Methods for solution-phase peptide synthesis and protection strategies therefor

Abstract

The disclosed invention relates generally to the field of peptide synthesis. A novel method of solution-phase peptide synthesis is disclosed that utilizes novel group-assisted purification protecting groups to facilitate efficient and scalable chemistry for synthetically producing peptides. The disclosed invention addresses shortcomings in the art by significantly reducing waste, increasing scalability, and producing high crude purity, largely avoiding post-synthetic purification.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application cross-references i) WO Application No. PCT / US2016 / 068112 and WO Publication No. WO2017112809A1, filed December 21, 2016, entitled "System and method for solution phase GAP peptide synthesis"; ii) WO Application No. PCT / US19 / 29569, filed April 29, 2019, published as WO No. , entitled "Method for Solution-Phase Peptide Synthesis"; and iii) U.S. Provisional Application No. 62 / 678,564, filed May 31, 2018, entitled "Protection Strategy for GAP Peptide Synthesis," which applications and publications are incorporated herein by reference in their entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] none. SEQUENCE LISTING, TABLE, OR COMPUTER LISTING COMPACT DISC APPENDIX REFERENCE

[0003] none. [Background technology]

[0004] Recent research efforts have made significant progress in the area of ​​purification chemistry with a particular focus on avoiding column chromatography and recrystallization. This work has been defined as group-assisted purification (GAP) chemistry / technology as chemistry for organic synthesis that avoids traditional purification methods, such as chromatography and / or recrystallization, by deliberately introducing fully functionalized groups into starting materials or into newly generated products. These GAP groups can often also be used as protecting groups to prevent undesired side reactions during the synthesis of the target molecule. Such work has the potential to encompass the entire field of synthetic organic chemistry.

[0005] Protecting groups are found in almost every complex synthesis where multiple functional groups are present. With regard to GAP chemistry, an ideal example would be one in which a semi-permanent protecting group introduced the necessary solubility properties required for GAP. However, most traditional protecting groups are non-polar and therefore do not produce the required GAP solubility for most substrates. If a protecting group is developed that produces reasonable solubility control, GAP chemistry could potentially be extended to all syntheses requiring the use of that protecting group. Several approaches have been utilized. Published patent application WO2014093723A2 teaches the protection of imines with chiral auxiliaries equipped with GAP, then using these chiral N-phosphoryl imines as electrophiles in asymmetric boron addition reactions. Purification was performed via the GAP process. This work is valuable in that it provides facile access to chiral α-boronic acid amines that could potentially be used to synthesize novel amino acid derivatives that could potentially be incorporated into novel peptide targets.

[0006] Effective protecting groups must be robust against a wide variety of conditions and must be added and removed in high yields. Protecting groups are used extensively in peptide synthesis, either in solid-phase or solution-phase approaches. In traditional peptide synthesis protection strategies, one of the most commonly used strategies is Fmoc / tBu. US Patent No. 8,383,770 B2 teaches the use of fluorenylmethoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) N-terminal protecting groups in solid-phase peptide synthesis (SPPS). This technique is well known and widely applied in the industry. The Fmoc group protects the N-terminus of the amino acid that is added to the growing peptide, and the tert-butyl (tBu)-based group protects the side chain of the same amino acid. The Fmoc group can be removed by the appropriate deprotection base, while the tBu group remains until acid-based global deprotection at the end of the synthesis. The Boc and Fmoc groups have been used for decades in all areas of peptide chemistry, with the preferred Fmoc group being almost entirely restricted to SPPS.

[0007] SPPS was developed by Merrifield in the 1960s and has become a standard protocol used by multiple scientific disciplines for research and manufacturing (see Figure 1A). The advantage of the polymeric support or resin is its ability to allow for easy purification of the growing peptide after each coupling / deprotection step, thus avoiding the use of column chromatography. The main disadvantage of SPPS is the difficulty in scaling up: many polymeric supports are expensive and account for a large proportion of the mass of the material handled. The coupling reaction in SPPS is also inefficient since it occurs at a solid-liquid interface. In addition, after each deprotection and coupling reaction, the resin must be washed with a solvent to remove any impurities generated from the previous reaction, which creates significant solvent waste that can be very problematic on a large scale.

[0008] However, examples of economically feasible Fmoc protection schemes for solution phase peptide synthesis (SolPPS) are rare and almost nonexistent in the literature. US Patent No. 5,516,891A provides one of the few examples of Fmoc-based SolPPS. Again, Fmoc peptide synthesis is almost entirely restricted to SPPS. This is because N-fluorenylmethylpiperidine (NFMP), which is difficult to remove without a polymeric support, is formed as a by-product during deprotection. The standard protocol for Fmoc deprotection is to stir the Fmoc peptide in a solution of dimethylformamide (DMF) or dichloromethane (DCM) with excess piperidine, deprotecting the Fmoc group in the process and forming NFMP. The '891 patent teaches the removal of this impurity by deprotecting with 4-aminomethylpiperidine (4AMP) instead of piperidine. This forms NFMP-CH2NH2 instead of NFMP, which can be extracted into water due to the presence of the extra amino group. The problem with this method is the high cost of using 4AMP, which is why this method is cost prohibitive and has not been accepted by the industry.

[0009] Another example of Fmoc-based SolPPS can be found in published patent application WO2017112809A1. This publication teaches the use of a C-terminal GAP protecting group, benzyldiphenylphosphine oxide (HOBnDpp), to control the solubility of the target peptide to allow selective precipitation after each successive coupling reaction (see Figure 1B). Solubility is controlled so that the growing peptide remains in an organic solvent such as ethyl acetate or DCM, and aqueous washes are performed to remove impurities; subsequent concentration of the organic solvent followed by mixing in an alkane solvent selectively precipitates the peptide product. Although this technique has adapted Fmoc / tBu chemistry to the solution phase in a much more economically feasible manner than the '891 patent, there are potential limitations inherent to the method. First, the GAP group is not acid labile, preventing convenient one-step global deprotection with the commonly used trifluoroacetic acid-based (TFA-based) or other acid cocktails; hydrogenation or hydrolysis is required to remove HOBnDpp from the peptide's C-terminus. In addition, as peptides continue to grow and change sequence, HOBnDpp is less able to maintain solubility control over the chain as it gets longer or as the sequence confers different solubility properties to the peptide. HOBnDpp is also prone to hydrolysis or accidental cleavage during synthesis. For example, during Fmoc deprotection of the second amino acid in a peptide sequence, diketopiperazine (DKP) formation facilitates the loss of HOBnDpp from the C-terminus. This cleaved HOBnDpp can remain in the reaction solution and react with activated amino acids during the coupling reaction, creating impurities that are difficult or impossible to remove. Moreover, due to current cleavage techniques (either hydrolysis or hydrogenation) for deprotection of the C-terminus after the protected peptide is synthesized, the C-terminus of the final peptide product is a carboxylic acid. Many therapeutic peptides need to be modified at the C-terminus to be an amide, cyclic peptide, thioester, etc., instead of this carboxylic acid, further limiting the application of the GAP peptide synthesis method.In addition, these cleavage reactions to remove HOBnDpp from the C-terminus are completely separate reactions from the global deprotection; they require specific conditions and work-up, which is an inconvenience that is both time-consuming and expensive. Summary of the Invention [Problem to be solved by the invention]

