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WO2003104249A1 - Novel linker for nucleotides - Google Patents

Novel linker for nucleotides Download PDF

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Publication number
WO2003104249A1
WO2003104249A1 PCT/KR2002/001456 KR0201456W WO03104249A1 WO 2003104249 A1 WO2003104249 A1 WO 2003104249A1 KR 0201456 W KR0201456 W KR 0201456W WO 03104249 A1 WO03104249 A1 WO 03104249A1
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WIPO (PCT)
Prior art keywords
linker
group
hydrocarbon chain
oligonucleotide
saturated
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Ceased
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PCT/KR2002/001456
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French (fr)
Inventor
Kyeong-Eun Jung
Sun Lee
Je-Geun Yoo
Dong-Won Shin
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Nexgen Biotechnologies Inc
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Nexgen Biotechnologies Inc
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Priority to AU2002321851A priority Critical patent/AU2002321851A1/en
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Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D211/00—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings
    • C07D211/04—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D211/06—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D211/08—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms
    • C07D211/18—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D211/20—Heterocyclic compounds containing hydrogenated pyridine rings, not condensed with other rings with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hydrocarbon or substituted hydrocarbon radicals directly attached to ring carbon atoms with substituted hydrocarbon radicals attached to ring carbon atoms with hydrocarbon radicals, substituted by singly bound oxygen or sulphur atoms
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/547—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom
    • C07F9/553—Heterocyclic compounds, e.g. containing phosphorus as a ring hetero atom having one nitrogen atom as the only ring hetero atom
    • C07F9/572—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/11—Compounds covalently bound to a solid support
    • 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
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
    • Y02P20/50—Improvements relating to the production of bulk chemicals
    • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

  • the present invention relates to novel linkers for nucleotides. More particularly, the present invention relates to novel linkers for tagging a fluorescent molecule as a reporter or quencher or linking a solid support to oligonucleotide and methods for preparing the same .
  • oligonucleotide synthesis is the phosphite-triester method synthesizing oligonucleotides on a solid support using nucleoside phosphoamidites .
  • the process contains the steps of deblocking, coupling, oxidation and capping .
  • oligonucleotides provide primers in cDNA synthesis and real-time PCR, templates for RNA transcription, linkers for plasmid manipulation and hybridization probes for diagnosis .
  • oligonucleotide enhances its applicability in various fields.
  • the most feasible method in current is attaching a label on the oligonucleotide using the phosphoamidite method.
  • the oligonucleotide synthesized is then labeled for chasing of oligonucleotide-target complex and quantitative analysis.
  • Such a specific labeling on oligonucleotide is pivotal process for DNA-probe synthesis
  • the synthesized oligonucleotides can be tagged with reporter etc., either by chemical and enzymatic methods, and a reporter or quencher can also be incorporated using usable material in the course of phosphoramidite method
  • linkers aforementioned exist in racemic mixture and then are very likely to generate 2 diastereomers upon linking to oligonucleotide, thereby leading to difficulty in purification of the synthetic oligonucleotides and complexity in analysis using the synthetic oligonucleotides.
  • Fig. la represents 4 sorts of 35S promoter sequences identified in cauliflower mosaic viruses
  • Fig. lb represents primers and probe designed on the basis of the 4 sorts of 35S promoters.
  • Fig. 2 represents intensity of fluorescence depending on the number of PCR cycles .
  • Fig. 3 represents quantification of transformed plants using standard curve obtained according to the present invention.
  • HCA represents a heterocyclic amine
  • n is an integer of 0-10;
  • X is linked to nitrogen atom of the heterocyclic amine and represents one selected from the group consisting of saturated C ⁇ -C ⁇ 0 hydrocarbon chain, saturated Ci-Cio hydrocarbon chain in which carbon atoms are substituted with 1-5 heteroatoms, and saturated C ⁇ -C ⁇ 0 hydrocarbon chain containing amide, ester, ether, amine, sulfonyl or combination thereof;
  • Rl is a hydrogen or hydroxyl-protecting group;
  • R2 is one selected from a group consisting of hydrogen, phosphoramidite group, H-phosphate and solid support; and
  • R3 is one selected from a group consisting of reporter, quencher, amino-protecting group, amino acid and peptide.
  • the heterocyclic amine centered in the linker according to the present invention is non-aromatic one comprising heterocyclic amine containing 5 atoms e.g. pyrrolidine or 6 atoms e.g. piperidine.
  • the heterocyclic amine can contain either one or more nitrogen atoms (e.g. piperazine) or other additional heterogeneous atom as well as one or more nitrogen atoms (e.g. morpholine) .
  • the linker is represented by formula (II)
  • n, X, Rl, R2 and R3 are the same as described in the formula I .
  • the linker is represented by the following formula (HI) .
  • n, X, Rl, R2 and R3 are the same as described in the formula I .
  • the linker of the present invention can be linked to any nucleotide in oligonucleotide with or without reporter molecule.
  • oligonucleotide used herein means substance comprising several nucleotides and may contain one or more derivatives of nucleotide and/or linker allowing for adequate gap.
  • nucleotide refers to ribonucleotide or deoxyribonucleotide containing adenine, cytosine, thymine, guanine, uracil and their derivatives.
  • the molecules incorporated into oligonucleotide by the linker of the present invention includes reporter, quencher, amino-protecting group, amino acid and peptide, but not limited to.
  • a reporter may comprise reporter molecule with oligonucleotide, permitting photometrical detection, linkage and quantification of oligonucleotide.
  • the reporter is a fluorescent reporter molecule.
  • the reporter can contain spacer not to hinder the function of the amine group.
  • the preferred fluorescent molecules includes 5- carboxyfluorescein (5-FAM) , 6-carboxyfluorescein (6-FAM) ,
  • the quencher may comprise quencher molecule with oligonucleotide, and can quench the function of reporter in a proximal position and restore the function of reporter in a distal position e.g. by separation from reporter.
  • the quencher is a fluorescent quencher molecule.
  • the reporter can contain spacer not to hinder the function of the amine group.
  • the preferred fluorescent quencher includes tetramethyl-6-carboxyrhodamine (T7AMRA) , tetrapropano-6- carboxyrhodamine (ROX) , cyanine, androquinone, nitrothiazole and nitroamidazole.
  • T7AMRA tetramethyl-6-carboxyrhodamine
  • ROX tetrapropano-6- carboxyrhodamine
  • cyanine cyanine
  • androquinone nitrothiazole and nitroamidazole.
  • the amino group in bases should be protected for prevention of acetylation or phosphorylation during oligonucleotide synthesis.
  • a protecting group stable in acid and removable conveniently in alkaline condition may be used.
  • benzyloxycarbonyl group is preferred. 5'-hydroxyl group should be protected during condensation, oxidation and capping steps and the protecting group is required to be removed by weak acid
  • DMT 4,4-dimethoxytrityl
  • pixyl 9-phenylxanthen-9-yl
  • Fmoc 9- fluorenylmethoxycarbonyl
  • n is an integer of 0-10. More preferably, n is an integer of 0-1, and the smaller n results in shorter-lengthen saturated hydrocarbon chain, which is linked to central heterocyclic amine, contributing to less steric hindrance and simpler linker structure .
  • X is C-C 8 hydrocarbon chain containing amide group and more preferably, X is C 6 hydrocarbon chain containing 1 amide group.
  • the amide group is likely to be prepared in easier manner, stable in condition of oligonucleotide synthesis and enable analysis to be easier.
  • X contains a heterogeneous atom
  • preferred is either 0 or N.
  • linker of the present invention is limitless; however, the representatives are incorporation of fluorescent molecule into oligonucleotide and application in oligonucleotide synthesis.
  • linker of the present invention being used for the purpose of incorporation of fluorescent molecules into oligonucleotide, one example of the present invention is represented by the following formula (IN) .
  • iPr represents isopropyl group.
  • linker represented by formula (IN) a reporter molecule or a quencher molecule is linked to in place of Fmoc, an amino-protecting group.
