Reversible Nucleotide Terminator Synthesis Without Chromatography

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Solution Overview

Problem

The standard synthesis of reversible nucleotide terminators for next-generation DNA sequencing involves multiple reaction steps, purification, and lyophilization, making the process costly and inefficient.

Innovation Solution

A method for producing deoxynucleotide triphosphates using in-situ introduction of the methyl-methylenedisulfide group (-CH2SSMe) without chromatographic purification, utilizing DMTSF as a donor of alkyl thiol groups, allowing for scalable and eco-friendly synthesis.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If standard synthesis method with multiple purification steps is used, then product purity is maintained, but production cost increases and productivity decreases

Engineering Contradiction:

Improveproduction efficiencyVSAvoidmanufacturing cost

Core Design Contradiction:

ProductivityVSEase of manufacture

Solution Approach 1:

The invention extracts and eliminates the chromatographic purification steps from the synthesis process. By using in-situ thio-alkyl group transfer, the method achieves sufficient product purity without requiring intermediate chromatographic purifications, thereby reducing manufacturing complexity and cost while maintaining productivity

Inventive Principle:

Principle #2Taking out (Extraction)

Solution Approach 2:

The invention merges the thio-alkyl group introduction step with the synthesis reaction step. The DMTSF reagent performs dual functions: introducing the thio-alkyl group and simultaneously acting as a protecting group, eliminating the need for separate purification and protection steps, thus improving both productivity and ease of manufacture

Inventive Principle:

Principle #5Merging (Combining)

2Productivity

If multiple reaction steps with purification are used, then product quality is ensured, but process time increases

Engineering Contradiction:

Improveproduction speedVSAvoidprocess duration

Core Design Contradiction:

ProductivityVSLoss of time

Solution Approach 1:

The invention performs preliminary protection of the 3'-OH group using DMTSF before the main synthesis reaction. This preliminary action prevents side reactions and eliminates the need for post-reaction purification steps, reducing overall process time while ensuring product quality

Inventive Principle:

Principle #10Preliminary action

Solution Approach 2:

The invention enables continuous synthesis by eliminating intermediate purification and lyophilization steps. The in-situ thio-alkyl group transfer allows the reaction to proceed continuously without interruption, significantly reducing process duration while maintaining product quality through the inherent selectivity of the reaction

Inventive Principle:

Principle #20Continuity of useful action

3Manufacturing precision

If chromatographic purification is performed at each step, then product purity is maximized, but device complexity and cost increase

Engineering Contradiction:

Improveproduct purityVSAvoidpurification equipment

Core Design Contradiction:

Manufacturing precisionVSDevice complexity

Solution Approach 1:

The invention replaces expensive and complex chromatographic purification equipment with simple filtration and concentration steps. The DMTSF reagent and its byproducts are designed to be easily removable through simple means, eliminating the need for sophisticated purification devices and reducing overall system complexity

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

4Productivity

If standard synthesis protocol is used, then product quality is maintained, but scalability is limited

Engineering Contradiction:

ImprovescalabilityVSAvoidproduct quality

Core Design Contradiction:

ProductivityVSManufacturing precision

Solution Approach 1:

The invention changes the reaction parameters by using DMTSF as both protecting group and thio-alkylating agent under mild conditions. This parameter change allows the reaction to be easily scaled up from laboratory to industrial production while maintaining product quality, as the reaction conditions remain consistent across different scales

Inventive Principle:

Principle #35Parameter changes

Applied Scientific Principles

This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.

Function Achieved in This Case

This method significantly reduces production costs by eliminating purification steps and lyophilization, increasing yield and reducing overall process time, enabling cost-effective upscale production of reversible nucleotide terminators.

Implementation Method 1

DMTSF (Dimethyl(methylthio)sulfonium tetrafluoroborate) or its analogues (1) which acts as a donor of alkyl thiol group (RS +(3) forming 3'-O-CH 2 SSR capped nucleotide

Methodology Applied

Scientific EffectAlkyl thiol group transfer: Chemical Bonding

Data Source

PatentEP4036098B1Synthesis of reversible nucleotide terminators by in-situ thio-alkyl group transfer for use in DNA sequencing by synthesis

Publication Date: 2026.01.28 MILTENYI BIOTEC BV & CO KG

Figure 1~2
Figure 3

AI summary

The invention is directed to a method for production of a deoxynucleotide triphosphate according to general formula (7) wherein B is a nucleobase and R is an alkyl group having 1 to 4 carbon atoms and n is an integer from 1 to 10 by reaction of reaction precursor of compound 6 wherein R1, R2 and R3 are independently H, O-Alkyl with alkyl residues having 1 to 4 carbon atoms or halogen and n is an integer from 1 to 10 with dialkyl(alkylthio)sulfonium salt (8B) wherein R4, R5 R6 are independently alkyl residues having 1 to 4 carbon atoms and X is selected from the group consisting of BP4-, F-, Cl-, Br-, I-, SO42-, SO32- in aqueous solution having a pH between 3 and 7.