JP3256539B2 - Solid support synthesis of 3'-tailed oligonucleotides via linking molecules - Google Patents

Description

BACKGROUND OF THE INVENTION The present invention relates to a method for synthesizing an oligonucleotide having a low molecular weight tail molecule bonded to the 3′-terminus of an oligonucleotide via a linking molecule. Oligonucleotides with attached low molecular weight tail molecules and intermediates utilized in the synthesis of such oligonucleotides.

Oligonucleotides have a variety of uses, including acting as primers for the polymerase chain reaction synthesis of DNA. Oligodeoxynucleotide (ODN)
Is conveniently synthesized on a solid support using the phosphite-triester synthesis method. See T. Atkinson et al. For details of such synthesis (T. Atkinson, M. Smith, Oligonucl
eatide Synthesis, Apractical Approach, MJGait (e
d.), IRL press, pp. 35-81 (1984)]. This document discloses detailed steps depending on the process conditions for the actual synthesis of oligonucleotides. In fact, the method described in this document is currently utilized in commercial oligonucleotide synthesizers available from various manufacturers.

Aseline et al. [Asselin eet.al (Proc.Natl.Acad.Sc.
i. 81 , 3297-3301 (1984))] discloses the synthesis of certain oligonucleotides. There, an intercalating agent is covalently attached to the oligodeoxynucleotide. The intercalating agent used was 2
-Methoxy-6-chloro-9-aminoacridine. The acridine molecule is covalently linked to the oligonucleotide via a methylene chain of 3 to 6 carbon atoms connecting the 3'-phosphate of the oligodeoxynucleotide to the 9-amino group of the acridine molecule. The authors of this study show that acridine-modified oligonucleotides can be intercalated (ie,
Acridine). The authors measured certain thermodynamic parameters and show that through these parameters the covalent attachment of the acridine ring strongly stabilizes the binding to the synthetic oligonucleotide complementary sequence. The melting temperature (Tm) of the oligonucleotide with the bound intercalating agent and complementary strand is higher than the melting temperature of a similar oligonucleotide without the intercalating agent and complementary strand. The authors conclude that these results clearly show that the presence of the intercalating agent strongly stabilizes the complex formed between the oligonucleotide and its complementary strand. ing.

In a similar study, Letsinger et al. (Letsinger et.a.
l. (Proc. Natl. Acad. sci. 86 , 6553-6655 (1989))),
A group of oligonucleotides having cholesteryl groups covalently linked at the phosphate linkage between the 3 'termini of the nucleotides has been prepared. Oligomers of various lengths have been synthesized. Oligomers having a cholesteryl moiety adjacent to either the 3 'terminus or both the 3' and 5 'termini are compared to oligomers not so substituted. These compounds were tested for their inhibitory effect on HIV-replication. Immobilization of one cholesteryl group adjacent to the 3 'end of the 20 oligonucleotides substantially increased the antimicrobial activity of the oligonucleotides. When the second cholesteryl group was immobilized on the 5 'end of the oligonucleotide, the antibacterial activity was reduced as compared with the monocholesteryl derivative oligomer.

The compounds of Rettsinger et al. Were first prepared by hand with a support-bound dinucleoside hydrogen phosphonate derivative. The cholesteryl group is then attached to the internucleotide phosphorus by oxidative phosphoramidation. Oligonucleotides use phosphoramidation, commercially available DNA
It was extended from the first dinucleoside by a synthesizer.

Commercially available controlled-perforated glass beads for oligonucleotide synthesis are available [CPG, Inc., Pierce Ch.
emical Co. and Sigma Chemical Co.]. As described above (Atkins and Smith), controlled pore glass beads (hereinafter CPG) are long-chain alkylamines, for example, having free amino groups available at their termini while being bound to CPG. Using an alkylamine
Modified by manufacturer. Various amide bonds are obtained with the terminal amine of the long-chain alkylamine on the CPG for attachment to the oligonucleotide that grows during its synthesis. An improvement in this synthesis was described by Pon et al. (Pon et. Al. Bio Techni
ques 6 (8), 768 (1988)). In this report, Pon et al. Pre-captured potentially contaminating side chains on long-chain alkylamine CPGs and replaced DEC (dicyclohexylcarbodiimide), a commonly used toxic coupling reagent, with DEC (a water-soluble carbodiimide 1).
-(3-dimethylaminopropyl) -3-ethylcarbodiimide or EDC) has been introduced.

[Nelson et.al (Nuc. Acids Research 17
(18), 7187-7194 (1989))] recently report the synthesis of oligonucleotides incorporating a 3 'terminal substituent. To produce an oligonucleotide with a 3'-terminal tail molecule, Nelson et al., Treated with succinic anhydride in the presence of DMAP (dimethylaminopyridine) to give a secondary hydroxy group N-Fmoc-O.
-DMT-amino-l, 2-propanediol was derived and subsequently treated with p-nitrophenol in DCC. The activated derivative is then immobilized on a long-chain alkylamine CPG carrier. The dimethoxytrityl protecting group is removed from the primary alcohol propanediol and the oligonucleotide is
The compound is synthesized stepwise from primary hydroxy groups while fixed on a carrier. The synthetic nucleotide was deprotected and separated from the CPG carrier. However, the purity of this 3'-amine modified oligonucleotide has not been proven. At this stage of the synthesis, the 3 'terminal substituent must still be attached to the oligonucleotide. Biotin-
Biotinylation with XX-NHS ester. After biotinylation, a second purification by both Sephadex and HPLC is required.

The above method of Nelson et al. Forms oligonucleotides with functional groups that can be derivatized and modified with a tail molecule forming reagent, and then repurified. However, derivatization with a tail molecule-forming agent occurs only after completion of oligonucleotide synthesis. Valuable oligonucleotides that are finally completed systematically through the steps of assembling nucleotides by nucleotides suffer losses due to incomplete reactions, side reactions, and the number of purification operations required after derivatization due to derivatization to such oligonucleotides. It is. In addition, this synthesis does not employ the Pon et al. Improvement described above.

(Overview of the Invention) The usefulness of oligonucleotides is described, for example, in Letsinger.
Cholesterol with 3'-tail molecule and Asseline et al.
And 3'-tail molecule with acridine oligonucleotide
By including a low molecular weight group at the 3 'end as in the above example
Thus, it can be improved. With 3'-tail molecule in serum
The stability of the oligonucleotide is also increased. example
For example, unmodified ODN may be 3'-oxonucleated in media containing serum.
Degraded rapidly by creatase. 3'-terminal phosphodi
Certain chemical modifications of the ester can prevent this degradation.
Wear. [Show et.al. (Nucl.Acids Res.19
(4), 747 (1991))] is between the last two nucleotides
The phosphodiester bond of phosphorothioate, phospho
Nuclear when converted to roamidate, or vice versa
The stability to zeolites was significantly improved. Nuclease stable
Other bonds that have been shown to improve the
Oxynucleotide derivatives and methylphosphonate induction
There are conductors.

In the first aspect of the present invention, it is a major object to provide an improved method for oligonucleotide synthesis having a low molecular weight tail molecule attached to the 3 'end of the oligonucleotide. Yet another object is to provide a method for the synthesis of oligonucleotides of low molecular weight linked to an oligomer via a linking molecule. Another object of the invention is at the 3 'end,
To provide an oligonucleotide derivatized with a phosphonate ester and a tail molecule attached to the phosphonate ester. Furthermore, the purpose is to provide a suitable linking molecule and support for the production of oligonucleotides. It is a further object to provide a linking molecule with a suitable low molecular weight molecule such that an oligonucleotide can be assembled.

A second aspect of the present invention discloses a linking molecule having a protected amino group that can be used for oligonucleotide synthesis.
The third aspect of the present invention provides a tail molecule adsorbent having an insertion group (intercalating group) that can be added to an oligonucleotide modified with an amino group.

A fourth aspect of the present invention discloses a linking molecule having a protected alkanol group that can be used for oligonucleotide synthesis.

These and other objects as will become apparent from the rest of the specification are achieved in an oligonucleotide synthesis method having a low molecular weight tail molecule attached to the 3 'end via a linking molecule. The method of the first embodiment is to select as a linking molecule a molecule having three independent functional groups, each exhibiting a different chemical reactivity, apart from the reactivity of the other two functional groups. Consists of The first functional group of the linking molecule is reacted with a low molecular weight tail molecule to attach the tail molecule to the linking molecule. The second functional group of the linking molecule is treated with succinic anhydride and the resulting carboxylic acid residue is reacted with the solid support to bind or bind to the linking molecule with a tail molecule attached to the solid support. Fix it.

Throughout the disclosure herein, the attachment of a linking molecule having a hydroxyl group (eg, R-OH) to a solid support, as described above,
Complete via succinate linkage. Thus, in the "support-OR" formula, the designation "support-O" includes such succinate linkages.

The first 3'-phosphoramidite nucleotide is then reacted with a third functional group on the linking molecule, linking the nucleotide to the linking molecule via the 3 'end. When the first nucleotide is attached to the linking molecule, the nucleotide binds to the tail molecule via the linking molecule and also attaches or is fixed to the solid support via the linking molecule. In addition, the 3'-phosphoramidite nucleotide is subsequently reacted with the 5 'end of the preceding nucleotide and the oligonucleotide's
Generate a synthetic oligonucleotide attached to the linking molecule at the 3 'end. As for the first nucleotide,
It binds to the tail molecule and the solid support as the synthetic nucleotide binds to the linking molecule via the 3 'end.
Thus, the growing oligonucleotide is bound to a solid support while further reacting with the oligonucleotide. The oligonucleotide with the tail molecule attached to the 3 'end via the linking molecule is then detached from the solid support by breaking the link between the second functional group of the linking molecule and the solid support. An oligonucleotide having a tail molecule attached to the 3'-end via a linking molecule is then isolated.

In a preferred embodiment of the first aspect of the present invention, the functional groups on the linking molecule include primary alcohols, secondary alcohols and amines. Reacting the tail molecule with an amine to bind the tail molecule to the linking molecule and reacting the solid support with the secondary alcohol to attach the linking molecule having the tail molecule to the solid support; The roamidite nucleotide is reacted with the primary alcohol to bind the linking molecule.

(2S, 4R) -4-hydroxy-2-hydroxymethylpyrrolidine (4-hydroxy-2S-trans) -2-pyrrolidinemethanol or trans-4-hydroxy-
L-prolinol) is particularly preferred as a linking molecule.

The tail molecule is a reporter group, an intercalating group,
It is screened as any one of a number of molecules of interest, including lipophilic groups, and cleavage groups (cleaving groups). A suitable lipophilic group is cholesterol. Suitable as reporter groups are biotin and fluorophores such as acridine, fluorescein, rhodamine, lysamine rhodamine B, malachite green, erythromycin,
Tetramethylrhodamine, eosin, pyrene, anthracene, 4-dimethylaminonaphthalene, 2-dimethylaminonaphthalene, 7-dimethylamino-4-methylcoumarin, 7-dimethylaminocoumarin, 7-hydroxy-
4-methylcoumarin, 7-hydroxycoumarin, 7-methoxycoumarin, 7-acetoxycoumarin, 7-diethylamino-3-phenyl-4-methylcoumarin, isoluminol, benzophenone, dansyl, dabsyl, mansyl, sulfofamine, 4-acetylamide -4'-stilbene-2,2'-disulfonate disodium salt and 4-
Benzamide-4'-stilbene-2,2'-disulfonate disodium salt. Suitable as intercalating groups are acridine, ellipticine, methidium, ethidium, phenan, phenanthroline, throline,
-Hydroxyethanethiolate-2,2'2 "-terpyridine-platinum (II) and quinoxaline. Also suitable as cleavage groups are EDTA ligands or porphyrins for binding iron (Fe) Ligands and phenanthroline (phenanthroline) ligands for binding copper (Cu).

For the reaction of the linking molecule with the amine, the tail molecule is selected to include a unique connecting group, or an additional connecting group is attached to the tail molecule via appropriate chemical synthesis. When an additional connecting group is used, it is used after linking to link the tail molecule to the connecting molecule like a unique connecting group. Regardless of whether intrinsic or additional connecting groups are used, connecting groups are those that react with the linking molecule to link the tail molecule to the linking molecule.

After binding the tail molecule to the linking molecule via the first functional group and before linking the linking molecule to the solid support,
The third functional group of the linking molecule is selectively protected. The linking molecule with the protected third functional group is then attached to the solid support via the second functional group.
The third functional group is then deprotected and the first phosphoramidite nucleotide is attached to the linking molecule via the deprotected functional group.

The object of the first aspect of the present invention is further achieved by an oligonucleotide derivative having a 3'-terminal hydroxyl group (hydroxy group). The phosphate ester is located on the 3'-terminal hydroxyl group and has the formula Or m and m 'are each independently a positive integer smaller than 11, n is 0 or 1, Q is a connecting group, and R is selected from the group consisting of a reporter group, an intercalating group, a lipophilic group and a cleavage group. Have.

Of this group, particularly preferred are structural formulas Is a compound of

Particularly preferred 3'-tailed oligonucleotides include oligonucleotides having either cholesterol or acridine attached to the 3 'end of the oligomer via a linking molecule. The cholesterol group is attached to the linking molecule utilizing a carbamate bond, and the acridine group, preferably 9-ethylacridine or another 9-alkylacridine, is an amide bond (actually the ethyl group of 9-ethylacridine is assumed). (In this case, an alkylamine bond).
The other tail molecule is linked to the linking molecule via a urea, thiourea or sulfonamide bond.

In attaching the tail molecule to the linking molecule, the connecting group Q preferably comprises alkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkyl, aryl, aryloxy, aralkyl, heterocyclic, heteroaryl, substituted aryl, substituted aralkyl. Selected from the group.

Further, an object of the first aspect of the present invention is that Or m and m 'are independently positive integers less than 11;
Or 1, Q is a connecting group, R is selected from the group consisting of a reporter group, an intercalating (inserting) group, a lipophilic group and a cleavage group, Y is H or dimethoxytrityl and X is a solid support A) a compound and a support for oligonucleotide synthesis comprising the compound. Useful supports include controlled pore glass supports derived from long chain alkylamines.

Of this group, particularly preferred are structural formulas Is a compound of

The purpose of the first aspect of the present invention is to further provide a structural formula Or m and m ′ are independently positive integers less than 11;
Or 1, Q is a connecting group, R is selected from the group consisting of a reporter group, an intercalating group, a lipophilic group and a cleavable group, and Y is H or dimethoxytrityl).

