METHOD FOR SYNTHESIZING DEOXYOLIGONUCLEOTIDES

Abstract: Deoxynucleoside phosphoramidites are prepared in good yield from doexynucleosides, bis-dialkylamino-phosphines, with the corresponding dialkylamine hydrotetrazolide or tetrazole as catalyst for the reaction. These phosphoramidites generated in situ lead to the direct synthesis of deoxyoligonucleotides on polymer supports.

Description: METHOD FOR SYNTHESIZING DEOXYOLIGONUCLEOTIDES The invention described herein was developed with funding received from the United States National Institutes of Health (GM25680) . The present invention relates to a new and novel method for the preparation of deoxynucleoside phos¬ phoramidites in situ from deoxynucleosides, bis- dial ylaminophosphines, and the corresponding dialkyl¬ amine hydrotetrazolide or tetrazole as catalyst for the reaction. These phosphoramidites lead to the direct synthesis of deoxyoligonucleotides on various polymer supports. Numerous prior attempts have been made to develop a successful' methodology for synthesizing sequence defined oligonucleσtides. One prior art technique has included the use of various organic polymers as supports during polynucleotide synthesis. Classically the major problems with many polymer supported synthesis strategies has been inherent in the nature of the polymer support. As a result, various prior art polymers used in such synthesis have proven inadequate for reasons such as: (1) slow diffusion rates of activated nucleotides into the support; (2) excessive swelling of various macropor- ous, low cross-linked support polymers; and (3) irreversible absorption of reagent onto the polymer. Modified inorganic polymers are known in the prior art, primarily for use as absorption materials, for example, in liquid chromatography. The attach¬ ment of nucleosidephosphates to silica gel using a trityl linking group is described in the prior art but the method is apparently applicable only to pyri idine nucleosides. The cleavage of the nucleo- side from the silica support can only be accomplished with acid to which the purine nucleosides are sensi¬ tive. The production of phosphotriester derivatives of oligothymidylates is described in the prior art by reaction of a phosphorodichloridite with a 5'-0 blocked thymidine and subsequent reaction of the product with a 3'-0 blocked ' thymidine. This is followed by oxidation of the resulting phosphite to a phosphate and removal of blocking groups to obtain the phosphotriesters. Unfortunately, the process requires separation and purification of products at each stage to ensure proper sequencing of the added nucleosides. Separation techniques including precipi¬ tation and washing of precipitates are necessary to implement each successive stage reaction. Prior to, the invention described in U.S. Patent No. 4,458,066, the stepwise synthesis of polynucleo- tides, and specifically oligonucleotides , remained a difficult and time consuming task, often rewarding the preparer with low yields of the desired compound. In this patented disclosure, the efficient stepwise synthesis of polynucleotides is described. This synthesis comprises a method of forming internucleo- tide bonds, that is bonds linking nucleosides in oligonucleotides or polynucleotides, by the reaction of halophosphoridites with suitably blocked nucleo- side or oligonucleotide molecules. The disclosure of this patent is incorporated in toto as descriptive of the present day methods used for the formation of polynucleotides and oligonucleotides. U.S. Patent No. 4,415,732 describes a new class of nucleoside phosphoramidites derived from saturated secondary amines, which are relatively stable. This permits isolation and storage of the compounds, at room temperature. These phosphoramidite compounds have been found to have excellent use in the forma¬ tion of the internucleotide bonds described in the earlier mentioned U.S. Patent. The disclosure of this patent is incorporated