Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/06—Pyrimidine radicals
  • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
  • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
  • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
  • C07H19/16—Purine radicals
  • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
  • Y02P20/00—Technologies relating to chemical industry
  • Y02P20/50—Improvements relating to the production of bulk chemicals
  • Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Definitions

• the present invention relates to the synthesis of oligonucleotides, and intermediates useful in the synthesis thereof.
• oligonucleotides are based on the selective formation of hybrids between antisense oligonucleotides and complementary nucleic acids, such as messenger RNAs (mRNAs) .
• mRNAs messenger RNAs
• Such hybrids inhibit gene expression by blocking protein translation.
• Successful inhibition of gene expression requires the antisense oligonucleotide to be nuclease resistant so that it can be transported through biological membranes and can hybridize selectively to a target complementary nucleic acid, thereby actively blocking protein translation.
• those bearing phosphorothioate internucleotide linkages are the most nuclease resistant and, therefore, are the most widely used.
• Oligonucleotides bearing phosphorothioate internucleotide linkages are typically prepared by sulfurization of a phosphite precursor which, in effect, substitutes a sulfur atom for one of the non-bridging oxygen atoms normally present in phosphodiesters . This substitution results in a stereogenic center at the phosphorus atom.
• sulfurization of oligonucleotide phosphodiesters prepared by conventional methods results in the formation of complex mixtures of diastereomers, since the precursors are typically diastereomeric with respect to phosphorus.
• the stereochemistry of the phosphorus center is important in imparting nucleolytic stability in the oligonucleotide.
• the most commonly used synthetic method for the synthesis of thioated oligonucleotides is the phosphoramidite method with stepwise sulfurization (see, e.g., U.S. Patent Nos . 4,415,732, 4,668,777, 4,973,679, 4,845,205, and 5,525,719).
• This method uses tricoordinated phosphorus precursors that normally produce products containing a mixture of different thioated oligonucleotide stereoisomers .
• the lack of stereoselectivity in the phosphoramidite process is primarily due to the non-stereoselective and non- stereospecific acid-catalyzed nucleophilic substitution reaction, which is typically required to effect substitution. Even when diastereomerically pure P-chiral precursors are used, the coupling reaction proceeds with full epimerization at phosphorus.
• Another attempt at controlling phosphorus stereochemistry in oligonucleotide synthesis involves a method for the stereospecific synthesis of thioated oligonucleotides utilizing tetracoordinated phosphorus precursors to accommodate base-catalyzed nucleophilic substitutions.
• this approach also has limited applicability because a different type of tetracoordinated phosphorus precursor must be used to generate a particular type of product, for example, phosphates, phosphorothioates and phosphoroselenoates .
• the structure of the desired product is determined by the structure of the tetracoordinated phosphorus precursor at the coupling step. Additionally, separation of the diastereomers is difficult, and these tetracoordinated phosphorus precursors also are hydrolytically unstable.
• R 1 is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, wherein R 1 is unsubstituted or substituted.
• R 2 and R 2' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, wherein R 2 and/or R 2' is unsubstituted or substituted.
• R 3 and R 3' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, wherein R 3 and/or R 3' is unsubstituted or substituted.
• R 2 or R 2' in combination with either of R 3 or R 3' , together with the carbon atoms to which they are bonded, form a cyclic substituent, which can be unsubstituted or substituted.
• R 4 is a protecting group or a solid support.
• R 5 is H or an alkyl, which is unsubstituted or substituted.
• R 6 is a protecting group, an amidoalkyl in which the nitrogen atom thereof is 2, 4, or 5 carbon atoms removed from the oxygen of OR 6 , an alkyl, an alkyl ketone, an alkenyl, an alkynyl, a cycloalkyl, an aryl, or an aralkyl, wherein R 6 is unsubstituted or substituted.
• R 15 is H or a protecting group.
• Q and Q 1 are the same or different and each is a nucleoside, a oligonucleotide comprising a nucleoside, or an oligomer comprising a nucleoside, wherein the nucleoside is of the formula:
• B is a labeling group, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl , an aralkyl, an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted, and E is H, a halogen, OR 13 , NHR 13 , NR 13 R 14 , wherein R 13 and R 14 are the same or different and each is H, a protecting group, an alkyl, or an acyl .
• X and X 1 are the same or different and each is 0, S, or Se, and n is an integer from 1 to about 300.
• Q can be the same or different in each of the units defined by n of formula (III), when n
• the present invention further provides a method of preparing a polymer, including the steps of:
• the method can further comprise repeating steps (a) through (c) , one or more times as necessary, until a polymer of specified length is obtained.
• Fig. 1 illustrates the synthesis of an N- acylphosphoramidite .
• Fig. 2 illustrates a solid phase synthesis of an oligonucleotide .
• Fig. 3 illustrates the solid phase stereocontrolled synthesis of phosphorothioate oligonucleotides.
• Fig. 4 illustrates the synthesis of various compounds of formula (I) .
• Fig. 5 illustrates the preparation of acyclic N- acylphosphoramidites and their application in solid phase syntheses .
• Fig. 6 illustrates an oligonucleotide synthesis using an alternative method.
• Fig. 7 illustrates the preparation of a particular oligonucleotide using either cyclic or acyclic N- acylphosphoramidites .
• Fig. 8A illustrates the structure of a standard phosphoramidite coupling reagent used in conventional nucleotide coupling reactions.
• Fig. 8B illustrates the structure of a P-chiral (S p ) N-acylphosphoramidite of the present invention.
• Fig. 8C illustrates the structure of a P-chiral (R p ) N-acylphosphoramidite of the present invention.
• Fig. 9A illustrates the structure of a standard phosphoramidite coupling reagent used in conventional nucleotide coupling reactions.
• Fig. 9B illustrates the structure of a P- diastereomeric (R P ,S P ) N-acylphosphoramidite of the present invention.
• Fig. 10A illustrates the HPLC chromatogram for a mixture of the four possible P-diastereomeric oligonucleotide phosphorothioate trimers d(C PS C ps C) prepared using standard phosphoramidite chemistry.
• Fig. 10B illustrates the HPLC chromatogram for the pure S p ,S p diastereomer of d(C ps C ps C) prepared in accordance with the present invention.
• Fig. IOC illustrates the HPLC chromatogram obtained by co-injecting the pure S p/ S p diastereomer of d(C PS C ps C) with the mixture containing all four possible P- diastereomers .
• Fig. 10D illustrates the HPLC chromatogram for the pure R P ,R P diastereomer of d(C ps C ps C), prepared in accordance with the present invention.
• Fig. 10E illustrates the HPLC chromatogram obtained by co-injecting the pure R P ,R P diastereomer of d(C ps C ps C) with the mixture containing all four possible P- diastereo ers .
• Fig. 11A illustrates the HPLC chromatogram for a mixture of the eight possible P-diastereomeric oligonucleotide phosphorothioate tetramers of d (C PS C PS C) prepared using standard phosphoramidite chemistry.
• Fig. 11B illustrates the HPLC chromatogram for the pure R p ,S p ,Rp diastereomer of d (C PS C PS C PS C) prepared in accordance with the present invention.
• Fig. 11C illustrates the HPLC chromatogram obtained by co-injecting the pure R P ,S P ,R P diastereomer of d(C ps C ps C ps C) with the mixture containing all eight possible P-diastereomers .
• Fig. 12A illustrates the HPLC chromatogram for the dimeric phosphodiester d(T P0 G) prepared under standard phosphoramidite coupling conditions in the absence of moisture .
• Fig. 12B illustrates the HPLC chromatogram for the product obtained in the preparation of dimeric phosphodiester d(T p0 G) under standard phosphoramidite coupling conditions in the presence of moisture (0.1% water) .
• Fig. 12C illustrates the HPLC chromatogram for the dimeric phosphodiester d(T P0 G) prepared by an N- acylphosphoramidite coupling reagent of the present invention in the absence of moisture.
• Fig. 12D illustrates the HPLC chromatogram for the product obtained in the preparation of dimeric phosphodiester d(T P0 G) by an N-acylphosphoramidite coupling reagent of the present invention in the presence of moisture (0.1% water).
• Fig. 13A illustrates a general example of a thermal cleavage of the bond linking an organic moiety to a non- bridging phosphate or phosphorothioate oxygen.
• Fig. 13B illustrates specific examples thermal cleavage of the bond linking an organic moiety to a non- bridging phosphate or phosphorothioate oxygen.
• DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated, at least in part, on the surprising and unexpected discovery that the utilization of N-acylphosphoramidites as a coupling vehicle, for example, with respect to the coupling of nucleoside-containing fragments, occurs without any epimerization at phosphorus.
• post-coupling reactions and transformations i.e., the synthetic steps that are carried out after step (b) of the method as set forth below
• oxidation, sulfurization, and deprotection occur without epimerization at the phosphorus atom.
• the present inventive method of synthesizing polymers has tremendous synthetic advantages that are unprecedented in the art, particularly with respect to the synthesis of oligonucleotides, in that it enables the facile production of P-chiral oligomeric or polymeric products, with complete control of stereochemistry with respect to the phosphorus atom. Moreover, stereochemistry can be controlled for tricoordinated and tetracoordinated phosphorus atoms .
