WO1994010128A1 - Novel photoreactive protecting groups - Google Patents

Novel photoreactive protecting groups Download PDF

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Publication number
WO1994010128A1
WO1994010128A1 PCT/US1993/010162 US9310162W WO9410128A1 WO 1994010128 A1 WO1994010128 A1 WO 1994010128A1 US 9310162 W US9310162 W US 9310162W WO 9410128 A1 WO9410128 A1 WO 9410128A1
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group
hydrogen
compound
alkyl
independently
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PCT/US1993/010162
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French (fr)
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Christopher P. Holmes
Dennis W. Solas
Benjang Kiangsoontra
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Affymax Technologies N.V.
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Priority to AU54490/94A priority Critical patent/AU5449094A/en
Publication of WO1994010128A1 publication Critical patent/WO1994010128A1/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/27Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups
    • C07C205/35Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • C07C205/36Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system
    • C07C205/37Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by etherified hydroxy groups having nitro groups and etherified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton to carbon atoms of the same non-condensed six-membered aromatic ring or to carbon atoms of six-membered aromatic rings being part of the same condensed ring system the oxygen atom of at least one of the etherified hydroxy groups being further bound to an acyclic carbon atom
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/39Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups
    • C07C205/42Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by esterified hydroxy groups having nitro groups or esterified hydroxy groups bound to carbon atoms of six-membered aromatic rings of the carbon skeleton
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/44Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by —CHO groups
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C205/00Compounds containing nitro groups bound to a carbon skeleton
    • C07C205/45Compounds containing nitro groups bound to a carbon skeleton the carbon skeleton being further substituted by at least one doubly—bound oxygen atom, not being part of a —CHO group
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07CACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C207/00Compounds containing nitroso groups bound to a carbon skeleton
    • C07C207/04Compounds containing nitroso groups bound to a carbon skeleton the carbon skeleton being further substituted by singly-bound oxygen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D307/00Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom
    • C07D307/77Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D307/78Benzo [b] furans; Hydrogenated benzo [b] furans
    • C07D307/79Benzo [b] furans; Hydrogenated benzo [b] furans with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to carbon atoms of the hetero ring
    • C07D307/80Radicals substituted by oxygen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D317/00Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/50Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to atoms of the carbocyclic ring
    • C07D317/54Radicals substituted by oxygen atoms
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D317/00Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D317/08Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3
    • C07D317/44Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems
    • C07D317/46Heterocyclic compounds containing five-membered rings having two oxygen atoms as the only ring hetero atoms having the hetero atoms in positions 1 and 3 ortho- or peri-condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D317/48Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring
    • C07D317/62Methylenedioxybenzenes or hydrogenated methylenedioxybenzenes, unsubstituted on the hetero ring 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 atoms of the carbocyclic ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07DHETEROCYCLIC COMPOUNDS
    • C07D319/00Heterocyclic compounds containing six-membered rings having two oxygen atoms as the only ring hetero atoms
    • C07D319/101,4-Dioxanes; Hydrogenated 1,4-dioxanes
    • C07D319/141,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems
    • C07D319/161,4-Dioxanes; Hydrogenated 1,4-dioxanes condensed with carbocyclic rings or ring systems condensed with one six-membered ring
    • C07D319/18Ethylenedioxybenzenes, not substituted on the hetero ring
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07HSUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02PCLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00Technologies relating to chemical industry
    • Y02P20/50Improvements relating to the production of bulk chemicals
    • Y02P20/55Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to the field of photosensitive protecting groups. More specifically, the invention provides ortho-nitrobenzylic groups for protecting carboxylic, amino, thiol, hydroxyl, and other moieties.
  • Chemical protecting groups are used during synthesis reactions to temporarily protect certain functional groups on a compound against undesired reactions. When a reaction sequence is complete, and protection is no longer necessary, the protective group is removed to restore the protected functional group to its natural activity.
  • Protective groups are removed by various procedures such as exposure to acidic or basic conditions.
  • One class of protective groups, the photolabile or photosensitive protecting groups, are removed by exposure to electromagnetic radiation of a prescribed wavelength.
  • nitrobenzyl derivatives When exposed to radiation (typically near ultra-violet), these compounds undergo an internal rearrangement that results in deprotection of the protected compound. In the process, the protected compound splits off an ortho-nitroso aldehyde or ketone, leaving a free hydroxyl, amino, or other previously protected group. See Cameron and Frechet, J. Am. Chem. Soc. (1991) 113 :4303-4313. incorporated herein by reference for all purposes.
  • Ortho-nitrobenzyl protected compounds have now found use in a variety of fields. For example, they have proven useful in Very Large Scale Immobilized Polymer Synthesis
  • VLSIPSTM VLSIPSTM which provides techniques of forming vast arrays of peptides and other polymer sequences using, for example, light-directed synthesis techniques.
  • the details of VLSIPSTM are disclosed in Pirrung et al., U.S. Patent No. 5,143,854
  • NVOC nitroveratryloxycarbonyl
  • NPOC nitropiperonyloxycarbonyl
  • MeNVOC alpha-methyl-nitroveratryloxycarbonyl
  • MeNPOC alpha- methyl-nitropiperonyl
  • benzylic form of each of these i.e. NV, NP, MeNV, and MeNP.
  • a "basis set" of protected monomers is used to synthesize a diverse collection of polymers.
  • the basis set is, for example, the twenty naturally occurring amino acids having protected alpha-amino groups. It is desirable to have each member of the basis set photolyze at approximately the same rate. Unfortunately, this is often not possible when a single protecting group (e.g. ortho- nitrobenzyloxycarbonyl) is used to protect each member of the basis set. For example, when two different amino acids protected with nitrobenzyloxycarbonyl groups are exposed to radiation of the same wavelength and intensity, they may be deprotected at substantially different rates. Thus, it is desirable to develop methods that "equalize" the photolysis rates of the basis set members.
  • the present invention provides new ortho-nitrobenzyl photosensitive protecting groups. These groups can be coupled to amino, carboxyl, hydroxyl, thiol and other moieties of compounds protected during certain chemical reactions.
  • the photosensitive groups of this invention can be used to protect functional groups of nucleosides, amino acids, or saccharides from unwanted side reactions during polymer synthesis.
  • the invention provides improved photoremovable protecting groups having the formula:
  • n is 0 or 1;
  • A is -C(O)-, -(CQ 1 Q 2 )-, or -C(S)-;
  • R 1 , Q 1 , and Q 2 are independently hydrogen, C 1 -C 8 alkyl, aryl, alkoxy, aryloxy, or carboxy; and
  • R 2 is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or oligonucleotide.
  • R 3 -R 6 are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C 1 -C 8 alkyl, aryl, or benzyl.
  • R 3 -R 6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic, or ether groups.
  • R 4 and R 5 are not both methoxy when R 1 , R 3 , and R 6 are hydrogen; (ii) R 1 is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R 3 -R 6 are hydrogen simultaneously; and (iii) R 1 is not 2-nitrophenyl or 3,4-dimethoxy-6-nitrophenyl when R 3 and R 6 are hydrogen and R 4 and R 5 are both either hydrogen or methoxy.
  • the amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone” groups (i.e. the amine and carboxyl groups that form the peptide bond).
  • Side chain functional groups found on natural amino acids include amines, carboxyl groups, thiols, hydroxy groups, imidazoles, amides, indole groups, etc.
  • the nucleoside, nucleoside analog, and oligonucleotide functional groups include the 2', 3', and 5' ribose hydroxy groups.
  • the purine and pyrimidine bases of nucleosides can be protected by the above protecting groups.
  • R 2 in the above compound structure can represent base exocyclic amine groups in some embodiments.
  • a particularly preferred class of photoremovable protecting groups has the formula:
  • R 2 is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an imidazole, an amide, a hydroxy group, or the like. Otherwise, R 1 , Q 1 , Q 2 , and R 3 -R 6 are the same as defined in the previous structure.
  • the functional group R 2 is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an oligonucleotide as described in connection with the above embodiment.
  • Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho- nitrobenzyloxymethyl groups such as ortho-nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides).
  • Other active groups that can replace the halo group include
  • the invention also includes basis sets of protected natural or unnatural amino or nucleic acids in which a
  • photoremovable protecting groups of the invention such that all of the protected amino or nucleic acids are deprotected at substantially the same rate in a particular solvent.
  • Fig. 1 schematically illustrates various reaction paths that can be employed to produce alcohols of the ortho-nitrobenzyl protecting groups of this invention
  • Fig. 2 schematically illustrates reaction paths to various protected compounds of this invention
  • Fig. 3 schematically illustrates reaction paths to various benzyloxycarbonyl protected compounds of this
  • Fig. 4 schematically illustrates reaction paths to various benzyl protected compounds of this invention
  • Fig. 5 schematically illustrates reaction paths to various benzyloxymethyl protected compounds of this invention.
  • Fig. 6 presents various compounds for which photolysis data is provided in Table V.
  • Monomer A member of the set of small molecules which are or can be joined together to form a polymer.
  • monomers have at least two different reaction sites (e.g. the amino and carboxyl groups of amino acids).
  • the set of monomers includes but is not restricted to, for example, the set of common L-amino acids, the set of D-amino acids, the set of synthetic and/or natural amino acids, the set of nucleotides and the set of pentoses and hexoses.
  • the particular ordering of monomers within a polymer is referred to herein as the "sequence" of the polymer.
  • the invention is described herein primarily with regard to the preparation of molecules containing sequences, of monomers such as amino acids, but could readily be applied in the preparation of other polymers.
  • Such polymers include, for example, both linear and cyclic polymers of nucleic acids, polysaccharides, phospholipids, and peptides having either ⁇ - , ⁇ -, or ⁇ -amino acids, heteropolymers in which a known drug is covalently bound to any of the above, polynucleotides, polyurethanes, polyesters, polycarbonates, polyureas, polyamides,
  • Functional group A group or moiety on a chemical compound that in some environments imparts certain chemical properties to the compound. More specifically, “functional group” refers to groups that are capable of undergoing
  • reaction such as oxidation or reduction under certain
  • functional group most often refers to a group on a monomer or polymer that can be protected by a photolabile or other group.
  • functional groups include the reactive sites on a monomer that participate in coupling reactions to produce a polymer.
  • amino acid functional groups include carboxyl and amino groups
  • nucleic acid functional groups include 2', 3', and 5' hydroxyl groups.
  • Protecting group A material which is chemically bound to a reactant functional group and which may be removed upon selective exposure to an activator such as
  • a protecting group prevents the protected functional group from undergoing undesired side reactions.
  • the amino group of glycine may be coupled to a protecting group to prevent the glycine amino group from reacting during a coupling reaction between the glycine carboxylic terminus and the amino terminus of a growing peptide.
  • photosensitive protecting groups include nitropiperonyl, nitroveratryl, and nitrobenzyl groups.
  • Other protective groups sensitive to acid or base include t-butyloxycarbonyl or fluorenylmethoxycarbonyl.
  • the 20 naturally occurring amino acids form one basis set and the four
  • the dimers of the 20 naturally occurring L-amino acids form a basis set of 400 monomers for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer. Furthermore, each of the sets may include protected members which are modified after synthesis.
  • Peptide A polymer in which the constituent monomers are amino acids joined together through amide bonds. Peptides are alternatively referred to as polypeptides.
  • the peptide may include the L-optical or D-optical isomers of ⁇ - , ⁇ - , or ⁇ -amino acids.
  • the peptide may include amino acids having unnatural side chains or other deviations from the naturally occurring amino acids. Such "peptides" have been the subject of much study recently, and are
  • Peptides are two or more amino acid monomers long, and often more than 20 amino acid monomers long. Standard abbreviations for amino acids are used (e.g., P for proline). These abbreviations are included in Stryer,
  • Radiation Energy which may be selectively applied including energy having a wavelength of between 10 -14 and 10 4 meters including, for example, electron beam radiation, gamma radiation, x-ray radiation, ultra-violet radiation, visible light, infrared radiation, microwave radiation, and radio waves. "Irradiation” refers to the application of radiation to a material.
  • the photosensitive protecting groups of this invention are typically photolyzed by electromagnetic
  • BOP benzotriazol-1-yloxytris-(dimethylamino) phosphonium hexafluorophosphate.
  • DCC dicyclohexylcarbodiimide
  • DIEA N,N-diisopropylethylamine.
  • HBTU 2-(1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate.
  • NMP N-methylpyrrolidone
  • NPOC 6-nitropiperonyloxycarbony1.
  • NV 6-nitroveratryl.
  • NVOC 6-nitroveratryloxycarbonyl.
  • TFA trifluoracetic acid
  • Protecting groups of the present invention can be used in conjunction with solid phase oligomer syntheses, such as peptide syntheses using natural or unnatural amino acids, nucleotide syntheses using deoxyribonucleic and ribonucleic acids, oligosaccharide syntheses, and the like.
  • solid phase oligomer syntheses such as peptide syntheses using natural or unnatural amino acids, nucleotide syntheses using deoxyribonucleic and ribonucleic acids, oligosaccharide syntheses, and the like.
  • the protecting groups of this invention are used to synthesize an array of polymers according to a VLSIPSTM process.
  • VLSIPSTM processes employ a substrate containing oligomers or other materials protected with photosensitive groups. Selected regions of the substrate are irradiated to create regions having reactive moieties that are essentially free of side products resulting from the protecting group.
  • the protecting groups serve two purposes: (1) protecting the substrate surface from unwanted reactions, and (2) blocking a reactive functional group of the monomer to prevent self-polymerization or other reaction. For instance, attachment of a protecting group to the amino terminus of an activated amino acid, such as an
  • N-hydroxysuccinimide-activated ester of the amino acid prevents the amino terminus of one monomer from reacting with the activated ester portion of another during peptide
  • the protecting group may be
  • the photolabile protecting group may be attached to the 2'- or 3'-hydroxyl group of a nucleoside.
  • the photosensitive protecting groups of this invention can be employed to protect side chain functionalities of amino acids during peptide syntheses employing conventional acid or base labile
  • the photolabile protecting groups protect any one or a combination of the following groups: the thiol group of cysteine; the basic residues (amine and
  • imidazole groups of lysine, arginine, and histidine; the acidic carboxy groups of aspartic and glutamic acids; the hydroxy groups of serine, threonine, and tyrosine; and the heterocyclic indole ring of tryptophan.
  • other amino acid side chains such as those occurring on non-natural amino acids can likewise be protected by the photosensitive groups of the present invention.
  • the purine and pyrimidine bases (and particularly the exocyclic amine groups) of nucleoside and nucleoside derivatives likewise can be protected with the photosensitive groups of this invention.
  • protecting groups are employed to protect a moiety on a molecule from reacting with another reagent.
  • the reaction to be blocked will be the formation of a covalent bond such as a peptide bond between two amine acid molecules.
  • the protecting groups of this invention can also block non-covalent reactions.
  • the photosensitive protecting groups of this invention can be used to "cage" (i.e. provide protection based upon steric
  • Protecting groups of the present invention preferably have the following general characteristics: they prevent selected reagents from modifying the group to which they are attached; they are stable (that is, they remain attached to the molecule) to the synthesis reaction
  • liberated byproducts of the photolysis reaction can be rendered
  • a suitable protecting group will depend, of course, on the chemical nature of the monomer unit and oligomer, as well as the specific reagents they are to protect against.
  • the removal rate of the protecting groups depends on the wavelength and intensity of the incident radiation, as well as the physical and chemical properties of the protecting group itself. Preferred protecting groups are removed at a faster rate and with a lower intensity of radiation. For example, at a given set of conditions, MeNVOC and MeNPOC are photolytically removed from the N-terminus of a peptide chain faster than their counterparts, NVOC and NPOC. Therefore for most applications, MeNVOC and MeNPOC are preferred over NVOC and NPOC.
  • the photosensitive protecting groups will be removable by radiation in the ultraviolet (UV) or visible portion of the electromagnetic spectrum.
  • the photosensitive protecting groups are sensitive to an electromagnetic spectral region that does not significantly overlap the absorption band of the monomers or polymers being protected.
  • Higher energy radiation i.e. low wavelength radiation
  • the four naturally occurring bases in deoxynucleosides absorb 250-280 nm light very strongly.
  • short wave ultra-violet light is known to photo-destruct DNA formation of thymine dimers.
  • the protecting groups will be removable by radiation in the near UV or visible portion of the spectrum.
  • each collection of monomers (basis sets) will have its own
  • characteristic absorption bands which can be avoided by choosing protecting groups that are sensitive to wavelengths well removed from the absorption band.
  • Preferred photochemical protecting groups of the present invention have the general formula:
  • n is 0 or 1;
  • A is -C(O)-, - (CQ 1 Q 2 ) - , or -C(S)-;
  • R 1 , Q 1 , and Q 2 are independently hydrogen, C 1 -C 8 alkyl, aryl, alkoxy, aryloxy, or carboxy; and
  • R 2 is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or oligonucleotide.
