Note: Descriptions are shown in the official language in which they were submitted.
~ 35
NUCLEOTIDE COMPOUND PREPARATION
A~D METHOD FOR PRODUCING THE SAME
BACKGROUND OF_TH~ INVENTION
This invention relates to a protected nucleotide
which is freeze-dried and a method for producing the
same. The protected nucleotide according -to this
invention is particularly suitable for use as the unit
fraction or "monomer" to be condensed in the synthesis
of an oligonucleotide according to the phosphotriéster
method (developed by us as disclosed in Nucleic Acids
Research, 8, 5507, 1980, and others).
An oligonucleotide, comprising a relatively small
number, for example, of about 4 to 30 of nucleotide
units bonded to each other to form a nucleotide chain
or strand is a constituent of the molecular chain of a
nucleic acid such as deoxyribonucleic acid (herein-
af~er called as DNA) or ribonucleic acid (hereinafter
called as RNA).
Since nucleic acids bear genetic information of
organisms, oligonucleotides forming the molecular
chains of nucleic acids constitute genes. As progress
in recombinant gene technique, various methods have
already been proposed for synthesis of oligonucleotides
-to be utilized therefor.
One of the methods ensuring production of an
oligonucleotide of a predetermined nucleotide sequence
- ~
--1--
~fæ~73~
is the solid phase synthesis according to the phos-
photriester method we have developed. This method is
disclosed in the references set forth below.
Tetrahedron Letters, 1979, 3636 (19791
Nucleic Acids Research, 8, 5473 (1980)
Nueleic Acids Research, 8, 5491 (1980)
Nucleic Acids Research, 8, 5507 (1980)
Nucleic Acids Research Symposium Series t
7, 281 (1980)
J. Am. Chem. Soc., 103 r 706 (1981)
Nueleic Aeids Researeh, 10, 197 (1981)
Aeeording to this method, a polymer support sueh as
polystyrene is modified to have a funetional group,
and this polymer is eaused to react with a protected
nueleoside to make the nueleoside bonded polymer
(hereinafter ealled the resin). Subsequently, the
proteeting group is removed from the resin (ordinarily
the proteeting group being for the terminal 5'~hydroxyl
group), and the resultant resin is eaused to reaet
with a proteeted nueleotide block (ordinarily the
terminal 3'-phosphate group is phosphodiester tri-
ethylammonium form), whereby this nueleotide bloek
is bonded to the 5l-hydroxyl group on the resin,
thus joining the nueleotide ehain. By repeating
a series of these operations, the oligonucleotide
with desired length sequenee ean be assembled.
In the final step or a step near the final step, the
oligonucleo-tide is isolated by cleavage from the resin.
The salient feature of this method resides in that
semi-automatic operations are possible.
Similarly as in the case of DNA synthesis in gene-
ral, also in the solid phase synthetic method, the mostimportant point to be borne in mind for obtaining the
desired product in a high yield is-that the condensa-
tion reaction must be carried out under absolutely
anhydrous conditions. This is because bonding between
the nucleotides oecurs through dehydrating condensa-
tion reaetion between phosphorie acid residues and
hydroxyl groups.
Anhydrous conditions during condensation reaetion
have been obtained in the prior art by, for example,
subjecting a mixture of a protected nucleotide bloek
and a resin to azeotropic distillation with pyridine.
As a result of a report [H. Ito, Y. Ike, S. Ikuta,
K. Itakura; Nucleic Aeids Researeh, 10, 1755, (1982)]
and our investigations, drying of a resin can be
aeeomplished b~; washing the resin with a volatile
solvent (e.g , anhydrous tetrahydrofuran) and sub-
sequently exposing the same to a stream of a gas
such as dry nitrogen.
However, as for the protected nucleotide bloek
to be condensed, the fully protected nucleo-tide
block (the compound [II] as hereinafter described)
--3--
1;21l473~;
must be partially deprotected when necessary to
convert the 3'-terminal phosphodiester compound,
and moreover the diester compound (the compound [I]
as hereinafter described) must be rendered anhy-
drous by repeated co-evaporation with pyridine.
Therefore, the phosphodiester compound [I] must
be prepared in situ. Further, even when the
phos?hodiester salt compound [I] is prepared
beforehand (usually stored as powder formed by
dropwise addition thereof into pentane), if it is
used directly as it is, yield will be poor.
