Belt reactor manufacturing method for oligomer synthesis

Abstract

Disclosed are methods and apparatus for synthesizing oligomers, such as oligonucleotides and peptides. The method uses a device including at least one deprotection station for performing a deprotection step, at least one coupling station for performing a coupling step, optionally one or more oxidation and / or thiolation stations for performing an oxidation or thiolation step, optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step. Multiple solid-phase materials for oligomer synthesis are moved to the stations via a conveyor device, where a fluid delivery device acts on the solid-phase materials to produce oligomers.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 482,675, filed February 1, 2023, the contents of which are incorporated by reference in their entirety.

[0002] The present disclosure relates to apparatus and methods for the solid phase synthesis of oligomers, such as oligonucleotides and peptides. [Background technology]

[0003] Oligonucleotides and peptides are biopolymers that play a central role in the biological, medical, and pharmaceutical industries (Wan et al., J. Med. Chem. 2016, 59, 21, 9645-9667). Therefore, major efforts have been devoted to improving their efficient synthesis, especially through solid-phase methodologies (Merrifield et al., "Solid Phase Synthesis" Science 1986, 232, 341-347;Itakura et al., "Synthesis and Use of Synthetic Oligonucleotides" Annual Review of Biochemistry 1984, 53(1), 323-356).

[0004] Common methods for generating oligonucleotides include the phosphoramidite, phosphotriester, or H-phosphonate methods (Verma et al., "Modified Oligonucleotides: Synthesis and Strategy for Users," Annu. Rev. Biochem. 1998, 67, 99-134). In these cases, the activated phosphorus atom of one nucleotide reacts with the 5'-hydroxy group of the sugar of the other nucleotide to form a new covalent bond, effectively linking both nucleotides together. The connection is further stabilized when the newly formed phosphite is oxidized to a phosphate. After deprotecting the 5'-hydroxy group of the newly introduced nucleotide, these steps can be repeated, thereby growing the nucleotide chain with each step until the desired sequence is assembled. To increase efficiency and throughput, solid-phase synthesis methodologies are often used.

[0005] Similar to oligonucleotide synthesis, peptides can be generated through a similar strategy (Jaradat, "Thirteen decades of peptide synthesis: key developments in solid-phase peptide synthesis and amide bond formation utilized in peptide ligation," Amino Acids 2018, 50, 39-68). The C-terminus of an N-protected amino acid can be activated using appropriate reagents known in the art and coupled to the N-terminus of another amino acid, resulting in the formation of a new amide bond. After deprotection of the N-terminus of the newly added amino acid, this process can be repeated with the same or different amino acids to grow the peptide chain until the desired sequence is prepared.

[0006] Solid-phase synthesis typically involves immobilizing organic molecules through covalent bonds to a solid-phase material and subsequently chemically manipulating them while maintaining their connection to the solid support (Doerwald, "Organic Synthesis on Solid Phase: Supports, Linkers, Reactions" Wiley-VCH: Weinheim, 2002). Conventional devices for synthesizing oligomers, such as oligonucleotides or peptides (i.e., solid-phase synthesizers), often use this strategy and incorporate a flow-through design. A column is packed with the solid-phase material, and a mobile phase containing the necessary reactants or reagents is passed through it. As the mobile phase passes through the column, chemical agents affect the desired manipulation of the organic molecules. While practical, such a setup has drawbacks, such as high cost and difficulties with scaling up the process.

[0007] Because the amount of oligonucleotide or peptide produced correlates with the amount of solid-phase material in the column, one way to increase production capacity is to increase the column size. However, due to physical constraints related to flow distribution, operating back pressure, and pore diffusion, the maximum column size is limited [Moore et al., "Overcoming Backpressure Problems during Solid-Phase Synthesis of Oligonucleotides" Org. Proc. Res. Dev. 2004, 8(2), 271-274]. Therefore, increasing production capacity is often achieved either by producing several batches sequentially or by using multiple columns to perform all steps in parallel. The latter results in an increase in the amount of piping, pumps, valves, and other materials required, which increases production costs and makes scale-up difficult. Summary of the Invention [Problem to be solved by the invention]

[0008] Thus, there remains a need for improved workflows for more streamlined, cost-effective, and scalable synthesis of oligonucleotides and peptides. The present disclosure provides, inter alia, a continuous manufacturing approach that overcomes the limitations of batch manufacturing and facilitates the production of biopolymers on a larger scale. [Means for solving the problem]

[0009] The present disclosure provides, inter alia, improved solid phase synthesizers for the continuous or quasi-continuous production of oligomers, as well as methods and systems for the continuous or quasi-continuous production of oligomers, such as oligonucleotides and peptides.

[0010] In some embodiments, an apparatus for oligomer synthesis is provided. In some embodiments, the apparatus includes at least one deprotection station for performing a deprotection step, at least one coupling station for performing a coupling step, optionally one or more oxidation and / or thiolation stations for performing an oxidation or thiolation step, optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step. In some embodiments, one or more solid phase materials are moved to each station via a conveyor device. At each station, a fluid delivery device dispenses reagents, reactants, or washing solutions onto the solid phase material to produce an oligomer (e.g., an oligonucleotide or peptide).

[0011] In some embodiments, an apparatus for synthesizing oligonucleotides is provided. The apparatus includes at least one deprotection station for performing a deprotection step, at least one coupling station for performing a coupling step, at least one oxidation station and / or thiolation station for performing an oxidation or thiolation step, optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step. One or more solid phase materials are moved to each station via a conveyor device. At each station, a fluid delivery device dispenses reagents, reactants, or washing solutions onto the solid phase material to produce oligonucleotides.

[0012] In some embodiments, there is provided a method of continuous or quasi-continuous oligonucleotide synthesis, comprising: (a) providing solid phase materials and equipment for oligonucleotide synthesis; The method includes (a) providing an apparatus comprising at least one deprotection station for performing a deprotection step; at least one coupling station for performing a coupling step; at least one oxidation station and / or thiolation station for performing an oxidation or thiolation step; optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step; the apparatus being configured to act on one or more solid phase materials at each station to produce oligonucleotides; and each station being at a fixed position within the apparatus and configured to deliver a reagent, reactant or wash solution to the mobile solid phase material via a fluid delivery device; and (b) initiating movement of the solid phase material through the stations of the apparatus.

[0013] In some embodiments, an apparatus for peptide synthesis is provided. The apparatus includes at least one deprotection station for performing a deprotection step, at least one coupling station for performing a coupling step, optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step. One or more solid phase materials are moved to each station via a conveyor device. At each station, a fluid delivery device delivers reagents, reactants, or wash solutions to the solid phase materials to produce the peptide.

[0014] In some embodiments, a method for continuous or quasi-continuous peptide synthesis is provided, comprising: (a) providing a solid phase material and an apparatus for peptide synthesis, the apparatus comprising at least one deprotection station for performing a deprotection step; at least one coupling station for performing a coupling step; optionally, one or more capping stations for performing a capping step; and optionally, one or more washing stations for performing a washing step; the apparatus configured to act on one or more solid phase materials at each station to produce peptides, each station being at a fixed position within the apparatus and configured to deliver reagents, reactants, or wash solutions to the mobile solid phase material via a fluid delivery device; and (b) initiating movement of the solid phase material through the stations of the apparatus.

[0015] In some embodiments, a solid phase belt is used as the solid phase material and is moved to each station via a conveyor device. In some embodiments, the solid phase belt is made up of linked cassettes. In some embodiments, the solid phase belt is made up of linked cassettes to form a continuous solid phase belt. In some embodiments, the cassettes are removable. In some embodiments, the flexible section of the solid phase belt or cassette is flat, folded, layered, wound, spirally arranged, or otherwise configured to increase surface area. In some embodiments, the solid phase belt comprises one or more of a polymer, glass, carbon, graphene, and graphene oxide. In some embodiments, the solid phase belt comprises a nonwoven material. In some embodiments, the solid phase material is treated to improve or add binding capability to one or more oligomers.

[0016] The following is a brief description of the drawings, which are presented for purposes of illustrating embodiments disclosed herein and are not intended to impose limitations on the disclosure. [Brief explanation of the drawings]

[0017] [Figure 1] FIG. 1 shows an exemplary method for synthesizing oligonucleotides using solid-phase particles using a multi-module belt reactor. [Figure 2] FIG. 2 shows an example of a single module of a belt reactor operating with solid phase particles and comprising several stations arranged horizontally. [Figure 3] Figure 3 shows an example of a single station within a module of a belt reactor. [Figure 4] Figure 4 shows an example of a single module of a belt reactor, with several stations arranged around a moving rotary cylinder. The solid phase material could be particles or a nonwoven fabric. [Figure 5] FIG. 5 shows an exemplary method for synthesizing oligonucleotides using a multi-module solid phase belt. [Figure 6] FIG. 6 shows an example of a single module of a belt reactor using a solid phase belt. [Figure 7] 1 shows an example of a single station within a module of a belt reactor operating with a solid phase belt. [Figure 8] FIG. 8 shows an example of a single module of a belt reactor that operates with cassettes and has several stations arranged horizontally. [Figure 9] FIG. 9 shows six examples of cassettes: (a) a single-sheet flexible band placed within a cassette; (b) a flexible band of several sheets of the same material placed within a cassette; (c) a flexible band of several sheets of two different materials placed within a cassette; (d) a single-sheet solid belt placed in a folded manner within a cassette; (e) a solid belt of several sheets of the same material placed in a folded manner within a cassette; and (f) two solid belts of several sheets of different materials placed in a folded manner within a cassette. DETAILED DESCRIPTION OF THE INVENTION

[0018] In order to better understand the details of this disclosure, certain terms will first be defined here. Additionally, the contents of all references, patents, and published patent applications cited throughout this application, as well as the figures, are hereby incorporated by reference in their entirety for all purposes. To the extent that a statement by reference contradicts a statement in this disclosure, the present disclosure will control.

[0019] definition As used herein, the singular terms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. As used herein, the term "and / or" refers to "either or both" of the elements so joined, i.e., elements that are present conjunctively in some cases and disjunctively in other cases. Thus, as a non-limiting example, "A and / or B," when used in conjunction with open-ended phrases such as "comprising," can refer in some embodiments to A only (which may include elements other than B); in other embodiments to B only (which may include elements other than A); in yet other embodiments to both A and B (which may include other elements); etc.

[0020] As used herein, "at least one" means one or more elements in a list of elements, but does not necessarily include at least one of each element specifically listed in the list of elements, and does not exclude any combination of elements in the list of elements.

[0021] As used herein, "about," when used in connection with an amount or ratio, includes values ​​of the specified amount and ratio that would be recognized by one of ordinary skill in the art as providing the desired function equivalent to that resulting from the specified amount and ratio. The term "about" can refer to an acceptable error for a particular value as determined by one of ordinary skill in the art, which error depends in part on how the value is measured or determined. In some embodiments, the term "about" means within 5%, 4%, 3%, 2%, 1%, or 0.5% of a given value or range.

[0022] As used herein, the term "agent" includes any chemical element or compound. Examples of agents include, but are not limited to, reactants, reagents, and wash solutions.

[0023] As used herein, the term "amino acid" refers to a molecule containing both an amine group and a carboxyl group. Suitable amino acids include, but are not limited to, both D- and L-isomers of naturally occurring amino acids, as well as non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. In some embodiments, a single "amino acid" may have multiple side chain moieties available per extended aliphatic or aromatic backbone scaffold. In some embodiments, one or more functional groups of an amino acid may be protected with a suitable protecting group.

[0024] As used herein, the term "belt reactor" refers to an apparatus that provides for the continuous production of solid-phase supported oligomers, e.g., biopolymers, peptides, and oligonucleotides, as they move along a transport system. A belt reactor can include one or more modules that operate on a solid-phase material (e.g., a solid-phase belt), which can be deposited directly on a conveyor device without the need for a reactor, or can be provided on a cassette, container, or other surface fixed to the conveyor device.