in toto herein. The current, phosphite triester methodology for deoxyoligonucleotide synthesis requires the conden¬ sation of deoxynucleoside phosphoramidites of the formula: D TO yB (FORMULA I) O wherein B may be 1-thyminyl; l-(N-4 benzoyl cyto- sinyl) ; 9-(N-2-isobutyrylquaninyl) ; or 9-(N-6-benzoyl- adeninyl) , wherein Z may be morpholino or N(lower alkyl)-, preferably (i-propyl)_; and wherein DMT is di-p-anisylphenyl methyl. These phosphoramidites are activated by tetra¬ zole, with the 5'-hydroxyl group of a deoxynucleoside or deoxyoligonucleotide attached covalently to a polymer support (see, for example, Caruthers, M. H. , Beaucage, S. L., Becker, C. , Efcavitch, J. . , Fisher, E. F. , Galluppi, G. , Goldman, R. A., de- Haseth, P. L., Martin, F. , Matteucci, M. D. and Stabinsky, Y. ,(1982) in Genetic Engineering, Setlow, J. and Hollaender, A. Eds., Vol. 4, pp. 1-17, Plenum Press, New York; Caruthers, M. H. (1982) in Chemical and Enzymatic Synthesis of Gene Fragments, A Labora¬ tory Manual, Gassen, H. G. and Lang, A. Eds., pp. 71-79. Verlag-Chemie, einheim, Federal Republic of Germany; Seliger, H. , Klein, S., Narang, C. , Seeman- Preising, B., Eiband, J.. and Hauel, N. (1982) in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, Gassen, H. G. and Lang, A. Eds., pp. 81-96; and innacker, E. and Dorper, T. (1982) in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, Gassen. H. G. and Lang. A. Eds., pp. 81-96.) These phosphoramidites can be prepared by existing methods (see, for example, Beaucage, S. L. and Caruthers, M. H. (1981) Tetrahedron Lett. 22, 1859-1862; and McBride, L. J. and Caruthers, M. H. (1983) ibid. 24, 245-248), from the appropriately protected deoxynucleosides of the following struc¬ tural formula: D (FORMULA II) wherein DMT and B are as defined for the compounds of Formula I above, and chlorphosphines of the following structural formula: CH- l O P ( FORMULA III ) wherein Z is a previously defined, and X may be halogen, preferably chlorine. These chlorophosphines, used in the preparation of the compounds of Formula I, are difficult to prepare and easily react with trace amounts of water. Moreover, the high reactivity of the chlorophosphines and the concomitant production- of insoluble amine hydrochloride salts preclude their use for any strategy involving the in situ generation of deoxynu¬ cleoside phosphoramidites for deoxyoligonucleotide synthesis on solid supports, (see, for example Fourrey, J. L. and Shire, D. (1981) Tetrahedron Lett. 22, 729-732) . More recently, Beaucage has reported in Tetra¬ hedron Letters (Vol. 25: 375-378) the in situ prepar¬ ation of deoxynucleoside phosphoramidite utilizing bis-(pyrrolidino)methoxyphosphine and 4,5-dichloro- imidazole as a mild acid catalyst. The present invention discloses similar results when the compound bis-(diisopropylamino)methoxyphosphine, that is a compound of the general Formula IV; α-ho p(Z z (FORMULA IV) wherein Z is N(CH 3 CH CH-),, and the compound bis- (morpholino)methoxyphosphine, that is the compound general Formula IV wherein 2 is morpholino, are activated with the corresponding amine hydrotetra- zolides of general formula: ( i Pr )2 H2 N.^ (FORMULA V) ; or ΪH ( FORMULA VI ) With the preparation of the present invention, the reaction is furthermore found to be catalytic in either tetrazole or the salt, as well as a greater selectivity of activation. This preparation has been successfully applied to the synthesis of deoxyoligo- nucleotides directly on a solid support via an in situ approach. It is, accordingly, an object of the present invention to disclose two novel bis-dialkylamino phosphine compounds. It is another object of the present invention to disclose a method for the preparation of deoxynucleo¬ side phosphoramidites by the reaction of suitably protected deoxynucleosides and bis-dialkylamino phosphines. It is still an object of the present invention to disclose a method for the preparation of