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WO2024227880A1 - Diamines à liaison covalente et leur utilisation - Google Patents

Diamines à liaison covalente et leur utilisation Download PDF

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Publication number
WO2024227880A1
WO2024227880A1 PCT/EP2024/062132 EP2024062132W WO2024227880A1 WO 2024227880 A1 WO2024227880 A1 WO 2024227880A1 EP 2024062132 W EP2024062132 W EP 2024062132W WO 2024227880 A1 WO2024227880 A1 WO 2024227880A1
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Prior art keywords
formula
residue
bond
carbon
integer
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Inventor
Lothar Just
Corinna GLEISER
Nikolas Pietrzik
Hubert Kalbacher
Gerhard Feil
Bernhard HIRT
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Eberhard Karls Universitaet Tuebingen
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Eberhard Karls Universitaet Tuebingen
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Priority claimed from EP23171315.7A external-priority patent/EP4272556A1/fr
Application filed by Eberhard Karls Universitaet Tuebingen filed Critical Eberhard Karls Universitaet Tuebingen
Publication of WO2024227880A1 publication Critical patent/WO2024227880A1/fr
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/02—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests containing liquids as carriers, diluents or solvents
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N25/00—Biocides, pest repellants or attractants, or plant growth regulators, characterised by their forms, or by their non-active ingredients or by their methods of application, e.g. seed treatment or sequential application; Substances for reducing the noxious effect of the active ingredients to organisms other than pests
    • A01N25/34—Shaped forms, e.g. sheets, not provided for in any other sub-group of this main group
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/34—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom
    • A01N43/36—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one nitrogen atom as the only ring hetero atom five-membered rings
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
    • A01P1/00—Disinfectants; Antimicrobial compounds or mixtures thereof

Definitions

  • the invention provides a product of formula (I) obtained by covalently binding diamines to a substrate via a linker, its use for interfering with the function and/or the structure of a polypeptide as well as a method for preparing said product comprising the step of reacting a compound of formula (II) with a linking agent and optionally reacting the obtained reaction product with a substrate.
  • the invention also relates to the use of a compound of formula (II) for preparing a product of formula (I).
  • the covalently bound diamines still provide biocidal activity, in particular activity in interfering with the function and/or the structure of a polypeptide.
  • the products of formula (I) provide a highly beneficial long-term stability with reduced risk of leaching and undesired rinse-off of the diamines and allow for an improved weatherability and hydrolysis resistance.
  • Derivatives of 1 ,3-diamino propanes have long been used for disinfecting purposes and are contained in formulations for microbial control.
  • Commercially available diamines with biocidal activity include “C12”, i.e. /V-dodecyl-1 ,3-diamino propane (C 15 H34N 2 ; molecular weight 242.45; CAS 5538-95-4) and “Bis-C12”, i.e. / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3- diamino propane (C 18 H 41 N 3 ; molecular weight 299.54; CAS 2372-82-9).
  • Biocidal agents are, however, often associated with a negative environmental toxicological potential. Therefore, binding those agents including their immobilization is of great interest, yet technologically challenging.
  • diamines like 1 ,3-diamino propane derivatives possess different physical characteristics such as solubility in aqueous and non-aqueous media and adhesive behaviour towards surfaces.
  • their physical and chemical features such as solubility and liquid phases (states of matter)
  • solubility and liquid phases can be of tremendous importance.
  • liquid phase and fluidic parameters are important for technical process management.
  • good solubility can cause problems like leaching or undesired rinse-off.
  • a frequently used method to circumvent the problem of leaching is covalent bond formation.
  • substrate and active ingredient are bound by at least one chemically strong bond reducing the risk of leaching and undesired rinse-off.
  • 5-oxo-pyrrolidine-2-carboxylic acid derivatives such as Py-C12 (5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine) and Py-C14 (5-/V-carboxamido-2- pyrrolidone-/V’-n-tetradecyl propane-1 , 3-diamine)
  • the possibility of coupling, i.e. covalent binding, via its heterocyclic oxo-pyrrolidine ring has been raised in WO 2016/016168 A1 and WO 2016/016167 A1.
  • covalently bound diamines like propylenediamines are expected to lose their protein denaturation and biocidal functionalities, respectively, once coupled.
  • diamines in particular 1 ,3-diamino propanes
  • diamines can be coupled, i.e. bound covalently via an acyclic secondary or primary amino group of the diamine in an efficient and reliable manner to numerous substrates and with beneficial long-term stability without losing their protein denaturation and biocidal functionalities.
  • diamines like propylenediamines coupled in this way even maintain protein denaturation and biocidal functionalities.
  • the present invention provides a product obtained by covalent coupling of a diamine backbone to a substrate via a linker.
  • This is reflected in the below formula (I) by the fact that either at least R 2 or R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S.
  • S is a substrate and L is a linker, wherein L can be separate from or can form part of S.
  • the below formula (I) also defines that this covalent bond is achieved via a primary and/or an acyclic secondary amine of the diamine backbone.
  • the product has a formula (I): formula (I).
  • m is an integer from 1 to 6, preferably m is 3.
  • R 1 is H.
  • R 2 and R 3 are each independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, , a heterocyclic compound, an organic acid, including a carboxylate, a sulfonate, a phosphate, or a substituent selected from
  • R 5 , R 6 or R 7 is NH 2 or NH-L-S, and the other are H.
  • n and n’ are each independently an integer from 1 to 25.
  • Either at least R 2 or R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S. with n and n’ each being independently an integer from 1 to 25.
  • the present invention provides a method for preparing a product of formula (I).
  • the method comprises a step (a) of reacting, preferably incubating, a compound of formula (II) formula (II) with m being an integer from 1 to 6, preferably m being 3; and
  • R 1 being H
  • R 2 and R 3 being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, a sulfonate, a phosphate, or a substituent selected from and with n and n’ being an integer from 1 to 25 and with
  • R 4 being independently selected from CH 2 -C 6 H 5 , with n and n’ being an integer from 1 to 25, with a linking agent and optionally with a reducing agent.
  • the method optionally further comprises a step (b) of reacting the product of step (a) with a substrate.
  • the present invention also provides a product of formula (I) preparable or prepared by the method described above and the use of a compound of formula (II) as described above for preparing a product of formula (I).
  • Figure 1 Common means of covalent solid phase coupling with different linking agents.
  • the squares are representing the substrate.
  • Figure 2 Unit derived from pentaerythritol for dendrimer build up for forming a dendrimer type linking agent.
  • Figure 3 /V-dodecyl-1 ,3-diamino propane (C12).
  • Figure 4 /V-(a-glutaminyl amido)-/V’-n-dodecyl-1 ,3 diamino propane (aGlu-C12).
  • Figure 5 /V-(y-glutaminyl amido)-/V’-n-dodecyl-1 , 3-diamino propane (yGlu-C12).
