Compositions and methods for chemical synthesis

Abstract

The present disclosure relates to chemical compositions that can include anchor molecules for chemical synthesis. GAP anchor molecules can include a GAP moiety, a linker moiety, and a spacer moiety. Anchor molecules can be used to synthetically produce peptides. Also disclosed is a novel method of solution-phase peptide synthesis that utilizes novel group-assisted purification protecting groups to facilitate efficient and scalable chemistry for synthetically producing peptides.

Description

[Technical Field]

[0001] All applications in which a foreign or domestic priority claim is identified in the application data sheet (whether filed together with this application or subsequently amended) are incorporated herein by reference under 37 CFR §1.57. This application cross-references: i) PCT application no. PCT / US16 / 68112, filed December 21, 2016, “System and method for solution phase GAP peptide synthesis”, ii) PCT application no. PCT / US19 / 29569, filed April 29, 2019, “Method for Solution-Phase Peptide Synthesis”, iii) PCT application no. PCT / US19 / 33296, filed May 21, 2019, “Method for Solution-Phase Peptide Synthesis and Protecting Strategies Thereof”, and iv) PCT application no. PCT / US20 / 15132, filed January 26, 2020, and these applications are incorporated herein by reference as examples. This application is described in PCT / US19 / 33296, PCT / US20 / 15132, U.S. Patent Application No. 62 / 678,564, and U.S. Patent Application No. 62 / 800,142, which are incorporated herein by reference as examples.

[0002] Description of federally funded research and development. none

[0003] References to sequence lists, tables, or computer lists in the Compact Disc appendix. none [Background technology]

[0004] Recent research efforts have made significant progress in the field of purification chemistry, particularly in avoiding column chromatography and recrystallization. This research is defined as group-assisted purification (GAP) chemistry / technique, which is the chemistry for organic synthesis that avoids conventional purification methods such as chromatography and / or recrystallization by intentionally introducing highly functionalized groups into starting materials or newly generated products. These GAP groups are also often used as protecting groups to prevent undesirable side reactions during the synthesis of target molecules. Such research has the potential to cover the entire field of organic synthesis chemistry.

[0005] Protecting groups are found in almost all complex synthesis involving multiple functional groups. In GAP chemistry, a semi-permanent protecting group that introduces the solubility characteristics required for GAP would be an ideal example. However, many conventional protecting groups are nonpolar and therefore cannot achieve the solubility required for GAP with most substrates. If a protecting group with appropriate solubility control can be developed, it may be possible to extend GAP chemistry to all synthesis requiring the use of such a protecting group. Several methods have been employed. Published patent application WO 2014093723 A2 teaches protecting imines with a chiral auxiliary possessing GAP and using these chiral N-phosphonylimines as electrophiles in asymmetric boronation reactions. Purification was performed by the GAP method. This study is valuable because it allows for the easy acquisition of chiral α-boronic acid amines, which may be used to synthesize novel amino acid derivatives and incorporate them into novel peptide targets.

[0006] Effective protecting groups need to be robust to a wide variety of conditions and can be added and removed in high yield. Protecting groups are widely used in peptide synthesis, either in solid-phase or solution-phase methods. One of the most commonly used protection strategies in conventional peptide synthesis is Fmoc / tBu. U.S. Patent No. 8,383,770B2 teaches the use of fluorenyl methoxycarbonyl (Fmoc) and tert-butyloxycarbonyl (Boc) N-terminal protecting groups in solid-phase peptide synthesis (SPPS). This technique is well known and widely applied in industry. The Fmoc group protects the N-terminus of the amino acid to be added to the growing peptide, while the tert-butyl (tBu) group protects the side chain of the same amino acid. The Fmoc group can be removed with a suitable deprotecting agent, and the tBu group remains until overall deprotection with acid is performed at the end of the synthesis. Boc and Fmoc groups have been used for decades in all areas of peptide chemistry, and preferred Fmoc groups are almost exclusively limited to SPPS.

[0007] Developed by Merrifield in the 1960s, SPPS has become a standard protocol used in multiple scientific fields for research and manufacturing (see Figure 1A). The advantage of polymer supports or resins lies in their ability to easily purify the grown peptide after each coupling / deprotection step, thereby avoiding the use of column chromatography. The main disadvantage of SPPS is its difficulty in scaling up; many polymer supports are expensive and account for a large portion of the mass of material being handled. Also, the coupling reaction in SPPS occurs at the solid-liquid interface, making it inefficient. Furthermore, after each deprotection and coupling reaction, the resin must be washed with a solvent to remove impurities from the previous reaction, which generates considerable solvent waste, a significant problem on a large scale.

[0008] However, economically feasible Fmoc protection schemes for solution-phase peptide synthesis (SolPPS) are rare, and virtually nonexistent in the literature. U.S. Patent 5,516,891A provides one of the few examples of Fmoc-based SolPPS. Here again, Fmoc peptide synthesis is almost exclusively limited to SPPS because N-fluorenylmethylpiperidine (NFMP) is generated as a byproduct during deprotection, and is difficult to remove without a polymer support. The standard protocol for Fmoc deprotection is to stir the Fmoc-peptide with excess piperidine in a solution of dimethylformamide (DMF) or dichloromethane (DCM) to deprotect the Fmoc group and form NFMP in the process. Patent '891 teaches that this impurity can be removed by deprotecting with 4-aminomethylpiperidine (4AMP) instead of piperidine. This results in the formation of NFMP-CH2NH2 instead of NFMP, and because of the presence of an extra amino group, it can be extracted in water. The problem with this method is the high cost associated with using 4AMP. Therefore, this method is expensive and not widely accepted in the industry.

[0009] Another example of an Fmoc-based SolPPS can be found in published patent application WO2017112809A1. This publication teaches controlling the solubility of a target peptide so that it can be selectively precipitated after each subsequent coupling reaction using benzyl diphenylphosphine oxide (HOBnDpp), a C-terminal GAP protecting group (see Figure 1B). The solubility is controlled so that the growing peptide remains in an organic solvent such as ethyl acetate or DCM, and impurities are removed by washing with water. The organic solvent is then concentrated and mixed with an alkane solvent, and the peptide product selectively precipitates. While this technique adapts Fmoc / tBu chemistry to the solution phase in a far more economically viable way than the '891 patent, this method has inherent potential limitations. First, the GAP group is not acid-unstable, and a one-step overall deprotection with commonly used trifluoroacetic acid systems (TFA systems) or other acidic mixtures is not possible; and hydrogenation or hydrolysis is required to remove HOBnDpp from the C-terminus of the peptide. Furthermore, as the peptide continues to grow and its sequence changes, HOBnDpp becomes increasingly unable to maintain control over solubility as the chain lengthens or as the sequence imparts different solubility characteristics to the peptide. HOBnDpp is also susceptible to hydrolysis and accidental cleavage during synthesis. For example, when deprotecting the second amino acid of the peptide sequence with Fmoc, the formation of diketopiperazine (DKP) makes it easy for HOBnDpp to disappear from the C-terminus. This cleaved HOBnDpp remains in the reaction mixture and reacts with activated amino acids during coupling reactions to produce impurities that are difficult or impossible to remove. Moreover, current cleavage techniques (either hydrolysis or hydrogenation) to deprotect the C-terminus after synthesizing the protected peptide result in a carboxylic acid at the C-terminus of the final peptide product. Many therapeutic peptides, conversely, require modification of the C-terminus with amides, cyclic peptides, thioesters, etc., instead of this carboxylic acid, further limiting the applicability of GAP peptide synthesis methods. Furthermore, these cleavage reactions to remove HOBnDpp from the C-terminus are entirely separate from overall deprotection, and they have the disadvantages of requiring specific conditions and workups, as well as being time-consuming and costly.

[0010] Due to these and other issues related to scale-up in SPPS, as well as the limitations of existing GAP peptide synthesis protection strategies, the industry has a need for a SolPPS method that is not only suitable for scale-up but also better suited to (i) various cleavage conditions, (ii) longer or more difficult sequences (in terms of solubility), and (iii) C-terminal modifications. [Overview of the project]

[0011] In one embodiment, the disclosure may include a chemical composition comprising a GAP component, a spacer component, and a linker component, wherein the spacer component is positioned between the GAP component and the linker component.

[0012] In another embodiment, the disclosure may include a method for synthesizing a peptide, the method comprising the steps of: attaching a first amino acid to an anchor molecule, the anchor molecule comprising a GAP component and a linker component, and the first amino acid being attached to the linker component of the anchor molecule; forming a peptide bond between a second amino acid and a first amino acid; and removing the anchor molecule from the first amino acid. In another embodiment, the disclosure may include a method for forming a composition comprising a GAP anchor, the method comprising the steps of: providing a GAP component; and attaching the GAP component to a linker component.