oligonu¬ cleotides utilizing the _in situ formation of deoxynu¬ cleoside phosphoramidites. The following examples are presented to enable the reader to obtain a greater and more thorough understanding of the present invention. EXAMPLE I Preparation of bis-(diisopropylamino)methoxyphosphine To 14 g (0.11 mol) of methyldichlorophosphite in 500 ml of diethylether at -10°C under a nitrogen atmosphere was added 96 g (0.95 mol, 9 eq) of diiso- propylamine over 1 h. The reaction was allowed to warm to room temperature and stirred an additional 16 h. Removal of the amine hydrochloride salt by filtra¬ tion and evaporation of the solvent gave a pale yellow liquid. The crude material was fractionally distilled twice from calcium hydride (to remove residual amine salts) to afford 21.5 g (77%) of the desired compound as a colorless liquid: b.p. 40 o -42°C. EXAMPLE II Preparation of (diisopropylaminomorpholino) ethoxy¬ phosphine To a mixture of 3.0 g (15.7 mmol) of (P-diiso- propylamino) , (P-Chloro) methoxyphosphine and 1.53 g (15.7 mmol, 1 eq) of triethylamine in 100 ml of diethylether at -10°C under a nitrogen atmosphere was added 1.32 g (15.7 mmol, 1 eq) of morpholine over 15 min. While still cold, the reaction mixture was poured into saturated aqueous sodium bicarbonate and the organic layer was then washed with brine and dried over sodium sulfate. Evaporation of the solvent followed by ' fractional distillation of the crude liquid -from calcium hydride afforded 1.91 g (77%) of the desired compound: b.p. 64-66°C. EXAMPLE III Preparation of bis-(morpholino)methoxyphosphine To 744 g (50 mmol) of methyldichlorophosphite in 200 ml of diethylether at -10°C under a nitrogen atmosphere was added 26.0 g (300 mmol, 6 eq) of morpholine over 1 h. The reaction mixture was allowed to warm to room temperature and stirred an additional 16 h. Removal of the amine salt by filtration and evaporation of the solvent gave a colorless liquid. The crude material was fraction¬ ally distilled from calcium hydride to afford 9.5 g (81%) of the desired compound: b.p. 82-84°C. The following Example IV shows a typical experi¬ mental procedure for the preparation of phosphor¬ amidites according to the present invention. Al¬ though the description is specific for the formation of the preparation of the compound of general Formula I wherein B is 1-thyminyl and Z is diisopropyl amino, the remaining compounds of general Formula I may be prepared along similar protocols by those skilled in the synthesis art. The 31P NMR spectral data for phosphoramidites prepared following the typical procedure given in Example IV are contained in Table I. EXAMPLE IV Preparation of Phosphoramidite To a mixture of 544 mg (1.0 mmol) of the 1-Thy- minyl substituted deoxynucleoside of Formula II and 86 mg (0.5 mmol, 0.5 eq) of the diisopropylamino tetrazole of Formula V in 5 ml of dry dichloromethane under a nitrogen atmosphere was added 282 mg (1.1 mmol, 1.1 eq) of bis-(diisopropylamino)methoxy¬ phosphine. After 1 h the reaction mixture was poured into saturated aqueous sodium bicarbonate and the organic layer was washed with brine and dried ever sodium sulf te. Evaporation of the solvent gave a white foam which was then taken up in 3 ml of dichlor¬ omethane and precipitated in 300 ml of cold hexanes (-78°C) . The resultant suspension was filtered cold and dried under vacuum to afford 700 mg (87%) of a 1:1 diastereomeric mixture of the desired diisopropyl phosphoramidite compound according to general Formula I, as an amorphous solid. TABLE I 31 P NMR SPECTRAL DATA FOR PHOSPHORAMIDITES SPECTRAL B DATA ( £) diisopropylamino 1-thyminyl 148.5;148.1 diisopropyamino 9- (N-6-benzoyladeninyl) 148.5;148.3 diisopropylamino 9-(N-2-isobutyrylguaninyl) 148.3;148.1 diisoprop lamino 1- (N-4-benzoylcytosinyl) 148.6; 148.1 morpholino 1-Thyminyl 143.4;143.2 morpholino 9-(N-6-benzoyladeninyl) 143.4 morpholino 9- (N-2-isobutyrylguaninyl) 143.7;143.2 morpholino 1- (N-4-benzoylcyrosinyl) 