[0010] Due to these and other challenges with scale-up in SPPS, as well as limitations in existing GAP peptide synthesis protection strategies, there is a need in the industry for SolPPS methods that are not only amenable to scale-up, but also that are more amenable to (i) a variety of cleavage conditions, (ii) longer or more difficult (from a solubility standpoint) sequences, and (iii) C-terminal modifications. [Means for solving the problem]

[0011] In one aspect, the invention disclosed herein comprises a protecting group for solution phase peptide synthesis, wherein the protecting group has a formula selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7 and 8. Chemical formula 1 is [ka] and; Chemical formula 2 is [ka] and; Chemical formula 3 is: [ka] and; Chemical formula 4 is [ka] and; Chemical formula 5 is [ka] and; Chemical formula 6 is [ka] and; Chemical formula 7 is [ka] and; Chemical formula 8 is: [ka] It is. Z is selected from the group consisting of -H, methyl (-Me) and methoxy (-OMe). Y is selected from the group consisting of -O-, -S- and -NH-. X is selected from the group consisting of -O-, -S- and -NH-. R is selected from the group consisting of known side chains of protected and unprotected amino acids. L is an abbreviation for 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), 4-((9-amino-9H-xanthen-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl").

[0012] In a second aspect, the disclosed invention includes a method of performing solution phase peptide synthesis, the method comprising several steps. The first step comprises attaching a first protecting group to a first amino acid to provide a first protected amino acid. The next step comprises performing a coupling reaction on the first protected amino acid or on a peptide formed by linking the first amino acid with another molecule. The first protecting group is the protecting group of claim 1.

[0013] In a third aspect, the disclosed invention includes a method for forming a protecting group for solution phase peptide synthesis, comprising coupling benzyldiphenylphosphine oxide (HOBnDpp), anilinediphenylphosphine oxide (NH2PhDpp) or a derivative thereof (i) directly to a linker molecule, (ii) via an amino acid to a linker molecule, or (iii) via an amino acid derivative to a linker molecule. [Brief description of the drawings]

[0014] The foregoing and other objects, features and advantages of the present disclosure will become apparent from the following description of embodiments 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 on illustrating the principles of the present disclosure. The figures are used as non-limiting examples and are intended only to represent preferred embodiments, without limiting the scope of the present disclosure. [Figure 1A] FIG. 1 illustrates a prior art solid phase peptide synthesis (SPPS) method. [Figure 1B] FIG. 1 shows the steps of the GAP peptide synthesis (GAP-PS) method, specifically the use of HOBnDpp for C-terminal protection. [Diagram 2] FIG. 1B illustrates a method for attaching the protecting group of FIG. 1B to the side chain of an amino acid residue, specifically to a serine in this non-limiting embodiment, followed by GAP-PS. [Diagram 3]As an example, we show a schematic for synthesizing a novel GAP molecule and conjugating it to a peptide, where HOBnDpp is reacted with a protected Rink amide-OH, and the resulting new chemical entity, Rink amide-GAP (RAG), is conjugated to the C-terminus of an amino acid. [Figure 4] 1 illustrates the attachment of a novel GAP molecule to the side chain of an amino acid. In this non-limiting example, the GAP molecule RAG is used to protect the side chain of aspartic acid, resulting in the synthesis of RAG-protected asparagine. Specifically, a coupling reaction is carried out to form an amide bond between the free amine on the RAG molecule and the carbonyl carbon of asparagine. [Diagram 5] 1 shows a non-limiting example of the synthesis of a novel GAP molecule. In this non-limiting example, HOBnDpp is coupled directly with protected HMPA-OH to form HMPA-GAP(HG) and via an intermediate (phenylalanine in one embodiment) to form the GAP molecule, HMPA-phenylalanine-GAP(HPG). Specifically, HOBnDpp can be coupled to the carboxylic acid moiety of protected HMPA-OH, or HOBnDpp can be first coupled to an amino acid and then coupled to HMPA to generate the GAP molecule HPG. [Figure 6] FIG. 6 illustrates the attachment of HPG from FIG. 5 to the C-terminus of a universal amino acid and, in a preferred embodiment of the present disclosure, to the side chain of aspartic acid. Specifically, this reaction is a coupling reaction that can be accomplished via the use of several different coupling reagents, in which the free alcohol of HPG couples with the carbonyl group of an activated carboxylic acid. [Figure 7] FIG. 1 shows a non-limiting example of the synthesis of a novel GAP molecule, anilinediphenylphosphine oxide ("NH2PhDpp"). [Figure 8]1 shows a non-limiting example of the synthesis of a novel GAP molecule, in which NH2PhDpp is reacted with a protected Rink amide-OH to obtain the target material. Specifically, NH2PhDpp can be coupled to the carboxylic acid moiety of the protected Rink amide-OH via a number of coupling reagents. [Figure 9] FIG. 1 shows a non-limiting example of the synthesis of a novel GAP molecule, in which NH2PhDpp is reacted with protected HMPA-OH, either directly or via an intermediate, to form the target molecule. Several different coupling reagents can be used to facilitate these reactions. [Figure 10] FIG. 1 shows, as a non-limiting example, compounds obtained upon attachment of an Fmoc-protected leucine to the C-terminus of the HPG GAP molecule. [Figure 11] FIG. 1 shows, as a non-limiting example, compounds resulting from side chain protection of glutamic acid with HPG, where the C-terminus of glutamic acid is protected with an allyl group, as a non-limiting example of a possible C-terminus protection strategy. [Figure 12] FIG. 1 shows, as a non-limiting example, the tetra-peptide, Fmoc-Glu(HPG)-Glu(HPG)-Tyr(tBu)-Leu-HPG, generated by the novel SolPPS method disclosed herein. [Figure 13] FIG. 1 shows the compound resulting from the attachment of RAG to the side chain of an amino acid, specifically aspartic acid in this non-limiting example, where the C-terminus of the aspartic acid is protected with an allyl group as a non-limiting example of a C-terminal protection strategy. [Figure 14] FIG. 1 shows, by way of non-limiting example, protected peptides generated by the SolPPS method disclosed herein. [Figure 15] FIG. 1 shows, as a non-limiting example, the peptide Bivalirudin fully protected with a novel GAP molecule. [Figure 16] FIG. 1 shows fully deprotected Bivalirudin, achieved via one-step global deprotection to remove all side chain and C-terminal protecting groups followed by Fmoc deprotection. [Figure 17] FIG. 1 shows a general methodology for protecting amines with novel GAP molecules. In this specific, non-limiting embodiment, HPG is used. [Figure 18] FIG. 1 shows, by way of non-limiting example, the myriad of molecules that can serve as reagents for the synthesis of the novel GAP molecules disclosed herein. [Figure 19] FIG. 1 shows a number of representative coupling reactions for synthesizing the novel GAP molecules disclosed herein. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Reference is made to specific features of the invention in the Summary of the Invention above, and in the Detailed Description and Claims below, as well as in the accompanying drawings. It should be understood that the disclosure of the invention herein includes all possible combinations of such specific features. For example, when a specific feature is disclosed in the context of a specific aspect or embodiment of the invention, or in a specific claim, that feature can also be used, to the extent possible, in combination with and / or in the context of other specific aspects and embodiments of the invention, and in the invention generally.

