Method for deprotection of at least one D-amino acid and / or D-amino acid derivative protected functional group

Abstract

The present invention relates to a method for deprotecting at least one D-amino acid and / or D-amino acid derivative protected function of at least one substrate having at least one D-amino acid and / or D-amino acid derivative protected function, to a D-stereospecific hydrolase enzyme, and to the use of the D-stereospecific hydrolase enzyme. Furthermore, the present invention relates to a process for the preparation of a compound comprising a deprotection process and the use of a D-amino acid and / or a D-amino acid derivative as a protecting group for a substrate function.

Description

TECHNICAL FIELD

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

[0002] The present invention also allows for a new protecting group strategy and the selective cleavage of affinity- or solubility-mediated functionalities in organic synthesis. BACKGROUND

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

[0004] On the other hand, unprotected functional groups with comparable or similar reactivity can lead to undesired side reactions, which in turn inevitably lead to a loss of yield and the formation of heterogeneous product mixtures, which often require expensive purification steps after the reaction has taken place.

[0005] The established chemical processes for protecting, in particular deprotecting, such functional groups often require reaction conditions that cannot guarantee the integrity of the reactants and / or products, and are therefore particularly (but not exclusively) unsuitable for unstable target structures. In addition, highly orthogonal protecting group strategies are required for multi-step syntheses, otherwise undesired side products accumulate at each individual reaction step, which prevents high synthetic yields even in a small number of reaction steps.

[0006] One deprotection strategy that has been widely adopted involves the use of L-amino acids as protecting agents, which can be cleaved enzymatically. However, due to the widespread use of L-amino acids in organic synthesis, the selectivity of the deprotection, i.e. the cleavage of the L-amino acid, is not high, which leads to the problems described above.

[0007] E. Barbayianni et al. (E. Barbayianni, I. Fotakopoulou, M. Schmidt, V. Constantinou-Kokotou, U.T. 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, .1021 / jo051004v) describe the use of hydrolytic enzymes, such as the esterase BS2 from Bacillus subtilis, and various lipases, such as CAL-A from Candida antarctica, for the selective hydrolysis of ester bonds used as protecting groups for carboxylic acids and hydroxyl groups. Similarly, F. Bellezza et al. (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, .1039 / B005512N) describe the use of this strategy with lipases such as CAL-A from Candida rugosa. Furthermore, H. Waldmann et al. (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, .1002 / ange.19971090620) discloses that the amino function can be masked by phenylacetic acid, the phenylacetic acid amide formed can be selectively recleaved again by penicillin G acylase.

[0008] Recent studies by M. Reille-Seroussi et al. (M. Reille-Seroussi, S. V. Mayer, W. Dörner, K. Lang, H. D. Mootz, “Expanding the genetic code with a lysine derivative bearing an enzymatically removable phenylacetyl group”, Chemical Communications, 2019, 55, pp. 4793-4796, .1039 / C9CC00475K) show that these phenylacetyl functions can also be selectively removed from the lysine side chain by selected sirtuins. Furthermore, A. G. Gum et al. (A. G. 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, .1002 / 1521-3765(20001016)6:20<3714::AID-CHEM3714>3.0.CO;2-Z) describe glucose- and galactose-based urethane protecting groups, which can be removed from selected functions by using glycosidases.

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

[0010] For example, penicillin G acylase for deprotection of the amino function exhibits a very high K MThe value, thus, it has a low specific activity for phenylacetic acid amides. In addition, the cleavage product leads to a very pronounced inhibition of the biocatalyst, which is accompanied by a loss of yield and a prolongation of the conversion, as investigated by W.B.L. Alkema et al. (W.B.L. Alkema, R. Floris, D.B. Janssen, “The Use of Chromogenic Reference Substrates for the Kinetic Analysis of Penicillin Acylases”, Analytical Biochemistry, 1999, 275, 1, pp. 47-53, : / / doi.org / 10.1006 / abio.1999.4300). For these reasons, this enzyme is not used for the preparation of the synthesis method.

[0011] The use of ethyl carbamate protecting groups based on glucose and galactose and their cleavage by glycosidases has also not been applied on a preparative scale. The sugar building blocks glucose and galactose contain reactive hydroxyl groups, which must be fully protected during the synthesis process due to their chemical reactivity. However, these protected sugar derivatives are not recognized by glycosidases, which requires an additional reaction step, i.e. the removal of the protecting group from the actual protecting group. This is again achieved by the use of lipases. This leads to a very complex process and, therefore, has not found any application in preparative synthesis.

[0012] Therefore, there is a need for new, highly selective deprotection strategies for protected functional groups, which require mild reaction conditions and are compatible with all reactants and chemical structures present in the preparative synthesis method for the preparation of the compound. SUMMARY

[0013] The present invention makes use of D-stereospecific hydrolases, which selectively recognize D-amino acids and / or derivatives thereof (but not the known L-amino acids) and hydrolytically cleave them from the target structure, e.g. as protecting groups or functional tags. Since D-amino acids and / or D-amino acid derivatives are, in principle, not distinguishable from their enantiomers, the L-amino acids, in terms of their reactivity under the achiral synthesis conditions of classical peptide chemistry, they are compatible with established synthesis strategies and, like natural L-amino acids, can be easily introduced into the target structure. The D-stereospecific hydrolases used in the present invention have a strict regioselectivity, chemoselectivity and enantioselectivity for the respective recognized D-amino acid and / or D-amino acid derivative. The cleavage occurs rapidly and quantitatively under mild reaction conditions without side reactions and is orthogonal to all previously established deprotection and synthesis methods.

[0014] The present invention relates to the protection of chemically reactive functional groups, such as amino, hydroxyl, carboxylic acid, thiol and selenohydryl functional groups, using D-amino acids and / or D-amino acid derivatives and their deprotection using D-stereospecific hydrolases. An important aspect of the present invention is the highly selective deprotection of D-amino acid and / or D-amino acid derivative protected functional groups by D-stereospecific hydrolases, which have substrate specificity for these D-amino acids and / or D-amino acid derivatives used as protecting groups. Since the deprotection reaction is enzyme catalyzed and subsequently occurs under mild reaction conditions, the method can be fully applied even in the case of chemically unstable substrates and target structures. Furthermore, the present invention is orthogonal to all established protecting group strategies, for example in peptide and carbohydrate chemistry, and expands the existing method repertoire so that complex multi-step syntheses or sequential reaction sequences can also be realized, for example when they are required in preparative synthesis methods.

[0015] Furthermore, the present invention not only allows the selective deprotection of reactive functional groups in substrates or products, but also cuts any functional groups from them in the same way, for example functional groups with solubility-mediated properties, or even affinity probes connected to target structures by D-amino acids and / or D-amino acid derivatives recognized by the specificity of the hydrolase.

[0016] In a first aspect, the present invention relates to a method for deprotecting at least one D-amino acid and / or D-amino acid derivative protected functional group of at least one substrate having at least one D-amino acid and / or D-amino acid derivative protected functional group using a D-stereospecific hydrolase, the method comprising: providing the at least one substrate, the D-stereospecific hydrolase and at least one solvent; mixing the at least one substrate, the D-stereospecific hydrolase and the at least one solvent to obtain a reaction mixture; and hydrolyzing the at least one D-amino acid and / or D-amino acid derivative protected functional group of the at least one substrate using the D-stereospecific hydrolase in the reaction mixture to obtain a D-amino acid and / or D-amino acid derivative and an at least partially deprotected substrate, wherein at least one deprotected functional group is deprotected from the D-amino acid and / or D-amino acid derivative.

[0017] In a second aspect, the present invention relates to a D-stereospecific hydrolase having the following features: at least one first 3 10 a helix-2 structure comprising the amino acid sequence SXXK; at least one first alpha-helix structure comprising the amino acid sequence YSN; and at least one first beta-sheet structure comprising the amino acid sequence HXG; wherein X is an amino acid selected from proteinogenic amino acids; wherein the first 3 10 - the helix-2 structure, the first alpha-helix structure and the first beta-sheet structure are closely arranged.

[0018] In a third aspect, the present application relates to the use of a D-stereospecific hydrolase for the deprotection of at least one D-amino acid and / or a functional group of a D-amino acid derivative of a substrate, preferably wherein the D-stereospecific hydrolase has the following features: at least one first 3 10 - the helix-2 structure comprising the amino acid sequence SXXK; at least one first alpha-helix structure comprising the amino acid sequence YSN; and at least one first beta-sheet structure comprising the amino acid sequence HXG; wherein X is an amino acid selected from proteinogenic amino acids; wherein the first 3 10 - the helix-2 structure, the first alpha-helix structure and the first beta-sheet structure are closely arranged, further preferably a D-stereospecific hydrolase having an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9.

[0019] In a fourth aspect, the present application provides a method of preparing 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, the method comprising: providing a precursor of 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, 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, a D-stereospecific hydrolase, and at least one first reagent; a 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, at least one first solvent, and at least one D-amino acid and / or D-amino acid derivative, to obtain a first reaction mixture; reacting the 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 with at least one D-amino acid and / or D-amino acid derivative, to obtain 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; optionally removing at least one first solvent from the first reaction mixture; optionally, in one or more further reaction steps, reacting the 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 with one or more further reagents, 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; mixing the 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, or the 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, at least one first reagent, and optionally at least one second solvent, to obtain a second reaction mixture; a precursor of 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, or a precursor of a reaction of 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, is reacted with 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 removing at least one second solvent from the second reaction mixture; mixing 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 at least one third solvent, and the D-stereospecific hydrolytic enzyme, to obtain a third reaction mixture; and 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; 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.

[0020] In a fifth aspect, the present invention relates to the use of a D-amino acid and / or D-amino acid derivative as a protecting group for a substrate functional group.

[0021] Further aspects and embodiments of the present invention are disclosed in the appended claims and can be derived from the following description, figures and examples, without limitation thereto. BRIEF DESCRIPTION OF DRAWINGS

[0022] The accompanying drawings are intended to describe embodiments of the present application and to provide a further understanding of the present application. Together with the description, they serve to explain the concepts and principles of the present application. Other embodiments and numerous

[0023] Figure 1-21 Results obtained in embodiments of the present application are shown. DETAILED DESCRIPTION

[0024] Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this application belongs.

[0025] Unless explicitly defined otherwise, or the context clearly dictates otherwise, all ranges disclosed herein are to be understood as being supplemented by the term "about." As used herein, the qualifier "about" or "approximately," and combinations and variations thereof, include not only the recited exact amount or value, but also amounts or values that are slightly different, which can result from, for example, manufacturing tolerances, measurement errors, wear, stresses applied to individual components, and combinations thereof.

[0026] As used herein, the term "substantially" means that the parameter, event, or condition described immediately thereafter occurs either entirely or in a great degree or to a significant extent. For example, the term "substantially" means that the parameter, event, or condition described immediately thereafter occurs 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% of the time, or means that a dimension or measurement is within 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 a reference dimension or measurement.

[0027] The use of the term "at least one" or "one or more" should be taken as including one as well as more than one. Additionally, the use of the phrase "at least one of X, Y and Z" should be interpreted as meaning X alone, Y alone, Z alone, as well as any combination of X, Y and Z.

[0028] The use of ordinal terms (i.e., "first," "second," "third," "fourth," etc.) is merely to distinguish two or more items, and does not imply a sequence or order or importance or any added significance to any item over another unless explicitly defined otherwise.

[0029] Unless explicitly defined to the contrary or context clearly dictates otherwise, all numbers or percentages in this application that relate to amounts are given in terms of weight (wt.%).

[0030] In this application, an enzyme is a protein that acts as a biological catalyst by accelerating a chemical reaction. The molecule on which an enzyme can act is called a substrate.

[0031] A carbohydrate, also called a sugar, is a biochemical compound whose structure includes carbon (C), hydrogen (H), and oxygen (O) atoms. Sugars include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. A disaccharide is a sugar formed when two monosaccharides are linked by a glycosidic bond. An oligosaccharide is a sugar formed when a small number (typically 3-10) of monosaccharides are linked by glycosidic bonds. Oligosaccharides can have linear or branched structures. A polysaccharide is a sugar containing a large number (at least 11) of monosaccharides linked by glycosidic bonds. Polysaccharides can have linear or branched structures.

[0032] A nucleotide includes a nucleoside and at least one phosphate. A nucleoside includes a nucleobase and a monosugar. The nucleobase can be, for example, selected from adenine, cytosine, guanine, thymine, uracil, and modifications thereof. According to certain embodiments, the monosugar can be selected from ribose and deoxyribose. If more than one phosphate is present, they are typically arranged in order, linked by P-O-P- bonds. The nucleotide is not particularly limited. According to certain embodiments, the nucleotide can be selected from adenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidine monophosphate (CMP), uridine monophosphate (UMP), and deoxythymidine monophosphate (dTMP).

[0033] An oligonucleotide is an oligomer formed from a small number (typically about 2-12) of nucleotides. The nucleotides are linked by phosphodiester bonds, particularly 3',5'-phosphodiester bonds. Oligonucleotides typically have linear structures. The nucleotides forming the oligonucleotide are not particularly limited and can be as described above.

[0034] A polynucleotide is an oligomer formed from a large number (at least 13) of nucleotides. The nucleotides are linked by phosphodiester bonds, particularly 3',5'-phosphodiester bonds. Polynucleotides typically have linear structures. The nucleotides are not particularly limited and can be as described above.

[0035] An amino acid is a compound having an amino group and a carboxyl group.

[0036] A peptide is a short chain of at least 2 amino acids linked by peptide bonds. An oligopeptide is a short chain of 2-10 amino acids linked by peptide bonds. A polypeptide is a long chain of 11-100 amino acids linked by peptide bonds. A protein is a long chain of at least 101 amino acids linked by peptide bonds.

[0037] Alpha-helices are common motifs in the secondary structure of proteins, enzymes, and the like, and are a right-handed helical conformation in which each backbone N-H group hydrogen bonds with the backbone C=0 group of an amino acid four residues before it in the protein sequence.

[0038] 3 10 A helix is a structure in which amino acids are arranged in a right-handed helix. Each amino acid corresponds to a 120° turn in the helix (i.e., there are three residues per turn of the helix, with the N-H group of the amino acid hydrogen bonding with the C=0 group of the amino acid three residues before it.

[0039] Beta-sheets are common motifs in the secondary structure of proteins, enzymes, and the like, and are composed of beta-strands (beta-chains) connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet fold. A beta-chain is a stretch of polypeptide chain, usually 3-10 amino acids in length, with the backbone in an extended conformation.

[0040] Unless otherwise indicated, units M in the present disclosure refer to mol / 1.