[0013] In another embodiment, the disclosure may include a method for synthesizing a peptide, the method comprising the steps of: attaching a GAP anchor to a first amino acid; forming a peptide bond between a second amino acid and the first amino acid; and removing the GAP anchor from the first amino acid, the GAP anchor is Compound 1 (wherein n=2 or 9 in the formula) [ka] , compound 2 [ka] , compound 3 [ka] Compound 4 (wherein R = H, Me, Et, or iPr) [ka] Compound 5 (where n=0 or 2 in the formula) [ka] Compound 6 (wherein n=0 or 2, and Y=O, NMe, NET, or N-iPr) [ka] Compound 7 (wherein n=0 or 2) [ka] Compound 8 (wherein n=0 or 2, and Y=O, NMe, NET, or N-iPr) [ka] , compound 9 [ka] Compound 10 (where n=0 or 2 in the formula) [ka] , and compound 11 (wherein n=0 or 2) [ka] It is selected from the group consisting of the following. The above and other purposes, features, and advantages of this disclosure will become apparent from the following description of embodiments illustrated in the accompanying drawings, where reference letters refer to the same parts from various perspectives. The drawings are not necessarily to scale and instead focus on illustrating the principles of this disclosure. The drawings are used as non-limiting examples and are intended solely to depict preferred embodiments without limiting the scope of this disclosure. [Brief explanation of the drawing]

[0014] [Figure 1A] Figure 1A illustrates a prior art method for solid-phase peptide synthesis (SPPS). [Figure 1B] Figure 1B shows the steps of the GAP peptide synthesis (GAP-PS) method, particularly illustrating the use of HOBnDpp for C-terminal protection. [Figure 2] Figure 2 illustrates a method in which the protecting group shown in Figure 1B is attached to the side chain of an amino acid residue, particularly serine in this non-limiting embodiment, and then GAP-PS is performed. [Figure 3] Figure 3 shows a schematic diagram illustrating the synthesis of a novel GAP molecule and its attachment to a peptide, as an example. HOBnDpp is reacted with protected Rink Amide-OH, and the resulting novel chemical compound Rink Amide-GAP (RAG) is attached to the C-terminus of an amino acid. [Figure 4] Figure 4 illustrates the attachment of a novel GAP molecule to the amino acid side chain. In this non-limiting example, the GAP molecule RAG is used to protect the side chain of aspartic acid, synthesizing RAG-protected asparagine. Specifically, a coupling reaction occurs, forming an amide bond between the free amine on the RAG molecule and the carbonyl carbon of asparagine. [Figure 5]Figure 5 illustrates a non-limiting example of the synthesis of a novel GAP molecule. In this non-limiting example, HOBnDpp can be directly linked to a protected HMPA-OH to form HMPA-GAP(HG), and also to form the GAP molecule HMPA-phenylalanineGAP(HPG) via an intermediate (phenylalanine in one embodiment). Specifically, HOBnDpp can be linked to the carboxylic acid portion of a protected HMPA-OH, or HOBnDpp can first link to an amino acid and then to HMPA to form the GAP molecule HPG. [Figure 6] Figure 6 illustrates the attachment of HPG from Figure 5 to the C-terminus of a common amino acid, and, in a preferred embodiment of this disclosure, to the side chain of aspartic acid. Specifically, this reaction is a coupling reaction that can be achieved using several different coupling reagents, in which the free alcohol of HPG binds to the carbonyl group of the activated carboxylic acid. [Figure 7] Figure 7 illustrates a non-limiting example of the synthesis of a novel GAP molecule, aniline diphenylphosphine oxide ("NH2PhDpp"). [Figure 8] Figure 8 illustrates a non-limiting example of the synthesis of a novel GAP molecule, in which NH2PhDpp is reacted with protected Rink Amide-OH to achieve the target entity. Specifically, NH2PhDpp can be coupled to the carboxylic acid moiety of protected Rink Amide-OH via a number of coupling reagents. [Figure 9] Figure 9 shows a non-limiting example of the synthesis of a novel GAP molecule, where NH2PhDpp reacts with protected HMPA-OH, either directly or via an intermediate, to form the target molecule. These reactions can be facilitated using several different coupling reagents. [Figure 10] Figure 10 shows a compound obtained by attaching an HPG GAP molecule to the C-terminus of Fmoc-protected leucine, as a non-limiting example. [Figure 11]Figure 11 depicts, as a non-limiting example, a compound obtained from side-chain protection of glutamate using HPG, where the C-terminus of glutamate is protected with an allyl group, as a non-limiting example of possible C-terminus protection strategies. [Figure 12] Figure 12 shows, as a non-limiting example, the tetrapeptide Fmoc-Glu(HPG)-Glu(HPG)-Tyr(tBu)-Leu-HPG obtained from the novel SolPPS method disclosed herein. [Figure 13] Figure 13 depicts compounds obtained by attaching RAGs to the side chains of amino acids, particularly aspartic acid in this non-limiting example, where the C-terminus of aspartic acid is protected by an allyl group, representing a non-limiting example of a C-terminus protection strategy. [Figure 14] Figure 14 shows, as a non-limiting example, a protected peptide obtained from the SolPPS method disclosed herein. [Figure 15] Figure 15 shows, as a non-limiting example, the peptide bivaliridine fully protected with a novel GAP molecule. [Figure 16] Figure 16 shows fully deprotected bivaliridine achieved by a one-step overall protection process that removes all protecting groups from the side chains and C-terminus, following Fmoc deprotection. [Figure 17] Figure 17 illustrates a general methodology for protecting amines with novel GAP molecules. In this particular non-limiting embodiment, HPG is used. [Figure 18] Figure 18 shows, as a non-limiting example, a number of molecules that could function as reagents for the synthesis of novel GAP molecules disclosed herein. [Figure 19] Figure 19 shows numerous representative coupling reactions for synthesizing the novel GAP molecules disclosed herein. [Figure 20A] Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 20B]Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 20C] Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 20D] Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 20E] Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 20F] Figures 20A to 20F show the structures of the H-Anchor molecule and its Gap, Linker, and Spacer components. [Figure 21A] Figures 21A and 21B depict exemplary structures of GAP anchors or H-Anchor molecules in accordance with this disclosure. [Figure 21B] Figures 21A and 21B depict exemplary structures of GAP anchors or H-Anchor molecules in accordance with this disclosure. [Figure 22] Figure 22 illustrates an exemplary structure of a GAP anchor (H-Anchor) according to an embodiment of the present disclosure. [Figure 23A] Figures 23A and 23B illustrate exemplary methods for synthesizing chemical compounds according to embodiments of the present disclosure. [Figure 23B] Figures 23A and 23B illustrate exemplary methods for synthesizing chemical compounds according to embodiments of the present disclosure. [Modes for carrying out the invention]

[0015] Specific features of the present invention are referenced in the above summary of the invention, the detailed description of the invention, the following claims, and the accompanying drawings. It should be understood that the disclosure of the invention herein includes all possible combinations of such specific features. For example, if a specific feature is disclosed in relation to a particular aspect or embodiment of the invention, or a particular claim, that feature may, to the extent possible, be used in combination with and / or in relation to other particular aspects and embodiments of the invention, and also in general use in the invention.

[0016] All compositions and / or methods disclosed and claimed herein can be manufactured and performed without excessive experimentation in light of this 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 in the compositions and / or methods, as well as in the processes or sequences of processes, can be applied without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications, which are 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 “comprising” and its grammatical equivalents are used herein to mean the presence of other components, ingredients, processes, etc., as such. For example, an article “comprising” (or “which comprises”) components A, B, and C may consist only of components A, B, and C, or it may contain not only components A, B, and C, but one or more other components.

[0018] Where this specification refers to a method comprising two or more defined steps, the defined steps may be performed in any order or simultaneously (unless the context excludes such possibility), and the method may include one or more other steps performed before any of the defined steps, between two of the defined steps, or after all of the defined steps (unless the context excludes such possibility).

[0019] In this specification, the term “at least” is followed by a number to indicate the start of a range that begins with that number (which may be a range with an upper limit or without an upper limit, depending on the variable being defined). For example, “at least 1” means 1 or greater than 1. In this specification, the term “maximum” is followed by a number to indicate the end of a range that ends with that number (which may be a range with a lower limit of 1 or 0, or without a lower limit, depending on the variable being defined). For example, “maximum 4” means 4 or less than 4, and “maximum 40%” means 40% or less than 40%. In this specification, “(initial value) ~ (to) (successor value)” or “(initial value) ~ (-) (successor value)” means a range where the lower limit is the initial value and the upper limit is the successor value. For example, 25 ~ 100 mm means a range with a lower limit of 25 mm and an upper limit of 100 mm.

[0020] The term "first" is used to distinguish one element from another, and does not mean that an element is the primary or first element in any given sequence of elements. For example, "first amino acid" does not mean that the amino acid is the first in a sequence of amino acids or the first amino acid to react. Rather, "first amino acid" simply indicates that the amino acid can be separated and distinguished from another amino acid, such as "second amino acid."