[0016] All of the compositions and / or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Although the compositions and methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods in the steps or sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

[0017] The term "comprises" and its grammatical equivalents are used herein to mean that other components, ingredients, steps, etc. are optionally present. For example, an article "comprising" (or "which comprises") components A, B, and C can consist of (i.e., contain only) components A, B, and C, or it can contain not only components A, B, and C, but also one or more other components.

[0018] When reference is made herein to a method that includes two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes that possibility), and the method may include one or more other steps that are performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes that possibility).

[0019] The term "at least" followed by a number is used herein to denote the beginning of a range starting from that number (which may be a range with or without an upper limit, depending on the variable being defined). For example, "at least 1" means 1 or more than 1. The term "up to" followed by a number is used herein to denote the end of a range ending at that number (which may be a range with 1 or 0 as its lower limit, or a range with no lower limit, depending on the variable being defined). For example, "up to 4" means 4 or less than 4, and "up to 40%" means 40% or less than 40%. When a range is given herein as "from (first number) to (second number)" or "from (first number) to (second number)", this means a range whose lower limit is the first number and whose upper limit is the second number. For example, 25 to 100 mm means a range whose lower limit is 25 mm and whose upper limit is 100 mm.

[0020] The term "first" is used to distinguish one element from another and is not intended to indicate that an element is the first or initial element in any given sequence of elements. For example, a "first amino acid" does not mean that the amino acid is first in a sequence of amino acids or that it is the very first amino acid to be reacted. Instead, a "first amino acid" indicates only that the amino acid is separate and distinct from another amino acid, such as a "second amino acid."

[0021] The term "coupling reaction" is generally used to refer to the formation of a bond between two component molecules that is facilitated by a "coupling reagent." In peptide chemistry, these coupling reactions can occur through many different mechanisms under many different reaction conditions that may entirely depend on the coupling reagent used. For example, a coupling reagent can "activate" a carboxylic acid of a component molecule, so that the carbonyl carbon becomes more prone to nucleophilic attack. A coupling reaction can result in the loss of a water molecule during the formation of a bond between two component molecules (see Chandrudu 2013, Mollica 2013, Shelton 2013, Amblard 2006, Bachem 2016).

[0022] In many types of protection schemes for peptide synthesis, a similar series of reactions occurs to grow the peptide chain. Generally, either the N- or C-terminus of each amino acid added to the chain is first protected, and the other end of the amino acid is free to participate in coupling reactions. After addition to the chain via the initial free end, a deprotection reaction is performed to free the protected N- or C-terminus to participate in subsequent coupling reactions to form a peptide bond with the adjacent amino acid. For example, in Fmoc / tBu-based peptide synthesis, the Fmoc group protects the N-terminus of the amino acid, and the side chain of the amino acid is protected with a tBu-based protecting group, including but not limited to butyl, trityl (triphenylmethyl), Boc (butyloxycarbonyl), Pbf (2,2,4,6,7-pentamethyl-2,3-dihydrobenzofuran-5-sulfonyl), Pmc (2,2,5,7,8-pentamethylchroman-6-sulfonyl) and Acm (acetamidomethyl). (Some amino acids do not require side chain protection because the side chain is naturally inert to the coupling and deprotection conditions.) The C-terminus of the first amino acid in the peptide sequence is connected to and protected by a resin or polymer in SPPS and a protecting group in SolPPS. The Fmoc / tBu peptide synthesis scheme is designed such that the Fmoc group at the N-terminus of an amino acid is base labile and upon treatment with the appropriate deprotection base, the Fmoc group is removed from the N-terminus without interfering with the C-terminal connection or side chain protection. When the deprotection reaction is performed, the N-terminus of the first amino acid is free, while the C-terminus and side chain are protected or otherwise inert. The second amino acid is then protected at the N-terminus with Fmoc and side chain protected or naturally inert, but is activated at the free C-terminus with a coupling reagent, such activation facilitating nucleophilic attack by the free N-terminus of the first amino acid at the activated carbonyl to form a peptide bond between the first and the adjacent amino acid. This process is repeated until the correct peptide sequence is realized. After Fmoc deprotection of the final amino acid, the peptide is still protected at the C-terminus and at the side chain.A global deprotection using a strong acid cocktail, such as a TFA-based cocktail, is then performed to remove all side chain protecting groups; in some cases the C-terminal resin or protecting group may also be cleaved.

[0023] The present disclosure addresses the shortcomings in the art by providing a method for solution-phase peptide synthesis. The method allows for a variety of global deprotection strategies, synthesis of longer or more difficult (in terms of solubility) peptide sequences, and C-terminal modifications in the target peptide, while maintaining solubility control of the target peptide and the overall synthesis process. By utilizing novel protection strategies at the C-terminus and side chains of the growing peptide, a SolPPS strategy is presented that is economically feasible and useful for commercial production of peptides.

[0024] Thus, a non-limiting objective of the present disclosure is to enable novel methods of SolPPS. In one embodiment, HOBnDpp, traditionally a C-terminal GAP protecting group, is used to directly protect the side chains of amino acid residues, including but not limited to serine, threonine, tyrosine, or cysteine, instead of or in addition to the C-terminus of the peptide chain. Other amino acid residues of the desired peptide can be protected using a number of different protection strategies, including but not limited to Fmoc / tBu, Boc / benzyl, Cbz (benzyloxycarbonyl) / tBu, Cbz / benzyl, etc. This enhanced protection strategy allows for the synthesis of longer and / or more difficult sequences while maintaining solubility control of the peptide chain. This strategy also allows for the use of different C-terminal protecting groups for solubility, deprotection, or post-synthetic C-terminal modification purposes. In another embodiment, the novel GAP molecules discussed below are used to protect the C-terminus and / or side chains of certain amino acids to enable peptide synthesis in solution in the C to N direction.

[0025] In another embodiment, the novel SolPPS method is accomplished via a specially designed C-terminal protection strategy, where the novel GAP molecule protecting group maintains the benefits of the original GAP peptide synthesis (GAP-PS) while allowing a wider range of cleavage conditions, synthesis of longer peptides, minimization of unintended or accidental deprotection, and C-terminal modification upon cleavage. Depending on the GAP molecule used, C-terminal deprotection can be accomplished under any of the following conditions: acidic conditions including but not limited to treatment with trifluoroacetic acid (TFA), hydrofluoric acid (HF), toluenesulfonic acid (TsOH), methanesulfonic acid (MsOH) or others, basic hydrolysis conditions including but not limited to metal hydroxides in either aqueous or alcoholic systems or biphasic systems with the addition of a phase transfer catalyst, and Pd 0 Or Pt 0 or Ru, molecular hydrogen over transition metal catalysts, transfer hydrogenation, or other hydrogen sources such as borohydride, aluminum hydride, silane, ammonium formate, etc. Depending on the specific GAP molecule, C-terminal amide formation or other post-synthetic modifications can also be accomplished during cleavage of the C-terminal GAP molecule to generate the desired peptide product while avoiding extra post-synthetic processing steps. These novel GAP molecules can remain stable against acid-catalyzed deprotection reactions, as well as hydrogenation-based and hydrolysis reactions, allowing for increased post-peptide synthesis recovery of GAP protecting groups. In addition, losses during C-terminal protection synthesis are also minimized by these novel GAP molecules.