[0025] As used herein, the term "module" refers to a portion of a belt reactor. A module can include one or more stations, where each module is configured to grow oligomers by one type of monomer or its pre-existing building block. Examples of monomers include nucleotides in oligonucleotide synthesis and amino acids in polypeptide synthesis. Examples of pre-existing building blocks include oligonucleotides in oligonucleotide synthesis and peptides in peptide synthesis. Modules can be arranged in a sequence to synthesize a specific oligomer sequence. Modules can be of different lengths and positioned at different distances from each other, depending on the target reaction and exposure time until the solid-phase material reaches the next module. A single module can be used more than once (e.g., multiple copies of the same type of module can exist in a belt reactor). Alternatively, a module can act on a solid-phase material more than once by feeding the solid-phase material back into the same module after the first pass or any subsequent passes.

[0026] As used herein, the term "cassette" refers to a segment of the solid-phase belt, which is linked together to other cassettes or spacers to form a continuous chain. Cassettes can be attached or detached independently from the remainder of the solid-phase belt at any position on the solid-phase belt. A single cassette can include a flexible band and a rigid frame that provides stability. The flexible band of the cassette can include a woven or nonwoven material, which exhibits functional groups on its surface that enable solid-phase synthesis of oligomers. Exemplary flexible band materials of the cassette include, but are not limited to, polymers, glass, carbon, graphene, and graphene oxide. The flexible band of the cassette can include a single sheet, multiple sheets of the same material, and / or multiple sheets of different materials. The configuration of the flexible band of the cassette can be flat, folded, layered, wound, spirally arranged, or otherwise configured to increase the surface area of ​​the flexible band. The rigid section of the cassette can include materials such as metal, polymer, wood, glass, and other materials that provide mechanical stability. The rigid section of the cassette can be configured to retain the shape of the flexible band that the cassette comprises. The rigid section of the cassette can be made of a material that is inert to the reagents and reactants to which the cassette is exposed during operation of the belt reactor. The cassette can further be configured to be sealable from the outside, thus creating a barrier between the flexible section of the cassette and the ambient atmosphere.

[0027] The term "catalyst" refers to a compound that accelerates the rate at which a chemical reaction occurs. A catalyst is not converted into a product itself or otherwise changed or consumed upon completion of the chemical reaction. After a catalyst has participated in one chemical reaction, it remains unchanged and can therefore participate in further chemical reactions and act on additional reagents or reactants to produce additional products.

[0028] As used herein, the term "conveyor device" refers to a portion of a belt reactor that transports solid phase material from one station to another. Conveyor devices can also be used to transport solid phase material from one module to another. Examples of such conveyor devices include, but are not limited to, conveyor belts and conveyor rollers. The device can be configured for continuous movement of the belt reactor and / or for periodic slowing or resting of the belt reactor.

[0029] As used herein, the term "modified nucleotide," "chemically modified nucleotide," or "chemically modified variant" is consistent with how the term is defined in the art and may include, but is not limited to, modified variants of DNA and RNA, such as 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleobases, bridged nucleotides, peptide nucleic acid monomers that form peptide nucleic acids (PNAs) [e.g., repeating N-(2-aminoethyl)glycine (AEG) groups], unlocked nucleotides, 2',3' seconucleotide mimics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (2'-internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-Me, 2'-fluoronucleotides, morpholino nucleotides, abasic nucleotides (also referred to as "abasic sites"), vinylphosphonate-containing nucleotides, and cyclopropylphosphonate-containing nucleotides. Commonly used modified nucleotides include 2'-modified nucleotides (i.e., nucleotides having a group other than -H or -OH at the 2' position of the five-membered sugar ring), including, but not limited to, 2'-O-methyl nucleotides (also called 2'-methoxy nucleotides), 2'-fluoro nucleotides (also called 2'-deoxy-2'-fluoro nucleotides), 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides (also called 2'-MOE nucleotides), 2'-amino nucleotides, and 2'-alkyl nucleotides.In some embodiments, modified nucleotides include modified nucleobases, such as 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, O-6-substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thio The oligonucleotides may include thymine, 2-thiocytosine, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, and 3-deazaadenine. In some embodiments, the oligonucleotide may include a modified internucleoside bond or backbone.Such modified linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, alkylphosphonates (e.g., methylphosphonates or 3'-alkylenephosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-aminophosphoramidate, aminoalkylphosphoramidate, or thionophosphoramidate), thionoalkylphosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with inverted polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short-chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short-chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and CH2 moieties. As known and described in the art, multiple modifications can be applied to a single nucleotide, and oligonucleotides can feature a variety of different types of nucleotides, including natural nucleotides, modified nucleotides, and combinations thereof.

[0030] As used herein, the term "monomer" refers to a single biological building block, such as a single nucleotide or a single amino acid. "Monomer" can be used in the context of an existing oligomeric sequence and can refer to a single building block therein, or it can refer to a new building block, such as a nucleotide or amino acid, that is to be added to a solid-bound monomer or oligomer.

[0031] As used herein, the term "nucleotide" includes conventional naturally occurring nucleotides as well as chemically modified variants thereof. Nucleotides may also be synthetically produced.

[0032] As used herein, the term "oligomer" refers to a molecule comprising at least two types of monomers and is intended to encompass biological polymers such as those comprising oligonucleotides or peptides.

[0033] As used herein, the term "oligonucleotide" refers to a compound that comprises natural nucleotides such as DNA and RNA, or their chemically modified variants.Examples include, but are not limited to: DNA, RNA, mRNA (messenger RNA), DNA and / or RNA hybrids (including their chemically modified variants), RNAi (RNA and / or chemically modified RNA that can induce RNA interference mechanism), siRNA (small interfering RNA), shRNA (short hairpin RNA), miRNA (microRNA), antisense RNA, ribozyme, catalytic DNA, RNA that induces triple helix formation, aptamer, and vector.

[0034] As used herein, the term "peptide" refers to a compound in which two or more amino acids are covalently linked together. The term "peptide" is intended to be used interchangeably with the terms "protein," "oligopeptide," and "polypeptide." Peptides may be composed of naturally occurring and / or synthetic amino acids.

[0035] As used herein, the term "quasi-continuous" refers to a series of operations in which the flow of product material (e.g., solid phase material or liquid feed) is essentially continuous for a certain period of time, but in which the flow may be interrupted for one or more periods. For example, if the operation of a belt reactor involves the use of cassettes, the process is generally continuous but may involve interruptions, for example, to move between or remove cassettes.

[0036] As used herein, the term "reactant" refers to an agent that includes a monomer or existing building block in an activated form, a protected form, or a form that allows the monomer or existing building block to be coupled to another monomer or oligomer through a chemical reaction. In the case of oligonucleotide synthesis, examples include, but are not limited to, nucleoside phosphoramidites, phosphotriesters, and H-phosphonates. In the case of peptide synthesis, examples include, but are not limited to, amino acids, partially protected amino acids, N-protected amino acids, and C-protected amino acids.

[0037] As used herein, the term "reagent" refers to a compound used to support, promote, or carry out a chemical reaction. Examples include, but are not limited to, activators, catalysts, oxidizing agents, thiolating agents, and capping agents.

[0038] As used herein, the term "solid-phase belt" refers to a type of solid-phase material comprising a flexible continuous band made of a permeable material, which exhibits functional groups on its surface that allow for solid-phase synthesis of oligomers. Examples of materials applicable to solid-phase belts include, but are not limited to, polymers, glass, carbon, graphene, and graphene oxide. Solid-phase belts can comprise either woven or nonwoven fabrics. Solid-phase belts can also be composed of interconnected cassettes that are tethered together to form a continuous chain. In some embodiments, the solid-phase belt comprises one or more interconnected cassettes. In some embodiments, the solid-phase belt comprises 10 or more interconnected cassettes. In some embodiments, the solid-phase belt comprises 50 or more interconnected cassettes.

[0039] As used herein, the term "solid phase material" refers to polymers, resins, controlled porous glass, graphene, graphene oxide, glass, and other materials that can be used as solid supports for solid-phase synthesis of oligomers, such as oligonucleotides or peptides. Examples of common solid phase materials include, but are not limited to, Nittophase®, Tentagel®, and controlled pore glass (CPG).

[0040] As used herein, the term "station" refers to a discrete segment of a module where a particular step is performed. Examples of such steps include, but are not limited to, deprotection, coupling, oxidation, thiolation, capping, and washing. A single module can contain one or more stations of the same type. Stations may vary in length, structure, and other variables to suit the specific requirements of the step performed in that station.

[0041] Abbreviation

[0042] [Table 1] JPEG2026506529000003.jpg252144 JPEG2026506529000004.jpg35144

[0043] Oligomer production using a belt reactor In some embodiments, the present disclosure provides methods and apparatus for continuous or quasi-continuous production of oligomers, such as oligonucleotides and peptides. The steps of the production method (which may be performed by the apparatus) include at least one deprotection station for performing a deprotection step, at least one coupling station for performing a coupling step, optionally one or more oxidation and / or thiolation stations for performing an oxidation or thiolation step, optionally one or more capping stations for performing a capping step, and optionally one or more washing stations for performing a washing step. One or more solid phase materials are moved between stations via a conveyor device, and at each station, a fluid delivery device provides reactants or reagents to the solid phase material.

[0044] In some embodiments, the belt reactor comprises one or more modules. The number of modules and their configuration can be adjusted based on the desired oligomer, production capacity, production rate, and / or other production parameters and requirements. In some embodiments, production occurs within a single module (e.g., all stations are present within the module for the preparation of the entire oligomer of interest). In some embodiments, production occurs within two or more modules (e.g., each module provides a portion of a method step, e.g., adding a single amino acid or nucleotide to an oligomer).

[0045] In some embodiments, the synthesis of an oligomer is obtained through the linear arrangement of modules, whereby each module adds one nucleotide or amino acid to the growing chain. In some embodiments, the synthesis of an oligomer is obtained through the linear arrangement of modules, whereby each module adds one nucleotide, amino acid, or pre-existing building block to the growing chain.

[0046] In some embodiments, oligomer synthesis is achieved through a nonlinear arrangement of modules, where each module adds one monomer or pre-existing building block to the growing chain, and one or more modules are used more than once. In some embodiments, oligonucleotide or peptide synthesis is achieved through a non-linear arrangement of modules, where each module adds one nucleotide, amino acid, or pre-existing building block to the growing chain, and one or more modules are used more than once. In some embodiments, a solid phase material passes through one or more modules more than once, where the solid phase material is re-fed to a previous module at least once to obtain the desired sequence. In some embodiments, multiple copies of a desired oligomer are produced in parallel, in the same module or in separate modules.

[0047] In some embodiments, two or more solid phase belts are acted upon within a module. In some embodiments, two or more solid phase belts are stacked one on top of the other within a module. In some embodiments, two or more solid phase belts are stacked horizontally next to each other within a module. In some embodiments, one or more fluid delivery devices simultaneously dispense reactants, reagents, or wash solutions to two or more stacked solid phase belts. In some embodiments, one or more fluid delivery devices sequentially dispense reactants, reagents, or wash solutions to two or more stacked solid phase belts, where the reactants, reagents, or wash solutions first permeate one belt and then permeate the other belt. In some embodiments, the reactants, reagents, or wash solutions are dispensed and collected by a first solid phase belt, and then dispensed by an additional solid phase belt within the same module.

[0048] In some embodiments, the modules comprise one or more stations arranged sequentially in a horizontal fashion. In some embodiments, the modules comprise one or more stations arranged sequentially in a stacked or one-above-the-other fashion. In some embodiments, the modules comprise one or more stations arranged sequentially around the circumference of a moving rotary cylinder.

[0049] In some embodiments, a buffer tank is used between two modules. In this case, solid phase material from the end of one module is collected in the buffer tank and fed to the start of the next module. In some embodiments, the buffer tank also serves as a reactor for performing a specific step. In some embodiments, the buffer tank performs a deprotection step. In some embodiments, separate reactors are used to perform specific steps in the sequence. In some embodiments, separate reactors are used to perform the deprotection step. In some embodiments, the buffer tank is used in a belt reactor with solid phase particles.

[0050] In some embodiments, a buffer tank is not used and the solid phase material is transferred directly from the end of one module to the beginning of the next module.

