JPH02264792A - Oligonucleotide derivative and synthetic raw material thereof - Google Patents

Abstract

NEW MATERIAL:A compound expressed by formula I [R is H, acyl or (substituted) phosphoryl; T is thymin-1-yl; Y1 to Y3 are H, OH or lower alkyloxy, etc.; B is thymin-1-yl, adenin-9-yl or guanin-9-yl, etc.; n is 1-100]. USE:Carrier for isolating poly(A)<+>-mRNA. Useful for promotion of efficiency in isolation of poly(A)<+>-mRNA as forming stable hybrid with poly(A) part. PREPARATION:The aimed substance is synthesized from nucleotide derivative expressed by formula II (X is monomethoxytrityl or dimethoxytrityl, etc.; Y is H or o-chlorophenyl phosphoric acid, etc.) using, e.g. DNA automatic synthesizer. Besides, the compound expressed by formula II is new substance and obtained from, e.g. ribothymidine as starting raw material through compounds expressed by formula III and formula IV.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to nucleotide derivatives that can be used to improve the efficiency of mRNA isolation operations that are commonly performed in the cloning of useful genes, and their synthesis. This invention relates to a nucleotide oligomer as a raw material and a nucleotide as a raw material for its synthesis. All of these substances are new compounds.

When rinsing five useful genes, all mRNA is isolated from the biological material in which the gene is expressed in the shell,
cDNA is synthesized from the mRNA, and the cloned gene is screened for the target gene f. In the operation to separate mRNA, mRNA uses poly(A> tail (poly(A)-mRNA)), and oligoboaoxythymidylic acid complementary to poly(A) is immobilized on cellulose etc. The so-called oligo (
dT)-cellulose column to utilize the hybridization cidin effect to obtain poly(A)-free RNA.
(Separation of poly(A>-RNA) is performed.

However, poly(A)-mRNA in this operation
The isolation yield is not necessarily sufficient, and in order to improve the efficiency of cloning, it has been strongly desired to discover an oligomer that forms a more stable hybrid with the poly(A) moiety.

Problem to be Solved by the Invention In the poly(A)-mRNA isolation operation, which greatly affects the efficiency of cloning a target useful gene, there is a technology that can replace the oligo(dT)-cellulose column and improve the yield. The development of a new oligomer that has the property of forming a more stable hybrid with poly(A) has been requested for 1 yen.

As a result of intensive research to solve the above problems, the present inventors have discovered that oligonucleotides (2°
-0-methylribonucleotide) was found to form more stable hybrids with complementary strand oligoribonucleotides, and reported that it is useful as a hybridization probe (H, Inoue et al., Nucle.
ic Ac1ds Ites, j5(15) p. 6131, 198/). Furthermore, as a result of a detailed study of the structural contribution of oligomers to hybrid stability, we found that 2
By using 2°-0-meb-5-methyluridylic acid as the constituent unit of the oligomer instead of °-0-methyluridylic acid, it is strongly bonded to the adenylate unit in the complementary strand, and the heat exchanger between the oligoribonucleotide and found that the stability was increased. Based on this finding, decariboadenylic acid and its complementary chain, deoxyribothymidylic acid.

2°-0-methyluridylic acid and 2°-0-methyl-5
- When each decamer of methyluridylic acid was synthesized and the thermal stability (Tm value) was measured, it was found that deca 2
It was discovered that °-0-methyl-5-methyluridylic acid has extremely excellent properties, and the present invention was completed.

The present invention will be explained in detail below.

The present invention relates to an oligonucleotide derivative represented by the following general formula (I).

In the formula, R is a hydrogen atom, an acyl group such as a carbonyl residue such as 0M cellulose or latex particles, a phosphoryl group,
or a phosphoryl group which may have a substituent, for example, a phosphoryl group having an ester bond between a compound having a hydroxyl group such as cellulose and IF5 acid, and ■ is thymine-
It represents a 1-yl group, and Yl, Y2 and Y3 may be the same as each other or may be stationery, and represent a hydrogen atom, a lower alkyloxy group such as a hydroxyl group, a methoxy group, an ethoxy group, or a lower alkyl group. It represents a silyl group, and B is a thymin-1-yl group or an adenine-9-yl group.

