A method for deprotecting a functional group protected with at least one D-amino acid and / or a D-amino acid derivative.

Abstract

The present invention relates to a method for deprotecting a functional group protected by at least one D-amino acid and / or D-amino acid derivative of at least one substrate having a functional group protected by at least one D-amino acid and / or D-amino acid derivative, a D-stereospecific hydrolase, and the use of this D-stereospecific hydrolase. Furthermore, the present invention relates to a method for producing a compound comprising this deprotection method, and the use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of a substrate.

Description

[Technical Field]

[0001] The present invention relates to a method for deprotecting a functional group protected by at least one D-amino acid and / or D-amino acid derivative of at least one substrate having a functional group protected by at least one D-amino acid and / or D-amino acid derivative, a D-stereospecific hydrolase, and the use of this D-stereospecific hydrolase. Furthermore, the present invention relates to a method for producing a compound comprising this deprotection method, and the use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of a substrate. In particular, the present invention enables the selective hydrolytic cleavage of D-amino acids and / or D-amino acid derivatives from monofunctional substrates, difunctional substrates, and polyfunctional substrates using a D-stereospecific hydrolase.

[0002] Furthermore, the present invention enables novel protective group strategies and selective cleavage of functional groups that mediate affinity or solubility in organic synthesis. [Background technology]

[0003] In particular, in the chemical synthesis of bifunctional or polyfunctional compounds, in order to ensure the selective reaction of two functional groups, it is usually necessary to temporarily protect any additional reactive groups present in the reactants.

[0004] On the other hand, unprotected functional groups with equivalent or similar reactivity can cause undesirable side reactions, inevitably leading to reduced yields and the formation of heterogeneous product mixtures, which generally necessitates costly purification steps after the reaction.

[0005] Established chemical processes for protecting, and especially deprotecting, such functional groups often require reaction conditions that do not reliably guarantee the integrity of reactants and / or products, making them unsuitable, particularly for (but not limited to) unstable target structures. Furthermore, especially in multi-step synthesis, the use of highly orthogonal protecting strategies is necessary. Otherwise, the accumulation of undesirable byproducts is expected in each individual reaction step, hindering high synthetic yields even with fewer reaction steps.

[0006] One widely used deprotection strategy involves using L-amino acids as enzymatically cleavable protective agents. However, because L-amino acids are widely used in organic synthesis, deprotection, i.e., cleavage of L-amino acids, is not very selective, which leads to the aforementioned problem.

[0007] E. Barbayianni et al. (Non-Patent Literature 1) describe hydrolases such as esterase BS2 from Bacillus subtilis and various lipases such as CAL-A from Candida antarctica that selectively hydrolyze ester bonds used as protective groups for carboxylic acids and hydroxyl groups. Similarly, F. Bellezza et al. (Non-Patent Literature 2) describe such a strategy using lipases such as CAL-A from Candida rugosa. Furthermore, H. Waldmann et al. (Non-Patent Literature 3) disclose that amino functionalities can be masked by phenylacetic acid, and the resulting phenylacetic acid amide can be selectively cleaved again by the enzyme penicillin G acylase.

[0008] Recent studies by M. Reille-Seroussi et al. (Non-Patent Document 4) have shown that these phenylacetyl functional groups can also be selectively removed from lysine side chains by selected sirtuins. Furthermore, A.G. Gum et al. (Non-Patent Document 5) have described glucose- and galactose-based urethane protecting groups that can be removed from selected functional groups by the use of glycosidases.

[0009] However, all of the above methods have certain disadvantages.

[0010] For example, the enzyme penicillin G acylase used for deprotection of amino functionality shows a very high K M value for the substrate used as a protecting group, so it shows only low specific activity against phenylacetamide. Furthermore, as investigated by W.B.L. Alkema et al. (Non-Patent Document 6), the cleaved product causes very significant inhibition of the biocatalyst, which is accompanied by not only a decrease in yield but also a delay in the conversion rate. For these reasons, this enzyme is not used in preparative synthetic approaches.

[0011] The use of glucose- and galactose-based urethane protecting groups and their cleavage by glycosidases are also not utilized on a preparative scale. Since glucose and galactose, the components constituting the sugar, contain reactive hydroxy groups, these must be fully protected during synthesis due to their chemical reactivity. However, these protected sugar derivatives are no longer recognized by glycosidases, so an additional reaction step, i.e., removal of the protective groups from the actual protecting groups, is required. This is further achieved by the use of lipases. As a result, a very complex process occurs, so these are not used at all in preparative synthesis.

[0012] Therefore, there is a need for a new, highly selective deprotection strategy for protected functional groups that requires mild reaction conditions and is compatible with all reactants and chemical structures present in the cautious synthetic methods used to produce chemical compounds. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] E. Barbayianni, I. Fotakopoulou, M. Schmidt, V. Constantinou-Kokotou, UT Bornscheuer, G. Kokotos G., "Enzymatic Removal of Carboxyl Protecting Groups: 2nd Cleavage of the Benzyl and Methyl Moieties," The Journal of Organic Chemistry, 2005, 70, 22, pp. 8730-8733, doi: 10.1021 / jo051004v [Non-Patent Document 2] F. Bellezza, A. Cipiciani, G. Cruciani, F. Fringuelli, "The importance of ester and alkoxy type functionalities for the chemo- and enantio-recognition of substrates by hydrolysis with Candida rugosa lipase," Journal of the Chemical Society, Perkin Transactions, 2000, 1, 24, pp. 4439-4444, doi: 10.1039 / B005512N [Non-Patent Document 3] H. Waldmann, A. Reidel, "The phenylacetyl group—the first enzymatically cleavable amino-protecting group in solution and on solid phase," Applied Chemistry, 1997, 109, 6, pp. 642-644, doi: 10.1002 / ange.19971090620 [Non-Patent Document 4] M. Reille-Seroussi, SV Mayer, W. Doerner, K. Lang, HD Mootz, "Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group," Chemical Communications, 2019, 55, pp. 4793-4796, doi: 10.1039 / C9CC00475K [Non-Patent Document 5] AG Gum, T. Kappes-Roth, H. Waldmann, "Labile Protecting Groups in Peptide Synthesis: Development of Glucose- and Galactose-Derived Urethanes," Chemistry - A European Journal, 2000, 20, pp. 3714-3721, doi: 10.1002 / 1521-3765(20001016)6:20<3714::AID-CHEM3714>3.0.CO;2-Z [Non-Patent Document 6] WBL Alkema, R. Floris, DB Janssen, "The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases," Analytical Biochemistry, 1999, 275, 1, pp. 47-53, doi: https: / / doi.org / 10.1006 / abio.1999.4300 [Overview of the project]

[0014] This invention utilizes the use of D-stereospecific hydrolases, for example as protecting groups or functional tags, to selectively recognize D-amino acids and / or their derivatives (instead of known L-amino acids) and hydrolytically cleave them from target structures. Since the reactivity of D-amino acids and / or D-amino acid derivatives under non-chiral synthetic conditions of classical peptide chemistry is in principle no different from the reactivity of their enantiomers, L-amino acids, they are compatible with already established synthetic strategies and can be readily introduced into target structures, similar to natural L-amino acids. The D-stereospecific hydrolases used in this invention are strictly regioselective, chemoselective, and enantioselective with respect to each recognized D-amino acid and / or D-amino derivative. Cleavage occurs rapidly and quantitatively without side reactions under mild reaction conditions and is orthogonal to all previously established deprotection and synthetic methods.

[0015] The present invention relates to the use of D-amino acids and / or D-amino acid derivatives for protecting chemically reactive functional groups such as amino functional groups, hydroxy functional groups, carboxylic acid functional groups, sulfhydryl functional groups, and selenohydryl functional groups, and for deprotecting them using D-stereospecific hydrolases. An important aspect of the present invention is the highly selective deprotection of functional groups (may be multiple) protected with D-amino acids and / or D-amino acid derivatives by substrate-specific D-stereospecific hydrolases for these D-amino acids and / or D-amino acid derivatives used as protecting groups. Since the deprotection reaction is enzymatically catalyzed and subsequently occurs under mild reaction conditions, this method can be fully applied even to chemically unstable substrates and target structures. Furthermore, the present invention is orthogonal to all existing protecting group strategies, for example in peptide and carbohydrate chemistry, and by extending the scope of existing methods, it is also possible to realize complex multi-step synthesis or sequential reaction sequences, for example, when required in prepared synthetic methods.

[0016] Furthermore, the present invention not only selectively deprotects reactive functional groups in the substrate or product, but also enables the cleavage of functional groups having solubility-mediated properties, or even any functional groups such as affinity probes linked to the target structure via D-amino acids and / or D-amino acid derivatives specifically recognized by hydrolase.

[0017] In a first aspect, the present invention relates to a method for deprotecting a functional group protected with at least one D-amino acid and / or a D-amino acid derivative of at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, using a D-stereospecific hydrolase, Prepare at least one substrate, a D-stereospecific hydrolase, and at least one solvent. A reaction mixture is obtained by mixing at least one substrate, a D-stereospecific hydrolase, and at least one solvent. In a reaction mixture, a D-stereospecific hydrolase is used to hydrolyze a functional group protected by at least one D-amino acid and / or D-amino acid derivative of at least one substrate, thereby obtaining a D-amino acid and / or D-amino acid derivative, and at least a partially deprotected substrate having at least one deprotected functional group deprotected from the D-amino acid and / or D-amino acid derivative. This includes methods.

[0018] In a second embodiment, the present invention has the following features: At least one first of three amino acids containing the amino acid sequence SXXK 10 - Helix-2 structure, At least one first α-helix structure containing the amino acid sequence YSN, and At least one first β-sheet structure containing the amino acid sequence HXG, (Here, X is an amino acid selected from the group consisting of proteinogenic amino acids) is characterized by the first 3 10 This invention relates to a D-stereospecific hydrolase in which a helix-2 structure, a first α-helix structure, and a first β-sheet structure are arranged in close proximity.

[0019] In a third aspect, the present invention relates to the use of a D-stereospecific hydrolase for deprotecting a functional group of a substrate protected with at least one D-amino acid and / or a D-amino acid derivative, preferably the D-stereospecific hydrolase having the following characteristics: At least one first of three amino acids containing the amino acid sequence SXXK 10 - Helix-2 structure, At least one first α-helix structure containing the amino acid sequence YSN, and At least one first β-sheet structure containing the amino acid sequence HXG, (Here, X is an amino acid selected from the group consisting of proteinogenic amino acids) is characterized by the first 3 10The -helix-2 structure, the first α-helix structure, and the first β-sheet structure are arranged in close proximity, and more preferably, the D-stereospecific hydrolase has an amino acid sequence selected from the following SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9, relating to use.

[0020] In a fourth embodiment, a method is provided for producing carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, the method being: The preparation of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivative precursors, or mixtures thereof, at least one D-amino acid and / or D-amino acid derivative, at least one first solvent, optionally at least one second solvent, optionally at least one third solvent, a D-stereospecific hydrolase, and at least one first reagent, A first reaction is obtained by mixing carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivative precursors, or mixtures thereof, at least one first solvent, and at least one D-amino acid and / or D-amino acid derivative. The method involves reacting a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative precursor, or mixture thereof with at least one D-amino acid and / or D-amino acid derivative to obtain a protected precursor, or mixture thereof, of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative. Optionally, remove at least one first solvent from the first reaction mixture, Optionally, a protected precursor of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof is reacted with one or more further reagents in one or more further reaction steps to obtain a reacted protected precursor of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof. A second reaction is obtained by mixing a carbohydrate, a carbohydrate derivative, a nucleotide, an oligonucleotide, a polynucleotide, a nucleotide derivative, an oligonucleotide derivative, a polynucleotide derivative, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a peptide intermediate, a peptide derivative, an oligopeptide derivative, a polypeptide derivative, or a mixture thereof, with at least one first reagent and optionally at least one second solvent. Protected precursors of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, or reacted protected precursors of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, with at least one first reagent to obtain protected carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, Optionally, remove at least one second solvent from the second reaction mixture, A third reaction is obtained by mixing a protected carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or a mixture thereof, optionally at least one third solvent, and a D-stereospecific hydrolase. The method involves deprotecting at least one D-amino acid and / or D-amino acid derivative from protected carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof to obtain carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof. Includes.

[0021] In a fifth aspect, the present invention relates to the use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of a substrate.

[0022] Further aspects and embodiments of the present invention are disclosed in the dependent claims and can be derived from the following description, drawings, and examples, but are not limited thereto.

[0023] The accompanying drawings illustrate embodiments of the present invention and should facilitate further understanding. For explanatory purposes, they serve to illustrate the concepts and principles of the present invention. Many other embodiments and advantages described can be derived in relation to the drawings. Elements in the drawings are not necessarily to scale. Identical, functionally equivalent, and operationally equivalent features and components are indicated by the same reference numeral in the drawings unless otherwise noted. [Brief explanation of the drawing]

[0024] [Figure 1] This figure shows the results obtained in this embodiment. [Figure 2] This figure shows the results obtained in this embodiment. [Figure 3] This figure shows the results obtained in this embodiment. [Figure 4] This figure shows the results obtained in this embodiment. [Figure 5] This figure shows the results obtained in this embodiment. [Figure 6] This figure shows the results obtained in this embodiment. [Figure 7] This figure shows the results obtained in this embodiment. [Figure 8] This figure shows the results obtained in this embodiment. [Figure 9] This figure shows the results obtained in this embodiment. [Figure 10] This figure shows the results obtained in this embodiment. [Figure 11a] This figure shows the results obtained in this embodiment. [Figure 11b] This figure shows the results obtained in this embodiment. [Figure 12] This figure shows the results obtained in this embodiment. [Figure 13] This figure shows the results obtained in this embodiment. [Figure 14] This figure shows the results obtained in this embodiment. [Figure 15a]This figure shows the results obtained in this embodiment. [Figure 15b] This figure shows the results obtained in this embodiment. [Figure 15c] This figure shows the results obtained in this embodiment. [Figure 16] This figure shows the results obtained in this embodiment. [Figure 17] This figure shows the results obtained in this embodiment. [Figure 18] This figure shows the results obtained in this embodiment. [Figure 19] This figure shows the results obtained in this embodiment. [Figure 20] This figure shows the results obtained in this embodiment. [Figure 21] This figure shows the results obtained in this embodiment. [Modes for carrying out the invention]

[0025] definition Unless otherwise defined, technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in the field of the present invention.

[0026] All scope disclosed herein should be considered supplemented by the term “about,” unless explicitly defined otherwise or the context makes otherwise clear. When used herein, modifiers such as “about,” “approximately,” and their combinations and variations are intended to include not only the exact quantity or value they modify, but also any slight deviations that may result from, for example, manufacturing tolerances, measurement errors, wear and abrasion, stresses on various parts, and combinations thereof.

[0027] As used herein, the term “substantially” means that the parameter, event, or situation described thereafter occurs entirely, or occurs to a considerable extent or degree. For example, “substantially” means that the parameter, event, or situation described thereafter occurs for at least 90% of the time, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99%, or that the dimension or measurement is within the range of at least 90%, or at least 91%, or at least 92%, or at least 93%, or at least 94%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% of the referenced dimension or measurement.

[0028] The use of the terms "at least one" or "one or more" will be understood to include not just one, but any quantity greater than one. Furthermore, the use of the phrase "at least one of X, Y, and Z" will be understood to include not only X alone, Y alone, and Z alone, but also any combination of X, Y, and Z.

[0029] The use of ordinal terms (i.e., "1st," "2nd," "3rd," "4th," etc.) is solely for the purpose of distinguishing between two or more items and, unless explicitly specified, should not be interpreted as signifying any order or sequence, the importance of one item to another, or any order of addition.

[0030] All numerical values ​​or percentages relating to the quantity of a substance in this application are expressed in weight percent unless otherwise clearly defined or the context makes otherwise.

[0031] In this application, an enzyme is a protein that functions as a biological catalyst by accelerating chemical reactions. The molecule on which an enzyme can act is called a substrate.

[0032] Carbohydrates, also known as sugars, are biochemical compounds having a structure containing carbon (C), hydrogen (H), and oxygen (O) atoms. Examples of sugars include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Disaccharides are sugars formed by the linkage of two monosaccharides via glycosidic bonds. Oligosaccharides are sugars formed by the linkage of a small number of monosaccharides, typically 3 to 10, via glycosidic bonds. Oligosaccharides can have a linear or branched structure. Polysaccharides are sugars containing a large number of at least 11 monosaccharides linked by glycosidic bonds. Polysaccharides can have a linear or branched structure.

[0033] A nucleotide comprises a nucleoside and at least one phosphate group. The nucleoside comprises a nucleic acid base and a monosaccharide. The nucleic acid base may be selected from the group consisting of, for example, adenine, cytosine, guanine, thymine, uracil, and their modifications. According to certain embodiments, the monosaccharide may be selected from the group consisting of ribose and deoxyribose. If there are two or more phosphate groups, they are usually arranged in a series and linked via POP bonds. The nucleotide is not particularly limited. According to certain embodiments, the nucleotide may be selected from the group consisting of adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and deoxythymidine monophosphate (dTMP).

[0034] Oligonucleotides are oligomers formed from a small number of nucleotides, typically 2 to 12. These nucleotides are linked by phosphodiester bonds, particularly 3',5'-phosphodiester bonds. Oligonucleotides usually have a linear structure. The nucleotides forming an oligonucleotide are not particularly limited and may be as described above.

[0035] A polynucleotide is an oligomer formed by a number of nucleotides, at least 13 in number. The nucleotides are linked by phosphodiester bonds, particularly 3',5'-phosphodiester bonds. Polynucleotides typically have a linear structure. The nucleotides are not particularly limited and may be as described above.

[0036] Amino acids are compounds that have both an amino group and a carboxylic acid group.

[0037] A peptide is a short chain of at least two amino acids linked by peptide bonds. An oligopeptide is a short chain of 2 to 10 amino acids linked by peptide bonds. A polypeptide is a long chain of 11 to 100 amino acids linked by peptide bonds. A protein is a long chain of at least 101 amino acids linked by peptide bonds.

[0038] The α-helix is ​​a common motif in the secondary structures of proteins, enzymes, and other proteins. It is a right-handed helix conformation in which all NH groups of the main chain are hydrogen-bonded to the C=O group of the amino acid located four residues prior in the protein sequence.

[0039] 3 10 - A helix is ​​a structure in which amino acids are arranged in a right-handed helix configuration. Each amino acid corresponds to a 120° rotation in the helix (i.e., a helix has 3 residues per rotation, and the NH group of an amino acid forms a hydrogen bond with the C=O group of the amino acid 3 residues prior).

[0040] A β-sheet is a common motif in the secondary structures of proteins, enzymes, and other proteins, and consists of beta strands (β-strands) that are connected laterally by hydrogen bonds of at least two or three main chains, generally forming a twisted, pleated sheet. A β-strand is a stretch of a polypeptide chain, typically 3 to 10 amino acids long, that has a main chain in an extended conformation.

[0041] In this disclosure, the unit M refers to mol / l unless otherwise specified.

[0042] Before describing the present invention in illustrative detail, it should be understood that the present invention is not limited to specific components of the method steps described herein, and that such methods may vary. It should also be understood that the terms used herein are intended solely to describe specific embodiments and are not intended to limit them. Where used herein and in the appended claims, singular nouns ("a", "an", and "the") include singular and / or plural references unless otherwise explicitly indicated by the context. For example, the term "a" used herein may be understood as a single entity or as "one or more" entities. Plural nouns also include singular and / or plural references unless otherwise explicitly indicated by the context. Furthermore, where parameter ranges are indicated by numerical delimiters, it should be understood that these ranges include these limits.

