Field of the Invention

The present invention relates to a method of linking nucleosides with a siloxane bridge comprising reacting a 3'-silylated nucleoside with an unprotected nucleoside. The present invention further relates to methods of synthesizing oligonucleotide analogs having at least one siloxane internucleoside bridge. Background of the Invention

The nucleic acids, RNA and DNA, represent naturally occurring oligonucleotideε. As used herein, the term "oligonucleotide" means homopolymer or heteropolymer sequences of nucleosides in which the nucleosides are linked with a phoεphodiester bridge.

Due to advances in chemical technology, oligonucleotideε comprising several hundred nucleosides or bases can now be synthetically produced. Oligonucleotide Synthesis: A Practical Approach, ed. by M.J. Gait, IRL Press, Washington, D.C. (1984) .

Synthetic oligonucleotides have great scientific and therapeutic utility. Synthetic oligodeoxynucleotides, for example, have widespread use in the field of recombinant DNA. Gait, supra at 1. In recent years, synthetic .oligonucleotides have been shown to have therapeutic potential as antiεense agents to inhibit gene expression. Ui.lman, E. and Pey an, A., Chemical Reviews, 90(4): 544-583 (1990).

An antisense agent is a compound that binds to or hybridizes with a nucleotide sequence in a target nucleic acid, RNA or DNA, to inhibit the function of said target nucleic acid. Because of their ability to hybridize with both RNA and DNA, antisense agents can interfere with gene expression at the level of transcription, RNA procesεing or tranεlation.

At the present time, however, the development of practical scientific and therapeutic applications of antisense technologies is hampered by a number of technical problems. See e.g. ; Klausner, A., Biotechnology. 8:303-304 (1990); Armstrong, L. , Business Week. March 5, 1990. Such problems include (1) degradation by endogenous nucleases, (2) the high cost of production, (3) lack of sequence specific hybridization to target nucleic acidε, (4) non- uniformity due to the presence of chiral phosphorous centers and (5) inadequate delivery to desired targets, for example, due to inappropriate solubility coefficients, membrane transport and cellular tracking. One approach to preparing antisenεe agents that are stable, nuclease resistant, inexpensive to produce and which can be delivered to and hybridize with nucleic acid targets throughout the body is to synthesize oligonucleotide analogs having modifications in the internucleoside bridges or linkages. As used herein, the phrase "oligonucleotide analog" refers to homopolymer or heteropolymer εeguences of nucleosides or analogs thereof with non- phosphodieεter internucleoεide linkages.

In general, two types of oligonucleotide analogs have been reported. The first type includeε those having modified phosphate linkages. The second type includes thoεe analogε having non-phosphate internucleoside linkages. Uhlmann, E., supra.

Representative non-phosphate internucleoside linkages include siloxane, carbamate, carboxymethyl esters, acetamidate, carbonate and thioetherε. Uhlmann, supra.

Of particular relevance to the present invention is the siloxane linkage or bridge. Nucleoside dimers and hexamerε having εiloxane internucleoside linkages and a ^' method ^" of syntheεizing

such polymerε have been reported by Ogilvie and Cormier. See, e.g.. Ogilvie, K.K. and Cormier, J.F., Tetrahedron Letterε. 26(35) :4159-4162 (1985); Cormier J.F. and Ogilvie, K.K. , Nucleic Acids Research, 16(10) :4583-4594 (1988).

According to this published method, a 5*- protected nucleoside is reacted with a silylating reagent to form a 3'-silylated nucleoside, which silylated nucleoside is then reacted with a protected nucleoside to produce a fully protected, 3',5*-silyl linked dinucleoside. The fully protected, εilyl linked dinucleoside is then deprctected at either terminal to carry out chain extension via another round of coupling with protected nucleosides. Certain problems are associated with this method. When thiε metho is employed to εyntheεize nucleoside polymerε, the desired end product is produced in low yields ranging from about 35% to about 46%. The low yield is attributed both to the production of undeεired byproducts, in particular, the 3 ',3'- εy metrical di er, Uhl an, E. supra at pg. 553, resulting from self conjugation of the nucleoside building blocks, and to significant loss of useful product resulting from polymer deprotection. The present invention provides a method of linking nucleosideε with a siloxane bridge while suppressing formation of the 3',3'-di er. This method comprises reacting a silylated nucleoside with an unprotected nucleoside in the presence of a hindered base catalyst. The use of unprotected nucelosides as compared to the procedure of Ogilvie and Cormier, has the advantages of increasing b th the yield of desired end-products and the efficiency of the synthesis (thereby reducing the cost) . A preferred embodiment of the present invention, in which the reaction is carried out in the

- A - presence of a εterically hindered base catalyst, provides the additional advantage that the formation of undesired 3*,3'-symmetrical di ers is minimized. Summary of the Invention The present invention provides a method of linking nucleosides with a siloxane bridge comprising reacting a 3'-silylated-S'-protected nucleoside with an unprotected nucleoside in the presence of a hindered base catalyst. The siloxane bridge has the formula:

R

I - O - Si - O - ;

I R where R is independently ^-C ₆ alkyl. In a preferred embodiment, R is methyl or isopropyl. Both the silylated nucleoside and the unprotected nucleoside may be either monomeric nucleosides or the 3' and 5' terminal nucleosides respectively of an oligonucleotide or oligonucleotide analog. Preferred monomeric nucleosides are thy idine, N°-benzoyldeoxyadenosine, N^-benzoyldeoxycytidine and N-isobutyldeox guanosine.

The reaction of the 3'-silylated-5'-protected nucleoside with the unprotected nucleoside preferably occurs in a neutral or alkaline, aprotic solution. In i preferred embodiment, the alkaline, aprotic solution comprises 2,6-di-tert-butyl-4-methylpyridine in a mixture of acetonitrile and dimethylformamide.

In another aspect, the present invention comprises a method of synthesizing an oligonucleotide analog having siloxane internucleoside linkages comprising the steps of:

a) silylating a 5 '-protected nucleoside with a bifunctional silylating reagent to form a 3 '-silylated- 5'-protected nucleoside; b) reacting the silylated nucleoside with an unprotected nucleoside; and c) repeating steps a) and b) to form said oligonucleotide analog.

Bifunctional silylating reagents utilized with the present invention have the formula I:

where R is independently C^-C ₆ alkyl; and R ¹ iε a leaving group. In a preferred embodiment, the silylating agent is symmetrical and R iε isopropyl or methyl and R ¹ iε a leaving group εuch as Cl or S0 ₂ CF ₃ .

