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EP3262058A1 - Composé pour la préparation de polypeptides - Google Patents

Composé pour la préparation de polypeptides

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Publication number
EP3262058A1
EP3262058A1 EP16708615.6A EP16708615A EP3262058A1 EP 3262058 A1 EP3262058 A1 EP 3262058A1 EP 16708615 A EP16708615 A EP 16708615A EP 3262058 A1 EP3262058 A1 EP 3262058A1
Authority
EP
European Patent Office
Prior art keywords
moiety
peptide
residue
muc1
compound
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP16708615.6A
Other languages
German (de)
English (en)
Inventor
Christian Friedrich Wilhelm BECKER
Claudia Bello
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universitaet Wien
Original Assignee
Universitaet Wien
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Universitaet Wien filed Critical Universitaet Wien
Publication of EP3262058A1 publication Critical patent/EP3262058A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
    • C07K1/026—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution by fragment condensation in solution

Definitions

  • the present invention relates to a compound comprising a protected or deprotected thiol moiety S enabling Native Chemical Ligation, a photolabile linker moiety L and at least one hydrophilic polymer moiety HP. Furthermore, the present invention relates to a method for production of a peptide conjugate P12 comprising a peptide moiety P1 and a second moiety P2 covalently bound with another via an amide bond, wherein the compound of the present invention employed for selectively forming the amide bond between P1 and P2.
  • glycosylation is known to play a key role in numerous biological and biochemical functions such as, e.g., protein folding, cellular differentiation, cell-cell communication, cell-matrix interaction and viral invasion.
  • Post- translational modifications such as glycosylation may play a crucial role in different types of biochemical recognition processes responsible for growth, development, infection, immune response, cell adhesion, signal transduction processes, formation and manifestation of neoplasia, cancer, metastases and autoimmune diseases.
  • Today, many peptides subjected to post-translational modifications such as glycopeptides are of great interest for pharmaceutical purposes.
  • such peptide may exemplarily serve as antibiotic, hormone and cytokine (e.g., juvenile human growth hormone, CD4 or tissue plasminogen activator), vaccine, artificial extracellular matrix, artificial glycocalyx or a coating for implants.
  • cytokine e.g., juvenile human growth hormone, CD4 or tissue plasminogen activator
  • vaccine e.g., artificial extracellular matrix, artificial glycocalyx or a coating for implants.
  • numerous of such peptides are of great interest for research purposes.
  • NCL Native Chemical Ligation
  • NCL-based methods known in the art merely partly solve the problems the experimenter is faced with.
  • only peptides that are well-soluble in aqueous buffers can be efficiency used in such NCL-based methods.
  • peptides which are purely soluble in aqueous environments can hardly be conjugated by NCL because one or both of the educts and/or intermediate products (i.e., the peptide moieties and/or the conjugate of the two educt peptide moieties conjugated with the auxiliary compound) may aggregate.
  • enzymatic modification of the peptides e.g., enzymatic glycosylation
  • a rather pure aqueous environment such as a buffer
  • solubility of peptides to be subjected to an NCL reaction is still a considerable issue, in particular when enzymatic modifications are intended to be included.
  • the polypeptide has to be conjugated to the hydrophilic polymer.
  • the hydrophilic polymer has to be cleaved off the polypeptide.
  • additional purification steps may be required for removing the hydrophilic polymer and cleaving reagents from the polypeptide.
  • an additional step of a conjugation reaction such as an NCL reaction (typically performed in another environment) is required and potentially even further purification steps.
  • these methods will include an undesired cysteinyl moiety into the polypeptide product.
  • the present invention relates to a compound comprising:
  • HP does not comprise primary amino groups or hydroxyl groups when the thiol moiety S is not covalently bound to a peptide moiety P1 .
  • the compound may be an auxiliary compound for preparing polypeptide products.
  • primary amino groups may, depending on the chemical environment of the compound, will occur in the form of -NH 2 or -NH 3 + .
  • hydrophilic polymer HP does not comprise primary amino groups or hydroxyl groups when the thiol moiety S is not covalently bound to a peptide moiety P1 may be understand in the broadest sense.
  • the hydrophilic polymer HP is free of primary amino groups (-NH 2 or -NH 3 + ) and hydroxyl groups (-OH).
  • the hydrophilic polymer HP may optionally comprise one or more primary amino groups and/or one or more hydroxyl groups.
  • the compound of the present invention not conjugated to a peptide moiety P1 comprises only one (a single) primary amino group and no hydroxyl group, wherein the one primary amino group most preferably forms part of the thiol moiety S. Accordingly, in one embodiment, the only primary amino group of the compound is the one comprised in the thiol moiety S.
  • the compound for preparing polypeptide products or a salt thereof comprises:
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP does not comprise primary amino groups or hydroxyl groups.
  • the compound for preparing polypeptide products or a salt thereof consists of:
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP does not comprise primary amino groups or hydroxyl groups.
  • the compound for preparing polypeptide products or a salt thereof comprises:
  • A2 one secondary amino group (-NH-) forming part of an amino acid moiety, in particular a glycyl (Gly) moiety (-NH-CH 2 -CO-),
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP may or may not comprise one or more primary amino group(s) and/or one or more hydroxyl group(s).
  • the compound for preparing polypeptide products or a salt thereof consists of:
  • A2 one secondary amino group (-NH-) forming part of an amino acid moiety, in particular a glycyl (Gly) moiety (-NH-CH 2 -CO-),
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP may or may not comprise one or more primary amino group(s) and/or one or more hydroxyl group(s).
  • the compound for preparing polypeptide products or a salt thereof comprises:
  • A2 one secondary amino group (-NH-) forming part of an carbamate group (- NH-CO-0-), in particular conjugated with a protecting group (e.g., an Fmoc protecting group),
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP may or may not comprise one or more primary amino group(s) and/or one or more hydroxyl group(s).
  • the compound for preparing polypeptide products or a salt thereof consists of:
  • A2 a secondary amino group (-NH-) forming part of a carbamate group (-NH- CO-0-), preferably conjugated with a protecting group, in particular an Fmoc protecting group,
  • photolabile linker moiety L is covalently bound to the at least one hydrophilic polymer moiety HP and to the thiol moiety S, and
  • HP may or may not comprise one or more primary amino group(s) and/or one or more hydroxyl group(s).
  • a first peptide moiety P1 conjugated with such compound could be very well conjugated to a second moiety P2 by means of Native Chemical Ligation (NCL).
  • NCL Native Chemical Ligation
  • the peptide moiety P1 conjugated with such compound could be modified enzymatically (as exemplified: glycosylated) prior to optionally be conjugated to a second moiety P2 by means of NCL.
  • al intermediate products are well-soluble in aqueous environments and can be easily purified in merely few steps. Thereby the procedure is less laborious.
  • conjugated with conjugated to or “bound to” may be understood interchangeably in the broadest sense as the covalent linkage of two or more molecular moieties with another.
  • polypeptide product as used in the context of the present invention as a modified or unmodified polypeptide strand that is the intermediate or final product of the present invention.
  • polypeptide and “peptide” may be understood interchangeably.
  • a (poly)peptide moiety my be understood as a peptide strand that is covalently bound.
  • a (poly)peptide may be understood in the broadest sense as a molecular structure comprising two or more amino acids that are conjugated with another via an amide bond.
  • a (poly)peptide is mainly composed of amino acids.
  • an amide bond may also be designated as "peptide bond” or "peptidic bond”.
  • a (poly)peptide may be a linear, cyclic or branched peptide.
  • a (poly)peptide may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10, more than 10, more than 15, more than 20, more than 25, more than 30, more than 40, more than 50, more than 75, more than 100, more than 200 or even more than 500 amino acids.
  • a (poly)peptide that bears more than one secondary structure element and/or a tertiary structure element may also be designated as "protein domain" or "protein".
  • the (poly)peptides in the context of the present invention are small (poly)peptides of less than 100 amino acids.
  • a (poly)peptide preferably consists of a single amino acid strand.
  • a (poly)peptide may also comprise two or even more strands covalently conjugated with another by any means such as e.g., by one or more disulfide bond(s).
  • the (poly)peptide (essentially) consists of natural L-amino acids.
  • the (poly)peptide may also comprise one or more non-natural amino acid(s) such as, e.g., D- amino acid(s), beta amino acid(s), methylated amino acid(s) (e.g., N-methylated amino acid(s)).
  • the (poly)peptide may even only consist of non-natural amino acids.
  • the (poly)peptide may also be a retro-inverso peptide, thus a (poly)peptide mainly comprising D-amino acids and a reverse amino acid sequence compared to the corresponding naturally occurring peptide consisting of L-amino acids.
  • the term "naturally occurring” may be understood as a sequence that has at least 70 %, preferably at least 80 %, more preferably at least 90 %, even more preferably at least 95 % and most preferably 100 % sequence identity with the amino acid sequence found in nature.
  • the amino acid moieties may be positively charged, negatively charged or neutral. Also a whole (poly)peptide may be positively charged, negatively charged or neutral. A (poly)peptide may be hydrophilic or hydrophobic. A peptide may be well-water soluble or poorly water-soluble. As used herein, the term “poorly water-soluble” refers to a peptide which solubility is less than 5 mg/ml, less than 4 mg/ml, less than 3 mg/ml, less than 2 mg/ml, less than 1 mg/ml, less than 0.5 mg/ml or even less than 0.25 mg/ml peptide per water.
  • a (poly)peptide may further bear any counter ion(s) known in the art, such as e.g., chloride ion(s), acetate ion(s), carbonate ion(s), hydrocarbonate ion(s), sodium ion(s), potassium ion(s), magnesium ion(s), and any ion(s) of the cleavage solution (e.g., TFA ion(s), bromide ion(s), perchlorate ion(s), ammonium ion(s)).
  • counter ion(s) known in the art, such as e.g., chloride ion(s), acetate ion(s), carbonate ion(s), hydrocarbonate ion(s), sodium ion(s), potassium ion(s), magnesium ion(s), and any ion(s) of the cleavage solution (e.g., TFA ion(s), bromide
  • a (poly)peptide may be covalently or non-covalently associated to traces of one or more scavenger(s), such as, e.g., triisopropylsilane (TIS), dithiothreitol (DTT), anisole, thioanisole or 1 ,2-ethanedithiol.
  • scavenger(s) such as, e.g., triisopropylsilane (TIS), dithiothreitol (DTT), anisole, thioanisole or 1 ,2-ethanedithiol.
  • a (poly)peptide of the present invention may further bear one or more modification(s).
  • a (poly)peptide may be amidated or capped at its C-terminus (e.g., by a non-amino acid moiety such as, e.g., a bead and/or a polymer, in particular a hydrophilic polymer).
  • a non-amino acid moiety such as, e.g., a bead and/or a polymer, in particular a hydrophilic polymer.
  • the terms "C-terminus”, “C-terminal end”, “carboxy terminus”, “carboxyterminus” and “carboxyterminal end” may be understood interchangeably.
  • the N-terminus may be capped (e.g., by an acetyl moiety, a methyl moiety, a pyroglutamyl moiety, a bead and/or or a polymer, in particular a hydrophilic polymer).
  • the terms "N-terminus”, “N-terminal end”, “amino terminus” and “amino terminal end” may be understood interchangeably.
  • the capping and/or amidation at one terminus or both termini may render the peptide more stable against exopeptidases.
  • the N-terminus of a (poly)peptide is capped by a hydrophilic polymer.
  • one or more amino acid residue(s) of the peptide may be lipidated, phosphorylated, sulfated, cyclized, oxidated, reduced, decarboxylated, acetylated, acylated, amidated, deamidated, biotinylated or bound to one or more other small molecule(s) and/or terpene(s).
  • a (poly)peptide may or may not form one or more intramolecular disulfide bond(s) or one or more intermolecular disulfide bond(s).
  • a (poly)peptide of the present invention may be labeled radioactively (e.g., by 3 H, 32 P, 35 S, 14 C, 99m Tc or lanthanides (e.g., 64 Gd)) or may be labelled with a spin label, such as one or more heavy isotopes, e.g., 13 C, detectable by Nuclear Magnetic Resonance (NMR).
  • NMR Nuclear Magnetic Resonance
  • a (poly)peptide of the present invention may be labeled by one or more small molecule dye(s) (e.g., Cy dye(s) (e.g., Cy3, Cy5, Cy5.5, Cy7), Alexa dye/s (e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750), VisEn dye(s) (e.g.
  • small molecule dye(s) e.g., Cy dye(s) (e.g., Cy3, Cy5, Cy5.5, Cy7)
  • Alexa dye/s e.g., Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 647, Alexa Fluor 680, Alexa Fluor 750
  • VisEn dye(s) e.g.
  • VivoTag680, VivoTag750 S dye(s) (e.g., S0387), DyLight fluorophore(s) (e.g., DyLight 750, DyLight 800), IRDye(s) (e.g., IRDye 680, IRDye 800), fluorescein dye(s) (e.g., fluorescein, carboxyfluorescein, fluorescein isothiocyanate (FITC)), rhodamine dye(s) (e.g., rhodamine, tetramethylrhodamine (TAMRA)) or HOECHST dye(s)) or one or more quantum dot(s).
  • S dye(s) e.g., S0387
  • DyLight fluorophore(s) e.g., DyLight 750, DyLight 800
  • IRDye(s) e.g., IRDye 680, IRDye 800
  • thiol moiety S as used herein may be understood in the broadest sense as any moiety comprising at least one protected or deprotected thiol group (-SA, wherein S represents sulfur and A represents hydrogen, a protecting group or a peptide moiety) and at least one primary or secondary amino group (-NHB, wherein N represents nitrogen, H represents hydrogen and B represents hydrogen, a peptide moiety P1 , an amino acid moiety, or a moiety activating the amino group).
  • a primary amino group represents -NH 2 or -NH 3 + , respectively.
  • a secondary amino group represents any bivalent - NH- moiety, which may optionally also form part of an amide group (-NH-CO-), a carbamate (-NH-CO-0-), an urate (-NH-CO-NH-), a hydrazine (NH-NH-) etc.
  • B is hydrogen.
  • the compound of the present invention comprises a primary amino group or a salt thereof (-NH 2 or -NH 3 + , respectively).
  • the compound of the present invention may be conjugated with an amino acid moiety, with more than one amino acid moieties consecutively conjugated with another or with a peptide moiety P1.
  • B is an amino acid moiety.
  • Particularly preferable in this context is an amino acid moiety that bears no chiral carbon.
  • B may particularly preferably be a glycine (Gly) moiety. Then, the whole compound may be designated as Aux-Gly.
  • B may also be another natural occurring amino acid moiety (e.g., alanine (Ala), cysteine (Cys), asparaginic acid (Asp), glutamic acid/glutamate (Glu), phenylalanine (Phe), histidine (His), isoleucine (Iso), lysine (Lys), leucine (Leu), methionine (Met), asparagine (Asn), proline (pro), glutamine (Gin), arginine (Arg), serine (Ser), threonine (Thr), valine (Val), tryptophane (Trp), tyrosine (Tyr)).
  • alanine Al
  • cysteine cysteine
  • Asparaginic acid Asp
  • Glu glutamic acid/glutamate
  • Glu glutamic acid/glutamate
  • Glu glutamic acid/glutamate
  • Glu glutamic acid/glutamate
  • Glu glutamic acid/gluta
  • B may also be a non-natural occurring amino acid moiety such as e.g. a D- amino acid, a beta amino acid, a gamma amino acid, a delta amino acid, an epsilon amino acid, or a methylated amino acid (e.g., an N-methylated amino acid).