  • the linker according to present invention is employed in the process of oligonucleotide synthesis, for typical example, phosphoramidite DNA synthesis, the linker can be attached to 3 '-end of nucleotide or oligonucleotide and employed instead of the protected deoxynucleoside phosphoramidite added into DNA synthesizer.
  • protected deoxynucleoside derivatized lcaa- CPG (long chain alkylamine-controlled pore glass) column which is generally employed in phosphoramidite DNA synthesis, can be successfully replaced with a column packed with resin having the linker of the present invention.
  • linker according to the present invention is an immobilized linker represented by the following formula (V).
  • n, X and Rl are the same as described in the formula ( I ) , Y is single bond, nucleotide or oligonucleotide; FS is functional spacer; SSM is solid support; and R3 is amino-protecting group, reporter or quencher.
  • non-limiting example includes long chain alkylamine CPG (lcca-CPG) , 3-amino-propyl silica gel, aminomethyl polystyrene resin and TengaGelTM
  • polyethylene glycol-TENTAcles grafted on low cross-linked , gelatinous polystyrene matrix polyethylene glycol-TENTAcles grafted on low cross-linked , gelatinous polystyrene matrix
  • the SSM is separated from final oligonucleotide and a functional spacer is located between the SSM and the central part of the linker.
  • the functional spacer includes any spacer useful for chemical synthesis of oligonucleotide, e.g., succinyl group and glutaryl group.
  • n 0 or 1
  • X is C 6 hydrocarbon chain containing 1 amide group
  • Y is single bond
  • FS is succinyl spacer
  • SSM is CPG
  • R3 is reporter or quencher
  • Rl is DMT in the linker of formula (V) .
  • linker of the formula (V) is represented by the following formula (VI) :
  • nucleotide synthesis employing the linker of the present invention can be performed by conventional methods described by Caruthers, M.H. Science, 230: 281-285 (1985), Itakura, K. et al . Ann . Rev. Biochem. 53; 323-356 (1984) and Hunkapiller, M. et al . Nature, 310: 105-111 (1984), and these publications are incorporated herein by reference.
  • the oligonucleotide with the linker of the present invention can be employed for real-time monitoring of DNA amplification, typically for real-time PCR.
  • the method for the real-time PCR is disclosed in U.S. Pat. 5,538,848, incorporated herein by reference .
  • the probe is represented by the following formula (VH) .
  • the linker according to the present invention is useful for tagging oligonucleotide with fluorescent molecule or linking solid resin to oligonucleotide.
  • the present linker can be prepared in pure enantiomer, removing the difficulty in purification and analysis caused by conventional linkers that are generally in racemic mixture.
  • Such pure enantiomer is prepared using natural-occurring chiral compound (e.g. proline with certain stereoisomeric property) .
  • the process for preparing the enantiomeric linker according to the present invention is exemplified in the following example.
  • scheme 1 shows a process for preparing the linker tagged with phosphoramidite according to the present invention.
  • the linker capable of attaching fluorescent material on oligonucleotide can be synthesized and linked either to 3 '-end or intra-position of oligonucleotide.
  • the elaborate process for preparing the linker tagged with phosphoramidite according to the present invention is described as follows: Scheme 1
  • a MeOH, acetylchloride; b: BnCl, tBu 4 NI , CH 3 CN; c: LAH, THF , N 2 ; d : Pd-C , EtOH, H 2 ; e : 6 -N-Fmoc- ⁇ -aminocaproic acid,
  • HOAT HOAT , HBTU, DIPEA
  • f DMTCI , DMAP , CH 2 C1 2 , TEA
  • g DMTCI , DMAP , CH 2 C1 2 , TEA
  • DIPEA CH 2 C1 2 , 2 -cyanoethyl -N, N-diisoprophylamino chl oropho sphor ami di t e
  • the compound 3 (11.2 g, 47.6 mmol) was added dropwise into 150 ml of THF containing LAH (2.16 g, 57.12 mmol) for 20 min at 0 ° C under nitrogen atmosphere and the mixture was stirred for 3 hrs at RT.
  • the excess LAH was treated with acetone and DW.
  • the gray precipitate yielded was removed with cellite and the remaining solution was concentrated.
  • the residual water was removed by use of constant boiling point with toluene, the remnant was vacuum-dried to obtain compound 4 (9.54 g, 97.0 %) .
  • the compound 4 (9.54 g, 46.0 mmol) was dissolved in 50 ml of absolute ethanol and 10% Pd-C (1.0 g) was added to, followed by stirring for 5 hrs at 25 ° C under 40 psi of H 2 gas.
  • Step 5 Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5- hydroxy methyl pyrrolidin-3-ol (6)
  • Step 6 Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT methyl-pyrrolidin-3 -ol (7)
  • the compound 6 (0.3 g, 0.66 mmol), DMTCl (0.22 g, 0.66 mmol) and DMAP (cat.) were dissolved in 2 ml of absolute CH 2 C1 2 , TEA (0.18 ml, 1.32 mmol) was added thereto at -20 ° C under nitrogen atmosphere and the mixture was stirred for 4 hrs. The mixture was then treated with methanol, diluted with CH 2 C1 2 (10 ml) , rinsed with NaCl solution (10 ml) , dried with Na 2 S0 4 and concentrated under vacuum.
  • Step 7 Synthesis of l-N-Fmoc-5-O-DMT methyl pyrrolidine- 3-0-2-cyanoehtyl-N,N-diisopropyl phosphoramidite (8)
  • the compound 7 (0.10 g, 0.13 mmol) was dissolved in 1 ml of CH 2 C1 2 , diisopropylethylamine (DIPEA; 67 ⁇ i , 0.39 mmol) and 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite (43 ⁇ lt , 0.20 mmol) were added and then the mixture was stirred for 1 hr at 0 ° C and for 2 hrs at RT subsequently. The reactant mixture was treated with methanol and concentrated under vacuum.
  • DIPEA diisopropylethylamine
  • 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite 43 ⁇ lt , 0.20 mmol
  • a succinic anhydride, DMAP, TEA, CH 2 C1 2 ; b: aminopropyl CPG, HOBT, HBTU, DIPEA; c: acetic anhydride/lutidine-THF, piperidine/DMF; and d: TAMRA NHS ester, acetic anhydride/lutidine-THF
  • Step 1 Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT- pyrrolidine-3-succinate (9)
  • the probe was applied to assay for quantitative analysis of transformed plants as follows: The plants were transformed with 35S promoter isolated from cauliflower mosaic virus.
  • primers for PCR For preparing primers for PCR to detect transformed plant containing the 35S promoter from of cauliflower mosaic virus, the adequate sequences for primers were selected referring to known sequences (GenBank accession
  • 3' -end indicate the conserved sequence.
  • the sequence comprising the conserved sequence is shown in SEQ ID N0:1.
  • the sequence comprising the conserved sequence was selected as amplified region in PCR.
  • the forward (SEQ ID NO: 2) and the reverse primers (SEQ ID NO: 3) were synthesized for amplification of the selected region.
  • Other primers hybridizable to the conserved sequence or its proximal region e.g., SEQ ID Nos:4, 5, 6 and 7 were synthesized and shown to be effective as a primer in Experimental Example 4.
  • the fraction of interest was lyophilized, dissolved in 1 ml of DW twice, lyophilized again and the residual TEAA salt was removed thoroughly. The remnant was dissolved in 1 ml of DW and the absorbance of UV was measured at 70 ° C.
  • the absorbance of natural nucleotide is as follows: dAMP, 15200: dCMP, 7700: TMP, 8830: dGMP, 11500: FAM, 20958: TAMRA, 31980.
  • composition of oligomers was confirmed by HPLC (Hewlett Packard, ODS hypersil, C-18; 20 mM K 2 HP0 4 , pH
  • the final nucleotide is 5'FAM-aag gaa agg cca teg ttg aag atg c-TAMRA-3' (SEQ ID NO: 8) .
  • Experimental Example 4 Real-Time PCR Using Probes Prepared in Experimental Example 3 3 PCR reactions per each unknown and standard sample were prepared. The total volume of each PCR reaction was 50 ⁇ i composed of 39.25 ⁇ i cocktail, 0.25 ⁇ i AmpliTaq GoldTM DNA polymerase (5 U//z£, Perkin-Elmer) , 0.5 ⁇ i AmpErase Uracil N-glycosylase (UNG, 1 U/ ⁇ i , Perkin-Elmer) , 10 ⁇ i DNA templates (unknown DNA or DNA for positive control) .
