JPS5980694A - Preparation of phosphate compound - Google Patents

Abstract

PURPOSE:To obtain a phosphoric acid compound of an OH-containing compound under mild conditions in high yield and economically advantageously, by metallizing OH of the OH-containing compound, reacting it with a phosphorylating agent. CONSTITUTION:An OH-containing compound (e.g., nucleotide or its derivative) is reacted with preferably 1 equivalent metallizing agent (e.g., dimethyllithium, NaH, t-butyllithium, etc.) based on 1 equivalent OH, OH is metallized, the prepared metal oxide is treated with preferably 1.1-1.2 equivalent phosphorylating agent [e.g., (C2H5O)2POCl, (o-ClC6H4O)2POCl, etc.] based on 1 equivalent OH, so that the OH-containing compound is phosphorylated to give a phosphate compound. USE:Useful for synthesizing oligonucleotide or polynucleotide for the purpose of genetic engineering, synthesizing phosphorus-containing agricultural chemical, synthesizing a phosphate compound as a ligand of catalyst, etc.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for phosphorylating a hydroxyl-functional compound, and further relates to a method for easily phosphorylating the hydroxyl group of a nucleoside in the synthesis of a nucleotide.

When synthesizing oligonucleotides, it is necessary to phosphorylate nucleosides using a phosphorylating agent (phosphoric acid derivative).

Traditionally, in this nucleic acid related field, over a long period of time,
Attention has been focused on the development of phosphorylating agents (phosphorylating agents) with strong phosphorylation ability.

The result 1. A mixed acid anhydride of sulfonic acid and phosphoric acid was discovered, and this reagent is currently widely used for nucleotide synthesis1. Hall u
No et ae, The 5 synthesis
of Nucleosides and Nucleots
ides, 69-239 (1977) J.

However, when this phosphorylating agent was used, it took a reaction time of usually 6 hours to overnight in pyridine at room temperature to complete the reaction, and an excessive amount of the phosphorylating agent had to be used. .

For example, the phosphorylating agent is usually used in an amount of 2 to 4 equivalents, and the chain length of the reagent becomes long, which may require 8 or more equivalents. Furthermore, phosphorylation occurs on the nitrogen atom of the nucleobase (
Because the reaction proceeds faster on the nitrogen atom of the amino group, it was necessary to preserve the amino group of the nucleobase in advance. Tenor, G, M0. Jiyanal, of,
The American Chemical Society, Volume 83, 15
9 pages.

1961 (Tener, GJ4. Journal
of the American Chemic, a7
Society, '83, 159 (1961)).

In response, mild phosphorylating agents such as phosphorochloridate or p-nitrophenyl phosphate have been investigated. These reagents are preferred because they are inexpensive.

However, the reaction time was long and strong reaction conditions were required, and the yield of the target compound was low.

On the other hand, a method is known in which a dinucleotide is synthesized by completely reacting a nucleoside having the following formula with a sodium salt of another nucleoside.

Nukarik. Buoy 1. Croateca, Chemica, Acta, 4
Volume 9, page 851, 1977 CS karic V, et al. + Croatica Chemica Acta,
49, 851 (1977) J.

However, this method requires very strong reaction conditions, and there are only reports using uridyl or thymidyl nucleosides, and there are no reports on other nucleosides that bind to nucleobases. ,.

The aim of the present invention is to provide a method that does not have the above-mentioned disadvantages and allows phosphorylation of nucleosides under mild conditions.

That is, 1. The method for producing a phosphoric acid compound of the present invention involves reacting a hydroxyl group-containing compound with a metallating agent to form a hydroxyl group-containing compound. Characterized by phosphorylating compounds.

The present invention will be explained in more detail below.

The unhydroxyl group-containing compound used in the present invention means an organic compound containing an alcoholic hydroxyl group and/or a phenolic hydroxyl group.

Hereinafter, this reaction will be explained in the case where a nucleoside or a derivative thereof is used as the hydroxyl group-containing compound, but even with other hydroxyl group-containing 16 compounds, the hydroxyl group can be converted to phosphoric acid f by the method of the present invention. It is possible to

When phosphorylating the hydroxyl group of the above nucleoside,
First, the hydroxyl group is activated as a metal oxide using a metalating agent, and then phosphorylated using a phosphorylating agent. As a representative example of this reaction.