  • B may also be a natural or non-natural occurring amino acid moiety conjugated in a non-natural way such as e.g. lysine conjugated via its epsilon amino group.
  • the amino acid residue may be protected or deprotected.
  • the amino acid residue may optionally also bear one or more dye moieties, e.g., one of those exemplified before. Pre-loading with an amino acid moiety as described herein may be subsequently easily extended by conjugating further amino acid moieties thereto. Then the compound of the present invention, wherein B is an amino acid residue, may act similar to a pre-loaded resin.
  • B is a consecutive sequence of amino acid moieties also designated as peptide moiety P1 as described in more detail herein.
  • P1 can also be an amino acid residue.
  • the thiol moiety S comprises one protected or deprotected thiol group and one primary or secondary amino group.
  • the at least one thiol group is protected and/or the at least one amino group is a primary amino group. More preferably, the at least one thiol group is protected and the at least one amino group is a primary amino group.
  • the thiol moiety S according to the present invention enables Native Chemical Ligation (NCL).
  • NCL Native Chemical Ligation
  • such consecutive atoms are bound with another and with the thiol group and the amino group via single bonds and are preferably not incorporated into a cyclic structure.
  • such consecutive atoms located between the thiol group and the amino group are carbon atoms, in particular between the thiol group and the amino group are one or more -CH 2 - group(s) and/or a -CH(R)- group, wherein R is any residue or binding site to a linker Y or a photolabile linker moiety L.
  • Y represents a spacer, it is preferably a spacer of not more than 5 carbon atoms, preferably a spacer selected from the group consisting of methylene, ethylene, propylene, butylene and pentylene.
  • the thiol group and the amino group are therefore linked with another via a flexible spacer of consecutive carbon atoms.
  • the thiol moiety S bears the following structure: AS-(CR a 2 )x-CR a #'-NHB, wherein each R a is independently from another H or a halogen (e.g. F, CI, Br or I), in particular is H.
  • a halogen e.g. F, CI, Br or I
  • A represents hydrogen or a protecting group
  • B represents nitrogen, H represents hydrogen and B represents hydrogen, a peptide moiety P1 , an amino acid moiety, or a moiety activating the amino group; and wherein #' represents the binding site to the photolabile linker moiety L or to a spacer Y binding to the photolabile linker moiety L;
  • thiol moiety S has the following structure:
  • the compound has the following structure of formula (I):
  • A represents a protecting group residue or hydrogen, preferably a protecting group residue selected from the group consisting of a ie f-butylsulfanyl residue, a 3-nitro- 2-pyridylsulfanyl moiety, a benzyl residue, a 4-methylbenzyl residue, a 4-methoxybenzyl residue, a 2,4,6-trimethoxybenzyl residue, a diphenylmethyl residue, a trityl residue, a tert- butyl residue, an acetamidomethyl residue, a trimethylacetamidomethyl residue, a 9- fluorenylmethyl residue, an allyloxycarbonylaminomethyl residue, and a 9H-xanthen-9-yl residue, in particular a tert-butylsulfanyl residue; wherein X represents a spacer moiety or a bond, preferably a substituted or unsubstituted Ci-5-alkylen spacer moiety,
  • the compound has the following structure of formula (la): wherein A represents a protecting group residue, in particular a ie f-butylsulfanyl residue; wherein X represents a substituted or unsubstituted Ci -5 -alkylen spacer moiety, in particular -CH 2 -;
  • L represents the photolabile linker moiety L
  • Y represents a bond or a substituted or unsubstituted Ci -5 -alkylen spacer moiety, in particular a bond;
  • HP represents a hydrophilic polymer moiety
  • the compound has the following structure of formula (lb):
  • A represents a protecting group residue, in particular a fe/f-butylsulfanyl residue; wherein X represents a substituted or unsubstituted Ci -5 -alkylen spacer moiety;
  • L represents the photolabile linker moiety L
  • HP represents a hydrophilic polymer moiety
  • the compound has the following structure of formula (lc):
  • A represents a protecting group residue, in particular a ie/f-butylsulfanyl residue; wherein L represents the photolabile linker moiety L;
  • photolabile linker moiety L may be any linker known in the art that is cleavable by irradiation of light.
  • the linker is cleavable by irradiation light in the UV-A range.
  • the UV-A range may be in the range of from 250-400 nm. Preferred structures of the photolabile linker moiety L are laid out below.
  • the photolabile linker moiety L has the following structure of formula (II):
  • R 1 may be any residue of up to 40 carbon atoms, preferably a residue selected from the group consisting of -OR 11 , hydrogen, halogen, -R 11 , -0-C(0)-R 11 , -NR 11 R 12 , - SiR 11 R 12 R 13 , -Ge R 11 R 12 R 13 , -SR 11 , -SOR 11 , and -S0 2 R 11 , wherein each of R 11 , R 12 and R 13 represents a residue selected from the group consisting of a substituted or unsubstituted linear, branched or cyclic Ci -2 o alkyl
  • a dye may be understood in the broadest sense as defined in the context of the polypeptide above. Accordingly, more preferably, the photolabile linker moiety L has the following structure of formula (I la):
  • # represents the binding site to the thiol moiety S
  • #' represents the binding site to Z.
  • the photolabile linker moiety L may be a linker based on a structure like Clnitrobenzyl, a 1 -(2-nitrophenyl)ethyl, a 3-hydroxy-2-naphthalenemethanol ether, a 3- hydroxy-2-naphthalenemethanol ester or a 4,4'-dimethoxytrityl ether.
  • hydrophilic polymer as used herein may be understood in the broadest sense as any polymeric molecule that has a hydrophilic surface that does (essentially) not comprise primary amino groups (-NH 2 or -NH 3 + ) or hydroxyl groups (-OH).
  • a polymeric molecule comprises more than one monomer.
  • the hydrophilic polymer may have a molecular mass of more than 500 Da, more than 1 ,000 Da, more than 1 ,500 Da, more than 2,000 Da, more than 5,000 Da or more than 10,000 Da.
  • the hydrophilic polymer may be a soluble polymer, a gel-like polymeric matrix, a bead, a surface, a bead coating or the coating of a surface.
  • the hydrophilic polymer of the present invention is a soluble polymer.
  • the at least one hydrophilic polymer moiety HP is/are selected from the group consisting of
  • PEG polyethylene glycol
  • PEI polyethylene imine
  • polyacrylic acid or derivatives thereof preferably a polymer of methacrylic acid or derivative thereof, more preferably a polymer of hydroxypropyl methacrylate or hydroxyethyl methacrylate (HEMA) or derivatives thereof, in particular a polymer of N-(2-hydroxypropyl) methacrylamide (HPMA) or derivatives thereof;
  • HEMA hydroxypropyl methacrylate or hydroxyethyl methacrylate
  • HPMA N-(2-hydroxypropyl) methacrylamide
  • the hydrophilic polymer is PEG or derivatives thereof.
  • the hydrophilic polymer is PEG or a derivative thereof of between 5 to 50 PEG units, even more preferably of between 10 to 40 PEG units, even more preferably of between 15 to 35 PEG units, in particular of between 20 to 30 PEG units.
  • the hydrophilic polymer is PEG or a derivative thereof of between 50 to 200 PEG units, preferably of 50 to 150 PEG units, in particular 50 to 100 PEG units.
  • hydrophilic polymers the term "derivative thereof” may be understood in the broadest sense as any substitution of said polymer.
  • the terminus of such hydrophilic polymer is capped by a protecting group by a dye.
  • the terminus of PEG may be capped by Fmoc, Boc or may be acylated (e.g., acetylated).
  • the hydrophilic polymer may be a bead having a hydrophilic surface or a bead having a coating comprising a hydrophilic polymer, in particular wherein the bead is a micro- or a nanobead.
  • the hydrophilic polymer may be a solid material or a surface coating comprising a hydrophilic polymer.
  • the hydrophilic polymer may optionally be the coating of a peptide microarray, the coating of a column matrix, in particular an affinity column matrix or the coating of an implant.
  • the hydrophilic polymer of the present invention may be conjugated with the peptide of the present invention via any functional group of the peptide.
  • the hydrophilic polymer may be conjugated with the N-terminus of the peptide, an amino acid residue side chain, or the C-terminus of the peptide.
  • any conjugation strategy may be used, such as, e.g., formation of an ester bond, formation of an amide bond, formation of a thioester bond, formation of an ether bond, formation of a thioether bond or formation of a disulfide bond.
  • the hydrophilic polymer is conjugated with the N- or the C-terminus of the peptide, more preferably the hydrophilic polymer is conjugated with the N- or the C- terminus of the peptide via the formation of an amide bond, more preferably the hydrophilic polymer is conjugated with the N-terminus of the peptide via the formation of an amide bond.
  • Such hydrophilic polymer may render the compound and its conjugates with peptide moieties more soluble in aqueous buffers. Further such conjugates may bear a lower tendency to interact with surfaces (e.g, plastic surfaces, glass surface and/or surfaces of column matrices) than the corresponding compound, peptide and/or conjugate not conjugated with a hydrophilic polymer.
  • interaction may be understood in the broadest sense as the stickiness and/or retention ability of the auxiliary compound, peptide and/or conjugate with or without being conjugated to at least one hydrophilic polymer when attached to a surface.
  • the hydrophilic polymer moieties are conjugated to the compound of the present invention by any means, in particular via the spacer Z.
  • Such spacer Z may have any bivalent structure that does neither disturb the NCL not the photo-induced cleavage of the compound.
  • the spacer Z has the following structure of formula (III)
  • #"-(CH 2 ) n -Z'-#'" (III), wherein #" represents the binding site to the photolabile linker moiety L or to the hydrophilic polymer moiety HP, in particular to the photolabile linker moiety L; wherein #"' represents the binding site to the hydrophilic polymer moiety HP or to the photolabile linker moiety L, in particular to the hydrophilic polymer moiety HP; wherein n represents and integer from 1 to 10, preferably wherein n is 1 , 2 or 3, in particular 3; wherein Z' represents a bivalent group or a bond, preferably wherein Z' is selected from the group consisting of -CO-NH-(CH 2 ) m -NH-CO-, -NH-CO-, -NH-(CH 2 ) m -NH-CO-, -NH-CO- (CH 2 ) m -NH-CO-, -CO-NH-(CH 2 ) m -CO-NH-, -NH-
  • Z' is selected from the group consisting of -CO-NH-(CH 2 ) m -NH-CO-, -NH-CO-, and NH-(CH 2 ) m -NH-CO-, and the other residues are selected as laid out above.
  • Z' is -CO-NH- (CH 2 ) m -NH-CO- and the other residues are selected as laid out above.
  • the spacer Z has the following structure of formula (III),
  • #" represents the binding site to the photolabile linker moiety L
  • #"' represents the binding site to the hydrophilic polymer moiety HP
  • n 1 , 2 or 3, in particular 3;
  • Z' is selected from the group consisting of -CO-NH-(CH 2 ) m -NH-CO-, -NH-CO-, and NH-(CH 2 ) m -NH-CO-;
  • m is 1 , 2 or 3, in particular 2.
  • the spacer Z has the following structure of formula (Ilia)
  • #"' represents the binding site to the hydrophilic polymer moiety HP
  • the compound preferably has the following structure
  • the compound has the following structure of formula (IV): wherein R 1 represents a linear or branched C1-2 0 alkoxy moiety or hydrogen, preferably - OCH3, hydrogen, -OCH 2 CH 3 , -0(CH 2 ) 2 CH 3 , or -OCH(CH 3 ) 2 , in particular -OCH 3 ; wherein Z represents a direct bond or a spacer moiety, in particular a spacer moiety as defined above; wherein A represents a protecting group residue or hydrogen; and wherein HP represents a hydrophilic polymer moiety, in particularly such as defined above.
  • the compound bears one of the following structures (V)- (VII):
  • R 1 represents a linear or branched Ci -20 alkoxy moiety or hydrogen, preferably - OCH 3 , hydrogen, -OCH 2 CH 3 , -0(CH 2 ) 2 CH 3 , or -OCH(CH 3 ) 2 , in particular -OCH 3 ; wherein n represents and integer from 1 to 10, preferably wherein n is 1 , 2 or 3, in particular 3; and wherein m represents an integer from 1 to 10, preferably wherein m is 1 , 2 or 3, in particular 2.
  • the compound bears one of the following structures (Va)-(Vlla):
  • y represents an integer from 5-50, preferably of from 10-40, more preferably of from 15-35, in particular of 20-30;
  • E represents a protecting group conjugated with the PEG via an amino or hydroxyl group, in particular Fmoc;
  • R 1 represents a linear or branched Ci -2 o alkoxy moiety or hydrogen, preferably - OCH 3 , hydrogen, -OCH 2 CH 3 , -0(CH 2 ) 2 CH 3 , or -OCH(CH 3 ) 2 , in particular -OCH 3 ;
  • n represents and integer from 1 to 10, preferably wherein n is 1 , 2 or 3, in particular 3;
  • n represents an integer from 1 to 10, preferably wherein m is 1 , 2 or 3, in particular 2.
  • the amine moiety comprised in the thiol moiety S of the compound may also form (together with the amino group it is bound to) an amino acid moiety, optionally bound to a peptide moiety P1.
  • the compound of the present invention bears a structure of the following formula (XIV):
  • the compound of the present invention may bear a structure of the following formulae (XV) or (XVI):
  • R 1 is defined as above, preferably is an alkoxy residue, in particular a methoxy residue
  • (PEG) 27 may have an unbound terminus or a terminus covered by a protecting group such as Fmoc.
  • the compound of the present invention may bear a structure of the following formulae (XVII) or (XIX): ssteu SS'Bu SS?E3 ⁇ 4j
  • R 1 , n and m are each independently from another is defined as above, preferably wherein R 1 is an alkoxy residue, in particular a methoxy residue;
  • (PEG) 27 may have an unbound terminus or a terminus covered by a protection
  • B represents a peptide moiety P1 , preferably a peptide moiety P1 characterized in that:
  • # v -CHR 2 -CO-# v (VIII), wherein # v represents the binding site to the compound of the present invention; wherein # vv represents the binding site to the remaining moieties of peptide moiety P1 (rest of P1 ); and wherein R 2 represents hydrogen or a substituted or unsubstituted Ci -5 -alkyl residue, preferably hydrogen, CH 3 , -CH 2 -OH or -CHOH-CH 3 , in particular hydrogen
  • the peptide moiety P1 bears a molecular mass of less than 10 kDa, more preferably less than 5 kDa, even more preferably less than 4 kDa, and most preferably less than 3 kDa.
  • the N-terminal amino acid moiety of the peptide moiety P1 is a glycyl (Gly) moiety.
  • the peptide moiety P1 bears the following sequence: GVTSAPDTRPAPGSTAPPAH (SEQ I D NO: 1 )
  • peptides moieties P1 of the peptide sequences disclosed in WO 2012/13977 to which an N-terminal G has been added may be used.
  • P1 As used herein, the rest of P1 is peptide moiety P1 without its N-terminal amino acid moiety (which is preferably a glycyl (Gly) moiety).