• N-acyl functionality of the N-acylphosphoramidite ring (which functions as a leaving group in coupling step (b) of the method as set forth below) is not labile under the coupling conditions utilized for the displacement thereof; rather, displacement occurs via a purely bimolecular nucleophilic mechanism. As such, there is no "scrambling" or epimerization of the phosphorus atom in the coupling step.
• the standard phosphoramidite approach presently utilized in the art involves displacement of an amino functionality on phosphorus, and requires acidic conditions for the displacement thereof.
• the phosphorus- nitrogen bond in a standard phosphoramidite is labile under acidic conditions (even when a mild acid such as tetrazole is used) , invariably resulting in epimerization of the phosphorus atom in the resulting coupled adduct.
• the compounds and methods of the present invention therefore, provide for the stereospecific substitution of tricoordinated phosphorus compounds under basic conditions.
• the monomeric compounds of the present invention preferably of formulae (I) and (II)
• the oligomeric compounds of the present invention preferably of formula (III)
• the compounds of the present invention are hydroxyl-protected monomer- O- (O-protected) - (N- acyl) phosphoramidites , or hydroxyl protected oligomer/polymer-O- (O-protected) - (N- acyl) phosphoramidites, exemplified by formulae (I) -(III).
• the compound is a hydroxyl- protected monomer-O- (N-acyl ) -1,3, 2-substituted oxazaphospholane (formula (I) ) , which can be isolated as the Rp or Sp chiral form, to be used in the synthesis of polymers containing stereogenic phosphorus centers of predetermined configuration in a site-specific manner.
• the present invention provides a compound of the formula :
• R 1 is an alkyl (e.g., a C-_-C 6 alkyl), an alkenyl (e.g., a C 2 -C 6 alkenyl), an alkynyl (e.g., a C 2 -C 6 alkynyl), a cycloalkyl (e.g., a C 3 -C 7 cycloalkyl), an aryl (e.g., phenyl or naphthyl) , or an aralkyl (e.g., benzyl, phenethyl, phenylpropyl , or the like) , wherein R 1 is unsubstituted or is substituted with one or more substituents, which are the same or different, selected from the group consisting of R 7 , OR 7 , SR 7 , NR 8 COR 7 , NR 8 CSR 7 , NR 8 C0 2 R 7 , NR 8 C(0)SR 7
• R 1 is an alkyl, which is preferably a C x -C 6 alkyl, that is unsubstituted or is substituted with one or more substituents, which are the same or different, selected from the group consisting of fluorine, OR 7 and SR 7 , wherein R 7 is an alkyl or an aryl.
• R 1 is a C-_-C 6 alkyl, it is more preferably a C 1 -C 3 alkyl which is unsubstituted or substituted with one or more fluorine atoms, for example, a methyl, optionally substituted with one or more fluorine atoms, most preferably fluoromethyl.
• R 1 is a alkyl substituted with OR 7 or SR 7 , R 1 is most preferably a methoxymethyl, a methylthiomethyl or a phenoxy .
• R 2 and R 2' can be any suitable substituent, preferably R 2 and R 2' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl or an aralkyl.
• R 2 or R 2' is an alkyl, it is preferably a alkyl.
• R 2 or R 2' is an alkenyl, it is preferably a C 2 -C 6 alkenyl.
• R 2 or R 2' is an alkynyl, it is preferably a C 2 -C 6 alkynyl.
• R 2 or R 2' is a cycloalkyl, it is preferably a C 3 -C 7 cycloalkyl.
• R 2 and/or R 2' can be unsubstituted or substituted with one or more substituents selected from the group consisting of OR 7 , CN, N0 2 , N 3 , and a halogen.
• R 2 and R 2' are hydrogen.
• R 3 and R 3' can be any suitable substituent, preferably R 3 and R 3' are the same or different and each is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl or an aralkyl.
• R 3 or R 3' is an alkyl, it is preferably a alkyl.
• R 3 or R 3' is an alkenyl, it is preferably a C 2 -C 6 alkenyl.
• R 3 or R 3' is an alkynyl, it is preferably a C 2 -C 6 alkynyl.
• R 3 or R 3' is a cycloalkyl, it is preferably a C 3 -C 7 cycloalkyl.
• R 3 and/or R 3' can be unsubstituted or substituted with one or more substituents selected from the group consisting of a trialkylsilyl, an aryldialkylsilyl, an alkyldiarylsilyl, CN, N0 2 , N 3 , a halogen, OR 7 , P (0) (OR 7 ) (OR 8 ) , COR 9 , CSR 9 , C0 2 R 9 , COSR 9 , CSOR 9 , CONR 8 R 9 , CSNR 8 R 9 , S0 2 R 9 , and S0 2 NR 8 R 9 , wherein R 7 is as defined herein.
• R 9 can be any suitable substituent
• R 9 preferably is H, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl or an aralkyl.
• R 9 is an alkyl, it is preferably a C- L -Cg alkyl.
• R 9 is an alkenyl, it is preferably a C 2 -C 6 alkenyl.
• R 9 is an alkynyl, it is preferably a C 2 -C 6 alkynyl.
• R 9 is a cycloalkyl, it is preferably a C 3 -C 7 cycloalkyl.
• R 9 can be unsubstituted or it can be substituted with one or more substituents selected from the group consisting of CN, N0 2 , N 3 , and a halogen.
• R 3 is a phenyl, R 2 , R 2' and R 3' all are H, and R 1 is selected from the group consisting of methyl, fluoromethyl, methoxymethyl and phenoxymethyl .
• R 3 is a vinyl, R 2 is H, and R 1 is selected from the group consisting of methyl, fluoromethyl, methoxymethyl and phenoxymethyl .
• R 3 and R 2 are both H, and R 1 is a methyl.
• R 2 and R 3 , R 2 and R 3' , R 2' and R 3 , and R 2' and R 3' can comprise a cyclic substituent.
• a combination of either of R 2 and R 2' with either of R 3 and R 3' can comprise a cyclic substituent.
• R 2 or R 2' and R 3 or R 3' together with the carbon atoms to which they are bonded, comprise a cyclic substituent of the formula:
• p is an integer from 0-6 and a-d are the same or different and each is selected from the group consisting of H, an alkyl, a nitro, an amino, a hydroxy, a thio, a cyano and a halogen.
• p is 1 and all of a-d are H.
• p is 1, all of a-d are H, and R 1 is fluoromethyl .
• R 4 is a protecting group or a solid support.
• R 5 is H or an alkyl, preferably a C 1 -C 3 alkyl, which can be unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of OR 7 , CN, N0 2 , N 3 , and a halogen, wherein R 7 is as defined herein.
• R 6 can be any suitable substituent
• R ⁇ preferably is a protecting group, an alkyl, an amidoalkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, an alkyl ketone, or an aralkyl.
• R 6 is an alkyl, it is preferably a C-_-C 6 alkyl.
• R 6 is an amidoalkyl, it is preferably an amidoalkyl, more preferably a C 1 -C 6 amidoalkyl, in which the nitrogen atom thereof is 2, 4, or 5 atoms removed from the oxygen atom of OR 6 , and is most preferably an amidoalkyl whose amide is easily hydrolyzed or cleaved and liberates an amine which is separated from the oxygen of OR 6 by 2 , 4, or 5 carbon atoms.
• R 6 is an alkenyl, it is preferably a C 2 -C 6 alkenyl.
• R 6 is an alkynyl, it is preferably a C 2 -C 6 alkynyl.
• R 6 is a cycloalkyl, it is preferably a C 3 -C 7 cycloalkyl.
• R 6 can be unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of CN, N0 2 , N 3 , and a halogen.
• R 15 is H or a protecting group.
• Q and Q 1 are the same or different and each is a nucleoside, an oligonucleotide comprising a nucleoside, or an oligomer comprising a nucleoside (e.g., an oligonucleotide or the like) .
• Q and/or Q 1 can be a natural nucleoside or a modified/unnatural nucleoside.
• Q and/or Q 1 also can be an oligomer comprising one or more natural or modified/unnatural nucleosides. Modified nucleosides can be obtained, for example, by any suitable synthetic method known in the art for preparing nucleosides, derivatives, or analogs thereof.
• Modified nucleosides include, but are not limited to, chemically modified nucleosides used as building blocks for "labeled” oligonucleotides, or suitable precursors or analogs used in the preparation of such modified nucleosides.
• chemically modified nucleosides are described, for example, in Smith et al . , Nucleosides & Nucleotides, 15(10), 1581-1594 (1996) ("Smith et al . " ) . Smith et al .
• nucleosides and oligomers which include such nucleosides
• the base ring is replaced by a carboxylic acid to which is appended various "labeling" groups (e.g., biotin, cholesterol, fluorenylmethoxycarbonyl (Fmoc) , and trifluoroacetyl) via a modified amide linker.
• Labeling groups e.g., biotin, cholesterol, fluorenylmethoxycarbonyl (Fmoc) , and trifluoroacetyl
• Modified nucleosides also include other chemically modified nucleosides, for example, nucleosides described in Smith et al . in which the base ring is replaced by a hydroxyethyl, a cyano, or a carboxylic acid (including esters and amides thereof) .
• Modified nucleosides further include nucleosides in which the base ring is replaced by a cyclic substituent, for example, an aryl, a cycloalkyl, a heterocycloalkyl, or a heteroaryl (other than a base naturally occurring in nucleosides) .
