4-SE-THYMIDINE DERIVATIVES, 4-SE-URID1NE DERIVATIVES, DI(2- CYANOETHYL) DISELENIDE, AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit to U.S. Provisional Patent Application No.

60/871 ,598, filed on December 22, 2006, the disclosure of which is expressly incoφorated herein by reference.

ACKNOWLEDGMENTS This invention was made with United States government support under the following grants: NIH GM069703 and NSF MCB-0517092. The United States government has certain rights to this invention.

BACKGROUND Nucleic acids (NAs) participate in many important biological functions in living systems, including genetic information storage, gene expression, and catalysis. ^{1 "4} Determination of 3-D structures can help in the understanding of the biological and molecular mechanisms of nucleic acids and their protein complexes. X-ray crystallography can be a powerful method for elucidation of the 3-D structures of the nucleic acids and protein complexes at the atomic level, although crystallization and heavy atom derivatization are major long-standing problems.

In recent years, the chemistry of or gano selenium compounds has attracted more and more attention. Organoselenium compounds have been used in many applications, including X-ray crystal structure determination of proteins and nucleic acids, self- assembled monolayers (SAMs), development of synthetic methodologies in organic chemistry, and cancer study and treatment. Selenium derivatization of proteins via selenium (Se) replacement of sulfur (S) in methionine has aided protein X-ray crystallography, which leads to structural determination of over two thirds of novel proteins using this Se derivatization strategy and multiwavelength anomalous dispersion (MAD).

However, selenium chemistry is relatively underdeveloped. Synthesis of the selenium-derivatized nucleotides and nucleic acids and other organo selenium compounds is in need of new synthesis, protection, and deprotection methods of the selenols and conversion of the selenols to selenides. There are also needs for new synthetic strategies in selenium functionality incorporation, protection, deprotection, and conversion of selenium nucleosides, nucleotides, phosphoramidites, triphosphates, and nucleic acids. As selenols can be readily oxidized to the corresponding diselenides, selenols are usually protected as the symmetrical diselenides. The diselenides can be later reduced to selenols again by strong reducing reagents. The use of strong reducing reagents, however, may not always be compatible with organoselenium molecules, and therefore new methods are needed. In addition, selenocyanates have been developed as the protected selenols, though the deprotection reaction can cause formation of HCN. It was also reported that selenols can be protected as the selenocarbamates, selenocarbonates, and selenoacetates. These protecting groups can be removed by bases. Unfortunately, these protecting groups generally do not give satisfactory results in the desired Pd/Cu-catalyzed reactions. ¹⁴

In addition, atom-specific mutagenesis can have utility in structural and functional studies of nucleic acids and their complexes with proteins. Structure studies of nucleic acid-protein complexes have revealed that a vast majority of the nucleobases do not contact with proteins, suggesting potential replacement of nucleobase oxygen with selenium without interfering with the native structure and function of the complexes. In addition, the recent study on 4-S thymidine (a size-expanded base) has indicated its enhanced replication efficiency though it causes slight destabilization in deoxyribonucleic acid (DNA) duplex (1.7 ⁰ C difference in melting temperatures). Accordingly, in would be desirable to provide new methods and reagents for synthesizing selenium nucleosides, nucleotides, phosphoramidites, triphosphates, and nucleic acids while.

SUMMARY The present disclosure encompasses a compound having the formula (I):

Formula (1) wherein Rj can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O, HSe, ciiselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl- O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oHgonucleotide, a 5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, alkyl-O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3'linked nucleotide, a 3'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R ₄ can be H or CH ₃ , and X can be an oxygen atom or selenium atom. The present disclosure also encompasses a compound having the formula (II):

Formula (II) wherein Ri can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O,

HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl-

O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a

5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, aikyl-O) ₃ Si-O, BzH-O ₅ DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3'linked nucleotide, a 3'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R4 can be H or CH ₃ , R ₅ can be CH ₂ CH ₂ CN, alkyl, Ar, alkyl-Se, Ar-Se, alkyl-S, or Ar-S, and X can be an oxygen atom or selenium atom.

The present disclosure also encompasses a process for producing a selenium- containing nucleic acid comprising providing a reagent selected from thymidine activated at the 4-position by an activating group and uridine activated at the 4-position by an activating group, displacing the activating group with sodium 2-cyanoethylselenide, displacing the 3' group of the thymidine with phosphoramidite to form a 4-Se thymidine phosphoramidite or 4-Se uridine phosphoramidite, and contacting the 4-Se thymidine phosphoramidite or 4-Se uridine phosphoramidite with a nucleotide, a nucleoside, a nucleotide phosphoramidite, an oligonucleotide, a nucleic acid, or combinations thereof.

The present disclosure additionally encompasses a process for replacing an oxygen in a nucleic acid with selenium comprising providing a nucleic acid and 2- cyanoethylselenide salt and displacing the oxygen with the 2-cyanoethylselenide salt. The present disclosure further encompasses a method for preparing a selenium- containing nucleic acid comprising providing an enzyme capable of a adding to nucleotide or oligonucleotide into a nucleotide, an oligonucleotide, or a nucleic acid chain, providing a nucleotide or oligonucleotide substrate of the enzyme and a selenium- containing nucleotide or a selenium-containing oligonucleotide or a selenium-containing nucleic acid chain of the formula I:

Formula (I) wherein R ₁ can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O, HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl- O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, alkyl-O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3'linked nucleotide, a 31inked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R ₄ can be H or CH ₃ . and X can be an oxygen atom or selenium atom, and contacting the enzyme with substrate and the selenium-containing nucleotide or selenium-containing oligonucleotide of formula I under conditions suitable for addition of the selenium-containing nucleotide or the selenium-containing oligonucleotide to the substrate.

The present disclosure further encompasses di(2-cyanoethyl) diselenide.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings or figures, which are incorporated in and constitute a part of this specification, illustrate several embodiments of this disclosure and together with the description serve to explain principles of certain embodiments of the invention.

FIG. 1 illustrates one embodiment of a method for synthesizing a compound having the Formula (I) and a selenium nucleic acid according to embodiments of the present disclosure.

FIG. 2 is the results of HPLC analysis of 5'-DMTr~GCG( ^Se T)ATACGC-3' according to an embodiment of the present disclosure after cleavage from the solid support and the deprotection of the bases and backbone.

FIG. 3 is the results of a UV comparison of pT (a), p ^Se T (b) and 5'-T- ^Se T-TT-3' (c) according to an embodiment of the present disclosure.

FIG. 4 provides a scheme for synthesis and application of di(2-cyanoethyl) diselenide according to an embodiment of the present disclosure.

FIGS. 5A-B are the results of HPLC and MS analyses of purified 5'GCG( ^Se T)ATACGC-3' according to an embodiment of the present disclosure. FIG. 6 is the results of LC-MS analysis of 5'- ^Se TT-3' according to an embodiment of the present disclosure.

FIG 7 is the results of LC-MS analysis of 5'DMTr- ^Se T ^Se TT-3 according to an embodiment of the present disclosure.

FIG. 8 is the results of UV thermostability analysis of the SeNA 5'- ATGG ^Se TGCTC-3' according to an embodiment of the present disclosure.

Other objects, features, and advantages of this invention will be apparent from the following detailed description, drawing, and claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS As summarized above, the present disclosure includes a compound having the formula (I) below:

Formula (I) wherein R ₁ can be H ₅ HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O,

HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl- O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite,

phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5 'linked nucleotide, a 5'linked seleno-nucleotide, a 5'Hnked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl- O, alkyl-O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O ₃ HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, trϊphosphoroselenoate, a 3'linked nucleotide, a 3'Iinked oligonucleotide, a 3 'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R ₄ can be H or CH ₃ , and X can be an oxygen atom or selenium atom.

In particular embodiments, R ₂ can be phosphoramidite, In another embodiment, R ₂ can be HO or TMS-O and R ₃ can be DMTr-O, In yet another embodiment, X can be an oxygen atom. In alternate embodiments, R ₂ can be H or OH and R4 can be H or CH ₃ . ϊn still other embodiments, R ₂ can be HO, TOM-O, ACE-O, CH ₃ - Se, or TBDMS-O and R ₃ can be DMTr-O or BzH-O.

In some embodiments, R ₂ can be a 5'linked nucleotide, a 5'linked seleno- nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain or a 5'linked seleno-nucleic acid chain, where any phosphorus atom of a bridging phosphate of the 5'linked seleno-nucleotide, the 5'linked seleno- oligonucleotide, or the 5'linked seleno-nucleic acid chain is bonded to non-selenium atoms. In other embodiments, R ₃ can be a 3'linked nucleotide, a 3'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, where any phosphorus atom of a bridging phosphate of the 3'linked seleno-nucleotide, the 3 'linked seleno-oligonucleotide, or the 3'linked seleno-nucleic acid chain is bonded to non- selenium atoms.

As used herein, the term "alky F ' refers to a paraffinic hydrocarbon group which may be derived from an alkane by removing one hydrogen from the formula, and in each instance includes linear and branched moieties. Although larger structures are suitable in the present disclosure, a typical cycloalkyl group can have from one to 20 carbon atoms. For example, alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, i- propyl, n-butyl, tert-butyl, sec-butyl, isobutyl, amyl. isoamyl, n-hexyl, n-heptyl, n- octyl,n- nonyl, n-decyL n-undecyl, n-dodecyl, 2-ethylhexyl, 2-methylhexyl, and the like.

As used herein, a 5' linked nucleotide, oligonucleotide or nucleic acid chain refers to a nucleotide, oligonucleotide or nucleic acid linked through the 5' position of the ribose or deoxyribose ring. Similarly, a 3 ^! linked nucleotide, oligonucleotide or nucleic acid chain refers to a nucleotide, oligonucleotide or nucleic acid linked through the 3' position of the ribose or deoxyribose ring.