The following formula: % formula % can be given. The above reaction is deoxyribonucleoside 1
It is applicable not only to the 1-phosphorylation of , but also to the 5'-phosphorylation of deoxyliponucleoside 3 or polybonucleoside 4 represented by the linear formula:

In formulas J~4, B is adenyl (Ad), guanyl (Gu
) or purine bases of these derivatives; cytosyl (Cy)
, uracil (Ur), thymidyl ('Th) or a pyrimidine base of these derivatives. B is adenyl, ♂-
In the case of protected (eg, methylated) uracil (Ur) or nucleosides that are uracil, high yields can be obtained using only stoichiometric amounts of R'M and the phosphorylating agent. B is N4-protected (e.g. benzylated) cytosyl (c
A good yield is also obtained for the nucleoside yB'').B
In the case of a nucleoside in which the mer is unprotected thymidyl or uridyl, the desired product can be obtained at a favorable rate of 1[7] by using two equivalents of the metallating reagent.

As a metal famine agent (R'M), mesityllithium (
MesIj), t,-butyllithium It-
C4H@Li), ■-butyllithium (2-C4I(,
Li), methyllithium (CHLi), hydrogen f arsenium (NaH), potassium l-butoxide (t-C
4H, OK) is exemplified. Preferably, MeaLi
, t -C4HgLt , t, -C4I(gOK
There is a T.

As the phosphorylating agent (R20) 2POX, for example,
(a -C11Co L O)2 POC11, (2-
CICaLO)2PO(OCOCF3), (C2Hs
O)z PO(OC6H4-2-NO2), (Cv H
IIO)2 POCJ? , (CsHsO) (p −N
O+Ca1140) POCl, (p -C#C5I(
<0) + PO (J etc. can be mentioned.
o, -CAlCa1(40)2POCd, (C2
HsO)2POC1, (CsHsO)2PO(OCeL
-p -NOz), (o-CIC+H40)+PO(
QC@H4-p-N02), etc.

This reaction is usually carried out in a solvent. Examples of solvents that can be used include tetrahydrofuran ('I'HF),
Examples include ether and dimethoxyethane.

Preferably 1, but ether solvent is even more preferable.
It is HF. Incidentally, a mixed solvent of these can also be used.

In this reaction, the nucleosides can be phosphorylated by further adding phosphoryl to the reaction mixture without isolating the metalmexide produced by metallation.

Metal and other agents ij: % per equivalent of hydroxyl group of nucleoside [
~, usually 1 to 1.1 equivalents, preferably 1 equivalent is used. However, in the case of male-uninsured thymidyl or uridyl nucleoside, use twice the equivalent amount.

Phosphoryl (5 agents are usually 1 to 3 equivalents per equivalent of the hydroxyl group of the nucleoside), preferably #i1.1 to 1.
Used for 2nd generation.

The reaction temperature is usually -78 to -50°C, preferably -78°C.
It is 0. The reaction time is usually 0.25 to 3. , hours, preferably 1 to 2 hours. Further, this reaction is preferably carried out in the absence of hydrogen ions.

After the reaction is completed, for example, extract the target product from one reaction mixture,
After drying this, the desired product is obtained by concentrating the resulting residue and subjecting it to total column chromatography.

The phosphorylation method of the present invention has the following advantages.

■ Reaction proceeds quickly even at low temperatures.

■ Nucleosides and phosphorylating agents may be used in chemically stoichiometric amounts.

0 By using THF gold as a solvent, post-treatment becomes easy.

■ Protection of the amine group of the adenyl nucleoside is not necessary.

(2) Cheap phosphorochloride and p-nitrophenyl phosphate, which are generally considered to have low reactivity, can also be used as phosphorylating agents.

As described above, this reaction is not only excellent in terms of ease of experimental operation, but also has advantages from an economic standpoint.

Furthermore, this reaction is applicable not only to the phosphorylation of nucleosides but also to the phosphorylation of other hydroxyl group-containing compounds.

The phosphorylation method of the present invention having the above advantages can be used, for example, in (J) nucleic acid science, especially genetic engineering.

■In the fields of organic synthesis and science, ■pesticide chemistry.

■Synthesis of oligo or polynucleotides for the purpose of genetic manipulation, ■Synthesis of organic synthesis reactions using organometallic complexes, especially phosphoric acid compounds as ligands for catalysts in catalytic reactions, ■Synthesis of effective pesticides containing phosphorus It is useful as a means.

Examples will be described below, but the scope of the present invention is not limited thereby.

(Phosphate ratio of Al nucleosides Example 1 5'-0-t-butyldimethylsilyl-/-deoxyadenosine-3'-0-di(o-chlorophenyl)phosne (obtained by mesityllithium or via thium alkoxide) method) +A mesityllithium (0,460 mmol) in THF
-ether-pentane (4:1:2) suspension.
-0-t-butyldimethylsilyl-2'-deoxyadenosine ((L460 mmol, 168.1 Qoki THF
(4III/!) The entire solution was added at 78°C and stirred for 15 minutes. The resulting white suspension of hedi(9-chlorophenyl)phosphorochloridate in THF) fBfi(0,50
6mmo1.0.886M.