  • Figure 6 /V’-(3-aminopropyl)-/V’-n-dodecyl-1 , 3-diamino propane (Bis-C12).
  • Figure 7 /V-oleyl- 1 ,3-diamino propane (Oleyl-C12).
  • Figure 10 Coupling of 5-/V-carboxamido-2-pyrrolidone-/V’-n-dodecyl propane-1 , 3- diamine (Py-C12) to NHS-activated carboxy groups on the surface of magnetic beads.
  • Figure 11 Coupling of /V-dodecyl-1 , 3-diamino propane (C12) to NHS-activated carboxy groups on the surface of magnetic beads.
  • Figure 12 Coupling of /V’-(3-aminopropyl)-/V’-n-dodecyl-1 , 3-diamino propane (Bis-
  • Figure 13 Coupling of /V-(a-glutaminyl amido)-/V’-n-dodecyl-1 , 3-diamino propane
  • FIG. 14 Diagram showing the results of a FRET-based endoprotease assay for determining the effect of Py-C12 (2 mM), C12 (500 .M), and Bis-C12 (100 jiM) coupled to NHS-activated beads (A) or not coupled (B) on collagenase activity. For comparison also a positive (aminoethanol- coupled beads, Pos.) and negative control were measured.
  • Figure 15 Diagram showing the results of a FRET-based endoprotease-assay for determining the stability of the coupling of Py-C12 (2 mM), C12 (500
  • Figure 16 Diagram showing the results of a FRET-based endoprotease-assay for determining the effect of aGlu-C12 ( range of 0.03 pmol to 0.59 pmol) on collagenase activity. For comparison, a positive (untreated collagenase, Pos.) and negative control were applied.
  • Figure 17 Diagram showing the results of a FRET-based endoprotease-assay for determining the effect of magnetic beads coupled to Py-Acetyl-C12 (5 mM and 10 mM) on collagenase activity compared to uncoupled Py- Acetyl-C12 (5 mM and 10 mM). For comparison, a positive (aminoethanol-coupled beads, Pos.) and negative control were applied.
  • Figure 18 Coupling methodology for /V-dodecyl-1 ,3-diamino propane (C12) to a polysaccharide backbone via Malaprade reaction.
  • Figure 19 Coupling methodology for / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-C12) to a polysaccharide backbone via Malaprade reaction.
  • Figure 20 Cellulose sheets coupled to C12 (left side), to Bis-C12 (middle) and a control sheet (right side without any coupled 1 ,3-diamino propane) after intensive washing steps and subsequent inoculation with a beta- galactosidase-expressing E. coli strain and incubation at 37 °C for 2 days.
  • Figure 23 MALDI-MS spectrum showing the characteristic peak for Di-(5- N- carboxamido-2-pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py- C12)-adduct to trichlorotriazine.
  • Figure 24 Schematic two step coupling of 5-/V-carboxamido-2-pyrrolidone-/ ⁇ /’-n- dodecyl propane-1 , 3-diamine (Py-C12) to cellulose using cyanuric chloride.
  • Figure 25 Schematic two step coupling of /V-dodecyl-1 ,3-diamino propane (C12) to cellulose using cyanuric chloride.
  • Figure 26 Schematic two step coupling of / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3- diamino propane (Bis-C12) to cellulose using cyanuric chloride.
  • Figure 27 Cellulose sheets coupled to Py-C12 (first from left), C12 (second from left), Bis-C12 (second from right) and a control (cellulose sheet without coupled 1 ,3-diamino propane, first from right) after intensive washing steps with water and subsequent inoculation with a cellulase solution at 37 °C for 28 days.
  • Figure 28 Cellulose sheets coupled to Py-C12 (first from left), C12 (second from left), Bis-C12 (second from right) and a control (cellulose sheet without coupled 1 ,3-diamino propane, first from right) after intensive washing steps with water and subsequent inoculation with a beta-galactosidase- expressing E. coli strain.
  • the invention is based on the finding that diamines, in particular 1 ,3-diamino propanes, can be covalently bound to a substrate via a linker with long-term stability and in particular without losing their biocidal activity.
  • the invention is based on the surprising finding that coupling via an acyclic amino function of the diamine backbone, preferably via a primary or an acyclic secondary amino function of the diamine backbone, enables advantageous long-term stability and biocidal effects.
  • acyclic amino functions were considered necessary for exerting the biocidal and protein denaturation functionalities.
  • linkage via such amino groups was expected to reduce the biocidal activity or a loss of biocidal activity.
  • a change in the spatial orientation when coupling an acyclic secondary amino group that is no head group was expected to further reduce the biocidal activity.
  • especially coupling to an acyclic secondary amino group that is no head group was associated with a loss of biocidal efficacy.
  • the invention relates to a product of formula (I): formula (I) with m being an integer from 1 to 6, preferably m is 3 and with
  • R 1 being H
  • R 2 and R 3 each being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, a sulfonate, a phosphate, or a substituent selected from with one of R 5 , R 6 and R 7 being NH 2 or NH-L-S, and the other being H, and with n and n’ each being independently an integer from 1 to 25, and with the proviso that either at least R 2 or R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S. with n and n’ each being independently an integer from 1 to 25.
  • the product of formula (I) can be present in form of a stereoisomer, a racemic mixture, a tautomer, a solvate such as a hydrate or a salt.
  • m is an integer from 1 to 6.
  • m is an integer from 2 to 4, more preferably it is an integer of 2 or 3, still more preferably “m” is 3 that is also referred to as “propylenediamines”.
  • n and “n”’ are each independently selected from an integer from 1 to 25.
  • n and “n”’ are independently selected from an integer from 1 to 15, further preferred from 1 to 12, still further preferred from 3 to 12 and still more preferred from 3 to 11.
  • R 2 and R 3 are independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide residue bound to the nitrogen via N-C-bond preferably via a carbon of the saccharide residue or via the carbon of a linker L’ attached to the saccharide via an ester or ether bond, a polyalcohol residue bound to the nitrogen via N-C-bond preferably via a carbon of a linker L’ attached to the polyalcohol via an ester or ether bond, a peptide residue bound to the nitrogen via an amide bond preferably via the carbon of the carboxyl group, a nucleic acid residue bound to the nitrogen via N-C-bond preferably via a carbon of a linker L’ attached to the nucleic acid residue via an ester or ether bond , an amino acid residue bound to the nitrogen via an amide bond preferably via the carbon of the carboxyl group, or an organic acid residue bound to the nitrogen via an amide bond preferably via the carbon of the carboxyl group
  • R 2 can be an amino acid or an amino acid derivative, wherein any enantiomer of the amino acid or amino acid derivative is encompassed.
  • R 2 may be a stereoisomer of a natural amino acid or a non-natural amino acid or derivative thereof.
  • R 2 may comprise a heterocyclic ring, in particular it can be an amino acid that forms a heterocyclic ring, preferably an oxo-pyrrolidine ring or a pyrrolidine ring.