As used herein, ''thymidine derivative" refers to an analog of thymidine, examples of which are discussed in reference to FIG. 1. As used herein, "uridine derivative" refers to an analog of uridine.

As used herein, "ACE" refers to the bis(2-acetoxyethoxy)methyl group of formula III:

Formula (III).

As used herein, "TOM" refers to the triisopropylsilyloxymethyl group of formula IV:

Formula (IV). As used herein, "DOD" refers to the bis(trimethylsiloxy)cyclododecyloxysilyl group of formula V:

Formula (V).

As used herein, BzH refers to the benzhydroxy-bis(trimethylsiloxyl)silyl group of formula VI:

Formula (VI),

The present disclosure also encompasses a method for producing the compound having Formula (I). In particular embodiments, the method comprises providing a thymidine derivative activated at the 4-position by an activating group and displacing the activating group with 2-cyanoethylselenide salt. The activating group can be a reactive moiety that readily reacts with a compound or reagent. In particular embodiments, the activating group may be instance triazolide. In other embodiments, the activating group may be any suitable activating group, such as Cl, Br ₅ Ms, Ts, and sulfonyl groups. In particular embodiments, the method may further comprise providing di(2-cyanoethyl) diselenide and reducing di(2-cyanoethyl) diselenide with a reducing agent to form 2- cyanoethylselenide salt. The 2-cyanoethylselenide salt may be, but is not limited to, sodium 2-cyanoethylselenide or 2-cyanoethylselenol. In some embodiments, the step of reducing can be performed in situ. In one embodiment, the reducing agent may be, but is not limited to, NaBH ₄ . Other suitable reducing agents include, but are not limited to, DTT, LiBH ₄ , NaBH ₃ CN, LiAlH ₄ , B ₂ H ₆ , Na, LiH, CaH ₂ , or NaH.

In some embodiments, the method may further comprise protecting the hydroxyl groups of the thymidine derivative with protecting groups. Suitable protecting groups for embodiments of the method include, but are not limited to, trimethylsilyl (TMS), dimethoxytrityl (DMT), orthoester. alkyl-O (such as methoxy, ethoxy, isopropyl, isobutyl etc.), acyl (may be any acyl group with from one to about 24 carbon atoms, including especially, acetyl), Bz (benzoyl), iPr (isoproponyl), iBu (isobutyryl), Ms (methylsulfonyl), TBDMS-O (tert-butyldimethylsilyloxyl), DMTr-O (dimethyltrityloxyl), 4, 4 '-dimethoxytrityl, and many others well known in the art. In particular embodiments, the protecting groups (also interchangeably referred to as blocking groups) are placed at one or more chemically reactive positions such as the hydroxyl groups of the thymidine

derivative to block reactivity with the reactive group at this position or these positions. The protecting groups are stable under the conditions of the desired reaction of the unprotected groups and may be removed by standard procedures, such as for instance, treatment with an acid or a base. By these means reactivity of groups that are not protected are available for reaction and the protected groups do not react.

In other embodiments, the method comprises providing a uridine derivative activated at the 4-position by an activating group and displacing the activating group with 2-cyanoethylselenide salt. It should be understood that embodiments disclosed throughout the present disclosure which describe thymidine or thymidine derivatives may be modified by a person of ordinary skill in the art to encompass analogous uridine or uridine derivatives and thus, such analogous embodiments are encompassed within the scope of the present disclosure.

FIG. 1 illustrates an embodiment of a method for producing the compound having the Formula (I). In this embodiment, a partially protected thymidine derivative 1 having DMTr as a protecting group and trimethylsilyl protection at its 3' -hydroxy 1 group is provided. The thymidine derivative 1 is activated at the 4-position via formation of triazolide to produce thymidine derivative 2, with (a) comprising TMS-Im and CH ₃ CN and (b) comprising triazole-POCl ₃ -Et ₃ N. A sodium 2-cyanoethylselenide, reduced in situ from a di(2-cyanoethyl) diselenide reagent with NaBFIf ₃ was introduced to the 4-position of thymidine derivative 2 by displacing the 4-triazolyl activating group to produce an embodiment of a thymidine derivative 3 having the Formula (I). 10% triethylamine in methanol can provide a mild condition for removing the 3'-TMS group. Embodiments of the di(2-cyanoethyl) diselenide reagent and methods of producing it are described in further detail below. In alternate embodiments (not shown), fluoride treatment can be used to remove the transient 3'-TMS group, but removal of the 2-canoethyl group may occur. Thus, in some embodiments, the 3'-TMS group may be first removed with a fluoride reagent, followed by the introduction of the sodium 2-cyanoethylselenide.

The present disclosure also encompasses a compound having the formula (II):

Se

R ₃ C H ₂ N "^O

^R ? ^R 1 Formula (II) wherein Ri can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O ₁ HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl-O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroseienoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a Slinked seleno-oligonucleotide, a 5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, aikyl-O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroseienoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3 'linked nucleotide, a 3 'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R ₄ can be H or CH ₃ , R ₅ can be H, CH ₂ CH ₂ CN ₅ alkyl, Ar, alkyl-Se, Ar-Se, alkyl-S, or Ar-S, and X can be an oxygen atom or selenium atom.

In some embodiments of Formula II, R ₂ can be a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain or a 5'linked seleno-nucleic acid chain, where any phosphorus atom of a bridging phosphate of the 5'linked seleno-nucleotide, the 5'linked seleno- oligonucleotide, or the 5'linked seleno-nucleic acid chain is bonded to non-selenium atoms. In other embodiments of Formula II, R ₃ can be a 3'linked nucleotide, a 3'linked oligonucleotide, a 3'linked nucleotide, a 31inked oligonucleotide, or a nucleic acid chain, where any phosphorus atom of a bridging phosphate of the 3'linked seleno-nucleotide, the 3'linked seleno-oligonucleotide, or the 3'linked seleno-nucleic acid chain is bonded to non-selenium atoms.

The present disclosure also encompasses a method for producing the compound having Formula (II). In particular embodiments, the method comprises providing a

thymidine derivative activated at the 4-position by an activating group, displacing the activating group with 2-cyanoethylselenide salt, and removing the 2-cyanoethyl group.

The present disclosure also includes a method for producing a selenium- containing nucleic acid. The method comprises providing a thymidine derivative activated at the 4-position by an activating group, displacing the activating group with sodium 2-cyanoethylselenide, converting the 3' group of the thymidine derivative with phosphoramidite reagent to form a 4-Se thymidine phosphoramidite, and contacting the 4-Se thymidine phosphoramidite with a nucleotide, a nucleoside, a nucleoside phosphoramidite, an oligonucleotide, a nucleic acid, a selenium-containing nucleotide, a selenium -containing nucleoside, a selenium-containing nucleoside phosphoramidite, a selenium-containing oligonucleotide, a Se-containing nucleic acid or combinations thereof.

In alternate embodiments, the process of producing the selenium-containing nucleic acid may further comprise providing more than one phosphoramidite coupling cycle. In other embodiments, the process of producing the selenium-containing nucleic acid may further comprise providing deprotection conditions to remove the nucleobase, sugar, and/or backbone protections, including the selenium protecting group, using a weak base (e.g., K ₂ CO ₃ methanol solution).

In particular embodiments, the activating group may be any activating group, such as for instance triazolide.

Embodiments of the method may further comprise removing the 2-cyanoethyl group. In such embodiments, the 2-cyanoethyl group may be removed from the 4-Se thymidine phosphoramidite or the selenium-containing nucleic acid.

In particular embodiments, the step of contacting comprises a solid phase reaction step. In some embodiments, the method may further comprise providing (2~cyanoethyl) diselenide and reducing di(2-cyanoethyl) diselenide with a reducing agent to form 2- cyanoethylselenide salt. The 2-cyanoethylselenide salt may be, but is not limited to. sodium 2-cyanoethylselenide or 2-cyanoethylselenol.

In other embodiments, the method may further comprise protecting the hydroxyl groups of the thymidine with protecting groups before the step of displacing the

activating group. Suitable protecting groups for use in the present disclosure include, but are not limited to, trimethylsilyl, dimethoxythrityl, BDMS ₅ TOM, ACE, BzH, combinations thereof, or any other protecting group described herein or known in the art. In some embodiments, the method may further comprise removing the protecting group from the 3 ^! position before the step of displacing the 3 'group of the thymidine derivative with phosphoramidite reagent.

FIG. 1 illustrates a particular embodiment of the method for producing a selenium-containing nucleic acid. As described above, FIG. 1 illustrates an embodiment of a method for producing the compound having the Formula (I). Thus, it should be understood that embodiments for the method for producing the compound having the Formula (I) may be incorporated into embodiments of the method for producing a selenium -containing nucleic acid of the present disclosure.

In FIG. 1, the thymidine derivative 3 having Formula (I) may be converted into a selenium-containing nucleic acid by reaction with 2-cyanoethyl N,N- diisopropylchlorophosphor-amidite and λζjV-diisopropylethylamine in CH ₂ Cb to form phosphoramidite 4, which also has Formula (I). The phosphoramidite 4 is then used in a solid phase synthesis of a selenium-containing oligonucleotide 5. It should be understood that the solid phase synthesis can include, but is not limited to, a coupling reaction, acetylation, I ₂ oxidation, and trichloroacetic acid treatment for removal of the 5'-DMTr group without causing deselenization. In some embodiments, potassium carbonate in methanol may be used to remove the 2-cyanoethyl group from the selenium when the nucleic acid is synthesized with phosphoramidites protected with fast deprotection groups.