[0051] In some embodiments, the number, type, and arrangement of stations within a module may vary depending on the desired oligomer sequence, desired purity level, potential cost, and other parameters. In some embodiments, there are two or more stations performing the same step within the same module. In some embodiments, one or more wash steps are performed after each of the deprotection, coupling, oxidation, thiolation, and / or capping steps. In other embodiments, some steps are performed sequentially without intermediate wash steps. In some embodiments, each module of the belt reactor includes a capping step to prevent the generation of oligomer sequences lacking nucleotides or amino acids. In other embodiments, no module includes a capping step. In some embodiments, each module of the belt reactor includes an oxidation or thiolation step. In other embodiments, no module includes an oxidation or thiolation step.

[0052] In some embodiments, the station lengths may be varied to accommodate differences in the reaction kinetics of the deprotection, coupling, oxidation, thiolation, capping, and / or washing steps. Increasing the station length may result in an increase in the time it takes for the solid phase material on the conveyor device or cassette to reach the next station. As a result, the solid phase material may be exposed to the reactants, reagents, or wash solutions dispensed at that station for a longer period of time. In some embodiments, operation of the belt reactor involves quasi-continuous movement of the conveyor device, thus allowing for longer reaction times for one or more reactions at one or more stations. Thus, increasing the length of a station performing a slower reaction can more significantly promote reaction completion and / or increase the purity of the synthesized oligomer. In some embodiments, increasing the station length and increasing the speed at which the conveyor belt transports the solid phase material can result in an increased amount of material throughput. In some embodiments, increasing the height and / or width of the solid phase material on the conveyor device can increase material throughput.

[0053] In some embodiments, the speed at which the conveyor device transports the solid phase material may be varied to accommodate differences in the reaction kinetics of each of the deprotection, coupling, oxidation, thiolation, capping, and / or washing steps. Slowing the speed of the conveyor device may result in an increase in the time it takes for the solid phase material on the conveyor device to reach the next station. As a result, the solid phase material may be exposed to the reactants, reagents, or wash solutions dispensed at that station for a longer period of time. Therefore, slowing the speed of the conveyor device at stations performing slower reactions may more significantly promote reaction completion and / or increase the purity of the synthesized oligomer. In some embodiments, increasing the speed at which the conveyor belt transports the solid phase material may result in an increase in the amount of material throughput.

[0054] In some embodiments, each step is performed in a separate station where the necessary reagents, reactants, or washes are dispensed onto the solid phase material on a conveyor device. The arrangement of the stations within the module corresponds to the sequence of actions required to affect the addition of monomers to the solid-bound oligomer. To synthesize an oligomer, the solid phase material is acted upon sequentially by the appropriate modules, each of which ligates one additional nucleotide or amino acid onto the growing oligomer chain.

[0055] In some embodiments, the belt reactor is configured with a number of modules corresponding to the total number of monomers in the desired oligomer. Each module can contain all the stations necessary for monomer addition (e.g., adding one nucleotide or amino acid to a growing oligomer). For example, to prepare an oligonucleotide of the sequence GAGA, a total of four modules are used, set up sequentially. The solid phase material is transferred from one module to the next in linear succession, with each module adding one nucleotide or amino acid to the sequence. In some embodiments, different modules can be exchanged in sequence to produce different oligomers. For example, to switch from the sequence GAGA to the sequence GGGA, different "G" and "A" production modules can be arranged in the reactor.

[0056] In another embodiment, the belt reactor is configured with a number of modules corresponding to the number of distinct monomers in the desired oligomer. For example, to prepare an oligonucleotide of the sequence GAGA, a total of two modules are used. After the solid phase material is acted upon by the first two modules, the solid phase material is re-fed to the start of the first module to obtain the desired sequence.

[0057] In some embodiments, the supply tank contains a reactant, reagent, or wash solution. The reactants and reagents may be present in neat form or as a solution in a suitable solvent. The supply tank can be connected to one or more fluid delivery devices through a coupling that provides fluid communication. Examples include, but are not limited to, tubing, piping, or functional equivalents thereof. If the same reagent, reactant, or wash solution is used at more than one station, the belt reactor can be configured to allow one supply tank to supply more than one station.

[0058] In some embodiments, the supply tank is replenished from a refill well, in which the reactants, reagents, and wash solutions can be heated, cooled, protected from light, agitated, or otherwise manipulated depending on the required storage conditions, stability, and specific properties of each agent.

[0059] In some embodiments, pumps are used to control the amount of reactant, reagent, or wash fluid dispensed. In some embodiments, supply tanks or refill wells are pressurized to dispense reactant, reagent, or wash fluid upon opening of valves. In some embodiments, one or more processors are programmed to control operation of pumps that supply reactants, reagents, and wash fluids from refill wells and supply tanks to the fluid delivery device.

[0060] In some embodiments, valves are used to control the flow of reactants, reagents, or wash fluids from supply tanks to one or more fluid delivery devices. The delivery of reactants, reagents, or wash fluids is controlled through the opening and closing of appropriate valves. In some embodiments, one or more processors are programmed to control the operation of valves that regulate the delivery of reactants, reagents, and wash fluids from the refill wells and supply tanks to the fluid delivery devices.

[0061] In some embodiments, one or more of the reactants, reagents, and wash fluids are mixed prior to dispensing through the fluid delivery device. Premixing can be achieved through an additional premix tank installed between the fluid delivery device and a refill well or supply tank. In some embodiments, one or more of the reactants, reagents, and wash fluids are dispensed through the same fluid delivery device within the station, thereby allowing one or more of the reactants, reagents, and wash fluids to mix upon release through the fluid delivery device.

[0062] In some embodiments, the fluid delivery device comprises a spray nozzle, a porous nozzle, a porous plate, or an open pipe for dispensing a reagent, reactant, or wash solution onto the solid phase material. The flow rate, outlet pressure, and spray angle of the fluid delivery device can be adjusted and controlled by process control. In some embodiments, the rate at which the reactant, reagent, or wash solution is dispensed onto the solid phase material is adjusted through controlling the operation of a pump or valve. The spray angle of the fluid delivery device can be adjusted through mechanical changes to the nozzle opening and can be configured to produce desired spray characteristics that enable optimal operation of the belt reactor with a particular conveyor width, conveyor speed, and solid phase material loading.

[0063] In some embodiments, a conveyor device transports the solid phase material. In some embodiments, the solid phase material, e.g., a solid phase belt, is integrated with the conveyor device. In some embodiments, the solid phase material, e.g., a solid phase belt, is disposed adjacent to (e.g., overlapping) the conveyor device. In some embodiments, the conveyor device transporting the solid phase material comprises rollers. In some embodiments, the conveyor device comprises a solid phase belt. In some embodiments, the conveyor device transporting the solid phase material is a conveyor belt. In some embodiments, the conveyor belt comprises a permeable material that allows liquid to pass through. In some embodiments, the conveyor belt functions as a filter. In some embodiments, the conveyor device comprises a filtration belt. In some embodiments, the conveyor device comprises a conveyor belt and a solid phase material (e.g., a solid phase belt). In some embodiments, the solid phase material is attached to the conveyor belt. In some embodiments, the solid phase material is deposited on the conveyor belt (e.g., overlapping the conveyor belt to form a layer of solid phase material). In some embodiments, the conveyor belt comprises a monolayer under the solid phase material. In some embodiments, the conveyor belt includes two layers that sandwich the solid phase material between its upper and lower sides. The material, width, length, thickness, tension, friction, porosity, and other properties of the conveyor belt can be selected based on, for example, the particular solid phase material, cassettes, reactants, reagents, wash solutions, scale, ambient temperature, ambient pressure, and / or humidity under which the belt reactor will operate. In some embodiments, the conveyor belt provides mechanical stability to the solid phase material being transported.

[0064] In some embodiments, the conveyor belt comprises a flat shape. In some embodiments, the conveyor belt comprises a folded (e.g., wavy) configuration. In some embodiments, the conveyor belt comprises an embossed surface. In some embodiments, the conveyor belt comprises a studded surface. In some embodiments, the conveyor belt comprises a diamond-shaped surface. In some embodiments, the conveyor belt comprises a sawtooth surface. In some embodiments, the conveyor belt comprises a grooved surface. In some embodiments, the conveyor belt comprises a cleated surface. In some embodiments, the conveyor belt comprises a curved surface. In some embodiments, the conveyor belt comprises a folded surface or a series of folded surfaces.

[0065] In some embodiments, the conveyor belt comprises a face coating. In some embodiments, the conveyor belt comprises a PVC coating. In some embodiments, the conveyor belt comprises a rubber coating. In some embodiments, the conveyor belt comprises an aramid coating. In some embodiments, the conveyor belt comprises a polyester coating. In some embodiments, the conveyor belt comprises a natural rubber coating. In some embodiments, the conveyor belt comprises a cellular foam coating. In some embodiments, the conveyor belt comprises a Lycra coating.

[0066] In some embodiments, the conveyor device is configured for continuous movement of the solid phase material. In some embodiments, the conveyor device is configured for continuous movement of the solid phase material with periodic slowing or pausing of the conveyor device. In some embodiments, the conveyor device periodically slows or stops to allow for removal of defective cassettes or solid phase material. In some embodiments, the conveyor device periodically slows or stops to allow a reaction at any given station to proceed before proceeding to the next station.

[0067] In some embodiments, the choice of material for the conveyor belt depends on the solid-phase material used, the concentration and type of reactants and reagents used, and other process parameters of the belt reactor. A variety of suitable materials for conveyor belts are known and can be used in the present disclosure. Examples include, but are not limited to, polystyrene, nylon, polyester, cotton, stainless steel, nitrile rubber, PVC (polyvinyl chloride), PE (polyethylene), PET (polyethylene terephthalate), PP (polypropylene), PU (polyurethane), PTFE (polytetrafluoroethylene), PEEK (polyether ether ketone), PA (polyamide), PVDF (polyvinylidene fluoride), ECTFE (ethylene chlorotrifluoroethylene), SBR (styrene butadiene rubber), EPDM (ethylene propylene diene monomer rubber), TPE (thermoplastic elastomer), and RMV (rubber-modified vinyl). In some embodiments, PEEK is a preferred material for the conveyor belt. In some embodiments, one or more processors are programmed to control the operation of the conveyor device.

[0068] In some embodiments, the station comprises one or more liquid collection zones that collect liquid that permeates the conveyor device. The liquid collection zones can comprise collection tanks mounted below the conveyor device that collect any fluid that separates from the solid phase material by gravity. Fluids collected in these collection zones can be reused or discarded. In some embodiments, separation of the fluid from the solid phase material is accelerated by applying a vacuum to the bottom of the conveyor device. In some embodiments, separation of the fluid from the solid phase material is accelerated by applying pressure to the top of the conveyor device.

[0069] In some embodiments, the fluid collection zones are configured for reuse of recovered reactants, reagents, or wash fluids. The number of fluid collection zones within a station is flexible and may vary from station to station.

[0070] In some embodiments, the recycled reactants, reagents, or wash fluids are recycled in a cocurrent or countercurrent manner. When configured for countercurrent recycling, fresh reagents, reactants, or wash fluids are supplied to the fluid delivery device closest to the end of the station. The fluids recycled in such collection zones are supplied to adjacent fluid delivery devices located further away from the end of the station. The fluids recycled in such collection zones are supplied to fluid delivery devices located even further away from the end of the station. As a result, when reactants or reagents are recycled and reused, their content in the recycled fluid decreases with each recycling / recycling cycle. In the case of recycled and reused wash fluids, the content of contaminants increases with each recycling / recycling cycle. At the same time, the amount of oligomers affected by the reactants, reagents, or wash fluids increases as the solid phase material progresses through the station.

[0071] When configured for parallel reuse, the fluid delivery device furthest from the end of the station is supplied with fresh reagents, reactants, or washes. Fluids collected in such collection zones are supplied to adjacent fluid delivery devices located closer to the end of the station. Fluids collected in such collection zones are supplied to fluid delivery devices located even closer to the end of the station.

[0072] In some embodiments, the number of recycling steps within a station corresponds to the number of separate collection zones within the station, and can be adjusted according to the specific requirements of each station.

