# US 2019/0375763 A1 · Reagents for reversibly protecting biological molecules

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- Number: US 2019/0375763 A1
- Document ID: US20190375763A1
- Status: Published application
- Kind: A1
- Application number: 16/314,824
- Filed: 2017-07-13
- Published: 2019-12-12
- Earliest claimed: 2016-07-13
- Assignees: Biomerieux; Universite De Caen Normandie
- Applicants: BioméRieux; Université De Caen Normandie
- Inventors: Sylvain URSUEGUI; Alain LAURENT; Ali LAAYOUN; Frédéric FABIS
- Web page: https://patentgrep.com/patent/US20190375763A1

## CPC

- C07D 498/04: Ortho-condensed systems
- C07D 498/00: Heterocyclic compounds containing in the condensed system at least one hetero ring having nitrogen and oxygen atoms as the only ring hetero atoms (4-oxa-1-azabicyclo [3.2.0] heptanes, e.g. oxapenicillins C07D503/00; 5-oxa-1-azabicyclo [4.2.0] octanes, e.g. oxacephalosporins C07D505/00; analogues thereof having ring oxygen atoms in other position C07D507/00)
- C07D 498/02: in which the condensed system contains two hetero rings
- C12N 9/1252: {DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase}
- C12P 19/34: Polynucleotides, e.g. nucleic acids, oligoribonucleotides
- C12Q 1/6848: characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- 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)}
- 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 concerns reagents for the reversible protection of biological molecules. It relates in particular to compounds derived from azaisatoic anhydride and their uses for the protection of biological molecules, particularly enzymes, in order to block their activity. The invention also relates to the biological molecules protected in this manner and to the methods for making use of these reagents.

## Claims

### Claim 1

Compound of the following formula (I):
wherein
X is a covalent bond or a C1-C4 alkyl,
Y is a nucleophilic group,
Z1, Z2, Z3 each represent, independently of one another, N or C,
R1 is H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocycle,
R2 is a thermolabile and/or acid-labile protecting group,
R3 is H, a substituted or unsubstituted C1-C12 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen, or a cyano group.

### Claim 2 (depends on 1)

Compound according to claim 1, of the following formula (II):

### Claim 3 (depends on 1)

Compound according to claim 1, wherein the thermolabile and/or acid-labile group R2 is selected from the tert-butoxycarbonyl (BOC), substituted or unsubstituted phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl, or citraconyl groups.

### Claim 4 (depends on 1)

Compound according to claim 1, wherein R1 is a methyl group.

### Claim 5 (depends on 1)

Compound according to claim 1, wherein R3 is iodine.

### Claim 6 (depends on 1)

Compound according to claim 1, wherein Z1 is N and Z2 is C.

### Claim 7 (depends on 1)

Compound according to claim 1, wherein one of the following structures:

### Claim 8 (depends on 1)

Method for preparing a protected biological molecule, comprising the placing of a compound according to claim 1 in contact with a biological molecule comprising one or more nucleophilic groups, under conditions permitting the acylation of one or more nucleophilic groups of the biological molecule in order to form a protected biological molecule.

### Claim 9 (depends on 8)

Protected biological molecule, obtainable by the method according to claim 8.

### Claim 10

Protected biological molecule of the following formula (III):
wherein
Biomol is a biological molecule
W is a nucleophilic group of the biological molecule,
X is a covalent bond or a C1-C4 alkyl,
Y is a nucleophilic radical,
Z1, Z2, Z3 each represent, independently of one another, N or C,
R1 is H, a substituted or unsubstituted C1-C6 alkyl group, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aryl group, or a substituted or unsubstituted heterocycle,
R2 is H or a thermolabile and/or acid-labile protecting group,
R3 is H, a substituted or unsubstituted C1-C12 alkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heterocycle, an acyl group, a substituted or unsubstituted alkenyl group, a halogen, or a cyano group.

### Claim 11 (depends on 9)

Protected biological molecule according to claim 9, wherein Biomol is selected from enzymes.

### Claim 12 (depends on 11)

Protected biological molecule according to claim 11, wherein Biomol is an enzyme intended for use in a nucleic acid polymerization reaction.

### Claim 13 (depends on 1)

Use of a compound according to claim 1 for the reversible inactivation of an enzyme.

### Claim 14 (depends on 13)

Use according to claim 13, wherein the enzyme is an enzyme intended for use in a nucleic acid polymerization reaction.

### Claim 15 (depends on 12)

Use of a protected biological molecule according to claim 12 in a nucleic acid amplification reaction.

### Claim 16 (depends on 9)

Method for the deprotection of nucleophilic groups of a protected biological molecule according to claim 9, the method comprising a step of cleaving the thermolabile and/or acid-labile group or groups R2, respectively by heat and/or acid treatment, and concomitant deprotection of the nucleophilic groups.

### Claim 17 (depends on 12)

Method for the polymerization of a nucleic acid, comprising (i) the use of a biological molecule protected according to claim 12, (ii) at least one step for the deprotection of the biological molecule, (iii) a nucleic acid polymerization step using the polymerase deprotected in step (ii).