In alternate embodiments (not shown), concentrated ammonia treatment may be used for deprotecting the 2-cyanoethyl groups after solid phase synthesis, but deselenization may occur.

In some embodiments, the synthesized selenium-containing nucleic acids can be purified twice by HPLC (DMTr-on and DMTr-off).

In particular embodiments, the methods of producing a selenium-containing nucleic acid have a high coupling yield per cycle (i.e., higher than 99% on average). For

example, in one embodiment, less than 2% short oligonucleotides can be formed in the synthesis of the decamer.

In alternate embodiments, the present disclosure provides an enzymatic process for preparing a selenium-containing nucleic acid, wherein the process has the following characteristics: the enzyme can be an DNA polymerase, an RNA polymerase, a terminal transferase, a reverse transcriptase, a DNA ligase or an RNA ligase and the nucleic acid template and primer, or top strand, can depend on the enzyme.

In one embodiment, a process for preparing a selenium-containing nucleic acid may comprise providing an enzyme capable of adding a nucleotide, an oligonucleotide, or a nucleic acid into a nucleotide, an oligonucleotide, or a nucleic acid chain, providing a nucleotide, oligonucleotide, or nucleic acid substrate of the enzyme and a selenium- containing nucleotide, a selenium-containing oligonucleotide or a selenium-containing nucleic acid chain of the Formula I:

Formula (I) wherein Ri can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester,

TBDMS-O, HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O, ACE-O, CH ₃ -Se, alkyl-O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, alkyl~O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3 'linked nucleotide, a 3'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R ₄ can be H or CH ₃ , and X can be an oxygen atom or selenium atom, deprotecting the selenium-

containing nucleotide, a selenium-containing oligonucleotide or a selenium-containing nucleic acid chain of the Formula I, providing a nucleic acid template and primer, and contacting the enzyme with substrate and the selenium-containing nucleotide or selenium-containing oligonucleotide of formula 1 under conditions suitable for addition of the selenium-containing nucleotide or the selenium-containing oligonucleotide to the substrate.

In particular embodiments, the deprotecting step may comprise deprotecing with a base (e.g., K ₂ CO ₃ methanol solution).

In an alternate embodiment, a process for preparing a selenium-containing nucleic acid may comprise providing an enzyme capable of adding a nucleotide, an oligonucleotide, or a nucleic acid into a nucleotide, an oligonucleotide, or a nucleic acid chain, providing a nucleotide, oligonucleotide, or nucleic acid substrate of the enzyme and a selenium-containing nucleotide, a selenium-containing oligonucleotide or a selenium-containing nucleic acid chain of the Formula Il :

Formula (II) wherein Ri can be H, HO, acyl-O, TOM-O, ACE-O, alkyl-O, orthoester, TBDMS-O, HSe, diselenide, or alkyl-Se, R ₂ can be H, HO, acyl-O, TOM-O ₅ ACE-O, CH ₃ -Se, alkyl-O, TBDMS-O, TMS-O, HSe, diselenide, alkyl-Se, phosphoramidite, phosphoroselenoamidite, phosphate, phosphoroselenoate, a 5'linked nucleotide, a 5'linked seleno-nucleotide, a 5'linked oligonucleotide, a 5'linked seleno-oligonucleotide, a 5'linked nucleic acid chain, or a 5'linked seleno-nucleic acid chain, R ₃ can be H, HO, acyl-O, alkyl-O) ₃ Si-O, BzH-O, DOD-O, alkyl-O, TBDMS-O, HSe, diselenide, alkyl-Se, DMTr-O, phosphate, phosphoroselenoate, diphosphate, diphosphoroselenoate, triphosphate, triphosphoroselenoate, a 3'linked nucleotide, a 3 'linked oligonucleotide, a 3'linked nucleotide, a 3'linked oligonucleotide, or a nucleic acid chain, R4 can be H or CH ₃ , R ₅ can be H, CH ₂ CH ₂ CN, alkyl, Ar, alkyl-Se, Ar-Se, alkyl-S, or Ar-S, and X can be

an oxygen atom or selenium atom, deprotecting the selenium-containing nucleotide, a selenium-containing oligonucleotide or a selenium-containing nucleic acid chain of the Formula I, providing a nucleic acid template and primer, and contacting the enzyme with substrate and the selenium-containing nucleotide or selenium-containing oligonucleotide of formula 1 under conditions suitable for addition of the selenium-containing nucleotide or the selenium-containing oligonucleotide to the substrate.

Table 1 illustrates embodiments particular of selenium-containing nucleic acids made in accordance with the present disclosure.

Table 1: DNAs Containing 4-Se-T

entry Se-oligonucleotides measured (calc.) m/z

1. 5' - ^Se TT-3 ^? [M-H] ^" : 609.0 (609.1) C ₂₀ Hi ₇ N ₄ Oi ₂ PSe: FW 610.1

2. 5' -DMTr- ^Se T ^Se TT-3' [M-H] ^" : 1279.0 (1279.1) C _{5 I} H ₅₈ N ₆ Os ₉ P ₂ Se ₂ : FW 1280.1

3. 5' -T ^Se TTT-3' [M+Hf: 1219.0 (1219.1) C ₄₀ H ₅₃ N ₈ O ₂₅ P ₃ Se: FW 1218.1

4. 5' -TT ^Se TTT-3' [M+H] ⁺ : 1523.0 (1523.2) C ₅₀ H ₆₆ N ₁₀ O ₃₂ P ₄ Se: FW 1522.2

5 5' -G ⁵⁶ TGTACACo' [M+H] ⁺ : 2476 (2474)

C ₇₈ H ₉₉ N ₃₀ O ₄₅ P ₇ Se: FW 2473

6. 5' -ATGG ⁵⁶ TGCTC-B' [M+Hf: 2792.5 (2793.4) C ₈₈ Hi ₁₂ N ₃₃ O ₅₃ P ₈ Se: FW 2792.4

7. 5' -GCG ^Se TATACGC-3' [M+Hf: 3091.6 (3092.0) C ₉₇ H ₁₂₃ N ₃₈ O ₅₇ P ₉ Se: FW 3091.0

As used herein a nucleic acid base can be any naturally occurring or synthetic base found in nucleic acids. Nucleic acid bases include adenine (A), thymine (T), uracil

(U), guanine (G) and cytosine (C), as well as other naturally occurring nucleic acid base derivatives, such as for example, inosine (hypoxanthine, 6-hydroxypurine) and xanthine

(2,6-dioxopurine).

FlG. 2 is the results of HPLC analysis of 5'-DMTr-GCG( ^Se T)ATACGC-3' according to an embodiment of the present disclosure after cleavage from the solid support and the deprotection of the bases and backbone (retention time: 21.0 minutes). FIG. 3 is the results of a UV comparison of pT (a), p ^Se T (b) and 5'-T- ^Se T-TT-3'

(c) according to an embodiment of the present disclosure. Without being bound by theory, it is believed that in FIG. 3, a red shift (over 100 nm) can be caused by the selenium modification on the 4-position of thymidine. The maximum absorption wavelength of the 4- ^Se T is 369 nm in aqueous solution, thereby leading to yellow colored DNAs containing the selenium modification. Probably due to much easier derealization of selenium electrons, this red shift is 34 nm higher than that of the sulfur modification at the same position (λ _max ^~ 335 nm). Furthermore, because the 4- ^Se T does not have absorption at 267 nm, the molar extinction coefficient constant of 4~ ^Se T (X ₃ Or= 10.7x10 ³ M ^"1 cm ^"1 ) can be determined by comparing ^e T with native T (λ ₂₆₇ - 9.49x10 M ^"1 cm ^" ) in 5'-T- ^Se T-TT-3' molecules. Embodiments of 4- ^Se U and 4- ^Se dU can be prepared in a process analogous to the process shown in FϊG.l and 4- ^Se U and 4- ^Se dU can lead to the colored Se-RNA and Se-DNA (such as yellow color). In embodiments where the Se- nucleic acid containing selenium at 4-position is chemically synthesized, 4- ^Se T, 4- ^Se rT, 4- ^Se U or 4- ^Se dU can be introduced to either DNA or RNA, and 4- ^Se T, 4- ^Se rT, 4- ^Se U or 4- ^Se dU can lead to colored nucleic acid (such as yellow color).

In particular embodiments, there can be non-significant perturbation from the selenium modification. In some embodiments, the interruption of the T-A base pair from the selenium modification and/or the duplex destabilization caused by the hydrogen bond disruption may be compensated by the better base stacking due to the selenium

derivatization, which is consistent with the stability of the duplexes containing the size- expanded bases.

The present disclosure additionally provides a method for replacing an oxygen in a nucleic acid with selenium comprising providing 2-cyanoethylselenide salt and displacing oxygen with 2-cyanoethylselenide salt. The 2-cyanoethylselenide salt may be, but is not limited to, sodium 2-cyanoethylselenide or 2-cyanoethylselenol,

In particular embodiments of the method, the step of displacing the oxygen comprises displacing the oxygen with an activating group and displacing the activating group with sodium 2-cyanoethylselenide. Suitable activating groups comprise any activating group described herein.

In some embodiments, the method for replacing an oxygen in a nucleic acid with selenium can comprise the methods for producing a selenium-containing nucleic acid discussed above.

Embodiments of the selenium incorporation methods of the present disclosure allow for achieving both incorporation and protection of the selenium functionality, simultaneously. The methods of the present disclosure provide a strategy for selenium incorporation and protection for solid-phase synthesis, synthesized the 4-Se thymidine phosphoramidite, and incorporated it into oligonucleotides with quantitative coupling yield. To further advance the field of nucleic acid X-ray crystallography, introduction of selenium to 4 position of thymine can reveal more insights on the base pairing, DNA duplex stability, and DNA polymerase interaction. By the UV-meiting study of embodiments of the present disclosure, it can be shown that the modification of 4-Se-T on DNA duplex does not cause significant structure change and destabilization disrupt the T-A base pair and the duplex stability, which promises the potential application in directly studying X-ray crystal structures of nucleic acids and their protein complexes via selenium MAD phasing.

