WO2010057961A1 - Peptide synthesis using enzymatic activation and coupling - Google Patents
Peptide synthesis using enzymatic activation and coupling Download PDFInfo
- Publication number
- WO2010057961A1 WO2010057961A1 PCT/EP2009/065512 EP2009065512W WO2010057961A1 WO 2010057961 A1 WO2010057961 A1 WO 2010057961A1 EP 2009065512 W EP2009065512 W EP 2009065512W WO 2010057961 A1 WO2010057961 A1 WO 2010057961A1
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- WIPO (PCT)
- Prior art keywords
- ester
- amino acid
- peptide
- terminal
- thioester
- Prior art date
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- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
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- VXIVSQZSERGHQP-UHFFFAOYSA-N chloroacetamide Chemical class NC(=O)CCl VXIVSQZSERGHQP-UHFFFAOYSA-N 0.000 description 1
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- IJKVHSBPTUYDLN-UHFFFAOYSA-N dihydroxy(oxo)silane Chemical compound O[Si](O)=O IJKVHSBPTUYDLN-UHFFFAOYSA-N 0.000 description 1
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- 239000002904 solvent Substances 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
- C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/10—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using coupling agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
Definitions
- the invention relates to a method for enzymatically synthesising a peptide.
- Peptides in particular oligopeptides have many applications, for instance as pharmaceutical, food or feed ingredient, agrochemical or cosmetic ingredient.
- peptides any chain of two or more amino acids.
- 'oligopeptides' is meant a peptide based on 2-200 amino acids, in particular based on 2-100, more in particular based on 2-50 amino acids, preferably any linear chain of 2-200 amino acids, more preferably of 2-100 or 2-50 amino acids.
- the term 'polypeptides' is used for peptides based on a higher number of amino acids than specified for oligopeptides.
- Chemo-enzymatic peptide synthesis which is defined for the purpose of the invention as the synthesis of peptides in which one or more peptidic bonds are formed by an enzymatic coupling reaction, has several advantages over chemical peptide synthesis. For instance, the cost-price in case of large scale production is lower due to the fact that no or limited amino acid side chain protection is required. Also, the process is more environmentally friendly.
- DCC dicyclohexyl carbodiimide
- EDC 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
- EDCI 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide methiodide
- DCI dicyclohexyl carbodiimide
- EDC 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride
- EDCI 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide methiodide
- enzyme- catalyzed couplings are devoid of racemisation (see for instance Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology", 1 st reprint, Ed. Wiley-VCH Verlag
- the carboxy component i.e. the component which is to be coupled on its C-terminus, bears a free carboxylic acid functionality, while in the kinetically controlled approach a reactive carboxy component is used, such as a primary n-alkyl ester.
- thermodynamic approach has three major disadvantages: i) the equilibrium is usually on the side of peptide bond cleavage so that the coupling yields are poor; ii) a large amount of enzyme is usually required; iii) the reaction rates are usually very low.
- alkyl esters are required as starting material but much less enzyme is required, the reaction time is significantly shorter, and, above all, the maximum obtainable yields are usually considerably higher. Therefore, for industrial application, an enzymatic peptide synthesis concept based on a kinetic approach, i.e. using an activated carboxy component, is most attractive (see for instance N. Sewald and H.-D. Jakubke, in: “Peptides: Chemistry and Biology", 1 st reprint, Ed. Wiley-VCH Verlag GmbH, Weinheim 2002, section 4.6.2).
- Chemo-enzymatic peptide synthesis can be stepwise performed in the C->N terminal direction or in the N->C terminal direction but can also entail the enzymatic coupling of fragments which have individually been synthesized using chemical synthesis or by a combination of chemical and chemo-enzymatic coupling steps. Chen et al. describe the coupling in "low aqueous" organic solvents with a protease which is active and stable in the organic solvent(J. Org. Chem. 1992, 57, 6960-65, Biomed. Biochim. Acta 1991 , 50, 181 , Bioorg. Med. Chem. Lett. 1991 , 1 , 445).
- D-amino acids and non-proteinogenic amino acids can be incorporated enzymatically with high efficiency and, additionally, no hydrolysis of the peptidic bonds in the fragments occurs except if there is a peptidic bond for which the coupling enzyme is specific (e.g. Arg-X bonds in the case of trypsin).
- the substrate mimetic approach is that substrate mimetics (such as Gp esters) require a laborious multi-step chemical synthesis which is difficult to scale up and by which often racemisation of the amino acid occurs; the substrate mimetics are also instable, and thus difficult to handle on a large scale and sometimes their solubility in aqueous solution is low.
- esters such as carbamoylmethyl (Cam) esters (as described for instance by T. Miyazawa et al. J. Chem. Soc, Perkin Trans. 1 , 2002, 390-395) or 2,2,2-trifluoroethyl esters (as described by A. Yan et al. Tetrahedron, 61 , 2005, 5933-5941 ).
- Cam carbamoylmethyl
- 2,2,2-trifluoroethyl esters as described by A. Yan et al. Tetrahedron, 61 , 2005, 5933-5941 .
- These esters are usually more easily prepared and more stable than the "real" substrate mimetics but still need chemical steps that are expensive and environmentally unfriendly. Further, the preparation of these esters often leads to some racemisation of the amino acid.
