Confirmation of nucleotide elongation chain

Abstract

A method and system for synthesizing oligonucleotides in a reaction chamber containing a solid support. A nucleotide monomer is selected for addition to the oligonucleotide. An inlet stream containing the selected monomer is fed into a reaction chamber (column) of the reaction chamber, providing the reaction chamber with conditions for oligonucleotide synthesis. The outlet stream from the reaction chamber can be drained and / or split or directed to an analysis station where the eluate is analyzed by mass spectrometry (MS) to reliably identify the monomer fed into the reaction chamber through the inlet. Based on the results of the MS analysis, it is determined whether the outlet stream contains the selected nucleotide monomer or a derivative thereof.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS none

[0002] The present disclosure relates to methods and systems for synthesizing oligonucleotides, such as RNA and DNA, and for monitoring such synthesis. [Background technology]

[0003] Solid-phase synthesis is a useful tool that can be used to prepare oligonucleotide products for many applications. Chemical synthesis of oligonucleotides is typically performed in a sequential manner, with one end of a growing nucleotide chain attached to a solid surface and reactive nucleotide monomers sequentially condensed using a repetitive synthesis cycle. The first step of the synthesis cycle typically involves the reaction of a protected nucleotide monomer with a hydroxyl group on the nucleoside-linked surface. After initially coupling a reactive phosphorus group to the hydroxyl on the surface, subsequent steps typically involve capping any unreacted hydroxyl groups and then oxidizing the reactive phosphorus intermediate. The final step of the cycle typically involves unblocking the hydroxyl group of the added nucleotide to prepare it for coupling with the next protected nucleotide monomer to be added. Upon completion of the nucleotide addition cycle, the desired oligonucleotide product is released from the solid phase, deprotected, and further utilized in biological applications.

[0004] The desired oligonucleotide synthesis provides the desired oligonucleotide product in high yield and purity, so that the completed synthesis composition contains only the desired oligonucleotide product. It is also desirable to be able to monitor the oligonucleotide synthesis in real time or near real time. This technology would be advantageous over current products and is not currently known to exist in the industry.

[0005] Alternative techniques for confirming the desired sequence of synthesized oligonucleotide products can be completed within the synthesis chamber, and the product removed from the synthesis column after cleavage and deprotection can be analyzed by high-resolution mass spectrometry (MS-MS) or next-generation sequencing (NGS). However, while MS can confirm the appropriate molecular weight of full-length synthetic oligonucleotides, it is often impossible to confirm the exact sequence, i.e., the order of monomer composition, of the full-length oligonucleotide product. Because oligonucleotides are long (30 mers or less) and some monomers are chemically modified, obtaining sequence information for the desired oligonucleotide product is severely limited. Furthermore, prior to these approaches, laborious and time-consuming chromatographic conditions are often required to separate the desired oligonucleotides from shorter oligonucleotides or other impurities. Furthermore, none of these approaches can be performed in real time. MS-MS techniques are limited by the length of the oligonucleotide sequence, with the signal-to-noise ratio decreasing significantly as the sequence length increases. Due to the polymerase function and requirements, NGS may not be able to "read" or identify chemically modified nucleotides. For some oligonucleotide therapeutics and other products, the inclusion of modified nucleotides is desirable.

[0006] Another approach uses online infrared (IR) analysis to identify sequences during synthesis. However, this technique has limitations in that it cannot easily distinguish between modified and unmodified nucleosides. Because the differences in the infrared spectra between single-atom changes, such as 2'-modified nucleotides, are extremely small, thorough visual comparisons are required to compare IR spectra. On the other hand, single-atom changes within the same 2'-modified nucleotide can be distinguished by directly measuring their molecular mass, even if the mass difference is only one unit, within the mass accuracy of the detector.

[0007] Therefore, there is a need to monitor synthesis (in-line) to verify the production of validated oligonucleotide sequences. There is also a need for methods and systems for real-time monitoring of oligonucleotide synthesis. Summary of the Invention

[0008] In one aspect of the present invention, there is provided a method for synthesizing oligonucleotides and monitoring their synthesis in real time, comprising the steps of selecting a nucleotide monomer for addition to an oligonucleotide, supplying an inlet stream to a reaction chamber for oligonucleotide synthesis, providing conditions in the reaction chamber for oligonucleotide synthesis, diverting an outlet stream from the reaction chamber to an analysis station, analyzing the outlet stream or a sample thereof by mass spectrometry (MS) in the analysis station, and determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof based on the results of the MS analysis.

[0009] In another aspect of the present invention, a system for synthesizing oligonucleotides is provided, the system comprising a reaction chamber including an inlet, a solid support for oligonucleotide synthesis, and an outlet, and a mass spectrometry (MS) device fluidly connected to the outlet of the reaction chamber.

[0010] These and other features and advantages of the methods and systems of the present invention will become apparent from the following detailed description taken in conjunction with the appended claims. [Brief explanation of the drawings]

[0011] [Figure 1] FIG. 1 shows a typical chemical synthesis of an oligonucleotide on a solid support. [Figure 2] FIG. 1 illustrates one embodiment of a system of the present invention. [Figure 3] FIG. 1 shows a flow chart for one embodiment of the method of the present invention. [Figure 4]FIG. 1 shows mass spectrum data of Sample 1-1 in Example 1. [Figure 5] FIG. 1 shows mass spectrum data of Sample 1-2 in Example 1. [Figure 6] FIG. 1 shows mass spectrum data of Sample 1-3 in Example 1. [Figure 7] FIG. 1 shows mass spectrum data of Samples 1-4 in Example 1. [Figure 8] FIG. 1 shows mass spectrum data of Samples 1-5 in Example 1. [Figure 9] FIG. 1 shows mass spectrum data of Samples 1-6 in Example 1. [Figure 10] FIG. 1 shows mass spectrum data of Samples 1-7 in Example 1. [Figure 11] FIG. 1 shows mass spectrum data of Samples 1-8 in Example 1. [Figure 12] FIG. 1 shows mass spectrum data of Sample 2-1 in Example 2. [Figure 13] FIG. 1 shows mass spectrum data of Sample 2-2 in Example 2. [Figure 14] FIG. 1 shows mass spectrum data of Sample 2-3 in Example 2. [Figure 15] FIG. 1 shows mass spectrum data of Sample 2-4 in Example 2. [Figure 16] FIG. 1 shows mass spectrum data of Sample 2-5 in Example 2. [Figure 17] FIG. 1 shows mass spectrum data of Sample 2-6 in Example 2. [Figure 18] FIG. 1 shows mass spectrum data of Sample 2-7 in Example 2. DETAILED DESCRIPTION OF THE INVENTION

[0012] Before describing various embodiments, it is to be understood that the teachings of the present disclosure are not limited to the particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present teachings will be limited only by the appended claims. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present teachings, some exemplary methods and materials are now described.

[0013] The methods and systems of the present invention can confirm the order of addition of each nucleotide monomer to a growing oligonucleotide in real time by using in-line mass spectrometry to analyze the exit stream from the reaction chamber of an oligonucleotide synthesizer. The methods and systems of the present invention solve some problems and offer some advantages. For example, they are the first to verify oligonucleotide synthesis in real time by in-line mass spectrometry (MS), providing the order of nucleotide monomer addition in real time. They enhance the accuracy of sequence verification by evaluating the characteristics of simple individual nucleotide monomers used in each cycle of synthesis, rather than the highly complex final oligonucleotide as a whole. The methods and systems of the present invention are independent of the length of the oligonucleotide or its chemical modifications. They are also more specific than existing online techniques. The methods and systems of the present invention generally do not require high-resolution mass spectrometry.

[0014] The methods and systems of the present invention can be used to synthesize oligonucleotides, such as oligoribonucleotides, oligodeoxyribonucleotides, and chimeric oligonucleotides. The desired oligonucleotides may contain ribonucleotides, deoxyribonucleotides, or mixtures thereof, and modified nucleotides (e.g., 2'-O-methyl, 2'-fluoro, LNA, 2'-MOE, 2'-CNEt, etc.). In some embodiments, a method for synthesizing or monitoring the synthesis of an oligonucleotide of a defined sequence is provided. The method includes the steps of: selecting chemicals to be added to the oligonucleotide; feeding an inlet stream into a reaction chamber (e.g., a column containing a solid support) for oligonucleotide synthesis; providing conditions in the reaction chamber for oligonucleotide synthesis; diverting the outlet stream from the reaction chamber to an analysis station; utilizing mass spectrometry (MS) in the analysis station to analyze the outlet stream at a specified time if the outlet stream contains an activated nucleotide phosphoramidite solution diverted from the synthesis apparatus after coupling; and determining whether the outlet stream contains a selected activated nucleotide monomer or a derivative thereof based on the analysis of the MS results.

[0015] Figure 1 is a schematic diagram of a typical chemical synthesis cycle for producing oligonucleotides on a solid support. To achieve synthesis, the first step in the synthesis cycle generally involves the reaction of a protected nucleotide monomer with a nucleoside attached to the surface of a solid support (bead or resin) or with a hydroxyl group on the surface. The hydroxyl group on the surface can be part of a cleavable universal linker (such as a Unylinker or succinic acid linker) or a non-cleavable surface linker. After initially coupling a reactive phosphorus group to the hydroxyl, subsequent steps typically involve capping any unreacted hydroxyl groups and subsequent oxidation of the reactive phosphorus intermediate (from a phosphite triester to a phosphate triester). Typically, oligonucleotides contain internucleotide linkages between the 5'- and 3'-hydroxyls of adjacent nucleotides, which can be phosphate or modified phosphorus groups (typically 2-cyanoethyl-protected phosphate groups). Under certain conditions, oxidation is performed before capping, especially when certain modified phosphorus groups are used, particularly when the oxidation reagent generates modified phosphorus groups such as phosphorothioates, boranophosphonates, or phosphoramidites. The final step in the synthesis cycle is usually to remove a protecting group (e.g., dimethoxytrityl group (DMT)) to deblock the 5'-terminal hydroxyl group of the growing oligonucleotide. The deblocked hydroxyl group can then be coupled to the next protected nucleotide phosphoramidite monomer fed into the reaction chamber under coupling conditions. Once coupling of the last nucleotide selected for the sequence is complete, the desired oligonucleotide product is released from the solid phase (resin), deprotected, purified, and utilized for further biological applications.