• a cyclic substituent for example, an aryl, a cycloalkyl, a heterocycloalkyl, or a heteroaryl (other than a base naturally occurring in nucleosides) .
• Q and/or Q 1 also include oligonucleotides, which can be natural or modified.
• Modified oligonucleotides include, for example, oligonucleotides containing a modified nucleoside (as described herein) , oligonucleotides containing a modified internucleotide linkage, or oligonucleotides having any combination of modified nucleosides and internucleotide linkages (even if a natural nucleoside is present in the oligomer chain) .
• Oligonucleotides whose nucleosides are connected via modified internucleotide linkages can be found, for example, in Waldner et al . , Bioorg . Med. Chem. Letters , 6, 19, 2363-2366 (1996) ( "Waldner et al . " ) , which describes the synthesis of oligonucleotides containing various amide internucleotide linkages.
• Q and Q 1 are the same or different and each is a nucleoside substituent (or an oligonucleotide comprising a nucleoside, a nucleoside, or an oligomer comprising a nucleoside) of the formula:
• B is a labeling group, an alkyl, an alkenyl, an alkynyl, a cycloalkyl, an aryl, a heteroaryl, a heterocycloalkyl, an aralkyl, an amino, an alkylamino, a dialkylamino, a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a protecting group, R 11 , OR 11 , NHR 11 , NR 1X R 12 , CN, N0 2 , N 3 , and a halogen, wherein R 11 and R 12 are the same or different and each is H, a protecting group, or an alkyl; and E is H, a halogen, OR 13 , NHR 13 , or NR 13 R 14
• B is an alkyl, preferably it is a alkyl.
• B is an alkenyl, it is preferably a C 2 -C 6 alkenyl.
• B is an alkynyl, preferably it is a C 2 -C 6 alkynyl.
• B is a cycloalkyl, preferably it is a C 3 -C 7 cycloalkyl.
• R 13 and/or R 14 is an alkyl, preferably it is a alkyl.
• the Q in the N- acylphosphoramidites of formulae (I) and (II) , and the Q and Q 1 in the intermediates obtained therefrom (formula (III) ) include nucleosides (natural and modified) and oligomers which include one or more of such nucleosides.
• Any suitable monomer-monomer, monomer-oligomer, oligomer- monomer, or oligomer-oligomer coupling reaction can be accomplished, stereospecifically, using the compounds and methods of the present invention.
• the N-acylphosphoramidite of formula (I) or (II) can be used to stereospecifically couple a suitably protected nucleoside (or even a suitably protected oligonucleotide) to an oligonucleotide.
• the N-acylphosphoramidite of the present invention can be attached to an oligomer such as, for example, an oligonucleotide (i.e., wherein Q is an oligonucleotide), as well as a monomer (i.e., wherein Q is a nucleoside) .
• the nucleophile which is coupled to an N-acylphosphoramidite of the present invention also can be monomeric or oligomeric.
• Q 1 also includes oligomers that contain, as a component thereof, a nucleoside substituent as described herein.
• the N-acyl group includes carbonyl (wherein X is O) , thiocarbonyl (wherein X is S) , and selenocarbonyl (wherein X is Se) .
• the N-acyl group is a carbonyl, wherein X is O.
• Examples of monomeric compounds of the present invention include compounds of the formulae:
• Stereospecific coupling reactions can be carried out successively "n" times, for example, starting with a nucleophile R 4 -0-Q 1 -OH (wherein R 4 and Q 1 are as defined herein) , and continuing thereafter, to provide an intermediate of formula (III) , wherein n is an integer from 1 to about 300. It will be appreciated that when a compound of formula (I) is reacted with a nucleophile R 4 -0-Q 1 -OH, then “R 4 " of formula (I) is represented by "R 15 " of formula (III) . When the protecting group R 15 is removed, then R 15 becomes a hydrogen.
• R 4 and R 15 desirably are not both solid supports in formula (III]
• R is hydrogen
• another coupling reaction can be carried out, and the process repeated successively, until a polymer of desired length or structure is obtained.
• the Q substituent of formula (I) can be the same or different, as desired, to obtain a variety of different combinations.
• Q can be the same or different in each of the units defined by n, when n is greater than 1.
• n is in the range of from about 3 to about 200; more preferably, n is in the range from about 10 to about 40; and most preferably n is in the range from about 15 to about 25.
• Q and/or Q 1 is a nucleoside substituent of the formula:
• R ,4 is advantageously a solid support or a protecting group.
• the protecting group is most preferably a 4 , 4 ' -dimethoxytrityl protecting group.
• alkyl means a straight-chain or branched-chain alkyl radical which, unless otherwise specified, contains from about 1 to about 20 carbon atoms, preferably from about 1 to about 10 carbon atoms, more preferably from about 1 to about 8 carbon atoms, and most preferably from about 1 to about 6 carbon atoms.
• alkyl radicals examples include methyl, ethyl, propyl, isopropyl, -n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, isoamyl, hexyl , octyl, dodecanyl, and the like.
• alkenyl means a straight-chain or branched-chain alkenyl radical, which has one or more double bonds and, unless otherwise specified, contains from about 2 to about 20 carbon atoms, preferably from about 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms, and most preferably from about 2 to about 6 carbon atoms .
• alkenyl radicals include vinyl, allyl, 1, 4-butadienyl, isopropenyl, and the like.
• alkynyl means a straight -chain or branched-chain alkynyl radical, which has one or more triple bonds and contains from about 2 to about 20 carbon atoms, preferably from about 2 to about 10 carbon atoms, more preferably from about 2 to about 8 carbon atoms, and most preferably from about 2 to about 6 carbon atoms.
• alkynyl radicals include ethynyl, propynyl (propargyl) , butynyl, and the like.
• alkylamino and dialkylamino mean an alkyl or a dialkyl amine radical, wherein the term “alkyl” is defined as above .
• alkylamino radicals include methylamino (NHCH 3 ) , ethylamino (NHCH 2 CH 3 ) , n- propylamino, isopropylamino, n-butylamino, isobutylamino, sec-butylamino, tert-butylamino, n-hexylamino, and the like.
• dialkylamino radicals include dimethylamino (N(CH 3 ) 2 ), diethylamino (N (CH 2 CH 3 ) 2 ) , di-n- propylamino, diisopropylamino, di-n-butylamino, diisobutylamino, di-sec-butylamino, di- tert-butylamino, di- ⁇ -hexylamino, and the like.
• cycloalkyl means a monocyclic alkyl radical, or a polycyclic alkyl which comprises one or more alkyl carbocyclic rings, which can be the same or different when the polycyclic radical has 3 to about 10 carbon atoms in the carbocyclic skeleton of each ring.
• the cycloalkyl has from about 4 to about 7 carbon atoms, more preferably from about 5 to about 6 carbons atoms.
• monocyclic cycloalkyl radicals include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclodecyl, and the like.
• polycyclic cycloalkyl radicals include decahydronaphthyl, bicyclo [5.4.0] undecyl, adamantyl , and the like.
• aryl refers to an aromatic carbocyclic radical, as commonly understood in the art, and includes monocyclic and polycyclic aromatics such as, for example, phenyl and naphthyl radicals, which radicals are, unless indicated otherwise, unsubstituted or substituted with one or more substituents selected from the group consisting of a halogen, an alkyl, an alkoxy, an amino, a cyano, a nitro, and the like.
• the aryl has one or more six-membered carbocyclic rings including, for example, phenyl, naphthyl, and biphenyl , and are unsubstituted or substituted as set forth herein.
• aralkyl means alkyl as defined herein, wherein an alkyl hydrogen atom is replaced by an aryl as defined herein.
• aralkyl radicals include benzyl, phenethyl, 1-phenylpropyl , 2 -phenylpropyl , 3-phenylpropyl, 1-naphthylpropyl , 2-naphthylpropyl, 3-naphthylpropyl, 3 -naphthylbutyl , and the like.
• heterocycle and heterocyclic refer to both heterocycloalkyls and heteroaryls .
• heterocycloalkyl means a cycloalkyl radical as defined herein (including polycyclics) , wherein at least one carbon of a carbocyclic ring is substituted with a heteroatom such as, for example, O, N, or S .
• the heterocycloalkyl optionally has one or more double bonds within a ring, and may be aromatic, but is not necessarily aromatic .
• the heterocycloalkyl preferably has 3 to about 10 atoms (members) in the carbocyclic skeleton of each ring, preferably from about 4 to about 7 atoms, more preferably from about 5 to about 6 atoms .
• heterocycloalkyl radicals include epoxy, aziridyl, oxetanyl, tetrahydrofuranyl , ribose, dihydrofuranyl, piperidinyl, piperazinyl, pyranyl , morpholinyl, and the like.
• heteroaryl means a radical defined by an aromatic heterocyclic ring as commonly understood in the art, including monocyclic radicals such as, for example, imidazole, thiazole, pyrazole, pyrrole, furane, pyrazoline, thiophene, oxazole, isoxazole, pyridine, pyridone, pyrimidine, cytosine, 5-methylcytosine, thymine, pyrazine, and triazine radicals, and polycyclics such as, for example, quinoline, isoquinoline, indole, purine, adenine, guanine, N 6 -methyladenine, and benzothiazole radicals, which heteroaryl radicals are unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a halogen, an alkyl, an alkoxy, an amino, a cyano, a
• heterocycloalkyl and the heteroaryl substituents can be coupled to the compounds of the present invention via a heteroatom, such as nitrogen (e.g., 1-imidazolyl) .