Thus, for obtaining a high yield, it must be
rendered anhydrous at the time of necessity by
way of azeotropic distillation with pyridine or
by another method.
The operation to make anhydrous the phos-
phodiester compound [I] at the time of necessity
has been one of the most troublesome operations
and has been considered difficult to practice
mechanically in the synthesis of DNA. This has
also been one of the causes of failure to obtain
stable high yield in the preparation of an
oligonucleotide.
SUMMARY OF THE INVENTION
We have made extensive studies and consequently
sucseeded in freeze-drying a phosphodiester compound [I] by
L4~3~
-the use of a suitable solvent. It was founcl -that
this compound can be stored Eor a long period as it is,
and can provide a stable high yield, even if it is used
directly for the condensation reaction. This inven-
tion is based on such discoveries.
More specifically, the present invention
relates to a freeze-dried preparation of a protected
nucleotide compound represented by the following
formula [I].
B'
~ ~R O
t 1 LI]
wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-
phate group;
R2 is a chemieal protecting ~roup for hydroxyl
group;
B' is a protected base selected fro~ guanine,
adenine, cytosine, uracil and thy-
mine;
A is a lower alkylamine; and
n is a natural number.
The method for preparing the freeze-dried prepa-
ration of the proteeted nucleotide eompound aecording
to the present invention comprises dissolving a powder form
--5--
7~3~
of a protectecl nucleotide compound represented by the
formula [I] shown below together with pyridine
in dioxane and subjecting the solution to Ereeze-
drying.
B'
~ ~R O
R2 t ~ - P - ~ O~-A [I]
wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-
phate group;
R2 is a chemical protecting group for hydroxyl
group;
B' is a protected base selected from guanine,
adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and
_ is a natural number.
Another method for preparing the freeze-dried
preparation of the protected nucleotide compound
according to the present invention comprises dis-
solving an oily form of a protected nucleotide
represented by the formula [I] shown below in dioxane
and subjecting the solution to freeze-drying.
lZ~ 35
~ ~-R O ~
R2 t ~ - P ~ OH-~ [I]
wherein: R is hydrogen or a protected hydroxyl group;
Rl is a chemical protecting group for phos-
phate group;
R2 is a chemical protecting group for hydroxyl
group;
L0 Bi is a protected base selected from guanine,
adenine, cytosine, uracil and thymine;
A is a lower alkylamine; and
n is a natural number.
In the above formula [I], when n is 2 or greater,
B', R and Rl in plural numbers may be either identical
or different.
The freeze-dried preparation according to the
present invention does not exist as a powder
of dense particles or mass but in the form typically
seen in freeze-dried products, namely, light and
1uffy ~owder with good solubility. This pre-
paration can be stored or transported in this state
by exercising only ordinary care to remove humidity
and to avoid contamination. The preparation can also
readily dissolved in solvents conventionally used in
the triester method such as pyridine.
~2~735
In the phosphotriester method of the prior art,
the nucleotide reagent (the formula [I]) has been
made anhydrous by azeotropic distillation with pyri-
dine immediately before use for condensation reaction.
This operation in actual practice is unexpectedly
complicated, and it has been practically difficult to
perform concurrently syntheses of a number of oligo-
nucleotides, as mentioned above. With the use of the
- reagent freeze-dried according to the present invention,
azeotropic distillation operation which has been
conducted in parallel with the condensation reaction
is no longer necessary, whereby it becomes possible
to carry out a number of condensation reactions at the
same time. Low yield due to erroneous opera-
tions in azeotropic distillation was also found to beavoided to give a stable yield. The yield per one
operation was also found to be equal to or higher than
that in the case of the azeotropic dlstillation
operation. As a result of affording such stable high
yield and simplification of the operations, it has
also become possible to reduce the reaction scale to
a great extent. As another advantage, mechanical
operation of the step of the condensation reaction,
which has been considered to be difficult by mechanical
operation, also becomes possible.
As a product similar to the preparation of the
present invention, there is the preparation disclosed
in Japanese Laid-Open Pa-tent Publication No.150700/
1982. However, the freeze-dried preparatlon describ-
ed in this Publication is that of the nucleotide
compound to be used ~or the phosphite method
[M. D. Matteucci, M. H. Caruthers : J. Am.