  • the composition of the reaction cocktail is : PCR reaction buffer (lOx, Roche), MgCl 2 (3.5 mM) , dATP (200 ⁇ M) , dCTP
  • the PCR reaction buffer contains 100 mM Tris/HCl, 400 mM KCl, 15 mM MgCl 2 , 10 mM DTT and 5 ⁇ i/ml BSA.
  • Figs. 2 and 3 The real-time PCR results are shown in Figs. 2 and 3. As shown in Fig. 2, the intense of fluorescence is increased in a sigmoidal pattern depending on the PCR cycles, which indicates the primers and probes of the present invention work their functions.
  • Fig. 3 shows the standard curve drawn based on the results of the positive and negative controls. As shown in Fig. 3, correlation coefficient of the standard curve is next to 1 (over 0.999) enhancing fidelity of the results on unknown samples. As indicated in Fig. 3, samples A and B are proved to have 0.35% and 0% of transformed plants. The above results are summarized in the following Table 2.
  • C t is threshold cycle number that indicates the number of PCR cycle showing overtly distinguished fluorescence from background.
  • the probe of the present invention is proven to get better sensitivity compared to Tagman probe.
  • the probe according to the present invention can be applied to real-time PCR with improved efficacy.

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Abstract

The present invention relates to a linker for oligonucleotide. The linker of this invention is useful for tagging a fluorescent molecule as a reporter or quencher or linking a solid support to oligonucleotide.

Description

NOVEL LINKER FOR NUCLEOTIDES
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to novel linkers for nucleotides. More particularly, the present invention relates to novel linkers for tagging a fluorescent molecule as a reporter or quencher or linking a solid support to oligonucleotide and methods for preparing the same .
DESCRIPTION OF THE RELATED ART
For recent years, automated DNA synthesizers have made breakthrough in molecular biology and biochemistry providing commercial linear DNA nucleotides with aimed sequences. Currently, the most general method for oligonucleotide synthesis is the phosphite-triester method synthesizing oligonucleotides on a solid support using nucleoside phosphoamidites . Generally, the process contains the steps of deblocking, coupling, oxidation and capping .
Applicability of oligonucleotides is limitless, e.g. DNA oligonucleotides provide primers in cDNA synthesis and real-time PCR, templates for RNA transcription, linkers for plasmid manipulation and hybridization probes for diagnosis .
Further modification of oligonucleotide enhances its applicability in various fields. Among several modifications of the oligonucleotide, the most feasible method in current is attaching a label on the oligonucleotide using the phosphoamidite method.
In general, the oligonucleotide synthesized is then labeled for chasing of oligonucleotide-target complex and quantitative analysis. Such a specific labeling on oligonucleotide is pivotal process for DNA-probe synthesis
(Goodchild, J. Bioconjugate Chem. , 1:165 (1990)).
Isotopes for labeling of nucleotides have been rapidly substituted by fluorescent materials owing to their hazardous and tricky property for treatment (Smith, L. M. et al., Nature, 321: 674-679 (1986); and Douglas, M. E. et al., BioMed . Chem. Lett . 4 (8) : 995-1000 (1994)). For labeling oligonucleotide with fluorescent materials, a variety of methods for oligonucleotide derivatives with amino or thiol group has been developed (Ono, K. et al . , Bioconjugate Chem. 4:499-508(1993); and Douglas, M. E. et al., BioMed . Chem. Lett . 4 (8) : 995-1000 (1994) ) .
The synthesized oligonucleotides can be tagged with reporter etc., either by chemical and enzymatic methods, and a reporter or quencher can also be incorporated using usable material in the course of phosphoramidite method
(Kempe, T. et al . , Nucleic Acid Res . 13:45-57 (1985); WO
91/17169; and Gibson, K. J. et al . , Nucleic Acid Res . 15:6455-6467 (1987)).
There are currently several types of linkers for tagging the reporter etc. on oligonucleotides. Nelson et al . have stepped their pioneer feet by developing a linker based on 2- (4-aminobutyl) -1, 3-propanoldiol capable to be covalently linked with 3 functional groups (Nucleic Acid Res . 20: 6253-6259 (1992)), and Mullah B. et al . have followed them with a improved linker introducing amide groups on their predecessors' creation (Nucleic Acid Res . 26: 1026-1031 (1998) ) .
The linkers aforementioned, however, exist in racemic mixture and then are very likely to generate 2 diastereomers upon linking to oligonucleotide, thereby leading to difficulty in purification of the synthetic oligonucleotides and complexity in analysis using the synthetic oligonucleotides.
Throughout this application, various patents and publications are referenced and citations are provided in parentheses. The disclosure of these patents and publications in their entitles are hereby incorporated by references into this application in order to more fully describe this invention and the state of the art to which this invention pertains.
SUMMARY OF THE INVENTION
Under such situation, the present inventors have made intensive studies to be from the shortcomings of the conventional linkers, and as a result, we have developed novel linkers to enable more convenient purification of and reliable analysis with oligonucleotide linked to the present linkers.
Accordingly, it is an object of this invention to provide a novel linker useful in modification such as labeling or synthesis of oligonucleotide.
Other objects and advantages of the present invention will become apparent from the detailed description to follow taken in conjunction with the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la represents 4 sorts of 35S promoter sequences identified in cauliflower mosaic viruses;
Fig. lb represents primers and probe designed on the basis of the 4 sorts of 35S promoters. Fig. 2 represents intensity of fluorescence depending on the number of PCR cycles .
Fig. 3 represents quantification of transformed plants using standard curve obtained according to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
In one aspect of this invention, there is provided a novel linker for oligonucleotide represented by the following formula (I) :
Figure imgf000005_0001
wherein HCA represents a heterocyclic amine containing
5 or 6 atoms; n is an integer of 0-10; X is linked to nitrogen atom of the heterocyclic amine and represents one selected from the group consisting of saturated Cι-Cι0 hydrocarbon chain, saturated Ci-Cio hydrocarbon chain in which carbon atoms are substituted with 1-5 heteroatoms, and saturated Cι-Cι0 hydrocarbon chain containing amide, ester, ether, amine, sulfonyl or combination thereof; Rl is a hydrogen or hydroxyl-protecting group; R2 is one selected from a group consisting of hydrogen, phosphoramidite group, H-phosphate and solid support; and R3 is one selected from a group consisting of reporter, quencher, amino-protecting group, amino acid and peptide.
The heterocyclic amine centered in the linker according to the present invention is non-aromatic one comprising heterocyclic amine containing 5 atoms e.g. pyrrolidine or 6 atoms e.g. piperidine. Furthermore, the heterocyclic amine can contain either one or more nitrogen atoms (e.g. piperazine) or other additional heterogeneous atom as well as one or more nitrogen atoms (e.g. morpholine) .
According to a preferred embodiment of the present invention (where the central heterocyclic amine is pyrrolidine), the linker is represented by formula (II)
Figure imgf000007_0001
wherein n, X, Rl, R2 and R3 are the same as described in the formula I .
According to another preferred embodiment of the present invention (where the central heterocyclic amine is piperidine) , the linker is represented by the following formula (HI) .
Figure imgf000007_0002
wherein n, X, Rl, R2 and R3 are the same as described in the formula I .
The linker of the present invention can be linked to any nucleotide in oligonucleotide with or without reporter molecule. The term "oligonucleotide" used herein means substance comprising several nucleotides and may contain one or more derivatives of nucleotide and/or linker allowing for adequate gap. The "nucleotide", as used herein, refers to ribonucleotide or deoxyribonucleotide containing adenine, cytosine, thymine, guanine, uracil and their derivatives.
The molecules incorporated into oligonucleotide by the linker of the present invention includes reporter, quencher, amino-protecting group, amino acid and peptide, but not limited to.
