3'-(2')Amino or thiolmodified, fluorescence coupled nucleoside and oligonucleotide, a method for their preparation and their use.

Labeled oligonucleotides have found extraordinarily numerous applications in genetic technology because their manipulation is easier than DNA samples traditionally used as hybridization samples, which are obtained by restriction digestion of natural genetic material.

Tagged oligonucleotides »which are used in the form of so-called antisense DNA oligonucleotides may intervene in cellular occurrences in a regulatory manner and thus become increasingly important for example for in vivo searches. * of protein expression. According to current knowledge the mechanism develops through effects of

<img file="PT99747A_D0001.tif" />

ADE - DNA, DNA - RNA and ASN - AEHj exchange however, this is not yet fully clarified »

Tagged oligoisiclotides are used in vitro *, for example, to identify gene fragments within a gene bank by probing and identifying tagged gene samples with the aid of tagged oligonucleotide.

To be able to perform such in vitro or in vivo assays, the oligonucleotides must be labeled, as already mentioned. Together with radioactive labeling by suitable isotopes, they are used as a non-radioactive fluorescence dye label, already derivatized, because they provide the possibility of easier and less hazardous manipulation.

Throughout this time, such a technique has also been successfully used for non-radioactive DNA sequencing. In this case preparations were made, which are based essentially on the Sanger process (F. Sanger, S. Nicklen and S. Coulson, Proc. Natl, Acad. Sci. USA 74,5465 (1977)).

The fluorescence label is either adapted at the 5 'oligonucleotide end (LE Hood, L. Smith &amp; G. Heiner, Nature 521, 674 (1986)) or at the base of the nucleus (J. J. Prober, GL Trainor and SJ). Dam, Science 258, 556 (1987)) (GL Trainor, Anal. Chem. 62, 418 (1990)). A decisive drawback of the last cited process is that the fluorescence label is introduced during synthesis, that is, during polymerization and in this case especially during enzymatic polymerization. This stage of the process has the consequence that only some polymerases can still be used for synthesis, that the acceptance of triphosphates is reduced due to the polymerases and that further substrate excess is required.

- 2 The introduction of the fluorescence label is not limited to Sâhger sequencing. Chemical sequencing according to Maxam-Gilbert with fluorescence labels is also known (II. Voss, 0. Dchwager, U. Wirkner, 3. Sproat, J. Zimmermann, A. 2Qsen.th.al, H * Erfle, J Stegemanr and W. Ansorge, Nucl. Acids Bes. 17, 2517 (1989))

Similarly, restriction fragment mapping with fluorescence detectors is also described (AV Oarrano, J. hamerdin, X, K. Ashwoeth, B., Watkins, E. Branscomb, Ϊ Slezak, S. Saff, P J. Jong, D. Keitb,

L ·. McBride, S., Iister, Kronick, Genomies 4, 129 (1989), and S. Brenner and E.i. Livak, Proc. Natl. Acad. Know. USA 86, 8902 (1989)).

We have now found that a fluorescence dye can be coupled to the 5 * - (2) position.⁴) (at the alpha or beta position) is a nucleoside, nucleotide or oligonucleotide through an amino or thiol group and that this compound may advantageously be used for the synthesis of a ribbon in the presence of a template ribbon or oligonucleotides as well as for the detection of genetic material * in vivo * and * in vitro *.

The invention therefore relates to

1. A process for the preparation of a compound of formula (I)

<img file="PT99747A_D0002.tif" />

in which

R¹ represents base 4e purine or 4e pyrazine;

At least one of R 1 and R 4 is a fluorescent dye attached via an amino or thiol group at the alpha or beta position and the other radical optionally represents a hydrogen atom, a hydroxyl group, or a hydroxyl group; protected methoxy, in the alpha or beta position;

n represents an AO number;

4· « «

S represents a protecting group at the 5 'position or phosphate, pyrophosphate, or triphosphate;

R^z represents oxygen, fluoromethylene, difluoromethylene or methylene;

represents a hydroxyl or methoxy group or a hydrogen atom at the alpha or beta position, where R 1 and R 2 may respectively have the same or different meanings;

Xδ X represent oxygen, sulfur, ITH or methylene, where X and Y may be respectively the same or different, characterized in that the OB group is derived from the solid, nucleotide or oligonucleotide nucleus, which is in position 5 * and / or 2 ', into an amino or thiol group, and then couple a fluorescent dye.

- 4 2 * The use of the compounds characterized in point 1. in

(a) synthesis of inverse tapes in the presence of a tape which serves as a template;

b) synthesis of defined oligonucleotides'

c) its detection in vivo * and in vitro and

d) detection of nucleic acids in vivo * and in vitro *.

In the following »the invention will be described in detail» especially »in its preferred embodiments. In addition» the invention is determined by the content of the claims.

