WO2006066260A2 - Compositions of and methods for producing phosphorus-chiral monomers and oligomers - Google Patents

Abstract

Described herein are phosphorus-containing monomers and oligomers and methods for making such monomers and oligomers. Each subunit of the oligomers may be the same or different. Furthermore, the phosphorus atom in the monomers and at least two of the phosphorus atoms in the oligomers are chiral, that is, attached to four different chemical groups in a desired configuration. The methods described herein, which use an intramolecular catalyst, permit the synthesis of oligomers in which each chiral phosphorus center is in a desired stereochemical configuration.

Description

COMPOSITIONS OF AND METHODS FOR PRODUCING PHOSPHORUS-CHIRAL

MONOMERS AND OLIGOMERS CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This Application claims the benefit of U.S. Provisional Patent Application Serial No. 60/637,301 filed December 17, 2004, incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The invention is in the field of oligomeric compositions of monomers wherein the monomelic units are joined together by chiral phosphorothioate linkages.

BACKGROUND OF THE INVENTION [0003] Most of the bodily states in multicellular organisms, including most disease states, are affected by proteins. Such proteins, either acting directly or through their enzymatic or other functions, contribute in major proportion to many diseases and regulatory functions in animals and man. Classical therapeutics has generally focused upon interactions with such proteins in an effort to moderate their disease-causing or disease-potentiating functions. In newer therapeutic approaches, modulation of the actual production of such proteins is desired. By interfering with the production of proteins, the maximum therapeutic effect might be obtained with minimal side effects. It is the general objective of such therapeutic approaches to interfere with or otherwise modulate gene expression which would lead to undesired protein formation. Generally, these therapeutic approaches employ nucleic acids and their chemically-modified counterparts as effectors of the therapeutic principle (see, Scherer, LJ. and JJ. Rossi, Approaches for the sequence-specific knockdown ofmRNA. Nature biotechnology, 2003. 21 : p. 1457-1465; Opalinska, J.B. and A.M. Gewirtz, Nucleic-acid therapeutics: Basic principles and recent applications. Nature Reviews. Drug Discovery, 2002. 1: p. 503-514). [0004] Nucleic acids and their chemically modified counterparts are also being developed to interact with biological targets other than nucleic acids. Aptamers are one such class, nucleic acids that are developed by an exponential enrichment (SELEX) process and are used in both diagnostic and therapeutic applications (see, Yan, A.C., et al., Aptamers: Prospect in therapeutics and biomedicine. Frontiers in Bioscience, 2005. 10: p. 1802-1827; Mayer, G. and A. Jenne, Aptamers in research and drug development. Biodrugs, 2004. 18: p. 351-359; Jayasena, S.D., Aptamers: An emerging class of molecules that rival antibodies in diagnostics. Clinical Chemistry, 1999. 45: p. 1628-1650).

[0005] Nucleic acids and their chemically-modified counterparts have also found use in diagnostic tests. Such diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components. As with the above gene expression inhibition, diagnostic use can take advantage of an nucleic acids ability to hybridize with a complementary strand of nucleic acid. Hybridization is the sequence specific hydrogen bonding of oligonucleotides via Watson-Crick and/or Hoogsteen base pairs to RNA or DNA. The bases of such base pairs are the to be complementary to one another. A particularly important area of diagnostic testing is the detection of genetic variation (see, Syvanen, A.-C, Accessing genetic variation: Genotyping single nucleotide polymorphisms. Nature reviews. Genetics, 2001. 2: p. 930-942).

[0006] Nucleic acids and to some extent their chemically-modified counterparts, are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of such other biological molecules. One particular use, the use of oligonucleotides as primers in the reactions associated with polymerase chain reaction (PCR), is important to an ever expanding commercial business. The use of such PCR reactions has increased and been extended into areas such as forensics, paleontology, evolutionary studies and genetic counseling. Primers are needed for each of these uses. Oligonucleotides, both natural and synthetic serve as the primers.

SUMMARY OF THE INVENTION [0007] Described herein are methods for the preparation of substantially diastereoisomerically-homogenous phosphorothioate oligonucleotides, and for intermediates useful in their preparation. Substantially diastereoisomerically-homogenous includes embodiments of oligonucleotides that are greater than about 95% diastereoisomerically- homogenous, greater than about 96% diastereoisomerically-homogenous, greater than about 97% diastereoisomerically-homogenous, greater than about 98% diastereoisomerically-homogenous, greater than about 99% diastereoisomerically-homogenous and greater than about 99.5% diastereoisomerically-homogenous. The stereospecific chemical synthesis methodology also relates to preparation of substantially diastereoisomerically-pure oligonucleotides containing phosphotriester linkages. Substantially diastereoisomerically-pure includes embodiments of oligonucleotides that are greater than about 95% diastereoisomerically-pure, greater than about 96% diastereoisomerically-pure, greater than about 97% diastereoisomerically-pure, greater than about 98% diastereoisomerically-pure, greater than about 99% diastereoisomerically-pure and greater than about 99.5% diastereoisomerically-pure. Described herein are also methods for the preparation of oligonucleotides with backbones composed of mixtures of any or all of chiral phosphorothioate diester, chirally-enriched phosphorothioate diester, racemic phosphorothioate diester, and phosphodiester, linkages, respectively, and to intermediates useful in their preparation. Oligonucleotide analogs with backbone compositions tailored to meet requirements of nucleic acid modulators such as improved resistance to nucleolytic degradation, hybridization properties, improved capacity to invoke enzyme-catalyzed degradation of an RNA target, and other factors that influence overall activity of a nucleic acid modulator, are provided. [0008] Described herein are stereospecific synthetic methodologies for the preparation of sequence-specific oligonucleotides having chiral phosphorothioate diester, chiral phosphorothioate or phosphate triester linkages. Also described herein are novel synthetic strategies in which a catalytic protecting group of a chiral phosphorothioate diester enables substantially complete stereospecificity in the condensation reaction with an incoming nucleophile. Such specificity includes greater than about 99% specificity, greater than about 99.9% specificity, and complete specificity. The efficiency of the reaction allows for the practical preparation of oligonucleotides in which chiral phosphorothioate diester linkages are part of the structure. [0009] In one aspect are sequence-specific oligonucleotides having substantially chirally- pure phosphorothioate linkages. In another aspect are sequence-specific oligonucleotides with backbones comprised of mixtures of any or all of the following type of functionalities: substantially chirally-pure phosphorothioate diester, chirally-enriched phosphorothioate diester, racemic phosphorothioate diester, and phosphodiester, linkages. In another aspect are oligonucleotides with substantially chirally-pure phosphorothioate linkages and oligonucleotides with backbones comprised of mixtures of any or all of the following type of functionalities: substantially chirally pure phosphorothioate diester, chirally enriched phosphorothioate diester, racemic phosphorothioate diester, and phosphodiester, linkages, that have antisense hybridizability against DNA and RNA sequences. In another aspect are oligonucleotides with substantially chirally pure phosphorothioate linkages and oligonucleotides with backbones comprised of mixtures of any or all of the following type of functionalities: substantially chirally-pure phosphorothioate diester, chirally-enriched phosphorothioate diester, racemic phosphorothioate diester, and phosphodiester, linkages, for use as nucleic acid modulators, hi another aspect are new methods for synthesizing sequence-specific oligonucleotides having substantially chirally-pure phosphorothioate triester, substantially chirally-pure phosphotriester, linkages, or any level of chiral enrichment thereof.

[0010] In further or additional aspects are methods, compositions, techniques and strategies for the production of oligonucleotides via solid phase synthetic methods as presented in any one of or any combination of Figures 1-9, as well as in the accompanying text. In further or additional aspects are methods, compositions, techniques and strategies for the production of oligonucleotides via solution phase synthetic methods as presented in any one of or any combination of Figures 1-9, as well as in the accompanying text. In further or additional aspects are building blocks, reagents, groups, moieties, reaction products and reactions for solid phase and/or solution phase synthetic methods as presented in any one of or any combination of Figures 1-9, as well as in the accompanying text. [0011] Presented herein are combination methods for selectively preparing compounds having a chiral phosphorus center. Such methods can occur on a solid support, in solution phase or in a combination thereof. Also presented herein are compounds having a chiral phosphorus center. Such compounds include monomers and oligomers of nucleosides and nucleotides. [0012] Described herein are stereospecific methods for preparing sequence-specific oligonucleotides having chiral phosph(orothio)ate linkages. In further embodiments, these methods comprise reacting a first synthon of Formula I:

wherein Q is independently O or S;

R¹ is a hydroxyl protecting group;

R² is a phosph(orothio)ate protecting group enabling intramolecular nucleophilic catalysis;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

B is a nucleobase; and n is an integer from 0 to 50; with a second synthon of Formula II:

wherein: R⁴ is hydroxyl protecting group or a linker connected to a solid support; and m is an integer from 0 to 50; for a time and under reaction conditions effective to form a third synthon of Formula III:

wherein D is the chiral phosph(orothio)ate linkage having the formula:

[0013] In further embodiments the chiral phosph(orothio)ate linkage is diastereomerically enriched. In further embodiments about 98% of the chiral phosph(orothio)ate linkage is in a single stereoisomeric form. In still further embodiments, the phosph(orothio)ate linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms. In further or alternative embodiments, the first and second synthons are in single stereoisomeric forms, substantially free of other stereoisomeric forms.

[0014] In further or alternative embodiments n is 0. In further or alternative embodiments, R¹ groups are subsequently removed to yield new second synthons for iterative synthesis, and phosph(orothio)ate protecting groups R² are removed after iterative synthesis is completed. In further or alternative embodiments of the present methods the oligomer of Formula III contains a plurality of phosph(orothio)ate linkages. In further or alternative embodiments about 98% of each phosph(orothio)ate linkage is in a single stereoisomeric form. In further or alternative embodiments about 98% of each phosphorothioate linkage is in a single stereoisomeric form. In further or alternative embodiments each phosph(orothio)ate linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms. In further or alternative embodiments each phosphorothioate linkage is in a single stereoisomeric form, substantially free of other stereoisomeric forms.

