Polynucleotide assay reagent and method.

Description: TECHNICAL FIELD The present invention relates to a polynucleotide diagnostic system and a method.

Background of the Invention Two common types of polynucleotide diagnostic systems have evolved together based on hybridization between complementary fragments of polynucleotide probes and single-stranded polynucleotide analysts. In the first type, this polynucleotide analysis is single-stranded and immobilized on a solid support such as a nitrocellulose filter. The carrier is then subjected to the reaction of the target sequence region of the analyte with a complementary reporter-labeled probe under complementary strand annealing conditions. After several washes to remove the unbound probe, the presence of a reporter on this solid support is analyzed. This system is not fully satisfactory, especially in medical situations where ease of measurement and sensitivity are required. The procedure used to immobilize a single-stranded polynucleotide material on this solid support is somewhat complex and time consuming. The sensitivity of this system is limited. This is because, in the normal case, each analyte molecule hybridizes to a single probe molecule, and each probe generally contains about 100 reporter moieties.

A second type of polynucleotide diagnostic system involves two analyte-specific probes that are complementary to each other in separate regions of this analyte polynucleotide. This first probe is linked to a solid support and this second probe is free in solution and has a large number of reporter molecules. In practice, this analyte is mixed with the two probes under the following conditions: Under this condition, the polynucleotide strand of the analyte is subjected to the annealing of complementary polynucleotide chains (this annealing includes annealing of both the immobilized probe and the probe with the reporter). It is possible to bind to a solid carrier via.

Although the two probe systems have eliminated the problem that the nucleic acid material to be tested should be immobilized on a solid support, the method still has a number of constraints. First of all, when the nucleic acid substance to be tested is derived from a double-stranded nucleic acid, hybridization between this analyte polynucleotide and its complementary strand is often between the analyte and the two probes. Compete with the hybridization of. Moreover, the two probe systems are inherently slower than the single probe system because they are more dependent on kinetics than the single probe system. Also, the need for two different probes increases the cost of this system. That is, in terms of test sensitivity, the two probe systems have the same limitations as a single probe system, as each analyte polynucleotide binds to a single "reporter" probe, as mentioned above. have.

Abstract of the Invention The general purpose of the present invention is to provide a diagnostic system and method that substantially overcomes the problems discussed above and the limitations associated with the prior art for use in detecting polynucleotides. To provide.

Another object of the present invention is a single-stranded polynucleotide analysis and a base-specific two-stranded polynucleotide in such a system under conditions that leave complementary polynucleotide strands in a single-stranded state. To provide a reagent having a polynucleotide-bound polymer adapted to form a chain structure. Therefore, the binding of this analyte does not conflict with the strand complementary to the analyte annealed in the measurement reaction mixture, under conditions where the reagent polymer and the analyte form a sequence-specific pair. Achieved.

Another object of the present invention is to provide a reporter adapted to bind to the backbone of an analyte polynucleotide by electrostatic force in such a system under conditions where the reporter does not bind to the reagent polymer. To supply. Therefore, this system has a very high signal level tolerance due to the advantage that a relatively large number of reporter molecules can bind to each polynucleotide analyzer molecule.

Furthermore, it is another object of the present invention to provide a rapid, convenient and highly sensitive method for diagnosing polynucleotides.

The present invention includes a diagnostic reagent used for detecting an analyte polynucleotide having a defined target base sequence. This reagent has a solid carrier, to which a large number of polynucleotide-bound polymers are linked. Each polymer constitutes a base-complementary recognition moiety sequence. Each recognized moiety can be hydrogen-bonded to the corresponding internal sequence base in the target region of this analyte under selected binding conditions. The unbranched, substantially stereoregular backbone (1) supports the recognition portion at a position where this recognition portion can hydrogen bond with a base in the corresponding target sequence, and (1) 2) Under the selected binding conditions, it has a backbone charge density that is substantially lower than the charge density of this analyte. In a more preferred embodiment of the invention, this recognition portion comprises a purine base and a pyrimidine base. This backbone consists of a series of backbones that are achiral and connected by an almost uncharged chain. In another embodiment of the invention, similar recognition portions include uncharged chains, stereoisomerically defined chains or achiral chains, with alternating negatively charged and achiral chains. It is prepared on the backbone that has become.

The diagnostic system for detecting the polynucleotide analyte has a limited target sequence containing diagnostic reagents, and the reporter binds to the charged backbone of the polynucleotide analyte by electrostatic attraction, but in substance. The backbone of the reagent polymer, which is largely uncharged or completely uncharged, has a polycationic tail that does not bind under preset binding conditions. One or more reporter groups attached to this polycationic tail are applied to generate a signal in which the presence of the reporter can be detected.

In the method of the present invention, the polynucleotide analyte is added to the diagnostic reagent under conditions such that the analyte is in a single chain shape and provides a sequence-specific pair between the analyte and the reagent polymer. After this analyte / polymer annealing reaction, the reagent is washed to remove unbound test material. The reagent and bound analyte are then subjected to reaction with the reporter under preset conditions. Under these conditions, the polycationic tail in the reporter electrostatically binds to the charged backbone of this polynucleotide, but is substantially uncharged or completely uncharged. Does not combine with. This reagent is washed again to remove unbound reporters. The presence and / or amount of reagent-bound analyte is determined in its original position by measuring the reporter signal associated with this reagent-bound analyte. Alternatively, the reporter is eluted from the reporter / analyte and the reporter signal of the eluate is measured.

These and other purposes and features of the invention will become even more apparent when the following detailed description of the invention is read in connection with the accompanying drawings.

Brief Description of Drawings Figure 1 shows the more preferred purine and pyrimidine structures used in forming the polymer molecules of the invention.

FIG. 2 shows the more preferred purine analog recognition moieties and pyrimidine analog recognition moieties used in forming this polymer molecule.

FIG. 3 shows a more preferred cyclic backbone moiety used in forming this polymer molecule.

FIG. 4 shows a more preferred acyclic backbone moiety used in forming this polymer molecule.

Fig. 5A and Fig. 5B are N₁...... N₂Two more preferred subunit assembly schemes for coupling types of subunit backbones are illustrated.

FIG. 6 shows the backbone structure of subunit dimer AA via DD formed according to the method exemplified in FIGS. 5A and 5B, and the method illustrated in FIGS. 7A and 7B. The two annular backbone structures formed according to are shown.

Figure 7A-7C shows N₁... Illustrates three more preferred subunit assembly schemes for coupling E-type subunit backbones. Then, FIG. 8 shows a more preferable acyclic backbone structure of the subunit dimer formed according to the method illustrated in FIGS. 7A-7C.

FIG. 9 shows the synthesis of tetranucleotide analogs with a methylphosphonate backbone chain instead of a phosphotriester chain.

FIG. 10 illustrates the addition reaction of spacer arm molecules to a solid carrier.

FIG. 11 shows a general method for synthesizing polyamines from N-methyl amino acids.

FIG. 12 shows a method for synthesizing a polyamine having a terminal primary amino group.

FIG. 13 shows the preparation of a quaternary ammonium cation tail having a terminal primary amino group.

FIG. 14 illustrates a reaction for the preparation of a plurality of charged enzyme reporters according to one embodiment of the present invention.

FIG. 15 illustrates a reaction for the preparation of a divalent fluorescent reporter according to one embodiment of the present invention.

FIG. 16 illustrates various components included in the diagnostic system and the method of the present invention.

Detailed Description of the Invention The diagnostic system of the present invention comprises a solid carrier reagent consisting of a solid carrier with a large number of nucleotide-bound polymer molecules. The polymeric compositions of the present invention are designed to bind to polynucleotides containing the target sequence of a base with selected binding affinities. This composition consists of non-monopolymerizable, substantially stereoregular molecules or species (in the form: Here, BR_iIs the backbone part B and the recognition part R_iA series of base-specific subunits containing. This recognition part R_iIs selected to specifically bind to the corresponding internal sequence bases in the target sequence by Watson / Crick base pairing. This subunit is primarily connected to an achiral and substantially uncharged bond through these backbone sections. The design and selection of subunits suitable for use in constructing this polymer species will be described in Section 1 below. The method of coupling subunits through an achiral chain is described in Section 2. The strategies involved in subunit synthesis, along with subunit protecting groups, are discussed in Section 2.

This polymer species is synthesized by successive subunit couplings to form polymer molecules of selected length and sequence. As described in Section 4, the length of this target sequence and polymer sequence is chosen to achieve the desired binding specificity. The binding affinity of this polymer for the target can be selectively altered by some of the strategies discussed in Section 4. This strategy involves the selection of cognitive parts. This recognition portion, along with the corresponding target base, forms a hydrogen bond paired with either three (for greater binding affinity) or two (for smaller binding affinity) bases. Can be. The resulting composition comprises a polymer species or polymer molecule. All of these have substantially the same subunit sequence and substantially the same type of inner subunit chain. Functionally speaking, all of this polymer species have substantially the same binding affinity for this target polynucleotide. Once the desired subunit sequence has been selected, a method for assembling this polymer is shown in Section 5.

This polymer composition is useful for the novel solid phase diagnostic system described in Sections 6-7. The system binds the analytical polynucleotide molecule to a carrier-bound polymer molecule and subsequently results in a number of polycationic reporter molecules to this polynucleotide in order to give an amplified reporter signal to each bound analytical molecule. Based on electrostatic addition.

I. subunit structure A. recognition part Subunit BR suitable for use in forming the polymer species of the present invention._iDesign includes a number of structural and / or stereochemical criteria. This criterion must be achieved not only by the backbone and recognition parts, but also by the chain between the two. The design requirements for this recognition part will be considered first.

The recognition portion of each subunit must supply two or three hydrogen bond groups held in a solid configuration. This configuration is compatible with hydrogen bonds to two or three Watson / Crick hydrogen bond sites on the specific internal sequence base of the target gene sequence. Under conditions of use, (1) the tautomeric state of this recognition portion should be relatively stabilized, and (1) to avoid unwanted mispairing between the recognition portion and the corresponding target base, and ( 2) This recognition portion should have a structure in which hydrogen bond groups are relatively fixed. Such immobilization is optimally provided by a ring structure with polar hydrogen bonding groups. Hydrogen-bonding groups of this polarity either form part of the ring or are added directly to the ring.

The structure of this more preferred recognition moiety includes structures of purines, purine analogs, pyrimidines, and pyrimidine analogs. This structure is designed to form Watson / Crick base pairs with selected polynucleotide bases via either two or three hydrogen bonds. The subunit group used in polymer synthesis contains at least two recognition moieties that are base specific for different polynucleotide bases. Preferably, there is one or more recognition portions for each of the four bases in the natural DNA or RNA nucleotide. Also, as you can see below, one base R of the recognition part_i(It can be base paired with a nucleotide base via two hydrogen bonds) and the second group R_jIt is desirable to supply (which can bind to the same base via three hydrogen bonds). FIG. 1 shows a typical purine-type recognition portion and a pyrimidine-type recognition portion. The purine structures 1 and 2 are designed to bind to a thymine base or a uracil base, structure 3-6 binds to a guanine base, structure 7-9 binds to a cytosine base, and structure 10-12 binds to an adenine base. Designed to do. Structures 1,4,5,6,8 and 10-12 are R adapted to bind to the corresponding internal sequence bases via two hydrogen bonds._iThis is the type part. The remaining structure is R_jPart of the type, which forms three hydrogen bonds on base pairs. As will be seen below, purine nucleosides and pyrimidine nucleosides are useful in synthesizing a variety of other subunits suitable for use in polymer synthesis. These subunits are modified with amine protecting groups if necessary. These can be obtained from commercially available raw materials or can be prepared according to known methods (eg, methods described or related to Example 1).

Many purine analog or pyrimidine analog structures are shown in FIG. Within these structures, the recognition portion is connected to the backbone via cyclic carbon. These structures have base pair specificity and hydrogen bond characteristics similar to the base pair specificity and hydrogen bond characteristics of the analog structure shown in FIG. The binding properties of the polymer containing the carbon-linked recognition moiety are not significantly different from the bonding properties of the polymer in which the recognition moiety is bonded with nitrogen (as shown in Fig. 1), but the subunit containing the carbon-linked moiety is included. Units are generally more difficult to synthesize than the subunits containing purines and pyrimidines performed. However, in the synthesis of certain subunits, for example the synthesis of the achiral acyclic backbone subunit described in Example 10, carbon-bonded recognition moieties provide a useful starting material.

B. Backbone part The backbone part of each subunit is the general formula N.₁...... E or N₁...... N₂Have. Where N₁And N₂Is a nucleophilic group and E is an electrophilic group. As discussed below, more preferred nucleophilic groups are amino groups, hydroxyl groups and hydrazino groups, based on the stability required for the resulting subunit-to-subunit linkage and the ease of subunit bonding. And more preferred electrophilic groups and / or electrophile-binding reagents are derivatives of carbonic acid, thiocarbonic acid, carboxylic acids and sulfonic acids.

The backbone portion can have either an annular structure or a non-annular structure. Although the total number of possible backbone structures is quite large, nonetheless, due to many factors, only a fairly limited number of structures deserve to be implemented in practice. The first steps to select a convenient backbone part are shown below.

As a first condition, it was considered that only these annular backbone portions consisted of or could be easily derived from deoxyribose or ribose. This constraint is a practical constraint, which reflects the difficulty of nascent synthesis of ring structures with many chiral centers and the correspondingly higher costs. In general, backbone moieties and subunit inter-unit chains that may be suitable for polymers were selected based on one or more of the following factors: feasibility of synthesis based on known reactions; available Expected ease of synthesis from a flexible starting material: simplicity of structure; and expected stability of the final backbone.

The first selection of convenient backbone parts and subunit-to-subunit chains was performed as follows. A spatial-filled CPK molecular model of dual DNA and RNA was constructed according to parameters determined by X-ray diffraction of B-type oligonucleotides and A-type oligonucleotides. In each of these constructed dual structures, one of the two sugar-phosphate backbones was removed. Then, if possible, each possible backbone was added to the site where the first sugar-phosphate backbone was removed in this base shape. Each polynucleotide / polymer dual structure obtained then has its Watson / Crick base pair homogeneity, possible torsional and angular strains in the polymer backbone, and the strains conferred on this nucleic acid strand. The degree and non-bonding interactions between and within the chains were investigated. Particular attention was paid to whether the amide moiety of the backbone containing each amide group could easily adopt a planar conformation.

These early studies proved that: Required unit backbone length (ie N₁...... N in E₁...... E interval or activated N₁...... N₂-N in the E subunit₁The E interval) is 5-7 atoms with respect to the backbone moiety that constitutes or derives from deoxyribose or ribose (cyclic backbone structure), with an optimal length of 6 atoms.

The subunit structures determined to be acceptable in the above model study were then evaluated for synthetic viability and stability of the aggregated polymer backbone. (The feasibility of this synthesis is based on or mediated by the actual synthesis of this subunit, based on the major synthetic reactions reported in the literature or the reactions performed on the model compounds.) (This aggregate. For the stability of polymer backbones, preliminary stability studies were generally performed using well-linked model compounds or subunit dimers.) These types of studies included a number of candidate backbone structures. Used to further limit. From this limited pool of candidates, the annular backbone structures AG shown in FIG. 3 were finally selected as a more preferred structure.

In this figure, N₁...... N₂Subunits AD containing cyclic backbone moieties of type are 2'-deoxyribonucleosides (structure A): 2'-deoxyribonucleosides with amino groups substituted at the 5'and 3'positions (structures B and C, respectively). ); And a morpholino derivative of ribonucleoside (Structure D). N₁... Subunits containing E-type backbone moieties are 2'-deoxyribonucleosides (structures E and F, respectively) in which these 5'positions are substituted with one or two carboxylic acids; and these 5'. It includes an N-aminomorpholino derivative of ribonucleoside whose position has been substituted with a carboxylic acid (Structure G) or an N-aminomorpholino derivative of ribonucleoside substituted with a sulfonic acid (Structure H).

In the same molecular model method applied to the acyclic (non-branched) backbone portion, the unit backbone in the range of 4-6 atoms with respect to the polymer conformation within the base-to-hydrogen bond to the single-stranded polynucleotide. Lengths were accepted, indicating that a 5-atom backbone length is optimal. This is in contrast to the cyclic backbone, where the unit backbone length of 6 atoms is optimal. In addition, this modeling study showed that: This circular recognition portion does not strictly interfere with the homogeneity of this complementary base, and without introducing an unwanted interaction between this recognition portion and the backbone, the linear backbone portion. Cannot be added directly to. When this recognition part was connected to this backbone part with a one-atom spacer, the bond distortion was minimized. Diatomic spacers are acceptable, but triatomic spacers are not. However, if the degree of freedom between this recognition part and the linear backbone increases, the diatomic spacer can mistakenly pair with the target base.

The structure IN of the acyclic backbone portion shown in FIG. 4 all contains a 1-atom methylene spacer between the backbone portion and the recognition portion. It was predicted that this structure would provide a good model for the preferred base pairs for complementary polynucleotides. Similar structures containing two carbon spacers have been shown to be acceptable, but bond conformation was unfavorable. The structure shown in the figure and two similar carbon spacer structures are generally more preferred acyclic backbone moieties due to their ease of synthesis. However, it should be noted that many other backbone parts are also quite feasible and suitable, although generally not easy to synthesize and / or not cheap to synthesize.

C. Backbone / recognition portion coupling As shown above, the chain or spacer connecting the backbone and recognition portion within this polymer subunit must meet certain design criteria. This criterion is effective in positioning this recognition area for base pairs that bind to polynucleotide bases. In the case of the cyclic backbone moiety shown in FIG. 3, this polymer is when this recognition moiety is added directly to the 1'carbon of the ribose structure or ribose-like structure or directly to the similar 1'position of the morpholino structure. Modeling studies show that the binding polymer within is most preferred. That is, this portion is added at the normal binding position with the correct stereoisomeric configuration of the purine or pyrimidine base for the ribose or deoxyribose group of the nucleoside. For acyclic structures, one and two carbon atom spacers are required to place this recognized portion in a preferred bond position. Single atom spacers are more preferred as discussed above.

Stereoregularity required for this polymer molecule to obtain a certain binding affinity for a polynucleotide target In order to achieve the configuration of the polymer, do all the bonds of this backbone / recognition portion have a certain chirality? Or it must be either achiral. If each bond has the same stereoisomeric configuration or chirality at a subunit position of all this polymer molecule, then for the definition here, this bond is considered to have a constant chirality. .. That is, although subsystems at different sequence positions can have different internal stereoisomeric configurations (including akiraity at the characteristic sequence positions), the bonds at the given sequence positions between the polymer molecules are , All have the same stereoisomerized configuration. The commonly defined chirality is achieved very easily by using the same stereoisomerized configuration for all subunits.

The most preferred binding occurs to this cyclic backbone moiety in nucleoside analogs with the natural D-type stereoisomeric configuration shown in FIG. This type of subunit is also readily synthesized using natural D-type nucleoside starting materials, as discussed below. With respect to FIG. 4, it can be seen that only structures I and L have a chiral chain between this backbone and the recognition portion. These subunits are easily synthesized in homochiral form by using homochiral starting materials as described. For all of the other acyclic structures shown in FIG. 4, this one-atom chain is added to the nitrogen atom of the backbone and is therefore achiral. Of course, this methylene spacer is also achiral in itself.

II. Protecting Group Strategy Because the compounds used to activate and / or couple this subunit have fairly high reactivity, it is possible to protect the nitrogen bound to the ring outside the ring of this recognition portion. , Generally desirable and often necessary. The choice of these protecting groups is primarily determined by the type of subunit-to-subunit bond used within the polymer assembled from these subunits. Second, it is determined by the relative reactivity of nitrogen to be protected.

Base-protected nucleosides are also useful starting reagents in many subunit synthesis reactions, such as those described below. The method of base-protecting a large number of very common ribonucleotides and deoxyribonucleotides of Example 1 is exemplified in Example 2. The methods detailed in this example are generally applicable to the formation of nucleosides with amine protecting groups.

If the subunit-to-subunit chain used is relatively stable to nucleophiles, especially ammonium hydroxide, common base protecting groups used in nucleic acid chemistry are suitable. Such non-nucleophilic insensitive subunit chains include carbamate chains, amide chains and sulfonamide chains. For this recognition, the corresponding nucleosensitive protecting groups include the benzoyl group for N4 in C, the benzoyl or p-nitrobenzoyl group for N6 in A, the acetyl or isobutyryl group for N2 in G, and 2 , There are N2, N6-bisisobutyryl groups for 6-diaminopurine residues. Removal of these groups is achieved by treatment with ammonium hydroxide after completion of the polymer assembly.

In contrast, if the interunit chain used is sensitive to nucleophiles such as ammonium hydroxide, suitable protecting groups can be removed via a β-elimination mechanism by a strong non-nucleophilic base. It is a protecting group. Such nucleophilic interunit chains include carbonate chains, ester chains, and, to a lesser extent, tricarbamate chains. For this recognition, suitable protecting groups that can be removed via β-desorption include 2- (4-nitrophenyl) ethoxycarbonyl groups or 2- (phenylsulfonyl) ethoxy for both N4 of C and N6 of A. Includes carbonyl groups and 9-fluorenylmethoxycarbonyl groups for N2 and N6 of G's N2 and 2,6-diaminopurine residues. Removal of these groups after completion of the polymer assembly is a strong non-nucleophilic base 1,8-diazabicyclo under severe anhydrous conditions.