[0016] Another aspect of the present disclosure provides a system for synthesizing or monitoring oligonucleotide synthesis. The system includes an automated oligonucleotide synthesizer, which includes a reaction chamber including at least one inlet for supplying reagents to a column on which oligonucleotide synthesis is performed, a solid-phase support contained in the column for oligonucleotide synthesis, and an outlet for discharging excess reagents, solvents, and wash solutions to waste. The system also includes a mass spectrometry (MS) device fluidly connected to the outlet of the reaction chamber. Reaction chambers suitable for solid-phase synthesis of oligonucleotides are described herein and in various publications.

[0017] FIG. 2 illustrates one embodiment of a system 200 of the present invention. A reaction chamber 202 for oligonucleotide synthesis has an inlet 204 and an outlet 206. The reaction chamber 202 contains a solid support compatible with solid-phase synthesis of oligonucleotides. The reaction chamber 202 can define a column containing the solid support, such that components of the inlet stream pass through the column as they move toward the outlet 206. A sample tee 208 receives the outlet stream from the outlet 206, so that the outlet stream is sent to a process waste 210 or for analysis. In some embodiments, some or most of the outlet stream is sent to the process waste 210, and a sample of the outlet stream is sent for analysis. A pump 212 provides a specific force to drive the outlet stream from the reaction chamber 202 toward the analytical components. For example, an isocratic pump can be used to continuously pump the outlet stream throughout the process. Initially, the outlet stream from the 202 reaction chamber is sent to waste 214, but at a desired time, for example during a column wash phase of the synthesis, a "start" signal is sent and a series of programmed tasks are executed to automatically collect a sample for retention and analysis. The programmed tasks may include switching the sample valve 216 so that the flow path from the isocratic pump 212 switches the outlet stream flow from waste 214 to analytical components, such as by sending the outlet stream or a sample thereof to a UHPLC-MS instrument.

[0018] A UHPLC-MS online sample manager 218 is used to collect samples from the flowing outlet stream, which may be placed in a sample vial for analysis and / or retention.

[0019] In some embodiments, immediately after sample generation, the system 200 automatically begins analyzing the sample. In some embodiments, the system also includes a dilution source 220 and a mobile phase pump 222 fluidly connected to an online sample manager 218. The system can also include an MS dilution source 224 and a flow rate control pump 226 fluidly connected to an MS instrument 230. The mobile phase pump 222 can control the flow of sample from the online sample manager 218 to the mass analyzer 230. During analysis, the sample may be automatically diluted online to adjust the concentration of the sample using the flow rate control pump 226 and valve 228 prior to MS detection. A computing device 232 is communicatively connected to the mass analyzer 230 to receive the results of the MS analysis. In certain embodiments, particularly for large-scale manufacturing, an automatic stop mechanism is implemented to abort oligonucleotide synthesis if the results indicate an error in identifying the expected monomer phosphoramidite, indicating that an incorrect base has been added to the growing oligonucleotide sequence.

[0020] 3 shows a flowchart for an embodiment of the method of the present invention. In 301, the online sample manager 218 and mass spectrometer 230 are started. In 302, the sample pump 212 is started. In 303, synthesis of the desired oligonucleotide in the reaction chamber 202 is initiated. In 304, after a conditioning period for oligo synthesis in the reaction chamber 202, a sampling signal is sent to the online sample manager 218. The sampling signal may be sent by the reaction chamber 202, a sensor, a computing device communicatively connected to the reaction chamber 202, or a sensor associated with the reaction chamber 202. In 305, the online sample manager collects a sample of the outlet stream containing the phosphoramidite. In 306, the online sample manager sends the sample to the mass spectrometer. In 307, the mass spectrometer provides the mass of the phosphoramidite or its derivative in real time. At 308, when synthesis has finished and sample collection and analysis has been completed, the system may run the pumps idly until another "start" signal is issued by the reaction chamber 202. This then wakes the system to begin another cycle of sample collection and analysis.

[0021] After synthesis is complete, the resulting sample data is analyzed. Sample / mass identification is ordered. All resulting sample data is collected by computing device 232 and compiled into a report that confirms the sequence of the oligonucleotide product by verifying the order of nucleotide monomers delivered to the reaction chamber. The report may also include a timestamp of the MS analysis results, delivery time from the inlet stream to the reaction chamber, and correlation of the timestamp with delivery time, residence time, or other data for confirmation or verification.

[0022] The methods and systems of the present invention can be used to synthesize any desired oligonucleotide product. In some embodiments, the oligonucleotide is an oligoribonucleotide (RNA). In some embodiments, the oligonucleotide is an oligodeoxyribonucleotide (DNA). In some embodiments, the oligonucleotide is a chimeric oligonucleotide, containing both ribonucleotides and deoxyribonucleotides or modified deoxy / ribonucleotides (including, but not limited to, 2'-OMe, 2'F, 2'-MOE, LNA, phosphorothioate, PNA, and phosphonate linkages). In some embodiments, the oligonucleotide is at least 15 nucleotides in length. In some embodiments, the oligonucleotide is at least 50 nucleotides in length. In some embodiments, the oligonucleotide is at least 70 nucleotides in length. In some embodiments, the oligonucleotide is at least 75 nucleotides in length. In some embodiments, the oligonucleotide is at least 100 nucleotides in length. In some embodiments, the oligonucleotide is at least 125 nucleotides in length. In some embodiments, the oligonucleotide is at least 150 nucleotides in length. In some embodiments, the oligonucleotide is from about 15 nucleotides to about 500 nucleotides in length. In some embodiments, the oligonucleotide is from about 40 nucleotides to about 300 nucleotides in length.

[0023] In some embodiments, the desired oligonucleotide is synthesized using a phosphoramidite-based method. In some embodiments, the synthesis method comprises a support-bound nucleoside having a 5'-DMT protecting group. In some embodiments, the synthesis method comprises a support-bound nucleoside having a 3'-DMT protecting group. In some embodiments, the synthesis method comprises a support-bound nucleoside having a 5'-silyl protecting group. In some embodiments, the synthesis method comprises a support-bound nucleoside having an oxidatively removable protecting group. In some embodiments, the synthesis comprises the steps of detritylation, coupling the support-bound nucleoside with a nucleoside phosphoramidite monomer, capping any unreacted 5'-hydroxyl groups, and phosphoramidite oxidation. In some embodiments, the oligonucleotide synthesis is automated. In some embodiments, the oligonucleotide is detritylated before performing the method of the present invention.

[0024] In some embodiments, the oligonucleotide comprises a phosphorus protecting group or a nucleobase protecting group. In some embodiments, the oligonucleotide comprises a phosphorus protecting group and a nucleobase protecting group. In some embodiments, the oligonucleotide is an RNA comprising a phosphorus protecting group, a nucleobase protecting group, and a 2'-hydroxyl protecting group.

[0025] In some embodiments, the selected nucleotide monomer is a ribonucleotide having a 2'-protecting group and a 5'-protecting group. For example, the ribonucleotide may have a 5'-DMT protecting group and a 2'-protecting group selected from the group consisting of a thionocarbamate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a t-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

[0026] In some embodiments, the selected nucleotide monomer contains a phosphorus-protecting group, and the phosphorus moiety of the oligonucleotide is bound to the phosphorus-protecting group. In some embodiments, the phosphorus moiety is a phosphate, phosphoramidite, or H-phosphonate group. In some embodiments, the phosphorus-protecting group is a methyl or cyanoethyl group (e.g., a beta-cyanoethyl group). Methyl groups may be removed using, for example, thiophenol or disodium 2-carbamoyl-2-cyanoethylene-1,1-dithiolate. Cyanoethyl groups may be removed using, for example, non-nucleophilic or hindered amines such as diethylamine, t-butylamine, or 1,8-diazabicycloundec-7-ene (DBU).

[0027] In some embodiments, the selected nucleotide monomer comprises a nucleobase protecting group. Any nucleobase in the oligonucleotide may comprise a nucleobase protecting group. In some embodiments, the nucleobase protecting group is acetyl, isobutyryl, benzoyl, or the like. For example, the protected nucleobase may be N 6 -benzoyl-A,N 6 -Isobutyryl-A,N 4 -Acetyl-C,N 4 -Isobutyryl-C, or N 2 -isobutyryl-G (containing an amidine protecting group such as dimethylacetamidine). In some embodiments, the nucleobase protecting group is removed by contacting the oligonucleotide with a polyamine. In some embodiments, the nucleobase protecting group is removed by contacting the oligonucleotide with a diamine such as 1,2-diaminoethane. In some embodiments, exposing the oligonucleotide to 1,2-diaminoethane at room temperature for 2 hours results in deprotection of the nucleobase. In some embodiments, the nucleobase protecting group is benzoyl, isobutyryl, acetyl, phenoxyacetyl, t-butylphenoxyacetyl, dimethylformamide, dimethylacetamidine, or the like. In some embodiments, the selected nucleotide monomer contains a modified nucleobase, for example, 5-methyl-C(m 5 C), 5-hydroxymethyl-C (5hmC), 5-methyl-U (m5 5-alkyl-pyrimidines such as N 6 -methyl-A (m6A) and other N 6 -Alkylpurine, pseudourin (Φ), 1-methylpseudourin (m 1 Φ, 2-thiouridine (2sU), 5-fluoro-U (5FU), 7-deazapurine, or any other known modified nucleobase used in oligonucleotide synthesis (see, for example, Hu, B., Zhong, L., Weng, Y. et al. Therapeutic siRNA: state of the art. Sig Transduct Target Ther 5, 101 (2020). https: / / doi.org / 10.1038 / s41392-020-0207-x. and Q. Chen, Y. Zhang, H. Yin. Recent advances in chemical modifications of guide RNA, mRNA, and donor template for CRISPR-mediated genome editing. Adv. Drug Deliv. Rev., 168 (2021), pp. 246-258).