[0041] Before exemplary detailed description of the present application is made, it is to be understood that the application is not limited to the specific components of the process steps of the methods described herein, as such methods can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. For example, the term "an" as used herein can be understood to mean a single entity or "one or more" entities. It will also be understood that the plural form includes the singular and / or plural referents, unless the context clearly dictates otherwise. Moreover, it will be understood that where given ranges by numerical limits are specified, the range is to be construed to include the limiting values.

[0042] As used herein, the terms "comprises", "comprising", "includes", "including", "has", "having" or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a process, method, article or apparatus that comprises a list of elements is not necessarily limited to only those elements but can include other elements not expressly listed or inherent to such process, method, article or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to an exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0043] In a first aspect, the present application provides a method for deprotection of at least one D-amino acid and / or D-amino acid derivative protected functional group of at least one substrate having at least one D-amino acid and / or D-amino acid derivative protected functional group using a D-stereospecific hydrolase, the method comprising: providing the at least one substrate, the D-stereospecific hydrolase and at least one solvent; mixing the at least one substrate, the D-stereospecific hydrolase and the at least one solvent to obtain a reaction mixture; and hydrolyzing the at least one D-amino acid and / or D-amino acid derivative protected functional group of the at least one substrate using the D-stereospecific hydrolase in the reaction mixture to obtain a D-amino acid and / or D-amino acid derivative and at least partially deprotected substrate, wherein at least one deprotected functional group is deprotected from the D-amino acid and / or D-amino acid.

[0044] In the method of the first aspect, the step of providing the different components, i.e. the at least one substrate, the D-stereospecific hydrolase and the at least one solvent, is not particularly limited. For example, the components can be provided prior to the provision of the different components with or without performing at least one further step, e.g. a synthesis step. Such further step can for example be selected from a chemical reaction - e.g. in a chemical synthesis, removing and / or isolating at least one component, and / or adding at least one component.

[0045] The step of mixing the different components is not particularly limited. The order in which the various components can be added is not particularly limited. For example, the at least one substrate and the at least one solvent can be added and mixed first, followed by the addition and mixing of the D-stereospecific hydrolase. However, this order can be changed as desired.

[0046] The mixing is not particularly limited and any known method of mixing can be used. The mixing can be performed in a suitable reaction vessel, which can also be the reaction vessel in which the hydrolysis is performed. The reaction vessel is not particularly limited and any known reaction vessel can be used as long as it allows the hydrolysis reaction to take place.

[0047] Hydrolysis is any chemical reaction in which a molecule of water ruptures one or more chemical bonds. Hydrolysis can be a substitution, elimination, and solvation reaction in which water is the nucleophile. In the method of the first aspect, the hydrolysis is catalyzed by the D-stereospecific hydrolase.

[0048] Here, the D-stereohydrolase can catalyze the hydrolysis of a bond between a D-amino acid and / or a D-amino acid derivative and at least one functional group of a substrate protected by the D-amino acid and / or the D-amino acid derivative. Thus, a water molecule can be introduced, which can be provided, for example, by at least one solvent. The water molecule can react with at least one D-amino acid and / or a functional group of the substrate protected by the D-amino acid and / or the D-amino acid derivative to obtain a D-amino acid and / or a D-amino acid derivative and a substrate at least partially deprotected (or, in case more than one substrate is provided, more than one substrate at least partially deprotected), wherein at least one deprotected functional group is deprotected from the D-amino acid and / or the D-amino acid derivative.

[0049] The D-amino acid is not particularly limited, as long as the D-amino acid comprises at least one carboxyl group, at least one amino group, and has a D-configuration. According to certain embodiments, the D-amino acid is selected from the group consisting of 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), D-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-gamma glutamic 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-azido-lysine, and thiooxo amino acids generated therefrom.

[0050] According to certain embodiments, 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 certain embodiments, 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 certain embodiments, 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).

[0051] By using one of these D-amino acids, the D-stereospecific hydrolytic enzyme can more effectively recognize the D-amino acid, and the deprotection of the functional group of the D-amino acid-protected substrate can be more effectively performed.

[0052] The D-amino acid derivative can include a D-amino acid having one or more functionalities as follows. The D-amino acid derivative can include a D-amino acid in a salt form, which is not particularly limited. The D-amino acid derivative can also include one or more of post-translational modification, artificial derivatization, and combinations thereof. According to certain embodiments, the one or more functionalities, post-translational modification, and / or artificial derivatization can be present at the N-terminus and / or C-terminus and / or side chain of the D-amino acid.

[0053] In the D-amino acid derivative, the D-amino acid is not particularly limited, as long as the D-amino acid includes at least one carboxyl group, at least one amino group, and has a D-configuration. According to certain embodiments, the D-amino acid can be selected from the group consisting of the above-described D-amino acids. According to certain embodiments, the one or more functionalities, post-translational modification, and / or artificial derivatization can be bonded to the D-amino acid through a bond selected from the group consisting of an ester bond and a carboxamide bond.

[0054] According to certain embodiments, the one or more functionalities can be selected from the group consisting of protecting functionalities (protecting groups), physico-chemical functionalities, analytical / preparative functionalities (e.g. labels), biological / therapeutic / medical functionalities, biochemical functionalities, and combinations thereof. According to certain embodiments, the one or more functionalities can be selected from the group consisting of protecting functionalities, physico-chemical functionalities, and analytical / preparative functionalities. According to certain embodiments, the one or more functionalities, also referred to as blocking functionalities, are derived from compounds which can be selected from the group consisting of allyl carbamates, 1,3-dioxanes, 1,3-dithianes, 1,3-dithiolanes, 1-chloroethyl carbamates, 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl, 2-(trimethylsilyl)-ethoxy-methyl acetal, 2,2,2-trichloroethyl carbonate, 2-alkyl-1,3-oxazolines, 2-methoxyethoxymethyl ether, 2-naphthyl methyl ether, sulfonamides such as 4-methoxybenzenesulfonamide, 4-methoxybenzyl ether, 9-fluorenylmethyl carbamate, acetamides, acetates, acetonides, allyl ethers, benzaldehyde acetal, benzoates, benzyl carbamates, benzyl esters, benzyl ethers, benzyl amines, benzylidene acetal, benzylidene amines, benzyloxy carbamates, benzyloxy methyl acetal, carbamates, carbonates, diethyl acetal, dimethyl acetal, di-tert-butyl dioxasilane, ethoxyethyl acetal, glycol acetal, formamides, methoxy methyl acetal, methoxy methyl amine acetal, methoxy methyl ether, methoxy propyl acetal, methyl carbamates, methyl esters, N-(4-methoxybenzyl)-indole, N,N-dimethyl hydrazones, neopentyl glycol acetal, phthalimides, neopentanoates, propionates, p-toluenesulfonamides, succinates, tert-butyl esters, tert-butyloxy carbamates, tert-butyl carbamates, tert-butyl esters, tert-butyl ethers, tert-butyl dimethyl silyl ethers, tert-butyl diphenyl silyl ethers, tetrahydropyranyl ethers, triethyl silyl ethers, trifluoroacetamides, triisopropyl ether, trimethyl silyl cyanide, trimethyl silyl ethers, triphenylmethyl amines, nitrophenyl carbamates, and any alkylating agents leading to formylation, methylation, ethylation, propylation, etc. According to certain embodiments, the one or more protecting functionalities are protecting groups selected from the group consisting of benzoyl (Bz), fluorenylmethoxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), acetyl (Ac), succinyl (Suc), benzyloxycarbonyl (Z), 2-azidomethylbenzyl (Ambz), 2-aminobenzoic acid (Abz), biotinyl groups, and mixtures thereof. By using D-amino acids with one or more protecting functionalities, the D-amino acids can be modified such that reactions with other components or compounds present in the reaction mixture can be prevented. This allows the use of D-amino acids under a wide range of reaction conditions. Furthermore, this allows the use of chemically less stable D-amino acids which can be modified with one or more of these protecting functionalities.Furthermore, the chemical properties of the substrate with the function protected by the D-amino acid derivative, such as chemical stability, solubility, compatibility with other components of the reaction mixture, and physical properties such as physical stability, boiling point, melting point, etc., can be adjusted.

[0055] According to certain embodiments, the one or more physico-chemical functionalities can be selected from dyes, such as fluorescent inorganic dyes, fluorescent organic dyes, non-fluorescent inorganic dyes, or non-fluorescent organic dyes; and spin probes such as organic free radicals. According to certain embodiments, the one or more physico-chemical functionalities can be selected from nitrophenol, aza-dyes, Alexa Fluor™ dyes, dyes prepared by dinitrobenzylation (dnp), etc. By using D-amino acids with one or more physico-chemical functionalities, the physico-chemical properties of the D-amino acids can be modified. This allows for a more complete characterization of the substrate with the function protected by the D-amino acid derivative. In particular, optical characterization can be simplified.

[0056] According to certain embodiments, the one or more analytical / preparative functionalities can include affinity fusion. According to certain embodiments, the one or more analytical / preparative functionalities can be selected from biotin, desulpho-biotin, Strepp-tag II, His-tag, c-Myc-tag, etc. By using D-amino acids with one or more analytical / preparative functionalities, analytical / preparative applications of the substrate with the function protected by the D-amino acid derivative can be simplified. In particular, characterization of the substrate with various analytical methods such as spectroscopic or chromatographic methods can be simplified. Furthermore, properties of the substrate with the function protected by the D-amino acid derivative can be adjusted in order to, for example, enhance purification of a reaction mixture comprising the substrate.

[0057] According to certain embodiments, the one or more biological / therapeutic / medical functionalities can include toxins targeting key intracellular pathways to induce cell death. According to certain embodiments, the one or more biological / therapeutic / medical functionalities can be selected from calicheamicin, monomethyl aurestatines, emtansine, doxorubicin, SN-38 (7-ethyl-10-hydroxy camptothecin), etc. By using D-amino acids with one or more biological / therapeutic / medical functionalities, biological / therapeutic / medical properties of the substrate with the function protected by the D-amino acid derivative can be enhanced.

[0058] According to certain embodiments, the one or more biochemical functionalities can be selected from propeptides, signal peptides, and the like. A propeptide is an inactive protein (or peptide) that can be converted to an active form through post-translational modification, such as cleaving a portion of the molecule or adding to another molecule. A signal peptide is a short peptide (typically 16-30 amino acids in length) that is present at the N-terminus of most newly synthesized proteins (or occasionally non- canonically at the C-terminus or internally) that enter the secretory pathway. By using D-amino acids with one or more biochemical functionalities, the biochemical properties of the substrate with the D-amino acid derivative-protected functionality can be enhanced. In particular, by using signal peptides as one or more functionalities of the intracellular pathway of the substrate, the protein with the D-amino acid derivative-protected functionality can be affected. Further, by using propeptides as one or more functionalities of the substrate (particularly a protein precursor) with the D-amino acid derivative-protected functionality, the substrate can be inactive and can be activated at a predetermined time through enzymatic activation.