[0043] As used herein, “comprises,” “comprising,” “includes,” “including,” “has,” “having,” or any other variation thereof are intended to include non-exclusive inclusion. For example, a process, method, article, or apparatus that includes a list of elements is not necessarily limited to those elements, but may include other elements not expressly enumerated or specific to such process, method, article, or apparatus. Furthermore, unless expressly stated otherwise, “or” refers to an inclusive “or” rather than an exclusive “or.” For example, the condition A or B is satisfied by one of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0044] In a first aspect, the present invention relates to a method for deprotecting a functional group protected with at least one D-amino acid and / or a D-amino acid derivative of at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, using a D-stereospecific hydrolase, Prepare at least one substrate, a D-stereospecific hydrolase, and at least one solvent. A reaction mixture is obtained by mixing at least one substrate, a D-stereospecific hydrolase, and at least one solvent. In a reaction mixture, a D-stereospecific hydrolase is used to hydrolyze a functional group protected by at least one D-amino acid and / or D-amino acid derivative of at least one substrate, thereby obtaining a D-amino acid and / or D-amino acid derivative, and at least a partially deprotected substrate having at least one deprotected functional group deprotected from the D-amino acid and / or D-amino acid. This provides a method that includes [something].

[0045] In the method of the first embodiment, the steps of preparing different components, namely at least one substrate, a D-stereospecific hydrolase, and at least one solvent, are not particularly limited. For example, the components may be prepared with or without performing at least one further step, such as a synthesis step, before preparing the different components. Such further steps may be selected from the group consisting of, for example, chemical reactions in chemical synthesis, removal and / or isolation of at least one component, and / or addition of at least one component.

[0046] The process of mixing different components is not particularly limited. The order in which various components may be added is not particularly limited. For example, at least one substrate and at least one solvent may be added and mixed first, and then a D-stereospecific hydrolase may be added and mixed. However, this order can be changed as needed.

[0047] The mixing is not particularly limited, and any known mixing method can be used. The mixing can be carried out in a suitable reaction vessel, in which hydrolysis can also be performed. The reaction vessel is not particularly limited, and any known reaction vessel can be used as long as the hydrolysis reaction can be carried out.

[0048] Hydrolysis is any chemical reaction in which a water molecule breaks one or more chemical bonds. Hydrolysis can be a substitution, elimination, or solvation reaction in which water acts as a nucleophile. In the method of the first embodiment, hydrolysis is catalyzed by a D-stereospecific hydrolase.

[0049] Here, the D-stereohydrolase can catalyze the hydrolysis of the bond between a D-amino acid and / or a D-amino acid derivative and a functional group of at least one substrate protected by the D-amino acid and / or D-amino acid derivative. This introduces water molecules, which may be provided by, for example, at least one solvent. The water molecules react with at least one D-amino acid and / or D-amino acid derivative-protected functional group of the at least one substrate to obtain a D-amino acid and / or D-amino acid derivative and at least partially deprotected substrate having at least one deprotected functional group deprotected from the D-amino acid and / or D-amino acid derivative (or, if two or more substrates are provided, two or more at least partially deprotected substrates).

[0050] D-amino acids are not particularly limited, as long as they contain at least one carboxylic acid group, at least one amino group, and have a D-stereoconfiguration. According to a particular embodiment, D-amino acids include D-alanine (D-ala), D-arginine (D-arg), D-asparagine (D-asn), D-aspartic acid (D-asp), D-cysteine ​​(D-cys), D-glutamic acid (D-glu), D-glutamine (D-gln), D-histidine (D-his), D-isoleucine (D-ile), D-leucine (D-leu), D-lysine (D-lys), D-methionine (D-met), D-phenylalanine (D-phe), D-proline (D-pro), D-serine (D-ser), D-threonine (D-thr), D-tryptophan (D-trp), D-tyrosine (D-tyr), and D - Selected from the group consisting of valine (D-val), D-homocysteine ​​(D-hcy), D-homoserine (D-hse), D-norleucine (D-nle), D-norvaline (D-nva), D-ornithine (D-orn), D-penicillamine (D-pen), D-pyroglutamic acid (D-pglu), D-gammaglutamic acid (D-gglu), D-citrulline (D-cit), D-2-aminobutyric acid (D-aba), D-isoaspartic acid (D-isoasp), D-selenocysteine ​​(D-sec), D-selenomethionine, D-methionine sulfoxide, D-methionine sulfone, D-azidrolynthetic acid, and thioxoamino acids derived therefrom.

[0051] According to a particular embodiment, the D-amino acid is selected from the group consisting of D-alanine (D-ala), D-arginine (D-arg), D-aspartic acid (D-asp), D-glutamic acid (D-glu), D-glutamine (D-gln), D-histidine (D-his), D-leucine (D-leu), D-lysine (D-lys), D-methionine (D-met), D-phenylalanine (D-phe), D-proline (D-pro), D-serine (D-ser), D-tryptophan (D-trp), D-tyrosine (D-tyr), and D-valine (D-val). According to a particular embodiment, the D-amino acid is selected from the group consisting of D-alanine (D-ala), D-glutamine (D-gln), D-histidine (D-his), D-leucine (D-leu), D-methionine (D-met), D-phenylalanine (D-phe), D-serine (D-ser), D-tryptophan (D-trp), D-tyrosine (D-tyr), and D-valine (D-val). According to a particular embodiment, the D-amino acid is selected from the group consisting of D-leucine (D-leu), D-methionine (D-met), D-phenylalanine (D-phe), D-tryptophan (D-trp), and D-tyrosine (D-tyr).

[0052] By using one of these D-amino acids, D-stereospecific hydrolases can recognize D-amino acids more efficiently, and thus more efficiently deprotect the D-amino acid-protected functional groups of the substrate.

[0053] Examples of D-amino acid derivatives include a single D-amino acid having one or more functional groups, as illustrated below. D-amino acid derivatives are not particularly limited, but may include D-amino acids in salt form. D-amino acid derivatives may also include one or more post-translational modifications, artificial derivatizations, and combinations thereof. According to certain embodiments, one or more functional groups, post-translational modifications, and / or artificial derivatizations may be present at the N-terminus and / or C-terminus and / or side chains of this D-amino acid.

[0054] In D-amino acid derivatives, the D-amino acid is not particularly limited, as long as it contains at least one carboxylic acid group, at least one amino group, and has a D-stereoconfiguration. According to certain embodiments, the D-amino acid may be selected from the above-mentioned D-amino acids. According to certain embodiments, one or more functional groups, post-translational modifications, and / or artificial derivatizations may be attached to the D-amino acid via bonds selected from the group consisting of ester bonds and carboxamide bonds.

[0055] According to certain embodiments, one or more functional groups may be selected from the group consisting of protective functional groups (protective groups), physicochemical functional groups, analytical / preparative functional groups, e.g., markers, biological / therapeutic / medical functional groups, biochemical functional groups, and combinations thereof. According to certain embodiments, one or more functional groups may be selected from the group consisting of protective functional groups, physicochemical functional groups, and analytical / preparative functional groups. According to certain embodiments, one or more protective functional groups (blocking functional groups (blocking functional groups) Functional substances (also called functional substances) include allyl carbamate, 1,3-dioxane, 1,3-dithiane, 1,3-dithiolane, 1-chloroethyl carbamate, 1-(4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)ethyl, 2-(trimethylsilyl)-ethoxymethyl acetal, 2,2,2-trichloroethyl carbonate, 2-alkyl-1,3-oxazoline, 2-methoxyethoxymethyl ether, 2-naphthylmethyl ether, sulfonamides such as 4-methoxybenzenesulfonamide, 4-methoxybenzyl ether, 9-fluorenylmethyl carbamate, acetamide, acetate, acetate ester, acetonide, allyl ether Tel, benzaldehyde acetal, benzoate, benzoic acid ester, benzyl carbamate, benzyl ester, benzyl ether, benzylamine, benzylidene acetal, benzylideneamine, benzyloxycarbamate, benzyloxymethyl acetal, carbamate, carbonate, diethyl acetal, dimethyl acetal, di-tert-butyldioxasilinane, ethoxyethyl acetal, ethylene glycol acetal, formamide, methoxymethyl acetal, methoxymethyl aminal, methoxymethyl ether, methoxypropyl acetal, methyl carbamate, methyl ester, N-(4-methoxybenzyl)-indole, N,The compounds are derived from compounds selected from the group consisting of N-dimethylhydrazone, neopentyl glycol acetal, phthalimide, pivalate ester, propyl ester, p-toluenesulfonamide, succinic acid, tert-butyl ester, tert-butoxycarbamate, tert-butylcarbamate, tert-butyl ester, tert-butyl ether, tert-butyldimethylsilyl ether, tert-butyldiphenylsilyl ether, tetrahydropyranyl ether, triethylsilyl ether, trifluoroacetamide, triisopropyl ether, trimethylsilyl cyanohydrin, trimethylsilyl ether, triphenylmethylamine, nitrophenylcarbamate, and any alkylating agent that results in formylation, methylation, ethylation, propylation, etc. According to certain embodiments, one or more protecting functional groups are protecting groups selected from the group consisting of benzoyl (Bz), fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), acetyl (Ac), succinic (Suc), benzyloxycarbonyl (Z), 2-azidomethylbenzyl (Ambz), 2-aminobenzoic acid (Abz), biotin (Biotin), and mixtures thereof. By using D-amino acids having one or more protecting functional groups, the D-amino acids can be modified to prevent them from reacting with other components or compounds present in the reaction mixture. This makes it possible to use D-amino acids under a wide range of reaction conditions. Furthermore, this makes it possible to use chemically less stable D-amino acids, which can be modified with one or more of these protecting functional groups. Furthermore, it is possible to adapt the chemical properties of the substrate having a functional group protected by a D-amino acid derivative, such as chemical stability, solubility, and compatibility with other components in the reaction mixture, as well as the physical properties of the substrate, such as physical stability, boiling point, and melting point.

[0056] According to certain embodiments, one or more physicochemical functional groups may be selected from the group consisting of dyes, such as fluorescent inorganic dyes, fluorescent organic dyes, non-fluorescent inorganic dyes, or non-fluorescent organic dyes, and spin probes such as organic radicals. According to certain embodiments, one or more physicochemical functional groups may be selected from the group consisting of nitrophenols, aza dyes, Alexa Fluor® dyes, dyes produced by dinitrobenzylation (DNP), etc. By using D-amino acids having one or more physicochemical functional groups, the physicochemical properties of D-amino acids can be modified. This enables more appropriate characterization of substrates having functional groups protected by D-amino acid derivatives. In particular, optical characterization can be simplified.

[0057] According to certain embodiments, one or more analytical / preparative functional groups may include affinity fusions. According to certain embodiments, one or more analytical / preparative functional groups may be selected from the group consisting of biotin, desthiobiotin, Strepp tag II, His tag, c-Myc tag, etc. By using D-amino acids having one or more analytical / preparative functional groups, the analytical / preparative use of substrates having functional groups protected by D-amino acid derivatives can be simplified. In particular, the characterization of these substrates by various analytical methods such as spectroscopic or chromatographic methods can be simplified. Furthermore, the properties of D-amino acid derivative-protected functional substrates can be adapted to improve the purification of reaction mixtures containing these substrates, for example.

[0058] In certain embodiments, one or more biological / therapeutic / medical functional groups may include toxins that target intracellular pathways important for inducing cell death. In certain embodiments, one or more biological / therapeutic / medical functional groups may be selected from the group consisting of calicheamycin, monomethyl aurestatin (aurestatines), emtansine, doxorubicin, SN-38 (7-ethyl-10-hydroxycamptothecin), etc. By using D-amino acids having one or more biological / therapeutic / medical functional groups, the biological / therapeutic / medical properties of substrates having functional groups protected by D-amino acid derivatives can be improved.

[0059] According to certain embodiments, one or more biochemical functional groups may be selected from the group consisting of propeptides, signal peptides, etc. Propeptides are inactive proteins (or peptides) that can be converted into an active form by post-translational modification, such as cleavage of part of the molecule or addition of another molecule. Signal peptides are short peptides (usually 16 to 30 amino acids long) that are located at the N-terminus (or sometimes atypically the C-terminus or inside) of most newly synthesized proteins destined for secretory pathways. By using D-amino acids having one or more biological functional groups, the biochemical properties of substrates having functional groups protected by D-amino acid derivatives can be improved. In particular, by using signal peptides as one or more functional groups in the intracellular pathway of a substrate, it is possible to affect proteins having functional groups protected by D-amino acid derivatives in particular. Furthermore, by using propeptides as one or more functional groups in a substrate, particularly a precursor of a protein having functional groups protected by D-amino acid derivatives, the substrate can be made inactive and then activated by enzymatic activation at a predetermined time.