[0015] In further or alternative embodiments the first synthon is formed by deprotecting a compound of Formula IV:

wherein R⁵ is a phosph(orothio)ate protecting group.

[0016] In further or alternative embodiments the compound of Formula IV is formed by reacting a condensing agent with a compound of Formula V:

and a (thio)phosphorylating reagent of Formula Via:

wherein R², R⁵, and Q are as defined above.

In further or alternative embodiments the thiophosphorylating reagent of Formula Via (Q = S) is in either of the single stereoisomeric forms of Formulas VIb and VIc, substantially free of other stereochemical forms:

In preferred embodiments of the method, first synthons are diastereomerically enriched, or in a single stereochemical form, substantially free of other stereochemical forms. Figure 7 summarizes formations and diastereomerical separations of further embodiments of first synthons.

[0017] In further or alternative embodiments the compound of Formula IV is formed by reacting a condensing agent with a compound of Formula VII:

and a phosph(orothio)ate protecting group R²OH enabling intramolecular catalysis. [0018] In further or alternative embodiments the first synthon is formed by reacting a compound of Formula VIII:

with a phosph(orothio)ate protecting group R²OH enabling intramolecular catalysis and contacting the resulting condensation product with an oxidizing agent or a sulfurizing agent. [0019] In further or alternative embodiments the first synthon is formed by reacting a condensing agent with a (thio)phosphonylating reagent of Formula IX:

and a compound of Formula V and contacting the resulting condensation product with an oxidizing agent or a sulfurizing agent.

[0020] In further or alternative embodiments the first synthon is formed by reacting a condensing agent with a (thio)phosphorylating reagent of Formula Xa:

and a compound of Formula V.

[0021] In further or alternative embodiments the first synthon is formed by reacting a (thio)phosphorylating reagent of Formula Xb:

wherein L is halogen, including Cl, or an azole substituent, including 3-nitro-l,2,4-triazolide, with a compound of Formula V and a phosph(orothio)ate protecting group R²OH enabling intramolecular catalysis. [0022] In further or alternative embodiments the reaction of first and second synthons is effected by the presence of a condensing reagent, the condensing reagent having one of the general Formulas XI, XII, or XIII:

R¹³ XIII

N=C=N

R¹⁴ wherein: R⁶-R¹⁰ are independently hydrogen, halogen, cyano, nitro, or alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring; and

L is halogen, including Cl, or an azole substituent, including 3-nitro-l,2,4-triazolide; and R¹¹ and R¹² are independently alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, or by being joined together forming a cyclic phosphoryl derivative with or without further ring-substitutions; and R¹³ and R¹⁴ are independently alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons. hi further or alternative embodiments the first synthon has one of the Formulas XIVa- XIVg:

wherein W has the formula:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, optionally substituted aryl, or optionally substituted alkyl having from one to 10 carbons; or wherein two adjacent R groups can together form an optionally substituted ring. [0023] An "alkoxy" group refers to a (alkyl)O- group, where alkyl is as defined herein. [0024] An "alkyl" group refers to an aliphatic hydrocarbon group. The alkyl moiety may be a "saturated alkyl" group, which means that it does not contain any alkene or alkyne moieties. The alkyl moiety may also be an "unsaturated alkyl" moiety, which means that it contains at least one alkene or alkyne moiety. An "alkene" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon double bond, and an "alkyne" moiety refers to a group consisting of at least two carbon atoms and at least one carbon-carbon triple bond. The alkyl moiety, whether saturated or unsaturated, may be branched, straight chain, or cyclic. [0025] The "alkyl" moiety may have 1 to 10 carbon atoms (whenever it appears herein, a numerical range such as "1 to 10" refers to each integer in the given range; e.g., "1 to 10 carbon atoms" means that the alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 10 carbon atoms, although the present definition also covers the occurrence of the term "alkyl" where no numerical range is designated). The alkyl group could also be a "lower alkyl" having 1 to 5 carbon atoms. The alkyl group of the compounds described herein may be designated as "Ci-C₄ alkyl" or similar designations. By way of example only, "Ci-C₄ alkyl" indicates that there are one to four carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, iso-propyl, n-butyl, iso- butyl, sec-butyl, and t-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, ethenyl, propenyl, butenyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like. [0026] The term "alkylamine" refers to the -N(alkyl)_xH_y group, where x and y are selected from the group x=l, y=l and x=2, y=0. When x=2, the alkyl groups, taken together, can optionally form a cyclic ring system.

[0027] The term "alkenyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a double bond that is not part of an aromatic group. That is, an alkenyl group begins with the atoms -C(R)=C-R, wherein R refers to the remaining portions of the alkenyl group, which may be the same or different. Non-limiting examples of an alkenyl group include -CH=CH,

-C(CH₃)=CH, -CH=CCH₃ and -C(CH₃)=CCH₃. The alkenyl moiety may be branched, straight chain, or cyclic (in which case, it would also be known as a "cycloalkenyl" group). [0028] The term "alkynyl" refers to a type of alkyl group in which the first two atoms of the alkyl group form a triple bond. That is, an alkynyl group begins with the atoms -C ≡ C-R, wherein R refers to the remaining portions of the alkynyl group, which may be the same or different. Non-limiting examples of an alkynyl group include -C ≡ CH, -C ≡ CCH₃ and - C ≡ CCH₂CH₃. The "R" portion of the alkynyl moiety may be branched, straight chain, or cyclic.

[0029] An "amide" is a chemical moiety with formula -C(O)NHR or -NHC(O)R, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). An amide may be an amino acid or a peptide molecule attached to a compound of Formula (I), thereby forming a prodrug. Any amine, hydroxy, or carboxyl side chain on the compounds described herein can be amidifϊed. The procedures and specific groups to make such amides are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety. [0030] The term "aromatic" or "aryl" refers to an aromatic group which has at least one ring having a conjugated π-electron system and includes both carbocyclic aryl (e.g., phenyl) and heterocyclic aryl (or "heteroaryl" or "heteroaromatic") groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. The term "carbocyclic" refers to a compound which contains one or more covalently closed ring structures, and that the atoms forming the backbone of the ring are all carbon atoms. The term thus distinguishes carbocyclic from heterocyclic rings in which the ring backbone contains at least one atom which is different from carbon.

[0031] As used herein, the term "contacting" means directly or indirectly causing placement together of moieties to be contacted, such that the moieties come into physical contact with each other. Contacting thus includes physical acts such as placing the moieties together in a container. The term "reacting" as used herein means directly or indirectly causing placement together of moieties to be reacted, such that the moieties chemically combine or transform. [0032] A "cyano" group refers to a -CN group.

[0033] The term "cycloalkyl" refers to a monocyclic or polycyclic radical that contains only carbon and hydrogen, and may be saturated, partially unsaturated, or fully unsaturated. Cycloalkyl groups include groups having from 3 to 10 ring atoms. Illustrative examples of cycloalkyl groups include the following moieties:

[0034] As used herein, the term "electron withdrawing" has its normal meaning as a chemical functionality which electronically or inductively causes the withdrawal of electron density form the moiety to which the electron withdrawing groups is attached. Representative electron withdrawing groups include nitro groups and halogens. Other electron withdrawing groups will be apparent to those of skill in the art.

[0035] The term "enabling" as used herein in regards to a protecting group "enabling" intramolecular nucleophilic catalysis refers to a protecting group that increases the reactivity of a phosphorus center to intramolecular nucleophilic reactions (e.g., in some way lowers the activation barrier of the phosphorus center to attack by a nucleophile). [0036] The term "ester" refers to a chemical moiety with formula -COOR, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon). Any hydroxy, or carboxyl side chain on the compounds described herein can be esterifϊed. The procedures and specific groups to make such esters are known to those of skill in the art and can readily be found in reference sources such as Greene and Wuts, Protective Groups in Organic Synthesis, 3^rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety. [0037] The term "halo" or, alternatively, "halogen" means fluoro, chloro, bromo or iodo. Preferred halo groups are fluoro, chloro and bromo.

[0038] The terms "haloalkyl," "haloalkenyl," "haloalkynyl" and "haloalkoxy" include alkyl, alkenyl, alkynyl and alkoxy structures, that are substituted with one or more halo groups or with combinations thereof. The terms "fluoroalkyl" and "fluoroalkoxy" include haloalkyl and haloalkoxy groups, respectively, in which the halo is fluorine. [0039] The terms "heteroalkyl" "heteroalkenyl" and "heteroalkynyl" include optionally substituted alkyl, alkenyl and alkynyl radicals and which have one or more skeletal chain atoms selected from an atom other than carbon, e.g., oxygen, nitrogen, sulfur, phosphorus or combinations thereof. [0040] The terms "heteroaryl" or, alternatively, "heteroaromatic" refers to an aryl group that includes one or more ring heteroatoms selected from nitrogen, oxygen and sulfur. An N- containing "heteroaromatic" or "heteroaryl" moiety refers to an aromatic group in which at least one of the skeletal atoms of the ring is a nitrogen atom. The polycyclic heteroaryl group may be fused or non- fused. Illustrative examples of heteroaryl groups include the following moieties:

[0041] The term "heterocycle" refers to heteroaromatic and heteroalicyclic groups containing one to four heteroatoms each selected from O, S and N, wherein each heterocyclic group has from 4 to 10 atoms in its ring system, and with the proviso that the ring of said group does not contain two adjacent O or S atoms. Non-aromatic heterocyclic groups include groups having only 4 atoms in their ring system, but aromatic heterocyclic groups must have at least 5 atoms in their ring system. The heterocyclic groups include benzo-fused ring systems. An example of a 4-membered heterocyclic group is azetidinyl (derived from azetidine). An example of a 5- membered heterocyclic group is thiazolyl. An example of a 6-membered heterocyclic group is pyridyl, and an example of a 10-membered heterocyclic group is quinolinyl. Examples of non- aromatic heterocyclic groups are pyrrolidinyl, tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, dihydropyranyl, tetrahydrothiopyranyl, piperidino, morpholino, thiomorpholino, thioxanyl, piperazinyl, azetidinyl, oxetanyl, thietanyl, homopiperidinyl, oxepanyl, thiepanyl, oxazepinyl, diazepinyl, thiazepinyl, 1,2,3,6- tetrahydropyridinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3- dioxolanyl, pyrazolinyl, dithianyl, dithiolanyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, 3-azabicyclo[3.1.0]hexanyl, 3- azabicyclo[4.1.0]heptanyl, 3H-indolyl and quinolizinyl. Examples of aromatic heterocyclic groups are pyridinyl, imidazolyl, pyrimidinyl, pyrazolyl, triazolyl, pyrazinyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, isothiazolyl, pyrrolyl, quinolinyl, isoquinolinyl, indolyl, benzimidazolyl, benzofuranyl, cinnolinyl, indazolyl, indolizinyl, phthalazinyl, pyridazinyl, triazinyl, isoindolyl, pteridinyl, purinyl, oxadiazolyl, thiadiazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, and furopyridinyl. The foregoing groups, as derived from the groups listed above, may be C- attached or N-attached where such is possible. For instance, a group derived from pyrrole may be pyrrol- 1-yl (N-attached) or pyrrol-3-yl (C-attached). Further, a group derived from imidazole may be imidazol-1-yl or imidazol-3-yl (both N-attached) or imidazol-2-yl, imidazol-4-yl or imidazol-5-yl (all C-attached). The heterocyclic groups include benzo-fused ring systems and ring systems substituted with one or two oxo (=0) moieties such as pyrrolidin-2-one. [0042] A "heteroalicyclic" group refers to a cycloalkyl group that includes at least one heteroatom selected from nitrogen, oxygen and sulfur. The radicals may be fused with an aryl or heteroaryl. Illustrative examples of heterocycloalkyl groups include:

and the like. The term heteroalicyclic also includes all ring forms of the carbohydrates, including but not limited to the monosaccharides, the disaccharides and the oligosaccharides.

[0043] The term "hybridization" shall mean hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding between complementary nucleobases. "Complementary" as used herein, refers to the capacity for precise pairing between two nucleobases. For example, adenine and thymine are complementary nucleobases which pair through the formation of hydrogen bonds. "Complementary" and "specifically hybridizable" refer to precise pairing or sequence complementarity between a first and a second nucleic acid- like oligomers containing nucleoside subunits. For example, if a nucleobase at a certain position of the first nucleic acid is capable of hydrogen bonding with a nucleobase at the same position of the second nucleic acid, then the first nucleic acid and the second nucleic acid are considered to be complementary to each other at that position. The first and second nucleic acids are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleobases which can hydrogen bond with each other. Thus, "specifically hybridizable" and "complementary" are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound described herein and a target RNA molecule. It is understood that an oligomeric compound described herein need not be 100% complementary to its target RNA sequence to be specifically hybridizable. An oligomeric compound is specifically hybridizable when binding of the oligomeric compound to the target RNA molecule interferes with the normal function of the target RNA to cause a loss of utility, and there is a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e. under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed.

[0044] The term "membered ring" can embrace any cyclic structure. The term "membered" is meant to denote the number of skeletal atoms that constitute the ring. Thus, for example, cyclohexyl, pyridine, pyran and thiopyran are 6-membered rings and cyclopentyl, pyrrole, furan, and thiophene are 5-membered rings.

[0045] An "isocyanato" group refers to a -NCO group. [0046] An "isothiocyanato" group refers to a -NCS group. [0047] A "mercaptyl" group refers to a (alkyl)S- group.

[0048] The term "moiety" refers to a specific segment or functional group of a molecule. Chemical moieties are often recognized chemical entities embedded in or appended to a molecule. [0049] The term "nucleobase" as used herein is intended to include naturally-occurring nucleobases (i.e., heterocyclic bases found in naturally occurring nucleic acids) and their non- naturally-occurring analogs. Thus, nucleobases described herein include naturally-occurring bases adenine (A), guanine (G), thymine (T), cytosine (C) and uracil (U), both in their unprotected state and bearing protecting or masking groups. Examples of nucleobase analogs include N⁴,N⁴-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N⁶ -methyladenine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxymethylaminomethyl uracil, inosine, N⁶- isopentyladenine, 1 -methyladenine, 2 -methyl guanine, 5-methylcytosine, N⁶-methyladenine, 7- methylguanine, 5-methylaminomethyl uracil, 5-methoxyaminomethyl-2-thiouracil, 5- methoxyuracil, pseudouracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-(l-propynyl)-4- thiouracil, 5-(l-propynyl)-2-thiouracil, 5-(l-propynyl)-2-thiocytosine, 2-thiocytosine, and 2,6- diaminopurine. Other suitable base analogs, for example the pyrimidine analogs 6-azacytosine, 6-azathymidine and 5-trifluoromethyluracil, may be found in Cook, D. P., et al, International Publication No. 92/02258, which is herein incorporated by reference. [0050] The terms "nucleophile" and "electrophile" as used herein have their usual meanings familiar to synthetic and/or physical organic chemistry. In general, a nucleophile or a nucleophilic group has a Pauling electronegativity less than the electrophile or electrophilic group with which it can react. In addition, an electrophilic group when bound to another group can render that group or portions of that group electrophilic, and thus susceptible to nucleophilic attack. Another way of viewing electophiles and nucleophiles is that electrophilic groups are electron-poor or electron-withdrawing relative to nucleophilic groups, which are correspondingly electron-rich or electron-donating.

[0051] The term "nucleoside" refers to a unit made up of a heterocyclic base and its sugar. The term "nucleotide" refers to a nucleoside having a phosphate group on its 3' or 5' sugar hydroxyl group

[0052] As used herein, the term "oligonucleotide" is intended to include both naturally occurring and non-naturally occurring (i.e., "synthetic") oligomers of linked nucleosides. Although such linkages generally are between the 3' carbon of one nucleoside and the 5' carbon of a second nucleoside (i.e., 3 '-5' linkages), other linkages (such as 2'-5' linkages) can be formed. Naturally occurring oligonucleotides are those which occur in nature; for example ribose and deoxyribose phosphodiester oligonucleotides having adenine, guanine, cytosine, thymine and uracil nucleobases. As used herein, non-naturally occurring oligonucleotides are oligonucleotides that contain modified sugar, internucleoside linkage and/or nucleobase moieties. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides. Thus, non-naturally occurring oligonucleotides include all such structures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target. [0053] The term "phosph(orothio)ate" refers to a substituent that can be either a phosphate group or a phosphorothioate group. In other words the term is equivalent to stating "phosphate or phosphorothioate."

[0054] The term "single bond" or "bond" refers to a chemical bond between two atoms, or two moieties when the atoms joined by the bond are considered to be part of larger substructure. [0055] A "sulfinyl" group refers to a -S(=O)-R, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon)

[0056] A "sulfonyl" group refers to a -S(=O)₂-R, where R is selected from the group consisting of alkyl, cycloalkyl, aryl, heteroaryl (bonded through a ring carbon) and heteroalicyclic (bonded through a ring carbon) [0057] A "thiocyanato" group refers to a -CNS group.

[0058] The term "optionally substituted" means that the referenced group may be substituted with one or more additional group(s) individually and independently selected from alkyl, cycloalkyl, aryl, heteroaryl, heteroalicyclic, hydroxy, alkoxy, aryloxy, mercapto, alkylthio, arylthio, cyano, halo, carbonyl, thiocarbonyl, isocyanato, thiocyanato, isothiocyanato, nitro, perhaloalkyl, perfluoroalkyl, silyl, and amino, including mono- and di-substituted amino groups, and the protected derivatives thereof. The protecting groups that may form the protective derivatives of the above substituents are known to those of skill in the art and may be found in references such as Greene and Wuts, above. [0059] The various phosphorus-containing compounds described herein may be present either as neutral compounds or as salts. If present as salts, the counter-ion will be any recognized counter-ion that can be used with phosphorus-containing compounds (including nucleotides and oligonucleotides). By way of example only, substituted ammonium salts, including tertiary alkyl ammonium cations, and tri-alkyl ammonium cations can be used as counter-ions to any oxygen anion present on any of the phosphorus-containing compounds described herein (see, e.g., the example seciton). Any anion present in any of the formula presented herein (including in the claims) can have as its counter-ion any cation that is used in the art for nucleotides and oligonucleotides.

[0060] Other objects, features and advantages of the methods and compositions described herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

All publications, patents and patent applications mentioned in this specification are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

[0061] A better understanding of the features and advantages of the present methods and compositions may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of our methods, compositions, devices and apparatuses are utilized, and the accompanying drawings of which:

[0062] FIG. 1 presents an illustrative example of the formation of an internucleotidic phosphate linkage under conditions of intramolecular nucleophilic catalysis. [0063] FIG. 2 presents an illustrative example of the formation of an Sp-configured internucleotidic phosphorothioate linkage under conditions of intramolecular nucleophilic catalysis.

[0064] FIG. 3 presents an illustrative example of the formation of an i?p-confϊgured internucleotidic phosphorothioate linkage under conditions of intramolecular nucleophilic catalysis.

[0065] FIG. 4 presents an illustrative example of the formation of an .ftp-configured internucleotidic phosphotriester linkage under conditions of intramolecular nucleophilic catalysis.

[0066] FIG. 5 presents an illustrative example of the formation of an Sp-confϊgured internucleotidic phosphotriester linkage under conditions of intramolecular nucleophilic catalysis.

[0067] FIG. 6 presents an illustrative example of the activation and hydrolytic regeneration of a first synthon under conditions of intramolecular nucleophilic catalysis.

[0068] FIG. 7 presents illustrative examples of formations and diastereomerical separations of first synthons. [0069] FIG. 8 shows the phosphorothioate diester region of a ³¹P-NMR spectrum recorded of the crude reaction mixture obtained in Example 5.