[0028] Synthesis of oligonucleotides may occur in the 3' to 5' direction, with the final protecting group being removed from the 5'-hydroxyl of the resulting oligonucleotide, or synthesis may occur in the 5' to 3' direction, with the final protecting group being removed from the 3'-hydroxyl of the resulting oligonucleotide.

[0029] The synthesized oligonucleotides can be treated with various basic amines (ammonia, ammonium hydroxide, methylamine, ethylenediamine, etc.) to optionally remove various protecting groups, nucleobase protecting groups, and, if present, base-labile 2'-hydroxyl protecting groups such as TC or PivOM, and to cleave linkers, typically succinic acid linkers or unylinkers, that attach the oligonucleotide to the solid support.

[0030] The 2'-hydroxyl protecting group can be removed using any suitable conditions. In some embodiments, the TC or PivOM protecting group is removed from the 2'-hydroxyl by contacting the oligoribonucleotide with a diamine such as 1,2-diaminoethane. In some embodiments, the PivOM protecting group is removed from the oligoribonucleotide by contacting the oligoribonucleotide with ammonia or an alkylamine. In some embodiments, contacting the oligoribonucleotide with a diamine, ammonia, or alkylamine simultaneously deprotects the 2'-hydroxyl group and cleaves the oligoribonucleotide from the solid support. In some embodiments, the ACE protecting group is removed by contacting the oligoribonucleotide with an acid.

[0031] After coupling the final nucleotide monomer into the desired oligonucleotide product sequence, the synthesized oligonucleotide is cleaved from the solid support, partially (for certain oligoribonucleotides with a remaining 2'-hydroxyl) or completely deprotected, and then eluted from the solid support in a buffer solution and exits the reaction chamber through an outlet. A typical buffer solution can contain 0.2 M sodium phosphate, 0.6 M sodium chloride, pH 7.4, and 10% dimethylformamide (loading buffer). In some embodiments, the oligonucleotide is treated with 1,2-diaminoethane, which removes the 2'-OH protecting group (e.g., TC or PivOM), nucleobase amine protecting groups (e.g., acetyl, benzoyl, isobutyryl, phenoxyacetyl, amidine), and phosphorus protecting groups (i.e., beta-cyanoethyl) from the oligonucleotide, simultaneously cleaving the oligonucleotide from the synthesis solid support before elution.

[0032] Nucleotide monomers contain reactive phosphorus or modified groups, such as phosphoramidites, H-phosphonates, or other reactive phosphorus groups, so they can be coupled to hydroxyl groups embedded on solid surfaces or growing oligonucleotides. Nucleotide monomers are derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), protected ribonucleosides (A, C, G, and U), or protected chemically modified nucleosides. The typical desired oligonucleotide product contains an internucleotide linkage between the 5'-hydroxyl and 3'-hydroxyl groups of adjacent nucleotides. However, it may also be desirable to form non-natural internucleotide linkages between either of the hydroxyl groups of adjacent nucleotides.

[0033] "Nucleotide" refers to a subunit of an oligonucleotide (DNA or RNA or their analogs) that contains a phosphate group, a sugar group, and a heterocyclic base, as well as analogs of such subunits. Other groups (e.g., protecting groups) can be attached to any component of the nucleotide. "Nucleoside" or "nucleoside moiety" refers to an oligonucleotide subunit that contains a sugar group and a heterocyclic base, as well as analogs of such subunits.

[0034] The terms "nucleoside" and "nucleotide" are intended to include those moieties that contain not only the known purine and pyrimidine bases, e.g., adenine (A), thymine (T), cytosine (C), guanine (G), or uracil (U), but also modified and other heterocyclic bases. Pyrimidine, purine, heterocyclic, and modified heterocyclic bases are referred to herein as "nucleobases." Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated or other heterocycles. Such modifications include, for example, diaminopurine and its derivatives, inosine and its derivatives, alkylated purines or pyrimidines, acylated purines or pyrimidines, thiolated purines or pyrimidines, or the addition of protecting groups such as acetyl, difluoroacetyl, trifluoroacetyl, isobutyryl, benzoyl, 9-fluorenylmethoxycarbonyl, phenoxyacetyl, dimethylformamidine, dibutylformamidine, and N,N-diphenylcarbamate. Furthermore, the terms "nucleoside" and "nucleotide" include moieties containing not only conventional ribose and deoxyribose sugars, but also other sugars. Modified nucleosides or nucleotides also include modifications of the sugar moiety, for example, replacement of one or more hydroxyl groups with halogen atoms or aliphatic groups, or functionalization such as ethers, amines, and the like. Modified nucleosides or nucleotides also include modifications of the internucleotide linkage or backbone moiety. "Analog" means a molecule having structural features recognized in the literature as being a mimetic, derivative, similar structure, or other similar term, and includes, for example, polynucleotides incorporating non-natural (usually not occurring in nature) nucleotides, non-natural nucleotide mimetics such as 2'-modified nucleosides, peptide oligonucleotides, oligomeric nucleoside phosphonates, and any polynucleotide to which substituents such as protecting groups or linking groups have been added.

[0035] "Oligonucleotide" refers to a compound containing multiple nucleoside moiety subunits linked by internucleotide bonds. Thus, this term also refers to a compound containing multiple nucleotide moiety subunits or residues. Oligonucleotides may contain ribonucleosides or deoxyribonucleosides, or mixtures thereof. Oligonucleotides may contain natural and / or unnatural nucleosides, nucleoside analogs, and modified nucleosides, and may contain natural (phosphodiester) and / or unnatural internucleotide bonds (e.g., amide bonds such as phosphorothioate, phosphonate, boranophosphonate, phosphoramidite, peptide nucleic acid (PNA)).

[0036] The inlet stream can be supplied to the reaction chamber by any suitable technique. Generally, the reaction chamber has one or more inlets connected to sources of reactive nucleotide monomers, other reagents for coupling and deprotection reactions, and one or more diluents. The inlet stream typically contains a selected nucleotide monomer intended for addition to the oligonucleotide being synthesized. However, the methods and systems of the present invention are believed to be able to reliably and quickly detect cases in which a selected nucleotide monomer is not present in the inlet stream when it should be, possibly due to mechanical failure or operational error. In some embodiments, the method also includes stopping the supply of the inlet stream to the reaction chamber if it is determined that the outlet stream does not contain the selected nucleotide monomer.

[0037] In some embodiments, the inlet stream is provided from one or more reservoirs fluidly connected to the inlet of the reaction chamber. The reservoirs can be connected to the inlet directly or indirectly, for example, via a series of conduits interposed by one or more valves. One of the reservoirs can be a source of the selected nucleotide monomer and can be fluidly connected to the inlet stream prior to the providing step, for example, by opening a valve disposed between the reservoir and the inlet. In some embodiments, the step of determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof is performed before the next nucleotide monomer is provided to the reaction chamber.

[0038] In some embodiments of the methods and systems, oligonucleotides are synthesized in a reaction chamber configured for solid-phase synthesis. In some embodiments, the reaction chamber contains a solid support, e.g., a planar substrate or a bead. In some embodiments, the solid support comprises a plurality of discrete resin pieces. In some embodiments, the plurality of discrete resin pieces comprises a plurality of resin beads.

[0039] In some embodiments, oligonucleotides are prepared by solid-phase synthesis. In such embodiments, synthesis is performed on a solid-phase support held between two filters in a column through which reagents and solvents can freely pass. In some embodiments, synthesis is performed on a planar surface. In some embodiments, synthesis is performed on a non-planar surface. In some embodiments, synthesis is performed on a surface of a support that does not allow the relevant reagents and chemicals of oligonucleotide synthesis to diffuse, absorb, or penetrate beyond the surface into the bulk of the support (in contrast to some polymeric oligonucleotide reaction chamber supports, where such diffusion and penetration are possible and oligonucleotide synthesis occurs within the bulk of the support). In some embodiments, synthesis is performed on a substantially smooth support surface that is at most superficially irregular, such that any irregularities are not of a magnitude sufficient to substantially affect the rate at which reagents are uniformly applied, mixed, or removed from the surface (in contrast to some controlled-pore glass oligonucleotide reaction chamber supports, which contain pores and irregularities that slow the application and removal of reagents). A solid, substantially smooth surface need not be flat, and includes, for example, a flat surface, an array of tubes, cylinders, depressions or wells, and combinations of these elements, as well as other designs exhibiting surface portions with the above attributes. For example, a substantially smooth surface includes a surface (or portion of a surface) that can be treated with an inkjet printhead.

[0040] In some embodiments, oligonucleotides are attached to a solid support such as controlled pore glass or a polymeric support, e.g., a polystyrene (PS) support. Suitable solid supports, which may be polymeric, can have a variety of forms and compositions and can be derived from naturally occurring, synthetically modified naturally occurring, or synthetic materials. Examples of suitable support materials include, but are not limited to, polysaccharides such as agarose (e.g., commercially available from Pharmacia under the trademarks Sepharose®) and dextran (e.g., commercially available from Pharmacia under the trademarks Sephadex® and Sephacryl®), polyacrylamide, polystyrene, polyvinyl alcohol, copolymers of hydroxyethyl methacrylate and methyl methacrylate, silica, Teflon, glass, and the like.

[0041] In some embodiments, oligonucleotide synthesis is carried out on a solid phase (e.g., CPG) containing a 3'-terminal nucleotide attached to the solid phase by a base-labile linker (such as a succinic acid linker or a universal support linker (e.g., Glen Research UnySupport or UnyLinker)). The synthesis cycle includes deprotection of the 5'-OH protecting group (e.g., trityl or DMT), coupling of a phosphoramidite in the presence of an activating agent, capping of unreacted hydroxyl groups with a base-labile protecting group (e.g., acetyl protecting group), and oxidation of the phosphite triester bond to a phosphate triester bond.

