Peptide synthesis method

Abstract

A method for deprotecting a protected amino acid during solid phase peptide synthesis (SPPS) includes removing a protecting group from a protected amino acid in a reactor using a deprotecting base present in the reactor in an amount greater than 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor. At least a portion of the deprotecting base evaporates into the upper interior (e.g., headspace) of the reactor during the removing step. The method also includes directing an inert gas through the reactor during the removing step to remove the evaporated deprotecting base from the interior (e.g., headspace) of the reactor.

Description

[Technical Field]

[0001] Cross-reference to priority application This application claims the benefit of pending U.S. patent application Ser. No. 63 / 442,216, filed January 31, 2023; pending U.S. patent application Ser. No. 63 / 452,550, filed March 16, 2023; pending U.S. patent application Ser. No. 63 / 452,674, filed March 16, 2023; pending U.S. patent application Ser. No. 63 / 521,623, filed June 16, 2023; pending U.S. patent application Ser. No. 63 / 532,041, filed August 10, 2023; and pending U.S. patent application Ser. No. 18 / 237,576, filed August 24, 2023, the entire disclosures of each of which are incorporated herein by reference in their entirety.

[0002] This application also claims the benefit of pending International Application No. PCT / US22 / 043806, filed September 16, 2022, the entire disclosure of which is also incorporated herein by reference in its entirety.

[0003] Technical Field The present disclosure relates to methods for deprotecting protected amino acids and / or protected peptides (e.g., as a step in peptide synthesis that includes a deprotection and coupling step, such as solid phase peptide synthesis, also referred to herein as SPPS, and / or liquid phase and / or solution phase peptide synthesis, also referred to herein as LPPS). [Background technology]

[0004] Solid-phase peptide synthesis (SPPS) has been the primary tool enabling peptide synthesis since its inception in 1963. SPPS dramatically simplified peptide production compared to solution-phase peptide synthesis (LPPS) by allowing for straightforward isolation of the product by simple filtration at each step, as opposed to more laborious extraction methods after each deprotection and coupling step. However, compared to LPPS, SPPS results in significant waste products from the successive washing steps between each deprotection and coupling step. Historically, approximately five washes were required between each step, resulting in 80-90% of the total waste generated from washing.

[0005] The use of microwave energy, and heating in general, was first applied to SPPS to accelerate synthesis time and improve purity by driving reaction steps toward completion. While successful in this regard, these early efforts did not fundamentally eliminate the need for washing in the process. Subsequent developments introduced a microwave-assisted, highly efficient SPPS method for 9-fluorenylmethyloxycarbonyl (Fmoc) SPPS, which eliminated washing after each coupling step. This was based on the insight that residual activated amino esters were quickly captured by the deprotecting base before insertion could occur. This approach eliminated half of the washings and shortened the overall cycle time of the method. It was later further demonstrated that the deprotecting base could be added directly to the post-coupling solution without any drainage. This "one-pot" deprotection and coupling method facilitates the deprotection process by allowing the reuse of both solvent and heat from the coupling solution, resulting in additional solvent, energy, and time savings.

[0006] Despite the above, the SPPS method still requires significant washing during each amino acid addition cycle, primarily due to the need to remove deprotection reagents, which can lead to the formation of undesired insertion and deletion impurities during the next coupling step. Typically, if residual base from deprotection contaminates the next coupling step, it will remove the Fmoc protecting group on the next amino acid, resulting in the undesired insertion of an additional amino acid onto the growing chain. Furthermore, the residual base can react with and consume the activated amino ester before reacting with the peptide terminus. This can result in both insertion and deletion of the next amino acid, potentially leading to impurities that are difficult to separate (e.g., by reverse-phase HPLC). For this reason, washing after the deprotection step was considered unavoidable. Summary of the Invention [Means for solving the problem]

[0007] The present disclosure relates to methods for deprotecting protected amino acids and / or protected peptides (e.g., as a step in peptide synthesis that includes a deprotection and coupling step, such as solid phase peptide synthesis, also referred to herein as SPPS, and / or liquid phase and / or solution phase peptide synthesis, also referred to herein as LPPS). The deprotection reaction removes the protecting group of the protected amino acid and / or protected peptide (deprotecting the amino acid and / or deprotecting the peptide) to prepare the amino acid and / or peptide for a coupling reaction with a second amino acid.

[0008] The terms "liquid phase peptide synthesis" and "solution phase peptide synthesis" may be used interchangeably herein and / or may be generally referred to herein as "LPPS."

[0009] In some embodiments, the present disclosure can help reduce the amount of solvent used in or eliminate post-deprotection washing steps, as opposed to the pre-deprotection steps of the SPPS method, which can, in some embodiments, reduce or eliminate washing steps (e.g., reducing or eliminating all post-coupling washing steps, reducing or eliminating all post-deprotection washing steps), providing significant waste reduction (e.g., up to a 95% reduction in overall waste) and associated cost and time savings.

[0010] In some embodiments, the present disclosure can help reduce the amount of solvent used in the extraction step after deprotection and before coupling, as opposed to the prior deprotection step of the LPPS method, which in some embodiments can reduce the amount of solvent used in the extraction step after deprotection and / or reduce or eliminate post-coupling wash steps, providing significant waste reduction and associated cost and time savings.

[0011] A method for deprotecting a protected amino acid (e.g., during solid phase peptide synthesis and / or solution phase peptide synthesis) includes removing a protecting group of a protected amino acid and / or a protected peptide in a reactor (e.g., in a batch SPPS and / or LPPS reactor) using a deprotecting base in an amount of about 5% by volume or less, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor.

[0012] In some embodiments, the deprotecting base may be present in the reactor in an amount of about 1 vol% to about 5 vol%, for example, about 2 vol% to about 5 vol%, for example, about 2 vol% to about 4.5 vol%, for example, about 3 vol% to about 4.5 vol%, or another example, about 3.5 vol% to about 4.5 vol%, based on the total volume (100 vol%) of the deprotecting reaction mixture (e.g., deprotecting reaction solution) in the reactor. In some embodiments, the deprotecting base may be present in the reactor in an amount of 0 to about 4.5 vol%, for example, greater than about 2 vol% to about 4.5 vol%, based on the total volume of the deprotecting reaction mixture (e.g., deprotecting reaction solution). In some embodiments, the deprotecting base may be present in the reactor in an amount of 0 to greater than about 4 vol%, for example, about 2 vol% to about 4 vol%, based on the total volume of the deprotecting reaction mixture (e.g., deprotecting reaction solution). In some embodiments, the deprotecting base can be present in the reactor in an amount of from 0 to about 3.5% by volume, for example, from about 2% to about 3.5% by volume, based on the total volume of the deprotecting reaction mixture (e.g., the deprotecting reaction solution). The amount of deprotecting base can be any value within the ranges described herein, including the endpoints (e.g., any value within the range of from greater than 0 to about 5% by volume) and all subranges within the ranges.

[0013] At least a portion (e.g., a majority) of the deprotected base evaporates (volatilizes) during the removing step (e.g., at least a portion, a majority, etc., of the deprotected base evaporates into the headspace of the reactor during the removing step). Methods for deprotecting a protected amino acid according to the present disclosure also include directing (e.g., continuously and / or intermittently directing) an inert gas through the reactor to aid in removing (e.g., to aid in flushing, evacuating, evacuating, replacing, replacing, purging, etc.) the evaporated (volatilized) deprotected base from the interior of the reactor (e.g., from the headspace of the reactor) during the protecting group removing step.

[0014] In some embodiments, the directing step can include introducing an inert gas into the upper interior of the reactor through a first opening disposed in the top of the reactor, and venting (flushing) the inert gas and venting the vaporized deprotected base from the upper interior of the reactor (e.g., from the headspace of the reactor) through a second opening also disposed in the top of the reactor.

[0015] In some embodiments, the directing step can include introducing an inert gas into a lower interior of the reactor through an opening disposed in the lower part of the reactor, and venting (flushing) the inert gas and evaporated deprotected base from an upper interior of the reactor (e.g., from the headspace of the reactor) through an opening disposed in the upper part of the reactor.

[0016] In some embodiments, the directing step can include introducing an inert gas into the upper interior of the reactor through a first opening disposed in the upper portion of the reactor, introducing an inert gas into the lower interior of the reactor through a second opening disposed in the lower portion of the reactor, and venting the inert gas and venting the evaporated deprotected base from the upper interior of the reactor (e.g., from the headspace of the reactor) through a third opening disposed in the upper portion of the reactor. In this embodiment, the inert gas introduced into the lower interior of the reactor can serve to agitate (bubble, stir, etc.) materials in the lower interior of the reactor (e.g., deprotection reaction mixture, e.g., deprotection reaction solution) and / or participate in headspace evacuation (flushing, purging, etc.) of evaporated deprotected base as described herein.

[0017] In some embodiments, the method may further comprise heating the protected amino acid and / or the protected peptide and / or the deprotected base during the step of removing the protecting group from the protected amino acid and / or the protected peptide. The heating step may be carried out at a temperature of, for example, about 40°C to about 120°C, for example, about 60°C to about 120°C, or for example, about 90°C to about 120°C. The heating step may be carried out using microwave radiation.

[0018] In exemplary embodiments of the deprotection methods disclosed herein, the deprotection methods can use a solid resin support (e.g., a protected amino acid and / or a protected peptide can be attached to a solid resin support) having a resin substitution of less than or about 0.35 mmol / g (e.g., <0.35 mmol / g), for example, less than or about 0.30 mmol / g (e.g., <0.30 mmol / g). In some embodiments, the deprotection method can utilize a solid resin support having a resin substitution of 0.10 mmol / g to 0.35 mmol / g, e.g., 0.15 mmol / g to 0.35 mmol / g, e.g., 0.10 mmol / g to 0.34 mmol / g, e.g., 0.15 mmol / g to 0.34 mmol / g, e.g., 0.20 mmol / g to 0.35 mmol / g, e.g., 0.20 mmol / g to 0.34 mmol / g, e.g., 0.20 mmol / g to 0.33 mmol / g. The amount of resin substitution can be any value within the ranges described herein, including the endpoints and all subranges within the ranges. In an exemplary embodiment, the resin can be a PEG-PS (polyethylene glycol-polystyrene) resin (e.g., a PEG-PS-based resin (Pro-Tide), a PS (polystyrene) resin, etc.).

[0019] The present disclosure also relates to methods for solid phase peptide synthesis (SPPS) and / or liquid phase peptide synthesis (LPPS). The SPPS and / or LPPS methods may include deprotecting a first protected amino acid and / or a protected peptide to provide a deprotected amino acid and / or a deprotected peptide, as described herein, and coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids, and / or coupling an amino acid to the deprotected peptide to form a second peptide from the deprotected peptide and the amino acid.

[0020] In some embodiments, the method does not include a washing step after the deprotection step and before the (next, consecutive) coupling step of the SPPS cycle.

[0021] In some embodiments, the method may include a washing step after the deprotection step of the SPPS cycle and before the (next, consecutive) coupling step using a washing composition (e.g., solvent). In some embodiments, the method may include a washing step after the deprotection step of the SPPS cycle and before the (next, consecutive) coupling step, using a washing composition (e.g., solvent) in an amount approximately equal to the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution). In some embodiments, the method may include a washing step after the deprotection step of the SPPS cycle and before the (next, consecutive) coupling step, using a washing composition (e.g., solvent) in an amount less than the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution). For example, the washing step may include washing with a washing composition (e.g., solvent) in an amount less than or about 1 / 2 of the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), and as another example, washing with a washing composition (e.g., solvent) in an amount less than or about 1 / 3 of the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution). In some embodiments, the washing step can include washing the inside of the reactor with a total volume of solvent that is two or less times the bed volume of the resin (e.g., the solid resin support present in the reactor) present in the reactor, for example, one or less times the bed volume of the resin present in the reactor.

[0022] In some embodiments, the method may include one or more extraction steps after the deprotection step of the LPPS cycle and before the (next, consecutive) coupling step using an extraction solvent (e.g., an aqueous solvent immiscible with the deprotection reaction product, e.g., the growing peptide chain). In some embodiments, the method may include one or more extraction steps after the deprotection step of the LPPS cycle and before the (next, consecutive) coupling step using a total volume of extraction solvent that is two times (2×) or less the total volume (total volume) of the deprotection reaction mixture in the reactor of that deprotection-coupling cycle (e.g., the total combined volume of extraction solvent for all extraction steps of a single deprotection-coupling cycle).

[0023] The present invention will now be described with reference to the accompanying drawings, in which embodiments of the invention are shown and in which like reference numerals may indicate the same or similar elements. The drawings are provided as examples and may be schematic and are not intended to be drawn to scale. Aspects of the present invention may be embodied in many different forms and should not be construed as being limited to the examples shown in the drawings. For clarity, not every component is shown in every figure, and not every component of each embodiment of the present invention is shown unless illustration is necessary to enable those skilled in the art to understand the invention. [Brief explanation of the drawings]

[0024] [Figure 1A] FIG. 1A is a cross-sectional view of an exemplary reactor, illustrating a method for deprotecting a protected amino acid (eg, in an SPPS method) according to an embodiment of the present disclosure. [Figure 1B] FIG. 1B is a cross-sectional view of another exemplary reactor, illustrating generally a method for deprotecting a protected amino acid (eg, in an SPPS method) according to another embodiment of the present disclosure. [Figure 2] Figure 2A is a schematic flow diagram illustrating selected portions of an exemplary peptide synthesis system (e.g., of an SPPS and / or LPPS system) according to an embodiment of the present disclosure, and Figure 2B is a schematic flow diagram illustrating selected portions of another exemplary peptide synthesis system (e.g., of an SPPS and / or LPPS system) according to another embodiment of the present disclosure. [Figure 3] FIG. 3 is a flow chart outlining the steps of a cycle of a conventional solid phase peptide synthesis (SPPS) method. [Figure 4-1] 4A and 4B are flow charts that outline the steps of a cycle of the SPPS method according to the present disclosure. [Figure 4-2] FIG. 4C is a flow chart that outlines the steps of a cycle of an LPPS method according to the present disclosure. [Figure 5A]FIG. 5A is a cross-sectional view of an exemplary reactor, schematically illustrating a method for deprotecting a protected amino acid (eg, in an LPPS method) according to an embodiment of the present disclosure. [Figure 5B] FIG. 5B is a cross-sectional view of another exemplary reactor, illustrating generally a method for deprotecting a protected amino acid (eg, in an LPPS method) according to another embodiment of the present disclosure. [Figure 6] Figure 6A is a schematic flow diagram illustrating selected portions of another exemplary peptide synthesis system (e.g., an SPPS and / or LPPS system) according to an embodiment of the present disclosure. Figure 6B is a schematic flow diagram illustrating selected portions of another exemplary peptide synthesis system (e.g., an SPPS and / or LPPS system) according to another embodiment of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0025] Exemplary embodiments are disclosed below. The above and other aspects, features, and advantages of the present invention will become apparent from the following detailed description of the embodiments. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. For example, features disclosed as part of one embodiment or example (e.g., one or more features of a method for deprotecting protected amino acids and / or protected peptides during peptide synthesis, including deprotection and coupling steps, solid-phase peptide synthesis (SPPS) methods, and / or liquid-phase and / or solution-phase peptide synthesis (LPPS) methods, etc.) can be used in the context of another embodiment or example to yield a further embodiment or example. The embodiments are provided for complete disclosure and to provide a complete understanding of the present invention to those skilled in the art. Well-known aspects may not be described in detail to avoid unnecessarily obscuring the present invention. Therefore, this detailed description should not be construed in a limiting sense, and other embodiments are intended to be within the spirit and scope of the present invention. The scope of the present invention is to be defined solely by the appended claims.

[0026] Embodiments of the present disclosure relate to methods and systems for deprotecting protected amino acids (e.g., deprotecting a protected amino acid; deprotecting a protected amino acid-derived unit of a peptide is also referred to herein as deprotecting a protected peptide, where a protected peptide refers to a peptide that includes a unit derived from a protected amino acid) as a process peptide synthesis (e.g., in solid-phase peptide synthesis and / or solution-phase peptide synthesis). In exemplary embodiments, the methods and systems are batch-based methods and systems.

[0027] Those skilled in the art will understand the meaning of the term amino acid. As used herein, the term amino acid in its broadest sense refers to an organic compound that contains both an amine functional group and a carboxylic acid functional group, and optionally also a side chain. Those skilled in the art will also understand that amino acid includes natural amino acids (proteinogenic amino acids) and / or non-proteinogenic amino acids, and will also understand the single-letter symbols used to identify them.

[0028] The methods of the present disclosure may be useful for producing peptides and / or proteins. The terms peptide and / or protein will also be understood by those skilled in the art. For example, as used herein, the term peptide and / or protein can refer to an amide derived from two or more amino acids (the same or different) by binding the carbonyl carbon of one amino acid to the nitrogen atom of another amino acid. As will be understood by those skilled in the art, peptides and proteins can be distinguished by chain length (e.g., peptides have a shorter chain length of amino acids (fewer amino acids) linked by chemical bonds compared to proteins). However, for ease of discussion, the term peptide will be used consistently throughout, and the present disclosure is not limited to the production of peptides (e.g., the methods described herein may be applicable to the production of peptides and / or proteins).

[0029] For ease of reference, the present disclosure relates to methods for deprotecting a protected amino acid (e.g., removing a protecting group of a protected amino acid). Those skilled in the art will understand that a protected amino acid can be part of a peptide, and that discussion herein of methods for deprotecting a protected amino acid also includes methods for deprotecting a protected peptide (e.g., removing a protecting group of a protected amino acid-derived unit of a peptide).

[0030] 1A is a schematic cross-sectional view of a reactor suitable for use in an amino acid deprotection and peptide synthesis method and system (e.g., SPPS method and system) according to an embodiment of the present disclosure. FIG. 1A also illustrates a schematic of a method (e.g., SPPS) according to one embodiment of the present disclosure for deprotecting a protected amino acid.

[0031] 1B is a schematic cross-sectional view of another reactor suitable for use in an amino acid deprotection and peptide synthesis method and system (e.g., SPPS method and system) according to an embodiment of the present disclosure. FIG. 1B also schematically illustrates a method (e.g., SPPS) according to one embodiment of the present disclosure for deprotecting a protected amino acid.

[0032] 5A and 5B are schematic cross-sectional views of a reactor suitable for use in amino acid deprotection and peptide synthesis methods and systems (e.g., LPPS methods and systems) according to embodiments of the present disclosure. FIGS. 5A and 5B also schematically illustrate methods (e.g., LPPS) according to embodiments of the present disclosure for deprotecting a protected amino acid. Those skilled in the art will appreciate that the reactor shown in FIGS. 5A and 5B can have many, if not most, of the same elements described herein for the reactor of FIGS. 1A and 1B, except that reactors for LPPS typically do not include filter 32 as described herein. As shown herein, the reactors of FIGS. 5A and 5B useful for LPPS do not include filter 32, and mixture 30, also described herein, can remain at the lower surface (e.g., bottom wall) of the reactor. The lower portion of the reactor in FIGS. 5A and 5B can also be configured as known in the art to allow extraction (removal) of a waste layer (e.g., an aqueous layer containing residual deprotected base) after deprotection while maintaining a product layer (e.g., an organic layer containing the growing peptide chain). Such reactors, including extraction mechanisms, suitable for use in LPPS processes are known in the art and will not be described in detail herein. Those skilled in the art will also understand that the reactors of Figures 5A and 5B are representative examples of reactors suitable for LPPS processes, the disclosure is not limited thereto, and other commercially available reactors suitable for LPPS processes may be used.

[0033] Unless otherwise indicated, elements shown in FIG. 1A have the same reference numbers as in other figures, including FIGS. 1B, 5A, and 5B.

[0034] As shown in Figures 1A and 1B (and Figures 5A and 5B), reactor 4 includes at least one sidewall 6 that extends around an interior 7 (e.g., hollow cavity) of reactor 4 (reactor interior 7 is also referred to herein as the outer interior space). The particular size and shape of reactor 4 are not limited. Reactors suitable for use in solid-phase peptide synthesis and / or solution-phase synthesis are well known in the art and are commercially available.

[0035] The size (internal volume) of the reactor is not limited. Exemplary reactor sizes can range from less than 1 liter to 40 liters or more, for example, but are not limited to, 10 ml, 30 ml, 125 ml, 1 liter, 3 liters, 5 liters, 8 liters, 10 liters, 15 liters, etc., up to 40 liters or more.

[0036] Reactor 4 further includes one or more openings. As a non-limiting example, Figure 1A (and Figure 5A) shows openings 10, 12, and 14 located in the upper portion (e.g., in the top wall) of reactor 4 and opening 16 located in the lower portion (e.g., in the bottom wall) of reactor 4. As another non-limiting example, Figure 1B (and Figure 5B) shows openings 200, 202, 204, 206, and 208 located in the upper portion (e.g., in the top wall) of reactor 4, and opening 16 located in the lower portion (e.g., in the bottom wall) of reactor 4. The openings (e.g., inlets, outlets, ports, etc.) allow for the introduction and / or removal of fluids and / or solids, such as reactants, solvents, gases, products (peptides), by-products, excess (residual) reactants, etc., as described in more detail herein.

[0037] Those skilled in the art will understand that reactor 4 is not limited to the number and / or locations of openings shown in Figures 1A and 1B (and Figures 5A and 5B), and that other reactor designs and configurations having fewer or more openings and / or different locations thereof can be used (e.g., the reactor can include fewer or more openings located in the top wall and / or bottom wall and / or side wall, etc.).

[0038] Fluids and / or solids may be introduced (e.g., moved, transported, directed, flushed, purged, drained, vented, etc.) into or out of reactor 4 via one or more flow paths (e.g., lines, passages, tubes, manifolds, etc.), such as flow paths 20, 22, and 24 in fluid communication with openings 10, 12, and 14, respectively, and flow path 26 in fluid communication with opening 16, as shown in FIG. 1A (and FIG. 5A).

[0039] Other non-limiting examples of flow paths are shown in FIG. 1B (and FIG. 5B) as flow paths 210, 212, 214, 216, and 218 in fluid communication with openings 200, 202, 204, 206, and 208, respectively, and flow path 26 in fluid communication with opening 16.

[0040] In some embodiments, reactor 4 can include at least one spray head (e.g., spray nozzle) or equivalent structure disposed within the reactor for adding (e.g., directing, feeding, spraying, etc.) fluids (e.g., solvent, reactants, and / or inert gas) to the reactor. The spray head can be a separate part (e.g., component, element, etc.) from the reactor (e.g., can be installed within and removed from the reactor) or can be an integrated part of the reactor.

[0041] As a non-limiting example, FIG. 1B (and FIG. 5B) schematically illustrates an embodiment including a spray head 220 disposed in the interior space 7 (also referred to herein as the outer interior space) of the reactor 4. The spray head 220 is positioned in the interior space 7 (also referred to herein as the outer interior space) of the reactor 4 and includes at least one sidewall 220a extending around the inner interior space. The spray head 220 includes a first portion (end) 220b proximate the opening 208 that is in fluid communication with the opening 208 (and the flow path 218), and a second portion (end) 220c distal to the opening 208. A plurality of holes 222 (e.g., ports, etc.) extend at least partially around the inner interior space and can be defined, for example, in the sidewall 220a. The inner interior space and the outer interior space are in fluid communication with each other through the plurality of holes.