[0060] One or more post-translational modifications are not limited, for example, (2-aminosuccinimyl)acidetic acid (Asp-Gly), (2S)-4-hydroxyleucine, (3R)-3-hydroxyarginine, (3R)-3-hydroxyasparagine, (3R)-3-hydroxyaspartate, (3R)-N4-methyl-3-hydroxy-D-asparagine, (3R,4R)-3,4-dihydroxyproline, (3R,4R)-4,5-dihydroxyisoleucine, (3R,4S)-3,4-dihydroxyproline, (3R,4S)-4-hydroxyisoleucine, (3S)-3-hydroxyaspara Gin, (3S)-3-hydroxyaspartate, (3S)-3-hydroxyhistine, (3S)-3-hydroxylysine, (3S)-3-methylglutamine, (3S,4R)-3,4-dihydroxyisoleucine, (4R)-4,5-dihydroxyleucine, (4R)-5-hydroxyleucine, (4R)-5-oxoleucine, (4S)-4,5-dihydroxyleucine, (4S)-thiazoline-4-carboxylic acid (Thr-Cys), (5R)-5-hydroxylysine, (5S)-5-hydroxylysine (E)-2,3-dehydrobutyrine, (E)-2,3-dehydrotyrosine, (Z)-2,3-dehydroaspartate, (Z)-2,3-dehydrobutyrine, (Z)-2,3-dehydrotyrosine, 1-(tryptophan-3-yl)-tryptophan (Trp-Trp)(interchain with W-...), 1-amino-2-propanone, 1-histidyl-3-tyrosine (His-Tyr), 1-thioglycine, 2-(3-methylbutanoyl)-5-hydroxyxazole-4-carbothothic acid (Leu-Cys) , 2-(4-guanibutanoyl)-5-hydroxyimazole (hydroxyimazole)-4-carbotionic acid (Arg-Cys), 2-(cysteine-S-ylcarbonyl)-3-methyl-4-(glutam-5-yloxy)methylindole (Glu-Cys), 2-(S-cysteinyl)-histidine (Cys-His), 2-(S-cysteinyl)-methionine (Cys-Met), 2-(S-cysteinyl)pyruvic acid O-phosphothioketal, 2,3-dehydroalanine (Cys),2,3-dehydroalanine (Ser), 2,3-dehydroalanine (Tyr), 2,3-dehydrobutyrine, 2,3-dehydrotyrosine, 2,4,5-topaquinone, 2-cysteinyl-6-hydroxytryptophan sulfoxide (Trp-Cys), 2-cysteinyl-D-allothreonine (Cys-Thr), 2-cysteinyl-D-phenylalanine (Cys-Phe), 2-cysteinyl-L-phenylalanine (Cys-Phe), 2-hydroxyproline, 2-iminomethyl-5-imazolinone (Gln- Gly), 2-iminomethyl-5-imazolinone (Glu-Gly), 2-iminomethyl-5-imazolinone (Met-Gly), 2-methylglutamine, 2-methylsulfonyltryptophan, 2-oxo-5,5-dimethylhexanoate, 2-oxobutanoic acid, 2-tetrahydro-2-pyryl-5-imazolinone (Lys-Gly), 3-(O4-tyrosyl)-valine (Val-Tyr), 3-(S-cysteinyl)-tyrosine (Cys-Tyr), 3-(S-cysteinyl)-tyrosine (Cys-Tyr), 3-(S-cysteinyl)-tyrosine (Tyr-Cys), 3,3-dimethylmethionine, 3,4,5-trihydroxyphenylalanine, 3,4-dihydroxyarginine, 3,4-dihydroxyphenylalanine, 3,4-dihydroxyproline, 3-bromotyrosine, 3-cysteinyl-aspartic acid (Cys-Asp), 3-farnesyl-2,N2-cyclotryptophan, 3-geranyl-2,N2-cyclotryptophan, 3'-histidyl-3-tyrosine (His-Tyr), 3-hydroxyasparagine, 3-hydroxyaspartate, 3-hydroxy-D-variol 3-hydroxyphenylalanine, 3-hydroxyproline, 3-hydroxypyrine (pyrine)-2,5-dicarboxylic acid (Ser-Cys) (containing S...), 3-hydroxypyrine (pyrine)-2,5-dicarboxylic acid (Ser-Ser) (containing C...), 3-hydroxytryptophan, 3-hydroxyvaline (Thr), 3-hydroxyvaline (Val), 3-methyl-D-valine, 3-methylisoleucine, 3-methylthioaspartic acid, 3-methylvaline, 3-nitrotyrosine, 3-oxoalanine (Cys),3-Oxoalanine (Ser), 3-phenyllacidtic acid, 3-prenyl-2,N2-cyclotryptophan, 4-(1-hydroxyethyl)-7-isoleucino-2-(threonine-O3-ylcarbonyl)-7,8-dihydroquinoline-8-ol (Ile-Thr), 4,5,5-trihydroxyleucine, 4,5-dihydroxylysine, 4-aspartyl phosphate, 4-carboxyglutamate, 4-cysteinylglutamic acid (Cys-Glu), 4-cysteinyltryptophylquinone (Cys-Trp), 4-hydroxyarginine, 4-hydroxyglutamic acid Glutamyl glutamate, 4-hydroxylysine, 4-hydroxyproline, 5-(methoxymethyl)thiazole-4-carboxylic acid (Val-Cys), 5-aminopiperein-2,5-dicarboxylic acid (Ser-Cys) (with S-...), 5-aminopiperein-2,5-dicarboxylic acid (Ser-Ser) (with C-...), 5-chlorotryptophan, 5-glutamyl 2-aminoadipic acid, 5-glutamyl dopamine, 5-glutamyl glutamate, 5-glutamylglycerylphosphorylethanolamine, 5-glutamyl Lysine, 5-glutamylhistamine, 5-glutamylN2-lysine, 5-glutamylN2-ornithine, 5-glutamylnorepinephrine, 5-glutamylpolyglutamate, 5-glutamylpolyglycine, 5-glutamylserotonin, 5-hydroxy-3-methylproline (Ile), 5-hydroxylysine, 5-imazolinone (Ala-Gly), 5-imazolinone (Asn-Gly), 5-imazolinone (Cys-Gly), 5-imazolinone (Lys-Gly), 5-imazolinone (Ser-Gly), 5-methylal Ginin, 5-methyloxazole-4-carboxylic acid (Cys-Thr), 5-methyloxazole-4-carboxylic acid (Ser-Thr), 5-methyloxazole-4-carboxylic acid (Thr-Thr), 5-methyloxazoline-4-carboxylic acid (Ser-Thr), 5-methylthiazole-4-carboxylic acid (Asn-Cys), 5-tyrosyl-5-aminotyrosine (Tyr-Tyr) (interchain with Y-...), 6-(S-cysteinyl)-8α-(pros-hystyl)-FAD(His-Cys),6-Bromotryptophan, 6-Chlorotryptophan, 7-Hydroxytryptophan, ADP-α-D-Ribosylarginine, ADP-Ribosylaspartic acid, ADP-Ribosylglutamic acid, ADP-Ribosylarginine, ADP-Ribosylasparagine, ADP-Ribosylcysteine, ADP-Ribosyldiphthalamide, ADP-Ribosylglycine, ADP-Ribosylhistine, ADP-Ribosylserine, ADP-Ribosyltyrosine, ADP-Riboxanized arginine, Alaninamide, Alanine derivatives, A Ranin isoaspartylcyclopeptide (Ala-Asn), allicin, aminomalonic acid (Ser), arginine amide, arginine derivative, asparagine amide, aspartate 1-(chondroitin 4-sulfate) ester, aspartic acid 1-[(3-aminopropyl)(5-adenosyl)phosphono]amide, aspartic acid 1-amide, aspartyl aldehyde, asymmetric dimethylarginine, β-decarboxylated aspartate, β-methyllanthionine (Cys-Thr), β-methyllanthionine (Thr-Cys), β-methyllanthionine Sulfoxe (Thr-Cys), blocked amino-terminated (Ala), blocked amino-terminated (Arg), blocked amino-terminated (Asn), blocked amino-terminated (Asp), blocked amino-terminated (Asx), blocked amino-terminated (Cys), blocked amino-terminated (Gln), blocked amino-terminated (Glu), blocked amino-terminated (Gly), blocked amino-terminated (Ile), blocked amino-terminated (Leu), blocked amino-terminated (Met), blocked amino-terminated (Pro), blocked amino-terminated (Ser) ), blocked amino-terminus (Thr), blocked amino-terminus (Val), blocked amino-terminus (Xaa), blocked carboxyl-terminus (Arg), blocked carboxyl-terminus (His), bromohistine, cholesterol glycine ester, Cis-14-hydroxy-10,13-dioxo-7-heptadecenoate aspartate, citrulline, C-linked (Man)hydroxytryptophan, C-linked (Man)tryptophan, cyclo[(prolylserine)-O-yl]cysteinate, cyclopeptide (Ala-Arg),Cyclopeptide (Ala-Ile), Cyclopeptide (Ala-Pro), Cyclopeptide (Arg-Cys) (interchain with C-...), Cyclopeptide (Asn-Gly), Cyclopeptide (Asp-Asn), Cyclopeptide (Cys-Arg) (interchain with R-...), Cyclopeptide (Cys-Ile), Cyclopeptide (Cys-Pro), Cyclopeptide (Glu-Asn), Cyclopeptide (Gly-Arg), Cyclopeptide (Gly-Asn), Cyclopeptide (Gly-Asp), Cyclopeptide (Gly-Pro), Cycl Cyclopeptide (His-Asn), Cyclopeptide (His-Asp), Cyclopeptide (His-Pro), Cyclopeptide (Ile-Lys), Cyclopeptide (Ile-Pro), Cyclopeptide (Leu-Leu), Cyclopeptide (Leu-Pro), Cyclopeptide (Leu-Trp), Cyclopeptide (Lys-Asp), Cyclopeptide (Met-Pro), Cyclopeptide (Phe-Pro), Cyclopeptide (Pro-Met), Cyclopeptide (Pro-Tyr), Cyclopeptide (Ser-Asn), Cyclopeptide (S er-Gly), cyclopeptide (Ser-Lys), cyclopeptide (Ser-Pro), cyclopeptide (Trp-Pro), cyclopeptide (Tyr-Pro), cyclopeptide (Val-Pro), CysO-cysteine ​​adduct, cysteinamide, cysteine ​​derivative, cysteine ​​methyl disulfe, cysteine ​​methyl ester, cysteine ​​persulfe, cysteinesulfenic acid (-SOH), cysteinesulfinic acid (-SO2H), cysteinesulfonic acid (-SO3H), cysteinyl- Selenocysteine ​​(Cys-Sec), cysteinyl-selenocysteine ​​(Sec-Cys), D-4-hydroxyvaline, D-alanine (Ala), D-alanine (Ser), D-alloisoleucine, D-asparagine, deaminated asparagine, deaminated glutamine, decarboxylated threonine, deoxyhypsin, diiodotyrosine, dimethylated arginine, diphosphoserine, diphosphothreonine, diphthamide, dityrosine (Tyr-Tyr) (interchain with Y-...), D-lacidtate, D-leucine, D-methionine, D-phenylalanine, D-serine (Cys), D-serine (Ser), D-threonine, D-tryptophan, D-valine, FMN phosphorylserine, FMN phosphorylthreonine, glutamate methyl ester (Gln), glutamate methyl ester (Glu), glutamic acid 1-amide, glutamine amide, glutamine derivative, glycinamide, glycine radical, glycyladenylate, glycylcysteine ​​dithioester (Cys-Gly) (interchain with G-...), glycylcysteine ​​di Thioester (Gly-Cys) (interchain with C-...), Glycylcysteine ​​thioester (Cys-Gly) (interchain with G-...), Glycylcysteine ​​thioester (Gly-Cys) (interchain with C-...), Glycyllysine isopeptide (Gly-Lys) (interchain with K-...), Glycyllysine isopeptide (Lys-Gly) (interchain with G-...), Glycylserine ester (Gly-Ser) (interchain with S-...), Glycylserine ester (Ser-Gly) (interchain with G-...), Glycylthreonine ester STER(Gly-Thr)(interchain with T-...), glycylthreonine ester(Thr-Gly)(interchain with G-...), GPI-anchored (amated) alanine, GPI-anchored asparagine, GPI-anchored aspartic acid, GPI-anchored carboxyl terminus, GPI-anchored cysteine, GPI-anchored glycine, GPI-anchored serine, GPI-anchored threonine, GPI-like anchor-bound alanine, GPI-like anchor-bound asparagine, GPI-like anchor-bound aspartate, GPI GPI-like anchor-linked glycine, GPI-like anchor-linked serine, histinamide, hydroxyarginine, hydroxyproline, hypsin, iodotyrosine, isoaspartylcysteine ​​isopeptide (Cys-Asn), isoaspartylglycine isopeptide (Asn-Gly), isoaspartylglycine isopeptide (Asp-Gly), isoaspartylglycine isopeptide (Gly-Asn), isoaspartylglycine isopeptide (Gly-Asp), isoaspartyllysine isopeptide (Asn-Lys) (K-...Interchain (with N-...), isoaspartyryllysine isopeptide (Lys-Asn), isoaspartyryllysine isopeptide (Lys-Asn) (interchain with N-...), isoaspartyryllysine isopeptide (Lys-Asp), isodityrosine (Tyr-Tyr), isoglutamylcysteine ​​thioester (Cys-Gln), isoglutamylglycine isopeptide (Gly-Glu), isoglutamyllysine isopeptide (Gln-Lys) (interchain with K-...), isoglutamyllysine isopeptide (Glu-Lys) (interchain with K-...), isoglutamyllysine isopeptide (Lys-Gln), isoglutamyllysine isopeptide (Lys-Gln) (interchain with Q-...), isoglutamyllysine isopeptide (Lys-Glu) (E-...(Interchain), isoleucinamide, isoleucine derivatives, L-alloisoleucine, lanthionine (Cys-Ser), lanthionine (Ser-Cys), leucinamide, leucine methyl ester, lysineamide, lysine derivatives, lysine methyl ester, lysine tyrosylquinone (Lys-Tyr), lysine tyrosylquinone (Tyr-Lys), lysinoalanine (Ser-Lys), lysino-D-alanine (Lys), methionine (R)- Sulfoxe, methionine (S)-sulfoxe, methionine amide, methionine derivative, methionine sulfone, methionine sulfoxe, methylhistine, murein peptoglycan-bound serine, N-(12-oxomyristoyl)cysteine, N,N-(cysteine-1,S-diyl)phenylalanine (Cys-Phe), N,N-(cysteine-1,S-diyl)serine (Cys -Ser), N,N,N-trimethylalanine, N,N,N-trimethylglycine, N,N,N-trimethylmethionine, N,N,N-trimethylserine, N,N-dimethylalanine, N,N-dimethylglycine, N,N-dimethylleucine, N,N-dimethylproline, N,N-dimethylserine, N-[(12R)-12-hydroxymyristoyl]cysteine, N2,N2-dimethylarginine, N2-acidetyl ) Arginine, N2-succinyltryptophan, N4,N4-dimethylasparagine, N4-methylasparagine, N4-methyl-D-asparagine, N5-[4-(SL-cysteinyl)-5-methyl-1H-imazol-2-yl]-L-ornithine (Arg-Cys)(interchain with C-...), N5-[4-(SL-cysteinyl)-5-methyl-1H-imazol-2-yl]-L-ornithine (Cys-Arg)(R-...(between the chains), N5-methylarginine, N5-methylglutamine, N6-(2-hydroxyisobutyryl)lysine, N6-(3,6-diaminohexanoyl)-5-hydroxylysine, N6-(ADP-ribosyl)lysine, N6-(β-hydroxybutyryl)lysine, N6-(pyroxal phosphate)lysine, N6-(retinylene)lysine, N6,N6,N6-trimethyl-5-hydroxylysine, N6,N6,N6-trimethyllysine, N6,N6-dimethyllysine, N6-1-carboxyethyllysine, N6-acidetyl Lysine, N6-Biotinyllysine, N6-Butyryllysine, N6-Carbamoyllysine, N6-Carboxylysine, N6-Clotonyllysine, N6-Formyllysine, N6-Glutaryllysine, N6-Lacidotoyllysine, N6-Lipoyllysine, N6-Malonyllysine, N6-Methylatedlysine, N6-Methyllysine, N6-Mureinpeptoglycanlysine, N6-Myristoyllysine, N6-Palmitoyllysine, N6-Poly(β-Hydroxybutyryl)lysine, N6-Poly(methylaminopropyl)lysine, N6-Propyl Onyllysine, N6-stearoyllysine, N6-succinyllysine, N-acidetyl (acidetyl)alanine, N-acidetyl (acidetyl)aspartate, N-acidetyl (acidetyl)cysteine, N-acidetyl (acidetyl)glutamate, N-acidetyl (acidetyl)glycine, N-acidetyl (acidetyl)isoleucine, N-acidetyl (acidetyl)methionine, N-acidetyl (acidetyl)proline, N-acidetyl (acidetyl)serine, N-acidetyl (ac Threonine (idetyl), N-acidetyl tyrosine, N-acidetyl valine, N-α-linked (Rha) arginine, N-β-linked (GlcNacid) arginine, N-carbamoylalanine, ND-glucuronoylglycine, N-formylglycine, N-formylmethionine, nitrated tyrosine, N-linked (DATDGlc) asparagine, N-linked (GalNacid) asparagine, N-linked (GalNacid...) (glycosaminoglycan) asparagine, N-linked (GalNacid...)) Asparagine, N-linked (Glc)(glycated) arginine, N-linked (Glc)(glycated) histine, N-linked (Glc)(glycated) isoleucine, N-linked (Glc)(glycated) lysine, N-linked (Glc)(glycated) valine, N-linked (Glc) arginine, N-linked (Glc) asparagine, N-linked (Glc...) arginine, N-linked (Glc...) asparagine, N-linked (GlcNacid) asparagine, N-linked (GlcNacid...)( (Complex) Arginine, N-linked (GlcNacid...) (Complex) Asparagine, N-linked (GlcNacid...) (High Mannose) Arginine, N-linked (GlcNacid...) (High Mannose) Asparagine, N-linked (GlcNacid...) (hybr) Arginine, N-linked (GlcNacid...) (hybr) Asparagine, N-linked (GlcNacid...) (Keratan Sulfate) Arginine, N-linked (GlcNa cid...) (keratin sulfate) asparagine, N-linked (GlcNacid...) (pausimannose) arginine, N-linked (GlcNacid...) (pausimannose) asparagine, N-linked (GlcNacid...) (polylacidtosaminoglycan) arginine, N-linked (GlcNacid...) (polylacidtosaminoglycan) asparagine, N-linked (GlcNac id...) Arginine, N-linked (GlcNacid...) Asparagine, N-linked (Hex) Arginine, N-linked (Hex) Asparagine, N-linked (Hex) Tryptophan, N-linked (Hex...) Arginine, N-linked (Hex...) Asparagine, N-linked (HexNacid) Arginine, N-linked (HexNacid) Asparagine, N-linked (HexNacid...) Arginine, N-linked (HexNacid...)) Asparagine, N-linked (Lacid)(glycated) lysine, N-linked (Man) tryptophan, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylleucine, N-methylmethionine, N-methylphenylalanine, N-methylproline, N-methylserine, N-methyltyrosine, N-myristoylglycine, N-palmitoylcysteine, N-palmitoylglycine, N-pirubate-2-iminylcysteine, N-pirubate-2-iminylvaline, O-(2-aminoethylphosphoryl)serine, O-(2-cholinephosphoryl O-(5'-phospho-DNA)-serine, O-(5'-phospho-DNA)-tyrosine, O-(5'-phospho-RNA)-serine, O-(5'-phospho-RNA)-tyrosine, O-(pantetheine 4'-phosphoryl)serine, O-(phosphoribosyl dephospho-coenzyme A)serine, O-(sn-1-glycerophosphoryl)serine, O3-poly(β-hydroxybutyryl)serine, O-8α-FAD-tyrosine, O-acidetyl serine, O-acidetyl threonine, O-α-linked (GlcNaci d) Threonine, O-AMP-serine, O-AMP-threonine, O-AMP-tyrosine, O-decanoylserine, O-decanoylthreonine, O-di-AMP-tyrosine, O-hexanoylserine, O-linked (Ara)hydroxyproline, O-linked (Ara...)hydroxyproline, O-linked (DADDGlc)serine, O-linked (DATDGlc)serine, O-linked (Fuc)serine, O-linked (Fuc)threonine, O-linked (Fuc...)serine, O-linked (Fuc...)threonine, O-linked (FucNacid) Serine, O-linked (FucNacid...) Serine, O-linked (Gal) Hydroxylysine, O-linked (Gal) Hydroxyproline, O-linked (Gal) Serine, O-linked (Gal) Threonine, O-linked (Gal...) Hydroxylysine, O-linked (Gal...) Hydroxyproline, O-linked (Gal...) Serine, O-linked (Gal...) Threonine, O-linked (GalNacid) Serine, O-linked (GalNacid) Threonine, O-linked (GalNacid) Tyrosine, O-linked (GalNacid...)..)(keratan sulfate)serine, O-linked (GalNacid...)(keratan sulfate)threonine, O-linked (GalNacid...)serine, O-linked (GalNacid...)threonine, O-linked (GalNacid...)tyrosine, O-linked (GATDGlc)serine, O-linked (Glc)hydroxylysine, O-linked (Glc)serine, O-linked (Glc)threonine, O-linked (Glc)tyrosine, O-linked (Glc...)serine, O-linked (Glc...)tyrosine, O-linked (GlcA)serine, O-linked (GlcNacid)hydroxyproline, . O-linked (GlcNacid) serine, O-linked (GlcNacid) threonine, O-linked (GlcNacid) tyrosine, O-linked (GlcNacid...) hydroxyproline, O-linked (GlcNacid...) serine, O-linked (GlcNacid...) threonine, O-linked (GlcNacid...) tyrosine, O-linked (GlcNacid1P) serine, O-linked (GlcNacid6P) serine, O-linked (Hex) hydroxylysine, O-linked (Hex) hydroxyproline, O-linked (Hex)Serine, O-linked (Hex)Threonine, O-linked (Hex)Tyrosine, O-linked (Hex...)Hydroxylysine, O-linked (Hex...)Hydroxyproline, O-linked (Hex...)Serine, O-linked (Hex...)Threonine, O-linked (Hex...)Tyrosine, O-linked (HexNacid)Hydroxyproline, O-linked (HexNacid)Serine, O-linked (HexNacid)Threonine, O-linked (HexNacid)Tyrosine, O-linked (HexNacid... Droxyproline, O-linked (HexNacid...) serine, O-linked (HexNacid...) threonine, O-linked (HexNacid...) tyrosine, O-linked (Man) serine, O-linked (Man) threonine, O-linked (Man...) (keratan sulfate) serine, O-linked (Man...) (keratan sulfate) threonine, O-linked (Man...) serine, O-linked (Man...) threonine, O-linked (Man1P) serine, O-linked (Man1P...) serine, O-linked O-linked (Man6P) threonine, O-linked (Man6P...) threonine, O-linked (Xyl) serine, O-linked (Xyl...) (chondroitin sulfate) serine, O-linked (Xyl...) (dermatan sulfate) serine, O-linked (Xyl...) (glycosaminoglycan) serine, O-linked (Xyl...) (glycosaminoglycan) threonine, O-linked (Xyl...) (heparan sulfate) serine, O-linked (Xyl...) (keratin sulfate) threonine, O-linked (Xyl...) Serine, ω-hydroxyceramide glutamate ester, ω-N-methylarginine, ω-N-methylated arginine, O-methylthreonine, O-octanoylserine, O-octanoylthreonine, O-palmitoreylserine, O-palmitoylserine, O-palmitoylthreonine, O-triAMP-tyrosine, O-UMP-histine, O-UMP-serine, O-UMP-threonine, O-UMP-tyrosine, oxazole-4-carboxylic acid (Cys-Ser), oxazole-4-carboxylic acid (Gly-Ser), oxazole-4-carboxylic acid (Ile-Ser), oxazole-4-carboxylic acid (Ser-Ser), oxazoline-4-carboxylic acid (Cys-Ser), pentaglysilmurein peptoglycan-bound alanine, pentaglysilmurein Peptoglycan-linked threonine, peptide (Met-Gly) (interchain with G-...), phenylalaninamide, phosphatylethanolamine-linked glycine, phosphatylserine-linked glycine, phosphoarginine, phosphocysteine, phosphohistine, phosphoserine, phosphothreonine, phosphotyrosine, polyADP-ribosyl aspartate, polyADP-ribosyl glutamate, proline 5-hydroxy-oxazole-4-carbothic acid (Pro-Cys), prolineamide, Pros-8α-FAD histine, Pros-methylhistine, Pros-phosphohistine, pyrine-2,5-dicarboxylic acid (Ser-Cys) (with S-...), pyrine-2,5-dicarboxylic acid (Ser-Ser) (C-...(containing), pyrrolone carboxylic acid, pyrrolone carboxylic acid (Glu), pyrroloquinoline quinone (Glu-Tyr), pyruvate (Cys), pyruvate (Ser), pyruvate (Tyr), S-(15-deoxy-Δ12,14-prostaglandin J2-9-yl)cysteine, S-(2,3-dicarboxypropyl)cysteine, S-(2-aminovinyl)-3-methyl-D-cysteine ​​(Thr-Cys), S-(2-aminovinyl)-D-cysteine ​​(Cys-Cys), S-(2-aminovinyl)-D-cysteine Cysteine ​​(Ser-Cys), S-(2-aminovinyl)-L-cysteine ​​(Cys-Cys), S-(2-succinyl)cysteine, S-(4-hydroxycinnamyl)cysteine, S-(coelenterazine-3a-yl)cysteine, S-(dipyrrolylmethanemethyl)cysteine, S-12-hydroxyfarnesylcysteine, S-4a-FMNcysteine, S-6-FMNcysteine, S-8α-FADcysteine, S-archaeolic cysteine, S-bacidillithiol cysteine Sulfe, S-carbamoylcysteine, S-cGMPcysteine, S-cyanocysteine, S-cysteinyl 3-(oxosulfanil)alanine (Cys-Cys), S-cysteinylcysteine, S-diacidylglycerolcysteine, serineamide, serinemicrosine E492 cellophore ester, S-farnesylcysteine, S-geranylgeranylcysteine, S-glutathionylcysteine, S-linked (Gal)cysteine, S-linked (Gal...)cysteine Stain, S-linked (Glc) cysteine, S-linked (Glc...) cysteine, S-linked (GlcNacid) cysteine, S-linked (GlcNacid...) cysteine, S-linked (Hex) cysteine, S-linked (Hex...) cysteine, S-linked (HexNacid) cysteine, S-linked (HexNacid...) cysteine, S-lysylmethionine sulfirimine (Lys-Met) (interchain with M-...), S-lysylmethionine sulfirimine (Met-Lys) (K-...(between the chains), S-methylcysteine, S-methylmethionine, S-nitrosocysteine, S-palmitreoylcysteine, S-palmitoylcysteine, S-poly(β-hydroxybutyryl)cysteine, S-ceranylcysteine, S-stearoylcysteine, sulfoserine, sulfothreonine, sulfotyrosine, symmetric dimethylarginine, tere-(1,2,3-trihydroxypropan-2-yl)histine, tere-8α-FADhistine, tere-8α-FMNhistine, tere -Methylhistine, tere-phosphohistine, thiazole-4-carboxylic acid (Arg-Cys), thiazole-4-carboxylic acid (Asn-Cys), thiazole-4-carboxylic acid (Cys-Cys), thiazole-4-carboxylic acid (Glu-Cys), thiazole-4-carboxylic acid (Gly-Cys), thiazole-4-carboxylic acid (Ile-Cys), thiazole-4-carboxylic acid (Phe-Cys), thiazole-4-carboxylic acid (Pro-Cys), thiazole-4-carboxylic acid (Ser-Cys), thiazole-4-carboxylic acid (Thr-Cys), thiazole-4-carboxylic acid (Val-Cys), thiazoline linkage to ring-open DNA debasement sites, thiazoline-4-carboxylic acid (Phe-Cys), threonine 5-hydroxy-oxazole-4-carbotionic acid (Thr-Cys), threoninamide, threonine methyl ester, threonyllysine isopeptide (Lys-Thr) (interchain with T-...), threonyllysine isopeptide (Thr-Lys The group can be selected from the following: (interchain with K-...), thyroxine, triiodothyronine, trithiocysteine ​​(Cys-Cys), tryptophanamide, tryptophan derivatives, tryptophan-tryptophylquinone (Trp-Trp), tryptophylquinone, tryptophyl-tyrosyl-methionine (Trp-Tyr) (with M-...), tryptophyl-tyrosyl-methionine (Tyr-Met) (with W-...), tyrosinamide, and valineamide.

