NZ621347B2 - Embodiments of a probe and method for targeting nucleic acids - Google Patents
Embodiments of a probe and method for targeting nucleic acids Download PDFInfo
- Publication number
- NZ621347B2 NZ621347B2 NZ621347A NZ62134712A NZ621347B2 NZ 621347 B2 NZ621347 B2 NZ 621347B2 NZ 621347 A NZ621347 A NZ 621347A NZ 62134712 A NZ62134712 A NZ 62134712A NZ 621347 B2 NZ621347 B2 NZ 621347B2
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- New Zealand
- Prior art keywords
- probe
- monomer
- human
- nucleic acid
- probe according
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- MSMNPNAQGHVRBA-UHFFFAOYSA-N chloro-[di(propan-2-yl)amino]phosphinite Chemical compound CC(C)N(C(C)C)P([O-])Cl MSMNPNAQGHVRBA-UHFFFAOYSA-N 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 229910000424 chromium(II) oxide Inorganic materials 0.000 description 1
- 238000010549 co-Evaporation Methods 0.000 description 1
- 230000004186 co-expression Effects 0.000 description 1
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- 230000001268 conjugating Effects 0.000 description 1
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- 239000000356 contaminant Substances 0.000 description 1
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- 238000007796 conventional method Methods 0.000 description 1
- ZIBSSYPUGNXJFJ-UHFFFAOYSA-N coronene-1-carbaldehyde Chemical compound C1=C2C(C=O)=CC3=CC=C(C=C4)C5=C3C2=C2C3=C5C4=CC=C3C=CC2=C1 ZIBSSYPUGNXJFJ-UHFFFAOYSA-N 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
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- 238000005661 deetherification reaction Methods 0.000 description 1
- 238000006392 deoxygenation reaction Methods 0.000 description 1
- 239000005549 deoxyribonucleoside Substances 0.000 description 1
- 230000001419 dependent Effects 0.000 description 1
- 238000010511 deprotection reaction Methods 0.000 description 1
- 238000005828 desilylation reaction Methods 0.000 description 1
- 230000001627 detrimental Effects 0.000 description 1
- 239000011903 deuterated solvents Substances 0.000 description 1
- SAIKDASRPDRSGZ-UHFFFAOYSA-O di(propan-2-yl)azanium;1,2,3-triaza-4-azanidacyclopenta-2,5-diene Chemical compound C1=NN=N[N-]1.CC(C)[NH2+]C(C)C SAIKDASRPDRSGZ-UHFFFAOYSA-O 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 150000004845 diazirines Chemical class 0.000 description 1
- 150000008049 diazo compounds Chemical class 0.000 description 1
- 229940043237 diethanolamine Drugs 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 150000002009 diols Chemical class 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- BVQAWSJMUYMNQN-UHFFFAOYSA-N dipyridophenazine Chemical compound C1=CC=C2C3=NC4=CC=CC=C4N=C3C3=CC=CN=C3C2=N1 BVQAWSJMUYMNQN-UHFFFAOYSA-N 0.000 description 1
- 201000008325 diseases of cellular proliferation Diseases 0.000 description 1
- OPCPRUQQEJNFIV-UHFFFAOYSA-N disodium;cyanoboron(1-) Chemical compound [Na+].[Na+].[B-]C#N.[B-]C#N OPCPRUQQEJNFIV-UHFFFAOYSA-N 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- WRZXKWFJEFFURH-UHFFFAOYSA-N dodecaethylene glycol Chemical compound OCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCOCCO WRZXKWFJEFFURH-UHFFFAOYSA-N 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000005670 electromagnetic radiation Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 201000003914 endometrial carcinoma Diseases 0.000 description 1
- 230000002708 enhancing Effects 0.000 description 1
- 230000002255 enzymatic Effects 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 102000015694 estrogen receptors Human genes 0.000 description 1
- 108010038795 estrogen receptors Proteins 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000002349 favourable Effects 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000011737 fluorine Substances 0.000 description 1
- YCKRFDGAMUMZLT-UHFFFAOYSA-N fluorine atom Chemical compound [F] YCKRFDGAMUMZLT-UHFFFAOYSA-N 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000003197 gene knockdown Methods 0.000 description 1
- 150000002338 glycosides Chemical class 0.000 description 1
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 1
- 239000004519 grease Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 125000005179 haloacetyl group Chemical group 0.000 description 1
- 125000004404 heteroalkyl group Chemical group 0.000 description 1
- 239000008241 heterogeneous mixture Substances 0.000 description 1
- 239000008079 hexane Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 150000002429 hydrazines Chemical class 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 150000002463 imido esters Chemical class 0.000 description 1
- QLNAVQRIWDRPHA-UHFFFAOYSA-N iminophosphane Chemical compound P=N QLNAVQRIWDRPHA-UHFFFAOYSA-N 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000006192 iodination reaction Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002513 isocyanates Chemical class 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 150000007527 lewis bases Chemical class 0.000 description 1
- WHXSMMKQMYFTQS-UHFFFAOYSA-N lithium Chemical compound [Li] WHXSMMKQMYFTQS-UHFFFAOYSA-N 0.000 description 1
- 229910052744 lithium Inorganic materials 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 229960003646 lysine Drugs 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 230000003211 malignant Effects 0.000 description 1
- 229920002106 messenger RNA Polymers 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- XPDWGBQVDMORPB-UHFFFAOYSA-N methyl trifluoride Chemical compound FC(F)F XPDWGBQVDMORPB-UHFFFAOYSA-N 0.000 description 1
- 238000007069 methylation reaction Methods 0.000 description 1
- 239000002679 microRNA Substances 0.000 description 1
- 229920001239 microRNA Polymers 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 238000002703 mutagenesis Methods 0.000 description 1
- 231100000350 mutagenesis Toxicity 0.000 description 1
- IMNFDUFMRHMDMM-UHFFFAOYSA-N n-heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000001613 neoplastic Effects 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 239000012299 nitrogen atmosphere Substances 0.000 description 1
- 229910000510 noble metal Inorganic materials 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 239000002853 nucleic acid probe Substances 0.000 description 1
- 239000002751 oligonucleotide probe Substances 0.000 description 1
- 238000000238 one-dimensional nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 239000011886 peripheral blood Substances 0.000 description 1
- 229940083251 peripheral vasodilators Purine derivatives Drugs 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 108010042024 pertuzumab Proteins 0.000 description 1
- IQZOCQOQNWTNOW-UHFFFAOYSA-N perylene-3-carbaldehyde Chemical compound C=12C3=CC=CC2=CC=CC=1C1=CC=CC2=C1C3=CC=C2C=O IQZOCQOQNWTNOW-UHFFFAOYSA-N 0.000 description 1
- 239000008194 pharmaceutical composition Substances 0.000 description 1
- GLUUGHFHXGJENI-UHFFFAOYSA-N piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000001184 potassium carbonate Substances 0.000 description 1
- 229910000027 potassium carbonate Inorganic materials 0.000 description 1
- 230000002335 preservative Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 102000003998 progesterone receptors Human genes 0.000 description 1
- 108090000468 progesterone receptors Proteins 0.000 description 1
- 230000002062 proliferating Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- NGDMLQSGYUCLDC-UHFFFAOYSA-N pyren-1-ylmethanol Chemical compound C1=C2C(CO)=CC=C(C=C3)C2=C2C3=CC=CC2=C1 NGDMLQSGYUCLDC-UHFFFAOYSA-N 0.000 description 1
- 239000002096 quantum dot Substances 0.000 description 1
- 230000000171 quenching Effects 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 102000027656 receptor tyrosine kinases Human genes 0.000 description 1
- 108091007921 receptor tyrosine kinases Proteins 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 239000003638 reducing agent Substances 0.000 description 1
- 230000022983 regulation of cell cycle Effects 0.000 description 1
- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 239000002342 ribonucleoside Substances 0.000 description 1
- 239000002336 ribonucleotide Substances 0.000 description 1
- 125000002652 ribonucleotide group Chemical group 0.000 description 1
- 125000000548 ribosyl group Chemical group C1([C@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 150000003335 secondary amines Chemical class 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Inorganic materials [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000007614 solvation Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 239000003381 stabilizer Substances 0.000 description 1
- 230000000087 stabilizing Effects 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 201000011549 stomach cancer Diseases 0.000 description 1
- 125000003107 substituted aryl group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000003871 sulfonates Chemical class 0.000 description 1
- 150000003463 sulfur Chemical class 0.000 description 1
- 230000004083 survival Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tBuOOH Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 1
- 125000000999 tert-butyl group Chemical group [H]C([H])([H])C(*)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- FGTJJHCZWOVVNH-UHFFFAOYSA-N tert-butyl-[tert-butyl(dimethyl)silyl]oxy-dimethylsilane Chemical compound CC(C)(C)[Si](C)(C)O[Si](C)(C)C(C)(C)C FGTJJHCZWOVVNH-UHFFFAOYSA-N 0.000 description 1
- 150000003512 tertiary amines Chemical class 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 229910052723 transition metal Inorganic materials 0.000 description 1
- 150000003624 transition metals Chemical class 0.000 description 1
- BBUQUBATODVRRV-UHFFFAOYSA-N triacetyloxyboron(1-) Chemical compound CC(=O)O[B-](OC(C)=O)OC(C)=O BBUQUBATODVRRV-UHFFFAOYSA-N 0.000 description 1
- 150000008648 triflates Chemical class 0.000 description 1
- 125000004044 trifluoroacetyl group Chemical group FC(C(=O)*)(F)F 0.000 description 1
- 125000001889 triflyl group Chemical group FC(F)(F)S(*)(=O)=O 0.000 description 1
- SIOVKLKJSOKLIF-CMDGGOBGSA-N trimethylsilyl (1E)-N-trimethylsilylethanimidate Chemical compound C[Si](C)(C)OC(/C)=N/[Si](C)(C)C SIOVKLKJSOKLIF-CMDGGOBGSA-N 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- 125000002264 triphosphate group Chemical class [H]OP(=O)(O[H])OP(=O)(O[H])OP(=O)(O[H])O* 0.000 description 1
- 125000002221 trityl group Chemical group [H]C1=C([H])C([H])=C([H])C([H])=C1C([*])(C1=C(C(=C(C(=C1[H])[H])[H])[H])[H])C1=C([H])C([H])=C([H])C([H])=C1[H] 0.000 description 1
- 210000004881 tumor cells Anatomy 0.000 description 1
- 229910052721 tungsten Inorganic materials 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 201000005112 urinary bladder cancer Diseases 0.000 description 1
- 230000003612 virological Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
- C07H19/06—Pyrimidine radicals
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6879—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for sex determination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/689—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for bacteria
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T436/00—Chemistry: analytical and immunological testing
- Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
- Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
- Y10T436/143333—Saccharide [e.g., DNA, etc.]
Abstract
The disclosure relates to a double stranded probe comprising a pair of monomers of the general formula as shown in the abstract figure and at least one nucleotide. The pair of monomers may be arranged in a manner that promotes thermoinstability of the probe complex, thus producing a probe capable of locating and/or detecting a target. The probe also may comprise one or more natural or non-natural nucleotides capable of Watson-Crick base pairing with an isosequential nucleic acid target. Particular disclosed embodiments concern a method of using the disclosed probe to target nucleic acids. In particular disclosed embodiments, the probe may be incubated with a target nucleic acid and then be detected. locating and/or detecting a target. The probe also may comprise one or more natural or non-natural nucleotides capable of Watson-Crick base pairing with an isosequential nucleic acid target. Particular disclosed embodiments concern a method of using the disclosed probe to target nucleic acids. In particular disclosed embodiments, the probe may be incubated with a target nucleic acid and then be detected.
Description
EMBODIMENTS OF A PROBE AND METHOD FOR
TARGETING NUCLEIC ACIDS
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of the earlier filing dates of U.S. provisional application
No. 61/509,336, filed on July 19, 2011, and U.S. provisional application No. 61/542,044, filed on
September 30, 2011, both of which prior applications are incorporated herein by reference in their
entirety.
FIELD
Disclosed herein are embodiments of a probe capable of targeting nucleic acids and
particular sequences thereof. Also disclosed are methods for using the disclosed probe to target
nucleic acids and sequences thereof.
ACKNOWLEDGMENT OF GOVERNMENT SUPPORT
This invention was made with government support under R01 GM088697 awarded by the
National Institute of General Medical Sciences, under P20 RR016448 awarded by the National
Institute of Health, and under a grant awarded by INBRE-Institute of Translational Health Sciences.
The government has certain rights in the invention.
BACKGROUND
The use of short exogenous antisense oligonucleotides (ONs) or siRNA to silence gene
expression on an RNA level has become an immensely popular approach to study fundamental
functions of genes, to detect genes of interest, and to design new drugs against diseases of genetic
origin. For development of high impact therapeutics with broad application, strategies that directly
target the gene offer a powerful alternative to conventional therapies.
Progress in sequence-selective targeting of double stranded DNA (dsDNA) has been
accomplished with minor groove binding polyamides or by DNA triple-helix-based approaches using
modified oligonucleotides or helix-invading peptide nucleic acids (PNAs). However, the utility of
these methods is limited by the sequence-dependent microstructure of the minor groove of DNA
duplexes (polyamides), by target sequence restrictions (triplex-based approaches), or by the necessity
of non-physiological salt concentrations (PNAs). An attractive alternative approach was introduced
with pseudo-complementary DNA (pcDNA), i.e., DNA duplexes containing modified purine and
pyrimidines that do not form stable base pairs with each other, while allowing hybridization to
natural complementary DNA. pcDNA is able to strand invade blunt ended duplexes containing
terminal mixed sequence target regions and this strategy has been extended into pseudo-
complementary PNA (pcPNA), which has been used to target mixed sequence internal target regions
6478592_1 (GHMatters) P95976.NZ ESTHERJ
of double stranded DNA. Unfortunately, the positively charged lysine residues commonly used to
increase pcPNA solubility and binding affinity may lead to self-inhibitory effects of strand invasion
at high probe concentrations. The requirement for low salt concentrations during pcPNA-mediated
strand invasion of mixed sequence dsDNA is a limitation for all experiments in biological media and
for numerous biotechnological applications. Development of alternative strategies for sequence
selective recognition of dsDNA at physiologically relevant salt concentrations is therefore highly
desirable.
SUMMARY
Particular disclosed embodiments concern a probe, comprising: a pair of monomers
comprising a first monomer having a formula
where each Y independently is selected from carbon, oxygen, sulfur, a triazole, and NR , wherein R
is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; V is selected from carbon,
b 1 2
oxygen, sulfur, and NR ; n ranges from 0 to 4; R and R are selected from hydrogen, aliphatic, aryl,
and a heteroatom-containing moiety; R is a heteroatom-containing functional group; R is selected
from any natural or non-natural nucleobase; R is selected from any aromatic moiety suitable for
intercalating within a nucleic acid; “optional linker” is selected from alkyl, amide, carbamate,
carbonate, urea, and combinations thereof; a second monomer having a formula
;
and at least one nucleotide selected from a natural nucleotide, a non-natural nucleotide, and
combinations thereof, wherein the at least one nucleotide typically is coupled to the first and/or
second monomer at R and/or R by a phosphate group.
In particular disclosed embodiments, the heteroatom-containing functional group is selected
a b a a b c d a b c a
from ether (R OR ), hydroxyl (R OH), silyl ether (R R R SiOR ), phosphine (PR R R ), thiol (R SH),
a b a b a a
thioether/sulfide (R SR ), disulfide (R SSR ), isothiocyanate (R NCS), isocyanate (R NCO), amine
a a b a b c a b
(NH , NHR , NR R ), amide (R NR C(O)R ), ester (R OC(O)R ), halogen (I, Br, Cl, F), carbonate
a b a a - a b
(R OC(O)OR ), carboxyl (R C(O)OH), carboxylate (R COO ), ester (R C(O)OR ), ketone
a b a a a b
(R C(O)R ), phosphate (R OP(O)OH ), phosphoryl (R P(O)(OH) ), sulfinyl (R S(O)R ), sulfonyl
6478592_1 (GHMatters) P95976.NZ ESTHERJ
a b a b a a a
(R SO R ), carbonothioyl (R C(S)R or R C(S)H), sulfino (R S(O)OH), sulfo (R SO H), amide
a b c a a + - a a
(R C(O)NR R ), azide (N ), nitrile (R CN), isonitrile (R N C ), and nitro (R NO ); R represents the
remaining monomer structure, which is attached to the abovementioned functional groups at the
b c d
position indicated; and R , R , and R independently are hydrogen, aliphatic, aryl, heteroaliphatic,
heteroaryl, and any combination thereof. In certain embodiemnts, R and R independently are
selected from a heteroatom functional group comprising phosphorous, sulfur, nitrogen, oxygen,
selenium, and/or a metal, more typically R and R independently are selected from a phosphate
group of the natural nucleotide, non-natural nucleotide, synthetic nucleotides, or combinations
thereof. In particular disclosed embodiments, R is selected from adenine, guanine, cytosine, uracil,
thymine, or any derivative thereof and the intercalator is an aromatic hydrocarbon or an aromatic
heterocycle. In particular disclosed embodiments, the intercalator is a hydrocarbon selected from
pyrene, coronene, perylene, anthracene, naphthalene and functionalized derivatives thereof; or may
be an aromatic heterocycle selected from a porphyrin, nucleobase, metal chelator, azapyrene, thiazole
orange, indole, pyrrole, and derivatives thereof.
Certain disclosed embodiments concern a probe having at least one monomer, and typically
two or more monomers. The monomers are generally incorporated within oligonucleotide strands,
and can be located anywhere in the oligonucleotide strand, including the first position, the last
position, and anywhere in between, and preferably are arranged in a final structure in an interstrand
zipper arrangement, such as a +1 zipper arrangement, as dicussed in more detail below. Certain
disclosed embodiments concern probes wherein either the first monomer or the second monomer has
a formula
, or
, or
6478592_1 (GHMatters) P95976.NZ ESTHERJ
In certain disclosed embodiments, either the first monomer or the second monomer has a
formula
, or
, or
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine(3H)-one or derivatives thereof.
Particular disclosed embodiments of the probe comprise monomers having any one of the
following structures:
6478592_1 (GHMatters) P95976.NZ ESTHERJ
e e e e
R O B R O B R O B
R O B
O O O
O O O
f O f f
OR OR OR OR
R O R O e
R O R O
OR N OR N f
OR N
OR N
OR N
Py Pery
R O R O
OR OR
Nap Py
R O O
OR N
6478592_1 (GHMatters) P95976.NZ ESTHERJ
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine(3H)-one or derivatives thereof, R is H,
DMTr, or phosphate, such as provided by a phosphodiester bond in an oligonucleotide and R is H,
(N(i-Pr) )P(OCH CH CN), or phosphate, such as provided by a phosphodiester bond in an
2 2 2
oligonucleotide; Nap refers to napthyl, such as 2-napthyl, Cor to coronenyl, such as coronenyl, Py
to pyrenyl, such as pyrenyl, pyrenyl and pyrenyl and Pery to peryleneyl, such as to
peryleneyl.
In particular disclosed embodiments, the at least one natural nucleotide may be selected
from adenine, guanine, cytosine, uracil, thymine and derivatives thereof, and the at least one non-
natural nucleobase may be selected from C5-functionalized pyrimidines, C6-functionalized
pyrimidines, C7-functionalized 7-deazapurines, C8-functionalized purines, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimidine(3H)-one or derivatives thereof. In certain
disclosed embodiments, the probe comprises at least one natural nucleotide, unnatural nucleotide, and
combinations thereof, which are selected to substantially match at least one nucleotide of a
corresponding nucleic acid sequence.
Particular disclosed embodiments concern a probe having the following formula:
’(B B …B )(XA)(B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r j 1 2
…B )
3’(C C …C )(DP)(C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r j 1 2
…C )
wherein B , B and B may be any natural or non-natural nucleotide, wherein m-s ranges from zero
1 2 m-s
to about 28; f, g, h, i and j range from 0 to 10, more likely 0 to 5, and typically 0 to 3; X is the first
monomer; A is the complement Watson-Crick base pairing nucleotide of P; C is any natural or non-
natural nucleotide capable of Watson-Crick base pairing with any one of B , B and B ; P is the
1 2 m-s
second monomer, and D is the complement Watson-Crick base pairing nucleotide of X. In certain
disclosed embodiments, the probe is selected to recognize a predetermined sequence of a nucleic acid
target, which may be single-stranded or double-stranded, more commonly double-stranded. In
certain disclosed embodiments, the probe may additionally comprise one or more monomers at any
given position that do not participate in base pairing, such as the following structures, wherein R is
H, DMTr, or phosphate, such as provided by a phosphodiester bond in an oligonucleotide and R is H,
(N(i-Pr) )P(OCH CH CN), or phosphate, such as provided by a phosphodiester bond in an
2 2 2
oligonucleotide, and R and R are, by way of example and without limitation, independently selected
from hydrogen, aliphatic, particularly alkyl, such as C1-C10 alkyl, cyclic, heterocyclic, aromatic,
heteroaromatic, amides, and carbamates .
6478592_1 (GHMatters) P95976.NZ ESTHERJ
R O OR
Particular disclosed embodiments concern a probe wherein the first monomer and the second
monomer are arranged in a manner that substantially weakens the duplex’s thermal stability. In
particular disclosed embodiments, the probe may have a thermal melting temperature which is
comparable (slightly lower, similar or slightly higher) to that of a corresponding (i.e., isosequential)
unmodified nucleic acid duplex, which typically does not comprise a monomer having the formulas
disclosed herein. The probe may comprise a pair of monomers wherein the first monomer and the
second monomer are arranged in a +n or -n interstrand zipper orientation, wherein n ranges from 0 to
about 10, more typically from 0 to about 2, more typically the first monomer and the second
monomer are arranged in a +n orientation, wherein n is 1. The probe may also comprise one or more
additional pairs of monomers; a signal generating moiety capable of being detected, selected from a
fluorophore, a member of a specific binding pair (e.g. biotin), a nanoparticle, a signal quenching
moiety, such as a quencher of fluorescence (e.g., Black Hole Quencher); permanent or inducible
crosslinking reagents (such as psoralen) capable of forming bonds between the probe and nucleic
acids, proteins, sugars, lipids and other biomolecules; a nucleic acid cargo (e.g., single-stranded
DNA, single-stranded RNA, double-stranded DNA, double-stranded RNA, plasmid, gene); and
combinations thereof .In particular disclosed embodiments, the secondary entity facilitates cell-
uptake and/or cellular compartmentalization and includes peptides [NLS, CPP, KDEL], and small
molecules with nuclear affinity (e.g. Hoechst-type dyes, ethidium, acridine and thiazole orange). The
probe may be used in solution, on a solid surface (e.g. multi-well plates; noble metal surfaces, such as
electrodes), or in combination with a colloidal material and/or nanomaterials (e.g. gold nanoparticles,
quantum dots).
Certain disclosed embodiments concern a method for detecting a target, comprising:
selecting a probe comprising a monomer having a formula
6478592_1 (GHMatters) P95976.NZ ESTHERJ
where each Y independently is selected from oxygen, sulfur, and NR , wherein R is selected from
hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; V is selected from carbon, oxygen, sulfur,
b 1 2
and NR ; n ranges from 0 to 4; R and R are selected from hydrogen, aliphatic, aryl, and a
heteroatom-containing moiety; R is a heteroatom-containing functional group; R is selected from
any natural or non-natural nucleobase; R is selected from any functional group suitable for coupling
to or associating with a nucleic acid and at least one natural nucleotide, unnatural nucleotide, and
combinations thereof; exposing a nucleic acid target to the probe; and detecting the probe. In
particular disclosed embodiments of the method, the probe is selected to substantially match a region
of the target nucleic acid, particularly double stranded nucleic acid target regions, which may or may
not comprise one or more polypurinestretches; more typically the nucleic acid is a mixed sequence of
nucleotides, structured nucleic acid, particularly double-stranded nucleic acid sequences (dsDNA),
even more particularly double stranded DNA, such as by way of example a mixed-sequence, hairpin
DNA targets, PCR amplicons, genomic DNA, etc. which are isosequential with the probe. In
particular disclosed embodiments, exposing the target to the probe comprises incubating the probe
with the nucleic acid target. In certain disclosed embodiments, the nucleic acid target is incubated
with an excess, such as about a 5-fold excess of the probe up to at least about a 5,000-fold excess of
the probe, more typically up to about a 500-fold excess of the probe, and even more typically about a
-fold excess of the probe to about a 200-fold excess of the probe. In particular disclosed
embodiments, the probe and probe-target (recognition) complex, is detected by fluorescence
spectroscopy, electrophoresis, absorption spectroscopy, fluorescence microscopy, flow cytometer,
and combinations thereof.
A person of ordinary skill in the art will appreciate that the target sequences for disclosed
probe embodiments can vary. For certain disclosed embodiments, the method is particularly useful
for gender determination in mammals. For example, the method can be used for gender
determination of ungulates and ruminates, particularly bovines, equines or porcines. For other
embodiments, the target is isosequential (relative to a probe) to double stranded DNA target regions,
including stems of molecular beacons, target regions embedded within PCR amplicons, target regions
embedded within circular or linearized plasmids, target regions embedded within genomic DNA, and
target regions embedded within microorganisms. The target also can be selected from a nucleic acid
sequence associated with a proliferative disorder, such as B cell and T cell leukemias, lymphomas,
breast cancer, colon cancer, and neurological cancers. In yet other embodiments, the target is
6478592_1 (GHMatters) P95976.NZ ESTHERJ
selected from a virus or other microorganism and the probe is used to detect and/or identify
the microorganism.
Kids comprising a nucleic acid probe also are disclosed. Such kits typically comprise a
probe comprising at least one disclosed monomer, and may comprise at least one bulge monomer.
Such kits also may include a sequence selected from SEQ ID NOs. 1-254. Certain kit embodiments
are particularly useful for gender determination in mammals.
The present invention as claimed herein is described in the following items 1 to 88;
1. A double stranded probe, comprising:
a pair of monomers comprising a first monomer having a formula
Linker
Optional
1 b b
where Y is selected from carbon, oxygen, sulfur, and NR , wherein R is selected from hydrogen,
2 3 4
aliphatic, aryl, heteroaliphatic, and heteroaryl; each of Y , Y , and Y independently is selected from
carbon, oxygen, sulfur, a triazole, oxazole, tetrazole, isoxazole, and NR , wherein R is selected from
hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; R and R are selected from hydrogen,
aliphatic, aryl, aryl aliphatic, and a heteroatom-containing moiety, or R is selected from a
heteroatom-containing functional group; R is a heteroatom-containing functional group; R is
selected from any natural or non-natural nucleobase; R is selected from an intercalator suitable for
intercalating within a nucleic acid selected from a hydrocarbon or an aromatic heterocycle; “optional
linker” is selected from linkers comprising alkyl linkers, amide linkers, carbonyl linkers, carbamate
linkers, carbonate linkers, urea linkers, and combinations thereof;
a second monomer having a formula
Linker
Optional
1 2 3 4 1 2 3 4 5
wherein Y , Y , Y , Y , R , R R , R , R , and “optional linker” are as stated for the first monomer; V
is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4; and
wherein the first monomer is positioned in a first strand of the double-stranded probe and the
second monomer is positioned in a second strand of the double stranded probe and wherein each of
the first strand and the second strand comprises at least one nucleotide selected from a natural
nucleotide, a non-natural nucleotide, and combinations thereof.
2. The probe according to item 1 wherein the heteroatom-containing moiety is
a b a a b c d a b c
selected from ether (R OR ), hydroxyl (R OH), silyl ether (R R R SiOR ), phosphine (PR R R ),
a a b a b a
thiol (R SH), thioether/sulfide (R SR ), disulfide (R SSR ), isothiocyanate (R NCS), isocyanate
a a a b a b c a b
(R NCO), amine (NH , NHR , NR R ), amide (R NR C(O)R ), ester (R OC(O)R ), halogen (I, Br, Cl,
a b a a - a b
F), carbonate (R OC(O)OR ), carboxyl (R C(O)OH), carboxylate (R COO ), ester (R C(O)OR ),
a b a a a b
ketone (R C(O)R ), phosphate (R OP(O)OH ), phosphoryl (R P(O)(OH) ), sulfinyl (R S(O)R ),
a b a b a a a
sulfonyl (R SO R ), carbonothioyl (R C(S)R or R C(S)H), sulfino (R S(O)OH), sulfo (R SO H),
a b c a a + - a a
amide (R C(O)NR R ), azide (N ), nitrile (R CN), isonitrile (R N C ), and nitro (R NO ); R
represents the remaining monomer structure, which is attached to the abovementioned functional
1 b c d
groups at the position indicated for R ; and R , R , and R independently are hydrogen, aliphatic, aryl,
heteroaliphatic, heteroaryl, and any combination thereof.
3. The probe according to item 1 or 2 wherein R and R independently are selected
from a heteroatom-containing functional group comprising phosphorous, sulfur, nitrogen, oxygen,
selenium, and/or a metal.
4. The probe according to item 1 or 2 wherein R and R independently are selected
from a phosphate group of a natural nucleotide, non-natural nucleotide, non-nucleosidic linker, or
combinations thereof.
. The probe according to claim 1 or 2 wherein R and R independently have a
formula
where each Y independently is selected from oxygen, sulfur, NR where R is selected from
hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and W is selected from phosphorus, SH, or SeH.
6. The probe according to claim 1 or 2 wherein R and R independently are
, or .
7. The probe according to item 1 or 2 where R and R independently have a formula
where W is phosphorus, and each Z independently is selected from ether, thioether, hydroxyl, and
NR .
8. The probe according to item 1 or 2 wherein R and R independently
are .
9. The probe according to any one of items 1 to 8 wherein R is selected from
adenine, guanine, cytosine, uracil, thymine, 2-thiouracil, 2,6-diaminopurine, inosine, 3-pyrrolo-[2,3-
d]-pyrimdine(3H)-one, or any derivative thereof.
. The probe according to item 1 wherein the intercalator is a hydrocarbon selected
from pyrene, coronene, perylene, anthracene, naphthalene, and functionalized derivatives thereof.
11. The probe according to item 1 wherein the intercalator is an aromatic heterocycle
selected from a porphyrin, nucleobase, metal chelator, azapyrene, thiazole orange, ethidium, an
indole, a pyrrole, a benzimidizole, and modified analogs thereof.
12. The probe according to item 1 wherein the second monomer has a formula
Linker
Optional
13. The probe according to item 1 wherein either the first monomer or the second
monomer has a formula
Linker
O Optional
.
14. The probe according to item 1 wherein the second monomer has a formula
Linker
Optional
. The probe according to item 1 wherein either the first monomer or the second
monomer has a formula
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof.
16. The probe according to item 1 wherein either the first monomer or the second
monomer has a formula
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof, and alk is
C1-C10 alkyl.
17. The probe according to item 1 wherein the second monomer has a formula
wherein B is selected from uracil, guanine, cytosine, adenine, or thymine.
18. The probe according to item 1 wherein either the first monomer or the second
monomer has a formula
19. The probe according to item 1 wherein either the first monomer or the second
monomer has any one of the following formulas:
R O O
OR O
N p P r Py
y Co
f O N
OR N OR
OR N
O y y
R O B
R O B
OR N NH f
OR N
2 OR N
R O B R O B
OR N N
N OR
N N y
e e e
R O B R O B R O B R O B
O O O O
O O O O
f f f f
OR OR OR
R O R O
OR OR
Nap Py
OR N f
OR N
R O O
OR N
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H,
DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or
2 2 2
phosphate.
20. The probe according to item 1 wherein the second monomer has any one of the
following formulas:
e moc
21. The probe according to item 1 wherein the at least one natural nucleotide is selected
from adenine, guanine, cytosine, uracil, and thymine.
22. The probe according to item 1 wherein the at least one non-natural nucleobase is
selected from C-5 functionalized pyrimidines, C6-functionalized pyrimidines, C7-functionalized 7-
deazapurines, C8-functionalized purines, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, deoxyinosine
and 3-(2’-deoxy- β-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimdine(3H)-one.
- 13a -
23. The probe according to item 1 wherein the at least one natural nucleotide, unnatural
nucleotide, and combinations thereof is selected to substantially match at least one nucleotide of a
corresponding nucleic acid sequence.
24. The probe according to any one of items 1 to 23 having a formula
’(B B …B )(XA)(B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r
XA) (B B …B )
j 1 2 s
3’(C C …C )(DP)(C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r
DP) (C C …C )
j 1 2 s
wherein B , B and B may be any natural, non-natural nucleotide, or a non-nucleosidic linker,
1 2 m-s
wherein m-r ranges from zero to about 28; f, g, h, i and j range from zero to 10; X is the first
monomer; A is the complement Watson-Crick base pairing nucleotide of P; C is any natural or non-
natural nucleotide capable of Watson-Crick base pairing with any one of B , B and B ; P is the
1 2 m-s
second monomer, and D is the complement Watson-Crick base pairing nucleotide of X.
. The probe according to any one of items 1 to 24 wherein the probe is selected to
recognize a predetermined sequence of a nucleic acid target.
26. The probe according to item 25 wherein the nucleic acid target is single-stranded or
double-stranded.
27. The probe according to any one of items 1 to 26 further comprising one or more
bulge monomers that do not participate in base pairing.
28. The probe according to item 27 wherein the one or more bulge monomers are
selected from
R O OR
- 13b -
29. The probe according to any one of items 1 to 28 wherein the first monomer and the
second monomer are arranged in a manner that substantially weakens the thermal stability of a duplex
comprising two strands of oligonucleotides comprising one or more of the first monomer and the
second monomer.
. The probe according to item 29 wherein the duplex has a thermal melting
temperature which is substantially similar to, or lower than, that of a corresponding unmodified
nucleic acid duplex.
31. The probe according to item 30 wherein the corresponding unmodified nucleic acid
duplex does not comprise a monomer having the formula of claim 1.
33. The probe according to item 1 wherein the first monomer and the second monomer
are arranged in a +n or -n zipper orientation, wherein n ranges from 0 to about 10.
33. The probe according to item 32 wherein the first monomer and the second
monomer are arranged in a +n orientation, wherein n is 1.
34. The probe according to any one of items 1 to 33 further comprising one or more
additional pairs of monomer pairs.
. The probe according to any one of items 1 to 34 further comprising a signal
generating moiety capable of being detected.
36. The probe according to item 35 wherein the signal generating moiety is selected
from a fluorophore, a member of a specific binding pair, a nanoparticle, and combinations thereof.
37. The probe according to item 36 wherein the member of a specific binding pair is
biotin.
38. The probe according to any one of items 1 to 37 further comprising a secondary
entity selected from a secondary entity that facilitates cell-uptake; a quencher; a crosslinking reagent
capable of forming bonds between the probe and nucleic acids, proteins, sugars, lipids or other
biomolecules; a nucleic acid cargo selected from single-stranded DNA, single-stranded RNA, double-
stranded DNA, double-stranded RNA, plasmid, or gene; and combinations thereof.
39. The probe according to any one of items 1 to 38 wherein the probe is used in
solution, on a solid surface, or in combination with a colloidal material.
- 13c -
40. A single stranded probe precursor when used to make the double stranded probe of
item 1, comprising at least one monomer selected from
e R O
f O OR
Co O y
R O B e
R O B
R N f
N O NH
P 2 OR N P
O R O B
OR N N OR N
e e e e
R O B R O B R O B
R O B
O O O
O O O
f O f f
OR OR OR OR
R O R O
f f f
R N f
OR OR
OR N
Nap Py C
R O O
OR N
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H,
DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or
2 2 2
phosphate; or
- 13d -
OR O
wherein B is selected from guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-diaminopurine,
inosine, or 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one; Nap is napthyl; Py is pyrenyl; R is H, DMTr, or
phosphate; and R is H, (N(i-Pr) )P(OCH CH CN), or phosphate.
2 2 2
41. The single stranded probe precursor according to item 40 further comprising a
second monomer selected from
e moc
e B O
OR N
f OR N
OR O O
Na P P
p y or O y
R O B
R O B
OR N NH f
OR N
2 OR N
R O B R O B
OR N N
N OR
N N y
e e e
R O B R O B R O B R O B
O O O O
O O O O
f f f f
OR OR OR
- 13e -
R O R O
OR OR
Nap Py
R N f
OR N
R O O
OR N
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H,
DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or
2 2 2
phosphate.
42. A single stranded probe precursor when used to make the double stranded probe of
item 1, wherein the single stranded probe precursor has a sequence selected from any one of SEQ ID
Nos. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, and 246-251.
43. A double stranded probe according to item 1 for use in associating the probe with a
nucleic acid target.
44. The probe according to item 43 wherein the double stranded probe is selected to
substantially match a target nucleic acid sequence.
45. The probe according to any one of items 43-44 wherein the nucleic acid target
comprises one or more polypurine units.
46. The probe according to any one of items 43-44 wherein the nucleic acid target does
not comprise a polypurine unit.
- 13f -
47. The probe according to any one of items 43-46 wherein the nucleic acid target is a
mixed-sequence, structured nucleic acid.
48. The probe according to any one of items 43-47 wherein the nucleic acid target is a
mixed-sequence, hairpin nucleic acid target.
49. The probe according to any one of items 43-48 wherein the nucleic acid target is
isosequential with the double stranded probe.
50. The probe according to any one of items 43-48 wherein the nucleic acid target is
incubated with the double stranded probe.
51. The probe according to any one of items 43-50 wherein the nucleic acid target is
incubated with about a 5-fold excess of the double stranded probe to a 5,000,000-fold excess of the
double stranded probe.
52. The probe according to any one of items 43-51 wherein the nucleic acid target is
incubated with about a 5-fold excess of the double stranded probe to about a 500-fold excess of the
double stranded probe.
53. The probe according to any one of items 43-52 wherein a double stranded probe-
nucleic acid target complex formed between the double stranded probe and the nucleic acid target is
detected by fluorescence spectroscopy, electrophoresis, absorption spectroscopy, flow cytometry, and
combinations thereof.
54. The probe according to any one of items 43-53 wherein the double stranded probe
is selected from SEQ. ID Nos. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, and
246-253.
55. The probe according to any one of items 43-54 wherein the nucleic acid target is
single-stranded or double-stranded.
56. The probe according to any one of items 43-55 wherein the double stranded probe
further comprises one or more bulge monomers that do not participate in base pairing.
57. The probe according to item 56 wherein the one or more bulge monomers are
selected from
- 13g -
OR or
58. The probe according to any one of items 43-57 wherein the monomer is positioned
in the double stranded probe in a manner that substantially weakens a duplex’s thermal stability.
59. The probe according to item 58 wherein the duplex has a thermal melting
temperature which is substantially similar to, or lower than, that of a corresponding unmodified
nucleic acid duplex.
60. The probe according to any one of items 43-59 wherein a first monomer and second
monomer are arranged in a +n or -n zipper orientation, wherein n ranges from 0 to about 10.
61. The probe according to item 60 wherein the first monomer and the second
monomer are arranged in a +n orientation, wherein n is 1.
62. The probe according to any one of items 43-61 wherein the nucleic acid target is a
DNA target selected from second insulin, PPAR gamma, and CEBP promoters.
63. The probe according to any one of items 43-62 wherein the double stranded probe
is used for gender determination.
64. The probe according to item 63 wherein the double stranded probe is used for
gender determination in mammals.
65. The probe according to item 64 wherein the mammals are ungulates.
66. The probe according to item 64 wherein the mammals are ruminants.
- 13h -
67. The probe according to item 64 wherein the mammals are bovines, equines or
porcines.
68. The probe according to any one of items 43-67 wherein the nucleic acid target is
isosequential (relative to a probe) double stranded DNA target regions, including stems of molecular
beacons, target regions embedded within PCR amplicons, target regions embedded within circular or
linearized plasmids, target regions embedded within genomic DNA, and target regions embedded
within microorganisms.
69. The probe according to any one of items 43-68 wherein the nucleic acid target is
selected from a nucleic acid sequence associated with B cell and T cell leukemias, lymphomas, breast
cancer, colon cancer, and neurological cancers.
70. The probe according to any one of items 43-68 wherein the nucleic acid target is
selected from the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC_000007, nucleotides
55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC_000008,
nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g.,
GENBANK™ Accession No. NC_000008, nucleotides 19841058-19869049), RB1 (13q14; e.g.,
GENBANK™ Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g.,
GENBANK™ Accession No. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC
(2p24; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides
151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC_000012,
complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession
No. NC_000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™ Accession
No. NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK
(2p23; e.g., GENBANK™ Accession No. NC_000002, complement,
nucleotides 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™ Accession
No. NC_000011, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession
No. NC_000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™
Accession No. NC_000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-
p31; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides 59019051-59022373),
TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NC_000017, complement,
nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No.
NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™
Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g.,
GENBANK™ Accession No. NC_000007, complement, nucleotides 13897379-13995289), EWS
(22q12.2; e.g., GENBANK™ Accession No. NC_000022, nucleotides 27994271-28026505); FLI1
(11q24.1-q24.3; e.g., GENBANK™ Accession No. NC_000011, nucleotides
- 13i -
128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC_000002,
complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™
Accession No. NC_000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™
Accession No. NC_000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g.,
GENBANK™ Accession No. NC_000019, complement, nucleotides 45431556-45483036), MYCL1
(1p34.2; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides
40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC_000002, nucleotides
60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC_000005,
complement, nucleotides 149413051-149473128).
71. The probe according to any one of items 43-68 wherein the nucleic acid target is
selected from a virus or other microorganism and the double stranded probe is used to detect and/or
identify the microorganism.
72. The probe according to any one of items 43-68 wherein the nucleic acid target is
selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular
parasite selected from Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba
histolytica, Giardia lamblia, Toxoplasma, Eimeria, Theileria, and Babesia.
73. The probe according to any one of items 43-68 wherein the nucleic acid target is
selected from human adenovirus A (NC_001460), human adenovirus B (NC_004001), human
adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266),
human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus
(V01109; GI:60851) human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human
coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus
OC43 ( NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472),
human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrovirus V9
(NC_004295), human foamy virus (NC_001736), human herpesvirus 1 (Herpes simplex virus type 1)
(NC_001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC_001798), human herpesvirus
3 (Varicella zoster virus) (NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1)
(NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human
herpesvirus 5 strain AD169 (NC_001347), human herpesvirus 5 strain Merlin Strain (NC_006273),
human herpesvirus 6A (NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7
(NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P
(NC_009333), human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2
(NC_001722), human metapneumovirus (NC_004148), human papillomavirus-1 (NC_001356),
human papillomavirus-18 (NC_001357), human papillomavirus-2 (NC_001352), human
papillomavirus-54 (NC_001676), human papillomavirus-61 (NC_001694), human
- 13j -
papillomavirus-cand90 (NC_004104), human papillomavirus RTRX7 (NC_004761), human
papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC_008189), human
papillomavirus type 103 (NC_008188), human papillomavirus type 107 (NC_009239), human
papillomavirus type 16 (NC_001526), human papillomavirus type 24 (NC_001683), human
papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human
papillomavirus type 34 (NC_001587), human papillomavirus type 4 (NC_001457), human
papillomavirus type 41 (NC_001354), human papillomavirus type 48 (NC_001690), human
papillomavirus type 49 (NC_001591), human papillomavirus type 5 (NC_001531), human
papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC_001593), human
papillomavirus type 60 (NC_001693), human papillomavirus type 63 (NC_001458), human
papillomavirus type 6b (NC_001355), human papillomavirus type 7 (NC_001595), human
papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC_001596), human
papillomavirus type 92 (NC_004500), human papillomavirus type 96 (NC_005134), human
parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC_003443), human
parainfluenza virus 3 (NC_001796), human parechovirus (NC_001897), human parvovirus 4
(NC_007018), human parvovirus B19 (NC_000883), human respiratory syncytial virus (NC_001781)
, human rhinovirus A (NC_001617), human rhinovirus B (NC_001490), human spumaretrovirus
(NC_001795), human T-lymphotropic virus 1 (NC_001436), human T-lymphotropic virus 2
(NC_001488), Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18),
Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS
virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), Herpes Simplex Virus (HSV),
Her1, Her2, Her3, Her4, EGFR1, EGFR2, EGFR3, EGFR4, ErbB-1, ErbB-2, ErbB-3 and ErbB-4.
74. The single stranded probe precursor according to item 40, for use in associating the
probe with a nucleic acid target.
75. A kit comprising the double stranded probe according to item 1.
76. The kit according to item 75 wherein the probe comprises at least one bulge
monomer.
77. The kit according to item 75 wherein the one or more bulge monomer is selected
from
- 13k -
OR or
78. The kit according to any one of items 75-77 comprising a sequence selected from
SEQ ID NOs. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, and 246-253.
79. The kit according to any one of items 75-78 for gender determination.
80. The kit according to item 79 for gender determination in mammals.
81. The kit according to item 80 for sexing mammalian embryos.
82. The kit according to item 81 where the mammal is an ungulate.
83. The kit according to claim 80 where the mammal is a bovine, equine or porcine.
84. An in vitro method for associating a probe with a nucleic acid target, comprising:
selecting a double stranded probe comprising a first monomer having a formula
Linker
Optional
1 b b
where Y is selected from carbon, oxygen, sulfur, and NR , wherein R is selected from
2 3 4
hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; each of Y , Y , and Y independently is
selected from carbon, oxygen, sulfur, a triazole, oxazole, tetrazole, isoxazole, and NR , wherein R is
selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; R and R are selected from
hydrogen, aliphatic, aryl, aryl aliphatic, and a heteroatom-containing moiety, or R is selected from a
heteroatom-containing functional group; R is a heteroatom-containing functional group; R is
selected from any natural or non-natural nucleobase; R is selected from an intercalator suitable for
- 13l -
intercalating within a nucleic acid selected from a hydrocarbon or an aromatic heterocycle; “optional
linker” is selected from linkers comprising alkyl linkers, amide linkers, carbamate linkers, carbonyl
linkers, carbonate linkers, urea linkers, and combinations thereof;
a second monomer having a formula
Linker
Optional
1 2 3 4 1 2 3 4 5
wherein Y , Y , Y , Y , R , R R , R , R , and “optional linker” are as stated for the first
monomer; V is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4;
wherein the first monomer is positioned in a first strand of the double-stranded probe and the
second monomer is positioned in a second strand of the double stranded probe and wherein each of
the first strand and the second strand comprises at least one nucleotide selected from a natural
nucleotide, unnatural nucleotide, and combinations thereof;
exposing the nucleic acid target to the double stranded probe; and
detecting the double stranded probe and/or a double stranded probe-nucleic acid target
complex.
85. The in vitro method according to item 84 wherein the double stranded probe further
comprises a second monomer having a formula selected from
Linker
Optional
wherein V is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4.
86. An in vitro method for associating a probe with a nucleic acid target, comprising:
selecting a single stranded probe comprising a monomer having a formula
OR N
f OR N
or O y
- 13m -
R O B
R O B
OR N NH f
OR N
2 OR N
R O B R O B
OR N N
N OR
N N y
e e e
R O B R O B R O B R O B
O O O O
O O O O
f f f f
OR OR OR
R O R O
OR OR
Nap Py
OR N f
OR N
R O O
OR N
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H,
DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or
2 2 2
phosphate; or
- 13n -
OR O
wherein B is selected from guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-diaminopurine,
inosine, or 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one; Nap is napthyl; Py is pyrenyl; R is H, DMTr, or
phosphate; and R is H, (N(i-Pr) )P(OCH CH CN), or phosphate; and at least one natural nucleotide,
2 2 2
unnatural nucleotide, and combinations thereof; exposing the nucleic acid target to the single
stranded probe; and
detecting the single stranded probe and/or a single stranded probe-nucleic acid target
complex.
87. The method of item 86 wherein the single stranded probe further comprises a
second monomer selected from
e moc
e B O
OR N
f OR N
OR O O
Na P P
p y or O y
R O B
R O B
R O B
OR N f
OR N NH
P 2 OR P
R O B R B
H Py
OR N OR N
- 13o -
e e e
R O B R O B R O B R O B
O O O O
O O O O
f f f f
OR OR OR
R O R O
OR OR
Nap Py
OR N f
R O O
OR N
wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-
diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H,
DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or
2 2 2
phosphate.
88. The double stranded probe according to item 1, wherein the double stranded probe has a
sequence selected from SEQ ID No. 252 or SEQ ID No. 253.
The foregoing and other objects, features, and advantages of the invention will become more
apparent from the following detailed description, which proceeds with reference to the accompanying
figures.
BRIEF DESCRIPTION OF THE DRAWINGS
is a schematic diagram that illustrates a particular embodiment of a method for
detecting a target using the disclosed probe.
- 13p -
is an image of an embodiment of the disclosed probe.
is a schematic drawing illustrating the concept of using bulge monomers.
- 13q -
is a schematic drawing illustrating probes with two +1 interstrand arrangements of
disclosed monmers (green) and one to four non-pairing bulged monomers (red loop), such as 402-4,
402-N and 402-9.
is an image of a thermal denaturation curve between an exemplary embodiment of
disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the
(two) probe strands and one of the (two) target strands.
is an image of a thermal denaturation curve between an exemplary embodiments of
disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the
(two) probe strands and one of the (two) target strands.
is an image of a thermal denaturation curve between an exemplary embodiment of
disclosed probes and a nucleic acid target illustrating characteristics of duplexes between one of the
(two) probe strands and one of the (two) target strands.
are absorption spectra obtained from an exemplary embodiment and duplexes
formed with a nucleic acid target.
are absorption spectra obtained from an exemplary probe embodiment and the
duplexes formed with a nucleic acid targets.
are fluorescence emission spectra of an exemplary probe embodiment in the presence
or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
are fluorescence emission spectra of an exemplary probe embodiment in the
presence or absence of a nucleic acid target.
is a schematic drawing of a target used in an exemplary method of using a disclosed
probe.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
is a schematic drawing of a target and exemplary embodiments of the disclosed
probe used in an exemplary method of using the probe.
is an image of a gel obtained using gel electrophoresis analysis of different
exemplary embodiments of the probe and their ability to bind to a particular target.
is schematic drawing of a target used to analyze the ability of the disclosed probe to
suppress gene expression.
is schematic drawing of exemplary embodiments of disclosed probes and the results
obtained using such probes to suppress gene expression in a particular target.
is schematic drawing of the results obtained from a dose-dependent study using
exemplary embodiments of the disclosed probe and a particular target.
is schematic drawing of an exemplary embodiment wherein the probe is used to
target a mixed-sequence, structured nucleic acid target.
is an image of a gel obtained from gel electrophoresis analysis of an exemplary
embodiment of the disclosed probe illustrating its ability to complex with a mixed-sequence,
structured nucleic acid target.
is an image of the results obtained using a variety of exemplary embodiments of the
disclosed probes as well as embodiments used as controls (e.g. single-stranded probe precursor and a
control with no probe) to target and form a complex with a mixed-sequence, structured nucleic acid
target.
are absorption spectra obtained using exemplary embodiments of a disclosed probe
comprising a triazole moiety.
are steady-state fluorescence emission spectra obtained using exemplary
embodiments of a disclosed probe comprising a triazole moiety.
is schematic drawing illustrating a putative mechanism of the disclosed probe
wherein universal hybridization occurs.
illustrates recognition of structured dsDNA targets by probes using electrophoretic
mobility shift assays: (a) schematic llustration of recognition process; (b) structures of dsDNA-
targets with isosequential (SL1) or non-isosequential (SL2 and SL3) stem regions (arrows denote
deviation points); (c), (d) and (e) recognition of SL1 using escalating excess of 126W2:126W5,
120Q2:120Q5 or D1:D2, respectively; (f) incubation of SL1 with 100-fold excess of single-stranded
126W2, 126W5, 120Q2 or 120Q5; (g) and (h) incubation of SL1-SL3 with 100-fold excess of
126W2:126W5 or 120Q2:120Q5, respectively. Probe-target incubation: 3 hours at 20 °C; 15% non-
denaturing PAGE; DIG: digoxigenin.
are dose-response curves for recognition of structured dsDNA SL1 by the
following probes (top to bottom): 126W2:126W5, 126X2:126X5, 126Y2:126Y5, 120Q2:120Q5,
120Y2:120Y5 (P2:P5), 124X6:124X8 (K6:K8) and 120’W6:120’W8 (M6:M8). For experimental
conditions see . Dose response curves are fitted to a logarithmic equation.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
illustrates the results of a control experiment. Incubation of SL1 with 100-fold
excess of single-stranded 120’W6 (M6), 120’W8 (M8), 120Y2 (P2) or 120Y5 (P5). For
experimental conditions and sequence of SL1, see .
illustrates the results of a control experiment. Incubation of SL1-SL3 with 100-
fold excess of a) 126X2:126X5, b) 126Y2:126Y5 or c) 124X6:124X8. For experimental conditions
and sequence of SL1-SL3, see .
illustrates targeting of structured dsDNA HP1 using different probes modified with
monomer 120Y (200-fold molar excess) as monitored by the gel mobility shift assay (electrophoretic
mobility shift assay; for concept see FIG 30). HP1: (5’-GGTATATATAGGC-(T10)-
GCCTATATATACC (34.4 nM) incubated at room temperature. HP1 alone (lane 1); HP1 incubated
with 200-excess of 120Y-P1 (lane 2), 120Y-P2 (lane 3), 120Y-P3 (lane 4), 120Y-P4 (lane 5), 120Y-
P5 (lane 6), 120Y-P6 (lane 7) or 120Y-P7 (lane 8).
illustrates targeting of structured dsDNA HP1 using various concentrations of a
selected probe (120Y-P4).
illustrates the results of a control experiment demonstrating the recognition
specificity of the disclosed probes.
provides proof for a proposed recognition mechanism, demonstrating the disclosed
mode of nucleic acid targeting using disclosed probes. Lane 1: DIG-labeled HP1 only; lane 2: DIG-
labeled ‘upper’ probe strand only (5’- GG(120Y)A(120Y)A TAT AGG C-DIG); lane 3: DIG-labeled
‘lower’ probe strand only (3’- DIG-CCA (120Y)A(120Y) ATA TCC G); lane 4: probe with DIG-
labeled upper strand (5’-GG(120Y)A(120Y)ATATAGGC-DIG + 3’-
CCA(120Y)A(120Y)ATATCCG) incubated with unlabeled structured target HP1; lane 5: probe with
DIG-labeled lower strand (5’-GG(120Y)A(120Y)ATATAGGC + 3’-DIG-
CCA(120Y)A(120Y)ATATCCG) incubated with unlabeled structured target HP1; lane 6: unlabeled
probe (5’-GG(120Y)A(120Y)ATATAGGC + 3’-CCA(120Y)A(120Y)ATATCCG) incubated with
labeled structured target HP1.
provides information that both strands of a double-stranded probe facilitate
recognition of dsDNA target regions. Lane 1: only HP1; lane 2: HP1 + upper strand of 120Y-P4
(i.e., 5’-GG(120Y)A(120Y)ATATAGGC); lane 3: HP1 + lower strand of 120Y-P4 (i.e., 3’-
CCA(120Y)A(120Y)ATATCCG); lane 4: HP1 + 120Y-P4. Probes are used in 200-fold molar excess
relative to structured target HP1. Incubated in Hepes buffer for 15 hours at room temperature.
is a gel illustrating the results of incubation of HP1 with increasing concentrations
of an isosequential and unmodified dsDNA probe. 5x-500x refers to molar fold excess of dsDNA
with respect to HP1
is a graph of linker chemistry versus invastion% for symmetric bulges illustrating
the results of targeting structured dsDNA (stem-loop) target using probe with one or more bulges,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in Hepes
buffer for 15 hours followed by electrophoresis, imaging, and quantification.
is a graph of linker chemistry versus invastion% for an up stranded bulge
illustrating the results of targeting structured dsDNA (stem-loop) target using probe with one or more
bulges where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in
Hepes buffer for 15 hours followed by electrophoresis, imaging, and quantification.
is a graph of linker chemistry versus invastion% for a down stranded bulge
illustrating the results of targeting structured dsDNA (stem-loop) target using probe with one or more
bulges where Dig-labeled structured dsDNA target was incubated with 200-fold excess of probe in
Hepes buffer for 15 hours followed by electrophoresis, imaging, and quantification.
is an illustration on the use of double-stranded probes with +1 interstrand zipper
arrangements that are additionally modified with a fluorophore-quencher pair. Binding to the nucleic
acid target results in generation of an optical signal, more commonly a fluorescent signal.
SEQUENCE LISTING
The nucleic acid sequences listed in the accompanying sequence listing are shown using
standard letter abbreviations for nucleotide bases, as defined in 37 C.F.R. 1.822. Only one strand of
each nucleic acid sequence is shown, but the complementary strand is understood as included by any
reference to the displayed strand. In the accompanying sequence listing:
SEQ ID NOs: 1-4, 11 and 240-245 are nucleotide sequences of structured dsDNA targets
with isosequential or non-isosequential stem regions.
SEQ ID NOs: 5-10, 12-239, 246-251 and 254 are nucleotide sequences of oligonucleotide
probes and target regions.
SEQ ID NOs: 252 and 253 are nucleotide sequences of double-stranded probes containing
a non-pairing bulge.
DETAILED DESCRIPTION
I. Terms
Unless otherwise noted, technical terms are used according to conventional usage.
Definitions of common terms in molecular biology may be found in Benjamin Lewin, Genes VII,
published by Oxford University Press, 2000 (ISBN 019879276X); Kendrew et al. (eds.), The
Encyclopedia of Molecular Biology, published by Blackwell Publishers, 1994 (ISBN 0632021829);
and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk
Reference, published by Wiley, John & Sons, Inc., 1995 (ISBN 0471186341); and other similar
references.
As used herein, the singular terms “a,” “an,” and “the” include plural referents unless context
clearly indicates otherwise. Similarly, the word “or” is intended to include “and” unless the context
6478592_1 (GHMatters) P95976.NZ ESTHERJ
clearly indicates otherwise. Also, as used herein, the term “comprises” means “includes.” Hence
“comprising A or B” means including A, B, or A and B.
A wavy line (“ ”) indicates a bond disconnection. A dashed line (“ ”) illustrates that
a bond may be formed at a particular position.
All nucleotide sizes or amino acid sizes, and all molecular weight or molecular mass values,
given for nucleic acids or polypeptides or other compounds are approximate, and are provided for
description.
Although methods and materials similar or equivalent to those described herein can be used
in the practice or testing of the present disclosure, suitable methods and materials are described
below.
All publications, patent applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict, the present specification, including
explanations of terms, will control. In addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
In order to facilitate review of the various examples of this disclosure, the following
explanations of specific terms are provided:
Aliphatic: Any open or closed chain molecule, excluding aromatic compounds, containing
only carbon and hydrogen atoms which are joined by single bonds (alkanes), double bonds (alkenes),
or triple bonds (alkynes). This term encompasses substituted aliphatic compounds, saturated
aliphatic compounds, and unsaturated aliphatic compounds.
Analog, Derivative or Mimetic: An analog is a molecule that differs in chemical structure
from a parent compound, for example a homolog (differing by an increment in the chemical structure,
such as a difference in the length of an alkyl chain), a molecular fragment, a structure that differs by
one or more functional groups, a change in ionization. Structural analogs are often found using
quantitative structure activity relationships (QSAR), with techniques such as those disclosed in
Remington (The Science and Practice of Pharmacology, 19th Edition (1995), chapter 28). A
derivative is a biologically active molecule derived from the base structure. A mimetic is a molecule
that mimics the activity of another molecule, such as a biologically active molecule. Biologically
active molecules can include chemical structures that mimic the biological activities of a compound.
Aromatic: A term describing conjugated rings having unsaturated bonds, lone pairs, or
empty orbitals, which exhibit a stabilization stronger than would be expected by the stabilization of
conjugation alone. It can also be considered a manifestation of cyclic delocalization and of resonance.
Aryl: A substantially hydrocarbon-based aromatic compound, or a radical thereof (e.g.
C H ) as a substituent bonded to another group, particularly other organic groups, having a ring
structure as exemplified by benzene, naphthalene, phenanthrene, anthracene, etc. This term also
encompasses substituted aryl compounds.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Aryl alkyl: A compound, or a radical thereof (C H for toluene) as a substituent bonded to
another group, particularly other organic groups, containing both aliphatic and aromatic structures.