A reporter may comprise reporter molecule with oligonucleotide, permitting photometrical detection, linkage and quantification of oligonucleotide. Preferably, the reporter is a fluorescent reporter molecule. Where amine group is linked to the reporter, the reporter can contain spacer not to hinder the function of the amine group.
The preferred fluorescent molecules includes 5- carboxyfluorescein (5-FAM) , 6-carboxyfluorescein (6-FAM) ,
2' ,4' , 1,4-tetrachlorofluroscein (TET) , 2 ' , 4 ' , 5' , 7' , 1, 4- hexachlorofluorescein (HEX) and 2 ' , 7 ' -dimethoxy-4 ' , 5 ' - dichloro-6-carboxyrhodamine (JOE) .
Furthermore, the quencher may comprise quencher molecule with oligonucleotide, and can quench the function of reporter in a proximal position and restore the function of reporter in a distal position e.g. by separation from reporter.
Preferably, the quencher is a fluorescent quencher molecule. Where amine group is linked to the reporter, the reporter can contain spacer not to hinder the function of the amine group.
The preferred fluorescent quencher includes tetramethyl-6-carboxyrhodamine (T7AMRA) , tetrapropano-6- carboxyrhodamine (ROX) , cyanine, androquinone, nitrothiazole and nitroamidazole.
It is prerequisite to protect functional groups (e.g. functional groups of 4 bases (e.g. amino group), phosphate group or 5-hydroxyl group) for permitting the aimed chemical reaction in the course of nucleotide synthesis.
To begin with, the amino group in bases should be protected for prevention of acetylation or phosphorylation during oligonucleotide synthesis. Generally, a protecting group stable in acid and removable conveniently in alkaline condition may be used.
For amino-protecting group in the linker of the present invention, benzyloxycarbonyl group is preferred. 5'-hydroxyl group should be protected during condensation, oxidation and capping steps and the protecting group is required to be removed by weak acid
(e.g. trichloroacetic acid: TCA) before the following nucleotide is added. As the hydroxyl-protecting group, 4,4-dimethoxytrityl (DMT) , 9-phenylxanthen-9-yl (pixyl) or 9- fluorenylmethoxycarbonyl (Fmoc) is recommendable and DMT works better than others .
According to another preferred embodiment of the present invention, n is an integer of 0-10. More preferably, n is an integer of 0-1, and the smaller n results in shorter-lengthen saturated hydrocarbon chain, which is linked to central heterocyclic amine, contributing to less steric hindrance and simpler linker structure .
According to another preferred embodiment of the present invention, X is C-C8 hydrocarbon chain containing amide group and more preferably, X is C6 hydrocarbon chain containing 1 amide group. The amide group is likely to be prepared in easier manner, stable in condition of oligonucleotide synthesis and enable analysis to be easier.
In the case that X contains a heterogeneous atom, preferred is either 0 or N.
The applicability of the linker of the present invention is limitless; however, the representatives are incorporation of fluorescent molecule into oligonucleotide and application in oligonucleotide synthesis. In the case for the linker of the present invention being used for the purpose of incorporation of fluorescent molecules into oligonucleotide, one example of the present invention is represented by the following formula (IN) .
Figure imgf000010_0001
wherein iPr represents isopropyl group.
In the linker represented by formula (IN) , a reporter molecule or a quencher molecule is linked to in place of Fmoc, an amino-protecting group. In the case that the linker according to present invention is employed in the process of oligonucleotide synthesis, for typical example, phosphoramidite DNA synthesis, the linker can be attached to 3 '-end of nucleotide or oligonucleotide and employed instead of the protected deoxynucleoside phosphoramidite added into DNA synthesizer.
Moreover, protected deoxynucleoside derivatized lcaa- CPG (long chain alkylamine-controlled pore glass) column, which is generally employed in phosphoramidite DNA synthesis, can be successfully replaced with a column packed with resin having the linker of the present invention. Another example of linker according to the present invention is an immobilized linker represented by the following formula (V).
Figure imgf000011_0001
wherein n, X and Rl are the same as described in the formula ( I ) , Y is single bond, nucleotide or oligonucleotide; FS is functional spacer; SSM is solid support; and R3 is amino-protecting group, reporter or quencher. In the formula (V) , non-limiting example includes long chain alkylamine CPG (lcca-CPG) , 3-amino-propyl silica gel, aminomethyl polystyrene resin and TengaGel™
(polyethylene glycol-TENTAcles grafted on low cross-linked , gelatinous polystyrene matrix) .
The SSM is separated from final oligonucleotide and a functional spacer is located between the SSM and the central part of the linker. The functional spacer includes any spacer useful for chemical synthesis of oligonucleotide, e.g., succinyl group and glutaryl group.
According to the most preferable embodiment of the present invention, n is 0 or 1, X is C6 hydrocarbon chain containing 1 amide group, Y is single bond, FS is succinyl spacer, SSM is CPG, R3 is reporter or quencher and Rl is DMT in the linker of formula (V) .
One example of the linker of the formula (V) is represented by the following formula (VI) :
Figure imgf000012_0001
The nucleotide synthesis employing the linker of the present invention can be performed by conventional methods described by Caruthers, M.H. Science, 230: 281-285 (1985), Itakura, K. et al . Ann . Rev. Biochem. 53; 323-356 (1984) and Hunkapiller, M. et al . Nature, 310: 105-111 (1984), and these publications are incorporated herein by reference.
The oligonucleotide with the linker of the present invention can be employed for real-time monitoring of DNA amplification, typically for real-time PCR. The method for the real-time PCR is disclosed in U.S. Pat. 5,538,848, incorporated herein by reference .
Where the oligonucleotide with the linker of the present invention is employed as probe in real-time PCR, the probe is represented by the following formula (VH) .
5.FAM_ Pr
Figure imgf000013_0001
As described above, the linker according to the present invention is useful for tagging oligonucleotide with fluorescent molecule or linking solid resin to oligonucleotide.
Moreover, the present linker can be prepared in pure enantiomer, removing the difficulty in purification and analysis caused by conventional linkers that are generally in racemic mixture. Such pure enantiomer is prepared using natural-occurring chiral compound (e.g. proline with certain stereoisomeric property) . The process for preparing the enantiomeric linker according to the present invention is exemplified in the following example.
It is obvious for ones skilled in the art that the following specific examples are intended to be illustrative pf the invention and should not be construed as limiting the scope of the invention as defined by appended claims.
EXAMPLE I: Synthesis of Linker Tagged with Phosphoramidite
The following scheme 1 shows a process for preparing the linker tagged with phosphoramidite according to the present invention. In scheme 1, the linker capable of attaching fluorescent material on oligonucleotide can be synthesized and linked either to 3 '-end or intra-position of oligonucleotide. Referring to scheme 1, the elaborate process for preparing the linker tagged with phosphoramidite according to the present invention is described as follows: Scheme 1
Figure imgf000014_0001
a: MeOH, acetylchloride; b: BnCl, tBu4NI , CH3CN; c: LAH, THF , N2 ; d : Pd-C , EtOH, H2 ; e : 6 -N-Fmoc-ε-aminocaproic acid,
HOAT , HBTU, DIPEA; f : DMTCI , DMAP , CH2C12 , TEA; and g :
DIPEA, CH2C12 , 2 -cyanoethyl -N, N-diisoprophylamino chl oropho sphor ami di t e
Step 1: Synthesis of Trans-4-hydroxy-L-proline methyl ester hydrochloride (2)
Trans-4-hydroxy-L-proline (1; 10 g, 76.3 mmol) was dissolved in 200 ml of methanol , acetyl chloride (7.6 ml, 106.8 mmol) was added therein and the mixture was refluxed with stirring up for overnight . The mixture was cooled to
RT (room temperature) and 500 ml of ether was added to form white crystal followed by filtration and drying under vacuum to yield the compound 2 (12.7 g, 87.6 %) : XH NMR (CDC13) 52.07-2.30 (m, 1H, C3-H), 2.33-2.50 (m, 1H, C3-H) , 3.29-3.34 (m, 2H, C2,C5-H), 3.45-3.50 (m, 1H, C5-H) , 3.84 (s, 3H, CO2CH3) , 4.43-4.60 (br, 1H, NH) , 4.62-4.72 (m, 1H, C4-H) ; Mass El m/z 146.80, 87.00, 68.75.
