High shear solid phase synthesis

Abstract

The present disclosure relates to solid phase synthesis of organic molecules, and in particular to efficient methods for synthesizing polymers, such as peptides, nucleotides or sugars, using solid phase synthesis.

Description

Technical Field

[0001] The present disclosure relates generally to the field of solid phase synthesis and methods of synthesizing peptides and other molecules using solid phase synthesis. Background Art

[0002] Peptide drugs have many applications. There are approximately 100 approved peptide drugs on the market, including several blockbuster drugs such as the somatostatin analog octreotide. The most popular and dominant areas for peptide drugs include HIV treatment, diabetes, cancer treatment, and applications are emerging in the fields of neurological disorders such as Alzheimer's disease and Parkinson's disease and autoimmune diseases such as lupus, rheumatoid arthritis and uveitis.

[0003] In the pharmaceutical industry, three main methods are used to prepare large quantities of peptides: recombinant technology; liquid phase chemical synthesis and solid phase peptide synthesis (SPPS). SPPS is the preferred method for manufacturing peptides due to low raw material costs and economies of scale. Solid phase chemical methods are faster and less expensive to produce relatively short peptides (usually up to about 50 amino acids long) on ​​a gram to 100Kg scale and are more suitable for early clinical development.

[0004] The SPPS method is a multi-step chain method involving two main types (i.e., stages or steps) of reactions: (i) removal of temporary protecting groups (deprotection) and (ii) amino acid coupling. These reactions occur one after another in sequence, with intermediate washing to remove unreacted reagents and soluble by-products. Typically, in solid phase synthesis, a series of building blocks are constructed on an insoluble polymer resin having a linking unit reactive to the building block. Since some building blocks, such as amino acids, contain at least two functional groups that react with each other (i.e., amino groups and carboxylate groups), one group (usually amino groups) is usually protected in advance so that the free carboxylate group reacts with the linking unit instead of reacting with another amino acid present. In order to allow the amino acid chain to continue, the amino group is deprotected, thereby producing a resin with the first amino acid, which can be used to react with a continuous N-protected amino acid. The process is repeated until the desired number of building blocks is obtained. The purpose of using an insoluble resin is to make a simple washing process possible, which only requires washing all excess soluble reagents and by-products while keeping the desired product attached to the insoluble resin. Therefore, the final synthetic step is to remove the side chain protecting groups and cleave off the building block chain (peptide) and separate it from the insoluble resin.

[0005] Therefore, the solid phase synthesis of polymer molecules generally includes the following synthesis steps: (a) swelling of functionalized resin polymer beads; (b) coupling the first protected monomer unit to the functionalized resin; (c) washing the excess first protected monomer unit; (d) deprotecting the resin connected to the first monomer formed in step (b) using a deprotecting agent to form a resin connected to the first deprotected monomer; and (e) washing the excess deprotecting agent. Steps (b)-(e) are then repeated for subsequent protected monomer cycles to form resin-polymer molecules. Therefore, steps (b)-(e) are considered to be one cycle, wherein the total solid phase synthesis generally includes multiple cycles. Finally, the desired product molecule is cut off from the resin and separated from the resin to obtain the final polymer product.

[0006] The resin used for solid phase synthesis is usually an insoluble polymer modified by chemically reactive functional groups. These functional groups are chemically suitable for coupling with the first monomer to be incorporated into the target product oligomer / polymer. The first monomer and other sequential monomers usually include at least two functional groups that react with each other. However, placing a protecting group on one of these functional groups limits the conversion to coupling the protected monomer unit to the resin, thereby forming a resin that is initially connected to the first monomer and connected to the monomeric construct after many similar cycles. Usually, in order to ensure that the reaction is completed, each protected monomer in a series of cycles is used in excess (relative to the molecule bound to the resin). Typically, a deprotecting agent is also used in excess (relative to the monomer molecule bound to the resin) to ensure that the reaction is completed.

[0007] Since Bruce Merrifield invented SPPS in 1963 (Merrifield, RB (1963) J. Am. Chem. Soc. 85, 2149-2154), many methods have been introduced to improve the technology. Most of these methods are based on chemical modification of coupling agents (Pattabiraman et al., Nature (2011) 480, 471-479). Other improvements include thermal methods such as microwaves and heating (Collins et al., (2014) Org. Lett. 16, 940-943). However, there are few studies on the effects of hydrodynamic parameters on yield and side reactions in SPPS.

[0008] The secondary and tertiary structures of polypeptides are particularly sensitive to shearing forces. When subjected to shearing, the secondary and / or tertiary structures of peptide molecules may be irreversibly changed, which may lead to loss of biological activity. If the peptide molecules are subjected to sufficient shearing forces, the primary structure, i.e., the amino acid sequence, may also be destroyed or interrupted. With this in mind, the method for synthesizing peptides generally utilizes procedures that minimize the process of exposing the peptide molecules to shearing stress. For example, EP0503683 discloses a "vortex" stirring mode that prevents resin agglomeration and allows total fluid-resin interaction without the need to use impeller-type mechanical stirring. According to the disclosure, under mechanical stirring, the shearing and resin wear caused by the impeller can break the resin beads into smaller and smaller particles, which will eventually clog the filter, thereby forcing the synthesis process to be interrupted. Using a vortex agitator, there is no impeller-type shearing or abrasion effect on the resin beads.

[0009] In his seminal paper (Merrifield, RB (1963) J.Am.Chem.Soc.85, 2149-2154), Merrifield used a magnetic stirrer to mix polymeric resin beads. It turns out that this method breaks the resin beads into small particles, which clogs the sintered glass filter and prevents filtration. In order to avoid this problem, Merrifield invented a manual device consisting of a reaction vessel, in which the reactants are mixed by shaking rather than stirring (Merrifield, RB Solid Phase Systhesis (Noble Lecture), Angewandte Chemie International Edition (1985) 24, 799-892). Since then, the production of peptides has generally been limited to gentle mixing methods such as vortex, nitrogen flow, rotation in a rotary evaporation rotor, rocking stirring, etc. In large-scale solid phase synthesis (SPS) glass reactors, mechanical stirrers are generally used to gently stir the resin beads at low speeds. The impeller of this stirrer is designed for stirring with very low shear rates.

[0010] Solid phase synthesis is commonly used for the synthesis of peptides and polypeptides. However, the method is by no means limited to the synthesis of such compounds, and the preparation of other oligomeric / polymeric molecules, such as modified peptides, peptidomimetics, oligonucleotides, peptide nucleic acid molecules, oligosaccharides and small organic molecules are all achieved by solid phase synthesis. In general, any organic polymer or molecule that requires selective protection / deprotection, coupling and cleavage steps can be synthesized by solid phase synthesis (e.g., Boncher et al., (2013) Mod. Chem. appl. 1: 113).

[0011] Solid phase oligonucleotide synthesis is usually carried out using the phosphoramidite method. In this method, the phosphoramidite structural unit is derived from a protected 2'-deoxynucleoside ribonucleoside or a chemically modified nucleoside. In order to obtain the desired oligonucleotide, the structural unit is sequentially coupled to the growing oligonucleotide chain in the desired order. The structural unit involved in this method is a 3'-O-(N,N-diisopropylphosphoramidite) derivative of a nucleoside (nucleoside phosphoramidite). Typically, a resin with an O protecting group is deprotected and coupled to an O protected nucleoside phosphoramidite to form a PO bond (wherein the phosphorus comes from the added structural unit, and the oxygen is initially part of the reactant bound to the resin). The next step usually involves a resin in which the unreacted end group is O to suppress the formation of by-products. Next, the phosphorus atom is oxygenated and the oxygen is deprotected so that the product reacts to further structural units. After the chain assembly is complete, the product is released from the solid phase into the solution, deprotected and collected. Typically, the synthetic oligonucleotide is a single-stranded DNA or RNA molecule with a length of about 15-25 bases.

[0012] The solid phase synthesis of oligosaccharides is more diverse because sugar units (i.e., monosaccharide units) can be linked to each other through various CO bonds at different positions of the sugar ring. However, the oligosaccharide SPS strategy shares the common feature of repeated deprotection-coupling cycles with other SPS methodologies. Seeberger and Haase (Chem. Rev., 2000, 100 (12), pp. 4349-4394) summarized different oligosaccharide SPS processes.

[0013] There is an unmet need for improved methods for large-scale synthesis of peptides with greater efficiency and speed and reduced side reactions. Summary of the invention

[0014] The present invention provides an improved method for solid phase synthesis of oligomers, such as peptides, oligosaccharides, oligonucleotides and other molecules. The present invention is based in part on the unexpected discovery that solid phase synthesis using high shear forces and manipulation speeds does not damage the solid support or the synthesized molecules, but improves synthesis efficiency, such as synthesis time, yield and purity.

[0015] The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods, which are exemplary and illustrative, and not limiting in scope. In various embodiments, one or more of the above-described problems are reduced or eliminated, while other embodiments are directed to other advantages or improvements.

[0016] According to one aspect, the present invention provides a method for solid phase stepwise synthesis of organic molecules comprising at least one mixing step involving application of high shear forces within the synthesis mixture, wherein the solid phase resin is in contact with a stirring device during all synthesis steps of the stepwise synthesis.

[0017] According to some embodiments, the method comprises the steps of: providing a reactor comprising a reaction chamber and a stirring device, the stirring device comprising an impeller having at least two blades rotatable about an axis; placing functionalized polymeric resin beads and at least one solvent in the reactor to provide a reaction mixture, wherein the reaction mixture is contacted with the rotatable blades; placing at least one reactant in the reaction chamber; and rotating the impeller at a speed of at least 600 rpm for a period of time while maintaining at least 3×10 3 s -1 A shear rate of 100° is used to perform at least one solid phase synthesis step.

[0018] According to some embodiments, the stirring device is a mechanical stirrer, and the rotation of the impeller is performed at a speed of 600 to 1000 rpm, maintaining at least 3×10 3 s -1 The shear rate.

[0019] According to other embodiments, the stirring device is a homogenizer, and the rotation of the impeller is performed at a speed of 5,000-30,000 rpm while maintaining at least 1×10 6 s -1 The shear rate.