Particularly preferred of this group are structural formulas Is a compound of

In each of the above structural formulas, m and m ′ are positive integers smaller than 11, that is, each independently 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Particularly preferred are compounds wherein m and m 'are 6 or less, ie, independently 1,2,3,4,5 or 6.

Another aspect of the claimed invention is to provide improved compounds and methods for the synthesis of the conventional carboxylic acid tetrafluorophenyl (TFP). Such activated ester derivatives are advantageously used for (1) the synthesis of acridine-CPG (which can also be used for the synthesis of 3′-tailed oligonucleotides),
(2) It can be used for modification after the synthesis of oligonucleotide having a 3'-amine-tail molecule, and (3) it can be used for modification of lignonucleotide of internal amine modification.

Embodiments of the present invention further include aminohexyl modified / advantageously used for the synthesis of 3'-tailed oligonucleotides.
Disclosed are a solid support and a hexanol-modified solid support.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows the time course of the swimming behavior of Paramecium after injecting antisense ODN39 with microsilidine.

FIG. 2 shows the biological effects of antisense ODN39-random or sense ODN.

FIG. 3 is a dose response curve of antisense ODN39 in an assay of the swimming behavior of paramesium.

Detailed description of the invention A.1.3 Modified solid support with 3'-tail molecule for direct synthesis of oligonucleotides with a tail-molecule (Method A) It has been found that an oligonucleotide having a low molecular weight tail molecule bonded to the 3 'end of the oligonucleotide can be synthesized. The linking molecule is carried to have three chemically distinct functional groups.
With such a linking molecule, a low molecular weight tail molecule is first attached to the linking molecule. The linking molecule with the low molecular weight tail molecule is then bound or immobilized on a solid support. The oligonucleotide is then synthesized. During synthesis, the oligonucleotide is fixed to a linking molecule, which is then fixed to a solid support system. Oligonucleotides are synthesized using standard phosphoramidite chemistry, either manually or on a DNA synthesizer. Upon completion of the synthesis of the oligonucleotide, the synthetic oligonucleotide having the linking molecule and the low molecular weight tail molecule attached thereto is cleaved from the solid support. This releases the oligonucleotide from the solid phase system. Oligonucleotides lead to having a low molecular weight tail molecule attached to the 3 'end via a linking molecule.

Using the above manufacturing steps, oligonucleotides with low molecular weight tail molecules attached to the 3 'end need only be subjected to a single purification step. Thus, an oligonucleotide having the molecule bound to the 3 'end can be easily and rapidly produced, as compared with the prior art.

The linking molecule with a suitable low molecular weight attached thereto is synthesized independently of oligonucleotide synthesis. Once the linking molecule with the attached tail molecule is made by reacting the tail molecule with the first functional group of the linking molecule, the combination of linking molecule and tail molecule is each 3 '
Due to the production of a number of different oligonucleotides with low molecular weight molecules attached to their ends, they can be regarded as suitable reagents for use in DNA synthesizers. This allows for the synthesis and storage of large quantities of combinations of linking molecules and tail molecules, if desired. In addition, other combinations of linking molecules with various other tail molecules are made in a similar manner. These can be used by non-organic chemists of oligonucleotide synthesis using a DNA synthesizer. Thus, even those who are unskilled in the synthetic organic chemistry department, as the first reagent in such an automatic DNA synthesizer for the synthesis of nucleotides with a selected low molecular weight tail molecule attached to the 3 'end. Some of the linking molecule-tail molecule combinations can be readily used.

In addition, many oligonucleotides that have a common sequence for many nucleotides and have different sequences for the remainder of the oligonucleotide but have the same 3'-tail molecule in question as the linking molecule are simply partially It can be produced by resolving the resulting solid support with the "common" oligonucleotide and linking it to the linking molecule and the tail molecule of interest. The synthesis of the different parts of the oligonucleotide is accomplished by loading the individual parts of the solid support with the common part of the oligonucleotide into a DNA synthesizer to complete the desired sequence of nucleotides.

Compound (2S, 4R) -4-hydroxy-2-hydroxymethylpyrrolidine, compound 2 of Step I, (4-hydroxy- (2S-trans) -2-pyrrolidinemethanol or trans-4-hydroxy-L-prolinol The same) serves as a particularly useful linking molecule to attach the molecule of interest to the 3 'end of the oligonucleotide. This compound is readily available from commercially available N-CBZ-L-hydroxyproline (sig
ma chemical Co.). Stanfield et al. [Stanfield et.al. (J. Org. Chem. 46 , 4799 (1981))
N-CBZ- using a method similar to that described by
Reduction of L-hydroxyproline is achieved using borane-tetrahydrofuran complex (Aldrich chemical Co.). This reduction proceeds while maintaining the optical purity, and compound 1 is obtained. The CBZ protecting group of compound 1 is easily removed using palladium-carbon reduction under a stream of hydrogen. This reaction proceeds smoothly and gives a quantitative yield of compound 2.

Compound 2 contains a primary hydroxyl group that is utilized as the base material from which oligonucleotides are synthesized. In addition, compound 2 contains a secondary alcohol group used to attach the solid support and a secondary amine used to attach the tail molecule of interest. Upon completion of oligonucleotide synthesis, the tail molecule of interest remains bound to the oligonucleotide via the linking molecule. After cleavage of the completed oligonucleotide containing the molecule of interest linked via the linking molecule from the solid support, the secondary hydroxyl groups of the linking molecule are essentially spatially retained and the linking molecule becomes the first nucleotide. Move away from near the phosphate bond to be bound. This results in increased stability of the linkage between the linking molecule and the 3 'terminal phosphonate group of the oligonucleotide.

Furthermore, since compound 2 is a single optical isomer and not a mixture of various stereoisomers, the bond between the 3 'end of the oligonucleotide and the linking molecule also gives rise to a single isomer, Does not produce a mixture of stereoisomers that affects properties. This is particularly important when attaching an intercalating group or other reactive group to the 3 'end of the oligonucleotide. This is because the optimal binding of the intercalating agent to double-stranded nucleotides and complementary DNA moieties has precise and important prerequisites for steric morphology.

Other linking molecules are structural formulas (Wherein m and m ′ are positive integers from 1 to 10)
It is. These linking molecules also have an amino group in the structure,
It also has a primary hydroxyl group and a secondary hydroxyl group. Two particularly useful linking molecules of this class are 3-amino-1,2-propanediol and 4-amino-1,3-butanediol (13) (see Step III).

The attachment of the tail molecule of interest to the linking molecule takes place completely independently of the oligonucleotide synthesis described above. The low molecular weight tail molecule of interest is attached to the linking molecule using organic chemistry techniques and reactions. The secondary amino group of the linking molecule can link the low molecular weight tail molecule of interest to the linking molecule, eg, compound 2, with a number of suitable connecting groups or, for example, an amide, carbamate, urea, thiourea, or sulfonamide linkage. It is used to join through any of the various joins. Given this disclosure, a linking molecule such as compound 2
Other suitable reactions between the secondary amine and the compound of interest with a suitable connecting or linking group will also be suggested to those skilled in the art.

For purposes of clarity herein and in the appended claims, confusion between the term "linking molecule" and the term "linking group" as used to attach a tail molecule to the linking molecule In order to avoid
The term group ") is used to indicate a connecting or linking group. Regardless of the name given to these groups, these groups link, connect, or attach the tail molecule to the linking molecule.

The molecule of interest to be the 3 'tail molecule of the oligonucleotide,
When thus connected to a linking molecule, the primary hydroxyl group on the linking molecule is preferably protected with a group such as, for example, a dimethoxytrityl group. This is conveniently achieved using dimethoxytrityl chloride (DMTrcl) in pyridine in the presence of 4-dimethylaminopyridine (DMAP). This selectively protects the primary alcohol group of the linking molecule. The secondary alcohol of the linking molecule is then converted to a succinic ester using succinic anhydride. The succinate of the linking molecule having the low molecular weight compound of interest is p-nitrophenol-D from Atkinson et al.
It is coupled to a controlled pore glass support using either the CC conventional method (supra) or preferably the modified DEC method of Pon et al. (Supra). DEC is currently the preferred method, as long as it is less toxic than DCC, because it is water soluble and eliminates the need to treat succinate with p-nitrophenol.

The linking molecule having the linked low molecular weight compound of interest, protected with a dimethoxytrityl group, is then linked or immobilized in a standard manner to a regulated pore glass support having linked, long chain alkylamine groups. Restricted pore glass supports derivatized with long-chain alkylamines are available [Pierce Chemical or Sigma Chemical].
These were previously activated with dichloroacetic acid using the method of Pont et al.
After reacting the linking molecule with the dimethoxytrityl group-protected linking molecule having the binding molecule of interest in pyridine to link the linking molecule to the solid support, the excess long-chain alkylamino group on the support is used. Is capped by acetylation with acetic anhydride.

The dimethoxytrityl group is removed from the primary alcohol of the linking molecule by treatment with 3% dichloroacetic acid in dichloroethane. The resulting regulated pore glass support, having the primary hydroxyl group of the deprotected linking molecule together with the low molecular weight tail molecule linked via the linking molecule, is ready for oligonucleotide synthesis. Producing also large amounts of solid supports with linking molecules and molecules of interest;
It can then be aliquoted for the synthesis of multiple oligonucleotides or stored for later use.
In any case, oligonucleotide synthesis starts from the primary hydroxyl group of the linking molecule using standard methods, for example using formamide chemistry on a DNA synthesizer, such as a Milligen DNA synthesizer.

Once the synthesis of the oligonucleotide is complete, the oligonucleotide is deprotected using standard methods for oligonucleotides synthesized on an automated DNA synthesizer. Oligonucleotides with a low molecular weight tail molecule attached to the 3 'end via a linking molecule are then cleaved from the solid support by standard methods of automated DNA synthesis using concentrated ammonia at room temperature in a conventional manner.

To purify an oligonucleotide having a low molecular weight tail molecule attached to the 3 'end via a linking molecule, only one purification is required. This can be conveniently performed using reverse phase high performance liquid (HPLC) chromatography. The low molecular weight tail molecule to be attached to the 3 'end of the oligonucleotide can be one of many molecules of biological interest. Included in this group can be reporter groups, intercalating (insertion) groups, lipophilic groups and cleavage groups. Of the lipophilic groups, currently particularly preferred is cholesterol. Particularly preferred at present for reporter groups are biotin and fluorophores, such as acridine, fluorescein, rhodamine, lisamine rhodamine B, malachite green, erythrosine, tetramethylrhodamine, eosin, pyrene, anthracene, 4-dimethylaminonaphthalene,
-Dimethylaminonaphthalene, 7-dimethylamino-4
-Methylcoumarin, 7-dimethylaminocoumarin, 7-
Hydroxy-4-methylcoumarin, 7-hydroxycoumarin, 7-methoxycoumarin, 7-acetoxycoumarin, 7-diethylamino-3-phenyl-4-methylcoumarin, isoluminol, benzophenone, dansyl,
Dabsyl, mansyl, sulfo-rhodamine, 4-acetamido-4'-stilbene-2,2'-disulfonate disodium salt, 4-benzamide-4'-stilbene-2,2'-disulfonate disodium salt . Particularly preferred for intercalating groups are acridine, ellipticine, methidium, ethidium, phenanthroline, 2-hydroxyethanethiolate-2,2 ', 2 "-terpyridine-platinum (II) and quinoxaline. Of the cleavage groups, particularly preferred are EDTA or porphyrin ligands for binding iron and phenanthroline ligands for binding copper.

For those tail molecules that do not contain a unique connecting group for attaching the tail molecule to the amino group of the linking molecule, the tail molecule can be reacted with a suitable reagent to provide an additional connection capable of reacting with the amine substituent of the linking molecule. Attach groups. Thus, regardless of whether the tail molecule has a unique connecting group capable of reacting with the amine of the linking molecule, or whether an additional connecting group must bind and react with the amino group of the linking molecule, The tail molecule is reacted with the amine group of the linking molecule to bind the tail molecule to the linking molecule.

The following formula: Or Z is selected from the following group in order to bind the tail molecule R via the linking molecule represented by the bond.

Wherein m and m ′ are independently selected as positive integers less than 11, n is selected as 0 or 1, and Q is a connecting group. The tail molecule R is selected from the group consisting of a reporter group, an intercalating group, a lipophilic group and a cleavage group, and Y is H or dimethoxytrityl.

As an example, cholesterol chloroformate is reacted with a linking molecule to attach the cholesterol group to the linking molecule via a carbamate connecting group. As a further example,
Reacting 9-acridinepropionic acid with the linking molecule produces a linking molecule with 9-ethylacridine linked via an amide bond (see Step II).

If cholesterol chloroformate as described above is used as a precursor of the tail molecule, n is 0, and thus there is no Q, and Z is It is. If 9-acridinepropionic acid as described above is used as a precursor of the tail molecule, n is 1, so Q is present and is an alkyl residue, ie, ethyl (step I).
I). In this example, Z is It is.

When n is 1 and Q is present, alkyl, alkoxy,
Alkoxyalkyl, alkenyl, cycloalkyl, aryl, aryloxy, aralkyl, heterocyclic, heteroaryl, substituted aryl and substituted aralkyl groups are suitable for use as connecting group Q.

Z is a structural formula Useful as precursor molecules for the preparation of compounds of the above formula when they are carbamates of is chloroformate. Z is a structural formula Useful as precursor molecules for the preparation of compounds of the above formula when they are ureas are isocyanates. Z is a structural formula For the preparation of compounds of the above formula in the case of thiourea of
Isothiocyanates are useful as precursor molecules. Z is a structural formula Sulfonyl halides are useful as precursor molecules for the preparation of compounds of the above formula where are sulfonamides of the formula

Various Sulfonyl Halide Precursor Tail Molecules
Available from r Probes, Inc., Eugene, Oregon. Such sulphonyl halides are aromatic sulphonyl halides, wherein the sulphonyl halide moiety is, for example, a connecting group (moiety) specific to one of the rings of the tail molecule of interest, such as a rhodamine, naphthalene, pyrene or anthracene ring. ). In such an example, n is 0 in the above formula, so there is no connecting group Q. Generally, the halide ion is chlorine or fluorine, but bromine and iodine may also be useful.

