Granted Patent B2

US 12,697,597 · App. 18/051,472 · Granted Aug 4, 2026

Inventors: Jeremy Lackey (Foster City, CA); David Dodd (San Francisco, CA); Stefan Pitsch (Stein am Rhein, CH)

Assignee: Twist Bioscience Corporation

B01J19/0046B01J2219/00587B01J2219/00596B01J2219/00722

Abstract

Claims (39)

1 . A method of synthesizing a polynucleotide comprising:

coupling at least one nucleoside attached to a solid support to a protected nucleoside, wherein the protected nucleoside comprises a protecting group, wherein the protecting group comprises a branched alkenylene, wherein the solid support comprises an addressable array, and wherein the addressable array comprises a plurality of loci;

contacting an amount of a reagent with the protected nucleoside, wherein the contacting results in deprotection of the protected nucleoside; and

repeating the coupling and contacting steps to synthesize the polynucleotide.

2 . The method of claim 1 , wherein the amount of the reagent is less than about 15 mol % of the protected nucleoside.

3 . The method of claim 1 , wherein the protecting group protects a 5′ hydroxyl of the protected nucleoside.

4 . The method of claim 1 , wherein the reagent comprises a transition metal catalyst.

5 . The method of claim 4 , wherein the transition metal catalyst comprises:

a zero oxidation state;

a Pd(0) catalyst;

comprises one or more phosphine ligands; and/or

Pd(Ph 3 ) 4 .

6 . The method of claim 1 , further comprising contacting the protected nucleoside with a nucleophile.

7 . The method of claim 1 , wherein the reagent is in contact with the protected nucleoside for less than about 10 minutes.

8 . The method of claim 1 , wherein the method further comprises capping, wherein capping comprises applying acetyl chloride or acetic anhydride to the solid support.

9 . The method of claim 1 , wherein the polynucleotide is 50-300 bases in length.

10 . The method of claim 1 , further comprising applying a voltage to a solvent in fluid communication with the protected polynucleotide, wherein the voltage is applied for a duration of 0.1 seconds to 10 seconds.

11 . The method of claim 1 , wherein the protected nucleoside comprises the formula:

wherein:

R1 is a branched alkenylene;

R2 is alkylene or alkenylene, each of which is independently unsubstituted or substituted;

each R3, R4, R5, R6, R7, R8, or R9 is independently H, OH, a halogen, an O-alkyl, an N-alkyl, an O-alkyl-O-alkyl, an azide; or

R3 and R6 are taken together to form a ring; and

B is a monocyclic or bicyclic C4-6 heterocyclic ring.

12 . The method of claim 11 , wherein one or both of R3 and R4 is:

F;

OCH 3 ; or

OCH 2 CH 2 OCH 3 .

13 . The method of claim 1 , wherein the reagent comprises a Pd(0) complex with negatively charged aryl-sulfonate containing phosphine ligands.

14 . The method of claim 13 , further comprising:

applying a voltage to one or more loci to electrophoretically attract the Pd(0) complex with negatively charged phosphine ligands to selected loci to deprotect the protected nucleoside.

15 . The method of claim 1 , wherein each polynucleotide of the plurality of polynucleotides synthesized has a different sequence from another polynucleotide of the plurality of polynucleotides.

16 . The method of claim 1 , further comprising:

selectively reducing a Pd(II) precursor at one or more loci of the addressable array to generate a Pd(0) catalyst;

contacting the protected nucleoside with the Pd(0) catalyst and a nucleophile to deprotect the protected nucleoside.

17 . The method of claim 1 , wherein the plurality of polynucleotides are synthesized at a rate of about 100 nucleotides per hour.

18 . The method of claim 6 , wherein the nucleophile is N,N-dimethylbarbiturate.

19 . The method of claim 1 , further comprising an oxidation step, wherein the oxidation step comprises treatment with iodine, water, and a base, wherein the base is selected from pyridine, lutidine, or collidine.

20 . The method of claim 1 , wherein the protecting group comprising a branched alkenylene is protecting a 3′ hydroxyl of the protected nucleoside.

Assignments (1)

Continuity (2)

References Cited (348)