[0042] Spray head 220 is configured such that fluid (e.g., an inert gas, solvent, etc., as discussed herein) directed into the inner interior space of spray head 220 through flow path 218 and opening 208 enters (e.g., is sprayed, directed, fed, etc.) the outer interior space 7 of reactor 4 through (e.g., through one, more than one, many, or all) of the plurality of holes 222. In some embodiments, spray head 220 can be configured such that fluid exiting at least one or more of the plurality of holes is directed (sprayed) against sidewall 6 of the reactor. An exemplary spray pattern is shown schematically (e.g., in an approximate manner) in FIG. 1B (and FIG. 5B ) by dashed line 224, with fluid exiting the plurality of holes 222 directed at a generally downward angle toward sidewall 6.

[0043] The present disclosure is not limited with respect to the particular spray head configuration, location within the reactor, spray pattern, and / or direction (angle) of the fluid exit holes of the spray head as shown in Figure 1B (and Figure 5B), and other spray head configurations, locations, spray patterns, and / or spray angles, etc. Spray heads suitable for use in SPPS methods and systems are known in the art and can be used in the present disclosure.

[0044] The present disclosure is not limited to a particular number and / or location of openings and flow paths, and thus a reactor can have one, two, three, four, or more openings and associated flow paths as desired. Furthermore, any series of flow paths and associated valves that serve to direct, permit, and / or block (e.g., close, restrict, etc.) the flow of fluids and / or solids can be used.

[0045] Reactor 4 contains a mixture of components, generally indicated at 30 in FIGS. 1A and 1B (and FIGS. 5A and 5B), located at the bottom of reactor 4. In FIGS. 1A and 1B, the mixture of components 30 can be retained on filter 32, located at the bottom of reactor 4. Filter 32 can also prevent solid materials present in the mixture (such as solid resin supports linked to growing peptide chains, as described in more detail herein) from entering flow path 26. As described and illustrated herein, the reactors of FIGS. 5A and 5B useful for LPPS may not include filter 32, and mixture 30 may be retained on the bottom surface (e.g., bottom wall) of the reactor.

[0046] For example, mixture 30 includes protected amino acids, i.e., amino acids that include at least one protecting group attached to a functional group, such as a terminal amine group, to protect against undesired reaction of the functional group. Protected amino acids can be part of a growing peptide chain linked to a solid resin support and / or other suitable support (e.g., a soluble tag), as understood and known in the art and discussed in more detail herein.

[0047] Suitable protecting groups for use in the method of the present disclosure are well known in the art.The example of the protecting group suitable for protecting amine or N-terminus includes, but is not limited to, fluorenylmethyloxycarbonyl (Fmoc) protecting group.See, for example, Chan and White, Fmoc solid phase peptide synthesis, a practical approach, Oxford University Press (2000).

[0048] The amino acid may also contain a side chain protecting group. Examples of side chain protecting groups include, but are not limited to, trityl, t-butyl, and / or 2,2,4,6,7 pentamethyldihydrobenzofuran-5-sulfonyl (Pbf) protecting groups, etc. Once the desired peptide chain length is obtained, the side chain protecting group may be removed.

[0049] The protected amino acid can be directly or indirectly attached to a solid support or other suitable support known in the art (e.g., a soluble tag of an LPPS). For example, the carboxy terminus of the protected amino acid can be attached to a solid support or other suitable support via a suitable linker. As another example, the carboxy terminus of the protected amino acid can be indirectly attached to a solid support or other suitable support, for example, the carboxy terminus of the protected amino acid can be coupled to the amine or N-terminus of another amino acid that is then linked to a solid support or other suitable support via a suitable linker (e.g., the amine or N-terminus of a single amino acid or the amine or N-terminus of an amino acid that is part of a growing peptide chain).

[0050] Any solid support known in the art can be used in the method of the present disclosure.In exemplary embodiments, the solid support is a solid resin support.Examples of solid resin support materials can include, but are not limited to, polystyrene (for example, resin forms such as microporous polystyrene resin, mesoporous polystyrene resin, macroporous polystyrene resin, etc.), glass, polysaccharides (for example, cellulose, agarose), polyacrylamide resin, polyethylene glycol and / or copolymer resin (for example, including polyethylene glycol, polystyrene, etc.).

[0051] In exemplary embodiments of the deprotection methods disclosed herein, the protected amino acids may be attached to a solid resin support having a resin substitution of less than or about 0.35 mmol / g (e.g., <0.35 mmol / g), for example, less than or about 0.30 mmol / g (e.g., <0.30 mmol / g). In some embodiments, the deprotection method is carried out at a resin substitution of 0.10 mmol / g to 0.35 mmol / g, for example, 0.15 mmol / g to 0.35 mmol / g, for example, 0.10 mmol / g to 0.34 mmol / g, for example, 0.15 mmol / g to 0.34 mmol / g, for example, 0.20 mmol / g to 0.35 mmol / g, for example, 0.20 mmol / g to 0.34 mmol / g, for example, 0.20 mmol / g to 0.34 mmol / g. Resins having a resin substitution of 0.33 mmol / g, e.g., 0.21 mmol / g to 0.35 mmol / g, e.g., 0.21 mmol / g to 0.34 mmol / g, e.g., 0.21 mmol / g to 0.33 mmol / g, e.g., 0.22 mmol / g to 0.35 mmol / g, e.g., 0.22 mmol / g to 0.34 mmol / g, e.g., 0.22 mmol / g to 0.33 mmol / g, can be used. The amount of resin substitution can be any value within the ranges described herein, including the endpoints and all subranges within the ranges. In an exemplary embodiment, the resin can be a PEG-PS (polyethylene glycol-polystyrene) resin (e.g., PEG-PS based resin (Pro-Tide), PS (polystyrene) resin, etc.). In an exemplary embodiment, the solid support is a medium-substituted (e.g., 0.2-0.3 mmol / g, 0.20-0.25 mmol / g, 0.21-0.3 mmol / g, 0.21-0.25 mmol / g, 0.22-0.3 mmol / g, 0.22-0.25 mmol / g, etc.) solid PEG-PS (polyethylene glycol-polystyrene) resin support (e.g., a PEG-PS-based resin (Pro-Tide)).

[0052] The solid support may have any suitable form, for example, the solid support may be in the form of beads, particles, fibers, and / or any other suitable form.

[0053] The present disclosure is not limited to the solid support materials described herein, and other types of supports and / or carriers and / or protecting group materials known in the art may also be used in the deprotection methods during peptide synthesis, including the deprotection and coupling steps described herein, SPPS methods, LPPS methods, etc.

[0054] In exemplary embodiments that include a deprotection step of the LPPS method, the carboxy or C-terminus of the amino acid and / or peptide may be linked to a suitable support and / or carrier and / or protecting group, and the N-terminus may also include a protecting group (e.g., an Fmoc protecting group).

[0055] As a non-limiting example, in some embodiments, soluble tags known in the art (e.g., soluble polymer tags (also called soluble resin tags) and / or soluble compound tags, etc.) may be used as carriers and / or supports and / or protecting groups (e.g., carboxyl or C-terminal protecting groups of amino acids and / or peptides) in deprotection methods during peptide synthesis, including deprotection and coupling steps, such as the LPPS methods described herein. In some embodiments of the present disclosure, protected amino acids (e.g., LPPS methods) may be attached to one or more soluble tags (e.g., soluble polymer tags, soluble compound tags, etc. known in the art) alone or as part of a (growing) peptide chain. Soluble tags (e.g., soluble polymer tags, soluble compound tags, etc.) may comprise polymers and / or compounds that can precipitate under certain conditions and dissolve under other conditions known in the art. Thus, as is known in the art, soluble tags (e.g., soluble polymer tags and / or soluble compound tags) useful in embodiments of the present disclosure (e.g., LPPS methods) can have the property of reversibly changing between a solid-phase state and a solution-phase state depending on conditions (e.g., solvents, reagents, temperature, etc.). Generally, in the solution-phase peptide synthesis methods of the present disclosure, the soluble tag and the amino acids and / or peptide chains linked thereto are in a solution or solution-phase state during the deprotection and / or coupling steps.

[0056] Exemplary soluble polymer tags and / or soluble compound tags can include one or more functional groups (e.g., alcohol functional groups, amine functional groups, etc.) that can react with the carboxy or amino terminus of an amino acid and / or peptide to link the amino acid and / or peptide to the soluble polymer tag and / or soluble compound tag. In exemplary embodiments of the present disclosure, the soluble tag is linked to the carboxyl (C-terminus) of the amino acid and / or peptide (which can also have a protected N-terminus, e.g., an Fmoc-protected N-terminus).

[0057] Soluble tags (e.g., soluble polymer tags, soluble compound tags, etc.) can be used in deprotection methods during peptide synthesis, including the deprotection and coupling methods described herein, LPPS methods, etc. (e.g., used in solution phase during the deprotection and / or coupling steps described herein). Soluble tags can employ solution-phase kinetics for reaction steps (e.g., the deprotection and / or coupling steps described herein), which can allow for the use of lower reagent equivalents.

[0058] Suitable soluble tags (e.g., soluble polymer tags, soluble compound tags, etc.) are known in the art and / or commercially available, and one of skill in the art will understand how to use them in deprotection methods during peptide synthesis, including deprotection and coupling steps, such as the LPPS methods described herein, including appropriate conditions to promote the solid-phase and / or solution-phase conditions in the deprotection and / or coupling steps.

[0059] For example, soluble compound tags can include aromatic compounds (e.g., benzyl compounds) with functional groups (e.g., alcohol and / or amine functional groups), hydrophobic groups (e.g., groups containing long-chain alkyl moieties), etc. Exemplary soluble compound tags can include, for example, hydrophobic benzyl alcohol or amine-type compounds as shown below: [ka] wherein R represents a group containing one or more atoms (e.g., oxygen, nitrogen, sulfur, etc.) that can react with the carboxy or amide terminus of an amino acid and / or peptide to link the polymer tag to the amino acid and / or peptide. Typical examples of soluble compound tags can include the compounds described above where R=OH or R=NH.

[0060] Those skilled in the art will understand how to couple amino acids and / or peptides to a support (e.g., a solid resin support and / or a soluble tag). Accordingly, a detailed discussion of methods known in the art for coupling amino acids and / or peptides to a solid support and / or a soluble tag is not provided.

[0061] 1A and 1B (and FIGS. 5A and 5B), mixture 30 also includes a deprotection reaction mixture (e.g., a deprotection reaction solution). The deprotection reaction mixture (e.g., a deprotection reaction solution) may include a mixture (e.g., which may include the total volume of liquid in the reactor) that includes a deprotection solution containing a deprotection base (e.g., a deprotection solution containing a deprotection base added to the reactor) and a coupling solution from a preceding coupling step (e.g., the undrained post-coupling solution remaining in the reactor from the preceding (previous) coupling step). In some embodiments, the deprotection reaction mixture (e.g., a deprotection reaction solution) may include additional liquid (e.g., additional solvent added to the reactor).

[0062] In this regard, as discussed in more detail herein, a method for deprotecting a protected amino acid according to the present disclosure can include adding a deprotection solution containing a deprotecting base to a reactor, where the amount of the deprotection solution containing a deprotecting base added to the reactor corresponds to the amount of the deprotection solution containing a deprotecting base (e.g., deprotection reaction solution) in the deprotection reaction mixture.

[0063] Also in this regard, methods for deprotecting protected amino acids according to the present disclosure can include a preceding coupling step (e.g., a coupling step before a step of removing a protecting group). In exemplary embodiments, the reactor can contain various components after completion of the coupling step. The components in the reactor after coupling are generally referred to herein as a post-coupling mixture.

[0064] For example, as understood in the art, the reactor may contain coupling solution after completion of the preceding coupling step. In exemplary embodiments, the methods of the present disclosure do not require a draining step (and / or washing step) after the preceding coupling step and before the next (e.g., consecutive) deprotection step (e.g., the deprotection step of the next SPPS and / or LPPS cycle) to remove (e.g., drain) the coupling solution from the reactor. When there is no draining step between the preceding coupling step and the consecutive deprotection step, the deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor includes the coupling solution remaining in the reactor from the preceding coupling step.

[0065] Thus, the post-coupling mixture may include a post-coupling solution (also referred to herein as a coupling solution). For example, the reactor may contain a coupling solution (e.g., an undrained post-coupling solution) remaining in the reactor from a previous coupling step. The components of a coupling solution (e.g., an undrained post-coupling solution) will be understood by those skilled in the art. For example, the coupling solution (e.g., an undrained post-coupling solution) may include solvent and residual (excess) coupling reagent and / or coupling reagent by-products from the preceding coupling reaction. Examples of residual (excess) coupling reagent and / or by-products thereof that may be present in the coupling solution may include, but are not limited to, residual (excess) activated amino acid, residual (excess) amino acid activator (e.g., DIC, as described herein), residual (excess) amino acid activator additive (e.g., Oxyma, as described herein), other residual (excess) coupling additive, unactivated protected amino acid, and / or by-products thereof. Those skilled in the art will understand the meaning of the terms coupling and / or post-coupling solution as used herein, and their definitions are not necessarily limited to the components described herein.

[0066] As another example, those skilled in the art will also understand that the post-coupling mixture in the reactor may contain solids, such as post-coupling reaction products (e.g., growing peptide chains comprising two or more amino acids coupled to a solid support resin and / or a soluble polymer support) that are not part of the coupling solution (e.g., the undrained post-coupling solution remaining in the reactor from the previous coupling step). However, in some embodiments, the post-coupling mixture may contain post-coupling reaction products in a solution or liquid phase (e.g., peptide chains comprising two or more amino acids coupled to a soluble tag in a solution or liquid phase). Thus, the deprotection reaction mixture (e.g., deprotection reaction solution) in some embodiments may also contain post-coupling reaction products in a liquid or solution phase. Again, those skilled in the art will understand the meaning of the term post-coupling reaction product as used herein, and its definition is not necessarily limited to the components described herein.

[0067] In exemplary embodiments, the disclosed method does not include a draining step between the preceding coupling step and the next deprotection step (e.g., the deprotection step of a subsequent SPPS and / or LPPS cycle) to remove (drain) the coupling solution from the reactor. If there is no draining step between the preceding coupling step and the subsequent deprotection step, the coupling solution remaining in the vessel from the preceding coupling step corresponds to the coupling solution of the deprotection reaction mixture (e.g., the deprotection reaction solution).

[0068] In exemplary embodiments, the disclosed method can include a drainage step between a preceding coupling step and a subsequent deprotection step (e.g., a deprotection step of a subsequent SPPS and / or LPPS cycle). When there is a drainage step between a preceding coupling step and a subsequent deprotection step, a portion of the coupling solution from the preceding coupling step (e.g., less than half, half, substantially all, all, etc.) can be drained from the reactor, and in some embodiments, the deprotection reaction mixture (e.g., deprotection reaction solution) can contain minimal, if any, coupling solution from the preceding coupling step (e.g., undrained post-coupling solution), depending on the amount of post-coupling solution drained from the reactor. When post-coupling solution is drained from the reactor between a preceding coupling step and a subsequent deprotection step, the volume percent of the deprotection base in the reactor can be the same as described in more detail herein (e.g., from 0 to greater than about 5 volume percent, based on the total volume (100 volume percent) of the deprotection reaction mixture (e.g., deprotection reaction solution). In some embodiments, much (e.g., half, substantially all, etc.) of the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution) can substantially correspond to the volume of deprotection solution added to the reactor, either alone or in combination with the volume of additional liquid (e.g., solvent) added to the reactor. For example, in some such embodiments in which substantially all (e.g., all) of the post-coupling solution is drained from the reactor before the next deprotection step, a deprotection solution containing greater than 0 to about 5% by volume of deprotecting agent, and optionally additional solvent, can be added to the drained reactor to provide the deprotection base in the reactor in an amount greater than 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor (in this case, the volume of the deprotection reaction mixture (e.g., deprotection reaction solution) can substantially correspond to the total volume of the deprotection solution and additional solvent, if any, added).As another example, in some embodiments in which substantially all (e.g., all) of the post-coupling solution is drained from the reactor before the subsequent deprotection step, a deprotection solution comprising greater than about 5% by volume of deprotecting agent and additional solvent can be added to the drained reactor to provide an amount of deprotection base in the reactor in an amount of greater than 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor (in this case, the volume of the deprotection reaction mixture (e.g., deprotection reaction solution) can substantially correspond to the total volume of the added deprotection solution and added solvent).

[0069] A method for deprotecting a protected amino acid and / or a protected peptide according to the present disclosure includes removing a protecting group of a protected amino acid and / or a protected peptide in a reactor, such as reactor 4, with a deprotecting base, where the deprotecting base is present in the reactor in an amount of greater than 0 to about 5% by volume, based on the total volume of the deprotection reaction mixture in the reactor (e.g., total volume of the deprotection reaction solution, total volume of liquid) (100% by volume). The deprotecting base reacts with the protected amino acid and / or the protected peptide to remove the protecting group, making the previously protected functional group (e.g., a terminal amine group) available for reaction (e.g., with one, two, or more consecutive amino acids to form a peptide chain).

[0070] The deprotection reaction mixture (e.g., deprotection reaction solution) can include a mixture (e.g., the total volume of liquid in the reactor) that includes a deprotection solution containing a deprotection base (e.g., a deprotection solution containing a deprotection base added to a reactor) and a coupling solution from a previous coupling step (e.g., the undrained post-coupling solution remaining in the reactor from the previous coupling step). In some embodiments, the deprotection reaction mixture (e.g., deprotection reaction solution) can also include additional liquid (e.g., solvent) added to the reactor and / or post-coupling reaction products in a liquid or solution phase. In some embodiments, the volume percent deprotection base of the deprotection reaction mixture (e.g., deprotection reaction solution) can be based on the total volume of liquid in the reactor (e.g., the deprotection reaction mixture or solution can include the total volume of liquid in the reactor). For example, in some embodiments, if the coupling solution from the preceding coupling step is not drained, the vol % deprotection base can be based on the total volume of liquid in the reactor, including the coupling solution from the preceding coupling step (e.g., the volume of the undrained post-coupling solution remaining in the reactor from the preceding coupling step, not including solids, e.g., not including the growing peptide chain attached to the solid support) and / or the volume of post-coupling reaction product that is in the liquid or solution phase, and the volume of the added deprotection solution containing the deprotection base (and the volume of additional solvent, if added to the reactor). In some other embodiments, if the coupling solution from the preceding coupling step is partially or substantially completely drained (e.g., less than half, half, substantially all, or all of the coupling solution is drained), the volume percent deprotection base may be based on the total volume of liquid in the reactor, including the volume of added deprotection solution containing the deprotection base (and the volume of additional solvent, if added to the reactor) and the remaining (e.g., greater than half, half, substantially none, or minimal (residual), or none) volume coupling solution and / or post-coupling reaction product (e.g., from the preceding coupling step) in liquid or solution phase.

[0071] In some embodiments, the deprotecting base may be present in the reactor in an amount of about 1 vol% to about 5 vol%, for example, about 2 vol% to about 5 vol%, for example, about 2 vol% to about 4.5 vol%, for example, about 3 vol% to about 4.5 vol%, or another example, about 3.5 vol% to about 4.5 vol%, based on the total volume of the deprotecting reaction mixture in the reactor (e.g., total volume of the deprotecting reaction solution, total volume of the liquid) (100 vol%). In some embodiments, the deprotecting base may be present in the reactor in an amount of 0 to about 4.5 vol%, for example, greater than about 2 vol% to about 4.5 vol%, based on the total volume of the deprotecting reaction mixture in the reactor (e.g., total volume of the deprotecting reaction solution, total volume of the liquid). In some embodiments, the deprotecting base may be present in the reactor in an amount of 0 to greater than about 4 vol%, for example, about 2 vol% to about 4 vol%, based on the total volume of the deprotecting reaction mixture in the reactor (e.g., total volume of the deprotecting reaction solution, total volume of the liquid). In some embodiments, the deprotecting base can be present in the reactor in an amount of from 0 to about 3.5 vol%, e.g., from about 2 vol% to greater than about 3.5 vol%, based on the total volume of the deprotecting reaction mixture in the reactor (e.g., total volume of the deprotecting reaction solution, total volume of liquid). The amount of deprotecting base can be any value within the ranges described herein, including the endpoints (e.g., any value within the range of from greater than 0 to about 5 vol%) and all subranges within the ranges.

[0072] The deprotection base used in the SPPS and / or LPPS method is typically a liquid at room temperature. Therefore, the deprotection base is typically added to the reactor as part of a deprotection solution containing the deprotection base and a suitable solvent. Alternatively, in some embodiments, the deprotection base may be added to the reactor neat.

[0073] In exemplary embodiments, the disclosed methods include adding a deprotection solution containing a deprotection base (alone or in combination with additional solvent) to a reactor under conditions sufficient to provide a desired amount of deprotection base in the reactor for removing the protecting group of the protected amino acid (e.g., to provide greater than 0 to about 5% by volume of deprotection base in the reactor, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., total volume of deprotection reaction solution, total volume of liquid) in the reactor). In exemplary embodiments, this may include adding a sufficient amount (volume) of the deprotection solution (and additional solvent, if necessary) to the reactor and / or adding a deprotection solution having a sufficient concentration of deprotection base to provide a desired amount (volume) of deprotection base in the reactor available for removing the protecting group of the protected amino acid (e.g., to provide greater than 0 to about 5% by volume of deprotection base in the reactor, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., deprotection reaction solution, liquid) in the reactor). The deprotection solution added to the reactor may contain a higher concentration of deprotection base than the resulting concentration of deprotection base in the reactor after the deprotection solution is added to the reactor.

[0074] As a non-limiting example, after completion of a small-scale coupling reaction, the reactor may contain about 3.5 mL of post-coupling solution (including solvent, residual (excess) activated amino acid, residual (excess) amino acid activator such as DIC, and / or residual (excess) amino acid activator additive such as Oxyma). The deprotection step may be initiated by adding 0.75 mL of 17% v / v pyrrolidine / DMF deprotection solution (0.1275 mL of pyrrolidine) directly to the undrained post-coupling solution to obtain a total volume of 4.25 mL of deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor containing about 3% by volume pyrrolidine based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution).

[0075] The deprotection solution added to the reactor can have a higher concentration of deprotection base compared to the concentration of deprotection base in the deprotection reaction mixture (e.g., deprotection reaction solution) after the deprotection solution is added to the reactor. Thus, a relatively small volume of deprotection solution can be added to the reactor to achieve a desired deprotection base concentration in the deprotection reaction mixture (e.g., deprotection reaction solution).