[0070] FIG. 9 shows the phosphorothioate diester region of a ³¹P-NMR spectrum recorded of the crude reaction mixture obtained in Example 6. DETAILED DESCRIPTION OF THE INVENTION

[0071] Since the first chemical synthesis of a dinucleotide half a century ago (see, Michelson, A.M. and A.R. Todd, Nucleotides part XXXII. Synthesis of a dithymidine dinucleotide containing a 3':5'-internucleotidic linkage. Journal of the Chemical Society, 1955: p. 2632-2638), the subject of oligonucleotide chemistry has undergone tremendous development. A handful of fundamentally distinct methods have been developed to address the need of nucleic acids and their chemically-modified counterparts in modern science. An excellent resource with accounts and protocols reflecting the current and developing state of the art of oligonucleotide synthesis as well as other aspects of the science of nucleic acids have been compiled (see, Beaucage, S. L., et al., eds. Current Protocols in Nucleic Acid Chemistry. 2000, John Wiley & Sons: New York).

[0072] Key to the success of chemical synthesis of nucleic acids has been the development of methods that afford close to quantitative formation of internucleotidic linkages during chain- assembly. The phosphoramidite approach (see, Beaucage, S. L. and R.P. Iyer, Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron, 1992. 48: p. 2223- 2311) is a highly efficient and robust method for oligonucleotide and oligonucleoside phosphorothioate synthesis. The phosphoramidite approach lends itself well to solid-phase synthesis of nucleic acids. Building blocks are repetitively added to a growing oligomer immobilized on a solid aupport. Each new chain-elongation step results in the initial formation of a phosphite triester linkage which is oxidized or optionally sulfurized to afford the corresponding phosph(orothio)ate linkages.

[0073] Other aspects of oligonucleotide synthesis include the development of novel protecting group strategies and efforts aimed at developing cost-efficient synthetic reagents and environmentally friendly reaction conditions and media. Recent progress of oligonucleotide synthesis in general, and of the phosphoramidite approach in particular has been reviewed (see, Tsukamoto, M. and Y. Hayakawa, Strategies useful for the chemical synthesis of oligonucleotides and related compounds. Frontiers in Organic Chemistry, 2005. 1: p. 3-40). A comparative perspective chronicling the development of various approaches to oligonucleotide synthesis has been published (see, Reese, CB. , The chemical synthesis ofoligo- and polynucleotides: A personal commentary. Tetrahedron, 2002. 58: p. 8893-8920). [0074] While chemical synthesis of DNA can be regarded as a more than a decade-old solved problem, the chemical synthesis of RNA has only recently seen substantial improvement and innovation (see, Marshall, W. S. and RJ. Kaiser, Recent advances in the high-speed solid phase synthesis of RNA. Current Opinion in Chemical Biology, 2004. 8: p. 222-229). Major improvements have been the development of novel and refined protecting group strategies that effectively address the requirements of a 2'-hydroxyl group protecting scheme. The advancements (see, Pitsch, S., et al., Reliable chemical synthesis of oligoribonucleotides (RNA) with 2 '-O-[(triisopropylsilyl)oxy]methyl(2 '-O-tom)-protectedphosphoramidites. Helvetica Chimica Acta, 2001. 84: p. 3773-3795; Scaringe, S.A., RNA oligonucleotide synthesis via 5 - silyl-2'-orthoester chemistry. Methods, 2001. 23: p. 206-217) in chemical synthesis of RNA have been fueled by ground-breaking discoveries of new roles of RNA within living systems and the emergence of new scientific fields of study (see, Dorsett, Y. and T. Tuschl, siRNAs: applications in functional genomics and potential as therapeutics. Nature Reviews. Drug Discovery, 2004. 3: p. 318-329; Tomari, Y. and P.D. Zamore, Perspectives: machines for RNAi. Genes & Development, 2005. 19: p. 517-529).

[0075] Of importance to the eventual success of nucleic acids as therapeutic agents from the chemist's point of view will be the successful introduction of various chemical modifications. Today's nucleotide chemists enjoy an expanding modification tool-box and reports in which these modifications are used to further our understanding of underlying biological phenomena are abundant. As our appreciation of phenomena critical to the implementation of nucleic acid based therapeutic approaches increases, avenues for strategic use of chemical modifications to improve or define pharmacologic properties can be expected to widen. [0076] Modifications of nucleic acids broadly fall into three categories, nucleobase modifications, carbohydrate modifications and modifications to the anionic phosphodiester backbones of oligonucleotides. In light of the fact that nucleic acids typically display high sequence-specificity as mediated by the information content of their nucleobases, modification strategies have generally focused on the backbone and carbohydrate aspects of oligonucleotides. These types of modifications typically confer higher metabolic stability to the oligonucleotide and improve its hybridization properties. [0077] The phosphorothioate backbone modification occupies a central role in nucleic acid based therapeutic approaches because of its ease of introduction via the commonly used phosphoramidite approach. Due to the diastereotopic nature (see, Cahn, R.S., C. Ingold, and V. Prelog, Specification of molecular chirality. Angewandte Chemie International Edition in English, 1966. 5: p. 385-415) of the internucleotidic linkage and the lack of stereocontrol during sulfurization, each phosphorothioate linkage is present in close to statistical diastereomeric ratio. The congregate number of diastereomers in a mixture increase exponentially with increasing number of phosphorothioate linkages. While studies and innovations point to a benefit of stereodefined phosphorothioate backbones over their close to stereorandom mixtures, their full potential is yet to be assessed. [0078] The impact of stereodefined phosphorothioate internucleotidic linkages on the elucidation of enzymatic reaction mechanisms (see, Eckstein, F., Nucleoside phosphor oihioates. Annual Review of Biochemistry, 1985. 54: p. 367-402; Eckstein, F., Developments in RNA chemistry, a personal view. Biochimie, 2002. 84: p. 841-848; Vortler, L.C.S. and F. Eckstein, Phosphorothioate modification of RNA for stereochemical and interference analyses. Methods in Enzymology, 2000. 317: p. 74-91) cannot be overstated and the wealth of information point to profound diastereoselectivity of phosphoryl-processing enzymes. Phosphate esters dominate the living world in a highly diastereoselective fashion over a wide range of functions (see, Westheimer, F.H., Why Nature chose phosphates. Science, 1987. 235: p. 1173-1178. The therapeutic potential of stereodefined phosphorothioate esters and the diastereoselective control that their use would result in warrants development of innovative methods for their preparation. [0079] Tetra-substituted phosphorus atoms bearing four different substituents can exist as two different isomers or enantiomers. Analogous to tetra-substituted carbon atoms bearing four different substituents, such chiral phosphorus centers can be either Rp or Sp. Compounds bearing two chiral phosphorus centers can exist as four different diastereomers: Rp-Rp, Rp-Sp, Sp-Rp, and Sp-Sp. Compounds bearing three chiral phosphorus centers can exist as eight different diastereomers, compounds with four chiral phosphorus centers as 16 diastereomers, and so on. Oligonucleoside phosphorothioates represent one class of compounds that contain tetra- substituted phosphorus atoms bearing four different substituents. [0080] The use of nucleoside phosphorothioates in the mechanistic study of phosphoryl transfer has been reviewed (see, Eckstein, F., Nucleoside phosphorothioates. Annual Review of Biochemistry, 1985. 54: p. 367-402; Vortler, L.C.S. and F. Eckstein, Phosphorothioate modification of RNA for stereochemical and interference analyses. Methods in Enzymology, 2000. 317: p. 74-91). Uses of oligonucleoside phosphorothioates include various nucleic acid based therapeutic approaches (see, Li, Z. -Y., et al., The effects of thiophosphate substitutions on native siRNA gene silencing. Biochemical and Biophysical Research Communications, 2005. 329: p. 1026-1030; Yang, X. and D. G. Gorenstein, Progress in thioaptamer development. Current Drug Targets, 2004. 5: p. 705-715; Kurreck, J., Antisense technologies. Improvement through novel chemical modifications. European Journal of Biochemistry, 2003. 270: p. 1628- 1644) and various nucleic acid molecular diagnostics and amplification protocols (see, Zhang, J. and K. Li, Single-base discrimination mediated by proofreading 3' phosphor othioate-modified primers. Molecular Biotechnology, 2003. 25: p. 223-227; Dean, F.B., et al., Rapid amplification ofplasmid and phage DNA using Phi29 DNA polymerase and multiply-primed rolling circle amplification. Genome Research, 2001. 11 : p. 1095-1099). Absent efficient and practical methods for the chiral preparation of internucleotidic linkages, applications of phosphorothioate nucleic acids have with only few exceptions involved mixtures of diastereoisomeric phosphorothioate nucleic acids.

[0081] The use of phosphorothioates in biochemical mechanistic studies shows that the biochemical fate of phosphorothioate nucleic acids is influenced by the stereochemical configuration of individual internucleotidic phosphorothioate linkages. For example, a study (see, Gilar, M., et al., Kinetics of phosphorothioate oligonucleotide metabolism in biological fluids. Nucleic Acids Research, 1997. 25: p. 3615-3620) on the metabolism of phosphorothioate nucleic acids revealed a metabolic profile consistent with stereoselectivity of the predominant metabolic enzymes (see, Gilar, M., et al., Impact of 3'-exonuclease stereoselectivity on the kinetics of phosphorothioate oligonucleotide metabolism. Antisense & Nucleic Acid Drug Development, 1998. 8: p. 35-42).

[0082] Since they exist as diastereomers, oligonucleoside phosphorothioates synthesized using known, automated techniques typically result in mixtures of R_? and Sp diastereomers at each individual phosphorothioate linkage (see, WiIk, A. and WJ. Stec, Analysis of oligo(deoxynucleosidephosphorothioate)s and their diastereomeric composition. Nucleic Acids Research, 1995. 23: p. 530-534; Murakami, A., et al., Separation and characterization of diastereoisomers of antisense oligodeoxyribonucleoside phosphorothioates. Analytical Biochemistry, 1994. 223: p. 285-290). Thus, a 21-mer oligonucleotide containing 20 asymmetric linkages has 2²⁰, i.e., 1,048,576 possible stereoisomers. It is possible that oligomers having diastereomerically enriched linkages could possess advantages in any or all individual steps of a oligonucleoside phosphorothioate-based therapeutic or diagnostic application. Accordingly, there is a need for such oligomers.

