AU618414B2 - Phosphorothioate and normal oligodeoxynucleotides with 5'-linked acridine - Google Patents

Description

OPI DATE 18/04/90 APPLN. ID 43179 89 AOJP DATE PCr E 24/05/90 PCT NUMBER PCT/US89/03973 PUiSH Ell,1A E*a E N OOPERATION TREATY (PCT) 10 4 0OPERA INTERNATIONAL APPLICATION (31) International Patent Classification 4 (11) Interntional Publication 'mber: WO 90/03383 C07H 21/00, G N 33/48 Al (43) International Publication Date: 5 April 1990 (05.04.90) e, IS (21) International Application Number: PCT/US89/03973 Gaithersburg, MD 20878 (US).

(22) International Filing Date: 19 September 1989 (19.09.89) (74) Agents: STERN, Marvin, R. et al.; Fleit, Jacobson, Cohn, Price, Holman Stern, The Jenifer Building, 400 Seventh Street, Washington, DC 20004 (US).

Priority data: 246,688 20 September 1988 (20.09.88) US (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European pa- (71) Applicant: THE UNITED STATES OF AMERICA, as re- tent), FR (European patent), GB (European patent), IT presented by THE SECRETARY, U.S. DEPARTMENT (European patent), JP, LU (European patent), NL (Eu- OF COMMERCE [US/US]; 5285 Port Royal Road, ropean patent), SE (European patent).

Springfield, VA 22161 (US).

(72)Inventors: COHEN, Jack, S. 6045 Rossmore Drive, Be- Published thesda, MD 20814 MORI, Kenya 8200 Wisconsin With international search report.

Avenue, Bethesda, MD 20814 LOKE, Shee, Loong Flat B, 7th Floor, Block 18, Baguio Villa, Victoria Road ZHANG, Xianghong 4932 Battery Lane Bethesda, MD 20814 NECKERS, Leonard, M. 5121 Wissioning Road, Bethesda, MD 20816 STEIN, Cy, A. 14637 Settlers Landing Way, (54)Title: PHOSPHOROTHIOATE AND NORMAL OLIGODEOXYNUCLEOTIDES WITH 5'-LINKED ACRIDINE (57) Abstract The present invention relates to an automated synthesis method of 5'-acridine linked oligonucleotides using phosphoramidite-linked acridine. These compounds are useful for inhibiting gene expression, and enable the kinetics of cellular uptake to be determined using fluorescence cell sorting.

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Field of the Invention The present invention relates to an automated synthesis method of 5'-acridine linked oligonucleotides using phosphoramidite-linked acridine. These compounds are useful for inhibiting gene expression, and enable the kinetics of cellular uptake to be determined using fluorescence cell sorting.

Background of the Invention Oligodeoxynucleotides, which are complementary to certain gene messages or viral sequences, are referred to as "anti-sense" compounds. These compounds have also been reported to have inhibitory effects against Rous sarcoma virus and human immunodeficiency virus, also referred to as HIV.

In the past several years, the use of oligodeoxynucleotides as anti-mRNA reagents has undergone remarkable definition and expansion, as reported by Helene, in Guschlbauer, W. DNA-licand Interactions: From Drugs to Proteins, Plenum, 1986; Heikkila et al., Nature, 328: 445-449 (1987); and Stein et al., Cancer Research, 1988, in press. Matsukura et al., in Proc.

Natl. Acad. Sci., USA 84:7706-7710 (1987) observed the inhibitory effect of phosphorothioate oligodeoxynucleotides on the cytopathic effect of the HIV virus on ATH8 cells. An anti-message S-oligonucleotide (28-m34) directed at the 5'-region of the HIV art/trs gene was able to inhibit p24 production almost completely. Interestingly, a homopolymer, S-dC 2 was effective at viral inhibition at a concentration as low as 1 micromolar. Recent studies by Majumdar, which are still in progress, have shown that this molecule can bind WO 90/03383 PCT/US89/039 73 2 still in progress, have shown that this molecule can bind to viral reverse transcriptase at low and high affinity binding sites. The normal oligo species, both sequence specific and homopolymers, were ineffective.