  • the compound has a structure of formula (lb): wherein R 2 represents hydrogen or a substituted or unsubstituted Ci -5 -alkyl residue, preferably hydrogen, CH 3 , -CH 2 -OH or -CHOH-CH 3 , in particular hydrogen; and
  • the compound may preferably have the structure of formula (lc):
  • R 2 represents hydrogen or a substituted or unsubstituted Ci -5 -alkyl residue, preferably hydrogen, CH 3 , -CH 2 -OH or -CHOH-CH 3 , in particular hydrogen;
  • the structure may be exemplarily as follows:
  • the molecule may intramolecularly rearrange, in particular when it is subjected to an aqueous environment:
  • the compound has the following structure of formula (IX):
  • R 1 represents a linear or branched Ci -2 o alkoxy moiety or hydrogen, preferably - OCH 3 , hydrogen, -OCH 2 CH 3 , -0(CH 2 ) 2 CH 3 , or -OCH(CH 3 ) 2 , in particular -OCH 3 ; wherein Z represents a direct bond or a spacer moiety, in particular a spacer moiety as defined above; wherein A represents a protecting group residue or hydrogen; wherein HP represents a hydrophilic polymer moiety; and wherein the rest of P1 represents the peptide moiety P1 without its N-terminal moiety - CHR 2 -CO-.
  • a herein represents a peptide moiety
  • the structure may be as follows:
  • the molecule may be restructured in an aqueous environment into:
  • polypeptide conjugate P12 i.e. P1 -P2
  • P1 -P2 the polypeptide conjugate P12
  • the compound bears one of the following structu (XII):
  • n represents and integer from 1 to 10, preferably wherein n is 1 , 2 or 3, in particular 3; wherein m represents an integer from 1 to 10, preferably wherein m is 1 , 2 or 3, in particular 2; and wherein the other residues A and R 1 as well as HP and P1 are defined as above.
  • A is a peptide moiety, the structure may be as follows:
  • these molecules may be restructured in an aqueous environment into:
  • polypeptide conjugate P12 may be cleaved off.
  • the compound of the present invention may be used for the production of a polypeptide product that is a polypeptide conjugate P12 comprising a peptide moiety P1 and a second moiety P2.
  • the present invention relates to a method for the production of a peptide conjugate P12 comprising a peptide moiety P1 and a second moiety P2 covalently bound with another via an amide bond, said method comprising the steps of:
  • step (iv) conjugating a second moiety P2 comprising a thioester with said peptide conjugate PC1 obtained from step (iii) by means of Native Chemical Ligation, thereby forming a peptide conjugate PC12.
  • step (iv) may be the a peptide conjugate PC12 having a structure according to formula (Xlllb):
  • Step (i) of providing a compound is shown in the example section below.
  • the compound may be produced and/or stored in a form wherein the amino group of the thiol moiety is protected by a protecting group. Then, this moiety is cleaved off in step (i).
  • the compound may then optionally be purified and optionally be dissolved in a suitable solvent.
  • the compound has a structure of formula (I) or (la), (IV), (V), (VI), (VII), (Va), (Vb) or (VI lb), wherein B is H and A is a protecting group.
  • the method preferably comprises the steps of:
  • A represents a protecting group residue, in particular a ie f-butylsulfanyl residue
  • X represents a substituted or unsubstituted Ci -5 -alkylen spacer moiety, in particular -CH 2 -;
  • L represents the photolabile linker moiety L
  • Y represents a bond or a substituted or unsubstituted Ci -5 -alkylen spacer moiety, in particular a bond;
  • HP represents a hydrophilic polymer moiety
  • step (i) wherein A, X, Y, L, Z, P1 and HP are defined as in step (i);
  • step (v) irradiating the compound comprising the peptide conjugate P12 obtained from step (iv) thereby cleaving said peptide conjugate P12 off the photolabile linker L and the hydrophilic polymer moiety HP obtaining a polypeptide conjugate P12 (P2-P1 ); and optionally
  • Step (vi) purifying the peptide conjugate P12 obtained from step (iv) or (v).
  • Step (ii) of adding the peptide moiety may be performed by any means known in the art. This step is described further below.
  • Step (iii) of cleaving off the protecting group from the thiol group may be performed by any means known for that purpose in the art. The person skilled in the art will know how to adapt such cleaving step to the protecting group used.
  • TCEP tris(2- carboxyethyl)phosphine
  • Step (iv) of conjugating a second moiety P2 to the peptide conjugate PC1 obtained from step (iii) is performed by Native Chemical Ligation. This is typically performed in an aqueous environment (e.g., in a buffer of pH 7-8). Exemplarily a NaPi buffer (sodium phosphate buffer) of pH 7.5 may be used at a temperature of 20-40°C. This exemplified further in the example section below.
  • the result us the method is the polypeptide conjugate P12.
  • the peptide may also be modified enzymatically.
  • the method further comprises the step (iiA) of modifying the peptide moiety P1 comprised in the compound obtained from step (ii) by means of one or more enzyme(s) prior to subjecting it to step (iii) or (iv).
  • the peptide conjugate PC1 may be provided in an aqueous buffer and admixed with a suitable amount of an enzyme and optionally cofactor(s) and educts and incubated in this mixture.
  • the one or more enzyme(s) is/are glycosyltransferase(s) and said step of modifying the peptide moiety P1 is glycosylating.
  • the peptide conjugate PC1 may be further subjected to one or more glycosylating step(s) by means of one or more glycosyltransferase(s). Then a glycosylated peptide (glycopeptide) is obtained.
  • glycosylated peptide and “glycopeptide” may be understood interchangeably in the broadest sense as any peptide that comprises one or more carbohydrate moiety/moieties.
  • the glycosylated peptide is a peptide that is glycosylated in a site-specific and/or stereospecific manner, more preferably in a site- specific and stereospecific manner.
  • carbohydrate moiety and “sugar moiety” may be understood interchangeably in the broadest sense as a molecular residue of any carbohydrate known in the art.
  • a carbohydrate moiety may comprise of one or more monosaccharide unit(s).
  • a monosaccharide unit may be any monosaccharide unit known in the art, such as, e.g., a triose (e.g., an aldotriose (e.g. glyceraldehyde) or a ketotriose (e.g., dihydroxyacetone)), a tetrose (e.g., an aldotetrose (e.g., erythrose, threose) or a ketotetrose (e.g., erythrulose)), a pentose (e.g., an aldopentose (e.g., ribose, deoxyribose, arabinose, xylose, lyxose) or a ketopentose (e.g., ribulose, xylulose)), a hexose (e.g., an aldohexose (e.g., al
  • Each monosaccharide unit may be a D- or an L-saccharide and may form a linear or cyclic (e.g., pyranose or furanose) structure.
  • one or more of the functional group(s) of a monosaccharide unit may be replaced by other residue(s) such as, e.g., amino group(s), carboxylic group(s), hydroxyl group(s) carbonyl group(s), hydrogen atom(s), halogen atom(s), alkyl group(s) (e.g., methyl group(s), ethyl group(s), propyl group(s)), aminoalkyl group(s) (e.g., aminomethyl group(s), aminoethyl group(s), aminopropyl group(s)), hydroxyalkyl group(s) (e.g., hydroxymethyl group(s), hydroxyethyl group(s), hydroxypropyl group(s)), one or more amino acid(s), lipid(
  • the employed glycosyltransferase(s) can still accept the carbohydrate moiety of which one or more of the functional group(s) is/are replaced by one or more other residues(s) as a substrate.
  • a monosaccharide unit is a pentose or a hexose, in particular a D- pentose or a D-hexose.
  • the carbohydrate moiety may be composed of one, two, three, four, five or more monosaccharide unit(s).
  • the carbohydrate moiety may be a linear, branched or cyclic carbohydrate moiety.
  • the monosaccharide units may be conjugated with another by an ether bond, a secondary amine, an ester bond or an amide bond.
  • a glycopeptide of the present invention may bear one or more glycosylation site(s).
  • a glycosylation site may be understood in the broadest sense as an amino acid moiety to which one or more saccharides may be conjugated with.
  • a glycosylation site may refer to an amino acid moiety in a sequential context of amino acids, typically, a sequential context of a consecutive amino acid strand, to which one or more carbohydrate moiety/moieties is/are conjugated in nature.
  • the glycosylation pattern of the glycopeptide of the present invention may be equal to the naturally occurring glycosylation pattern or may be different.
  • glycosylation pattern may be understood in the broadest sense as the location and number of glycosylated sites of the peptide, the type(s) of conjugated monosaccharide(s), the number of conjugated monosaccharide(s) and/or the sequential orientation of the conjugated monosaccharide(s) in the carbohydrate moiety/moieties.
  • a carbohydrate moiety may be conjugated to an asparagyl or glutamyl residue via an amide bond or an ester bond, to a serinyl or threonyl residue via an ether bond, or to a cysteinyl residue via a thioether bond.
  • glycosylation may be understood in the broadest sense as the conjugation of one or more carbohydrate moiety/moieties to the peptide.
  • a glycosyltransferase specific for a particular carbohydrate moiety is used to conjugate said carbohydrate moiety with the peptide.
  • said glycosyltransferase may be removed.
  • one or more further glycosylation step(s) are conducted.
  • a glycosylation step may be understood as a reaction cycle.
  • a further glycosyltransferase specific for the conjugation of a further carbohydrate moiety is used to conjugate said further carbohydrate moiety with the peptide moiety P1 or with a carbohydrate moiety already conjugated with the peptide moiety P1 . More preferably, in a further glycosylation step, a further glycosyltransferase specific for the conjugation of a further carbohydrate moiety is used to conjugate said further carbohydrate moiety with the carbohydrate moiety already conjugated with the peptide moiety P1 and, thereby, elongates said carbohydrate moiety conjugated with the peptide moiety P1. Then, the carbohydrate moiety conjugated with the peptide moiety P1 grows with each glycosylation step.
  • a glycosyltransferase conjugates a further monosaccharide unit to the growing carbohydrate moiety conjugated with the peptide moiety P1.
  • glycosyltransferase As used in the context of the present invention, the terms “glycosyltransferase”, “glycosylating enzyme” and “glycosylation enzyme” may be understood interchangeably in the broadest sense as any enzyme that can conjugate one or more carbohydrate moiety/moieties to the peptide moiety P1 and/or to the one or more carbohydrate moiety/moieties conjugated to the peptide moiety P1 of the present invention.
  • P2 may optionally also be glycosylated.
  • the glycosylation may, exemplarily, be further performed after NCL to obtain a peptide conjugate such as exemplarily MUC1 (T)-Aux-MUC1 (T). Therefore, notably, such glycosyltransferase may also or alternatively be able to conjugate one or more carbohydrate moiety/moieties to the peptide moiety P2 and/or to the one or more carbohydrate moiety/moieties conjugated to the peptide moiety P2 of the present invention.
  • a glycosyltransferase is an enzyme of enzyme classification (EC) class 2.4 that acts as a catalyst for the transfer of one or more monosaccharide(s), disaccharide(s), trisaccharide(s), oligosaccharide(s) or polysaccharide(s) from activated donor molecules to specific acceptor molecules (i.e., the peptide moiety P1 ).
  • EC enzyme classification
  • Glycosyltransferases using nucleotide diphospho-carbohydrates, nucleotide monophospho-carbohydrates and carbohydrate phosphates include hexosyltransferases (EC 2.4.1 ), pentosyltransferases (EC 2.4.2) and transferases of other glycosyl groups (EC 2.4.99).
  • glycosyltransferase of the present invention may be any glycosyltransferase known in the art such as members of the families of glucosyltransferases, glucuronosyltransferases, galactosyltransferases, galacturonosyltransferases, fucosyltransferases, mannosyltransferases, N-acetylglucosaminyltransferases and N- acetylgalactosaminyltransferases (EC 2.4.1 ), xylosyltransferases, arabinosyltransferases and ribosyltransferases (EC 2.4.2), and sialyltransferases (EC 2.4.99).
  • glycosyltransferase known in the art such as members of the families of glucosyltransferases, glucuronosyltransferases, galactosyltransferases, galacturonosyltransfer
  • glycosyltransferase catalyses a site-specific glycosylation.
  • the glycosyltransferase(s) of the present invention may be O-glycosyltransferase(s), N- glycosyltransferase(s) or S-glycosyltransferase(s), preferably O-glycosyltransferase(s) or N-glycosyltransferase(s), more preferably O-glycosyltransferase(s).
  • the method of the present invention further comprises the step of removing the glycosyltransferase(s) and/or reaction component(s) of the step of glycosylating the polymer-conjugated peptide moiety P1 by means of one or more glycosyltransferase(s). More preferably, the method of the present invention further comprises the step of removing the glycosyltransferase(s) and reaction component(s).
  • the step of removing the one or more enzyme(s) and/or reaction component(s) is performed by chromatography and/or by precipitating the polymer-conjugated peptide, preferably by gel permeation chromatography (GPC) and/or precipitating the polymer-conjugated peptide, more preferably by a combination of GPC and precipitating the polymer-conjugated peptide (e.g. by a combination of microspin GPC and precipitating the polymer-conjugated peptide) or by precipitating as the only purification step, in particular by precipitating as the only purification step.
  • GPC gel permeation chromatography
  • the step of removing the one or more enzyme(s) and/or reaction component(s) consists of precipitating the polymer-conjugated peptide.
  • removing may be understood in the broadest sense as the decrease of the content of glycosyltransferase(s) and/or reaction component(s) by any means.
  • removing means that the content of glycosyltransferase(s) and/or reaction component(s) is decreased at least 10fold, more preferably at least 50fold, even more preferably at least 10Ofold, even more preferably at least 250fold, even more preferably at least 500fold and most preferably at least 1 ,000fold in comparison to the concentration of the reaction batch of the preceding the step of glycosylating the polymer- conjugated peptide moiety P1 by means of one or more glycosyltransferase(s) (glycosylation batch).
  • reaction component may be understood in the broadest sense as any component that may be used for performing the step of glycosylating the polymer- conjugated peptide moiety P1 .
  • Said component(s) may be, e.g., one or more carbohydrate protecting group(s), one or more protected or unprotected carbohydrate moiety/moieties not conjugated with the peptide moiety P1 , one or more cofactor(s) suitable for the employed glycosyltransferase(s), one or more side product(s) generated by the glycosyltransferase(s) and/or one or more precursor(s) usable by the employed glycosyltransferase(s).
  • a cofactor suitable for the employed glycosyltransferase(s) may be, but may not be limited to a nucleotide triphosphate (e.g., adenosine triphosphate (ATP), guanosine triphosphate (GTP), uracil triphosphate (UTP)), lipids, vitamin(s) (e.g., biotin) and/or metal ions (e.g., iron ions, cobalt ions, magnesium ions).
  • a nucleotide triphosphate e.g., adenosine triphosphate (ATP), guanosine triphosphate (GTP), uracil triphosphate (UTP)
  • lipids e.g., vitamin(s) (e.g., biotin) and/or metal ions (e.g., iron ions, cobalt ions, magnesium ions).
  • ATP adenosine triphosphate
  • GTP guanosine triphosphate
  • a side product generated by the glycosyltransferase(s) may be, but may not be limited to phosphate, pyrophosphate, uracil diphosphate (UDP), cytosine diphosphate (CDP), guanosine diphosphate (GDP), uracil monophosphate (UMP), cytosine monophosphate (CMP), uracil and/or cytosine.