[0059] The one or more of the post-translational modifications can be selected, for example, from (2-aminosuccinimidyl)acetic acid (Asp-Gly), (2S)-4-hydroxyleucine, (3R)-3-hydroxyarginine, (3R)-3-hydroxyasparagine, (3R)-3-hydroxyaspartic acid, (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-hydroxyasparagine, (3S)-3-hydroxyaspartic acid, (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-dehydroaspartic acid, (Z)-2,3-dehydrobutyrine, (Z)-2,3-dehydrotyrosine, 1-(tryptophan-3-yl)-tryptophan (Trp-Trp) (with W-...interchain), 1-amino-2-propanone, 1-histidyl-3-tyrosine (His-Tyr), 1-thioglycine, 2-(3-methylbutanoyl)-5-hydroxyoxazole-4-thiocarbonic acid (Leu-Cys), 2-(4-guanidinobutanoyl)-5-hydroxyimidazole-4-thiocarbonic acid (Arg-Cys), 2-(cysteinyl-S-carbonyl)-3-methyl-4-(glutamyl-5-oxo)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-imidazolone (Gln-Gly), 2-iminomethyl-5-imidazolone (Glu-Gly), 2-iminomethyl-5-imidazolone (Met-Gly), 2-methylglutamine, 2-methylsulfonyltryptophan, 2-oxo-5,5-dimethylhexanoate, 2-oxobutyric acid, 2-tetrahydro-2-pyryl-5-imidazolone (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-hydroxyaspartic acid, 3-hydroxy-D-valine, 3-hydroxyphenylalanine, 3-hydroxyproline, 3-hydroxypyrine-2,5-dicarboxylic acid (Ser-Cys) (containing S-...), 3-hydroxypyrine-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-phenyllactic acid, 3-prenyl-2,N2-cyclotryptophan, 4-(1-hydroxyethyl)-7-isoleucyl-2-(threonine-O3-ylcarbonyl)-7,8-dihydroxyquinoline-8-ol (Ile-Thr), 4,5,5-trihydroxyleucine, 4,5-dihydroxylysine, 4-aspartylphosphate, 4-carboxyglutamic acid, 4-cysteinyl-glutamic acid (Cys-Glu), 4-cysteinyl-tryptophylquinone (Cys-Trp), 4-hydroxyarginine, 4-hydroxyglutamic acid, 4-hydroxylysine, 4-hydroxyproline, 5-(methoxymethyl)thiazole-4-carboxylic acid (Val-Cys), 5-amino-pipereine-2,5-dicarboxylic acid (Ser-Cys) (containing S-...), 5-amino-pipereine-2,5-dicarboxylic acid (Ser-Ser) (containing C-...), 5-chlorotryptophan, 5-glutamyl 2-aminoadipic acid, 5-glutamyl dopamine, 5-glutamyl glutamic acid, 5-glutamyl glycerophosphoethanolamine, 5-glutamyl glycine, 5-glutamyl histamine, 5-glutamyl N2-lysine, 5-glutamyl N2-ornithine, 5-glutamyl norepinephrine, 5-glutamyl polyglutamic acid, 5-glutamyl polyglycine, 5-glutamyl serotonin, 5-hydroxy-3-methylproline (Ile), 5-hydroxylysine, 5-imidazolinone (Ala-Gly), 5-imidazolinone (Asn-Gly), 5-imidazolinone (Cys-Gly), 5-imidazolinone (Lys-Gly), 5-imidazolinone (Ser-Gly), 5-methylarginine, 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-amino tyrosine (Tyr-Tyr) (with Y-...Interchain), 6-(S-cysteine)-8α-(pros-histyl)-FAD (His-Cys), 6-bromotryptophan, 6-chlorotryptophan, 7-hydroxytryptophan, ADP-α-D-ribosylarginine, ADP-ribosylaspartic acid, ADP-ribosylglutamate, ADP-ribosylarginine, ADP-ribosylasparagine, ADP-ribosylcysteine, ADP-ribosyldiphtheria amide, ADP-ribosylglycine, ADP-ribosylhistidine, ADP-ribosylserine, ADP-ribosyltyrosine, ADP-ribosylarginine, alanine amide, alanine derivatives, alanine isaspartic ring Peptide (Ala-Asn), allysine, aminomalonic acid (Ser), arginine amide, arginine derivatives, asparagine amide, aspartic acid 1-(chondroitin 4-sulfate)-ester, aspartic acid 1-[(3-aminopropyl)(5-adenosyl)phosphonyl]amide, aspartic acid 1-amide, aspartic aldehyde, asymmetric dimethylarginine, β-decarboxylated aspartic acid, β-methyllanothionine (Cys-Thr), β-methyllanothionine (Thr-Cys), β-methyllanothionine sulfoxide (sulfoxe) (Thr-Cys), closed amino terminus (Ala), closed amino terminus (Arg), closed amino terminus (Asn), closed amino terminus (Asp), closed amino terminus (Asx), closed amino terminus (Cys), closed amino terminus (Gln), closed amino terminus (Glu), closed amino terminus (Gly), closed amino terminus (Ile), closed amino terminus (Leu), closed amino terminus (Met), closed amino terminus (Pro), closed amino terminus (Ser), closed amino terminus ( Thr), closed amino terminus (Val), closed amino terminus (Xaa), closed carboxyl terminus (Arg), closed carboxyl terminus (His), bromohistidine, cholesterol glycine ester, cis-14-hydroxy-10,13-dioxo-7-heptadecenoic acid aspartate ester, citrulline, C-linked (Man) hydroxytryptophan, C-linked (Man) tryptophan, cyclic [(prolylserine)-O-yl]cysteine, cyclic peptide (Ala-Arg), cyclic peptide (Ala-Ile), cyclic peptide (Ala-Pro), cyclic peptide (Arg-Cys) (with C-... chain), cyclic peptide (Asn-Gly), cyclic peptide (Asp-Asn), cyclic peptide (Cys-Arg) (with R-...Interchain), Cyclic Peptide (Cys-Ile), Cyclic Peptide (Cys-Pro), Cyclic Peptide (Glu-Asn), Cyclic Peptide (Gly-Arg), Cyclic Peptide (Gly-Asn), Cyclic Peptide (Gly-Asp), Cyclic Peptide (Gly-Pro), Cyclic Peptide (His-Asn), Cyclic Peptide (His-Asp), Cyclic Peptide (His-Pro), Cyclic Peptide (Ile-Lys), Cyclic Peptide (Ile-Pro), Cyclic Peptide (Leu-Leu), Cyclic Peptide (Leu-Pro), Cyclic Peptide (Leu-Trp), Cyclic Peptide (Lys-Asp), Cyclic Peptide (Met-Pro), Cyclic Peptide (Phe-Pro), Cyclic Peptide (Pro-Met), Cyclic Peptide (Pro-Tyr), Cyclic Peptide (Ser-Asn), Cyclic Peptide (Ser-Gly), Cyclic Peptide (Ser-Lys), Cyclic Peptide (Ser-Pro), Cyclic Peptide (Trp-Pro), Cyclic Peptide (Tyr-Pro), Cyclic Peptide (Val-Pro), CysO-Cysteine Adduct, Cysteine Amide, Cysteine Derivative, Cysteine Disulfe, Cysteine Methyl Ester, Cysteine Persulfe, Cysteine Sulfenic Acid (-SOH), Cysteine Sulfenic Acid (-SO2H), Cysteine Sulfonic Acid (-SO3H), Cysteinyl-Selenocysteine (Cys-Sec), Cysteinyl-Selenocysteine (Sec-Cys), D-4-Hydroxyvaline, D-Alanine (Ala), D-Alanine (Ser), D-Alloisoleucine, D-Asparagine, Deamated Asparagine, Deamated Glutamine, Decarboxylated Threonine, Deoxyhypusine, Diiodotyrosine, Dimethylated Arginine, Diphospho Serine, Diphospho Threonine, Diphtheramide, 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 Phosphoserine, FMN Phosphothreonine, Glutamate Methyl Ester (Gln), Glutamate Methyl Ester (Glu), Glutamic Acid 1-Amide, Glutamine Amide, Glutamine Derivative, Glycine Amide, Glycine Radical, Glycyl Adenylic Acid, Glycyl Cysteine Disulfide (Cys-Gly) (Interchain with G-...), Glycyl Cysteine Disulfide (Gly-Cys) (Interchain with C-...), Glycyl Cysteine Sulfide (Cys-Gly) (Interchain with G-...), Glycyl Cysteine Sulfide (Gly-Cys) (Interchain with C-...), Glycyl Lysine Isopetide (Gly-Lys) (Interchain with K-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...), Glycyl Lysine Isopetide (Lys-Gly) (Interchain with G-...),Gly-Ser (interchain with S-...), Gly-Ser (interchain with S-...), Gly-Thr (interchain with T-...), Gly-Thr (interchain with G-...), G-anchored amated alanine, G-anchored amated asparagine, G-anchored amated aspartic acid, G-anchored amated carboxy terminus, G-anchored amated cysteine, G-anchored amated glycine, G-anchored amated serine, G-anchored amated threonine, GPI-like-anchored amated alanine, GPI-like-anchored amated asparagine, GPI-like-anchored amated aspartic acid, GPI-like-anchored amated glycine, GPI-like-anchored amated serine, histidine amide, hydroxy arginine, hydroxy proline, hypusine, iodotyrosine, isoadipyl cysteine isopeptide (Cys-Asn), isoadipyl glycine isopeptide (Asn-Gly), isoadipyl glycine isopeptide (Asp-Gly), isoadipyl glycine isopeptide (Gly-Asn), isoadipyl glycine isopeptide (Gly-Asp), isoadipyl lysine isopeptide (Asn-Lys) (interchain with K-...), isoadipyl lysine isopeptide (Lys-Asn), isoadipyl lysine isopeptide (Lys-Asn) (interchain with N-...), isoadipyl lysine isopeptide (Lys-Asp), isodityrosine (Tyr-Tyr), isoglutamyl cysteine thioester (Cys-Gln), isoglutamyl glycine isopeptide (Gly-Glu), isoglutamyl lysine isopeptide (Gln-Lys) (interchain with K-...), isoglutamyl lysine isopeptide (Glu-Lys) (interchain with K-...), isoglutamyl lysine isopeptide (Lys-Gln), isoglutamyl lysine isopeptide (Lys-Gln) (interchain with Q-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...), isoglutamyl lysine isopeptide (Lys-Glu) (interchain with E-...),chain), isoleucine amide, isoleucine derivative, L-allo-isoleucine, lanthionine (Cys-Ser), lanthionine (Ser-Cys), leucine amide, leucine methyl ester, lysine amide, lysine derivative, lysine methyl ester, lysine tyrosylquinone (Lys-Tyr), lysine tyrosylquinone (Tyr-Lys), lysylalanine (Ser-Lys), lysyl-D-alanine (Lys), methionine (R)-sulfoxide, methionine (S)-sulfoxide, methionine amide, methionine derivative, methionine sulfone, methionine sulfoxide, methylhistidine, Murein peptoglycan amated 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-acidetylarginine, N2-succinyltryptophane, N4,N4-dimethylasparagine, N4-methylasparagine, N4-methyl-D-asparagine, N5-[4-(S-L-cysteinyl)-5-methyl-1 H-imidazol-2-yl]-L-ornithine (Arg-Cys) (with C-... interchain), N5-[4-(S-L-cysteinyl)-5-methyl-1 H-imidazol-2-yl]-L-ornithine (Cys-Arg) (with R-...N5-methylarginine, N5-methylglutamine, N6-(2-hydroxyisobutyryl)lysine, N6-(3,6- diaminohexanoyl)-5-hydroxylysine, N6-(ADP-ribosyl)lysine, N6-(beta-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-acetyllysine, N6-biotinyllysine, N6-butyryllysine, N6-carbamoyllysine, N6- carboxylysine, N6-crotonyllysine, N6-formyllysine, N6-glutaryllysine, N6-lacidtoyllysine, N6-lipoyllysine, N6-malonyllysine, N6-methylated lysine, N6-methyllysine, N6- murein peptoglycan lysine, N6-myristoyllysine, N6-palmitoyllysine, N6-poly(beta- hydroxybutyryl)lysine, N6-poly(methylaminopropyl)lysine, N6-propionyllysine, N6- stearoyllysine, N6-succinyllysine, N-acetylalanine, N-acetyl aspartic acid, N-acetyl cysteine, N-acetyl glutamic acid, N-acetyl glycine, N-acetylisoleucine, N-acetylmethionine, N- acetylproline, N-acetylserine, N-acetythreonine, N-acetytyrosine, N-acetyvaline, N-alpha- linked (Rha)arginine, N-beta-linked (GlcNacid)arginine, N-carbamoylalanine, N-D- glucuronoylglycine, N-formylglycine, N-formylmethionine, nitrotyrosine, N-linked (DATDGlc)asparagine, N-linked (GalNacid)asparagine, N-linked (GalNacid...) (glycosaminoglycan)asparagine, N-linked (GalNacid...) asparagine, N-linked (Glc) (glycosylated)arginine, N-linked (Glc) (glycosylated)histidine, N-linked (Glc) (glycosylated)isoleucine, N-linked (Glc) (glycosylated)lysine, N-linked (Glc) (glycosylated)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...) (complex)glutamine, N-linked (GlcNacid...) (complex)histidine, N-linked (GlcNacid...) (complex)isoleucine, N-linked (GlcNacid...) (complex)lysine, N-linked (GlcNacid...) (complex)proline, N-linked (GlcNacid...) (complex)serine, N-linked (GlcNacid...) (complex)threonine, N-linked (GlcNacid...) (complex)tyrosine, N-linked (GlcNacid...) (complex)valine, N-linked (GlcNacid...)arginine, N-linked (GlcNacid...)asparagine, N-linked (GlcNacid...)glutamine, N-linked (GlcNacid...)histidine, N-linked (GlcNacid...)isoleucine, N-linked (GlcNacid...)lysine, N-linked (GlcNacid...)proline, N-linked (GlcNacid...)serine, N-linked (GlcNacid...)threonine, N-linked (GlcNacid...)tyrosine, N-linked (GlcNacid...)valine, N-linked (GlcNacid)arginine, N-linked (GlcNacid)asparagine, N-linked (GlcNacid)glutamine, N-linked (GlcNacid)histidine, N-linked (GlcNacid)isoleucine, N-linked (GlcNacid)lysine, N-linked (GlcNacid)proline, N-linked (GlcNacid)serine, N-linked (GlcNacid)threonine, N-linked (GlcNacid)tyrosine, N-linked (GlcNacid)valine, N-linked (Glc)arginine, N-linked (Glc)asparagine, N-linked (Glc)glutamine, N-linked (Glc)histidine, N-linked (Glc)isoleucine, N-linked (Glc)lysine, N-linked (Glc)proline, N-linked (Glc)serine, N-linked (Glc)threonine, N-linked (Glc)tyrosine, N-linked (Glc)valine, N-linked (Glc...)arginine, N-linked (Glc...)asparagine, N-linked (Glc...)glutamine, N-linked (Glc...)histidine, N-linked (Glc...)isoleucine, N-linked (Glc...)lysine, N-linked (Glc...)proline, N-linked (Glc...)serine, N-linked (Glc...)threonine, N-linked (Glc...)tyrosine, N-linked (Glc...)valine, N-linked (GlcNacid)arginine, N-linked (GlcNacid)asparagine, N-linked (GlcNacid)glutamine, N-linked (GlcNacid)histidine, N-linked (GlcNacid)isoleucine, N-linked (GlcNacid)lysine, N-linked (GlcNacid)proline, N-linked (GlcNacid)serine, N-linked (GlcNacid)threonine, N-linked (GlcNacid)tyrosine, N-linked (GlcNacid)valine, N-linked (Glc...)arginine, N-linked (Glc...)asparagine, N-linked (Glc...)glutamine, N-linked (Glc...)histidine, N-linked (Glc...)isoleucine, N-linked (Glc...)lysine, N-linked (Glc...)proline, N-linked (Glc...)serine, N-linked (Glc...)threonine, N-linked (Glc...)tyrosine, N-linked (Glc...)valine, N-linked (GlcNacid)arginine, N-linked (GlcNacid)asparagine, N-linked (GlcNacid)glutamine, N-linked (GlcNacid)histidine, N-linked (GlcNacid)isoleucine, N-linked (GlcNacid)lysine, N-linked (GlcNacid)proline, N-linked (GlcNacid)serine, N-linked (GlcNacid)threonine, N-linked (GlcNacid)tyrosine, N-linked (GlcNacid)valine, N-linked (Glc...)arginine, N-linked (Glc...)asparagine, N-linked (Glc...)glutamine, N-linked (Glc...)histidine, N-linked (Glc...)isoleucine, N-linked (Glc...)lysine, N-linked (Glc...)proline, N-linked (Glc...)serine, N-linked (Glc...)threonine, N-linked (Glc...)tyrosine, N-linked (Glc...)valine, N-linked (GlcNacid)arginine, N-linked (GlcNacid)asparagine, N-linked (GlcNacid)glutamine, N-linked (GlcNacid)histidine, N-linked (GlcNacid)isoleucine, N-linked (GlcNacid)lysine, N-linked (GlcNacid)proline, N-linked (GlcNacid)serine, N-linked (GlcNacid)threonine, N-linked (GlcNacid)tyrosine, N-linked (GlcNacid)valine, N-linked (Glc...)arginine, N-linked (Glc...)asparagine, N-linked (Glc...)glutamine, N-linked (Glc...)histidine, N-linked (Glc...)isoleucine, N-linked (Glc...)lysine, N-linked (Glc...)proline, N-linked (Glc...)serine, N-linked (Glc...)threonine, N-linked (Glc...)tyrosine, N-linked (Glc...)valine, N-linked (GlcNacid)arginine, N-linked (GlcNacid)asparagine, N-linked (GlcNacid)glutamine, N-linked (GlcNacid)histidine, 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 (GlcNacid...) (keratan sulfate) asparagine, N-linked (GlcNacid...) (paucimannose) arginine, N-linked (GlcNacid...) (paucimannose) asparagine, N-linked (GlcNacid...) (polylacidtosaminoglycan) arginine, N-linked (GlcNacid...) (polylacidtosaminoglycan) asparagine, N-linked (GlcNacid...) 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 (HexNacid...) tryptophan, N-linked (HexNacid...) (complex) arginine, N-linked (HexNacid...) (complex) asparagine, N-linked (HexNacid...) (complex) tryptophan, N-linked (HexNacid...) (complex) serine, N-linked (HexNacid...) (complex) threonine, N-linked (HexNacid...) (complex) tyrosine, N-linked (HexNacid...) (complex) cysteine, N-linked (HexNacid...) (complex) proline, N-linked (HexNacid...) (complex) glutamic acid, N-linked (HexNacid...) (complex) glutamine, N-linked (HexNacid...) (complex) aspartic acid, N-linked (HexNacid...) (complex) asparagine, N-linked (HexNacid...) (complex) arginine, N-linked (HexNacid...) (complex) histidine, N-linked (HexNacid...) (complex) lysine, N-linked (HexNacid...) (complex) isoleucine, N-linked (HexNacid...) (complex) leucine, N-linked (HexNacid...) (complex) methionine, N-linked (HexNacid...) (complex) phenylalanine, N-linked (HexNacid...) (complex) valine, N-linked (HexNacid...) (complex) alanine, N-linked (HexNacid...) (complex) citrulline, N-linked (HexNacid...) (complex) glycine, N-linked (HexNacid...) (complex) serine, N-linked (HexNacid...) (complex) threonine, N-linked (HexNacid...) (complex) tyrosine, N-linked (HexNacid...) (complex) cysteine, N-linked (HexNacid...) (complex) proline, N-linked (HexNacid...) (complex) glutamic acid, N-linked (HexNacid...) (complex) glutamine, N-linked (HexNacid...) (complex) aspartic acid, N-linked (HexNacid...) (complexAsparagine, 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-pyruvate 2-imino-cysteine, N-pyruvate 2-imino-valine, O-(2-aminoethylphosphoryl)serine, O-(2-cholinephosphoryl)serine, O-(5'-phosphate-DNA)-serine Acid, O-(5'-phosphate-DNA)-tyrosine, O-(5'-phosphate-RNA)-serine, O-(5'-phosphate-RNA)-tyrosine, O-(pantoyl thioethylamine 4'-phosphoryl)serine, O-(phosphoribosyl dephosphoryl-coenzyme A)serine, O-(sn-1-glycerophosphoryl)serine, O3-poly(β-hydroxybutyryl)serine, O-8α-FAD tyrosine, O-acetylserine, O-acetylthreonine, O-α-linked (GlcNacid)threonine, O-AMP-serine, O-AMP-threonine, O-A MP-Tyrosine, O-decylserine, O-decylthreonine, 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...) (keratin sulfate) serine, O-linked (GalNacid...) (keratin sulfate) threonine, O-linked (GalNacid...) serine, O-linked (GalNacid...) threonine, O-linked (GalNacid...)) tyrosine, O-linked (GATD Glc) 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 (GlcNacid 1P) serine, O-linked (GlcNacid 6P) 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...) hydroxyproline, 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 (Man 1P) serine, O-linked (Man 1P...) serine, O-linked (Man 6P) threonine, O-linked (Man 6P...) 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...) (keratan sulfate) threonine, O-linked (Xyl...)) Serine, omega-hydroxynorvaline glutamate, omega-N-methylarginine, omega-N-methylated arginine, O-methylthreonine, O-octanoylserine, O-octanoylthreonine, O-palmitoleoylserine, O-palmitoylserine, O-palmitoylthreonine, O-tri-AMP-tyrosine, O-UMP-histidine, 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), pentaglycyl murein peptoglycan amated alanine, pentaglycyl murein peptoglycan amated threonine, peptide (Met-Gly) (with G-... interchain), phenylalanine amide, phosphatidylethanolamine amated glycine, phosphatidylserine amated glycine, phosphoarginine, phosphocysteine, phosphohistidine, phosphoserine, phosphothreonine, phosphotyrosine, PolyADP-ribosyl aspartic acid, PolyADP-ribosyl glutamic acid, proline 5-hydroxy-oxazole-4-thiocarbonic acid (Pro-Cys), proline amide, Pros-8 alpha-FAD histidine, Pros-methylhistidine, Pros-phosphohistidine, pyr-2,5-dicarboxylic acid (Ser-Cys) (with S-...), pyr-2,5-dicarboxylic acid (Ser-Ser) (with C-...), pyrroline carboxylic acid, pyrroline carboxylic acid (Glu), pyrroloquinoline quinone (Glu-Tyr), pyruvic acid (Cys), pyruvic acid (Ser), pyruvic acid (Tyr), S-(15-deoxy-delta 12,14-prostaglandin J2-9-yl) cysteine, S-(2,3-dicarboxypropyl) cysteine, S-(2-aminoethenyl)-3-methyl-D-cysteine (Thr-Cys), S-(2-aminoethenyl)-D-cysteine (Cys-Cys), S-(2-aminoethenyl)-D-cysteine (Ser-Cys), S-(2-aminoethenyl)-L-cysteine (Cys-Cys), S-(2-succinyl) cysteine, S-(4-hydroxycinnamoyl) cysteine, S-(coelenterazine-3a-yl) cysteine, S-(dipyrromethanemethyl) cysteine, S-12-hydroxyfarnesyl cysteine, S-4a-FMN cysteine, S-6-FMN cysteine, S-8a-FAD cysteine, S-archaeol cysteine, S-bacidillithiol cysteine disulfide, S-carbamoyl cysteine, S-cGMP-cysteine, S-cyanide cysteine, S-cysteinyl 3-(oxothioalkyl)alanine (Cys-Cys), S-cysteinyl cysteine, S-diglycerol cysteine, serine amide, serophore ester of serinamycin E492, S-farnesyl cysteine, S-geranylgeranyl cysteine, S-glutathione cysteine, S-linked (Gal) cysteine, S-linked (Gal...) cysteine, 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-lysyl-methionine sulfimine (Lys-Met) (with M-... interchain), S-lysyl-methionine sulfimine (Met-Lys) (with K-... interchain), S-methionine sulfimine (M-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionine sulfimine (Met-... interchain), S-methionineS-methylcysteine, S-methylmethionine, S-nitrosocysteine, S-palmitoleoylcysteine, S- palmitoylcysteine, S-poly(beta-hydroxybutyryl)cysteine, S-selenocysteine, S-stearoylcysteine, Sulfhydrylserine, Sulfhydrylthreonine, Sulfhydryltyrosine, Sym-dimethylarginine, Tele-(1,2,3-trihydroxypropan-2-yl)histidine, Tele-8a-FAD histidine, Tele-8a-FMN histidine, Tele-methylhistidine, Tele-phosphohistidine, 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 open DNA abasic site, Thiazoline-4-carboxylic acid (Phe-Cys), Threonine 5-hydroxy-oxazole-4-carbonthionic acid (Thr-Cys), Threonine amide, Threonine methyl ester, Threonyl lysine isopeptide (Lys-Thr) (with T-... interchain), Threonyl lysine isopeptide (Thr-Lys) (with K-... interchain), Thyroxine, Triiodothyronine, Trithiocysteine (Cys-Cys), Tryptamine amide, Tryptamine derivative, Tryptaminyl-tryptophyl-methionine (Trp-Trp) (with M-...), Tryptaminyl-tryptophyl-methionine (Trp-Tyr) (with W-...), Tryptophan amide, Valine amide.