[5,4, O] Achieved by the treatment of Undec-7-en (DBU).

For temporary nucleophilic protection of this backbone moiety (generally N1 of the above structure), a general polymer assembly strategy uses selected backbone protecting groups that are easily removed by mild acids. One major criterion for the selection of protecting groups is that these groups are sufficiently stable, but the conditions required for removal are not stable enough to impair the growth polymer. A major problem with polymers collected from cyclic backbone moieties is the significant acid sensitivity of glycosidic bonds that link protected purine residues to C1 in these backbone moieties. The second criterion for the selection of this backbone protecting group is that this protecting group is easily introduced. Based on the above, the following backbone protecting groups are more preferred; di (p-methoxy) trityl groups for primary hydroxyl groups; p-methoxytrityl groups for primary amine groups; and secondary amine groups (in the morpholino type backbone moiety). A phenylisopropoxycarbonyl group for (as in). These protecting groups can be easily removed by treatment with 0.2 M dichloromethane in dichloromethane.

III. Subunit synthesis A. Cyclic backbone portion Subunits having a deoxyribonucleoside subunit structure (Structure A in FIG. 3) can be obtained from commercially available raw materials or as described in Example I by the method of the literature. Can be prepared. This subunit contains the following ribosides and deoxyribosides and is identified according to the number of structures of the recognition moiety obtained in FIG. 1: adenosine and deoxyadenosine (structure 1); 2,6-diaminopurine riboside and Deoxyriboside (Structure 2); Citodin and deoxycitodine (Structure 3); 4-methoxy-2-pyrimidineone deoxyriboside (Structure 4); 2-Hydroxy-5-methylpyrimidine deoxyriboside (Structure 5); 2- Hydroxypyrimidineriboside (Structure 6); Guanosine and Deoxyguanosine (Structure 7); Inosine and Deoxyinosine (Structure 8); Thioguanosine and Deoxythioguanosine (Structure 9); Urazine and Deoxyuridine (Structure 10); Timidin and 5 -Methyluridine (Structure 11); and 5-halosine and 5-halodeoxyuridine (Structure 12).

The 5'-amino-2', 5'-dideoxyribonucleoside (Structure B in FIG. 3) is prepared according to the method detailed in Example 3. Simply put, this selected deoxyribonucleoside (protected with a base if necessary) is subjected to the reaction with triphenylphosphine, carbon tetrabromide and lithium azide, with the corresponding 5'-azidonucleoside. It is formed. It is then reduced by hydrogenation in the presence of palladium on the carbon catalyst. This nucleoside can be obtained as in Example 1 and can be base protected as in Example 2. The stereochemistry of this reactant nucleoside is conserved in the formation of 5-aminonucleoside analogs.

Other reduction methods are used to reduce the azide group of 5'-azido-5-bromouridine, as described in Example 3.2. Here, uncatalyzed hydrogenation is necessary to prevent the separation of bromine atoms in the ring. When forming this 5'-amine guanosine compound, the azide is placed at the 5'position by first tosylating the 5'hydroxyl group and then substituting with the azide, as detailed in Example 3.2.

The 3'-amino-2', 3'-dideoxyribonucleoside (structure C in FIG. 3) is prepared according to the method detailed in Example 4. Simply put, thymidine protected by a hydroxyl group at the 5'position is tosylated at the hydroxyl group at the 3'position and then intramolecularly substituted with a dioxybase substituent. The resulting ring is immediately ring-opened by treatment with azide to produce an azide analog with an accurate stereoisomeric shape. This analog can be converted to the selected base analog by reaction of the azide compound with the selected base or pyrimidine base. This latter may optionally be base protected against subsequent subunit coupling reactions, as described below. This thymidine or other azide analog is then reduced to produce the desired 3'-amine nucleoside. The stereochemistry of this thymidine starting material is conserved in synthesis.

The synthesis of this morpholinotype subunit derivative shown in structure D of FIG. 3 is detailed in Example 5 for a variety of different recognition moieties. Simply put, the selected nucleosides (base-protected as needed) are dissolved in ammonium salts such as ammonium diborate and then subjected to reaction with sodium periodate in a transient manner. 2', 3'-dialdehyde is formed. It is then ring-closed on the ammonium ion to form a morpholino ring with hydroxyl groups at the 2'and 4'positions. The compound is then treated with sodium cyanoborohydride to remove the hydroxyl groups on this ring. The nitrogen in this ring is preferably protected as 2-phenylisopropylcarbamate for subsequent subunit coupling. The stereochemistry of this nucleoside starting material is retained.

The deoxyribose subunit structure having an acetic acid group at the 5'-position (structure F in FIG. 3) can be prepared according to the general synthetic scheme described in Example 6. This scheme details the synthesis of acetic acid compounds at the 5'position represented by structure F. Here, the selected deoxyribonucleoside whose 3'position is protected by a hydroxyl group is a Wittig reagent having carbon at the 2nd position in order to convert the aldehyde to the 5'position and then give an unsaturated acetic acid derivative. It is processed. Reduction of this side chain olefin gives the desired compound with acetic acid at the 5'position. This reaction preserves the stereochemistry of the starting nucleoside material.

This similar 5'-formate compound (Structure E in FIG. 3) is obtained by treating the above 5'nucleoside aldehyde with a single carbon Wittich reagent and hydrolyzing the resulting enol to the corresponding formaldehyde. It is formed by forming a derivative, which is oxidized to the desired acid. This reaction preserves the stereochemistry of the starting nucleoside material.

B. Subunit synthesis-Acyclic backbone The 5-atom chain amino acid subunit shown in Structure I of FIG. 4 is prepared according to the general method outlined in Examples 7-9. Simply put, (S) -5-carboxypyrrolidone was converted to the corresponding stereochemically pure (S) -5-tosylmethyl-2-pyrrolidone by known methods. It is then subjected to reaction with the selected purine or pyrimidine to form the corresponding (S) -5-methylpurine (or methylpyrimidine) pyrrolidone. This substitution reaction retains its initial stereospecificity, and the subunits formed as a result are identical to this backbone carbon at the binding site of this recognition portion.

The pudding used for subunit synthesis preferably has an electron-withdrawing group at the 6-position in order to reduce the reactivity of the ring nitrogen at the 1- and 3-positions, that is, to minimize the coupling of pyrrolidone to the ring nitrogen. Includes. This pyrrolidone-derived purine or pyrimidine may then be converted to an amine derivative and optionally base protected prior to the ring-opening step described below. Examples 7.1 and 7.2 detail how cytosine pyrrolidone and its base-protected derivatives are formed. The adenosine pyrrolidone formed in Examples 7.3-7.5 uses 6-chloropurine as a starting material. This corresponding pyrrolidone undergoes azide formation and is converted to an adenosine derivative, as described. This adenosine is base protected before ring opening as in Example 7.6. Similar methods are used to generate base-protected guanine pyrrolidones starting from 2-amino-6-chloropurine, as described in Examples 7.7-7.9. Similar methods are also used for the synthesis of 2,6-diaminopurine pyrrolidone, inosine pyrrolidone and 2-hydroxypyrimidine pyrrolidone, as detailed in Example 7.

The pyrrolidone is then processed through a series of reactions that cleave the pyrrolidone ring with a t-BOC-protected amine to form amino acids. Examples 8.1-8.4 describe the synthesis of such t-BOC amino acids with a large number of selected recognition moieties. In the final step (Example 5), this t-BOC protecting group may be removed to form the corresponding amino acid shown in Structure I of FIG. This amino acid can be used directly for subunit coupling according to the following reaction. On the other hand, this t-BOC protected compound can be activated at its acid group for use in the subunit coupling reaction.

The subunit forming method shown by structure J in FIG. 4 is outlined in Examples 10.1 and 10.2 to form the sitodine analog subunit and the uridine analog subunit, respectively. Simply put, when forming pyrimidine-type subunits, a 6-membered heterocycle, such as pyrimidine or nicotinate, is a reactive methylene at the carbon ring site that can be successfully added to the backbone. Is modified to include. .. As detailed in Example 10.1, this cytosine analog is formed by reacting 5-amino-5-bromopyrimidine to form the corresponding 5-position carboxylaldehyde. As in Example 12, this uridine analog is formed by converting the 6-hydroxynicotinate ester to the corresponding benzylic alcohol. This compound is converted to an aldehyde by the reaction in the presence of manganese dioxide.

The aldehyde compound is then subjected to reaction with the desired amino acid, such as 4-aminobutyric acid. The reaction is carried out with amine groups that produce unstable imines that are reduced for stable amine subunit formation. This secondary amine can be protected by the t-BOC group against subunit coupling involving activation of acid groups, as described in Example 10.1. In backbone nitrogen, a base is added to the backbone through a methylene spacer, making this subunit lacking a chiral center and therefore not stereoregular.

To form the subunit structure shown in K in FIG. 4, a C-carbon backbone analog of the above compound was prepared with 3-aminopropionic acid, which compound was further treated with nitrogen oxides. The corresponding N-nitroso compound is formed. This compound is H in the presence of iron salts₂It is reduced by IPd in order to obtain the hydradinoic acid subunit.

III. Backbone Coupling Reaction The coupling reaction used to form the polymer molecules of the invention is the step of binding one selected subunit or subunit sequence to another selected subunit or subunit sequence. Reaction. To make this argument, this coupling reaction is generally the selected recognition part R.₁Single subunit BR with₁Is the same or different selected recognition part R₂Another single subunit BR with₂Will be described for coupling with to form a dimer of the following shape.

Here R₁And R₂Has the specific sequence shown.

A: Subunit coupling; N₁...... N₂Backbone configuration N₁...... N₂A common method of coupling subunits with a backbone configuration is illustrated in Figures 5A and 5B. As recalled from Section I above, N₁And N₂Is a nucleophilic backbone group such as a hydroxyl group or an amine group. This group can be activated by the electrophilic group E and the activated N₁-E backbone group or N₂-E backbone groups are formed. These groups are then the second N₁...... N₂Reacting with the backbone part of the type N₁...... N₂-E-N₁...... N₂It forms a backbone-bonded dimer. This type of subunit backbone is specifically described above and is the annular backbone of structures AD in FIG. In structures A and B, this activated N₂The nucleophilic group is a 3'hydroxyl group, a 5'hydroxyl group in structure C, and a 6'-hydroxyl group corresponding to the 5'-hydroxyl group of structure C in structure D.

In the more preferred coupling method illustrated in FIG. 5A, the selected recognition portion R₁The subunits with are activated by the electrophilic group E. The asterisk in this recognition area indicates the required base protection. As can be seen from this figure, this selected subunit is N₁Protected by nucleophiles, (a) N₂Only nucleophiles are activated. And (b) this activated subunit guarantees that it cannot polymerize on its own. In the structures A, C and D of FIG. 3, this backbone protecting group is a 5'hydroxyl group, which is preferably an acid unstable group such as dimethoxytrityl (DMTO).

It has the activation reagent shown in Fig. 5A and the following general formula: Here, X is oxygen or sulfur, and C is an active electrophilic group that can react with a nucleophilic group (for example, oxygen of a hydroxyl group or nitrogen of an amine) and replace with Y to form an activated subunit. Is.

Activators (eg, bis-p-nitrophenyl carbonate) provide carbonyl-activated subunits (X = 0) and are used in forming carbonate and carbamate subunit chains. Similarly, activators such as thiocarbonyl-di- (1,2,4-triazole) (X = S) are used in forming the thiocarbamate chain.

The subunit activation reaction involving the carbonyl-activated subunit is detailed in Example 11 for the 5'-protected 2'-deoxynucleoside shown in FIG. 4A; In Example 12, the 5'-protected aminonucleoside shown in B of FIG. 4; in Example 13, the N-protected morpholinotype subunit shown in D of FIG. 4 is detailed. ing. The general reaction conditions used for the activation of hydroxyl groups in these structures are generally applicable; subunit activation reactions involving thiocarbonyl activating subunits relate to the 5'-aminonucleoside shown in B. The morpholinotype subunits shown in Example 14 and in D of FIG. 3 are shown in Example 15.

Following this activation reaction, the activation complex is purified by conventional methods such as silica gel chromatography. It is then subjected to reaction with the second subunit. Selected recognition part R of this subunit₂Form a recognition portion in the next sequence in the finished polymer. This coupling reaction preferably has an active group of backbone N.₂Can react with amine groups, but N₁It is carried out under mild conditions that cannot react with hydroxyl groups. Therefore, this method is suitable for coupling the types of subunits shown in structures BD in FIG. 4, but not for the subunits of structure A. The advantage of this coupling method is the second subunit-which is the amine N₁Nucleophiles and hydroxyl groups N₂Containing the nucleophilic group of-is the first activated subunit and this N₁Coupling only via amine, hence N₂There is no need to protect the backbone group. Therefore, the resulting dimer subunit is N, as shown in the figure.₂-E-N₁Coupled through binding.

(A) Free N of this second subunit₂The above steps of activating the nucleophile, separating this activated species from the activator, and coupling the activated compound with the subunits in the next sequence where the backbone is unprotected. This oligomer can be extended by repeating. This method is particularly used to form short oligomeric blocks by solution methods suitable for solid phase block assembly (described below).

The second coupling method outlined in FIG. 5B is more preferred for forming the polymer by the continuous addition of subunits in the solid phase. This second method differs from the first method in that polymer growth occurs by adding activated excess subunits to the existing subunits or polymer chains. Rather, in the first method, the addition of unactivated subunits to the activated strand results in polymer growth. In Fig. 5B, the recognition part is R.₁The subunits (second column in Figure 5B) indicate the subunits or subunit chains that exist. This subunit is a free N₁Backbone nucleophile and N₂It has a nucleophilic group. This N₂The nucleophilic group is protected by a relatively acid-stable bond to the solid support or by linking to a subunit chain that binds to the solid support. N like this₂Methods of forming a protected annular backbone subunit are commonly described in Examples 12-15. This first subunit, the most recently added subunit of the growing polymer, is immediately subjected to reaction with the activated subunit that becomes the subunit in the next sequence in the polymer. This activated subunit is N₂N by protecting groups that are activated at the backbone site and are relatively acid-labile₁The part is protected. This subunit is prepared by the method described above in connection with FIG.

As can be seen in Fig. 5B, this coupling reaction causes N.₂-The activated second subunit is N₁-Attached to the first protected subunit, N₂-E-N₁Both are coupled via binding to form a compound in which both free backbone nucleophilic sites are protected. This compound is treated immediately, for example by reacting with an acid, and the acid-labile N on the last subunit added.₁The protecting group is deprotected. This procedure is repeated to build the desired sequence polymer.

From the above, this N₂The activated subunit, which forms the subunit in the last sequence, is its N₁Protected at the backbone site, thereby N₂It must allow selective activation at the site and prevent self-polymerization of the activated compound. Also, this N₁-The deprotected subunit can be coupled with the activated subunit. This subunit is N₂ Activated subunit and its N₁N to allow selective reaction with the part₂The site will be protected. Therefore, since this reactive subunit retains one backbone site together, this method is not only for the coupling of nucleosides (Structure A in FIG. 4), but also for amines and hydroxyl group backbone nucleophiles. It is also suitable for coupling subunits with an annular backbone that includes both (eg, structures BD in this figure). The reaction method used to couple the subunits of structure A via carbonate binding is detailed in Example 12. Simply put, the subunit containing the backbone moiety protected at the 5'position is activated by the hydroxyl group at the 3'position, and with other subunits (or growth chains) that protect the hydroxyl group at the 3'position. Subject to reaction. This coupling reaction is carried out in the presence of a catalyst required to form a carbonate bond, such as N-methylimidazole or N, N-dimethylaminopyridine. The coupling of subunits, including the cyclic backbone of structures B through D, is a milder, catalytic, as described above for the first method, where a carbamate or thiocarbamate bond between the subunits is formed. No coupling conditions are suitable.

The advantage of this second coupling method over polymer formation by the addition of solid phase subunits is that a substantially excess of the number of moles of activated subunits grows during the addition of each polymer. It is added to the carrier-bound polymer that is present, resulting in a very high proportion of subunits that are coupled to the carrier-bound polymer. This ensures that most carrier-bound polymers contain the complete sequence of desired subunits. Compared to the first method, if this growing polymer is activated with the last subunit added, the efficiency of subunit addition is limited by the efficiency of the activation step.

FIG. 6 shows a dimer structure formed by the coupling of the annular backbone subunits shown in FIGS. 4A to DD. The nucleoside subunits in structure A in this figure are bound by carbonate (Y = O). In the remaining three structures BD, the subunits are linked by carbamate (Y = O) or thiocarbamate (Y = S) bonds. As can be seen from this figure, all subunit chains are uncharged and achiral. That is, it does not contain a chiral center. Upon attachment, this chain is stable in aqueous media. This is judged from the polymer's ability to withstand hydrolysis for a long period of time in a neutral aqueous solution.

According to another important feature of the invention, this structure exhibits the correct Watson / Crick base pairing with complementary polynucleotides when bound to form a polymer.

Finally, according to another important feature of the invention, the polymer formed from this subunit is stereoregular. This involves (a) using a natural nucleoside or nucleoside derivative or a synthetic nucleoside with a natural stereoisomeric configuration as the subunit, and (b) linking the subunits by an achiral inter-subunit chain. Achieved by. Examples of coupling methods are detailed in Examples 11-15.

B. subunit coupling; N₁...... E backbone three-dimensional arrangement N₁... A general coupling method for subunits with an E-backbone configuration is illustrated in FIGS. 7A, 7B and 7C. As recalled from Section I above, N₁Is a nucleophilic backbone group such as a hydroxyl or amine group and E is an electrophilic group such as a carboxyl or sulfonyl group. This N₁When activated, the second N₁...... Reacting with the E-type backbone, N₁...... E-N₁... E-backbone bond dimer can be formed. The subunit backbones of this type, particularly the backbones described above, are the annular backbone structures E to H in FIG. 3 and the non-annular backbone structures I to N in FIG. In the annular structures E and F, this N₁The nucleophilic group is the hydroxyl group at the 3'position, and in structures G and H, it is an amine added to the nitrogen at the 3'position on the morpholino ring. In all cyclic structures, this E group is a carboxyl group or a corresponding sulfonyl group added to the 5'position of the ribose ring or the 6'position of the morpholino ring. In all the structures shown in FIG. 4, this N₁The groups and E groups are a backbone amine or hydrazine and a carboxyl group or a sulfonic acid group at the partial end of the backbone, respectively.

This first coupling method is illustrated in FIG. 7A and is similar to the method described above in relation to FIG. 5A. Here, the first N₁-The protected subunit is activated and then directly coupled with the backbone-unprotected subunit. This subunit forms the subunits in the next sequence in the growing polymer. This subunit N₁P₁The protecting group is preferably a cleaving linker attached to a solid support or an acid-labile protecting group such as the t-butoxycarbonyl group. N of annular backbone structure₁-The method of protection is similar to the procedure described above for the annular structures A to D. Similar methods are effective in protecting amine nitrogen in acyclic backbone structures. On the other hand, if this polymer can be constructed on a solid phase carrier, the N of this backbone, as described below.₁The site is N with the addition of subunits to the growth polymer.₁It can be protected by binding to a solid support of the part. This growth polymer is produced by the activated electrophilic group E of the subunit added last.

In this activation reaction, the activated portion or the terminal portion of the chain is N.₁...... Designed to form EX. Here, X is described above in relation to FIG. This activated subunit has almost the same reactivity with the backbone nucleophile as the subunit activated by the previously described method. That is, this activation is N₁...... N₂It is designed to produce activated coupling groups that are similar to the type of backbone. However, in that case, this reactive carbonyl or sulfonyl group is supplied by the backbone itself rather than the carbonyl or sulfonyl group of the activation reagent, as in the method shown in FIG. This activation is N in the presence of p-nitrophenol when the backbone electrophilic group is a carbonyl group.₁This can be easily achieved by reacting the E-type subunit with the carbodiimide. Alternatively, if this backbone is a sulfonyl, it can be achieved by reacting this subunit with a carbodiimide in the presence of imidazole, 1,2,4-triazole or tetrazole. The subunit activation reaction involving the carbonyl electrophilic group is detailed in Examples 16 and 17. The general reaction conditions used to activate this N-amino-morphylino group and the carbonyl group of the linear chain backbone are generally the other N shown in Figures 3 and 4.₁...... Applicable to E-type backbone structure.

Following this activation reaction, the activation complex is purified by conventional methods such as silica chromatography. Or in the case of carrier-bound and activated chains, they are simply washed. Then the selected recognition part R₂Is subjected to reaction with a second subunit such that forms the next recognition moiety in the sequence in the complete polymer. This coupling reaction gives a dimer as shown in the figure. This dimer subunit is therefore E-N₁It is coupled via a bond. As mentioned above, both subunits must be properly base protected during the activation and coupling reactions.

The polymer can be elongated by repeating the above steps. This step involves (a) activating the free E electrophilic group in the second subunit, (b) separating the activated species from this activator, and (c) this activity. Coupling of the compound with the subunits in the next sequence, which has an unprotected backbone.