Useful sulfonyl halides are sulforhodamine (“Je
xas Red "sold by Molecular Probes, Inc.). Also useful are lisamine rhodamine B sulfonyl chloride, lisamine rhodamine B
Sulfonyl fluoride, 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride), 2-dimethylaminonaphthalene-5-sulfonyl chloride, 2-dimethylaminonaphthalene-6-sulfonyl chloride, 6- (N-methylanilino) -naphthalene -2-sulfonyl chloride (mansyl chloride), 1-pyrenesulfonyl chloride, 2-anthracene sulfonyl chloride, 5-dimethylaminonaphthalene-1-sulfonyl fluoride (dansyl fluoride), and 4-dimethylamino-azobenzene-4 '
-Would be sulfonyl chloride (Dabsil chloride).

Various isothiocyanate-based precursor tail molecules are useful for creating thiourea bonds between the tail molecule and the linking molecule. As with the sulfonyl halides described above, the isothiocyanate is generally present as a unique connecting group on the aromatic ring of the tail molecule, where n is 0 and therefore Q is absent. Such isothiocyanates are also known as Molecula
Available from r Probes, Inc.

Suitable isothiocyanates to react with the linking molecule are fluorescein-5-isothiocyanate, fluorescein-6-isothiocyanate, tetramethylrhodamine-5- (and -6) -isothiocyanate, rhodamine X isothiocyanate, malachite green isothiocyanate , Eosin-5-isothiocyanate,
Erythrosine-5-isothiocyanate, 7-diethylamino-3- (4'-isothiocyanatephenyl) -4
-Methylcoumarin, p- (5-dimethylaminonaphthalene-1-sulfonyl) aminophenylisothiocyanate, N- (4- (6-dimethylamino-2-benzofuranyl) phenylisothiocyanate hydrochloride, 1-pyreneisothiocyanate, -Anthracene isothiocyanate, 4-dimethylaminonaphthyl-1-isothiocyanate, 9-acridine isothiocyanate, 4-isoluminol isothiocyanate, 4-dimethylaminophenylazophenyl-4'-isothiocyanate, benzophenone-4-isothiocyanate 4,4′-diisothiocyanate stilbene-2,2′-disulfonate disodium salt, 4,4′-diisothiocyanate dihydrostilbene-2,2′-disulfonate disodium salt, 4-acetylamide-4 '-Isothiocyan Tosuchiruben 2,2'-disulfonic acid disodium salt, and 4-Benz amides 4 '
-Isothiocyanate stilbene-2,2'-disulfonate disodium salt.

Other useful precursor molecules for the tail molecule, including those where n is 1 and therefore a connecting group Q is present, are tetrafluorophenyl (TFP) ester, 5- (and 6-) carboxyfluorescein diacetate succini Midyl ester, 7-dimethylaminocoumarin-4-acetic acid, 7-amino-4-methylcoumarin-3-acetic acid, 7-diethylaminocoumarin-3-carboxylic acid, 7-hydroxycoumarin-4-acetic acid, 7-hydroxy-4 -Methylcoumarin-3
Acetic acid, 7-hydroxy-coumarin-3-carboxylic acid,
7-methoxycoumarin-3-carboxylic acid, 7-carboxymethoxy-4-methylcoumarin, 7-acetoxycoumarin-3-carboxylic acid, acridone-2-acetic acid, acridone-10-acetic acid, 9-anthracenepropionic acid, 1 −
Includes pyrinbutanoic acid (pyrenebutyric acid) and N- (5-dimethylaminonaphthalene-1-sulfonyl) glycine (dansylglycine).

EDTA • Fe (II) has been used as a cleavage group with oligonucleotides. The EDTA molecule is bound to the base of the uridine nucleoside. A carboxyl terminated chain was extended from the uracil group to which EDTA was attached. This method provides EDTA-bound nucleosides, while oligonucleotides incorporating such nucleosides are:
Since EDTA is present on the base, it can be interfered by the EDTA molecule, thereby interrupting primary base binding between the oligomer and the complementary DNA strand. By use of the linking molecule of the present invention, the EDTA group is extended from the linking molecule away from the nucleotide bases and, therefore, to a less interferent position due to the first base pairing on the complementary strand of DNA.

The linking molecule is reacted with an alkyl isocyanate such as, for example, ethyl isocyanate acetate, and then ethylenediamine and EDTA-triethylester-N-
Treatment with the hydroxysuccinimide ester will serve to attach the EDTA group to the linking molecule via an amide bond. In this case, the additional connecting group is EDTA
Used to form a bond with the linking molecule of the cleavage group. The conditions for the cleavage reaction were Dreyer (Proc. Natl. Acad. Sci. 8
2, in such a way as indicated by 968 to 972 (1985)), in aqueous solution, is initiated by the addition of suitable oxidizing agents such as Fe (II) and dithiothreitol.

Further, when the cleavage group, phenanthroline-Cu (I) complex, is bonded, 1,10-phenanthroline is added to 5 or 6
Aminated at the position. The amine is then succinylated. The resulting terminal carboxylate is, for example, N
-Activated by conversion to a hydroxysuccinimide ester and used for the reaction of the linking molecule with an amine or with an additional connecting group attached to the linking molecule. Cleavage with this reagent is described in Francois et al. (Biochemistry 2
7 is initiated by the addition of copper sulfate and mercaptopropionic acid in the same manner as 2272 to 2276 (1988)).

Similarly, the secondary amino substituent of ellipticine is succinylated directly with succinic anhydride in pyridine, and then D in tetrahydrofuran (THF) for attachment to the linking molecule.
Activated to N-hydroxysuccinimide ester (NHS ester) with CC (1,3-dicyclohexylcarbodiimide). Quinoxaline requires amination of its ring prior to succinylation and activation, in a manner similar to periphenanthroline.

Bithion having a long-chain spacer is also commercially available from Molecular Probes as succinimidyl ester. This product, 6- (6-biotinoylamino) hexanoylamino) hexanoic succinimide ester, is also referred to as biotin-XX-succinimide ester. In the same manner as the phenanthroline succinimide ester described above, it reacts with the amino group of the linking molecule and attaches a biotin tail molecule to the linking molecule.

In a similar manner to Dervan et al. (J. Am. Chem. Soc. 100 , 1968 (1978)), p-carboxymethidium can be prepared for coupling to a linking molecule via its carboxyl group. Similarly, ethidium is produced.

A.2. Synthesis of acridine TFP ester and improved production method of acridine CPG 9-Acridine alkanoic acid was strongly bound to DNA (ST
ahenaka et al., Anal. Sci. 4 , 481 (1988)), with a variety of different chain lengths. Acridine-modified ODNs have strict geometrical requirements for efficient insertion of the acridine molecule between DNA duplex base pairs, but these geometrical conditions are not predictable. difficult. Therefore, it is preferred to have a linking chain length that can be easily modified. In addition, the length of various bonds is evaluated with respect to bond strength. The strength of the binding of an oligonucleotide (ODN) to its complementary nucleic acid strand is readily determined by thermal denaturation (Tm) studies.

Acridine alkanoic acids with various alkyl chain lengths are described in H. Jense and L. Howland (J. Am. Chem. Soc. 48 , 1926
(1989)) by heating the corresponding aliphatic dicarboxylic acid with diphenylamine and zinc chloride. For example, a condensation reaction with adipic acid yielded 45 g (16% yield) of 5- (9-acridinyl) pentanoic acid. The two alkyl chain lengths used for the production of the acridine-CPG differ from the other 3'-acridine tail molecules.
ODN (U. Asseine et al., Proc. Natl. Acad. Sci. USA 81 , 3297
(1984)) was chosen to approximate the previously reported length to give an optimal Tm.

One example of this aspect of the invention is acridine-CPG
An improved method for the synthesis of is provided. This improvement uses an active ester derivative of acridinyl carboxylic acid as a precursor molecule.

Various acridinyl carboxylic acid active esters have been evaluated as potential precursors for acridine-CPG (10,23). N-hydroxysuccinimide (NHS) esters and p-nitrophenyl esters were prepared by activating a carboxylic acid with dicyclohexylcarbodiimide (DCC) and then reacting with N-hydroxysuccinimide or p-nitrophenol. . However, these condensation reactions are difficult to carry out because both the starting carboxylic acid and the by-product dicyclohexylurea (DCU) are insoluble in organic solvents commonly used for DCC activation. Furthermore, these reactions were not completed and the desired ester could not be easily purified by silica gel column chromatography due to its instability. In contrast, TFP (tetrafluorophenyl) ester is suitably reactive with the nucleophilic amino group of trans-4-hydroxy-L-prolinol 2 and is sufficiently stable to allow purification by flash chromatography.

A representative method for activating a carboxylic acid to a TFP ester will be described. TFP ester 19 or 20 initially activates the carboxylic acid with 2-fluoromethylpyridinium tosylate (FMPT), thereby forming an unstable intermediate, which is then converted to 2,3,5, It is prepared by reacting with 6-tetrafluorophenol to form a stable product (see Step IV, Method a). This reaction does not produce insoluble DCU, and the insoluble acridinyl carboxylic acid becomes soluble as the reaction proceeds.
The resulting homogeneous mixture was purified by flash chromatography after removing the solvent. FMPT is a particularly preferred activator for carboxylic acids that are less soluble in polar aprotic organic solvents (such as ether, THF, DMF and acetonitrile).

The present invention also discloses improved reagents for the production of TFP esters. In summary, treatment of 2,3,5,6-tetrafluorophenol with trifluoroacetic anhydride gives TFP trifluoroacetate 18. This improved reagent 18 is
Reaction with a carboxylic acid in the presence of triethylamine, as shown in IV, Method b, gives the TFP ester. For example, 9-acridinylpropanoic acid is converted to TFP in methylene chloride.
Treatment with trifluoroacetate 18 and triethylamine gives the desired acridinyl TFP ester 19.

In addition, other carboxylic acids are also suitable for reaction with TFP trifluoroacetate 18. In this regard, representative carboxylic acids include N-CBZ-L-phenylalanylglycine, protoporphyrin IX, 3-amino-9-ethylcarbazole succinimide, and the like. Advantages of the disclosed reagents 18 and methods of using them include the following. (1) TFP trifluoroacetate is easily produced from inexpensive starting materials. (2) No expensive condensation reagent is required to produce TFP esters using this synthesis method. (3) Improved purification of the TFP ester product because trifluoroacetate is the only by-product of the disclosed reaction.

In particular, in a preferred method (Example XXVIII, Method 2)
The acridine-CPG support (10 and 23) was prepared from trans-4-hydroxy-L-prolinol 2 and the appropriate acridinylpropionic or pentanoic acid TFP ester (19 or 20) as shown in Step V. Manufactured.
Reaction of the purified TFP ester 19 or 20 with aminodiol 2 gives a quantitative yield of the key intermediate amide product 7 or 21. This method (Method 2) involves the intermediate N
-Obtain more reproducible results than when preparing diol amide 7 directly from acridine carboxylic acid via methylpyridinium ester (Example IX, Method 1, Step II)
reference). The primary hydroxyl group of 7 or 21 is selectively protected as DMTr ether under standard conditions, giving 8 or 22 in good yield. The remaining secondary hydroxyl groups are succinylated and the resulting carboxylic acid is fixed to a controlled pore glass support (LCAA-CPG) with a long chain alkylamine to give the desired ACr-CPG (1
0 or 23). Each of these GPGs has a degree of DMTr of 1
8.5 μmol / g and 20.6 μmol / g.

B. 3'-amine modified ODN using acridine TFP ester
Post-Synthetic Modification of (Method B) 3′-Tail Molecule Oligonucleotides are Synthesized Directly from Solid Supports Covalently Linked with 3′-Tail Molecules (Method A,
See paragraphs A.1 and A.2.) Alternatively, OD with 3'-tail molecule
N is synthesized using a special solid support that incorporates a protected nucleophilic amino or thiol group. The conjugate group is then post-synthesized with a suitable electrophile to remove the conjugated group to such a 3'-
Introduced into ODN with tail molecule. In this claimed aspect, post-synthetic modification according to Method B is advantageously used;
Sensitive 3 'that cannot remain under the synthesis conditions required for Method A
-Introduce a tail molecule. Further, the advantages of Method B relate to the large number of electrophilic conjugate groups utilized in the corresponding starting materials. In addition, method B is advantageously used for the production of small amounts of modified ODN.

Two methods (method A and method B) disclosed herein for the production of ODNs with a 3'-tail molecule include acridine TFP.
A comparison was made by studying the reaction of esters 19 and 20 with a 3'-amine-tailed ODN. More specifically,
11 units of ODN with a 3'-amine-tail molecule complementary to the initiation codon region of the mRNA corresponding to the hepatitis B surface antigen protein were produced. Such a 3'-tailed ODN with improved target nucleic acid binding would be advantageously used as an antisense oligonucleotide.

C. Modified Support for the Synthesis of 3'-Amine-tailed ODNs An embodiment of the present invention further comprises a 3'-amine
An improved solid support for the synthesis of amine-tailed ODNs is provided. A particularly preferred support in this regard is aminohexyl-modified CPG (AH-CPG) prepared using 6-aminohexan-1-ol.

Currently, modification of the 3'-end of ODN is performed using commercially available supports (eg, "Amine-ON-CPG" by Clonrech Laboratories, Inc., Palo Alto, CA). However, as noted above, such a support (corresponding to the support disclosed by Nelson et al., Supra) shows that at least two distinctly different 3'-tailed ODN products (about 1: 1) were obtained by PAGE analysis. Has significant additional disadvantages, including generation. In addition, 3'-
Derivatization of the amine tail molecule with various active esters always resulted in low yield, and HPLC (High Performance Liquid Chromatography)
Analysis shows that less than 50% of the starting 3'-amine-tailed ODN is reacted.

The suggested mixed products are: (1) the transfer of the phosphate moiety of the first nucleotide from O to N during the synthesis of ODN on a commercially available solid support; (2) the FMOC moiety of the tail molecule during ODN synthesis. Deprotection and subsequent capping with acetic anhydride and / or (3) formation of cyclic phosphate between the primary secondary hydroxyl groups of the tail molecule, with concomitant loss of ODN. In all cases, the resulting 3'-tailed ODN can subsequently form distinct species according to PAGE analysis without reacting with the active ester.

The modified solid supports herein overcome these disadvantages associated with commercial solid supports for modification of the 3 'end of ODN. Other advantageous features provided by the claimed modified solid support include (1) a unique amine protecting group that also serves as a CPG binding site and (2) a dimethoxytrityl protected hydroxyl group. Furthermore, these modified supports can be adapted to all methods used in commercial DNA synthesizers.

