JPS59163397A - Preparation of phosphoric acid compound - Google Patents

Abstract

PURPOSE:To phosphorylate OH and to obtain a phosphoric acid compound by a simple operation in improved selectivity for functional group economically advantageously, by treating an OH-containing compound with an aluminizing agent to give an aluminum alkoxide, reacting it with a phosphorylating agent. CONSTITUTION:An OH-containing compound (e.g., nucleoside, its derivative, etc.) is treated with an aluminizing agent {e.g., a combination of a compound shown by the formula Al]N(R<1>)2]3 (R<1> is H, or 1-5C alkyl) and a compound shown by the formula ArOH[Ar is (substituted)aryl, etc.} to give an aluminum alkoxide, which is reacted with a phosphorylating agent [e.g, (C2H5O)2POCl, etc.]. USE:Useful as a method for synthesizing nucleotide, a phosphoic acid compound of a ligand of catalyst, etc.

Description

Detailed Description of the Invention [Technical Field of the Invention] The present invention relates to a method for phosphorylating a hydroxyl group-containing compound, and more particularly, to a method for easily and selectively phosphorylating a hydroxyl group of a nucleoside in the synthesis of a nucleotide. .

[Technical background of the invention and its problems]

In the field of genetic engineering, oligo9 nucleotides have become an increasingly important research subject in recent years.

The basic reaction for its chemical synthesis is phosphorylation of nucleosides. In order for this reaction to proceed easily, the following reaction formula (1
) or (2), two methods can be considered in principle: activation of a phosphorylating agent that is an electrophile, or activation of a nucleoside that is a nucleophile.

X-08 (hAr Ar = aryl group X = CI
, 0C1lH4-p -NO2(R3=alkyl M*
R'=nucleoside residue) Conventionally, in this nucleic acid-related field, attention has been focused on the development of phosphorylating agents (phosphorylating agents) having strong phosphorylation ability for a long time.

As a result, a mixed acid anhydride of sulfonic acid and phosphoric acid was discovered, and this reagent is currently widely used for nucleotide synthesis.

However, when this phosphorylating agent is used, its action is too strong, and phosphorylation occurs more on the nitrogen atom of the nucleobase (the nitrogen atom of the amino group) than on the oxygen atom of the nucleoside (the oxygen atom of the hydroxyl group). In order for the reaction to proceed quickly, it was necessary to protect the amine group of the nucleobase in advance. In addition, during the reaction, either the phosphorylating agent or the nucleoside must be used in excess;
Moreover, the sulfonic acid conductor (5ulfon) required for activation
ylchlorides, 5ulfonolides
s) had the disadvantage that it was extremely expensive. Mizuno et al., Synthesis of nucleosides and nucleotides, 69-239
Page, 1977 (Mizuno et at, Th
e5ynthesis of Nucleoside
sand Nucleotides, 69-239
(1977) ) and Tenor So M 1. Journal of the American Ordinance Chemical Society,
Volume 83.

p. 159, 1961 (Tener, G. M., J.
our own of the American Che
mical 5ociety, eup 3,159 (196
See 1)).

In contrast, milder phosphorylating agents such as phosphorochloridate or p-nitrophenyl phosphate have been discovered. 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 sonucleotide is synthesized by reacting a nucleoside having p-nitrophenol as a leaving group with a sodium salt of another nucleoside, as shown in the following formula: etc. Skalitsk buoy 1. Croatian Chemiriki Acta, Volume 49.

851 pages, 1977 C5karic V, eta/
,,CroaticaChemicaActa,
49, 851 (1977)).

However, this method requires extremely strong reaction conditions, and there are only reports using urituru or thymidyl nucleosides, and there are no reports on nucleosides containing other nucleobases.

[Purpose of the invention]

An object of the present invention is to provide a method that does not have the above-mentioned drawbacks and can easily and selectively phosphorylate hydroxyl groups of nucleosides.

[Summary of the invention]

The present inventors believe that by using a metal that has a greater affinity with acid purple atoms than with nitrogen atoms, as shown by the following formula (3): px = phosphorylating agent, M = metal, metal alkoxides and metal It was thought that the equilibrium of the amide was shifted to the alkoxide side, 0-selective activation was achieved, and the O-phosphorylated product was selectively obtained. Generally, bond dissociation energy provides a good measure of the affinity between atoms. As shown below,
Aluminum is believed to have a greater affinity for acid formation than nitrogen.

Bond dissociation energy (KJmot) ht-o: 512.1 ± 4.2 At-N: 297 ± 96 Bee Soeiba Dagtugian et al., The Technical OpQ Chemical Physics, Vol. 62.

1824 pages, 1975 CP, J, Dagdigi
an etal The Journal of Ch.
epical Physics, 62.

1824 (1975));

Dissociation energy and spectrum of diatomic molecules.

1968 CA, G., Gaydon, Diss.
ociationEnergies and 5pec
tra of Diaatomic Molecules
.

(1968)) etc.

Based on the above speculation, the present inventors completed the present invention by first converting the nucleoside into aluminum alkoxide and then phosphorylating it.

The production method of the present invention is characterized in that a hydroxyl group-containing compound is treated with an aluminizing agent, and then the obtained aluminum alkoxide is treated with a phosphorylating agent, thereby phosphorylating the hydroxyl group of the hydroxyl group-containing compound.

The present invention will be explained in more detail below.

The hydroxyl 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 using a nucleoside or a derivative thereof as the hydroxyl group-containing compound, but the hydroxyl group of other hydroxyl group-containing compounds can also be phosphorylated by the method of the present invention. be.

In order to phosphorylate the hydroxyl group of a nucleoside in this reaction, the hydroxyl group is first activated as aluminum oxide using an aluminizing agent, and then phosphorylated using a phosphorylating agent. A typical example of this reaction is the following formula: %Formula%. The above reaction is deoxyliponucleoside 1
Not limited to the 3'-phosphorylation of It also applies to 5'-phosphorylation of nucleoside 4.