Q, 57ml) was added dropwise at -78CK. After about 2 minutes, it became a clear, pale yellow solution. After stirring for 15 minutes at -78°C, the reaction mixture was dissolved in dichloromethane (20t/).
Dilute with water-dichloromethane C40ml1-20ml
) and extracted. Drain the aqueous layer in dichloromethane (
The organic layer was washed with saturated brine (20 tons/XI, 10*/X2), and then dried over magnesium sulfate. Concentrated, pale yellow syrup <336.
49) was obtained.

], ], ]] HNMR using 2.2-tetrachloroene as an internal standard and comparing the area intensity with Cs I-1.
I was disappointed. (Yield: 97%) When stored for 2 weeks in 20 tll of candy, crystallization stopped. This was washed with ether to obtain the title compound as white crystals (276.0η, 90%).

Rf (chloroform-methanol-ethyl acetate-10:
1:1)”0.36 mp i 14-1],
5C' HNMR (J, CDC&, CHC/2c
Hc 4 = 5.94 ppmGO,07(s,
6H,5iC)b), 0.87(s, 9H,5
iC(CHa)s).

2.78-2.96 (m, 2H, C≦H,), 3
．． 88 (brs, IH.

C/)(), 3.92 (brs, ]H,Cs'
H), 4.36-4.48 (m.

lH,cjI(), 5.44-5.62(m, IH
, CaH), 6.46 (brs.

2H,-Nl(p) 6.51 f t , (6,
46).

J=6.5J(,z,C,'10,7.04-7.52
(m, 811 atff 11) 8゜1j (s,
IH, 2-H), 8.33(s, IH.

8-H) I3CNMEt(δ, CDCl3. CDCl+ ”
76.9ppm) knee 6.00. −5.89(Si
CJb), 17.81 (SiC(CHs)s).

25.43 (SiC(CH3)3), 39.19 (d,
Jpocc-4,4Hz.

C-2'), 62.72 (C-5'), 80.
72 (d, , Ipoc”6.6Hz.

C-3'), 83.80 (C-1'), 85.
5] (d, Jpocc-6,6Hz.

C-4') 119.38 (C-5-), 121.
88 (d, J=3.2Hz, aromatic-C
), 124.9 (d, J=7.0Hz Aromatic-C), 126. ]6. ]27.63,130.
31 (A07 Teitsuku C), 138.02 (C-
8).

145.78 (d, J”6.6Hz, aromatic-
c).

149, ]0 (C-4), 152.57 (C-2), 1
55.59 (C-6)-Otori R (CHC13, Cm G 354 (L3490 (NH,), J640 (nucleobase in-plane), 1290 (>P-0) UV (Me Oli, l"I CnI): λm
ax=259.8nm i-1,64XIO' element cumulative analysis: C16C16) Si total 11 values to I2 c:so,
4sqb; N: 10s5Hs; H5*15% actual value
C: 50.22q6; N: 10.54%; I5.15%
4A Newman Era fr;s,,f) Rahedron Letters, page 4839, 1976 [Neunann
, us et al, Tetrahedron
Lemmuers.

4839 (1976) J Example 2 5'-〇-mu-butyldimethylsilyl-2'-deoxy (method via sodium alkoxide obtained with sodium hydrogen) g1 water g1 509b oil suspension of arsenium,
(11,6!, 0.242mmol)'e dry
The suspension was suspended in hexane (Q, 7 rnl x 3), decanted, the supernatant was removed with a syringe, and the residue was dried under reduced pressure. To this, 5'-0-t-butyldimethylsilyl-2-deoxyadenosine (83.9 mV, 0:23
5 mmol) of THF (2 ml) -10
After stirring for 30 minutes, the reaction mixture was di- and di-(
THF of σ-chlorophenyl) phosphorochloridate
Solution (0.34*1.0°760M.

0.258 mmol) t-78CK was added and stirred for 1 hour. The reaction mixture was diluted with total dichloromethane (15%/) and poured into a water-dichloromethane (30ml-15am/) mixture for extraction. The aqueous layer was dichloromethane (15 m/X
I, 10dX, 2), the combined organic layer was washed with a fully saturated saline solution (20t/), dried over magnesium sulfate, concentrated, and a candy-like substance (77.7W) was obtained. , 1.2.2-tetrachloroethane was used as an internal standard and the area intensity was compared with that of C3'HNM.
R was quantified. (72%) Example 3 5'-0-t-diptyldimethylsilyl-2'-deoky-111111nie-11-11----------------------
----------Synthesis of 1-111-1111-11-1cyadefcin-3'-0-diethyl phosphate 5'-0-t-butyldimethylsilyl-2'-deoxy Adenosine (75,2+'IP, 0.206 mmol
) of dry THF (2ffI) g solution, add potassium 1-
THF bath solution of butoxide (0.40a/, 0.51
M, 0.206 mmol)'e-78c, 5
Stir for a minute. .