[0083] Six or more nucleotides units are generally necessary for an oligonucleotide to be of use in applications involving hybridization. More nucleoside units generally provide better performance, often as many as 10 to 30. Because it has not been possible to stereochemically resolve more than two or three adjacent phosphorus linkages, the effects of induced chirality in the phosphorus linkages of chemically synthesized oligonucleotides has not been well assessed heretofore. This is because with few limited exceptions, the sequence-specific phosphorothioate oligonucleotides obtained utilizing known automated synthetic techniques have been mixtures with little to no diastereomeric excess. [0084] The oxathiaphospholane method (see, Guga, P. and WJ. Stec, Synthesis of phosphorothioate oligonucleotides with stereodefined phosphorothioate linkages, in Current Protocols in Nucleic Acid Chemistry, S. L. Beaucage, et al., Editors. 2003, John Wiley & Sons, Inc. p. 4.17.1-4.17.28; Stec, WJ., et al., Diastereomers of nucleoside 3'-O-(2-thio-l,3,2- oxathia(selena)phospholanes: Building blocks for stereocontrolled synthesis of oligo(nucleoside phosphorothioate)s. Journal of the American Chemical Society, 1995. 117: p. 12019-12029; Stec, WJ. , et al., Novel route to oligo(deoxyribonucleoside phosphorothioates) : Stereocontrolled synthesis ofP-chiral oligo(deoxyribonucleoside phosphorothioates). Nucleic Acids Research, 1991. 19: p. 5883-5888; Stec, WJ., et al., Deoxyribonucleoside 3'-O-(2-thio- and 2-oxo-spiro- 4, 4-pentamethylene-l , 3, 2 -oxathiaphospholane) s: Monomers for stereocontrolled synthesis of oligo(deoxyribonucleosidephosphorothioate)s and chimeric PS/PO oligonucleotides. Journal of the American Chemical society, 1998. 120: p. 7156-7167) has been successful for the preparation of oligonucleoside phosphorothioates with defined stereochemistry. However, it suffers from disadvantages, such as the non-trivial preparation of substantially diastereomerically pure oxathiaphospholane, and the difficulty in synthesizing and isolating satisfactorily-pure oligonucleotides of adequate lengths for therapeutic applications.

[0085] Other methods for the preparation of oligonucleoside phosphorothioates with defined or enriched stereochemistry include versions of the established phosphoramidite methodology (see, Beaucage, S. L. and M.H. Caruthers, Deoxynucleosidephosphoramidites: A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Letters, 1981. 22: p. 1859- 1862). Both versions (see, Iyer, R.P., et al., Solid-phase stereoselective synthesis of oligonucleoside phosphorothioates: The nucleoside bicyclic oxazaphospholidines as novel synthons. Tetrahedron Letters, 1998. 39: p. 2491-2494; Iyer, R.P., et al., A novel nucleoside phosphoramidite synthon derived from lR,2S-ephedrine. Tetrahedron: Asymmetry, 1995. 6: p. 1051-1054; WiIk, A., et al., Deoxyribonucleoside cyclic N-acylphosphoramidites as a new class of monomers for the stereocontrolled synthesis of oligothymidylyl- and oligodeoxycytidylyl- phosphorothioates. Journal of the American Chemical Society, 2000. 122: p. 2149-2156 make use of cyclic phosphoramidites derived from natural products). [0086] Methods devised and applied to the synthesis of dinucleoside phosphorothioates with enriched or stereodefined chirality at the internucleotidic phosphorothioate linkage are more prevalent (see, Aimer, H., T. Szabo, and J. Stawinski, A new approach to stereospecific synthesis of P-chiral phosphorothioates. Preparation of diastereomeric dithymidyl-(3'-5)' phosphorothioates. Chemical Communications, 2004: p. 290-291; Jin, Y., G. Biancotto, and G. Just, A stereoselective synthesis of dinucleotide phosphorothioates using chiral phosphoramidites as intermediates. Tetrahedron Letters, 1996. 37: p. 973-976; Jin, Y. and G. Just, Stereoselective synthesis of dithymidine phosphorthioates using xylose derivatives as chiral auxiliaries. Journal of Organic Chemistry, 1998. 63: p. 3647-3654; Lu, Y. and G. Just, Stereoselective synthesis of Rp- and Sp-dithymidine phosphorothioates via chiral indoloxazaphosphorine intermediates derived from tryptophan. Angewandte Chemie International Edition in English, 2000. 39: p. 4521-4524; Oka, N., T. Wada, and K. Saigo, Diastereocontrolled synthesis of dinucleoside phosphorothioates nusing a novel class of activators, dialkyl(cyanomethyl) ammonium tetrafluoroborates. Journal of the American Chemical Society, 2002. 124: p. 4962-4963; Wada, T., et al., Stereocontrolled synthesis of dithymidine phosphorothioates by use of a functionalized 5 '-protecting group bearing an imidazole residue. Nucleosides and Nucleotides, 1998. 17: p. 351-364; Wang, J.C. and G. Just, Indol-oxazaphosphorine precursors for stereoselective synthesis of dinucleotide phosphorothioates. Journal of Organic Chemistry, 1999. 64: p. 8090-8097). [0087] Whereas existing methodologies for oligonucleoside phosphorothioate synthesis providing close to stereorandom or undefined phosphorothioate linkages allow for efficient construction of oligomeric products of lengths suitable for therapeutic and diagnostic applications, the methodologies mentioned above, including the oxathiaphospholane method, all present practical limitations for the construction of oligonucleoside phosphorothioates with stereodefined phosphorothioate linkages and of lengths suitable for therapeutic or diagnostic applications. [0088] Factors influencing practicability include varying degrees of stereospecificity of repetitive coupling steps during chain-elongation resulting in chiral enrichment rather than stereodefinition of individual phosphorothioate linkages. Other factors limiting practicability include sub-optimal repetitive chain-elongation efficiencies, access to requisite monomers, sensitivity to the presence of adventitious moisture during repetitive chain-elongation. [0089] Limited practicability of methods for stereospecific phosphorothioate synthesis has presented inevitable obstacles to stringent and rational design of oligonucleoside phosphorothioates for therapeutic and diagnostic applications. Oligonucleoside phosphorothioates as therapeutic drugs or components of molecular diagnostic protocols are amenable to pharmacological characterization, which in the case of the prior diastereomeric mixtures quickly becomes contrived and inexact.

[0090] Of great benefit to the field of nucleic acid based therapeutics and molecular diagnostic protocols would be the advent of stereodefined oligonucleoside phosphorothioates that could address issues relevant to pharmacological function or assay performance. [0091] It would therefore be of great advantage to provide a practical method for producing oligonucleoside phosphorothioates having phosphorothioate linkages with defined stereochemical configuration. Furthermore, it would be of great benefit if the practical method would allow for production of oligonucleotide containing any desired combination of stereodefϊned phosphorothioate and achiral phosphodiester linkages.

[0092] Described herein are methods for the synthesis of phosphorothioate compounds having diastereomerically enriched phosphorothioate linkages, and to intermediates useful in their preparation.

[0093] In one aspect, methods for the preparation of phosph(orothio)ate linkages comprise reacting a first synthon of Formula I:

wherein:

Q is independently O or S;

R¹ is a hydroxyl protecting group;

R² is a phosph(orothio)ate protecting group enabling intramolecular nucleophilic catalysis;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group; B is a nucleobase; and n is an integer from 0 to 50; with a second synthon of Formula II:

wherein:

R⁴ is hydroxyl protecting group or a linker connected to a solid support; and m is an integer from 0 to 50; for a time and under reaction conditions effective to form a third synthon of Formula III:

wherein D is the chiral phosph(orothio)ate linkage having the formula:

94] The first synthons are phosph(orothio)ates having the general Formula XV:

R²-W XV wherein R² have one of the general Formulas XVIa-XVIg:

and wherein W has the formula:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring. [0095] The reaction of first and second synthons is conducted in the presence of a condensing reagent. The structure of the condensing reagent is chosen such that the condensing reagent activates the desired position of the negatively-charged phosphorothioate diester of the first synthon. Condensing reagents of the general Formulas XI and XII are chosen when a chemoselective activation of the oxygen atom of the negatively-charged phosphorothioate diester of the first synthon is desired, to yield a third synthon, whose newly- formed phosphorothioate triester linkage is diastereomerically enriched at phosphorus. Condensing reagents of the general Formula XIII are chosen when a chemoselective activation of the sulfur atom of the negatively- charged phosphorothioate diester of the first synthon is desired, to yield a third synthon, whose newly formed phosphotriester linkage is diastereomerically enriched at phosphorus. [0096] Accordingly, in further or alternative embodiments of the methods described herein, first synthons are diastereomerically enriched, including in a single stereochemical form, substantially free of other stereochemical forms. As used herein, the term chemoselective has its normal meaning as a process in which one chemical feature reacts faster or slower than another, resulting in a predominance of the favored product. As used herein, the term stereoselective has its normal meaning as a process in which one stereoisomer is produced or destroyed more rapidly than another, resulting in a predominance of the favored stereoisomer. [0097] Intra- and intermolecular nucleophilic catalysis is important in phosphoryl transfer reactions, both in chemical synthesis and in biochemical transformations. The general rate enhancement observed when applying intramolecular nucleophilic catalysis to the synthesis of a range of phosphoryl- and phosphonyl-containing oligomeric products have allowed chemical synthesis of oligomers of lengths generally required for therapeutic and diagnostic applications (see, Efimov, V. A., et al., Application of new catalytic phosphate protecting groups for the highly efficient phosphotriester oligonucleotide synthesis. Nucleic Acids Research, 1986. 14: p. 6525-6540; Froehler, B.C. and M.D. Matteucci, l-Methyl-2-(2-hydroxyphenyl)imidazole: A catalytic phosphate protecting group in deoxyoligonucleotide synthesis. Journal of the American Chemical Society, 1985. 107: p. 278-279; Rejman, D., M. Masojidkova, and I. Rosenberg, Nucleosidyl-O-methylphosphonates: A pool of monomers for modified oligonucleotides. Nucleosides, Nucleotidses & Nucleic Acids, 2004. 23: p. 1683-1705; Sproat, B.S., P. Rider, and B. Beijer, Highly efficient oligodeoxyribonucleotide synthesis using fully base protected phosphodiester building blocks carrying 2-(l -methylimidazol-2-yl) phenyl protection of the phosphate. Nucleic Acids Research, 1986. 14: p. 1811-1824; Szabό, T., A. Kers, and J. Stawinski, A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogs. Nucleic Acids Research, 1995. 23: p. 893-900). While not wishing to be bound by a particular theory, it is believed that the rate enhancement originates from the rapid formation of a highly reactive cyclic phosphorus-containing intermediate after initial reaction of a condensing reagent and a phosphoryl or phosphonyl moiety of a first synthon. In further or alternative embodiments of this process, the cyclic intermediate formed during intramolecular nucleophilic catalysis is either a 5- or a 6-membered cyclic intermediate. The highly reactive intermediate species contains a good leaving group in the form of the intramolecular catalyst, which is displaced by the nucleophile of a second synthon intended for covalent bond formation with the phosphoryl or phosphonyl moiety of a first synthon resulting in a third synthon. After (iterative) synthesis of a desired third synthon is completed, the catalytic protecting group(s) can be removed. The overall process is depicted in Figure 1. [0098] Use of the intramolecular nucleophilic catalysis concept to synthesize chiral phosph(orothio)ate triesters is novel. In these processes, first synthons are diastereomerically enriched, including in single diastereochemical forms, substantially free of other stereochemical forms. Figures 2-5 depict these processes with some illustrative embodiments of the processes as defined above.