Both the 5' protected nucleoεide and the unprotected nucleoεide are independently thy idine, N°-benzoyldeoxyadenosine, N ^A -benzoyldeoxycytidine or N ² -iεobutyldeoxyguanoεine. Preferably, both εteps a) and b) occur in an alkaline, aprotic εolution and, more preferably in a solution comprising 2 , 6-di-tert-butyl-4-methylpyridine in a mixture of acetonitrile and dimethylformamide, more preferably about a 1:1 (v/v) mixture of said solvents. The present invention still further provides a solid phase method of synthesizing oligonucleotide analogs having siloxane internucleoside linkages comprising the steps of: a) attaching a 5 '-protected n cleo ide " -. solid support; b) deprotecting the attached nucleoside;

c) reacting the deprotected nucleoεide with.a 3*-εilylated-5'-protected nucleoside in the presence of a base catalyst and a neutral or basic aprotic solvent; d) capping the ^* unreacted nucleosides; e) repeating steps b) , c) and d) until an oligonucleotide analog of desired length is formed; and f) removing the formed oligonucleotide analog from the solid support.

The siloxane internucleoside linkages of the oligonucleotide analog have the formula II.

R

I [nucleoside] — Si — [nucleoεide] II R

In the εolid phase method, the base catalyst used in accordance with the present invention iε preferably a hindered baεe and, more preferably 2,6-di- tert-butyl-4-methylpyridine. Generally, the baεe, imidazole, is also present as a εtabilizer for the silylated intermediate. Its presence also helpε to improve the yield. A preferred aprotic solvent iε a mixture of acetonitrile and dimethlyformamide, more preferably about a 1:1 (v/v) mixture. The formed oligonucleotide analog iε preferably removed from the solid support by cleaving with aqueous ammonia in isopropanol and acetonitrile. Detailed Description of the Invention Nucleosides linked with a siloxane bridge are synthesized by reacting a 3 '-silylated-5 ¹ -protected nucleoside with an unprotected nucleoside.

Siloxane bridges contemplated by the present invention are dialkylεilyl linkageε of the formula: R

- O - Si - O - ;

I

R

where each R iε independently C ₁ -C ₆ alkyl.

In a preferred embodiment, R iε isopropyl or methyl.

The 3'-silylated-5'-protected nucleoside can be made in accordance with procedures Icnown and readily apparent to those of skill in the art. In a preferred embodiment, a 3 ^» -hydroxyl-5'-protected nucleoside iε reacted with a bifunctional silylating reagent in an alkaline, aprotic solution. The protecting group at the 5'-carbon hydroxyl group is selected on the basis of its acid lability. Suitable protecting groups are known and readily apparent to those of skill in the oligonucleotide εyntheεiε art. Preferred protecting groups are tritylε, particularly monomethoxytrityl and dimethoxytrityl. Most preferred is dimethoxytrityl

(DMT) .

Bifunctional silylating reagents contemplated by the preεent invention have the formula:

R I

R ¹ - Si - R ¹ ;

I R I where each R iε independently C ₁ -C ₆ alkyl; and

R iε a leaving group. In a preferred embodiment, the εilylating reagent iε symmetrical and R iε isopropyl or methyl and ^' P ¹ iε Cl or S0 ₂ CF ₃ .

The alkaline aprotic solution preferably comprises a proton acceptor, such as a base, dissolved in an aprotic solvent. Suitable bases and solvents are known and readily apparent to those of skill in the art. A preferred base is a hindered base exemplified by 2,6- άi-tert-butyl-4-methylpyridine. A preferred solvent iε a mixture of dimethylforma ide (DMF) and acetonitrile (CH _j CN) .

Nucleosides utilized with the present invention include both oxy- and deoxy-nucleotides. The

purine and pyrimidine moieties of such nucleosideε are optionally protected at exocyclic amino groupε. A preferred protecting group for the exocyclic amino groups of adenine and cytosine iε the benzoyl moiety. A preferred protecting group for the exocyclic amino group of guanine is the iεobutyl moiety. Optionally, guanine may also be protected at the O ⁶ position.

The 3'-silylated nucleoεide used in the method of the present invention may be a nucleoside monomer or the 3'- terminal nuceloside of an oligonucleotide or oligonucleotide analog. The 3' ,5'- unprotected nucleoεide used in the method of the present invention may be a nucleoεide monomer or the 5'- terminal nucleoεide of an oligonucleotide or oligonucleotide analog in which both the 3'- and 5'- terminal nucleoεideε are unprotected. The oligonucleotide analog can have any type of internucleoside linkage.

Alternatively, the method of the present invention iε employed to εyntheεize oligonucleotide analogs having only siloxane internucleoside linkages. Synthesis of such oligonucleotide analogs proceeds in accordance with modified solution phaεe or solid phase εynthetic proceεεes, Gait, εupra.

In a preferred εolution phaεe method, the reaction of a monomeric 3'-εilylated-5'-protected nucleoside with a first monomeric unprotected nucleoside is followed by reaction of the 5'-protected silyl linked (3* to 5 ¹ ) dinucleoside product (nucleoside dimer) with a bifunctional silylating reagent and a second unprotected monomeric nucleoside to form a 5'-protected, silyl linked trimer (trinucleoside) . The length of the chain iε extended by repeating theεe reaction steps until an oligonucleotide analog (nucleoside polymer) of deεired length iε achieved. Alternatively, and preferably, chain extension or elongation proceeds by isolating the 5 ' -protected,

εilyl linked polymerε as they are formed, removing the 5'-protecting group and uεing such unprotected nucleoside polymers in place of the unprotected monomeric nucleosides. In this way, chain elongation proceeds in a more rapid and efficient εtoichiometric manner (i.e. trimer + dimer, trimer + tri er) .

In a preferred embodiment, oligonucleotide analogs having εiloxane internucleoεide linkages are synthesized by a modified solid phase synthetic process employing the linking method of the present invention.

The initial step in solid phase syntheεiε iε attachment of a 5'-protected nucleoεide to a εolid εupport, preferably a controlled pore glaεε (CPG) support. The nucleoεide iε preferably attached to the CPG εupport via a εuccinate linkage at the 3 '-hydroxyl position of the nucleoεide. Other means of attaching nucleosides to εolid εupportε are known and readily apparent to thoεe of skill in the oligonucleotide syntheεiε art. Alεc, such 5 '-protected nucleoεideε linked to a CPG εupport are commercially available.