- Cam esters can be prepared by treatment of the Cs salts of the acyl donor with 2-chloroacetamide and Tfe esters can be prepared using carbodiimide chemistry.
- the Cam and Tfe esters are usually coupled to amino acid or peptide nucleophiles using cheap and readily available proteases such as papain and subtilisin.
- D-amino acids can be incorporated into peptides (Pro has not been incorporated yet).
- Such methods may, e.g., provide additional methodology that increases flexibility in peptide synthesis and/or allow the synthesis of specific peptides that are not or relatively difficult to obtain using known methodology. For instance, it is still a challenge to enzymatically couple various amino acids in a good yield within an acceptable time.
- the present invention relates to a method for enzymatically synthesising a peptide, comprising enzymatically preparing an ester or a thioester from (i) an N-terminal protected amino acid, an N-terminal protected amino acid C-terminal ester, an N- terminal protected peptide, or an N-terminal protected peptide C-terminal ester and (ii) an alcohol represented by the formula HO-CX 2 -Z respectively a thiol represented by the formula HS-CX 2 -Z each X independently representing a halogen atom or a hydrogen atom; and
- Z being selected from the group of sp 3 -hybridised carbons comprising at least two substituents comprising a heteroatom directly attached to the sp 3 - hybridised carbon and sp 2 -hybridised carbons comprising one or two substituents comprising a heteroatom directly attached to the sp 2 -hybridised carbon,
- the preparation of the ester or thioester being carried out in a reaction medium comprising 2 wt. % water or less based on the weight of liquids in the reaction medium;
- the term 'C-terminal protected' is used herein to indicate that a C- terminal carboxyl-group is provided with a protective group, generally substantially protecting the carboxyl group from being coupled to an amine group of another molecule.
- the C-terminal protective group may be a C-terminal ester whereby the C-terminal carboxyl group is at least substantially protected from being coupled to an amine under peptide synthesis conditions used.
- a t-alkyl group is a commonly used protective group.
- N-terminal protected' is used herein to indicate that an N- terminal amine group is provided with a protective group, generally at least substantially protecting the N-terminal amine group from participating in coupling of a C-terminal carboxyl group to the N-terminal amine group.
- the ester or thioester prepared in a method of the invention may respectively be referred to as an 'activated ester' or an 'activated thioester', as such ester is capable of participating in the coupling reaction.
- the free C-terminal carboxylic acid or the ester which may be used for the preparation of the activated ester is not capable to participate in the coupling, or at least has a substantially lower reactivity in the coupling reaction, e.g. less than half of the reactivity, less than one tenth of the reactivity or less than one hundredth of the reactivity of the activated ester that is enzymatically prepared in the method of the invention.
- Typical examples of difficult to couple amino acids or amino acid residues are D-amino acids or amino acid residues thereof and other non-proteinogenic amino acids or amino acid residues thereof.
- Further examples include sterically hindered amino acids, e.g. valine and isoleucine, or amino acid residues thereof.
- esterification or thioesterification may be referred to hereinafter as an esterification or thioesterification, respectively.
- This term includes the case wherein the preparation of the (thio)ester involves the reaction of an N-terminal protected amino acid or peptide C-terminal ester with the (thio)alcohol.
- the method of the invention is advantageous in that it offers the possibility to use a method of the invention for the coupling of various amino acids or various peptides differing in the terminal amino acid residue that is to participate in the coupling reaction, including proline and non-proteinogenic amino acids or peptides containing proline or a non-proteinogenic terminal amino acid residue to take part in the coupling.
- the method of the invention is advantageous in that the activated (thio)esters are synthesized in high yields without racemisation or other side reactions in an environmentally friendly way, i.e. not producing stoichiometric amounts of waste compounds.
- the method of the invention is advantageous in that it offers a high stability and/or activity of the enzyme or enzymes used in the method.
- the method of the invention is advantageous in that the extent of hydrolysis of the enzymatically prepared ester is small (within a typical time frame for achieving the preparation of the (thio)ester and the coupling), at least in several embodiments no detectable hydrolysis of the ester has been observed.
- the method of the invention is advantageous in that the extent of hydrolysis of the enzymatically prepared peptide is low; at least in several embodiments no detectable hydrolysis of the enzymatically prepared peptide has been observed.
- the method of the invention is advantageous in that it offers a high overall reaction rate, i.e. a high conversion rate from the N- protected amino acid or peptide starting compounds to synthesised peptides, leading to relatively short reaction times to achieve a specific yield.
- a method of the invention allows coupling of an amino acid or peptide to another amino acid or peptide, without needing a large excess of one of the coupling partners in order to obtain the synthesised peptide in an acceptable yield based on the other coupling partner within a relatively short time.
- the molar ratio of the N-terminal protected amino acid, the N-terminal protected amino acid C-terminal ester, the N-terminal protected peptide, or the N-terminal protected peptide C-terminal ester from which the (activated) (thio)ester is prepared to the optionally C-terminal protected amino acid or peptide usually is chosen in the range of 2:1 to 1 :3, in particular in the range of 1 :1 to 1 :2, preferably in the range of 1 :1 to 1 :1.5. In a specifically preferred method, said molar ration is in the range of 1 :1 to 1 :1.1.