  • R 3 -R 6 are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy
  • R 3 -R 6 may also be a cyclic bridge between adjacent substituents.
  • Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic (which together with the first benzene ring forms a derivative of naphthalene, quinoline, anthracene, etc.), or ether groups.
  • R 4 and R 5 are not both methoxy when R 1 , R 3 , and R 6 are hydrogen; and (ii) R 1 is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R 3 -R 6 are hydrogen simultaneously.
  • compounds having hydroxyl substituents on the phenyl ring, and/or alkoxy or aryloxy substituents on the benzyl carbon may be too reactive for certain applications, and therefore are generally less preferred.
  • amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone” groups (i.e. the amine and carboxyl groups that form the peptide bond).
  • backbone groups i.e. the amine and carboxyl groups that form the peptide bond.
  • Those of skill in the art will recognize that a variety of other functional groups can be protected by the above photosensitive groups. Specific examples include hydroxy and amino groups on saccharides, hydroxy groups on steroids, and amino groups attached to linkers on a substrate surface (see PCT Publication No.
  • biotin and biotin analogs can be caged with the above photosensitive groups to prevent binding with avidin.
  • the protecting group can be coupled through the biotin urea groups.
  • carboxyl, amino, and hydroxy groups of amino acids and nucleosides can, in principle, be protected with either benzyl or benzyloxycarbonyl protecting groups
  • a primary amine group e.g. those found on most natural amino acids
  • the amine nitrogen must be further protected for some applications, if the amine is not further protected during peptide synthesis using VLSIPSTM, the free amine hydrogen may react with other amino acids activated with coupling reagents such as BOP. If, however, a second protecting is employed, no free hydrogen atoms are present on the amine to act as a reaction sites.
  • the additional protection is provided by C 1 -C 6 alkyl, aryl, acyl, or benzyl groups. More preferably, the additional protective group is methyl or acetyl.
  • Such compounds are readily synthesized by methods well-known in the chemical arts.
  • the photoprotected amino acid may be reacted with a reagent known to methylate amino acid nitrogen moieties, such as methyl iodide, to form the desired protected amino acid.
  • a particularly preferred class of protecting groups is the 6-nitrobenzyloxymethyl groups which have exhibited very rapid photolysis rates and have the general formula:
  • R 2 is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an imidazole, an amide, a hydroxy group, and the like.
  • R 1 , Q 1 , Q 2 and R 3 -R 6 are the same as defined in the above structure.
  • the functional group R 2 is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an
  • photosensitive groups can also be used to protect functional groups found on a variety of compounds such as carbohydrates, lipids, biotin analogs, linker molecules, etc.
  • Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho-nitrobenzyloxymethyl compounds such as ortho-nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides).
  • Other active groups that can replace the halo group include hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.
  • Another class of preferred protecting groups includes compounds in which R 5 and R 6 are both alkyloxy groups or together form a cyclic bridge acetal or ketal.
  • R 1 is hydrogen or methyl and R 3 and R 4 are hydrogen.
  • R 3 and R 4 are hydrogen.
  • R 4 and R 5 are alkoxy groups or together form the cyclic acetal or ketal.
  • R 3 and R 6 will then be hydrogen.
  • Such compounds are made by standard methods such as those described in Greene, Protective Groups in Organic
  • R 4 and R 5 or R 5 and R 6 form a methylene acetal or an acetonide: (-O-CH 2 -O- , or -O-C(CH 3 ) 2 -O-).
  • Y 1 and Y 2 are independently C 1 -C 8 alkyl groups, or fused to form a cyclic bridge acetal or ketal.
  • photoprotecting groups of the invention may also include compounds where adjacent benzene ring substituents form a ring having the formula -O-CRR'-O- or -O-CRR'-CR"R'''-O-, where R, R', R", and R"' can independently be hydrogen, C 1 -C 8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl carboxylic acid ester (i.e. -C(O)O-alkyl), or carbonyl.
  • R and R'-O- cyclic orthoesters
  • R and R' are alkoxy, aryloxy, or carboxy
  • cyclic ethers -O-CRR'-CR"R"'-O- where R, R', R", and R"" are selected from the group hydrogen, alkyl, aryl, benzyl, alkoxy, aryloxy, or carboxy.
  • R and R' together or R" and R'" together may be a carbonyl oxygen (e.g. forming the following bridges: -O-C(O)CH 2 -O- or -O-CH 2 C(O)-O-).
  • a preferred cyclic ether has R, R', R", and R" hydrogen (i.e., -O-CH 2 -CH 2 -O-).
  • Preferred substitution patterns are those at the R 4 and R 5 , and R 5 and R 6 positions:
  • Still other preferred compounds are those where R 5 and R 6 are methoxy.
  • R 3 is dimethylamino and R 4 is hydrogen:
  • R 4 is methoxy and R 5 is dimethylamino, or, conversely, where R 4 is dimethylamino and R 5 is methoxy:
  • an amine protected amino acid may have the following structure:
  • R is the side chain of a natural or unnatural amino acid
  • X is a photoremovable protecting group according to this invention
  • Y is an activated carboxylic acid derivative.
  • the activated ester, Y is preferably a reactive derivative having a high coupling efficiency, such as an acyl halide, mixed anhydride, N-hydroxysuccinimide ester, perfluorophenyl ester, or urethane protected acid, and the like.
  • Other activated esters and reaction conditions are well known (See Atherton et al., "Solid Phase Peptide Synthesis" 1989, IRL Press, London, incorporated herein by reference for all purposes).
  • the photoreactive protecting groups of the present invention are useful for protecting both natural and unnatural nucleosides or nucleotides. More specifically, the 2', 3', and/or 5' hydroxyl functions of such compounds. Such compounds can therefore have the general structure:
  • R 1 is a purine or pyrimidine base or analog thereof (for example, adenine, cytosine, thymine, guanine, uracil, 6-ketoadenine, or an analog thereof).
  • R 2 -R 4 may be one of the commonly used protecting groups (see below), or a photoreactive group as described above. Typically, one position will be protected with a photoreactive protecting group while the other
  • position (s) will be (a) protected or substituted with one of the commonly used protecting groups, or (b) activated with one of the commonly reactive groups. However, more than one position may be protected with a photoreactive protecting group of the invention.
  • R 2 , R 3 , and R 4 these may be hydrogen, hydroxy, an oligonucleotide or one of the commonly used protecting groups: alkoxy, tetrahydropyranyl, ⁇ -benzoylpropionyl, acetyl, or silyl. They can also be -NRR', -OP (O) (OR”) (OR"'), -OP(O)O 2 -2 , -OP(O)O 2 H-, -OP (O) (OR”) (NRR'), or -OP (OR”) (NRR' ) (i.e.
  • R and R' are independently selected from the group consisting of C 1 -C 3 alkyl, aryl, benzyl, or acetyl
  • R" and R' are selected independently from the group consisting of C 1 -C 3 alkyl, aryl, benzyl, or C 1 -C 3 cyanoalkyl.
  • R 2 or R 3 also together form a cyclic acetal, ketal, orthoester, or ether.
  • R 4 can be triphenylmethyl, di-p-methoxytrityl, dimethylpropanoyl.
  • An especially preferred photoreactive protecting group for use with nucleosides and nucleotides has the
  • a desired polypeptide will not be contaminated as a result of cross reactions between sidechains which have inadvertently become unprotected and other monomers or the sidechains of other amino acids. Since the removal of the photoreactive protecting groups of the present invention typically creates an acidic environment, care must be taken to choose sidechain protecting groups which are moderately acid stable.
  • the term "acid stable” means that the sidechain protecting group in question is not removed in appreciable amounts under conditions where the effective pH is less than about 7. Determining stability will depend on the particular amino acid sidechain as well as the sidechain protecting group which is chosen. For lysine, it has been found that ⁇ -amino moieties protected with BOC (t- butyloxycarbonyl) tend to degrade over time in 5mM sulfuric acid. Thus, a protecting group for the ⁇ -amino group of this amino acid should be less acid labile than BOC. Generally, trityl protecting groups are also too sensitive to protect ⁇ - amino groups.
  • One preferred protecting group for the ⁇ -amino acid of lysine is dimethoxybenzyloxycarbonyl.
  • Another preferred protecting group is FMOC (9- fluorenylmethyloxycarbonyl).
  • trityl is not preferred with respect to lysine, trityl is stable enough to be used with cystine, asparagine, and glutamine. Generally, however, asparagine and glutamine do not require protection. With respect to
  • the protected compounds of the present invention often can be prepared via a process employing a benzyl alcohol of the protecting group.
  • a benzyl alcohol of the protecting group Various synthetic routes to some useful benzyl alcohols are shown in Fig. 1.
  • R, R', and R" represent hydrogen, methyl, C 1 to C 8 alkyl, and other groups in accordance with the
  • Fig. 1 depicts a different reaction pathway, employing different reagents.
  • Typical reagents for suitable use in the various reaction paths are as follows: (a) RC(O)Cl and aluminum chloride; (b) DMF and POCl 3 ; (c) R'I or R"I or MeBr 2 or EtBr 2 and potassium carbonate; (d) concentrated nitric acid or other nitration agent; (e) NaBH 4 ; and (f) RMgBr or RLi.
  • Fig. 2 displays reaction paths for making various protected compounds from benzyl alcohols.
  • reaction path (a) the alcohol is reacted with ClC(Z)OR' and triethylamine or ClC(Z)SR' and
  • triethylamine produces a protected carboxylic acid.
  • Fig. 3 schematically depicts some general reactions that can be employed to produce benzyloxycarbonyl (and
  • substitution on the phenyl ring R denotes substitution on the benzylic carbon
  • Z denotes an oxygen or sulfur atom.
  • Typical reagents employed in the noted routes are as follows: (a) C(O)Cl 2 or C(S)Cl 2 ; (b) R'OH and triethyl amine; (c) R'SH and triethyl amine; (d) R'R"NH; and (e) H 2 NC(O)R'
  • the appropriate benzyl alcohol can be reacted with phosgene or other agent to produce an activated benzyloxycarbonyl derivative of the protecting group.
  • phosgene or other agent to produce an activated benzyloxycarbonyl derivative of the protecting group.
  • These compounds are then used to produce the above-described benzyloxycarbonyl-protected groups (e.g. n is 1 and A is -C(O)- in the above generic structures).
  • These groups are preferably coupled to the amino nitrogen of a natural or unnatural amino acid; or the 2', 3', or 5' oxygen of a natural or unnatural nucleoside or nucleotide using standard methods, for example, reacting the amino group of an amino acid or a ribose hydroxyl moiety of a nucleoside with the desired activated benzyloxycarbonyl derivative.
  • activated protecting groups have the general formula: where X is halogen, mixed anhydride, phenoxy, p-nitrophenoxy, N-hydroxysuccinimide, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.
  • R 1 is hydrogen, C 1 -C 8 alkyl, aryl, alkoxy, aryloxy, or carboxy.
  • R 3 -R 6 are selected
  • R 3 -R 6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups. Compounds falling within this formula are readily formed using known standard techniques such as those described in March, Advanced Organic Chemistry.
  • Fig. 4 schematically depicts various reaction pathways from a benzyl alcohol starting material to benzyl-protected compounds via an activated benzyl intermediate.
  • X denotes substitution on the phenyl ring
  • R denotes substitution on the benzylic carbon.
  • Typical reagents used in the various reaction steps are as follows: (a) SOCl 2 ; (b) R'OH and triethylamine; (c) R'SH and
  • the carboxy terminus of an amino acid protected with a benzylic photoactivatable group can be formed by esterifying the carboxy group with an activated benzyl derivative of the protecting group.
  • the amino terminus of an amino acid or a hydroxy group of a nucleoside (or nucleoside derivative) protected with a benzylic group can be formed by alkylating the amino or hydroxy group with an activated benzyl derivative of the protecting group.
  • benzyoxycarbonyl groups are more preferred for protecting amine groups of amino acids and hydroxy groups of nucleosides.
  • a benzylic form of a photoreactive protecting group is used to protect the amino nitrogen of an amino acid, that nitrogen may have to be further protected with an alkyl, aryl, benzyl, or acyl group.
  • R 1 is hydrogen, C 1 -C 8 alkyl, aryl, alkoxy, aryloxy, or carboxy.
  • R 3 -R C are selected
  • R 3 -R 6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups.
  • Another method for generating compounds protected with benzylic protecting groups is to react the benzylic alcohol derivative of the protecting group with an activated ester of the compound to be protected.
  • an activated ester of the amino acid is reacted with the alcohol derivative of the protecting group.
  • activated esters suitable for such uses include halo-formate, mixed anhydride, imidazolyl formate, acyl halide, and also include formation of the activated ester in situ the use of common reagents such as DCC and the like. See Atherton et al. (previously
  • nucleotides having activating groups attached to the 5'-hydroxyl group have the general formula:
  • Y is a halogen atom, a tosyl, mesyl, trifluoromethyl, azido, or diazo group, and the like.
  • Y is a halogen atom, a tosyl, mesyl, trifluoromethyl, azido, or diazo group, and the like.
  • Benzyloxymethyl-protected compounds can be synthesized using standard techniques known in the chemical arts.
  • 6-nitroveratryloxymethyl chloride NV0MCl
  • 6-nitroveratryloxymethyl chloride NV0MCl
  • 6-nitroveratryl alcohol upon exposure to gaseous HCI and paraformaldehyde following the methods
  • benzyloxymethylether Primary and secondary amines as well as carboxylic acids can protected in an analogous manner. Examples of reactions to produce benzyloxymethyl- protected compounds via a chloromethyl ether intermediate are schematically presented in Fig. 5. As in Figs. 2 to 4, X denotes substitution on the phenyl ring and R denotes substitution on the benzylic carbon.
  • Typical reagents that can be used in the various reaction steps are as follows: (a) CH 2 O and HCl; (b) R'OH and triethyl amine; (c) R'SH and triethylamine; (d) R"R'NH; (e) H 2 NC(O)R' and triethylamine; and (f) R'C(O)OH and triethylamine.
  • the benzaldehyde compound was dissolved in 40 mL of 70% HN0 3 cooled to 0°C. After stirring for 30 min., the reaction mixture was poured into cold water and extracted with EtOAc. The organic phase was washed (sat NaHCO 3 ), dried
  • the alcohol was dissolved in 20 mL of CH 2 Cl 2 and treated with DMAP (0.26 g, 0.0018 mol) and Ac 2 O (1.5 g, 0.15 mmol) for 18 hours.
  • the reaction mixture was partitioned between CH 2 Cl 2 and 3 N HCI.
  • the organic phase was washed (sat. NaHCO 3 ), dried (MgSO 4 ), and was evaporated to give 1.4 g of a mixture of the two regioisomers as a light yellow solid.
  • the crude nitrophenyl compound was dissolved in 25 mL of anhydrous THF and treated with NaBH 4 (400 mg; 10.6 mmol) at room temperature. The solution darkened to dark brown after 2 min. After stirring for 30 min., 10 mL of EtOH was added and the stirring continued for an additional 30 min. The solution was partitioned between EtOAc and sat. NH 4 Cl, and the organic phase was dried (MgSO 4 ), and evaporated to afford a brown oil.
  • NaBH 4 400 mg; 10.6 mmol
  • the crude benzyl alcohol was taken up in 20 mL of CH 2 Cl 2 and 6 mL of pyridine and was treated with 2 mL of Ac 2 O and 10 mg of DMAP for 14 hours.
  • the reaction mixture was partitioned between EtOAc and 1 N HCI, and the organic phase was dried (MgSO 4 ), and concentrated to give a brown oil.
  • MeNPOC-L-Phe-OH as a white solid, MP 144-154°C (decomposes).
  • Alpha-methyl-6-nitropiperonyl alcohol was prepared as follows. All anhydrous reagents, methylene chloride, pyridine, triethylamine, and diisopropylethyl amine were obtained from Aldrich Chem Co. N-4-isobutyryl-2'-deoxycytidine, N6-phenoxyacetyl-2'-deoxyadenosine, N2-phenoxyacetyl-2'-deoxyguanosine, 2'-deoxythymidine, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite were purchased from Sigma Chem Co. 4,5-methylenedioxy-2-nitroacetophenone was purchased from Lancaster Synthesis and 1.93M phosgene in toluene was from Fluke Chem Co.
  • Alpha-methyl-6-nitropiperonyloxycarbonyl chloride was prepared as follows. Phosgene (500ml, 1.93M in toluene) was added to a rapidly stirring solution of (20 g, 0.095 mol) in 700ml anhydrous tetrahydrofuran . After stirring overnight at room temperature the mixture was evaporated to dryness in vacuo resulting in a thick brown oil. Tituration with hexane yielded 20 g (73%) of ⁇ -methyl-6-nitropiperonyloxycarbonyl chloride as a yellow-brown solid.
  • 1 H NMR (CDCl 3 ) 1.7 (d, 3H) CH 3 ; 6.15 (m, 2H) O-CH 2 -O; 6.5 (q, 1H) CH; 7.05 and 7.55 (s, 2H) aromatic.