Complementary: The natural binding of polynucleotides under permissive salt and
temperature conditions by base-pairing. Complementarity may exist when only some of the nucleic
acids bind, or when total complementarity exists between the nucleic acids.
Conjugating, joining, bonding or linking: Joining one molecule to another molecule to
make a larger molecule. For example, making two polypeptides into one contiguous polypeptide
molecule, or covalently attaching a hapten or other molecule to a polypeptide, such as an scFv
antibody. In the specific context, the terms include reference to joining a ligand, such as an antibody
moiety, to an effector molecule. The linkage can be either by chemical or recombinant means.
“Chemical means” refers to a reaction between the antibody moiety and the effector molecule such
that there is a covalent bond formed between the two molecules to form one molecule.
Coupled: The term "coupled" means joined together, either directly or indirectly. A first
atom or molecule can be directly coupled or indirectly coupled to a second atom or molecule. A
secondary antibody provides an example of indirect coupling. Coupling can occur via covalent, non-
covalent, and ionic bond formation.
Derivative: In chemistry, a derivative is a compound that is derived from a similar
compound or a compound that can be imagined to arise from another compound, for example, if one
atom is replaced with another atom or group of atoms. The latter definition is common in organic
chemistry. In biochemistry, the word is used for compounds that at least theoretically can be formed
from the precursor compound.
Deviation from additivity (DA): The DA value for a probe ONX:ONY is defined as:
DA ≡ ΔT (ONX:ONY) – [ΔT (ONX:DNA X) + ΔT (DNA Y:ONY)], where ONX:ONY
ONX:ONY m m m
is a double-stranded probe with certain interstrand zipper arrangements of disclosed monomers and
‘DNA X’ and ‘DNA Y’ are the complementary single-stranded nucleic acid targets of ONX and
ONY, respectively. The term “Thermal advantage” is significantly related to DA, i.e., TA = - DA.
Displace (ment) (ed): A reaction in which an atom, radical, or molecule (anionic or
neutral) replaces another in a compound.
Double Stranded Nucleic Acid: An oligonucleotide containing a region of two or more
nucleotides having a double stranded motif.
Epitope: An antigenic determinant. These are particular chemical groups or contiguous or
non-contiguous peptide sequences on a molecule that are antigenic, that is, that elicit a specific
immune response.
Fluorescence: The emission of light by a substance that has absorbed light or other
electromagnetic radiation of a different wavelength.
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Fluorophore: A functional group of a molecule which causes the molecule to be
fluorescent. Typically, the functional group can absorb energy of a specific wavelength and re-emit
energy at a different (but equally specific) wavelength.
Human epidermal growth factor receptor (Her) family: A family of structurally related
proteins, including at least Her1, Her2, Her3 and Her4 (aka EGFR1, EGFR2, EGFR3 and EGFR4,
respectively, or ErbB-1, ErbB-2, ErbB-3 and ErbB-4, respectively). Her1, Her2 and Her4 are
receptor tyrosine kinases; although Her3 shares homology with Her1, Her2 and Her4, Her3 is kinase
inactive. Included in the Her family is p95, a truncated form of Her2 lacking portions of the Her2
extracellular domain (see, e.g., Arribas et al., Cancer Res., 71:1515-1519, 2011; Molina et al.,
Cancer Res., 61:4744-4749, 2001).
The human epidermal growth factor family of receptors mediate cell growth and are
disregulated in many types of cancer. For example Her1 and Her2 are upregulated in many human
cancers, and their excessive signaling may be critical factors in the development and malignancy of
these tumors. See, e.g., Herbst, Int. J. Radiat. Oncol. Biol. Phys., 59:21–6, 2004; Zhang et al., J.
Clin. Invest. 117 (8): 2051–8, 2007. Receptor dimerization is essential for Her pathway activation
leading to receptor phosphorylation and downstream signal transduction. Unlike Her1, -3 and -4,
Her2 has no known ligand and assumes an open conformation, with its dimerization domain exposed
for interaction with other ligand-activated Her receptors.
Approximately 30% of breast cancers have an amplification of the Her2 gene or
overexpression of its protein product. Her2 overexpression also occurs in other cancer types, such as
ovarian cancer, stomach cancer, and biologically aggressive forms of uterine cancer, such as uterine
serous endometrial carcinoma. See, e.g., Santin et al., Int. J. Gynaecol. Obstet., 102 (2): 128–31,
2008. Her2-containing homo- and hetero-dimers are transformation competent protein complexes.
Trastuzumab, a humanized antibody that prevents Her2 homodimerization is used to treat certain
Her2 overexpressing cancers, including breast cancer. Additionally, the level of Her2 expression in
cancer tissue is predictive of patient response to Her2 therapeutic antibodies (e.g., Trastuzumab).
Because of its prognostic role as well as its ability to predict response to Trastuzumab, tumors (e.g.,
tumors associated with breast cancer) are routinely checked for overexpression of Her2.
The Her pathway is also involved in ovarian cancer pathogenesis. Many ovarian tumor
samples express all members of the Her family. Co-expression of Her1 and Her2 is seen more
frequently in ovarian cancer than in normal ovarian epithelium, and overexpression of both receptors
correlates with poor prognosis. Preferred dimerization with Her2 (Her1/Her2, Her2/Her3) and
subsequent pathway activation via receptor phosphorylation have also been shown to drive ovarian
tumor cell proliferation, even in the absence of Her2 overexpression. Pertuzumab, a humanized
antibody that prevents Her2 dimerization (with itself and with Her3) has been shown to provide
therapeutic benefit to patients with Her2 and/orHer3 expressing ovarian cancer.
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Examples of Her1 amino acid sequence include NCBI/Genbank accession Nos. NP_005219.2,
CAA25240.1, AAT52212.1, AAZ66620.1, BAF83041.1, BAH11869.1, ADZ75461.1, ADL28125.1,
BAD92679.1, AAH94761.1. Examples of, Her2, amino acid sequences include NCBI/Genbank
accession BAJ17684.1, P04626.1, AAI67147.1, NP_001005862.1, NP_004439.2, AAA75493.1,
AAO18082.1. Examples of Her3 amino acid sequences include NCBI/Genbank accession Nos.
NP_001973.3, P21860.1, AAH82992.1, AAH02706.1, AAA35979.1. Examples of Her4 amino acid
sequences include NCBI/Genbank accession Nos., AAI43750, Q15303.1, NP_005226.1,
NP_001036064.1, AAI43748.1.
Heteroaliphatic: An aliphatic group, which contains one or more atoms other than carbon
and hydrogen, such as, but not limited to, oxygen, sulfur, nitrogen, phosphorus, chlorine, fluorine,
bromine, iodine, and selenium.
Homology: As used herein, “homology” refers to a degree of complementarity. Partial
homology or complete homology can exist. Partial homology involves a nucleic acid sequence that
at least partially inhibits an identical sequence from hybridizing to a target nucleic acid.
Homopolymer: This term refers to a polymer formed by the bonding together of multiple
units of a single type of molecular species, such as a single monomer (for example, an amino acid).
Interstrand Zipper Nomenclature (+1/-1, etc…): The “interstrand zipper arrangement”
nomenclature is used to describe relative arrangement between two monomers positioned on
opposing strands in a duplex. The number ‘n’ describes the distance measured in number of base
pairs and has a positive value if a monomer is shifted toward the 5’-side of its own strand relative to a
second reference monomer on the other strand. Conversely, n has a negative value if a monomer is
shifted toward the 3’-side of its own strand relative to a second reference monomer on the other
strand.
Isolated: An “isolated” microorganism (such as a virus, bacterium, fungus, or protozoan)
has been substantially separated or purified away from microorganisms of different types, strains, or
species. Microorganisms can be isolated by a variety of techniques, including serial dilution and
culturing.
An “isolated” biological component (such as a nucleic acid molecule, protein or organelle)
has been substantially separated or purified away from other biological components in the cell of the
organism in which the component naturally occurs, such as other chromosomal and extra-
chromosomal DNA and RNA, proteins, and organelles. Nucleic acids and proteins that have been
“isolated” include nucleic acids and proteins purified by standard purification methods. The term
also embraces nucleic acids and proteins prepared by recombinant expression in a host cell, as well as
chemically synthesized nucleic acids or proteins, or fragments thereof.
Leaving Group: A molecular fragment that departs with a pair of electrons after heterolytic
bond cleavage. Leaving groups can be anions or neutral molecules. Common anionic leaving groups
− − −
can include halides, such as Cl , Br , and I , and sulfonate esters, such as para-toluenesulfonate
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(TsO ), trifluoromethanesulfonate (TfO ), Common neutral molecule leaving groups can include
H O, NH , alcohols, and gases (N , O , CO , CO, and SO ).
2 3 2 2 2 2
Lewis acid: A chemical substance that can accept a pair of electrons from a Lewis base, B,
which acts as an electron-pair donor, forming an adduct, AB as given by the following: A+:B →
A—B.
Linker: As used herein, a linker is a molecule or group of atoms positioned between two
moieties.
Lower alkyl: Any aliphatic chain that contains 1-10 carbon atoms.
Modified: As used herein, “modified” refers to an oligonucleotide that has a non-natural
composition, in that it comprises one or more synthetic nucleobases which can pair with a natural
base.
Molecule of interest or Target: A molecule for which the presence, location and/or
concentration is to be determined.
Nucleobase: As used herein, “nucleobase” includes naturally occuring nucleobases as well
as non-natural nucleobases. A person of ordinary skill in the art will recognize that “nucleobase”
encompasses purine and pyrimidine derivatives, as well as heterocyclic derivatives and tautomers
thereof.
Nucleophile: A reagent that forms a chemical bond to its reaction partner (the electrophile)
by donating both bonding electrons. A molecule or ion with a free pair of electrons can act as
nucleophile.
Nucleotide: Phosphorylated nucleosides are “nucleotides,” which are the molecular
building-blocks of DNA and RNA.
Nucleoside: A glycoside of a heterocyclic base. The term "nucleoside" is used broadly as to
include non-naturally occurring nucleosides, naturally occurring nucleosides as well as other
nucleoside analogues. Illustrative examples of nucleosides are ribonucleosides comprising a ribose
moiety as well as deoxyribo-nucleosides comprising a deoxyribose moiety. With respect to the bases
of such nucleosides, it should be understood that this may be any of the naturally occurring bases, e.g.
adenine, guanine, cytosine, thymine, and uracil, as well as any modified variants thereof or any
possible unnatural bases.
Oligonucleotide: A plurality of joined nucleotides joined by native phosphodiester bonds.
An oligonucleotide is a polynucleotide of between at least 2 and about 300 nucleotides in length.
Typically, an oligonucleotide is a polynucleotide of between about 5 and about 50 nucleotides. An
oligonucleotide analog refers to moieties that function similarly to oligonucleotides but have non-
naturally occurring portions. For example, oligonucleotide analogs can contain non-naturally
occurring portions, such as altered sugar moieties or inter-sugar linkages, such as a phosphorothioate
oligodeoxynucleotide. Functional analogs of naturally occurring polynucleotides can bind to RNA or
DNA, and include locked nucleic acid (LNA) and peptide nucleic acid (PNA) molecules.
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Pharmaceutically acceptable salt: Pharmaceutically acceptable salts are more soluble in
aqueous solutions than the corresponding free acids and bases from which the salts are produced;
however, salts having lower solubility than the corresponding free acids and bases from which the
salts are produced may also be formed. Pharmaceutically acceptable salts are typically
counterbalanced with an inorganic base, organic base, or basic amino acid if the salts are positively
charged; or the salt is counterbalanced with an inorganic acid, organic acid, or acidic amino acid if
they are negatively charged. Pharmaceutically acceptable salts can also be zwitterionic in form.
Salts can be formed from cations such as sodium, potassium, aluminum, calcium, lithium,
magnesium, zinc, and from bases such as ammonia, ethylenediamine, N-methyl-glutamine, lysine,
arginine, ornithine, choline, N,N'-dibenzylethylenediamine, chloroprocaine, diethanolamine,
procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)aminomethane,
and tetramethylammonium hydroxide. Other elements capable of forming salts are well-known to
those of ordinary skill in the art, e.g. all elements from the main groups I to V of the Periodic Table
of the Elements, as well as the elements from the subgroups I to VIII. Any chemical compound
recited in this specification may alternatively be administered as a pharmaceutically acceptable salt
thereof. “Pharmaceutically acceptable salts” are also inclusive of the free acid, base, and.
Descriptions of suitable pharmaceutically acceptable salts can be found in Handbook of
Pharmaceutical Salts, Properties, Selection and Use, Wiley VCH (2002), which we herein
incorporate by reference.
Polypeptide: A polymer in which the monomers are amino acid residues that are joined
together through amide bonds. When the amino acids are α-amino acids, either the L-optical isomer
or the D-optical isomer can be used. The terms “polypeptide” or “protein” as used herein are
intended to encompass any amino acid sequence and include modified sequences such as
glycoproteins. The term “polypeptide” is specifically intended to cover naturally occurring proteins,
as well as those which are recombinantly or synthetically produced.
The term “residue” or “amino acid residue” includes reference to an amino acid that is
incorporated into a protein, polypeptide, or peptide.
Protecting Group: A moiety that can be introduced into a molecule by chemical
modification of a functional group. Protecting groups often are used to protect one functional group
in order to obtain chemoselectivity in a chemical reaction with a different functional group. Suitable
protecting groups are well known to those of ordinary skill in the art and can include aryl groups,
aliphatic groups, heteroaliphatic groups, heteroaryl groups.
Protein: A molecule, particularly a polypeptide, comprised of amino acids.
Purified: The term “purified” does not require absolute purity; rather, it is intended as a
relative term. Thus, for example, a purified compound is one that is isolated in whole or in part from
other contaminants. Generally, substantially purified peptides, proteins, conjugates, oligonucleotides,
or other active compounds for use within the disclosure comprise more than 80% of all
6478592_1 (GHMatters) P95976.NZ ESTHERJ
macromolecular species present in a preparation prior to admixture or formulation of the peptide,
protein, conjugate or other active compound with a pharmaceutical carrier, excipient, buffer,
absorption enhancing agent, stabilizer, preservative, adjuvant or other co-ingredient in a complete
pharmaceutical formulation for therapeutic administration. More typically, the peptide, protein,
conjugate or other active compound is purified to represent greater than 90%, often greater than 95%
of all macromolecular species present in a purified preparation prior to admixture with other
formulation ingredients. In other cases, the purified preparation may be essentially homogeneous,
wherein other macromolecular species are not detectable by conventional techniques.
Quantum Yield: A measure of the efficiency of the fluorescence process. The “quantum
yield” of a radiation-induced process indicates the number of times that a defined event occurs per
photon absorbed by the system.
Reactive Groups: Formulas throughout this application refer to "reactive groups," which
can be any of a variety of groups suitable for underogoing a chemical transformation as described
herein. For example, the reactive group might be an amine-reactive group, such as an isothiocyanate,
an isocyanate, an acyl azide, an NHS ester, an acid chloride, such as sulfonyl chloride, aldehydes and
glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides,
anhydrides, and combinations thereof. Suitable thiol-reactive functional groups include haloacetyl
and alkyl halides, maleimides, aziridines, acryloyl derivatives, arylating agents, thiol-disulfide
exchange reagents, such as pyridyl disulfides, TNB-thiol, and disulfide reductants, and combinations
thereof. Suitable carboxylate-reactive functional groups include diazoalkanes, diazoacetyl
compounds, carbonyldiimidazole compounds, and carbodiimides. Suitable hydroxyl-reactive
functional groups include epoxides and oxiranes, carbonyldiimidazole, N,N'-disuccinimidyl
carbonates or N-hydroxysuccinimidyl chloroformates, periodate oxidizing compounds, enzymatic
oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone-reactive functional groups include
hydrazines, Schiff bases, reductive amination products, Mannich condensation products, and
combinations thereof. Active hydrogen-reactive compounds include diazonium derivatives, Mannich
condensation products, iodination reaction products, and combinations thereof. Photoreactive
chemical functional groups include aryl azides, halogenated aryl azides, benzophonones, diazo
compounds, diazirine derivatives, and combinations thereof.
Sample: A biological specimen from a subject, such as might contain genomic DNA, RNA
(including mRNA), protein, or combinations thereof. Examples include, but are not limited to,
peripheral blood, urine, saliva, tissue biopsy, surgical specimen, amniocentesis samples and autopsy
material.
Single nucleotide polymorphism: A nucleic acid sequence variation occurring when a
single nucleotide in the genome (or other shared sequence) differs between members of a species or
paired chromosomes in an individual. For example, two sequenced nucleic acid fragments from
different individuals, AAGCCTA to AAGCTTA, contain a difference in a single nucleotide.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Substantially complementary: As used herein, “substantially complementary refers to the
oligonucleotides of the disclosed methods that are at least about 50% homologous to target nucleic
acid sequence they are designed to detect, more preferably at least about 60%, more preferably at
least about 70%, more preferably at least about 80%, more preferably at least about 90%, more
preferably at least about 90%, more preferably at least about 95%, most preferably at least about
99%.
Thermal advantage: Thermal Advantage (TA) is defined as TA (ONX:ONY) ≡ T
(ONX:DNA X) + T (DNA Y:ONY) - T (ONX:ONY) – Tm (DNA X: DNA Y), where ONX:ONY
is a double-stranded probe with certain interstrand zipper arrangements of disclosed monomers,
ONX:DNA X and DNA Y:ONY are the duplexes between individual probe strands and nucleic acid
targets, and DNA X: DNA Y is the double-stranded nuclei acid target. A large, positive TA-value
signifies significant potential for probe ONX:ONY to target DNA X:DNA Y. The term “Deviation
from Additivity” is significantly related to ‘TA’ by the equation: TA = - DA (highly positive TA or
highly negative DA values demonstrate significant targeting potential).
Transition metal: Any of the metallic elements within Groups 3 to 12 in the Periodic Table
that have an incomplete inner electron shell and that serve as transitional links between the most and
the least electropositive in a series of elements.
II. INTRODUCTION
Disclosed herein is a probe for targeting nucleic acids and particular sequences thereof. In
particular disclosed embodiments, the probe comprises one or more pairs of monomers capable of
intercalating with one or more nucleic acid targets. In particular disclosed embodiments, the probe
comprises one or more pairs of monomers comprising a first monomer and a second monomer
arranged on opposite nucleic acid strands. Certain disclosed embodiments concern a probe wherein
one or more monomers are arranged in a manner that promotes thermal instability of the probe and
increases the probe’s ability to detect a target.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
III. EMBODIMENTS OF DISCLOSED PROBES
A. Monomers
In particular disclosed embodiments, the disclosed probe may comprise one or more
monomers capable of coupling with a nucleic acid. In particular disclosed embodiments, each
monomer independently may have a Formula 1, illustrated below.
Formula 1
With reference to Formula 1, each Y may independently be selected from oxygen, sulfur, a triazole,
oxazole, tetrazole, isoxazole, and NR , wherein R is selected from hydrogen, aliphatic, aryl,
heteroaliphatic, and heteroaryl, and V may be selected from carbon, oxygen, sulfur, and NR , wherein
R is as previously recited. The variable “n” may range from 0 to 4; more typically, n is 1 or zero. A
person of ordinary skill in the art will recognize that when n is 1 or greater, V may or may not be
bound to Y, as indicated by the dashed line connecting these two variables in Formula 1. R may be
selected from hydrogen, aliphatic, such as alkyl, more typically lower alkyl, such as methyl, ethyl,
propyl, butyl, etc., alkenyl, alkynyl, aryl, aryl aliphatic, such as aryl alkyl, and a heteroatom-
containing moiety. The heteroatom-containing moiety may be selected from, but not limited to, ether
a b a a b c d a b c a
(R OR ), hydroxyl (R OH), silyl ether (R R R SiOR ), phosphine (PR R R ), thiol (R SH),
a b a b a a
thioether/sulfide (R SR ), disulfide (R SSR ), isothiocyanate (R NCS), isocyanate (R NCO), amine
a a b a b c a b
(NH , NHR , NR R ), amide (R NR C(O)R ), ester (R OC(O)R ), halogen (I, Br, Cl, F), carbonate
a b a a - a b
(R OC(O)OR ), carboxyl (R C(O)OH), carboxylate (R COO ), ester (R C(O)OR ), ketone
a b a a a b
(R C(O)R ), phosphate (R OP(O)OH ), phosphoryl (R P(O)(OH) ), sulfinyl (R S(O)R ), sulfonyl
a b a b a a a
(R SO R ), carbonothioyl (R C(S)R or R C(S)H), sulfino (R S(O)OH), sulfo (R SO H), amide
a b c a a + - a
(R C(O)NR R ), azide (N ), nitrile (R CN), isonitrile (R N C ), and nitro (R NO ). With reference to
all the heteroatom-containing moieties disclosed herein, R represents the remaining monomer
structure, which is attached to the abovementioned functional groups at the position indicated for R ;
b c d
and R , R , and R independently are hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and any
combination thereof. The optional linker may be selected from alkyl, amide, carbamate, carbonate,
urea, and combinations thereof
R may be selected from hydrogen, aliphatic, aryl, and any one of the heteroatom-containing
moieties described herein. In particular embodiments, R may be selected from a protecting group
known to those of ordinary skill in the art, such as, but not limited to, 4,4’-dimethoxytrityl, trityl, 9-
arylthioxanthenyl, mesyl (Ms), tosyl (Ts), besoyl (Bs), trifluoromethane (CF ), and
6478592_1 (GHMatters) P95976.NZ ESTHERJ
trifluoromethanesulfonyl. In certain disclosed embodiments, R may be one or more nucleotides or
monomers.
R typically may be a heteroatom-containing functional group. In particular disclosed
embodiments, the heteroatom may be selected from phosphorous, sulfur, nitrogen, oxygen, selenium,
and/or a metal. Certain disclosed embodiments utilize R substituents having a formula
where Y is selected from oxygen, sulfur, NR where R is selected from hydrogen, aliphatic, aryl,
heteroaliphatic, heteroaryl, and W is selected from phosphorus, SH, or SeH. Certain species of R
substituents include, without limitation, the following:
, or .
In other disclosed embodiments, R has a formula
where W is phosphorus, and each Z independently is selected from ether, thioether, hydroxyl, and
NR . In particular disclosed embodiments, R may be
.
R may be selected from any natural or non-natural nucleobase. In particular disclosed
embodiments, R is a natural nucleobase selected from uracil, adenine, thymine, cytosine, guanine. A
person of ordinary skill in the art will recognize that R may also be any non-natural, or synthetically
developed nucleobase including those presently known or developed in the future. In particular
disclosed embodiments, R may be selected from C-5 functionalized pyrimidines, C6-functionalized
pyrimidines, C7-functionalized 7-deazapurines, C8-functionalized purines, 2,6-diaminopurine, 2-
thiouracil, 4-thiouracil, deoxyinosine and 3-(2’-deoxy-β-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimdine-
2-(3H)-one.
R may be an intercalator capable of intercalating within a nucleic acid. In particular
disclosed embodiments, R may be any moiety capable of intercalating with single stranded nucleic
acids, double stranded nucleic acids, and/or triple stranded nucleic acids. In particular disclosed
embodiments, R may be a planar moiety capable of maintaining a flat orientation when inserted into
a nucleic acid. R may be a hydrocarbon selected from pyrene, coronene, perylene, anthracene,
naphthalene, and functionalized derivatives thereof; or an aromatic heterocycle, such as a porphyrin,
a nucleobase (such as pyrimidines, purines, size-expanded nucleobases), a metal chelator (such as
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phenanthroline, DPPZ), an azapyrene, thiazole orange, ethidium, a diazobenzene, an indole, a
pyrrole, benzimidizoles, and modified analogs thereof. R may be modified to include various
aliphatic, aryl, or heteroatom-containing functional groups.
Particular disclosed embodiments concern a probe comprising one or more monomers
selected from any one of Formulas 3-5.
Formula 3
Formula 4
Formula 5
In certain disclosed embodiments, the probe may comprise one or more monomers having
any one of Formulas 6 and 7. With reference to Formulas 6 and 7, B may be selected from uracil,
adenine, thymine, guanine, cytosine and 2-thiouracil with or without common protecting groups, and
R may be selected from napthyl, pyrenyl, coronenyl, CH -pyrenyl, CH -coronenyl,
CO-pyrenyl, COCH -pyrenyl, CH -perylenyl; CH (7-neopentylpyrenyl), CH (6-
2 2 2 2
bromopyrenyl), CH (8-bromopyrenyl), CH (8-methylpyrenyl), CH (7-tert-butyl
2 2 2
methoxypyrenyl), CH -pyrenyl and CH -pyrenyl.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Formula 6
Formula 7
Formula 8
Formula 9
Exemplary working embodiments include the following compounds:
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Particular disclosed embodiments concern using one or more monomers that do not
participate in base pairing when either forming the probe or when the probe is reacted with a target.
The monomers that do not participate in base pairing are herein referred to as “bulge monomers.” In
particular disclosed embodiments, the bulge monomers may comprise one or more aliphatic, abasic
sites, heteroalkyl groups, natural/modified nucleotides, and combinations thereof. One or more of the
bulge monomers may be included in the probe.
B. Single-Stranded Probe Precursor
Particular disclosed embodiments concern a single-stranded probe precursor wherein one or
more of the disclosed monomers are contained within a single-stranded oligonucleotide. In certain
6478592_1 (GHMatters) P95976.NZ ESTHERJ
disclosed embodiments, the single-stranded probe precursor may comprise one or more monomers
arranged sequentially or in a manner wherein one or more natural or non-natural nucleotides are
located between two or more monomers.
In particular disclosed embodiments, a single-stranded probe may serve as a precursor to a
duplex comprising one or more of the disclosed monomers, or it may be used in the disclosed method
discussed herein. Certain disclosed embodiments concern a single-stranded probe having a general
Formula 9, illustrated below. The disclosed single-stranded probe precursor may comprise locked
monomers, unlocked monomers, or combinations thereof.
5’(B B …B )(XA)(B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XV) (B B
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r j 1 2
…B )
Formula 9
With reference to Formula 9, B B , B may be any natural or non-natural nucleotide presently
1 2 m-s
known or discovered in the future, wherein m-s may range from zero to about 28. In particular
disclosed embodiments, f, g, h, i and j may range from 0 to 10, more typically 0 to 5, and even more
typically 0 to 1. Each X independently may be selected from any of the disclosed monomers and A
may be a Watson-Crick base pairing nucleotide, or derivative thereof, which is capable of coupling
with a complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a target. In
particular disclosed embodiments, each X independently may be a monomer having any one of
formulas 1-7 and A may be selected from uracil, adenine, guanine, thymine, cytosine, and derivatives
thereof. In particular disclosed embodiments, the single-stranded probe precursor may have a
Formula 9 wherein the variables are defined according to any one of the following: m = 2, n = 5, o =
p = q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed monomers comprising a uracil
nucleobase, and A is adeninyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j
= 0, X is any one of the disclosed monomers comprising a uracil nucleobase, and A is adeninyl
DNA nucleotide; m = 2, n = 9, o = p = q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed
monomers comprising a uracil nucleobase, and A is adeninyl DNA nucleotide; m = 4, n = 7, o = p
= q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed monomers comprising a uracil
nucleobase, and A is adeninyl DNA nucleotide; m = 6, n = 5, o = p = q = r = s = 0, f = g = h = i = j
= 0, X is any one of the disclosed monomers comprising a uracil nucleobase, and A is adeninyl
DNA nucleotide; m = 8, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed
monomers comprising a uracil nucleobase, and A is adeninyl DNA nucleotide; m = 2, n = 0, o = 7,
p = q = r = s = 0, f = 1, g = h = i = j = 0, X is any one of the disclosed monomers comprising a uracil
nucleobase, and A is adeninyl DNA nucleotide; m = 2, n = 2, o = 5, p = q = r = s = 0, f = 1, g = h =
i = j = 0, X is any one of the disclosed monomers comprising a uracil nucleobase, and A is adenin
yl DNA nucleotide; m = 2, n = 4, o = 3, p = q = r = s = 0, f = 1, g = h = i = j = 0, X is any one of the
disclosed monomers comprising a uracil nucleobase, and A is adeninyl DNA nucleotide; m = 2, n
6478592_1 (GHMatters) P95976.NZ ESTHERJ
= o = p = q = r = s = 0, f = g = h = 1, i = j = 0, X is any one of the disclosed monomers comprising a
uracil nucleobase, and A is adeninyl DNA nucleotide; m = 2, n = 13, o = p = q = r = s = 0, f = g = h
= i = j = 0, X is any one of the disclosed monomers comprising a uracil nucleobase, and A is adenin-
9-yl DNA nucleotide; m = 2, n = 0, o = 11, p = q = r = s = 0, f = 1, g = h = i = j = 0, X is any one of
the disclosed monomers comprising a uracil nucleobase, and A is adeninyl DNA nucleotide; m = 2,
n = 7, o = 4, p = q = r = s = 0, f = 1, g = h = i = j = 0, X is any one of the disclosed monomers
comprising a uracil nucleobase, and A is adeninyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s
= 0, f = g = h = i = j = 0, X is any one of the disclosed monomers comprising an adenine nucleobase,
and A is thyminyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X is
any one of the disclosed monomers comprising a cytosine nucleobase, and A is guaninyl DNA
nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed
monomers comprising a guanine nucleobase, and A is cytosinyl DNA nucleotide; m = 4, n = 3, o =
p = q = r = s = 0, f = g = h = i = j = 0, X is any one of the disclosed monomers comprising an adenine
nucleobase, and A is a guaninyl DNA nucleotide; and m = 4, n = 3, o = p = q = r = s = 0, f = g = h
= i = j = 0, X is any one of the disclosed monomers comprising an cytosine nucleobase, and A is an
adeninyl DNA nucleotide.
One or more bulge monomers also may be inserted at any position within a single stranded
or double stranded probe. Probes may be additionally modified with one or more non-pairing
modifications (a non-pairing modification is anything that can be incorporated internally within an
oligonucleotide), which serves to decrease the thermostability of the probe, which facilitates the
dsDNA-recognition reaction. The following provides examples of structures of non-pairing bulges
that have been incorporated into disclosed probe embodiments.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
O P O
O P O
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C. Probe Duplex
Particular disclosed embodiments concern a probe that can be used to identify, locate, bind,
and/or modify a target. The probe may be a duplex comprising two strands of oligonucleotides
comprising one or more pairs of the disclosed monomers. In particular disclosed embodiments, the
one or more pairs of the disclosed monomers are arranged in a manner that substantially decrease the
duplex’s thermal stability, thereby providing the probe with the ability to bind to the target. Without
being limited to a single theory of operation, it is currently believed that arranging the monomers in
the particular manner disclosed herein provides the probe with sufficient energy to dissociate (or
denature) into two strands that then couple with the target (.
The probe may comprise more than one pairs of monomers; for example, the probe may
comprise anywhere from 1 pair of monomers to about 5 pairs of monomers. In particular disclosed
embodiments, a pair of monomers can comprise two monomers, having any of the formulas disclosed
herein, that are located on opposite strands of the probe (e.g. opposite strands of the duplex). In
particular disclosed embodiments, a first monomer may be positioned at any location on one of the
probe strands, with the second monomer of the pair being positioned at a particular location relative
to the first monomer on the other probe strand. Certain disclosed embodiments concern a probe
having one or more pairs of monomers arranged in a (+/-)n zipper arrangement, wherein n can range
from 0 to about 10; more typically from 0 to about 3; even more typically from at least 1 to about 2.
Particular disclosed embodiments concern a probe having at least one pair of monomers arranged in a
+n zipper arrangement. Without being limited to a particular theory of operation, it currently is
believed that certain arrangements of the interstrand monomers result in destabilization of the probe,
such as illustrated in FIGS. 1-2. In particular disclosed embodiments, the pair of monomers may be
arranged in a –n zipper arrangement Exemplary embodiments of the disclosed probe typically
comprise a pair of monomers comprising a first monomer and a second monomer arranged in a (+1)
interstrand zipper arrangement. Other exemplary embodiments concern a probe comprising two pairs
of monomers, with the first monomer and the second monomer of each pair being arranged in a (+1)
interstrand zipper arrangement and each pair of monomers being separated by at least 0 to about 10
natural or non-natural nucleotides or bulge monomers.
Particular disclosed embodiments of the probe may have a Formula 10, illustrated below.
The disclosed probe may comprise locked monomers, unlocked monomers, and combinations
thereof.
’(B B …B )(XA)(B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r j 1 2
…B )
3’(C C …C )(DP)(C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C
1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r j 1 2
…C )
Formula 10
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With reference to Formula 10, B , B …B may be any natural or non-natural nucleotide, presently
1 2 m-s
known or discovered in the future, wherein m-s may range from zero to about 28. In particular
disclosed embodiments, f, g, h, i and j may range from 0 to 1,000, such as 0 to 900, such as 0 to 800,
such as 0 to 700, such as 0 to 600, such as 0 to 500, such as 0 to 400, such as 0 to 300, such as 0 to
200, such as 0 to 100, such as 0 to 50, typically 0 to 10, and even more typically 0-5 X may be
selected from any of the disclosed monomers and A may be a Watson-Crick base pairing nucleotide,
or derivative thereof, or another disclosed monomer, which is capable of coupling with a
complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a target. In particular
disclosed embodiments, X may be a monomer having any one of Formulas 1-7, and A may be
selected from nucleotides with uracil, adenine, guanine, thymine or cytosine nucleobases, or
nucleotides with pseudocomplementary nucleobases (e.g., 2-thiouracil, 2,6-diaminopurine, inosine, 3-
pyrrolo-[2,3-d]-pyrimidine(3H)-one). C may be any natural or non-natural nucleotide, presently
known or discovered in the future, that is capable of Watson-Crick base pairing with any one of B ,
B …B . “B” and “C” also can be pseudocomplementary base pairs that do not form strong (or any
2 m-s
base pairs); P may be selected from any of the disclosed monomers, and D may be a Watson-Crick
base pairing nucleotide, or derivative thereof, or another disclosed monomer, which is capable of
coupling with a complementary Watson-Crick base pairing nucleotide, or derivative thereof, in a
target. In particular disclosed embodiments, P may be a monomer having any one of Formulas 1-7,
and D may be selected from uracil, adenine, guanine, thymine, and cytosine.
Particular disclosed embodiments concern a probe having a Formula 10, wherein the
variables are defined according to any one of the following: m = 2, n = 5, o = p = q = r = s = 0, f = g
= h = i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y are adeninyl DNA
nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X = P are monomers with a uracil
nucleobase, and V = Y are adeninyl DNA nucleotide; m = 2, n = 9, o = p = q = r = s = 0, f = g = h =
i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m
= 4, n = 7, o = p = q = r = s = 0, f = g = h = i = j = 0, X = P are monomers with a uracil nucleobase,
and V = Y are adeninyl DNA nucleotide; m = 6, n = 5, o = p = q = r = s = 0, f = g = h = i = j = 0, X
= P are monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m = 8, n = 3,
o = p = q = r = s = 0, f = g = h = i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y
are adeninyl DNA nucleotide; m = 2, n = 0, o = 7, p = q = r = s = 0, f = 1, g = h = i = j = 0, X = P
are monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m = 2, n = 2, o =
, p = q = r = s = 0, f = 1, g = h = i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y
are adeninyl DNA nucleotide; m = 2, n = 4, o = 3, p = q = r = s = 0, f = 1, g = h = i = j = 0, X = P
are monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m = 2, n = o = p
= q = r = s = 0, f = g = h = 1, i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y are
adeninyl DNA nucleotide; m = 2, n = 13, o = p = q = r = s = 0, f = g = h = i = j = 0, X = P are
monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m = 2, n = 0, o = 11,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
p = q = r = s = 0, f = 1, g = h = i = j = 0, X = P are monomers with a uracil nucleobase, and V = Y are
adeninyl DNA nucleotide; m = 2, n = 7, o = 4, p = q = r = s = 0, f = 1, g = h = i = j = 0, X = P are
monomers with a uracil nucleobase, and V = Y are adeninyl DNA nucleotide; m = 4, n = 3, o = p =
q = r = s = 0, f = g = h = i = j = 0, X = P are monomers with an adenine nucleobase, and V = Y are
thyminyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X = W are
monomers with a cytosine nucleobase, and V = Y are guaninyl DNA nucleotide; m = 4, n = 3, o = p
= q = r = s = 0, f = g = h = i = j = 0, X = W are monomers with a guanine nucleobase, and V = Y are
cytosinyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j = 0, X is a monomer
with an adenine nucleobase, W is a monomer with a cytosine nucleobase, Y is a thyminyl DNA
nucleotide and V is a guaninyl DNA nucleotide; m = 4, n = 3, o = p = q = r = s = 0, f = g = h = i = j
= 0, X is a monomer with a cytosine nucleobase, W is a monomer with a thymine nucleobase, Y is a
guaninyl DNA nucleotide and V is an adeninyl DNA nucleotide.
FIGS. 3A and B illustrates the concept of using bulge monomers. With reference to FIGS.
3A and B green denotes disclosed the intercalator-functionalized monomers; red represents a non-
nucleosidic linker. One or more bulge monomers can be added to any of the embodiments defined by
the above formula.
Exemplary embodiments of the disclosed probe are provided in Table 1. A person of
ordinary skill in the art will recognize that any of the disclosed embodiments of the probe can be
made from two single-stranded precursors that are combined (hybridized) to form the probe. The
embodiments disclosed in Table 1 concern a probe wherein X is a monomer disclosed herein and R
is defined as a uracil or thymine nucleobase; however a person of ordinary skill in the art would
recognize that any of the disclosed monomers may be used.
Table 1
Exemplary Embodiments of Certain Disclosed Probes and Corresponding Target
Probe Target region
’-d(GTG AXA TGC) 5’-GTG ATA TGC
3’-d(CAC TAX ACG 3’-CAC TAT ACG
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’-d(GGX ATA TAT AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA XAT ATA TCC G) 3’-CCA TAT ATA TCC G
’-d(GGT AXA TAT AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA TAX ATA TCC G) 3’-CCA TAT ATA TCC G
’-d(GGT ATA XAT AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA TAT AXA TCC G) 3’-CCA TAT ATA TCC G
’-d(GGT ATA TAX AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA TAT ATA XCC G) 3’-CCA TAT ATA TCC G
’-d(GGX AXA TAT AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA XAX ATA TCC G) 3’-CCA TAT ATA TCC G
’-d(GGX ATA XAT AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA XAT AXA TCC G) 3’-CCA TAT ATA TCC G
’-d(GGX ATA TAX AGG C) 5’-GGT ATA TAT AGG C
3’-d(CCA XAT ATA XCC G) 3’-CCA TAT ATA TCC G
’-d(G GXA XAT AAG CAG C) 5’-G GTA TAT AAG CAG C
3’-d(C CAX AXA TTC GTC G) 3’-C CAT ATA TTC GTC G
’-GGT AXA XAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAX AXA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA XAX AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT AXA XCC G 3’-CCA TAT ATA TCC G
’-GGX AXA XAX AGG C 5’-GGT ATA TAT AGG C
3’-CCA XAX AXA XCC G 3’-CCA TAT ATA TCC G
Additional exemplary embodiments are disclosed in Table 2. The embodiments disclosed in Table 2
concern a probe wherein the monomer in each strand (e.g. the strands designated as 5’ and 3’) may
have any one of formulas 1-7, wherein the R moiety is selected from adenine (A), cytosine (C),
guanine (G), uracil (U), or thymine (T).
Table 2
Exemplary Embodiments of Certain Disclosed Probes and Corresponding Target Regions
Probe Target region
’-d(GTG AAT TGC) 5’-GTG AAT TGC
3’-d(CAC TTA ACG) 3’-CAC TTA ACG
’-d(GTG AAG TGC) 5’-GTG AAG TGC
3’-d(CAC TTC ACG) 3’-CAC TTC ACG
’-d(GTG AAC TGC) 5’-GTG AAC TGC
3’-d(CAC TTG ACG) 3’-CAC TTG ACG
’-d(GTG ACG TGC) 5’-GTG ACG TGC
3’-d(CAC TGC ACG) 3’-CAC TGC ACG
’-d(GTG ACC TGC) 5’-GTG ACC TGC
3’-d(CAC TGG ACG) 3’-CAC TGG ACG
’-d(GTG ACA TGC) 5’-GTG ACA TGC
3’-d(CAC TGT ACG) 3’-CAC TGT ACG
’-d(GTG AGC TGC) 5’-GTG AGC TGC
3’-d(CAC TCG ACG) 3’-CAC TCG ACG
’-d(GTG AGA TGC) 5’-GTG AGA TGC
3’-d(CAC TCT ACG) 3’-CAC TCT ACG
’-d(GTG ATT TGC) 5’-GTG ATT TGC
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3’-d(CAC TAA ACG) 3’-CAC TAA ACG
’-d(GTG ATA TGC) 5’-GTG ATA TGC
3’-d(CAC TAT ACG) 3’-CAC TAT ACG
Additional working embodiments of probes are provided below in Table 3 with 0-
arrangements.
Table 3
Working Embodiments of Probes with 0-Arrangement
Probe Target Region
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 5’-GGT ATA TAT AGG C
3’-CCA TAT ATA TCC G 3’-CCA TAT ATA TCC G
Tables 4-6 outline additional working embodiments of certain exemplary probes targeting
different DNA regions, i.e., second insulin [INSB], PPAR gamma and CEBP promotors.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 4
Probes Targeting Second Insulin Promoter [INSB]
Probe Target region
’-G GTA TAT AAG CAG CAC A 5’-G GTA TAT AAG CAG CAC A
3’-C CAT ATA TTC GTC GTG T 3’-C CAT ATA TTC GTC GTG T
’-G GTA TAT AAG CAG CAC A 5’-G GTA TAT AAG CAG CAC A
3’-C CAT ATA TTC GTC GTG T 3’-C CAT ATA TTC GTC GTG T
’-AGG AAG GTA TAT AAG CA 5’-AGG AAG GTA TAT AAG CA
3’-TCC TTC CAT ATA TTC GT 3’-TCC TTC CAT ATA TTC GT
’-ACT ATA GAA TAC TCA AG 5’-ACT ATA GAA TAC TCA AG
3’-TGA TAT CTT ATG AGT TC 3’-TGA TAT CTT ATG AGT TC
Table 5
Additional Examples of Probes for PPAR Gamma
Probe Target region
’-CCC ACG TTA GCA GTT 5’-CCC ACG TTA GCA GTT
3’-GGG TGC AAT CGT CAA 3’-GGG TGC AAT CGT CAA
’-CCC ACG TTA GCA GTT 5’-CCC ACG TTA GCA GTT
3’-GGG TGC AAT CGT CAA 3’-GGG TGC AAT CGT CAA
’-AGA CAA AAC ACC AGT 5’-AGA CAA AAC ACC AGT
3’-TCT GTT TTG TGG TCA 3’-TCT GTT TTG TGG TCA
’-AGA CAA AAC ACC AGT 5’-AGA CAA AAC ACC AGT
3’-TCT GTT TTG TGG TCA 3’-TCT GTT TTG TGG TCA
’-CTA CAT TGT CTC GCC 5’-CTA CAT TGT CTC GCC
3’-GAT GTA ACA GAG CGG 3’-GAT GTA ACA GAG CGG
’-CTA CAT TGT CTC GCC 5’-CTA CAT TGT CTC GCC
3’-GAT GTA ACA GAG CGG 3’-GAT GTA ACA GAG CGG
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’-CGT CAT CGT GCT CGC 5’-CGT CAT CGT GCT CGC
3’-GCA GTA GCA CGA GCG 3’-GCA GTA GCA CGA GCG
Table 6
Additional Examples of Probes for CEBP
Probe Target region
’-CGG ACC ACG TGT GTG 5’-CGG ACC ACG TGT GTG
3’-GCC TGG TGC ACA CAC 3’-GCC TGG TGC ACA CAC
’-CGG ACC ACG TGT GTG 5’-CGG ACC ACG TGT GTG
3’-GCC TGG TGC ACA CAC 3’-GCC TGG TGC ACA CAC
’-GTC AGT GGG CGT TGC 5’-GTC AGT GGG CGT TGC
3’-CAG TCA CCC GCA ACG 3’-CAG TCA CCC GCA ACG
’-GTC AGT GGG CGT TGC 5’-GTC AGT GGG CGT TGC
3’-CAG TCA CCC GCA ACG 3’-CAG TCA CCC GCA ACG
’-CCT CTA TAA AAG CGG 5’-CCT CTA TAA AAG CGG
3’-GGA GAT ATT TTC GCC 3’-GGA GAT ATT TTC GCC
’-CCT CTA TAA AAG CGG 5’-CCT CTA TAA AAG CGG
3’-GGA GAT ATT TTC GCC 3’-GGA GAT ATT TTC GCC
Additional working embodiments of probes that may be used for gender determination in
animals, more commonly, in bovine, are shown in Table 7, where Cy3 is a Cy3 fluorophore;
underlined A/C/G/T are monomers; and underlined B is a bulged (non-pairing) monomer .
Table 7
Bovine Series
Probe Target Region
’-AGC CCT GTG CCC TG 5’-AGC CCT GTG CCC TG
3’-TCG GGA CAC GGG AC 3’-TCG GGA CAC GGG AC
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’-CCT GTG CCC TG 5’-CCT GTG CCC TG
3’-GGA CAC GGG AC 3’-GGA CAC GGG AC
’-CCT GTG CCC TG 5’-CCT GTG CCC TG
3’-GGA CAC GGG AC 3’-GGA CAC GGG AC
’-AGC CCT GTG CCC TG 5’-AGC CCT GTG CCC TG
3’-TCG GGA CAC GGG AC 3’-TCG GGA CAC GGG AC
’-CTG AAGC CCT GTG CCC TG 5’-CTG AGC CCT GTG CCC TG
3’-GAG TCG GGA CAC GGG AC
3’-GAG TCG GGA CAC GGG AC
’-AGC CCT GTG CCC TG 5’-AGC CCT GTG CCC TG
3’-TCG GGA CAC GGG AC 3’-TCG GGA CAC GGG AC
’-AGC CCT GTG CCC TG
'-Cy3 AGC CCT GTG B CCC TG 3’-TCG GGA CAC GGG AC
3'- TCG GGA CAC B GGG AC Cy3
’-AGC CCT GTG CCC TG
'-Cy3 AGC CCT GTG B CCC TG 3’-TCG GGA CAC GGG AC
3'- TCG GGA CAC GGG AC Cy3
’-AGC CCT GTG CCC TG
'-Cy3 AGC CCT GTG CCC TG 3’-TCG GGA CAC GGG AC
3'- TCG GGA CAC B GGG AC Cy3
IV. TARGETS
Particular disclosed embodiments concern targeting and binding to a particular target using
the disclosed probe. In particular disclosed embodiments, the target may be a nucleic acid, such as,
but not limited to, single-stranded DNA (and derivatives thereof), double-stranded DNA (and
derivatives thereof), and any combinations thereof. Particular disclosed embodiments concern
targeting isosequential (relative to the probe) double stranded DNA target regions, including: stems
of molecular beacons, target regions embedded within PCR amplicons, target regions embedded
within circular or linearized plasmids, target regions embedded within genomic DNA (crude,
purified, cell culture, in vivo, embryos, etc.), target regions embedded within microorganisms, and
the like. This list of targets is meant to be exemplary and is not intended to be limiting. In particular
disclosed embodiments, the target may be selected by identifying an RNA target, such as those
pursued in antisense/siRNA/anti-miRNA clinical trials and pre-clinical trials (e.g. those used in
modulation of gene expression and/or identification of biomarkers) and design a target comprising
the corresponding DNA to this particular RNA target. Solely by way of example, specific targets
include linearized plasmids (e.g. against T7 promotor, as illustrated by FIGS. 18-20); circular
6478592_1 (GHMatters) P95976.NZ ESTHERJ
plasmids (e.g. against insulin B promotor in circular plasmids, as illustrated by FIGS. 21-23);
genomic DNA; structured dsDNA targets (FIGS 24-26 and FIGS 30-42).
A target nucleic acid sequence can vary substantially in size. Without limitation, the nucleic
acid sequence can have a variable number of nucleic acid residues. For example a target nucleic acid
sequence can have at least about 2 nucleic acid residues, typically at least about 10 nucleic acid
residues, or at least about 20, 30, 50, 100, 150, 500, 1000 residues.
In specific, non-limiting examples, a protein is produced by a target nucleic acid sequence
(e.g., genomic target nucleic acid sequence) associated with a neoplasm (for example, a cancer).
Numerous chromosome abnormalities (including translocations and other rearrangements,
amplification or deletion) have been identified in neoplastic cells, especially in cancer cells, such as B
cell and T cell leukemias, lymphomas, breast cancer, colon cancer, neurological cancers and the like.
Therefore, in some examples, at least a portion of a protein is produced by a nucleic acid sequence
(e.g., genomic target nucleic acid sequence) that is amplified or deleted in at least a subset of cells in
a sample.
Oncogenes are known to be responsible for several human malignancies. For example,
chromosomal rearrangements involving the SYT gene located in the breakpoint region of
chromosome 18q11.2 are common among synovial sarcoma soft tissue tumors. The t(18q11.2)
translocation can be identified, for example, using the disclosed probe. In other examples, a protein
produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is selected that is
a tumor suppressor gene that is deleted (lost) in malignant cells. In particular disclosed embodiments,
this type of target may also be detected, identified, the expression of the gene reduced and/or the gene
modified using disclosed probe embodiments. For example, the p16 region (including D9S1749,
D9S1747, p16(INK4A), p14(ARF), D9S1748, p15(INK4B), and D9S1752) located on chromosome
9p21 is deleted in certain bladder cancers. Chromosomal deletions involving the distal region of the
short arm of chromosome 1 (that encompasses, for example, SHGC57243, TP73, EGFL3, ABL2,
ANGPTL1, and SHGC-1322), and the pericentromeric region (e.g., 19p13-19q13) of chromosome 19
(that encompasses, for example, MAN2B1, ZNF443, ZNF44, CRX, GLTSCR2, and GLTSCR1) are
characteristic molecular features of certain types of solid tumors of the central nervous system.
The aforementioned examples are provided solely for purpose of illustration and are not
intended to be limiting. Numerous other cytogenetic abnormalities that correlate with neoplastic
transformation and/or growth are known to those of ordinary skill in the art. Target proteins that are
produced by nucleic acid sequences (e.g., genomic target nucleic acid sequences), which have been
correlated with neoplastic transformation and which are useful in the disclosed methods, also include
the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC_000007, nucleotides
55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC_000008,
nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g.,
GENBANK™ Accession No. NC_000008, nucleotides 19841058-19869049), RB1 (13q14; e.g.,
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GENBANK™ Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g.,
GENBANK™ Accession No. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC
(2p24; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides
151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC_000012,
complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession
No. NC_000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™ Accession
No. NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK
(2p23; e.g., GENBANK™ Accession No. NC_000002, complement,
nucleotides 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™ Accession
No. NC_000011, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession
No. NC_000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™
Accession No. NC_000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32-
p31; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides 59019051-59022373),
TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NC_000017, complement,
nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No.
NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™
Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g.,
GENBANK™ Accession No. NC_000007, complement, nucleotides 13897379-13995289), EWS
(22q12.2; e.g., GENBANK™ Accession No. NC_000022, nucleotides 27994271-28026505); FLI1
(11q24.1-q24.3; e.g., GENBANK™ Accession No. NC_000011, nucleotides
128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC_000002,
complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™
Accession No. NC_000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™
Accession No. NC_000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g.,
GENBANK™ Accession No. NC_000019, complement, nucleotides 45431556-45483036), MYCL1
(1p34.2; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides
40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC_000002, nucleotides
60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC_000005,
complement, nucleotides 149413051-149473128).
In other examples, a target protein is selected from a virus or other microorganism
associated with a disease or condition. Detection of the virus- or microorganism-derived target
nucleic acid sequence (e.g., genomic target nucleic acid sequence) in a cell or tissue sample is
indicative of the presence of the organism. The disclosed probe may be used to detect and/or identify
these types of targets. Also, a gene encoding a critical enzyme for the survival of a microorganism
can be targeted by disclosed probe embodiments, which can cause the death of the microorganism.
For example, the target protein can be selected from the genome of an oncogenic or pathogenic virus,
a bacterium or an intracellular parasite (such as Plasmodium falciparum and other Plasmodium
6478592_1 (GHMatters) P95976.NZ ESTHERJ
species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, and Giardia lamblia, as
well as Toxoplasma, Eimeria, Theileria, and Babesia species).
In some examples, the target protein is produced from a nucleic acid sequence (e.g.,
genomic target nucleic acid sequence) from a viral genome. Exemplary viruses and corresponding
genomic sequences may selected from the following (GENBANK™ RefSeq Accession No. in
parentheses): human adenovirus A (NC_001460), human adenovirus B (NC_004001), human
adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266),
human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus
(V01109; GI:60851) human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human
coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus
OC43 ( NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472),
human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrovirus V9
(NC_004295), human foamy virus (NC_001736), human herpesvirus 1 (Herpes simplex virus type 1)
(NC_001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC_001798), human herpesvirus
3 (Varicella zoster virus) (NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1)
(NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human
herpesvirus 5 strain AD169 (NC_001347), human herpesvirus 5 strain Merlin Strain (NC_006273),
human herpesvirus 6A (NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7
(NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P
(NC_009333), human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2
(NC_001722), human metapneumovirus (NC_004148), human papillomavirus-1 (NC_001356),
human papillomavirus-18 (NC_001357), human papillomavirus-2 (NC_001352), human
papillomavirus-54 (NC_001676), human papillomavirus-61 (NC_001694), human
papillomavirus-cand90 (NC_004104), human papillomavirus RTRX7 (NC_004761), human
papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC_008189), human
papillomavirus type 103 (NC_008188), human papillomavirus type 107 (NC_009239), human
papillomavirus type 16 (NC_001526), human papillomavirus type 24 (NC_001683), human
papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human
papillomavirus type 34 (NC_001587), human papillomavirus type 4 (NC_001457), human
papillomavirus type 41 (NC_001354), human papillomavirus type 48 (NC_001690), human
papillomavirus type 49 (NC_001591), human papillomavirus type 5 (NC_001531), human
papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC_001593), human
papillomavirus type 60 (NC_001693), human papillomavirus type 63 (NC_001458), human
papillomavirus type 6b (NC_001355), human papillomavirus type 7 (NC_001595), human
papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC_001596), human
papillomavirus type 92 (NC_004500), human papillomavirus type 96 (NC_005134), human
parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC_003443), human
6478592_1 (GHMatters) P95976.NZ ESTHERJ
parainfluenza virus 3 (NC_001796), human parechovirus (NC_001897), human parvovirus 4
(NC_007018), human parvovirus B19 (NC_000883), human respiratory syncytial virus (NC_001781)
, human rhinovirus A (NC_001617), human rhinovirus B (NC_001490), human spumaretrovirus
(NC_001795), human T-lymphotropic virus 1 (NC_001436), human T-lymphotropic virus 2
(NC_001488).
In certain examples, the target protein is produced from a nucleic acid sequence (e.g.,
genomic target nucleic acid sequence) from an oncogenic virus, such as Epstein-Barr Virus (EBV) or
a Human Papilloma Virus (HPV, e.g., HPV16, HPV18). In other examples, the target protein
produced from a nucleic acid sequence (e.g., genomic target nucleic acid sequence) is from a
pathogenic virus, such as a Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a
Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), or a
Herpes Simplex Virus (HSV). Other targets contemplated by the present disclosure include Her1,
Her2, Her3 and Her4 (e.g. EGFR1, EGFR2, EGFR3 and EGFR4, respectively, or ErbB-1, ErbB-2,
ErbB-3 and ErbB-4, respectively).
V. METHOD OF MAKING MONOMERS
Particular disclosed embodiments concern a method of making the disclosed monomers. In
certain disclosed embodiments, the method may concern synthesizing locked monomers (e.g.
monomers having Formulas 1, 2, or 3); and in other disclosed embodiments, the method may concern
synthesizing unlocked monomers (e.g. monomers having Formulas 1, 2, or 4).
In particular disclosed embodiments, the monomer may be a locked monomer, which may
be synthesized from a precursor 2 using the synthetic sequence illustrated in Scheme 1.
Scheme 1
According to Scheme 1, precursor nucleoside 2 may be converted to an N2’-functionalized
locked nucleic acid using a number of chemical transformations. Treating precursor nucleoside 2
may be converted to azide 4 using methods known to those of ordinary skill in the art, such as by
converting the YH group of precursor nucleoside 2 to a leaving group, such as a mesylate, triflate,
and/or tosylate, and displacing the leaving group using a nucleophilic azide compound, such as
6478592_1 (GHMatters) P95976.NZ ESTHERJ
sodium azide. Azide 4 can then be converted to locked nucleoside 6 using a tandem Staudinger
reaction (iminophosphorane formation)/intramolecular nucleophilic substitution sequence. Protection
and protecting group manipulation of N2’ nucleoside 6 followed by deprotection ultimately provides
locked nucleoside 10 in a number of steps.
In particular disclosed embodiments, the locked nucleoside 10 may be converted to a
monomer suitable for implementation into the disclosed probe. Scheme 2 illustrates the conversion
of locked nucleoside 10 to such a monomer.
Scheme 2
With reference to Scheme 2, the locked nucleoside 10 can be converted to protected
nucleoside 20. N2’ functionalization of protected nucleoside 20 using a variety of substituents can
carried out using conditions known to a person of ordinary skill in the art as being suitable for
coupling an amine with various functional groups. These conditions include, for example, reductive
amination with aromatic aldehydes (ArCHO) using trisacetoxyborohydride or acylation using
aromatic carboxylic acids (ArCOOH) using 1-ethyl(3-dimethylaminopropyl)carbodiimide (EDC)
or 2-(1Hazobenzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate methanaminium
(HATU) as a coupling reagent. N2’ functionalized nucleoside 22 can comprise an optional linker and
an R moiety selected from any of the R moieties disclosed herein. After functionalization of the
YH group of N2’ functionalized nucleoside 22 using methods known to those having ordinary skill in
the art, such as by base-mediated substitution, a monomer 24 can be made. Monomer 24 is suited for
further incorporation into a nucleic acid sequence.
An exemplary method of making the disclosed monomers is illustrated below in Schemes 3
and 4. See, N. K. Andersen, J. Wengel and P. J. Hrdlicka, “N2’-Functionalized 2’-Amino-α-L-LNA
Adenine Derivatives – Efficient Targeting of Single Stranded DNA,” Nucleosides Nucleotides
Nucleic Acids, 2007, 26, 1415-1417, which is incorporated herein by reference.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Bz Bz
MsO O
MsO O MsO O
OBn b OBn
OAc OH
Bz Bz Bz
A A A
MsO O MsO O O
OBn OBn N OBn N
MsO MsO
36 38 40
Bz A
A A O
OH N
OBn N
OH N
MsO MsO
42 44
Bz A
OH N
OH N
DMTrO
Scheme 3 Reagents and conditions: a) BSA, TMSOTf, A , an. 1,2-DCE, reflux 70h; b) 1)
Guanidinium nitrate, NaOMe, MeOH, DCM, 30min, rt. or 2) ½ sat. NH /MeOH, 0 C,1.5h; c)
(CF SO ) O pyridine, an. DCM -78 C, 3h; d) NaN , 15-C-5, an. DMF, 40 C, 16h; e) 2M aq. NaOH,
3 2 2 3
PMe , THF, rt. 16h; f) (CF CO) O, an. pyridine, an CH Cl , 0 C, 2h; g) BCl , an. CH Cl , -78 C to
3 3 2 2 2 3 2 2
rt., 17h; h) NaOBz, 15-crown-5, an. DMF, 90 C, 5h,; i) 2M aq. NaOH, 1,4-diooxane/water, 0 C, 2h;
o Bz
j) DMTrCl, an. pyridine, 0 C to rt., 23h. Key: A = 6-N-benzoyl-adenine. 1,2-DCE = 1,2-
dichloroethane
According to Scheme 3, conversion of diacetate 30 to the desired β-nucleoside 32 was
carried out using a one-pot reaction, under modified Vorbrüggen conditions, with in-situ persilylation
of the benzoyl protected adenine nucleobase using N,O-bis(trimethylsilyl)acetamide (BSA) and
TMSOTf at reflux in 1,2-dichloroethane. Nucleoside 32 was then subjected to chemoselective O2’-
deacetylation using half saturated methanolic ammonia to afford nucleoside 34. Nucleoside 34 was
reacted with trifluoromethanesulfonic anhydride to facilitate formation of the O2’-triflate 36 which
was subsequently without intermediate purification treated with sodium azide and 15-crown-5 in
anhydrous DMF to afford azide 38. IR spectroscopy verified the presence of the azide functionality
(sharp band at 2115 cm ) and provided along with NMR and HRMS-MALDI, evidence for the
proposed structure of azide 38. Azide 38 was converted into the desired bicyclic nucleoside 40 in
80% yield using a one-pot tandem Staudinger/intramolecular nucleophilic substitution reaction.