~ In formulas 1 to 4, B is adenyl (Ade), guanyl (G
purine base of ua) or these derivatives; cytosyl (C
yt), uranium A, (lJra), thymisol (Thy
) or the birimidun base of these derivatives. In the present invention, 0-selective phosphorylation can be performed without protecting the amino group and imide group of a nucleobase, so even if the nucleoside of an N-protected nucleobase is selectively phosphorylated, only the hydroxyl group can be selectively phosphorylated. can be phosphorylated.

As the aluminizing agent, for example, the following formula (1), (II
): At[N(R”h]s+ArOH(I)LiA
t(N(R)2)4 +ArOH(II) (wherein R
1 and R2 each represent a frozen candy atom or an alkyl group having 1 to 5 carbon atoms; and Ar in (■) and (...) each represents a substituted or unsubstituted aryl group. In addition, in formula (1), 7kl (
The combination of NCR'')g)3 and ArOH means Al
A combination of [N(R')2]s and ArOH, At(:
A combination of N(R)2)2(OAr) and ArOH,
and At[N'('R)2:] (OAr)2 alone. Furthermore, in formula (II), L i A L
The combination of [N(R2)2]4 and ArOH is Li
Combination of At[N(Rh)4 and ArOH, Li
Combination of AtCN(R2)z )3 (OAr) and ArOH, LiAt[N(R2)z )2 (OAr)2
and ArOH, and LiAt(:N(R2)z
) (OAr)s alone. Examples of the alkyl group having 1 to 5 carbon atoms for R1 and R2 include methyl group, ethyl group, n-propyl group, isozorobyl group, n-butyl group,
Examples include 1-butyl group and n-pentyl group, but methyl group is most preferred. As Ar in formula (I),
For example o -CH3C3H4-1o -CLCsHA-
1p-NO2C6H4-1C6H5-12C4-4NO
2CsH3-1p-C4C6L-1p-c and C6Hji,
p-CH3C3H4,2,4-, (NO2)2C8H3
- etc., o-C4CsH4-1p -N0
2C6H4-12-Cl-4-NOgCaHs and the like are preferred. Equation (Ar in the loss is, for example, Cs1(5
”, o czc6)14-1p -CLCeHa-1
o CH3C5Hdi, p -CH2C6H4-1o
-N02C6H4-1p -N02C6H4-12,6
((C)h)3C)zC6H3-12,4-(N011
)2 C6H5-12-Cl-4-N0zCsHs
-1p -CH30Ca H4-12-naphthyl etc., p -N02C6H4-12-Ct 4
- N02C6H3-1p -CtcsHji, o -C
LCsH<- etc. are preferred.

As a phosphorylating agent, for example, the following formula (4): % formula % (wherein R5 represents an alkyl group having 1 to 10 carbon atoms or a substituted or unsubstituted aryl group; X represents a halogen atom or a substituted or unsubstituted aryl group) represents an oxy group). R5 carbon number 1-10
Examples of the alkyl group include methyl group, ethyl group, n
-Propyl group, isozolobyl group, n-butyl group, t-butyl group, n-pentyl group, n-hexyl group, n-hebutyl group, n-octyl group, n-nonyl group, n-decyl group, etc. However, methyl group and ethyl group are most preferred.

The substituted or unsubstituted group for R5 is, for example, o-C1CeH<-1C6H5-1p -N02C6H
4-12-C1-4-NO2C11H3-, etc., but ethyl group, C5Hs-1 and 0-C4CsH4-
is preferred.

Examples of the atom of X include a chlorine atom and a fluorine atom, with a chlorine atom being the most preferred.

Examples of the substituted or unsubstituted aryloxy group for X include o -CtCsH40-, p -N02C11H4
0-,2,4-(NO2)2C6H30-,2-Ct-
4-N02C8 几-郎 is mentioned, but 2-Ct-4-
N02C6H3-1p -N02CsH40-, 2, 4
-(NO2)2C6H50- and the like are preferred. Specific examples of phosphorylating agents include (CzHsO) 2
P OCLl(C2H50)2PO(OC6H3”2.
4-N02), (0-C4CsH40)zPOcLs
(o-C4CsH40)2PO(OCgH32-CL-
4-NOJ(C2H50)'2PO('0C11H4P
-NO2), (C11H50)2PO(OC6H3
-2-CL-4-NO2), (CeHsO)zPO
ctl(CaHsO)2PO(OC6H4~J)-NO
2), (CaHsO)zPO(OCaH3-2,4-N
O2), (C6H50)2PO(OC6H3-2-
(C1-4-NOx) etc., but (C2H3O
)2 POCLl(CsHsO)2POC4゜(o −
CL Ca H40) 2 POCts (C copy HsO
)2PO(OCg&-2-(1j-4Nl), (Cg
HsO)zPO(OCsH3-2-Ct-4-NO2)
, (CzHsO)gPO(OCeH32,4-NO
2) , (CsHsO)2PO(OCsH3-2,4
-NO2) is preferred.

This reaction passes through different intermediates depending on the type of aluminizing agent used.

(1) When At[N(CH3)2)a+Ar0Hk is used (wherein, Ar + R5+ X and B have the same meanings as above). In this reaction, alkoxyaryloxyaluminum AL (OA r )20R is used as an intermediate.
(R represents all nucleoside residues).

When carrying out the reaction, Li At(:N(C
Using Hs )2 )4, At(N(C)I3)2 )3 is obtained by subjecting it to HC1k reaction. still,
The aluminizing agent is 1IAt[N(CH3h h
(OAr)k is formed and TAt[N(CH3)2 )
(OAr)z' is formed, which reacts with the hydroxyl group of the nucleoside. Therefore, as aluminizing agents, ALcN(CHs)232 (OAr) and ArOH
and i:r:x ratio, or At[N(CL
)2) (OAr)2 can also be used alone. The above reaction is usually carried out in a solvent.