One possible explanation for these observations may lie in the nuclease stability of the phosphorothioate diester bond, as described by Eckstein in Ann. Rev.

Biochem 54:367-402 (1985). However, actual comparisons of the rates of digestion of normal oligonucleotides versus the S-oligonucleotides have not been reported, nor have measurements of normal oligo-PS-oligo duplex thermal stability. The synthesis and some properties of the two stereoisomers (Sp and Rp) of an oligonucleotide with a single PS substitution have been reported by LaPlanche et al. in Nucl. Acids Res. 14:9081-9093 (1986), but the very widely used automated synthesis is not stereospecific.

Indeed, formidable obstacles remain before these compounds are truly useful on a clinical basis. For example, at current prices, 1.0 gram of a 28-mer S-oligo may cost in excess of $50,000. Theoretically, shorter J oligos would be less expensive. However, sequences much shorter than 15-mers lose specificity, and S-oligo/RNA hybrids may have melting temperatures below the assay temperature of 37 0 C. Helene and co-workers, Asseline et al., EMBO Journal 3:795-800 (1984); Asseline et al., Proc. Natl. Acad. Sci USA 81:3297-3301 (1984); Helene et al., Guschlbauer, op cit.; and Toulme et al., Proc. Natl.

Acad. Sci. USA 83:1227-1231 (1986) have defined a series of oligomers that contain an intercalating acridine moiety covalently linked at the 3' end and some linked at the 5' end. These workers noted a substantial increase in melting temperature for the shorter oligonucleotides (n<12) that they considered to arise from binding stabilization due to the acridine intercalation.

o WO 90/03383 P~/US89/03973 3 Helene et al., in European patents 0 169 787, 02 14 908, and 0 117 777, disclose modified oligonucleotides bonded to intercalation groups for cleavage of RNA and DNA, for prevention of replication or development of viruses, or for detection and purification of particular DNA and RNA sequences. These oligonucleotides are prepared by fusing an intercalation group by a covalent bond. The compounds are prepared by known methods, particularly by a phosphotriester synthesis. In the process disclosed, the chain of nucleotides is first prepared, the groups not entering into the reaction being protected during the reaction, after which the protecting groups are eliminated to obtain the final products. For example, a 3'-phosphodiester nucleotide is coupled with the hydroxyl derivative of the intercalating agent.

Unfortunately, this synthetic method is rather complicated, and is not easily adaptable to the standard automated synthesizer processes.

Other workers have attempted to incorporate fluorescent markers in oligonucleotides. However, these methods have not been suited to large-scale production of the products.

Sproat et al., PCT WO 87/07611, disclose a process for labelling oligonucleotides using fluorescent dyes.

The labelling is effected by converting the oligonucleotide into its 5'-[HS-(Y)z]-derivative, and the latter is reacted with a derivative of a fluorescent dye, which together with the 5-derivative forms an oligonucleotide of a fluorphore oligonucleotide.

Yamane et al., European patent 0 251 283, disclose a method for preparing a poly-labelled oligonucleotide derivative by using the sequence used for forming the basic skeleton of the compound and/or introduction of substituents thereon. One method includes synthesizing -Y WO 90/03383 PCrUS89/03973

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Ii 4 an aminoalkylated oligonucleotide, then introducing a polylysine into the compounds and labelling the polylysine with a labelling substance. Alternatively, the aminoalkylated oligonucleotide can be bonded with a polylysine previously labelled with a labelling substance.

Inoue et al., European patent 0 235 301, disclose a method for forming pyridopyrimidine oligonucleotides which are fluorescent and can form a base pair with guanidine or adenine. The compounds can be synthesized by standard methods. All of the compounds are said to be fluorescent and display the common characteristics of the natural pyrimidine bases.