  • UDP uracil diphosphate
  • CDP cytosine diphosphate
  • GDP guanosine diphosphate
  • UMP uracil monophosphate
  • CMP cytosine monophosphate
  • a precursor usable by the employed glycosyltransferase(s) may be, but may not be limited to one or more glycosyl moiety/moieties such as, e.g., one or more carbohydrate-phosphate(s), one or more carbohydrate-pyrophosphate(s), one or more carbohydrate-triphosphate(s), one or more nucleotide-diphosphate carbohydrate(s) (e.g., a uracil-diphosphate carbohydrate(s) (UDP-carbohydrate(s)), guanosin-diphosphate carbohydrate(s) (GDP-carbohydrate(s))), nucleotidephosphate carbohydrate(s) (e.g., cytosine-monophosphate carbohydrate(s) (CMP-carbohydrate(s))), one or more lipid- linked carbohydrate donor(s) (e.g., terpenoid-linked carbohydrate
  • buffer component(s) such as, e.g., salts, buffer substances (e.g., phosphate ions, hydrogenphosphate ions, TAPS (3- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ propanesulfonic acid), Bicine (N,N-bis(2- hydroxyethyl)glycine), Tris(tris(hydroxymethyl)methylamine), Tricine (N- tris(hydroxymethyl)methylglycine), TAPSO ([N-Tris(hydroxymethyl)methylamino]-2- hydroxypropanesulfonic acid), HEPES (2-hydroxyethyl-1 -piperazineethanesulfonic acid), TES (2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethanesulfonic acid), MOPS (3-(N- morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid), Cacodylate (d
  • detergent may be understood interchangeably with “surfactant”.
  • a detergent may be every detergent used in the art such as, e.g., Triton (X-100, CF-54), Nonidet P-40, CHAPS (3-[(3-Cholamidopropyl)dimethylammonio]-1 -propanesulfonate), a polysorbate (glyceryl laurate, polyoxyethylene glycol sorbitan alkyl ester, e.g., polysorbate 20, polysorbate 21 , polysorbate 40, polysorbate 60, polysorbate 61 , polysorbate 65, polysorbate 80, polysorbate 81 , polysorbate 85, polysorbate 120), a poloxamer (block copolymer of polyethylene glycol and polypropylene glycol), ADS (ammonium dodecyl sulfate), SLES (sodium lauryl ether
  • the one or more detergent(s) is/are Triton, Nonidet P-40, CHAPS, a polysorbate
  • reaction component(s) may also be residuals of the step of providing a peptide moiety P1 conjugated with one or more hydrophilic polymer(s) still present when performing the glycosylating step.
  • Said component(s) may be, e.g., one or more amino acid protecting group(s), one or more protecting group(s) of the hydrophilic polymer(s), one or more coupling agent(s), one or more scavenger(s), one or more cleaving agent(s), one or more protected or deprotected amino acid(s) not conjugated with the peptide moiety P1 , one or more protected or deprotected hydrophilic polymer(s) not conjugated with the peptide moiety P1 , one or more cofactor(s) suitable for the employed of glycosyltransferase(s), one or more side product(s) generated by the glycosyltransferase(s) and/or one or more precursor(s) usable by the employed glycosyltransfer
  • the glycosyltransferase(s) and/or reaction component(s) may be removed by any method(s) known in the art such as, e.g., one or more chromatographic method(s), one or more filtration method(s), one or more electrophoretic method(s), one or more precipitation-based method(s) and/or one or more dialysis method(s).
  • the step of removing the glycosyltransferase(s) and/or reaction component(s) does not require any toxic agent(s) such as, e.g., acetonitrile and TFA. Therefore, purification may preferably be performed with an aqueous solution such as a buffer, more preferably a buffer suitable for the glycosyltransferase(s) of the next glycosylating step as shown in the examples.
  • aqueous solution such as a buffer, more preferably a buffer suitable for the glycosyltransferase(s) of the next glycosylating step as shown in the examples.
  • removing the glycosyltransferase(s) and/or reaction component(s) is performed by a combination of not more than three different methods, more preferably a combination not more than two different methods or even not more than a single method.
  • a chromatographic method may be any chromatographic method known in the art such as, e.g., gel permeation chromatography (GPC), high performance liquid chromatography (HPLC), reversed phase HPLC (RP-HPLC), fast protein liquid chromatography (FPLC), Flash Chromatography (flash), Rapid Refluid Liquid Chromatography (RRLC), Rapid Separation Liquid Chromatography (RSLC), Ultra Fast Liquid Chromatography (UFLC), reversed phase UFLC (RT-UFLC), Ultra Performance Liquid Chromatography (UPLC) or reversed phase UPLC (RT-UPLC).
  • the column may be a prepacked column and/or a spin column usable by the employment of a centrifugal force.
  • the step of removing the glycosyltransferase(s) and/or reaction component(s) after one glycosylating cycle is performed by a single chromatographic step and/or a single precipitating step. That means that only one column and/or one precipitation step is used. That may lead to higher yields of the glycosylated peptide moiety P1 in comparison to a multistep procedure in which more than one chromatographic steps and/or more than one precipitating steps are used. This leads to a particular improvement of yields when there are more than one glycosylation steps, thus, more than one glycosylating cycles.
  • the chromatographic matrix may preferably be a bead matrix.
  • the beads may be spherical or may bear any other shape known in the art to conduct chromatographic matrix.
  • the beads may be of any material known in the art to be useful for preparing chromatographic material such as, e.g., silica, sugar-based bead material (e.g., agarose, sepharose), plastic bead material (e.g., polystyrene).
  • the beads may bear a neutral, a positive or a negative zeta potential.
  • the column may be a flow- through device or may be used batch-wise.
  • removing the glycosyltransferase(s) and/or reaction component(s) is performed by a chromatographic method using an aqueous buffer as solvent. More preferably removing the glycosyltransferase(s) and/or reaction component(s) is performed by gel permeation chromatography (GPC).
  • GPC gel permeation chromatography
  • the terms "gel permeation chromatography”, “molecular sieve chromatography”, “gel filtration” and “size exclusion chromatography” may be understood interchangeably in the broadest sense as a chromatographic method for separating molecules via size.
  • the GPC matrix has a pore size in which several molecules that fit in (i.e., the polymer-conjugated peptide moiety P1 ) are retarded and that high- molecular weight components (i.e., glycosyltransferase(s)) pass by. It will be understood that in the context of the present invention the pore-size is selected accordingly.
  • the GPC matrix has a hydrophilic surface such as, e.g., cross-linked polysaccharide material (e.g, a cross-linked dextran gel (e.g., Sephadex material such as, e.g., Sephadex G-10 or Sephadex G-15)).
  • a cross-linked polysaccharide material e.g, a cross-linked dextran gel (e.g., Sephadex material such as, e.g., Sephadex G-10 or Sephadex G-15)
  • cross-linked polysaccharide material e.g, a cross-linked dextran gel (e.g., Sephadex material such as, e.g., Sephadex G-10 or Sephadex G-15)
  • a chromatographic method is a fast chromatographic method such, e.g., a chromatographic method working with a pressure that is higher than ambient pressure.
  • This pressure may be obtained by using a pump and/or by centrifuging the column.
  • the higher pressure may typically lead to a higher flow rate. Consequently, the tailing of the elution profile is minimized and the peptide moiety P1 is obtainable faster, in a higher purity, a higher yield and higher concentrated. Undesired interaction with the column matrix and surface is minimized.
  • removing the glycosyltransferase(s) and/or reaction component(s) is performed by centrifuging a microspin GPC column as used in the examples.
  • an aqueous buffer may be any aqueous buffer suitable for the glycosyltransferase(s) used in the context of the present invention such as, e.g., phosphate buffered saline (PBS), a HEPES buffer, a Tris buffer, a TAPS buffer, a Bicine buffer, a Tricine buffer, a TAPSO buffer, a TES buffer, a MOPS buffer, a PIPES buffer, a Cacodylate buffer, an SSC buffer, an MES buffer, an acetate buffer or culture medium.
  • PBS phosphate buffered saline
  • HEPES buffer HEPES buffer
  • Tris buffer Tris buffer
  • TAPS buffer Tris buffer
  • Bicine buffer Tricine buffer
  • TAPSO buffer Tricine buffer
  • TES buffer Tricine buffer
  • MOPS buffer Tricine buffer
  • PIPES buffer a PIPES buffer
  • Cacodylate buffer an SSC buffer
  • an MES buffer an acetate buffer or culture medium
  • a filtration method may be any filtration method known in the art, such as, e.g, dead-end filtration or cross-flow filtration. Filtration may be conducted batch-wise of in a continuous flow method. As used herein, the terms “cross-flow filtration”, “crossflow filtration”, “tangential flow filtration” or “tangential filtration” may be understood interchangeably.
  • the filter may be of any material known in the context of filtration in the art, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material.
  • the filter may be hydrophobic or hydrophilic.
  • the surface of the filter may be neutral or positively charged or negatively charged.
  • the filtration method is a filtration method using an aqueous buffer as solvent.
  • an electrophoretic method may be used.
  • an electrophoretic method may be any electrophoretic method known in the art such as, e.g., gel electrophoresis or capillary (CE) electrophoresis.
  • the electrophoretic method is an electrophoretic method using an aqueous buffer as solvent.
  • a precipitation-based method may be any precipitation- based method known in the art such as, e.g., salting in, salting out or precipitation by adding one or more organic solvents (e.g, acetone, diethylether, ethanol, methanol, dichloromethane).
  • organic solvents e.g, acetone, diethylether, ethanol, methanol, dichloromethane.
  • precipitation is performed by adding a solvent in which only one or some of the component(s) is/are well-soluble and the other component(s) is/are not or poorly soluble and precipitate.
  • the supernatant and/or the pellet may be used further.
  • precipitation may be diethyl ether precipitation.
  • a dialytical method may be used.
  • Dialysis is based on diffusion and osmosis, respectively, and is well-known in the art. Dialysis is widely used in protein purification and is also used to provide an artificial replacement for lost kidney function in people with renal failure.
  • the membrane may be of any material, such as, e.g., plastic (e.g., nylon, polysterene), metal, alloy, glass, ceramics, cellophane, cellulose, or composite material.
  • the membrane may be hydrophobic or hydrophilic.
  • the surface of the membrane may be neutral or positively charged or negatively charged.
  • dialysis is dialysis based on an aqueous buffer as solvent. As described above, most preferably, chromatography is a single chromatographic step, thus, the use of a single column only.
  • microspin GPC may be understood in the broadest sense as any spin column packed with a GPC matrix that is usable in a centrifugal tube and has a maximal loading volume of less than 15 ml, less than 10 ml, less than 5 ml, less than 4 ml, less than 3 ml, less than 2 ml, less than 1 ml, less than 0.5 ml, or less than 0.25 ml.
  • the microspin GPC column may be used in a centrifuge and subjected to a centrifugal force of at least 100 x g, at least 1 ,000 x g, at least 2,500 x g, at least 5,000 x g, at least 7,500 x g, at least 10,000 x g, at least 15,000 x g, at least 25,000 x g, or at least at least 50,000 x g, depending on the GPC matrix.
  • the glycosylated peptide moiety P1 is in contact with the column matrix for a comparably short time. The person skilled in the art will understand that this may increase yield and purity of the eluted glycosylated peptide moiety P1 . A tailing in the elution profile is minimized.
  • the peptide conjugate obtained in step (ii) may be prepared by any means known in the art for adding a peptide to a structure like the compound.
  • step (ii) of adding a peptide moiety P1 to the amino group covalently bound to the thiol moiety S of the compound is:
  • step (iia) providing an unbound peptide P1 and conjugating said peptide P1 to the compound of step (i) that is optionally is attached to a solid phase; or (iib) synthesizing the peptide moiety P1 on the compound of step (i) by sequentially conjugating amino acid building blocks, preferably wherein said compound is attached to a solid phase, in particular by means of Fmoc- or Boc-based solid phase peptide synthesis (SPPS).
  • SPPS solid phase peptide synthesis
  • the peptide P1 may be provided by chemical synthesis, obtained from a biotechnological method and/or extracted from a natural source.
  • the peptide P1 is provided by chemical synthesis.
  • chemical synthesis may refer to SPPS, liquid phase peptide synthesis or a combination of both.
  • the synthesis typically bases on the stepwise coupling of amino acids bearing protected side chains (orthogonal protecting groups).
  • the peptide strand grows from the C- terminus to the N-terminus.
  • the most common methods base on at least two different types of protecting groups that are cleavable under at least two different conditions, such as, e.g., the fluorenyl-9-methoxycarbonyl/tert- butanyl- (Fmoc/tBu) protecting group scheme (Sheppard Tactics) or the tert- butoxycarbonyl/benzyl- (Boc/Bzl) protecting group scheme (Merrifield Tactics).
  • Fmoc/tBu fluorenyl-9-methoxycarbonyl/tert- butanyl-
  • Boc/Bzl tert- butoxycarbonyl/benzyl-
  • the peptide P1 may be also provided by conjugating two or more peptide strand(s) with another by any conjugation method known in the art such as, e.g., Native Chemical Ligation (NCL), Click Chemistry, Maleimide-Thiol Conjugation, enzymatic conjugation, biochemical protein ligation and/or soluble handling conjugation.
  • the peptide P1 may be obtained from a biotechnological method.
  • biotechnological methods are well-known in the art such as, e.g., overexpression and/or heterologous expression, in particular heterologous expression based on cloning of one or more gene(s) in bacteria, insect cells, mammalian cells or yeast cells.
  • the peptide may further be extracted by any means known in the art.
  • the peptide may be extracted from a natural source by any means known in the art.
  • the peptide P1 may be purified by any means known in the art, such as, e.g., one or more chromatographic method(s), one or more filtration method(s), one or more electrophoretic method(s), one or more precipitation-based method(s), one or more dialysis method(s) or a combination of two or more thereof.
  • the natural source may be any biological material such as, e.g., bacterial material, plant material, animal material or fungal material, such as e.g. tissue, liquids or secretion(s).
  • the peptide obtained from a natural source may also be digested or partly digested by one or more protease(s). It will be understood by a person skilled in the art, that the aforementioned methods for providing a peptide may also be combined with another. In particular a peptide obtained from a biotechnological method or a natural source may further be purified and/or modified by chemical means known in the art.
  • solid support in the context of peptide synthesis may be understood interchangeably in the broadest sense as any solid matrix known for peptide synthesis in the art.
  • the solid support may be, but may not be limited to, chloromethyl resin (Merrifield resin), 4-benzyloxybenzyl alcohol resin (Wang resin), (2,4-dimethoxy)benzhydrylamine resin (Rink amide resin), 2,4- dialkoxybenzyl resin (super acid-sensitive resin, SASRIN®), 2-chlorotrityl resin, alpha- chlorotritylchloride resin (Barlos resin), benzhydrylamine resin (BHA resin), chloromethyl resin, hydroxymethylbenzoic acid resin (HMBA resin), 4-hydroxymethyl-3- methoxyphenoxybutyric acid resin (HMPB resin), hydroxycrotonoyl aminomethyl resin (HYCRAM resin), MBHA resin, oxime resin, 4-(hydroxymethyl)phenylaceta
  • HMBA resin hydroxymethylbenzoic acid resin
  • a protecting group may be any protecting group known in the art such as, e.g., an amino-protecting group of the urethane type (e.g., benzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl (Z(OMe)), 2- nitrobenzyloxycarbonyl (Z(2-N0 2 )), 4-nitrobenzyloxycarbonyl (Z(N0 2 )), chlorobenzyloxycarbonyl (Z(CI), Z(2-CI), Z(3-CI), Z(2,4-CI)), 3,5- dimethoxybenzyloxycarbonyl (Z(3,5-OMe), alpha,alpha-dimethyl-3,5- dimethoxybenzyloxycarbonyl (Ddz), 6-nitroveratryloxycarbonyl (Nvoc), 4-(phenyldiazenyl)- benzyl (Z), 4-methoxybenzyloxycarbonyl (Z), 4-methoxybenzyloxycarbonyl (Z(OMe
  • the peptide P1 may be conjugated to the compound by any means known in the art.