This second coupling method is illustrated in FIG. 7B. This method is directly similar to the method described above in relation to Figure 5B. Here, the first subunit with an E-protected backbone is the activated E group and the protected N.₁It is subjected to a reaction with a second subunit having a nucleophilic group, and is subjected to E-N.₁A dimer is formed that is linked by binding and that both free backbone end groups are protected. If this polymer is formed by solid phase synthesis, this P₂Protecting "groups" form a chain of acid stability with the solid support. This N₁-The protected subunit is prepared and activated as described above. The coupling conditions generally follow those used in the cyclic subunit coupling reaction described above.

In the third coupling method shown in Fig. 7C, this N₁-Protected and E-unprotected subunits (or polymer units) are both subjected to the reaction in the presence of suitable E-activating reagents such as carbodiimides, as indicated.

FIG. 8 is generated by coupling annular and non-annular backbone subunits (these are shown by EK and EE to KK in FIGS. 3 and 4, respectively). Shows a dimer structure. The nucleoside subunits of the structure FF in FIG. 3 are linked by an ester bond. Retaining the cyclic backbone structure, the subunits GG are linked via a hydrazide or sulfonylhydrazide bond. All of the acyclic backbone subunits shown in I-I, JJ and KK are linked via an amide bond (E = carbonyl) or a sulfonamide bond (E = sulfonyl). As in the cyclic backbone subunit structure described above, all subunit chains are in an aqueous medium, as inferred from the known stability of ester, hydrazide and amide bonds in solution. It's fairly stable.

According to another important feature of the invention, this structure exhibits complementary polynucleotides and Watson / Crick base pairs when linked to form a polymer.

Finally, the N mentioned above₁...... N₂All N shown with backbone subunit₁The E-backbone structure is stereoregular. This is (a) the homomorality of the addition of the recognition portion to the backbone portion (E to H in Fig. 3 and the I and L properties or achiral in Fig. 4 (J, K, M and N in Fig. 4). By nature and (b) achiral subunit chain.

C. Subunit coupling; alternating charged and uncharged achiral chains In some applications, it is desirable to introduce one or more charged achiral subunit chains into the polymer molecule. As further described below, the charge of this backbone can be used to promote the solubility of the polymer in many solutions or to adjust the target binding constant of the polymer. A charged achiral backbone chain, eg, a polymer regularly placed between the second or third subunits, is suitable for novel diagnostic systems such as those described below. For the purposes currently being discussed, polymer units consisting of two or more subunits that are internally linked by an achiral uncharged chain can be linked by a charged achiral chain. These uncharged units are formed by one of the methods described in Sections A and B above.

The dimer subunits that can be linked are selected as follows. Free N of one dimer₂The group is another dimer free N₁It can be coupled to a group via a properly charged achiral chain. This desirable chain is the free N of one subunit.₂Free N of the group and another subunit₁It is a phosphodiester bond that requires both groups to be hydroxyl groups. As can be seen in FIG. 3, under this condition, a dimer formed from one of the subunits of structure A and structure B (both of which are free N).₂A dimer in which (having a hydroxyl group) is formed from subunits of structure A, C, or D (these are all free N).₁It is suitable if it binds to (having a hydroxyl group).

In a typical coupling method, N₁The protected dimer A is N via a phosphodiester bond, as shown in FIG.₂It is retained during a series of reactions induced in reactive phosphoester coupling species linked to hydroxyl groups. More preferred coupling methods are detailed in US patent application "Polynucleotide Measuring Reagents and Methods" serial number 712,396 (filed March 15, 1985). This method alternates between uncharged stereospecific methylphosphonate bonds and charged achiral phosphodiester bonds for the formation of tetramers. Simply put, this illustrated method first forms a nucleotide dimer linked by a methylphosphonate bond, and the method linked by a methylphosphonate bond first forms a nucleotide dimer linked by a methylphosphonate bond, and the dimer is isolated in stereoisomeric form. , And activate one of the stereoisomeric dimers to N₁ Protected reactive N₂Includes the formation of-(p-chlorophenyl-2-cyanoethyl) -phosphate. This activated dimer is then subjected to a reaction with a second dimer, which is alternately linked by an uncharged, stereoisomeric chain and a charged achiral inter-subunit chain. Form a tetramer.

This phosphodiester bonding method involves solution-type polymer synthesis or carrier-type polymer synthesis according to the general methods outlined in Sections A and B for polymer couplings involving achiral but uncharged bonding reactions. Any combination of methods may be used. As shown, this phosphodiester bond can be used to couple dimeric or multimeric units that are themselves coupled by uncharged achiral bonds. Alternatively, this bond can be formed for a coupling of dimeric units linked by an uncharged, chiral but stereoisomeric bond (a separate stereoisomeric form of the dimer to which methylphosphonate is linked). ..

For the diagnostic applications described below, it is generally more preferable to introduce a chain that is as little charged as it takes to obtain sufficient polymer solubility in an aqueous medium. This promotion of solubility is generally achieved with 1-3 charged backbone chains per polymer of about 20 subunits, preferably no more than about 1 charged chain per 4 subunits. As defined here, an achiral backbone chain contains less backbone chains than about one charged backbone chain per dimer unit, preferably one or a few backbones per tetramer unit. If it contains a chain, it is considered to be substantially uncharged.

IV. Consideration of Polymer Targeting The design considerations applied in preparing the polynucleotide-bound polymer used in the present invention are governed by the nature of the target analyte and the reaction conditions under which this analyte can be validated.

The first consideration is the selected non-monopolymerizable target base sequence, whereas the polymer is indicated. This target sequence is preferably specific for the analyte to be tested. That is, this sequence is calculated to occur only with a limited probability (eg, 1% or less) in a test mixture containing a large number of known sequence bases. The probability of appearance of a known n-base target sequence is approximately 1/4._nIs. That is, the known n-base target sequence is 4ⁿIt would be estimated to appear about once in a polymer containing a single base. Therefore, the probability P that a known n-base sequence occurs in a polynucleotide containing all N unique sequence bases is approximately P = N / 4.ⁿIs. For illustration purposes, the probability P for a 9-base target sequence to be found in a 20 kilobase nucleotide is approximately 20 × 10.³/2×10⁵That is, it is 0.08, and the probability that a 16-base target sequence exists is about 20 × 10.³/4.3×10⁹That is, 0.0000047. From these calculations, a polymer with 9-16 recognition moieties specific for 9-16 constant base target sequences is a test mixture containing only the viral genome (this largest complex is about 400 K). It has been found that it should have high specificity for the target sequence in (corresponding to the unique sequence base of).

In a similar type of calculation, 12 to 16 subunit polymers are viral or bacterial in a test mixture containing only viral and bacterial genomic material (maximum genomic size is about 5000 kilobases). It has been shown that it can provide sufficient specificity for the target sequence of the virus. In addition, 16 to 22 subunit polymers are sufficient for the target sequence in a polynucleotide mixture containing animal genomic DNA material (genome size is about 5 billion base pairs of unique sequence DNA). It has also been shown that it can provide unique specificity.

The binding affinity of this polymer / analyte, and in particular the temperature at which this polymer binds well to the target sequence (melting temperature or T)._m) Depends on (a) the length of the polymer, (b) the number of hydrogen bonds that can be formed between the recognition moiety and the base in the sequence of the corresponding analysis target sequence, and (c) the charge density of the backbone. It can be changed selectively. From many studies in model homopolymer double-stranded, the melting temperature of the oligonucleotide double-stranded is in the range of 10-20 bp and is added if this complementary base can form two hydrogen bonds. It is known that the temperature increases by about 6 ° C per base pair and at 3 ° C, when this base can form three hydrogen bonds. Therefore, the length of the target sequence initially selected to ensure high binding specificity with this polymer is extended to obtain the desired melting temperature with the complementary base polymer under the selected assay conditions. May be done. Also, as illustrated above, if the recognition moiety used in constructing this polymer is a standard nucleobase, then this target sequence is to achieve relatively high polymer / analysis melting temperatures. , The ratio of cytosine base added to guanine base may be selected to be high. Alternatively, this target sequence may be selected to increase the proportion of adenine base plus thymine base in order to achieve a relatively low melting temperature.

The charge density of the backbone of this polymer should be substantially lower than the charge density of this polynucleotide analysis. This is because, as already mentioned, the polycationic reporter molecule is preferentially attached to the analyte under conditions where the reporter does not attach to the polymer. This requirement is met if the spacing between adjacent negative charges on the polymer backbone is at least twice the spacing between the negative charges on the adjacent phosphodiester bonds of the analyte. The charge density of this backbone also has an important effect on the melting temperature of the polymer / analyte. In particular, under low salt concentrations, the repulsion between the charges between the analyte and the polymer backbone can also act to reduce the temperature at which the two can be annealed. Therefore, in general, polymers with a less charged backbone will (1) have a higher melting temperature of the analyte / polymer, and (2) will be less dependent on this melting temperature for salinity. Would be expected to show. Based on these considerations, uncharged and achiral polymers, such as some of the polymers described above in connection with Figures 6 and 8, are alternately charged achiral phosphodiester bonds and achirals. Wider salt concentrations than partially charged and sterically limited polymers, such as polymers formed from uncharged chains, which are either chiral or chiral, and broader salt concentrations than polymers in diagnostic methods. Will allow the polymer / analytical object annealing reaction to be performed in the diagnostic method.

V. Polymer assembly method A. Geometric assembly method According to the factors discussed in Section IV, after selecting the desired polymer length and sequence of recognition moieties, the general subunit coupling detailed above. This polymer is collected using a procedure. One method of collecting polymers is to first prepare a suitable set of dimers, then concatenate the selected dimers to form a tetramer, and then concatenate them to form an octamer. And so on. This method is performed in solution and substantially follows the method described above in connection with FIGS. 5A and 7A. It should be noted that not all couplings need to be done between oligomers of the same size. For example, often in the final coupling, to get a 20-mer, connect a 16-mer and a tetramer, or to get a 24-mer, connect a 16-mer and an octamer. Is desirable.

A particular advantage of this assembly method is that each coupling product has approximately twice the mass of its precursor, making it easy to purify each coupling product. Example 18 below describes in detail the aggregates formed by this method of polymers in which 8 subunits of carbamate are linked.

B. Stepwise assembly on solid support One preferred method for polymer synthesis is stepwise assembly on solid support. Here, the first subunit in the polymer is its N₂It is added to the solid support via the backbone group. Typically, a solid support such as glass beads derived by a long chain linker that is cleaveable and preferably acid stable is used as the carrier material. As described in Example 19, this solid support is N, such as the 5'hydroxyl group of a nucleoside.₂Prepared for the addition of nucleophilic groups. These glass beads are preferably acid-labile N₁Protecting groups and activated N₂It is subjected to a reaction with a subunit having a group or E backbone group, as detailed in Section III. Coupling of various types of subunits to glass bead carriers is generally described in Examples 20-22.

After coupling to the carrier of this second subunit (or polymer unit that can be assembled in solution), all unreacted linker nucleophiles are properly capped, such as p-nitrophenyl acetate. Capped by the addition of reagents. The carrier is then washed and filtered. This N₁Protecting groups on the terminal subunit are typically removed by acid treatment. After neutralization, this carrier is then free N₂It is subjected to reaction with an excess of subunits (or polymer units) in the next sequence that are activated in the backbone moiety. The activated excess subunit maximizes the number of large number of carrier-binding subunits whose chains are extended. That is, one of the features of this solid carrier assembly method is that high coupling efficiency is required at each subunit addition stage. The concentration of the added and activated subunits is chosen to maximize this efficiency. Chain elongation is continued after addition of each subunit, with capping of the missing sequence, until a polymer of the desired length and sequence is obtained. This common method is used in Example 20 for the preparation of polymers linked with 14 subunit carbonates, in Example 21 for the synthesis of polymers linked with 19 subunits, and 19 It is shown in Example 22 for a set of polymers to which the subunit amides are linked.

After the addition of subunits in this last sequence, this polymer has a terminal N₁It may be capped with a suitable charged or uncharged group coupled to the group. If this backbone assembly is done solely on uncharged achiral chains, then this capping reagent is preferably a charged group that gives the polymer a terminal charge. After capping the polymer, the polymer is cleaved by treatment with a nucleophile, such as ammonium hydroxide. The polymer is then purified, for example, by anion exchange chromatography. This collected and purified polymer is added to the solid support by the methods discussed below.

The polymer design principles discussed above are in Example 18 for synthesis in solution from pre-assembled dimer units, in Examples 20-23 for the preparation of various uncharged achiral chains, and , Example 24 illustrates the preparation of alternating charged types of polymers.

VI. Diagnostic Reagent A. Solid Carrier Reagent The reagent of the present invention is formed from the above substances by coupling a large number of bound polymers to a solid carrier. On the other hand, the polymer may be synthesized on a carrier in a stepwise or block form, as described above for carbamate-linked polymers. The polymer may be coupled directly to the carrier. (Or may be formed directly on the carrier.) For example, the polymer is via OH end groups on the polymer to OH reactive groups on solid carriers such as activated agarose, cellulose or the like. It may be coupled. However, direct coupling often places subunits close to this carrier too close to the carrier, resulting in the inability to complement complementary base binding between this analyte and this adjacent subunit recognition moiety. Therefore, the polymer molecules are preferably linked to the solid support, respectively, via a spacer arm. The spacer arm is adapted to distance the polymer from the surface region of the carrier so that the bond between the polymer and the target sequence of the analyte is not substantially impeded.

The spacer arm is preferably an unbranched chain having a total chain length of at least 6 atoms. The spacer arm then has suitable reactive end groups at one end for binding to the carrier and at the other end for binding to the polymer. Different types of spacer arms, especially chains containing different lengths and hydrophilic carbons, are known as reactive groups used in spacer arm coupling reactions. Example 25 below describes the synthesis of an aminohexyl spacer arm, its methylphosphonate / phosphodiester mixture polymer binding to the 5'end, and its coupling to a solid support. Example 26 below details the preparation of polymer carriers with multiple surface-bonded linear spacer arms for use in forming diagnostic reagents with polymers linked with carbamates. The method of Example 26 is illustrated in FIG. The carrier shown in this figure is aminomethylated polystyrene, which is first subjected to a reaction with succinic anhydride to form a carboxylated derivative. Activation of this carrier with di (succinimide) carbonate and reaction with 6-aminohexanol yields a 13-atom terminal hydroxyl spacer arm as shown.

More preferred solid carrier, agarose, cellulose, nitrocellulose, latex, polystyrene, and other commercially available elongate carrier substances or particulate bead support material (this the Re may be based on or activation of reactive surface groups Has) is included. These groups can be effectively coupled to polymer molecules, especially via spacer arms that bind to the polymer. The concentration of polymer coupled to the surface of this carrier is as described below, in this diagnostic method, when a suitable sample volume of test material is mixed with the carrier, 10 polymer molecules are added to the analyte molecule. It is preferable to select so as to have a molar excess of ~ 1000 times. A method of coupling a polymer bound to a target with agarose beads via an aminohexyl spacer arm from Example 25 is described in the same Example.

B. Reporter The reporter in the diagnostic system of the present invention consists of two parts: (1) This reporter is on the diagnostic reagent. A signal at which the presence of a polycationic moiety or tail designed to electrostatically bind to a fully charged polynucleotide, and (2) a reporter, can be detected, provided that it does not bind to a less charged binding polymer. One or more reporter groups attached to this tail adapted to occur. Polycationic, as the term is used herein, includes tails having multiple cationic groups with two or more suitable spacings.

This cationic tail is preferably configured to supply at least two positively charged groups. These groups are spaced apart to bind adjacent negative charges on the sugar-phosphate backbone of the polynucleotide analysis. At the same time, the spacing between adjacent charges on this cationic tail is such that electrostatic binding to a reagent polymer backbone containing more than half the charge of this reporter is energetically unfavorable. For example, in the polymer described above, which alternates between uncharged phosphate chains and charged phosphate chains, the distance between adjacent negative charges in this polymer backbone is approximately the distance in the polynucleotide backbone. It is double. Therefore, binding between substantially all of the charged groups of this cationic tail and the alternating negative charges of this polymer backbone would result in an energetically unfavorable distortion of this polymer backbone. Will need. When this polymer backbone has alternating charged / uncharged configurations, the adjacent positive charge spacing of this cationic tail creates a strong electrostatic bond between this polynucleotide backbone and this reporter tail. It can be evaluated that it should be narrowed down to the minimum interval of supply. Of course, if this reagent polymer has no more than one negative charge density per two subunit chains, or if this reagent polymer has an uncharged backbone, the charge spacing requirements within this reporter are of little importance. It disappears.

This cationic reporter when the polymer backbone has alternating charged / uncharged configurations, eg, alternating charged and uncharged merphosphonate chains. Is most effective when the cationic portion of the polymer cannot hydrogen bond with this uncharged chain (eg, the double-bonded oxygen of the methylphosphonate chain) between the fully charged polynucleotide and the polymer. It is also noted that it can be identified by. For this reason, it is generally advantageous to use quaternary ammonium ions for the cationic groups in such reporters.

The number of catio moieties in this reporter tail is 2 to 8 or more, depending on the total electrostatic attraction required for the reporter to bind to the polynucleotide analysis under binding conditions where the reporter does not bind to the reagent polymer backbone. May change up to. For reporters with relatively large reporter groups, such as enzymes, many electrostatic bonds may be required to attach this reporter tail to the analysis backbone. Reporter tails with multiple spaced cationic moieties are particularly suitable for use with the following reagents: This reagent does not charge its polymer backbone and therefore cannot form electrostatic bonds with this tail. Methods for constructing cationic tails suitable for use in the present invention are outlined in Figures 11-13. First, for FIG. 11, N-methyl amino acids are N-protected with di-t-butyl dicarbonate (BOC) and activated with carbonyldiimidazole. It is then coupled to dimethylamide via the amide chain. After unprotection, this amide is subjected to reaction with the amino acids from above activated by N-protected carbonyldiimidazole to form the diamide compound shown in the center of Figure 11. This protecting group-removing reaction and the N-protected and diimidazole-activated amino acid reaction are repeated until a polyamide of the desired length is formed. Details of this synthetic reaction will be described in Examples 28-30.

Reporter groups are typically very easily added to the primary amine group in this polycation. To add a primary amine to the secondary amine terminal of the compound in Figure 11, amino acids such as 4-aminobutyric acid are protected with BOC, as illustrated in Figure 12, with diimidazole according to some suitable method. It is subjected to reaction with activated and unprotected polyamines. The resulting BOC-protected compound is then treated with trifluoroacetic acid to remove this BOC group and then reduced by reaction with borane-tetrahydrofuran. These methods are described in Examples 31-32.

FIG. 13 shows the reaction scheme for the conversion of polyamines to polyquaternary ammonium salts as synthesized above. This method includes protection of the terminal amine followed by formation of a polyquaternary ammonium salt by reaction with methyl iodide, and deprotection with trifluoroacetic acid. The details of this method are shown in Example 33 below.

One or more reporter groups in this reporter may be present when the reporter is attached to the polynucleotide backbone, or when the reporter is eluted from the analyte, or after the reporter is eluted from the analyte. In either, it should be possible to (1) generate a signal that is easily attached to the cationic tail and (2) that it is easily detected. Small reporter groups containing chromophores (nitroaniline or other strongly adsorbed dyes) and fluorophores (dansyl-based or rhodamine-based fluoromolecules) are suitable and are suitable for visual inspection by a photometer or in the case of dyes. It has an advantage that it can be easily detected by. Radioisotope reporter groups may provide an advantage in diagnostic sensitivity. However, reporter detection will be more complex and expensive. Stable paramagnetic molecules such as nitroxide spin label can also be used. The binding of this reporter to the polynucleotide is detected by the spread of the adsorption line properties of the electron spin resonance (esr) of the immobilized paramagnetic species.

Another class of suitable reporter groups includes ligand molecules, and preferably small antigen molecules. This antigen molecule can specifically bind to the anti-ligand molecule with high affinity. Typical ligand / anti-ligand pairs include antigen / antibody, lectin / hydrocarbon, and biotin / avidin. This anti-ligand molecule is part of a signal-producing binding conjugate, which also contains a signal-producing group such as a chromophore, fluorophore, or enzyme. This signal-generating group can detect the presence of a ligand group when ligand / anti-ligand binding occurs.

The reporter may have an enzyme reporter moiety, especially if the reporter tail has more than one cationic group, as mentioned above. Typical classes of enzymes include oxidoreductases such as luciferase, glucose oxidase, galactose oxidase and catalase, hydrolases such as various phosphatases, and glycosyl hydrolysis such as β-galactosidase, peptidase and lyase. There is an enzyme.

The one or more reporter groups are typically coupled to the amine of the polycationic tail, preferably the primary amine, according to known coupling methods. In the more preferred method illustrated in FIG. 15, the polyamine is subjected to a reaction with a suitable bifunctional coupling reagent such as 4-fluoro-3-nitrophenyl azide, followed by an enzyme-like reporter group. Is coupled with. Various bifunctional reagents for coupling amines to reactive reporter groups such as amines, carboxyl groups, OH groups and salhydral groups are well known. A detailed method for forming an alkaline phosphatase reporter group and a tetracationic reporter is shown in Example 38. In another more preferred method illustrated in FIG. 15, a reporter is prepared by reacting bis-3,3'-aminopropylmethylamine with an amine and a reactive dansyl group. The reaction scheme in this figure also shows how the amino portion of this reporter tail can be fully alkylated before or after coupling the reporter to this tail. Details of this method are shown in Example 34.