Chem. Soc., 103, 3185 (1981)]. The freeze-dried
preparation of the present invention is of course
different as a compound from that of this prior in-
vention. Furthermore, it can be stated that it could
not be anticipated from this prior art that a freeze-
dried specimen can be prepared by the present invention
in the form of a l:l salt of a nucleotide and a lower
alkylamine and also that it has become possible, due
to the fact that the salt is one with a lower alkyl-
amine, that is, that a lower alkylamine has a boiling
point of about 80C or lower under atmospheric pressure,
whereby excessive amount thereof can be easily removed
in the pyridine azeotropi~ distillation step or in
the freeze-drying step to prepare a l:l salt.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings:
FIG. l and FIG. 4 show chromatographs on Sephadex
G-50 column;
FIGS. 2 and 5 show HPLC patterns of oligonucleo-
tides having dimethoxytrityl group; and
FIGS, 3 and 6 show HPLC patterns of the desired
~2~73S
oligonucleotides.
Column~ Bondapak C 18 (Waters)
- Eluant: acetonitrile -0.02M EDAA buffer (pH 7.8)
: Concentration gradient: as shown in the dxawings
Flow rate: 2 ml/min.
Chart speed: 10 mm/minO
Temperature: 50C
DETAILED DESCRIPTION OF THE INVENTION
Nucleotide compound [I]
The nucleotide compound as the freeze-dried pre-
paration according to the present invention is shown
by the foregoing formula [I~.
In the above formula, the symbol ~ is conven-
tionally used for showing a residue of the rebose
portion of a nucleotide from which 2'-, 3'- and 5'-
hydroxyl groups and the base are removed. More speci-
fically, it has the followiny structure:
~
When R is hydrogen in the formula [I], this
ribose unit is 2'-deoxyribosyl, and the compound of
the formula [I] forms a DNA chain. When R is a
hydroxyl group or a protected hydroxyl group, this
ribose unit is ribosyl, and the compound of the
--10--
.:
lZl 4735
formula [I] forms a RNA chain. When the degree of
polymerization n is 2 or higher, the two or more of
R may be the same or different, and therefore the
compound of the formula [I] can be a hybrid of DNA
chain (s) and RNA chain (s).
B' is a protected base. This generally
includes, as the base adenine (A), guanine (G),
cytosine (C), thymine (T) or uracil (U). When the
compound of the formula [I] forms a DNA chain, the
base is A, G, C or T, while it is A, G, C or U when
the eompound forms a RNA chain. The protecting group
is usually an acyl group, and illustrative of the
protected base are N6-benzoyladenine, N-isobutyryl-
guanine, N6-benzoylcytosine, thymine and uracil. As
clearly seen from thymine and uracil themselves as
examples of s', thymine and uracil generally require
no protection. They themselves stand protected with-
out any foreign proteeting group. Thus, "the pro-
tected thymine" and "the protected uracil" are
inelusive also of those having no specific protecting
group.
In this context, the bases in the formula [I],
whieh are as specified above, may of course be modified
with groups other than acyl groups falling within the
"protecting groups" as deseribed above, and the present
invention is clearly valid for DNA or RNA in which
changes generally seen in this kind of bases, such as
73~
methylation or partial conversion of amino groups
into carbonyl groups when B is cytosine or adenine
have occurred. Accordingly, the compounds in
which the bases are thus changed also belong to
the cope of the present invention.
The group Rl, the group R2 and the group R
when showing a protected hydroxyl group may be those
known as the protecting groups for phosphate group
and hydroxyl group in the art of nucleic acid syn-
thesis, specific examples together with those of
the protecting groups for the above bases being dis-
closed in "Journal of the Society of Organic Syn-
thetic Chemistry, Japan", Vol. 36, No. 9, pages 723-
731, (1978), "Synthesis of Nucleosides and Nucleo-
tides" (Maruzen, Japan 1977), "Organic Chemistry of
Nucleic Acids" (Kagaku Dojin, Japan 1979), "Nucleic
Acids" (Asakura Shoten, Japan 1979), Tetrahedron, 34,
3143 (1978), Journal of the Society of Synthetic
Organic Chemistry, Japan 34, 723 (1978), "Kagaku
no Ryoiki" (Domain of Chemistry), 33, 566 (1979),
and other review publications and textbooks. The
protective groups particularly preferred in the
present in~Jention are o-chlorophenyl group and p-
chlorophenyl group as the group Rl, mono-methoxy-
trityl group and di-m~thoxytrityl group as the group
R2, and hydrogen and hydroxyl group protected with
o-nitrophenyl as the group R.
