Oligonucleotide derivatives

An oligonucleotide derivative having an amino group protected with an eliminatable group bonded through a phosphate group and a spacer with an appropriate length to the 5'-end of an oligonucleotide protected suitably at the 3'-hydroxyl group and the base moiety of the nucleotide, and an immobilized oligonucleotide derivative having a Sepharose carrier bonded to the amino group in place of said protective group are disclosed. Methods for production of these derivatives are also disclosed.

BACKGROUND OF THE INVENTION 
1. Field of the Art 
n relates to oligonucleotide derivatives having amino groups introduced 
through intermediary straight or branched alkylene groups into the 
5'-phosphate groups of oligonucleotide of a certain length, to an 
immobilized oligonucleotide bound to a carrier at the amino group moiety, 
and to a method for production of them. 
In the field of biochemistry, purification of vital polymers is one of the 
important tasks of research, and a great deal of effort by a large number 
of researchers have been made in the past therefor. For this object, 
affinity chromatography techniques and electrophoresis using primarily 
polyacrylamide gel have been developed and appreciably utilized. 
Many vital polymers have inherent properties to bind or interact 
specifically with specific substances. Affinity chromatography may be said 
to be a method utilizing skillfully the principle of biological 
discrimination possessed by vital polymers. 
Today, when the affinity technique is undergoing rapid progress, it is 
being widely utilized for purification and separation of various vital 
substances, including, as a matter of course, proteins, enzymes, and also 
lipids, hormones, vitamins, and receptors. 
Above all, affinity chromatography with the use of a nucleic acid as ligand 
is expected to be widely utilized in the future in various applications, 
including isolation of nucleic acids or proteins which are also important 
in molecular biology. Also, for the purpose of efficient isolation, it is 
of great interest to develop a crosslinking method between ligands and 
carriers. 
2. Prior Art 
From such a point of view, among the affinity chromatographies using 
carriers having nucleic acids bound thereto, the most widely utilized is 
the method in which RNA containing poly (A) at the 3'-end is isolated by 
the use of an oligo (dT)-cellulose or a poly (U)-agarose column [Ono, M., 
Kondo, T., Kawakami, M : J. Biochem., 81, 941 (1977)]. 
Poly (U), Poly (dA)-cellulose, etc. are used in the method wherein the base 
moiety of nucleotide is bound to a carrier activated with BrCN, etc., and 
therefore the resultant bound product is stable due to binding formed at 
multiple sites therebetween, while, on the other hand, it involves a 
drawback in that its adsorption capacity is weakened, because the base 
moieties necessary for affinity activity are used for binding with the 
carrier [Lindberg, U., Persson, T. : Eur. J. Biochem., 31, 246 (1972)]. 
Also, in the case of oligo (dT)-cellulose, binding between the hydroxyl 
groups of a carrier and the phosphoric acid groups of an oligonucleotide 
is said to be accomplished by the use of, for example, DCC 
(dicyclohexylcarbodiimide), but it involves the problems of non-specific 
adsorption and lack of reproducibility of adsorption capacity. 
Other than the proposals of immobilized homopolymers of nucleotide as 
described above, there are several proposals in which DNA obtained from 
natural resources is immobilized [Anderson, J. N., Monahan J. J., 
O'Malley, B. W.: J. Biol. Chem., 252, 5789 (1977)], but there has, insofar 
as we are aware, been no report in the past that an oligonucleotide of a 
certain length having any desired base sequence has been bound to a 
carrier only at a specific position to be successfully immobilized. 
Under these circumstances, if an oligonucleotide having any desired base 
sequence could be bound to a carrier at a specific site, such a technique 
would be useful not only for isolation and purification of a mRNA 
according to affinity chromatography utilizing the immobilized nucleotide 
homopolymer but also for isolation and purification of a mRNA having a 
specific base sequence. Further, its applicability for purificarion of 
various nucleic acid related enzymes recognizing specific base sequences 
may also be considered. 
A large number of researches have also been carried out on affinity 
carriers by using mono- or di-nucleotides as ligand, and the results of 
some of them are now commercially available. However, the sites at which 
the nucleotide is bound to the carrier through an intermediary spacer are 
mostly the base moieties thereof.sup.(a). There are also some products in 
which the nucleotide is bound to the sites other than base 
moieties.sup.(b), but to the best of the present inventors' knowledge, 
such products involve the drawbacks of a large number of steps required 
for synthesis of the starting ligand and cumbersome procedures over the 
entire synthesis. Also, none of the methods can be used for 
oligonucleotide. 
(a) Lee, C. Y., Lappi, D. A., Wermuth, B., Everse, J., Kaplan, N. O.: Arch. 
Biochem. Biophs., 168, 561 (1974); Ishiwata, K., Yoshida, H. : J. 
Biochem., 83, 783 (1978); Japanese Patent Laid-Open Nos. 25795/1977, 
101396/1978, 133283/1978 and 36277/1980. 
(b) Jervis, L., Pettit, N. M.: J. Chromatog., 97, 33 (1974); Lamed, R., 
Levin, Y., Wilchek, M. : Biochem. Biophys. Acta., 304, 231 (1973); Janski, 
A., Oleson, A. E. : Anal. Biochem., 71, 471 (1976). 
SUMMARY OF THE INVENTION 
Gist 
In view of the state of the art as described above, the present inventors 
have developed an immobilized oligonucleotide which is useful in 
purification of nucleic acids and is utilizable for affinity resins, and a 
of producing the same. 
The present inventors have previously developed a method of synthesizing a 
completely protected oligonucleotide according to the solid-phase 
synthetic method. The present inventors have found a method for 
immobilization, which comprises introducing a functional group capable of 
being bound with another carrier into the 5'-hydroxyl group of the 
objective compound synthesized by the solid-phase synthetic method, so as 
to be bound at said functional group to the carrier. According to this 
method, the present inventors have succeeded in synthesizing effectively 
an immobilized oligonucleotide, in which an oligonucleotide having any 
desired base sequence is bound at a specific position to a carrier. 
The present invention concerns immobilized oligonucleotide derivatives, 
which can be used also as affinity resins as well as a plural number of 
oligonucleotide derivatives which can be used as intermediates thereof, 
and a method for production thereof. 
More specifically, the oligonucleotide derivatives according to the present 
invention can be represented by the following formulae (2), (4) and (5). 
The method of producing the oligonucleotide derivatives represented by the 
following formulae (2), (4) and (5), as expressed conceptionally and 
comprehensively, comprises causing a compound (1) to react with a compound 
(0) to produce a compound (2), while, on the other hand, condensing a 
compound (3') obtained by the nucleic acid synthetic method with a 
compound (2') which is a derivative of the compound (2) from which the 
protective group R.sup.4 of the 3'-phosphate has been eliminated to 
produce a compound (4), and removing all the protective groups from this 
compound to produce a compound (5). 