The compounds of general formula X are synthesized substantially by known literature procedures (L1 Gait, Oligonucleotide Synthesis, JL-Press, Oxford 1984).

For dye coupling in position 3¹ and / or 2 'starts from a compound of general formula II' preferably 'from a nueleoxide

<img file="PT99747A_D0003.tif" />

II compound of the general formula II, wherein R *, R²B?, B *, R ^, R ^, R ^, X, Y and n have the above meanings, and the substituents may be the same or less and B / or R representam represent hydrogen, a hydroxyl group or a protected hydroxyl or methoxy group; The dye is coupled via the hydroxyl group at the 5 'and / or 2' position by the introduction of an amine or thiol.

For the introduction of azide, a leaving group is inserted at the 5 '- (2 *) position. As a leaving group, preferably a triylate or mesylate or tosylate group is employed. By means of a nucleophilic attack with azide, preferably lithium azide, azide is introduced. The nucleophilic attack of a thiolate or thiolate with the protected 3-atom provides the protected thiol or thiol.

The stereochemistry of the desired nucleotide sugar cluster is best obtained by S2 substitutions.

Amino group can be readily obtained by azide and subsequent reduction to amine (Lit. VII, S. llungall and B. X. Letsinger, J. Org. Chern. Vol.

40, 11, 1659 (3-975). For this operation, Staidinger's reaction with triphenylphosphine and water is preferred (lit. D. May and JW Sngels, Nucl. Acids Kes. 17, 15, 5975 ( 1989)).

All compounds were tested for constitution and configuration by nuclear magnetic resonance, elemental analysis, ultraviolet spectrum (BV), infrared spectrum (IR), etc.

After polycondensation is over, the 5 * (2 *) amino or thiol group of the sugar cluster at the 5 'end of the DNA can react with a fluorescence dye θ **.

<img file="PT99747A_D0004.tif" />

reactive by known methods in the literature (Sunkapiller, STucl. Acids, Res., 13, 2399, 1985; Rodges RR et al. Biochemistry, Vol. 28, 261 (1989)).

Alternatively, the amino group can also be obtained by the ilitsunobu reaction (Synthesis, Volume 1, 1981, page 1) and according to the reaction described by Tamamoto et al. (J. Chem. Soc. Perk. Trans. X, 1, 306, 1980).

coupling of the fluorescence dye via the thiol group is carried out analogously. However, instead of the azide group, a thiol group in the protected or unprotected form is introduced.

Coupling of the fluorescence dye to the free amino group or thiol function of the nucleoside or oligonucleotide nueleoside, alternatively can also be performed only after use. For example, it is possible to carry out the reaction with the fluorescence dye after completion of the AD sequence sequence reaction. or ARS, marking the total reaction mixture with the fluorescence dye.

As fluorescence dyes, in principle all commercially available dyes which react with an amino or thiol group, preferably fluoresceins, rodaainas, Texas red (4-fluorine-7-nitrobenzofuran), are suitable, c &amp; marinas »fluorescamines, succinylfluorescins and dansilas.

The derivatized reaction mixture may be separated by gel electrophoresis and detected by photometry or laser spectroscopy (H. Swerdlow and R. Gesterland, Ruel. Acids Res. 18, page 1415 (1990)). &lt;ASCohen et al., PHAS US. 85, 9660 (1988)), for example, capillaries filled with

Detection is then preferably carried out at the outlet of the canals (H. Swerdlow, S. I7u, H. Herke, D. Dovichi, Gromatography 516, 61 (1990)).

Starting from a compound f and the general formula (I), which has in position 5^f With triphosphate, the double stranded primary DNA (primer) can be synthesized which? and a ribbon which serves as a template with the aid of a polymerase, i.e. an enzyme, which synthesizes in the presence of suitable substrates a faithful complementary sequence sequence in the presence of the four nucleoside triphosphates, preferably with the aid of Ip polymerase or Taq polymerase, AP-Polymerase I and Reverse Trascriptase * As 5 'protecting groups, trityl, methoxytrityl or dimethoxytrityl groups are used (Gait, Oligonueleotide Synthesis, JKL-Press, Oxford 1984).

The end of the synthesis may be determined specifically, by use of a 3 * - (2 ') amino-modified nucleotide A, O, G, T of formula (I). This is of particular importance for the synthesis of reverse strands of DNA in the presence of a template strand and thus also for ABB strand sequencing, as the use of a modified nucleotide guarantees a very long reaction interruption. base specific.

The synthesis of nucleosides, nucleotides and oligonucleotides of ARK is done analogously.

In addition, derivatizable oligonucleotide synthesis is possible with the aid of a starting nucleotide, which has been modified with amino or thio at the 5 * - (2 *) end and fixed to a polymeric support *

As oligonucleotides are considered all DNA and ASN nucleotides prepared in the traditional sense, preferably, however, having a length of a 100, especially 12 * 50 nucleotides (chemical synthesis) or a length of up to about 5,000 nucleotides (enzymatic synthesis), depending on the effectiveness of the polymerase used.