[0026] In another embodiment, any of the novel GAP molecules described above are used to protect the side chains of various amino acids, including but not limited to aspartic acid, cysteine, serine, threonine, lysine, glutamic acid, asparagine and glutamine, instead of or in addition to the C-terminus of a peptide. This strategy allows for protection of reactive side chains while simultaneously controlling the solubility of the target peptide.

[0027] In another embodiment, methods for synthesizing these novel GAP molecules and their derivatives are presented.As a non-limiting example, HOBnDpp is attached to the carboxylic acid moiety of a linker molecule, either directly or via an intermediate, an example of which can be seen in Figure 18, and then the resulting novel GAP protecting group is attached to the C-terminus of a peptide.As another non-limiting example, aniline diphenylphosphine oxide (i.e., NH2PhDpp) is coupled in a similar manner with different linkers to realize novel GAP protecting groups that can be used in a similar manner.

[0028] In another embodiment, any of these different strategies discussed above are utilized in different combinations to allow for the best possible synthesis strategy for a given peptide. As a non-limiting example, when used appropriately, these strategies allow for the synthesis of longer peptides entirely in solution, with C-terminal deprotection and C-terminal amide formation being accomplished in a one-step reaction along with side chain deprotection.

[0029] Therefore, a non-limiting objective of the present disclosure is to provide a method for SolPPS. In designing this method, it was clear that the method should seek to maintain the advantages of other solution-phase methods such as GAP-PS, such as avoiding the difficult distillation and purification strategies that hinder large-scale production. Enhanced protection strategies will need to be designed to maintain solubility control of the target peptide to allow separation of the product from generated amino acids and deprotection protocol impurities, while also addressing the difficulties in the art.

[0030] In a non-limiting exemplary embodiment of the present disclosure, the novel SolPPS method can begin with the direct attachment of HOBnDpp to the C-terminus of the first amino acid in a given sequence (see FIG. 1B). As an alternative or in addition to protecting the C-terminus of the first amino acid with HOBnDpp, attachment of HOBnDpp to the side chain of an amino acid residue, such as, but not limited to, serine, threonine, tyrosine and / or cysteine, can also be performed with subsequent incorporation into the growing peptide to better control the solubility and protect the side chain of certain amino acids (see FIG. 2). To accomplish side chain protection with HOBnDpp, HOBnDpp is first reacted with a halogenating agent (e.g., PBr3, POBr3, (COCl)2, SOCl2, etc.) in the presence of a base (e.g., triethylamine, diisopropylethylamine, sodium bicarbonate, etc.) to provide either IBnDpp, ClBnDpp or BrBnDpp, depending on the halogenating agent used. The reaction can take place in a variety of conditions, depending on the halogenating agent and base used in the reaction. By way of example, the reaction can be carried out at pressures from 1 to 100 bar, at temperatures from -78°C to 100°C, and in monophasic or biphasic reaction media. This halogenated compound can then be reacted with an amino acid bearing a primary protecting group (Pg) at the N-terminus and a secondary protecting group (Pg') at the C-terminus to effectively protect the amino acid side chain. Peptide synthesis can then be carried out in the C to N direction if a GAP molecule protects the C-terminus of the peptide instead of or in addition to GAP protection of the side chain of one or more amino acids integrated into the peptide sequence; alternatively, peptide synthesis can be carried out or in the N to C direction if HOBnDpp or other GAP molecules protect only the amino acid side chains. A myriad of protection strategies for N-terminus and side chain protection may be possible.Potential N- or C-terminal protecting groups can include, but are not limited to, Cbz, Fmoc, Boc, methyl, Bn (benzyl), or any of the GAP molecules discussed herein, and side chain protecting groups can include, but are not limited to, tBu, Acm, trityl, Boc, Pbf, Pmc, Fm (fluorenylmethyl), and any of the GAP molecules discussed herein.

[0031] In another non-limiting exemplary embodiment, the novel SolPPS method can begin with a novel GAP molecule (here, RAG) that is used for C-terminal protection, allowing both acid-labile C-terminal protection of the peptide and C-terminal amide formation upon cleavage of the RAG from the peptide, as seen in Figure 3. Once C-terminal protection with RAG is accomplished, peptide synthesis can be carried out in the C to N direction utilizing a myriad of N-terminal and side chain protection strategies, such as, but not limited to, the Fmoc / tBu strategy. To synthesize RAG, HOBnDpp can be coupled with the protected Rink amide-OH via a number of coupling reagents, including but not limited to, to provide the GAP molecule RAG. Examples of suitable coupling reagents include TFFH ("N-((dimethylamino)fluoromethylene)-N-methylmethanaminium hexafluorophosphate"), TBTU ("1-((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide tetrafluoroborate"), HBTU ("1-((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazole 3-oxide hexafluorophosphate"), EDCI ("3-(((ethylimino)methylene)amino)-N,N-dimethylpropan-1-amine"), or COMU ("(1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate"), and the like. The coupling reaction can occur under a variety of conditions, depending on the specific coupling reagent used. By way of example, the reaction can be carried out at ambient pressure, room temperature, with gentle stirring with TBTU. Once the protected RAG molecule is synthesized, a deprotection reaction (e.g., Fmoc deprotection reaction) can occur, freeing the amine on the Rink amide to participate in a coupling reaction to a carboxylic acid such as may be found at the C-terminus of an amino acid. RAGs can alternatively or additionally be attached to the side chains of amino acid residues, including but not limited to aspartic acid and glutamic acid (see FIG. 4), allowing for enhanced protection or solubility control upon addition of those amino acids to a growing sequence. Upon cleavage of the RAG from the side chain of an amino acid, the amide forms of those side chains (here, asparagine and glutamine, respectively) can be generated (see FIG. 4).

[0032] In another non-limiting exemplary embodiment, the novel SolPPS method can start with a novel GAP molecule (here, (Rink amide)-phenylalanine-GAP, i.e., RPG) used for C-terminal protection. The RPG can be synthesized similarly to FIG. 5. Specifically, HOBnDpp can be coupled with Fmoc-protected phenylalanine to form Fmoc-phenylalanine-GAP (i.e., Fmoc-F-GAP), and after Fmoc deprotection, the now free N-terminus of the GAP-protected phenylalanine can be coupled with the carboxylic acid moiety of a protected Rink amide-OH (the molecular structure of the protected Rink amide-OH can be seen in FIG. 3). The resulting structure can be deprotected at the N-terminus of the Rink amide molecule, and the free N-terminus can then participate in a coupling reaction to form a peptide bond with the first amino acid in a given peptide sequence. Peptide synthesis can then proceed in a C to N direction, with the RPG providing the C-terminal protection of the growing peptide. RPG as a C-terminal protecting group can be used in combination with any of the disclosed GAP molecules that provide side chain protection, or with any other protection strategies available for N-terminus and side chain protection. RPG can also be used to protect the side chain of an amino acid in the same way that RAG is used, in addition to or instead of C-terminal protection.