[0073] In some embodiments, a pressure gradient is used at one or more stations to facilitate the flow of reactants, reagents, or wash solutions through the conveyor device, for example, by applying a vacuum to an area below the conveyor device and / or through applying a positive pressure to an area above the conveyor device.

[0074] In some embodiments, the time the solid phase material is exposed to the reactants, reagents, or washes can be adjusted. If longer exposure is desired, a pressure gradient can be applied gradually and / or only at the later stations, thereby increasing the time the solid phase material is exposed to the reactants, reagents, or washes dispensed at that station. In some embodiments, the time the solid phase material is exposed to the reactants, reagents, or washes can also be adjusted by increasing the spacing between stations. Increasing the spacing between stations increases the time it takes for the solid phase material on the conveyor device to reach the next station. As a result, the solid phase material can be exposed to the reactants, reagents, or washes dispensed at that station for a longer period of time. Therefore, increasing the length of the station performing the slow reaction can more significantly promote completion of the reaction and / or increase the purity of the synthesized oligomer.

[0075] In some embodiments, the process control device monitors and controls some or all parts of the belt reactor, including, but not limited to, devices for creating pressure gradients, flow meters, valves, conveyor devices, fluid delivery devices, and analytical devices. In some embodiments, the process control device comprises software that monitors and / or controls the delivery of reagents and reactants and / or the movement of solid phase materials. In some embodiments, the software runs on a computing device in electrical communication with the belt reactor apparatus.

[0076] In some embodiments, analytical devices are used to monitor station products or intermediates. In some embodiments, the analytical devices are in electronic communication with process control devices. Examples include, but are not limited to, UV spectrometers, IR spectrometers, ATR spectrometers, Raman spectrometers, NMR spectrometers, LCMS, mass spectrometers, light monitors, pH meters, nephelometers, turbidimeters, refractometers, temperature monitors, and / or devices configured to measure conductivity or count particles.

[0077] In some embodiments, the analytical device can act as a quality control during oligomer synthesis, for example, by directly inspecting the material as it passes by the analytical device or by extracting a sample for inspection by the analytical device. If a defective or unsatisfactory product or intermediate is detected, some or all of the defective material can be discharged to a separate waste tank. In some embodiments, the system may allow all or a subset of the material to be discarded without having to waste the entire batch of material.

[0078] In some embodiments, the reactants and / or reagents collected in the collection zone are analyzed by one or more analytical devices to determine the progress of the reaction. If the progress of the reaction falls below a certain threshold, the conveyor speed is slowed down and / or more reactants or reagents are dispensed to allow longer exposure of the solid phase material to the reactants and / or reagents. In some embodiments, if the progress of the reaction is determined to fall below a certain threshold, the pressure gradient at that station is adjusted, thus allowing longer exposure times.

[0079] In some embodiments, the oligomers produced at the stations are analyzed by one or more analytical devices, and if the product quality falls below a certain threshold, an alarm is sent to process control and some or all of the sample is discarded to a waste tank.

[0080] In some embodiments, the wash fluid collected in the collection zone is analyzed by one or more analytical devices to determine the presence and levels of residual reactants, reagents and / or impurities. If the level of residual reactants and / or reagents or impurities is above a certain threshold, the conveyor speed is slowed down and more wash fluid is dispensed to allow for a longer and more rigorous wash process.

[0081] Process Sequence In some embodiments, a solid phase material is used that includes nucleotides or amino acids already bound to it. In some embodiments, a solid phase material is used that does not have pre-bound nucleotides or amino acids. In some embodiments, the solid phase material is fed directly onto a conveyor device, e.g., a conveyor belt, for oligomer synthesis. The conveyor device, e.g., a conveyor belt, transports the solid phase material continuously through all modules and stations therein. In some embodiments, the solid phase material is transported through the conveyor device without the need for a conveyor belt.

[0082] In some embodiments, when using a solid phase material with pre-bound nucleotides or amino acids, the solid phase material is deposited onto a conveyor device and transported to the first module. In some embodiments, the first module begins with a deprotection station, where one or more reagents are dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the one or more reagents for a period of time, for example, until excess fluid is drained through the conveyor device.

[0083] In some embodiments, prior to further processing, for example, if the solid phase material has been pre-bound to nucleotides or amino acids, the solid phase material leaves the deprotection station and enters an auxiliary washing station, where a wash solution is dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the wash solution for a period of time, for example, until excess fluid is drained through a conveyor device.

[0084] In some embodiments, when pre-bound nucleotides or amino acids are used, the solid phase material leaves the auxiliary wash station and enters a coupling station, where reactants and, optionally, one or more reagents are dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the reactants and reagents (if used) for a period of time, for example, until excess fluid is drained through a conveyor device.

[0085] In some embodiments, the solid phase material is fed directly onto a conveyor device for oligomer synthesis, which transports the solid phase material continuously through all modules and stations therein.

[0086] In some embodiments, when using solid phase materials without pre-bound nucleotides or amino acids, after deposition on the conveyor device, the solid phase material may be transferred to a coupling station where reactants and, optionally, one or more reagents are dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the reactants and reagents (if used) for a period of time, for example, until excess fluid is drained through the conveyor device.

[0087] In some embodiments, the solid phase material leaves the coupling station and enters a first wash station, where a wash solution is dispensed from a fluid delivery device onto the solid phase material, and the solid phase material remains exposed to the wash solution for a period of time, for example, until excess fluid is drained through a conveyor device.

[0088] In some embodiments, the solid phase material leaves the first wash station and enters an oxidation station, where an oxidizing agent is dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the oxidizing agent for a period of time, for example, until excess fluid is drained through a conveyor device.

[0089] In some embodiments, the solid phase material leaves the oxidation station and enters a second washing station, where a washing solution is dispensed from a fluid delivery device onto the solid phase material, and the solid phase material remains exposed to the washing solution for a period of time, for example, until excess fluid is drained through a conveyor device.

[0090] In some embodiments, the solid phase material leaves the second washing station and enters a capping station, where a mixture of capping agent and catalyst is dispensed onto the solid phase material from a fluid delivery device. In some embodiments, the solid phase material leaves the second washing station and enters a capping station, where a capping agent is dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the capping mixture for a period of time, for example, until excess fluid is drained through a conveyor device.

[0091] In some embodiments, the solid phase material leaves the capping station and enters a third washing station, where a washing solution is dispensed onto the solid phase material from a fluid delivery device. The solid phase material remains exposed to the washing solution for a period of time, for example, until excess fluid is drained through a conveyor device and collected in a collection tank. In some embodiments, the end of the third washing station marks the end of the first module, where the solid phase material is collected in a buffer tank or moved directly to the second module.

[0092] In some embodiments, the solid phase material from the buffer tank is fed into a reactor containing a reagent for deprotecting the newly added nucleotide or amino acid, and the solid phase material remains exposed to the deprotection reagent for a period of time before being fed onto a conveyor device and transported to the next module.

[0093] In some embodiments, one or more additional stations may be positioned before, interposed between, or placed after the steps outlined above so that additional or alternative wash solutions, reactants, and / or reagents can be delivered to the solid phase material.

[0094] In some embodiments, the belt reactor includes one or more stations for cleaving the generated oligomers from the solid phase material. Cleavage can be accomplished in a variety of ways depending on the linker used. Examples include, but are not limited to, chemical cleavage reactions, changes in pH, changes in temperature, or application of radiation.

[0095] In some embodiments, the synthesized oligomers are subjected to further purification, separation, and / or other processes after cleavage from the solid phase material. A variety of methods for post-synthesis processing are known in the art and can be used in this step.

[0096] In some embodiments, parameters controlling the reaction time and / or output from a belt reactor for a given solid phase material include belt speed, solid phase height, and / or solid phase width. Thus, scale-up during operation can be achieved in a variety of ways, including, but not limited to, increasing the belt speed, increasing the loading of solid phase material on the belt, and / or increasing the belt width. The values ​​of any of these variables can depend on the desired oligomer, its purity, and other process parameters.

[0097] Without being bound by theory, the present disclosure offers several advantages over conventional solid-phase synthesizers, such as higher material throughput, higher conversion rates and yields, higher purity, better product quality, lower manufacturing costs, better manufacturing flexibility, reduced amounts of reactants, reagents, or solvents used, faster reaction kinetics, easier scalability, fewer failure sequences, and a lower risk of errors ruining product batches. In some embodiments, recycling and reuse of reactants, reagents, and washing solutions results in reduced amounts of reactants, reagents, and washing solutions required, facilitating higher conversion rates and yields of desired oligomers. In some embodiments, a continuous manufacturing model for solid-phase oligomer synthesis facilitates reduced manufacturing costs and increased production speed. In some embodiments, the use of liquid-specific supply lines eliminates the need for cleaning steps between process steps, thus reducing manufacturing time and liquid loss. In some embodiments, the adaptation of a fluid delivery system eliminates the need for high-precision pumps to dose liquids, thus reducing the cost of manufacturing oligomers. In some embodiments, the use of a functionalized solid phase belt as the solid phase material reduces the need for reactants, reagents, or wash solutions to diffuse through the particle pores of the solid phase material. Without being bound by theory, this can provide several advantages over traditional solid phase synthesizers, such as lower operating costs, facilitating higher oligomer loading densities, and enabling faster reactions, higher throughput, better product quality, and / or fewer failed sequences. In some embodiments, the use of a solid phase belt provides several advantages over alternative solid phase materials. In some embodiments, the use of a solid phase belt is more cost-effective than other solid phase materials. In some embodiments, the use of a solid phase belt partially or completely eliminates problems arising from pressure drop in existing solid phase materials. In some embodiments, the use of a solid phase belt partially or completely eliminates problems arising from pore diffusion.In some embodiments, the use of a solid phase belt eliminates problems arising from swelling of the solid phase material in certain solvents, and in some embodiments, the use of a solid phase belt results in higher loading of oligomers compared to other solid phase materials.

[0098] In some embodiments, the belt reactor can be configured for peptide synthesis, which uses the same principles and processes as oligonucleotide synthesis but with different reactants, reagents, and / or wash solutions. For example, a method for solid-phase peptide synthesis can include the following sequence of steps: (1) coupling an N-protected amino acid to a suitable solid-phase material; (2) deprotecting the α-amino group of the amino acid using an appropriate reagent, depending on the protecting group used; (3) contacting the solid-phase-bound amino acid with a mixture of the N-protected amino acid reactant and a suitable reagent to enable the formation of a peptide bond between the α-amino terminus of the solid-phase-bound amino acid and the carboxyl terminus of the amino acid reactant; (4) repeating steps 1-3 until the desired peptide length is reached; (5) optionally, incorporating a capping step after the coupling step to block the termini of unreacted amino acids from reacting in the next coupling step; and (6) optionally, incorporating a washing step into the sequence to remove residual reactants or reagents from the solid-phase material. (7) A step of cleaving the peptide from the solid phase material, and optionally, a purification and / or drying step may be performed. Each step may be performed in a station of the belt reactor. Thus, different solid phase materials, linkers, reactants, reagents, and / or wash solutions may be used, and the stations and process parameters may be adjusted to suit the peptide synthesis (e.g., by spacing the stations to suit different reaction times for each step in the peptide synthesis).

[0099] solid phase material As used herein, solid phase materials can include different materials, forms and configurations.

[0100] In some embodiments, the solid phase material is a commercially available solid support such as Nittophase® or Tentagel®. However, there are other suitable solid phase materials known in the art that can be used in the present disclosure. Examples include, but are not limited to, macroporous polystyrene polymers, cross-linked polystyrene polymers, and controlled pore glass. In some embodiments, preferred solid phase materials have high loadings and contain functional hydroxyl groups. It may be preferable to use macroporous polystyrene resin as the solid phase material in some cases. In some embodiments, the solid phase material comprises a polymeric material in the form of particles with internal pores. In some embodiments, the solid phase material comprises glass in the form of particles with internal pores. In some embodiments, the solid phase material comprises carbon in the form of particles with internal pores.