[0060] Post-translational modification (PTM) is a covalent and usually enzymatic modification of a protein after its biosynthesis.

[0061] According to certain embodiments, the one or more artificial derivatization can be selected from a polymer, a chemical chelator, a polyamino carboxylate, deferoxamine, deferasirox, deferiprone, and the like.

[0062] The polymer is not particularly limited, and can be selected from, for example, polyethylene glycol, dextran, and the like.

[0063] According to certain embodiments, the chemical chelator, which is not particularly limited, can include a cyclic polyamine. The cyclic polyamine can be selected from, for example, cyclen, cyclam, and the like.

[0064] According to certain embodiments, the polyaminocarboxylate salt can be selected from (2,2',2"-(1,4,7-triazonane-1,4,7-triyl)-triacetic acid (N0TA), 2,2',2",2"'-(1,4,7,10-tetraazododecane-1,4,7,10-tetrayl)-tetraacetic acid (DOTA), 1,4,8,11-tetraazotetradecane-1,4,8,11-tetraacetic acid (TETA), and the like.

[0065] By using one of these D-amino acid derivatives, the properties of the substrate with the D-amino acid derivative-protected functional group can be adjusted as detailed above. By using a combination of one or more of the functionalization, post-translational modification, and artificial derivatization, the properties of the substrate with the D-amino acid derivative-protected functional group can be adjusted efficiently, selectively, and precisely.

[0066] In the method, the at least one substrate is not particularly limited as long as it has at least one D-amino acid and / or D-amino acid derivative-protected functional group (the functional group is not limited), and can be one substrate or more than one substrate, for example, two or more, three or more, and the like, which can be mixed. According to certain embodiments, one substrate is used in the method. In general, the suitable substrate can be any organic compound, for example, for the synthesis of low-molecular-weight active ingredients.

[0067] According to certain embodiments, the substrate can be selected from 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; and a mixture thereof; in particular wherein the substrate has at least two functional groups. According to certain embodiments, the substrate can be selected from a carbohydrate, a carbohydrate derivative, and a mixture thereof; in particular wherein the substrate has at least two functional groups. In a particular embodiment, the substrate can be selected from an amino acid; a peptide; an oligopeptide; a polypeptide; a protein; a peptide intermediate; a peptide derivative; an oligopeptide derivative, a polypeptide derivative; and a mixture thereof; in particular wherein the substrate has at least two functional groups. According to certain embodiments, the amino acid, the peptide, the oligopeptide, the polypeptide, and the peptide intermediate can be an L-amino acid, an L-peptide, an L-oligopeptide, an L-polypeptide, and an L-peptide intermediate.

[0068] According to certain embodiments, the amino acid, the peptide, the oligopeptide, the polypeptide, the protein, and the peptide intermediate can be an enzyme precursor.

[0069] In the present application, the carbohydrate is not particularly limited. The carbohydrate can include monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The monosaccharide is not particularly limited, and can be selected from, for example, di-, tri-, tetra-, penta-, and hexoses. Among them, the di-saccharide can include glycolaldehyde, the tri-saccharide can be selected from, for example, glyceraldehyde and dihydroxyacetone, the tetra-saccharide can be selected from, for example, erythrose, threose, and erythrulose, the penta-saccharide can be selected from, for example, ribose, arabinose, xylose, lyxose, deoxyribose, ribulose, and xylulose, and the hexa-saccharide can be selected from, for example, allose, altrose, glucose, mannose, gulose, idose, galactose, talose, and fructose. The disaccharide is a sugar formed when two monosaccharides are linked by a glycosidic bond. For example, the disaccharide can be selected from cellobiose, gentiobiose, sucrose, lactose, trehalose, and maltose. The oligosaccharide is a sugar formed when a small number (typically 3 to 10) of monosaccharides are linked by a glycosidic bond. The oligosaccharide can have a linear or branched structure. For example, the oligosaccharide can be selected from fructooligosaccharide, raffinose, and galactooligosaccharide. The polysaccharide is a sugar containing a large number (at least 11) of monosaccharides linked by a glycosidic bond. The polysaccharide can have a linear or branched structure. For example, the polysaccharide can be selected from glycogen, starches such as amylose and amylopectin, pectin, chitin, callose, and cellulose.

[0070] The carbohydrate derivative can be based on a carbohydrate, as described above, except that one or more chemical groups can be introduced into the carbohydrate and / or one or more functional groups of the carbohydrate can be substituted with at least one chemical group and / or the carbohydrate is in the form of a salt. According to certain embodiments, one or more hydroxyl groups of the carbohydrate can be substituted with at least one other chemical group, which is not particularly limited, and if there is more than one chemical group, the groups can be the same or different. The one or more chemical groups can be selected from, for example, hydrogen, halogen, alkyl, alkenyl (e.g., vinyl, allyl, etc.), alkynyl, aromatic, ester, carboxyl, acetyl, ether, ketone, aldehyde, amine, amide, imine, imide, acetamide, acetal, hemiacetal, nitrile, thiol, sulfide, and phosphine.

[0071] According to certain embodiments, the one or more chemical groups can be selected from hydrogen, halogen, alkyl, alkenyl, alkynyl, ester, carboxyl, acetyl, ether, ketone, aldehyde, amine, and acetamide. According to certain embodiments, the alkyl group can have 1 to 20 carbon atoms. According to certain embodiments, the alkenyl group can have 1 to 20 carbon atoms. According to certain embodiments, the alkynyl group can have 1 to 20 carbon atoms.

[0072] In the present application, nucleotides, oligonucleotides and polynucleotides are not particularly limited. Nucleotide derivatives include one or more modifications. Nucleotide derivatives can be in the form of a salt. Nucleotide derivatives can include modified nucleobases. Modified nucleobases can be based on the above-mentioned nucleobases, except that one or more chemical groups can be introduced into the above-mentioned nucleobases and / or one or more functional groups of the above-mentioned nucleobases can be substituted by at least one chemical group, for example selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, aromatic, amine, amide, imine, imide, acetylamino, thiol. The same derivatization can be applied to oligonucleotides and polynucleotides.

[0073] Amino acids that can be used as a substrate are not particularly limited, preferably L-amino acids. According to certain embodiments, the amino acids can be selected from the group consisting of proteinogenic amino acids and non-proteinogenic amino acids. Proteinogenic 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 pyrrolysine (pyl, O). Non-proteinogenic amino acids are not limited and can for example be selected from the group consisting of carnitine, GABA (gamma-aminobutyric acid), levothyroxine, hydroxyproline, 2-amino isobutyric acid, gamma-aminobutyric acid, ornithine, citrulline and selenomethionine.

[0074] According to certain embodiments, the peptide in the present application can be an L-peptide. The amino acids forming the peptide are not particularly limited. According to certain embodiments, the amino acids forming the peptide are selected from the group consisting of alanine (ala), lysine (lys) and tyrosine (tyr). The oligopeptide can likewise be an L-oligopeptide. The amino acids forming the oligopeptide are not particularly limited. The polypeptide can be an L-polypeptide. The amino acids forming the polypeptide are not particularly limited. Likewise, the amino acids forming the protein are not particularly limited.

