[0001] This application includes material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent disclosure, as it appears in the Patent and Trademark Office files or records, but otherwise reserves all copyright rights whatsoever.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application cross-references WO Application No. WO2017112809A1,“System and method for solution phase gap peptide synthesis,” and US Provisional Patent Application No. 62/667,591,“Method for Solution-Phase Peptide Synthesis.”

TECHNICAL FIELD

[0003] The present disclosure relates in general to the field of peptide synthesis. In general, the system provides for solution-phase peptide synthesis methods in organic solvents, some of which are immiscible with aqueous solutions, alkane solvents, or both, that allow for purification with minimal chromatography, recrystallization, or polymer supports, and allows for high overall yield and purity. The disclosed systems and methods support a wide variety of scenarios and include various products and services.

STATEMENT OF FEDERALLY FUNDED RESEARCH

[0004] None.

BACKGROUND OF THE DISCLOSURE

[0005] In the fields of synthetic and organic chemistry, many of the solvents utilized raise serious environmental and public health concerns due to their toxicities. Specifically, in peptide synthesis, solvents such as dichloromethane and DMF are used in massive quantities to manufacture peptide therapeutics on large scales. These chemicals constitute most of the waste generated in these processes. In response, recent research efforts have made significant advancements in adapting existing synthetic methodologies to“green” solvents and purification techniques. Additionally, there is a need in the industry for peptide synthesis technologies that are amenable to large-scale syntheses; this generally would require a deviation from the traditional solid-phase peptide synthesis (SPPS) methodologies. On a larger scale, a one-pot reaction with minimal concentrations, distillations, and purification steps is very desirable. Such attributes provide the cornerstones of this non-traditional solution-phase peptide synthesis (SolPPS) technology, and such a SolPPS technology with these attributes that utilizes exclusively green solvents offers significant advancements to the peptide synthesis industry.

[0006] Current peptide synthesis methodologies largely revolve around SPPS. Developed by Merrifield in the 1960’s, SPPS has become a standard protocol used by multiple scientific disciplines for research and manufacturing (See FIG. 1A). The advantages of the polymer support lie in its ability to allow facile purification of the growing peptide after each

coupling/deprotection step, which avoids the use of column chromatography. SPPS is amenable to a wide range of protecting and coupling strategies, with the Fmoc//Bu strategy being among the most popular in the industry. U.S. Patent No. 8,383,770 B2 teaches the use of the Fmoc and Boc N-terminus protecting groups in SPPS. Boc and Fmoc groups have been used for decades in all areas of peptide chemistry, and the preferred Fmoc group is almost entirely restricted to solid phase. The key disadvantage of SPPS lies in the difficulty of scale-up: many polymer supports are expensive, and occupy the majority of the mass of the material to be worked with. Examples of economically feasible Fmoc protection schemes in solution are scarce, with few examples in the literature at all. [0007] An example of an attempt at adapting Fmoc-based protecting strategies to a SolPPS method is seen in U.S. Patent No. 5,516,891 A. Again, the Fmoc peptide synthesis is almost entirely restricted to SPPS due to the formation of N-fluorenylmethylpiperidine (NFMP) as a side product during deprotection, which is difficult to remove without polymer supports. The standard protocol for Fmoc deprotection is to stir the Fmoc-peptide in a solution of DMF or DCM with excess piperidine, deprotecting the Fmoc group and forming NFMP in the process. The‘891 patent teaches removal of this impurity by deprotecting with 4-aminomethylpiperidine (4AMP) instead of piperidine. This forms NFMP-CH2NH2 instead of NFMP, which due to the presence of the extra amino group, can be extracted into water. The problem with this method lies in the high cost of using 4AMP. Per Sigma Aldrich, 4AMP costs $3.80 per gram, while piperidine only costs $0.12 per gram. This is why this method is cost prohibitive, and why it has not been accepted by the industry.