In addition, embodiments of colored DNAs and RNAs of the present disclosure having an oxygen replaced with selenium allow direct visualization of DNA and RNA. Due to the derealization of the selenium electrons in one embodiment, the UV spectra of the 4-Se thymidine nucleotides (λ _max = 369 nm) reveal a red shift over 100 nm as

compared to non-modified thymidine nucleotide (λ _max = 267 nm). Such nucleic acid visualization may be useful in nucleic acid function study, DNA and RNA chip (e.g., microchip) and nanotechnology applications, and DNA-protein interaction study. Furthermore, embodiments of the selenium derivatization of the present disclosure will largely facilitate X-ray crystal structure studies of nucleic acids and nucleic acid-protein complexes.

These embodiments of colored DNA and RNAs may thus be used to visualize target (e.g., diseased, non-disease, and pathogen) DNA and RNA. Therefore, embodiments of the present disclosure may include methods for visualizing a target DNA, RNA, or other nucleic acid by probing the targeted DNA, RNA, or other nucleic acid with any colored nucleic acid disclosed in the present disclosure. The visualization may be achieved via immobilization of the targeted DNA, RNA, or other nucleic acid or immobilization of the Se-derivatived colored oligonucleotide or nucleic acids, and via hybridization between the target nucleic acid and any colored nucleic acid disclosed in the present disclosure.

The present disclosure further provides a di(2-cyanoethyl) diselenide reagent for use in methods of the present disclosure. Di(2-cyanoethyl) diselenide can be a stable liquid with a light orange color and which can be purified by flash chromatography or distillation. In particular embodiments, selenium introduction described in the methods above at the 4 position of thymidine can be achieved by di(2-cyanoethyl) diselenide, which allows simultaneous incorporation and protection of the selenium functionality. In some embodiments, di(2-cyanoethyl) diselenide can be reduced with a reducing agent to 2- cyanoethylselenide salt. The 2-cyanoethylselenide salt may be, but is not limited to, sodium 2-cyanoethylselenide or 2-cyanoethylselenol. 2-cyanoethylselenide salt can react with an alkylating reagent to generate the corresponding cyanoethyl-protected selenol via a S _N 2 substitution. Though the cyanoethyl-protected selenol can be stable under acidic conditions in some embodiments, this protecting group can be removed by weak base treatment. Thus, in particular embodiments, in situ generated selenols can be

simultaneously converted to desired selenides in the presence of an alkylating reagent in the deprotection reaction using weak bases.

In addition, the present disclosure provides a method for making the di(2- cyanoethyl) diselenide reagent. In one embodiment, the method for producing the di(2-cyanoethyl) diselenide comprises providing diselenide salt or diselenol and alkylating the diselenide salt or diselenol with 3-bromopropionitrile. The 2-cyanoethylselenide salt may be, but is not limited to, sodium 2-cyanoethyiseIenide or 2-cyanoethyIselenol.

In some embodiments, the step of providing the diselenide salt comprises partially reducing selenium metal with sodium borohydride. Other suitable reducing agents include, but are not limited to, DTT, LiBH ₄ , NaBH ₃ CN, LiAlH ₄ , B ₂ H ₆ , Na, LiH, CaH ₂ , or

NaH.]

FIG. 4 illustrates an embodiment of the method for producing the di(2- cyanoethyl) diselenide of the present disclosure. Selenium metal 1 is reduced with NaB H ₄ to disodium diselenide 2, using ethanol as solvent. A minimum amount of

NaBH ₄ can be used to avoid formation of sodium selenide, which later leads to the formation of undesired di-2-cyanoethyl selenide. This reduction reaction can be performed under argon to prevent rapid oxidation of the diselenide back to selenium metal. The generated brown color disodium diselenide 2 is then immediately alkylated, without work-up or purification, with excess 3-bromoproponitrile to create di(2- cyanoethyl) diselenide 3.

In particular embodiments, a satisfied yield of di(2-cyanoethyl) diselenide (72%) can be obtained after two reactions: the NaBH ₄ reduction and the alkylation reactions.

Embodiments of the di(2-cyanoethyl) diselenide of the present disclosure may be used to produce selenides and other diselenides. FIG. 4 further illustrates an embodiment of the synthesis of selenides and diselenides using di(2-cyanoethyl) diseienide. In this embodiment, di(2-cyanoethyl) diselenide can be rapidly reduced with NaBH ₄ in ethanol solution, indicated by the color change from light orange to colorless within five minutes.

In particular embodiments, the cyano group may not be reduced under this reductive condition when an appropriate amount Of NaBH ₄ is used. In embodiments where a large

excess of NaBH ₄ may be used, the cyano group can be partially reduced to the imine group. This over-reduction problem can be easily avoided by dropwise adding a solution of NaBH ₄ dissolved in ethanol into di(2-cyanoethyl) diselenide 3 until the diselenide solution just turns colorless. The formed sodium 2-cyanoethyl selenide or 2-cyanoethyl selenol 4, can be converted quantitatively to a 2-cyanoethyl selenide 5 when an alkylating compound (R ^! -X) is added after the reduction.

For instance, the 2-cyanoethyl selenol 4 can be a strong nucleophile which reacts with halogenated alkyl compounds by displacing the halogens via a S _N 2 reaction. Though the generated 2-cyanoethyl selenide 5 can be stable under acidic conditions, the 2-cyanoethyl selenide 5 (NCCH ₂ CH ₂ -Se-R ¹ , equivalent to a protected selenol) can be easily deprotected to give a corresponding selenol 6 (H-Se-R ¹ ) under a mild condition, such as a weak base (K ₂ CO ₃ methanol solution), which removes the 2-cyanoethyl group in two hours. The deprotection reaction, conducted under argon to prevent the formation of the diselenide, is quantitative. Stronger bases, such as ammonia and NaOH, can also be used for the deprotection with high yields. In the presence of another alkylating reagent (R ² -X) during this mild deprotection using K ₂ COa, the generated selenol 6 (R'-Se-H) can be quantitatively converted to a selenide 7 (R^Se-R ² ). The alkylation reaction can be performed under argon to prevent rapid oxidation of the selenol to the corresponding diselenide 8 (R ¹ -Se-Se-R ¹ ). The formed diselenide 8 (R ¹ -Se-Se-R ¹ ) ^19"20 can also be converted to the selenide 7 (R ¹ -Se-R ² ) after the NaBH ₄ reduction and alkylation.

Embodiments of the di(2-cyanoethyl) diselenide provide a reagent for site- specific incorporation of selenium into nucleotide and nucleic acid molecules for X-ray crystal structure determination. Specifically, embodiments of the di(2-cyanoethyl) diselenide reagent may be capable of use for selenium functionality incorporation, protection and deprotection of selenol, and conversion to selenides. In particular, embodiments of the di(2-cyanoethyl) diselenide reagent can be useful for incorporating 2-cyanoethyl-protected selenol functionality into molecules containing halogens or other leaving groups. The cyanoethyl protection can be removed with a weak base to generate the corresponding selenol in situ, which can then be conveniently converted to a selenide in the presence of an alkylating reagent during the deprotection. Using this strategy, the

selenium functionality incorporation and protection are achieved simultaneously, and the deprotection and conversion of the protected selenols are also accomplished in one step, which is convenient.

Other embodiments are further illustrated below in the examples which are not to be construed in any way as imposing limitations upon the scope of this disclosure. On the contrary, it is to be clearly understood that resort may be had to various other embodiments, modifications, and equivalents thereof which, after reading the description therein, may suggest themselves to those skilled in the art without departing from the scope of this disclosure and the appended claims. EXAMPLES

In Examples 1-16 below, the solvents and reagents were used without purification unless mentioned otherwise, Triethylamine (TEA) was dried over KOH (s) and distilled under argon. When necessary, solid reagents were dried under high vacuum. Reactions with compounds sensitive to air or moisture were performed under argon. Solvent mixtures are indicated as volume/volume ratios. Thin layer chromatography (TLC) was run on Merck 60 F ₂₅₄ plates (0.25 mm thick; the R _f values are for the compounds being synthesized), and visualized under UV-light or by a Ce-Mo staining solution (polyphosmolybdate), 25 g; CE(SO ₄ ) ₂ -4H ₂ O, 10 g; H ₂ SO ₄ , 60 mL. cone; H ₂ O, 940 mL) with heating. Preparative TLC was performed using Merck 60 F2 ₅ 4 pre-coated plates (2 mm thick). Flash chromatography was performed using Fluka silica gel 60 (mesh size 0.040-0.063 mm) using a silica gelxrude compound weight ratio of ca. 30:1 (or using Al ₂ O ₃ ). ¹ H, ¹³ C and ³¹ P-NMR spectra were recorded using Bruker-300 or 400 (300 or 400 MHz). All chemical shifts (δ) are in ppm relative to tretramethylsilane and all coupling constants (J) are in Hz. High resolution mass spectrum (HRMS) analysis was performed at Scripps Center for Mass Spectrometry, California.