[0042] The reaction chamber may contain or be connected to devices that provide multiple functions to accomplish oligonucleotide synthesis, such as delivery of multiple reagents in liquid form from a reagent storage container to the reaction chamber; delivery of multiple reagents to a container that can be used to mix the reagents before delivery to the reaction chamber; argon purging at a constant flow rate using a needle valve to maintain an inert atmosphere over all storage containers and the reaction chamber; permitting metered delivery of reagents to ensure specified delivery volumes; a mechanism for stirring and mixing the resin and solvent inside; a UV detector connected to the outlet to detect UV absorption of the outlet stream leaving the reaction chamber; and a device for detecting the UV absorption of the outlet stream leaving the reaction chamber throughout the synthesis. a pressure sensor that detects the pressure applied to the reaction chamber; a pump that connects the solvent reservoir to the reaction chamber and subsequently to a waste line, supplying solvent to the reaction chamber and passing it directly to waste; a pump that allows flow from the outlet of the reaction chamber back to the inlet of the reaction chamber, allowing circulation of solvent within the reaction chamber; and one or more specific configurations of pumps and valves where reactive nucleotide monomers are delivered to the reaction chamber through one valve block and pump, and reagents are delivered to the reaction chamber through another valve block and pump, to prevent cross-contamination and to clean the valves and lines before delivery of the next reagent and monomer.

[0043] The present invention involves providing an inlet stream to a reaction chamber and providing conditions within the reaction chamber for oligonucleotide synthesis. Such conditions may be established before, during, or after the inlet stream is provided to the reaction chamber, and thus exist for at least a portion of the time the contents of the inlet stream are in the reaction chamber. Conditions for oligonucleotide synthesis include the selection and concentration of nucleotide monomers, the selection and concentration of reagents, increasing or decreasing temperature, etc. In some embodiments, the conditions are adapted or selected to accomplish one or more steps of a method for synthesizing oligonucleotides. For example, the conditions may result in a method including one or more activation steps, one or more coupling steps, one or more capping steps, one or more oxidation steps, one or more detritylation steps, one or more cleavage steps, and / or one or more deprotection steps.

[0044] In some embodiments, the synthesis methods of the present invention also include one or more of the following steps: attaching or coupling a first nucleotide to a solid support directly or indirectly through the 5'-OH of a nucleoside already attached to a solid support via a linker; removing the 5'-protecting group (e.g., DMT) from the first nucleotide; activating the 3'-phosphoramidite of the incoming selected monomer; coupling the selected nucleotide monomer to the 5'-OH of the first nucleotide, whereby the selected nucleotide monomer becomes the second nucleotide of the growing oligonucleotide attached to the first nucleotide attached to the solid support by a phosphite triester; oxidizing the phosphite triester to form a phosphate triester; and capping any unreacted 5'-OH nucleotides, such as by acetylation.

[0045] The methods and systems of the present invention may also include a step of splitting the outlet stream from the reaction chamber to an analysis station. The timing of splitting the outlet stream can be selected based on the synthesis cycle, and typically the outlet stream is split for analysis before or after the expected completion of the cycle. The outlet stream, or a sample of the outlet stream, can then be analyzed by mass spectrometry in the analysis station. If MS analysis reveals that the outlet stream contains selected nucleotide monomers or derivatives thereof, this confirms that the selected monomers were indeed delivered to the reaction chamber in the proper order and as intended by the defined sequence of the oligonucleotide being synthesized.

[0046] In some embodiments, MS analysis determines that derivatives of selected monomers are present in the outlet stream. Expected derivatives in the outlet stream include nucleotide monomers that have lost one or more protecting groups, such as phosphorus protecting groups (e.g., methyl or cyanoethyl groups), or that exist in activated forms (i.e., where diisopropylamine has been replaced with a tetrazole activator or ethylthiotetrazole (ETT)), or where the phosphoramidite has been hydrolyzed to the corresponding H-phosphonate.

[0047] In some embodiments, the method includes recording one or more time points associated with the synthesis of oligonucleotides, the operation of the reaction chamber, or related equipment. For example, the method can include time-stamping the results of the MS analysis and recording the delivery times at which various inlet streams were delivered to the reaction chamber. The method can also include associating the delivery times of the inlet streams with the timestamps and using the timestamps and associated delivery times to determine whether selected nucleotide monomers were present in the inlet streams. In some embodiments, the method can include calculating the nucleotide sequence of the oligonucleotides synthesized in the reaction chamber using the timestamps and associated delivery times. In some embodiments, delivery times are recorded for each time the composition of the inlet stream changes. In some embodiments, the delivery times are associated with timestamps used in the MS analysis results, such as by associating the timestamps with delivery times when the timestamps are separated by a selected time interval. The interval can be predetermined, fixed, or variable.

[0048] In some embodiments, one or more timestamps and one or more delivery times are used to determine whether a selected nucleotide monomer has been present in the inlet flow.In some embodiments, the method also includes a residence time corresponding to the measured or estimated time that the reactant has been present in the reaction chamber.The association between the timestamp and the delivery time can be based on or correlated with the residence time.

[0049] In some embodiments, synthesis of a desired oligonucleotide is confirmed using a timestamp and associated delivery time to calculate the nucleotide sequence of the oligoribonucleotide synthesized in the reaction chamber.

[0050] In some embodiments, the recorded time points are used as a quality control measurement or for validation of the oligonucleotide sequence. In some embodiments, the method further comprises dispensing or allowing the synthesized oligonucleotide if the calculated nucleotide sequence matches the predetermined nucleotide sequence. In some embodiments, the method further comprises withholding or removing the synthesized oligonucleotide if the calculated nucleotide sequence does not match the predetermined nucleotide sequence. In some embodiments, the inlet stream is not analyzed for the selected nucleotide monomers prior to the feeding step, since analysis of the outlet stream is sufficient as a quality control measurement.

[0051] In some embodiments, the methods of the present invention are current Good Manufacturing Practice (cGMP) compliant manufacturing methods that include appropriate quality controls to produce a desired oligonucleotide product. In some embodiments, the methods of the present invention are for the synthesis of a desired oligonucleotide product that is suitable for use as an active pharmaceutical ingredient (API) and is suitable for clinical use. In some embodiments, the present disclosure provides methods and systems for controlling the quality of oligonucleotides produced by solid phase synthesis, including cGMP compliant manufacturing methods.

[0052] In some embodiments, the methods further include comparing the results of MS analysis of the outlet stream or a sample thereof with one or more predetermined MS patterns. In some embodiments, the MS analysis includes evaluating the spectroscopic results of fragments having masses between 250 and 1000 Da. For example, if the selected nucleotide monomer is 793.3, and MS detects a fragment having a mass between 792.3 and 794.3 Da, the outlet stream is determined to contain the selected nucleotide monomer.

[0053] In some embodiments, the method further comprises one or more steps for purifying the oligonucleotide. For example, the method can include synthesizing the oligonucleotide on a solid support; reacting the oligonucleotide with an orthoester linker to form an oligonucleotide-orthoester linker conjugate, where the orthoester linker comprises an affinity tag; cleaving the oligonucleotide-orthoester linker conjugate from the solid support used for synthesis; isolating the oligonucleotide-orthoester linker conjugate using a chromatographic method or affinity capture method; and cleaving the orthoester linker from the oligonucleotide-orthoester linker conjugate, thereby releasing the purified oligonucleotide. Further details and teachings regarding the purification of oligonucleotides can be found in Dellinger et al., U.S. Patent Application No. 2020 / 0181124.

[0054] The methods and systems of the present invention also use or include a mass spectrometry (MS) device. MS devices can generally include an ion detector, a mass analyzer, and a detector.

[0055] A mass analyzer may be any device configured to separate, sort, or filter analyte ions based on their mass (e.g., mass-to-charge ratio, or m / z ratio). Examples of mass analyzers include, but are not limited to, multipole electrode structures (e.g., mass filters, ion traps), time-of-flight (TOF) analyzers, electrostatic analyzers (ESAs), and magnetic sector analyzers. A mass analyzer may include a system of multiple mass analyzers, particularly when ion fragmentation is desired. By way of example, a mass analyzer may be a tandem MS or MS n As another example, the mass analyzer may include a mass filter followed by a collision cell or other ion fragmentation device, followed by another mass filter or analyzer.

[0056] An MS device includes a detector configured to analyze a sample by measuring the mass-to-charge ratio of ions received by the detector. The results may be presented as a mass spectrum, a plot of intensity as a function of mass-to-charge ratio. Mass spectrometry is useful for distinguishing components of complex mixtures. In some embodiments, the MS device is selected from the group consisting of a time-of-flight mass spectrometer, a single quadrupole (SQ) LC / MSD, a triple quadrupole (TQ) LC / MS, and a quadrupole time-of-flight (TOF / Q-TOF).

[0057] In some embodiments, the MS analysis is performed with a low-resolution detector. The instrument can be configured for direct infusion mass spectrometry, liquid chromatography / mass spectrometry, gas chromatography / mass spectrometry, ion mobility / mass spectrometry, supercritical fluid chromatography / mass spectrometry, or any combination thereof. In some embodiments, the outlet stream is analyzed by liquid chromatography / mass spectrometry (LC / MS) or ultra-high performance liquid chromatography (UHPLC). In some embodiments, the MS instrument is a low-resolution MS instrument. As used herein, "low resolution" refers to analysis by mass spectrometry that results in a mass-to-charge ratio of two decimal places.