  • 5'-O-( ⁇ -methyl-6-nitropiperonyloxycarbonyl)-2'-deoxynucleosides was prepared as follows. N-protected deoxynucleoside (34 mmol) was evaporated twice from 100ml anhydrous pyridine and dried in vacuo. The resultant residue was dissolved in 150 ml anhydrous pyridine, cooled to 0°C in an ice water bath, and 37 mmol added. After stirring for 15 min. at 0°C the ice bath was removed and the reaction stirred for 4-5 hours at room temperature. The mixture was
  • 5'-O-( ⁇ -methyl-6-nitropiperonyloxycarbonyl)-2'-deoxynucleoside 3'-O-(2-cyanoethyl)-N,N'-diisopropylaminophosphites were prepared as follows. 5'-protected deoxynucleoside (10 mmol) was dissolved in 75 ml of methylene chloride. Diisopropylethylamine (30 mmol) was added and the solution cooled to 0°C in an ice water bath. 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (25 mmol) was slowly added to the cooled mixture. The ice bath was removed and the reaction stirred for 2 hours at room temperature.
  • regioisomers was used without further purification.
  • the crude nitrobenzaldehyde was reduced Py treating a solution of the aldehyde (501 mg; 2.37 mmol) in 25 mL of anhydrous THF with NaBH 4 (185 mg; 4.89 mmol) cooled to 15 °C. After stirring the solution for 10 min., 3 mL of MeOH was added to aid solubility. The reaction mixture was stirred an additional 30 min. and was then partitioned between EtOAc and sat. NH 4 Cl. The organic phase was washed (sat. NaCl), dried (MgSO 4 ), and evaporated to give 0.56 g of a 1:1 mixture of the two regioisomers as a white crystalline solid. The alcohols were used without further purification.
  • Nitroveratryloxymethylchloride (NV0M-Cl) was prepared as follows. To a suspension/solution of 5g (0.0235 mol) of NV-OH in 50 ml of toluene was added 1.0g (0.033 mol) dry powdered paraformaldehyde [(CH 2 O) n ] with vigorous
  • the oil was placed on a vacuum line whereupon yellow crystals began to come out of the oil.
  • the semi-solid was then triturated 3 times with 40 ml of hexane each time decanting off the hexane.
  • the yellow crystalline residue was then dried in vacuum at room temperature. The yield was 5.67 g on 92%.
  • NVOM ⁇ -estradiol-benzylether was prepared as follows. To a mixture of 400 mg (1.105 mmol) of ⁇ -estradiol- 3-benzyl ether and 434 mg (1.66 mmol) of NVOM-Cl in 5 ml of CH 2 Cl 2 was added 213 mg (1.65 mmol) of DIE. After stirring overnight at room temperature the mix was with H 2 O, 0.1N HCl, dried (Na 2 SO 4 ) and solvent removed. The yield was 584 mg or 90%. NVOM protected 2-deoxyadenosine was prepared by an analogous method.
  • photoreactive groups of this invention require two or more photons for photolysis.
  • Such compounds may be useful during light-directed synthesis on substrates to prevent unwanted deprotections caused by the diffraction or light around the edges of the mask (see the discussion of binary synthesis strategies above).
  • Compounds useful for this type of "multiple quantum" deprotection include those in which a first photon alters the photoactive isomerization, redox reaction, cyclization, or deprotection of a functional group on the protecting group itself. Upon exposure to the second photon, the altered protecting group is then removed.
  • a deprotection is shown below:
  • Removal of the protecting group is accomplished by irradiation to separate the reactive group and the degradation products derived from the protecting group. Not wishing to be bound by theory, it is believed that irradiation of NVOC- and MeNVOC-protected oligomers, for example, occurs by the
  • NVOC-AA 3,4-dimethoxy-6-nitrosobenzaldehyde + CO 2 + AA
  • AA represents the N-terminus of an amino acid
  • the degradation product is a
  • nitrosobenzaldehyde while the degradation product for MeNVOC is a nitrosophenyl ketone. It is believed that the product aldehyde from NVOC degradation reacts with free amines to form a Schiff base (imine) that affects the remaining polymer synthesis.
  • Preferred photoremovable protecting groups react slowly or reversibly with the oligomer on the support.
  • scavengers or other reagents are added to the reaction mixture to react with and render harmless photolysis byproducts that might otherwise react with a growing oligomer.
  • Suitable scavengers (which are often nucleophiles) will be known to those of skill in the art. Specific examples include acids, bisulfites, hydroxy-containing compounds, amines, etc.
  • t 1 2 is the time in seconds required to remove 50% of the starting amount of protecting group.
  • the photolysis was carried out in the indicated solvent with 362/364 nm-wavelength irradiation having an intensity of 10 mW/cm 2 , and the concentration of each protected phenylalanine was 0.10 mM.
  • the table shows that deprotection of NVOC-, MeNVOC-, and MeNPOC-protected phenylalanine proceeded faster than the deprotection of NBOC. Furthermore, it shows that the
  • a 0.1mM solution of each of the four 5'-protected nucleosides prepared in Example 5, MeNPoc-dT, MeNPoc-dC , MeNPoc-dG , and MeNPoc-dA was prepared in dioxane.
  • a 200 ⁇ L aliquot of one of the four deoxynucleoside solutions was irradiated with 350-450nm light at 14.5 mW/cm 2 (Oriel) in a narrow path (2mm) quartz cuvette for a given period of time x 1 .
  • a fresh aliquot of the 0.1mM solution was irradiated as before for a different time x 2 .
  • Four or five time points were collected for each MeNPoc- deoxynucleosides.
  • the deprotection rate (or photolysis half life) of a protected compound is a function of the particular
  • photosensitive protecting group as well as the amino or nucleic acid functional group being protected. It is also a function of the solvent in which photolysis is performed and neutralize photolysis decomposition products. As illustrated in the above Tables, the deprotection rate can vary
  • VLSIPSTM it is desirable that the protected compounds are deprotected at substantially the same rate to prevent unwanted side reactions between the byproducts described above and the unprotected amino acids.
  • monomers that deprotect earliest are exposed to reactive photolysis byproducts until the substrate is washed. If all oligomers on the substrate deprotect at the same rate, the time of exposure will be minimized.
  • VLSIPSTM it is also desirable to minimize the exposure time so that stray light from an illuminated region does not deprotect compounds in adjacent regions. To ensure a minimal exposure time, it is preferred to match the various photoreactive protecting groups of the invention with different amino acids (or other monomer types) so that all of the amino acids will be deprotected at substantially the same rate in a particular solvent.
  • Basis sets can be created for any class or subclass of compounds, most notably the natural or unnatural amino acids and the natural or unnatural nucleic acids.
  • “At substantially the same rate” is defined herein to mean that the half-life for deprotection of the most rapidly deprotected compound in the basis set is within about 25% of the compound with the slowest deprotection half-life in a particular solvent. Preferably, the differences between half-lives is about 10% and most preferably about 5%.
  • One preferred class of compounds for forming a basis set is a plurality of the twenty naturally-occurring amino acids.
  • Another preferred class is a subset of the D-isomers of the twenty naturally-occurring amino acids.
  • Yet another include ⁇ amino acids and amino acids whose side chains have been altered in order to adjust the stearic bulk,
  • Still another preferred class is formed by the common nucleosides adenosine, thymidine, guanosine, cytidine, and inosine in addition to their 2', or 3'-deoxy analogs and their 2', 3', or 5'-phosphates.
  • "Phosphate” is meant herein to include the phosphotriesters (-OP(OR") (OR”')), phosphoramidites (-OP(OR") (NRR'), where R-R"" are described above), as well as phosphate anions (-OP(O)O 2 H-, and -OP(O)O 2 -2 ).
  • dioxane dioxane
  • acetonitrile is acetonitrile

Abstract

Photoremovable protecting groups for the creation of large scale chemical diversity are disclosed. Orthonitrobenzyl groups containing various phenyl-ring substituents, benzylic carbon substituents, and benzylic carbon extensions protect nucleoside and amino acid functional groups, including hydroxyl, amino, and carboxyl groups. Phenyl-ring substituents include cyclic orthoesters, acetals, ketals, substituted amines, and cyclic ethers.

Description

NOVEL PHOTOREACTIVE PROTECTING GROUPS
FIELD OF THE INVENTION
The present invention relates to the field of photosensitive protecting groups. More specifically, the invention provides ortho-nitrobenzylic groups for protecting carboxylic, amino, thiol, hydroxyl, and other moieties.
BACKGROUND OF THE INVENTION
Chemical protecting groups are used during synthesis reactions to temporarily protect certain functional groups on a compound against undesired reactions. When a reaction sequence is complete, and protection is no longer necessary, the protective group is removed to restore the protected functional group to its natural activity. Protective groups are removed by various procedures such as exposure to acidic or basic conditions. One class of protective groups, the photolabile or photosensitive protecting groups, are removed by exposure to electromagnetic radiation of a prescribed wavelength.
The properties and uses of some photolabile protecting compounds have been reviewed. See, McCray et al., Ann. Rev, of Biophys. and Biophys. Chem. (1989) 18 :239-270, which is incorporated herein by reference. A particularly useful class of photoremovable protecting groups is the ortho
nitrobenzyl derivatives. When exposed to radiation (typically near ultra-violet), these compounds undergo an internal rearrangement that results in deprotection of the protected compound. In the process, the protected compound splits off an ortho-nitroso aldehyde or ketone, leaving a free hydroxyl, amino, or other previously protected group. See Cameron and Frechet, J. Am. Chem. Soc. (1991) 113 :4303-4313. incorporated herein by reference for all purposes.
Examples of known ortho-nitrobenzyl photoremovable protecting groups are described in, for example, Patchornik,
J. Amer. Chem. Soc. (1970) 92:6333, Amit et al., J. Org. Chem. (1974) 39:192, Pillai, Synthesis (Jan. 1980) 1-26, and E.P. Application 046 083, all of which are incorporated herein by reference. Known members of this class include ortho- nitrobenzyl and ortho-nitrobenzyloxycarbonyl protected
compounds.
Figure imgf000004_0001
Figure imgf000004_0002
Patchornik and others have reported some phenyl-substituted ortho-nitrobenzyl groups for protecting amino acid functional groups. For example, the phenyl ring has been substituted methoxy groups at the 3 and 4 positions (nitroveratryl). In other known compounds, one of the benzylic hydrogens has been replaced with a methyl or phenyl (substituted and
unsubstituted) group.
Ortho-nitrobenzyl protected compounds have now found use in a variety of fields. For example, they have proven useful in Very Large Scale Immobilized Polymer Synthesis
(VLSIPS™) which provides techniques of forming vast arrays of peptides and other polymer sequences using, for example, light-directed synthesis techniques. The details of VLSIPS™ are disclosed in Pirrung et al., U.S. Patent No. 5,143,854
(see also PCT Application No. WO90/15070), Fodor et al., PCT Publication No. WO92/10092, and Fodor et al., Science (1991) 251:767-777. all incorporated herein by reference for all purposes.
Some preferred photosensitive groups for protecting amino acids and nucleosides during VLSIPS™ have been described in PCT Publication No. W092/10092. These include
nitroveratryloxycarbonyl (NVOC), nitropiperonyloxycarbonyl (NPOC), alpha-methyl-nitroveratryloxycarbonyl (MeNVOC), alpha- methyl-nitropiperonyl (MeNPOC), and the benzylic form of each of these (i.e. NV, NP, MeNV, and MeNP).
Figure imgf000005_0001
In addition to VLSIPS™, ortho-nitrobenzyl photolabile protecting groups have been proposed for use in photolithography for electronic device fabrication (see e.g. Reichmanis, et al., J. Polymer Sc. Polymer Chem. Ed. (1985) 23:1-8, incorporated herein by reference for all purposes), and monitoring transport of various molecules and ions in biological milieus (see e.g. Kaplan et al., PNAS USA (1987) 85:6571-6575, incorporated herein by reference for all
purposes).
In VLSIPS™, a "basis set" of protected monomers is used to synthesize a diverse collection of polymers. The basis set is, for example, the twenty naturally occurring amino acids having protected alpha-amino groups. It is desirable to have each member of the basis set photolyze at approximately the same rate. Unfortunately, this is often not possible when a single protecting group (e.g. ortho- nitrobenzyloxycarbonyl) is used to protect each member of the basis set. For example, when two different amino acids protected with nitrobenzyloxycarbonyl groups are exposed to radiation of the same wavelength and intensity, they may be deprotected at substantially different rates. Thus, it is desirable to develop methods that "equalize" the photolysis rates of the basis set members.
Although some ortho-nitrobenzyl photoremovable protecting groups have proven useful, additional
photoremovable protecting groups having various structures and photolysis rates are desirable. SUMMARY OF THE INVENTION
The present invention provides new ortho-nitrobenzyl photosensitive protecting groups. These groups can be coupled to amino, carboxyl, hydroxyl, thiol and other moieties of compounds protected during certain chemical reactions. For example, the photosensitive groups of this invention can be used to protect functional groups of nucleosides, amino acids, or saccharides from unwanted side reactions during polymer synthesis.
According to one aspect, the invention provides improved photoremovable protecting groups having the formula:
Figure imgf000006_0001
In this structure, n is 0 or 1; A is -C(O)-, -(CQ1Q2)-, or -C(S)-; R1 , Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; and R2 is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or oligonucleotide. R3-R6 are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl. R3-R6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic, or ether groups.
In preferred novel embodiments, when n is 0 or A is
-C(O)- (i) R4 and R5 are not both methoxy when R1, R3, and R6 are hydrogen; (ii) R1 is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R3-R6 are hydrogen simultaneously; and (iii) R1 is not 2-nitrophenyl or 3,4-dimethoxy-6-nitrophenyl when R3 and R6 are hydrogen and R4 and R5 are both either hydrogen or methoxy.
The amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone" groups (i.e. the amine and carboxyl groups that form the peptide bond). Side chain functional groups found on natural amino acids include amines, carboxyl groups, thiols, hydroxy groups, imidazoles, amides, indole groups, etc. The nucleoside, nucleoside analog, and oligonucleotide functional groups include the 2', 3', and 5' ribose hydroxy groups. In addition, the purine and pyrimidine bases of nucleosides can be protected by the above protecting groups. For example, R2 in the above compound structure can represent base exocyclic amine groups in some embodiments.
A particularly preferred class of photoremovable protecting groups has the formula:
Figure imgf000007_0001
Here, R2 is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an imidazole, an amide, a hydroxy group, or the like. Otherwise, R1, Q1 , Q2, and R3-R6 are the same as defined in the previous structure. In preferred embodiments, the functional group R2 is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an oligonucleotide as described in connection with the above embodiment. Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho- nitrobenzyloxymethyl groups such as ortho-nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides). Other active groups that can replace the halo group include
hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.
The invention also includes basis sets of protected natural or unnatural amino or nucleic acids in which a
plurality of amino or nucleic acids are protected by
photoremovable protecting groups of the invention such that all of the protected amino or nucleic acids are deprotected at substantially the same rate in a particular solvent.
A further understanding of the nature and advantages of the inventions herein may be realized by reference to the remaining portions of the specification and the attached drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 schematically illustrates various reaction paths that can be employed to produce alcohols of the ortho-nitrobenzyl protecting groups of this invention;
Fig. 2 schematically illustrates reaction paths to various protected compounds of this invention;
Fig. 3 schematically illustrates reaction paths to various benzyloxycarbonyl protected compounds of this
invention;
Fig. 4 schematically illustrates reaction paths to various benzyl protected compounds of this invention; and Fig. 5 schematically illustrates reaction paths to various benzyloxymethyl protected compounds of this invention.
Fig. 6 presents various compounds for which photolysis data is provided in Table V.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
CONTENTS
I. Definitions
II. Function of Protecting Groups III. Preferred Ortho-nitrobenzyl Protecting Groups
IV. Choice of Sidechain Protecting Groups
V. Synthesis of Protected Compounds
1. Example
2. Example
3. Example
4. Example
5. Example
6. ExaMple
7. Example
VI . Multiple-quantum Protecting Groups VII. Photolysis of Protected Group Compounds
VIII. Basis Sets of Photoprotected Amino or Nucleic Acids
IX. Conclusion
1. Definitions
Certain terms used herein are intended to have the following general definitions A. Monomer: A member of the set of small molecules which are or can be joined together to form a polymer.