Examples of usable solvents include, for example, tetrahydrof 5
:y(THF)IN ether, somethoxyethane, and dichloromethane. Preferably, it is an ether solvent, and more preferably TI(F).

Incidentally, a mixed solvent of these can also be used.

This reaction is usually carried out by adding a nucleoside to an At[N(CH3)2h solution and then adding ArOHk.
The order of these additions is not particularly limited; for example, they may be added at all times. Thereafter, the nucleoside can be phosphorylated by further adding a phosphorylating agent to the reaction mixture without isolating the aluminum alkoxide produced by aluminization.

The aluminizing agent At[N(CH3)213 is
Usually 1.0 to 1 per equivalent of hydroxyl group of nucleoside
．． One equivalent, preferably 1.0 equivalent, is used, and ArOH is usually used in twice this equivalent. The phosphorylating agent is generally used in an amount of 1 to 1.5 equivalents, preferably 1.1 to 1.2 equivalents, per 1 equivalent of the hydroxyl group of the nucleoside.

The reaction temperature is usually 0 to 20°C, preferably 10 to 20°C.
It is. The reaction time is usually 5 to 24 hours, preferably 1
It is 0 to 20 hours.

(II) LiAt1:N(CHa)Z:14
When +ArOHfc is used, this reaction is expressed, for example, by the following formula: (wherein Ar r R5r X and B are as defined above). In this reaction, lithium alkoxytriaryloxydialuminate Li[At(OA
r) Generate sOR:II (R represents all nucleoside residues). Note that the aluminizing agent is once LiAt
[:N(CHs)2)3(OAr) and L
iALcN(CHshlh'(OAr)z', then LiAt[N(CHs)2)(OAr
) sk is formed, which reacts with the hydroxyl group of the creoside. Therefore, as an aluminizing agent, L
iAt[N(CH!I)2]s (OAr) and Ar
OH in a ratio of 1:2 or L i At
[N(CHs) zMOAr, l+arOH and 1=
or LiAt(N(CHs h ))
It is also possible to use (OAr)3 alone. The above reaction is usually carried out in a solvent. Examples of solvents that can be used include tetrahydrofuran (THF), ether, somethoxyethane, and dichloromethane.

Preferably an ether solvent, more preferably T
It is HF. Incidentally, a mixed solvent of these can also be used.

In this reaction, usually LiAt[:N((Jb)z)
This is carried out by adding the nucleoside to the solution and then adding ArOH, but the order of these additions is not particularly limited, and for example, they may all be added at the same time. Thereafter, the nucleoside can be phosphorylated by further adding a phosphorylating agent to the reaction mixture without isolating the aluminum alkoxide produced by aluminization.

LiAtCN(CH3)2]4 which is an aluminizing agent
is usually 1.0 per equivalent of hydroxyl group of nucleoside.
~1.1 equivalents, preferably 1.0 equivalents are used, ArO
H is usually used in an amount equivalent to three times this amount. The amount of the phosphorylating agent is usually 1.0 to 1.0 to 1 equivalent of the hydroxyl group of the nucleoside.
1.5 equivalents, preferably 1.1 to 1.2 equivalents are used.

The reaction temperature is usually -20 to 60°C, preferably -20 to 60°C.
It is 0°C. The reaction time is usually 3 to 108 hours, preferably 3 to 10 hours.

After the above reactions (1) and (II) are completed, for example, the target product is extracted from the reaction mixture, dried and concentrated, and the resulting residue is subjected to column chromatography. The objective is obtained.

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

〔Effect of the invention〕

The method for producing a phosphoric acid compound of the present invention has the following advantages.

■ Nucleosides, aluminating agents, and phosphorylating agents may be used in haso-stoichiometric amounts.

This means that thymidyl, which has an acidic proton in its nucleobase,
Also applies to uracil and guanyl nucleosides.

(2) In this reaction, the desired phosphorous triester is obtained without protecting the active amino group of adenyl, cytosyl, or nucleoside.

■ Inexpensive phosphorochloridate, 2,4-sonitrophenyl phosphate, which is generally considered to have low reactivity,
2-chloro-4-nitrophenyl phosphate and the like can also be used as phosphorylating agents.

(2) Post-treatment of the reaction is easy, especially since THFe can be used as a solvent.

As described above, the present invention is not only excellent in terms of ease of experimental operation and selectivity of functional groups in reactions, but also has advantages from an economical standpoint.

The phosphorylation method of the present invention, which has the above-mentioned advantages, can be used, for example, in the fields of ■nucleic acid chemistry, especially genetic engineering, ■organic synthesis chemistry, and ■pesticide chemistry.■Synthesis of oligos or polynucleotides for the purpose of genetic manipulation. , organic synthesis reactions using O organometallic complexes, synthesis of phosphoric acid compounds as ligands for catalysts in immediate catalytic reactions, and (1) useful as a means of synthesizing effective agricultural chemicals containing phosphorus.

[Examples of the Invention] A. Method Synthesis Example 1 via Alkoxydiarylokidialuminum Preparation (Procedure A) Unless otherwise specified, alkoxydiaryloki7aluminum was obtained from a nucleoside and phenol by the following production method. Lithium tetrakis (trimethylamino)
Aluminate (0,22, mmol) in THF (0,
3 ml) 0.74 M solution ThTHF diluted with approx. Hydrogen chloride (0.22 m mo
l) DMEo,09-solution (2,40M)'fr:
added. After stirring for 5 minutes, nucleoside (0.22 mmol) in THF2Wd! was added to the resulting brown solution. Solution at 0℃
I added it. After continued stirring at 20-27°C for 15-30 minutes, the reaction mixture was concentrated with a vacuum pump to obtain a residue, which was added with THF 2 tnl and phenol (0,4
4 mmol) of THF U, 4-0.6 ml solution was added. The solution was stirred at 20-27° C. for 1-1.5 hours and concentrated again to give a foam or gum. This T HF 2 tnl solution was used in the next step without purification. Thus, alkoxydiaryloxydialuminum was obtained by adding nucleosides followed by phenol.