[0076] In some embodiments, a deprotection solution added to a reactor having a "high" or "higher" concentration of deprotection base, as used herein, can include a deprotection solution containing solvent and about 10% or more by volume of deprotection base, about 15% or more by volume of deprotection base, about 20% or more by volume of deprotection base, about 25% or more by volume of deprotection base, about 30% or more by volume of deprotection base, about 40% or more by volume of deprotection base, and less than 50% by volume of deprotection base, based on the total volume of the deprotection solution. In some embodiments, the deprotection solution added to the reactor can include about 10% to about 40% by volume of deprotection base, for example, about 10% to about 30% by volume, or as another example, about 10% to about 25% by volume of deprotection base, based on the total volume of the deprotection solution. In some embodiments, the deprotection solution added to the reactor may comprise a deprotection base in an amount of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49% by volume, based on the total volume of the deprotection solution (100% by volume). Further, according to some embodiments, the deprotection base may be present in an amount ranging from about any of the amounts recited above to about any other of the amounts recited above. In some other embodiments, the deprotection solution added to a reactor having a "high" or "higher" concentration of deprotection base may comprise a deprotection solution comprising solvent and deprotection base in an amount of about 50, 61, 52, 53, 54, 55, 56, 57, 58, 59, 60, 71, 62, 63, 64, 65, 66, 67, 68, 69, 70, 81, 72, 73, 74, 75, 76, 77, 78, 79, 80, 91, 82, 83, 84, 85, 86, 87, 88, 89, 90, 100, 92, 93, 94, 95, 96, 97, 98, 99, or 100% by volume, based on the total volume of the deprotection solution (51% by volume). Furthermore, according to some embodiments, the deprotection base can be present in an amount from about any of the amounts recited above to about any other of the amounts recited above.

[0077] However, the present disclosure is not limited to the use of a deprotection solution having a relatively high concentration of deprotection base; methods may also include adding a deprotection solution to a reactor having a concentration of deprotection base of less than about 10% by volume (e.g., the deprotection solution added to the reactor can include a solvent and a deprotection base, and the deprotection base is present in an amount of greater than 0% by volume to about 10% by volume, e.g., greater than 0% by volume to about 5% by volume, based on the total volume of the deprotection solution), so long as the amount of deprotection solution added to the reactor is selected to provide a desired amount of deprotection base (e.g., greater than 0% by volume to about 5% by volume) relative to the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution) as defined herein. In some embodiments, the deprotection solution added to the reactor can include about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10% by volume of deprotection base, based on the total volume (100% by volume) of the deprotection solution. Furthermore, according to some embodiments, the deprotection base can be present in an amount from about any of the amounts recited above to any other amount recited above.

[0078] The deprotecting base may be an organic base. In some embodiments, the deprotecting base may have a boiling point of less than or about 107°C. In some embodiments, the deprotecting base may have a boiling point of less than or about 107°C, and / or the difference between the deprotecting reaction temperature as discussed herein and the boiling point of the deprotecting base may be less than or about 50°C, e.g., less than or about 25°C, e.g., less than or about 15°C. For example, the difference between the deprotecting reaction temperature and the boiling point of the deprotecting base may be in the range of about 1°C to about 50°C. For example, the difference between the deprotecting reaction temperature and the boiling point of the deprotecting base may be in the range of about 15°C to about 50°C. For example, the difference between the deprotecting reaction temperature and the boiling point of the deprotecting base may be in the range of about 1°C to about 35°C. As another example, the difference between the deprotecting reaction temperature and the boiling point of the deprotecting base may be in the range of about 1°C to about 25°C. In some embodiments, the deprotecting base can have a boiling point of less than or equal to about 107° C., and / or the difference between the deprotection reaction temperature and the boiling point of the deprotecting base can be about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50° C. The difference between the deprotection reaction temperature and the boiling point of the deprotecting base can be any value within the ranges described herein, including the endpoints (e.g., any value in the range of about 1° C. to about 50° C.), with all subranges therein also disclosed. In some embodiments, the deprotecting base can have a boiling point of less than or equal to about 107° C., and / or the deprotecting step can be carried out at a temperature of at least about 35° C. below the boiling point of the deprotecting base used. Examples of organic bases suitable for use as deprotecting bases can include, but are not limited to, piperidine and / or pyrrolidine. Other organic bases that provide the deprotecting function without interfering with other steps in the process, growing peptide chain, or system can be suitable as well.

[0079] Examples of solvents that may be part of the deprotection solution (and / or may be added separately to the reactor) may include, but are not limited to, dimethylformamide (DMF), dimethylacetamide (DMA), N-methylpyrrolidinone (NMP), native and / or non-liprotoxin solvents, etc., and combinations and / or mixtures thereof. Examples of native and / or non-liprotoxin solvents may include, but are not limited to, N-formylmorpholine (NFM), N-butylpyrrolidinone (NBP), alkoxybenzene-based solvents (e.g., dimethoxybenzene-based solvents such as anisole, 1,3-dimethoxybenzene, etc.), etc., and combinations and / or mixtures thereof.

[0080] Referring again to FIGS. 1A and 1B (and FIGS. 5A and 5B), mixture 30 typically initially resides in the lower interior of the reactor. At least a portion of the deprotection base present in mixture 30 evaporates during the removal step (e.g., at least a portion of the deprotection base evaporates into the upper interior of the reactor during the removal step). For example, the deprotection base may have a boiling point lower than the deprotection reaction temperature / reactor temperature during deprotection, and / or a boiling point lower than the boiling point of a solvent that may be present in the reactor (e.g., a solvent (e.g., a deprotection reaction solution) present in the deprotection reaction mixture), such as dimethylformamide (DMF) and N-methylpyrrolidinone (NMP). Thus, the deprotection base may volatilize or evaporate during the deprotection method.

[0081] In some embodiments, the deprotection step (e.g., deprotection reaction step) can be carried out without heat (e.g., at room temperature), as long as the deprotection conditions (type of base, time, etc.) are selected to facilitate evaporation of the deprotection base (e.g., into the headspace of the reactor).

[0082] More typically, in some embodiments, the deprotection step / method may be carried out with heat (e.g., the deprotection step / method may include heating the protected amino acid and / or protected peptide and / or deprotecting base (e.g., heating a deprotection solution containing the deprotecting base) and / or other liquids (e.g., additional solvent, remaining coupling solution, etc.) and / or reactor, etc.). The protected amino acid and / or protected peptide and / or deprotecting base and / or other liquids and / or reactor, etc. may be heated before and / or during the step of removing protecting groups from the protected amino acid and / or protected peptide. For example, the method may include heating the protected amino acid and / or protected peptide and / or deprotecting base and / or other liquids, e.g., additional solvent, before and / or during delivery to reactor 4, and / or heating the protected amino acid and / or protected peptide and / or deprotecting base and / or other liquids in reactor 4 before and / or during the deprotection reaction (e.g., by heating the reactor before and / or during the step of removing protecting groups from the protected amino acid and / or protected peptide). Heating during solid phase peptide synthesis can be useful, for example, to accelerate the rate of deprotection and thereby reduce the time required to synthesize the peptide.

[0083] The heating temperature of the heating step may vary. In some embodiments, the heating step (e.g., deprotection step / heating during the method) may be performed at a temperature of about 40°C to about 120°C, e.g., about 50°C to about 120°C, for example, about 60°C to about 120°C, for example, about 70°C to about 120°C, for example, about 80°C to about 120°C, for example, about 90°C to about 120°C, for example, about 80°C to about 110°C, or for example, about 90°C to about 110°C, but is not limited thereto. In certain embodiments, the heating step (e.g., deprotection step / heating during the method) may be performed at a temperature of about 60°C to about 120°C, e.g., about 90°C to about 120°C, for example, about 90°C to about 110°C, but is not limited thereto. The temperature may be any value within the ranges described herein, including the endpoints (e.g., any value within the range of about 40°C to about 120°C), and all subranges within the ranges are also disclosed.

[0084] 1A and 1B (and FIGS. 5A and 5B) schematically illustrate a heating process in which a heat source 40 heats reactor 4 and mixture 30. In certain embodiments, heat source 40 includes a microwave source 42 positioned to direct microwave radiation 44 through a waveguide 46 attached to a microwave cavity (not shown) containing reactor 4. Microwave power can be adjusted as known in the art to provide a reaction temperature and / or reaction time (e.g., without limitation, about 40 seconds to about 8 minutes to provide a deprotection temperature as described herein, and, as another non-limiting example, to provide a deprotection reaction time in the range of about 10 seconds to about 15 minutes).

[0085] In embodiments utilizing microwave energy to heat the reactants, reactor 4 may be formed from a material transparent to microwave radiation, such as, but not limited to, glass, Teflon, and / or polypropylene.

[0086] Microwave sources are well known in the art and can include, for example, magnetrons, klystrons, and / or solid-state diodes. Microwave sources, waveguides, and microwave cavities suitable for solid-phase peptide synthesis methods and systems are well known in the art and commercially available (e.g., systems commercially available from CEM Corporation, as described herein). Thus, one of ordinary skill in the art will know how to use the same in solid-phase peptide synthesis methods and systems without undue experimentation.

[0087] However, the present disclosure is not limited to the use of a microwave source as the heat source, and other types of heat sources known in the art for solid phase peptide synthesis can be used.

[0088] Despite the benefits of heating, high temperatures during the deprotection step can present various challenges for peptide synthesis.

[0089] For example, the organic amine used in the deprotection reaction may have a relatively low boiling point compared to the boiling point of the solvent used in the deprotection reaction and / or the temperature of the deprotection step. Piperidine has a boiling point of about 106°C, and pyrrolidine has a boiling point of about 87°C. In contrast, the solvent dimethylformamide (DMF) has a boiling point of about 153°C, and the solvent N-methylpyrrolidinone (NMP) has a boiling point of about 200°C. Also in contrast, as described herein, the deprotection reaction may be carried out at elevated temperatures, such as, but not limited to, up to about 120°C, e.g., from about 90°C to about 120°C, or as another example, from about 90°C to about 110°C.

[0090] The reactor may exhibit a temperature continuum during processing, with the upper portion being at a lower temperature than the lower portion. Because the deprotection base may have a boiling point lower than the boiling point of other components present in the reactor, such as the solvent, and / or lower than the reaction temperature, the deprotection base may volatilize (evaporate) to the upper portion (e.g., headspace) of the reactor and then condense on the upper portion of the reactor wall and / or the upper wall of the reactor. The rate / amount of volatilization (evaporation) may also increase, for example, when the reactants are bubbled (e.g., with an inert gas such as nitrogen) during deprotection to help mix the reactants.

[0091] Volatilization of the deprotection base can be particularly problematic when using pyrrolidine. Pyrrolidine may be desirable as a deprotection base because it can provide faster deprotection than piperidine. As a five-membered ring (vs. a six-membered piperidine ring), the carbon atom of pyrrolidine bends back more from the nitrogen atom, facilitating easier attack for deprotection. However, because pyrrolidine has a lower boiling point than piperidine, significant evaporation and subsequent condensation can occur during the deprotection process, thereby limiting its use, notably in the synthesis of long peptides.

[0092] In the method of the present disclosure, the heating step can volatilize (evaporate) the deprotection base (e.g., pyrrolidine) in the deprotection reaction mixture (e.g., deprotection reaction solution) upward from the bottom of reactor 4 to the top of reactor 4 (e.g., into the headspace above mixture 30).

[0093] Residual deprotecting base remaining in the reactor during subsequent solid-phase peptide synthesis steps (e.g., subsequent coupling steps) (e.g., residual deprotecting base condensed on the top of the reactor walls and / or on the upper wall of the reactor) can be problematic. Residual deprotecting base can, for example, prematurely remove the protecting group from an amino acid that is already coupled to the deprotected amino acid. This can result in undesired insertion into the peptide chain. Residual deprotecting base can also reduce the activated amino acid by reacting with the amino acid, resulting in deletion of the peptide chain.

[0094] Thus, conventional SPPS methods required a washing step after deprotection and before coupling (e.g., to help remove residual deprotected base to minimize or prevent its participation in subsequent solid phase peptide synthesis steps, such as the subsequent coupling step).

[0095] In contrast to conventional SPPS methods, the disclosed methods can eliminate washing steps between the deprotection and coupling steps and / or help reduce the amount of solvent required for washing steps between the deprotection and coupling steps of the SPPS method.

[0096] Furthermore, as is known in the art, conventional LPPS methods require an extraction step after deprotection and before coupling. The extraction step involves adding a suitable extraction solvent, such as water, to a reactor containing the peptide product (e.g., growing peptide chain) and residual deprotected base. This results in the formation of separate layers, including an organic layer containing the peptide product and an aqueous layer containing waste products, such as residual deprotected base. The waste layer (e.g., aqueous layer containing residual base) can be drained from the reactor using techniques known in the art, while maintaining the organic product layer containing the growing peptide chain in the reactor. The extraction method is used to separate the peptide product from waste products, such as residual deprotected base, to minimize or prevent base involvement in subsequent liquid-phase peptide synthesis steps, such as the subsequent coupling step.

[0097] Nevertheless, conventional LPPS methods typically require several extraction steps and / or significant amounts of extraction solvent to remove residual base, and even then, the remaining organic layer containing the peptide product may be contaminated with waste products (e.g., may contain more deprotected base than desired, etc.).

[0098] In contrast to conventional LPPS methods, in some embodiments, the LPPS methods of the present disclosure can help reduce the volume (amount) of extraction solvent required after deprotection and before the next sequential coupling step, and / or can help remove a significant portion of the residual deprotected base remaining from the reactor after deprotection is complete.

[0099] To help eliminate a wash step between the deprotection step and the coupling step, and / or to help reduce the amount of solvent required for the wash step between the deprotection step and the coupling step in an SPPS method, and / or to help reduce the amount of solvent used in the extraction step after deprotection and before the next consecutive coupling step in an LPPS method, the disclosed methods use small amounts of deprotection base as described herein, and / or direct an inert gas through the interior of the reactor (e.g., directing an inert gas through the upper interior or headspace of the reactor) during the deprotection step (e.g., to help flush, evacuate, vent, displace, replace, purge, etc.) to help remove evaporated (volatilized) deprotection base from the interior of the reactor (e.g., from the headspace of the reactor) (e.g., continuously and / or intermittently).

[0100] The methods of the present disclosure may also generally facilitate the production of peptides with acceptable purity levels for downstream applications.

[0101] In some embodiments, directing the inert gas through the interior of the reactor can include directing (introducing, feeding, flowing, etc.) the inert gas into the upper interior of the reactor through one or more openings (inlet ports) located at the top of the reactor such that the inert gas flows through the upper interior of the reactor and exits the upper interior of the reactor through one or more other openings (outlet ports) located at the top of the reactor. In this manner, the inert gas can flow through the upper interior of the reactor containing the evaporated deprotected base (e.g., through a headspace) and aid in removing (e.g., flushing, evacuating, venting, replacing, replacing, purging, etc.) the evaporated deprotected base from the upper interior of the reactor (e.g., from the headspace above mixture 30) through one or more other openings (outlet ports) located at the top of the reactor.

[0102] In some embodiments, directing the inert gas through the interior of the reactor can include directing (introducing, feeding, flowing, etc.) the inert gas into the lower interior of the reactor through one or more openings (inlet ports) located at the bottom of the reactor, whereby the inert gas flows upward from the lower interior of the reactor into / through the upper interior of the reactor and out of the upper interior of the reactor through one or more other openings (outlet ports) located at the top of the reactor. In this manner, the inert gas can flow upward from the lower interior of the reactor (e.g., through a vascular headspace) into / through the upper interior of the reactor containing the evaporated deprotected base and aid in removing (e.g., flushing, evacuating, venting, replacing, replacing, purging, etc.) the evaporated deprotected base from the upper interior of the reactor (e.g., from the headspace above mixture 30) through one or more other openings (outlet ports) located at the top of the reactor.

[0103] In some embodiments, the step of directing the inert gas through the interior of the reactor can include directing (directing, feeding, flowing, etc.) the inert gas into both the upper and lower interiors of the reactor through one or more openings (inlet ports) located at the top and bottom of the reactor, respectively, whereby the inert gas flows through the upper interior of the reactor (including flowing from the lower interior of the reactor to / upward through the upper interior of the reactor, as appropriate) and exits the upper interior of the reactor through one or more other openings (outlet ports) located at the top of the reactor. In this manner, the inert gas may flow through the upper interior of the reactor containing the evaporated deprotected base (e.g., through the headspace), and optionally flow upwardly from the lower interior of the reactor to / through the upper interior of the reactor containing the evaporated deprotected base (e.g., through the headspace), to aid in removing (e.g., flushing, evacuating, venting, replacing, replacing, purging, etc.) the evaporated (volatilized) deprotected base from the upper interior of the reactor (e.g., from the headspace above mixture 30) through one or more other openings (exit ports) located at the top of the reactor.

[0104] In some embodiments, the inert gas can be continuously directed through the reactor as a continuous flow. In some embodiments, the inert gas can be directed through the reactor as an intermittent (e.g., pulsed) flow.

[0105] In some embodiments, the inert gas introduced into and / or flowing through the reactor can have a pressure of about 1 psi to about 25 psi. In some embodiments, the inert gas introduced into and / or flowing through the reactor (e.g., flowing through the reactor headspace containing the volatilized deprotected base; and / or bubbling through mixture 30 and / or flowing upward through mixture 30 in the reactor) can have a pressure of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 psi. In some embodiments, the inert gas introduced into and / or flowing through the reactor can have a pressure ranging from about any of the aforementioned pressure values ​​to about any other of the aforementioned pressure values. The pressure of the inert gas, including the inert gas introduced into and / or flowing through the upper interior of the reactor and / or introduced into and / or flowing through the lower interior of the reactor (including introduced into and / or flowing through mixture 30, e.g., bubbling through the mixture and / or flowing generally upward into the upper interior of the reactor), can be any value within the ranges described herein, including the endpoints and all subranges therein (e.g., any value in the range of about 1 to about 25 psi).

[0106] In some embodiments, the pressure of the inert gas introduced into and / or flowing through the reactor may be greater than about 25 psi. As a non-limiting example, in some large-scale production methods (e.g., large-scale microwave peptide synthesizer production methods) including a deprotection step according to the present disclosure (e.g., including one or more deprotection steps using a deprotection base in an amount of greater than 0 to about 5% by volume, based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), as described herein, and / or using an inert gas to flush volatilized deprotection base from the reactor (e.g., from the headspace inside the reactor), also described in more detail herein), the inert gas can have a pressure of from about 1 psi to about 100 psi, for example, greater than 25 psi or about 100 psi, for example, from about 50 psi to about 95 psi, or as another example, from about 75 psi to about 95 psi. In some embodiments, large-scale manufacturing methods including a deprotection step according to the present disclosure may use a reactor having a size (e.g., internal volume) of 3 liters or more (e.g., 3 liters, 8 liters, 10 liters, 15 liters, etc., up to 40 liters or more), and / or may have a synthesis scale of about 25 mmol or more (e.g., about 25 mmol or more, about 50 mmol or more, about 100 mmol or more, about 200 mmol or more, about 250 mmol or more, about 500 mmol or more, etc.), and / or may provide peptide amounts per batch of up to about 500 grams or more (e.g., about 500 grams or more, about 1 kg or more, etc.), and / or may have a variety of deprotection cycle times (e.g., from about 8 minutes to about 15 minutes, about 10 minutes, etc.).

[0107] In some embodiments where the inert gas is introduced into the reactor through both a first opening located at the top of the reactor (the top opening) and a second opening located at the bottom of the reactor (the bottom opening), the inert gas flowing into and / or through the top opening and / or upper interior of the reactor may have a higher psi than the inert gas flowing into and / or through the bottom opening and / or lower interior of the reactor.

[0108] The inert gas may also be directed through the interior of the reactor (e.g., through the upper interior or headspace) during the deprotection step at a flow rate based on the time rate at which the inert gas substantially replaces the headspace gas volume. More specifically, the inert gas flow rate may be an amount (volume) of inert gas that allows for the substantial replacement of the volume of gas in the headspace region of the reactor with the inert gas (e.g., the substantial replacement of the volume of volatilized deprotected base in the headspace region of the reactor) within a selected period (time rate). For example, the inert gas flow rate may be an amount (volume) of inert gas that allows for the substantial replacement of the volume of gas in the headspace region of the reactor (e.g., the volume of volatilized deprotected base in the headspace region) every about 1 to 20 (20) seconds, e.g., about 5 to 10 (10) seconds. Those skilled in the art will know how to determine and calculate the appropriate inert gas flow rate to replace (displace) the volume of headspace gas (volatilized deprotected base) in the reactor within a time frame (time rate) without undue experimentation.

[0109] Without wishing to be bound by any explanation or theory, it is currently believed that directing an inert gas source into and / or through the headspace during deprotection can induce a high air exchange rate in the gas above the deprotection reaction mixture (e.g., the deprotection reaction solution) and / or other reactants, products, etc., within the lower interior of the reactor (e.g., in the headspace gas containing the volatilized deprotection base), allowing the inert gas to displace the volatilized deprotection base from the reactor. This can shorten the residence time of the volatilized deprotection base within the reactor, allowing the volatilized deprotection base to be removed more quickly with less condensation on the sidewalls and / or top wall of the vessel. This, in turn, can help reduce the amount of residual deprotection base remaining in the reactor after the deprotection step is complete. The inert gas can also exert a downward force on droplets (e.g., condensed deprotection base) on the sidewall of reactor 4, thereby blowing the droplets toward the mixture 30 at the bottom of reactor 4.

[0110] Because the deprotection method uses a small amount of deprotection base (less than or about 5% by volume of the deprotection base (e.g., deprotection reaction solution) based on the total volume of the deprotection reaction mixture), the deprotection base (e.g., pyrrolidine) can be essentially completely removed from the reactor upon completion of the deprotection step. For example, without being bound by any theory or explanation, it is presently believed that in such embodiments, the deprotection base can be substantially completely evaporated during the heating step, and / or the volatilized deprotection base can be substantially completely removed from the headspace using inert gas flushing, each as described herein. Also, without being bound by any theory or explanation, it is presently believed that in such embodiments, the residual amount of deprotection base remaining after completion of the deprotection step is small enough to minimize problems associated with the presence of residual deprotection base in the subsequent coupling step, even without a post-deprotection step washing step and / or even with the use of reduced amounts of washing liquid (e.g., solvent) in the post-deprotection step washing step and / or the use of reduced amounts of extraction solvent in the post-deprotection step in LPPS methods, as described in more detail herein.

[0111] Thus, in exemplary embodiments, the deprotection methods described herein can remove a substantial portion of the deprotection base used in the deprotection step from the reactor (e.g., by evaporation). As used herein, a "substantial portion of the deprotection base" can include, but is not limited to, at least a majority (e.g., more than half), at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 99%, or more of the deprotection base used in the deprotection step from the reactor. For example, in some embodiments, the deprotection methods described herein can remove at least about 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% of the deprotection base used in the deprotection step from the reactor. Furthermore, according to some embodiments, the amount of deprotection base removed from the reactor can be from about any of the amounts recited above to about any other of the amounts recited above.

[0112] In contrast to conventional approaches, the disclosed methods may also facilitate the production of peptides with acceptable purity levels for downstream applications, since the amount of residual deprotected base can be reduced.

[0113] The inert gas can be nitrogen. The present disclosure is not limited to the use of nitrogen as the inert gas, and other inert gases, such as noble gases, can be used that do not chemically interfere with solid phase peptide synthesis reactions and systems.