EQU HO(N' .sub.p.sbsb.x).sub.m R.sup.3 ( 0) 
EQU R.sup.2 --NH--R.sup.1 --OH (1) 
EQU R.sup.2 --NH--R.sup.1 --.sub.p.sbsb.x (N' .sub.p.sbsb.x).sub.m R.sup.3 ( 2) 
EQU R.sup.2 --NH--R.sup.1 --.sub.p.sbsb.x (N' .sub.p.sbsb.x).sub.m H (2') 
EQU HO(N' .sub.p.sbsb.x).sub.n N' OCOR.sup.4 ( 3') 
EQU R.sup.2 --NH--R.sup.1 --.sub.p.sbsb.x (N' .sub.p.sbsb.x).sub.m+n N' 
OCOR.sup.4 ( 4) 
EQU NH.sub.2 --R.sup.1 --.sub.p (N.sub.p).sub.m+n NOH (5) 
In the above formulae, the respective symbols have the meanings set forth 
below: 
N': a nucleoside having a base residue selected from the group consisting 
of adenine, guanine, cytosine and thymine, acylated to a necessary extent 
[acyl groups may be, for example, those from lower aliphatic 
mono-carboxylic acids (C.sub.2 -C.sub.4) such as acetyl, isobutyryl, or 
those from aromatic carboxylic acids such as benzoyl, anisoyl], from which 
3'-and 5'-oxygens in the riboside skeleton have been removed, that is A, 
G, C and T, respectively, namely: 
##STR1## 
wherein B' represents a base residue as mentioned above acylated to a 
necessary extent, provided that the plural number of N' may be the same or 
different with respect to the base moiety and/or the acyl moiety when m or 
m+n (as described hereinafter) is 2 or more; the term "to a necessary 
extent" means a necessary extent required in nucleic acid synthesis, and 
therefore no acylation is necessary when the base is thymine; specific 
examples of acyl groups are benzoyl for adenine and cytosine and 
isobutyryl for guanine. 
N: a nucleoside having the above base residue not protected, from which the 
3'- and 5'-oxygens in the riboside skelton have been removed; provided 
that plural number of N when m+n (as described hereinafter) is 2 or more 
may be the same or different. 
p.sub.x : phospho-triester bond, namely: 
##STR2## 
R.degree. is a phenyl group or a substituted phenyl group such as 
o-chlorophenyl group or p-chlorophenyl group. 
p: phospho diester bond, namely: 
##STR3## 
R.sup.1 : a straight or branched divalent hydrocarbon group, e g., C.sub.2 
-C.sub.20 straight or branched alkylene group. 
R.sup.2 : a protecting group for amino group which is substitutent stable 
during elimination of R.sup.3 group and eliminatable while permitting the 
oligonucleotide moiety to remain stable, e.g., trifluoroacetyl group 
(Tfa-) or o-nitrophenylsulphenyl group (Nps-). 
R.sup.3 : a protecting group for phosphate group which is substituent 
easily eliminatable under the conditions where all other protective groups 
are stable and capable of forming the terminal phosphoric acid triester 
bonding into a free phosphoric acid diester bonding, e.g., cyanoethyl 
group (CE), trichloroethyl group, phosphoroamidate group, etc. 
COR.sup.4 : a protecting group for 3'-hydroxyl group conventionally used 
for oligonucleotide synthesis; 
R.sup.4 being, for example, a lower (about C.sub.1 -C.sub.3) alkyl group 
(e.g., methyl) or a lower alkyl- or lower alkoxy (about C.sub.1 
-C.sub.3)-substituted or non-substituted phenyl group (e.g., phenyl or 
methoxyphenyl) accordingly acetyl, benzoyl or anisoyl as COR.sub.4, in the 
nucleotide liquid-phase synthesis, or a carrier for nucleotide synthesis 
with intermediary spacer, such as polystyrene derivatives, silica gel 
derivatives or polyacrylamide derivatives, in the nucleotide solid-phase 
synthesis. 
R.sup.5 : a protecting group for 5'-hydroxyl group conventionally used for 
oligonucleotide synthesis, for example, substituted (e.g., 
dimethoxy-substituted) or unsubstituted (trityl group). 
m: an integer 0 to 6 (preferably 1 to 4) 
n: an integer 0 to 40 (preferably 0 to 20). 
In the above formulae, among p or p.sub.x or HO or O, those positioned at 
the right side of N' or N or the bracket including these represent those 
bonded to the 3'-hydroxyl group of the nucleoside, while those on the left 
side thereof represent those bonded to the 5'-hydroxyl group of the 
nucleoside. 
The immobilized oligonucleotide according to the present invention is 
represented by the following formula (6). 
The method of producing an immobilized oligonucleotide represented by the 
following formula (6) according to the present invention comprises causing 
a compound (5) to react with a Sepharose derivative capable of being bound 
with an amino group at the amino group of the oligonucleotide derivative 
to produce a compound (6). 
EQU NH.sub.2 --R.sup.1 --.sub.p (N.sub.p).sub.m+n NOH (5) 
EQU [Sepharose]--NH--R.sup.1 --.sub.p (N.sub.p).sub.m+n NOH (6) 
wherein the respective symbols have the following meaings: N, N', px, p, 
R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5, m and n are the same as those 
defined above; [Sepharose] is a residue of Sepharose derivative capable of 
being bound with an amino group. 
To represent more specifically the various formulae as symbolized above to 
be used in the present invention, the formula (4), for example, can be 
represented as follows: 
##STR4## 
In the present invention, to speak of the compound (4), the representation 
by the formula (4) may be used interchangeably with that of the formula 
(4a). 
Advantageous Effect 
According to the present invention, it is possible to synthesize an 
immobilized oligonucleotide useful also as an affinity resin, comprising 
an oligonucleotide with a certain length and having any desired base 
sequence bonded at a specific site to a carrier, and the bonding 
unattainable by the method of the prior art can be attained to produce a 
resin of improved quality by the method of the present invention. 
This is because a primary amino group has been introduced in an 
oligonucleotide as a functional group for binding the oligonucleotide with 
the carrier. That is, the following meritorious effects can be considered 
to be realized by the functional group. 
(1) The functional group has higher reactivity with other functional groups 
(hydroxyl groups, phosphoric acid groups and amino groups at the base 
moieties). 
(2) Therefore, even when a mixture of deprotected oligonucleotides is used 
without purification for condensation with the carrier, selective binding 
at this position is possible by employment of suitable reaction and other 
conditions. 
Also, as the result, it has become possible to immobilize by a simple step 
and moreover effectively an oligonucleotide having any desired base 
sequence which has been synthesized according to any of the solid-phase 
methods and the liquid-phase methods. 
Further, by avoiding binding at the base moiety which interferes with the 
adsorption activity, the immobilized oligonucleotide obtained by the 
present invention has excellent adsorption capacity. 
Accordingly, the oligonucleotide-Sepharose according to this invention is 
superior by far in adsorption capacity, reproducibility, selectivity, and 
durability to those of the prior art [oligo (dT)-cellulose and poly 
(U)-agarose].

(a) Purification conditions by Sephadex G-50 column: 
Column: Sephadex G-50 
Column volume: 1.5 cm.times.120 cm 
Eluant: 50 mM TEAB buffer, pH 7.5 
Fraction amount: 35 droplets/fraction 
(b) Analytical conditions by HPLC: 
Column: .mu.-Bondapak C18 (Waters) 
Eluant: CH.sub.3 CN in 0.02 M EDAA buffer (pH 7.8) 
Gradient: as shown in the drawings 
Flow rate: 2 ml/min. 
Chart speed: 10 mm/min. 
Temperature: 50.degree. C. 
(c) Assay conditions by affinity chromatography column: 
Washing solution: 0.5 M NaCl, 10 mM Tris-HCl 
Eluant: 10 mM Tris-HCl (pH 7.5) 
Fraction amount: 15 droplets (350 .mu.l) 
Application of vital test sample is indicated by A and initiation of 
elution by B. 
DETAILED DESCRIPTION OF THE INVENTION 
Reaction Scheme 
The present invention can be comprehended as a link in the production of an 
immobilized oligonucleotide, having an oligonucleotide with a base 
sequence capable of synthesis bound to a carrier, and the reaction 
starting from the oligonucleotide synthesized by the solid phase method 
along its best mode from this point may be illustrated as in FIG. 1. 
The symbols in this reaction scheme have the following meanings. 