Protecting nucleoside 40 with a trifluoroacetyl group using trifluoroacetic anhydride in anhydrous
dichloromethane and anhydrous pyridine facilitated formation of nucleoside 42 in 70% yield.
Nucleoside 42 was subsequently subjected to benzylic ether cleavage conditions using BCl in
anhydrous dichloromethane affording debenzylated nucleoside 44 in yields of 65-87%.
Debenzylation was followed by exchanging the methanesulfonyl protecting group at C-5’ with a
benzoyl protecting group. The reaction was carried out under anhydrous conditions using sodium
6478592_1 (GHMatters) P95976.NZ ESTHERJ
benzoate and 15-crown-5 in DMF affording nucleoside 46 in isolated yields of 70 – 83%. Subjecting
nucleoside 46 to sodium hydroxide in water and 1,4-dioxane cleaved both the 5’-benzoyl and
trifluroacetic acid protecting groups. Purification of the amino diol afforded target nucleoside 48 in
60-80% isolated yield. The hydroxyl group of nucleoside 48 was subsequently protected at the 5’-
position by 4,4’-dimethoxytrityl (DMTr) to afford the DMTr-protected nucleoside 50.
Scheme 4 illustrates an exemplary method of converting a DMTr-protected nucleoside 50 to
different exemplary embodiments of the disclosed monomers.
Scheme 4 Reagents and conditions: a) Fmoc-Cl, an. pyridine, rt. 6h, 51%; b) 2-cyanoethyl-N,N’-
(diisopropyl)-phosphoamidochloridite, N-methylimidazole, DIPEA, CH Cl , rt, 4h, 47%; c) 1-
pyrenecarboxaldehyde, NaBH(OAc) , 1,2-DCE, rt., 17h, 68%.; d) 2-cyanoethyl-N,N’-(diisopropyl)-
phosphoamidochloridite, 20% DIPEA in CH Cl , rt. 21h, 51%; e) 1-pyrenecarboxylic acid,
EDCHCl, CH Cl , rt. 45h, 64%, or 1-pyrenecarboxylic acid, HATU, DIPEA, DMF, 0°C to rt, 5h,
74%; f) 2-cyanoethyl-N,N’-(diisopropyl)-phosphoamidochloridite, 20% DIPEA in CH Cl , rt. 22h,
67%; g)1-pyreneacetic acid, EDCHCl, CH Cl , rt, 3.5h, 79%; h) 2-cyanoethyl-N,N’-(diisopropyl)-
phosphoamidochloridite, DIPEA, CH Cl , rt, 71%.
According to Scheme 4, monomer 51W was synthesized from nucleoside 50 using 9-
fluorenylmethoxycarbonyl chloride (Fmoc-Cl) in anhydrous pyridine at 0°C for 6h, and isolated in
51% yield. Nucleoside 51W was converted into the corresponding amidite 52W for use during
oligonucleotide synthesis using 2-cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite and
diisopropylethylamine in anhydrous dichloromethane.
Pyrenylmethyl derivative 51X was synthesized from nucleoside 50 via reductive amination,
using pyrene carboxaldehyde and sodium triacetoxy borohydride in 1,2-dichloroethane at room
temperature for 24h, and was isolated in 60% yield. The functionalized nucleoside 51X was
converted into the corresponding amidite 52X for use during oligonucleotide synthesis, using 2-
6478592_1 (GHMatters) P95976.NZ ESTHERJ
cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite and 20% diisopropylethylamine in
anhydrous dichloromethane.
The amide bond of 51Y was formed via a 1-ethyl(3-dimethyl-amino-propyl)-
carbodiimide hydrochloride (EDCHCl) mediated coupl ing. The reaction was performed by
dissolving bicyclic nucleoside 50 in anhydrous dichloromethane and adding pyrenecarboxylic acid
and EDCHCl. The reaction mixture was stirred at ro om temperature for 45h. The reaction mixture
was subsequently subjected to standard aqueous workup and purification, isolating nucleoside 51Y in
64% yield. Nucleoside 51Y was converted into the corresponding phosphoramidite 52Y using 2-
cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite and 20% diisopropylethylamine in
anhydrous dichloromethane. Similar
In addition, bicyclic nucleoside 50 was converted to monomer 51Z by adding
pyrenecarboxylic acid and EDCHCl. The reaction mi xture was stirred at room temperature for 2.5h
and was subjected to standard aqueous workup and purification, isolating nucleoside 51Z in 79%
yield. Nucleoside 51Z was converted into the corresponding phosphoramidite 52Z using 2-
cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite and diisopropylethylamine in anhydrous
dichloromethane.
Also disclosed are embodiments wherein the R moiety is selected to be thymine. An
exemplary method for synthesizing monomers having R = thymine is illustrated below in Scheme 5.
See, T. S. Kumar, A. S. Madsen, M. E. Østergaard, S. P. Sau, J. Wengel and P. J. Hrdlicka ,
”Functionalized 2′-Amino-α-L-LNA - Directed Positioning of Intercalators for DNA Targeting”, J.
Org. Chem. 2009, 74, 1070-1081, which is incorporated herein by reference.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Scheme 5
With reference to Scheme 5, O5′-tritylated bicyclic nucleoside 54, which may be obtained
from commercially available diacetone-α-D-glucose in 5% overall yield over seventeen steps
involving eight chromatographic purification steps, was used as a suitable starting material for the
synthesis of N2′-functionalized 2′-amino-α-L-LNA phosphoramidites 58Q-58Z. The disclosed
monomers are selected to probe the available structural space in nucleic acids and fall into two
groups based on the nature of the N2’-moiety (e.g. monomers with small non-aromatic units
[monomers 58Q, 58S, and 58V] or with aromatic units [monomers 58W-58Z]). Sodium
triacetoxyborohydride mediated reductive amination of secondary amine 54 with acetaldehyde or 1-
pyrenecarbaldehyde provided tertiary amines 56S and 56W in 48% and 67% yield, respectively.
Chemoselective N-acylation of amino alcohol 54 was achieved using two different strategies.
Treatment of amino alcohol 54 with slight excess of acetic anhydride followed by selective O3′-
deacylation using dilute methanolic ammonia provided nucleoside 56V in excellent 88% yield over
two steps. EDC-mediated coupling of amino alcohol 54 with 1-pyrenylcarboxylic acid, 1-
pyrenylacetic acid or 4-(1-pyrenyl)butyric acid afforded nucleosides 56X, 56Y and 56Z in 62%, 86%
and 63% yield, respectively. A HATU-mediated coupling procedure were used in order to improve
the yield of 56X to 90%. Disappearance of H NMR signals of the exchangeable 3′-OH protons upon
D O addition confirmed the N2′-functionalized constitution of nucleosides 56S-56Z, which
6478592_1 (GHMatters) P95976.NZ ESTHERJ
subsequently were converted to the corresponding phosphoramidites 58S-58Z using 2-cyanoethyl
N,N′-(diisopropyl)-phosphoramidochloridite and diisopropylethyl amine (Hünig’s base). While
amidites 58S-58Y were obtained in good to excellent yields (60-90 %), 58Z is obtained in 36% yield.
The yield of 58X was improved using bis-(N,N-diisopropylamino)cyanoethoxyphosphine in
dichloromethane with diisopropylammonium tetrazolide as an activator.
Other disclosed embodiments concern unlocked monomers and a method of making these
monomers. In particular disclosed embodiments, the unlocked monomers may be made in
approximately three steps from a particular compound. A particular disclosed embodiment of a
method of making the unlocked monomers is illustrated in Scheme 6.
Scheme 6
According to Scheme 6, bicyclic nucleoside 60 may be converted to nucleoside 62 via
methods known to those of ordinary skill in the art, such as Lewis acid-mediated ring-opening and/or
heat-catalyzed nucleophilic addition. In particular disclosed embodiments, an alcohol, thiol, or amine
may be used to open the ring illustrated in Scheme 6. These reagents may be combined with a Lewis
acid, such as a borane, and heat to facilitate functionalization and conversion of the bicyclic
nucleoside 60 into nucleoside 62. Subsequently, nucleoside 62 can be converted to protected
nucleoside 64, which may then be further protected to produce monomer 66. Also according to
Scheme 6, nucleoside 62 may be converted to a compound having a di-functionalized Y2’ moiety at
the C2’ position, such as compound 68, which can be made by methods known in the art, such as via
ring-opening and functional group manipulation. In order to make monomer 70, compound 68 can be
exposed to coupling conditions known to those of ordinary skill in the art, such as, but not limited to
activated couplings and base mediated couplings. In particular disclosed embodiments, activating
6478592_1 (GHMatters) P95976.NZ ESTHERJ
agents, such as N-ethyl-N′-(3-dimethylaminopropyl)carbodiimide (EDC), 1-ethyl(3-
dimethylaminopropyl)carbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC),
carbonyl diimidazole (CDI), 1-hydroxybenzotriazole (HOBt), 1-hydroxyaza-benzotriazole
(HOAt), and o-(7-azabenzotriazolyl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU)
may be used in the activated coupling. In certain disclosed embodiments, coupled nucleosides may
also be obtained by treating nucleoside 68 with a base, such as an aliphatic amine base (e.g.
triethylamine, 1,8-diazabicycloundecene, 1,4-diazabicyclo[2.2.2]octane, and
diisopropylethylamine), and an electrophile, such as a compound comprising an R moiety and a
leaving group, wherein the leaving group may be selected from a halide, a mesylate, a triflate, and a
tosylate. Monomer 70 is made via base-mediated functionalization. In certain embodiments, an
optional linker is inserted between Y and R and/or R .
Sulfur analogs can be used in the general approach illustrated in Scheme 6. An example of
this approach is illustrated below in Scheme 6A where a sulfur nucleophile is introduced using a
nucleophile R YH and a base (e.g., NaH).
N NH
N N O
HO O HO O
NaphCH SH, NaH
DMA, 88%
OH OH S
Scheme 6A
In particular disclosed embodiments, the monomer may be synthesized using the methods
illustrated in Scheme 7, below. A person of ordinary skill in the art will recognize that the methods
of Scheme 7 are exemplary only and are not intended to be limiting.
Scheme 7A
6478592_1 (GHMatters) P95976.NZ ESTHERJ
According to Scheme 7A, 2,2’-anhydrouridine 70 is treated with neat phenols, such as 2-
naphtol and 1-pyrenol, to afford O2’-arylated uredines 72W and 72X in 25% and 44% yield,
respectively. This method was adapted in order to obtain nucleosides 72Y and 72Z by treating
bicyclic nucleoside 70 with tris(pyreneylmethyl) or tris(coroneneylmethyl) borate - generated in
situ upon addition of pyrenylmethanol or coronenylmethanol to borane. This modification
afforded nucleosides 72Y and 72Z in reproducible yields. Subsequent O5’-dimethoxytritylation
afforded nucleosides 74W-74Z in 47-78% yield, which upon treatment with 2-cyanoethyl-N,N-
diisopropylchlorophosporamidite (PCl-reagent) and diisopropylethylamine (Hünig’s base) provided
target phosphoramidites 76W-76Z in 74-78% yield (Scheme 7).
Scheme 7B provides additional examples of monomers obtained via this method.
N NH
HO N HO N O
DMTrCl, pyridine, DMAP
O O X O
BH .THF, DMSO, rt, 14h
160 C, 3h O
OH OH
200W
: R = R" = X = H, R' = neopentyl (20%)
200X : R' = R" = H, R or X = Br (17%)
200Y : R' = R" = X = H, R = CH (20%)
200Z : R = X = H, R' = Bu, R" = OMe (21%)
DMTrO N O DMTrO N O
PCl, DIPEA, CH Cl
rt, 3h
OH N O
204W : R = R" = X = H, R' = neopentyl (81%)
202W
: R = R" = X = H, R' = neopentyl (64%)
204X : R' = R" = H, R or X = Br (82%)
202X : R' = R" = H, R or X = Br (85%)
204Y : R' = R" = X = H, R = CH (78%)
202Y : R' = R" = X = H, R = CH (86%)
204Z : R = X = H, R' = Bu, R" = OMe (80%)
202Z
: R = X = H, R' = Bu, R" = OMe (70%)
Scheme 7B
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Other particular disclosed embodiments of making the disclosed monomers are illustrated in
Schemes 8 and 9. With reference to Scheme 8, and concerning the exemplary conversion of 128 to
130W, certain embodiments used O2’ alkylation, typically using a haloalkane and base, such as
arylmethylhalide and sodium hydride.
Scheme 8
Scheme 9
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Exemplary embodiments of the disclosed monomer may also be obtained using the methods
illustrated in Scheme 10. According to Scheme 10, methylamine nucleoside 80 (made from 5’-O-
dimethoxytrityl-2,2’-anhydrouridine in ~73% yield over three steps) may undergo direct N2’-
alkylation using pyrenylmethyl chloride afforded the desired product 82Q in 46% yield.
Interestingly, reductive amination of 80 using 1-pyrenecarbaldehyde and sodium
triacetoxyborohydride or sodium cyanoborohydride failed to afford 82Q in acceptable yields due to
prominent formation of the corresponding cyclic N2’,O3’-hemiaminal ether. While formation of this
byproduct was avoided by prior protection of the O3’-position of 80 as a TBDMS-ether, the
increased steric bulk resulted in low yields during the subsequent reductive amination.
Scheme 10
Still with reference to Scheme 10 HATU-mediated coupling between nucleoside 80 and 1-
pyrenecarboxylic acid afforded N2’-acylated nucleoside 82S in 78% yield, while EDC-mediated
coupling between nucleoside 80 and 1-pyreneacetic acid furnished 82V in 83% yield. Subsequent
O3’-phosphitylation of 82Q/82S/82V using similar conditions as for the synthesis of 76W-76Z
afforded phosphoramidites 84Q/84S/84V only in moderate yields (42-57%), presumably due to the
increased steric bulk at the N2’-position.
With reference to Scheme 11 reductive amination of 220 using an aromatic aldehyde (e.g.,
1-pyrenecarbaldehyde, 3-perylenecarbaldehyde or 1-coronencarbaldehyde) affords 222 in 43-95%
yield. Subsequent N-methylation of 222 via reductive amination affords 224 in 89-99% yield (note
224X = 82Q & 226X = 84Q). Subsequent O3’-phophitylation (e.g., using cyanoethyl N,N-
diisopropyl-chloro-phosphoramidite, i.e., PCl-reagent) provides 226 in 62-90% yield.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
NH NH
N O N O
DMTrO O DMTrO O
R-CHO, NaBH(OAc) ,
CH O, NaBH(OAc) ,
1,2-DCE
1,2-DCE
OH NH OH HN
NH NH
N O N O
O Cyanoethyl N,N-
DMTrO DMTrO O
diisopropyl-chloro-
phosphoramidite, DIPEA
OH N O N
N P Me
Perylene Coronene
Pyrene
N NH
N N O
HO HO
NaphCH SH, NaH
DMA, 88%
OH OH S
70 240
Scheme 11
Other disclosed embodiments concern monomers comprising a triazole moiety, wherein the
triazole moiety allows the nucleoside to be coupled with a variety of R moieties. Schemes 12 and 13
illustrate an exemplary embodiment of making a monomer comprising a triazole moiety. Scheme 12
illustrates an exemplary method for making the necessary coupling reagents used in producing the
triazole moieties, and Scheme 13 illustrates how these coupling reagents are ultimately used in
triazole synthesis.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Scheme 12
With reference to Scheme 12, 2,2,2-trifluoro-N-(propynyl)acetamide 96, 1-ethynylpyrene
98 and N-(propynyl)pyrenecarboxamide 100 were prepared according to methods readily
known in the art. In contrast, 1-(pyrenyl)-propynone 92 and 4-(pyrenyl)-butyne 94
were obtained via substantially different methods. According to Scheme 12, nucleophilic addition of
MgC≡CTMS (generated in situ from trimethylsilylacetylene and MeMgBr in THF) to pyrene
carboxaldehyde followed by desilylation using potassium carbonate provided 90 in 58% yield.
Subsequent Jones oxidation afforded 92 in 75% yield. Similarly, nucleophilic addition of
HC≡CCH ZnBr (generated in situ from propargyl bromide and activated zinc in THF) to pyrene
carboxaldehyde, followed by deoxygenation of the resultant homopropargyl alcohol using
trifluoroboron etherate and triethylsilane, afforded 94 in 31% yield.
Scheme 13
With reference to Scheme 13, room temperature reactions between 102 and terminal alkynes
92-100 provided the corresponding triazoles 104V-104Z in robust yields (60-83%), except for the
reaction involving 1-ethynylpyrene 98, which required heating (75 °C) to afford nucleoside 104W in
% yield (Scheme 2). Nucleosides 104V-104Z were subsequently converted into phosphoramidites
106V-106Z (51-67% yield) using 2-cyanoethyl-N,N,N’,N’-tetraisopropylphosphordiamidite (PN2-
reagent) and 1H-tetrazole as an activator.
Other disclosed embodiments concern monomers comprising pseudocomplimentary
nucleobases (n particular those involving C2-thiouracil as the nucleobase moiety). With reference to
Scheme 14, protecting group manipulations on nucleoside 82Q (= 224X) converted it to nucleoside
260 in 55% yield. Base-mediated anhydronucleoside formation and nucleophilic cleavage hereof,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
provided 262 in 52% yield. O5’-protection hereof provided 264, which upon treatment with a sulphur
source produced 2-thiouracil derivative 266. Subsequent O3’-phosphitylation using standard
conditions, afforded amidite 268 as a suitable building block for oligonucleotide synthesis.
NH NH
i) (CH CO) O, pyr.
3 2 N OEt
N O N O
ii) AcOH, H O HO O
DMTrO O MsO O
iii) MsCl, pyr:CH Cl DMTr-Cl, pyr
2 2 NaHCO , EtOH, 80 °C
OH N
OH N OAc N
Me Me Me
Py Py Py
82Q = 224X 260
N OEt
DMTrO O
DMTrO O PCl, DIPEA,
DMTrO O
CH Cl
H S, pyr
2 2 2
OH N
OH N
Py Me
Py N P Me
Scheme 14
VI. METHOD OF MAKING DISCLOSED PROBE EMBODIMENTS
Disclosed embodiments concern a method of making a probe capable of recognizing a
target, particularly a double stranded DNA target. In particular disclosed embodiments, the probe is a
nucleic acid duplex that comprises at least one pair of monomers that are capable of reducing the
thermal stability of the duplex and thus promote dissociation of two strands of the duplex.
In certain disclosed embodiments, the probe may be able to identify an isosequential nucleic
acid sequence. For example, the probe may be made by first identifying a desired target, such as a
particular nucleic acid sequence, and then constructing the probe to be a complement to such
sequence by developing a probe having nucleotides capable of Watson-Crick base pairing with the
target. The probe may be modified with at least one pair of monomers, whereas the isosequential
target lacks such a modification.
In particular disclosed embodiments, the probe is constructed by converting one or more of
the monomers disclosed herein to an oligonucleotide, wherein the monomer is coupled with one or
more natural or non-natural nucleobases to form a modified oligonucleotide. The probe may be
designed as a double stranded DNA probe (e.g. monomers and natural and/or non-natural
nucleobases bound together via phosphate moieties), a double stranded phosphorothioate-DNA probe
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(e.g. monomers and natural and/or non-natural nucleobases bound together via one or more
phosphorothioate moieties), a triazole-linked DNA or RNA probe, an unmodified RNA probe,
modified RNA probe and/or other non-natural DNA/RNA strands now known or hereafter discovered
or made.
Scheme 14 illustrates a particular disclosed embodiment of a method for making the probe.
According to Scheme 14, a monomer may be incorporated into an oligonucleotide using methods
known to a person of ordinary skill in the art, such as by using a nucleic acid synthesizer. With
reference to Scheme 15, monomer 110 may be converted into oligonucleotide 112, wherein the wavy
lines indicate the position at which one or more natural or non-natural nucleotides and/or additional
identical or different monomers may be coupled. The transformation illustrated in Scheme 14 can be
obtained by any methods known to those of ordinary skill in the art, such as by using an activator,
such as an imidazole, triazole, or tetrazole compound, and an oxidant, such as iodine or a peroxide
compound. Particular embodiments utilize dicyanoimidazole as the activator. Examples of peroxide
compounds include, but are not limited to, hydrogen peroxide or tert-butyl hydrogen peroxide.
Scheme 15
Particular disclosed embodiments may concern making the disclosed probe according to the
method illustrated in Scheme 16.
DNA synthesizer
DMTrO O
O P O
(i-Pr) N O(CH )CN
58'W: R = CH Py
126W: R = CH Py
58'X:
R = C(O)Py
126X:
R = C(O)Py
58'Y:
R = C(O)CH Py
2 126Y: R = C(O)CH Py
58'Z: R = C(O)CH CH CH Py
2 2 2
126Z: R = C(O)CH CH CH Py
2 2 2
6478592_1 (GHMatters) P95976.NZ ESTHERJ
DMTrO B P O B
synthesizer
(i-Pr) N O O
P R R
76W : R = Nap, B = uracil 120W : R = Nap, B = uracil
76X : R = Py, B = uracil 120X : R = Py, B = uracil
76Y : R = CH Py, B = uracil
120Y : R = CH Py, B = uracil
76Z : R = CH Cor, B = uracil
120Z : R = CH Cor, B = uracil
76'W: R = CH Py, B = adenine-Bz 120'W:
R = CH Py, B = adenine
76'X: R = CH Py, B = thymine 120'X:
R = CH Py, B = thymine
140X: R = CH Py, B = cytosine-Bz 140'X
: R = CH Py, B = cytosine
140Y: R = CH Py, B = guanine-iBu 140'Y: R = CH Py, B = guanine
DMTrO N O
DMTrO N O
DNA synthesizer
O O O
204W : R = R" = X = H, R' = neopentyl
Monomer 208W : R = R" = X = H, R' = neopentyl
204X : R' = R" = H, R or X = Br 208X
Monomer : R' = R" = H, R or X = Br
204Y
: R' = R" = X = H, R = CH Monomer 208Y : R' = R" = X = H, R = CH
204Z : R = X = H, R' = Bu, R" = OMe Monomer 208Z : R = X = H, R' = Bu, R" = OMe
6478592_1 (GHMatters) P95976.NZ ESTHERJ
NH NH
N O N O
DMTrO O O O
DNA synthesizer
O N O O N
N P Me P Me
226Y: R = Pery 228Y: R = Pery
226Z: R = Cor
228Z: R = Cor
DMTrO O
DNA synthesizer
N P Intercalator
Intercalator
DMTrO O
DNA synthesizer
P Me
N P Me R
Scheme 16
6478592_1 (GHMatters) P95976.NZ ESTHERJ
According to Scheme 16, intercalator-functionalized phosphoramidites 120W-120Z, 120Q-
120V, 122W-122Z, and 124W-124Y (a person of ordinary skill in the art will realize that this can
apply to all disclosed monomer embodiments) were incorporated into oligonucleotides via machine-
assisted solid-phase DNA synthesis using an activator, such as an activator selected from 4,5-
dicyanoimidazole and 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole, for time periods ranging from
about 1 minute to about 40 minutes; more typically from about 10 minutes to about 35 minutes.
Particular embodiments concern probes simultaneously comprising one or more of the
disclosed monomers and one or more non-pairing or bulge monomers. According to Scheme 17
below, non-pairing or bulge monomers 402-4, 402-9 and 402-N were incorporated into
oligonucleotides via machine-assisted solid-phase DNA synthesis as recommended by commercial
vendors and/or in an equivalent manner as described for the disclosed intercalator-functionalized
monomers.
N(iPr)
DNA synthesizer
DMTrO P
400-4
402-4
N(iPr)
DNA synthesizer
CN P
DMTrO
O O O O
400-9
402-9
O O O
DMTrO P CN O
DNA synthesizer
N(iPr)2
400-N
FMoc 402-N
Scheme 17
In order to determine the efficiency of probes disclosed herein, the thermal affinity of the
probe toward complementary DNA or RNA targets can be evaluated via UV thermal denaturation
experiments using medium salt buffers that mimic physiological ionic strengths ([Na ] = 110 mM,
Tables 8 and 9). In particular disclosed embodiments, denaturation curves may display sigmoidal
monophasic transitions, such as those exemplary embodiments illustrated in FIGS. 4-5. Changes in
thermal denaturation temperatures (T -values) of modified duplexes are discussed relative to T -
values of unmodified reference duplexes, unless otherwise mentioned.
Certain embodiments entail single-stranded probes comprising the disclosed monomers. It
currently is believed that certain disclosed monomers result in significantly increased thermal affinity
toward single-stranded nucleic acid targets, more commoncly single-stranded DNA. For example, 9-
mer oligonucleotides (ONs), which are modified with locked monomers 126W-Z, display
6478592_1 (GHMatters) P95976.NZ ESTHERJ
extraordinary thermal affinity toward complementary DNA relative to unmodified ONs (ΔT up to
+19.5 °C, Table 8).
Without being limited to one theory of operation, incorporation of monomers with short
linkers appears to result in greater duplex stabilization than monomers with long linkers
(126X>126Y>>126Z); still without being limited to one operation of theory, monomers with acyl
linkers seem preferred over those with alkyl linkers (126X>126W). Significantly similar trends seem
to be observed for ONs modified with N2'-pyrene-functionalized 2'-amino-α-L-LNA adenine
monomers 124X and 124Y (Table 9).
Control ONs that are singly modified with 2'-oxy-α-L-LNA thymine monomer O (i.e.,
without an intercalator) only display moderately increased thermal affinity toward DNA
complements (see Table 8). ONs modified with unfunctionalized 2’-amino-α-L-LNA thymine
monomer N (i.e., without an intercalator) also display low thermal affinity toward DNA (see Table
8).. This suggests that the intercalators of the disclosed monomers have stabilizing roles.
Without being limited to a single theory, it is currently believed that the observed trends in
DNA duplex thermostabilization of probes comprising monomers 120Q-120V indicate that
monomers where the intercalator moiety is attached via N2’-alkyl linkers are preferred over those
with N2’-alkanoyl linkers (compare ΔT /mod for 120S1-120S5 = -6.0 to +4.0 °C with data for
120Q1-120Q5, Table 8) and that extending the N2’-alkanoyl linker between the furanose and
intercalator moiety as in oligonuleotides modified with 2’-N-(pyrenylmethycarbonyl)-2’-
aminouridine monomer 120V, partially reverses the detrimental effects of N2’-acylation on DNA
duplex thermostability (monomer 120S → monomer 120V, ΔT /mod = -0.5 to +6.5 °C, Table 8).
Also, without being limited to a single theory of operation, it is currently believed that the rigid 2’-N-
alkanoyl linker positions the intercalator in an unsuitable position for affinity-enhancing intercalation
and/or that increased solvation of the linker stabilizes the single-stranded state rendering
hybridization less energetically favorable.
Without being limited to a single theory of operation, it is currently believed that the
observed trends in DNA affinity of ONs modified with unlocked monomers, such as monomer 120Q
or 120Y are substantially similar to those obtained with locked monomers, such as 126W-126Z
(Table 8). Significantly similar trend is observed for probes modified with monomers 120’W and
124X bearing adenines as nucleobases (Table 9).
Table 8
ΔT values of duplexes between probes comprised of certain disclosed monomers and
complementary DNA.
ΔT /°C
ON Duplex B = O N 126W 126X 126Y 126Z 120Q 120S 120V 120Y
+2.5 -2.0 +7.0 +10.0 +10.5 +0.5 +5.0 -6.0 -0.5 +5.0
B1 5′-GBG ATA TGC
6478592_1 (GHMatters) P95976.NZ ESTHERJ
D2 3′-CAC TAT ACG
B2 5′-GTG ABA TGC
+6.0 +0.5 +14.0 +19.0 +15.5 +6.0 +14.0 +3.0 +6.0 +12.5
D2 3′-CAC TAT ACG
′-GTG ATA BGC
+3.0 -1.0 +10.5 +14.0 +11.5 - - - - +8.0
D2 3′-CAC TAT ACG
D1 5′-GTG ATA TGC
+3.5 -0.5 +6.5 +10.5 +10.0 +0.5 +1.5 -6.0 +1.0 +3.5
3′-CAC BAT ACG
′-GTG ATA TGC
+8.0 +2.5 +15.5 +19.5 +16.5 +6.5 +13.0 +4.0 +6.5 +11.5
B5 3′-CAC TAB ACG
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); T values
m m m m
determined as the first derivative maximum of denaturation curves (A vs T) recorded in medium salt buffer
([Na ] = 110 mM, [Cl ] = 100 mM, pH 7.0 (NaH PO /Na HPO )), using 1.0 µM of each strand. T values are
2 4 2 4 m
averages of at least two measurements within 1.0 °C; A = adeninyl DNA monomer, C = cytosinyl DNA
monomer, G = guaninyl DNA monomer, T = thyminyl DNA monomer. “-“ = not determined.
Table 9
T Values of Duplexes Between Probes Comprised of Certain Disclosed Monomers and
Complementary DNA
ΔT /°C
ON Duplex B = 124X 124Y 120’W
′-GTG BTA TGC
+5.0 +11.0 +4.5
D2 3′-CAC TAT ACG
B7 5′-GTG ATB TGC
+7.0 +14.0 +8.5
3′-CAC TAT ACG
′-GTG ATA TGC
+6.5 +11.5 +8.5
B8 3′-CAC TBT ACG
D1 5′-GTG ATA TGC
+5.5 +12.0 +6.5
3′-CAC TAT BCG
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table 8
m m m
for experimental conditions.
In particular disclosed embodiments, the results of thermal DNA affinity of other monomers
may vary from ΔT /mod = -13.0 °C to +20.0 °C (Table 10). Also suggested by these results is the
theory that increasing the intercalator surface area leads to additional increases in DNA duplex
thermostability (compare data for probes comprising monomers 120Y and 120Z, Tables 8 and 10)
and that shortening the linker between the furanose and intercalator moiety results in markedly lower
DNA-affinity (compare data for probes comprising monomers 120Y and 120X, Tables 8 and 10).
Furthermore, it is currently believed that concomitant reduction in aromatic surface area results in
6478592_1 (GHMatters) P95976.NZ ESTHERJ
additionally decreased DNA duplex thermostability (compare data for probes comprising monomers
120X and 120W, Table 10).
Table 10
T Values of Duplexes Between Probes Comprised of Certain Disclosed
Monomers and Complementary DNA
T [ΔT /mod] (°C)
ON Duplex B = T 120W 120X 120Z
B1 5′-GBG ATA TGC 29.5 21.5 26.5 34.0
D2 3′-CAC TAT ACG [-8.0] [−3.0] [+4.5]
B2 5′-GTG ABA TGC 29.5 24.5 33.5 49.5
D2 3′-CAC TAT ACG [-5.0] [+4.0] [+20.0]
B3 5′-GTG ATA BGC 29.5 24.5 26.0 40.5
D2 3′-CAC TAT ACG [-5.0] [−3.5] [+11.0]
D1 5′-GTG ATA TGC 29.5 16.5 26.0 36.0
B4 3′-CAC BAT ACG [-13.0] [−3.5] [+6.5]
D1 5′-GTG ATA TGC 29.5 24.5 30.5 45.5
B5 3′-CAC TAB ACG [-5.0] [+1.0] [+16.0]
D1 5′-GTG ATA TGC 29.5 25.5 47.0
B6 3′-CAC BAB ACG [−2.0] [+8.8]
B7 5′-GBG ABA BGC 29.5 25.5
ND ND
D2 3′-CAC TAT ACG [−1.3]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table
m m m
8 above for experimental conditions.
Certain embodiments entail double-stranded probes with interstrand zipper arrangements of disclosed
monomers such as those shown in Table 11, which result in destabilization of the probe. The impact
on duplex thermostability upon hybridization of two single-stranded probes that are each modified
with one or more of the disclosed monomers can be additive, more-than-additive or less-than-additive
relative to the correspondingly singly modified duplexes. This can be readily assessed by the term
‘deviation from additivity’ (DA) for a probe ONX:ONY, defined as:
DA ≡ ΔT (ONX:ONY) – [ΔT (ONX:DNA X) + ΔT (DNA Y:ONY)]
ONX:ONY m m m
where ONX:ONY is a duplex with an interstrand monomer arrangement and ‘DNA X’ and ‘DNA Y’
are the complementary DNA of ONX and ONY, respectively. It follows that DA ~ 0 °C for additive
impacts, DA >> 0 °C for more-than-additive impacts, and DA << 0 °C for less-than-additive impacts
(see also definitions). The term thermal advantage, TA, is strongly related to DA, i.e., TA = - DA (see
also definitions). Double-stranded probes may display a largely positive TA (or largely negative
DA). This energy difference between probe-target duplexes on one side and double-stranded nucleic
acid targets (more commonly dsDNA) and probes on the other side, may provide the driving force for
recognition of double-stranded target regions (more commonly dsDNA target regions), via the
method shown in
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Particular embodiments entail double-stranded probes with +1 interstrand zipper
arrangements (see also definitions) of disclosed monomers as shown in Tables 11 and 12, which
display significant dsDNA-targeting potential as evidenced by the highly negative DA-values (DA
between -40 °C and -12 °C). Other embodiments entail double-stranded probes with +2 zipper
arrangements of intercalator-modified monomers, which also display negative DA-values, although
the values are generally less prominent than probes with +1 zipper monomer arrangements. Probes
with other interstrand zipper arrangements (e.g., +4, -1 and -3) display less regular and/or prominent
dsDNA-targeting potential as indicated by DA-values ranging from moderately negative to
moderately positive (DA between -8.5 °C and +4 °C). Without being limited to a single theory of
operation, probes with +1 interstrand arrangements of disclosed monomers display significant
potential for targeting of isosequential dsDNA regions, and enable targeting of isosequential double-
stranded nucleic acid regions, more commonly double-stranded DNA, via the method outlined in
Control duplexes with two conventional 2'-oxy-α-L-LNA thymine monomer O or 2’-amino-
α-L-LNA thymine monomer N in +1 arrangements, display DA values ~0 °C. This implies that the
dsDNA-targeting potential is generated by the active involvement of the intercalators moieties.
Table 11
Δ Δ Δ ΔT And DA Values for Double-Stranded Probes with Certain Interstrand Zipper
Arrangements of Disclosed Monomers
ΔT [DA]/°C
ON Zipper Duplex B = O N 126W 126X 126Y 126Z 120Q 120S 120V 120Y
+11.0 +1.0 +25.0 +28.5 +26.0 +8.5 +19.5 -3.5 +9.0 +16.5
B1 5′-GBG ATA TGC
3′-CAC TAB ACG
[+0.5] [+0.5] [+2.5] [-1.0] [-1.0] [+1.5] [+1.5] [-5.5] [+3.0] [0.0]
+8.0 -1.5 0.0 +6.5 +12.5 -1.0 -1.5 -17.5 -2.0 -6.0
B1 5′-GBG ATA TGC
B4 3′-CAC BAT ACG
[+2.0] [+1.0] [-13.5] [-14.0] [-8.0] [-2.0] [-8.0] [-5.5] [-2.5] [-14.5]
+16.0 -5.5 +2.5 -1.5 +1.0 -5.5 -2.0 -10.0 +0.5 -2.0
B2 5′-GTG ABA TGC
B5 3′-CAC TAB ACG
[+2.0] [-8.5] [-27.0] [-40.0] [-31.0] [-18.0] [-29.0] [-17.0] [-12.0] [-26.0]
+7.0 -5.5 +15.5 +26.0 +26.5 +9.5 +13.0 -8.5 +5.5 +10.5
B2 5′-GTG ABA TGC
B4 3′-CAC BAT ACG
[+2.5] [-5.5] [-5.0] [-3.5] [+1.0] [+3.0] [-2.5] [-5.5] [-2.5] [-5.5]
B3 5′-GTG ATA BGC +8.5 -2.5 +18.0 +25.0 +29.5
-1 - - - - -
B5 3′-CAC TAB ACG [-2.5] [-4.0] [-8.0] [-8.5] [+1.5]
′-GTG ATA BGC +8.0 -2.5 +18.0 +28.5 +22.0
-3 - - - - -
3′-CAC BAT ACG [+1.5] [-1.0] [+1.0] [+4.0] [+0.5]
ΔT = change in T values relative to unmodified reference DNA duplex D1:D2 (T ≡ 29.5 °C); see Table 8
m m m
for experimental conditions.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 12
Δ Δ Δ ΔT And DA Values for Double-Stranded Probes with Certain
Interstrand Zipper Arrangements of Disclosed Monomers.
ΔT [DA]/°C
ON Zipper Duplex B = 124X 124Y 120’W
+12.0 +13.0 +10.0
′-GTG BTA TGC
B9 3′-CAC TAT BCG
[+1.5] [-10.0] [-1.0]
-7.0 -8.0 -7.5
B6 5′-GTG BTA TGC
3′-CAC TBT ACG
[-18.5] [-30.5] [-20.5]
-5.0 -7.0 -7.0
B7 5′-GTG ATB TGC
B9 3′-CAC TAT BCG
[-17.5] [-33.0] [-22.0]
+14.0 +23.5 +15.0
B7 5′-GTG ATB TGC
B8 3′-CAC TBT ACG
[+0.5] [-2.0] [-2.0]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table
m m m
8 for experimental conditions.
Certain embodiments entail double-stranded probes with ‘mixed’ interstrand arrangements
of disclosed monomers. Double-stranded probes with ‘mixed’ interstrand arrangements of 124X and
124Y are one representative example of this embodiment (e.g., one strand modified with 2'-amino-α-
L-LNA monomer 124X, the other strand modified with 2'-amino-α-L-LNA monomer 124Y, Table
13). Significantly negative DA values are observed for these double-stranded probes. For example,
probes with ‘mixed’ +1 zippers display DA values between -19.5 °C and -23.0 °C (Table 13).
Without being limited to a single operation of theory, probes with +1 zippers composed of different
monomers, display significant dsDNA-targeting potential and enable targeting of dsDNA-regions as
described in FIG 1.
Table13
Δ ΔT And DA Values for Double-Stranded Probes with ‘Mixed’
Interstrand Arrangements of Monomer 124X and Monomer 124Y
ON Zipper Duplex ΔT [DA]/°C
+13.0
124X6 5′-GTG KTA TGC
124Y9 3′-CAC TAT LCG
[-4.0]
′-GTG LTA TGC
124Y6
+3 +11.0
124X9 3′-CAC TAT KCG
6478592_1 (GHMatters) P95976.NZ ESTHERJ
[-5.5]
-3.0
124X6 5′-GTG KTA TGC
3′-CAC TLT ACG
124Y8
[-19.5]
-4.0
′-GTG LTA TGC
124Y6
124X8 3′-CAC TKT ACG
[-21.5]
-4.0
124X7 5′-GTG ATK TGC
3′-CAC TAT LCG
124Y9
[-23.0]
′-GTG ATL TGC
124Y7
124X9 3′-CAC TAT KCG
[-19.5]
+22.0
124X7 5′-GTG ATK TGC
3′-CAC TLT ACG
124Y8
[+3.5]
+18.0
′-GTG ATL TGC
124Y7
124X8 3′-CAC TKT ACG
[-2.5]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table
m m m
8 for experimental conditions.
Certain embodiments entail double-stranded probes where one strand is modified with one
or more monomers comprising a thymine nucleobase, and the other strand is modified is with one or
more monomers comprising an adenine nucleobase. Particular embodiments of a double-stranded
probe where one strand is modified with a thymine monomer (126W or 126X) and the other strand is
modified is with an adenine monomer (124X or 124Y) are given in (Tables 14-17 ). Probes with 0 or
+2 interstrand monomer arrangements generally display significantly negative DA-values (DA-values
between -22 °C to -10 °C), indicating significant potential for targeting of double-stranded nucleic
acid targets, more commonly dsDNA, via the method shown in FIGS 1-2. Probes with -2 interstrand
arrangements display DA-values ~0 °C. Without being limited to a single theory of operation, probes
with 0, +1 or +2 interstrand zipper arrangements of disclosed monomers may display significant
potential for targeting of double-stranded nucleic acids, more commonly dsDNA, and may enable
targeting of dsDNA as shown FIG 1.
Table 14
Δ Δ Δ ΔT And DA Values for Selected Double-Stranded Probes with ‘Mixed’ Interstrand Zippers
Comprised of Monomer 126WAnd Monomer 124X
ON Zipper Duplex ΔT [DA]/°C
+9.5
′-GTG A(126W)A TGC
126W2
124X9 3′-CAC TAT (126X)CG
[-10.0]
6478592_1 (GHMatters) P95976.NZ ESTHERJ
124X6 -4.0
′-GTG (126X)TA TGC
3′-CAC (126W)AT ACG
126W4 [-15.5]
126W2 0.0
′-GTG A(126W)A TGC
3′-CAC T(126X)T ACG
124X8 [-20.5]
126W3 -4.5
′-GTG ATA (126W)GC
3′-CAC TAT (126X)CG
124X9 [-20.5]
126W3 +17.5
′-GTG ATA (126W)GC
3′-CAC T(126X)T ACG
124X8 [+0.5]
124X7 +14.5
′-GTG AT(126X) TGC
3′-CAC (126W)AT ACG
126W4 [+1.0]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table
m m m
8 for experimental conditions.
Table 15
Δ ΔT And DA Values for Selected DNA Duplexes with ‘Mixed’ Interstrand Zippers
Comprised of Monomer 126X And Monomer 124Y
ON Zipper Duplex ΔT [DA]/°C
+12.0
126X2
′-GTG A(126X)A TGC
3′-CAC TAT (124Y)CG
124Y9 [-19.0]
124Y6 +11.5
′-GTG (124Y)TA TGC
3′-CAC (126X)AT ACG
[-10.0]
126X4
+12.5
126X2
′-GTG A(126X)A TGC
3′-CAC T(124Y)T ACG
124Y8 [-18.0]
126X3 +6.5
′-GTG ATA (126X)GC
3′-CAC TAT (124Y)CG
[-19.5]
124Y9
+24.5
126X3
′-GTG ATA (126X)GC
3′-CAC T(124Y)T ACG
124Y8 [-1.0]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table 8
m m m
for experimental conditions.
Table 16
Δ Δ Δ ΔT and DA Values for Selected DNA Duplexes with ‘Mixed’ Interstrand Zippers
Comprised of Monomer 126X) and Monomer 124X
ON Zipper Duplex ΔT [DA]/°C
6478592_1 (GHMatters) P95976.NZ ESTHERJ
126X2 +12.0
′-GTG A(126X)A TGC
3′-CAC TAT (124X)CG
124X9 [-12.5]
124X6 -2.0
′-GTG (124X)TA TGC
3′-CAC (126X)AT ACG
126X4 [-17.5]
126X2 +6.0
′-GTG A(126X)A TGC
3′-CAC T(124X)T ACG
124X8 [-19.5]
124X7 +6.0
′-GTG AT(124X) TGC
3′-CAC TA(126X) ACG
126X5 [-20.5]
126X3 +21.5
′-GTG ATA (126X)GC
3′-CAC T(124X)T ACG
124X8 [+1.0]
124X7 +19.5
′-GTG AT(124X) TGC
3′-CAC (126X)AT ACG
126X4 [+2.0]
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table 8
m m m
for experimental conditions.
Table 17
Δ Δ Δ ΔT And DA Values for Selected DNA Duplexes with ‘Mixed’ Interstrand
Zippers Comprised of Monomer 126W and Monomer 124Y
ON Zipper Duplex ΔT [DA]/°C
126W2 +11.0
′-GTG A(126W)A TGC
3′-CAC TAT (124Y)CG
[-15.0]
124Y9
+0.5
126W3
′-GTG ATA (126W)GC
3′-CAC TAT (124Y)CG
124Y9 [-22.0]
126W3 +24.0
′-GTG ATA (126W)GC
3′-CAC T(124Y)T ACG
[+2.0]
124Y8
ΔT = change in T values relative to unmodified reference duplex D1:D2 (T ≡ 29.5 °C); see Table 8
m m m
for experimental conditions.
Additional embodiments include double-stranded probes with +1 interstrand zipper
arrangements of monomers 208W-Z (Table 18).
Table 18
Thermal Denaturation Properties and dsDNA-Targeting Potential of Certain Disclosed Probes
B = 5’-GTG ATA 5’-GTG ABA 5’-GTG ABA 5’-GTG ATA TA
TGC TGC TGC TGC
6478592_1 (GHMatters) P95976.NZ ESTHERJ
3’-CAC TAT 3’-CAC TAB 3’-CAC TAT 3’-CAC TAB
ACG ACG ACG ACG
o o o o o
T ( C) T [ΔT ]( C) T [ΔT ]( C) T [ΔT ]( C) C
m m m m m m m
120Y 29.5 27.5[-2.0] 42.0 [+12.5] 41.0 [+11.5] +26.0
208W 29.5 25.0 [-4.5] 40.0 [+10.5] 38.0 [+8.5] +23.5
208X 29.5 27.0 [-2.5] 45.0 [+15.5] 45.0 [+15.5] +33.5
208Y 29.5 29.0 [-0.5] 46.5 [+17.0] 45.5 [+16.0] +33.5
208Z 29.5 22.0 [-7.5] 36.0 [+6.5] 35.0 [+5.5] +19.5
see Table 8 for experimental conditions.
With reference to Table 18 above, thermal denaturation temperatures for model DNA duplex target
(column 2 of 6), probe (column 3 of 6), probe-target duplex involving upper (column 4 of 6) or lower
probe strand (column 5 of 6) are given. Thus, probes with +1 interstrand zipper arrangement of the
monomers (column 3) display similar or lower thermostability than model DNA duplex target
(column 2; note negative delta Tm values in column 3), while probe-target duplexes (columns 4 and
) may be greatly stabilized (note highly positive delta Tm values). Without being limited to a single
theory of operation, probes with +1 interstrand arrangements of monomers 120Y/208X/208Y/208Z
display significantly positive thermal advantage (TA) values and, thus, significant potential for
targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method outlined in
FIGS 1-2.
Additional particular embodiments entailing double-stranded probes with +1 interstrand
zipper arrangements of certain disclosed monomers are given below in Table 19. Without being
limited to a single theory of operation, probes with +1 interstrand arrangements of monomers 228X-Z
display significantly positive thermal advantage (TA) values and, thus, significant potential for
targeting of double-stranded nucleic acid targets, more commonly dsDNA, via the method outlined in
FIGS 1-2.
Table 19
Exemplary Embodiments of Double-stranded Probes with +1 Interstrand Zipper
Arrangements Comprising Certain Disclosed Monomers
Sequence Probe Upper probe Lower probe dsDNA target TA [°C]
T [°C] strand strand T [°C]
vs DNA vs DNA
T [°C]
T [°C]
6478592_1 (GHMatters) P95976.NZ ESTHERJ
’ – GTG A(228X)A TGC 29.5 43.5 42.5 29.5 +27.0
3’ – CAC TA(228X) ACG
’ – GTG A(228Y)A TGC 32.5 48.0 51.5 29.5 +37.5
3’ – CAC TA(228Y) ACG
’ – GTG A(228Z)A TGC 41.0 50.5 49.5 29.5 +29.5
3’ – CAC TA(228Z) ACG
see Table 8 above for experimental conditions.
Additional examples of working embodiments of probes based on monomer 120Y are
provided below in Table 20. Table 20 provides thermal denaturation temperatures for a model DNA
duplex target (column 6 of 7), probe (column 5 of 7), probe-target duplexes [i.e., model products
from dsDNA recognition] involving upper (column 3 of 7) or lower probe strand (column 4 of 7).
Probes with one +1 interstrand zipper arrangement of the monomers (first four entries) display
similar or lower thermostability than unmodified dsDNA (representing the target; note negative delta
Tm values in column 5), while probe-target duplexes (columns 3 and 4) display far greater
stabilization (note highly positive delta Tm values). As previously indicated, probes with other
interstrand arrangements may display less positive (or even negative) TA values and may therefore
display less dsDNA-targeting potential (i.e., entriesin this Table). Probes with two or more +1
interstrand arrangements of the disclosed monomers (bottom six entries), may display very high TA-
values, suggesting significant potential for targeting of double-stranded targets, more commonly,
dsDNA target regions, via the method shown in FIGS 1-2.
Table 20
Exemplary Probes Based on Monomer 120Y (= T)
Probe Zipper Upper probe Lower Probe dsDNA TA
strand probe T [ΔT ]( C) target
vs DNA strand T ( C) ( C)
T [ΔT ]( C) vs DNA
T [ΔT ]( C)
’-GGT ATA TAT AGG C +1 44.5 [+7.0] 47.5 [+10.0] 36.5 [-1.0] 37.5
+18.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +1 47.5 [+10.0] 48.5 [+11.0] 36.5 [-1.0] 37.5
+22.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +1 48.5 [+11.0] 47.5 [+10.0] 36.5 [-1.0] 37.5 +22.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +1 47.5 [+10.0] 46.5 [+9.0] 35.5 [-2.0] 37.5
+21.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +3 44.5 [+7.0] 48.5 [+11.0] 54.0 [+16.5] 37.5 +1.5
6478592_1 (GHMatters) P95976.NZ ESTHERJ
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +5 44.5 [+7.0] 47.5 [+10.0] 55.0 [+17.5] 37.5 -0.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +7 44.5 [+7.0] 46.5 [+9.0] 53.0 [+15.5] 37.5 +0.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +3 47.5 [+10.0] 47.5 [+10.0] 57.0 [+19.5] 37.5 +0.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -1 47.5 [+10.0] 47.5 [+10.0] 53.0 [+15.5] 37.5 +4.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +5 47.5 [+10.0] 46.5 [+9.0] 56.0 [+18.5] 37.5 +0.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C +3 48.5 [+11.0] 46.5 [+9.0] 56.0 [+18.5] 37.5 +1.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -1 48.5 [+11.0] 48.5 [+11.0] 55.0 [+17.5] 37.5 +4.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -3 48.5 [+11.0] 47.5 [+10.0] 57.0 [+19.5] 37.5 +1.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -1 47.5 [+10.0] 47.5 [+10.0] 54.0 [+16.5] 37.5 +3.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -3 47.5 [+10.0] 48.5 [+11.0] 57.0 [+19.5] 37.5 +1.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C -5 47.5 [+10.0] 47.5 [+10.0] 57.0 [+19.5] 37.5 +0.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 2x +1 51.5 [+14.0] 55.5 [+18.0] 40.0 [+2.5] 37.5 +29.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 2x +1 53.5 [+16.0] 56.5 [+19.0] 49.0 [+11.5] 37.5 +23.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 2x +1 52.5 [+15.0] 55.5 [+18.0] 49.0 [+11.5] 37.5 +21.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 2x +1 55.5 [+18.0] 55.5 [+18.0] 45.0 [+7.5] 37.5 +28.5
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 2x +1 54.5 [+17.0] 54.5 [+17.0] 47.5 [+10.0] 37.5 +24.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 4x +1 65.5 [+28.0] 67.5 [+30.0] 50.0 [+12.5] 37.5 +45.5
3’-CCA TAT ATA TCC G
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Particular embodiments concern probes with 0-arrangements of disclosed monomers, in
some particular cases, monomers 120Y and 120”W. Such probes may display variable TA values
ranging from -1 °C to +27 °C, demonstrating that said probes may display significant potential for
targeting of double-stranded nucleic acid targets, more commonly dsDNA targets, via the method
outlined in FIGS 1-2.
Table 21
Thermal Denaturation Properties of Certain Probes with 0-Zipper Interstrand Arrangements
Where T is 120Y and A is 120’W
Probe Upper Lower Probe dsDNA TA
probe strand probe strand target
vs DNA vs DNA T [ΔT ]( C)
T ( C) ( C)
T [ΔT ]( C) T [ΔT ]( C)
m m m m
’-GGT ATA TAT AGG C 44.5 [+7.0] 34.0 [-3.5] 38.0 [+0.5] 37.5 +3.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 47.5 [+10.0] 31.0 [-6.5] 40.0 [+2.5] 37.5 +1.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 48.5 [+11.0] 31.0 [-6.5] 40.0 [+2.5] 37.5 +2.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 47.5 [+10.0] 31.0 [-6.5] 40.0 [+2.5] 37.5 +1.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 51.5 [+14.0] 27.0 [-10.5] 41.0 [+3.5] 37.5 ±0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 53.5 [+16.0] 26.0 [-11.5] 41.0 [+3.5] 37.5 +1.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 52.5 [+15.0] 26.0 [-11.5] 42.0 [+4.5] 37.5 -1.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 55.5 [+18.5] 26.0 [-11.5] 42.0 [+4.5] 37.5 +2.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 54.5 [+17.5] 26.0 [-11.5] 42.0 [+4.5] 37.5 +1.0
3’-CCA TAT ATA TCC G
’-GGT ATA TAT AGG C 65.5 [+28.0] 43.0 [+5.5] 44.0 [+6.5] 37.5 +27.0
3’-CCA TAT ATA TCC G
Tables 22-24 outline thermal denaturation properties of additional working embodiments of
yet further probes comprising unlocked monomers targeting different DNA regions, i.e., second
insulin [INSB], PPAR gamma and CEBP promotors). Yet again, the following thermal denaturation
6478592_1 (GHMatters) P95976.NZ ESTHERJ
temperatures are given: model DNA duplex target (column 5 of 6), probe (column 4 of 6), probe-
target duplex [product from dsDNA recognition] involving upper (column 2 of 6) or lower probe
strand (column 3 of 6). Probes with one or more +1 interstrand zipper arrangement of the monomers
display variable thermostability (ranging from strongly destabilized to moderately stabilized relative
to unmodified dsDNA (note delta Tm values from -18 to +8 C, column 4). Probe-target duplexes
(columns 2 and 3) are greatly stabilized (note highly positive delta Tm values). All probes display
positive thermal advantage (TA). Probes with two or more +1 interstrand monomers arrangements
display larger TA values (and thus, greater dsDNA-targeting potential) than probes with a single +1
interstrand monomers arrangement. Without being limited to a single theory of operation, probes with
more than one +1 zipper arrangement of monomers facilitate dsDNA-targeting.
Table 22
Probes Targeting Second Insulin Promoter [INSB] Where Y = 120Y and X = 120Q
Probe Upper probe Lower probe Probe DNA target TA
strand strand duplex
vs DNA vs DNA T [ΔT ]( C)
T ( C) ( C)
T [ΔT ]( C) T [ΔT ]( C)
m m m m
’-G GYA TAT AAG CAG CAC A 58.5 [+6.5] 60.5 [+8.5] 56.0 [+4.0] 52.0 +11.0
3’-C CAY ATA TTC GTC GTG T
’-G GYA YAT AAG CAG CAC A 64.5 [+12.5] 65.5 [+13.5] 60.0 [+8.0] 52.0 +18.0
3’-C CAY AYA TTC GTC GTG T
’-AGG AAG GYA YAT AAG CA 61.5 [+12.0] 64.0 [+14.5] 53.0 [+3.5] 49.5 +23.0
3’-TCC TTC CAY AYA TTC GT
’-ACY AYA GAA TAC TCA AG 57.5 [+12.5] 56.5 [+11.5] 48.0 [+3.0] 45.0 +21.0
3’-TGA YAY CTT ATG AGT TC
’-G GXA TAT AAG CAG CAC A 60.5 [+8.5] 62.5 [+10.5] 54.0 [+2.0] 52.0 +17.0
3’-C CAX ATA TTC GTC GTG T
’-G GXA XAT AAG CAG CAC A 65.0 [+13.0] 66.0 [+14.0] 56.0 [+4.0] 52.0 +23.0
3’-C CAX AXA TTC GTC GTG T
’-AGG AAG GXA XAT AAG CA 62.0 [+12.5] 64.0 [+14.5] 49.0 [-0.5] 49.5 +27.5
3’-TCC TTC CAX AXA TTC GT
’-ACX AXA GAA TAC TCA AG 58.5 [+13.5] 57.0 [+12.0] 44.0 [-1.0] 45.0 +26.5
3’-TGA XAX CTT ATG AGT TC
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 23
Additional Examples of probes for PPAR Gamma
Where T = 120Y; A = 120’W; and C = 140’X
Probe Upper probe Lower Probe dsDNA TA
strand probe strand target
vs DNA vs DNA T [ΔT ]( C)
T ( C) ( C)
T [ΔT ]( C) T [ΔT ]( C)
m m m m
’-CCC ACG TTA GCA GTT 69.0 [+12.0] 68.0 [+11.0] 54.0 [-3.0] 57.0 +26.0
3’-GGG TGC AAT CGT CAA
’-CCC ACG TTA GCA GTT 71.0 [+14.0] 68.0 [+11.0] 57.0 [±0] 57.0 +25.0
3’-GGG TGC AAT CGT CAA
’-AGA CAA AAC ACC AGT 65.0 [+12.0] 60.0 [+7.0] 54.0 [+1.0] 53.0 +18.0
3’-TCT GTT TTG TGG TCA
’-AGA CAA AAC ACC AGT 65.0 [+12.0] 58.0 [+5.0] 46.0 [-7.0] 53.0 +24.0
3’-TCT GTT TTG TGG TCA
’-CTA CAT TGT CTC GCC 66.0 [+10.0] 69.0 [+13.0] 56.0 [±0] 56.0 +23.0
3’-GAT GTA ACA GAG CGG
’-CTA CAT TGT CTC GCC 64.0 [+8.0] 68.0 [+12.0] 59.0 [+3.0] 56.0 +17.0
3’-GAT GTA ACA GAG CGG
’-CGT CAT CGT GCT CGC 73.0 [+9.0] 75.0 [+11.0] 62.0 [-2.0] 64.0 +22.0
3’-GCA GTA GCA CGA GCG
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 24
Additional Examples of Probes for CEBP
Where T = 120Y; A=120’W; C=140’X; and G=140’Y
Probe Upper Lower Probe dsDNA TA
probe strand probe strand target
vs DNA vs DNA T [ΔT ]( C)
T ( C) ( C)
T [ΔT ]( C) T [ΔT ]( C)
m m m m
’-CGG ACC ACG TGT GTG 67.5 [+6.0] 68.5 [+7.0] 46.5 [-15.0] 61.5 +28.0
3’-GCC TGG TGC ACA CAC
’-CGG ACC ACG TGT GTG 69.5 [+8.0] 70.0 [+8.5] 44.5 [-17.0] 61.5 +33.5
3’-GCC TGG TGC ACA CAC
’-GTC AGT GGG CGT TGC 70.5 [+9.5] 73.0 [+11.5] 61.5 [±0] 61.5 +20.5
3’-CAG TCA CCC GCA ACG
’-GTC AGT GGG CGT TGC 74.5 [+13.0] 70.5 [+9.0] 60.5 [-1.0] 61.5 +23.0
3’-CAG TCA CCC GCA ACG
’-CCT CTA TAA AAG CGG 64.5 [+14.0] 62.5 [+12.0] 59.5 [+9.0] 50.5 +17.0
3’-GGA GAT ATT TTC GCC
’-CCT CTA TAA AAG CGG 63.5 [+13.0] 64.5 [+14.0] 47.5 [-3.0] 50.5 +30.0
3’-GGA GAT ATT TTC GCC
The following Tables 25-27 describe the thermal denaturation properties of probes that may
be used for gender determination of individual cells or multicellular assemblies from certain animals
and humans; more commonly somatic cells, sperm cells or embryos from certain animals and
humans; even more commonly, somatic cells, sperm cells or embryos from bovine.