Example 1 By procedure A, 5'-0-t-butyltmethylsilyl-
2'-deoxyadenosine (80,8■, 0.22mm
ol) and 0-chlorophenol (56,7!.

0.44 mmol) (!-Alkoxydiaryloxydialuminum was prepared from its THF2-solution di,so-chlorophenylphosphorochlorite)
(89.5Tq, 0.27mmol)
A 4 m suspension of THFl was added at 0°C. 20
After stirring for 4 hours at
, 5mJX2). The combined extracts were washed with 10 ml of 10% brine and then dried. When this was concentrated, 0.189 g of a colorless gum was obtained. Its IHNMR
Based on the spectrum analysis, the yield of the title compound was found to be 67%.

The measurement data is shown below.

Rf (CHsOH-AC'OEt-CHCIs,
1:1:10.

v7v) 0.36; mp 114-115°C;
, IR (CHClg) 3490.3400 (FH2),
and 1290c+y+-"(P'=0); UV(
CHsOH) 260nm (e16700); lHNM
R(CDC1a)δ0.08 (6H,s, 5i(
CHs)z), 0.89(9H,5i-t-Ci
He) +2.87 (2H, m, 2H2z, )t3.
90 (2H, d-: like + J=3.1Hz + 2H
5/l) + 4.43 (IH, m, 7H4/),
5.55 (IH, m, Hsz), 5.87 (21
-1, br 8 , FH2), 6.52 (IH, uniform (, J=7.0 Hz, H4,), 7.05
-7.57 (8H, m, 2C6H4C1).

8.11(1)1+s, Hz) t8.34(FH
, s + Hs ): 13CNMR (CDC13) δ-6
,0,-5,9(St(CH3)2).

17.8 (SiC(CHa)3), 25.4 (Si
C(CH3)3).

39.2 (d, J-"4Hz,,C2,), 62.
7 (Cs, ), 80.6 (d, J==4Hz 103'
), 83.8 (C1t), 85.5 (d.

J=7Hz, C4/), 119.4(Cs), 121.
9(d,,X,=2Hz,C6H4C1), 125.
0 (d, J=7Hz +' C6H4C1).

126.2.127.6.130.3 (C6H4C1
), 1'38.0 (Cs).

145.8 (d, J=7Hz, C6H4C1), 1
49.1 (C4).

152.6 (C2), 155.6 (Cs).

Actual value: C, 50, 22; H, 5, 15; N, 10.5
4%.

Calculated value: (CzsH34NsOsSfPCh)C, 5
0,45:H.

5.14:N, 10.51%.

Example 2 5'-0-t-butyldimethylsilyl-
2'-deoxyadenodine (68,5, 0.20m
mol) and 0-chlorophenol (51,4■.

0.40 mmol) was prepared from alkoxy7soaryluoxinealuminum. A suspension of the THFI; 5-dissolved f51c no-o-chlorophenylphosphorochloridate (8), Oq, 0.24 mmol) in 0.25 m JIMi of anhydrous dichloromethane was added at 0°C.

After stirring at 0°C for 15 hours and then at 27°C for 14 hours, the same treatment as in Example 1 (!) was carried out. This was purified by silica gel column chromatography to obtain 79.1+wg (79.1+wg) of the desired product as a colorless rubber. 62%).

Bf (CH30HCHC13, -1: 5, V'
/V)0.60:mp 131℃;IR(CHCI
s) 3560.3420(N&) and 1385cr
n-1 (P=O): UV (CHsOH) 272.2°C
m (g8880): 'HNMR (CDCIg) δ0.0
6 (6H.

s+ Si(CHa)2)IO-87(9
H+ s t Sit C4Hg).

1.90-2.38.2.56-2.88 (2H, ea
ch m, 2H2Q+3.16 (2H, m, 2H5
'), 4.33 (IH, m, H4'), 5.3
5 (IH, m, H3/), 5.80 (IH, d
, J = 7.5 Hz + Hs) + 6.37 (IH, uniform, J = 7.5 I(Z l )ill ) ,
7.00-7.50 (IOH, m, NH2' 2C
8H4C1), 7.77 (IH.

d, J=7.5 Hz, Ha); C
NMR (CDC13, CDCl5=76.9) δ-5,
8, -5, 7 (S i (C city) 2), 18-0 (Si
C(CJL+)3), 25.6(5iC(C
H3) 3), 4t), 0 (d.

J=3Hz, C2'), 63.0 (C5') +80.
8(d, J=7Hz.

C4'), 85.4 (d, J=4Hz, C3'), 85
．． 8 (Cl.

94.7 (Cs), 121.3 (d, J=2Hz,
C6H4C1).

125.3 (d, J=8Hz, CsH4C1), 126
．． 3,127.8°130.6 (C6H4C1), 14
0.2 (C6), 146.0 (d.

J=7H3,C6H4C1), 155.5(C2), 1
65.7 (C4).

Actual value: C, 50,25; H, 5,35; N, 6.42
%.

Calculated value; (Cz7Hs4C1sNsChPSi)C
, 5U, 47:H.

5.33:N, 6.54%.

Example 3 By procedure A, N3-methyl-2',3'-0-ingropylidene uriso>(5'9.8 ml, 0.2
0 m 'mol) and 0-chlorophenol (51,4
(2)+0.40 mmol) and alkoxysoaryloxoaluminium was prepared. That TH, F2.
(41,4
■, 0.24 mmol) was added at 0°C. After stirring at 0° C. for 4 hours, the reaction mixture was poured into a 0.5 M aqueous sodium hydroxide solution and extracted with dichloromethane (20 ix 3).