MSNT: mesitylenesulfonyl nitrotriazolide 
TMG-Oxime: 0.5 M tetramethylguanidium pyridine-2-aldoxime in dioxane/water 
(9:1) 
In the following description, specific compounds (1) to (6) are to be 
explained in this order on the basis of this reaction scheme. 
Concerning chemical synthesis of nucleotides or nucleic acids, a number of 
textbooks and reviews have already been published. Accordingly, for 
details, other than those in the following description, relating to the 
kinds of protecting groups, their introduction or removal as well as 
condensation and other features in the synthesis of 
deoxyoligoribonucleoside according to the present invention, reference is 
made to, for example, H. Kossel, H. Seliger: PROGRESS IN THE CHEMISTRY OF 
ORGANIC NATURAL PRODUCTS, Vol. 32, p.297, Springer, Wien (1975) and 
Tetrahedron, Vol. 34, 3143 (1978). 
Compound (1) 
The compound (1) is represented by the formula (1): 
EQU R.sup.2 --NH--R.sup.1 --OH (1) 
The compound (1) can be obtained by introduction of R.sup.2 as a protecting 
group for the amino group of .omega.-amino alcohol (NH.sub.2 --R.sup.1 
--OH). 
R.sup.1 is a straight or branched divalent hydrocarbon, suitably a C.sub.2 
-C.sub.20, preferably C.sub.2 -C.sub.12, alkylene group. As .omega.-amino 
alcohols, those of C.sub.2 -C.sub.12 are commercially available. 
R.sup.2 is a protecting group, which is stable under the eliminating 
conditions of R.sup.3 group (--CE) (e.g., in Et.sub.3 N-pyridine-H.sub.2 O 
1:3:1) or phosphorylating conditions, [e.g., in 
pyridine-1-methylimidazole, or in DMAP (dimethylaminopyridine) in pyridine 
(hereinafter referred to as Py in some cases)], and is further capable of 
being eliminated while the oligonucleotide moiety remains stable. 
If possible, it is more convenient that the protecting group R.sup.2 be one 
which can be eliminated at the same time under such conditions for 
removing the protecting groups of the oligonucleotide as in, for example, 
conc. ammonia water. 
Specific examples of the protecting group of the amino group are 
trifluoroacetyl group which can be removed by conc. ammonia water and 
o-nitrophenylsulphenyl group which can be removed by a weak acid or 
mercaptoethanol. 
Compound (2) 
(1) Definition: 
The compound (2) is a novel substance represented by the formula (2): 
EQU R.sup.2 --NH--R.sup.1.sub.p.sbsb.x (N' .sub.p.sbsb.x).sub.m R.sup.3 (2) 
The definitions of the substituents in the compound (2) and preferable 
examples are as set forth above. 
(2) Synthesis: 
The compound (2) can be prepared by bonding the 5'-hydroxyl group of the 
oligonucleotide derivative represented by the following formula (0) to the 
compound of the above formula (1) through a phosphate group. This bonding 
can be obtained by phosphorylating the 5'-hydroxyl group of the compound 
(0) with a bivalent phosphorylating agent (e.g., phospho-di-triazolide, 
phospho-dichloride or phospho-bibenzo-triazolide) and then carrying out 
the reaction with the compound (1) under condensing conditions (preferably 
in the presence of 1-methyl-imidazole). Specific examples of the reaction 
conditions are set forth in the experimental examples presented below. 
EQU HO(N' .sub.p.sbsb.x).sub.m R.sup.3 (0) 
Compound (3) 
The compound (3) is represented by the formula (3): 
EQU R.sup.5 --O(N' .sub.p.sbsb.x).sub.n N'OCOR.sup.4 (3) 
The compound (3 ) is an oligonucleotide completely protected in a broad 
sense, and it may be synthesized according to any method. 
The oligonucleotide of the compound (3) is protected at its 3'-end by 
R.sup.4 through the carbonyl group. That is, this hydroxyl group is 
acylated. Definition of R.sup.4 and examples thereof are as given above. 
When the compound (3) is synthesized according to the solid-phase method 
(to be described in detail hereinafter), COR.sup.4 is suitably a carrier 
having an appropriate spacer (e.g., polystyrene derivative, polyacrylamide 
derivative, etc.). As to a polystyrene resin as COR.sup.4, see Chem. Rev. 
77, 183 (1977); and Forsuchr. Chem. Org. Naturstoff, 32, 297 (1975); as to 
a polyamide resin, see J. Am. Chem. Soc., 98, 8514 (1976); Nucleic Acids 
Research 4, 1135 (1977); ibid. 4, 4391 (1977); ibid. 6, 1265 (1979); and 
Tetrahedron Letters, 1979, 1819. 
The compound (3) can be synthesized according to any method suited for the 
purpose. Generally speaking, as synthesizing methods for an 
oligonucleotide such as the compound (3), there are the triester method, 
the phosphite method and respective solid-phase and liquid-phase methods, 
but it is preferable to use the solid-phase method developed by the 
present inventors. Details of the solid-phase synthesizing method are 
described in Tetrahedron Letters 1979, 3635 (1979); Nucleic Acids Research 
8, 5473 (1980); ibid. 8, 5491 (1980); ibid. 8, 5507 (1980); and Nucleic 
Acids Research Symposium Series 7, 281 (1980). 
Compound (3') 
The compound (3') corresponds to the compound (3) from which the protective 
group R.sup.5 at the 5'-end has been removed. 
For removing only the 5'-protecting group of the compound (3), when R.sup.5 
is a group conventionally used, the method in which the compound (3) is 
treated in a 1.0 M isopropanol-methylene chloride solution of 
benzenesulfonic acid, acetic acid or zinc bromide, or some other method 
may be used. 
Compound (4) 
(1) Definition: 
The compound (4) is a novel substance represented by the formula (4): 
EQU R.sup.2 --NH--R.sup.1 --.sub.p.sbsb.x (N' .sub.p.sbsb.x).sub.m+n 
N'OCOR.sup.4 (4) 
The definitions of the substituents in the compound (4) and preferable 
examples are as described above. 
(2) Synthesis: 
The compound (4) can be obtained by eliminating the R.sup.3 group in the 
compound (2) and the R.sup.5 group in the compound (3), respectively and 
then causing the reaction of the both compounds between the 3'-phosphate 
group on the compound (2) and the 5'-hydroxyl group on the compound (3) in 
the presence of a condensing agent. 
The R.sup.3 group of the starting compound (2) is an easily eliminatable 
group, and the 3'-phosphate of the oligonucleotide after deprotection may 
be PO.crclbar. (free form) or in the form of a suitable salt. As the 
R.sup.3 group, a cyanoethyl group is generally used, and typical examples 
of salts are tertiary amine salts, for example, triethylammonium salt. 
The other starting compound corresponding to the compound (3) from which 
R.sup.5 has been removed, namely, the compound (3'), is as described 
above. 
Condensation is conducted preferably in the presence of a condensing agent. 
Typical examples of condensing agents which can be used in this step are 
tosyl chloride, mesitylene sulfonyl chloride, mesitylene sulfonyl 
tetrazolide (MSTe) and mesitylene sulfonyl nitrotriazolide (MSTN). As for 
specific examples of the reaction conditions, see the Experimental 
Examples set forth below. 
Compound (5) 
(1) Definition: 
The compound (5) is a novel substance represented by the formula (5): 
EQU NH.sub.2 --R.sup.1 --.sub.p (N.sub.p).sub.m+n NOH (5) 
The definition of the substituent in the compound (5) and preferable 
examples are as given above. 