[0075] A peptide intermediate is a compound present during the synthesis of a peptide, oligopeptide, polypeptide, protein or enzyme. According to certain embodiments, a peptide intermediate can comprise a peptide which can be modified with one or more chemical groups, in particular protecting groups. According to certain embodiments, the functional groups of the amino acids can be protected with protecting groups to prevent undesired reactions of these functional groups. According to certain embodiments, the N-terminus of the peptide can be modified with one or more chemical groups, in particular protecting groups. The protecting groups are not particularly limited. According to certain embodiments, the protecting groups can be selected from the group consisting of benzoyl (Bz), fluorenylmethyloxycarbonyl (Fmoc), tert-butyloxycarbonyl (Boc), acetyl (Ac), succinyl (Suc), benzyloxycarbonyl (Z), 2-azidomethylbenzyl (Ambz), 2-aminobenzoic acid (Abz), a biotin group and mixtures thereof. A peptide intermediate can also comprise a linker, for example a linker between the peptide intermediate and a resin. The linker is not particularly limited and can for example be selected from the group consisting of a Wang linker, a Rink linker, a Merrifield linker, a 4-methyldiphenylamine linker (MBHA), a phenylacetamidomethyl (PAM) linker, a nitrobenzyl linker and an allyl linker. A Wang linker can comprise a p-alkyloxybenzyl ester linker. A Rink linker can comprise a 4-(2',4'-dimethoxyphenyl-4-hydroxymethyl-phenoxy) linker and a 4-(2',4'-dimethoxyphenyl-4-methylamido-phenoxy) linker. A Merrifield linker can comprise a benzyl linker. The resin is not particularly limited and can for example be selected from polystyrene.

[0076] In a peptide derivative and / or oligopeptide derivative and / or polypeptide derivative, one or more chemical groups can be introduced into the peptide and / or one or more functional groups of the peptide can be substituted with at least one chemical group, for example as given above for a carbohydrate derivative. Furthermore, the peptide derivative can be a peptide in salt form.

[0077] According to certain embodiments, the method can further comprise adding a buffer to the reaction mixture, wherein the pH value of the reaction mixture can be in the range of 3-13. The buffer can be provided in addition to the at least one substrate having at least one D-amino acid and / or a D-amino acid derivative protected functional group, the D-stereospecific hydrolase and the at least one solvent. The buffer can be provided and mixed with the at least one substrate having at least one D-amino acid and / or a D-amino acid derivative protected functional group, the D-stereospecific hydrolase and the at least one solvent.

[0078] The buffer is not particularly limited and can be selected, for example, from among acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium phosphate (NaPi) buffer, potassium phosphate (KPi) buffer, 2-(4-(2-hydroxyethyl)piperazin-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 (Bis-Tris) buffer, 1,3-bis-(tris-(hydroxymethyl)-methylamino)-propane (Bis-Tris propane) buffer, N-(2-acetylamino)iminodiacetic acid (ADA) buffer, N-(2-acetylamino)-2-aminoethanesulfonic acid (ACES) buffer, acetylglycine buffer, 2-hydroxy-3-morpholino propanesulfonic acid (MOPSO) buffer, N,N-bis-(2-hydroxyethyl)-2-amino-ethanesulfonic 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-hydroxy-propanesulfonic acid) (HEPPSO) buffer, piperazine-N,N'-bis-(2-hydroxypropanesulfonic acid) (POPSO) buffer, N-(tris(hydroxymethyl)-methyl)-glycine (Tricine) buffer, glycylglycine buffer, glycine buffer, (bis(2-hydroxyethyl)-amino)acetic acid (BICINE) buffer, N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid (TAPS) buffer, 2-amino-2-methyl-1,3-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-methylpropan-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 increase the effectiveness of the hydrolysis step and can enhance the activity of the D-stereospecific hydrolase in the hydrolysis step. According to certain embodiments, the pH value of the reaction mixture can be in the range of 3-13, preferably in the range of 5-11, more preferably 7-10, more preferably 7-9. By carrying out the deprotection at such a pH value in the above-mentioned range, the effectiveness of the hydrolysis step can be increased and the activity of the D-stereospecific hydrolase in the hydrolysis step can be enhanced. The concentration of the buffer in the reaction mixture is not limited and can be, for example, in the range of 1 mmol to 1 mol, more preferably 10 mmol to 500 mmol, more preferably 15 mmol to 300 mmol, more preferably 20 mmol to 200 mmol, based on the reaction mixture. By carrying out the deprotection with a buffer concentration in the above-mentioned range, the pH value can be precisely controlled. Furthermore, the effectiveness of the hydrolysis step can be increased and the activity of the D-stereospecific hydrolase in the hydrolysis step can be enhanced.

[0079] According to certain embodiments, the functional group of the protected substrate can be selected from the group consisting of amino, carboxyl, hydroxyl, thiol and selenohydril, for example amino, hydroxyl, thiol and / or selenohydril, in particular the D-amino acid and / or the D-amino acid derivative can be bound to the substrate via a carbonyl group. In the hydrolysis step, for example a water molecule provided by the solvent can react with at least one functional group of the above-mentioned protected substrate to form the D-amino acid and / or the D-amino acid derivative and the at least partially deprotected substrate, wherein at least one deprotected functional group is deprotected from the D-amino acid and / or the D-amino acid derivative.

[0080] The concentration of the substrate in the reaction mixture is not particularly limited and can 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, even more preferably 100 µM to 1 mM, based on the reaction mixture. By carrying out the deprotection with a substrate concentration in the above-mentioned range, the hydrolysis reaction proceeds more effectively.

[0081] The concentration of the D-stereospecific hydrolytic enzyme in the reaction mixture is not limited and can be, for example, in the range of 1 nM - 200 µM, preferably 100 nM - 100 µM, more preferably 500 nm - 50 µM, more preferably 750 nm - 10 µM, based on the reaction mixture. By carrying out the deprotection with a D-stereospecific hydrolytic enzyme concentration in the above ranges, the hydrolysis reaction proceeds more efficiently.

[0082] The at least one solvent is not particularly limited but preferably comprises at least water. The solvent can be a mixture of at least two solvents. According to certain embodiments, the at least one solvent can be selected from 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, such as selected from water, alcohols, nitriles, sulfoxides and mixtures thereof, such as selected from water, DMSO (dimethyl sulfoxide), DMF (dimethylformamide), acetonitrile, methanol and mixtures thereof. According to certain embodiments, the at least one solvent can be water. Using these solvents, the effectiveness of the hydrolysis step and the activity of the D-stereospecific hydrolytic enzyme in the hydrolysis step can be enhanced.

[0083] The concentration of the at least one solvent can be in the range of 0.1 - 90 %, preferably 0.5 - 70 %, more preferably 1 - 50 %, even more preferably 2 - 35 %, even more preferably 2.5 - 10 %, based on the volume of the entire solvent.

[0084] According to certain embodiments, the hydrolysis step can be carried out at a reaction temperature in the range of 0 °C - 50 °C, preferably 5 °C - 40 °C, more preferably 15 °C - 35 °C, more preferably 22 °C - 32 °C, even more preferably about 30 °C. By carrying out the deprotection at a temperature in the above ranges, the hydrolysis reaction proceeds more efficiently. Furthermore, side reactions can be prevented.

[0085] The first aspect of the method may further include adding at least one additive to the reaction mixture. In addition to at least one substrate having a functional group protected by 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 also be provided. There are no particular limitations on the additive, provided that it is compatible with the compounds present in the reaction mixture. In particular, the enzyme used is compatible with the additive. According to some embodiments, at least one additive may be selected from salts and organic compounds. Salts may be added to enhance the solubility of the substrate and may be selected from NaCl, KCl, LiCl, CaCl2, MgCl2, NaBr, AgCl, MnCl2, MgSO4, MnSO4, ZnSO4, (… ) Mixtures thereof. According to certain embodiments, NaCl may be preferred. Similarly, the organic compound is not limited and may be selected, for example, from alkanes, alkenes, alkynes, alcohols, aldehydes, ketones, esters, ethers, amines, amides, imides, nitriles, thiols, their salts, and mixtures thereof, such as from alcohols, amines, amides, thiols, their salts, and mixtures thereof. According to certain embodiments, the organic compound may be selected from DTT (dithiothreitol), DTT derivatives, 2-mercaptoethanol, 2-mercaptoethanol derivatives, urea, urea derivatives, guanidine chloride, guanidine chloride derivatives, and mixtures thereof. According to certain embodiments, the additive may be selected from NaCl, KCl, LiCl, CaCl2, MgCl2, and mixtures thereof; DTT, DTT derivatives, 2-mercaptoethanol, 2-mercaptoethanol derivatives, urea, urea derivatives, guanidine chloride, guanidine chloride derivatives, and mixtures thereof, such as from NaCl, DTT, 2-mercaptoethanol, urea, guanidine chloride, and mixtures thereof. By adding at least one additive, the solubility of one or more compounds in the reaction mixture can be enhanced. The concentration of at least one organic compound is not limited and can 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 some embodiments, the concentration of the salt (e.g., NaCl) can be in the range of 1 mM to 5 M, more preferably 1 mM to 2.5 M. Deprotection with additive concentrations within the above ranges can further enhance the solubility of one or more compounds in the reaction mixture.

[0086] In the method of the first aspect, in some embodiments, the D-stereospecific hydrolase may have the following characteristics: At least one first 3 10- a helix-2 structure comprising the amino acid sequence SXXK; at least one first alpha-helical structure comprising the amino acid sequence YSN; and at least one first beta- sheet structure comprising the amino acid sequence HXG; wherein X is an amino acid selected from the group consisting of protein amino acids; wherein the first 3 10 - the helix-2 structure, the first alpha-helical structure and the beta-sheet structure are closely packed.

[0087] According to certain embodiments, the 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), and valine (val, V).

[0088] According to certain embodiments, the first 3 10 - the helix-2 structure is an N-terminal extension of the alpha-helix-2.

[0089] According to certain embodiments, the first 3 10 - the helix-2 structure can be at the N-terminus of the alpha-helix-2. 10 - the helix-2 structure comprises the amino acid sequence SXXK at its terminus.

[0090] According to certain embodiments, the first 3 10- The amino acids S and K of the amino acid sequence SXXK in the helix-2 structure can directly participate in the hydrolytic cleavage of at least one D-amino acid and / or D-amino acid derivative-protected functional group of a substrate to yield a D-amino acid and / or D-amino acid derivative and a D-amino acid and / or D-amino acid derivative at least partially deprotected substrate. According to certain embodiments, the S of the amino acid sequence SXXK can participate in binding a substrate having at least one D-amino acid and / or D-amino acid derivative-protected functional group during hydrolysis. According to certain embodiments, the S of the amino acid sequence SXXK that can participate in binding a substrate having at least one D-amino acid and / or D-amino acid derivative-protected functional group during hydrolysis can be an activated S. In this structure, the serine has an -OH group that can act as a nucleophile, attacking the carbonyl carbon of the substrate's scissile peptide bond. In addition, a pair of electrons on the lysine nitrogen can accept a hydrogen from the serine -OH group, thereby coordinating the attack of the peptide bond. According to certain embodiments, X in the amino acid sequence SXXK can be selected from the group consisting of I, K, L, V, and combinations thereof.

[0091] According to certain embodiments, the YSN motif of the first alpha-helix structure can be located at the N-terminus of alpha-helix-6. In this structure, the tyrosine has an -OH group that can activate a water molecule that is postulated to attack the Ser-substrate ester intermediate bond. The asparagine can support this activation through an electrostatic network produced by E, D, or Q.

[0092] According to certain embodiments, the HXG motif of the first beta-sheet structure can participate in the formation of an oxyanion hole. The oxyanion hole is a pocket in the enzyme active site that stabilizes the transition state negative charge on the deprotonated oxygen. This pocket can be composed of backbone amides or positively charged residues. According to certain embodiments, X in the sequence HXG can be selected from the group consisting of G, N, R, and S. According to certain embodiments, the HXG motif can participate in stabilizing a substrate having at least one D-amino acid and / or D-amino acid derivative-protected functional group. The histidine therein is a central part of the electrostatic network that allows the coordination and order of the reaction. The glycine can participate in the formation of the oxyanion hole, which allows the nucleophilic attack of the serine described above.

[0093] According to certain embodiments, further, the D-stereospecific hydrolase described above can be characterized by a binding pocket for a substrate having at least one D-amino acid and / or D-amino acid derivative-protected functional group.

[0094] According to certain embodiments, at least one second 3 10- the helix structure can be involved in the region for substrate recognition. This structure can allow the D-stereospecific hydrolase to interact with the substrate. Alternatively or additionally, it can allow the D-stereospecific hydrolase to interact with the correct coordination of the substrate in the binding pocket.

[0095] According to certain embodiments, the first 3 10 - the helix-2 structure, the first a-helix structure and the first b-sheet structure are closely arranged such that all fragments are close enough to interact with a substrate having at least one D-amino acid and / or D-amino acid derivative protected functional group.

[0096] According to certain embodiments, the D-stereospecific hydrolase can have an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9. According to certain embodiments, the D-stereospecific hydrolase can have an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 7, No. 8 and No. 9. .

[0097] The present application also discloses the above-mentioned amino acid sequences derived from the amino acid sequences of SEQ ID No. 1-9, the corresponding sequences with the initial amino acid M that can be removed during post-translational modification (SEQ ID No. 1A-9A), and the native amino acid sequences derived from the amino acid sequences of SEQ ID No. 1-9, i.e. with the amino acid sequences of SEQ ID No. 1B-9B. The corresponding sequences can also be used in the present application and are also respectively covered according to certain embodiments. For the sake of brevity, they are not repeated when discussing other aspects of the present application, but are covered as embodiments using or relating to the corresponding aspects with the amino acid sequences of SEQ ID No. 1-9.

[0098] According to certain embodiments, the D-stereospecific hydrolytic enzyme can catalyze the hydrolytic cleavage of at least one D-amino acid and / or a D-amino acid derivative-protected functional group of a substrate to obtain a D-amino acid and / or a D-amino acid derivative and a D-amino acid and / or a D-amino acid derivative at least partially deprotected substrate.

[0099] The method of the first aspect allows for the selective deprotection of at least one D-amino acid and / or a D-amino acid derivative-protected functional group of a substrate. The method of the first aspect further allows for the fast and quantitative cleavage of a D-amino acid and / or a D-amino acid derivative from a D-amino acid and / or a D-amino acid derivative-protected functional group of a substrate. The method allows for a side reaction-free deprotection under mild reaction conditions and the method can be orthogonal to all previously established deprotection and synthesis methods. Furthermore, such a method can be fully applied even in the case of chemically unstable substrates and target structures.