[0101] In some embodiments, the solid phase material is made from one or more of a polymer, a resin, controlled pore glass, graphene, graphene oxide, and glass. In some embodiments, the solid phase material is made from a polymeric material. In some embodiments, the solid phase material is made from a resin. In some embodiments, the solid phase material is made from graphene. In some embodiments, the solid phase material is made from graphene oxide. In some embodiments, the solid phase material is made from reduced graphene oxide. In some embodiments, the solid phase material is made from glass.

[0102] In some embodiments, the solid phase material comprises a solid phase belt. In some embodiments, the solid phase material comprises nonwoven fibers. In some embodiments, the preferred material for the nonwoven fibers is glass. In some embodiments, the preferred material for the nonwoven fibers is polystyrene. In some embodiments, the preferred fiber diameter for the nonwoven fibers is 1-10 μm. In some embodiments, the preferred fiber diameter for the nonwoven fibers is 0.2-1 μm. Such fibers can be produced through electrospinning (with or without air blowing) or centrifugal spinning [Bhardwaj et al., "Electrospinning: A fascinating fiber fabrication technique" Biotechnology Advances 2010, 28(3), 325-347; Agarwal et al., "Use of electrospinning technique for biomedical applications" Polymer 2008, 49(26), 5603-5621].

[0103] In some embodiments, the solid phase material is treated to improve or add binding capability to one or more of amino acids, peptides, nucleotides, and oligonucleotides. In some embodiments, the solid phase material is modified to enable solid phase oligomer synthesis. In some embodiments, the treatment of the solid phase material increases the presence of hydroxy groups on the solid phase material. In some embodiments, the treatment of the solid phase material is achieved through one or more of plasma treatment, irradiation, coating, UV irradiation, chemical grafting, and annealing. In some embodiments, the treatment of the solid phase material is achieved through plasma treatment. In some embodiments, the treatment of the solid phase material is achieved through irradiation. In some embodiments, the treatment of the solid phase material is achieved through coating. In some embodiments, the treatment of the solid phase material is achieved through chemical grafting. In some embodiments, the treatment of the solid phase material is achieved through annealing. In some embodiments, the treatment of the solid phase material through irradiation comprises UV irradiation.

[0104] In some embodiments, the oligomer is directly attached to the solid phase material. In some embodiments, the oligomer is attached to the solid phase material via a linker. Various linkers that can be used in belt reactors are known in the art. Examples include, but are not limited to, Wang linkers, Rink linkers, Sheppard linkers, phenylfluorenyl linkers, oxazolidine linkers, acetal linkers, methacrylic linkers, DDE linkers, vinyl sulfone linkers, REM linkers, sulfone linkers, photolabile linkers, aryl sulfonic acid linkers, and cysteine ​​linkers.

[0105] In some embodiments, the solid phase material is in a form selected from particles, fibers, woven fibers, nonwoven fabrics, felts, fabrics, gels, flat surfaces, tapes, membranes, foams, extrudates, and extruded materials. In some embodiments, the solid phase material comprises fibers. In some embodiments, the solid phase material comprises fibers, preferably with a width of 0.2 to 10 μm. In some embodiments, the solid phase material comprises woven fibers. In some embodiments, the solid phase material comprises nonwoven fabrics. In some embodiments, the solid phase material comprises felts. In some embodiments, the solid phase material comprises fabrics. In some embodiments, the solid phase material comprises gels. In some embodiments, the solid phase material comprises flat surfaces. In some embodiments, the solid phase material comprises tapes. In some embodiments, the solid phase material comprises membranes. In some embodiments, the solid phase material comprises foams. In some embodiments, the solid phase material comprises extruded materials. In some embodiments, the solid phase material comprises a mixture of two or more of particles, fibers, woven fibers, nonwoven fabrics, felts, fabrics, gels, flat surfaces, tapes, membranes, foams, and extruded materials.

[0106] In some embodiments, the solid phase material is a nonwoven material. In some embodiments, the nonwoven material is modified with linkers known in the art to enable solid phase oligomer synthesis. In some embodiments, the linkers are disposed within the pores of the solid phase material. In some embodiments, the linkers are disposed on the surface of the fibers, resulting in increased mass transfer of reagents, reactants, or wash solutions through the solid phase material due to elimination of pore diffusion. In some embodiments, the use of nonwoven materials can shorten reaction times and / or achieve higher throughput. In some embodiments, the cost of the nonwoven material is lower than the cost of other solid phase materials.

[0107] Several nonwoven materials can be used in the present disclosure, including, but not limited to, polystyrene, nylon, polyester, cotton, nitrile rubber, PVC (polyvinyl chloride), PE (polyethylene), PET (polyethylene terephthalate), PP (polypropylene), PU (polyurethane), PTFE (polytetrafluoroethylene), PEEK (polyether ether ketone), PA (polyamide), PVDF (polyvinylidene fluoride), ECTFE (ethylene chlorotrifluoroethylene), SBR (styrene butadiene rubber), EPDM (ethylene propylene diene monomer rubber), TPE (thermoplastic elastomer), and RMV (rubber modified vinyl).

[0108] In some embodiments, the solid fiber, woven fiber, nonwoven fabric, felt, fabric, gel, flat surface, tape, membrane, foam, or extruded material is contained within a flexible section of a cassette transported on a conveyor device. In some embodiments, the flexible section of the cassette is configured as a single layer with the rigid section of the cassette. In some embodiments, the flexible section of the cassette is configured as two or more layers with the rigid section of the cassette. In some embodiments, two or more layers of the flexible section of the cassette are made of the same type of solid phase material. In some embodiments, two or more layers of the flexible section of the cassette are made of different solid phase materials.

[0109] In some embodiments, the multiple layers of the flexible section of the cassette are flat, folded, wound, spiral, star, laminated, or a mixture of any of the foregoing. In some embodiments, the arrangement of the flexible section of the cassette increases the loading of oligomer per surface area of ​​the conveyor device. In some embodiments, the arrangement of the flexible section of the cassette increases the density of oligomer chains per cassette.

[0110] In some embodiments, the use of cassettes in solid phase manufacturing allows for quasi-continuous manufacturing. In some embodiments, the use of cassettes allows for continuous manufacturing of oligomers while retaining the advantages of individual belt units. In some embodiments, the use of cassettes in solid phase manufacturing allows for particular cassettes to be discarded during a run. In some embodiments, the use of cassettes in solid phase manufacturing allows for cassettes containing defective solid phase material to be selected and discarded without affecting the remaining solid phase material in other cassettes.

[0111] In some embodiments, the cassette is configured to seal the flexible section from the exterior. In some embodiments, sealing the flexible section of the cassette allows for temporary or permanent storage of the solid-bound oligomers. In some embodiments, sealing the cassette prevents the solid-bound oligomers from being exposed to moisture, oxygen, light, or other undesirable conditions that may adversely affect the solid-bound oligomers.

[0112] Manufacturing Reagents and Reactants In some embodiments, one or more stations are configured to chemically modify the last monomer added to an oligomer sequence. For oligonucleotide synthesis, examples include, but are not limited to, removing a protecting group from the last nucleotide added to an oligonucleotide sequence, ligating a new monomer to the nucleotide, oxidizing a phosphite to a phosphate moiety in the nucleotide, converting a phosphite triester to a phosphorothioate in the nucleotide, and capping the remaining functional group of the nucleotide. For peptide synthesis, examples include, but are not limited to, removing a protecting group from the last amino acid added to a peptide sequence, ligating a new monomer to the amino acid, and capping the remaining functional group of the amino acid.

[0113] In some embodiments, the deprotection step involves feeding the solid-phase material to a deprotection station, where the protecting group is removed from the solid-bound monomer or oligomer. A variety of protecting groups and different deprotection conditions are available in the art. The protecting group and corresponding deprotection conditions are selected based on the nucleotide or amino acid involved and other process parameters. Examples of protecting groups for use in oligonucleotide synthesis include, but are not limited to, trityl and DMT protecting groups. Examples of protecting groups for use in peptide synthesis include, but are not limited to, Bn, Boc, BOM, TBS, TBDPS, Cbz, Fmoc, DMB, SEM, Aloc, tert-butyl, DMT, trityl, and benzoyl protecting groups. The use of DMT as a protecting group for oligonucleotide synthesis and Fmoc as a protecting group for peptide synthesis may be preferred in some cases.

[0114] In some embodiments, the deprotection step involves dispensing one or more reagents onto a solid-phase material to cleave the protecting groups from the solid-bound oligomer. The reagents used for deprotection may be dispensed in neat form or as a solution in an appropriate solvent. The selection of the deprotection reagent, amount, and concentration may depend on the specific protecting group, the nucleotide or amino acid being treated, and other process parameters, and may be adjusted as needed. Several reactants are known in the art and can be used in this step. Examples of reagents for cleaving acid-labile protecting groups include, but are not limited to, acetic acid, dichloroacetic acid, trichloroacetic acid, trifluoroacetic acid, CSA, PTSA, mineral acids, and other applicable organic acids. The use of trifluoroacetic acid may be preferred in some cases. Examples of reagents for cleaving base-labile protecting groups include, but are not limited to, pyridine, triethylamine, methylamine, imidazole, ammonia, and other applicable organic bases. The use of imidazole may be preferred in some cases.

[0115] In some embodiments, the coupling step comprises dispensing reactants and / or one or more reagents onto a solid phase material to grow a nucleotide chain.The reagents may be dispensed in the form of a neat solution or in a solution of a suitable solvent.Several reactants are known in the art and can be used in this step.For oligonucleotide synthesis, examples include, but are not limited to, phosphoramidites, phosphotriesters, and H-phosphonates.The use of phosphoramidite reactants may be preferred in some cases.

[0116] In some embodiments, the coupling step involves dispensing reactants and, optionally, one or more reagents onto a solid-phase material to grow a peptide chain. The reactants and / or reagents may be dispensed in neat form or as a solution in a suitable solvent. Several reactants are known in the art and can be used in this step. Examples include, but are not limited to, N-protected amino acids and C-protected amino acids. Depending on the terminus anchored to the solid phase, the C-terminus, N-terminus, and / or one or more side chain functional groups, as appropriate, can be protected with appropriate protecting groups to facilitate selective reaction at unprotected sites. Examples of reagents for coupling amino acids include, but are not limited to, COMU, DCC, DIC, EDC, HBTU, TBTU, HATU, TATU, BOP, PyBOP, HOAt, and HOBt. The use of T3P in combination with COMU may be preferred in some cases.

[0117] In some embodiments, one or more of the reagents used in the coupling step comprises a catalyst. The catalyst may be dispensed in neat form or as a solution in an appropriate solvent. For solid-phase oligonucleotide synthesis, examples of catalysts include, but are not limited to, 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, 5-mercapto-tetrazole, 1-hydroxybenzotriazole, 3-nitro-1H-1,2,4-triazole, pyridinium trifluoroacetate, imidazolium triflate, imidazolium perchlorate, imidazolium tetrafluoroborate, N-(methyl)imidazolium triflate, N-(phenyl)imidazolium triflate, N-(phenyl)imidazolium perfluoroborate. chlorate, N-(phenyl)imidazolium tetrafluoroborate, N-(p-acetylphenyl)imidazolium triflate, 2-(phenyl)imidazolium triflate, 4-(methyl)imidazolium triflate, 4-(methyl)imidazolium tosylate, 4-(methyl)imidazolium trifluoroacetate, 4-(phenyl)imidazolium triflate, 4-(phenyl)imidazolium trifluoroacetate, benzimidazolium triflate, benzimidazolium tetrafluoroborate, N-(methyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium perchlorate, diisopropylammonium tetrazolide, and 4,5-dicyanoimidazole. The use of 5-ethylthio-1H-tetrazole may be preferred in some cases.

[0118] In some embodiments, the washing step involves removing residual reagents or reactants from the solid phase material by washing with a suitable liquid. The liquid may comprise a single solvent or a mixture of solvents. Several solvents are known in the art and can be used in this step. Examples include, but are not limited to, acetonitrile, dichloromethane, dichloroethane, chloroform, dimethoxyethane, N,N-dimethylformamide, N,N'-dimethylacetamide, N-methyl-2-pyrrolidone, N-formylmorpholine, N-butylpyrrolidinone, cyclopentyl methyl ether, methyl tert-butyl ether, diethyl ether, tetrahydrofuran, toluene, 2-methyltetrahydrofuran, ethyl acetate, dimethyl carbonate, dioxane, methanol, ethanol, isopropanol, water, and dimethyl sulfoxide. The use of acetonitrile may be preferred in some cases.