• heteroaryls as defined herein, are not necessarily “aromatic” in the same context as phenyl is aromatic, although heteroaryls nonetheless demonstrate physical and chemical properties associated with aromaticity, as the term is understood in the art .
• nucleoside includes all modified and naturally occurring nucleosides, including all forms of furanosides found in nucleic acids. Naturally occurring nucleosides include, for example, adenosine, guanosine, cytidine, thymidine, and uridine .
• Nucleoside “derivatives” or “analogs” include synthetic nucleosides as described herein. Nucleoside derivatives also include nucleosides having modified base moieties, with or without protecting groups. Such analogs include, for example, deoxyinosine, 2,6- diaminopurine-2 ' -deoxyriboside, 5-methyl-2 ' - deoxycytidine, and the like.
• the base rings most commonly found in naturally occurring nucleosides are purine and pyrimidine rings .
• Naturally occurring purine rings include, for example, adenine, guanine, and N 6 - methyladenine .
• Naturally occurring purine rings include, for example, cytosine, thymine, and 5-methylcytosine.
• nucleoside derivatives include other purine and pyrimidine derivatives, for example, halogen-substituted purines (e.g., 6-fluoropurine) , halogen-substituted pyrimidines, N 6 -ethyladenine, N 6 - (alkyl) -cytosines, 5-ethylcytosine, and the like.
• halogen-substituted purines e.g., 6-fluoropurine
• pyrimidines for example, halogen-substituted pyrimidines, N 6 -ethyladenine, N 6 - (alkyl) -cytosines, 5-ethylcytosine, and the like.
• oligonucleotide as used herein includes linear oligomers of natural or modified nucleosides, and modified ologonucleotides, as described herein. Oligonucleotides include deoxyribonucleosides, ribonucleosides and anomeric forms thereof, and the like. Oligonucleotides are typically linked by phoshodiester bonds, or the equivalent thereof, ranging in size from a few monomeric units (e.g., 3 or 4) to several hundred monomeric units.
• the oligonucleotides of the present invention are oligomers of naturally-occurring nucleosides ranging in length from about 12 to about 60 monomeric units, and more preferably, from about 15 to about 30 monomeric units.
• an oligonucleotide is represented by a sequence of letters, such as "AGTC" it will be appreciated that the nucleotides are in the 5 ' -3 ' orientation from left to right.
• Phosphorus linkages between nucleosidic monomers include phosphodiester bonds and analogs of phosphodiester bonds, such as phoshorothioate, phosphoroselenoate, alkylphosphonate, and phosphoramidate .
• the monomers of the oligonucleotides of the present invention are linked by phosphodiester, phosphorothioate, methanephosphonate, or phosphoramidate linkages .
• oligomer comprising a nucleoside as utilized herein means an oligomer in which at least one of the monomeric units comprises nucleoside, and at least one of the other monomeric units is not a nucleoside.
• one of the monomeric units in the oligomer can be an amino acid, an organic spacer (e.g., an aliphatic or aromatic spacer, an alkylene glycol, or the like), or a carbohydrate (e.g., a sugar).
• one of the non-nucleoside units of the oligomer can itself be oligomeric, for example, a peptide, an oligosaccharide, a polyalkylene glycol, or the like. Any suitable protecting group (sometimes referred to as a blocking group) can be utilized in accordance with the present invention.
• protecting group means a substituent, a functional group, a salt, a ligand, or the like, which is bonded (e.g., via covalent bond, ionic bond, or complex) to a potentially reactive functional group and prevents the potentially reactive functional group from reacting under certain reaction conditions.
• Potentially reactive functional groups include, for example, amines, carboxylic acids, alcohols, double bonds, and the like.
• the protecting group is stable under the reaction conditions for which the protecting group is employed, and also can be removed under reasonably mild deprotection conditions. It will be appreciated that the protecting group to be used in accordance with the present invention depends on the type of substituent that is being protected.
• protecting group for each of a phosphite oxygen, a phosphate oxygen, an amine, a thiol, a hydroxyl, and the like. It will also be appreciated that the choice of protecting groups will depend on other factors such as, for example, the reaction conditions employed in a particular synthetic step, the pH, the temperature, and the relative reactivities of the reactants and/or products.
• Protecting groups for hydroxyls include, for example, silyl ethers (e.g., trimethylsilyl , triethylsilyl , tert-butyldimethylsilyl , dimethylphenylsilyl, and diphenylmethylsilyl) , benzyl carbonates, trityl, monomethoxytrityl, dimethoxytrityl, esters (e.g., acetate, benzoate, and the like), pixyl, tert-butyloxycarbonyl, 9- fluorenylmethyloxycarbonyl (Fmoc), a tetrahydropyranyl group, and the like.
• silyl ethers e.g., trimethylsilyl , triethylsilyl , tert-butyldimethylsilyl , dimethylphenylsilyl, and diphenylmethylsilyl
• benzyl carbonates
• preferred protecting groups include, for example, pixyl, acetyl, 9- fluorenylmethyloxycarbonyl (Fmoc) , t-butyldimethylsilyl (TBDMS) , trityl, monomethoxytrityl ( "MMT” or “MMTr” ) , dimethoxytrityl (“DMT” or “DMTr” ) , and the like.
• Protecting groups for nitrogen include, for example, amides (e.g., trifluoroacetyl, benzoyl, and isobutyryl) , carbamates (e.g., tert-butyloxycarbonyl and N- benzyloxycarbonyl) , trityl, and the like.
• suitable protecting groups can include amides, for example, benzoyl, isobutyryl, and the like.
• carboxyl means any functional group with a carbonyl backbone, and includes functional groups such as, for example, a carboxylic acid, an esters (e.g., ethoxycarbonyl) , and amides (e.g., benzamido) .
• Solid supports are commonly known in the art and include, for example, organic solid supports (e.g., crosslinked polystyrene) and inorganic solid supports.
• the solid support is inorganic, and is more preferably a silica support.
• the solid support includes all linkers, spacers, arms, and other moieties (organic or inorganic) known in the art for manipulating attachment to a solid support. It will also be appreciated that the solid support can be bonded to the molecule directly, without using any of the aforesaid linkers, spacers, arms, or other connecting moieties.
• the monomeric units in the polymers prepared in accordance with the present invention are connected via phosphorus diester linkages, for example, phosphate or chiral phosphate (P-chiral) linkages, as desired.
• the compounds and methods of the present invention are not limited to the synthesis of polymers having only phosphorus-linked monomeric units.
• the compounds of the present invention also can be used to introduce one or more phosphorus-linked units into a polymer having another type of linkage in the structure thereof, for example, a carbonate, a urea, an ester, an ether, or any suitable combination thereof.
• the compound of the present invention is a cyclic N-acylphosphoramidite of the formula :
• R 1 -R 4 , B, and E are as defined herein.
• R 1 , R 2 , R 2' , R 3 and/or R 3' can be selected which have a structure that facilitates removal of the organic moiety remaining on the non-bridging phosphate or phosphorothioate oxygen after coupling has been carried out in accordance with the present invention.
• R 1 advantageously can be an alkyl group substituted with a suitably positioned nucleophile that is protected with an easily removable protecting group (e.g., a "latent" internal nucleophile) .
• the nucleophile on R 1 is positioned such that it can intramolecularly attack (and therefore cleave) the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen.
• R 1 is trifluoroacetamido
• the trifluoroacetyl group can be easily removed, thereby liberating an amine (nucleophile) which, in turn, intramolecularly cleaves the organic moiety from the non- bridging phosphate, phosphorothioate or phosphoroselenoate oxygen as illustrated below in Scheme 1.
• Scheme 1 Other suitably protected “latent” nucleophiles (in addition to an amine, as illustrated in Scheme 1) can be used in this fashion with respect to R 1 , for example, a protected hydroxyl (e.g., an ester or a silyl ether) or a protected thiol (e.g., acetylthio) .
• a protected hydroxyl e.g., an ester or a silyl ether
• a protected thiol e.g., acetylthio
• R 1 , R 2 , R 2' , R 3 , and/or R 3' can be advantageously chosen to promote cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen by other mechanisms.
• the properties of R 1 , R 2 , R 2' , R 3 , R 3' , R 5 and/or R 6 can promote cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen.
• R 2 and/or R 2' is an electron-withdrawing group, an olefin, or an aryl (e.g., phenyl) substituent, such groups will promote cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen via elimination (i.e., because these groups promote elimination and formation of a double bond between the carbons bearing R 2 and R 3 ) .
• R 3 , R 3' and/or R 6 advantageously can be a group that promotes cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen.
• R 3 and/or R 3' and/or R 3' can be an electron-withdrawing group, an olefin, an aryl substituent (e.g., phenyl), or an aralkyl (e.g., benzyl), to promote cleavage via elimination.
• R 6 when an acyclic N-acylphosphoramidite is used (formula (II) ) , R 6 can be a group that promotes cleavage via displacement of an external nucleophile (e.g., when R 6 is methyl) or via elimination (e.g., when R 6 is cyanoethyl, 4-nitrophenyl , nitroethyl, or the like) .