Guanine-9-yl group, cytosine-9-yl group.

It represents a bocyl-1-yl group or a hyboxanthine-9-yl group, and n represents an integer of 1 to 100, but those having an average chain length of n-10 to 40 can be used practically.

Incidentally, in the derivative of the general formula CI), R is an acyl group as described above (which may also be referred to as a carrier) or a phosphoryl group having a substituted U<which may also be referred to as a carrier). Some are compounds that are directly used for mRNA separation operations, and are compounds in which R is a hydrogen atom or a phosphoryl group without a substituent (oligonucleotide).
not only can be used as a raw material for the synthesis of the former compound, but also
No matter how the former compound was synthesized, 11
The former compound fulfills its central function as a constituent part of the former compound.

The present invention also relates to a nucleotide derivative represented by the following general formula (I).

In the formula, X represents a monomethoxytriple group, a dimethoxytrityl group, or a phosphoryl group which may have a substituent such as an 0-chlorophenyl group, and Y represents a hydrogen atom,
0-Chlorphenyl phosphoric acid, -1] (OCI-13)
-N-<CF+ (CH3) 2) 2
.

-P (OC82CI-12ON) -N-(CH(CH
3)2')2 Also Gt-GO-(CH,).

C0NH-(CH2'), -(CPGII form) (here, m and n are each an integer of 1 to 1o), provided that X is a phosphoryl group which may have a substituent When , Y is a hydrogen atom. The compound of general formula (I[) can be used as a raw material for the synthesis of the compound of general formula (1).

Next, examples of methods for synthesizing these compounds will be explained.

In order to synthesize the oligo(2°-O-methyl-5-methyluridylic acid) moiety of the present invention, first, its constituent nucleoside, 2°-0-methyl-5-methyluridine (compound 3), is synthesized as described below. (Fig. 1), and then by a conventional method (for example, H. Yoshikawa et al.
, Tetrahedron 1ett,, 5065
(1967)), phosphorus L2 group was introduced directly into the 5'-position hydroxyl group to obtain 2'-0-methyl-5-methyl-5'-uridylic acid, which was then converted into di-hexylcarbodiimide (D
A method of polymerization using a condensing agent such as CC) can be adopted. Alternatively, Compound 3 is converted into a 3°-phosphoramidate derivative (Compound 5) as described below, and prepared according to a known method (S, 5hibaraha et al,, H
ucleic acids Rcs,, 15(11),
4403 (1987)) and can be easily synthesized using DNA Automation Synthesis i (Model 38OA, manufactured by Applied Biosystems).

On the other hand, in order to produce oligo(2°-0-methyl-5-methyluridylic acid) derivatives, the above-mentioned DCC polymerization reaction of 2°-〇-methyl-5-methyl-5°-uridylic acid is carried out using the known method. method (P, T, Gi Ih
Am, JAC5, 88, 4982 (1964)), it is convenient to react cellulose and the like at the same time to obtain a cellulose derivative and the like. On the other hand, after introducing phosphorus wIW into the 5' terminal water wi group of oligo (2°-0-methyl-5-methyluridylic acid) having a 5° terminal water Rj4 synthesized by the automatic synthesizer described above, It is also possible to react with cellulose etc.

On the other hand, the oligo with 5゛ terminal water Milk (2゛-
〇-Methyl-5-methyluridylic acid) and CM-cellulose or latex particles, etc. are bonded using a condensing agent (DCC, etc.) to form a cellulose or latex derivative (
In, Niuribayashi et al.

NuCleiCAc1ds 5VllpO3iul 5
19.61 (1988)).

Compound 3 can be synthesized using ribothymidine as a starting material, for example, after first obtaining compound 1 (see FIG. 1 and Examples 1 to 3). Incidentally, compound 1 was synthesized by converting the 3' and 5' hydroxyl groups of ribothymidine to known methods (tl, Inouc).
et al., Nuclic Acids Symp.
osiun 5eries, N(116,165(
1985), Beast ■.