Example 1: Synthesis of 4-(l,2,4-triazoI-l-yI)-5'-0-(4,4'-dimethoxytriphenyJmethyl)- 3'O-trimethylsiIyl thymidine

Phosphorus oxychloride (0.92 mL, 10 mmol) was added to a solution of 1,2,4- triazole (2.77 g, 40 mmol) in dry acetonitrile (32 mL) under argon, and the reaction was

stirred for 45 minutes at room temperature. Dry triethylamine (11.2 mL, 79 mmol) was then injected into the reaction and the reaction was stirred for another 45 minutes. Without exposing to air, the suspension was filtered directly into a round bottle flask (100 mL) containing a thymidine derivative 1 as in FIG. 1 (1.82 g, 3.35 mmol) reacted with 1- (trimethylsilyl)imidazole (0.98 ml, 6.67 mmol) in dry acetonitrile (15 mL) under argon. The reaction mixture was allowed to run for 1 hr at room temperature. The formation of iV ⁴ - triazolide of having the formula of thymidine derivative 2 in FlG. 1 was revealed by a fluorescent spot on TLC (5% MeOH in CH ₂ Cl ₂ , product R/=0.25). After the reaction was complete, the reaction mixture was poured into water (150 mL), and extracted with ethylacetate (3x 80 mL). The combined organic layer was washed with saturated NaCl (100 mL) and dried over MgSO ₄ before evaporation. After loading the crude product on a silica gel column equilibrated with hexanes/CFϊ2Cl ₂ (1:1), the column was eluted with hexanes/CH ₂ Cl ₂ (1 : 1), pure CH ₂ Cl ₂ , and then with a methanol/CH ₂ Cl ₂ gradient (0, 0.5% and 1% methanol in CH ₂ Cl ₂ ). After solvent evaporation and dry on high vacuum, the pure compound having the formula of thymidine derivative 2 in FIG. 1 was obtained as a white foam product (1.94 g, 87% yield).

¹ H-NMR (300 MHZ, CDCl ₃ ) δ: 0.61 [s, 9H, (CH ₃ ) ₃ Si], 1.98 (s, 3 H, CH ₃ ), 2.26- 2.35 and 2.63-2.71 (2x m, 2H, H-2'), 3.30-3.60 (2x dd, J =2.7. 1 1.1 Hz, 2H. H-5'), 3.79 (s, 6H, 2xCH ₃ O), 4.07-4.10 (m, IH, H-3'), 4.46-4.52 (q, J =4.8 Hz, IH, H-4'), 6.30 (t, J = 6.3 Hz, IH, H-I'), 6.84 (dd, J -1.5, 9.0 Hz, 4H aromatic), 7.22-7.42 (m, 9H, aromatic), 8.09 (s, IH, H-6), 8.42 (s, IH, H-Im), 9.29 (s, IH, H-Im).

¹³ C-NMR (100 MHz, CDCl ₃ ) δ: -0.06 (CH ₃ -Si), 16.54 (CH ₃ ), 42.20 (C-2'), 55.26 (OCH ₃ ), 62.12 (C-5'), 70.58 (C-3'), 86.88, 135.31, 135.33, 144.23, 158.16 (Ar-C), 87.16 (C-4'), 87.65 (C-I '), 105.71 (C-5), 1 13.29, 1 13.31 , 127.14, 128.02, 128.13, 130.06, 130.07 (Ar-CH), 146.70 (C-6), 153.36 (CH-Im), 153.95 (C-2), 158.79 (C-4).

HRMS (ESI-TOF): molecular formula C ₃₆ H ₄₁ N ₅ O ₆ Si; [M+Na] ⁺ : 690.2724 (calc. 690.2718).

Example 2: Synthesis of 4-(2-cyanoethyl)seleno-5'-0-(4,4'- dimethoxytriphenylmethyl) thymidine

The NaBH ₄ suspension (250 mg NaBH ₄ in 5 mL of EtOH) was injected into a flask containing di(2-cyanoethyl) diselenide [(NCCH ₂ CH ₂ Se) ₂ , 0.45 mL, d= 1.8 g/mL, 3.0 mmol] and ethanol (8 mL) on an ice bath and under argon. After injection of the NaB H ₄ suspension for 15 minutes, the ice bath was removed. The yellow color of the diseienide disappeared in approximately 15 minutes, giving an almost colorless solution of sodium selenide (NCCH ₂ CH ₂ SeNa). A solution of the compound having the formula of thymidine derivative 2 in FIG. 1 from Example 1 (1.34 g. 2 mmol) in the THF (4 mL) was injected to the solution of sodium selenide. After the selenium incorporation was completed in 15 minutes (monitored on TLC, 5% MeOH in CH ₂ Cl ₂ , product Ry= 0.58), water (100 mL) was added to the reaction flask. The solution was adjusted to pH 7-8 using CH ₃ COOH (10%), and was then extracted with ethyl acetate (3 x 100 mL). The organic phases were combined, washed with NaCl (sat., 100 mL), drived over MgSO ₄ (s) for 30 minutes, and evaporated to minimum volume under reduced pressure. Deprotecting solution (5 mL, 10% triethylamine in methanol) was added to the crude product, and the reaction was stirred for 4 hours to completely remove the 3'-TMS group (monitored on TLC, 5% MeOH in CH ₂ Cl ₂ , product Ry= 0.46). The deprotecting solution was evaporated under reduced pressure. The crude product was then dissolved in methylene chloride (5 mL) and purified on a silica gel column equilibrated with hexanes/methylene chloride (1 :1). The column was eluded with a gradient of methylene chloride (CH ₂ Cl ₂ , 0.5%, 1%, and 2% MeOH in CH ₂ Cl ₂ , 500 mL each). After solvent evaporation and dry on high vacuum, the pure compound having the formula of thymidine derivative 3 in FIG. 1 was obtained as a white foamy product (1.07 g, 81% yield).

¹ H-NMR (400 MHz, CDCl ₃ ) δ: 1.62 (s, 3H, CH ₃ ), 2.27-2.35 and 2.71-2.77 (2x m, 2H, H-2'), 3.00 (dd, J = 6.5 and 6.7 Hz, 2H, CH2-CN), 3.32 (br, IH, OH), 3.37-3.41 (m+t, J= 6.7 Hz, 3H, lH-5\ CH ₂ -Se), 3.52 (dd, Jy. _r = 10.6 Hz, J ₅ - _-4 > = 3.0 HZ, IH, IKS'), 3.81 (s, 6H, 2x OCH ₃ ), 4.17-4.22 (m, IH, H-3'), 4.57-4.62 (m, IH, H-4'), 6.34 (t, J _v .

_T = 6.3 Hz, IH, H-I'), 6.85 (dd, J = 8.8 Hz, 4H, aromatic), 7.23-7.42 (m, 9H, aromatic), 7.91 (s, IH, H-6).

¹³ C-NMR (100 MHz, CDCl ₃ ) δ: 14.30 (CH ₃ ), 18.73 (CH ₂ -CN), 20.53 (CH ₂ -CH ₂ - CN), 42.17 (C-2'X 55.29 (OCH ₃ ), 63.18 (c-5'), 71.71 (C-3'), 86.90, 135.37, 135.41, 144.36, 158.73 (Ar-C), 86.73 (C-4'), 87.01 (C-I '), 113.31, 127.17, 128.03, 128.10, 130.07 (Ar-CH), 1 14.38 (C-5), 1 19.00 (CN), 137.56 (C-6), 153.35 (C-2), 177.20 (C-4).

HRMS (MALDl-TOF): molecular formula, C ₃₄ H ₃₅ N ₃ O ₆ Se; [M+Na] ⁺ : 684.1583 (calc. 684.1583).

Example 3: Another Synthesis of 4-(2-cyanoethyl)seleno-5'-0-(4,4'- dimethoxytriphenylmethy!) thymidine

Et ₃ N-3HF (7.5 mL, 1 M in THF, 7.5 mmol) was added to a solution of the compound having the formula of thymidine derivative 2 in FIG. 1 from Example 1 (4.98 g, 7.47 mmol) in the THF (10 mL). The reaction was completed after stirring under argon for 15 minutes (monitored by TLC). The solvent of the reaction was removed by rotary evaporation and the crude intermediate was then dried on high vacuum. The NaBH ₄ suspension (933 mg NaBH ₄ in 10 mL of EtOH) was injected into a flask containing di(2-cyanoethyl) diselenide [(NCCH ₂ CH ₂ Se) ₂ , 1.66 mL, d= 1.8 g/mL, 1 1.2 mmol, 3 eq.] and ethanol (10 mL) on an ice bath and under argon. After adding the NaBH ₄ suspension and stirring it for 15 minutes, the ice bath was removed. The brown- yellow color of the diselenide disappeared in approximately 15 minutes, giving an almost colorless solution of sodium selenide (NCCH ₂ CH ₂ SeNa). The sodium selenide solution was then injected into the flask containing the crude intermediate under argon. After the selenium incorporation reaction was completed in about 45 to 60 minutes (monitored on TLC, 5% MeOH in CH ₂ Cl ₂ , product R/= 0.58), CH ₂ Cl ₂ (100 mL) was added to the reaction flask, followed by addition of NaCl solution (100 mL, saturated) and addition of CH ₃ COOH (5%) to adjust the pH to 7-8. After removing the organic phase, the aqueous phase was extracted again with CH ₂ Cl ₂ (2 x 100 mL). The organic phases were combined, washed with NaCl (sat., 50 mL), dried over MgSO ₄ (s) for 25 minutes, and evaporated to minimum volume under reduced pressure. The crude product was

dissolved in CH ₂ Ch (5 mL) and purified on a silica gel column equilibrated with CH ₂ Cl ₂ and eluded with a gradient (CH ₂ Cl ₂ , 0.5%, 1.0%, 2.0% MeOH in CH ₂ Cl ₂ , 400 mL each). After solvent evaporation and dry on high vacuum, the pure compound having the formula of thymidine derivative 3 in FIG. 1 was obtained as a white foamy product (4.20 g, 85% yield).