[0114] In certain embodiments, as shown in FIG. 1A (and FIG. 5A ), a method can include providing pressurized inert gas from an inert gas source (such as the inert gas source designated as 100 in FIGS. 2A and 6A ) through flow path 20 and directing the pressurized inert gas (e.g., directing the pressurized inert gas generally downward) through opening 10 to an upper portion of the interior of reactor 4 and into a headspace above mixture 30 (comprising a deprotection reaction mixture (e.g., a deprotection reaction solution) and a protected amino acid) disposed within the lower interior of the reactor. As the pressurized inert gas flows from the upper portion of the interior of reactor 4 through the headspace, the inert gas purges (e.g., removes, flushes, replaces, exchanges, vents, vents, etc.) the volatilized deprotected base from the interior of reactor 4 through opening 14 and into flow path 24. In this manner, the pressurized inert gas substantially displaces the volatilized deprotected base from the headspace of the reactor. This can reduce residence time and minimize condensation of the deprotected base on the walls of the reactor.

[0115] Gas flows (movements) within reactor 4 include an upward flow of volatilized deprotected base (e.g., pyrrolidine) from mixture 30 in the lower interior of reactor 4 to the headspace above the mixture (e.g., into the upper interior of reactor 4), a downward flow of inert gas into the headspace (upper interior portion) of reactor 4, and purging (e.g., removing, flushing, displacing, venting, venting, replacing, etc.) of volatilized deprotected base and inert gas from the headspace (upper interior portion) of reactor 4, as shown schematically by arrows in FIG. 1A (and FIG. 5A).

[0116] In certain embodiments, as shown in FIG. 1B (and FIG. 5B ), the method can include supplying (e.g., directing) pressurized inert gas from an inert gas source (such as the inert gas source designated as 100 in FIGS. 2B and 6B ) through a flow path 218, an opening 208, an inner interior space of a spray head 220, and an exit opening 222 into the outer interior space 7 of the reactor 4 (e.g., into the headspace above the mixture 30). FIG. 1B (and FIG. 5B ) also shows an embodiment in which the spray head 220 directs (e.g., sprays) the inert gas through the opening 222 at an angle (e.g., a spray pattern) indicated generally by dashed line 224 toward the sidewall 6 of the reactor 4. This can promote a cleaning effect, allowing the inert gas to contact the sidewall and "wash" condensed deprotected base toward the lower reactants inside the reactor 4.

[0117] As the pressurized inert gas flows through the headspace of reactor 4, it purges (e.g., removes, flushes, displaces, replaces, vents, vents, etc.) the volatilized deprotected base from reactor 4 through opening 206 and into flow path 216. Again, the pressurized inert gas effectively displaces the volatilized deprotected base from the headspace of the reactor, thereby reducing residence time and minimizing condensation of the deprotected base on the walls of the reactor.

[0118] FIG. 1B (and FIG. 5B) schematically illustrates gas flow (movement) within reactor 4 with arrows and dashed lines, including flow of volatilized deprotected base (e.g., pyrrolidine) from mixture 30 in the lower interior of reactor 4 into the headspace above the mixture (e.g., into the upper interior of reactor 4), flow of inert gas from spray head 220 through openings 222 into the headspace (e.g., the upper interior portion) of the reactor (e.g., inclined flow toward sidewall 6), and purging (e.g., flushing, replacing, venting, removing, venting, etc.) of the volatilized deprotected base and inert gas from the headspace (e.g., the upper interior) of reactor 4.

[0119] In certain embodiments, as discussed herein, the method can include introducing (e.g., directing) an inert gas into a lower interior of reactor 4 in addition to, or instead of, introducing (e.g., directing) an inert gas into the upper interior (e.g., into the headspace) of the reactor. For example, with reference to FIGS. 1A and 1B (and FIGS. 5A and 5B ), the method can include directing pressurized inert gas from an inert gas source (which, if present, may be the same or different from the inert gas source introduced into the upper interior of the reactor) through flow path 26 and through opening 16 into the lower interior of reactor 4. The pressurized inert gas can flow from the lower interior of the reactor (e.g., generally upwardly through mixture 30) to / through the upper interior (e.g., headspace) of reactor 4 containing the volatilized (evaporated) deprotected base. As the inert gas flows upward (e.g., through the headspace), it can purge (e.g., flush, displace, replace, vent, vent, remove, etc.) volatilized deprotected base from the upper interior (e.g., headspace) of reactor 4 through opening 14 and into flow path 24. Again, in this manner, the pressurized inert gas can displace volatilized deprotected base from the headspace of the reactor, thereby reducing residence time and minimizing condensation of deprotected base on the walls of the reactor.

[0120] The pressurized inert gas introduced into the lower interior of the reactor may additionally or alternatively agitate (mix, bubbling, etc.) the mixture 30 .

[0121] In some embodiments, the method can include introducing (directing) both a first pressurized inert gas into the upper interior (e.g., headspace) of the reactor containing the vaporized deprotected base and a second pressurized inert gas into the lower interior of the reactor. As a non-limiting example, with reference to FIGS. 1A and 1B (and 5A and 5B), the method can include directing the first pressurized inert gas into the upper interior of reactor 4 through a first opening disposed in the upper part of the reactor, such as opening 10 in FIG. 1A (and 5A) or openings 208 and 222 in FIG. 1B (and 5B), and directing the second pressurized inert gas into the lower interior of reactor 4 through a second opening disposed in the lower part of the reactor, such as opening 16 in FIGS. 1A and 1B (and 5A and 5B). The first pressurized inert gas can flow through the upper interior of the reactor containing the vaporized deprotected base (e.g., through the headspace). The second pressurized inert gas may flow through mixture 30 to agitate (stir, bubble, etc.) mixture 30 and / or may flow generally upward from the lower interior of the reactor through mixture 30 to / through the upper interior (e.g., headspace) of the reactor containing the vaporized deprotected base. The first pressurized inert gas and optionally the second pressurized inert gas may purge (e.g., flush, replace, exchange, vent, evacuate, remove, etc.) the vaporized deprotected base from the upper interior (e.g., from the headspace) of the reactor through a third opening located at the top of the reactor, such as opening 14 or 206 in Figures 1A and 1B (and Figures 5A and 5B).

[0122] A first inert gas (also referred to herein as an overhead inert gas) introduced into and / or flowing through the upper interior of reactor 4 (e.g., the headspace above mixture 30) (e.g., a first inert gas introduced through a first opening located at the top of the reactor, such as opening 10 in FIG. 1A (and FIG. 5A), or openings 208 and 222 in FIG. 1B (and FIG. 5B)) can have a higher pressure than a second inert gas introduced into and / or flowing through the lower interior of reactor 4 (upward, if necessary, to the headspace). As a non-limiting example, the first (overhead) inert gas introduced into and / or flowing through the upper interior of reactor 4 (e.g., flowing through the headspace) can have a pressure of from about 1 psi to about 25 psi. In some embodiments, the first (overhead) inert gas introduced into and / or flowing through the upper interior of reactor 4 can have a pressure of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 psi. Furthermore, according to some embodiments, the first (overhead) inert gas introduced into and / or flowing through the upper interior of reactor 4 can have a pressure ranging from about any of the above pressure values ​​to any other of the above pressure values.

[0123] As a non-limiting example, the second inert gas introduced into and / or flowing through the lower interior of reactor 4 (and optionally toward the headspace) can have a pressure that is less than the pressure of the first (overhead) inert gas introduced into and / or flowing through the upper interior of reactor 4. For example, the second inert gas may have a pressure of from about 1 psi to about 25 psi, so long as the pressure of the second inert gas is less than the pressure of the first inert gas. In embodiments in which inert gas is introduced only into the lower interior of the reactor (no inert gas is introduced into the upper interior of the reactor), the inert gas may also have a pressure of from about 1 psi to about 25 psi.

[0124] In a non-limiting example, a first (overhead) inert gas introduced into and / or flowing through the upper interior (e.g., headspace) of reactor 4 can have a pressure of about 15 psi, and a second inert gas introduced into and / or flowing through the lower part of reactor 4 (optionally upward into the headspace) can have a pressure that is less than the pressure of the first (overhead) inert gas introduced into and / or flowing through the upper part of reactor 4, such as a pressure of about 5 psi.

[0125] Thus, this method may allow for the use of deprotecting bases with relatively low boiling points at higher temperatures to accelerate reaction times while minimizing (reducing) the adverse effects associated with the use of low-boiling, easily volatile reactants.

[0126] After deprotection is complete, the inert gas flow may be stopped and the coupling step may be carried out using methods known in the art.

[0127] The present disclosure also relates to solid-phase and / or liquid-phase peptide synthesis methods that include one or more deprotection steps as described in more detail herein (e.g., using about 5% by volume or less of the deprotection base (e.g., the deprotection reaction solution) based on the total volume of the deprotection reaction mixture, and / or using an inert gas to flush the volatilized deprotection base from the reactor (e.g., from the headspace inside the reactor), as described in more detail herein). The solid-phase and / or liquid-phase peptide synthesis methods of the present disclosure may further include one or more coupling steps (e.g., may include one or more deprotection coupling cycles). Coupling steps of solid-phase and / or liquid-phase peptide synthesis and systems for carrying them out are generally known in the art and therefore will not be described in detail herein.

[0128] Conventional SPPS methods require multiple washing steps between the deprotection step and the coupling step (e.g., after deprotection and before coupling) to remove residual deprotection base. In some embodiments of the present disclosure (e.g., SPPS methods including a deprotection step described herein), a washing solution (e.g., a solvent such as, but not limited to, dimethylformamide (DMF), methanol, and / or isopropanol) can be added to the reactor for the post-deprotection washing step. The washing step can include a single washing step, or repeated washing steps (e.g., repeated 2, 3, 4, 5, etc. times).

[0129] However, washing steps may require the use of large amounts of solvent, which may require solvent recovery and disposal, etc. This may increase material costs and peptide synthesis time, reduce efficiency, etc. Furthermore, multiple washing steps may be less effective in preventing undesired reactions and reducing impurities, especially as peptide length increases, making it difficult to synthesize peptides of acceptable purity for downstream applications.

[0130] In contrast to conventional methods, in some embodiments, the present disclosure relates to an SPPS method comprising a deprotection step followed by a coupling step, wherein the SPPS method does not include a washing step after the deprotection step and before the associated coupling step. In other words, the method of the present disclosure can eliminate one or more washing steps (e.g., all washing steps) between a deprotection step and its associated coupling step (i.e., the coupling step immediately following the deprotection step). This can provide advantages such as improved process efficiency, energy savings, reduced amounts of solvents required in the SPPS method, reduced material costs, and reduced solvent disposal issues.

[0131] For example, the SPPS method of the present disclosure can include a series of deprotection-coupling cycles, wherein one or more (e.g., all) washing steps are eliminated (e.g., there are no washing steps) between the deprotection step and the coupling step of at least one of the deprotection-coupling cycles of the SPPS method. In other examples, the SPPS method of the present disclosure can include a series of deprotection-coupling cycles, wherein one or more (e.g., all) washing steps are eliminated (e.g., there are no washing steps) between the deprotection step and the coupling step of more than one of the deprotection-coupling cycles of the SPPS method, for example, for half of the deprotection-coupling cycles, for example, for a majority of the deprotection-coupling cycles, and as another example, for all of the deprotection-coupling cycles.

[0132] In yet other embodiments, the present disclosure relates to an SPPS method comprising a deprotection step followed by a coupling step, wherein the SPPS method includes one or more washing steps (e.g., 1, 2, 3, 4, 5, etc. washing steps) using a washing composition (e.g., a solvent) after the deprotection step and before the associated coupling step. However, in contrast to conventional washing steps, the washing step(s) of this embodiment may use reduced amounts of solvent compared to conventional SPPS methods.

[0133] In some embodiments, the washing step after deprotection and before coupling can include washing the interior of the reactor one or more times (e.g., 1, 2, 3, 4, 5, etc.) with a washing composition (e.g., solvent) in an amount (volume) that is about the same as the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution). Additionally, the SPPS method can include a series of deprotection-coupling cycles, where one or more deprotection-coupling cycles (e.g., half, most, or all) include one or more washing steps (e.g., 1, 2, 3, 4, 5, etc. washing steps) between the deprotection and coupling steps, and the washing steps use an amount (volume) of a washing composition (e.g., solvent) in an amount (volume) that is about the same as the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution).

[0134] In some embodiments, the washing step after deprotection and before coupling can include washing the inside of the reactor with a washing composition (e.g., a solvent) in an amount (volume) less than the total volume of the deprotection reaction mixture (e.g., a deprotection reaction solution). For example, the washing step can include washing the inside of the reactor one or more times (e.g., once, twice, three times, four times, five times, etc.) with a washing composition (e.g., a solvent) in an amount (volume) less than or about half the total volume of the deprotection reaction mixture (e.g., a deprotection reaction solution). As another non-limiting example, the method can include washing the inside of the reactor one or more times (e.g., once, twice, three times, four times, five times, etc.) with a washing composition (e.g., a solvent) in an amount (volume) less than or about one-third the total volume of the deprotection reaction mixture (e.g., a deprotection reaction solution). In some embodiments, the method can include washing the interior of the reactor one or more times (e.g., once, twice, three times, four times, five times, etc.) with a cleaning composition (e.g., solvent) in an amount (total volume) that is two or less times the bed volume of the resin (e.g., a solid support resin described herein present in the reactor) present in the reactor, e.g., one or less times the bed volume of the resin present in the reactor. One skilled in the art will understand that the term "bed volume" refers to the area of ​​the reactor that is occupied (taken up) by the resin (e.g., a solid support resin described herein present in the reactor), and that a total volume of solvent that is two or less times the bed volume of the resin present in the reactor, e.g., one or less times the bed volume of the resin present in the reactor, refers to the volume of liquid (solvent) that fills this same area (e.g., two or less times the bed volume of the resin present in the reactor, e.g., one or less times the bed volume of the resin present in the reactor).Furthermore, the SPPS method can include a series of deprotection-coupling cycles, wherein one or more deprotection-coupling cycles (e.g., half, most, or all) include one or more washing steps (e.g., 1, 2, 3, 4, 5, etc. washing steps) between the deprotection step and the coupling step, and the washing steps use an amount (volume) of a washing composition (e.g., a solvent) that is less than the total amount (total volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) (e.g., less than or about 1 / 2 of the total volume of the deprotection reaction mixture; as another example, less than or about 1 / 3 of the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution); and as another example, an amount (total volume) of two or less times the bed volume of the resin present in the reactor, for example, one or less times the bed volume of the resin present in the reactor.

[0135] If used, the cleaning composition (cleaning solution) may include a solvent such as, but not limited to, dimethylformamide (DMF), methanol, and / or isopropanol.

[0136] Conventional LPPS methods require one or more extraction steps between the deprotection step and the coupling step (e.g., after deprotection and before coupling) to remove residual deprotection base. In some embodiments of the present disclosure (e.g., LPPS methods including a deprotection step described herein), an extraction solvent (e.g., an aqueous solvent) can be added to the reactor for one or more extraction steps after deprotection. The extraction step may include a single extraction step, or the extraction step may be performed repeatedly (e.g., two, three, four, five, etc. times).

[0137] However, extraction steps may require the use of large amounts of solvent, which may require solvent recovery and disposal, etc. This may increase material costs and peptide synthesis time, reduce efficiency, etc. Furthermore, multiple extraction steps may be less effective in preventing undesired reactions and reducing impurities, especially as peptide length increases, making it difficult to synthesize peptides of acceptable purity for downstream applications.

[0138] An exemplary embodiment of the present disclosure relates to an LPPS method comprising a deprotection step followed by a coupling step, wherein the LPPS method comprises one or more extraction steps (e.g., extraction steps 1, 2, 3, 4, 5, etc.) using an extraction solvent (e.g., water) after the deprotection step and before the associated (next) coupling step. The extraction step according to the disclosed method comprises adding a suitable extraction solvent, such as water, to a reactor after the deprotection step and before the associated (next) coupling step. The reactor contains a peptide product (e.g., a growing peptide chain) and residual deprotected base. Upon addition of the extraction solvent to the reactor (and optional shaking of the vessel), separate layers are formed, including an organic layer containing the peptide product and an aqueous layer containing waste products, such as residual deprotected base. The waste layer (e.g., an aqueous layer containing residual base) can be formed and drained from the reactor using techniques known in the art, while maintaining the organic product layer containing the growing peptide chain in the reactor. However, in contrast to conventional extraction processes, the extraction step of this embodiment can use a reduced amount of solvent compared to conventional LPPS methods.

[0139] In some embodiments, the LPPS method includes one or more extraction steps (e.g., 1, 2, 3, 4, 5, etc. extraction steps) after the deprotection step and before the next consecutive coupling step, and the total combined volume of extraction solvent used in all extraction steps associated with a deprotection coupling cycle (total volume of extraction solvent for all extraction steps in a single deprotection coupling cycle) is 2 times (2×) or less than the total volume (total volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) for the deprotection step of that deprotection coupling cycle. Additionally, the LPPS method can include a series of deprotection-coupling cycles, wherein one or more deprotection-coupling cycles (e.g., half, most, or all) include an extraction step (e.g., extraction step 1, 2, 3, 4, 5, etc.) after the deprotection step and before the next consecutive coupling step, and the total volume of extraction solvent used in all extraction steps associated with a deprotection-coupling cycle (the total combined volume of extraction solvent for all extraction steps in a single deprotection-coupling cycle) is two times (2×) or less than the total volume (total volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) for the deprotection step of that deprotection-coupling cycle.

[0140] When a washing and / or extraction step is used, in some embodiments, the washing solution (e.g., solvent) and / or extraction solvent (e.g., aqueous solvent) can be introduced into the reactor through a suitable opening into the upper interior of the reactor, such as opening 10 in FIG. 1A (and FIG. 5A), and / or using a different or the same spray head (e.g., spray head 220 in FIGS. 1B and 5B) used to introduce inert gas into the reactor during the deprotection step described herein. As a non-limiting example, as shown in FIG. 1B (and FIG. 5B), the method can include supplying (e.g., directing) solvent from a solvent source (not shown in FIG. 1B or FIG. 5B) through flow path 218, opening 208, the inner interior space of spray head 220, and exit opening 222 into the outer interior space 7 of reactor 4. As also shown schematically in FIG. 1B (and FIG. 5B ), in some embodiments, spray head 220 can direct (e.g., spray) solvent toward sidewall 6 of reactor 4 through opening 222 at an angle (e.g., spray pattern) shown schematically by dashed line 224, which can facilitate washing deprotected base condensed on the sidewall downward toward the lower interior of reactor 4.

[0141] If a washing step in an SPPS process and / or an extraction step in an LPPS process is included, the washing solution and / or extraction waste layer (e.g., the aqueous layer containing the deprotecting base) may then be removed in a draining step, after which the coupling step may begin according to known methods.

[0142] In some embodiments, a peptide synthesis method (e.g., a solid phase peptide synthesis method and / or a liquid or solution phase peptide synthesis method) can include deprotecting a first amino acid (e.g., removing a protecting group of the first protected amino acid) to form a deprotected amino acid, coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids, and repeating the deprotection and coupling steps to form a peptide comprising the first, second, and consecutive amino acids; the deprotection and coupling steps are carried out in a reactor (e.g., in a reactor such as batch reactor 4 described in more detail herein); one or more deprotection steps use a deprotection base in an amount of about 5% by volume or less, based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), as described in more detail herein; and / or As described in more detail herein, one or more deprotection steps may utilize an inert gas purging (e.g., headspace flushing) step to aid in (e.g., remove) the evaporated deprotected base from the interior of the reactor (e.g., from the headspace).

[0143] In some embodiments, a peptide synthesis method (e.g., a solid phase peptide synthesis method and / or a liquid or solution phase peptide synthesis method) can include deprotecting a protected peptide (e.g., removing a protecting group of the protected peptide) to form a deprotected peptide, coupling an amino acid to the deprotected peptide to form a second peptide from the deprotected peptide and the amino acid, and repeating the deprotection and coupling steps to form a peptide comprising a first, second, and consecutive plurality of amino acids; the deprotection and coupling steps are carried out in a reactor (e.g., in a reactor such as batch reactor 4 described in more detail herein); one or more deprotection steps use a deprotection base in an amount of about 5% by volume or less, based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), as described in more detail herein; and / or As described in more detail herein, one or more deprotection steps may utilize an inert gas purging (e.g., headspace flushing) step to aid in (e.g., remove) the evaporated deprotected base from the interior of the reactor (e.g., from the headspace).

[0144] In the SPPS method including the deprotection step described herein, the solid phase peptide synthesis method may not include a washing step between one or more deprotection steps and coupling steps in the deprotection coupling cycle of the SPPS method. In other SPPS methods including the deprotection step described herein, the solid phase peptide synthesis method may include one or more washing steps between one or more deprotection steps and coupling steps in the deprotection coupling cycle of the SPPS method, and the washing step uses an amount of washing solution (e.g., solvent) described in more detail herein (e.g., an amount that is approximately the same as and / or less than the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution) in the deprotection step, for example, less than half or about half the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution); as another example, an amount that is less than or about one-third the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution); as another example, an amount (e.g., total volume) of solvent that is two or less times the bed volume of the resin present in the reactor, for example, an amount that is one or less times the bed volume of the resin present in the reactor). For example, the solid phase peptide synthesis method may omit one or more (e.g., all) washing steps between one or more deprotection steps and coupling steps of a deprotection-coupling cycle of the SPPS method. As another example, the solid phase peptide synthesis method may include one or more washing steps using a reduced amount of washing liquid (e.g., solvent) between the deprotection step and coupling step of one or more deprotection-coupling cycles of the SPPS method, as described in more detail herein (e.g., using a volume that is about the same as and / or less than the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), for example, less than half or about half the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), as another example, less than about one-third or about one-third the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), as another example, using an amount (e.g., total volume) of solvent that is two times or less than the bed volume of the resin present in the reactor, for example, an amount of solvent that is one time or less than the bed volume of the resin present in the reactor).

[0145] In the LPPS method including a deprotection step described herein, the liquid phase peptide synthesis method can include one or more extraction steps between the deprotection step and the successive coupling step of one or more deprotection-coupling cycles of the LPPS method, where the extraction step uses an amount of extraction solvent (e.g., water) described in more detail herein (e.g., using a total volume of extraction solvent for one deprotection-coupling cycle that is two times or less the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution) for the deprotection step of that deprotection-coupling cycle).

[0146] Solid phase peptide synthesis and / or solution phase peptide synthesis methods can further include activating chemical groups on the second (and consecutive) amino acids prior to coupling using methods and agents known in the art to prepare the second (and consecutive) amino acids for coupling with the first (and consecutive) amino acids.

[0147] Prior to the coupling step, an amino acid activating agent (amino acid activator) may be used to activate the amino acid (e.g., convert the acid group of the amino acid into an activated form). Any suitable amino acid activating agent may be used. Examples of amino acid activating agents include, but are not limited to, carbodiimides and / or onium salt activators. In some embodiments, the amino acid activating agent comprises a carbodiimide, such as, but not limited to, N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and the like, and combinations thereof. In certain embodiments, the amino acid activator includes onium activators such as, but not limited to, benzotriazol-1-yloxytripyrrolidinophosphonium hexafluorophosphate (PyBOP), O-(benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU), 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HATU), 1-[(1-(cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylaminomorpholino)]uronium hexafluorophosphate (COMU), and combinations thereof.

[0148] An amino acid activator additive (amino acid activator additive) can also be used to activate the amino acid prior to the coupling step. Any suitable amino acid activator additive may be used. Examples of amino acid activator additives include, but are not limited to, benzotriazole additives such as 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), and 6-chloro-1-hydroxybenzotriazole (6-Cl-HOBt); ethyl(hydroxyimino)cyanoacetate (Oxyma); 1-hydroxy-2,5-pyrrolidinedione (NHS), and the like, and combinations thereof.