Step 2: Synthesis of l-Benzyl-4-hydroxy-pyrrolidine-2- carboxylic acid methyl ester (3)
The above-prepared compound 2 (12.7 g, 66.6 mmol), K2C03 (26.4 g, 199.8 mmol), BnCl (6.5 ml, 56.6 mmol) and tBu4NI (cat.) were dissolved with dried CH2CN (100 ml) and refluxed with stirring up for overnight . The mixture was cooled to RT, diluted with 200 ml of DW, extracted three times with 50 ml of AcOEt and washed with DW. The final concentrate in oil phase was obtained by drying organic phase with MgS04. The concentrate was separated through a column chro atography (MC:MeOH = 20:1/15:1) and colorless oily compound 3 (10.57 g, 67.5%) was yielded: XH NMR
(CDC13) 52.03-2.11 (m, IH, C3-H) , 2.20-2.29 (m, IH, C3-H) ,
2.46 (dd, IH, J=3.82, 10.14 Hz, C5-H) , 3.32 (dd, IH,
J=5.62, 10.14 Hz, C5-H) , 3.60 (m, IH, C2-H), 3.65 (s, 3H,
CO2CH3) , 3.66 (d, IH, J=12.87 Hz, CH2Ph) , 3.89 (d, IH,
J=12.87 Hz, CH2Ph) , 4.44 (m, IH, C4-H) , 7.24-7.32 (m, 5H,
Ph) ; Mass El m/z 235.20, 176.10, 91.15.
Step 3: Synthesis of 1-Benzyl-5-hydroxymethyl-pyrrolidin- 3-ol (4)
The compound 3 (11.2 g, 47.6 mmol) was added dropwise into 150 ml of THF containing LAH (2.16 g, 57.12 mmol) for 20 min at 0 °C under nitrogen atmosphere and the mixture was stirred for 3 hrs at RT. The excess LAH was treated with acetone and DW. The gray precipitate yielded was removed with cellite and the remaining solution was concentrated. The residual water was removed by use of constant boiling point with toluene, the remnant was vacuum-dried to obtain compound 4 (9.54 g, 97.0 %) . The compound 4 was employed without further purification in the subsequent reaction: DH NMR (CDC13) 51.77-1.90 (m, IH, C3-H) , 2.07-2.22 (m, IH, C3-H), 2.38 (dd, IH, J=4.88, 10.17 Hz, C5-H) , 3.04-3.13 (m, IH, CH2OH) , 3.24 (dd, IH, J=5.70, 10.17 Hz, C5-H) , 3.41 (dd, IH, J=2.03, 11.39 Hz, C2-H) , 3.48 (d, J=13.02 Hz, CH2Ph) , 3.67 (dd, IH, J--3.26, 10.99 Hz, CH2OH) , 3.99 (d, IH, J=13.02 Hz, CH2Ph) , 4.28-4.80 (m, IH, C4-H) , 7.25-7.33 (m, 5H , Ph) ; Mass El m/ z 207 . 35 , 176 . 15 , 90 . 85 .
Step 4: Synthesis of 5-hydroxymethyl-pyrrolidin-3-ol (5)
The compound 4 (9.54 g, 46.0 mmol) was dissolved in 50 ml of absolute ethanol and 10% Pd-C (1.0 g) was added to, followed by stirring for 5 hrs at 25°C under 40 psi of H2 gas. The residual Pd-C was discarded by filtration and raw compound 5 (4.8 g, 88.9%) was prepared by concentration under vacuum: 1H NMR (D20) δl.93-2.14 (m, IH, C3-H) , 2.15-2.25 (m, IH, C3-H) , 3.34-3.40 (m, IH, C5-H) , 3.46-3.54 (m, IH, C5-H) , 3.71-3.81 (m, IH, CH20H) , 3.94- 4.02 (m, IH, CH2OH) , 4.05-4.10 (m, IH, C2-H), 4.69-4.78 (m, IH, C4-H) .
Step 5: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5- hydroxy methyl pyrrolidin-3-ol (6)
The compound 5 (0.3 g, 2.5 mmol), 6-N-Fmoc-ε- aminocaproic acid (0.88 g, 2.5 mmol), l-hydroxy-7- azabenzotriazole (HOAT) (0.34 g, 2.5 mmol) and 2- (1H- benzotriazol-1-yl) -1, 1, 3, 3-tetramethyl uronium hexafluorophosphate (HBTU) (0.95 g, 2.5 mmol) were dissolved in 1 ml of DMF, diisopropylethyl amine (0.44 ml,
2.5 mmol) was added dropwise therein at 25°C under nitrogen atmosphere, followed by stirring for 2.5 hrs. The reaction mixture was concentrated under vacuum, dissolved in 100 ml of chloroform and washed with 5% HC1 (50 ml) , DW (50 ml) and NaCl solution (50 ml) consecutively. The organic phase was dried with MgS04, vacuum-dried to prepare oily residual and 2 ml of ethanol was then added to, followed by standing the resultant in a freezer. The precipitate thus obtained was dried to yield compound 6 (0.98 g,
86.7%): XH NMR (CDC13) δl.27-1.35 (m, 2H, CH2-CH2NHFmoc) , 1.41-1.50 (m, 2H, CH2-CH2NHFmoc) , 1.55-1.64 (m, 4H, CH2-
CH2NHFmoc) , 2.10 (dd, IH, J=7.50, 13.59 Hz, C3-H), 2.13-
2.32 (m, 2H, C3,C5-H), 3.08-3.16 (m, 2H, CH2-CH2NHFmoc) ,
3.40-3.49 (m, 3H, C2-H, CH20DMT) , 3.59-3.65 (m, IH, C5-H) ,
4.14 (dd, IH, J=6.77, 13.53 Hz, C4-H) , 4.25-4.33 (m, 2H, NHCQ2CH2) , 4.93 (s, IH, NH) , 5.34 (d, IH, J=8.21 Hz, Fmoc),
7.19-7.26 ( , 2H, Fmoc), 7.30-7.35 (m, 2H, Fmoc), 7.50-
7.53 (m, 2H, Fmoc), 7.67-7.70 (m, 2H, Fmoc); Mass FAB+ cal .
452.2311 found 453.2390 (+H) .
Step 6: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT methyl-pyrrolidin-3 -ol (7)
The compound 6 (0.3 g, 0.66 mmol), DMTCl (0.22 g, 0.66 mmol) and DMAP (cat.) were dissolved in 2 ml of absolute CH2C12, TEA (0.18 ml, 1.32 mmol) was added thereto at -20 °C under nitrogen atmosphere and the mixture was stirred for 4 hrs. The mixture was then treated with methanol, diluted with CH2C12 (10 ml) , rinsed with NaCl solution (10 ml) , dried with Na2S04 and concentrated under vacuum. The remnant was separated by column chromatography (50:1 = MC:MeOH, containing TEA 5%) to obtain compound 7 (0.32 g, 64.2%): x_i NMR (CDC13) δl.12-1.17 (m, 2H, CH2- CH2NHFmoc) , 1.20-1.33 (m, 2H, CH2-CH2NHFmoc) , 1.34-1.48 (m, 2H, CH2-CH2NHFmoc) , 1.49-1.60 (m, 2H, CH2-CH2NHFmoc) , 1.83- 1 . 91 (m, IH, C3 -H) , 2 . 09 - 2 . 33 (m, 2H , C3 , C5 -H) , 2 . 99 - 3 . 08
(m, 2H, CH2-CH2NHFmoc) , 3.32-3.36 (m, IH, C5-H) , 3.55-3.60
(m, IH, C2-H) , 3.65-3.66 (brs, 8H, CH2ODMT, PhOCH3) , 4.01-
4.12 (m, IH, C4-H) , 4.26-4.31 (m, 2H, NHC02CH2) , 5.16 (s, IH, Fmoc), 5.20-5.22 (br, IH, NH) , 6.67-6.73 (m, 4H, DMT),
7.05-7.17 (m, 9H, DMT), 7.22-7.29 (m, 4H, Fmoc), 7.48-7.51
(m, 2H, Fmoc), 7.62-7.66 (m, 2H, Fmoc); Mass FAB+ cal .
