Synthesis of N3- and 2-NH2-substituted 6,7-diphenylpterins and their use as intermediates for the preparation of oligonucleotide conjugates designed to target photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene.

Two 17-mer oligodeoxynucleotide-5'-linked-(6,7-diphenylpterin) conjugates, 2 and 3, were prepared as photosensitisers for targeting photooxidative damage to a 34-mer DNA oligodeoxynucleotide (ODN) fragment 1 representing the chimeric bcr-abl gene that is implicated in the pathogenesis of chronic myeloid leukaemia (CML). The base sequence in the 17-mer was 3'G G T A G T T A T T C C T T C T T5'. In the first of these ODN conjugates (2) the pterin was attached at its N3 atom, via a -(CH2)3OPO(OH)- linker, to the 5'-OH group of the ODN. Conjugate 2 was prepared from 2-amino-3-(3-hydroxypropyl)-6,7-diphenyl-4(3H)-pteridinone 10, using phosphoramidite methodology. Starting material 10 was prepared from 5-amino-7-methylthiofurazano[3,4-d]pyrimidine 4 via an unusual highly resonance stabilised cation 8, incorporating the rare 2H,6H-pyrimido[6,1-b][1,3]oxazine ring system. In the characterisation of 10 two pteridine phosphazenes, 15 and 29, were obtained, as well as new products containing two uncommon tricyclic ring systems, namely pyrimido[2,1-b]pteridine (20 and 24) and pyrimido[1,2-c]pteridine (27). In the second ODN conjugate the linker was -(CH2)5CONH(CH2)6OPO(OH)- and was attached to the 2-amino group of the pterin. In the preparation of 3, the N-hydroxysuccinimide ester 37 of 2-(5-carboxypentylamino)-6,7-diphenyl-4(3H)-pteridinone was condensed with the hexylamino-modified 17-mer. Excitation of 36 with near UV light in the presence of the single-stranded target 34-mer, 5'T G A C C A T C A A T A A G14 G A A G18 A A G21 C C C T T C A G C G G C C3' 1 caused oxidative damage at guanine bases, leading to alkali-labile sites which were monitored by polyacrylamide gel electrophoresis. Cleavage was observed at all guanine sites with a marked preference for cleavage at G14. In contrast, excitation of ODN-pteridine conjugate 2 in the presence of 1 caused oxidation of the latter predominantly at G18, with a smaller extent of cleavage at G15 and G14 (in the double-stranded portion) and G21. These results contrast with our previous observation of specific cleavage at G21 with ruthenium polypyridyl sensitisers, and suggest that a different mechanism, probably one involving Type 1 photochemical electron transfer, is operative. Much lower yields were found with the ODN-pteridine conjugate 3, perhaps as a consequence of the longer linker between the ODN and the pteridine in this case.

[1]  E. Oliveros,et al.  Singlet oxygen (^1Δ_g) production by pterin derivatives in aqueous solutions , 2003 .

[2]  J. Kelly,et al.  Targeting of photooxidative damage on single-stranded DNA representing the bcr-abl chimeric gene using oligonucleotide-conjugates containing [Ru(phen)_3]^2+-like photosensitiser groups , 2002, Photochemical & photobiological sciences : Official journal of the European Photochemistry Association and the European Society for Photobiology.

[3]  Y. Hiraku,et al.  Photohydrolysis of Methotrexate Produces Pteridine, Which Induces Poly-G–specific DNA Damage Through Photoinduced Electron Transfer¶ , 2002, Photochemistry and photobiology.

[4]  B. Nordén,et al.  Formation of DNA Triple Helices by an Oligonucleotide Conjugated to a Fluorescent Ruthenium Complex , 2002, Chembiochem : a European journal of chemical biology.

[5]  M. Morita,et al.  Synthesis of new heterocondensed pteridines , 2001 .

[6]  Gunther Hartmann,et al.  Antisense therapy in oncology: new hope for an old idea? , 2001, The Lancet.

[7]  F. Garcia-Pichel,et al.  A Novel Prokaryotic UVB Photoreceptor in the Cyanobacterium Chlorogloeopsis PCC 6912 , 2000, Photochemistry and photobiology.

[8]  K. Nakatani,et al.  Mapping of Highest Occupied Molecular Orbitals of Duplex DNA by Cobalt-Mediated Guanine Oxidation , 2000 .

[9]  N. Boutonnet,et al.  Ru‐Labeled Oligonucleotides for Photoinduced Reactions on Targeted DNA Guanines , 1999 .

[10]  M. Fontecave,et al.  Method for Preparing New Flavin Derivatives: Synthesis of Flavin−Thymine Nucleotides and Flavin−Oligonucleotide Adducts , 1997 .

[11]  J. Maier,et al.  Inhibition of pteridine biosynthesis eliminates blue-light dependent stimulation of red-light saturated photosynthesis in Laminaria saccharina (L.) Lamouroux. , 1997, Journal of photochemistry and photobiology. B, Biology.

[12]  S. Kawanishi,et al.  Photoinduced hydroxylation of deoxyguanosine in DNA by pterins: sequence specificity and mechanism. , 1997, Biochemistry.

[13]  D. Brault,et al.  Chlorin-Oligonucleotide Conjugates: Synthesis, Properties, and Red Light-Induced Photochemical Sequence-Specific DNA Cleavage in Duplexes and Triplexes†,‡ , 1996 .