  • # vv represents the binding site to the remaining moieties of peptide moiety P1 ;
  • R 2 represents hydrogen or a substituted or unsubstituted Ci -5 -alkyl residue, preferably hydrogen, CH 3 , -CH 2 -OH or -CHOH-CH 3 , in particular hydrogen,
  • conjugation is preferably performed by incubation with iodoacetic acid. This is exemplified further in the example section.
  • the final product obtained by the method may be the polypeptide conjugate P12 conjugated to the compound. In most cases, however, the indented final product will be the released unbound polypeptide conjugate P12.
  • the method comprises the further step (v) of irradiating the compound comprising the peptide conjugate P12 obtained from step (iv) thereby cleaving said peptide conjugate P12 off the photolabile linker L and the hydrophilic polymer moiety HP.
  • the wavelength and light intensity may be adapted to the individual photolabile linker L. Most typically, this will be performed in the UV-A range.
  • the polypeptide conjugate P12 may also be purified. Therefore, in a preferred embodiment, the method further comprises the step (vi) of purifying the peptide conjugate P12 obtained from step (iv) or (v). In a more preferred embodiment, purification is performed by one or more chromatographic method(s) and/or one or more precipitation step(s), in particular gel permeation chromatography (GPC), fast protein liquid chromatography (FPLC), reversed phase high performance liquid chromatography (RP-HPLC) and/or diethyl ether precipitation.
  • GPC gel permeation chromatography
  • FPLC fast protein liquid chromatography
  • RP-HPLC reversed phase high performance liquid chromatography
  • diethyl ether precipitation diethyl ether precipitation
  • the second moiety P2 used in the method may be any moiety bearing an alpha-thioester moiety.
  • the second moiety P2 is a peptide moiety, a peptide analogue moiety, a protein moiety, a lipid moiety, a polymeric scaffold moiety, a surface of a device, an oligonucleotide or a dye moiety.
  • the second moiety P2 is a peptide moiety.
  • the surface of a device may exemplarily be a microarray coated with moieties comprising alpha-thioester moieties.
  • An oligonucleotide may be a natural oligonucleotide (e.g., consisting of a ribonucleic acid (RNA) strand or a deoxyribonucleic acid (DNA) strand) or may be an analogue of such natural oligonucleotide consisting of or comprising non-natural moieties such as, e.g., comprising xeno nucleic acid (XNA) (e.g., glycol nucleic acid (GNA), threose nucleic acid (TNA) and/or hexose nucleic acid (HNA)), locked nucleic acid (LNA), peptide nucleic acid (PNA) and/or morpholinos.
  • XNA xeno nucleic acid
  • GNA glycol nucleic acid
  • TAA threose nucleic acid
  • HNA hexose nucleic acid
  • LNA locked nucleic acid
  • the second moiety P2 being a peptide moiety may be a peptide moiety P2 bearing the same sequence or a peptide moiety P2 bearing another sequence than the peptide moiety P1 .
  • the second peptide moiety P2 may be a glycopeptide or a non-glycosylated peptide.
  • the second peptide moiety P2 may be a peptide moiety of less than 20 amino acids, more than 20 amino acids, more than 50 amino acids, more than 100 amino acids, more than 150 amino acids, more than 200 amino acids or even more than 250 amino acids.
  • the peptide moieties P1 and P2 are conjugated with another by head-to-tail.
  • the second peptide moiety P2 may bear a sequence derived from the same naturally occurring peptide.
  • the peptide moiety P1 may be conjugated to another functional peptide such as, e.g., a fluorescent protein (e.g., (enhanced) green fluorescent protein (e)GFP, red fluorescent protein (RFP), yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) or mCherry), a cell-penetrating peptide (e.g., a polyarginine (e.g., R 7 , Rs or R 9 ), a penetrating peptide, a Chariot peptide, a lactoferrin-derived peptide, a HIV Tat peptide, a SynB1 peptide, a Buforin peptide, a Magainin peptide, a HIV-gp41 peptide or a Kaposi FGF signal sequence peptide), or a reporter group (e.g., a hist
  • the obtained polypeptide conjugate P12 may be purified by any means known in the art, e.g., by one or more chromatographic method(s), one or more filtration method(s), one or more electrophoretic method(s), one or more precipitation-based method(s) and/or one or more dialysis method(s).
  • purifying the polypeptide conjugate P12 is performed by GPC, FPLC, RP-HPLC and/or diethyl ether precipitation. More preferably, purifying the polypeptide conjugate P12 is performed by GPC, FPLC and/or RP-HPLC and diethyl ether precipitation.
  • diethyl ether precipitation is preferably performed with cooled diethyl ether, such as, e.g., diethyl ether at 4°C, -20°C or -80°C.
  • the cooled diethyl ether may be added to the broth containing the polypeptide conjugate P12.
  • the polypeptide conjugate P12 may then be precipitated.
  • the reaction tube containing diethyl ether and polypeptide conjugate P12 is incubated at a low temperature such as, e.g., at 4°C, -20°C or -80°C.
  • a polypeptide conjugate P12-containing pellet may be obtained by centrifugating the reaction tube.
  • the diethyl ether solution may optionally further contain other organic solvents such as, e.g., ethanol.
  • ethanol is first added to the reaction containing the polypeptide conjugate P12, mixed, and diethyl ether is added before incubation at a low temperature such as, e.g., at 4°C, -20°C or -80°C.
  • the diethyl ether solution may be discarded and/or evaporated.
  • the obtained polypeptide conjugate P12-containing pellet may be dried or dissolved in a suitable solvent (e.g., an aqueous buffer or an organic, polar solvent (e.g.
  • the polypeptide conjugate P12 may first be purified by precipitation and subsequently purified by a chromatographic method or vice versa.
  • polypeptide conjugate P12 may also be subjected to a further step of preserving the polypeptide conjugate P12.
  • polypeptide conjugate P12 may be understood in the broadest sense as any means for preparing a storable polypeptide conjugate P12 and elongating shelf-life of said polypeptide conjugate P12.
  • preserving the polypeptide conjugate P12 may be drying or freeze-drying.
  • freeze-drying As used herein, the terms “freeze-drying”, “lyophilization” and “cryodesiccation” may be understood interchangeable in the broadest sense as the removal of evaporable residuals from the polypeptide conjugate P12 in the frozen state.
  • the polypeptide conjugate P12 is first dissolved in an aqueous solution, preferably in water, a water/acetonitrile mixture or a buffer with extensive volatile components.
  • the sample is then frozen (e.g., preferably at -80° or liquid nitrogen) and, finally, the sample is dehydrated.
  • the freeze-dried polypeptide conjugate P12 may be stored at any suitable temperature such as, e.g., at room temperature, at -20°, at -80°C or in liquid nitrogen.
  • the polypeptide conjugate P12 may be dissolved in any suitable solvent (e.g., an aqueous buffer or an organic, polar solvent (e.g.
  • the polypeptide conjugate P12 may be frozen such as, e.g., at -20°, -80°C or in liquid nitrogen.
  • the polypeptide conjugate P12 may be frozen in the dry state or dissolved in a suitable solvent.
  • the polypeptide conjugate P12 may be thawed.
  • the polypeptide conjugate P12 may further be preserved by adding one or more preservative agent(s) such as, e.g., sodium azide (NaN 3 ) and/or benzoic acid.
  • polypeptide conjugate P12 may be dissolved in an organic solvent such as, e.g., DMSO, DMF, acetonitrile, ethanol, methanol or a mixture of two or more thereof.
  • organic solvent such as, e.g., DMSO, DMF, acetonitrile, ethanol, methanol or a mixture of two or more thereof.
  • one or more reducing agent(s) may be added to the stored polypeptide conjugate P12 such as, e.g. traces of a thiol.
  • the present invention further relates to a polypeptide conjugate P12 obtained from the method of the present invention or salts thereof, in particular pharmaceutically acceptable salts thereof.
  • the polypeptide conjugate P12 obtained from the method of the present invention is typically an extensively pure polypeptide. Therefore, said polypeptide conjugate P12 is particularly well usable for any means a particularly pure polypeptide conjugate P12 may be used for, in particular for one or more pharmaceutical purpose(s).
  • said polypeptide conjugate P12 or a pharmaceutically acceptable salt thereof may be used for a pharmaceutical purpose such as, e.g., as antibiotic, hormone, cytokine, vaccine, artificial extracellular matrix, artificial glycocalyx and/or coating for an implant.
  • said polypeptide conjugate P12 or a pharmaceutically acceptable salt thereof may be used as a vaccine, in particular a mixed vaccine, thus, a vaccine in that several polypeptide moieties of a polypeptide conjugate P12 of the present invention and at least a part of the one or more carbohydrate moiety/moieties conjugated with said polypeptide conjugate P12 in common serve as an antigen.
  • Said antigen may be recognizable by an immune cell receptor or an antibody or fragment thereof.
  • Said polypeptide conjugate P12 may also serve as a haptene.
  • Said polypeptide conjugate P12 or a pharmaceutically acceptable salt thereof may be used in a method for the treatment or prevention of a disease such as, e.g., cancer, thrombosis, myocardial infarction, viral or bacterial infection and/or stroke.
  • a disease such as, e.g., cancer, thrombosis, myocardial infarction, viral or bacterial infection and/or stroke.
  • the polypeptide conjugate P12 of the present invention may be used in a method for the prevention of a viral or bacterial infection or for the treatment of cancer by immune therapy.
  • said polypeptide conjugate P12 or a salt thereof may be used for scientific research, in particular research dealing with cell-cell interaction, cell-matrix interaction, neoplastic cells, cancer, thrombosis, myocardial infarction, viral or bacterial infection and/or stroke.
  • the peptide obtainable by the method of the present invention may also be used in a screening library or on a microarray for detecting novel interaction partners of enzymatically modified polypeptides(s) (e.g, glycopeptides(s)) or characterizing interaction pattern(s) of such polypeptides with cells or other peptides.
  • the peptide may further be immobilized on an affinity chromatographic matrix and used for affinity chromatography.
  • such polypeptide conjugate P12 or a salt thereof may be a short mucin-type O-glycopeptide.
  • said mucin-type O-glycopeptide is carrying the ⁇ -, T-, ST- or STn-antigen or combinations thereof, in particular Tn-, T- or STn-antigen O-glycans or combinations thereof usable as a cancer vaccine.
  • Scheme 1 shows a schematic representation of the auxiliary mediated chemoenzymatic approach, including all glycans installed on MUC1 peptides.
  • Scheme 2 shows the basic principle of using the compound of the present invention in a method of the present invention. It is demonstrated that an efficient approach for the preparation of site-specifically modified proteins has been established and applied to the synthesis of native homogeneously glycosylated MUC1 analogues. A photo-cleavable PEGylated auxiliary mediates the enzymatic site-specific O-glycosylation and subsequent ligation of MUC1 peptides and is eventually cleanly removed by UV irradiation.
  • Scheme 3 shows a schematic representation of the synthesis of an auxiliary-MUC1 peptide conjugate.
  • the MUC1 peptide (MUC1 (3Tn-3Ac)) carries three Tn antigens at specific positions.
  • the synthesis of the conjugate does not involve iodoacetylation of the peptidyl resin and S N 2 reaction, instead the auxiliary 8 is first converted into an auxiliary-glycine conjugate 10, which is in turn coupled to the N- terminus of the MUC1 (3Tn-3Ac) peptidyl resin via HATU-mediated coupling generating the conjugate Aux-MUC1 (3Tn-3Ac).
  • Figure 1 shows the synthesis of the photocleavable ligation auxiliary, a) Methyl 4-chloro butanoate, K 2 C0 3 , Bu 4 NI, CH 3 CN, reflux, 80%; b) HN0 3 ,CH 3 COOH, 0°C to rt, 93%; c) MePPh 3 Br, TMS 2 NNa, THF, -10°C to 0°C, 65%; d) AD-mixa, ' ⁇ / ⁇ 2 ⁇ 2 / ⁇ 2 ⁇ ,90%; e) SOCI 2 , NEt 3 ,CH 2 CI 2 , 0°C, f) NaN 3 , DMF, 60°C; g) PPh 3 , H 2 0, THF, 70°C; h) Boc 2 0, DMAP, CH 3 CN, 0°C, 63% (over four steps); i) PPh 3 , DIAD, CH 3 COSH, THF, 0°C to
  • Figure 2 shows the sequential glycosylation of peptide conjugate Aux-MUCI (Tn) gives peptide conjugates Aux-MUC1(T) and Aux-MUCI (ST).
  • MUC1 Peptide consisting of the tandem repeat sequence of Mucin 1 VTSAPDTRPAPGSTAPPAH (SEQ ID NO: 2), the O- glycan on the side chain of Thr(14) are indicated in brackets.
  • Tn GalNaca
  • T Gal31 - 3GalNAca
  • ST Neu5Aca2-3Gal31 -3GalNaca.
  • Left panel HPLC chromatogram and deconvoluted mass spectra of starting material Aux-MUCI (Tn) and of Aux-MUC1 (T) and Aux-MUCI(ST) after precipitation. The peak at 2 min is the injection peak.
  • Right panel ESI-MS spectra of the conjugates.
  • FIG. 3 shows an HPLC chromatogram of the NCL reaction with non-glycosylated Aux- MUC1 -OH and MUC1 -SR and ESI-MS of the ligation product.
  • MUC1 Peptide consisting of the tandem repeat sequence of Mucin 1 VTSAPDTRPAPGSTAPPAH (SEQ ID NO: 2), Aux-MUCI -OH: auxiliary conjugate-MUC1 peptide.
  • MUC1 -SR MUC1 peptide thioester.
  • MUC1 -Aux-MUCI ligation product, calculated mass: 5580 Da, found: 1861 .0 Da ([M+3H] 3+ ),1396.0 Da ([M+4H] 4+ ), 1 1 16.9 Da ([M+5H] 5+ ), 931 .0 Da ([M+6H] 6+ ), 798.2 Da ([M+7H] 7+ ), 698.6 Da ([M+8H] 8+ ), 621 .1 Da ([M+9H] 9+ ).
  • Asterisk indicates the ligation product (21 .92 min), peak at 23.03 min: thiol deprotected Aux-MUC1 -OH, peak at 24.48 min: Aux-MUC1 -OH (with thiol protected as ie f-butyldisulfanyl) peak at 9.27 min: MUC1 - SR, injection peak at 2 min.
  • Figure 4 shows HPLC chromatograms of the NCL reaction between Aux-MUCI (Tn) and MUC1 -SR and ESI-MS of the ligation product. Calculated mass: 5783 Da, found: 1929.0 Da ([M+3H] 3+ ),1447.0 Da ([M+4H] 4+ ), 1 157.5 Da ([M+5H] 5+ ), 965.1 Da ([M+6H] 6+ ), 827.2 Da ([M+7H] 7+ ), 723.8 Da ([M+8H] 8+ ). Asterisk indicates the ligation product.
