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WO2025056712A1 - Acides aminés de silanetriol et leurs utilisations - Google Patents

Acides aminés de silanetriol et leurs utilisations Download PDF

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Publication number
WO2025056712A1
WO2025056712A1 PCT/EP2024/075550 EP2024075550W WO2025056712A1 WO 2025056712 A1 WO2025056712 A1 WO 2025056712A1 EP 2024075550 W EP2024075550 W EP 2024075550W WO 2025056712 A1 WO2025056712 A1 WO 2025056712A1
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Prior art keywords
amino acid
polypeptide
alkyl
solid
silanetriol
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Inventor
Philippe Marliere
Louis FENSTERBANK
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Scientist of Fortune SA
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Scientist of Fortune SA
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F7/00—Compounds containing elements of Groups 4 or 14 of the Periodic Table
    • C07F7/02—Silicon compounds
    • C07F7/08—Compounds having one or more C—Si linkages
    • C07F7/0834—Compounds having one or more O-Si linkage
    • C07F7/0836—Compounds with one or more Si-OH or Si-O-metal linkage
    • 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
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y601/00—Ligases forming carbon-oxygen bonds (6.1)
    • C12Y601/01—Ligases forming aminoacyl-tRNA and related compounds (6.1.1)

Definitions

  • the templated biosynthesis of informational polymers proceeds by exclusively incorporating the C, H, N, O, P subset in nucleic acids (as catalyzed by RNA and DNA polymerases) and the C, H, N, O, S subset in proteins (as catalyzed by ribosomes).
  • the only known exception to the restricted specification of informational bioelements is selenium, the incorporation of which occurs in some proteins of certain species. Elucidation of the enzymatic machinery that underlies the biosynthesis and coding of selenocysteinyl-tRNA, thereby endowing proteins with the selenol catalytic group, represented a major advance in biochemical and evolutionary understanding.
  • a polypeptide comprising the amino acid according to any one of embodiments 1 to 4. 6.
  • the solid comprises a functional group that allows for the formation of the siloxane bond.
  • polypeptide according to embodiment 8 wherein the functional group that allows for the formation of the siloxane bond is comprised in a silanol, a silanediol, a silanetriol, a silicic acid, a silicate salt, a boric acid, a borate salt, or a phosphate salt of phosphoric acid.
  • the solid comprises or consists of glass, concrete, ceramics, zeolites, and/or clays.
  • the polypeptide according to any one of embodiments 5 to 10 wherein the polypeptide is an enzyme. 12.
  • a siloxane (Si-O) cluster comprising the amino acid according to any one of embodiments 1 to 4 and/or the polypeptide according to any one of embodiments 5 to 13. 15.
  • siloxane (Si-O) cluster according to embodiment 14, wherein the siloxane cluster comprises at least one siloxane bond, in particular wherein the siloxane bond comprises the structure (Si-O-Si), (Si-O-B) or (Si-O-P).
  • siloxane bond comprises the structure (Si-O-Si), (Si-O-B) or (Si-O-P).
  • An aminoacylated tRNA comprising the amino acid according to any one of embodiments 1 to 4. 20.
  • PG 1 is Cbz group
  • PG 2 is C 1-6 alkyl
  • each R is ethyl. 22.
  • a method of producing a polypeptide comprising a silanetriol amino acid at a specified position in a cell comprising: a) growing, in an appropriate medium, the cell, wherein the cell comprises a nucleic acid that encodes the polypeptide, and wherein the nucleic acid comprises at least one selector codon; and b) providing the amino acid according to any one of embodiments 1 to 4; wherein the cell further comprises: c) a nucleic acid sequence encoding an orthogonal aminoacyl-tRNA synthetase (O-RS), and, d) an orthogonal tRNA (O-tRNA), that recognizes the selector codon, wherein said O—RS aminoacylates the O-tRNA with the amino acid according to any one of embodiments 1 to 4.
  • O-RS orthogonal aminoacyl-tRNA synthetase
  • O-tRNA orthogonal tRNA
  • a method of immobilizing a polypeptide on a solid comprising the steps of: a) providing a solid comprising a functional group that allows for the formation of a siloxane bond; b) contacting the solid of step (a) with the polypeptide according to any one of embodiments 5 to 13; c) immobilizing the polypeptide on the solid.
  • a method of equipping a solid with a biological agent comprising the steps of: a) providing a solid comprising a functional group that allows for the formation of a siloxane bond; b) equipping the solid with a biological agent by contacting the solid of step (a) with the amino acid according to any one of embodiments 1 to 4 or the polypeptide according to any one of embodiments 5 to 13. 28.
  • the functional group that allows for the formation of a siloxane bond is comprised in a silanol, a silanediol, a silanetriol, a silicic acid, a silicate salt, a boric acid, a borate salt, or a phosphate salt of phosphoric acid.
  • the solid comprises or consists of glass, concrete, ceramics, zeolites, and/or clays. That is, the present invention is based on the surprising finding that the silanetriol functional group can be incorporated into amino acids.
  • silanetriol amino acids serve as nitrogen sources for bacteria expressing appropriate aminotransferases.
  • the transamination products, silanetriol oxo acids will likely give rise to aldehydes and acyl-CoA thioesters through the action of thiamine enzymes and in turn generate an unprecedented cascade of secondary metabolites.
  • a swell of metabolic innovation is to be expected from the synthetic availability of silanetriol amino acids and their propensity to undergo the enzymatic conversions of canonical amino acids.
  • silanetriol amino acid according to the invention can be linked to suitable molecules or materials via siloxane bonds, preferably when incorporated in a peptide or protein.
  • This has various potential applications ranging from the immobilization of polypeptides, viral particles or cells on solid surfaces or the functionalization of otherwise inert materials, including inert mineral materials.
  • DETAILED DESCRIPTION OF THE INVENTION As mentioned before, the present invention relates, in one embodiment, to an amino acid comprising a silanetriol functional group. It will be understood that salt forms of said amino acid are also encompassed by the present invention.
  • a silanetriol functional group is a group according to formula -Si(OH) 3 .
  • amino acid refers to a compound comprising at least one - COOH group and one -NH 2 group (or their ionized forms, in particular wherein -COO- group and -NH 3 - are present at the same time in one molecule, which can be also referred to as zwitterionic form).
  • the amino acid as referred to herein, may be an -amino acid, that is, a compound comprising a moiety according to formula: H 2 N COOH . It is further preferred that -amino acid includes a moiety according to formula:H 2 N COOH .
  • the amino acid is an -amino acid, as referred to herein, preferably comprising a moiety according to formula term “amino acid” may also refer to a monovalent or divalent radical derived from an “amino acid” as described herein above, wherein preferably an amino group and/or a carboxylic acid group may serve as point(s) of attachment, preferably through an amide bond.
  • amino acid may refer to an amino acyl moiety attached to the rest of the molecule, e.g., through a -CO- group formed from its carboxylic acid group, and/or through a -NH- group formed from its amino group.
  • the amino acid of the present invention comprises a silanetriol (-Si(OH)3) functional group and a moiety according to formulaH 2 N COOH , which preferably is a moiety according to formulaH 2 N COOH .
  • the amino acid of the present invention is an amino acid (or, more generally speaking, a compound) according to formula (I): or its salt.
  • Z is C 0-6 alkylene, wherein one or more, preferably non-adjacent, -CH 2 - groups can be independently replaced by -O-, -S-, -NH-, -N(C 1-4 alkyl)-, -CO-, -CONH-, -NHCO-, -CON(C 1- 4 alkyl)-, -N(C 1-4 alkyl)CO-, -Si(OH) 2 -, -Si(CH 3 ) 2 -, -SiF 2 -, -SO 2 -, or -PO 2 H- and/or one or more hydrogen atoms can be independently replaced by C 1-4 alkyl, -OH, -O(C 1-4 alkyl), -SH, -S(C 1-4 alkyl), -NH 2 , -NH(C 1-4 alkyl), -N(C 1-4 alkyl)(C 1-4 alkyl).