[0119] In some embodiments, the oxidation step involves dispensing one or more reagents onto the solid phase material to oxidize the phosphite esters to phosphate ester moieties. The oxidizing reagents may be dispensed in neat form or as a solution in a suitable solvent. Several oxidizing agents are known in the art and can be used in this step. Examples include, but are not limited to, iodine, tert-butyl hydroperoxide, and 10-camphorsulfonyl oxaziridine. The use of iodine may be preferred in some cases.

[0120] In some embodiments, the thiolation step involves dispensing one or more reagents onto a solid phase material to perform thiolation. The thiolation reagents may be dispensed in neat form or as a solution in a suitable solvent. Several thiolation agents are known in the art and can be used in this step. Examples include, but are not limited to, DDTT, 3H-1,2-benzodithiol-3-one 1,1-dioxide, xanthan hydride, and N,N,N',N'-tetraethylthiuram disulfide. The use of xanthan hydride may be preferred in some cases.

[0121] In some embodiments, the capping step involves dispensing one or more reagents onto the solid phase material to cap unreacted functional groups, thereby preventing them from further reaction. The capping reagent may be dispensed in neat form or as a solution in a suitable solvent. Several capping agents are known in the art and can be used in this step. Examples include, but are not limited to, acetic anhydride, acetyl chloride, propionic anhydride, isobutyric anhydride, phenoxyacetic anhydride, and tert-butylphenoxyacetic anhydride. The use of acetic anhydride may be preferable in some cases.

[0122] In some embodiments, one or more of the reagents in the coupling step include a catalyst. The catalyst may be provided in combination with another capping reagent, one or both of which may be dispensed in neat form or as a solution in an appropriate solvent. Several catalysts and capping reagents are known in the art and can be used in this step. Examples of catalysts include, but are not limited to, N-methylimidazole, 4-dimethylaminopyridine, and pyridine. Examples of capping reagents include, but are not limited to, acetic anhydride, acetic acid chloride, propionic anhydride, isobutyric anhydride, phenoxyacetic anhydride, and tert-butylphenoxyacetic anhydride. The use of NMI and acetic anhydride may be preferred in some cases.

[0123] Example layout Figure 1 shows an overview of an exemplary belt reactor setup. The illustrated belt reactor includes three modules (1), each with seven stations. The deprotection step is performed in reactor (2) containing solid-phase particles (3). Upon completion of the reaction, the solid-phase material is deposited in the first module. Excess reactants, reagents, or wash solutions are collected in a waste tank (4), and a buffer tank (5) is used to collect the solid-phase particles midway between modules. Supply tanks (6-16) deliver reagents, reactants, or wash solutions to each station through pipes, tubing, or their functional equivalents (14). For clarity, only select pipes, tubing, or their functional equivalents are shown. Supply tank (8) delivers a solution containing a reactant that causes the incorporation of C into the oligomer; supply tank (9) delivers a solution containing a reactant that causes the incorporation of G into the oligomer; and supply tank (10) delivers a solution containing a reactant that causes the incorporation of T into the oligomer. The process control (17) monitors and controls the operation of all essential parts of the belt reactor, such as opening / closing of valves (18), speed of conveyor devices and dispensing of fluids.

[0124] Figure 2 shows an example of a module with seven stations, each responsible for performing a specific step in oligomer synthesis (where each step is shown in a box above the station). In the reactor (1), the deprotection step is performed. Upon completion, the solid-phase particles (2) are deposited onto a conveyor device (3), which moves the solid-phase particles through each station. At each station, a fluid delivery device (4-10) dispenses reagents, reactants, or wash solutions onto the solid-phase material. A vacuum is applied below the conveyor device to increase the flow of fluid through the solid-phase particles. One or more collection zones (11) are present for the recovery of reactants, reagents, or wash solutions.

[0125] Figure 3 shows an example of a station. Solid phase particles (1) pass through the station via a conveyor device (2). Supply tanks (3) supply reactants, reagents, and / or wash solutions to a fluid delivery device (4), which dispenses them onto the solid phase particles. Excess reactants, reagents, or wash solutions are collected by liquid collection zones (5-7) and fed back to the fluid delivery device.

[0126] Figure 4 shows an example of a rotary-configured module with seven stations, each responsible for performing a specific step in oligomer synthesis. Solid-phase material (1) is deposited from a buffer tank (4) onto a conveyor device (2) on a moving rotary cylinder (3) and passes through each station as the rotary cylinder rotates (5). At each station, fluid delivery devices (6-12) dispense reagents, reactants, or wash solutions onto the solid-phase material. A vacuum is applied underneath the conveyor device to increase the fluid flow through the solid-phase material. One or more collection zones (13) are present for the recovery of reactants, reagents, or wash solutions. At the end of the module, the acted-on solid-phase material (14) is removed from the conveyor device.

[0127] Figure 5 shows an overview of an exemplary setup for a belt reactor operating using a solid-phase belt as the solid-phase material. The illustrated belt reactor includes three modules (1), each with seven stations. The solid-phase belt (2) unrolls from a reel (3) and is fed to the first station of the first module. Individual stations then act on the solid-phase belt to grow oligomer chains. Excess reactants, reagents, or wash solutions are collected in a waste tank (4). Supply tanks (5-14) supply reagents, reactants, or wash solutions to each station through pipes, tubing, or their functional equivalents (15). For clarity, only a subset of the pipes, tubing, or their functional equivalents that can be used in the disclosed method are illustrated in Figure 5. A feed tank (7) provides a solution containing reactants that cause the incorporation of C into the oligomer; a feed tank (8) provides a solution containing reactants that cause the incorporation of G into the oligomer; a feed tank (9) provides a solution containing reactants that cause the incorporation of T into the oligomer. A process control (16) monitors and controls the operation of all essential parts of the belt reactor, such as opening / closing of valves (17), speed of the conveyor device and dispensing of fluids.

[0128] Figure 6 shows an example of a single module of a belt reactor operating using a solid phase belt (1) as the solid phase material. The solid phase belt includes chemical modifications on its surface. A linker (2) is configured to connect growing oligomers (3) to the solid phase belt. The solid phase belt is unwound from its reel (4) and deposited onto a conveyor device (5). The parts of a belt reactor using a solid phase belt as the solid phase material can be substantially similar to those outlined in Figures 1-3, except for the differences due to different solid phase materials.

[0129] Figure 7 shows an example of a single station of a belt reactor operating with a solid-phase belt as the solid-phase material. The solid-phase belt (1) passes through the station via a conveyor device (2). Supply tanks (3) supply reactants, reagents, and / or wash solutions to a fluid delivery device (4), which dispenses them onto the solid-phase belt. Excess reactants, reagents, or wash solutions are collected by liquid collection zones (5-7) and fed back to the fluid delivery device.

[0130] Figure 8 shows an example of a single module having eight stations arranged horizontally inside a belt reactor and operating with a cassette comprising a flexible section (1) and a rigid frame (2) configured for coupling to an oligomer. The operating principle of all essential parts of the belt reactor can be substantially similar to that outlined in Figures 1-3, except for the differences resulting from the use of cassettes.

[0131] FIG. 9 shows six examples of cassettes: (a) a single-sheet flexible band (1) arranged with a rigid section (2) to form a single cassette; (b) several-sheet flexible bands (3) of the same material arranged with a rigid section (2) to form a single cassette; (c) several-sheet flexible bands arranged with a rigid section (2) to form a single cassette, where the material of one flexible band (4) is different from the material of the other flexible bands (5); (d) a single-sheet flexible band (6) arranged with a rigid section (2) to form a single cassette; (e) several-sheet flexible bands (7) arranged in a folded manner with a rigid section (2) to form a single cassette; and (f) several-sheet flexible bands arranged in a folded manner with a rigid section (2) to form a single cassette, where the material of one flexible band (8) is different from the material of the other flexible bands (9). [Example]

[0132] The following examples are presented to illustrate possible embodiments and are not intended to limit the scope of the disclosure. [Example 1]

[0133] Oligonucleotide synthesis An example of a belt reactor disclosed herein is described below for the synthesis of oligonucleotides. Oligonucleotides are produced using the apparatus and processes described herein. A process control device is configured to sequentially control the movement of solid-phase material on a conveyor device through stations and the delivery of appropriate amounts of reactants, reagents, and wash solutions to the solid-phase material to synthesize the desired oligonucleotide. Reactants, reagents, and wash solutions are collected and recycled in a countercurrent manner at all stations.

[0134] (1) A solid phase material containing macroporous polystyrene resin is deposited onto a permeable conveyor belt and transported to the first module for ligation of nucleotides to the solid phase material. As the solid phase material passes continuously through the first station, a fluid containing a solution of nucleoside phosphoramidites and 5-(4-nitrophenyl)-1H-tetrazole in acetonitrile is dispensed from a spray nozzle onto the solid phase material on the conveyor belt. A vacuum is applied below the conveyor belt to increase the flow of fluid through the solid phase material. The recovered fluid is supplied to another spray nozzle within the same station, and recycled fluid is dispensed onto the previous portion of the solid phase material.

[0135] (2) As the solid phase material leaves the coupling station along the conveyor belt and enters the first washing station, a washing solution comprising acetonitrile is dispensed onto the solid phase material to remove residual reactants and reagents.

[0136] (3) Entering the oxidation station, a fluid containing iodine, pyridine, and water in acetonitrile is dispensed from a spray nozzle onto the solid phase material to oxidize the phosphites to phosphates.

[0137] (4) After leaving the oxidation station, the solid phase on the conveyor belt enters a washing station, where a wash solution containing acetonitrile is dispensed onto the solid phase material to remove residual reagents.

[0138] (5) The solid phase material enters a capping station where a fluid containing a solution of acetic anhydride and N-methylimidazole in acetonitrile is dispensed onto the solid phase material.

[0139] (6) The solid phase material then moves along a conveyor belt to a washing station, where a wash solution containing acetonitrile is dispensed onto the solid phase material to remove residual reagents.

[0140] (7) The solid phase material leaves the first module and is collected in a buffer tank.

[0141] (8) A solution of dichloroacetic acid in acetonitrile is added to the tank to remove the DMT protecting group from the 5'-oxygen on the nucleotide added in step 1.

[0142] (9) The solid-phase material is fed into a new module, starting with a wash station, to remove residual reagents from the previous step. The solid-bound oligonucleotide is further grown by subjecting it to steps 1 through 9 in either the same or a different module. Once the desired oligonucleotide sequence has been prepared, the oligonucleotide can be cleaved from the solid support and subjected to further post-synthesis procedures. [Example 2]

[0143] Peptide synthesis An example of a belt reactor disclosed herein is described below for the synthesis of peptides. Peptides are produced using the apparatus and processes described herein. A process control device is configured to sequentially control the movement of solid-phase material on a conveyor device through stations and the delivery of appropriate amounts of reactants, reagents, and wash solutions to the solid-phase material to synthesize the desired peptide. The use of the counterflow principle described above is applied to all stations.

[0144] (1) A solid-phase material containing Rink amide resin with pre-bound amino acids is deposited onto a permeable conveyor belt and transported to the first module for coupling N-protected amino acids to the solid-phase material. As the solid-phase material passes continuously through the first station, a fluid containing an N-Fmoc-protected amino acid, EDC, and HOBt in DMF is dispensed from a spray nozzle onto the solid-phase material on the conveyor belt. A vacuum is applied below the conveyor belt to increase the fluid flow. The recovered fluid is fed to another spray nozzle within the same station, which dispenses recycled fluid onto the previous portion of the solid-phase material.

[0145] (2) As the solid phase material leaves the coupling station and enters the first washing station, a washing solution containing DMF is dispensed onto the solid phase material to remove residual reactants and reagents.