(2) Synthesis: 
The compound (5) can be prepared by eliminating the COR.sup.4 group, the 
R.sup.2 group, the acyl group on the base and the protective groups 
(usually aryl groups, for example, o-chlorophenyl group) in the phospho 
triester in the compound (4) while the oligonucleotide remains stable. 
The COR.sup.4 group and o-chlorophenyl group in phospho triester is 
preferably eliminated by the use of a TMG-Oxime solution. Other protecting 
groups (R.sup.2 group and acyl group at the base moiety) may also be 
removed by carrying out an alkali treatment (conc. ammonia water). The 
TMG-Oxime solution refers to 0.5 M tetramethylguanidium 
pyridine-2-aldoxamate in dioxane/water (9:1). 
When R.sup.2 is Tfa-, it can be eliminated by ammonia treatment, but when 
it is Nps-, further treatment with mercaptoethanol is necessary. When 
other protective groups are employed, still another treatment may also be 
considered, provided that the oligonucleotide moiety remains stable. 
As for the specific examples of the reaction conditions, see the 
Experimental Examples set forth below. 
Compound (6) 
(1) Definition: 
The compound (6) is a novel substance represented by the formula (6): 
EQU [Sepharose]--NH--R.sup.1 --.sub.p (N.sub.p).sub.m+n NOH (6) 
The definition of the substituent in the compound (6) and preferable 
examples thereof are as mentioned above. 
(2) Synthesis: 
The compound (6) can be prepared by condensation of the compound (5) with a 
Sepharose derivative capable of binding with amino group. A condensing 
agent may be necessary or unnecessary depending on the kind of the 
Sepharose derivative to be bonded. 
Sepharose as its chemical entity is agarose and is available from Pharmacia 
Fine Chemicals, U.S.A. This material, in spite of its chemical entity 
being agarose, is conventionally called "Sepharose" and is well known to 
those skilled in the art. For example, see "Affinity Chromatography", 
Elsevier Scientific Pub. Co., Amsterdam (1978); Agric. Biol. Chem. 80, 409 
(1976). Some examples of Sepharose derivatives which can be used in the 
present invention are enumerated below. 
Cyanogen bromide activated Sepharose: 
##STR5## 
Activated CH Sepharose: 
##STR6## 
Epoxidized Sepharose: 
##STR7## 
CH Sepharose: 
EQU agarose--NH--(CH.sub.2).sub.5 --COOH 
AH Sepharose: 
EQU agarose--NH--(CH.sub.2).sub.5 --NH.sub.2 
Among these derivatives, the first three, especially the first two, are 
preferred from the standpoint that no condensing agent (e.g., DCC) is 
required for their use. 
The reaction between the compound (5) and the Sepharose derivative can be 
carried out according to any suitable method which enables the reaction of 
the primary amino group extending from the 5'-end with a group in the 
Sepharose derivative capable of reacting with that amino group (e.g., 
carboxyl group) such as formation of amide bonding through dehydration. 
Such a method is basically known in the art. Details of this method are 
set forth in the Experimental Examples presented hereinafter. 
When there is a possibility of a reaction occurring at groups other than 
the desired primary amino group extending from the 5'-end depending on the 
kind of the Sepharose derivative employed and/or the condensing 
conditions, the other groups of the compound (5) may be protected. 
Accordingly, the expression "causing an oligonucleotide to react with a 
Sepharose derivative capable of binding with amino group at the amino 
group of the oligonucleotide derivative" used in the present invention is 
also inclusive of a case wherein the compound (5) is protected in such a 
manner, and this expression is also inclusive of a case wherein the 
Sepharose derivative is in the form of its functional derivative. 
(3) Assay of binding amount and adsorption capacity: 
The amount of the compound (5) and Sepharose bound is shown by the amount 
of the compound (5) bound per 1 mg of Sepharose or the amount adsorbed 
when using HOA.sub.13 or HOT.sub.13 as a material to be adsorbed, which is 
expressed in terms of OD unit. 
Also, for comparison between the compound (6) synthesized according to the 
present invention and the carrier synthesized according to the method of 
the prior art [oligo(dT)-cellulose or oligo(dA)-cellulose (both of which 
are commercial products)], similar adsorption tests were conducted. 
Similarly, as confirmation of formation of the compound (6) (namely 
determination of the binding amount), examination of bonding of the 
oligonucleotide having no amino group extending from the 5'-phosphate 
group according to the present invention such as tridecaadenylic 
acid(HOA.sub.13), tridecathymidylic acid (HOT.sub.13) is important for 
establishing the bond positions. 
As a result, it is possible to obtain a compound (6) which exhibits a 
binding amount approximating a level of 0.06 OD/mg, and it can be seen 
that all of the compounds (5) can be bonded irrespective of their base 
sequences. Also, because there occurs no bonding between an 
oligonucleotide having no amino group and a carrier whatsoever, it can be 
seen that the compound (5) undergoes no bonding at its base moiety but 
only through its primary amino group. 
On the other hand, according to the experiments by the present inventors, 
among the commercially available resins, an adsorption capacity of about 
0.010 to 0.037 OD/mg was sometimes assayed in oligo(dT)-cellulose, but no 
reproducible value was obtained when the same assay was repeated again. On 
the other hand, there exists substantially no adsorption capacity in 
oligo(dA)- cellulose [which may explain the fact known in the art that 
there is no oligo(dA)-cellulose of good quality, as compared with 
oligo(dT)-cellulose]. 
Thus, the compound (6) of the present invention may be said to be an 
affinity carrier of improved quality, which is bound to a carrier only 
through the primary amino group existing at the tip of the spacer newly 
developed, entirely free from non-specific bonding at other portions 
(e.g., amino group on the base moiety) and enables bonding of an 
oligonucleotide having any synthesizable base sequence to a carrier. 
EXPERIMENTAL EXAMPLES 
A. Compound (1) [Synthesis of R.sup.2 --NH--R.sup.1 --OH] 
Example 1 - 1 [the case where R.sup.1 =Hex (i.e., C.sub.6 H.sub.12 --), 
R.sup.2 =Tfa] 
(1) Reagent: 
6-Aminohexanol (1.17 g, 10 mmol) 
Trifluoroacetyl thioethyl (Tfa-SEt) (1.80 ml, 14.4 mmol) 
Dioxane (15 ml) 
(2) Synthesis: 
6-Aminohexanol (in an amount as shown above) was dissolved in dioxane (15 
ml), and trifluoroacetyl thioethyl (Tfa-SEt) (in an amount as shown above) 
was gradually added to the resultant solution, and the reaction was 
carried out at room temperature overnight. After the reaction, the mixture 
was concentrated and the residue dissolved in ether, after which 
extraction is carried out three times with water. The ether layer was 
dried over anhydrous sodium sulfate and concentrated. The residue was 
dissolved with addition of ether, and pentane was added for 
crystallization to produce the compound (1 - 1) as the powdery product. 
Yield: 1.40 g (70%) 
Example 1-2 [the case where R.sup.1 =Et (i.e., --CH.sub.2 CH.sub.2 --), 
R.sup.2 =Nps] 
The procedure in Example 1--1 was carried out with the use of 
2-aminoethanol (NH.sub.2 --EtOH) and o-nitrophenylsulphenylchloride 
(Nps--Cl). 
Example 1-3 [the case where R.sup.1 =Hex, R.sup.2 =Nps] 
The procedure in Example 1--1 was carried out with the use of 
2-aminohexanol (NH.sub.2 --HexOH) and o-nitrophenylsulphenyl chloride 
(Nps--Cl). 
