoms of X1 forms a 3-membered ring; or X2 is —CH2—CH<, —NH—CH< or —CH2—N< wherein the two carbon atoms or the carbon and the nitrogen atom together with two adjacent ring atoms of X1 form a 4-membered ring; or X2 is —CH2—CH2—CH<, —NH—CH2—CH<, —CH2—NH—CH<, —CH2—CH2—N<,
wherein the three carbon atoms or the two carbon atoms and the nitrogen atom together with two adjacent ring atoms of X1 form a 5-membered ring;
or an acid or base addition salt thereof.

### Claim 23 (depends on 1)

The compound or salt of claim 1 that is a compound of formula
or an acid or base addition salt thereof.

### Claim 24 (depends on 1)

A process for preparing a target polypeptide having one or more than one cyclooctynyl or trans-cyclooctenyl analog group, the process comprising:
a) providing a translation system comprising:
(i) an aminoacyl tRNA synthetase, or a polynucleotide encoding it;
(ii) a compound or salt of claim 1;
(iii) a tRNA having an anticodon to a selector codon, or a polynucleotide encoding said tRNA; and
(iv) a polynucleotide encoding the target polypeptide and comprising one or more than one selector codon(s),
wherein the aminoacyl tRNA synthetase (i) is capable of specifically acylating the tRNA (iii) with the compound or salt (ii);
b) allowing translation of the polynucleotide (iv) thereby incorporating the compound (ii) into the target polypeptide at the position(s) encoded by the selector codon(s).

### Claim 25 (depends on 24)

The process of claim 24, wherein said translation system is a cell expressing said aminoacyl tRNA synthetase.

### Claim 26 (depends on 24)

The process of claim 24, wherein said aminoacyl tRNA synthetase is a pyrrolysyl tRNA synthetase.

### Claim 27 (depends on 26)

The process of claim 26, wherein said pyrrolysyl tRNA synthetase comprises the amino acid sequence set forth in SEQ ID NO:1 or 2.

### Claim 28

A polypeptide comprising one or more than one residue of formula II
wherein:
X1 has the formula
wherein:
Y1, Y2, Y3, Y4, Y5, Y6 independently are —CH2—, —NH—, —S— or —O— provided that at least 4 of Y1, Y2, Y3, Y4, Y5, Y6 are —CH2— or;
R2 is hydrogen, halogen, C1-C4-alkyl, CF3, CN, C1-C4-alkoxy, —O—CF3, C2-C5-alkanoyloxy, C1-C4-alkylaminocarbonyloxy or C1-C4-alkylthio;
X2 is —CH2—, —O—, —S—, —NH—, —C(O)—, —OC(O)—, —C(O)O—, —NH—C(O)— or —C(O)—NH— or
X2 is >CH— or >N— wherein the carbon or the nitrogen atom together with two adjacent ring atoms of X1 forms a 3-membered ring, or
X2 is —CH2—CH<, —NH—CH< or —CH2—N< wherein the two carbon atoms or the carbon and the nitrogen atom together with two adjacent ring atoms of X1 form a 4-membered ring, or
X2 is —CH2—CH2—CH<, —NH—CH2—CH<, —CH2—NH—CH<, —CH2—CH2—N<,
wherein the three carbon atoms or the two carbon atoms and the nitrogen atom together with two adjacent ring atoms of X1 form a 5-membered ring;
X3 is C1-C6-alkylene, —(CH2—CH2—O)m—, —(CH2—O)p— or a single bond;
X4 is —NH—, —C(O)—NH—, —NH—C(O)—, —NH—CH(NH2)—, —CH(NH2)—NH—, —NH—C(NH)—NH—, —C(O)—NH—CH(NH2)—, —C(O)—NH—C(NH)—NH—, NH—CH(NH2)—C(O)— or —NH—C(NH)—NH—C(O)—;
Z1 is —O— or —NH—;
n is an integer from 1 to 4;
m is an integer from 1 to 6; and
p is an integer from 1 to 6.

### Claim 29 (depends on 1)

A kit for preparing a polypeptide having one or more than one cyclooctynyl or trans-cyclooctenyl analog group, comprising the compound or salt of claim 1.