Cohen et al., in Serial No. 07/159,017, filed February 22, 1988, disclose oligodeoxynucleotides which can be used for treating a variety of tumors and retroviruses. This application is incorporated herein by reference.

At present, a variety of approaches for polynucleotide synthesis are available. These approaches can be characterized based on several criteria. First, the synthesis is usually carried out either on a solidphase substrate or in solution. Solid-phase synthesis relies on sequential addition of mononucleotides to a growing chain attached at one end to the substrate. The solid phase allow easy separation of the reactants, but the method requires excess quantities of reactants and usually provides only small quantities, less than 1 mg, or the desired sequence. Solution phase synthesis, while it requires lesser amounts of the expensive reagents and can provide larger quantities of the product sequence, requires isolation and purification of the intermediate product after every addition. Virtually all automated polynucleotide systems rely on solid-phase synthesis.

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WO 90/03383 PCT/US89/03973 Reactor systems for producing oligomers or polymers of oligonucleotides or oligodeoxynucleotides and their derivatives have become convenient ways of synthesizing these compounds relatively inexpensively. The solidphase reactor systems currently in use employ either a tight bed column, a loose bed column, a batch reactor, or a tubular reactor. Such a system is disclosed in Urdea et al., U.S. Patent No. 4,483,964, which is hereby incorporated by reference.

Summary of the Invention It is an object of the present invention to overcome the aforementioned deficiencies in the prior art.

It is another object of the present invention to provide a synthesis method for a series of phosphorothioate oligodeoxynucleotide analogs.

It is a further object of the present invention to provide an automated method for making anti-message inhibitors of gene expression.

It is yet another object of the present invention to provide an automated method for making compounds for the treatment of HIV infection.

It is still another object of the present invention to provide a synthetic method for making an oligonucleotide or chemically modified analog thereof with a fluorescent label attached.

It is still a further object of the present invention to provide a synthesis of a phosphoramidite with a linked fluorescent acridine group and its addition in an automated DNA synthesizer to the 5' end of a synthetic oligodeoxynucleotide or analog thereof.

The present invention provides a phosphoramidite synthesis involving a simple, automated, high yield method for fluorescently tagging the 5' end of an oligodeoxynucleotide. The reaction scheme is shown in 3T WO 90/03383 PCY!US89/03973 6 Figure 1. The 5'-acridine linked molecules, both PO and PS, are readily purified by reverse phase chromatography.

Two major peaks are observed, each containing acridine, but it can be shown by 1H and 31 P NMR that the desired compound invariably elutes at longer retention time. The nature of the faster-eluting component is not clear at this time.

cells take up homo-oligodeoxynucleotides of thymidine as monitored by flow cytometric analysis.

Thus, it appears that there is an energy-dependent transport mechanism for the oligodeoxynucleotides. Thus, these compounds are useful as markers and inhibitors of gene expression.

Brief Description of the Drawings Figure 1 shows a scheme for the synthesis of linked acridine oligodeoxynucleotides using a linked phosphoramidite in an automatic synthesizer.

Figure 2 shows a proton NMR spectrum at 400 MHz and 0 C of the aromatic region of 5'-acridine-dT 7 Figure 3 shows the difference in melting temperatures between normal and phosphorothioate 14-m34 oligodeoxynucleotides with different GC content.

Figure 4 shows the effect of temperature on cellular uptake of free acridine and 5'-acridine-dT 2 cells.

Figure 5A and 5B show the cellular uptake of normal dT and S-dT oligodeoxynucleotides.

Detailed Description of the Invention The 5'-acridine linked oligodeoxynucleotides can be synthesized in an automated synthesizer using phosphoramidite acridine. These compounds are important, because certain phosphorothioate oligodeoxynucleotide analogs, unlike their normal congeners, have been found to exhibit significant anti-HIV activity, as reported by Matsukura et al, Proc. Natl. Acad. Sci. USA 84: 7706-7710 WO 90/03383 PCT/US89/03973 7 (1987).

