# US 8,785,151 B2 · Biosynthetically generated pyrroline-carboxy-lysine and site specific protein modifications via chemical derivatization of pyrroline-carboxy-lysine and pyrrolysine residues

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- Number: US 8,785,151 B2
- Document ID: US8785151B2
- Status: Granted
- Kind: B2
- Application number: 13/125,547
- Filed: 2009-10-23
- Published: 2011-10-27
- Granted: 2014-07-22
- Earliest claimed: 2008-10-24
- Applicants: Bernhard Geierstanger; Weijia Ou; Susan E. Cellitti; Tetsuo Uno; Tiffany Crossgrove; Hsien-Po Chiu; Jan Grunewald; Xueshi Hao
- Inventors: Bernhard Geierstanger; Weijia Ou; Susan E. Cellitti; Tetsuo Uno; Tiffany Crossgrove; Hsien-Po Chiu; Jan Grunewald; Xueshi Hao
- Web page: https://patentgrep.com/patent/US8785151B2

## CPC

- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C07D 207/20: with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
- C07D 207/48: Sulfur atoms
- C07K 5/0215: {containing natural amino acids, forming a peptide bond via their side chain functional group, e.g. epsilon-Lys, gamma-Glu}
- 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)}
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)

## Abstract

Disclosed herein is pyrroline-carboxy-lysine (PCL), a pyrrolysine analogue, which is a natural, biosynthetically generated amino acid, and methods for biosynthetically generating PCL. Also disclosed herein are proteins, polypeptides and peptides that have PCL incorporated therein and methods for incorporating PCL into such proteins, polypeptides and peptides. Also disclosed herein is the site-specific derivatization of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein. Also disclosed herein is the crosslinking of proteins, polypeptides and peptides having PCL or pyrrolysine incorporated therein.

## Claims

### Claim 1

A compound having the structure of Formula (II):
R1-(BB)n-R2 (II)
wherein:
R1 is H or an amino terminus modification group;
R2 is OH or a carboxy terminus modification group;
n is an integer from 1 to 5000;
each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (D-1), and a pyrrolysine analogue amino acid residue having the structure of Formula (F 1):
wherein:
R5 is selected from H, —OH, —NO2, halo, C1-8alkyl, halo-substituted-C1-8alkyl, hydroxy-substituted-C1-8alkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX1;
R6 is H or C1alkyl;
A is a phenyl optionally substituted with 1 to 3 substituents independently selected from —OH, —NO2, halo, C1-8alkyl, halo-substituted-C1-8alkyl, hydroxy-substituted-C1-8alkyl, aryl, heteroaryl, heterocycloalkly, or cycloalkyl and -LX1;
L is selected from a bond, C1-8alkylene, halo-substituted-C1-8alkylene, hydroxy-substituted-C1-8alkylene, C2-8alkenylene, halo-substituted-C2-8alkenylene, hydroxy-substituted-C2-8alkenylene,
—O(CR11R12)k—, —S(CR11R12)k, —S(O)k(CR11R12)k—, —O(CR11R12)k—NR11C(O)—, —O(CR11R12)kC(O)NR11—, —C(O)—, —C(O)(CR11R12)k—, —C(S)—, —C(S)(CR11R12)k—, —C(O)NR11—, —NR11C(O)—, —NR11(CR11R12)k—, —CONR11(CR11R12)k—, —N(R11)CO(CR11R12)k—, —C(O)NR11(CR11R12)k—, —NR11C(O)CR11R12)k—, where each R11 and R12 are independently H, C1-8alkyl, halo-substituted-C1-8alkyl, or hydroxy-substituted-C1-8alkyl, and k is an integer from 1 to 12, and
X1 is selected from a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a cytotoxic compound, an affinity label, a peptide, a protein, an antibody or antibody fragment, a carbohydrate, a DNA, a RNA, phosphorothioate-modified DNA, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a spin label, a fluorophore, a radioactive moiety a photocaged moiety, biotin, a biotin analogue, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, an immunogenic hapten, an immune response potentiator, a solid support, —CH2CH2—(OCH2CH2O)p—OX2, —O—(CH2CH2O)pCH2CH2—X2, and any combination thereof, wherein p is 1 to 10,000 and X2 is H, a C1-8alkyl, a protecting group or a terminal functional group, and
wherein:
at least one BB in the compound of Formula (II) is a pyrrolysine analogue amino acid residue having the structure of Formula (D-1).