- a method of the invention is in particular advantageous in that it has been found possible to enzymatically prepare activated C-terminal esters or thioesters of an N-terminal protected amino acid or an N-terminal protected peptide, in particular C-terminal carbamoylmethyl (Cam) esters or C-terminal 2,2,2-trifluoroethyl (Tfe) esters of an N-terminal protected amino acid or an N-terminal protected peptide, as part of a chemo-enzymatic peptide synthesis.
- Cam carbamoylmethyl
- Tfe 2,2,2-trifluoroethyl
- Cam and Tfe esters can be prepared enzymatically in a high yield using a protease or a lipase, and, even better, that the activation step with the protease or lipase can be performed simultaneously with the peptidic bond formation step using a protease giving the condensation product (the peptide to be synthesised) without noticeable side reactions in a yield of more than 90%, even when using equimolar amounts of the amino acids or peptides to be coupled, i.e. without needing an excess of the amino acid or peptide acting as the nucleophile.
- a method according to the invention in particular offers a relatively simple process because it is possible to carry out (thio)esterification in a so-called 'one-pot' process.
- (thio)esterification and coupling take place without carrying out an isolation step (of the prepared ester or thioester) in between these reactions.
- the preparation of the ester or thioester is carried out in the presence of optionally C-terminal protected amino acid or optionally C-terminal protected peptide with which the ester or thioester that is prepared is to be coupled.
- the (thio)esterification and coupling take place sequentially, i.e. the optionally C-terminal protected amino acid or the optionally C-terminal protected peptide is added after the ester or thioester has been prepared.
- the preparation of the (thio)ester is carried out using a lipase or esterase, and thereafter an enzyme is added (usually a protease) for catalysing the coupling reaction.
- an enzyme usually a protease
- Such approach may in particular be followed in case the enzyme catalysing the coupling is detrimental to the preparation of the (thio)ester, e.g. because it has an adverse effect on the activity or stability of the enzyme catalysing the preparation of the (thio)ester.
- the enzyme catalysing the (thio)esterification and the enzyme catalysing the coupling may be the same, in particular the same lipase, esterase, or protease.
- subtilisin Carlsberg or Candida antarctica lipase B may be used for such an embodiment.
- N-terminal protected amino acid or N-terminal protected peptide that is to be (thio)esterified may in principle be any amino acid, proteinogenic or non- proteinogenic or any peptide, based on proteinogenic and/or non-proteinogenic amino acids.
- the invention relates to a method according to the invention, wherein for the enzymatic preparation of the ester or thioester (i) an N-terminal protected amino acid or an N-terminal protected peptide is reacted with (ii) the alcohol represented by the formula HO-CX 2 -Z, respectively the thiol represented by the formula HS-CX 2 -Z.
- N-terminal protected amino acid or peptide may be represented by a compound of formula I.
- N-terminal protecting group is those N-protecting groups which can be used for the synthesis of (oligo)peptides. Such groups are known to the person skilled in the art.
- suitable N-protecting groups include carbonyl type protective groups, for instance 'Cbz' (i.e. benzyloxycarbonyl), 'Boc' (i.e. t-butyloxycarbonyl), 'For' (i.e. formyl), Fmoc (i.e. 9-fluorenylmethoxycarbonyl) and 'PhAc' (i.e. phenacetyl).
- the groups For or PhAc may be introduced and cleaved enzymatically using the enzymes Peptide Deformylase or PenG acylase, respectively. Chemical cleavage methods are generally known in the art.
- n is an integer of at least 1.
- n may in particular be at least 2, at least 3, at least 4, at least 5, at least 6, at least 8, at least 9 or at least 10.
- n may in particular be 100 or less, 75 or less, 50 or less, 25 or less, 20 or less 15 or less or 10 or less, e.g. 5 or less.
- Each R A and each R B independently represent H, or an organic moiety, preferably an amino acid side chain. Thus, it is not required that R A is the same in all n amino acid units. Similarly, it is not required that R B is the same in all n amino acid units.
- amino acid side chain is meant any proteinogenic or non-proteinogenic amino acid side chain.
- the reactive groups in the amino acid side chains may be protected by amino acid side chain protecting groups or may be unprotected. Suitable protecting groups for the side chains of the amino acids are known to a man skilled in the art.
- all or a part of the acidic or alkaline side groups may be provided with a protective group in order to improve the solubility of the amino acid or peptide, if desired.
- Proteinogenic amino acids are the amino acids that are encoded by the genetic code. Proteinogenic amino acids include: alanine, valine, leucine, isoleucine, serine, threonine, methionine, cysteine, asparagine, glutamine, tyrosine, tryptophan, glycine, aspartic acid, glutamic acid, histidine, lysine, arginine, proline and phenylalanine.
- Non-proteinogenic amino acids may in particular be selected amongst D-amino acids, phenylglycine, DOPA (3,4-dihydroxy-L-phenylalanine), beta-amino acids, 4-fluoro-phenylalanine, or alfa-alkylated amino acids.
- the N-terminal protected amino acid or peptide used for the preparation of the ester or thioester that is to take part in the coupling reaction is a C-terminal ester different from the (thio)ester that is to be prepared, for instance a t-alkyl ester or another ester which generally does not show substantial activity in an enzymatic coupling reaction for the synthesis of peptides, or at least a lower activity than the (thio)ester that is prepared.