[0061] Post-translational modifications (PTMs) are covalent and generally enzymatic modifications of proteins after protein biosynthesis.

[0062] According to a particular embodiment, one or more artificial derivatives may be selected from the group consisting of polymers, chemical chelating agents, polyaminocarboxylates, deferoxamine, deferasirox, deferipone, and the like.

[0063] The polymer is not particularly limited and may be selected from the group consisting of, for example, polyethylene glycol, dextran, and the like.

[0064] According to certain embodiments, the chemical chelating agent is not particularly limited, but examples include cyclic polyamines. Cyclic polyamines can be selected from the group consisting of, for example, cyclenes, cyclams, and the like.

[0065] According to a particular embodiment, the polyaminocarboxylate may be selected from the group consisting of (2,2',2''-(1,4,7-triazacyclononane-1,4,7-triyl)-triacetic acid (NOTA), 2,2',2'',2'''-(1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)-tetraacetic acid (DOTA), 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA), etc.

[0066] By using one of these D-amino acid derivatives, the properties of a substrate having a functional group protected by a D-amino acid derivative can be adapted as detailed above. By using one or more functional groups, post-translational modifications, and artificial derivatizations in combination, the properties of a substrate having a functional group protected by a D-amino acid derivative can be adapted effectively, selectively, and precisely.

[0067] In this method, the substrate is not particularly limited, as long as it has a functional group (the functional group is not limited) protected by at least one D-amino acid and / or a D-amino acid derivative. It may be one substrate or more substrates that can be mixed, such as two or more, for example, three or more substrates. According to a particular embodiment, one substrate is used in this method. Generally, suitable substrates can be any organic compound used, for example, in the synthesis of low molecular weight active ingredients.

[0068] According to certain embodiments, the substrate may be selected from the group consisting of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, and mixtures thereof, in particular the substrate having at least two functional groups. According to certain embodiments, the substrate may be selected from the group consisting of carbohydrates, carbohydrate derivatives, and mixtures thereof, in particular the substrate having at least two functional groups. In specific embodiments, the substrate may be selected from the group consisting of amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, and mixtures thereof, in particular the substrate having at least two functional groups. According to certain embodiments, amino acids, peptides, oligopeptides, polypeptides, and peptide intermediates may be L-amino acids, L-peptides, L-oligopeptides, L-polypeptides, and L-peptide intermediates.

[0069] According to certain embodiments, amino acids, peptides, oligopeptides, polypeptides, proteins, and peptide intermediates can be enzyme precursors.

[0070] In the present invention, carbohydrates are not particularly limited. Examples of carbohydrates include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. Monosaccharides are not particularly limited and can be selected from the group consisting of disaccharides, trisaccharides, tetrasaccharides, pentoses, and hexoses. Among these, glycolaldehyde is an example of a disaccharide; trisaccharides can be selected from the group consisting of glyceraldehyde and dihydroxyacetone, for example; tetrasaccharides can be selected from the group consisting of erythrose, threose, and erythrulose, for example; pentoses can be selected from the group consisting of ribose, arabinose, xylose, lyxose, deoxyribose, ribulose, and xylulose, for example; and hexoses can be selected from the group consisting of allose, altrose, glucose, mannose, gros, idose, galactose, talose, and fructose. Disaccharides are sugars formed by linking two monosaccharides by glycosidic bonds. For example, disaccharides can be selected from the group consisting of cellobiose, genthiobiose, sucrose, lactose, trehalose, and maltose. Oligosaccharides are sugars formed by linking a small number of monosaccharides, typically 3 to 10, by glycosidic bonds. Oligosaccharides can have a linear or branched structure. For example, oligosaccharides can be selected from the group consisting of fructooligosaccharides, raffinose, and galactooligosaccharides. Polysaccharides are sugars containing a large number of monosaccharides, at least 11, linked by glycosidic bonds. Polysaccharides can have a linear or branched structure. For example, polysaccharides can be selected from the group consisting of glycogen, starch such as amylose and amylopectin, pectin, chitin, callose, and cellulose.

[0071] Carbohydrate derivatives may be based on the above-mentioned carbohydrates, except that one or more chemical groups may be introduced into the carbohydrate, and / or one or more functional groups of the carbohydrate may be substituted by at least one chemical group, and / or the carbohydrate may be in the form of a salt. According to certain embodiments, one or more hydroxyl groups of the carbohydrate may be substituted by at least one other chemical group, which is not particularly limited, and if two or more chemical groups are present, they may be the same or different. One or more chemical groups may be selected from the group consisting of, for example, hydrogen, halogen, alkyl, alkenyl groups, such as vinyl groups, allyl groups, etc., alkynyl groups, aromatic groups, ester groups, carboxylic acid groups, acetyl groups, ether groups, keto groups, aldehyde groups, amine groups, amide groups, imine groups, imide groups, acetylamino groups, acetal groups, hemiacetal groups, nitrile groups, thiol groups, sulfide groups, and posphine groups.

[0072] According to certain embodiments, one or more chemical groups may be selected from the group consisting of hydrogen, halogen, alkyl group, alkenyl group, alkynyl group, ester group, carboxylic acid group, acetyl group, ether group, keto group, aldehyde group, amine group, and acetylamino group. According to certain embodiments, alkyl groups may have 1 to 20 carbon atoms. According to certain embodiments, alkenyl groups may have 1 to 20 carbon atoms. According to certain embodiments, alkynyl groups may have 1 to 20 carbon atoms.

[0073] In the present invention, nucleotides, oligonucleotides, and polynucleotides are not particularly limited. Nucleotide derivatives include one or more modifications. Nucleotide derivatives may be in the form of salts. Nucleotide derivatives may include modified nucleic acid bases. Modified nucleic acid bases may be based on the above nucleic acid bases, except that one or more chemical groups may be introduced into the above nucleic acid bases, and / or one or more functional groups of the above nucleic acid bases may be substituted with at least one chemical group selected from the group consisting of, for example, hydrogen, halogen, alkyl, alkenyl, alkynyl, aromatic, amine, amide, imine, imide, acetylamino, and thiol groups. The same derivatization can be applied to oligonucleotides and polynucleotides.

[0074] The amino acids that can be used as substrates are not particularly limited, but are preferably L-amino acids. According to certain embodiments, the amino acids may be selected from the group consisting of proteinogenic amino acids and non-proteinogenic amino acids. Protein amino acids can be selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), valine (val, V), selenocysteine ​​(sec, U), and pyrrolicin (pyl, O). Non-proteinogenic amino acids are not limited and may be selected from the group consisting of, for example, carnitine, GABA (gamma-aminobutyric acid), levothyroxine, hydroxyproline, 2-aminoisobutyric acid, gamma-aminobutyric acid, ornithine, citrulline, and selenomethionine.

[0075] Peptides within the scope of the present invention may be L-peptides according to certain embodiments. The amino acids that form the peptide are not particularly limited. According to certain embodiments, the amino acids that form the peptide are selected from the group consisting of alanine (ala), lysine (lys), and tyrosine (tyr). Oligopeptides may similarly be L-oligopeptides. The amino acids that form the oligopeptides are not particularly limited. Polypeptides may be L-polypeptides. The amino acids that form the polypeptide are not particularly limited. Similarly, the amino acids that form proteins are not particularly limited.

[0076] Peptide intermediates are compounds present in the synthesis of peptides, oligopeptides, polypeptides, proteins, or enzymes. In certain embodiments, peptide intermediates may include peptides that can be modified by one or more chemical groups, particularly protecting groups. In certain embodiments, the functional groups of amino acids can be protected by protecting groups to prevent undesirable reactions of these functional groups. In certain embodiments, the N-terminus of a peptide may be modified by one or more chemical groups, particularly protecting groups. The protecting groups are not particularly limited. In certain embodiments, the protecting groups may be selected from the group consisting of benzoyl groups (Bz), fluorenylmethyloxycarbonyl groups (Fmoc), tert-butyloxycarbonyl groups (Boc), acetal groups (Ac), succinic acid groups (Suc), benzyloxycarbonyl groups (Z), 2-azidomethylbenzyl groups (Ambz), 2-aminobenzoic acid groups (Abz), biotin groups, and mixtures thereof. The peptide intermediate may further include, for example, a linker, which is a link between the peptide intermediate and the resin. The linker is not particularly limited and may be selected from the group consisting of, for example, Wang linkers, Rink linkers, Merrifield linkers, 4-methylbenzhydrylamine linkers (MBHA), phenylacetamidomethyl (PAM) linkers, nitrobenzyl linkers, and allyl linkers. Examples of Wang linkers include p-alkoxybenzyl ester linkers. Examples of Rink linkers include 4-(2',4'-dimethoxyphenyl-4-hydroxymethylphenoxy) linkers and 4-(2',4'-dimethoxyphenyl-4-methylamidephenoxy) linkers. Examples of Merrifield linkers include benzyl linkers. The resin is not particularly limited and may be selected from the group consisting of, for example, polystyrene.

[0077] In peptide derivatives and / or oligopeptide derivatives and / or polypeptide derivatives, as shown above for carbohydrate derivatives, for example, one or more chemical groups may be introduced into the peptide, and / or one or more functional groups of the peptide may be substituted with at least one chemical group. Furthermore, peptide derivatives may be peptides in the form of salts.

[0078] According to certain embodiments, the above method may further include adding a buffer to the reaction mixture, where the pH of the reaction mixture may be in the range of 3 to 13. A buffer may be prepared in addition to at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, a D-stereospecific hydrolase, and at least one solvent. The buffer can be prepared and mixed with at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, a D-stereospecific hydrolase, and at least one solvent.

[0079] The buffer is not particularly limited and can be, for example, acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium phosphate (NaPi) buffer, potassium phosphate (KPi) buffer, 2-(4-(2-hydroxyethyl)-piperazine-1-yl)-ethane-1-sulfonic acid (HEPES) buffer, tris-(hydroxymethyl)-aminomethane (Tris) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane (Bi s-Tris) buffer, 1,3-bis-(tris-(hydroxymethyl)-methylamino)-propane (Bis-Tris propane) buffer, N-(2-acetamide)iminodiacetic acid (ADA) buffer, N-(2-acetamide)-2-aminoethanesulfonic acid (ACES) buffer, acetamidoglycine buffer, 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer, N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, 3-(N-morpholino)-propanesulfonic acid (MOPS) Buffers, 2-((1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl)-amino)ethane-1-sulfonic acid (TES) buffer, cholamine hydrochloride, 4-(N-morpholino)-butanesulfonic acid (MOBS) buffer, 3-N-bis-(hydroxyethyl)-amino-2-hydroxy-propanesulfonic acid (DIPSO) buffer, 3-((1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl)-amino)-2-hydroxypropane-1-sulfonic acid (TAPSO) buffer, triethanolamine buffer, 4-(2-hydroxyethyl)-piperazine-1-(2-hydroxypropanesulfonic acid) (HEPPSO) buffer, piperazine-N,N'-bis-(2-hydroxypropanesulfonic acid) (POPSO) buffer, N-(tri(hydroxymethyl)-methyl)-glycine (tricine) buffer, glycylglycine buffer, glycinamide buffer, (bis(2-hydroxyethyl)-amino)acetic acid (BICINE) buffer, N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid (TAPS) buffer, 2-amino-2-methyl-1,The buffers can be selected from the group consisting of 3-propanediol (AMPD) buffer, N-tris(hydroxymethyl)-methyl-4-aminobutanesulfonic acid (TABS) buffer, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) buffer, (2-amino-2-methylpropane-1-ol)(AMP) buffer, and 3-(cyclohexylamino)propane-1-sulfonic acid (CAPS) buffer. According to certain embodiments, the buffer is selected from the group consisting of acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium phosphate (NaPi), N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid (TAPS) buffer, and 3-(cyclohexylamino)propane-1-sulfonic acid (CAPS) buffer. By using these buffers, the pH value can be kept constant. A constant pH value can improve the efficiency of the hydrolysis step and increase the activity of D-stereospecific hydrolase in the hydrolysis step. According to certain embodiments, the pH value of the reaction mixture may be in the range of 3 to 13, preferably in the range of 5 to 11, more preferably in the range of 7 to 10, and more preferably in the range of 7 to 9. By performing deprotection at such a pH value within the above range, the efficiency of the hydrolysis step can be improved and the activity of D-stereospecific hydrolase in the hydrolysis step can be increased. The concentration of the buffer solution in the reaction mixture is not limited and may range from 1 mmol to 1 mol, more preferably from 10 mmol to 500 mmol, more preferably from 15 mmol to 300 mmol, and more preferably from 20 mmol to 200 mmol, depending on the reaction mixture. By performing deprotection using a buffer solution within the above range, the pH value can be precisely controlled. Furthermore, the efficiency of the hydrolysis process can be improved, and the activity of D-stereospecific hydrolases in the hydrolysis process can be enhanced.

[0080] According to certain embodiments, the functional group of the protected substrate may be selected from amino groups, carboxyl groups, hydroxyl groups, sulfhydryl groups, and selenohydryl groups, for example, amino groups, hydroxyl groups, sulfhydryl groups, and / or selenohydryl groups, in particular D-amino acids and / or D-amino acid derivatives may be bonded to the substrate via carbonyl groups. During the hydrolysis step, for example, water molecules provided from a solvent may react with at least one of the above-mentioned functional groups of the protected substrate to form a D-amino acid and / or D-amino acid derivative and a partially deprotected substrate having at least one deprotected functional group deprotected from the D-amino acid and / or D-amino acid derivative.

[0081] The concentration of the substrate in the reaction mixture is not particularly limited and may be, for example, in the range of 100 nM to 1 M, preferably 1 μM to 100 mM, more preferably 5 μM to 50 mM, more preferably 10 μM to 10 mM, and even more preferably 100 μM to 1 mM, depending on the reaction mixture. By performing deprotection using a substrate concentration within the above range, the hydrolysis reaction proceeds more efficiently.

[0082] The concentration of D-stereospecific hydrolase in the reaction mixture is not limited and may be, for example, in the range of 1 nM to 200 μM, preferably 100 nM to 100 μM, more preferably 500 nM to 50 μM, and more preferably 750 nM to 10 μM, depending on the reaction mixture. By performing deprotection using a concentration of D-stereospecific hydrolase within the above range, the hydrolysis reaction proceeds more efficiently.

[0083] The solvent is not particularly limited, but preferably includes at least water. The solvent may be a mixture of at least two solvents. According to a particular embodiment, at least one solvent may be selected from the group consisting of water, alcohols such as methanol, ethanol, propanol, isopropanol, butanol, isobutanol, and mixtures thereof, aldehydes such as acetaldehyde, propionaldehyde, butyraldehyde, and mixtures thereof, ketones such as acetone, butanone, 2-pentanone, 3-pentanone, 3-methylbutanone, and mixtures thereof, esters such as methyl acetate, ethyl acetate, and mixtures thereof, amides such as DMF (dimethylformamide), formamide, and mixtures thereof, nitriles such as acetonitrile, propionitrile, isobutyronitrile, and mixtures thereof, sulfoxides such as DMSO (dimethyl sulfoxide), and mixtures thereof, for example, it may be selected from the group consisting of water, alcohols, nitriles, sulfoxides, and mixtures thereof, for example, it may be selected from the group consisting of water, DMSO (dimethyl sulfoxide), DMF (dimethylformamide), acetonitrile, methanol, and mixtures thereof. According to certain embodiments, at least one solvent may be water. These solvents can be used to enhance the efficiency of the hydrolysis process and the activity of D-stereospecific hydrolases in the hydrolysis process.

[0084] The concentration of at least one solvent may be in the range of 0.1% to 90%, preferably 0.5% to 70%, more preferably 1% to 50%, even more preferably 2% to 35%, and even more preferably 2.5% to 10%, based on the total volume of the solvent.

[0085] According to a particular embodiment, the hydrolysis step can be carried out at a reaction temperature within the range of 0°C to 50°C, preferably 5°C to 40°C, more preferably 15°C to 35°C, more preferably 22°C to 32°C, and even more preferably about 30°C. By performing deprotection at a temperature within the above range, the hydrolysis reaction proceeds more efficiently. Furthermore, side reactions can be prevented.

[0086] The method of the first embodiment may further include adding at least one additive to the reaction mixture. In addition to at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, a D-stereospecific hydrolase, and at least one solvent, the additive may be prepared. The additive is not particularly limited, as long as it is compatible with the compounds present in the reaction mixture. In particular, the enzyme applied is compatible with the additive. According to certain embodiments, at least one additive may be selected from the group consisting of salts and organic compounds. Salts can be added to increase the solubility of the substrate and can be selected from the group consisting of NaCl, KCl, LiCl, CaCl2, MgCl2, NaBr, AgCl, MnCl2, MgSO4, MnSO4, ZnSO4, (NH4)2SO4, and mixtures thereof. According to certain embodiments, NaCl may be preferred. Similarly, the organic compound may be selected from, for example, the group consisting of alkanes, alkenes, alkynes, alcohols, aldehydes, ketones, esters, ethers, amines, amides, imides, nitriles, thiols, their salts, and mixtures thereof. According to a particular embodiment, the organic compound may be selected from the group consisting of DTT (dithiothreitol), DTT derivatives, 2-mercaptoethanol, 2-mercaptoethanol derivatives, urea, urea derivatives, guanidinium chloride, guanidinium chloride derivatives, and mixtures thereof. According to certain embodiments, the additive may be selected from the group consisting of NaCl, KCl, LiCl, CaCl2, MgCl2, and mixtures thereof, DTT, DTT derivatives, 2-mercaptoethanol, 2-mercaptoethanol derivatives, urea, urea derivatives, guanidinium chloride, guanidinium chloride derivatives, and mixtures thereof, for example, from the group consisting of NaCl, DTT, 2-mercaptoethanol, urea, guanidinium chloride, and mixtures thereof. By adding at least one additive, the solubility of one or more compounds in the reaction mixture can be increased.The concentration of at least one organic compound is not limited and may be in the range of 1 mM to 5 M, for example, 1 mM to 2.5 M, more preferably 1 mM to 1 M, more preferably 1 mM to 0.5 M, more preferably 1 mM to 0.25 M, and even more preferably 1 mM to 0.1 M. According to a particular embodiment, the concentration of a salt, for example NaCl, may be in the range of 1 mM to 5 M, more preferably 1 mM to 2.5 M. By performing deprotection using additive concentrations within the above ranges, the solubility of one or more compounds in the reaction mixture can be further increased.