[0100] In a second aspect of the present application, a D-stereospecific hydrolytic enzyme is provided. The D-stereospecific hydrolytic enzyme is characterized as for the method of the first aspect, which is incorporated here. The D-stereospecific hydrolytic enzyme of the second aspect has the following characteristics: at least one first 3 10 - a helix-2 structure comprising the amino acid sequence SXXK; at least one first alpha-helix structure comprising the amino acid sequence YSN; and at least one first beta-sheet structure comprising the amino acid sequence HXG; wherein X is an amino acid selected from the group consisting of proteinogenic amino acids; wherein the first 3 10- the helix-2 structure, the first alpha-helical structure and the beta-sheet structure are closely packed. According to certain embodiments, the D-stereospecific hydrolase has an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9, or has a sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 7, No. 8 and No. 9, as indicated above.

[0101] In a third aspect of the application, there is provided the use of a D-stereospecific hydrolase in the deprotection of at least one D-amino acid and / or a functionally protected D-amino acid derivative of a substrate. According to certain embodiments, the D-stereospecific hydrolase is a D-stereospecific hydrolase of the second aspect.

[0102] According to certain embodiments, the deprotection can comprise the same steps and features as described in the first aspect of the application for the deprotection of at least one D-amino acid and / or a functionally protected D-amino acid derivative of a substrate.

[0103] According to certain embodiments, the D-stereospecific hydrolase can have an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9. According to certain embodiments, the D-stereospecific hydrolase can have an amino acid sequence selected from the group consisting of SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 7, No. 8 and No. 9.

[0104] The D-stereospecific hydrolases, in particular the D-stereospecific hydrolases of the application, have strict regioselectivity, chemoselectivity and enantioselectivity for the respective recognized D-amino acid and / or D-amino acid derivative. Thus, the use of the D-stereospecific hydrolases allows for the selective cleavage of at least one D-amino acid and / or D-amino acid derivative from the D-amino acid and / or functionally protected D-amino acid derivative of a substrate.

[0105] In a fourth aspect of the application, there is provided a method of preparing 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, the method comprising: providing a 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 a mixture 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 hydrolytic enzyme, and at least one first reagent; mixing a 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 a mixture thereof, at least one first solvent, and at least one D-amino acid and / or D-amino acid derivative, to obtain a first reaction mixture; reacting a 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 a mixture thereof, with at least one D-amino acid and / or D-amino acid derivative, to obtain 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 a mixture thereof; optionally removing at least one first solvent from the first reaction mixture; optionally, in one or more further reaction steps, reacting the 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 a mixture thereof, with one or more further reagents, 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 a mixture thereof; a precursor of 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, or a precursor of a reacted 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, at least one first reagent, and optionally at least one second solvent, to obtain a second reaction mixture; reacting a precursor of 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, or a precursor of a reacted 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, with 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 removing at least one second solvent from the second reaction mixture; 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 mixture thereof, optionally at least one third solvent, and a D-stereospecific hydrolytic enzyme, to obtain a third reaction mixture; and deprotecting at least one D-amino acid and / or D-amino acid derivative from 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 to obtain 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.

[0106] The step of providing the different components is not particularly limited. According to certain embodiments, the components can be provided prior to providing the different components with or without performing at least one synthesis step. According to certain embodiments, the synthesis step can be selected from the group consisting of a chemical reaction, removing at least one component, and adding at least one component.

[0107] The step of mixing the different components is not particularly limited. The order in which the 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.

[0108] For the (optional) reaction step, the reaction vessel is not particularly limited and any known reaction vessel can be used.

[0109] The optional removal of at least one first solvent and / or at least 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 second solvent can be removed by evaporation.

[0110] The step of reacting a 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 with at least one D-amino acid and / or D-amino acid derivative to obtain a precursor of 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 is not particularly limited. According to certain embodiments, the functional group of the precursor can be selected from the group consisting of an amino group, a carboxyl group, a hydroxyl group, a thiol group, and a selenol group. According to certain embodiments, the functional group can be an amino group, a hydroxyl group, a thiol group, and / or a selenol group, and the D-amino acid and / or D-amino acid derivative can bind to the substrate via a carbonyl group.

[0111] The step of deprotection can comprise the same steps and features as the deprotection of at least one D-amino acid and / or D-amino acid derivative protected functional group of the substrate described in the method of the first aspect of the present application. According to certain embodiments, the step of deprotection can further comprise the addition of 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 can be selected from the buffers described in the first aspect of the present application. According to certain embodiments, the step of deprotection can further comprise the addition of one or more additives to the reaction mixture. The additive is not particularly limited as long as it is compatible with the compounds present in the reaction mixture. According to certain embodiments, the additive can be selected from the additives described in the first aspect of the present application.

[0112] 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 is not particularly limited and can be the same as described in the first aspect of the present application.

[0113] The at least one first solvent, the at least second solvent, and the at least third solvent are not particularly limited. They can be the same or different, but according to certain embodiments, they are different and can be the same as the at least one solvent described in the first aspect of the present application.

[0114] Furthermore, the D-amino acid and the D-amino acid derivative are not particularly limited and can be selected from the D-amino acids and D-amino acid derivatives described in the first aspect of the present application.

[0115] The D-stereospecific hydrolase is also not limited and can have the same features as the D-stereospecific hydrolases described in the first and second aspects of the present application.

[0116] The at least one first reagent is not particularly limited. According to certain embodiments, the at least one first reagent can be selected from a precursor of an introduced 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.

[0117] The precursor of an introduced chemical group is not particularly limited.

[0118] The chemical group can be selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, aromatic, ester, carboxyl, acetyl, ether, ketone, aldehyde, amine, amide, imine, imide, acetylamino, acetal, hemiacetal, nitrile, thiol, sulfide, and phosphine.

[0119] The base is not particularly limited. According to certain embodiments, the base can be selected from the group consisting of organic and inorganic bases. According to certain embodiments, the base can be selected from the group consisting of amines, carbonates, carbamates, hydroxides, oxides, and ammonia, such as amines, carbonates, hydroxides, and ammonia.

[0120] The acid is not particularly limited. According to certain embodiments, the acid can be selected from the group consisting of organic and inorganic acids. According to certain embodiments, the acid can be selected from the group consisting of carboxylic acids, sulfuric acid, hydrochloric acid, phosphoric acid, and nitric acid. The carboxylic acid can be selected from the group consisting of formic acid and acetic acid.

[0121] The reducing agent is not particularly limited. According to certain embodiments, the reducing agent can be selected from the group consisting of metals such as sodium or lithium, hydrogen, lithium aluminum hydride, Red-Al (NaAlH2(OCH2CH2OCH3)2), diborane, sodium borohydride, Fe compounds including Fe 2+ (II) such as ferrous sulfate (II), Sn compounds including Sn 2+ ions such as stannous chloride (II), sulfites, dithiothionates, thiothionates, hydrazine, diisobutylaluminum hydride (DIBAL-H), oxalic acid, formic acid, ascorbic acid, reducing sugars such as erythrose, phosphites, hypophosphites, DTT (dithiothreitol), carbon monoxide, and tris-2-carboxyethylphosphine (TCEP).

[0122] The oxidizing agent is not particularly limited. According to certain embodiments, the oxidizing agent can be selected from the group consisting of oxygen, nitrogen oxides, peroxides (such as hydrogen peroxide, hydrogen peroxide derivatives), manganates, chromates (such as PCC (pyridinium chlorochromate) or PDC (pyridinium dichromate)), bromates, hypochlorites, and TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical or (2,2,6,6-tetramethylpiperidin-1-yl)oxyl radical).

[0123] 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 is not particularly limited and can be described as in the first aspect of the present application.

[0124] This method of preparation allows for modeling of the biological activity or biochemical functionality of the compound during the synthesis of the compound. For example, by introducing one or more functionalities, post-translational modifications and / or artificial derivatization to the D-amino acid, the chemical and physical properties such as solubility or compatibility with other compounds can be altered, as described above. This includes therapeutic agents that can be inactivated by modification of the D-amino acid and / or D-amino acid derivative and lead to the production of prodrugs. Thus, the therapeutic agent can be introduced as an inactive precursor and, after accumulation in the tissue, can be activated by cleavage of the D-amino acid and / or D-amino acid derivative.

[0125] The functionality of the D-amino acid and / or D-amino acid derivative can further block catalytically active residues or prevent structure-induced interactions. This enables the formation of structural elements or the interaction of different amino acid side chains to be impeded, thus enabling the activity of the enzyme or the binding of the interaction partner to the target structure to be modulated. After cleavage of the D-amino acid and / or D-amino acid derivative, the formation of the active protein or the intended interaction can take place.

[0126] By using D-stereospecific hydrolytic enzymes, at least one D-amino acid and / or D-amino acid derivative-protected functional group of the substrate can be cleaved easily at any time in the method of synthesis of the compound. This means that at least one D-amino acid and / or D-amino acid derivative can be introduced into the substrate at any time during the synthesis of the compound and can be cleaved at any time after the introduction. Thus, the chemical and physical properties of the substrate can be precisely, selectively and efficiently adjusted depending on the reaction conditions required at the particular time of the synthesis of the compound and the reaction system required.

[0127] In a fifth aspect of the application, the use of D-amino acids and / or D-amino acid derivatives as protecting groups for the functional groups of substrates is provided.

[0128] The D-amino acids, D-amino acid derivatives, functional groups and substrates are not particularly limited and can be as described above.

[0129] The above embodiments can be combined in any combination, if appropriate. Further embodiments and implementations of the application also include combinations of features not explicitly mentioned above or below with respect to the examples of the application. In particular, the skilled person will also add individual aspects as improvements or supplements to the individual basic forms of the application. Examples

[0130] Preparation and characterization of D-stereospecific hydrolases

[0131] Preparations of D-stereospecific hydrolases with SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9 were performed in E. coli (BL21 (DE3) RIL, Novagen) under standard conditions (growth conditions: LB-medium, kanamycin, 37°C, continuous shaking; protein biosynthesis induction: 0.1 M IPTG and temperature shift to 30°C). E. coli

[0132] The isolated yields of these D-stereospecific hydrolases are shown in Table 1 after expression in E. coli and subsequent purification by ion exchange (SP-Sepharose or Supelco 650 M, 0.1 M HEPES, 0.1 M NaCI, pH 7.0 (SEQ ID No. 1, No. 2, No. 3, i.e. Paenidases 6.0); protein elution: 0.1 M TRIS pH 9.0) and final gel permeation chromatography (HiLoad 16 / 60 Superdex 75 prep grade column, 0.1 M HEPES, 0.01 M NaCI, pH 8.0).

[0133] Specific activity A of D-stereospecific hydrolytic enzyme s

[0134] The specific activities A of the D-stereospecific hydrolases with SEQ ID No. 4, No. 6, No. 7, No. 8 and No. 9 were tested on selected esterified D-amino acid derivatives s (s -1 ).

[0135] ​In these experiments, a NaPi buffer was used, which was based on a concentration of 0.1 M of the reaction mixture. The pH value of the reaction mixture was 8.0. As an example, the substrate Bz-D-Phe-OMe (benzoyl-D-phenylalanine-methyl ester) was used. As a comparative example, the substrates Bz-L-Phe-OMe (benzoyl-L-phenylalanine-methyl ester) and Bz-Gly-OGp (benzoyl-glycyl-4-guanidinophenyl ester) were used. The concentration of the substrates was 2 mM. The concentration of the enzyme was in the range of 2-400 nM. The concentration was based on the reaction mixture. Water including 1 % DMF (v / v) was used as solvent. The activity measurement was a discontinuous measurement by UPLC (gradient 0% B => 60% B (v / v), eluent: A water (0.1 % (v / v) trifluoroacetic acid), B acetonitrile (0.1 % (v / v) trifluoroacetic acid)), flow rate 0.5 ml / min, detection wavelength 254 nm, temperature 40°C. At specific time points (0 to 5 min) a sample volume of 25 mΐ was taken and stopped with 25 mΐ 50 % (v / v) acetic acid.A s is defined as the conversion of pmol substrate per pmol enzyme per time scale and is given in kat / mol.

[0136] In this case, the D-stereospecific hydrolase catalyzes the hydrolysis of each ester bond, resulting in an alcohol group and a carboxyl group. The specific activity A s refers to the rate at which the specific D-stereospecific hydrolase catalyzes the hydrolysis of each ester bond.

[0137] The specific activity A s of the D-stereospecific hydrolases having SEQ ID No. 4, No. 6, No. 7, No. 8 and No. 9 was determined as shown in Table 2.

[0138] As can be seen from Table 2, the D-stereospecific hydrolases tested exhibit a high A s when using substrates containing D-amino acids, while they exhibit a low A s when using substrates containing L-amino acids. Thus, the D-stereospecific hydrolases tested selectively recognize and convert D-amino acids or D-amino acid derivatives.

[0139] In further experiments, the specific activity A s (mkat / mol) of the D-stereospecific hydrolases having SEQ ID No. 2, No. 4, No. 6, No. 7, No. 8 and No. 9 was determined for selected peptides protected with D-amino acid derivatives. In this case, the N-terminus of the peptide was protected with a D-amino acid derivative.

[0140] In these experiments, in addition to the above-mentioned conditions, a NaPi buffer based on the concentration of the reaction mixture of 0.1 M was used. The pH value of the reaction mixture was 8.0. The peptide had the amino acid sequence of AKAKY(NO2) (SEQ ID No. 10), wherein Y was modified with a NO2 group to generate 3-nitrotyrosine. The concentration of the D-amino acid derivative-protected peptide based on the reaction mixture was 100 µM. In addition, DTT was added based on the reaction mixture at a concentration of 10 mM. Furthermore, NaCl was added based on the reaction mixture at a concentration of 150 mM. The concentration of each D-stereospecific hydrolase based on the reaction mixture was 1 nM - 1 µM. As D-amino acid derivative, the D-amino acid derivatives shown in Table 3 below were used. The reaction temperature was 30°C. Water was used as solvent.

[0141] The evaluation was carried out with discontinuous measurement by UPLC (gradient 0% B => 20% or 40% or 60% B (v / v), eluent: A water (0.1 % (v / v) trifluoroacetic acid), B acetonitrile (0.1 % (v / v) trifluoroacetic acid)), flow rate 0.5 ml / min, detection wavelength 360 nm, temperature 40°C. At certain time points (0 to 5 min) a sample volume of 25 µl was taken, which was stopped with 25 µl 50 % (v / v) acetic acid. All values represent the average of duplicate determinations. The deviation was always below 10%.

