US20120258891A1 - Diarylsulfide backbone containing photolabile protecting groups - Google Patents

Diarylsulfide backbone containing photolabile protecting groups Download PDF

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US20120258891A1
US20120258891A1 US13/433,373 US201213433373A US2012258891A1 US 20120258891 A1 US20120258891 A1 US 20120258891A1 US 201213433373 A US201213433373 A US 201213433373A US 2012258891 A1 US2012258891 A1 US 2012258891A1
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Klaus-Peter Stengele
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Nimblegen Systems GmbH
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Priority to US14/061,297 priority Critical patent/US20140051605A1/en
Priority to US14/935,516 priority patent/US20160060286A1/en
Priority to US15/425,329 priority patent/US10150791B2/en
Priority to US16/171,970 priority patent/US11001602B2/en
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    • C07C323/18—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and singly-bound oxygen atoms bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
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    • C07C323/31—Thiols, sulfides, hydropolysulfides or polysulfides substituted by halogen, oxygen or nitrogen atoms, or by sulfur atoms not being part of thio groups containing thio groups and nitrogen atoms, not being part of nitro or nitroso groups, bound to the same carbon skeleton having the sulfur atom of at least one of the thio groups bound to a carbon atom of a six-membered aromatic ring of the carbon skeleton
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    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/16—Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
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    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/60—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
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    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
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    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
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    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
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    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present disclosure relates to photoactivable protecting groups (PLPGs). More specifically, the present disclosure relates to the use of PLPGs in biomolecule synthesis.
  • PLPGs photoactivable protecting groups
  • Photolabile protecting groups play an important role in blocking functional groups present in nucleosides, nucleotides, sugars and amino acids, which are used for the synthesis of biomolecules, e.g. nucleic acids and their derivatives, proteins, peptides and carbohydrates. Additionally, PLPG have the advantage that deprotection of the protected functional group can be performed simply via light exposure. Therefore, PLPG provide the basis for the photolithography based spatially resolved synthesis of oligonucleotides or peptides on solid supports. The major advantage of this technique is that high resolution microarrays can be produced.
  • PLPG used for oligonucleotide synthesis include, but are not limited to, ⁇ -methyl-6-nitropiperonyl-oxycarbonyl (MeNPOC), and 2-(2-nitrophenyl)-propoxycarbonyl (NPPOC).
  • MeNPOC ⁇ -methyl-6-nitropiperonyl-oxycarbonyl
  • NPOC 2-(2-nitrophenyl)-propoxycarbonyl
  • PLPG used for photolithography based peptide synthesis are for example nitroveratryloxycarbonyl (NVOC) and 2-nitrobenzyloxycarbonyl (NBOC).
  • Previous PLPG synthesis used light at a wavelength of approximately 365 nm or shorter for the deprotection of the protected functional groups.
  • Light sources which are suitable to generate such wavelength are e.g. mercury arc lamps, excimer lasers, UV-LEDs and frequency multiplied solid-state lasers. Such light sources are characterized by high purchase costs, provide limited luminous power and have a short life-time leading to high overall costs of operation. Since some of the above mentioned light sources contain hazardous substances, e.g. mercury, and appropriate actions to secure occupational safety and proper disposal are necessary, further increasing the costs.
  • Optical devices used for the photolithography based synthesis of oligonucleotides or peptides are primarily designed for the visible wavelength range of approximately 380 to 780 nm. Such devices carry an antireflective or protective antiscratch coating optimized for transparency for the respective visible wavelength range.
  • the near UV wavelength of 365 nm used for the deprotection of the functional groups protected with PLPG require optical devices which are optimized for near UV wavelengths. Since most of the optical devices are optimized for the use with visible light, such optimization often comprises removing the coating intended for the use with visible light from the optical devices and/or coating the optical device with materials intended for use with near UV or UV light.
  • DNA or peptide microarrays might be of low quality due to undefined lengths of the synthesized DNA strands and peptides, respectively.
  • PLPG are presented herein which are suitable for the deprotection of the functional groups using visible light. Consequently, harmless and cost-effective light sources as well as regular optical elements can be used for the photolithography based oligonucleotide and peptide synthesis.
  • the compounds of the present disclosure may be used for a variety of different applications.
  • the disclosure is directed to the use of the compounds as photoactivable protecting groups using maskless photolithography.
  • the compounds are used for the maskless photolithography based DNA array synthesis as intermediate or permanent OH-protecting group in nucleoside derivatives at the 3′-OH end or the 5′-OH end.
  • the compounds are useful for maskless photolithography based peptide array synthesis as NH-protecting group in amino acids.
  • the compounds are useful for the maskless photolithography based peptide array synthesis as COOH-protecting group in amino acids and/or for the maskless photolithography based synthesis of carbohydrates as OH-protecting group and/or for orthogonal protecting group strategy as SH-protecting group.
  • the compounds are used for maskless photolithography having a wavelength of 374 to 405 nm, for example a wavelength of 390 nm.
  • the disclosure is directed to a method for the synthesis of a diarylsulfide backbone containing photolabile protecting group as described above comprising the steps of
  • FIG. 1 demonstrates half-lives of examples of PLPG according to the disclosure in various solvents used at a wavelength of 390 nm. Light exposure was performed for various time periods.
  • FIG. 2 demonstrates half-lives of examples of PLPG according to the disclosure in various solvents used at a wavelength of 404 nm. Light exposure was performed for various time periods.
  • FIG. 3 demonstrates certain UV absorption characteristics of PLPG.
  • FIG. 4 exemplifies synthesis pathways of disulfide-PLPG-amino acids.
  • FIG. 5 exemplifies synthesis pathways of disulfide-PLPG-nucleotides.
  • FIG. 6 exemplifies synthesis pathways of further PLPG according to the disclosure.
  • FIG. 7 exemplifies an alternative synthesis pathway of PLPG without further alkyl substituents.
  • FIG. 8 shows a microarray scan of a peptide array containing the target sequence of an anti-V5 antibody synthesized according to the disclosure using disulfide-PLPG-amino acids.
  • unsubstituted is used herein as known to the expert skilled in the art and refers to a hydrocarbon chain which fully consists of carbon and hydrogen.
  • substituted is used herein as known to the expert skilled in the art and refers to a replacement of a chemical group or substituent (typically H or OH) with a functional group, and particularly contemplated functional groups include electrophilic groups (e.g., C(O)—OR, C(X)—OH, etc.), nucleophilic (e.g., —NH2, —OH, —SH, —NC, etc.), ionic groups (e.g., —NH3-), polar groups (e.g., —OH), non-polar groups (e.g., aryl, alkyl, alkenyl, alkynyl, etc.), and halogens (e.g., —F, —Cl), and combinations thereof.