Combined organic layer 'kO', 5M aqueous sodium hydroxide solution 5rn1. and then with 5 m of 10% saline.

After drying and concentrating, 100 μg of a rubber-like product was obtained, which contained 94% (!·HN MR analysis) of the title compound.

Rf(CHsOHC'HCl511:10.v/V)U
, 33;”HNMR (CDCIg) δ1.35
(9H, m, 2P (JCH2CH3'CCHs)
, 1.58 (3H,s, CCHs),
3.95-4.22 (7H, m, 2POC Danz CHs
+ Hzt, H3/ + H4/)
+4.88 (2H, m, '1H5t), 5.7
0(IH, d, J=7.5H7+, H5') + 5
−78 (I HHd + , r==i−2H7I H
tl) +7.38 (LH,'d, J=7.5Hz,
Ha), 9.52 (II-1,, brs, Nu).

Example 4 Lithium tetrakis(dimethylamino)aluminate (
0.20 mmol) of THE'0.25-solution (0
, 81M) was diluted with THFlrnt. To this, 0-chlorophenol (51,4'q, 0.4 (l
A solution of 40 +++ l of THFo (mmol) was evaporated at 0°C. After stirring for 0.5 h at 20°C, the reaction mixture was heated to ca.
It was concentrated to 5 ml and then diluted with THF], 5-ml. This solution was passed through a cross-linked stainless steel tube at 0°C for 2 hours.
',3'-0-isopropylidenecytidine hydrochloride (63
, 8 ■.

0.199 mylol) into a suspension. 20
After stirring for 1.2 hours at °C, the reaction mixture was evaporated to dryness. To the obtained residue, THF], 5M and soethyl phosphorochloridate (41.4W, (J, 24 mmol)
) was added at -40°C. After stirring at -40 to 0°C for 20 hours, 1 1/2 0.5M aqueous sodium hydroxide solution was added to the reaction mixture.
The reaction was completely stopped by adding dichloromethane (20M
, 10+++A'X2). The combined extract layers were washed with 10% edible water (5 m/x2), dried and concentrated to obtain a pale yellow foam. Washing with ether gave the pure target product (56.8 cm, 74%).

Bf (CHsOHCHCIg, 1:10,
v/v, bidirectional expansion 0.21, HNMR (CI)
C13) δ1.10-1.14 (9H, m, CCHs
, 2POCH2CH3), 1.52 (3H.

s, CCHs), 3.90-4.44 (7H, m, '2
POC & CHa.

H2/ , H3/ , H4t )+5.02 (:
? Hr dd + J-7,219,2Hz 2H5
/ ), 5.46 (iH,br 5IHI/ ), 6
．． 00 (IH+d, J=7.2Hz, Hs), 7. ．． 2
9 (IH, d, J=7.2Hz).

8.30 (2H,brs,NHz),C
NMR (CDCIg) δ16.1 (d, J=
7H2, POCH2CH3), 25.3.27.1 (C
(CHs)z), 64.1 (d, J=7Hz
, POCH2CH3).

67.3 (d, J=5', 5 Hz
, Cst), 82.2(03)),
84.9 (C2/), 86.7. (d, J=8Hz
lc4/ ), 95.7 (C1/) 197.6 (C
s),], 13.7(C(CHs)2), 143.9(
Cs).

155.6 (Cz), 166.7 (C4).

Regarding Examples 1 to 4 above, the reaction conditions, yields, etc. are collectively listed in Table 1. In the table, the number of nucleoside (B) represents the following compound.

~ 4 (TBDMS and B have the same meanings as above) In addition, (Ura) in Example 3 refers to ocide of the following formula 20.

Examples 5 to 8 In the same manner as in the above examples, the effects of ArO groups were investigated in the case where various alkoxydiaryloxydialuminum gold pieces were used. The reaction conditions, yield, etc. are listed in Table 2, and Example 3 is also listed. The nucleosides used in these Examples are all compounds represented by the following formula:

B, Lithium alkoxytriaryloxyal Synthesis Example 2 % K Unless otherwise noted, 1 equivalent of nucleoside and 3
Lithium J aluminate consisting of an equivalent amount of phenol was obtained as follows. Lithium tetrakis(trimethylamino)alumina) (0.20 mmo) was placed in a 30 mm flask equipped with a three-way cock and filled with argon gas
l) THFo, 25M solution-Q (0.81M)
I put it in. Nucleosides (0.20 mmol) were added to this solution in THF3 under argon gas pressure through a cross-linked stainless steel tube in a container equipped with a rubber cap.
．． The O-solution was added dropwise. After stirring for 10 minutes at 0°C, the resulting suspension was evaporated to dryness using a full vacuum pump. To the residue, THF2rn1. and phenol (0,60m
m01) in THFo, 5-0.6 m solution was added at 'tO<0>C. Obtained solution'fr: 1-2FI at 20-25℃
Stirred for # and concentrated again to give a foam, which was dissolved in THF2-. T)I of this aluminate
The F bath solution was used in the next step without purification.

Example 9 To investigate the effect of the functional group ArO in lithium aluminate, an experiment was conducted according to the general procedure described below.

By operation B, 5'-0-t-butyltmethylsilyl-
2'-deoxyadenosine (0.20 mmol) and p-
Nitro7 x ) -le (0,50 mmol) THF2m7 of lithium aluminate obtained from TOKA! In the solution, so-0-chlorophenylphosphorochloroliso-) (0,2
A solution of 2 mmol) of anhydrous dichloromethane was added at 0°C. The reaction progress was followed by TLC. 27℃
After stirring for several hours, the reaction mixture was poured into a mixture of 20 tons of 0.5M aqueous sodium hydroxide and 10 tons of dichloromethane. Dichloromethane (15 m x 2.5 m
6×2). Add 1 part of the combined organic layer to saturated saline solution.
After washing at 0 m, drying and concentrating yielded a rubbery substance. The yield of the target product was determined by total IHNMR analysis (82%). A rubber-like target product was obtained by silica gel column chromatography using methanol-4:acid-ethyl-chloroform (1:1:20°V/V).