The melting temperatures for acridine-thymidine base pairs of phosphorothioate oligodeoxynucleotides are significantly depressed relative to normal oligodeoxynucleotides, while GC-containing phosphorothioate oligodeoxynucleotides show much less depression in the melting temperature. The melting temperatures of S-dT oligomers with poly-rA are reduced relative to the duplexes with normal dA oligomers. These results provide a rational basis for the S-d(CG) sequences as anti-message inhibitors of gene expression.

During the automated synthesis of 5'-acridine linked oligothymidylates using phosphoramidite-linked acridine, it was noted that the 6-chloro substituent on acridine was replaced by thiophenol. Small increases of melting temperature were found for the 5-methylene linked acridine derivative as compared with the compounds with three and five methylene groups linked to normal and phosphorothioate dTn (with n=3-40) on duplex formation with the equivalent dAn. It was found that these fluorescently labelled oligodeoxynucleotides were taken up more rapidly than the longer compounds, and the normal oligodeoxynucleotides were taken up more rapidly than the S-oligodeoxynucleotides. This temperature dependence of the cellular uptake suggests an energy dependent process, and a possible membrane receptor for the oligodeoxynucleotides.

Preparation of N-(6-chloro-2-methoxy-acridinyl)-0methoxy-diisopropyl-aminophosphinyl 3-aminopropan(l)ol and The 3-aminopropanol, 5-aminopentanol, and 6,9dichloro-2-methoxy acridine were purchased from Aldrich Chemical Co. The 6-chloro-2-methoxy(hydroxyalkylamino)- 9-acridine was prepared via the method of Asseline et al.

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11 4 1* .~i in Proc. Natl. Acad. Sci. USA, oE. cit. The phosphoramidite was prepared by a modification of the procedure of Connally. 6-chloro-2-methoxy-9-(3hydroxypropyl)amino acridine (318 mg, 1 mmol) or the hydroxypentylamino derivative (346 mg, 1 mmol) or the hydroxypentylamino derivative (346 mg, 1 mmol) was dissolved in 2 ml of CH 2 Cl 2 N-ethyl-diisopropylamine (380 microL, 2 mmol) was then added. N,N-diisopropylmethyl-phosphonamidic chloride (194 microL, 1 mmol) was added over a period of about five minutes. After a further thirty minutes, thin layer chromatography on silica gel (hexane:ethyl acetate:triethylamine, 10:10:1) showed complete reaction (Rf of the starting material 0.1, Rf of the product 0.8, m=3; Rf 0.75, m=5. Five ml 15 of CH 2 Cl 2 was added, and the mixture was extracted with 2 x 5 ml portions of 5% NaHCO 3 and 5 ml of saturated NaCl.

The organic phase was dried over sodium sulfate and evaporated to a yellow oil. This was dissolved in a small volume of 9:1 hexane/trimethylamine and purified chromatographically over a column of silica gel (10 x 2cm) using this solvent mixture. The product eluted as a single spot in the above thin layer chromatographic system, and had the correct proton NMR spectrum. In addition, the 3 1P NMR spectrum showed a single peak, 25 147.331 and 147.171 Yields of about were typical.

Preparation of oligodeoxyribonucleotides 0-cyanoethyl phosphoramidite was obtained from Glen Research (Herndon, VA). All of the normal oligodeoxynucleotides were synthesized on the Applied Biosystems 380B DNA Synthesizer, and were purified by HPLC reverse phase chromatography (PRP-1) column. An extra round of synthesis was carried out using a 100 mM solution of the acridinyl phosphoramidites L- r WO 90/03383 PCT/US89/03973 9 in acetonitrile. Following this final coupling, the methyl phosphate protecting groups were removed with thiophenol (total thirty minute exposure), and cleavage from the resin was effected with concentrated aqueous ammonia. The automated synthesis of the phosphorothioate oligodeoxynucleotides was accomplished by modification of the method of Stec., as reported by Stein et al., Nucl.