  • electrophilic groups e.g., C(O)—OR, C(X)—OH, etc.
  • nucleophilic e.g., —NH2, —OH, —SH, —NC, etc.
  • protecting group is used herein as known to the expert skilled in the art and refers to a substituent, functional group, ligand, or the like, which is bonded (e.g., via covalent bond, ionic bond, or complex) to a potentially reactive functional group and prevents the potentially reactive functional group from reacting under certain reaction conditions.
  • Potentially reactive functional groups include, for example, amines, carboxylic acids, alcohols, double bonds, and the like.
  • Photo labile protecting groups include, but are not limited to, 2-Nitrobenzyloxycarbonyl (NBOC), 2-nitrophenyl-ethyloxycarbonyl (NPEOC), 2-(3,4-methylenedioxy-2-nitrophenyl)-propyloxy-carbonyl (MeNPPOC), 2-(3,4-methylenedioxy-2-nitrophenyl)-oxycarbonyl (MeNPOC), 2-(2-nitrophenyl)-propyloxycarbonyl (NPPOC), dimethoxy-benzo-inylyl-oxycarbonyl (DMBOC), 2-(2-nitrophenyl)-ethylsulfonyl (NPES), (2-nitrophenyl)-propylsulfonyl (NPPS), and the like.
  • NBOC 2-Nitrobenzyloxycarbonyl
  • NPEOC 2-nitrophenyl-ethyloxycarbonyl
  • MeNPPOC 2-(3,4-methylenedioxy-2-nitrophenyl
  • aryl is used herein as known to the expert skilled in the art and refers to an aromatic residue consisting solely of hydrogen and carbon atoms, such as a phenyl (C6H5-), naphthyl (C10H7-) pyrenyl- or anthracenyl (C14H9-) residue.
  • the aryl can be substituted or unsubstituted with e.g.
  • alkyl groups such as methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl, or alkoxy- such as methoxy-ethoxy- or isopropoxy- or halogen atoms, such as bromide, chloride, or fluoride.
  • heteroaryl is used herein as known to the expert skilled in the art and refers to a cyclic aromatic group having five or six ring atoms wherein at least one ring atom is selected from the group consisting of oxygen, sulfur, and nitrogen, and the remaining ring atoms are carbon.
  • the heteroaromatic ring may form a fused heteroaromatic system together with other aryl- or heteroaryl-rings such as benzothiophene, benzimidazole, pteridine or alloxazine.
  • alkyl is used herein as known to the expert skilled in the art and refers to a univalent residue consisting only of carbon and hydrogen atoms.
  • the alkyls form homologous series with the general formula CnH2n+1.
  • the alkyl can be a straight or branched alkyl, for example the alkyl can be a secondary alkyl which is branched with the central carbon atom linked to two carbon residues or a tertiary alkyl which is branched with the central carbon atom linked to three carbon residues.
  • the letter A in the group -A-O— represents a “fragmentation linker” comprising from 1 to 2 linearly, covalently connected atoms such as methylene- or ethylene-.
  • fragmentation linker is used herein as known to the expert skilled in the art and relates to a moiety which is used as a moiety in photochemistry that effects the light-induced fission of the PLPG by transforming the primary photoprocess into a chemical cleavage reaction.
  • the divalent group -A- refers to a linking group which connects the functional group R2 with the nitrophenyl-chromophore.
  • the 1 to 2 atom chain of the linking group A can be fully comprised of hydrogen and carbon atoms in form of a substituted or unsubstituted, branched or linear, saturated or unsaturated hydrocarbon chain.
  • the hydrocarbon chain can also be branched having one or more alkyl groups, wherein the alkyl group can be methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl.
  • Such a hydrocarbon chain can also be substituted by e.g. halogen atoms. Accordingly, from 1 hydrogen atom to 3 hydrogen atoms of the respective hydrocarbon chain can be substituted through e.g. halogen.
  • branched in context with the definition of the term linking group is used herein as known to the expert skilled in the art and refers to the presence of a side-chain at the main chain of the molecule or moiety.
  • a branched linking group can be a hydrocarbon chain as defined above having one or more alkyl groups as side chain, wherein the alkyl group is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, or tert-butyl; in some embodiments the alkyl group is a methyl or ethyl group.
  • the branched hydrocarbon chain represented by A from one to all carbon atoms can have one or more alkyl groups as defined above.
  • saturated in context with the definition of the term linking group is used herein as known to the expert skilled in the art and relates to a linking group in which all members of the group are connected to the respective adjacent atom(s) through single bonds. Accordingly, a saturated hydrocarbon chain is represented by the formula —(CH2)n- with n being an integer ranging from 1 to 2.
  • Typical functional groups are hydroxyl, carboxyl, aldehyde, carbonyl, amino, azide, alkynyl, thiol and nitril.
  • solid support is used herein as known to the expert skilled in the art and refers to any insoluble and rigid or semi-rigid inorganic or organic material, often having a large surface area to which surface organic molecules can be attached through bond formation or absorbed through electronic or static interactions such as through bond formation through a functional group.
  • biomolecule is used herein as known to the expert skilled in the art and refers to any organic molecule that is produced by a living organism or to any artificially produced derivatives of such compounds, including large polymeric molecules such as proteins, polysaccharides, carbohydrates, lipids, nucleic acids and oligonucleotides as well as small molecules such as primary metabolites, secondary metabolites, and natural products.
  • nucleic acid is used herein as known to the expert skilled in the art and refers to a macromolecule composed of chains of monomeric nucleotides, wherein each nucleotide consists of three components: a nitrogenous heterocyclic base, which is either a purine or pyrimidine; a pentose sugar; and a phosphate group.
  • natural amino acid is used herein as known to the expert skilled in the art and refers to one of the 20 canonical amino acids used for protein biosynthesis as well as all amino acids which can be incorporated into proteins during translation (including pyrrolysine and selenocysteine).
  • the 20 canonical amino acids include histidine, alanine, valine, glycine, leucine, isoleucine, aspartic acid, glutamic acid, serine, glutamine, asparagine, threonine, arginine, proline, phenylalanine, tyrosine, tryptophan, cysteine, methionine and lysine.
  • non-natural amino acid is used herein as known to the expert skilled in the art and refers to organic compounds that are not among those encoded by the standard genetic code or incorporated into proteins during translation. Furthermore, the term “non-natural amino acid” refers to organic compounds that do not occur naturally.