Example 10 By procedure B, 5'-0-t-butyltmethylsilyl-
2'-deoxythymidine (80,8~, 0.22mm
oL) and p-nitrophenol (92.2 mg.

0.66 mmol) and karitium alumina were prepared. So0-chlorophenylphosphorochloro1Jf-) (99.9 towns) was added to the THF 2m solution.

0.30 mmol) of THFo, 88m solution was
Added dropwise at °C. After stirring for 10 h at 0 °C, the reaction mixture was diluted with dichloromethane 20- and then diluted with 10%
The reaction was stopped by adding saline. This was then thoroughly shaken, and the resulting emulsion was centrifuged (2000 cprn x 5
After separation (min), the layers were separated, and the aqueous layer was extracted with dichloromethane (20 rnl x 2.10 mt x 2). The combined extract layers were dried and concentrated to yield 0.299 yellow combs. The yield of the target product was 88% according to 1H NMR analysis.

town (acetone CHzC12H1: 61 v/v)
0-67 *mp95-96℃; IR (CHCIg)
3390(CONH)and1290crn(P-0
); UV (CHsOH) 266.4℃m (ε10800
), HNMR (CDCIg) δ0.08 (6H.

s y Si (CH3) 2 ) s O-8,8
(9H+ s t S s t C4H9) +1
．． 90 (3H, d, J=0.3 Hz, CH
sC=), 2.00-2-70 (2H* m r
Z H2t ) + 3.86 (2HHbr s
12 Hs l) +4-32 (IH, m, Ha')
,5.34(]H+m+[3')16.36(IH,d
d, J=4.2, 7.5 Hz
, Hl).

7.08-7.60 (9H, m, us, 2C11H
4C1), 9.56 (IH.

brs, Nu); CNMR (CDCIs, CDCl
5=76.9)a -5,7,-5,6(St (CH
s)2), 12.3(CHsC=).

18.1(SiC(CH3)+), 25.7(SiC(
CH3)3), 39.3(d, J=4I(z, Cz/)
, 63.1 (Csz), 80.8 (d, J=7 Hz'
+ Cs l) + 84-3' (Cs l) +
85-3 (d + J-6Hz +C4/), 1
10.9 (Cs), 121.4 (d, J=2Hz.

Cr, HiCl), 125.3 (d, J=9
FLz, C6H4C1), 126.4°127
．． 9. 130.6 (C6H4C1), 134.6 (C
s).

146.0 (d, J=7Hz, C6H4C1), 150
．． 2 (Cz).

163.6 (C4).

Actual value: C, 5o, 97 SR, 5, 26; N, 4.2
5%.

Calculated value: (CzsHasClxNzOsPSi)C
, 51, 14:H.

5.37:N, 4.26%.

Example 11 By procedure B, 5'-0-t-butyltmethylzolyl-
2'-deoxycytidine (75,2■, 0.22mm
ol) and p-nidraphenol (92,211Iy
.

Lithium aluminate was prepared from 0.66 mmol). Add so-0-chlorophenylphosphorochloroliso-h (77,6+oy,
0.23 mmol) of TI-IFl, 25-resistant solution was added dropwise at 0°C. After stirring at 20°C for 6 hours,
The reaction mixture was poured into 0.5M aqueous sodium hydroxide solution 20- and extracted with dichloromethane (20PX2.5mX2). The combined organic layers were washed with 10 ml of lθ% brine, dried and concentrated to obtain a gum-like substance.

The yield was found to be 73% from its HNMR analysis.
+ v/v) k silica gel column chromatography (5 ml, U, 9 x 13 crn) yielded the desired product (97.7 mq, 6
8.9%).

Bf (CH30HCHCl5, 1:5, v/
v) 0.60:mp131℃:IR(CHCIg)
3560.3420(NHZ) and 1385m
(P-0);UVICHsOH)272.2℃m(ε
8880) ; “HNMR (CDCIs) δ0
．． 06 (6H, town.

8i (CHI)2), 0.87 (9H+s
*5t-t-C4H9) yl, 90-2.38
, 2゜56-2.88 (2H, each m,
2] E1m/) 13.16 (2H, m, 2H++z),
4.33 (IH, m, H4z) + 5-35 (I H+
rn + HsI) + 5-80 (II(+d
+ J=7.5 nz.

Hs), 6.37 (II(, uniformly, J
= 7.5 Hz, Ml/) 17.00-7.5
0 (101(, m, NH2,2CsH4CI), 7.
773 (IH, d, J=7.5Hz, Ha); CNMR (
CDCIs.

CDC1g=76. 'l) δ-5,8,-5,7(
Si(CHs)z).

18.0(SiC(CHs)s), 25.6(SiC(
CHsh).

40.0 (d, J=3Hz, Cz/), 63.0 (Cs
l), 80.8 (d, J=7Hz, C4/) 1
85.4 (d I J = 4Hz lc3/) 185.8
(C1/), 94.7 (Cs ), 12], 3 (d
, J=2Hz.

CsH4C1), 125.3(d', J=8Hz, Cs
H4C1).

126.3. 127.8. 130.6 (0gH4
C1), 40.2 (Cg), 146.0 (d, J=
7Hz, CaH4C1), 155.5 (C2), 165
．． 7 (04).

Actual value: C, 50, 25: H, 5, 35: N, 6.42
%.

Calculated value: (CaH4C1 5.33: N, 6.54%. .