  • FIG. 5 shows HPLC runs of the NCL reaction between Aux-MUCI(ST) and MUC1 -SR and ESI-MS of the starting material and ligation product (from bottom to top):
  • Figure 6 shows HPLC runs of the crude (A) and isolated (B) final product MUC1 -G- MUC1(ST) and the corresponding ESI-MS (C). Calculated mass: 4355 Da, found: 1452.5 Da ([M+3H] 3+ ),1089.8 Da ([M+4H] 4+ ), 871 .8 Da ([M+5H] 5+ ), 727.0 Da ([M+6H] 6+ ), 623.4 Da ([M+7H] 7 . Asterisk indicates the umprotected ligation product.
  • FIG. 7 shows HPLC runs and mass spectra of glycosylated Aux-MUC1 -NHNH 2 , precursors of glycosylated peptide thioesters and the corresponding ESI-MS spectra (from bottom to top):
  • Aux-MUC1 (Tn)-NHNH 2 calculated mass: 4073 Da, found: 1358.9 Da ([M+3H] 3+ ),1019.3 Da ([M+4H] 4+ ), 815.8 Da ([M+5H] 5+ ), 680.0 Da ([M+6H] 6+ ), 583.0 Da ([M+7H] 7+ ),
  • Figure 8 shows a example for a protected compound of the present invention.
  • Figure 9 shows an HPLC chromatogram and ESI-MS spectrum (major peak in the chromatogram) of Fmoc protected peptide MUC1 .
  • Figure 10 shows an HPLC chromatogram and ESI-MS spectrum of Fmoc protected MUC1 (Tn) peptide (GalNAc protected as Ac3). Calculated Mass for C107H153N25O37:
  • Figure 11 shows an HPLC chromatogram and ESI-MS spectrum of IAc-MUC1 peptide. Calculated mass for CsoH ⁇ sl l ⁇ C ⁇ : 1996.8 Da, found: 1000.0 Da ([M+2H] 2+ ), 666.9 Da ([M+3H] 3+ ), 500.4 Da ([M+4H] 4+ ).
  • Figure 12 shows an HPLC chromatogram and ESI-MS spectrum (peak at 6.58 min) of IAc-MUC1 (Tn) peptide.
  • Figure 13 shows an HPLC chromatogram and ESI-MS spectrum (peak at 26.45 min, asterisk) of Aux(Fmoc)MUC1. Calculated Mass for C1 MH166N28O35S2: 2551.2 Da, found:
  • Figure 15 shows an HPLC chromatogram and ESI-MS spectrum of Aux-MUC1. Calculated Mass for C173H283N29O64S2: 3854.9 Da, found: 1929.7 Da ([M+2H] 2+ ), 1286.4 Da ([M+3H] 3+ ), 965.2 Da ([M+4H] 4+ ), 772.4 Da ([M+5H] 5+ ), 643.8 Da ([M+6H] 6+ ), 552.0 Da ([M+7H] 7+ ).
  • Figure 16 shows an HPLC chromatogram and ESI-MS spectrum of Aux-MUCI (Tn).
  • Figure 18 shows an HPLC chromatogram and ESI-MS spectrum of MUC1 -SR peptide thioester. Calculated mass: 1952.8 Da, found: 977.8 Da ([M+2H] 2+ ), 652.1 Da ([M+3H] 3+ ),489.6 Da ([M+4H] 4+ ).
  • Figure 19 shows an HPLC chromatogram and ESI-MS spectrum of Aux-MUCI (Tn)- NHNH 2 (A) and of Aux(NH 2 )-MUC1(Tn)-NHNH 2 (B).
  • Aux-MUC1(Tn)-NHNH 2 calculated mass: 4073 Da, found 1358.6 Da ([M+3H] 3+ ),1019.3 Da ([M+4H] 4+ ), 815.8 Da ([M+5H] 5+ ), 680.0 Da ([M+6H] 6+ ), 583.0 Da ([M+7H] 7+ ).
  • Aux-MUCI (Tn) calculated mass: 4059 Da, found: 1354.0 Da ([M+3H] 3+ ),1015.7 Da ([M+4H] 4+ ), 812.8 Da ([M+5H] 5+ ), 677.8 Da ([M+6H] 6+ ), 580.9 Da ([M+7H] 7+ );
  • Aux-MUC1(T) calculated:4222 Da, found: 1408.5 Da ([M+3H] 3+ ),1056.7 Da ([M+4H] 4+ ), 845.4 Da ([M+5H] 5+ ), 704.6 Da ([M+6H] 6+ ).
  • Figure 21 shows HPLC traces and ESI-MS spectra of sequential glycosylation of Aux ( NH2)-MUC1 (Tn)-NHNH 2 and precipitation.
  • Aux ( NH2)-MUC1 (Tn)-NHNH 2 calculated mass: 3852 Da, found: 1284.6 Da ([M+3H] 3+ ), 964.0 Da ([M+4H] 4+ ), 771 .3 Da ([M+5H] 5+ ), 642.9 Da ([M+6H] 6+ ), 551 .3 Da ([M+7H] 7+ );
  • Aux(NH 2 )-MUC1 (T)-NHNH 2 calculated mass: 4013 Da, found: 1339.2 Da ([M+3H] 3+ ).1004.5 Da ([M+4H] 4+ ), 803.5 Da ([M+5H] 5+ ), 669.9 Da ([M+6H] 6+ ), 574.5 Da ([M+7H] 7+
  • Figure 22 shows HPLC chromatograms of crude Aux-MUC1(ST)-NHNH 2 and ESI-MS of purified product. Calculated mass: 4637 Da, found: 1546.7 Da ([M+3H] 3+ ),1 160.4 Da ([M+4H] 4+ ), 928.3 Da ([M+5H] 5+ ), 773.7 Da ([M+6H] 6+ ), 663.4 Da ([M+7H] 7+ ).
  • FIG. 23 shows HPLC chromatograms of the deprotection reaction of Aux-MUCI(ST) at different time points and ESI-MS of the starting material (Aux-MUCI (ST) (A), calculated mass: 4513 Da, found: 1505.2 Da ([M+3H] 3+ ),1 129.3 Da ([M+4H] 4+ ), 903.5 Da ([M+5H] 5+ ), 753.2 Da ([M+6H] 6+ )), of the desired Aux-MUCI(ST) with umprotected thiol ((B), calculated mass: 4425 Da, found: 1476.2 Da ([M+3H] 3+ ), 1 107.0 Da ([M+4H] 4+ ), 886.1 Da ([M+5H]5+), 738.5 Da ([M+6H] 6+ )) and of the dimer MUC1 -Aux(SS)Aux-MUC1 ((C), calculated mass: 8848 Da, found: 1770.5 Da (
  • Figure 24 shows the photorelease of MUC1 -G-MUC1 peptide.
  • Figure 25 shows the photorelease of MUC1 -G-MUC1 (Tn) peptide.
  • HPLC of the crude after irradiation and of the purified unprotected product (indicated by asterisk, purification and final analysis were done using two different HPLC columns) and ESI-MS of pure MUC1 -G-MUC1 (Tn).
  • calculated mass 3901 Da, found: 1302.0 Da ([M+3H] 3+ ), 976.4 Da ([M+4H] 4+ ), 781.3 Da ([M+5H] 5+ ), 651 .3 Da ([M+6H] 6+ )), 558.5 Da ([M+7H] 7+ ).
  • Figure 26 shows an HPLC chromatogram and an ESI-MS spectrum of MUC1 (Tn 7 )- NHNH 2 peptide hydrazide. Calculated mass: 2047 Da, found: 1024.1 Da ([M+H] + ), 683.2 Da ([M+2H] 2+ ), 512.7 Da ([M+3H] 3+ ). Asterisk indicates the desired peptide hydrazide.
  • Figure 27 shows an HPLC chromatogram and an ESI-MS spectrum of MUC1(Tn 7 )-SR peptide thioester.
  • Figure 29A 5987 Da, found: 1498.2 Da ([M+4H] 4+ ), 1 198.1 Da ([M+5H] 5+ ), 998.9 Da ([M+6H] 6+ ), 856.4 Da ([M+7H] 7+ ), 749.4 Da ([M+8H] 8+ ), calculated mass for MUC1(T 7 )- Aux-MUC1 (T): 6312 Da, found: 1579.1 Da ([M+4H] 4+ ), 1263.3 Da ([M+5H] 5+ ), 1053.0 Da ([M+6H] 6+ ), 902.9 Da ([M+7H] 7+ ), 702.2 Da ([M+9H] 9+ ).
  • Asterisk indicate compound MUC1 (Tn 7 )-Aux-MUC1 (Tn) (left) or compound MUC1(T 7 )-Aux-MUC1 (T) (right).
  • Figure 29B 5765 Da, found: 1442.2 Da ([M+4H] 4+ ), 1 154.0 Da ([M+5H] 5+ ), 961 .8 Da ([M+6H] 6+ ), 824.6 Da ([M+7H] 7+ ), 721 .7 Da ([M+8H] 8+ ), 641 .4 Da ([M+9H] 9+ ), calculated mass for MUC1(T 7 )-Aux-MUC1 (T) : 6089Da, found: 1523.3 Da ([M+4H] 4+ ), 1218.9 Da ([M+5H] 5+ ), 1015.8 Da ([M+6H] 6+ ), 870.9 Da ([M+7H] 7+ ), 762.2 Da ([M+8H] 8+ ), 677.6 Da([M+9H] 9+ ).
  • Figure 30 shows an a total ion current (TIC) chromatogram (A), HPLC chromatogram (B) and mass spectrum (C, 27.2-29.3 min of the TIC) of the mixture of conjugate MUC1(ST 7 )- Aux-MUCI(ST) and MUC1(T 7 )-Aux-MUC1 (ST) / MUC1(ST 7 )-Aux-MUC1(T).
  • TIC total ion current
  • Figure 31 shows a mass spectrum (28.0-28.2 min of the TIC ( Figure 30A)) of the mixture of conjugate MUC1(T 7 )-Aux-MUC1(ST) / MUC1(ST 7 )-Aux-MUC1 (T)
  • Figure 33 shows an HPLC chromatogram of the crude product Aux-MUC1(3Tn-3Ac) obtained after coupling of the Aux-Gly to the N-terminus of the peptide containing three Tn antigens still protected as acetates.
  • the MS spectrum in the figure correspond to the desired product indicated by the asterisk.
  • the peak at 1 1 .1 min is the PEGylated peptide (a small amount of MUC1 (3Tn-3Ac) was not coupled to the auxiliary, as a consequence the activated PEG then reacted with the free N-terminus of the peptide).
  • the peak at 8.9 min corresponds to the peptide modified with a tetramethyl guanidinium group (generated from excess HATU used as activating agent for the coupling).
  • Fmoc- Thr(GalNAc-Ac 3 )-OH was purchased fromshire Research (Ottawa, Canada), UDP-Gal and CMP-Neu5Ac from Merck (Darmstadt, Germany). /V,/V-Dimethylformamide (DMF), Methanol, dichloromethane (DCM), acetonitrile (ACN) and trifluoroacetic acid (TFA) were obtained from Biosolve (Valkenswaard, The Netherlands). Fmoc-PEG 2 7-COOH was from Polypure (Oslo, Norway). All other chemicals were obtained from Sigma-Aldrich at the highest purity available and used without further purification.
  • auxiliary 8 missing the PEG moiety, was installed first and subsequently PEGylated on the resin.
  • the synthesis of the auxiliary was modified accordingly by introducing an Fmoc protecting group on the free primary amine instead of the PEG ( Figure 1 , m).
  • peptide hydrazide could be an ideal masked peptide thioester sufficiently stable to allow enzymatic glycosylation and subsequent conversion into the desired glycosylated peptide thioester.
  • the same peptidyl resin provided access both to the peptide acid and to the peptide hydrazide.
  • MUC1 -NHNH 2 was incubated with 5% (v/v) hydrazine monohydrate in methanol overnight.
  • Protected MUC1 -NHNH 2 was obtained in 85% yield and deprotection of the side chains in solution by treatment with a mixture of TFA/TIS/H 2 0 (92.5:5:2.5) gave the desired MUC1 -NHNH 2 ( Figure 17, ESI).
  • Treatment with NaN0 2 followed by addition of MesNa and a RP-HPLC purification gave the MesNa thioester MUC1 -SR ( Figure 18, ESI) in 14% overall yield.
  • the glycosylated peptide conjugates were linked to MUC1 -SR via NCL to demonstrate all advantages of the auxiliary.
  • Non-glycosylated Aux-MUC1 was used to establish and optimize the ligation conditions for which the buffer composition was essential.
  • the thiol group of the auxiliary was deprotected via incubation with TCEP at 24°C for 6h before addition of MUC1 -SR.
  • Optimized NCL conditions for MUC1 peptides that give 65% conversion after two days of incubation are NaPi buffer (pH 7.5) at 30°C with Aux-MUC1 at 8mM concentration and a 2.5-fold excess of MUC1 -SR (Figure 3).
  • peptide hydrazides are potentially useful thioester precursors that remain unaffected in glycosylation reactions in which peptide thioesters quickly hydrolyze.
  • Hydrazine-induced cleavage of Aux-MUCI (Tn) peptidyl resin gave protected Aux- MUC1 (Tn)-NHNH 2 ( Figure 19A, ESI) that, after acidic deprotection in solution, was successfully used in sequential glycosylation reactions giving the corresponding Aux- MUC1(ST)-NHNH 2 hydrazide with similar yields as found for Aux-MUC1(ST)-OH ( Figure 7).
  • This peptide thioester was used in NCL with Aux-MUCI (Tn) to access MUC1 (Tn 7 )-Aux-MUC1 (Tn) ( Figures 28 and 29-left).
  • This conjugate was efficiently used in the glycosylation- precipitation approach leading to a peptide (MUC1 (T 7 )-Aux-MUC1 (T), Figure 29) consisting of two MUC1 tandem repeats and containing each a T antigen at different positions.
  • sialylated MUC1(ST 7 )-Aux-MUC1(ST) was obtained ( Figure 30 and 31 ).
  • MUC1 (T)-Aux-MUC1 (T) and MUC1 (ST)-Aux-MUC1 (ST) where obtained (with the T and ST antigen at position 14 in both MUC1 segments) and the auxiliary was fully converted into a glycine residue (G) by irradiation with 365 nm light generating MUC1 (T)-G-MUC1 (T) and MUC1 (ST)-G-MUC1 (ST) ( Figure 32).
  • the conjugation of the auxiliary to the peptide via SN2 reaction is the limiting step in the method. Indeed, while giving conversions that are higher that 50% with a non-modified MUC1 peptide or a peptide containing modifications far away from the N-terminus, the yields drop in the case of peptides carrying the modifications close to the N-terminus.
  • i-butylglyoxalate was prepared via periodate oxidation of di-i-butyl-L- tartrate and directly used in a reductive amination reaction with auxiliary 8.
  • Aux-Gly(tBu) 9 was obtained in 52% yield after purification via flash column chromatography on silica gel (ca 20% of the starting material can be recovered unreacted after the reductive amination). Removal of the tBu group by acidic treatment, followed by purification and freeze-drying, gave the desired product Aux-Gly 10 in 91 % yield (scheme 3).
  • MUC1 3Tn-3Ac
  • MUC1 3Tn-3Ac
  • the peptidyl resin was incubated with a solution of Aux-Gly 10, HATU and DIEA in DMF. After 2h incubation the resin was washed and a test cleavage confirmed that the coupling reaction proceeded with a very high conversion (>90%).