[0087] In the method of the first embodiment, the D-stereospecific hydrolase has the following characteristics in a particular embodiment: At least one first of three amino acids containing the amino acid sequence SXXK 10 - Helix-2 structure, At least one first α-helix structure containing the amino acid sequence YSN, and At least one first β-sheet structure containing the amino acid sequence HXG, (Here, X is an amino acid selected from the group consisting of proteinogenic amino acids) is characterized by the first 3 10 The -helix-2 structure, the first α-helix structure, and the first β-sheet structure can be arranged in close proximity.

[0088] According to a particular embodiment, the protein amino acid may be selected from the group consisting of alanine (ala, A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine ​​(cys, C), glutamine (gln, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine (val, V).

[0089] According to a particular embodiment, 310 The -helix-2 structure is the N-terminal extension of α-helix-2.

[0090] According to certain embodiments, at least one first 3 10 -helix-2 structure may include the amino acid sequence SXXK at the terminus of the 3 10 -helix-2 structure.

[0091] According to certain embodiments, the S and K amino acids of the amino acid sequence SXXK in at least one first 3 10 -helix-2 structure may be directly involved in hydrolytically cleaving at least one functional group of the substrate protected by a D-amino acid and / or D-amino acid derivative to obtain a D-amino acid and / or D-amino acid derivative and a substrate at least partially deprotected by a D-amino acid and / or D-amino acid derivative. According to certain embodiments, the S of the amino acid sequence SXXK may be involved in the binding to a substrate having at least one functional group protected by a D-amino acid and / or D-amino acid derivative during hydrolysis. According to certain embodiments, the S of the amino acid sequence SXXK that may be involved in the binding to a substrate having at least one functional group protected by a D-amino acid and / or D-amino acid derivative during hydrolysis may be activated S. In this structure, serine has an -OH group, which acts as a nucleophile and attacks the carbonyl carbon of the substrate of the scissile peptide bond. Further, the electron pair on the lysine nitrogen can receive a hydrogen from the serine-OH group, thus regulating the attack on the peptide bond. According to certain embodiments, the X in the acid sequence SXXK may be selected from the group consisting of I, K, L, V, and combinations thereof.

[0092] According to a particular embodiment, the YSN motif of the first α-helix structure may be located at the N-terminus of α-helix-6. In this structure, tyrosine is presumed to attack the Ser-substrate ester intermediate bond because it has an -OH group that can activate a water molecule. Asparagine can assist in this activation via an electrostatic network generated by E, D, or Q.

[0093] According to certain embodiments, the HXG motif of the first β-sheet structure may be involved in the formation of an oxyanion hole. The oxyanion hole is a pocket in the enzyme's active site that stabilizes the negative charge of the transition state of deprotonated oxygen. This pocket may consist of an amide or a positively charged residue of the main chain. According to certain embodiments, X in the sequence HXG may be selected from the group consisting of G, N, R, and S. According to certain embodiments, the HXG motif may be involved in the stabilization of a substrate having a functional group protected by at least one D-amino acid and / or a D-amino acid derivative. The histidine therein is the central part of the electrostatic network that enables the coordination and sequencing of the reaction. Glycine may be involved in the formation of the oxyanion hole, thereby enabling the nucleophilic attack of serine described above.

[0094] According to a particular embodiment, the features of the D-stereospecific hydrolase described above can provide a binding pocket for a substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative.

[0095] According to a particular embodiment, further comprising at least one second of three amino acids selected from E, D, and Q 10 - The helix structure may be involved in the substrate recognition region. Such a structure may enable interaction between the D-stereospecific hydrolase and the substrate. Alternatively or additionally, this may enable interaction between the D-stereospecific hydrolase and the correct coordination of the substrate in the binding pocket.

[0096] According to a particular embodiment, the first 3 10 The -helix-2 structure, the first α-helix structure, and the first β-sheet structure are positioned in close proximity, so that all fragments are close enough to interact with a substrate having a functional group protected by at least one D-amino acid and / or a D-amino acid derivative.

[0097] According to a particular embodiment, the D-stereospecific hydrolase may have an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. According to a particular embodiment, the D-stereospecific hydrolase may have an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0098] Sequence ID 1: ASLQLSQTKELLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGGVLLRKGYGFAGTNKLNRPDSKTRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLQSEFTRSGDVTIEQTIEELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLSGISYSDYVSEHFLTPLGMKNSGTATPATPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDTALRAGKVVSEQSLEAMYTPHSDKNYGYGWIALGLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 2: TIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNHPDAKSRIASLTKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGNVTIEQTIAELRTKQLKYEPGTTYLYSNNGYVLLAYVL EQLSGESYADYVSEHFLTPLGMKNSGTATPATATIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSKQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 3: ASLQLSQTKEFLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNRPDAKSRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGDVTIEQTIAELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLAGESYADYVNEHFLTPLGMKNSGTATPESPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSEQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 4: SSVQTSTQRDRNSVKQAVRDTLQLGFPGILAKTSEGGKTWSYAAGVANLSSKKPMKTDFRFRIGSVTKTFTATVVLQLAEENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIAEYTRSKSFDLMDTKKSYRAEELVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE TVTGNSYAEIEIENRIIEPLELSNTFLPGNSSVIPGTKHARGYIQLDGASEPKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGEAALGRYGLGIYETKLPNGVSIWGHGGSIPGFVTFAGGTLGGKHTLAVNLNSLNAESPDPFKNILLAEFSK; Sequence ID 5: SSLQTSTQSDRTSVKKAMRDELQLGYPGILAKISKGGKTWSYTAGVADLKTKKPMKADFRFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYVNSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQINGASELKDVTYINPGSSDGDMISTADDLNKFFSCLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGAVPGFSTFAGGTLGGKHTLAINSNSLNLNNPEVFKNILLAEFRK; Sequence ID 6: SSLQTSTQSDRTSVKKAIRDELQLGYPGILAQISKGGKTWSYTAGIADLRTKKPMKADFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYINSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQLDGASELKDVTYINPGSSDGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGGVLGFSTFAGGTLGGKHTLAINSNSFNINNPESFKNVLIAEFSK; Sequence ID 7: NSLQTSTQRDRNSVKEAMRDTLKLGYPGILAKTSEGGKTWSYAAGVADLSNKKAMKTDFRFRIGSVTKTFTATVVLQLAGENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIANYTRSKDFNMMDTKKSYTAEGLVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE KVTGNSYAEEIENRIIEPLELANTFLPGNSSVIPGTKHARGYIQLDGASETKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGVGELGDSGLGIFKMKLSNGESIWGHGGTIPGFLTFAGGTLGGKHTLAVNLNSLKADTPDPFKNILLAEFSK; Sequence ID 8: SSLQTNTQRDRTSVKQAMRDTLQLGYPGILAKTSEDGKTWGYAAGIADLRTKKPMKTDFRFRIGSVTKTFTATVVLQLVGENRLKLDDYIEKWLPGVIQGNGYDGNKITIREILNHTSGIAEYSRSKDVDFTDTKKSYTAEELVKMGISLPPDFAPGKGWSYSNTGYVLLGILIE KVTGNSYAEEVENRIIEPLELSNTFLPGNSSVIPGTNHARGYVQPDGASELKDVTYYNPSAGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGKEGIDGYGLGIYETKLPSGVSIWGHSGGILGFTTLVGGKLGGKHTLVVNWNSLGRTGSPNPFKNILLAEFSK; Sequence ID 9: NSSQEPNQKNRNGWKQVMQETIQIGAPGVLAKTSNKGKVNSYTAGVADLITKKPVKSDFRIGSVTKTFTATTVLQLVGENRVQLDDPIEKWLPGLVQGNGYDGNQITIRQLLNHTSGIAEYLKSKDADVMNSKKTYTAEEIVKIGLSLPPDFSPGKDWLYSNTGYVILGMLIEK ITGNNYAEEIEKRIIEPLDLPNTFLPGNSPVIPGKNHARGYVKMEETGELKDITYYNPSLANAAGDMISNADDDLNKFFSSLLGGKLLKERELKEMLTTVPVEGKGVGDGYGLGIYETKLPNGVSVWGHGGSIPGFMTFAGGVIGGKHTFAVNVNSLGPVDILTQFDKMMQVEFNK.

[0099] Also disclosed are the amino acid sequences from which the above amino acid sequences having SEQ ID NOs: 1 to 9 originate, each sequence having an initiating amino acid M that can be removed during post-translational modification (SEQ ID NOs: 1A to 9A), and the natural amino acid sequences from which the amino acid sequences having SEQ ID NOs: 1 to 9 originate, i.e., the amino acid sequences having SEQ ID NOs: 1B to 9B. Each of these sequences may be used in the present invention and is also included according to certain specific embodiments. For brevity, these will not be repeated when discussing other aspects of the present invention and will be included as with the embodiments of each respective embodiment that use or relate to the amino acid sequences having SEQ ID NOs: 1 to 9.

[0100] Sequence ID 1A: MASLQLSQTKELLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGGVLLRKGYGFAGTNKLNRPDSKTRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLQSEFTRSGDVTIEQTIEELRTKQLKYEPGTTYLYSNNGY VLLAYVLEQLSGISYSDYVSEHFLTPLGMKNSGTATPATPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDTALRAGKVVSEQSLEAMYTPHSDKNYGYGWIALGLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ Sequence ID 1B: MTGIRGKKIAGWFNRHTVARGALIGAIVSAFAGAGGAVAAAQPSSKVEAAQPASSKVIAVEVNGKAVEWNVQPLIKDGTTFVPLREAGKAAAGNIEWDGKTQTATIKVNGDVIVHRAGTSVVTVDGLAMNMSAPSLNIKGTLMLPLRSVTDALKASLQLSQTKELLTIRIQTDAVTKYKEDAAIDAYLKGEGFSGMALVAKDGGVLLRKGYGFAGTNKLNRPDSKTRIASITKSFTAASIMQ LVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLQSEFTRSGDVTIEQTIELRTKQLKYEEPGTTYLYSNNGYVLLAYVLEQLSGISYSDYVSEHFLTPLGMKNSGTATPATPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDTALRAGKVVSEQSLEAMYTPHSDKNYGYGWIALGLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ sequent number 2A: MTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNHPDAKSRIASLTKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGNVTIEQTIAELRTKQLKYEEPGTTYLYSNNGYVLLAYV LEQLSGESYADYVSEHFLTPLGMKNSGTATPATATIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSKQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ sequent number 2B: MLNGFQVWQSCRFSFKRRRMRKRRSWHGMTGIKGKKIAGWLNRHTVARSALIGIASVTAFAGVGGASVAAQTGSKGSKVEAVHPASSKAIAVEVNGKAVAWNVQPLIKDDTTFVPLREAGKAAAGNIEWDGKTKTATIKVNGDVIVHQAGTSVVTVDGLAMMSMSAPSLNIKGTLMLPLRSVTDALKASLQLSQTKELLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNHPDAKSRIASLTKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGNVTIEQTIAELRTKQLKYEPGTTYLYSNNGYVLLAYVLEQLSGESYADYVSEHFLTPLGMKNSGTATPATATIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSKQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ sequence number 3A: MASLQLSQTKEFLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKNLNRPDAKSRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGDVTIEQTIAELRTKQLKYEEPGTTYLYSNNGY VLLAYVLEQLAGESYADYVNEHFLTPLGMKNSGTATPESPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSEQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ sequence number 3B: MTGIRGKIAGWFNGHTVARSALIGMAAVTAFAGAGSAAAAQPSSQVEAVQPASSKAIAVEVNGKAVVWNVQPLIKDGTTFVPLREAGKAAAGNIEWDGKTQTATIKVNGDVIVHPAGTSVITVDGLAMNMASAPSLNIKGTLMPLRSVTDALKASLQLSQTKEFLTIRIQTAVTKYGKEDAAIDAYLKEGEFSGMALVAKDGEVLLRKGYGFSGTNKLNRPDAKSRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGDVTIEQTIAELRTKQLKYEPGTTYLYSNNGYVLLAYVLEQLAGESYADYVNEHFLTPLGMKNSGTATPESPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSEQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ sequence number 4A: MSSVQTSTQRDRNSVKQAVRDTLQLGFPGILAKTSEGGKTWSYAAGVANLSSKKPMKTDFRFRIGSVTKTFTATVVLQLAEENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIAEYTRSKSFDLMDTKKSYRAEELVKMGISMPPDFAPGKSWSYSNTGYVLLGILIETVTGNSYAEEIENRIIEPLELSNTFLPGNSSVIPGTKHARGYIQLDGASEPKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGEAALGRYGLGIYETKLPNGVSIWGHGGSIPGFVTFAGGTLGGKHTLAVNNLNSLNAESPDPFKNILLAEFSK sequence number 4B: MKTRSQITCASLALLIAGSSLLYTTQTLIVKAEPTQSVSSSVQTSTQRDRNSVKQAVRDTLQLGFPGILAKTSEGGKTWSYAAGVANLSSKKPMKTDFRFRIGSVTKTFTATVVLQLAEENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIAEYTRSKSFDLMDTKKSYRAEELVKMGISMPPDFAPGKSWSYSNTGYVLLGILIETVTGNSYAEEIENRIIEPLELSNTFLPGNSSVIPGTKHARGYIQLDGASEPKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGEAALGRYGLGIYETKLPNGVSIWGHGGSIPGFVTFAGGTLGGKHTLAVNNLNSLNAESPDPFKNILLAEFSK sequence number 5A: MSSLQTSTQSDRTSVKKAMRDELQLGYPGILAKISKGGTWSYTAGVADLKTKKPMKADFRFRIGSVTKTFIATVLLQLSGENRLNLDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYVNSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGILIEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSVIPGTKHARGYLQINGASELKDVTYINPGSSDGDMISTADDLNKFFSCLLGGKLLKEQQLKQMLTTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGAVPGFSTFAGGTLGGKHTLAINSNSLNLNNPEVFKNILLAEFRK sequence number 5B: KTRSKITCASLALLIGGSSLLYITPTSVVKAEPTQNVSSSLQTSTQSDRTSVKKAMRDELQLGYPGILAKISKGGKTWSYTAGVADLKTKKPMKADFRFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYVNSKDFDIMDTKKSYTAEFVKMGISLPPDF APGKGWSYSNTGYVLLGILIEKVTGNSYAEEVENRIIEPLDLSLNTFLPGNSSVIPGTKHARGYLQINGASELKDVTYINPGSSDGDMISTADDLNKFFSCLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGAVPGFSTFAGGTLGGKHTLAINSNSLNLNNPEVFKNILLAEFRK SEQ ID NO:6A: MSSLQTSTQSDRTSVKKAIRDELQLGYPGILAQISKGKTWSYTAGIADLRTKKPMKADFRFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYINSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGI LIEKVTGNSYAEEVENRIIEPLDSNTFLPGNSSVIPGTKHARGYLQLDGASELKDVTYINPGSSDGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTNREGTGYGLILEIKLPNGVSVWGHRGGVLGFSTFAGGTLGGKHTLAINSNSFNINNPESFKNVLIAEFSK sequence number 6B: MKTRSKITCASLALLIGGSSLLYITPTSVVKAEPTQNVSSSLQTSTQSDRTSVKKAIRDELQLGYPGILAQISKGGTWSYTAGIADLRTKKPMKADFRFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYINSKDFDIMDTKKSYTAEEFVKMGISLPPD FAPGKGWSYSNTGYVLLGILIEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQLDGASELKDVTYINPGSSDGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGGVLGFSTFAGGTLGGKHTLAINSNSFNINNPESFKNVLIAEFSK SEQ ID NO:7A: MNSLQTSTQRDRNSVKEAMRDTLKLGYPGILACTSEGGKTWSYAAGVADLSNKKAMKTDFRFRIGSVTKTFTATVVLQLAGENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIANYTRSKDFNMDTKKSYTAEGLVKMGISMPPDFAPGKSWSYSNTGYVLLGILI EKVTGNSYAEEIENRIIEPLELANTFLPGNSSVIPGTKHARGYIQLDGASETKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGVGELGDSGLGIFKMKLSNGESIWGHGGTIPGFLTFAGGTLGGKHTLAVNLNSLKADTPFKNILLAEFSK SEQ ID NO:7B: MKKHSQITCTGLALLIIIGGSLFYTTPTSSVKAESSIQNVSNSLQTSTQRDRNSVKEAMRDTLKLGYPGILAKTSEGGKTWSYAAGVADLSNKKAMKTDFRFRIGSVTKTFTATVVLQLAGENRLNLDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIANYTRSKDFNMMDTKKSYTAEGLVKMGISMPPDF APGKSWSYSNTGYVLLGILIEKVTGNSYAEEIENRIIEPLELANTFLPGNSSVIPGTKHARGYIQLDGASETKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGVGELGDSGLGIFKMKLSNGESIWGHGGTIPGFLTFAGGTLGGKHTLAVNLNSLKADTPDPFKNILLAEFSK Sequence ID 8A: MSSLQTNTQRDRTSVKQAMRDTLQLGYPGILAKTSEDGKTWGYAAGIADLRTKKPMKTDFRFRIGSVTKTFTATVVLQLVGENRLKLDDYIEKWLPGVIQGNGYDGNKITIREILNHTSGIAEYSRSKDVDFTDTKKSYTAEELVKMGISLPPDFAPGKGWSYSNTGYVLLGILI EKVTGNSYAEEVENRIIEPLELSNTFLPGNSSVIPGTNHARGYVQPDGASELKDVTYYNPSAGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGKEGIDGYGLGIYETKLPSGVSIWGHSGGILGFTTLVGGKLGGKHTLVVNWNSLGRTGSPNPFKNILLAEFSK Sequence ID 8B: MAQIVGIDRYLCTGALIYMREGFSLSLVRKSGTVSKHLYNMNRGEECLMKIRSQITCASLALLIAGSSLLYTTPTSIVKAEPTQNVSSSLQTNTQRDRTSVKQAMRDTL QLGYPGILAKTSEDGKTWGYAAGIADLRTKKPMKTDFRFRRIGSVTKTFTATVVLQLVGENRLKLDDYIEKWLPGVIQGNGYDGNKITIREILNHTSGIAEYSRSKDVDF TDTKKSYTAEELVKMGISLPPDFAPGKGWSYSNTGYVLLGILIEKVTGNSYAEEVENRIIEPLELSNTFLPGNSSVIPGTNHARGYVQPDGASELKDVTYYNPSAGSSA GDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGKEGIDGYGLGIYETKLPSGVSIWGHSGGILGFTTLVGGKLGGKHTLVVNWNSLGRTGSPNPFKNILLAEFSK Sequence ID 9A: MNSSQEPNQKNRNGWKQVMQETIQIGAPGVLAKTSNKGKVNSYTAGVADLITKKPVKSDFRFRIGSVTKTFTATTVLQLVGENRVQLDDPIEKWLPGLVQGNGYDGNQITIRQLLNHTSGIAEYLKSKDADVMNSKKTYTAEEIVKIGLSLPPDFSPGKDWLYSNTGYVILGMLIE KITGNNYAEEIEKRIIEPLDLPNTFLPGNSPVIPGKNHARGYVKMEETGELKDITYYNPSLANAAGDMISNADDDLNKFFSSLLGGKLLKERELKEMLTTVPVEGKGVGDGYGLGIYETKLPNGVSVWGHGGSIPGFMTFAGGVIGGKHTFAVNVNSLGPVDILTQFDKMMQVEFNK Sequence ID 9B: MKTRNQIKLASLAVLLAGTTLSTPGFTVKAESTQNISNSSQEPNQKNRNGWKQVMQETIQIGAPGVLAKTSNKGKVNSYTAGVADLITKKPVKSDFR FRIGSVTKTFTATTVLQLVGENRVQLDDPIEKWLPGLVQGNGYDGNQITIRQLLNHTSGIAEYLKSKDADVMNSKKTYTAEEIVKIGLSLPPDFSPG KDWLYSNTGYVILGMLIEKITGNNYAEEIEKRIIEPLDLPNTFLPGNSPVIPGKNHARGYVKMEETGELKDITYYNPSLANAAGDMISNADDLNKFFSSLLGGKLLKERELKEMLTTVPVEGKGVGDGYGLGIYETKLPNGVSVWGHGGSIPGFMTFAGGVIGGKHTFAVNVNSLGPVDILTQFDKMMQVEFNK

[0101] According to a particular embodiment, a D-stereospecific hydrolase can catalyze the hydrolytic cleavage of a functional group protected by at least one D-amino acid and / or D-amino acid derivative of a substrate, thereby obtaining a D-amino acid and / or D-amino acid derivative and a substrate at least partially deprotected by a D-amino acid and / or D-amino acid derivative.