Example 4: Synthesis of 4-(2~cyanoethyl)seleno-5'-0-(4,4'- dimethoxytriphenylmethyl)-thymidine 3'-O-(2-cyanoethyI)</πsopropylamino phosphoramidite To the flask (25 mL) was added a compound containing the compound having the formula of thymidine derivative 2 in FIG. 1 from Example 3 (453 mg., 0.68 mmol) under argon, dry methylene chloride (2.5 mL), N,N-diisopropylethylamine (0.17 mL, 1.03 mmol, 1.5 eq.), and 2-cyanoethyl N,N-diisopropyl-chlorosphosphoramidite (195 mg, 0.83 mmol, 1.2 eq.) were added sequentially. The reaction mixture was stirred at -10 ⁰ C in an ice-salt bath under argon for 10 minutes, followed by removal of the bath. The reaction was completed in 45 minutes at room temperature (indicated by TLC, 5% MeOH in CH ₂ Cl ₂ , product R/ = 0.63 and 0.68), generating a mixture of two diastereisomers. The reaction was then quenched with NaHCO ₃ (2 mL, sat.), stirred for 5 minutes, and the product was then extracted with CH ₂ Cl ₂ (3 x 5 mL). The combined organic layer was washed with NaCl (10 ml, sat.) and dried over MgSO ₄ (s) for 20 minutes, followed by filtration. The solvent was then evaporated under reduced pressure and the crude product was re-dissolved in CH ₂ Cl ₂ (2 mL). This solution was added drop-wise to petroleum ether (100 mL) under vigorous stirring, generating a white precipitate. The petroleum ether solution was decanted. The crude product was re-dissolved again in CH ₂ C1?/Hexanes (1 :1). The column was eluded with a gradient of methylene chloride and ethyl acetate [CH ₂ Cl ₂ to CH ₂ Cl ₂ /Et0Ac (7:3)]. After solvent evaporation and dry over high vacuum, the pure compound having the formula of thymidine derivative 4 in FIG. 1 (538 mg) was obtained as a white foamy product (92% yield).

¹ H-NMR (400 MHz, CDCl ₃ , two sets of signals from a mixture of two diastereomers) δ: 0.85-1.20 (m, 24H, 8x CH ₃ -ipr), 1.55 and 1.57 (2x s, 6H ₅ 2x CH ₃ ),

2.30-2.38 and 2.70-2.82 (2x m, 4H. 2x H-2'), 2.34 and 2.64 (2x t, J - 6.4 Hz ₅ 4H, 2x O- CH ₂ -CH ₂ -CN), 3.00-3.04 (m, 4H, 2x Se-CH ₂ -CH ₂ -CN) ₃ 3.32-3.44 (m, 6H ₅ 2x H-5\ 2x Se-CH ₂ ), 3.52-3.64 (m, 8H. 4x CH-ipr, 2x 0-CH ₂ -CH ₂ -CN), 3.73-3.84 (m, 2H, 2x H-5'), 3.82 and 3.83 (2x s, 12H, 4x OCH ₃ ), 4.18-4.24 (m. 2H, 2x H-3'), 4.63-4.72 (m, 2H ₃ 2x H-4'), 6.28 and 6.32 (2x t, J _v . _τ = 6.3 Hz, 2H, 2x H-I'), 6.83-3.88 (m, 8H, aromatic), 7.27-7.43 (m, 18H, aromatic), 7.82 and 7.94 (2x s, 2H, 2x H-6).

¹³ C-NMR (100 MHz, CDCl ₃ , two sets of signals from a mixture of two diastereomers) δ: 14.18 (CH ₃ ), 18.78 (Se-CH ₂ -CH ₂ -CN), 20.15 and 20.23 (0-CH ₂ -CH ₂ - CN), 20.43 (Se-CH ₂ -CH ₂ -CN), 24.48, 24.52, 24.55, 24.59, 24.67 (CH ₃ -iρr), 40.85, 40.89 and 41.17, 41.19 (C-T), 43.17, 43.25, 43.29, 43.38 (CH-ipr), 55.27 and 55.31 (OCH ₃ ), 58.15 and 58.34 (0-CH ₂ -CH ₂ -CN), 62.30 and 62.65 (C-5') ₃ 72.31 and 72.87 (C-3'), 85.68 and 85.86 (C-4') _s 86.74 and 86.76 (C-I '), 86.87, 135.28, 135.31, 144.25, 158.77 (Ar-C), 113.27, 127.18, 127.23, 128.00, 128.18, 128.26, 130.13, 130.19, 130.21 (Ar-CH), 114.04 and 1 14.08 (C-5), 117.38 and 117.53 (0-CH ₂ -CH ₂ -CN), 119.03 (Se-CH ₂ -CH ₂ - CN), 137.41 and 137.44 (C-6), 153.10 (C-2), 176.98 (C-4).

HRMS (MALDI-TOF): molecular formula, C ₄₃ H ₅₂ N ₅ O ₇ PSe; [M+H] ⁺ : 862.2836 (calc. 862.2842).

Example 5: Synthesis of the Se-derivattøed nucleic acids (SeNA) containing 4-Se-T DNA oligonucleotides were synthesized chemically on a 1.0 μmol scale using an

ABI392 DNA/RNA Synthesizer. The concentration of the Se-nucleoside phosphoramidites was identical to that of the conventional phosphoramidites (0.1 M in acetonitrile). Coupling was carried out using a 5-(benzylmercapto)-lH-tetrazole (5- BMT) solution (0.3 M) in acetronitrile. The coupling time for the Se-nucleoside phosphoramidites was 25 seconds. The 5'-detritylation was done using 3% trichloroacetic acid in methylene chloride. Syntheses were performed on control pore glass (CPG-500) immobilized with the appropriate nucleoside through a succinate linker (Glen Research). All the oligonucleotides were prepared with DMTr-on and the phosphoramidites protected with fast deprotection groups on the nucleobases. After synthesis, the Se-DNA oligonucleotides were cleaved from the solid support and fully

deprotected by K ₂ CO ₃ in methanol (50 raM) overnight at room temperature. The SeNAs were then purified twice by HPLC with DMTr-on and DMTr-off. The DMTr group was removed by 3% TCA in 3 minutes without causing deselenization,

FIGS. 5A-B are the results of HPLC and MS analyses of purified 5'GCG( ^Se T)ATACGC-3' according to an embodiment of the present disclosure, FIG. 6A is the results of the HPLC analysis of the sample analyzed on a Zorbax SB-C 18 column (4.6 x 250), eluted (1 ml/min) with a linear gradient from buffer A (2OmM triethylammonium acetate, pH 7.1) in 30 minutes (retention time is 11.52 minutes). FIG. 6B is the results of the MALDI-MS analysis of the molecular formula: C ₉₇ Hi ₂₃ N ₃₈ O ₅₇ P ₉ Se: [M+H] ⁺ : 3091.6 (calculated: 3092.0). HPLC analysis and purification

The DNA oligonucleotides were analyzed and purified by reverse phase high performance liquid chromatography (RP-HPLC) with both DMTr-on and DMTr-off (FIG. 5A). Purification was carried out using a 21.2x250 mm Zorbax, RX-C8 column at a flow rate of 10 mL/min, Buffer A consisted of 2OmM triethylammonium acetate (TEAAc, pH 7.1 , RNase-free water), while buffer B contained 50% aqueous acetronitrile and 20 mM TEAAc, pH 7.1. Similarly, analysis was performed on a Zorbax SB-C 18 column (4.6x250 mm) at a flow of 1.0 mL/min using the same buffer system. The DMTr-on oligonucleotides were eluded with up to 90% buffer B in 25 minutes in a linear gradient, while the DMTr-off oligonucleotides were eluted with up to 40% of buffer B in a linear gradient in the same period of time. The collected fractions were lyophilized; the purified compounds were re-dissolved in RNase-free water. The pH was adjusted to 7.0 after the final purification of the Se-oligonucleotides without the DMTr group. The Se- oligonucleotides were analyzed by MS (Figure 5B, 6, 7). FIGS. 6 and 7 show LC-MS analysises of SeNAs. FIG. 6 is the results of LC-MS analysis of 5'- ^Sc TT-3' with the molecular formula: C ₂₀ Hi ₇ N ₄ Oi ₂ PSe: [M-H] ^' (609.0, calculated 609.1) according to an embodiment of the present disclosure. FIG 7 is the results of LC-MS analysis of 5'DMTr- ^Se T ^Se TT-3' with the molecular formula: C _{5 I} H ₅₈ N ₆ O] ₉ P ₂ Se ₂ : [M-H] ^" (1279.0, calculated 1379.1) according to an embodiment of the present disclosure.

FIG. 8 is the results of UV thermostability analysis of the SeNA 5'- ATGG ^Se TGCTC-3' according to an embodiment of the present disclosure before heating (a) and after heating at 65°C for 2 hours (b) in buffer [5OmM NaCl, 10 mM NaH ₃ PO ₄ - Na ₂ HPO ₄ (pH 6.5), 0.1 mM EDTA, 1OmM MgCl ₂ ]. This thermostability study by UV indicated that the 4-Se functionality is stable at 65 ⁰ C over an hour without noticeable decomposition. In addition, in the UV-melting study, the melting temperature of 5'- ATGG ^Se TGCTC-3' and 5'-GAGCACCAT-3' (42.7 ⁰ C) is almost the same as that of the native duplex (42.5 ⁰ C), suggesting non-structural perturbation of the selenium modification. Thermostability Study of the SeNA containing 4-Se-T

The SeNA containing 4-Se-T (5 '-ATGG ³⁰ TGCTC-S') was heated at 65 ⁰ C for two hour (FIG. 8). Only less than 10% was decomposed, which shows that the 4-Sε-T is relatively stable in aqueous solution. Crystal Structure Determination of the Se-DNA Containing 4-Se-T The crystallization conditions for the Se-DNA (5'-G-dU _Se -G- ^Se T-A-C-A-C-3\ self-complementary), where the dUs _e (2'-Se-dU) was used to facilitate the crystal growth, were screened using the Nucleic Acid Mini Screen kit (Hampton Research, with 24 diversified crystallization buffers). DTT was used to prevent the oxidative deselenization of the 4-Se thymidine. The Se-DNA crystallized in three days in buffer #8, #17, and #21 of the Hampton kit. All crystals gave quality diffraction. The structure of the Se-DNA crystal, grown under buffer #17, was determined at 1.50 A resolution via Se MAD phasing. The determined Se-DNA structure (1.50 A resolution, FIG. 2) is superimpo sable over the native structure (2.0 A resolution) with the same tetragonal space group, indicating that this Se derivatization did not alter the native structure significantly. The data collection, phasing and refinement statistics of the determined Se- DNA structure (2NSK) are listed here in Table 2 and Table 3.