  • Z is C 0-6 alkylene, wherein one or more, preferably non-adjacent, -CH 2 - groups can be independently replaced by -O-, -S-, -NH-, -N(C 1-4 alkyl)-, -CO-, -CONH-, -NHCO-, -CON(C 1-4 alkyl)-, -N(C 1-4 alkyl)CO-, -Si(OH) 2 -, -Si(CH 3 ) 2 -, or -SiF 2 - and/or one or more hydrogen atoms can be independently replaced by C 1-4 alkyl, -OH, -O(C 1-4 alkyl), -SH, -S(C 1-4 alkyl), -NH 2 , -NH(C 1-4 alkyl), -N(C 1-4 alkyl)(C 1-4 alkyl), preferably by C 1-4 alkyl, -OH, -SH, and -
  • Z is C 0-6 alkylene, wherein one or more, preferably non-adjacent, -CH 2 - groups can be independently replaced by -O-, -S-, -NH-, -N(C1-4 alkyl)-, -CONH-, -NHCO-, - CON(C 1-4 alkyl)-, or -N(C 1-4 alkyl)CO-, and/or one or more hydrogen atoms can be independently replaced by C1-4 alkyl, -OH, -O(C1-4 alkyl), -SH, -S(C1-4 alkyl), -NH2, -NH(C1-4 alkyl), -N(C1-4 alkyl)(C1-4 alkyl), preferably by C1-4 alkyl, -OH, -SH, and -NH2, more preferably by C1-4 alkyl, or -OH.
  • Z is C 0-6 alkylene, wherein one or more, preferably non-adjacent, -CH 2 - groups can be independently replaced by -O-, -S-, -NH-, -N(C 1-4 alkyl)-, -CONH-, or -NHCO-, and/or one or more hydrogen atoms can be independently replaced by C1-4 alkyl, -OH, -O(C1-4 alkyl), -SH, -S(C 1-4 alkyl), -NH 2 , -NH(C 1-4 alkyl), -N(C 1-4 alkyl)(C 1-4 alkyl), preferably by C 1-4 alkyl, - OH, -SH, and -NH 2 , more preferably by C 1-4 alkyl, or -OH.
  • Z is C 0-6 alkylene, wherein one or more, preferably non-adjacent, -CH 2 - groups can be independently replaced by -O-, -S-, -NH-, -N(C1-4 alkyl)-, or -CONH-, and/or one or more hydrogen atoms can be independently replaced by C1-4 alkyl, -OH, -O(C1-4 alkyl), -SH, -S(C1-4 alkyl), -NH2, -NH(C1-4 alkyl), -N(C1-4 alkyl)(C1-4 alkyl), preferably by C1-4 alkyl, -OH, -SH, and -NH2, more preferably by C1-4 alkyl, or -OH.
  • Z is C0-6 alkylene, wherein one or more hydrogen atoms can be independently replaced by C1-4 alkyl, -OH, -O(C1-4 alkyl), -SH, -S(C1-4 alkyl), -NH2, -NH(C1-4 alkyl), -N(C1-4 alkyl)(C1-4 alkyl), preferably by C1-4 alkyl, -OH, -SH, and -NH2, more preferably by C1-4 alkyl, or -OH.
  • Z is C0-6 alkylene.
  • said alkylene is a linear alkylene.
  • Z are methylene (-CH2-), ethylene (in particular -CH2CH2-) and propylene (in particular -CH2CH2CH2-).
  • X and Y are each independently hydrogen, halogen, -CN, C1-4 alkyl, -OH, -O(C1-4 alkyl), -(C1-4 alkylene)-OH, -SH, -S(C1-4 alkyl), -(C1-4 alkylene)-SH, -NH2, -NH(C1-4 alkyl) or -N(C1-4 alkyl)(C1-4 alkyl), -COOH, -CONH2, -CONH(C1-4 alkyl), -CON(C1-4 alkyl)(C1-4 alkyl), -SO3H or - PO3H2.
  • X and Y are each independently hydrogen, halogen, -CN, C1-4 alkyl, -OH, -O(C1-4 alkyl), -(C 1-4 alkylene)-OH, -SH, -S(C 1-4 alkyl), -(C 1-4 alkylene)-SH, -NH 2 , -NH(C 1-4 alkyl) or -N(C 1-4 alkyl)(C 1-4 alkyl), -COOH, -CONH 2 , -CONH(C 1-4 alkyl), or -CON(C 1-4 alkyl)(C 1-4 alkyl).
  • X and Y are each independently hydrogen, -CN, C 1-4 alkyl, -OH, -O(C 1-4 alkyl), -(C 1-4 alkylene)-OH, -SH, -S(C 1-4 alkyl), -(C 1-4 alkylene)-SH, -NH 2 , -NH(C 1-4 alkyl) or -N(C 1-4 alkyl)(C 1-4 alkyl).
  • X and Y are each independently hydrogen, halogen, -CN, -OH, -SH, or - NH 2 , wherein it is preferred that at least one of X and Y is hydrogen, unless X and Y are both - F.
  • the amino acid of formula (I) may be an amino acid according to formula (Ia): , or its salt.
  • n is an integer ranging from 1 to 7.
  • n can be 1, 2, 3, 4, 5, 6 or 7.
  • n is an integer ranging from 1 to 5.
  • n is an integer ranging from 1 to 3.
  • n may be 1 and –(CH 2 ) n – as defined in formula (Ia) is -CH 2 -.
  • n may be 2, and –(CH 2 ) n – as defined in formula (Ia) is -CH 2 CH 2 -or -CH(CH 3 )-.
  • n may be 3, and –(CH 2 ) n – as defined in formula (Ia) is -CH 2 CH 2 CH 2 -, -CH(CH 3 )CH 2 -, -CH 2 CH(CH 3 )-, or - CH(CH 2 CH 3 )-.
  • –(CH 2 ) n – is -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -.
  • the amino acid of formula (Ia) is an amino acid of formula (Ib): or its salt.
  • n and –(CH2)n– in formula (Ib) are as defined for formula (Ia).
  • Particularly preferred compounds of formula (Ib) are the following compounds, or their salts: It is to be understood that the scope of the invention embraces all salts, including pharmaceutically acceptable salt forms of the amino acids of the present invention, in particular of the compounds of formula (I), which may be formed, e.g., by protonation of an atom carrying an electron lone pair which is susceptible to protonation, such as an amino group, with an inorganic or organic acid, or as a salt of an acid group (such as a carboxylic acid group) with a physiologically acceptable cation.
