JP2003508406A - Nucleoside analog - Google Patents

Abstract

(57) [Summary] Structural formula (I) Wherein, X is CH or N, Y is -CO -, - CONW -, - O -, - S -, - SO -, - SO 2 -, - NWCO -, - NW- or -OCO-, W Are the same or different at different places on the molecule, each H or alkyl or aryl or Rp or -Ln-Rp, Ln is a linker group, Rp is a reporter moiety, and Q is a sugar or sugar analog or nucleic acid backbone or backbone analog Provided that at least one reporter moiety Rp is present] provides a good enzyme substrate, nucleoside triphosphate.

Description

Detailed Description of the Invention

INTRODUCTION The present invention relates to compounds suitable for use as nucleoside analogs, and polynucleotide chains comprising the nucleoside analogs.

Nucleic acids have been handled in vitro in a wide range of research and diagnostic techniques. The method comprises DNA or RN for the purpose of determining label or base sequence identity.
By means of A polymerase, reverse transcriptase or terminal transferase enzymes,
It may include the synthesis of nucleic acid probes. Labels often include incorporation of nucleotides that are chemically labeled or of a particular chemical composition to make them detectable. Nucleic acid probes made in this way can be used to determine the presence of nucleic acid targets having complementary sequences by means of hybridization of the probe to its target.

In WO94 / 21658, TI Kalman describes novel nucleoside or nucleotide analogs with a 4-acetylimidazolin-2-one base and their use for the inhibition of virus-encoded reverse transcriptase. .

In Z Naturforsch B, 1986, 41b (12), 1571-9, T Fukuda et al.
The effect of incorporation of a nucleoside analog having an imidazolin-2-one base as both T and G of the A duplex is described.

In Tetrahedron Letters, 40 (1999), 835-838, E Bedu et al, prepared a nucleoside analog having a 4-amidoimidazolin-2-one base and used as a cytosine analog in a triple helix-forming oligonucleotide. Is described.

Purine and pyrimidine base nucleosides and nucleotides derivatized with reporter groups are known and have been used extensively for labeling DNA or RNA and other molecular biology applications. However, these molecules are often not good enzyme substrates. There continues to be a need for labeled nucleoside analogs where triphosphate is a good enzyme substrate.

[0007] Description of the invention   According to the present invention, the structural formula

[Chemical 5] Wherein, X is CH or N, Y is -CO -, - CONW -, - O -, - S -, - SO -, - SO 2 -, - NW
CO-, -NW- or -OCO-, W are the same or different at different positions on the molecule, and each is H or alkyl or aryl or Rp or -Ln-Rp, Ln is a linker group, Rp is a reporter moiety, and Q is A sugar or sugar analog or nucleic acid backbone or backbone analog, provided that at least one reporter moiety Rp is present.

[0008]   Q is

[Chemical 6] [In the formula, Z is O, S, Se, SO, NW or CH ₂ , R ¹ , R ² , R ³ and R ⁴ are the same or different, and each is H, OH, F, NH ₂ ,
N _3, O-hydrocarbyl or Rp or -Ln-Rp, ^{R 5} is OH, SH or NH ₂ or mono-, - di - or tri - phosphate or thiophosphate or corresponding boranophosphate, or ^{R 2} and ^{R 5,} Is a phosphoramidite or other group for incorporation into a polynucleotide chain, Rp or -Ln-Rp], or Q is a modified sugar structure acyclic sugar below.

[Chemical 7] Morpholino backbone

[Chemical 8] Or Q is a nucleic acid backbone (eg LNA) consisting of sugar-phosphate repeats or modified sugar-phosphate repeats (Koshkin et al, 1998, Tetr.
ahedron 54, 3607-30) or a backbone analog such as a peptide or polyamide nucleic acid (PNA) (Nielsen et al, 1991, Science 254, 1497-1500).
).

The reporter moiety Rp is attached to W or Y with or without a linker group Ln.
Is present in Y when contains W and always has at least one atom between the reporter and the base ring. When Q is a sugar or sugar analog or modified sugar, these compounds are nucleotide analogs or nucleoside analogs. When Q is a nucleic acid backbone or backbone analog, these compounds are referred to herein as nucleic acids or polynucleotides.

Nucleoside analogs can be chemically or enzymatically incorporated into oligomeric or polymeric nucleic acid (DNA or RNA) strands, and when incorporated in this way are complementary strands or in appropriate nucleic acid strands. It is a molecule that can form base pairs with nucleotide residues in a base stack.

In the context of the present invention, a nucleotide is a naturally occurring compound consisting of a heterocyclic base and a sugar moiety containing a phosphate. Nucleosides are the corresponding compounds in the absence of phosphate. Nucleotide analogs and nucleoside analogs are analog compounds having different bases and / or different sugar moieties. A nucleoside analog is a compound that is capable of forming part of a nucleic acid (DNA or RNA or PNA) chain, which is capable of base pairing with a complementary strand in a suitable nucleic acid strand or a base in a stack of bases. Nucleoside analogs may be specific by pairing with only one complementary nucleotide; or base pairing with one or more natural bases, eg pyrimidine (T / C) or purine (A / G). Can be degenerate; or can be common by base pairing with each of the natural bases with minor differences; or it can pair with other analogs or itself.

In one preferred embodiment of the present invention, the base analog binds to a sugar moiety such as ribose, deoxyribose or dideoxyribose to form a nucleoside analog. When the group R ⁵ is triphosphate, the nucleoside triphosphate analog of the invention can be incorporated into the nucleic acid chain by enzymatic means.

The reporter moiety Rp can be any one of a variety. It is a radioisotope that makes the nucleoside analog readily detectable, such as 32-P or 33-P or 35S incorporated into a phosphate or thiophosphate or phosphoramidite or H-phosphonate group, or 3-H or 14 -C
Or it may be 125-I. It can be a stable isotope or a specific chemical moiety suitable for detection by mass spectrometry. (Alternatively, the compound as a whole may be suitable for detection by mass spectrometry.) It may be a signal moiety such as an enzyme, a hapten, a fluorophore, a chemiluminescent group, a Raman label or an electrochemical label.

The reporter moiety may be a solid surface to which the nucleoside analogue is attached and by which means is distinguishable from nucleoside analogues which are not so immobilized. Reporter moieties include reactive groups, nucleophilic groups such as NH ₂ , OH, COOH, C
It may be ONH ₂ , ONH ₂ , SH or thiophosphate or hydrazine or hydrazide, or an electrophilic group, such as an active ester or aldehyde or maleimide, whereby the signal moiety and / or the solid surface of the nucleoside analogue to the nucleic acid chain. It can be combined before or after incorporation. Such reporter groups are known and are well documented in the literature.

The linker group Ln is a fixed or flexible, saturated or unsaturated chain of 1 to 60 or more carbon, nitrogen, oxygen, phosphorus and / or sulfur atoms, as is known in the art. . Preferably, the linker group is attached to the 4-triazole ring (when X is N) or 4-imidazole ring (when X is CH) of the nucleoside analog moiety by an alpha carbonyl group, such as an amide or amine group. ing. Preferably, the linker group is attached to the reporter moiety by an amide bond.

To avoid the risk of steric hindrance, the linker preferably has at least 3 chain atoms, for example — (CH ₂ ) _n- , where n is at least 3.

There may be two (or more) reporter moieties, for example a signal moiety and a solid surface, or a signal moiety different from the hapten, or two fluorescent signal groups, which serve as donor and acceptor. Various forms of these arrangements may be useful for separation or detection purposes.

Purine and pyrimidine nucleoside derivatives labeled with a reporter moiety are known in the art and are well documented in the literature. Labeled nucleoside derivatives have the advantage of being easily detectable during sequencing or other molecular biology methods.

R ¹ , R ² , R ² and R ⁴ can each be H, OH, F, NH ₂ , N ₃ , O-alkyl or a reporter moiety. Thus, ribonucleosides and deoxyribonucleosides and dideoxyribonucleosides are considered together with other nucleoside analogs. These sugar substituents may contain a reporter moiety, in place of or in addition to one or two present at the base.

R ⁵ is OH or mono-, di- or tri-phosphate or -thiophosphate or the corresponding boranophosphate. From the nucleoside (R ⁵ is OH) it is readily possible to make the corresponding nucleotide (R ⁵ is triphosphate) by literature methods. Alternatively, one of R ² and R ⁵ can be a phosphoramidite or H-phosphonate or methylphosphonate or phosphorothioate or amide, or suitable for solid surfaces such as hemisuccinate controlled pore glass. Can be a binding, or
It may be another group for incorporation into the polynucleotide chain, generally by chemical means. The use of phosphoramidites and related derivatives in oligonucleotide synthesis is known and described in the literature.

In the novel nucleoside analogues aimed at by the invention, at least one reporter moiety is preferably present in the base analogue and / or optionally in the sugar or phosphate moiety. The reporter moiety is a sugar moiety of a nucleoside analog, which is described in literature methods (for example, J. Chem. Soc. Chem. Commun. 1990, 1547-8;
Med. Chem., 1988, 31. 2040-8). Reporter moieties in the form of isotope labels are described in literature methods (Analytical Biochemistry, 214, 338-340, 1993; WO 95 /
15395) to be incorporated into the phosphate group.

When R ⁵ is triphosphate, the nucleoside analog is DNA or R
It can be used for enzymatic incorporation into NA. The invention comprises, in another aspect, a polynucleotide chain comprising at least one residue of a nucleoside analogue, as defined.

The nucleoside analogs of the invention are useful for labeling DNA or RNA, or incorporation into oligonucleotides or PNAs. The reporter moiety binds to a position that does not significantly adversely affect the physical or biochemical properties of the nucleoside analog, particularly its ability to be incorporated into single-stranded or double-stranded nucleic acid.

Templates containing incorporated nucleoside analogs of the invention may be suitable for replication in nucleic acid synthesis. When the reporter moiety of the incorporated nucleoside analog comprises a linker group, the signal moiety can be inserted into the incorporated nucleoside analog by attaching it via the end of the linker group or other reactive group.

The nucleoside analog triphosphates of the present invention can be included to extend the 3'end of a nucleic acid strand by an enzyme such as terminal transferase in a template-independent manner. The ends of the nucleoside analog triphosphates made in this way can be detected directly in the absence of a reporter label by the use of antibodies to the nucleoside analog. When included in an oligonucleotide or nucleic acid, this analog acts on the basis of nucleic acid modifying enzymes such as ligases or restriction endonucleases.

The nucleoside analogues of the present invention may also be used in any existing application using natural nucleic acid probes labeled with haptens, fluorophores or other reporter groups such as Southern blots, dot blots and polyacrylamides. It can also be used in agarose gel-based methods, or solution hybridization assays, and other assays in microtiter plates or tubes, or in oligonucleotide or nucleic acid assays such as on microchips. The probe may be detected with a hapten that binds to the base analog, or an antibody that targets the base analog itself, which is advantageous in avoiding additional chemical modifications. The antibody used in this method is usually labeled with a detectable group such as a fluorophore or an enzyme. Fluorescent detection can also be used when the base analog itself is fluorescent or there is a fluorophore attached to the nucleoside analog.

RNA is a very versatile biological molecule. Some laboratory experiments have shown that in vitro selection methods from RNA libraries that bind to proteins with high affinity and properties that are not normally involved in RNA binding, including some antibodies, from short RNs.
It has been shown that it can be used to isolate the A molecule (Gold, Allen, Bink.
ley, et al, 1993, 497-510 in The RNA World, Cold Spring Harbor Press, Col
d Spring Harbor NY, Gold, Polisky, Unlenbeck, and Yarus, 1995, Annu.
Rev. Biochem. 64: 763-795, Tuerk and Gold, 1990, Science 249: 505-510, J.
oyce, 1989, Gene 82: 83-87, Szostak, 1992, Trends Biochem. Sci 17: 89-93.
, Tsai, Kenan and Keene, 1992, PNAS 89: 8864-8868, Tsai, Kenan and Keene
, 1992, PNAS 89: 8864-8868, Doudna, Cech and Sullenger, 1995, PNAS 92: 2
355-2359). Some of these RNA molecules have been proposed as drug candidates for the treatment of diseases such as myasthenia gravis and several other autoimmune diseases.

The basic principle involves adding an RNA library to a protein or molecule of interest. Wash to remove unbound RNA. RNA that binds to the protein is then specifically eluted. The RNA is then reverse transcribed and amplified by PCR. The DNA, with, 2 'modification to impart modified nucleotides (nuclease resistant, for example, 2'F, 2'NH _2, either 2'OCH ₃ and / or C5 modified pyrimidines and / or C8 modified purines ) To transfer.
Those molecules found to bind the protein of interest or other molecule are cloned and sequenced to find consensus sequences. The consensus sequence is taken and used to deliver short oligonucleotide therapy.

The base analogs described here, when converted to nucleoside triphosphates or nucleoside phosphoramidites, clearly increase the molecular diversity available for this selection step. This can lead to oligonucleotides with increased binding affinity for targets not available from current building blocks.

The use of triphosphate nucleotide analogs containing a 5-membered heterocycle such as pyrrole has been demonstrated to serve as a substrate for enzymatic uptake (WO97 /
28176). The nucleotide base analog, pyrrole-3,4-dicarboxamide, is a particularly good substrate. The corresponding base analog, pyrrole-3-carboxamide, is also a substrate, but there is a clear reduction in effect compared to dicarboxamide.
This explains that even though the same group is presented on the hydrogen-bonding surface, subtle structural changes have the apparent effect of altering the ability of the analog to act as a substrate. These effects are not yet predictable. Both the pyrrole mono and dicarboxamide analogs also differ in that they displace all natural bases with varying efficiencies.
It is also a degenerate, for example, pyrrole-3,4-dicarboxamide is A
It also changes T and G and allows extension from it, but it also changes T and G and acts as a terminator.

When the linker group and reporter were introduced at the 4-position of the base analog pyrrole-3-carboxamide, a clear reduction in the ability to act as a substrate was observed. Similar results have been observed in an equivalent system with the modification of pyrrole-3,4-dicarboxamide.

The nucleotide analogs of the invention have several advantages other than those described above for enzymatic uptake. Analogs act in a non-denaturing form and are excellent specific T-substitutions when X = CH. Furthermore, when the linker arm and reporter group are present, they are still substrates for good enzymatic uptake and act in a specific manner, see Example 2.

Direct enzymatic uptake is only one aspect of enzymatic recognition and tolerance that must be considered. Attachment of the linker arm or analog itself can affect the ability of the enzyme to the extent of either analog or analog readthrough,
To date, these properties are unpredictable. The base analog difluorotoluene is
An enzyme that puts the base on the opposite side of the extending complementary strand interrupts the read-through (P
roc. Natl. Acad. Sci. USA (1997), 94, 10506-11). The common base 5-nitroindole (WO 97/28176) is easily read through by the enzyme and the inverted base when included in the oligo. Since 5-nitroindole is a common base, it appears to be ambiguous with respect to the inverted base during formation of the strand complementary to the template.

The nucleotide analogs of the present invention have the advantage as phosphoramidites because they are selectively placed at positions within a DNA oligo through chemical synthesis. At that position, the presence of the linker and reporter group proves to allow it to read through and place the base in the opposite of the analog, see Examples 4A-4G. In comparable experiments with common bases such as 5-nitroindole, the introduction of linkers and reporters was found to have a detrimental effect on readthrough ability. In addition, further experiments show that the enzyme still treats the analog of the invention as a T specific base substitution by placing the A base opposite the analog in the extending complementary strand. , Examples 5A-5C. The combined properties allow the selective insertion of reporter groups at defined positions, which allows the production of complementary strands of unambiguous sequence.

DETAILED DESCRIPTION OF THE INVENTION Example 1 Preparation of 1- (2′-deoxyribose-1′-yl) imidazolidin-2-one-4-carboxylic acid (2)

[Chemical 9] This was done according to the method of Otter et al. (J Org Chem, 1969, 34, 1390). The acid was purified by reverse phase HPLC.

[Table 1]

[Chemical 10]

Preparation of 4 "-(N'-tert-butoxycarbonylamino) butyl 1- (2'-deoxyribose-1'-yl) imidazolidin-2-one-4-carboxamide (3) Under nitrogen atmosphere Carboxylic acid (30 mg, 0.12 mmol) and 4- (N-tert-butoxycarbonylamino) -1-aminobutane (25 mg, in dry DMF (1.5 ml).
0.14 mmol) was added to diphenylphosphoryl azide (39 mg) in dry DMF (0.5 ml).
, 0.14 mmol) followed by dry triethylamine (0.04 ml). The mixture was allowed to stir at room temperature for 60 hours. The solvent was removed in vacuo to give a pale yellow solid which was purified by preparative tlc (RP18, 1: 1 ethanol: water) then reverse phase HPLC,
Obtained 18.6 mg of the desired amide as a colorless oil.

[Table 2]

4 ″-(N′-tert-butoxycarbonylamino) butyl 1- (2′-deoxyribose-1′-yl) imidazolidin-2-one-4-carboxamido-5′-triphosphate (4) Preparation of Nucleoside (3) (18.4 mg, 0.04 mmol) was dissolved in a 1: 1 mixture of trimethyl phosphate and triethyl phosphate (2 ml) under argon atmosphere.
The mixture was cooled to 0 ° C. in an ice bath, phosphoryl chloride (17 μl) was added dropwise and the mixture was cooled to 0 °.
Mix for 2 hours at ° C. Tributylammonium prophosphate (0.44 ml of a 0.5M solution in dry DMF) was added immediately followed by tributylamine (50 μl).
The mixture was stirred at room temperature for 15 minutes and 1M triethylammonium bicarbonate (5m
Quenched by addition of l). The mixture was stirred for 1 hour then the solvent removed in vacuo. The mixture was purified by ion exchange chromatography followed by reverse phase chromatography to give a colorless solid. The λ _max (H ₂ O) 264 nm, ¹ Hnmr and ^{3 1} Pnmr (D ₂ O) were consistent with the desired material, but the compound was shown to be contaminated with triethylammonium pyrophosphate.

4 ″ -aminobutyl 1- (2′-deoxyribose-1′-yl) imidazoline-
2-one-4-carboxamide-5'-triphosphate trifluoroacetic acid salt (5
) Tert-Butoxycarbonyl protected amine (6.6 μmol) was added to trifluoroacetic acid (1 m
l) at room temperature for 1.5 hours. The solvent was removed in vacuo to give the ammonium salt as a colorless solid. ¹ H nmr showed a lack of the 9-proton singlet of the tert-butoxycarbonyl group, but was otherwise consistent with the desired structure.

4 "-(6"'-(fluorescein-5""-(and 6 ""-) carboxamidohexanamide) butyl 1-deoxyribose-1'-yl) imidazolin-2-one-4- Preparation of carboxamide-5'-triphosphate (6) The amine salt (5) was dissolved in anhydrous DMSO to give N, N-diisopropyethylamine (5).
μl) and 6- (fluorescein-5- (and 6-) carboxamidohexanoic acid NHS ester (3.6 mg) .The mixture was kept stirring for 20 h and the mixture was purified by ion exchange chromatography. _max 486 nm, ¹ H n
mr was consistent with the predicted structure.

Example 2 A primer extension assay was used to evaluate compounds (4,5, and 6) as substrates for Klenow fragment DNA polymerase I (EFK). Assay is 2
A ³³ P 5 ′ end labeled 15mer primer hybridized to a tetramer template was used. The primer and template sequences are:

[Chemical 11]

1 picomole ³³ P-labeled primer was hybridized with 2 picomoles of template in × 2 Klenow buffer. To this, 4 μM dNTPαS or 40 μM (4
Or 5), 20 μM (6) or 160 μM (4 or 5) or 4 μM dNT
Either PαS and either 40 μM (4 or 5) or 160 μM (4 or 5) mixtures were added. One unit EFK and 2 mU (4 or 5) inorganic pyrophosphate was used per reaction. Primer alone, primer + template + enzyme, primer + template + enzyme + 4 μM dNTPαS controls were also performed. 37 reactants
Incubated at 0 ° C. for 3 minutes (4 or 5) or 10 minutes (6). The reaction was then stopped by the addition of formamide / EDTA stop solution. Reactive products were separated on a 10% polyacrylamide 7M urea gel. After exposure to Kodak Biomax autoradiography film, analog uptake was tested by comparison of primer alone or primer + template + enzyme with control reactions using 4 μM dNTPαS.

This indicates that compounds (4, 5 and 6) were good substrates for EFK and each was incorporated into template 1 above instead of dTTP. With either mold,
No uptake of dCTP was seen.

[0043] Example 3 Synthesis of imidazolidin-2 (3H) -one-4-carboxamide Synthetic scheme: Synthesis of imidazolidin-2 (3H) -one-4-carboxylic acid nucleoside

[Chemical 12]

Preparation of amines for coupling with imidazolidin-2 (3H) -one-4-carboxylic acid nucleosides

[Chemical 13]

[Chemical 14]

[Chemical 15]

[Chemical 16]

Examples of synthetic steps required to convert amines and acids (2) into phosphoramidites for oligonucleotide synthesis

[Chemical 17]

[0046] Structures of other manufactured phosphoramidites

[Chemical 18]

Of 4-carboxy-1- (2′-deoxy-3 ′, 5′-di (tert-butyldimethylsilyloxy) ribose-1′-yl) imidazolidin-2 (3H) -one (7) Production The crude reaction product from the production of acid (2), carried out on a 33 mmol scale, was dried with DMF (
50 ml), treated with imidazole (10.2 g, 150 mmol) and tert-butyldimethylsilyl chloride (11.3 g, 75 mmol) and the mixture was stirred at room temperature for 1 h. TLC (1: 1 methanol: water, on RP18 tlc plate)
It showed that some starting material remained. A further aliquot of tert-butyldimethylsilyl chloride was added until the starting material was consumed. The mixture was then heated at 60 ° C. for 16 hours. The mixture was poured into water (200 ml) then ethyl acetate (
Three times 200 ml), the combined extracts are washed with 2M sodium hydroxide solution, then dried (MgSO ₄ ), filtered and evaporated to chromatograph, the desired material being 10% methanol in dichloromethane. Eluting with silyl ether
(3.7 g, 25% in two steps from 5-bromouridine) was obtained as a yellow foam.

[Table 3]

Preparation of 4-trifluoroacetamidobutyric acid (8) 4-Aminobutyric acid (5.16 g, 50 mmol) was dissolved in methanol (50 ml) and ethyl trifluoroacetate (7.81 g, 55 mmol) and triethylamine (5) were added. .57
g, 55 mmol). The solid gradually went into solution over 1.5 hours and the mixture was stirred for a further 0.5 hours. The solvent is removed in vacuo, the residue is taken up in ethyl acetate and 2
It was washed with M hydrochloric acid and brine. The organics were dried (MgSO _4), filtered and the desired amide evaporated to give a colorless solid (8.15g, 82%).

[Table 4]

Preparation of O (N-succinimidyl) 4-trifluoroacetamidobutanoate (9) 4-Trifluoroacetamidobutyric acid in anhydrous acetonitrile (25 ml) under nitrogen
(2.1 g, 10.5 mmol) and O- (N-succinimidyl) -N, N, N ', N'-
Tetramethyluronium tetrafluoroborate (3.2 g, 11 mmol) was added to N, N
Treated with diisopropylethylamine (1.42 g, 11 mmol). The mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo and the residue was chromatographed with ether: ethyl acetate to give the desired ester as a colorless solid (2.66g, 90%).

[Table 5]

Preparation of N-3- (tert-butoxycarbonylamino) propyl 4-trifluoroacetamidobutanamide (10) O- (N-succinimidyl) 4-trifluoro in anhydrous tetrahydrofuran (5 ml) under an atmosphere of argon. Acetamide butanoate (834 mg, 2.8 mmol)
3-tert-butoxycarbonylamino-1-aminopropane (500 mg, 2.
9 mmol). The mixture was stirred at room temperature for 16 hours. Remove the solvent in vacuo,
The residue was partitioned between ether and water. The organics were washed with 2M hydrochloric acid, and brine, then dried (MgSO _4), filtered and evaporated to give the desired amide as a colorless oil that solidified on standing (570mg, 57%).

[Table 6]

Preparation of N-3-aminopropyl 4-trifluoroacetamidobutanamide hydrochloride (11) N- (3-tert-butoxycarbonylamino) propyl 4-trifluoroacetamidobutanamide (570 mg, 1.6 mmol) , 4M hydrogen chloride in dioxane (5 ml) and stirred at room temperature for 1.5 hours. The solvent was removed in vacuo and the resulting oily material dried in vacuo to give the desired amine salt as a thick gum which was used without further purification.

[Table 7]

Preparation of methyl 4- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) butanoate (14) Anhydrous pyridine (5 ml) was added to methyl 4-aminobutanoate (0.43 g, 3 .1m
mol) and 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonyl chloride (1 g, 3.1 mmol) were added under argon atmosphere. The mixture was stirred at room temperature for 1 hour. The solvent was removed in vacuo and the solid partitioned between ethyl acetate and pH 6 citrate buffer. The organics were separated and the aqueous layer was extracted with ethyl acetate (2 x 100ml). The combined extracts were dried (MgSO _4), filtered, orange solid of the desired sulfonamide was evaporated (1.06 g, 80%) was obtained as a.

[Table 8]

N-3- (N′-tert-butoxycarbonyl) aminopropyl 4- (4-N, N-
Preparation of dimethylaminophenyl) azobenzenesulfonamide (16) This sulfonamide, in a form similar to 14, was prepared from 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonyl chloride (1 g, 3.1 mmol) and mono ( t
ert-butoxycarbonyl) -1,3-propanediamine (0.49 g, 2.8 mmol)
Was manufactured using. The sulfonamide (0.82 g, 63%) was isolated as an orange solid.

[Table 9]

Preparation of Methyl 6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanoate (20) This sulfonamide, in a form similar to 14, was treated with 4- (4-N, N Prepared using -dimethylaminophenyl) azobenzenesulfonyl chloride (1.03 g, 3.2 mmol) and methyl 6-aminohexanoate hydrochloride (0.58 g, 3.2 mmol), the sulfonamide (810 mg, 59 %) As an orange solid.

[Table 10]

[0055] N-3-aminopropyl 4- (4- (4-N, N-dimethylaminophenyl) azo
Production of benzenesulfonamide) butanamide (15)   Methyl 4- (4- (4-dimethylaminophenyl) azobenzenesulfonami
De) Butanoate (1 g, 2.47 mmol) in 1,3-diaminopropane (10 ml)
Once dissolved, the mixture was heated under nitrogen at 100 ° C. for 3 hours. Remove the solvent in vacuo and
The body was partitioned between pH 6 citrate buffer and chloroform. Dry organics (MgSO 4
_Four), Filtered and evaporated to give an orange solid (0.95 g, 86%);

[Table 11]

Preparation of N-6-aminohexyl 6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide (21) Methyl 6- (4- (4-N, N-dimethyl) Aminophenyl) azobenzenesulfonamide) hexanoate (0.72 g, 1.7 mmol) was melted and stirred 1,6
Added to diaminohexane (10.7 g, 92 mmol) at 50 ° C. 77 of the mixture
Stirred at 6 ° C for 6 hours then allowed the mixture to cool to room temperature. Then melt the mixture,
It was poured into pH 6 citrate buffer, the solid was extracted with chloroform and the extract was washed with water (3 x
150 ml), then washed with pH 6 citrate buffer, dried (MgSO ₄ ), filtered and evaporated. The obtained solid was suspended in chloroform / pH 6 buffer and collected by filtration. The solid was washed with water and air dried to give the amine (580 mg, 66%) as an orange solid.

[Table 12]

Preparation of N-3-aminopropyl 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide (22) N- (3-N ′-(tert-butoxycarbonyl) in dichloromethane (10 ml). Aminopropyl) 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide (1.1 g, 2.4 mmol) was treated with 4M hydrogen chloride in 1,4-dioxane (4 ml) at room temperature. The mixture was stirred for 16 hours and the solvent removed in vacuo. The residue was treated with saturated sodium hydrogen carbonate, and the obtained solid was dissolved in ethanol. The ethanolic solution was evaporated and dried in vacuo to give the amine (0.86g, quantitative) as an orange solid.

[Table 13]

Preparation of N-3-aminopropyl 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide hydrochloride (17) N- (3-N ′-(tert-butoxycarbonyl) aminopropyl) 4 -(4-N,
N-Dimethylaminophenyl) azobenzenesulfonamide (0.82 g, 1.78)
mmol) was suspended in 1,4-dioxane (3 ml) and treated with 4M hydrogen chloride in 1,4-dioxane (2 ml). The mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo and the resulting gum co-evaporated with ethanol and dried in vacuum. The amine hydrochloride was used without further purification.

[Table 14]

Preparation of N-3- (N-4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propyl 4-trifluoroacetamidobutanamide (18) Unpurified N- (3-aminopropyl) 4- (4-N, N-dimethylaminophenyl
) Azobenzenesulfonamide hydrochloride (1.78 mmol) and O- (N-
Succinimidyl) 4-trifluoroacetamidobutanoate (0.63 g,
2.1 mmol) was suspended in anhydrous tetrahydrofuran (20 ml) under nitrogen at room temperature. The mixture was treated with triethylamine (0.36 g, 3.6 mmol), stirred for 4 hours and another aliquot of O- (N-succinimidyl) 4-trifluoroacetamidobutanoate (0.2 g, 0.6 mmol) was added. Addition, the mixture was stirred for 1 hour, then the solvent was removed in vacuo. The residue was chromatographed, eluting the desired amide with ethyl acetate followed by 5% methanol in dichloromethane with N-hydroxysuccinimide. The solid was taken up in ethyl acetate and washed with pH 6 citrate buffer,
Dry (MgSO ₄ ), filter and evaporate to give the desired amide (690 mg, 65%) as an orange powder.

[Table 15]

N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonyl)
Preparation of aminopropyl 4-aminobutanamide (19) N-3- (N-4- (4-N, N) in methanol: water (1: 1) (40 ml) under nitrogen.
-Dimethylaminophenyl) azobenzenesulfonamide) propyl 4-trifluoroacetamidobutanamide (0.68 g, 1.25 mmol) was treated with potassium carbonate. The mixture was heated at 60 ° C. for 3 hours. The solvent was removed in vacuo. The residue was washed with chloroform / pH 6 citrate buffer, then ethanol. The resulting solid was air dried and combined with an ethanol wash to give an orange solid (0.54 g, 96
%).

[Table 16]

[0061] N-3- (4-trifluoroacetamidobutanoyl) aminopropyl 1- (3 '
, 5'-di (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-
Preparation of (yl) imidazolidin-2 (3H) -one-4-carboxamide (23)   1- (3 ', in anhydrous N, N-dimethylformamide (5 ml) under an atmosphere of argon.
5'-di (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-
Ile) imidazolidin-2 (3H) -one-4-carboxylic acid (628 mg, 1.3 mmol)
And N- (3-aminopropyl) 4-trifluoroacetamide butanamide
Hydrochloride (388 mg, 1.3 mmol) was added to triethylamine (263 mg, 2.6 m)
mol) and diphenylphosphoryl azide (385 mg, 1.4 mmol) at room temperature
It was The mixture was stirred for 16 hours. Add diphenylphosphoryl azide (300mg)
The mixture was stirred for a further 8 hours. The solvent was removed in vacuo and the residue was taken up in ethyl acetate.
Wash with water, 2M hydrochloric acid and 2M sodium hydrogen carbonate, and dry (MgSO4).
_Four), Filtered, evaporated, then diluted with 5% methanol in dichloromethane.
Matography was performed to give the desired material as a pale yellow gum (440 mg, 48%).
I got it.

[Table 17]

N-3- (4- (4- (4-dimethylaminophenyl) azobenzenesulfonamide
) Butanoyl) aminopropyl 1- (3 ', 5'-bis (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide This amide was prepared by a method similar to 23 to obtain anhydrous N, N-dimethylformamide (
1- (3 ', 5'-bis (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxylic acid in 10 ml)
(0.9 g, 1.9 mmol), N- (3-aminopropyl) 4- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) butanamide (0.9 g, 2
mmol), triethylamine (0.20 g, 2 mmol) and diphenylphosphoryl azide (0.61 g, 2.2 mmol). The amide (0.82 g, 48%) was obtained as an orange gum with accompanying undetermined impurities after chromatography with dichloromethane: methanol (100: 5).

[Table 18]

N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propyl 1- (3 ′, 5′-bis (tert-butyldimethylsilyloxy) -2′-
Preparation of Deoxyribose-1′-yl) imidazolidin-2 (3H) -one-4-carboxamide This amide was prepared by a method similar to that of 23 to obtain anhydrous N, N-dimethylformamide (
1- (3 ', 5'-bis (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxylic acid in 10 ml)
(710 mg, 1.5 mmol), N-3-aminopropyl 4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide (361 mg, 1 mmol), triethylamine (101 mg, 1 mmol) and diphenylphosphoryl azide (303 mg, 1 .1
mmol). Amide (280 mg) was added to dichloromethane: methanol (10
Obtained with impurities after chromatography eluting at 0: 5).

[Table 19]

N- (4- (3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propylamino) -4-oxobutyl 1- (3 ′, 5′-di (tert-butyl) Preparation of dimethylsilyloxy) -2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide This amide was prepared according to the method used for the preparation of 23 to give anhydrous N, N-dimethyl. 1- (3 ', 5'-bis (tert-butyldimethylsilyloxy) in formamide (10 ml)
-2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-
Carboxylic acid (710 mg, 1.5 mmol), N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propyl 4-aminobutanamide (4
Prepared using 46 mg, 1 mmol), triethylamine (101 mg, 1 mmol) and diphenylphosphoryl azide (303 mg, 1.1 mmol). The product was obtained as an orange oil after chromatography eluting with 5% methanol in dichloromethane. It was taken up in dichloromethane (80 ml), washed with water (2 × 100 ml), dried (MgSO ₄ ) and evaporated to give the product as an orange oil (500 m).
g, 55%).

[Table 20]

N-6- (6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (2′-deoxy-3 ′, 5′-di (tert-
Butyldimethylsilyloxy) ribose-1′-yl) imidazolidine-2 (3H)
Preparation of 4-one-4-carboxamide N-6-aminohexyl 6 in anhydrous N, N-dimethylformamide (10 ml)
-(4- (4-N, N-Dimethylaminophenyl) azobenzenesulfonamide) hexanamide (580 mg, 1.1 mmol) and 1- (3 ', 5'-di (tert-butyldimethylsilyloxy) -2'-Deoxyribose-1'-yl) imidazolidine-2 (
Treatment of 3H) -one-4-carboxylic acid (800 mg, 1.7 mmol) with bromotripyrrolidinephosphonium hexafluorophosphate (800 mg, 1.7 mmol) and triethylamine (222 mg, 2.2 mmol) at room temperature under an argon atmosphere. did. The mixture was stirred for 64 hours. The solvent was removed in vacuo and the reaction mixture was pre-adsorbed on silica gel and chromatographed by eluting the desired amide with dichloromethane 5% methanol to give the desired material (720 mg) as 3 molar equivalents of tripyrrolidine phosphor. Obtained with amide.

[Table 21]

N-3- (4-trifluoroacetamidobutanoyl) aminopropyl 1- (2 ′
-Deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (24) Preparation N- (3- (4-trifluoroacetamidobutanoyl) aminopropyl) 1-
(3 ', 5'-di (tert-butyldimethylsilyloxy) -2'-deoxyribose-
1′-yl) imidazolidin-2 (3H) -one-4-carboxamide (440 mg,
0.62 mmol) and ammonium fluoride (137 mg, 3.7 mmol) were added to methanol (
20 ml) and refluxed together for 48 hours. The solvent was evaporated and the residue was chromatographed to give the product in 15% methanol in dichloromethane as a colorless solid (1
25 mg, 42%).

[Table 22]

N-3- (4- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) -butanoyl) aminopropyl 1- (2′-deoxyribose-1′-
Preparation of (yl) imidazolidin-2 (3H) -one-4-carboxamide This nucleoside was treated with N- (3- (
4- (4- (4-dimethylaminophenyl) azobenzenesulfonamide) butanoyl) aminopropyl 1- (3 ', 5'-di (tert-butyldimethylsilyloxy)-
2'-Deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (0.8 g, 0.9 mmol) and ammonium fluoride (1.2 g, 32 mm).
ol). The reaction mixture was purified by chromatography and the desired nucleoside (300 mg, 50%) was eluted with 7.5% methanol in dichloromethane as an orange oil.

[Table 23]

N-4- (3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propylamino) -4-oxobutyl 1- (2′-deoxyribose-
Preparation of 1'-yl) imidazolidin-2 (3H) -one-4-carboxamide This nucleoside was treated with N- (4- (
3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide)
Propylamino) -4-oxobutyl 1- (3 ', 5'-di (tert-butyldimethylsilyloxy) -2'-deoxyribose-1'-yl) imidazolidine-2 (3H)
Prepared using -one-4-carboxamide (500 mg, 0.55 mmol) and ammonium fluoride (200 mg, 11.2 mmol). The reaction mixture was purified by chromatography and the desired nucleoside (0.18 g, 49%) was eluted with 10% methanol in dichloromethane as an orange solid.

[Table 24]

N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamido) propyl 1- (2′-deoxyribose-1′-yl) imidazolidine-2 (3
Preparation of H) -4-carboxamide This was treated in the same manner as 24 with N-3- (4- (4-N,
N-dimethylaminophenyl) azobenzenesulfonamide) propyl 1- (2 '
-Deoxy-3 ', 5'-di (tert-butyldimethylsilyloxy) ribose-1'-
Prepared using (yl) imidazolidine-2 (3H) -4-carboxamide (280 mg) and ammonium fluoride (240 mg, 6.5 mmol). The desired nucleoside (
110 mg, 0.19 mmol) was isolated as an orange solid by chromatography eluting with 10% methanol in dichloromethane.

[Table 25]

N-6- (6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (2′-deoxyribose-1′-yl)
Preparation of imidazolidin-2 (3H) -one-4-carboxamide This was treated in the same manner as 24 with N- (6- (6- (4- (4
-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (2'-deoxy-3 ', 5'-di (tert-butyldimethylsilyloxy) ribose-1'-yl) imidazolidine- Prepared using 2 (3H) -one-4-carboxamide (720 mg) and ammonium fluoride (240 mg, 6 mmol). The desired nucleoside (260 mg, 32% over 2 steps) was isolated as an orange solid by chromatography eluting with 10% methanol in dichloromethane.

[Table 26]

N-3- (4-trifluoroacetamidobutanoyl) aminopropyl 1- (2 ′
-Deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl)
Preparation of imidazolidin-2 (3H) -one-4-carboxamide (25) N- (3- (4-trifluoroacetamidobutanoyl) in dry pyridine (2 ml)
) Aminopropyl) 1- (2′-deoxyribose-1′-yl) imidazolidine-
2 (3H) -one-4-carboxamide (110 mg, 0.23 mmol) was added to 4,4'-dimethoxytrityl chloride (93 mg, 0.27 mmol) in dichloromethane (1 ml).
And treated with 4-N, N-dimethylaminopyridine (10 mg). The mixture was stirred at room temperature for 6 hours. Dimethoxytrityl chloride was gradually added to tlc (9: 1).
Dichloromethane: methanol) was added until no starting material remained. The solvent was removed in vacuo and the desired product was chromatographed to 100%.
: 10: 1 dichloromethane: methanol: triethylamine to elute and isolate,
Trityl ether (180 mg, 80%) was obtained as a colorless solid with 2 molar equivalents of triethylamine.

[Table 27]

N-3- (4- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) -butanoyl) aminopropyl 1- (2′-deoxy-5 ′-(4,4′-
Preparation of dimethyltrityloxy) ribose-1′-yl) imidazolidin-2 (3H) -one-4-carboxamide This was done in the same manner as 25 in N- (3- (4- (4)) in anhydrous pyridine (2 ml). (4- (4
-N, N-dimethylaminophenyl) azobenzenesulfonamide) -butanoyl)
Aminopropyl 1- (2'-deoxyribose-1'-yl) imidazolidine-2
(3H) -one-4-carboxamide (300 mg, 0.48 mmol) and 4,4'-dimethoxytrityl chloride (134 mg, 0.4 mmol) in dichloromethane (4 ml).
Was manufactured using. Further 4,4'-dimethoxytrityl chloride (200 mg)
Was added. The desired material is dichloromethane: methanol: triethylamine (
100: 10: 1) chromatographed to give 330 mg (74%) of an orange oil with 1 molar equivalent of triethylamine impurity;

[Table 28]

N-4- (3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propylamino) -4-oxobutyl 1- (2′-deoxy-5 ′-(4
, 4'-Dimethoxytrityloxy) ribose-1'-yl) imidazolidine-2 (3
Preparation of (H) -one-4-carboxamide This was done in a similar manner to 25 in N- (4- (3- (4- (
4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propylamino) -4-oxobutyl 1- (2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (170 mg) , 0.25 mmol) and 4,4'-dimethoxytrityl chloride (134 mg, in dichloromethane (4 ml).
It was prepared using 0.4 mmol). Two more additions of 4,4'-dimethoxytrityl chloride (40 and 36 mg, respectively) were added. The desired material was isolated by chromatography with dichloromethane: methanol: triethylamine (100: 4: 1) and the gum was triturated with ether to give the desired material (139 mg, 57%) as an orange solid.

[Table 29]

N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propyl 1- (2′-deoxy-5 ′-(4,4′-dimethoxytrityloxy)
Preparation of ribose-1′-yl) imidazolidin-2 (3H) -one-4-carboxamide This compound was prepared according to the method used for the preparation of 25 to give N in anhydrous pyridine (2 ml).
-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide
) Propyl 1- (2'-deoxyribose-1'-yl) imidazolidine-2 (3H
) -4-Carboxamide (110 mg, 0.19 mmol) and 4,4'-dimethoxytrityl chloride (79 mg, 0.23 mmol) in dichloromethane (4 ml). Further 4,4'-dimethoxytrityl chloride (20, 16, 13 and 13 mg) was added. The desired material (99 mg, 58%) was isolated after chromatography with dichloromethane: methanol: triethylamine (100: 10: 1).

[Table 30]

N- (6- (6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (2′-deoxy-5 ′-(4,4′- Preparation of dimethoxytrityloxy) ribose-1′-yl) imidazolidin-2 (3H) -one-4-carboxamide This was prepared according to the method used for the preparation of 25 to give N-6- in anhydrous pyridine (2 ml).
(6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide)
Hexanamido) hexyl 1- (2'-deoxyribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (200 mg, 0.27 mmol) and 4,4'- in dichloromethane (4 ml). Dimethoxytrityl chloride (110 mg,
It was prepared using 0.32 mmol). Further 4,4'-dimethoxytrityl chloride
(36, 35, 25, 23, and 20 mg) were added. Chromatography eluted the desired material (220 mg, 75%) with dichloromethane: methanol: triethylamine (100: 5: 1) along with triethylamine and methanol impurities.

[Table 31]

[0076] N-3- (4-trifluoroacetamidobutanoyl) aminopropyl 1- (3 '
-(2-Cyanoethyl) diisopropylaminophosphityl-2'-deoxy-5 '
-(4,4'-Dimethoxytrityloxy) ribose-1'-yl) imidazolidine-
Preparation of 2 (3H) -one-4-carboxamide (26)   N- (3- (4-trifluoroacetamidobutanoyl) aminopropyl) 1-
(2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-ii
) Imidazolidine-2 (3H) -one-4-carboxamide (180 mg, 0.18 m
mol) was dissolved in anhydrous tetrahydrofuran (3 ml) under an argon atmosphere. Solution
N, N-diisopropylethylamine (71 mg, 0.55 mmol), and then (2
-Cyanoethyl) diisopropylchlorophosphoramidite (65 mg, 0.27 mmo
In l), it was treated with ice cooling. The mixture was stirred at room temperature. After 10 minutes, tlc (9
: 1 dichloromethane: methanol) showed that starting material remained, and
N, N-diisopropylethylamine (48 mg, 0.36 mmol) and (2-sia
Noethyl) diisopropylchlorophosphoramidite (40 mg, 0.18 mmol) was added.
Added After 30 minutes, tlc showed no starting material left. Ethyl acetate
(25 ml) was added to the reaction and the solution was washed with brine (10 ml) and dried (MgSO4).
_Four), Filtered and evaporated. The oil obtained was taken up in dichloromethane (1.5 ml).
Add solids to a cold, stirring ice-cold 40-60 petroleum ether to precipitate a solid.
. The product was dried under vacuum and used without further purification. δ_P(CD_ThreeCN) 147
.95 and 148.17.

N- (3- (4- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) -butanoyl) aminopropyl 1- (3 ′-(2-cyanoethyl) diisopropylaminophosphityl Oxy-2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl) imidazolidin-2 (3H) -one-4
-Preparation of carboxamide (27) This phosphoramidite was treated with N- (3- (4- (4- (4
-Dimethylaminophenyl) azobenzenesulfonamide) -butanoyl) aminopropyl 1- (2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl) imidazolidine-2 (3H)- On-4-carboxamide (1
38 mg, 0.14 mmol) and (2-cyanoethyl) diisopropylaminochlorophosphoramidite (50 mg, 0.21 mmol) and diisopropylethylamine (5
Prepared using 4 mg, 0.42 mmol). Furthermore, (2-cyanoethyl) diisopropylaminochlorophosphoramidite (50 mg) and diisopropylethylamine
(54 mg) was needed to ensure complete reaction. The product was obtained by precipitation as an orange solid (150 mg). δ _P (CD ₃ CN) 148.04, 14
8.27.

N- (4- (3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propylamino) -4-oxobutyl 1- (3 ′-(2-cyanoethyl)
Diisopropylaminophosphityloxy) -2′-deoxy-5 ′-(4,4′-dimethoxytrityloxy) ribose-1′-yl) imidazolidin-2 (3H) -one-4-carboxamide (28) Production This phosphoramidite was treated with N- (4- (3- (4
-(4-N, N-Dimethylaminophenyl) azobenzenesulfonamido) propylamino) -4-oxobutyryl 1- (2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl ) Imidazolidine-2 (3H) -one-4
Prepared using carboxamide (139 mg, 0.14 mmol) and (2-cyanoethyl) diisopropylaminochlorophosphoramidite (50 mg, 0.21 mmol) and diisopropylethylamine (54 mg, 0.42 mmol). Further (2-cyanoethyl) diisopropylaminochlorophosphoramidite (50 mg) and diisopropylethylamine (54 mg) were required to ensure complete reaction. The product was obtained by precipitation as an orange solid (139 mg). δ _P (CD ₃
CN) 147.98 and 148.22.

N-6- (6- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (3 ′-(2-cyanoethyldiisopropylaminophosphityloxy)- Preparation of 2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (29) This phosphoramidite was N- (6- (6- (
4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) hexanamide) hexyl 1- (2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl) imidazo Using lysine-2 (3H) -one-4-carboxamide (111 mg, 0.11 mmol), (2-cyanoethyl) diisopropylaminochlorophosphoramidite (38 mg, 0.16 mmol) and diisopropylethylamine (41 mg, 0.32 mmol) Manufactured. Further (2-cyanoethyl) diisopropylaminochlorophosphoramidite (38 mg, 0.16 mmol) and diisopropylethylamine (52 mg, 0.32 mmol) were needed to ensure complete reaction. The product was obtained as an orange solid (105 mg) after precipitation. δ _P (CD ₃ CN) 148.05 and 148.26.

N-3- (4- (4-N, N-dimethylaminophenyl) azobenzenesulfonamide) propyl 1- (3 ′-(2-cyanoethyl) diisopropylaminophosphityloxy) -2′-deoxy- Preparation of 5 '-(4,4'-dimethoxytrityloxy) ribose-1'-yl) imidazolidin-2 (3H) -one-4-carboxamide (30) This phosphoramidite was prepared in the same manner as in 26. Therefore, N-3- (4- (4
-N, N-dimethylaminophenyl) azobenzenesulfonamido) propyl 1
-(2'-deoxy-5 '-(4,4'-dimethoxytrityloxy) ribose-1'-
Ill) imidazolidin-2 (3H) -one-4-carboxamide (80 mg, 0.09 m
mol), (2-cyanoethyl) diisopropylaminochlorophosphoramidite (4
3 mg, 0.18 mmol) and diisopropylethylamine (47 mg, 0.36 mmol)
Was manufactured using. Further, (2-cyanoethyl) diisopropylaminochlorophosphoramidite (43 mg, 0.18 mmol) and diisopropylethylamine (47
mg, 0.36 mmol) was added to ensure complete reaction. The product was obtained as an orange solid (75 mg) after precipitation. δ _P (CD ₃ CN) 147.99 and 148.23.

Example 4 Preparation of Templates Containing Imidazolidine-2 (3H) -one-4-carboxamide Base Analogues Imidazolidine-2 (3H)-each having the same sequence but used in their synthesis.
Five nucleotides differing only in on-4-carboxamide phosphoramidite were prepared.

Oligonucleotide sequence 5'-ACT GXA AGG GGA TCC TCT AGA GTC G
AC CTG CA-3 ′ (where X is imidazolidin-2 (3H) -one-4-carboxamide nucleotide) Template 1: Synthetic using phosphoramidite 26 Template 2: using phosphoramidite 27 Synthetic Template 3: Synthetic Using Phosphoramidite 28 Template 4: Synthetic Using Phosphoramidite 29 Template 5: Synthesized Using Phosphoramidite 30

The phosphoramidite prepared as described above was dissolved in Cruahcem DNA reagent grade acetonitrile to make a 1 millimolar concentration solution. The natural base phosphoramidite was obtained from Amersham Pharmacia Biotech and dissolved in DNA grade acetonitrile immediately prior to oligonucleotide synthesis according to the manufacturer's instructions. The synthetic phosphoramidite was dissolved in DNA reagent grade acetonitrile to make a 1 millimolar concentration solution, while 29 was dissolved in 1: 1 tetrahydrofuran: acetonitrile.

Oligonucleotides were synthesized in 3 steps on an ABI 394 DNA synthesizer. The first 27 bases were synthesized using a pre-programmed 0.2 μM CE cycle (DMT On) on a 1000 A CPG A column from ABI.

In the next step imidazolidin-2 (3H) -one-4-carboxamide base was added using a manual cycle (DMT On) and the synthetic phosphoramidite was used for 6 minutes using this cycle. The DABSYL labeled analog (27-30) was reacted for a binding time of 12 minutes. Finally, the last 4 bases were preprogrammed with 0.2 μM CE cycle (D
MT Off).

The oligonucleotide was cleaved from the CPG support using ammonia and the base was deprotected by heating the ammonia solution at 57 ° C. for 18 hours. The crude oligonucleotide was PAGE purified and desalted using a NAP-5 column using water as the eluent.

Read Through Experiments Recognition of the imidazolidin-2 (3H) -one-4-carboxamide nucleobase analogs in the polymerase enzyme in the oligonucleotides was performed in the following read through experiments. Using the above-mentioned oligonucleotide as a template, a 25-mer primer (5'-F
AM-TGC AGG TCG ACT CTA GAG GAT CCC C
-3 ') (supplied by Oswell) was used.

Example 4A Primer (7 μl 46 μM aqueous solution), Template 1 (7 μl 100 μM aqueous solution) 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Thermosequenase II® (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds after incubation. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

The reaction was analyzed on an 8% denaturing polyacrylamide gel and analyzed using Molecular Dynamics Fluor
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore Th
It was shown that ermosequenase II (registered trademark) reads through the base analog of the template oligonucleotide.

Example 4B Primer (7 μl of 46 μM aqueous solution), Template 2 (7 μl of 100 μM aqueous solution), 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Thermosequenase II® (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

The hybridization mixture (50 μl) and premix were mixed, the reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds later. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

The reaction was analyzed on an 8% denaturing polyacrylamide gel and analyzed using Molecular Dynamics Fluor
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore Th
It was shown that ermosequenase II (registered trademark) reads through the base analog of the template oligonucleotide.

Example 4C Primer (7 μl of 46 μM aqueous solution), Template 3 (7 μl of 100 μM aqueous solution), 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Thermosequenase II® (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds after incubation. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

Reactions were analyzed on an 8% denaturing polyacrylamide gel and tested on Molecular Dynamics Fluor
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore Th
It was shown that ermosequenase II (registered trademark) reads through the base analog of the template oligonucleotide.

Example 4D Primer (7 μl of 46 μM aqueous solution), Template 4 (7 μl of 100 μM aqueous solution), 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Thermosequenase II® (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds after incubation. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

The reaction was analyzed on an 8% denaturing polyacrylamide gel and analyzed using Molecular Dynamics Fluor
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore Th
It was shown that ermosequenase II (registered trademark) reads through the base analog of the template oligonucleotide.

Example 4E Primer (7 μl of 46 μM aqueous solution), Template 5 (7 μl of 100 μM aqueous solution), 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Thermosequenase II® (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C., 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds later. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

Reactions were analyzed on an 8% denaturing polyacrylamide gel and tested on Molecular Dynamics Fluor.
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore Th
It was shown that ermosequenase II (registered trademark) reads through the base analog of the template oligonucleotide.

Example 4F Primer (7 μl 46 μM aqueous solution), Template 2 (7 μl 100 μM aqueous solution), 1
A hybridization mixture consisting of 0 × Thermopol buffer (Biolabs) (21 μl) and double distilled water (35 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of Bst polymerase (Biolabs) (8 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds after incubation. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

The reaction was analyzed on an 8% denaturing polyacrylamide gel and analyzed using Molecular Dynamics Fluor.
Imaged on the imager. This is because the full-length primer is formed in the reaction and therefore B
It was shown that st polymerase is reading through the base analog of the template oligonucleotide.

Example 4G Primer (7 μl of 46 μM aqueous solution), Template 2 (7 μl of 100 μM aqueous solution), 5
X KGB buffer (250 mM Tris acetate, 17.5 mM magnesium acetate,
125 mM potassium glutamate, 10% glycerol, pH 7.9) (28 μl)
A hybridization mixture consisting of and double distilled water (28 μl) was heated to 95 ° C. for 10 minutes and allowed to cool slowly to room temperature. A premix of TTS DNA polymerase (as described in PCT Application No. PCT / US96 / 20225) (20 U, 5 μl), dNTPs (5 μl of 8 mM solution) and water (40 μl) was prepared.

Hybridization mixture (50 μl) and premix were mixed, reaction mixture was incubated at 72 ° C. and 10 μl sample was pre-incubated, 30, 60, 90, 120, 150, 180, 210, 240 and 600 seconds later. I took it. The reaction sample was quenched with EDTA (2 μl of 50 mM solution, pH 8). Orange G (5 μl) in 80% formamide was added and the mixture heated at 95 ° C. for 3 minutes. Primer alone, primer + template, primer + template + enzyme,
Primer + enzyme + dNTP, template + enzyme + dNTP, primer + template + dN
A TP control was also performed, and the mixture was incubated at 72 ° C for 300 seconds.

Reactions were analyzed on an 8% denaturing polyacrylamide gel and tested on Molecular Dynamics Fluor.
Imaged on the imager. This is because the full length primer is formed in the reaction and therefore T
It was shown that TS was reading through the base analog of the template oligonucleotide.

Example 5 Identification of Base Complementary Pairs for Imidazolidine-2 (3H) -one-4-carboxamide Base Analogues

Example 5A Four reactions were set up to identify base complementary pairs to the imidazolidin-2 (3H) -one-4-carboxamide base analog. To the hybridization mixture prepared in Example 4B (5 μl) were added: i) Thermosequenase II® (1 μl), dTTP (1 μl of 8 mM solution) and water (13 μl) premix ii) Thermosequenase II® (1 μl), dTTP (1 μl of 8 mM solution), d
A premix of ATP (1 μl of 8 mM solution) and water (12 μl) iii) Thermosequenase II® (1 μl), dTTP (1 μl of 8 mM solution), d
GTP (1 μl of 8 mM solution) and water (12 μl) premix iv) Thermosequenase II® (1 μl), dTTP (1 μl of 8 mM solution), d
Premix of CTP (1 μl of 8 mM solution) and water (12 μl).

The reaction was incubated at 72 ° C. for 5 minutes and EDTA (4 μl of 50 mM solution, p
Quenched with H8). These reactions were run alongside the controls in Example 4B. Reactions were analyzed on an 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluor imager.

Only reaction ii showed the product of the primer being extended by 3 bases, while the remaining 3 reactions (i, iii and iv) showed only the addition of 2 thymidine bases. In these reactions, only dATP was imidazolin-2 (3H) -one-4-
It is a base complement of a carboxamide base analog, thus indicating that the analog behaves as a thymidine analog.

Example 5B Four reactions were set up to identify base complementary pairs to the imidazolidin-2 (3H) -one-4-carboxamide base analog. To the hybridization mixture prepared in Example 4B (5 μl) were added: i) Bst polymerase (1 μl), dTTP (1 μl of 8 mM solution) and water (13 μl).
Ii) Bst polymerase (1 μl), dTTP (1 μl of 8 mM solution), dATP (1 μl of 8 mM solution) and water (12 μl) premix iii) Bst polymerase (1 μl), dTTP (1 μl of 8 mM solution), dGTP (1 μl
Premix of Bst polymerase (1 μl), dTTP (1 μl of 8 mM solution), dCTP (1 μl of 8 mM solution) and water (12 μl).

The reaction was incubated for 5 minutes at 72 ° C. and EDTA (4 μl of 50 mM solution, p
Quenched with H8). These reactions were run alongside the controls in Example 4B. Reactions were analyzed on an 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluor imager.

Only reaction ii showed the product of the primer being extended by 3 bases, while the remaining 3 reactions (i, iii and iv) showed only the addition of 2 thymidine bases. In these reactions, only dATP was imidazolin-2 (3H) -one-4-
It is a base complement of a carboxamide base analog, thus indicating that the analog behaves as a thymidine analog.

Example 5C Four reactions were set up to identify base complementary pairs to the imidazolidin-2 (3H) -one-4-carboxamide base analog. To the hybridization mixture prepared in Example 4B (5 μl) were added: i) TTS polymerase (1 μl), dTTP (1 μl of 8 mM solution) and water (13 μl).
l) premix ii) TTS polymerase (1 μl), dTTP (1 μl of 8 mM solution), dATP (1 μl)
Premix of 1 (8 mM solution) and water (12 μl) iii) TTS polymerase (1 μl), dTTP (1 μl 8 mM solution), dGTP (1
Premix of μl 8 mM solution) and water (12 μl) iv) TTS polymerase (1 μl), dTTP (1 μl 8 mM solution), dCTP (1 μl)
1 premix of 8 mM solution) and water (12 μl).

The reaction was incubated at 72 ° C. for 5 minutes and EDTA (4 μl of 50 mM solution, p
Quenched with H8). These reactions were run alongside the controls in Example 4B. Reactions were analyzed on an 8% denaturing polyacrylamide gel and imaged on a Molecular Dynamics Fluor imager.

Only reaction ii showed the product due to the primer being extended by 3 bases, while the remaining 3 reactions (i, iii and iv) showed only the addition of 2 thymidine bases. In these reactions, only dATP was imidazolin-2 (3H) -one-4-
It is a base complement of a carboxamide base analog, thus indicating that the analog behaves as a thymidine analog.

Example 6 Synthesis of Triazolone Analogs Synthesis of T analogs using 1,2,4-triazol-3-one-5-carboxamide can be accomplished using Scheme 1. The synthesis of compound 3 is known (T
J Schwan and RL White, J Heterocycl. Chem., 1975, 771). This compound is
It can be glycosylated with 1- □ -chloro-3,5-ditoluoyl-2-deoxyribose. Removal of the toluoyl protecting group and reprotection as the silyl ether, followed by amidation with propylenediamine gives the extended amine (5). from here,
Triphosphate was converted from its amine by its tert-butoxycarbamate.
It can be prepared by protection as e), removal of the silyl protecting group and phosphorylation. The phosphoramidite extends the amine with a TFA-protected 4-aminobutyric acid NHS ester,
It can be prepared by removing the silyl protecting group, dimethoxytritylation and phosphorylation.

[Chemical 19]

The invention is not limited to the embodiments and examples described above, which can be used both in the structure and in the process steps without departing from the invention.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁷ Identification code FI theme code (reference) G01N 33/53 G01N 33/566 33/566 33/58 A 33/58 C12N 15/00 A (81) designation Country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG) , CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZW) ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AG, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, BZ, A, CH, CN, CR, CU, CZ, DE, DK, DM, DZ, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW, MX, MZ, NO, NZ, PL, PT, RO , RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, TZ, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor William Jonathan Cummins UK 23.4 Jay, Hartfordshire, Tring, Thorntree Drive 5 (72) Inventor Robert James Domet Nearn UK, ARL 4.9 YouN, Hartfordshire , St Albans, Springwood Walk No. 16 F-term (reference) 2G045 DA12 DA13 DA14 FB02 FB07 FB08 4B024 AA01 AA11 BA80 CA05 HA08 4B063 QA01 QQ42 QQ52 QR08 QR31 QR42 QR58 QR62 QX02 4C057 AA03 AA18 LL02 BB02 CC03 DD03 CC03 DD06LL LL21 LL60 MM04

Claims (15)

[Claims] 1. A structural formula: Wherein, X is CH or N, Y is -CO -, - CONW -, - O -, - S -, - SO -, - SO 2 -, - NW
CO-, -NW- or -OCO-, W are the same or different at different positions on the molecule, and each is H or alkyl or aryl or Rp or -Ln-Rp, Ln is a linker group, Rp is a reporter moiety, and Q is A sugar or sugar analogue or nucleic acid backbone or backbone analogue, provided that at least one reporter moiety Rp is present]. 2. Q is [In the formula, Z is O, S, Se, SO, NW or CH ₂ , R ¹ , R ² , R ³ and R ⁴ are the same or different, and each is H, OH, F, NH ₂ ,
N _3, O-hydrocarbyl or Rp or -Ln-Rp, ^{R 5} is OH, SH or NH ₂ or mono-, - di - or tri - phosphate or thiophosphate or corresponding boranophosphate, or ^{R 2} and ^{R 5,} Is a phosphoramidite or other group for incorporation into a polynucleotide chain, or a reporter moiety], or Q is an acyclic sugar consisting of one of the following modified sugar structures: Morpholino backbone [Chemical 4] The compound according to claim 1. 3. A compound according to claim 1 or 2 wherein the reporter moiety Rp is absent from Q. 4. Any of claims 1 to 3, wherein the linker group Ln is a fixed or flexible, saturated or unsaturated chain of 1 to 60 carbon, nitrogen, oxygen, phosphorus and / or sulfur atoms. The compound described in. 5. The compound according to any one of claims 1 to 4, wherein the reporter moiety Rp is a signal moiety or solid surface, or a reactive group which allows the signal moiety or solid surface to bind to a nucleoside or nucleotide analogue. . 6. The compound according to claim 5, wherein the reactive group is NH ₂ , OH, COOH, CONH ₂ , ONH ₂ , SH or thiophosphate or hydrazine or hydrazide, or active ester or aldehyde or maleimide. 7. A nucleoside analog comprising the compound according to any one of claims 1 to 6. 8. A nucleotide analog comprising the compound according to any one of claims 2 to 6. 9. The nucleotide analogue according to claim 8, wherein R ⁵ is triphosphate. 10. A polynucleotide comprising the nucleoside analog of claim 7. 11. A nucleic acid backbone (LNA) in which Q consists of a sugar-phosphate repeat or a modified sugar-phosphate repeat, or a peptide or polyamide nucleic acid (
The polynucleotide according to claim 10, which is a backbone analog such as PNA). 12. A chain extension method comprising reacting the polynucleotide according to claim 10 or 11 with a primer in the presence of a polymerase. 13. The chain extension method of claim 12, wherein the primer is selected to hybridize to a portion of the polynucleotide chain that is free of nucleoside analogs. 14. A method for detecting a nucleic acid comprising the compound according to any one of claims 1 to 6, which comprises a step of detecting the presence of the reporter moiety Rp. 15. The method according to claim 14, wherein the reporter moiety is a radioisotope, a stable isotope, a signal moiety or a specific chemical moiety suitable for detection by spectroscopy, in particular mass spectroscopy.