[0149] Additionally, in exemplary embodiments, the solid phase peptide synthesis and / or solution phase peptide synthesis methods can include applying microwave energy during one or more of the solid phase peptide synthesis and / or solution phase peptide synthesis steps, e.g., during the deprotection and / or coupling steps.

[0150] In exemplary embodiments, the solid phase peptide synthesis method can further comprise the step of cleaving the peptide from the solid phase resin after the deprotection step, optional washing step, and / or coupling step.

[0151] In exemplary embodiments, the solution phase peptide synthesis method can further include cleaving the peptide from the support and / or carrier and / or protecting group material (e.g., from a soluble tag) after the deprotection, extraction and / or coupling steps.

[0152] Those skilled in the art will understand how to bond or couple amino acids to form a chain.Methods and agents for cleaving peptides from solid-phase resins and / or LPPS supports and / or carriers and / or protective group materials (e.g., from soluble tags) are also well known in the art.Therefore, detailed discussion of methods known in the art for bonding amino acids and / or cleaving peptides to form peptides from solid-phase resins and / or LPPS supports and / or carriers and / or protective group materials (e.g., from soluble tags) is not provided.

[0153] The scale of solid phase peptide synthesis and / or solution phase peptide synthesis, including the deprotection methods disclosed herein, is not limited and can include, for example, research and / or manufacturing (e.g., large) scale solid phase peptide synthesis and / or solution phase peptide synthesis.

[0154] In exemplary embodiments, the deprotection methods disclosed herein (e.g., including one or more deprotection steps described herein, using a deprotection base in an amount greater than 0 to about 5% by volume, based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), and / or flushing the volatilized deprotection base from the reactor (e.g., from the headspace inside the reactor) with an inert gas, as described in further detail herein) can be used in large-scale systems incorporating the ability to heat the reaction solution (e.g., a 25 mmol / L large-scale Liberty PRO system available from CEM Corporation is approximately 125 grams of 0.2 mmol / g resin). In such embodiments, the amount of base (e.g., pyrrolidine) can be used at a very low concentration, as low as 2.5% by volume (the standard is 20%), much less than typically used, which can facilitate the reduction and / or elimination of washing. Also, in some of these embodiments, the deprotection conditions can be performed at variable times and temperatures to facilitate more complete deprotection and base removal until sufficient removal is achieved. An example of this is 90°C for 10 minutes.

[0155] In another exemplary embodiment, the deprotection method disclosed herein comprises: a method for deprotecting protected amino acids during solid phase peptide synthesis (SPPS) and / or liquid phase peptide synthesis (LPPS), comprising a deprotection step and a coupling step, The deprotection method is removing the protecting groups of the protected amino acids and / or protected peptides in the reactor with a deprotecting base, wherein at least a portion of the deprotecting base evaporates into the upper interior of the reactor during the removing step; directing an inert gas through the reactor to assist in removing vaporized deprotection base from the interior of the reactor during the step of removing the protecting group; The deprotection process may be carried out under one or more of the following conditions: (a) In the SPPS method, the deprotection method may use a solid resin support having a resin substitution of less than or about 0.35 mmol / g, such as less than or about 0.30 mmol / g, for example, 0.10 mmol / g to 0.35 mmol / g, for example, 0.15 mmol / g to 0.35 mmol / g, for example, 0.20 mmol / g to 0.35 mmol / g, such as 0.10 mmol / g to 0.34 mmol / g, for example, 0.15 mmol / g to 0.34 mmol / g, for example, 0.20 mmol / g to 0.34 mmol / g, for example, 0.20 mmol / g to 0.33 mmol / g; and / or (b) the deprotection method may use a deprotection base (e.g., pyrrolidine) in an amount of from 0 to more than about 5% by volume, for example, from 0 to more than about 3.5% by volume, or less, based on the total volume (100% by volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) in the reactor as defined herein; and / or (c) the deprotection method may be carried out at a temperature greater than 30°C, such as greater than 50°C, or alternatively, greater than 70°C, or alternatively, at a temperature between 30°C and 120°C; and / or (d) the directing step may include directing an inert gas through a first opening disposed at the top of the reactor and out of a second opening into the reactor to aid in removing the evaporated deprotected base from the interior of the reactor (e.g., to purge, vent, etc. the evaporated deprotected base from the headspace); and / or (e) In the SPPS method, the deprotection method can optionally include washing the inside of the reactor after the deprotection step with a total volume of solvent that is 2 times or less the bed volume of the resin present in the reactor, for example, 1 time or less the bed volume of the resin present in the reactor; and / or (f) the total time of the deprotection reaction (e.g., the total time of the deprotection reaction for one deprotection coupling cycle of the SPPS method) may be 1.5 hours or less, for example, from about 30 seconds to about 1 hour, for example, from about 30 seconds to about 10 minutes, or as another example, from about 10 minutes to about 1 hour; and / or (g) the boiling point of the deprotecting base is less than 107°C; and / or (h) the difference between the deprotection reaction temperature and the boiling point of the deprotecting base may be less than or about 50°C, e.g., less than or about 25°C, e.g., less than or about 15°C, e.g., the difference between the deprotection reaction temperature and the boiling point of the deprotecting base may be in the range of 15°C to 50°C; and / or (i) In the LPPS method, the total volume of the extraction solvent in a single deprotection coupling cycle may be two times or less than the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution) in the deprotection step; and / or (j) The deprotection may be carried out at a temperature at least 35° C. below the boiling point of the deprotecting base and / or for a time of 1.5 hours or less.

[0156] In an exemplary embodiment, the method includes heating the protected amino acid and the deprotecting base during the step of removing a protecting group from the protected amino acid, wherein the deprotecting base is pyrrolidine, the protected amino acid is directly or indirectly attached to a solid PEG-PS (polyethylene glycol-polystyrene) resin support, the resin can have a resin substitution of 0.2 mmol / g to 0.3 mmol / g, e.g., 0.20 mmol / g to 0.25 mmol / g, the protecting group of the protected amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group, and the heating step is performed at a temperature of about 60°C or greater.

[0157] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for deprotecting a protected peptide during liquid-phase peptide synthesis, including a deprotection step and a coupling step, wherein the deprotection method comprises removing the protecting group of the protected peptide in the liquid phase in a reactor containing a deprotecting base at a temperature at least 35°C lower than the boiling point of the deprotecting base for 1.5 hours or less, thereby providing a deprotected peptide whose C-terminus is protected. The deprotecting base is present in the reactor in an amount of 0 to greater than about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and most of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group of the protected peptide. The method may further include directing an inert gas through the reactor to assist in removing the evaporated deprotected base from the interior of the reactor during removal of the protecting group of the protected peptide, and extracting the residual deprotected base from the deprotection reaction mixture after removing the protecting group and prior to successive coupling steps of one or more deprotection coupling cycles of the liquid phase peptide synthesis with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture of the deprotection coupling cycle. In some embodiments, the directing step may include directing the inert gas into the upper interior of the reactor through a first opening disposed at the top of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed at the top of the reactor.

[0158] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for deprotecting a protected peptide during liquid-phase peptide synthesis, including a deprotection step and a coupling step. The deprotection step involves removing the protecting group of the protected peptide in the liquid phase in a reactor containing a deprotecting base at a temperature at least 35°C lower than the boiling point of the deprotecting base for 1.5 hours or less, thereby providing a deprotected peptide whose C-terminus is protected. The deprotecting base is present in the reactor in an amount of 0 to greater than about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and most of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group of the protected peptide. The deprotection step may also include the step of introducing an inert gas into the upper interior of the reactor through a first opening located at the top of the reactor and evacuating the inert gas and evaporated deprotecting base from the upper interior of the reactor through a second opening located at the top of the reactor. In some embodiments, the method may further comprise, after removing the protecting group and prior to successive coupling steps of one or more deprotection coupling cycles of solution phase peptide synthesis, extracting residual deprotected base from the deprotection reaction mixture with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture of that deprotection coupling cycle.

[0159] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for liquid phase peptide synthesis (LPPS), comprising deprotecting a protected peptide in a liquid phase, wherein the C-terminus of the protected peptide is protected in a reactor to provide a deprotected peptide. The deprotection comprises removing the protecting group of the protected peptide using a deprotecting base at a temperature at least 35°C lower than the boiling point of the deprotecting base for 1.5 hours or less, wherein the deprotecting base is present in the reactor in an amount of 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and most of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group; and directing an inert gas through the reactor to remove the evaporated deprotecting base from the interior of the reactor during removal of the protecting group. LPPS may further include, after removing the protecting group and prior to successive coupling steps of one or more deprotection coupling cycles of liquid phase peptide synthesis, extracting the residual deprotected base from the deprotection reaction mixture with a total volume of an extraction solvent that is two times or less the total volume of the deprotection reaction mixture of the deprotection coupling cycle, coupling an amino acid to the deprotected peptide to form a peptide from the deprotected peptide and the amino acid, and repeating the deprotection, extraction, and coupling to form a peptide comprising one or more additional consecutive amino acids. In some embodiments, the directing step may include directing an inert gas into the upper interior of the reactor through a first opening disposed at the top of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed at the top of the reactor.

[0160] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for liquid phase peptide synthesis (LPPS), comprising deprotecting a protected peptide in a liquid phase, wherein the C-terminus of the protected peptide is protected in a reactor to provide a deprotected peptide. The deprotection may include removing a protecting group of the protected peptide using a deprotecting base at a temperature at least 35° C. lower than the boiling point of the deprotecting base for 1.5 hours or less, wherein the deprotecting base is present in the reactor in an amount of 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and most of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group; introducing an inert gas into the upper interior of the reactor through a first opening disposed at the top of the reactor; and venting the inert gas and the evaporated deprotecting base from the upper interior of the reactor through a second opening disposed at the top of the reactor to remove the deprotecting base evaporated from the reactor during removal of the protecting group. The LPPS method further includes coupling an amino acid to the deprotected peptide to form a peptide from the deprotected peptide and the amino acid, and repeating the deprotection and coupling to form a peptide comprising one or more additional consecutive amino acids. In some embodiments, the LPPS method can further include, after removing the protecting group and prior to successive coupling steps of one or more deprotection coupling cycles of solution phase peptide synthesis, extracting residual deprotected base from the deprotection reaction mixture with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture of the deprotection coupling cycle.

[0161] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for deprotecting a protected amino acid during solid phase peptide synthesis (SPPS), comprising a deprotection step and a coupling step. The deprotection method comprises removing a protecting group of a protected amino acid attached to a solid resin support in a reactor having a size of at least 3 liters with a deprotecting base to provide a deprotected amino acid. The deprotecting base is present in the reactor in an amount of 0 to greater than about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and at least a portion of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group of the protected amino acid. The deprotection method further comprises directing an inert gas having a pressure of about 50 psi to about 95 psi through the reactor to remove the evaporated deprotecting base from the interior of the reactor during removal of the protecting group of the protected amino acid.

[0162] Other exemplary embodiments of the present disclosure may include, but are not limited to, a method for solid-phase peptide synthesis having a synthesis scale of about 25 mmol or greater. The method may include deprotecting a first protected amino acid to provide a deprotected amino acid, and coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids. The deprotection includes removing a protecting group of the protected amino acid in a reactor having a size of at least 3 liters using a deprotecting base, wherein the deprotecting base is present in the reactor in an amount of 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and at least a portion of the deprotecting base evaporates into the upper interior of the reactor during the removal of the protecting group; and directing an inert gas having a pressure of about 50 psi to about 95 psi through the reactor to remove the deprotecting base that evaporates from the interior of the reactor during the removal of the protecting group.

[0163] The present disclosure also relates to systems for solid-phase and / or liquid-phase peptide synthesis. Figure 2A (and Figure 6A) are schematic flow diagrams showing selected portions of an exemplary solid-phase and / or liquid-phase peptide synthesis system according to an embodiment of the present disclosure. Figure 2B (and Figure 6B) are schematic flow diagrams showing selected portions of another exemplary solid-phase and / or liquid-phase peptide synthesis system according to another embodiment of the present disclosure.

[0164] Generally, elements shown in both Figures 1A and 2A (and Figures 5A and 6A) are given the same reference numbers. Similarly, generally, elements shown in both Figures 1B and 2B (and Figures 5B and 6B) are given the same reference numbers. Also, unless otherwise indicated, elements shown in both Figures 2A and 2B (and Figures 6A and 6B) are given the same reference numbers.

[0165] The peptide synthesis system of Figures 2A and 2B (and Figures 6A and 6B) is generally designated 2. Peptide synthesis system 2 of Figures 2A and 2B (and Figures 6A and 6B) is suitable for use in the SPPS and / or LPPS methods described herein. Peptide synthesis system 2 includes a reactor 4 as discussed herein. Peptide synthesis system 2 also includes a plurality of reagent vessels positioned upstream of reactor 4 in fluid communication with reactor 4.

[0166] For example, as shown in Figures 2A and 2B (and Figures 6A and 6B), system 2 can include a plurality of solid supports and / or supports suitable for LPPS processes (e.g., soluble tag) vessels 50a, 50b, and 50c in fluid communication with flow path 52, which fluidly connects the solid supports and / or supports for the LPPS vessels to reactor 4, for delivering solid supports (e.g., solid resins with protected amino acids attached) and / or supports for LPPS (e.g., soluble tags) from the solid supports and / or supports for the LPPS (e.g., soluble tag) vessels to reactor 4. Flow paths 51a, 51b, and 51c fluidly connect the solid supports and / or supports for the LPPS (e.g., soluble tag) vessels 50a, 50b, and 50c, respectively, to flow path 52.

[0167] In certain embodiments, flow path 52 can be in direct fluid communication with reactor 4, for example, via opening 12 or 204 in FIG. 1A or FIG. 1B (and FIG. 5A or FIG. 5B), respectively. In certain embodiments, as shown schematically in FIG. 2A (and FIG. 6A), the system can include a rotary valve 140 that can rotate between multiple positions (e.g., two positions) to fluidly connect reactor 4 with solid supports and / or supports for LPPS (e.g., soluble tag) vessels 50a, 50b, and 50c through opening 12, flow path 22, and flow path 52. In certain other embodiments, as shown schematically in FIG. 2B (and FIG. 6B), rotary valve 140 can rotate between multiple positions (e.g., two or more positions) to fluidly connect reactor 4 with solid supports and / or supports for LPPS (e.g., soluble tag) vessels 50a, 50b, and 50c through opening 204, flow path 214, and flow path 52. During the deprotection steps described herein, rotary valve 140 may be closed to flow path 52 (and flow path 152), as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B).

[0168] As another example, in certain embodiments, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), system 2 can include multiple amino acid containers 60a, 60b, and 60c in fluid communication with channel 62 (FIGS. 2A and 6A) or channel 212 (FIGS. 2B and 6B), which fluidly connects the amino acid containers and reactor 4, for delivering protected amino acids from the amino acid containers to reactor 4. Channels 61a, 61b, and 61c fluidly connect amino acid containers 60a, 60b, and 60c with channel 62 (FIGS. 2A and 6A) or channel 212 (FIGS. 2B and 6B), respectively. In some embodiments, channel 62 and / or channel 212 can be in direct fluid communication with reactor 4, e.g., via openings 10 or 202 in FIG. 1A or FIG. 1B (and FIGS. 5A and 5B), respectively. In some embodiments, the system may include one or more additional valves and / or flow paths, as shown schematically in Figures 2A and 2B (and Figures 6A and 6B) and described in more detail below. During the deprotection steps described herein, opening 10 or 202 may be closed to flow path 62 or flow path 212 of Figures 2A and 2B (and Figures 6A and 6B).

[0169] As another example, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), system 2 can include a deprotection base reservoir 70 in fluid communication with a flow path 72 fluidly connecting the deprotection base reservoir and reactor 4 for delivering a deprotection base (e.g., as part of a deprotection solution described herein) from the deprotection base reservoir to reactor 4. In some embodiments, flow path 72 can be in direct fluid communication with reactor 4, for example, via opening 16 in FIGS. 1A and 1B (and FIGS. 5A and 5B). In some embodiments, the system can include one or more additional valves and / or flow paths, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B) and described in more detail below. During the deprotection step described herein, opening 16 can be closed to flow path 72 in FIGS. 2A and 2B (and FIGS. 6A and 6B).

[0170] As yet another example, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), system 2 can include a solvent container 80 in fluid communication with a flow path 82 fluidly connecting the solvent container and reactor 4 for delivering solvent from the solvent container to reactor 4. In some embodiments, flow path 82 can be in direct fluid communication with reactor 4, e.g., via opening 10 or 208 in FIGS. 1A or 1B (and FIGS. 5A or 5B), respectively. In some embodiments, the system can include one or more additional valves and / or flow paths, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B) and described in more detail below. FIG. 2B (and FIG. 6B) also schematically illustrates an embodiment in which solvent can be introduced into reactor 4 using at least a portion of the same flow path used to introduce an inert gas, e.g., via flow path 218, opening 208, spray head 220, and multiple openings 222, as described in more detail herein. During the deprotection steps described herein, opening 10 or 208 may be closed to channel 82 of Figures 2A and 2B (and Figures 6A and 6B).

[0171] As yet another example, as shown schematically in FIGS. 6A and 6B, for an LPPS process, system 2 can include an additional solvent vessel (e.g., extraction solvent vessel), such as solvent vessel 80a, that can be in fluid communication with flow path 82a fluidly connecting the extraction solvent vessel and reactor 4 to deliver the extraction solvent from the solvent vessel to reactor 4. In some embodiments, flow path 82a can be in direct fluid communication with reactor 4, e.g., via openings 10 or 208 in FIGS. 1A or 1B (and FIGS. 5A or 5B), respectively. In some embodiments, the system can include one or more additional valves and / or flow paths, as shown schematically in FIGS. 6A and 6B and described in more detail below. FIG. 6B also schematically illustrates an embodiment in which solvent can be introduced into reactor 4 using at least a portion of the same flow paths used to introduce inert gas, e.g., via flow path 218, opening 208, spray head 220, and multiple openings 222, as described in more detail herein. During the deprotection steps described herein, openings 10 or 208 may be closed to channel 82a of Figures 6A and 6B, respectively.

[0172] As yet another example, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), system 2 can include an additional reagent container 90, which can be, for example, an activator container, in fluid communication with a flow path 92 ( FIGS. 2A and 6A ) or a flow path 210 ( FIGS. 2B and 6B ) that fluidly connects the additional reagent container and reactor 4 for delivering an additional reagent, such as an activator, from the additional reagent container to reactor 4. In some embodiments, flow path 92 or 210 can be in direct fluid communication with reactor 4, for example, via opening 10 or 200 in FIGS. 1A and 1B (and FIGS. 5A and 5B ), respectively. In some embodiments, the system can include one or more additional valves and / or flow paths, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B ) and described in more detail below. During the deprotection steps described herein, opening 10 or 200 may be closed to channel 92 or channel 210 of Figures 2A and 2B (and Figures 6A and 6B).

[0173] Those skilled in the art will understand that the number of reactors, solid support containers, amino acid containers, deprotection base containers, solvent containers, and / or other reagent containers and associated flow paths, as well as the manner in which these elements are connected, can vary and are not limited to the depictions in FIGS. 2A and 2B (and FIGS. 6A and 6B). Those skilled in the art will also understand that the system can include other containers, such as containers and associated flow paths for extraction solvents (e.g., to deliver extraction solvent to the reactors), associated valves, pathways, containers, etc., for removing and / or disposing of extraction waste from the reactors, etc. Those skilled in the art will also understand that a peptide synthesis system can include various subsystems associated with the above-mentioned containers, flow paths, and / or reactor(s) (including, e.g., flow paths, valves, filters, gauges, monitors, controllers, etc.) to direct the flow of materials into and / or out of the containers and / or reactor(s) at appropriate stages of solid-phase and / or solution-phase peptide synthesis methods. Such subsystems are well known in the art and will not be described in detail herein.

[0174] System 2 is also associated with a heating source (not shown), such as a microwave source, and associated elements, such as a microwave guide and / or microwave cavity, for heating reactor 4, as described herein. Heating sources, including microwave heating sources and associated elements, such as a microwave guide and / or microwave cavity, and their use in solid phase peptide synthesis and / or solution phase peptide synthesis methods and systems, are also well known in the art and will not be described in greater detail herein.

[0175] System 2 is shown generally as operating in an amino acid deprotection step and / or peptide deprotection step of solid-phase peptide synthesis and / or solution-phase peptide synthesis, as described herein with reference to Figures 1A and 1B (and Figures 5A and 5B). In this state of operation, reactants including protected amino acids and / or protected peptides and deprotected bases have already been delivered to (and / or are already present in) reactor 4. The reactor may also contain a coupling solution from a preceding coupling step that combines with the deprotection solution to form a deprotection reaction mixture (e.g., a deprotection reaction solution), all as described herein.

[0176] The protected amino acid and / or protected peptide can be attached to a solid support and / or a support suitable for LPPS (e.g., a soluble tag). In some embodiments, the protected amino acid and / or protected peptide can be attached directly to the solid support and / or LPPS support (e.g., a soluble tag) (e.g., the solid support and / or LPPS support (e.g., a soluble tag) can be delivered to reactor 4 via flow paths 52 of one or more of vessels 50a, 50b, and 50c). In some embodiments (e.g., after a preceding coupling step), the protected amino acid and / or protected peptide can be attached directly or indirectly to the solid support and / or LPPS support (e.g., a soluble tag) (e.g., it can be attached to another amino acid or growing peptide chain, and the other amino acid or peptide is attached to the solid support and / or LPPS support (e.g., a soluble tag)).

[0177] System 2 further includes an inert gas source 100 positioned upstream of reactor 4 in fluid communication with reactor 4. In certain embodiments, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), a flow path 102 fluidly connects inert gas source 100 and reactor 4 and delivers inert gas provided by inert gas source 100 to the upper interior of reactor 4 via opening 10 ( FIGS. 1A and 5A ) or opening 208 ( FIGS. 1B and 5B ) described herein. In certain embodiments, system 2 includes a valve 104 in fluid communication with flow path 102 positioned between inert gas source 100 and opening 10 ( FIGS. 1A and 5A ) or opening 208 ( FIGS. 1B and 5B ), wherein valve 104 has an open position and a closed position relative to flow path 102.

[0178] Valve 104 is shown in an open position in FIGS. 2A and 2B (and FIGS. 6A and 6B) relative to flow path 102. In the open position, valve 104 allows a flow of pressurized inert gas from inert gas source 100 to enter the upper interior (e.g., reactor headspace) of reactor 4 through flow path 102, flow path 20 (FIGS. 1A and 5A) or flow path 218 (FIGS. 1B and 5B), and opening 10 (FIGS. 1A and 5A) or opening 208 and spray head 220 (FIGS. 1B and 5B). In this manner, the system can continuously and / or intermittently direct an overhead source of pressurized inert gas to the upper interior of reactor 4 during the heating / deprotection step to purge volatilized deprotected base from the reactor headspace, according to methods described herein.