Diethyl phosphorochloridate (38,7
W, 0.233 mmol)'e was added at -78°C, and stirred at -780 for 1 hour and then at -40°C for 1 hour. The reaction mixture was poured into water (20 mg) and extracted with dichloromethane (20 mg). Aqueous layer Odichloromethane (2
After washing with 0*/X1.5×2), drying on magnesium sulfate1. Ta. Concentrate under reduced pressure to obtain the title compound f 100. ]
"F was obtained as a colorless oil.

(90,4%) 'HNMR quantification is performed using 1.1.2.2 as an internal standard substance.
-Tetrachloroethane (5,49 ppm)? : 2-H of the base of adenosine used (nucleoside 8.19 ppm,
The area intensity was determined by comparing the area intensity with nucleotide (8.13 ppm).

Rf (methanol:chloroform=1:5)=
0.51 (S M = 0.43)' HNMR (δ, CDC15, CHClm = 7
．． 25 ppm): 0.04(s, 6H, 5iC
)Ta), 0.85(s, 9H,5iC((
J(s)s).

1.31 (dt, J=0.9, 7.2Hz, POC
Hs ), 2.6 2.8 (m, 2H, C2'H2)
, 3.85 (br'8.LH, (4'H) , 3.9
5 (brs.

IH, Cg'H), 3.92 4.34 (m, 5H, P
OCHy, C*'H) 4.96-5.16 (m, IH
,Cg'H,,) ,6.21(brs, 2H,-
NH! ) 16.45 (t, J””6.8Hz,
IH, C1'H), 8.07 (s, IH,2
-H).

8.29 (s, IH, 8-H) ”CNMR (δ, CDC4, CDCl5 =76.9
ppm) knee 6.00°-5,91 (SiCHs),
15.67 (d, Jpocc=7.6Hz, POC
I(s).

17-81 (S I CC), 25-43. (St
O(C)L+X+39.00(d, Jpoc
c=4.4Hz, (4'), 62.67(Cs
'Hs) v 63.64 (d, Jpoc==6.6H
z, POCHt), 77.63 (d, Jpoc=
5.5Hz.

Cs'), 83, 75 (C,'), 85.5
8 (d, Jpocc=5.5Hz, C4').

119.33 (C-s), 137.97 (C-s),
149.05 (C-4) 152.52 (C-2),
155.54(C-6)IR((J(CA'3.cR
): 3540.3405 (1%) 1630.1595 (nucleobase).

1250(p-0) UV(MeOIl); λmax-259.8nrnj-1.04X10'Example 4 Synthesis of 5'-0-t-butyldimethylsilyl-/-deoxysulphate (lithiation of t-butyllithium 5'-0-t-particles 1 True 2 in a 30M eggplant flask heated and dried under reduced pressure and replaced with argon.
-deoxyadenosine (73,111111,0,20
0 mmo/ THF residue C1,5#I/) at -78℃
A hexane solution of monobutyllithium (0,390M
, 0.513WLl.

Q, 200 mmo/) was added dropwise. -10 at 780
Stir for a minute. Di(0-6 di-phenyl)phosphorochloridate was dissolved in THF' to the cloudy reaction mixture.
(0,760M, 0.29 shoulder z, 0.22 mmo/)'
fr was added dropwise and stirred at -78°C for 1 hour. The resulting clear bath solution was diluted with dichloromethane (20) and poured into 10% saline-dichloromethane (40 mg-20 mg) for extraction. Wash the aqueous layer with dichloromethane (20*gX2),
The combined seven layers were washed with brine and dried over magnesium sulfate. Crude product obtained by concentration (0,155E)
Silica gel column chromatography (6q6' water.

4F) and acetone-dichloromethane (l:4) ~
Acetone-dichloromethane-methanol (1:4:
o, 1 to 1:4:1) to give the title compound (12)
, 0■, 91qb) was obtained.

Example 5 ■(2,6-Simethoxyphenyllithium (5tocl)
csoln, ) Method using Connector B] 5'-Q-t-Butyldimethylsilyl-! in a 39*/ eggplant flask that was dried under reduced pressure and replaced with argon. -Deoxyadenosine (62,8N, 0.172mmoA')
2,6- in THF g solution (2s+g) at -780
THF solution of cymethoxyphenyl lithium (0.19M
, 0.90d, 0.172mmol) k dripping 7.1
Stir for 5 minutes.