143.7;143.4 The ease of phosphine preparation is exemplified by the direct procedure used for the production of bis- (diisopropylamino)methoxyphosphine. Excess diisopropyl- amine was added to a solution of methyldichlorophosphite. Removal of the amine salt and fractional distillation of the crude liquid afforded a 77% yield of the ' desired product. The bis-aminophosphine is very stable when stored at -10°C; even with repeated sampling of the phosphine, the 31p NMR spectrum was unchanged after one month. When oxygen was bubbled through a solution of the bis-aminophosphine in dichloromethane for 24 h, the 31P NMR indicated approximately 8% degradation and 11% hydrolysis. The bis-morpholinomethoxyphosphine was prepared in a similar manner (80% yield) and had stability comparable to that obtained for the bis-(diisopropylamino)methoxy¬ phosphine. The mixed (diisopropylaminomorpholino) methoxyphosphine could not be obtained cleanly in the same one pot procedure as above; however, treatment of the mixed (diisopropylaminochloro)methoxyphosphine with one equivalent of morpholine and triethylamine gave a 77% yield of methoxyphosphine contaminated with about 2% of the thermodynamically more stable bis-(diisopropylamino) methoxyphosphine. The reaction of the starting deoxynucleosides of general Formula II with bis-(diisopropylamino)methoxy¬ phosphine gave the appropriate diisopropylamino phosphor¬ amidites of general Formula I in 82% to 92% isolated yield after precipitation from cold hexanes. The typical preparation of phosphoramidites depicted in Example IV required the addition of 1.1 equivalents of the diisopropyl phosphine to a mixture of the nucleoside and 0.5 equivalents of the catalyst (general Formula V) in dichloromethane. The TLC showed the reaction to be complete in about 20 min. After an aqueous work-up and precipitation of the material in cold hexanes, the product was obtained in 87% yield as an amorphous solid. The 31P NMR spectrum showed two signals corresponding to a 1:1 diastereo eric mixture of phosphoramidites. Furthermore, the spectral data of the phosphoramidites, prepared according to the present invention, was found to be identical with that of control samples of -phosphor¬ amidites prepared according to the procedure of McBride and Caruthers, and reported in Tetrahedron Letters, 22: 1859-1862. Phosphoramidites prepared using the procedure according to the present invention were tested as syn- thons by constructing d(GGGAATTCCC) , a self-complementary segment containing the EcoRI recognition sequence. The deoxyoligonucleotide was synthesized and deprotected using standard procedures according to Caruthers in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, and isolated by gel electrophoresis in a 57% yield. The average coupling yield (measured spectrophotometrically from the dimethoxytrityl cation, 4 Λ v 498 n , €= 7.2 x 10 ) was greater than 95%. After end-labeling with \ηr- P] ATP and T4-kinase, the self-com¬ plementary segment was found to be degraded completely with EcoRI indicating that the synthesis was satisfactory (See Green, P. J. , et al (1975) J. Mol. Biol. 99:237). Bis-(diisopropylamino) ethoxyphosphine was also tested as part of an _in situ synthesis protocol. Phos¬ phoramidites according to general Formula I were each prepared as 0.1M solutions in dry acetonitrile containing bis-(diisopropylamino)methoxyphosphine (1.0 eq) and the diisopropylamine tetrazolides of general Formula V (0.5 eq) . The segment d(GGGAATTCCC) was then prepared in 50% isolated yield (average coupling yield was 94%) using the following procedure. EXAMPLE V To a suspension of the appropriately der vatized silica support (See Chow, F. , Ke pe, T. , and Palm, G. (1981) Nucleic Acids Res. 9:2807-2817) in 250 L of a 0.4 M solution of tetrazole in ac ' etonitrile was added 450 L (45 mol, 20 eq) of a 0.1 M solution of the appropriate deoxynucleoside phosphoramidite generated in situ. The