[0020] The stirring device according to the invention is a stirrer, such as a mechanical stirrer or a homogenizer, comprising at least two rotatable blades. According to the invention, any stirrer capable of stirring a solid phase synthesis reaction and generating the above-mentioned rotation speed and shear rate can be used.

[0021] According to some embodiments, the homogenizer is a rotor-stator homogenizer.

[0022] According to some embodiments, the stirring device and the reaction chamber are substantially coaxial. According to some embodiments, the mechanical stirrer and the reaction chamber are substantially coaxial. According to some embodiments, the homogenizer and the reaction chamber are substantially coaxial.

[0023] According to some embodiments, any stirring device disclosed herein, such as a mechanical stirrer or a homogenizer, comprises a generally elongated body aligned along an axis. According to some embodiments, the reaction chamber is three-dimensional and comprises an opening edge for inserting the stirring device. According to some embodiments, the opening edge is two-dimensional, so that the stirring device is inserted through a position substantially in the center of the opening edge. In this way, according to some embodiments, the reaction chamber and the homogenizer are substantially coaxial.

[0024] The term "solid phase synthesis" (SPS) refers to one or a series of chemical reactions used to prepare a single compound or a library of molecularly diverse compounds, wherein the chemical reactions are carried out on a compound that is bound to a solid support material via an appropriate linker. Thus, according to the solid phase synthesis method, in some or all of the synthesis steps, the compound or its precursor is attached to a solid support. SPS is commonly implemented in the production of peptides, polysaccharides, and polynucleotides.

[0025] According to some embodiments, the organic molecules formed in the method are polymeric organic molecules.

[0026] According to some embodiments, the method comprises the steps of:

[0027] (a) providing a reactor, the reactor comprising a reaction chamber and a stirring device, the stirring device comprising an impeller having at least two blades rotatable about an axis;

[0028] (b) placing functionalized polymeric resin beads and at least one solvent in a reactor to provide a reaction mixture, wherein the reaction mixture is contacted with a rotatable blade;

[0029] (c) placing at least one protected monomer organic molecule into a reaction chamber and rotating an impeller and at least one coupling agent to form a coupling product of the protected monomer organic molecule and the resin;

[0030] (d) washing excess of the protected monomeric organic molecules; and

[0031] (e) placing at least one deprotecting agent in the reaction chamber and rotating the impeller to remove at least one protecting group from the coupling product to form a coupling product of the deprotected monomer organic molecule and the resin, thereby completing a cycle in the solid phase synthesis of the polymerized organic molecule;

[0032] wherein the impeller in at least one of steps (c) and (e) is rotated for a period of time at a speed of at least 600 rpm while maintaining a rotation speed of at least 3×10 3 s -1 Optionally, steps (c) to (e) are repeated for multiple cycles.

[0033] According to some embodiments, there is provided a method for performing at least one cycle in a solid phase synthesis of a polymeric organic molecule, the method comprising the steps of:

[0034] (a) providing a reactor comprising a reaction chamber and a stirring device, wherein the stirring device comprises an impeller having at least two blades rotatable about an axis;

[0035] (b) placing functionalized polymeric resin beads and at least one solvent in a reactor to provide a reaction mixture, wherein the reaction mixture is contacted with a rotatable blade;

[0036] (c) placing at least one protected monomer organic molecule and at least one coupling agent into a reaction chamber and rotating an impeller to form a coupling product of the protected monomer organic molecule and the resin;

[0037] (d) washing excess of the protected monomeric organic molecules; and

[0038] (e) placing at least one deprotecting agent in the reaction chamber and rotating the impeller to remove at least one protecting group from the coupling product to form a coupling product of the deprotected monomer organic molecule and the resin, thereby completing a cycle in the solid phase synthesis of the polymerized organic molecule;

[0039] wherein the impeller in at least one of steps (c) and (e) is rotated for a period of time at a speed of at least 600 rpm while maintaining a rotation speed of at least 3×10 3 s -1 Optionally, steps (c) to (e) are repeated for multiple cycles.

[0040] According to some embodiments, the method further comprises the step of washing the excess deprotecting agent, thereby separating the coupling product of the deprotected monomer organic molecule and the resin.

[0041] According to some embodiments, step (d) comprises washing the excess protected monomer organic molecules, thereby separating the excess protected monomer organic molecules from the coupling product of the protected monomer organic molecules and the resin. According to some embodiments, step (d) also comprises discarding the separated excess protected monomer organic molecules.

[0042] According to some embodiments, step (d) comprises washing the excess of the protected monomer organic molecules, thereby separating the excess of the protected monomer organic molecules from the coupling products of the protected monomer organic molecules and the resin in the reaction chamber. According to some embodiments, step (d) comprises washing the excess of the protected monomer organic molecules, thereby separating the excess of the protected monomer organic molecules from the coupling products of the protected monomer organic molecules and the resin, so that the coupling products remain in the reaction chamber.

[0043] According to some embodiments, steps (c) and (e) are both performed at a speed of at least 600 rpm for a period of time while maintaining a speed of at least 3×10 3 s -1 The shear rate.

[0044] According to some embodiments, steps (c) to (e) are repeated for at least 2, at least 3, at least 4, at least 5 or at least 7 cycles.

[0045] According to some embodiments, the method further comprises the step of cleaving the coupling product of step (e) from the resin, thereby forming a polymeric organic molecule and completing its solid phase synthesis.

[0046] According to some embodiments, the method further comprises the step of isolating the polymerized organic molecules from the reaction mixture.

[0047] It should be understood that the term "monomer organic molecule" refers to any organic molecule that can form a corresponding polymeric organic molecule by sequential or cyclic coupling (e.g., solid phase synthesis) with other organic molecules. Typically, the monomer organic molecule in SPS may include natural or synthetic structural units, such as amino acids, nucleotides, nucleosides, and monosaccharides, or derivatives or analogs thereof. However, as used herein, the term "monomer organic molecule" also includes dimers, trimers, etc., which can serve as structural units in SPS. Therefore, the method provided herein includes, for example, solid phase synthesis, in which the resin combined with the monomer amino acid is first coupled with a dipeptide, then coupled with a tripeptide, and then cut off to form a hexapeptide.

[0048] According to some embodiments, the monomeric organic molecule is selected from amino acids, peptides, sugars, nucleotides and nucleosides. Thus, the protected monomeric organic molecule is selected from N-protected amino acids, N-protected peptides, O-protected sugars, O-protected nucleotides and O-protected nucleosides. According to some embodiments, the monomeric organic molecule is selected from amino acids, monosaccharides and nucleosides. According to some embodiments, the monomeric organic molecule is an amino acid.

[0049] Therefore, it should also be understood that the term "polymeric organic molecule" refers to any organic molecule that can be formed by sequential or cyclic coupling (e.g., in solid phase synthesis) with monomeric organic molecules. The properties of the polymeric organic molecule depend on the characteristics of the monomeric organic molecules. Typically, the polymeric organic molecules prepared in SPS can include natural / biological compounds, such as (polypeptide) peptides, polynucleotides, and polysaccharides.

[0050] As used herein, the term "cycle" refers to a series of steps that can be repeated multiple times. These steps are collectively referred to as a single cycle. Thus, "cyclic" refers to a method in which at least some of the steps are repeated in a cyclic manner.

[0051] As used herein, the term "plurality" refers to an integer equal to or greater than two.

[0052] According to some embodiments, at least one step of solid phase synthesis is coupling a monomer to one of: a polymeric resin; an oligomer chain connected to the polymeric resin; and an additional monomer connected to the polymeric resin; wherein the monomer and the additional monomer are each independently selected from a sugar, an amino acid, and a nucleoside; and wherein the oligomer comprises at least one of a sugar, an amino acid, a nucleotide, and a nucleoside. According to some embodiments, the sugar is a monosaccharide.

[0053] According to some embodiments, at least one step of the solid phase synthesis is coupling the amino acid to a polymeric resin or to an amino acid or peptide chain attached to a polymeric resin.

[0054] According to some embodiments, at least one step of the solid phase synthesis is coupling the nucleoside to one of: a polymeric resin; a polynucleotide chain attached to a polymeric resin; a nucleoside attached to a polymeric resin; and a nucleotide attached to a polymeric resin.

[0055] According to some embodiments, at least one step of the solid phase synthesis is coupling a sugar to one of: a polymeric resin; a polysaccharide chain attached to a polymeric resin; and a monosaccharide attached to a polymeric resin. According to some embodiments, the sugar coupled in the step is a monosaccharide.

[0056] According to some embodiments, at least one step of the solid phase synthesis comprises the removal of protecting groups.

[0057] According to some embodiments, at least one step of the solid phase synthesis is selected from coupling of amino acids to resin, coupling of nucleosides to resin, coupling of sugars and removal of protecting groups. Each possibility represents a separate embodiment of the present invention.

[0058] According to some embodiments, at least one step of the solid phase synthesis is selected from coupling of amino acids to resins and removal of protecting groups.

[0059] According to some embodiments, the method comprises at least two steps of coupling a monomer to a resin and at least two steps of removing a protecting group, wherein the monomer is selected from the group consisting of a sugar, an amino acid and a nucleoside.

[0060] According to some embodiments, the method comprises at least two steps of coupling the amino acid to the resin and at least two steps of removing the protecting groups.

[0061] According to some embodiments, the method comprises at least two steps of coupling the sugar to the resin and at least two steps of removing the protecting groups.

[0062] According to some embodiments, the method comprises at least two steps of coupling the nucleoside to the resin and at least two steps of removing the protecting groups.

[0063] According to some embodiments, at least one step of the solid phase synthesis is cleavage of the synthesized molecule from the solid phase resin.

[0064] The method of the present invention can be used to synthesize any organic molecule that can be synthesized on a solid support.

[0065] According to some embodiments, organic molecule synthesis comprises multiple steps.

[0066] According to some embodiments, the solid phase synthesis method is used to synthesize a polymeric organic molecule selected from the group consisting of peptides, polypeptides, modified peptides, peptidomimetics, oligonucleotides, peptide nucleic acid molecules and oligosaccharides. Each possibility represents a separate embodiment of the present invention.