A THF bath solution of di(0-chlorophenyl)phosphorochloridate (0,772M, 9.
245ml, 0.189mmol) was added at -78°C and stirred for 1 hour. Sumi, da F? The reaction mixture, which became lf, was diluted with dichloromethane (20 mr), and the t'L
f: Saturated saline-dichloromethane (3omt-1oy)
The mixture was poured and extracted. The aqueous layer was dichloromethane (20
110yxl.times.2) and the combined organic layers were dried over magnesium sulfate. A concentrated pale yellow candy-like substance (0,13F) was observed by total 1H NMR. (88ch) Chi B Matsu Queen Dai Double 0 E et al. Journal of the American Chemical Threat Society, No. 1 ()
() volume 17304, 1978 [McEwen, W.
, E, etal, Journal of the A
r11Ar119ricanChe! 5ociety
+100.7304 (] 978) ) @ (in 5
2,6-DMPL synthesized in itu (method for using as t-lithio ratio agent) 1.3-tumetquinbenzene THF solution
mg, 0.207 mmo1.0.96 M) in THF
(6sv)) and cooled to % 0C. Add to this solution a hexane vinegar solution of nibbutyl lithium (0.15ml 1.0.
207 mmo#, 1, 38 M) was added dropwise. After stirring the resulting solution at OC for 5 hours.

-5'-Q-t-7'' methyldimethylsilyl- at -78C
/-deoxyadenosy (75,7!, 0.207m
mol) of TfJF solution (2we) was added in a stainless steel tube.

Stir for 15 minutes. , diC was added to the resulting cloudy reaction mixture.
o-chlorophenyl)phosphorochloridate T T(
, F solution (0.31 mg, 0.227 min, 0.
724M) t-Dropped at -78°C. A clear solution appeared after 10 minutes.

After stirring at -780 for 2 hours, the reaction mixture was diluted with dichloromethane ('30-), and saturated brine (30 mg) was added.
and extracted. water f%-2 dichloromethane (20I!
! The combined organic layers were dried over magnesium sulfate and concentrated to obtain a pale yellow syrupy substance (o, 17F), which was subjected to IINMR measurement.
Qh) -C Lambert G.I. et al., Journal Opsha Organic Φ Chemistry, Vol. 47, p. 3350, 1
982 (Lambert, G. 1. etal.

Journal of Organic Che
Mistry, Kei 7, 3350 (1982)) Example 6 5'-0-TBDMS-2'-deoxyadenosine-3
Synthesis of '-〇-di(9-chlorophenyl)phosphate (method using methyllithium) A reaction tube was used, which was heated and dried under reduced pressure, replaced with argon, and replaced with Cebu f Murano (-) and through which Ar was flowed.

5 tsl suX 7 in Lasco, 5'-0-T)
3 DMS-2'-deoxyadenosine (73, I
Q, 0.200 mmol) of Tl[F solution'k
This was transferred to a reaction tube under argon pressure using a stainless steel tube made of MMI, and cooled to -780 for 70 minutes. Add methyllithium (0.74M, 0.271W) here.

0.200 mmol) was added dropwise onto an ether solution. 15
After stirring for a minute, the cloudy reaction mixture was diluted at -78°C (kl).
0-Nitrophenyl) phosphorochloride in THF 1 (0,724M, 0.22 rq, 0.3
0 mmo/)+i was added.

The reaction mixture was diluted with dichloromethane (30 ml) and then poured into saturated brine (30 d) for extraction. The aqueous layer was washed with dichloromethane (20 mg, 10 dX2), and the organic layer was dried on M, g S04. +ta. The crude product (0
, 16F) was quantified by HNMR. (90%) Example 7 Sheadefusin-3'-0-di<9-10 Rofunil) (
Method using 5ee-butyllithium) A THF solution of 5'-0-t-butyldimethylsilyl in a 30 mA eggplant flask that was heated to dryness under reduced pressure and replaced with argon.
A suspension of sec-butyllithium in cyclohexane (1.OIM, 19ml!, 0.19ml) in 2#L/)
It was added dropwise at 78C and stirred for 2 hours. , a solution of di(9-chlorophenyl)phosphorochloridate in THF (0.77
2M. 0. , 27 hours. 0.209mmot)'(# -
It was added dropwise at 78 C and stirred for 2 hours. The slightly cloudy reaction mixture was diluted with dichloromethane (30PR1) and
The aqueous layer was poured into saturated brine (30 ffe) and extracted, and the aqueous layer was dissolved in dichloromethane (20 ml'.) Orqi x 2
> and the combined organic layers were dried over magnesium sulfate.