TABLE 1 
______________________________________ 
Example Product Yield 
______________________________________ 
1-1 R.sup.1 = Hex, 
R.sup.2 = Tfa 
70% 
1-2 R.sup.1 = Et, 
R.sup.2 = Nps 
79% 
1-3 R.sup.1 = Hex, 
R.sup.2 = Nps 
72% 
______________________________________ 
B. Compound (2) [Synthesis of R.sup.2 --NH--R.sup.1 --.sub.p.sbsb.x 
(N'.sub.p.sbsb.x).sub.m R.sup.3 ] 
Example 2 - 1 [the case where R.sup.1 =Hex, R.sup.2 =Tfa, N'=A.sub.Bz, m=2 
and R.sup.3 =CE] 
(1) Reagent 
HO A.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x CE (Bz is N.sup.6 -benzoyl 
group) (800 mg, 0.71 mmol) 
o-Chlorophenyl phosphoroditriazolide (1.0 mmol) in Dioxane (6.0 ml) 
Compound (1 - 1) (300 mg, 1.4 mmol) 
1-Methyl-imidazole (115 mg, 1.4 mmol) 
(2) Synthesis: 
To HOA.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x CE (in an amount as shown 
above) made anhydrous by azeotropy with Py was added a solution of 
o-chlorophenyl phosphoroditriazolide (in an amount as shown above) in 
dioxane (in an amount as shown above), and the reaction was carried out 
for 2 hours. The progress of the reaction was checked by TLC (CHCl.sub.3 
--MeOH=14:1), and the compound (1 - 1) (in an amount as shown above) and 
1-methyl-imidazole (in the amount shown above) were then added to the 
mixture, and the reaction was carried out for 2 hours. The progress of the 
reaction was checked by TLC, and then water was added to decompose 
excessive triazolide. The solvent was evaporated off. The residue was 
dissolved in CHCl.sub.3, washed with water, 0.5 M-NaH.sub.2 PO.sub.4, 
saturated NaHCO.sub.3 and 5% NaCl aqueous solution and thereafter dried 
over anhydrous sodium sulfate. The CHCl.sub.3 layer was concentrated and 
purified through a silica gel short column (eluant : 0-4% 
MeOH/CHCl.sub.3). The desired product was collected, concentrated, and the 
concentrate was added dropwise into pentane to obtain a powdery compound 
(2 - 1). 
Yield: 610 mg (57%) 
Examples 2--2 to 2- 6 
The procedure in Example 2- 1 was carried out with the use of materials 
listed in Table 2 to obtain the results shown in Table 2 below. 
TABLE 2 
______________________________________ 
Exam- 
ple Product Yield 
______________________________________ 
2-1 R.sup.1 = Hex, R.sup.2 = Tfa, .sub.--N' = A.sup.Bz **, m = 
57% 
R.sup.3 = CE 
2-2 R.sup.1 = Hex, R.sup.2 = Tfa, .sub.--N' = A.sup.Bz, m 
86%, 
R.sup.3 = CE 
2-3 R.sup.1 = Hex, R.sup.2 = Tfa, .sub.--N' = T, m = 2, R.sup.3 = 
72% 
2-4 R.sup.1 = Hex, R.sup.2 = Tfa, .sub.--N' = T, m = 1, R.sup.3 = 
95% 
2-5 R.sup.1 = Hex, R.sup.2 = Nps, .sub.--N' = T, m = 2 
46% 
R.sup.3 = CE 
2-6 R.sup.1 = Pen*, R.sup.2 = Tfa, .sub.--N' = G.sup.iBu, m 
51%, 
R.sup.3 = CE 
______________________________________ 
*Pen = --C.sub.5 H.sub.10 
**A, T and G mean the groups --N derived from nucleoside, when the base i 
adenine, thymine and guanine, respectively; Bz indicates benzoyl and iBu 
isobutyryl. 
C. Compound (3) [Synthesis of R.sup.5 --O(N'.sub.p.sbsb.x).sub.n 
N'OCOR.sup.4 ] 
Example 3 - 1(the case where N'=A.sup.Bz, n=12, R.sup.5 =DMTr, and R.sup.4 
=-- -- .circle.Ps *) 
(1) Reagent: 
DMTr--OA.sup.Bz OCO-- -- .circle.Ps (300 mg, 0.033 mmol) 
DMTr--OA.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x .crclbar.Et.sub.3 
N.sym.H (150 mg, 0.1 mmol) 
MSNT (150 mg, 0.5 mmol) 
FNT *) DMTr is dimethoxytrityl, -- -- .circle.Ps is --(CH.sub.2).sub.2 
CONHCH.sub.2 -- -- .circle.Ps ( .circle.Ps is polystyrene). 
(2) Synthesis: 
DMTr--OA.sup.Bz OCO-- -- .circle.Ps was sampled (in the amount shown 
above), (1) washed with isoPrOH--CH.sub.2 Cl.sub.2 (15:85 v/v, 10 
ml.times.3), (2) detritylated with 0.1 M ZnBr.sub.2 solution in 
isoPrOH--CH.sub.2 Cl.sub.2 (15:85 v/v, 8 ml.times.4, total 20 minutes), 
(3) washed with isoPrOH--CH.sub.2 Cl.sub.2 (15:85 v/v, 10 ml.times.3), (4) 
washed with pyridine (Py.) (10 ml.times.3), and then (5) treated with 
Py--Et.sub.3 N--H.sub.2 O (3:1:1 v/v, 10 ml, 30 minutes) to be made 
anhydrous, which step was followed by addition of a Py solution of 
DMTr--OA.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x .crclbar.Et.sub.3 
N.sym.H (in the amount shown above) for azetropy with Py, to be made 
completely anhydrous. (6) To the resultant mixture were added MSNT (in the 
amount shown above) and anhydrous Py (2 ml), and the reaction was carried 
out with shaking for 90 minutes. After (7) washing with Py (10 
ml.times.3), (8) the reaction was carried out with addition of Ac.sub.2 
O--Py (1:9 v/v, 10 ml) containing a catalytic amount of 
dimethylaminopyridine (DMAP) for 10 minutes to protect the unreacted 
5'-hydroxyl groups. (9) By washing with Py (10 ml.times.3), the first 
condensation was completed. 
This procedure was repeated similarly 6 times to obtain the desired 
compound (3 - 1) (tridecaadenic acid). 
The yields by quantitative determination of trityl groups for respective 
reactions were found to be 89%, 83%, 80%, 79%, 81% and 90%, respectively. 
Overall yield: 34%. 
Example 3 - 1' (Deprotection) 
DMTr-O(A.sup.Bz.sub.p.sbsb.x).sub.12 A.sup.Bz OCO-- --Ps (15 mg) was 
sampled in a centrifugal precipitating tube, and a solution of 0.5 M 
TMG-Oxime in pyridine-H.sub.2 O (9:1 v/v) (100 .mu.l ) was added thereto, 
after which the mixture was left to stand at room temperature for 24 
hours. To this mixture was added conc. ammonia (2.5 ml), and the resultant 
mixture was left to stand in sealed state at 50.degree. C. overnight. The 
resin was filtered off, and the filtrate was concentrated, dissolved in 
water, and extracted three times with ether. The aqueous layer was 
concentrated and desalted with Sephadex G-50 (1.5.times.120 cm) [eluant: 
0.05 M-TEAB (triethylammonium bicarbonate) buffer, pH 7.5]. The elution 
pattern as shown in FIG. 2. 
The portions of the peaks obtained were collected, concentrated and treated 
with 80% acetic acid (2 ml, 10 minutes) to obtain tridecaadenylic acid 
(HOA.sub.13). The purity of this product was checked by HPLC 
(.mu.-Bondapak C-18), and the elution pattern obtained is shown in FIG. 3. 