73
:
_ is any desired natural number. Generally,
is about l -to 10, preferably about l to 6, parti~
eularly about l to 3.
A is a lower alkylamine. The term "lower alkyl"
means an alkyl having about l to 4 carbon atoms.
Typieal examples of lower alkylamines are triethyl-
amine, diisopropylamine, dimethylamine, t-butylamines,
sec-butylamines, n-butylamines, n-propylamines, and
isopropylamines, trlethylamine being preferable.
Production of the compound [I] can be carried
out also aceording to any suitable method for syn-
thesis o:E nucleic acids described in the above
references or others. A typical example is as
- described below. First, the compound of the formula
[II] is synthesized aceording to the above references
and is also commereially available.
~ ~R O ~
t ~ ORl [II]
wherein: R3 is a ehemical proteeting group for phos-
phate group whieh ean be depro-
teeted under the eonditions where other
proteeting groups are all stable, usual-
ly a eyanoethyl group; and
R, Rl, B' and n have the same respeetive
-13-
7~i
meanings as defined ahove.
The compound of the formula [II] is treated
with a lower alkylamine, preferably triethylamine
in pyridine or in pyridine-water to remove the
protecting group R3 to form compound [I~. The
compound [I] thus formed is obtained in an oily
form by distilling off the excessive lower alkyla-
mine, pyridine and water from the reaction product.
This oily compound ~I] is stored and used for the
DNA synthesis as it is, or as a powder form pre-
pared by dropwise addition of the oily form into
hexane. The oily form of the compound ~I~ may
be considered to be oily, because components
other than the compound [I], particulariy pyri-
dine, are contained.
Freeze-drying
Preparation of Solution
The essential solvent is dioxane. However,
the compound [I], when it is a powder form, is dis-
solved in dioxane at a slow rate, and therefore itis preferably disolved in a mixture of pyridine and
dioxane. In this case, if the amount of pyridine is
excessive, freeze-drying of the solution cannot be
effectively carried out. Accordingly, it i
-14-
lZ~4735
desirable to use pyridine in a volume of 2 to 10
v/v % oE dioxane. In prepariny a solution of
the compound ~I], pyridine may be previously
mixed with dioxane before dissolving the compound
[I] therein, or alternatively the compound [I]
may first be dissolved in pyridine and the re-
sultant solution further dissolved iII dioxane.
The latter procedure can en~oy better the ad-
vantage thanks to use of tne pyridine. Thus,
the expression l'dissolved in dioxane together
- with pyridine" used in the present invention
is inclusive of both of the procedures described
above.
In the case where the compound [I] is in a
oily form, an amply high dissol~ing rate can be
obtained without using pyEidine in com~ination~
In either of the cases described above, the
essential solvent is dioxane, but a small amount
o another solvent or a lower alkylamine can be
used in combination therewith without departing
from the spirit of the invention, and the scope
of claims should also be understood in this context.
Accordingly, the use of a small amount of pyri-
dine in the case when the compound [I] is in a
oily form is within the scope of the present invention.
The concen-tration of the compound [I] in the
solution may be appropriately determined in view of
-15-
73~
its viscosity or convenience in the freeze-drying
step. More specifically, it is ordinarily of the
order of, for example, 10 to 100 mg/ml.
Freeze-drying Step
Except for the point that the solute is the
compound [I] and the solvent is dioxane (and a small
amount of pyridine), the freeze drying operation
is not different from the conventional one.
- Generally speaking, this step would comprise
freezing by cooling the solution (ordinarily of the
order of -30C or lower) and sublimation of the frozen
solvent by application of a reduced pressure (ordi-
narily of the order of several mmHg or lower).
With regard to other necessary information
about freeze drying, reference may be made to text-
books such as Shin Jikken Kagaku Koza "New Course of
Experimental Chemistry) 1 [I] 459" (~laruzen, Japan
1975)-
Utilization of FrPeze-Dried Preparation
The freeze-dried preparation of the compound
~I] according to the present invention not oly has
good solubility in pyridine but is also equal
to or better than the preparations of -the
prior art (dried by azeotropic dïstillation
immediately before use) in reactivity at the
condensation step.
Accordingly, the freeze-dried preparation, as
~16-
~2~35
long as it is stored under moistureproof conditions,
can be used advantageously as the nucleotide unit
for the DNA synthesis withou~ azeotropic drying.