[0033] In another non-limiting example, the novel SolPPS method can utilize different novel GAP molecules, such as HG or HPG, for C-terminal protection. The synthesis of these GAP molecules can be seen in FIG. 5, and these molecules can be attached to the C-terminus of peptides to allow for acid-labile C-terminal protection without forming a C-terminal amide upon cleavage (see FIG. 6). Instead of or in addition to protecting the C-terminus of an amino acid, HG and / or HPG can also be attached to the side chains of amino acid residues, including but not limited to aspartic acid, cysteine, serine, threonine, tyrosine, and glutamic acid, to aid in solubility control (see FIG. 6). Peptide synthesis can then proceed in the C to N direction utilizing a myriad of protection strategies, such as, but not limited to, the Fmoc / tBu strategy.

[0034] In another non-limiting exemplary embodiment, the novel SolPPS method can begin with the synthesis of aniline diphenylphosphine oxide (i.e., NH2PhDpp), as seen in FIG. 7. Specifically, the synthesis of NH2PhDpp can begin with 4-bromonitrobenzene and be activated with N-butyllithium (nBuLi) in an ethereal solvent at ultra-low temperature (-100 to -30°C) and room pressure to form an organolithium reagent. This reagent can then be reacted in situ with the slow addition of chlorodiphenylphosphine, which will yield NH2PhDpp after phosphine oxidation with hydrogen peroxide and nitro group reduction with H2 and 10% Pd / C. This molecule can then be coupled, in a non-limiting example, to the Rink amide-OH (see FIGS. 8, 18), and the resulting GAP molecule can be coupled to the C-terminus of a peptide as in FIG. 3 and / or to the side chains of aspartic acid or glutamic acid as in FIG. 4. For aspartic acid or glutamic acid, a protected asparagine or glutamine is generated, respectively. In another non-limiting example, another novel GAP molecule can be synthesized via reaction of NH2PhDpp with the carboxylic acid moiety of a linker (here, HMPA), either directly or via an amine-based intermediate, as seen in Figure 9. The resulting GAP molecule can then be attached to the C-terminus of a peptide and / or to the side chains of aspartic acid, cysteine, serine, threonine, and glutamic acid, as seen in Figure 6. Peptide synthesis can then proceed in the C to N direction, utilizing a myriad of protection strategies, such as, but not limited to, the Fmoc / tBu strategy.

[0035] For all of the above non-limiting examples, HOBnDpp, NH2PhDpp, or any other GAP molecule can be attached to the C-terminus or side chain of an amino acid residue in a number of different solvents, including but not limited to dichloromethane, ethyl acetate, isopropyl acetate, methyltetrahydrofuran, tetrahydrofuran, and propylene carbonate. For any of the coupling reactions described herein, whether for GAP molecule synthesis or attachment of the GAP molecule to the first amino acid of a peptide being synthesized, a myriad of different coupling reagents can be used, including but not limited to TFFH, TBTU, HBTU, COMU, or EDCI.

[0036] As a non-limiting exemplary embodiment and using any of the examples discussed above or any combination thereof, SolPPS can then be accomplished in the C to N direction, such as by Fmoc / tBu chemistry. Deprotection of the Fmoc group for the first and subsequent amino acids can be performed using a myriad of deprotection reagents, including diethylamine, DBU (diazabicycloundecene), piperidine, tert-butylamine and other alkylamines, and additional coupling reactions can be performed to grow the peptide chain. After each coupling reaction, a different quenching agent can be used to neutralize the excess activated amino acid and impart the desired solubility characteristics to the resulting molecule. In a non-limiting example, decylamine can be used to quench the activated amino acid and increase the solubility of the resulting molecule in alkane solvents. Depending on the reaction solvent, liquid-liquid extraction techniques or selective precipitation can then be performed to purify the reaction mixture. The novel GAP molecules disclosed herein allow for solubility control and enable novel SolPPS methods. Here, selective precipitation or extraction effectively removes amino acid and fulvene impurities (i.e., NFMP). After the last amino acid of the desired peptide sequence is coupled, in a non-limiting example, a global deprotection with an appropriate TFA cocktail can be performed to remove the acid-labile GAP molecule from the C-terminus and appropriate side chain; such deprotection can also remove other acid-labile side chain protecting groups, such as tBu-based side chain protecting groups. This novel SolPPS method allows for a one-step global deprotection to generate the native peptide sequence, avoiding additional reactions such as hydrogenation or hydrolysis to remove the C-terminal protecting group. In addition, when a GAP molecule such as RAG is used, a C-terminal amide can be formed during TFA cleavage, successfully avoiding post-synthesis reactions that would normally be required for such modifications. In addition, these novel GAP molecules disclosed herein remain stable during peptide synthesis, without accidental hydrolysis or removal due to DKP formation.

[0037] In one non-limiting embodiment, these novel GAP molecules can be used to protect the amine-based side chains of certain amino acid residues, including but not limited to the N-terminus and lysine of any given peptide. In a non-limiting example, HPG and diimidazole carbonyl are dissolved in a suitable reaction solvent, such as DCM, and gently stirred at room temperature and pressure for 1 hour or more. An amine is then added to the reaction mixture at room temperature and pressure to generate the HPG-protected carbamate product (see FIG. 17).

[0038] In another non-limiting embodiment, the novel SolPPS method can begin with the synthesis of a novel GAP molecule protecting group, where HOBnDpp, NH2PhDpp or derivatives thereof can be reacted directly with the carboxylic acid moiety of any given linker molecule, examples of which can be seen in Figure 18 and in the table below. [Table 1]

[0039] Examples of such reactions can be seen in Figures 3, 8, 9. These resulting GAP molecules can then be coupled to any given amino acid to facilitate C-terminal protection (as in Figure 3), side chain protection (as in Figure 4) or N-terminal protection (similar reactions to those seen in Figure 17). In another non-limiting example, new GAP molecules can be synthesized by reacting HOBnDpp or a derivative thereof with any given amino acid, followed by reaction with any given linker molecule at the carboxylic acid moiety, as can be seen in Figures 5 and 9. The resulting GAP molecules can be used for C-terminal protection (as in Figure 3), side chain protection (as in Figure 4) or N-terminal protection (similar reactions to those seen in Figure 17).

[0040] The synthesis strategy of the pharmacologically interesting biologically active peptide Bivalirudin is shown as a non-limiting preferred embodiment of the novel method of SolPPS disclosed herein. Figure 6 depicts a schematic for attaching the novel GAP molecule HPG to the C-terminus of an unspecified amino acid, according to which C-terminal protection with Fmoc-Leucine-OH forms the compound shown in Figure 10. In this particular embodiment, HPG is selected as the C-terminal protecting group to allow post-synthetic acid-labile C-terminal deprotection without facilitating C-terminal amide formation. Following this initial step, standard Fmoc / tBu chemistry is performed to couple Fmoc-Tyr(tBu)-OH to form an Fmoc-protected dipeptide. The side chain of Fmoc-Glu-O allyl is then protected with HPG before incorporation into the growing peptide, according to the schematic shown in Figure 6 for amino acid side chain protection, resulting in the compound shown in Figure 11. Allyl deprotection of the C-terminus of Fmoc-Glu(HPG)-Oallyl is performed by treatment with Pd(PPh3)4 and triisopropyl or phenylsilane (see Isidro-Llobet 2009), followed by activation of the resulting molecule and coupling with a peptide. This process is repeated once more to attach the next HPG-protected glutamic acid residue to generate the Fmoc-protected tetra-peptide shown in Figure 12. Standard Fmoc / tBu peptide synthesis is performed to add proline and isoleucine, and the procedure discussed above is followed to add the next two glutamic acid residues, followed by phenylalanine. An aspartic acid residue is protected with HPG and added following the schematic shown in Figure 6 in the same way as glutamic acid, followed by the addition of a glycine residue.