[0142] In this case, the D-stereospecific hydrolase catalyzes the hydrolysis of each carboxamide bond between the D-amino acid derivative and the peptide, resulting in an amine group and a carboxyl group, here for example f is D-Phe (source: IUPAC-IUB Commission on Biochemical Nomenclature (CBN), A One-Letter Notation for Amino Acid Sequences, 1968, Arch. Biochem. Biophys. 125(3), i-v (l968)). The specific activity A s refers to the rate at which a particular D-stereospecific hydrolase catalyzes the hydrolysis of each amide or carboxamide bond.

[0143] The specific activity A of the D-stereospecific hydrolases having SEQ ID No. 2, No. 4, No. 6, No. 7, No. 8 and No. 9 on selected D-amino acid derivative-protected peptides sAs shown in Table 3. In this regard, it should be noted that (Bz-f) in AAK(Bz-f)AKY(N02) (SEQ ID No. 18) is located on the NH2of the lysine chain, not within the peptide chain, so that the peptide chain produced after cleavage thereof is AAKAKY(N02) (SEQ ID No. 137), i.e. the same as after cleavage of Bz-fAAKAKY(N02) (SEQ ID No. 21). The products produced from Bz-fCAKAKY(N02) (SEQ ID No. 11), Z-fCAKAKY(N02) (SEQ ID No. 12), Suc-fCAKAKY(N02) (SEQ ID No. 13) Ac-fCAKAKY(N02) (SEQ ID No. 14), Boc-fCAKAKY(N02) (SEQ ID No. 15), Abz-fCAKAKY(N02) (SEQ ID No. 16) Fmoc-fCAKAKY(N02) (SEQ ID No. 17), fCAKAKY(N02) and (SEQ ID No. 19) are CAKAKY(N02) (SEQ ID No. 169).The other products after cleavage of the protected D-Phe from SEQ ID No. 20-38 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. 144), WAKAKY(NO2) (SEQ ID No. 145), RAKAKY(NO2) (SEQ ID No. 146), KAKAKY(NO2) (SEQ ID No. 147), HAKAKY(NO2) (SEQ ID No. 148), DAKAKY(NO2) (SEQ ID No. 149), EAKAKY(NO2) (SEQ ID No. 150), QAKAKY(NO2) (SEQ ID No. 151), NAKAKY(NO2) (SEQ ID No. 152), MAKAKY(NO2) (SEQ ID No. 153), PAKAKY(NO2) (SEQ ID No. 154).

[0144] As can be seen from Table 3, all tested D-stereospecific enzymes have activity on the selected substrates. Thus, D-stereospecific enzymes are able to convert peptides having at least one functional group protected with a D-amino acid or a D-amino acid derivative. This is the case even if no protecting group is present.

[0145] In further experiments, the specific activities A s (mkat / mol) of D-stereospecific hydrolases having SEQ ID No. 4, No. 7, No. 8 and No. 9 on selected D-amino acid derivative protected peptides were tested 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 duplicate determinations. The deviation was always below 10%.

[0146] As can be seen from Table 4, the concentration of the substrate to be applied can be changed without deteriorating the activity of the D-stereospecific hydrolase. Thus, the D-stereospecific hydrolase can be applied to various synthetic conditions.

[0147] Subsite mapping experiments with respect to S1 '-subsite

[0148] Subsite mapping experiments were performed to characterize the S1 '-binding pocket. To this end, Bz-Gly-OGp and Bz-D-Phe-OMe were used as acyl donors and selected D- and L-amino acid amides (X / x aa NH2, x represents a D-amino acid and X represents an L-amino acid) as acyl acceptors. Activity measurements were performed in a discontinuous assay by HPLC (gradient 0% B => 20% or 40% or 60% B (v / v), eluent: A water (0.1 % (v / v) trifluoroacetic acid), B acetonitrile (0.1 % (v / v) trifluoroacetic acid)), flow rate 0.5 ml / min, detection wavelength 220 nm and 254 nm, respectively, temperature 40 °C. At specific time points (0 to 5 min) a sample volume of 25 μΙ was taken and stopped with 25 μΙ 50% (v / v) acetic acid. In the experiments, a NaPi buffer was used based on a concentration of 0.1 M of the reaction mixture. The pH value of the reaction mixture was 8.0. The concentration of the acyl acceptor (amino acid amide or pentapeptide) was 10 mM 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 including 1 % DMF (v / v) was used as solvent.

[0149] The respective model reactions (aminolysis reactions) are shown below (formula 1 and 2). In formula 1 and 2, DHy represents the respective D-stereospecific hydrolase as described below.

[0150] The reciprocal partition value is used to determine the efficiency of such reactions (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. .1021 / bi00180a020. PMID: 8155642):

[0151] The reciprocal partition value (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having SEQ ID No. 6 is shown in Figure 1 The reciprocal partition value (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having SEQ ID No. 7 is shown in Figure 2 The reciprocal partition value (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having SEQ ID No. 8 is shown in Figure 3 The reciprocal partition value (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolase having SEQ ID No. 9 is shown in Figure 4

[0152] In Figures 1-4 , the black bars represent Bz-D-Phe-OMe and the white bars represent Bz-Gly-OGp.

[0153] The smaller the reciprocal value (1 / p), the higher the reaction rate of the hydrolysis reaction, the higher the rate of the aminolysis reaction, reflecting the S1' specificity.

[0154] As can be seen from Figures 1-4 , the use of D-amino acids as x aa (x aa ​The aminolysis reaction of the D-amino acid (or D-amino acid derivative at the P1' position) is preferred because 1 / p increases for all tested D-stereospecific enzymes. Thus, the D-stereospecific enzyme specifically recognizes the D-amino acid or D-amino acid derivative when the D-amino acid or D-amino acid derivative is located at the P1' position. When an L-amino acid (Xaa) is used, the 1 / p value decreases mainly, which highlights the high S'1 subsite selectivity of the D-stereospecific hydrolytic enzyme for the hydrolysis of the functional group of the D-amino acid or D-amino acid derivative of the substrate, especially in the case of a single amino acid or its derivative.

[0155] Using all D / L peptides (x aa aaaGly, / X aa Similar experiments were performed using Bz-Gly-OGp as acyl donor and the following D- / L-amino acid amides as acyl acceptors instead of the above D- / L-amino acid amides. The varying D- / L-amino acid was located at the P1' position. Again, Bz-Gly-OGp was used as acyl donor. The respective model reactions (hydrolysis reactions) are shown below (formula 4A and 4B). In formula 4, DHy represents the D-stereospecific hydrolytic enzyme.

[0156] In the experiments, a NaPi buffer was used based on the concentration of the reaction mixture of 0.1 M. The pH value 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 the D-stereospecific hydrolytic enzyme was 500 nM. Water including 1 % DMF was used as solvent.

[0157] The following SEQ ID are applicable: XAAAG (SEQ ID No. 106), xaaaG (SEQ ID No. 107), Bz-GXAAAG (SEQ ID No. 170), Bz-GxaaaG (SEQ ID No. 171).

[0158] The reciprocal partition values (1 / p) of the S1' subsite mapping of the D-stereospecific hydrolytic enzymes with SEQ ID No. 7 (white bars), 8 (light grey bars) and 9 (dark grey bars) are shown in Figure 5The following sequences are labeled as educt and product, respectively: FAAAG (SEQ ID No. 108), faaaG (SEQ ID No. 109), Bz-GFAAAG (SEQ ID No. 110), Bz-GfaaaG (SEQ ID No. 111), LAAAG (SEQ ID No. 112), laaaG (SEQ ID No. 113), Bz-GLAAAG (SEQ ID No. 114), Bz-GlaaaG (SEQ ID No. 115), MAAAG (SEQ ID No. 116), maaaG (SEQ ID No. 117), Bz-GMAAAG (SEQ ID No. 118), Bz-GmaaaG (SEQ ID No. 119), RAAAG (SEQ ID No. 120), raaaG (SEQ ID No. 121), Bz-GRAAAG (SEQ ID No. 122), Bz-GraaaG (SEQ ID No. 123), AAAAG (SEQ ID No. 124), aaaaG (SEQ ID No. 125), Bz-GAAAAG (SEQ ID No. 126), Bz-GaaaaG (SEQ ID No. 127), GAAAG (SEQ ID No. 128), gaaaG (SEQ ID No. 129), Bz-GGAAAG (SEQ ID No. 130), Bz-GgaaaG (SEQ ID No. 131), PAAAG (SEQ ID No. 132), paaaG (SEQ ID No. 133), Bz-GPAAAG (SEQ ID No. 134), Bz-GpaaaG (SEQ ID No. 135).

[0159] In contrast to single amino acid derivatives, D-stereospecific enzymes specifically recognize L-amino acid derivatives in the P1' position when using peptides as substrates. This allows the enzyme to cleave D-L amino acid bonds in the case of longer peptides.

[0160] Experiments with fluorescent substrates

[0161] In further experiments, hydrolysis reactions using D-stereospecific hydrolases with SEQ ID No. 4, No. 6, No. 7, No. 8, No. 9 were carried out with an internally quenched fluorescent substrate (IQFS) (Abz-AAX / x aaFAAK(DNP)-OH) (Abz = 2-aminobenzoic acid, DNP = 2,4-dinitrophenol). The cleavage by the IQFS liberates the fluorescent signal and can be monitored by continuous fluorescence measurements (0.1 M NaPi, pH 8.0, 1% DMSO (v / v), 5-1000 nM enzyme, excitation wavelength 320 nm emission wavelength 420 nm, reaction volume 100 μΐ, 96-well microtiter plate, time scale 2 h). The respective model reactions are shown below (formula 5). In formula 5, DHy stands for the respective D-stereospecific hydrolytic enzyme.

[0162] The following SEQ ID apply: Abz-AAXFAAK(DNP)-OH (SEQ ID No. 172), Abz-AAxFAAK(DNP)-OH (SEQ ID No. 173), FAAK(DNP)-OH (SEQ ID No. 155).

[0163] Apparent specific activity (A s,app) as shown in Table 5. In Table 5, the values of D-configuration amino acids (Xaa) are on the left side and the values of L-configuration amino acids (Xaa) are on the right side. The sequences in Table 5 are as follows: AAFFAAK (SEQ ID No. 74), AAfFAAK (SEQ ID No. 75), AAWFAAK (SEQ ID No. 76), AAwFAAK (SEQ ID No. 77), AAYFAAK (SEQ ID No. 78), AAyFAAK (SEQ ID No. 79), AAAFAAK (SEQ ID No. 80), AAaFAAK (SEQ ID No. 81), AAVFAAK (SEQ ID No. 82), AAvFAAK (SEQ ID No. 83), AALFAAK (SEQ ID No. 84), AAlFAAK (SEQ ID No. 85), AASFAAK (SEQ ID No. 86), AAsFAAK (SEQ ID No. 87), AAMFAAK (SEQ ID No. 88), AAmFAAK (SEQ ID No. 89), AAHFAAK (SEQ ID No. 90), AAhFAAK (SEQ ID No. 91), AAKFAAK (SEQ ID No. 92), AAkFAAK (SEQ ID No. 93), AARFAAK (SEQ ID No. 94), AArFAAK (SEQ ID No. 95), AAQFAAK (SEQ ID No. 96), AAqFAAK (SEQ ID No. 97), AAEFAAK (SEQ ID No. 98), AAeFAAK (SEQ ID No. 99), AADF AAK (SEQ ID No. 100), AAdFAAK (SEQ ID No. 101), AAPFAAK (SEQ ID No. 102), AApFAAK (SEQ ID No. 103), AAGFAAK (SEQ ID No. 104), AAgFAAK (SEQ ID No. 105), wherein, respectively, in each sequence, the first A is protected by Abz and the last K is protected by DNP-OH. Furthermore, FAAK(DNP)-OH (SEQ ID No. 155) was obtained as a product.

[0164] As can be seen from Table 5, the activity of the D-stereospecific hydrolases is higher using substrates containing D-amino acids compared to substrates containing L-amino acids, similar to the above.

[0165] In Table 6, the respective catalytic parameters of the hydrolysis reaction of the D-stereospecific hydrolases with SEQ ID No. 8 and No. 9 using the fluorescence-quenched substrate (Abz-AAX / xaa FAAK(DNP)-OH) are shown. The sequences in Table 6 are as follows: AAfFAAK (SEQ ID No. 68), AAwFAAK (SEQ ID No. 69), AAyFAAK (SEQ ID No. 70), AAlFAAK (SEQ ID No. 71), AAmFAAK (SEQ ID No. 72), AAhFAAK (SEQ ID No. 73), wherein in each sequence, respectively, the first A is protected by Abz and the last K is protected by DNP-OH.

[0166] Similar experiments were performed using the D-stereospecific hydrolase with SEQ ID No. 3 and the substrate Abz-AAX / x aa FAAK(DNP)-OH. The specific activities (As) are shown in Table 7.

[0167] Experiments with respect to S2-preference of D-stereospecific hydrolases

[0168] In further experiments, the S2-preference of the D-stereospecific hydrolases using the D-stereospecific hydrolases with SEQ ID No. 8 and No. 9 was investigated using the substrate Abz-AX / x aa fAAAK(DNP)-OH, as above with IQFS. The respective model reactions are shown below (Equation 6), wherein AAKK(DNP)-OH (SEQ ID No. 156) is obtained as product. In Equation 6, DHy again stands for the D-stereospecific hydrolase.

[0169] The following SEQ ID apply: Abz-AXfAAAK(DNP)-OH (SEQ ID No. 174), Abz-AxfAAAK(DNP)-OH (SEQ ID No. 175), AAKK(DNP)-OH (SEQ ID No. 156).

[0170] In Table 8, the specific activities A s(kat / mol). In Table 8, the amino acid in the P2 position is printed in bold, the cleavage site is marked with an arrow.

[0171] In Table 9, the specific activity and kinetic parameters of the D-stereospecific enzymes are shown.

[0172] As can be seen from Tables 8 and 9, the D-stereospecific enzymes specifically recognize D-amino acids or D-amino acid derivatives when the D-amino acid or the D-amino acid derivative is located in the P2 position, which highlights the broad variability of the application of the D-stereospecific enzymes.

[0173] Experiments with respect to S1 '-preference of D-stereospecific hydrolases

[0174] In further experiments, the S1 '-preference of the D-stereospecific hydrolases using the substrates Abz-AAfx aa The S1 '-preference of the D-stereospecific hydrolases using the D-stereospecific hydrolases with SEQ ID No. 8 and No. 9 was investigated.

[0175] In Table 10, the specific activity A s (kat / mol). In Table 10, the amino acid in the P1'position is printed in bold, the cleavage site is marked with an arrow.

[0176] In addition to Abz-AAf, the following products were obtained after cleavage: MAAK (SEQ ID No. 157), FAAK (SEQ ID No. 158), VAAK (SEQ ID No. 159), LAAK (SEQ ID No. 160), AAKK (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), HAAK (SEQ ID No. 168).