ODN with 3'-amine-tail molecule is preferably CP on a DNA synthesizer.
G Removed from support. The claimed support has no adjacent diol and therefore has a 3'-amine-tailed molecule.
Elimination of ODN from the support with ammonium hydroxide is equivalent to O
Compatible with the conservation of the aminohexyl group at the 3 'end of the DN. Conversely, because amine-ON-CPG contains vicinal diols, elimination of ODN from this support with ammonium hydroxide results in significant loss of tail molecules from ODN.

U. Asseline and NTThuong (Trt. Lete. 31 , 81-84)
(1990)) reported a solid support for introducing a 3'-aminohexyl tail molecule into the ODN. Disadvantages associated with this solid support include: (1) a seven-step synthetic method for making the support (see the four-step method described herein); Data analysis suggesting that only low yields of solid support are obtained, including (3) lack of data suggesting that a theoretical yield of ODN with 3'-tail molecule of this solid support is obtained .

To make the claimed solid support, the preferred 6-
In addition to the aminohexan-1-ol reagent, other aminoalkanols are commercially available and suitable for producing modified CPGs according to the present invention. ODs with 3'-amine tail molecules of various lengths by substituting aminoalkanols of various alkyl chain lengths for 6-aminohexan-1-ol
N is synthesized by the methods disclosed herein. In contrast, aminodiols of various lengths are generally not commercially available, and therefore solid supports with a range of alkyl chain lengths cannot be easily produced by the method of Nelson et al. Therefore, it has various 3'-amine tail molecules
ODN cannot be easily synthesized using amine-ON-CPG or a variant thereof. In addition, other spacer groups (ie,
Wherein R is alkyl, aryl, arylalkyl, heteroalkyl or heteroaryl) is inserted between the amino group of the claimed modified solid support and the hydroxyl group protected by DMTr. In a preferred embodiment, R is (CH ₂ ) n and n
Is 1 to 10. In particular, in a preferred embodiment, R is (CH
₂ ) It is ₆ .

Oligonucleotides having a 3'-amine tail molecule can be efficiently synthesized using the modified solid support of the present invention. Such 3'-amine-tailed oligonucleotides are contrasted with similar 3'-tailed ODNs produced using commercially available amine-ON-CPG supports. Representative modified solid supports of the claimed invention obtained high yields of ODN with 3'-amine tail molecule as a single product by both HPLC and PAGE. 3 'made from the modified solid support claimed
Subsequent derivatization of the ODN with an amine-tail molecule was completed rapidly and gave a quantitative yield of 3'-modified ODN without the need for HPLC purification.

D. Reaction of Acridine TFP Ester with Intramolecular Amine-Modified ODN
Provide a means to introduce Such a large number of internal intercalating groups can result in increased binding of the ODN to the target nucleic acid strand, but may have strict mathematical requirements for effective intercalation. The expected increase in binding affinity of nucleic acids due to intercalating groups can be neutralized or reduced by steric effects that disrupt the normal Watson-Crick base pairing of the duplex.

The claimed method involves using an acridine TFP ester to produce a double-modified ODN flanking either one or two base pairs of the smallest duplex formed between the ODN and the target. According to the present invention, internally modified ODNs are designed that have a linkage that optimizes the interaction between the intercalating group and the target nucleic acid strand. For example, the 5-aminopropyl-deoxyuridine group was selectively introduced into ODN, and the effect of one and two internal acridine modifications on Tm was studied. For a single internal amine modification, ODN was reacted with acridine TFP ester 19 or 20 to determine the effect of chain length on coupling efficiency (Tm). The acridine-contracted ODN with five carbon-length bonds increased the Tm by 5.9 ° C compared to the unmodified control, while the three carbon-length bonds increased 1.1 ° C compared to the same control. It just increased. When two internal acridine modifications were tested, optimized bond lengths ranged from 45.5 ° C (unmodified ODN) to 56.2 ° C (two-way inserted ODN).
An increase in Tm was observed. In addition, numerous internal acridine modifications can improve the stability of ODN to cell consumption and nuclease digestion.

E. Improved stability of ODN with 3'-tail molecule in cell culture (hexanol-CPG) Unmodified ODN is rapidly degraded by 3'-exonuclease in serum-containing media. Certain chemical modifications of the 3'-terminal phosphodiester linkage (i.e., changing the last two internuclease phosphodiester linkages to phosphorothioates, phosphoramidates, or reverse conjugates)
Significantly improves the stability of ODN to nucleases. Other chemical linkages can provide increased nuclease stability, such as α-deoxynucleotide derivatives (C. Cazenave et al., Nucl. Acids Res. 15 , 10507 (1987)) and methylphosphonate derivatives. Since the intercalating group is a bulky 3'-substituent, it is also
The terminal phosphodiester bond can be protected from 3'-exonucleases (E. Uhlmann and A. Pyman, C.
hem. Rev. 90 , 543 (1990)). For example, 3'-cholesterol, 3'-acridine- and 3'-hexylamine-tailed ODNs were examined in cell culture assays and showed increased stability compared to unmodified ODNs.

In one embodiment of the present invention, 3 ′ using a modified solid support
Disclosed are methods for synthesizing oligonucleotides that prevent degradation by exonucleases. This modified solid support advantageously synthesizes ODNs free of other 3'-modifications and ODNs useful for in vivo evaluation of structure-activity relationships of other 3'-modifications such as cholesterol or acridine. Can be used. 3'-Modified ODN made by this method
Does not interfere with hybridization, so the modified solid supports described herein are a suitable substitute for the immobilized, DMTr-protected nucleosides currently used in ODN synthesis. Disadvantages associated with these fixed nucleosides are their high cost and the four types of
Includes the need to have a CPG. Conversely, within the scope of the claimed method wherein the 3'-terminal nucleoside is added as a phosphoramidite, the modified solid support can be used as a universal reagent for the production of ODN sequences. Furthermore, in contrast to the 5'-hydroxyl group protected by the DMTr of the immobilized nucleoside, the DMTr on the bond protruding from the solid support
The hydroxyl group protected by is not inhibited. Therefore, the hydroxyl groups protected by DMTr on the modified solid support have increased access to bulky phosphoramidite reagents and can provide high yields of ODN products.

The modified solid support of this aspect of the invention comprises a support and a DMTr.
Characterize the R group that connects the hydroxyl group protected with (ie, alkyl, aryl, arylalkyl, heteroalkyl or heteroaryl). In a preferred embodiment, R is 2 to 10 n in (CH ₂₎ n. In particular, in a preferred embodiment, R is (CH ₂ ) ₆ . Typically, the preparation of a modified solid support, hexanol-CPG, is shown in Step VII. Key intermediate in the synthesis of hexanol-CPG,
O- (4,4'-dimethoxytrityl) -1,6-hexanediol 37 was obtained from F. Seela and K. Kaiser (Nucl. Acids Res.
15 , 3113 (1987)) by treating hexanediol with DMTr-Cl. Succinylation of the intermediate followed by immobilization on a long chain alkylamine controlled pore glass support (LCAA-CPG) gives hexanol-CPG.

"Antisense" oligodeoxynucleotides with a 3'-hexanol-tail molecule have been studied in models of biological systems. These antisense ODNs had a remarkable effect on the swimming behavior of Parameciur tetraurelia. Wild-type cells and cells treated with random or sense ODN had no effect.

The examples described below correspond to the reaction series of steps I to VIII. In steps I and III, the identification "Cho
l "represents the cholesteryl group. Step I shows the synthesis of cholesterol-CPG6, Step II shows the acridine-CPG1
Step III describes the synthesis of cholesterol-CPG17, Step IV describes the synthesis of acridine TFP esters 19 and 20, Step V illustrates an improved synthesis of acridine-CPGs 10 and 23, Step VI Is aminohexyl-CP
It describes the synthesis of G30, step VII describes the synthesis of hexanol-CPG38, and step VIII shows the structure of the 3'-acridine tail molecule.

Example I (2S, 4R) -N-benzyloxycarbonyl-4-hydroxy-2-hydroxymethylpyrrolidine (1) CBZ hydroxyproline (4.76 g, 18 mmol, Sigma Chemical)
al Co.) in an ice-cooled solution of anhydrous tetrahydrofuran (20 mL) was added a 1 M borane-tetrahydrofuran complex in tetrahydrofuran solution (45 mL, Alrich). After stirring at 0 to 50 ° C for 15 minutes and then at room temperature for 4.5 hours under argon, methanol (50 mL) was added to the reaction mixture to stop the reaction. After 30 minutes, the reaction solution was concentrated and the remaining colorless syrup was purified by flash chromatography (3.5 × 23 cm, silica) using a gradient method (gradient elution method) with methanol-methylene chloride. The product was eluted with 10% methanol. Removal of the solvent from the fraction containing the pure product gave the above (1) (2.18 g, 46
% Yield) was obtained as a colorless syrup.

TLC (95: 5 / methylene chloride: methanol), Rf = 0.16. IR (neat) 3600-3100 (br), 2940,1680,1
420, and ^{1355cm -1. 1 H-NMR (} CDCl 3) 7.36 (s, 5H), 5.17 (s, 2H), 4.50
(M, 2H), 3.67 (m, 4H), 2.09 (m, 3H). Analytical value (as C ₁₃ H ₁₇ NO ₄ .0.3H ₂ O) Calculated value: C, 60.83; H, 6.91; N, 5.46. Observed value: C, 60.85; H, 6.88; N, 5.36. Example II ( 2S, 4R) -4-Hydroxy-2-hydroxymethylpyrrolidine (2) (trans-4-hydroxy-L-prolinol) CBZ hydroxyprolinol (1) (1.92 g, 7.6 mmol)
A solution of in methanol (50 mL) was stirred with 10% palladium on carbon (320 mg) under a hydrogen balloon. After 16 hours, no starting material remained as indicated by thin layer chromatography (9: 1 base methylene: methanol). The reaction mixture was filtered through celite (washed with methanol) and the filtrate was concentrated to give the desired product (2) quantitatively as a yellow syrup. When this syrup is dissolved in ethanol, it becomes 0.
A 5M stock solution (15.2 mL) was obtained.

IR (neat) 3600-3100 (br), 2920, 1530 and 1410
^{cm -1. 1 H NMR (D} 2 O) 4.40 (m, 1H), 3.60 (m, 3H), 3.02 (d,
of d, 1H, J = 12.4,4.8Hz), 2.77 (d of t, 1H, J = 12.4,1.
8Hz), 1.84 (m, 1H), 1.60 (m, 1HO). Example III (2R, 4R) -N-cholesteryloxycarbonyl-4-
Hydroxy-2-hydroxymethylpyrrolidine (3) A solution of cholesterol chloroformate (1.48 g, 3.3 mmol) in methylene chloride (8 mL) was added to a 0.5 M solution of hydroxyprolinol (2) in ethanol (7.6 mL, 3.8 mmol). The mixed solution was stirred at room temperature for 1.5 hours. The cloudy reaction was poured into ice water (100 mL) and the heterogeneous mixture was extracted with hot ethyl acetate (3 × 150 mL). The collected organic layer was washed with brine, dried over magnesium sulfate, and concentrated. The solid residue was treated with methanol-hexane: ethyl acetate (1: 1)
(Gradient) and purified by flash chromatography (4 × 15 cm silica). The product was eluted with 10% methanol. Removal of the solvent from the fractions containing pure product provided (3) (1.42 g, 81% yield) as a white solid.

TLC. (95: 5 / methylene chloride: methanol), Rf = 0.10 10% sulfuric acid - product and spraying heated with methanol analysis values colored in black (C ₃₃ H ₅₅ as NO ₄₎ Calculated: C, 74.81 H, 10.46; N, 2.64. Found: C, 74.74; H, 10.33; N, 2.50. Example IV (2S, 4R) -N-cholesteryloxycarbonyl-4-
Hydroxy-2-dimethoxytrityloxymethylpyrrolidine (4) Triethylamine (0.52) was added to a solution of diol (3) (1.42 g, 2.68 mmol) in dry pyridine (27 mL) with stirring.
4 mL), 4-dimethylaminopyridine (16.5 mg), and dimethoxytrityl chloride (1.10 g, 3.23 mmol) were added. After stirring for 4.5 hours under a stream of argon, the solvent was removed from the reaction mixture, and the remaining pyridine was distilled off together with toluene. The residue was partitioned between ether (100mL)-water (40mL). The aqueous layer was extracted with ether (80 mL), and the collected organic layers were washed with brine, dried over sodium sulfate, and concentrated. Ethyl acetate-hexane (gradient)
And purified by flash chromatography (4.5 × 20 cm silica). The product was eluted with 2: 1 / hexane: ethyl acetate immediately after obtaining a yellow impurity. Removal of the solvent from the fraction containing the pure product gives (4)
(1.33 g, 60% yield) was obtained as a pale yellow foamy solid.

TLC (95: 5 / methylene chloride: methanol), Rf = 0.49. Sprayed with 10% sulfuric acid-methanol, the product turned orange.

_{^{1 H NMR (CDCl 3) 7.26}} (m, 9H), 6.81 (d, 4H, J = 8.8H
z), 5.30 (m, 1H), 4.50 (m, 2H), 4.15 (m, 1H), 3.78
(S, 6H), 3.7-3.0 (m, 4H), 2.4-0.6 (m, 46H). .. Analysis (as C ₅₄ H ₇₃ NO ₆₎ Calculated: C, 77.94; H, 8.84 ; N, 1.68 Found: C, 77.26; H, 8.82 ; N, 1.56 Example V (2S, 4R) - N-cholesteryloxycarbonyl-4-
Succinyloxy-2-dimethoxytrityloxymethylpyrrolidinone (5) Succinic anhydride (443 mg, 4.43 mmol) was stirred in a solution of alcohol (4) (1.22 g, 1.47 mmol) in dry pyridine (12 mL).
l) and dimethylaminopyridine (89 mg, 0.73 mmol) were added and the reaction mixture was stirred under argon for 26 hours. The solvent was removed and residual pyridine was distilled off together with toluene.
The residue was dissolved in chloroform (40 mL), washed with brine, dried over sodium sulfate and concentrated to give the product (5)
Was quantitatively obtained as a beige foamy solid.

The product turned orange when sprayed with TLC (95: 5 / methylene chloride: methanol), Rf = 0.32.10% sulfuric acid-methanol.

Example VI cholesterol -CPG support (6) The method of publication described succinylated cholesterol derivative (5) (RTPon et Biotechn. 6, 768 (1988)) pore glass was controlled by the long-chain alkyl amine in the support (LCAA
-CPG, Sigma).