Harkiewicz, J., Chem.
) 24-25 (1979)), and then protected with a tetraisopropyldisiloxane-1,3-diyl group according to a known method (H, Rnouc et al., supra, Telegram (1987)). It can be obtained by selectively protecting the 3N position by treatment with benzoyl chloride in the presence of triethylamine in a suitable solvent.

Also, 2°-〇-methyl-5-methyluridine-3°-
Phosphoramidite derivative (compound 5) and 2°-
〇-Mediru 5-methyluridine-3゛-CPG derivative (compound 6) can be produced by a known method (S, 5hiba
Hara et at, above mentioned telegram, L.J.HcB
rid13 et at,, Tetrahedron
Iett, 24, 245 (1983)) (see Figure 2 and Examples 4 and 5). In addition to the phosphoramidite derivative (compound 5) synthesized here, a compound having a cyanoethyl group as a protecting group for subg4r11 was also synthesized using the same method (S, 5hibah
ara et at, the aforementioned telegram, N, D, 5hin
ha et al, Nucleic Ac1ds R
QS, 12°4539 (1984) 6).

The important point regarding the present invention is that oligo(2°-〇-methyl-5-methyluridyl M)- is an oligo(2°-〇-methyl-5-methyluridyl The present invention is based on the discovery that it has the property of forming an extremely stable hybrid with riboadenylic acid (oligo(rA)).

This point will be explained in detail below. That is, in order to actually measure the thermal stability (Tm value) of those hybrids, decariboadenyl acid was purified by a known method (p,
Ek et al., Biochemistry, 2
5.7840 (198B)) and deca(2°-0-methyl-5-methyluridylic acid) was synthesized according to Example 6) using compounds 5 and 6 prepared according to Examples 4 and 5. Using an automatic synthesizer as usual (S, 5h
ibahara et al., supra)) was synthesized (Example 7). On the other hand, the oligomer for comparative experiments, decadeochitimidylic acid, was synthesized by deca(2°-0-methyluridyl) using commercially available reagents in a conventional manner. acid) by a known method (
Each of these DNAs was synthesized using an automatic DNA synthesizer according to the above-mentioned telegram of Hibahara et al.

After fixing each oligonucleotide by a conventional method, it was used as a sample for thermal stability measurement. The Tm value of each hybrid was measured using a Beckman DU-8 according to a conventional method.
Measured using a B-type spectrophotometer (Figure 3 and Example 8)
reference). As a result, the Tm values of each hybrid were 18.9°C, 21.0"C, and 31.1°C, and the hybrid of deca(2°-0-methifle-5-methyluridylic acid) and decariboadenyl acid It was discovered that the Tm value was about 10°C higher than that of other hybrids, and it was extremely stable.

From the above, oligo(2°-0-methyl-5-methyluridylic acid), as its cellulose conjugate, is a novel poly(A)-mRNA isolation alternative to the currently commercially available oligo((fT)-cellulose. It is fully expected that it can be used as a carrier for various purposes.

Hereinafter, the present invention will be specifically explained with reference to Examples.

1 dustman The present invention will be further explained below with reference to Examples.

Example 1 (N3-Benzoyl-5-sethyl-3',
5' -0-(tQtraisol)rot)yl
-disilOXane -1,3-dll/I)ur
Synthesis of idine (compound 1)) 5-14ethyl-1,3-O-(tetraisO
I) I'0DVldiSi 1OXane-1,3-d
iyl) uridine (IG, 67 g, 33.
3 mmol) was dissolved in 250 d of methylene chloride, triethylamine (6,3 d, 45.2 ma+of) was added, and benzoyl chloride (4,4 d, 37.9
n+mol) was added dropwise. After stirring at room temperature overnight and confirming the disappearance of the raw materials, add 0.01 NHCl, water, NaHCO3
The reaction solution was washed with an aqueous solution and then with water. tIII@N
a After drying with SO4, the solvent was distilled off under reduced pressure, and the residue was subjected to silica gel chromatography (C-200, 4009,
Compound 1 was purified as a foam with 13
．． 699 (67, 9%) was obtained.

IJV (MeOH): λwax 256rv, λs
in 225ne.