VII. Diagnostic Methods This section describes the use of the diagnostic system described above for the detection of polynucleotide analytes. Is this analyte normally present in single-stranded form, such as messenger-RNA (mRNA), ribosomal RNA (rRNA), or single-stranded RNA or DNA viral genomes; or double-stranded viral RNA, RNA / DNA viral replication intermediates, and double-stranded or overlapping analyzes such as double DNA derived from viruses, bacteria, eukaryotic cells, which are present under physiological conditions. is there. The considerations and principles used in selecting the target sequence in the analyte polynucleotide to which the reagent polymer is targeted are discussed in Section I.

When making this diagnosis, the sample to be analyzed is typically collected by conventional clinical sample collection techniques. If this analyte is a polynucleotide of viral or bacterial origin, removing longer non-analyzed cellular material is often useful as a treatment for the first sample. For example, when determining the presence of viral pathogens, it is often useful to filter the sample through a membrane with a hole size of 0.2 microns to remove bacterial material. When testing for the presence of bacterial pathogens, it is often useful to filter the sample through a membrane with a hole size of 1.0 micron to also remove eukaryotic cells that may be present in the sample. Typical sample preparation methods are described in Examples 36 and 37 for the diagnosis of viral pathogens.

In order to prepare this sample analyte material in a form suitable for binding to this reagent polymer, this sample should be treated to release the nucleic acid of the organism. Surfactants and / or chaotropic salts are generally suitable for organisms lacking cell membranes. Alkali (when the analyte is DNA) or suitable enzymes (eg, lysozyme against many bacteria) can be used for organisms with cell membranes. If desired, this free nucleic acid can be conveniently separated from proteins and other contaminants by adding a chaotropic salt (sodium trichloroacetate) followed by selective precipitation of the nucleic acid with ethanol.

One effective method for releasing and isolating nucleic acids from viral or cellular components is described by the present inventor in Anal Biochem (1983) 133: 79. The method detailed in Example 36 involves suspending the sample in 4M trichloroacetic acid, 100 mM EDTA to denature and solubilize the proteinaceous material, followed by the addition of an equal volume of cold ethanol to release the nucleic acid material. Is included in the precipitation of the protein in a salt solution. Carrier polynucleotides, such as polyuridylic acid, may be added to facilitate nucleic acid precipitation. After a brief cooling, the precipitated nucleic acid is pelleted by centrifugation. The nucleic acid is then washed to remove salts.

This nucleic acid fraction is resuspended in annealing buffer containing a divalent cationic chelating agent at the selected salt concentration. Annealing buffers containing about 100 mM or less monovalent salts (eg NaCl) and about 10 mM chelating agents (eg EDTA) are generally suitable for binding normally present polynucleotides in the form of double-stranded DNA. There is. Salt concentrations significantly below 100 mM are difficult to achieve accurately. Higher salt concentrations, especially those above about 0.5 M, tend to form double strands between complementary polynucleotide strands, and for binding to this analyte polynucleotide between the reagent polymer and the complementary strand. Conflict arises. Double-stranded formation between complementary polynucleotides is also inhibited by chelating agents. This chelating agent is Mg in the annealing mixture.⁺²Ion and Ca⁺⁺It acts to separate the ions. If this analyte is RNA, it may be advantageous to treat the sample with DNase to avoid competition for binding of the dissociated DNA to the reagent polymer.

This annealing reaction is preferably carried out at a temperature about 5 to 15 ° C. lower than the melting temperature of the analyte / polymer double structure formed during the reaction. This annealing temperature is preferred for accurate base-sequence pairing between the analyte target sequence and the polymer. As shown above, when this analyte polynucleotide is normally present as a double-stranded structure with its complementary strand, its reaction temperature is comparable to that of the analyte competing for the desired pairing of the analyte and the reagent polymer. A reaction temperature higher than the melting temperature of the original polynucleotide double strand is preferred to avoid unwanted pairing with the complementary polynucleotide. For annealing buffer concentrations of about 100 mM monovalent cation concentration, annealing temperatures in the range of 24-60 ° C are generally suitable. As previously noted, this exact optimum annealing temperature is a function of the length of the polymer to which it binds, the ratio of A + T to C + G of its recognition portion, and this salt concentration in partially charged polymers.

As discussed above, depending on the structural characteristics of this polymer, the melting temperature of this analyte / polymer double structure is, for example, 37 ° C. in an annealing buffer such as 100 mM monovalent salt. It can be substantially higher than the preferred reaction temperature. In such cases, the melting temperature of the polymer / analyte double structure may be lowered to the desired temperature by adding a modifier such as formamide to add the melting temperature of the polymer / analyte double structure. Can be convenient. To determine the amount of formamide required to achieve the desired melting temperature, the melting curves of the polymer and the target sequence, which would be a synthetic oligonucleotide with the analyte target sequence, are many different denaturants according to conventional methods. Determined by concentration. When formamide is added, it is typically done with a denaturant volume in the range of about 5-50%. From this determination, the amount of formamide required in the annealing buffer to achieve a melting temperature about 5-15 ° C. above the desired reaction temperature is determined.

The analyte sample in this reaction buffer is preferably added to the diagnostic reagent under conditions where the binding polymer of this reagent is present in excess of 10-1000 times the molar concentration of the analyte molecule in the assay mixture. .. The polymer / polynucleotide annealing reaction is carried out at a selected reaction temperature for a period of substantially sufficient annealing of the polymer / analyte, typically between 10 minutes and 3 hours. This solid support reagent is washed once or more to remove unbound material.

The reporter is then added to the washed reagent under preselected conditions for binding the reporter molecule to the fully charged backbone of the reagent bound to the reagent. The spatial / charged characteristics of this reporter that allow selective binding of this reporter to the polynucleotide backbone are discussed above. If this reagent polymer has a negatively charged backbone, one important factor is the salt concentration of the binding medium. At very low salinity, the electrostatic interaction is stronger. It can result in unwanted binding of the reporter to the widely spaced anionic groups of the reagent polymer. Conversely, at very high salt concentrations, electrostatic charge shielding can interfere with the reporter's desired binding to the narrowly spaced anionic groups of the analysis backbone. The optimum salt concentration of this binding medium is the concentration of the maximum or near maximum monovalent salt that allows the reporter to bind sufficiently electrostatically to the fully charged analyte backbone. This optimum salt concentration can be determined by equilibrium dialysis. In this equilibrium dialysis method, the binding of a diffusible reporter to a non-diffusible polynucleotide is measured as a function of the salt concentration of this suspended medium. Optimal ionic strength conditions for reporter binding to this analyte are also readily determined by reporter binding to immobilized polynucleotides under low salt concentration conditions and reporter elution by salt gradients. obtain.

The binding components in this diagnostic system are shown in Figure 16 below to function in the diagnostic method of the present invention. Here, S is a solid carrier in which many bonded polymers are bonded to the surface thereof via a spacer arm indicated by a serrated line. The "m" and "p" subunit chains represent an uncharged, sterically defined methylphosphonate chain and a charged phosphodiester chain, respectively. This reporter has a divalent cationic tail and an R reporter group. For illustrative purposes, each polymer has a sequence of recognition moieties complementary to selected 16-base target sequences in Herpes simplex virus, type I and type 2, as described in Example 21. A base-complementary bond to the target sequence in the Herpes simplex analyte polynucleotide of one of these polymers has been shown.

The reporter shown in this figure is a divalent cationic reporter from Example 34. Each reporter is adapted to bind to a pair of adjacent phosphodiester bonds in a polynucleotide by electrostatic attraction via electrostatic attraction with adjacent phosphodiester bonds. This reporter molecule is shown in their expected binding configuration. Here, virtually any phosphodiester chain is paired with a reporter cation, and this reporter molecule is lined up head-to-head and tail-to-tail. By estimating the maximum density of bound reporter molecules, it can be evaluated that the N-base analyzer can bind up to about N / 2 reporter molecules. More generally, this assay method allows reagent binding of hundreds of thousands or more of reporter molecules, depending on the size of the analyte and the relative number of both reporter moieties and cationic groups per reporter. Binding to each molecule of the analyte easily occurs. Therefore, if the detection of the analyte is generally based on one or several probes per analyzer molecule, then each probe typically contains about 100 reporter moieties, thus making it an existing type of polynucleotide. The sensitivity of 2- to 4-digit detection is higher than that of based diagnosis.

After reaction with this reporter solution (typically 1-2 minutes at room temperature), the reagent is washed to remove unbound reporters. It can then be evaluated directly against the bound reporter. In determining the amount of reporter bound to this reagent, especially in the case of fluorescent or color-developing reporter groups, this reporter is eluted from this reagent with a high salinity solution and then the eluent is evaluated for the reporter. It may be desirable to do so. Other types of reporter groups, such as enzymes, can be easily evaluated by a reporter bound to this reagent or a reporter eluted from the reagent. Methods for detecting a variety of different reporter groups as mentioned above are well known. Examples 36 and 37 illustrate diagnostic methods based on the detection of fluorescent reporters and enzyme reporters, respectively.

From the above, it can be understood how the present invention achieves various purposes and features. The design of the reagent polymer allows this diagnostic reagent to be readily adapted for the detection of any ribopolynucleotide or deoxyribopolynucleotide with a unique base pair sequence. In addition, this reagent polymer can be adapted to bind to a constant target sequence with high sequence specificity at a suitable temperature.

For detection of double-stranded polynucleotides, this reaction is performed under low salt concentration and / or denaturation conditions, which interferes with competition with complementary polynucleotide strands for reagents that bind to reagents. sell. Therefore, this diagnostic system offers the advantages of prior art solid support, single probe polynucleotide diagnostic systems. The advantage is that the competition with complementary strands is eliminated, but the diagnostic system avoids a rather difficult step in adding the single-stranded test nucleic acid to the solid support. At the same time, this system avoids the problems associated with the previously described dual probe polynucleotide diagnostic system. The problem is that the detection of the analyte is based on pseudo-first-order kinetics and requires only one sequence-specific binding polymer.

According to another important feature of the present invention, the binding of the reporter to this analyte is electrostatically independent of the sequence rather than the sequence-specific binding as with existing types of polynucleotide diagnostics. It is based on interaction. Therefore, the reporter itself can be prepared relatively inexpensively and can react with the analyte immediately and under a wide range of conditions. And, most importantly, this reporter can bind to this analysis at densities ranging from multiple reporter moieties per polynucleotide subunit in this analysis. Therefore, the sensitivity of this diagnostic system can be on the order of 2-4 orders of magnitude or more higher than existing tests. This is because existing tests rely on the detection of one or a few probes, including a limited number of reporter portions per molecule of analysis.

The following examples illustrate the preparation and use of a assay system configured according to a particular embodiment of the invention. However, these examples never limit the scope of the invention.

Example 1 Preparation of ribonucleosides and deoxyribonucleosides The following nucleosides are obtained from Sigma Chemical Co., St. Louis, MO: deoxyuridine, deoxyguanosine, thymidine, deoxyadenosine, deoxycytidine, 5-bromodeoxyuridine, deoxy. Inosin, 2,6-diamino-9- (2-deoxy-β-)_D-Erythro-pentoflanosyl) 9H-purine (2,6-diaminopurine deoxyriboside), uridine, guanosine, 5-methyluridine, adenosine, cytidine, 5-bromouridine, inosine.

2,6-diamino-9- (β-)_DRivofuranosyl) -9H-purine (2,6-diaminopurine riboside) is obtained from Pfaltz and Bauer, Inc., Division of Aceto Chemical Co., Inc., Waterbury, CT.

The following nucleosides are prepared by the methods in the literature listed below: 1- (2-deoxy-β-)._D-Erythro-pentoflanosyl-2-pyrimidineone (2-hydroxypyrimidine deoxyriboside) was described by John Wiley and Sons, Inc. in Nucleic Acid Chemisty; LB Townsend and RSTipson, eds (1976), P. Kohler, E. et al. Prepared by the method of Volz, U. Sequin and C. Tamm.

1- (2-deoxy-β-_D-Erythro-pentoflanosyl) -4-methoxy-2-pyrimidinone is prepared by the following method: 1- (3', 5'-di-O-benzoyl-2-deoxy-β-_D-Erythro-pentoflanosyl-4-methylthio-2-pyrimidinone

[Prepared by the method of D. Cech and A. Holy of Collection of Czechoslov Chem Comm (1977) 42: 2246] is treated with 200 ml of 0.2 M sodium methoxide solution. The treated solution is left at room temperature overnight. This solution was then added to Dowex 50x8 (Dowex 50x8; H).⁺Neutralize with mold) and filter. Dissolve the residue in 100 mL of water, extract with 2.50 mL of ether, and evaporate the aqueous phase. The residue is an amorphous substance and is used directly in the next reaction.

2-Amino-9- (2-Deoxy-β-)_D-Erythro-pentoflanosyl) -1,9-dihydro-6H-purine-6-thione (deoxythioguanosine) was prepared by the method of RHIwamoto, EMActon, and L. Goodman of J Med Chem (1963) 6: 684. Will be done.

1- (β-)_D-Riboflanosyl) -2-pyrimidineone (2-hydroxypyrimidineriboside) is prepared by the method of U. Niedballa and H. Vorbruggen of J Org Chem (1974) 39: 3668.

1- (2-deoxy-β-_D-Riboflanosyl) -4-methoxy-2-pyrimidinone (2-hydroxypyrimidine deoxyriboside) is prepared by the method of R. Wightman and D. Holy in the Collection of Czechoslov Chem Comm (1973) 38: 1381.

2-Amino-9- (β-)_D-Riboflanosyl) -1,6-dihydro-6H-purine-6-thione (thioguanosine) was prepared by the method of JJ Fox, I. Wempen, A. Hampton and IL Doerr of J Amer Chem Soc (1958) 80: 1669. To.

Example 2 Preparation of Base-Protected Nucleotides Dimethoxytrityl chloride, N-benzoyladenosine, N-benzoyl-2'-deoxyadenosine, N-benzoylcytidine, N-benzoyl-2'-deoxycytidine and N-isobutyryl-2' -Deoxyguanosine is obtained from Sigma Chemicals, St. Louis, Missouri. 9-Fluorenylmethoxycarbonylchloride (FMOC chloride), trimethylchlorosilane, anhydrous isobutyric acid, 4-nitrobenzoyl chloride, and all organic solvents (for reaction and chromatography) are obtained from Aldrich Chemical Co., Milwaukee, Wisconsin. Be done. Silica gel is obtained from EM Science, Cherry Hill, New Jersey.

2.1 Guanosine N-2 9-fluorenylmethoxycarbonyl derivative is prepared by the following general procedure for protection of the amino group of a nucleoside: guanosine (1 mmol) suspended in pyridine (5 mL) and trimethylchlorosilane. Treat with (5 mmol). After stirring this solution for 15 minutes, 9-fluorenylmethoxycarbonyl chloride (5 mmol) is added and the solution is kept at room temperature for 3 hours. Cool the reaction solution in an ice bath and add water (1 mL). After stirring for 5 minutes, concentrated ammonia (1 mL) is added, and the reaction solution is stirred for 15 minutes. Evaporate this solution to near dryness and dissolve the residue in chloroform (10 mL). The solution is washed with sodium bicarbonate solution (5 mL, 10%), dried over sodium sulfate and evaporated. The residue is evaporated with toluene several times and the product is chromatographed on silica gel using a methanol gradient (0-50%) in methylene chloride.

N-isobutyryl guanosine is prepared by the method of Letsinger and Miller of JAmer Chem Soc (1969) 91: 3356.

N-Acetyl guanosine is obtained by the method of CB Reese and RSS affhill of J Chem Soc Perkin Trans (1972) 1: 2937.

2.2 Deoxyguanosine N-2 9-fluorenylmethoxycarbonyl derivatives are prepared by the methods of J. Heikkla and J. Chattopadhyaya of Acta Chem Scand (1983) B 37: 263.

N-2 acetyl derivatives are obtained from Research Plus Inc., Bayonne, NJ.

2.3 Deoxyadenosine N-6 2- (4-nitrophenyl) ethoxycarbonyl derivatives are prepared by the methods of F. Himmelsbach and W. Pfleiderer of Tetrahedron Lett (1983) 24: 3583.

N-6 4-nitrobenzoyl-2'-deoxyadenosine is prepared by the above method for preparing FMOC-guanosine, except that 4-nitrobenzoyl chloride is used instead of FMOC chloride.

N-6 2 (Phenylsulfonyl) ethoxycarbonyl derivative is a 2- (Phenylsulfonyl) -ethylchloroformate as an acylating agent.

Preparation of FMOC guanosine, except that N-methylimidazole or pyridine was used as the solvent using [obtained by the method of N. Balgobin, s. Josephson and B. Chattopadhyaya of Tetrahedron Lett (1981) 22: 3667]. Prepared by.

2.4 Adenosine N-6 2- (4-nitrophenyl) -ethoxycarbonyl derivatives are prepared by the methods of F. Himmelsbach and W. Pfleiderer of Tetrahedron Lett (1983) 24: 3583.

N-6 4-nitrobenzoyl adenosine is prepared by the method of preparing FMOC-guanosine except that 4-nitrobenzoyl chloride was used instead of FMOC chloride.

The N-6 2- (phenylsulfonyl) ethoxycarbonyl derivative is a 2- (phenylsulfonyl) -ethylchloroformate as an acylating agent.

Preparation of FMOC guanosine, except that N-methylimidazole or pyridine was used as the solvent using [obtained by the method of N. Balgobin, s. Josephson and B. Chattopadhyaya of Tetrahedron Lett (1981) 22: 3667]. Prepared by.

2.5 Deoxycytidine N-4 2- (4-nitrophenyl) -ethoxycarbonyl derivatives are prepared by the methods of F. Himmelsbach and W. Pfleiderer of Tetrahedron Lett (1983) 24: 3583.

The N-6 2- (phenylsulfonyl) ethoxycarbonyl derivative is a 2- (phenylsulfonyl) -ethylchloroformate as an acylating agent.

Preparation of FMOC guanosine, except that N-methylimidazole or pyridine was used as the solvent using [obtained by the method of N. Balgobin, s. Josephson and B. Chattopadhyaya of Tetrahedron Lett (1981) 22: 3667]. Prepared by.

2.6 Cytidine N-4-2- (4-nitrophenyl) -ethoxycarbonyl derivatives are prepared by the methods of F. Himmelsbach and W. Pfleiderer of Tetrahedron Lett (1983) 24: 3583.

The N-6 2- (phenylsulfonyl) ethoxycarbonyl derivative is a 2- (phenylsulfonyl) -ethylchloroformate as an acylating agent.

Preparation of FMOC guanosine, except that N-methylimidazole or pyridine was used as the solvent using [obtained by the method of N. Balgobin, s. Josephson and B. Chattopadhyaya of Tetrahedron Lett (1981) 22: 3667]. Prepared by.

2.7 2.6-Diaminopurine riboside N-2, N-6 bis (9-fluorenylmethoxycarbonyl) derivatives of 2,6-diaminopurine riboside are prepared by conventional methods.

N-2, N-6-bis (isobutyryl) derivatives are prepared by conventional methods.

2.8 2,6-diaminopurine-2'-deoxyriboside The bis-N-2,N-6- (9-fluorenylmethoxycarbonyl) derivative of 2,6-diaminopurine-2'-deoxyriboside is a conventional procedure. Prepared by.

2.9 Thioguanosine N-2 9-fluorenylmethoxycarbonyl derivative of thioguanosine is prepared by a conventional method.

2.10 2'-Deoxythioguanosine N-2 9-fluorenylmethoxycarbonyl derivative of 2'-deoxythioguanosine is prepared by a conventional method.

Example 3 Preparation of 5'-amino-2', 5-dideoxyribonucleoside subunits Carbon tetrabromide, sodium azide, p-toluenesulfonyl chloride (tosyl lolide), triphenylphosphine and 10% palladium-charcoal are Aldrich. Purchased at Chem Co. Lithium azide is available at Kodak Laboratory and Specialty Chemicals.

3.1 General Procedures The nucleosides used in this example are thymidine, N6-benzoyl-2'-deoxyadenosine, N4-benzoyl-2'-deoxycitodine, and 2'-deoxyinosine, 2-hydroxy-pyrimidine-2. N2-N6-bisisobutyryl derivatives of -deoxyriboside and 2,6-diaminopurine-2 -deoxyriboside (see Example 1). The desired dried nucleoside (1 mmol) is weighed and added to the reaction vessel containing triphenylphosphine (1.01 mmol) and lithium azide (5 mml). After suspending the solid in DMF, carbon tetrabromide (1.0 mmol) is added to the container. The solution is stirred at room temperature for 24 hours. After stopping the reaction with methanol, the solution is evaporated to dryness. The residue is subjected to silica gel chromatography and eluted with a methanol / chloroform mixture to give the desired 5'-azido-2', 5'-deoxynucleoside.

This 5'-azidone nucleoside (1 mmol) is dissolved in ethanol. Hydrogenation is carried out for 10 hours at a hydrogen pressure of 35 psi in the presence of a catalytic amount of 10% palladium-carbon. The solution is filtered and evaporated under reduced pressure to give a crude solid. This solid content is purified by adding a suitable solvent and kneading.