Following attachment of the first nucleoside to the εolid support, chain elongation occurε via the εequential εteps of removing the 5 '-hydroxyl protecting group from the attached nucleoside, adding a 5'- protected-3 '-silylated nucleoside together with an activating reagent, and capping the unreacted chains.

The protecting group at the 5 •-hydroxyl position of the attached nucleosides iε removed with acid, preferably trichloroacetic acid. The activation step occurε in the presence of an added silylated nucleoside and a hindered base activating reagent. A preferred activating reagent is a hindered base such as, 2, 6-di-tert-butyl-4- methylpyridine. A preferred solvent is a mixture of acetonitrile and DMF. Unreacted chains are terminated

or capped with capping reagents such as acetic anhydride and N-methyl imidazole.

After the desired oligonucleotide chain assembly is complete, the chains are separated from the εolid εupport and the protecting groups are removed by conventional methods. Gaits, supra at pp 67-70.

Preferably, completed chains are cleaved from the εolid εupport by a solution of aqueous ammonia in isopropanol and acetonitrile. Using the solid phaεe synthetic procedures set forth above, 3',5'-silyl linked nucleoεide polymerε or oligonucleotide analogε having siloxane internucleoside linkages of any desired length can be prepared.

Those skilled in the art will appreciate that other means of synthesizing oligonucleotideε can be modified in an analogous manner to produce oligo¬ nucleotide analogs having siloxane internucleoside linkages. Similarly, those skilled in the art will appreciate that the methods of the present invention can be used in conjunction with known methodε of preparing oligonucleotideε or analogs thereof having other typeε of internucleoside linkages to prepare oligonucleotide analogs having mixtures of siloxane and such other linkages. The method of the preεent invention can be utilized to synthesize a variety of oligonucleoside sequenceε comprising baseε which, when substituted for the naturally occurring bases in DNA and RNA, enable oligonucleotide analogs in which they are incorporated to hybridize with target segments of DNA or RNA. Suitable bases include adenine (A) , cytidine (C) , guanine (G) , uracil (U) , thymine (T) and modifications thereof, as for example, 5-bromo or 5-iodouracil, 5- methyl cytosine, isocytosine (2-amino-4-oxopyrimidine) , isoguanine (2-oxo-6-amino purine) , Inoεine (6-oxo purine) , 5-vinyl uracil and 5-vinylcytoεine.

The following examples further illustrate the invention and are not to be conεtrued as limiting of the specification and claims in any way. Example 1: Synthesis of 5'-0-dimethoxytrityl-3 '-0-

(5 '-dimethylεilyl-3 '-O-acetylthymidyl) thymidine

A solution of 5'-O-dimethoxytrityl thymidine (7.35 mmol, 4.0 g) in CH ₂ Cl ₂ (40ml) and triethylamine (16.16 mmol, 2.2 ml) was slowly cannulated into a solution of dichlorodi ethylsilane (7.35 mmol, 0.948 g, 0.89 ml) in CH ₂ C1 ₂ (100 ml) at -40°C (dry ice-CH ₃ CN) and εtirred at -40"C for 3 hours. A solution of 3 '-O- acetylthymidine (3.5 mmol, 1.0 g) and triethylamine

(16.16 mmol, 2.2 ml) in CH ₂ C1 ₂ (25 ml) was added and the reaction was εtirred at 0°C for 3 hourε. The reaction waε quenched by adding 5% aqueouε NaHC0 ₃ (25 ml) . The organic layer waε washed with brine (2 X 25 ml) and dried over Na ₂ SO, . Crude product (5.3 g) waε purified by column chro atography (Si0 ₂ , gradient of ethyl acetate/hexaneε) .

Iεolated yield: 670mg, 22%. Rf 0.23(7:3 EtOAc.-Hexaneε) . ¹ H NMR (300MHz, CDC1 ₃ ) <5 9.02 (ε, 1 H, NH) , 8.95 (ε, 1 H, NH) , 7.64 (ε, 1 H) , 7.50 (ε, 1 H) ,

7.41-7.27 ( , 9 H) , 6.84 (d, J = 7.7 Hz, 4 H) , 6.38 ( , 2H) , 5.19 (d, J = 5.6 Hz, 1 H) , 4.61 (d, J = 2.5 Hz, 1 H) , 4.04 (ε, 2H) , 3.87 (ε, 2H) , 3.79 (ε, 6H) , 3.39 (ABq, J = 10.4 ¥.7 , Δv = 67-3 Hz, 2H) , 2,41-2.26 (m, 4 H) , 2.09 (s, 3 H) , 1.89 (ε, 3 H) , 1.50 (ε, 3 H) , ' 16 (s, 3 H) , 0.15 (s, 3 H) . FABMS (TG/G, 5% HOAc) : (M+H) ⁺ = 884.6.

Example 2: Synthesis of 5'-O-dimethoxytrityl-3 '-O-

(5 '-O- _^ iεopropylsilyl;_hymid/l) thv idine

Method A. (Hindered baεe catalyst) A solution of 5 '-O-dimethoxytritylthymidine (0.92 mmol, 0.5 g) and 2, 6-di-tert-butyl-4-methylpyridine (0.23

mmol, 47 mg) in DMF (4 ml) was added to a solution of _. 2,6-di-tert-butyl-4-methylpyridine (1.0 mmol, 0.2 g) and diisopropylsilylbistriflate (1.0 mmol, 0.30 ml) in CH ₃ CN (5 ml) at -40"C. After 30 in at -40 ^* C, the reaction waε allowed to warm to room temperature. Imidazole (1.0 mmol, 70 mg) waε added, followed by the addition of unprotected thymidine (0.8 mmol, 193 mg) . The reaction was stirred for 1 hour and then added dropwise to a vigorously εtirred icewater mixture (500 ml) . The resulting mixture waε then εtirred for 30 min. and filtered. Crude product waε chromatographically purified.