[0179] In contrast, when valve 104 is in a closed position relative to flow path 102, valve 104 can prevent the flow of pressurized inert gas from the inert gas source through flow path 102, flow path 20 (Figures 1A and 5A) or flow path 218 (Figures 1B and 5B), and opening 10 (Figures 1A and 5A) or opening 208 and spray head 220 (Figures 1B and 5B).

[0180] The system may also include a flow path 106 fluidly connecting the inert gas source 100 and an opening 16 in the bottom of the reactor 4. The system may also include a valve 108 in fluid communication with the flow path 106 disposed between the inert gas source 100 and the opening 16, the valve 108 having an open position and a closed position relative to the flow path 106.

[0181] When in an open position relative to flow path 106 (e.g., as shown in FIGS. 2A and 2B and 6A and 6B ), valve 108 allows the flow of pressurized gas from inert gas source 100 through flow path 106, flow path 26, and opening 16 to the lower interior of reactor 4. In this manner, in deprotection methods described herein (e.g., when valve 108 is open as needed), pressurized inert gas can be directed to the lower reactor to agitate (e.g., stir, bubble) the reactants and / or flush evaporated deprotected base from the headspace of the reactor. The closed position of second valve 108 relative to flow path 106 prevents the flow of pressurized inert gas from inert gas source 100 through flow path 106, flow path 26, and opening 16 to the lower interior of reactor 4.

[0182] In certain embodiments, flow paths 102 and 106 may be fluidly connected to a single inert gas source 100 via a pressure regulator designated 110 in Figures 2A (and 6A) and 2B (and 6B). Alternatively, flow paths 102 and 106 may fluidly connect the reactor to at least two different inert gas sources.

[0183] If present, pressure regulator 110 may be located downstream from inert gas source 100 and upstream from valves 104 and 108. In these embodiments, pressure source 100 directs inert gas to pressure regulator 110, which supplies pressurized inert gas to flow path 106 having a higher pressure ("high pressure" inert gas) than the inert gas supplied to flow path 102 ("low pressure" inert gas). For example, but not by way of limitation, pressure regulator 110 may supply "high pressure" inert gas to flow path 102 having a pressure of about 1 psi to about 25 psi. Pressure regulator 110 may also supply "low pressure" inert gas having a pressure that is less than the pressure of the "high pressure" inert gas supplied to flow path 102.

[0184] Pressure regulators are also well known in the art, and one skilled in the art will understand how to use one in system 2 to provide high pressure and low pressure inert gas as discussed herein.

[0185] As also shown schematically in Figures 2A and 2B and 6A and 6B (and Figures 1A and 1B and 5A and 5B), in exemplary embodiments, the system can include flow path 24 (Figures 1A / 5A and 2A / 6A) or flow path 216 (Figures 1B / 5B and 2B / 6B) located downstream from opening 14 (Figures 1A and 5A) or opening 206 (Figures 1B and 5B) of reactor 4. Flow paths 24 and 216 function as gaseous waste flow paths that allow gaseous waste (e.g., volatilized deprotection base, inert gas, etc.) to be purged from the upper interior (headspace) of reactor 4 during the deprotection methods described herein.

[0186] In certain embodiments, the system includes a valve 120 in fluid communication with flow path 24 or flow path 216. Valve 120 has an open position and a closed position with respect to flow path 24 or flow path 216. The open position of valve 120 allows gas flow from the upper interior of reactor 4 through opening 14 or opening 206 and flow path 24 or flow path 216 to an exhaust and / or waste collection zone, such as a waste container (not shown), to allow purging / evacuation of gas from reactor 4. The closed position of valve 120 prevents gas flow from the upper interior of the reactor through opening 14 or opening 206.

[0187] 2A and 2B (and FIGS. 6A and 6B) show a particular embodiment of the system in an operational state in which both valve 104 and valve 120 are in the open position relative to channel 102, channel 20 or 218, and channel 24 or 216, respectively. This corresponds to the position used during the (heated) deprotection method described herein. The simultaneous open positions of valves 104 and 120 for flow path 102, flow path 20 or 218, and flow path 24 or 216, respectively, allow an overhead supply of pressurized inert gas to flow continuously and / or intermittently through reactor 4 (e.g., into vessel 4 through opening 10 in the vessel headspace and out of vessel 4 through separate opening 14, or, as another example, into vessel 4 through opening 208, spray head 220 and opening 222 toward sidewall 6, into the vessel headspace and out of vessel 4 through separate opening 206) to purge (flush) volatilized reactants present in the headspace during the (heated) deprotection step.

[0188] In certain embodiments, system 2 can include valve 122 in series with valve 104 and a flow path 124 positioned between and fluidly connecting valve 104 and valve 122. Valve 122 is in fluid communication with flow path 102 and has an open position and a closed position relative to flow path 102. As shown in Figures 2A (and 6A) and 2B (and 6B), when valve 122 is present and valve 122 and valve 104 are in an open position relative to flow path 102, inert gas source 100, pressure regulator 110, flow path 102, valve 104, flow path 124, valve 122, flow path 20 (Figures 2A and 6A) or flow path 218 (Figures 2B and 6B), opening 10 (Figures 1A / 5A / 2A / 6A) or opening 208 and spray head 220 (Figures 1B / 5B / 2B / 6B), and reactor 4 can be fluidly connected (in fluid communication).

[0189] In certain embodiments, valve 104 may be a rotary valve that can rotate between multiple positions to fluidly connect a selected one of multiple flow paths with reactor 4. As a non-limiting example, FIG. 2A illustrates rotary valve 104 that can rotate between four positions to be in fluid communication with flow paths 102, 62, 82, or 92, depending on the open or closed position of the valve. As another non-limiting example, FIG. 6A illustrates rotary valve 104 that can rotate between five positions to be in fluid communication with flow paths 102, 62, 82, 82a, or 92, depending on the open or closed position of the valve. For example, FIG. 2A schematically illustrates rotary valve 104 in an open position relative to flow path 102 but in a closed position relative to flow paths 62, 82, and 92. For example, FIG. 6A schematically illustrates rotary valve 104 in an open position relative to flow path 102 but in a closed position relative to flow paths 62, 82, 82a, and 92. As another non-limiting example, FIG. 2B illustrates a rotary valve 104 that can rotate between two positions to fluidly communicate with flow paths 102 or 82, depending on the open or closed position of the valve. As another non-limiting example, FIG. 6B illustrates a rotary valve 104 that can rotate between three positions to fluidly communicate with flow paths 102, 82, or 82a, depending on the open or closed position of the valve. For example, FIG. 2B schematically illustrates rotary valve 104 in an open position relative to flow path 102 but in a closed position relative to flow path 82. For example, FIG. 6B schematically illustrates rotary valve 104 in an open position relative to flow path 102 but in a closed position relative to flow paths 82 and 82a. The open and closed positions of rotary valve 104 for different flow paths can be selected depending on the stage of the peptide synthesis method, the reactants delivered to reactor 4, etc. Rotary valve 104 (and other valves described herein) can be operated as known in the art.

[0190] Other valves in the system may be rotary valves. For example, as described herein, in certain embodiments, system 2 may include a flow path 124 positioned between and fluidly connecting valve 104 and valve 122. In this embodiment, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), valve 122 in series with valve 104 may be rotated between multiple positions (e.g., valve 122 between two positions in FIGS. 2A and 2B (and FIGS. 6A and 6B), and valve 104 between four positions shown schematically in FIG. 2A, five positions shown schematically in FIG. 6A, two positions shown schematically in FIG. 2B, and three positions shown schematically in FIG. 6B) to, for example, fluidly connect inert gas source 100 and reactor 4 via flow path 102, flow path 124, flow path 20 or flow path 218, and opening 10 or opening 208. Alternatively, as shown in FIG. 2A, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions and four positions, respectively) to fluidly connect, for example, one or more amino acid containers 60a, 60b, and 60c to reactor 4 via flow path 62, flow path 124, flow path 20, and opening 10; solvent container 80 to reactor 4 via flow path 82, flow path 124, flow path 20, and opening 10; or reagent container 90 to reactor 4 via flow path 92, flow path 124, flow path 20, and opening 10. Alternatively, as shown in FIG. 6A, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions and five positions, respectively) to fluidly connect, for example, one or more amino acid containers 60a, 60b, and 60c to reactor 4 via flow path 62, flow path 124, flow path 20, and opening 10; solvent container 80 to reactor 4 via flow path 82, flow path 124, flow path 20, and opening 10; extraction solvent container 80a to reactor 4 via flow path 82a, flow path 124, flow path 20, and opening 10; or reagent container 90 to reactor 4 via flow path 92, flow path 124, flow path 20, and opening 10.Also, in some embodiments, as shown in Figure 2B, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions) to fluidly connect, for example, solvent container 80 and reactor 4 via flow path 82, flow path 124, flow path 218, and opening 208 and spray head 222. Also, in some embodiments, as shown in Figure 6B, valve 122 in series with valve 104 can be rotated between multiple positions (e.g., two positions and three positions, respectively) to fluidly connect, for example, solvent container 80 and reactor 4 via flow path 82, flow path 124, flow path 218, and opening 208 and spray head 222; or extraction solvent container 80a and reactor 4 via flow path 82a, flow path 124, flow path 218, and opening 208 and spray head 222.

[0191] As another example, in certain embodiments, as shown schematically in FIGS. 2A and 2B (and FIGS. 6A and 6B), valve 108 may be a rotary valve that rotates between multiple positions (e.g., three positions) to fluidly connect reactor 4 through opening 16, flow path 26, and flow path 72, flow path 106, or flow path 132 with deprotection base container 70, inert gas source 100, or waste container 130, respectively, depending on the position of valve 108. For example, FIGS. 2A and 2B (and FIGS. 6A and 6B) schematically show rotary valve 108 in an open position relative to flow path 106 but in a closed position relative to flow paths 72 and 132. This may be the position of rotary valve 108 during the heating and / or deprotection methods described herein, where low-pressure inert gas is directed (bubbled) into the bottom of reactor 4 to agitate the reactants and / or flush evaporated deprotection base from the headspace of the reactor.

[0192] As another example, as described herein, in certain embodiments, as shown generally in Figure 2A (and Figure 6A), the system can include a rotary valve 140 that can rotate between multiple positions (e.g., two positions) to fluidly connect reactor 4 with solid support and / or LPPS support (e.g., soluble tag) containers 50a, 50b, and 50c through opening 12, flow channel 22, and flow channel 52. Alternatively, rotary valve 140 can rotate between multiple positions (e.g., two positions) to fluidly connect reactor 4 with opening 12, flow channel 22, and flow channel 152, which is in fluid communication with multiple flow channels 151a, 151b, and 151c, which in turn fluidly connects flow channel 152 with multiple product containers 150a, 150b, and 150c, respectively. This allows product (e.g., peptides and / or peptides linked to solid supports and / or LPPS supports (e.g., soluble tags)) to pass from reactor 4 to vessels 150a, 150b, and 150c. Again, one skilled in the art will understand that the number of product vessels 150a, 150b, and 150c and corresponding flow channels 151a, 151b, and 151c can vary and are not limited to the number shown in FIG. 2A (and FIG. 6A). FIG. 2A (and FIG. 6A) schematically shows rotary valve 140 in a closed position relative to flow channels 52 and 152 (e.g., in a closed position relative to flow channels 52 and 152 during the deprotection step described herein).

[0193] As another example, as described herein, in certain embodiments, as shown generally in Figure 2B (and Figure 6B), the system can include a rotary valve 140 that can rotate between multiple positions (e.g., two positions) to fluidly connect reactor 4 with solid support and / or LPPS support (e.g., soluble tag) containers 50a, 50b, and 50c through opening 204, flow channel 214, and flow channel 52. Alternatively, rotary valve 140 can rotate between multiple positions (e.g., two positions) to fluidly connect reactor 4 with opening 204, flow channel 214, and flow channel 152, again fluidly connecting flow channel 152 with multiple flow channels 151a, 151b, and 151c, which in turn fluidly connect flow channel 152 with multiple product containers 150a, 150b, and 150c, respectively. Again, this allows product (e.g., peptides and / or peptides linked to solid supports and / or LPPS supports (e.g., soluble tags)) to pass from reactor 4 to vessels 150a, 150b, and 150c. Again, one skilled in the art will appreciate that the number of product vessels 150a, 150b, and 150c and corresponding channels 151a, 151b, and 151c can vary and are not limited to the number shown in Figure 2B (and Figure 6B). Figure 2B (and Figure 6B) schematically illustrates rotary valve 140 in a closed position relative to channels 52 and 152 (e.g., in a closed position relative to channels 52 and 152 during the deprotection steps described herein).

[0194] Peptide synthesis system 2 may also include one or more fluid channels, vents, containers, valves, controllers, etc., for example, to remove waste products (e.g., excess reactants, solvents, extraction waste, etc.) from the peptide synthesis system. The waste products may be in gaseous, liquid, and / or solid form, and one of skill in the art will understand appropriate types of fluid channels and containers for removing waste products from the peptide synthesis system. For example, in certain embodiments, as described herein, waste container 130 may be fluidly connected to reactor 4 via fluid channel 132 and rotary valve 108 when in an appropriate open position that allows the passage of waste products (e.g., extraction waste) from reactor 4 to waste container 130. FIGS. 2A and 2B (and FIGS. 6A and 6B) schematically show rotary valve 108 in a closed position relative to fluid channel 132 (e.g., in a closed position relative to fluid channel 132 during a deprotection step described herein). As another example, in certain embodiments, as described herein, an open position of valve 120 can allow gas flow from the upper interior (headspace) of reactor 4 through opening 14 or 206 and flow path 24 or 216 to a waste collection zone, such as an exhaust and / or waste container (not shown), to allow purging / exhaust of gas from reactor 4 (e.g., during a deprotection step described herein). Figures 2A and 2B (and Figures 6A and 6B) schematically show valve 120 in an open position relative to flow path 24 or 216 (e.g., in an open position relative to flow path 24 or 216 during a deprotection step described herein).

[0195] Thus, in general, Figures 2A and 2B (and Figures 6A and 6B) depict exemplary systems for delivering solvents, reactants (amino acids, deprotecting bases, activating agents, extraction solvents, etc.), solid phase resins, LPPS supports (e.g., soluble tags), gases, etc. from their respective sources to reactor 4, and for further delivering products and by-products (peptides, gaseous, liquid, and / or solid waste, etc.) from reactor 4 to their respective destinations. It will be understood that the particular flow paths and valve locations are illustrative rather than limiting of the present disclosure.

[0196] Repeating at least partially from above, a peptide synthesis system typically includes at least one controller operatively associated with, for example, numerous electrical components of the system (e.g., microwave sources, sensors, and solenoids and / or other electrically operated valves). The at least one controller may include one or more computers, computer data storage devices, programmable logic devices (PLDs), and / or application-specific integrated circuits (ASICs). A suitable computer may include one or more of a central processing unit or processor, computer hardware integrated circuits or memory, a user interface, peripherals or device interfaces for interfacing with other electrical components of the system, and / or any other suitable features. The controller may communicate with each of the electrical components of the system via appropriate signal communication paths. Representative signal communication paths associated with the controller are schematically illustrated in Figures 2A and 2B (and Figures 6A and 6B), and are designated by the numerals *2 (signal communication paths) and *1 (controller), respectively. The methods of the present disclosure may be controlled (e.g., at least partially controlled) in response to the execution of a computer-based algorithm operatively associated with at least one controller *1.

[0197] Solid-phase peptide synthesis methods, including batch-based methods, are known, and therefore this disclosure does not provide detailed information thereon. See, for example, the pioneering work R.B. Merrifield (1963) "Solid Phase Peptide Synthesis I, The Synthesis of a Tetrapeptide," J. Am. Chem. Soc. 85(14), 2149-2154. Therefore, a detailed discussion of solid-phase peptide synthesis methods is not provided.

[0198] Systems suitable for performing solid phase peptide synthesis, including batch-based methods, are also known. Exemplary systems for performing solid phase peptide synthesis include, for example, the LIBERTY line of instruments commercially available from CEM Corporation of Matthews, NC.

[0199] Liquid phase peptide synthesis methods, including batch-based methods, are also known, and therefore this disclosure does not provide detailed information or a detailed discussion thereof. Systems suitable for performing liquid phase peptide synthesis, including batch-based methods, are also known and commercially available.

[0200] See also exemplary United States patents dealing with the subject matter of solid phase peptide synthesis (including exemplary systems and / or methods), including, but not limited to, U.S. Patent Nos. 7,393,920; 7,550,560; 7,563,865; 7,939,628; 7,902,488; 7,582,728; 8,153,761; 8,058,393; 8,426,560; 8,846,862; 9,211,522; 9,669,380; 10,052,607; 10,308,677; 10,125,163; 10,858,390; and 10,239,914, the contents of each of which are incorporated herein by reference in their entirety.

[0201] The deprotection and / or SPPS and / or LPPS methods of the present disclosure may be used as part of an SPPS and / or LPPS method that does not include (eliminates) washing and / or draining after each coupling step, and / or adds the deprotection base directly to the coupling solution from the preceding coupling step without draining after coupling, as disclosed, for example, in U.S. Patent Nos. 10,308,677; 10,125,163; 10,858,390; and 10,239,914. Such SPPS methods are sometimes commonly referred to as "high-efficiency SPPS (HE-SPPS)."

[0202] Figure 3 is a flow chart outlining the steps of a cycle of a conventional solid phase peptide synthesis (SPPS) method, generally designated 300. Deprotection step 302 is performed in a reactor, as known in the art, by adding a deprotection solution containing a deprotecting base to the reactor. The deprotection solution is then drained (step 304), followed by a wash step 306, which is performed repeatedly, typically five times, by adding a wash solution (e.g., methanol or isopropanol) to the vessel. The wash solution is then removed in a second drain step 308, after which a coupling step 310 is performed in the reactor. The coupling solution is then removed in a third drain step 312, followed by a second wash step 314, again typically five times, followed by a fourth drain step 316.

[0203] Thus, Figure 3 shows the draining and washing steps that follow each of the deprotection and coupling steps of a conventional SPPS cycle. It will be understood that Figure 3 is a schematic diagram, and that there are many details about one SPPS cycle that could be added, but Figure 3 illustrates sufficient concepts for one skilled in the art to understand both it and the present invention.

[0204] In contrast, FIGS. 4A and 4B are flowcharts that generally illustrate steps in a cycle of an SPPS method according to an exemplary embodiment of the present disclosure, broadly designated 400a and 400b, respectively, as described in more detail herein.

[0205] FIG. 4A schematically illustrates a deprotection step / deprotection-coupling cycle of an SPPS method according to an exemplary embodiment of the present disclosure described in more detail herein, in which a deprotection solution containing a deprotecting base is added to the coupling solution from the previous coupling cycle without a wash or drain step between the coupling step of the previous cycle and the addition of the deprotection solution for the deprotection step of the subsequent cycle. FIG. 4A also schematically illustrates the optional draining of any liquid remaining in the vessel after deprotection (e.g., solvent, residual deprotection agent, if any). FIG. 4A further schematically illustrates that the wash step after each deprotection step (before the next coupling step) according to exemplary embodiments of the present disclosure described herein can be eliminated. Accordingly, FIG. 4A schematically illustrates an exemplary embodiment of the present disclosure in which the wash step following the deprotection step of an SPPS cycle (and also the wash and / or drain steps following the coupling step) can be eliminated.

[0206] FIG. 4B schematically illustrates a deprotection step / deprotection-coupling cycle of an SPPS method according to another exemplary embodiment of the present disclosure, described in more detail herein, in which a deprotection solution containing a deprotection base is added to the coupling solution from the previous coupling cycle without a wash or drain step between the coupling step of the previous cycle and the addition of the deprotection solution of the deprotection step of the subsequent cycle. FIG. 4B also schematically illustrates the optional draining of any liquid remaining in the vessel after deprotection (e.g., solvent, residual deprotection agent, if any). FIG. 4B further schematically illustrates one or more wash steps after each deprotection step and before the next coupling step, followed by one or more additional optional drain steps. However, in contrast to conventional SPPS methods, such as those schematically illustrated in FIG. 3, FIG. 4B illustrates that any post-deprotection wash steps may use a reduced amount of solvent according to another exemplary embodiment described herein. Accordingly, FIG. 4B schematically illustrates an exemplary embodiment of the present disclosure in which the volume of wash fluid (e.g., solvent) may be reduced relative to the volume of solvent used after deprotection in conventional SPPS methods.

[0207] FIG. 4C is a flow chart generally illustrating a deprotection step / deprotection coupling cycle of an LPPS method, broadly designated 400c, according to another exemplary embodiment of the present disclosure, described in more detail herein. A deprotection solution containing a deprotection base is added to a reactor, with or without a wash and / or drainage step between the coupling step of the previous deprotection-coupling cycle and the deprotection step of the successive cycle (e.g., the coupling solution from the preceding coupling cycle may or may not be present in the reactor to which the deprotection solution is added). FIG. 4C also schematically illustrates one or more extraction steps after deprotection, in which an extraction solvent (e.g., an aqueous solvent) is added to the reactor after deprotection and before the next successive coupling to form a waste layer containing residual deprotection agent remaining in the reactor after deprotection, which is removed (e.g., drained) from the reactor. FIG. 4C further schematically illustrates one or more drainage steps for removing waste (e.g., the aqueous waste layer containing residual deprotection base). 4C shows that the post-deprotection extraction step can use a reduced amount of extraction solvent according to other exemplary embodiments described herein, compared to the volume of extraction solvent used after deprotection in a conventional LPPS method. Thus, FIG. 4C shows a schematic diagram of extracting residual deprotected base from a reactor after removing a protecting group and before the successive coupling steps of a deprotection coupling cycle, in which the total volume of extraction solvent used in all extraction steps related to the deprotection coupling cycle (the total combined volume of extraction solvent in all extraction steps of a single deprotection coupling cycle) is 2 times (2x) or less than the total volume (total volume) of the deprotection reaction mixture (e.g., deprotection reaction solution) in the deprotection step of the deprotection coupling cycle.

[0208] The following examples are provided for illustrative purposes only and should not be construed as limiting the invention in any way. The examples demonstrate that even small amounts of deprotecting base can result in essentially complete deprotection and scavenging of protecting groups (e.g., Fmoc protecting groups), leaving only residual base that can be small enough to minimize problems in the subsequent coupling step. [Example]

[0209] Example 1 low base concentration, Post-deprotection washing can be and without, and with and without headspace flushing Analysis of one-pot synthesis of JR 10mer using The JR 10-mer is synthesized using solid-phase peptide synthesis using a commercially available automated microwave peptide synthesizer (e.g., Liberty PRIME 2.0 from the Liberty line of microwave peptide synthesizers available from CEM Corporation, Matthews, NC) on a 0.1 mmol scale. PEG-PS resin (e.g., Rink Amide ProTide Resin LL available from CEM Corporation) or PS resin (e.g., Fmoc-Rink Amide MBHA PS available from CEM Corporation) is used as the solid-phase resin support, and the coupling reaction is carried out in the presence of an Fmoc-protected amino acid (AA).

[0210] The deprotection reaction is carried out by adding a pyrrolidine / dimethylformamide (DMF) deprotection reagent (deprotection solution) to the non-drained post-coupling mixture (coupling solution). The concentration of pyrrolidine (e.g., the volume percent of pyrrolidine in the reactor based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), including the added pyrrolidine / DMF deprotection solution and the non-drained coupling solution from the preceding coupling reaction) is set forth in Table 1 below. The microwave power is adjusted to provide a deprotection temperature of 110°C and a deprotection reaction time, as also shown in Table 1 below.