  • Exemplary base addition salts comprise, for example: alkali metal salts such as sodium or potassium salts; alkaline earth metal salts such as calcium or magnesium salts; zinc salts; ammonium salts; aliphatic amine salts such as trimethylamine, triethylamine, dicyclohexylamine, ethanolamine, diethanolamine, triethanolamine, procaine salts, meglumine salts, ethylenediamine salts, or choline salts; aralkyl amine salts such as N,N-dibenzylethylenediamine salts, benzathine salts, benethamine salts; heterocyclic aromatic amine salts such as pyridine salts, picoline salts, quinoline salts or isoquinoline salts; quaternary ammonium salts such as tetramethylammonium salts, tetraethylammonium salts, benzyltrimethylammonium salts, benzyltriethylam
  • Exemplary acid addition salts comprise, for example: mineral acid salts such as hydrochloride, hydrobromide, hydroiodide, sulfate salts (such as, e.g., sulfate or hydrogensulfate salts), nitrate salts, phosphate salts (such as, e.g., phosphate, hydrogenphosphate, or dihydrogenphosphate salts), carbonate salts, hydrogencarbonate salts, perchlorate salts, borate salts, or thiocyanate salts; organic acid salts such as acetate, propionate, butyrate, pentanoate, hexanoate, heptanoate, octanoate, cyclopentanepropionate, decanoate, undecanoate, oleate, stearate, lactate, maleate, oxalate, fumarate, tartrate, malate, citrate, succinate, adipate, gluconate, glycolate, nic
  • Preferred salts of the amino acids of the present invention or the compounds of formula (I) include a hydrochloride salt, a hydrobromide salt, a mesylate salt, a sulfate salt, a tartrate salt, a fumarate salt, an acetate salt, a citrate salt, and a phosphate salt.
  • a particularly preferred salt of the amino acids of the present invention or the compound of formula (I) is a hydrochloride salt.
  • the present invention also specifically relates to the amino acid of the present invention or the compound of formula (I), including any one of the specific compounds of formula (I) described herein, in non-salt form or in a zwitterionic form.
  • the scope of the invention embraces the amino acids of the present invention or the compounds of formula (I) in any solvated form, including, e.g., solvates with water (i.e., as a hydrate) or solvates with organic solvents such as, e.g., methanol, ethanol, isopropanol, acetic acid, ethyl acetate, ethanolamine, DMSO, or acetonitrile. All physical forms, including any amorphous or crystalline forms (i.e., polymorphs), of the amino acids of the present invention or the compounds of formula (I) are also encompassed within the scope of the invention.
  • the invention embraces the isolated optical isomers of the amino acids or the compounds according to the invention as well as any mixtures thereof (including, in particular, racemic mixtures/racemates and non-racemic mixtures).
  • the racemates can be resolved by physical methods, such as, e.g., fractional crystallization, separation or crystallization of diastereomeric derivatives, or separation by chiral column chromatography.
  • the individual optical isomers can also be obtained from the racemates via salt formation with an optically active acid followed by crystallization.
  • the present invention further encompasses any tautomers of the amino acids of the present invention or the compounds of formula (I). It will be understood that some compounds may exhibit tautomerism. In such cases, the formulae provided herein expressly depict only one of the possible tautomeric forms.
  • the formulae and chemical names as provided herein are intended to encompass any tautomeric form of the corresponding compound and not to be limited merely to the specific tautomeric form depicted by the drawing or identified by the name of the compound.
  • the scope of the invention also embraces amino acids of the present invention or compounds of formula (I), in which one or more atoms are replaced by a specific isotope of the corresponding atom.
  • the invention encompasses amino acids of the present invention or compounds of formula (I), in which one or more hydrogen atoms (or, e.g., all hydrogen atoms) are replaced by deuterium atoms (i.e., 2 H; also referred to as “D”). Accordingly, the invention also embraces amino acids of the present invention or compounds of formula (I) which are enriched in deuterium.
  • Naturally occurring hydrogen is an isotopic mixture comprising about 99.98 mol-% hydrogen-1 ( 1 H) and about 0.0156 mol-% deuterium ( 2 H or D).
  • the content of deuterium in one or more hydrogen positions in amino acids of the present invention or compounds of formula (I) can be increased using deuteration techniques known in the art.
  • a compound of formula (I) or a reactant or precursor to be used in the synthesis of the compound of formula (I) can be subjected to an H/D exchange reaction using, e.g., heavy water (D 2 O).
  • H/D exchange reaction e.g., heavy water (D 2 O).
  • deuteration techniques are described in: Atzrodt J et al., Bioorg Med Chem, 20(18), 5658-5667, 2012; William JS et al., Journal of Labelled Compounds and Radiopharmaceuticals, 53(11-12), 635-644, 2010; Modvig A et al., J Org Chem, 79, 5861-5868, 2014.
  • the content of deuterium can be determined, e.g., using mass spectrometry or NMR spectroscopy.
  • amino acid of the present invention or the compound of formula (I) is not enriched in deuterium. Accordingly, the presence of naturally occurring hydrogen atoms or 1 H hydrogen atoms in the amino acids of the present invention or the compounds of formula (I) is preferred. In one embodiment, particularly preferred are amino acids of the present invention or the compounds of formula (I) of the present invention, wherein the nitrogen atom in the amino group of the amino acid is 15 N-labeled. Enrichment in the 15 N can be achieved, for example, by starting from a starting material enriched in this isotope at a particular position.
  • the present invention provides a method for obtaining an amino acid of formula (Ia), wherein n is an integer ranging from 2 to 7, the method comprising the step of reacting the compound of formula (II): with HSi(OR)3 in the presence of a Karstedt catalyst.
  • PG1 and PG2 are protecting groups for -NH2 and -COOH groups, respectively.
  • the skilled person is capable of selecting suitable protecting groups that do not interfere with the reaction with HSi(OR)3, and at the same time can be removed efficiently to yield the desired product.
  • Information and teaching with regard to the selection of particular protecting groups can be found in Greene's Protective Groups in Organic Synthesis, by Peter G. M. Wuts, and Theodora W.
  • Particularly suitable PG 1 is Cbz group.
  • Cbz group is known to the skilled person, and is a group according to formula:
  • Particularly suitable PG 2 is C 1-6 alkyl, for example ethyl or methyl, preferably methyl.
  • each R is independently of each other selected from C 1-6 alkyl.
  • Preferably, each R is ethyl.
  • the method further comprises the step(s) of deprotecting - Si(OH)3, -NH2 and -COOH groups in the obtained product.
  • the compounds of the present invention can be obtained following the alkylation of Schiff base derivatives of glycine by the O'Donnell method seemed worth exploring (M. J. O’Donnell, J. M. Boniece, S. E. Earp, Tetrahedron Letters 1978, 19, 2641– 2644.), as shown in scheme 1b.
  • the present invention further provides a method for obtaining an amino acid of formula (I), the method comprising the step of reacting LG-Z-CXY-Si(OR)3, with PGN-CH2-COO- PG2 pretreated with a base capable of generating PGN-CH--COO-PG2 carbanion.
  • Z, X and Y in LG-Z-CXY-Si(OR)3 are as in formula (I) hereinabove.
  • LG in LG-Z-CXY-Si(OR)3 is a leaving group.
  • the skilled person is capable of selecting appropriate leaving group.
  • One preferred non-limiting example of the leaving group LG is iodo group (-I)
  • PGN in LG-Z-CXY-Si(OR)3 is a protected -NH2 group.
  • PG 2 in LG-Z-CXY-Si(OR) 3 is a protecting group for -COOH group.
  • PG 2 is C 1-6 alkyl. Particularly suitable C 1-6 alkyl is tert-butyl.
  • rach R is independently of each other selected from C 1-6 alkyl and C 2-6 alkenyl.
  • each R is allyl
  • the method further comprises the step(s) of deprotecting Si(OH)3, -NH2 and -COOH groups in the obtained product. This can be achieved, for example, by refluxing the compound for at least 30 minutes, preferably at least 1 hour, more preferably at least 2 hours, still more preferably for at least 4 hours (or about 4 hours) in 6N HCl in water.
  • the present invention relates to a polypeptide comprising the amino acid of the present invention.
  • polypeptide refers to a polymer of two or more amino acids linked via amide bonds that are formed between an amino group of one amino acid and a carboxylic acid group of another amino acid.