Acids Research, 1988, in press.

Briefly, the standard iodine oxidation is substituted by a sulfurization step by using a 10% solution of elemental sulfur in CS 2 /pyridine/triethylamine (45:45:10).

Before and after the oxidation step, the column is washed repeatedly with a 1:1 solution of carbon disulfide and pyridine to remove any residual sulfur. The phosphorothioate oligodeoxynucleotides were purified by reverse phase HPLC as above, although the percent organic phase (acetonitrile) is higher. Samples were detritylated at room temperature in 3% acetic acid, extracted with ethyl acetate, and lyophilized.

Melting Temperatures Poly-rA and poly-rI were obtained from Pharmacia.

All optical measurements were made on a Shimadzu-UV-160 recording spectrophotometer coupled to a CPS Controller thermostat. Values of absorbance were recorded at 260 nm in 10 mM sodium cacodylate/140 mM NaCl buffer, pH All duplexes were formed in 1:1 mixtures of a strand with its complement. All of the samples were pre-melted at 75-98 0 C to destroy secondary structures, and then were allowed to equilibrate thermally. Each melt curve is composed of a minimum of twenty individual temperature points.

NMR Measurements NMR spectra were recorded on a Varian XL-400 spectrometer at 400 MHz for H and 162 MHz for 3P at 220C.

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WO 90/03383 PCT/US89/03973 Chemical shifts were measured with respect to internal TMS and external TMP, respectively. The recycle times were 2-3 seconds and the number of scans was 64-200 for 1H and up to 3000 for 31p. Integration was performed using the Varian program.

Enzyme Kinetics S1 nuclease and P1 nuclease were obtained from BRL.

Bovine spleen phosphodiesterase and Fnake venom phosphodiesterase were obtained from Pharmacia. All of the reactions were run in a total volume of one ml at 37 0 C. The value of absorbance was measured at Aax. Sl nuclease (1000 microns/ml) was diluted 1:10 in reaction buffer, which consisted of 30 mM sodium acetate (pH 50 mM NaC1, 1 mM zinc acetate, and 5% (v/v) glycerol. The final concentration of enzyme was 100u/ml. P1 nuclease (40 u/ml) was diluted 1:10 in reaction buffer, which consisted of 50 mM sodium acetate (pH 5.3).

The final enzyme concentration was 4 u/ml. Bovine spleen phosphodiesterase was dissolved in water (0.04 u/AL), and added (1 u/ml) to a solution containing 125 mM succinate- HC1, pH 6.5. Snake venom phosphodiesterase (46 u/mg solid) was dissolved in 500 pL water. One gL of this solution was added to the reaction mixture, which contained 100 mM Tris-HCl, pH 8.9, 100 mM NaC1, and 14 mM MgC 12 Data Analysis All data was analyzed using the MLAB program on the DEC PDP10 computer of the NIH Computer Center. A simple exponential was fitted to the nuclease digestion data (absorption vs. time), and a sigmoidal curve of the form.

OD(T) K) Eb/(l-K) where, OD(T) is optical density at any temperature T, ea and eb are the maximum and minimum values of the absorption, K exp[ H(T-Tm)RTm 2 and H is the van't I: i u I~ WO 90/03383 PC'/US89/03973 11 Hoff enthalpy, was fitted to the melting curves (normalized absorption vs. temperature).