[0102] The method of the first embodiment enables selective deprotection of a functional group protected by at least one D-amino acid and / or D-amino acid derivative of a substrate. The method of the first embodiment further enables rapid and quantitative cleavage of D-amino acids and / or D-amino acid derivatives from the functional group protected by the D-amino acid and / or D-amino acid derivative of the substrate. This method enables deprotection without side reactions under mild reaction conditions, and this method may be orthogonal to all previously established deprotection and synthesis methods. Furthermore, this method can be fully applied even to chemically unstable substrates and target structures.

[0103] A second aspect of the present invention provides a D-stereospecific hydrolase, characterized with respect to the method of the first aspect referenced herein. The D-stereospecific hydrolase of the second aspect is characterized by the following features: At least one first of three amino acids containing the amino acid sequence SXXK 10 -Helix-2 structure, at least one first α-helix structure containing the amino acid sequence YSN, and at least one first β-sheet structure containing the amino acid sequence HXG, where X is an amino acid selected from the group consisting of proteinogenic amino acids, and the first 3 10 The -helix-2 structure, the first α-helix structure, and the first β-sheet structure are arranged in close proximity. According to a particular embodiment, the D-stereospecific hydrolase has an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9, as shown above, or has an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 7, 8, and 9.

[0104] A third aspect of the present invention provides the use of a D-stereospecific hydrolase to deprotect a functional group of a substrate protected with at least one D-amino acid and / or a D-amino acid derivative. According to a particular embodiment, the D-stereospecific hydrolase is the D-stereospecific hydrolase of the second aspect.

[0105] According to certain embodiments, deprotection may include the same steps and features as deprotection of a functional group protected with at least one D-amino acid and / or D-amino acid derivative of a substrate described in the first aspect of the present invention.

[0106] According to a particular embodiment, the D-stereospecific hydrolase may have an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. According to a particular embodiment, the D-stereospecific hydrolase may have an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9.

[0107] D-stereospecific hydrolases, particularly the D-stereospecific hydrolases of the present invention, are strictly regioselective, chemoselective, and enantioselective with respect to each recognized D-amino acid and / or D-amino derivative. Therefore, the use of D-stereospecific hydrolases makes it possible to selectively cleave at least one D-amino acid and / or D-amino acid derivative from a functional group protected by a D-amino acid and / or D-amino acid derivative of the substrate.

[0108] A fourth aspect of the present invention provides a method for producing carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, the method being: The preparation of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivative precursors, or mixtures thereof, at least one D-amino acid and / or D-amino acid derivative, at least one first solvent, optionally at least one second solvent, optionally at least one third solvent, a D-stereospecific hydrolase, and at least one first reagent, A first reaction is obtained by mixing carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivative precursors, or mixtures thereof, at least one first solvent, and at least one D-amino acid and / or D-amino acid derivative. The method involves reacting a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative precursor, or mixture thereof with at least one D-amino acid and / or D-amino acid derivative to obtain a protected precursor, or mixture thereof, of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative. Optionally, remove at least one first solvent from the first reaction mixture, Optionally, a protected precursor of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof is reacted with one or more further reagents in one or more further reaction steps to obtain a reacted protected precursor of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof. A second reaction is obtained by mixing a carbohydrate, a carbohydrate derivative, a nucleotide, an oligonucleotide, a polynucleotide, a nucleotide derivative, an oligonucleotide derivative, a polynucleotide derivative, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a peptide intermediate, a peptide derivative, an oligopeptide derivative, a polypeptide derivative, or a mixture thereof, with at least one first reagent and optionally at least one second solvent. Protected precursors of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, or reacted protected precursors of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, with at least one first reagent to obtain protected carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, Optionally, remove at least one second solvent from the second reaction mixture, A third reaction is obtained by mixing a protected carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or a mixture thereof, optionally at least one third solvent, and a D-stereospecific hydrolase. The method involves deprotecting at least one D-amino acid and / or D-amino acid derivative from protected carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof to obtain carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof. Includes.

[0109] The process of preparing different components is not particularly limited. According to certain embodiments, components can be prepared with or without performing at least one synthesis step before preparing the different components. According to certain embodiments, the synthesis step may be selected from the group consisting of a chemical reaction, removal of at least one component, and addition of at least one component.

[0110] The process of mixing different components is not particularly limited. The order in which various components can be added in such mixing is also not particularly limited. The mixing is not particularly limited, and any known mixing method can be used.

[0111] For any reaction step, the reaction vessel is not particularly limited, and any known reaction vessel can be used.

[0112] The removal of at least one first solvent and / or at least a second solvent is not particularly limited. For example, any known evaporation method can be used. According to certain embodiments, at least one solvent and / or at least a second solvent can be removed by evaporation.

[0113] The process of reacting a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative precursor, or mixture thereof with at least one D-amino acid and / or D-amino acid derivative to obtain a protected precursor, or mixture thereof of a carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof is not particularly limited. According to a particular embodiment, the functional group of the precursor may be selected from the group consisting of an amino group, a carboxylic acid group, a hydroxyl group, a sulfhydryl group, and a selenohydryl group. According to a particular embodiment, the functional group may be an amino group, a hydroxyl group, a sulfhydryl group, and / or a selenohydryl group, and the D-amino acid and / or D-amino acid derivative can be bound to the substrate via a carbonyl group.

[0114] The deprotection step may include the same steps and features as the deprotection of a functional group protected with at least one D-amino acid and / or D-amino acid derivative of a substrate as described in the method of the first aspect of the present invention. According to certain embodiments, the deprotection step may further include adding a buffer to the reaction mixture to obtain a desired pH value of the reaction mixture. The buffer is not particularly limited. According to certain embodiments, the buffer may be selected from the buffers described in the first aspect of the present invention. According to certain embodiments, the deprotection step may further include adding one or more additives to the reaction mixture. The additives are not particularly limited as long as they are compatible with the compounds present in the reaction mixture. According to certain embodiments, the additives may be selected from the additives described in the first aspect of the present invention.

[0115] Carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof are not particularly limited and may be the same as those described in the first embodiment of the present invention.

[0116] The first solvent, the second solvent, and the third solvent are not particularly limited. They may be the same or different, depending on a particular embodiment, and may be the same as the first solvent described in the first aspect of the present invention.

[0117] Furthermore, the D-amino acids and D-amino acid derivatives are not particularly limited and may be selected from the D-amino acids and D-amino acid derivatives described in the first aspect of the present invention.

[0118] The D-stereospecific hydrolase is not limited and may have the same characteristics as the D-stereospecific hydrolase described in the first and second embodiments of the present invention.

[0119] The first reagent is not particularly limited to at least one type. According to a particular embodiment, the first reagent may be selected from the group consisting of a precursor for introducing a chemical group, a base, an acid, a reducing agent, an oxidizing agent, a carbohydrate, a carbohydrate derivative, a nucleotide, an oligonucleotide, a polynucleotide, a nucleotide derivative, an oligonucleotide derivative, a polynucleotide derivative, an amino acid, a peptide, an oligopeptide, a polypeptide, a protein, a peptide intermediate, a peptide derivative, an oligopeptide derivative, a polypeptide derivative, or a mixture thereof.

[0120] The precursor into which the chemical group is introduced is not particularly limited.

[0121] The chemical group may be selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, aromatic, ester, carboxylic acid, acetyl, ether, keto, aldehyde, amine, amide, imine, imide, acetylamino, acetal, hemiacetal, nitrile, thiol, sulfide, and phosphine groups.

[0122] The base is not particularly limited. According to certain embodiments, the base may be selected from the group consisting of organic bases and inorganic bases. According to certain embodiments, the base may be selected from the group consisting of amines, carbonates, carbamates, hydroxides, oxides, and ammonia, for example, amines, carbonates, hydroxides, and ammonia.

[0123] The acid is not particularly limited. According to certain embodiments, the acid may be selected from the group consisting of organic acids and inorganic acids. According to certain embodiments, the acid may be selected from the group consisting of carboxylic acids, sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. The carboxylic acid may be selected from the group consisting of formic acid and acetic acid.

[0124] The reducing agent is not particularly limited. According to certain embodiments, the reducing agent may be a metal such as sodium or lithium, hydrogen, lithium aluminum hydride, Red-Al (NaAlH2(OCH2CH2OCH3)2), diborane, sodium borohydride, iron(II) sulfate, etc. 2+ Fe compounds containing ions, Sn such as tin(II) chloride 2+ The following can be selected from the group consisting of ions-containing Sn compounds, sulfites, dithionates, thiosulfates, hydrazine, diisobutylaluminum hydride (DIBAL-H), reducing sugars such as oxalic acid, formic acid, ascorbic acid, and erythrose, phosphates, hypophosphates, DTT (dithiothreitol), carbon monoxide, and tris-2-carboxyethylphosphine hydrochloride (TCEP).

[0125] The oxidizing agent is not particularly limited. According to certain embodiments, the oxidizing agent may be selected from the group consisting of oxygen, nitrogen oxides, peroxides such as hydrogen peroxide and hydrogen peroxide derivatives, manganese salts, chromates such as PCC (pyridinium chlorochromate) or PDC (pyridinium dichromate), bromates, chlorites, and TEMPO(2,2,6,6-tetramethylpiperidine-1-yl)oxyl or (2,2,6,6-tetramethylpiperidine-1-yl)oxydanil).

[0126] Carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof are not particularly limited and may be as described in the first embodiment of the present invention.

[0127] This manufacturing method makes it possible to model the biological activity or biochemical functional groups of a compound during its synthesis. For example, by introducing one or more functional groups, post-translational modifications, and / or artificial derivatization to a D-amino acid to obtain a D-amino acid derivative, chemical and physical properties such as solubility or compatibility with other compounds can be altered as described above. This includes therapeutic agents that may be modified with D-amino acids and / or D-amino acid derivatives, and which are then inactivated to form prodrugs. As a result, therapeutic agents can be introduced as inactive precursors, and after accumulation in tissues, they can be activated by cleavage of the D-amino acids and / or D-amino acid derivatives.

[0128] D-amino acids and / or functional groups derived from D-amino acids can further block catalytically active residues or prevent structure-inducing interactions. This makes it possible to prevent the formation of structural elements or interactions between different amino acid side chains, thereby regulating enzyme activity or the binding of interaction partners to target structures. Following cleavage in D-amino acids and / or D-amino acid derivatives, the formation of active protein chemical species or intended interactions may occur.

[0129] By using D-stereospecific hydrolases, functional groups protected by at least one D-amino acid and / or D-amino acid derivative of a substrate can be easily cleaved at any point in the synthesis procedure of the compound. That is, at least one D-amino acid and / or D-amino acid derivative can be introduced into the substrate at any point in the synthesis of the chemical compound, and cleaved at any point after its introduction. Therefore, the chemical and physical properties of the substrate can be precisely, selectively, and effectively adapted to the required reaction conditions and reaction system at a specific point in the synthesis of the chemical compound.

[0130] A fifth aspect of the present invention provides the use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of a substrate.

[0131] The D-amino acids, D-amino acid derivatives, functional groups, and substrates are not particularly limited and may be as described above.

[0132] The above embodiments can be combined as appropriate and arbitrarily. Further embodiments and implementations of the present invention include combinations of features described above or below with respect to the embodiments of the present invention that are not explicitly mentioned. In particular, those skilled in the art may add individual embodiments as improvements or supplements to each of the basic embodiments of the present invention. [Examples]

[0133] Preparation and characterization of D-stereospecific hydrolases D-stereospecific hydrolases having SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9 were prepared in E. coli under standard conditions (growth conditions: LB medium, kanamycin, 37°C, continuous shaking; induction of protein biosynthesis: 0.1 M IPTG, and temperature shift to 30°C).

[0134] Table 1 shows the isolated yields of these D-stereospecific hydrolases after expression in E. coli and subsequent purification by ion exchange (SP-Sepharose or Supelco 650M, 0.1M HEPES, 0.1M NaCl, pH 7.0 (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, i.e., Paenidases 6.0), protein elution: 0.1M TRIS (pH 9.0)) and final gel permeation chromatography (HiLoad 16 / 60 Superdex 75 prep-grade column, 0.1M HEPES, 0.01M NaCl, pH 8.0).

[0135] [Table 1]

[0136] D-Stereospecific hydrolase specific activity A s Specific activity A of D-stereospecific hydrolases having SEQ ID NOs: 4, 6, 7, 8, and 9 against esterified selected D-amino acid derivatives s (s -1 ) was tested.

[0137] In these experiments, a NaPi buffer with a concentration of 0.1 M based on the reaction mixture was used. The pH of the reaction mixture was 8.0. As an example, the substrate Bz-D-Phe-OMe (benzoyl-D-phenylalanine-methyl ester) was used. For comparative examples, the substrates Bz-L-Phe-OMe (benzoyl-L-phenylalanine-methyl ester) and Bz-Gly-OGp (benzoyl-glycyl-4-guanidinophenyl ester) were used. The substrate concentration was 2 mM. The enzyme concentration ranged from 2 nM to 400 nM. The concentrations were based on the reaction mixture. Water containing 1% DMF (vol / vol) was used as the solvent. Activity measurements were performed discontinuously at 40°C with a flow rate of 0.5 ml / min and a detection wavelength of 254 nm via UPLC (gradient 0% B → 60% B (volume / volume), eluent: A water (0.1% (volume / volume) trifluoroacetic acid), B acetonitrile (0.1% (volume / volume) trifluoroacetic acid)). A 25 μl sample volume was taken at specific time points (0 min to 5 min) and quenched with 25 μl of 50% (volume / volume) acetic acid. s kat / mol is defined as the number of μmol of substrate converted per μmol of enzyme on a timescale, and is expressed as kat / mol.

[0138] In this case, the D-stereospecific hydrolase catalyzes the hydrolysis of each ester bond, thereby yielding the alcohol group and the carboxylic acid group. Specific activity A s This refers to the rate at which a specific D-stereospecific hydrolase catalyzes the hydrolysis of each ester bond.

[0139] Specific activity A of D-stereospecific hydrolase having SEQ ID NOs. 4, 6, 7, 8, and 9 s This is shown in Table 2.

[0140] [Table 2]

[0141] As shown in Table 2, the D-stereospecific hydrolases tested showed high A when substrates containing D-amino acids were used. s This indicates that when a substrate containing L-amino acids is used, the A value is low. s This demonstrates that the tested D-stereospecific hydrolase selectively recognizes and converts D-amino acids or D-amino acid derivatives.

[0142] In further experiments, the specific activity A of D-stereospecific hydrolases having SEQ ID NOs. 2, 4, 6, 7, 8, and 9 against peptides protected with selected D-amino acid derivatives was observed. s (mkat / mol) was tested. In this case, the N-terminus of the peptide is protected by a D-amino acid derivative.

[0143] In these experiments, which used conditions different from those described above, a NaPi buffer with a concentration of 0.1 M based on the reaction mixture was used. The pH of the reaction mixture was 8.0. This peptide had the amino acid sequence AKAKY(NO2)(SEQ ID NO 10), where Y was modified with an NO2 group to 3-nitrotyrosine. The concentration of the peptide protected with the D-amino acid derivative was 100 μM based on the reaction mixture. Furthermore, DTT was added at a concentration of 10 mM based on the reaction mixture. Furthermore, NaCl was added at a concentration of 150 mM based on the reaction mixture. The concentrations of each D-stereospecific hydrolase were 1 nM to 1 μM based on the reaction mixture. The D-amino acid derivatives used were those shown in Table 3 below. The reaction temperature was 30°C. Water was used as the solvent.

[0144] Evaluation was performed using discontinuous measurements at 40°C with a flow rate of 0.5 ml / min via UPLC (gradient 0% B → 20%, 40%, or 60% B (volt / volt), eluent: A water (0.1% (volt / volt) trifluoroacetic acid), B acetonitrile (0.1% (volt / volt) trifluoroacetic acid)) and a detection wavelength of 360 nm. A 25 μl sample volume was taken at specific time points (0 to 5 minutes) and quenched with 25 μl of 50% (volt / volt) acetic acid. All values ​​represent the average of two consecutive measurements. The deviation was always less than 10%.

[0145] In this case, the D-stereospecific hydrolase catalyzes the hydrolysis of the carboxamide bonds between the D-amino acid derivative and the peptide, thereby yielding the amine group and the carboxylic acid group, where, for example, f is D-Phe (Source: IUPAC-IUB Commission on Biochemical Nomenclature (CBN), Single-letter notation for amino acid sequences, 1968, Arch. Biochem. Biophys. 125(3), iv (1968)). Specific activity A s This refers to the rate at which a specific D-stereospecific hydrolase catalyzes the hydrolysis of the respective amide or carboxamide bond.

[0146] Specific activity A of D-stereospecific hydrolases having SEQ ID NOs. 2, 4, 6, 7, 8, and 9 against peptides protected with selected D-amino acid derivatives sThis is shown in Table 3. In this regard, it should be noted that in AAK(Bz-f)AKY(NO2)(SEQ ID NO 18), (Bz-f) is located at NH2 of the lysine chain, not inside the peptide chain, so the resulting peptide chain after cleavage is AAKAKY(NO2)(SEQ ID NO 137), which is the same as the cleavage result of Bz-fAAKAKY(NO2)(SEQ ID NO 21). From Bz-fCAKAKY(NO2) (SEQ ID NO. 11), Z-fCAKAKY(NO2) (SEQ ID NO. 12), Suc-fCAKAKY(NO2) (SEQ ID NO. 13), Ac-fCAKAKY(NO2) (SEQ ID NO. 14), Boc-fCAKAKY(NO2) (SEQ ID NO. 15), Abz-fCAKAKY(NO2) (SEQ ID NO. 16), Fmoc-fCAKAKY(NO2) (SEQ ID NO. 17), and fCAKAKY(NO2) (SEQ ID NO. 19), the product obtained is CAKAKY(NO2) (SEQ ID NO. 169). Other products after cleavage of protected D-Phe, from SEQ ID NO 20 to SEQ ID NO 38, are GAKAKY(NO2)(SEQ ID NO 136), AAKAKY(NO2)(SEQ ID NO 137), SAKAKY(NO2)(SEQ ID NO 138), TAKAKY(NO2)(SEQ ID NO 139), LAKAKY(NO2)(SEQ ID NO 140), VAKAKY(NO2)(SEQ ID NO 141), IAKAKY(NO2)(SEQ ID NO 142), FAKAKY(NO2)(SEQ ID NO 143), YAKAKY(NO2)(SEQ ID NO 143) The sequence numbers are 144), WAKAKY(NO2)(sequence number 145), RAKAKY(NO2)(sequence number 146), KAKAKY(NO2)(sequence number 147), HAKAKY(NO2)(sequence number 148), DAKAKY(NO2)(sequence number 149), EAKAKY(NO2)(sequence number 150), QAKAKY(NO2)(sequence number 151), NAKAKY(NO2)(sequence number 152), MAKAKY(NO2)(sequence number 153), and PAKAKY(NO2)(sequence number 154).

[0147] [Table 3]

[0148] As can be seen from Table 3, all D-stereospecific enzymes tested exhibit activity with respect to the selected substrates. Therefore, D-stereospecific enzymes can convert peptides having at least one functional group protected by a D-amino acid or D-amino acid derivative. This is true even in the absence of the protecting group.