[0021] The term "coupling reaction" is generally used to refer to the formation of a bond between two constituent molecules, which can be facilitated by a "coupling reagent." In peptide chemistry, these coupling reactions can occur in many different mechanisms under many different reaction conditions, which can be entirely dependent on the coupling reagent used. For example, a coupling reagent can "activate" the carboxylic acid of a constituent molecule so that the carbonyl carbon becomes more susceptible to nucleophilic attack. In coupling reactions, water molecules may be lost when forming a bond between two constituent molecules.

[0022] In many types of protection schemes for peptide synthesis, similar reactions are repeated to grow the peptide chain. Generally, either the N-terminus or C-terminus of each amino acid added to the chain is initially protected, while the other end of the amino acid is free to participate in the coupling reaction. After the amino acid is initially added to the chain via the unprotected end, a deprotection reaction is performed, releasing the protected N-terminus or C-terminus and allowing it to participate in the subsequent coupling reaction to form a peptide bond with the next amino acid. For example, in Fmoc / tBu peptide synthesis, the N-terminus of an amino acid is protected by an Fmoc group, and the side chain of the amino acid is protected by a tBu protecting group, which is 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 their side chains are naturally inert to coupling and deprotection conditions). The C-terminus of the primary amino acid in the peptide sequence is protected by being attached to a resin or polymer in SPPS, or to a protecting group in SolPPS. The Fmoc / tBu peptide synthesis scheme is designed so that the Fmoc group on the N-terminus of an amino acid is base-unstable, and by treatment with an appropriate deprotecting agent, the Fmoc group can be removed from the N-terminus without interfering with C-terminus attachment or side chain protection. During the deprotection reaction, the N-terminus of the primary amino acid is freed, while the C-terminus and side chain are protected or spontaneously inactivated. Next, the following amino acid, whose N-terminus is protected with Fmoc and whose side chain is protected or spontaneously inactivated, is activated at its free C-terminus using a coupling reagent. This activation facilitates nucleophilic attack by the free N-terminus of the primary amino acid at the activated carbonyl, forming a peptide bond between the primary and subsequent amino acids. This process is repeated until a suitable peptide sequence is obtained. After Fmoc deprotection of the final amino acid, the peptide remains protected at its C-terminus and side chain.Next, overall deprotection is performed using a strong acid mixture such as a TFA-based mixture, removing all protecting groups from the side chains, and in some cases, cleaving the resin or protecting group at the C-terminus.

[0023] This disclosure addresses the shortcomings of the technique by providing a method for solution-phase peptide synthesis that enables various overall deprotection strategies, synthesis of longer or more difficult (solubility-wise) peptide sequences, and C-terminal modification on target peptides, while maintaining solubility control of the target peptide and the entire synthesis process. By utilizing novel protection strategies for the C-terminus and side chains of the growth peptide, we present an economically feasible SolPPS strategy useful for the commercial production of peptides.

[0024] Therefore, enabling novel methods for SolPPS is a non-limiting object of this disclosure. In one embodiment, the conventional C-terminal GAP protecting group HOBnDpp 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 a peptide chain. Other amino acid residues of the desired peptide can be protected using many different protection strategies, including but not limited to Fmoc / tBu, Boc / benzyl, Cbz(benzyloxycarbonyl) / tBu, Cbz / benzyl, etc. This enhanced protection strategy enables the synthesis of longer and / or more difficult sequences while maintaining control of the solubility of the peptide chain. This strategy also allows the use of different C-terminal protecting groups for the purposes of solubility, deprotection, or post-synthesis C-terminal modification. In another embodiment, the novel GAP molecules described below are used to protect the C-terminus and / or side chains of specific amino acids, enabling C-to-N peptide synthesis in solution.

[0025] In another embodiment, the novel SolPPS method is achieved by a specially designed C-terminal protection strategy, and the novel GAP molecular protecting group allows for a wider range of cleavage conditions, synthesis of longer peptides, minimal unintentional or accidental deprotection, and modification of the C-terminus during cleavage, while maintaining the advantages of the original GAP peptide synthesis (GAP-PS). Depending on the GAP molecule used, C-terminal deprotection includes, but is not limited to, treatment with trifluoroacetic acid (TFA), hydrofluoric acid (HF), toluenesulfonic acid (TsOH), methanesulfonic acid (MsOH), etc., and basic hydrolysis conditions including, but not limited to, aqueous or alcoholic metal hydroxides, or two-phase systems using phase-transfer catalysts, and Pd 0 or Pt 0 This can be achieved by any of the following reduction conditions, including but not limited to hydrogen molecules on a transition metal catalyst such as Ru, mobile hydrogenation, or other hydrogen sources such as boron hydride, aluminum hydride, silane, or ammonium formate. Furthermore, depending on the specific GAP molecule, C-terminal amide formation and other post-synthesis modifications can be performed during the cleavage of the C-terminal GAP molecule, allowing for the production of the desired peptide product while eliminating extra post-synthesis processing steps. These novel GAP molecules are stable to acid-catalyzed deprotection reactions, as well as hydrogenation and hydrolysis reactions, and can increase the recovery rate of the GAP protecting group after peptide synthesis. Moreover, the loss of the C-terminal protecting group during synthesis is minimized by these novel GAP molecules.

[0026] In another embodiment, any of the novel GAP molecules described above is 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 the peptide. This strategy allows for the 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 directly or via an intermediate to the carboxylic acid portion of a linker molecule, an example of which is shown in Figure 18, and the resulting novel GAP protecting group is then attached to the C-terminus of a peptide. As yet another non-limiting example, aniline diphenylphosphine oxide (i.e., NH2PhDpp) is similarly bound to a different linker and realizes a novel GAP protecting group that can be used in a similar manner.

[0028] In another embodiment, any of these different strategies described above may be used in different combinations to enable the best synthetic 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 achieved in a single reaction step along with side-chain deprotection.

[0029] Therefore, providing a method for SolPPS is a non-limiting object of this disclosure. In designing this method, it was clear that it should be required to retain the advantages of other solution-phase methods such as GAP-PS, such as avoiding difficult distillation and purification strategies that would hinder large-scale production. The enhanced protection strategy would need to be designed to maintain control over the solubility of the target peptide, while also addressing the shortcomings of the art, and to allow for the separation of the product from the resulting amino acids and impurities of the deprotection protocol.

[0030] In non-limiting exemplary embodiments of this disclosure, the novel SolPPS method can begin with the direct attachment of HOBnDpp to the C-terminus of a primary amino acid in a given sequence (see Figure 1B). Alternatively, or in addition to protecting the C-terminus of a primary amino acid with HOBnDpp, but not limited to, attachment of HOBnDpp to the side chains of amino acid residues such as serine, threonine, tyrosine, and / or cysteine, and subsequent incorporation into the growing peptide, can also be performed to better control solubility and protect the side chains of specific amino acids (see Figure 2). To achieve 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 obtain IBnDpp, ClBnDpp, or BrBnDpp depending on the halogenating agent used. This reaction can be carried out under a variety of conditions depending on the halogenating agent and base used in the reaction. For example, this reaction can be carried out at a pressure of 1 to 100 bar, a temperature of -78°C to 100°C, and in a monophase or biphase reaction medium. Next, the halogenated compound can be reacted with an amino acid having 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 chains. If the GAP molecule protects the C-terminus of the peptide, instead of protecting the side chains of one or more amino acids incorporated into the peptide sequence with GAP, or in addition to this, peptide synthesis can be carried out in the C-to-N direction. Alternatively, if HOBnDpp or another GAP molecule protects only the amino acid side chains, peptide synthesis can be carried out in the N-to-C direction. Numerous protection strategies are possible for protecting the N-terminus and side chains. Potential N-terminal or C-terminal protecting groups may include, but are not limited to, Cbz, Fmoc, Boc, methyl, Bn (benzyl), or any of the GAP molecules discussed herein. Side-chain protecting groups may 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, as shown in Figure 3, begins with the use of a novel GAP molecule (here, RAG) for C-terminal protection, which can enable both C-terminal protection of acid-unstable peptides and C-terminal amide formation by cleavage of RAG from the peptide. Once C-terminal protection by RAG is achieved, peptide synthesis proceeds from C to N, and a multitude of N-terminal and side-chain protection strategies, such as the Fmoc / tBu strategy, can be utilized, but are not limited to. To synthesize RAG, HOBnDpp is protected via a number of coupling reagents, including, but not limited to, TFFH ("N-((dimethylamino)fluoromethylene)-N-methylmethaneaminium hexafluorophosphate"), TBTU ("1-((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazole-3-oxidetetrafluoroborate"), HBTU ("1-((dimethylamino)(dimethylimino)methyl)-1H-benzo[d][1,2,3]triazole-3-oxidehexafluorophosphate"), EDCI ("3-(((ethylimino)methylene)amino)-N,N-dimethylpropane-1-amine"), or COMU ("(1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium hexafluorophosphate"), and Rink. RAG molecules can be bonded to amide-OH to provide GAP molecules. The coupling reaction can be carried out under various conditions depending on the specific coupling reagent used. For example, this reaction can be carried out at atmospheric pressure and room temperature with gentle stirring using TBTU. Once a protected RAG molecule is synthesized, a deprotection reaction (e.g., Fmoc deprotection) occurs, releasing the amine on the Rink Amide, allowing it to participate in coupling reactions with carboxylic acids, such as those present at the C-terminus of amino acids. RAGs can be attached to the side chains of amino acid residues, including but not limited to aspartic acid and glutamic acid (see Figure 4), to enhance protection and solubility control when adding these amino acids to the growth sequence.When the RAG is cleaved from the side chains of amino acids, amide forms of those side chains (in this case, asparagine and glutamine, respectively) are produced (see Figure 4).