[0177] From the experiments on sub-site mapping, S2-preference and S1 '-preference, it can be seen that the enzymes are active regardless of which position the D-amino acid or the D-amino acid derivative is located. Thus, the D-stereospecific hydrolases can be variably used with substrates having at least one D-amino acid or D-amino acid derivative-protected functional group.

[0178] Experiments on the influence of different additives and different reaction conditions on the activity of D-stereospecific hydrolases

[0179] In further experiments, the influence of the pH value on the activity of the D-stereospecific hydrolases SEQ ID No. 4, No. 6, No. 7, No. 8 and No. 9 was investigated. As substrate, the peptide Abz-AAfFAAK(DNP)-OH was used. The pH value was varied by using different buffers (acetate 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 s ) of each enzyme as shown in Figures 6-10 ( 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) is calculated by dividing the activity at the different pH values by the corresponding activity of each D-stereospecific enzyme at pH 8.0 (used as standard conditions).

[0180] As can be seen from Figures 6-10 , the D-stereospecific hydrolases are active in a very broad pH range. Thus, the D-stereospecific hydrolases can be variably used under different synthesis conditions.

[0181] Furthermore, experiments were carried out to investigate the influence of the addition of different additives in different concentrations to the reaction mixture on the activity of the D-stereospecific hydrolases. The concentration of each additive varied between 0 and 2.5 M. As additives, urea, guanidine hydrochloride, NaCl and ß-mercaptoethanol (ß-ME) were used. The additives were added to the reaction mixture comprising the D-stereospecific hydrolase and the peptide Abz-AAfFAAK(DNP)-OH. As D-stereospecific hydrolases, the D-stereospecific hydrolases having SEQ ID No. 4, No. 6, No. 7, No. 8 and No. 9 were used.

[0182] In Figure 11a and 11b , the relative activity of the D-stereospecific hydrolase having SEQ ID No. 9 was shown when the additives urea or guanidine hydrochloride were added.

[0183] In Figures 12-14 ​In the example, when different concentrations of additives such as urea, guanidine hydrochloride, NaCl, or β-mercaptoethanol are added, it is shown that the result has SEQ ID No. 8 ( Figure 12 No. 6 Figure 13 ) and No. 4 ( Figure 14 The relative activity of D-stereospecific hydrolases.

[0184] In this case, relative activity is calculated by dividing the activity when different concentrations of additives are added by the maximum activity of each D-stereospecific enzyme.

[0185] Table 11 shows that the relative specific activity of each enzyme is still 50% ( ) or 10% The concentration of additive (M) in the reaction mixture is used as the reference.

[0186] As shown in Table 11, D-stereospecific hydrolases retain their activity even with the addition of different additives to the reaction mixture. Therefore, D-stereospecific hydrolases can be used variably under different synthetic conditions.

[0187] In addition, experiments were conducted to investigate the effect of solvent on the activity of D-stereospecific hydrolases.

[0188] DMSO, DMF, methanol, and ACN (acetonitrile) were used as solvents. Solvent concentrations varied between 0 and 20% v / v (DMF, acetonitrile) and 0 and 30% v / v (DMSO, methanol). Solvents of varying concentrations were added to the reaction mixture comprising the D-stereospecific hydrolase and the peptide Abz-AAfFAAK(DNP)-OH, based on the entire solvent volume. The remaining solvent was water. D-stereospecific hydrolases having SEQ ID No. 4, No. 6, No. 7, No. 8, and No. 9 were used as the D-stereospecific hydrolase.

[0189] exist Figures 15a-15c In the study, the relative activities (relative to A) of the D-stereospecific hydrolase with SEQ ID No. 9 were shown when DMSO (15a), ACN (15b), and DMF (15c) were added. s ).

[0190] exist Figures 16-19 In the example, when DMSO, ACN, methanol, and DMF are added, the result shows SEQ ID No. 4 ( Figure 19 No. 6 Figure 18 ), No. 7 Figure 17) and No. 8 ( Figure 16 ) of the D-stereospecific hydrolytic enzymes (relative A s ).

[0191] In this case, the relative activity was calculated by dividing the activity when different concentrations of solvents were added by the maximum activity of each D-stereospecific enzyme.

[0192] In Table 12, the effect of selected organic solvents on the activity of D-stereospecific hydrolytic enzymes is shown. The concentration of the organic solvent (v / v) at which the relative specific activity of each enzyme was still 50% ( ) or 10% ( ) is shown.

[0193] As can be seen from Figures 15 a-15 c, 16-19 and Table 11, the D-stereospecific hydrolytic enzymes are active even if solvents other than water are added to the reaction mixture. The D-stereospecific hydrolytic enzymes are active even if the concentration of the other solvent is 35% (v / v). Thus, the D-stereospecific hydrolytic enzymes can be used variably under different synthesis conditions.

[0194] Alignment of D-stereospecific hydrolases isolated and used

[0195] In Figure 20 , the alignment of the D-stereospecific hydrolytic enzymes isolated and used is shown.

[0196] In the alignment, conserved amino acids are highlighted in red, amino acids with similar functions are written in red, and conserved regions are marked with a box. The numbering is the complete numbering based on the consensus sequence. In addition, the secondary structure elements (a-a-helix, η-3 10 -helix, β-β-fold, TT-β-turn) are labeled according to SEQ ID No. 6, the crystal structure of which was obtained with a resolution of 1.46 Å, as described in Figure 21The crystal was obtained by mixing 0.1 ml of the protein solution with 0.1 ml of the precipitant solution 0.1 M imidazole, 0.1 M MES pH 6.5, 30 mM MgCI2, 30 mM CaCI2, 12.5% (v / v) MPD, 12.5% (w / v) polyethylene glycol 1000 and 12.5% (w / v) polyethylene glycol 3350 at 20°C by hanging-drop vapour-diffusion crystallization. Crystals appeared within 14-21 days and were flash-frozen without any additional cryo-protectant. Data collection was performed at -172°C on beamlines 14.1 and 14.2 at BESSY synchrotron (Helmholtz Zentrum, Berlin). The crystal belongs to the monoclinic space group P21. Shown is the structure with the conserved motives SXXK and YXN (A). In addition shown is the HRG-motive, which is in beta-strand 13, and alpha-helices 2 and 6. Also shown in detail are the surface and the putative binding site and the co-crystallized phosphate (B), and the orientation of the phosphate in the active site and the distance to the catalytically important side chains (C). Additional crystal structure results of a soaking experiment with the substrate Bz-D-Arg-OMe confirm the binding of the substrate and the mechanism (data not shown).

[0197] Alignments were performed using MultAlign and visualized using ESPript3 (Corpet, 1988; Robert & Gouet, 2014).

[0198] From Figure 20 As can be seen, all sequences of the isolated enzymes have a 3 10 - helix 2 structure, an alpha-helix structure comprising the amino acid sequence YSN (alpha-helix-6 structure) and a beta-strand structure comprising the amino acid sequence HXG (beta-strand-13).

[0199] The use of D-amino acids and / or D-amino acid derivatives, e.g. as protecting groups or linkers for affinity probes or solubility tags, is compatible with standard synthesis protocols in peptide and carbohydrate chemistry and can thus easily be applied to established synthesis protocols without additional effort. In contrast to e.g. penicillin acylases, the biocatalysts used here are very efficient for the reactions considered, e.g. with specificity constants up to 7 10 6 M -1 s -1This allows quantitative conversion at low enzyme concentrations in very short reaction times. Using standard molecular biology and protein chemistry protocols, these enzymes can be expressed in high yields and are tolerant to a wide range of additives and organic solvents. Furthermore, the used biocatalysts do not require co-factors or stabilizers, are stable and catalytically active in a broad pH range.

[0200] Experiments with different substrates using D-stereospecific hydrolases

[0201] To demonstrate that the D-stereospecific hydrolases of the present application can also be used for non-protein substrates, experiments were performed with different substrates, similar to the above, with the following conditions: 100 µM substrate, 10-50 nM enzyme, 100 mM NaPi pH=8, 150 mM NaCl, 30°C. The assessment was based on UPLC at 254 nM. Ahx-NH2= amino hexanoic acid amide, Glc = Glucosamine. The results are shown in Table 13.

[0202] Regarding the sequence listing, note that ID No. 1-9 in the sequence listing corresponds to SEQ ID No. 1-9, ID No. 10-27 in the sequence listing corresponds to SEQ ID No. 1A, 1B, 2A, 2B, 3A, 3B, 4A, 4B, 5A, 5B, 6A, 6B, 7A, 7B, 8A, 8B, 9A, 9B, ID No. 28-193 in the sequence listing corresponds to SEQ ID No. 10-175.

Claims

1. A method for deprotecting a functional group protected by at least one D-amino acid and / or a D-amino acid derivative of at least one substrate using a D-stereospecific hydrolase, said at least one substrate having at least one functional group protected by a D-amino acid and / or a D-amino acid derivative, the method comprising: - Provide at least one substrate, a D-stereospecific hydrolase, and at least one solvent; - The at least one substrate, the D-sterile-specific hydrolase, and the at least one solvent are mixed to obtain a reaction mixture; and - Hydrolyze at least one D-amino acid and / or D-amino acid derivative protected functional group of the at least one substrate using a D-stereospecific hydrolase in the reaction mixture to obtain a D-amino acid and / or D-amino acid derivative and at least partially deprotected substrate, wherein at least one deprotected functional group is deprotected from the D-amino acid and / or D-amino acid derivative.

2. The method of claim 1, wherein the at least one substrate is selected from 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; particularly, wherein the substrate has at least two functional groups.

3. The method according to any one of the preceding claims further comprises adding a buffer solution to the reaction mixture, wherein the pH value of the reaction mixture is in the range of 3-13.

4. The method according to any one of the preceding claims, wherein the functional group is selected from amino, carboxyl, hydroxyl, mercapto, and selenhydride.

5. The method of claim 4, wherein the functional group is amino, hydroxyl, mercapto, and / or selenhydride, and the D-amino acid and / or D-amino acid derivative is bound to the substrate via a carbonyl group.

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

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

8. The method according to any one of the preceding claims, wherein the at least one solvent comprises at least water.

9. The method of claim 3, wherein the buffer is selected from acetate buffer, 2-(N-morpholino)ethanesulfonic acid (MES) buffer, sodium phosphate (NaPi) buffer, potassium phosphate (KPi) buffer, 2-(4-(2-hydroxyethyl)piperazin-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 (Bis-Tris) buffer, 1,3-bis-(tris-(hydroxymethyl)-methylamino)-propane (Bis-Tris propane) buffer, N-(2-acetamino)iminodiacetic acid (ADA) buffer, N-(2-acetamino)-2-aminoethanesulfonic acid (ACES) buffer, acetaminoglycine 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 Buffers for: 2-((1,3-dihydroxy-2-(hydroxymethyl)-propyl-2-yl)-amino)acetyl-1-sulfonic acid (TES) buffer, choline chloride hydrochloride, 4-(N-morpholino)-butyric acid (MOBS) buffer, 3-N-bis-(hydroxyethyl)-amino-2-hydroxy-propanesulfonic acid (DIPSO) buffer, 3-((1,3-dihydroxy-2-(hydroxymethyl)-propyl-2-yl)-amino)-2-hydroxypropane-1-sulfonic acid (TAPSO) buffer, triethanolamine buffer, 4-(2-hydroxyethyl)-piperazine-1-(2-hydroxy-propanesulfonic acid) (HEPPSO) buffer, and piperazine-N,N′-bis-(2-hydroxypropanesulfonic acid). (POPSO) buffer, N-(tris(hydroxymethyl)-methyl)-glycine (Tricine) buffer, glycylglycine buffer, glycamide buffer, (bis(2-hydroxyethyl)-amino)acetic acid (BICINE) buffer, N-tris(hydroxymethyl)-methyl-3-aminopropanesulfonic acid (TAPS) buffer, 2-amino-2-methyl-1,3-propanediol (AMPD) buffer, N-tris(hydroxymethyl)-methyl-4-aminobutyric 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-methylprop-1-ol) (AMP) buffer, and 3-(cyclohexylamino)prop-1-sulfonic acid (CAPS) buffer.

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

11. A D-stereospecific hydrolase, having the following characteristics: At least one first 3 10 - Helix-2 structure, which contains the amino acid sequence SXXK; At least one first α-helix structure comprising 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 protein amino acids; Among them, the first 3 10 The first helical structure, the first α-helical structure, and the first β-fold structure are closely arranged.

12. The D-stereospecific hydrolase according to claim 11, wherein the D-stereospecific hydrolase has an amino acid sequence selected from SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8 and No. 9: 。 13. Use of a D-stereospecific hydrolase in the deprotection of at least one D-amino acid and / or D-amino acid derivative protected functional group of a substrate, preferably wherein said D-stereospecific hydrolase has the following characteristics: At least one first 3 10 - Helix-2 structure, which contains the amino acid sequence SXXK; At least one first α-helix structure comprising 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 protein amino acids; Among them, the first 3 10 The helix-2 structure, the first α-helix structure, and the first β-sheet structure are closely arranged, and further preferred are D-stereospecific hydrolases having amino acid sequences selected from SEQ ID No. 1, No. 2, No. 3, No. 4, No. 5, No. 6, No. 7, No. 8, and No. 9: 。 14. A method for preparing 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, said method comprising: Provides 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 precursors of mixtures thereof. At least one D-amino acid and / or D-amino acid derivative, At least one first solvent, At least one optional second solvent, At least one optional third solvent, D-stereospecific hydrolases, and At least one first reagent; A mixture 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 a precursor of a mixture thereof, at least one first solvent, and at least one D-amino acid and / or a D-amino acid derivative, to obtain a first reaction mixture; To react a precursor of 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 D-amino acid and / or a D-amino acid derivative to obtain a protected 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 a precursor thereof. Optionally, at least one first solvent is removed from the first reaction mixture; Optionally, in one or more further reaction steps, 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 precursor of a mixture thereof is reacted with one or more further reagents to obtain a reacted 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 precursor of a mixture thereof. A mixture of 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 a mixture thereof, or a precursor thereof, at least one first reagent, and optionally at least one second solvent, to obtain a second reaction mixture; A precursor of 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, or a precursor thereof, is reacted with 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 a mixture thereof. Optionally, at least one second solvent is removed from the second reaction mixture; A mixture of 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 containing at least one third solvent and a D-stereospecific hydrolase, to obtain a third reaction mixture; and Deprotecting at least one D-amino acid and / or a D-amino acid derivative from 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 to obtain 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.

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

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