The solid phase method for synthesis of an
oligonucleotide by the use of a freeze-dried nucleo-
tide compound is speci~ically described by referring
to an example as set forth below, by which the
present invention is not limited.
About 50 to 60 mg of a polystyrene resin
having a nucleoside bonded thereto is placed in
a reactor equipped with a stop cock and glass
~ilter and washed thoroughly with an isopropanol-
methylene chloride (15 : 85, y/v) solution.
The resin is then treated with 1 molar zinc bromide
in isopropanol-methylene chloride (15 : 85, v/v
solution for 5 minutes 4 times. After detrity-
lation with ~inc bromide solution, the resin ;s
washed with an isopropanol-methylene chloride
(15 : 85, v/v) solution, then with pyridine, and
the resin is dried by means of a vacuum pump.
Th2 detritylation method may be replaced by a
method in WhiCh benzenesulfonic acid or trichloro-
acetic acia is employed.
Alternatively, drying o~ ~he resin may ~e
carried out by washing with a volatile organic sol-
vent and subsequent drying with a dry gas.
On the other hand, the nucleotide reagent to be
'73~i
used for condensation is prepared by adding mesity-
lenesulEonyl nitrotriazolide (hereinafter abbreviated
as MSNT) (about 30 mg) to the freeze-dried prepara-
tion of the nucleotide block (about 30 mg) of
the present invention and dissolving the mixture in
anhydrous pyridine (about 400 ~1) under stirring.
This reagent is added quickly to the dried
resin, and the reaction is carried out for 60
minutes. After the reaction, the resin is washed
with pyridine, and the unreacted 5'-hydroxyl group
is protected through acetylation. As the acetylating
agent, 0.5 ml of an acetic anhydride-pyridine ~1 :5)
solution and 0.5 ml of dimethylaminopyridine (herein-
after abbreviated as DMAP)-pyridine solution are
added in combination, and the reaction is carried out
for 5 minutes. After washing with pyridine, the
following cycle is initiated. This operation (S-teps
1 - 9 in Table 1) is repeated to extend successively
the chain length.
-18-
~Z~3S
Table 1
Reaction Operations
Ouantity Time Number of
Step Reagent or Solvent of solv- (min ) repeti-
ent (ml) . tion
_ _
1 iso-PrOH-CH2C12 1 2 5
: 2 1.0 mol ZnBr2/ 1 5 4
iso-PrOH-CH2C12
: 3 iso-PrOH-CH2C12 1 1 3
4 Dry pyridine 1 1 5
Vacuum drying _ ca.l0
6 Nucleotide +MSNT/ 0.5 60
dry pyridine
7 Pyridine 1 1 3
8 Ac2O-pyridine(1:5) (0.5 5 1
DMAP-pyridine O.5
(200 mg/10 ml)
9 Pyridine 1 1 3
*) based on 50 to 60 mg of resin
- 25
--19--
.
_xperimental Examples
Example l
_
About 30 mg of a compound [II] represented by
the formula
DMTr -O ~ -O - P - O-CE
ORl
wherein: DMTr is a dimethoxytrityl group;
Rl is an o-chlorophenyl group;
T is thymine; and
CE is a cyanoethyl group,
was treated with l ml of a pyridine-triethylamine-
water (3:1:1, v/v) solution at room temperature for
15 minutes to remove the cyanoethyl group. The
solvent was evaporated, and excessive triethylamine
and water were completely evaporated by pyridine
azeotropic distillation. The resultant oily product
was dissolved in 600 ~1 (microliters) of dioxane and
freeze-dried to obtain the freeze-dried preparation
of the triethylamine salt of the desired compound
[I]-
Example 2
About 30 mg of the compound [II] was treated
with 1 ml of diethylamine-pyridine (1:9, v/v) at room
temperature for 30 minutes. The solvent was removed,
and excessive diethylamine was evaporated by pyridine
-20-
~2~4~
azeotropic distillation. The oily product obtained
was d~ssolved in 600 ~1 of dioxane, and, following
the procedure described in Example 1, the freeze-
dried preparation of the diethylamine salt of the
compound [I] was obtainedO
Similar results were also obtained when substi-
tuting n-butylamine or diisopropylamine for diethyl-
amine.