  • the amino acids comprised in the polypeptide may be selected from the 20 standard proteinogenic -amino acids (i.e., Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val), which can be present as the L-isomer or the D-isomer, and are preferably all present as the L-isomer, but also from non-proteinogenic and/or non-standard -amino acids (such as, e.g., ornithine, citrulline, homolysine, pyrrolysine, 4-hydroxyproline, -methylalanine (i.e., 2-aminois
  • the polypeptide may be unmodified or may be modified, e.g., at its N-terminus, at its C-terminus and/or at a functional group in the side chain of any of its amino acid residues (particularly at the side chain functional group of one or more Lys, His, Ser, Thr, Tyr, Cys, Asp, Glu, and/or Arg residues).
  • modifications may include, e.g., the attachment of any of the protecting groups described for the corresponding functional groups in: Wuts PG & Greene TW, Greene’s protective groups in organic synthesis, John Wiley & Sons, 2006.
  • Such modifications may also include the covalent attachment of one or more polyethylene glycol (PEG) chains (forming a PEGylated peptide), the glycosylation and/or the acylation with one or more fatty acids (e.g., one or more C 8-30 alkanoic or alkenoic acids; forming a fatty acid acylated peptide or protein).
  • PEG polyethylene glycol
  • fatty acids e.g., one or more C 8-30 alkanoic or alkenoic acids; forming a fatty acid acylated peptide or protein.
  • modified peptides or proteins may also include peptidomimetics, provided that they contain at least two amino acids that are linked via an amide bond (formed between an amino group of one amino acid and a carboxyl group of another amino acid).
  • polypeptide may, e.g., be present as a linear molecular chain (forming a linear peptide) or may form one or more rings (corresponding to a cyclic peptide).
  • the polypeptide may also form oligomers consisting of two or more identical or different molecules.
  • polypeptide may also refer to a monovalent radical derived from a “polypeptide” as described herein above, wherein preferably an amino group or a carboxylic acid group, in particular the N-terminal amino group or the C-terminal carboxylic acid group, serves as point of attachment, preferably through an amide bond.
  • the invention relates to a viral particle comprising the polypeptide according to the invention, i.e., a polypeptide comprising a silanetriol amino acid.
  • the polypeptide comprising the silanetriol amino acid is comprised in the capsid of the viral particle.
  • the invention relates to a cell comprising the polypeptide according to the invention, in particular wherein the polypeptide according to the invention is displayed on the surface of the cell.
  • the cell may be any cell, i.e., may be eukaryotic or prokaryotic.
  • the cell is a microbial cell, in particular a bacterial cell or a yeast cell.
  • the cell is an E. coli cell and the polypeptide displayed on the surface of the E. coli cell is a variant of the protein OmpC comprising a silanetrial amino acid according to the invention.
  • the polypeptide may be an enzyme.
  • enzyme as used herein, generally refers to a polypeptide which acts as a catalyst to induce a chemical change in compounds or “substrates”.
  • the other molecule has to comprise a functional group that allows for the formation of a siloxane bond.
  • Functional groups that allow for the formation of a siloxane bond may be comprised, without limitation, in silanols, including silanediols, silanetriols and silicic acids, or salts thereof.
  • the silicic acid may be orthosilicic acid (Si(OH) 4 ), metasilicic acid (SiO(OH) 2 ) , pyrosilicic acid (OSi(OH) 3 ) 2 ), or disilicic acid (Si 2 O 3 (OH) 2 ).
  • the molecule comprising the functional group that allows the formation of a siloxane bond is orthosilicic acid (Si(OH) 4 ). Condensation of molecules comprising these functional groups with a silanetriol amino acid according to the invention will result in the formation of a siloxane bond (Si-O-Si).
  • siloxane bond is to be interpreted broadly and encompasses further bonds comprising silicon-oxygen linkages.
  • the silanetriol amino acid according to the invention may condensate with boric acid (B(OH)3) or borate salts, resulting in the formation of borosiloxane bond (Si-O-B).
  • the invention relates to the polypeptide according to the invention, wherein the functional group that allows for the formation of a siloxane bond is comprised in a silanol, a silanediol, a silanetriol, a silicic acid, a silicate salt, boric acid, a borate salt, or a phosphate salt of phosphoric acid.
  • polypeptides comprising a silanetriol amino acid according to the invention can undergo formation of a siloxane bond with materials comprising silicic acid molecules or silicate salts.
  • the naturally occurring substances are inorganic and have a crystal structure or are amorphous.
  • the mineral material comprises a silicic acid or a silicate salt that can undergo formation of a siloxane bond with the silanetriol group of the amino acid according to the invention.
  • amino acids comprising a silanetriol functional group may be used for the immobilization of biological agents onto otherwise inert mineral surfaces for various purposes including, without limitation, purification or biocatalysis, such as heterogenous catalysis.
  • the amino acid comprising the silanetriol functional group may be comprised, without limitation, in a polypeptide, an enzyme, a virus particle or a cell.
  • amino acids comprising a silanetriol functional group may be used for functionalizing inert mineral surfaces or particles.
  • functionalizing an otherwise inert mineral surface or particle with the amino acid according to the invention may confer the surface or particle with biologically active functional groups (COOH, NH2).
  • mineral surfaces or particles may be functionalized with macromolecules (polypeptides, proteins, enzymes, protein complexes), viral particles or even entire cells comprising the amino acid according to the invention.
  • macromolecules polypeptides, proteins, enzymes, protein complexes
  • viral particles or even entire cells comprising the amino acid according to the invention.
  • amino acids, macromolecules, viral particles and cells may also be referred to as biological agents.
  • the invention relates to a solid surface or particle covalently linked to the amino acid according to the invention or the polypeptide according to the invention, wherein the amino acid or the polypeptide is covalently linked to the surface or the particle via a siloxane bond, in particular wherein the siloxane bond comprises the structure (Si-O-Si), (Si-O-B) or (Si-O-P).
  • the invention relates to the solid surface or particle according to the invention, wherein the surface or particle comprises a molecule selected from the group consisting of: a silanol, a silanediol, a silanetriol, a silicic acid, a silicate salt, a boric acid, a borate salt, and a phosphate salt of phosphoric acid.
  • the invention relates to the solid surface or particle according to the invention, wherein the surface or particle comprises or consists of glass, silica, concrete, ceramics, zeolites, and/or clays.
  • amino acids comprising a silanetriol functional group may be used in functionalizing buildings or parts of buildings with biological agents.
  • biological agents comprising or consisting of the amino acid or the peptide according to the invention may be coupled to parts of a building that comprise a functional group that allows formation of a siloxane bond, such as, without limitation, elements made of glass or concrete. This may have various applications, including strategies to make buildings more resilient or to improve the air quality in buildings.
  • amino acids comprising a silanetriol functional group may be used in functionalizing the interior of pipes, such as concrete pipes, with biological agents. That is, biological agents comprising or consisting of the amino acid or the peptide according to the invention may be coupled to the interior wall of a pipe if the interior wall of the pipe comprises a functional group that allows formation of a siloxane bond.
  • a functional group may inherently be comprised in concrete pipes.
  • pipes may be coated with a material that allows formation of a siloxane bond with the silanetriol amino acid according to the invention.
  • pipes may be functionalized with enzymes or cells that assist in wastewater treatment.
  • the invention relates to a siloxane (Si-O) cluster comprising the amino acid according to the invention and/or the polypeptide according to the invention.
  • a siloxane cluster is defined as a molecular structure composed of silicon (Si), oxygen (O), and occasionally other elements (e.g., B or P), typically forming a three-dimensional network.