Examples 3 - 2' to 3 - 6' 
As already reported, in the following papers, various compounds (3) were 
synthesized by repeating the procedure in Example 3 - 1. The yield 
obtained per condensation is about 85% on an average. 
Tetrahedron Letters 1979, 3635 (1979) 
Nucleic Acids Research 8, 5473 (1980) 
Nucleic Acids Research 8, 5491 (1980) 
Nucleic Acids Research 8, 5507 (1980) 
Nucleic Acids Research Symposium Series 7, 281 (1980) 
J. Am. Chem. Soc., 103, 706 (1981) 
Nucleic Acids Research 10, 197 (1981) 
D. Compound (4) [Synthesis of R.sup.2 --NH--R.sup.1 --O.sub.p.sbsb.x 
(N'.sub.p.sbsb.x).sub.m+n N'OCOR.sup.4 ] 
Example 4 - 1 (R.sup.1 =Hex, R.sup.2 =Tfa, N=A.sup.Bz, m=2, n=12, R.sup.4 
=-- -- .circle.Ps ) 
(1) Reagent: 
Compound (3 - 1) (DMTr--O(A.sup.Bz.sub.p.sbsb.x).sub.12 A.sup.Bz OCO-- -- 
.circle. Ps ) (115 mg, 3.45 .mu.mol) 
Compound (2 - 1) [Tfa--NH--Hex.sub.p.sbsb.x (A.sup.Bz.sub.p.sbsb.x).sub.2 
CE](60 mg, 0.4 mmol) 
MSNT (60 mg, 0.2 mmol) 
(2) Synthesis: 
The compound (3 - 1) (DMTr--O(A.sup.Bz.sub.p.sbsb.x).sub.12 A.sup.Bz OCO-- 
-- .circle. Ps ) was sampled (in the amount shown above), swelled well 
with isoPrOH--CH.sub.2 Cl.sub.2 (15:85 v/v, 10 ml.times.3) and then 
detritylated with a solution of 1 M--ZnBr.sub.2 in isoPrOHCH--CH.sub.2 
Cl.sub.2 (15:85 v/v, 5 ml.times.6, 30 minutes). The resin was washed with 
isoPrOH--CH.sub.2 Cl.sub.2 (15:85 v/v, 5 ml.times.3) and then with Py (5 
ml.times.3). On the other hand, the compound (2 -1) 
[Tfa--NH--Nex--.sub.p.sbsb.x (A.sup.Bz.sub.p.sbsb.x).sub.2 CE] (in the 
amount shown above) was sampled and subjected to decyanoethylation by 
treatment with Py--Et.sub.3 N--H.sub.2 O (3:1:1, 3 ml, 15 minutes). After 
evaporation of the solvent, the residue was subjected twice to azeotropy 
with Py. This was then dissolved in Py, and the resultant solution was 
added to the previous resin, the mixture being azeotroped with Py to be 
made completely anhydrous. MSNT (in the amount shown above) and anhydrous 
Py (15 ml) were added to this mixture, and the reaction was carried out 
with shaking for 90 minutes. After the reaction, the resin was washed with 
Py and MeOH then dried to produce the compound (4 - 1). 
Yield: 120 mg. 
Example 4 - 2 
Similarly as in Example 4 - 1, with the use of the compound (3 - 2) 
[DMTr--O(T.sub.p.sbsb.x).sub.12 TOCO-- -- .circle.Ps ] and the compound (2 
- 3) [Tfa--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.2 CE], the compound 
(4 - 2) [Tfa--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.14 TOCO-- -- 
.circle.Ps ] was synthesized. 
Example 4 - 3 
Similarly as in Example 4 - 1, with the use of the compound (3 - 3) 
[DMTr--O(A.sup.Bz.sub.p.sbsb.x).sub.9 A.sup.Bz OCO-- -- .circle.Ps ] and 
the compound (2 - 1) [Tfa--NH--Hex--.sub.p.sbsb.x 
(A.sup.Bz.sub.p.sbsb.x).sub.2 CE], the compound (4 - 3) 
[Tfa--NH--Hex--.sub.p.sbsb.x (A.sup.Bz.sub.p.sbsb.x).sub.11 A.sup.Bz OCO-- 
-- .circle.Ps ] 
was synthesized. 
Example 4 - 4 Similarly as in Example 4 - 1, with the use of the compound 
(3 - 4) 
[DMTr--OG.sup.iBu.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x 
G.sup.iBu.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x 
A.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
T.sub.p.sbsb.x - 
T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x C .sup.Bz OCO-- -- .circle.Ps ] 
and the compound (2 - 3) 
[Tfa--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.2 CE], the compound (4 - 
4) 
[Tfa--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.2 G.sup.iBu.sub.p.sbsb.x 
GiBu.sub.p.sbsb. x G.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x 
A.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
T.sub.p.sbsb.x T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
C.sup.Bz OCO-- -- .circle.Ps ] 
was synthesized. 
Example 4 - 5 
Similarly as in Example 4 - 1, with the use of the compound (3 - 2) 
[DMTr--O(T.sub.p.sbsb.x).sub.12 TOCO-- -- .circle.Ps ] and the compound (2 
- 5) [Nps--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.2 CE], the compound 
(4 - 5) [Nps--NH--Hex--.sub.p.sbsb.x (T.sub.p.sbsb.x).sub.14 TOCO-- -- 
.circle. ] was synthesized. 
Example 4 - 6 
Similarly as in Example 4 - 1, with the use of the compound (3 - 4) 
[DMTr--OG.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x 
G.sup.iBu.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x - 
T.sub.p.sbsb.x T.sub.p.sbsb.x T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
A.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub. p.sbsb.x 
T.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x A.sup.Bz OCO-- -- .circle.Ps ] 
and the compound (2 - 6) 
[Tfa--NH--Pen--.sub.p.sbsb.x (G.sup.iBu.sub.p.sbsb.x).sub.2 CE], the 
compound (4 - 6) 
[Tfa--NH--Pen--.sub.p.sbsb.x (G.sup.iBu.sub.p.sbsb.x).sub.2 
G.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x - 
A.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
T.sub.p.sbsb.x T.sub.p.sbsb.x T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb. x 
A.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x G.sup.iBz.sub.p.sbsb.x 
T.sub.p.sbsb.x - 
A.sup.Bz.sub.p.sbsb.x A.sup.Bz OCO-- -- .circle.Ps ] was synthesized. 
Example 4 - 7 
Similarly as in Example 4 - 1, with the use of the compound (3 - 6) 
[DMTr--OG.sup.iBu.sub.p.sbsb.x T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
G.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
T.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x - 
A.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x C.sup. 
Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x TOCO-- -- 
.circle.Ps ] 
and the compound (2 - 6) 
[Tfa--NH--Pen--.sub.p.sbsb.x (G.sup.iBu.sub.p.sbsb.x).sub.2 CE], the 
compound (4 - 7) 
[Tfa--NH--Pen--.sub.p.sbsb.x (G.sup.iBu.sub.p.sbsb.x).sub.2 
G.sup.iBu.sub.p.sbsb.x T.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x - 
G.sup.iBu.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
T.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x A.sup.Bz.sub.p.sbsb.x 
C.sup.Bz.sub.p.sbsb.x G.sup.iBu.sub.p.sbsb.x C.sup.Bz.sub.p.sbsb.x 
A.sup.Bz.sub.p.sbsb.x - 
G.sup.iBu.sub.P.sbsb.x TOCO-- -- .circle.Ps ] 
was synthesized. 