As before, probes display thermostabilities that range from significantly lower to
moderately higher than corresponding unmodified double-stranded DNA targets (note delta Tm
values from -13 C to +9; column 4), while probe-target duplexes (column 2 and 3) are significantly
more thermostable (range from +5 to +24 C). Accordingly, all of the probes (which have between
two to five +1 zipper monomer arrangements) display significantly positive TA-values suggesting
significant potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 25
Thermal Denaturation Properties of Exemplary Probes
Where T = 120Y; A = 120’W; C = 140’X and G = 140’Y
Probe Upper Lower Probe dsDNA TA
probe strand probe target
vs DNA strand T [ΔT ]( C
vs DNA ) T ( C) ( C)
T [ΔT ]( C)
T [ΔT ]( C
’-AGC CCT GTG CCC TG 69.5 [+9.0] 74.5 [+14.0] 58.0 [-2.5] 60.5 +25.5
3’-TCG GGA CAC GGG AC
’-CCT GTG CCC TG 59.5 [+9.0] 65.5 [+15.0] 48.0 [-2.5] 50.5 +26.5
3’-GGA CAC GGG AC
’-CCT GTG CCC TG 59.0 [+8.5] 64.0 [+13.5] 47.0 [-3.5] 50.5 +25.5
3’-GGA CAC GGG AC
’-AGC CCT GTG CCC TG 69.5 [+9.0] 75.5 [+15.0] 61.5 [+1.0] 60.5 +23.0
3’-TCG GGA CAC GGG AC
74.0 [+8.0] 78.0 [+12.0] 57.0 [-9.0] 66.0 +29.0
’-CTG AGC CCT GTG CCC TG
3’-GAG TCG GGA CAC GGG AC
70.0 [+9.5] 80.0 [+19.5] 60.0 [-0.5] 60.5 +29.5
’-AGC CCT GTG CCC TG
3’-TCG GGA CAC GGG AC
Yet another working example of a particular embodiment is provided in Table 26 below,
which shows thermal denaturation properties and TA-values for probes modified with unlocked
monomer 120Q. Similar patters as seen for other disclosed monomers are observed, i.e., probes
display relatively low thermostability while probe-target duplexes are significantly more
thermostable. Probes containing one or more +1 zipper arrangement of unlocked monomer 120Q
therefore display significantly positive TA-values and therefore significant potential for targeting of
double-stranded nucleic acid targets, more commonly dsDNA targets.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 26
Thermal Denaturation Properties and TA-Values for Probes
Modified with Unlocked Monomer 120Q
‘lower’
‘upper’
probe
probe Target
TA Probe strand
Probe strand DNA
(°C) Tm (°C) vs
vs DNA Tm (°C)
Tm (°C)
Tm (°C)
' - GG120Q ATA TAT AGG C
22.0 33.5 45.5 47.5 37.5
3' - CCA 120Q AT ATA TCC G
' - GGX 120Q A120Q A TAT AGG C
.0 43.5 51.5 54.5 37.5
3' - CCA 120Q A120Q ATA TCC G
' - GG120Q A120Q A 120Q A120Q AGG C
56.5 39.5 66.5 67 37.5
3' - CCA 120Q A120Q A120Q A 120Q CC G
Particular embodiments entail double-stranded probes with certain zipper arrangements of
monomers comprising so-called pseudo-complementary nucleobases (e.g., such as 2-thiouracil, 2,6-
diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine(3H)-one), more commonly, +1 zipper
arrangements of monomers comprising pseudo-complementary nucleobases, even more commonly,
+1 zipper arrangements of monomers such as 270. Examples of working examples of these particular
embodiments are given in Table 27 below.
Further particular embodiments entail double-stranded probes with certain zipper
arrangements (more commonly +1 zippers) of monomers comprising nucleobases where, in addition,
the nucleotide opposite of the disclosed monomer comprising a pseudo-complementary nucleobase,
is a nucleotide or disclosed monomer comprising a pseudo-complementary nucleobase (e.g., such as
2-thiouracil, 2,6-diamonopurines, inosine and pyrrolo-[2,3-d]-pyrimidine(3H)-one). For a
representative working examples, please see entries 2 and 4 in Table 29 below, where D is a DNA
monomer with a 2,6-diaminopurine nucleobase (i.e., 2,6-diaminopurine-2’-deoxyriboside). With
reference to Table 27 below, it observed that double-stranded probes with -1 or +1 zipper
arrangements of monomer 270 display positive TA-values, and therefore significant potential for
targeting of double-stranded nucleic acid targets via the method disclosed in FIGS 1-2, more
commonly, dsDNA. With further reference to Table 27 below, it is observed that double-stranded
probes with -1 or +1 zipper arrangements of monomer 270, where, in addition, the nucleotide
opposite of monomer 270 is D display positive TA-values, and therefore significant potential for
targeting of double-stranded nucleic aicd targets via the method disclosed in FIGS 1-2, more
commonly, dsDNA.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 27
Double-Stranded Probes with -1 Or +1 Zipper Arrangements of Monomer 270
Probe dsDNA target Upper probe Lower probe Probe TA
T [°C] strand strand T
vs DNA vs DNA [°C]
T [°C] T [°C]
’ – GTG A(270)A TGC 29.5 41.0 40.5 28.5 +23.5
3’ – CAC TAS ACG
’ – GTG A(270)D TGC 29.5 45.0 45.0 29.5 +31.0
3’ – CAC TD(270) ACG
’ – GTG A(270)A TGC 29.5 41.0 32.0 39.5 +4.0
3’ – CAC (270)AT ACG
’ – GTG D(270)A TGC 29.5 42.5 33.0 29.0 +17.0
3’ – CAC (270)DT ACG
Particular embodiments entail double-stranded probes simultaneously comprising one or
more arrangements of disclosed monomers and one or more non-pairing monomers ( ).
Working examples of such embodiments are provided below. Table 28 concerns thermal
hybridization properties and dsDNA-targeting potential of 18-mer probes containing non-pairing
bulges with a general structure:
5’- GG(120Y) GGT CAA L CTA TC(120Y) GGA
3’- CCA (140’X)CA GGT L GAT AGA (140’X)CT
where “L” denotes the non-pairing monomer; in particular working examples, L is selected from
monomers 402-4, 402-9 and 402-N. With reference to Table 28 below, “4”, “9” and “N” denotes a
single incorporation of 402-4, 402-9 and 402-N, respectively. “444”, “999” and “NNN” denotes three
consecutive incorporations of 402-4, 402-9 and 402-N, respectively. Still with reference to Table 28
below, “Up”, “down” and “both” in the third column, denotes whether bulged monomer(s) were
included only in the upper probe strand, only in the lower probe strand or in both probe strands,
respectively.
With continued reference to the data in Table 28 below, introduction of one or two non-
pairing bulges into double-stranded probes, greatly reduces probe thermostability relative to
corresponding to double-stranded probes that do not contain non-pairing bulges, denoted control
probes (note, Tm values for probes are lower than the 60.5 °C observed for the control probe [i.e.,
bulge = none] shown in the first entry, column 3). While the stability of the probe-target duplexes
also is decreased relative to the control probe (note that Tm values in column 4 and 5 are similar or
lower than the Tm values in column 3), probes comprising one or two non-pairing bulges display
positive TA-values; in the working examples shown below, several of the probes comprising one or
two non-pairing bulges display significantly similar or larger TA-values than the control probe
shown in entry 1, suggesting significant potential for targeting of double-stranded nucleic acid targets
via the method disclosed in FIGS 1-2, more commonly dsDNA.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 28
Thermal Denaturation Properties and TA-Values of Duplexes Involving
Probes Comprising Non-Pairing Monomers
Used bulge Strand(s) with Probe Upper Lower TA
Bulge(s) probe strand probe strand
vs DNA vs DNA
T [˚C] T [˚C] T [˚C] [˚C]
m m m
None - 60.5 68.5 65.5 14.5
Up 47.5 60.0 65.5 19.0
Down 51.5 68.5 58.0 16.0
Both 40.5 60.0 58.0 18.5
9 Up 45.0 55.5 65.5 17.0
Down 46.5 55.0 68.5 19.0
Both 44.5 55.5 53.5 5.5
N Up 54.0 64.5 65.5 17.0
Down 58.0 68.5 63.0 14.5
Both 52.0 64.5 63.0 16.5
444 Up 39.0 53.5 65.5 22.0
Down 44.0 68.5 53.0 18.5
Both 37.0 53.0 53.5 10.5
999 Up 43.0 56.0 65.5 19.5
Down 44.0 68.5 53.0 18.5
Both 44.0 53.0 56.0 6.0
Up 47.5 59.0 65.5 18.0
Down 52.0 68.5 58.0 15.5
Both 41.0 59.0 58.0 17.0
Further working examples of this embodiment are shown in Table 29 below, which
illulstrates the thermal hybridization properties and dsDNA-targeting potential of 13-mer double-
stranded probes comprising monomer 120Y ( = T) and non-pairing monomer 402-9 (= 9).
Introduction of one or two non-pairing monomer 402-9 near the termini of the probes, results in
greatly reduced probe thermostability (note that all Tm values are lower than the 45 C observed for
the control probe shown in the first entry, column 2). While the stability of the probe-target duplexes
also is decreased relative to the control probe (note that Tm values in columns 3 and 4 are the same or
lower than the Tm value in column 3 of entry 1), all probe except for one, positive TA-values.
Without being limited to a theory or operation, this suggests that probes with one or more bulges
6478592_1 (GHMatters) P95976.NZ ESTHERJ
toward probe termini, display significant potential for targeting of double-stranded nucleic acid
targets, more commonly dsDNA, via the method shown in FIGS 1-2.
Table 29
Thermal Hybridization Properties and dsDNA-Targeting Potential
of 13-Mer Double-Stranded Probes Comprising Monomer 120Y
Sequence Probe Upperprobe Lower TA
strand vs DNA probe strand vs (˚ C)
'- GGT ATA TAT AGG C 45.0 55.5 55.5 28.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 44.0 46.5 38.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 44.5 43.5 35.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 <15 <15 -22.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 28.5 44.0 43.5 21.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 32.5 44.5 46.5 21.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 31.5 44.0 55.5 30.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 33.0 44.5 55.5 29.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 35.0 55.5 46.5 29.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C 28.5 55.5 43.5 33.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 <15 55.5 18.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 55.5 <15 18.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 <15 46.5 9.0
3'- CCA TAT ATA TCC G
6478592_1 (GHMatters) P95976.NZ ESTHERJ
'- GGT ATA TAT AGG C <15 <15 43.5 6.0
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 44.0 <15 6.5
3'- CCA TAT ATA TCC G
'- GGT ATA TAT AGG C <15 44.5 <15 7.0
3'- CCA TAT ATA TCC G
Table 30
Thermal Denaturation Properties of exemplary probes
Where T = 120Y; A = 120’W; C = 140’X and G = 140’Y
Lower
Upper probe
dsDNA probe strand strand Probe
target vs DNA vs DNA Tm TA
Probe
Tm (°C) Tm (°C) Tm (°C) (°C) (°C)
'-Cy3 AGC CCT GTG 9 CCC TG 60.5 51.5 57.5 26.5 22
3'- TCG GGA CAC 9 GGG AC Cy3
'-Cy3 AGC CCT GTG 4 CCC TG 60.5 61.5 62.5 34.5 29
3'- TCG GGA CAC 4 GGG AC Cy3
'-Cy3 AGC CCT GTG N CCC TG 60.5 67 70 45.5 31
3'- TCG GGA CAC N GGG AC Cy3
'-Cy3 AGC CCT GTG 9 CCC TG 60.5 51.5 74 29.5 35.5
3'- TCG GGA CAC GGG AC Cy3
'-Cy3 AGC CCT GTG CCC TG 60.5 69.5 57.5 32.5 34
3'- TCG GGA CAC 9 GGG AC Cy3
'-Cy3 AGC CCT GTG 4 CCC TG 60.5 61.5 74 50.5 24.5
3'- TCG GGA CAC GGG AC Cy3
'-Cy3 AGC CCT GTG CCC TG 60.5 69.5 62.5 41.5 30
3'- TCG GGA CAC 4 GGG AC Cy3
'-Cy3 AGC CCT GTG N CCC TG 60.5 67 74 60 20.5
3'- TCG GGA CAC GGG AC Cy3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
'-Cy3 AGC CCT GTG CCC TG 60.5 69.5 70 47 32
3'- TCG GGA CAC N GGG AC Cy3
Table 30 above shows additional working examples of this embodiment. Hybridization
properties and dsDNA-targeting potential of probes comprising non-pairing monomers 4 (402-4), 9
(402-9) or N (402-N) bulges designed for sexing of bovine cells are shown in Table 30 where A =
120’W, C = 140’X, G = 140’Y, and T = 120Y.
VII. METHOD OF USING DISCLOSED PROBE EMBODIMENTS
In particular disclosed embodiments, the disclosed probe is capable of associating with a
target. The disclosed probe comprises at least one pair of monomers that are arranged in a +/-n
zipper arrangement, which allows the monomers to affect the thermostability of the probe. Certain
zipper arrangements, i.e., -1, 0, +1 and +2 zipper arrangements but more commonly +1 zipper
arrangements provides the probe with sufficient thermodynamic instability to cause the two strands of
the probe duplex to dissociate in the presence of a significantly complementary double-stranded
nucleic acid target, more commonly dsDNA, and thereby associate with a target nucleic acid. In
particular disclosed embodiments, the two separated strands of the probe will partake in Watson-
Crick base pairing with the nucleotides of the target nucleic acid, more commonly the two strands of
a dsDNA. In particular disclosed embodiments, the probe may be used in diagnostic techniques, such
as identification of biomarkers, oncogenes, gender-specific genes, etc., and/or direct detection of
double-stranded DNA in living cells, embryos, organs and tissues and/or induction of site-specific
mutagenesis, recombination or repair of genomic DNA and site-specific modulation of gene
expression (i.e. up- or down regulation). The probe is not limited to use in these techniques, as this
list is meant only to be exemplary and not limiting. In particular disclosed embodiments, the probe
may be pre-annealed using methods known to those of ordinary skill in the art. The pre-annealed
probe may then be added to a target, such as a double stranded DNA target.
Particular disclosed embodiments concern using the disclosed probe to inhibit transcription.
The disclosed probe may be designed to have at least one pair of monomers. In particular disclosed
embodiments, the probe is designed to have two pairs of monomers, wherein the two pairs may be
identical or different, and are separated by zero or more natural or non-natural nucleotides, such as
disclosed monomers herein. In particular disclosed embodiments, a pair of monomers comprises at
least two unlocked or locked monomers functionalized with an intercalator and arranged in a +1
zipper arrangement.
The probe may be designed to target a particular isosequential nucleic acid target – whether
synthetic or biological, such as any of those disclosed herein. In certain disclosed embodiments, the
nucleic acid target may be the SP6 and T7 promoters on PGEM-Teasy plasmids that may or may not
overlap with a transcription start site. With reference to this particular disclosed embodiment, the
pGEM-T-Easy vector containing insb-cDNA was linearized with either SpeI or SacII and used for in
6478592_1 (GHMatters) P95976.NZ ESTHERJ
vitro transcription reactions to synthesize cRNA driven by T7 or SP6 promoters, respectively (). The linearized plasmids were incubated with dsDNA-targeting agents as follows: either positive
control (commercial Zorro LNA), targeting the SP6 promoter, or the disclosed probe selected to
target the SP6 or T7 promoter (). Following incubation to facilitate binding of the positive
control and the probe, in vitro transcription was initiated by incubating with ribonucleotide
triphosphates, buffer, and T7 or SP6 polymerases. cRNA products were reverse transcribed to
cDNA. Primers designed to detect a 240 base insert amplicon were used in end-point PCR and the
product was resolved by gel electrophoresis. The results of this particular disclosed embodiment are
illustrated in wherein lanes 1 and 9 illustrate the DNA ladder (100 bp increments); lane 2
illustrates the T7-driven product formed in SpeI digested plasmid in the absence of either the control
or the probe; lane 3 illustrates the SacII digested plasmid, which does not yield T7-driven product;
lane 4 illustrates that a particular embodiment of the probe binds to the T7 promoter and prevents
formation of T7-driven product in SpeI digested plasmid; and lane 5 illustrates that the SP6-driven
product is formed in SacII digested plasmid in the absence of a either the positive control or the
probe. Also referring to , lanes 6-8 illustrate that Zorro LNA (ln 6), one embodiment of the
disclosed probe (ln 7) or a different embodiment of the disclosed probe (ln 8) bind to the SP6
promoter and prevent formation of SP6-driven product in SacII treated plasmid. Other targets are
contemplated by the disclosed method, such as gene knockdown in live cell lines targeting
chromosomal progesterone receptor, estrogen receptors, and any other biologically relevant targets.
In particular disclosed embodiments, the disclosed probe may be used for animal sexing, such as
sexing of ungulates, ruminates, and more particularly bovines, equines and porcines, as the probe
may be designed to selectively targeting gender-specific DNA regions. Examples of gender-specific
DNA regions are known in the art. See, for example, WO 2009079456 and Brown, Kim H., Irvin R.
Schultz, J. G. Cloud, and James J. Nagler (2008) “Aneuploid Sperm Formation in Rainbow Trout
Exposed to the Environmental Estrogen 17a-ethynylestradiol PNAS (USA), 105:19786-19791, both
of which are incorporated herein by reference. In particular disclosed embodiments, the probe may
be designed to comprise one or more pairs of the disclosed monomers and have a sequence of
nucleotides that is isosequential with a particular gene of a target cell that is specific for certain
genetic traits, such as gender. In certain disclosed embodiments, the probe comprises one or more
pairs of disclosed monomers selected from embodiments of monomers disclosed herein, such as
those of Scheme 16, and may contain zero or more non-pairing monomers such as 402-4, 402-9 or
402-9. In particular disclosed embodiments, the probe may be used to determine the gender of
animals and cells (in particular sperm cells), organs, tissues and embryos thereof. Particular
embodiments enable gender determination of unadulterated early-stage embryos from animals used in
food production and sport breeding.
In another disclosed embodiment of using the disclosed probe, a transfected plasmid in-cell
assay was performed. Beta TC-6 cells (ATCC, CRL-11506) were co-transfected with [pGL4.10
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(luc2/-374insb)] and an internal transfection control vector [pGL4.74 (hRluc/TK) (). A probe
comprising at least one pair of locked monomers targeting the insb promotor was transfected 24h
after plasmid co-transfection. In this particular disclosed embodiment, 2 μg of the probe per well was
used (6-well plate format). The cells were harvested 24h after probe addition (90-100% confluency)
and assayed for Firefly and Renilla luciferase (enzyme) activity using a dual luciferase assay system
(Promega) to determine the efficacy and specificity of probe-mediated antigene activity. Firefly
luciferase activity was normalized to Renilla luciferase activity to correct for transfection variation.
Normalized Firefly luciferase activity is expressed relative to a scrambled control probe.
Experiments conducted in triplicate were replicated on three independent occasions. For the dose-
response study, cells were transfected in triplicate with 0.1, 1.0 or 5.0 μg the particular probe per
well. With reference to this transfection study and , a particular embodiment of the probe (i.e.
a probe comprising only one pair of locked monomers) did not show a detectable decrease in
luciferase activity. Two different probes comprising more than one pair of locked monomers did
display an effect (10-30% reduction in luciferase activity). Also explored were dose-dependent
studies, which illustrated that particular embodiments of the probe displayed efficacy at the highest
dose, particular embodiments displayed dose-response effect, and other embodiments displayed
evidence of inversed efficacy with increasing dose. These results are illustrated in .
In particular disclosed embodiments, the probe may be used to target an isosequential
double-stranded DNA duplex or structured analogs hereof. For example, two strands of an
isosequential target duplex may be connected through a linker, such as a polynucleotide, to produce a
duplex having a hairpin configuration (). In exemplary embodiments, the linker comprises
ten thymidines, with the stem of the hairpin target being the primary region recognized by the probe.
In particular disclosed embodiments, the nucleic acid target may or may not comprise one or more
polypurine units. In certain disclosed embodiments, the intramolecular nature of the target duplex of
the hairpin structure increases the T value of the duplex, rendering it a more challenging target than
linear dsDNA targets. The ability of the probe to recognize and invade (or associate with) the
complex may be analyzed using an electrophoretic mobility shift assay.
In exemplary embodiments, the ability of a (+1) interstrand zipper probe comprising at least
one pair of monomers was used to target a hairpin target. According to , hairpin invasion by
the disclosed probe resulted in a probe-target complex having a slower migration rate, such as that
illustrated in lanes A-D of . As illustrated in , the concentration of the probe added to
the target, affects the ability of the probe-target complex to be formed. In particular disclosed
embodiments, an excess of about 5-fold to about 500-fold of the disclosed probe may result in probe-
target complex formation; even more typically, an excess of from about 5-fold to about 50-fold of the
disclosed probe will result in significant probe-target complex formation. According to ,
lanes A-E represent samples in which a varying excess of the probe is used, with lane A representing
a 500-fold excess of the probe, lane B representing a 100-fold excess of the probe, lane C
6478592_1 (GHMatters) P95976.NZ ESTHERJ
representing a 50-fold excess of the probe, lane D representing a 10-fold excess of the probe, and lane
E representing a 5-fold excess of the probe. In particular disclosed embodiments, any of the
disclosed monomers may be incorporated into the probe.
In exemplary embodiments, monomers 124X, 124Y, 126W, 126X, 126Y, 126Z, 120Q, 120S, 120V,
120Y, and 120’W were used to form a (+1) interstrand zipper within the probe. Working examples of
these embodiments are given in FIG 30. Thus, probes with +1 arrangements of the disclosed
monomers, recognize a structured and digoxigenin-labeled dsDNA target comprised of an
isosequential double-stranded target region, which is linked on one side by a single-stranded T loop
(SL1, b). The unimolecular nature of this target (SL) structures leads to extensive
thermostabilization of the double-stranded region [T (SL1) = 56.0 °C vs T (D1:D2) = 29.5 °C].
Incubation of 126W2:126W5 with the structured dsDNA target SL1 (3h at 20 °C) results in the
formation of a recognition complex with lower electrophoretic mobility on 15% non-denaturing
PAGE gels than SL1 (c). A clear dose-response trend is observed; a trace of complex
formation is observed when 5-fold excess of 126W2:126W5 is used, while a 100-fold excess of
126W2:126W5 results in ~48% recognition (c; Table 31, where for all except 124X and
120’W the probe is:
’-GTG ABA TGC
3’-CAC TAB ACG
where B is the disclosed monomer. For all 124X and 120’W the probe is:
5’-GTG BTA TGC
3’-CAC TBT ACG
where B is the disclosed monomer; ). Similar results are observed for probe 120Q2:120Q5
(FIG 30 D, Table 31, FIG 31) and all other evaluated probes (Table 31, FIG 31), except for
120S2:120S5 and 120V2:120V5 (Table 31). This includes probes with +1 interstrand arrangements
of locked thymine monomers 126X and 126Y, unlocked uracil-based monomers 120Y and 120Q, and
adenine-based monomers 124X and 120’W.Thus, in exemplary embodiments, monomers 120S and
120V did not produce a probe-target complex, and monomer 124X was found to be less efficient to
invade the hairpin duplex, while the other monomers exhibited substantial probe-target complex
formation. Without being limited to a single theory of operation, it is currently believed that the
reactivity of monomers 120S, 120V and 124X could be attributed to formation of less thermally
stable probe-target duplexes. In exemplary embodiments, the hairpin invasion reached a saturation
point at 25μM of the probe, wherein about 50% to about 75% of the hairpin was targeted. Additional
results of the percentage of hairpin invasion using 5μM of the probe are summarized in Table 31,
below.
Table 31
Efficiency of Hairpin Invasion by Various Invader
Duplex at 100 Fold Excess of Probe
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Monomer % of invasion
126W 48
126X
126Y 38
120Q
120S <5
120V
120Y 47
124X
120’W 41
average of three independent measurements
In particular disclosed embodiments, the ability of the disclosed probe to invade, or
associate with, the target may be evaluated by comparing its reactivity with that of control probes.
For example, incubation of the mixed-sequence hairpin target with an unmodified isosequential DNA
duplex did not show any complex formation, even with up to a 500-fold excess of the unmodified
DNA duplex. In addition, incubation of the hairpin target with the either single-stranded probe
precursor that comprises a double-stranded probe did not exhibit substantial complex formation
(FIGS. 26 D & E). Furthermore, the sequence specificity of the probe may be determined by
methods known to those of ordinary skill in the art. In exemplary embodiments, singly and doubly
mutated, but perfectly base paired hairpins were targeted. In these particular embodiments, the
probe-target complex was not observed, even at a 100-fold (5uM) excess of the probe, thereby
demonstrating high target specificity (FIGS. 26 A & B).
Specific examples of control experiments were performed to validate the approach and
evaluate the binding specificity (e-h): a) addition of up to 500-fold excess of unmodified
DNA duplex D1:D2 to the structured DNA target SL1 (3 hours, 20 °C) fails to produce a recognition
complex, which underlines the critical role of +1 interstrand arrangements of the disclosed monomers
for dsDNA-targeting (e); b) addition of 100-fold excess of single-stranded
126W2/126W5/120Q2/120Q5/120P2/120P5/120’W6/120’W8 to structured DNA target SL1 (3
hours, 20 °C) fails to yield a recognition complex, which demonstrates that both strands of a probe
enable dsDNA-recognition (f; ); and c) addition of 100-fold excess of probes
126W2:126W5, 120Q2:120Q5, 126X2:126X5, 120Y2:120Y5 or 124X6:124X8 to structured DNA
targets SL2 or SL3 (3h, 20 °C) featuring fully base-paired but non-isosequential stem regions [one or
two base pair deviations relative to probes; T (SL2) = 57.0 °C; T (SL3) = 64.5 °C], failed to form a
recognition complex, which demonstrated that the approach is very specific (g and 30h; ).
VIII. WORKING EXAMPLES
General Experimental Section: Unless otherwise noted, reagents and solvents were
commercially available, of analytical grade and used without further purification. Petroleum ether of
the distillation range 60-80 C was used. Solvents were dried over activated molecular sieves:
6478592_1 (GHMatters) P95976.NZ ESTHERJ
acetonitrile and THF (3Å); CH Cl , 1,2-dichloroethane, N,N’-diisopropylethylamine and anhydrous
DMSO (4Å). Water content of “anhydrous” solvents was verified on Karl-Fisher apparatus.
Reactions were conducted under argon whenever anhydrous solvents were used. Reactions were
monitored by TLC using silica gel coated plates with a fluorescence indicator (SiO -60, F-254) which
were visualized a) under UV light and/or b) by dipping in 5% conc. H SO in absolute ethanol (v/v)
followed by heating. Silica gel column chromatography was performed with Silica gel 60 (particle
size 0.040–0.063 mm) using moderate pressure (pressure ball). Evaporation of solvents was carried
out under reduced pressure at temperatures below 45 °C. After column chromatography, appropriate
fractions were pooled, evaporated and dried at high vacuum for at least 12h to give the obtained
products in high purity (>95%) as ascertained by 1D NMR techniques. Chemical shifts of H NMR
13 31
(500 MHz), C NMR (125.6 MHz), and/or P NMR (121.5 MHz) are reported relative to deuterated
solvent or other internal standards (80% phosphoric acid for P NMR). Exchangeable (ex) protons
were detected by disappearance of signals upon D O addition. Assignments of NMR spectra are
based on 2D spectra (HSQC, COSY) and DEPT-spectra. Quaternary carbons are not assigned in C
NMR but verified from HSQC and DEPT spectra (absence of signals). MALDI-HRMS spectra of
compounds were recorded on a Q-TOF mass spectrometer using 2,5-dihydroxybenzoic acid (DHB)
as a matrix and polyethylene glycol (PEG 600) as an internal calibration standard.
9-[2-O-AcetylO-benzylO-methanesulfonylC-methanesulfonyloxy-methyl]-α α α α-L-threo-
pentofuranosylN-benzoyladenine (32). Benzoylated adenine (28. 6 g, 120 mmol) and coupling
sugar 30 α/β (40.6 g, 80 mmol) were co-evaporated with 1,2-DCE (2 × 150 mL), redissolved in
anhydrous 1,2-DCE (270 mL), and BSA (49.2 mL, 0.20 mol) was added. The heterogeneous mixture
was heated at reflux until becoming homogeneous. After cooling to rt. TMSOTf (43.1 mL, 0.24 mol)
was added and the reaction mixture heated at reflux for 70h. After cooling to rt. the mixture was
poured into sat. aq. NaHCO /crushed ice (500 mL, 1:1, v/v). To control effervescence additional
crushed ice (400 mL) was added, and the mixture was stirred for 30 min during which a precipitate
was formed. The precipitate was removed by filtration and the filtrate was washed with CH Cl (1.5
L). The combined organic phase was washed with sat. aq. NaHCO (2 × 500 mL) and the aqueous
phase back-extracted with CH Cl (2 × 500 mL). The organic phase was evaporated to afford a crude
yellow foam, which was purified by silica gel column chromatography (0-10% i-PrOH in CH Cl ,
v/v) to afford target nucleoside 32 as a white foam (38.3 g, 70%). R = 0.4 (5% MeOH in CH Cl ,
f 2 2
+ + 1
v/v). MALDI-HRMS m/z 712.1380 ([M + Na] , C H N O S Na Calc. 712.1354); H NMR (300
29 31 5 11 2
MHz, CDCl ) δ 8.97 (s, 1H), 8.81 (s, 1H), 8.37 (s, 1H), 8.03 (d, 2H, J = 8.1 Hz), 7.50-7.65 (m, 5),
7.29-7.38 (m, 5H), 6.45 (d, 1H, J = 2.6 Hz), 5.95 (t, 1H, J = 2.4 Hz), 4.78-4.82 (d, J = 11.7 Hz, 1H),
4.63-4.67 (m, 3H), 4.38-4.43 (m, 4H), 3.28-3.32 (d, 1H, J = 10.6 Hz), 3.0 (s, 3H), 2.96 (s, 3H), 2.16
13 Bz Bz
(s, 3H); C NMR (75.5 MHz, CDCl ) δ 169.5, 164.6, 152.9 (A ), 151.6, 149.7, 141.2 (A ), 135.7,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
133.4, 132.8, 128.9, 128.8, 128.7, 128.6, 128.5, 128.4, 128.0, 127.8, 123.0, 87.3 (C1’), 86.0 (C4’),
81.3 (C3’), 79.4 (C2’), 73.3 (CH -Ph), 67.4 (C5’), 65.5 (C5”), 37.7 (OMs), 37.6 (OMs), 20.7 (Ac).
9-(3-O-BenzylO-methanesulfonylC-methanesulfonyloxymethyl-α α-L-threo-pentofuranosyl)-
6-N-benzoyladenine (34)
Small scale procedure using guanidinium nitrate.
A stock solution of guanidine/guanidinium nitrate (G/GHNO ) was prepared according to the
published procedure by dissolving guanidinium nitrate (4.91 g, 40.2 mmol) in a mixture of
MeOH:CH Cl (450 mL, 9:1, v/v) and adding NaOMe (0.24 g, 4.5 mmol). Fully protected nucleoside
32 (4.66 g, 6.7 mmol) was dissolved in an aliquot of the prepared G/GHNO solution (450 mL) and
the mixture was stirred at rt. for 30 min whereupon sat. aq. NH Cl (200 mL) was added, resulting in
formation of a white precipitate. The formed precipitate was filtered off and washed repeatedly with
CH Cl until sugar residues were undetectable in the filtrate by TLC. The filtrate was concentrated
under reduced pressure, the aqueous phase washed with CH Cl (5 × 200 mL) and the combined
organic phase evaporated, affording a yellowish solid. The crude solid was purified by silica gel
column chromatography (0-4% MeOH in CH Cl , v/v) affording alcohol 34 (3.84 g, 88%) as a white
foam. R : 0.5 (10% MeOH in CH Cl , v/v).
f 2 2
Large scale method using ½ sat. methanolic ammonia.
Fully protected nucleoside 32 (30.42 g, 44 mmol) was dissolved in MeOH (706 mL) and the solution
cooled to 0 °C, whereupon sat. methanolic ammonia (353 mL) was added. The reaction mixture was
stirred at rt. for 2.5h whereupon the reaction mixture was evaporated and the obtained residue co-
evaporated with abs. EtOH:toluene (2 × 500 mL, 1:1, v/v). The solid residue was purified by silica
gel column chromatography (0-2.5% MeOH in CH Cl , v/v) affording target alcohol 34 as a white
foam (23.81 g, 84%). R : 0.5 (10% MeOH in CH Cl , v/v). MALDI-HRMS m/z 670.1234 ([M + Na] ,
f 2 2
C H N O S Na Calc. 647.1248); H NMR (300 MHz, CDCl ) δ 9.21 (s, 1H), 8.47 (s, 1H), 8.29
27 29 5 10 2 3
(s, 1H), 7.97 (d, 2H, J = 7.7 Hz), 7.61-7.32 (m, 9H), 6.11 (d, 1H, J = 6.2 Hz), 5.28 (d, 1H, J = 9.2
Hz), 4.96 (d, 1H, J = 11.7 Hz), 4.75 (d, 1H, J = 11.3 Hz), 4.61 (d, 1H, J = 10.6 Hz), 4.45-4.25 (m,
13 Bz
4H), 3.03 (s, 3H), 2.90 (s, 3H). C NMR (75.5 MHz, CDCl ) δ 165.2, 152.4 (A ), 151.1, 148.6,
141.9 (A ), 136.8, 133.2, 133.0, 128.9, 128.6, 128.3, 127.8, 121.7, 87.6 (C1’), 83.0 (C4’), 81.9
(C3’), 78.6 (C2’), 73.2 (CH -Ph), 68.4 (C5’), 68.2 (C5”), 37.7 (OMs), 37.4 (OMs).
1-(2-AzidoO-benzylC-methanesulfonyloxymethylO-methanesulfonyldeoxy-α α α α-L-
erythro-pentofuranosyl)benzoyl-adenineyl (36, 38): Alcohol 34 (18. 6 g, 30 mmol) was co-
evaporated with pyridine (50 mL), and subsequently dissolved in an. CH Cl (200 mL). The mixture
was then cooled to -78 °C, and pyridine (7.3 mL, 90 mmol) and trifluoromethanesulfonyl anhydride
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(9.90 mL, 60 mmol) was added. The mixture was stirred for 3h during which it was allowed to heat
to rt. Hereafter crushed ice (50 mL) was added. The organic phase was washed with sat. aq. NaHCO
(2 × 50 mL), evaporated to dryness and co-evaporated with abs. EtOH (2 × 50 mL) affording a crude
yellowish/brown solid which was used in the next step without further purification. The crude solid,
tentatively assigned as triflate derivative 36, was dissolved in DMF (300 mL), and NaN (19.6 g, 0.3
mol) and 15-crown-5 (6.0 mL, 30.2 mmol) was added. The reaction mixture was stirred at rt. for 15h,
where analytical TLC showed approximately 50% conversion. Subsequently the mixture was heated
to 50 °C for 8h, whereupon analytical TLC showed full conversion. After cooling to rt., the mixture
was filtered to remove excess NaN . The filtrate was washed repeatedly with EtOAc and the
combined organic phase was concentrated under reduced pressure. The concentrated mixture was
taken up in brine (200 mL) and EtOAc (200 mL). The phases were separated and the aqueous phase
extracted with EtOAc (4 × 100 mL). The combined organic phase was evaporated under reduced
pressure affording a crude dark brown solid. The crude solid was then purified by silica gel column
chromatography (0-90% EtOAc in petroleum ether, v/v) affording azide 38 (17.9 g, 89% over 2
steps) as a white solid. R : 0.4 (EtOAc). IR: 2115.6 cm (-N ). MALDI-HRMS m/z 695.1295 ([M +
+ + 1
Na] , C H N O S Na Calc. 695.1313); H NMR (300 MHz, DMSO-d ) δ 11.30 (s, 1H, NH, ex),
27 28 8 9 2 6
Bz Bz
8.79 (s, 1H, A ), 8.54 (s, 1H, A ), 8.05 (d, 2H, J = 7.0 Hz, Bz), 7.53-7.67 (m, 3H, Bz), 7.35-7.47
(m, 5H, Bn), 6.74 (d, 1H, J = 4.4 Hz, H-1’), 5.20 (t, 1H, J = 4.8 Hz, H-2’), 4.89 (d, 1H, J = 5.1 Hz, H-
3’), 4.79-4.83 (d, 1H, J = 11.7 Hz, H-5’ ), 4.74-4.78 (d, 1H, J = 11.7 Hz, H-5’ ), 4.69-4.72 (d, 1H, J =
11.4 Hz, CH -Ph), 4.47-4.51 (d, 1H, J = 11.4 Hz, CH -Ph), 4.42 (s, 2H, H-5’’), 3.28 (s, 3H, OMs),
13 Bz Bz
3.24 (s, 3H, OMs). C NMR (75.5 MHz, DMSO-d ) δ 151.9 (A ), 150.4, 142.7 (A ), 136.9, 133.2,
132.5, 128.5, 128.0, 81.9 (C1’), 81.7 (C3’), 80.1 (C2’), 73.5 (CH -Ph), 68.3 (C5’), 61.9 (C5”), 36.98
(OMs), 36.94 (OMs).
(1R,3R,4S,7R)(6-Benzoyladenineyl)benzyloxymethanesulfonyloxy-methyloxa
azabicyclo[2.2.1]heptane (40): Azide derivative 38 (35.18 g, 51.8 mmol) was dissolved in THF
(500 mL) cooled to 0 °C and aq. NaOH (2M, 38.9 mL, 77.8 mmol) and trimethylphosphine (1M in
THF, 77.8 mL, 77.8 mmol) was added. The reaction mixture was stirred for 21h during which it was
allowed to heat to rt. subsequently, the reaction mixture was evaporated to dryness. The resulting
crude residue was taken up in brine (200 mL) and EtOAc (200 mL), and the aqueous phase was
washed with MeOH:CH Cl (3 × 200 mL, 2:8, v/v). The combined organic phases was evaporated to
dryness and the crude purified by silica gel column chromatography (0-5% MeOH in CH Cl , v/v)
affording bicyclic nucleoside 40 as a yellow/brown solid (24.2 g, 85%). R : 0.3 (EtOAc). MALDI-
+ 13
HRMS m/z 573.1517 ([M + Na] , C H N O SNa Calc. 573.1527); C NMR data is not in
26 26 6 6
accordance with previous data. The corrected data are given here. H NMR (500 MHz, DMSO-d ) δ
Bz Bz
11.20 (s, 1H, NH, ex), 8.77 (s, 1H, A ), 8.73 (s, 1H, A ), 8.06 (d, 2H, J = 7.0 Hz, Bz), 7.53-7.67 (m,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
3H, Bz), 7.29-7.47 (m, 5H, Ph), 6.52 (d, 1H, J = 1.8 Hz, H-1’), 4.72-4.76 (d, 1H, J = 11.7 Hz,
CH Ph), 4.62-4.67 (d, 1H, J = 11.7 Hz, CH Ph), 4.57-4.60 (d, 1H, J = 11.7 Hz, H-5 ’), 4.49-4.53 (d,
2 2 a
1H, J = 11.7 Hz, H-5 ’), 4.44 (s, 1H, H-3’), 3.92 (m, 1H, H-2’), 3.24-3.30 (m, 1H, H5 ” overlap with
H O)3.23 (s, 3H, OMs), 3.10-3.13 (d, 1H, J = 9.89 Hz, H-5 ’’). C NMR (125 MHz, DMSO-d ) δ
2 b 6
Bz Bz
165.5, 152.0, 151.4 (A ), 150.0, 143.1 (A ), 137.8, 133.3, 132.3 (Ar), 128.4 (Ar), 128.2 (Ar), 127.6
(Ar), 127.5 (Ar), 125.0, 87.1, 84.3 (C-1’), 80.3 (C-3’), 71.0 (CH -Ph), 66.8 (C-5’), 59.8 (C-2’), 51.1
(C-5”), 36.8 (Ms).
(1R,3R,4S,7R)(6-Benzoyladenineyl)benzyloxymethanesulfonyloxy-methyl
trifluoroacetyloxaazabicyclo[2.2.1]heptane (42): Amine derivative 40 (8.68 g, 15.8 mmol)
was co-evaporated with pyridine (2 × 20 mL) and dissolved in anhydrous CH Cl (200 mL).
Anhydrous pyridine (5.09 mL, 63 mmol) was added, the mixture cooled to 0 °C, and trifluoroacetic
acid anhydride (4.45 mL, 31.5 mmol) was added. The reaction mixture was stirred for 2h whereupon
crushed ice (50 mL) was added. The organic phase was washed with sat. aq. NaHCO (2 × 50 mL),
and the aqueous phase was back-extracted with CH Cl (2 × 100 mL) and MeOH:CH Cl (100 mL,
2 2 2 2
2:8, v/v). The combined organic phase was evaporated to dryness and co-evaporated with toluene (50
mL) and once with abs. EtOH:toluene (50 mL, 1:1, v/v). The crude residue was purified by silica gel
column chromatography (0-100% EtOAc in petroleum ether, v/v) affording the fully protected
nucleoside 42 as a white foam (6.27 g, 62%). R : 0.5 (10% MeOH:EtOAc, v/v). Physical data for the
mixture of rotamers: MALDI-HRMS m/z 647.1541 ([M + H] , C H F N O S⋅H Calc. 647.1530); H
28 25 3 6 7
NMR (500 MHz, DMSO-d ) δ 11.21 (s, 0.5H, NH, ex), 11.19 (s, 0.5H, NH, ex), 8.78 (s, 0.5H, A ),
Bz Bz Bz
8.76 (s, 0.5H, A ), 8.64 (s, 0.5H, A ), 8.60 (s, 0.5H, A ), 8.05 (d, 2H, J = 7.0 Hz, Bz), 7.52-7.67
(m, 3H, Bz), 7.31-7.41 (m, 5H, Ph), 6.83 (d, 0.5H, J = 1.1 Hz, H-1’), 6.80 (d, 0.5H, J = 1.1 Hz, H-1’),
.26 (s, 0.5H, H-2’), 5.17 (s, 0.5H, H-2’), 4.84 (s, 1H, H-3’), 4.82 (s, 0.5H, H-3’), 4.62-4.79 (m, 4H,
CH Ph, H5”), 4.53 (d, 0.5H, J = 10.6 Hz, H-5’), 4.35 (d, 0.5H, J = 12.1 Hz, H-5’), 4.07 (d, 0.5H, J =
.6 Hz, H-5’), 3.90 (d, 0.5H, J = 12.1 Hz, H-5’), 3.28 (s, 3H, OMs). C NMR (125 MHz, DMSO-
Bz Bz
d ) δ 165.5, 155.2 (q, J = 36.6 Hz, COCF ), 154.8 (q, J = 36.6 Hz, COCF ), 151.8 (A ), 151.6 (A ),
6 3 3
Bz Bz
151.5, 150.3, 150.26, 141.2 (A ), 141.0 (A ), 137.1, 137.0, 133.2, 132.4 (Ar), 128.4 (Ar), 128.38
(Ar), 128.36 (Ar), 128.32 (Ar), 127.9 (Ar), 127.8 (Ar), 127.5 (Ar), 125.4, 125.2, 115.5 (q, J = 288
Hz, CF ), 115.2 (q, J = 288 Hz, CF ), 86.15 (C-4’), 86.14 (C-4’), 84.9 (C-1’), 84.8 (C-1’), 83.9 (C-
2’), 79.1 (C-3’), 77.4 (C-3’), 71.6 (CH Ph), 65.3 (C-5”), 65.0 (C-5”), 63.0 (C-2’), 61.4 (C-2’), 53.2
(C-5’), 53.07 (C-5’) 53.05 (C-5’), 37.0 (OMs). F NMR (376 MHz, DMSO-d ) δ -71.3, -72.1.
(1R,3R,4S,7R)(6-Benzoyladenineyl)hydroxymethanesulfonyloxy-methyl
trifluoroacetyloxaazabicyclo[2.2.1]heptane (44): Fully protected nucleoside 42 (19.63 g,
.4 mmol) was co-evaporated with 1,2-DCE (3 × 100 mL) and dissolved in anhydrous CH Cl (610
6478592_1 (GHMatters) P95976.NZ ESTHERJ
mL). The solution was cooled to -78 °C, followed by addition of BCl (1M solution in hexanes, 370
mL, 370 mmol). The reaction mixture was stirred for 17h during which it was allowed to heat to rt.
The reaction mixture was subsequently cooled to 0 °C and crushed ice (800 mL) was added. The
phases were separated and the organic phase was washed with sat. aq. NaHCO (2 × 300 mL). The
aqueous phase was back-extracted with EtOAc (4 × 500 mL), and the combined organic phase was
evaporated under reduced pressure. The resulting residue was purified using silica gel column
chromatography (0-20% MeOH in CH Cl , v/v) affording target alcohol 44 as a white solid (14.65 g,
87%). R : 0.3 (50% acetone in CH Cl , v/v). Physical data for the mixture of rotamers: MALDI-
f 2 2
HRMS m/z 579.0871 ([M + Na] , C H F N O S⋅Na Calc. 579.0880); H NMR (300 MHz, DMSO-
21 19 3 6 7
Bz Bz Bz
d ) δ 11.21 (s, 1H, NH, ex), 8.76 (s, 0.5H, A ), 8.75 (s, 0.5H, A ), 8.61 (s, 0.5H, A ), 8.56 (s, 0.5H,
A ), 8.04 (d, 2H, J = 7.7 Hz, Bz), 7.51-7.67 (m, 3H, Bz), 6.83 (d, 0.5, J = 1.37 Hz, H-1’), 6.80 (d,
0.5H, J = 1.37 Hz, H-1’), 6.73 (d, 0.5H, J = 4.12 Hz, 3’-OH, ex), 6.69 (d, 0.5H, J = 4.12 Hz, 3’-OH,
ex), 4.91 (br s, 0.5H, H-2’), 4.81 (d, 0.5H, J = 4.12 Hz, H-3’), 4.76 (d, 0.5H, J = 4.12 Hz, H-3’), 4.70
(br s, 0.5H, H-2’), 4.60-4.67 (m, 2H, H-5”), 4.46 (d, 0.5H, J = 10.3 Hz, H-5’), 4.26 (d, 0.5H, J = 11.7
Hz, H-5’), 4.03 (d, 0.5H, J = 10.3 Hz, H-5’), 3.86 (d, 0.5H, J = 11.7 Hz, H-5’), 3.29 (s, 3H, OMs);
C NMR (75.5 MHz, DMSO-d ) δ 165.5, 155.3 (q, J = 36.6 Hz, COCF ), 155.1 (q, J = 36.6 Hz,
Bz Bz Bz Bz
COCF ), 151.7 (A ), 151.5 (A ), 150.3, 150.2, 141.2 (A ), 141.0 (A ), 133.3, 132.4, 128.4, 125.4,
125.2, 115.5 (q, J = 288 Hz, CF ), 115.2 (q, J = 288 Hz, CF ), 87.0 (C-4’), 85.7 (C-4’), 84.7 (C-1’),
83.8 (C-1’), 72.5 (C-3’), 70.6 (C-3’), 65.7 (C-5”), 65.4 (C-5”), 65.3 (C-2’), 63.6 (C-2’), 52.7 (C-5’),
52.6 (C-5’), 36.97 (OMs). F NMR (376 MHz, DMSO-d ) δ -71.1, -72.1.
(1S,3R,4S,7R)(6-Benzoyladeninyl)benzoylmethylhydroxytrifluoro-acetyloxa
azabicyclo[2.2.1]heptane (46): Alcohol 44 (5.8 g, 10.4 mmol) was dissolved in DMF (100 mL)
whereupon NaOBz (2.99 g, 20.8 mmol) and 15-crown-5 (2.07 mL, 10.4 mmol) were added. The
mixture was heated to 90 °C and stirred for 5h, then allowed to cool to rt. and stirred for an additional
18h. Subsequently the mixture was concentrated in-vaccuo and taken up in EtOAc and brine. The
phases were separated and the aqueous phase extracted with EtOAc (4 × 200 mL). The combined
organic phase was evaporated under reduced pressure affording a dark brown solid material which
was purified by silica gel column chromatography (0-3.5% i-PrOH in CHCl , v/v) to afford target
benzoyl protected alcohol 46 (5.05 g, 83%) as a white foam. R = 0.4 (10% i-PrOH in CHCl , v/v).
Physical data for the mixture of rotamers: MALDI-HRMS m/z 605.1337 ([M + Na] ,
C H F N O ⋅Na Calc. 605.1367); H NMR (300 MHz, DMSO-d ) δ 11.18 (br s, 1.7H, NH , ex),
27 21 3 6 6 6 a,b
Bz Bz Bz Bz
8.77 (s, 1H, A ), 8.76 (s, 0.7H, A ), 8.67 (s, 0.7H, A ), 8.63 (s, 1H, A ), 8.09-8.18 (m, 4H,
a b b b
Bz), 8.02-8.09 (m, 4H, Bz), 7.51-7.76 (m, 13.5H, Bz), 6.89 (d, 1H, J = 1.65 Hz, H1’ ), 6.86 (d, 0.7H,
J = 1.65 Hz, H1’ ), 6.67 (d, 1H, J = 4.12 Hz, 3’-OH , ex), 6.63 (d, 0.7H, J = 4.12 Hz, 3’-OH , ex),
b a b
4.89-4.96 (m, 2.4H, H3’ , H2’ ), 4.74-4.82 (dd, 2H, J = 6.04 Hz, J = 12.65 Hz, H5” ), 4.68-4.72 (m,
a,b b a
6478592_1 (GHMatters) P95976.NZ ESTHERJ
1H, H2’ ), 4.58-4.66 (dd, 1.4H, J = 6.04 Hz, J = 12.65 Hz, H5” ), 4.55 (d, 0.7H, J = 10.7 Hz, H5’ ),
a b b
4.38 (d, 1H, J = 11.53 Hz, H5’ ), 4.10 (d, 0.7H, J = 10.7 Hz, H5’ ), 3.93 (d, 1H, J = 11.53 Hz, H5’ ).
a b a
13 Bz Bz
C NMR (75.5 MHz, DMSO-d ) δ 166.5, 165.3, 155.1 (q, COCF ), 151.9 (A ), 151.7 (A ), 151.1,
Bz Bz
140.3(A ), 140.1(A ), 134.5, 134.4, 133.5 (Bz), 131.9 (Bz), 129.55 (Bz), 129.54 (Bz), 129.18,
129.17, 128.7 (Bz), 128.4 (Bz), 128.2 (Bz), 125.4, 125.2, 115.5 (q, CF ), 87.3 (C-4’), 86.0 (C-4’),
84.4 (C-1’ ), 83.6 (C-1’ ), 72.9 (C-3’ ), 71.0 (C-3’ ), 65.3 (C-2’ ), 63.6 (C-2’ ), 60.7 (C-5” ), 60.2 (C-
b a a b b a a
” ), 53.0 (C-5’ ), 52.9 (C-5’ ). F NMR (376 MHz, DMSO-d ) δ -71.1, -72.0.
b a b 6
(1S,3R,4S,7R)(6-Benzoyladeninyl)hydroxyhydroxymethyloxa
azabicyclo[2.2.1]heptane (48): Alcohol 46 (3.41 g, 5.85 mmol) was dissolved in 1,4-dioxane:H O
(225 mL, 2:1, v/v) and cooled to 0 °C, whereafter aq. NaOH (2M, 17.5 mL, 35 mmol) was added.
The reaction mixture was stirred for 2h, whereupon sat. aq. NH Cl (25 mL) was added, and the
mixture was then evaporated to dryness. The resulting crude was adsorbed on silica gel and purified
by silica gel column chromatography (0-20% MeOH in CH Cl , v/v) to afford target amino diol 48 as
a white solid (1.33 g, 60%). R = 0.4 (20% MeOH in CH Cl , v/v). MALDI-HRMS m/z 405.1294 ([M
f 2 2
+ + 1
+ Na] , C H N O Na Calc. 405.1282); H NMR (300 MHz, DMSO-d ) δ 11.17 (br s, 1H, NH,
18 18 6 4 6
Bz Bz
ex), 8.73 (s, 1H, A ), 8.71 (s, 1H, A ), 8.05 (d, 2H, J = 8.1 Hz, Bz), 7.49-7.67 (m, 3H, Bz), 6.44 (d,
1H, J = 1.92 Hz, H-1’), 5.70 (d, 1H, J = 4.0 Hz, 3’OH, ex), 4.82 (t, 1H, J = 5.5 Hz, 5’-OH, ex), 4.30
(d, 1H, J = 4.0 Hz, H-3’), 3.69 (d, 2H, J = 5.5 Hz, H-5’), 3.51 (s, 1H, H-2’), 3.15 (d, 1H, J = 10.6 Hz,
H -5’’), 2.96 (d, 1H, J = 10.6 Hz, H -5’’); C NMR (75.5 MHz, DMSO-d ) δ 165.5 (C=O), 152.0,
a b 6
Bz Bz
151.2 (A ), 149.8, 143.2 (A ), 133.3, 132.3 (Bz), 128.4 (Bz), 125.2, 91.3 (C-4’), 83.9 (C-1’), 73.4
(C-3’), 62.2 (C-5”), 58.3 (C-2’), 50.6 (C-5’).
(1R,3R,4R,7R)(6-Benzoyladeninyl)hydroxy(4,4’-dimethoxytrityloxy-methyl)oxa
azabicyclo[2.2.1]heptane (50): Diol 48 (2.47 g, 6.5 mmol) was co-evaporated with anhydrous
pyridine (2 × 50 mL), redissolved in anhydrous pyridine (130 mL) and cooled to 0 °C. DMTrCl (3.25
g, 2.7 mmol) was added, and the reaction mixture stirred for 23h during which it was allowed to heat
to rt. Subsequently MeOH (20 mL) was added, and the mixture was diluted with EtOAc (200 mL)
and washed with sat. aq. NaHCO (2 × 20 mL). After separating the two phases, the aqueous phase
was back-extracted with EtOAc (4 × 50 mL), and the combined organic phase was evaporated to
dryness. The resulting residue was purified by silica gel column chromatography (0-8% MeOH and
1% pyridine in CH Cl , v/v) and the resulting product was co-evaporated with abs. EtOH:Toluene (2
× 50 mL, 1:1, v/v) affording nucleoside 50 as a white solid (1.51 g, 34%). R : 0.2 (7% MeOH in
+ + 1
CH Cl , v/v). MALDI-HRMS m/z 707.2609 ([M + Na] , C H N O Na Calc. 707.2589). H NMR
2 2 39 36 6 4
Bz Bz
(500 MHz, DMSO-d ) δ 11.15 (br s, 1H, NH, ex), 8.78 (s, 1H, A ), 8.73 (s, 1H, A ), 8.06 (d, 2H, J
= 7 Hz, Bz), 7.52 – 7.69 (m, 3H, Bz), 7.18 – 7.46 (m, 9H, DMTr), 6.86-6.93 (m, 4H, DMTr), 6.54 (d,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
1H, J = 1.92 Hz, H-1’), 5.66 (d, 1H, J = 4.67 Hz, 3’-OH, ex), 4.39 (d, 1H, J = 4.94 Hz, H-3’), 3.74 (s,
6H, 2 × OMe), 3.48-3.51 (m, 1H, H-2’), 3.31 (d, 1H, J = 10.7 Hz, H-5’ ), 3.26 (d, 1H, J = 10.7 Hz, H-
’ ), 3.16-3.21 (m, 2H, H-5” ), 2.89 (br s, 1H, 2’-NH, ex). C NMR (125 MHz, DMSO-d ) δ 165.5,
b a,b 6
Bz Bz
158.0, 152.2, 151.3 (A ), 149.9, 144.8, 143.3 (A ), 135.5, 135.4, 133.4, 132.3 (Ar), 129.7 (Ar),
129.68 (Ar), 128.4 (Ar), 128.39 (Ar), 127.8 (Ar), 127.7 (Ar), 126.5 (Ar), 125.1, 113.1(Ar), 89.6
(C4’), 85.1 (C1’), 84.0, 74.0 (C-3’), 62.2 (C-2’), 61.2 (C5”), 54.9 (OMe), 51.2 (C5’).
*A trace impurity of pyridine was identified in C NMR (149.5 ppm).
(1S,3R,4S,7R)(6-benzoyladeninyl)hydroxy(4,4’-dimethoxytrityl-oxy-methyl)(9’-
fluorenylmethoxycarbonyl)oxaazabicyclo[2.2.1]heptane 51W: Amino Alcohol 50 (200 mg,
0.29 mmol) was co-evaporated in anhydrous pyridine (2 x 2 mL) and re-dissolved in anhydrous
pyridine (1.5mL). This was cooled to 0°C whereupon 9’-fluorenylmethyl chloroformate (100mg, 0.38
mmol) was added and allowed to reach rt while stirring for 6h. It was then diluted with EtOAc (30
mL) and washed with sat. aq. NaHCO (30 mL). The aqueous phase was back extracted with EtOAc
(25 mL) and the combined organic phases were evaporated to dryness and co-evaporated with 2:1
EtOH:Toluene (2 x 6 mL). The resulting crude was purified by silica gel column chromatography
(30-100% EtOAc in Petroleum Ether, v/v) to afford target nucleoside 51W as a white foam (136 mg,
51%). R = 0.4 (EtOAc). Physical data for the mixture of rotamers: MALDI-HRMS m/z 929.3241
+ + 1
([M + Na] , C H N O Na Calc. 929.3269); H NMR (300 MHz, DMSO-d ) δ 11.29 (br s, 2H, ex),
54 46 6 8 6
Bz Bz Bz Bz
8.74 (s, 1H, A ), 8.72 (s, 1H, A ), 8.55 (s, 1H, A ), 8.49 (s, 1H, A ), 8.02-8.08 (m, 4H, Bz), 7.77 –
7.88 (m, 6H, Bz), 7.09 – 7.69 (m, 40H, DMT, Fmoc), 6.90-6.95 (m, 8H, DMT), 6.80 (d, 1H, J = 1.65
Hz, H-1’), 6.73 (d, 1H, J = 1.65 Hz, H-1’), 6.25 (d, 1H, J = 4.39 Hz, 3’-OH, ex), 6.22 (d, 1H, J = 4.39
Hz, 3’-OH, ex), 4.50 (br s, 1H, H-2’), 4.45 (br s, 1H, H-2’), 4.22 (d, 1H, J = 6.86, CH Fmoc), 4.15 (d,
1H, J = 6.86, CH Fmoc), 3.87 (d, 1H, J = 6.86, CH Fmoc), 3.83 (d, 1H, J = 6.86, CH Fmoc), 3.73-
2 2 2
3.76 (m, 14H, OCH , CH (Fmoc)), 3.70 (d, 1H, J = 10.5 Hz, H-5’), 3.62 (d, 1H, J = 10.5 Hz, H-5’),
3.34-3.43 (m, 6H, H-5”, H-5’). C NMR (75.5 MHz, DMSO-d ) δ 165.6, 158.1, 154.7, 154.6, 151.8
Bz Bz Bz Bz
(A ), 151.5 (A ), 150.3, 150.2, 144.7, 143.7, 143.6, 143.5, 142.8, 141.3 (A ), 141.2 (A ), 140.6,
140.4, 140.3, 135.3, 135.2, 133.3, 132.4, 129.7, 128.8, 128.4, 127.9, 127.7, 127.6, 127.5, 127.2,
127.1, 126.9, 126.7, 125.4, 125.2, 125.1, 124.9, 124.6, 121.3, 119.98, 113.2, 88.9 (C-4’), 88.4 (C-4’),
85.4 (C-(Ph) , DMT), 84.9 (C-1’), 84.7 (C-1’), 72.5 (C-3’), 72.0 (C-3’), 66.7 (CH -fmoc), 63.9 (C-
2’), 63.4 (C-2’), 60.6 (C-5”), 60.56 (C-5”), 54.96 (OCH ), 52.7 (C-5’), 52.6 (C-5’), 46.4 (CH, fmoc),
45.9 (CH, fmoc).
FMOC protected nucleoside phosphoramidite 52W: Nucleoside 51W (225 mg, 0.25 mmol) was
co-evaporated with an. 1,2-DCE (2 × 4 mL) and re-dissolved in anhydrous CH Cl (3.5 mL).