The candy-like substance (0.13#) obtained by concentration was subjected to IHNMR quantification. (45 t) Example 8 Mesityllithium (0,203 mmoA!) in THF
-N in a suspension of ether-pentane (approximately 4:1:2)
-Methyl-2',3'-isoglopylideneuridine (0
,203mmol, 60.7n1! Rino THF (3m
) solution was added at -78°C and stirred for 15 minutes. A THF solution of di(9-chlorophenyl)phosphorochloridate (0,223 mmoA!, 0.8
86M, 0.25-) was added thereto, and the mixture was stirred at -78°C for 1 hour. The reaction mixture was diluted with dichloromethane (10ml) and poured into a mixture of water-dichloromethane (20ml-10ml) for extraction. Aqueous layer total dichloromethane (10dX1
, 5-x2), and the combined organic layer was washed with saturated saline (i
After washing with 0 m/), it was dried over magnesium sulfate. The colorless candy-like substance (0,151I) obtained by concentration was subjected to silica gel column chromatography (6% water content).

4.5 f, 0.9 cmφx l Q ctc
) Sochloromethane ~ dichloromethane: acetone (
=3:1) to obtain the title compound. (110,
1■, 90ch) Rf (acetone-sochloromethane/=1nia) = 0.63 (SM: fl, 18)' HNMR: (δ, CI) C1s, CuO2C
uO2=5.94ppm)3,22(8,34(,N
-CB5) 14.324.56 (m, 3H, CB
'1(,C3')i.

C4'H) 4-724.82 (m* 2H, CB
'H! ), 5.62 (d, J-79Hz, I
H, 5-H) 5.70 (d, J=1.8Hz, IH, C
%H) 7.0-7.5(m, 9H+6I(+Al1ffWtickH)Is CNMR: (δ, C
DCl5, CDC15=76.9ppm) (d, J
poc=6.6Hz, Cs')80.34(Ct'),
84.14 (Cs') 84.80 (d, Jpocc =
7.7H2IC 4, 94.59 (C+'), 1
01.56(CI+)ICC), 125.02(
d, J=7.7Hz, aromatic C), 126
．． 17.127.63, 130.31 (Aromatic C), 139.00 (Co), 145.88
(d, J=7.6Hz, aromatic C), 1
50.42 (C2).

162.04 (C4) 1, R (CHCls 1cm): 1715, 1670 (nucleobase), 1290 (
P=O) MeOH: λ1nB) (= 258, 9sm, ε= 1.0
6 x 10' Example 9 t-butyl ram (0,234 mmall) t7
) <Tanthane solution e, 3'-c>-t-butyltmethylsilyl-27-zooxyadenosy/(0,234mm
o#, 85.5”f)ノTHF (3+nol)
It was added to the solution at -78°C and stirred for 30 minutes. A THF solution of so(dan-chlorophenyl)phosphorochloridate (0,257 mmol, 0.692
M, 0.37 ml) was added thereto, and the mixture was stirred at -78°C for 1 hour. The reaction mixture was diluted with dichloromethane (10ml) and diluted with saturated brine-dichloromethane (2ofne-10ml).
It was poured into the mixture of 1) and extracted. Drain the aqueous layer in dichloromethane (
10 m/XI, 5 m/X2), and the combined organic layer was washed with saturated brine (10 tnt) and dried over magnesium sulfate. Colorless candy-like Qll obtained by concentration) (
0,23f) by silica gel column chromatography (
The mixture was subjected to 6 hours of water (5.5 rQ, 9αφ x 14 degrees) and eluted with dichloromethane-acetone (10:1 to 5:2) to give the title compound. (144■, 92%) Rf
(acetone-dichloromethane=5:2)=0.39
(SM O, 23)' HNMR (δ, CDCl3
, TMS=O, Oppm): 0.10 (s r 6H
+5iCHs)+0-90(s r 9H+S
iC(CHs)s) +2.242.96 (m
, 2H+ c,'n,, ), 4-124-30
(m, lH2C4'I■), 4.50 - 4.78
(m, 3H, (, 乍Is, Cs'H), 6.34(
t) r8+ 2H+1'JHffi) + 6.43
(d, J-G, OHz, LH, CI'H)
.

7.0-7.48 (m, 8H, aromatic H)
7.98(s, IH, 2-H), 8.32(
s, 11L8-H)" CNMR (δ, CDCl5, C
DCl5 =76.9ppmC-4,98,-4,88
(SiCHs), 17.72 (SiC9), 2
5.57 (SiC(Cru)s), 40.22 (Ct
'), 68.82 (d, Jpoc=6.6Hz, Cs
') 7233 (Cs'), 84.63 (C+')
, 85.31(d, Jpocc = 8.2I
Iz, C4'), 120.31 (Cw).

121.35 (d, J=2.2Hz, atffmatic C).

125.41 (d, J=7.7Hz, aromatic C
).

126.06. 127.63. 130.50 (Aromatic C), 138.95 (Cs), 146
゜42(d, J=e,, tHz, aromatic C
), 149.59 (C4).