[0041] In another preferred embodiment, the novel GAP molecule RAG is reacted with Fmoc-Asp-O allyl to generate the compound Fmoc-Asp(RAG)-O allyl shown in Figure 13. Allyl deprotection of the compound is performed, followed by activation and coupling to a peptide chain to generate the Fmoc-protected peptide shown in Figure 14. Standard Fmoc / tBu peptide synthesis is then performed to add four glycines, a proline, an arginine, another proline, and a D-phenylalanine to generate the fully protected Bivalirudin peptide as shown in Figure 15.

[0042] In this particular embodiment, after Fmoc deprotection of the fully protected Bivalirudin, a one-step global deprotection with a TFA cocktail (i.e., 90% TFA, 5% HO, 5% triisopropylsilane (TIPS)) can be performed to cleave all of the side chain protecting groups (including the new GAP molecule) and the HPGC terminal protecting group (see FIG. 16). During this global deprotection, the RAG-protected aspartic acid is also converted to a deprotected asparagine. The molecule resulting from the global deprotection with a TFA cocktail is native Bivalirudin.

[0043] General procedure for Fmoc deprotection and coupling: For the new SolPPS method carried out in propylene carbonate (PC): To Fmoc-(AA)n-OBnDpp, Fmoc-(AA)n-HPG, Fmoc-(AA)n-RAG, or any amino acid C-terminally protected by any GAP protecting group pre-dissolved in propylene carbonate (200 mM), the deprotection base and octanethiol are added, followed by stirring with an alkane bilayer at room temperature for 15 minutes. The reaction mixture is then washed twice (x2) with fresh alkane solvent, then three times with saturated aqueous ammonium chloride, and then dried. In separate propylene carbonate solutions, 3.0 equivalents (eq.) of TBTU or TFFH, 3.0 eq. of Fmoc-AA-OH, and 3.0 eq. of diisopropylethylamine (DIPEA) are added, and the reaction is stirred for 7 minutes. This solution is then added to the pre-dried PC solution containing H-(AA)n-(GAP molecules) and allowed to couple with stirring for 10-60 minutes. An excess of a quenching agent is then added, such as a long-chain (having 10 to 18 carbons linearly linked, i.e., C10-C18) aliphatic thiol, long-chain (C10-C18) aliphatic alcohol, long-chain (C10-C18) aliphatic amine, long-chain (C10-C18) aliphatic selenol, aliphatic polyamine or aliphatic polyalcohol. The reaction mixture is then washed three times with saturated aqueous ammonium chloride and then dried to yield the extended peptide in PC solution. This process is repeated as necessary to produce peptides with the desired sequence and length. Regarding the novel SolPPS method carried out in dichloromethane or ethyl acetate: to Fmoc-(AA)n-OBnDpp, Fmoc-(AA)n-HPG, Fmoc-(AA)n-RAG, or any amino acid C-terminally protected by any GAP protecting group pre-dissolved in DCM or ethyl acetate (100 mM), the deprotection base is added, followed by stirring at room temperature for 10 minutes. The reaction mixture is then washed three times with saturated aqueous ammonium chloride and three times with saturated aqueous sodium bicarbonate, and then dried.In a separate DCM or ethyl acetate solution, 2.0 eq. TBTU or TFFH, 2.0 eq. Fmoc-AA-OH and 5.0 eq. DIPEA are added and the reaction is stirred for 7 minutes. This solution is then added to the pre-dried DCM or ethyl acetate solution containing H-(AA)n-(GAP molecule) and allowed to couple with stirring for 10-60 minutes. An excess of a quenching agent such as a long chain (C10-C18) aliphatic thiol, a long chain (C10-C18) aliphatic alcohol, a long chain (C10-C18) aliphatic amine, a long chain (C10-C18) aliphatic selenol, an aliphatic polyamine or an aliphatic polyalcohol is then added. The reaction mixture is then washed three times with saturated aqueous ammonium chloride and then dried to yield the extended peptide in DCM or ethyl acetate solution. This process is repeated as necessary to generate peptides with the desired sequence and length.

[0044] General procedure for EDCI coupling: The reaction mixture containing the deprotected N-terminus of the amino acid or peptide is cooled in an ice bath. Then, 2 eq of the amino acid to be coupled with such free N-terminus is added to the reaction mixture, followed by 2 eq of EDCI. The resulting mixture is removed from the ice bath and stirred for 1 hour, and then an excess of a quenching agent, such as a long-chain (C10-C18) aliphatic thiol, a long-chain (C10-C18) aliphatic alcohol, a long-chain (C10-C18) aliphatic amine, a long-chain (C10-C18) aliphatic selenol, an aliphatic polyamine or an aliphatic polyalcohol, is added. The reaction mixture is then washed three times with saturated aqueous ammonium chloride solution and three times with saturated aqueous sodium bicarbonate solution, and then dried.

[0045] Functionality may be distributed, in whole or in part, among multiple components in a manner now known or to become known. Thus, countless combinations are possible in achieving the functions, features and preferences described herein. Moreover, the scope of the present disclosure encompasses conventionally known ways of performing the features described, as well as variations and modifications that may be made to the methods, compositions or compounds described herein, as now and in the future will be understood by those skilled in the art.

[0046] Furthermore, the embodiments of the methods shown and described in this disclosure as diagrams, schematics or flow charts (e.g., figures) are provided as examples to provide a more complete understanding of the technology. The disclosed methods are not limited to the operations and logical flows presented herein. Alternative embodiments are contemplated in which the order of various operations is changed or sub-operations described as being part of a larger operation are performed independently. Although various embodiments have been described for purposes of this disclosure, such embodiments should not be considered to limit the teachings of this disclosure to those embodiments. Various changes and modifications may be made to the elements and operations described above to obtain results that remain within the scope of the systems and methods described in this disclosure. Additional Embodiments

[0047] Additional embodiments of the claimed invention include:

[0048] 1. A protecting group for solution phase peptide synthesis, wherein the protecting group has a formula selected from the group consisting of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7 and formula 8; Formula 1 is [ka] and; Chemical formula 2 is [ka] and; Formula 3 is [ka] and; Formula 4 is [ka] and; Formula 5 is [ka] and; Formula 6 is [ka] and; Formula 7 is [ka] and; Chemical formula 8 is [ka] It is. [In the formula, Z is selected from the group consisting of -H, a methyl group (-Me), and a methoxy group (-OMe); Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is an abbreviation for 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), 4-((9-amino-9H-xanthen-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl").

[0049] 2. The protecting group of item 1, wherein the protecting group is selected from the group consisting of formula 1, formula 4, formula 5 and formula 7; and R is the side chain of an amino acid.