  • non-natural amino acids include amino acids or analogs of amino acids, but are not limited to, the D-isostereomers of amino acids, citrulline, homocitrulline, homoarginine, hydroxyproline, homoproline, ornithine, 4-amino-phenylalanine, cyclohexylalanine, ⁇ -aminoisobutyric acid, N-methyl-alanine, N-methyl-glycine, norleucine, N-methyl-glutamic acid, tert-butylglycine, ⁇ -aminobutyric acid, tert-butylalanine, 2-aminoisobutyric acid, ⁇ -aminoisobutyric acid, 2-aminoindane-2-carboxylic acid, selenomethionine, dehydroalanine, lanthionine, ⁇ -amino butyric acid, and derivatives thereof wherein the amine nitrogen has been mono- or di-alkylated.
  • peptide is used herein as known to the expert skilled in the art and refers to organic compounds made of amino acids arranged in a linear chain and joined together by peptide bonds between the carboxyl and amino groups of adjacent amino acid residues.
  • amino group is used herein as known to the expert skilled in the art and refers to primary (—NH2), secondary (—NHR1), or tertiary (—N R1 R2), and in cationic form, may be quaternary (—N R1 R2 R3).
  • amino groups include, but are not limited to, —NH2, —NHCH3, —NHC(CH3)2, —N(CH3)2 and —N(CH2CH3)2.
  • cyclic amino groups include, but are not limited to, aziridino, azetidino, pyrrolidino, piperidino, piperazino, morpholino, and thiomorpholino.
  • maskless photolithography is used herein as known to the expert skilled in the art and refers to a technique for the synthesis of DNA- or peptide-microarrays without the use of photographic masks.
  • the maskless photolithography uses an array of optical switching elements that are individually addressable and operable under software control. Examples for such optical switching elements are micro mirror devices.
  • One example of a micro mirror device is the Digital Light Processor (DLP) from Texas Instruments, Inc.
  • the disclosure is directed to photolabile protecting groups containing a diarylsulfide chromophore having the general formula:
  • A is selected from the group consisting of —CH 2 —, —CH 2 —CH 2 —, —CH(CH 3 )—, —CH(CH 3 )—CH 2 —, and R1 is an unsubstituted or substituted aryl- or heteroaryl-group, and R3 is H, a methyl group or an ethyl group, and wherein R2 is H, forms a phosphoramidite, H-phosphonate or phosphate triester, or wherein R2 is
  • R4 is H, or OR4 forms a phosphoramidite, H-phosphonate or phosphate triester and wherein R5 is H, OH, a halogen or XR6, wherein X is O or S and R6 is H, an alkyl-group, aryl-group, or OR6 forms a phosphoramidite, phosphodiester, phosphotriester or H-phosphonate or an acetal or a silicone moiety, and wherein B is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl, hypoxanthin-9-yl, 5-methylcytosinyl-1-yl, 5-amino-4-imidazolecarboxylic acid-1-yl or 5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is adenine, cytosine or guanine the primary amino
  • R7 is a natural amino acid, a non-natural amino acid or an amino acid derivative forming an urethan bond to formula Ib or wherein formula IV represents the carboxy function of a natural amino acid, a non-natural amino acid or an amino acid derivative, forming an ester bond to formula Ib.
  • R1 is a phenyl-group, a tert-butyl-phenyl group, a 1- or 2-naphthyl-group, a 2-pyridyl-group an aminophenyl-group, an N-alkylaminophenyl-group, an N-Acylaminophenyl-group, a carboxyphenyl-group, a phenylcarboxylic ester or an amide, and/or A is —CH(CH 3 )—CH 2 — and/or R2 is a phosphoramidite or —P(OCH 2 CH 2 CN)(N-iPr 2 ) and/or R4 is H and/or R5 is H and/or R7 is a natural amino acid.
  • B is selected from the group consisting of adenine, cytosine, guanine, thymine or uracil, or when B is adenine, cytosine or guanine the protecting group is typically selected from phenoxyacetyl-, 4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or dimethylformamidino-residues, when B is adenine the protecting group is typically selected from benzoyl- or p-nitro-phenyl-ethoxy-carbonyl-(p-NPPOC)-residues, when B is guanine the protecting group is typically selected from isobutyroyl-, p-nitrophenylethyl (p-NPE) or p-NPEOC-residues and when B is cytosine the protecting group is typically selected from benzoyl-, isobutyryl- or p-NPEOC-res
  • R1 is a phenyl-group, a tert-butyl-phenyl group, a 1- or 2-naphthyl-group, a 2-pyridyl-group, A is —CH(CH 3 )—CH 2 — and R3 is H or an ethyl group.
  • A is selected from the group consisting of —CH2-, —CH 2 —CH 2 —, —CH(CH 3 )—, —CH(CH 3 )—CH 2 —, —CH2-CH(Alky,Aryl)- and —CH(CH3)-CH(Alkyl, Aryl)-
  • R1 is an unsubstituted or substituted aryl- or heteroaryl-group or a condensed aryl- or heteroaryl-group
  • R3 is H, a methyl group or an ethyl group
  • R4 is H, an alkyl-group, aryl-group, or OR4 forms a phosphoramidite, H-phosphonate or phosphate triester and
  • R5 is H, OH, a halogen or XR6, wherein X is O or S and R6 is an alkyl-group, aryl-group, or OR6 forms a phosphitamide-group, phosphodiester, phosphotriester or H-phosphonate or an acetal or a silicone moiety and
  • B is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl, hypoxanthin-9-yl, 5-methylcytosinyl-1-yl, 5-amino-4-imidazolecarboxylic acid-1-yl or 5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is adenine, cytosine or guanine the primary amino group optionally has a protecting group or when B is thymine or uracil at the O 4 position is optionally a protecting group,
  • R7 is a natural amino acid, a non-natural amino acid or an amino acid derivative, including but not limited to ⁇ - or ⁇ -amino acids, forming an urethan bond to formula Ia,
  • formula IV represents the carboxy function of a natural amino acid, a non-natural amino acid or an amino acid derivative, forming an ester bond to formula Ia, including but not limited to ⁇ - or ⁇ -amino acids.
  • compounds according to formula Ia are used, characterized in that R1 is a phenyl-group, a tert-butyl-phenyl group, a 1- or 2-naphthyl-group or a 2- or 4-pyridyl-group, A is —CH(CH 3 )—CH 2 —, R4 is H and R5 is H, R4 is H and R5 is OH or OSi(Alkyl3).
  • compounds according to formula Ia are used, characterized in that B is selected from the group consisting of adenine, cytosine, guanine, thymine, 5-methylcytosineor uracil.
  • compounds according to formula Ia are used, characterized in that, when B is adenine, cytosine or guanine the protecting group is phenoxyacetyl-, 4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or dimethylformamidino-residues, when B is adenine the protecting group is a benzoyl-residue, when B is guanine the protecting group is a isobutyroyl-residue and when B is cytosine the protecting group is benzoyl- or isobutyroyl-residues.
