US 12,281,134 B2Granted

Electrochemically-cleavable linkers

AssigneeMicrosoft Technology Licensing, LLC

InventorsBichlien Nguyen, Jake Smith, Karin Strauss

Life of this patent

Estimating the term…

Loading patent…

Abstract

This disclosure provides electrochemically-cleavable linkers with cleavage potentials that are less than the redox potential of the solvent in which the linkers are used. In some applications, the solvent may be water or an aqueous buffer solution. The linkers may be used to link a nucleotide to a bound group. The linkers include a cleavable group which may be one of a methoxybenzyl alcohol, an ester, a propargyl thioether, or a trichloroethyl ether. The linkers may be cleaved in solvent by generating an electrode potential that is less than the redox potential of the solvent. In some implementations, an electrode array may be used to generate localized electrode potentials which selectively cleave linkers bound to the activated electrode. Uses for the linkers include attachment of blocking groups to nucleotides in enzymatic oligonucleotide synthesis.

Claims21

Dependent claims

A compound having a structure P—Y-L1-C1-C2-L2-L3-nucleotide, wherein:

P is a bound group that is a peptide, a linked nucleotide, a fluorophore, or a water-soluble group;
Y is optional, if present Y is a bound group attachment group with the structure:
α-S-β, or α-NH2-β, wherein α represents a point of attachment to P and β represents a point of attachment to L1 or C1;
L1 is optional, if present L1 is a flexible extension with the structure one or more of:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein β represents a point of attachment to Y or P and χ represents a point of attachment to C1;
C1 is a cleavable group with the structure:
wherein X1 is 1 to 4 ring substituents selected from the group consisting of a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubstituted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, and a halogen and X2 is hydrogen, a methyl group, an ethyl group, or an isopropyl group,
or
wherein x represents a point of attachment to L1, Y, or P and δ represents a point of attachment to C2;
C2 is an extension of C1 with the structure:
wherein R2 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, δ represents a point of attachment to C1 and ε represents a point of attachment to L2 or L3;
L2 is optional, if present L2 is flexible extension with the structure:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein ε represents a point of attachment to C2 and ϕ represents a point of attachment to L3 and wherein at least one of L1 or L2 is present, and
L3 is a nucleotide attachment group with the structure:
wherein ϕ represents a point of attachment to C1, C2, or L2 and γ represents a point of attachment to the nucleotide.

12 more elements

A compound having a structure P—Y-L1-C1-C2-L2-L3-nucleotide, wherein:

P is a bound group that is a peptide that is an enzyme, a linked nucleotide comprising at least one of DNA, RNA, or a synthetic nucleotide having a universal base, or a linked nucleotide that is complementary to the nucleotide;
Y is optional, if present Y is a bound group attachment group with the structure:
α-S-β, or α-NH2-β, wherein α represents a point of attachment to P and β represents a point of attachment to L1 or C1;
L1 is optional, if present L1 is a flexible extension with the structure one or more of:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein β represents a point of attachment to Y or P and χ represents a point of attachment to C1;
C1 is a cleavable group with the structure:
wherein X1 is 1 to 4 ring substituents selected from the group consisting of a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubstituted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, and a halogen and X2 is hydrogen, a methyl group, an ethyl group, or an isopropyl group,
wherein χ represents a point of attachment to L1, Y, or P and δ represents a point of attachment to C2, L2, or L3;
C2 is optional, if present C2 is an extension of C1 with the structure:
wherein R2 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, δ represents a point of attachment to C1 and ε represents a point of attachment to L2 or L3;
L2 is optional, if present L2 is flexible extension with the structure:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein ε represents a point of attachment to C1 or C2 and ϕ represents a point of attachment to L3 and wherein at least one of L1 or L2 is present, and
L3 is a nucleotide attachment group with the structure:
wherein ϕ represents a point of attachment to C1, C2, or L2 and γ represents a point of attachment to the nucleotide.

11 more elements

A compound having a structure P—Y-L1-C1-C2-L2-L3-nucleotide, wherein:

P is a bound group that is a peptide, a linked nucleotide, a fluorophore, or a water-soluble group;
Y is optional, if present Y is a bound group attachment group with the structure:
α-S—B, or α-NH2-β, wherein α represents a point of attachment to P and β represents a point of attachment to L1 or C1;
L1 is optional, if present L1 is a flexible extension with the structure one or more of:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein β represents a point of attachment to Y or P and χ represents a point of attachment to C1;
C1 is a cleavable group wherein x represents a point of attachment to L1, Y, or P and δ represents a point of attachment to C2, and C1 and C2 have one of the following structures:
C1 is
wherein X1 is 1 to 4 ring substituents selected from the group consisting of a hydroxyl group, an ether group with an alkyl group having 1 to 3 carbon atoms, an amine group which is unsubstituted or substituted with one or two alkyl groups having 1 to 2 carbon atoms, an alkyl group having 1 to 2 carbon atoms, and a halogen and X2 is hydrogen, a methyl group, an ethyl group, or an isopropyl group and C2 is
wherein in C2δ represents a point of attachment to C1 and ε represents a point of attachment to L2 or L3;
L2 is optional, if present L2 is flexible extension with the structure:
wherein n is 1-20, R1 is hydrogen or a substituted or unsubstituted straight or branched alkyl group having 1 to 6 carbon atoms, wherein ε represents a point of attachment to C1 or C2 and ϕ represents a point of attachment to L3 and wherein at least one of L1 or L2 is present, and
L3 is a nucleotide attachment group with the structure:
wherein ϕ represents a point of attachment to C2 or L2 and γ represents a point of attachment to the nucleotide.

10 more elements

Dependencies are inferred from the claim text.

Facts

Application: 16/781,987Filed 4 Feb 2020
Published: US 2021/0238577 A15 Aug 2021
Granted: 22 Apr 2025
Term ends: Not estimatedThe source records don’t support an estimate.

Classification13 codes

C07H 19/10
Pyrimidine radicals: with the saccharide radical esterified by phosphoric or polyphosphoric acids
G01N 33/547
Synthetic resin: with antigen or antibody attached to the carrier via a bridging agent
C12P 19/34
Nucleotides: polynucleotides, e.g. nucleic acids, oligoribonucleotides
C12Q 1/6834
Hybridisation assays: enzymatic or biochemical coupling of nucleic acids to a solid phase