[0099] The success of these processes stems from the intramolecular nature of the rate- enhancing nucleophilic catalysis. Following chemoselective activation of first synthons, a catalytic phosphorothioate protecting group displaces the activated phosphorothioate ligand with inversion of configuration forming a cyclic reactive intermediate which reacts with the nucleophile of a second synthon, the ring-opening occuring with inversion of configuration. The overall processes occurs with retention of configuration, the spatial arrangement of ligands around the pseudo-tetrahedral phosphorus center is conserved and the chemoselectively activated ligand is replaced by the nucleophile of a second synthon. [00100] After (iterative) synthesis of a desired third synthon is completed, the catalytic protecting group(s) can be removed. In Figures 1-5 this removal occurs with retention of configuration of phosphorus stereochemistry. The use of other embodiments described herein may lead to a process in which the final removal of catalytic protecting group(s) from a third synthon occurs with inversion of configuration of phosphorus stereochemistry. Also in the preparation of first synthons may a phosph(orothio)ate protecting group be removed with concomitant inversion of configuration of phosphorus stereochemistry.

[00101] Figure 7 depicts a non-limiting set of chemical transformations and diastereochemical separations of useful in the preparation of first synthons. The scheme includes processes that occur with either retention or inversion of configuration of the chiral phosphorus linkage. In Figures 4 and 5 the removal of the catalytic protecting group results in loss of chirality resulting in an achiral phosphorus linkage. Embodiments of the present invention in which activation of the sulfur atom of a first synthon' s phosphorothioate di ester anion preferably occur in solution- phase approaches. The "fat" or "hollow" arrows in Figure 7 indicate separation or purification steps; the thin solid arrows represent chemical transformations; the dotted arrow signifies a reaction that can occur with either retention or inversion at the phosphorus center, depending upon the identity of the R⁵ group.

[00102] The stereospecific production of phosphotriester linkages of Figures 4 and 5 facilitates solution phase production of third synthons in which the third synthons are converted to new first and/or second synthons. In so doing the solution phase production benefits from the formation of only one phosphotriester stereochemical configuration thereby simplifying intermediate purification, characterization, and their conversion into new first and second synthons as required by a solution phase production strategy.

[00103] The methods and compositions described herein allow the use of configurationally- stable first synthons for the preparation of oligonucleoside phosphorothioates with defined stereochemistry. The phosph(orothio)ate diester moieties of first synthons are also hydrolytically stable and the presence of adventitious water during reaction between first and second synthons may result in hydrolysis of the reactive cyclic intermediates. In cases where the desired third synthon contains a newly-formed stereodefined phosphorothioate linkage, this hydrolysis occurs stereospecifically with inversion of configuration of phosphorus stereochemistry, consuming condensing reagent and effectively regenerating the first synthon while depleting adventitious water as illustrated in Figure 6. A consequence of such hydrolyses is that it allows for recovery of excess first synthons used.

[00104] The methods and compositions described herein can also be used to produce analogs of phosphorothioates, including stereodefined phosphoroselenoates and isotopically-defined phosphates. For example, requisite phosphoroselenoate containing first synthons can be formed by reacting a compound of Formula V with a selenophosphorylating reagent of Formula XVII:

wherein R² and R⁵ are as defined above. [00105] R¹ and R⁴ can each be a hydroxyl protecting group. Protecting groups are chemical functional groups that can be selectively appended to and removed from functionalities, such as hydroxyl groups and carboxyl groups. These groups are present in a chemical compound to render such functionality inert to chemical reaction conditions to which the compound is exposed. One protecting group for R¹ is the dimethoxytrityl group. A comprehensive introduction to protecting groups, conditions for their introduction and removal can be found in (see, Greene, T. W. and P.G.M. Wuts, Protective Groups in Organic Synthesis. 3rd ed. 1999: Wiley-Interscience). Typically, protecting groups are removed at the end of the iterative synthesis.

[00106] R⁴ may alternatively be a linker connected to a solid support. Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Pat. Nos. 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418; and Koster U.S. Pat. Nos. 4,725,677 and Re. 34,069. Linkers are short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in Oligonucleotides And Analogues. A Practical Approach, Eckstein, F. Ed., IRL Press, N. Y., 1991.

[00107] The compounds described herein have up to 50 nucleobases in length, with 10 to 30 nucleobases being one embodiment, and 15 to 25 nucleobases being a further or alternative embodiment.

[00108] In further or alternative embodiments the phosph(orothio)ate linkage produced by the methods described herein is diastereomerically enriched. The term "diastereomerically enriched" denotes the predominance of one stereochemical form over the other. In further or alternative embodiments the phosph(orothio)ate linkage is about 98% in a single stereochemical form. In further or alternative embodiments the phosphorothioate linkage is about 98% in a single stereochemical form. In further or alternative embodiments the phosph(orothio)ate linkage is in a single stereochemical form, substantially free of other stereochemical forms. The term "substantially diastereomerically pure" refers to a material in which at least about 95% of the molecules (e.g., oligonucleotides) have the same diastereomeric configuration. Of course the term also encompasses materials in which about 96%, about 97%, about 98%, about 99%, and about 99.5% of the molecules have the same diastereomeric configuration. One illustrative method for determining diastereomeric purity of the phosphorus-chiral compounds described herein is by ³¹P nmr spectroscopy. [00109] The employment of a first synthon with a predetermined diastereomeric enrichment will lead to the formation of a new third synthon with a diastereomeric enrichment reflecting the diastereomeric enrichment of the first synthon. For example, if a phosphorothioate linkage is sought to be produced in a 1:1 ratio of Rp- and Sp-configurations, respectively, first synthons of no diastereomerically enrichment can be used. [00110] Using the methods and compositions described herein, oligonucleoside phoph(orothio)ates can be produced by solid-phase based methods, by solution phase methods, or by combination methods thereof. These methods can encompass strategies in which newly formed phosph(orothio)ate linkages results from activating a phosph(orothio)ate diester functionality covalently attached to a 5'-hydroxyl of a synthon and further reaction with a nucleophile of a synthon other than a 5'-hydroxyl. Furthermore, inverted, 3 '-3' and 5 '-5', linkages can be obtained by the use of appropriate synthons.

[00111] During particular solid-phase based methods, new second synthons are formed by removal of the 5'-hydroxyl protecting group R¹ under conditions which will depend upon the chemical identity of the specific R¹ group. After removal of the protecting group, the 5'- hydroxyl becomes the nucleophile of a new second synthon in the iterative process. Libraries of dimeric and higher synthons may be prepared and stored to facilitate the iterative synthesis of desired nucleobase sequences.

[00112] The methods described herein can be carried out in any suitable vessel which provides efficient contacting between the first and second synthons, and the condensing reagent. The reaction vessel used should be resistant to the components of the reaction mixture. Glass- lined vessels would be suitable for this purpose. Additional vessel materials will be apparent to those skilled in the art based on this disclosure.

[00113] The methods described herein are performed in the presence of a solvent. Solvents suitable for use in the present methods will be readily apparent to those skilled in the art, once having been made aware of the present disclosure.

[00114] In general, embodiments of the method uses an excess of one synthon to the other, and an excess of condensing reagent to the first synthon, effectively driving the condensation reaction to completely consume one of the synthons. Illustrative embodiments of solid-phase based methods employ excesses of the first synthon to the second synthon from about 1 to 50, and excesses of condensing reagent to first synthon from about 1 to 50.

[00115] The method can be conducted under an inert atmosphere, and be carried out in a dry atmosphere. Any suitable inert gas may be employed, such as nitrogen, helium and argon. The method is carried out at temperatures ranging between about -20 ⁰C, and about 40 ⁰C. Reaction time is generally from about 30 seconds to about 30 minutes, with reaction times from about one minute to about 10 minutes being one embodiment.

[00116] Product can be recovered by any of several methods known to those of skill in the art. Products can be recovered by chromatography. When using solid-phase based strategies, purification is carried out after removal of the oligonucleotide from the solid support using methods known in the art. [00117] The methods and compositions described herein are further illustrated by way of the following examples. These examples are illustrative only and are not intended to limit the scope of the appended claims.

ILLUSTRATIVE EXAMPLES [00118] Pyridine, acetonitrile, and tert-butylamine were re fluxed with CaH₂ and then distilled and stored over molecular sieves (4A). Triethylamine was refluxed with CaH₂ and then distilled and stored over CaH₂ (triethylamine). Methylene chloride was freshly distilled from CaH₂.