[0032] In another non-limiting exemplary embodiment, the novel SolPPS method can begin with the use of a novel GAP molecule (here, (Rink Amide)-phenylalanine-GAP, i.e., RPG) for C-terminus protection. RPG can be synthesized as shown in Figure 5. Specifically, HOBnDpp is bonded to Fmoc-protected phenylalanine to form Fmoc-phenylalanine-GAP (i.e., Fmoc-F-GAP), and after Fmoc deprotection, the now-liberated N-terminus of the GAP-protected phenylalanine is bonded to the carboxylic acid moiety of the protected Rink Amide-OH (the molecular structure of the protected Rink Amide-OH can be seen in Figure 3). The resulting structure can then deprotect the N-terminus of the Rink Amide molecule, and the liberated N-terminus can be used in a coupling reaction to form a peptide bond with the primary amino acid of a given peptide sequence. Subsequently, peptide synthesis can proceed from C to N while RPG protects the C-terminus of the growing peptide. As a C-terminal protecting group, RPG can be used in conjunction with any of the disclosed GAP molecules that provide side-chain protection, or with other protection strategies available for N-terminal and side-chain protection. In addition to, or instead of C-terminal protection, RPG can also be used to protect the side chains of amino acids, similar to how RAG is used.

[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 Figure 5, and these molecules can be attached to the C-terminus of peptides to enable acid-unstable C-terminal protection without forming a C-terminal amide during cleavage (see Figure 6). Instead of protecting the C-terminus of amino acids, or in addition to doing so, 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 assist in controlling solubility (see Figure 6). Peptide synthesis can then proceed from C to N using a number of protection strategies, including 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 shown in Figure 7. Specifically, the synthesis of NH2PhDpp can begin with 4-bromonitrobenzene, and at ultra-low temperatures (-100 to -30°C) and room pressure, in an ether-based solvent, N-butyllithium (nBuLi) can be activated to form an organolithium reagent. Upon slow addition of chlorodiphenylphosphine, this reagent reacts in situ, and after phosphine oxidation with hydrogen peroxide and reduction of the nitro group using H2 and 10% Pd / C, NH2PhDpp can be obtained. This molecule can then, in a non-limiting example, be attached to Rink Amide-OH (see Figures 8 and 18), and the resulting GAP molecule can be attached to the C-terminus of a peptide as in Figure 3 and / or to the side chain of aspartic acid or glutamic acid as in Figure 4 (creating protected asparagine or glutamine, respectively). In another non-limiting example, another novel GAP molecule may be synthesized via a direct or amine-mediated reaction between NH2PhDpp and the carboxylic acid moiety of a linker (here, HMPA), as seen in Figure 9. The resulting GAP molecule can then be attached to the C-terminus and / or side chains of aspartic acid, cysteine, serine, threonine, and glutamic acid of a peptide, as seen in Figure 6. The peptide synthesis can then proceed from C to N, utilizing, but not limited to, a multitude of protection strategies such as the Fmoc / tBu strategy.

[0035] For all of the non-limiting examples above, HOBnDpp, NH2PhDpp, or any other GAP molecule can be attached to the C-terminus or side chain of amino acid residues 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 the synthesis of GAP molecules or for the attachment of GAP molecules to the initial amino acids of the synthesized peptide, countless different coupling reagents, including but not limited to TFFH, TBTU, HBTU, COMU, or EDCI, can be used.

[0036] As an exemplary, non-limiting embodiment, and in any or a combination thereof of the examples described above, SolPPS can then be achieved in the C-to-N direction, for example, by using Fmoc / tBu chemistry. Deprotection of the Fmoc group from the primary and subsequent amino acids can be carried out with a multitude of deprotection reagents, including diethylamine, DBU (diazabicycloundecene), piperidine, tert-butylamine, and other alkylamines, and additional coupling reactions can be carried out to grow the peptide chain. After each coupling reaction, different quenching agents can be used to neutralize excess activated amino acids and give the resulting molecule the desired solubility properties. In a non-limiting example, decylamine can be used to quench the activated amino acids and increase the solubility of the resulting molecule in alkane solvents. The reaction mixture can then be purified by liquid-liquid extraction or selective precipitation, depending on the reaction solvent. The novel GAP molecules disclosed herein enable a novel SolPPS method that allows for controlled solubility and effective removal of amino acid and flubene impurities (i.e., NFMPs) by selective precipitation or extraction. In non-limiting examples, after the last amino acid of the desired peptide sequence is bonded, overall deprotection with a suitable TFA mixture can be performed to remove acid-unstable GAP molecules from the C-terminus and suitable side chains. Such deprotection can also remove other acid-unstable side-chain protecting groups, such as tBu-based side-chain protecting groups. This novel SolPPS method avoids additional reactions such as hydrogenation or hydrolysis to remove C-terminal protecting groups, allowing the native peptide sequence to be obtained in a single-step overall deprotection. Furthermore, using GAP molecules such as RAG allows for the formation of a C-terminal amide during TFA cleavage, successfully avoiding post-synthetic reactions typically required for such modifications. Moreover, these novel GAP molecules disclosed herein remain even more 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 N-terminus of any given peptide, as well as the amine side chains of certain amino acid residues, including but not limited to lysine. 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 at least one hour. Then, the amine is added to the reaction mixture at room temperature and pressure to obtain the HPG-protected carbamate product (see Figure 17).

[0038] In another non-limiting embodiment, the novel SolPPS method can begin with the synthesis of a novel GAP molecular protecting group, HOBnDpp, NH2PhDpp, or a derivative thereof, which can react directly with the carboxylic acid moiety of any given linker molecule, examples of which can be seen in Figure 18 and the list below. HMPA: 4-(hydroxymethyl)phenoxyacetyl HMPB: 4-(hydroxymethyl)phenoxybutanoyl HMB: 4-(hydroxymethyl)benzoyl MMB: 4-(mercaptomethyl)benzoyl MMPA: 4-(mercaptomethyl)phenoxyacetyl AMPA: 4-(aminomethyl)phenoxyacetyl DMPPA: 4-(3,3-dimethyl-3-hydroxypropyl)phenoxyacetyl Rink Amide: 2-(4-(amino(2,4-dimethoxyphenyl)methyl)phenoxy)acetyl Xanthenyl:4-((9-amino-9H-xanthene-3-yl)oxy)butanoyl TCA: 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]anulen-3-yl)pentanoyl 2-Chlorotrityl:3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl

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

[0040] A synthetic strategy for the pharmacologically interesting and biologically active peptide bivalirudin is presented as a non-limiting preferred embodiment of the novel SolPPS method disclosed herein. Figure 6 depicts a schematic diagram for attaching the novel GAP molecule HPG to the C-terminus of an unspecified number of amino acids, and according to this schematic, C-terminal protection of Fmoc-leucine-OH is performed to form the compound shown in Figure 10. In this particular embodiment, HPG is selected as the C-terminal protecting group to allow deprotection of the acid-unstable C-terminus after synthesis without promoting the formation of a C-terminal amide. Following this initial step, standard Fmoc / tBu chemistry is performed to attach Fmoc-Tyr(tBu)-OH to form an Fmoc-protected dipeptide. Then, according to the schematic diagram of amino acid side-chain protection shown in Figure 6, the side chain of Fmoc-Glu-OAllyl is protected with HPG before incorporation into the growing peptide to obtain the compound shown in Figure 11. Allyl deprotection of the C-terminus of Fmoc-Glu(HPG)-OAllyl is performed by treating it with Pd(PPh3)4 and triisopropyl or phenylsilane, activating the resulting molecule, and binding it to the peptide. Repeating this process once more to attach the next HPG-protected glutamate residue yields the Fmoc-protected tetrapeptide shown in Figure 12. Standard Fmoc / tBu peptide synthesis is performed, proline and isoleucine are added, the above procedure is followed, the next two glutamate residues are added, and then phenylalanine is added. The aspartate residue is protected with HPG and added in the same way as glutamate according to the schematic diagram shown in Figure 6, and then a glycine residue is added.