LCAA-CPG (5.0 g) was stirred with 3% dichloroacetic acid-methylene chloride (100 mL) for 3 hours. The CPG was filtered through a 30 mL volumetric glass frit and washed with chloroform (150 mL) and ether (150 mL). The obtained solid was dried under reduced pressure, and dried pyridine (50 m
L), succinylated cholesterol derivative (5) (932m
g, 1 mmol), triethylamine (0.4 mL), 1-ethyl-
It was mixed with 3- (3-dimethylaminopropyl) carbodiimide (1.92 g, 10 mmol) and 4-dimethylaminopyridine (60 mg). The mixture was placed in an orbital mixer and stirred at 100 rpm for 38 hours. The CPG was filtered through a 30 mL sintered glass filter, washed with pyridine (50 mL), methanol (100 mL), chloroform (50 mL) and ether (50 mL) and dried under reduced pressure. The remaining amino groups on the CPG were removed using dry pyridine (15 mL) and acetic anhydride (2.0 m
The support was capped by stirring during L). Two hours later, the CPG was filtered, washed as above, and dried under reduced pressure to obtain the product (6) (5.0 g).
This method is described in the publication (T. Atkinson and M. Smit
h, Oligonuclcotide Synthesis, a Practical Approach,
Analyzed for dimethoxytrityl content according to MJ Gait, M, J. (ed.), IRL press (1984), p48) and found that the CPG support had a loading of 17.6 μmol / g.

Example VII Synthesis of Oligonucleotide with 3 'Cholesterol Tail Molecule from Cholesterol-CPG Cholesterol-CPG in an amount corresponding to the dimethoxytrityl content (1 micromol) was filled with an empty column (for example, Applied
Biosystem Inc. or Cruachem column).
Oligonucleotides with the base sequence CTCCATGTTCGTCACA were prepared using standard phosphoramidite chemistry to make Milligen DN
Manufactured on an A synthesizer. The 5'-DMTr protecting group was left. Oligonucleotides were cleaved from CPG and deprotected by treatment with concentrated ammonia (2 mL) at room temperature for 3 days. The supernatant was injected directly onto a PRP-1 reverse phase high performance liquid chromatography column (eluted with a 20-100% acetonitrile-triethylammonium acetate (pH 7.5) gradient). The product is collected in portions and lyophilized to give 5'-DMTr
A protected product was obtained. The DMTr group was removed by treatment with 80% acetic acid at room temperature for 16 hours and repurified by HPLC. The collected product was analyzed at UV 260 nm and found to contain 0.54 mg of product. When the method was repeated separately, the DMTr group was removed with 3% DCA while in the synthesizer, increasing the yield of oligonucleotide.

Example VIII (2R, 4R) -N- (9-acridinepropanamidyl)
-4-Hydroxy-2-hydroxymethylpyrrolidine (7) {1- [3- (9-acridinyl) -1-oxopropyl] -5-hydroxymethyl- (3R-trans) -3-
Pyrrolidinol; Step II｝ 9-Acridinepropionic acid (125.5 mg, 0.5 mmol) (H. Jensen and LJHowl and J. Am. Chem. Soc. 48 , 1926 (1989)) in methylene chloride (1.5 mL), ethyl To a slurry of diisopropylamine (0.104 mL, 0.6 mmol) was added 2-fluoro-1-methylpyridinium tosylate (FMPT) (0.6 mmol) with stirring. After 1.5 hours at room temperature, the cloudy brown solution was added with stirring to a solution of hydroxyprolinol (2) (0.5 mmol) in ethanol cooled in an ice-salt bath. After continuing stirring at room temperature for 1 hour, methanol (10 mL) was added to the reaction mixture to stop the reaction, and the mixture was concentrated. The residue was purified by flash chromatography (1.5 × 24 cm silica) using methanol-methylene chloride (gradient). Removal of the solvent from the fractions containing the pure product gave (7) (112 mg, 64
% Yield) was obtained as a yellow foamy solid.

TLC (90: 10 / methylene chloride: methanol), Rf = 0.50 The product appeared as a yellow spot emitting blue fluorescence.

IR (KBr) 3400 (br) , 1620,1440,1070cm -1. 1 H NMR (CDCl 3) 8.30 (d, 4H, J = 9.4Hz), 7.82 (t, 2
H, J-6.6Hz), 7.62 (m, 2H), 4.31 (m, 2H), 4.05 (t, 2H,
J = 8.2Hz), 3.70 (m, 1H), 3.50 (m, 2H), 3.22 (d, 2H, J
= 1.4Hz), 2.78 (m, 2H), 2.03 (m, 1H), 1.64 (m, 1H). Analytical value (as C ₂₁ H ₂₂ N ₂ O ₃ .0.5H ₂ O) Calculated value: C, 70.18; H, 6.45; N, 7.79. Observed value: C, 70.41; H, 6.45; N, 7.68. IX (2S, 4R) -N- (9-acridinepropanamidyl)
4-Hydroxy-2-dimethoxytrityloxymethylpyrrolidine (8) Diol (7) (100 mg, 0.285 mmol) in pyridine (2.5
mL) solution, 4-dimethylaminopyridine (6.4 mg), triethylamine (0.13 mL), and dimethoxytrityl chloride (154 mg) were added with stirring. After stirring for 16 hours under a stream of argon, the solvent was removed from the mixture,
Residual pyridine was still distilled off with methylene chloride; the residue was dissolved in methylene chloride (5 mL), washed with water (2 × 3 mL), then with brine (3 mL), dried over sodium sulfate and the solvent was removed. . The residue was purified by flash chromatography using methanol-1: 1 / hexane: ethyl acetate (gradient). The fractions containing the pure product were collected and the solvent was removed (8) (123 mg, 66%
Yield) was obtained as a yellow foamy solid.

TLC (45: 45: 10 / hexane: ethyl acetate: methanol), Rf = 0.26. The product appeared as a yellow spot emitting blue fluorescence and turned orange when sprayed with 10% sulfuric acid-methanol.

IR (KBr) 3400 (br) , 1620,1440,1070cm -1. 1 H NMR (CDCl 3) 8.17 (m, 4H), 7.75 (t, 2H, J = 7.0H
z), 7.57-7.09 (m, 9H), 6.82 (d, 2H, J = 8.8Hz), 6.66
(M, 2H), 4.56 (m, 2H), 3.65-2.90 (m, 2H), 2.69 (m, 2H)
H), 2.15 (m, 1H), 1.95 (m, 1H). Analytical value (as C ₄₂ H ₄₀ N ₂ O ₅・ 0.5H ₂ O) Calculated value: C, 76.23; H, 6.24; N, 4.23. Observed value: C, 76.53; H, 6.67; N, 3.80. X (2S, 4R) -N- (9-acridinepropanamidyl)
4-Succinyloxy-2-dimethoxytrityloxymethylpyrrolidine (9) To a stirred solution of alcohol (8) (120 mg, 0.184 mmol) in dry pyridine (1.5 mL) was added 4-dimethylaminopyridine (11.2 mg). The mixture was stirred under argon for 40 hours before removing the solvent. Residual pyridine was distilled off with toluene and removed. Chloroform (3m
L), washed with brine, dried over sodium sulfate and concentrated to give the product (9) (128 mg, 93% yield) as a yellow foamy solid.

TLC (95: 5 / methylene chloride: methanol), Rf = 0.13. The product appeared as a blue fluorescent yellow spot and turned orange when sprayed with 10% sulfuric acid-methanol.

Example XI Acridine-CPG support (10) Porous glass support with succinylated acridine derivative (9) controlled with long-chain alkylamine using method described above for cholesterol-CPG support (6) To solid. LCAA-CPG with acid wash in round bottom flask
(0.85 g) with dry pyridine (8.5 mL), triethylamine (0.068 mL), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (325 mg, 1.7 mmol), and 4-dimethylaminopyridine (10.2 mg). After mixing, the mixture was stirred under a stream of argon for 19 hours. The CPG was filtered, washed with pyridine, methanol, chloroform and ether, and then dried under reduced pressure. Residual amino groups on the CPG were capped by stirring the support in dry pyridine (2.5 mL) and acetic anhydride (0.34 mL). After 2 hours, the CPG was filtered, washed as above and dried under reduced pressure to give the product (10) (0.85 g). This material was analyzed for dimethoxytrityl content and found that the CPG support had a loading of 18.5 μmol / g.

Example XII Synthesis of Oligonucleotide (10 ′) with 3′-Acridine-Tail Molecule from Acridine-CPG (10) An amount of acridine-CPG (10) corresponding to dimethoxytrityl content (1 micromol) was packed in an oligonucleotide synthesis column. did. Oligonucleotides having the base sequence 5'-CTCTCCATCTTCGTCACA were produced on a Milligen DNA synthesizer using standard phosphoramidite chemistry. The resulting oligonucleotide was cleaved from the CPG and deprotected by treatment with concentrated ammonia (2 mL) at 40 ° C. for 24 hours. The supernatant was injected directly onto a PRP-1 reverse phase high performance liquid chromatography column (20% to 100% acetonitrile-triethylammonium acetate, eluting with a pH 7.5 gradient) and the product (10 ') was collected in one fraction. Oligonucleotide product (10 ') that is fluorescent when lyophilized
(1.99 mg) (determined by VU 260 nm) was obtained as a pale yellow solid.

Example XIII 4-N-benzyloxycarbonyl-3-hydroxybutyric acid (11) 4-amino-3-hydroxybutyric acid (5.0 g, 42.0 mmol, Si
gma Chemical Co.) was dissolved in a solution of sodium hydroxide (3.7 g) in water (35 mL), and CBZ chloride (7.88 g, 46.2 mmol) was added dropwise to the ice-cooled solution over 20 minutes. The mixture was stirred in ice for 2 hours and then washed with ether (50 mL) to remove excess CBZ chloride. The aqueous layer was acidified with 3N HCl (20 mL) and extracted with ethyl acetate (4 × 50 mL). The extracts were combined, dried over magnesium sulfate, and the solvent was distilled off under reduced pressure to obtain a colorless syrup-like substance, which was immediately crystallized. Recrystallization from chloroform gives the desired product (11) (4.
70 g, 44% yield) were obtained as white crystals (mp 94-95 ° C).

_{^{1 H NMR (CDCl 3) 7.35}} (s, 5H), 6.70 (s, 2H), 5.70
(S, 1H), 5.15 (s, 2H), 4.35−3.95 (m, 1H), 3.25−3.0
0 (m, 2H), 2.50 (d, 2H, J = 6.5 Hz). Example XIV 1M borane-tetrahydrofuran (Aldrch chemical Co.) obtained by cooling a solution of 1-N-benzyloxycarbonyl-2,4-butanediol (12) acid (11) (4.57 g, 18.0 mmol) in dry tetrahydrofuran (18 mL) was cooled with ice. .) Was added dropwise with stirring under an argon atmosphere. After the addition was complete, the mixture was stirred at room temperature for 30 minutes and then 10%
The reaction was stopped by adding methanol (36 mL). The solvent was removed under reduced pressure, and the residue was washed with ethyl acetate (80 mL) with 1.5 N hydrochloric acid, water and a saturated aqueous solution of sodium hydrogen carbonate. After drying over potassium carbonate, the solvent was distilled off under reduced pressure to obtain a white solid. Recrystallization from benzene-hexane gave the desired product (12) (1.69 g, 38% yield) as white crystals (mp 8
0.5-82 ° C).

_{^{1 H NMR (CDCl 3) 7.35}} (s, 5H), 5.50 (t, 1H, J = 6.0H
z), 5.10 (s, 2H), 3.75 (t, 2HJ = 6.0), 4.10-2.65 (m,
5H), 1.60 (q, 2H, J = 6.0 Hz). Example XV 1-amino-2,4-butanediol (13) Step III: CBZ protected aminodiol (12) (1.69
g, 7.06 mmol) were mixed with palladium hydroxide-carbon (800 mg), ethanol (100 mL), and 1,4-cyclohexadiene (20 mL), and the mixture was heated under reflux for 16 hours. Thin layer chromatography (10% methanol-methylene chloride) showed no starting material remaining. The mixture was filtered through celite on a semi-solid glass funnel (washed with ethanol) and the solvent was removed in vacuo to give the desired product (13) (0.84 g).
Was obtained as a yellow syrup. Dilution to 7 mL with ethanol provided a 1 M stock solution of aminodiol (13).

_{^{1 H NMR (D 2 O)}} 4.80 (s, 4H), 3.80 (t, 2H, J = 6.0H
z), 4.00-3.65 (m, 1H), 3.40-2.50 (m, 2H), 1.95-1.
55 (m, 2H). Example XVI 1-N-cholesteryloxycarbonyl-2,4-butanediol (14) Aminodiol (13) (6.0 mmol) in ethanol (6.0 mmol)
cholesterol chloroformate (2. mL) in ice-cold solution.
25 g, 5.00 mmol) in methylene chloride (5 mL) was added.
The mixture was removed from the ice bath, stirred for 1.5 hours under a stream of argon, and then poured into ice water (150 g). The mixture was extracted with ethyl acetate (2 × 150 mL) and the extract was washed with water (2 × 100 mL).
L), washed with brine (1 × 100 mL), dried over magnesium sulfate, and evaporated. Dissolve the solid residue in tetrahydrofuran (10 mL) and use a silica gel column (3.5 × 1
5 cm, packed with 1: 1 hexane: ethyl acetate). After elution with 1: 1 hexane: ethyl acetate (300 mL), the product was eluted using 45:45:10 hexane: ethyl acetate: methanol. Removal of the solvent gave a sticky white solid (14) (2.04 g, 79%).

_{^{1 H NMR (CDCl 3) 5.38}} (d, 1H), 5.14 (t, 1H), 4.50
(M, 1H), 4.00-3.70 (m, 3H), 3.60-3.00 (m, 2H), 2.4
0-2.20 (m, 2H), 2.10-0.60 (m, 3H). Example XVII 1-N-cholesteryloxycarbonyl-2-hydroxy-4-dimethoxytrityloxybutane (15) Diol (14) (1.74 g, 3.35 mmol) was added to a stirred solution of dry pyridine (25 mL) in dimethoxytrityl chloride (1). .
36 g, 4.02 mmol), triethylamine (0.655 mL) and 4-dimethylaminopyridine (20.5 mg) were added. The reaction mixture was stirred for 16 hours under a stream of argon, and then water (25 mL)
And ether (75 mL). The aqueous layer was separately extracted with ether (75 mL), and the extracts were combined, washed with water and brine, dried over sodium sulfate, and the solvent was distilled off. The residue was chromatographed on a silica gel column (3.5 × 13 cm, packed with 4: 1 hexane: ethyl acetate). Fractions containing the product (contaminated with yellow impurities) were collected and evaporated to give a yellow foamy solid (2.22 g). Repeated chromatography using 5% methanol-methylene chloride and 4: 1 hexane: ethyl acetate provided the pure product (15) as a pale yellow foamy solid.