Table 2: X-ray data and phasing statistics for the Se-DNA (5'-G-dU _Se -G- ^Se T-A-C-A-C-

3', 2NSK)

Data collection and phasing peak inflection remote reference

Wavelength, A 0.9795 0.9798 0.9400 1.100

Resolution range, A 40.0-1.5 40.0-1.5 40.0-1.5 40.0-1.5

(last shell) (1.55-1.5) (1.55-1.5) (1.55-1.5) (1.55-1.5)

Unique reflections 3736 (357) 3741 (352) 3743 (354) 3718 (352)

Completeness, % 99.1 (100.0) 99.2 (100.0) 99.2 (100.0) 98.3 (100.0) '" 8.8 (27.6) 6.3 (26.8) 6.3 (27.9) 4.8 (27.2)

<I/σ(ϊ)> 15.3 (6.8) 14.9 (6.3) 14.6 (5.9) 14.7 (5.6)

Redundancy 13.0 (13.6) 13.1 (13.6) 13.2 (13.7) 12.7 (12.2)

R-CuIHs (Friedel) 0.485 0.371 (0.428)

(0.306) (0.279)

Phasing power 2.31 (4.98) 4.37 (6.24) (4.79) λs/λ ₄ (Friedel)

Figure of merit (Friedel) 0.473 0.578 (0.438)

(0.548) (0.584)

Overall flcπtre nf meril

R _merge =σ|I-<I>|/σI

Table 3: X-ray refinement and model statistics for the Se-DNA (5'G-dU _Se G- ^Se T-A-C-A- C-3\ 2NSK)

Refinement and model

Resolution range, A (last shell) 15.82 - 1.50 (1.59 - 1.50) Number of reflections 3531 (492)

Rwork, % 19.5 (19.5) Rfree, % 21.2 (19.9)

Number of atoms

Nucleic Acid (single) 160

Heavy atom 2 (Se)

Water 36

R.m.s. deviations

Bond length, A 0.01 1

Bond angle, 1.8

Average B -factors A ²

All atoms 24.0

Wilson plot 17.4

Overall anistoropic B-values

B-11/B22/B33 -1.5 I/-1.51/3.02

Bulk solvent correction

Solvent density, e/ A ³ 0.34

B-factors, A ² 47.6

Coordinates error (c.-v.), 5A

Esd. From Luzzatt plot. A 0.16

Esd. From SIGMAA, A 0.08

Example 6: Synthesis of Di(2-cyanoethyl) diselenide

A solution of dioxane:ethanol (4:1, 100 mL) was injected into a flask containing selenium metal (7.9 g, 100 mmol; FW-79) and NaBH ₄ (2.7 g) under argon. After stirring the dark suspension for 1 hr, 3-bromopropionitrile (12.4 mL, 150 mmol, 1.5 equiv.; MW=I 34, d=1.62 g/mL) was added dropwise in an ice bath. The reaction was stirred for 1 hr before it was poured into a beaker containing water (400 mL). The suspension (yellow-orange) in the beaker was adjusted to pH 7 and then extracted with ethyl acetate (3x200 mL). The combined organic phases were washed with NaCl (sat., 100 mL) and then dried over MgSO ₄ (s). After evaporation of the solvent under reduced pressure, the crude product was purified on silica gel column equilibrated with CHiC^/hexane (30:70). The gradient was run using CH ₂ Cl ₂ in hexane (150 mL each, 30%, 40%, 50%, 60%, 70%,

and 80%). The eluted product was light orange. The solvents were evaporated under reduced pressure (using rotary evaporator, don't use high vacuum) and co-evaporated with methanol twice (2x 30 niL). The light orange product was obtained (9.65 g, 72% yield). ¹ H NMR (300 MHz, CDCl ₃ ) δ: 2.84 (t, J = 7.2 Hz, 2H, CH ₂ Se), 3.04 (t, J = 7.2

Hz ₅ 2H, CH ₂ CN); ¹³ C NMR (75 MHz, CDCl ₃ ) δ: 18.65 (CH ₂ Se), 21.53 (CH ₂ CN), 118.27 (CN); HRMS (MALDI-FTMS): molecular formula, C ₆ HgN ₂ Se ₂ ; [M (with ⁸⁰ Se)+Na] ⁺ : 290.8912 (calc. 290.8916). When a large excess Of NaBH ₄ was used in the reaction, colorless di-2-cyanoethyl selenide was also isolated. ¹ H NMR (300 MHz, CDCl ₃ ) δ: 2.91 (t, J = 7.1 Hz, 2H, CH ₂ Se), 3.53 (t, J = 7.1

Hz, 2H, CH ₂ CN);

¹³ C NMR (75 MHz, CDCl ₃ ) δ: 21.03 (CH ₂ Se), 23.27 (CH ₂ CN), 117.42 (CN); HRMS (MALDI-FTMS): molecular formula, C ₆ H ₈ N ₂ Se: [M (with ⁸⁰ Se) +Na] ⁺ : 210.9746 (calc. 210.9750).

Example 7: Synthesis of Benzyl 2-cyanoethyl selenide

Ethanol (5 ml) was added to NaBH ₄ (317 mg) placed in an airtight flask under an argon balloon. The supernate of the mixture was added into a round bottom flask (100 ml, on an ice bath) containing di (2-cyanoethyl) diselenide (1.48 mL, 2.67 g, 10 mmol, d=1.8 g/mL) under an argon balloon. After stirring for 5 minutes, the reaction turned colorless from light orange color. Benzyl chloride (0.75 mL, 821 mg, 6.5 mmol, d-1.1 g/mL) was then injected into the reaction. After completion in 30 minutes (monitored on TLC, hexanes: methylene chloride= 9: 1; the product R _f = 0.61), H ₂ O (50 mL) was added to quench the reaction, and the pH was adjusted to 7 with 10% acetic acid. The crude product was extracted three times with ethyl acetate (50 mL each time), and the organic phases were combined and dried over MgSO ₄ (S). After removal of the solvent using rotary evaporator under reduced pressure, the crude product was purified by several preparative TLC plates (hexanes: methylene chloride= 8:2). An oil product (1.412 g) was obtained (97% yield).

¹ H NMR (CDCl ₃ ) δ: 2.48 (t, J = 7.2 Hz, 2H, CH ₂ -Se), 2.55 (t, J = 7.2 Hz, 2H, CH ₂ -CN), 3.83 (s, 2H, CH ₂ -ph), 7.18-7.32 (m, 5H, aromatic protons);

¹³ C NMR (CDCl ₃ ) δ: 17.19 (NCCH ₂ CH ₂ Se), 19.41 (CH ₂ -CN), 27.80 (CH ₂ -ph), 1 18.91 (CN), 127.22 (p-ar.-C), 128.78 (o-ar.-C), 128.94 (m-ar.-C), 138.28 (ar. C-CH ₂ - Se); MS (ESI): 91 (benzyl) ⁺ , 169, 225 (M+H) ^τ ;

HRMS (MALDI-FTMS): molecular formula, C ₁₀ H _n NSe; (M with ⁸⁰ Se) ⁺ : 225.0054 (calc. 225.0057).

Example 8; Synthesis of 2-Cyanoethyl 2-phenylethyI selenide The synthesis (85% yield) is analogous to the synthesis of Example 7.

¹ H NMR (CDCI ₃ ) δ: 2.62-2.81 (m, 4H, Se-CH ₂ -CH ₂ CN), 2.94 (t, J = 7.4 Hz, 2H ₅ Se-CH ₂ -CH ₂ -Ph), 3.04 (t, J= 7.4 Hz, 2H, Se-CH ₂ -CH ₂ -Ph), 7.18-7.37 (m, 5H, aromatic); ¹³ C NMR (CDCl ₃ ) δ: 17.57 (NCCH ₂ CH ₂ Se), 19.64 (CH ₂ -CN), 25.95 (CH ₂ CH ₂ - ph), 36.94 (CH ₂ -ph), 118.87 (CN), 126.59 (p-ar.-C), 128.42 (o-ar.-C), 128.58 (m-ar.-C), 140.53 (ar. C-CH ₂ -Se);

HRMS (MALDI-FTMS): molecular formula, C _n Hi ₃ NSe; (M with ^S0 Se) ^T : 239.0209 (calc. 239.0213).

Example 9: Synthesis of 2-Cyanoethyl (l-naphthyl)ro ethyl selenide The synthesis (81% yield) is analogous to the synthesis of Example 7.