- the preparation of the (thio)ester may be referred to as a trans(thio)esterification.
- the compound that is to be reacted with an alcohol or thiol that is to be prepared is an ester, it is typically an ester of an amino acid or peptide as described above.
- ester may be represented by the formula Il
- This ester typically is less reactive in the coupling reaction than the ester or thioester that is enzymatically prepared in the method of the invention.
- the R c group is not an electron withdrawing group, or is less electron withdrawing than the CX 2 -Z group of the alcohol or thiol used in the preparation of the ester or thioester.
- R c usually is a hydrocarbon group or a hydrocarbon group comprising a substituent at a carbon in the gamma position relative to the ester or more remote from the ester moiety.
- R c may be an alkyl group, in particular a C1-C6 alkyl group, more in particular a methyl group, an ethyl group, a primary propyl group, a secondary propyl group, a primary butyl group, a secondary butyl group, or a tertiary butyl group.
- R A , R B , and n are as identified above.
- the alcohol HO- X 2 -Z or thiol HS-X 2 -Z used in the preparation of the (thio)ester generally is an activating alcohol or thiol.
- An activating alcohol or thiol is an alcohol or thiol which provides an activated C-terminal carboxy (thio)ester group after (thio)esterification, i.e. a carboxy (thio)ester group that can take part in the coupling reaction.
- the alcohol is represented by the formula HO-CX 2 -Z
- the thiol is represented by the formula HS-CX 2 -Z.
- each X independently represents a halogen atom (F, Cl, Br, I) or a hydrogen atom.
- a halogen atom F, Cl, Br, I
- a hydrogen atom it is preferably F. Good results have been achieved in a method of the invention wherein both X's are hydrogen.
- Z represents an electron withdrawing group. Without being bound by theory it is believed that this is advantageous with respect to the coupling reaction rate.
- Z represents an sp 2 or sp 3 hybridised carbon atom comprising one or more substituents comprising a heteroatom. It is in general difficult or at least laborious to chemically prepare C-terminal (thio)esters of N-terminal protected amino acids or N- terminal protected peptides and such an alcohol or thiol. Thus, the present invention is further in particular advantageous in that a relatively simple method is provided for preparing such esters.
- Z is selected from the group of sp 3 - hybridised carbons comprising one substituent comprising a heteroatom, which heteroatom is directly bound to the sp 3 -hybridised carbon.
- an N- terminal protected amino acid trifluoroethylester or an N-terminal protected peptide C- terminal 2,2,2-trifluoroethylester can be prepared enzymatically from an N-terminal protected amino acid or peptide and subsequently be used in an enzymatic coupling method, and even more that it is possible to accomplish this in a one-pot process with a good yield.
- Z is selected from the group of sp 2 -hybridised carbons comprising at least one substituent comprising a heteroatom, which heteroatom is directly bound to the sp 2 -hybridised carbon.
- R 1 may in particular be selected from the group of amine groups, such as -NH 2 , NHCH 3 or N(CH 3 ) 2 , (optionally) C-terminal protected alpha-amino acids or (optionally) C-terminal protected peptides, preferably -NH 2 ; -OH or a salt thereof such as an alkali or ammonium salt; alkyl or aryl groups; substituted alkyl or aryl groups, in particular substituted alkyl or aryl groups comprising one or more halogen substituents; -OR 2 ; -SR 2 .
- amine groups such as -NH 2 , NHCH 3 or N(CH 3 ) 2 , (optionally) C-terminal protected alpha-amino acids or (optionally) C-terminal protected peptides, preferably -NH 2 ; -OH or a salt thereof such as an alkali or ammonium salt; alkyl or aryl groups; substituted alkyl or
- the R 2 group usually is a hydrocarbon or a substituted hydrocarbon, in particular an alkyl, a substituted alkyl (such as an alkyl comprising one or more halogen substituents) an unsubstituted aryl group or a substituted aryl group (such as an aryl comprising one or more halogen substituents).
- the alkyl or substituted alkyl in R 1 or R 2 may in particular comprise 1-6 carbon atoms; the unsubstituted or substituted aryl group may in particular comprise 5-18 carbon atoms in the ring or rings, more in particular 6-12.
- the optionally C-terminal protected amino acid or optionally C- terminal protected peptide that is to be coupled with the (thio)ester may in principle be any amino acid, proteinogenic or non-proteinogenic, or any peptide, based on proteinogenic and/or non-proteinogenic amino acids.
- the optionally C-terminal protected amino acid or peptide may be represented by a compound of formula III:
- n each R A and each R B are as defined above.
- Q represents an amide group or an OR moiety (see below).
- the amide group may be represented by the formula NR 1 R 2 in which R 1 and R 2 may each individually represent any (substituted) alkyl or (substituted) aryl group.
- R 1 and R 2 may each individually represent any (substituted) alkyl or (substituted) aryl group.
- one out of R 1 and R 2 is a H atom and the other a (substituted) alkyl group.
- R 1 and R 2 are both H.
- R may represent a C-terminal protective group or a cation, for instance a monovalent cation, such as a tri- or tetrasubstituted ammonium ion or an alkaline metal cation or a H.
- R is a C- terminal protective group this may in particular be an optionally substituted alkyl group.