Anhydrous DIPEA (195 μL, 1.12 mmol) and N-methylimidazole (16 µL, 0.20 mmol) were added
followed by dropwise addition of 2-cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite (111 μL,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
0.50 mmol). The reaction mixture stirred at rt. for 4h whereupon the crude was evaporated to dryness
and the resulting residue was purified by silica gel column chromatography (0-2% MeOH in CH Cl ,
v/v, initially built in 0.5% Et N) and precipitated from CH Cl /Petroleum Ether to afford target
3 2 2
amidite 52W as a white foam (126 mg, 46%). Physical data for the mixture of rotamers: R = 0.5 (5%
MeOH in CH Cl , v/v); HiResESI m/z 1129.4356 ([M + Na] , C H N O PNa Calc. 1129.4348);
2 2 63 63 8 9
P NMR (121 MHz, CDCl ) δ 150.32, 150.26, 150.12, 149.48.
(1R,3R,4S,7R)(6-Benzoyladeninyl)hydroxy(4,4’-dimethoxytrityl-oxy-methyl)
(pyrenyl)methyloxaazabicyclo[2.2.1]heptane (51X): Nucleoside 50 (300 mg, 0.44 mmol)
was co-evaporated with anhydrous 1,2-DCE (2 × 10 mL), suspended in anhydrous CH Cl (4.4 mL),
and pyrenecarboxaldehyde (171 mg, 0.74 mmol) and NaBH(OAc) (186 mg, 0.88 mmol) was added.
The resulting suspension was stirred at rt. for 17h whereupon sat. aq. NaHCO (4 mL) was added and
the mixture subsequently diluted with CH Cl (20 mL). The two phases were separated and the
organic phase was washed with sat. aq. NaHCO (20 mL). The resulting aqueous phase was back-
extracted with EtOAc (2 × 20 mL). The combined organic phase was evaporated to dryness, and the
resulting residue was purified by silica gel column chromatography (0-99% EtOAc and 1% pyridine
in petroleum ether, v/v) affording target nucleoside 51X as a white foam (268 mg, 68%). R = 0.4
+ + 1
(EtOAc). MALDI-HRMS m/z 921.3326 ([M + Na] , C H N O Na Calc. 921.3371). H NMR (300
56 46 6 6
Bz Bz
MHz, DMSO-d ) δ 11.13 (s, 1H, NH, ex), 8.46 (s, 1H, A ), 8.36 (s, 1H, A ), 7.89-8.16 (m, 12H,
Ar), 7.51-7.76 (m, 9H, Ar), 7.12-7.36 (m, 14H, Ar), 6.82 (s, 3H, DMT), 6.78 (s, 3H, DMT), 6.42 (s,
1H, H-1’), 6.07 (s, 1H, 3’-OH, ex), 4.66 (d, 1H, J = 12.5 Hz, CH Py), 4.51 (s, 1H, H-3’), 4.45 (d, 1H,
J = 12.5 Hz, CH Py), 3.64 (s, 6H, 2 × OMe), 3.57 (s, 1H, H-2’), 3.42 (d, 1H, J = 9.6 Hz, H-5’), 3.17
(d, 1H, J = 9.6 Hz, H-5’). The signals from H-5’’ were not identifiable due to signal overlap with
13 Bz
water; C NMR (75.5 MHz, CDCl ) δ 164.1, 158.6, 151.3 (A ), 150.5, 149.6, 148.3, 144.4, 141.8
(A ), 136.1, 135.5, 135.4, 133.7, 132.6, 132.3, 131.1, 130.7, 130.5, 130.0, 129.1, 128.7, 128.0,
127.8, 127.6, 127.2, 127.1, 127.0, 126.8, 125.7, 125.0, 124.8, 124.6, 124.4, 124.1, 123.8, 122.9,
122.6, 113.2 (DMT), 90.2 (C-4’) 86.5 (C-(Ph) DMT), 86.1 (C-1’), 65.2 (C-3’), 61.2 (C-2’), 59.2 (C-
”), 57.6 (C-5’), 55.2 (OCH ), 29.6 (CH Py).
(1S,3R,4R,7S)(6-Benzoyladeninyl)[2-cyanothoxy(diisopropylamino)-phosphinoxy]
(4,4’-dimethoxytrityloxymethyl)(pyrenyl)methyloxaazabicyclo[2.2.1]heptane (52X):
Nucleoside 51X (220 mg, 0.24 mmol) was co-evaporated with 1,2-DCE (2 × 10 mL), dissolved in
% DIPEA in anhydrous CH Cl (4.4 mL, v/v) and 2-cyanoethyl-N,N’-(diisopropyl)-
phosphoramidochloridite (0.12 mL, 0.54 mmol) was added. The reaction mixture was stirred at rt. for
21h, whereupon additional 2-cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite (0.05 mL, 0.06
mmol) was added. The reaction mixture was then stirred for 4h followed by addition of abs. EtOH (2
mL). Subsequently the mixture was diluted with CH Cl (10 mL) and the organic phase was washed
6478592_1 (GHMatters) P95976.NZ ESTHERJ
sequentially with sat. aq. NaHCO (10 mL) and brine (10 mL). After separation of the two phases the
organic phase was evaporated to dryness and the resulting residue purified by silica gel column
chromatography (0-60% EtOAc and 1% pyridine in petroleum ether, v/v). The resulting product was
co-evaporated with abs. EtOH:toluene (2 × 10 mL, 1:1, v/v) affording a white solid which was then
precipitated from HPLC-grade EtOAc/n-hexane affording pure amidite 52X as a white solid (136 mg,
51%). R : 0.7 (5% MeOH in CH Cl , v/v). HiResESI m/z 1099.4642 ([M + Na] , C H N O PNa
f 2 2 65 63 8 7
Calc.1099.4630). P NMR (121 MHz, CDCl ) δ 151.3, 149.0.
(1S,3R,4S,7R)(6-Benzoyladeninyl)hydroxy(4,4’-dimethoxytrityl-oxy-methyl)
(pyrenyl)carbonyloxaazabicyclo[2.2.1]heptane (51Y): Amino alcohol 50 (407 mg, 0.59
mmol) was co-evaporated with anhydrous 1,2-DCE (2 × 10 mL), dissolved in anhydrous CH Cl
(11.9 mL), and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochlorid (EDC· HCl, 226 mg,
1.19 mmol) and 1-pyrenylcarboxylic acid (293 mg, 1.19 mmol) was added. The reaction mixture was
stirred at rt. for 45h, whereafter it was diluted with CH Cl (50 mL) and washed with water (20 mL).
The two phases were separated and the aqueous phase back-extracted with CH Cl (3 × 50 mL). The
combined organic phase was evaporated under reduced pressure and the resulting residue was
purified by silica gel column chromatography (0-99% EtOAc and 1% pyridine in petroleum, v/v).
The resulting product was co-evaporated with abs. EtOH:toluene (2 × 50 ml, 1:1, v/v) affording
target nucleoside 51Y as a yellow solid (343 mg, 64%). Physical data for the mixture of rotamers: R :
0.5 (5% MeOH: CH Cl , v/v); MALDI-HRMS m/z 935.3138 ([M + Na] , C H N O Na Calc.
2 2 56 44 6 7
935.3164). Unknown impurity is identified at 6.22 ppm in H-NMR. H NMR (300 MHz, DMSO-d )
Bz Bz Bz
δ 11.21 (s, 1H, NH, ex), 11.20 (s, 1H, NH, ex), 8.55 (s, 1H, A ), 8.53 (s, 1H, A ), 8.45 (s, 1H, A ),
8.40 (s, 1H, A ), 7.97 – 8.23 (m, 10H, Ar), 7.56 – 7.84 (m, 7H, Ar), 7.18 – 7.44 (m, 10H, Ar), 7.09
(s, 1H, Ar), 7.06 (s, 1H, Ar), 6.82 – 6.90 (m, 4H, DMTr), 6.50 (d, 1H, J = 2.2 Hz, H-1’), 6.40 (d, 1H,
J = 1.8 Hz, H-1’), 6.15 (d, 1H, J = 4.0 Hz, 3’-OH, ex), 6.07 (d, 1H, J = 3.7 Hz, 3’-OH, ex), 4.42 –
4.77 (m, 4H, H-3’, H-5’), 3.73 (s, 12H, 2 × OMe), 3.66 (s, 2H, H-2’), 3.41 – 3.44 (d, 1H, J = 13.6 Hz,
H-5’’ ), 3.15 – 3.20 (d, 2H, J = 13.6 Hz, H-5’’ ); C NMR (75.5 MHz, DMSO-d ) δ 165.5, 157.9,
a b 6
157.7, 151.7, 150.98, 149.8, 148.2, 144.7, 142.8, 140.1, 135.4, 135.3, 133.5, 133.1, 132.3, 130.5,
130.2, 129.9, 129.6, 128.8, 128.6, 128.4, 127.8, 127.6, 127.5, 127.3, 127.2, 126.9, 126.5, 126.3,
125.9, 125.5, 124.9, 124.8, 124.78, 124.2, 123.8, 123.7, 123.1, 113.1, 112.7, 92.0, 90.3, 85.1, 84.7,
79.8, 75.3, 74.7, 66.4, 61.2, 59.1, 58.6, 58.3, 58.2, 54.6.
(1S,3R,4S,7R)(6-Benzoyladeninyl)hydroxy(4,4’-dimethoxytrityl-oxy-methyl)
(pyrenyl)carbonyloxaazabicyclo[2.2.1]heptanes (51Y): Pyrenecarboxylic acid (127
mg, 0.50 mmol), 2-(1HAzabenzotriazolyl)-1,1,3,3-tetramethyluronium hexafluorophosphate
(HATU, 166 mg, 0.44 mmol), and diisopropylethylamine (DIPEA, 0.15 mL, 0.87 mmol) were
6478592_1 (GHMatters) P95976.NZ ESTHERJ
dissolved in anhydrous DMF (3 mL) and allowed to stir at rt for 1h then cooled to 0°C. Amino
alcohol 50 (230 mg, 0.34 mmol) was co-evaporated with anhydrous 1,2-DCE (2 × 3 mL) and
redissolved in anhydrous DMF (3 mL) and added to the cooled reaction mixture. After 5 h stirring,
the mixture was diluted with EtOAc (30 mL) and washed with water (3 x 20 mL). The organic layer
was evaporated to dryness and the resulting crude purified by silica gel column chromatography (0 –
4% MeOH in CH Cl , v/v) affording compound 51Y as a white solid (227 mg, 74%). Physical data
for the mixture of rotamers: R : 0.5 (5% MeOH: CH Cl , v/v); MALDI-HRMS m/z 935.3138 ([M +
f 2 2
+ + 1
Na] , C H N O Na Calc. 935.3164). H NMR (500 MHz, DMSO-d ) δ 11.32 (br s, 1.3H, NH,
56 44 6 7 6
Bz Bz Bz
ex), 8.85 (s, 0.3H, A ), 8.79 (s, 1.3H, A ), 8.75 (s, 0.3H, A ), 8.02-8.39 (m, 13.7H, Ar), 7.82 (m,
0.6H, Ar), 7.49-7.75 (m, 6H, Ar), 7.10-7.42 (m, 9.7H, Ar), 6.93-6.98 (m, 4H, DMT ), 6.92 (d, 0.3H, J
= 1.92 Hz, H-1’ ), 6.72-6.78 (m, 1.2H, DMT ), 6.54 (d, 0.3H, J = 3.57 Hz, 3’-OH , ex), 6.48 (d, 1H, J
b b b
= 4.94 Hz, 3’-OH , ex), 6.44 (s, 1H, H-1’ ), 5.22 (s, 0.3H, H-2’ ), 4.87 (d, 0.3H, J = 3.56 Hz, H-3’ ),
a a b b
4.61 (d, 1H, J = 5.21 Hz, H-3’ ), 4.36 (d, 1H, J = 12.08 Hz, H-5’ ) 4.02 (d, 1H, J = 12.08 Hz, H-5’ ),
a a a
3.77 (s, 7H, 2 x (OCH ) , H-2’ ), 3.69 (d, 0.3H, J = 10.43 Hz, H-5’ ), 3.66 (s, 1H, (OCH ) ), 3.64 (s,
3 a a b 3 b
1H, (OCH ) ), 3.47 (s, 2H, H5” ), 3.39 (d, 0.3H, J = 10.43 Hz, H-5’ ), 3.18 (s, 0.6H, H-5” ). C
3 b a b b
NMR (125 MHz, DMSO-d ) δ 169.5, 169.1, 165.8, 165.6, 158.0, 157.9, 152.0, 151.6, 151.23,
Bz Bz
151.21, 150.3, 144.7, 144.5, 141.5 (A ), 141.4 (A ), 135.4, 135.2, 135.15, 135.11, 133.48, 132.5
(Ar), 132.4 (Ar), 131.3, 130.9, 130.6, 130.5, 130.4, 130.1, 129.9 (Ar), 129.8 (Ar), 129.7 (Ar), 129.6
(Ar), 128.8 (Ar), 128.7, 128.6 (Ar), 128.5 (Ar), 128.49 (Ar), 128.47, 128.2 (Ar), 128.1 (Ar), 127.9
(Ar), 127.8 (Ar), 127.7 (Ar), 127.6 (Ar), 127.1 (Ar), 126.9 (Ar), 126.7 (Ar), 126.6 (Ar), 126.5 (Ar),
125.9 (Ar), 125.8 (Ar), 125.6 (Ar), 125.5 (Ar), 124.8 (Ar), 124.2 (Ar), 124.0, 123.9 (Ar), 123.5,
123.3, 123.1, 113.2 (DMT), 113.06 (DMT), 113.04 (DMT), 88.7 (C-4’ ), 88.5 (C-4’ ), 85.4 (C-(Ph) ,
a b 3
DMT ), 85.3 (C-(Ph) , DMT ), 85.0 (C-1’ ), 84.7 (C-1’ ), 72.5 (C-3’ ), 71.8 (C-3’ ), 65.6 (C-2’ ),
a 3 b b a a b b
62.8 (C-2’ ), 60.5 (C-5” ), 60.0 (C-5” ), 55.0 (OCH ), 54.8 (OCH ), 54.0 (C-5’ ), 52.2 (C-5’ ).
a a b 3 3 b a
(1S,3R,4S,7R)(6-Benzoyladeninyl)[2-cyanothoxy(diisopropylamino)-phosphinoxy]
(4,4’-dimethoxytrityloxymethyl)(pyrenyl)carbonyloxaazabicyclo[2.2.1]heptane
(52Y): Nucleoside 51Y (300 mg, 0.33 mmol) was co-evaporated with 1,2-DCE (2 × 5 mL),
dissolved in anhydrous CH Cl (2.7 mL) and anhydrous DIPEA (0.66 ml), and 2-cyanoethyl-N,N’-
(diisopropyl)-phosphoramidochloridite (0.11 mL, 0.5 mmol) was added. The reaction mixture stirred
at rt. for 22h. whereafter abs. EtOH (2 mL) was added. The mixture was subsequently taken up in
CH Cl (50 mL) and washed with sat. aq. NaHCO (50 mL) and brine (50 mL). The combined
2 2 3
aqueous phase was back-extracted with CH Cl (2 × 20 ml) and the combined organic phase was
evaporated under reduced pressure. The resulting residue was purified by silica gel column
chromatography (0-99% EtOAc and 1% pyridine in petroleum ether, v/v) and the resulting product
co-evaporated with abs. EtOH:toluene (2 × 50 ml, 1:1, v/v) and precipitated from HPLC-grade
EtOAc/n-hexane to afford target amidite 52Y as a white foam (247 mg, 67%). Physical data for the
6478592_1 (GHMatters) P95976.NZ ESTHERJ
mixture of rotamers: R = 0.5 (EtOAc); HiResESI m/z 1113.4462 ([M + Na] , C H N O PNa
f 65 61 8 8
Calc.1113.4422); P NMR (121 MHz, CDCl ) δ 152.3, 151.8, 150.2.
(1S,3R,4S,7R)(6-Benzoyladeninyl)hydroxy(4,4’-dimethoxytrityl-oxy-methyl)
(pyrenyl)acetyloxaazabicyclo[2.2.1]heptane 51Z: Amino alcohol 50 (250 mg, 0.37 mmol)
was co-evaporated with anhydrous 1,2-DCE (2 × 5 mL), dissolved in anhydrous CH Cl (10 mL),
and 1-ethyl(3-dimethylaminopropyl)carbodiimide hydrochlorid (EDC·HCl, 108 mg, 0.55 mmol)
and 1-pyreneacetic acid (145 mg, 0.55 mmol) was added. The reaction mixture was stirred at rt. for
2.5h, whereafter it was diluted with CH Cl (20 mL) and washed with water (2 x 10 mL). The two
phases were separated and the aqueous phase back-extracted with CH Cl (10 mL). The combined
organic phase was evaporated under reduced pressure and the resulting residue was purified by silica
gel column chromatography (0-4% MeOH in CH Cl , v/v) affording target nucleoside 51Z as a
white solid (266 mg, 79%). Physical data for the mixture of rotamers: R : 0.5 (10% MeOH:CH Cl ,
f 2 2
+ + 1
v/v); MALDI-HRMS m/z 949.3321 ([M + Na] , C H N O Na Calc. 949.3320). H NMR (500
57 46 6 7
Bz Bz
MHz, DMSO-d ) δ 11.29 (br s, 1.5H, NH, ex), 8.96 (s, 1H, A ), 8.73 (s, 0.5H, A ), 8.71 (s, 1H,
6 a b
Bz Bz
A ), 8.55 (s, 0.5H, A ), 7.92-8.33 (m, 16H, Py, Bz), 7.83 (m, 1H, Py, Bz), 7.60-7.79 (m, 4.5H, Py,
Bz), 7.44-7.54 (m, 3.5H, DMT), 7.20-7.40 (m, 10.5H, DMT), 6.88-6.98 (m, 6H, DMT), 6.82 (d,
0.5H, J = 1.65 Hz, H-1’ ), 6.79 (d, 1H, J = 1.37 Hz, H-1’ ), 6.31 (d, 0.5H, J = 4.39 Hz, 3’-OH , ex),
b a b
6.19 (d, 1H, J = 4.39 Hz, 3’-OH , ex), 5.07 (s, 0.5H, H-2’ ), 4.71-4.74 (m, 1.5H, H-2’ , H-3’ ), 4.67
a b a b
(d, 1H, J = 10.43, H-5’ ), 4.63 (d, 1H, J = 4.39 Hz, H-3’ ), 4.57 (d, 1H, J = 16.2 Hz, CH Py ), 4.38
1a a 2 a
(d, 1H, J = 10.2 Hz, CH Py ), 4.11 (d, 0.5H, J = 16.2 Hz, CH Py ), 3.95-4.04 (m, 1.5H, H-5’ , H-
2 a 2 b 1b
’ ), 3.66-3.77 (m, 9.5H, OCH , H-5’ ), 3.34-3.51 (m, 3.5H, H-5” , CH Py ). C NMR (125 MHz,
2a 3 2b a,b 2 b
Bz Bz
DMSO-d ) δ 172.7, 169.8, 169.6, 165.5, 165.4, 158.1, 158.0, 151.9, 151.7 (A ), 151.5, 151.4 (A ),
Bz Bz
150.5, 150.2, 144.7, 144.6, 141.7 (A ), 141.5 (A ), 135.5, 135.2, 133.5, 133.4, 132.5, 130.8, 130.7,
130.6, 130.3, 130.2, 130.1, 129.9, 129.88, 129.82, 129.78, 129.77, 129.74, 129.68, 129.67, 129.66,
129.62, 129.4, 129.2, 128.9, 128.8, 128.7, 128.6, 128.5, 128.5, 128.2, 127.8, 127.77, 127.72, 127.4,
127.3, 127.27, 127.25, 127.1, 126.9, 126.8, 126.7, 126.6, 126.2, 126.0, 125.9, 125.4, 125.3, 125.2,
125.0, 124.9, 124.8, 124.76, 124.72, 124.6, 124.5, 124.4, 124.3, 124.0, 123.9, 123.8, 123.78, 123.76,
123.72, 123.6, 123.2, 113.2 (DMT), 89.0 (C-4’), 88.4 (C-4’), 85.47 (C-(Ph) ), 85.40 (C-(Ph) ), 84.7
(C-1’ ), 84.6 (C-1’ ), 72.9 (C-3’ ), 72.0 (C-3’ ), 64.5 (C-2’ ), 61.8 (C-2’ ), 60.9 (C-5” ), 60.8 (C-5” ),
a b b a b a a b
55.0 (OCH ), 54.8 (OCH ), 52.8 (C-5’ ), 52.3 (C-5’ ), 38.7, 37.8 (CH Py ), 37.7 (CH Py ). *Trace
3 3 a b 2 a 2 b
impurities of residual 1-pyreneacetic acid were identified in CNMR (C=O at 172 ppm, CH at 38.7
ppm, plus extra pyrene peaks, couples to impurities in HNMR (4.35 ppm is extra CH Py peak that
couples to 38.7 in HSQC).
(1S,3R,4S,7R)(6-Benzoyladeninyl)[2-cyanothoxy(diisopropylamino)-phosphinoxy]
(4,4’-dimethoxytrityloxymethyl)(pyrenyl)acetyloxaazabicyclo[2.2.1]heptane (52Z):
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Nucleoside 51Z (235 mg, 0.25 mmol) was co-evaporated with an. 1,2-DCE (2 × 5 mL) and re-
dissolved in anhydrous CH Cl (5 mL). Anhydrous DIPEA (220 μL, 1.27 mmol) was added followed
by dropwise addition of 2-cyanoethyl-N,N’-(diisopropyl)-phosphoramidochloridite (115 μL, 0.51
mmol). The reaction mixture stirred at rt. for 22h. whereafter abs. EtOH (1 mL) was added. The
crude was evaporated to dryness and the resulting residue was purified by silica gel column
chromatography (0-2% MeOH in CH Cl , v/v) and precipitated from CH Cl /Petroleum Ether to
2 2 2 2
afford target amidite 52Z as a white foam (203 mg, 71%). Physical data for the mixture of rotamers:
R = 0.6 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1149.4358 ([M + Na] , C H N O PNa
f 2 2 66 63 8 8
Calc. 1149.4399); P NMR (121 MHz, CDCl ) δ 150.39, 150.31, 150.26, 148.09.
Synthesis of probes with locked monomers 124W-124Y: Syntheses of probes containing
incorporations of locked phosphoramidites 52W, 52X, and 52Y were performed on an automated
DNA synthesizer (0.2 µmol scale) using the following hand coupling conditions (activator; coupling
time; approximate coupling yield): Monomer 124W (pyridinium hydrochloride; 30 min; ~82%),
monomers 124X and 124Y (pyridinium hydrochloride; 15 min; ~95%). The probes were deprotected
using 32% aq. NH at 55 °C for 2h. Purification of probes (till at least 75% purity) was performed by
RP-HPLC (DMT-ON), followed by detritylation (80% aq. AcOH, 20 min) and precipitation (abs.
EtOH, −18 °C, 12 h). RP-HPLC purification of oligonucleotides was performed using a Waters Prep
LC 4000 system equipped with an Xterra MS C18-column (10 µm, 300 mm × 7.8 mm). A
representative RP-HPLC gradient protocol for purification of oligonucleotides with DMT-ON is to
use an isocratic hold of 100% A-buffer for 5 min followed by a linear gradient to 55% B-buffer over
75 min at a flow rate of 1.0 mL/min (A-buffer: 95% 0.1 M NH HCO , 5% CH CN; B-buffer: 25%
4 3 3
0.1 M NH HCO , 75% CH CN). The composition of the probes was verified by MALDI-MS
4 3 3
analysis (Table 32) whereas the purity (>80%, unless stated otherwise) was verified by ion-exchange
HPLC using a LaChrom L-7000 system (VWR International) equipped with a Gen-Pak Fax column
(100 mm × 4.6 mm). A representative protocol involves the use of an isocratic hold of 95% A-buffer
for 5 min, followed by a linear gradient to 70% B-buffer over 41 min at a flow rate of 0.75 mL/min
(A-buffer: 25 mM Tris-Cl, 1 mM EDTA, pH 8.0; B-buffer: 1 M NaCl).
Table 32
MS-data of representative single-stranded probe modified with adenine monomers
124X (= K) or 124Y (= L).
ONs Sequence Calc. m/z Found m/z
124X6 5’-GTG KTA TGC 2995 2995
124X7 5’-GTG ATK TGC 2995 2993
6478592_1 (GHMatters) P95976.NZ ESTHERJ
124X8 3’-CAC TKT ACG 2924 2923
124X9 3’-CAC TAT KCG 2924 2923
124Y6 5’-GTG LTA TGC 3009 3008
124Y7 5’-GTG ATL TGC 3009 3009
124Y8 3’-CAC TLT ACG 2937 2937
124Y9 3’-CAC TAT LCG 2937 2936
Thermal Affinity of probes modified with monomers 124W/X/Y: The thermal affinity of probes
toward complementary DNA or RNA targets and of probe duplexes was evaluated via UV thermal
denaturation experiments ([Na ] = 110 mM, Tables 33 and 34. Changes in thermal denaturation
temperatures (T -values) of modified duplexes are discussed relative to T -values of unmodified
reference duplexes, unless otherwise stated. Single-stranded probes that are modified with monomer
124W show similar thermal affinity toward single-stranded DNA as toward toward single-stranded
RNA targets. Single-stranded probes that are modified with 124X and 124Y show higher thermal
affinity toward single-stranded DNA than toward single-stranded RNA targets (Tables 33 and 34).
This DNA selectivity makes these monomers useful as probes for targeting double stranded DNA.
Table 33
T -Values of Duplexes Between B6-B11 and Complementary DNA Targets
T [ΔT /mod] (°C)
ON Duplex B= T 124W 124X 124Y
B6 5’-GTG BTA 27.5 27.0 32.5 38.5
D2 TGC [-0.5] [+5.0] [+11.0]
3’-CAC TAT
B7 5’-GTG ATB 27.5 28.0 34.5 41.5
D2 TGC [+0.5] [+7.0] [+14.0]
3’-CAC TAT
B10 5’-GTG BTB 27.5 26.5 32.5 41.0
D2 TGC [-0.5] [+2.5] [+6.8]
3’-CAC TAT
D1 5’-GTG ATA 27.5 27.5 34.0 39.0
B8 TGC [+0.0] [+6.5] [+11.5]
3’-CAC TBT
D1 5’-GTG ATA 27.5 27.5 33.0 29.5
B9 TGC [+0.0] [+5.5] [+12.0]
3’-CAC TAT
6478592_1 (GHMatters) P95976.NZ ESTHERJ
D1 5’-GTG ATA 27.5 25.5 32.5 41.0
TGC [-1.0] [+2.5] [+6.8]
3’-CAC TBT
ΔT = change in T ‘s relative to unmodified reference duplex; T ’s determined as the maximum of
m m m
the first derivative of melting curves (A vs T) recorded in medium salt buffer ([Na ] = 110mM, [Cl
] = 100mM, pH 7.0 (NaH PO /Na HPO )), using 1.0 µM of each strand. T ’s are averages of at least
2 4 2 4 m
two measurements within 1.0 ˚C; A = adeninyl DNA monomer, C = cytosinyl DNA monomer,
G = guaninyl DNA monomer, T = thyminyl DNA monomer. ND = not determined.
Table 34
T -Values of Duplexes Between B6-B11 and Complementary RNA Targets
T [ΔT /mod] (°C)
ON Duplex T 124W 124X 124Y
’-GTG BTA 26.0 27.0 25.0 27.5
R2 TGC [+1.0] [-1.0] [+1.5]
3’-CAC UAU
B7 5’-GTG ATB 26.0 27.0 27.0 31.0
TGC [+1.0] [+1.0] [+5.0]
3’-CAC UAU
’-GTG BTB 26.0 27.5 24.5 30.5
R2 TGC [+0.8] [-0.8] [+2.3]
3’-CAC UAU
R1 5’-GUG AUA 26.0 25.5 24.0 27.0
UGC [-0.5] [-2.0] [+1.0]
3’-CAC TBT
’-GUG AUA 26.0 28.0 27.0 31.0
B9 UGC [+2.0] [+1.0] [+5.0]
3’-CAC TAT
R1 5’-GUG AUA 26.0 28.0 24.5 31.5
UGC [+1.0] [-0.8] [+2.8]
3’-CAC TBT
For conditions of thermal denaturation experiments, see Table 33.
The Watson-Crick specificity of single-stranded probes modified with monomers 124W,
124X and 124Y (B8-series) was evaluated using DNA (Table 35) or RNA targets (Table 36) that
contain mismatched nucleobases opposite to the modification sites. All monomers show excellent
discrimination in terms of T .
Table 35
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Discrimination of Mismatched DNA Targets By Probes Modified with Monomers 124W-Y
DNA: 5′-GTG ABA TGC
T [°C] ΔT [°C]
ON Sequence B = T A C G
D2 3’-CAC TAT ACG 27.5 -21.0 -16.5 -7.5
3′-CAC T124WT
124W8 27.5 -20.0 -17.0 -16.0
124X8 3′-CAC T124XT ACG 34.0 -15.5 -7.0 -14.5
3′-CAC T124YT ACG -21.0 -11.0 -13.5
124Y8 39.0
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully matched
duplexes are shown in bold. ΔT = change in T relative to fully matched DNA:DNA duplex.
Table 36
Discrimination of Mismatched RNA Targets by Probes Modified with Monomers 124W-Y.
RNA: 5′-GUG ABA UGC
T [°C] ΔT [°C]
ON Sequence B = T A C G
D1 3’-CAC TAT ACG 26.0 -16.0 -15.5 -11.0
3′-CAC T124WT
124W8 25.5 -13.5 -13.0 -11.5
3′-CAC T124XT ACG -10.5 -8.0 -10.0
124X8 24.0
124Y8 3′-CAC T124YT ACG 27.0 -11.0 -11.0 -8.5
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully matched
duplexes are shown in bold. ΔT = change in T relative to fully matched RNA:DNA duplex.
The specificity of doubly-modified single-stranded probes against centrally mismatched
DNA targets was also evaluated (Table 37). Decreased mismatch discrimination compared
to unmodified reference strand D1 was observed.
Table 37
Discrimination of Mismatched DNA Targets by Probes Modified with Monomers 124X/Y
DNA : 3’-CAC TBT ACG
T [°C] ΔT [°C]
ON Sequence A T C G
D1 5’-GTG ATA TGC 27.5 -16.5 -16.5 -8.0
124X10 5’-GTG 124XT124X TGC 32.5 +2.0 +2.0 +0.5
124Y10 5’-GTG 124YT124Y TGC 41.0 -2.0 -5.0 -2.0
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully
matched duplexes are shown in bold. ΔT = change in T relative to fully matched DNA:DNA
duplex. ND = not determined
Absorption maxima in the 300-400 nm region for single-stranded probes modified with monomers
124X and 124Y in the presence or absence of complementary DNA/RNA targets are given below
(Table 38).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 38
Absorption Maxima in The 300-400 Nm Region for Single-Stranded Probes Modified with
Monomers 124X And 124Y in The Presence or Absence of Complementary DNA/RNA
Targets.
λ [Δλ ] (nm)
max max
B= 124X 124Y
ON Sequence ss +DNA +RNA ss +DNA +RNA
B6 5’-GTG BTA TGC 348 350 [+2] 350 [+2] 349 352 [+3] 351 [+2]
B7 5’-GTG ATB TGC 348 350 [+2] 349 [+1] 349 353 [+4] 351 [+2]
B10 5’-GTG BTB TGC 347 349 [+2] 348 [+1] 349 351 [+2] 351 [+2]
B8 3’-CAC TBT ACG 348 350 [+2] 348 [+0] 347 353 [+6] 352 [+5]
B9 3’-CAC TAT BCG 348 350 [+2] 350 [+2] 350 351 [+1] 350 [+0]
B11 3’-CAC TBT BCG 348 350 [+2] 348 [+0] 348 352 [+4] 353 [+5]
Measurements were performed using a spectrophotometer and quartz optical cells with a 1.0 cm
path length. Buffer conditions are as for thermal denaturation experiments.
General protocol for coupling between 1 and phenols (ArOH) to prepare 72W/72X (description
for ~1.33 mmol scale): The appropriate phenol and 2,2’-anhydrouridine 70 were placed in a sealed
pressure tube (specific quantities of substrates and reagents given below) and heated (165 °C for
72W; 175 °C for 72X) until analytical TLC indicated full conversion (~2h). The resulting crude was
purified by silica gel column chromatography (2-4% MeOH in CH Cl , v/v) to afford nucleoside
72W/72X (yields specified below).
2’-O-(Napthyl)uridine (72W): A mixture of 2,2’-anhydrouridine 70 (1.00 g, 4.42 mmol) and 2-
napthol (2.40 g, 22.1 mmol) were reacted and purified as described above to afford nucleoside 72W
(0.41 g, 25 %) as a light brown solid. R : 0.4 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z
f 2 2
+ . + 1
393.1039 ([M+Na] , C H N O Na , Calc. 393.1057); H NMR (DMSO-d ) δ 11.34 (s, 1H, ex,
19 18 2 6 6
NH), 8.03 (1H, d, J = 8.1 Hz, H6), 7.82-7.85 (ap d, 2H, Nap), 7.73-7.75 (1H, d, J = 8.3 Hz, Nap),
7.44-7.48 (ap t, 1H, Nap), 7.41 (d, 1H, J = 2.5 Hz, Nap), 7.34-7.37 (ap t, 1H, Nap), 7.23-7.25 (dd,
1H, J = 9.1 Hz, J = 2.5 Hz, Nap), 6.14 (d, 1H, J = 4.9 Hz, H1’), 5.68 (d, 1H, J = 8.1 Hz, H5), 5.46 (d,
1H, ex, J = 6.4 Hz, 3’-OH), 5.25 (t, 1H, ex, J = 5.2 Hz, 5’-OH), 5.02 (ap t, 1H, H2’), 4.43-4.46 (m,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
1H, H3’), 4.02-4.05 (m, 1H, H4’), 3.74-3.78 (m, 1H, H5’), 3.65-3.69 (m, 1H, H5’); C NMR
(DMSO-d ) δ 162.9, 155.4, 150.5, 140.4 (C6), 133.9, 129.2 (Nap), 128.7, 127.4 (Nap), 126.6 (Nap),
126.4 (Nap), 123.8 (Nap), 118.9 (Nap), 108.9 (Nap), 102.0 (C5), 86.4 (C1’), 85.2 (C4’), 79.3 (C2’),
68.2 (C3’), 60.4 (C5’).
2’-O-(Pyrenyl)uridine (72X): A mixture of 2,2’-anhydrouridine 70 (0.30 g, 1.33 mmol) and 1-
pyrenol (0.86 g, 3.97 mmol) were reacted and purified as described above to afford nucleoside 72X
(0.26 g, 44 %) as a pale yellow solid. R : 0.4 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z
f 2 2
+ . + 1
467.1217 ([M+Na] , C H N O Na , Calc. 467.1214); H NMR (DMSO-d ) δ 11.36 (s, 1H, ex,
20 2 6 6
NH), 8.45 (d, 1H, J = 9.3 Hz, Py), 8.20-8.24 (m, 3H, Py), 8.14 (d, 1H, J = 9.3 Hz, Py), 7.99-8.09 (m,
4H, H6, Py), 7.86 (1H, d, J = 8.5 Hz, Py), 6.35 (d, 1H, J = 4.6 Hz, H1’), 5.68 (d, 1H, J = 8.2 Hz, H5),
.63 (br s, 1H, ex, 3’-OH), 5.30 (br s, 1H, ex, 5’-OH), 5.22-5.25 (ap t, 1H, H2’), 4.55-4.56 (m, 1H,
H3’), 4.19-4.21 (m, 1H, H4’), 3.81-3.84 (ap d, 1H, H5’), 3.72-3.75 (ap d, 1H, H5’); C NMR
(DMSO-d ) δ 162.9, 151.7, 150.5, 140.3 (C6), 131.0, 130.9, 127.1 (Py), 126.4 (Py), 125.6 (Py),
125.2, 125.0 (Py), 124.8 (Py), 124.5 (Py), 124.3 (Py), 123.9, 121.1 (Py), 120.1, 111.9 (Py), 102.0
(C5), 86.6 (C1’), 85.4 (C4’), 80.9 (C2’), 68.5 (C3’), 60.4 (C5’).
General protocol for coupling between 1 and arylmethyl alcohol (ArCH OH) for the
preparation of 72Y/72Z (description for ~44.2 mmol scale): The appropriate aromatic alcohol
(ArCH OH), NaHCO and 1.0M BH in THF were placed in a pressure tube, suspended in anhydrous
2 3 3
DMSO and stirred under an argon atmosphere at rt until effervescence ceased (~10 min). At this
point, 2,2’-anhydrouridine 70 was added (specific quantities of substrates and reagents given below),
the pressure tube was purged with argon and sealed, and the reaction was heated at ~160 °C until
analytical TLC indicated full conversion (~3h). At this point, the reaction mixture was poured into
water (200 mL), stirred for 30 min and diluted with EtOAc (500 mL). The organic phase was washed
with water (4 × 200 mL), evaporated to dryness, and the resulting crude purified by silica gel column
chromatography (2-4%, MeOH in CH Cl , v/v) to afford a residue, which was precipitated from cold
acetone to obtain nucleosides 72Y/72Z (yields specified below).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
2’-O-(Pyrenyl-methyl)uridine (72Y): 2,2’-Anhydrouridine 70 (10.00 g, 44.2 mmol), pyren
ylmethanol (20.5 g, 88.4 mmol), NaHCO (0.73 g. 8.80 mmol), 1.0 M BH in THF (24.5 mL, 22.0
mmol) and anhydrous DMSO (40 mL) were mixed, reacted, worked up, and purified as described
above to afford nucleoside 72Y (5.04 g, 25 %) as a white solid. R : 0.4 (10 % MeOH in CH Cl , v/v);
f 2 2
+ + 1
MALDI-HRMS m/z 458.1480 ([M] , C H N O , Calc. 458.1472); H NMR (DMSO-d ): δ 11.29
26 22 2 6 6
(s, 1H, ex, NH), 8.37-8.39 (d, 1H, J = 9.3 Hz, Py), 8.29-8.31 (m, 2H, Py), 8.24-8.26 (d, 1H, J = 7.7
Hz, Py), 8.17-8.19 (m, 3H, Py), 8.06-8.12 (m, 2H, Py), 7.82 (d, 1H, J = 8.4 Hz, H6), 6.04 (d, 1H, J =
5.1 Hz, H1’), 5.43-5.50 (m, 2H, H5, CH Py), 5.37 (d, 1H, ex, J = 5.7 Hz, 3’-OH), 5.28-5.30 (d, 1H, J
= 12.0 Hz, CH Py), 5.11 (t, 1H, ex, J = 4.9 Hz, 5’-OH), 4.26-4.31 (m, 1H, H3’), 4.18-4.21 (m, 1H,
H2’), 3.96-3.97 (m, 1H, H4’), 3.64-3.68 (m, 1H, H5’), 3.59-3.62 (m, 1H, H5’); C NMR (DMSO-d )
δ 162.9, 150.6, 140.1 (C6), 131.4, 130.7, 130.2, 128.7, 127.4 (Py), 127.3 (Py), 127.0 (Py), 126.2 (Py),
125.3 (Py), 124.5 (Py), 124.0 (Py), 123.8, 123.5 (Py), 101.7 (C5), 86.2 (C1’), 85.4 (C4’), 80.9 (C2’),
69.8 (CH Py), 68.5 (C3’), 60.6 (C5’).
2’-O-(Coronenyl-methyl)uridine (72Z): 2,2’-Anhydrouridine 70 (1.40g, 6.19 mmol), coronen
ylmethanol (4.08 g, 12.4 mmol), NaHCO (0.104 g. 1.24 mmol), 1.0 M BH in THF (3.5 mL, 3.1
mmol) and anhydrous DMSO (40 mL) were mixed, reacted, worked up and purified as described
above with a minor modification. The precipitate that formed upon pouring the reaction mixture into
water was collected by filtration, washed with water (3 × 100 mL) and purified by column
chromatography to afford nucleoside 72Z (450 mg, 13 %) as a pale yellow solid. R : 0.4 (10 %
6478592_1 (GHMatters) P95976.NZ ESTHERJ
+ . +
MeOH in CH Cl , v/v); MALDI-HRMS m/z 579.1537 ([M+Na] , C H N O Na , Calc. 579.1532);
2 2 34 24 2 6
H NMR (DMSO-d ) δ 11.30 (br d, ex, 1H, J = 1.9 Hz, NH), 9.13-9.15 (d, 1H, J = 8.7 Hz, Cor), 8.93-
9.03 (m, 10H, Cor), 7.82 (d, 1H, J = 8.0 Hz, H6), 6.18 (d, 1H, J = 4.9 Hz, H1’), 5.85-5.88 (d, 1H, J =
12.1 Hz, CH Cor), 5.67-5.69 (d, 1H, J = 12.1 Hz, CH Cor), 5.52 (d, 1H, ex, J = 5.5 Hz, 3’-OH), 5.38
(dd, 1H, J = 8.0 Hz, 1.9 Hz, H5), 5.11 (ap t, 1H, ex, 5’-OH), 4.37-4.43 (m, 2H, H2’, H3’), 4.03-4.06
(m, 1H, H4’), 3.63-3.71 (m, 2H, H5’); C NMR (DMSO-d ) δ 162.9, 150.6, 140.1 (C6), 132.1,
128.2, 128.1, 127.9, 127.4, 126.5, 126.23 (Cor), 126.20 (Cor), 126.1 (Cor), 126.0 (Cor), 122.5 (Cor),
121.8, 121.5, 121.4, 121.23, 121.17, 101.7 (C5), 86.3 (C1’), 85.5 (C4’), 81.1 (C2’), 70.7 (CH Cor),
68.6 (C3’), 60.6 (C5’).
General DMTr-protection protocol for the preparation of 74W-74Z (description for ~2.2 mmol
scale): The appropriate nucleoside 72 (specific quantities given below) was co-evaporated twice
with anhydrous pyridine (15 mL) and redissolved in anhydrous pyridine. To this was added 4,4’-
dimethoxytritylchloride (DMTrCl) and N,N-dimethylaminopyridine (DMAP), and the reaction
mixture was stirred at rt until TLC indicated complete conversion (~14h). The reaction mixture was
diluted with CH Cl (70 mL) and the organic phase sequentially washed with water (2 × 70 mL) and
sat. aq. NaHCO (2 × 100 mL). The organic phase was evaporated to near dryness and the resulting
crude co-evaporated with absolute EtOH and toluene (2:1, v/v, 3 × 6 mL) and purified by silica gel
column chromatography (0-5%, MeOH in CH Cl , v/v) to afford nucleoside 74 (yields specified
below).
2’-O-(Napthyl)-5’-O-(4,4’-dimethoxytrityl)uridine (74W): Nucleoside 72W (150 mg, 0.40
mmol), DMTrCl (240 mg, 0.60 mmol) and DMAP (~6 mg) in anhydrous pyridine (7 mL) were
mixed, reacted, worked up and purified as described above to afford nucleoside 74W (120 mg, 47%)
as a pale yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 695.2379 ([M+Na] ,
f 2 2
. + 1
C H N O Na Calc. 695.2364); H NMR (DMSO-d ) δ 11.40 (d, 1H, ex, J = 2.1 Hz, NH), 7.83-
40 36 2 8 , 6
7.86 (m, 3H, H6, Nap), 7.68 (d, 1H, J = 8.2 Hz, Nap), 7.24-7.45 (m, 13H, DMTr, Nap), 6.90-6.92 (d,
4H, J = 7.1 Hz, DMTr), 6.06 (d, 1H, J = 3.2 Hz, H1’), 5.51 (d, 1H, ex, J = 7.1 Hz, 3’-OH), 5.38-5.40
(m, 1H, H5), 5.12-5.14 (m, 1H, H2’), 4.51-4.55 (m, 1H, H3’), 4.14-4.17 (m, 1H, H4’), 3.75 (s, 6H, 2
× CH O), 3.32-3.41 (m, 2H, H5’); C NMR (DMSO-d ) δ 162.9, 158.1, 155.5, 150.3, 144.6, 140.6
(C6), 135.4, 135.2, 133.9, 129.8 (Ar), 129.1 (Nap), 128.8, 127.9 (Ar), 127.7 (Ar), 127.5 (Nap), 126.8
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(Ar), 126.6 (Nap), 126.4 (Ar), 123.8 (Ar), 119.0 (Ar), 113.2 (Ar), 109.0 (Ar), 101.8 (C5), 87.6 (C1’),
85.9, 82.6 (C4’), 79.1 (C2’), 68.5 (C3’), 62.7 (C5’), 55.0 (CH O).
74X
2’-O-(Pyrenyl)-5’-O-(4,4’-dimethoxytrityl)uridine (74X): Nucleoside 72X (230 mg, 0.52
mmol), DMTrCl (0.30 g, 0.78 mmol) and DMAP (~9 mg) in anhydrous pyridine (8 mL) were mixed,
reacted, worked up and purified as described above to afford nucleoside 74X (0.30 g, 78%) as a light
yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 769.2504 ([M+Na] ,
f 2 2
. + 1
C H N O Na , Calc. 769.2520); H NMR (DMSO-d ): δ 11.32 (s, 1H, ex, NH), 8.49 (d, 1H, J = 9.2
46 38 2 8 6
Hz, Py), 8.20-8.24 (m, 3H, Py), 8.14 (d, 1H, J = 9.2 Hz, Py), 8.08-8.10 (d, 1H, J = 9.1 Hz, Py), 7.99-
8.04 (m, 2H, Py), 7.86-7.89 (m, 2H, H6, Py), 7.43-7.45 (m, 2H, DMTr), 7.24-7.36 (m, 7H, DMTr),
6.90-6.92 (m, 4H, DMTr), 6.25 (d, 1H, J = 3.2 Hz, H1’), 5.69 (d, 1H, ex, J = 6.8 Hz, 3’-OH), 5.38 (d,
1H, J = 8.2 Hz, H5), 5.35-5.37 (m, 1H, H2’), 4.61-4.65 (m, 1H, H3’), 4.34-4.37 (m, 1H, H4’), 3.74
(s, 6H, 2 × CH O), 3.46-3.50 (m, 1H, H5’), 3.38-3.41 (m, 1H, H5’); C NMR (DMSO-d ) δ 162.9,
158.1, 151.7, 150.3, 144.6, 140.5 (C6), 135.4, 135.1, 131.1, 131.0, 129.78 (DMTr), 129.76 (DMTr),
127.9 (DMTr), 127.1 (Py), 126.8 (DMTr), 126.4 (Py), 125.6 (Py), 125.3, 125.1 (Py), 124.9, 124.5
(Py), 124.3 (Py), 124.0, 121.2 (Py), 120.1, 113.2 (DMTr), 112.3 (Py), 101.7 (C5), 87.8 (C1’), 85.9,
82.7 (C4’), 80.7 (C2’), 68.7 (C3’), 62.7 (C5’), 55.0 (CH O).
2’-O-(Pyrenyl-methyl)-5’-O-(4,4’-dimethoxytrityl)uridine (74Y): Nucleoside 72Y (1.02 g,
2.20 mmol), DMTrCl (1.29 g, 3.30 mmol) and DMAP (~18 mg) in anhydrous pyridine (20 mL) were
mixed, reacted, worked up and purified as described above to afford 74Y(1.20 g, 72 %) as pale
6478592_1 (GHMatters) P95976.NZ ESTHERJ
yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 783.2698 ([M+Na] ,
f 2 2
. + 1
C H N O Na , Calc. 783.2677); H NMR (DMSO-d ) δ 11.36 (d, 1H, ex, J = 1.9 Hz, NH), 8.41-
47 40 2 8 6
8.43 (d, 1H, J = 9.3 Hz, Py), 8.30-8.32 (m, 2H, Py), 8.14-8.25 (m, 5H, Py), 8.07-8.10 (t, 1H, J = 7.7
Hz, Py), 7.63 (d, 1H, J = 8.1 Hz, H6), 7.17-7.34 (m, 9H, DMTr), 6.82-6.86 (m, 4H, DMTr), 6.02 (d,
1H, J = 3.9 Hz, H1’), 5.48-5.50 (d, 1H, J = 12.1 Hz, CH Py), 5.45 (d, 1H, ex, J = 6.3 Hz, 3’-OH),
.36-5.38 (d, 1H, J = 12.1 Hz, CH Py), 5.13 (dd, 1H, J = 8.1 Hz, 1.9 Hz, H5), 4.34-4.38 (m, 1H,
H3’), 4.21-4.24 (m, 1H, H2’), 4.05-4.09 (m, 1H, H4’), 3.71 (s, 3H, CH O), 3.69 (s, 3H, CH O), 3.20-
3.24 (m, 2H, H5’); C NMR (DMSO-d ) δ 162.8, 158.08, 158.06, 150.4, 144.5, 140.0 (C6), 135.3,
135.0, 131.3, 130.7, 130.2, 129.71 (DMTr), 129.66 (DMTr), 128.7, 127.8 (DMTr), 127.6 (DMTr),
127.4 (Py), 127.3 (Py), 127.0 (Py), 126.7 (DMTr), 126.2 (Py), 125.3 (Py), 124.5 (Py), 124.0, 123.8,
123.4 (Py), 113.20 (DMTr), 113.17 (DMTr), 101.4 (C5), 87.1 (C1’), 85.9, 83.1 (C4’), 80.6 (C2’),
69.9 (CH Py), 68.7 (C3’), 62.8 (C5’), 55.0 (CH O).
74Z
2’-O-(Coronenyl-methyl)-5’-O-(4,4’-dimethoxytrityl)uridine (74Z): Nucleoside 72Z (250 mg,
0.45 mmol), DMTrCl (262 mg, 0.67 mmol) and DMAP (~15 mg) in anhydrous pyridine (6 mL) were
mixed, reacted, worked up and purified as described above to afford nucleoside 74Z (245 mg 63%)
as a yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 881.2824 ([M+Na] ,
f 2 2
. + 1
C H N O Na , Calc. 881.2839); H NMR (DMSO-d ) δ 11.40 (br d, 1H, ex, J = 1.9 Hz, NH), 9.14-
55 42 2 8 6
9.16 (d, 1H, J = 8.8 Hz, Cor), 8.96-9.02 (m, 9H, Cor), 8.88-8.90 (d, 1H, J = 8.5 Hz, Cor), 7.63 (d, 1H,
J = 8.1 Hz, H6), 7.29-7.31 (d, 2H, J = 7.4 Hz, DMTr), 7.11-7.22 (m, 7H, DMTr), 6.74-6.79 (m, 4H,
DMTr), 6.18 (d, 1H, J = 4.1 Hz, H1’), 5.87-5.90 (d, 1H, J = 12.6 Hz, CH Cor), 5.75-5.78 (d, 1H, J =
12.6 Hz, CH Cor), 5.59 (d, 1H, ex, J = 6.3 Hz, 3’-OH), 5.06 (dd, 1H, J = 8.1 Hz, 1.9 Hz, H5), 4.46-
4.50 (m, 1H, H3’), 4.40-4.43 (m, 1H, H2’), 4.15-4.18 (m, 1H, H4’), 3.63 (s, 3H, CH O), 3.58 (s, 3H,
CH O), 3.32-3.34 (m, 1H, H5’), 3.25-3.27 (m, 1H, H5’); C NMR (DMSO-d ) δ 162.8, 158.02,
157.97, 150.4, 144.4, 140.0 (C6), 135.3, 135.0, 132.2, 129.7 (DMTr), 129.6 (DMTr), 128.3, 128.2,
128.0, 127.7 (DMTr), 127.59 (DMTr), 127.56, 126.61 (DMTr), 126.59, 126.41 (Cor), 126.36 (Cor),
126.3 (Cor), 126.23 (Cor), 126.21, 126.19, 126.1 (Cor), 122.6 (Cor), 121.9, 121.6, 121.5, 121.4,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
121.35, 121.28, 113.13 (DMTr), 113.09 (DMTr), 101.4 (C5), 87.1 (C1’), 85.9, 83.2 (C4’), 80.7 (C2’),
70.7 (CH Cor), 68.8 (C3’), 62.8 (C5’), 54.9 (CH O), 54.8 (CH O).
2 3 3
General phosphitylation protocol for the preparation of 4W-4Z (description for ~1 mmol scale):
The appropriate nucleoside 74 (specific quantities of substrates and reagents given below) was co-
evaporated with anhydrous 1,2-dicholoroethane (4 mL) and redissolved in anhydrous CH Cl . To this
was added N,N-diisopropylethylamine (DIPEA) and 2-cyanoethyl-N,N-
diisopropylchlorophosporamidite (PCl-reagent) and the reaction mixture was stirred at rt until TLC
indicated complete conversion (~3h), whereupon abs. EtOH (2 mL) and CH Cl (20 mL) were
sequentially added to the solution. The organic phase was washed with sat. aq. NaHCO (10 mL),
evaporated to near dryness, and the resulting residue purified by silica gel column chromatography
(40-70% EtOAc in petroleum ether, v/v) to afford the corresponding phosphoramidite 76 (yields
specified below).
76W
2’-O-(Napthyl)-3’-O-(N,N-diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-
dimethoxytrityl)uridine (76W): Nucleoside 74W (100 mg, 0.15 mmol), DIPEA (106 μL, 0.59
mmol) and PCl-reagent (66 μL, 0.23 mmol) in anhydrous CH Cl (1.5 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 76W (95 mg, 74 %) as a white
foam. R : 0.8 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 895.3462 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calc. 895.3448); P NMR (CDCl ) δ 151.0, 150.9.
49 53 4 9 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-2’-O-(pyrenyl)-5’-O-(4,4’-
dimethoxytrityl)uridine (76X): Nucleoside 74X (0.28 g, 0.38 mmol), DIPEA (268 μL, 1.50
mmol) and PCl-reagent (167 μL, 0.75 mmol) in anhydrous CH Cl (2.5 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 76X (0.27 g, 76 %) as a white
foam. R : 0.8 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 969.3608 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calc. 969.3604); P NMR (CDCl ) δ 149.8, 149.4.
55 55 4 9 3
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-2’-O-(pyrenyl-methyl)-5’-O-(4,4’-
dimethoxytrityl)uridine (76Y): Nucleoside 74Y (0.58 g, 0.76 mmol), DIPEA (0.53 mL, 3.05
mmol) and PCl-reagent (340 μL, 1.53 mmol) in anhydrous CH Cl (5 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 76Y (0.56 g, 76 %) as a white
foam. R : 0.8 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 983.3767 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calc. 983.3761); P NMR (CDCl ) δ 150.3, 150.2.
56 57 4 9 3
2’-O-(Coronenyl-methyl)-3’-O-(N,N-diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-
dimethoxytrityl)uridine (76Z): Nucleoside 74Z (240 mg, 0.28 mmol), DIPEA (200 μL, 1.11 mmol)
and PCl-reagent (125 μL, 0.56 mmol) in anhydrous CH Cl (6 mL) were mixed, reacted, worked up
and purified as described above to afford phosphoramidite 76Z (230 mg, 78 %) as a light yellow
foam. R : 0.8 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1081.3864 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calc. 1081.3917); P NMR (CDCl ) δ 150.2.
64 59 4 9 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
7-neopentylpyreneylmethyl alcohol: Pyrene (2.0 g, 9.9 mmol) was added into anhydrous CH Cl
containing a mixture of anhydrous AlCl (1.3 g, 9.0 mmol) and 2,2,2-trimethylacetylchloride (0.97
mL, 7.9 mmol) at 0°C. The yellow crude product was purified using silica gel column
chromatography to afford 7-(2,2,2-trimethylacetyl)pyrene (1.4 g, 50%) as a bright yellow solid. This
intermediate was then reduced to 7-neopentylpyrene (1.4 g, 98%) in the presence of
trifluoroaceticacid (3.6 mL, 49 mmol) and triethylsilane (4.7 mL, 29.0 mmol). R : 0.8 (10% EtOAc in
petroleum ether, v/v). To a stirred solution of 7-neopentylpyrene (0.9 g, 3.3 mmol) and
dichloromethylmethylether (0.4 mL, 4.3 mmol) in anhydrous CH Cl (20 mL) at 0°C was added a
solution of titaniumtetrachloride (0.7 mL, 6.3 mmol). The mixture was poured into a large amount of
ice water and extracted with CH Cl . The crude was purified by silica gel column chromatography
(40% benzene in petroleum ether, v/v) to afford 7-neopentylpyrenecarboxaldehyde (0.9 g, 91%) as
a yellow solid. This intermediate was then reduced to 7-neopentylpyreneylmethylalcohol (0.9 g,
99%) using sodium borohydride (0.1 g, 2.66 mmol) in tetrahydrofuran (50 mL).
6/8-bromopyreneylmethylalcohol: To a solution of 1-bromopyrene (2.6 g, 9.25 mmol) and
dichloromethylmethylether (1.08 mL, 12.0 mmol) in anhydrous CH Cl
(60 mL) at 0°C was added a solution of titaniumtetrachloride (1.98 mL,
17.5 mmol). The mixture was poured into a large amount of ice water and
extracted with CH Cl . The crude was purified by silica gel column
chromatography (40% benzene in petroleum ether, v/v) to afford a mixture
of 6- and 8-bromopyrenecarboxaldehyde (1.4 g, 49%) as a yellow solid.
This intermediate was then reduced to a mixture of 6- and 8-bromopyreneylmethylalcohol (1.4 g,
99%) as using sodium borohydride (0.17 g, 4.53 mmol) in tetrahydrofuran (80 mL).
8-methylpyreneylmethylalcohol: To a stirred solution of 1-methylpyrene (4.3 g, 19.8 mmol) and
dichloromethylmethylether (2.34 mL, 25.8 mmol) in anhydrous CH Cl
(150 mL) at 0°C was added a solution of titaniumtetrachloride (4.26 mL,
37.7 mmol). The mixture was poured into a large amount of ice water and
extracted with CH Cl . The crude was purified by silica gel column
chromatography (40% benzene in petroleum ether, v/v) to afford 8-
methylpyrenecarboxaldehyde (4.0 g, 83%) as a yellow solid. This intermediate was then reduced
to 8-methylpyreneylmethylalcohol (4.0 g, 99%) in the presence of sodium borohydride (0.74 g,
19.6 mmol) in tetrahydrofuran (100 mL).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
7-tert-Butylmethoxypyreneylmethylalcohol: 7-tert-butyl
methoxypyrenecarboxaldehyde (obtained as described in: Yamoto et al.
Organic Preparation and Procedure International. 1997, 29, 3) was
reduced to 7-tert-butylmethoxypyreneylmethylalcohol (2.1 g, 99%)
using sodium borohydride (0.26 g, 6.94 mmol) in tetrahydrofuran (30 mL).
General protocol for coupling between O2,O2’-anhydronucleoside 70 and arylmethyl alcohol
(ArCH OH) for the preparation of 200W-Z (description for ~44.2 mmol scale). The appropriate
aromatic alcohol (ArCH OH), NaHCO and 1.0M BH in THF were placed in a pressure tube,
2 3 3
suspended in anhydrous DMSO and stirred under an argon atmosphere at room temperature until
effervescence ceased (~10 min). At this point, nucleoside 70 was added (specific quantities of
substrates and reagents given below), the pressure tube was purged with argon and sealed, and the
reaction was heated at ~160 °C until analytical TLC indicated full conversion (~3h). At this point, the
reaction mixture was poured into water (200 mL), stirred for 30 min and diluted with EtOAc (500
mL). The organic phase was washed with water (4 × 200 mL), evaporated to dryness, and the
resulting crude purified by silica gel column chromatography (2-4%, MeOH in CH Cl , v/v) to afford
a residue, which was precipitated from cold acetone to obtain nucleosides 200 (yields specified
below).
2’-O-(7-neopentylpyreneylmethyl)uridine (200W). Nucleoside 70 (0.40 g, 1.8 mmol), 7-
neopentylpyreneylmethanol (1.07 g, 3.5 mmol), NaHCO (0.03 g. 0.35 mmol), 1.0 M BH in THF
(0.9 mL, 0.9 mmol) and anhydrous DMSO (3 mL) were mixed, reacted, worked up, and purified as
described above to nucleoside 200W (0.18 g, 20 %) as a white solid. R : 0.4 (10 % MeOH in CH Cl ,
f 2 2
v/v).