The synthesis of oligonucleotides is carried out starting from the starting-support nucleotide complex in the traditional sense, i.e. in the 5 '© direction, enables the synthesis of an oligonucleotide with a defined sequence.

Attachment of the starting nucleotide to commercial media is accomplished by a spacer which can be dissociated after synthesis for example by the known succinic acid binding in the literature or by ligation with urethane (Efimov et al., ITuel. Acids. Ses. 11, 8569, 1985))

After synthesis, the oligonucleotide must be dissociated from the support with a suitable reagent. Modification with a fluorescent dye of any kind immediately follows.

Oligonucleotides thus synthesized and modified at the 5 ** end (2 ') can then be used for the detection of, for example, complementary oligonucleotides.

The use of the process according to the present invention combines two advantages with each other:

1) In the preparation of a labeled reverse strand, the interruption of the strand by the modified nucleotide at the 5 '- (2 *) end with amino or thiol occurs simultaneously. 2> Marking of the same with the fluorescence dye at position 5 * - (2 *) is possible »in addition, a hazard-free detection»

<img file="PT99747A_D0005.tif" />

2) In the preparation of finely defined oligonucleotides it is possible, by labeling the starting nucleotide coupled to a support and also modified in 5 * - (2 *) with amino or thiol, to perform an accurate synthesis of the linked oligonucleotide with the possibility of labeling. fluorescence and thus for detection.

The following examples serve to further clarify the invention,

Examples

1. J · - or 2 * -Aaino- or azido-nucleoside-5 *-anomeric triphosphates

Example 1

Summary of 5^,triphosphate-3'-amino-3'-soxyriboside thymidine;

Reaction scheme

<img file="PT99747A_D0006.tif" />

* - ^ onophosphate-3 * azido-3 * deoxyriboside thymidine

3'-AzidObimidine (Sigma) (160 mg, 0.6 mmol) is dissolved under stirring in 10 mL of triethyl phosphate. With a loading funnel, 0.2 ml of POOl and 6 ml of triethyl phosphate are added to the solution at 4 ° C. After 24 hours, neutralize with 5θ of saturated NaHCG solution and extract, respectively, twice with 60 ml of toluene and 100 ml of ether. The aqueous phase is diluted with distilled water to 500 ml and placed in an exchange column

<img file="PT99747A_D0007.tif" />

anion (W Sephadex A-25 · 50 s 2.5 en), which was exchanged by passing 200 ml of 0.2 K TBK buffer (tri.ethylammonium bicarbonate buffer; pH 7.5 justly), with HOOj as counterion. It is then purified by passing 500 ml of distilled water and eluted first with 200 ml of T3K 0.1 II buffer and then with a linear gradient of TBE buffer.

0.1-0.2 M (total volume at gradient 1350 ml). The chromatogram shows a peak, the maximum product, and the product was eluted at a concentration of TBK buffer from 0.12 to 0.15 Ii. The positive 20 ml fractions are combined according to the thin layer chromatogram and concentrated by drying after freezing. Triethylammonium bicarbonate is removed by repeated addition of ethanol. The product is presented as a white crystalline triethylammonium salt.

Yield 220 mg (5θ> 2%); molecular weight 651.81; R_f (ammonia, iso-propyl water 10 ϊ 73: 20) 0.405 300 ΜΗζ- ^ Η-ΜΐΝ (DgO); 1.36 (t, 3H, CH₅); 1.90 (s, 3H, CH₅) δ 2.50 (m, 4H, 2 *, 2H); 5.18-3.50 (dd, 2H, 0H₂); 4.10 (α, 1H, 4MI); 4.3 (m, 1H,

3'-H); 6.31 (t, 1H, 1¹-H); 7.7 (s, 1Ξ, 6-H) δ 11.50 (s, 1H, JSH), 300 æS-^5Ip-Su (HjPO)₄ 85%, external, D ^O): 1.23 (s, IP) 5'-Triphosphate-3 * azido-3 * deoxyribeside thymidine

5 * -monophosphate-3 * -azido-3 *, 2 * · deoxyriboside thymine is dissolved as a triethylammonium salt (130 mg, 0.2 mmol) in 6 ml absolute DtiP (dimethylformamide) and To the solution, at 25 DEG C., N ', R'-carbonyl diimidazole (162.15 mg, 1 mmol) in 3 ml of Dl₃ is added. absolute under agitation. After 2 hours of reaction time, 1 ml of absolute methanol is added, stirred for a further 15 minutes and methanol is distilled off. To the residue is added 5 ml of a 0.2 L pyrophosphate solution. tri-n-butylammonium in DPI and stir overnight at room temperature. The precipitate consisting of imidazole pyrophosphate is separated by filtration, washed with 20 ml DMF and the filtrate concentrated on a rotary evaporator. The residue is dissolved in 2 0 0 ml of 0.1 TB TBK buffer and the solution is introduced into a separate column.