  • diarylsulfide chromophore containing PLPG which can be used for the photolithography based oligonucleotide and peptide synthesis have the structures:
  • micro mirror devices are used to perform a spatial selective exposure of the oligonucleotide and peptide microarrays to visible light in order to deprotect nucleotides and amino acids, respectively, in the exposed areas during the synthesis process. Deprotection of nucleotides and amino acids, respectively, lead to the release of the next linkage site for the respective next nucleotide or amino acid. The next nucleotide or amino acid which should be coupled to the released linkage site within the specific areas is simply added by its provision within a solvent plus an activating reagent which is poured onto the array.
  • This strategy is repeated until oligonucleotides and oligopeptides, respectively, of the desired lengths and design are obtained.
  • This strategy it is possible to produce highly dense microarrays of at least 10,000 features per cm 2 .
  • This strategy may produce microarrays with even higher densities in the range of 100,000 to 500,000 features per cm 2 .
  • Various embodiments of the PLPG according to the disclosure can be removed by using visible light in a range from 375 nm to 420 nm, in the range from 390 to 405 nm, or in the range from 390 nm and 404 nm, respectively. These wavelengths can be generated using light sources which are much less expensive as compared to light sources necessary to perform deprotection in the near UV range at approximately 365 nm.
  • solid state lasers within the range from 375 nm to 420 nm, alternatively 390 nm and 404 nm, are used as light sources to remove the PLPG according to the disclosure.
  • LEDs (light emitting diodes) with sufficient emission within the range from 375 nm to 420 nm, or 390 nm and 404 nm, are used as light sources to remove the PLPG according to the disclosure.
  • LEDs are one example of light sources that are low cost products as they are produced in high quantities, e.g. for the use in Blu-ray Players, that are useful in conjunction with the PLPG of the disclosure.
  • micro mirror devices are used which are optimized for the use of visible light in the range of 375 nm to 420 nm, or in the range of 390 to 410 nm, or in the range of 390 nm and 404 nm, respectively.
  • the coating of the micro mirror devices remain on the devices in order to be used with visible light.
  • Devices that are used for UV- or near UV-light have to be optimized for that purpose, i.e. the coating on the micro mirror elements has to be removed by polishing.
  • LCD displays or a beam splitter can be used as virtual masks between the light source and the synthesis area.
  • Photolithographic synthesis of the oligonucleotides and peptides, respectively, can be performed on a support, such as a solid support.
  • the support can be made of any material known by the skilled person used for such a purpose, such as plastic, silicon, diamond carbon or glass. Examples of the material are optical grade polyolefin or optical grade microscope glass slides.
  • the support can be provided in any form, such as beads, gels, plates, membranes, slides or chips.
  • the support can be transparent or non-transparent, in various embodiments the support exhibits at least 30%, at least 60%, at least 90% light transmission at a wavelengths of between 375 nm to 410 nm.
  • the PLPG according to the disclosure can be used in any process for oligonucleotide synthesis known by the skilled person where protected nucleosides or nucleotides are necessary.
  • the PLPG-nucleotides as described herein can be used for the synthesis of oligonucleotides in solution, further the PLPG-nucleotides as described herein can be used for the synthesis of oligonucleotides on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. In some examples the synthesis can be performed by using photolithographic techniques, such as maskless techniques wherein a micro mirror device is used to expose light to spatial selected features on a microarray as explained above.
  • Solvents known by the skilled person can be used during oligonucleotide synthesis, such as acetonitrile.
  • Embodiments of the PLPG associated with nucleosides or nucleotides for oligonucleotide synthesis can be used in a concentration within the solvents of 1 mmol/L to 100 mmol/L, in a concentration of 10 mmol/L to 40 mmol/L, or in a concentration of 25 mmol/L.
  • the PLPG used with nucleosides or nucleotides can be used in connection with sensitizing agents known by the skilled person, which increase the effectiveness of the deprotection reaction.
  • sensitizing agents are benzophenone, xanthone and thioxanthone derivates, like e.g. thioxanthen-9-one, alkylthioxanthen-9-ones, as for example isopropylthioxanthen-9-one, 2-ethylthioxanthen-9-one, 2-chloro-thioxanthen-9-one, 1,4-dimethoxythioxanthen-9-one.
  • Oligonucleotide microarrays can be used for a variety of purposes, including but not limited to sequence capturing, comparative genomic hybridization (CGH), CHIP-chip analysis, DNA-methylation analysis, gene expression analysis and comparative genome sequencing.
  • CGH comparative genomic hybridization
  • CHIP-chip analysis DNA-methylation analysis
  • gene expression analysis gene expression analysis and comparative genome sequencing.
  • compounds according to formula Ia are used in the maskless photolithography based DNA array synthesis as intermediate or permanent OH-protecting group in nucleoside derivatives at the 3′-OH end or the 5′-OH end as carbon ester, wherein the synthesis can be performed in 3′-5′-direction or in 5′-3′-direction.
  • the nucleotide carries a phosphoramidite group on its 3′-end, which can be reacted with a free —OH group on the solid support to form a stable elongated oligonucleotide.
  • all PLPG are removed and the oligonucleotide still bound to the solid support has a free 5′-OH.
  • the nucleotide carries a phosphoramidite group on its 5′-end, which can be reacted with any free —OH group on the solid support to form a stable elongated oligonucleotide.
  • all PLPG are removed and the oligonucleotide still bound to the solid support has a free 3′-OH.
  • oligonucleotides that exhibit a free 3′-OH may be used for enzymatic reactions for detection, labeling, capping or elongation by ligation or enzymatic polymerization.
  • the PLPG according to the disclosure can further be used in any process for peptide synthesis known by the skilled person where protected amino acids are necessary.
  • the amino acids can be non-natural amino acids, amino acid derivatives and/or natural amino acids.
  • the PLPG as described herein can be used for the synthesis of oligopeptides in solution, or the PLPG as described herein can be used for the synthesis of oligopeptides on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. For example, the synthesis can be performed by using photolithographic techniques, such as techniques where a micro mirror device is used to expose visible light to spatial selected features on a microarray as explained above.
  • solvents known by the skilled person can be used during peptide synthesis
  • Polar solvents like dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), acetonitrile (MeCN) or isopropanol are examples of solvents that can be used.
  • Said solvents can contain certain additives, such as imidazole, hydroxylamine and water. Imidazole can be added at various concentrations, for example 0.1% to 3% (v/v), 0.5% to 1.5% (v/v), or 1% (v/v).
  • Hydroxylamine can be added at concentrations of 0.1% to 3% (v/v), 0.2% to 1% (v/v), or 1% (v/v). Water can be added at concentrations of 0.1% to 20% (v/v 1% to 17% (v/v), or 1% (v/v).