2’-O-(6/8-bromopyreneylmethyl)uridine (200X). Nucleoside 70 (0.7 g, 3.1 mmol), 6/8-
bromopyreneylmethanol (1.9 g, 6.2 mmol), NaHCO (0.05 g. 0.62 mmol), 1.0 M BH in THF (1.6
mL, 1.55 mmol) and anhydrous DMSO (10 mL) were mixed, reacted, worked up, and purified as
described above to afford nucleoside 200X (0.28 g, 17 %) as a white solid. R : 0.4 (10 % MeOH in
CH Cl , v/v).
2’-O-(8-methylpyreneylmethyl)uridine (200Y). Nucleoside 70 (2.0 g, 8.84 mmol), 8-
methylpyreneylmethanol (4.3 g, 17.7 mmol), NaHCO (0.15 g. 1.77 mmol), 1.0 M BH in THF
(4.9 mL, 4.42 mmol) and anhydrous DMSO (10 mL) were mixed, reacted, worked up, and purified
as described above to afford nucleoside 200Y (0.87 g, 20 %) as a white solid. R : 0.4 (10 % MeOH in
CH Cl , v/v).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
2’-O-(3-methoxytert-butylpyreneylmethyl)uridine (200Z). Nucleoside 70 (0.36 g, 1.57
mmol), 3-methoxytertiarbutylypyreneylmethanol (1.0 g, 3.14 mmol), NaHCO (26.0 mg. 0.31
mmol), 1.0 M BH in THF (0.8 mL, 0.78 mmol) and anhydrous DMSO (8 mL) were mixed, reacted,
worked up, and purified as described above to afford nucleoside 200Z (0.2 g, 21 %) as a white solid.
R : 0.4 (10 % MeOH in CH Cl , v/v).
f 2 2
General DMTr-protection protocol for the preparation of 202V-Z (description for ~2.2 mmol
scale). The appropriate nucleoside 200 (specific quantities of substrates and reagents given below)
was co-evaporated twice with anhydrous pyridine (15 mL) and redissolved in anhydrous pyridine. To
this was added 4,4’-dimethoxytritylchloride (DMTrCl) and N,N-dimethylaminopyridine (DMAP),
and the reaction mixture was stirred at room temperature until TLC indicated complete conversion
(~14h). The reaction mixture was diluted with CH Cl (70 mL) and the organic phase sequentially
washed with water (2 × 70 mL) and sat. aq. NaHCO (2 × 100 mL). The organic phase was
evaporated to near dryness and co-evaporated with absolute EtOH and toluene (2:1, v/v, 3 × 6 mL)
and the resulting crude purified by silica gel column chromatography (0-5%, MeOH in CH Cl , v/v)
to afford nucleosides 202 (yields specified below).
’-O-(4,4’-dimethoxytrityl)-2’-O-(7-neopentylpyreneylmethyl)uridine (202W). Nucleoside
200W (0.13 g, 0.25 mmol), DMTrCl (0.14 g, 0.37 mmol) and DMAP (~1 mg) in anhydrous pyridine
(3 mL) were mixed, reacted, worked up and purified as described above to afford 202W (0.13 g, 64
%) as pale yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v).
f 2 2
’-O-(4,4’-dimethoxytrityl)-2’-O-(6/8-bromopyreneylmethyl)uridine (202X). Nucleoside 200X
(0.21 g, 0.39 mmol), DMTrCl (0.23 g, 0.58 mmol) and DMAP (~2 mg) in anhydrous pyridine (7 mL)
were mixed, reacted, worked up and purified as described above to afford 202X ( 0.28g, 85 %) as
pale yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v).
f 2 2
’-O-(4,4’-dimethoxytrityl)-2’-O-(8-methylpyreneylmethyl)uridine (202Y). Nucleoside 200Y
(0.7 g, 1.48 mmol), DMTrCl (0.86 g, 2.20 mmol) and DMAP (~4 mg) in anhydrous pyridine (10 mL)
were mixed, reacted, worked up and purified as described above to afford 202Y (0.9 g, 78 %) as pale
yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v).
f 2 2
5’-O-(4,4’-dimethoxytrityl)-2’-O-(pyreneylmethyl)uridine (202Z). Nucleoside 200Z (0.18 g,
0.33 mmol), DMTrCl (0.19 g, 0.49 mmol) and DMAP (~2 mg) in anhydrous pyridine (7 mL) were
mixed, reacted, worked up and purified as described above to afford 202Z ( 0.2 g, 70 %) as pale
yellow foam. R : 0.6 (5%, MeOH in CH Cl , v/v).
f 2 2
General phosphitylation protocol for the preparation of 204V-Z (description for ~1 mmol
scale). The appropriate nucleoside 202 (specific quantities of substrates and reagents are given
below) was co-evaporated with anhydrous 1,2-dicholoroethane (4 mL) and redissolved in anhydrous
6478592_1 (GHMatters) P95976.NZ ESTHERJ
CH Cl . To this was added DIPEA and 2-cyanoethyl-N,N-diisopropylchlorophosporamidite (PCl-
reagent) and the reaction mixture was stirred at rt until TLC indicated complete conversion (~3h),
whereupon abs. EtOH (2 mL) and CH Cl (20 mL) were sequentially added to the solution. The
organic phase was washed with sat. aq. NaHCO (10 mL), evaporated to near dryness, and the
resulting residue purified by silica gel column chromatography (40-70% EtOAc in petroleum ether,
v/v) to afford the corresponding phosphoramidite 204 (yields specified below)
’-O-(4,4’-dimethoxytrityl)-2’-O-(7-neopentylpyreneylmethyl)uridine-3’-O-(2-cyanoethyl)-
N,N-diisopropylphosphoramidite (204W). Nucleoside 202W (0.11 g, 0.13 mmol), DIPEA (0.1 mL,
0.53 mmol) and PCl-reagent (60 μL, 0.26 mmol) in anhydrous CH Cl (3 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 204W (0.11 g, 81 %) as a
white foam. R : 0.8 (5% MeOH in CH Cl , v/v).
f 2 2
’-O-(4,4’-dimethoxytrityl)-2’-O-(6/8-bromopyreneylmethyl)uridine-3’-O-(2-cyanoethyl)-
N,N-diisopropylphosphoramidite (204X). Nucleoside 202X (0.25 g, 0.29 mmol), DIPEA (021 mL,
1.19 mmol) and PCl-reagent (133 μL, 0.59 mmol) in anhydrous CH Cl (5 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 204X (0.26 g, 82 %) as a white
foam. R : 0.8 (5% MeOH in CH Cl , v/v).
f 2 2
5’-O-(4,4’-dimethoxytrityl)-2’-O-(8-methylpyreneylmethyl)uridine-3’-O-(2-cyanoethyl)-N,N-
diisopropylphosphoramidite (204Y). Nucleoside 202Y (0.65 g, 0846 mmol), DIPEA (0.59 mL,
3.35 mmol) and PCl-reagent (374 μL, 1.77mmol) in anhydrous CH Cl (10 mL) were mixed, reacted,
worked up and purified as described above to afford phosphoramidite 204Y (0.6 g, 78 %) as a white
foam. R : 0.8 (5% MeOH in CH Cl , v/v)
f 2 2
’-O-(4,4’-dimethoxytrityl)-2’-O-(3-methoxytertiarybutylpyreneylmethyl)uridine-3’-O-(2-
cyanoethyl)-N,N-diisopropylphosphoramidite (204Z). Nucleoside 202Z (0.15 g, 0.18 mmol),
DIPEA (126µL, 0.71 mmol) and PCl-reagent (79 μL, 0.35 mmol) in anhydrous CH Cl (5 mL) were
mixed, reacted, worked up and purified as described above to afford phosphoramidite 204Z (0.15 g,
80 %) as a white foam. R : 0.8 (5% MeOH in CH Cl , v/v).
f 2 2
6478592_1 (GHMatters) P95976.NZ ESTHERJ
2’-O-(Pyrenyl-methyl)adenosine (130W): An adenosine-functionalized nucleoside 128 (1.60 g,
.98 mmol), pyrenylmethylchloride (1.0 g, 3.9 mmol), NaH (0.36 g, 14.9 mmol), and anhydrous
DMSO (30 mL) were mixed, reacted, worked up, and purified as described above to afford
nucleoside 2W (0.48 g, 17%) as a pale yellow solid. R 0.29 (10% MeOH in CH Cl , v/v); MALDI-
f 2 2
+ + 1
HRMS m/z 504.1649 ([M+Na] , C H N O .Na , Calcd 504.1642); HNMR (DMSO-d ) δ 8.32(s,
27 23 5 4 6
1H, adenine), 7.95–8.29 (m, 10H, pyrene, adenine), 7.27 (s, 2H, NH ), 6.14 (d, 1H, H1’, J = 6.3 Hz),
.48 (d, 1H, ex, J = 5.1 Hz, 3’-OH), 5.39-5.45 (m, 2H, CH -Py, 5’-OH (ex)), 5.17-5.20 (d, 1H, J =
11.7 Hz, CH Py), 4.81 (t, 1H, J = 5.1 Hz, 11.1 Hz, H2’), 4.50-4.54 (m, 1H, H3’), 4.08-4.11 (m, 1H,
H4’), 3.68-3.75 (m, 1H, H5’), 3.57-3.65 (m, 1H, H5’); C NMR (DMSO-d ) δ 156.1, 152.3, 148.9,
139.6, 131.2, 130.6, 130.6, 130.1, 128.7, 127.2, 127.2, 127.0, 126.1, 125.2, 125.2, 124.3, 123.8,
123.7, 123.3, 119.3, 86.7, 86.1, 80.8, 69.9, 69.1, 61.5.
2’-O-(Pyrenyl-methyl)cytidine (130X): Cytidine 132 (10.0 g, 41.1 mmol), pyren
ylmethylchloride (6.8 g, 27.1 mmol), NaH (2.47 g, 102.8 mmol), and anhydrous DMSO (150 mL)
were mixed, reacted, worked up, and purified as described above to afford nucleoside 130X (3.5 g,
19%) as a pale yellow solid. R 0.3 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z 480.1532
f 2 2
+ + 1
([M+Na] , C H N O .Na , Calcd 480.1529); HNMR (DMSO-d ) δ 8.06-8.49 (m, 9H, Py), 7.90 (d,
26 23 3 5 6
1H, cytosine), 7.15 (s, 2H, NH ), 6.08 (d, 1H, H1’), 5.67 (d,1H, cytosine), 5.41-5.43 (d, 1H, CH2Py),
5.24 (d, 1H, CH Py), 5.22 (d, 1H, 3’-OH), 5.07-5.11 (m, 1H, 5’-OH), 4.18-4.22 (m, 1H, H3’), 4.06-
4.11 (m, 1H, H2’), 3.93-3.95 (m, 1H, H4’), 3.53-3.74 (m, 2H, H5’); C NMR (DMSO-d ) δ 165.6,
155.1, 141.0, 131.6, 130.7, 130.5, 130.2, 128.6, 127.4, 127.3, 127.2, 126.9, 126.1, 125.2, 124.5,
123.9, 123.8, 123.6, 93.8, 89.7, 87.5, 84.2, 81.7, 77.2, 72.9, 69.7, 68.2, 60.1.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
2’-O-(Pyrenyl-methyl)guanosine (130Y): Guanosine 134 (3.0 g, 10.6 mmol), pyren
ylmethylchloride (1.86 g, 7.4 mmol), NaH (0.64 g, 26.5 mmol), and anhydrous DMSO (50 mL) were
mixed, reacted, worked up, and purified as described above to afford nucleoside 130Y (0.90 g, 17%)
as a white solid. R 0.29 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z 520.1608 ([M+Na] ,
f 2 2
C H N O .Na , Calcd 520.1591); HNMR (DMSO-d ) 10.6 (s, 1H, NH), 8.0-8.35 (m, 9H, Py), 7.97
27 23 5 5 6
(s, 1H, H7-adenine), 6.37 (s, 2H, NH ), 5.97 (d, 1H, H1’), 5.40-5.43 (dd, 2H, CH Py), 5.20 (d, 1H,
ex, 3’-OH), 5.08 (t, 1H, ex, 5’-OH), 4.61-4.63 (m, 1H, H2’), 4.44-4.46 (m, 1H, H3’), 4.00-4.02 (m,
1H, H4’), 3.60-3.68 (m, 2H, H5’); C NMR (DMSO-d ) δ 156.6, 153.6, 151.1, 135.2, 131.2, 130.6,
130.2, 128.7, 127.3, 127.0, 126.2, 125.2, 125.1, 124.4, 123.8, 123.7, 123.5, 116.7, 86.1, 84.5, 81.3,
69.8, 69.0, 61.3
2’-O-(Coronenyl-methyl)cytidine (130Z): Cytidine 132 (0.70 g, 2.88 mmol), coronen
ylmethylchloride (0.66 g, 1.99 mmol), NaH (0.17 g, 7.19 mmol), and anhydrous DMSO (20 mL)
were mixed, reacted, worked up, and purified as described above to afford nucleoside 130Z (0.16 g,
10%) as a pale yellow solid. R 0.3 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z 578.1679
f 2 2
+ + 1
([M+Na] , C H N O .Na , Calcd 578.1686); H-NMR (DMSO-d ) δ 9.01-9.17 (m, 11H, Cor), 7.94
34 25 3 5 6
(d, 1H, cytosine), 7.20 (s, 2H, NH ), 6.08 (d, 1H, H1’), 5.85 (d,1H, cytosine), 5.68-5.78 (m, 2H,
CH Cor), 5.22 (d, 1H, 3’-OH), 5.07-5.11 (m, 1H, 5’-OH), 4.18-4.22 (m, 1H, H3’), 4.13-4.15 (m, 1H,
H2’), 3.90-3.93 (m, 1H, H4’), 3.58-3.88 (m, 2H, H5’).
136W
2’-O-(Pyrenyl-methyl)-N-benzoyl-adenosine (136W): Nucleoside 130W (0.40g, 0.83 mmol)
was dried by co-evaporation with pyridine (5 mL) and re-dissolved in anhydrous pyridine (10 mL)
followed by addition of trimethylsilyl chloride (0.42 mL, 3.32 mmol). After stirring for 30 min at rt,
benzoyl chloride (0.14 mL, 1.25 mmol) was added and the mixture was stirred for additional 5h.
Aqueous NH (28%, 10 mL) was added and stirred for additional 1h and the reaction mixture was
6478592_1 (GHMatters) P95976.NZ ESTHERJ
concentrated to near dryness. The residual material was re-dissolved in CH Cl (150 mL) and washed
with water (2 x 75 mL). The organic phase was evaporated to near dryness and the product was
purified by silica gel column chromatography (1-3% MeOH in CH Cl , v/v), to afford nucleoside
136W (0.245 g, 50%) as pale yellow solid. R 0.56 (10% MeOH in CH Cl , v/v); MALDI-HRMS
f 2 2
+ + 1
m/z 608.1910 ([M+Na] , C H N O .Na , calcd 608.1904); H NMR (DMSO-d ) δ 11.09 (s, 1H,
34 27 5 5 6
amide), 8.62(s, 1H, adenine), 8.57(s, 1H, adenine), 7.99–8.28(m, 11H, benzoyl, Py), 7.56-7.70 (m,
3H, benzoyl), 6.25(d, 1H, H1’, J = 6 Hz), 5.58(d, 1H, J = 5.1 Hz, ex, 3’-OH), 5.46-5.50 (d, 1H, J =
11.4 Hz, CH Py), 5.17–5.24 (m, 2H, 1 ex, 5’-OH, CH Py), 4.86 (m, 1H, H2’), 4.52-4.57 (m, 1H,
H3’), 4.10-11 (m, 1H, H4’), 3.59-3.74 (m, 2H, H5’). C NMR (DMSO-d ) δ 165.4, 151.8, 151.4,
150.2, 142.77, 133.4, 132.4, 131.1, 131.1, 130.7, 130.6, 130.1, 128.7, 128.4, 128.4, 128.1, 127.4,
127.3, 127.2, 127.1, 126.1, 125.6, 125.2, 125.2, 124.3, 123.8, 123.7, 123.3, 86.6, 85.9, 80.8, 70.1,
69.0, 61.3.
136X
2’-O-(Pyrenyl-methyl)-N-benzoyl-cytidine (136X): Benzoic anhydride (1.68 g, 7.45 mmol) was
added to the solution of nucleoside 130X (3.1 g, 6.77 mmol) in anhydrous DMF (100 mL). The
reaction mixture was stirred at 60°C until TLC indicated complete conversion (~6h). The reaction
mixture was diluted with CH Cl (150 mL), and the organic phase was sequentially washed with
water (2 x 60 mL) and satd aq NaHCO (2 x 100 mL). The organic phase was dried over Na SO ,
3 2 4
evaporated to near dryness, and the resulting crude was purified by silica gel column chromatography
(0-2%, MeOH in CH Cl , v/v) to afford nucleoside 136X (2.1 g, 55%) as pale yellow solid. R 0.6
2 2 f
(10% MeOH in CH Cl , v/v); MALDI-HRMS m/z 584.1785 ([M+Na] , C H N O .Na , calcd
2 2 33 27 3 6
584.1790); HNMR (DMSO-d ) δ 11.16 (s, 1H, amide), 8.15-8.45 (m, 9H, Py), 8.00-8.03 (m, 3H,
benzoyl, cytosine), 7.62-7.66 (m, 1H, benzoyl), 7.52-7.55 (m, 2H, benzoyl), 7.44 (d, 1H, cytosine),
6.13 (d, 1H, H1’), 5.43-5.53 (dd, 2H, CH Py), 5.30 (d, 1H, ex, 3’-OH), 5.19 (t, 1H, 5’-OH), 4.24-
4.27 (m, 1H, H3’), 4.17-4.18 (m, 1H, H2’), 4.02-4.04 (m, 1H, H4’), 3.76-3.80 (m, 1H, H5’), 3.63-
3.67 (m, 1H, H5’); C NMR (DMSO-d ) δ 167.2, 162.9, 154.5, 144.8, 133.1, 132.7, 132.6, 131.8,
131.5, 130.6, 130.3, 130.2, 129.2, 128.7, 128.4, 127.4, 127.3, 126.2, 125.2, 124.5, 123.9, 123.7,
123.6, 96.0, 88.1, 84.7, 82.6, 81.7, 76.3, 73.0, 69.9, 67.8, 59.7
6478592_1 (GHMatters) P95976.NZ ESTHERJ
136Y
2’-O-(Pyrenyl-methyl)-N-isobutyryl-guanosine (136Y): Trimethylsilyl chloride (0.5 mL, 3.92
mmol) was added to a solution of nucleoside 130Y (0.26 g, 0.52 mmol), which was dried by
coevaporation with pyridine three times, in pyridine (6 mL). After stirring 2h at rt, isobutyrylchloride
(0.15 ml, 1.57 mmol) was added drop wise at 0°C for 10 min. Water (2 mL) was added to the
reaction mixture at 0°C and stirred for another 10 min. The solution was stirred for another 5 min at
rt, and aq NH (28%, 5 mL) was added to the solution. After stirring for another 15 min at room
temperature, the solution was concentrated to near dryness. The residual material was re-dissolved in
CH Cl (100 mL) and washed with water (2 x 60 mL). The organic phase was dried over Na SO ,
2 2 2 4
evaporated to near dryness and the crude was purified by silica gel column chromatography (1-3%
MeOH in CH Cl , v/v), to afford nucleoside 136Y (0.220 g, 74 %) as pale yellow solid. R 0.56 (10%
2 2 f
MeOH in CH Cl , v/v); MALDI-HRMS m/z 590.2008 ([M+Na] , C H N O .Na , calcd 590.2010);
2 2 31 29 5 6
HNMR (DMSO-d ) 11.8 (s, 1H, amide), 11.3 (s, 1H, NH), 8.01-8.27 (m, 9H, Py), 7.95 (s, 1H, H7-
guanine), 5.93 (d, 1H, H1’), 5.43-5.46 (m, 2H, 1 ex, 3’-OH, CH Py), 5.20 (d, 1H, ex, 3’-OH), 5.06-
.16 (m, 2H, 1 ex, 5’-OH, CH Py), 4.65-4.67 (m, 1H, H2’), 4.45-4.48 (m, 1H, H3’), 4.01-4.03 (m,
1H, H4’), 3.61-3.66 (m, 2H, H5’), 2.56-2.61 (m, 1H, isobutyryl), 1.00-1.04 (dd, 6H,isobutyryl); C
NMR (DMSO-d ) δ 179.8, 154.5, 148.5, 147.8, 137.2, 131.1, 130.6, 130.1, 128.7, 127.3, 127.1,
127.0, 126.1, 125.2, 125.1, 124.3, 123.8, 123.6, 123.5, 86.5, 84.6, 81.6, 70.2, 69.2, 61.3, 34.5, 18.7,
18.6
General DMTr-Protection Protocol for the preparation of 138W-138Y [Description for ~3.6
mmol Scale]: The appropriate nucleoside 136 (specific quantities given below) was coevaporated
twice with anhydrous pyridine (15 mL) and redissolved in anhydrous pyridine. To this were added
4,4’-dimethoxytritylchloride (DMTrCl) and N,N-dimethylaminopyridine (DMAP), and the
reaction mixture was stirred at rt until TLC indicated complete conversion (~14h). The reaction
mixture was diluted with CH Cl (100 mL), and the organic phase was sequentially washed with
water (2 x 50 mL) and satd aq NaHCO (2 x 100 mL). The organic phase was evaporated to near
dryness, and the resulting crude coevaporated with absolute EtOH and toluene (2: 1, v/v, 3 x 10 mL)
and purified by silica gel column chromatography (0-5%, MeOH in CH Cl , v/v) to afford nucleoside
138 (yields specified below).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
138W
’-O-(4,4’-dimethoxytrityl)-2’-O-(Pyrenyl-methyl)-N-benzoyl-adenosine (138W). Nucleoside
136W (0.24 g, 0.41 mmol), DMTrCl (0.27 g, 0.70 mmol), and DMAP (~12 mg) in anhydrous
pyridine (6 mL) were mixed, reacted, worked up, and purified as described above to afford
nucleoside 138W (0.3 g, 71%) as a pale yellow foam. R 0.78 (10% MeOH in CH Cl , v/v); MALDI-
f 2 2
+ + 1
HRMS m/z 910.3225 ([M+Na] , C H N O .Na , calcd 910.3211); H NMR (DMSO-d ) δ 11.12 (s,
55 45 5 7 6
1H, amide), 8.50 (s, 1H, adenine), 8.46 (s, 1H, adenine), 8.00–8.29 (m, 11H, benzoyl, Py), 7.64-7.69
(m, 1H, benzoyl), 7.55-7.60 (m, 2H, benzoyl), 7.35 (d, 2H, J = 9 Hz, DMTr), 7.14–7.27 (m, 7H,
DMTr), 6.81 (d, 4H, J = 8.4 Hz, DMTr), 6.27 (d, 1H, J = 5.7 Hz, H1’), 5.56 (d, 1H, ex, J = 5.7 Hz,
3’-OH), 5.50 (d, 1H, J = 11.7 Hz, CH Py), 5.28 (d, 1H, J = 11.7 Hz, CH Py), 4.97-5.00 (m, 1H, H2’),
4.61-4.65 (m, 1H, H3’), 4.20-4.24 (m, 1H, H4’), 3.70 (s, 6H, 2 × CH O), 3.26-3.28 (m, 2H, H5’). C
NMR (DMSO-d ) δ 165.5, 158.0, 151.8, 151.3, 150.3, 144.7, 135.5, 135.4, 133.4, 132.4, 131.1,
131.1, 130.7, 130.6, 130.1, 129.6, 129.6, 128.8, 128.7, 128.4, 128.4, 128.1, 128.1, 127.7, 127.6,
127.4, 127.3, 127.2, 127.1, 126.6, 126.1, 125.7, 125.2, 124.3, 123.9, 123.7, 123.3, 113.1, 86.3, 85.5,
84.2, 79.8, 70.1, 69.2, 63.5, 54.9.
NHBz
DMTrO
OH O Py
138X
’-O-(4,4’-dimethoxytrityl)-2’-O-(Pyrenyl-methyl)-N-benzoyl-cytidine (138X). Nucleoside
136X (2.0 g, 3.56 mmol), DMTrCl (2.07 g, 5.34 mmol), and DMAP (~20 mg) in anhydrous pyridine
(50 mL) were mixed, reacted, worked up, and purified as described above to afford nucleoside 138X
(2.0 g, 65%) as a pale yellow foam. R 0.78 (10% MeOH in CH Cl , v/v); MALDI-HRMS m/z
f 2 2
+ + 1
886.3087 ([M+Na] , C H N O .Na , calcd 886.3098); HNMR (DMSO-d ) δ 11.22 (s, 1H, amide),
54 45 3 8 6
847-8.49 (d, 1H, Py), 8.21-8.30 (m, 6H, H6 and Py), 8.16 (s, 2H, Py), 8.00-8.05 (m, 3H, benzoyl and
Py), 7.63-7.65 (m, 1H, benzoyl), 7.52-7.55 (m, 2H, benzoyl), 7.24-7.39 (m, 9H, DMTr), 7.04 (d, 1H,
H5), 6.86-6.90 (m, 4H, DMTr), 6.15 (d, 1H, H1’), 5.56 (bs, 2H, CH Py), 5.37 (d, 1H, ex, 3’-OH),
4.41-4.45 (m, 1H, H3’), 4.16-4.20 (m, 2H, H2’and H4’), 3.73 (bs, 6H, 2 x CH O), 3.38-3.41 (m, 1H,
H5’), 3.29-3.34 (m, 1H, H5’); C NMR (DMSO-d ) δ 167.1, 163.1, 158.1, 154.3, 144.3, 135.5,
135.1, 133.1, 132.6, 131.4, 130.6, 130.2, 129.7, 129.6, 128.7, 128.4, 127.8, 127.7, 127.5, 127.2,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
127.8, 126.1, 125.2, 124.5, 123.9, 123.8, 123.6, 113.2, 96.0, 88.7, 85.9, 82.3, 81.5, 69.9, 68.0, 61.8,
54.9.
138Y
5’-O-(4,4’-dimethoxytrityl)-2’-O-(Pyrenyl-methyl)-N-isobutyryl-guanosine (138Y).
Nucleoside 136Y (0.20 g, 0.35 mmol), DMTrCl (0.20 g, 0.53 mmol), and DMAP (~10 mg) in
anhydrous pyridine (5 mL) were mixed, reacted, worked up, and purified as described above to afford
nucleoside 138Y (0.27 g, 89%) as a pale yellow foam. R 0.8 (10% MeOH in CH Cl , v/v); MALDI-
f 2 2
+ + 1
HRMS m/z 892.3325 ([M+Na] , C H N O .Na , calcd 892.3317); HNMR (DMSO-d ) 11.8 (s, 1H,
52 47 5 8 6
amide), 11.3 (s, 1H, NH), 8.25-8.32 (m, 3H, Py), 7.99-8.15 (m, 7H, H7-guanine and Py), 7.32-7.34
(m, 2H, DMTr), 7.18-7.25 (m, 7H, DMTr), 6.79-6.82 (m, 4H, DMTr), 5.97 (d, 1H, H1’), 5.46-5.51
(m, 2H, 1 ex, 3’-OH, CH Py), 5.22-5.25 (d, 1H, CH Py), 4.71-4.73 (m, 1H, H2’), 4.45-4.48 (m, 1H,
H3’), 4.11-4.13 (m, 1H, H4’), 3.70-3.71(bd, 6H, 2 x CH O), 3.18-3.28 (m, 2H, H5’), 2.59-2.65 (m,
1H, isobutyryl), 1.04-1.06 (dd, 6H,isobutyryl); C NMR (DMSO-d ) δ 179.8, 158.0, 154.5, 148.5,
147.8, 144.6, 137.1, 135.4, 135.3, 131.12, 130.7, 130.6, 130.1, 129.6, 128.7, 127.7, 127.6, 127.3,
127.2, 127.1, 127.0, 126.6, 126.1, 125.2, 125.1, 124.4, 123.8, 123.7, 123.4, 120.1, 113.1, 85.6, 85.0,
84.2, 80.8, 70.3, 69.3, 63.9, 54.9, 34.6, 18.7, 18.6.
General Phosphitylation Protocol: [Description for ~2.3 mmol Scale]
The appropriate nucleoside (specific quantities of substrates and reagents given below) was co-
evaporated twice with anhydrous 1,2-dicholoroethane (20 mL) and redissolved in anhydrous CH Cl
(25 mL). To this was added N,N-diisopropylethylamine (DIPEA) and 2-cyanoethyl-N,N-
diisopropylchlorophosporamidite (PCl-reagent) and the reaction mixture was stirred at rt until TLC
indicated complete conversion (~3h), whereupon abs. EtOH (4 mL) and CH Cl (50 mL) were
sequentially added to the solution. The organic phase was washed with sat. aq. NaHCO (20 mL),
evaporated to near dryness, and the resulting residue purified by silica gel column chromatography
(40-70% EtOAc in petroleum ether, v/v) to afford the corresponding phosphoramidite (yields
specified below).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
76’W
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-2’-O-(pyrenyl-methyl)-5’-O-(4,4’-
dimethoxytrityl)-N-benzoyl-adenosine (76’W). Nucleoside 138W (0.25 g, 0.28 mmol), DIPEA (74
μL, 0.43 mmol) and PCl-reagent (95 μL, 0.43 mmol) in anhydrous CH Cl (6 mL) were mixed,
reacted, worked up and purified as described above to afford phosphoramidite 76’W (0.26 g, 82 %)
as a white foam. R : 0.75 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1110.4295 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calcd 1110.4289); P NMR (CDCl ) δ 150.7, 150.8.
64 62 7 8 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
140X
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-2’-O-(pyrenyl-methyl)-5’-O-(4,4’-
dimethoxytrityl)-N-benzoyl-cytidine (140X). Nucleoside 138X (2.0 g, 2.31 mmol), DIPEA (1.65
mL, 9.26 mmol) and PCl-reagent (1.03 mL, 4.63 mmol) in anhydrous CH Cl (25 mL) were mixed,
reacted, worked up and purified as described above to afford phosphoramidite 140X (2.3 g, 93 %) as
a white foam. R : 0.67 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1086.4183 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calcd 1086.4177); P NMR (CDCl ) δ 149.7, 150.1.
63 62 5 9 3
140Y
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-2’-O-(pyrenyl-methyl)-5’-O-(4,4’-
dimethoxytrityl)-N-isobutyryl-guanosine (140Y). Nucleoside 138Y (0.26 g, 0.29 mmol), DIPEA
(213 μL, 1.19 mmol) and PCl-reagent (133 μL, 0.59 mmol) in anhydrous CH Cl (7 mL) were mixed,
reacted, worked up and purified as described above to afford phosphoramidite 140Y (0.25 g, 78 %) as
a white foam. R : 0.7 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1092.4389 ([M+Na] ,
f 2 2
. + 31
C H N O P Na , Calcd 1092.4395); P NMR (CDCl ) δ 150.3, 150.4.
61 64 7 9 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
DMTrO
DMTrO DMTrO
Py-CHO, NaBH(OAc) O
O 3 O
1,2-DCE
OH N Py
OH NH O N
N2', O3'-hemiaminal ether
Scheme 15
Cyclic N2’,O3’-hemiaminal ether: The structure of the cyclic N2’,O3’-hemiaminal ether byproduct
(Scheme 15) is supported by the following H NMR observations (500 MHz, DMSO-d , results not
shown): a) appearance of the hemiaminal ether proton as a singlet at 6.07 ppm; b) absence of an
exchangeable signal corresponding to a 3’-OH group, c) absence of a signal corresponding to a CH -
group linking the O2’-position and the pyrene moiety, and, d) appearance of the H1’-signal as a
singlet at 6.13 ppm indicating formation of a restricted North type furanose conformation unlike what
is observed for 6Q (H1’ signal appearing as a doublet at 6.43 ppm, J = 8.2 Hz). Despite formation of
a new stereocenter, we only observed one set of signals, suggesting that only one of the diastereomers
of the cyclic N2’,O3’-hemiaminal ether byproduct is formed.
2’-Amino-2’-deoxy-2’-N-methyl-2’-N-(pyrenyl-methyl)-5’-O-(4,4’-dimethoxytrityl)uridine
(82Q): Nucleoside 80 (200 mg, 0.36 mmol) was co-evaporated with anhydrous 1,2-dichloroethane
(2 × 4 mL) and redissolved in anhydrous THF (5 mL). Pyrene‐1‐ylmethylchloride (205 mg, 0.37
mmol) and triethylamine (0.52 mL, 3.73 mmol) were added and the reaction mixture was heated at
reflux for two days, whereupon the solvent was evaporated off. The crude residue was taken up in a
mixture CHCl and sat. aq. NaHCO (50 mL, 3:2, v/v) and the layers were separated. The aqueous
phase was extracted with CHCl (2 × 20 mL) and the combined organic phase was evaporated to
dryness. The resulting residue was purified by silica gel column chromatography (0‐1.25 %
MeOH/CH Cl , v/v) to afford nucleoside 82Q as a yellow foam (129 mg, 46 %). R : 0.5 (60%, EtOAc
2 2 f
+ . + 1
in petroleum ether, v/v); MALDI-HRMS m/z 774.3156 ([M+H] C H N O H , Calc 774.3174); H
48 43 3 7
6478592_1 (GHMatters) P95976.NZ ESTHERJ
NMR (DMSO-d ) δ 11.41 (d, 1H, ex, J = 1.7 Hz, NH), 8.50 (d, 1H, J = 9.1 Hz, Py), 8.01‐8.29 (m,
8H, Py), 7.63 (d, 1H, J = 8.2 Hz, H6), 7.20‐7.38 (m, 9H, DMTr), 6.85‐6.89 (m, 4H, DMTr), 6.43 (d,
1H, J = 8.2 Hz, H1’), 5.56 (d, 1H, ex, J = 5.2 Hz, 3’‐OH), 5.43 (dd, 1H, J = 8.3 Hz, 1.7 Hz, H5),
4.41-4.50 (m, 3H, CH Py, H3’), 4.06-4.08 (m, 1H, H4’), 3.71 (s, 3H, CH O), 3.70 (s, 3H, CH O),
2 3 3
3.44-3.48 (dd, 1H, J = 8.3 Hz, 8.1 Hz, H2’), 3.28‐3.31 (m, 1H, H5’, overlap with H O), 3.16‐3.20
(dd, 1H, J = 10.6 Hz, 3.6 Hz, H5’), 2.33 (s, 3H, CH N); C NMR (DMSO-d ) δ 162.7, 158.08,
158.07, 150.6, 144.5, 140.2 (C6), 135.4, 135.1, 132.7, 130.7, 130.3, 130.2, 129.71 (DMTr), 129.67
(DMTr), 129.2, 128.0 (Py), 127.8 (DMTr), 127.6 (DMTr), 127.3 (Py), 126.9 (Py), 126.8 (Py), 126.7
(DMTr), 126.1 (Py), 125.01, 124.98 (Py), 124.4 (Py), 124.1, 124.0 (Py), 123.9, 113.20 (DMTr),
113.17 (DMTr), 102.1 (C5), 85.9, 85.1 (C4’), 83.4 (C1’), 71.3 (C3’), 67.8 (C2’), 64.1 (C5’), 57.4
(CH Py), 55.0 (CH O), 38.6 (CH N).
2 3 3
HO N
2’-Amino-2’-deoxy-2’-N-methyl-2’-N-(pyrenyl-carbonyl)-5’-O-(4,4’-dimethoxytrityl)uridine
(82S): Nucleoside 80 (150 mg, 0.27 mmol) was co‐evaporated with anhydrous 1,2-dichloroethane (2
× 5 mL), dissolved in anhydrous DMF (4.5 mL) and added to a pre-stirred (1h at rt) solution of 1-
pyrenecarboxylic acid (100 mg, 0.40 mmol), O-(7-azabenzotriazoleyl)-N,N,N′,N′-
tetramethyluronium hexafluorophosphate (HATU, 125 mg, 0.32 mmol) and DIPEA (0.12 mL, 0.70
mmol) in anhydrous DMF (4.5 mL). The reaction mixture was stirred for 17 h, whereupon it was
diluted with EtOAc (50 mL) and sequentially washed with sat. aq. NaHCO (20 mL) and H O (2 × 20
mL). The organic phase was evaporated to dryness and the resulting residue purified by silica gel
column chromatography (0‐2% MeOH in CH Cl , v/v) to afford nucleoside 82S as a white foam (164
mg, 78 %). R = 0.4 (5% MeOH in CH Cl , v/v). MALDI-HRMS m/z 788.2985 ([M+H] ,
f 2 2
C H N O ·H , calc. 788.2966); H NMR (CDCl ) δ 8.65 (br s, 1H, NH, ex), 7.97-8.28 (m, 9H, Py),
48 41 3 8 3
7.85 (d, 1H, J = 8.3 Hz, H6), 7.20-7.44 (m, 9H, DMTr), 6.80-6.90 (m, 4H, DMTr), 6.78 (d, 1H, J =
6.0 Hz, H1’), 5.42-5.48 (m, 1H, H5), 4.80-4.90 (m, 2H, H2’, H3’), 4.28-4.43 (m, 1H, H4’), 3.77 (s,
6H, CH O), 3.48-3.63 (m, 2H, H5’), 2.98 (s, 3H, NCH ), traces of a second rotamer are observed; C
NMR (CDCl ) δ 174.4, 162.9, 159.0, 150.5, 144.5, 140.0 (C6), 135.6, 135.5, 132.3, 131.4, 131.0,
130.44 (DMTr), 130.42 (DMTr), 129.4 (Py), 128.7 (Py), 128.5 (DMTr), 128.3 (DMTr), 127.4, 126.7
(Py), 126.1 (Py), 126.0 (Py), 125.0 (Py), 124.8, 124.7, 124.3 (Py), 113.6 (DMTr), 113.3 (DMTr),
6478592_1 (GHMatters) P95976.NZ ESTHERJ
103.2 (C5), 87.3, 86.6 (C4’), 85.3 (C1’), 71.8 (C3’/C2’), 65.8 (C2’/C3’), 63.0 (C5’), 55.5 (CH O),
38.5 (NCH ). A trace impurity of grease was observed at 29.9 ppm.
2’-Amino-2’-deoxy-2’-N-methyl-2’-N-(pyrenyl-methylcarbonyl)-5’-O-(4,4’-
dimethoxytrityl)uridine (82V): Nucleoside 80 (158 mg, 0.28 mmol) was co-evaporated with 1,2-
dichloroethane (2 × 5 mL) and subsequently dissolved in anhydrous CH Cl (8 mL). To this was
added 1‐ethyl‐3‐(3‐dimethylamino‐propyl) carbodiimide hydrochloride (EDCHCl, 73 mg, 0.38
mmol) and pyrene‐1‐ylacetic acid (108 mg, 0.41 mmol). The reaction mixture was stirred under argon
at rt for 3h whereupon the reaction mixture was diluted with CH Cl (30 mL) and sequentially
washed with sat. aq. NaHCO (20 mL) and H O (3 × 15 mL). The organic phase was evaporated to
dryness and the resulting residue was purified by silica gel column chromatography (0‐3 %
i‐PrOH/CH Cl , v/v) to afford a rotameric mixture (2:5 by HNMR) of nucleoside 82V (187 mg, 83
%) as a brown foam. R : 0.5 (5%, i-PrOH in CH Cl , v/v); MALDI-HRMS m/z 801.3078 ([M] ,
f 2 2
C H N O , calc. 801.3045); H NMR (DMSO-d ) δ 11.52 (d, 1H, J = 1.7 Hz, NH ), 11.46 (d,
49 43 3 8 6 (A)
0.4H, J = 1.7 Hz, NH ), 8.05‐8.31 (m, 11.2H, Py + Py ), 7.89 (d, 1H, J = 7.8 Hz, Py ), 7.82 (d,
(B) (A) (B) (A)
0.4H, J = 7.8 Hz, Py ), 7.67‐7.70 (m, 1.4H, H6 + H6 ), 7.13-7.43 (m, 12.6H, DMT ),
(B) (A) (B) (A+B)
6.78‐6.87 (m, 5.6H, DMT ), 6.42 (d, 1H, J = 8.0 Hz, H1’ ), 6.31 (d, 0.4H, J = 5.5 Hz, H1’ ),
(A+B) (A) (B)
.92 (d, 0.4H, ex, J = 4.9 Hz, 3’‐OH ), 5.76 (d, 1H, ex, J = 4.9 Hz, 3’‐OH ), 5.38 (dd, 1H, J = 8.2
(B) (A)
Hz, 1.7 Hz, H5 ), 5.33 (dd, 0.4H, J = 8.2 Hz, 1.7 Hz, H5 ), 5.11-5.17 (m, 1H, H2’ ), 4.80-4.86
(A) (B) (A)
(m, 0.4H, H2’ ), 4.58-4.63 (d, 1H, J = 16.5 Hz, CH Py ), 4.51-4.56 (d, 0.4H, J = 16.5 Hz,
(B) 2 (A)
CH Py ), 4.37-4.49 (m, 2.4H, 1 x CH Py , H3’ , H3’ ), 4.30-4.37 (d, 0.4H, J = 16.5 Hz,
2 (B) 2 (A) (A) (B)
CH Py ), 4.10‐4.16 (m, 1.4H, H4’ + H4’ ), 3.68 (s, 2.4H, CH O ), 3.64 (s, 3H, CH O ), 3.62
2 (B) (A) (B) 3 (B) 3 (A)
(s, 3H, CH O ), 3.35-3.37 (m, 0.4H, H5’ ), 3.32 (s, 3H, CH N ), 3.29-3.32 (m, 1H, H5’ ,
3 (A) (B) 3 (A) (A)
overlap with H O), 3.23-3.27 (dd, 0.4H, J = 10.6 Hz, 2.6 Hz, H5’ ), 3.15-3.19 (dd, 1H, J = 10.6 Hz,
2 (B)
2.6 Hz, H5’ ), 3.03 (s, 1.2H, CH N ); C NMR (DMSO-d ) δ 172.2, 172.1, 162.81, 162.78,
(A) 3 (B) 6
158.06, 158.03, 158.02, 150.5, 150.4, 144.6, 144.3, 140.2 (C6 ), 140.1 (C6 ), 135.4, 135.3, 135.2,
(B) (A)
135.0, 130.7, 130.62, 130.56, 130.3, 130.2, 129.71 (DMTr), 129.69 (DMTr), 129.1, 128.1 (Py-CH ),
127.9 (Py-CH ), 127.8 (DMTr), 127.73 (DMTr), 127.70 (DMTr), 127.3 (Py), 127.14 (Py), 127.10
(Py), 126.73 (Py), 126.66 (DMTr), 126.1 (Py), 125.1 (Py), 125.0 (Py), 124.9 (Py), 124.5 (Py), 124.1,
124.0, 123.85 (Py), 123.76 (Py), 113.2 (DMTr), 113.1 (DMTr), 102.2 (C5 ), 102.0 (C5 ), 85.9,
(A) (B)
6478592_1 (GHMatters) P95976.NZ ESTHERJ
85.7, 85.5 (C1’ ), 85.2 (C4’ ), 84.8 (C4’ ), 83.2 (C1’ ), 70.5 (C3’ ), 69.3 (C3’ ), 63.8
(B) (A) (B) (A) (A) (B)
(C5’ ), 63.7 (C5’ ), 62.1 (C2’ ), 59.0 (C2’ ), 54.92 (CH O ), 54.87 (CH O ), 54.85
(A) (B) (B) (A) 3 (B) 3 (B)
(CH O ), 54.82 (CH O ), 38.1 (CH Py ), 37.8 (CH -Py ), 34.2 (CH N ), 31.4 (CH N ).
3 (A) 3 (A) 2 (A) 2 (B) 3 (A) 3 (B)
84Q
2’-Amino-2’-deoxy-2’-N-methyl-2’-N-(pyrenyl-methyl)-3’-O-(N,N-diisopropylamino
cyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)uridine (84Q): Nucleoside 82Q (135 mg,
0.18 mmol) was co-evaporated with CH CN (2 × 4 mL) and redissolved in anhydrous CH CN (2.5
mL). To this was added DIPEA (153 μL, 0.87 mmol) and PCl-reagent (78 μL, 0.35 mmol). The
reaction mixture was stirred at rt for 4h, whereupon it was cooled on an ice bath and abs. EtOH (3
mL) was added. The solvent was evaporated off and the resulting residue purified by silica gel
column chromatography (0‐40 % EtOAc in petroleum ether, v/v; column built in 0.5 % Et N) to
afford nucleoside 84Q as a white foam (97 mg, 57 %). R : 0.3 (40% EtOAc in petroleum ether, v/v);
+ . + 31
MALDI-HRMS m/z 996.4046 ([M+Na] , C H N O P Na , Calc. 996.4077); P NMR (CDCl ) δ
57 60 5 8 3
151.0, 149.8.
(i-Pr) N
2’-Amino-2’-deoxy-2’-N-methyl-3’-O-(N,N-diisopropylaminocyanoethoxyphosphinyl)-2’-N-
(pyrenyl-carbonyl)-5’-O-(4,4’-dimethoxytrityl)uridine (84S): Nucleoside 82S (219 mg, 0.28
mmol) was co-evaporated with anhydrous 1,2-dicholoroethane (2 × 2 mL) and redissolved in
anhydrous CH Cl (2 mL). To this was added DIPEA (58 μL, 0.33 mmol) followed by dropwise
addition of PCl-reagent (74 μL, 0.33 mmol). After stirring at rt for 2h, CH Cl (10 mL) was added
6478592_1 (GHMatters) P95976.NZ ESTHERJ
and the reaction mixture stirred for additional 10 min whereupon the solvent was evaporated under
reduced pressure. The resulting residue was purified by silica gel column chromatography (1
column: 0‐40 % EtOAc in petroleum ether, v/v; 2 column: 0‐4 % MeOH in CH Cl , v/v) to afford a
rotameric mixture of phosphoramidite 84S as a white foam (138 mg, 49 %). R : 0.8 (5% MeOH in
+ . + 31
CH Cl , v/v); MALDI-HRMS m/z 1010.3865 ([M+Na] , C H N O P Na , Calc. 1010.3870); P
2 2 57 58 5 9
NMR (CDCl ) δ 151.8, 151.2, 150.8.
(i-Pr) N
2’-Amino-2’-deoxy-2’-N-methyl-3’-O-(N,N-diisopropylaminocyanoethoxyphosphinyl)-2’-N-
(pyrenyl-methylcarbonyl)-5’-O-(4,4’-dimethoxytrityl)uridine (84V): Nucleoside 82V (0.30 g,
0.37 mmol) was co-evaporated with anhydrous 1,2-dicholoroethane (2 × 3 mL) and redissolved in
anhydrous CH Cl (4 mL). To this was added DIPEA (0.32 mL, 1.84 mmol) followed by dropwise
addition of PCl-reagent (0.16 mL, 0.74 mmol). After stirring for 1.5 h at rt, CH Cl (10 mL) was
added and the reaction mixture stirred for additional 10 min whereupon the solvent was evaporated
under reduced pressure. The resulting residue was purified by silica gel column chromatography
(0‐60 % EtOAc in petroleum ether, v/v) to afford phosphoramidite 84V as a bright yellow foam (153
mg, 42 %). R : 0.4 (60% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z 1024.4037 ([M+Na] ,
. + 31
C H N O P Na , Calc. 1024.4026); P NMR (CDCl ) δ 150.6, 150.5.
58 60 5 9 3
General procedure for 222: Coevaporated starting material ( 1 mmol) in anhydrous 1,2-
dichloroethane (2 x 5 mL) and redissolved in anhydrous 1,2-dichloroethane (5 mL). To this was
added NaBH(OAc) (1.5-7 eq) and RCHO (1.5 eq). Allowed to stir at rt under an argon atmosphere
for 5-22 h whereupon the reaction mixture was diluted with CH Cl (50 mL) washed with sat. aq.
NaHCO (2 x 30 mL) and H O (30 mL). Back-extracted aqueous phase with CH Cl (2 x 20 mL),
3 2 2 2
dried over Na SO and evaporated to dryness. The crude was then purified via silica gel column
chromatography isolate products (43-95%).
General procedure for synthesis of 224: Coevaporated starting material (1 mmol) in anhydrous 1,2-
dichloroethane (2 x 10 mL) and redissolved in anhydrous 1,2-dichloroethane (10 mL). To this was
added NaBH(OAc) (10 eq) and CH O (1.5 eq) and was stirred at rt under argon atmosphere for 4-7 h
whereupon the reaction mixture was diluted with CH Cl (50 mL) washed with sat. aq. NaHCO (2 x
2 2 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
mL) and the aqueous back-extracted with CH Cl (2 x 20 mL), dried over Na SO and evaporated
2 2 2 4
to dryness. The crude was then purified via silica gel column chromatography isolate products (89-
99%).
General procedure for synthesis of 226: Coevaporated starting material (1 mmol) in anhydrous 1,2-
dichloroethane (10 mL) and redissolved in an. CH Cl (10 mL). To this was added DIPEA (4-5 eq)
followed by dropwise addition of “PCl” reagent (2-3 eq) and allowed to stir under argon atmosphere
at rt for 2-4 h whereupon the reaction mixture was quenched with cold EtOH (1 mL) and evaporated
to dryness. The crude was purified by silica gel column chromatography and precipitated in CH Cl
and petroleum ether to afford the desired phoshoramidite (62-90%).
Synthesis of 240. Naphthymethylthiol (616 mg, 3.53 mmol) and sodium hydride (42 mg, 1.77
mmol) were dissolved in an. DMA (3 mL) and stirred under argon atm for 10 min whereupon
O2,O2’-anhydrouridine 70 (200 mg, 0.88 mmol) was added. After stirring for 48 h, the reaction
mixture was diluted with EtOAc (30 mL), neutralized with acetic acid (1 drop) and washed with H O
(2 x 15 mL). The aqueous was back-extracted with CH Cl (3 x 15 mL) and the organic layers
evaporated to dryness. The crude product was purified by silica gel column chromatography (0-7%
MeOH/CH Cl , v/v) to give nucleoside 240 (311 mg, 88%). R = 0.3 (10% MeOH in CH Cl ); H
2 2 f 2 2
NMR (DMSO-d ) 11.33 (br s, 1H, NH, ex), 7.77-7.89 (m, 3H, Ar), 7.61-7.68 (m, 2H, Ar),7.39-7.54
(m, 3H, Ar, H6), 6.09 (d, 1H, J = 8.23 Hz, H1’), 5.65 (d, 1H, J = 5.21 Hz, ex, 3’-OH), 5.34 (d, 1H, J
= 7.96 Hz, H5), 5.05 (t, 1H, J = 5.21, ex, 5’-OH), 4.15-4.20 (m, 1H, H3’), 3.81-3.94 (m, 3H, SCH ,
H4’), 3.50-3.58 (m, 2H, H5’), 3.36 (dd, 1H, J = 8.5 Hz, 5.5 Hz, H2’).
Synthesis and purification of single-stranded probes modified with monomers 120W/X/Y/Z,
120Q/S/V, 120’W, 120’X, 140’X, 140’Y, 208W/X/Y/Z, 228Y/Z and 244: Synthesis of modified
probes was performed on a DNA synthesizer using 0.2 μmol scale succinyl linked LCAA-CPG (long
chain alkyl amine controlled pore glass) columns with a pore size of 500Å. Standard protocols for
incorporation of DNA phosphoramidites were used. A ~50-fold molar excess of modified
phosphoramidites in anhydrous acetonitrile (at 0.05 M) was typically used during hand-couplings
(performed to conserve material) except with 76Z (~70-fold molar excess in anhydrous CH Cl , at
0.07M). Moreover, extended oxidation (45s) and coupling times were typically used (0.01 M 4,5-
dicyanoimidazole as activator, 15 min for monomers 84V/76W/76Y, 35 min for monomer 76Z; 0.01
M 5-(bis-3,5-trifluoromethylphenyl)-1H-tetrazole [Activator 42], 15 min for monomers
84Q/84S/84V). Probes modified with monomers 120’W, 120’X, 140’X, 140’Y, 208W/X/Y/Z,
228Y/Z and 244, were made in an equivalent manner. Cleavage from solid support and removal of
protecting groups was typically accomplished upon treatment with 32% aq. ammonia (55 °C, 24 h).
Purification of all modified oligonucleotides was performed to minimum 80% purity using either of
6478592_1 (GHMatters) P95976.NZ ESTHERJ
two methods: a) overall synthesis yield >80%: cleavage of DMT using 80% aq. AcOH, followed by
precipitation from acetone (-18 °C for 12-16 h) and washing with acetone, or b) overall synthesis
yield <80%: purification of oligonucleotides by RP-HPLC as described below, followed by
detritylation and precipitation.
Purification of the crude oligonucleotides was performed on a HPLC system equipped with
an XTerra MS C18 pre-column (10 μm, 7.8 x 10 mm) and a XTerra MS C18 column (10μm, 7.8 x
150 mm). The identity of synthesized oligonucleotides was established through MALDI-MS/MS
analysis (Table 35-36) recorded in positive ions mode on a Quadrupole Time-Of-Flight Tandem
Mass Spectrometer equipped with a MALDI source using anthranilic acid as a matrix (Tables 39-51),
while purity (>80%) was verified by RP-HPLC running in analytical mode.
Table 39
MALDI-MS of Representative Single-Stranded Probes Modified with Monomers 120W/X/Y/Z
ONs Sequence Calc. m/z [M] Found m/z [M+H]
120W1 5’-G120W G ATA TGC 2880 2881
120W2 5’-GTG A120WA TGC 2880 2881
120W3 5’-GTG ATA 120WGC 2880 2881
120W4 3’-CAC 120WAT ACG 2809 2810
120W5 3’-CAC TA120W ACG 2809 2810
’-G120XG ATA TGC 2954 2955
120X1
’-GTG A120XA TGC 2954 2955
120X2
120X3 5’-GTG ATA 120XGC 2954 2955
120X4 3’-CAC 120XAT ACG 2883 2884
120X5 3’-CAC TA120XACG 2883 2884
120X6 3’-CAC 120XA120XACG 3085 3086
120X7 5’-G120XG A120XA 120XGC 3358 3359
’-G120YG ATA TGC 2968 2969
120Y1
120Y2 5’-GTG A120YA TGC 2968 2969
120Y3 5’-GTG ATA 120YGC 2968 2969
3’-CAC 120YAT ACG 2897 2898
120Y4
3’-CAC TA120Y ACG 2897 2898
120Y5
120Y6 3’-CAC 120YA120Y ACG 3113 3114
120Y7 5’-G120YG A120YA 120YGC 3400 3401
’-G120ZG ATA TGC 3066 3067
120Z1
120Z2 5’-GTG A120ZA TGC 3066 3067
120Z3 5’-GTG ATA 120ZGC 3066 3067
3’-CAC 120ZAT ACG 2995 2996
120Z4
120Z5 3’-CAC TA120Z ACG 2995 2996
3’-CAC 120ZA120Z ACG 3309 3310
120Z6
6478592_1 (GHMatters) P95976.NZ ESTHERJ
120Z7 5’-G120ZG A120ZA 120ZGC 3694 3695
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 40
MALDI-MS of Representative Single-Stranded Probes Modified with Monomers 120Q/S/V
ONs Sequence Calc. m/z [M] Found m/z [M+H]
’-G120QG ATA TGC 2982 2983
120Q1
120Q2 5’-GTG A120QA TGC 2982 2983
120Q4 3’-CAC 120QAT ACG 2911 2912
3’-CAC TA120Q ACG 2911 2912
120Q5
3’-CAC 120QA120Q ACG 3140 3141
120Q6
120S1 5’-G120SG ATA TGC 2996 2997
120S2 5’-GTG A120SA TGC 2996 2997
120S4 3’-CAC 120SAT ACG 2925 2926
3’-CAC TA120S ACG 2925 2926
120S5
120S6 3’-CAC 120SA120S ACG 3168 3169
120V1 5’-G120VG ATA TGC 3009 3010
’-GTG A120VA TGC 3009 3010
120V2
120V4 3’-CAC 120VAT ACG 2938 2939
120V5 3’-CAC TA120V ACG 2938 2939
120V6 3’-CAC 120VA120VACG 3195 3196
Table 41
MS-Data of Representative Single-Stranded Probes Modified with Monomer 120’W (= M).