1H-kawa(coct3>δE)l)lnia, 99-7.
29 (1,6H, blZOVI QrOIjl), C
6-H).

5.76 (d, IH, J-111Z.

C1°-H), 1.97(d, 311.J=1.2H
2°C5-CH3).

1.12-1.06 (m, 28H, TIPDS a
roup).

Example 2 (N3-Benzoyl-5-nethyl-2'-0
-methyl-3'5' -0-(tetraiso
propyldisiloxane-1,3-diyl
) uridine (Compound 2) synthesis> Compound 1 obtained in Example 1 (13,697,22,6 mmol)
was dissolved in 80ml of benzene, and silver oxide (14.7g, 63ml) was dissolved in 80ml of benzene.
．． 3 uo l) and add about 10
It took a while. Methyl iodide (65,Od, 1045mmo
1) was added and stirred at 45°C for 3 days. The reaction mixture was evaporated, and the solvent was distilled off under reduced pressure. CHC13=H20't
' Separate the liquid, dry am with Na2SO4, and then perform silica gel column chromatography (C-200, 300g, C
HCl3) to give compound 2 as a foam at 6.7
59 (48,2%) was obtained. Furthermore, the fractions that could not be separated were dyed and silica gel chromatography (C-200, 2G03, hexane:ethyl acetate-4) was performed.
:1) to f6 and foamy substance (compound 2) to 3.96
! ? (28.4%) was obtained.

UV (HeOH): λmax 252tv, λwin
225 nt Example 3 (5-Nethyl-2'-0-sethylurid
Synthesis of ine (compound 3)) Compound 2 (6,99rJ, 11.3mmol) was dissolved in 100d of dioxane, and cNH40H (12,2
ml of the mixture was added, and the mixture was stirred at air temperature for 5 hours. After confirming the disappearance of the raw materials, the reaction solution was concentrated and separated with CHCl3-820.

The organic layer was washed with water, an aqueous NaHCO3 solution, and water in this order, then dried with NaSO4, and after distilling off the solvent under reduced pressure, it was crystallized from hequinane to obtain a crystalline compound of 5.469 (93.9%). Ta.

m, p,: 116.5-118.0°C mass m/e: 471 (14+-1sopro
pyl radical) elemental analysis: C23++4282
Calculated value as 07 S12: C, 53, 70; H
, 8, 17; N, 5.4b Experimental value: C, 54, 53,
H, 8,13; N, 5.68 This compound (4,00 S,
7.811101) in Tetrahydro7ran 50-1 and
Add uoride (3.2m, 3.1w5ol),
The mixture was stirred at air temperature for 4.5 hours. Pyridine: Water: Me
01-I J3: 1:1 (v/V) solution for 100ml!
dilute the reaction solution by adding Oowcx 50
(pyridinium type) was added to neutralize the mixture. After filtering the resin, the reaction solution was concentrated and separated with CHCl3-1-120. After concentrating the aqueous layer to dryness, it was crystallized from ethanol to obtain 1.859 (87.2%) of Compound 3.

UV (He'll): λwax 266nm,
λsin 233nm.

1, p. :197. O~198.0℃m
ass m/e: 2γ2 (14") elemental analysis"C11'16'2 Calculated value as 0G:
C, 48,53; H, 5,88; H, 10,29 Experimental value: C, 48,36; H, 5,96; N, 10.37
'II-NHR(DH3O-d6)δpp17.78(
d, 111. J-1,2Hz, CB-H).

5,86(d, 111. J-5,4Hz, CI'
-It).

3.33 (s, 311.02'-CH3).

1.78 (S, 3N, C5-CH3).

Incidentally, there is a report that Compound 3 was isolated from urine (C, A, Vol, 1G1, 68654a (
(1984), isolation from urine has its own limitations on the amount that can be isolated, and it is extremely advantageous to produce according to Examples 1 to 3 above.

Example 4 (Synthesis of methoxy-5'-dimethoxytrityl-2'-〇-methyl-5-methyl-uridine-310-(N,N-diisopropylamino)phosphine (compound 5)) Compound 3 (0,544 tJ, 2.0 mmol After pyridine azeotropy, dimethoxytritylchloride (0,829,2,4 mmol) dissolved in 5-pyridine was added and stirred at air temperature for 4 hours. After confirming the disappearance of the raw material, methanol 1111 was added. The reaction was stopped. The solvent was removed under reduced pressure]; the layers were separated with CHC13-H, 20, and the t1 layer was purified with NaSO4, silica gel column chromatography (C-200, 407, ~4%). MeOH/C
After purification with HCl3) and powdering from n-hexane, 0.92 g (80%) of compound 4 was obtained.

This compound 4 (2811910, 49 Kasumi ■ 01) was dissolved in distilled methylene chloride with pyridine and diisopropylethylamine (0.35ad!, 2.0mm).
ol) was added. The atmosphere was replaced with argon, and then chloro-N, N-diisopr was added.
opylaiinoiethoxyphosphine
(0.12ad, 0.61101) was added dropwise over about 1 minute. Leave it at room temperature for about 60 minutes and check that the raw materials have disappeared.
(Silica gel TLC method: FIJ Fn, ethyl acetate,
Raw material R (Wi O, 43, II!i part R, value 0.7),
Approximately 15 mL of ethyl acetate was added to dilute the reaction solution.

After washing with a saturated aqueous NaHCO3 solution and saturated brine, the mixture was dried over anhydrous sodium sulfate and concentrated to obtain a residue. Furthermore, silica gel column chromatography (C-300,
2.5 g of ethyl acetate) to give 0.349 (94%) of compound 5 as a foam.

Example 5 (Synthesis of nucleoside resin) Compound 4 (0,179,3,01101) was azeotroped with pyridine, dissolved in 3 m of methylene chloride, and mixed with succinic anhydride (47.8 μl, 4.5 uol) and dimethylamino Pyridine (55.5q, 0.46imo) was added and stirred at a variable temperature for 2 hours. After confirming the disappearance of the raw materials by TLC, the reaction solution was washed with 0.5M KHPO4 water. The solvent was distilled off under reduced pressure. , the residue was purified by silica gel column chromatography (C-300, 109), powdered with normal 4-san, 0.18 g (0.17 mol, 8
8.9%) of 3'-(2'-0-Hethl/+-5
'-0-dimctoxytrityl-5-1
cthyluridinyl) -monosucct
I got nate.

The entire amount was dissolved in DMF3ae, pentachlorophenol (87,111I!F, about 1.2 equivalents) and dicyclohexylcarbodiimide (66,0μ, about 1.2 equivalents) were added, and the mixture was stirred at room temperature overnight. After confirming the disappearance of the raw materials by TLC and separating the resulting precipitate with P, the solvent was distilled off under reduced pressure. Furthermore, benzene was added to the residue, the precipitated insoluble matter was separated from P, the P solution was concentrated, and then powdered from n-hexane.
ntach+oropheny+ -3'(2'-
0-methyl-5'-0-dimethoxy
trityl=5-18thyILIridiny1
)-3uCCinate was obtained. Yield gL0.23 g,
0.249 mg+o I, yield 92.3%.

Cono compound (75,3I!rg, 82 μmol) in CP
G resin (100μ1iol NH2/g, 0.24I
J) triethylamine (15J11)-DMF (3
m) and shaken overnight at room temperature. The reaction solution was passed through, and the resin was washed successively with DMF, pyridine, and methylene chloride, and then dried under reduced pressure.

It was determined by a conventional method, and the loading amount was 1 μm01/29.
It was confirmed that it was 4■.

Add 0.1M dimethylaminopyridine/pyridine 4.5- and 0.5m acetic anhydride for 10 minutes! ffi shaken.

The reaction solution was filtered, and the resin was washed successively with pyridine and methylene chloride, and then dried under reduced pressure to obtain Compound 6.

Example 6 (Synthesis of decariboadenylic acid (γA 10wer))
A known method (E, Ohtsuka et al.

Tetrahedron, 40.47 (1984)) N6-penzoyl-5'-〇-dimethoxytrityl-2'-0-tetrahydropyranyl adenosine was synthesized according to a known method (L, J, McBri
dectal.

Tetrahedron Letters, 24.
245 (1983)) Therefore, 3' -0-(N, N-
diisopropylamino)-methoxyphosphoramidite derivatives.

That is, N6-penzoyl-5'-〇-dimethoxytrityl-2'-0-tetrahydropyranyladenosine (5
76.3q, 766.5 μmol) was made anhydrous by pyridine azeotrope, and then dichloromethane 1ai! dissolved in. After adding a nitrogen gas stream of diisopropylethylamine (0.49rd) to this solution, chloro-
Methoxy-N,N-diisopropylaminophosphine (
0.17d) was added gradually. After leaving for 1 hour, a thin layer O
After confirming the disappearance of the raw materials by mudgraphy, 10 d of ethyl acetate was added to dilute the solution. This diluted solution was washed with a saturated aqueous sodium hydrogen carbonate solution and then with saturated brine, and the ethyl acetate layer was concentrated to dryness under reduced pressure. The obtained residue was dissolved in ethyl acetate and subjected to column chromatography packed with 10 g of silica gel (C-300) (mobile phase: ethyl acetate).
It was purified by Eluted NG-penzoyl-5'-
〇-dimethoxytriethyl-2'-〇-tetrahydropyranyladenosine-3'-0-(N.

The pure fractions of N-diisopropylamino)methoxyphosphoramidite (compound 7) were collected and concentrated to dryness, giving a concentration of 0.67
g (733.9 μmol) of compound 7 was obtained in a yield of 95.7%.

Next, a solid phase support (R
, K. 1erzek etal, , Biochem
istry.

25, 7840-7846 (1986) as a starting material, and D together with an acetonitrile solution of compound 7 dissolved to a concentration of 20 μmol/140IIi.
The chain elongation reaction was performed using an automatic NA synthesis m<7 model 380A manufactured by Pride Biosystems. The dedimethoxytritylation reaction prior to each condensation cycle was carried out at 1%
A dichloric acid/methylene chloride solution was used. The ammonia aqueous solution of decaadenylic acid, which was cut out from the solid support after the reaction and had a partial retention capacity of 1 m3, was heated at 60° C. for 5 hours. This solution was then concentrated and purified by column chromatography packed with reverse phase silica gel (Prel F'AK-500/C-18, manufactured by Waters).

That is, 501M t'-ethylamine-carbonate buffer (1) H7,5 was applied with a linear concentration gradient of 5% to 35% acetonitrile as a mobile phase, hereinafter abbreviated as TEAB. ), and dimethoxytrityl group and 2
A pure fraction of decaadenylic acid having a tetrahydropyranyl group at the water w1 group at the ' position was collected and concentrated to dryness. Next, this residue was dissolved in 10 d of a 0.01 N aqueous hydrochloric acid solution and
It was adjusted to i-12.0 and left at room temperature for 24 hours. The reaction solution was neutralized with diluted aqueous ammonia, washed with ethyl acetate, concentrated, and subjected to gel column chromatography packed with Sephadex G-25 (mobile phase: 0.I
Desalting was performed by performing MTEAB).

The completely deprotected decaadenylic acid was finally purified by high performance liquid chromatography using reverse phase silica gel (YMCPack ODS, C-18 (manufactured by Yamamura Kagaku)) to obtain 6.5700 units of decaadenylic acid, approximately 50 nIO+. was isolated with an overall yield of 5%.

The obtained decaadenylic acid was subjected to an anion exchange column (DE
Analysis was performed by high performance liquid chromatography using AE-28W), and it was confirmed that a nearly single peak was obtained.

Example 7 (Deca(2'-0-methyl-5-methyluridylic acid) i.e. m5[Jm 10mar (Synthesis D>2'-0-methyl-5-methyluridine obtained in Example 5 (1 μ5o
Using the solid phase support bound with l) as a starting material, Example 4.
An acetonitrile solution (20 μmof/
140 l11), chain elongation reaction was carried out in the same manner as in Example 6 according to a conventional method (in this case, dimethyl tritylation was carried out using a 3% trichloroacetic acid dichloromethane solution). Deca(2'-0-methyl-5
-Methyl uridylic acid) was prepared in the same manner as in Example 6 using a reversed phase silica gel column.

Next, a portion of this was taken, the solvent was distilled off under reduced pressure, 1 d of 80% acetic acid aqueous solution was added, and the reaction solution was allowed to stand for 10 minutes at room temperature. After the reaction solution was distilled off under reduced pressure, it was further azeotroped with water under reduced pressure. Acetic acid was removed by The residue was dissolved in water, washed with ethyl acetate, evaporated under reduced pressure, and subjected to high performance liquid chromatography using the same reversed-phase silica gel used in Example 6 to give IIIJL/7.28.
00 units of deca(2'-0-methyl-5-methyluridylic acid) were obtained.

The obtained oligomer was subjected to an anion exchange column (DEAE-
Analysis by high performance liquid chromatography using 28W) gave a single peak, confirming its purity.

In addition, 1 liter of decadeoxythymidyl (
dT 10mer) wo20.8400-L knit, 7
Ka(2'-〇-methyluridylic acid) (Us 10m
ar) obtained 15.4700 units.

Example 8 (Ti measurement experiment) The amount of each decaoligonucleotide obtained in Examples 6 and 7 was determined by the following ε value (calculated value).

m-” knee order-γA 10111Or (complementary strand): 123
400d 10mer (control): 816
00m Un 10mer (present invention):
96800UmlOmer (control):
968007A 10a+er(0,20TOD, 1
．． 6nmol) and dTlomer, 15U11101m
+3r, LJI 1011er (7) each 1.
A volume equivalent to 6 tvol was taken in an Eppendorf dupe and dried under reduced pressure. Each buffer (0.01M cacodylate sodium salt, 0.IM NaCl, 11M E
DTA, E)H7,0) dissolved in 300p1,
Using a Beckman IJDLI-88 spectrophotometer, the change in absorbance was measured three times at a temperature increase rate of 2° C./win, and the average value thereof was determined to be the T+e value for each of these three hybrids.

The results are shown in Table 1 below (Figure 3).

Table 1 shows an overview of the synthesis of Compound 5. FIG. 3 shows absorbance changes with respect to temperature changes for three types of hybrids (Example 8).

Incidentally, the structural formula of each hybrid is as follows.

In the formula, N means 5-MetMIuracil.

Heia 1 no 1: (1): 5' r (AAAA
AAAAAA), 3'3' (TTTTTTTT
TT) d 5' hybrid ■, 5' r (AAA
AAAAAAAAA) 3'3' (UtlUUUUU
ULtU) i 5' high frit ■: 5' r (AA
AAAAAAAAA) 3'3' (NHNNNHN
NNN)m 5'

[Brief explanation of drawings]

Figures 1 and 2 show Examples 1 to 31 (embedded patent attorney Masao Shimokoshi), respectively.

Claims (1)

[Claims] (1) An oligonucleotide derivative represented by the following general formula (I). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (I) In the formula, R represents a hydrogen atom, an acyl group, or a phosphoryl group that may have a substituent, and T represents a thymine 1-yl group. Y_1, Y_2 and Y_3 may be the same or different and represent a hydrogen atom, a hydroxyl group, a lower alkyloxy group or a lower alkylsilyl group, and B is thymine-1 -yl group, adenine-9-yl group, guanine-9-yl group, cytosine-9-yl group
yl group, uracil-1-yl group or hypoxanthine-9
-yl group, and n represents an integer of 1 to 100. A nucleotide derivative represented by the following general formula (II). ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) In the formula, X represents a monomethoxytrityl group, a dimethoxytrityl group, or a phosphoryl group that may have a substituent, and Y represents a hydrogen atom, an o- Chlorphenyl phosphate, -P(
OCH_3)-N-(CH(CH_3)_2)_2,-
P(OCH_2CH_2CN)-N-(CH(CH_3
)_2)_2 or -CO-(CH_2)_m-CONH
-(CH_2)_n- (CPG derivative) (where m and n are each an integer of 1 to 10). However, when X is a phosphoryl group which may have a substituent, Y is a hydrogen atom.