  • These clusters comprise one or more siloxane bonds (e.g., Si-O- Si, Si-O-B, Si-O-P) and may have various organic or inorganic groups attached to the silicone (or B or P) atoms.
  • the siloxane cluster may be formed between two or more amino acids and/or peptides according to the invention.
  • the siloxane cluster may comprise additional binding partners that can contribute to the formation of the siloxane cluster.
  • the additional binding partner comprises silanols, including silanediols, silantriols and silicic acids, or salts thereof, boric acid, borate salts, or phosphate salts of phosphoric acid.
  • the siloxane cluster may be formed between one or more amino acids according to the invention and a surface or particle comprising a functional group that allows for the formation of a siloxane bond.
  • the siloxane cluster may be formed between one or more polypeptides according to the invention and a surface or particle comprising a functional group that allows for the formation of a siloxane bond.
  • the polypeptide may also be part of a larger entity, such as a viral particle or a cell.
  • the siloxane cluster may be formed between one or more amino acids and/or peptides according to the invention, and a glass particle or surface.
  • the silanetriol functional group of the amino acid or peptide according to the invention may be used to immobilize polypeptides, enzymes, viral particles or cells on a glass particle or surface.
  • glass particles or surfaces may be functionalized with the amino acid or polypeptide according to the invention.
  • An example for the functionalization of glass surfaces with silanetriols has been provided by Spirk at al. (ACS Appl Mater Interfaces, 2010, 2(10):2956-62.
  • the glass surface may be a glass surface of a laboratory equipment, such as, without limitation, a test tube, a flask, a beaker or a microscope slide.
  • the glass surface may be a window of a building or a vehicle, including cars, busses, trains, etc.
  • the siloxane cluster according to the invention may be formed between one or more amino acids and/or peptides according to the invention, and a surface made of a mineral material and comprising a functional group that allows for the formation of a siloxane bond, including, without limitation concrete, ceramics, zeolites, and clays.
  • the siloxane cluster may be formed between one or more amino acids and/or peptides according to the invention, and the interior of a concrete pipe or a pipe that has been coated with a material comprising a function group that allows formation of a siloxane bond.
  • the amino acid according to the invention can directly undergo a condensation reaction with mineral materials comprising a suitable functional group.
  • these surfaces may further be coated with another material to facilitate formation of the siloxane cluster, such as a material comprising the reactive groups disclosed herein.
  • the invention relates to a method of immobilizing a polypeptide on a solid, the method comprising the steps of: a) providing a solid comprising a functional group that is suitable for the formation of a siloxane bond; b) contacting the solid of step (a) with the polypeptide according the invention; c) immobilizing the polypeptide to the solid.
  • the invention relates to a method of equipping a solid with a biological agent, the method comprising the steps of: a) providing a solid comprising a functional group that allows for the formation of a siloxane bond; b) equipping the solid with a biological agent by contacting the solid of step (a) with the amino acid according to the invention or the polypeptide according to the invention.
  • the invention relates to the method according to the invention, wherein the solid is a surface or a particle.
  • the invention relates to the method according to the invention, wherein the functional group that allows for the formation of a siloxane bond is comprised in a silanol, a silanediol, a silanetriol, a silicic acid, a silicate salt, a boric acid, a borate salt, or a phosphate salt of phosphoric acid.
  • the invention relates to the method according to the invention, wherein the solid comprises or consists of glass, silica, concrete, ceramics, zeolites, and/or clays. The skilled person is aware of conditions that are suitable for the formation of siloxane bonds.
  • the invention relates to an aminoacylated tRNA comprising the amino acid according to the invention.
  • the aminoacylated tRNA comprising the amino acid according to the invention may serve as substrate for a ribosome and may thus be used to incorporate the amino acid according to the invention into a polypeptide.
  • Any one of the silanetriol amino acids disclosed herein may be attached to a tRNA.
  • the amino acid according to the invention is attached to a tRNA via its carboxy group, as known in the art.
  • Attachment of an amino acid according to the invention to a tRNA may be achieved with an engineered aminoacyl-tRNA synthetase that specifically recognizes the amino acid according to the invention, as described, inter alia, by Krahn et al. (Enzymes, 2020, 48:351- 395. doi: 10.1016/bs.enz.2020.06.004) or in the appended Example 4.
  • the flexizyme approach may be used to attach an amino acid according to the invention to a tRNA as described, inter alia, by Lee et al. (Nat Commun, 2019, 10(1):5097. doi: 10.1038/s41467- 019-12916-w).
  • the tRNA may be any tRNA.
  • the tRNA is an orthogonal tRNA.
  • the tRNA may comprise one or more mutations in the acceptor stem and/or the anticodon region to allow site specific incorporation into a polypeptide by the ribosome in a cell; see Krahn et al. (Enzymes, 2020, 48:351-395. doi: 10.1016/bs.enz.2020.06.004).
  • the invention relates to a method of producing a polypeptide comprising a silanetriol amino acid at a specified position in a cell, the method comprising: a) growing, in an appropriate medium, the cell, wherein the cell comprises a nucleic acid that encodes the polypeptide, and wherein the nucleic acid comprises at least one selector codon; and b) providing the amino acid according to the invention; wherein the cell further comprises: c) a nucleic acid sequence encoding an orthogonal aminoacyl-tRNA synthetase (O-RS), and, d) an orthogonal tRNA (O-tRNA), that recognizes the selector codon, wherein said O—RS aminoacylates the O-tRNA with the amino acid according to the invention.
  • OF-RS orthogonal aminoacyl-tRNA synthetase
  • O-tRNA orthogonal tRNA
  • the skilled person is well aware of methods to introduce non-natural amino acids into polypeptides with an orthogonal tRNA/aminoacyl-tRNA synthetase (O-tRNA/O-RS) pair.
  • O-tRNA/O-RS orthogonal tRNA/aminoacyl-tRNA synthetase
  • the introduction of non-natural amino acids into polypeptides with an O-tRNA/O-RS pair is disclosed in WO 2010/037062, which is incorporated herein by reference in its entirety.
  • the translational components used in the present invention are typically derived from non- eukaryotic organisms.
  • the orthogonal O-tRNA can be derived from a non- eukaryotic organism, e.g., an archaebacterium, such as Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, or the like, or a eubacterium, such as Escherichia coli, Thermus thermophilus, Bacillus stearothermphilus, or the like, while the orthogonal O-RS can be derived from a non-eukaryotic organism, e.g., Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Py
  • eukaryotic sources can also be used, including but not limited to, plants, algae, protists, fungi, yeasts, animals (e.g., mammals, insects, arthropods, etc.), or the like.
  • the individual components of an O-tRNA/O-RS pair can be derived from the same organism or different organisms. In one embodiment, the O-tRNA/O-RS pair is from the same organism. Alternatively, the O-tRNA and the O-RS of the O-tRNA/O-RS pair are from different organisms.
  • the O-tRNA, O-RS or O-tRNA/O-RS pair can be selected or screened in vivo or in vitro and/or used in a cell, e.g., a non-eukaryotic cells (such as E. coli cell), or a eukaryotic cell, to produce a polypeptide with an amino acid according to the invention.
  • a cell e.g., a non-eukaryotic cells (such as E. coli cell), or a eukaryotic cell, to produce a polypeptide with an amino acid according to the invention.
  • a eukaryotic cell can be from a variety of sources, including but not limited to, a plant (e.g., complex plant such as monocots, or dicots), an algae, a protist, a fungus, a yeast (including but not limited to, Saccharomyces cerevisiae), an animal (including but not limited to, a mammal, an insect, an arthropod, etc.), or the like.