Iεolated yield 70%. Rf 0.45 (5% MeOH/EtOAc) . ^H NMR (300 MHz, CDC1 ₃ ) δ 9.85 (ε, 1 H, NH) , 9.44 (ε, 1 H, NH) , 7.64 (ε, 1 H) , 7.41-7.24 (m, 10 H) , 6.84 (d, J = 7.8 Hz, 4 H) , 6.33 ( , 2 H) , 4.65 (ε, 1 H) , 4.43 (d, J = 2.2 Hz, 1 H) , 4.11 (d, J = 2.56, 1 H) , 4.00 (d, J = 3.36, 1 H) , 3.93 (ABq, J = 3.7 Hz, 11.0 Hz, Δv = 24.6, 2 H) , 3.79 (s, 6 H) , 3.39 (ABq, J = 3.0 Hz, 10.8 Hz, Δv = - 49.5 Hz, 2 H) , 2.50-2.38 (m, 2 H) , 2.30-2.07 (m, 2 H) , 1.88 (ε, 3 H) , 1.56 (ε, 3 H) , 1.05-0.98 ( , 14 H) . ³ C NMR (CDC1 ₃ ) δ 164.84, 164.80. 159.40, 151.72, 151.25, 144.89, 136.16, 136.01, 135.86, 130.60, 128.58, 127.75, 113.80, 112.10, 111.42, 87.48, 87.38, 85.76, 85.48, 73.90, 71.70, 63.82, 63.48, 60.75, 55.57, 41.71, 40.85, 21.24, 17.52, 17.47, 17.39, 14.35, 12.69, 12.18, 12.10, 11.85. FABMS (TG/G) : (M+H) ⁺ = ^** 899.

Anal. Calcd for C^ ₇ H ₅₈ N ₄ 0 ₁₂ Si: C, 62.79; H, 6.50; N, 6.23; MW, 898. Found: C, 61.83; H, 6.53; N, 6.23.

Method B. (Unhindered baεe catalyst) A solution of 5 '-O-dimethoxytritylthymidine (6.65 mmol, 3.54 g) and imidazole (13.3 mmol, 0.88 g) in DMF (18 ml) was added slowly via a dropping funnel to a solution of dichlorodiisopropylεilane (6.62 mmol, 1.22 g, 1.20 ml) in DMF (4.5 ml) at -40°C (dry ice-CH ₃ CN) . The reaction

was stirred at -40"C for 1 hour. A solution of unprotected thymidine (6.65 mmol, 1.61 g) and imidazole (1.33 mmol, 0.9 g) in DMF (15 ml) was added via a dropping funnel. The reaction was stirred at -40 ^~ C for 1 hour then warmed to room temperature overnight. The reaction mixture was then added dropwise to a vigorouεly stirred ice-water mixture (1 L) and stirred for 30 min. The mixture was filtered to yield the product as a white εolid which waε air dried and εubjected to column chromatography (Si0 ₂ , gradient of 60% to 100% EtOAc/hexaneε) . Isolated yield: 1.47g, 25%.

Example 3: Syntheεiε of N°-benzoyl-2 '-deoxy-5'-0- dimethoxytrityl-3 '-0-(5'- diisopropylsilylthyr.idyl adenosine

N ⁶ -Benzoyl-2 '-deoxy-5'-0- dimethoxytrityladenoεine (5 mmol, 3.28 g) and imidazole (10 mmol, 0.68 g) were dissolved in DMF (15 ml) and added slowly via a dropping funnel to a solution of dichlorodiisopropylsilane (5 mmol, 0.9 ml) in DMF (1.5 ml) at -40 ^C C (dry ice-CH ₃ CN) . The reaction was εtirred at -40"C for 1 hour and a solution of thymidine (7.5 mmol, 1.81 g) and imidazole (7.5 mmol, 0.51 g) in DMF (20 ml) waε added via a dropping funnel. The reaction waε εtirred at -40 ^C C for 1 hour and then warmed to room temperature overnight. The reaction mixture waε then added dropwise to a vigorously stirred ire-water mixture (1 L) and εtirred for 30 min. The precipitate waε filtered and dried to give a white solid (5.8 g) , which was purified by preparative TLC (1 mm Si0 ₂ , 3% MeOH/EtOAc) .

Isolated yield: 50 g, 38%. Rf 0.32 (2% MeOH/EtOAc) . ¹ H NMR (300 MHz, CDC1 ₃ ) δ 9.99 (ε, 1 H, NH) , 8.76 (ε, 1 H) , 8.22 (ε, 1 H) , 8.09 (d, J = 7.8, 2

H) , 7.55-7.13 ( , 13 H) , 6.74 ( , 4 K) , 6.42 (t, J = 5.8 Hz, 1 H) , 6.25 (t, J = 6.0, 1 H) , 4.96 (d, J = 5.4 Hz, 1

H) , 4.48 (s, 1 H) , 4.19 (d, J = 3.8 Hz, 1 H) , 3.94 (ε, 1 H) , 3.83-3.72 (m, 2 H) , 3.72 (s, 6 H) , 3.38 (d, J = 3.4 HZ, 2 H) , 2.83-2.76 (m, 1 H) , 2.60-2.52 (m, 1 H) , 2.45- 2.38 (m, 1 H) , 2.05-1.95 (m, 1 H) , 1.79 (S, 3 H) , 0.95 (m, 14 H) . FABMS (TG/G) : (M-H) ^* = 1011.

Example 4: Synthesis of N^-benzoyl-2'-deoxy-5'-O- dimethoxytrityl-3 '-O-(5 ^» -O- diisopropylsilylthvmidγl) cytidine

The procedure described above in Example 3 was employed for the εyntheεiε of the title compound. Purification by preparative TLC (1 m, Si0 ₂ , 3% MeOH/EtOAc) gave pure product. Eεti ated yield: 70%. Rf 0.40 (2%

MeOH/EtOAc) . ¹ H NMR (CDC1 ₃ ) <5 9.54 (ε, 1 H, NH) , 8.21 (d, J = 7.6 Hz, 1 H) , 7.94 (d, J = 8.2 Hz, 2 H) , 7.59- 7.24 (m, 14 H) , 6.83 (d, J = 8.4 Hz, 4 H) , 6.25 ( , 2 H) , 4.58 (ε, 1 H) , 4.46 (ε, 1 H) , 4.17 (ε ^' , 1 H) , 4.07- 3.80 (m, 3 H) , 3.77 (ε, 6 H) , 3.39 (ABq, J = 3.0 Hz,

10.9 Hz, Δv = 29.1, 2 H) , 2.84-2.78 ( , 1 H) , 2.46-2.41 (m, 1 H) , 2.14-1.79 ( , 2 H) , 1.84 (ε, 3H) , 0.98 (m, 14 H) . FABMS (NBA) : (M-H) ^" = 987.