Example 3
1~ Production of freeze-dried preparations
Dimethoxytrityl-di-nucleotides of the formula
` Bl B2
l 1l
DMTr - O ~ -O - P - O ~ -O - P - OH-Et3N ,
\ ORl OR
wherein: DMTr is a dimethoxytrityl group;
Rl is an o-chlorophenyl group; and
Bi and B2 are each a protected base
selected from N-benzoyladenine (A),
N-benzoylcytosine (C), N-isobutyryl-
guanine (G) and thymine (T),
were processed into freeze-dried preparations as des-
cribed below.
In the following examples, these dinucleotides are
called as AC or TG by referring to its bases.
35 mg of each of (1) [CA], (2) [GG], (3) [CA],
-21-
~L,fC~ L~L73~
(4) [TA], (5) [TA], (6) [AA], and (7) [TC] was dis-
solved in 50 ~1 of anhydrous pyridine, respectively,
and frozen with addition of 800 ~1 of anhydrous dioxane
at -50C, and drying was carried out by sublimation
of the solvent under a reduced pressure of 3 mmHg.
The freeze-dried dinucleotides (1) through (7) were
stored in a dessicator.
2) Production of an oligonucleotide
Dimethoxytrityladenosine resin (60 mg, 6.6 ~mol)
was treated in a reactor according to the operations
in Table 1 as shown above. That is to say, isopropanol-
methylene chloride washing, detritylation with 1 molar zinc
bromide, isopropanol-methylene chloride washing, pyridine
washing and drying of the resin by means of a vacuum
pump were conducted. To this dried resin was added a
pyridine solution (400 ~1) of the dinucleotide (1) [CA]
and MSNT (30 mg), and reaction was carried out for 60
minutes. After the reaction, pyridine washing,
acetylation with acetic anhydride-pyridine-dimethyl-
aminopyridine (1: 9 :catalytic amount) and pyridinewashing were conducted to complete one cycle of con-
densation. This procedure was repeated for successive
condensation of the dinucleotides (2) [GG], (3) [CA], (4)
[TA], (5) [TA], (6) [AA], and (7) [TC], thereby to
synthesize a pentadecanucleotide d (TCAATATACAGGCAA).
The average yield was 95%, and the overall yield 70%.
This is a yield which is ~igher than that
-22-
~LZ~73~
of the prior art method according to pyridine azeo-
tropic distillation.
The synthesized oligonucleotide was deprotected
in a conventional manner and purified. More specifi-
cally, about 10 mg of the resin was treated at roomtemperature overnight with 100 ~1 of a solution of 0.5
M tetramethylguanidine-pyridine-2-aldoxime in dioxane-
water (9 : 1, v/v) and thexeafter treated at 50~C over-
night with addition of 2.5 ml of conc. ammonia water.
The resin was filtered off, the filtrate was concentrat-
ed and subjected to rough purification on Sephadex
G-50 (1.5 x 120 cm~ with an eluent of 0.05 M TEAB
- (triethylammonium bicarbonate) buffer (pH 7.5) (FIG. 1).
The peak fraction was collected, and the trityl
derivative was purified by high performance li~uid
chromatography (HPLC) through a reversed phase column
(FIG. 2). After detrityla~ion with 80~ acetic acid,
purification was conducted again through a reversed
phase column to obtain the desired pentadecanucleotide
of high purity in a high yield (FIG. 3).
Example 4
1) Production of freeze-dried preparations
35 mg of each of the dinucleotide reagents (1) [CA],
(2) [TG], (3) [CA], (4) [TA], (5) [TA], (6) [AA], and
(7) [TC] was dissolved in anhydrous pyridine (50 ~1)
and freeze-dried with addition of anhydrous dioxane
(800 ~1).
-23-
, . . .
2) Production of an oligonucleotlde
Dimethoxytrityladenosine resin (60 mg, 6.6 mmol)
was placed in a reactor and condensations were suc-
cessively conducted in the order of the nucleotide
reagents (1) - (2) - (3) ~ (4) - (5) - (6) - (7) to
synthesi~e a pentadecanucleoside d (TCAATATACATGCAA).
The average yield was 95%, and the overall yield
70%. AS compared with the method of the prior art,
the yield was less deviantly scattered among the cycles
and was hi~h.
The desired pentadecanucleotide was purified
similarly as in the preceding Examples, whereupon a
product of high purity was obtained in a high yield
(FIG. 4 to FIG. 6).
-24-