[0058] In some embodiments, the outlet stream or a sample thereof is purified using high-performance liquid chromatography (HPLC) prior to analysis by mass spectrometry. The outlet stream is typically introduced into the HPLC as a mixture containing multiple components. In some embodiments, the methods of the present invention can separate nucleotide monomers from other components of the outlet stream, which may be of similar size. The other components may be of any type. In some embodiments, the outlet stream or a sample thereof is purified using reverse-phase HPLC, for example, by using a reverse-phase column (e.g., a C5 or C18 hydrocarbon column). In some embodiments, the outlet stream or a sample thereof is purified on a normal-phase HPLC column. The HPLC system can include an injector, a pump, and an HPLC column. In some embodiments, the HPLC system is connected to a triple quadrupole LC-MS, an orbitrap LC-MS, an ion trap LC-MS, or a TOF LC-MS. In some embodiments, the methods and systems of the present invention include an ultra-high performance liquid chromatography mass spectrometer (UHPLC-MS) fluidly connected to the reaction chamber.

[0059] In some embodiments, the system also includes an online sample manager between the outlet of the reaction chamber and the MS instrument. The online sample manager receives the outlet stream from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument. The system may also include one or more pumps between the reaction chamber and the online sample manager. The system may also include one or more valves between the online sample manager and the MS instrument.

[0060] Other components of the systems of the invention may include one or more computing devices connected to the reaction chamber and / or its associated components. The one or more computing devices connected to the MS instrument may be the computing devices connected to the reaction chamber or may be separate, independent computing devices. For example, the invention may include one or more computing devices communicatively connected to the reaction chamber, the online sample manager, and / or the MS instrument, the one or more computing devices including a processor and a storage medium. The processor is configured to execute instructions stored on the storage medium to determine whether an outlet stream from the reaction chamber contains a selected nucleotide monomer or derivative thereof based on data from the MS instrument. The one or more computing devices may also be configured to create, maintain, regulate, or otherwise control conditions within the reaction chamber, and / or start and / or stop the supply of an inlet stream to the reaction chamber, and / or activate and / or deactivate the online sample manager. In some embodiments, the instructions stored on the computer medium include instructions for various experimental functions and / or instrument operations, such as operating a liquid chromatography instrument. Such instructions may be integrated with or separate from the operating instructions for the reaction chamber and / or the MS instrument.

[0061] In some embodiments, the system also includes a sensor at the outlet of the reaction chamber, which detects the presence or absence of nucleotide monomers in the diluent or the presence or absence of other components of the inlet stream. The sensor may be communicatively coupled to one or more computing devices, which may also be configured to direct the outlet stream to and / or activate an online sample manager if the sensor detects the presence or absence of nucleotide monomers or another component in the outlet stream.

[0062] As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has individual components and features which may be readily separated or combined with any of the features of some of the other embodiments without departing from the scope or spirit of the inventive teachings. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0063] It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Defined terms are inclusive of the technical and scientific meaning of the defined terms as commonly understood and accepted in the art of the present teachings.

[0064] The term "valve" generally includes any structure that can be adjusted (e.g., by flipping a switch or turning on and off) to change the flow path to, from, and / or through the structure. Generally, valves are substantially fluid-tight to prevent loss of fluid from the flow path. One example of a suitable valve (e.g., a first, second, third, or fourth valve) is a rotary valve, such as a rotary valve including a stator and a rotor. A rotary valve includes a stator and a rotor, and one or both of the stator and rotor can rotate to different rotary valve positions. The stator and rotor have surfaces adjacent to each other, and one or both are configured to rotate relative to the other. The inlet and outlet of the valve in this embodiment are passages or through-holes in the stator. The rotor includes switchable fluid paths, which may be grooves in the rotor surface. By rotating the stator and / or rotor, fluid paths connect different valve inlets to valve outlets. Another example of a suitable valve is a diaphragm valve. Typical valve materials include metallic materials, which may or may not be inert. Valves should have low dead volumes to allow for short flush times and to avoid sample entrapment.

[0065] The term "conduit" generally includes any structure configured to define a flow path for a fluid to travel from one point (e.g., a conduit inlet) to another point (e.g., a conduit outlet), although a conduit can also deliver fluid to intermediate points. A conduit may be flexible, rigid, or both, to some extent or in part. A conduit may be relatively long or short, and / or linear or nonlinear, so long as it provides a flow path from one component (e.g., a gas source) to another (e.g., a vent). For example, a conduit may be a long length of tubing, a short fitting, or a manifold with multiple inlets and / or outlets. Typically, a conduit has an inlet and an outlet, but in some embodiments, a conduit may have multiple inlets and / or outlets; for example, a conduit with two or more inlets converges or joins into one outlet, and a conduit with one inlet diverges or splits into two or more outlets. Conduits are often described by their length and internal diameter (id), which can be used to calculate the volume of the conduit. Conduits come in a wide variety of shapes, including circular, rectangular, square, D-shaped, trapezoidal, or other polygonal cross sections. Conduits may also have varying shapes (e.g., a rectangular cross section in one section and a trapezoidal cross section in another section).

[0066] The term "coupled" means that two components are fluidly coupled, physically coupled, and / or communicatively coupled. The term "fluidly coupled" means that two components are in fluid communication and includes a direct connection between the two components as well as an indirect connection where one or more other components are in the flow path between the two components. For example, a first component and a second component are fluidly coupled if the outlet of the first component is physically connected to the inlet of the second component, if a conduit connects the first and second components, or if one or more intervening components, such as valves, pumps, or other structures, are between the two components when fluid flows from the first component to the second component or vice versa. Components may be physically coupled in any suitable manner, such as by using ferrules, brazing, and other methods. Generally, a physical connection that is fluid-tight and / or minimizes dead volume is desirable in devices of the present invention. "Communicatively coupled" means that two components can exchange information, such as by transmitting and receiving communication data. The components may be communicatively connected via a wired or wireless communication network. A wired network generally includes lines for wired communication, conforms to known communication standards such as Ethernet, and processes communication data sent to and received from external computing devices via a network such as the Internet. A wireless network (such as WiFi) generally includes communication via radio waves or other waves, possibly via an antenna. A wireless network generally conforms to known communication standards such as IEEE 802.11, and processes communication data sent to and received from external components.

[0067] As used herein, the term "linker" means a hydrocarbyl chain (e.g., (C-C) alkylene, (C-C) alkenylene, (C-C) alkynylene) optionally substituted with substituents or interrupted by other atoms, such as represented by -(CHR')a-Wb-(CHR')c-Vd-(CHR')e-, where W and V are independently -O-, -S-, or -NR'-, R' is H or (C-C6) alkyl, a, b, c, d, and e are independently integers from 0 to 10, preferably 0 to 6, or preferably 0 to 3, and the sum of a, b, c, d, and e is preferably an integer from 2 to 6. The hydrocarbyl chain may be interrupted by -OR", -O-CO-R", -NR'-R", -NR'-CO-R", -CO-NR'-R", -CO-R", or combinations thereof, where R' and R" are independently H or (C1-C6)hydrocarbyl.

[0068] A "thionocarbamate protecting group" refers to a protecting group containing a thionocarbonyl having a nitrogen and oxygen bonded to the thionocarbonyl carbon atom: --OC(S)N--.

[0069] The term "electron-withdrawing group" refers to a chemical group that withdraws electrons from a reaction center. Non-limiting examples of electron-withdrawing groups (EWGs) are halogens (e.g., fluorine and chlorine), haloalkyls (e.g., CH2Cl, CF3, etc.), nitrile (-RCN), carbonyl (-COR), sulfonyl (-SO3R), ammonium (N+R3), and nitro groups (-NO2).

[0070] The term "chromatographic method" means a method for separating one or more compounds that involves the use of a stationary phase and a mobile phase or eluent that passes through or across the stationary phase. Non-limiting examples of chromatographic methods include fluorous affinity purification, high performance liquid chromatography, and gas chromatography.

[0071] The term "alkyl" refers to straight-chain or branched alkyl groups, preferably having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons. Examples of such alkyl groups include methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl, and the like. In some embodiments, the alkyl group may be a cycloalkyl group.

[0072] The alkyl group may be unsubstituted or substituted as defined above. The term "halo-substituted alkyl" refers to an alkyl group substituted with one or more halogen atoms. Non-limiting examples include trifluoromethyl, trifluoroethyl, pentafluoroethyl, 2,2,2-trichloroethyl, chloromethyl, and the like. The halo-substituted alkyl group may be unsubstituted or substituted as defined above. The term "fluorine-substituted alkyl" refers to an alkyl group substituted with one or more fluorine atoms.

[0073] The term "alkenyl" preferably refers to a straight-chain or branched hydrocarbon having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons and one or more carbon-carbon double bonds. Non-limiting examples of alkenyl groups include ethenyl, 1-propenyl, 2-propenyl (allyl), iso-propenyl, 2-methyl-1-propenyl, 1-butenyl, and 2-butenyl. An alkenyl group may be unsubstituted or substituted with one or more suitable substituents, as defined above. The term "halo-substituted alkenyl" refers to an alkenyl group substituted with one or more halogen atoms. The term "fluorine-substituted alkenyl" refers to an alkenyl group substituted with one or more fluorine atoms.

[0074] The term "alkynyl" preferably refers to a straight-chain or branched hydrocarbon having 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 carbons and having one or more carbon-carbon triple bonds. Alkynyl groups include, but are not limited to, ethynyl, propynyl, and butynyl. Alkynyl groups can be unsubstituted or substituted with one or more suitable substituents, as defined above. The term "halo-substituted alkynyl" refers to an alkynyl group substituted with one or more halogen atoms. The term "fluorine-substituted alkynyl" refers to an alkynyl group substituted with one or more fluorine atoms.

[0075] "Carbocycle" or "carbocyclic" means a saturated (i.e., cycloalkyl), partially unsaturated (e.g., cycloalkenyl, cycloalkadienyl, etc.), or aromatic ring having 3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and up to about 20 carbon atoms as a polycycle. Monocyclic carbocycles have 3 to 6 ring atoms, and even more typically 5 or 6 ring atoms. Bicyclic carbocycles have, for example, 7 to 12 ring atoms arranged as a bicyclo[4,5], [5,5], [5,6], or [6,6] system, or 9 or 10 ring atoms arranged as a bicyclo[5,6] or [6,6] system, or spiro-fused rings. Non-limiting examples of monocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl, 1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and phenyl. Non-limiting examples of bicyclic carbocycles include naphthyl.