  • useful solvents are DMSO, DMSO+1% imidazole, NMP+0.5% hydroxylamine, MeCN+1% H 2 O, MeCN+1% H 2 O+1% imidazole, isopropanol+1% imidazole, isopropanol+12% H 2 O+1% imidazole.
  • the PLPG associated to amino acids for peptide synthesis can be used in a various concentrations within the solvents, such concentrations such as 0.1 mmol/L to 0.5 mmol/L, 0.2 mmol/L to 0.4 mmol/L, or the PLPG can be used in a concentration of 0.3 mmol/L.
  • the PLPG associated to amino acids can be used in connection with sensitizing agents known by the skilled person, which increase the effectiveness of the deprotection reaction.
  • Oligopeptide microarrays can be used for a variety of purposes, including but not limited to screening of antibody libraries, quantitative or qualitative analysis of biological samples, biomarker discovery, enrichment of scarce proteins, depletion of high abundant proteins, analysis of protein-protein-interactions, analysis of DNA-protein-interactions or RNA-protein-interactions.
  • compounds according to formula Ia are used for the maskless photolithography based peptide array synthesis as NH-protecting group in amino acids as urethan.
  • the PLPG is used as NH-blocked free acid, activated ester, acid halogenide, anhydride, intermolecular or intramolecular as N-carboxy-anhydride (NCA).
  • the compounds according to formula Ia are used for the maskless photolithography based peptide array synthesis as COOH-protecting group in amino acids as ester for inverse direction of synthesis.
  • the PLPG according to the disclosure can further be used in any process known by the skilled person where protected sugars are necessary.
  • the sugars used can be compounds, such as aldohexoses and aldopentoses.
  • the PLPG as described herein can be used for the synthesis of carbohydrates, glycoproteins and proteoglycans in solution.
  • the PLPG as described herein can be used for the synthesis of carbohydrates, glycoproteins and proteoglycans on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. For example, the synthesis can be performed by using photolithographic techniques, such as techniques where a micro mirror device is used to expose visible light to spatial selected features on a microarray as explained above.
  • Carbohydrate microarrays can be used for a variety of purposes, including but not limited to analysis of saccharide-protein-interactions, high-throughput analysis of proteins and cells, and analysis of glycans and their molecular interactions.
  • the compounds according to formula Ia are used for the maskless photolithography based synthesis of carbohydrates, glycoproteins, proteoglycans, and the like, as OH-protecting group as ether.
  • the compounds according to formula Ia are used as SH-protecting group for orthogonal strategies as ether, ester or thiocarbonate.
  • the compounds according to formula Ia are used as photoactivable protecting groups for releasing an biologically active structure for the initiation of a polymerase reaction or a ATP-dependent biochemical conversion.
  • the present disclosure further relates to the use of the compound according to formula Ia, characterized in that light is used for the maskless photolithography having a wavelength of 375 to 405 nm. Some embodiments use a wavelength of 390 nm.
  • the present disclosure further relates to a method for producing the diarylsulfide backbone containing PLPG which can be used for the photolithography based oligonucleotide and peptide synthesis, wherein the method comprises the following steps:
  • A is selected from the group consisting of —CH 2 —, —CH 2 —CH 2 —, —CH(CH 3 )—, —CH(CH 3 )—CH 2 —, and R1 is an unsubstituted or substituted aryl- or heteroaryl-group, and R3 is H, a methyl group or an ethyl group, and wherein R2 is
  • R4 is H, forms a phosphoramidite, H-phosphonate or phosphate triester
  • R5 is H, OH, a halogen or XR6, wherein X is O or S and R6 is H, an alkyl-group, aryl-group, or OR6 forms a phosphoramidite, phosphodiester, phosphotriester, H-phosphonate or an acetal or silicone moiety
  • B is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl, hypoxanthin-9-yl, 5-methylcytosinyl-1-yl, 5-amino-4-imidazolecarboxylic acid-1-yl or 5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein when B is adenine, cytosine or guanine the primary amino group optionally has
  • R7 is a natural amino acid, a non-natural amino acid or an amino acid derivative forming an urethan bond to formula Ia, or wherein formula IV represents the carboxy function of a natural amino acid, a non-natural amino acid or an amino acid derivative, forming an ester bond to formula Ia.
  • the present disclosure further relates to diarylsulfide chromophore containing PLPG which can be used for the photolithography based oligonucleotide and peptide synthesis having the structure
  • A is selected from the group consisting of —CH2-, —CH 2 —CH 2 —, —CH(CH 3 )—, —CH(CH 3 )—CH 2 —, —CH2-CH(Alky,Aryl)- and —CH(CH3)-CH(Alkyl, Aryl)-
  • R1 is an unsubstituted or substituted aryl- or heteroaryl-group or a condensed aryl- or heteroaryl-group
  • R3 is H, a methyl group or an ethyl group
  • R2 is H, forms a phosphoramidite, H-phosphonate or phosphate triester, or
  • R4 is H, an alkyl-group, aryl-group, or OR4 forms a phosphoramidite, H-phosphonate or phosphate triester and
  • R5 is H, OH, a halogen or XR6, wherein X is O or S and R6 is an alkyl-group, aryl-group, or OR6 forms a phosphitamide-group, phosphodiester, phosphotriester or H-phosphonate or an acetal or a silicone moiety and
  • B is selected from the group consisting of adenine, cytosine, guanine, thymine, uracil, 2,6-diaminopurine-9-yl, hypoxanthin-9-yl, 5-methylcytosinyl-1-yl, 5-amino-4-imidazolecarboxylic acid-1-yl or 5-amino-4-imidazolecarboxylic acid amide-3-yl, wherein
  • B when B is adenine, cytosine or guanine the primary amino group optionally has a protecting group or when B is thymine or uracil at the O 4 position is optionally a protecting group,
  • R7 is a natural amino acid, a non-natural amino acid or an amino acid derivative, including but not limited to ⁇ - or ⁇ -amino acids, forming an urethan bond to formula Ib,
  • formula IV represents the carboxy function of a natural amino acid, a non-natural amino acid or an amino acid derivative, forming an ester bond to formula Ib, including but not limited to ⁇ - or ⁇ -amino acids.
  • compounds according to formula Ib are used, characterized in that R1 is a phenyl-group, a tert-butyl-phenyl group, a 1- or 2-naphthyl-group, an aminophenyl-group, an N-alkylaminophenyl-group, an N-Acylaminophenyl-group, a carboxyphenyl-group, a phenylcarboxylic ester, an amide or a 2- or 4-pyridyl-group, A is —CH(CH 3 )—CH 2 —, R2 is a phosphoramidite or —P(OCH 2 CH 2 CN)(N-iPr 2 ), R3 is H or an ethyl group, R4 is H and R5 is H, R4 is H and R5 is OH or OSi(Alkyl3).