[0050] 3. The protecting group of item 1, wherein R is selected from the group consisting of -H, methyl, -CH2SH, -CH2CH2COOH, -CH2COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH3)(CH2CH3), -CH2CH2CH2CH2NH2, -CH2CH2CH2NH2, isobutyl, -CH2CH2SCH3, -CH2CONH2, -CH2CH2CONH2, propyl group [wherein the 1-position of the propyl group is bonded to the same position as the R group and the 3-position of the propyl group is bonded to the nitrogen], -CH2CH2CH2N=C(NH2)2, -CH2OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl and 3-(1H-indolyl)methyl.

[0051] 4. A method for carrying out liquid phase peptide synthesis, comprising: attaching a first protecting group to a first amino acid to provide a first protected amino acid, the first protecting group being the protecting group of item 1; performing a coupling reaction on the first protected amino acid or on a peptide formed by linking the first amino acid with another molecule; Includes.

[0052] 5. The method of item 4, wherein the first protecting group is attached to the C-terminus of the first amino acid.

[0053] 6. The method of item 4, wherein a first protecting group is attached to the side chain of the first amino acid.

[0054] 7. The method of item 4, wherein the first protecting group is attached to the N-terminus of the first amino acid.

[0055] 8. The method of item 4, comprising coupling a second protected amino acid to the first protected amino acid, wherein the second protected amino acid comprises a second protecting group and a second amino acid, the second amino acid comprises a side chain, the second protecting group is attached to the side chain of the second amino acid, and the second protecting group has a chemical formula selected from the group consisting of Chemical Formula 1, Chemical Formula 2, Chemical Formula 3, Chemical Formula 4, Chemical Formula 5, Chemical Formula 6, Chemical Formula 7, and Chemical Formula 8.

[0056] 9. The method of claim 8, wherein the step of attaching a first protecting group includes attaching the first protecting group to the C-terminus of the first amino acid, the N-terminus of the first amino acid, or a side chain of the first amino acid.

[0057] 10. A method for forming a protecting group for solution phase peptide synthesis, comprising the steps of: coupling benzyldiphenylphosphine oxide (HOBnDpp), anilinediphenylphosphine oxide (NH2PhDpp) or a derivative thereof (i) directly to the linker molecule, (ii) via an amino acid to the linker molecule, or (iii) via an amino acid derivative to the linker molecule; Includes.

[0058] 11. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 1: [ka] is defined as follows: [In the formula, R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0059] 12. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 2: [ka] is defined as follows: [In the formula, L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0060] 13. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 3: [ka] is defined as follows: [In the formula, L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0061] 14. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, wherein the protecting group is represented by formula 4: [ka] is defined as follows: [In the formula, R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0062] 15. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 5: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0063] 16. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 6: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0064] 17. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 7: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0065] 18. The method of claim 10, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 8: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0066] 19. A protecting group for solution phase peptide synthesis, wherein the protecting group has a formula selected from the group consisting of formula 1, formula 2, formula 3, formula 4, formula 5, formula 6, formula 7 and formula 8; Formula 1 is [ka] and; Chemical formula 2 is [ka] and; Formula 3 is [ka] and; Formula 4 is [ka] and; Formula 5 is [ka] and; Formula 6 is [ka] and; Formula 7 is [ka] and; Chemical formula 8 is [ka] It is. [In the formula, Z is selected from the group consisting of -H, a methyl group (-Me), and a methoxy group (-OMe); Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is an abbreviation for 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), 4-((9-amino-9H-xanthen-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl").

[0067] 20. The protecting group of item 19, wherein the protecting group is selected from the group consisting of formula 1, formula 4, formula 5 and formula 7; and R is a side chain of an amino acid.

[0068] 21. The protecting group according to any one of items 19 and 20, wherein R is selected from the group consisting of -H, methyl, -CH2SH, -CH2CH2COOH, -CH2COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH3)(CH2CH3), -CH2CH2CH2CH2NH2, -CH2CH2CH2NH2, isobutyl, -CH2CH2SCH3, -CH2CONH2, -CH2CH2CONH2, a propyl group [wherein the 1-position of the propyl group is bonded to the same position as the R group and the 3-position of the propyl group is bonded to the nitrogen], -CH2CH2CH2N=C(NH2)2, -CH2OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl and 3-(1H-indolyl)methyl.

[0069] 22. A method for carrying out liquid phase peptide synthesis, comprising: attaching a first protecting group to the first amino acid to provide a first protected amino acid, the first protecting group being the protecting group of claim 1; performing a coupling reaction on the first protected amino acid or on a peptide formed by linking the first amino acid with another molecule; Includes.

[0070] 23. The method of item 22, wherein the first protecting group is attached to the C-terminus of the first amino acid.

[0071] 24. The method of any of items 22 and 23, wherein a first protecting group is attached to the side chain of the first amino acid.

[0072] 25. The method of any one of items 22, 23 and 24, wherein the first protecting group is attached to the N-terminus of the first amino acid.

[0073] 26. The method according to any one of items 22, 23, 24 and 25, comprising coupling a second protected amino acid to the first protected amino acid; The second protected amino acid comprises a second protecting group and a second amino acid, the second amino acid comprises a side chain, the second protecting group is attached to the side chain of the second amino acid, and the second protecting group has a chemical formula selected from the group consisting of: 1, 2, 3, 4, 5, 6, 7, and 8.

[0074] 27. The method of any one of items 22, 23, 24, 25 and 26, wherein the step of attaching a first protecting group comprises attaching the first protecting group to the C-terminus of the first amino acid, the N-terminus of the first amino acid, or a side chain of the first amino acid.

[0075] 28. A method for forming a protecting group for solution phase peptide synthesis, comprising the steps of: coupling benzyldiphenylphosphine oxide (HOBnDpp), anilinediphenylphosphine oxide (NH2PhDpp) or a derivative thereof (i) directly to the linker molecule, (ii) via an amino acid to the linker molecule or (iii) via an amino acid derivative to the linker molecule; Includes.

[0076] 29. The method of claim 28, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 1: [ka] is defined as follows: [In the formula, R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0077] 30. The method of any of items 28 and 29, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 2: [ka] is defined as follows: [In the formula, L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0078] 31. The method according to any one of items 28, 29 and 30, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 3: [ka] is defined as follows: [In the formula, L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0079] 32. The method according to any one of items 28, 29, 30 and 31, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 4: [ka] is defined as follows: [In the formula, R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0080] 33. The method according to any one of items 28, 29, 30, 31 and 32, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 5: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0081] 34. The method according to any one of items 28, 29, 30, 31, 32 and 33, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 6: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0082] 35. The method according to any one of items 28, 29, 30, 31, 32, 33 and 34, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 7: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; R is selected from the group consisting of known side chains of protected amino acids and known side chains of unprotected amino acids; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl.

[0083] 36. The method according to any one of items 28, 29, 30, 31, 32, 33, 34 and 35, wherein the coupling step comprises: [ka] with L-OH to form a protecting group, the protecting group being represented by formula 8: [ka] is defined as follows: [In the formula, Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; X is selected from the group consisting of -O-, -S- and -NH-; L is selected from the group consisting of HMPA, HMPB, HMB, MMB, MMPA, AMPA, DMPPA, Rink amide, xanthenyl, TCA and 2-chlorotrityl. Related References

[0084] The following references are incorporated herein by reference, by way of example:

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[0104] Merrifield, R. B. Solid Phase Peptide Synthesis. I. The Synthesis of a Tetrapeptide. J. Am. Chem. Soc. 1963, 85, 2149.