  • a plant e.g., complex plant such as monocots, or dicots
  • an algae e.g., complex plant such as monocots, or dicots
  • a protist e.g., a protist, a fungus, a yeast (including but not limited to, Saccharomyces cerevisiae)
  • an animal including but not limited to, a mammal, an insect, an arthropod, etc.
  • Compositions of cells with translational components of the present invention are also a feature of the present invention. See also
  • polypeptide of interest with an amino acid according to the invention in a host cell
  • Suitable bacterial promoters are well known in the art.
  • Bacterial expression systems for expressing a polypeptide of interest are available in, including but not limited to, E.
  • coli Bacillus sp., Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, and Salmonella. Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. A tRNA and/or RS and/or a polypeptide of interest may be utilized and/or expressed in any number of suitable expression systems including, for example, yeast, insect cells, mammalian cells, and bacteria.
  • orthogonal refers to a molecule (e.g., an orthogonal tRNA (O-tRNA) and/or an orthogonal aminoacyl tRNA synthetase (O-RS)) that is used with reduced efficiency by a system of interest (e.g., a translational system, e.g., a cell).
  • Orthogonal refers to the inability or reduced efficiency, e.g., less than 20% efficient, less than 10% efficient, less than 5% efficient, or e.g., less than 1% efficient, of an orthogonal tRNA and/or orthogonal RS to function in the translation system of interest.
  • an orthogonal tRNA in a translation system of interest is aminoacylated by any endogenous RS of a translation system of interest with reduced or even zero efficiency, when compared to aminoacylation of an endogenous tRNA by an endogenous RS.
  • an orthogonal RS aminoacylates any endogenous tRNA in the translation system of interest with reduced or even zero efficiency, as compared to aminoacylation of the endogenous tRNA by an endogenous RS.
  • a second orthogonal molecule can be introduced into the cell that functions with the first orthogonal molecule.
  • an orthogonal tRNA/RS pair includes introduced complementary components that function together in the cell with an efficiency (e.g., about 50% efficiency, about 60% efficiency, about 70% efficiency, about 75% efficiency, about 80% efficiency, about 85% efficiency, about 90% efficiency, about 95% efficiency, or about 99% or more efficiency) to that of a corresponding tRNA/RS endogenous pair.
  • efficiency e.g., about 50% efficiency, about 60% efficiency, about 70% efficiency, about 75% efficiency, about 80% efficiency, about 85% efficiency, about 90% efficiency, about 95% efficiency, or about 99% or more efficiency
  • the term “selector codon” refers to codons recognized by the O-tRNA in the translation process and not recognized by an endogenous tRNA.
  • the O-tRNA anticodon loop recognizes the selector codon on the mRNA and incorporates its amino acid, e.g., an amino acid according to the invention, at this site in the polypeptide.
  • Selector codons can include but are not limited to, e.g., nonsense codons, such as, stop codons, including but not limited to, amber, ochre, and opal codons; four or more base codons; rare codons; codons derived from natural or unnatural base pairs and/or the like.
  • a selector codon can also include one of the natural three base codons, wherein the endogenous system does not use (or rarely uses) said natural three base codon. For example, this includes a system that is lacking a tRNA that recognizes the natural three base codon, and/or a system wherein the natural three base codon is a rare codon.
  • a suppressor tRNA is a tRNA that alters the reading of a messenger RNA (mRNA) in a given translation system, e.g., by providing a mechanism for incorporating an amino acid into a polypeptide chain in response to a selector codon.
  • mRNA messenger RNA
  • a suppressor tRNA can read through a codon including but not limited to, a stop codon, a four base codon, or a rare codon.
  • the term “medium” or “media” includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents.
  • the term may encompass medium in which the host cell has been grown, e.g., medium into which a culture or whole organism or cell is growing, and the medium may have a non- natural amino acid included to support growth of replication deficient organisms or cells, including medium either before or after a proliferation step.
  • nucleic acid refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides, or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides.
  • oligonucleotide analogs including PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like).
  • PNA peptidonucleic acid
  • analogs of DNA used in antisense technology phosphorothioates, phosphoroamidates, and the like.
  • a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated.
  • degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res.
  • polypeptides comprising the amino acid according to the invention may also be synthesized chemically as known in the art, for example as disclosed by Tan et al. (J Am Chem Soc, 2020, doi: 10.1021/jacs.0c09664) The following definitions apply throughout this specification and claims, unless explicitly indicated to the contrary.
  • hydrocarbon group refers to a group consisting of carbon atoms and hydrogen atoms.
  • alkyl refers to a monovalent saturated acyclic (i.e., non-cyclic) hydrocarbon group which may be linear or branched. Accordingly, an “alkyl” group does not comprise any carbon-to-carbon double bond or any carbon-to-carbon triple bond.
  • a “C1- 5 alkyl” denotes an alkyl group having 1 to 5 carbon atoms.
  • Preferred exemplary alkyl groups are methyl, ethyl, propyl (e.g., n-propyl or isopropyl), or butyl (e.g., n-butyl, isobutyl, sec-butyl, or tert-butyl).
  • alkyl preferably refers to C1-4 alkyl, more preferably to methyl or ethyl, and even more preferably to methyl.
  • alkenyl refers to a monovalent unsaturated acyclic hydrocarbon group which may be linear or branched and comprises one or more (e.g., one or two) carbon- to-carbon double bonds while it does not comprise any carbon-to-carbon triple bond.
  • C 2-5 alkenyl denotes an alkenyl group having 2 to 5 carbon atoms.
  • alkenyl groups are ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, or prop-2-en-1-yl), butenyl, butadienyl (e.g., buta-1,3-dien-1-yl or buta-1,3-dien-2-yl), pentenyl, or pentadienyl (e.g., isoprenyl).
  • alkenyl preferably refers to C 2-4 alkenyl.
  • alkylene refers to an alkanediyl group, i.e.
  • C 1-5 alkylene denotes an alkylene group having 1 to 5 carbon atoms
  • C0-3 alkylene indicates that a covalent bond (corresponding to the option “C 0 alkylene”) or a C 1-3 alkylene is present.
  • Preferred exemplary alkylene groups are methylene (-CH 2 -), ethylene (e.g., -CH 2 -CH 2 - or -CH(-CH3)-), propylene (e.g., -CH2-CH2-CH2-, -CH(-CH2-CH3)-, -CH2-CH(-CH3)-, or -CH(-CH3)- CH 2 -), or butylene (e.g., -CH 2 -CH 2 -CH 2 -CH 2 -).
  • alkylene preferably refers to C1-4 alkylene (including, in particular, linear C1-4 alkylene), more preferably to methylene or ethylene, and even more preferably to methylene.
  • halogen refers to fluoro (-F), chloro (-Cl), bromo (-Br), or iodo (-I).
  • bond and “covalent bond” are used herein synonymously, unless explicitly indicated otherwise or contradicted by context.
  • the terms “optional”, “optionally” and “may” denote that the indicated feature may be present but can also be absent. Whenever the term “optional”, “optionally” or “may” is used, the present invention specifically relates to both possibilities, i.e., that the corresponding feature is present or, alternatively, that the corresponding feature is absent.
  • X is optionally substituted with Y
  • X may be substituted with Y
  • a component of a composition is indicated to be “optional”, the invention specifically relates to both possibilities, i.e., that the corresponding component is present (contained in the composition) or that the corresponding component is absent from the composition.
  • Various groups are referred to as being “optionally substituted” in this specification. Generally, these groups may carry one or more substituents, such as, e.g., one, two, three or four substituents. It will be understood that the maximum number of substituents is limited by the number of attachment sites available on the substituted moiety.