ONs Sequence Calc. m/z Found m/z
120’W6 5’-GTG MTA TGC 2984 2984
’-GTG ATM TGC 2984 2984
120’W7
120’W8 3’-CAC TMT ACG 2913 2913
120’W9 3’-CAC TAT MCG 2913 2914
Table 42
MS-Data of Representative Single-Stranded Probes Modified with Monomer 208W/X/Y/Z
ONs expected mass observed mass
[M+H]
’-GTG A(208W)A TGC 3038 3039
3’-CAC TA(208W) ACG 2967 2968
’-GTG A(208X)A TGC 3046 3046
3’-CAC TA(208X) ACG 2975 2975
’-GTG A(208Y)A TGC 2982 2983
3’-CAC TA(208Y) ACG 2911 2912
’-GTG A(208Z)A TGC 3054 3056
3’-CAC TA(208Z) ACG 2983 2984
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 43
MS-Data of Representative Single-Stranded Probes Modified with Monomer 228Y/Z
Probe Calc [M] Found [M+1]
’ – GTG A(228Y)A TGC 3031.6 3032.4
3’ – CAC TA(228Y) ACG 2960.6 2961.4
’ – GTG A(228Z)A TGC 3079.6 3079.6
3’ – CAC TA(228Z) ACG 3008.6 3010.5
Table 44
MS-Data of Additional Single-Stranded Probes Modified with Monomer 120Y
Probes Expected Observed
mass [M+H]
’-GG(120Y) ATA TAT AGG C 4227 4228
3’-CCA (120Y)AT ATA TCC G 4107 4108
’-GGT A(120Y)A TAT AGG C 4227 4228
3’-CCA TA(120Y) ATA TCC G 4107 4108
’-GGT ATA (120Y)AT AGG C 4227 4228
3’-CCA TAT A(120Y)A TCC G 4107 4108
’-GGT ATA TA(120Y) AGG C 4227 4228
3’-CCA TAT ATA (120Y)CC G 4107 4108
’-GG(120Y) A(120Y)A TAT AGG C 4443 4444
3’-CCA (120Y)A(120Y) ATA TCC G 4323 4324
’-GG(120Y) ATA (120Y)AT AGG C 4443 4444
3’-CCA (120Y)AT A(120Y)A TCC G 4323 4324
’-GG(120Y) ATA TA(120Y) AGG C 4443 4444
3’-CCA (120Y)AT ATA (120Y)CC G 4323 4324
’-GGT A(120Y)A (120Y)AT AGG C 4443 4444
3’-CCA TA(120Y) A(120Y)A TCC G 4323 4324
’-GGT ATA (120Y)A(120Y) AGG C 4443 4444
3’-CCA TAT A(120Y)A (120Y)CC G 4323 4324
’-GG(120Y) A(120Y)A (120Y)A(120Y) AGG C 4875 4876
3’-CCA (120Y)A(120Y) A(120Y)A (120Y)CC G 4755 4757
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 45
MS-Data of Additional Single-Stranded Probes Modified with Monomer 120’W
Probe Expected Observed
mass Mass [M+H]
3’-CC(120’W) TAT ATA TCC G 4121 4122
3’-CCA T(120’W)T ATA TCC G 4121 4122
3’-CCA TAT (120’W)TA TCC G 4121 4122
3’-CCA TAT AT(120’W) TCC G 4121 4123
3’-CC(120’W) T(120’W)T ATA TCC G 4351 4352
3’-CC(120’W) TAT (120’W)TA TCC G 4351 4352
3’-CC(120’W) TAT AT(120’W) TCC G 4351 4353
3’-CCA T(120’W)T (120’W)TA TCC G 4351 4353
3’-CCA TAT (120’W)T(120’W) TCC G 4351 4352
3’-CC(120’W) T(120’W)T (120’W)T(120’W) TCC G 4812 4813
Table 46
MS-Data of Even Further Additional Single-Stranded Probes Modified with Monomer 120Y
Probe Expected Observed
mass Mass
[M+H]
’-G-G(120Y)A-TAT-AAG-CAG-CAC-A 5440 5441
3’-C-CA(120Y)-ATA-TTC-GTC-GTG-T 5364 5365
’-G-G(120Y)A-(120Y)AT-AAG-CAG-CAC-A 5657 5658
5580 5582
3’-C-CA(120Y)-A(120Y)A-TTC-GTC-GTG-T
’-AGG-AAG-G(120Y)A-(120Y)AT-AAG-CA 5721 5722
5515 5516
3’-TCC-TTC-CA(120Y)-A(120Y)A-TTC-GT
’-G-G(120Y)A-(120Y)AT-AAG-CAG-C 4741 4742
4643 4684
3’-C-CA(120Y)-A(120Y)A-TTC-GTC-G
’-AC(120Y)-A(120Y)A-GAA-TAC-TCA-AG 5616 5617
5620 5621
3’-TGA-(120Y)A(120Y)-CTT-ATG-AGT-TC
’-AC(120Y)-A(120Y)A-GAA-TAC-TC 4660 4661
4722 4723
3’-TGA-(120Y)A(120Y)-CTT-ATG-AG
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 47
MS-Data of Even Further Single-Stranded Probes Modified with Monomer 120Q
Probe Calc [M] Found [M+H]
' – GG(120Q) ATA TAT AGG C 4240.8 4242.5
3' - CCA (120Q)AT ATA TCC G 4120.8 4122.6
' - GG(120Q) A(120Q)A TAT AGG C 4469.9 4471.5
3' - CCA (120Q)A(120Q) ATA TCC G 4349.9 4351.6
' - GGT A(120Q)A (120Q)AT AGG C 4469.9 4471.9
3' - CCA TA(120Q) A(120Q)A TCC G 4349.9 4351.4
' - GG(120Q) A(120Q)A (120Q)A(120Q) AGG C 4928.1 4929.7
3' - CCA (120Q)A(120Q) A(120Q)A (120Q)CC G 4808.1 4810.5
Table 48
MS-Data of Yet Even Further Additional Single-Stranded Probes Modified with Monomer
120Q
Probe Expected Observed
mass Mass
[M+H]
’-G-G(120Q)A-TAT-AAG-CAG-CAC-A 5454 5455
5377 5378
3’-C-CA(120Q)-ATA-TTC-GTC-GTG-T
’-G-G(120Q)A-(120Q)AT-AAG-CAG-CAC-A 5683 5684
5607 5608
3’-C-CA(120Q)-A(120Q)A-TTC-GTC-GTG-T
5747 5749
’-AGG-AAG-G(120Q)A-(120Q)AT-AAG-CA
5542 5543
3’-TCC-TTC-CA(120Q)-A(120Q)A-TTC-GT
’-AC(120Q)-A(120Q)A-GAA-TAC-TCA-AG 5642 5646
5646 5648
3’-TGA-(120Q)A(120Q)-CTT-ATG-AGT-TC
Table 49
MS-Data of Even Further Additional Single-Stranded Probes Modified with
Monomer 120Y, 120’W and 140’X
Probe Expected Observed
mass Mass
[M+H]
’-CC(140’X) ACG T(120Y)A GCA GTT 4971 4973
5046 5047
3’-GGG (120Y)GC AA(120Y) CGT CAA
4971 4972
’-CCC ACG T(120Y)A G(140’X)A GTT
5046 5047
3’-GGG TGC AA(120Y) CG(120Y) CAA
’-AGA CAA AA(140’X) AC(140’X) AGT 5020 5021
5009 5010
3’-TCT GTT TTG (120Y)GG (120Y)CA
6478592_1 (GHMatters) P95976.NZ ESTHERJ
’-AGA (140’X)AA AA(140’X) ACC AGT 5020 5021
5009 5011
3’-TCT G(120Y)T TTG (120Y)GG TCA
’-CTA (140’X)AT (120Y)GT CTC GCC 4922 4923
5109 5110
3’-GAT G(120Y)A A(140’X)A GAG CGG
’-CTA C(120’W)T (120Y)GT CTC GCC 4922 4923
5123 5124
3’-GAT GT(120’W) A(140’X)A GAG CGG
4963 4965
’-CGT (140’X)AT CG(120Y) GCT CGC
5070 5071
3’-GCA G(120Y)A GCA (140’X)GA GCG
Table 50
MS-Data of Yet Even Further Additional Single-Stranded Probes Modified with Monomers
120Y, 120’W, 140’X and 140’Y
Probes Expected Observed
mass Mass
[M+H]
’-CGG ACC ACG (120Y)G(120Y) GTG 5038 5040
4995 4996
3’-GCC TGG TGC A(140'X)A (140'X)AC
’-CGG AC(140'X) ACG TG(120Y) GTG 5052 5053
4981 4982
3’-GCC TGG (120Y)GC ACA (140'X)AC
’-GT(140'X) AG(120Y) GGG CGT TGC 5083 5084
4950 4951
3’-CAG (120Y)CA (140'X)CC GCA ACG
’-GT(140'X) AG(120Y) GGG CG(120Y) TGC 5299 5299
5181 5182
3’-CAG (120Y)CA (140'X)CC GCA (120’W)CG
’-CCT C(120Y)A (120Y)AA AAG CGG 4990 4991
5012 5013
3’-GGA GA(120Y) A(120Y)T TTC GCC
’-CC(120Y) C(120Y)A (120Y)AA AAG CGG 5206 5208
5242 5244
3’-GGA (140’Y)A(120Y) A(120Y)T TTC GCC
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 51
MS-Data of Yet Even Further More Additional Single-Stranded Probes
Modified with Monomer 120Y, 120’W, 140’X and 140’Y
’-Cy3 labeled probes Expected Observed
mass mass
[M+H]
’-AGC CC(120Y) G(120Y)G CCC TG 5522 5523
3’-TCG GGA (140’X)A(140’X) GGG AC 5495 5496
’-CC(120Y) G(120Y)G CCC TG 5390 5391
3’-GGA (140’X)A(140’X) GGG AC 6075 6076
’-CC(120Y) GTG CC(140’X) TG 5713 5714
3’-GGA (140’X)AC GGG (120’W)C 5922 5923
’-(120’W)GC CC(120Y) GTG CC(140’X) TG 5508 5509
3’-T(140’X)G GGA (140’X)AC GGG (120’W)C 6520 5621
6697 6696
’-C(120Y)G (120’W)GC CC(120Y) G(120Y)G CCC (120Y)G-3’
6940 6941
3’-GA(140’Y) T(140’X)G GGA (140’X)A(140’X) GGG A(140’X)-5’
’-(120’W)GC CC(120Y) G(120Y)G CCC (120Y)G-3’ 5599 5600
3’-T(140’X)G GGA (140’X)A(140’X) GGG A(140’X)-5’ 5739 5740
Thermal Denaturation Studies involving probes modified with 120W/X/Y/Z, 120Q/S/V, 120’W,
120’X, 140’X, 140’Y, 208W/X/Y/Z, 228Y/Z and 244: Concentrations of oligonucleotides were
estimated using the following extinction coefficients for DNA (OD/μmol): G (12.01), A (15.20), T
(8.40), C (7.05); for RNA (OD/μmol): G (13.70), A (15.40), U (10.00), C (9.00); for fluorophores
(OD/μmol): naphthalene (3.75), pyrene (22.4), and coronene (36.0). Each strand was thoroughly
mixed and denatured by heating to 70-85 °C followed by cooling to the starting temperature of the
experiment. Quartz optical cells with a path length of 1.0 cm were used. Thermal denaturation
temperatures (T values [°C]) of duplexes (1.0 µM final concentration of each strand) were measured
on a UV/VIS spectrophotometer equipped with a 12-cell Peltier temperature controller and
determined as the maximum of the first derivative of the thermal denaturation curve (A vs. T)
recorded in medium salt buffer (T buffer: 100 mM NaCl, 0.1 mM EDTA, and pH 7.0 adjusted with
mM Na HPO and 5 mM Na HPO ). The temperature of the denaturation experiments ranged
2 4 2 4
from at least 15 °C below T to 20 °C above T (although not below 1°C). A temperature ramp of
0.5 °C/min was used in all experiments. Reported T -values are averages of two experiments within
± 1.0 °C.
In particular disclosed embodiments, single-stranded probes modified with monomers
120W/X/Y/Z or 120 Q/S/V, display varying trends in thermal affinity toward complementary RNA
6478592_1 (GHMatters) P95976.NZ ESTHERJ
(ΔT /mod = -12.0 °C to +10.5 °C, Table 52 Substantial DNA-selectivity, defined as ΔΔT (DNA-
RNA) = ΔT (vs DNA) - ΔT (vs RNA) > 0 °C, is observed for these probes (Table 53). The DNA-
selectivity of probes modified with unlocked monomers 120Y and 120Z is especially pronounced and
comparable to that of probes modified with locked monomer 126W (Table 53). This suggests that
interesting DNA-targeting applications are possible. DNA-selective hybridization is typically
observed for oligonucleotides modified with intercalating moieties, since intercalation favors the less
compressed helix geometry of DNA:DNA duplexes. In contrast, the majority of probes modified with
monomers 120X or 120W display much lower DNA selectivity, indicating that intercalative binding
modes are less prominent than in probes modified with monomer 120Y and 120Z (Table 53).
Table 52
T -Values of Duplexes between Probe Modified with Monomers 120W/X/Y/Z/Q/S/V Or 126W
and Complementary RNA Targets
T [ΔT /mod] (°C)
ON Duplex B = T 120W 120X 120Y 120Z 120Q 120S 120V 126W
′-GBG ATA TGC 2 26.5 21.0 22.5 24.5 21.5 22.0 14.0 20.0 27.0
R2 3′-CAC UAU ACG [-5.5] [-4.0] [−2.0] [−5.0] [-4.5] [-12.5] [-6.5] [+0.5]
B2 5′-GTG ABA TGC 26.5 16.0 22.5 30.5 37.0 29.0 18.0 28.0 31.5
R2 3′-CAC UAU ACG [-10.5] [-4.0] [+4.0] [+10.5] [+2.5] [-8.5] [+1.5] [+5.0]
B3 5′-GTG ATA BGC 26.5 18.0 14.5 26.5 30.5 28.0
ND ND ND
R2 3′-CAC UAU ACG [-8.5] [-12.0] [±0.0] [+4.0] [+1.5]
R1 5′-GUG AUA UGC 24.5 16.5 16.5 20.0 22.5 17.5 9.0 16.0 23.5
B4 3′-CAC BAT ACG [-8.0] [-8.0] [−4.5] [−2.0] [−7.0] [-15.5] [-8.5] [−1.0]
R1 5′-GUG AUA UGC 24.5 19.5 21.5 27.0 30.5 27.0 16.0 26.5 32.0
B5 3′-CAC TAB ACG [-5.0] [-3.0] [+2.5] [+6.0] [+2.5] [-8.5] [+2.0] [+7.5]
R1 5′-GUG AUA UGC 24.5 14.5 24.0 11.0 20.0 19.0
ND ND ND
B6 3′-CAC BAB ACG [-5.0] [-0.3] [-6.8] [-2.3] [-2.8]
′-GBG ABA BGC 26.5 15.5 27.5
ND ND ND ND ND ND
D2 3′-CAC UAU ACG [-3.7] [+0.3]
Table 53
DNA-Selectivity of Probes Modified With Monomers 120W/X/Y/Z/Q/S/V or 126W
ΔΔT (DNA-RNA) [°C]
ON Duplex B = 120W 120X 120Y 120Z 120Q 120S 120V 126W
B1 5′-GBG ATA TGC
-2.5 +1.0 +7.0 +9.5 +9.5 +6.5 + 6.0 +6.5
3′-CAC TAT ACG
′-GTG ABA TGC
+5.5 +8.0 +8.5 +9.5 +11.5 +11.5 +4.5 +9.0
D2 3′-CAC TAT ACG
B3 5′-GTG ATA BGC
+3.5 +8.5 +8.0 +7.0 ND ND ND +9.0
3′-CAC TAT ACG
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D1 5′-GTG ATA TGC
+5.0 +4.5 +8.0 +8.5 +8.5 +9.5 +9.5 +7.5
B4 3′-CAC BAT ACG
′-GTG ATA TGC
0 +4.0 +9.0 +10.0 +10.5 +12.5 +4.5 +8.0
B5 3′-CAC TAB ACG
D1 5′-GTG ATA TGC
ND +6.0 +14.5 +31.0 +18.5 ND +13.0 ND
3′-CAC BAB ACG
B7 5′-GBG ABA BGC
ND +7.0 +20.5 ND ND ND ND ND
D2 3′-CAC TAT ACG
DNA selectivity defined as ΔΔT (DNA-RNA) = ΔT (vs DNA) - ΔT (vs RNA).
m m m
The Watson–Crick specificity the disclosed probes may also be determined. In particular
disclosed embodiments, modified oligonucleotides may be evaluated using nucleic acid targets with
mismatched nucleotides opposite of the monomer-incorporation site. Certain disclosed embodiments
concern using single-stranded probes and their ability to associate with DNA (Table 54) or RNA
targets (Table 55) comprising mismatched nucleotides opposite of the incorporation site.
The B2-series probes generally displayed reduced DNA target specificity relative to
reference strand D1. For example, 120Y2 and 120Z2 discriminate dT-mismatches very poorly
relative to D1 (compare ΔT -values for D1/120Y2/120Z2 against targets with a dT-mismatch, Table
X) while dC- and dG-mismatches almost are as efficiently discriminated as with D1. Particular
disclosed embodiments of oligonucleotides comprising locked monomers had a similar specificity
profile but discriminated the dT-mismatch even more poorly. Interestingly, 120Q2 displays
substantially better discrimination of dC- and dT-mismatches than 120Y2, 120Z2 and 126W2
rendering it as the most specific of the high-affinity DNA-targeting modifications. In particular
disclosed embodiments, even minor changes in linker chemistry and length have marked influence on
target specificity. For example, 120S2 displays similar DNA target specificity as
120Y2/120Z2/126W2, while 120V2 discriminates DNA mismatches more poorly. Similarly, 120W2
exhibits very poor target specificity, while 120X2 displays much higher target specificity than
120W2 or 120Y2 (Table 54).
Table 54
Discrimination of Mismatched DNA Targets by the B2-series of Single-stranded Probes.
DNA: 3′-CAC TBT ACG
T [°C] ΔT [°C]
B = A C G T
ON Sequence
D1 5′-GTG ATA TGC 29.5 −16.5 −9.5 −17.0
120W2 5′-GTG A(120W)A TGC 24.5 −11.0 +2.0 −3.5
′-GTG A(120X)A TGC <−23.5 −7.0 −13.0
120X2 33.5
120Y2 5′-GTG A(120Y)A TGC 42.0 −13.0 −5.0 −6.5
′-GTG A(120Z)A TGC −13.5 −6.0 −7.0
120Z2 49.0
120Q2 5′-GTG A(120Q)A TGC 43.5 −22.0 −3.5 −12.0
′-GTG A(120S)A TGC −11.5 −9.0 −8.5
120S2 32.5
6478592_1 (GHMatters) P95976.NZ ESTHERJ
120V2 5′-GTG A(120V)A TGC 35.5 −11.5 +1.5 −3.5
126W2 5′-GTG A(126W)A TGC 43.5 −12.5 −5.5 −3.5
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully matched
duplexes are shown in bold. ΔT = change in T relative to fully matched DNA:DNA duplex.
Table 55
Discrimination of Mismatched RNA Targets by the B2-Series of Single-Stranded Probes
RNA: 3′-CAC UBU ACG
T [°C] ΔT [°C]
B = A C G U
ON Probe
′-GTG ATA TGC <−16.5 −4.5 <−16.5
D1 26.5
120W2 5′-GTG A(120W)A TGC 16.0 −1.0 +0.5 +6.0
′-GTG A(120X)A TGC −11.5 −5.0 <−12.5
120X2 22.5
120Y2 5′-GTG A(120Y)A TGC 31.0 −17.5 −3.5 -9.5
′-GTG A(120Z)A TGC −12.0 -9.0 -13.0
120Z2 37.0
120Q2 5′-GTG A(120Q)A TGC 29.0 −16.5 −0.5 −13.0
′-GTG A(120S)A TGC <−8.5 −6.0 −6.0
120S2 18.0
120V2 5′-GTG A(120V)A TGC 28.0 −16.5 −1.0 −7.5
′-GTG A(126W)A TGC −12.0 −1.0 -4.5
126W2 31.5
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully matched
duplexes are shown in bold. ΔT = change in T relative to fully matched DNA:RNA duplex.
In other disclosed embodiments, the specificity of oligonucleotides with two modifications
positioned as next-nearest neighbors (B6-series) were evaluated against nucleic acid (e.g. DNA)
targets with a single central mismatched nucleotide opposite of the central 2’-deoxyadenosine residue
(Table 56). Interestingly, this probe design generally resulted in improved mismatch discrimination
relative to reference strand D2; A- and G-mismatches are particularly efficiently discriminated.
Table 56
Discrimination of Mismatched DNA Targets by B6-Series and Reference Strands
DNA : 5’-GTG ABA ACG
T [°C] ΔT [°C]
ON Sequence B = T A C G
3’-CAC TAT ACG -17.0 -15.5 -9.0
D2 29.5
120X6 3’-CAC (120X)A(120X) ACG 25.5 <-15.5 -14.0 -14.5
120Y6 3’-CAC (120Y)A(120Y) ACG 43.5 -24.0 -17.0 -14.0
120Z6 3’-CAC (120Z)A(120Z) ACG 47.0 -19.5 -13.0 -11.0
120Q6 3’-CAC (120Q)A(120Q) ACG 43.5 -21.5 -10.5 -13.5
120V6 3’-CAC (120V)A(120V) ACG 37.0 -14.5 -13.5 -11.0
6478592_1 (GHMatters) P95976.NZ ESTHERJ
For conditions of thermal denaturation experiments, see Table 33 above. T -values of fully matched
duplexes are shown in bold. ΔT = change in T relative to fully matched DNA:DNA duplex.
In particular disclosed embodiments, the fluorescence characteristics of the disclosed probe can be
examined, wherein an observed optical signal provides information regarding the probe and/or target.
In particular disclosed embodiments, absorption and steady state fluorescence emission spectra of a
probe comprising one or more of the disclosed monomers, such as monomers
120Q/120S/120V/120X/120Y, in the presence or absence of complementary or mismatched nucleic
acid targets (e.g. DNA/RNA) were recorded to gain additional insight into the binding modes of the
intercalator (e.g. pyrene) moieties and/or presence of a matched or mismatched target. A person of
ordinary skill in the art will recognize that intercalation of pyrene moieties is known to induce
bathochromic shifts of pyrene absorption peaks. Particular embodiments of the disclosed probe, such
as those modified with monomers 120Q/120S/120Y, display significant bathochromic shifts upon
hybridization with nucleic acid (e.g. DNA) targets (average Δλ = 2.8-5.0 nm vs 2.5 nm,
respectively, Table 58 and FIG 8. Hybridization with complementary RNA leads to smaller
bathochromic shifts (average Δλ = 2.0-4.5 nm, Table 58). Probes 120X1-120X5 display distinctly
smaller hybridization-induced bathochromic shifts than the other pyrene-functionalized
oligonucleotides (average Δλ ~0.6 nm, Table 57 and FIG 7), which substantiates the trends from
thermal denaturation studies indicating that intercalation of the pyrene moiety is a less important
binding mode for monomer 120X.
Table 57
Absorption Maxima in the 320-360 Nm Region for Probes Modified with Monomers 120X-120Z
Or 126W in the Presence or Absence of Complementary DNA/RNA Targets
λ [Δλ ] (nm)
max max
B= 120X 120Y 120Z 126W
ON Sequence ss +DNA +RNA ss +DNA +RNA ss +DNA +RNA ss +DNA +RNA
B1 5’-GBG ATA TGC 349 352[+3] 351[+2] 350 353[+3] 352[+2] 347 348[+1] 348[+1] 348 350[+2] 349[+1]
B2 5’-GTG ABA TGC 353 352[-1] 352[±0] 348 353[+5] 352[+4] 347 348[+1] 348[+1] 347 351[+4] 349[+2]
B3 5’-GTG ATA BGC 352 352[±0] 352[±0] 350 353[+3] 352[+2] 348 348[±0] 348[±0] 348 351[+3] 350[+2]
B4 3’-CAC BAT ACG 350 352[+2] 352[+2] 350 352[+2] 352[+2] 347 348[+1] 348[+1] 348 350[+2] 348[±0]
B5 3’-CAC TAB ACG 353 352[-1] 352[-1] 349 353[+4] 352[+3] 347 348[+1] 347[±0] 348 350[+2] 349[+1]
Measurements were performed at room temperature (monomer 126W), 10 °C (monomer 120Z) and
7 °C (monomer 120X and 120Y) using a spectrophotometer and quartz optical cells with a 1.0 cm
path length. Buffer conditions are as for thermal denaturation experiments. Values in italics are for
duplexes with low thermostability (partial duplex dissociation at experimental temperature possible).
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 58
Absorption Maxima in the 320-360 Nm Region for Probes Modified with Monomers
120Q/120S/120V in ihe Presence or Absence of Complementary DNA/RNA Targets
λ [Δλ ] (nm)
max max
B= 120Q 120S 120V 126W
ON Sequence ss +DNA +RNA ss +DNA +RNA ss +DNA +RNA ss +DNA +RNA
B1 5’-GBG ATA TGC 349 353[+4] 351[+2] 347 351[+4] 348[+1] 348 352[+4] 352[+4] 348 350[+2] 349[+1]
B2 5’-GTG ABA TGC 348 353[+5] 351[+3] 346 349[+3] 349[+3] 347 352[+5] 353[+6] 347 351[+4] 349[+2]
B4 3’-CAC BAT ACG 349 354[+5] 349[±0] 348 350[+2] 348[±0] 349 353[+4] 352[+3] 348 350[+2] 348[±0]
3’-CAC TAB ACG 348 354[+6] 352[+4] 346 348[+2] 350[+4] 347 352[+5] 352[+5] 348 350[+2] 349[+1]
Measurements were performed at 5 °C.
Steady-state fluorescence emission spectra: Steady-state fluorescence emission spectra of
oligonucleotides modified with pyrene-functionalized monomers 120Q/120S/120V/120X/120Y and
the corresponding duplexes with complementary DNA/RNA targets were recorded in non-
deoxygenated thermal denaturation buffer (each strand 1.0 μM) and obtained as an average of five
scans using an excitation wavelength of λ = 350 nm, excitation slit 5.0 nm, emission slit 2.5 nm and
a scan speed of 600 nm/min (FIGS 9-17). Experiments were determined at 5 ºC to ascertain maximal
hybridization of probes to DNA/RNA targets. Solutions were heated to 80 ºC followed by cooling to
ºC over 10 min.
The steady-state fluorescence emission spectra (λ = 350 nm; λ = 360-600 nm; T = 5 °C)
ex em
display the two expected vibronic bands I and III at λ = 382 ± 3 nm and 402 ± 3 nm, respectively, as
well as a small shoulder at ~420 nm (FIGS. 9-17). Hybridization of oligonucleotides modified with
monomers 120Q/120S/120V/120X/120Y with complementary targets, and DNA in particular, results
in reduced fluorescence emission intensity (FIGS. 9-17). This trend is indicative of pyrene
intercalation as nucleobase moieties are known to quench pyrene fluorescence via photoinduced
electron transfer (PET) with guanine and cytosine moieties being the strongest quenchers. In
agreement with this, duplexes involving the 120Y/120Q/120V-series (intercalation, important
binding mode) display far lower emission intensity than those involving the 120X-series
(intercalation, less important binding mode).
On balance, the data from the thermal denaturation and optical spectroscopy studies (DNA-
selectivity; mismatch discrimination; UV-vis; fluorescence) suggest that intercalation of the attached
hydrocarbon is a possible binding mode for all studied monomers. Intercalation is least important for
6478592_1 (GHMatters) P95976.NZ ESTHERJ
monomers 120W and 120X, while being a dominant binding mode for monomers 120Y, 120Z and
120Q.
Additional working examples of embodiments involving probes modified with monomers
228Y/Z.
Particular embodiments involve probes modified with monomers 228Y and 228Z. With reference to
Table 59 below, it is observed that single-stranded probes modified with monomers 228Y and 228Z
display significantly increased thermal affinity toward nucleic acid targets, more commonly single-
stranded DNA. This property will facilitate targeting of double-stranded nucleic acid targets, more
commonly dsDNA, via the approach shown in FIG 1.
Table 59
Additional Examples of Thermal Denaturation Temperatures of DNA Duplexes
Modified with Monomers 228Y or 228Z
T (ΔT /mod)
Sequence B = 228Y 228Z
’ – GTG ABA TGC 48.0 50.5
3’ – CAC TAT ACG (+18.5) (+21.0)
’ – GTG ATA TGC 51.5 49.5
3’ – CAC TAB ACG (+22.0) (+20.0)
’ – GTG ATA TGC 41.0 37.0
3’ – CAC BAT ACG (+11.5) (+7.5)
’ – GBG ATA TGC 41.0 37.0
3’ – CAC TAT ACG (+11.5) (+7.5)
’ – GTG ATA TBC 45.5 43.5
3’ – CAC TAT ACG (+16.0) (+14.0)
’ – GTG ATA TGC 54.0 54.0
3’ – CAC BAB ACG (+12.3) (+12.3)
Particular embodiments involve double-stranded probes with certain interstrand zipper arrangements
of monomers 228Y and/or 228Z. With reference to Table 60 below, it is observed that double-
stranded probes +1 and +2 interstrand zipper arrangements display relatively low thermostability,
which coupled with the high DNA affinity that each probe strands displays (Table 59), results in
pronounced potential for targeting of double-stranded nucleic acid targets, more commonly dsDNA
via the method outlined in FIG 1.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 60
Additional Examples of Double-Stranded Probes with Various
Interstrand Zipper Arrangements of Monomer 228Y or 228Z
T (ΔT /mod)
Sequence B = 228Y 228Z
’ – GTG ABA TGC 32.5 41.0
3’ – CAC TAB ACG (+1.5) (+5.8)
’ – GTG ABA TGC 29.5 62.5
3’ – CAC BAT ACG (±0) (+16.5)
’ – GBG ATA TGC 34.5 43.5
3’ – CAC BAT ACG (+2.5) (+6.8)
’ – GBG ATA TGC 54.0 61.5
3’ – CAC TAB ACG (+12.3) (+16.0)
General click reaction protocol for preparation of 104V-104Z (description for ~6 mmol scale):
’-O-Dimethoxytrityl-2'-azido-2’-deoxyuridine 102 and the appropriate alkyne 92-100 were added to
a mixture of THF/t-BuOH/H O (3:1:1, v/v/v) along with sodium ascorbate and CuSO ⋅5H O (reagent
2 4 2
quantities, and solvent volumes are specified below). The reaction mixture was stirred under a
nitrogen atmosphere until analytical TLC indicated full conversion (reaction times and temperatures
specified below) whereupon it was diluted with EtOAc (10 mL). The organic phase was successively
washed with sat. aq. NaHCO (20 mL) and brine (20 mL), dried over anhydrous Na SO and
3 2 4
evaporated to dryness. The resulting crude was purified by silica column chromatography (eluent
specified below) to afford the corresponding nucleoside 2 (yield specified below).
1-(Pyrenyl)-propynol (90): Trimethylsilylacetylene (1.0 mL, 7.00 mmol) was added to
MeMgBr in THF (1M, 4.0 mL, 4.00 mmol) under an argon atmosphere and stirred at rt for 1h. At this
point, pyrenecarboxaldehyde (0.70 g, 3.00 mmol) was added and the reaction mixture was stirred
at rt for another 2h. Sat. aq. NH Cl (~1 mL) was added and the mixture was extracted with EtOAc (2
x 20 mL). The combined organic phase was dried over Na SO and evaporated to dryness. The
resulting crude [assumed to be 1-(pyrenyl)trimethylsilyl-propynol] was dissolved in
CH Cl and MeOH (10 mL, 1:1, v/v) and stirred with K CO (0.50 g, 3.62 mmol) at rt for 2h. The
2 2 2 3
reaction mixture was then diluted with CH Cl (10 mL) and successively washed with brine (20 mL)
and water (20 mL). The organic phase was dried over Na SO and evaporated to dryness. The
resulting crude was purified by silica gel column chromatography (0-50% EtOAc in petroleum ether,
v/v) to afford 90 (0.47 g, 58%) as a white solid material. R = 0.3 (25% EtOAc in petroleum ether,
+ + 1
v/v); ESI-HRMS m/z 279.0783 ([M+Na] , C H O⋅Na , calc 279.0780); H NMR (DMSO-d ) δ 8.59
19 12 6
(d, 1H, J = 10.0 Hz, Py), 8.35-8.29 (m, 4H, Py), 8.26 (d, 1H, J = 10.0 Hz, Py), 8.20-8.16 (m, 2H, Py),
6478592_1 (GHMatters) P95976.NZ ESTHERJ
8.09 (t, 1H, J = 7.5 Hz, Py), 6.41-6.39 (d, 1H, ex, J = 5.0 Hz, OH), 6.34-6.31 (dd, 1H, J = 5.0 Hz, 2.5
Hz, HC(OH)), 3.60 (d, 1H, J = 2.5 Hz, HC≡C); C NMR (DMSO-d ) δ 135.0, 130.7, 130.5, 130.1,
127.34, 127.31 (Py), 127.28 (Py), 127.25 (Py), 126.2 (Py), 125.3 (Py), 125.2 (Py), 124.65 (Py),
124.59 (Py), 124.1, 123.8, 123.7 (Py), 85.5, 76.6 (HC≡C), 60.8 (HC(OH)).
1-(Pyrenyl)-propynone (92): The Jones reagent (2.67 M CrO in 3M H SO , 1.0 mL, 2.67
3 2 4
mmol) was added to a solution of alcohol 90 (180 mg, 0.67 mmol) in acetone (10 mL), and the
reaction mixture was stirred under an ambient atmosphere at rt for 2h, whereupon it was diluted with
EtOAc (20 mL), neutralized by drop-wise addition of 6M NaOH (1.0 mL) under stirring, and
sequentially washed with water (30 mL) and sat. aq. NaHCO (30 mL). The organic phase was dried
over Na SO , evaporated to dryness, and the resulting crude purified by silica gel column
chromatography (0-20% EtOAc in petroleum ether, v/v) to furnish 92 (130 mg, 75%) as a brightly
yellow solid material. R = 0.6 (50% EtOAc in petroleum ether, v/v); ESI-HRMS m/z 277.0626
+ + 1
([M+Na] , C H O⋅Na , calc 277.0624); H NMR (CDCl ) δ 9.48 (d, 1H, J = 10.0 Hz), 8.94 (d, 1H,
19 10 3
J = 8.0 Hz), 8.28-8.23 (m, 3H), 8.19-8.14 (m, 2H), 8.07-8.02 (m, 2H), 3.53 (s, 1H); C NMR
(CDCl ) δ 179.3, 135.8, 132.2 (Py), 131.31, 131.25 (Py), 131.14, 131.05 (Py), 130.6, 128.4, 127.35
(Py), 127.29 (Py), 127.1 (Py), 126.8 (Py), 124.97 (Py), 124.96, 124.95, 124.2 (Py), 124.1, 82.6, 80.1
(HC≡C).
4-(Pyrenyl)-butyne (94): An oven-dried flask was charged with pyrenecarboxaldehyde
(230 mg, 1.00 mmol) and activated zinc (100 mg, 1.50 mmol) and placed under an argon atmosphere.
Anhydrous THF (5 mL) and propargyl bromide (0.20 mL, 1.79 mmol) were added and the reaction
mixture was stirred at 45 °C for 4h. Sat. aq. NH Cl (1 mL) was added and the mixture was extracted
with EtOAc (2 x 20 mL). The organic phase was washed with brine (20 mL) and evaporated to
dryness. The resulting crude was purified by silica gel column chromatography (0-30% EtOAc in
petroleum ether, v/v) to afford a crude white solid material (145 mg), which H NMR suggested to be
a ~9:1 mixture of the desired 1-(pyrenyl)-butynol and the corresponding allene isomer.
Et SiH (0.20 mL, 1.25 mmol) and boron trifluoride etherate (0.20 mL, 1.62) were added to a solution
of the crude mixture in CH Cl (5 mL), which then was stirred at rt for 1h. The reaction mixture was
diluted with CH Cl (20 mL) and sat. aq. NaHCO (2 mL), and successively washed with brine (20
2 2 3
mL) and water (20 mL). The organic phase was dried over Na SO , evaporated to dryness under
reduced pressure, and the resulting crude purified by silica gel column chromatography (0-3% EtOAc
in petroleum ether, v/v) to afford 94 (80 mg, 31%) as a white solid material. R = 0.5 (5% EtOAc in
+ + 1
petroleum ether, v/v); ESI-HRMS m/z 277.0973 ([M+Na] , C H ⋅Na , calc 279.0988); H NMR
14
(CDCl ) δ 8.27-8.25 (d, 1H, J = 9.5 Hz, Py), 8.17-8.14 (m, 2H, Py), 8.12-8.10 (m, 2H, Py), 8.01 (ap s,
2H), 8.00-7.96 (t, 1H, J = 8.0 Hz, Py), 7.92-7.90 (d, 1H, J = 7.5 Hz, Py), 3.59 (t, 2H, J = 7.7 Hz,
6478592_1 (GHMatters) P95976.NZ ESTHERJ
CH CH C≡CH), 2.72 (dt, 2H, J = 7.7 Hz, 2.5 Hz, CH C≡CH), 2.03 (t, 1H, J = 2.5 Hz, HC≡C); C
2 2 2
NMR (CDCl ) δ 134.7, 131.6, 131.1, 130.5, 128.9, 127.8 (Py), 127.7 (Py), 127.5 (Py), 127.1 (Py),
126.1 (Py), 125.31, 125.27 (Py), 125.2, 125.1 (Py), 125.0 (Py), 123.2 (Py), 84.0, 69.6 (HC≡C), 32.8
(CH CH C≡CH), 21.0 (CH C≡CH).
2 2 2
104V
’-O-(4,4’-Dimethoxytrityl)-2’-C-[4-(2,2,2-trifluoroacetamidomethyl)-1H-1,2,3-triazolyl]-2'-
deoxyuridine (104V): Nucleoside 102 (0.40 g, 0.70 mmol), 2,2,2-trifluoro-N-(prop
ynyl)acetamide Av (105 mg, 0.70 mmol), sodium ascorbate (70 mg, 0.35 mmol), CuSO ⋅5H O (5 mg,
0.02 mmol) and THF/t-BuOH/H O (5 mL) were mixed, reacted (14h at rt), worked up and purified
(50-100% EtOAc in petroleum ether, v/v) as described above except that the organic phase was
successively washed with brine and water. Nucleoside 104V (0.42 g, 83%) was obtained as a yellow
solid material. R = 0.3 (80% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z 745.2225
+ + 1
([M+Na] , C H F N O ⋅Na , calc 745.2204); H NMR (DMSO-d ) δ 11.40 (d, 1H, ex, J = 2.0 Hz,
34 3 6 8 6
H3), 10.02 (t, 1H, J = 6.0 Hz, NHCOCF ), 8.01 (s, 1H, Tz), 7.81 (d, 1H, J = 8.0 Hz, H6), 7.43-7.22
(m, 9H, DMTr), 6.93-6.88 (m, 4H, DMTr), 6.42 (d, 1H, J = 4.5 Hz, H1’), 5.79 (d, 1H, ex, J = 6.0 Hz,
3’-OH), 5.50 (dd, 1H, J = 7.0 Hz, 4.5 Hz, H2’), 5.45 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 4.52 (m, 1H,
H3’), 4.47 (d, 2H, J = 5.5 Hz, CH NHCO), 4.24-4.20 (m, 1H, H4’), 3.75 (s, 6H, CH O), 3.38-3.30
(m, 2H, H5’ – partial overlap with H O); C NMR (DMSO-d ) δ 162.8, 158.09, 158.08, 156.2 (q,
J = 36 Hz, COCF ), 150.1, 144.6, 142.4, 140.5 (C6), 135.3, 135.1, 129.7 (DMTr), 127.8 (DMTr),
CF 3
127.7 (DMTr), 126.7 (DMTr), 124.5 (Tz), 115.8 (q, J = 288 Hz, CF ), 113.2 (DMTr), 101.9 (C5),
CF 3
87.1 (C1’), 85.8, 83.2 (C4’), 68.8 (C3’), 64.5 (C2’), 62.8 (C5’), 55.0 (CH O), 34.5 (CH NHCO); F-
NMR (DMSO-d ) δ -74.2.
104W
6478592_1 (GHMatters) P95976.NZ ESTHERJ
’-O-(4,4’-Dimethoxytrityl)-2’-C-[4-(pyreneyl)-1H-1,2,3-triazolyl]-2'-deoxyuridine
(104W): Nucleoside 102 (0.28 g, 0.49 mmol), 1-ethynylpyrene 98 (130 mg, 0.58 mmol), sodium
ascorbate (200 mg, 1.00 mmol), CuSO ⋅5H O (25 mg, 0.10 mmol) and THF/t-BuOH/H O (10 mL)
4 2 2
were mixed, reacted (7h at 75 °C), worked up and purified (40-70% EtOAc in petroleum ether, v/v)
as described above to provide nucleoside 104W (140 mg, 35%) as an off-white solid material. R =
0.5 (80% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z 820.277 ([M+Na] , C H N O ⋅Na ,
48 39 5 7
calc 820.274); H NMR (DMSO-d ) δ 11.46 (d, 1H, ex, J = 1.5 Hz, NH), 8.87 (d, 1H, J = 9.0 Hz,
Py), 8.80 (s, 1H, Tz), 8.41-8.33 (m, 4H, Py), 8.27 (d, 1H, J = 9.2 Hz, Py), 8.26-8.22 (m, 2H, Py); 8.12
(t, 1H, J = 7.5 Hz, Py), 7.91 (d, 1H, J = 8.0 Hz, H6), 7.48-7.20 (m, 9H, DMTr), 6.96-6.90 (m, 4H,
DMTr), 6.65 (d, 1H, J = 5.0 Hz, H1’), 5.95 (d, 1H, ex, J = 6.0 Hz, 3’-OH), 5.69 (dd, 1H, J = 7.0 Hz,
.0 Hz, H2’), 5.54 (dd, 1H, J = 8.0 Hz, 1.5 Hz, H5), 4.69-4.64 (m, 1H, H3’), 4.40-4.36 (m, 1H, H4’),
3.76 (s, 6H, CH O), 3.46-3.36 (m, 2H, H5’); C NMR (DMSO-d ) δ 162.9, 158.2, 150.3, 145.7,
144.7, 140.8 (C6), 135.4, 135.2, 130.9, 130.6, 130.3, 129.78 (DMTr), 129.76 (DMTr), 128.0 (Py),
127.9 (DMTr), 127.73 (DMTr), 127.67 (Py), 127.5, 127.3 (Py), 127.0 (Py), 126.8 (DMTr), 126.4
(Py), 125.7 (Tz), 125.5 (Py), 125.16, 125.15 (Py), 125.09 (Py), 124.8 (Py), 124.3, 123.9, 113.3
(DMTr), 102.1 (C5), 87.4 (C1’), 85.9, 83.4 (C4’), 69.1 (C3’), 64.9 (C2’), 63.1 (C5’), 55.0 (CH O).
DMTrO
OH N
104X
’-O-(4,4’-Dimethoxytrityl)-2’-C-[4-(pyreneylcarbonyl)-1H-1,2,3-triazolyl]-2'-
deoxyuridine (104X): Nucleoside 102 (0.28 g, 0.49 mmol), 1-(pyrenyl)-propynone 92 (140
mg, 0.55 mmol), sodium ascorbate (200 mg, 1.00 mmol), CuSO ⋅5H O (25 mg, 0.10 mmol) and
THF/t-BuOH/H O (10 mL) were mixed, reacted (5h at rt), worked up and purified (40-90% EtOAc in
petroleum ether, v/v) as described above to provide nucleoside 104X (0.25 g, 60%) as yellow solid
material. R = 0.4 (80% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z 848.267 ([M+Na] ,
C H N O ⋅Na , calc 848.270); H NMR (DMSO-d ) δ 11.46 (br s, 1H, ex, NH), 8.96 (s, 1H, Tz),
49 39 5 8 6
8.51-8.28 (m, 8H, Py), 8.17 (t, 1H, J = 7.5 Hz, Py), 7.83 (d, 1H, J = 8.0 Hz, H6), 7.44-7.21 (m, 9H,
DMTr), 6.93-6.88 (m, 4H, DMTr), 6.55 (d, 1H, J = 5.0 Hz, H1’), 5.90 (d, 1H, ex, J = 5.0 Hz, 3’-OH),
.68 (dd, 1H, J = 7.0 Hz, 5.0 Hz, H2’), 5.53 (dd, 1H, J = 8.0 Hz, 2.0 Hz, H5), 4.64-4.58 (m, 1H, H3’),
4.31-4.26 (m, 1H, H4’), 3.74 (s, 6H, CH O), 3.40-3.30 (m, 2H, H5’); C NMR (DMSO-d ) δ 188.3,
162.9, 158.1, 150.2, 147.2, 144.6, 140.8 (C6), 135.4, 135.2, 133.0, 131.8, 131.5 (Tz), 130.6, 130.0,
129.74 (DMTr), 129.72 (DMTr), 129.4 (Py), 129.1 (Py), 128.9, 127.9, 127.84 (DMTr), 127.80 (Py),
6478592_1 (GHMatters) P95976.NZ ESTHERJ
127.7 (DMTr), 127.2 (Py), 126.8 (Py), 126.7 (DMTr), 126.5 (Py), 126.1 (Py), 124.01 (Py), 123.98
(Py), 123.8, 123.5, 113.2 (DMTr), 102.0 (C5), 87.4 (C1’), 85.8, 83.3 (C4’), 69.0 (C3’), 65.0 (C2’),
63.0 (C5’), 55.0 (CH O).
104Y
’-O-(4,4’-Dimethoxytrityl)-2’-C-[4-{2-(pyreneyl)ethyl}-1H-1,2,3-triazolyl]-2'-
deoxyuridine (104Y): Nucleoside 102 (0.34 g, 0.60 mmol), 4-(pyrenyl)-butyne 94 (160 mg,
0.63 mmol), sodium ascorbate (0.25 g, 1.25 mmol), CuSO ⋅5H O (31 mg, 0.12 mmol) and THF/t-
BuOH/H O (10 mL) were mixed, reacted (2h at rt), worked up and purified (50-100% EtOAc in
petroleum ether, v/v) as described above to provide nucleoside 104Y (0.33 g, 67%) as a white solid
material. R = 0.3 (80% EtOAc in petroleum ether, v/v); MALDI-HRMS m/z 848.3046 ([M+Na] ,
C H N O ⋅Na , calc 848.3055); H NMR (DMSO-d ) δ 11.44 (s, 1H, ex, NH), 8.40 (d, 1H, J = 9.0
50 43 5 7 6
Hz, Py), 8.30-8.19 (m, 4H, Py), 8.13 (ap s, 2H, Py), 8.06 (t, 1H, J = 8.0 Hz, Py), 8.01 (s, 1H, Tz),
7.95 (d, 1H, J = 8.0 Hz, Py), 7.82 (d, 1H, J = 8.0 Hz, H6), 7.44-7.41 (m, 2H, DMTr), 7.36-7.23 (m,
7H, DMTr), 6.94-6.90 (m, 4H, DMTr), 6.44 (d, 1H, J = 5.0 Hz, H1’), 5.79 (d, 1H, ex, J = 6.0 Hz, 3’-
OH), 5.49-5.45 (m, 2H, H5, H2’), 4.54-4.49 (m, 1H, H3’), 4.27-4.22 (m, 1H, H4’), 3.75 (s, 6H,
CH O), 3.72-3.66 (m, 2H, CH CH ), 3.40-3.30 (m, 2H, H5’), 3.19-3.14 (m, 2H, CH CH ); C NMR
3 2 2 2 2
(DMSO-d ) δ 162.8, 158.12, 158.11, 150.2, 145.8, 144.6, 140.5 (C6), 135.6, 135.4, 135.1, 130.8,
130.3, 129.7 (DMTr), 129.4, 128.0, 127.8 (DMTr), 127.7 (DMTr), 127.5 (Py), 127.4 (Py), 127.3
(Py), 126.7 (DMTr), 126.5 (Py), 126.1 (Py), 124.93 (Py), 124.88 (Py), 124.8 (Py), 124.2, 124.1, 123.4
(Tz), 123.2 (Py), 113.2 (DMTr), 102.0 (C5), 87.1 (C1’), 85.9, 83.3 (C4’), 68.9 (C3’), 64.3 (C2’), 62.9
(C5’), 55.0 (CH O), 32.6 (CH CH ), 27.3 (CH CH ).
3 2 2 2 2
104Z
5’-O-(4,4’-Dimethoxytrityl)-2’-C-[4-(pyreneyl)carboxamidomethyl-1H-1,2,3-triazolyl]-2'-
deoxyuridine (104Z): Nucleoside 102 (0.40 g, 0.70 mmol), N-(propynyl)pyrenecarboxamide
6478592_1 (GHMatters) P95976.NZ ESTHERJ
100 (200 mg, 0.71 mmol), sodium ascorbate (50 mg, 0.25 mmol), CuSO ⋅5H O (5 mg, 0.02 mmol)
and THF/t-BuOH/H O (5 mL) were mixed, reacted (8h at rt), worked up and purified (50-100%
EtOAc in petroleum ether, v/v) as described above except that the organic phase was successively
washed with brine and water. Nucleoside 104Z (0.49 g, 83%) was obtained a yellow solid material.
+ + 1
R = 0.2 (EtOAc); MALDI-HRMS m/z 877.2979 ([M+Na] , C H N O ⋅Na , calc 877.2956); H
f 50 42 6 8
NMR (DMSO-d ) δ 11.43 (s, 1H, ex, H3), 9.26 (t, 1H, ex, J = 6.0 Hz, NHCO), 8.53-8.52 (d, 1H, J =
9.5 Hz, Ar), 8.36-8.34 (m, 3H, Ar), 8.27-8.22 (m, 3H, Ar), 8.17-8.11 (m, 3H, Ar, Tz), 7.85 (d, 1H, J
= 8.5 Hz, H6), 7.44-7.43 (m, 2H, DMTr), 7.35-7.24 (m, 7H, DMTr), 6.93-6.89 (m, 4H, DMTr), 6.50
(d, 1H, J = 4.7 Hz, H1’), 5.87 (d, 1H, ex, J = 5.5 Hz, 3’-OH), 5.56 (dd, 1H, J = 7.0 Hz, 4.7 Hz, H2’),
5.47 (d, 1H, J = 8.0 Hz, H5), 4.71 (d, 2H, J = 6.0 Hz, CH NHCO), 4.58-4.54 (m, 1H, H3’), 4.30-4.25
(m, 1H, H4’), 3.75 (s, 6H, CH O), 3.41-3.32 (m, 2H, H5’); C NMR (DMSO-d ) δ 168.8, 162.9,
158.13, 158.12, 150.2, 144.7, 144.6, 140.5 (C6), 135.4, 135.2, 131.6, 131.5, 130.7, 130.2, 129.8
(DMTr), 128.3 (Ar), 128.1 (Ar), 127.9 (DMTr), 127.8, 127.7 (DMTr), 127.1 (Ar), 126.8 (DMTr),
126.5 (Ar), 125.7 (Ar), 125.5 (Ar), 125.2 (Ar), 124.7 (Ar), 124.3 (Ar), 124.2 (Tz), 123.7, 123.6, 113.2
(DMTr), 102.0 (C5), 87.1 (C1’), 85.9, 83.3 (C4’), 69.0 (C3’), 64.5 (C2’), 62.9 (C5’), 55.0 (CH O),
.0 (CH NHCO).
General phosphitylation protocol for preparation of 106V-106Z (description for ~3 mmol
scale): The appropriate nucleoside 104 was co-evaporated with anhydrous CH Cl (5 mL) and
redissolved in anhydrous CH Cl (reagent quantities and solvent volumes are specified below). To
this was added N,N-diisopropylethylamine (DIPEA), 0.45 M tetrazole in CH CN and 2-cyanoethyl-
N,N,N’,N’-tetraisopropylphosphordiamidite (PN2-reagent). The reaction mixture was stirred at rt
until analytical TLC indicated complete conversion (reaction time specified below) whereupon cold
abs. EtOH (0.5 mL) was added. The reaction mixture was evaporated to dryness and the resulting
residue was purified by silica gel column chromatography (eluent specified below). The crude
material was triturated from cold petroleum ether to afford phosphoramidite 106 (yields specified
below).
DMTrO
(i-Pr) N O
NHTFA
106V
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)-2’-C-[4-
(2,2,2-trifluoroacetamidomethyl)-1H-1,2,3-triazolyl]-2'-deoxyuridine (106V): Nucleoside
6478592_1 (GHMatters) P95976.NZ ESTHERJ
104V (0.29 g, 0.40 mmol), DIPEA (0.10 mL, 0.57 mmol), tetrazole in CH CN (0.45 M, 1.0 mL, 0.45
mmol), PN2-reagent (0.15 mL, 0.46 mmol) and anhydrous CH Cl (1 mL) were mixed, reacted (3h),
worked up and purified (50-90% EtOAc in petroleum ether, v/v) as described above except that: a)
the reaction mixture was extracted with EtOAc (5 mL) after addition of EtOH, followed by drying of
the organic phase over anhydrous Na SO and evaporation to dryness under reduced pressure and b)
trituration was not performed. Phosphoramidite 106V (0.24 g, 67%) was obtained as a white solid
material. R = 0.3 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 945.3322 ([M+Na] ,
f 2 2
+ 31 19
C H F N O ⋅Na , calc 945.3283); P NMR (CDCl ) δ 152.0, 149.8; F NMR (CDCl ) δ -75.7.
44 50 3 8 9 3 3
106W
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)-2’-C-[4-
(pyreneyl)-1H-1,2,3-triazolyl]-2'-deoxyuridine (106W): Nucleoside 104W (230 mg, 0.29
mmol), DIPEA (0.10 mL, 0.57 mmol), tetrazole in CH CN (0.45 M, 1.0 mL, 0.45 mmol), PN2-
reagent (0.20 mL, 0.62 mmol) and anhydrous CH Cl (2 mL) were mixed, reacted (4h), worked up
and purified (0-4% MeOH/CH Cl , v/v) as described above to afford 106W (180 mg, 62%) as a white
powder. R = 0.35 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1020.3855 ([M+Na] ,
f 2 2
+ 31
C H N O P⋅Na , calc 1020.3826); P NMR (CDCl ) δ 152.0, 150.5.
57 56 7 8 3
106X
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)-2’-C-[4-
(pyreneylcarbonyl)-1H-1,2,3-triazolyl]-2'-deoxyuridine (106X): Nucleoside 104X (150 mg,
0.18 mmol), DIPEA (0.10 mL, 0.57 mmol), tetrazole in CH CN (0.45 M, 0.6 mL, 0.27 mmol), PN2-
reagent (0.12 mL, 0.37 mmol) and anhydrous CH Cl (2 mL) were mixed, reacted (3.5h), worked up
and purified (0-4% MeOH/CH Cl , v/v) as described above to afford 106X (110 mg, 59%) as a
6478592_1 (GHMatters) P95976.NZ ESTHERJ
yellow solid material. R = 0.4 (5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1048.3779
f 2 2
+ + 31
([M+Na] , C H N O P⋅Na , calc 1048.3775); P NMR (CDCl ) δ 152.4, 150.9.
58 56 7 9 3
106Y
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)-2’-C-[4-{2-
(pyreneyl)ethyl}-1H-1,2,3-triazolyl]-2'-deoxyuridine (106Y): Nucleoside 104Y (0.33 g, 0.40
mmol), DIPEA (0.10 mL, 0.57 mmol), tetrazole in CH CN (0.45 M, 1.5 mL), PN2-reagent (0.25 mL,
0.78 mmol) and anhydrous CH Cl (2 mL) were mixed, reacted (3.5h), worked up and purified (0-4%
MeOH/CH Cl , v/v) as described above to afford 106Y (210 mg, 51%) as a white powder. R = 0.45
2 2 f
(5% MeOH in CH Cl , v/v); MALDI-HRMS m/z 1048.4147 ([M+Na] , C H N O P⋅Na , calc
2 2 59 60 7 8
1048.4139); P NMR (CDCl ) δ 151.6, 150.6.
106Z
3’-O-(N,N-Diisopropylaminocyanoethoxyphosphinyl)-5’-O-(4,4’-dimethoxytrityl)-2’-C-[4-
(pyreneyl)carboxamidomethyl-1H-1,2,3-triazolyl]-2'-deoxyuridine (106Z): Nucleoside
104Z (0.36 g, 0.42 mmol), DIPEA (0.10 mL, 0.57 mmol), tetrazole in CH CN (0.45 M, 1.0 mL, 0.45
mmol), PN2-reagent (0.15 mL, 0.46 mmol) and anhydrous CH Cl (1 mL) were mixed, reacted (3h),
worked up and purified (50-90% EtOAc in petroleum ether, v/v) as described above except that: a)
the reaction mixture was extracted with EtOAc (5 mL) after addition of EtOH, followed by drying of
the organic phase over anhydrous Na SO and evaporation to dryness under reduced pressure and b)
trituration was not performed. Phosphoramidite 106Z (0.28 g, 67 %) was obtained as a white solid
material. R = 0.3 (5% MeOH in CH Cl v/v); MALDI-HRMS m/z 1077.3984 ([M+Na] ,
f 2 2,
+ 31
C H N O P⋅Na , calc 1077.4035); P NMR (CDCl ) δ 151.9, 150.1.
59 59 8 9 3
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Synthesis and purification of single-stranded probes modified with monomers 122V/W/X/Y/Z:
Synthesis of modified oligodeoxyribonucleotides was performed on an Expedite 8909 DNA
Synthesizer using 0.2 μmol scale succinyl linked LCAA-CPG (long chain alkyl amine controlled pore
glass) columns with a pore size of 500Å. Standard protocols for incorporation of DNA
phosphoramidites were used. A ~50-fold molar excess of modified phosphoramidites in anhydrous
acetonitrile (at 0.05 M) was used during hand-couplings using the conditions specified in the main
manuscript. Moreover, extended oxidation (45s) was employed during hand-couplings. Cleavage
from solid support and removal of protecting groups was accomplished upon treatment with 32% aq.
ammonia (55 °C, 20 h). Purification of all modified oligonucleotides was performed by ion-pair
reverse phase HPLC as described below followed by detritylation (80% aq. AcOH) and precipitation
from acetone (-18 °C for 12-16h).
Purification of crude oligonucleotides was performed on a Varian Prostar HPLC system
equipped with an XTerra MS C18 pre-column (10 μm, 7.8 x 10 mm) and an XTerra MS C18 column
(10 μm, 7.8 x 150 mm) using a 0.05 mM TEAA (triethylammonium acetate) buffer - 25%
water/acetonitrile (v/v) gradient. The identity of synthesized oligonucleotides was established through
MALDI-MS/MS analysis recorded in positive ions mode on a Quadrupole Time-Of-Flight Tandem
Mass Spectrometer (Q-TOF Premiere) equipped with a MALDI source (Waters Micromass LTD.,
U.K) using anthranilic acid as a matrix (Table 61, while purity (>80%) was verified by RP-HPLC
running in analytical mode.
Table 61
MALDI-Tof MS-Analysis of Single-Stranded Probes Modified with 122W/X/Y/Z
Sequence Calc.(M+H) Expt. (M+H)
'-CGCAA A122WA AACGC 4188.8 4188.9
'-CGCAA C122WC AACGC 4140.8 4140.9
'-CGCAA G122WG AACGC 4220.8 4220.8
'-CGCAA T122WT AACGC 4170.8 4170.8
'-GCGTT 122WAT TTGCG 4223.8 4223.8
'-GCGTT TA122W TTGCG 4223.8 4223.9
'-GCGTT 122WA122W TTGCG 4476.8 4477.0
'-CGCAA A122XA AACGC 4216.8 4216.9
'-CGCAA C122XC AACGC 4168.8 4169.0
'-CGCAA G122XG AACGC 4248.8 4248.8
'-CGCAA T122XT AACGC 4198.8 4198.9
'-GCGTT 122XAT TTGCG 4251.8 4251.9
'-GCGTT TA122X TTGCG 4251.8 4251.9
'-GCGTT 122XA122X TTGCG 4532.8 4532.9
'-CGCAA A122YA AACGC 4216.8 4216.9
'-CGCAA C122YC AACGC 4168.8 4169.0
'-CGCAA G122YG AACGC 4248.8 4248.9
'-CGCAA T122YT AACGC 4198.8 4198.9
'-GCGTT 122YAT TTGCG 4251.8 4251.9
'-GCGTT TA122Y TTGCG 4251.8 4251.9
6478592_1 (GHMatters) P95976.NZ ESTHERJ
'-GCGTT 122YA122Y TTGCG 4532.9 4533.1
'-CGCAA A122ZA AACGC 4245.8 4245.9
'-CGCAA C122ZC AACGC 4197.8 4197.9
'-CGCAA G122ZG AACGC 4277.8 4278.1
'-CGCAA T122ZT AACGC 4227.8 4227.9
'-GCGTT 122ZAT TTGCG 4280.8 4280.9
'-GCGTT TA122Z TTGCG 4280.8 4280.8
'-GCGTT 122ZA122Z TTGCG 4590.9 4590.9
’-GTG ATA 122VGC 2835.5 2835.9
’-GTG A122VA TGC 2835.5 2835.9
’-G122VG ATA TGC 2835.5 2835.8
’-GTG ATA 122ZGC 3063.6 3063.9
’-GTG A122ZA TGC 3063.6 3063.9
’-G122ZG ATA TGC 3063.6 3063.8
Thermal Denaturation Studies involving probes modified with monomers 122V/W/X/Y/Z:
Concentrations of oligonucleotides were estimated using the following extinction coefficients
(OD/μmol): dG (12.01), dA (15.20), dT (8.40), dC (7.05); rG (13.70), rA (15.40), rU (10.00), rC
(9.00); 122V (19.96), 122W (31.08), 122X (35.60), 122Y (27.62) and 122Z (30.95) [values for
monomers 122V-122Z were estimated through A measurements of the corresponding
phosphoramidites in 1% aq. DMSO solutions]. Each strand was thoroughly mixed and denatured by
heating to 80-85 °C followed by cooling to the starting temperature of the experiment. Quartz optical
cells with a path length of 10 mm were used. Thermal denaturation temperatures (T values [°C]) of
duplexes (1.0 µM final concentration of each strand) were measured on a Cary 100 UV/VIS
spectrophotometer equipped with a 12-cell Peltier temperature controller and determined as the
maximum of the first derivative of the thermal denaturation curve (A vs. T) recorded in medium
salt buffer (T -buffer: 100 mM NaCl, 0.1 mM EDTA, and pH 7.0 adjusted with 10 mM Na HPO and
m 2 4
mM Na HPO ). The temperature of the denaturation experiments ranged from at least 20 °C below
T to 20 °C above T . A temperature ramp of 0.5 °C/min was used in all experiments. Reported T -
m m m
values are averages of two experiments within ± 1.0 °C.
Certain embodiments pertain probes modified with monomers 122W/X/Y/Z. Thermal
denaturation studies for duplexes between such probes and matched or mismatched DNA targets are
disclosed in Tables 62 and 63. The examples provided therein, demonstrate that probes modified with
monomers 122W/X/Y/Z, display universal thermal hybridization properties, i.e., they display
significantly similar thermal affinity toward matched and mismatched nucleic acid targets, more
commonly single-stranded nucleic acid targets, even more commonly single-stranded DNA and/or
single-stranded RNA.