  • a heterocyclic ring refers to a saturated or unsaturated ring that incorporates one or more heteroatoms, i.e. O, N and/or S.
  • Preferred amino acids or amino acid derivatives are pyroglutamic acid (5-oxopyrrolidine- 2-carboxylic acid) or a derivative thereof or glutamic acid or a derivative thereof.
  • Preferred pyroglutamic acid or derivatives thereof correspond to general formula (III): formula (III) with R 8 and R 9 being selected from with p being an integer from 0 to 20 and with X being O or S.
  • R 8 and R 9 are both H.
  • Preferred glutamic acid or derivatives thereof have a structure of formula (IV) formula (IV) with R 5 or R 7 being NH 2 or NH-L-S and the other of R 5 , R 6 and R 7 being H.
  • one of R 3 and R 4 is selected from L-S, H, and the other is selected from
  • one of R 3 and R 4 is L-S, H or n and the other is , with n and n’ being independently selected from an integer from 3 to 12, still more preferred from 3 to 11.
  • one of R 3 and R 4 is L-S or H and the other is , with n being an integer between 4 to 12, preferably 8 to 12, most preferably 11.
  • R 2 is H, L-S or an amino acid or an amino acid derivative, wherein the amino acid or amino acid derivative is selected from the group consisting of: er from 0 to 20 and with X being O or S and with one of R 5 , R 6 and R 7 being NH 2 or NH-L-S, and the other being H, and with the proviso that R 2 or R 3 is L-S or one of R 5 , R 6 and R 7 is NH-L-S.
  • R 2 is with R 8 and R 9 being both H, and with R 3 being L-S and R 4 being , with n being an integer from 8 to 12.
  • R 2 is with R 5 or R 7 being NH 2 or NH-L-S and the other of R 5 , R 6 , and R 7 being H, and with R 3 being H or L-S and R 4 being , with n being an integer from 8 to 12, with the proviso that either R 3 is L-S or R 5 or R 7 is NH-L-S.
  • R 2 is L-S with R 3 being selected from
  • R 2 is L-S, and one of R 3 and R 4 is , with n being an integer of 11 and the other is H.
  • R 2 is H
  • R 3 is L-S
  • R 4 is , with n being an integer of 11 . of 11.
  • R 2 is L-S
  • one of R 3 and R 4 is n and the other is , with n being an integer of 7 to 11 , preferably an integer of 7 or 11.
  • R 2 is L-S, one of R 3 and R 4 is n and the other being an integer of 8 and n’ being an integer of 7.
  • R 3 is
  • L-S and R 4 is , with n being an integer of 1 to 25, preferably of 11.
  • the product of formula (I) comprises a diamine backbone.
  • This term as used herein refers to the component of the product that is defined by the above formula (II) excluding the L-S substituent.
  • L-S substituent within the structure of the compound of formula (II)
  • the diamine backbone is preferably a 1 ,3-diamino propane backbone, i.e. m is preferably 3.
  • the diamine backbone is covalently bound to a substrate via a linker.
  • This is reflected in the above formula (I) by the fact that either at least one of R 2 and R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S.
  • the above formula (I) defines that this covalent bond is via a primary and/or an acyclic secondary amine of the diamine backbone.
  • the linker preferably increases the distance between the diamine backbone and the substrate and may also be used to adjust the spatial orientation and structural arrangement, respectively, of the diamine backbone.
  • product encompasses any form like an article, a conjugate, a compound and so on without any limitation regarding its size or dimensions.
  • linker between diamine backbone and substrate. It is especially preferred that the linker retains the functionality of the diamine and optionally induces a certain type of structural arrangement.
  • a linker is derived from a chemical molecule comprising at least one functional group or it is derived from a functional unit that can link a substrate (e.g. a macromolecular structure, or a surface) and an active or functional substance based on chemical reactions (e.g. addition to epoxides and active esters).
  • Linking or linkage as used herein means covalently connecting atoms or atom groups.
  • Linker as used herein encompasses a molecule with specific features, like a tether to a functional group enabling covalent coupling, single atoms or even covalent bonds, preferably two-centre two-electron bond types.
  • the linker according to the present invention can be derived from a functional group or it can be derived from a molecule comprising at least one functional group that is or is not already attached to or forms part of the substrate.
  • the term linker thus encompasses embodiments in which the functional group or molecule is naturally present on the substrate and likewise in which it is generated by chemical or physical modifications on the substrate.
  • linkers are derived from functional groups that are attached to a substrate (e.g. through chemical modification of the surface) and that can be reacted with at least one primary or acyclic secondary amino group of the diamine.
  • the linker is derived from a function group forming part of the substrate, in particular being naturally present on the substrate, whose functional group can be reacted with at least one primary or acyclic secondary amino group of the diamine.
  • a linker is the residue obtainable by reacting a linking agent optionally coupled to a substrate with a primary or acyclic secondary amino group of the diamine backbone.
  • An intermediate product is a linking agent coupled to a substrate, which still comprises a functional group allowing bond formation with a primary or acyclic secondary amino group of the diamine.
  • An alternative intermediate product is a linking agent coupled to a primary or acyclic secondary amino group of the diamine backbone, which still comprises a functional group allowing bond formation with the substrate.
  • linking agents Commercially available linking agents, substrates and intermediate products of linking agents coupled to a substrate can be applied, according to the present invention.
  • nitrile CNj, aryl sulfonyl halide, alkyl sulfonyl halide, aldehyde, ketone, dialdehyde, epoxide, di- and polydentate epoxy ether (e.g.
  • crosslinkers and hardeners of the state of the art halogenated /V-heterocycle, aryl halide (such as aryl chloride), alkyl halide (such as alkyl chloride), carboxylic acid ester, imidoester, carbodiimide, double or triple bond, hydroxyl, peroxide, carboxylate, carbonate, amine, anhydride, ester, ether, thiol or sulfonate.
  • linkers can be divided into permanent and labile linkers; the latter can be cleaved again by a specific reaction, liberating the covalently bound structures.
  • any combinations of linkers L, diamines, and substrates are possible.
  • Fig. 1 shows exemplary covalent solid phase couplings of different linking agents, wherein the squares represent the substrate.
  • the linking agent is a /V-hydroxysuccinimide (NHS)-activated ester.
  • the NHS-activated ester is coupled to the substrate, in particular comprising a polymer, more preferably forming NHS-activated magnetic beads as intermediates, namely as linking agents coupled to substrate.
  • the molar rate of covalently bound molecules is an important technical parameter, often called ..degree of loading".
  • a special form of linking agent is used that has several anchoring units, thus multiplying or even diversifying the possible degree of loading.
  • multifunctionalized linking agents and linkers, respectively like dendrimer type linking agents and linkers, respectively, are preferably applied further, increasing the biocidal activity.