Frequently, monomers have at least two different reaction sites (e.g. the amino and carboxyl groups of amino acids). The set of monomers includes but is not restricted to, for example, the set of common L-amino acids, the set of D-amino acids, the set of synthetic and/or natural amino acids, the set of nucleotides and the set of pentoses and hexoses. The particular ordering of monomers within a polymer is referred to herein as the "sequence" of the polymer. The invention is described herein primarily with regard to the preparation of molecules containing sequences, of monomers such as amino acids, but could readily be applied in the preparation of other polymers. Such polymers include, for example, both linear and cyclic polymers of nucleic acids, polysaccharides, phospholipids, and peptides having either α- , β-, or ω-amino acids, heteropolymers in which a known drug is covalently bound to any of the above, polynucleotides, polyurethanes, polyesters, polycarbonates, polyureas, polyamides,
polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, polyacetates, or other polymers which will be apparent to those of skill in the art. Methods of cyclization and polymer reversal of polymers are disclosed in copending application Serial No. 796,727 (Attorney Docket No. 11509-51), filed on the same date as the present application, entitled "POLYMER REVERSAL ON SOLID SURFACES," incorporated herein by reference for all purposes.
B. Functional Group: A group or moiety on a chemical compound that in some environments imparts certain chemical properties to the compound. More specifically, "functional group" refers to groups that are capable of undergoing
reaction such as oxidation or reduction under certain
conditions. As used in the context of this invention, functional group most often refers to a group on a monomer or polymer that can be protected by a photolabile or other group. Typically, functional groups include the reactive sites on a monomer that participate in coupling reactions to produce a polymer. For example, amino acid functional groups include carboxyl and amino groups; nucleic acid functional groups include 2', 3', and 5' hydroxyl groups. Other common
functional groups encountered in the context of this invention include side chain groups (thiol, imidazole, indole, etc.) on amino acids; purine/pyrimidine groups (e.g. exocyclic amines) on nucleic acids; amine or carboxyl groups on linkers; and hydroxyl and amine groups on carbohydrates or lipids.
C. Protecting group: A material which is chemically bound to a reactant functional group and which may be removed upon selective exposure to an activator such as
electromagnetic radiation. A protecting group prevents the protected functional group from undergoing undesired side reactions. For instance, the amino group of glycine may be coupled to a protecting group to prevent the glycine amino group from reacting during a coupling reaction between the glycine carboxylic terminus and the amino terminus of a growing peptide. Examples of photosensitive protecting groups include nitropiperonyl, nitroveratryl, and nitrobenzyl groups. Other protective groups sensitive to acid or base include t-butyloxycarbonyl or fluorenylmethoxycarbonyl.
D. Basis Set: A set of protected reactants such as monomers used in, for example, a synthesis step in the
formation of a polymer array. For example, the 20 naturally occurring amino acids form one basis set and the four
naturally occurring deoxyribonucleic acids form another basis set. As a further example, the dimers of the 20 naturally occurring L-amino acids form a basis set of 400 monomers for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of a polymer. Furthermore, each of the sets may include protected members which are modified after synthesis. E. Peptide: A polymer in which the constituent monomers are amino acids joined together through amide bonds. Peptides are alternatively referred to as polypeptides. In the context of this specification it should be appreciated that the peptide may include the L-optical or D-optical isomers of α- , β- , or ω-amino acids. In addition, the peptide may include amino acids having unnatural side chains or other deviations from the naturally occurring amino acids. Such "peptides" have been the subject of much study recently, and are
discussed by Fauchère, J-L, in Advances in Drug Design, Vol. 15, Testa ed., (1986), which is incorporated herein by reference for all purposes. Peptides are two or more amino acid monomers long, and often more than 20 amino acid monomers long. Standard abbreviations for amino acids are used (e.g., P for proline). These abbreviations are included in Stryer,
Biochemistry, Third Ed., 1988, which is incorporated herein by reference for all purposes.
F. Radiation: Energy which may be selectively applied including energy having a wavelength of between 10-14 and 104 meters including, for example, electron beam radiation, gamma radiation, x-ray radiation, ultra-violet radiation, visible light, infrared radiation, microwave radiation, and radio waves. "Irradiation" refers to the application of radiation to a material. The photosensitive protecting groups of this invention are typically photolyzed by electromagnetic
radiation in the ultraviolet or visible regions of the spectrum.
Abbreviations : The following frequently used abbreviations are intended to have the following meanings:
BOC: t-butyloxycarbonyl.
BOP: benzotriazol-1-yloxytris-(dimethylamino) phosphonium hexafluorophosphate.
DCC: dicyclohexylcarbodiimide.
DCM: dichloromethane; methylene chloride.
DDZ : dimethoxydimethylbenzyloxy.
DIEA: N,N-diisopropylethylamine.
DMAP: 4-dimethylaminopyridine.
DMF: dimethyl formamide.
DMT: dimethoxytrityl.
DTT: dithiothreitol
FMOC: fluorenylmethyloxycarbonyl.
HBTU: 2-(1H-benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate.
HOBT: 1-hydroxybenzotriazole.
NBOC: 2-nitrobenzyloxycarbonyl.
NMP: N-methylpyrrolidone.
NPOC : 6-nitropiperonyloxycarbony1.
NV: 6-nitroveratryl.
NVOC: 6-nitroveratryloxycarbonyl.
TFA: trifluoracetic acid.
THF: tetrahydrofuran.
II. Function of Protecting Groups
Protecting groups of the present invention can be used in conjunction with solid phase oligomer syntheses, such as peptide syntheses using natural or unnatural amino acids, nucleotide syntheses using deoxyribonucleic and ribonucleic acids, oligosaccharide syntheses, and the like. In a
particularly preferred embodiment, the protecting groups of this invention are used to synthesize an array of polymers according to a VLSIPS™ process. Such processes employ a substrate containing oligomers or other materials protected with photosensitive groups. Selected regions of the substrate are irradiated to create regions having reactive moieties that are essentially free of side products resulting from the protecting group.
The protecting groups serve two purposes: (1) protecting the substrate surface from unwanted reactions, and (2) blocking a reactive functional group of the monomer to prevent self-polymerization or other reaction. For instance, attachment of a protecting group to the amino terminus of an activated amino acid, such as an
N-hydroxysuccinimide-activated ester of the amino acid, prevents the amino terminus of one monomer from reacting with the activated ester portion of another during peptide
synthesis. Alternatively, the protecting group may be
attached to the carboxyl group of an amino acid to prevent reaction at this site. Most protecting groups can be attached to either the amino or the carboxyl group of an amino acid, and the nature of the chemical synthesis will dictate which reactive groups will require a protecting group. Analogously, attachment of a protecting group to the 5 '-hydroxyl group of a nucleoside or nucleoside derivative during oligonucleotide synthesis using for example, phosphoramidite coupling
chemistry, prevents the 5'-hydroxyl of one nucleoside from reacting with the 3'-activated phosphate-triester of another . In other embodiments, the photolabile protecting group may be attached to the 2'- or 3'-hydroxyl group of a nucleoside.
In alternative embodiments, the photosensitive protecting groups of this invention can be employed to protect side chain functionalities of amino acids during peptide syntheses employing conventional acid or base labile
protecting groups such as BOC (t-butyloxycarbonyl) or FMOC (fluorenylmethoxycarbonyl) on the alpha backbone amine. In some preferred embodiments, the photolabile protecting groups protect any one or a combination of the following groups: the thiol group of cysteine; the basic residues (amine and
imidazole groups) of lysine, arginine, and histidine; the acidic carboxy groups of aspartic and glutamic acids; the hydroxy groups of serine, threonine, and tyrosine; and the heterocyclic indole ring of tryptophan. Of course, other amino acid side chains such as those occurring on non-natural amino acids can likewise be protected by the photosensitive groups of the present invention. Further, the purine and pyrimidine bases (and particularly the exocyclic amine groups) of nucleoside and nucleoside derivatives likewise can be protected with the photosensitive groups of this invention.
Regardless of the specific type of compound being synthesized, protecting groups are employed to protect a moiety on a molecule from reacting with another reagent.
Typically, the reaction to be blocked will be the formation of a covalent bond such as a peptide bond between two amine acid molecules. However, the protecting groups of this invention can also block non-covalent reactions. For example, the photosensitive protecting groups of this invention can be used to "cage" (i.e. provide protection based upon steric
hindrance) biotin or a biotin analog, thus preventing binding with avidin. Useful applications of this strategy are
described in Barrett et al., PCT Publication No. WO 91/07087, incorporated herein by reference for all purposes.
Protecting groups of the present invention preferably have the following general characteristics: they prevent selected reagents from modifying the group to which they are attached; they are stable (that is, they remain attached to the molecule) to the synthesis reaction
conditions; they are removable under conditions that do not adversely affect the structure to which they are attached; and once removed, they do not react appreciably with the surface or surface-bound oligomer. In some embodiments, liberated byproducts of the photolysis reaction can be rendered
unreactive toward the growing oligomer by adding a reagent that specifically reacts with the byproduct. The selection of a suitable protecting group will depend, of course, on the chemical nature of the monomer unit and oligomer, as well as the specific reagents they are to protect against.
The removal rate of the protecting groups depends on the wavelength and intensity of the incident radiation, as well as the physical and chemical properties of the protecting group itself. Preferred protecting groups are removed at a faster rate and with a lower intensity of radiation. For example, at a given set of conditions, MeNVOC and MeNPOC are photolytically removed from the N-terminus of a peptide chain faster than their counterparts, NVOC and NPOC. Therefore for most applications, MeNVOC and MeNPOC are preferred over NVOC and NPOC.
In many embodiments, the photosensitive protecting groups will be removable by radiation in the ultraviolet (UV) or visible portion of the electromagnetic spectrum. In preferred embodiments, the photosensitive protecting groups are sensitive to an electromagnetic spectral region that does not significantly overlap the absorption band of the monomers or polymers being protected. Higher energy radiation (i.e. low wavelength radiation) is generally more likely to damage monomers such as amino acids and nucleosides. For example, the four naturally occurring bases in deoxynucleosides absorb 250-280 nm light very strongly. In addition, short wave ultra-violet light is known to photo-destruct DNA formation of thymine dimers. Thus, in most preferred embodiments, the protecting groups will be removable by radiation in the near UV or visible portion of the spectrum. Of course, each collection of monomers (basis sets) will have its own
characteristic absorption bands which can be avoided by choosing protecting groups that are sensitive to wavelengths well removed from the absorption band.
Ill. Preferred Ortho-nitrobenzyl Protecting Groups
Preferred photochemical protecting groups of the present invention have the general formula:
Figure imgf000017_0001
In this structure, n is 0 or 1; A is -C(O)-, - (CQ1Q2 ) - , or -C(S)-; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; and R2 is a functional group of a molecule such as a natural or unnatural amino acid or peptide, a nucleoside, nucleoside analog, or oligonucleotide. R3-R6 are selected independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy
(e.g., CH3 C(O)O-) nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl. R3-R6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, fused aromatic (which together with the first benzene ring forms a derivative of naphthalene, quinoline, anthracene, etc.), or ether groups.
In preferred novel embodiments, when n is 0 or A is -C(O)- (i) R4 and R5 are not both methoxy when R1, R3, and R6 are hydrogen; and (ii) R1 is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R3-R6 are hydrogen simultaneously.
Further, compounds having hydroxyl substituents on the phenyl ring, and/or alkoxy or aryloxy substituents on the benzyl carbon may be too reactive for certain applications, and therefore are generally less preferred.
The amino acid (and peptide) functional groups protected as described include side chain groups as well as "backbone" groups (i.e. the amine and carboxyl groups that form the peptide bond). Those of skill in the art will recognize that a variety of other functional groups can be protected by the above photosensitive groups. Specific examples include hydroxy and amino groups on saccharides, hydroxy groups on steroids, and amino groups attached to linkers on a substrate surface (see PCT Publication No.
WO92/10092, previously incorporated by reference). In
addition, certain molecules can be "caged" to provide steric protection against binding. For example, biotin and biotin analogs can be caged with the above photosensitive groups to prevent binding with avidin. The protecting group can be coupled through the biotin urea groups.
Although carboxyl, amino, and hydroxy groups of amino acids and nucleosides can, in principle, be protected with either benzyl or benzyloxycarbonyl protecting groups
(i.e. n=0 and 1, and ignoring the possibility that A is -CH2- or -C(S)-), certain couplings are preferred. Specifically, amino acid carboxyl groups preferably are protected with benzyl form photosensitive groups (n=0), while amino acid amine groups and nucleoside hydroxy groups preferably are protected with benzyoxycarbonyl form of the photosensitive groups (n=1).
When a primary amine group (e.g. those found on most natural amino acids) is protected by a benzylic group (n=0) of the above structure, the amine nitrogen must be further protected for some applications,. For example if the amine is not further protected during peptide synthesis using VLSIPS™, the free amine hydrogen may react with other amino acids activated with coupling reagents such as BOP. If, however, a second protecting is employed, no free hydrogen atoms are present on the amine to act as a reaction sites. Preferably, the additional protection is provided by C1-C6 alkyl, aryl, acyl, or benzyl groups. More preferably, the additional protective group is methyl or acetyl. Such compounds are readily synthesized by methods well-known in the chemical arts. For example, following the coupling of the above-described photoprotective group to an amino acid (made, e.g., by using an activated benzyl derivative of the photoprotective group), the photoprotected amino acid may be reacted with a reagent known to methylate amino acid nitrogen moieties, such as methyl iodide, to form the desired protected amino acid.
A particularly preferred class of protecting groups is the 6-nitrobenzyloxymethyl groups which have exhibited very rapid photolysis rates and have the general formula: Here, R2 is a molecule functional group that may be one of the following: an amine, a carboxyl group, a thiol, an imidazole, an amide, a hydroxy group, and the like.
Otherwise, R1, Q1, Q2 and R3-R6 are the same as defined in the above structure. In preferred embodiments, the functional group R2 is found on a natural or unnatural amino acid or peptide, a nucleoside, a nucleoside analog, or an
oligonucleotide. Both side chain and backbone groups may be protected. As in the above general embodiment, the
photosensitive groups can also be used to protect functional groups found on a variety of compounds such as carbohydrates, lipids, biotin analogs, linker molecules, etc.
Preferred reactive compounds employed to make protected compounds having the above structure include activated ortho-nitrobenzyloxymethyl compounds such as ortho-nitrobenzyloxymethyl halides (e.g. ortho-nitrobenzyloxymethyl chlorides). Other active groups that can replace the halo group include hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like.
Another class of preferred protecting groups includes compounds in which R5 and R6 are both alkyloxy groups or together form a cyclic bridge acetal or ketal. In
particularly preferred embodiments R1 is hydrogen or methyl and R3 and R4 are hydrogen. In alternative preferred
embodiments, R4 and R5 are alkoxy groups or together form the cyclic acetal or ketal. Preferably, R3 and R6 will then be hydrogen. Such compounds are made by standard methods such as those described in Greene, Protective Groups in Organic
Synthesis, Wiley 1981, which is incorporated herein by reference for all purposes. One example is where R4 and R5 or R5 and R6 form a methylene acetal or an acetonide: (-O-CH2-O- , or -O-C(CH3)2-O-). In the structures below, Y1 and Y2 are independently C1-C8 alkyl groups, or fused to form a cyclic bridge acetal or ketal.
Figure imgf000021_0003
Figure imgf000021_0004
In addition to acetals and ketals, the
photoprotecting groups of the invention may also include compounds where adjacent benzene ring substituents form a ring having the formula -O-CRR'-O- or -O-CRR'-CR"R'''-O-, where R, R', R", and R"' can independently be hydrogen, C1-C8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl carboxylic acid ester (i.e. -C(O)O-alkyl), or carbonyl. Specific examples include cyclic orthoesters (-O-CRR'-O-, where R and R' are alkoxy, aryloxy, or carboxy) or cyclic ethers (-O-CRR'-CR"R"'-O- where R, R', R", and R"" are selected from the group hydrogen, alkyl, aryl, benzyl, alkoxy, aryloxy, or carboxy). In addition, R and R' together or R" and R'" together may be a carbonyl oxygen (e.g. forming the following bridges: -O-C(O)CH2-O- or -O-CH2C(O)-O-). These compounds are readily synthesized by known methods (see, e.g., Greene). A preferred cyclic ether has R, R', R", and R" hydrogen (i.e., -O-CH2-CH2-O-). Preferred substitution patterns are those at the R4 and R5, and R5 and R6 positions:
Figure imgf000021_0001
Figure imgf000021_0002
Still other preferred compounds are those where R5 and R6 are methoxy. In a specific preferred embodiment R3 is dimethylamino and R4 is hydrogen:
Figure imgf000022_0002
Figure imgf000022_0001
Yet still other preferred compounds are formed when R4 is methoxy and R5 is dimethylamino, or, conversely, where R4 is dimethylamino and R5 is methoxy:
Figure imgf000022_0003
Figure imgf000022_0004
As noted, the photoremovable protecting groups of the present invention can be attached to an activated ester of a natural or unnatural amino acid or peptide at the amino terminus. For use in VLSIPS™ or other peptide synthesis techniques, an amine protected amino acid may have the following structure:
Figure imgf000022_0005
where R is the side chain of a natural or unnatural amino acid, X is a photoremovable protecting group according to this invention, and Y is an activated carboxylic acid derivative. The activated ester, Y, is preferably a reactive derivative having a high coupling efficiency, such as an acyl halide, mixed anhydride, N-hydroxysuccinimide ester, perfluorophenyl ester, or urethane protected acid, and the like. Other activated esters and reaction conditions are well known (See Atherton et al., "Solid Phase Peptide Synthesis" 1989, IRL Press, London, incorporated herein by reference for all purposes).