<img file="PT99747A_D0008.tif" />

Sephadex A-25 anion killer loaded with HCO_-. Purifi_# The product is dried by passing 200 ml of distilled water and eluted with a linear BE buffer gradient at 0 to 0.5% (total gradient volume 2000 ml). The eromatograms appear at the maximum, the first of which is caused by the monophosphate and the second by the product. The product was eluted at a T3K buffer concentration of 0.50 to 0.55 ll. The 20 ml positive fractions are combined and concentrated by lyophilization. Triethylammonium bicarbonate is removed by repeated addition of ethanol. The product is presented as a white crystalline triethylammonium salt.

Yield 92 mg (50.4%): Molecular Weight: 911.95; % (ammonia: isoprop .: water 10: 00: 20): 0.14; 500 (Η ^ Ρ0₄ 85% external, D ~ 0): -10, ≤4 (d,²tf_pp(22.1 Hz, IP, alpha-P atom); -11.45 (d, tf = 21.9 Hz, IP, gaaa-P atom): -22.99 (t, 5tf-_D * 20.5 Hz, IP, beta-P atom).

JrJr * -Triphosphate-5'-amino-5 * -deoxyriboside thymidine

5'-Triphosphate-5'-azido-5 ', 2-deoxyribaside thymine (68 mg, 0.075 mmol) is dissolved in 5 ml of dioxane-distilled water (2: 1) and under stirring at room temperature. added and triphenylphosphine (200 mg, 0.75 mmol). The reaction mixture is stirred at 25 ° C for 50 hours and then the solvent is removed. The residue is dissolved in 50 ml of distilled water and extracted three times with 50 ml of ether respectively. Dilute the aqueous phase to 150 ml and place the solution on a charged Sephadex A-25 anion exchange column. Purify the product with 200 ml of distilled water and elute with linear gradient of 0 to 0.5 S PBK buffer (2,000 ml gradient volume).

The eromatogram has three maximums, the first of which represents monophosphate compound 6, the second maximum product and the third maximum educt 7. The product is eluted at a T3K buffer concentration of 0.40 to 0.42 II . The 25 ml positive fractions are pooled and concentrated by lyophilization. Triethylammonium bicarbonate is thinned by the addition of ethanol. The product is a white amorphous triethylammonium salt.

<img file="PT99747A_D0009.tif" />

Yield: 57 mg (85.7%); molecular weight 885.90; R_f(ammonia: isoprop .: water 10: 70: 20): 0.08; 500 mHzi-RuiH (P₂O): 1.50 (t, 52.0H₃); 1.92 (s, 52.02%); 2.64 (m, 42.2 ', 2MI): 5.19-5.21 (dd, 22, G2₂); 4.26 (m, 12.4^r-2); 4.41 (m, 12.5'-H); 6.54 (t, 6.74 Hz, 1H, 1H); 7-69 (s, 1H,

6-2); 11.50 (a, 12, RH). 500 LSz-⁵¹P-S2w (85% external H ^ PC ^) »D_O0); -10.75 (d, gg Hz, IP; alpha-P atom);

is it 3? .P

-11.45 (d, δ * 21.9 2z, IP, gamma-P atom); -22.05 (t, x PP? Jpp? 20.4 Hz, IP, beta-P atom).

Example 2

Synthesis of l- (3^r-amino-2 ·, 5 * -dideoxy-5 *-triphosphate-beta-0-threopentofuranosyl) -thymine Reaction scheme

T

<img file="PT99747A_D0010.tif" />

<img file="PT99747A_D0011.tif" />

1- (5'-Azido-2 *, 5'-dideoxy-N, Q-dimethoxytrityl-beta-D-tre opentofuran sil) -thymine

To a solution of 5 * - * dimethoxytrityldimidine (obtained see U.Gait, Oligonucleotide synthesis, ISL Press (1984), page 27) (5.98 g, 11 mmol), triphenylphosphine (3.076 g, 11.73 mmol) and azide of lithium (5.1 S, 55 mmol) in 57 ml of water-free DuP, with stirring is added carbon tetrabromide (11.91 mmol, 4.10 g). The preparation is stirred for 56 hours at room temperature (ΤΛ) and then 15 ml of methanol is added. The product is then precipitated into 800 ml of ice-cold distilled water. The precipitate is taken up in chloroform and purified by flesh chromatography (ethyl acetate: n-hexane * 5: 1). Chlorine R value