[0076] "Heteroalkyl," "heteroalkenyl," and "heteroalkynyl" refer to alkyl, alkenyl, and alkynyl groups, respectively, in which one or more carbon atoms are replaced with a heteroatom such as O, N, or S. Carbons within an alkyl, alkenyl, or alkynyl group can be independently replaced with a heteroatom (O, N, or S), meaning a primary, terminal, or internal carbon. For example, when the carbon atom of an alkyl group attached to a parent molecule is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkoxy group (e.g., -OCH), an amine alkyl group (e.g., -NHCH, -N(CH), or a thioalkyl group (e.g., -SCH), respectively. When a non-terminal carbon atom of an alkyl group that is not bonded to the parent molecule is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is an alkyl ether (e.g., -CHCH-O-CH), an alkylamine (e.g., -CHNHCH, -CHN(CH)), or a thioalkyl ether (e.g., -CH-S-CH). When a terminal carbon atom of an alkyl group is replaced with a heteroatom (e.g., O, N, or S), the resulting heteroalkyl group is a hydroxyalkyl group (e.g., -CHCH-OH), an aminoalkyl group (e.g., -CHNH), or an alkylthiol group (e.g., -CHCH-SH). The heteroalkyl, heteroalkenyl, or heteroalkynyl group may have, for example, 1 to 24 carbon atoms. A C1-C6 heteroalkyl group refers to a heteroalkyl group having 1 to 6 carbon atoms. A "substituted heteroalkyl," "substituted heteroalkenyl," or "substituted heteroalkynyl" is a heteroalkyl, heteroalkenyl, or heteroalkynyl, as defined herein, in which one or more hydrogen atoms are replaced with a non-halogen substituent, as defined in the definition of "substituted."

[0077] The term "aryl," as commonly understood in the art, refers to an unsubstituted or substituted aromatic carbocyclic substituent having 3 to 7 carbon atoms as a monocycle, 7 to 12 carbon atoms as a bicycle, and up to about 20 carbon atoms as a polycycle, e.g., phenyl, naphthyl, anthracyl, indanyl, and the like. The term aryl is understood to be used for cyclic substituents containing 4n+2 electrons in a plane according to Hückel's rule. Aryl groups can be unsubstituted or substituted with one or more suitable substituents, as defined above. The term "halo-substituted aryl" refers to an aryl substituted with one or more halogen atoms or halogen-containing substituents. The term "fluorine-substituted aryl" refers to an aryl substituted with one or more fluorine atoms or fluorine-containing substituents.

[0078] "Arylalkyl" refers to a cyclic alkyl group in which one of the hydrogen atoms bonded to a carbon atom, typically a terminal carbon atom, is replaced with an aryl group. Typical arylalkyl groups include, but are not limited to, benzyl, 2-phenylethan-1-yl, naphthylmethyl, 2-naphthylethan-1-yl, naphthobenzyl, 2-naphthophenylethan-1-yl, and the like. The arylalkyl group may contain 6 to 24 carbon atoms, e.g., the alkyl and aryl carbon atoms add up to 6 to 24 carbon atoms. The aryl group may be unsubstituted or substituted with one or more suitable substituents, as defined above.

[0079] The term "carbonyl" refers to a substituent containing a carbon double-bonded to oxygen. Examples of such substituents include aldehydes, ketones, carboxylic acids, esters, amides, carbonates, and carbamates. A carbonyl group can be unsubstituted or substituted with one or more suitable substituents, as defined above.

[0080] The term "amino" refers to a nitrogen-containing moiety. Non-limiting examples of amino groups are NH2- (primary), RHN- (secondary), and RN (tertiary), where R is alkyl, alkenyl, alkynyl, aryl, heterocycle, or heteroaryl. The RHN- and RN groups can be unsubstituted or substituted as defined above.

[0081] The term "heteroaryl" refers to a monocyclic or bicyclic 5- or 6-membered ring system, wherein the heteroaryl group is unsaturated and satisfies Hückel's rule. Non-limiting examples of heteroaryl groups include furanyl, thiophenyl, pyrrolyl, pyrazolyl, imidazolyl, 1,2,3-triazolyl, 1,2,4-triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, 1,3,4-oxadiazol-2-yl, 1,2,4-oxadiazol-2-yl, 5-methyl-1,3,4-oxadiazole, 3-methyl-1,2,4-oxadiazole, pyridinyl, pyrimidinyl, pyrazinyl, triazinyl, benzofuranyl, benzothiophenyl, indolyl, quinolinyl, isoquinolinyl, benzimidazolyl, benzoxazolinyl, benzothiazolinyl, quinazolinyl, and the like. Heteroaryl groups can be unsubstituted or substituted as defined above.

[0082] The term "heterocycle" or "heterocyclic" means a monocyclic, bicyclic, or tricyclic moiety containing 1 to 4 heteroatoms selected from O, N, and S. Heterocyclic groups optionally contain one or more double bonds. Heterocyclic groups include, but are not limited to, azetidinyl, tetrahydrofuranyl, imidazolidinyl, pyrrolidinyl, piperidinyl, piperazinyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, thiomorpholinyl, tetrahydrothiazinyl, tetrahydro-thiadiazinyl, morpholinyl, oxetanyl, tetrahydrodiazinyl, oxazinyl, oxathiazinyl, indolinyl, isoindolinyl, quinuclidinyl, chromanyl, isochromanyl, and benzoxazinyl. Non-limiting examples of monocyclic saturated or partially saturated ring systems include tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, imidazolidin-l-yl, imidazolidin-2-yl, imidazolidin-4-yl, pyrrolidin-l-yl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-l-yl, piperidin-2-yl, piperidin-3-yl, piperazin-l-yl, piperazin-2-yl, piperazin-3-yl, 1,3-oxazolidin-3-yl, iso-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 11-, 12-, 13-, 14-, 15-, 16-, 17-, 18-, 19-, 20-, 21-, 22-, 23-, 24-, 25-, 26-, 27-, 28-, 29-, 30-, 31-, 32-, 33-, 34-, 35-, 36-, 37-, 38-, 39-, 40-, 41-, 42-, 43-, 44-, 45-, 46-, 47-, 48-, 49-, 50-, 51-, 52-, 53-, 54-, 55-, 56-, 57-, 58-, 59-, 60-, 61-, 62-, 63-, 64-, 65-, 66-, 67-, 68-, 69-, 70-, 71-, 72-, 73 Thiazolidine, 1,3-thiazolidin-3-yl, 1,2-pyrazolidin-2-yl, 1,3-pyrazolidin-1-yl, thiomorpholin-yl, 1,2-tetrahydrothiazin-2-yl, 1,3-tetrahydrothiazin-3-yl, tetrahydrothiadiazin-yl, morpholin-yl, 1,2-tetrahydrodiazin-2-yl, 1,3-tetrahydrodiazin-1-yl, 1,4-oxazin-2-yl, and 1,2,5-oxathiazin-4-yl. The heterocyclic group may be unsubstituted or substituted by one or more suitable substituents, as defined above.

[0083] "Halogen" or "halo" means fluorine, chlorine, boron, and iodine.

[0084] As used herein and in the appended claims, in addition to its ordinary meaning, the terms "substantial" or "substantially" mean within the limits or degree acceptable to one of ordinary skill in the art. For example, "substantially canceled" means that one of ordinary skill in the art would consider the cancellation acceptable.

[0085] As used herein and in the appended claims, in addition to their ordinary meaning, the terms "approximately" and "about" mean within acceptable limits or amounts to those of ordinary skill in the art. The term "about" generally means plus or minus 15% of the indicated number. For example, "about 10" indicates a range of 8.5 to 11.5. For example, "about the same" means that one of ordinary skill in the art would consider the compared items to be the same.

[0086] In this disclosure, numerical ranges are inclusive of the numbers defining the range. It is recognized that chemical structures and formulas may be extended or expanded for illustrative purposes.

[0087] When a range of the number of atoms in a structure is given (e.g., C1-C 24 Alkyl, C2-C 24 Alkenyl, C2-C 24 It is expressly contemplated that, for example, a group consisting of 1 to 24 carbon atoms (e.g., C1 to C6), a substituent can be described by any carbon atom within a subrange, or by any individual number of carbon atoms within the indicated range. 24 ), 1 to 6 carbon atoms (e.g., C1 to C6), 1 to 4 carbon atoms (e.g., C1 to C4), 1 to 3 carbon atoms (e.g., C1 to C3), or 2 to 24 carbon atoms (e.g., C2 to C 24), where appropriate, includes alkyl groups having any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 carbon atoms, as well as any subrange thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon atoms, 1-13 carbon atoms, 1-14 carbon atoms, 1-15 carbon atoms, 1-24 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, and the like, where appropriate, includes alkyl groups having any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, and 24 carbon atoms, as well as any subrange thereof (e.g., 1-2 carbon atoms, 1-3 carbon atoms, 1-4 carbon atoms, 1-5 carbon atoms, 1-6 carbon atoms, 1-7 carbon atoms, 1-8 carbon atoms, 1-9 carbon atoms, 1-10 carbon atoms, 1-11 carbon atoms, 1-12 carbon atoms, 1-13 carbon atoms, 1-14 carbon atoms, 1-15 carbon atoms, 1-24 carbon atoms, 2-3 carbon atoms, 2-4 carbon atoms, 2-5 carbon atoms, 2-6 carbon atoms, and carbon atoms, 2-7 carbon atoms, 2-8 carbon atoms, 2-9 carbon atoms, 2-10 carbon atoms, 2-11 carbon atoms, 2-12 carbon atoms, 2-13 carbon atoms, 2-14 carbon atoms, 2-15 carbon atoms, 2-16 carbon atoms, 3-4 carbon atoms, 3-5 carbon atoms, 3-6 carbon atoms, 3-7 carbon atoms, 3-8 carbon atoms, 3-9 carbon atoms, 3-10 carbon atoms, 3-11 carbon atoms, 3-12 carbon atoms, 3-13 carbon atoms, 3-14 carbon atoms, 3-15 carbon atoms, 3-16 carbon atoms, 3-17 carbon atoms, 3-18 carbon atoms, 3-19 carbon atoms, 3-20 carbon atoms, 3-21 carbon atoms, 3-22 carbon atoms, 3-23 carbon atoms, and / or 3-24 carbon atoms).