[0008] Another example of Fmoc-based SolPPS can be seen in published patent application WO2017112809A1. This patent teaches the use of a C-terminus group-assisted purification (GAP) protecting group to control the solubility of the target peptide to allow for selective precipitation after each successive coupling reaction. While this technology adapted Fmoc/tBu chemistry to solution-phase in a much more economically feasible manner, there are potential limitations inherent in the method. Namely, this method requires precipitation to remove the fulvene impurity (NFMP). While this method of removing NFMP is potentially more cost- effective than using 4AMP like in the‘891 patent, precipitations such as these can be problematic on a large scale, potentially limiting the overall scalability of the method.

[0009] Because of these issues with scale up in SPPS, as well as the common necessity of toxic solvents such as dichloromethane and DMF, there is a need in the industry for both SolPPS methods that are more amenable to scale up, and green SolPPS methods that pose less risks to the environment and public health. One example of a SolPPS method run exclusively in green solvents is seen in a 2017 issue of Green Chemistry. There, Lawrenson et. al. succeeded in performing amino acid coupling and deprotection reactions in propylene carbonate (PC), a green solvent. The coupling reagents HOBt and EDC were used, and the Boc-protected amino acids and subsequent peptides were successfully deprotected with a number of different acidic conditions. While this research proved that solution-phase peptide synthesis with different protection strategies could be performed in PC, it failed to address and resolve many of the issues that hinder and increase the cost of large-scale peptide manufacturing. Specifically, Lawrenson et. al.’s method required short-path distillation to remove PC from the peptides after the coupling reactions; such distillation was required after Boc deprotection as well. Because of these shortcomings, such a peptide synthesis technique would not be as amenable to upscale as one that avoids distillations. There is, therefore, an apparent need in the art to develop an economically feasible and green SolPPS system capable of overcoming these limitations.

SUMMARY OF THE DISCLOSURE

[0010] The present disclosure addresses failings in the art by providing greener systems and methods for peptide synthesis utilizing reactions which occurs in solution phase, without the mass waste of polymer supports, but retains all of the purification benefits of SPPS as an alternative to both traditional SolPPS as well as SPPS, affording advantages of both methods. By utilizing the solubility properties of green solvents in conjunction with solubility characteristics of protected peptides, a SolPPS strategy is presented that is economically feasible and useful for the commercial production of peptides. [0011] It is therefore an object of the present disclosure to enable SolPPS in a number of solvents while avoiding distillation and accomplishing purification of the peptide products through liquid-liquid extraction techniques. Such green SolPPS may be achieved with a protection strategy that lends protected amino acids and potentially resulting peptides solubility in the reaction solvent and/or either aqueous solutions or alkane solvents. A number of different protection strategies are possible, including, but not limited to, Fmoc//Bu, Boc/benzyl, Cbz, etc. Many of these strategies are currently used in both SPPS and SolPPS methods but are commonly restricted to toxic/non-green solvents in both phases. It is therefore also an object of the present disclosure to provide an amount of target peptide in high yield and high purity via utilization of such a protecting group strategy (such as Fmoc//Bu or GAP -PS as an example) in entirely green solvents while avoiding distillation and relying entirely on liquid-liquid extraction methods for a one-pot purification process. Various amino acids protected in different ways have shown high solubility and coupling reaction efficiency in green solvents such as PC.

[0012] In one aspect, an adaptation of the group assisted purification peptide synthesis method to propylene carbonate is provided. As see in WO Application No. WO2017112809A1, the GAP method allows for a high yield with high purity using the Fmoc//But strategy with solution-phase peptide synthesis (SolPPS). In one embodiment, the present invention enhances the scalability of the Group-Assisted Purification (GAP) method by adapting it to run in PC, avoiding the post coupling precipitation step and purifying the target peptide through a series of liquid-liquid extractions.

[0013] In another aspect, an adaptation of GAP peptide synthesis is presented wherein the chemistry is run in a DMF/DCM solvent mixture. The DMF component of the solvent mixture prevents solubility in alkane solvents, while the DCM component of the mixture prevents solubility in aqueous systems. This allows for a similar tri-layer extraction method.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The foregoing and other objects, features, and advantages of the disclosure will be apparent from the following description of embodiments as illustrated in the accompanying drawings, in which reference characters refer to the same parts throughout the various views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating principles of the disclosure:

FIG. 1A depicts a prior art process of Solid Phase Peptide Synthesis (SPPS).