[0041] In another preferred embodiment, the novel GAP molecule RAG is reacted with Fmoc-Asp-OAllyl to produce the compound Fmoc-Asp(RAG)-OAllyl, shown in Figure 13. This compound is then deprotected by allylation, activated, and attached to a peptide chain to obtain the Fmoc-protected peptide shown in Figure 14. Subsequently, a standard Fmoc / tBu peptide synthesis is performed, adding four glycines, proline, arginine, another proline, and D-phenylalanine to obtain a fully protected bivalirudin peptide, as shown in Figure 15.

[0042] In this particular embodiment, after Fmoc deprotection of fully protected bivalirudin, a one-step global deprotection using a TFA mixture (i.e., 90% TFA, 5% H2O, 5% triisopropylsilane (TIPS)) can be performed to cleave all side-chain protecting groups (including the novel GAP molecule) as well as the protecting group at the HPG C-terminus (see Figure 16). During this global deprotection, the RAG-protected aspartic acid is also converted to deprotected asparagine. The molecule obtained from global deprotection using the TFA mixture is native bivalirudin.

[0043] General procedure for Fmoc deprotection and coupling: Novel SolPPS method performed in propylene carbonate (PC): Add a deprotecting agent and octanthiol to Fmoc-(AA)n-OBnDpp, Fmoc-(AA)n-HPG, Fmoc-(AA)n-RAG, or any amino acid with its C-terminus protected by a GAP protecting group, pre-dissolved in propylene carbonate (200 mM), and then stir in the alkane bilayer for 15 minutes at room temperature. The reaction mixture is then washed twice (×2) with fresh alkane solvent, then three times with saturated ammonium chloride aqueous solution, and dried. To the separated propylene carbonate solution, add 3.0 equivalents (eq) of TBTU or TFFH, 3.0 equivalents of Fmoc-AA-OH, and 3.0 equivalents of diisopropylethylamine (DIPEA), and stir the reaction for 7 minutes. Add this solution to the previously dried PC solution containing H-(AA)n-(GAP molecule) and bond while stirring for 10-60 minutes. Next, an excess of a quenching agent such as a long-chain (10-18 carbon atoms linked in a linear chain, 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 is added. The reaction mixture is then washed three times with saturated aqueous ammonium chloride solution and dried to obtain the extended peptide in the PC solution. This step is repeated as needed to produce peptides with the desired sequence and length. For a novel SolPPS method performed in dichloromethane or ethyl acetate: A deprotecting agent is added to Fmoc-(AA)n-OBnDpp, Fmoc-(AA)n-HPG, Fmoc-(AA)n-RAG, or an amino acid whose C-terminus is protected with any GAP protecting group, pre-dissolved in DCM or ethyl acetate (100 mM), followed by stirring at room temperature for 10 minutes. Next, the reaction mixture is washed three times with saturated ammonium chloride aqueous solution and three times with saturated sodium bicarbonate aqueous solution, and then dried. To the separated DCM or ethyl acetate solution, 2.0 equivalents of TBTU or TFFH, 2.0 equivalents of Fmoc-AA-OH, and 5.0 equivalents of DIPEA are added, and the reaction is stirred for 7 minutes. This solution is added to the previously dried DCM or ethyl acetate solution containing H-(AA)n-(GAP molecule), and the mixture is bonded while stirring for 10 to 60 minutes.Next, an excess of a quenching agent such as a long-chain (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 is added. The reaction mixture is then washed three times with saturated ammonium chloride aqueous solution and dried to obtain the extended peptide in DCM or ethyl acetate solution. This process is repeated as needed to produce peptides with the desired sequence and length.

[0044] General procedure for EDCI coupling: The reaction mixture containing the deprotected N-terminus of an amino acid or peptide is cooled in an ice bath. Subsequently, two equivalents of the amino acid to be bonded to such a free N-terminus are added to the reaction mixture, followed by the addition of two equivalents 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, long-chain (C10-C18) aliphatic alcohol, long-chain (C10-C18) aliphatic amine, long-chain (C10-C18) aliphatic selenol, aliphatic polyamine, or aliphatic polyalcohol is added. The reaction mixture is then washed three times with saturated aqueous ammonium chloride and three times with saturated aqueous sodium bicarbonate, and then dried.

[0045] In another embodiment, the disclosure may include a chemical composition comprising a group-assisted purification (GAP) component and a linker component. In one embodiment, the GAP component may be any molecule exhibiting the properties of the GAP molecule or anchor described herein, such as a GAP molecule capable of precipitation from a nonpolar organic solvent. For example, the GAP component may have sufficient polarity to be insoluble in nonpolar organic solvents such as petroleum ether, hexane, or heptane. In another example, the GAP component may include polar bonds sufficient to insoluble the GAP component in nonpolar organic solvents, such as a phosphate-oxygen bond (phosphine oxide), a sulfur-oxygen bond, a nitrogen-oxygen bond, or any other polar bond sufficient to insoluble the GAP component in nonpolar organic solvents. In another embodiment, the GAP component may include a moiety that contributes to π-π stacking, such as an aromatic moiety; for example, the GAP component may include one or more phenyl substituents. In another embodiment, the GAP component may be any molecule suitable for excluding itself from nonpolar organic solvents. In another embodiment, the GAP component may include a nucleophilic moiety capable of participating in coupling reactions. For example, the GAP component may include an alcohol, amine, thiol, or other nucleophilic moiety. In another embodiment, the GAP component can impart its solubility properties to the molecule to which it is attached. In another embodiment, the GAP component may include a hydrocarbon moiety, preferably containing at least six carbon atoms. In yet another embodiment, the GAP component may include an alkyl moiety, preferably containing 6 to 30 carbon atoms.

[0046] Preferably, the GAP component includes a polar covalent bond having a significant charge distribution. In one embodiment, the significant charge distribution may result from a mismatch in the size of the p orbitals of the bonding atom, and in another embodiment, the significant charge distribution may result from the hybridization of all of one p orbital of the bonding atom, resulting in reduced π overlap, which may cause charge separation. In another embodiment, the GAP component may further include one or more aromatic moieties to enable π-π stacking, thereby promoting crystallization during precipitation and reducing water solubility. In another embodiment, the GAP component may further include additional alkyl substituents that can further reduce aqueous solubility. In another embodiment, the GAP component may include a nucleophilic moiety that can participate in nucleophilic substitution reactions, such as coupling reactions, which are known in the art, particularly with respect to peptide synthesis. In one embodiment, the GAP component may include a free alcohol, an amine, or other nucleophilic moiety that can participate in nucleophilic substitution reactions. In another embodiment, the GAP component is preferably between 250 and 1250 Daltons.

[0047] For example, in one embodiment shown in Figure 20C, the GAP component [ka] teeth, [ka] It can include...

[0048] In the formula, A 1 These are S, O, NH, NMe, NET, NBn, NPh, -C(O)-, and -(CH2) j -Selected from the group consisting of (wherein "j" is defined below); A 2 These are S, O, NH, NMe, NET, NBn, NPh, -C(O)-, and -(CH2) k -Selected from the group consisting of (wherein "k" is defined below); A 3is selected from the group consisting of S, O, NH, NMe, NEt, NBn, and NPh; R 4 is -(CH2) m -H (where "m" is as defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R 5 is -(CH2) p -Me (where "p" is as defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R 6 = Dpp; Dpop; or Dap, where

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

Chemical formula

[0049] The components discussed herein can be attached to other components or molecules, as indicated by the dashed lines above.

[0050] In one embodiment, the linker component may include any of the linker molecules discussed herein. For example, the linker component may be substantially in any form of the linker molecules discussed herein, as depicted in Figure 18. In another embodiment, the linker component may include a molecule having a first and a second chemical moiety. In one embodiment, the first moiety may form a bond with a GAP component or a spacer component (discussed further below). For example, the first moiety may include an electrophile that can participate in nucleophilic substitution reactions such as coupling reactions. In another example, the first moiety may be a carbonyl moiety, and in yet another example, the first moiety may be a carboxylic acid moiety. In yet another embodiment, the second moiety may form a bond with an amino acid. For example, the second moiety may be a free alcohol, an amine, or another nucleophilic moiety. In yet another example, the second moiety may participate in coupling reactions. In yet another example, the second moiety may form a bond with the C-terminus of an amino acid. In one embodiment, the linker component may be a molecule capable of linking the GAP component to another molecule, and in another embodiment, the linker component may be a molecule capable of linking the spacer component to which the GAP component is attached to a target molecule. In another embodiment, the linker component may be configured such that, if a charge is generated on an atom adjacent to the nucleophilic moiety, that charge can be stabilized via resonance or conjugation. For example, with respect to the RAG molecule discussed herein, the linker component (Rink Amide in this example) can generate a charge on the benzyhydryl carbon upon cleavage of the carbon-nitrogen bond, leaving nitrogen attached to the target molecule. The linker component (and therefore the GAP anchor containing the linker component) may include conjugation that can stabilize the charge generated on the benzyhydryl carbon such that the cleavage reaction for removing the GAP anchor from the target molecule is energetically favorable. In another embodiment, the charge generated on the carbon adjacent to the nucleophilic moiety may be stabilized by the fact that the carbon atom is a trisubstituted (tertiary) carbon.In another embodiment, the linker component can be attached to a target molecule via a bond that is easily cleaved under hydrolysis conditions known in the art.