mixture was allowed to stand five minutes. The solution was then removed by filtration and the silica was washed with dry acetonitrile. After acetylation, oxidation, and detritylation in the usual manner, (See Caruthers in Chemical and Enzymatic Synthesis of Gene Fragments, A Laboratory Manual, the cycle was repeated until the synthesis was complete. Quantitative cleavage at the EcoRI site demonstrated that the In situ method outlined in the proceeding example can be used satisfactorily for the synthesis of DNA segments. In summary, the present invention demonstrates that very stable dialkylamino phosphines according to general Formula IV can be used as phosphitylating reagents to form deoxynucleoside phosphoramidites cleanly and in good yields. The reactions are catalytic in either tetrazole or the corresponding amine hydrotetrazolides. These reactions have furthermore been found to be selective to the formation of only 3'-deoxynucleoside phosphoramidites without concurrent hydrolysis to phosphonus acid or synthesis of the 3'-3 * dinucleoside phosphit-3. This selectivity renders these reagents attractive for the in situ generation of phosphoramidites useful ' for DNA synthesis on solid supports. From the foregoing description, one skilled in the art can easily ascertain the essential characteristics of my invention and without departing from the spirit and scope thereof, can make various changes and/or modifica¬ tions to the invention for adapting it to various usages and conditions. Accordingly, such changes and modifica¬ tions are properly intended to be within the full range of equivalents of the following claims. Having thus described my invention and the manner and process of making and using it in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most closely connected, to make and use the same, and having set forth the best modes for carrying out my invention:

Claims: I CLAIM: 1. A compound of the formula: wherein X is selected from the group consisting of morpholino and (lower alkyl)-. 2. The compound of Claim 1 wherein X is morpholino. 3. The compound of Claim 1 wherein X is N(CH 3 CH CH.,)-,. 4. A method for the preparation of a compound of the formula: n CH 3 CT F ^Z wherein B is selected from 1-thyminyl; l-(N-4 benzoyl- cytosinyl) ; 9-(N-2-isobutyrylguaninyl) ; and 9-(N-6-ben- zoyladeninyl) , wherein Z is selected from morpholino and N(lower alkyl) 2 ; and wherein DMT is di-p-anisylphenyl- methyl; which comprises reacting a first compound of the form la: with a second compound of the formula; CH-30 P( Z 5. The method according to Claim 4 which further comprises reacting said compounds in the presence of a catalytic compound selected from the group 6. The method according " to Claim 5 wherein N (lower alkyl) 2 of said first and second compound is N(CH, CH CH,) 2 , and wherein the catalytic compound is 7. The method according to Claim 5 wherein Z is morpho¬ lino, and wherein is 8. A method for the preparation of deoxyoligonucleo- tides which comprises (a) reacting a compound of the formula DM wherein B is selected from 1-thyminyl; l-(N-4 benzoyl- cytosinyl) ; 9-(N-2-isobutyrylguaninyl) ; and 9-(N-6-ben- zoyladeninyl) , and wherein DMT is di-p-anisylphenyl- methyl, with a compound of the formula wherein Z is selected from N(CH_ CH CH 3 ) 2 and morpholino to form a reaction mixture; (b) adding said reaction mixture to a suspension of appropriately derivatized support material; (c) allowing said reaction mixture-suspension to react for an appropriate length of time; and (d) isolating said support material from * said reaction-mixture suspension. 9. The method according to Claim 8 which further comprises adding a catalytic compound from the group to the reaction mixture of step (a) .

Inventors: CARUTHERS MARVIN H, CARUTHERS, MARVIN, H.
Applicants: UNIVERSITY PATENTS INC, UNIVERSITY PATENTS, INC.
Application year: 1985
Main classification: C07H15
IPC codes: C07B61, C07F9, C07H19, C07H21