  • the linker comprises a dendrimeric structure, i.e.
  • the linking agent preferably comprises several repeating units and/or carries more than one functional group so that one linking agent can be coupled to more than one compound of formula (II).
  • the linking agent is 1 ,1 ,1-tris-(hydroxymethyl)-propane triglycidyl ether (TMPTGE).
  • the linking agent is cyanuric chloride (trichlorotriazine). Cyanuric chloride is shown in Fig. 21. Cyanuric chloride is a trident linking agent capable of cross-linking substrates, e.g. polysaccharide fibres, cellulose fibres, polyols. It can be used for forming dendrimer type linking agents.
  • Cyanuric chloride is a well-known and widely applied linking agent for polysaccharides, e.g. for cellulose.
  • At least one of R 2 and R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S as defined above.
  • one of R 2 and R 3 is L-S or one of R 5 and R 7 is NH-L-S.
  • L-S can be derived from NHS-activated magnetic beads as linking agent coupled to a substrate.
  • the linker L’ may be employed to further attach a saccharide residue, a polyalcohol residue or a nucleic acid residue to at least one primary or acyclic secondary amino group of the diamine, wherein the linker is bound to the nitrogen via N-C- bond.
  • Definitions for linkers given above are also valid for linker L’, however in this embodiment, the linker L’ is bound to the respective residue via a functional group derived from an ether or ester.
  • Suitable linkers L’ may be selected from ...
  • a substrate may be any macromolecular structure, or surface.
  • the substrate comprises reactive chemical groups that allow the coupling of the linking agent to the substrate or the surface of the substrate has been treated such as activated, e.g. by plasma activation (or plasma functionalization), by generating free radicals and/or by anchoring functional groups, such as ligands, whose functional groups include, for example, hydroxyl, carbonyl, peroxyl, carboxyl, amino or amines forming the linking agent for linkage to the diamine backbone.
  • substrate encompasses any material including absorbents, adsorbents, hygroscopic and porous materials, such as fabrics (textiles), e.g. cotton, wool, hemp, silk, polyester, polyamide; building materials, e.g. straw, glass wool, rock wool, silicone, gypsum, lime, cement, concrete, bricks, porous and absorbent stones, clay; glues and gels, biological and synthetic sponges for cleaning and medical applications, porous and sintered metals, porous and absorbent ceramics, biological tissues, natural and processed biomaterials, e.g. extracellular matrix molecules, scaffolds, bone, wood, leather, paper, cardboard, resins, plastics and composite materials.
  • fabrics e.g. cotton, wool, hemp, silk, polyester, polyamide
  • building materials e.g. straw, glass wool, rock wool, silicone, gypsum, lime, cement, concrete, bricks, porous and absorbent stones, clay
  • glues and gels e.g. porous and sintered metals,
  • the term also includes polymers, such as, e.g. polyamides, polyurethane, polypeptides, polyols or polyesters.
  • the polyols include polysaccharides, in particular cellulose and chitosan.
  • the substrate is selected from a synthetic sponge, a fabric, leather, bone tissue or wood.
  • the substrate is a fabric and the fabric comprises cellulose, silk or a polyester, more preferably the fabric is cotton, i.e. the fabric comprises cellulose and may additionally comprise waxes, fats, pectins and/or water.
  • the substrate is in an embodiment selected from metal, glass, ceramic, polymer, textile and/or natural substance such as wood.
  • the substrate can comprise a polysaccharide, such as cellulose.
  • the substrate may also be a powdered polysaccharide, such as chitosan and carboxymethylcellulose.
  • the substrate can also be a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, sulfonate, and phosphate.
  • the substrate can also comprise a polymer forming magnetic particles or magnetic beads, in particular magnetic beads comprising carboxy groups. Said functionalized magnetic beads can be activated with NHS, thus forming NHS-activated magnetic beads as linking agent coupled to the substrate.
  • a further group of substrates are polyalcohols.
  • Polyalcohols are an important group of substrates.
  • Polyalcohols may also comprise different ligands (i.e. oligoligand polysaccharides) that may act as linkers and linking agents, respectively.
  • a substrate can also be a specially treated surface that allows the coupling of diamines to the substrate.
  • Surface activation and modification for the covalent binding of diamines to many substrates and materials such as metals, glass, ceramics, polymers, textiles and natural materials may be carried out by plasma treatment.
  • the surface can be activated by generating free radicals and by anchoring functional groups, such as hydroxyl, carbonyl, peroxyl, carboxyl, or amines.
  • the present invention provides a method for preparing a product of formula (I) as defined above, comprising the steps of:
  • R 1 being H
  • R 2 and R 3 being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, sulfonate and phosphate, or a substituent selected from with one of R 5 , R 6 and R 7 being NH 2 , and the other being H, and with n and n’ being an integer from 1 to 25 and with R 4 being selected from CH 2 -C 6 H 5 ,
  • R 2 and R 3 are independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide residue bound to the nitrogen via N-C-bond preferably via a carbon of the saccharide residue or via the carbon of a linker L’ attached to the saccharide via an ester or ether bond, a polyalcohol residue bound to the nitrogen via N-C-bond preferably via a carbon of a linker L’ attached to the polyalcohol via an ester or ether bond, a peptide residue bound to the nitrogen via an amide bond preferably via the carbon of the carboxyl group, a nucleic acid residue bound to the nitrogen via N-C-bond preferably via a carbon of a linker L’ attached to the nucleic acid residue via an ester or ether
  • the compound of formula (II) can be a stereoisomer, a racemic mixture, a solvate, such as a hydrate or a salt.
  • the compound of formula (II) is selected from the group consisting of / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-C12), /V-oleyl-1 ,3-diamino propane (Oleyl-C12), /V-dodecyl-1 ,3-diamino propane (C12), 5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-C12), /V-(a-glutaminyl amido)-/V’-n- dodecyl propane-1 , 3-diamine (aGlu-C12) and /V-(y-glutaminyl amido)-/V’-n-dodecyl propane-1 , 3-diamine (yGlu-C12) or mixtures thereof.
  • the compound of formula (II) is Py-C12, referred to as 5-/V-carboxamido-2-pyrrolidone-/ ⁇ /’-n-dodecyl propane-1 , 3-diamine.
  • the compound of formula (II) is reacted with a linking agent optionally bound to a substrate or naturally present on the substrate, and optionally a reducing agent.
  • the linking agent reacts at least with a primary or an acyclic secondary amino group of the diamine of formula (II).
  • compounds of formula (II) are reacted with a linking agent bound to a substrate or naturally present on the substrate.
  • the linking agent can be bound to the substrate forming NHS-activated magnetic beads.
  • said NHS-activated magnetic beads react with at least one primary or acyclic secondary amino group of the diamine of formula (II).
  • compounds of formula (II) are reacted with a linking agent and reacted with a substrate and optionally reacted with a reducing agent.