Table 62
T -Values of Duplexes Between Probes Modified with Monomers
122W/X/Y/Z and Complementary or Centrally Mismatched DNA Targets
6478592_1 (GHMatters) P95976.NZ ESTHERJ
T (ΔT ) Mismatch ΔT
m m m
[°C] [°C]
Probe B A C G T
’-CGCAA ATA AACGC 48.5 -10.0 -5.0 -9.0
’-CGCAA CTC AACGC 55.5 -13.5 -9.5 -9.0
’-CGCAA GTG AACGC 55.5 -13.0 -9.5 -
.0
’-CGCAA TTT AACGC 48.5 -11.0 -9.0 -
11.0
’-CGCAA A(122W)A AACGC 48.0 (- +1.0 +1. +1.5
0.5) 5
’-CGCAA C(122W)C AACGC 53.5 (- +0.5 +2. +2.5
2.0) 0
’-CGCAA G(122W)G AACGC 51.5 (- +1.0 -4.5 0.0
4.0)
’-CGCAA T(122W)T AACGC 47.0 (- +2.5 -0.5 +2.0
1.5)
’-CGCAA A(122X)A AACGC 46.5 (- +1.0 +0. +1.0
2.0) 5
’-CGCAA C(122X)C AACGC 52.0 (- -1.5 0.0 -0.5
3.5)
’-CGCAA G(122X)G AACGC 52.5 (- +0.5 -7.0 -0.5
3.0)
’-CGCAA T(122X)T AACGC 44.5 (- +1.0 -1.0 0.0
4.0)
’-CGCAA A(122Y)A AACGC 49.5(+1.0 +1.5 0.0 +1.0
’-CGCAA C(122Y)C AACGC 50.5 (- -5.0 -1.0 -2.5
.0)
’-CGCAA G(122Y)G AACGC 53.0 (- +1.5 -3.5 +0.5
2.5)
’-CGCAA T(122Y)T AACGC 44.5 (- -2.0 -2.0 -1.5
4.0)
’-CGCAA A(122Z)A AACGC 47.0 (- -5.5 -2.5 -4.0
1.5)
’-CGCAA C(122Z)C AACGC 51.5 (- -6.5 -1.0 -4.0
4.0)
’-CGCAA G(122Z)G AACGC 52.0 (- -2.0 -6.0 -4.0
3.5)
’-CGCAA T(122Z)T AACGC 45.5 (- -4.5 -5.0 -4.0
3.0)
T ’s determined as maximum of the first derivative of denaturation curves (A vs T) recorded in
m 260
T -buffer ([Na ] = 110 mM, [Cl ] = 100 mM, pH 7.0 (NaH PO /Na HPO )) using 1.0 µM of each
m 2 4 2 4
strand. T ’s are averages of at least two measurements within 1.0 °C. “ΔT ” = change in T relative
m m m
to unmodified reference duplex. “Mismatch ΔT ” = change in T relative to fully matched duplex (B
= A). “Avg Mismatch ΔT seq” = average of all three “Mismatch ΔT ”-values for a given probe.
“Avg Mismatch ΔT series” = average of all twelve “Mismatch ΔT ”-values of all four studied
sequences within a monomer series. “±” denotes standard deviation. DNA targets: 3’-GCGTT TBT
TTGCG, 3’-GCGTT GBG TTGCG, 3’-GCGTT CBC TTGCG and 3’-GCGTT ABA TTGCG.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Table 63
Additional T -Values of Duplexes Between Probes Modified with Monomers 122W/X/Y/Z and
Complementary or Centrally Mismatched DNA Targets.
DNA: 5’-CGCAA ABA AACGC
T (ΔT ) [°C] Mismatch ΔT [°C]
m m m
Probe B=
T A C G
3’-GCGTT TAT TTGCG 48.5 -10.0 -10.0 -5.5
3’-GCGTT TA122W TTGCG 46.5 (-2.0) -17.0 -9.0 -12.0
3’-GCGTT 122WAT TTGCG 41.0 (-7.5) -11.5 -10.0 -5.5
3’-GCGTT 122WA122W TTGCG 35.0 (-13.5) nt -3.5 Nt
3’-GCGTT TA122X TTGCG 44.0 (-4.5) -9.0 -7.5 -5.0
3’-GCGTT 122XAT TTGCG 42.0 (-6.5) -10.5 -11.5 -6.0
3’-GCGTT 122XA122X TTGCG 36.5 (-12.0) -2.5 -4.5 +0.5
3’-GCGTT TA122Y TTGCG 40.0 (-8.5) -7.0 -7.0 -5.0
3’-GCGTT 122YAT TTGCG 42.5 (-6.0) -6.0 -2.5 -7.0
3’-GCGTT 122YA122Y TTGCG 34.5 (-14.0) -4.5 -3.5 -4.5
3’-GCGTT TA122Z TTGCG 42.5 (-6.0) -7.0 -8.5 -7.5
3’-GCGTT 122ZAT TTGCG 44.0 (-4.5) -11.5 -12.0 -9.0
3’-GCGTT 122ZA122Z TTGCG 39.5 (-9.0) -2.5 -0.5 -3.5
Conditions and definitions as described in footnote of Table above. “nt” = no transition.
Steady-state fluorescence emission spectra involving probes modified with monomers
122V/W/X/Y/Z: Spectra of oligonucleotides modified with pyrene-functionalized monomers
122W/122X/122Y/122Z and the corresponding duplexes with complementary or mismatched
DNA/RNA targets were recorded in non-deoxygenated thermal denaturation buffer (each strand 1.0
μM) using an excitation wavelength of λ = 350 nm for 122W/122Y/122Z or λ = 400 nm for 122X,
ex ex
excitation slit 5.0 nm, emission slit 5.0 nm and a scan speed of 600 nm/min. Experiments were
performed at ambient temperature (~20 °C).
Determination of quantum yields involving probes modified with monomers 122V/W/X/Y/Z:
Relative fluorescence emission quantum yields (Φ ) of modified nucleic acids (SSP or duplex) were
determined using the following equation: Φ (NA) = [Φ (std)/α(std)] × [IFI (NA) / A (NA)] ×
F F ex
[n(NA)/n(std)] where Φ (std) is the fluorescence emission quantum yield of standard; α(std) is the
slope of the integrated fluorescence intensity vs. optical intensity plot made for the standard; IFI
(NA) is the integrated fluorescence intensity (λ = 360-510 nm for monomer 122W/122Y/122Z; λ
em em
6478592_1 (GHMatters) P95976.NZ ESTHERJ
= 425-625 nm for monomer 122X; λ = 360-600 nm for standards); A (NA) is the optical density
em ex
of the sample at the utilized excitation wavelength (λ = 350 nm for monomer 122W/122Y/122Z;
λ = 400 nm for monomer 122X; λ = 350 nm for standards; optical densities of all solutions at the
ex ex
excitation wavelengths were between 0.01 and 0.10); n(NA) and n(std) are refractive indexes of
solvents used for sample and standard respectively (n = 1.33, n = 1.36, and n = 1.43).
water ethanol cyclohexane
The validity of this method under our experimental set-up was ascertained by determining
the quantum yield of anthracene in ethanol with respect to 9,10-diphenylanthracene in cyclohexane
(Φ = 0.86). The measured value of Φ = 0.28 is in excellent agreement with the reported value of
(Φ = 0.27). Subsequently, the literature value for anthracene in ethanol was used as the standard for
determination of quantum yields of SSPs and duplexes.
Optical spectroscopy studies involving probes modified with monomers 122V/W/X/Y/Z. UV-Vis
absorption spectra of oligonucleotides modified with monomers 122W-122Z were recorded in
absence or presence of complementary or centrally mismatched DNA targets, in order to gain
additional insights into the mechanism that governs the observed universal hybridization
characteristics (); hybridization-induced intercalation of pyrene moieties is known to induce
subtle bathochromic shifts.
Single-stranded probes modified with monomer 122W display a single unstructured
maximum in the pyrene region (λ ~ 351 nm, ), while duplexes with complementary,
mismatched or abasic DNA targets display two resolved maxima at ~351 nm and ~365 nm. The lack
of defined peaks for the single stranded probes (SSPs) precludes analysis of bathochromic shifts.
Single-stranded probes modified with monomer 122X display two broad and virtually equally intense
peaks which renders exact determination of absorption maxima unfeasible (λ ~ 385 nm and ~ 415
nm, ). Hybridization with complementary DNA results in subtle bathochromic shifts, while
more pronounced shifts are observed upon hybridization with mismatched or abasic DNA. The
pyrene maxima are red-shifted relative to those of unconjugated pyrenes chromophores, which
suggests electronic coupling between the pyrene and triazole moieties. Single-stranded probes
modified with monomer 122Y or monomer 122Z), on the other hand, have structured absorption
spectra with two maxima in the ‘normal’ region (i.e., λ ~333/348 nm and ~332/346 nm,
respectively, ). Hybridization of these probes with complementary, mismatched or abasic
DNA target strands results in subtle bathochromic shifts (Δλ between +1 and +3 nm, ,
Table 64. Thus, the absorption data are consistent with the hypothesis that the pyrene moieties of
monomers 122W-122Z intercalate into the duplex core upon hybridization with DNA targets.
Table 64
Pyrene Absorption Maxima of Oligonucleotides Modified With 122Y or 122Z in the
Absence (SSP) or Presence of Matched (M) or Centrally Mismatched (MM) DNA Targets.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
λ /nm (λ )
max max
Sequence
SSP +M (A) +MM (C) +MM (G) +MM (T)
’-CGCAA A(122Y)A AACGC 348 349 (+1) 349 (+1) 349 (+1) 349 (+1)
’-CGCAA C(122Y) C AACGC 347 349 (+2) 349 (+2) 350 (+3) 350 (+3)
’-CGCAA G(122Y) G AACGC 348 349 (+1) 349 (+1) 349 (+1) 349 (+1)
’-CGCAA T(122Y) T AACGC 346 349 (+3) 349 (+3) 349 (+3) 349 (+3)
’-CGCAA A(122Z)A AACGC 346 348 (+2) 349 (+3) 349 (+3) 349 (+3)
’-CGCAA C(122Z)C AACGC 346 349 (+3) 349 (+3) 349 (+3) 347 (+1)
’-CGCAA G(122Z)G AACGC 347 348 (+1) 350 (+3) 349 (+2) 350 (+3)
’-CGCAA T(122Z)T AACGC 346 348 (+2) 348 (+2) 349 (+3) 349 (+3)
Recorded in T -buffer at T = 20 °C using 1.0 µM of each strand. SSP = single-stranded probe.
Nucleotide opposite of modification is mentioned in parenthesis. “M” = matched, “MM” =
mismatched.
Next, steady-state fluorescence emission spectra and fluorescence emission quantum yields
were determined for oligonucleotides modified with 122W/X/Y/Z in absence or presence of
complementary or centrally mismatched DNA targets ( and Table 65).
Single-stranded probes modified with monomer 122W display two structured emission
peaks at λ ~ 390 nm and 405 nm (). The single-stranded probe with a central A122WA-
context has higher fluorescence quantum yield than SSPs in other contexts (Φ = 0.27 vs
0.07/0.05/0.05, Table 65). The spectra of the corresponding duplexes with complementary DNA
have a similar shape and sequence dependency, affirming that the pyrene moiety is in close contact
with the neighboring nucleobases (, Table 65). The extensive decreases in fluorescence
quantum yield (Table 65) upon hybridization with matched or mismatched DNA targets further
corroborate this hypothesis. The probe with T122WT-context exhibits considerably smaller changes,
presumably since the fluorophore interacts with the neighboring and only weakly quenching adenine
moieties upon target binding ().
Fluorescence emission spectra of single-stranded probes modified with monomer 122X and
the corresponding duplexes with complementary or mismatched DNA targets display broad and
unstructured emission peaks with maxima at λ ~ 490 nm (). SSPs are strongly quenched
with the probe having the G(122X)G-context) displaying the lowest intensity (Φ < 0.04, Table 65).
Quantum yields are markedly increased upon hybridization of the probes with A(122X)A- and
T(122X)T-sequence contexts with complementary/mismatched DNA targets (Table 65). In contrast,
probes with C(122X)C- and G(122X)G-sequence contexts display hybridization-induced decreases in
fluorescence intensity (Table 65). One interpretation of these observations is that the conjugated
pyrene moiety of monomer 122X intercalates into the base stack where it is quenched by neighboring
cytosine and guanine moieties (but not quenched by adenine and thymine moieties.
The fluorescence emission spectra of single-stranded probes modified with monomer 122Y
and the corresponding duplexes with matched or mismatched DNA targets, display two well-resolved
6478592_1 (GHMatters) P95976.NZ ESTHERJ
pyrene peaks at λ ~380 nm and 400 nm, with an additional shoulder at λ ~ 420 nm ().
em em
Very low quantum yields are observed (Φ < 0.03, Table 65), except for the single-stranded probe
with A(122Y)A sequence context. Hybridization of single-stranded probes modified with monomer
122Y with complementary or mismatched DNA targets generally results in decreased fluorescence
intensity, which is consistent with an intercalating binding mode for the pyrene moiety.
The fluorescence emission spectra of single-stranded probes modified with monomer 122Z
the corresponding duplexes with matched or mismatched DNA targets display an unstructured peak
at λ ~ 410 nm with a weaker shoulder at λ ~ 390 nm (). The quantum yields of SSPs
em em
range from moderate to high and closely align with the previously discussed quenching trends of
nucleobases (Φ = 0.05-0.58, Table 65). Hybridization with matched or mismatched DNA targets
generally results in decreases or minor increases in quantum yields and intensity (Table 65).
Table 65
Relative Fluorescence Emission Quantum Yield (Φ Φ Φ Φ ) of Single-Stranded Probes Modified with
Monomer 122W/X/Y/Z in the Absence (SSP) or Presence of Matched (M) or
Centrally Mismatched (MM) DNA Targets.
Sequence SSP +M (A) +MM (C) +MM (G) +MM (T)
’-CGCAA A(122W)A AACGC 0.27 0.08 0.09 0.06 0.08
’-CGCAA C(122W)C AACGC 0.07 0.02 0.02 0.02 0.01
’-CGCAA G(122W)G AACGC 0.05 0.03 0.01 0.02 0.01
’-CGCAA T(122W)T AACGC 0.05 0.07 0.06 0.06 0.04
’-CGCAA A(122X)A AACGC 0.02 0.25 0.33 0.10 0.25
’-CGCAA C(122X)C AACGC 0.02 <0.01 <0.01 <0.01 <0.01
’-CGCAA G(122X)G AACGC <0.01 <0.01 <0.01 <0.01 <0.01
’-CGCAA T(122X)T AACGC 0.04 0.16 0.35 0.04 0.32
’-CGCAA A(122Y)A AACGC 0.09 0.02 0.02 0.02 0.03
’-CGCAA C(122Y)C AACGC 0.01 0.02 <0.01 <0.01 <0.01
’-CGCAA G(122Y)G AACGC 0.03 0.01 0.01 0.01 <0.01
’-CGCAA T(122Y)T AACGC 0.01 <0.01 <0.01 <0.01 <0.01
’-CGCAA A(122Z)A AACGC 0.58 0.52 0.78 0.29 0.79
’-CGCAA C(122Z)C AACGC 0.24 0.15 0.17 0.15 0.19
’-CGCAA G(122Z)G AACGC 0.05 0.04 0.02 0.02 0.03
’-CGCAA T(122Z)T AACGC 0.27 0.57 0.58 0.31 0.52
Relative to quantum yield of anthracene in ethanol (0.27). Recorded in T -buffer at T = 20 °C
using 1.0 µM concentration of each strand and λ = 350 nm and λ = 360-510 nm (monomers
ex em
122W, 122Y and 122Z) or λ = 400 nm and λ = 425-625 nm (monomer 122X). Nucleotide
ex em
opposite of modification is mentioned in parenthesis.
Very similar quantum yields are observed for the four duplexes between a particular probe
and matched/mismatched DNA targets Collectively, these observations indicate a) that the
fluorophore is in a similar electronic environment within the duplex core regardless of the nucleotide
6478592_1 (GHMatters) P95976.NZ ESTHERJ
opposite of the monomer, and b) that the opposing nucleotide is not strongly involved in base pairing
and possibly even pushed into an extrahelical position (). Along the lines, it is interesting to
note that placement of pyrene-functionalized C-glycosides in DNA duplexes opposite of abasic sites,
which are generated via enzyme-mediated extrahelical flipping of the opposing nucleotide, is known
to be stabilizing.
Universal hybridization – RNA targets: A representative subset of modified oligonucleotides
(TBT/CBT-contexts) was studied with respect to thermal denaturation, absorption and fluorescence
properties with complementary/mismatched RNA targets. Briefly described: a) incorporation of
monomer 122W or 122X into oligonucleotides results in similar decreases in thermal affinity toward
complementary RNA as toward DNA, while oligonucleotides modified with monomers 122Y or
122Z are more destabilizing (Table 66); b) oligonucleotides modified with monomers 122W or 122X
display robust universal hybridization characteristics (compare ‘mismatch ΔT ’-values for
ON6/ON8/ON12 and ON2/ON4, Table 66), while oligonucleotides modified with monomers 122Y
or 122Z do not; c) pyrene absorption spectra of duplexes between modified oligonucleotides and
complementary or centrally mismatched RNA targets are very similar to those of the corresponding
DNA duplexes;; and d) hybridization of modified oligonucleotides to RNA targets results in very
similar changes in fluorescence intensity as with DNA targets.
Thus, the results indicate that the universal RNA hybridization characteristics of
oligonucleotides modified with monomer 122W/122X also are be governed by a similar mechanism
as universal DNA hybridization ().
Table 66
T -Values of Duplexes Between Centrally Modified Oligonucleotides and
Complementary or Centrally Mismatched RNA Targets.
T (ΔT ) [°C] Mismatch ΔT [°C]
m m m
ON Sequence B= A C G U
2 5’-CGCAA CTC AACGC 51.5 -15.5 -3.0 -13.5
4 5’-CGCAA TTT AACGC 40.5 -19.0 -3.5 -17.0
6 5’-CGCAA C122WC AACGC 47.0 (-4.5) +1.0 +0.0 +0.5
8 5’-CGCAA T122WT AACGC 42.0 (+1.5) +3.0 +0.5 +1.5
12 5’-CGCAA T122XT AACGC 38.0 (-2.5) +1.0 +0.0 +1.0
14 5’-CGCAA C122YC AACGC 43.5 (-8.0) -2.0 -5.0 -4.0
16 5’-CGCAA T122YT AACGC 36.0 (-4.5) +1.5 -1.0 -1.0
18 5’-CGCAA C122ZC AACGC 44.5 (-7.0) -3.0 -8.0 -7.0
5’-CGCAA T122ZT AACGC 36.5 (-4.0) -12.0 -7.5 -13.0
RNA targets: 3’-GCGUU GBG UUGCG and 3’-GCGUU ABA UUGCG.
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Synthesis of 260. Co-evaporated 82Q (3.40g, 4.39 mmol) with anhydrous 1,2-dicholoethane (2 x 30
mL), redissolved in anhydrous pyridine (55 mL) and cooled to 0°C. Dimethylaminopyridine
(DMAP, 55 mg, 0.44 mmol) was added followed by dropwise addition of acetic anhydride (1.25 mL,
13.18 mmol). After stirring at rt for 12 h, the reaction mixture was diluted with EtOAc (150 mL) and
washed with H O (80 mL) and sat. aq. NaHCO (80 mL). The product was co-evaporated with
EtOH:toluene (2:1, 3 x 30 mL) and suspended in AcOH:H O (4:1, 55 mL) and stirred overnight. The
reaction mixture was then evaporated to dryness and purified via silica gel column chromatography
(0-5% MeOH/CH Cl , v/v). The appropriate fractions were pooled and evaporated to dryness. The
product was then coevaporated in anhydrous pyridine:CH Cl (1:1, 2 x 30 mL), redissolved in
pyridine:CH Cl (1:1, 44 mL) and cooled to -20°C. Methanesulfonyl chloride (MsCl, 0.75 mL, 9.62
mmol) was added dropwise over 30 min and allowed to stir at -20°C for 2h whereupon the crude was
diluted with CH Cl (80 mL) and washed with sat. aq. NaHCO3 (50 mL). The aqueous layer was
back-extracted with CH Cl (3 x 15 mL) and the organic layers were evaporated to dryness via co-
evaporation with EtOH:toluene (2:1, 3 x 30 mL). The crude was purified by silica gel column
chromatography (0-3% MeOH/CH Cl , v/v) to afford nucleoside 260 (1.43 g, 55%) as a white foam.
+ . +
R : 0.4 (5%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 614.1600 ([M+Na] , C H N O S Na ,
f 2 2 30 29 3 8
Calc. 614.1568); ); H NMR (DMSO-d ) δ 11.48 (d, IH, ex, J = 2.20 Hz, NH), 8.03-8.36 (m, 8H, py),
7.93 (d, 1H, J = 7.96 Hz, py), 7.73 (d, 1H, J = 8.23 Hz, H6), 6.39 (d, 1H, J = 7.41, H1’), 5.67 (dd, 1H,
J = 2.20 Hz, 8.23 Hz, H5), 5.39 (dd, 1H, J = 3.57 Hz, 6.56 Hz, H3’), 4.33-4.38 (m, 5H, CH Py, H4’,
H5’), 3.83 (dd, 1H, J = 6.59 Hz, 7.41 Hz, H2’), 3.20 (s, 3H, CH (Ms)), 2.33 (s, 3H, NCH ), 2.13 (s,
3H, CH (Ac)); C NMR (DMSO-d ) δ 169.7, 162.7, 150.5, 140.6 (C6), 131.9, 130.7, 130.3, 130.2,
129.1, 127.7 (py), 127.3 (py), 127.0 (py), 126.8 (py), 126.1 (py), 125.1 (py), 124.4 (py), 124.2, 123.8,
123.7 (py), 102.6 (C5), 83.7 (C5), 79.9 (C4’), 71.7 (C3’), 69.1 (C5’), 64.9 (C2’), 57.5 (CH Py), 37.7
(NCH ), 36.8 (CH , Ms), 20.9 (CH , Ac).
3 3 3
Synthesis of 262. Nucleoside 260 (770 mg, 1.30 mmol) was coevaporated in anhydrous 1,2-
dicholoethane (3 x 6 mL) and suspended in absolute EtOH (25 mL) whereupon NaHCO (275 mg,
3.25 mmol) was added and the suspension was heated to reflux under Argon atmosphere for 4 days.
The reaction mixture was then diluted with CH Cl and the salts were filtered and washed with
CH Cl . The CH Cl layer was evaporated to dryness and purified via silica gel column
2 2 2 2
chromatography (0-7% MeOH/CH Cl , v/v) to afford nucleoside 262 (338 mg, 52%) as a white foam.
+ . +
R : 0.3 (10%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 522.1985 ([M+Na] , C H N O Na ,
f 2 2 29 29 3 5
Calc. 522.1999); ); H NMR (DMSO-d ) δ 8.40 (d, 1H, J = 9.33 Hz, Py), 8.24-8.29 (m, 2H, Py), 8.21
(d, 1H, J = 7.96 Hz, Py), 8.13-8.16 (m, 2H, Py), 8.05-8.11 (m , 2H, Py), 7.97 (d, 1H, J = 7.96 Hz, Py),
7.92 (d, 1H, J = 7.68 Hz, H6), 6.34 (d, 1H, J = 8.51 Hz, H1’), 5.82 (d, 1H, J = 7.68 Hz, H5), 5.53 (d,
1H, ex, J = 4.94 Hz, 3’-OH), 5.11 (t, 1H, ex, J = 5.21 Hz, 5’-OH), 4.42-4.52 (m, 3H, CH Py, H3’),
4.11-4.28 (m, 2H, CH CH ), 3.97-4.02 (m, 1H, H4’), 3.57-3.63 (m, 2H, H5’), 3.47 (dd, 1H, J = 5.21
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Hz, 8.51 Hz, H2’), 2.36 (s, 3H, NCH ), 1.07 (t, 3H, J = 7.14 Hz, CH CH ); C NMR (DMSO-d ) δ
3 2 3 6
169.3, 155.0, 138.1 (C6), 132.7, 130.7, 130.3, 130.1, 129.0, 127.5 (Py), 127.3 (Py), 126.9 (Py), 126.8
(Py), 126.1 (Py), 125.02 (Py), 125.00 (Py), 124.4 (Py), 124.1, 123.8, 123.6 (Py), 108.4 (C5), 87.6
(C4’), 85.0 (C1’), 71.2 (C3’), 68.8 (C2’), 64.2 (CH , OEt), 61.7 (C5’), 57.1 (CH Py), 39.0 (NCH ),
2 2 3
13.6 (CH , OEt).
Synthesis of 264. Compound 262 (315 mg, 0.63 mmol) was coevaporated with anhydrous 1,2-
dicholoethane (2 x 5 mL) and redissolved in anhydrous pyridine (6 mL). Added DMTr-Cl (260 mg,
0.76 mmol) and DMAP (8 mg, 0.06 mmol) and let stir at ambient temperature for 14 h whereupon the
crude mixture was diluted with CHCl (80 mL) and washed with sat. aq. NaHCO (30 mL) and H O
3 3 2
(30 mL). The aqueous layer was back-extracted with CHCl (3 x 15 mL) and the combined organic
layers dried over Na SO and evaporated to dryness. The crude was purified by silica gel column
chromatography (0-2.5 % MeOH/CH Cl , v/v) to afford nucleoside 264 (445 mg, 88%) as a pale
orange foam. R : 0.7 (10%, MeOH in CH Cl , v/v); MALDI-HRMS m/z 824.3319 ([M+Na] ,
f 2 2
. + 1
C H N O Na , Calc. 824.3306); ); H NMR (DMSO-d ) δ 8.40 (d, 1H, J = 9.33 Hz, Py), 8.24-8.30
50 47 3 7 6
(m, 2H, Py), 8.18 (d, 1H, J = 7.96 Hz, Py), 8.14 (s, 2H, Py) 8.03-8.11 (m, 2H, Py), 7.99 (d, J = 7.68
Hz, Py), 7.68 (d, 1H, J = 7.68 Hz, H6), 7.19-7.39 (m, 9H, DMTr), 6.83-6.90 (m, 4H, DMTr), 6.32 (d,
1H, J = 7.96 Hz, H1’), 5.59-5.64 (m, 2H, 3’-OH, H5), 4.51 (m, 2H, CH Py), 4.42-4.48 (m, 1H, H3’),
4.08-4.24 (m, 3H, H4’, CH CH ), 3.71 (s, 3H, OCH ), 3.70 (s, 3H, OCH ), 3.51-3.58 (m, 1H, H2’),
2 3 3 3
3.31-3.35 (m, 1H, H5’ ), 3.14-3.20 (m, 1H, H5’ ), 2.42 (s, 3H, NCH ), 1.05 (t, 3H, CH CH ); C
A B 3 2 3
NMR (DMSO-d ) δ 169.2, 158.09, 158.08, 154.9, 144.5, 138.1 (C6), 135.3, 135.0, 132.6, 130.7,
130.2, 130.1, 129.7 (Ar), 129.6 (Ar), 129.0, 127.8 (Ar), 127.6 (Ar), 127.5 (Ar), 127.3 (Ar), 126.9
(Ar), 126.8 (Ar), 126.7 (Ar), 126.1 (Ar), 125.0 (Ar), 124.4 (Ar), 124.1, 123.9, 123.6 (Ar), 113.2 (Ar),
113.1 (Ar), 108.1 (C5), 85.9, 85.7 (C4’), 85.3 (C1’), 71.0 (C3’), 68.1 (C2’), 64.2 (CH CH ), 63.9
(C5’), 57.1 (CH Py), 55.0 (OCH ), 38.8 (NCH ), 13.6 (CH CH ).
2 3 3 2 3
Synthesis of 266. Nucleoside 264 (435 mg, 0.54 mmol) was flushed with argon and cooled to 0°C
over a freezing bath. A solution of 1,1,3,3-tetramethylguanidine (TMG, 0.68 mL, 5.42 mmol) in
anhydrous pyridine (10 mL) was flushed with argon and cooled to 0°C over a freezing bath. After
cooling, the pyridine solution was saturated with hydrogen sulfide for 1 h while maintaining the
temperature of the bath below 0°C. The solution was then transferred to the pre-cooled flask
containing nucleoside 264 and allowed to reach room temperature while stirring for 72 h whereupon
the mixture was diluted with EtOAc (100 mL) and washed with conc. aq. NaHCO (50 mL) and H O
(50 mL). The aqueous layer was then back-extracted with CH Cl (3 x 20 mL) and the combined
organic layers were evaporated to dryness and co-evaporated with EtOH:toluene (2:1, 3 x 15 mL).
The crude was then purified via silica gel column chromatography (0-70% EtOAc/ petroleum ether,
v/v) to afford nucleoside 268 (353 mg, 82%) as a white foam. R : 0.8 (80%, EtOAc in petroleum
6478592_1 (GHMatters) P95976.NZ ESTHERJ
+ . + 1
ether, v/v); MALDI-HRMS m/z 812.2765 ([M+Na] , C H N O S Na , Calc. 812.2797);; H NMR
48 43 3 6
(DMSO-d ) δ 12.76 (br d, 1H, ex, J = 1.65 Hz, NH), 8.50 (d, 1H, J = 9.33 Hz, Py), 8.01-8.30 (m, 8H,
Py), 7.74 (d, 1H, J = 8.23 Hz, H6), 7.18-7.41 (m, 10H, DMTr, H1’), 6.81-6.91 (m, 4H, DMTr), 5.61
(dd, 1H, J = 1.65 Hz, 8.23 Hz, H5), 5.54 (d, 1H, ex, J = 4.94, 3’-OH), 4.42-4.58 (m, 3H, CH Py,
H3’), 4.06-4.11 (m, 1H, H4’), 3.71 (s, 3H, OCH ), 3.70 (s, 3H, OCH ), 3.45-3.53 (m, 1H, H2’), 3.30-
3.37 (m, 1H, H5’ ), 3.15-3.24 (m, 1H, H5’ ), 2.43 (s, 3H, NCH ); C NMR (DMSO-d ) δ 176.6,
A B 3 6
159.0, 158.1, 144.5, 140.8 (C6), 135.3, 135.0, 132.6, 130.7, 130.3, 130.2, 129.7 (Ar), 129.6 (Ar),
129.2, 128.0 (Ar), 127.9 (Ar), 127.6 (Ar), 127.3 (Ar), 126.9 (Ar), 126.8 (Ar), 126.7 (Ar), 126.1 (Ar),
125.0 (Ar), 124.9, 124.4 (Ar), 124.1, 123.9 (Ar), 123.8, 113.24 (Ar), 113.21 (Ar), 106.9 (C5), 88.0
(C1’), 86.0, 85.3 (C4’), 70.9 (C3’), 68.6 (C2’), 63.9 (C5’), 57.6 (CH Py), 55.0 (OCH ), 39.2 (NCH ).
2 3 3
Synthesis of 268. Nucleoside 266 (150 mg, 0.19 mmol) was added to a flame dried 10 mL round-
bottom flask and dissolved in an. CH Cl (2 mL) whereupon anhydrous diisopropylethylamine
(DIPEA, 165 µL, 0.95 mmol) and 2-cyanoethyl diisopropylchlorophosphoramidite (PCl-reagent, 85
µL, 0.38 mmol) were added. The reaction mixture was stirred at ambient temperature under an argon
atmosphere for 3.5 h whereupon it was quenched with 1 mL cold EtOH, evaporated to dryness, and
purified by silica gel column chromatography (0-55% EtOAc/petroleum ether (v/v) followed by
precipitation in cold petroleum ether to afford nucleoside 268 (153 mg, 81%) was a white foam. R :
+ . +
0.6 (50%, EtOAc in Pet Ether, v/v); MALDI-HRMS m/z 1012.3843 ([M+Na] , C H N O PS Na ,
57 60 5 7
Calc. 1012.3887); P NMR (CDCl ) δ 150.9, 149.6.
Synthesis of single-stranded probes modified with monomer 270. Briefly, the monomer was
introduced into probes using compound 268, MeCN as solvent ansd DCI as an activator (15 min
hand-coupling, 90-99% yield). 10% tert-butylhydroperoxide (tBuOOH) in an. MeCN was used as an
oxidizer (2 x 5 min oxidation, 10:87:3, tBuOOH, MeCN, H O) whereas 2,6-aminopurine 2’-
deoxyriboside monomers were coupled using MeCN and 4,5-dicyanoimidazole activator, 15 min, 95-
99%. Strands were cleaved from solid support 15-17h, rt, NH OH. Purified via RP-HPLC, with DMT
cleavage and precipitation overnight in freezer.
Probes modified with non-pairing (or bulged) monomers 402-4, 402-N or 402-9. Particular
embodiments pertain to probes that are modified with monomers 402-4, 402-N or 402-9. These
monomers were incorporated into probes (e.g., oligonucleotides) using 400-4, 400-N or 400-9 as
suggested by commercial vendors. The composition of the probes was verified by MALDI-MS
analysis (Table 67) whereas the purity (>80%, unless stated otherwise) was verified by ion-exchange
HPLC using a LaChrom L-7000 system (VWR International) equipped with a Gen-Pak Fax column
(100 mm × 4.6 mm). A representative protocol involves the use of an isocratic hold of 95% A-buffer
6478592_1 (GHMatters) P95976.NZ ESTHERJ
Claims (1)
- CLAIMS : 1. A double stranded probe, comprising: a pair of monomers comprising a first monomer having a formula Linker Optional 1 b b where Y is selected from carbon, oxygen, sulfur, and NR , wherein R is selected from hydrogen, 2 3 4 aliphatic, aryl, heteroaliphatic, and heteroaryl; each of Y , Y , and Y independently is selected from carbon, oxygen, sulfur, a triazole, oxazole, tetrazole, isoxazole, and NR , wherein R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; R and R are selected from hydrogen, 10 aliphatic, aryl, aryl aliphatic, and a heteroatom-containing moiety, or R is selected from a heteroatom-containing functional group; R is a heteroatom-containing functional group; R is selected from any natural or non-natural nucleobase; R is selected from an intercalator suitable for intercalating within a nucleic acid selected from a hydrocarbon or an aromatic heterocycle; “optional linker” is selected from linkers comprising alkyl linkers, amide linkers, carbonyl linkers, carbamate 15 linkers, carbonate linkers, urea linkers, and combinations thereof; a second monomer having a formula Linker Optional 1 2 3 4 1 2 3 4 5 wherein Y , Y , Y , Y , R , R R , R , R , and “optional linker” are as stated for the first monomer; V is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4; and 20 wherein the first monomer is positioned in a first strand of the double-stranded probe and the second monomer is positioned in a second strand of the double stranded probe and wherein each of the first strand and the second strand comprises at least one nucleotide selected from a natural nucleotide, a non-natural nucleotide, and combinations thereof. 25 2. The probe according to claim 1 wherein the heteroatom-containing moiety is a b a a b c d a b c selected from ether (R OR ), hydroxyl (R OH), silyl ether (R R R SiOR ), phosphine (PR R R ), a a b a b a thiol (R SH), thioether/sulfide (R SR ), disulfide (R SSR ), isothiocyanate (R NCS), isocyanate a a a b a b c a b (R NCO), amine (NH , NHR , NR R ), amide (R NR C(O)R ), ester (R OC(O)R ), halogen (I, Br, Cl, a b a a - a b F), carbonate (R OC(O)OR ), carboxyl (R C(O)OH), carboxylate (R COO ), ester (R C(O)OR ), a b a a a b 30 ketone (R C(O)R ), phosphate (R OP(O)OH ), phosphoryl (R P(O)(OH) ), sulfinyl (R S(O)R ), a b a b a a a sulfonyl (R SO R ), carbonothioyl (R C(S)R or R C(S)H), sulfino (R S(O)OH), sulfo (R SO H), a b c a a + - a a amide (R C(O)NR R ), azide (N ), nitrile (R CN), isonitrile (R N C ), and nitro (R NO ); R represents the remaining monomer structure, which is attached to the abovementioned functional 1 b c d groups at the position indicated for R ; and R , R , and R independently are hydrogen, aliphatic, aryl, 5 heteroaliphatic, heteroaryl, and any combination thereof. 3. The probe according to claim 1 or 2 wherein R and R independently are selected from a heteroatom-containing functional group comprising phosphorous, sulfur, nitrogen, oxygen, selenium, and/or a metal. 4. The probe according to claim 1 or 2 wherein R and R independently are selected from a phosphate group of a natural nucleotide, non-natural nucleotide, non-nucleosidic linker, or combinations thereof. 15 5. The probe according to claim 1 or 2 wherein R and R independently have a formula where each Y independently is selected from oxygen, sulfur, NR where R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, heteroaryl, and W is selected from phosphorus, SH, or SeH. 6. The probe according to claim 1 or 2 wherein R and R independently are S P O P , or . 7. The probe according to claim 1 or 2 where R and R independently have a formula where W is phosphorus, and each Z independently is selected from ether, thioether, hydroxyl, and NR . 8. The probe according to claim 1 or 2 wherein R and R independently 30 are . 9. The probe according to any one of claims 1 to 8 wherein R is selected from adenine, guanine, cytosine, uracil, thymine, 2-thiouracil, 2,6-diaminopurine, inosine, 3-pyrrolo-[2,3- d]-pyrimdine(3H)-one, or any derivative thereof. 5 10. The probe according to claim 1 wherein the intercalator is a hydrocarbon selected from pyrene, coronene, perylene, anthracene, naphthalene, and functionalized derivatives thereof. 11. The probe according to claim 1 wherein the intercalator is an aromatic heterocycle selected from a porphyrin, nucleobase, metal chelator, azapyrene, thiazole orange, ethidium, an 10 indole, a pyrrole, a benzimidizole, and modified analogs thereof. 12. The probe according to claim 1 wherein the second monomer has a formula Linker Optional 15 13. The probe according to claim 1 wherein either the first monomer or the second monomer has a formula Linker O Optional 14. The probe according to claim 1 wherein the second monomer has a formula Linker Optional 15. The probe according to claim 1 wherein either the first monomer or the second monomer has a formula wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof. 16. The probe according to claim 1 wherein either the first monomer or the second 10 monomer has a formula O N R wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof, and alk is C1-C10 alkyl. 17. The probe according to claim 1 wherein the second monomer has a formula wherein B is selected from uracil, guanine, cytosine, adenine, or thymine. 20 18. The probe according to claim 1 wherein either the first monomer or the second monomer has a formula 19. The probe according to claim 1 wherein either the first monomer or the second monomer has any one of the following formulas: e R O R O O OR O O Na P P p y or y R O R O N O OR N N OR R O B e R O B R N f N O NH P 2 OR N P O R O B OR N N OR N e e e e R O B R O B R O B R O B O O O O O O f O f f OR OR OR OR R O R O OR OR Nap Py R N f OR N R O O OR N wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H, 5 DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or 2 2 2 phosphate. 20. The probe according to claim 1 wherein the second monomer has any one of the following formulas: e moc 10 . 21. The probe according to claim 1 wherein the at least one natural nucleotide is selected from adenine, guanine, cytosine, uracil, and thymine. 15 22. The probe according to claim 1 wherein the at least one non-natural nucleobase is selected from C-5 functionalized pyrimidines, C6-functionalized pyrimidines, C7-functionalized 7- deazapurines, C8-functionalized purines, 2,6-diaminopurine, 2-thiouracil, 4-thiouracil, deoxyinosine and 3-(2’-deoxy- β-D-ribofuranosyl)pyrrolo-[2,3-d]-pyrimdine(3H)-one. 20 23. The probe according to claim 1 wherein the at least one natural nucleotide, unnatural nucleotide, and combinations thereof is selected to substantially match at least one nucleotide of a corresponding nucleic acid sequence. 24. The probe according to any one of claims 1 to 23 having a formula 5’(B B …B )(XA)(B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )(XA) (B B …B )( 1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r XA) (B B …B ) j 1 2 s 3’(C C …C )(DP)(C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )(DP) (C C …C )( 1 2 m 1 2 n f 1 2 o g 1 2 p h 1 2 q i 1 2 r DP) (C C …C ) j 1 2 s 5 wherein B , B and B may be any natural, non-natural nucleotide, or a non-nucleosidic linker, 1 2 m-s wherein m-r ranges from zero to about 28; f, g, h, i and j range from zero to 10; X is the first monomer; A is the complement Watson-Crick base pairing nucleotide of P; C is any natural or non- natural nucleotide capable of Watson-Crick base pairing with any one of B , B and B ; P is the 1 2 m-s second monomer, and D is the complement Watson-Crick base pairing nucleotide of X. 25. The probe according to any one of claims 1 to 24 wherein the probe is selected to recognize a predetermined sequence of a nucleic acid target. 26. The probe according to claim 25 wherein the nucleic acid target is single-stranded 15 or double-stranded. 27. The probe according to any one of claims 1 to 26 further comprising one or more bulge monomers that do not participate in base pairing. 20 28. The probe according to claim 27 wherein the one or more bulge monomers are selected from OR r 29. The probe according to any one of claims 1 to 28 wherein the first monomer and 25 the second monomer are arranged in a manner that substantially weakens the thermal stability of a duplex comprising two strands of oligonucleotides comprising one or more of the first monomer and the second monomer. 30. The probe according to claim 29 wherein the duplex has a thermal melting temperature which is substantially similar to, or lower than, that of a corresponding unmodified nucleic acid duplex. 5 31. The probe according to claim 30 wherein the corresponding unmodified nucleic acid duplex does not comprise a monomer having the formula of claim 1. 33. The probe according to claim 1 wherein the first monomer and the second monomer are arranged in a +n or -n zipper orientation, wherein n ranges from 0 to about 10. 33. The probe according to claim 32 wherein the first monomer and the second monomer are arranged in a +n orientation, wherein n is 1. 34. The probe according to any one of claims 1 to 33 further comprising one or more 15 additional pairs of monomer pairs. 35. The probe according to any one of claims 1 to 34 further comprising a signal generating moiety capable of being detected. 36. The probe according to claim 35 wherein the signal generating moiety is selected 20 from a fluorophore, a member of a specific binding pair, a nanoparticle, and combinations thereof. 37. The probe according to claim 36 wherein the member of a specific binding pair is biotin. 25 38. The probe according to any one of claims 1 to 37 further comprising a secondary entity selected from a secondary entity that facilitates cell-uptake; a quencher; a crosslinking reagent capable of forming bonds between the probe and nucleic acids, proteins, sugars, lipids or other biomolecules; a nucleic acid cargo selected from single-stranded DNA, single-stranded RNA, double- stranded DNA, double-stranded RNA, plasmid, or gene; and combinations thereof. 39. The probe according to any one of claims 1 to 38 wherein the probe is used in solution, on a solid surface, or in combination with a colloidal material. 40. A single stranded probe precursor when used to make the double stranded probe of 35 claim 1, comprising at least one monomer selected from OR N f OR N or O y R O B R O B OR N NH f OR N 2 OR N R O B R O B OR N N N OR N N y e e e R O B R O B R O B R O B O O O O O O O O f f f f OR OR OR R O R O O O R OR N f OR OR Nap Py C R O O OR N wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H, 10 DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or 2 2 2 phosphate; or OR O wherein B is selected from guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-diaminopurine, inosine, or 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one; Nap is napthyl; Py is pyrenyl; R is H, DMTr, or phosphate; and R is H, (N(i-Pr) )P(OCH CH CN), or phosphate. 2 2 2 41. The single stranded probe precursor according to claim 40 further comprising a second monomer selected from e moc e B O OR N f OR N OR O O Na P P p y or O y R O B R O B OR N NH f OR N 2 OR N R O B R O B OR N N N OR N N y e e e R O B R O B R O B R O B O O O O O O O O f f f f OR OR OR R O R O OR OR Nap Py R N f OR N R O O OR N wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- 5 diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H, DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or 2 2 2 phosphate. 42. A single stranded probe precursor when used to make the double stranded probe of 10 claim 1, wherein the single stranded probe precursor has a sequence selected from any one of SEQ ID Nos. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239 and 246-251. 43. A double stranded probe according to claim 1 for use in associating the probe with a nucleic acid target. 44. The probe according to claim 43 wherein the double stranded probe is selected to substantially match a target nucleic acid sequence. 45. The probe according to any one of claims 43-44 wherein the nucleic acid target 20 comprises one or more polypurine units. 46. The probe according to any one of claims 43-44 wherein the nucleic acid target does not comprise a polypurine unit. 47. The probe according to any one of claims 43-46 wherein the nucleic acid target is a mixed-sequence, structured nucleic acid. 48. The probe according to any one of claims 43-47 wherein the nucleic acid target is a 5 mixed-sequence, hairpin nucleic acid target. 49. The probe according to any one of claims 43-48 wherein the nucleic acid target is isosequential with the double stranded probe. 10 50. The probe according to any one of claims 43-48 wherein the nucleic acid target is incubated with the double stranded probe. 51. The probe according to any one of claims 43-50 wherein the nucleic acid target is incubated with about a 5-fold excess of the double stranded probe to a 5,000,000-fold excess of the 15 double stranded probe. 52. The probe according to any one of claims 43-51 wherein the nucleic acid target is incubated with about a 5-fold excess of the double stranded probe to about a 500-fold excess of the double stranded probe. 53. The probe according to any one of claims 43-52 wherein a double stranded probe- nucleic acid target complex formed between the double stranded probe and the nucleic acid target is detected by fluorescence spectroscopy, electrophoresis, absorption spectroscopy, flow cytometry, and combinations thereof. 54. The probe according to any one of claims 43-53 wherein the double stranded probe is selected from SEQ. ID Nos. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, and 246-253. 30 55. The probe according to any one of claims 43-54 wherein the nucleic acid target is single-stranded or double-stranded. 56. The probe according to any one of claims 43-55 wherein the double stranded probe further comprises one or more bulge monomers that do not participate in base pairing. 57. The probe according to claim 56 wherein the one or more bulge monomers are selected from OR or 58. The probe according to any one of claims 43-57 wherein the monomer is positioned 5 in the double stranded probe in a manner that substantially weakens a duplex’s thermal stability. 59. The probe according to claim 58 wherein the duplex has a thermal melting temperature which is substantially similar to, or lower than, that of a corresponding unmodified nucleic acid duplex. 60. The probe according to any one of claims 43-59 wherein a first monomer and second monomer are arranged in a +n or -n zipper orientation, wherein n ranges from 0 to about 10. 61. The probe according to claim 60 wherein the first monomer and the second 15 monomer are arranged in a +n orientation, wherein n is 1. 62. The probe according to any one of claims 43-61 wherein the nucleic acid target is a DNA target selected from second insulin, PPAR gamma, and CEBP promoters. 20 63. The probe according to any one of claims 43-62 wherein the double stranded probe is used for gender determination. 64. The probe according to claim 63 wherein the double stranded probe is used for gender determination in mammals. 65. The probe according to claim 64 wherein the mammals are ungulates. 66. The probe according to claim 64 wherein the mammals are ruminants. 67. The probe according to claim 64 wherein the mammals are bovines, equines or porcines. 68. The probe according to any one of claims 43-67 wherein the nucleic acid target is 5 isosequential (relative to a probe) double stranded DNA target regions, including stems of molecular beacons, target regions embedded within PCR amplicons, target regions embedded within circular or linearized plasmids, target regions embedded within genomic DNA, and target regions embedded within microorganisms. 10 69. The probe according to any one of claims 43-68 wherein the nucleic acid target is selected from a nucleic acid sequence associated with B cell and T cell leukemias, lymphomas, breast cancer, colon cancer, and neurological cancers. 70. The probe according to any one of claims 43-68 wherein the nucleic acid target is 15 selected from the EGFR gene (7p12; e.g., GENBANK™ Accession No. NC_000007, nucleotides 55054219-55242525), the C-MYC gene (8q24.21; e.g., GENBANK™ Accession No. NC_000008, nucleotides 128817498-128822856), D5S271 (5p15.2), lipoprotein lipase (LPL) gene (8p22; e.g., GENBANK™ Accession No. NC_000008, nucleotides 19841058-19869049), RB1 (13q14; e.g., GENBANK™ Accession No. NC_000013, nucleotides 47775912-47954023), p53 (17p13.1; e.g., 20 GENBANK™ Accession No. NC_000017, complement, nucleotides 7512464-7531642)), N-MYC (2p24; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 151835231-151854620), CHOP (12q13; e.g., GENBANK™ Accession No. NC_000012, complement, nucleotides 56196638-56200567), FUS (16p11.2; e.g., GENBANK™ Accession No. NC_000016, nucleotides 31098954-31110601), FKHR (13p14; e.g., GENBANK™ Accession 25 No. NC_000013, complement, nucleotides 40027817-40138734), as well as, for example: ALK (2p23; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 29269144-29997936), Ig heavy chain, CCND1 (11q13; e.g., GENBANK™ Accession No. NC_000011, nucleotides 69165054..69178423), BCL2 (18q21.3; e.g., GENBANK™ Accession No. NC_000018, complement, nucleotides 58941559-59137593), BCL6 (3q27; e.g., GENBANK™ 30 Accession No. NC_000003, complement, nucleotides 188921859-188946169), MALF1, AP1 (1p32- p31; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides 59019051-59022373), TOP2A (17q21-q22; e.g., GENBANK™ Accession No. NC_000017, complement, nucleotides 35798321-35827695), TMPRSS (21q22.3; e.g., GENBANK™ Accession No. NC_000021, complement, nucleotides 41758351-41801948), ERG (21q22.3; e.g., GENBANK™ 35 Accession No. NC_000021, complement, nucleotides 38675671-38955488); ETV1 (7p21.3; e.g., GENBANK™ Accession No. NC_000007, complement, nucleotides 13897379-13995289), EWS (22q12.2; e.g., GENBANK™ Accession No. NC_000022, nucleotides 27994271-28026505); FLI1 (11q24.1-q24.3; e.g., GENBANK™ Accession No. NC_000011, nucleotides 128069199-128187521), PAX3 (2q35-q37; e.g., GENBANK™ Accession No. NC_000002, complement, nucleotides 222772851-222871944), PAX7 (1p36.2-p36.12; e.g., GENBANK™ Accession No. NC_000001, nucleotides 18830087-18935219), PTEN (10q23.3; e.g., GENBANK™ Accession No. NC_000010, nucleotides 89613175-89716382), AKT2 (19q13.1-q13.2; e.g., 5 GENBANK™ Accession No. NC_000019, complement, nucleotides 45431556-45483036), MYCL1 (1p34.2; e.g., GENBANK™ Accession No. NC_000001, complement, nucleotides 40133685-40140274), REL (2p13-p12; e.g., GENBANK™ Accession No. NC_000002, nucleotides 60962256-61003682) and CSF1R (5q33-q35; e.g., GENBANK™ Accession No. NC_000005, complement, nucleotides 149413051-149473128). 71. The probe according to any one of claims 43-68 wherein the nucleic acid target is selected from a virus or other microorganism and the double stranded probe is used to detect and/or identify the microorganism. 15 72. The probe according to any one of claims 43-68 wherein the nucleic acid target is selected from the genome of an oncogenic or pathogenic virus, a bacterium or an intracellular parasite selected from Plasmodium species, Leishmania (sp.), Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Toxoplasma, Eimeria, Theileria, and Babesia. 20 73. The probe according to any one of claims 43-68 wherein the nucleic acid target is selected from human adenovirus A (NC_001460), human adenovirus B (NC_004001), human adenovirus C (NC_001405), human adenovirus D (NC_002067), human adenovirus E (NC_003266), human adenovirus F (NC_001454), human astrovirus (NC_001943), human BK polyomavirus (V01109; GI:60851) human bocavirus (NC_007455), human coronavirus 229E (NC_002645), human 25 coronavirus HKU1 (NC_006577), human coronavirus NL63 (NC_005831), human coronavirus OC43 ( NC_005147), human enterovirus A (NC_001612), human enterovirus B (NC_001472), human enterovirus C (NC_001428), human enterovirus D (NC_001430), human erythrovirus V9 (NC_004295), human foamy virus (NC_001736), human herpesvirus 1 (Herpes simplex virus type 1) (NC_001806), human herpesvirus 2 (Herpes simplex virus type 2) (NC_001798), human herpesvirus 30 3 (Varicella zoster virus) (NC_001348), human herpesvirus 4 type 1 (Epstein-Barr virus type 1) (NC_007605), human herpesvirus 4 type 2 (Epstein-Barr virus type 2) (NC_009334), human herpesvirus 5 strain AD169 (NC_001347), human herpesvirus 5 strain Merlin Strain (NC_006273), human herpesvirus 6A (NC_001664), human herpesvirus 6B (NC_000898), human herpesvirus 7 (NC_001716), human herpesvirus 8 type M (NC_003409), human herpesvirus 8 type P 35 (NC_009333), human immunodeficiency virus 1 (NC_001802), human immunodeficiency virus 2 (NC_001722), human metapneumovirus (NC_004148), human papillomavirus-1 (NC_001356), human papillomavirus-18 (NC_001357), human papillomavirus-2 (NC_001352), human papillomavirus-54 (NC_001676), human papillomavirus-61 (NC_001694), human papillomavirus-cand90 (NC_004104), human papillomavirus RTRX7 (NC_004761), human papillomavirus type 10 (NC_001576), human papillomavirus type 101 (NC_008189), human papillomavirus type 103 (NC_008188), human papillomavirus type 107 (NC_009239), human papillomavirus type 16 (NC_001526), human papillomavirus type 24 (NC_001683), human 5 papillomavirus type 26 (NC_001583), human papillomavirus type 32 (NC_001586), human papillomavirus type 34 (NC_001587), human papillomavirus type 4 (NC_001457), human papillomavirus type 41 (NC_001354), human papillomavirus type 48 (NC_001690), human papillomavirus type 49 (NC_001591), human papillomavirus type 5 (NC_001531), human papillomavirus type 50 (NC_001691), human papillomavirus type 53 (NC_001593), human 10 papillomavirus type 60 (NC_001693), human papillomavirus type 63 (NC_001458), human papillomavirus type 6b (NC_001355), human papillomavirus type 7 (NC_001595), human papillomavirus type 71 (NC_002644), human papillomavirus type 9 (NC_001596), human papillomavirus type 92 (NC_004500), human papillomavirus type 96 (NC_005134), human parainfluenza virus 1 (NC_003461), human parainfluenza virus 2 (NC_003443), human 15 parainfluenza virus 3 (NC_001796), human parechovirus (NC_001897), human parvovirus 4 (NC_007018), human parvovirus B19 (NC_000883), human respiratory syncytial virus (NC_001781) , human rhinovirus A (NC_001617), human rhinovirus B (NC_001490), human spumaretrovirus (NC_001795), human T-lymphotropic virus 1 (NC_001436), human T-lymphotropic virus 2 (NC_001488), Epstein-Barr Virus (EBV) or a Human Papilloma Virus (HPV, e.g., HPV16, HPV18), 20 Respiratory Syncytial Virus, a Hepatitis Virus (e.g., Hepatitis C Virus), a Coronavirus (e.g., SARS virus), an Adenovirus, a Polyomavirus, a Cytomegalovirus (CMV), Herpes Simplex Virus (HSV), Her1, Her2, Her3, Her4, EGFR1, EGFR2, EGFR3, EGFR4, ErbB-1, ErbB-2, ErbB-3 and ErbB-4. 74. The single stranded probe precursor according to claim 40, for use in associating 25 the probe with a nucleic acid target. 75. A kit comprising the double stranded probe according to claim 1. 76. The kit according to claim 75 wherein the probe comprises at least one bulge 30 monomer. 77. The kit according to claim 75 wherein the one or more bulge monomer is selected from OR or 78. The kit according to any one of claims 75-77 comprising a sequence selected from SEQ ID NOs. 5-10, 12-33, 38-65, 72-85, 94-105, 112-129, 136-218, 220-239, and 246-253. 79. The kit according to any one of claims 75-78 for gender determination. 80. The kit according to claim 79 for gender determination in mammals. 10 81. The kit according to claim 80 for sexing mammalian embryos. 82. The kit according to claim 81 where the mammal is an ungulate. 83. The kit according to claim 80 where the mammal is a bovine, equine or porcine. 84. An in vitro method for associating a probe with a nucleic acid target, comprising: selecting a double stranded probe comprising a first monomer having a formula Linker Optional 1 b b where Y is selected from carbon, oxygen, sulfur, and NR , wherein R is selected from 2 3 4 20 hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; each of Y , Y , and Y independently is selected from carbon, oxygen, sulfur, a triazole, oxazole, tetrazole, isoxazole, and NR , wherein R is selected from hydrogen, aliphatic, aryl, heteroaliphatic, and heteroaryl; R and R are selected from hydrogen, aliphatic, aryl, aryl aliphatic, and a heteroatom-containing moiety, or R is selected from a heteroatom-containing functional group; R is a heteroatom-containing functional group; R is 25 selected from any natural or non-natural nucleobase; R is selected from an intercalator suitable for intercalating within a nucleic acid selected from a hydrocarbon or an aromatic heterocycle; “optional linker” is selected from linkers comprising alkyl linkers, amide linkers, carbamate linkers, carbonyl linkers, carbonate linkers, urea linkers, and combinations thereof; a second monomer having a formula Linker Optional 1 2 3 4 1 2 3 4 5 wherein Y , Y , Y , Y , R , R R , R , R , and “optional linker” are as stated for the first monomer; V is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4; wherein the first monomer is positioned in a first strand of the double-stranded probe and the second monomer is positioned in a second strand of the double stranded probe and wherein each of 10 the first strand and the second strand comprises at least one nucleotide selected from a natural nucleotide, unnatural nucleotide, and combinations thereof; exposing the nucleic acid target to the double stranded probe; and detecting the double stranded probe and/or a double stranded probe-nucleic acid target complex. 85. The in vitro method according to claim 84 wherein the double stranded probe further comprises a second monomer having a formula selected from Linker Optional wherein V is selected from carbon, oxygen, sulfur, and NR ; and n ranges from 0 to 4. 86. An in vitro method for associating a probe with a nucleic acid target, comprising: selecting a single stranded probe comprising a monomer having a formula OR N f OR N or O y R O B R O B OR N NH f OR N 2 OR N R O B R O B OR N N N OR N N y e e e R O B R O B R O B R O B O O O O O O O O f f f f OR OR OR R O R O OR OR Nap Py OR N f OR N R O O OR N wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H, DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or 2 2 2 10 phosphate; or OR O wherein B is selected from guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6-diaminopurine, inosine, or 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one; Nap is napthyl; Py is pyrenyl; R is H, DMTr, or phosphate; and R is H, (N(i-Pr) )P(OCH CH CN), or phosphate; and at least one natural nucleotide, 2 2 2 5 unnatural nucleotide, and combinations thereof; exposing the nucleic acid target to the single stranded probe; and detecting the single stranded probe and/or a single stranded probe-nucleic acid target complex. 10 87. The method of claim 86 wherein the single stranded probe further comprises a second monomer selected from e moc e B O OR N f OR N OR O O Na P P p y or O y R O B R O B R O B OR N f OR N NH P 2 OR P R O B R B H Py OR N OR N e e e R O B R O B R O B R O B O O O O O O O O f f f f OR OR OR R O R O OR OR Nap Py OR N f R O O OR N wherein B is selected from uracil, guanine, cytosine, adenine, thymine, 2-thiouracil, 2,6- diaminopurine, inosine, 3-pyrrolo-[2,3-d]-pyrimdine(3H)-one, or any derivative thereof; R is H, DMTr, or phosphate; R is peryleneyl or coronenyl; and R is H, (N(i-Pr) )P(OCH CH CN), or 2 2 2 phosphate. 88. The double stranded probe according to claim 1, wherein the double stranded probe has a sequence selected from SEQ ID No. 252 or SEQ ID No. 253.
Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
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US201161509336P | 2011-07-19 | 2011-07-19 | |
US61/509,336 | 2011-07-19 | ||
US201161542044P | 2011-09-30 | 2011-09-30 | |
US61/542,044 | 2011-09-30 | ||
PCT/US2012/047442 WO2013013068A2 (en) | 2011-07-19 | 2012-07-19 | Embodiments of a probe and method for targeting nucleic acids |
Publications (2)
Publication Number | Publication Date |
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NZ621347A NZ621347A (en) | 2016-03-31 |
NZ621347B2 true NZ621347B2 (en) | 2016-07-01 |
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