Formiomethanol 9: 1 = * 0.52. (Cu): 2100 azide; yield 80%, 4.95 S '1- (5'-Amino-2 *, 3'-dideoxy-5'-0-dimethoxytrityl-beta-D-threopento furanoxyl) -timine

To a stirred solution of 1- (5'-azido-2 ', 5'-dideoxy-5'-D-dimethoxytrityl-beta-D-threopentofuranosyl) thymine (0.5 0.8 mmol) 0.15 triphenylphosphine (1.3 g, 4.94 mmol) and allowed to react for 4 hours. To hydrolyze phosphinimin 3 ml of distilled water is added and stirring is continued for an additional 3 hours. After removal of the solvent by rotary distillation, the residual oil is purified by flash chromatography (chloroform imethanol 99: 1 + 1% triethylamine). Value in same eluent * 0.27. The amine may be stained violet by treatment with ninhydrin in the thin layer chromatography (OD) assay, Kendimento 87.5% $ 0.42 g. The product has the corresponding spectrum.

Section for 1- (3 * -amino-2 *, * -dideoxy-5¹triphosphate-beta-D-threopentofuranosyl) thiazide

To continue the reaction to obtain 5'-triphosphate, the dimethoxytrityl group is removed analogously to the procedure described in M.ôait, Oligonucleotide synthesis, IBL Press page 49 (1984) and the triphosphate coupling is performed at the 5 'position as described in the Example

1.

Preparation of 2 * -amino-2'-deoxy-5^ftriphosphate uridine Reaction scheme

- 14 (2,2'-Anhydro-1-beta-arabinofuranosyl) -uracil

To a solution of 19 S (77.8 mmol) of uridine and 22.2 g (103.6 mmol) of diphenyl carbonate in 75 ml of absolute dimethylformamide are added 0.5 g (5 * 93 mmol) of sodium hydrogen carbonate. After complete dissolution the clear colorless liquid is heated to a temperature of 150 ° C for 30 minutes. After allowing to cool, the cold solution is poured out into absolute ether. The ether is separated by decantation of the precipitate formed and the crude product is recrystallized from 1650 ml of methanol. After drying, a white crystalline paddle is obtained.

Melting point: 259 ° C, HS: 227; 0.31 in methylene chloride / methanol S: 2: yield: 9.22 g (52.3%):

AHz corresponds to literature data.

* -Azido-2 * ~ deoxyluridine

1.16 g (10 mmol) of (2,2 * -aniaro-1-beta-arabinofuranosyl) uracil and 5.5 g (71.7 mmol) of lithium azide are stirred in 25 ml of absolute (1,3-dimethyl-3,4,5,6-tetrahydro-2- (1H) -pyrimidinol) previously in a container. The suspension is heated to 120 ° C and the reaction solution stained black. After 3 days, the black solution is diluted with 80 ml of water and extracted 2 times with 100 ml of methylene chloride. The combined organic phases are washed four more times with water and concentrated on a rotary evaporator. The oily solid residue is purified by column chromatography on silica gel (8: 2 methylene chloride / methanol). After concentration of the black oily residue by distillation on a rotary evaporator, it is further purified twice on silica gel base (acetone: methanol 8: 3 and acetone: ethyl acetate (1: 1). A single colorless glass is obtained by thin layer chromatography (methylene chloride / methanol 8: 1 * 0.61). Yield 2.035 S (30%)} IR (chloroform film): 2120 δΛ LiS (PAB): 270. The product has the expected spectrum.

Obtaining 2'-amiro-2'-d.esoxy-5

triphosphate uridine is carried out by selective 3'-acylation of the hydroxyl function. The introduction of the 5'-triphosphate group is then carried out analogously to Example 1. After deacylation at the 5 'position the azide is reduced to amine by reaction with triphenylphosphine (as in Example 2).

Example 4

Synthesis of 3'-amino-2 *, 3 * -dideoxy-5 *-triphosphate adenosine Reaction scheme

<img file="PT99747A_D0012.tif" />

A suspension of 920 is cooled to 30 ° C

<img file="PT99747A_D0013.tif" />

by the procedure described in Nishino et al., Nucleo tides 1986, 5,159) in 30 ml of absolute dichloromethane (containing pyridine al) and 5 ml of a solution of trifluoroethanesulfonic acid and dichloroebano ( 10 ° C and volume), lentamsnbe, and dropwise »After the cooling bath is removed» the solution is allowed to warm and to add 1 · 1 of water after 5 hours. In addition to the water and wash it in the "organic phase", remove the remaining residue, dissolve the remaining residue with 5 ml of methanol and add it to the sodium bicarbonate. 2 hours at 4 ° C, neutralize it to 10% ethyl ether, the solvent is distilled off and purified by flash chromatography (silica gel) (chloroform methanol 9: 3). »&« Dlaento * U0 stg (1,4 & amoles) *?% '> *