[0211] Table 1 further indicates whether post-deprotection washes and / or headspace flushes are used. For samples in Table 1 where "Headspace Flushing" is indicated as "On," a stream of nitrogen gas is directed through the headspace of the reactor to purge headspace gas from the reactor in accordance with embodiments of the present disclosure described herein (e.g., directing a pressurized stream of nitrogen gas into the reactor through an inlet port such as shown in FIGS. 1A and 1B and exiting the reactor through an outlet port such as an exhaust port such as shown in FIGS. 1A and 1B). For examples in Table 1 where "Headspace Flushing" is indicated as "Off," headspace flushing as described herein is not used. For examples in Table 1 where "Post-Deprotection Washes" are used, the wash step includes washing twice with 4 mL of DMF after each deprotection step.

[0212] After the synthesis of the JR 10-mer is completed, the JR 10-mer is cleaved from the solid phase and the crude purity of the resulting JR 10-mer is analyzed, the results of which are also reported in Table 1 below. [Table 1-1] [Table 1-2]

[0213] The results of JR peptide synthesis demonstrate that high purity results can be obtained without cleaning when headspace flushing as described herein is used at each deprotection step. For example, without being bound by any explanation or theory and without limiting the scope of the present invention, it is currently believed that directing an inert gas (nitrogen gas) through the reactor (e.g., entering the reactor through an inlet port as shown in Figures 1A and 1B, exiting the reactor through the headspace, and exiting the reactor through an outlet port (exhaust port)) can result in both a higher gas exchange rate over the deprotection solution and a top-down flow that pushes condensate back into the reactor and then reheats it.

[0214] Furthermore, without being bound by any explanation or theory and without limiting the scope of the present invention, Example 1 is presently believed to demonstrate that pyrrolidine base, which has a lower boiling point (87°C) compared to piperidine (106°C), can be significantly evaporated in the Fmoc removal step, and even small amounts (as low as 2% by volume) can be used to result in essentially complete deprotection and scavenging of the Fmoc group, leaving only residual base that is small enough to minimize problems in the subsequent coupling step. This method is presently believed to enable the high-purity synthesis of JR with a complete cycle waste of only 4.25 mL per amino acid and a total cycle time of approximately 3.5 minutes on a typical 0.1 mmol research scale.

[0215] Recently, more straightforward solvent exchanges have been explored for SPPS. The above experiments also evaluated the disclosed method using a straightforward solvent alternative. To evaluate this, JR sequences are synthesized using N-butylpyrrolidinone (NBP), which completely replaces DMF under both control conditions using post-deprotection washes and no-wash conditions (Table 1, entries 16 and 17). While NBP exhibits reduced purity compared to DMF, it still successfully produces targets with relatively high purity under both wash-based and no-wash conditions. This result demonstrates that the disclosed method can also function with solvent alternatives to DMF.

[0216] Example 2 After deprotection Cleaning none and headspace flushing A protective composition having a low base concentration was used 65-74 ACP, liraglutide, and 1-42 Analysis of one-pot synthesis of β-amyloid sequences Other well-known difficult sequences, i.e. 65-74 ACP (acyl carrier protein), 1-42β-amyloid and liraglutide (entries 1, 2, and 3 in Table 2 below) are investigated next. Sequences are synthesized using solid-phase peptide synthesis using a commercially available automated microwave peptide synthesizer (e.g., Liberty PRIME 2.0 from the Liberty line of microwave peptide synthesizers available from CEM Corporation of Matthews, NC) at the 0.1 mmol scale. 65-74 ACP and liraglutide were synthesized on Fmoc-Gly-Wang-ProTide resin (0.24 meq / g substitution), 1-42 β-amyloid is synthesized on Fmoc-Ala-Wang-ProTide resin (0.23 meq / g substitution). The coupling reaction is carried out in the presence of an Fmoc-protected amino acid (AA).

[0217] Liraglutide synthesis is further investigated in a 0.1 mmol-scale simulated run in a 35 mL reactor (entry 4 in Table 2 below), using the deprotection and coupling conditions for the large-scale (25 mmol) production method previously developed in a 15 L reactor. This method uses a low-temperature deprotection and coupling method limited to 80 °C. To test the robustness of this method at production scale, the deprotection step is extended to 8 min at 80 °C. As reported in Table 2, this method yields liraglutide with a crude purity consistent with previous results obtained with the wash method.

[0218] The deprotection reaction is carried out by adding a pyrrolidine / dimethylformamide (DMF) deprotection reagent (deprotection solution) to the undrained post-coupling mixture (coupling solution). The concentration of pyrrolidine (e.g., the volume percent of pyrrolidine in the reactor based on the total volume of the deprotection reaction mixture (e.g., deprotection reaction solution), including the added pyrrolidine / DMF deprotection solution and the undrained coupling solution from the preceding coupling reaction) is 3% by volume. A pyrrolidine concentration of 3% by volume is selected as the midpoint utilization of the method for the synthesis of these sequences. Microwave power is adjusted to provide the deprotection temperature and deprotection reaction time, as also shown in Table 2 below.

[0219] After synthesis of the sequence is complete, the sequence is cleaved from the solid phase and the crude purity of the resulting sequence is analyzed. The results are reported in Table 2 below. Specifically, the column in Table 2 labeled "Crude Purity (No Wash)" reports the crude purity of peptides produced using the deprotection method according to embodiments of the present disclosure described herein (including headspace flushing and no post-deprotection wash). For comparison, the column in Table 2 labeled "Crude Purity (Wash-Based)" reports the crude purity of peptides produced using a wash-based deprotection method (no headspace flushing and post-deprotection wash). [Table 2]

[0220] 65-74 ACP, 1-42 The results for the β-amyloid and liraglutide sequences also show that high purity results can be obtained without washing when headspace flushing as described herein is used at each deprotection step.

[0221] The preceding Examples 1 and 2 demonstrate that embodiments of the present disclosure, including the deprotection step described herein, can provide improved methods for solid-phase peptide synthesis that can eliminate one or more (e.g., all) washing steps (e.g., post-deprotection washing steps). In some embodiments, the method can use a small amount of deprotection base (e.g., about 3-4% by volume of pyrrolidine) for Fmoc removal; and / or base removal from the deprotection solution by heating (e.g., microwave heating at 80-110°C); and / or nitrogen purging (headspace flushing), resulting in sufficiently low residual base that washing is not necessary before adding the next amino acid. Examples 1 and 2 demonstrate significant robustness, even with longer, more challenging sequences, such as liraglutide. Thus, the method can provide significant savings in solvent and time.

[0222] Example 3 No-clean manufacturing-scale synthesis In preparation for testing the no-wash method at manufacturing scale, deprotection and coupling conditions are investigated on a 25 mmol scale by synthesizing liraglutide at a research scale (0.1 mmol) in a 35 mL reactor, as discussed above in Example 2. This method uses lower temperature conditions for deprotection (80°C for 8 minutes) and coupling (80°C for 5 minutes). Also, as reported above in Example 2, this method yields liraglutide of crude purity consistent with previous results obtained with wash-based methods at research and manufacturing scale.

[0223] Having established high enantiomeric and chromatographic purity using large-scale reaction conditions for the 0.1 mmol synthesis, a 25 mmol wash-free synthesis of liraglutide was performed on a Liberty PRO large-scale microwave peptide synthesizer. Based on the results of an initial optimization experiment involving six amino acid couplings, exemplary conditions for the 25 mmol wash-free synthesis include, but are not limited to, (i) a 2.5% pyrrolidine concentration in the reactor, (ii) 10 minutes for deprotection at 90 °C and 5 minutes for coupling at 80 °C, and (iii) 85 psi of nitrogen pressure with headspace flushing during each deprotection. A four-equivalent excess of the normal amino acid was used for coupling, with only two equivalents of Fmoc-Lys(palmitoyl-Glu-OtBu)-OH. Under these conditions, the 25 mmol wash-free synthesis of liraglutide generated 28.4 L of total waste compared to 139.7 L from the wash-based 25 mmol run, representing an overall waste reduction of approximately 80%. Liraglutide samples from the wash-based and no-wash 25 mmol syntheses show very similar (77-78%) crude purity. These results confirm the general applicability of the no-wash methodology for research-scale as well as production-scale synthesis of peptides. Future use of this technique in large-scale peptide drug manufacturing will help reduce the large amount of waste generated from SPPS.

[0224] The potential for epimerization using this new no-wash method is assessed by measuring the presence of D-amino acids in liraglutide samples. Liraglutide samples synthesized by both the research and production-scale no-wash method are analyzed using established methods (CAT GmbH). See Gerhardt, J.; Nicholson, GJ, Validation of a GC-MS Method for Determination of the Optical Purity of Peptides. GmbH, CAT, Ed.). Results from the crude and corresponding purified samples are then compared to a commercially available sample of liraglutide (Victoza®), as shown in Table 3. The results demonstrate very low levels of epimerization, with the stereochemistry of each amino acid in the liraglutide sequence being controlled to well over 99.5% using this manufacturing method. This demonstrates that all epimerization-related impurities are below the critical 0.5% limit for the newly identified peptide-related impurities. Meeting this limitation is necessary for the application of synthetic peptides as an alternative to approved peptide drugs of recombinant deoxyribonucleic acid (rDNA) origin and for abbreviated new drug applications (ANDAs). The results demonstrate that the disclosed method can be used not only in research and development, but also in manufacturing processes that require stringent purity standards. [Table 3-1] [Table 3-2]

[0225] Example 4 Protein synthesis The capabilities of this method were then further tested by synthesizing two proteins, proinsulin and barstar, with sequence lengths exceeding 80 amino acids. Linear synthesis of long sequences by SPPS is challenging due to the repetitive accumulation of impurities and increased susceptibility to aggregation. The 86-mer sequence of proinsulin and the 89-mer sequence of barstar were selected for synthesis, as they had previously been synthesized using fast-flow methods with overall yields of 1% and 2%, respectively. The fast-flow approach advantageously offers extremely fast synthesis times of only approximately 2.5 minutes per amino acid cycle at small synthesis scales (0.035 mmol for proinsulin; 0.027 mmol for barstar). However, this method requires a large excess of amino acids (approximately 100 equivalents) and wash solvent (approximately 90 mL per amino acid).

[0226] To account for the potential increased synthetic difficulty of these longer sequences, a higher coupling concentration was used with 10 equivalents of amino acid, and deprotection (2 min) and coupling (4.5 min) times were extended. The pyrrolidine concentration was also increased (3.8% for the no-wash method, compared to 6.8% for the more conservative 3 × 4 mL wash method) to quench the larger excess of activated amino acid. These conditions result in a cycle time of approximately 7.3 min per amino acid and a total waste volume of 5.5 mL per amino acid at a 0.1 mmol synthesis scale. Using this no-wash method, both proteins were obtained with crude purity similar to that achieved using washes. Crude proinsulin and barstar samples were then purified by reverse-phase HPLC, resulting in overall yields of 2.4% and 3.4%, respectively. The purified protein samples were identified by deconvoluted mass spectra showing 9395 Da and 10210 Da for proinsulin and barstar, respectively. These protein synthesis examples demonstrate that the disclosed method can be robust for producing highly pure results, even with long, challenging sequences.

[0227] The above demonstrates a no-wash method for solid-phase synthesis of peptides and proteins. The disclosed method may not adversely affect peptide purity relative to wash controls, and when combined with high-temperature reaction conditions, for example, can provide high purity and rapid synthesis times. Compared to traditional SPPS, the disclosed method can reduce waste generation by up to 95%.

[0228] The above also demonstrates the successful application of the disclosed method at a 25 mmol scale in a large-scale microwave peptide synthesizer, and that the disclosed method can be easily scaled. This may enable larger reactor sizes (e.g., up to 15 liters) in which the disclosed method can be applied to synthesis scales exceeding 200 mmol per batch. At these larger scales, hundreds of liters of solvent can be saved per batch of synthesized peptide. Furthermore, by significantly reducing solvent as a material cost, the disclosed method can provide a significant boost to overcoming the cost barriers associated with the use of more expensive and potentially less efficient raw solvents for SPPS.

[0229] method synthesis (a) Peptides: All peptides were synthesized using automated microwave synthesis conditions on a 0.1 mmol scale CEM Liberty PRIME 2.0 system using a one-pot coupling / deprotection methodology. See, for example, U.S. Pat. No. 10,239,914; Singh, SK; Collins, JM, New Developments in Microwave-Assisted Solid Phase Peptide Synthesis. In Peptide Synthesis: Methods and Protocols, Hussein, WM; Skwarczynski, M.; Toth, I., Eds. Springer US: New York, NY, 2020; 95-109. Method details, including reaction times, temperatures, and deprotection reagent concentrations, are listed in Tables 1 and 2. Coupling is performed using Fmoc-amino acid (1.0 mL, 0.5 M in DMF, 5 equiv.), DIC (1.0 mL, 0.75 M in DMF, 7.5 equiv.), and Oxyma (1.5 mL, 0.26 M in DMF, 4 equiv.) at room temperature for 30 s, followed by 105 °C for 60 s. The Fmoc deprotection step using 3% by volume pyrrolidine (see, for example, Example 2) is initiated by adding 0.75 mL of pyrrolidine / DMF (17% v / v) directly to the non-draining post-coupling solution (optimization experiments are performed by adding 0.75 mL of 11.3–25% v / v pyrrolidine / DMF as described in Table 1). A headspace flush pressure of 15 psi is used during the deprotection step. The wash-based method uses 2 × 4 mL DMF post-deprotection washes. Cycles involving deprotection coupling (no-wash case) or deprotection-wash coupling (wash-based case) runs are automatically performed for every amino acid residue in the peptide sequence. JR-10mers are synthesized on Fmoc-Rink Amide ProTide™ LL resin (0.20 meq / g substitution) or Fmoc-Rink Amide MBHA PS resin (0.33 meq / g substitution). 65-74 ACP and liraglutide were synthesized on Fmoc-Gly-Wang-ProTide resin (0.24 meq / g substitution), 1-42β-amyloid is synthesized on Fmoc-Ala-Wang-Portside resin (0.23 meq / g substitution).

[0230] (b) No-Wash Production-Scale Liraglutide Synthesis: Liraglutide was synthesized on a 25 mmol scale using Fmoc-Gly-Wang-ProTide resin (0.24 meq / g substitution) in a 3 L reactor of a Liberty PRO microwave peptide synthesizer. Coupling was performed at 80 °C for 5 min using Fmoc-amino acid (200 mL, 0.5 M in DMF), DIC (50 mL, 4 M in DMF), and Oxyma (225 mL, 0.33 M in DMF). After draining the coupling mixture, an Fmoc deprotection step was performed at 90 °C for 10 min by adding 50 mL of pyrrolidine / DMF (15% v / v), followed by additional DMF (250 mL), to obtain a final concentration of 2.5% pyrrolidine in the reactor. To facilitate the directed flushing of headspace gas during each deprotection step, 85 psi of nitrogen pressure was directed through a spray head on top of the reactor. Fmoc-Lys(palmitoyl-Glu-OtBu)-OH is coupled using a 2 equivalent excess in a wash-based coupling cycle, while all other amino acid residues in the sequence do not use a wash after the deprotection and coupling steps. Fmoc-His(Boc)-OH is coupled using a 2 x 30 min method at 40 °C. The wash-based cycle on the 25 mmol scale uses 4 x 650 mL of DMF and 1 x 800 mL of DMF for the post-deprotection wash. Cycles involving deprotection coupling (no wash) or deprotection-wash coupling (wash-based) runs are automatically performed for all amino acid residues in the peptide sequence.

[0231] (c) Protein: Proteins are synthesized using a one-pot coupling / deprotection methodology on a 0.10 mmol scale CEM Liberty PRIME 2.0 system using automated microwave synthesis conditions. See, for example, U.S. Pat. No. 10,239,914; Singh, SK; Collins, JM, New Developments in Microwave-Assisted Solid Phase Peptide Synthesis. In Peptide Synthesis: Methods and Protocols, Hussein, WM; Skwarczynski, M.; Toth, I., Eds. Springer US: New York, NY, 2020; 95-109. Coupling is performed using Fmoc-amino acid (2.0 mL, 0.5 M in DMF), DIC (1.0 mL, 2.0 M in DMF), and Oxyma (1.75 mL, 0.50 M in DMF) at room temperature for 30 seconds, followed by 4 minutes at 90 °C. The Fmoc deprotection step is performed at 110°C for 2 minutes and is initiated by adding 0.75 mL of pyrrolidine / DMF (28% v / v) directly to the undrained post-coupling solution. A headspace flush pressure of 15 psi is used during the deprotection step. The wash-based method uses 3 x 4 mL DMF post-deprotection washes. Cycles involving deprotection coupling (no-wash case) or deprotection-wash coupling (wash-based case) runs are automatically performed for every amino acid residue in the peptide sequence. Proinsulin 86-mer and barstar 89-mer proteins are synthesized on Fmoc-Rink Amide ProTide™ LL resin (0.18 meq / g substitution).

[0232] Resin cutting (a) Peptide: After synthesis, the peptidyl resin was washed with DCM (3 × 5 mL). Cleavage was carried out with 5 mL of freshly prepared cleavage cocktail [TFA / TIS / H2O / DODT (92.5 / 2.5 / 2.5 / 2.5)] at 38 °C for 30 min. The TFA solution was collected by filtration, and ice-cold ethyl ether was added, followed by centrifugation at 3500 rpm for 5 min to obtain the crude peptide as a white pellet.

[0233] (b) Protein: After synthesis, the peptidyl resin was washed with DCM (3 × 15 mL). After the addition of 7.5 mL of [TFA / TIS / HO / DODT (6 / 0.5 / 0.5 / 0.5)], cleavage was carried out at room temperature for 5 h using the slow cleavage method, with 4 mL of TFA added every hour over 3 h. After the third addition (final concentration: TFA / TIS / HO / DODT (18 / 0.5 / 0.5 / 0.5)), cleavage was allowed to proceed for an additional 2 h. The TFA solution was collected by filtration, and ice-cold ethyl ether was added, followed by centrifugation at 3900 rpm for 3 min to obtain the crude peptide as a white pellet.

[0234] analysis After dissolving the pellet in 10% acetic acid / deionized water, all peptides were lyophilized overnight. Lyophilized aliquots of peptides were taken up in deionized water (approximately 2 mg / mL peptide concentration), and clear solutions were obtained by adding acetonitrile, ammonium hydroxide (up to 1%), or acetic acid (up to 9%), followed by sonication. Protein samples (barstar and proinsulin) were dissolved in a solution of HO / ACN / AcOH (8:1:1) by sonication for 1 hour. The peptide / protein solution was analyzed on a Vanquish UHPLC system (Thermo Fisher; Waltham, MA, USA) equipped with a Waters ACQUITY UPLC BEH C8 reversed-phase column (100 x 2.1 mm i.d., 1.7 μm, 130 Å; Waters Corporation, Milford, MA, USA) coupled to an Exactive™ Plus Orbitrap™ mass spectrometer (Thermo Fisher; Waltham, MA, USA) via an ESI source (operated in positive polarity mode). Deconvoluted mass spectra of proteins are obtained using UniDec (Universal Deconvolution) Version 6.0.1, developed by Marty et al., Marty, MT; Baldwin, AJ; Marklund, EG; Hoshberg, GKA; Benesch, JLP; Robinson, CV, Bayesian Deconvolution of Mass and Ion Mobility Spectra: From Binary Interactions to Polydisperse Ensembles. Anal. Chem. 2015, 87(8), 4370-4376. Analytical runs are performed using 0.05% trifluoroacetic acid in water (A) and 0.05% trifluoroacetic acid in acetonitrile (B) with a gradient elution of 10–70% B at a flow rate of 0.5 mL / min. 1-42 For all peptides except β-amyloid, the column and autosampler are maintained at 40° C. and 24° C., respectively. 1-42β-Amyloid is analyzed on a Waters ACQUITY UPLC BEH C8 reversed-phase column (100 × 2.1 mm i.d., 1.7 μm, 130 Å; Waters Corporation, Milford, MA, USA) with a column temperature of 70°C and a flow rate of 0.6 mL / min on a Waters Acquity RP-UPLC system equipped with a PDA detector coupled to a 3100 Single Quad mass spectrometer.

[0235] purification Lyophilized protein samples (barstar and proinsulin) are dissolved (barstar: 6.4 mg / mL in water containing 0.2% ammonium hydroxide and 10 mM DTT; proinsulin: diluted to 8 mg / mL in 6 M GdnHCl containing 0.1% ammonium hydroxide and 100 mM DTT and sonicated for 1 h at 40 °C). Lyophilized liraglutide is dissolved in 20% acetonitrile (8.1 mg / mL). Prior to purification, samples are filtered through 0.45 µm regenerated cellulose syringe filters (Phenomenex; Torrance, CA, USA).

[0236] Purification is completed on a CEM Prodigy HPLC system, which includes an integrated heating system (column oven and mobile phase heater) that allows for highly efficient, high-temperature operation. Barstar and proinsulin purification is performed at 60 °C using a Waters Protein XBridge C4 column (19 × 150 mm, 5 μm, 300 Å; Waters Corporation, Milford, MA, USA) with a mobile phase consisting of 0.1% trifluoroacetic acid in water (A) and 0.1% trifluoroacetic acid in acetonitrile (B). Liraglutide purification is performed using the same conditions but on a Waters XBridge C8 column (19 × 150 mm, 5 μm, 130 Å).

[0237] Optimized gradient conditions were determined by first injecting approximately 10 mg of crude sample with a 10-70% B screening gradient (20 min gradient; approximately 3% B / CV) at a flow rate of 27 mL / min. The target peak retention time was then used to calculate the optimized focusing gradient for each purification (using CEM focusing gradient calculation software, version 1.1.673.1159). Protein samples were purified using a 25 min focused gradient (proinsulin: 27-39% B; barstar: 39-51% B), while liraglutide was purified using an 18 min focused gradient (46-55% B).

[0238] Exemplary embodiments have been disclosed above. However, the present invention is not limited to such embodiments. In the foregoing, descriptions of sequences of steps or other operations are presented for purposes of example, not for the purpose of limiting the scope of the disclosure (e.g., steps or operations may be performed in a different order than described, and steps and operations may be omitted and / or added, where appropriate). The drawings are schematic and, therefore, are not necessarily drawn to scale. Unless otherwise indicated, certain terms have been used in a generic and descriptive sense and not for purposes of limitation.

[0239] Numerical values ​​provided throughout this disclosure may be approximate, and for each range specified in this disclosure, all values ​​within the range (including endpoints) and all subranges within the range are also disclosed. Those skilled in the art will also readily understand that in different embodiments of the features of the present disclosure, reasonably different engineering tolerances, precision, and / or accuracy (e.g., with respect to numerical values) may be applicable and may be appropriate to achieve desired results. Thus, those skilled in the art will readily understand the meaning, usage, etc. of terms such as "substantially," "about," "approximately," etc., herein. As a non-limiting example, the term "about" can indicate that the numerical value may vary by plus or minus 25%, e.g., plus or minus 20%, e.g., plus or minus 15%, e.g., plus or minus 10%, e.g., plus or minus 5%, e.g., plus or minus 4%, e.g., plus or minus 3%, e.g., plus or minus 2%, e.g., plus or minus 1%, e.g., plus or minus less than 1%, e.g., plus or minus 0.5%, e.g., plus or minus less than 0.5%, including all values ​​and subranges therebetween for each of the above ranges.