As noted, the photoreactive protecting groups of the present invention are useful for protecting both natural and unnatural nucleosides or nucleotides. More specifically, the 2', 3', and/or 5' hydroxyl functions of such compounds. Such compounds can therefore have the general structure:
Figure imgf000023_0001
X may be oxygen or sulphur. R1 is a purine or pyrimidine base or analog thereof (for example, adenine, cytosine, thymine, guanine, uracil, 6-ketoadenine, or an analog thereof). R2-R4 may be one of the commonly used protecting groups (see below), or a photoreactive group as described above. Typically, one position will be protected with a photoreactive protecting group while the other
position (s) will be (a) protected or substituted with one of the commonly used protecting groups, or (b) activated with one of the commonly reactive groups. However, more than one position may be protected with a photoreactive protecting group of the invention.
With respect to R2, R3, and R4, these may be hydrogen, hydroxy, an oligonucleotide or one of the commonly used protecting groups: alkoxy, tetrahydropyranyl, β-benzoylpropionyl, acetyl, or silyl. They can also be -NRR', -OP (O) (OR") (OR"'), -OP(O)O2 -2, -OP(O)O2H-, -OP (O) (OR") (NRR'), or -OP (OR") (NRR' ) (i.e. phosphoramidite) where R and R' are independently selected from the group consisting of C 1-C3 alkyl, aryl, benzyl, or acetyl, and R" and R'" are selected independently from the group consisting of C1-C3 alkyl, aryl, benzyl, or C1-C3 cyanoalkyl. R2 or R3 also together form a cyclic acetal, ketal, orthoester, or ether. In some preferred embodiments, R4 can be triphenylmethyl, di-p-methoxytrityl, dimethylpropanoyl. These compounds and their methods of synthesis are well-known in the art (see, e.g., Fuhrhop and Penzlin, Organic Synthesis Concepts, Methods, and Starting Materials, Verlag Chemie 1983, or Gait, Oligonucleotide
Synthesis a Practical Approach, IRL Press 1984, both of which are incorporated herein by reference for all purposes).
An especially preferred photoreactive protecting group for use with nucleosides and nucleotides has the
formula:
Figure imgf000024_0001
IV. Choice of Sidechain Protecting Groups
It will be appreciated by those skilled in the art that functional groups other than those directly involved in coupling during polymer synthesis may have to be protected. It will also be appreciated that such protecting groups should not be removable under coupling conditions. Thus, for
example, a desired polypeptide will not be contaminated as a result of cross reactions between sidechains which have inadvertently become unprotected and other monomers or the sidechains of other amino acids. Since the removal of the photoreactive protecting groups of the present invention typically creates an acidic environment, care must be taken to choose sidechain protecting groups which are moderately acid stable.
Herein, the term "acid stable" means that the sidechain protecting group in question is not removed in appreciable amounts under conditions where the effective pH is less than about 7. Determining stability will depend on the particular amino acid sidechain as well as the sidechain protecting group which is chosen. For lysine, it has been found that ∈-amino moieties protected with BOC (t- butyloxycarbonyl) tend to degrade over time in 5mM sulfuric acid. Thus, a protecting group for the ∈-amino group of this amino acid should be less acid labile than BOC. Generally, trityl protecting groups are also too sensitive to protect ∈- amino groups. One preferred protecting group for the ∈-amino acid of lysine is dimethoxybenzyloxycarbonyl. Another preferred protecting group is FMOC (9- fluorenylmethyloxycarbonyl). These groups are well-known in the art as are the techniques for forming the appropriately protected lysine amino groups.
Although trityl is not preferred with respect to lysine, trityl is stable enough to be used with cystine, asparagine, and glutamine. Generally, however, asparagine and glutamine do not require protection. With respect to
histidine, protection of the imidazole nitrogen is needed during addition to the polymer in order to prevent self-condensation and racemization. However, once the histidine is bound to the substrate, protection is no longer required. FMOC and trityl are examples of suitable protecting groups for the imidazole nitrogen of histidine. It will be appreciated by those of skill in the art that various other protecting groups may be used depending upon the application. V. Synthesis of Protected Compounds
The protected compounds of the present invention often can be prepared via a process employing a benzyl alcohol of the protecting group. Various synthetic routes to some useful benzyl alcohols are shown in Fig. 1. In the displayed benzyl alcohols, R, R', and R" represent hydrogen, methyl, C1 to C8 alkyl, and other groups in accordance with the
structures presented above. Each arrow shown in Fig. 1 depicts a different reaction pathway, employing different reagents. Typical reagents for suitable use in the various reaction paths are as follows: (a) RC(O)Cl and aluminum chloride; (b) DMF and POCl3; (c) R'I or R"I or MeBr2 or EtBr2 and potassium carbonate; (d) concentrated nitric acid or other nitration agent; (e) NaBH4 ; and (f) RMgBr or RLi. Fig. 2 displays reaction paths for making various protected compounds from benzyl alcohols. In the structures shown, X denotes substitution on the phenyl ring, R denotes substitution on the benzylic hydrogen, and Z denotes an oxygen or sulfur atom. In reaction path (a), the alcohol is reacted with ClC(Z)OR' and triethylamine or ClC(Z)SR' and
triethylamine to form the protected thiol or alcohol shown. The reactants (other than the benzyl alcohol of the protecting group) and the products for the various other reactions are as follows: (b) reaction with ClCH2OR and triethylamine produces a protected alcohol; (c) reaction with Z=C=NR' or ClC(Z)NR'R" produces a protected amine; (d) reaction with ClCH2SR' and triethylamine produces a protected thiol; and (e) reaction with R'C(O)OH, DCC, and DMAP or with R'C(O)Cl and
triethylamine produces a protected carboxylic acid.
Fig. 3 schematically depicts some general reactions that can be employed to produce benzyloxycarbonyl (and
thiocarbonyl) protected functional groups. A shown, a benzyl alcohol is reacted through a chloroformate intermediate to produce the desired compounds. In Fig. 3, X denotes
substitution on the phenyl ring, R denotes substitution on the benzylic carbon, and Z denotes an oxygen or sulfur atom.
Typical reagents employed in the noted routes are as follows: (a) C(O)Cl2 or C(S)Cl2; (b) R'OH and triethyl amine; (c) R'SH and triethyl amine; (d) R'R"NH; and (e) H2NC(O)R'
In one specific embodiment, the appropriate benzyl alcohol can be reacted with phosgene or other agent to produce an activated benzyloxycarbonyl derivative of the protecting group. These compounds are then used to produce the above-described benzyloxycarbonyl-protected groups (e.g. n is 1 and A is -C(O)- in the above generic structures). These groups are preferably coupled to the amino nitrogen of a natural or unnatural amino acid; or the 2', 3', or 5' oxygen of a natural or unnatural nucleoside or nucleotide using standard methods, for example, reacting the amino group of an amino acid or a ribose hydroxyl moiety of a nucleoside with the desired activated benzyloxycarbonyl derivative. Examples of such activated protecting groups have the general formula: where X is halogen, mixed anhydride, phenoxy, p-nitrophenoxy, N-hydroxysuccinimide, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like. R1 is hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy. R3-R6 are selected
independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of C1-C8 alkyl, aryl, or benzyl. R3-R6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups. Compounds falling within this formula are readily formed using known standard techniques such as those described in March, Advanced Organic Chemistry. 3rd ed., Wiley 1985, or Carey and Sundberg, Advanced Organic Chemistry Part B: Reactions and Synthesis, 2nd ed., Plenum 1984, both of which are incorporated herein by reference for all purposes. It should be noted that the corresponding benzyloxythiocarbonyl (employing the -C(S)-group in place of -C(O)-) can be prepared by analogous
methods.
Fig. 4 schematically depicts various reaction pathways from a benzyl alcohol starting material to benzyl-protected compounds via an activated benzyl intermediate. In the compounds shown, X denotes substitution on the phenyl ring and R denotes substitution on the benzylic carbon. Typical reagents used in the various reaction steps are as follows: (a) SOCl2; (b) R'OH and triethylamine; (c) R'SH and
triethylamine; (d) R"R'NH; and (e) H2NC(O)R' and
triethylamine; (f) R'C(O)OH and triethylamine.
In a specific embodiment, the carboxy terminus of an amino acid protected with a benzylic photoactivatable group can be formed by esterifying the carboxy group with an activated benzyl derivative of the protecting group.
Likewise, the amino terminus of an amino acid or a hydroxy group of a nucleoside (or nucleoside derivative) protected with a benzylic group can be formed by alkylating the amino or hydroxy group with an activated benzyl derivative of the protecting group. As noted above, benzyoxycarbonyl groups are more preferred for protecting amine groups of amino acids and hydroxy groups of nucleosides. Further, if a benzylic form of a photoreactive protecting group is used to protect the amino nitrogen of an amino acid, that nitrogen may have to be further protected with an alkyl, aryl, benzyl, or acyl group.
Examples of activated benzyl derivatives have the general formula:
Figure imgf000028_0001
where X is halogen, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido, and the like. R1 is hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy. R3-RC are selected
independently from among the groups hydrogen, alkoxy, aryloxy, benzyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, or a group having the formula -NR'R" where R' and R" are selected independently from the group consisting of C1-C8 alkyl, aryl, or benzyl. R3-R6 may also be a cyclic bridge between adjacent substituents. Exemplary bridges include acetal, ketal, orthoester, thioester, phenyl, or ether groups.
Another method for generating compounds protected with benzylic protecting groups is to react the benzylic alcohol derivative of the protecting group with an activated ester of the compound to be protected. For example, to protect the carboxyl terminus of an amino acid, an activated ester of the amino acid is reacted with the alcohol derivative of the protecting group. Examples of activated esters suitable for such uses include halo-formate, mixed anhydride, imidazolyl formate, acyl halide, and also include formation of the activated ester in situ the use of common reagents such as DCC and the like. See Atherton et al. (previously
incorporated by reference) for other examples of activated esters.
To protect the 5'-hydroxyl group of a nucleic acid with a benzylic protecting group, a derivative having a 5'- activated carbon is reacted with the alcohol derivative of the protecting group. Examples of nucleotides having activating groups attached to the 5'-hydroxyl group have the general formula:
Figure imgf000029_0001
where Y is a halogen atom, a tosyl, mesyl, trifluoromethyl, azido, or diazo group, and the like. Similarly, the 2'- and 3'- hydroxyl moieties of nucleotide, nucleosides and
derivatives thereof may be protected.
Benzyloxymethyl-protected compounds can be synthesized using standard techniques known in the chemical arts. For example, 6-nitroveratryloxymethyl chloride (NV0MCl) is readily made from 6-nitroveratryl alcohol upon exposure to gaseous HCI and paraformaldehyde following the methods
described in Organic Syntheses, Vol. 6, pp. 101-103 (1988), which is incorporated herein by reference. Reaction of NVOMCl with the 5'-hydroxyl group of a nucleoside to form the
protected 5'-NVOM nucleoside is readily performed using techniques such as those described in Greene, or by Corey, et al., in Tetrahedron Letters, No. 11, pp. 809-812 (1976), which is incorporated herein by reference. For example, the hydroxy group to be protected is reacted with the activated form of the benzyloxymethyl protecting group in the presence of a tertiary amine to form the protected compound, a
benzyloxymethylether. Primary and secondary amines as well as carboxylic acids can protected in an analogous manner. Examples of reactions to produce benzyloxymethyl- protected compounds via a chloromethyl ether intermediate are schematically presented in Fig. 5. As in Figs. 2 to 4, X denotes substitution on the phenyl ring and R denotes substitution on the benzylic carbon. Typical reagents that can be used in the various reaction steps are as follows: (a) CH2O and HCl; (b) R'OH and triethyl amine; (c) R'SH and triethylamine; (d) R"R'NH; (e) H2NC(O)R' and triethylamine; and (f) R'C(O)OH and triethylamine.
If excess activated benzyloxymethyl compound is reacted with a primary amine to be protected, an amine having two protecting groups will be formed. Such protected
compounds require two photons to be completely deprotected.
Figure imgf000030_0001
Figure imgf000030_0002
A solution of 2,3-dihydroxy benzaldehyde (1.38 g, 0.01 mol), KF (2.90 g, 0.05 mol), and CH2Br2 (1.91 g, 0.011 mol) in 30 mL of DMF was heated to 110-120°C for 2 hours. The reaction mixture was cooled to room temperature and
partitioned between ether and water. The organic phase was washed (sat. NaCl), dried (MgSO4), and evaporated to give 1.46 g of a light brown oil. Chromatography on silica gel with 10% EtOAc in hexanes afforded 1.14 g of
2,3-methylenedioxybenzaldehyde as a light yellow solid.
The benzaldehyde compound was dissolved in 40 mL of 70% HN03 cooled to 0°C. After stirring for 30 min., the reaction mixture was poured into cold water and extracted with EtOAc. The organic phase was washed (sat NaHCO3), dried
(MgSO4), and evaporated to give 1.2 g of a yellow solid which was used without further purification. The solid was taken up in 25 mL of anhydrous THF and treated with NaBH4 (466 mg, 0.12 mol) at room temperature for 1 hour. The reaction mixture was partitioned between sat. NH4Cl and EtOAc. The organic phase was dried (MgSO4) and evaporated to afford 1.18 g of the benzyl alcohol as a light yellow solid, which was used without further purification.
The alcohol was dissolved in 20 mL of CH2Cl2 and treated with DMAP (0.26 g, 0.0018 mol) and Ac2O (1.5 g, 0.15 mmol) for 18 hours. The reaction mixture was partitioned between CH2Cl2 and 3 N HCI. The organic phase was washed (sat. NaHCO3), dried (MgSO4), and was evaporated to give 1.4 g of a mixture of the two regioisomers as a light yellow solid. Chromatography on silica gel with 10% hexanes in CH2Cl2 afforded 0.15 g of the less polar compound 1 as a light yellow solid and 0.40 g of the more polar compound 2 as a light yellow solid and 0.69 g of a mixture of the two.
Figure imgf000031_0001
A solution of veratrole (2.00 g; 14.5 mmol) and propionyl chloride (1.4 mL; 16.1 mmol) in 15 mL of CS2 cooled to 0°C was treated with AlCl3 (2.00 g; 15.0 mmol). After stirring for 30 min., the mixture was allowed to warm to room temperature and turned deep red in color. The reaction mixture was quenched after an additional 4.5 hours by cooling to 0°C and slowly adding water. The residue was partitioned between EtOAc and 1 N HCI, and the organic phase washed (sat. NaCl), dried (MgSO4), and concentrated to yield 3.00 g of a yellow solid. The crude ketone was used without further purification.
The crude ketone was dissolved in 15 mL of glacial acetic acid and added to 100 mL of 70% HNO3 cooled to 0°C. The reaction mixture darkened in color within 5 min. After stirring for 20 min., the mixture was allowed to warm to room temperature for an additional 1 hour. The mixture was poured into cold water, and was extracted with EtOAc. The organic phase was washed (sat,. NaCl), dried (MgSO4), and evaporated to give a yellow solid. Recrystalization from MeOH/H2O afforded 1.716 g (50% yield for two steps) of 3 as tan crystals.
Figure imgf000032_0001
l,4-Benzodioxan-6-yl methyl ketone (1.00 g; 5.61 mmol) was dissolved in 25 mL of 70% HNO3 cooled to 0°C. The reaction mixture darkened in color within 5 min. The solution was warmed to room temperature after 5 min and was quenched /after an additional 2.5 hours by pouring into cold water and was extracted with EtOAc. The organic phase was washed (sat. NaCl, sat NaHCO3), dried (MgSO4), and evaporated to give a tan solid which was used without further purification.
The crude nitrophenyl compound was dissolved in 25 mL of anhydrous THF and treated with NaBH4 (400 mg; 10.6 mmol) at room temperature. The solution darkened to dark brown after 2 min. After stirring for 30 min., 10 mL of EtOH was added and the stirring continued for an additional 30 min. The solution was partitioned between EtOAc and sat. NH4Cl, and the organic phase was dried (MgSO4), and evaporated to afford a brown oil.
The crude benzyl alcohol was taken up in 20 mL of CH2Cl2 and 6 mL of pyridine and was treated with 2 mL of Ac2O and 10 mg of DMAP for 14 hours. The reaction mixture was partitioned between EtOAc and 1 N HCI, and the organic phase was dried (MgSO4), and concentrated to give a brown oil.
Chromatography on silica gel with 100% CH2Cl2 gave 0.68 g of a yellow oil, which was shown to be a mixture of three compounds by NMR analysis. Chromatography of this mixture on silica gel with 25% EtOAc in hexanes afforded 255 mg of pure 5 as a light yellow solid, MP 82-85°C, and an additional 238 mg of slightly impure 5 as a light yellow solid.