FIG. IB depicts a step of the GAP Peptide Synthesis (GAP -PS) process, specifically the use of a benzyl-type protecting group for C-terminus protection.

FIG. 2 depicts a process for development of a protecting group utilized in FIG. IB. FIG. 3 depicts a schematic for testing the orthogonality and GAP capability of the protecting group of FIG. 2.

FIGS. 4A-4B each depicts a schematic for the process of attaching the protecting group of FIG. 2 to various amino acids.

FIG. 5 depicts a schematic for the synthesis of a bivalirudin fragment using the protecting group of FIG. 2 for purposes of the exemplary non-limiting example of peptide synthesis of the present invention.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0015] While the making and using of various embodiments of the present disclosure are discussed in detail below, it should be appreciated that the present disclosure provides many applicable inventive concepts that can be embodied in a wide variety of specific contexts, goods, or services. The specific embodiments discussed herein are merely illustrative of specific ways to make and use the disclosure and do not delimit the scope of the disclosure.

[0016] All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this disclosure pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0017] The present disclosure will now be described more fully hereinafter with reference to the accompanying drawings, which form a part hereof, and which show, by way of illustration, specific example embodiments. Subject matter may, however, be embodied in a variety of different forms and, therefore, covered or claimed subject matter is intended to be construed as not being limited to any example embodiments set forth herein; example embodiments are provided merely to be illustrative. Likewise, a reasonably broad scope for claimed or covered subject matter is intended. Among other things, for example, subject matter may be embodied as methods, compositions, or systems. Accordingly, embodiments may, for example, take the form of methods, compositions, compounds, materials, or any combination thereof. The following detailed description is, therefore, not intended to be taken in a limiting sense.

[0018] Throughout the specification and claims, terms may have nuanced meanings suggested or implied in context beyond an explicitly stated meaning. Likewise, the phrase“in one

embodiment” as used herein does not necessarily refer to the same embodiment and the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment. It is intended, for example, that claimed subject matter include combinations of example embodiments in whole or in part. [0019] In general, terminology may be understood at least in part from usage in context. For example, terms, such as“and”,“or”, or“and/or,” as used herein may include a variety of meanings that may depend at least in part upon the context in which such terms are used.

Typically,“or” if used to associate a list, such as A, B or C, is intended to mean A, B, and C, here used in the inclusive sense, as well as A, B or C, here used in the exclusive sense. In addition, the term“one or more” as used herein, depending at least in part upon context, may be used to describe any feature, structure, or characteristic in a singular sense or may be used to describe combinations of features, structures or characteristics in a plural sense. Similarly, terms, such as“a,”“an,” or“the,” again, may be understood to convey a singular usage or to convey a plural usage, depending at least in part upon context. In addition, the term“based on” may be understood as not necessarily intended to convey an exclusive set of factors and may, instead, allow for existence of additional factors not necessarily expressly described, again, depending at least in part on context.

[0020] In designing this method, it was apparent that the method should seek to overcome the issues seen in traditional SPPS methods and other SolPPS methods, such as arduous distillation and purification strategies that hinder large scale productions. Liquid-liquid extraction methods would need to be designed to leverage solubility characteristics of the target peptide, generated amino acid impurities, and deprotection protocol impurities to overcome these issues. It was also apparent that the method should attempt to identify at least one embodiment that utilizes entirely green solvents for reactions and wash protocols.

[0021] It is therefore an embodiment of the present disclosure to provide a method of performing solution-phase peptide synthesis (SolPPS), wherein the method comprises the steps of:

dissolving an amino acid protected with a protecting strategy in a reaction solvent that is immiscible with both aqueous solutions and alkane solvents; performing coupling and deprotection reactions on the dissolved amino acid; and performing aqueous washes, alkane solvent washes, or a combination thereof after each coupling and deprotection reaction to selectively purify or extract the target peptide resulting from such coupling and deprotection reactions.

[0022] In another embodiment, the present invention discloses a method of peptide synthesis wherein the protecting strategy is selected from a group consisting of: Fmoc/tBu, Boc/benzyl, Cbz, GAP, Nvoc, Nitrobenzyl, or azide.