[0149] Further experiments revealed that the specific activity A of D-stereospecific hydrolases having SEQ ID NOs: 4, 7, 8, and 9 against peptides protected with selected D-amino acid derivatives was s The (mkat / mol) test was performed as described above, except that the concentration of the D-amino acid derivative-protected peptide was 1 mM based on the reaction mixture. The results are shown in Table 4. All values ​​represent the average of two sets of measurements. The deviation was always less than 10%.

[0150] [Table 4]

[0151] As can be seen from Table 4, the concentration of the applied substrate can be varied without reducing the activity of the D-stereospecific hydrolase. Therefore, the D-stereospecific hydrolase can be applied to a variety of synthetic conditions.

[0152] Subsite mapping experiment for S1' subsite Subsite mapping experiments were performed to characterize the S1' binding pocket. For this purpose, Bz-Gly-OGp and Bz-D-Phe-OMe were used as acyl donors, and selected D-amino acid amides and L-amino acid amides (X / x) were used. aa-NH2 (where x represents a D-amino acid and X represents an L-amino acid) was used as the acyl acceptor. Activity was measured using discontinuous measurements at 40°C with a flow rate of 0.5 ml / min via HPLC (gradient 0% B → 20%, 40%, or 60% B (volt / volt), eluent: A water (0.1% (volt / volt) trifluoroacetic acid), B acetonitrile (0.1% (volt / volt) trifluoroacetic acid)) and detection wavelengths of 220 nm and 254 nm, respectively. A 25 μl sample volume was taken at specific time points (0 to 5 minutes) and quenched with 25 μl of 50% (volt / volt) acetic acid. In this experiment, a NaPi buffer with a concentration of 0.1 M based on the reaction mixture was used. The pH value of the reaction mixture was 8.0. The concentration of the acyl acceptor was 10 mM (amino acid amide or pentapeptide) based on the reaction mixture. The concentration of the acyl donor (Bz-Gly-OGp or Bz-Phe-OMe) was 1 mM based on the reaction mixture. The concentration of the D-stereohydrolase was 500 nM. Water containing 1% DMF (vol / vol) was used as the solvent.

[0153] The respective model reactions (aminolysis reactions) are shown below (Equations 1 and 2). In Equations 1 and 2, DHy represents the respective D-stereospecific hydrolases described below.

[0154] Formula 1: TIFF2026522795000005.tif13170

[0155] Formula 2: TIFF2026522795000006.tif14170

[0156] The efficiency of such a reaction is determined using the reciprocal partition value (Equation 3) (Source: Schellenberger V, Turck CW, Rutter WJ, "Role of the S' subsites in serine protease catalysis. Active-site mapping of rat chymotrypsin, rat trypsin, alpha-lytic protease, and cercarial protease from Schistosoma mansoni." Biochemistry. 1994 Apr 12;33(14):4251-7. doi: 10.1021 / bi00180a020. PMID: 8155642). TIFF2026522795000007.tif12170 (Formula 3) p: Partition value [N]: Total nucleophile concentration v H : Hydrolysis reaction rate v A : Aminolysis reaction rate

[0157] Figure 1 shows the reciprocal fraction (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having sequence number 6. Figure 2 shows the reciprocal fraction (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having sequence number 7. Figure 3 shows the reciprocal fraction (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having sequence number 8. Figure 4 shows the reciprocal fraction (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having sequence number 9.

[0158] In Figures 1 to 4, the black bars represent Bz-D-Phe-OMe, and the white bars represent Bz-Gly-OGp.

[0159] The smaller the reciprocal value (1 / p), the higher the reaction rate of the hydrolysis reaction and the higher the rate of the amination reaction, which reflects S1' specificity.

[0160] As can be seen in Figures 1 to 4, each amide (x aa -NH2, x at the D-amino acid position at the P1' position aa A desorption reaction using D-amino acids is preferred because the 1 / p value is large for all D-stereospecific enzymes tested. Therefore, D-stereospecific enzymes specifically recognize D-amino acids or D-amino acid derivatives when they are at the P1' position. The 1 / p value is for L-amino acids (X aa This is mainly reduced when using ), which highlights the high S'1 subsite selectivity of D-stereospecific hydrolases for the hydrolysis of functional groups protected by D-amino acids or D-amino acid derivatives of substrates, especially in the case of a single amino acid or its derivative.

[0161] Instead of the above D / L-amino acid amide, use total D / L peptide (x aa aaaGly, / X aa Similar experiments were conducted using AAAG) as the acyl receptor. Various D- / L-amino acids are located at the P1' position. Bz-Gly-OGp was again used as the acyl donor. The respective model reactions (hydrolysis reactions) are shown below (Equations 4A and 4B). In Equation 4, DHy represents a D-stereospecific hydrolase.

[0162] In this experiment, a NaPi buffer with a concentration of 0.1 M based on the reaction mixture was used. The pH of the reaction mixture was 8.0. The concentration of the acyl acceptor was 10 mM based on the reaction mixture. The concentration of the acyl donor was 1 mM based on the reaction mixture. The concentration of D-stereohydrolase was 500 nM. Water containing 1% DMF was used as the solvent.

[0163] Formula 4: TIFF2026522795000008.tif27170

[0164] The following sequence numbers apply: XAAAG (sequence number 106), xaaaG (sequence number 107), Bz-GXAAAG (sequence number 170), Bz-GxaaaG (sequence number 171).

[0165] Figure 5 shows the reciprocal division values ​​(1 / p) of the S1' subsite mapping of D-stereospecific hydrolases having sequence numbers 7 (white bar), 8 (light gray bar), and 9 (dark gray bar). The following sequences are shown as the starting materials (educts) and products, respectively: FAAAG (sequence number 108), faaaG (sequence number 109), Bz-GFAAAG (sequence number 110), Bz-GfaaaG (sequence number 111), LAAAG (sequence number 112), laaaG (sequence number 113), Bz-GLAAAG (sequence number 114), Bz-GlaaaG (sequence number 115), MAAAG (sequence number 116), maaaG (sequence number 117), Bz-GMAAAG (sequence number 118), Bz-GmaaaG (sequence number 119), RAAAG (sequence number 120), r aaaG (sequence number 121), Bz-GRAAAAG (sequence number 122), Bz-GraaaG (sequence number 123), AAAAG (sequence number 124), aaaaG (sequence number 125), Bz-GAAAAG (sequence number 126), Bz-GaaaaG (sequence number 127), GAAAG (sequence number 128), gaaaG (sequence number 129), Bz-GGAAAG (sequence number 130), Bz-GgaaaG (sequence number 131), PAAAG (sequence number 132), paaaG (sequence number 133), Bz-GPAAAG (sequence number 134), Bz-GpaaaG (sequence number 135).

[0166] In contrast to single amino acid derivatives, D-stereospecific enzymes specifically recognize the L-amino acid derivative at the P1' position when a peptide is used as a substrate. This allows the enzyme to cleave the DL-amino acid bond in the case of longer peptides.

[0167] Experiments using fluorescence-generating substrates In further experiments, hydrolysis reactions using D-stereospecific hydrolases with SEQ ID NOs: 4, 6, 7, 8, and 9 were performed with an internally quenched fluorescence-generating substrate (IQFS) (Abz-AAX / x aa The reaction was carried out using FAAK(DNP)-OH) (Abz = 2-aminobenzoic acid, DNP = 2,4-dinitrophenol). A fluorescent signal was released upon cleavage of IQFS, which could be monitored by continuous fluorescence measurement (0.1 M NaPi, pH 8.0, 1% DMSO (volume / volume), 5 nM to 1000 nM enzyme, excitation wavelength 320 nm, emission wavelength 420 nm, reaction volume 100 μl, 96-well microtiter plate, time scale 2 hours). Each model reaction is shown below (Equation 5). In Equation 5, DHy represents the respective D-stereospecific hydrolase.

[0168] Formula 5: TIFF2026522795000009.tif13170

[0169] The following sequence numbers apply: Abz-AAXFAAK(DNP)-OH (sequence number 172), Abz-AAxFAAK(DNP)-OH (sequence number 173), FAAK(DNP)-OH (sequence number 155).

[0170] Apparent specific activity (A s,app Table 5 shows the D-stereoconfiguration of amino acids (x aa The values ​​for ) are on the left, and the L-stereoconfiguration amino acid (X aaThe values ​​for ) are on the right. The sequences in Table 5 are as follows: AAFFAAK (sequence number 74), AAfFAAK (sequence number 75), AAWFAAK (sequence number 76), AAwFAAK (sequence number 77), AAYFAAK (sequence number 78), AAyFAAK (sequence number 79), AAAFAAK (sequence number 80), AAaFAAK (sequence number 81), AAVFAAK (sequence number 82), AAvFAAK (sequence number 83), AALFAAK (sequence number 84), AAlFAAK (sequence number 85), AASFAAK (sequence number 86), AAsFAAK (sequence number 87), AAMFAAK (sequence number 88), AAmFAAK (sequence number 89), AAHFAAK (sequence number 90), AAhF In each of the following sequences, AAK (sequence number 91), AAKFAAK (sequence number 92), AAkFAAK (sequence number 93), AARFAAK (sequence number 94), AArFAAK (sequence number 95), AAQFAAK (sequence number 96), AAqFAAK (sequence number 97), AAEFAAK (sequence number 98), AAeFAAK (sequence number 99), AADFAAK (sequence number 100), AAdFAAK (sequence number 101), AAPFAAK (sequence number 102), AApFAAK (sequence number 103), AAGFAAK (sequence number 104), and AAgFAAK (sequence number 105), the first A is protected by Abz in each sequence, and the last K is protected by DNP-OH. Furthermore, FAAK(DNP)-OH (sequence number 155) is obtained as a product.

[0171] [Table 5]

[0172] As can be seen from Table 5, D-stereospecific hydrolase exhibits higher activity when using a substrate containing D-amino acids compared to a substrate containing L-amino acids, similar to the above.

[0173] Table 6 shows the catalytic parameters for hydrolysis reactions using a quenching fluorescence-generating substrate (Abz-AAX / xaa FAAK(DNP)-OH) along with D-stereospecific hydrolases having SEQ ID NOs. 8 and 9. The sequences in Table 6 are as follows: AAfFAAK (SEQ ID NOs. 68), AAwFAAK (SEQ ID NOs. 69), AAyFAAK (SEQ ID NOs. 70), AAlFAAK (SEQ ID NOs. 71), AAmFAAK (SEQ ID NOs. 72), and AAhFAAK (SEQ ID NOs. 73). In each sequence, the first A is protected by Abz, and the last K is protected by DNP-OH.

[0174] [Table 6]

[0175] Similar experiments were conducted using D-stereospecific hydrolase with Sequence ID No. 3 and the substrate Abz-AAX / x aa The test was performed using FAAK(DNP)-OH. Specific activity (A s ) are shown in Table 7.

[0176] [Table 7]

[0177] Experiments on the S2 preference of D-stereospecific hydrolases In further experiments, the S2 preference of D-stereospecific hydrolases having SEQ ID NOs. 8 and 9 was determined with respect to IQFS as described above for substrate Abz-AX / x aa The investigation was conducted using fAAAK(DNP)-OH. The respective model reactions are shown below (Equation 6). Here, AAAK(DNP)-OH (SEQ ID NO: 156) is obtained as the product. In Equation 6, DHy again represents D-stereospecific hydrolase.

[0178] Formula 6: TIFF2026522795000013.tif12170

[0179] The following sequence numbers apply: Abz-AXfAAAK(DNP)-OH (sequence number 174), Abz-AxfAAAK(DNP)-OH (sequence number 175), AAAK(DNP)-OH (sequence number 156).

[0180] Table 8 shows the specific activity A of D-stereospecific enzymes. s (kat / mol) is shown. In Table 8, amino acids at the P2 position are shown in bold, and the cleavage site is indicated by an arrow.

[0181] [Table 8]

[0182] Table 9 shows the specific activity and kinetic parameters of the D-stereospecific enzyme.

[0183] [Table 9]

[0184] As can be seen from Tables 8 and 9, D-stereospecific enzymes specifically recognize D-amino acids or D-amino acid derivatives when they are located at the P2 position, which highlights the wide range of applications for D-stereospecific enzymes.

[0185] Experiments on the S1' preference of D-stereospecific hydrolases In further experiments, the S1' preference of D-stereospecific hydrolases having SEQ ID NOs. 8 and 9 was determined using the substrate Abz-AAfx aa The investigation was conducted using AAK(DNP)-OH.

[0186] Table 10 shows the specific activity A of D-stereospecific enzymes. s (kat / mol) is shown. In Table 10, amino acids at the P1' position are shown in bold, and the cleavage site is indicated by an arrow.

[0187] [Table 10]

[0188] In addition to Abz-AAf, the following products are obtained after cleavage: MAAK (SEQ ID NO: 157), FAAK (SEQ ID NO: 158), VAAK (SEQ ID NO: 159), LAAK (SEQ ID NO: 160), AAAK (SEQ ID NO: 161), GAAK (SEQ ID NO: 162), PAAK (SEQ ID NO: 163), SAAK (SEQ ID NO: 164), NAAK (SEQ ID NO: 165), EAAK (SEQ ID NO: 166), RAAK (SEQ ID NO: 167), and HAAK (SEQ ID NO: 168).

[0189] Experiments involving subsite mapping, S2 preference, and S1' preference demonstrate that the enzyme is active regardless of the position of the D-amino acid or D-amino acid derivative. Therefore, D-stereospecific hydrolases can be used in a variety of applications with substrates having a functional group protected by at least one D-amino acid or D-amino acid derivative.

[0190] Experiments on the effects of various additives and reaction conditions on the activity of D-stereospecific hydrolases In further experiments, the effect of pH on the activity of D-stereospecific hydrolases of SEQ ID NOs: 4, 6, 7, 8, and 9 was investigated. The peptide Abz-AAfFAAK(DNP)-OH was used as the substrate. pH values ​​were varied using various buffers (acetic acid buffer: pH 4.0-5.5, MES buffer: pH 5.5-7.0, NaPi buffer: pH 6.5-8.0, TAPS buffer: pH 7.5-9.5, CAPS buffer: pH 9.5-11.5). The relative activity (rel. A) of each enzyme was measured. s The results are shown in Figures 6 to 10 (Figure 6: SEQ ID NO: 9, Figure 7: SEQ ID NO: 8, Figure 8: SEQ ID NO: 7, Figure 9: SEQ ID NO: 6, Figure 10: SEQ ID NO: 4). Relative activity is calculated by dividing the activity at various pH values ​​by the corresponding activity of each D-stereospecific enzyme at pH 8.0 (used as a standard condition).

[0191] As can be seen from Figures 6 to 10, D-stereospecific hydrolases exhibit activity over a wide pH range. Therefore, D-stereospecific hydrolases can be used in a variety of ways under various synthetic conditions.

[0192] Furthermore, experiments were conducted to investigate the effects of adding various additives at different concentrations based on the reaction mixture on the activity of D-stereospecific hydrolase. The concentration of each additive was varied between 0 M and 2.5 M. Urea, guanidine hydrochloride, NaCl, and β-mercaptoethanol (β-ME) were used as additives. The additives were added to a reaction mixture containing D-stereospecific hydrolase and the peptide Abz-AAfFAAK(DNP)-OH. D-stereospecific hydrolases with SEQ ID NOs: 4, 6, 7, 8, and 9 were used.

[0193] Figures 11a and 11b show the relative activity of the D-stereospecific hydrolase having SEQ ID NO: 9 when the additive urea or guanidine hydrochloride is added.

[0194] Figures 12 to 14 show the relative activity of D-stereospecific hydrolases having SEQ ID NO: 8 (Figure 12), SEQ ID NO: 6 (Figure 13), and SEQ ID NO: 4 (Figure 14) when the additives urea, guanidine hydrochloride, NaCl, or β-mercaptoethanol are added at various concentrations.

[0195] In this case, the relative activity is calculated by dividing the activity when additives of various concentrations are added by the maximum activity of each D-stereospecific enzyme.

[0196] In Table 11, 50% of the relative specific activity of each enzyme ( * ) or 10% ( ** This shows the concentration (M) of the additive based on the reaction mixture in which ) still exists.

[0197] [Table 11]

[0198] As can be seen from Table 11, the D-stereospecific hydrolase shows activity even when various additives are added to the reaction mixture. Therefore, the D-stereospecific hydrolase can be used variously under various synthesis conditions.

[0199] Furthermore, an experiment was conducted to investigate the effect of the solvent on the activity of the D-stereospecific hydrolase.

[0200] As solvents, DMSO, DMF, methanol, and ACN (acetonitrile) were used. The concentration of the solvent was varied between 0% (volume / volume) and 20% (volume / volume) for DMF and acetonitrile, and between 0% (volume / volume) and 30% (volume / volume) for DMSO and methanol. The solvent was added to the reaction mixture containing the D-stereospecific hydrolase and the peptide Abz-AAfFAAK(DNP)-OH at various concentrations based on the total volume of the solvent. The remaining solvent was water. As the D-stereospecific hydrolase, the D-stereospecific hydrolase having SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9 was used.

[0201] In FIGS. 15a to 15c, the relative activity (rel.A s ) of the D-stereospecific hydrolase having SEQ ID NO: 9 when DMSO (15a), ACN (15b), and DMF (15c) are added is shown.

[0202] In FIGS. 16 to 19, the relative activity (rel.A s ) of the D-stereospecific hydrolase having SEQ ID NO: 4 (FIG. 19), SEQ ID NO: 6 (FIG. 18), SEQ ID NO: 7 (FIG. 17), and SEQ ID NO: 8 (FIG. 16) when DMSO, ACN, methanol, and DMF are added is shown.

[0203] The relative activity in this case is calculated by dividing the activity when a solvent having various concentrations is added by the maximum activity of each D-stereospecific enzyme.

[0204] In Table 12, the effect of the selected organic solvent on the activity of the D-stereospecific hydrolase is shown. The concentration (volume / volume) of the organic solvent at which 50% ( * ) or 10% ( ** ) of the relative specific activity of each enzyme still remains is shown.

[0205]

Table 12

[0206] As can be seen from FIGS. 15a to 15c, FIGS. 16 to 19, and Table 11, the D-stereospecific hydrolase shows activity even when a solvent other than water is added to the reaction mixture. The D-stereospecific hydrolase shows activity even when the concentration of other solvents is 35% (volume / volume). Therefore, the D-stereospecific hydrolase can be used variously under various synthetic conditions.

[0207] Alignment of the isolated and used D-stereospecific hydrolase In FIG. 20, the alignment of the isolated and used D-stereospecific hydrolase is shown.

[0208] In this alignment, conserved amino acids are highlighted in red, amino acids with similar functions are written in red, and conserved regions are framed. The numbering is based on the complete numbering of the consensus sequence. Furthermore, secondary structure elements (α-α helix, η-3 10The helix, β-β sheet, and TT-β turn are labeled according to Sequence ID No. 6. Their crystal structures were obtained at a resolution of 1.46 Å and are shown in Figure 21. To obtain the crystals, Sequence ID No. 6 was concentrated to 120 μM in 0.1 M phosphate buffer (pH 8.0) and 0.1 M NaCl, and crystallized by hanging-drop vapor diffusion at 20°C by mixing with equivolute precipitating agent solutions: 0.1 M imidazole, 0.1 M MES (pH 6.5), 30 mM MgCl2, 30 mM CaCl2, 12.5% ​​(vol / vol) MPD, 12.5% ​​(weight / vol) polyethylene glycol 1000, and 12.5% ​​(weight / vol) polyethylene glycol 3350. Crystals appeared within 14 to 21 days and were rapidly frozen without any additional cryoprotective agents. Data was collected at -172°C at BESSY synchrotron beamlines 14.1 and 14.2 (Helmholtz Zentrum, Berlin). The crystals belong to the monoclinic space group P21. Structure (A) with conserved motifs SXXK and YXN is shown. Furthermore, the HRG motif in β-sheet 13 and α-helix 2 and α-helix 6 are shown. In addition, details of the surface, presumed binding sites, and co-crystallized phosphates (B) are shown, as well as the orientation of the phosphate at the active site and the distance to the side chains important for catalytic activity (C). Additional crystal structure results from immersion experiments using the substrate Bz-D-Arg-OMe confirmed binding to the substrate and its mechanism (data not shown).