- it is a t-alkyl group, although in principle it also may be any other protective ester as known to a man skilled in the art.
- the t-alkyl may in principle be any protective tertiary alkyl group.
- the t-alkyl is selected from the group of t-butyl (2-methyl- 2-propyl), t-pentyl (2-methyl-2-butyl) and t-hexyl (2,3-dimethyl-2-butyl). Good results have in particular been achieved with t-butyl.
- Q represents an amide group or an OR moiety in which R is a protective group. However, at least in some embodiments such side-reactions are adequately avoided otherwise.
- (thio)esterification of the amino acid or peptide is catalysed by an enzyme, as is the coupling of the resultant (thio)ester to another peptide or amino acid.
- an enzyme any enzyme known in the art capable of catalyzing one or both of these reactions can be used.
- recombinant enzymes originating from a first organism, but actually produced in a (genetically modified) second organism are specifically meant to be included as enzymes from that first organism.
- organisms from which an enzyme used in a method of th e invention may be derived include Trichoderma species, such as from Trichoderma reesei; Rhizopus species, such as from Rhizopus oryzae; Bacillus species, such as from Bacillus licheniformis, Bacillus subtilis Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus alkalophilus, Bacillus halodurans ; Aspergillus species, such as from Aspergillus oryzae or Aspergillus niger; Streptomyces species, such as from Trichoderma reesei; Rhizopus species, such as from Rhizopus oryzae; Bacillus species, such as from Bacillus licheniformis, Bacillus subtilis Bacillus amyloliquefaciens, Bacillus clausii, Bacillus lentus, Bacillus alkalophilus, Bacillus hal
- Streptomyces caespitosus or Streptomyces griseus Candida species; fungi; Humicola species; Rhizoctonia species; Cytophagia; Mucor species; and animal tissue, in particular from pancreas, such as from porcine pancreas, bovine pancreas or sheep pancreas. It will be clear to the average person skilled in the art that use can also be made of mutants of naturally occurring (wild type) enzymes in a method according to the invention.
- Mutants of wild-type enzymes can for example be made by modifying the DNA encoding the wild-type enzymes using mutagenesis techniques known to the person skilled in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene shuffling, etc.) so that the DNA encodes an enzyme that differs by at least one amino acid from the wild-type enzyme or so that it encodes an enzyme that is shorter compared to the wild-type and by effecting the expression of the thus modified DNA in a suitable (host) cell.
- Mutants of the enzyme may have improved properties, for instance with respect to one or more of the following aspects: substrate scope, activity, stability, organic solvent resistance, temperature profile, synthesis/hydrolysis ratio and side reaction profile.
- a lipase and/or a protease are used to catalyse at least one of said reactions.
- a suitable lipase or esterase is selected from enzymes classifiable under EC 3.1.
- a carboxylic ester hydrolase (E. C. 3.1.1 ) or a thiolester hydrolase (E. C. 3.1.2) may be used.
- a lipase is used selected from the group of lipases from Candida. Good results, in particular with respect to catalysing the (thio)esterification, have been achieved with Candida antarctica lipase, in particular with Candida antarctica lipase B.
- a suitable protease is selected from enzymes classifiable under EC
- Proteases can be further divided into two subclasses, the exopeptidases, acting only near the terminus of a peptide, and the endopeptidases that act internally in a peptide.
- the enzymatic preparation of the ester or thioester is catalysed by a lipase or an esterase; and the enzymatic coupling is catalysed by a protease.
- the exopeptidases acting at the N-terminus of a peptide release a single amino acid (aminopeptidases E. C. 3.4.1 1 ) or dipeptides (dipeptidases, E. C. 3.4.13) or both dipeptides and tripeptides (dipeptidyl-peptidases or tripeptidyl- peptidases, E. C. 3.4.14).
- the peptidases acting at the C-terminus of a peptide are called carboxypeptidases and liberate a single amino acid (carboxypeptidases, E. C. 3.4.16-18) or a dipeptide (peptidyl-dipeptidases, E. C. 3.4.15).
- the carboxypeptidases are grouped according to their catalytic mechanism, e.g. serine-type carboxypeptidases (E. C. 3.4.16), metallocarboxypeptidases (E. C. 3.4.17), cysteine- type carboxypeptidases (E. C. 3.4.18) or cleave terminal amino acids that are substituted, cyclised, or that have isopeptide bonds (peptide bonds involving the side chain) (omega-peptidases, E. C. 3.4.19).
- catalytic mechanism e.g. serine-type carboxypeptidases (E. C. 3.4.16), metallocarboxypeptidases (E. C. 3.4.17), cysteine- type carboxypeptidases (E. C. 3.4.18) or cleave terminal amino acids that are substituted, cyclised, or that have isopeptide bonds (peptide bonds involving the side chain) (omega-peptidases, E. C. 3.4.19).
- an endopeptidase may be used, especially an endopeptidase selected from the group of serine endopeptidases (E. C. 3.4.21 ), cysteine endopeptidases (E. C. 3.4. 22), aspartic endopeptidases (E. C. 3.4.23) and metalloendopeptidases (E. C. 3.4.24).
- subtilisins particularly suitable is a serine endopeptidase selected from the group of subtilisins. Such enzyme has been found particularly suitable for catalysing the coupling.