[0067] As used herein, the term "polymeric organic molecule" refers to any molecule consisting of connected monomeric units, as long as the molecule includes at least two chemically connected monomeric units. The term "polymeric molecule" is intended to include short oligomers, such as but not limited to dimers, trimers and tetramers, and oligomers of about maximum 50 monomers and long polymers including up to about one hundred monomeric units. According to some embodiments, the polymeric organic molecule comprises 2-50, 2-30, 3-25 or 3-25 monomeric units. Preferably, the polymeric molecule is a biopolymer, particularly a polynucleotide, a polysaccharide, a polypeptide or a hybrid thereof. The hybrid molecule according to the present invention includes but is not limited to peptide-nucleic acid molecules (PNA), glycoproteins and proteoglycans.

[0068] As used herein, the terms "polypeptide" and "oligopeptide" are well known in the art and are used to refer to a series of connected amino acid molecules. The term is intended to include short peptide sequences (such as but not limited to tripeptides) and longer protein sequences. Similarly, the term "polynucleotide" or "oligonucleotide" refers to a short or long oligomer of connected nucleotides, and the term "polysaccharide" refers to a short or long oligomer of connected sugar units. As used herein, the term "hybrid" refers to oligomers and polymers with at least two types of monomers. For example, a hybrid oligomer can include sugars, amino acids, nucleotides and / or nucleosides as structural unit monomers.

[0069] In other embodiments, solid phase synthesis methods are used to synthesize peptides, polypeptides, modified peptides or peptidomimetics.

[0070] According to some embodiments, the methods of the invention are used to synthesize combinatorial libraries or arrays. According to some embodiments, combinatorial libraries or arrays of peptides are generated using the methods of the invention.

[0071] As used herein, the term "amino acid" refers to an organic acid that contains both a protected or unprotected amino group (NHPG or NH2) and an acidic carboxyl group (COOH). Typically, amino acids include α-amino acids. These include, but are not limited to, the 25 amino acids that have been identified as protein components. Amino acids contain at least one carboxyl group and one primary or secondary amino group on the amino acid molecule. Amino acids include proteinogenic amino acids such as alanine, valine, leucine, isoleucine, norleucine, proline, hydroxyproline, phenylalanine, tryptophan, methionine, methionine, glycine, serine, threonine, cysteine, cystine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, hydroxylysine, ornithine, arginine, histidine, penicillamine, etc. The term "amino acid" is intended to include unprotected amino acids and protected amino acids.

[0072] According to some embodiments, the amino acid comprises a protected amino acid. According to some embodiments, the amino acid comprises an N-protected amino acid.

[0073] As used herein, the term "N-protected amino acid" refers to an amino acid in which the amino group (NH2) is protected by an amino protecting group and is therefore protected from participating in chemical reactions that may occur during the coupling reaction. Since the most abundant amino acids in various fields of biology and medicine are α-amino acids, N-protected amino acids generally include an amino protecting group covalently linked to an α-amine. However, the present invention also encompasses solid phase synthesis, which uses less commonly used structural units, such as β-amino acids, in which the amino group is separated from the carboxyl group by two carbon atoms. Therefore, N-protected amino acids also include β-amino acids, in which the amino protecting group is covalently linked to the β-amine. According to some embodiments, the N-protected amino acid is selected from α-N-protected amino acids and β-N-protected amino acids. According to some embodiments, the N-protected amino acid is an α-N-protected amino acid. It should be understood that "α-N-protected amino acid" and "β-N-protected amino acid" refer to an α-amino acid comprising an amino protecting group covalently linked to its α-nitrogen atom; and a β-amino acid comprising an amino protecting group covalently linked to its β-nitrogen atom.

[0074] As used herein, the term "amino protecting group" refers to a protecting group that retains an amino group or an amino acid that would otherwise be modified by a chemical reaction involving an amino-containing compound (e.g., an amino acid). Non-limiting examples of such protecting groups include formyl or lower alkanoyl groups having 2-4 carbon atoms, such as acetyl or propionyl; trityl or substituted trityl groups, such as monomethoxytrityl and dimethoxytrityl, such as 4,4'-dimethoxytrityl; trichloroacetyl; trifluoroacetyl; silyl; phthaloyl; (9-fluorenylmethoxycarbonyl) or "FMOC" groups; alkoxycarbonyl, such as tert-butyloxycarbonyl (BOC); or other protecting groups derived from halogenated carbonates, such as C6-Cn aryl lower alkyl carbonates. When preparing polypeptides by solid phase synthesis techniques, the FMOC group is typically used.

[0075] As used herein, the term "nucleoside" refers to a compound consisting of any pentose or modified pentose moiety connected to a specific portion of a heterocyclic base, a tautomer, or a derivative thereof (e.g., position 9 of purine, position 1 of pyrimidine, or an equivalent position of a heterocyclic base derivative). Nucleosides may include unreactive or reactive phosphorus substituents, such as phosphoramidites. Examples include, but are not limited to, ribonucleosides comprising a ribose moiety and deoxyribonucleosides comprising a deoxyribose moiety, each of which may be substituted with a phosphorus-containing substituent such as a phosphoramidite. According to some embodiments, a nucleoside may be a nucleoside drug analog. Examples of ribonucleosides include, but are not limited to, adenosine, guanosine, 5-methyluridine, uridine, 5-methylcytosine, cytosine, inosine, xanthosine, and whitinoside, each of which may be substituted with a phosphorus-containing substituent. Examples of deoxyribonucleosides include, but are not limited to, deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine, deoxycytidine, deoxyinosine, and deoxyxanthine nucleosides, each of which may be substituted with phosphorus-containing substituents. The term "nucleoside" is intended to include unprotected nucleosides and protected nucleosides.

[0076] As used herein, the term "nucleotide" refers to a nucleoside with a phosphate ester substituted on the 5'-position or the equivalent position of a nucleoside derivative. In the case where a phosphate analog such as phosphoramidite exists as a substituent, the molecule is generally referred to as a nucleoside rather than a nucleotide. However, in polynucleotide synthesis, a nucleoside structural unit with a phosphorus group (such as phosphoramidite) can ultimately be converted into a corresponding phosphate ester, which causes the term to be changed from a nucleoside to a nucleotide in the reaction. Therefore, when discussing chemical transformation and sequence in this article, "nucleotide" and "nucleoside" should be broadly interpreted to include the phosphorus derivatives of nucleosides.

[0077] According to some embodiments, the nucleoside comprises a protected nucleoside. According to some embodiments, the nucleoside comprises an O-protected nucleoside. According to some embodiments, the nucleoside comprises a 5'-O-protected nucleoside.

[0078] The term "O-protected nucleoside" as used herein refers to a nucleoside in which at least one hydroxyl group (OH) is protected by an oxygen protecting group and is therefore protected from chemical reactions that may occur during the coupling reaction. Therefore, the term "5'-O-protected nucleoside" refers to a ribonucleoside or deoxyribonucleoside in which the 5' hydroxyl group is protected by an oxygen protecting group. According to some embodiments, the nucleoside is a ribonucleoside that includes an oxygen protecting group on its 5' hydroxyl group and the 2' hydroxyl group. According to some embodiments, the nucleoside is a deoxyribonucleoside that includes an oxygen protecting group on its 5' hydroxyl group.

[0079] As used herein, the term "oxygen protecting group" refers to a protecting group that retains an oxygen atom or hydroxyl group that would otherwise be modified by a chemical reaction involving an oxygen-containing compound (e.g., a nucleoside or a carbohydrate). Non-limiting examples of such protecting groups include trityl or substituted trityl groups, such as monomethoxytrityl and dimethoxytrityl (DMT) groups, such as 4,4'-dimethoxytrityl; silyl ethers, such as trimethylsilyl and tert-butyldimethylsilyl; esters, such as acetates and haloacetates; lower alkyl groups that may be substituted with halogen atoms or cyano groups; benzyl groups that may have substituents; and phenyl groups that may have substituents. When preparing polypeptides using solid phase synthesis techniques, trityl derivatives, particularly DMT, are typically used.

[0080] As used herein, the terms "sugar" and "saccharide" are interchangeable and refer to compounds containing one or more monosaccharide groups. As used herein, the term "monosaccharide" refers to the most basic unit of carbohydrates. Monosaccharides are the basic units of carbohydrates that cannot be further hydrolyzed into simpler compounds. They are the simplest form of sugar and are generally colorless, water-soluble, and crystalline solids. Some monosaccharides have a sweet taste. Examples of monosaccharides include glucose, fructose, and ribose. Monosaccharides are the building blocks of disaccharides (such as sucrose and lactose) and polysaccharides (such as cellulose and starch). With a few exceptions (e.g., deoxyribose), monosaccharides have the chemical formula: C x (H2O) y , traditionally x ≥ 3. According to the number of carbon atoms x they contain, monosaccharides can be divided into: trisaccharides (3), tetrasaccharides (4), pentoses (5), hexoses (6), heptoses (7), etc. In aqueous solution, if monosaccharides have more than four carbon atoms, they exist as rings. According to some embodiments, the sugar is a monosaccharide. According to some embodiments, preferred monosaccharides are pentoses and / or hexoses.

[0081] According to some embodiments, the monosaccharide comprises a protected monosaccharide. According to some embodiments, the monosaccharide comprises an O-protected monosaccharide.

[0082] As used herein, the term "O-protected monosaccharide" refers to a monosaccharide in which one of its hydroxyl groups (OH) is protected by an oxygen protecting group and is therefore protected from participating in chemical reactions that may occur during the coupling reaction. According to some embodiments, the monosaccharide is selected from pentoses and hexoses. According to some embodiments, the monosaccharide is a pentose. According to some embodiments, the monosaccharide is a hexose. According to some embodiments, the monosaccharide comprises a single free hydroxyl group, wherein the remaining hydroxyl groups thereof comprise a protecting group. According to some embodiments, the monosaccharide is a hexose comprising four O-protected hydroxyl groups and one free hydroxyl group. According to some embodiments, the monosaccharide is a pentose comprising three O-protected hydroxyl groups and one free hydroxyl group.