Flow Cytometric Analysis of Cellular Accumulation of Acridine-labelled Oligodeoxynucleotides Oligodeoxynucleotides of various lengths with acridine (dT 7 dT 1 2 dT 15 and dT 2 0 were incubated with cells at a final concentration of 0.2-0.5 micromolar in RPMI 1640 medium containing 10% fetal calf serum and antibiotics. At the times indicated, 100,000 cells were removed from the culture, washed three times in phosphate buffered saline, and analyzed by flow cytometry using a Becton Dickinson Facstar instrument. Intracellular acridine was excited by the 488 nm line of an argon laser set at 300 mW, and the resultant emitted fluorescence of individual cells was recorded. Since the log amplified fluorescence of the populations was unimodal, the data are expressed as the median fluorescence of a population calculated using Consort 30 software supplied with the Facstar.

Characterization of Acridine Containing Oligomers The reaction scheme used in the synthesis of the Sacridine-linked oligodeoxynucleotides is shown in Figure 1. The products were characterized by HPLC, UV H, and 3P NMR spectroscopy. All products gave a single major peak in 31p NMR spectrum. The UV and 1 H NMR spectra were inconsistent with a simple acridine linked compound.

SThe peak observed does not correspond to the 6-chloro compound reported by Asseline et al., o2. cit., 1984a.

The relative areas of the aromatic proton resonances 30 consistently indicated an extra aromatic moiety present in the product, as shown in Figure 2. Treatment of the acridine precursor, with no nucleotide attached, with thiophenol under the conditions in the synthesizer gave a product that also contained an extra aromatic group.

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WO 90/03383 PC/US89/03973 This was therefore considered to be a replacement reaction of thiophenol for the 6-chloro substituent on acridine, cf. Figure 1. Notwithstanding this thiophenol substitution, the products obtained were found suitable for use in the melting studies and the cellular uptake studies described below.

Melting Temperatures Melting temperatures of duplexes of oligo-dT of various lengths (n=12,15,20), both all-PS and normal, with poly-rA were determined, as shown in Table I. The melting temperatures of duplexes of poly-rI with oligodC (15 and 28-mers) as well as 5-methyl-oligo-dC (28mers), both PS and normal, were also determined.

Table I Melting Temperature of 5' Acridine Oligo-dT with Poly-rA Li PO-Oligo m Tm AH Tm O-dT12 32 42 O-dT12-Acr 3 37 37 5 39 43 7 O-dT15 39 54 3 43 41 4 45 50 6 45 65 O-dT20-Acr 3 49 60 4 52 70 7 As shown in Table II, temperature values about corresponding PO complexes.

PS-Oligo m Tm AH Tm S-dT12 <12 S-dT12-Acr 3 <12 5 <12 S-dT15 22 38 S-dT15-Acr 3 20 40 -2 5 22 46 0 S-dT20 29 49 S-dT20-Acr 3 30 24 -1 5 30 29 -1 the S-oligos have melting 10-12 0 C lower than the Surprisingly, the melting temperature for 5-methyl S-dC 28 42 0 C, AH 80, is about equal to that of the non-methylated normal congener. A comparison of the melting temperatures for all-PS and normal 15-mers with their complementary oligos as a function of GC content showed a minimum difference at WO 90/03383 PCT/US89/03973 13 about 50% content, as shown in Figure 3.

Table II Melting Temperatures of Oligo-dC with Poly-rl PO-Oliqo Tm AH PS-Olico Tm AH Tm O-dC15 29 83 S-dC15 20 85 9 0-dC28 41 107 S-dC28 31 92 S-5Me-dC28 42 Melting temperatures of duplexes of poly-rA and oligo-dT of various lengths containing the 5' linked acridine derivative (normal and PS) were compared to unmodified phophorothioate oligodeoxynucleotides of identical length, as shown in Table I. In each case, following the precedent of Asseline et al (op. cit., 1984b), the link between the intercalator and the oligo contained either three or five methylene groups. The melting temperatures for poly-rA duplexes with S-oligos with n<12 could not be obtained because of their very low melting temperatures. In the cases studies, dT 12 dT 15 and dT 20 there was, on the average, a 40 increment in melting temperature with a 5' linked modified acridine with m=3, and a 70 increment for m=5. Values of delta H were not changed appreciably from those with normal oligos. However, for the all-PS-oligos, (dT 15 dT 20 essentially no change in melting temperature was observed for either m=3 or 5 as compared to the unmodified PS oligo. For the S-dT poly-rA duplex, a marked decrease in AH was seen (49 kcal/mol to 29 kcal/mol).