• an external nucleophile e.g., when R 6 is methyl
• elimination e.g., when R 6 is cyanoethyl, 4-nitrophenyl , nitroethyl, or the like
• R 6 can be a group that promotes cleavage via intramolecular nucleophilic displacement (by the mechanism illustrated in Scheme 1) , for example, when R 6 is an amidoalkyl whose amide can be hydrolyzed to liberate an amine that is separated from the oxygen of OR 6 by 2, 4, or 5 carbon atoms.
• amidoalkyl R 6 groups can include, for example, (CH 2 ) m N (R 8 ) COCH 2 F, (CH 2 ) m N(R 8 )COCHF 2 , and (CH 2 ) m N (R 8 ) C0CF 3 , wherein m is 2, 4, or 5, and R 8 is H or alkyl.
• R 2 , R 3 , R 2' , R 3' , R 5 and/or R 6 can be substituted with a silyl group that promotes elimination when the silyl group is removed.
• R 6 can be a 2- (tri-organosilyl) - ethyl group, or R 3 and/or R 3' can be a tri- organosilylmethyl group.
• Preferred silyl groups include, for example, trialkylsilyl (e.g., trimethylsilyl and triethylsilyl) , aryldialkylsilyl (e.g., dimethylphenylsilyl) , and alkyldiarylsilyl (e.g., methyldiphenylsilyl) groups.
• trialkylsilyl e.g., trimethylsilyl and triethylsilyl
• aryldialkylsilyl e.g., dimethylphenylsilyl
• alkyldiarylsilyl e.g., methyldiphenylsilyl
• R 1 , R 2 , R 2' , R 3 and/or R 3' can be chosen to promote thermal cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen.
• Cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen is indicated by the dotted lines shown in Figs. 13A and 13B.
• Thermal cleavage can be advantageous in that the use of harsh chemicals, such as ammonium hydroxide, is avoided.
• thermal cleavage provides a mild alternative that can be desirable for use in monomeric, oligomeric, or polymeric compounds with chemically labile substituents.
• R 1 , R 2 , R 2' , R 3 , and R 3' , or of R 1 , R 5 , or R 6 can be chosen to promote thermal cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen.
• R 3 can be a substituent that makes the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen more labile, e.g., an electron withdrawing group or a cation-stabilizing group, e.g., an aryl, preferably a phenyl.
• An example of such a thermal cleavage is generally illustrated in Fig. 13A. Specific examples of thermal cleavage are shown in Fig. 13B.
• R 1 is an alkyl, R 2 , R 2' , and R 3' all are H, and R 3 is H or an aryl.
• R 1 is methyl, R 2' and R 3' are H, and R 3 is H or phenyl.
• R 1 is methyl, and R 2 , R 2' , R 3 and R 3 ' all are H (e.g., as shown in Fig. 13B) .
• thermal cleavage of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen can be accomplished under fairly mild conditions.
• thermal cleavage in the two systems shown in Fig. 13B i.e., wherein X is O or S
• thermal cleavage in the two systems shown in Fig. 13B can be carried out to completion in about 80 minutes at about 80° C.
• the present invention further provides a method of preparing a polymer, including the steps of:
• steps (a) through (c) can be repeated, one or more times as necessary, until a polymer of specified length is obtained.
• the method of the present invention further comprises the step of the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen atom (e.g., by aminolysis or thermal cleavage) , after step (a) , (b) , (c) or (d) .
• cleavage of the bond linking the organic moiety to the non-bridging phosphate, phoshphorothioate or phosphoroselenoate oxygen atom can be done at any stage after any of steps (a) - (d) , it is preferably carried out after step (c) or (d) .
• the bond linking the organic moiety to the non-bridging phosphate, phoshphorothioate or phosphoroselenoate oxygen atom is cleaved thermally, for example, as illustrated in Figs. 13A and 13B.
• the N-acylphosphoramidite used in step (a) is a compound of formula (I) or (II) , wherein R 4 is a protecting group.
• the N-acylphosphoramidite is a P-chiral N-acylphosphoramidite.
• the resulting adduct also is P-chiral, since the coupling reaction (step (a)) occurs with stereospecificity.
• reaction of the resulting adduct of step (a) with an oxidizing, a sulfurizing, or a selenizing agent (step (b) ) occurs stereospecifically, that is, without any epimerization at phosphorus.
• step (a) sulfurization of the P- diastereomerically pure adduct of step (a) , obtained by using a P-diastereomerically pure N-acylphosphoramidite, results in a P-diastereomerically pure adduct.
• sulfurization reactions are applied to adducts prepared from standard phosphoramidite coupling chemistry, the phosphorothioate products obtained thereby contain a mixture of phosphorus stereoisomers (i.e., they are not stereopure) because the phosphorus adducts prepared via standard phosphoramidite chemistry contain a mixture of stereoisomers .
• standard phosphoramidite coupling reactions are not stereospecific .
• the present invention stereospecifically produces P-chiral coupling adducts and, thus, provides access to oligonucleotides which are stereochemically pure at phosphorus (e.g., oligonucleotide phosphorothioates) .
• any suitable base can be used in coupling step (a) including, for example, inorganic and organic bases.
• the base used in step (a) is a relatively non-nucleophilic base, which is more preferably a relatively non-nucleophilic amine base such as, for example, tetramethylguanidine (TMG) .
• TMG tetramethylguanidine
• the coupling conditions of the present invention are carried out under basic conditions. As a result, the use of an acid in the coupling reaction is avoided, and the P-diastereomerically pure adduct formed in step (a) does not epimerize. Since the coupling reaction of step (a) occurs with complete stereospecificity, the stereochemical purity with respect to phosphorus can be governed by the stereochemical purity of the N-acylphosphoramidite used therein.
• the method of the present invention further includes the step of capping the unreacted nucleophilic group after step (b) or (c) .
• Capping is usually done as a prophylactic measure to prevent the unreacted nucleophilic groups, left over from prior condensation reactions, from reacting in subsequent condensation cycles. Capping promotes synthetic advantages such as, for example, preventing the formation of undesirable side products.
• the nucleophile or oligomeric adduct, if steps (a) - (c) are repeated at least once) is a sugar hydroxyl, capping typically involves acylation of the unreacted sugar hydroxyls.
• the reaction in step (a) leads to formation of a tricoordinated P-chiral product, thereby enabling, in step (b) , the formation of a P-chiral product.
• Deprotection of the preferred tetracoordinated P-chiral products can provide a P-chiral polymer of predetermined chirality and length.
• the nucleophile utilized in the method of the present invention is a nucleoside, an oligonucleotide, or a derivative thereof
• step (a) utilizes a P-chiral N- acylphosphoramidite
• step (b) comprises sulfurization. Repeating the steps (a) - (c) can be continued as many times as desired, until a polymer of a particular length and chirality is obtained.
• step (a) formation of a tricoordinated P- chiral product in step (a) can be achieved by using any suitable P-chiral N-acylphosphoramidite, most preferably a P-chiral analog of compound (I) or (II) .
• P-chiral N- acylphosphoramidites can be obtained by any suitable method such as, for example, chiral synthesis, chromatographic resolution, or any suitable combination thereof . Chromatographic separation of a mixture of P- chiral isomers can be facilitated, for example, if the monomeric subunit of the N-acylphosphoramidite is a chiral molecule, as illustrated, for example, in Scheme 2.
• R 1 and B are as defined herein
• P-chiral products having any desired phosphorus stereochemistries can be stereospecifically prepared simply by selecting the appropriate P-chiral N-acylphosphoramidite and using it in accordance with the method of the present invention.
• the present invention provides new chemistry for synthesizing oligonucleotides and related polymers having phosphate or phosphate analogue linkages.
• the present invention provides a method for synthesizing polymers having a predetermined sequence of P-chirality along the polymer backbone.
• P-chiral oligonucleotides obtained by the method of the present invention can be employed as hybridization probes, therapeutic agents, e.g., selective protein expression inhibitors, and the like.
• the methods and compounds of the present invention offer other unique advantages, such as moisture stability.
• the N-acylphosphoramidites of the present invention are far more stable to moisture under the coupling conditions of step (a) than are the conventional phosphoramidite synthons for which mild acid conditions are required.
• Moisture instability is a major disadvantage inherent in oligonucleotide synthesis using standard phosphoramidite chemistry.
• standard phosphoramidite precursors hydrolytically degrade, rapidly, upon contact with moisture under standard (acidic) conditions which are required to accomplish a coupling reaction.
• acid-promoted phosphoramidite nucleoside couplings typically are carried out in a scrupulously moisture-free environment, particularly if the target polymer comprises a large number of monomeric units .
• the requirement of maintaining an essentially water- free environment dramatically increases the cost and complexity oligonucleotide synthesis using standard phosphoramidite chemistry. Since the N-acylphosphoramidites of the present invention undergo hydrolytic degradation sluggishly, or not at all, under the coupling conditions of step (a) , the problem of competitive hydrolytic cleavage has essentially been eliminated. As such, the method of the present invention need not be carried out in a scrupulously water-free environment.
• the nucleophile is attached to a solid support. Accordingly, the present invention provides a novel approach to solid phase synthesis, in particular, the synthesis of oligonucleotides and related polymers, using N- acylphosphoramidites, for example, hydroxyl-protected monomeric-O- (O-protected) - (N-acyl ) phosphoramidites .