3.3 5'-Azidouridine Derivative The 5'-azido derivative of 5-bromo-2'-deoxyuridine is prepared by the above method. 5'-Azido-t-bromo-2'-deoxyuridine (1 mmol) is treated with triphenylphosphine (1.5 mmol) in pyridine at room temperature for 2 hours. Concentrated ammonia (1 mL) is added to this reaction vessel. After 14 hours, the solvent is removed under vacuum. The residue is dissolved in THF and this solution is added to hexane. The precipitate is collected and the solid component is kneaded with a suitable solvent to give 5'-amino-5-bromo-2'-deoxyuridine.

3.5 5'-Azidoguanosine 5'-Amino-2', 5'-Dideoxyguanosine Other preparations are protected with protected 2'-deoxyguanosine (N-2-acetyl or N-2-FMOC derivative). (1 mmol) is treated with tosyl lolide (1.3 mmol) in pyridine at 0 ° C. overnight. This solution is evaporated to dryness and the residue is dissolved in chloroform. The resulting solution is washed with aqueous sodium bicarbonate solution and dried over sodium sulfate. This solution is evaporated and the residue is subjected to silica gel chromatography and eluted with a methanol / chloroform mixture. The resulting tosylate (1 mmol) is treated with sodium azide (6 mmol) in DMF at 80-100 ° C. for several hours. The solvent is removed with a rotary evaporator, the residue is subjected to silica gel chromatography and eluted with a mixed chloroform / methanol solvent. This azide is reduced to the desired amine using the above technique.

The base nitrogen protective groups for 2'-deoxycytidine, 2'-deoxyadenosine, 2'-deoxyguanosine, and 2,6-diaminopurine deoxyriboside are against 2'-deoxycytidine and 2'-deoxyadenosin. Example 2 is a 2- (phenylsulfonyl) ethoxycarbonyl group and a 9'-fluorenylmethoxycarbonyl (FMOC) group to protect 2'-deoxyguanosine and 2,6-diaminopurine deoxyriboside. Used as shown in.

Example 4 Preparation of 3'-amino-2', 3'-dideoxyribonucleoside subunit Thymidine was converted to 5'-O-acetyl-3'-azido-2', 3'-dideoxythymidine, which was converted to J. Converted to 3'-azido-2', 3'-dideoxyadenosine and 3'-azido-2', 3'-dideoxyguanosine by the method of M. Imazawa and F. Eckstein of Org Chem (1978) 43:30 44. The base portion of 3'-azidoadenosine is protected as a benzoyl derivative or a 2- (phenylsulfonyl) ethoxycarbonyl derivative (shown in Example 1). N-2 of 3'-azidoguanosine is protected as an acetyl derivative or FMOC derivative (see Example 1). Reduction of these 3'-azido groups to 3'-amino-2'-3'-dideoxyribonucleosides is made as shown in Example 3.

Example 5 Preparation of Morpholine-Type Subunits Derived from Ribonucleosides Ammonium cyanoborohydride, sodium m-sodium periodate, sodium cyanoborohydride, uridine, N4-benzoylcytidine, and N6-benzoyladenosine are described in Sigma Chemical Co., Ltd. Obtained at. N2-isobutyryl guanosine is prepared by the general procedure of Letsinger and Miller (JAmer Chem SoC (1969) 91: 3356). 2-Phenyl-2-propanol and bis (p-nitrophenyl) carbonate are obtained from Aldrich Chemical Co.

The morpholine derivatives of uridine are 1 mmol of uridine and ammonium diborate, (NH₄)₂B₂O₇Prepared by dissolving 2 mmol and in 5 ml of water. Add a solution of 1.2 mmol of sodium m-periodate in 5 ml of water while stirring under an ice bath and avoiding direct sunlight. After 90 minutes, add 0.2 ml of 1,2-propanediol and continue stirring at room temperature for 10 minutes. Then, add a solution of 0.25 g of sodium cyanoborohydride in 3 ml of water with sufficient stirring in a well-ventilated hood, and stir the mixture at room temperature for 4 hours. Next, the reaction mixture is concentrated in a warm water bath under vacuum to form an oil, which is redispersed with a minimum amount of methanol, laminated on a silica gel chromatography column, and eluted with methanol / 1% triethylamine. The morpholine-type product is eluted from the column with a sharp band. This product moves much more slowly than the original ribonucleoside and its oxidation products. This morpholine-type product has almost the same UV spectrum as the original ribonucleoside. However, the mass spectrum is as low as 17 daltons (determined by mass spectrum analysis by fast atom bombardment activation).

The product is then reacted with 2-phenylisopropylphenyl carbonate to protect the nitrogen of the morpholine. The desired active carbonate is prepared and reacted with the morpholine subunit according to the method of Sandberg and Ragmarsson of Int J Peptide Protein Res (1974) 6:111. Finally, as needed (subunit The hydroxyl group at the position corresponding to the original 5'(depending on the method of incorporating the assembly into the polymer) can be activated by the following method. The method is to dissolve the N-protected subunit in the smallest volume of dry dimethylformamide, bis (p-nitrophenyl) carbonate 2 equivalents and triethylamine, N-methylimidazole, or N, N-dimethylaminopyridine 0.2. Is to add an equal amount. After holding at room temperature for 3 hours, the reaction mixture is concentrated under vacuum in a warm bath. This concentrated syrup is resuspended in a small volume of dichloromethane, laminated on a silica gel column and eluted with a dichloromethane / ether (1: 1 volume ratio) mixture containing 0.2% by volume of N, N-dimethylaniline. .. In this solvent system, activation products move substantially slower than p-nitrophenols and bis (p-nitrophenyl) carbonates, but much faster than unreacted starting materials. The activated product can be easily visually observed on a silica gel TLC plate by spraying ammonia. By spraying ammonia, yellow nitrophenolate ions appear in 1-2 minutes.

Other ribonucleosides (base protected as in Example 2 if necessary) are converted to the corresponding morpholine derivatives in a basically similar manner. However, appropriate amounts of methanol should be added, depending on the solubility of the protected ribonucleoside prior to the initial periodic oxidation step.

When morpholine-type subunits are attached via a thiocarbomet bond, suitable base protecting groups for the usual starting ribonucleosides are: cytidines and adenosines to phenylsulfonylethoxycarbonyl groups, and guanosines. Is a 9-fluorenylmethoxycarbonyl group.

Example 6 Preparation of 5'-acetic acid-2', 5'-dideoxyribonucleoside subunits Triethylamine, pyridine / sulfur trioxide complex, p-nitrophenol, dicyclohexylcarbodiimide, and 40% dimethylamine aqueous solution are obtained with Aldrich.

Methylene chloride and dimethyl sulfoxide (1/1, v / v; 10 mL) of 3'-O-tert-butyldimethylsilyl-N-2- (phenylsulfonyl) etoshikicarbonyladenosine (1 mmol) prepared in Example 12.1. Dissolve in 0 ° C. To this is added triethylamine (3 mmol) and a pyridine / sulfur trioxide complex (3 mmol). The mixture is stirred at this temperature for 1 hour and washed with water. Dry over sodium sulphate and evaporate under reduced pressure. The residue is dissolved in dry pyridine (10 mL) and treated with vezyloxycarbonylmethylenetriphenylphosphorane (1.5 mmol) at 37 ° C. for 24 hours. The mixture is then evaporated to dryness under reduced pressure, the residue is dissolved in methylene chloride and washed with dilute HCl and water. Next, the organic phase is dried over sodium sulfate, and the solvent is distilled off under reduced pressure. The residue is subjected to silica chromatography using a methanol gradient (0-20%) in chloroform.

The unsaturated ester (1 mmol) obtained in the previous step is dissolved in ethyl acetate / ethanol (40 mL, 1 / 1, v / v) containing cyclohexanediene (5 mL) and 10% palladium-carbon (100 mg), and this suspension Stir vigorously for 5 to 24 hours under a nitrogen stream. The mixture is filtered and the solvent is evaporated under reduced pressure. The residue is subjected to silica gel chromatography using a methanol gradient in chloroform (5-50%).

Approximately 20% excess of p-nitrophenol is added to the 0.2-0.5 M ethyl acetate (or methylene chloride) solution of the acid obtained in the previous step. Add a calculated amount of dicyclohexylcarbodiimide to this solution at 0 ° C. After 0.5 hours, the solution was warmed to room temperature and kept for 1 hour. The separated dicyclohexylurea is filtered and washed with a solvent. The mixture of the filtrate and the washing liquid is evaporated under reduced pressure and dried. The ester can be used as is in the coupling reaction or can be purified on a short column of silica using an isopropanol gradient in chloroform (0-50%).

A similar series of reactions can be performed with other well-protected 2'-deoxynucleosides.

When this active ester (1 mmol) is treated in 40% aqueous dimethylamine solution (2 mmol) and methanol, N-protected 3'-O-tert-butyldimethylsilyl-2', 5'-dideoxyadenosine-5'- N, N-dimethylamide of acetic acid is produced. The solvent is evaporated under reduced pressure to remove and the residue is desorbed and silylated using 2M HF / 1M tert-butylammonium fluoride (TBAF) reagent. This TBAF reagent is described by BLGaffney and RA Jones in Tetrahedron Lett (1982) 23: 2257. The deprotected nucleoside obtained in the previous step is added to 2M HF / 1M tert-butylammonium fluoride (TBAF1 mmol) in pyridine. After stirring for 24 hours, the reaction solution is separated into methylene and sodium bicarbonate aqueous solution. The organic layer is washed with water, dried over sodium sulfate, evaporated and dried. The residue is purified by silica chromatography using a methanol gradient (5-25%) in methylene chloride.

Example 7 Preparation of Purine and Pyrimidine Pyrrolidone Potassium t-butoxide, anisoyl chloride, 6-chloropurine, 10% palladium-charcoal, phthaloyl chloride, 2-amino-6-chloropurine, 20% aqueous solution of tetraethylammonium hydroxide, 25% aqueous solution of trimethylamine, 2-pyrimidineone, imide N-bromosuccinate, trifluoroacetic acid, 4-nitrophenol, and N, N-discusimidyl carbonate are obtained with Aldrich. Lithium azides are available in Eastman Kodak, Rochester, New York. C18 reverse phase silica gel is obtained from Whatman, Hillsboro, Oregon.

7.1. (S) -5

[(4-Amino-2-oxopyrimidinyl) -methyl] -Preparation of pyrrolidone (hereinafter referred to as cytosine pyrrolidone) Cytosine (2.2 mmol) is dissolved in dimethyl sulfoxide (2 mL) containing potassium tert-butoxide (2 mmol). .. This solution is added to a flask containing (S) -5- (tosyloxymethyl) -2-pyrrolidone (1 mmol). This (S) -5- (tosyloxymethyl) -2-pyrrolidone is prepared by the method of E, Hardegger and H. Ott. (Helv Chim Acta (1955) 38: 312). After dissolution, the mixture is stirred at 25 ° C. for 6 hours. The mixture is neutralized with acetic acid (2 mmol) and evaporated under reduced pressure. The residue is taken in dimethylformamide (5 mL) and evaporated under reduced pressure. This process is repeated 3 times.

7.2. Preparation of N-4 anisoylcytosine pyrrolidone The mixture obtained in the previous step is dissolved in pyridine or N-methylimidazole (10 mL) and treated with anisoyl chloride (2.8 mmol) at room temperature. After stirring for 2 hours, stop the reaction with ice (0.5 g) of the mixture. After 5 minutes, add 1 mL of 29% ammonium. After keeping at room temperature for 15 minutes, the solution is dissolved in ethyl acetate (15 mL) and washed twice with brine (15 mL). Combine with the washing liquid and wash with ethyl acetate (2 x 30 mL). The organic layer is combined, dried over sodium sulfate, and evaporated under reduced pressure. The residue is evaporated several times with toluene and the resulting residue is subjected to silica gel chromatography on a methanol gradient (0-20%) in methylene chloride.

7.3.6-Preparation of chloropurine pyrrolidone This compound is prepared by the method of alkylation of cytosine. The difference from the alkylation conditions for cytosine is that the alkylation is done on 6-chloropurine and the mixture is heated to 35-95 ° C. for several hours. The mixture is cooled to room temperature, neutralized with acetic acid (2 mmol) and evaporated under reduced pressure. Shake this with a mixture of 20% methanol / chloroform and 10% sodium bicarbonate. The aqueous layer is washed with chloroform, the combined organic layer is dried over sodium sulfate, and evaporated under reduced pressure. The residue is subjected to silica gel chromatography using a methanol gradient in chloroform (0-25%).

7.4.6 Preparation of azidopurinpyrrolidone 6-chloropurinepyrrolidone (1 mmol) is treated in dimethyl sulfoxide (2 mL) containing lithium azide (2 mmol) at 30-100 ° C. for several hours. After treatment, the mixture is evaporated under reduced pressure and dissolved in chloroform. Wash with 10% sodium bicarbonate, dry the organic layer over sodium sulfate and concentrate under reduced pressure. The residue is subjected to silica chromatography using a methanol gradient in chloroform (0-25%).

7.5. Preparation of adenosine pyrrolidone Hydrogenate the azide obtained in the above example. This hydrogenation is carried out by applying 30 lbs of hydrogen pressure to a solution of the azide (1 mmol) in ethanol (10 mL) containing palladium-carbon (100 mg, Pd 10 wt%) and shaking for 24 hours. The solution is filtered through Celite and the filtrate is evaporated. The residue is used directly in the next step.

7.6. Preparation of N-6 phthaloyl adenosine pyrrolidone A phthaloyl group is introduced at the N-6 position by acylating adenosine pyrrolidone (1 mmol) with phthaloyl chloride (1.4 mmol). This reaction is carried out by using the same method as the above-mentioned anisoylization of cytidine, except that ammonia is not added to the reaction solution.

7.7.2-Preparation of amino-6-chloropurine pyrrolidone Pyrrolidone tosylate is alkylated with 2-amino6-chloropurine by the method shown in the preparation of 6-chloropurine derivatives.

7.8. Preparation of guanine pyrrolidone The chloropurine pyrrolidone (1 mmol) obtained in the previous step is dissolved in diglyme (10 mL) and a 25% trimethylamine aqueous solution (2 mL). The solution is stirred at room temperature for 5 hours, water (10 mL) is added and the mixture is concentrated to 10 mL under reduced pressure. Acetic acid (2 mL) was added, and the mixed solution was evaporated under reduced pressure to give an oily substance. The residue containing tetraethylammonium acetate is used directly in the next step.

7.9. Preparation of N-2 acetylguanine pyrrolidone The guanine pyrrolidone (1 mmol) obtained in the previous step is reacted with acetic anhydride (5 mL) for 2 hours under reflux. The solvent is evaporated and the residue is evaporated with dimethylformamide. The residue is dissolved in ethanol (5 mL) saturated with ammonia at 0 ° C. and the solution is stirred at this temperature for 2 hours. The solvent is evaporated under reduced pressure and the residue is purified by short column silica chromatography using a methanol gradient in chloroform (5-50%).

7.10.2-Preparation of Amino-6-Azidopurine Pyrrolidone This compound is prepared from 2-amino-6-chloropurine pyrrolidone using the method of Example 7.4. The difference from the method of Example 7.4. Is that the reaction requires a longer time and is carried out at a higher temperature.

7.11.2, Preparation of 6-diaminopurine pyrrolidone This compound is prepared in the same manner as adenosine pyrrolidone by reduction of 2-amino-6-azido derivative.

7.12. N-2 Acetyl N-6 Preparation of phthaloyl 2,6-diaminopurine pyrrolidone Acetyl group is added by the same method as the above preparation of N-2 acetylguanine pyrrolidone except that the treatment with methanolic ammonia is carried out for 8 hours. be introduced. Evaporate the solvent and phthaloylate the residue as in the case of the adenosine derivative.

7.13. Preparation of inosine pyrrolidone 6-chloropurine pyrrolidone (1 mmol) was converted to an inosine derivative by the method used for the preparation of a guanine pyrrolidone derivative. The compound is purified by silica chromatography using a methanol gradient in chloroform (5-25%).

7.14.2-Preparation of hydroxypyrimidine pyrrolidone This compound is obtained by alkylation of 2-pyrimidine by the method used in the preparation of 6-chloropurine derivatives.

Example 8 Preparation of Purine and Pyrimidine T-BOC Amino Acids The following general procedure for cytosine pyrrolidone can be applied to all properly protected pyrrolidone derivatives. Its product, referred to as t-BOC acid, is formed by cleavage of the pyrrolidone ring to the acid and t-BOC amine.

8.1. Preparation of Cytosine t-BOC Acid N-4-anisoil cytosine pyrrolidone (1 mmol) is dissolved in methylene chloride containing di-tert-butyl dicarbonate (2 mmol), triethylamine (1 mmol) and dimethylaminopyridine (1 mmol). Let (0.5 M solution). The solution is stirred at room temperature for 10 hours. Volatile substances are removed and the residue is purified on silica using a methanol gradient (0-20%) in chloroform. The residue is dissolved in tetrahydrofuran (0.2M solution) and then lithium hydroxide is added (as a 3 mmol, 1M solution). After holding at room temperature for 5 hours, the solution is neutralized by adding 10% acetic acid (3 mmol) and the solvent is removed under reduced pressure. The residue was dissolved in 9N ammonia and stirred at room temperature for 1-10 hours. The solvent is removed under reduced pressure and the residue is purified by chromatography on C18 reverse phase silica gel using a mixture of water and methanol (or trifluoroethanol).

8.2. Preparation of uracil t-BOC acid This is prepared from cytosine t-BOC acid by the following procedure: Cytosine t-BOC acid (1 mmol) is dissolved in acetic acid aqueous solution and treated with sodium sulfite (1 mmol) at 4 ° C. To do. The mixture is stirred for 1 hour and evaporated to dryness under reduced pressure. The product is purified by chromatography on C18 reverse phase silica gel with a mixture of water and methanol (or trifluoroethanol).

8.3.5 Preparation of bromouracil t-BOC acid This compound is obtained from uracil t-BOC acid by the following procedure: uracil t-BOC acid (1 mmol) is dissolved in DMF (3 ml) and N-bromosuccinimide Treat with (1.2 mmol). The solution is left at room temperature for 16 hours. After removing the solvent under reduced pressure, the product is purified by chromatography on C18 reverse phase silica gel using a mixture of water and methanol (or trifluoroethanol).

8.4. Preparation of N-Protected t-BOC Acids This common technique for acylating recognition moieties and out-of-ring amino groups also applies to the protection of adenosine derivatives.

The cytosine t-BOC acid (1 mmol) obtained in the previous step is dissolved in N-methylimidazole or pyridine (5 mL) and treated with chlorotrimethylsilane (5 mmol). After holding at room temperature for 15 minutes, the solution is 2 mmol of 2- (4-nitrophenyl) ethylchloroformate.

Treat with [obtained by the method of F. Himmelsbach and W. Pfleiderer (Tetrahedron Lett (1983) 24: 3583)]. The reaction was kept at room temperature for 8 hours, then cooled in an ice bath and water (1 mL) was added. After 5 minutes, add 1 mL of concentrated ammonia and stir the mixture at room temperature for 15 minutes. The reaction solution is then evaporated to dryness and the residue is dissolved in water. The solution is acidified with 2M hydrochloric acid and extracted with chloroform. The combined organic extract is dried over sodium sulfate and evaporated under reduced pressure. The residue is purified by silica chromatography using a methanol gradient (5-50%) in chloroform.

For protection of guanine and 2,6-diaminopurine derivatives, the above procedure was modified and the reaction was carried out in pyridine to give the acylating reagent 9-fluorenylmethylchloroformate.

Example 9 Preparation of Purine and Pyrimidine Amino Acids The following techniques apply to all derivatives. It removes the t-BOC group and frees the primary amino group of the backbone.

T-BOC acid (1 mmol) was dissolved in 5 mL of methylene chloride containing 20% trifluoroacetic acid and stirred at room temperature for 30 minutes. Toluene (3 mL) is added and the solvent is evaporated under reduced pressure to give the amino acid trifluoroacetic acid salt. This is used directly for the coupling reaction.

Example 10 Preparation of subunits for assembly of achiral acyclic polynucleotide analogs 10.1 Preparation of cytidine analogs Lithium borohydride, di-tert-butyl dicarbonate and bis (triphenylphosphine) palladium II chloride are available. Obtained from Aldrich Chemical Co., Milwaukee, WI. 2-Amino-5-bromopyrimidine is T. Nishikawa

It is prepared by the method of [Chem.Pharm.Bull.9:38 (1961)].

For 2-amino-5-bromopyrimidine, the extracyclic amino group is benzoylated by the general method of Example 2 using benzoyl chloride. The product benzamide (25 mmol) is added to a 45 mL pressure vessel containing triethylamine (10 mL), benzene (10 mL) and bis (triphenylphosphine) palladium II chloride (0.375 mmol). The container is blown with argon, sealed and pressurized to 600 psi with carbon monoxide and then to 1200 psi with hydrogen. Heat the reaction vessel to 145 ° C in an oil bath with stirring. After all gas absorption is complete, the reactants are cooled and the gas is slowly evacuated. After the addition of anhydrous ether, the reaction mixture is filtered and evaporated under vacuum. The product was then isolated by silica gel chromatography using an isopropanol gradient in methylene chloride (0-20%).