  • “Reacting” in general means mixing the components, in particular in the presence of a reaction medium, in particular it means incubating the components in a reaction medium at a certain temperature and for a certain time.
  • a reaction in which the compound of formula (II), the linking agent, the substrate and optionally the reducing agent are reacted may be any form of carbonyl-amine condensation, preferably a Malaprade reaction.
  • Malaprade reaction compounds containing two hydroxyl groups, or a hydroxyl and an amino group, attached to adjacent carbon atoms, undergo cleavage of the carbon-carbon bond when treated with periodic acid to yield aldehyde groups.
  • the compound of formula (II) is reacted under condensation with an aldehyde group and optionally the condensation product is reduced with a reducing agent.
  • the method using the Malaprade reaction is especially suitable for 1 ,4- and 1 ,6-linked polysaccharides as substrates.
  • Exemplary Malaprade reactions are depicted in Fig. 18 and 19.
  • a polysaccharide is cleaved with periodate, reacted with the diamine of formula (II) and subsequently reduced with a reducing agent.
  • the reducing agent is selected from the group of hydrogen or hydride evolving compounds, preferably boron species, preferably borohydrides and borane amines, preferably sodium cyanoborohydride or mixtures thereof.
  • the ratio of substrate : linking agent : compound of formula (II) is preferably between 1 :2:2 and 1 :1.5: 1.5.
  • the ratio of substrate : linking agent : compound of formula (II) is selected according to desired product specifications, as are the degree of loading, solubility, layer thickness etc. and depends on the chemical features of the substrate to be coated. Ratios applied herein vary between 1 :2:2 and 1 :1.5: 1.5.
  • the method for preparing the product of formula (I) is preferably carried out in a basic reaction medium.
  • the pH of the reaction medium is preferably within the range of 7.5 to 13, more preferably within the range of 8 to 10, most preferably within the range of 8.5 to 9.5, further preferred the pH is about 8.5.
  • the 1 ,3-diamino propane is incubated with the linking agent coupled to a substrate, such as with NHS-activated magnetic beads, for 30 minutes to 10 h, preferably for 1 to 3 hours, more preferably for about 2 hours at a temperature of 10 °C to 30 °C, preferably at about 20 °C to 25 °C, preferably at room temperature.
  • a substrate such as with NHS-activated magnetic beads
  • At least one washing step is performed, e.g. with water optionally with borate, and optionally at least one quenching step with a quenching solvent, e.g. aminoethanol.
  • a quenching solvent e.g. aminoethanol.
  • cellulose substrate is treated with the linking agent, in particular cyanuric chloride, followed by incubation with the diamine for at most 1 hour, in particular for 10 minutes to 40 minutes, more preferably for about 30 minutes at a temperature of 10 °C to 30 °C, preferably at about 20 °C to 25 °C, preferably at room temperature, preferably followed by at least one washing step.
  • the linking agent in particular cyanuric chloride
  • samples are incubated with the compound of formula (II) for at least 30 minutes to 40 hours, preferably for 10 hours to 30 hours, more preferred for about 20 hours preferably at a temperature of 20 °C to 70 °C, more preferred 40 °C to 60 °C and most preferred at about 50 °C.
  • the method of the present invention leads to a covalent binding of the primary or acyclic secondary amino group of the compound of formula (II) to the substrate via the linker.
  • the covalent binding preferably does not lead to a change of the spatial orientation of the compound of formula (II) and avoids leaching from the substrate.
  • the product of formula (I) provides long term stability which is intended to mean a reduced risk of leaching and rinse-off of the diamine backbone and biocide, respectively, and prevents enzymatic degradation of the product of formula (I).
  • Preferred is less than about 20 %, preferably less than about 10 %, leaching and rinse-off and/or enzymatic degradation of the product of formula (I) after about 2 weeks, preferably after about 3 weeks and more preferably after about 4 weeks.
  • the covalent binding of at least one primary or acyclic secondary amino group of the diamine backbone, especially of an acyclic secondary amino group does not interfere with the biocidal activity of the compound of formula (II). More preferably, the biocidal activity is maintained or even increased by covalently binding the compound of formula (II) to the substrate compared with the compound of formula (II), which is not coupled to a substrate.
  • Biocidal activity as used herein, in particular means that the compounds interfere with the function and/or the structure of at least one polypeptide, in particular a protein. I.e. the present invention specifically refers to the ability of the diamine backbone of the product of formula (I) to still interfere with the function and/or the structure of at least one polypeptide as a specific form of biocidal activity.
  • “interfering with the function and/or the structure of a polypeptide” means a targeted change in function and/or structure of a polypeptide.
  • This change includes an increase, i.e. a positive change, or a decrease, i.e. a negative change.
  • the change in function and/or structure of a polypeptide is a decrease, i.e. a negative change of its function and/or structure.
  • it is a decrease of the function of a polypeptide, i.e. a decrease of the enzyme activity, in particular a denaturation of the polypeptide.
  • the polypeptide is an enzyme and the decrease of its function is an inhibition of enzyme activity.
  • polypeptide The function of a polypeptide is linked to its three-dimensional form, which includes secondary, tertiary, and, where applicable, quaternary structures. Interfering with its structure may also be referred to as denaturation, i.e. a process in which the polypeptide loses, at least in part, its three-dimensional form.
  • polypeptide is an enzyme
  • the relevant function might be referred to as an “activity”, and loss thereof might be referred to as “inhibiting” or “inactivating”.
  • polypeptide is not a prion.
  • the product of formula (I) is used for inhibiting an enzyme, i.e. for inhibiting enzyme activity, in particular by polypeptide-denaturation.
  • This “use of the product of formula (I) for inhibiting an enzyme, i.e. for inhibiting enzyme activity” is based on the biocidal activity of the diamine backbone, more specifically its ability to interfere with the function and/or the structure of a polypeptide.
  • the decrease or inhibition of enzyme activity refers to a decrease in activity of the enzyme of at least about 30 %, more preferably of at least about 50 %, preferably of at least about 70 %, more preferably of at least about 80 %, and most preferably of at least about 90 %.
  • the inhibition of enzyme activity, in particular of protease activity by polypeptide-denaturation, of the diamine backbone of the product of formula (I) is at least maintained.
  • the inhibition of enzyme activity namely the decrease in activity of the enzyme exerted by the product of formula (I), more specifically by the diamine backbone and coupled compound of formula (II), respectively, is at least 80 % of the enzyme inhibition determined with the same amount in relation to the compound of formula (II) in uncoupled form and under the same test conditions.
  • the inhibition of enzyme activity, in particular of protease activity is at least 90 %, further preferred at least 100 % of the enzyme inhibition exerted by compound (II) in uncoupled form, still further preferred at least 110 %.