• the nucleophile is attached to a solid support
• the nucleophile is preferably a compound of the formula:
• Q is a nucleoside, an oligonucleotide comprising a nucleoside, or an oligomer comprising a nucleoside, wherein the nucleoside is of the formula:
• B and E are as defined herein, or an oligomer which includes one of these nucleosides as a component thereof, and R 4 is the solid support.
• the nucleophile is a monomer.
• the nucleophile is a monomer and is attached to a solid phase support through a linking group that will resist cleavage in the presence of a base, for example, a base used in step (a) , thereby allowing the resulting oligomer/polymer to remain attached to the solid support throughout each successive coupling step.
• Q is preferably a nucleoside of the formula:
• Q is a nucleoside substituent having a defined stereochemistry, and is represented by the formula : wherein B and E are as defined herein.
• a cyclic N- acylphosphoramidite of formula (I) is used to effect the desired coupling, and is represented by the formula:
• R 1 -R 4 , B, and E are as defined herein.
• B is a purine, a pyrimidine, adenine, guanine, cytosine, uracil, or thymine, wherein B is unsubstituted or substituted with one or more substituents, which are the same or different, selected from the group consisting of a protecting group, R 11 , OR 11 , NHR 11 , NR 1X R 12 , CN, N0 2 , N 3 , and a halogen, wherein R 11 and R 12 are as defined herein.
• R 1 is an alkyl, which is unsubstituted or substituted with one or more substituents selected from the group consisting of fluorine, OR 7 and SR 7 , wherein R 7 is an alkyl or an aryl. More preferably, R 1 is a alkyl, which is unsubstituted or substituted with one or more fluorine atoms. Still more preferably, R 1 is a methyl, which is unsubstituted or substituted with one or more fluorine atoms, and is most preferably fluoromethyl.
• R 2 , R 2' , R 3 , or R 3 ' is a vinyl group, a phenyl or a .benzyl.
• R 4 is a 4 , 4 ' -dimethoxytrityl group.
• Oxidizing agents that can be used in the context of the present invention include any suitable reagent that can oxidize a tricoordinated phosphorus atom, particularly a phosphite, to provide a phosphorus atom having a valence of higher than three, preferably a tetracoordinated phosphorus atom such as, for example, a phosphate, or an equivalent thereof.
• Suitable oxidizing agents include, for example, I 2 /H 2 0, peroxides, such as tert- butylhydroperoxide , and the like.
• Sulfurizing agents include any suitable reagent that can sulfurize a tricoordinated phosphorus atom, particularly a phosphite, to provide a phosphorus atom with a valence of greater than three, preferably a tetracoordinated phosphorus atom such as, for example, a phosphorothioate, or an equivalent thereof.
• Suitable sulfurizing agents include, for example, 3H-1,2- benzodithiol-3-one 1,1-dioxide ( "Beaucage Reagent"), phenylacetyl disulfide, bis (0,0- diisopropoxyphosphinothioyl) disulfide, and the like.
• Selenizing agents include any suitable reagent that can selenize a tricoordinated phosphorus atom, particularly a phosphite, to provide a phosphorus atom having a valence of greater than three, preferably a tetracoordinated phosphorus atom such as a phosphoroselenoate, or an equivalent thereof.
• Suitable selenizing agents include, for example, potassium selenocyanate (KSeCN) or elemental selenium.
• the present invention also provides an alternative method to the synthesis of unmodified oligonucleotides and to the non-stereospecific synthesis of oligonucleotide analogues.
• the alternative method of the present invention comprises:
• X and R ⁇ R 3' are as defined herein, and W is a leaving group amenable to nucleophilic displacement, to produce an adduct of the nucleophile and the synthon, which is an N-acylphosphoramidite having a tricoordinated phosphorus atom;
• the method further comprises the step of capping unreacted nucleophilic groups after step (iv) or (v) , as discussed herein. It is further preferred to attach the first monomer (i.e., the nucleophile in the first coupling reaction of a synthesis) to a solid phase support through a linking group that will resist cleavage, when in the " presence of the base used in step (iv) . It is preferred that W is a leaving group that can be displaced by a monomer of the formula R 4 -0-Q-OH or R 4 -0-Q 1 -OH, wherein R 4 , Q, and Q 1 are as defined herein.
• W is halogen, a dialkylamino having from 2 to about 8 carbon atoms (e.g., dimethylamino, diethylamino, N-methyl-N-isopropylamino, and the like) , or a cyclic amine substituent having from 2 to about 6 carbon atoms (e.g., pyrrolidinyl, piperidinyl, morpholinyl, aziridinyl, and the like), wherein one or more carbon atoms of the dialkylamino and cyclic amine substituents are unsubstituted or substituted with one or more heteroatoms, which are the same or different.
• a dialkylamino having from 2 to about 8 carbon atoms e.g., dimethylamino, diethylamino, N-methyl-N-isopropylamino, and the like
• a cyclic amine substituent having from 2 to about 6 carbon atoms (e.g., pyrroli
• step (iii) and (iv) enable the formation of the tricoordinated P-chiral product and, preferably, step (v) causes formation of the tetracoordinated P-chiral product in a stereospecific manner. Moreover, further deprotection preferably gives either a P-achiral or a P-chiral polymer of predetermined length.
• suitably protected nucleosides comprise unmodified and/or modified nucleosides.
• Step (v) preferably comprises oxidation and/or sulfurization.
• N-acylphosphoramidite of formula (I) is used.
• the resulting product of steps (a) - (c) , (a) - (d) , (iii), or (iii) - (v) is a compound of formula (III) .
• step (c) or step (iv) any desired number of subsequent coupling steps can be performed, typically requiring deprotection (step (c) or step (iv) ) prior to subsequent coupling reactions, wherein each monomeric unit defined by "n" is the same or different, and the substituents R ⁇ R, R 15 , X, Q 1 , and Q are as defined herein.
• Compounds of formula (III) are useful in the synthesis of polymers, particularly phosphodiester- linked polymers, more particularly P-chiral phosphodiester- linked polymers, which can be obtained from (III) via cleavage of the 2-amidoethoxy fragment (i.e., the bond linking the organic moiety to the non-bridging phosphate, phosphorothioate or phosphoroselenoate oxygen atom) , as described herein.
• Oligomers and polymers synthesized in accordance with a preferred aspect of the present invention are typically represented by the formula:
• n is in the range from about 3 to about 200; more preferably, n is in the range from about 10 to about 40; and most preferably in the range from about 15 to about 25.
• Q, X, and Y, or any combination thereof can be the same or different when n is 1, and can be the same or different in each of the units defined by n when n is greater than 1.
• R 4 is preferably a hydrogen or a hydroxyl protecting group such as, for example, a 4,4'- dimethoxytriphenylmethyl (DMTr) , 4-methoxy- triphenylmethyl (MMTr) , pixyl, acetyl, 9- fluorenylmethyloxycarbonyl (Fmoc) , t-butyldimethylsilyl (TBDMS) , and the like.
• R 4 is a reporter group such as, for example, an amine, a mercapto, a phosphate, a phosphorothioate, and the like.
• Reporter groups preferably contain an active moiety for further reaction with radioactive label such as, for example, 32 P- phosphate, 125 I-iodinated Bolton-Hunter reagent, and the like, or a non-radioactive label such as, for example, fluorescein isothiocyanate (FITC) , dansyl chloride, and the like, or any other biologically active group such as, for example, biotin, digoxigenin, and the like. Reporter groups can be introduced by means known to those skilled in the art including, for example, introduction of appropriate linkers, spacers, arms, or other reagents used for manipulating the distance between the reporter group and the polymer .
• radioactive label such as, for example, 32 P- phosphate, 125 I-iodinated Bolton-Hunter reagent, and the like
• FITC fluorescein isothiocyanate
• dansyl chloride and the like
• Reporter groups can be introduced by means known to those skilled in the art including,
• Y is an OH (or suitable salt thereof) .
• P-chiral polymers of the present invention are * of formula (IIIA) above, wherein X and Y, or any combination thereof, can be the same or different in any of the units being defined by n. More preferably, P-chiral oligonucleotides prepared in accordance with the present invention are of the formula:
• B is preferably a natural or a synthetically modified nucleic base, or B is a synthetic analog or reporter group, preferably a reporter group comprising a carboxyl, an alkyl, or an alkylamine .
• E 1 is preferably a 3 ' -hydroxyl (optionally protected) , and E is preferably a hydrogen, a halogen, a hydroxyl, or an appropriately protected hydroxyl, an amine, or an appropriately protected amine, or the like.
• n is in the range from about 3 to about 200, but is more preferably in the range from about 12 to about 60.
• the P-chiral oligonucleotides of the invention can include linkages, for example, 5'-3', 5'-2 ⁇ ⁇ '-S', 3 ' -3 • , 2 ' -2 ' , and 3'-2' linkages, between nucleosides by the appropriate selection of Q and Q 1 , as defined herein.
• W is halogen, a dialkylamino having from 2 to about 8 carbon atoms, or a cyclic amine substituent having from 2 to about 6 carbon atoms, wherein at least one carbon of the alkyl groups in the dialkylamino and cyclic amine substituents is optionally substituted with one or more heteroatoms, which are the same or different. More preferably W is Cl, dialkylamino, or a cyclic amino. Most preferably, W is diethylamino .