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[0106] Shelton, P. T.; Jensen, K. J. Linkers, Resins, and General Procedures for Solid-Phase Peptide Synthesis. In Peptide Synthesis and Applications, 2nd Edition, Jensen, K. J.; Shelton, P. T.; Pedersen, S. L., Eds. Humana Press Inc: Totowa, 2013; Vol. 1047, pp 23-41.

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[0112] Spinella, M.; De Marco, R.; Belsito, E. L.; Leggio, A.; Liguori, A. The dimethylsulfoxonium methylide as unique reagent for the simultaneous deprotection of amino and carboxyl function of N-Fmoc-α-amino acid and N-Fmoc-peptide esters. Tetrahedron 2013, 69, 2010-2016.

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Claims

1. A compound having a formula selected from the group consisting of: 1, 2, 3, 4, 5 and 6; Chemical formula 1 is 【Chemistry 1】 and Chemical formula 2 is 【Chemistry 2】 and Chemical formula 3 is 【Chemistry 3】 and Chemical formula 4 is 【Chemistry 4】 and Chemical formula 5 is 【Chemistry 5】 and Chemical formula 6 is 【Chemistry 6】 The compound according to claim 1, [In the formula, Z is selected from the group consisting of -H, a methyl group (-Me), and a methoxy group (-OMe); Y is selected from the group consisting of -O-, -S-, and -NH-; R is -H, methyl, -CH ２ S-H, -C-H ２ CH ２ COOH, -CH ２ COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH ３ ) (CH ２ CH ３ ), -CH ２ CH ２ CH ２ CH ２ N.H. ２ , -CH ２ CH ２ CH ２ N.H. ２ , isobutyl, -CH ２ CH ２ SCH ３ , -CH ２ CONH ２ , -CH ２ CH ２ CONH ２ , -CH ２ CH ２ CH ２ N=C(NH ２ ) ２ , -CH ２ or R is selected from the group consisting of OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl, and 3-(1H-indolyl)methyl, or R together with the carbon atom bonded to O═C and the NH bonded to said carbon atom forms a pyrrolidine ring; L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), 4-((9-amino-9H-xanthen-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl").

2. 2. The compound according to claim 1, wherein the compound is selected from the group consisting of formula 1, formula 4 and formula 5.

3. 1. A method for performing liquid phase peptide synthesis comprising the steps of: coupling a first compound to a first amino acid to provide a first protected amino acid, said first compound being a compound according to claim 1; performing a coupling reaction on the first protected amino acid or on a peptide formed by linking the first amino acid with another molecule; The method according to claim 1, further comprising:

4. 4. The method of claim 3, wherein the first compound is attached to the C-terminus of the first amino acid.

5. 4. The method of claim 3, wherein the first compound is attached to a side chain of the first amino acid.

6. 4. The method of claim 3, wherein the first compound is attached to the N-terminus of the first amino acid.

7. 4. The method of claim 3, comprising coupling a second protected amino acid to the first protected amino acid, wherein the second protected amino acid comprises a second compound and a second amino acid, the second amino acid comprises a side chain, the second compound is attached to the side chain of the second amino acid, and the second compound has a formula selected from the group consisting of:

8. 8. The method of claim 7, wherein the step of attaching the first compound comprises attaching the first compound to the C-terminus of the first amino acid, the N-terminus of the first amino acid, or the side chain of the first amino acid.

9. 1. A method of forming a compound, the method comprising: 【Chemistry 7】 with L-OH to form the compound, the compound having the formula 1: 【Chemistry 8】 wherein: R is -H, methyl, -CH ２ S-H, -C-H ２ CH ２ COOH, -CH ２ COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH ３ ) (CH ２ CH ３ ), -CH ２ CH ２ CH ２ CH ２ N.H. ２ , -CH ２ CH ２ CH ２ N.H. ２ , isobutyl, -CH ２ CH ２ SCH ３ , -CH ２ CONH ２ , -CH ２ CH ２ CONH ２ , -CH ２ CH ２ CH ２ N=C(NH ２ ) ２ , -CH ２ or R is selected from the group consisting of OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl, and 3-(1H-indolyl)methyl, or R together with the carbon atom bonded to O═C and the NH bonded to said carbon atom forms a pyrrolidine ring; L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

10. 1. A method of forming a compound, the method comprising: 【Chemistry 9】 with L-OH to form the compound, the compound having the formula 2: 【Chemistry 10】 wherein: L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

11. 1. A method of forming a compound, the method comprising: 【Chemistry 11】 with L-OH to form the compound, the compound having the formula 3: 【Chemistry 12】 wherein: L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

12. 1. A method of forming a compound, the method comprising: 【Chemistry 13】 with L-OH to form the compound, the compound having formula 4: 【Chemistry 14】 wherein: R is -H, methyl, -CH ２ S-H, -C-H ２ CH ２ COOH, -CH ２ COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH ３ ) (CH ２ CH ３ ), -CH ２ CH ２ CH ２ CH ２ N.H. ２ , -CH ２ CH ２ CH ２ N.H. ２ , isobutyl, -CH ２ CH ２ SCH ３ , -CH ２ CONH ２ , -CH ２ CH ２ CONH ２ , -CH ２ CH ２ CH ２ N=C(NH ２ ) ２ , -CH ２ or R is selected from the group consisting of OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl, and 3-(1H-indolyl)methyl, or R together with the carbon atom bonded to O═C and the NH bonded to said carbon atom forms a pyrrolidine ring; L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

13. 1. A method of forming a compound, the method comprising: 【Chemistry 15】 with L-OH to form the compound, the compound having the formula 5: 【Chemistry 16】 wherein: Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; R is -H, methyl, -CH ２ S-H, -C-H ２ CH ２ COOH, -CH ２ COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH ３ ) (CH ２ CH ３ ), -CH ２ CH ２ CH ２ CH ２ N.H. ２ , -CH ２ CH ２ CH ２ N.H. ２ , isobutyl, -CH ２ CH ２ SCH ３ , -CH ２ CONH ２ , -CH ２ CH ２ CONH ２ , -CH ２ CH ２ CH ２ N=C(NH ２ ) ２ , -CH ２ or R is selected from the group consisting of OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl, and 3-(1H-indolyl)methyl, or R together with the carbon atom bonded to O═C and the NH bonded to said carbon atom forms a pyrrolidine ring; L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

14. 1. A method of forming a compound, the method comprising: 【Chemistry 17】 with L-OH to form the compound, the compound having the formula 6: 【Chemistry 18】 wherein: Z is selected from the group consisting of -H, -Me, and -OMe; Y is selected from the group consisting of -O-, -S-, and -NH-; L is selected from the group consisting of 4-(hydroxymethyl)phenoxyacetyl ("HMPA"), 4-(hydroxymethyl)phenoxybutanoyl ("HMPB"), 4-(hydroxymethyl)benzoyl ("HMB"), 4-(mercaptomethyl)benzoyl ("MMB"), 4-(mercaptomethyl)phenoxyacetyl ("MMPA"), 4-(aminomethyl)phenoxyacetyl ("AMPA"), 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl ("DMPPA"), 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl ("Rink amide"), xanthenyl, 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulen-3-yl)pentanoyl ("TCA"), and 2-chlorotrityl.

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