Figure imgf000033_0001
Piperonal (21.65 g; 129 mmol) was slowly added to 175 mL of 70% HNO3 cooled to 10°C. The dark reaction mixture was allowed to warm to room temperature and stirred for 1 hour total. The resultant slurry was poured into cold water and filtered to give a light yellow solid. Recrystalization from MeOH/H2O afforded 21.46 g of light yellow crystals having a melting point of 108-112°C (80% yield).
To a solution of the nitroacetophenone (14.7 g; 70.4 mmol) in 500 mL of MeOH and 100 mL of CHCl3 was added NaBH4
(2.71 g; 71.6 mmol). After stirring for 5 hours, the solution was partitioned between CHCl3 and sat. NH4Cl. The organic phase was dried (MgSO4), and evaporated to give a yellow solid. Recrystalization from MeOH/H2O afforded 14.24 g of the alcohol as light yellow crystals (96% yield).
A solution of the nitropiperonyl alcohol (1.00 g; 4.74 mmol) in 25 mL of THF was treated with phosgene (8 mL of
Figure imgf000034_0001
1.93 M in toluene; 15.4 mmol) at room temperature for 12 hours. The excess phosgene and solvent were removed under reduced pressure to give a tan solid, which was used without any further purification.
A solution of the crude chloroformate in 10 mL of dioxane was added to a solution of L-phenylalanine (540 mg; 3.27 mmol) and NaHCO3 (680 mg; 8.09 mmol) in 15 mL of H2O and 5 mL of dioxane. The reaction mixture was a light brown, homogeneous solution. The solution was stirred at room temperature for 26 hours and then was partitioned between ether and 0.1 N NaOH. The ether phase was discarded and the aqueous phase was acidified to pH 2 with 3 N HCl and was extracted with CHCl3. The organic phase was dried (MgSO4) and evaporated to give a white solid. Chromatography on silica gel with 15% MeOH in CHCl3 afforded 1.11 g (84% yield) of
MeNPOC-L-Phe-OH as a white solid, MP 144-154°C (decomposes).
Example 5
Alpha-methyl-6-nitropiperonyl alcohol was prepared as follows. All anhydrous reagents, methylene chloride, pyridine, triethylamine, and diisopropylethyl amine were obtained from Aldrich Chem Co. N-4-isobutyryl-2'-deoxycytidine, N6-phenoxyacetyl-2'-deoxyadenosine, N2-phenoxyacetyl-2'-deoxyguanosine, 2'-deoxythymidine, and 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite were purchased from Sigma Chem Co. 4,5-methylenedioxy-2-nitroacetophenone was purchased from Lancaster Synthesis and 1.93M phosgene in toluene was from Fluke Chem Co.
1H NMR spectra were measured on a Varian Gemini 300 MHz Spectrometer and are reported in ppm. Mass Spectra were obtained from the U. C. Berkeley Mass Spectrometry Laboratory.
4,5-methylenedioxy-2-nitroacetophenone (30 g, 0.144 mol) was dissolved in 200ml ethanol and 10ml of
tetrahydrofuran. Sodium borohydride (5.5 g, 0.144 mol) was added to the stirred suspension over a 5 min. period. The suspension was stirred for an additional hour at room
temperature, poured into 1.2L of ice water, and acidified to pH 2 with 1N HCl. The yellow solid was collected by
filtration, washed with water, and dried in vacuo. The solid was recrystallized from 1:1 toluene/hexane to yield 22.8 g (75%) of α-methyl-6-nitropiperonyl alcohol. 1H NMR (CDCl3): 1.55 (d, 3H) CH3; 5.45 (q, 1H) CH; 6.1 (m, 2H) O-CH2-O; 7.3 and 7.5 (s, 2H) aromatic. MS
Alpha-methyl-6-nitropiperonyloxycarbonyl chloride was prepared as follows. Phosgene (500ml, 1.93M in toluene) was added to a rapidly stirring solution of (20 g, 0.095 mol) in 700ml anhydrous tetrahydrofuran . After stirring overnight at room temperature the mixture was evaporated to dryness in vacuo resulting in a thick brown oil. Tituration with hexane yielded 20 g (73%) of α-methyl-6-nitropiperonyloxycarbonyl chloride as a yellow-brown solid. 1H NMR (CDCl3) : 1.7 (d, 3H) CH3; 6.15 (m, 2H) O-CH2-O; 6.5 (q, 1H) CH; 7.05 and 7.55 (s, 2H) aromatic.
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-2'-deoxynucleosides was prepared as follows. N-protected deoxynucleoside (34 mmol) was evaporated twice from 100ml anhydrous pyridine and dried in vacuo. The resultant residue was dissolved in 150 ml anhydrous pyridine, cooled to 0°C in an ice water bath, and 37 mmol added. After stirring for 15 min. at 0°C the ice bath was removed and the reaction stirred for 4-5 hours at room temperature. The mixture was
concentrated in vacuo to an oil and taken up in 100 ml of methylene chloride. The solution was washed successively with saturated sodium bicarbonate (25 ml), water (25 ml), and saturated sodium chloride (25 ml). The organic phase was dried over anhydrous sodium sulfate and the solvent removed by rotary evaporation. The reaction product was purified by silica gel chromatography, eluting with 10% (v/v) methanol in methylene chloride. Fractions containing the major product were pooled and dried in vacuo.
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-2'-deoxythymidine: yield 60%; Rf= 0.47 (silica, methylene chloride/methanol 9:1); 1H NMR (CDCl3): 1.7 (d, 3H) CH3 ;
1.9 (s, 3H) CH3; 2.0-2.4 (m, 2H) 2'-CH2 ; 4.1-4.5 (m, 4H) 3'- H, 4'-H, 5'-CH2; 6.1 (m, 2H) O-CH2-O; 6.3 (m, 2H) 1'-H, CH (benzyl); 7.0 and 7.5 (s and m, 2H) aromatic; 7.35 (s, 1H) 6H. MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N6- phenoxyacetyl-2'-deoxyadenosine: yield 52%; Rf= 0.69
(silica, methylene chloride/methanol 9:1); 1H NMR (CDCl3): 1.65 (d, 3H) CH3; 2.6 and 2.9 (m, 2H) 2'-CH2; 4.2-4.4 (m, 4H) 3'-H, 4'-H, 5'-CH2 ; 4.9 (d, 2H) CO-CH2-O; 6.25 (m, 1H) 1'-H; 6.5 (q, 1H) CH (benzyl); 7.05 (m, 4H) aromatic (PAC), aromatic (nitrobenzyl); 7.35 (m. 2H) aromatic (PAC); 7.45 (s, 1H) aromatic (nitrobenzyl); 8.25 and 8.75 (d, 2H) 2H, 8H; 9.4 (bs, 1H) NH.
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N2- phenoxyacetyl-2'-deoxyguanosine: yield 70%; Rf= 0.57
(silica, methylene chloride/methanol 6:1); 1H NMR
(CDCl3/CD3OD): 1.6 (dd, 3H) CH3; 2.4-2.8 (m, 2H) 2'-CH2; 4.2-4.6 (m, 4H) 3'-H, 4'-H, 5'-CH2; 4.7 (s, 2H) CO-CH2-O;
6.1-6.4 (m, 4H) O-CH2-O, 1'-H, CH (nitrobenzyl); 6.9-7.15 (m, 4H) aromatic (PAC), aromatic (nitriobenzyl); 7.3-7.42 (m, 3H) aromatic (PAC), aromatic (nitrobenzyl); 8.1 (s, 1H) 8H. MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N4-isobutyryl-2'-deoxycytidine: yield 60%; Rf= 0.56 (silica, methylene chloride/methanol 9:1); 1H NMR (CDCl3) : 1.2 (dd, 6H) 2CH3 (ibu); 1.65 (dd, 3H) CH3, 2.1 (m, 1H) 2'-H; 2.5-2.8 (m, 2H) 2'-H, CH (ibu); 4.2-4.5 (m, 4H) 3'-H, 4'-H, 5'-CH2; 6.1 (m, 2H) O-CH2-O; 6.3 (m, 2H) 1'-H, CH (benzyl);
7.0 and 7.49 (d, 2H) aromatic; 7.45 and 8.0 (dd, 2H) 5H, 6H; 8.35 (bs, 1H) NH. MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-2'-deoxynucleoside 3'-O-(2-cyanoethyl)-N,N'-diisopropylaminophosphites were prepared as follows. 5'-protected deoxynucleoside (10 mmol) was dissolved in 75 ml of methylene chloride. Diisopropylethylamine (30 mmol) was added and the solution cooled to 0°C in an ice water bath. 2-cyanoethyl-N,N'-diisopropylchlorophosphoramidite (25 mmol) was slowly added to the cooled mixture. The ice bath was removed and the reaction stirred for 2 hours at room temperature.
Anhydrous methanol (5 ml) was added and the reaction stirred for an additional 30 min. at room temperature. The mixture was diluted with methylene chloride (150 ml) and washed with saturated sodium bicarbonate (50 ml) followed by water (50 ml). The organic phase was dried over anhydrous sodium sulfate and the solvent removed in vacuo. The product was purufied by silica gel chromatography, eluting with 45:45:10 ethyl acetate/methylene chloride/triethylamine. Fractions containing the major product were pooled and dried in vacuo.
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-2'- deoxythymidine 3'-O-(2-cyanoethyl)-N,N'- diisopropylphosphoramidite: yield 80%; Rf= 0.58 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine); 1H NMR (CDCl3): 1.1-1.3 (m, 12H) CH3 (isopropyl); 1.65 (d, 3H) CH3 (nitrobenzyl); 1.9 (s, 3H) CH3; 2.1-2.3 (m, 1H) 2'-H; 2.4-2.7 (m, 3H) 2'-H, CH2-CN; 3.5-3.9 (m, 4H) NH (isopropyl), P-O-P-CH2; 4.1-4.5 (m, 4H) 3'-H, 4'-H, 5'-CH2; 6.15 (s, 2H) O-CH2-O; 6.3 (m, 2H) 1'-H, CH (nitrobenzyl); 7.0 and 7.5 (s, 2H) aromatic; 7.3 (m, 1H) 6H; 8.4 (bs, 1H) NH (thymidine). MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N4-isobutyryl-2'-deoxycytidine 3'-O-(2-cyanoethyl)-N,N'-diisopropylphosphoramidite: yield 85%; Rf= 0.52 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine); 1H NMR (CDCl3): 1.1-1.3 (m, 18H) CH3 (isopropyl), CH3 (ibu);
1.65 (m, 3H) CH3 (nitrobenzyl); 2.1 (m, 1H)1 2'-H; 2.55-2.8 (m, 3H) 2'-H, CH2-CN; 3.5-3.9 (m, 4H) NH (isopropyl), P-O-CH2; 4.2-4.5 (m, 4H) 3'-H, 4'-H, 5'-CH2; 6.1-6.35 (m, 4H) O-CH2-O, 1'-H, CH (nitrobenzyl); 7.0 and 7.5 (d, 2H) aromatic; 7.4 and 7.95 (m, 2H) 5-H, 6H; 8.25 (bs, 1H) NH (amide). MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N6-phenoxyacetyl-2'-deoxyadenosine 3'-O-(2-cyanoethyl)-N,N'-diisopropylphosphoramidite: yield 81%; Rf= 0.50 (silica, 45:45:10 ethyl acetate/methylene chloride/triethylamine); 1H NMR (CDCl3): 1.1-1.3 (m, 12H) CH3 (isopropyl); 1.65 (m, 3H) CH3 (nitrobenzyl); 2.6-3.0 (m, 4H) 2'-H, CH:-CN; 3.6-3.9 (m, 4H) NH (isopropyl), P-O-CH2; 4.3-4.6 (m, 4H) 3'-H, 4'-H, 5'- CH2; 4.7 (2s, 2H) CO-CH2-O; 6.1 (m, 2H) O-CH2-O; 6.3 ( m, 1H) 1'-H; 6.5 (m, 1H) CH (nitrobenzyl); 7.1 (m, 4H) aromaitc (nitrobenzyl), aromatic (PAC); 7.35 (m, 2H) aromatic (PAC); 7.45 (s, 1H) aromatic (nitrobenzyl); 8.2 and 8.8 (s, 2H) 2H, 8H; 9.4 (bs, 1H) NH (amide). MS
5'-O-(α-methyl-6-nitropiperonyloxycarbonyl)-N2- phenoxyacetyl-2'-deoxyguanosine 3'-O-(2-cyanoethyl)-N,N'- diisopropylphosphoramidite: yield 75%; Rf= 0.55 (silica, 9:1 methylene chloride/methanol); 1H NMR (CDCl3) : 1.3 (d, 12H) CH3 (isopropyl); 1.6 (dd, 3H) CH3 (nitrobenzyl); 2.5-2.9 (m, 4H) 2'-H, CH2-CN; 3.55-3.95 (m, 4H) NH (isopropyl), P-O-CH2; 4.3-4.6 (m, 4H) 3'-H, 4'-H, 5'-CH2; 4.7 (2s, 2H) CO-CH2-O; 6.1-6.35 (m, 4H) O-CH2-O, 1'-H, CH (nitrobenzyl); 6.95-7.15 (m, 4H) aromaitc (nitrobenzyl), aromatic (PAC); 7.35-7.45 (m, 3H) aromatic (PAC), aromatic (nitrobenzyl); 7.9 (s, 1H) 8H. MS
Figure imgf000038_0001
2,3-Dimethoxybezaldehyde (1.00 g; 6.02 mmol) was added to 10 mL of 70% HNO3 cooled to 0°C. The solution was warmed to room temperature after 5 min and stirring was continued for an additional 10 min. The reaction mixture was poured into cold water and filtered to give 1.16 g of an off-white solid precipitate. The crude 1:1 mixture of
regioisomers was used without further purification. The crude nitrobenzaldehyde was reduced Py treating a solution of the aldehyde (501 mg; 2.37 mmol) in 25 mL of anhydrous THF with NaBH4 (185 mg; 4.89 mmol) cooled to 15 °C. After stirring the solution for 10 min., 3 mL of MeOH was added to aid solubility. The reaction mixture was stirred an additional 30 min. and was then partitioned between EtOAc and sat. NH4Cl. The organic phase was washed (sat. NaCl), dried (MgSO4), and evaporated to give 0.56 g of a 1:1 mixture of the two regioisomers as a white crystalline solid. The alcohols were used without further purification.
A solution of the crude alcohols (0.56 g) in 10 mL of CHCl3 was treated with 5 mL of pyridine and 2 mL of Ac2O for 3 hours at room temperature. The reaction mixture was partitioned between CHCl3 and 1 NHCl, and the organic phase was dried (MgSO4) and evaporated to give .6 g of a colorless oil. Chromatography on silica gel with 25% EtOAc in hexanes afforded 314 mg of 6 as a white solid (52% yield for the two steps) and 261 mg of 7 as a white solid (43% yield for the two steps).
Figure imgf000039_0001
Nitroveratryloxymethylchloride (NV0M-Cl) was prepared as follows. To a suspension/solution of 5g (0.0235 mol) of NV-OH in 50 ml of toluene was added 1.0g (0.033 mol) dry powdered paraformaldehyde [(CH2O)n] with vigorous
stirring. HCl(g) was bubbled into the solution (no cooling) until all of the solid had gone into solution (30 min) at which time the solution turned dark green with a black aqueous layer coating the walls of the flask. The mixture was then allowed to stand at room temperature for approximately 30 min and the green solution was then decanted off the black aqueous layer. To the toluene solution was added 30 ml of toluene and 20 ml of hexane followed by 2.5 g of MgSO4 (anhydrous). The solution was stirred at approximately 10°C for 15 min, filtered, and the filtrate rotary evaporated (at approximately 30°C) to an amber oil. The oil was placed on a vacuum line whereupon yellow crystals began to come out of the oil. The semi-solid was then triturated 3 times with 40 ml of hexane each time decanting off the hexane. The yellow crystalline residue was then dried in vacuum at room temperature. The yield was 5.67 g on 92%.
Figure imgf000040_0001
An NVOM β-estradiol-benzylether was prepared as follows. To a mixture of 400 mg (1.105 mmol) of β-estradiol- 3-benzyl ether and 434 mg (1.66 mmol) of NVOM-Cl in 5 ml of CH2Cl2 was added 213 mg (1.65 mmol) of DIE. After stirring overnight at room temperature the mix was with H2O, 0.1N HCl, dried (Na2SO4) and solvent removed. The yield was 584 mg or 90%. NVOM protected 2-deoxyadenosine was prepared by an analogous method.