Example.5: Syntheεiε of 3 '-Silylated-N ⁴ -benzoyl-2 '- deoxy-5 '-O-dimethoxytritylcytidine

Method A. A εolution of N^-benzoyl-Σ '-deoxy- 5'-O-dimethoxytritylcytidine (0.4 mmol, 260 mg) and imidazole (0.8 mmol, 52 mg) in CH ₃ CN (1.6 ml) waε slowly added via a syringe to a solution of dichlorodiisopropylεilane (0.4 mmol, 72 μl) in CH ₃ CN (0.4 ml) at -40"C (dry ice-CH ₃ CN) . The reaction mixture waε allowed to stir at -40"C for 30 min, then at room temperature for 30 min. The reaction mixture waε filtered, and the product (3 '-O-εilylated cytidine) iεolated by column chromatography (Si0 ₂ , gradient of 60% EtOAc/Hex to 100% EtOAc to 1% MeOH/EtOAc) .

Yield: lOO g, 28%. Rf 0.71 (0.5% MeOH/EtOAc) . Also isolated was 50 mg of a 3 '-3' symmetrical C-C dimer. ¹ H NMR (300 MHz, CDC1 ₃ ) δ 8.43 (d, J - 7.6 Hz, 1 H) , 7.88 (d, J = 8.2 Hz, 2 H) , 7.60-7.26 (m, 13 H) , 6.87 (d, J = 8.4 HZ, 4 H) , 6.25 (t, J = 6.5 Hz, 1 H) , 4.71

(m, 1 H) , 4.10 (m, 1 H) , 3.80 (ε, 6 H) , 3.48 (ABq, J = 3 Hz, 11 Hz, Δv 30 Hz, 2 H) , 2.67 (m, 1 H) , 2.35 (m, 1 H) , 0.97 ( , 14 H) . FABMS (TG/G) : (M+H) ⁺ = 884.6.

Method B. To minimize formation of the 3*,3' εymmetrical dimer, a εolution of N ^ά -benzoyl-2 '-deoxy- 5 '-O-dimethoxytritylcytidine (3.08 mmol, 2.0 g) in DMF/CH ₃ CN (5ml/2ml) waε added dropwiεe at -40°C (dry ice-CH ₃ CN) to a εolution of diisopropylsilylbistriflate (3.38 mmol, 1.0 ml) and 2 , 6-di-tert-butyl-4- ethylpyridine (3.38 mmol, 700 mg) in CH ₃ CN (8 ml) . The reaction mixture was εtirred at -40 ^C C for 30 min. and the product purified by TLC.

Example 6: Detritylation of 5 '-O-dimethoxytrityl- 3 ' -O- (5 '-0' -diiεopropylsilylthymidyl) thyridine

A εolution of the title compound (22 mmol, 200 mg) in CK ₂ C1 ₂ (4 ml) waε added to 3% trichloroacetic acid in CH ₂ C1 ₂ (6 ml) . The bright orange εolution waε εtirred at room temperature for 10 min. The reaction mixture waε poured into 5% aqueouε NaHC0 ₃ (5 ml) and extracted into 5% Ke-O ^' /EtO r.. The organic layrr war- waεhed with brine (10 ml) and dried over Na ₂ S0 ₄ . Crude product was purified by column chromatography (Si0 ₂ gradient of 60:40 EtOAc/Hex to 10% MeOH/EtOAc) .

Isolated yield: 90 mg, 70%. Rf 0.40 (10% MeOH/EtOAc) . ¹ H NMR (300 MHz, CD ₃ 0D) δ 7.54 (ε, 1 H) , 7.28 (ε, 1 H) , 6.03 ( , 2 H) , 4.46 (m, 1 H) , 4.18 (m, 1 H) , 3.82-3.69 (m, 3 H) , 3.50 (m, 4 H) , 2.06-1.97 ( , 4 H) , 1.62 (ε, 6 H) , 0.85 (m, 14 H) . FABMS (TG/G) : (M+H) ⁺ = 597.3.

Example 7: Synthesis of 5'-O-dimethoxytrityl-3 '-0-

[5'-0-diisopropylsilylthymidyl-3•-O-(5•- O-diisopropylsilylthymidyl) ] thymidine Chem. Abstr. Index name is Thymidine,

5*-0-[bis(l-methylethyl)silylene]-5•-0- dephoεphinicothymidyly-(5' .fwdarw.3')- 5'-0-[bis(l-methylethyl)silylene]-5•-0- dephoεphinicothymidylyl-(5' .fwdarw.3')- 5'-0-rbiε(4-methoxyphenyl')phenylmethvπ-

Method A. A solution of 5'-0- dimethoxytritylthymidine (14.7 mmol, 8.0 g) and imidazole (29.94 mmol, 2.0 g) in DMF (40 ml) was added slowly via a dropping funnel to a εolution of dichlorodiisopropylεilane (14.7 mmol, 2.72 g, 2.64 ml) in DMF (10 ml) at - 0"C (dry ice-CH ₃ CN) . The reaction waε εtirred at -40°C for 1 hour. A εolution of thymidine (14.7 mmol, 3.56 g) and imidazole (29.4 mmol, 2.0 g) in DMF (40 ml) waε added via a dropping funnel. The reaction waε εtirred at -40°C for 1 hour then reaction cannulated under N ₂ to a solution of dichlorodiisopropylεilane (14.7 mmol, 2.72 g, 2.64 ml) in DMF (10 ml) at -40°C. The reaction mixture waε εtirred at -40'C for 1 hour. A εolution of thymidine

(14.7 mmol, 3.56 g) and imidazole (29.4 mmol, 2.0 g) in DMF (40 ml) waε added and the reaction waε εtirred at -40°C for 1 hour. The reεulting mixture waε added dropwiεe to a vigorouεly-εtirred ice/water mixture. (2 L) and stirred for 30 min. The precipitate was filtered and air-dried. The white εolid (27 g) was subjected to column chromatography (150 g Si0 ₂ , gradient of 60% to 100% EtOAc/hexaneε) to give pure product. Isolated yield: 1.8 g, 10%. Method B. A solution of 5'-0- dimethoxyltrityl-3 ^» -0-(5'-O-diiεopropylεilylthymidyl) thymidine (1.11 mmol, 1.0 g) and 2,6-di-tert-butyl-4- methylpyridine (0.28 mmol, 60 mg) in DMF (3 ml) waε added via a syringe to a solution of

diisopropylsilylbistriflate (1.22 mmol, 0.504 g, 0.360 ml) and 2,6-di-tert-butyl-4-methylpyridine (1.22 mmol, 0.2 ^' 5 g) in CH ₃ CN (3 ml) at -40*C (dry ice-CH ₃ CN) . The reaction waε stirred for 1 hour at -40 ' C . A solution of imidazole (1.22 mmol, 0.16 g) in CH ₂ CN (2.5 ml) was added and the reaction warmed to room temperature. A solution of thymidine (1.11 mmol, 0.269 g) in DMF (2 ml) was added and the reaction εtirred for 1 hour and then added dropwise to a vigorouεly-εtirred ice/water mixture (1 L) and εtirred for an additional 30 min. The precipitate was filtered and air-dried to give a white solid (1.5 g) , which waε triturated with hexanes (20 ml) to give pure product.