Chlorophoriomethanol 9%; 3 '0.49' ΙΠ '459 * * - sdo-2', 5 * -dideoxyiadenosinn

Cool to -53 ° C a solution of 920 mg (2 ml) of iίθ-bemsoi1-9- (3-O-bs ”soil-α-30α-beba-3-treopentofuranosyl) adenine and 23 ml dichloroaotaro Absolute (at 2 m pyridine). Then, 5 x 8 (5 mmoles) of a triluoroethanesulphonic acid anhydride solution are added as Absolute. The cooling bath is removed, stirred for a further 23 minutes and 980 g (23 mmol) of lithium acid is added to 20 ml of 327 µl solution. After 2 hours of stirring at 2A salts are added. 50 ml of water and 150 ml of chloroform, stir the organic flask and wash with distilled water. Aluine-the solvent and hot-evaporate and treat overnight with ammonia methanol solution to remove protection base group. After further removal of the solvent, flash chromatography (chloroform: methanol $ p; 9) is purified by silica gel. Yield: 4 * 0 mg (1.6 honey, nsp) of crystalline foot. white »IR: 2,100 ciT¹ asido group »* -V2ino-2 *, 5 • -asides: i-5 * -ta? ifb3fâto-aden-3sina

Obtaining 5'-triphosphate is carried out analogously to the procedure described in Example 1. Then, the azide is reduced by obtaining outputs as in Example 2.

Example 5

Synthesis of 3'-amino-2 *, 3'-dideoxy-5 * triphosphate guanosine

Reaction scheme

<img file="PT99747A_D0014.tif" />

Obtaining 5'-sjaino-2 ', 3 * -didesoxy-5'ρ

triphosphate-guanosine is made from Δ-isobutyryl-5 * -0-benzoyl-2 * -d3S0xiguancBina, which can be obtained by the process of Nishino et al. (Lucleosides &amp; Hueleobides 5, ± 59, 1986). The nucleoside reaction to obtain the azide is carried out analogously to that of Example 4. It should be noted in this case that treatment with the ammoniacal methanol solution is not performed, since the elimination of the isobutyryl protecting group only takes place after the introduction of the triphosphate group and before the reduction of azide to amine. The triphosphate reaction in guanosine is also carried out by the procedure mentioned in Example 1. The amine reduction is described in Example 2.

2. Synthesis of 5 * -amino oligomers and subsequent 5 ♦ -fluorescence labeling.

Example 6

Synthesis of a 20-nucleotide oligomer 3¹-H ^ -Tl'2TTϊ¹ϊ2TTΪTT2ST2m-5^, takes place from 3^r-aaino-3^,5'-dimethoxytrityl-protected deoxythymidine.

This compound is obtained according to the description of Example 1. In this case, the starting point is the A3 * (deoxyriboside thymidine) az' -3 (dimethoxytrityl protected) which can be obtained from this compound. of A3T and dimethoxytrityl chloride by the tI.Gait (Qligonucleotide Synthesis,

13L Press 1984, page 27).

Thereafter, the compound is reduced thereto. triphenylphosphine as described in Example 1. The next operation is to obtain the support material. To est? 200 mg 5 '“^ - (4₁4'-di3ethoxytrityl) -5^tamino-5'-deoxythymidine in 700 µl absolute pyridine in a container. To this solution are added 45 mg DBA? (dimethylaminopyridine) and then 40 mg succinic acid anhydride. After standing overnight, the remaining succinic acid anhydride is hydrolyzed by the addition of 10 æl of water, evaporated three times with toluene and the residue taken up in 12 ml of methylene chloride. with 4 ml of cold (10%) citric acid and twice with 4 ml of water. The organic phase is dried over sodium sulfate, the volume is concentrated by evaporation of the solvent and the substance is dissolved with 1 ml of methylene chloride. This solution is slowly added dropwise with stirring. in 50 ml of n-hexane. The precipitate formed is filtered off under suction and dried at 40 ° C in oil pump vacuum. The continuation of the OPG-based support material is carried out according to standard procedures and to obtain the 12-nucleotide oligomer proceeds with the standard cycle (A3I 58OA User Bullstin, Number 56, Nulho). 1986) in an ABI 580a DNA synthesizer by the phosphoramidide process. After deprotection and decoupling of 5 *^OKAmino-oligonucleotide of the support is purified and characterized according to οε standard methods. In this case, better base-based coupling processes are best achieved for support binding. It is advantageous to replace the acid amide to be dissociated with a urethane function as described in Bfimov (Nucleic Acid Ses. 11, 8569, 1985).

<img file="PT99747A_D0015.tif" />

Synthesis of a 3'-amino terminus of 3'-H nucleotide oligomer is sealed₂N-AOAOOOAATTOTGAAAATGGAT-5 * proceeding according to the description of Example 1. Purification and characterization are performed by standard procedures.