• the synthon precursor 5 (Fig. 1) is synthesized by first refluxing a mixture of acrolein (1) , trimethylsilyl cyanide, and catalytic amounts of zinc iodide according to the procedure reported by Gardrat et al. (J. Heterocyclic Chem . 1990, 27, 811). Reduction of the resulting nitrile 2 with LiAlH 4 in Et 2 0 afforded amino-alcohol 3. Heating 3 with a slight excess (1.1 molar equiv) of ethyl fluoroacetate at 120 °C until all ethyl alcohol has distilled off gave the hydroxylated amide 4 in 88% yield (b.p.
• Nucleoside cyclic acylphosphoramidite 7 was prepared by the reaction of a suitably protected nucleoside 6 with equimolar amounts of 5 and lH-tetrazole in anhydrous dichloromethane for 4 h at ambient temperature. Following evaporation of the reaction mixture, the residue is purified using a short silica gel column chromatography . The nucleosidic synthon 7 is rapidly eluted with a solution of acetonitrile : chloroform (1:2 v/v) . Removal of the eluent under reduced pressure afforded 7 as a white foam. The nucleoside cyclic acylphosphoramidite 9 is prepared in a similar manner from nucleoside 8 and compound 5.
• Example 2 This example illustrates a solid phase synthesis in accordance with the present invention.
• the general reaction scheme is illustrated in Fig. 2, in which nucleoside cyclic acylphosphoramidite 7 (Fig. 1) is specifically applied to the manual solid-phase synthesis of a decanucleotide (dC 10 ) .
• a solid support is denoted in Figs. 2 and 3 by a darkened sphere with "S" in the center.
• the first nucleoside monomer was attached to long chain alkylamine controlled pore glass (LCAA-CPG) to generate 10 has been modified.
• the attachment of the leader nucleoside to LCAA-CPG is accomplished via a sarcosine succinyl linkage according to the method of Brown et al . (J " . Chem . Soc . Chem . Commun . , p . 891-893 (1989)).
• Stepwise DMTr analysis indicated that each coupling yield proceeded with high efficiency, typically 90% or greater.
• the content of the column was then transferred into a glass vial, and treated with concentrated ammonium hydroxide for 10 h at 55 °C .
• the crude oligomer was characterized by reversed phase (RP) HPLC and polyacrylamide gel electrophoresis (PAGE) . Both techniques indicated that dC 10 was prepared in a yield consistent with that determined by the stepwise DMTr analysis.
• crude dC 10 was cleanly hydrolysed by snake venom phosphbdiesterase and alkaline phosphatase to 2 ' -deoxycytidine with no evidence of either partially deprotected nucleotides or nucleobase modifications.
• the synthon 7 (Fig. 1) must first be separated into its Rp and Sp diasteroisomers (see Fig. 3) . This is accomplished by chromatography on functionalized silica
• a diastereomeric mixture of nucleosidic N- acylphosphoramidite 7 was chromatographically separated into its Rp and Sp isomers 7Rp and 7Sp, respectively.
• Each P-chiral isomer was coupled with nucleophilic monomer 10 (Fig. 2), using the conditions of Example 2, to provide P-chiral adducts.
• the coupling reactions are stereospecific .
• Sulfurization of the resulting adducts results in the formation of the llSp and llRp isomers, as illustrated in Fig. 3. Deprotection of the solid support and the 2-amidoethoxy fragment from the sulfurized products is therefore expected to provide stereochemically pure Rp and Sp oligonucleotide products.
• the oxidant in the oxidation step is replaced by a sulfur-transfer reagent such as 3H- 1, 2-benzodithiol-3 -one 1,1-dioxide, phenylacetyl disulfide, bis (O, O-diisopropoxyphosphinothioyl) disulfide, and the like.
• a capping step should be performed after the sulfur transfer step.
• This example illustrates the preparation of various nucleosidic N-acylphosphoramidites of the present invention, wherein the N-acyloxazaphospholane moiety is introduced at different hydroxyls of a differentially protected nucleoside core.
• the reaction schemes are illustrated generally in Fig. 4. Using the procedure of Example 1, nucleophilic monomers 12, 14, 16, and 18 were coupled to synthon 5 using tetrazole, to provide nucleosidic N- acylphosphoramidites 13, 15, 17, and 19, respectively.
• the resulting nucleosidic N-acylphosphoramidites can be used as a vehicle for one or more coupling reactions, to provide oligomer or polymer products.
• the resulting nucleosidic N-acylphosphoramidites can be separated into their Rp and Sp isomers prior to their use as coupling reagents.
• the phospholane moiety of nucleosidic N-acylphosphoramidites 13, 15, 17, and 19 are attached to either the 3 ' - or 5 ' - hydroxyl in the case of 2 ' -deoxyribonucleosides or, additionally, to the 2'- hydroxyl in the case of ribonucleosides .
• This example illustrates the preparation of acyclic N-acylphosphoramidites, and methods of using them in the context of the present invention.
• the nucleoside acylphosphoramidites can be applied in a manner similar to that described in Examples 2 and 3 , and Figs . 2 and Fig. 3.
• the reaction scheme is illustrated generally in Fig. 5.
• a solid support is denoted in Fig. 5 by a darkened sphere with "S" in the center.
• the non-nucleosidic chlorophosphoramidite derivative 20 is condensed with a suitable N-methylamide (21) to generate the acylphosphoramidite 22.
• Reaction of 22 with suitably protected nucleosides 6 (Fig. 1) in the presence of 1H- tetrazole affords the corresponding nucleoside 3 ' - acylphosphoramidites 23 as a mixture of P- diastereoisomers .
• These amidites are activated under basic conditions and are expected to be useful in solid- phase oligonucleotide synthesis in a manner similar to that shown in Fig. 2.
• ribonucleotide syntheses can be used in accordance with the present invention for ribonucleotide syntheses, and are expected to work in the same manner as the cyclic species, for example, 13, 15, 17, and 19 (Fig. 4) .
• FIG. 6 A solid support is denoted in Figs. 6 and 7 by a darkened sphere with "S" in the center.
• the strategy was demonstrated by reacting non- nucleosidic cyclic N-acylphosphoramidite 5 (Fig. 1) and acylphosphoramidite 22 (Fig. 5) with the functionalized solid-support-bound 10 (Fig. 2) in the presence of 1H- tetrazole to generate 25 and 26, respectively, as shown in Fig. 6.
• step 1 the appropriate CPG-bound nucleoside is detritylated in accordance with a standard procedure.
• step 2 5 ' -O-Dimethoxytrityl-3 ' -O- ( 5 -phenyl-3 -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' - 0- deoxyribonucleoside derivatives (5 mg, ca . 5 ⁇ mol) are dissolved in acetonitrile (200 ⁇ L) . Tetramethylguanidine (TMG, 4 ⁇ l, ca. 30 ⁇ mol) is subsequently added and the mixture is applied to the synthesis column.
• TMG Tetramethylguanidine
• step 3 a standard oxidation or sulfurization reaction is carried out after the reaction of step 2 is continued for 5 min.
• Steps 2 and 3 are repeated to optimize the yield for a particular synthesis cycle. Steps 2 and 3 need not be performed more than once for a particular synthesis cycle. However, yields are typically improved (e.g., resulting in nearly 100% overall yield) if steps 2 and 3 are repeated within a particular synthesis cycle. Optionally, steps 2 and 3 can be repeated three or more times, as desired, to optimize the yield for a particular synthesis cycle even further. In step 4, the synthesis cycle is concluded with a capping step. Synthesis cycles can be repeated until the designed sequence length is obtained.
• step 5 the synthetic oligonucleotide is subjected to post -synthesis cleavage from the support, and deprotection.
• This example describes the synthesis of a dinucleotide, particularly T P0 T. The following steps were used in the present example.
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems).
• Step 2 5 ' -O-Dimethoxytrityl-3 ' -O- (5-phenyl-3 -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' -O-deoxythymidine (5 mg, ca. 5 ⁇ mol) and tetramethylguanidine (TMG, 4 ⁇ l, ca. 30 ⁇ mol) in acetonitrile (200 ⁇ l) were added to the column and reacted for 5 min., followed by washing with acetonitrile (3 mL, 30s) .
• TMG tetramethylguanidine
• Step 3 The resulting product was treated with iodine/water/pyridine/tetrahydrofuran (Applied Biosystems DNA synthesis reagent) , (500 ⁇ l, 30s) , followed by washing with acetonitrile (3 ml, 30s) .
• Step 5 The dinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, lOh, 55 °C) .
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure and analyzed by RP-HPLC.
• This example describes the synthesis of P- diasteriomerically pure phosphorothioate [R p ]-C PS C. The following steps were used in the present example.
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems).
• Step 3 The resulting product was treated with 3H- 1, 2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in acetonitirile (w/v) ) , 3 min., followed by washing with acetonitirile (3 mL, 30s) .
• Step 5 The dinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, 10 h, 55°C) .
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure and analyzed by RP-HPLC.
• This example describes the synthesis of a P- diastereomerically pure phosphorothioate-linked trinucleotide (trimer) , [R p , R p ] C PS C PS C .
• trimer phosphorothioate-linked trinucleotide
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems DNA synthesis reagent) , (3 mL, 1 min) , followed by washing with acetonitirile (3 mL, 30s) .