  • the “optionally substituted” groups referred to in this specification carry preferably not more than two substituents and may, in particular, carry only one substituent. Moreover, unless defined otherwise, it is preferred that the optional substituents are absent, i.e. that the corresponding groups are unsubstituted.
  • the substituent groups comprised in the compounds of the present invention may be attached to the remainder of the respective compound via a number of different positions of the corresponding specific substituent group. Unless defined otherwise, the preferred attachment positions for the various specific substituent groups are as illustrated in the examples.
  • the terms “a”, “an” and “the” are used interchangeably with “one or more” and “at least one”.
  • compositions comprising “a” compound of formula (I) can be interpreted as referring to a composition comprising “one or more” compounds of formula (I).
  • the term “comprising” (or “comprise”, “comprises”, “contain”, “contains”, or “containing”), unless explicitly indicated otherwise or contradicted by context, has the meaning of “containing, inter alia”, i.e., “containing, among further optional elements, ...”. In addition thereto, this term also includes the narrower meanings of “consisting essentially of” and “consisting of”.
  • a comprising B and C has the meaning of “A containing, inter alia, B and C”, wherein A may contain further optional elements (e.g., “A containing B, C and D” would also be encompassed), but this term also includes the meaning of “A consisting essentially of B and C” and the meaning of “A consisting of B and C” (i.e., no other components than B and C are comprised in A).
  • the invention will be illustrated in the following examples. These are not to be construed as limiting in any way, but merely serve the purpose of illustrating the embodiments of the present invention.
  • BRIEF DESCRIPTION OF THE DRAWINGS Fig.1 Silanetriol amino acids: background and opportunities.
  • Fig.2 29 SiNMR evidence for siloxane crosslinking by Silamate II upon increasing concentration. Other parameters such as temperature, pH and time are identical (pH ⁇ 2, 20°C, 0 day).
  • Fig.12 Growth curves of bacterial strains Acinetobacter baylyi ADP1 and Pseudomonas putida KT2440 with Silaglutamate, in comparison with Glutamate and Ammonium Chloride.
  • Fig.13 Improved growth on Silaglutamate of bacterial strains Acinetobacter baylyi ADP1 and Pseudomonas putida KT2440 in serial cultures. (The x-axis shows time in minutes and the y- axis shows optical density at 600 nanometers. Three iterative inoculations are shown for each of the two strains.)
  • ATA T7684-10mg, Lot # 097H1003
  • Pyridoxal phosphate P3657-1G; Source: SLCL8133
  • the enzyme powder and le Pyridoxal phosphate were stored at -18 °C before use.
  • General procedure for hydrosilylation To a solution of alkene 2 or 7 (1 equiv.) in tetrahydrofuran (0.1 M) at room temperature were added triethoxysilane (1.5 equiv.) and 0.01 equiv.
  • Triethanolamine buffer solution preparation to a 30 mL beaker equipped with a magnetic stirrer, HPLC grade water (10.0 mL), triethanolamine (0,250 g), s(4 mg) were added. Under gentle stirring at rt, concentrated HCl was added dropwise. After pH reached 7.5 (indicated by a pH meter), the total volume was then adjusted to 15.0 mL by adding HPLC grade water. The buffer solution was used directly for transamination.
  • the reaction mixture was allowed to slowly warm-up to room temperature and stirred for 16 h.
  • the reaction was quenched with a saturated aqueous ammonium chloride solution (100 mL) and the aqueous layer was extracted with diethyl ether (2 x 50 mL).
  • the combined organic layers were washed with a saturated aqueous sodium chloride solution (50 mL), dried over magnesium sulfate, filtered and evaporated to dryness to afford to an orange oil.
  • the crude material was purified by flash chromatogaphy on silica gel to afford to the expected compound 12 (colorless oil, 2.16 g, 93% yield).
  • the ledbpgp2s 2D sequence combining constant time, bipolar pulse, stimulated echo, and the longitudinal eddy current delay method, was used (Wu, D. ; Chen, A. ; Johnson Jr, C.S. J. Magn. Reson.
  • the averaged diffusion coefficient is equal to 1.056x10-10 m2/s, while from DOSY recorded with the same sample after letting it evolve for 16 days (Fig.7) results in an averaged diffusion coefficient equal to 4.137x10-10 m2/s.
  • the factor four is clearly indicative of a depolymerization pathway from an oligomeric structure to the free silanetriol acid.
  • Transamination reactions between silanetriol amino acids (I-III) and the oxoglutarate a) Triethanolamine buffer solution preparation To a 30 mL beaker equipped with a magnetic stirrer, HPLC grade water (10.0 mL), triethanolamine (0,250 g), PLP (4 mg) were added. Under gentle stirring at rt, concentrated HCl was added dropwise.
  • Example 1 Synthesis of silanetriol amino acids The inventors contemplated two reaction classes to forge the key carbon-silicon bonds of silanetriol amino acids I-III. The first consisted of the transition metal-catalyzed hydrosilylation, the second of the alkylation of a glycyl anion by a silyl electrophile (sila- alkylation).
  • Example 2 Structural and biochemical analysis of silanetriol amino acids These syntheses could be scaled up to gram quantities. This made it possible to conduct structural as well as biochemical studies. Focusing on Silamate II, the inventors investigated the effect of pH ( ⁇ 2 and >11) duration (0, 8 and 17 days) and concentration (0.01 to 0.22 M) on condensation processes by the silanetriol group (see SI). Multinuclear NMR studies were carried out.
  • the enzyme (according to the vendor Sigma Aldrich) is of bacterial origin and shows an elevated activity in converting oxoglutarate to L- glutamate using a wide spectrum of L-amino acids.
  • Application of the enzymatic assay to the three silanetriol amino acids I-III resulted in the formation of a product chromatographically indistinguishable from glutamate (as detected by ninhydrin after TLC).
  • MS analysis corroborated these observations by showing the formation of an entity with the mass of glutamate and also, except in the reaction of III, of entities whose masses corresponded to silanetriol oxo acids in the proportions expected for the natural isotopes of silicon ( 28 Si, 29 Si, and 30 Si).
  • Example 3 Utilization of the silanetriol amino acid Silaglutamate as a nitrogen source by two species of Proteobacteria Silaglutamate (also designated as Silamate), as shown below, is the S enantiomer of the compound (HO) 3 Si-CH 2 -CH 2 -CH(NH 2 )-CO-OH. It corresponds to the substitution of the lateral carboxylic group (CO2H) of glutamate with silanetriol (Si(OH)3.
  • Two reference bacterial strains were used, ADP1 for Acinetobacter baylyi and KT2440 for Pseudomonas putida.
  • MSN The same defined culture medium, designated MSN, was used for all species, the composition of which was as follows: Na gluconate: 10 millimolar; citric acid: 4 millimolar; MgSO4: 1 millimolar; K2HPO4: 50 millimolar; nitrilotriacetic acid: 10 micromolar; CaCl2: 3 micromolar; FeCl3: 3 micromolar; MnCl2: 1 micromolar; ZnCl2: 0.1 micromolar; CoCl2: 0.1 micromolar; NiCl2: 0.1 micromolar; CrCl3: 0.1 micromolar; CuCl2: 0.1 micromolar; Na2MoO4: 0.1 micromolar; Na2SeO3: 0.1 micromolar.
  • a nitrogen source was added at a concentration of 10 millimolar, composed of either NH4Cl, Glutamate (L) or Silaglutamate (L). The final pH reached 7.3.
  • Agar at a concentration of 15 g/L was added to prepare solid medium plates.
  • Acinetobacter baylyi and Pseudomonas putida were able to partially utilize this silanetriol amino acid. These results are shown in Figure 12.