[0083] The term "free" hydroxyl used herein refers to an unprotected OH chemical moiety. Similarly, the term "free" amine used herein refers to an unprotected NH2 chemical moiety. Typically, "free" hydroxyl and / or amine can react in a reaction such as a coupled reaction, and the corresponding protecting group will not undergo similar chemical reactions under similar conditions. According to some embodiments, the method is used for solid phase synthesis to comprise an organic molecule comprising a total of 2-50, 2-20, 3-15, 3-10 or 3-6 residues selected from amino acid residues, nucleotide residues and sugar residues. According to some embodiments, the peptide comprises 2-50, 2-20, 3-15, 3-10 or 3-6 amino acid residues. According to some embodiments, polysaccharides comprise 2-50, 2-20, 3-15, 3-10 or 3-6 monosaccharide residues. According to some embodiments, polynucleotides comprise 2-50, 2-20, 3-15, 3-10 or 3-6 nucleoside residues.

[0084] As used herein, the term "coupling", "coupling process" or "coupling step" refers to the process of forming a bond between two or more molecules, such as two monomer units. The bond can be a covalent bond, such as a peptide bond, a glycosidic bond or a phosphodiester bond.

[0085] A peptide bond is a chemical bond formed between two molecules when the carboxyl group of one coupled molecule reacts with the amino group of another coupled molecule, thereby releasing a water (H2O) molecule. This is a dehydration synthesis reaction (also called a condensation reaction) and usually occurs between amino acids. The resulting -C(=O)NH- bond is called a peptide bond, and the resulting molecule is an amide.

[0086] A glycosidic bond is a chemical covalent bond formed between a hemiacetal or hemiketal group of a sugar and a second compound, such as a hydroxyl group of a second sugar. Glycosidic bonds can be named α- or β- based on the relative stereochemistry (R or S) of the terminal position (i.e., C1 of the sugar). Typically, polysaccharides are formed by the formation of a glycosidic bond between the C1 of a first sugar and an oxygen atom derived from the hydroxyl group of a second sugar.

[0087] Phosphodiester bonds refer to covalent phosphate ester bonds between residues in a polynucleotide chain. It occurs when two oxygen atoms in phosphoric acid form two ester bonds. Phosphodiester bonds form the backbone of nucleic acid chains. In DNA and RNA, phosphodiester bonds are bonds between the 3' carbon atom of one sugar molecule and the 5' carbon atom of another sugar molecule (deoxyribose in DNA and ribose in RNA).

[0088] The term "deprotection" as used herein refers to the removal of at least one protecting group. For example, deprotection includes removing the amino protecting group from the protected amino acid. More specifically, according to some embodiments, deprotection includes replacing the FMOC protecting group connected to the amino group of the protected amino acid with a hydrogen atom, thereby forming a basic NH2 group in the deprotected amino acid. Alternatively, when applying a protecting group on an oxygen atom, deprotection includes replacing the protecting group connected to the oxygen of the protected nucleoside with a hydrogen atom, thereby forming a free OH group in the deprotected nucleoside. Deprotection can be one or more protecting groups. For compounds with n protecting groups, deprotection will produce the same compound with at least one less protecting group, i.e., the product compound will include n-1 to 0 protecting groups.

[0089] The term "deprotected" refers to a compound that has undergone removal of at least one protecting group. The term includes compounds that have undergone removal of all protecting groups and compounds that have undergone removal of some protecting groups. For example, amino acids, such as lysine, arginine, aspartic acid, histidine, glutamic acid, serine, threonine, cysteine, tyrosine, etc., may include two protecting groups, a first protecting group covalently linked to the α-nitrogen and a second protecting group covalently linked to a reactive group of the side chain. In this case, a deprotected amino acid can be defined as an amino acid after only one protecting group has been removed or after both protecting groups have been removed.

[0090] The term "unprotected" refers to a compound that has undergone the removal of all protecting groups, or that does not initially include a protecting group. In other words, an unprotected compound does not include a protecting group.

[0091] According to some embodiments, the method comprises the step of placing at least two reactants into a reaction chamber.

[0092] According to some embodiments, the at least one reactant is selected from the group consisting of: a deprotecting agent, a coupling agent, a sugar, a nucleoside and an amino acid. Each possibility represents a separate embodiment of the present invention.

[0093] According to some embodiments, the at least one reactant is selected from the group consisting of: a deprotecting agent, a coupling agent, and an amino acid.

[0094] According to some embodiments, the N-protected amino acid is an α-N-protected amino acid. According to some embodiments, the N-protected amino acid comprises a protecting group covalently linked to its α-nitrogen. According to some embodiments, the N-protected amino acid comprises a protecting group selected from the group consisting of an Fmoc protecting group and a tBoc protecting group. According to some embodiments, the protecting group is selected from the group consisting of an Fmoc protecting group and a tBoc protecting group.

[0095] According to some embodiments, the N-protected amino acid is an Fmoc protected amino acid. According to some embodiments, the protecting group is an Fmoc protecting group. According to some embodiments, the N-protected amino acid comprises an Fmoc group covalently linked to its α-nitrogen.

[0096] According to some embodiments, the at least one reactant further comprises a reagent capable of removing an Fmoc group.

[0097] According to some embodiments, the reagent capable of removing the Fmoc group comprises a base.

[0098] According to some embodiments, the base comprises an amine.

[0099] According to some embodiments, the amine is selected from the group consisting of piperidine, morpholine, piperazine, dicyclohexylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, 1,8-diazabicycloundec-7-ene, pyrrolidine, cyclohexylamine, ethanolamine, diethylamine, trimethylamine, ammonia, tributylamine, 1,4-diazabicyclo[2.2.2]octane, hydroxylamine, tris(2-aminoethyl)amine, and combinations thereof.

[0100] According to some embodiments, the O-protected nucleoside comprises a protecting group selected from benzyl, diphenylmethyl, triphenylmethyl and derivatives thereof. Each possibility represents a separate embodiment of the present invention. According to some embodiments, the O-protected nucleoside is a dimethyltrityl (DMT) protected nucleoside. According to some embodiments, the O-protected nucleoside is a 5' dimethyltrityl (DMT) protected nucleoside.

[0101] According to some embodiments, the at least one reactant further comprises a reagent capable of removing a DMT group. The reagent capable of removing a DMT group is typically an acidic compound, such as but not limited to dichloroacetic acid and / or trichloroacetic acid.

[0102] According to some embodiments, the O-protected monosaccharide comprises at least one protecting group selected from silyl ether, ester, benzyl, diphenylmethyl, triphenylmethyl and acetal, each of which may be substituted with one or more halogen, cyano and / or nitro groups. Each possibility represents a separate embodiment of the present invention.

[0103] According to some embodiments, the time period is in the range of 1-600, 10-300, 15-210, 5-60, 10-30, 30-180 or 60-120 minutes.

[0104] According to some embodiments, the reactants further comprise a coupling agent. Specifically, carbodiimides are considered to be useful coupling agents for forming amide bonds (e.g., peptide bonds), while various azole compounds are useful catalysts for forming PO bonds (e.g., phosphodiester bonds); and various agents promote the formation of glycosidic bonds, depending on the polysaccharide structure to be formed.

[0105] According to some embodiments, the coupling agent comprises a carbodiimide.

[0106] According to some embodiments, placing at least one reactant into the reaction chamber comprises gradually placing at least one reactant into the reaction chamber. According to some embodiments, placing the carbodiimide into the reaction chamber comprises gradually placing the carbodiimide into the reaction chamber.

[0107] According to some embodiments, the carbodiimide is selected from the group consisting of: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and combinations thereof.

[0108] According to some embodiments, the carbodiimide comprises ethyl-3-(3-dimethylaminopropyl)carbodiimide.

[0109] According to some embodiments, the reactant further comprises 1-hydroxybenzotriazole (HOBt).

[0110] According to some embodiments, the coupling agent includes an azole catalyst. According to some embodiments, the azole catalyst is selected from the group consisting of: 1H-tetrazole, 2-ethylthiotetrazole, 2-benzylthiotetrazole, 4,5-dicyanoimidazole, and combinations thereof.

[0111] According to some embodiments, the method further comprises placing at least one solvent into the reactor.

[0112] As used herein, the term "solvent" refers to a fluid medium, particularly a liquid medium, in which a chemical transformation occurs. The term is used broadly to include liquid media that dissolve the reactants involved in the transformation as well as media in which some of the reactants are insoluble. Thus, it is understood that in solid phase synthesis as described herein, the reactants are soluble in the solvent, while the functionalized polymeric resin is not.

[0113] According to some embodiments, the at least one solvent is selected from the group consisting of water, dimethylformamide, dichloromethane, N-methyl-2-pyrrolidone, dimethylacetamide, and combinations thereof.

[0114] According to some embodiments, the solvent comprises N-methyl-2-pyrrolidone.

[0115] According to some embodiments, the solvent comprises water.

[0116] According to some embodiments, the solvent includes N-methyl-2-pyrrolidone and water.

[0117] According to some embodiments, the method comprises an initial step of swelling the polymeric resin beads in a solvent. According to some embodiments, the swelling step is performed after the step of placing the beads and the solvent in a reactor; and before the step of placing the reactants in the reaction chamber.

[0118] According to some embodiments, the concentration of the polymeric resin in the solvent is 5-25% w / w, 5-20% w / w, 5-15% w / w, or 8-12% w / w.

[0119] According to some embodiments, the swelling step comprises mixing the polymeric resin beads in the solvent for a specified period of time. According to some examples, the period of time is in the range of 1-60 minutes, 2-45 minutes, or 4-30 minutes.

[0120] According to some embodiments, the rotational speed is in the range of 600-1400 rpm. According to some embodiments, the rotational speed is in the range of 600-1200 rpm. According to some embodiments, the rotational speed is in the range of 600-1000 rpm. According to some embodiments, the rotational speed is in the range of 600-900 rpm. According to some embodiments, the rotational speed is in the range of 600-800 rpm. According to some embodiments, the rotational speed is in the range of 600-700 rpm.