Isolated yield: 1.05 g, 76%. Rf 0.38 (5% MeOH/EtOAc) . ¹ H NMR (300 MHz, CDC1 ₃ ) δ 7.62 (s, 1 H) ,

7.36-7.22 (m, 11 H) , 6.80 (d, J = 7.7, 4 K) , 6.36-6.22 (m, 3 H) , 4.62-4.54 (m, 2 H) , 4.45 (m, 1 H) , 4.06-3.83 (m, 7 H) , 3.75 (ε, 6 H) , 3.36 (ABq, J = 10.0Hz, Δv = 47.6 Hz, 2 H) , 2.45-2.30 ( , 3 H) , 2.28-2.14 (m, 1 H) , 2.13-2.00 ( , 2 H) , 1.85 (ε, 3 H) , 1.81 (ε, 3 H) , 1.48 (ε, 3 H) , 0.98 (m, 28 H) . ¹³ C NMR (CDC1 ₃ ) δ 164.54, 164.45, 164.33, 159.00, 151.12, 150.99, 144.40, 135.73, 135.51, 135.40, 130.15, 128.16, 113.37, 111.54, 111.32, 111.07, 87.50, 87.02, 85.23, 85.02, 73.35, 72.98, 71.24, 63.28, 63.02, 55.15, 41.33, 40.64, 40.25, 17.04, 17.00, 16.92, 12.26, 11.70, 11.59, 11.53, 11.47. FABMS (TG/G): (M+H) ⁺ = 1252.5.

Anal. Calcd. for C ₆₃ H _a( .N ₆ 0. ₁₇ Si ₂ : C, 60.38; H, 6.71; N, 6.71; MW, 1253.6. Found: C, 60.44; H, 6.84; N, 6.56.

Exa.r.plc C: f^crAtyl tion of 5 ^■ -0-άiκ'ethoxytιi y ^' ',

A solution of 5 '-O-dimethoxytrityl trimer (0.638 mmol, 0.80 g) in CH ₂ C1 ₂ (12 ml) was added to 3% (v/v) trichloroacetic acid/CH ₂ Cl ₂ (14 ml) . The bright orange solution was εtirred at room temperature for 1

hour and then poured into 5% agueouε NaHC0 ₃ (15 -ml) and extracted into 5% MeOH/EtOAc. The organic layer waε washed with brine (20 ml) and dried over Na _j SO^. The product was purified by column chromatography (Si0 ₂ , gradient of EtOAc/MeOH 100% to 95%) .

Isolated yield: 420 mg, 70%. Rf 0.50 (10% MeOH/EtOAc) . 'H NMR (300 MHz, CD ₃ OD) δ 7.55 (s, 1 H) , 7.27 (m, 2 H) , 6.05 (m, 3 H) , 4.47 (m, 2 H) , 4.16 (m, 1 H) , 3.87-3.70 (m, 7 H) , 3.50 (m, 2 H) , 2.14-1.96 (m, 6 H) , 1.62 (ε, 9 H) , 0.92 (ffi, 28 H) . FABMS (TG/G) : (M-H) ^' = 950.

Example 9: Syntheεiε of 5'-O-dimethoxytrityl-3 '-0-

[5'-O-diisopropylsilylthymidyl-3•-O- { (5'-O-diiεopropylεilylthymidyl)-3'-0-

(5'-O-diiεopropylεilylthymidyl) }] thymidine (Tetranucleotide Analog)

A εolution of 5'-O-dimethoxytrityl trimer (20 μmol, 25 mg) and 2,6-di-tert-butyl-4-methylpyridine (40 μmol, 8.25 mg) in DMF (200 μl) was added εlowly via a syringe to a solution of diiεopropylεilylbiεtriflate (20 μmol, 6 μl) and 2,6-di-tert-butyl-4-methylpyridine (20 μmol, 4.1 mg) in DMF (100 μl) in a 5 ml round bottom flaεk cooled to -40°C (dry ice-CH ₃ CN) . The reaction waε εtirred for 1 hour and a solution of thymidine (20 μmol, 4.84 mg) and imidazole (40 μmol, 2.7 mg) in DMF (200 μl) was then added. Stirring continued at -40"C for 30 min. The reaction was warmed to room temperature. Aqueous NaHC0 ₃ (1 ml of a 5% solution) was added and the reaction mixture extracted into CHC1 ₃ (2 X 5 ml) , washed with brine (l ml) and dried over Na ₂ S0 ₄ . Product was purified by preparative reverse-phase HPLC [Ultrasphere ODS, 20% 0.01 M triethylammonium acetate pH 7.5, 80% CH ₃ CN] .

Isolated yield: 10 mg, 31%. Rf 0.16 (5% MeOH/EtOAc) . ¹ H NMR (300 MHz, CDC1 ₃ ) δ 7.65 (ε, 1 H) , 7.39-7.26 (m, 12 H) , 6.85 (d, J ^* = 7.7 Hz, 4 H) , 6.38-

6.27 (m, 4 H) , 4.66-4.45 (m, 4 H) , 4.10-3.85 (m, 10 H) , 3.80 (ε, 6 H) , 3.39 (ABq, J = 11 Hz, Δv = 45 Hz, 2 H) , 2.45-2.08 (m, 8 H) , 1.92 (ε, 3 H) , 1.90 (s, 3 H) , 1.87 (ε, 3 H) , 1.56 (ε, 3 H) , 1.03 (m, 42 H) . FABMS (TG/G): (M+H) ⁺ = 1608.3; (M+Na) ⁺ = 1630.0.

Example 10: Synthesis of pentathymidyl nucleotide analog with silyl linkages A εolution of 5 '-dimethoxytrityl trimer (58.4 μmol, 73 mg) and 2, 6-di-tert-butyl-4-methylpyridine (29.2 μmol, 6 mg) in DMF (600 μl) waε added εlowly via a εyringe to a εolution of diiεopropylεilylbiεtriflate (58.4 μmol, 17.5 μl) and 2, 6-di-tert-butyl-4- ethylpyridine (58. μmol, 12 mg) in DMF (300 μl) in a 5 ml round bottom flask cooled to -40 ^C C (dry ice-CH ₃ CN) . The reaction was εtirred for-1 hour and a εolution of the detritylated dimer (55.5 μmol, 50.6 mg) and imidazole (100 μ oi, 6.75 mg) in DMF (300 μl) waε added. The reaction waε εtirred for 1 hour at -40"C, then warmed to room temperature. Aqueous NaHCO- (2 ml of a 5% solution) waε added and the mixture waε extracted into CHC1 ₃ (2 x 10 ml) , washed with brine (2 ml) and dried over Na ₂ SO _ώ to yield product. FABMS (TG/G): (M+H) ⁺ = 1961. Fragment ions at

1900, 1607, 1252, 898 and 544. FABMS (NBA) : (M-H) ^' = 1961.