_{^{1 H NMR (CDCl 3) 7.45-7.10}} (m, 9H), 6.80 (d, 4H, J
= 8.8Hz), 5.38 (d, 1H), 5.05 (brt, 1H), 4.50 (m, 1
H), 3.90-3.80 (m, 2H), 3.80 (s, 6H), 3.50-2.95 (m,
4H), 2.30 (m, 2H), 2.10-0.60 (m, 43H). Analysis (C ₃₃ as H ₇₃ NO ₆₎ Calculated:. C, 77.62; H, 8.97; N, 1.17 Found:. C, 77.41; H, 8.97; N, 1.60 Example XVIII 1-N-cholesteryloxycarbonyl Carbonyl-2-succinyloxy-4-dimethoxytrityloxybutane (16) To a stirred solution of alcohol (15) (415 mg, 0.506 mmol) in dry pyridine (2 mL) was added 4-dimethylaminopyridine (3
0 mg) and succinic anhydride (48 mg, 0.48 mmol) were added and the mixture was stirred for 24 hours under a stream of argon. Thin layer chromatography (5% methanol-methylene chloride) showed traces of unreacted starting material. The solvent was removed and the residual pyridine was distilled off with toluene (2 × 10 mL). The remaining yellow foamy solid (16) was used for conversion to p-nitrophenyl ester and immobilization on CPG.

Example XIX Cholesterol-CPG support (17) Succinylated cholesterol derivative (16) was
nson and Smith (Oligonuclcotide Synthesis, a Pract
ical Approach, MJGait, (ed.) IRL press, pp47-49
(1984)) and immobilized on a porous glass support (LCAA-CPG; Sigma) controlled with a long-chain alkylamine. Crude succinate (16) (105 mg) was dissolved in dry dioxane (10 mL) along with p-nitrophenol (16 mg) and dry pyridine (0.05 mL). Dicyclohexylcarbodiimide (52 mg, 0.25 mmol) was added and the mixture was stirred at room temperature for 15 minutes, then cooled in a refrigerator for 16 hours. The crude p-nitrophenyl ester solution was filtered through a small amount of celite to remove DCU, and the filtrate was directly LCAA-CPG in dimethylformamide (DMF) (1.5 mL).
(0.50 g). Ethyl diisopropylamine (0.1
mL) was added and the mixture was stirred in a stream of argon for 18 hours. The CPG was filtered through a semi-solid glass funnel, and dimethylformamide (3 × 10 mL) and methanol (3 × 10 mL)
And ether (3 × 10 mL). Derived CP
G was dried on a vacuum pump, then treated with dry pyridine (1.5 mL) and acetic anhydride (0.2 mL) and capped. After stirring for 3 hours under argon, the CPG was filtered, washed with methanol (3 × 10 mL) and ether (3 × 10 mL),
Cholesterol-CPG (17) when dried with a vacuum pump
(0.46 g) was obtained. The CPG was analyzed for DMTr content according to the protocol described by Gait and found to have a CPG support loading of 24 μmol / g.

Example XX Cholesterol-CPG (17) to 3'-Cholesterol-
Synthesis of 5'DMTr-thymidine with tail molecule Cholesterol-CP for oligonucleotide synthesis
To test the stability of G (17), a single thymidine residue was added to a solid support (41.6 mg, 1 micromol). Milligen DN with standard phosphoramidite coupling chemistry
We made full use of the A synthesizer. The 5'-dimethoxytrityl protecting group on the thymidine helps to isolate and confirm the product,
Not removed. The CPG was washed well with acetonitrile (8 mL) to remove traces of unreacted DMTr-thymidine phosphoramidite and other non-covalently bound impurities. The CPG was dried with a vacuum pump and analyzed for DMTr content as described above. As a result, it was found that the filling degree was 27 micromol / g. Thymidine with 3'-cholesterol tail molecule is a support (20mg) in a 5mL reaction bottle (Teflon liner)
Separated by treatment with concentrated ammonia (10 mL) at 44 ° C. for 24 hours. Supernatant (pasteur pipette)
, And the support was washed with methanol (3 x 2 mL) and 3 '
-Thymidine with cholesterol tail molecule was isolated. Washings were combined, solvent removed (rotary evaporator / vacuum pump) and analyzed by thin layer chromatography on silica gel plates using 7: 1: 1: 1: 1 ethyl acetate: acetone: methanol: water: acetic acid. One major spot (Rf = 0.63) containing DMTr was colored orange when sprayed with 10% sulfuric acid-methanol. Only traces of DMTr-thymidine (Rf = 0.84) were detected, thus indicating stability of cholesterol binding. The CPG support after ammonia treatment for 24 hours showed a DMTr content of 1.4 μmol / g.

Ammonia treatment of DMTr-thymidine derivatized cholesterol-CPG at 44 ° C. for 5 hours gave the same thin layer chromatography results as treatment for 24 hours. The CPG support after 5 hours of ammonia treatment showed a DMTr content of 3.2 μmol / g.

The oligonucleotide (10 ') obtained from Example XII with an acridine 3'-tail molecule was studied for its melting temperature properties in the presence of a complementary oligonucleotide chain. The presence of the acridine intercalating agent increased the Tm (melting temperature) by about 4 ° C. compared to a similar oligonucleotide without the 3′-tail molecule acridine.

A suitable reporter group attached to the 3 'tail molecule of a suitable oligonucleotide is useful for confirming (identifying) the presence of the oligonucleotide. The fluorescence, chemiluminescence, or other properties of such reporter groups serve to non-radioactively label these nucleotides. Thus, nucleotides in a sample of interest, such as a biological sample, can be identified by the presence of fluorescence or other similar properties. For example, a lipophilic tail group, such as a 3 'oligonucleotide with a cholesterol tail molecule, helps the permeability of the oligonucleotide to the cell membrane, thus increasing the intracellular concentration of the oligonucleotide or promoting the permeability of the cell membrane. Achieved. A cleavage group at the 3 'end of the oligonucleotide binds the oligonucleotide to such a complementary sequence and has the complementary sequence of the oligonucleotide
It can aid in the site-specific cleavage of DNA. Such use may relate to gene identification, isolation, and the like.

Other tail molecules or conjugates will be chosen based on both other biological and physical properties. Such other biotail molecules may include appropriately blocked synthetic peptides, puromycin, digoxigenin, and the like. Other tail molecules with useful physical properties include spin-labeled compounds, DTPA chelators, phospholipids, di- and trinitrophenyl groups, crosslinking reagents such as alkylating agents, azidobenzene, psoralen, iodoacetamide, Including azidoprofflavin and azidouracil.

Example XXI 2,3,5,6-tetrafluorophenyltrifluoroacetate (18) Trifluoroacetic anhydride (28 mL, 0.2 mol) was added to 2,3,5,6-
Tetrafluorophenol (27.1 g, 0.163 mmol) was added dropwise with stirring. Boron trifluoride ether adduct (0.2m
l) was added and the mixture was heated to reflux overnight. The residual solution was distilled at normal pressure to remove trifluoroacetic anhydride and trifluoroacetic acid. The desired product (18) (32.2 g, 75% yield) was collected as a colorless liquid at 45 ° C. (18 mm).

d = 1.52 g / mL IR (CHCl ₃ ) 3010,1815,1525,1485,1235,1180,1110,95
5cm ^-1 . Analytical value (as C ₈ HO ₂ F ₇ ) Calculated: C, 36.66; H, 0.38; F, 50.74. Found: C, 36.31; H, 0.43; F, 50.95. Example XXII 2, 3,5,6-tetrafluorophenyl 3- (9-acridinyl) propionate (19) Method a: 3- (9-acridinyl) propionic acid (400 m
g, 1.59 mmol) and triethylamine (0.22 mL) in methylene chloride (20 mL) were added 2-fluoromethylpyridinium tosylate (FMPT) (496 mg, 1.75 mmol) and the mixture was stirred at room temperature for 15 minutes. 2,3,5,6-Tetrafluorophenol (317 mg, 1.91 mmol) and triethylamine (0.22 mL) were added and the mixture was stirred for 15 minutes. The heterogeneous mixture was evaporated and the residue was flash chromatographed with 1: 1 hexane: ethyl acetate (2: 1).
× 29 cm silica). The fractions containing the pure product are combined and evaporated to dryness to give the desired product (1
9) (250 mg, 39% yield) was obtained as a pale yellow solid.

183-185 ° C; TLC (1: 1 hexane: ethyl acetate), Rf = 0.70, blue fluorescence under long wavelength ultraviolet (UV) IR (KBr) 3075, 1790, 1520 and 1095 cm ^-1 ; ¹ H NMR ( CDCL ₃ ) 8.29 (d, 4H, J = 8.0 Hz), 7.82 (t, 2H,
J = 7.2Hz), 7.64 (t, 2H, J = 8.6Hz), 7.04 (m, 1H), 4.13
(T, 2H, J = 8.7Hz), 3.16 (t, 2H, J = 8.7Hz).

Analysis (C ₂₂ H ₁₃ NO ₂ as F ₄₎ Calculated: C, 66.17; H, 3.28 ; N, 3.51; F, 19.03 Found: C, 66.01; H, 3.11 ; N, 3.33; F, 19.13.

Method b: 3- (9-acridinyl) propionic acid (251m
g, 1 mmol) in methylene chloride (10 mL) was added triethylamine (200 μL, 1.4 mmol) and TFP trifluoroacetate 18 (200 μL). After stirring for 2 days under argon, the heterogeneous mixture was filtered through celite and the filtrate was evaporated to dryness. Residue in 1: 1 hexane:
Flash chromatography using ethyl acetate (2
X 25 cm silica). The fractions containing the product were collected and evaporated to dryness. The residue was repurified by flash chromatography (2 × 25 cm silica) using methylene chloride. The pure product eluted with 5% ethyl acetate-methylene chloride as a yellow band. The fractions containing the pure product are combined and evaporated to dryness to give 19 (67 m
g, 17% yield) as a pale yellow solid.

Example XXIII 2,3,5,6-tetrafluorophenyl 5- (9-acridinyl) pentanoate (20) The same procedure as in Example XII (described above) was used for the preparation of 20. 5- (9-acridinyl) pentanoic acid (262 mg, 1 mmo
l) gave 20 (180 mg, 42% yield) as a pale yellow solid.

 Melting point 122-124 ° C TLC (1: 1 hexane: ethyl acetate), Rf = 0.63, blue fluorescence at long wavelength UV.

IR (KBr) 3075,2960,1790,1515,1105,955 and 750cm
_{^{-1; 1 H NMR (CDCL 3}} ) 8.26 (d, 4H, J = 9.2Hz), 7.92 (t, 2H,
J = 6.8Hz), 7.58 (t, 2H, J = 8.8Hz), 7.00 (m, 1H), 3.71
(T, 2H, J = 7.8Hz), 2.78 (t, 2H, J = 6.4Hz), 2.05 (m, 4
H).

Analysis (C ₂₄ H ₁₇ NO ₂ as F ₄₎ Calculated: C, 67.45; H, 4.01 ; N, 3.28; F, 17.78 Found: C, 67.18; H, 3.87 ; N, 3.02; F, 17.67.

Example XXIV 1- [3- (9-acridinyl) -1-oxopropyl] -5-hydroxymethyl- (3R-trans) -3-
Pyrrolidinol (7) Step V: Acridine TFP ester 19 (206 mg, 0.52 mmol)
Of methylene chloride (8 mL) with aminodiol 2 (0.5 mL)
7 mmol) and triethylamine (86 μL) were added dropwise to an ice-cold solution of ethanol (1.14 mL). The mixture was stirred at room temperature for 18 hours and concentrated. The residue was purified by flash chromatography using 5% methanol in methylene chloride to give the desired product 7 (181 mg, 100% yield) as a yellow foamy solid.

TLC (9: 1 methylene chloride: methanol), Rf = 0.50, yellow spot emitting blue fluorescence at long wavelength UV IR (KBr) 3400 (br), 1620, 1440 and 1070 cm ^-1 ; ¹ H NMR (CDCl ₃ ) 8.30 (D, 4H, J = 9.4Hz), 7.82 (t, 2H,
J = 6.6Hz), 7.62 (m, 2H), 4.31 (m, 2H), 4.05 (t, 2H, J
= 8.2Hz), 3.70 (m, 1H), 3.50 (m, 2H), 3.22 (d, 2H, J =
1.4Hz), 2.78 (m, 2H), 2.03 (m, 1H), 1.64 (m, 1H).

Analytical values (as C ₂₁ H ₂₂ N ₂ O ₃ .0.5H ₂ O) Calculated: C, 70.18; H, 6.45; N, 7.79 Found: C, 70.41; H, 6.45; N, 7.68.

UV (pH 7.2) λmax = 252 nm (ε101,000), 260 nm (ε64
00), 356 nm (ε6200).

Excitation at 355 nm, emission at 460 nm Fluorescence (pH 7.2) Example XXV 1- [5- (9-Acridinyl) -1-oxopentyl] -5-hydroxymethyl- (3R-trans) -3-
Pyrrolidinol (21) A similar method as described in Example XXIV was used for the preparation of 21. 2 from acridine TFP ester 20 (1.28 g, 3.00 mmol)
1 (1.04 g, 92% yield) was obtained as a pale yellow foamy solid.

TLC (45:45:10 hexane: ethyl acetate: methanol),
Rf = 0.14 Yellow spot emitting blue light at long wavelength UV, IR (CHCl ₃ ) 3350 (br), 3010, 2930, 1610 and 1435 cm
^-1 ; ¹ H NMR (CDCl ₃ ) 8.22 (d, 4H, J = 9.0 Hz), 7.77 (t, 2H,
J = 6.6Hz), 7.56 (t, 2H, J = 7.8Hz), 4.27 (m, 2H), 3.7
−3.4 (m, 6H), 2.3−1.6 (m, 10H).