[0146] (3) The solid phase material enters a capping station where a fluid containing a solution of acetic anhydride and pyridine in DMF is dispensed onto the solid phase material.

[0147] (4) The solid phase material on the conveyor belt then enters a washing station, where a washing solution containing DMF is dispensed onto the solid phase material to remove residual reactants and / or reagents.

[0148] (5) The solid phase material leaves the first module and is collected in a buffer tank.

[0149] (6) A solution of piperidine in DMF is added to the tank to remove the Fmoc protecting group from the amine group of the newly coupled amino acid.

[0150] (7) The solid-phase material is fed into a new module, starting with a washing station, to remove residual piperidine from the previous step. The solid-bound peptide is further grown by subjecting it to steps 1-7 in another module. Once the desired peptide sequence is obtained, the peptide is cleaved from the solid support and subjected to further post-synthesis procedures. [Example 3]

[0151] Oligonucleotide synthesis using a solid-phase belt Oligonucleotides are produced using the apparatus and process described herein. A process control device is configured to sequentially control the movement of a solid-phase material on a conveyor device through stations and the delivery of appropriate amounts of reactants, reagents, and wash solutions to the solid-phase material to synthesize the desired oligonucleotides. The reactants, reagents, and wash solutions are collected and recycled in a countercurrent manner at all stations. The solid-phase material is a solid-phase belt, which includes a polymer material functionalized to allow coupling of oligonucleotides to the solid-phase belt. In this example, a solid-phase belt without pre-bound nucleotides is used. A permeable filtration belt including PEEK acts as a conveyor device, providing additional mechanical support to the solid-phase belt and assisting in the transport of the solid-phase belt through the belt reactor.

[0152] (1) The solid-phase belt is unfolded from its storage configuration and fed into the first module of the belt reactor. In this first module, a first nucleotide is to be coupled to the solid-phase belt. As the solid-phase belt passes continuously through the first station of the first module, a fluid containing a solution of nucleoside phosphoramidite and 5-(4-nitrophenyl)-1H-tetrazole in acetonitrile is dispensed onto the solid-phase belt from a spray nozzle. A vacuum is applied to the underside of the filter belt to increase the flow of fluid through the solid-phase belt and the filter belt. The recovered fluid is fed to a different spray nozzle within the same station, which dispenses recycled fluid onto the solid-phase belt in a previous section of the first station. When the solid-phase belt reaches the end of the first station, the coupling reaction is complete, and the first nucleotide is coupled to the solid-phase belt.

[0153] (2) As the solid phase belt leaves the coupling station and enters the first washing station, a washing solution containing acetonitrile is dispensed onto the solid phase belt to remove residual reactants and reagents. When the solid phase belt reaches the end of the first station, the reactants, reagents, and other impurities from the coupling station have been washed off the solid phase belt. The solid phase belt leaves the first washing station.

[0154] (3) As the solid-phase belt enters the oxidation station, a fluid containing iodine, pyridine, and water in acetonitrile is dispensed onto the solid-phase belt from a spray nozzle to oxidize the newly created phosphite ester bonds to phosphate esters. Upon reaching the end of the oxidation station, the oxidation reaction is complete and the solid-phase belt exits the oxidation station.

[0155] (4) After leaving the oxidation station, the solid phase belt enters a second washing station, where a washing solution containing acetonitrile is dispensed onto the solid phase material, effectively removing any residual reagents and other impurities from the oxidation station. When the solid phase belt reaches the end of the second washing station, all impurities have been washed away. The solid phase belt leaves the second washing station.

[0156] (5) The solid phase belt enters a capping station, where a fluid containing acetic anhydride and N-methylimidazole in acetonitrile is dispensed onto the solid phase material. As the solid phase belt approaches the end of the capping station, any remaining hydroxy groups have been converted to ethyl esters, thus preventing undesired further reaction with reactants and reagents added in further stations. The solid phase belt leaves the capping station.

[0157] (6) The solid phase belt is transported to the third washing station. Upon entering the third washing station, a washing solution containing acetonitrile is dispensed onto the solid phase material to remove residual reagents. Upon reaching the end of the third washing station, all impurities from the previous washing station have been washed away. The solid phase belt then leaves the third washing station.

[0158] (7) The solid phase belt enters the deprotection station, where an acetonitrile solution containing dichloroacetic acid is dispensed onto the solid phase belt to remove the DMT protecting groups from the 5'-oxygens of the nucleotides added in step 1. When the solid phase belt reaches the end of the deprotection station, all protecting groups have been removed from the newly added nucleotides of step 1. The solid phase belt leaves the deprotection station.

[0159] (8) The solid phase belt is transported to the fourth wash station. Upon entering the fourth wash station, a wash solution containing acetonitrile is dispensed onto the solid phase material to remove residual reagents and other impurities. Upon reaching the end of the fourth wash station, all impurities from the previous station have been washed away. The solid phase belt then leaves the fourth wash station.

[0160] (9) The solid phase belt leaves the first module and is transported by a filter belt to the next module of the belt reactor.

[0161] (10) The solid-bound oligonucleotides are further grown by subjecting the solid-phase belt to steps 1-9 of additional modules of either the same or different type.

[0162] (11) Once the desired oligonucleotide sequence has been prepared, the solid-phase belt leaves the last washing station of the last module of the belt reactor, and instead of repeating steps 1 to 9, the oligonucleotide thus prepared is cleaved from the solid support and subjected to further post-synthesis steps, as appropriate.

Claims

1. 1. An apparatus for oligomer synthesis comprising: at least one deprotection station; at least one coupling station; Optionally, one or more oxidation and / or thiolation stations; Optionally, one or more capping stations; and Optionally, one or more washing stations Equipped with the apparatus is configured to act on one or more mobile solid phase materials at each station to produce oligomers; each station being at a fixed position within the apparatus and configured to deliver a reagent, reactant or wash solution to the mobile solid phase material via a fluid delivery device; Device.

2. The apparatus of claim 1 , wherein at least one deprotection station performs the step of deprotection.

3. 10. The apparatus of claim 1, wherein at least one coupling station performs the step of coupling.

4. The apparatus of claim 1 , wherein one or more oxidation and / or thiolation stations perform an oxidation or thiolation step.

5. The apparatus of claim 1 , wherein one or more capping stations perform the capping step.

6. The apparatus of claim 1 , wherein one or more cleaning stations perform the cleaning step.

7. 7. The device of claim 1, wherein the oligomer is bound to a solid phase material.

8. 8. A device according to any one of claims 1 to 7, wherein the oligomers are attached to the solid phase material via a linker, preferably by solid phase coupling.

9. 9. An apparatus according to any one of claims 1 to 8, configured to act on one or more solid phase materials in parallel.

10. 10. The apparatus of claim 1, wherein the one or more fluid delivery devices comprise a spray nozzle, a porous nozzle, a porous plate, or an open pipe.

11. 11. The apparatus of any one of claims 1 to 10, wherein the one or more solid phase materials are configured to pass through the station via a conveyor device.

12. 12. The apparatus of any one of claims 1 to 11, wherein the one or more solid phase materials are configured to pass stepwise through the stations via a conveyor device.

13. 13. Apparatus according to any one of claims 1 to 12, wherein the conveyor device comprises a conveyor belt.

14. The apparatus of claim 13 , wherein the conveyor belt comprises a permeable material.

15. 15. The apparatus of any one of claims 1 to 14, including a pressure gradient across the conveyor belt to increase the flow of reagents, reactants or wash solutions through the permeable material.

16. 16. The apparatus of claim 15, wherein the pressure gradient is achieved by applying a vacuum to the underside of the conveyor belt.

17. 16. The apparatus of claim 15, wherein the pressure gradient is achieved by applying a positive pressure to the upper surface of the conveyor belt.

18. 18. An apparatus according to any one of claims 1 to 17, configured to recover excess reagents, reactants or wash solutions.

19. 19. The device of any one of claims 1 to 18, wherein excess reagents, reactants or wash solutions are collected in a collection tank after passing through the solid phase material.

20. 20. The apparatus of claim 18 or 19, wherein the recovered reagent, the recovered reactant, or the recovered wash solution can be reused within the same station.

21. 20. The device of claim 18 or 19, wherein the recovered reagent, the recovered reactant, or the recovered wash solution can be reused in different stations.

22. 22. The apparatus of any one of claims 18 to 21, wherein the recovered reagent, recovered reactant, or recovered wash solution is configured to be fed back to the fluid delivery device.

23. 23. The apparatus of claim 22, wherein the fluid delivery device is configured to provide countercurrent recycling of reagents, reactants and / or wash solutions.

24. 23. The apparatus of claim 22, wherein the fluid delivery device is configured to provide co-current recycling of reagents, reactants and / or wash solutions.

25. 25. The apparatus of any one of claims 1 to 24, wherein one or more stations within a module are positioned at a distance from each other selected to result in different reaction kinetics of the deprotection, coupling, oxidation, thiolation, and capping steps.

26. 26. The apparatus of any one of claims 1 to 25, further comprising a product analysis device.

27. 27. The apparatus of claim 26, wherein the product analysis device is configured to provide a signal indicating whether the solid phase material should be retained, partially retained, or discarded.

28. 28. An apparatus according to any one of claims 1 to 27, wherein two or more stations performing the same process and using the same composition of reactants, reagents and washing solutions are operably linked to a tank containing the reactants, reagents or washing solutions.

29. 29. The apparatus of any one of claims 1 to 28, wherein the one or more solid phase materials are deposited onto a conveyor device.

30. 30. The apparatus of any one of claims 1 to 29, wherein the one or more solid phase materials comprise particles made from one or more of a controlled pore glass material, cross-linked polystyrene, and polystyrene.

31. 31. The apparatus of any one of claims 1 to 30, wherein the one or more solid phase materials comprise a solid phase belt.

32. 32. The apparatus of any one of claims 1 to 31, wherein the solid phase belt comprises one or more materials selected from polymer, carbon, glass, graphene, and graphene oxide.

33. 33. The device of any one of claims 1 to 32, wherein one or more solid phase materials have been modified to increase or add to the presence of functional groups on the solid phase material.

34. 34. The device of any one of claims 1 to 33, wherein the one or more solid phase materials comprise particles, fibers, woven fibers, nonwoven fibers, nonwoven fabrics, felts, fabrics, gels, flat surfaces, tapes, membranes, foams, and extrudates.

35. 35. The device of any one of claims 1 to 34, wherein the one or more solid phase materials comprise a nonwoven material.

36. 36. The device of claim 35, wherein the nonwoven material comprises polystyrene, nylon, polyester, cotton, nitrile rubber, polyvinyl chloride, polyethylene, polyethylene terephthalate, polypropylene, polyurethane, polytetrafluoroethylene, polyether ether ketone, polyamide, polyvinylidene fluoride, ethylene chlorotrifluoroethylene, styrene butadiene rubber, ethylene propylene diene monomer rubber, thermoplastic elastomer, and / or rubber-modified vinyl.

37. 37. The apparatus of any one of claims 1 to 36, wherein the shape of the solid phase material is flat, folded, star-shaped, coiled, or spiral.

38. 35. The device of claim 34, wherein a fiber, woven fiber, nonwoven fabric, felt, fabric, gel, flat surface, tape, membrane, foam, or extrusion is contained within the flexible section of the cassette.

39. 40. The device of claim 38, wherein the fibers, woven fibers, nonwoven fabric, felt, fabric, gel, flat surface, tape, membrane, foam, or extrudate are arranged in a configuration that increases the surface area of ​​the fibers, woven fibers, nonwoven fabric, felt, fabric, gel, flat surface, tape, membrane, foam, or extrudate.

40. 40. The device of claim 38 or 39, wherein the configuration of the fiber, woven fiber, nonwoven, felt, fabric, gel, flat surface, tape, membrane, foam, or extrudate is selected from folded, layered, wound, and spiral.

41. 9. The device of claim 8, wherein the linker is configured to release the oligomer from the solid support by one or more chemical cleavage reactions or by a change in pH and / or temperature.