Nuclease Susceptibilities 5 normal, m=3, was studied with regard to DNase sensitivity, as reported in Table III. DNases employed were the predominantly endonuclease Sl, the exoand endonuclease P1, snake venom phosphodiesterase (SVP), and bovine spleen phosphodiesterase (BSP), which required a free 5'-OH group. The nuclease digestions proceeded WO 90/03383 PCT/US89/03973 14 with virtually identical rates for the unmodified and acridine linked oligos (S1, P1, SVP), but was about fold slower for the acridine-oligo when BSP was used (tl/2=855 seconds vs. 1.95 x 104 seconds for Table III Nuclease Digestion of Oligodeoxynucleotides (t 1/2 sec) Olicomer DNaseS1 SNaseP1 SVPa BSPa dTs 1 21 124 18 855 15 23 85 23 19500 aAbbreviations: SVP=snake venom phosphodiesterase; BSP=bovine spleen phosphorodiesterase.

Accumulation of 5'-acridine Labelled Oligos by Cells The increase in cellular fluorescence associated with accumulation of 5'-acridine-dT 2 (PO, m=3) as compared to free acridine is depicted in Figure 4. It is clear that the oligo accumulates intracellularly over several hours, and that this process occurs at 37 0 C but not at 4 0 C. Free acridine aminopropanol, which enters cells by diffusion, accumulates equally well at either 370 or 40. When accumulation of normal oligos of different lengths was o^ examined, it was apparent that the 5'-acridine-dT 7 was taken up more rapidly than dT 15 or dTz 2 as shown in Figure The 5'-acridine-SdT 7 was taken up much more slowly than normal dT 7 as seen in a comparison of Figure 5A with Figure As shown in Figure 1, the phosphoramidite synthesis of the present invention provides a simple, automated, high yield method for fluorescently tagging the 5' end of Ii 30 an oligodeoxynucleotide. Because when the molecule is subjected to base-deblocking conditions, aqueous ammonia, 60 0 C, for 10 hours, the acridine is cleaved from the oligodeoxynucleotide. Therefore, this method is primarily suited for hoiuo-oligos of thymidine. The -i WO 90/03383 PC/US89/03973 replacement of the ring chloride by thiophenol at the 6 position under mild conditions is novel, and appears to be aided by the presence of thymidine in the mixture.

The 5'-acridine linked molecules, both PO and PS, are readily purified by reverse phase chromatography. Two major peaks are observed, each containing acridine, but it can be shown by H and 31 P NMR that the desired compound invariably elutes at longer retention times.

A series of melt temperature measurements of poly-rA duplexes were performed with the acridine linked homooligodeoxynucleotides of thymidine, both PO and PS. The change in absorbance at 260 nm was used; this is a composite band, consisting of absorption from both the oligo and acridine chromophores. With n>12, this band becomes substantially oligo in character. In contrast to previous observations, in which oligos were taggged at the 3' and the 5' ends with a somewhat different acridine derivative (cf. Asseline et al., ops. cits.), the results shown herein gave smaller elevations of melting temperature. For example, for n=12, Asseline et al.

noted an approximately 14° increase in melting temperature, m=5, whereas Table I shows that with a different modified acridine there was observed only a increment. Furthermore, in similar experiments with normal 5'acridine-dT 7 there was little if any apparent increase in melting temperature, while Asseline et al.