  • compounds according to formula Ib are used, characterized in that B is selected from the group consisting of adenine, cytosine, guanine, thymine, 5-methylcytosine or uracil.
  • compounds according to formula Ib are used, characterized in that, when B is adenine, cytosine or guanine the protecting group is phenoxyacetyl-, 4-tert-butyl-phenoxyacetyl-, 4-isopropyl-phenoxyacetyl- or dimethylformamidino-residues, when B is adenine the protecting group is a benzoyl-residue, when B is guanine the protecting group is a isobutyroyl-residue and when B is cytosine the protecting group is benzoyl- or isobutyroyl-residues.
  • micro mirror devices are used to perform a spatial selective exposure of the oligonucleotide and peptide microarrays to visible light in order to deprotect nucleotides and amino acids, respectively, in the exposed areas during the synthesis process. Deprotection of nucleotides and amino acids, respectively, lead to the release of the next linkage site for the respective next nucleotide or amino acid. The next nucleotide or amino acid which should be coupled to the released linkage site within the specific areas is simply added by its provision within a solvent plus an activating reagent which is poured onto the array.
  • Certain embodiments of the PLPG according to the disclosure can be removed by using visible light in a range from 375 nm to 420 nm, while certain embodiments can be removed in the range from 390 to 405 nm. Certain embodiments are removed via deprotection at wavelengths of 390 nm and 404 nm, respectively. Both wavelengths can be generated using light sources which are much less expensive as compared to light sources necessary to perform deprotection in the near UV range at approximately 365 nm. In some embodiments, solid state lasers within the range from 375 nm to 420 nm, or within the range of 390 nm and 404 nm, are used as light sources to remove the PLPG according to the disclosure.
  • LEDs (light emitting diodes) with sufficient emission within the range from 375 nm to 420 nm, or within the range of 390 nm and 404 nm, are used as light sources to remove the PLPG according to the disclosure.
  • LEDs are low cost products as they are produced in high quantities, e.g. for the use in Blu-ray Players.
  • micro mirror devices are used, which are optimized for the use of visible light in the range of 375 nm to 420 nm, or in the range of 390 to 410 nm, or in the range of 390 nm to 404 nm, respectively.
  • the coating of the micro mirror devices remain on the devices in order to be used with visible light.
  • Devices that are used for UV- or near UV-light have to be optimized for that purpose, i.e. the coating on the micro mirror elements has to be removed by polishing.
  • LCD displays or a beam splitter can be used as virtual masks between the light source and the synthesis area.
  • Photolithographic synthesis of the oligonucleotides and peptides, respectively, can be performed on a support, for example a solid support.
  • the support can be made of any material known by the skilled person used for such a purpose. Examples of materials useful for the support is are plastic, silicon, diamond carbon or glass. In some embodiments, plastic or glass is used as a support. In certain embodiments, as the material is optical grade polyolefin or optical grade microscope glass slides.
  • the support can be provided in any form, such as beads, gels, plates, membranes, slides or chips.
  • the support can be transparent or non-transparent. In some embodiments, the support exhibits at least 30%, at least 60%, or at least 90% light transmission at a wavelengths of between 375 nm to 410 nm.
  • the PLPG according to the disclosure can be used in any process for oligonucleotide synthesis known by the skilled person where protected nucleosides or nucleotides are necessary.
  • the PLPG-nucleotides as described herein can be used for the synthesis of oligonucleotides in solution, or the PLPG-nucleotides as described herein can be used for the synthesis of oligonucleotides on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. For example, the synthesis can be performed by using photolithographic techniques, such as maskless techniques wherein a micro mirror device is used to expose light to spatial selected features on a microarray as explained above.
  • Solvents known by the skilled person can be used during oligonucleotide synthesis, such as acetonitrile.
  • the PLPG associated to nucleosides or nucleotides for oligonucleotide synthesis can be used at various concentrations within the solvents, such as in the range of 1 mmol/L to 100 mmol/L, 10 mmol/L to 40 mmol/L, or at a concentration of 25 mmol/L.
  • the PLPG associated with nucleosides or nucleotides can be used in connection with sensitizing agents known by the skilled person, which increase the effectiveness of the deprotection reaction.
  • sensitizing agents useful for this disclosure are benzophenone, xanthone and thioxanthone derivates, like e.g. thioxanthen-9-one, alkylthioxanthen-9-ones, as for example isopropylthioxanthen-9-one, 2-ethylthioxanthen-9-one, 2-chloro-thioxanthen-9-one, 1,4-dimethoxythioxanthen-9-one.
  • Oligonucleotide microarrays can be used for a variety of purposes, including but not limited to sequence capturing, comparative genomic hybridization (CGH), CHIP-chip analysis, DNA-methylation analysis, gene expression analysis and comparative genome sequencing.
  • CGH comparative genomic hybridization
  • CHIP-chip analysis DNA-methylation analysis
  • gene expression analysis gene expression analysis and comparative genome sequencing.
  • compounds according to formula Ib are used in the maskless photolithography based DNA array synthesis as intermediate or permanent OH-protecting group in nucleoside derivatives at the 3′-OH end or the 5′-OH end as carbon ester, wherein the synthesis can be performed in 3′-5′-direction or in 5′-3′-direction.
  • the nucleotide carries a phosphoramidite group on its 3-end, which can be reacted with a free —OH group on the solid support to form a stable elongated oligonucleotide.
  • all PLPG are removed and the oligonucleotide still bound to the solid support has a free 5′-OH.
  • the nucleotide carries a phosphoramidite group on its 5′-end, which can be reacted with any free —OH group on the solid support to form a stable elongated oligonucleotide.
  • all PLPG are removed and the oligonucleotide still bound to the solid support has a free 3′-OH.
  • the PLPG according to the disclosure can further be used in any process for peptide synthesis known by the skilled person where protected amino acids are necessary.
  • the used amino acids can be non-natural amino acids, amino acid derivatives and/or natural amino acids PLPG as described herein can be used for the synthesis of oligopeptides in solution, or the PLPG as described herein can be used for the synthesis of oligopeptides on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. For example, the synthesis can be performed by using photolithographic techniques, such as techniques where a micro mirror device is used to expose visible light to spatial selected features on a microarray as explained above.
  • solvents known by the skilled person can be used during peptide synthesis.
  • polar solvents like dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), acetonitrile (MeCN) or isopropanol can be used.
  • Said solvents can contain certain additives, such as imidazole, hydroxylamine and water. Imidazole can be added at concentrations of 0.1% to 3% (v/v), 0.5% to 1.5% (v/v), or imidazole can be added at a concentration of 1% (v/v).