• Step 2 [Sp] - -Benzoyl-5 ' -0-dimethoxytrityl-3 ' -O- ( 5 -phenyl -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' -O- deoxycytidine (Fig. 8B, 5 mg, ca. 5 ⁇ mol) and tetramethylguanidine (TMG,4 ⁇ l, oxazaphospholanyl-2 '- O- (5 -phenyl-3 -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' -0- deoxycytidine (5 mg, ca.
• Step 3 The resulting product from step 2 was treated with 3H-1 , 2 , -benzodithiol-3 -one 1,1-dioxide (1%
• Step 4 The resulting product from step 3 was capped with acetic anhydride/lutidine/tetrahydrofuran (Applied Biosystems D ⁇ A synthesis reagent) , (1 mL) , mixed with 1-methylimidazole/tetrahydrofuran (Applied Biosystems D ⁇ A synthesis reagent), (1 mL) , 2 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 5 The trinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, 10 h, 55°C) .
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure to provide the P- diastereomerically pure phosphorothioate-linked trinucleotide (trimer) , [R P , R p ] C PS C PS C .
• the product obtained in accordance with the present invention was analyzed by RP-HPLC (Fig. 10D) .
• the trimer C PS C PS C also was prepared using a standard phosphoramidite coupling reagent (Fig. 8A) .
• the HPLC of C PS C PS C obtained using the standard phosphoramidite coupling reagent is shown in Fig.
• This example describes the synthesis of a P- diastereomerically pure phosphorothioate-linked trinucleotide (trimer), [S P , S p ] C PS C PS C .
• trimer phosphorothioate-linked trinucleotide
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3 mL, 1 min.), followed by washing with acetonitrile (3 mL, 30s) .
• Step 2 [Rp] -i ⁇ -Benzoyl-5 ' -O-dimethoxytrityl-3 ' -O- (5 -phenyl-3 -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' -O- deoxycytidine (Fig. 8C, 5 mg, ca. 5 ⁇ mol) and tetramethylguanidine (TMG, 4 ⁇ l, ca. 30 ⁇ mol) in acetonitrile (200 ⁇ l) were added to the column and reacted for 5 min., followed by washing with acetonitrile (3 mL, 30s) .
• TMG tetramethylguan
• Step 3 The resulting product from step 2 was treated with 3H-1 , 2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in acetonitrile (w/v) ) , 3 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 4 The resulting product from step 3 was capped with acetic anhydride/lutidine/tetrahydrofuran (Applied Biosystems D ⁇ A synthesis reagent), (1 mL) , 2 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 5 The trinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, 10 h, 55°C).
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure and analyzed by RP-HPLC.
• the product obtained in the present example was analyzed by RP-HPLC (Fig. 10B) .
• the trimer C PS C PS C also was prepared using a standard phosphoramidite coupling reagent (Fig. 8A) .
• the HPLC of the C PS C PS C obtained using the standard phosphoramidite coupling reagent is shown in Fig. 10A and contained, as expected, a mixture of all four possible P-diastereomers (indicated by RR, SR, RS, and SS) .
• the trimer prepared in accordance with the present invention produced only one P- diastereomer (SS) .
• This example describes the synthesis of a P- diastereomerically pure phosphorothioate-linked tetramer [Rp, Sp,Rp] -C PS C PS C PS C. The following steps were used in the present example .
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3 mL, 1 min.), followed by washing with acetonitrile (3 mL, 30s) .
• Step 2 [S p ] -.- -benzoyl-5 ' -O-dimethoxytrityl-3 ' -0- (5- phenyl-3 -N- fluoroacetyl) -1,3,2, -oxazaphospholanyl-2 ' - 0- deoxycytidine (5 mg, ca. 5 ⁇ mol) and tetramethylguanidine (TMG, 4 ⁇ l, ca. 30 ⁇ mol) in acetonitrile (200 ⁇ l) were added to the column and reacted for 5 min., followed by washing with acetonitrile (3 mL, 30s) .
• TMG tetramethylguanidine
• Step 2' [Rp] -N 1 -Benzoyl-5 ' -O-dimethoxytrityl-3 ' -O- (5-phenyl-3 -N- fluoroacetyl) -1,3, 2 -oxazaphospholanyl-2 ' - O- deoxycytidine (Fig. 8C, 5 mg, ca. 5 ⁇ mol) and tetramethylguanidine (TMG, 4 ⁇ l, ca . 30 ⁇ mol) in acetonitrile (200 ⁇ l) ) were added to the column and reacted for 5 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 3 The resulting product from step 2 or 2 ' was treated with 3H-1 , 2-benzodithiol-3 -one 1,1-dioxide (1% Beaucage Reagent in acetonitrile (w/v) ) , 3 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 4 The resulting product from step 3 was capped with acetic anhydride/lutidine/tetrahydrofuran (Applied Biosystems D ⁇ A synthesis reagent) , (1 mL) , mixed with 1- methylimidazole/tetrahydrofuran (Applied Biosystems D ⁇ A synthesis reagent) , (1 mL) , 2 min. , followed by washing with acetonitrile (3 mL, 30s) .
• Step 5 The trinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, 10 h, 55 °C) .
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure and analyzed by RP-HPLC.
• the product obtained in the present example was analyzed by RP-HPLC (Fig. 11B) .
• the tetramer C PS C PS C PS C also was prepared using a standard phosphoramidite coupling reagent (Fig. 8A) .
• the HPLC of the C PS C PS C PS C obtained using the standard phosphoramidite coupling reagent is shown in Fig. 11A and contained, as expected, a mixture of all eight possible P-diastereomers (indicated by RRR, SRR, RRS, SRS, RSR, SSR, RSS, and SSS) .
• the tetramer prepared in accordance with the present example produced only one P-diastereomer (RSR) .
• No other P-diastereomers were present by HPLC, even in trace a amounts.
• Co-injection of the tetramer prepared in the accordance with the present invention and the P-diastereomeric mixture obtained by the standard phosphoramidite method (Fig. 11C) confirmed that the product obtained in accordance with the present invention was indeed [Rp, Sp, Rp] -C PS C PS C PS C .
• This example further demonstrates that the N- acylphosphoramidites of the present invention can predictably produce oligomers that are P- diastereomerically pure.
• This example describes the synthesis of a P- diastereomerically pure phosphorothioate-linked undecamer, [all R p ]-(T ps ) 11 T (eleven nucleoside units in the oligonucleotide chain) .
• the following steps were used in the present example.
• Step 1 The bound nucleoside was treated with 3% trichloroacetic acid/dichloromethane (Applied Biosystems DNA synthesis reagent), (3 mL, . 1 min.), followed by washing with acetonitrile (3 mL, 30s) .
• Step 2 [S p ] -5' -O-Dimethoxytrityl-3 ' -O- (5-phenyl-3- N-fluoroacetyl) -1,3, 2-oxazaphospholanyl-2 ' -O- deoxythymidine (5 mg, ca . 5 ⁇ mol) and tetramethylguanidine (TMG, 4 ⁇ l, ca . 30 ⁇ mol) in acetonitrile (200 ⁇ l) were added to the column and reacted for 5 min., followed by washing with acetonitrile (3 mL, 30s) .
• TMG tetramethylguanidine
• Step 3 The resulting product from step 2 was treated with 3H-1, 2-benzodithiol-3-one 1,1-dioxide (1% Beaucage Reagent in acetonitrile (w/v) ) , 3 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 4 The resulting product from step 3 was capped with acetic anhydride/lutidine/tetrahydrofuran (Applied Biosystems DNA synthesis reagent), (1 mL) , 2 min., followed by washing with acetonitrile (3 mL, 30s) .
• Step 5 The trinucleotide was cleaved from the support by treatment with concentrated aqueous ammonium hydroxide solution (1 mL, 10 h, 55°C) .
• the ammoniacal solution obtained in the final step was concentrated under reduced pressure and analyzed by RP-HPLC.
• the product obtained in the present example exhibited only one peak by HPLC.
• HPLC system used in this example cannot chromatographically separate all possible P-diastereomers for an oligomer of this length, it is believed that the product obtained in the present example is indeed P-diastereomerically pure.
• Example 13 This example demonstrates the hydrolytic stability of the N-acylphosphoramidites of the present invention (Fig. 9B) , relative to the hydrolytic stability of standard phosphoramidites (Fig. 9A) .
• the hydrolytic stability for each type of reagent was determined under reaction conditions normally employed for each type of coupling reagent.
• Samples of the dinucleotide d(T P0 G) were prepared by a standard coupling method using the phosphoramidite of Fig. 9A.
• Samples of d(T P0 G) also were prepared by a coupling reaction in accordance with the present invention using the N-acylphosphoramidite of Fig. 9B .
• Each coupling method was performed in the absence of moisture and in the presence of moisture (0.1% water) .
• the products were analyzed by HPLC.
• the HPLC chromatogram of the product obtained via the standard phosphoramidite reagent (Fig. 9A) in a moisture-free environment is shown in Fig. 12A.
• Fig. 12B The HPLC chromatogram of the product obtained via the N-acylphosphoramidite (Fig. 9B) in a moisture-free environment is shown in Fig. 12C.
• the product obtained using the N-acylphosphoramidite of Fig. 9B in the presence of 0.1% moisture is shown in Fig. 12D.
• the target product is indicated in the HPLC chromatogram by a peak labeled d(T P0 G), corresponding to the dinucleotide.
• Hydrolysis of the coupling reagent i.e., hydrolytic instability
• dG Hydrolysis of the coupling reagent

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