VI. Multiple-guantum Protecting Groups
In most of the above described compounds, only a single photon is required (in theory) to photolyze a single molecule of the compound. However, some photoreactive groups of this invention require two or more photons for photolysis. Such compounds may be useful during light-directed synthesis on substrates to prevent unwanted deprotections caused by the diffraction or light around the edges of the mask (see the discussion of binary synthesis strategies above). Compounds useful for this type of "multiple quantum" deprotection include those in which a first photon alters the photoactive isomerization, redox reaction, cyclization, or deprotection of a functional group on the protecting group itself. Upon exposure to the second photon, the altered protecting group is then removed. One example of such a deprotection is shown below:
Figure imgf000041_0001
It will be appreciated by those skilled in the art that the compound shown in the example above is but one of many compounds which require two photons to become
deprotected.
VII. Photolysis of Protected Group Compounds
Removal of the protecting group is accomplished by irradiation to separate the reactive group and the degradation products derived from the protecting group. Not wishing to be bound by theory, it is believed that irradiation of NVOC- and MeNVOC-protected oligomers, for example, occurs by the
following reaction schemes:
NVOC-AA -> 3,4-dimethoxy-6-nitrosobenzaldehyde + CO2 + AA MeNVOC-AA-> 3,4-dimethoxy-6-nitrosoacetophenone + CO2 +AA where AA represents the N-terminus of an amino acid or
oligomer.
Along with the unprotected amino acid, other 3,4-dimethoxy-6-nitrosophenylcarbonyl compound, which can react with nucleophilic portions of the oligomer to form unwanted secondary reactions. In the case of an NVOC- protected amino acid, the degradation product is a
nitrosobenzaldehyde, while the degradation product for MeNVOC is a nitrosophenyl ketone. It is believed that the product aldehyde from NVOC degradation reacts with free amines to form a Schiff base (imine) that affects the remaining polymer synthesis. Preferred photoremovable protecting groups react slowly or reversibly with the oligomer on the support.
Not wishing to be bound by theory, it is believed that the product ketone from irradiation of a MeNVOC-protected oligomer reacts at a slower rate with nucleophiles on the oligomer than the product aldehydes from irradiation of the same ΝVOC-protected oligomer. Although not unambiguously determined, it is believed that this difference in reaction rate arises from radical stabilization. However, it may also be due to the difference in general reactivity between aldehydes and ketones towards nucleophiles due to steric and electronic effects.
In some preferred embodiments, scavengers or other reagents are added to the reaction mixture to react with and render harmless photolysis byproducts that might otherwise react with a growing oligomer. Suitable scavengers (which are often nucleophiles) will be known to those of skill in the art. Specific examples include acids, bisulfites, hydroxy-containing compounds, amines, etc.
Because photolysis is a first order process, the protected compound must be illuminated for nine half-lives to effect 100% removal. Very fast deprotection rates are
therefore desired because they lessen the overall exposure time, reducing the effects of stray light and the total time necessary to synthesize a polymer.
The photolysis of Ν-protected L-phenylalanine in solution having different photoremovable protecting groups was analyzed, and the results are presented in the following table: Photolysis of Protected L-Phe-OH t1 2 in seconds
Solvent NBOC NVOC MeNVOC MeNPOC
Dioxane 1288 110 24 19
5mM H2SO4/Dioxane 1575 98 33 22
The half life, t1 2 is the time in seconds required to remove 50% of the starting amount of protecting group. The photolysis was carried out in the indicated solvent with 362/364 nm-wavelength irradiation having an intensity of 10 mW/cm2, and the concentration of each protected phenylalanine was 0.10 mM.
The table shows that deprotection of NVOC-, MeNVOC-, and MeNPOC-protected phenylalanine proceeded faster than the deprotection of NBOC. Furthermore, it shows that the
deprotection of the two derivatives that are substituted on the benzylic carbon, MeNVOC and MeNPOC, were photolyzed at the highest rates in both dioxane and acidified dioxane.
Another photolysis study was performed on various MeNPOC nucleosides. The t 1 2 rates for the disappearance of starting material are tabulated below and are normalized to a photolysis intensity of 10 mW/cm2 power using the 350-450 nm dichroic reflector on a Hg(Xe) arc lamp. DTT was intended to act as scavenger for the nitrosoketone byproduct and was included in the study to see if it had any effect on the photolysis rates.
Table II
(t 1 2 in seconds)
Nucleoside Dioxane Acid/Diox CH3CN DTT/CH3CN
NVOC-Ac 93.4
MeNPOC-A(PheAc) 32.1 decomp. 73.1 74.7
MeNPOC-C(i-Bu) 31.3 39.0 80.6 90.5
MeNPOC-G(PheAc) 37.9 decomp. 80.7 79.4
MeNPOC-G(iPrPheAc) 41.0 decomp. 93.1 88.0 In another photolysis study, a 0.1mM solution of each of the four 5'-protected nucleosides prepared in Example 5, MeNPoc-dT, MeNPoc-dC , MeNPoc-dG , and MeNPoc-dA was prepared in dioxane. A 200 μL aliquot of one of the four deoxynucleoside solutions was irradiated with 350-450nm light at 14.5 mW/cm2 (Oriel) in a narrow path (2mm) quartz cuvette for a given period of time x1. A fresh aliquot of the 0.1mM solution was irradiated as before for a different time x2. Four or five time points were collected for each MeNPoc- deoxynucleosides.
Each time point was analyzed for loss of starting material at 280 nm on a nucleosil 5-C8 HPLC column using a mobile phase of 60% (v/v) CH3CN in water containing 0.1% (v/v) TFA. MeNPOC-dT required a mobile phase of 70% (v/v) methanol in water. The integrated peak area of the remaining 5'- MeNPoc-2'-deoxynucleoside was calculated. A plot of natural logarithm of area vs. time resulted in a straight line of slope k where t1 2= 0.639/k.
Figure imgf000044_0001
In other photolysis experiments, the deprotection of nitroveratryloxymethyl protected compounds was shown to compare favorably with certain NVOC and MeNPOC compounds.
Figure imgf000044_0002
Figure imgf000045_0001
Still other photolysis results are shown below. In the second solvent, 5mM H2SO4 is used and in the third solvent 5mM DTT is used. The photolysis radiation was provided at a wavelength of 365 nm and an intensity of 10 mW/cm2. The listed values in the chart are t1 2 in seconds. The various compounds are shown in Fig. 6.
Figure imgf000046_0001
VIII. Basis Sets of Photoprotected Amino or Nucleic Acids
The deprotection rate (or photolysis half life) of a protected compound is a function of the particular
photosensitive protecting group as well as the amino or nucleic acid functional group being protected. It is also a function of the solvent in which photolysis is performed and neutralize photolysis decomposition products. As illustrated in the above Tables, the deprotection rate can vary
substantially for the basis set of naturally occurring deoxynucleosides, each protected with the same photosensitive group. It has also been observed that the set of NVOC- protected amino acids (naturally occurring) have photolysis half lives ranging from 70-150 seconds.
In VLSIPS™, it is desirable that the protected compounds are deprotected at substantially the same rate to prevent unwanted side reactions between the byproducts described above and the unprotected amino acids. In other words, monomers that deprotect earliest are exposed to reactive photolysis byproducts until the substrate is washed. If all oligomers on the substrate deprotect at the same rate, the time of exposure will be minimized. In VLSIPS™, it is also desirable to minimize the exposure time so that stray light from an illuminated region does not deprotect compounds in adjacent regions. To ensure a minimal exposure time, it is preferred to match the various photoreactive protecting groups of the invention with different amino acids (or other monomer types) so that all of the amino acids will be deprotected at substantially the same rate in a particular solvent. Sets of such rate-matched photoprotected amino acids or nucleosides are hereinbelow referred to as "basis sets". It will be appreciated that basis sets can be created for any class or subclass of compounds, most notably the natural or unnatural amino acids and the natural or unnatural nucleic acids.
"At substantially the same rate" is defined herein to mean that the half-life for deprotection of the most rapidly deprotected compound in the basis set is within about 25% of the compound with the slowest deprotection half-life in a particular solvent. Preferably, the differences between half-lives is about 10% and most preferably about 5%.
One preferred class of compounds for forming a basis set is a plurality of the twenty naturally-occurring amino acids. Another preferred class is a subset of the D-isomers of the twenty naturally-occurring amino acids. Yet another include β amino acids and amino acids whose side chains have been altered in order to adjust the stearic bulk,
hydrophobicity, or electronic character of the corresponding natural amino acid. Such amino acid analogs have found increasing utility in drug research (see, e.g., Fauchère).
Still another preferred class is formed by the common nucleosides adenosine, thymidine, guanosine, cytidine, and inosine in addition to their 2', or 3'-deoxy analogs and their 2', 3', or 5'-phosphates. "Phosphate" is meant herein to include the phosphotriesters (-OP(OR") (OR"')), phosphoramidites (-OP(OR") (NRR'), where R-R"" are described above), as well as phosphate anions (-OP(O)O2H-, and -OP(O)O2 -2).
Typically, the choice of protecting groups will depend on the character of the solvent 'Which, as described above, has an important effect on the rate of deprotection. One
preferred solvent for both amino acids and nucleosides is dioxane. Another preferred solvent is acetonitrile.
IX. Conclusion
The above description is illustrative and not restrictive. Many variations of the invention will become apparent to those of skill in the art upon review of this disclosure. Merely by way of example, while the invention is illustrated primarily with regard to peptide and nucleotide synthesis, the invention is not so limited. The scope of the invention should, therefore, be determined not with reference to the above description, but instead should be determined with reference to the appended claims along with their full scope of equivalents.

Claims

WHAT IS CLAIMED IS:
1. A compound having the formula:
Figure imgf000049_0001
wherein n is 0 or 1; A is -C(O)-, -(CQ1Q2)-, or -C(S)- ; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R2 is a functional group of a molecule selected from the group, consisting of natural or unnatural amino acids and peptides, nucleosides, nucleoside analogs, and oligonucleotides; R3-R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro,
alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals, ketals, orthoesters, thioesters, fused aromatic groups, and ether groups; provided that when n is 0 or A is -C(O)- (i) R4 and R5 are not both methoxy when R1, R3, and R6 are hydrogen; (ii) R1 is not hydrogen, methyl, phenyl, or 2-nitrophenyl when R3-R6 are hydrogen simultaneously; and (iii) R1 is not 2-nitrophenyl or 3,4-dimethoxy-6-nitrophenyl when R3 and R6 are hydrogen and R4 and R5 are both either hydrogen or methoxy.
2. The compound of claim 1, wherein said functional group is a carboxyl, hydroxyl, thiol or amino group.
3. The compound of claim 1, wherein R2 has the formula -NRR' where R is selected from the group consisting of hydrogen, C1-C6 alkyl, aryl, acyl, or benzyl, and R' is the residue of a natural or unnatural amino acid.
4. The compound cf claim 3, wherein R is methyl.
5. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R5 are hydrogen, and R4 and R5 are methoxy.
6. The compound of claim 1 having the following structure:
Figure imgf000050_0001
wherein R1 is hydrogen or methyl, R3 and R6 are hydrogen, and Y1 and Y2 are independently C1-C8 alkyl groups, or together form a cyclic bridge acetal or ketal.
7. The compound of claim 6, wherein R4 and R5 form a methylenedioxy acetal ring.
Figure imgf000050_0002
8. The compound of claim 1 having the following structure:
Figure imgf000050_0003
wherein R1 is hydrogen or methyl, R3 and R4 are hydrogen, and Y1 and Y2 are independently C1-C8 alkyl or together form a cyclic bridge acetal or ketal.
9. The compound of claim 8, wherein R5 and R6 form a methylenedioxy acetal ring.
Figure imgf000051_0001
10. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R6 are hydrogen, and R4 and R5 together form a ring, the compound having the structure
Figure imgf000051_0002
wherein Y is -CRR'- or -CRR '-CR"R"-, and where R, R', R" and R'" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl ester, R and R' together are the oxygen of a carbonyl group, and R" and R'" together are the oxygen of a carbonyl group .
11. The compound of claim 10 , wherein Y is -CRR '-CR "R "'- and R, R ' , R " and R'" are hydrogen .
Figure imgf000051_0003
12. The compound of claim 10, wherein Y is -CRR'- and R is hydrogen and R' is alkoxy, aryloxy, carboxy, or alkyl ester.
13. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R4 are hydrogen, and R5 and R6 together form a ring, the compound having the structure
Figure imgf000052_0001
wherein Y is -CRR'- or -CRR'-CR"R'"-, and where R, R', R" and R'" are selected independently from the group consisting of hydrogen, C1 C8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl ester, R and R' together are the oxygen of a carbonyl group, and R" and R'" together are the oxygen of a carbonyl group.
14. The compound of claim 13, wherein Y is -CRR'-CR"R"- and R, R', R" and R'" are hydrogen.
Figure imgf000052_0002
15. The compound of claim 13, wherein Y is -CRR'- and R is hydrogen and R' is alkoxy, aryloxy, carboxy, or alkyl ester.
16. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R6 are hydrogen, and R5 and R6 are methoxy.
17. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 is dimethylamino, R4 is hydrogen, and R5 and R6 are methoxy.
18. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R6 are hydrogen, and R4 is dimethylamino, and R5 is methoxy.
19. The compound of claim 1, wherein R1 is hydrogen or methyl, R3 and R6 are hydrogen, and R4 is methoxy, and R5 is dimethylamino.
20. The compound of claim 1 wherein R2 has the formula -NRR' where R is either hydrogen, methyl, or acetyl, and R' is a natural or unnatural amino acid residue.
21. The compound of claim 1 wherein n is 1 and A is -CH2-.
22. The compound of claim 21, wherein said functional group is carboxyl, amino, thiol, amide or hydroxyl.
23. A compound having the formula:
Figure imgf000053_0001
wherein R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R2 is a molecule functional group selected from the group consisting of amines, carboxyl groups, thiols, imidazoles, amides, and hydroxy groups; R3-R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals. ketals, orthoesters, thioesters, fused aromatic groups, and ether groups.
24. The compound of claim 23 wherein R1, Q1, and Q2 are hydrogen.
25. The compound of claim 23 wherein the functional group is part of a molecule selected from the group consisting of nucleosides, nucleoside analogs, oligonucleotides, amino acids, and peptides.
26. The compound of claim 23 having the following structure:
Figure imgf000054_0002
27. A compound having the formula:
Figure imgf000054_0001
wherein R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R10 is a group selected from the group consisting of halo, hydroxyl, tosyl, mesyl, trifluoromethyl, diazo, azido; R3-R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro,
alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals, ketals, orthoesters, thioesters, fused aromatic groups, and ether groups.
28. The compound of claim 27 wherein R10 is a halide.
29. The compound of claim 28 wherein R1, Q1, and Q2 are all hydrogen; R10 is chloro; R3 and R6 are both hydrogen; and R4 and R5 are both methoxy.
30. A compound having the formula:
Figure imgf000055_0001
wherein X is selected from the group consisting of oxygen or sulphur; R1 is a purine, a pyrimidine, or an analog thereof; and R2, R3, and R4 are each independently hydrogen, hydroxy, oligonucleotide, alkoxy, tetrahydropyranyl, β-benzoylpropionyl, acetyl, a bridge between adjacent
substituents which together form an acetal, ketal, orthoester, or cyclic ether, a photolabile group, -NRR', -OP(O)O2 -2, -OP(O)O2H-, -OP(O)(OR")(OR"), -OP(OR")(NR"R'") where R and R' are independently selected from the group consisting of C1-C3 alkyl, aryl, benzyl, or acetyl, and R" and R"' are selected independently from the group consisting of C1-C3 alkyl, aryl, benzyl, or C1-C3 cyanoalkyl; provided that at least one of R2-R4 is a photoreactive compound and wherein said photolabile group having the formula:
Figure imgf000055_0002
wherein n is 0 or 1 ; A is -C (O) - , - ( CQ1Q2 ) - , or - aryl, alkcxy, aryloxy, or carboxy; R6-R9 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro,
alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals, ketals, orthoesters, thioesters, fused aromatic groups, and ether groups.
31. The compound of claim 30, wherein R4 is said photolabile group.
32. The compound of claim 30, wherein R2 is said photolabile group.
33. The compound of claim 30, wherein R3 is said photolabile group.
34. The compound of claim 30, wherein X is oxygen and said photolabile group has the formula:
Figure imgf000056_0001
35. A basis set of protected amino acids, comprising a plurality of natural or unnatural amino acids, each
independently coupled through either an amine or carboxyl group to at least one photolabile group having the formula:
Figure imgf000056_0002
wherein n is 0 or 1; A is -C(O)-, -(CQ1Q2)-, or - C(S)-; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R3-R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro,
alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals, ketals, orthoesters, thioesters, fused aromatic groups, and ether groups; wherein said photolabile groups are chosen so that each member of the basis set photolyzes at substantially the same rate upon exposure to radiation in a particular solvent.
