CA2244924A1 - Non-nucleotide phosphorous ester oligomers - Google Patents

Abstract

A phosphorus ester oligomer having structure (I) wherein A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl; B1 and B2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labelling group, a protecting group, a phosphoramidate or a phosphomonoester; R1 can be the same or different in each monomeric unit, and in at least one of the non-nucleotide monomeric units, R1 is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic functionality; (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label, and n is at least one. Preferred R1 moieties include condensation products of heterocyclic diols, alicyclic diols, and polycyclic diols. Also the non-nucleotide monomers thereof, combinatorial library mixtures of the oligomers and the use of the oligomers as selective target-binding compounds.

Description

NON-Nn~CL~Oll~ P~OS~hOKU~ ESTER OLIGOIDERS

This application is a continuation-in-part of U.S.
serial no. 08/374,040, ~iled on January 18, 1995.

This invention relates to the field o~ oligomeric molecules ( 1l oligomers") cont~;n~ng non-nucleotide phosphorus esters, which are useful as mixtures, particularly in the form of comh~n~torial li~raries, to screen ~or binding activity ~o biologically signi~icant targets, including protein targets. Esters with high a~finity have value as drug or diagnostic candidates, and' such combinatorial libraries thereo~ have value as products used to identi~y such candidates.

A variety o~ phosphorus ester oligomers have been reported in the literature. Non-nucleo~ide phosphorus esters have been incorporated into oligonucleotides as linker groups which connect two separate oligonucleotide sequences. For example, oligonucleotides cont~in;ng a single non-nucleotide hexaethylene glycol phosphodiester have been described (Durand et al., 1990). The, oligonucleotide reyions of the molecule were base paired to ~oxm a duplex structure, and the non-nucleotide phosphorus ester ~unctioned as a loop to connect the two strands.
aromatic non-nucleotide phosphodiester has also bee incorporated into an oligonucleotide as a l~nke~ group (Salunkhe et al., 1992), and the non-nucleosides 1,2 dideoxyribose and l-phenyl-1,2-dideoxyribose have been incorporated into oligonucleotides ~or enzymatic studies.

SUBSmVTE Stl EET (RULE 26) Molecules contA~ n~n~ multiple non-nucleotide phosphorus esters have al80 been reported. For example, several 1,3-propanediol phosphodiester groups have been incorporated into oligonucleotides (~ichardson and Schepartz, 1991) to act as tethers of varying lengths which connect two separate oligonucleotide sequences. The tethers enabled the oligonucleotide sequences to bind to non-contiguous regions of an RNA target. Diethylene-glycol phosphodiester and deca-ethylene-glycol phosphodiester moieties have also been incorporated into oligonucleotides for similar purposes ~Cload and Sch~pA~tz, 1991).

Non-nucleotide phosphorus ester oligomers have been used for other ~unctions. For example, oligomeric phosphorus esters possessing substituted alkyl substituents have been synthesized and used as ~ire retardants (Hardy and Jaffe 1980). Oligomeric phosphorus esters o~ phenolic compounds have also been described as fireproofing agents (Sase et al., 1988).

In relation to drug discovery, non-nucleotide compounds have been used in combination with nucleotides in a combinatorial m~nn~ to search for compounds which bind to biological targets. Co...~o~lds possessing various backbone elements such as ethylene glycol phosphate, hydroxyproline phosphate, or PNA ("peptide nucleic acid") have been used, and a twelve residue p~n~m~ comhin~torial library cont~; n i n~ substituted ethylene glycol phosphate residues in cQ~h~n~tion with nucleotide residues was reported ~Bcker, 1994). Other groups have described the assembly of combinatorial libraries o~ oligomers made ~rom a set of monomers consisting of a side chain and a uniform h~khon~ element (Zuckermann et al., 1994). The application o~ combinatorial libraries to drug discovery has recently been extensively reviewed ~Gallop et al., 1994, Gordon et al., 1994).

SUE~STITUTE StlEE T (RULE 26) W O97128168 PCT~US97/01060 The desirability o~ introducing conformational constraint into potential drug c~n~ teS is a well understood principle in medicinal chem;~try. The conc~rt of designing combinatorial libraries of constr~i n~
molecules has recently been described, wherein rigid small molecules act as a frame onto which various p~n~nt functions can be attached. Alternati~ely, the fl~hi-;ty of members of an oligomer library can be limited by the use o$ intrinsically constrained amide linking groups, as in the case of PNA or N-substituted glycine libraries (ZuckPr~n et al., 1994). In the area of antisense oligonucleotides, the phosphodiester linkages have been substituted with acetal groups (Matteucci et al., 1993), and the regular deoxyribose element of the barkhon~ has been replaced with a 5-3-ethano-bridged rigid analog, both with the objective of intro~nc~ n~ conformational constraint. In the latter case, the oligonucleotides bound with higher a~finity to their cognate sequences than did conventional oligonucleotides (L~m~nn et al., 1993).