Analysis _{(C 23 H 26 N 2 O} 3 · 0.75H 2 O ) Calculated values: C, 70.48; H, 7.07 ; N, 7.15 Found: C, 70.33; H, 6.89 ; N, 7.15.

Example XXVI For the preparation of 1- [5- (9-acridinyl) -1-oxopentyl] -5- [bis (4-methoxyphenyl) phenylmethoxy] methyl- (3R-trans) -pyrrolidinol (22) 22 A method similar to that described in Example IX was used. Diol 21 (103 mg, 0.272 mmol) to 22 (78 mg, 42%
Yield) was obtained as a pale yellow foamy solid.

TLC (45:45:10 hexane: ethyl acetate: methanol),
Rf = 0.41 A yellow spot that emits blue fluorescence and turned orange when sprayed with 10% sulfuric acid / methanol.

IR (KBr) 3350 (br), 2930, 1610, 1510 and 1250c
m ^-1 ; ¹ H NMR (CDCl ₃ ) 8.24 (d, 4H, J = 8.8 Hz), 7.76 (m, 2
H), 7.53 (m, 2H), 7.35-7.10 (m, 9H), 6.79 (m, 4H),
4.7-4.3 (m, 2H), 4.08 (m, 1H), 3.9-3.3 (m, 11H), 3.
10 (m, 2H), 2.32-1.71 (m, 6H).

Example XXVII Acridine-CPG support (23) The same procedure as described in Examples X and XI was used for the preparation of 23. Alcohol 22 (58 mg, 80 μmol) and LCAA-
CPG (0.39g) to CPG support with DMTr loading of 20.6μmol / g
Of 23 was obtained.

Example XXVIII Direct synthesis of 3'-tailed oligonucleotides (24-26) using CPG supports 6, 10 and 23 Complementary to the initiation codon region of the mRNA corresponding to hepatitis B surface antigen protein (5'-TCCATGTTCGT) Oligonucleotides with 3'-tail molecules having specific sequences were synthesized using CPG supports 6, 10 and 23. Such 3'-tailed ODNs with improved binding can be advantageously used as antisense oligonucleotides.

The synthesis of 3'-tailed oligonucleotides having the sequence 5'-TCCATGTTCGT was performed using solid supports 6 and 10, respectively, as described in Examples VII and XII.

The synthesis of the same oligonucleotide with a 3′-tail molecule using the solid support 23 was the same as the method described in Example XII, except that the solid support 23 was used instead of the solid support 10.

Example XXIX Preparation of 3'-aminohexyl-CPG (AH-CPG) a. 1,3-Dioxo-2- (6-hydroxyhex-1-
Yl) -isoindole-5-carboxylic acid (27) 6-aminohexane-1-ol (11.7 g, 10 mmol) and 1,2,4-benzenetricarboxylic anhydride (7.68 g, 4 mmol)
l) mix 175 for 15 minutes or until steam generation stops
Heated to 225225 ° C. The melt was poured into water to give a white crystalline solid. The solid was collected by vacuum filtration and dried in vacuo (vacuum) overnight to give an analytical purity of 27 (9.1 g, 78%).

127-135 ° C ¹ H NMR (DMSO-d ₆ ): δ 8.344 (1 H, d, H-7 [6]);
8.208 (1H, s, H-4); 7.967 (1H, d, H-6 [7]); 3.57
8 (2H, t, H-1 '[6']; 3.365 (2H, t, H-6 '[1']); 1.7
-1.2 (8H, m, H- 2 ', 3', 4 ', 5'); analysis _{(C 15 H 17 N 1 O} 5 ) Calculated value: C, 61.85; H, 5.88 ; N, 4.81; Found: C, 61.56; H, 6.12; N, 4.98.

b. 1,3-Dioxo-2- (6-dimethoxytrityloxyhex-1-yl) isoindole-5-carboxylic acid (28) 27 (2.91 g, 10 mmol) was added to pyridine (50 mL) and triethylamine (1.9 mL). ), Dimethoxytrityl chloride (7.11 g, 20 mmol) and 4-dimethylaminopyridine (63 mg) were added. The mixture was stirred overnight and then evaporated to dryness. The residue was dissolved in methylene chloride (100 mL), and the obtained solution was washed with ice-cooled 1 M citric acid (100 mL) and then with water (100 mL), dried over sodium sulfate, and evaporated. The resulting hard syrup 27 was used without further purification.

Triethylamine was added to a mixture of cp-nitrophenyl 1,3-dioxo-2- (6-dimethoxytrityloxyhex-1-yl) isoindole-5-carboxylic acid (29) 28 (2.4 g, 4 mmol) in methylene chloride. Was. After the solution was cooled in an ice water bath, p-nitrophenyl chloroformate was added, and the mixture was stirred at 4 ° C for 5 hours.
After adding 4-dimethylaminopyridine to the cooled solution, the solution was allowed to rise at room temperature. After stirring overnight at room temperature, the solution was repeatedly extracted once with a saturated sodium bicarbonate solution (5 × 10 mL) and then with ice-cold 1M citric acid. The organic layer was dried over sodium sulfate and evaporated to dryness. The residue was purified on a silica gel column (2: 1) using toluene-ethyl acetate (5: 1) as eluent.
(9 × 150 mm).
Fractions containing pure material were pooled and evaporated to dryness to give an analytical purity of 29 (1.7 g, 60%).

Analytical values (as C ₄₂ H ₃₈ N ₂ O ₉ ) Calculated: C, 70.58; H, 5.36; N, 3.92; Found: C, 70.57; H, 5.22; N, 3.84.

d. 3'-Amine CPG support (AH-CPG) (30) The long chain alkylamine CPG was activated with 3% dichloroacetic acid-methylene chloride. 29 g (143 mg,
0.2 mmol) in pyridine (10 mL) and triethylamine (1 m
L) and the reaction mixture was gently stirred for 24 hours. The remaining amine was capped by treating with acetic anhydride (0.5 mL) and gently stirring for 24 hours. The CPG was filtered, washed with pyridine (1 × 5 mL) and then with acetonitrile (3 × 5 mL) and air-dried. The obtained AH-CPG30 was vacuum dried at room temperature for 2 days. Analysis of AH-CPG by treatment with perchloric acid showed a filling degree of 20 μmmol / g. The AH-CPG support (50 mg, 1 μmol) was used as a standard 1
Packed in a μmol synthesizer column.

Example XXX Synthesis of Amine-Modified Oligonucleotides (32,33) ODN was prepared on a 1 micromolar scale, using two different CPGs, utilizing standard β-cyanoethyl phosphoramidite coupling chemistry (Atkinson & Smith, supra). Manufactured from support. AH- prepared as described above in Example XXIX
CPG30 was used to synthesize 32. Clon Tech Lab, Inc
“Amine-ON” CPG obtained from (palo Alto, CA) is 33
Was used to synthesize

The amine-modified ODNs 32 and 33 having the sequence 5'-TCCATGTTCGT were Mill along with the protocol supplied by the manufacturer.
Manufactured using either igen7500 or Applied Biosystems Model 380B. After deprotection with ammonia, the tritiated ODN is separated from the ammonia solution by Hamilton PR.
HPLC purification by direct injection on a P-1 column (305 × 7.0 mm) to purify the amine-modified ODN product 32 or 33 with 20-45% acetonitrile-0.1 M TEAA (pH 7.5) (primary gradient)
And eluted in 20 minutes (flow rate = 4 mL / min). Appropriate fractions were combined and concentrated to dryness using a savant speed-vac. The residue was detritylated with 80% acetic acid (500 μL, 28 ° C., 70 minutes), precipitated with 3 M sodium acetate (100 μL) and 1-butanol (4 mL), centrifuged, and ethanol (1 mL)
Wash, centrifuge, evaporate to dryness and sterile distilled water (1m
L). ODN32 or 33 concentration was determined from UV absorption at 260 nm. All ODN concentrations were measured in pH 7.2 PBS (9.2 mM disodium phosphate, 0.8 mM monosodium phosphate, 0.131 M saline). Extinction coefficient of each ODN nearest model (CRCantor et al, Biopolymers 9, 1059 (1970) ) are determined using, it was used to calculate the theoretical concentration (μg / mL) ratio of A _260. Purified ODN 32 or 33 is 5-45%
Dy using acetonitrile-TEAA (primary gradient)
Analysis was performed by HPLC (flow rate = 1 mL / min) on an amax C-18 column. ODN purity was confirmed by denaturing PAGE analysis.

Example XXXI Synthesis of ODN (32a) with 3'-acridine-tail molecule ODN32 with 3'-amine-tail molecule analyzed by analytical C-18 HPLC shows one peak and PAGE shows only one band Was. Isolated 3′-amine-modified ODN32 [100 μg (29 nm
ol)] in water (100 μL) and 1.0 M borate buffer (50 μL).
M, pH 8.3). 5.0 mg of freshly prepared 1: 1 M-pyrrole: acetonitrile of acridine TFP ester 19
/ mL solution (220 μL, 1.1 mg, 3.0 μmol) was added and the heterogeneous mixture was shaken at room temperature. The progress of the reaction was monitored by HPLC. Reaction of ODN32 with amine-tail molecule with acridine TFP ester 19 was completed within 1 hour. 13 minutes of product
To more easily separate from the 14 minute impurities, the crude reaction mixture was added to water (1.5 mL) and cut through an ultrafiltration membrane (Centricon-3 microconcentrator; Amicon) cut at 3000 MW to a final volume of 100100 μL. Previously purified by centrifugation. The solution is treated with 5% to 45% acetonitrile-0.4%.
Use a 1M TEAA (primary gradient) on a Dyanamax C-18 column (0.75 × 25 cm) for 20 minutes (efflux rate = 1 mL / min).
Purified by HPLC. The product was collected in one fraction and concentrated on a Speed-Vac. Sterile distilled water (100
[mu] L) was added, acridine from playing - determine the concentration of the modified ODN32a from A ₂₆₀ measurements. The theoretical yield for 29 nmol is
At 108 μg, the actual yield was 39 μg. The purity of the ODN product 32a was quantified by HPLC and PAGE.

Since some loss of ODN32a occurred during ultrafiltration, another method was used to purify the acridine-modified ODN32a. With all starting 3'-hexylamine-tail molecules
Since ODN32 reacted, purification of the acridine-modified ODN32a was performed using Sephadex (trade name) G-25 (NAP-25 column, pharmac
a, Piscataway, NJ) through a pre-packed column containing
Achieved by gel filtration. Equilibrate the column with water (25 mL), then apply the crude mixture and apply the acridine-modified
ODN was eluted with water (0.5 mL fraction). Fractions containing pure product (as revealed by C-18 HPLC) were combined and concentrated on a Speed-Vac. The pale yellow solid residue 32a was regenerated with water (200 μL) and analyzed with 260 nm UV light. The concentration was measured as 108 μg / 200 μL. Theoretical yield is 108
μg.

Example XXXII ODN with 3'-acridine-tail molecule using amine-OnCPG
Synthesis of (33a, 33b) Using the same reaction conditions described above in Example XXXI, HP
ODN33 with 3'-amine-tail molecule for LC purification was treated with acridine TFP ester (19 or 20) in pH 8.3 borate buffer.
After shaking for several hours, HPLC analysis showed that about 70% of ODN33 with 3'-amine tail molecule remained unreacted. PAGA analysis of the crude reaction mixture showed that only the least mobile zone was completely reacted. Further treatment of 19 with borate buffer did not increase the extent of the reaction. After 22 hours, the mixture was concentrated and low molecular weight impurities were removed by centrifugation through a 3,000 MW cut-off ultrafiltration membrane. The pool is purified by C-18 HPLC and concentrated to ODN33a or 33b
Was obtained as a single peak according to C-18 HPLC and as a single band according to PAGE.

Table I shows the modified and unmodified oligonucleotide 5'-TC
Indicates the properties of CATGTTCGT.

^a The sequence of 20 units ODN (target) is 5'-GTGACGAACATGGAGAA
CAT. 11 units ODN (control) is 5'-TCCATGTTCGT.

^b Calculated concentration of ODN giving 1.00 absorption units at 260 nm ^c % yield of ODN isolated from 1 μmol CPG, 32a, 33a and 3
The yield of 3b is based on μmol of ODN with starting amine tail.

^d Purified by C-18 HPLC ^e Purified by gel filtration ^f Elution time; ^h Rm using HPLC system ^g 35-80% acetonitrile (30 min) gradient shown in Fig. 1 Rm is relative ratio of migration distance to bromophenol blue ⁱ temperature step VIII of the midpoint of the maximum slope of the Rm product published ^j Tm is a ₂₆₀ vs temperature ODN25,26,32a, 33a, 3, incorporated 33b '
-Shows the structure of the acridine tail molecule. AH-CPG is amine-O
Compared to the yield obtained with NCPG, with 3'-acridine-tail molecule
An improved yield of ODN was obtained.

Example XXXIII Synthesis of Internal Amine-Modified ODNs (34-36) Internal amine modifications were performed by KJ Gibson and SJ Benkovi
c. With the 5'-DMTr-3'-0-cyanoethyldiisopropyl phosphoramidite derivative of 5-phthalimidopropyl-2'-deoxyuridine as described by (Nucl, Acido Res. 15 , 6455 (1987)). Introduced according to the protocol described in Example XXX. In summary, OD
N5'-TCCATGTTCGT was internally modified with a, b, and / or c, as shown below.

Position a (34), Position a + b (35) or Position a + c
In (36) three different ODNs were prepared by amine modification.

Example XXXIV Internally Modified Acridine ODN (34a, 34b, 35a, 35b, 36a, 36b)
Internally amine-modified ODNs 34, 35, 36 were prepared according to Example XXXI
Reacting with acridine TFP ester 19 to produce ODN products 34a, 35a and 36a, respectively, according to the method described in
Or reacted with acridine TFP ester 20 to produce ODN products 34b, 35b and 36b, respectively.

Example XXXV Heat denaturation experiment To determine the effect on the binding affinity of the 3'-tail molecule,
Tm experiments were performed using the various 3'-modified ODNs described above. Thermal melting curves are obtained by tracking the change in A ₂₆₀ of the aqueous solution containing equimolar amounts of the appropriate complementary unmodified 20 units ODN target and ODN with the selected 3'-tailed (5'-GTGACGAACATGGAGAACAT) . The 20 unit ODN target provides a nucleotide overhang, which may affect the interaction of the ODN with the 3'-modification. Such interactions will be equally important in real biological targets. For these experiments, the target was DNA
And not a similar biological target, RNA. If acridine is now efficiently inserted between base pairs of the miniduplex by hybridization with the target strand, the Tm of the acridine-modified ODN target duplex can be increased. Unmodified 11 unit ODN was used as a control for each Tm study.

