RU2382047C1 - Reagents for internal fluorescent control when determing genotype and subtype of hepatitis c virus on oligonucleotide microchip and method of producing said reagents - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: reagents are phosphoramidites of fluorescent dyes. Method of producing said reagents involves reaction of corresponding activated ethers of indocarbocyanine dyes with a functional amino-linker, with subsequent reaction of the obtained product with 2-cyanethyl-N,N,N',N'-tetraisopropylphosphoramidite.

EFFECT: design of a novel method of producing novel reagents containing indocarbocyanine dyes which fluoresce in a narrow wavelength range with maximum on 550-555 nm, which allows for avoiding superposition of the internal control fluorescence signal on signals of detected samples which are marked with indocarbocyanine dyes with maximum emission in the 650-660 nm range.

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Description

The invention relates to the field of molecular diagnostics, molecular biology, virology and bioorganic chemistry. New reagents are proposed for introducing internal fluorescence control in the method of identifying the genotype and subtype of hepatitis C virus (HCV) using a specialized oligonucleotide microchip (biochip). New reagents are reactive phosphoramidites of fluorescent dyes (Figure 1), analogues of cyanine dye Cy3. The internal fluorescence control on microchips is carried out by introducing oligonucleotides labeled with new reagents into the composition of the samples. Indocarbocyanine dyes, which are part of the reagents, fluoresce in a narrow wavelength range with a maximum at 550-555 nm. This avoids the imposition of a fluorescent internal control signal on the signals of the detected samples, which are marked with dyes of the indodicarbocyanine series with an emission maximum in the range of 650-660 nm.

Biochips for identifying the HCV genotype and subtype are a substrate containing many discrete elements, each of which has a unique oligonucleotide probe immobilized having a genotype-specific or subtype-specific sequence of a fragment of the NS5B region of the HCV genome.

The method for determining the HCV genotype and subtype is based on two-stage PCR to obtain a fluorescently labeled, predominantly single-stranded fragment of the NS5B region, followed by hybridization of this fragment on a biochip containing a set of specific discriminating oligonucleotides. The studied fragment of the NS5B region forms perfect hybridization duplexes only with the corresponding (fully complementary) oligonucleotides. With all other oligonucleotides, the studied DNA fragment gives an imperfect duplex. Discrimination of perfect and imperfect duplexes is performed by comparing the fluorescence intensities of the cells in which the duplexes formed. The identification of the HCV genotype and subtype is carried out by identifying the cells in which perfect hybridization duplexes have formed.

The use of reagents for internal fluorescence control allows the concentration of specific oligonucleotides immobilized in biochip elements to be estimated and hybridization fluorescence signals to be normalized taking into account the concentration of oligonucleotides. The normalization of fluorescence signals is used to accurately calculate the discriminatory relationships between cells in which perfect and imperfect hybridization duplexes have formed, which is necessary to uniquely determine the genotype and subtype of HCV.

The present invention contemplates novel reagents for introducing an indocarbocyanine fluorescent label into synthetic oligonucleotides used in biochip assays. Such reagents can be used for fluorescence labeling of oligonucleotides used in various fields of molecular biology and medicine.

Synthetic oligonucleotides can be labeled with cyanine dyes either after automatic synthesis or during automatic synthesis on a DNA synthesizer. The first method is widely known and based on the use of activated derivatives of dyes - succinimide esters, nitrophenyl ethers, etc. In this way, a fluorescent label can be attached to a synthetic oligonucleotide containing an amino group. Another method is based on the use of phosphoramidites of cyanine dyes and allows fluorescence labeling of oligonucleotides during their synthesis. In addition, the introduction of a label in this case does not require the presence of an amino group. Moreover, the amino group can be introduced into the oligonucleotide independently, along with a fluorescent label. The latter circumstance is very important for the synthesis of control fluorescently labeled oligonucleotide samples.

There are a number of phosphoramidites of cyanine dyes. Both hydrophobic and hydrophilic phosphoramidites based on indocyanines with the number of atoms in the polymethine chain from 3 to 7 are described [1-7]. Some of these compounds can only serve as terminators in oligonucleotide synthesis, while others allow chain synthesis to continue after they are turned on.

Of this series of compounds, only one phosphoramidite reagent is known (Figure 2), which allows the labeling of oligonucleotides during their automatic synthesis with an indocarbocyanine dye with spectral characteristics acceptable for internal control purposes. This product is supplied by Glen Research under license from Amersham Corporation. This phosphoramidite contains an indocarbocyanine nucleus and two functional side chains attached to nitrogen atoms. The presence of a hydroxypropyl fragment protected by a 4-methoxy-triphenylmethyl (MMTr) group in the phosphoramidite structure allows, if necessary, the introduction of a fluorophore into an arbitrary position of the oligonucleotide (except for the 3 'position). There are no hydrophilic substituents in the molecule that could increase the solubility of the dye in water. The release of fluorescently labeled oligonucleotides, i.e. the removal of protective groups, after automatic synthesis, is carried out under mild conditions in order to avoid destruction of the cyanine fragment.

The above phosphoramidite has two significant drawbacks that complicate its use for the purpose of fluorescent labeling of control oligonucleotide samples. First, it contains an MMTr protecting group, which is not standard for oligonucleotide synthesis. This leads to the need to adjust the synthesis protocol and the impossibility of an adequate assessment of the degree of inclusion of the fluorescent label during synthesis. Secondly, the hydrophobic nature of the dye leads to an increased tendency of the samples to self-associate, which negatively affects the results of the analysis.

The basis of the invention is the synthesis of indo-carbocyanine dye phosphoramidites containing a hydrophilic substituent and a standard 4,4'-dimethoxy-triphenylmethyl group (DMTr), characterized by predetermined spectral characteristics and allowing fluorescence labeling during automatic synthesis of the oligonucleotide as at the 5 'end in the middle of the chain.

The problem is solved in that the hydrophilic sulfosubstituted indocarbocyanin is attached to a functional linker containing a primary hydroxyl group protected by a DMTr fragment (Figure 3). The resulting conjugate is subsequently converted to reactive phosphoramidite. For the synthesis of a functional linker, 1,2,6-hexanetriol is used. The vicinal diol is selectively protected with an isopropylidene group, after which the hydroxyl group at position 6 is transformed into an amine via the azide step. The primary hydroxyl of the diol system is selectively protected by the DMTr group after removal of the isopropylidene protection. The synthesis of phosphoramidite after conjugation of the dye with a functional linker is carried out using 2-O-cyanoethyl-N, N, N ', N'-tetraisopropylphosphoramidite in the presence of pyridinium tetrazolid.

Using the obtained hydrophilic sulfosubstituted phosphoramidite, fluorescent control oligonucleotides are also synthesized, which also contain an amino group for immobilization on hepatitis C genotyping chips. Modified oligonucleotides are released by concentrated aqueous ammonia under mild conditions.

Biochips for identifying the genotype and subtype of hepatitis C virus are obtained by photoinitiated polymerization of microdrops containing the necessary monomers and fluorescently labeled oligonucleotide according to the method described previously [8].

Example 1

3,3,3 ', 3'-Tetramethyl-5-sulfo-1-ethyl-1'-N- [6-0- (4,4'-dimethocrystyl) -5-hydroxyhexyl] carboxamidopentyl indocarbocyanine

CH ₃CH₂). ¹³C NMR (DMSO-d₆, 100MHz) δ 173.9, 173.6, 171.5, 157.9, 149.9, 145.9, 145.1, 141.8, 141.3, 140.6, 140.0, 135.9, 135.8, 129.6, 128.6, 127.7, 127.6, 126.4, 126.2, 125.2, 122.4, 119.9, 113.0, 111.5, 110.3, 102.7, 102.4, 84.9, 68.9, 67.6, 54.9, 48.9, 48.8, 47.4, 45.6, 43.7, 35.1, 33.5, 33.3, 29.2, 27.4, 27.3, 26.7, 25.7, 25.3, 25.2, 24.8, 24.4, 22.4, 12.2. MS (MALDI): m/z=968.4 (M⁺), рассч. для C₅₈H₆₉N₃O₈S 968.24.To a solution of carbocyanine (R = H, R ¹ = C ₂ H ₅ ) (100 mg, 0.18 mmol) and N-hydroxysuccinimide (30 mg, 0.27 mmol) in anhydrous dimethylformamide (2 ml) was added dicyclohexylcarbodiimide (50 mg, 0.24 mmol) . Additional portions of dicyclohexylcarbodiimide (20 mg, 0.097 mmol) were added every 12 hours to complete conversion. The progress of the reaction was monitored by reverse phase TLC. Then the mixture was filtered and a solution of linker (150 mg, 0.345 mmol) and iPr ₂ EtN (50 μl, 0.287 mmol) in dimethylformamide (300 μl) were added to it. After 20 minutes, the reaction mixture was diluted with ethyl acetate (30 ml). The precipitate of the dye conjugate was separated by centrifugation and dried. Yield 130 mg (73.9%). ¹ H NMR (DMSO-d ₆ , 400 MHz) δ 8.35 (1 H, t, J13.5, β-CH), 6.86-7.95 (20 H, m, DMTr and dye aromatics), 6.51 (2H, d, J13.5 , α, α'-CH), 4.00-4.20 (6H, m., CH ₃ CH ₂ N ⁺ , CH ₂ CH ₂ N ⁺ , CH ₂ NHCO), 3.73 (6H, s, OCH ₃ ), 3.50-3.51 (1H, m, CH OH), 2.70-3.05 (2H, m, CH ₂ ODMTr), 2.04 (2H, t, J7.16, CH ₂ CONH), 1.69 and 1.70 (12H, s, 4 × CH ₃ ) 1.15-1.61 (15H, m,