¹ H NMR (CDCl ₃ ) δ: 2.53 (t, J = 7.0 Hz, 2H, NC-CH ₂ -CH ₂ -Se), 2.66 (t, J = 7.0 Hz, 2H, NC-CH ₂ -CH ₂ -Se), 4.36 (s, 2H, CH ₂ -C ₁₀ H ₇ ), 7.2-8.1 (m, 7H, aromatic);

¹³ C NMR (CDCl ₃ ) δ: 17.77 (NCCH ₂ CH ₂ Se), 19.48 (CH ₂ -CN), 24.47 (CH ₂ - Ci ₀ H ₉ ), 1 18.91 (CN), 123.88, 125.20, 126.14, 126.22, 127.01, 128.39, 128.92, 130.96, 133.57, and 134.15 (1OC, aromatic);

HRMS (ESI-TOF): molecular formula, Cj ₄ Hi ₃ NSe; [M (with ⁸⁰ Se)+Na] ⁺ : 298.0099 (calc. 298.0105).

Example 10: Synthesis of 2-Cyanoethyl 3~(l,3-dioxoisoindoiin-2-yl)propyl selenide

The synthesis (97% yield) is analogous to the synthesis of Example 7. In this reaction, mixture of methanol and toluene (1 :9) was used to dissolve N-(3- bromopropyl )phthal imide . ¹ H NMR (CDCl ₃ ) δ: 2.04-2.1 1 (m, 2H, N-CH ₂ -CH ₂ -CH ₂ -Se), 2.69-2.86 (m, 6H,

N-CH ₂ -CH ₂ -CH ₂ -Se-CH ₂ -CH ₂ -CN), 3.81 (t, J = 6.8 Hz, 2H, N-CH ₂ -CH ₂ -CH ₂ -Se), 7.72- 7.89 (m, 4H, aromatic);

¹³ C NMR (CDCl ₃ ) δ: 17.8 (NCCH ₂ CH ₂ Se), 20.1 (CH ₂ -CN), 21.6 (N-CH ₂ -CH ₂ - CH ₂ ), 29.4 (N-CH ₂ -CH ₂ -CH ₂ -Se), 38.0 (N-CH ₂ -CH ₂ -CH ₂ -Se), 123.3 (CN), 132.4, 133.8 and 134.1 (C, aromatic), 169.2 (C=O); ESI-MS: molecular formula, C] ₄ Hi ₄ N ₂ O ₂ Se; (M with ⁸⁰ Se) ⁺ : 322.0218 (calc. 322.0221).

When a large excess of NaBH ₄ was used, reduction of the nitrile to the corresponding imine was also observed,

¹ H NMR (CDCl ₃ ) δ: 1.99-2.08 (m, 2H, N-CH ₂ -CH ₂ -CH ₂ -Se), 2.66-2.78 (m. 6H, N-CH ₂ -CH ₂ -CH ₂ -Se-CH ₂ -CH ₂ -CH=NH), 3.51 (t, J = 6.8 Hz, 2H, N-CH ₂ -CH ₂ -CH ₂ -Se), 4.05 (br, IH ₅ NH), 5.76 (s, IH, Se-CH ₂ -CH ₂ -CH=NH), 7.43-7.62 (m, 4H, aromatic). ESI- MS: molecular formula, C ₁₄ Hj ₆ N ₂ O ₂ Se; (M with ⁸⁰ Se) ⁺ : 324.0371 (calc. 324.0377).

Example 11: Synthesis of Dibenzyl selenide A methanol solution of potassium carbonate (0.05M, 6 mL, 300 μmols, thoroughly purged with argon) was injected into a round bottom flask containing benzyl (2-cyanoethyl) selenide (5a, 35.0 mg, 155 μmols) under argon, followed by injection of benzyl chloride (38.9 mg, 307 μmols, 2, eq.). When the reaction was completed over a few hours (monitored on analytical TLC, hexanes: methylene chloride= 8:2), the solvent was evaporated under reduced pressure and the crude product was purified by preparative TLC (hexanes: methylene chloride- 7:3). An oil product was obtained (39.4 mg, 97% yield).

¹ H NMR (CDCl ₃ ) δ: 3.71 (s, 4H, CH ₂ -ph), 7.19-7.32 (m, 1OH, aromatic protons); ¹³ C NMR (CDCl ₃ ) δ: 27.2 (CH ₂ -ph), 127.2 (p-ar.-C), 128.7 (o-ar.-C), 128.9 (m- ar.-C), 138.2 (ar. C-CH ₂ -Se); MS (ESI): 91 (benzyl) ^' , 171, 260 and 262 (M ⁺ ).

HRMS (MALDI-FTMS): molecular formula, C _!4 H ₁₄ Se; (M with ⁸⁰ Se) ⁺ : 262.0264 (calc. 262.0261).

Example 12: Synthesis of Benzyl 2-phenylethyl selenkle The synthesis (95% yield) is analogous to the synthesis of Example 11.

¹ H NMR (CDCl ₃ ) δ: 2.74 (t, J = 7.3 Hz, 2H, CH ₂ -Se), 2.92 (t, J = 7.3 Hz, 2H, CH ₃ CH ₂ -Ph) ₃ 3.78 (s, 2H, CH ₂ -ph), 7.17-7.36 (m, 1OH, aromatic);

¹³ C NMR (CDCl ₃ ) δ: 26.1 (CH ₂ CH ₂ -ph), 27.9 (CH ₂ -ph) 38.0 (CH ₂ CH ₂ -ph), 126.3 ancil26.7 (p-ar.-C), 128.4 and 128.7 (o-ar.-C), 129.6 and 129.9 (m-ar.-C), 139.4 and 141.2 (ar. C-CH ₂ );

HRMS (MALDI-FTMS): molecular formula, C ₁₅ H ₁₆ Se; (M with ⁸⁰ Se) ⁺ : 276.0419 (calc. 276.0417).

Example 13: Synthesis of Benzyl (l-naphthyl)methyl selenide The synthesis (84% yield) is analogous to the synthesis of Example 1 1.

¹ H NMR (CDCl ₃ ) δ: 3.71 (s, 2H, CH ₂ -C ₁₀ H ₇ ), 4.12 (s, 2H, CH ₂ -Ph) 7.19-7.89 (m, 12H ₅ aromatic);

¹³ C NMR (CDCl ₃ ) δ: 25.30 (CH ₂ -Ci ₀ H ₇ ), 28.72 (CH ₂ -ph), 124.27, 125.48, 126.1 1, 126.29, 127.04, 127.10, 128.09, 128.37, 128.66, 128.88, 129.26, 129.34, 131.53, 134.35, 134.91, and 139.36 (16C, aromatic);

HRMS (MALDI-FTMS): molecular formula, C ₁₈ H ₁₆ Se; (M with ⁸⁰ Se) ⁺ : 312.0415 (calc. 312.0417). Example 14: Synthesis of Dibenzyl diselenide

A methanol solution of potassium carbonate (0.05M, 6 mL, 300 μmols) was injected into a round bottom flask containing benzyl (2-cyanoethyl) selenide (5a, 20.0 mg, 89 μmols), which was open to air. When the reaction was completed over night

(monitored on analytical TLC, hexanes: methylene chloride= 7:3, R _f = 0.38), the solvent was evaporated under reduced pressure and the crude product was purified by preparative

TLC (hexanes: methylene chloride- 6:4). A solid product was obtained (28.1 mg, 92% yield).

¹ H NMR (CDCl ₃ ) δ: 3.86 (s, 4H, CH ₂ -ph), 7.23-7.38 (m, 1OH, aromatic protons);

¹³ C NMR (CDCl ₃ ) δ: 32.58 (CH ₂ -ph), 127.09 (p-ar.-C), 128.43 (o-ar.-C), 129.01 (m-ar.-C), 138.18 (ar. C-CH ₂ -Se); MS (ESI): 91 (benzyl) ⁺ , 169, 181, 262, and 342 (M+H)"; HRMS (MALDl-FTMS): molecular formula, Cj ₄ Hi ₄ Se ₂ ; (M with ⁸⁰ Se) ⁺ :

341.9428 (calc. 341.9426).

Example 15: Di-phenylethyl disefenide

A synthesis of di-phenylethyl diselenide was performed. ¹ H NMR (CDCl ₃ ) δ: 3.06 (t, J = 7.6 Hz, 4H, CH ₂ -Se), 3.18 (t, J = 7.6 Hz, 4H,

CH ₂ -ph), 7.22-7.37 (m, 1OH, aromatic);

¹³ C NMR (CDCl ₃ ) δ: 30.72 (CH ₂ -Se), 37.54 (CH ₂ -ph), 126.39 (p-ar.-C), 128.50

(o-ar.-C), 128.54 (m-ar.-C), 140.79 (ar. C-CH ₂ -Se); HRMS (MALDI-FTMS): molecular formula, Ci ₆ H ₁₈ Se ₂ ; (M with ⁸⁰ Se) ⁺ : 369.9736 (calc. 369.9739).

Example 16: Synethsis of Diselenides and Selenides

Benzyl chloride was first used as an alkylating reagent for both alkylation steps (4 to 5 and 5 to 7 in FIG. 4). In some embodiments, quantitative yields were obtained for both alkylation steps: the first alkylation reaction and the second alklylation when K ₂ CO ₃ was used as the deprotecting reagent. If strong bases, such as ammonia and NaOH, were used as the deprotecting reagents in the second step, reduced yields of the selenide formation were observed because these strong bases reacted with benzyl chloride.

Several other alkylating reagents were used in the investigation, and satisfactory yields were obtained for both alkylation reactions in each case, as shown in Table 4. The selenizing reagent, the intermediates, and the final selenides were synthesized and fully characterized by ¹ H-NMR, ¹³ C-NMR, and HR-MS.

Table 4. Synthesis of Diselenides and Selenides.

conversion reduction or deprotection T alkylation or yield (%

Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only.

The following references are generally related to various aspects of the present invention, including X-ray crystallography, heavy-atom derived function, organoselenium chemistry, and nucleic acid chemistry.

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