ODN was prepared as a 2 μM solution in PBS pH 7.2. Gilford
Gilford System2600UV with 2527 thermo programmer
-A VIS spectrophotometer was used. Temperature control quartet (cuv
ette) with a temperature rise of 0.5 ° C / min.
The sample was heated from 15 ° C to 85 ° C. Tm was measured using the ultimate derivative. For replication experiments, Tms was given within 0.5 ° C. One representative experimental data is shown in Table I in Example XXXII.
It is listed in.

Compared to unmodified ODN, 3′-cholesterol-tailed O
Was observed a significant increase in the melting temperature of the DN24, this is reported previously (RLLetsingen et al Proc 86, 655
3 (1989)) was significantly different from the similar study of ODN with 3'-cholesterol-tail molecule. In the Tm studies described herein, the overhang of the complementary target can affect the Tm, as well as the interaction of the target nucleic acid with the lipophilic tail molecule.

ODNs 25 and 26 showed equivalent binding affinity for the target ODN (ie, comparable Tm). No surprising difference was observed between the Tm of ODNs 25 and 26 (ie, the length of the 3- and 5-carbon bonds), but ODN33a and 32a were compared to the ΔTm of ODN25, 26, and 33b. And resulted in only a small increase in Tm. These data indicate that the strength of the bond, the reliability of the stereochemistry, and / or the sequence-dependent intercalation effect
Suggest that this can be affected.

Table II provides the Tm data obtained for the unmodified ODN and the internally modified ODN (both 1 and 2 internal modifications). Determine the Tm of the unmodified and internally modified ODN, and
The difference between the DNTm and the Tm for each internal modification to the ODN was calculated (ΔTm (° C.)). The difference between the calculated Tm is -2.1 ° C to +10.7
° C. The length of the 5-carbon bond resulted in a large Tm increase (ie, 34b, 35b, 36b)
The increase is not easy to predict. Inclusion of a second intercalating acridine group had an additional effect on increasing Tm. These data indicate that the bond length, sequence-dependent intercalation effects, and / or hydrogen bonding of amide bonds (generated through an amine modification reaction with acridine TFP ester) can affect Tm. Indicated.

Example XXXVI O- (4,4'-dimethoxytrityl) -1,6-hexanediol (37) DMTr-cl (3.38 g, 10 mmol) and diisopropylethylamine (3.45 mL, 20 mmol) in dry pyridine (50 mL) 1,6-hexanediol (5.91 g, 50 mmol) was added to the solution. After stirring for 4 hours, 5% sodium hydrogen carbonate (85 mL)
Was added to stop the reaction, and the mixture was diluted with methylene chloride (2 × 100 m
L). The organic layer was washed with water (2 × 100 mL), then with brine (100 mL), dried over sodium sulfate, filtered, and concentrated. The residue was purified by flash chromatography (3.5 × 17 cm silica) using methylene chloride. Fractions containing the pure product were combined and concentrated to give 37 (1.40 g) as a yellow syrup.

TLC (95: 5 methylene chloride: methanol), Rf = 0.40, yellow spot: this turned orange when sprayed with 10% sulfuric acid-methanol.

_{^{1 H NMR (CDCl 3) 7.5-7.1}} (m, 9H), 6.81 (m, 4H), 3.8
0 (s, 3H), 3.77 (s, 3H), 3.64 (q, 2H, J = 6.6Hz), 3.02
(Q, 2H, J = 6.0 Hz), 1.7-1.2 (m, 8H).

Analytical values (as C ₂₇ H ₃₂ O ₄ .0.7H ₂ O) Calculated: C, 74.87; H, 7.77 Found: C, 74.61; H, 7.36.

Example XXXVII Hexanol-CPG (38) Alcohol 37 (530 mg, 1.26 mmol) in dry pyridine (10
Succinic anhydride (380 mg, 3.80 mmol) and DMAP (76 mg) were added to the stirred solution in (mL). The mixture was stirred under argon for 23 hours and evaporated to dryness. Residual pyridine was azeotropically distilled with toluene. The residue was dissolved in chloroform (30 mL), washed with brine, dried over sodium sulfate and concentrated to give a quantitative yield of succinate as a yellow foamy solid.

TLC (95: 5 methylene chloride: methanol), Rf = 0.19, product appeared as a yellow spot and turned orange when sprayed with 10% sulfuric acid-methanol.

LCAA-CPG (1.0 g) acid-washed to the crude succinylated derivative (104 mg, 0.200 mmol), dry pyridine (10 mL),
Triethylamine (0.080 mL), EDC (380 mg, 2.0 mmol)
And DMAP (12 mg) were added and the mixture was stirred under argon for 46 h. The CPG was filtered, washed with pyridine, methanol, chloroform and ether, and then dried under vacuum. Residual amino groups on the CPG were capped by stirring the support in dry pyridine (3.5 mL) and acetic anhydride (0.46 mL). After 3 hours, the CPG was filtered, washed with methanol, chloroform and ether, and dried in vacuo to give product 38 (1.0 g). This CPG was analyzed for DMTr content and found to be 26.
It was found to have a filling degree of 2 μmol / g.

Example XXXVIII Synthesis of calmodulin antisense ODN (39) with 3′-hexanol-tail molecule Hexanol-C corresponding to DMTr content (38 mg, 1 μmol)
Replace PG38 with an empty ODN synthesis column (American Bisnetecs, Inc.)
Was filled. 24 units (5 ′ TAATTATTCAGCCATTTATTAGTT) having a sequence complementary to the initiation codon region (−12 to +12) of the calmodulin mRNA of Paramecium (Paramecium)
Manufactured using hexanol-CPG38, an ABI380B DNA synthesizer utilizing standard phosphoramidite chemistry, and a protocol supplied by the manufacturer. The 5'-DMTr protecting group was not removed. The ODN was cleaved from the support and then deprotected by treatment with 30% aqueous ammonia (2 ml) at 40-44 ° C for 24 hours. The supernatant was directly subjected to PRP-1 reverse phase HPLC.
Inject into a column (305 x 7.0 mm) and use 20-45% acetonitrile / 0.1 M TEAA (pH 7.5) (primary gradient)
Elution was performed at a flow rate of 1 mL / min per minute. The product was collected in one fraction and concentrated on a Speed-Vac to give the 5'-DMTr protected product. DMTr group is added with 80% acetic acid (300μL).
Removed by treatment at 80 ° C for 80 minutes, and 2.4M sodium chloride (100
μL) and 1′-butanol (4 mL).
ODN with tail molecule was precipitated. The mixture was centrifuged, washed with ethanol (1 mL), further centrifuged and evaporated to dryness. The remaining white solid was regenerated with sterile distilled water (1 mL) and filtered through a 0.2 μm filter. ODN39 concentration is 26
3.75 mg / mL, as determined from UV absorption at 0 nm.
The theoretical yield for 1 μmol is 7.49 mg. Purified ODN
39 was analyzed by HPLC on a Dynamax C-18 column (0.75 × 25 cm) using 5-45% acetonitrile / TEAA (primary gradient), eluting for 20 minutes (eluent rate = 1 mL / min). One major peak eluted at 9.9 minutes (> 95% purity). ODN39 purity was confirmed by PAGE denaturation analysis (one band).

Example XXXIX Biological Action of ODN39 with Hexanol-Tail Molecule Paramecium tetraurel
The swimming behavior of ia) has been used as a biological model system to study the antisense effects of injected ODN39. Paramecium tetrauria
swimming behavior elia) a series of ion channels (voltage dependence of Ca ² + and K + channels and C in the cell membrane
It is controlled by the action of a ² + dependence of Na + and K + channels). These ion channels work in tandem to produce action potentials that cause ciliary movement to reverse. This reversal causes the paramesium tetraurea to swim backwards. The regulatory protein calmodulin plays a pivotal role in regulating membrane potential and swimming behavior in Paramesium tetraurea.

The 3'-hexanol-tail molecule antisense ODN39 has been used to suppress calmodulin gene expression in Paramesium tetraurea. This antisense OD
N39 was injected into the cytoplasm (cell cytoplasm) with a microsyringe and the behavior of the cells was monitored over time. The injected cells were analyzed by incubation in a solution testing the function of the Na + channel. Cells of the wild-type strain swim backwards in such a solution for about 15 seconds; individual cells injected with antisense ODN39 showed a much reduced behavioral response. After 15-20 hours, the backward swimming time of the injected cells dropped to a minimum of about 2 seconds (FIG. 1). Normal backward swimming behavior was restored when cells injected with ODN39 at time 0 were subsequently injected with calmodulin 15 hours later. Injection of ODN with 24 units-3'-hexanol-tail molecule showing either random or sense sequence did not change backward swimming (FIG. 2).

The 3′-hexanol-tail molecule calmodulin antisense ODN39 was effective even at a concentration as low as 0.42 mg / mL (56 μM) (FIG. 3). In contrast, calmodulin antisense ODN without 3'-modification had no effect even at a high concentration of 6.1 mg / mL (835 μM). Thus, the 3'-tail molecule antisense ODN39 showed at least 15 times greater potency (titer) than similar unmodified ODN. This increase in potency is due to 3'-exonuclease degradation against 3'-tailed ODNs.
Due to its improved stability. Assuming an injection volume of 10 pL and a paramesium-tetraurea volume of 200 pL, the minimum cytoplasmic concentration of ODN39 for the observable biological effect is
It was calculated to be 2.8 μM.

While the preferred embodiment of the invention has been described, it has been described by way of illustration only, and other specific examples, modifications, and improvements will be apparent to those skilled in the art. The present invention
It is not limited to the preferred embodiment but is set forth in the following claims.

────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Mayer, Rich B. Jr. USA, 98072 Washington, Woodinville, NE Wanhand Dread and Seventy Sixth Place, No. 15411 (72) Inventor Petrie, Charles Earl United States, 98072 Washington, Woodinville, N.W. Thi Six vinegar play vinegar, No. 12117 (58) investigated the field (Int.Cl. ^7, DB name) C07H 21/04 C12Q 1/68 CA (STN ) RE ISTRY (STN)

Claims (8)

(57) [Claims] 1. Synthesis of an oligonucleotide having a low molecular weight tail linked to its 3 ′ end via a linking molecule: (a) having a primary alcohol, a secondary alcohol and an amine as a functional group (B) a low molecular weight tail molecule (the tail molecule is a reporter group,
Selected from an intercalator group, a lipophilic group and a cleavage group), and reacting the amino group of the linking molecule with a low molecular weight tail molecule to bind the linking molecule and the tail molecule; Immobilizing the secondary alcohol group of the linking molecule on the solid support and binding the linking molecule having a binding tail molecule to the solid support; and (d) three of the first 3 'phosphoramidite nucleotides. ′ End is reacted with the primary alcohol group of the linking molecule to bind the first nucleotide to the linking molecule via its 3 ′ end (the binding of the first nucleotide to the linking molecule is (E) linking one nucleotide to the tail molecule via the linking molecule, and linking the first nucleotide to the solid support via the linking molecule.), (E) The 'phosphoramidite nucleotide is reacted sequentially with the 5' end of the preceding nucleotide to form a synthetic oligonucleotide attached to the linking molecule at the 3 'end of the oligonucleotide (the 3' end of such a synthetic oligonucleotide). Attachment to the linking molecule at the terminus involves linking the oligonucleotide to the tail molecule at the 3 'end of the oligonucleotide via the linking molecule, and attaching the oligonucleotide to the solid support and the continuation of the additional nucleotides. (F) An oligonucleotide having a tail molecule linked to its 3 ′ end via a linking molecule is separated from the solid support by a second functional group of the linking molecule and the oligonucleotide. Dissociating by cleavage of the link between the solid supports, and then (g) removing the amine group of the linking molecule Isolating an oligonucleotide having a tail molecule linked to its 3 'end through the oligonucleotide. 2. The derivative oligonucleotide obtained after step (g), wherein the phosphate ester located at the 3 ′ end is of the formula: Or [In the formula, Z is m and m ′ are each independently a positive integer less than 11,
n is 0 or 1, Q is a connecting group, and R is selected from the group consisting of a reporter group, an intercalator group, a lipophilic group, and a cleavage group. ] The method according to claim 1, comprising an oligonucleotide represented by the following formula: 3. The compound obtained after step (c), which is bound to a solid support, has the formula: Or [Wherein, Z is m and m ′ are each independently a positive integer less than 11,
n is 0 or 1, Q is a connecting group, and R is a member selected from the group consisting of a reporter group, an intercalator group and a lipophilic group and a cleavage group, Y is hydrogen or dimethoxytrityl, and X is a solid support. is there. The method according to claim 1, having a structure represented by the following formula: 4. The compound obtained after step (b) has the formula: Or Wherein Z is m and m ′ are each independently a positive integer less than 11,
n is 0 or 1, Q is a connecting group, and R is selected from the group consisting of a reporter group, an intercalator group, a lipophilic group and a cleavage group, and Y is hydrogen or dimethoxytrityl. The method according to claim 1, wherein the method has a structure represented by the following formula: 5. The method according to claim 1, wherein the solid phase support for oligonucleotide synthesis has the formula: Wherein R is alkyl, aryl, arylalkyl,
Heteroalkyl or heteroaryl. The method according to claim 1, having a structure represented by the following formula: 6. The derivative oligonucleotide obtained after step (g), wherein the phosphate ester located at the 3 ′ end is represented by the formula: [Wherein, Z is n is 0 or 1, Q is a connecting group, and R is selected from the group consisting of a reporter group, an intercalator group, a lipophilic group, and a cleavage group. ] The method according to claim 1 or 2, comprising an oligonucleotide represented by the formula: 7. The compound obtained after step (c) and bound to a solid support, has the formula: Wherein Z is n is 0 or 1, Q is a connecting group, and R is a member selected from the group consisting of a reporter group, an intercalator group and a lipophilic group and a cleavage group, Y is hydrogen or dimethoxytrityl, and X is a solid support. is there. The method according to claim 1, having a structure represented by the following formula: 8. The compound obtained after step (b) has the formula: Wherein Z is n is 0 or 1, Q is a connecting group, and R is selected from the group consisting of a reporter group, an intercalator group, a lipophilic group and a cleavage group, and Y is hydrogen or dimethoxytrityl. The method according to claim 1, having a structure represented by the following formula: