# US 2014/0155286 A1 · Modified tyrosine amino acids, peptides containing them, and their use for detecting hydrolase enzyme activity

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- Number: US 2014/0155286 A1
- Document ID: US20140155286A1
- Status: Published application
- Kind: A1
- Application number: 14/118,920
- Filed: 2012-05-18
- Published: 2014-06-05
- Earliest claimed: 2011-05-20
- Assignees: PamGene B.V.
- Applicants: Pamgene B.V.
- Inventors: Robby Ruijtenbeek; Jeroen van Ameijde; Robertus Matthias Joseph Liskamp
- Web page: https://patentgrep.com/patent/US20140155286A1

## CPC

- G01N 33/573: for enzymes or isoenzymes
- C07C 305/24: of non-condensed six-membered aromatic rings
- C07F 9/08: Esters of oxyacids of phosphorus {(C07F9/062 takes precedence)}
- C07F 9/12: with hydroxyaryl compounds
- C12Q 1/42: involving phosphatase
- 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)
- C07C: ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)
- 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

## Abstract

The present invention relates to a method and tools which allow the kinetic monitoring of hydrolase enzyme activity. The present invention also aims to provide synthetic peptides and peptide residues modified such that they can be used in assays for monitoring hydrolase enzyme activity, and preferably phosphatase activity. Also claimed are modified tyrosine amino acid derivatives of formula (IV), wherein Y is chosen from —POy2− with y equal to 2 or 3 and esters thereof, —SOz− with z equal to 2 or 3 and esters thereof, a carbohydrate or carbohydrate derivatives; and; wherein X1 and/or X2 is selected from —N3, —NO2, —CI, or —CN, —SCN, a C1 to C8 alkyl, or H, wherein either X1 or X2 is not H.

## Claims

### Claim 1

A modified tyrosine amino acid according to formula IV
wherein Y is chosen from —POy2− with y equal to 2 or 3 and esters thereof; and;
wherein X1 and/or X2 is selected from —N3, —NO2, or H, wherein either X1 or X2 is not H.

### Claim 2 (depends on 1)

A modified tyrosine amino acid according to claim 1 wherein Y is —PO32−.

### Claim 3 (depends on 1)

A modified tyrosine amino acid according to claim 1 wherein X1 is —NO2.

### Claim 4 (depends on 1)

A synthetic peptide comprising between 2 and 100 amino acids, characterized therein that said peptide has incorporated at least one modified tyrosine amino acid according to claim 1.

### Claim at position 5

5-6. (canceled)

### Claim 7

Method for the manufacturing of a double modified tyrosine monomer, comprising the steps of:
(a) modifying tyrosine monomers with a first protecting group on the amine residue, thereby obtaining a modified tyrosine monomer according to formula VII;
wherein X1 and/or X2 is selected from —N3, —NO2, —Br, or H, wherein either X1 or X2 is not H, and wherein P1 is a first protecting group;
(b) modifying the modified tyrosine monomers obtained from step (a) by esterification of the carboxyl group, thereby obtaining a modified tyrosine monomer according to formula VIII;
wherein P2 is a second protecting group;
(c) modifying the modified tyrosine monomers obtained from step (b) by providing the remaining hydroxyl group with a functional group provided with secondary protecting groups, thereby obtaining a modified tyrosine monomer according to formula IX;
wherein Y is chosen from —PO32− with y equal to 2 or 3 and esters thereof; and;
wherein said Y group is provided with n P3 groups, wherein P3 is a third protecting group and n is an integer selected from 1 or 2.

### Claim 8 (depends on 7)

Method according to claim 7, wherein said protecting groups P1, P2 and P3 are orthogonal and chosen from phthalimido, nitrobenzenesulphonyl, fluorenylmethyloxycarbonyl (Fmoc), tert-Butyloxycarbonyl (Boc), allyloxycarbonyl (Aloc), carboxybenzyl (Cbz), allyl, C1 to C8 alkyl, benzyl, (2-phenyl-2-trimethylsilyl)ethyl (PTMSE), phenyl, succinimidyl, pentafluorophenyl, pentachlorophenyl, 4-nitrophenyl, t-butyl and/or 2-cyanoethyl.

### Claim 9

A modified tyrosine monomer according to formula IX
wherein P1, P2 and P3 are orthogonal protecting groups selected from phthalimido, nitrobenzenesulphonyl, fluorenylmethyloxycarbonyl (Fmoc), tert-Butyloxycarbonyl (Boc), allyloxycarbonyl (Aloc), carboxybenzyl (Cbz), allyl, C1 to C8 alkyl, benzyl, (2-phenyl-2-trimethylsilyl)ethyl (PTMSE), phenyl, succinimidyl, pentafluorophenyl, pentachlorophenyl, 4-nitrophenyl, t-butyl and 2-cyanoethyl;
wherein X1 and/or X2 is selected from —N3, —NO2, or H, wherein either X1 or X2 is not H;
wherein Y is chosen from —PO32−with y equal to 2 or 3 and esters thereof; and
wherein said Y group is provided with n P3 groups, wherein P3 is a third protecting group and n is an integer selected from 1 or 2.

### Claim 10 (depends on 9)

Modified tyrosine monomer according to claim 9, wherein Y(P3)n is according to formula XI
wherein P3a and/or P3b are chosen from a protecting group selected from benzyl, allyl, t-butyl, C1 to C8 alkyl and/or 2-cyanoethyl.

### Claim 11

(canceled)

### Claim 12 (depends on 1)

A method for detecting hydrolase enzyme activity in a sample, said method comprising the step of:
contacting said sample with one or more modified tyrosine amino acids according to claim 1;
or contacting said sample with one or more synthetic peptides comprising between 2 and 100 amino acids, characterized therein that said peptide has incorporated therein at least one of said modified tyrosine amino acids
detecting the product of the hydrolase enzyme activity on said amino acids or peptides, and
optionally detecting the inhibition of hydrolase enzyme activity on said amino acids or peptides.

### Claim 13 (depends on 12)

A method according to claim 12 wherein said hydrolase enzyme activity is phosphatase activity and wherein said modified tyrosine amino acids or synthetic peptides comprise at least one nitro phosphate tyrosine residue.

### Claim 14 (depends on 1)

An array comprising two or more synthetic peptides having incorporated at least one modified tyrosine amino acid according to claim 1.

### Claim 15 (depends on 12)

The method according to claim 12 wherein said hydrolase enzyme activity is selected from the group consisting of EC 3.1, EC 3.2 and EC 3.3.