[0051] Preferably, the linker component can enable a composition containing the linker component and the GAP component to be removably bonded to another molecule via a second portion of the linker component. For example, the first portion of the linker component can participate in bonding with either the GAP component or the spacer component, and such bonding can be stable under a multitude of conditions, such as acid and base deprotection reactions known in the art (i.e., global deprotection using TFA, hydrolysis reactions, etc., as discussed herein). In this way, the composition is resistant to degradation and can maintain its chemical structure under many conditions. Preferably, the bond formed between the second portion of the linker component and another molecule may be unstable to reactions in which the bond of the first portion is stable. In this way, the composition can be cleaved from another molecule at the second portion of the linker component while maintaining the bond between the first portion and either the spacer portion or the GAP component. In one embodiment, this functionality (i.e., the orthogonality of the bond of the second portion to the bond of the first portion) may be made possible by the intrinsic structure of the linker component, such as those described in Figures 19 and 22A. For example, the linker component can be specifically designed to exhibit this functionality, such as by using linker molecules known in the art, examples of which can be found in Figures 19 and 22A. In another embodiment, the bond between the first portion and either the spacer component or the GAP component, and the bond between the second portion and another molecule, can be cleaved under the same conditions.

[0052] For example, the linker component can be exemplified by HMPA-OH, as shown in Figure 5. HMPA-OH comprises a first and a second chemical moiety. The first moiety of HMPA-OH is a carboxylic acid vulnerable to nucleophilic attack—thus, a nucleophilic moiety (e.g., the nucleophile of the GAP component, or the N-terminus of an amino acid) can form a bond with the first moiety of HMPA-OH via nucleophilic substitution. The second moiety of HMPA-OH is a benzyl alcohol, which itself is a nucleophilic moiety capable of attacking electrophiles, such as the carbonyl carbon at the C-terminus of an amino acid, to similarly form a bond via nucleophilic substitution. Furthermore, the structure of HMPA-OH allows the bond formed in the second moiety of the linker component to be cleaved under strongly acidic conditions. Preferably, the bond formed in the first moiety (such as HOBnDpp, or the bond with an amino acid, as depicted in Figure 5) remains stable under such conditions.

[0053] In one embodiment, a linker component can adhere to two other components (i.e., a GAP component, another linker component, a spacer component, or any combination thereof). [ka] As can be seen in Figure 20A, [ka] It can include...

[0054] In the formula, X is Y (defined below), -C(O)-, and -(CH2) a - (wherein "a" is defined below) is selected from the group consisting of; Y is selected from the group consisting of S, NH, NMe, NET, NBn, NPh, and O; R 1 is, -(CH2) b -H (wherein "b" is defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl are selected from the group; R 2 is, -(CH2)c -H (wherein "c" is defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl are selected from the group; a, b, c, and n are separate numerical values ​​selected from the group consisting of any integer between 0 and 30.

[0055] In one embodiment, a linker component can adhere to three other components (i.e., a GAP component, another linker component, a spacer component, or any combination thereof). [ka] As can be seen in Figure 20A, [ka] It can include...

[0056] In the formula, X is Y (defined below), -C(O)-, and -(CH2) a - (wherein "a" is defined below) is selected from the group consisting of; Y is selected from the group consisting of S, NH, NMe, NET, NBn, NPh, and O; R 1 is, -(CH2) b -H (wherein "b" is defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl are selected from the group; R 2 is, -(CH2) c -H (wherein "c" is defined below), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl are selected from the group; a, b, c, and n are separate numerical values ​​selected from the group consisting of any integer between 0 and 30.

[0057] In another embodiment, the disclosure may include a chemical composition comprising a GAP component, a linker component (linker), and a spacer component (spacer). In one embodiment, the spacer may be any molecule capable of facilitating the linkage between the GAP component and the linker component. For example, the spacer component may include a first portion that can be used to bind to the GAP component and a second portion that can be used to bind to the linker component. In one embodiment, the spacer may be an amino acid having a C-terminus and an N-terminus, the C-terminus of which can bind to a nucleophilic moiety of the GAP component and the N-terminus of which can bind to an electrophilic moiety of the linker. In another embodiment, the spacer may impart a desired solubility property to the entire chemical composition. For example, the spacer may include an alkyl chain, an amino acid, a polar organic moiety, or any other chemical moiety exhibiting a desired solubility property. In another embodiment, the spacer component may provide sufficient non-conjugated physical separation between the linker component and the GAP component, such as via a series of sp3 hybridized carbon atoms, so that the GAP component and the linker component are electronically separated from each other. Such separation can prevent one component from negatively influencing the other in a way that could interfere with the properties exhibited by each component disclosed herein. For example, a spacer component can prevent the chemical-electronic properties of the GAP component from interfering with the conjugation found in the linker component, such as a conjugation that allows the bond formed by the second part of the linker to be cleavable under known conditions. In another example, a spacer can be placed between a part of the GAP and a linker component that can participate in coupling or other bond-forming reactions with each other, thereby providing a distance between them that reduces steric hindrance that could make the reaction inefficient.

[0058] In one embodiment, a linker component can adhere to two other components (i.e., a GAP component, a linker component, another spacer component, or any combination thereof). [ka] As can be seen in Figure 20B, [ka] It can include...

[0059] In the formula, Z 1 These are S, O, NH, NMe, NET, NBn, NPh, -C(O)-, and -(CH2) d -Selected from the group consisting of (wherein "d" is defined below); Z 2 These are S, O, NH, NMe, NET, NBn, NPh, -C(O)-, and -(CH2) e -Selected from the group consisting of (wherein "e" is defined below); R 3 These include H, methyl, ethyl, propyl, isopropyl, benzyl, isobutyl, sec-butyl, tert-butyl, 2-(methylmercapto)ethyl, and the following structures [ka] It is selected from the group consisting of the following.

[0060] In the formula, d, e, f, g, h, and i are separate numerical values ​​selected from the group of integers between 0 and 30.

[0061] In one embodiment, the disclosure may include group-assisted purification (GAP) anchor molecules. For example, a GAP anchor (anchor molecule) (GAP molecule) (GAP protecting group) may be the product of the reaction shown in Figure 19. In another embodiment, the GAP anchor may be RAG, HPG, or NH2PhDpp, as described herein and shown in Figures 3, 5, and 7, respectively. In another embodiment, the GAP anchor may be the molecule defined in Figures 20A-20F, 21A-21B, and 22. In another embodiment, the GAP anchor may be a GAP component. In another embodiment, the GAP anchor may be a molecule comprising a GAP component and a linker component, and in another embodiment, the GAP anchor may include a GAP component, a linker component, and a spacer component. For example, a GAP anchor may incorporate any number of GAP components, linker components, or spacer components. Preferably, the GAP anchor (H-Anchor) may take the form shown in Figure 20A. H-Gap, H-Linker-Gap, [ka] , H-Linker-Spacer-Gap or [ka] This type of structure is collectively known as an H-Anchor molecule, where H is the element hydrogen, and Linker, Spacer, and Gap molecules are defined as follows:

[0062] Preferably, different combinations of GAP, linker, and spacer components can be used to achieve specific purposes, as illustrated in Figures 20A-20C. For example, the HPG molecule can be an example of a GAP anchor (H-Anchor) discussed herein. As seen in Figure 5, the linker component may be HMPA-OH, which can attach to a GAP component (e.g., OBnDpp) via a spacer component (e.g., phenylalanine) to form HPG, an exemplary GAP anchor. The linker component may include HMPA-OH having an electrophilic moiety (i.e., carbonyl) that can participate in nucleophilic substitution reactions and a nucleophilic moiety. When the nucleophile of the GAP component (i.e., HOBnDpp) attacks the carbonyl, a bond can be formed via nucleophilic substitution. Subsequently, the nucleophilic moiety of HMPA-OH (i.e., benzyl alcohol) can act as a nucleophile itself and attack an electrophilic moiety, such as the C-terminus of an amino acid. Alternatively, a GAP component bound to a spacer (e.g., phenylalanine) can bind to HMPA-OH to form a GAP anchor HPG.

[0063] For example, as shown in Figure 20D, a GAP anchor (H-Anchor) is, [ka] It can include...

[0064] In this embodiment, the H-Anchor (GAP anchor) is composed of the following specific components [ka] Includes the H-Linker-Spacer-GAP format.

[0065] In the formula, Y=O;R 1 =H;R 2 =H;X=Pararth O;Z 1 =-(CH2)-, where d=1;f=8;R 3 =H;g=0;Z 2=-C(O)-;A 1 =NH;A 2 = Para position O; and R 6 = Dpp.

[0066] In another embodiment, as shown in Figure 20E, the H-Anchor is [ka] It can include...

[0067] In this embodiment, the GAP anchor is the following GAP component [ka] It is an H-Gap format that has [a specific feature].