[0121] In other embodiments, the homogenizer is used to maintain the rotation speed in the range of 5,000-30,000. According to some embodiments, the homogenizer is used to maintain the rotation speed in the range of 10,000-30,000.

[0122] According to some embodiments, mixing the polymeric resin beads in the solvent for a specified period of time comprises maintaining a shear rate of at least 3×10 3 s -1 .

[0123] In other embodiments, mixing the polymeric resin beads in the solvent for a specified period of time comprises maintaining a shear rate of at least 1×10 6 s -1 .

[0124] According to some embodiments, mixing the polymeric resin beads in the solvent for a specified period of time includes maintaining a shear stress of at least 1.5 N / m 2 According to some embodiments, the shear stress is 1.5-5, 1.8-3.8, 1.8-3.0 or 1.8-2.4 N / m 2 within the range.

[0125] In other embodiments, a homogenizer is used to maintain 500-2000 N / m 2 According to some embodiments, a homogenizer is used to maintain a shear stress of 750-1500 or 900-1200 N / m 2 of shear stress.

[0126] A functionalized polymeric resin according to the present invention is any polymeric resin comprising reactive groups that can be coupled to organic molecules.

[0127] The reactive groups of the polymeric resins according to the present invention include, but are not limited to, amino groups, hydroxycarboxyl groups, carboxyl derivative groups such as acyl halides, halogen groups and pseudohalogenated groups such as sulfonate derivatives.

[0128] According to some embodiments, the functionalized resin comprises residues selected from amino acid residues, nucleotide residues, nucleoside residues and sugar residues. According to some embodiments, the functionalized resin comprises amino acid residues. According to some embodiments, the functionalized resin comprises sugar residues. According to some embodiments, the functionalized resin comprises nucleotide residues.

[0129] According to some embodiments, the functionalized polymeric resin beads comprise polystyrene-divinylbenzene.

[0130] According to some embodiments, the resin comprising polystyrene-divinylbenzene is selected from 1% DVB-PS chloromethylated resin and Rink Amide Tentagel resin.

[0131] According to some embodiments, the resin is a 1% DVB-PS chloromethylated resin.

[0132] According to some embodiments, the resin is Rink Amide Tentagel resin.

[0133] According to some embodiments, the coupling capacity of the polymeric resin beads is 0.2-0.6 mmol / g, 0.3-0.5 mmol / g or 0.35-0.45 mmol / g.

[0134] According to some embodiments, the polymeric resin beads have a particle size in the range of 20-200, 50-100, 60-90, 65-85, 70-80, or 73-77 μm.

[0135] According to some embodiments, the mechanical agitator comprises at least three blades rotatable about an axis. According to some embodiments, the mechanical agitator comprises three blades rotatable about an axis. According to some embodiments, the blades rotatable about an axis rotate upward at a first angle. According to some embodiments, the first angle is in the range of 20-40°, 25-35°, or 30-35°.

[0136] According to some embodiments, the rotatable blade is rotated downward at a second angle. According to some embodiments, the second angle is in the range of 30-50°, 35-45° or 40-45°.

[0137] Certain embodiments of the present disclosure may include some, all, or none of the above advantages. One or more technical advantages will be apparent to those skilled in the art based on the drawings, descriptions, and claims included herein. In addition, although specific advantages have been listed above, various embodiments may include all, some, or none of the listed advantages.

[0138] In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by reference to the drawings and by study of the following detailed descriptions. BRIEF DESCRIPTION OF THE DRAWINGS

[0139] Embodiments of exemplary embodiments are described below with reference to the accompanying drawings. In the accompanying drawings, identical structures, elements or components appearing in more than one drawing are generally labeled with the same number in all drawings in which they appear. Alternatively, elements or components appearing in more than one figure may be labeled with different numbers in different drawings in which they appear. The sizes of the components and features shown in the drawings are generally selected for convenience and clarity of representation and are not necessarily shown to scale. These figures are listed below.

[0140] Figure 1A is a photograph of a high shear SPS (HS-SPS) apparatus according to some embodiments.

[0141] Figure 1B is a close-up photograph of a HS-SPS apparatus according to some embodiments.

[0142] Figure 2 is a photograph of a magnetic stirrer apparatus.

[0143] Figure 3 is a photograph of a homogenizer stirrer device (device 3).

[0144] Figure 4A is a photograph of TentaGel resin beads before being rotated in water in HS-SPS apparatus 1. The agitator consists of a small impeller with three 3.8 cm long blades rotating upward at an angle of 32.2°.

[0145] Figure 4B is a close-up photograph of TentaGel resin beads after spinning in water for 30 minutes in HS-SPS apparatus 1.

[0146] Figure 4C is a photograph of TentaGel resin beads after spinning in water for 30 minutes in HS-SPS apparatus 1.

[0147] Figure 5A is a photograph of TentaGel resin beads before spinning in water in HS-SPS apparatus 1.

[0148] Figure 5B is a photograph of TentaGel resin beads after spinning in water for 42 seconds in HS-SPS apparatus 1.

[0149] Figure 5C is a photograph of TentaGel resin beads after spinning in water for 1.1 minutes in HS-SPS apparatus 1.

[0150] Figure 5D is a photograph of TentaGel resin beads after spinning in water for two minutes in HS-SPS apparatus 1.

[0151] Figure 5E is a photograph of TentaGel resin beads after spinning in water for three minutes in HS-SPS apparatus 1.

[0152] Fig. 5F is a photograph of TentaGel resin beads after being rotated in water in HS-SPS apparatus 1 for four hours.

[0153] Fig. 6A is a photograph of TentaGel resin beads before spinning in water on a magnetic stirrer.

[0154] Figure 6B is a photograph of TentaGel resin beads after being rotated in water on a magnetic stirrer for two hours.

[0155] Figure 6C is a photograph of TentaGel resin beads after being rotated in water on a magnetic stirrer for 18 hours.

[0156] Fig. 7Ais a photograph of TentaGel resin beads before spinning in NMP in HS-SPS unit 2. BOLA-mini impeller agitator with three 5 cm long blades (h = 35 cm, spinning downward at an angle of 41.1°).

[0157] Figure 7B is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for 24 hours.

[0158] Fig. 8A is a photograph of TentaGel resin beads without solvent.

[0159] Figure 8B is a photograph of TentaGel resin beads immediately after contact with NMP.

[0160] Figure 8C is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for 30 minutes.

[0161] Fig. 9A is a photograph of TentaGel resin beads before spinning in NMP in HS-SPS apparatus 2.

[0162] Fig. 9B is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for 30 seconds.

[0163] Fig. 9C is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for one minute.

[0164] Fig.9D is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for 1.3 minutes.

[0165] Fig.9E is a photograph of TentaGel resin beads after two minutes of spinning in NMP in HS-SPS apparatus 2.

[0166] Fig.9F is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for 2.3 minutes.

[0167] Figure 9G is a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for three minutes.

[0168] Figure 9His a photograph of TentaGel resin beads after spinning in NMP in HS-SPS apparatus 2 for four minutes.

[0169] Fig.10 It is a graph showing the progress % of Fmoc cutoff using a mechanical stirring device at 700 rpm with 5% piperidine (filled circles); a shaking device with 5% piperidine (open triangles); a shaking device with 20% piperidine solution (filled triangles); a mechanical stirring device at 100 rpm with 5% piperidine (open circles); a fixed reactor device without mixing with 5% piperidine (open squares). Errors are standard deviations between three independent experiments performed. DETAILED DESCRIPTION

[0170] In the following description, various aspects of the present disclosure will be described. For illustrative purposes, specific configurations and details are set forth in order to provide a thorough understanding of the different aspects of the present disclosure. However, it will be apparent to those skilled in the art that the present disclosure may be practiced without presenting the specific details herein. In addition, well-known features may be omitted or simplified to avoid confusing the present disclosure.

[0171] The method of the present invention is suitable for synthesizing any organic molecule that can be synthesized on a solid support. This includes polymeric molecules such as peptides and polynucleotides. The method is particularly suitable for molecules produced using multiple synthesis steps and requiring orthogonal protection of reactive groups.

[0172] The methods of the present invention provide at least one improvement in synthesis parameters including, but not limited to, synthesis time, synthesis yield, reduced by-product formation, and reduced racemization rate.

[0173] Without wishing to be bound by any mechanism of action, these improvements may be due to the application of high shear forces during the synthetic steps, eliminating the accumulation of reactants or intermediates in the reaction mixture and maintaining the reaction mixture with improved homogeneity.

[0174] Some mixing devices that can be used according to the invention have not been used or disclosed for use in organic synthesis before. These include, for example, homogenizers (rotor-stator and other types of homogenizers), which are commonly used, for example in biology, to disperse tissues.

[0175] The rotor-stator homogenizer uses a high-speed, close-fitting rotor inside a toothed stator. The sample to be homogenized is drawn into the center of the rotor and passes through the narrow gap between the rotor and stator where it is mixed, accelerated, and squeezed.

[0176] A fundamental understanding of the kinetic energy of the fluid as well as the shear and compressive stresses and forces applied to the reaction medium is crucial for the successful scale-up of synthetic processes. However, to date, there have been few studies on the effects of fluid dynamic parameters on yields and side reactions in SPS. Some important fluid dynamic parameters include "shear rate", "shear stress" and "shear force".

[0177] The term "shear rate" measured in reciprocal seconds (SI units) refers to the rate at which progressive shear deformation is applied to a material. As used herein, shear rate refers to the rate at which deformation is applied to polymeric resin beads within a reaction mixture during mixing. Typically, the shear rate of a fluid flowing between two parallel plates (one plate moving at a constant speed and the other plate stationary) is defined as:

[0178]

[0179] where γ is the shear rate; v is the speed of the moving plate (in s -1 units); h is the distance between the two parallel plates. For simple shear conditions, it is simply the gradient of velocity in the flowing material.