The 2-benzamide pyrimidine-5-carboxyaldehyde (1 mmol) obtained in the previous step is dissolved in ethanol (5 mL) and treated with 4-aminobutyric acid (1 mmol) and triethylamine (1 mmol). After stirring for 1 hour, the mixture is hydrogenated at 30 psi with 10% palladium-carbon (100 mg). After the absorption of hydrogen is completed, the reaction solution is filtered and the solvent is removed under reduced pressure. The residue is dissolved in methanol saturated with ammonia at 0 ° C. and stirred at room temperature for 10 hours. After evaporating the solvent, the residue is purified by chromatography on C18 silica gel using a methanol-water mixture buffered with ammonium acetate (0.2M) to pH 7.

The amino acid (1 mmol) obtained in the previous step is dissolved in ethanol (3 mL) containing di-tert-butyl dicarbonate (1 mmol). Triethylamine (1.5 mmol) is added slowly at room temperature. After the carbon dioxide generation is finished, the solvent is removed under vacuum and the residue is used directly for the next reaction.

The extracyclic amino group of the t-BOC amino acid obtained in the previous step is acylated by the general method of Example 2 using 2- (phenylsulfonyl-ethoxycarbonyl chloride), the product of which is a methanol gradient in chloroform ( It can be purified by silica gel chromatography using 0-50%).

The 2- (phenylsulfonyl) -ethoxycarbonyl-protected BOC amino acid obtained in the previous step was converted to p-nitrophenyl or N-hydroxysuccimidoyl active ester by the method in Examples 11.3 and 11.4.

10.2. Preparation of uridine analogs Ethyl 6-hydroxynicotinate, H.Gault, J.Gilbert, and D.Briaucourt

Prepared by the method of [CRAcad.Sci., Paris, Ser.C, 266: 131 (1968)]. Lithium borohydride is obtained from Aldrich. Manganese dioxide, Vereshchagin et al.

Prepared by the method of [Zhurnal Arganicheskoi Khimil, 8: 1129 (1972)]. Dowex resin is obtained from Bio-Rad Laboratories, Richmond, CA.

A solution of ethyl 6-hydroxynicotinate (49 mmol) in tetrahydrofuran (10 mL) is added dropwise to a suspension of lithium borohydride (100 mmol) in tetrahydrofuran (THF) (75 mL) at room temperature under a nitrogen atmosphere. The viscous mixture is stirred at 25 ° C. for 16 hours. Then 60 ml of 20% acetic acid is added to the cooled suspension. THF is evaporated under reduced pressure and the residual aqueous solution is applied to an ion exchange column (Dowex-50W-X8) to remove lithium ions. Carefully wash the column with 250 mL of water and then evaporate. Boric acid is removed from the resulting residue by repeated evaporation with methanol. The product is purified by distillation under reduced pressure.

The benzic alcohol (66 mmol) prepared in the previous step is slowly treated (with heat generation) with an ether (100 ml) slurry. After holding at room temperature for 12 hours, the mixture is filtered, the solvent removed under reduced pressure and the residue is distilled in vacuo to give 6-hydroxynicotinaldehyde.

The aldehyde obtained in the previous step is reacted with 4-aminobutyric acid in the same manner as in the preparation of the cytidine analog in Example 11.1.

The amino acid obtained in the previous step was converted to the corresponding BOC-derivative in the same manner as in the preparation of the cytidine analog in Example 10.1.

The BOC-amino acid obtained in the previous step is converted to p-nitrophenyl or N-hydroxysuccimidoyl active ester by the method in Examples 11.3 and 11.4.

Example 11 Preparation of 5'-protected, 3'-activated 2'-deoxynucleoside 11.12'-deoxynucleoside 5'-O-dimethoxytrityl derivative For preparation of 2'-deoxynucleoside 5'-O-dimethoxytrityl derivative The following method is common.

Dissolve N-2 9-fluorenylmethoxycarbonyl-2'-deoxyguanosine (1 mmol) in anhydrous pyridine (2 mL) and keep at 0 ° C. Dimethoxytrityl chloride (1.2 mmol) is added in 0.15 mmol increments over 8 hours. After another 2 hours, methanol (0.5 mL) is added and the solution is concentrated under reduced pressure. The residue is treated with a mixture of sodium bicarbonate (2 mL, 0.5%) and methylene chloride (5 mL).

Extract with methylene chloride (2 x 5 mL) in the aqueous layer, dry the combined organic layer with sodium sulfate, and evaporate. The residue is evaporated with toluene several times and the product is purified by silica gel chromatography using a methanol gradient (0-10%) in methylene chloride.

11.2.5'-dimethoxytrityl-2'-deoxynucleoside 3'-0- (4-nitrophenyl) carbonate 5'-0-dimethoxytrityl-2-deoxycytidine 3'-0- (4-nitrophenyl) carbonate The preparation method is described in the following operation method, which is a general preparation method for 4-nitrophenyl carbonate. Bis (4-nitrophenyl) carbonate and N, N-dimethylaminopyridine are obtained from Sigma.

5'-0-dimethoxytrityl-N-2- (4-nitrophenyl) -ethoxycarbonyl-2'-deoxycytidine (1 mmol) was dissolved in anhydrous dimethylformamide (5 mL) at room temperature and bis (4-nitrophenyl). ) Treat with carbonate (2 mmol) and then evaporate under reduced pressure. The residue is dissolved in dimethylformamide (5 mL) and N, N-dimethylaminopyridine (0.1 mmol) is added. Evaporate the yellow solution and allow to react for 1 hour. The reaction mixture is dissolved in chloroform (10 mL) and washed with aqueous hydrochloric acid solution (5 mL, 0.1 M). The organic layer is washed twice with aqueous sodium hydroxide solution (5 mL, 0.1 M) and water (2 mL), dried over sodium sulfate, and evaporated. The residue is placed on a silica gel column and eluted with an isopropanol gradient (0-10%) in chloroform. Fractions containing the product are collected, evaporated, dissolved in chloroform (10 mL) and repeated washing with aqueous sodium hydroxide solution to remove traces of 4-nitrophenol. The organic layer is washed with water, dried over sodium sulfate and evaporated. The product is uniform according to TLC and is used directly in the preparation of dimerized carbonate.

It is sometimes better to use N-methylimidazole as a catalyst rather than N, N-dimethylaminopyridine for the preparation of nucleoside carbonates containing FMOC groups.

11.3. N-Protected t-BOC p-Nitrophenyl Ester Activated ester for subunit binding is prepared by the following two procedures. This is common in all t-BOC acid derivatives.

Add about 20% excess of p-nitrophenol to a 0.2-0.5 M ethyl acetate (or methylene chloride) solution of t-BOC acid obtained in Example 8.4. At 0 ° C., a calculated amount of dicyclohexylcarbodiimide is added to the solution. After 0.5 hours, bring the mixture to room temperature and hold for 1 hour. The separated dicyclohexylurea is filtered and washed with a solvent. Combine the filtrate and washing solution, evaporate under reduced pressure and dry. This ester can be used directly in the coupling reaction. Alternatively, it can be purified with an isopropanol gradient in chloroform (0-50%) on a short column of silica.

11.4 N-Protected-t-BOC N-Hydroxysuccimidyl Ester In an acetonitrile (5 mL) solution of t-BOC acid (1 mmol) obtained in Example 8.4., Pyridine (1 mmol) and N, N'-disk Add cymidyl carbonate (1 mmol). The solution is stirred at room temperature for 3 hours. The solvent is removed under reduced pressure and the residue is dissolved in chloroform. The organic phase is washed with 0.1 M HCl and water, dried over sodium sulphate, evaporated to dryness. This ester is pure enough for direct use.

Example 12 Formation of Carbonate Intermolecular Subunit Bonds The method for preparing cytidineyl- (3'-5') -cytidine carbonate is described in the following procedure. This is a common procedure for the formation of carbonate intermolecular subunits and the deprotection of oligocarbonates. Note that the group protecting the base must be 2- (4-nitrophenyl) ethoxycarbonyl, 2- (phenylsulfonyl) ethoxycarbonyl, or FMOC. tert-Butyldimethylsilyl chlorite is obtained from Aldrich.

12.1 Preparation of 2'-deoxynucleoside 3'-O-tert-butyldimethylsilyl derivative 3'-O-tert-butyldimethylsilyl-N-2- (4-nitrophenyl) preparation of ethoxycarbonyl-2'-deoxycytidine The method is shown below. This is a common procedure for the preparation of 2'-deoxynucleoside-3'-O-tert-butyldimethylsilyl derivatives.

In a solution of 5'-O-dimethoxytrityl-N-4- (4-nitrophenyl) -ethoxycarbonyl-2'-deoxycytidine (1 mmol) and imidazole (3.5 mmol) in 10 mL of dry dimethylformamide or tetrahydrofuran. A dry DMF (10 mL) solution of tert-butyldimethylsilyl chloride (3 mmol) is added dropwise with stirring. After stirring the reaction solution for 1 hour, the reaction is stopped with ice and extracted with methylene chloride. Rinse with water and dry over sodium sulphate. The organic layer is evaporated to dryness under reduced pressure and purified by silica gel chromatography using methylene chloride as an eluent.

To the methylene chloride (5 mmol) solution of the 5'-O-dimethoxytrityl derivative (1 mmol) obtained in the previous step, a methanol (15% v / v) -containing methylene chloride (10 mL, 1 M) solution of zinc bromide is added. The reaction mixture is stirred for 30 minutes, the reaction is stopped with ice and extracted with methylene chloride. The organic layer is washed with water, dried over sodium sulfate, and evaporated to dryness under reduced pressure. The product is purified by silica gel chromatography using a methanol gradient in methylene chloride (0-10%).

5'-O-dimethoxytrityl-N-2- (4-nitrophenyl) ethoxycarbonyl-2'-deoxycytidine-3'-O- (4-nitrophenyl) carbonate (1.5 mmol) and 3'-O-tert -Butyldimethylsilyl-N-4- (4-nitrophenyl) ethoxycarbonyl-2'-deoxycytidine (1 mmol) is dissolved in dimethylformamide (5 mL) and the solvent is removed under reduced pressure. This is repeated twice. The residue is re-dissolved in dimethylformamide (5 mL) and treated with N, N'-dimethylaminopyridine (0.5 mmol) or N-methylimidazole (1 mmol). The solvent is removed by evaporation under reduced pressure and the reaction is allowed to stand overnight at room temperature. Dissolve the residue in chloroform (20 mL), wash with aqueous HCl (2 mL, 0.1 M), water (2 mL), aqueous NaOH sodium solution (2 x 2 mL, 0.2 M), water (2 mL), and dry the organic layer over sodium sulfate. And evaporate. The residue is purified on silica gel using an isopropanol gradient in chloroform (0-30%). The isolated product is uniform on TLC (and 400 MHz NMR).

Residues, BLGaffney and RA Jones

Desilylation is performed with a 2M HF / 1M tert-butylammonium fluoride (TBAF) reagent according to the description in [Tetrahedron Lett (1982) 23: 2257]. The deprotected nucleoside obtained in the previous step is added to pyridine (TBAF 1 mmol) containing 2M HF / 1M tert-butylammonium fluoride. After stirring for 24 hours, the reaction solution is partitioned between methylene chloride and an aqueous sodium bicarbonate solution. The organic layer is washed with water, dried over sodium sulfate, evaporated and dried. The residue is purified by silica chromatography using a methanol gradient in methylene chloride (5-25%).

The 3'-hydroxydinucleoside carbonate is converted to 3'-(4-nitrophenyl) -carbonate by the method of Example 11. These derivatives can react with the 5'-hydroxyl groups of nucleosides or oligonucleoside carbonates to prepare higher-grade oligonucleoside carbonates.

Example 13 Formation of Carbamate Intermolecular Subunit Bonds Bis (p-nitrophenyl) carbonate, p-anisoil diphenylmethyl chloride, N, N-dimethylaniline (DMA), and triethylamine are obtained from Aldrich Chemical Co.

The desired 5'-amino 2', 5'-dideoxyribonucleoside (using an acetyl or benzoyl group for base protection as needed) prepared in Example 3 above is p in pyridine. -Treat with anisoyl diphenylmethyl chloride (1.2 mmol). After 12 hours, the solvent is evaporated and the residue is dissolved again in chloroform. This solution is LVDS₃Wash once with aqueous solution and water, and Na₂SO₄To dry. The solvent is evaporated and the residue is subjected to silica gel chromatography to elute with a chloroform / methanol / 1% triethylamine mixture.

Evaporate 5'-tritylated aminonucleoside (1 mmol) twice from DMF. DMF is then used as the solvent and treated with bis (p-nitrophenyl) carbonate (2 mmol) in the presence of a catalytic amount of triethylamine (or N, N-diethylaminopyridine, or N-methylimidazole). After 3 hours, the solvent is evaporated and the residue is dissolved in chloroform. This solution was washed twice with 0.01N aqueous sodium hydroxide solution, once with water, and then Na.₂SO₄To dry. This solvent is removed with a rotary evaporator. The residue is subjected to silica gel chromatography, first eluted with a chloroform / 0.1% DMA mixture, then eluted with a chloroform / methanol / 0.1% DMA solvent system. Collect appropriate fractions, evaporate and dry. Dissolve the residue in the minimum amount of THF and add this solution to the excess amount of hexane. Precipitated activated nucleosides are filtered and collected and dried under vacuum.

The target 5'-aminonucleoside (1.1 mmol) is evaporated twice with DMF as in Example 3 above. Activated nucleosides (1 mmol) are added to the reactor and the solids are dissolved in DMF. Concentrate the solution in small volumes and leave overnight. The solvent is completely removed under vacuum and the residue is dissolved in chloroform. The solution is washed twice with 0.01N aqueous sodium hydroxide solution, once with water and then dried over solid sodium sulfate. The solvent is removed on a rotary evaporator and the residue is chromatographed on silica gel and eluted with a suitable methanol / chloroform / 1% triethylamine solvent system. Combine the fractions containing the dimer nucleoside and evaporate and dry. Dissolve the residue in the minimum amount of THF and add this solution to hexane. Collect the precipitate and dry it under vacuum.

Example 14 Formation of Bonds Between Thiocarbamate subunits N, N-dimethylaminopyridine (DMAP) is purchased from Aldrich Chemical Co.

The formation of the subunit-linked thiocarbamate of the 5'-aminonucleoside prepared in Example 3 is carried out in the same manner as described in Example 13 above. The differences in the operation method are as follows. The 5'-aminonucleoside used was a 2- (phenylsulfonyl) ethoxycarbonyl (for deoxycytidine and deoxyadenosine) or 9-FMOC (for guanosine and 2,6-diamino-2'-dioxyribomid) groups as a base-protecting moiety. Has. Methods for preparing these molecules are described above (Example 3). Activation of 5'-tritylaminonucleoside (1 mmol) is carried out in DMF in the presence of triethylamine (2 mmol) and DMAP (catalytic amount) p-nitrophenylchlorothioformate (Monatsber Deut Akad Wiss Berlin (1964) 6: 897 G (Prepared by the method of Hilgetag and r. Phillippson). In the coupling step, this activated monomer (1 mmol) and the desired 5'-aminonucleoside (1.1 mmol) are used. DMAP or N-methylimidazole is used as the catalyst, and DMF is used as the solvent. In the aqueous treated product in this step, washing with 0.01N sodium hydroxide aqueous solution is omitted and washing is performed with water.

Example 15 Formation of carbamate and thiocarbamate intermolecular subunit bonds between morpholine-type subunits N-methylimidazole is purchased from Aldrich Chemical Co.

The desired dry N'-protected, 5'-free alcohol morpholine-type nucleoside prepared in Example 5 (using acetyl or benzoyl groups for base protection as needed) under anhydrous conditions. Treat with bis- (p-nitrophenyl) carbonate and triethylamine (or DMAP or N-methylimidazole) (catalytic amount) in DMF. The solution is stirred for 3 hours, then evaporated to dryness. The residue is dissolved in chloroform, and the solution is washed twice with 0.01N aqueous sodium hydroxide solution and once with water, and then Na.₂SO₄To dry. The solvent is removed with a rotary evaporator, the residue is subjected to silica gel chromatography and eluted with a suitable chloroform / isopropanol / 0.1% DMA mixture. Fractions containing the desired activated nucleoside are combined and evaporated to dissolve the resulting solid in the minimum amount of THF. This THF solution is added to an excess amount of hexane, and the obtained precipitate is collected and dried.

After evaporating twice with DMF, the desired 5'-free hydroxy, N'-freemorpholinic nucleoside (using the same base protecting group as listed in this example) (1.1 mmol) was added. Treat with a DMF solution of 5'-(p-nitrophenoxycarbonyl) morpholine subunit (1 mmol). Reduce the volume of the reaction solution under vacuum to reduce the volume. If necessary, add N-methylimidazole or tolethylamine into the catalytic dose reaction vessel. The resulting solution is left at room temperature overnight. Evaporate the residual solvent and dissolve the residue in chloroform. The chloroform solution was washed twice with 0.01N aqueous sodium hydroxide solution, once with water, and Na.₂SO₄To dry. This solvent is removed with a rotary evaporator, the residue is subjected to silica gel chromatography, and eluted with a mixed solvent of chloroform / methanol. Combine the fractions containing the desired dimer and dry to evaporate. Dissolve the residue in the minimum amount of THF and add this solution to the excess amount of hexane. Collect the precipitate and dry it under vacuum.

The morpholine subunit bound by the thiocarbamate moiety is prepared in connection with this example above by modifying the procedure as follows. The protecting groups for the base are 1- (phenylsulfonyl) ethoxycarbonyl groups (for deoxycytidine and deoxyadenosine) or 9-FMOC groups (for deoxyguanosine and 2,6-diamino-2'-deoxyriboside). The preparation of these molecules is described above (Example 5). N-protected morpholine nucleosides (1 mmol) are active in DMF with p-nitrophenylchlorothioformate (1.2 mmol) in the presence of triethylamine (2 mmol) and DMAP or N-methylimidazole (catalytic amount). To be transformed. The active monomer (1 mmol) and the desired N'-freemorpholin nucleoside (1.1 mmol) are used in the coupling step. The solution is heated to 35-40 degrees with DMAP or N-methylimidazole as the catalyst and DMF as the solvent. For the water-based treated products in these steps, the method of washing with water is adopted instead of washing with 0.01N NaOH aqueous solution.

Example 16 Preparation of Ester Subunit Bonds N, N-dimethylaminopyridine and N-methylimidazole are obtained from Aldrich.

The binding of the two subunits is carried out by the reaction of a 5'-N, N-acetamide-3'-hydroxy derivative with a 3'-silylated-5'-activated ester. These are prepared in the same manner as in Example 6. The reagent (1 mmol each) is evaporated together with dimethylformamide several times, then dissolved in dimethylformamide (5 mL) and treated with N, N-dimethylaminopyridine (0.2 mmol) or N-methylimidazole (1 mmol). .. The solvent is removed under reduced pressure and the reaction is allowed to stand at room temperature for 2 hours. The residue is dissolved in chloroform, washed with dilute HCl, water, the organic layer is dried over sodium sulphate, and the solvent is evaporated under reduced pressure. The residue is purified by silica gel chromatography using a methanol gradient in chloroform.

The chain may be desilylated as in Example 6 and extended by treating the resulting 3'-free hydroxy compound as shown in the previous section with an activating ester.

Example 17 Formation of t-BOC amide bond dimer Fully protected t-BOC N-hydroxysucciimideyl ester or p-nitrophenyl ester (1 mmol) was prepared according to Examples 11.3 and 11.4 and dried dimethylformamide. Ester and dry together several times. The protected amino acid trifluoroacetic acid salt (1 mmol) prepared in Example 9 is treated in the same manner. The two components are separately dissolved in dry dimethylformamide (2 mL), mixed and treated with diisopropylethylamine (1.0 mmol). The solution is stirred at room temperature for 1 hour and then removed by evaporating the solvent under reduced pressure. This residue is dissolved in chloroform and washed with dilute hydrochloric acid. The organic layer is dried over sodium sulfate and evaporated to dryness under reduced pressure. The residue is purified by silica gel chromatography using a methanol gradient (15-50%) in chloroform. The t-BOC dimer acid can also be purified by C18 reverse phase chromatography using a water-methanol (trifluoroethanol) mixture.

17. Preparation of 1t-BOC Dimer Activated Ester The acid obtained in the previous step is converted to N-hydroxysucciimideyl ester or p-nitrophenyl ester using the general method of Examples 11.3 and 11.4.

17.2 Preparation of t-BOC dimer amino acid trifluoroacetate The t-BOC dimer acid is treated with trifluoroacetate according to the general procedure of Example 9.

17.3 Preparation of t-BOC tetramer acid The t-BOC dimer activated ester and the trifluoroacetic acid dimer acid are combined by a general procedure to obtain t-BOC tetramer acid. For purification, the most effective method is to use C18 reverse phase silica gel chromatography to elute a mixture of water and methanol (or trifluoroethanol) with a pH adjusted to 7.0 with triethylammonium acetate. In this way, chains of any length can be prepared by combining the activated ester with the free amine component.