[0088] As disclosed herein, several ranges of values ​​are provided. Unless expressly stated otherwise, each intervening value between the upper and lower limits of that range is understood to be disclosed to the tenth of the unit of the lower limit. Each smaller range between any stated or intervening value within a stated range and any other stated or intervening value within that stated range is also encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded, and each range in which either, either, or both limits are included in the smaller range is also encompassed within the invention, subject to any explicitly excluded limits within the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also encompassed within the invention.

[0089] All patents and patent publications mentioned herein are expressly incorporated by reference.

[0090] As used in the specification and the appended claims, the terms "a," "an," and "the" include both singular and plural references unless the context clearly dictates otherwise. Thus, for example, "a device" includes one device and multiple devices.

[0091] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. [Example]

[0092] General method DNA and RNA strands are chemically synthesized by sequentially conjugating each nucleotide. As each nucleotide is added, a portion of the nucleotide solution is diverted from the synthesizer's column outlet to an online sample manager or other equivalent sample preparation and injection device with a mass spectrometer. Adjustments to the sample concentration and / or ionic strength are performed by programming the online sample manager for automatic dilution before or after sample injection. This produces a constant volume of sample in real time that can be used for identification testing. The sample is then automatically prepared and injected into a mass spectrometer detector (MSD) for mass confirmation.

[0093] The synthesis was carried out using an AKTA Oligopilot equipped with multiple delivery locations for different amidite solutions. The synthesis support was loaded onto a 2 cm x 2.54 cm synthesis column to form the reaction chamber for oligonucleotide synthesis. Each amidite was prepared at a concentration of 200 mM in ACN.

[0094] A sample stream is continuously taken from the outlet of the reaction chamber for analysis by mass spectrometry to identify the amidites and to confirm the order in which the amidites are added.

[0095] A sample tee in the reaction chamber downstream of the outlet splits the outlet flow, with one branch of the tee directed to process waste and the other to a sample pump and then to the sample valve of the online sample manager. To minimize vertical backmixing, the sample tee is located close to the outlet from the reaction chamber.

[0096] A sample pump installed between the sample branch of the tee and the mass spectrometer provides a continuous, stable sample flow to the mass spectrometer throughout the synthesis period. The sample pump is located close to the sample tee to minimize the tubing length between the sample tee and the pump and maintain adequate priming pressure for reliable pump function. The pump moves the sample to the mass spectrometer through a post-pump tube (lengths up to 30 meters have been evaluated) of narrow-bore tubing that runs across the laboratory. The narrow-bore tubing between the pump and mass spectrometer maintains sample integrity by minimizing holdup volume in the tubing and minimizes backmixing caused by longitudinal diffusion over long distances.

[0097] The sample valve in the online sample manager typically diverts the flow from the pump to a sample waste located in the online sample manager. Upon receiving an electrical signal from the reaction chamber, the sample valve diverts the sample flow to collect the sample. The signal is controlled by a specific command inserted into the reaction chamber method code. The flow rate of the sample pump is used to time the arrival of the sample of interest to coincide with the sample signal from the reaction chamber. The collected sample may be analyzed immediately or at a later time by mass spectrometry.

[0098] By providing a steady continuous sample stream from a point at the outlet of the reaction chamber, samples can be collected at any time to identify outlet streams from steps other than coupling.

[0099] [Example 1] In this example, the nucleotide sequence of an oligonucleotide containing eight nucleotides, including modified nucleotides, was confirmed. An eight-base ribonucleic acid (RNA) oligonucleotide sequence consisting of both 2'-fluoro and 2'-methoxy RNA nucleotides was synthesized using an NCEV system connected to a reaction chamber. The standard nucleic acid solid-phase synthesis method involves 1) detritylation, 2) coupling, 3) oxidation, and 4) capping. The solid-phase support used was CPG, preloaded with 3'-DMT-5'dT. The synthesis reagents used are listed in Table 1. All washing steps were performed with acetonitrile.

[0100] [Table 1]

[0101] The reaction chamber setup was the same as that listed in the General Methods Information section.

[0102] Sample preparation and analysis were performed using a customized Agilent 1290U HPLC consisting of two analytical pumps (sample and mobile phase), an online sample manager (AS) with external valves and custom rotor seals, and an Agilent 6135XT single quadrupole MSD. Samples were automatically diluted with acetonitrile (ACN) using the AS at the time of sample collection (prior to sample injection). The mobile phase pump solution was 0.1% formic acid + 50% ACN. The instrument parameters used are listed in Table 2.

[0103] [Table 2]

[0104] The nucleotides were conjugated in the following order: 3'-fAfAmCfCmGfAfGmA-5'. Each nucleotide adduct was collected and analyzed. The experimental data are shown in Table 3 and Figures 4-11. The detected species is the product of quenching the activated amidite with water to form an H-phosphonate species (Scheme 1). Water is added to the synthesis matrix to prepare the sample for mass spectrometry measurement.

[0105] Scheme 1 shows the formation of a phosphoramidite quenched species.

[0106] [ka]

[0107] [Table 3]

[0108] Figure 4 shows the mass spectral data and theoretical chemical structure (fA) of Sample 1-1. Figure 5 shows the mass spectral data and theoretical chemical structure (fA) of Sample 1-2. Figure 6 shows the mass spectral data and theoretical chemical structure (mC) of Sample 1-3. Figure 7 shows the mass spectral data and theoretical chemical structure (fC) of Sample 1-4. Figure 8 shows the mass spectral data and theoretical chemical structure (mG) of Sample 1-5. Figure 9 shows the mass spectral data and theoretical chemical structure (fA) of Sample 1-6. Figure 10 shows the mass spectral data and theoretical chemical structure (fG) of Sample 1-7. Figure 11 shows the mass spectral data and theoretical chemical structure (mA) of Sample 1-8.

[0109] Each position in the 8-mer RNA sequence was confirmed. Nucleotides in H-phosphonate protected positions were confirmed.

[0110] [Example 2] In this example, the nucleotide sequence of an oligonucleotide containing seven nucleotides, including modified nucleotides, was confirmed. A seven-base ribonucleic acid (RNA) oligonucleotide sequence consisting of both 2'-fluoro and 2'-methoxy RNA nucleotides was synthesized using an NCEV system connected to a reaction chamber. Solid-phase synthesis was performed as described in Example 1. Samples were automatically diluted after sample collection using a flow control device.

[0111] The reaction chamber setup was the same as that listed in the General Methods section.

[0112] Sample preparation and analysis were performed using a customized Agilent 1290U HPLC consisting of three analytical pumps (sample, mobile phase, and flow control pumps), a custom online sample manager (AS) with flow control devices and external valves, and an Agilent 6135XT single quadrupole MSD. Samples were collected near the AS. After sample injection, the sample was diluted with 0.1% formic acid + 50% ACN by the flow control device. The mobile phase pump solution was 0.1% formic acid + 50% ACN. The instrument parameters used are listed in Table 4.

[0113] [Table 4]

[0114] The nucleotides were conjugated in the following order: 3'-fAmCfCmGfAfGmA-5'. Each nucleotide adduct was collected and analyzed. The experimental data are shown in Table 5 and Figures 12-18.

[0115] [Table 5]

[0116] Figure 12 shows the mass spectral data and theoretical chemical structure (fA) of Sample 2-1. Figure 13 shows the mass spectral data and theoretical chemical structure (mC) of Sample 2-2. Figure 14 shows the mass spectral data and theoretical chemical structure (fC) of Sample 2-3. Figure 15 shows the mass spectral data and theoretical chemical structure (mG) of Sample 2-4. Figure 16 shows the mass spectral data and theoretical chemical structure (fA) of Sample 2-5. Figure 17 shows the mass spectral data and theoretical chemical structure (fG) of Sample 2-6. Figure 18 shows the mass spectral data and theoretical chemical structure (mA) of Sample 2-7.

[0117] Each position in the 7-mer RNA sequence was confirmed. Nucleotides in H-phosphonate protected positions were confirmed.

[0118] Experimental embodiment Before describing various exemplary embodiments, it is to be understood that the teachings of the present disclosure are not limited to particular embodiments described, as such may, of course, vary. Exemplary embodiments provided by the presently disclosed subject matter include, but are not limited to, the following:

[0119] Embodiment 1. A method for synthesizing oligonucleotides and monitoring the synthesis in real time, comprising the steps of: selecting a nucleotide monomer for addition to an oligonucleotide; feeding an inlet stream into a reaction chamber for oligonucleotide synthesis; providing conditions in the reaction chamber for oligonucleotide synthesis; diverting an outlet stream from the reaction chamber to an analysis station; analyzing the outlet stream or a sample thereof by mass spectrometry (MS) in the analysis station; and determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof based on the results of the analysis by MS.

[0120] Embodiment 2. The method of embodiment 1, further comprising the step of stopping the supply of the inlet stream to the reaction chamber if the outlet stream is determined to not contain the selected nucleotide monomer.

[0121] Embodiment 3. The method of embodiment 1 or embodiment 2, further comprising the step of connecting a source of selected nucleotide monomers to the inlet stream prior to the feeding step.

[0122] Embodiment 4. The method of any one of embodiments 1 to 3, wherein the determining step is performed before the next nucleotide monomer is provided to the reaction chamber.

[0123] Embodiment 5. The method of any of embodiments 1-4, further comprising the steps of time-stamping the MS analysis results, associating the time-stamp with the delivery time of the inlet stream to the reaction chamber, and using the time-stamp and the associated delivery time to determine whether the selected nucleotide monomer was present in the inlet stream.

[0124] Embodiment 6. The method of embodiment 5, comprising using the timestamp and associated delivery time to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber.

[0125] Embodiment 7. The method of embodiment 6, further comprising distributing or licensing the synthesized oligonucleotide if the calculated nucleotide sequence matches a predetermined nucleotide sequence.

[0126] Embodiment 8. The method of embodiment 6, further comprising withholding or removing the synthesized oligonucleotide if the calculated nucleotide sequence does not match the predetermined nucleotide sequence.

[0127] Embodiment 9. The method of any one of embodiments 1 to 8, wherein the outlet stream or a sample thereof is analyzed by liquid chromatography / mass spectrometry (LC / MS).

[0128] Embodiment 10. The method of any one of embodiments 1 to 9, wherein the MS analysis is performed on a low resolution detector.

[0129] Embodiment 11. The method of any one of embodiments 1 to 10, wherein prior to the feeding step, the inlet stream is not analyzed for the selected nucleotide monomer.

[0130] Embodiment 12. The method of any one of embodiments 1 to 11, wherein the oligonucleotide comprises ribonucleotides, deoxyribonucleotides, or a mixture thereof.

[0131] Embodiment 13. The method of embodiment 12, wherein the selected nucleotide monomer is a 3'-phosphoramidite ribonucleotide having protecting groups at the 2'-, 5'-, and nucleobase positions.

[0132] Embodiment 14. The method of embodiment 13, wherein the ribonucleotide has a 5'-dimethyltrityl protecting group and a 2'-protecting group selected from the group consisting of a thionocarbamate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a t-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

[0133] Embodiment 15. The method of any one of embodiments 1 to 14, wherein the selected nucleotide monomer is 793.3 Da, and if MS detects a fragment having a molecular weight between 792.3 and 794.3 Da, it is determined that the outlet stream contains the selected nucleotide monomer.

[0134] Embodiment 16. The method of any one of embodiments 1 to 15, wherein the selected nucleotide monomer comprises a modified nucleobase.

[0135] Embodiment 17. A system for synthesizing oligonucleotides, comprising a reaction chamber comprising an inlet, a solid support for oligonucleotide synthesis, and an outlet, and a mass spectrometry (MS) device fluidly connected to the outlet of the reaction chamber.

[0136] Embodiment 18. The system of embodiment 17, further comprising an online sample manager between the outlet of the reaction chamber and the MS instrument, whereby the online sample manager receives the outlet stream or a sample thereof from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument.

[0137] Embodiment 19. The system of embodiment 17 or 18, further comprising a sample pump between the reaction chamber and the online sample manager.

[0138] Embodiment 20. A system described in any one of embodiments 17 to 19, further comprising a flow control valve between the online sample manager and the MS device.

[0139] Embodiment 21. A system according to any one of embodiments 17 to 20, wherein the solid support is a planar substrate or a bead.

[0140] Embodiment 22. The system of any one of embodiments 17-21, further comprising one or more computing devices communicatively coupled to the reaction chamber, the online sample manager, and the MS instrument, wherein the one or more computing devices comprise a processor and a storage medium, and wherein the processor is configured to execute instructions stored on the storage medium to determine, based on data from the MS instrument, whether the outlet stream from the reaction chamber contains a selected nucleotide monomer or a derivative thereof.

[0141] Embodiment 23. The system of embodiment 22, wherein the one or more computing devices are further configured to control conditions within the reaction chamber, to start and / or stop the supply of an inlet stream to the reaction chamber, and / or to activate and / or deactivate an online sample manager.

[0142] Embodiment 24. The system of any one of embodiments 17 to 23, further comprising a sensor at the outlet of the reaction chamber, which sensor detects the presence or absence of nucleotide monomers in the diluent.

[0143] Embodiment 25. The system of claim 24, wherein the sensor is communicatively connected to one or more computing devices, and wherein the one or more computing devices are further configured to direct the outlet stream to an online sample manager and / or activate the online sample manager if the sensor detects the presence of nucleotide monomers in the outlet stream.

[0144] Embodiment 26. A system according to any one of embodiments 17 to 25, wherein the MS instrument is a low-resolution MS instrument.

[0145] In view of this disclosure, it will be appreciated that methods and systems can be implemented in accordance with the teachings of the present invention. Furthermore, the various components, materials, structures, and parameters are included by way of illustration and example only, and not in a limiting sense. In view of this disclosure, the teachings of the present invention can be implemented in other applications, and components, materials, structures, and apparatus for implementing these applications can be determined so long as they fall within the scope of the appended claims.

Claims

1. selecting a nucleotide monomer for addition to the oligonucleotide; providing an inlet stream to a reaction chamber for oligonucleotide synthesis; providing conditions in said reaction chamber for oligonucleotide synthesis; diverting an outlet stream from the reaction chamber to an analysis station; analyzing the outlet stream or a sample thereof by mass spectrometry (MS) at the analysis station; determining whether the outlet stream contains the selected nucleotide monomer or a derivative thereof based on the results of the MS analysis; 1. A method for synthesizing oligonucleotides and monitoring said synthesis in real time, comprising:

2. 10. The method of claim 1, further comprising the step of stopping the supply of the inlet stream to the reaction chamber if the outlet stream is determined to not contain the selected nucleotide monomer.

3. 10. The method of claim 1, further comprising the step of connecting a source of said selected nucleotide monomer to said inlet stream prior to said feeding step.

4. The method of claim 1 , wherein the determining step is performed before a next nucleotide monomer is provided to the reaction chamber.

5. time-stamping the MS analysis results; Associating the timestamp with the delivery time of the inlet stream to the reaction chamber; and using the timestamp and the associated delivery time to determine whether the selected nucleotide monomer was present in the inlet stream. The method of claim 1 further comprising:

6. 6. The method of claim 5, comprising using the timestamp and the associated delivery time to calculate the nucleotide sequence of the oligonucleotide synthesized in the reaction chamber.

7. 7. The method of claim 6, further comprising distributing or licensing the synthesized oligonucleotide if the calculated nucleotide sequence matches a predetermined nucleotide sequence.

8. 7. The method of claim 6, further comprising withholding or removing the synthesized oligonucleotide if the calculated nucleotide sequence does not match the predetermined nucleotide sequence.

9. The method of claim 1 , wherein the outlet stream or a sample thereof is analyzed by liquid chromatography / mass spectrometry (LC / MS).

10. The method of claim 1 , wherein the MS analysis is performed on a low resolution detector.

11. 10. The method of claim 1, wherein prior to said feeding step, said inlet stream is not analyzed for said selected nucleotide monomer.

12. The method of claim 1 , wherein the oligonucleotide comprises ribonucleotides, deoxyribonucleotides, or a mixture thereof.

13. 13. The method of claim 12, wherein the selected nucleotide monomers are 3'-phosphoramidite ribonucleotides having protecting groups at the 2'-, 5'-, and nucleobase positions.

14. 14. The method of claim 13, wherein the ribonucleotide has a 5'-dimethyltrityl protecting group and a 2'-protecting group selected from the group consisting of a thionocarbamate (TC) protecting group, a bis(2-acetoxyethoxy)methyl (ACE) protecting group, a t-butyldimethylsilyl (TBDMS) protecting group, a triisopropylsilyloxymethyl (TOM) protecting group, a pivaloyloxymethyl (PivOM) protecting group, and a 2-cyanoethoxymethyl (CEM) protecting group.

15. 2. The method of claim 1, wherein the selected nucleotide monomer is 793.3 Da, and if the MS detects a fragment having a molecular weight of 792.3 to 794.3 Da, the outlet stream is determined to contain the selected nucleotide monomer.

16. 10. The method of claim 1, wherein the selected nucleotide monomer comprises a modified nucleobase.

17. a reaction chamber including an inlet, a solid support for oligonucleotide synthesis, and an outlet; a mass spectrometry (MS) device fluidly connected to the outlet of the reaction chamber; A system for synthesizing oligonucleotides, comprising:

18. 20. The system of claim 17, further comprising an online sample manager between the outlet of the reaction chamber and the MS instrument, whereby the online sample manager receives the outlet stream or a sample thereof from the reaction chamber and delivers the outlet stream or a sample thereof to the MS instrument.

19. 20. The system of claim 17, further comprising a sample pump between the reaction chamber and the online sample manager.

20. 20. The system of claim 17, further comprising a flow control valve between the online sample manager and the MS device.

21. 18. The system of claim 17, wherein the solid support is a planar substrate or a bead.

22. 18. The system of claim 17, further comprising one or more computing devices communicatively coupled to the reaction chamber, the online sample manager, and the MS instrument, wherein the one or more computing devices comprise a processor and a storage medium, and wherein the processor is configured to execute instructions stored on the storage medium to determine, based on data from the MS instrument, whether an outlet stream from the reaction chamber comprises a selected nucleotide monomer or a derivative thereof.

23. wherein the one or more computing devices control conditions within the reaction chamber; to start and / or stop the supply of an inlet stream to the reaction chamber; and / or 23. The system of claim 22, further configured to activate and / or deactivate the online sample manager.

24. 24. The system of claim 23, further comprising a sensor at the outlet of the reaction chamber, wherein the sensor detects the presence or absence of nucleotide monomers in the diluent.

25. the sensors are communicatively connected to one or more of the computing devices; 25. The system of claim 24, wherein one or more of the computing devices are further configured to direct the outlet stream to the online sample manager and / or activate the online sample manager if the sensor detects the presence of nucleotide monomers in the outlet stream.

26. 18. The system of claim 17, wherein the MS device is a low-resolution MS device.

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