  • Such enzyme activities include those of oxidoreductases, such as dehydrogenases, peroxidases, dioxygenases; transferases, such as methyltransferase, kinases, aminotransferases polymerases; hydrolases, such as esterases, glycosylases, peptidases/proteinases; lyases such as dehydratases; isomerases, such as racemases, cis-trans isomerases, intramolecular oxidoreductases, intramolecular transferases; or ligases.
  • oxidoreductases such as dehydrogenases, peroxidases, dioxygenases
  • transferases such as methyltransferase, kinases, aminotransferases polymerases
  • hydrolases such as esterases, glycosylases, peptidases/proteinases
  • lyases such as dehydratases
  • isomerases such as racemases, cis
  • the enzyme is selected from an oxidoreductase, a transferase, a hydrolase or an isomerase.
  • the enzyme is selected from an oxidoreductase, preferably from a dioxygenase and/or a peroxidase, more preferably from cyclooxygenase and/or a myeloperoxidase.
  • the enzyme is selected from a hydrolase, preferably from an esterase, lipase, peptidase/proteinase and/or glycoside hydrolase, more preferably from a nuclease, a phospholipase, a serinprotease, a cellulase, a laminarinase and/or a xylanase, further preferably from a ribonuclease (RNAse), a deoxyribonuclease (DNAse), thrombin, an elastase and/or a collagenase.
  • RNAse ribonuclease
  • DNAse deoxyribonuclease
  • thrombin an elastase and/or a collagenase.
  • the present invention is directed to the product of formula (I) prepared by a method as described above. Furthermore, the invention also relates to the use of a compound of formula (II) as described above for preparing a product of formula (I) as described above.
  • the product of formula (I) as described above for interfering with the function and/or the structure of a polypeptide, in particular an enzyme, further preferred a oxidoreductase, a transferase, a hydrolase or a isomerase and wherein the polypeptide is not a prion.
  • the invention relates to the use of the product of formula (I) as described above for inhibiting an enzyme, i.e. for inhibiting enzyme activity, in particular by polypeptide denaturation.
  • the enzyme is selected from an oxidoreductase, more preferably from a dioxygenase and/or a peroxidase, further preferably from cyclooxygenase and/or a myeloperoxidase.
  • the enzyme is selected from a hydrolase, more preferably from an esterase, lipase, peptidase/proteinase and/or glycoside hydrolase, further preferably from a nuclease, a phospholipase, a serinprotease, a cellulase, a laminarinase and/or a xylanase, still further preferably from a ribonuclease (RNAse), a deoxyribonuclease (DNAse), thrombin, an elastase and/or a collagenase.
  • RNAse ribonuclease
  • DNAse deoxyribonuclease
  • thrombin an elastase and/or a collagenase.
  • Embodiment 1 A product of formula formula (I) with m being an integer from 1 to 6,
  • R 1 being H
  • R 2 and R 3 being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, an organic acid, including a carboxylate, a sulfonate, a phosphate, or a substituent selected from ker and S being a substrate, with one of R 5 , R 6 and R 7 being NH 2 or NH-L-S, and the other being H, and with n and n’ being independently an integer from 1 to 25 and with the proviso that either at least R 2 or R 3 is L-S and/or at least one of R 5 , R 6 and R 7 is NH-L-S and with R 4 being selected from
  • I n with n and n’ being an integer from 1 to 25.
  • Embodiment 2 The product of embodiment 1 , wherein R 2 is H, L-S, or an amino acid or an amino acid derivative, wherein the amino acid or amino acid derivative is selected from the group consisting of: r from 0 to
  • R 5 , R 6 or R 7 being NH 2 or NH-L-S, and the other being H
  • R 3 being selected from with R 4 being selected from and with the proviso that R 2 or R 3 is L-S or one of R 5 , R 6 and R 7 is NH-L-S.
  • Embodiment 3 The product of embodiment 2, wherein R 2 is an amino acid that is pyroglutamic acid of formula (III) with R 8 and R 9 both being H, and with R 3 being L-S and R 4 being , with n being an integer from 8 to 12.
  • Embodiment 4 The product of embodiment 2, wherein R 2 is with R 5 or R 7 being NH 2 or NH-L-S and the other of R 5 , R 6 , and R 7 being H, and with R 3 being H or L-S and R 4 being , with n being an integer from 8 to 12, with the provision that either R 3 is L-S or R 5 or R 7 is NH-L-S.
  • Embodiment 5 The product of embodiment 2, wherein R 2 is L-S and R 3 is selected from 11.
  • Embodiment 7 The product of any of embodiments 1 to 6, wherein the substrate comprises a polymer, glass, ceramic, metal or textile.
  • Embodiment 8 The product of embodiment 7, wherein the substrate comprises a polymer selected from a polysaccharide or a polyalcohol.
  • Embodiment 9 The product of embodiment 8, wherein the substrate comprises cellulose.
  • Embodiment 10 A method for preparing a product of formula (I) of any of embodiments 1 to 9, comprising the steps of:
  • R 2 and R 3 being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, sulfonate, phosphate or a substituent selected from with one of R 5 , R 6 and R 7 being NH 2 , and the other being H, and with n and n’ being an integer from 1 to 25 and with R 4 being selected from
  • n and n’ being an integer from 1 to 25, with a linking agent and optionally with a reducing agent; and (b) optionally reacting the product of step (a) with a substrate.
  • Embodiment 11 The method of embodiment 10, wherein the compound of formula (II) is selected from the group consisting of / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3- diamino propane (Bis-C12), /V-oleyl-1 ,3-diamino propane (Oleyl-C12), /V-dodecyl-
  • Embodiment 12 The method of any of embodiments 10 or 11 , wherein the compound of formula (II) is selected from the group consisting of 5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-C12), /V-dodecyl-1 ,3-diamino propane (C12), / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-012), and /V-(a-glutaminyl amido)-/V’-n-dodecyl propane-1 , 3-diamine (aGlu-012).
  • the compound of formula (II) is selected from the group consisting of 5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-
  • Embodiment 13 The method of any of embodiments 10 to 12, wherein the compound of formula (II) is 5-/V-carboxamido-2-pyrrolidone-/ ⁇ /’-n-dodecyl propane-1 , 3-diamine (Py-C12).
  • Embodiment 15 The method of any of embodiments 10 to 14, wherein the linking agent is /V-hydroxysuccinimide (NHS)-active ester, or trichlorotriazine.
  • the linking agent is /V-hydroxysuccinimide (NHS)-active ester, or trichlorotriazine.
  • Embodiment 16 The method of any of embodiments 10 to 15, wherein the linking agent comprises a dendrimer and/or more than one functional group for bond formation with the compound of formula (II).
  • Embodiment 17 A product of formula (I) prepared by a method according to any of embodiments 10 to 16.
  • Embodiment 18 Use of a product of formula (I) of any of embodiments 1 to 9 for interfering with the function and/or the structure of a polypeptide.
  • Embodiment 19 The use of embodiment 18, wherein the polypeptide is an enzyme selected from an oxidoreductase, a transferase, a hydrolase or an isomerase.