Example 11: Solid-phase εyntheεiε of thymidine decanucleotide

A. Syntheεiε of the monomeric synthetic unit. Diisopropylεilylbiεtriflate (2 mmol, 0.60 ml) waε r.« :ed Vi a syringe t a t- lutic.i or 2, 6-cli-tert-b tyl- 4-methylpyridine (2 mmol, 0.41 g) in CH ₃ CN (5 ml) in a 100 ml round bottom flaεk under N ₂ . The clear εolution waε cooled to -40 ^C C (dry ice-CH ₃ CN) and a εolution of 5 '-O-dimethoxytritylthymidine (1.84 mmol, 1.0 g) and

2,6-di-tert-butyl-4-methylpyridine (0.46 mmol, 94 mg) in DMF (5 ml) was added dropwise via a syringe over 10 min. The reaction was stirred at -40°C for 1 hour. A solution of imidazole (3.7 mmol, 250 mg) in DMF (4 ml) was added, followed by dilution with CH ₃ CN (5 ml) to a final concentration of 0.1 M.

B. Synthesis Of The Decanucleotide. The mixture was warmed to room temperature and employed in a solid-phase automated synthesis of the decanucleotide. ^~ H NMR (300 MHz, CD ₃ 0H) δ 1.55-1.41 (m, 10

H) , 6.23-6.33 (m, 10 H) , 4.69 (s, br, 9 H) , 4.40 (s, br, 1 H) , 4.10-3.40 ( , 19 H) , 3.25-3.08 (m, 1 H) , 2.55-2.20 (m, 20 H) , 1.87 (s, br, 27 H) , 1.30 (ε, br, 3 H) , 1.08 (m, 126 H) . FABMS (TG/G): (M-H) ^" = 3434.2; (M+H) ^÷ = 3433.7. FABMS (NBA): (M-H) ^" = 3435.2.

M . F . C ₁₅₄ H _24S N ₂ o0 ₅₀ ; Sig. Calc : MW, 3432 . 5 .

Example 12: Synthesiε of 5'-0-dimethoxytrityl-3 '-O-

(5'-0-dimethoxytrityl-3 '-θ-(5'-θ- diisopropylsilylthy idyl)thymidine 3'-

N,N-diisopropyl(2- cyanoethyl)phosphoramidite

5 ⁱ -o-dimethoxytrityl dimer (0.1 mmol, 90 mg) was coevaporated from tetrahydrofuran/pyridine (6 ml) (ratio 2:1) twice, dissolved in THF (500 μl) and added dropwise via a syringe to a εtirred solution of 4- dimethylaminopyridine (4 mg) , diiεopropylethylamine (distilled from CaH ₂ , 0.4 mmol, 87 μl) and 2-cyanoethyl- N,N-diisopropylchlorophoεphoramidite (0.15 mmol, 28.77 μl) in THF (500 μl) under N ₂ flow at room temperature. The reaction waε stirred for 2 hour. To remove trace amounts of 5*-O-dimethoxytrityl-3 '-O-(5'-O- diisopropylsilylthymidyl)thymidine, additional 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.025 mmol, 5 μl) was added. The reaction waε εtirred 1 hour and added to EtOAc (10 ml, pre-washed with 5 ml brine) , washed with brine (2 X 2 ml) and dried over Na ₂ S0 ₄ .

Crude product was purified by column chromatography (Si0 ₂ , 1:1 EtOAc/hexanes) .

Isolated yield: 82 mg, 74.5%. Rf 0.70 (1% MeOH/EtOAc) . ^α H NMR (300 MHz, CDC1 ₃ ) δ 8.96 (s, br, 1 H, NH) , 7.64 (ε, 1 H) , 7.40-7.22 (m, 10 H) , 6.84 (d, J = 8.8, 4 H) , 6.40 (t, J = 6.5 Hz, 1 H) , 6.27 (t, J = 6.5 Hz, 1 H) , 4.67 (m, 1 H) , 4.53 (m, 1 H) , 4.10 (m, 2 H) , 3.93 (m, 1 H) , 3.87 (m, 1 H) , 3.80 (s, 6 H) , 3.61 (m, 2 H) , 3.39 (ABq, J = 4 Hz, 10 Hz, Δv = 42 Hz, 2 H) , 2.64 (m, 2 H) , 2.53-2.05 (m, 4 H) , 1.86 (s, 3 H) , 1.51 (s, 3 H) , 1.26 (m, 2 H) , 1.17 (m, 12 H) , 1.01 (m, 14 H) . ¹³ C NMR (CDC1 ₃ ) <5 163.58, 163.44, 158.72, 150.14, 144.18, 135.47, 135.25, 129.99, 127.99, 127.16, 117.58, 113.26, 111.16, 111.02. 110.94, 86.96, 86.23, 85.82, 85.74, 84.90, 84.74, 84.64, 73.41, 73.22, 63.40, 62.89, 62.70, 58.25, 58.17, 57.98, 57.87, 55.23, 43.36, 43.17, 41.47, 39.69, 39.47, 24.52, 24.44, 22.96, 20.42, 20.32, 17.24, 17.05, 12.44, 11.92, 11.71, 1 ^* 1.57. ³¹ P NMR (CDC1 ₃ , referenced to H ₃ P0 ₄ ) <5 149.16; IR (KBr) 3192, 3058, 2965, 2932, 2868, 2838, 2246, 1693, 1608, 1510, 1465, 1398, 1382, 1365, 1322, 1289, 1274, 1251, 1199, 1179, 1158, 1129, 1084, 1064, 1035, 1003, 978, 913, 885, 828, 812, 794, 773, 756, 727, 702 cm ^"1 . FABMS (NBA) : (M+H) ⁺ = 1099. Anal. Calcd. for C ₅₆ H ₇₅ N ₆ 0 ₁₃ PSi: C , 61.19; Η,

6.88; N, 7.64; MW, 1100. Found: C, 60.60; H, 6.87; N, 7.60.