  • Hydroxylamine can be added at concentrations of 0.1% to 3% (v/v), 0.2% to 1% (v/v), or hydroxylamine can be added at a concentration of 1% (v/v).
  • Water can be added at concentrations of 0.1% to 20% (v/v), 1% to 17% (v/v), or water can be added at a concentration of 1% (v/v).
  • useful solvents are DMSO, DMSO+1% imidazole, NMP+0.5% hydroxylamine, MeCN+1% H 2 O, MeCN+1% H 2 O+1% imidazole, isopropanol+1% imidazole, isopropanol+12% H 2 O+1% imidazole.
  • the PLPG associated with amino acids for peptide synthesis can be used in a concentration within the solvents of 0.1 mmol/L to 0.5 mmol/L, 0.2 mmol/L to 0.4 mmol/L, or the PLPG can be used at a concentration of 0.3 mmol/L.
  • the PLPG associated with amino acids can be used in connection with sensitizing agents known by the skilled person, which increase the effectiveness of the deprotection reaction.
  • Oligopeptide microarrays can be used for a variety of purposes, including but not limited to screening of antibody libraries, quantitative or qualitative analysis of biological samples, biomarker discovery, enrichment of scarce proteins, depletion of high abundant proteins, analysis of protein-protein-interactions, analysis of DNA-protein-interactions or RNA-protein-interactions.
  • compounds according to formula Ib are used for the maskless photolithography based peptide array synthesis as NH-protecting group in amino acids as urethan.
  • the PLPG is used as NH-blocked free acid, activated ester, acid halogenide, anhydride, intermolecular or intramolecular as N-carboxy-anhydride (NCA).
  • the compounds according to formula Ib are used for the maskless photolithography based peptide array synthesis as COOH-protecting group in amino acids as ester for inverse direction of synthesis.
  • the PLPG according to the disclosure can further be used in any process known by the skilled person where protected sugars are necessary.
  • the sugars used can be compounds, such as aldohexoses and aldopentoses.
  • the PLPG as described herein can be used for the synthesis of carbohydrates, glycoproteins and proteoglycans in solution.
  • the PLPG as described herein can be used for the synthesis of carbohydrates, glycoproteins and proteoglycans on a solid support.
  • the synthesis can be performed by any standard method known in the state of the art. For example, the synthesis can be performed by using photolithographic techniques, such as techniques where a micro mirror device is used to expose visible light to spatial selected features on a microarray as explained above.
  • Carbohydrate microarrays can be used for a variety of purposes, including but not limited to analysis of saccharide-protein-interactions, high-throughput analysis of proteins and cells, analysis of glycans and their molecular interactions,
  • the compounds according to formula Ib are used for the maskless photolithography based synthesis of carbohydrates, glycoproteins, proteoglycans, and the like, as OH-protecting group as ether.
  • the compounds according to formula Ib are used as SH-protecting group for orthogonal strategies as ether, ester or thiocarbonate.
  • the compounds according to formula Ib are used as photoactivable protecting groups for releasing an biologically active structure for the initiation of a polymerase reaction or a ATP-dependent biochemical conversion.
  • the present disclosure further relates to the use of the compound according to formula Ib, characterized in that light is used for the maskless photolithography having a wavelength of 375 to 405 nm, for example 390 nm.
  • the present disclosure further relates to a method for producing the diarylsulfide backbone containing PLPG which can be used for the photolithography based oligonucleotide and peptide synthesis, wherein the method comprises the following steps:
  • solvents dimethylsulfoxide (DMSO), n-methylpyrrolidone (NMP), acetonitrile (MeCN) and isopropanol were used. Imidazole, hydroxylamine or water were added to the solvents as depicted in the table. In case of an irradiation wavelength of 390 nm ( FIG.
  • UV absorption for different PLPG at the wavelengths commonly used is depicted in FIG. 3 .
  • the appropriate derivatives of phenylalanine with the PLPG according to the disclosure were dissolved at a concentration of 1 mg/mL in UV grade methanol. UV spectra were recorded in a scanning photometer and absorption values were taken at the given wavelengths. Molar extinction coefficients were calculated from the molecular weight using Lambert Beers law. Deprotection speed of any PLPG is approximately the product of triplett quantum yield times molar extinction coefficient. It may thus be estimated, that PhS-phenylalanine deprotects 15 times more efficient as BTA-phenylalanine and about 25 times more efficient as NPPOC-phenylalanine at an irradiation wavelength 390 nm.
  • Target-epitope (H)G K P I P N P L L G L D S T-(OH)
  • Peptide features on the array were synthesized in a pattern of varying density on a Roche Nimblegen Maskless Array Synthesizer according to the synthesis scheme in FIG. 8 , at a light dose of:
  • the array was incubated with anti-V5-antibody (labeled with Cy-3 fluorescent dye), obtained from Sigma in the manufacturers recommended buffer system at 1:10 000 dilution (0.1 ⁇ g/mL) overnight at room temperature. After washing with buffer and drying the array was scanned at the appropriate filter setting in a Roche Nimblegen MS 200 fluorescent scanner at 2 ⁇ m resolution. Images were analyzed in Nimblescan and Genepix (Molecular Dynamics) software packages.
  • results show excellent signal intensity with a maximum at about 21 s irradiation at 390 nm as shown in FIG. 8 c , indicating complete photodeprotection at less than 2.000 mW*s, whereas NPPOC-amino acids are insufficiently deprotected at 390 nm and do not give signals attributable to the peptides made.
  • the corresponding synthesis pathway is depicted in FIG. 4 a .
  • a few drops of bromine are added to a mixture of 1902.6 g 1,4-diethylbenzene and 26 g of iron powder. The mixture is stirred at ambient temperature until HBr evolution starts. Then the mixture is cooled in an ice bath and further 2288 g of bromine are added under vigorous stirring over a period of approximately 5 h. Then the ice bath is removed and the mixture is stirred over night at ambient temperature. The reaction mixture is washed with water, saturated NaHCO 3 solution and again with water. The crude product is diluted with toluene, concentrated and distilled in vacuum (approximately 5 mbar/82-84° C.).
  • FIG. 4 c The corresponding synthesis pathway is depicted in FIG. 4 c .
  • a mixture of 1000 g 2,5-diethyl-4-nitro-bromobenzene, 418.8 g paraformaldehyde, 1090 mL triton B (40% in methanol) and 5.2 L DMSO is heated for 2 h at 80 to 90° C. The heating is switched off and the mixture is stirred for further 4 h. 400 mL of acetic acid are added.
  • the mixture is diluted with water to a volume of approximately 15 L and extracted twice with 2 L of toluene.
  • the toluene extract is washed twice with 1 L of water and then concentrated in vacuum.