Example 12 5'-0-t-gtyltsmethylsilyl-2'-deoxyguanosine was dehydrated by azeotropic distillation using pyridine (0.7 m/3 times). By operation B, this 5'-0
-t-butyldimethylsilyl-2'-deoxyguanosine (84.6W, 0.22 mmol) and p-
Nitrophenol (92.6■, 6.67mmol)
Lithium alumina was obtained from The THF2 m
l solution 1c, so-0-chlorophenylphosphorochlori f -) (99,91119, 0,30mm
The THFO088-solution was added dropwise at kO<0>C. After stirring for 6 hours at 0°C, followed by 11 hours at 20°C, the reaction mixture was diluted with 20 m of dichloromethane and then quenched by the addition of 15 m6 of 10qb brine. After shaking this mixture, the resulting emulsion was separated into layers by centrifugation (2000 cpm x 5 min).
separated. The aqueous layer was dichloromethane (20dX2 + 1
0 ml x 2). The combined organic layers were dried and concentrated to obtain a residue (0.32F).・
Analysis of this by K''H NMR revealed that the target product had been produced in a yield of 69%.The crude product was mixed with acetone-dichloromethane (1:12.v/v).

followed by methanol-chloroform (3:40
A colorless solid was obtained by subjecting it to silica gel column chromatography (4, Of, 0.9X11 crn) using a silica gel (90.7 mm, 60%).
).

(CHCl3)3300.3500(NH2), 129
0crn (P=0); 1HNMR (CDC13)
δ0.07 (6H, s.

5i(CHs)2), 0.89(9H,s, 5i-t
C4H9)+2-75 (2H+ m + 2 H2
t) + 3.87 (2H+m+2Hs
l) +4.37 (IHlm, H4/), 5.50 (I
Hlm, H3/) 16.30 (IH, uniform r J ”
7, OH7+ Ht t ) + 6-50 (2
H+br S , NH2) 17.03-7.53 (8
H+m, 2°C6H4C1)7.80 (IH+8
+H8) 412.03 (in, br s,
NH).

Example 13 By procedure B, 2′,3′-0-isoglopylideneadeno,cy (61,5q”, 0.20 mmol)
and 0-chlorophenol (77,1■+, (7,60m
T of lithium aluminate obtained from
Diethyl phosphorochloridate (35 ttL, 0.24 mmol) k-3 in 51 nl solution of HFl
It was 0 degrees Celsius.

After stirring for 3 hours at −20° C., the reaction mixture was diluted with 20 μl of dichloromethane and then washed with 201 rlt of 0.5′M aqueous sodium hydroxide solution. Drain the aqueous layer in dichloromethane (2
0m/, 5m7! X2), and then the combined extracted layers were washed with 5M hydroxide solution and brine, dried, and concentrated to give colorless crystals (
86.1■, 97%).

R-L (Mean-AcOgt, 1:33)
0.19:mp 139-141℃:IR(CHCI
g) 3490.3400 (IH2) and 1290m
(P=0): UV (CH30H) 259.2Hm (
g17300):1HNMR(CDCa)δ
1.28 (6H, m, 2P'0CH2CH3),
1.40, 1.63 (6H.

each s, C(CH3)2) + 3.7-
4.6 (7H, m, 2POCH2CH8, kht
, ■3/ H4/ ), 5.10(II(, dd
.

J=3.6, 7.5 Hz, Hsz), 5.40(
IH, dd, J-”3.0, 7.5 Hz, H5
/), 5.68 (2H, br 8tNH2) 16.14
(IH, d, J=2.5 Hz, Hlt),
7.94 (IH18, H2), 8.34 (LH+8
1H8).

Actual measurements: C, 15,76; H, 5,82; N, 15.
76%.

Calculated value: (C17H26N507P)C, 46,05
;H+ 5.91:N, 15.80%.

Example 14 By operation B, 2',3'-0-inglobylidene
Lysine (56.9■, 0.20 mmol
) and 〇-ri olov x/-ru (77, IQ', 0.6
A total of lithium aluminate was prepared from 0 mn1o1). Diethylphosphorochloridate (41.4w, 0.24 mmol) was added to the THFl, 5-solution.
) 2L20℃T Addition L f,=.

After stirring at -20°C for 6 hours, the reaction mixture was added with 10% brine.
5- was added to stop the reaction. The aqueous layer was extracted with dichloromethane (201 nl, 10 m×2).

After drying and concentrating the combined organic layers, a rubber-like substance was obtained.
Lahta. The crude product was dissolved in acetone-chloromethane (4:
By subjecting it to silica gel column chromatography (5f), the desired product was obtained as a colorless foam (73.I Tng.

87%).

R((CHsOHCHCl5.1 :10.v/v)O
-33; 1HNM, R (CDC13) δ1. '35 (9
H, m, 2POCHxCHs.

CCH3), 1.58 (,3H,S, CCH
3), 3.95-4.22 (7H, m, 2POCH
2CH31H2/lH3/, H4/)+4-88(2H
, m, 2Hsy), 5.70 (II-Ld, J=7.
5 nZlus), 5.78 (IH, d, J-”],
, 2 Hz, )ltl), 7.38(IH,d, J=
7.5 Hz r Ha), 9.52 (IH+ br
81NH).

Example 15 By procedure B, N3-methyl-2',3'-0-isozolobylipidoneuridine (60.0, 0.20
mmol) and O-chlorophenol (77,1q
.

Lithium aluminate was prepared from 0.60 mmol) (!:).To the 1.5 ml THF solution was added diethyl phosphorochloridate (51,3q, 0.297
mmol) was added at 20°C. After stirring at −20° C. for 3 hours, 5 ml of a 0.5 M aqueous solution of IJ hydroxide was poured into the reaction mixture to stop the reaction. The aqueous layer was extracted with dichloromethane (20mAX2.10n/). The combined extract layers were washed with brine and then dried. This was concentrated to give a foam (146η). Preparative TLC using ethyl acetate gave the pure target product (81-93%).

R((CH30H-CHC13, 1: 19, v/
v, two-way dust 8) 0.44: ”HNMR(C
DCIs) δ1.35°1.37 (9H,s absorption and t, J=6.5Hz+CCH3,2
POCH2CH3), 1.60 (3H,s, CCH3)
.