36. The basis set of claim 35, wherein said particular solvent is dioxane.
37. A basis set of protected nucleosides or nucleoside analogs comprising a plurality of nucleosides or nucleoside analogs, each independently coupled to a
photolabile group having the formula:
Figure imgf000057_0001
wherein n is 0 or 1; A is -C(O)-, - (CQ1Q2) - , or -C(S)-; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R3-R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro,
alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl, and cyclic bridges between adjacent substituents where the bridges are selected from the group consisting of acetals, ketals, orthoesters, said photolabile groups are chosen so that each member of the basis set photolyzes at substantially the same rate upon exposure to radiation in a particular solvent.
38. The basis set of claim 37, wherein said particular solvent is acetonitrile.
39. The basis set of claim 37, wherein said nucleosides are selected from the group consisting of 2'- deoxynucleosides, 3'-deoxynucleosides and 2', 3'- dideoxynucleosides.
40. The basis set of claim 37, wherein said nucleosides include a phosphate containing group selected from the group consisting of -OP(O)O2 -2, -OP(O)O2H-, -OP(OR")(OR"'), and -OP (OR") (NR"R'"), wherein R and R' are independently C1- C3 alkyl, aryl, benzyl, or acetyl, and R" and R'" are selected independently from the group consisting of C1-C3 alkyl, aryl, benzyl; or C1-C3 cyanoalkyl.
41. The basis set of claim 40, wherein said phosphorous containing group is located at a position selected from the group consisting of the 2'-carbon, the 3'-carbon and the 5'-carbon positions of said nucleoside or nucleoside analog.
42. A compound having the formula:
Figure imgf000058_0001
wherein n is 0 or 1; A is -C(0)-, -(CQ1 Q2)-, or -C(S)-; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R2 is a molecule functional group selected from the group consisting of amines, carboxyl R4 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl; and wherein Y is -CRR'- or -CRR'-CR"R'"-, and where R, R', R" and R'" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl ester, R and R' together are the oxygen of a carbonyl group, and R" and R'" together are the oxygen of a carbonyl group.
43. The compound of claim 42 wherein R1, Q1, and Q2 are hydrogen.
44. The compound of claim 42 wherein the functional group is part of a molecule selected from the group consisting of nucleosides, nucleoside analogs, oligonucleotides, amino acids, and peptides.
45. A compound having the formula:
Figure imgf000059_0001
wherein n is 0 or 1; A is -C(O)-, -(CQ1Q2)-, or -C(S)-; R1, Q1, and Q2 are independently hydrogen, C1-C8 alkyl, aryl, alkoxy, aryloxy, or carboxy; R2 is a molecule functional group selected from the group consisting of amines, carboxyl groups, thiols, imidazoles, amides, and hydroxy groups; R3 and R6 are independently hydrogen, alkoxy, aryloxy, benzyloxy, acyloxy, nitro, alkylthio, arylthio, hydroxyl, halogen, -NR'R" where R' and R" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, or benzyl; and R'" are selected independently from the group consisting of hydrogen, C1-C8 alkyl, aryl, benzyl, alkoxy, aryloxy, carboxy, alkyl ester, R and R' together are the oxygen of a carbonyl group, and R" and R'" together are the oxygen of a carbonyl group.
46. The compound of claim 45 wherein R1 , Q1 , and Q2 are hydrogen.
47. The compound of claim 45 wherein the functional group is part of a molecule selected from the group consisting of nucleosides, nucleoside analogs, oligonucleotides, amino acids, and peptides.
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Cited By (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5489678A (en) * 1989-06-07 1996-02-06 Affymax Technologies N.V. Photolabile nucleoside and peptide protecting groups
WO1996018634A2 (en) * 1994-12-16 1996-06-20 Wolfgang Pfleiderer Nucleoside derivatives with photolabile protective groups
US5585069A (en) * 1994-11-10 1996-12-17 David Sarnoff Research Center, Inc. Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis
US5846396A (en) * 1994-11-10 1998-12-08 Sarnoff Corporation Liquid distribution system
US5959098A (en) * 1996-04-17 1999-09-28 Affymetrix, Inc. Substrate preparation process
EP0967217A2 (en) * 1998-06-22 1999-12-29 Affymetrix, Inc. (a California Corporation) Reagents and methods for solid phase synthesis and display
US6022963A (en) * 1995-12-15 2000-02-08 Affymetrix, Inc. Synthesis of oligonucleotide arrays using photocleavable protecting groups
US6147205A (en) * 1995-12-15 2000-11-14 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
DE19938092A1 (en) * 1999-08-12 2001-02-22 Epigenomics Gmbh Nucleoside derivatives and process for their preparation
DE19952113A1 (en) * 1999-10-29 2001-05-03 Nigu Chemie Gmbh Nucleoside derivatives with photolabile protecting groups
US6239273B1 (en) 1995-02-27 2001-05-29 Affymetrix, Inc. Printing molecular library arrays
US6300063B1 (en) 1995-11-29 2001-10-09 Affymetrix, Inc. Polymorphism detection
US6331439B1 (en) 1995-06-07 2001-12-18 Orchid Biosciences, Inc. Device for selective distribution of liquids
WO2002020150A2 (en) * 2000-09-11 2002-03-14 Affymetrix, Inc. Photocleavable protecting groups
WO2000061594A3 (en) * 1999-04-08 2002-04-04 Deutsches Krebsforsch Nucleoside derivatives with photo-unstable protective groups
US6706875B1 (en) 1996-04-17 2004-03-16 Affyemtrix, Inc. Substrate preparation process
US6800439B1 (en) 2000-01-06 2004-10-05 Affymetrix, Inc. Methods for improved array preparation
US6806361B1 (en) 2000-03-17 2004-10-19 Affymetrix, Inc. Methods of enhancing functional performance of nucleic acid arrays
US6833450B1 (en) 2000-03-17 2004-12-21 Affymetrix, Inc. Phosphite ester oxidation in nucleic acid array preparation
US6924094B1 (en) 1996-02-08 2005-08-02 Affymetrix, Inc. Chip-based species identification and phenotypic characterization of microorganisms
US6953663B1 (en) 1995-11-29 2005-10-11 Affymetrix, Inc. Polymorphism detection
US7005259B1 (en) 2000-06-01 2006-02-28 Affymetrix, Inc. Methods for array preparation using substrate rotation
US7108968B2 (en) 1998-04-03 2006-09-19 Affymetrix, Inc. Mycobacterial rpoB sequences
EP1721993A2 (en) 1996-04-04 2006-11-15 Affymetrix, Inc. (a Delaware Corporation) Kits and methods for the detection of target nucleic acids with help of tag nucleic acids
JP2008214343A (en) * 2007-02-07 2008-09-18 Samsung Electronics Co Ltd Photodegradable compound, substrate for oligomer probe array coupled with the photodegradable compound, oligomer probe array and method for producing the same
EP2365098A2 (en) 2002-06-20 2011-09-14 Affymetrix, Inc. Antireflective coatings for high-resolution photolithographic synthesis of dna array
WO2011107747A3 (en) * 2010-03-05 2011-12-29 Medical Research Council Genetically encoded photocontrol
US8101737B2 (en) 2004-12-31 2012-01-24 Affymetrix, Inc. Parallel preparation of high fidelity probes in an array format
US8133987B2 (en) 2004-12-31 2012-03-13 Affymetrix, Inc. Primer array synthesis and validation
EP2267165A3 (en) * 1997-07-28 2012-10-10 Gen-Probe Incorporated Nucleic acid sequence analysis
US8343710B1 (en) 2005-03-11 2013-01-01 The Regents Of The University Of Colorado, A Body Corporate Photodegradable groups for tunable polymeric materials
US9180196B2 (en) 2005-03-11 2015-11-10 The Regents Of The University Of Colorado, A Body Corporate Photodegradable groups for tunable polymeric materials
US10472364B2 (en) 2016-09-09 2019-11-12 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US10570167B2 (en) 2016-12-22 2020-02-25 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US11078228B2 (en) 2018-06-21 2021-08-03 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
CN114805432A (en) * 2022-03-03 2022-07-29 西北工业大学 Novel photocatalytic-cleavable carboxylic acid protecting group and preparation method of amino acid derivative thereof
EP4249495A1 (en) * 2022-03-21 2023-09-27 ATDBio Limited Method for the purification of polynucleotides and analogues thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof

Cited By (68)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5489678A (en) * 1989-06-07 1996-02-06 Affymax Technologies N.V. Photolabile nucleoside and peptide protecting groups
US5755942A (en) * 1994-11-10 1998-05-26 David Sarnoff Research Center, Inc. Partitioned microelectronic device array
US5863708A (en) * 1994-11-10 1999-01-26 Sarnoff Corporation Partitioned microelectronic device array
US5585069A (en) * 1994-11-10 1996-12-17 David Sarnoff Research Center, Inc. Partitioned microelectronic and fluidic device array for clinical diagnostics and chemical synthesis
US5643738A (en) * 1994-11-10 1997-07-01 David Sarnoff Research Center, Inc. Method of synthesis of plurality of compounds in parallel using a partitioned solid support
US5681484A (en) * 1994-11-10 1997-10-28 David Sarnoff Research Center, Inc. Etching to form cross-over, non-intersecting channel networks for use in partitioned microelectronic and fluidic device arrays for clinical diagnostics and chemical synthesis
US5858804A (en) * 1994-11-10 1999-01-12 Sarnoff Corporation Immunological assay conducted in a microlaboratory array
US5846396A (en) * 1994-11-10 1998-12-08 Sarnoff Corporation Liquid distribution system
AU692658B2 (en) * 1994-12-16 1998-06-11 Wolfgang Pfleiderer Nucleoside derivatives with photolabile protective groups
US5763599A (en) * 1994-12-16 1998-06-09 Wolfgang Pfleiderer Nucleoside derivatives with photolabile protective groups
WO1996018634A3 (en) * 1994-12-16 1996-08-22 Wolfgang Pfleiderer Nucleoside derivatives with photolabile protective groups
WO1996018634A2 (en) * 1994-12-16 1996-06-20 Wolfgang Pfleiderer Nucleoside derivatives with photolabile protective groups
US6239273B1 (en) 1995-02-27 2001-05-29 Affymetrix, Inc. Printing molecular library arrays
US6667394B2 (en) 1995-02-27 2003-12-23 Affymetrix, Inc. Printing oligonucleotide arrays
US6331439B1 (en) 1995-06-07 2001-12-18 Orchid Biosciences, Inc. Device for selective distribution of liquids
US7674587B2 (en) 1995-11-29 2010-03-09 Affymetrix, Inc. Polymorphism detection
US6953663B1 (en) 1995-11-29 2005-10-11 Affymetrix, Inc. Polymorphism detection
US6300063B1 (en) 1995-11-29 2001-10-09 Affymetrix, Inc. Polymorphism detection
US6586186B2 (en) 1995-11-29 2003-07-01 Affymetrix, Inc. Polymorphism detection
US8095323B2 (en) 1995-11-29 2012-01-10 Affymetrix, Inc. Polymorphism detection
US6566515B1 (en) 1995-12-15 2003-05-20 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
US6147205A (en) * 1995-12-15 2000-11-14 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
US6022963A (en) * 1995-12-15 2000-02-08 Affymetrix, Inc. Synthesis of oligonucleotide arrays using photocleavable protecting groups
US7470783B2 (en) 1995-12-15 2008-12-30 Affymetrix, Inc. Photocleavable protecting groups and methods for their use
US6924094B1 (en) 1996-02-08 2005-08-02 Affymetrix, Inc. Chip-based species identification and phenotypic characterization of microorganisms
US7252948B2 (en) 1996-02-08 2007-08-07 Affymetrix, Inc. Chip-based speciation and phenotypic characterization of microorganisms
EP1721993A2 (en) 1996-04-04 2006-11-15 Affymetrix, Inc. (a Delaware Corporation) Kits and methods for the detection of target nucleic acids with help of tag nucleic acids
US6706875B1 (en) 1996-04-17 2004-03-16 Affyemtrix, Inc. Substrate preparation process
US6307042B1 (en) 1996-04-17 2001-10-23 Affymetrix, Inc. Substrate preparation process
US5959098A (en) * 1996-04-17 1999-09-28 Affymetrix, Inc. Substrate preparation process
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US7108968B2 (en) 1998-04-03 2006-09-19 Affymetrix, Inc. Mycobacterial rpoB sequences
EP0967217A2 (en) * 1998-06-22 1999-12-29 Affymetrix, Inc. (a California Corporation) Reagents and methods for solid phase synthesis and display
EP0967217B1 (en) * 1998-06-22 2005-12-21 Affymetrix, Inc. (a California Corporation) Reagents and methods for solid phase synthesis and display
WO2000061594A3 (en) * 1999-04-08 2002-04-04 Deutsches Krebsforsch Nucleoside derivatives with photo-unstable protective groups
US6756492B1 (en) 1999-04-08 2004-06-29 Deutsches Krebsforschungszentrum Stiftund Des Offentlichen Rechts Nucleoside derivatives with photo-unstable protective groups
DE19938092A1 (en) * 1999-08-12 2001-02-22 Epigenomics Gmbh Nucleoside derivatives and process for their preparation
WO2001012642A2 (en) * 1999-08-12 2001-02-22 Epigenomics Ag Nucleoside derivatives and a method for producing same
WO2001012642A3 (en) * 1999-08-12 2001-06-07 Epigenomics Ag Nucleoside derivatives and a method for producing same
DE19952113A1 (en) * 1999-10-29 2001-05-03 Nigu Chemie Gmbh Nucleoside derivatives with photolabile protecting groups
US6750335B2 (en) 1999-10-29 2004-06-15 Nigu Chemie Gmbh Nucleoside derivatives with photolabile protective groups
US6800439B1 (en) 2000-01-06 2004-10-05 Affymetrix, Inc. Methods for improved array preparation
US6806361B1 (en) 2000-03-17 2004-10-19 Affymetrix, Inc. Methods of enhancing functional performance of nucleic acid arrays
US6833450B1 (en) 2000-03-17 2004-12-21 Affymetrix, Inc. Phosphite ester oxidation in nucleic acid array preparation
US7005259B1 (en) 2000-06-01 2006-02-28 Affymetrix, Inc. Methods for array preparation using substrate rotation
WO2002020150A3 (en) * 2000-09-11 2003-03-13 Affymetrix Inc Photocleavable protecting groups
WO2002020150A2 (en) * 2000-09-11 2002-03-14 Affymetrix, Inc. Photocleavable protecting groups
EP2365098A2 (en) 2002-06-20 2011-09-14 Affymetrix, Inc. Antireflective coatings for high-resolution photolithographic synthesis of dna array
EP2497837A1 (en) 2002-06-20 2012-09-12 Affymetrix, Inc. Antireflective coatings for high-resolution photolithographic synthesis of DNA array
US8338585B2 (en) 2004-12-31 2012-12-25 Affymetrix, Inc. Parallel preparation of high fidelity probes in an array format
US8729251B2 (en) 2004-12-31 2014-05-20 Affymetrix, Inc. Parallel preparation of high fidelity probes in an array format
US8101737B2 (en) 2004-12-31 2012-01-24 Affymetrix, Inc. Parallel preparation of high fidelity probes in an array format
US8133987B2 (en) 2004-12-31 2012-03-13 Affymetrix, Inc. Primer array synthesis and validation
US8338093B2 (en) 2004-12-31 2012-12-25 Affymetrix, Inc. Primer array synthesis and validation
US8343710B1 (en) 2005-03-11 2013-01-01 The Regents Of The University Of Colorado, A Body Corporate Photodegradable groups for tunable polymeric materials
US9180196B2 (en) 2005-03-11 2015-11-10 The Regents Of The University Of Colorado, A Body Corporate Photodegradable groups for tunable polymeric materials
JP2008214343A (en) * 2007-02-07 2008-09-18 Samsung Electronics Co Ltd Photodegradable compound, substrate for oligomer probe array coupled with the photodegradable compound, oligomer probe array and method for producing the same
CN102939375A (en) * 2010-03-05 2013-02-20 医药研究委员会 Genetically encoded photocontrol
JP2013521269A (en) * 2010-03-05 2013-06-10 メディカル リサーチ カウンシル Genetically encoded light control
WO2011107747A3 (en) * 2010-03-05 2011-12-29 Medical Research Council Genetically encoded photocontrol
US11208414B2 (en) 2016-09-09 2021-12-28 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US10472364B2 (en) 2016-09-09 2019-11-12 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US10570167B2 (en) 2016-12-22 2020-02-25 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US11034715B2 (en) 2016-12-22 2021-06-15 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
US11078228B2 (en) 2018-06-21 2021-08-03 Calithera Biosciences, Inc. Ectonucleotidase inhibitors and methods of use thereof
CN114805432A (en) * 2022-03-03 2022-07-29 西北工业大学 Novel photocatalytic-cleavable carboxylic acid protecting group and preparation method of amino acid derivative thereof
EP4249495A1 (en) * 2022-03-21 2023-09-27 ATDBio Limited Method for the purification of polynucleotides and analogues thereof
WO2023180237A1 (en) * 2022-03-21 2023-09-28 ATDBio Limited Method and compound

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