  • the crude product is purified by chromatography (silica gel, gradient: iso-hexane to iso-hexane/EtOAc 30%).
  • FIG. 4 d The corresponding synthesis pathway is depicted in FIG. 4 d .
  • a mixture of the reactants and DMF was stirred at 140-160° C. for 5 h. After cooling to 110° C., the solvent is removed by distillation under vacuum. The residue was treated with approximately 2.5 L water and extracted with approximately 1 L dichlormethane. The organic phase was washed with dilute NaOH and water, then evaporated to dryness in vacuo, further distilled with an azeotropic toluene/ethanol-mixture and purified by column chromatography on silica gel in 5 to 30% ethylacetate in hexanes.
  • the material is pure for further use without purification.
  • the corresponding synthesis pathway is depicted in FIG. 4 f .
  • 5.81 g glycine und 18.1 g Na 2 CO 3 are dissolved in 190 mL water and 60 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 29.4 g PhSNPPOC-Cl in 90 mL THF. Stirring is continued for 20 min.
  • THF was evaporated and the solution adjusted to pH 11.
  • the solution is extracted twice with approximately 500 mL Hexane/Ethylacetate 1:1, the pH is adjusted to 2.5 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by column chromatography on silica gel with methanol in dichlormethane (0 to 3%).
  • the corresponding synthesis pathway is depicted in FIG. 4 g .
  • 8.6 g proline and 17.5 g Na 2 CO 3 are dissolved in 1000 mL water and 1000 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 28.5 g PhSNPPOC-Cl in 200 mL THF. Stirring is continued for 20 min.
  • THF was evaporated and the solution adjusted to pH 11.
  • the solution is extracted twice with approximately 500 mL ethylacetate, the pH is adjusted to 2.5 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by column chromatography on silica gel with methanol in dichlormethane (0 to 2%).
  • the corresponding synthesis pathway is depicted in FIG. 4 h .
  • 9.97 g isoleucin and 26.8 g Na 2 CO 3 are dissolved in 300 mL water and 200 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 28.9 g PhSNPPOC-Cl in 90 mL THF. Stirring is continued for 20 min.
  • THF was evaporated and the pH of the solution adjusted to 9.5.
  • the solution is extracted twice with approximately 500 mL hexane/ethylacetate 1:1, the pH is adjusted to 3.2 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by column chromatography on silica gel with methanol in dichlormethane (0 to 2%).
  • the corresponding synthesis pathway is depicted in FIG. 4 j .
  • 12.7 g asparagine and 19.8 g Na 2 CO 3 are dissolved in 1000 mL water and 1000 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 32.2 g PhSNPPOC-Cl in 200 mL THF. Stirring is continued for 20 min.
  • THF was evaporated.
  • the solution is extracted twice with approximately 500 mL ether, the pH is adjusted to 2 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by crystallization from ethylacetate.
  • the corresponding synthesis pathway is depicted in FIG. 4 k .
  • 12.1 g leucine and 21.5 g Na 2 CO 3 are dissolved in 250 mL water and 200 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 35 g PhSNPPOC-Cl in 50 mL THF. Stirring is continued for 20 min.
  • THF was evaporated.
  • the solution is extracted twice with approximately 300 mL hexane/ethylacetate 1:1, the pH is adjusted to 3 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by column chromatography on silica gel with methanol in dichlormethane (0 to 3%).
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 30 g PhSNPPOC-Cl in 200 mL THF. Stirring is continued for 20 min. THF was evaporated. The pH is adjusted to 2 with dilute HCl and extracted with approximately 500 mL ethylacetate. The organic phase is washed with approximately 500 mL Water and evaporated to dryness. The product is purified by column chromatography on silica gel with methanol in dichlormethane (0 to 1%).
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 30 g PhSNPPOC-Cl in 200 ml THF. Stirring is continued for 20 min. THF was evaporated. The pH is adjusted to 2 with dilute HCl and extracted with approximately 500 mL ethylacetate. The organic phase is washed with approximately 500 mL water and evaporated to dryness. The product is purified by column chromatography on silica gel with 1% methanol in dichlormethane.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 30 g PhSNPPOC-Cl in 100 mL THF. Stirring is continued for 20 min. THF was evaporated. The pH is adjusted to 2 with dilute HCl and extracted with approximately 500 mL ethylacetate. The organic phase is washed with approximately 500 ml water and evaporated to dryness. The product is purified by column chromatography on silica gel with 1% methanol in dichlormethane.
  • the crystals from above, 20 g, and 12.3 g Na 2 CO 3 were dissolved in approximately 800 mL water and 700 mL THF.
  • the solution is stirred in an ice-bath and is dropwise treated with a solution of 20 g PhSNPPOC-Cl in 100 mL THF. Stirring is continued for 20 min.
  • THF was evaporated.
  • the pH is adjusted to 1.5 with dilute HCl and extracted with approximately 500 mL ethylacetate.
  • the organic phase is washed with approximately 500 mL water and evaporated to dryness.
  • the product is purified by column chromatography on silica gel with 0-1% methanol in dichlormethane and acetic acid (0.01%).
  • the raw product was suspended in 400 mL hydrobromic acid (48%) and heated for 0.5 h to boiling (3-ethyl-4-nitro-aniliniumbromide starts to crystallize, which is associated with a significant increase of the reaction volume).
  • the mixture was cooled to room temperature under stirring and subsequently cooled to 5° C. on ice.
  • the suspension was removed by suction, resuspended in 200 mL cold hydrobromic acid (48%) and filtered again, followed by washing on the nutsch filter with approximately 50 mL cold hydrobromic acid (48%).
  • the humid product of the previous approach was suspended in a solution of 250 mL hydrobromic acid (48%) in 400 mL of water.
  • a solution of 107.6 g NaNO 2 was added dropwise to 550 mL water on ice, in that the temperature of the mixture did not exceed 12° C.
  • the mixture was stirred for 30 min at 0° C. and filtered.
  • Diazoniumsalt-solution ca. 1.45 mol Copper powder 84.9 g CuSO 4 ⁇ 5H 2 O 212.3 g Hydrobromic acid (48%) 670 mL
  • the diazoniumsalt-solution was added dropwise to a mixture of 84.9 g copper powder, 212.3 g CuSO 4 ⁇ 5H 2 O and 670 mL hydrobromic acid (48%) on ice, in that the temperature of the mixture did not exceed 15° C.
  • the mixture was stirred over night at room temperature, filtered and the organic phase was separated. The aqueous phase was extracted with dichloromethane. The combined organic phases were filtered under usage of a thin layer of silica gel and then evaporated to dryness in vacuo. 167.8 g of raw product was yielded.
  • the distillation residue was distilled in high vacuum (Temp.: 155° C., Head-Temp.: 85° C.).

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