- subtilisins are known in the art, see e.g. US 5,316,935 and the references cited therein.
- Subtilisin A is a commercially available subtilisin from Novozymes. Particularly preferred is subtilisin Carlsberg, which when used in combination with a lipase, such as Candida antarctica lipase, has been found particularly advantageous with respect to synthesising a peptide with good yield in a relatively short time.
- Alcalase® is a suitable source for subtilisin Carlsberg. This product is available from Novozymes (Bagsvaerd, Denmark). Alcalase® is a cheap and industrially available proteolytic enzyme mixture produced by Bacillus licheniformis (containing subtilisin Carlsberg as a major enzyme component).
- the enzyme such as Alcalase®
- the enzyme may be provided by the supplier as a liquid, in particular an aqueous liquid.
- the enzyme is preferably first isolated from undesired liquid, for instance excess water, or alcohols that cause an undesired side-reaction. This may suitably be accomplished by precipitating, usually followed by separation of the solid from the liquid, and/or drying. Precipitation may be accomplished using the alcohol, such as t-butanol, or an alcohol or thiol used in the method of the invention. In case another alcohol or thiol is used, care should be taken that such alcohol or thiol does not interfere adversely with the (thio)esterification reaction or the coupling reaction.
- At least one of the enzymes is immobilised on a solid support. At least in some embodiments this may result in an increased yield of synthesised peptide after a relatively short reaction time, in particular in a one-pot process wherein both an enzyme for the catalysis of the (thio)esterification and a different enzyme for the catalysis of the coupling are used.
- (thio)esterification in particular in case a lipase, such as Candida antarctica lipase, and a protease, such as subtilisin Carlsberg, are used.
- a lipase such as Candida antarctica lipase
- a protease such as subtilisin Carlsberg
- Alcalase cross-linked enzyme aggregates Alcalase-CLEAs
- Alcalase-CLEAs Alcalase cross-linked enzyme aggregates
- at least two different enzymes are used, at least one catalysing the preparation of the (thio)ester and at least one other catalysing the coupling, wherein the different enzymes are immobilised on the same support.
- thiol or alcohol used for the preparation of the (thio)ester present in large excess with respect to the amino acid or peptide that is to be esterified.
- the molar ratio of said thiol or alcohol to said amino acid or peptide can be 50:1 or less, preferably
- the ratio is at least 1 :1.
- said molar ratio is at least 3:1 , in particular at least 5:1 , in order to allow the (thio)esterification to proceed at an advantageous rate.
- a method of the invention is carried out under substantially non- aqueous conditions.
- a small amount of water may be desired, depending upon the enzyme(s), to enable the enzyme(s) to properly perform a desired catalytic activity.
- a trace of water e.g. of at least 0.005 wt. %, based on the liquid phase, may be desired.
- the water concentration may be at least 0.01 wt. % or at least 0.03 wt. %.
- a desired upper limit for the water concentration depends on the specific enzyme(s), the alcohol or thiol used, the nature of the peptide to be synthesised (e.g. size, the amino acids upon which the peptide is based), the desired final conversion and the desired reaction rate.
- the reaction medium usually contains, at least at the beginning of the activation or (trans)esterification less than 2.0 wt. % water, based on the weight of liquids in the reaction medium.
- the reaction medium may be dispersed in a second liquid phase or another liquid phase may be dispersed in the reaction medium.
- the specified water content is based on the weight of liquid in the phase wherein the (trans)esterification or activation reaction - at least predominantly - takes place.
- the water concentration may be less than 1.0 wt. % or less, at least at the beginning of the (thio)esterification.
- the method may be carried out in a medium containing 0.5 wt. % or less water, in particular 0.2 wt. % or less water, more in particular 0.1 wt. % or less water, at least at the beginning of the reaction, whilst still retaining substantial desired enzyme activity and a low, or even undetectable, undesired hydrolysis.
- an improved peptide synthesis rate has been observed in a reaction medium having a very low water content, in particular in a reaction medium having a water content of less than 0.5 wt. % or less than 0.1 wt. %, compared to a method carried out in a reaction medium comprising 1-2.5 % water.
- water that is formed, in particular during (thio)esterification may be removed continuously or intermittently.
- removal may be accomplished in a manner known in the art. Good results have in particular been achieved using molecular sieves.
- evaporation such as azeotropic removal using vacuum or distillation.
- the pH used (in as far as a pH exists in the chosen reaction medium) may be chosen within wide limits, as long as a pH is chosen at which the enzyme shows sufficient activity.
- a pH is usually known for the enzyme to be used and may be based on its known hydrolytic activity in an aqueous solution, or can be routinely determined, making use of a known substrate for the enzyme under known reaction conditions. It may in particular be chosen to be about neutral.
- alkaline or acidic conditions may be used, depending on the enzyme.
- the pH may be adjusted using an acid and/or a base or buffered with a suitable combination of an acid and a base. Suitable acids and bases are in particular those soluble in the reaction medium, e.g. from the group of ammonia and alcohol-soluble acids, e.g. acetic acid and formic acid.
- the temperature used is not critical, as long as a temperature is chosen at which the enzyme(s) used show sufficient activity and stability.
- a temperature is usually known for the enzyme(s) to be used or can be routinely determined, making use of a known substrate for the enzyme(s) under known reaction conditions.