CH ₃ CH ₂ ). ¹³ C NMR (DMSO-d ₆ , 100MHz) δ 173.9, 173.6, 171.5, 157.9, 149.9, 145.9, 145.1, 141.8, 141.3, 140.6, 140.0, 135.9, 135.8, 129.6, 128.6, 127.7, 127.6, 126.4, 126.2, 125.2, 122.4, 119.9, 113.0, 111.5, 110.3, 102.7, 102.4, 84.9, 68.9, 67.6, 54.9, 48.9, 48.8, 47.4, 45.6, 43.7, 35.1, 33.5, 33.3, 29.2, 27.4, 27.3, 26.7, 25.7, 25.3, 25.2, 24.8, 24.4, 22.4, 12.2. MS (MALDI): m / z = 968.4 (M ⁺ ), calc. for C ₅₈ H ₆₉ N ₃ O ₈ S 968.24.

Example 2

3,3,3 ', 3'-Tetramethyl-5-sulfo-1-ethyl-1'-N- {6-O- [4,4'-dimethoxytrityl] -5-O - [(N, N-diisopropylamino ) (2-cyanoethoxy) phosphinyl] hexyl} carboxamidopentyl indocarbocyanine

СН ₃СН₂), ¹³С NMR (DMSO-d₆, 100MHz) δ173.9, 173.7, 171.5, 158.0, 149.9, 146.0, 144.9, 141.8, 141.2, 140.6, 140.1, 135.7, 135.6, 129.6, 128.6, 127.7, 126.5, 126.2, 125.2, 122.4, 119.9, 113.0, 111.5, 110.3, 102.7, 102.4, 85.2, 65.6, 58.0, 57.8, 55.0, 54.8, 48.8, 45.7, 43.7, 42.5, 42.4, 35.1, 33.0, 32.7, 29.1, 27.4, 27.3, 26.7, 25.7, 25.5, 24.9, 24.3, 24.3, 24.2, 24.1, 22.6, 21.9, 21.7, 19.7, 19.7, 19.0, 12.1. ³¹P NMR (DMSO-d₆, 162MHz) δ 150.2. MS (MALDI): m/z=1168.4 (M⁺), рассч. для C₆₇Н₈₆N₅O₉S 1168.47.The dye conjugate conjugate (60 mg, 62 μmol) was dissolved in 300 μl of anhydrous MeCN. Tetrazole (22 mg, 310 μmol), dry pyridine, (30 ml) and molecular sieves (4Å; approx. 5% by volume) were added to the solution. After 30 minutes, 2-cyanoethyl N, N, N ′, N′-tetraisopropylphosphoramidite (92 μl, 310 μmol) was added to the mixture with vigorous stirring. Phosphitylation took place in 15 minutes according to TLC. Then, cold saturated aqueous NaHCO ₃ (30 ml) was added to the reaction mixture and the product was extracted with methylene chloride (30 ml). The organic layer was dried over Na ₂ SO ₄ , the solution was concentrated under reduced pressure. Ethyl acetate (30 ml) was added to the residue. The precipitate of the product was separated by centrifugation and dried. Yield 60 mg (86%). ¹ N NMR (DMSO-d ₆ , 400 MHz), selective signals δ 8.36 (1H, t, J13.4, β-CH), 6.85-7.82 (20Н, m, DMTr and aromatics), 6.52 (2Н, d, J13 .4, α, α'-CH), 4.10-4.20 (4Н, m., CH ₃ CH ₂ N ⁺ , CH ₂ CH ₂ N ⁺ ), 3.73 (6Н, s, OCH ₃ ), 2.04 (2Н, m , CH ₂ CONH), 1.69 and 1.70 (12H, s, 4 × CH ₃ ), 0.96-1.62 (15H, m,

CH ₃ CH ₂ ), ¹³ C NMR (DMSO-d ₆ , 100 MHz) δ173.9, 173.7, 171.5, 158.0, 149.9, 146.0, 144.9, 141.8, 141.2, 140.6, 140.1, 135.7, 135.6, 129.6, 128.6, 127.7 , 126.5, 126.2, 125.2, 122.4, 119.9, 113.0, 111.5, 110.3, 102.7, 102.4, 85.2, 65.6, 58.0, 57.8, 55.0, 54.8, 48.8, 45.7, 43.7, 42.5, 42.4, 35.1, 33.0, 32.7, 29.1 , 27.4, 27.3, 26.7, 25.7, 25.5, 24.9, 24.3, 24.3, 24.2, 24.1, 22.6, 21.9, 21.7, 19.7, 19.7, 19.0, 12.1. ³¹ P NMR (DMSO-d ₆ , 162MHz) δ 150.2. MS (MALDI): m / z = 1168.4 (M ⁺ ), calc. for C ₆₇ H ₈₆ N ₅ O ₉ S 1168.47.