Example 18 Geometric assembly of 8-subunit carbonate-bound polymer DEAE cellulose is purchased from Sigma Chemical Co. The prepared TLC plate is a product of EM Science and is purchased from VWR Scientific. HPLC equipment, columns and feeders are available from Beckman Instruments, Inc.

The desired carbamate-bound dimer (0.2 mmol) prepared by the method described in Example 12 is treated with 4 mL of a mixed solution of methanol / THF / glacial acetic acid (1: 1: 1) at room temperature for 12 hours. Remove the solvent under vacuum and dissolve the residue in the minimum amount of THF. This THF solution is added to a large volume of hexane, and the obtained precipitate is collected and dried. The acetate is dissolved in a THF / ethanol (4/1) mixture and DEAE cellulose (0.8 mmol base) is added to this vessel. After stirring for 20 minutes, the heterogeneous mixture is filtered and the filtrate is evaporated and dried. Dissolve the residue in a minimum amount of THF / ethanol (4: 1) and add this solution to hexane. Collect the solids and dry. Repeat the precipitation operation again.

Evaporate the desired N-5'-trityldimer (0.1 mmol) (prepared in Example 12) twice with DMF, then triethylamine (or DMAP or N-methylimidazole) (catalytic amount) with DMF as the solvent. ), Treated with bis (p-nitrophenyl) carbonate (2 mmol). After 3 hours, the solvent is evaporated and the residue is dissolved in chloroform. This solution was washed twice with 0.01N aqueous sodium hydroxide solution and once with water, then Na.₂SO₄To dry. The solvent is removed on a rotary evaporator and the residue is chromatographed on silica gel and eluted with chloroform / isopropanol or methanol / 0.1% DMA mixture. Combine appropriate fractions, evaporate and dry. The residue is dissolved in a minimum amount of THF and the solution is added to an excess amount of hexane. The precipitated activated dimer is filtered and collected and dried under vacuum.

The prepared 5'-aminodimane nucleoside (0.11 mmol) is evaporated twice with DMF. This activation dimer (0.1 mmol) is added to the reaction vessel to dissolve the solid in DMF. Concentrate the solution in small volumes and leave overnight. The solvent is completely removed under vacuum and the resulting residue is dissolved in chloroform. This solution is washed twice with 0.01N aqueous sodium hydroxide solution and once with water, then twice with aqueous sodium sulfate solution and once with water, and then dried over sodium sulfate. The solvent is removed on a rotary evaporator and the residue is placed on a prepared TLC plate and eluted with a suitable methanol / chloroform / 1% triethylamine solvent system. Elute the band containing the tetramer nucleoside and evaporate the solvent. Dissolve the residue in THF to a minimum amount and add this solution to hexane. The precipitate is collected and dried under vacuum.

Using the operation method mentioned in this example, the following conversion is performed. The desired N-5'-trityl etrama nucleoside (0.06 mmol) is treated with THF / methanol / glacial acetic acid (1/1/1) and purified to give the 5'-aminotetramanucleoside (0.05 mmol). Another N-5'-trityltetramanucleoside (0.05 mmol) is activated with bis (p-nitrophenyl) carbonate and then purified. The two tetramers are combined, processed and further purified by prepared TLC plate or HPLC chromatography. The substance obtained by chromatography is isolated as a solid, dissolved in the minimum amount of THF, and precipitated with hexane. The octamer nucleoside is collected and dried.

To use the octamer below, it is formed as follows. The N-5'-trityloctamanucleoside is treated with THF / methanol / glacial acetic acid (1/1/1) and purified under the conditions listed above. 5'-Aminooctamanucleoside (0.01 mmol) is treated with succinic anhydride (0.02 mmol) in pyridine for 2 hours at room temperature. The solvent is removed with a rotary evaporator and the residue is evaporated with ethanol several times. Dissolve the residue in THF / ethanol (3/1) and add to the hexane / benzene mixture (2/1). Collect and dry the solids. N-5'-succinylated octamanucleoside (0.01 mmol) is treated with pyridine / concentrated ammonia (1/1) (1 mL). After leaving overnight, the solvent is removed with a rotary evaporator and the residue is evaporated several times with ethanol. The crude solid is purified using reverse phase (RP-18) HPLC. Evaporate the eluate, add the residue to DMSO and precipitate with a hexane / benzene (1/1) solution. Collect solids and dry.

Example 19 Preparation of a cleavable linker bound to a carrier suitable for stepwise and stepwise / block assembly of polymers: carrier activation and linker binding Long chain alkylamine derivatization controlled pore glass (Cat. No. 24875) is obtained from Pierce Chemical Co. 2,2-sulfonyldiethanol and dicyclohexylcarbodiimide are obtained from Aldrich Chemical Co.

The amino group of the glass carrier (about 40 mmol per gram of carrier) is reacted with succinic anhydride substantially by the method of Matteucci and Caruthers (JAmer Chem Soc (1981) 103; 3185) to obtain a free carboxylic acid terminal. .. Dry 1/10 mol of 2,2-sulfonyl diethanol (60 wt% aqueous solution) as follows. First, it is mixed with 3 times the volume of dimethylformamide (DMF) and concentrated under vacuum in a warm water bath to make a thick syrup. Next, add the same volume of DMF and concentrate to make a thick syrup, and repeat this operation once more. Next, dry DMF is added to finally obtain 2M of sulfonyldiethanol. A mixed solution of 1 ml of sulfonyl diethanol and 0.2 g of dicyclohexylcarbodiimide is added to 1 g of a dry-controlled porous glass carrier having a carboxylic acid residue, and the slurry is shaken back and forth and left and right (without stirring) and mixed overnight at room temperature. Then, the glass carrier is thoroughly washed with methanol and dried.

Example 20 Preparation of 14-subunit carbonate-binding polymer for conserved sequences on AIDS virus chromosomes: stepwise assembly on solid carriers: addition of first subunit and extension of chain N-methyl Imidazole, 4- (N, N-dimethylamino) pyridine (DMAP), and 1,8-diazabicyclo

[5,4,0] Undec-7-en (DBU) is obtained from Aldrich Chemical Co. Methyl p-nitrophenyl carbonate is prepared from methyl chloroformate and p-nitrophenol.

The 2'-deoxycitidine subunit prepared in Example 4.2 (N4 is the p-nitrophenetoxycarbonyl moiety, 5'oxygen is the di (p-methoxy) trityl moiety (DMT), and 3'oxygen is the p-. A fully dried controlled porous glass carrier (1 mmol N-methylimidazole or 1 mmol) prepared in Example 19 in which (with a nitrophenoxycarbonyl moiety) (0.1 mmol) is dissolved in a minimum volume of dry tetrahydrofuran (THF) or dimethylformamide. Add any of 0.1 mmol DMAP) and shake the slurry back and forth and left and right (without stirring) to mix for 2 hours. The slurry is then filtered and the solids washed with THF.

Cap the unreacted hydroxyl groups by adding 2 ml of THF containing 1 M methyl p-nitrophenyl carbonate and 0.5 M DMAP and mixing at room temperature for 20 minutes. The glass carrier is then washed with THF and filtered.

The carrier is washed with dichloromethane to remove dimethoxytrityl at the 5'end and then treated with 5 ml of 0.2 M dichloroacetic acid dichloromethane solution at room temperature for 5 minutes. The glass carrier is then washed with dichloromethane and filtered.

Next, the subunits prepared in Example 1 and activated in Example 2 are added in the same manner and in the following order: G, A, T, A, A, C, A, T, T, T, T. , T, C, (G is protected by the FMOC subunit, A and C are protected by the p-nitrophenetoxycarbonyl moiety).

Example 21 Preparation of 19-subunit carbamate-binding polymer for conserved sequences on the AIDS virus chromosome. Stepwise assembly of oligomeric blocks on a solid support: addition of the first subunit and elongation of the chain.

The 5'-amino-2', 5'-dideoxycitidine subunit (0.2 mmol) prepared in Example 3 (where N4 has a benzoyl moiety and 5'-amine has a p-methoxytrityl moiety. , 3'Oxygen has a p-nitrophenoxycarbonyl moiety) is dissolved in a minimum amount of dry THF and added to 0.5 g of a dry controlled pore glass carrier with a cleavable linker prepared in Example 19. A catalyst (1 mmol of N-methylimidazole or 0.1 mmol of DMAP) is added, and the slurry is shaken back and forth and left and right for 2 hours to mix. The slurry is then filtered and the solids are thoroughly washed with THF.

The mono-p-methoxytrityl is washed with dichloromethane to remove it, followed by treatment with 5 ml of a solution of 0.2 M dichloroacetic acid in dichloromethane at room temperature for 1 minute. The carrier is then washed with dichloromethane and filtered. Easily wash with 3 ml of THF containing 1% volume of diisopropylethylamine to convert the 5'-amino terminus to a free amine. This is washed with THF and filtered.

3'-p-nitrophenoxycarbonyl-activated dimer with 5'-AG-3'sequence (prepared in Example 13) 0.1 mmol (guanine N2 is protected by an acetyl moiety, adenine N6 is a benzoyl Alternatively, it is dissolved in a minimum amount of dry THF (protected by a p-nitrobenzoyl moiety), added to the glass carrier and mixed for 2 hours (no catalyst is added to this reaction and the next coupling step). The slurry is then washed with THF and filtered.

Cap the unreacted amine moiety by adding 2 ml of THF containing 2M of p-nitrophenylacetic acid and mixing at room temperature for 20 minutes. Then, the glass carrier is washed with THF and filtered.

The 5'-terminal mono-methoxytrityl is removed with dichloroacetic acid as described above.

The activated dimer subunit prepared in Example 17 (where the extracellular nitrogen of cytosine is protected by a benzoyl moiety and the extracellular nitrogen of A is protected by a benzoyl or p-nitrobenzoyl moiety). In the same way, add in the following order: A-T, C-A, T-A, T-T, T-T, AC, AC, C-A (5'to 3). ).

Subunits with extracyclic basic nitrogen protecting groups that can be removed by strong non-nucleophilic bases (eg, p-nitrophenetoxycarbonyl or phenylsulfonylethoxycarbonyl for A and C and FMOC for G) are also treated as described above. It can be used by method to assemble a polymer. However, polymers with one of these protecting groups are usually deprotected by treating with DBU rather than with ammonium hydroxide, usually with polymers with either of these protecting groups. ..

Example 22 Preparation of 19-subunit amide-binding polymer for conserved sequences on the AIDS virus chromosome. Stepwise assembly of oligomeric blocks on solid carriers: addition of first subunit and extension of chains Cytosine-containing acyclic backbone subunits prepared in the same manner as in Example 11.3 (where cytosine N4 is 2 (p-nitro). It has a phenyl) ethoxycarbonyl moiety, the amine of the backbone has a t-butoxycarbonyl moiety, and the carboxyl is in the form of a p-nitrophenyl ester) is dissolved in a minimum volume of dry THF. This is added to 0.5 g of a dry controlled pore glass carrier prepared in Example 19 and having a cleavable linker. A catalyst (1 mmol N-methylimidazole or 0.1 mmol DMAP) is added, the carrier is mixed for 2 hours, filtered and the solids are thoroughly washed with THF.

The carrier is washed with dichloromethane, followed by treatment with 5 ml of methylene chloride containing 20% trifluoroacetic acid at room temperature for 30 minutes to remove t-BOC. The carrier is washed with dichloromethane and filtered. The amino terminus attached to the carrier is simply washed with 3 ml of THF containing 1% by volume diisopropylethylamine to convert to a free amine, which is further washed with THF.

Activated dimer subunit having the sequence of (amino-terminal) -GA (carboxy-terminal) prepared in Example 17 (where N2 of guanine has an FMOC moiety and N6 of adenine is p-nitro. It has a phenetoxycarbonyl moiety, the amino terminus of the back-bon has a t-BOC moiety, and the carboxy terminus is in the form of a p-nitrophenyl ester) 0.1 mmol is dissolved in a minimum volume of dry THF. In addition to the glass carrier, mixing is carried out for 2 hours (no catalyst is added in this reaction and subsequent coupling steps). The carrier is washed with THF.

Add 2 ml of THF containing 2M p-nitrophenyl acetate and mix at room temperature for 20 minutes to cap any unreacted amine moiety. The glass carrier is then washed with THF.

As mentioned above, the terminal t-BOC moiety is removed with TFA and the carrier is neutralized.

Subsequently, the dimer subunits prepared in Example 17 are added in the following order: (C-terminal) TA, AC, AT, T-T, T-T, CA, C-A, AC (N-terminal).

Extracyclic nitrogen protecting group

The protecting group can also be removed by a suitable nucleophile (eg, benzoyl for C, benzoyl or nitrobenzoyl for A, and acetyl or isobutyl for G]. However, polymers with any of these protecting groups are usually deprotected by treatment with ammonium hydroxide rather than with DBU.

Example 23 Preparation of nucleoside 3'-O- (p-chlorophenyl-2-cyanoethyl) -phosphoric acid Preparation of nucleoside with protecting group of 23.13'thymidine, N-benzoyldeoxyadenosine, N-benzoyldeoxycytidine, N- Isobutyryldeoxyguanosine and their 5'-O- (di-p-methoxytrityl) 5'-ODMT-nucleoside) derivatives are obtained from Pharmacia PL biochemicals (Piscataway, NJ). Beta-benzoylpropionic acid and dicyclohexylcarbodiimide (DCCD) are obtained from distributors.

The selected 5'-O-dimethoxytrityl nucleoside (1 mmol) is reacted with β-benzoylpropionic acid (3 mmol) and DCCD (4 mmol) in 6 ml of pyridine. The reaction mixture is stirred at 25 ° C. for 3 hours, then 1.5 ml of water is added and the mixture is stirred at 25 ° C. for 5 hours. The reaction mixture is filtered, the filtrate is evaporated, and then pyridine is removed from the residue by several co-evaporation with ethanol. The purified residue is taken up in ethyl acetate and chromatographed on silica gel with ethyl acetate. The concentrated ethyl acetate eluate is treated with hexane to precipitate the resulting 3'-O-β-benzoylpropionyl-nucleoside (3'-OβB-nucleoside).

23.2 Activation of nucleosides P-chlorophenyl phosphorodichlorate is obtained from Aldrich (Milwaukee, WI). Benzenesulfonic acid, tetrahydrofuran, triethylamine, and 3-hydroxypropanenitrile are obtained from distributors.

A solution of p-chlorophenylphosphologitriazolide is prepared by a continuous reaction of 3 mmol of triazole in 10 ml of tetrahydrofuran with 3 mmol of triethylamine and 1.5 mmol of phosphorodichlorate p-chlorophenyl. After a 10 minute reaction at 25 ° C., 1 mmol of the selected 5'-ODMT nucleoside in 2 ml of dry pyridine is added to the reaction mixture. After standing at 25 ° C. for a maximum of 2 hours, the mixture is treated with a solution of benzenesulfonic acid (4 mmol) and triethylamine (4 mmol) in 3 ml anhydrous pyridine, followed by the addition of 3-hydroxypropanenitrile (2 mmol). The reaction mixture is concentrated to 4 ml under vacuum and left at 25 ° C. for up to 2 hours and then at 4 ° C. for up to 24 hours. The reaction is terminated by pouring the mixture into 20 ml 5% aqueous sodium bicarbonate solution at 0 ° C., extracting completely with chloroform, drying the extract with sodium sulfate and removing the solvent under reduced pressure. The 5'-ODMT nucleoside 3'-O- (p-chlorophenyl- (2-cyanoethyl))-phosphate product is obtained by chromatography on silica gel with continuous elution of ether, ethyl acetate, and tetrahydrofuran.

Example 24 Tetramer Preparation with Alternating Loaded and Unloaded Bonds Methyl phosphonodichlorate is obtained from Aldrich.

Benzenesulfonyl tetrazolides are prepared according to Stawinski et al, Nucleic Acids Research (1977) 4: 353.

24.1 Synthesis of Dimer By reacting 1,2,4-triazole (3 mmol) and triethylamine (3 mmol) in 20 ml of anhydrous tetrahydrofuran at 25 ° C for 2 to 10 hours in the presence of methyl phosphonodichlorate (1.2 mmol). , Prepare a methylphosphonoditriazolide solution. Following filtration, a 10 ml dry pyridine solution of the selected 5'-ODMT nucleoside (1 mmol) is added to the filtrate, the mixture is concentrated to 8 ml and left at 25 ° C. for up to 2 hours. To the solution is added benzenesulfonyl tetrazolide (1.2 mmol) and the selected 3'-OβB nucleoside. After keeping warm at 25 ° C for 2.5 hours, stop the reaction by adding 20 ml of 50% sodium bicarbonate at 78 ° C, and completely extract with chloroform. The solvent is dried over sodium sulfate and evaporated. The reaction may also start with the 3'-O-PO- (OpCP) (OCE) -protected nucleoside phosphate from Example II and proceed substantially as described above.

24.2 Separation of Methyl Dimer of Methyl Phosphonate Acetate: Using a tetrahydrofuran mixed solution (0 to 100% tetrahydrofuran) or a chloroform / methanol mixed solution (0 to 20% methanol), use a large glass column packed with silica gel 60. The steric isomer dimer produced in the previous section is separated by silica gel column chromatography performed at atmospheric pressure. The faster moving stereoisomer fractions are combined and precipitated from the tetrahydrofuran solution by adding hexane. The slower moving steric isomers are no longer used.

24.3 Phosphodiester / Methyl Phosphonate-Linked Tetramer First and second sterically specific methyl phosphonate dimers with selected dimer bonds and 3'OβD and 5'ODMT protecting groups, respectively. Is prepared as in Examples 24.1 and 24.2, respectively. Mesitylene sulfonyl tetrazolide is prepared according to Stawinski, et al, Nuc Acids Res (1977) 4: 353.

The first dimer of the 5'ODMT protecting group is removed to obtain a pure 5'hydroxy compound. The solvent is evaporated by removing the cyanoethyl group of the second dimer by treating the 1 mmol dimer with pyridine solution (8.5 ml), water (2.9 ml) and triethylamine (2.9 ml) at 25 ° C. for 1 hour. To obtain 3'hydroxynucleotides.

Dissolve 5'-OH dimer (1 mmol) and 3'-hydroxynucleotide dimer (1 mmol) in 8 ml of pyridine, add mesitylene sulfonyl tetrazolide (3-6 mmol) and treat at 25 ° C. for 3.5 hours. By doing so, a dimer condensation reaction is carried out. The reaction mixture is added to 4 ml of cold 50% pyridine aqueous solution and poured into 80 ml of sodium bicarbonate aqueous solution. The reaction product is completely extracted with chloroform, dried over sodium sulfate and evaporated with an organic solvent, and then purified with silica gel using a methanol / chloroform mixture. The eluted product is precipitated with hexane. Tetramers with 3'-O-P0- (OpCP) (OCE) protecting groups start with a dimer with 3'-O-P0- (OpCP) (OCE) groups in a substantially similar manner. May be prepared by.

Example 25 Adhesion of Polymer to Solid Support by Linker Arm 25.1 Preparation of Bifunctional Hexane Linker Arm Dowex 50X pyridinium resin is obtained from Bio-Rad (Richmond, CA) and 6-aminohexanol, dimethylformamide and 3-. Hydroxypropanenitrile is obtained from the distributor. 6- (2-Methylsulfonyl) -ethyl p-nitrophenyl carbonate is prepared according to Eberle, et al, Helvetica Chimica Acta (1975) 58: 2106.

Bifunctional hexanelinker arm 6- (2-methylsulfonyl-ethoxycarbonylamino) -hexanol is 6-aminohexanol (1 mmol) in 1 ml of dimethylformamide 2- (methylsulfonyl) ethyl p-nitrophenyl carbonate Prepare by reacting with (1 mmol) at 25 ° C for up to 4 hours. The reaction mixture is poured into water and completely extracted with benzene, the benzene is washed with water, the organic solvent is then dried over sodium sulfate and evaporated to give the carbamate alcohol. This is purified by silica gel chromatography using a methanol / chloroform solvent mixture.

Preparation of oligonucleotide analogs with a linker arm of 25.25'-O- (6-aminohexyl) Triethylammonium bicarbonate and tetrabutylammonium fluoride are available from distributors. Fully protected oligonucleotide analogs are prepared by the methyl phosphonate condensation scheme described in Example VI. Dissolve the oligonucleotide (1 mmol) in 10 ml of chloroform: methanol (7: 3, v / v) containing 2% benzenesulfonic acid and leave at 0 ° C. for about 40 minutes. The reaction mixture is washed with 5% sodium bicarbonate solution and then with water. The chloroform layer is dried over sodium sulphate, filtered and evaporated under reduced pressure. The substance is dissolved in chloroform, and the residue is chromatographed on silica gel using a mixed solution of chloroform / methanol to obtain a 5'-hydroxy oligonucleotide compound.

A solution of 6- (2-methylsulfonyl) -ethoxy-carbonylamino) -hexylmethylphosphonyltriazolide (5 mmol) in pyridine (10 ml) is described in Example 24.1. It is prepared by treatment with the methylphosphonoditriazolide thus prepared. To this solution, add the 5'hydroxy compound (1 mmol) from the previous section and benzenesulfonyl tetrazolide (4 mmol). Continue the reaction and finish as in Example 24.1.