152.76 (Cz), 155.64 (Ce)I
R (CHC11, *, Confucian): 3540, 3
400 (NHs), 1630, 1585 (nucleobases).

1290 (P20) tJV (MeOH): λ+nax = 260.2 nm, ε= 1
．． 31
203 mmo, g, 62, 41 ng) of THF (
5 m/liter of the suspension was added at -78°C and stirred for 3 hours.

A solution of di(dan-chlorophenyl)phosphorochloridate in THF (0,223 mmol, 40.886 M
, 0.25 mg) and stirred at -78°C for 4 hours. The reaction mixture was diluted with dichloromethane (20me) and poured into a mixture of water-dichloromethane (30rnl - 10ml) for extraction. The aqueous layer was diluted with dichloromethane (3t) m4
xl, 15m/! X2), and the combined organic layer was washed with saturated brine (20 mg) and dried over magnesium sulfate. A pale yellow gum-like substance obtained by concentration (o,
2oy) to 7 Licadal column chromatography (
6chi water content, 5.0ri, 0.9cIlφ×12crn
) and eluted with benzene-acetone (10:1 to 3:1) to give the title compound (109μ, 88%). mp1
38-139℃ Rf (chloroform-methanol-acetic acid
10:1:1) =0.28 (SM:□0.
14)'HNMR(δ, CDCl5, TMS=0
, 0 ppm) :C4'H) , 5.14 (d
d, J=2.0, 6.2Hz, Ill, C
4'H), 5.39(dd, J=2.2,6.2H
z, IH, C5'll), 6.14 (d, J=2.4n
z tcI'H) + 6.55 (brs, 2H
,NH,), 7.0-7.44 (m.

8H, aromatic H), 7.90 (s, I
H, 2-H).

s, 29 (s, IH, 8-H) 18CNMR (δ, CDCl5, CDCl5 = 7
6.9 ppm) :Hz, Cs'), 81.
46 (Ct')j83.99 CCm'), 84
．． 92 (d.

Jpocc = 8.8Hz, C: ), 9(1
,78(C'+) + 114.45 aromatic C
), 125.36 (d, J=7.7Hz.

Elfftic C), 126.02, 127.
53,130.41 (Aromatic C), 139
．． 44 (Cm), 146.37 (d + J=6
．． 6Hz, 7 o Machic C), 149.
20 (C4).

152.91 (Cz), 155.74 (Cs) IR
(CuCl2.c+++-”): 3540.3400 (NH,), 1630.1585 (
Nucleic acid base) 1290 (, > P = 0) UV (MeOH): λmax = 261.2 nm, g = 1.28xlO'
Elemental analysis: Ct5Ht4NsO□PCA! ! Calculated value:
C, 49,36%: N, 11.51%; H, 3,98
% actual value: C, 49,18%; N, 11.38%: H
,4,00% or more of the examples, reagents, reaction conditions,
The yield etc. are listed in the following table, and other examples are also listed in the same table.

TBOMS=S i (CH3) 2- t -C+
HO old size ■ -0■ To 0
0 ■ 0Q pus no 0 tozuV″4 -sl --1-sl -1-5l-s?-s
l -sl ■ ■ 0 0
Q OΦ ■Murder, 8-2 mz mz size? -f2
-P? Size? −? 2.4-7 Synthesis of dinucleotide Reference Example 1 Synthesis of dinucleotide 5"0-t~butyldimethylsilyl-2'-deoxyadenosine-3'-0-p-2 was performed using the following formula using this cooker. Synthesis of trophenylphenyl phosphate (1) Mesityllithium (0,180 m mob) in THF-
5'- in a suspension of ether-pentane (approximately 4:1:2)
0-t-butyldimethylsilyl-2'-deoxyadenosine (0,180 mmol, 65.8 mV) in THF
(1,5 td) solution was added at -78°C, and after stirring for 10 minutes, a THF solution of p-nitrone enylphenyl phosphorochloritate *3 (0,216 mmo/, 0.
785M, 0.275+++/) was added. -78℃
After stirring for 1 hour, the mixture was diluted with dichloromethane and a saturated aqueous ammonium chloride solution was added. The organic layer was separated, the aqueous layer was extracted twice with dichloromethane, the organic layers were combined, washed with saturated brine, and dried over anhydrous magnesium sulfate. The oil obtained by concentration under reduced pressure was subjected to silica gel column chromatography (6% water content, 5v) and eluted with dichloromethane-acetone (10:0 to 5:5) to give 1 (65, 4■, 57%).