  • a new PEGylated ligation auxiliary that efficiently supports the quantitative enzymatic glycosylation of peptides in solution and can mediate NCL reactions.
  • Addition of a PEG polymer to the previously described ligation auxiliary turns the auxiliary-peptide conjugates into excellent substrates for the site-specific enzymatic glycosylation approach we previously described (Bello et al., 2014) and allows further simplification of the intermediate purification step.
  • the auxiliary-modified (and glycosylated) peptides can be used in NCL reactions with other MUC1 tandem repeat peptides carrying a C-terminal othioester.
  • Controlling thioester generation and deprotection of the thiol group within the auxiliary also allows the controlled extension of each building block in C- and N-terminal direction.
  • This approach to create a library of site- selectively O-glycosylated MUC1 variants with different glycosylation pattern for a detailed study of the role of these patterns in MUC1 function.
  • this approach is not limited to the synthesis of glycosylated MUC1 analogues, but it is in principle applicable to the synthesis of many larger, posttranslationally modified proteins, only limited by the availability of the suitable chemistry or enzymes that introduce the desired PTMs.
  • LC-MS liquid chromatography-mass spectrometry
  • LC-MS Waters AutoPurification HPLC/MS system (3100 Mass Detector, 2545 Binary Gradient Module, 2767 Sample Manager and 2489 UVA isible Detector) was used. Mass spectra were acquired by electrospray ionization (ESI-MS) operating in positive ion mode. Separation was achieved with a Kromasil 300-5-C4 or 300-5-C18 column (50 ⁇ 4.6 mm, 5 ⁇ particle size) at a flow rate of 1 mL/min running a linear gradient from 5% to 65% of (ACN + 0.05 % TFA) in (ddH 2 0 + 0.05 % TFA) in 10 min.
  • ESI-MS electrospray ionization
  • Crude peptides or peptide-auxiliary conjugates were purified by reverse phase-high performance liquid chromatography (RP-HPLC) on Waters AutoPurification HPLC/MS system. According to the amount and hydrophobicity of the peptides to be purified, different columns were used: Kromasil 300-10-C4 column (250 ⁇ 21 .2 mm, 10 ⁇ particle size), Kromasil 300-10-C4 column (250 ⁇ 10 mm, 10 ⁇ particle size). Detection for all the chromatographic methods occurred at 214 nm and 280nm wavelengths.
  • MUC1(ST7)-Aux-MUC1 (ST) conjugate was analyzed by RP-HPLC/HR-ESI-TOF on a Dionex Ultimate 3000 (Thermo Scientific) capillary HPLC coupled with a maXis HD ESI- QTOF (Bruker) instrument.
  • An Agilent Technologies Zorbax 300SB C18 column (150x 0.3 mm, 3.5 ⁇ particle size) was used for optimal separation with a flow rate of 6 ⁇ /min and a gradient of 10% to 60% buffer B in buffer A in 40 min (in this case buffer A: ddH20 with 0.1 % v/v formic acid, buffer B: ACN with 0.1 % v/v formic acid)
  • Reactions were monitored by thin layer chromatography (Merck silica gel 60F254 plates). Detection and staining were carried out by UV light (254 nm) and KMn04 [KMn0 4 (3g), K 2 C0 3 (20g), AcOH (0.25 ml) and H 2 0 (300ml)] or Pancaldi reagent [(NH 4 ) 6 Mo0 4 (21 g), Ce(S0 4 ) 2 (1 g), H2S04 (31 ml) and H 2 0 (470 ml)]. Purification of products was performed by flash column chromatography (silica gel 60, 230-400 mesh, 0.04-0.063 mm, Macherey- Nagel (Dijren, Germany)).
  • Vanillin (10.0 g, 66mmol) was dissolved in anhydrous acetonitrile (140 ml) under argon.
  • Anhydrous K 2 C0 3 (18.4 g, 133mmol, 2 eq) was added, followed by tetrabutylamonium iodide (4.4 g, 13 mmol, 0.2 eq) and methyl 4-chlorobutyrate (9.5 ml, 1 1.3 g, 83 mmol, 1.3 eq).
  • the pink suspension was heated at reflux giving a yellow solution that was stirred at reflux for 18h.
  • the carbonate was filtered off and the solvent was evaporated under reduced pressure.
  • the obtained solution was added dropwise to the solution of ylide at 0°C.
  • the purple solution was stirred at 0°C for 5 min, then at room temperature for 18h.
  • the solvent was evaporated under reduced pressure.
  • Water and chloroform were added to the crude and the two phases were separated.
  • the aqueous phase was extracted with chloroform.
  • the collected organic layers were washed with brine, dried over MgS0 4 and the solvent was evaporated under reduced pressure.
  • AD-mix a (10.2g) was dissolved in l BuOH/CH 2 CI 2 /H 2 0 (1 :1 :2, 68ml) at room temperature. The mixture was stirred at room temperature until the reagent was completely dissolved, then the orange solution was cooled down to 0°C. Methyl 4-(2-methoxy-5-nitro-4- vinylphenoxy)butanoate (2.1 g, 7.1 mmol) was added and the solution was stirred at room temperature for 18h. After consumption of the starting material (TLC ethyl acetate/pentane 4:1 ) sodium sulfite (10.2g) was added and the suspension was stirred at room temperature for 1 h. Water was added, followed by ethyl acetate.
  • Methyl 4-(4-(1 ,2-dihydroxyethyl cyclic sulfite)-2-methoxy-5-nitrophenoxy)butanoate was obtained as yellow solid (2.96g, 7.9 mmol, 88% yield) and used in the following reaction without any further purification.
  • the crude (2.96 g, 7.9 mmol) was dissolved in DMF, sodium azide (1.28g, 19.7 mmol, 2.5 eq) was added and the solution was stirred at 60°C. After consumption of the starting material (TLC ethyl acetate/pentane 4:1 ) the DMF was partially removed under reduced pressure. Water was added and the solution was extracted with diethyl ether.
  • the reaction was stirred at 0°C for 1 h (followed by TLC CH 2 CI 2 /MeOH 95:5 + 1 % NH 4 OHaq) and then the solvent was evaporated under reduced pressure.
  • the crude was dissolved in CH 2 CI 2 , water was added and the two phases were separated. The aqueous phase was extracted with CH 2 CI 2 , the collected organic phases were washed with brine, dried over MgS04 and the solvent was evaporated under reduced pressure.
  • Triphenylphosphine (661 .5 mg; 2.52 mmol, 2.1 eq) was dissolved in anhydrous THF (19.5 ml) at 0°C under argon atmosphere.
  • Diisopropyl azodicarboxylate (0.55 ml, 2.79 mmol, 2.4 eq) was added and the solution was stirred at 0°C for 30 min. A white precipitate formed.
  • Methyl 4-(4-(N-ie f-butyloxycarbonyl (2-amino-1 -hydroxyethyl)-2-methoxy-5- nitrophenoxy)butanoate 8 (505.3 mg, 1.17 mmol) was dissolved in anhydrous THF (24ml) and the obtained solution was added dropwise to the solution of triphenylphosphine at 0°C. Neat thioacetic acid (0.18 ml, 2.55 mmol, 2.2 eq) was then added dropwise and the dark red solution was stirred at 0°C for 5 min, then at room temperature for 16h.
  • Methyl 4-(4-(2-(acetylsulfanyl)-1 -((ie f-butoxycarbonyl)amino)ethyl)-2-methoxy-5- nitropheno-xy)butanoate (101 mg, 0.21 mmol) was dried under high vacuum for 2h and then dissolved in anhydrous THF (825 ⁇ ) and MeOH (225 ⁇ ), previously degassed. Sodium methoxide (500 ⁇ , 0.5M solution in MeOH) was added dropwise and the solution was stirred at room temperature for 10 min.
  • Ester 5 (288 mg, 0.54 mmol) and HOBt were dissolved in extra-dry acetonitrile in a heat- dried two-neck round-bottom flask under argon atmosphere.
  • Ethylenediamine (1 ml, 14.9 mmol, 27 eq)
  • zirconium (IV) ie f-butoxide 230 ⁇ , 0.59 mmol, 1 .1 eq
  • the solution was stirred at room temperature for 18h.
  • Fmoc-PEG 2 7-OH (476 mg, 0.31 mmol, 1 .5 eq) was dissolved in a solution of HATU (0.5 M in acetonitrile, 420 ⁇ ).
  • DIEA 72,4 ⁇ , 2 eq was added and the solution was diluted with acetonitrile (0.7 ml) and mixed with a solution of ie f-butyl (1 -(4-(4-((2-aminoethyl)amino)- 4-oxobutoxy)-5-methoxy-2-nitrophenyl)-2-(ie f-butyldisulfanyl)ethyl)carba-mate (6) (1 18 mg, 0.21 mmol) in dry acetonitrile (1 .5 ml).
  • Mucin peptide MUC1 (VTSAPDTRPAPGSTAPPAH (SEQ ID NO: 2) was synthesized on a solid support using the fluorenylmethoxycarbonyl (Fmoc) strategy4.
  • Fmoc fluorenylmethoxycarbonyl
  • Preloaded TentaGel® R PHB-His(Trt)-Fmoc resin (TentaGel® R PHB-His(Trt)-Fm resin) (1 g) was used as the solid support for the synthesis of the peptides on a 0.2 mmol scale.
  • the resin was loaded into the reaction vessel of an automated peptide synthesizer (PTI Tribute), and swollen in DMF.
  • PTI Tribute automated peptide synthesizer
  • the amino acids used in the synthesis were carrying the following orthogonal side-chain protecting groups: Thr(O'Bu), Ser(O'Bu), Arg(Pbf), Asp(O'Bu). Double couplings were performed after coupling of each proline residue.
  • Mucin peptide MUC1 (Tn) (VTSAPDTRPAPGST(GalNAc-Ac3)APPAH) was synthesized manually until Ser(13) on a solid support using the fluorenylmethoxycarbonyl (Fmoc) strategy.4 Preloaded TentaGel® R PHB-His(Trt)-Fmoc, resin (1 g) was used as the solid support for the synthesis of the peptide on a 0.2 mmol scale.
  • N-terminal Fmoc deprotection was performed by incubating the resin twice with a piperidine solution (20% v/v in DMF) for 3 and 7 min, respectively. After washing with DMF, the resin was incubated for 30 min with the pre-activated amino acid (2.5 eq, activated with 2.4 eq HBTU (0.5M, DMF) and 5.0 eq DIEA, 3 min) and washed again with DMF before starting a new deprotection step. Coupling and deprotection reaction of Ala(15) were monitored by ninhydrine test.
  • Fmoc- Thr(GalNAc-Ac 3 )-OH 160 mg, 0.24 mmol, 1 .2 eq
  • DMF 477 ⁇
  • HATU 91 mg, 0.24 mmol, 1 .2 eq
  • DIEA 87 ⁇ , 0.5 mmol,2.5 eq
  • the resin was incubated with the solution of activated modified amino acid for 2h and then washed with DMF. Coupling and deprotection of Fmoc-Thr(GalNAc-Ac 3 )-OH were monitored by ninhydrine test, as well as the coupling of the following serine (Ser(13)).
  • the resin was washed with DMF, MeOH and DCM, dried under vacuum and transferred into the reaction vessel of the Fmoc-synthesizer (PTI Tribute) and the synthesis was continued automatically.
  • PTI Tribute Fmoc-synthesizer
  • Mucin peptide MUC1(Tn 7 ) (VTS AP DT(G a I N Ac -Ac3 ) RP APG ST AP PAH ) was synthesized manually on a solid support using the fluorenylmethoxycarbonyl (Fmoc) strategy.
  • Fmoc fluorenylmethoxycarbonyl
  • Preloaded TentaGel® R PHB-His(Trt)-Fmoc, resin (0.5g) was used as the solid support for the synthesis of the peptide on a 0.1 mmol scale.
  • N-terminal Fmoc deprotection was performed by incubating the resin twice with a piperidine solution (20% v/v in DMF) for 3 and 7 min, respectively.
  • the resin was incubated for 30 min with the pre-activated amino acid (2.5 eq, activated with 2.4 eq HBTU (0.5M, DMF) and 5.0 eq DIEA, 3 min) and washed again with DMF before starting a new deprotection step. Coupling and deprotection reaction of Arg(8) were monitored by ninhydrine test.
  • Fmoc- Thr(GalNAc-Ac3)-OH 80 mg, 0.12 mmol, 1 .2 eq was dissolved in DMF (238.5 ⁇ ) and activated with HATU (45.5 mg, 0.12 mmol, 1 .2 eq) and DIEA (43.5 ⁇ , 0.25 mmol, 2.5 eq) for 2 min.
  • the resin was incubated with the solution of activated modified amino acid for 2h and then washed with DMF.
  • Coupling and deprotection of Fmoc-Thr(GalNAc-Ac3)-OH were monitored by ninhydrine test, as well as the coupling of the following aspartic acid (Asp(6)).
  • the sequence was completed by coupling the remaining amino acids following the standard coupling and deprotection procedure.
  • the peptidyl-resin was then washed, dried and directly used for the preparation of the corresponding peptide hydrizide (page 38 and following).
  • peptides MUC1 and MUC1(Tn) after the last coupling, the resin was washed with DCM and dried under vacuum.
  • the peptidyl resins were used in the subsequent reactions after performing a test cleavage to verify the efficiency of the synthesis: 10 mg of peptidyl- resin were incubated with a mixture of TFA TIS/H 2 0 (92.5:5:2.5) for 2h at room temperature. The free peptide was precipitated by addition of cold diethyl ether and centrifugation.
  • the resin (0.4566 g) was swollen in DMF for 1 h, then the Fmoc protecting group was removed by incubating the resin twice with a solution of piperidine (20% v/v in DMF) for 3 and 7 min respectively. After extensive washing with DMF, the resin was incubated with a solution of iodoacetic acid (47 mg, 0.23 mmol) and DIC (35.7 ⁇ , 0.23 mmol) in DMF (992 ⁇ ) for 30 min. After removal of the supernatant and washing with DMF, the coupling was repeated for 20 min.
  • the peptide resin was washed with DMF, DCM and MeOH, dried under vacuum and a test cleavage was performed: 10 mg of the resin were incubated with a mixture of TFA/TIS/H 2 0 (92.5:5:2.5) for 2h at room temperature. The free peptide was precipitated by addition of cold diethyl ether and centrifugation. Finally, the supernatant was removed and the peptide washed with cold diethyl ether, dissolved in a mixture of H2O/CH 3 CN 1 :1 and analyzed by RP-HPLC and ESI-MS.
  • the resin (0.450 g) was swollen in DMF for 1 h, then the Fmoc protecting group was removed by incubating the resin twice with a solution of piperidine (20% v/v in DMF) for 3 and 7 min respectively. After extensive washing with DMF, the resin was incubated with a solution of iodoacetic acid (37 mg, 0.18 mmol) and DIC (28 ⁇ , 0.18 mmol) in DMF (792 ⁇ ) for 30 min. After removal of the supernatant and washing with DMF, the coupling was repeated for 20 min.