[00119] 5'-0-(tert-butyldiphenylsilyl)thymidine (see, Larsen, E., et al., A new and easy synthesis od silylated furanoid glycate in one step from nucleosides. Synthesis, 1994: p. 1037- 1038), 3'-O-(tert-butyldiphenylsilyl)thymidine (see, Szabό, T., et al., Molecular and crystal structure of Sp-thymidin-3'-yl 4-thiothymidin-5'-yl methylphosphonate. Nucleic Acids Research, 1993. 21 : p. 3921-3926), 2-chloro-5,5-dimethyl-2-oxo-l,3,2-dioxaphosphinane (see, Patois, C, L. Ricard, and P. Savignac, 2-Alkyl-5,5-dimethyl-l,3,2-dioxaphosphorinan-2-ones a-lithiated carbanions. Synthesis, stability, and conformation. Journal of the chemical society, Perkin Transactions 1, 1990: p. 1577-1581), and 4-methoxy-2-pyridine methanol 1-oxide (see, Rejman, D., J. Erbs, and I. Rosenberg, Large-scale synthesis of a key catalytic reagent for phosphorus protection in building blocks for isopolar phosphonate oligonucleotide preparation. Organic Process Research & Development, 2000: p. 473-476) were synthesized according to literature procedures. 9-Fluorenemethyl H-phosphonate was prepared via transesterification of diphenyl H- phosphonate with 9-fluorenemethanol, analogously to other alkyl H-phosphonate monoesters (see, Kers, A., et al., Studies on aryl H-phosphonates; Part 2: A general method for the preparation of alkyl H-phosphonate monoesters. Synthesis, 1995: p. 427-430). Nucleosides, snake venom phosphodiesterase (SVPD, Crotalus atrox) and nuclease Pl were purchased from Sigma. All isolated compounds were of purity >98% (¹H NMR spectroscopy).

Example 1 4-Methoxy-l-oxido-2-picolyl 9-fluorenemethyl phosphorothioate, triethylammonium salt (1)

1

[00120] 9-Fluorenemethyl phosphonic acid (0.43 g, 1.2 mmol) and 4-methoxy-2-pyridine methanol 1-oxide (0.19 g, 1.2 mmol) were coevaporated with added pyridine/triethylamine (4:1 v/v, 10 mL), followed by pyridine (2 x 10 mL). The mixture was dissolved in pyridine (10 mL) and 2-chloro-5,5-dimethyl-2-oxo-l,3,2-dioxaphosphinane (0.44 g, 2.4 mmol) was added during stirring. The reaction was allowed to stand for 10 min. Water (43 μL, 2.4 mmol) was added and after an additional minute elemental sulfur (0.15 g, 4.8 mmol) was added to the reaction mixture. After 2 h the reaction mixture was concentrated in vacuo and the resulting oily residue was dissolved in chloroform (50 mL), washed with triethylammonium bicarbonate buffer (1 M, 50 mL) followed by aqueous Na₂S₂O₃ (sat., 50 mL). The organic phase was dried over Na₂SO₄ and the solvent was evaporated. Silica gel column chromatography using a stepwise gradient of MeOH (0-25%) in CHCl₃ containing 0.1% triethylamine to afford phosphorothioate as a white solid (0.40 g, 63%).

Example 2 5'-(9-(tert-Butyldiphenylsilyl)thymidin-3'-yl 9-fluorenylmethyl 1 -oxido-4-methoxy-2-picolyl phosphorothioate, [<Sp]-isomer (2a) and [i?p]-isomer (2b)

[00121] 4-Methoxy-l-oxido-2-picolyl 9-fluorenemethyl phosphorothioate, triethylammonium salt (0.36 g, 0.68 mmol) and 5'-O-(tert-butyldiphenylsilyl)thymidine (0.39 g, 0.81 mmol) were dissolved in pyridine (5.6 mL) and 2-chloro-5,5-dimethyl-2-oxo-l,3,2-dioxaphosphinane (0.37 g, 2.0 mmol) was added. After 40 minutes the reaction was complete. The mixture was poured into EtOAc (50 mL) and washed with aqueous NaCl (sat., 50 mL). The organic phase was dried over Na₂SO₄ and the solvent was evaporated. Silica gel column chromatography using a stepwise gradient of Pr¹OH (0-10%) in CHCl₃ containing 0.1 %o HOAc gave faster eluting isomer 2a [0.29 g, 48%; R_f = 0.48 in CHC1₃/CH₃OH (9:1, Wv)], slower eluting isomer 2b [0.11 g, 18%; Rf = 0.37 in CHCI3/CH3OH (9:1, Wv)] and a mixed fraction of isomers (0.14 g, 23%) for a total yield of 89%. Correlation with enzymatic hydrolyses of dinucleotides stemming from these products (see Examples below) allows the assignment of Sp- and ^-configuration to faster eluting isomer 2a and slower eluting isomer 2b, respectively. Note the ligand priority change (see, Cahn, R.S., C. Ingold, and V. Prelog, Specification of molecular chirality. Angewandte Chemie International Edition in English, 1966. 5: p. 385-415) which results in a Rp <→ Sp change in designating chirality when transforming 2a and 2b to 5a and 5b, respectively.

Example 3

[Sp]S '-0-(tert-Butyldiphenylsilyl)thymidin-3 '-yl 1 -oxido-4-methoxy-2-picolyl phosphorothioate, triethylammonium salt (3 a)

[00122] [Sp]S '-O-Ctert-Butyldiphenylsily^thymidin-S'-yl 9-fluorenylmethyl l-oxido-4- methoxy-2-picolyl phosphorothioate 2a (1.13 g, 1.27 mmol) was dissolved in pyridine/tert- butylamine (9:1, v/v, 50 mL) and the reaction mixture was stirred at room temperature for 15 min. The solvent was evaporated in vacuo and the residue was partitioned between CHCl₃ (2 x 100 mL) and triethylammounium bicarbonate buffer (1 M, 100 mL). The organic phase was dried over Na₂SO₄ and the solvent was evaporated. Residual pyridine was removed by evaporation of added MeCN (2 x 50 mL). Silica gel chromatography using a stepwise gradient of MeOH (0-25%) in CHCl₃ containing triethylamine (0.1%) afforded the diester 3a (0.87 g, 85 %) as a white foam.

Example 4

[Λp]-5'-O-(tert-Butyldiphenylsilyl)thymidin-3'-yl l-oxido-4-methoxy-2-picolyl phosphorothioate, triethylammonium salt (3b)

[00123] [Λ_P]-5'-O-(tert-Butyldiphenylsilyl)thymidin-3'-yl 9-fluorenylmethyl l-oxido-4- methoxy-2-picolyl phosphorothioate 2b (0.49 g, 0.55 mmol) was dissolved in pyridine/tert- butylamine (9:1, v/v, 20 mL) and the reaction mixture was stirred at room temperature for 15 min. The solvent was evaporated in vacuo and the residue was partitioned between chloroform (2 x 50 mL) and triethylammounium bicarbonate buffer (1 M, 50 mL). The organic phase was dried over Na₂SO₄ and the solvent was evaporated. Residual pyridine was removed by evaporation of added MeCN (2 x 50 mL). Silica gel chromatography using a stepwise gradient of MeOH (0- 25%) in CHCI3 containing triethylamine (0.1 %) afforded the diester 3b (0.33 g, 74 %) as a white foam.

Example 5

[Sp]S '-O-(tert-Butyldiphenylsilyl)thymidin-3 '-yl 3 '-O-(tert-butyldiphenylsilyl)thymidin-5 '-yl l-oxido-4-methoxy-2-picolyl phosphorothioate (4a)

[00124] [.Sp]-S '-O-(tert-Butyldiphenylsilyl)thymidin-3 '-yl 1 -oxido-4-methoxy-2-picolyl phosphorothioate, triethylammonium salt 3a (0.71 g, 0.87 mmol) and V-iO-tert- butyldiphenylsilyl)thymidine (0.63 g, 1.3 mmol) were coevaporated with CH₂Cl₂ZMeCN (1:1, v/v, 2 x 10 mL) and dissolved in CH₂Cl₂ (7.2 mL). Pyridine (0.21 mL, 2.6 mmol) was added followed by 2-chloro-5,5-dimethyl-2-oxo-l,3,2-dioxaphosphinane (0.48 g, 2.6 mmol). After 15 minutes the reaction was poured into triethylammonium bicarbonate buffer (1 M, 100 mL) and extracted with CH₂Cl₂ (2 x 20 mL). The organic layer was dried over Na₂SO₄, filtered and the solvent was evaporated. Column chromatography using CHCl₃ as an eluent with a gradient of Pr¹OH (0-10 %) afforded the protected TT-dimer 4a (0.70 g, 68%) as a white foam.

Example 6

[Rp] -5 ' -0-(tert-Butyldiphenylsilyl)thymidin-3 ' -yl 3 ' -0-(tert-butyldiphenylsilyl)thymidin-5 ' -yl l-oxido-4-methoxy-2-picolyl phosphorothioate (4b)

[00125] [i?p]-5'-O-(tert-Butyldiphenylsilyl)thymidin-3'-yl l-oxido-4-methoxy-2-picolyl phosphorothioate, triethylammonium salt 3b (0.33 g, 0.40 mmol) and 3'-(0-tert- butyldiphenylsilyl)thymidine (0.29 g, 0.60 mmol) were coevaporated with CH₂Cl₂/MeCN (1:1, v/v, 2 x 4 mL) and dissolved in CH₂Cl₂ (3.3 mL). Pyridine (0.1 mL, 1.2 mmol) was added followed by 2-chloro-5,5-dimethyl-2-oxo-l,3,2-dioxaphosphinane (0.22 g, 1.2 mmol). After 15 minutes the reaction was poured into triethylammonium bicarbonate buffer (1 M, 50 mL) and extracted with CH₂Cl₂ (2 x 10 mL). The organic layer was dried over Na₂SO₄, filtered and the solvent was evaporated. Column chromatography using CHCl₃ as an eluent with a gradient of Pr¹OH (0-10 %) afforded the protected TT-dimer 4b (0.35 g, 73%) as a white foam.