'HNMR(δ, CDCIs, TMS=O,O
PPM): 0-08 (FI+ 6 H+ Si
-C) Is) , 1.89 (s + 9 H+ S
i-C-C%), 2,52 2.98 (m,
2H2Ct'H) 13.68 3.94(m, 2I
-I,C,'H), 4.20-4.44(m,,IH
, C4'H).

5.228-554(,IH,C,'H), 6.17(
brs, 2H, N)5).

Synthesis of xyadenosine (2) 3'-0
-t-Butyldimethylsilyl-2'-deoxyadenosine (0,100 mmol, 36.5'f) in THF (
LOml) solution was added at -78°C and stirred for 10 minutes. 1 (0,102m mol, 65.4
'F) THF (1,0m) solution was added dropwise, -7
After stirring at 8° C. for 1 hour and at -50° C. for 3 hours, the mixture was diluted with dichloromethane, poured into water-dichloromethane, and the organic layer was separated.

Extract the aqueous layer three times with dichloromethane and combine with the organic layer.%
It was washed with saturated brine and dried over anhydrous magnesium sulfate.

The oil obtained by concentration under reduced pressure was purified by silica gel column chromatography (6q6 water-containing, 52) [dichloromethane-acetone-methanol (8:2:0 to 6:4:
O~6:4:l)] Colorless oil 2 (60,1
! , 69%) was obtained as an approximately 1:1 noastereomer concentration.

This norastereomer was obtained by HPL using an ODS column.
It was separated and fractionated at C.

'HNM'R, (δ, CIJC7,, TMS=Op
pm): Shorter retention time 0.06, 0.09 (er, ch s, 12fi, 5i
-CHs), 0.87°0.90(each s, 1
8H,5i-C-eHs), 2.26-2.98(
m, 4H, Cp'H), 3.68-3.85(m
, 2H,C,'H-OSf).

4.02-4.48 (m, 4H, C, 'H-OP, C4
'H), 4.58-4.78(m, IH,C,'H
-O8i), 5.12-5.30(m, IH,
Cs'H-+-+p), 6.20-6.56 (m
, 6H,C+'H,NH2), 7.12-7.3
8 (m, 5H, ao'v tick H), 7.97,
8.08(e a c h s + 2 H+ Ct
H) r' 27 (s + 2 H + C3H) Longer retention time 0.04,0 old (each s, 12H, 5i-CH,
), 0.84°0.91 (each s, 181
(,Si-C-CHs), 2.28-3.02(
m, 4H, (4'H), 3.52-3.90 (+n,
2H, Cs'H-O8l).

5.06-5.25 (m, 2H, Cs'H-OP)
, 5.28-5.48 (m.

2H, C4'H), 5.585.76 (m,
IH, Cs'H-O81), 6.10-6-32
(m, I Ht CH'H-OP)! 618
(b r −s r 4 )i, I'J4(!).

6-28652 (m, 2H, C+'H) + 7
．． 127-44 (m, 5H+7'o-r tick H) +7.98, 8.04 (eaclls, 2H.

Ct H) * & 26 (s, 2 He Cs
H) If this reaction were carried out without isolating 1, 2 would be obtained at a rate of 8% of 0%.

Reference example 2 173.1 mV (0,200 mmol) anhydrous THF
2 ml of the solution was cooled to −78° C. under an atmosphere of t −B
uLi (0.48M.

pentane solution) 0.420 mg (0.20 m mol
) was added dropwise over 5 minutes. Cool to -95°C and use 0.605 mJ (2,28 mmo) of toluene solution (0,377 M) of 2.
/) was added dropwise, and the mixture was stirred at -78°C for 1 hour to obtain a solution of 3. Anhydrous THF solution 2- of 480.4 μm (0,220 mmol) was cooled to -78°C under an Ar atmosphere.
t-BuLi in pentane solution (0.48M) 0
．． 458 mg (0,220 mmol) was added dropwise over 5 minutes. The THF solution of 3 prepared above was added dropwise to this solution, and the mixture was stirred at -50°C for 5 hours. 10% brine was added, the temperature was returned to room temperature, and extraction was performed with methylene chloride. The organic layer was washed with saturated U water and saturated brine in that order, and dried over anhydrous magnesium sulfate. The oil obtained by concentration under reduced pressure was purified by silica dull column chromatography to obtain 5 as a colorless solid at 139W (8
0%) obtained.

Claims (1)

[Claims] (1) It is characterized by reacting a metalating agent with a hydroxyl group-containing compound to fully metalate the hydroxyl groups, and then allowing a phosphorylating agent to act on the obtained metal oxide to phosphorylate the hydroxyl group-functionalized compound. A method for producing a phosphoric acid compound. (Claim 1) The hydroxyl group-functionalized compound is a nucleoside or a derivative thereof, and the reaction f is carried out in 1m.
A method for producing a phosphoric acid compound as described in .