note a 230 increase in the melting temperature for 3'acridine-dTa. In the present system, an increase in melting temperature was still observed for n=20 (70, In addition, there was no observed increase in melting temperature for all of the PS compounds (S-dT 15 S-dT 20 tested in similar experiments. These results appear to indicate that 5'-linked acridines of the type synthesized here are in fact very weak intercalators. This may be 9 li- i I:i-.U"I I-- 1 I P WO 90/03383 PpUS89/03973 16 reflective of the critical nature of the bulkier substituents bound to the acridine. It is also possible that m=5 is an insufficient number of methylene groups for maximal intercalation with this particular acridine moiety. It is not clear why S-oligo poly-rA duplexes derive no stabilization from a 5' linked acridine, although the evidence may indicate that no intercalation is occurring. It is possible that the greater Van der Waal's radius of sulfur versus oxygen blocks the approach of the already weak intercalator to its binding sites.

The difference in melting temperature between the normal and S-oligo decreases as the GC-content is increased, as shown in Figure 3, to a minimum at about GC content. Additionally, the most active anti-HIV S-oligos so far identified have a high GC content (cf.

Matsukura et al., op. cit.).

The synthesis method of the present invention can be applied to all four bases. The thiophosphate side reactions can be avoided by using other substituted acridines.

One of the major problems with the use of normal oligodeoxynucleotides in cellular systems is their Snuclease sensitivity. S-oligodeoxynucleotides, however, are highly nuclease resistant. Capping the 5' end of an oligodeoxynucleotide with a modified acridine greatly modifies its sensitivity towards the 5'exonuclease bovine spleen phosphodiesterase, as expected, but does not change sensitivity towards Sl and P1 nucleases, or towards snake venom phosphodiesterase.

Homo-oligos of thymidine are taken up by HL60 cells as monitored by flow cytometric analysis. No intracellular fluorescence could be seen in dead cells, suggesting that accumulation of these compounds is an energy-dependent process. In support of this hypothesis, WO 90/03383 PCT/US89/03973 17 it was found that penetration of free acridine into cells is temperature independent (40 vs. 370), while acridine-dT 12 does not penetrate at as shown in Figure 4.

The rate of cellular uptake of 5'acridine-S-dT 7 appears to be slower than that of normal acridine-dT 20 and is probably at or below the limit of detectability for about 48 hours, as shown in Figure 5. After this point, digestion of the oligodeoxynucleotide and formation of free acridine may become significant. Soligos have also been found to prevent the uptake of fluorescently tagged normal oligos.

The fluorescent group on the oligodeoxynucleotide enables one to determine the kinetics of cellular uptake using fluorescence cell sorting. Additionally, this fluorescent group makes it possible to monitor the inhibitory effects of other substances on cellular uptake, metabolism effects, and release of oligos, related nucleotide derivatives such as plasmids, and naked RNA molecules.

The foregoing description of the specific embodiments I will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and therefore such adaptations and modifications are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of Slimitation.

Claims (5)

1. A method for fluorescently tagging the 5' end of an oligodeoxynucleotide or a chemically modified analog thereof comprising: linking a phosphoramidite to a 6-substituted acridine and reacting said phosphoramite-acridine with an oligodeoxynucleotide to form a fluorescently marked oligodeoxynucleotide.

2. The method according to claim 1 wherein the

6-substituent on the acridine is chlorine. 3. The method according to claim 1 wherein the 6-substituent on the acridine is thiophenol. 4. The method according to claim 1 wherein the process is conducted in an automated nucleotide synthesis reactor. The method according to claim 1 wherein the oligodeoxynucleotide is a GC-containing phosphorothioate oligodeoxynucleotide. 6. The method according to claim 1 wherein the oligodeoxynucleotide is an S-oligodeoxynucleotide.

7. The method according to claim 1 wherein the Soligodeoxynucleotides contain from about 10 to about mers.

8. The method according to claim 1 wherein the oligodeoxynucleotide is a homo-oligomer of thymidine. -A