[0180] In a stirred tank, the following correlation was found [Perez et al., Chem. Eng. J. 124, 2006, 1; and Metzner and Otto AIChE Journal, 1957, 3, 3]:

[0181] γ=k i N

[0182] where k i is the impeller constant; N is the stirring speed (i.e. the rotation speed of the impeller), expressed in s -1 is the unit; τ is the shear stress in Pascals (Newtons per square meter); P is the power input in Watts, which depends on the torque of the impeller and its speed (N); and V is the volume of the fluid in the tank.

[0183] The term "shear stress" measured in reciprocal seconds refers to the component of stress that is coplanar with the cross-section of the material. As used herein, shear stress refers to the component of stress exerted on the polymeric beads that is coplanar with their cross-section. Typically, shear stress is generated by a force vector component parallel to the cross-section.

[0184] As a measure of stress, shear stress is measured as force per unit area (in SI units: N / m 2 )

[0185] τ=F / A'

[0186] where τ is the shear stress; F is the applied force; and A is the cross-sectional area of ​​the material, the area parallel to the applied force vector (ie, the cross-sectional area of ​​the bead).

[0187] With reference to other fluid dynamic parameters, shear stress can also be derived from shear rate by the following formula: where μ is the dynamic viscosity of the fluid.

[0188] The term "viscosity" refers to the hydrodynamic property of a fluid, describing its ability to resist gradual deformation due to shear stress or tensile stress. Viscosity is caused by collisions between adjacent particles moving at different speeds in a fluid. As used herein, viscosity refers to the viscosity of a reaction mixture containing solvents, beads, and other added reagents.

[0189] The term "shear force" refers to a force acting in a direction parallel to the surface or planar cross-section of an object. As used herein, shear force refers to a force acting in a direction parallel to the surface or planar cross-section of a polymeric bead during mixing. The shear force Fs can be derived from the shear stress because it consists of the overall shear stress (τ) over the surface area (A) of the object.

[0190]

[0191] As a heterogeneous reaction (i.e., the protected amino acid is in solution and the resin is not), the reaction carried out in the SPS cycle is a diffusion-controlled reaction that is highly affected by stirring or agitation. As mentioned above, gentle mixing methods (e.g., vortexing, nitrogen flow, rotary evaporator rotor, agitation by rocking) are generally used in large-scale production of peptides to maintain low shear stress on the polymeric beads, thereby avoiding their damage. In contrast to the gentle mixing methods in solid phase synthesis, the mixing methods used in the production of small molecules in solution are generally carried out at high rotation speeds.

[0192] The effects of gentle mixing, which is commonly employed in SPS, have not been well documented and characterized. There are two main parameters that may be critical in the steps of the SPS cycle (i.e., coupling of protected structural units; deprotection and washing). First, the compressive and shear stresses applied to the resin beads may cause them to break, and as a result, the separation process will be cumbersome due to slow filtration speeds and possible filter clogging. Secondly, the rates of coupling and deprotection reactions and washing steps may be affected by bead breakage. In addition, secondary parameters arise from the interaction between the solid phase (resin beads) and the liquid phase. A better distribution of the resin beads is caused by the circulation flow rate of the beads in the medium, which can increase the mass transfer between the liquid agglomerates and the solid surface, thereby accelerating the reaction. In specific reactions, it can reduce the generation of impurities and improve the impurity distribution of the final product.

[0193] Although many exemplary aspects and embodiments have been discussed above, those skilled in the art will recognize certain modifications, additions and sub-combinations thereof. It is therefore intended that the claims and claims hereafter introduced be interpreted as including all such modifications, additions and sub-combinations within their true spirit and scope.

[0194] Example

[0195] Example 1 - Resin Bead Stability Check

[0196] Equipment and configuration

[0197] To characterize and optimize the method for industrial applications, the effects of equipment operation and geometry (i.e., the fluid dynamics of the system) on the method were estimated. The study evaluated the stability of the polymerized resin beads after mixing and compared three mixing methods:

[0198] (i) A hybrid method including a HS-SPS device consistent with some embodiments of the present invention Figure 1A and 1B ).

[0199] (ii) Mixing method using a magnetic stirrer ( Figure 2 ).

[0200] (iii) Mixing method using a homogenizer ( Figure 3 ).

[0201] The HS-SPS apparatus consisted of a reactor comprising a 250 mL beaker (6.8 cm in diameter and 10 cm in height) equipped with a mechanical stirrer and a high shear stress device. The reactor also included 4 baffles, each with a thickness of 0.6 cm. Two mechanical stirrers were examined:

[0202] 1. Agitator, consisting of a small impeller with three 3.8 cm long blades, rotating upward at an angle of 32.2°. The device including this agitator will be referred to as "HS-SPS Device 1" or "Device 1" hereinafter.

[0203] 2. A BOLA-mini impeller agitator with three 5 cm long blades, an impeller height of 35 cm, and a downward rotation at an angle of 41.1°. The apparatus including this agitator will be referred to as "HS-SPS Apparatus 2" or "Apparatus 2" hereinafter.

[0204] Such equipment, in different configurations, has been widely used in the fine chemicals and active pharmaceutical ingredients (API) industries at all volume levels, however similar devices were not considered suitable for methods that generally employ solid phase synthesis, in particular solid phase peptide synthesis.

[0205] The magnetic stirring device consists of a 250 ml round bottom flask and an oval or octagonal magnetic stirrer. Geometry of the magnetic stirrer:

[0206] Length = 25 mm;

[0207] Width = 10 mm;

[0208] Circumference = 43 mm;

[0209] Diameter = 12 mm;

[0210] Weight = 6.6189 g;

[0211] Distance between parallel bands = 5 mm.

[0212] Figure 3 The homogenizer apparatus (referred to as "HS-SPS Apparatus 3" or "Apparatus 3") consisted of a 250 mL beaker flask (6.8 cm in diameter and 10 cm in height) equipped with Polytron PT 6100, PT-DA3020 / ZEC. The homogenizer included the following geometry:

[0213] Homogenizer diameter = 2 cm;

[0214] Round window diameter = 1 cm;

[0215] Number of teeth = 14;

[0216] Number of windows = 14;

[0217] Window height (from the bottom) = 0.3 cm;

[0218] Window width = 0.2 cm

[0219] Z(window)=0.8cm

[0220] Two PS-DVB (polystyrene-divinylbenzene) based resins were used: (i) conventional PS-1% DVB (polystyrene composed of 1% divinylbenzene monomer) chloromethylated resin (Merrifield resin); (ii) Rink Amide Tentagel resin (a modified PS-1% DVB polymer grafted with ethylene oxide monomers, including Rink Amide linkers). Specifically, the resin was purchased from RAPP POLYMER's "TentaGel HL RAM, 12023', with a capacity of 0.4 mmol / g and a particle size of 75 μm.

[0221] The mechanical stability of the resins was tested in two directions. Shear stress was applied to a mixture of the two resins in N-methylpyrrolidone (NMP) and water solvents for different periods of time. The concentration of beads in the solvent was set to 10% w / w, 12.5 g beads in 125 g solvent.

[0222] The stability of the resin beads was checked under a high power microscope to detect any damage. The microscope used to observe and measure the beads was a Zeiss scope A.1AxioCam iCc3 microscope.

[0223] result

[0224] First, HS-SPS apparatus 1 was examined with water as solvent and TentaGel resin. Rotating the impeller at 1450 rpm produced a turbid solution due to air entering the vessel. Only at about 600-700 rpm did the solution appear turbid and no vortexes were produced, so the maximum impeller speed was set to this speed.

[0225] The beads were rotated in water at room temperature for 24 hours in a mechanical stirrer of HS-SPS apparatus 1. Samples were taken every 30 minutes during the first 8 hours and a final sample was taken after 24 hours. As can be seen in Figure 4, the diameter of the beads remained essentially unchanged. Therefore, in order to test the swelling of the beads, the kinetics were tested under a microscope at small time intervals without any stirring.

[0226] like Figure 5A -F As can be seen, the beads maintained a flawless round shape before and after 24 hours of stirring in HS-SPS apparatus 1. In addition, the beads swelled as expected (Table 1).

[0227] Table 1: Swelling of TentaGel beads in water over several hours in HS-SPS apparatus 1

[0228] Time (minutes) Average diameter of beads (μm) 0 95.45 42 seconds 102.12 1.1 106.74 2 107.08 3 106.77 4 107.74

[0229] In the next stage of the experiment, after the beads were stirred in water for 24 hours, their durability under stirring under high shear stress was tested in a homogenizer apparatus. In this experiment, half of the initial solution used in the HS-SPS apparatus 1 experiment was stirred in the homogenizer apparatus at 23,000 rpm for an additional 5 minutes, resulting in only minimal loss of bead shape.

[0230] Next, the durability of the beads stirred by the HS-SPS apparatus 1 and the homogenizer apparatus was examined using a magnetic stirring apparatus. After stirring for 24 hours and after one week, the samples were taken out and observed under a microscope.

[0231] The above procedure was performed on TentaGel beads in water using HS-SPS apparatus 1, and was repeated on TentaGel beads in NMP using HS-SPS apparatus 2, and on polystyrene beads in NMP using HS-SPS apparatus 2 (polystyrene in water is irrelevant since this polymer swells poorly under aqueous conditions). In all cases, shear stress was measured using a dynamometer (dynamometer FH 10 from PCE Instruments). Table 2 summarizes the stability of the different beads corresponding to the stirring time, apparatus and stress employed.

[0232] Table 2: Sensitivity of various resins to shear stress and operating rate.

[0233]

[0234] 1 Using HS-SPS device 1; 2 A HS-SPS apparatus 2 was used.

[0235] Table 2 and Figure 7A-7B It was shown that surprisingly, the resin beads were not damaged even under high shear rate operation using the HS-SPS device or the homogenizer device. The only configuration that damaged the resin beads was the use of a magnetic stirring device. Fig. 6A -C shows the structural difference of the resin before and after stirring in the magnetic stirring device. Obviously, after the beads were stirred in the magnetic stirring device for 2 hours, their structure was significantly damaged ( Figure 6B ), whereas after 18 h under the same conditions, the beads were observed to be completely smeared, virtually causing their disappearance ( Figure 6C ). Unexpectedly, these findings are in stark contrast to the results obtained in HS-SPS Apparatus 1 and HS-SPS Apparatus 2 under similar conditions. As shown in Table 2, both TentaGel beads and Merrifield resin beads remained stable for more than 24 hours under high shear rate stirring in different solvents. For example, stirring TentaGel beads with NMP solvent in HS-SPS Apparatus 2 had no significant effect on the structure of the beads, which can be seen by comparing the TentaGel beads before stirring in HS-SPS Apparatus 2 ( Fig. 7A ) and after stirring ( Figure 7B ) after microscopic examination.

[0236] Without wishing to be bound by any theory or mechanism, the unexpected variation between magnetic and mechanical stirring stems from the different levels of compressive stress applied by each device. The HS-SPS device primarily applies shear stress to the medium at different levels. Similarly, the magnetic stirring device applies the same kind of shear stress. However, since the magnetic bar stirrer is located at the bottom of the flask, it also applies a significant amount of compressive stress. The direction of the stress tension may be the primary cause of resin failure in magnetic stirring vessels.

[0237] Example 2 - Resin Bead Swelling Examination

[0238] Swelling of the dried resin beads is performed at the initial stage of the solid phase peptide synthesis process. NMP, DMF (dimethylformamide) and DCM (dichloromethane) are considered good swelling solvents for polystyrene-divinylphenyl resins and are commonly used in SPSS processes, although TentaGel resins are also known to swell to some extent in water. Swelling is a direct function of the nature of the solvent, the interaction and compatibility with the resin, and the operating conditions in the equipment. In a commonly used shaker reaction vessel, the swelling step is usually completed within one to two hours.

[0239] The swelling kinetics of the polymerized beads were tested in an agitated tank by adding the beads to the solvent in the tank and stirring at 600-700 rpm for two hours. Much like the stability evaluation experiments, the swelling kinetics of TentaGel beads in water were evaluated using HS-SPS Apparatus 1, the swelling kinetics of TentaGel beads in NMP solvent were evaluated using HS-SPS Apparatus 2, and the swelling kinetics of Merrifield resin beads in NMP were evaluated only using HS-SPS Apparatus 2.

[0240] Samples were taken every 30 seconds and examined under a microscope. For each sample, the average diameter of the beads was measured using the microscope's software. Since the beads swelled in NMP for a short time, it was decided to test the swelling under a microscope at smaller time intervals without agitation to observe the effect of the solvent itself in real time. A picture was taken every 30 seconds for the first two minutes, and then every minute for the remaining two minutes. This procedure was performed on the same samples mentioned above. The diameter growth of the beads was measured at different times under two types of operation: (1) directly under a microscope with the resin added to the solvent film without agitation; (2) in a mechanical stirring device with a high shear stress device. The results are shown in Table 3.

[0241] Table 3: Swelling kinetics of two resins.

[0242]

[0243] NR - Not relevant

[0244] It was found that both resins swelled to half their maximum extent immediately upon contact with NMP. When immersed in NMP, Tentagel swelled to a diameter of approximately 150 μm within a few seconds, whereas when stirred in HS-SPS apparatus 2, it swelled to a diameter of 300 μm within 30 minutes. Figure 8 shows the starting beads ( Fig. 8A ); the same beads immediately after contact with NMP ( Figure 8B ); Beads after mechanical stirring for 30 minutes in NMP in HS-SPS apparatus 2 ( Figure 8C ).from Fig. 9A -H It can be further concluded that no damage to the resin was observed in this study. The average diameter of the beads increased as expected over time (Table 4).

[0245] Table 4: Swelling of TenraGel resin in NMP using HS-SPS apparatus.

[0246]

[0247] From the above results, it can be concluded that the resin can be swollen for a short time, thereby reducing the total time of the synthesis.

[0248] Example 3 - Coupling and racemization workup

[0249] As mentioned above, peptide synthesis is usually carried out in the solid phase by using a shaking table apparatus. In order to demonstrate the feasibility of the high shear solid phase peptide synthesis (HS-SPPS) method, the two methods were compared using tripeptide synthesis as a model. The transformation selected was the coupling reaction of an amino-protected amino acid with a dipeptide attached to a resin to form a resin containing a tripeptide. Specifically, the model peptide to be synthesized was Fmoc-L-His-Phe-Gly-NH2. Therefore, the model reaction involved the free amine residue of a resin containing glycine (Gly) and phenylalanine (Phe) units (H2N-Phe-Gly-resin) with a histidine unit (Fmoc-His(Trt)OH; N2N-Phe-Gly-resin) protected on its heterocyclic nitrogen by a trityl group (trityl-Ph3C) and on its primary amine by Fmoc (fluoroalkenylmethoxycarbonyl) protection. α -Fmoc-N (im)1-trityl-L-histidine; CAS NO 109425-51-6). It should be noted that coupling of Fmoc-His(Trt)OH to H2N-Phe-Gly-resin using coupling agents such as DIPC (N,N'-diisopropylcarbodiimide), DCC (N,N'-dicyclohexylcarbodiimide) and EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) in the absence of additives or base is known to cause racemization and result in reduced yields of tripeptides. In other words, coupling of Fmoc-L-His(Trt)OH is considered challenging, mainly due to the increased formation of diastereomeric peptide byproducts containing D-histidine.

[0250] In order to monitor the rate and racemization degree of the coupling reaction, two pure diastereomeric peptides (i.e., Fmoc-L-His-Phe-Gly-NH2 and Fmoc-D-His-Phe-Gly-NH2) were prepared separately using a known coupling method. The two pure peptides were checked in HPLC. The inspection showed two independent peaks, which also did not overlap with the HPLC signal of the starting material, so the conversion and racemization of the tripeptide product could be tracked simultaneously.

[0251] In the experiment, the coupling of Fmoc-His(Trt)OH with H2N-Phe-Gly-resin was tested in the presence of EDC in NMP solvent (without base or additives) using (i) a conventional shaking table apparatus, and (ii) HS-SPS apparatus 2. Afterwards, an aliquot was taken for "small cutting" (as described in Falb et al., The Journal of Peptide Research, (1999) 53, 507-517) of the product from the resin, and the sample conversion and racemization were checked using HPLC. The results are summarized in Table 5.

[0252] Table 5: Conversion and racemization of Fmoc-His(Trt)OH coupling to H2N-Phe-Gly-resin using shaker and HS-SPPS conditions without additives.

[0253]

[0254] 1 EDC was added gradually using a micro syringe. 2 No coupling was performed and the symmetrical histidine anhydride was formed. 3 Histidine anhydride was immediately added to the resin suspension.

[0255] As is evident from Table 5, reaction 2, in which EDC was added to a mixture of His(Trt)OH and H2N-Phe-Gly-resin in NMP, did not provide any target tripeptide within 90 min.

[0256] Since no product was formed in reaction 2, a symmetrically protected histidine anhydride was prepared separately and added to a suspension of H2N-Phe-Gly-resin in NMP (reaction 3). This method allowed 67% conversion after 1.5 hours of reaction, although almost half of the product was the undesired D diastereoisomer racemic product.

[0257] Surprisingly, under similar reaction conditions of mixing the reactants with EDC in NMP, stirring rather than shaking in a HS-SPS apparatus provided 70% conversion, providing only 8.5% of the D-diastereomer (reaction 1). Without wishing to be bound by any mechanism, this suggests that due to the possibility in the process of the present invention (which is not possible in other processes) of gradually adding EDC, the coupling reaction is more efficient, faster and reduces racemization and other side reactions. This may be due to the elimination of accumulation of reactants or intermediates in the reaction mixture. The fact that the conversion is the same (58.4%) under HS-SPS conditions and on a shaker and does not reach 100% reflects the known fact that the reaction of the active ester 4 with the peptide-resin is diffusion controlled and therefore cannot be accelerated by stirring.

[0258] Similar experiments were performed using HS-SPS apparatus 2 (reaction 4) and adding HOBt (hydroxybenzotriazole) as an additive using a shaker (reaction 5). The reactions were monitored and the results are provided in Table 6.

[0259] Table 6: Conversion and racemization of Fmoc-His(Trt)OH coupled to H2N-Phe-Gly-resin in the presence of additives under shaking and HS-SPPS conditions.

[0260]

[0261] 1 Add DIC gradually using a micro syringe for 5 min. 2 Immediate addition of DIC results in the formation of the isomer with D-histidine.

[0262] As is evident from Table 6, reaction 6, where EDC and HOBt were added to a mixture of His(Trt)OH and H2N-Phe-Gly-resin in NMP in a shaker, proceeded within 90 minutes to form the product, but with a racemization rate of 5.5%.

[0263] Surprisingly, similar reaction conditions of mixing the reactants with EDC and HOBt in NMP with stirring rather than shaking in a HS-SPS apparatus provided 60% conversion without observation of the D-diastereomer (Reaction 4).

[0264] Example 4 - Cleavage of Protecting Groups

[0265] To evaluate the effect of the hybrid approach on the reaction rates occurring on the solid support, the reaction to cleave the primary amine protecting group FMOC was monitored. Fmoc removal typically occurs multiple times during peptide extension in Fmoc-based SPPS and is performed using large amounts of piperidine (20% v / v) in DMF or NMP. Piperidine is associated with acute and chronic health effects, including irritation to the eyes and skin and damage to mucous membranes. As a result, large amounts of toxic piperidine waste are generated in both research and industrial facilities.

[0266] Therefore, the progress of Fmoc cleavage was monitored under different conditions by spectrophotometric quantification of the amount of dibenzofullerene released. The rate of removal of Fmoc from Fmoc Rink amide resin was determined using a 5%-20% piperidine solution in NMP in (i) a conventional shaker, (ii) a mechanical stirrer at 100 RPM, (iii) a mechanical stirrer at 700 RPM; and (iv) a fixed reactor. The amount of dibenzofullerene was quantified and normalized compared to the Fmoc content of the resin determined by Fmoc quantitative detection.