E. Compound (5) [Synthesis of NH.sub.2 --R.sup.1 --.sub.p (N.sub.p).sub.m+n 
OH] 
Example 5 - 1 
The compound (4 - 1) [Tra--NH--Hex--.sub.p.sbsb.x 
(A.sup.Bz.sub.p.sbsb.x).sub.14 A.sup.Bz --OCO-- -- .circle.Ps ] (15 mg) 
was sampled in a centrifugal precipitating tube, and a solution of 0.5 M 
TMG-Oxime in pyridine--H.sub.2 O (9:1 v/v) (100 ml) was added thereto, the 
mixture then being left to stand at room temperature for 24 hours. Then, 
after addition of conc. ammonia water (2.5 ml) thereto, the mixture was 
left to stand in a sealed state at 50.degree. C. overnight. The resin was 
filtered off, and the filtrate was concentrated, dissolved in water and 
extracted three times with ether. The aqueous layer was concentrated and 
thereafter subjected to desalting purification through Sephadex G - 50 
(1.5.times.120 cm) (eluant: 50 mM TEAB buffer, pH 7.5). The elution 
pattern is shown in FIG. 4. 
The peaks were collected and concentrated, and the purity of the compound 
(5 - 1) obtained was assayed by HPLC (.mu.-Bondapak C18). Its elution 
pattern is shown in FIG. 7. 
Example 5 - 2 
Similarly as in Example 5 - 1, the compound (4 - 2) was deprotected to 
synthesize the compound (5 - 2) [NH.sub.2 --Hex--.sub.p (T.sub.p).sub.14 
TOH. Its elution pattern is shown in FIG. 5 and FIG. 7. 
Example 5 - 3 
Similarly as in Example 5 - 1, the compound (4 - 3) was deprotected to 
synthesize the compound (5 - 3) [NH.sub.2 --Hex--.sub.p (A.sub.p).sub.11 
AOH]. 
Example 5 - 4 
Similarly as in Example 5 - 1, the compound (4 - 4) was deprotected to 
synthesize the compound (5 - 4) 
[NH.sub.2 --Hex--.sub.p (T.sub.p).sub.2 G.sub.p G.sub.p G.sub.p A.sub.p 
A.sub.p G.sub.p C.sub.p T.sub.p T.sub.p C.sub.p C.sub.p COH]. Its elution 
pattern is shown in FIG. 6 and in FIG. 9. 
Example 5 - 5 
Similarly as in Example 5 - 1, the compound (4 - 6) was deprotected to 
synthesize the compound (5 - 5) 
[NH.sub.2 --Pen--.sub.p (G.sub.p).sub.2 G.sub.p A.sub.p A.sub.p G.sub.p 
C.sub.p T.sub.p T.sub.p T.sub.p C.sub.p A.sub.p C.sub.p G.sub.p T.sub.p 
A.sub.p AOH]. 
Example 5 - 6 
Similarly as in Example 5 - 1, the compound (4 - 7) was deprotected to 
synthesize the compound (5 - 6) 
[NH.sub.2 --Pen--.sub.p (G.sub.p).sub.2 G.sub.p T.sub.p C.sub.p G.sub.p 
A.sub.p C.sub.p T.sub.p A.sub.p A.sub.p C.sub.p G.sub.p C.sub.p A.sub.p 
G.sub.p TOH]. 
F. Compound (6) [Synthesis of [Sepharose]--NH--R.sup.1 --.sub.p 
(N.sub.p).sub.m+n --NOH] 
Example 6 - 1 
(1) Reagent: 
BrCN-activated Sepharose 4B (40 mg) 
Compound (5 - 1) [NH.sub.2 --Hex--.sub.p (A.sub.p).sub.14 AOH (4.0 OD) 
(2) Reaction: 
The BrCN-activated Sepharose 4B was sampled (in the amount shown above), 
washed with 1 mM--HCl and further with a solution of 0.5 M--NaCl and 0.1 
M--NaHCO.sub.3 (pH 8.3), and the compound (5 - 1) (in the amount shown 
above) in a solution of 0.5 M-NaCl and 0.1 M--NaHCO.sub.3 (pH 8.3) (200 
.mu.l) was added thereto. While under gentle stirring, the reaction was 
carried out overnight at room temperature. After the reaction, the mixture 
was subjected to filtration, and the resin was washed with 10 mM-Tris-HCl 
(pH 7.5) and 0.5 M-NaCl, 10 mM-Tris-HCl (pH 7.5). 
(3) Assay of adsorption capacity: 
A half amount (20 mg) of this resin was sampled, and affinity 
chromatography was conducted with the use of synthetic tridecathymidylic 
acid for assay of the adsorption amount. 
Adsorption amount: 1.14 OD/20 mg resin (0.057 OD/mg) (FIG. 10) 
(4) Determination of the binding site: 
In place of the compound (6 - 1), tridecaadenyl (HO/A.sub.13) (crude 
product) was employed to carry out a similar operation. Substantially no 
binding was found, and none was detected when adsorption capacity was 
assayed. 
The results of the affinity chromatography column are shown in FIG. 11, 
which indicates that the adsorption capacity is substantially 0 OD/15 mg 
(0 OD/mg). 
(5) Conclusion: 
From the above results, it can be appreciated that no reaction occurs at 
all on the amino group at the adenine moiety. Therefore, bonding of the 
compound (6 - 1) to the carrier may be said to have occurred entirely at 
the amino group extended from the 5'-phosphate group. 
Example 6 - 2 
(1) Reagent: 
Activated CH-Sepharose 4B (30 mg) 
Compound (5 - 1) [NH.sub.2 --Hex--.sub.p (A.sub.p).sub.14 AOH](4.0 OD) 
(2) Reaction and assay of adsorption capacity: 
The activated CH-Sepharose 4B (in the amount shown above) was sampled and 
washed thoroughly with 1 mM HCl. After the resin was washed quickly with 
0.5 M-NaCl, 0.1 M-NaHCO (pH 8.3), the compound (5 - 1) (in the amount 
shown above) in a solution of 0.5 M-NaCl and 0.1 M-NaHCO.sub.3 (pH 8.3) 
(160 .mu.l ) was added thereto, and the reaction was carried out under 
gentle shaking at room temperature for 3 hours. After the reaction, the 
mixture was filtered, and the resin was washed with 10 mM-Tris-HCl (pH 
7.5) and 0.5 M-NaCl, 10 mM-Tris-HCl (pH 7.5). 
For the resin, the adsorption amount was determined with the use of 
tridecathymidylic acid and calculated similarly as in Example 6 - 1 (FIG. 
12). 
Adsorption amount: 0.62 OD/15 mg (0.042 OD/mg) 
(3) Determination of binding site: 
Adsorption capacity was assayed by carrying out the same reaction as in 
Example 6 - 1. 
Adsorption capacity: substantially 0 OD/15 mg (0 OD/mg) 
(4) Conclusion: 
Similarly as in Example 6 - 1, binding of 0.042 OD/mg may be said to have 
occurred entirely at the amino groups extended from the 5'-phosphate 
group. 
Commercially available oligo(dA)-cellulose are said to be bound at the base 
moiety of adenine, but under the condensing conditions employed, it 
appears that binding at the adenine base moiety, considered as one 
possibility, did not really occur at all. 
Example 6 - 3 
(1) Reagent: 
BrCN-activated Sepharose 4B (30 mg) 
Compound (5 - 2) (3.43 OD) 
HOT.sub.13 (2.47 OD) 
(2) Reaction and assay of the amount bound: 
From the results in Example 6 - 1, the oligothymidylic acid (HOT.sub.13) 
having no amino group was considered to be further less reactive than 
oligoadenylic acid (HOA.sub.13) and unreactive with BrCN-activated 
Sepharose, and, therefore, for making easier analysis by HPLC, the 
reaction was carried out with addition of HOT.sub.13 as internal reference 
substance. 