Example 3

Synthesis of Oligonucleotides

Oligonucleotides were synthesized on an automatic DNA synthesizer ASM-102U (Biosset Ltd., Russia) or ABI 3400 (Applied Biosystems, Forster City, CA, USA) using a 0.2 μmol phosphoramidite method.

The release of fluorescently labeled oligonucleotides was carried out in concentrated aqueous ammonia at 25 ° C for 8 h.

The oligonucleotides were purified using reverse phase chromatography on a Hypersil column (5 mm; 4.6 × 250 mm) in an acetonitroil gradient (0-50%) in 0.05 M TEAA (pH 7.0). The flow rate is 1 ml / min.

Nucleotide sequences of control oligonucleotides

GCC TGT CGA GC (C / T) GC oligonucleotide for identification of HCV genotype 1

TAC AGG CG (C / T) TG (C / T) CGC oligonucleotide for identification of HCV genotype 2

AC TGA GAG CGA CAT SS oligonucleotide for identification of HCV subtype 1a

ATA A (A / G) G TCG STS ASA GAG oligonucleotide for identification of the HCV lb subtype

TAC ACT CGC TGA CTG AGA oligonucleotide for identification of the HCV subtype 2a

TAC ACT CGC TCA CTG AGA oligonucleotide for identification of the HCV subtype 2b

Brief Description of the Figures

Figure 1. New phosphoramidite reagents.

Figure 2. Commercially available phosphoramidite reagent with acceptable spectral characteristics for internal control purposes.

Figure 3. Synthesis of linker and phosphoramidites, R = H, SO ₃ H; R ¹ = alkyl C1-C3. Reaction conditions: (i) acetone, p-toluenesulfonic acid, chloroform, boiling with azeotropic distillation of water; (ii) MsCl in pyridine, 25 ° C, 12 hours; (iii) LiN ₃ in DMF, 12 hours, 70 ° C; (iv) 80% AcOH, 15 min, 60 ° C; (v) DMTrCl in pyridine, 25 ° C, 12 hours; (vi) Ph ₃ P in pyridine; (vii) activated dye esters in DMF, 25 ° C, 2 hours; (viii) 2-cyanoethyl-N, N, N ', N'-tetraisopropylphosphoramidite, tetrazole, pyridine in MeCN, 25 ° C, 30 min.

Literature

1. Kvach M.V., Gontarev SV., Prokhorenko I.A., Stepanova I.A., Shmenay V.V., Korshun V.A. Izvestia AN, Chemical Series, 2006, 154-158.

2. Anderson, E. Synthesis of a Carbocyanine Phosophoramidite and its use in Oligonucleotide Labeling; International Conference on Nucleic Acid Medical Applications, Jan. 25-29, Cancun, Mexico, 1993.

3. Narayanan, N., Little, G., Lugade, A., Gibson, J., Prescott, C, Raghavachari, R., Reimen, K., Roemer, S., Sutter, S., Draney D. New NIR Dyes: Synthesis, Spectral Properties and Applications in DNA Analyzes; Daehne, S .; Resch-Genger, U.,

Wolfbeis, O.S. Eds. Near-infrared Dyes for High Technology Applications, Kluwer Academic Publishers, 1998, 141-158.

4. Michael M.A., Farooqui F., Reddy M.P., Li H. U.S. Patent 7,230,117, 2007.

5. Brush C.K., Anderson E.D. U.S. Patent 5,556,959, 1996.

6. Brush C.K., Anderson E.D. U.S. Patent 5,808,044, 1998.

7. Reddy M.P., Farooqui F., Michael M.A. U.S. Patent 6,331,632, 2001.

8. Rubina A.Yu., Pan'kov S.V., Dementieva E.I., Pen'kov D.N., Butygin A.V., Vasiliskov V.A., Chudinov A.V., Mikheikin A.L., Mikhailovich V.M., Mirzabekov A.D. Anal. Biochem. 2004, V.325, 92-106.

Claims (2)

1. Phosphoramidite reagents of the formula

where R = H, SO ₃ H; R ¹ = alkyl C1-C3. 2. A method for synthesizing phosphoramidite of formula (1) according to claim 1, characterized in that the method is carried out by reacting a functional aminolinker of formula (2) with the corresponding activated esters of indocarbocyanine dyes, after which the resulting dye conjugates of formula (3) are reacted with 2-cyanethyl -N, N, N ', N'-tetraisopropylphosphoramidite to obtain the phosphoramidite reagent of formula (1) according to claim 1

where R = H, SO ₃ H; R ¹ = alkyl C1-C3; DMTr is a dimethoxytrityl group.

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