3.32 (3H, s, NCHa), 3.92-4.44
(7H, m.

2 POCH2CH3, H2/, Hs l,
H41), 4.84 (2H+ m.

2 Hz), 5.78 (IH, d, J=8.] Hz
, Hs), 5.79 (IH, d, J=1.2Hz,
Hlt), 7.36 (IH, d, J = 8.1 Hz,
Ha).

Regarding the above Examples 9 to 15, the reaction conditions, yields, etc. are listed in Table 3. The number of nucleoside (B) in the table has the same meaning as above for the male. In addition, in Example 15 (Ura
) have the same meaning as above.

Examples 16 to 24 In the same manner as in the above examples, the effects of ArO groups when used in various lithium aluminates were investigated. The reaction conditions, yield, etc. are listed in Table 4, and Example 9 is also listed. The nucleosides used in these Examples are all compounds represented by the following formula: and the phosphorylating agent used is di-0-chlorophenylphosphorochloridate.

Examples 25 to 30 In the same manner as in the above examples, various mononotium aluminates were used to investigate their reactivity. The reaction conditions, yield, etc. are listed in Table 51, and the fruit (Example 15) is also listed.The nucleosides used in these Examples are all compounds represented by the following formula: It is.

By synthesis operation B of Reference Example, 5'-0-t-glythyl-2'-deoxyadenosine (73,IN, 0.20 mmol
) and 2-chloro-4-nitrophenol (104,1■
.

ㅅ(2-chloro-4-nitrophenyl)phenylphosphate 17) THF (2
m7ri IP! Add the suspension at 0°C and incubate at 0°C to 10°C for 4 hours.
．． Stirred for 5 hours. This reaction mixture was prepared in Operation B (from 3
'-0-t-butyl-2'-deoxyadenosine (58
,5m? , 0.16 mmol) and 2-chloro-4-ditrophenol (90.31 ng, 0.52
lithium aluminate obtained from THF (
2mg) solution at 0°C, and stirred at 20°C for 36 hours. The reaction mixture was diluted with dichloromethane (2 oy) and poured into a 0.5 M aqueous solution of IJ hydroxide (15 ml) for extraction. The aqueous layer was dichloromethane (20ml!

After washing at 10 m/×2), the combined conditioned layer was washed at 10 m/×2
Washed with brine (10 m/liter), dried over anhydrous magnesium sulfate, and concentrated to give a pale yellow foam.

With this high-speed liquid flow, internal aUS
Quantification was performed using 2,4-dut-butylphenol as a substance. The target product is a mixture of soastereomers (5:6)
It was found that the product was produced at a yield of 30%.

HPLC (cosmosil) ODS-5
μmlλ=260nm, flow rate== 1 m1. m1n
-”, pressure = 1.27X107Pa, solvent = H2O
-CH30H, 1:4.7, v/v).

Ac0Et CHCl + 1: 1 * 10
r v/v) 0.21': IR(CHCL
3) 3500, 3410 (NHZ) and 1280m
”(P=0): UV (CHaOH) 260nm (g
26000): "HNMR (CD Cl g ) δU,
06, 0.09 (12H, two S.

2Si (CH3)2 ), 0,87.0.90 (1
8H, two s' s.

2Si-t-C4H9), 2.26-2.98 (
4H, m, 4H2/ ).

3.82 (2H, m, 2Hs10Si) 4.02-4
．． 48 (4H.

m) 2H4/ + 2H570P) H4,65
(L H+ 111 r H310SI) +5.2
2 (IH, m, HsloP), 6.20 6.56 (6
H, m.

2H1/, 2NH2L7.12 7.38 (5H, m+
csHs)+7.97,8.08(2H, two s'
s + 2 H2) + 8.27 (2Hrs, 2Hs
); FDMS (m/z) 868 (M). Other isomers: 1
:10. v/v) 0.22: IR (CHCL3) 350
0.

3410(NH2)and1290crrI (P-
0); UV (CHaOH) 260 nm (t 27
000), HNMR (CDC1a) δ0.04,
0.11(12H, two s, 25i(CH3)2
).

0.84, 0.91 (18H, two s, 2
S 1-t-C4H9) 12.28-3.02 (4
H,'m,4H2/),'3.74 (21t(,
m'.

2HszO8i), 4.18 (2H,m, 2Hst
OP), 4.39 (2H.

m, 2H4/), 4.68 (IH, m+HstO8i
), 5.22 (IH.

m, HslOP), 6.18 (4H, br S,
2NH2), 6.41 (2H,, m, 2H1/ )+
7.1.2-7.44 (5H+ m, C6esar7
．． 98.8.04 (2I (, two s, 2H2),
8.26 (2H.

+s, 2H++); FDMS (m/z)868 (
M).

Claims (3)

[Claims] (1) A phosphoric acid compound characterized in that an aluminizing agent is allowed to act on a hydroxyl group-containing compound, and then a phosphorylating agent is allowed to act on the obtained aluminum alkoxide, thereby oxidizing the hydroxyl group (, IJ) of the hydroxyl group-containing compound. Production method. (2) The aluminizing agent is a compound represented by the following formula: (% formula %) (wherein R1 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms) and the following formula: (wherein, 2. The method for producing a phosphoric acid compound according to claim 1, wherein Ar represents a substituted or unsubstituted aryl group. (3) The aluminizing agent is a compound represented by the following formula: (% formula %)) (wherein, R2 represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms) and the following formula: (wherein, Ar is Claim 1jR is a combination of compounds represented by (representing a substituted or unsubstituted aryl group)
A method for producing a phosphoric acid compound according to item 1. f41 Claim 1 in which the hydroxyl group-containing compound is a nucleoside or a derivative thereof, and the reaction is carried out in a solvent.
A method for producing a phosphoric acid compound as described in .