754.3618 found 777.3514 (+Na) .
Step 7: Synthesis of l-N-Fmoc-5-O-DMT methyl pyrrolidine- 3-0-2-cyanoehtyl-N,N-diisopropyl phosphoramidite (8)
The compound 7 (0.10 g, 0.13 mmol) was dissolved in 1 ml of CH2C12, diisopropylethylamine (DIPEA; 67 μi , 0.39 mmol) and 2-cyanoethyl-N,N-diisopropylchloro phosphoramidite (43 βlt , 0.20 mmol) were added and then the mixture was stirred for 1 hr at 0°C and for 2 hrs at RT subsequently. The reactant mixture was treated with methanol and concentrated under vacuum. The remnant was resuspended in AcOEt, rinsed with NaHC03 and NaCl solution consecutively, and concentrated under vacuum after desiccation with NaHS04. The remnant was separated to obtain compound 8 (0.076 g, 58.8%) through column chromatography (Hex:AcOEt = 3:2, containing TEA 2%) : ^Η NMR (CDC13) δl.10-1.15 (m, 2H, CH2-CH2NHFmoc) , 1.16 (d, 6H, J=0.9Hz, iPr) , 1.18 (d, 6H, J=1.0Hz, iPr) , 1.21-1.31 (m, 2H, CH2-CH2NHFmoc) , 1.30-1.46 (m, 2H, CH2-CH2NHFmoc) , 1.46- 1.57 (m, 2H, CH2-CH2NHFmoc) , 1.80-1.90 (m, IH, C3-H) , 2.07- 2.32 (m, 2H, C3,C5-H), 2.64 (t, 2H, J=5.9Hz, 0CH2CH2CN) , 2.95 (m, IH, iPr) , 2.96-3.07 (m, 2H, CH2-CH2NHFmoc) , 3.12
(m, IH, iPr) , 3.30-3.34 (m, IH, C5-H) , 3.54-3.59 (m, IH,
C2-H) , 3.65-3.66 (brs, 8H, CH2ODMT, PhOCH3) , 3.75 (m, 2H,
OCH2CH2CN) , 4.00-4.11 (m, IH, C4-H) , 4.24-4.30 (m, 2H, NHCO2CH2) , 5.15 (s, IH, Fmoc), 5.20-5.22 (br, IH, NH) ,
6.66-6.73 ( , 4H, DMT), 7.05-7.17 (m, 9H, DMT), 7.22-7.29
( , 4H, Fmoc), 7.48-7.51 (m, 2H, Fmoc), 7.62-7.66 (m, 2H, Fmoc); Mass FAB+ cal . 968.4853 found 991.4754 (+Na) .
Example II : Synthesis of Linker Attached to CPG
The process for preparing the linker attached to CPG
(controlled pore glass) according to the present invention is schematically described in the following scheme 2.
According to scheme 2, a linker attached to solid support can be obtained.
Scheme 2
Figure imgf000020_0001
a: succinic anhydride, DMAP, TEA, CH2C12; b: aminopropyl CPG, HOBT, HBTU, DIPEA; c: acetic anhydride/lutidine-THF, piperidine/DMF; and d: TAMRA NHS ester, acetic anhydride/lutidine-THF
Step 1: Synthesis of 1-N- (N-Fmoc-6-aminohexanoyl) -5-O-DMT- pyrrolidine-3-succinate (9)
The compound 7 (0.10 g, 0.13 mmol), succinic anhydride (17 g, 0.17 mmol) and DMAP (cat.) were dissolved in absolute CH2C12 (0.5 ml), TEA (18 μϋ , 0.13 mmol) was added thereto and the mixture was stirred overnight. The reacted mixture was diluted with 5 ml of CH2C12 and rinsed with 5% citric acid solution (5 ml) and NaCl solution (5 ml) sequentially. The organic phase was dried with MgS0 , the residual after concentration under vacuum was separated to yield compound 9 (0.079 g, 71.8 %) through column chromatography (CH2Cl2:MeOH = 50:1/20:1, 5% TEA contained) : αH NMR (CDC13) δl.38-1.48 (m, 4H, CH2_- CH2NHFmoc) , 1.55-1.66 ( , 4H, CH2-CH2NHFmoc) , 1.97-2.04 (m, IH, C3-H), 2.09-2.24 ( , 2H, C3,C5-H), 2.46-2.48 (m, 4H, CH2CH2CQ2H) , 3.04-3.11 (m, 2H, CH2-CH2NHFmoc) , 3.38-3.48 (m, IH, C5-H) , 3.54-3.60 (m, IH, C2-H) , 3.69 (brs, 8H, CHzODMT, PhOCH3) , 4.13-4.15 (m, IH, C4-H), 4.28-4.33 (m, 2H, NHCQ2CH2) , 5.18-5.23 (m, IH, Fmoc), 5.32 (br, IH, NH) , 6.70-6.75 (m, 4H, DMT), 7.10-7.23 (m, 9H, DMT), 7.26-7.30 (m, 4H, Fmoc), 7.50-7.53 (m, 2H, Fmoc), 7.66-7.69 (m, 2H, Fmoc); Mass FAB+ cal . 854.3778 found 877.3679 (+Na) . Step 2: CPG Attachment (10)
The compound 9 (0.025 g, 0.029 mmol), aminopropyl CPG
(164 mg, 89.2 μ mol/g amine loading, 14.6 μ mol), 1- hydroxybenzotriazole (HOBT) (3.9 mg, 29 μ mol) and 2-(lH- benzotriazol-1-yl) -1, 1, 3, 3, -tertramethyl uronium hexafluorophosphate (HBTU) (11 mg, 29 μ mol) were dissolved in absolute DMF (4 ml), diisopropylethylamine (DIPEA) (8.4 μi , 49.3 μ mol) was added under nitrogen atmosphere, and the mixture was stirred with vibrational stirrer for 3 days. Solid support was rinsed with DMF (5 ml x 3) and CH3CN (2 ml x 2) sequentially, and dried overnight under vacuum to prepare compound 10 (158 mg, 71.6 μ mol/g) .
Step 3: Removal of Fmoc group (11) With vibrational stirring, the compound 10 (158 mg,
71.6 μ mol/g) was capped in acetic anhydride/lutidine-THF
(10 % solution, 3 ml) for 1 hr and washed with CH3CN (3 ml x 3) . Fmoc group was detached by treating residual 4 times with 20% piperidine/DMF (4 ml) for 10 min. The detachment of Fmoc was continuously checked by UN (302 nm) spectrophotometer . The residual was washed with DMF (4 ml x 3) and dried under vacuum, thereby obtaining compound 11 (150 mg, 71.6 μ mol/g, 10.7 μ mol) .
Step 4 : Synthesis of Compound 12
The compound 11 (150 mg, 71.6 μ mol/g, 10.7 μ mol) , TAMRA Ν-hydroxysuccinimide (ΝHS) ester (30 μi , 57 μ mol) and TEA (15 μl , 171 μ mol) were stirred for 3 days in absolute DMF (4 ml) . The reacted mixture was rinsed with
DMF (1 ml x 3) and then CH3CN (1 ml x 2) . The residual was capped in acetic anhydride/lutidine-THF (10%, 2 ml) for 1 hr with vibrational stirring, washed with CH3CN (5 ml x 3) and vacuum-dried for 3 days to obtain the final compound
12 (145 mg, 71.6 μ mol/g, 10.4 μ mol) .
In an effort to evaluate the applicability of the oligonucleotide tagged with the linker of the present invention as probe, the probe was applied to assay for quantitative analysis of transformed plants as follows: The plants were transformed with 35S promoter isolated from cauliflower mosaic virus.