[0209] Alignment was performed using MultAlign, and visualization was performed using ESPript3 (Corpet, 1988, Robert & Gouet, 2014).

[0210] As can be seen from Figure 20, all sequences of the isolated enzyme contain the amino acid sequence SXXK. 10 - It has a helix 2 structure, an α-helix structure containing the amino acid sequence YSN (α-helix-6 structure), and a β-sheet structure containing the amino acid sequence HXG (β-sheet-13).

[0211] For example, the use of D-amino acids and / or D-amino acid derivatives as protecting groups or linkers for affinity probes or soluble tags is compatible with standard synthetic protocols in peptide and carbohydrate chemistry and can therefore be easily applied to already established synthetic protocols without additional effort. In contrast to penicillin acylase, for example, the biocatalysts used herein are highly effective in the reactions considered, for example, up to 7 × 10⁻⁶. 6 M -1 s -1 It possesses a specificity constant. This allows for quantitative conversion at low enzyme concentrations with very short reaction times. These enzymes can be expressed in high yield using standard molecular biology and protein chemistry protocols and are resistant to a wide range of additives and organic solvents. Furthermore, the biocatalysts used do not require cofactors or stabilizers, are stable over a wide pH range, and exhibit catalytic activity.

[0212] Experiments using various substrates with D-stereospecific hydrolase To demonstrate the usefulness of the D-stereospecific hydrolase of the present invention for non-proteinaceous substrates, experiments were conducted using various substrates under the following conditions, similar to those described above: 100 μM substrate, 10 nM to 50 nM enzyme, 100 mM NaPi (pH=8), 150 mM NaCl, 30°C. Evaluation was based on ULC at 254 nm (nM). Ahx-NH2 = aminohexanoic acid amide, Glc = glucosamine. The results are shown in Table 13.

[0213] [Table 13]

[0214] Regarding the sequence listing, please note that numbers 1-9 in the sequence listing correspond to sequence numbers 1-9, numbers 10-27 correspond to sequence numbers 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 6A, 6B, 7A, 7B, 8A, 8B, 9A, 9B, and numbers 28-193 correspond to sequence numbers 10-175.

Claims

1. A method for deprotecting a functional group protected with at least one D-amino acid and / or a D-amino acid derivative of at least one substrate having a functional group protected with at least one D-amino acid and / or a D-amino acid derivative, using a D-stereospecific hydrolase, The preparation of at least one substrate, the D-stereospecific hydrolase, and at least one solvent, A reaction mixture is obtained by mixing the at least one substrate p, the D-stereospecific hydrolase, and the at least one solvent. In the reaction mixture, the D-stereospecific hydrolase is used to hydrolyze the functional group protected by at least one D-amino acid and / or D-amino acid derivative of the at least one substrate to obtain a D-amino acid and / or D-amino acid derivative and at least a partially deprotected substrate having at least one deprotected functional group deprotected from the D-amino acid and / or D-amino acid derivative. Methods that include...

2. The method according to claim 1, wherein the at least one substrate is selected from the group consisting of carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, and mixtures thereof, and in particular the substrate has at least two functional groups.

3. The method according to claim 1 or 2, further comprising adding a buffer to the reaction mixture, wherein the pH value of the reaction mixture is in the range of 3 to 13.

4. The method according to any one of claims 1 to 3, wherein the functional group is selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, a sulfhydryl group, and a selenohydryl group.

5. The method according to claim 4, wherein the functional group is an amino group, a hydroxyl group, a sulfhydryl group, and / or a selenohydryl group, and the D-amino acid and / or D-amino acid derivative is bonded to the substrate via a carbonyl group.

6. The method according to any one of claims 1 to 5, wherein the concentration of the substrate in the reaction mixture is in the range of 100 nM to 1 M based on the reaction mixture.

7. The method according to any one of claims 1 to 6, wherein the concentration of the D-stereospecific hydrolase in the reaction mixture is in the range of 1 nM to 200 μM based on the reaction mixture.

8. The method according to any one of claims 1 to 7, wherein the at least one solvent includes at least water.

9. The buffers mentioned above include acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium phosphate (NaPi) buffer, potassium phosphate (KPi) buffer, 2-(4-(2-hydroxyethyl)-piperazine-1-yl)-ethane-1-sulfonic acid (HEPES) buffer, tris-(hydroxymethyl)-aminomethane (Tris) buffer, piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES) buffer, and bis-(2-hydroxyethyl)-imino-tris-(hydroxymethyl)-methane (Bis-Tris). Buffers, 1,3-bis-(tris-(hydroxymethyl)-methylamino)-propane (Bis-Trispropane) buffer, N-(2-acetamide)iminodiacetic acid (ADA) buffer, N-(2-acetamide)-2-aminoethanesulfonic acid (ACES) buffer, acetamidoglycine buffer, 2-hydroxy-3-morpholinopropanesulfonic acid (MOPSO) buffer, N,N-bis-(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES) buffer, 3-(N-morpholino)-propanesulfonic acid (MOPS) buffer, 2 - ((1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl)-amino)ethane-1-sulfonic acid (TES) buffer, cholamine chloride hydrochloride, 4-(N-morpholino)-butanesulfonic acid (MOBS) buffer, 3-N-bis-(hydroxyethyl)-amino-2-hydroxy-propanesulfonic acid (DIPSO) buffer, 3-((1,3-dihydroxy-2-(hydroxymethyl)-propan-2-yl)-amino)-2-hydroxypropane-1-sulfonic acid (TAPSO) buffer, triethanolamine buffer, 4-( 2-Hydroxyethyl)-piperazine-1-(2-hydroxypropanesulfonic acid) (HEPPSO) buffer, piperazine-N,N'-bis-(2-hydroxypropanesulfonic acid) (POPSO) buffer, N-(tri(hydroxymethyl)-methyl)-glycine (tricine) buffer, glycylglycine buffer, glycinamide buffer, (bis(2-hydroxyethyl)-amino)acetic acid (BICINE) buffer, N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid (TAPS) buffer, 2-amino-2-methyl-1,The method according to claim 3, selected from the group consisting of 3-propanediol (AMPD) buffer, N-tris(hydroxymethyl)-methyl-4-aminobutanesulfonic acid (TABS) buffer, N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid (AMPSO) buffer, N-cyclohexyl-2-aminoethanesulfonic acid (CHES) buffer, 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid (CAPSO) buffer, (2-amino-2-methylpropane-1-ol) (AMP) buffer, and 3-(cyclohexylamino)propane-1-sulfonic acid (CAPS) buffer.

10. The method according to any one of claims 1 to 9, wherein the hydrolysis is carried out at a reaction temperature in the range of 0°C to 50°C.

11. The following features: At least one first of three amino acids containing the amino acid sequence SXXK １０ - Helix-2 structure, At least one first α-helix structure containing the amino acid sequence YSN, and At least one first β-sheet structure containing the amino acid sequence HXG, (Here, X is an amino acid selected from the group consisting of proteinogenic amino acids) characterized by the first 3 １０ A D-stereospecific hydrolase wherein the -helix-2 structure, the first α-helix structure, and the first β-sheet structure are arranged in close proximity.

12. The D-stereospecific hydrolase is an amino acid sequence selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9: Sequence ID 1: ASLQLSQTKELLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGGVLLRKGYGFAGTNKLNRPDSKTRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLQSEFTRSGDVTIEQTIEELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLSGISYSDYVSEHFLTPLGMKNSGTATPATPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDTALRAGKVVSEQSLEAMYTPHSDKNYGYGWIALGLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 2: TIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNHPDAKSRIASLTKSFTAASIMQLVEQGKLSLTDPSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGNVTIEQTIAELRTKQLKYEPGTTYLYSNNGYVLLAYVL EQLSGESYADYVSEHFLTPLGMKNSGTATPATATIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSKQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 3: ASLQLSQTKEFLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNRPDAKSRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGDVTIEQTIAELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLAGESYADYVNEHFLTPLGMKNSGTATPESPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSEQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 4: SSVQTSTQRDRNSVKQAVRDTLQLGFPGILAKTSEGGKTWSYAAGVANLSSKKPMKTDFRFRIGSVTKTFTATVVLQLAEENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIAEYTRSKSFDLMDTKKSYRAEELVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE TVTGNSYAEIEIENRIIEPLELSNTFLPGNSSVIPGTKHARGYIQLDGASEPKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGEAALGRYGLGIYETKLPNGVSIWGHGGSIPGFVTFAGGTLGGKHTLAVNLNSLNAESPDPFKNILLAEFSK; Sequence ID 5: SSLQTSTQSDRTSVKKAMRDELQLGYPGILAKISKGGKTWSYTAGVADLKTKKPMKADFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYVNSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQINGASELKDVTYINPGSSDGDMISTADDLNKFFSCLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGAVPGFSTFAGGTLGGKHTLAINSNSLNLNNPEVFKNILLAEFRK; Sequence ID 6: SSLQTSTQSDRTSVKKAIRDELQLGYPGILAQISKGGKTWSYTAGIADLRTKKPMKADFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYINSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQLDGASELKDVTYINPGSSDGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGGVLGFSTFAGGTLGGKHTLAINSNSFNINNPESFKNVLIAEFSK; Sequence ID 7: NSLQTSTQRDRNSVKEAMRDTLKLGYPGILAKTSEGGKTWSYAAGVADLSNKKAMKTDFRFRIGSVTKTFTATVVLQLAGENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIANYTRSKDFNMMDTKKSYTAEGLVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE KVTGNSYAEEIENRIIEPLELANTFLPGNSSVIPGTKHARGYIQLDGASETKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGVGELGDSGLGIFKMKLSNGESIWGHGGTIPGFLTFAGGTLGGKHTLAVNLNSLKADTPDPFKNILLAEFSK; Sequence ID 8: SSLQTNTQRDRTSVKQAMRDTLQLGYPGILAKTSEDGKTWGYAAGIADLRTKKPMKTDFRFRIGSVTKTFTATVVLQLVGENRLKLDDYIEKWLPGVIQGNGYDGNKITIREILNHTSGIAEYSRSKDVDFTDTKKSYTAEELVKMGISLPPDFAPGKGWSYSNTGYVLLGILIE KVTGNSYAEEVENRIIEPLELSNTFLPGNSSVIPGTNHARGYVQPDGASELKDVTYYNPSAGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGKEGIDGYGLGIYETKLPSGVSIWGHSGGILGFTTLVGGKLGGKHTLVVNWNSLGRTGSPNPFKNILLAEFSK; Sequence ID 9: NSSQEPNQKNRNGWKQVMQETIQIGAPGVLAKTSNKGKVNSYTAGVADLITKKPVKSDFRFRIGSVTKTFTATTVLQLVGENRVQLDDPIEKWLPGLVQGNGYDGNQITIRQLLNHTSGIAEYLKSKDADVMNSKKTYTAEEIVKIGLSLPPDFSPGKDWLYSNTGYVILGMLIE KITGNNYAEEIEKRIIEPLDLPNTFLPGNSPVIPGKNHARGYVKMEETGELKDITYYNPSLANAAGDMISNADDDLNKFFSSLLGGKLLKERELKEMLTTVPVEGKGVGDGYGLGIYETKLPNGVSVWGHGGSIPGFMTFAGGVIGGKHTFAVNVNSLGPVDILTQFDKMMQVEFNK The D-stereospecific hydrolase according to claim 11, having the properties of the D-stereospecific hydrolase described in claim 11.

13. The use of a D-stereospecific hydrolase for deprotecting a functional group of a substrate protected with at least one D-amino acid and / or a D-amino acid derivative, wherein the D-stereospecific hydrolase has the following characteristics: At least one first of three amino acids containing the amino acid sequence SXXK １０ - Helix-2 structure, At least one first α-helix structure containing the amino acid sequence YSN, and At least one first β-sheet structure containing the amino acid sequence HXG, (Here, X is an amino acid selected from the group consisting of proteinogenic amino acids) characterized by the first 3 １０ The -helix-2 structure, the first α-helix structure, and the first β-sheet structure are arranged in close proximity, and more preferably, the D-stereospecific hydrolase has an amino acid sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, and 9: Sequence ID 1: ASLQLSQTKELLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGGVLLRKGYGFAGTNKLNRPDSKTRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLQSEFTRSGDVTIEQTIEELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLSGISYSDYVSEHFLTPLGMKNSGTATPATPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDTALRAGKVVSEQSLEAMYTPHSDKNYGYGWIALGLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 2: TIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNHPDAKSRIASLTKSFTAASIMQLVEQGKLSLTDPSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGNVTIEQTIAELRTKQLKYEPGTTYLYSNNGYVLLAYVL EQLSGESYADYVSEHFLTPLGMKNSGTATPATATIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSKQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 3: ASLQLSQTKEFLTIRIQTDAVTKYGKEDAAIDAYLKGEGFSGMALVAKDGEVLLRKGYGFSGTNKLNRPDAKSRIASITKSFTAASIMQLVEQGKLSLTDPVSKFVTGIPRGDDITIHMLLSHTSGLPSEFTRSGDVTIEQTIAELRTKQLKYEPGTTYLYSNNGYV LLAYVLEQLAGESYADYVNEHFLTPLGMKNSGTATPESPTIQGYILQKNNEWAAAPYYVSQSGTGTLYSTVDDLLKWDQALRAGKVVSEQSLEAMYTPHSDKNYGYGWIAINLNGEKGVFHNGSGSGYATGMLRNLDSGMTVILLGNHAGMDMTKLLQQVHKLAAEQ; Sequence ID 4: SSVQTSTQRDRNSVKQAVRDTLQLGFPGILAKTSEGGKTWSYAAGVANLSSKKPMKTDFRFRIGSVTKTFTATVVLQLAEENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIAEYTRSKSFDLMDTKKSYRAEELVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE TVTGNSYAEIEIENRIIEPLELSNTFLPGNSSVIPGTKHARGYIQLDGASEPKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGEAALGRYGLGIYETKLPNGVSIWGHGGSIPGFVTFAGGTLGGKHTLAVNLNSLNAESPDPFKNILLAEFSK; Sequence ID 5: SSLQTSTQSDRTSVKKAMRDELQLGYPGILAKISKGGKTWSYTAGVADLKTKKPMKADFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYVNSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQINGASELKDVTYINPGSSDGDMISTADDLNKFFSCLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGAVPGFSTFAGGTLGGKHTLAINSNSLNLNNPEVFKNILLAEFRK; Sequence ID 6: SSLQTSTQSDRTSVKKAIRDELQLGYPGILAQISKGGKTWSYTAGIADLRTKKPMKADFRIGSVTKTFIATVLLQLSGENRLNLDDSIEKWLPGVIQGNGYDGNQITIRQILNHTSGIADYINSKDFDIMDTKKSYTAEEFVKMGISLPPDFAPGKGWSYSNTGYVLLGIL IEKVTGNSYAEEVENRIIEPLDLSNTFLPGNSSVIPGTKHARGYLQLDGASELKDVTYINPGSSDGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTNREGTGYGLGILEIKLPNGVSVWGHRGGVLGFSTFAGGTLGGKHTLAINSNSFNINNPESFKNVLIAEFSK; Sequence ID 7: NSLQTSTQRDRNSVKEAMRDTLKLGYPGILAKTSEGGKTWSYAAGVADLSNKKAMKTDFRFRIGSVTKTFTATVVLQLAGENRLNLDDSIEKWLPGVIQGNGYDDKQITIRQLLNHTSGIANYTRSKDFNMMDTKKSYTAEGLVKMGISMPPDFAPGKSWSYSNTGYVLLGILIE KVTGNSYAEEIENRIIEPLELANTFLPGNSSVIPGTKHARGYIQLDGASETKDVTYYNPSMGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGVGELGDSGLGIFKMKLSNGESIWGHGGTIPGFLTFAGGTLGGKHTLAVNLNSLKADTPDPFKNILLAEFSK; Sequence ID 8: SSLQTNTQRDRTSVKQAMRDTLQLGYPGILAKTSEDGKTWGYAAGIADLRTKKPMKTDFRFRIGSVTKTFTATVVLQLVGENRLKLDDYIEKWLPGVIQGNGYDGNKITIREILNHTSGIAEYSRSKDVDFTDTKKSYTAEELVKMGISLPPDFAPGKGWSYSNTGYVLLGILIE KVTGNSYAEEVENRIIEPLELSNTFLPGNSSVIPGTNHARGYVQPDGASELKDVTYYNPSAGSSAGDMISTADDLNKFFSYLLGGKLLKEQQLKQMLTTVPTGKEGIDGYGLGIYETKLPSGVSIWGHSGGILGFTTLVGGKLGGKHTLVVNWNSLGRTGSPNPFKNILLAEFSK; Sequence ID 9: NSSQEPNQKNRNGWKQVMQETIQIGAPGVLAKTSNKGKVNSYTAGVADLITKKPVKSDFRFRIGSVTKTFTATTVLQLVGENRVQLDDPIEKWLPGLVQGNGYDGNQITIRQLLNHTSGIAEYLKSKDADVMNSKKTYTAEEIVKIGLSLPPDFSPGKDWLYSNTGYVILGMLIE KITGNNYAEEIEKRIIEPLDLPNTFLPGNSPVIPGKNHARGYVKMEETGELKDITYYNPSLANAAGDMISNADDDLNKFFSSLLGGKLLKERELKEMLTTVPVEGKGVGDGYGLGIYETKLPNGVSVWGHGGSIPGFMTFAGGVIGGKHTFAVNVNSLGPVDILTQFDKMMQVEFNK To have, to use.

14. A method for producing carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, The carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivative precursors, or mixtures thereof, At least one D-amino acid and / or a D-amino acid derivative, At least one first solvent, Optionally, at least one second solvent, Optionally, at least one third solvent, D-stereospecific hydrolase, and Prepare at least one type of first reagent, A first reaction mixture is obtained by mixing the carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative precursor, or mixture thereof, the at least one first solvent, and the at least one D-amino acid and / or D-amino acid derivative. The method involves reacting the aforementioned carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof with at least one D-amino acid and / or a D-amino acid derivative to obtain protected precursors of the aforementioned carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof. Optionally, remove the at least one first solvent from the first reaction mixture, Optionally, the protected precursors of the carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof may be reacted with one or more further reagents in one or more further reaction steps to obtain the reacted protected precursors of the carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof. The process involves mixing the protected precursors of the carbohydrates, carbohydrate derivatives, nucleotides, oligonucleotides, polynucleotides, nucleotide derivatives, oligonucleotide derivatives, polynucleotide derivatives, amino acids, peptides, oligopeptides, polypeptides, proteins, peptide intermediates, peptide derivatives, oligopeptide derivatives, polypeptide derivatives, or mixtures thereof, with at least one first reagent and optionally at least one second solvent to obtain a second reaction mixture. The protected precursor of the carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof, or the reacted protected precursor of the carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof, and the at least one first reagent to obtain a protected carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof, Optionally, remove the at least one second solvent from the second reaction mixture, The protected carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof, optionally the at least one third solvent, and the D-stereospecific hydrolase are mixed to obtain a third reaction mixture. Deprotecting at least one D-amino acid and / or D-amino acid derivative from the protected carbohydrate, carbohydrate derivative, nucleotide, oligonucleotide, polynucleotide, nucleotide derivative, oligonucleotide derivative, polynucleotide derivative, amino acid, peptide, oligopeptide, polypeptide, protein, peptide intermediate, peptide derivative, oligopeptide derivative, polypeptide derivative, or mixture thereof to obtain the carbohydrate, the carbohydrate derivative, the nucleotide, the oligonucleotide, the polynucleotide, the nucleotide derivative, the oligonucleotide derivative, the polynucleotide derivative, the amino acid, the peptide, the oligopeptide, the polypeptide, the protein, the peptide intermediate, the peptide derivative, the oligopeptide derivative, the polypeptide derivative, or mixture thereof, Methods that include...

15. Use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of substrates.

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