  • the peptide resin was washed with DMF, DCM and MeOH, dried under vacuum and a test cleavage was performed: 10 mg of the resin were incubated with a mixture of TFA/TIS/H 2 0 (92.5:5:2.5) for 2h at room temperature. The free peptide was precipitated by addition of cold diethyl ether and centrifugation. Finally, the supernatant was removed and the peptide washed with cold diethyl ether, dissolved in a mixture of H2O/CH 3 CN 1 :1 and analyzed by RP-HPLC and ESI-MS.
  • the iodoacetylated peptidyl MUC1 resin (247 mg) was swollen in CH 3 CN (600 ⁇ ) for 2h. DIEA (17 ⁇ , 0.1 mmol) was added followed by the auxiliary (8) (59.0 mg, 0.1 mmol in 300 ⁇ CH 3 CN). The suspension was stirred at 28°C for 18h, then the supernatant was removed (the unreacted auxiliary was recovered by recrystallization) and the resin washed with DMF, DCM and MeOH. A test cleavage was performed to check the efficiency of the S N 2 reaction. 10 mg of the resin were incubated with a mixture of TFA TIS/H 2 0 (92.5:5:2.5) for 2h at room temperature.
  • a test cleavage was performed to check the efficiency of the S N 2 reaction: 10 mg of the resin were incubated with a mixture of TFA TIS/H2O (92.5:5:2.5) for 2h at room temperature. The free peptide was precipitated by addition of cold diethyl ether and centrifugation. Finally, the supernatant was removed and the peptide washed with cold diethyl ether, dissolved in a mixture of H 2 0/CH 3 CN 1 :1 and analyzed by RP-HPLC and ESI-MS: the major peak in the chromatogram correspond to the desired product, some unreacted iodoacetylated peptide could still be detected, together with a few side products. After test cleavage, the remaining Aux-peptidyl resin conjugate was directly used in the following PEGylation step. PEGylation of Aux(Fmoc)MUC1 peptidyl resin and cleavage from the solid support
  • Aux(Fmoc)MUC1 peptidyl resin was swollen in DMF for 1 h.
  • the Fmoc protecting group on the auxiliary was removed by incubating the peptidyl resin twice with a solution of piperidine (20% v/v in DMF) for 3 min and 7 min respectively.
  • Fmoc- PEG2 7 -COOH 130 mg, 83.8 mol, 1.37 eq
  • HATU 149.5 ⁇ (0.5M in DMF/CH 3 CN 3:2), 71 .9 mol, 1 .19 eq) and then DIEA (26 ⁇ , 149.6 mol, 2.5eq) was added.
  • the deprotected peptidyl resin was washed with DMF and then incubated with the solution of activated PEG for 20h at room temperature under shaking. The supernatant was removed and the resin was washed extensively with DMF, DCM and MeOH and dried under vacuum. The reaction was checked by performing a test cleavage.
  • the PEGylated auxiliary-MUC1 conjugate was cleaved from the resin: the peptidyl resin was incubated with a mixture of TFA/TIS/H 2 0 (1 .5 ml, 92.5:5:2.5) for 3h at room temperature. The free conjugate was precipitated by addition of cold diethyl ether and recovered by centrifugation.
  • Aux(Fmoc)MUC1 (Tn) peptidyl resin was swollen in DMF for 1 h.
  • the Fmoc protecting group on the auxiliary was removed by incubating the peptidyl resin twice with a solution of piperidine (20% v/v in DMF) for 3 min and 7 min respectively.
  • Fmoc- PEG 27 -COOH 146 mg, 94.8 ⁇ , 1.37 eq
  • the deprotected peptidyl resin was washed with DMF and the incubated with the solution of activated PEG for 20h at room temperature under shaking. The supernatant was removed and the resin was washed extensively with DMF, DCM and MeOH and dried under vacuum. The reaction was monitored by performing a test cleavage.
  • the PEGylated auxiliary-MUC1 conjugate was cleaved from the resin: the peptidyl resin was incubated with a mixture of TFA/TIS/H 2 0 (2 ml, 92.5:5:2.5) for 3h at room temperature. The free conjugate was precipitated by addition of cold diethyl ether and collected by centrifugation.
  • the conjugate was dissolved in CH 3 CN/H 2 0 1 :1 (1 % TFA) and freeze-dried.
  • the crude product was dissolve in a 8% v/v solution of ⁇ 2 ⁇ 2 ⁇ 20 in CH 3 CN (3 ml), stirred at room temperature for 18h and then freeze dried (monitoring by HPLC-MS: the glycan was completely deprotected, a side product could be detected accounting for the Aux- MUC1 (Tn) conjugate without Fmoc on the terminal amine of the PEG chain).
  • the desired pure Aux-MUCI (Tn) was obtained (9.2 mg, 2.3 ⁇ , 4% yield, based on peptide synthesis scale) after purification of the crude by RP-HPLC (C4 column, gradient: 5-45% buffer B in buffer A in 45 min, flow-rate: 20 ml/min, buffer A: ddH 2 0 + 0.05% TFA, buffer B: CH 3 CN + 0.05% TFA).
  • the crude peptide hydrazide was dissolved in 5.5 ml of buffer 1 (6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH3) at -10°C. A 0.2M aqueous solution of NaN0 3 (1 .2ml), was added dropwise and the solution was stirred at -10°C for 30min. A solution of MesNa (0.45M in buffer 2 (6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH7.5), 440 ⁇ ) was added and the reaction mixture was stirred at -10°C for 10 min and then at room temperature for 20 min.
  • buffer 1 6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH3
  • the pure thioester (21 mg, 1 1 ⁇ , 14% yield,) was obtained after purification by rp-HPLC (the reaction mixture was directly used for injection without any dilution. C4 column, gradient 5- 30% buffer B in buffer A in 45min, flow: 20ml/min. 6.7 mg of hydrolyzed peptide thioester were recovered as well).
  • Aux-MUCI (Tn) peptidyl resin (50mg) was swollen in 5% (v/v) H 2 NNH 2 -H20 in MeOH (1 ml) and shaken at room temperature for 18h. The supernatant was collected, the resin was washed with MeOH (0.2 ml) and the washing solution was added to the supernatant. The solution was neutralized with acetic acid and then the solvent was evaporated under reduced pressure. The crude was dissolved in buffer A (ddH 2 0 + 0.1 % TFA, 0.4 ml) and freeze-dried.
  • the crude lyophilized product was dissolved in TFA/TIS/H20 (0.5 ml, 92.5:5:2.5) and rotated at room temperature for two hours.
  • the deprotected Aux- MUC1 (Tn)-NHNH 2 was precipitated by addition of cold diethyl ether and recovered by centrifugation. After washing with cold diethyl ether and recovery by centrifugation, the crude product was dissolved in H 2 0/CH 3 CN 1 :1 and freeze-dried.
  • the respective protein coding regions were amplified from human Mammalian Gene Collection clones to truncate NH2-terminal transmembrane sequences and append an NH 2 -terminal fusion peptide of a tobacco etch virus (TEV) protease cleavage site and flanking Gateway attB recombination site sequences to facilitate Gateway cloning using a two-step adapter PCR method.
  • PCR products were gel purified and cloned into the pDONR221 plasmid vector using Gateway BP Clonase II (Invitrogen) and confirmed by DNA sequencing.
  • Expression constructs were generated using the Gateway LR Clonase recombination with the pDONR221 entry clones a Gateway adapted version of the pGEn2 mammalian expression vector (pGEn2- DEST).
  • the resulting expression constructs encode fusion proteins comprised of an NH 2 - terminal signal sequence, an 8xHis tag, an AviTag recognition site, the "superfolder” GFP coding region, the 7 amino acid recognition sequence of the tobacco etch virus (TEV) protease, followed by the corresponding enzyme catalytic domain.
  • Recombinant enzyme expression was accomplished by transient transfection of HEK293f cells (FreeStyle 293-F cells, Life Technologies, Grand Island, NY) as previously described.
  • C1 GALT1 secreted expression of the enzyme was facilitated by co-expression with the selective chaperone C1 GALT1 C1.
  • the cultures were harvested, clarified by centrifugation, and the recombinant product was purified by Ni-NTA Superflow (Qiagen, Valencia, CA) chromatography as described.
  • the eluted recombinant enzyme preparation was concentrated to ⁇ 1 mg/ml by ultrafiltration with a 10-kDa molecular mass cutoff membrane (Millipore, Billerica, MA) and used directly for enzymatic modification of glycopeptide substrates.
  • the conjugate (Aux-MUCI (Tn) or Aux-MUC1(Tn)-NHNH 2 (with or without Fmoc), reaction scale 50-30C ⁇ g, final concentration (fc): 0.24 mM) was dissolved in an aqueous solution of TRIS buffer (fc: 75mM, pH7.5), Triton X-100 (fc: 0.06%), MnCI2 (fc: 10 mM), UDP-Gal (fc: 2mM) at room temperature.
  • the enzyme, human C1 GalT1 was added and the reaction was shaken at 37°C for 6h (reaction monitored by HPLC-MS).
  • the reaction mixture 50 ⁇ was added to 450 ⁇ of EtOH.
  • reaction scale 50-30C ⁇ g, final concentration (fc): 0.24 mM) MgCI 2 (fcl OmM) and CMP-Neu5Ac (fc 2mM) were added, followed by the enzyme, human ST3Gal1 , and the reaction mixture was incubated for 4h at 37°C (reaction monitored by HPLC-MS) under gentle shaking.
  • the reaction mixture (50 ⁇ ) was added to 450 ⁇ of EtOH.
  • Et 2 0 (1 ml) was added and the solution was incubated for 18h at -80°C.
  • the conjugate carrying a sialyl-T antigen was recovered by centrifugation (15000g ⁇ 30min), dried under the fume-hood (30 min at room temperature) and, in the case of Aux- MUC1(ST), directly used in the ligation reaction. Recovery: 90% (Aux-MUCI(ST), Figure 20B and C), 93% (Aux-MUC1(ST)-NHNH 2 ) and 80% (Aux (N H2 ) -MUC1(ST)-NHNH 2 ) ( Figure 21 ).
  • a 0.32 M solution of ascorbic acid (0.25 ⁇ , 5eq) was added followed by 1 ⁇ of a 0.06M solution of MesNa in buffer 2 (6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH7.5) and stirred at room temperature for 20 min.
  • the solution has been diluted with 60 ⁇ of ddH 2 0 and directly used for RP-HPLC analysis and purification (63% conversion).
  • the collected material was used for ESI analysis (direct injection).
  • auxiliary-peptide conjugate (0.08-0.25 ⁇ ) was dissolved in degassed ligation buffer (0.2M NaPi buffer pH 8.5, 35mM TCEP, final pH 7.5. Concentration of conjugate ranging from 5mM to 8.5 mM) and the solution was shaken at 24°C for 5h to allow deprotection of the thiol group (in the case of Aux-MUCI (ST) 36h incubation at 30°C and further addition of TCEP (20eq in total) were needed to achieve deprotection of the tert- butyldisulfanyl group ( Figure 23)).
  • the crude ligation solution was diluted with degassed H 2 0 or H 2 0/CH 3 CN 7:3 (final concentration: 0.5-1 .0 mM) containing TCEP (1 eq) in a glass tube under argon and irradiated with a UV-A lamp (UV reactor) for 30 min.
  • the unprotected ligation product was obtained in all cases in high conversion and recovered pure after HPLC purification.
  • MUC1 (Tn 7 ) peptidyl resin (108mg, ca 55 ⁇ peptide) was swollen in DMF for 1 h. N- terminal Fmoc protecting group was removed by incubating the resin twice with a 20% v/v solution of piperidine in DMF (3 and 7 min respectively). The resin was washed with DMF, DCM and MeOH and dried, then it was incubated with a 5% (v/v) ⁇ 2 ⁇ 2 ⁇ 2 ⁇ in CH 3 CN/H2O 1 :1 (2.5ml) for 18h under shaking. The supernatant was removed and the resin washed with CH 3 CN/H 2 0 1 :1 .
  • the collected solutions containing the desired fully protected peptide hydrazide were neutralized with TFA and freeze-dried.
  • the crude was dissolved in TFA/TIS/H 2 0 (2 ml, 92.5:5:2.5) and shaken at room temperature for 2h.
  • the umprotected peptide hydrazide was precipitated with cold diethyl ether and recovered by centrifugation. After washing with diethyl ether and centrifugation, the crude was dissolved in H 2 0/CH 3 CN 1 :1 and freeze-dried.
  • the crude peptide hydrazide (6mg, 2.3 ⁇ ) was dissolved in 1 13 ⁇ of buffer 1 (6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH3) at -10°C. A 0.2M aqueous solution of NaN0 3 (22 ⁇ ), was added dropwise and the solution was stirred at -10°C for 20min. A solution of MesNa (0.45M in buffer 2 (6M Gnd-HCI, 0.2M Na 2 HP0 4 , pH7.5), 66 ⁇ ) was added and the reaction mixture was stirred at -10°C for 5 min. A solution of ascorbic acid (2.2mM, 13 ⁇ ) was added and the solution was stirred at room temperature for 10 min.
  • the pure thioester (2.1 mg, 1 ⁇ , 43% yield,) was obtained after purification by RP-HPLC (the reaction mixture was diluted with ddH 2 0 (total volume 3.5ml) and directly used for injection. C18 column, gradient 5- 45% buffer B in buffer A in 45min, flow: 10ml/min).
  • the isolated ligation product MUC1(Tn 7 )-Aux-MUC1 (Tn) was directly used in the galactosylation reaction following the general procedure for galactosylation described above.
  • the only modification to the procedure was the use of a higher amount of UDP-Gal (4mM final concentration) because of the different stoichiometry of the reaction.
  • TCEP (1 ⁇ of a 70mM aqueous solution) was added to reduce the disulfide that the free thiol on the auxiliary was forming. After 4h the starting material was converted into the desired product (Figure 29, left). Ethanol (393 ⁇ ) was added and then, after vortexing, diethyl ether (870 ⁇ ).
  • the solution was incubated at -80°C overnight. After centrifugation (30min x 15000g, 5°C) the supernatant was removed and the solid product dried at room temperature under the fume hood.
  • the recovered MUC1(T 7 )-Aux-MUC1 (T) was dissolved in ddH 2 0 (19 ⁇ + 1 ⁇ of 70mM TCEP solution) and analyzed by RP-HPLC ( Figure 29, right). Recovery after precipitation: 49%.
  • the mass of a conjugate carrying one sialyl-T and one T antigen can be also detected ( Figure 31 C and 32).
  • the difference between the product and the partially sialylated conjugate consist in one sugar unit and that the sugar in these conjugate has a minimal influence on the ionization of the compound (as it can be seen in Figure 20A, UV trace and ionization intensity correlate very well) it can be estimated that the partially sialylated conjugate represent less than 10% of the mixture.
  • the masses of the conjugates MUC1(T 7 )-Aux-MUC1 (T) and MUC1 (Tn 7 )-Aux-MUC1 (Tn) are barely detectable indicating that the glycosylation reactions are almost quantitative. Recovery after precipitation: 38%. References WO2012/139777,

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Abstract

La présente invention concerne un composé comprenant un fragment thiol S protégé ou déprotégé permettant une ligature chimique native, un fragment de liaison L photolabile et au moins un fragment polymère hydrophile HP. En outre, la présente invention concerne un procédé de production d'un conjugué peptidique P12 comprenant un fragment peptidique P1 et un second fragment P2 liés de façon covalente l'un à l'autre par l'intermédiaire d'une liaison amide, le composé de la présente invention étant utilisé pour former sélectivement la liaison amide entre P1 et P2.
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