Example 7 [7?p]-Thymidin-3'-yl thymidin-5'-yl phosphorothioate, sodium salt (5a)

[00126] [S_P]-5'-O-(te^Butyldiphenylsilyl)thymidin-3'-yl V-O-(tert- butyldiphenylsilyl)thymidin-5'-yl l-oxido-4-methoxy-2-picolyl phosphorothioate 4a (0.70 g, 0.60 mmol) was dissolved in pyridine/triethylamine/thiophenol (1:1:1, v/v/v) and mixture stirred at room temperature for 2 h. Silica gel chromatography using a stepwise gradient of MeOH (0- 10%) in chloroform containing 0.1 %o triethylamine gave the corresponding phosphorothioate diester. Then, the obtained compound (0.32 g, 0.28 mmol) and tetrabutylammonium fluoride trihydrate (0.27 g, 0.84 mmol) were dissolved in THF (0.26 mL) and stirred at room temperature over night. H₂O (5 mL) was added and the solution was washed with ethyl ether (3 x 2 mL). The aqueous phase was evaporated to near dryness and the product was passed through an ion- exchange column Dowex 50W-X2, 100 - 200 mesh (dry), strongly acidic cation exchange, ion form Na⁺. The product was passed through a gel filtration column Sephadex GlO and lyophilized to give TT-dimer 5a (142 mg, 40%).

Example 8 [iSp]-Thymidin-3'-yl thymidin-5'-yl phosphorothioate, sodium salt (5b)

[00127] [Rp]S '-O-(tert-Butyldiphenylsilyl)thymidin-3 '-yl 3 '-O-(tert- butyldiphenylsilyl)thymidin-5'-yl l-oxido-4-methoxy-2-picolyl phosphorothioate 4a (0.20 g, 0.18 mmol) was dissolved in pyridine/triethylamine/thiophenol (1:1:1, v/v/v) and mixture stirred at room temperature for 2 h. Silica gel chromatography using a stepwise gradient of MeOH (0- 10%) in chloroform containing 0.1 %o triethylamine gave of the corresponding phosphorothioate diester. Then, the obtained compound (0.10 g, 0.088 mmol) and tetrabutylammonium fluoride trihydrate (81 mg, 0.26 mmol) were dissolved in THF (0.26 mL) and stirred at room temperature over night. H₂O (5 mL) was added and the solution was washed with ethyl ether (3 x 2 mL). The aqueous phase was evaporated to near dryness and the product was passed through an ion- exchange column Dowex 50W-X2, 100 - 200 mesh (dry), strongly acidic cation exchange, ion form Na⁺. The product was passed through a gel filtration column Sephadex GlO and lyophilized to give TT-dimer 5b (46 mg, 44%).

Example 9 Enzymatic digestion of Compounds 5 a and 5b

[00128] The following stock solutions were prepared: dinucleoside phosphorothioate 5a or 5b (5 mg, 0.009 mmol) was dissolved in buffer A [0.250 mL; 30 mM (NH₄)₂SO₄ and 0.44 mM ZnSO₄] and in buffer B [0.250 mL;50 mM Tris-HCl and 0.2 mM MgCl₂]. Nuclease Pl (1 mg ) was dissolved in buffer A (0.5 mL) and snake venom phosphodiesterase (SVPD, 1.6 mg), in buffer B (0.5 mL).

The enzymatic digestion was carried out by mixing a sample of 5a or 5b in buffer A (0.05 mL) with nuclease Pl in buffer A (0.05 mL) or with SVPD in buffer B (0.05 mL), and the reaction mixtures were incubated at 37°C over night. TLC, Pr¹OH /ammonia/water (7:2:1, v/v/v) revealed that nuclease Pl hydrolyzed the isomer 5b and snake venom phosphodiesterase (SVPD) hydrolyzed the isomer 5a. This identified (see, Burgers, P.M.J, and F. Eckstein, Absolute configuration of the diastereomers of adenosine 5'-O-(l-thiotriphosphate): consequences for the stereochemistry of polymerization by DNA-dependent RNA polymerase from Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America, 1978. 75: p. 4798-4800; Potter, B.V.L., B.A. Connolly, and F. Eckstein, Synthesis and configurational analysis of a dinucleoside phosphate isotopically chiral at phosphorus. Stereochemical course of penicillium citrum nuclease Pl reaction. Biochemistry, 1983. 22: p. 1369-1377) configurations of the phosphorus centers in 5a and 5b as being Rp and Sp, respectively. [00129] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. Variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Claims

CLAIMSWe claim:

1. A method for preserving the stereochemistry of a chiral phosphorus center undergoing substitution with an intermolecular nucleophile comprising performing an activation reaction on the chiral phosphorus center with a condensing reagent followed by intramolecular nucleophilic displacement of the activated phosphorus portion and then performing a second substitution reaction on the chiral phosphorus center by intermolecular nucleophilic displacement, wherein the second substitution reaction reforms the intramolecular nucleophile to form a product preserving the original stereochemistry of the chiral phosphorus center.

2. The method of claim 1 wherein the chiral phosphorus center is a phosphorothioate diester.

3. The method of claim 1 wherein the chiral phosphorus center is a phosphoroselenoate diester.

4. The method of claim 2 wherein the activation reaction activates the oxygen anion portion of the phosphorothioate diester.

5. The method of claim 2 wherein the activation reaction activates the sulfur portion of the phosphorothioate diester.

6. The method of claim 3 wherein the activation reaction activates the oxygen anion portion of the phosphoroselenoate diester.

7. The method of claim 3 wherein the activation reaction activates the selenium portion of the phosphoroselenoate diester.

8. The method of claim 2 wherein the phosphorothioate diester has the structure

W W

I I I o o

R²— O P=S or R²— O— P=S I θ o Ξ θ O wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has the structure

wherein:

R¹ is a hydroxyl protecting group; R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group; Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the nucleophile of the second substitution reaction has the structure

wherein:

R is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁴ is hydroxyl protecting group or a linker connected to a solid support;

Q is O or S;

B is a protected nucleobase; m is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis has a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring.

9. A compound having the structure w W I o O

R²— O P=S R²— O— P=S

I Θ ϊ θ o O wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has the structure

wherein:

R¹ is a hydroxyl protecting group; R² is a phosph(orothio)ate protecting group; R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis has a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring.

10. The method of claim 2 wherein the phosphorothioate diester has the structure

wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis;

R⁵ is a phosphorothioate protecting group; and wherein the nucleophile of the second substitution reaction has the structure

wherein:

R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁴ is hydroxyl protecting group or a linker connected to a solid support;

B is a protected nucleobase; m is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis has a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring.

11. A compound having the structure o O

R2 — OMlMP I -=S R²— O— P=S i

O δ I I R⁵ wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; and R⁵ is a phosphorothioate protecting group.

12. A compound having the structure

W W

I I

I I O O

I P=S or R²— O»-P=S

1 o o

I I R⁵ R⁵ wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; R⁵ is a phosphorothioate protecting group; W has the structure

wherein:

R¹ is a hydroxyl protecting group;

R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring.

13. The method of claim 4 wherein the phosphorothioate diester has the structure w I o

R²— O— P=S

I e o wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has the structure

wherein:

R¹ is a hydroxyl protecting group; R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group; Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the nucleophile of the second substitution reaction has the structure

wherein: R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁴ is a linker connected to a solid support;

Q is O or S;

B is a protected nucleobase; m is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring; wherein the phosphorothioate diester is diastereomerically enriched.

14. The method of claim 2 wherein the phosphorothioate diester has the structure w w

I I o o

R²— O" "P=S R²— O— P=S o o^θ wherein:

R »2 is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has a structure selected from:

wherein:

R¹ is a hydroxyl protecting group; R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group; R⁵ is a phosph(orothio)ate protecting group; Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the nucleophile of the second substitution reaction has a structure selected from

wherein:

R¹ is a hydroxyl protecting group;

R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁵ is a phosph(orothio)ate protecting group;

Q is O or S;

B is a protected nucleobase; m is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring.

15. A compound having the structure w W o o

R²— O P=S R²— O— P=S

1 Θ Ξ θ

O O wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has a structure selected from:

wherein:

R¹ is a hydroxyl protecting group;

R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁵ is a phosph(orothio)ate protecting group;

Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring; wherein all chiral phosphorus centers are substantially diastereomerically pure.

16. A compound having the structure

W W

I I O o

I R²— O P=S or R²— O—P=S

1

O ό

I

wherein: R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis;

R⁵ is a phosphorothioate protecting group. W has a structure selected from:

wherein:

R² is a phosph(orothio)ate protecting group;

R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁵ is a phosph(orothio)ate protecting group;

Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substututed aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring; wherein all chiral phosphorus centers are substantially diastereomerically pure.

17. A compound having the structure

wherein:

R² is a phosphorothioate protecting group enabling intramolecular nucleophilic catalysis; W has a structure selected from:

wherein:

R _>2 is a phosph(orothio)ate protecting group; R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

R⁵ is a phosph(orothio)ate protecting group; Q is O or S;

B is a protected nucleobase; n is an integer from 0 to 50; and wherein the phosphorothioate protecting group enabling intramolecular nucleophilic catalysis have a structure selected from:

and R¹⁵-R⁵⁰ are independently hydrogen, alkoxy, alkylamino, halogen, cyano, nitro, alkyl having from one to 10 carbons, substituted alkyl having from one to 10 carbons, aryl, substituted aryl, ester groups, or by being joined together and together with the carbon atoms to which they are attached, form a substituted or unsubstituted ring; wherein all chiral phosphorus centers are substantially diastereomerically pure.

18. A compound having a structure selected from:

wherein:

R² is a phosph(orothio)ate protecting group or H; R³ is H, a protected hydroxyl, a 2'-substituent group or a protected 2'-substituent group;

B is a nucleobase; n is an integer from 2 to 100; wherein all chiral phosphorus centers are substantially diastereomerically pure and at least two of the phosphorus centers are chiral.

19. The method of claim 1 wherein the product formed is a substantially diastereomerically pure oligonucleotide.

20. The method of claim 1, further comprising recovery and reuse of excess synthon containing the chiral phosphorus center.