[0068] In the formula, A 1 =O;R 4 =H;R 5 =-(CH2) p -Me, and p=0;A 2 = Para-position -(CH2) k -and k=0; and R 6 = Dpp.

[0069] In another example, a GAP anchor is, [ka] It can include...

[0070] This H-Anchor molecule contains the following specific components: [ka] Includes the H-Linker-Spacer-Gap format.

[0071] In the formula, Y=O;R 1 =H;R 2 =H;X=para-order O;n=1;Z 1 =NH;f=0;R 3As defined above, h=2;g=0;Z 2 =-C(O)-;A 1 =O;R 4 =H;R 5 =-(CH2) p -Me, and p=0;A 2 = Para-position -(CH2) k -and k=0;R 6 =Dpp; and R 3 = [ka] That is the case.

[0072] In another embodiment, the present disclosure is as shown in Figures 21A and 21B. [ka] It may contain chemical compositions such as the following.

[0073] In the formula, Y is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; R 1 H, -(CH2) a -H (wherein "a" is selected from the group consisting of integers from 0 to 30), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R 2 is, -(CH2) b -H (wherein "b" is an integer from 1 to 30), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl are selected from the group; R 6 C y H (2y+1) (In the formula, "y" is an integer between 1 and 30), YC y H (2y+1) (wherein "y" is an integer from 1 to 30, and Y is as defined above), selected from the group consisting of (2-ethyl)hexyl and isooctyl; L is hydrogen or [ka] It is one of the following:

[0074] In the formula, Z is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; X is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; n is selected from the group consisting of any integer from 0 to 30; R 3 H, -(CH2) c -H (wherein "c" is selected from the group consisting of any integer from 0 to 30), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R 4 H, -(CH2) d -H (wherein "d" is selected from the group consisting of any integer from 0 to 30), -CCl3, -CF3, phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; [ka] R is an attachment point to other components; 5 teeth, [ka] It can be.

[0075] During the ceremony, [ka] is the attachment point to L; e is selected from the group consisting of integers from 0 to 30; and e, f, and g are selected from the group consisting of integers from 0 to 30.

[0076] In another embodiment, the disclosure is shown in Figure 22. [ka] This may include chemical compositions such as anchor molecules disclosed herein.

[0077] In one embodiment, the GAP, linker, and spacer components can be considered as three distinct chemical entities that can confer different useful properties to a given GAP anchor. For example, the GAP component may be a GAP molecule containing a polar bond (e.g., phosphine oxide) and an aromatic component, which ultimately gives the GAP anchor polar organic properties with the potential for π-π stacking, which can help the GAP anchor (and the attached target molecule) to precipitate selectively from nonpolar solvents. In another embodiment, this can help the GAP anchor exclude itself from aqueous and nonpolar solvents. In one embodiment, in the same GAP anchor, a linker component, as seen in Figures 19 and 22A, can confer the anchor the ability to selectively remove it from a desired substrate, as with the RAG and HPG anchors discussed herein, which can be removed, for example, from the C-terminus or N-terminus or side chain of an amino acid or peptide. For example, the linker component may be specifically designed to form a stable bond at one location and an unstable bond at another location. This orthogonality is ultimately reflected in the GAP anchor, and together the GAP and linker components thus impart to the GAP anchor polar organic properties suitable for group-assisted purification chemistry, and the GAP anchor can further cleave under conditions suitable for the linker component itself. In another embodiment, in the same GAP anchor, a spacer component can impart further desired properties to the GAP anchor. For example, the spacer may be highly organic or nonpolar, increasing the overall solubility of the GAP anchor in nonpolar solvents; in another example, the spacer may be highly polar, resulting in a more polar GAP anchor and therefore lower solubility in nonpolar organic solvents.

[0078] In accordance with the principles of this disclosure, GAP anchors can be specially formulated to assist in the synthesis of a given molecule having specific properties. For example, as further discussed herein, a desired peptide may be synthesized with the assistance of a GAP anchor. The GAP component of the anchor may have properties discussed herein that help to solubilize the anchor (and the attached peptide) in organic solvents, and the GAP component may also be suitable for π-π stacking to assist the peptide in selective precipitation, such as via dropwise precipitation. A spacer component may bind to the GAP component, and may be, for example, an amino acid, and solubility can be further controlled by providing multiple attachment sites to different aforementioned components and additional sites as needed to achieve the desired properties. A linker component may be used to bind to the spacer to form a stable product and may further have a free portion that can participate in reactions such as coupling reactions. Preferably, the bond formed by the free portion of the linker component may be unstable to TFA deprotection known in the art, such as as discussed herein.

[0079] Different GAP components can offer different advantages; for example, more polar or charged substituents can increase solubility in polar solvents and decrease solubility in nonpolar solvents; more polar bonds can increase solubility in polar organic solvents; more organic or fatty substituents can increase solubility in organic solvents while decreasing solubility in aqueous solvents; more π bonds and aromatic substituents are superior for precipitation by π-π stacking, and larger GAP components can exhibit a greater solubility control effect on larger target molecules. Similarly, different spacer components can offer different advantages. For example, smaller spacers can have a less overall impact on the solubility properties of the GAP anchor, while still providing assistance for the GAP component to adhere to the linker component and form a stable compound; more polar spacers can increase the solubility of the anchor in polar solvents; more organic or fatty spacers can increase solubility in organic solvents; longer spacers can reduce the possibility of steric hindrance at the binding site between a given GAP component and linker component; and smaller spacers, compared to larger spacers, can enable a higher overall product load of the target substrate and the GAP anchor by reducing the molecular weight of the GAP anchor. Similarly, different linker components can offer different advantages, such as different cleavage conditions from the target substrate (i.e., acid-catalyzed, base-catalyzed, etc.), or C-terminal modification during cleavage of the GAP anchor from a peptide (e.g., the RAG molecule discussed herein). Furthermore, different linkers can provide varying degrees of stability during peptide synthesis; for example, some linkers may be highly resistant to premature hydrolysis or diketopiperazine (DKP) formation, while others may be less resistant. Also, depending on the site of the linker, different linkers can enhance solubility in different solvents; more organic linkers enhance anchor solubility in organic solvents, while more polar linkers enhance solubility in polar or polar organic solvents. Those skilled in the art will understand that GAP anchors can incorporate the advantages of these different components to create anchors specifically tailored to a given chemical synthesis strategy.

[0080] In another embodiment, the disclosure may include a method for synthesizing a peptide, the method comprising attaching a first amino acid to an anchor molecule, the anchor molecule comprising a group-assisted purification (GAP) component and a linker component, the first amino acid being attached to the linker component of the anchor molecule, forming a peptide bond between a second amino acid and the first amino acid, and removing the anchor molecule from the first amino acid. The method may further include an anchor comprising a spacer component positioned between the GAP component and the linker component. In one embodiment, the spacer component may be an amino acid. In another embodiment, the disclosure may include a method for synthesizing a peptide using the GAP molecules disclosed herein, such as those shown in Figures 21A, 21B, and 22. In one embodiment, the peptide synthesis method may enable improved control of solubility for a target peptide by using an anchor molecule that influences the properties of each component. An anchor molecule, as discussed herein, can be attached to the C-terminus, N-terminus, or side chain of a given amino acid for incorporation into a peptide. Those skilled in the art will understand that by using such a method, peptides of a desired length containing a desired number of amino acids can be synthesized.

[0081] In another embodiment, the present disclosure relates to, for example, the method shown in Figure 23A. [ka] This could include methods for synthesizing GAP anchors, such as the following.

[0082] In another embodiment, a method for synthesizing H-Anchor molecules (anchor molecules) is, for example, as shown in Figure 23B, [ka] It can include...

[0083] In one embodiment, each component (i.e., GAP, spacer, and linker component) can be considered a reactant in a given synthetic strategy. For example, in Figure 23A, compound 3 can be considered a GAP component, i.e., a group-supported purification molecule. Compound 4 can be considered a spacer component that, when bound to the GAP component, can facilitate binding with a linker component such as compound 6. The final obtained GAP anchor, compound 7, can then be reduced at the free linker moiety to provide a benzyl alcohol that can participate in nucleophilic substitution reactions, such as coupling reactions known in the art with respect to peptide chemistry. In another example, as shown in Figure 23B, the GAP component can take the form of either ClPPh2 or compound 2. The spacer component can be an oleylamine, which, when bound to the GAP component, can form a GAP-spacer molecule such as compound 3. This can then be bound to a linker component such as compound 4 to finally obtain a GAP anchor such as compound 5.

[0084] Additional embodiments of the claimed invention include the following:

[0085] 1. A protecting group for solution-phase peptide synthesis, wherein the protecting group has a chemical formula selected from the group consisting of chemical formulas 1, 2, 3, 4, 5, 6, 7, and 8. In the formula, chemical formula 1 is [ka] Therefore, chemical formula 2 is [ka] Therefore, chemical formula 3 is [ka] Therefore, chemical formula 4 is [ka] Therefore, chemical formula 5 is [ka] Therefore, chemical formula 6 is [ka] Therefore, chemical formula 7 is [ka] Therefore, chemical formula 8 is [ka] And 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 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") Selected from the group consisting of "Amide", 4-((9-amino-9H-xanthene-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]anulen-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl"), Protecting group.