42. 42. The device of claim 8 or 41, wherein the linker comprises a Wang linker, a Sheppard linker, a Rink linker, a phenylfluorenyl linker, an oxazolidine linker, an acetal linker, a methacrylic linker, a DDE linker, a vinylsulfone linker, a REM linker, a sulfone linker, a photolabile linker, an arylsulfonic acid linker, a cysteine ​​linker, and / or a traceless linker.

43. 43. The apparatus of any one of claims 1 to 42, wherein the buffer tank is configured to collect one or more solid phase materials at the end of one module and supply it to the start of another module.

44. 44. The apparatus of claim 43, wherein the buffer tank is configured to act as a reactor.

45. 45. The apparatus of any one of claims 1 to 44, wherein the reagents for the capping station comprise acetic anhydride, acetyl chloride, propionic anhydride, isobutyric anhydride, phenoxyacetic anhydride, and / or tert-butylphenoxyacetic anhydride.

46. 46. ​​The device of any one of claims 1 to 45, wherein the reagent further comprises a catalyst.

47. 47. The apparatus of claim 46, wherein the catalyst comprises N-methylimidazole, 4-dimethylaminopyridine and / or pyridine.

48. 48. The apparatus of any one of claims 1 to 47, wherein the cleaning liquid comprises one or more of acetonitrile, dichloromethane, dichloroethane, chloroform, dimethoxyethane, N,N-dimethylformamide, N,N'-dimethylacetamide, N-methyl-2-pyrrolidone, N-formylmorpholine, N-butylpyrrolidinone, cyclopentyl methyl ether, methyl tert-butyl ether, diethyl ether, tetrahydrofuran, toluene, 2-methyltetrahydrofuran, ethyl acetate, dimethyl carbonate, dioxane, methanol, ethanol, isopropanol, water, and dimethyl sulfoxide.

49. 49. An apparatus according to any one of claims 1 to 48, wherein the reagents delivered at the deprotection station are suitable for removing protecting groups.

50. 50. The device of any one of claims 1 to 49, wherein the oligomer comprises an oligonucleotide.

51. 51. The apparatus of claim 50, wherein the reactants for the coupling station comprise nucleoside phosphoramidites, phosphotriesters and / or H-phosphonates.

52. 52. The apparatus of claim 50 or 51, wherein the reactants further comprise an activator.

53. The activator may be 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, 5-mercapto-tetrazole, 1-hydroxy-benzotriazole, 3-nitro-1H-1,2,4-triazole, pyridinium trifluoroacetate, imidazolium triflate, imidazolium perchlorate, imidazolium tetrafluoroborate, N-(methyl)imidazolium triflate, N-(phenyl)imidazolium triflate, N-(phenyl)imidazolium perchlorate, N-(phenyl)imidazolium tetrafluoroborate 53. The device of claim 52, comprising at least one of: N-(p-acetylphenyl)imidazolium triflate, N-(p-acetylphenyl)imidazolium triflate, 2-(phenyl)imidazolium triflate, 4-(methyl)imidazolium triflate, 4-(methyl)imidazolium tosylate, 4-(methyl)imidazolium trifluoroacetate, 4-(phenyl)imidazolium triflate, 4-(phenyl)imidazolium trifluoroacetate, benzimidazolium triflate, benzimidazolium tetrafluoroborate, N-(methyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium perchlorate, diisopropylammonium tetrazolide, and / or 4,5-dicyanoimidazole.

54. 54. The apparatus of any one of claims 40 to 53, wherein the reagents for the oxidation station include iodine, tert-butyl hydroperoxide, and / or 10-camphorsulfonyloxaziridine.

55. 54. The apparatus of any one of claims 40 to 53, wherein the reagents for the thiolation station comprise 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione, 3H-1,2-benzodithiol-3-one 1,1-dioxide, 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione, xanthan hydride, and / or N,N,N',N'-tetraethylthiuram disulfide.

56. 50. The device of any one of claims 1 to 49, wherein the oligomer comprises a peptide.

57. 57. The apparatus of claim 56, wherein the reactants for the coupling station comprise amino acids and one or more activating agents.

58. The activating agent may be benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, O-(benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), 58. The device of claim 57, comprising O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, propylphosphonic anhydride, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.

59. 50. The apparatus of any one of claims 1 to 49, wherein two or more stations act simultaneously on different portions of the solid phase material.

60. 60. A method for preparing an oligomer, comprising synthesizing the oligomer using an apparatus according to any one of claims 1 to 59.

61. 1. A method for quasi-continuous oligomer synthesis comprising: (a) providing an apparatus for solid phase material and oligomer synthesis, said apparatus comprising: at least one deprotection station; at least one coupling station; Optionally, one or more oxidation and / or thiolation stations; Optionally, one or more capping stations; and Optionally, one or more washing stations Equipped with the apparatus is configured to act on one or more solid phase materials at each station to produce oligomers; each station being at a fixed position within the apparatus and configured to deliver a reagent, reactant or wash solution to the mobile solid phase material via a fluid delivery device; To prepare and (b) initiating movement of the solid phase material through a station of the apparatus to produce oligomers; Including, method.

62. 62. The method of claim 61, wherein at least one deprotection station performs the deprotection step.

63. 62. The apparatus of claim 61, wherein at least one coupling station performs the coupling step.

64. 62. The apparatus of claim 61, wherein one or more oxidation and / or thiolation stations perform an oxidation or thiolation step.

65. 62. The apparatus of claim 61, wherein one or more capping stations perform the capping step.

66. 62. The apparatus of claim 61, wherein one or more washing stations perform the washing step.

67. 67. The method of any one of claims 61 to 66, further comprising attaching the oligomer to a solid phase material via a linker, preferably by solid phase coupling.

68. 68. The method of any one of claims 61 to 67, further comprising depositing the solid phase material onto a conveyor device.

69. 69. The method of any one of claims 61 to 68, wherein a conveyor device moves the solid phase material through the stations.

70. 70. The method of any one of claims 61 to 69, wherein deprotecting comprises contacting the protected oligomer with a reagent suitable for deprotection to cause removal of the protecting group.

71. 71. The method of any one of claims 61 to 70, wherein coupling comprises contacting the oligomer with a reactant and optionally one or more reagents to form a bond between the oligomer and the reactant.

72. 72. The method of any one of claims 61 to 71, wherein washing comprises contacting the oligomer with a wash solution to remove residual reagents or reactants from the solid phase material.

73. 73. The method of any one of claims 61 to 72, further comprising delivering a reagent, reactant or wash solution to the solid phase material via a spray nozzle, a porous nozzle, a porous plate or an open pipe.

74. 74. The method of any one of claims 61 to 73, further comprising applying a pressure gradient to the conveyor device to increase the flow of reagents, reactants or wash solutions.

75. 75. The method of claim 74, further comprising applying a pressure gradient by applying a vacuum to the underside of the conveyor belt.

76. 75. The method of claim 74, further comprising applying a pressure gradient by applying a positive pressure to an upper surface of the conveyor belt.

77. 77. The method of any one of claims 91 to 76, further comprising recovering excess reagents, reactants or wash solutions.

78. 78. The method of any one of claims 61 to 77, further comprising collecting excess reagents, reactants or wash fluid in a collection tank after the excess reagents, reactants or wash fluid has passed through the solid phase material.

79. 79. The method of claim 77 or 78, wherein recovering comprises feeding back the recovered reagent, recovered reactant, or recovered wash solution to the fluid delivery device.

80. 79. The method of claim 77 or 78, wherein recovering comprises providing the recovered reagent, recovered reactant, or recovered wash solution to one or more additional fluid delivery devices.

81. 81. The method of claim 79 or 80, wherein feeding the one or more further fluid delivery devices is countercurrent feeding.

82. 81. The method of claim 79 or 80, wherein feeding the one or more further fluid delivery devices is co-current feeding.

83. 83. The method of any one of claims 621 to 82, further comprising releasing the oligomer from the solid support by one or more chemical cleavage reactions or by a change in pH and / or temperature.

84. 84. The method of any one of claims 61 to 83, further comprising monitoring the oligomers via a product analysis device configured to provide a signal indicating whether the solid phase material should be retained or discarded.

85. 85. The method of claim 84, wherein the monitoring occurs at one or more stations.

86. 86. The method of any one of claims 61 to 85, further comprising retaining or discarding the solid phase material based on a signal from the product analysis device.

87. 87. The method of any one of claims 61 to 86, further comprising simultaneously preparing multiple oligomers.

88. further comprising collecting one or more solid phase materials in a buffer tank at the end of one module and feeding it to the start of another module; 88. The method of any one of claims 61 to 87.

89. 89. The method of claim 88, further comprising carrying out the reaction in a buffer tank.

90. 90. The method of any one of claims 61 to 89, further comprising releasing the oligomer from the solid phase material.

91. 91. The method of any one of claims 61 to 90, further comprising depositing the one or more solid phase materials onto a conveyor device.

92. 92. The method of any one of claims 61 to 91, wherein the oligomer comprises an oligonucleotide.

93. 93. The method of any one of claims 61 to 92, wherein coupling comprises contacting the nucleotide with a reactant comprising a nucleoside phosphoramidite, a phosphotriester and / or an H-phosphonate.

94. 94. The method of claim 93, wherein the reactants further comprise an activator.

95. The activator may be 1H-tetrazole, 5-(4-nitrophenyl)-1H-tetrazole, 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, 5-methylthio-1H-tetrazole, 5-ethylthio-1H-tetrazole, 5-benzylthio-1H-tetrazole, 5-mercapto-tetrazole, 1-hydroxy-benzotriazole, 3-nitro-1H-1,2,4-triazole, pyridinium trifluoroacetate, imidazolium triflate, imidazolium perchlorate, imidazolium tetrafluoroborate, N-(methyl)imidazolium triflate, N-(phenyl)imidazolium triflate, N-(phenyl)imidazolium perchlorate, N-(phenyl)imidazolium tetrafluoroborate 95. The method of claim 94, comprising the step of preparing a fluorocarbon polymer comprising: N-(p-acetylphenyl)imidazolium triflate, 2-(phenyl)imidazolium triflate, 4-(methyl)imidazolium triflate, 4-(methyl)imidazolium tosylate, 4-(methyl)imidazolium trifluoroacetate, 4-(phenyl)imidazolium triflate, 4-(phenyl)imidazolium trifluoroacetate, benzimidazolium triflate, benzimidazolium tetrafluoroborate, N-(methyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium triflate, 2-(phenyl)benzimidazolium perchlorate, diisopropylammonium tetrazolide, and / or 4,5-dicyanoimidazole.

96. 96. The method of any one of claims 61 to 95, wherein the step of oxidizing comprises contacting the oligonucleotide with an oxidizing agent to cause conversion of the phosphite triester to a phosphate triester.

97. 97. The method of any one of claims 61 to 96, wherein thiolation comprises contacting the oligonucleotide with a thiolation reagent to cause conversion of a phosphite triester to a phosphorothioate.

98. 98. The method of any one of claims 61 to 97, wherein capping comprises contacting the oligonucleotide with one or more reagents to cause conversion of one or more remaining free hydroxy groups to an ester.

99. 99. The method of any one of claims 96 to 98, wherein the oligomer comprises a peptide.

100. 92. The method of any one of claims 61 to 91, wherein the reactant comprises an amino acid.

101. The activating agent may be benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, (1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate, N,N'-dicyclohexylcarbodiimide, diisopropylcarbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate, O-(benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate), 101. The method of claim 100, comprising O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate, 1-hydroxy-7-azabenzotriazole, 1-hydroxybenzotriazole, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate, propylphosphonic anhydride, O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate, O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.

102. 102. The method of any one of claims 61-91 and 99-101, wherein capping comprises contacting the peptide with one or more reagents to cause conversion of one or more remaining amines to amides.

103. 103. The method of any one of claims 61-91 and 99-102, wherein capping comprises contacting the peptide with one or more reagents to cause conversion of one or more remaining carboxylic acid groups to an ester.

104. 104. The method of any one of claims 61 to 103, which is carried out continuously.

105. 99. An oligonucleotide produced according to the method of any one of claims 61 to 98.

106. 105. A peptide produced according to the method of any one of claims 61-91 and 99-104.

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