[0240] As used herein, the phrase "and / or" includes any and all combinations of one or more of the associated listed items (e.g., it can refer to elements that are conjunctive in some embodiments and elements that are disjunctive in other embodiments), and in some embodiments, optionally in combination with other elements not specifically identified by the phrase "and / or." As non-limiting examples, "A and / or B" can refer, in some embodiments, to A without B; in some embodiments, to B without A; in some embodiments, to both A and B; etc.

[0241] As used herein, the phrase "at least one," referring to a list of one or more elements, can refer to at least one element selected from any one or more of the elements in the list of elements, but does not necessarily include at least one of each and every element specifically listed in the list of elements, and does not exclude any combinations of elements in the list of elements. In some embodiments, elements, whether related to the specifically identified elements or not, may optionally be present other than the elements specifically identified in the list of elements to which the phrase "at least one" refers. As a non-limiting example, "at least one of A and B" ("at least one of A or B," "at least one of A and / or B") can refer, in one embodiment, to at least one, optionally more than one, A, with no B present (and optionally including elements other than B); in some embodiments, to at least one, optionally more than one, A, with no A present (and optionally including elements other than A); in some embodiments, to at least one, optionally more than one, A, and at least one, optionally more than one, B (and optionally including other elements), etc.

[0242] As used herein, the indefinite articles "a" and "an" refer to at least one thing ("a" and "an" can refer to singular and / or plural elements).

Claims

1. 1. A method for deprotecting a protected peptide during solution phase peptide synthesis comprising a deprotection step and a coupling step, said deprotection method comprising: removing a protecting group from the protected peptide in a liquid phase, wherein the C-terminus of the protected peptide is protected in a reactor containing the deprotecting base at a temperature at least 35° C. lower than the boiling point of the deprotecting base for 1.5 hours or less to provide a deprotected peptide; the deprotection base is present in the reactor in an amount of from 0 to greater than about 5 volume percent, based on the total volume (100 volume percent) of the deprotection reaction mixture in the reactor; removing the protecting groups of the protected peptide in a liquid phase, wherein a majority of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting groups of the protected peptide; directing an inert gas through the reactor to aid in removing vaporized deprotection base from the interior of the reactor during removal of the protecting group of the protected peptide; and extracting residual deprotected base from the deprotection reaction mixture after removing the protecting group and before a subsequent coupling step with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture.

2. 2. The method of claim 1, wherein the directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

3. 10. The method of claim 1, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

4. 10. The method of claim 1, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

5. 10. The method of claim 1, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

6. 10. The method of claim 1, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

7. 10. The method of claim 1, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

8. 10. The method of claim 1, comprising removing the protecting group of the protected amino acid using microwave radiation at a temperature of about 90°C to about 120°C.

9. 10. The method of claim 1, wherein the deprotecting base has a boiling point of less than 107°C.

10. 2. The method of claim 1, wherein the deprotecting base is pyrrolidine.

11. 1. A method for deprotecting a protected peptide during solution phase peptide synthesis comprising a deprotection step and a coupling step, said deprotection method comprising: removing a protecting group from the protected peptide in a liquid phase, wherein the C-terminus of the protected peptide is protected in a reactor containing the deprotecting base at a temperature at least 35° C. lower than the boiling point of the deprotecting base for 1.5 hours or less to provide a deprotected peptide; the deprotection base is present in the reactor in an amount of from 0 to greater than about 5 volume percent, based on the total volume (100 volume percent) of the deprotection reaction mixture in the reactor; removing the protecting groups of the protected peptide in a liquid phase, wherein a majority of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting groups of the protected peptide; directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor; and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

12. 12. The method of claim 11, further comprising extracting residual deprotected base from the deprotection reaction mixture after removing the protecting group and before the subsequent coupling step with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture.

13. 12. The method of claim 11, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

14. 12. The method of claim 11, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

15. 12. The method of claim 11, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

16. 12. The method of claim 11, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

17. 12. The method of claim 11, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

18. 12. The method of claim 11, comprising removing the protecting group of the protected amino acid using microwave radiation at a temperature of about 90°C to about 120°C.

19. 12. The method of claim 11, wherein the deprotecting base has a boiling point of less than 107°C.

20. 12. The method of claim 11, wherein the deprotecting base is pyrrolidine.

21. 1. A method for liquid phase peptide synthesis comprising: deprotecting the protected peptide in the liquid phase, wherein the C-terminus of the protected peptide is protected, to provide a deprotected peptide in a reactor; The deprotection step comprises: removing the protecting group of the protected peptide using the deprotecting base at a temperature at least 35° C. below the boiling point of the deprotecting base for 1.5 hours or less; the deprotection base is present in the reactor in an amount of from 0 to greater than about 5 volume percent, based on the total volume (100 volume percent) of the deprotection reaction mixture in the reactor; during removal of the protecting group, a majority of the deprotecting base evaporates into the upper interior of the reactor; directing an inert gas through the reactor to remove evaporated deprotection base from the interior of the reactor during removal of the protecting group; extracting residual deprotected base from the deprotection reaction mixture after removing the protecting group and before subsequent coupling steps with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture; coupling an amino acid to said deprotected peptide to form a peptide from said deprotected peptide and said amino acid.

22. 22. The method of claim 21 , wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening located at an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed at an upper portion of the reactor.

23. 22. The method of claim 21, comprising repeating the deprotection, extraction and coupling to form a peptide comprising one or more further consecutive amino acids.

24. 22. The method of claim 21, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

25. 22. The method of claim 21, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

26. 22. The method of claim 21, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

27. 22. The method of claim 21, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

28. 22. The method of claim 21, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

29. 22. The method of claim 21, comprising removing the protecting group of the protected amino acid using microwave radiation at a temperature of about 90°C to about 120°C.

30. 22. The method of claim 21, wherein the deprotecting base has a boiling point of less than 107°C.

31. 22. The method of claim 21, wherein the deprotecting base is pyrrolidine.

32. 1. A method for liquid phase peptide synthesis comprising: deprotecting the protected peptide in the liquid phase, wherein the C-terminus of the protected peptide is protected, to provide a deprotected peptide in a reactor; removing the protecting groups of the protected peptide using the deprotecting base at a temperature at least 35° C. lower than the boiling point of the deprotecting base for 1.5 hours or less, wherein the deprotecting base is present in the reactor in an amount of greater than 0 to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor, and wherein most of the deprotecting base evaporates into the upper interior of the reactor during the removal of the protecting groups; directing an inert gas into the upper interior of the reactor through a first opening disposed in the upper portion of the reactor; venting the inert gas and the evaporated deprotection base from the upper interior of the reactor through a second opening disposed at the top of the reactor to remove the evaporated deprotection base from the reactor during the removal of the protecting group; coupling an amino acid to said deprotected peptide to form a peptide from said deprotected peptide and said amino acid; A method comprising:

33. 33. The method of claim 32, further comprising extracting residual deprotected base from the deprotection reaction mixture after removing the protecting group and prior to successive coupling steps with a total volume of extraction solvent that is two times or less the total volume of the deprotection reaction mixture.

34. 33. The method of claim 32, comprising repeating the deprotection and coupling to form a peptide comprising one or more additional consecutive amino acids.

35. 33. The method of claim 32, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

36. 33. The method of claim 32, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

37. 33. The method of claim 32, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

38. 33. The method of claim 32, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

39. 33. The method of claim 32, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

40. 33. The method of claim 32, comprising removing the protecting group of the protected amino acid using microwave radiation at a temperature of about 90°C to about 120°C.

41. 33. The method of claim 32, wherein the deprotecting base has a boiling point of less than 107°C.

42. 33. The method of claim 32, wherein the deprotecting base is pyrrolidine.

43. 1. A method for deprotecting a protected amino acid during solid phase peptide synthesis (SPPS), comprising a deprotection step and a coupling step, the deprotection method comprising: adding a deprotection solution to the reactor comprising a sufficient amount of deprotection base to provide from greater than 0 to about 5 volume percent of deprotection base in the reactor, based on 100% by volume of the total volume of the deprotection reaction mixture in the reactor, wherein the volume percent of deprotection base in the deprotection solution added to the reactor is greater than the volume percent of deprotection base in the deprotection reaction mixture after the deprotection solution has been added to the reactor, and the volume percent of deprotection base in the deprotection solution added to the reactor is about 30% by volume or less, based on the total volume of the deprotection solution added to the reactor; removing the protecting groups of the protected amino acids in the reactor with the deprotecting base to obtain deprotected amino acids, wherein at least a portion of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting groups of the protected amino acids; and during removal of the protecting group of the protected amino acid, directing an inert gas through the reactor to remove vaporized deprotected base from the interior of the reactor.

44. 44. The method of claim 43, comprising a coupling step preceding removal of the protecting group of the protected amino acid, wherein the reactor contains a post-coupling solution following completion of the preceding coupling step, wherein the post-coupling solution is not drained from the reactor after the preceding coupling step and prior to removal of the protecting group of the protected amino acid, and wherein the deprotection reaction mixture comprises a mixture of a deprotection solution comprising the deprotecting base and the post-coupling solution from the preceding coupling step.

45. 44. The method of claim 43, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

46. 44. The method of claim 43, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

47. 44. The method of claim 43, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

48. 44. The method of claim 43, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

49. 44. The method of claim 43, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

50. 44. The method of claim 43, wherein said directing comprises directing an inert gas into a lower interior of the reactor through a first opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

51. 44. The method of claim 43, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, directing an inert gas into a lower interior of the reactor through a second opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a third opening disposed in an upper portion of the reactor.

52. 44. The method of claim 43, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

53. 44. The method of claim 43, comprising heating the protected amino acid and the deprotected base during removal of the protecting group from the protected amino acid.

54. 54. The method of claim 53, comprising heating the protected amino acid and the deprotected base at a temperature of about 60°C to about 120°C.

55. 55. The method of claim 54, comprising heating the protected amino acid and the deprotected base using microwave radiation at a temperature of about 90°C to about 120°C.

56. 54. The method of claim 53, wherein the deprotecting base has a boiling point of less than 107°C, and the difference between the temperature to which the protected amino acid and the deprotecting base are heated during removal of the protecting group from the protected amino acid and the boiling point of the deprotecting base is in the range of about 1°C to about 50°C.

57. 44. The method of claim 43, wherein the deprotecting base is pyrrolidine.

58. 44. The method of claim 43, wherein the volume percent of the deprotection base in the deprotection solution added to the reactor is about 25 volume percent or less, based on the total volume of the deprotection solution added to the reactor.

59. 44. The method of claim 43, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.10 mmol / g to 0.35 mmol / g.

60. 60. The method of claim 59, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.20 mmol / g to 0.30 mmol / g.

61. 54. The method of claim 53, the deprotecting base is pyrrolidine; the protected amino acid is attached to a solid PEG-PS (polyethylene glycol-polystyrene) resin support having a resin substitution of 0.20 mm / g to 0.25 mmol / g; the protecting group of the protected amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group; and The method wherein the heating is at a temperature of about 60° C. or greater.

62. 1. A method for solid phase peptide synthesis comprising: deprotecting the first protected amino acid to provide an unprotected amino acid; coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids; The deprotection step comprises: adding a deprotection solution comprising a deprotection base to a reactor containing the first protected amino acid, the deprotection solution being added to the reactor in an amount sufficient to provide greater than 0 to about 5 volume % of the deprotection base in the reactor, based on a total volume (100 volume %) of the deprotection reaction mixture in the reactor, the volume % of the deprotection base in the deprotection solution added to the reactor being greater than the volume % of the deprotection base in the deprotection reaction mixture after the deprotection solution has been added to the reactor, and the volume % of the deprotection base in the deprotection solution added to the reactor being about 30 volume % or less, based on the total volume of the deprotection solution added to the reactor; removing the protecting group of the first protected amino acid in the reactor with the deprotecting base, wherein at least a portion of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group; directing an inert gas through the reactor to remove vaporized deprotecting base from the interior of the reactor during removal of the protecting group.

63. 63. The method of claim 62, comprising repeating the deprotection and coupling to form a peptide comprising the first, second, and one or more consecutive amino acids.

64. 63. The method of claim 62, comprising a coupling step preceding the deprotection step, wherein the reactor contains a post-coupling solution following completion of the preceding coupling step, wherein the post-coupling solution is not drained from the reactor after the preceding coupling step and before the deprotection step, and wherein the deprotection reaction mixture comprises a mixture of a deprotection solution comprising the deprotection base and the post-coupling solution from the preceding coupling step.

65. 63. The method of claim 62, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

66. 63. The method of claim 62, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

67. 63. The method of claim 62, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

68. 63. The method of claim 62, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

69. 63. The method of claim 62, wherein said directing comprises directing an inert gas into a lower interior of the reactor through a first opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

70. 63. The method of claim 62, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, directing an inert gas into a lower interior of the reactor through a second opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a third opening disposed in an upper portion of the reactor.

71. 63. The method of claim 62, wherein the inert gas is nitrogen gas and the directing comprises continuously directing the nitrogen gas through the reactor.

72. 63. The method of claim 62, comprising heating the protected amino acid and the deprotected base during removal of the protecting group from the protected amino acid.

73. 73. The method of claim 72, comprising heating the protected amino acid and the deprotected base using microwave radiation at a temperature of about 60°C to about 120°C.

74. 73. The method of claim 72, wherein the deprotecting base has a boiling point of less than 107°C, and the difference between the temperature to which the protected amino acid and the deprotecting base are heated during removal of the protecting group from the protected amino acid and the boiling point of the deprotecting base is in the range of about 1°C to about 50°C.

75. 63. The method of claim 62, wherein the deprotecting base is pyrrolidine.

76. 63. The method of claim 62, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.10 mmol / g to 0.35 mmol / g.

77. 77. The method of claim 76, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.20 mmol / g to 0.30 mmol / g.

78. 73. The method of claim 72, the deprotecting base is pyrrolidine; the protected amino acid is directly or indirectly attached to a solid PEG-PS (polyethylene glycol-polystyrene) resin support having a resin substitution of 0.20 mm / g to 0.25 mmol / g; the protecting group of the protected amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group; and The method wherein the heating is at a temperature of about 60° C. or greater.

79. 63. The method of claim 62, wherein said method does not include washing between the deprotection and coupling steps of one or more deprotection-coupling cycles of said solid phase peptide synthesis method.

80. 63. The method of claim 62, further comprising washing the interior of the reactor between the deprotection step and the coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method with a washing composition in an amount approximately equal to the total volume of the deprotection reaction mixture.

81. 63. The method of claim 62, further comprising washing the interior of the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method with a washing composition in an amount less than the total volume of the deprotection reaction mixture.

82. 82. The method of claim 81, further comprising washing the interior of the reactor with a washing composition in an amount less than or about half the total volume of the deprotection reaction mixture between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

83. 82. The method of claim 81, further comprising washing the interior of the reactor with a washing composition in an amount of less than or about one-third of the total volume of the deprotection reaction mixture between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

84. 63. The method of claim 62, comprising washing the inside of the reactor with a solvent in an amount less than twice the bed volume of resin present in the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

85. 85. The method of claim 84, comprising washing the inside of the reactor with a solvent in an amount of less than 1 bed volume of resin present in the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

86. 1. A method for deprotecting a protected amino acid during solid phase peptide synthesis (SPPS), comprising a deprotection step and a coupling step, the deprotection method comprising: removing a protecting group of a protected amino acid attached to a solid resin support in a reactor having a size of at least 3 liters with a deprotecting base to provide a deprotected amino acid; the deprotection base is present in the reactor in an amount of from 0 to greater than about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor; At least a portion of the deprotecting base evaporates into the upper interior of the reactor during removal of the protecting group of the protected amino acid. removing the protecting group of said protected amino acid coupled to a solid resin support in a reactor having a size of at least 3 liters with a deprotecting base to provide a deprotected amino acid; and directing an inert gas having a pressure of about 50 psi to about 95 psi through the reactor to remove vaporized deprotected base from the interior of the reactor while removing the protecting group of the protected amino acid.

87. 87. The method of claim 86, comprising a coupling step preceding removal of the protecting group of the protected amino acid, wherein the reactor contains a post-coupling solution following completion of the preceding coupling step, wherein the post-coupling solution is not drained from the reactor after the preceding coupling step and prior to removal of the protecting group of the protected amino acid, and wherein the deprotection reaction mixture comprises a mixture of a deprotection solution comprising the deprotecting base and the post-coupling solution from the preceding coupling step.

88. 87. The method of claim 86, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

89. 87. The method of claim 86, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

90. 87. The method of claim 86, wherein the deprotection base is present in the reactor in an amount of about 2% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

91. 87. The method of claim 86, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

92. 87. The method of claim 86, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

93. 87. The method of claim 86, wherein said directing comprises directing an inert gas into a lower interior of the reactor through a first opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

94. 87. The method of claim 86, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, directing an inert gas into a lower interior of the reactor through a second opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a third opening disposed in an upper portion of the reactor.

95. 87. The method of claim 86, wherein the inert gas is nitrogen gas, and the method comprises continuously directing the nitrogen gas through the reactor.

96. 87. The method of claim 86, comprising heating the protected amino acid and the deprotected base during removal of the protecting group from the protected amino acid.

97. 97. The method of claim 96, comprising heating the protected amino acid and the deprotected base at a temperature of about 60°C to about 120°C.

98. 98. The method of claim 97, comprising heating the protected amino acid and the deprotected base using microwave radiation at a temperature of about 90°C to about 120°C.

99. 97. The method of claim 96, wherein the deprotecting base has a boiling point of less than 107°C, and the difference between the temperature to which the protected amino acid and the deprotecting base are heated during removal of the protecting group from the protected amino acid and the boiling point of the deprotecting base is in the range of about 1°C to about 50°C.

100. 87. The method of claim 86, wherein the deprotecting base is pyrrolidine.

101. 87. The method of claim 86, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.10 mmol / g to 0.35 mmol / g.

102. 102. The method of claim 101, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.20 mmol / g to 0.30 mmol / g.

103. the deprotecting base is pyrrolidine; the protected amino acid is attached to a solid PEG-PS (polyethylene glycol-polystyrene) resin support having a resin substitution of 0.20 mm / g to 0.25 mmol / g; the protecting group of the protected amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group; and The heating is carried out at a temperature of about 60°C or higher.

97. The method of claim 96.

104. 1. A method for solid phase peptide synthesis having a synthesis scale of about 25 mmol or greater, comprising: deprotecting the first protected amino acid to provide an unprotected amino acid; coupling a second amino acid to the deprotected amino acid to form a peptide from the first and second amino acids; The deprotection step comprises: removing the protecting group of the protected amino acid in a reactor having a size of at least 3 liters with a deprotecting base, wherein the deprotecting base is present in the reactor in an amount of 0 to about 5 volume % based on the total volume (100 volume %) of the deprotection reaction mixture in the reactor, and at least a portion of the deprotecting base evaporates into the upper interior of the reactor during the removal of the protecting group; and directing an inert gas having a pressure of about 50 psi to about 95 psi through the reactor during removal of the protecting group to remove vaporized deprotected base from the interior of the reactor.

105. 105. The method of claim 104, comprising repeating the deprotection and coupling to form a peptide comprising the first, second, and one or more consecutive amino acids.

106. 105. The method of claim 104, comprising a coupling step preceding the deprotection step, wherein the reactor contains a post-coupling solution following completion of the preceding coupling step, wherein the post-coupling solution is not drained from the reactor after the preceding coupling step and before the deprotection step, and wherein the deprotection reaction mixture comprises a mixture of a deprotection solution comprising the deprotection base and the post-coupling solution from the preceding coupling step.

107. 105. The method of claim 104, wherein the deprotection base is present in the reactor in an amount of about 1% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

108. 105. The method of claim 104, wherein the deprotection base is present in the reactor in an amount of about 2% to about 5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

109. 105. The method of claim 104, wherein the deprotection base is present in the reactor in an amount of about 3% to about 4.5% by volume, based on the total volume (100% by volume) of the deprotection reaction mixture in the reactor.

110. 105. The method of claim 104, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

111. 105. The method of claim 104, wherein said directing comprises directing an inert gas into a lower interior of the reactor through a first opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a second opening disposed in an upper portion of the reactor.

112. 105. The method of claim 104, wherein said directing comprises directing an inert gas into an upper interior of the reactor through a first opening disposed in an upper portion of the reactor, directing an inert gas into a lower interior of the reactor through a second opening disposed in a lower portion of the reactor, and venting the inert gas and evaporated deprotected base from the upper interior of the reactor through a third opening disposed in an upper portion of the reactor.

113. 105. The method of claim 104, wherein the inert gas is nitrogen gas and the directing comprises continuously directing the nitrogen gas through the reactor.

114. 105. The method of claim 104, comprising heating the protected amino acid and the deprotected base during removal of the protecting group from the protected amino acid.

115. 115. The method of claim 114, comprising heating the protected amino acid and the deprotected base using microwave radiation at a temperature of about 60°C to about 120°C.

116. 115. The method of claim 114, wherein the deprotecting base has a boiling point of less than 107°C, and the difference between the temperature to which the protected amino acid and the deprotecting base are heated during removal of the protecting group from the protected amino acid and the boiling point of the deprotecting base is in the range of about 1°C to about 50°C.

117. 105. The method of claim 104, wherein the deprotecting base is pyrrolidine.

118. 105. The method of claim 104, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.10 mmol / g to 0.35 mmol / g.

119. 119. The method of claim 118, wherein the protected amino acid is attached to a solid resin support having a resin substitution of 0.20 mmol / g to 0.30 mmol / g.

120. the deprotecting base is pyrrolidine; the protected amino acid is directly or indirectly attached to a solid PEG-PS (polyethylene glycol-polystyrene) resin support having a resin substitution of 0.20 mm / g to 0.25 mmol / g; the protecting group of the protected amino acid is a 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group; and The heating is carried out at a temperature of about 60°C or higher. The method of claim 114.

121. 105. The method of claim 104, wherein said method does not include washing between the deprotection and coupling steps of one or more deprotection-coupling cycles of said solid phase peptide synthesis method.

122. 105. The method of claim 104, further comprising washing the interior of the reaction vessel between the deprotection step and the coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method with a washing composition in an amount approximately equal to the total volume of the deprotection reaction mixture.

123. 105. The method of claim 104, further comprising washing the interior of the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method with a washing composition in an amount less than the total volume of the deprotection reaction mixture.

124. 124. The method of claim 123, further comprising washing the interior of the reactor with a washing composition in an amount less than or about half the total volume of the deprotection reaction mixture between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

125. 124. The method of claim 123, further comprising washing the interior of the reactor with a washing composition in an amount of less than or about one-third of the total volume of the deprotection reaction mixture between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

126. 105. The method of claim 104, comprising washing the inside of the reactor with a solvent in an amount less than twice the bed volume of resin present in the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

127. 127. The method of claim 126, comprising washing the inside of the reactor with a solvent in an amount of less than 1 bed volume of resin present in the reactor between a deprotection step and a coupling step of one or more deprotection-coupling cycles of the solid phase peptide synthesis method.

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