Experimental Example 1: Isolation of Plant DNA DNA from several plants including non-transformed Korean native maize, transformed maize (BT-11 maize, Fluka) , non-transformed Korean native soybean, transformed soybean (Roundup Ready Soybean, Fluka) and unknown maize or soybean, was isolated respectively using the method described by Edwards K. , et al . (Nucleic Acids Research, 19: 1349(1991)). Firstly, 200 μl of extraction buffer (200 mM Tris-Cl, pH 7.5, 250 mM NaCl, 25 mM EDTA and 0.5% SDS) was added to plant tissue and the tissue was crushed with a pestle. And then, additional 200 μi of extraction buffer was added and was centrifuged for 10 min. 300 μi of the extract were transferred into a tube, 300 μi of chloroform: isoamylalcohol (24:1) were added thereto, then inverted and centrifuged for 5 min. at 13,000 rpm. The supernatant was transferred into a new microtube and mixed with 300 μi of isopropanol. After centrifugation, the precipitated DNA was rinsed twice with 70% ethanol and resuspended with 50 μi of ddH20. Each isolated DNA was diluted to 50 ng/μi, and serial dilution was performed to indicated in Table 1. The diluents in Table 1 are prepared as a positive control for real-time RCR in Experimental
Example 4.
Since plant DNA sample with known concentration must be highly purified for fidelity of standard curve, DNA samples with 99.99% purity were employed as positive control in the instant Examples .
Table 1
Figure imgf000024_0001
Experimental Example 2 : Construction of Primers
For preparing primers for PCR to detect transformed plant containing the 35S promoter from of cauliflower mosaic virus, the adequate sequences for primers were selected referring to known sequences (GenBank accession
No. AJ251014, X84105, AF044029 and X04879) . The known sequences were aligned and the conserved sequences were identified (Fig. la and lb) . In Fig. la and lb, TBI251014,
Astdnabv, Af044029 and Arcamvpr indicate sequence of
GenBank accession No. AJ251014, X84105, AF044029 and
X04879, respectively and the shaded sequences adjacent to
3' -end indicate the conserved sequence. The sequence comprising the conserved sequence is shown in SEQ ID N0:1. Considering application to DNAs from various plants, the sequence comprising the conserved sequence was selected as amplified region in PCR. And then, the forward (SEQ ID NO: 2) and the reverse primers (SEQ ID NO: 3) were synthesized for amplification of the selected region. Other primers hybridizable to the conserved sequence or its proximal region (e.g., SEQ ID Nos:4, 5, 6 and 7) were synthesized and shown to be effective as a primer in Experimental Example 4.
Experimental Example 3: Preparation of Probe for Real-Time PCR Using Linker Attached to CPG To prepare the probe to be employed in PCR of the present invention, the adequate region of template DNA which is hybridized to the probes was selected. The probe was designed in such a manner that it may be located between the forward and reverse primers and more adjacent to the forward primer to evaluate amplified DNA more rapidly .
All probes employed in Example were synthesized at 1 μ mol scale in trityl-on using Expedite 8909 DNA/PNA
Synthesizer (PerSeptive Biosystems) according to instructions of manufacturer. The compound 12 was employed for the incorporation of TAMRA quencher at 3' -end. And for the incorporation of FAM at 5' -end, 5-fluorescein phosphoramidite ( [ (3' , 6' -dipivaloylfluoresceinyl) ] -6- carboxyamidohexyl) -1-0- (2-cyanoethyl) - (N,N-diisopropyl) - phosphoramidite, Glen Research) was used. As nucleotides added, dAbz, dCbz, dGdmf and T phosphoramidite (ABI) were used. The condensation of 5' -end FAM was done for 3 min. whereas the general duration of condensation was 1 min. Solid support and protecting groups were heated in t- butylamine:methanol :DW (1:1:2) at 65°C for 3 hrs, isolated and lyophilized. The residual was dissolved in 1 ml of triethylammonium acetate (TEAA, pH 7, 100 mM) and purified through reverse phase HPLC (Hamilton PRP-1, 300 mm x 7 mm, 18-38% acetonitrile/100 mM TEAA, pH 7 , UV monitor at 260 nm and 560 nm) . The fraction of interest was lyophilized, dissolved in 1 ml of DW twice, lyophilized again and the residual TEAA salt was removed thoroughly. The remnant was dissolved in 1 ml of DW and the absorbance of UV was measured at 70°C. For calculation of concentration from absorbance, the absorbance of natural nucleotide is as follows: dAMP, 15200: dCMP, 7700: TMP, 8830: dGMP, 11500: FAM, 20958: TAMRA, 31980.
The composition of oligomers was confirmed by HPLC (Hewlett Packard, ODS hypersil, C-18; 20 mM K2HP04, pH
5.6(A), MeOH(B), 100% A:40% B, 20 min.) and Laser
Desorption molecular weight analysis after enzymatic treatment of alkaline phosphatase and snake venom phosphodisesterase. The final nucleotide is 5'FAM-aag gaa agg cca teg ttg aag atg c-TAMRA-3' (SEQ ID NO: 8) .
Experimental Example 4: Real-Time PCR Using Probes Prepared in Experimental Example 3 3 PCR reactions per each unknown and standard sample were prepared. The total volume of each PCR reaction was 50 μi composed of 39.25 μi cocktail, 0.25 μi AmpliTaq Gold™ DNA polymerase (5 U//z£, Perkin-Elmer) , 0.5 μi AmpErase Uracil N-glycosylase (UNG, 1 U/μi , Perkin-Elmer) , 10 μi DNA templates (unknown DNA or DNA for positive control) . The composition of the reaction cocktail is : PCR reaction buffer (lOx, Roche), MgCl2 (3.5 mM) , dATP (200 μM) , dCTP
(200 μM) , dGTP (200 μM) , dUTP (400 μM) , forward primer (300 nM) , reverse primer (300 nM) and probe (200 nM) . The PCR reaction buffer contains 100 mM Tris/HCl, 400 mM KCl, 15 mM MgCl2, 10 mM DTT and 5 μi/ml BSA.
0.25 μi AmpliTaq Gold™ DNA polymerase and 0.5 μi AmpErase uracil N-glycosylase (UNG) were freshly mixed in 39.25 μi PCR cocktail at the brink before PCR. And then, the mixture of PCR cocktail and enzymes was aliquot in each tube with each template DNA. The template DNAs added were positive controls (i.e. 10 //! of 1, 3, 5, 10 or 50% transformed maize or soybean DNA with concentration of 50 ng/μl) , negative control (10 μi of DW, non-transformed maize or soybean DNA with concentration of 50 τig/μi) or 10 μi of unknown DNA A or B with concentration of 50 ng/μi , which were obtained in Experimental Example 1. Thereafter, pre-denaturation at 50°C for 2 min. and denaturation at 95 °C for 10 min. were done consecutively, and total 40 cycles of PCR was done in which each cycle is composed of the denaturation step at 95°C for 15 sec, annealing and extension step at 64°C for 1 min. Real-time PCR was performed in Rotor Gene 2000 (Corbett) under checking of copy number of amplified template DNA using florescence measurement and the PCR result was also analyzed using the program equipped in the PCR device.
The real-time PCR results are shown in Figs. 2 and 3. As shown in Fig. 2, the intense of fluorescence is increased in a sigmoidal pattern depending on the PCR cycles, which indicates the primers and probes of the present invention work their functions.
Fig. 3 shows the standard curve drawn based on the results of the positive and negative controls. As shown in Fig. 3, correlation coefficient of the standard curve is next to 1 (over 0.999) enhancing fidelity of the results on unknown samples. As indicated in Fig. 3, samples A and B are proved to have 0.35% and 0% of transformed plants. The above results are summarized in the following Table 2.
Table 2
Figure imgf000029_0001
In Table 1, Ct is threshold cycle number that indicates the number of PCR cycle showing overtly distinguished fluorescence from background.
Experimental Example 5 : Comparison of Taqman Probe and Probe of the Present Invention in their Efficacy
Each real-time PCR was performed using the probes in Experimental Example 3 and Taqman probe (ABI) in the same manner as Experiment 4 to compare their efficacy.
The results of comparison are summarized in Table 3. Table 3
Figure imgf000030_0001
As shown in Table 3 , the probe of the present invention is proven to get better sensitivity compared to Tagman probe. In a result, the probe according to the present invention can be applied to real-time PCR with improved efficacy.