[0086] 2. The protecting group described in Section 1, wherein the protecting group is selected from the group consisting of chemical formulas 1, 4, 5, and 7, and R is the side chain of an amino acid.

[0087] 3. A protecting group as described in Section 1, wherein R is selected from the group consisting of -H, methyl, -CH2SH, -CH2CH2COOH, -CH2COOH, benzyl, 4-(1H-imidazolyl)methyl, -CH(CH3)(CH2CH3), -CH2CH2CH2NH2, -CH2CH2CH2NH2, isobutyl, -CH2CH2SCH3, -CH2CONH2, -CH2CH2CONH2, a propyl group in which 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 nitrogen, -CH2CH2CH2N=C(NH2)2, -CH2OH, 1-hydroxyethyl, isopropyl, 4-hydroxybenzyl, and 3-(1H-indolyl)methyl.

[0088] 4. A method for performing solution-phase peptide synthesis, wherein the method is The present invention provides a first protected amino acid by attaching a first protecting group to a first amino acid, wherein the first protecting group is the protecting group of claim 1. The coupling reaction involves performing a coupling reaction on a first protected amino acid or a peptide formed by bonding the first amino acid to another molecule, Methods that include...

[0089] 5. The method according to Section 4, wherein the first protecting group is attached to the C-terminus of the first amino acid.

[0090] 6. The method according to Section 4, wherein the first protecting group is attached to the side chain of the first amino acid.

[0091] 7. The method according to Section 4, wherein the first protecting group is attached to the N-terminus of the first amino acid.

[0092] 8. The said method The process involves binding the second protected amino acid to the first protected amino acid. Includes, The second protected amino acid comprises a second protecting group and a second amino acid, the second amino acid comprising a side chain, the second protecting group bonded to the side chain of the second amino acid, and the second protecting group having a chemical formula selected from the group consisting of chemical formulas 1, 2, 3, 4, 5, 6, 7, and 8. The method described in Section 4.

[0093] 9. The method according to Section 8, wherein the step of attaching the 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 the side chain of the first amino acid.

[0094] 10. A method for forming a protecting group for solution-phase peptide synthesis, wherein the method is Benzyl diphenylphosphine oxide (HOBnDpp), aniline diphenylphosphine oxide (NH2PhDpp), or derivatives thereof, are to be attached to the linker molecule (i) directly, (ii) via an amino acid, or (iii) via an amino acid derivative. Methods that include...

[0095] 11. The bonding process is [ka] This includes bonding to L-OH to form a protecting group, wherein the protecting group is of chemical formula 1 [ka] Defined by, 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. The method described in Section 10.

[0096] 12. The bonding process is [Chemical formula] involves binding to L-OH to form a protecting group, where the protecting group is represented by Chemical Formula 2 [Chemical formula] defined by, 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; The method described in Section 10.

[0097] 13. The binding step [Chemical formula] involves binding to L-OH to form a protecting group, where the protecting group is represented by Chemical Formula 3 [Chemical formula] defined by, 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; The method described in Section 10.

[0098] 14. The binding step [Chemical formula] involves binding to L-OH to form a protecting group, where the protecting group is represented by Chemical Formula 4 [Chemical formula] defined by, in the formula R is selected from the group consisting of the known side chains of protected amino acids and the 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. The method described in Section 10.

[0099] 15. The bonding process is [ka] This includes bonding to L-OH to form a protecting group, wherein the protecting group is of chemical formula 5 [ka] Defined by, 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. The method described in Section 10.

[0100] 16. The bonding process is [ka] This includes bonding to L-OH to form a protecting group, wherein the protecting group is of chemical formula 6 [ka] Defined by, 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, the method described in section 10.

[0101] 17. The coupling step is

Chem.

Chem.

[0102] 18. The coupling step is

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Claims

1. A GAP anchor compound comprising a GAP component, a spacer component, and a linker component, wherein the spacer component is positioned between the GAP component and the linker component, and the GAP anchor compound is Formula A 【Chemistry 1】 , formula B 【Chemistry 2】 , formula C 【Transformation 3】 , formula D 【Chemistry 4】 , formula F 【Transformation 5】 , formula G 【Transformation 6】 , formula H 【Transformation 7】 Equation I 【Transformation 8】 , and formula J 【Chemistry 9】 Selected from the group consisting of, in the formula, Y is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; R １ However, H, -(CH ２ ) ａ -H (wherein "a" is selected from the group consisting of integers from 0 to 30), -CCl ３ , -CF ３ Selected from the group consisting of phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R ２ is -(CH ２ ), -H (where "b" is an integer from 1 to 30), -CCl ｂ ), -CF ３ ), phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; selected from the group consisting of ３ ​ R ６ However, C ｙ H （２ｙ＋１） (In the formula, "y" is an integer between 1 and 30), YC ｙ H （２ｙ＋１） (wherein "y" is an integer from 1 to 30, and Y is as defined above), selected from the group consisting of (2-ethyl)hexyl and isooctyl; L, Formula 1 【Chemistry 10】 , Equation 2 【Chemistry 11】 , Equation 3 【Chemistry 12】 , Equation 4 【Chemistry 13】 , Equation 5 【Chemistry 14】 , Equation 6 【Chemistry 15】 , Equation 7 【Chemistry 16】 , Equation 8 【Chemistry 17】 , and formula 9 [Chemistry 18] Selected from the group consisting of, During the ceremony, Z is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; X is selected from the group consisting of S, O, NH, NMe, NET, and N-iPr; n is selected from the group consisting of integers from 0 to 30; R ３ However, H, -(CH ２ ) ｃ -H (wherein "c" is selected from the group of integers from 0 to 30), -CCl ３ , -CF ３ Selected from the group consisting of phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; R ４ However, H, -(CH ２ ) ｄ -H (wherein "d" is selected from the group of integers from 0 to 30), -CCl ３ , -CF ３ Selected from the group consisting of phenyl, isopropyl, tert-butyl, chlorophenyl, dichlorophenyl, methoxyphenyl, and dimethoxyphenyl; 【Chemistry 19】 However, it is an attachment point to any of equations A, B, C, D, F, G, H, I, or J. R ５ but, Equation i 【Chemistry 20】 , formula ii 【Chemistry 21】 , formula iii 【Chemistry 22】 , formula iv 【Chemistry 23】 , formula v 【Chemistry 24】 , formula vi 【Chemistry 25】 , formula vii 【Chemistry 26】 , formula viii 【Chemistry 27】 , formula ix 【Chemistry 28】 , expression x 【Chemistry 29】 , formula xi 【Transformation 30】 , formula xi 【Chemistry 31】 , formula xiii 【Chemistry 32】 , formula xiv 【Transformation 33】 , formula xv 【Transformation 34】 , and formula xvi 【Chemistry 35】 Selected from the group consisting of, in the formula, 【Transformation 36】 However, it is an attachment point to any of equations 3, 6, or 7; e is selected from the group consisting of integers from 0 to 30; f is selected from the group consisting of integers from 0 to 30; g is selected from the group consisting of integers from 0 to 30; i is selected from the group consisting of integers from 0 to 30, or L is 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") A GAP anchor compound selected from the group consisting of "Amide", 4-((9-amino-9H-xanthene-3-yl)oxy)butanoyl ("xanthenyl"), 5-(5-amino-10,11-dihydro-5H-dibenzo[a,d][7]annulene-3-yl)pentanoyl ("TCA"), and 3-(4-(chloro(2-chlorophenyl)(phenyl)methyl)phenyl)propanoyl ("2-chlorotrityl")).

2. Compound 1 (wherein n = 2 or 9) 【Chemistry 37】 , compound 2 【Transformation 38】 , compound 3 【Chemistry 39】 Compound 4 (wherein R = H, Me, Et, or iPr) 【Chemistry 40】 Compound 5 (wherein n = 0 or 2) 【Chemistry 41】 Compound 6 (wherein n = 0 or 2, and Y = O, NMe, NET, or N-iPr) 【Chemistry 42】 Compound 7 (wherein n = 0 or 2) 【Chemistry 43】 Compound 8 (wherein n = 0 or 2, and Y = O, NMe, NET, or N-iPr) 【Chemistry 44】 Compound 10 (wherein n = 0 or 2) 【Chemistry 45】 , and compound 11 (wherein n = 0 or 2) 【Chemistry 46】 A GAP anchor compound according to claim 1, selected from the group consisting of the following.

3. A method for synthesizing peptides, wherein the method is The GAP anchor compound according to claim 1 or 2 is attached to a first amino acid, Forming a peptide bond between the second amino acid and the first amino acid, The removal of the GAP anchor compound from the first amino acid, including, method.

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