A rapidly growing new industry has developed around the preparation and use of ~o~;n~torial libraries o~ a wide variety of compounds, including polypeptides, small molecules and oligonucleotides. Comr~nies in this industry sell such ~omhi n~torial libraries to pharmaceutical, diagnostic and other markets as research reagents suitable for screening and also provide services for co~r~n;es which do not have the facilities for in house screening o~ such libraries. The market ~m~n~ for these combinatorial libraries has been an encouraging ~mon~tration that their utility is well recognized by those skilled in the art.

Combinatorial libraries o~ oligomers having potential for h; n~; n~ to proteins and other biological targets are valuable products for use in drug screening and other research by pharmaceutical and diagnostic industry members and by investigators in the biological and medical arts.
They provide a convenient and rich source of candidates for SllBSTlTlJTE SHEET ~RULF 26) W O 97/28168 PCTrUS97/01060 modulating biologically active agents, particularly proteins, through their binding potential. Thus these products constitute a pool of readily accessible and sophisticated oligomeric constructs and libraries that satisfies this market and thereby makes structures available to research programs which would not otherwise have access to the broad spectrum of candidates they provide.

One aspect of the invention provides a phosphorus ester oligomer of monnm~ric units, which oligomer has the structure:

B, Pll--O ~- OF~1 B2 - A -n wherein A can be the same or different in each ~nom~ric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkyl~m~ nn ~ substituted or unsubstituted aryl ~mi nn and Ami no~lkyl;
Bl and B2 can be the same or di~ferent and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
Rl can be the same or different in each mo~o~ric unit, and in at least one of the non-nucleotide monomeric units, R~ is independently selected from the group consisting of a con~n~ation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine-or pyrimidine-substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic functionality (iv) a SUBSTITUTE SHEET(RULE 26) W O 97/28168 PCT~US97/01060 diol attached to a ring substituted-anionic ~unctionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any o~ which can ~urther include a detectable label; and n is at least one.

Preferred embo~;m~nts o~ the above aspect include the ~ollowing. Pre~erably n = 2-2~, and more pre~erably 1-6.
Preferably the hydrogen bond donor c~nt~i n~ a hydro~yl, amide, imide or thiol group or is a basic compound. The ba~ic compound can cont~;n an amine moiety or a substituted or unsubstituted thiazole. Preferred hydrogen bond acceptors include an amine or ether moiety. Examples include the 5-substituted 2-hydlo~ylllethyl-3-hy~ro~y--tetrahydro~urans and bis~hydroxyalkyl)-substituted heterocycles or substituted or unsubstituted theophyllines.
Preferred hydrophobic functionalities are selected from aromatic rings, alkanes, cycloAlk~nPc and aromatic rings fused to Al k~nefi . Particularly preferred hy~lv~hobic functionalities include those wherein R, is selected from a substituted ~lk~n~, a 3,3-disubstituted 3-amino-1,2-propanediol, a suhstituted or unsubstituted alicyclic ring wherein the ring size is from 4-12, a 3-substituted indole, a substituted or unsubstituted hydroxyalkyl phenol and an alicyclic dic~hoxylic acid. Preferred anionic functionalities include those wherein the anionic functionality is a mono-or dic~hn~ylic acid moiety. More particularly pre~erred moieties include tricyclononene dicalboxylic acids and cyclopentane acetic acids. Preferred cationic functionalities include bis(hydroxyalkyl)-substituted nitrogen-cnnt~1ning heterocycles. More particularly preferred cationic functionalities include substituted alkanes and 3,3-disubstituted 3-amino-1,2-prop;~n~;ols. Another pre~errred ~mho~lm~nt of the above aspect is an oligomer wherein in at least one of the mon~ -~iC units R~ is a heterocyclic, an alicyclic or a polycyclic ring system, preferably con~n~n~ an indole, thiazole, imidazole, pyridine, purine or pyrimidine ring.