[0267] Fig.10 Graph showing the % progress of Fmoc cleavage (measured by dibenzofullerene formation) over time for the following apparatus configurations and piperidine amounts: mechanical stirring apparatus at 700 rpm with 5% piperidine (solid circles); shaker apparatus with 5% piperidine (open triangles); shaker apparatus with 20% piperidine solution (solid triangles); mechanical stirring apparatus at 100 rpm with 5% piperidine (open circles); fixed reactor apparatus with 5% piperidine and no mixing (open squares). Errors are standard deviations between three independent experiments performed.

[0268] like Fig.10As can be seen, UV measurements of dibenzofullerene formation showed that after 10 minutes of reaction in a shaker with 20% piperidine / NMP, 95% Fmoc removal was achieved (solid triangles), but only 60% cut-off was obtained after 10 minutes using the same device with 5% piperidine / NMP (open triangles). Surprisingly, almost the same cut-off curve as that achieved with 20% piperidine / NMP in a shaker was achieved with 5% piperidine / NMP in two separate mechanical stirring devices (700-solid circles; and 100 rpm-open circles) at both high and low rpm mechanical stirring rates. As a control, Fmoc was removed in a fixed reactor with 5% piperidine / NMP. After 20 minutes of reaction, the amount of Fmoc removed was only 20%, which is significantly less than the amount of Fmoc removed by overhead stirring (open squares). Without wishing to be bound by any theory or mechanism of action, it is assumed that the penetration of the reagent into the solid support depends on the mixing properties of the solution. The experiments demonstrated that the reaction rate was much faster with a mechanical stirrer using a 5% solution than with a shaker using the same solution. This suggests that the higher diffusion rate achieved by mechanical stirring allows for the maintenance of a high local concentration of piperidine while rapidly dispersing the dibenzofullerene product. Thus, stirring has the combined effect of increasing diffusion into the beads and penetration into the interior of the beads.

[0269] The description of the foregoing specific embodiments will so fully reveal the general nature of the invention that others can easily modify and / or adapt various applications of such specific embodiments by applying existing knowledge without undue experimentation and without departing from the general concept, and therefore, such adaptations and modifications should and are intended to be understood as being within the equivalent meaning and scope of the disclosed embodiments. It should be understood that the words or terms used herein are for the purpose of description and not limitation. The devices, materials and steps for performing the various disclosed functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for performing at least one cycle of solid phase synthesis of an organic molecule, the method comprising the steps of: i. Providing a reactor comprising a reaction chamber and a stirring device, wherein the stirring device comprises an impeller having at least two blades rotatable about an axis; ii. placing the functionalized polymeric resin beads and at least one solvent in a reactor to provide a reaction mixture, wherein the reaction mixture is contacted with a rotatable blade; iii. placing at least one reactant into the reaction chamber; iv. Rotate the impeller at 600-700 rpm for a period of time while maintaining 1.8-2.4 N / m 2 Shear stress; Thereby at least one cycle of solid phase synthesis of organic molecules is performed.

2. The method of claim 1, wherein the stirring device is a mechanical stirrer, wherein the mechanical stirrer comprises at least three blades rotatable about an axis.

3. The method of claim 1, wherein the rotatable blades rotate upward at a first angle in the range of 20-40°.

4. The method of claim 3, wherein the rotatable blades rotate upward at a first angle in the range of 25-35°.

5. The method of claim 4, wherein the rotatable blades rotate upward at a first angle in the range of 30-35°.

6. The method of claim 1, wherein the functionalized polymeric resin beads comprise polystyrene-divinylbenzene.

7. The method of claim 6, wherein the resin is selected from the group consisting of 1% DVB-PS chloromethylated resin and Rink Amide Tentagel resin.

8. The method of claim 1, wherein the functionalized polymeric resin beads comprise a coupling capacity of 0.2-0.6 mmol / g.

9. The method of claim 1, wherein the functionalized polymeric resin beads have a particle size of 20-200 μm.

10. The method of claim 1, wherein the organic molecule is a polymer comprising a molecule selected from the group consisting of a peptide chain, a nucleotide chain, and a sugar.

11. The method of claim 1, wherein the organic molecule comprises a peptide chain.

12. The method of claim 10 for cycling in solid phase synthesis of polymeric organic molecules, wherein the method comprises the steps of: (a) providing a reactor comprising a reaction chamber and a stirring device, the stirring device comprising an impeller having at least two blades rotatable about an axis; (b) placing functionalized polymeric resin beads and at least one solvent in a reactor to provide a reaction mixture, wherein the reaction mixture is contacted with a rotatable blade; (c) placing at least one protected monomer organic molecule and at least one coupling agent into a reaction chamber and rotating an impeller to form a coupling product of the protected monomer organic molecule and the resin; (d) washing excess of the protected monomeric organic molecules; and (e) placing at least one deprotecting agent in the reaction chamber and rotating the impeller to remove at least one protecting group from the coupling product to form a coupling product of the deprotected monomer organic molecule and the resin, thereby completing a cycle in the solid phase synthesis of the polymerized organic molecule; The impeller in at least one of steps (c) and (e) is rotated for a period of time at a speed of 600-700 rpm while maintaining a speed of 1.8-2.4 N / m 2 of shear stress, optionally, wherein steps (c) to (e) are repeated for a plurality of cycles.

13. The method of claim 12, wherein both steps (c) and (e) are performed at a rotation speed of 600-700 rpm for a period of time while maintaining a speed of 1.8-2.4 N / m 2 of shear stress.

14. The method of claim 10, wherein the at least one reactant is selected from the group consisting of a deprotecting agent, a coupling agent, and a protected monomeric organic molecule.

15. The method of claim 1, wherein the at least one reactant is gradually introduced into the reaction chamber.

16. The method of claim 10, wherein the at least one reactant comprises a coupling agent and a protected monomeric organic molecule, thereby performing at least one coupling cycle of solid phase synthesis of organic molecules.

17. The method of any one of claims 12 and 16, wherein the protected monomeric organic molecule is selected from the group consisting of N-protected amino acids, O-protected nucleosides, and O-protected sugars.

18. The method of claim 17, wherein the protected monomeric organic molecule is an α-N-protected amino acid.

19. The method of claim 18, wherein the α-N-protected amino acid comprises an α-nitrogen atom covalently linked to a protecting group selected from the group consisting of Fmoc and tBoc.

20. The method of claim 18, wherein the α-N-protected amino acid comprises an α-nitrogen atom covalently linked to an Fmoc protecting group.

21. The method of claim 10, wherein the reactants further comprise at least one deprotecting agent.

22. The method of any one of claims 12 and 21, wherein the at least one deprotecting agent is a reagent capable of removing an Fmoc group.

23. The method of claim 22, wherein the reagent capable of removing the Fmoc group comprises a base.

24. The method of claim 23, wherein the base comprises an amine.

25. The method of claim 24, wherein the amine is selected from the group consisting of piperidine, morpholine, piperazine, dicyclohexylamine, N,N-diisopropylethylamine, 4-dimethylaminopyridine, 1,8-diazabicycloundec-7-ene, pyrrolidine, cyclohexylamine, ethanolamine, diethylamine, trimethylamine, ammonia, tributylamine, 1,4-diazabicyclo[2.2.2]octane, hydroxylamine, tris(2-aminoethyl)amine, and combinations thereof.

26. The method of any one of claims 12 and 14, wherein the coupling agent comprises a carbodiimide.

27. The method of claim 26, wherein the carbodiimide is gradually added to the reaction mixture.

28. The method of claim 27, wherein the carbodiimide is selected from the group consisting of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, and combinations thereof.

29. The method of claim 14, wherein the reactant further comprises 1-hydroxybenzotriazole (HOBt).

30. The method of claim 12, wherein step (c) comprises placing at least one protected monomer organic molecule, at least one carbodiimide and HOBt into a reaction chamber and rotating an impeller to form a coupling product of the protected monomer organic molecule and the resin.

31. The method of any one of claims 1 and 12, wherein the at least one solvent is selected from the group consisting of water, dimethylformamide, dichloromethane, N-methyl-2-pyrrolidone, dimethylacetamide, and combinations thereof.

32. The method of claim 31, wherein the at least one solvent comprises N-methyl-2-pyrrolidone.

33. The method of claim 1 further comprising the steps of washing the reaction mixture and filtering the polymeric resin beads.

34. The method of claim 1, further comprising repeating steps (ii) to (iv) at least once more, thereby performing at least one more cycle of solid phase synthesis of the organic molecule.

35. The method of claim 34, comprising at least two steps of coupling an amino acid to the resin and at least two steps of removing protecting groups.

36. The method of any one of claims 10 and 12, further comprising the step of cleaving the polymeric organic molecule from the polymeric resin.

37. The method of claim 1 including an initial step of swelling the polymeric resin beads in at least one solvent.

38. The method of claim 12, wherein step (b) comprises swelling the polymeric resin beads in the reaction mixture.

39. The method of any one of claims 37 and 38, wherein swelling the polymeric resin beads comprises mixing the polymeric resin beads in the solvent at a rotation speed of 600-700 rpm for a specified period of time.

40. The method of claim 39, wherein the mixing of the polymeric resin beads in the solvent for a specified period of time comprises maintaining a pressure of 1.8-2.4 N / m 2 of shear stress.

41. The method of claim 39, wherein the designated period of time is 1-60 minutes.

42. The method of claim 41, wherein the designated period of time is 2-45 minutes.

43. The method of claim 42, wherein the designated period of time is 4-30 minutes.

44. The method of any one of claims 1 and 12, wherein: The period of time is in the range of 1-600 minutes.

45. The method of claim 44, wherein: The period of time is in the range of 10-300 minutes.

46. ​​The method of claim 44, wherein The period of time is in the range of 5-60 minutes.

47. The method of claim 44, wherein: The period of time is in the range of 10-30 minutes.

48. The method of claim 44, wherein: The period of time is in the range of 15-210 minutes.

49. The method of claim 44, wherein: The period of time is in the range of 30-180 minutes.

50. The method of claim 44, wherein: The period of time is in the range of 60-120 minutes.

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