- the temperature may be at least 0 0 C, in particular at least 15 0 C or at least 25 0 C.
- the temperature may preferably be at least 40 0 C.
- a desired maximum temperature depends upon the enzyme(s). In general such maximum temperature is known in the art, e.g.
- the temperature is usually 70 0 C or less, in particular 60 0 C or less or 45 0 C or less. However, in particular if one or more enzyme(s) from a thermophilic organism are used, the temperature may be chosen higher, for example up to 90°C.
- subtilisin in particular subtilisin Carlsberg (e.g. in Alcalase®) the temperature may advantageously be in the range of 25-60 0 C. In case a combination of Alcalase® and CaI-B is used the temperature may advantageously be in the range 30-55 0 C.
- the invention further relates to all possible combinations of different embodiments and/or preferred features according to the method according to the invention as described herein.
- TLC Thin layer chromatography
- 3A molsieves (8 to 12 mesh, Acros) were activated under reduced pressure at 200 0 C and t-butanol ('BuOH) was stored on these molsieves.
- 'BuOH was pre-heated to a liquid (45°C) before use.
- the gradient program was: 0-25 min linear gradient ramp from 5% to 98% eluent B and from 25.1-30 min with 5% eluent B (eluent A: 0.5 mL/L methane sulfonic acid (MSA) in H 2 O, eluent B 0.5 mL/L MSA in acetonitrile).
- eluent A 0.5 mL/L methane sulfonic acid (MSA) in H 2 O
- MSA methane sulfonic acid
- MSA methane sulfonic acid
- Alcalase-CLEA was purchased from CLEA-Technologies and contained 3.5 wt% water; the activity was 650 AGE units per gram (with 1 AGE unit catalyzing the formation of 1 ⁇ mol N-acetyl-glycine from N-acetyl-glycine ethyl ester at 40 0 C and pH 7.5).
- This Alcalase-CLEA was treated as follows before use: 1 g Alcalase- CLEA was suspended in 20 mL 'BuOH and crushed with a spatula. After filtration, this procedure was repeated with 20 mL MTBE.
- CaI-B Novozymes, lipase Novozym-435 from Candida Antarctica, batch n. LC200204 was used without any pre-treatment.
- Dipeptide reference compounds were chemically synthesized as follows: 1.0 mmol of amino acid amide HCI salt or amino acid 'Bu-ester HCI salt was dissolved in a mixture of 10 mL CHCI 3 and 30 mL EtOAc containing 355 ⁇ L diisopropylethylamine (DIPEA, 2.1 mmol, 2.1 eq). This solution was added to a solution (0°C) of 1 mmol N-Cbz-protected amino acid (1 eq), 207 mg N-(3- dimethylaminopropyl)-N'-ethylcarbodiimide.HCI (EDCI.
- DIPEA diisopropylethylamine
- N-Cbz-protected amino acid methyl esters were synthesized as follows: 100 mL MeOH was cooled to -80°C with a bath of dry-ice in acetone followed by dropwise addition of 4 ml. SOCI 2 (5.5 mmol, 3.7 eq). Subsequently, 1.5 mmol of the Cbz-protected amino acid was added and after removal of the bath the mixture was stirred at ambient temperature for 5 h. Volatiles were evaporated in vacuo and the remaining volatiles of the residue co-evaporated with MeOH (3x). The resulting residue was dried overnight in vacuo at ambient temperature yielding the pure N-Cbz-protected amino acid methyl ester.
- N-Cbz-protected amino acid carbamoylmethyl (Cam) or trifluoroethyl (Tfe) esters was performed as follows: 1 mmol of the N-Cbz- protected amino acid was dissolved in a mixture of 30 ml. EtOAc and 5 mmol of carbamoylmethanol (CamOH) or 2,2,2-trifluoroethanol (TfeOH) (5 eq). Subsequently, 207 mg EDCI. HCI (1.1 mmol, 1.1 eq), 150 mg HOAt (1.2 mmol, 1.2 eq), 292 ⁇ l_ triethylamine (TEA, 1.1 mmol, 1.1 eq) were added.
- Example 1 Enzymatic synthesis of dipeptides starting from Me, Cam and Tfe esters using alcalase-CLEA
- Alcalase-CLEA 300 mg Alcalase-CLEA was added to a mixture of 3 mL MTBE and 200 mg 3A molsieves. Subsequently, 100 mg N-Cbz-protected amino acid ester and 1.0 equiv. of C-terminal protected amino acid were added. The mixture was shaken at 5O 0 C at 150 rpm for 16h. After filtration, the solids were washed with 10 mL EtOAc (3 x).
- Protocol 1 Synthesis of Cam or Tfe esters using alcalase-CLEA
- 300 mg Alcalase-CLEA was added to a mixture of 3 ml. MTBE, 200 mg 3A molsieves, 200 mg of carbamoylmethanol or 2,2,2-trifluoroethanol and 50 mg N- Cbz-protected amino acid.
- the mixture was shaken at 5O 0 C at 150 rpm for 72 h. After filtration, the solids were washed with 10 ml. EtOAc (3 x).