Example .13: Synthesis of 5 '-O-dimethoxytri yl-3 '-O- [5 '-O-diisopropylsilylthymidyl-3 '-O (5'-

O-diiεopropylεilylthymidyl) ]thymidine- 3 '-N,N-diiεopropyl(2-cyanoethyl) - phosphoramidite (Trimer) 5 '-Diirethoxvtrityl-trimer (0.44 mmol, 550 mg) waε coevaporated rrom THF (20 lj/pyr (10 ml) twice, dissolved in CH ₂ C1 ₂ (2 ml) and added dropwise via a syringe to a εtirred εolution of 4-dimethylaminopyridine

(20 mg) , diiεopropylethylamine (distilled from CaH ₂ ; 1.69 mmol, 370 μl) and 2-cyanoethyl N,N- diiεopropylchlorophoεphoramidite (0.64 mmol, 120 μl) in CH ₂ C1 ₂ (2.0 ml) under N ₂ flow at O'C. The reaction mixture waε then brought to room temperature, εtirred . for 1 hour, poured into EtOAc (prewashed with 25 ml brine; 50 ml) , washed with brine (2 X 20 ml) and dried over Na ₂ S0 ₄ . Crude product was purified by column chromatography (10 g Si0 ₂ , EtOAc) . Isolated yield: 320 mg, 64%. Rf 0.76 (EtOAc). H NMR (300 MHz, CDCl ₃ ) 5.7.63 (s, 1 H) , 7.41-7.22 ( , 11 H) , 6.83 (d, J = 7.8 Hz, 4 H) , 6.40-6.24 (a, 3 K) , 4.67-4.54 ( , 3 H) , 4.13-3.85 (m, 7 H) , 3.75 (ε, 6 H) , 3.55 (m, 2 H) , 3.48-3.28 ( , 2 H) , 2.74 (t, J = 6 Hz, 2 H) 2.45-2.03 (m, 6 H) , 1.88 (ε, 3 H) , 1.83 (ε, 3 H) ,

1.50 (ε, 3 H) , 1.28-1.13 ( , 14 H) , 1.00 ( , 28 H) . FABMS (NBA): (M-H) ^" = 1453.

Example 14: Syntheεiε of 5 '-0-dimethoxytrityl-3 '-0- (3 '-0-diiεopropylεilyl-5'-0- dimethoxytritylthy idyl) thymidine

(3 ' .3 '-dimer)

In the preparation of a 3 ',5' thymidine- thymidine dimer, uεing the εilylation procedure deεcribed in Example 14, a 3 ',3 '-dimer waε obεerved aε a major by-product. The title compound was isolated from crude reaction product (800 mg) by column chromatography (Si0 ₂ , gradient of 60% to 90% EtOAc/hexanes) . Rf 0.41 (60 % ETOAC/Hex) . ¹ H NMR (300 MHz,

CDC1 ₃ ) δ 9.95 (ε, br, 1 H, NH) , 8.63 (s, br, 1 H, NH) ,

7.51 (S, 2 H) , 7.35-7.15 (m, 18 H) , 6.77 (d, J = 8.7 Hz, 8 H) , 6.34 (m, 2 H) , 4.57 (d, J = 4.3 Hz, 2 H) , 3.91 (ε, 2 H) , 3.71 (ε, 12 H) , 3.24 (ABq, J = 2.8 Hz, 10.7 Hz, Δv = 54 Hz, 4 H) , 2.46 ( , 2 H) , 2.26 ( , 2 H) , 1.46 (s, 6 H) , 0.88 (m, 14 H) . FABMS (NBA): (M-H) ^' = 1200.1.

Example 15: Synthesis of N ⁶ -benzoyl-2 '-deoxy-5 '-o- dimethoxytrityl-3 '-O-(3 '-O- diisopropylεilyl-N ⁶ -benzoyl-2 '-deoxy 0-dimethoxytrityladenosyl)adenosine

In the preparation of the 3 ',5' adenosine- thymidine dimer, a 3 ',3' dimer was observed as a major byproduct of the silylation step. A portion of this crude product (100 mg) was purified by preparative TLC (1mm Si0 ₂ , 3% MeOH/EtOAc) to give pure material.

Rf 0.45 (90% EtOAc/hexanes) . 'E NMR (300 MHz, CDC1 ₃ ) δ 8.60 (ε, 2 H) , 8.14 (ε, 2 H) , 8.00 (d, J = 8 Hz, 2 H) , 7.62 (ε, 2 H) , 7.57-7.04 (m, 22 H) , 6.73 (d, J = 9, 8 H) , 6.38 ( , 2 H) , 4.79 (m, 2 H) , 4.14 (m, 2 H) , 3.68 (ε, 12 H) , 3.52-3.24 (m, 4 H) , 2.86 ( , 2 H) , 2.51 (m, 2 H) , 0.98 ( , 14 H) . FABMS (NBA): (M+H) ⁺ = 1428.3, (M-H) ^" = 1426.4.

Example 16: Synthesis of N ^4" benzoyl-2 'deoxy-5 '-0- dimethoxytrityl-3 '-0-(3 '-O- diisopropylεilyl- N ⁴ -benzoyl-2 '-deoxy- 5 '-O- dimethoxytritylcytidyl)cytidine.

In the silylation of N ⁴ -benzoyl-2 '-deoxy-5'- O-dimethoxytritylcytidine, using the procedure deεcribed by Example 15, a 3 ',3' dimer waε obεerved as a major by¬ product. This dimer was a isolated from crude reaction mixture by column chromatography (Siθ ₂ , gradient of 60% to 100% EtOAc/Hex to 1% EtOAc/MeOH) to yield the title compound.

Rf 0.31 (1% MeOH/EtOAc) . ^X H NMR (300 MHz, CDC1 ₃ ) δ 8.82 (ε, br, 1 H, NH) , 8.28 (d, J = 8 Hz, 1 H) , 8.25 (d, J = 8 Hz, 1 H) , 7.89 (m, 4 H) , 7.63-7.24 (m, 26 H) , 6.85 ( , 8 H) , 6.23 (m, 2 H) , 4.57 (m, 2 H) , 4.20 (m, 2 H) , 3.80 (ε, 12 H) , 3.50-3.23 (m, 4 H) , 2.60 (m, 2 H) , 2.15 (m, 2 H) , 0.96 (m, 14 H) . FABMS (NBA): (M+H) ⁺ = 1379.2, (M-H) ^' = 1378.2.