Claims

What is claimed is :
1. A linker for oligonucleotide represented by the following formula ( I ) :
wherein HCA represents a heterocyclic amine containing 5 or 6 atoms; n is an integer of 0-10; X is linked to nitrogen atom of the heterocyclic amine and represents one selected from the group consisting of saturated CL-CIO hydrocarbon chain, saturated CL-CIO hydrocarbon chain in which carbon atoms are substituted with 1-5 heteroatoms, and saturated d-Cio hydrocarbon chain containing amide, ester, ether, amine, sulfonyl or combination thereof; Rl is a hydrogen or hydroxyl-protecting group; R2 is one selected from a group consisting of hydrogen, phosphoramidite group, H-phosphate and solid support; and R3 is one selected from a group consisting of reporter, quencher, amino-protecting group, amino acid and peptide.
2. The linker for oligonucleotide according to claim 1, wherein the linker is represented by the following formula (II) or (HI) :
Figure imgf000032_0001
Figure imgf000032_0002
wherein n is an integer of 0-10; X is linked to nitrogen atom of the heterocyclic amine and represents one selected from the group consisting of saturated Cι-Cι0 hydrocarbon chain, saturated Cx-Cio hydrocarbon chain in which carbon atoms are substituted with 1-5 heteroatoms, and saturated Cι-Cι0 hydrocarbon chain containing amide, ester, ether, amine, sulfonyl or combinations thereof; Rl is a hydrogen or hydroxyl-protecting group; R2 is one selected from a group consisting of hydrogen, phosphoramidite group, H-phosphate and solid support; and R3 is one selected from a group consisting of reporter, quencher, a ino-protecting group, amino acid and peptide.
3. The linker for oligonucleotide according to claim 1, wherein the n is 0-1.
4. The linker for oligonucleotide according to claim 1, wherein the X is C4-C8 hydrocarbon chain containing amide group.
5. The linker for oligonucleotide according to claim 4, wherein the X is C6 hydrocarbon chain containing 1 amide group .
6. The linker for oligonucleotide according to claim 1, wherein the Rl is hydroxyl-protecting group.
7. The linker for oligonucleotide according to claim 6, wherein the Rl is dimethoxytrityl group.
8. The linker for oligonucleotide according to claim 2, wherein the linker is represented by the following formula
(IV):
Figure imgf000033_0001
wherein iPr represents isopropyl group.
9. The linker for oligonucleotide according to claim 2, wherein the linker is represented by the following formula (V):
Figure imgf000034_0001
wherein n is an integer of 0-10; X is linked to nitrogen atom of the heterocyclic amine and represents one selected from the group consisting of saturated Ci-Cio hydrocarbon chain, saturated Cι-C10 hydrocarbon chain in which carbon atoms are substituted with 1-5 heteroatoms, and saturated Cι-C10 hydrocarbon chain containing amide, ester, ether, amine, sulfonyl or combinations thereof; Rl is a hydrogen or hydroxyl-protecting group; Y represents single bond, nucleotide or oligonucleotide; FS represents functional spacer; SSM represents solid support; and R3 is one selected from a group consisting of amino-protecting group, reporter and quencher.
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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006031461A3 (en) * 2004-09-09 2007-01-18 Isis Pharmaceuticals Inc Pyrrolidinyl groups for attaching conjugates to oligomeric compounds
US8569515B2 (en) 2008-11-27 2013-10-29 Roche Diagnostics Operations, Inc. Directed synthesis of oligophosphoramidate stereoisomers
US9688758B2 (en) 2012-02-10 2017-06-27 Genentech, Inc. Single-chain antibodies and other heteromultimers
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
US10106612B2 (en) 2012-06-27 2018-10-23 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
IL264221A (en) * 2016-07-15 2019-02-28 Am Chemicals Llc Non-nucleosidic solid supports and phosphoramidite building blocks for oligonucleotide synthesis
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
US10633460B2 (en) 2010-12-23 2020-04-28 Roche Diagnostic Operations, Inc. Binding agent
US10982007B2 (en) 2010-12-23 2021-04-20 Roche Diagnostics Operations, Inc. Detection of a posttranslationally modified polypeptide by a bivalent binding agent
US11421022B2 (en) 2012-06-27 2022-08-23 Hoffmann-La Roche Inc. Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
US11618790B2 (en) 2010-12-23 2023-04-04 Hoffmann-La Roche Inc. Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714606A (en) * 1994-01-11 1998-02-03 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US6316626B1 (en) * 1997-09-05 2001-11-13 Isis Pharmaceuticals, Inc. Amine compounds and combinatorial libraries comprising same

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5714606A (en) * 1994-01-11 1998-02-03 Isis Pharmaceuticals, Inc. Pyrrolidine-containing monomers and oligomers
US6316626B1 (en) * 1997-09-05 2001-11-13 Isis Pharmaceuticals, Inc. Amine compounds and combinatorial libraries comprising same

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DATABASE CA [online] SWAYZE ERIC E.: "The solid phase synthesis of trisubstituted 1,4-diazabicyclo(4.3.0)nonan-2-one scaffolds: on bead monitoring of heterocycle forming reactions using 15N NMR", Database accession no. 1997:790867 *
TETRAHEDRON LETTERS, vol. 38, no. 50, 1997, pages 8643 - 8646 *

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Publication number Priority date Publication date Assignee Title
WO2006031461A3 (en) * 2004-09-09 2007-01-18 Isis Pharmaceuticals Inc Pyrrolidinyl groups for attaching conjugates to oligomeric compounds
US8569515B2 (en) 2008-11-27 2013-10-29 Roche Diagnostics Operations, Inc. Directed synthesis of oligophosphoramidate stereoisomers
US8853132B2 (en) 2008-11-27 2014-10-07 Roche Diagnostics Operations, Inc. Directed synthesis of oligophosphoramidate stereoisomers
US9994646B2 (en) 2009-09-16 2018-06-12 Genentech, Inc. Coiled coil and/or tether containing protein complexes and uses thereof
US10106600B2 (en) 2010-03-26 2018-10-23 Roche Glycart Ag Bispecific antibodies
US10982007B2 (en) 2010-12-23 2021-04-20 Roche Diagnostics Operations, Inc. Detection of a posttranslationally modified polypeptide by a bivalent binding agent
US10633460B2 (en) 2010-12-23 2020-04-28 Roche Diagnostic Operations, Inc. Binding agent
US11618790B2 (en) 2010-12-23 2023-04-04 Hoffmann-La Roche Inc. Polypeptide-polynucleotide-complex and its use in targeted effector moiety delivery
US9688758B2 (en) 2012-02-10 2017-06-27 Genentech, Inc. Single-chain antibodies and other heteromultimers
US10106612B2 (en) 2012-06-27 2018-10-23 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
US11407836B2 (en) 2012-06-27 2022-08-09 Hoffmann-La Roche Inc. Method for selection and production of tailor-made highly selective and multi-specific targeting entities containing at least two different binding entities and uses thereof
US11421022B2 (en) 2012-06-27 2022-08-23 Hoffmann-La Roche Inc. Method for making antibody Fc-region conjugates comprising at least one binding entity that specifically binds to a target and uses thereof
US10633457B2 (en) 2014-12-03 2020-04-28 Hoffmann-La Roche Inc. Multispecific antibodies
US11999801B2 (en) 2014-12-03 2024-06-04 Hoffman-La Roche Inc. Multispecific antibodies
IL264221A (en) * 2016-07-15 2019-02-28 Am Chemicals Llc Non-nucleosidic solid supports and phosphoramidite building blocks for oligonucleotide synthesis
EP3484908A4 (en) * 2016-07-15 2020-04-08 AM Chemicals Llc Non-nucleosidic solid supports and phosphoramidite building blocks for oligonucleotide synthesis
US10781175B2 (en) 2016-07-15 2020-09-22 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates
US11447451B2 (en) 2016-07-15 2022-09-20 Am Chemicals Llc Solid supports and phosphoramidite building blocks for oligonucleotide conjugates

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