  • Embodiment 20 The use of embodiment 19, wherein the polypeptide is an enzyme selected from an oxidoreductase or a hydrolase.
  • Embodiment 21 The use of embodiment 20, wherein the oxidoreductase is selected from a dioxygenase or a peroxidase.
  • Embodiment 22 The use of embodiment 20, wherein the hydrolase is selected from a ribonuclease (RNAse), a deoxyribonuclease (DNAse), thrombin, an elastase and/or a collagenase.
  • RNAse ribonuclease
  • DNAse deoxyribonuclease
  • thrombin an elastase and/or a collagenase.
  • Embodiment 23 The use of embodiments 18 to 22, wherein the use of the product of formula (I) is for inhibiting an enzyme and wherein the enzyme inhibition of the product of formula (I) is at least 80 % of the enzyme inhibition of the uncoupled compound of formula (II).
  • Embodiment 24 Use of any of embodiments 18 to 23, wherein R 2 is an amino acid, and wherein the amino acid is pyroglutamic acid of formula (III) formula (III) with R 8 and R 9 being H and wherein one of R 3 and R 4 is with n being 11 and the other being H.
  • Embodiment 25 Use of a compound of formula (II): formula (II) with m being an integer from 1 to 6 and
  • R 1 being H and with
  • R 2 and R 3 being independently selected from H, a polar protic or polar aprotic substituent independently selected from a saccharide, a polyalcohol, a nucleic acid, an amino acid or an amino acid derivative, a peptide, a heterocyclic compound, an organic acid, including a carboxylate, sulfonate, phosphate or a substituent selected from with one of R 5 , R 6 or R 7 being NH 2 , and the other being H, and with n and n’ being an integer from 1 to 25 and with
  • R 4 being selected from and with n and n’ being an integer from 1 to 25, for preparing a product of formula (I) of any of claims 1 to 8.
  • Example 1 Coupling of 5-/V-carboxamido-2-pyrrolidone-Af-n-dodecyl propane-1 ,3- diamine (Py-C12), A/-dodecyl-1,3-diamino propane (C12), A/’-(3-aminopropyl)-/V’-n- dodecyl-1,3-diamino propane (Bis-C12) and A/-(a-glutaminyl amido)-/V’-n-dodecyl- 1,3 diamino propane (aGlu-C12) to NHS-activated carboxy groups on the surface of magnetic beads
  • Coupling method The 5-/V-carboxamido-2-pyrrolidone-/ ⁇ /’-n-dodecyl propane-1 , 3- diamine (Py-C12), /V-dodecyl-1 ,3-diamino propane (C12), /V’-(3-aminopropyl)-/ ⁇ /’-n- dodecyl-1 ,3-diamino propane (Bis-C12) and / ⁇ /-(a-glutaminyl amido)-/V’-n-dodecyl-1 ,3- diamino propane (aGlu-C12) was coupled to /V-hydroxysuccinimide (NHS)-activated magnetic beads according to the information of the manufacturer (PierceTM NHS- Activated Magnetic Beads; ThermoFisher Scientific).
  • Fig. 10 shows the coupling experiment of Py-C12 to NHS-activated carboxy groups on the surface of magnetic beads as an intermediate product of a linking agent coupled to a substrate.
  • the grey-shaded circles represent the magnetic beads.
  • Fig. 11 shows the coupling experiment of C12 to NHS-activated carboxy groups on the surface of magnetic beads as an intermediate product of a linking agent coupled to a substrate.
  • the grey-shaded circles represent the magnetic beads.
  • Fig. 12 shows the coupling experiment of Bis-C12 to NHS-activated carboxy groups on the surface of magnetic beads as an intermediate product of a linking agent coupled to a substrate.
  • the grey-shaded circles represent the magnetic beads.
  • Fig. 13 shows the coupling of aGlu-012 to NHS-activated carboxy groups on the surface of magnetic beads as an intermediate of a linking agent coupled to a substrate.
  • the grey- shaded circles represent the magnetic beads.
  • Example 2 Testing of the functional protein denaturing effect of coupled 5-N- carboxamido-2-pyrrolidone-A/’-n-dodecyl propane-1, 3-diamine (Py-C12), N- dodecyl-1,3-diamino propane (C12) and A/’-(3-aminopropyl)-/V’-n-dodecyl-1,3- diamino propane (Bis-C12) in comparison to 5-/V-carboxamido-2-pyrrolidone-/V’-n- dodecyl propane-1, 3-diamine (Py-C12), A/-dodecyl-1,3-diamino propane (C12) and A/’-(3-aminopropyl)-/V’-n-dodecyl-1,3-diamino propane (Bis-C12) not coupled
  • the beads were separated from the collagenase on a magnetic stand and washed with 100 pl MOPS buffer (pH 7.0). The supernatant containing the collagenase was transferred to a 96 well plate and mixed with 10 pl of P-Check substrate. Collagenase activity was determined by the detection of the change of fluorescence intensity (RFU) in a kinetic mode every 60 seconds for at least 30 minutes on a Cytation 5 Multi-Mode-Reader (BioTek, Germany).
  • REU fluorescence intensity
  • the preparation was washed three times with 50 mM MOPS/2 mM CaCI 2 (pH 7.0; wash buffer) by centrifugation (400 x g) for 30 minutes on an Amicon® Ultra-4* Centrifugal Filter Unit (10.000 NMWL; Merck Millipore, Germany).
  • 400 ml wash buffer was added to the Py-C12-, C12- or Bis-C12-free collagenase concentrate on the filter membrane.
  • 90 pl of the concentrated both treated and untreated collagenase was taken up with the aid of a pipette, transferred to a new vessel and mixed with 10 pl of P-Check substrate.
  • Collagenase activity was determined by the detection of the change of fluorescence intensity (RFU) in a kinetic mode every 60 seconds for at least 30 minutes on a Cytation 5 Multi-Mode-Reader (BioTek).
  • Example 3 Testing of the functional stability of coupling of 5-/V-carboxamido-2- pyrrolidone-A/’-n-dodecyl propane-1, 3-diamine (Py-C12), A/-dodecyl-1,3-diamino propane (C12) and Af-(3-aminopropyl)-/V’-n-dodecyl-1,3-diamino propane (BisCi 2)
  • Example 4 Testing of the functional protein denaturing effect of coupled N-(a- glutaminyl amido)-/V’-n-dodecyl propane-1 ,3-diamine (aGlu-C12)
  • Collagenase activity was determined by the detection of the change of fluorescence intensity (RFU) in a kinetic mode every 60 seconds for at least 30 minutes on a Cytation 5 Multi-Mode-Reader (BioTek). Up to 0.27 pM aGlu-C12 lead to an about 90 % reduction of the collagenase activity in comparison to the untreated collagenase (positive control) (Fig. 16).