The residue is chromatographed on silica gel with a chloroform / methanol solvent mixture. The protected oligonucleotide (1 mmol) is treated in 14 ml of 20% acetic acid / pyridine with 4 ml of hydrazine hydrate at 25 ° C. for 16-24 hours. After evaporation, the residue is treated in 30 ml tetrahydrofuran / pyridine / water (8: 1: 1, v / v / v) with a solution containing 0.017 M tetrabutylammonium fluoride at 25 ° C. for 24 hours. The solution is then treated with 50% concentrated ammonium hydroxide in 60 ml of pyridine at 4 ° C. for 10 hours. The resulting oligomer is prepared on a reverse phase HPLC column using a water / acetonitrile mixture in 0.1 M ammonium acetate buffer, pH 5.8 as the chromatography solvent.

Attachment of 25.35'-O- (aminohexyl) -oligonucleotide analogs to homologous carriers Affigel 10 is obtained from Bio-Rad (Richmond, CA). The 10 ml filled gel is washed with isopropyl alcohol (3 bed volume) and then ice-cold deionized water (3 bed volume). 5'-O- (6-aminohexyl) -oligonucleotide (150 mmol) is prepared as described in Example 26. Oligonucleotide (150M) in 0.1M sodium bicarbonate buffer, pH 8.0, 5ml is slurried by adding a washed gel at 25 ° C. for 1 to 4 hours. The mixture is then treated with 1 ml of 1 M ethanolamine: hydrochloric acid buffer (pH 8.0) to cover any unreacted active ester on the gel. The gel is washed with water until all buffers have been removed and stored at 4 ° C. in the presence of 0.02% sodium azide.

Example 26 Preparation of Reagent Carrier Aminomethylated polystyrene is prepared as described by BAMukhitdinova, EEEighozin, and GA Makhmudova, Izv Akad Nauk Kaz SSR., Ser Khim (1980) 48. Dissuccinimide bicarbonate and 6-aminohexanol are obtained from Aldrich.

Aminomethylated polystyrene (1 g, 3.0 meq / g) is suspended in water / acetonitrile (10 ml, 3: 1 v / v), succinic anhydride (20 mmol) is added, and the mixture is treated at 4 ° C. The pH of the solution is kept at 6.0 by adding 20% NaOH. Keep the pH of the solution constant and continue the reaction for 24 hours. The beads are filtered, washed with 2N HCl, water (until the pH of the wash is neutral) and dehydrated by repeated washing with dioxane).

The carrier (with fixed acidic groups) from the previous section is reacted with dissuccinimide bicarbonate (20 mmol) in acetonitrile (10 ml) at 25 ° C. for 24 hours. The carrier is isolated by filtration and the carrier with immobilized succinimide ester is thoroughly washed with acetonitrile.

The carrier from the above is suspended in dimethylformamide (10 ml), 6-aminohexanol (20 mmol) is added, and the mixture is reacted at 25 ° C. for 24 hours. The carrier is isolated by filtration and the carrier with fixed alcohol chains is thoroughly washed with dimethylformamide.

Example 27 Procedure for Coupling Aminonucleosides to Carriers N-methylimidazole, tetrabutylammonium chloride, tetrabutylammonium fluoride, and imidazole are obtained from Aldrich. Bis (p-nitrophenyl) carbonate is obtained from Sigma (St. Louis, MO).

The supported alcohol (10 mmol) is reacted in dimethylformamide (30 ml) at 25 ° C. for 3 hours with the addition of a coupling reagent such as carbonyldiimidazole (50 mmol). The activated carrier is isolated by filtration and washed thoroughly with dimethylformamide. Thoroughly wash the carrier with 30 ml dimethylformamide. The carrier is resuspended in 30 ml of dimethylformamide and treated with the addition of the selected polycarbamate polymer (50 mmol) prepared as in Example 21. After 3 hours, the supporting alcohol is filtered and washed thoroughly with dimethylformamide.

The probe selected was specific for a target sequence containing residues 8441-8456 of the ORF-2 gene of ARV-2 and was an etiologic factor for AIDS (Sanchez-Pescador et al, Science (1985) 227). : 484) is synthesized by coupling to a gel with the following 5'-aminonucleotides (early introduction order): C, T, G, C, T, C, C, C, A, C, C, C , C, A, T, C, where C, G, and A are N- (β- (trimethylsilyl) ethoxycarbonyl) protected 5'-amino-2', 5'-dideoxycytidine, guanosine and adenosine, respectively. And T represents 5'-amino-2', 5'-dideoxythymidine.

Supported polycarbamate (10 mmol) is suspended in acetonitrile (30 mmol) and treated with tetrabutylammonium chloride (3 mmol per 1meq protecting group) and potassium fluoride dihydrate (4 mmol per 1meq protecting group), 50. Heat at ° C for 12 hours. At the end of this time, add water (30 ml) and wash the supporting probe thoroughly with water. The protecting group may be removed with tetrabutylammonium fluoride in tetrahydrofuran (3 mmol per 1meq protecting group) under similar conditions.

Example 28 Protection of amino groups as BOC derivative 4- (N-tert-butoxycarbonyl-N-methylamino) -butyl acid 4- (methylamino) -butyl acid is obtained from Aldrich. Di-tert-butyl dicarbonate is obtained from Pierce (Rockford, IL).

Dissolve 4- (methylamino) -butyl acid (10 mmol) in dioxane / water (30 ml, 2/1) and add 1N NaOH (10 ml). The solution is cooled to 0 ° C. and di-tert-butylcarbonate (11 mmol) is added with stirring. After 30 minutes at 25 ° C, remove dioxane under reduced pressure and add potassium hydrogensulfate to adjust the pH of the solution to pH-2.5. The aqueous phase is completely extracted with ethyl acetate, organic layers are added and dried over sodium sulfate. Removal of the solvent under reduced pressure gives the free acid, which is purified by recrystallization from chloroform / hexane or chromatography on silica gel with a mixed chloroform / methanol solution.

Example 29 General procedure for introduction of terminal dimethylamino group, activation of acid by conversion to imidazole, and general procedure for coupling with amine N, N'-carbonyldiimidazole is obtained from Aldrich.

A solution of a BOC-protected amino acid (or oligopeptide acid, 1 mmol) is treated with carbonyldiimidazole (1 mmol) at 20 ° C. for 6 hours. At this time, a DMF solution (1 ml) of dimethylamine (excess) or oligopeptide acid (1 mmol) prepared as in Example 30 is added at 20 ° C. and the reaction is then stirred at 25 ° C. for 12 hours. The solvent is removed under reduced pressure and the residue is chromatographed on silica gel with methanol / chloroform to give N, N-dimethyl4- (N-tert-butoxycarbonyl-N-methylamino) -butyramide.

Example 30 General Procedure for Removal of BOC Protecting Group for N, N-Dimethyl4- (Methylamino) -Butylamide Trifluoroacetic acid is obtained from Aldrich. Dissolve N, N-dimethyl4- (N-tert-butoxycarbonyl-N-methylamino) -butyramide (Example XV) in 5 ml of trifluoroacetic acid (BOC-protected oligopeptide, 1 mmol) at 25 ° C. Stir for 1 hour. Remove the solvent and divide the residue into 1N NaOH / saturated NaCl (1/10) and chloroform. The aqueous phase was completely extracted with chloroform, and the added organic layer was dried over sodium sulfate and evaporated under reduced pressure to obtain a free amine. It can be used directly in subsequent coupling reactions and can be purified on silica gel using a methanol / chloroform mixture containing 1% triethylamine.

Polyamides of various lengths may be prepared using the coupling procedure and the BOC unprotected sequence. For example, 4- (N-tert-butoxycarbonyl -N- methylamino) - butyryl imidazolinium de and N, N-dimethyl-4- (methylamino) - by reaction with butyramide is monoamide dimer produced. Removal of the BOC group and coupling with other imidazolide equivalents produces the trimer diamide, a process that is repeated until the desired length is obtained.

Instead, 4- (N-tert-butoxycarbonyl-N-methylamino) -butyrylimidazolide is reacted with 4- (methylamino) -butyl acid, and the resulting dimeric acid is N, Activate with N-carbonyldiimidazole and couple with free aminooligoamide.

Example 31 General Procedures for Reduction of Amide Bonds to Amines AG-50 and Dowex-50W are obtained from Bio-Rad (Richmond, CA) to remove oligomeric polyamides to be reduced to remove BOC groups. , Treat with trifluoroacetic acid as in the general procedure. After isolation and purification, free amine (1 mmol) in tetrahydrofuran (1 ml) is titrated into a solution of borane (2 mmol per amide residue) in tetrahydrofuran (3 ml) at 25 ° C. The colorless solution is refluxed for 1 hour, cooled to 0 ° C. and titrated with 6N HCl (1 ml). After all gas elution is complete, the solution is evaporated under reduced pressure to remove hydrochloric acid, the remaining aqueous solution is applied to AG-50 ion exchange resin, and the column is washed with water (10 ml) and sodium acetate (0.1-2.0 M). ) And salt (0.1-2.0M) in a pH 5 buffer to purify by eluting the polyamine. After collecting and evaporating the fraction containing the desired product, the residue was dissolved in 1N HCl (4 ml). , And put on the Dowex-50W ion exchange column. After washing with water and 2M HCl, the product is 6N It is recovered as hydrochloride by eluting with HCl and removing the solvent under reduced pressure.

The primary amino group of the polyamine is protected as a BOC-derivative and is exchanged for an ammonium salt with a methyl iodide as in Example 33.

Example 33 General procedure for incorporation of terminal primary amino groups into polyamines 4-Aminobutyl acid is replaced with a BOC-derivative. This is reacted with carbonyldiimidazole as in Example 28 and treated with the dimeric amide from Example 29. Cleavage and reduction of the BOC-group as in Example 31 yields polyamines.

The primary amino group of the polyamine is protected as a BOC-derivative as in the general procedure and is replaced with an ammonium salt using a methyl iodide as in Example 33.

Example 33 General conversion of polyamines to poly (tetraammonium) salt (3-aminopropyl-)-dimethyl (3- (5'-dimethylamino-1-naphthalene-sulfonylamino) -propyl) -ammonium chloride Procedure Methyl iodide and ethyl diisopropylamine are obtained from Aldrich.

Add 1NNaOH (1 ml) to a solution of bis (3-aminopropyl) methylamine (1 mmol) in dioxane / water (3 ml, 2/1). While stirring at 0 ° C., di-tert-butyl dicarbonate (1.1 mmol) is added, and the mixture is stirred at 25 ° C. for 30 minutes. The basic solution is thoroughly extracted with chloroform, and the combined organic phase is dried over sodium sulfate and evaporated. The product is dried by evaporation from anhydrous dimethylformamide (5 ml). The residue is dissolved in DMF and the solution is treated with methyl iodide (12 mmol) at 25 ° C. for 1 hour. The solvent is evaporated and the residue is dissolved in trifluoroacetic acid (5 ml). After 1 hour, the solvent is evaporated and the residue is dissolved in pyridine (5 ml). Ethyldiisopropylamine (2 mmol per 1 mmol of primary, secondary, and tertiary amine functional groups) is added to this solution, and the solution is mixed with 5-dimethylamino-1-naphthalene-sulfonyl at 0 ° C. A dansyl group is introduced by treating with chloride (0.9 mmol) (obtained as in Example 20) and then allowing the mixture to stand overnight at 4 ° C. The reaction product is purified by ion exchange chromatography and isolated as hydrochloride, as in the previous section.

Alternatively, the danicylation reporter may be treated directly with methyl iodide in dimethylformamide and purified by ion exchange chromatography as described above.

Example 34 General Procedure for Introducing Fluorofore 5-Dimethylamino-1-naphthalene-sulfonyl chloride is obtained from Aldrich.

At 0 ° C., 5-dimethylamino-1-naphthalene-sulfonyl chloride (0.85 mmol) is added to a solution of starting polyamine (1 mmol) in pyridine (5 ml) and the mixture is stirred at 4 ° C. overnight. Pyridine is then removed at 25 ° C. under reduced pressure, the residue is dissolved in 1N HCl (5 ml) and run on an AG-50W ion exchange column. After washing with water (10 ml), the product is eluted with a pH 5 buffer consisting of sodium acetate (0.1-2.0M) and salt (0.1-2.0M). After collecting and concentrating fractions containing the product, the residue is dissolved in water (10 ml), placed on a Dowex-50W (Bio-Rad) ion exchange column and washed with water (10 ml) and 0.5N HCl (5 ml). , And finally the product is eluted with 6N HCl. Hydrochloride of the product is obtained by removal of the solvent under reduced pressure.

Example 35 Preparation of Diagnostic Reagents for Detection of Herpes Simplex, Type I and II The 16-base sequence 5'-GCGGGGCTGCGTTCGG-3' from positions 462 to 477 of the 2gD gene of herpes simplex virus, type I and II was selected. Contains target sequences (Lasky & Downbenko, DNA (1984) 3:23). Complementary reagent polymers consisting of alternating methyl phosphate phosphodiester bonds are constructed substantially according to the methods of Example 23 and Example 24.

The polymer is replaced with a 5'-O- (6-aminohexyl) -oligonucleotide analog and is coupled to the Affi-Gel 10 solid support material via an aminohexyl spacer arm by conventional coupling methods.

The melting temperature of the polymer / analyte duplex is determined using synthetic oligonucleotides with the target sequence of 5'-GCGGGGCTGCGTTCGG-3'. This target sequence, which can be purchased or constructed by conventional methods, is mixed with a substantially equimolar amount of polymer in annealing buffer (10 mM EDTA, 100 mM sodium phosphate, pH 7.2). Before adhering to the solid carrier). The solution is heated to 90 ° C. and slowly cooled to room temperature for annealing. The temperature is then slowly increased and the absorbance is recorded as a function of temperature. The melting temperature (Tm) shall be the temperature at which the total absorbance change is half.

Example 36 Detection of Herpes Simplex Types I and II Analytical samples taken as skin wounds in the infected area were placed in 0.1 ml of EDTA-surfactant solution (10 mM EDTA, 1% w / v sodium dodecyl sulfate, pH 7.0). It is suspended, easily homogenized with a microtissue crusher (Kontes # K-885470), and the homogenate is centrifuged with a microfilter (VWR Scientific # 28151-807). Add 0.5 ml of 4.5M sodium trichloroacetate and add 0.6 ml ethanol within a few seconds. The formulation is placed in an ice bath for 30 minutes and then centrifuged at 10,000 g for 5 minutes. Carefully decant the supernatant and discard, and gently wash the tube with 80% aqueous ethanol. 0.05 ml annealing buffer (10 mM) of pelleted material (often invisible) Resuspend in EDTA, 100 mM sodium phosphate, pH 7.2) and add to the appropriate amount of diagnostic reagent from Example 21 (reagent polymer / analytical product molar ratio estimated to be greater than 100). Annealing is performed at a temperature 8 ° C. lower than the polymer / target duplex Tm (predetermined as described in Example 35) for 30 minutes, then the diagnostic reagents are washed 3 times with a 2 ml volume of annealing buffer. .. This cleaning is performed centrifugally with a microfilter for convenience. After washing, the diagnostic reagent is suspended in 0.2 ml of a reporter solution containing 1 mg of fluorescent diquaternary ammonium reporter (this synthesis is described in Example 34). The reporter solution contains only the appropriate concentration of NaCl, which is predetermined as described above. The diagnostic reagents are then washed 3 times in a 2 ml volume with a reporter-free binding solution. Finally, the reporter is eluted from the diagnostic reagent with 0.1 ml of 2M NaCl, and the eluate is assessed for fluorescence with a spectrophotometer. Fluorescence is drawn from the control sample that does not contain the analyte to obtain a quantitative measurement proportional to the analyte present in the initial sample.

Example 37 Preparation of Diagnostic Reagent for AIDS Virus Detection The 16 nucleotide sequence 5'-GATGGGGTGGGAGCAG-3'at position 8441-8456 of the ORF-2 gene of the AIDS-related retrovirus (ARV-2) contains the selected target sequence. (Sanchez-Pescador, et al, Science (1985) 227: 484). Complementary polymers with subunits attached to carbamate are substantially constructed by the methods detailed in Example 21. Simply put, the 2'-deoxyribonucleosides dA, dC and dG are protected as in Example 10. These protected nucleosides and thymidines are then converted to 5'-amino derivatives. The polymer carrier was then prepared as in Example 25 or Example 26, and the subunits were substantially attached to this carrier by the methods described in these Examples, and the carrier ... CTGCTCCCACCCCATC-3. A carrier-bound protected polymer with a sequence is obtained. In the final step, the protecting group of the base is removed to obtain the desired carbamate binding diagnostic reagent.

The melting temperature of the polymer / analyte is determined as follows.

RNA transcripts are prepared from single-stranded polynucleotides containing sequences complementary to the target sequence. This target-containing RNA is suspended in annealing buffer (10 mM EDTA, 100 mM sodium phosphate, pH 7.0) and added to the diagnostic reagents described above. Warm the mixture to 90 ° C. and slowly cool to room temperature for annealing. The diagnostic reagent is then placed on a small water jacket chromatography column through which the annealing buffer is slowly pumped. Slowly raise the temperature while monnuring the outflow of free RNA. Free temperature (Tr) is the temperature at which RNA is eluted from the column. This Tr value often differs from the corresponding Tm value determined by the dark color shift method described in Example 21 by 1 ° C to several ° C.

Example 38 Preparation of Enzymatic Reporter for Use in Systems with Uncharged Diagnostic Reagents Polycationic tails with a primary amine at one end were prepared as described in Examples 14-18. Its structure is as follows: H₂N (CH₂)₄ N (CH₂)₄ N (CH₃)₂ CH₃₃ 1 mmol of this product is reacted with excess 4-fluoro-3- in the dark at room temperature for 24 hours.

Under low light conditions, the solvent is removed under reduced pressure and the solid is milled with hexane to remove unreacted phenyl azide. The solid is then suspended in 10 ml of 0.1 M NaCl, and cacodylic acid is added to reduce the pH to 7.2. Next, 5 mg of alkaline phosphatase (Sigma Chem Co., # P5778) is added to 1 ml of the tail solution described above, and a high flow rate of 366 nm light is applied for 1 hour. The resulting reporter is dialyzed against a large volume of buffer (0.1 M NaCl, 0.05 M sodium cocadylate, pH 7.0) for 18 hours to remove cationic tails that do not bind to the enzyme. Bovine serum albumin (1% w / v) and sodium azide (0.02% w / v) are added to stabilize the enzymatic moiety of this tetracationic reporter. Store this reporter solution in the dark at 4 ° C.

Example 39 Detection of AIDS Virus Centrifuge 5 ml of blood suspected to contain AIDS virus and dilute 2 ml of cell-free serum into 8 ml of 4.5 M sodium trichloroacetate containing 0.1 mg polyadenyl acid (Sigma Chem Co., # P9403). Add. After a few seconds, add 10 ml of ethanol and cool the formulation in an ice bath for 30 minutes, then centrifuge at 10,000 g for 5 minutes. Carefully decant the supernatant and discard. The pelleted nucleic acid (often invisible) is washed with 80% aqueous ethanol, drained well and resuspended in 0.1 ml annealing buffer (10 mM EDTA, 100 mM sodium phosphate, pH 7.2). This is added to the appropriate amount of diagnostic reagent from Example 37. (The polymer / target molar ratio is estimated to be above 100). Annealing is performed at a temperature 7 ° C. lower than the polymer / target duplex Tr (predetermined as in Example 37) for 1 hour, then the diagnostic reagents are washed 3 times with a 2 ml volume of annealing buffer. This cleaning is performed centrifugally with a microfilter for convenience.

After washing, the diagnostic reagent is suspended in 0.2 ml of the prepared enzymatic-tetracationic reporter solution of Example 38. After 30 seconds, the diagnostic reagent is washed 3 times with a volume of 2 ml of binding solution without reporter. Then, the diagnostic reagent is suspended in a developing solution (15 mM p-nitrophenyl phosphate, 0.5 mM MgCl, 1.0 M diethanolamine, pH 9.8) and kept warm at 37 ° C. for 3 hours. The p-nitrophenol produced by the reporter is quantified with a spectrophotometer. The corresponding absorbance of the control sample lacking the analyte is subtracted to provide a quantitative measurement proportional to the analyte present in the initial sample.

Although the present invention has been described with respect to a particular embodiment, it will be appreciated that various changes and modifications can be made without departing from the present invention. For example, diagnostic reagents may include a wide variety of carrier-binding polymers, where various are designed to bind to different selected target sequences in polynucleotide chains from different analytes. Alternatively, the diagnostic reagent may include two carrier-bound polymers, each of which is designed to bind to different complementary strands of the duplex analyte. These large amounts of reagents provide the detectability of one or more analytes in a single diagnostic test and also offer the potential to double the detection sensitivity of a single duplex analyte.

Continuation of front page (56) References JP-A-60-36496 (JP, A) JP-A-59-206766 (JP, A) JP-A-61-227785 (JP, A) JP-A-60-93355 (JP, A) , A) Nucleic Aads Research, V.I. 12, No. 8 (1984) P.M. 3435 Jour. of Biologica l Chem. V. 255, No. 20 (1980) P.I. 9659