Example 8

Subsequent derivatization of the 3'-amino terminus oligomer 3 is performed with fluorescein isothiocyanate. 50 mmol of the 3'-amino-olisonuclide thidium are dissolved in a 2.5 µl Eppendorf flask of a 500 ns sodium hydrogen carbonate solution. Add 20 æl of a 300 mh (Sigma) om ^ WQG EITO solution. After δ hours of reaction time at room temperature, the reaction mixture is purified by passing it in a 20 N ammonium acetate solution through a column with Sephadex G-2. 0 oligoaero

3'-EITO was analyzed by an analytical HPLO assay for both fluorescence detection and OT detection. The Hi'1.0 test results were also verified by capillary electrophoresis analysis (Dionex firm).

Fluorescein isothiocyanate formula »8

<img file="PT99747A_D0016.tif" />

0E

Both 3'-amino-oligo 'are reacted

Example 9 mere from Examples 1 and 2, according to the process described in Example 3, with rhodamine isocyanate and com. tetramethylrodamine isocyanate. The 5'-fluorescence labeled oligomers are analyzed by both analytical HPLC assay for fluorescence detection as well as UV detection. The results of the IIPLO assay were also verified by capillary electrophoresis assays (Dionex firm).

Formula of dyes rhodamine isothiocyanate to tetramethyl rhodamine isocyanate

<img file="PT99747A_D0017.tif" />

example 10

Both 3'-aminG-oligomers of Examples 1 and 2 are reacted according to the procedure described in Example 5 with the coumarin derivative whose formula is represented. 0 labeled oligomer of 5^sLightness is analyzed by an analytical HPLO assay for both fluorescence detection and UV detection. HPLC assay results are also verified by capillary electrophoresis assays (Firm Dionex).

Coumarin Dye Molecule Formula

<img file="PT99747A_D0018.tif" />

5. Introduction of β-polyase modified nucleotides and analytics of sequencing assays.

To exhaust the acceptance of aminotiphosphates by common sequencing enzymes, corresponding termination solutions were prepared and polymerases were tested (Klenow, Boehringer; Taq, Amersh.asi; T7; Pharmacia; Sequenase, USB). In this case, the incorporation ratios and the termination properties of the triphosphates were analyzed both classical and by fluorescence dye labeling.

Example 11

The 5'-triphosphate-3'-amino-3'-deoxyribosidio-thymidine, obtained as described in Example 1, was replaced by 2 *, 3 * -dideoxy - '* - triphosphate-lysidins. the termination solutions of all the above polymerases. Respectively, equimolar dNIP / termItor ratios were experienced ten times higher and ten times lower. Sequencing was performed according to standard procedures. In these exams it was

<img file="PT99747A_D0019.tif" />

It has been found that (a) the 3'-amino-nucleodide acts terminatively on the enzymes used, (b) the insertion ratios of T7, Saq and sequenase are identical to the usual dideoxytherainator, and (c) a sequence can be performed. smooth sequencing with the amino nucleotide.

Example 12

Compound 5 was reacted^,~ 3'-Amino-6'-deoxyribosido-thiaidine-viphosphate with fluorescein isothroeianate to obtain 5'-'-niphosphate-3^,-amino-3'-cesoxyribosido-5-fluorescein isothiocyanate thymidine.

XTum Bppe Button »dorf dissolves 5^rtriphosphate-3^f-amino-3 *, 2 * deoxyribaside thymine (2.5 mg, 2.8 µmoles) in 200 µl of distilled water and add 200 µl of (pH 9) buffer. To the daylight reaction mixture involving the aluminum foil container and 100 µl pipette, 80 µl of a fluorescein isothiocyanate solution (10 mg in 1 ml DJ.1) is added.¹). After 10 minutes of reaction time at room temperature, the educt is reacted quantitatively as shown by the thin-capped eromatography assay. Purification is by gel filtration. The Sephadex ti-10 column is protected from the daytime action by foil wrapping; The entire reaction mixture is then introduced into the column material and eluted with a flow rate of 1 ml / min of water. 0 The chromatogram has two maximums, the first maximum is produced by the product and the second by the unreacted dye. ϋ volume of each fraction corresponds to 5 ml; In all, 24 fractions are obtained. According to the chroatogram, fraction 4 is positive. This is confirmed by thin layer eromatography. Concentrate the solvent by lyophilization and store the substance at -80 ° C. Yield 3.32 mg (95%); molecular weight; 1,274.32; B_f (ammonia; isopropanol: water 10; TB * € 0); 0.62; fluorescence emission spectrum; the wavelength of the irradiated light corresponds to 420 na. The maximum emission of the compound is 514.8 nm. A 2.5 mm solution in distilled water was used. The maximum emission of 2.5 mia solution of the non-derivatized dye in distilled water corresponds to a wavelength of 519.4 nm.

5¹ 3-amino-3-amino-3-isoxyriboside-5 * -U-fluorescein-thiidine isothiocyanate was exchanged for 2 ', 3 * -did 3Soxy-5^,-thiocytate thymidine. This was done in the termination solutions of all the above polymerases. They were tried, respectively. equimolar proportions, ten times higher and ten smaller voices of dUS? / terminator. Sequencing was performed according to standard pxOceder ways. In these assays, alpha-Sd / VTP incorporation was detected autoradiographically. It is found that a) the β-fluorescence labeled nucleotide acts to terminate the chain on the enzymes used; b) the incorporation ratios are identical with 17, 1aq and sequenced to the usual dideoxyterminators in the case of ten times higher concentrations due to the sterically hindered dye radical; and o) trouble-free sequencing can be performed with the 3 '-fluoroscence-terminated terminator.

Example 13

Fluorescein-thymidine 5'-triphosphate-3'-amino-3'-deoxyriboside-3'-XT-isothiocyanate was replaced by 2 ', 3'-dide' 3Oxy-5 '^,phosphate-thymidine. This is consistent with 17 θ laq polymerase termination solutions. Ten times higher dUTP / terminator ratios were used. Sequences were performed by standard procedures. Success was successfully performed on a commercial ADH sequencer,

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Example 14

Summary of 5^,triphosphate ~ 3^,-thio.ilyl Reaction * deoxyriboside thymidine

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3 * -0-Limetoxytrityl-3'-g-benzoyl thiotinidine

Sulfur introduction at the 3 * position is accomplished by replacing a leaving group (mesylate) with sodium thiobenzoate.

The synthesis of sodium thiobenzoate was accomplished by adding 10% NaOH to an ice-cold solution of thiobenzoic acid (10 g) in 15 ml of water until the solution was alkaline. The pH is then adjusted to pH 7 with aqueous thiobenzoic acid, cooled to -0 ° C and the solution filtered to remove solid residues. With ethanol, the yellow salt is dried under vacuum over phosphorus pentoxide.

Stir with a solution of ^^ - O- ^ imethoxytriethyl-3^t-O-methanesulfonyl-2'-deoxyixyl thymidine (8.45 mmol) (synthesis of mesylate: killer, Pox (1964) J. Org. Ohem. 29,

1772) Sodium β thiobenzoate (33 mmol) in DfFe (30 ml) for 4 hours at 100 ° C. The reaction mixture is extracted with dichloromethane and the organic phase is washed with saturated TaOH solution and saturated water solution. After drying the organic phase over sodium sulfate, evaporate twice with toluene and purify the oily crude product by flash chromatography. (Silica gel 60H, chloroform / methanol 0-10%), yielding 60% ; The product has the spectrum of 3TAS. As expected, 5'-dimethoxytrityl protecting group is eliminated for 5'-position phosphorylation according to procedures known in the literature (H.Gait, Oligorucleotide bythesis, J.L-Press, Oxford 1934). Thereafter, 5'-triphosphate is obtained by the method of jSckstein and Luâwig (J.Org. Ohesi, 1939, Vol, 54, ss 3,631) with salicylphosphorchloride (Alarich) and pyrophosphate treatment. 0 thiol is released by reaction with NaOH 10 II in argon saturated ethanol (H. Osstick, IToleic Acids Hesearch 18, 4, 329),

The 3'-thio-5'-triphosphate thymidine was coupled according to the procedure described in Example 8 with iodoacetamidofluorescein (molecular samples) and at this stage 30% yield is obtained with fluorescence-labeled thionucleotide,

Example 15

Synthesis of 1- (3'-thio-2 *, 3 '* - dideoxy-5 *-triphosphate-beta-D-treopefttofuranyl) thymine

Reaction foam

From 5 * -ΕΕϊη-0-, 3 * -0-ηΐΘ3ϋ-ύί3ΐdine is replaced, as described in Example 14, with. sodium thiobenzoate. Thereafter, the 5% disassociation of the protecting group is performed by introducing 5'-triphosphate, thiol release and iodoacetamidoyluorescein binding analogously to the procedures described in Example 14.

Example 16

Synthesis of a 3'-beta-EgE-fll 10-Nucleotide Oligomer⁵EE MT T-3 »

The synthesis of a 5'-beta-H1 N-TTT TTT TTT T-5 'oligomer is carried out from 1- (3'-amino-2' -.^r-didesoxy-6 · -PETr-beta-S-ts / eopentofuranosyl) -tisine, which is reacted by the process described in Example 6 to obtain the support material for the phosphoramidite process.

Subsequent fluorescence labeling of 3α-amino-oligonucleotide with fluorescein isothiocyanate is performed according to Example 8.