### Claim 2 (depends on 1)

The compound of claim 1, wherein ring A is a phenyl substituted with -LX1.

### Claim 3 (depends on 1)

The compound of claim 1, wherein each BB is independently selected from an amino acid residue, a pyrrolysine analogue amino acid residue having the structure of Formula (D-2);
wherein,
R5 is selected from H, —OH, —NO2, halo, C1-8alkyl, halo-substituted-C1-8alkyl, hydroxy-substituted-C1-8alkyl, aryl, heteroaryl, heterocycloalkly or cycloalkyl and -LX1;
R6 is H or C1alkyl;
when present each R7 is independently selected from —OH, —NO2, halo, C1-8alkyl, halo-substituted-C1-8alkyl, hydroxy-substituted-C1-8alkyl, aryl, heteroaryl, heterocycloalkly, or cycloalkyl and -LX1;
L is selected from a bond, C1-8alkylene, halo-substituted-C1-8alkylene, hydroxy-substituted-C1-8alkylene, C2-8alkenylene, halo-substituted-C2-8alkenylene, hydroxy-substituted-C2-8alkenylene,
—O(CR11R12)k—, —S(CR11R12)k, —S(O)k(CR11R12)k—, —O(CR11R12)k—NR11C(O)—, —O(CR11R12)kC(O)NR11—, —C(O)—, —C(O)(CR11R12)k—, —C(S)—, —C(S)(CR11R12)k—, —C(O)NR11—, —NR11C(O)—, —NR11(CR11R12)k—, —CONR11(CR11R12)k—, —N(R11)CO(CR11R12)k—, —C(O)NR11(CR11R12)k—, —NR11C(O)CR11R12)k—, where each R11 and R12 are independently H, C1-8alkyl, halo-substituted-C1-8alkyl, or hydroxy-substituted-C1-8alkyl, and k is an integer from 1 to 12, and
X1 is selected from a water-soluble polymer, a polyalkylene glycol, a poly(ethylene glycol), a derivative of poly(ethylene glycol), a sugar, a lipid, a cytotoxic compound, an affinity label, a peptide, a protein, an antibody or antibody fragment, a carbohydrate, a DNA, a RNA, phosphorothioate-modified DNA, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a spin label, a fluorophore, a radioactive moiety, a photocaged moiety, biotin, a biotin analogue, a TLR2 agonist, a TLR4 agonist, a TLR7 agonist, a TLR9 agonist, a TLR8 agonist, a T-cell epitope, a phospho-lipid, a LPS-like molecule, an immunogenic hapten, an immune response potentiator, a solid support, —CH2CH2—(OCH2CH2O)p—OX2, —O—(CH2CH2O)pCH2CH2—X2, and any combination thereof, wherein p is 1 to 10,000 and X2 is H, a C1-8alkyl, a protecting group or a terminal functional group.

### Claim 4 (depends on 3)

The compound of claim 3, wherein R7 is -LX1.

### Claim 5 (depends on 1)

The compound of claim 1, wherein X1 is a sugar, a polyethylene glycol, a fluorophore, an immunomodulator, a ribonucleic acid (RNA), a deoxyribonucleic acid (DNA), a protein, a peptide, a biotin, a phospholipid, a TLR7 agonist, an immunogenic hapten, a T-cell epitope, a lipid, a carbohydrate, phosphorothioate-modified DNA, a saccharide, a disaccharide, an oligosaccharide, a polysaccharide, a spin label, an immune response potentiator, a cytotoxic compound or a solid support.

### Claim 6 (depends on 5)

The compound of claim 5, wherein L is a poly(alkyleneglycol), a poly(ethyleneglycol), C1-8alkylene, halo-substituted-C1-8alkylene, hydroxy-substituted-C1-8alkylene,

### Claim 7 (depends on 6)

The compound of claim 6, wherein R6 is H.