[0023] In another embodiment, the present invention discloses a method of peptide synthesis wherein the coupling and deprotection reactions are performed such that the peptide is synthesized in the C to N direction.

[0024] In another embodiment, the present invention discloses a method of peptide synthesis wherein the coupling and deprotection reactions are performed such that the peptide is synthesized in the N to C direction.

[0025] In another embodiment, the present invention discloses a method of peptide synthesis wherein the peptide remains in the reaction solvent, and the aqueous and alkane solvent washes remove impurities generated during the coupling and deprotection reactions from the reaction solvent.

[0026] In an exemplary embodiment of the present disclosure, a novel SolPPS method begins with the choice of a green reaction solvent that is immiscible with both aqueous solutions and alkane solvents: for this example, propylene carbonate (PC). This characteristic is vital to avoiding distillations and other purification strategies because it allows for a wider range of wash protocols to take advantage of different solubility properties of different impurities in the reaction mixture. As an example, Fmoc-protected amino acids show a high solubility in PC with the addition of 1 eq. of mild organic base. Boc protected amino acids and deprotected amino acids show similar solubility as well. Benzyl diphenylphosphine oxide (BnDppOH) as seen in the GAP -PS method also shows an affinity for the solvent, thereby providing a reaction solvent for attaching the GAP protecting group to an Fmoc-protected amino acid. BnDppOH can be attached to the amino acid in PC via a number of different coupling reagents, including EDCI and TFFH. Subsequently, deprotection of the Fmoc group can be carried out with a myriad of deprotection reagents, including diethylamine, DBU, piperidine, and tertbutylamine, and additional coupling reactions can be performed. After each coupling reaction, different quenching agents can be used to nullify excess activated amino acids and lend desired solubility characteristics to the resulting molecule. For example, decylamine could be used to quench activated amino acid and increase the resulting molecule’s solubility in alkane solvents.

Additionally, after Fmoc deprotection, deprotected excess amino acid is often rendered water- soluble and can be removed from the PC via aqueous washes. Similarly, fulvene impurities, such as NFMP created by piperidine Fmoc deprotection, can be removed from the PC by alkane solvent washes. Boc deprotection, as well as /Bu type global deprotection, can be performed with TFA as well.

[0027] The principles discussed herein may be embodied in many different forms. The preferred embodiments of the present disclosure will now be described where for completeness, reference should be made at least to the Figures.

[0028] EXAMPLE 1

[0029] For a first application of a new SolPPS method, capabilities in handling an Fmoc//Bu SolPPS strategy are tested. The target peptide of interest for this non-limiting example is a fragment of bivalirudin, a pharmacologically interesting, biologically active peptide. FIGS. 4A- 4B depict a schematic for the process of attaching the GAP protecting group of FIG. 2 to the amino acid leucine, and synthesis of the bivalirudin fragment is illustrated in FIG. 5. Compound 6 is first treated with 30% tertbutylamine (TBA) in PC for 15 minutes, with 7 eq. octane thiol added as a scavenging agent, to remove the Fmoc group, followed by multiple alkane solvent washes to remove the excess TBA, and then multiple (about three) saturated ammonium chloride washes to remove amino acid impurities while simultaneously concentrating the PC solution. In a separate pot, the next Fmoc amino acid (3 eq. relative to peptide) is dissolved in PC, 3 eq. of organic base such as DIPEA or 2,4,6-TMP is added, followed by 3 eq. of coupling reagent such as TFFH or TBTU to activate the Fmoc amino acid. The activated amino acid solution is then added to the dried peptide solution in PC to effect the coupling. After coupling for 10 - 60 minutes, the reaction mixture is washed with a quenching agent, such as long-chain (C10-C18) aliphatic thiols, long-chain (C10-C18) aliphatic alcohols, long-chain (C 10-08) aliphatic amines, long-chain (C10-C18) aliphatic selenols, aliphatic polyamines, aliphatic polyalcohols, primary or secondary amines, or primary or secondary alcohols for an hour. Subsequently, the reaction mixture is washed about three times with saturated ammonium chloride solution and dried. The crude product after coupling has a high degree of purity, with only trace amounts (if any) of impurities from the deprotection and coupling reactions. These same steps are repeated with different Fmoc amino acids to grow the peptide chain. The novel SolPPS method can easily remove any impurities simply by washing the reaction mixture with combinations of aqueous solutions and alkane solvents. This allows for the elimination of the precipitation step from the traditional GAP -PS method. Additionally, because PC is somewhat soluble in aqueous solutions, the reaction mixture volume can be concentrated during the same liquid-liquid extraction protocols, completely avoiding evaporation, distillation, or other more arduous means of concentration.