- Protocol 2 Synthesis of Cam or Tfe esters using CaI-B
- Example 3 Enzymatic synthesis of dipeptides usinq Cam or Tfe alcohol as additive
- Protocol 1 Synthesis of dipeptides using Cam or Tfe alcohol as additive with alcalase- CLEA.
- Alcalase-CLEA 300 mg Alcalase-CLEA was added to a mixture of 3 ml. MTBE, 200 mg 3A molsieves and 200 mg of the appropriate alcohol. Subsequently, 50 mg N-Cbz- protected amino acid and 1 equiv. of amino acid amide were added. The mixture was shaken at 5O 0 C with 150 rpm for 16 h.
- Protocol 2 Synthesis of dipeptides using Cam or Tfe alcohol as additive with CaI-B.
- Protocol 3 Synthesis of dipeptides using Cam or Tfe alcohol as additives with Alcalase-CLEA and CaI-B.
- Example 4 Enzymatic synthesis of Tfe esters of di- and tripeptides using CaI-B.
- % P area% Tfe ester product / (area% starting compound + area% Tfe ester product).
- the reaction mixture of the esterification of Cbz-Ala-Ala to Cbz-Ala- Ala-OTfe was worked up as follows.
- the reaction mixture obtained after 7 days was filtrated and the solids were washed with 10 mL EtOAc (3 x).
- the volatiles in the resulting residue were removed by coevaporation with 20 ml. toluene (2 x) and with 20 ml. CHCI 3 (2 x).
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CN200980146237.8A CN102216324B (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
EP09752858A EP2365982A1 (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
JP2011543777A JP2012509089A (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
BRPI0921081A BRPI0921081A2 (en) | 2008-11-19 | 2009-11-19 | peptide synthesis using activation and enzymatic coupling. |
CA2744243A CA2744243A1 (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
US13/129,663 US8883444B2 (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
EA201100802A EA019536B1 (en) | 2008-11-19 | 2009-11-19 | Peptide synthesis using enzymatic activation and coupling |
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WO2013135786A1 (en) * | 2012-03-14 | 2013-09-19 | Dsm Fine Chemicals Austria Nfg. Gmbh & Co Kg | Enzymatic peptide synthesis |
US20150037840A1 (en) * | 2012-02-29 | 2015-02-05 | Enzypep B.V. | Side-chain protected oligopeptide fragment condensation using subtilisins in organic solvents |
EP3299085A1 (en) | 2016-09-26 | 2018-03-28 | Enzypep B.V. | Peptide coupling molecule |
US11492374B2 (en) | 2020-06-25 | 2022-11-08 | Humanwell Pharmaceutical US | Peptides for treatment of medical disorders |
US12036286B2 (en) | 2021-03-18 | 2024-07-16 | Seagen Inc. | Selective drug release from internalized conjugates of biologically active compounds |
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EP3204401A1 (en) * | 2014-10-10 | 2017-08-16 | Enzypep B.V. | Peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio |
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JPS5272885A (en) * | 1975-10-23 | 1977-06-17 | Sagami Chem Res Center | Preparation of peptide |
JPH06237784A (en) * | 1993-02-18 | 1994-08-30 | Daiwa Kasei Kk | Method for synthesizing oligopeptide |
NL1005901C2 (en) * | 1997-04-25 | 1998-10-27 | Dsm Nv | Method for the esterification of amino acids and peptides. |
WO2009047354A1 (en) * | 2007-10-12 | 2009-04-16 | Dsm Ip Assets B.V. | Chemo-enzymatic synthesis of a c-terminal thioester of an amino acid or a peptide |
WO2009047311A1 (en) * | 2007-10-12 | 2009-04-16 | Dsm Ip Assets B.V. | Chemo-enzymatic peptide synthesis via c-terminal ester interconversion |
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Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150037840A1 (en) * | 2012-02-29 | 2015-02-05 | Enzypep B.V. | Side-chain protected oligopeptide fragment condensation using subtilisins in organic solvents |
US9598714B2 (en) * | 2012-02-29 | 2017-03-21 | Enzypep B.V. | Side-chain protected oligopeptide fragment condensation using subtilisins in organic solvents |
WO2013135786A1 (en) * | 2012-03-14 | 2013-09-19 | Dsm Fine Chemicals Austria Nfg. Gmbh & Co Kg | Enzymatic peptide synthesis |
EP3299085A1 (en) | 2016-09-26 | 2018-03-28 | Enzypep B.V. | Peptide coupling molecule |
US11492374B2 (en) | 2020-06-25 | 2022-11-08 | Humanwell Pharmaceutical US | Peptides for treatment of medical disorders |
US12036286B2 (en) | 2021-03-18 | 2024-07-16 | Seagen Inc. | Selective drug release from internalized conjugates of biologically active compounds |
Also Published As
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KR20110088519A (en) | 2011-08-03 |
EP2365982A1 (en) | 2011-09-21 |
US8883444B2 (en) | 2014-11-11 |
EA019536B1 (en) | 2014-04-30 |
CN102216324A (en) | 2011-10-12 |
BRPI0921081A2 (en) | 2015-12-15 |
JP2012509089A (en) | 2012-04-19 |
US20120129214A1 (en) | 2012-05-24 |
EA201100802A1 (en) | 2011-12-30 |
CN102216324B (en) | 2014-02-12 |
CA2744243A1 (en) | 2010-05-27 |
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