The reaction was carried out according to the procedure in Example 6 - 1. 
From the HPLC pattern of the solution before the reaction, the compound (5 
- 2) was found to be about 3.2 OD, HOT.sub.13 about 2.1 OD and unknown 
substances about 0.6 OD (5.9 OD as total), but after the reaction the 
compound (5 - 2) was found to be about 2.1 OD, HOT.sub.13 about 2.0 OD and 
unknown substances about 0.5 OD (4.9 OD as total) (FIG. 14), indicating 
that most of the Sepharose reacted with the compound (5 - 2). 
Bound amount: 1.1 OD/30 mg (0.037 OD/mg). 
(3) Assay of adsorption capacity: 
Adsorption capacity was assayed through an affinity column with the use of 
dA.sub.11 (crude product, containing 56% impurities). 
(1) As a result of applying crude dA.sub.11 (0.55 OD, dA.sub.11 
=corresponding to 0.24 OD), only the desired product can be purified and 
substantially completed (FIG. 15(A)). 
Non-adsorbed portion : 0.34 OD 
Adsorbed portion: 0.23 OD 
(2) As the result of adsorption and elution of crude dA.sub.11 (0.95 OD, 
dA.sub.11 =corresponding to 0.41 OD), the non-adsorbed portion was 0.73 OD 
and the adsorbed portion 0.27 OD. As a consequence, the column employed 
was found to have an adsorption capacity of 0.27 OD, which was about a 
half of the bound amount calculated from HPLC. This may be considered to 
be a loss during recovery of the reaction mixture, and it can be explained 
if the residual OD after crosslinking is considered to have been about 5.2 
OD. 
The HPLC pattern at the adsorbed portion is shown in FIG. 16. It can be 
seen that this dA.sub.11 is very pure. 
(3) Among the above non-adsorbed portions (0.73 OD, dA.sub.11 =0.15 OD), 
the portion of 0.31 OD (dA.sub.11 corresponding to 0.06 OD) was eluted 
again through the column. The non-adsorbed portion was 0.28 OD and the 
adsorbed portion 0.05 OD. 
From the HPLC pattern of the non-adsorbed portion, it can be seen that the 
non-adsorbed portion contained no dA.sub.11 whatsoever (FIG. 17). 
The results of the above chromatography are listed in FIG. 15. 
Examples 6 - 4 to 6 - 8 
A procedure similar to Example 6 - 3 was carried out to obtain the results 
shown in Table 6 shown below. The results in Example 6 - 4 corresponding 
to those in FIG. 14 in Example 6 - 3 are also shown in FIG. 18. 
Comparative Example 1 
(1) Resin: 
Commercially available oligo(dA)-cellulose (P-L Biochemicals Lot No. 
115577) 
(2) Assay of adsorption capacity: 
The above resin was sampled in an amount of 20 mg and, after being caused 
to swell with a 0.5 M-NaCl, 10 mM-Tris-HCl (pH 7.5) solution, was packed 
in a column and thereafter washed with 10 mM-Tris-HCl (pH 7.5) and 0.5 
M-NaCl, 10 mM-Tris-HCl (pH 7.5) solutions. For this column, the adsorption 
capacity was assayed with the use of a synthetic tridecathymidylic acid 
(HOT.sub.13). 
Eluant: 10 mM Tris-HCl (pH 7.5) 
One fraction: 15 droplets (350 .mu.l ) 
(1) When 2.1 ml of a solution of HOT.sub.13 with 1.33 OD in 0.5 M-NaCl, 10 
mM-Tris-HCl solution was applied, almost no adsorption thereof occurred. 
(2) The eluate from the above (1) was recovered and adsorption was 
attempted again. No adsorption whatsoever occurred. 
(3) A column was newly prepared, and assay was similarly conducted. With 
regard to the above three points, the results are shown in FIG. 19. 
(3) Conclusion: 
In spite of its being a commercially available resin for adsorption, it has 
almost no adsorption capacity. 
It has been known in the art that there is no oligo(dA)-cellulose of good 
quality, as compared with oligo(dT)-cellulose, and these results may be 
construed to support this fact. 
Comparative Example 2 
(1) Resin: 
Commercially available oligo(dT)-cellulose (P-L Biochemicals Lot No. 
675130) 
(2) Assay of adsorption capacity: 
Similarly as in Comparative Example 1, assay was conducted with the use of 
HOA.sub.13. The results calculated from FIG. 20 are shown in Table 5 
below. 
TABLE 5 
______________________________________ 
Amount of vital Amount Adsorption 
test sample adsorbed capacity 
(.mu.l) (OD/20 mg) (OD/mg) 
______________________________________ 
FIG. 20-A 
4.3 OD/200 0.73 0.037 
FIG. 20-B 
3.0 /350 0.41 0.021 
FIG. 20-C 
1.5 /700 0.31 0.016 
FIG. 20-D 
1.7 /350 0.19 0.010 
FIG. 20-E 
1.8 /350 0.26 0.013 
______________________________________ 
(3) Conclusion: 
Depending on the difference in amount or concentration of the vital test 
sample, or by repeating adsorption and elution, the adsorption capacity 
varies, whereby no reproducible result can be obtained. 
The above results are summarized in Table 6. 
TABLE 6 
__________________________________________________________________________ 
Sample Bound amount 
Adsorption capacity 
Carrier 
Oligo (dN) 
OD/mg 
HPLC Test sample 
OD/mg 
Column 
__________________________________________________________________________ 
Example 
6-1 a Compound(5-1) HOT.sub.13 
0.057 
FIG. 10 
6-1 a HOA.sub.13 HOT.sub.13 
0 FIG. 11 
6-2 b Compound(5-1) HOT.sub.13 
0.047 
FIG. 12 
6-2 b HOA.sub.13 HOT.sub.13 
0 FIG. 13 
6-3 a Compound(5-2) 
0.037 
FIG. 14 
HOA.sub.13 
0.037 
FIG. 15 
6-4 b Compound(5-2) 
0.049 
FIG. 18 
HOA.sub.13 
0.049 
6-5 a Compound(5-4) 
0.004 * ** 
6-6 b Compound(5-4) 
0.016 * ** 
6-7 a Compound(5-5) 
0.024 *** 
6-8 a Compound(5-6) 
0.035 **** 
Commercial 
product 
1 c -- HOT.sub.13 
0 FIG. 19 
2 d -- HOA.sub.13 
0.010 
FIG. 20 
-0.037 
__________________________________________________________________________ 
a = BrCNactivated Sepharose 4B; 
b = activated CHSepharose 4B 
(a and b are produced by Pharmacia Fine Chemicals, U.S.A.); 
c = Oligo (dA)cellulose (Lot No. 115577); 
d = Oligo (dT)cellulose (Lot No. 675130); 
*.sup.5' HOG.sub.p G.sub.p G.sub.p A.sub.p A.sub.p G.sub.p C.sub.p T.sub. 
T.sub.p C.sub.p C.sub.p COH.sup.3' ; 
**not adsorbed due to selfcomplementarity, unmeasurable; 
***.sup.5' HOT.sub.p T.sub.p A.sub.p C.sub.p G.sub.p T.sub.p G.sub.p 
A.sub.p A.sub.p A.sub.p G.sub.p C.sub.p T.sub.p T.sub.p 
****.sup.5' HOA.sub.p C.sub.p T.sub.p G.sub.p C.sub.p G.sub.p T.sub.p 
T.sub.p A.sub.p G.sub.p T.sub.p C.sub.p G.sub.p A.sub.p COH.sup.3