[14]  H. Sugiyama,et al.  Theoretical Studies of GG-Specific Photocleavage of DNA via Electron Transfer: Significant Lowering of Ionization Potential and 5‘-Localization of HOMO of Stacked GG Bases in B-Form DNA , 1996 .

[15]  R. Lipman,et al.  An unnatural folate stereoisomer is catalytically competent in DNA photolyase. , 1996, Biochemistry.

[16]  P. Galland.  FORTY YEARS OF BLUE‐LIGHT RESEARCH AND NO ANNIVERSARY , 1992 .

[17]  W. Ried,et al.  Fused Imidazo-, Pyrimido- and 1,3-Diazepino(1,2-c)pteridines from o- Aminopyrazinecarbonitriles. , 1992 .

[18]  M. S. Jorns,et al.  Direct evidence for singlet-singlet energy transfer in Escherichia coli DNA photolyase. , 1992, Biochemistry.

[19]  F. Müller,et al.  Energy Transfer from a Lumazine (= Pteridine‐2,4(1H,3H)‐dione) Chromophore to Bathophcnanthroline‐ruthenium(II) Complexes during Hybridization Processes of DNA , 1991 .

[20]  F. Müller,et al.  Energy Transfer within Oligonucleotides from a Lumazine (= Pteridine‐2,4(1H,3H)‐dione) Chromophore to Bathophenanthroline‐ruthenium(II) Complexes , 1991 .

[21]  W. Bannwarth,et al.  Formation of carboxamides with N,N,N′,N′-tetramethyl (succinimido) uronium tetrafluoroborate in aqueous / organic solvent systems , 1991 .

[22]  M. S. Jorns,et al.  Chromophore function and interaction in Escherichia coli DNA photolyase: reconstitution of the apoenzyme with pterin and/or flavin derivatives. , 1990, Biochemistry.

[23]  F. Müller,et al.  Bathophenanthroline-ruthenium(II) complexes as non-radioactive labels for oligonucleotides which can be measured by time-resolved fluorescence techniques , 1988 .

[24]  P. Galland,et al.  THE ROLE OF PTERINS IN THE PHOTORECEPTION AND METABOLISM OF PLANTS , 1988 .

[25]  P. Boyle,et al.  Synthesis and properties of 7-alkoxyfurazano[3,4-d]pyrimidines and their use in the preparation of 4-alkoxypteridines and N3-substituted pterins , 1985 .

[26]  R. Santus,et al.  PHOTOPHYSICAL AND PHOTOSENSITIZING PROPERTIES OF 2‐AMINO‐4 PTERIDINONE: A NATURAL PIGMENT , 1981 .

[27]  T. Sugimoto,et al.  Studies on Biologically Active Pteridines. II. Synthesis of Some 2-Pyrimidinyl- and 2-Pteridinyl-amino Acids , 1979 .

[28]  牧 敬文.  Reduction of the amidine C=N bond of 7-aminofurazano(3,4-d)-pyrimidines with sodium borohydride. , 1976 .

[29]  E. Lovett,et al.  N3,O4-Ethylene-1-methyluracilium methanesulfonate. Uracil-derived heteronuclear stabilized cation , 1975 .

[30]  E. Lovett,et al.  Role of Michael adducts in pyrimidine chemistry. Reactions of 3-(.beta.-methanesulfonyloxyethyl)-1-methyluracil with bases , 1975 .

[31]  D. Abernethy,et al.  The synthesis of 2,4-diketo-5-phenyl- 5 -7-oxa-1,3-diazabicyclo(4.4.0)decane and 2,4-diketo-3-phenyl- 5 -7-oxa-1,5-diazabicyclo(4.4.0)decane. , 1972, The Journal of organic chemistry.

[32]  G. P. Beardsley,et al.  A new, general synthesis of 2-,8-, and 9-substituted adenines. , 1971, The Journal of organic chemistry.

[33]  U. Schwenk,et al.  Reaktionen mit Isobutyraldehyd, III1) 4‐Hydroxy‐2.8‐diisopropyl‐3.3.9.9‐tetramethyl‐perhydro‐pyrimido[4.3‐b] [1.3]oxazin‐6‐on , 1970 .

[34]  R. Angier,et al.  Pteridine Chemistry. IV. Structure of the Reaction Products Obtained from Acrylonitrile and 2-Amino-4-hydroxypteridines , 1959 .

[35]  Jean Cadet,et al.  Effects of duplex stability on charge-transfer efficiency within DNA , 2004 .

[36]  J. Knutson,et al.  Fluorescence properties of pteridine nucleoside analogs as monomers and incorporated into oligonucleotides. , 1997, Analytical biochemistry.

[37]  J. Cadet,et al.  Oxidative damage to DNA: formation, measurement, and biological significance. , 1997, Reviews of physiology, biochemistry and pharmacology.

[38]  Alan R. Katritzky,et al.  Comprehensive Heterocyclic Chemistry IV , 1996 .

[39]  A. McPhail,et al.  Novel Syntheses of Tricyclic, N -Aryl, Pyridine- and Pyrazine-Fused Pyrimidones , 1993 .

[40]  P. Boyle,et al.  Properties and reactions of pteridines carrying functionalised side chains at the 3-position. , 1993, Advances in experimental medicine and biology.

[41]  G. Zigeuner,et al.  Über kondensierte Heterocyclen aus β-Isothiocyanatoketonen und Aminoalkoholen bzw. Diaminen , 1976 .

[42]  G. M. Badger,et al.  The chemistry of heterocyclic compounds , 1961 .