  • Serial culture by reinoculation at one-hundredth dilution in MSN Silaglutamate medium gave rise to an improvement in growth, for each of the two species Acinetobacter baylyi and Pseudomonas putida. These results are shown in Figure 13.
  • Example 4 Incorporation of silaspartate by a reprogrammed strain of Escherichia coli in the outer membrane protein OmpC and immobilisation of cells of the reprogrammed strain in silica gel
  • Silaspartate is the S enantiomer of the compound (HO)3Si-CH2-CH(NH2)-CO- OH. It corresponds to the substitution of the lateral carboxylic group (CO2H) of aspartate with silanetriol (Si(OH)3.
  • silanetriol Si(OH)3.
  • Plasmid construction This section describes the construction of a two plasmids system that allows the incorporation of the non-canonical amino acid Silaspartate in response to the amber codon into the OmpC protein expressed in a reprogrammed E. coli strain.
  • the construction begins with the plasmid pTECH-MmPylRS(IPYE) (Addgene plasmid #104073) which contains the pylS pylT genes specifying the PylRS tRNAPyl orthogonal translation system and a chloramphenicol resistance gene.
  • the MmPylRS(IPYE) gene is replaced with a pylS* gene encoding MaPylRSIFGFF by standard cloning techniques.
  • the latter is a pyrrolysyl-tRNA synthetase (PylRS) variant from Methanomethylophilus alvus carrying the following mutations: L125I, Y126F, M129G, V168F, and Y206F. This variant is active toward a range of histidine analogues (https://onlinelibrary.wiley.com/doi/full/10.1002/pro.4640).
  • the PylRSIFGFF gene is under the control of a very strong constitutive lpp promoter.
  • the tRNAPyl gene pylT is under the control of the proK promoter.
  • the final constructed plasmid is called pTECH-MaPylRSIFGFF.
  • the plasmid pET28-OmpC-Y232TAG is constructed by cloning the ompC gene from E. coli into the widely used pET system containing a kanamycin resistance gene.
  • the OmpC gene is first amplified by PCR from E. coli genome and then cloned into linearized pET28 backbone using Gibson Assembly (NEBuilder® HiFi DNA AssemblyMaster Mix, New England Biolabs).
  • the amber stop codon (TAG) is introduced in the ompC gene at the position corresponding to Y232, by using the Q5TM Site-Directed Mutagenesis Kit (New England Biolabs).
  • the Y232 mutation site corresponds to an exposed location in one of the eight external loops of OmpC (Spicer CD, Triemer T, Davis BG. Palladium-mediated cell-surface labeling. J Am Chem Soc. 2012 Jan 18;134(2):800-3).
  • the expression of OmpC-Y322TAG is controlled by an IPTG inducible T7 promoter. Protein expression Incorporation of Silaspartate in response to the amber codon UAG is OmpC-Y232TAG using the engineered Pyl/tRNACUAPyl system.
  • Cells of an E. coli strain bearing a chromosomal deletion of the ompC gene, BL21(DE3) ompC, are co-transformed with the two plasmids pET28-OmpC-Y232TAG and pTECH-MaPylRSIFGFF.
  • the gene deletion is done following a procedure well known in the art (Datsenko KA.; Wanner BL. Proceedings of the National Academy of Sciences, 2000, vol.97 (12), 6640-6645).
  • Protein expression is carried out in either minimal media or rich media supplemented with the required antibiotics kanamycin (50 g/mL) and chloramphenicol (37 g/mL), the inducer IPTG (0.1-1 mM), and in the presence or absence of Silaspartate (3 mM). Cultures are incubated with shaking at 37 °C for 16 h, and then 150 ⁇ L are taken and analysed by SDS-PAGE.
  • Sol–gel synthesis of silica is based on the strong tendency of orthosilicic or silicic acid to the condensation reaction: (OH) 3 Si-OH + HO-Si(OH) 3 (OH) 3 Si-O-Si(OH) 3 + H 2 O, (1) as a result of which siloxane bridges Si-O-Si are formed.
  • the two silicon atoms are bonded covalently to each other via oxygen.
  • the remaining three silanol groups Si-OH at each silicon atom can in turn participate in further condensation reactions with (OH) 3 Si-OH + (OH) 3 Si-O-Si(OH) 3 + HO-Si(OH) 3 (OH)3Si-O-Si(OH)2-O-Si(OH)2-O-Si(OH)3 + H2O, (2) which lead to polymerization and the formation of polysilicic acids.
  • oligomeric reaction products form sol particles. Their flocculation, which promotes the formation of covalent siloxane bonds between them, leads to a sol–gel transition with the formation of a three-dimensional network structure.
  • the silanetriol group of Silaspartate (R-CH2-(OH)2Si-OH), as incorporated in the OmpC protein and displayed at the surface of E. coli cells, can substitute for silicic acid in condensation reactions at any stage and thereby covalently bond bacteria to the polysilicic sol-gel through siloxane bridges: R-CH2-(OH)2Si-OH + (OH)3Si-O-Si(OH)3 + HO-Si(OH)3 R-CH2-(OH)2Si-O-Si(OH)2-O-Si(OH)2-O-Si(OH)3 + H2O (3) E.
  • coli BL21(DE3) ompC cells co-transformed with pET28-OmpC-Y232TAG and pTECH- MaPylRSIFGFF are grown in LB medium containing kanamycin (50 g/mL) and chloramphenicol (37 g/mL).
  • OmpC expression is triggered by adding 3 mM Silaspartate and 1 mM IPTG, and cell growth is continued at 37 °C with shaking for 16 h. Then 1 mL of culture is collected by centrifugation at 10,000 rpm for 20 min and washed with 1 ml of sterile PBS (pH 7.4).
  • the washed cells are resuspended in a water–glycerol (10 wt%) phosphate buffer solution (cells 10 9 ml 1 ).
  • the formation of silica gels encapsulating the E. coli cells is initiated by means of acidification down to pH 7.
  • the mixture is homogenized under mild stirring. Gelation occurs within about 2 min at room temperature.
  • Wet gels and aqueous bacteria suspensions are kept in their mother solution and aged in a closed flask at 20 °C. Aged gels are then dispersed in a phosphate buffer solution and washed to remove non-immobilised cells. Transmission electron micrographs show that the cellular integrity of bacteria is maintained within the gel.

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Abstract

La présente invention concerne un acide aminé comprenant un groupement fonctionnel silanetriol (-Si(OH)3), ainsi que des polypeptides comprenant un acide aminé comprenant un groupement fonctionnel silanetriol (-Si(OH)3). De plus, la présente invention concerne des procédés d'obtention d'acides aminés comprenant des groupes fonctionnels silanetriol (-Si(OH)3) et des procédés d'immobilisation de polypeptides comprenant des groupes fonctionnels silanetriol (-Si(OH)3) sur un solide.
PCT/EP2024/075550 2023-09-12 2024-09-12 Acides aminés de silanetriol et leurs utilisations Pending WO2025056712A1 (fr)

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Citations (3)

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US7642077B2 (en) * 2003-12-08 2010-01-05 Genencor International, Inc. Biocomposite comprising co-precipitate of enzyme, silicate and polyamine
WO2010037062A1 (fr) 2008-09-26 2010-04-01 Ambrx, Inc. Vaccins et micro-organismes dépendant de la réplication d'acide aminé non naturels
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Patent Citations (3)

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US7642077B2 (en) * 2003-12-08 2010-01-05 Genencor International, Inc. Biocomposite comprising co-precipitate of enzyme, silicate and polyamine
WO2010037062A1 (fr) 2008-09-26 2010-04-01 Ambrx, Inc. Vaccins et micro-organismes dépendant de la réplication d'acide aminé non naturels
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