[0030] General methods: All solvents were ACS grade and used without additional

purification. LCMS analysis was conducted using a Thermo-Fisher LCMS system equipped with gradient elution. Fmoc and Boc protected amino acids were purchased from SigmaAldrich and/or Oakwood Chemical and used directly for coupling.

[0031] General procedure for Fmoc deprotection and coupling: For GAP -PS run in propylene carbonate: To Fmoc-(AA)n-OBnDpp pre-dissolved in propylene carbonate (200 mM) is added deprotection base and octane thiol, followed by stirring at room temperature for 15 minutes with an alkane bilayer. Reaction mixture is then washed X2 with fresh alkane solvent, and then washed X3 with saturated ammonium chloride aqueous solution, and then dried. In a separate propylene carbonate solution is added 3.0 eq of TBTU or TFFH, 3.0 eq Fmoc-AA-OH, and 3.0 eq. DIPEA and the reaction is stirred for 7 min. This solution is then added to the previously dried PC solution containing H-(AA)n-OBnDpp and allowed to couple with stirring for 10-60 min. An excess of quenching agent (ammonia solution or sodium hydroxide solution can be used in this preferred embodiment) was then added, such as long-chain (C 10-08) aliphatic thiols, long-chain (C10-C18) aliphatic alcohols, long-chain (C 10-08) aliphatic amines, long-chain (00-08) aliphatic selenols, aliphatic polyamines, aliphatic polyalcohols, primary or secondary amines, or primary or secondary alcohols. The reaction mixture is then washed X3 with saturated ammonium chloride aqueous solution and then dried to afford the elongated peptide in PC solution. This process is repeated as necessary to generate the peptide with the desired sequence and length (i Compound 9, MS (ESI): m/z calcd for [C56H75N4011P + H]+: 1011.5, found: 1011.8). [0032] Further GAP Groups and Attachment Methods

[0033] FIGS. 4A-4B each depicts a schematic for the process of attaching the protecting group of FIG. 2 to various amino acids.

[0034] Those skilled in the art will recognize that the methods and systems of the present disclosure may be implemented in many manners and as such are not to be limited by the foregoing exemplary embodiments and examples. In other words, functional elements being performed by single or multiple components, in various combinations of hardware and software or firmware, and individual functions, may be distributed among various software applications at either the client level or server level or both. In this regard, any number of the features of the different embodiments described herein may be combined into single or multiple embodiments, and alternate embodiments having fewer than, or more than, all of the features described herein are possible.

[0035] Functionality may also be, in whole or in part, distributed among multiple components, in manners now known or to become known. Thus, myriad combinations are possible in achieving the functions, features, and preferences described herein. Moreover, the scope of the present disclosure covers conventionally known manners for carrying out the described features as well as those variations and modifications that may be made to the processes, composition, or compounds described herein as would be understood by those skilled in the art now and hereafter.

[0036] Furthermore, the embodiments of methods presented and described as diagrams, schematics or flowcharts in this disclosure (such as the Figures) are provided by way of example in order to provide a more complete understanding of the technology. The disclosed methods are not limited to the operations and logical flow presented herein. Alternative embodiments are contemplated in which the order of the various operations is altered and in which sub-operations described as being part of a larger operation are performed independently. While various embodiments have been described for purposes of this disclosure, such embodiments should not be deemed to limit the teaching of this disclosure to those embodiments. Various changes and modifications may be made to the elements and operations described above to obtain a result that remains within the scope of the systems and processes described in this disclosure.

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