  • REU fluorescence intensity
  • Example 5 Testing of the functional protein denaturing effect of coupled 5-N- carboxamido-2-pyrrolidone-A/’-acetamido-A/’-n-dodecyl propane-1 ,3-diamine (Py- Acetyl-C12), in comparison to 5-/V-carboxamido-2-pyrrolidone-/V’-acetamido-/V’-n- dodecyl propane-1, 3-diamine (Py-Acetyl-C12) not coupled: Confirming the binding site at the acyclic secondary amine of the diaminesby acetylation of Py-C12 at this site
  • Example 6 Coupling of C12 and Bis-C12 to cellulose scaffolds by condensation reaction and testing their biocidal efficacy
  • cellulose sheets were coupled to /V-dodecyl-1 ,3-diamino propane (C12) (Fig. 18) and / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-C12) (Fig. 19).
  • the Malaprade reaction followed by reductive aldimine condensation was used for covalent binding of diamino propane derivatives.
  • cellulose sheets were inoculated with a p-galactosidase-expressing E. coli strain.
  • cellulose sheets coupled to C12 or Bis-C12 showed a pronounced biocidal activity (Fig. 20).
  • Fig. 20 shows that cellulose scaffolds coupled to C12 and Bis-C12 exhibit biocidal activity.
  • round sterile cellulose sheets were treated with sodium periodate (100 mM) for 1 h at room temperature, followed by intensive washing steps. Thereafter, samples were incubated with /V-dodecyl-1 ,3-diamino propane (C12) and / ⁇ /’-(3- aminopropyl)-/V’-n-dodecyl-1 ,3-diamino propane (Bis-C12), respectively, (5 mM), each for 20 h at 37 °C. As a control, the samples were treated with water.
  • Example 7 Testing the reactivity of 5-/V-carboxamido pyrrolidone-A/’-n-dodecyl propane-1, 3-diamine (Py-C12) towards cyanuric chloride
  • Cyanuric chloride (Fig. 21) was treated with 3 eq. of Py-C12 in chloroform as an inert solvent at ambient temperature for three days while stirring. Analytical samples were collected and any reactive species within were quenched with methanol. Subsequent analysis with mass spectrometry (MALDI-MS) revealed a disubstituted reaction product of triazine and Py-C12 (Fig. 22). These findings clearly indicate that the acyclic secondary amino group reacts with cyanuric chloride yielding a disubstitute depending on the molar ratios and reaction conditions at ambient temperatures.
  • MALDI-MS mass spectrometry
  • Fig. 23 shows the MALDI-MS spectrum demonstrating the characteristic peak fordi-(5- N-carboxamido-2-pyrrolidone-N’-n-dodecyl propane-1 , 3-diamine (Py-C12)-adduct to tri chlorotri azine.
  • Example 8 Two-step coupling procedure of 5-/V-carboxamido-2-pyrrolidone-/V’-n- dodecyl propane-1, 3-diamine (Py-C12), A/-dodecyl-1,3-diamino propane (C12) and A/’-(3-aminopropyl)- A/’-n-dodecyl-1,3-diamino propane (Bis-C12) to cellulose
  • Py-C12 Two-step coupling procedure of 5-/V-carboxamido-2-pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-C12) and cyanuric chloride
  • Example 9 Inactivation of cellulase by coupled Py-C12, C12 and Bis-C12 to cellulose sheets
  • cellulose sheets were covalently bound to 5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-C12), /V-dodecyl-1 ,3-diamino propane (C12) and / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-C12). Cyanuric chloride was used for covalent binding. To evaluate the effect of a covalent bond, cellulose sheets were intensively washed and then incubated with the enzyme cellulase.
  • cellulose sheets covalently bound to Py-C12, C12, or Bis-C12 were protected from cellulase digestion due to enzyme inhibition of Py-C12, C12 and Bis-C12 (Fig. 27).
  • Fig. 27 shows that cellulose scaffolds covalently bound to Py- C12, C12 and Bis-C12 inhibit cellulase activity and maintain the structural integrity.
  • Round sterile cellulose sheets (8 mm in diameter) were incubated with the linking agent cyanuric chloride (5 % (w/v) in dioxane/xylene (w/w) 1 :1) for 30 min at room temperature, followed by intensive washing steps.
  • Example 10 Testing the biocidial efficacy of coupled Py-C12, C12 and Bis-C12 to cellulose scaffolds
  • cellulose sheets were covalently bound to 5-/V-carboxamido-2- pyrrolidone-/V’-n-dodecyl propane-1 , 3-diamine (Py-C12), /V-dodecyl-1 ,3-diamino propane (C12), or / ⁇ /’-(3-aminopropyl)-/ ⁇ /’-n-dodecyl-1 ,3-diamino propane (Bis-C12).
  • Cyanuric chloride was used for covalent binding of Py-C12, C12 and Bis-C12 as described below.
  • cellulose sheets were inoculated with a p-galactosidase- expressing E. coli strain. In contrast to the control, cellulose sheets covalently bound to Py-C12, C12, or Bis-C12 showed biocidal activity (Fig. 28).
  • Fig. 28 shows that cellulose scaffolds coupled to Py-C12, C12 and Bis-C12 exhibit a pronounced biocidal activity.
  • round sterile cellulose sheets (8 mm in diameter) were incubated with the linking agent cyanuric chloride (1 % (w/v) in dioxane/xylene (w/w) 1 :1) for 30 min at room temperature, followed by intensive washing steps.
  • cellulose sheets were inoculated with a p-galactosidase-expressing E. coli strain and incubated at 37 °C for 7 days.
  • the biocidal effect of the sample coupled to Py-C12, C12 and Bis-C12 is clearly visible. Blue coloured bacterial colonies appear only on the control sample.

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Abstract

L'invention concerne un produit de formule (I) ayant une structure diamine, en particulier une structure 1,3-diamino propane, lié de manière covalente à un substrat par l'intermédiaire d'un lieur. La liaison covalente se fait par l'intermédiaire d'un groupe amino secondaire primaire ou acyclique de la structure diamine, de préférence de la structure 1,3-diamino propane. L'invention concerne également sa préparation à partir d'un composé de formule (II) et l'utilisation du produit de formule (I) pour interférer avec la fonction et/ou la structure d'un polypeptide, de préférence une enzyme. De préférence, la diamine accouplée fournit toujours une activité biocide. Les produits de formule (I) offrent une stabilité à long terme avantageuse et donc un risque réduit de lixiviation et d'élimination indésirable de la structure diamine et des biocides, respectivement.
PCT/EP2024/062132 2023-05-03 2024-05-02 Diamines à liaison covalente et leur utilisation Pending WO2024227880A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
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WO2016016168A1 (fr) 2014-07-30 2016-02-04 Eberhard Karls Universitaet Tuebingen Medizinische Fakultaet Composition pour moduler l'activité de protéines qui ne sont pas des protéines de structure
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