SUBSl~TUTE SHEET (RULE 26) W O 97/28168 PCT~US97tO1060 The alicyclic ring system preferably contains a cyclopentane or cy~looctane ring, and can be substituted with at least one carboxylic acid moiety. The polycyclic ring system preferably contains a bicyclic or tricyclic ring or is a polycyclic arene.The bicyclic ring sy~tem is preferably a bicyclic alkane, such as tricyclo~o~Pne. The polycyclic arene is preferably diphenyl bicyclooctane.

Another aspect of the invention provides a phosphorus ester oligomer of monor?riC units, which oligomer has the structure:

E~l Rl--~--I OR1 B2 - A -n wherein A can be the same or different in each mon~m~ric unit and each is indep~nAently selected from the group consisting of o~yy~ll, sulfur, lower alkyl, substituted or unsubstituted alkyl~m; no, substituted or unsubstituted aryl;~mino and ~m; no~llkyli B~ and B2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling y~ou~, a protecting group, a phosphoramidate or a phosphomn~oester;
R~ can be the same or different in each mono~ic unit and in at least one of the mnn~m~riC units is independently selected from the group consi~ting of (i~ an aliphatic acyclic hydrocarbon diol wherein the diol groups are non-vicinal or are substituted;
~ ii) a purine- or pyrimidine-substituted variant of the diols of (i) or of aliphatic acyclic hydrocarbon vicinal diols;
(iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety;
~c~

SUBSTITVTE SHEET(RULE 26) , W O 97/28168 ~ Y//01060 (iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;
(v) a hydroxy or hydroxyalkyl substituted tetrahydrofuran;
(vi) an indole-substituted acyclic aliphatic diol;
(vii) an aromatic ring or ring system having two substitutions independently selected ~rom the group consisting o~ hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independentl~ selected ~rom the group consisting of hydroxy or hydroxyalkyl; and (ix) a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, any of which can ~urther include a detectable label; and n is at least 1, preferably 2-20, and more preferably 2-6.

- Another aspect of the invention provides a non-nucleotide ~n~_ -~ic unit having the structure:

~ Y
X,- O Rl--O--P~

wherein X~ is a protecting group;
X2 is a branched or unbranched lower alkyl group or a substituted or unsubstituted alkoxy group;
Y is a branched or unbranched lower alkyl group; and R, is independently selected from the group consisting o~ a con~nsation product o~ (i) a non-vicinal diol attached to a hydrogen bond donor ~unctionality; (ii3 a hydrogen bond acceptor selected from an ether, a purine--or pyrimidine-substituted 1,2-diol or a disubstituted SU8STITVTE SHEET (RULE 26) W O 97/28168 PCTrUS97/01060 heterocycle; (iii~ a non-vicinal diol attached to a hydrophobic ~unctionality or a vicinal diol attached to an - aliphatic or alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic ~unctionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label.

Another aspect of the invention provides a non-nucleotide m~nomeric unit having the structure:
y ~N--Y
X, O R,--O- P~

wherein Xl is a protecting group;
X2 is a branched or unbr~nrh~ lower alkyl group or a substituted or unsubstituted alkoxy group;
Y is a branched or unbranched lower alkyl group; and Rl is a cnn~ncation product of:
(i) an aliphatic acyclic diol wherein the diol hydroxyl groups are non-vicinal or are substituted;
(ii) a purine- or pyrimidine-substituted variants of the diols of (i) or of aliphatic acyclic hydrocarbon vicinal diols;
(iii) an acyclic ~l;rh~tic diol having an amino group with at least one hydrogen substitution moiety;
(iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;
(v) a hydroxy or hydroxyalkyl substituted tetrahydro~uran;
(vi) an indole substituted acyclic aliphatic diol;

S~S I I I UTE SH E~T (RULE 26) WO97/28168 ~CT~S97/01060 (vii) an aromatic ring or ring system having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independently selected from the group consisting o~ hydroxy or hydroxyalkyl; and (ix) a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, any of which can further include a detectable label.

Another aspect of the invention provides a non-nucleotide monomeric unit having the structure:

X- O ~--O 1~ O' wherein X is a protecting group;
R~ is indepen~ntly selected ~rom the group consisting o~ a con~pn~tion product of (i) a non-vicinal diol attached to a hydrogen bond donor ~unctionality; (ii) a hydrogen ~ond acceptor selected ~rom an ether, a purine-or pyrimidine-substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic h~d,~hobic ~unctionality (iv) a diol att~ch~ to a ring-substituted anionic ~unctionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any o~ which can ~urther include a detectable label.

Another aspect o~ the invention provides a non-nucleotide m~nom~ric unit having the structure:
D' SVBSTITUTE SHEET ~RULE Z6) W O 97/28168 PCTrUS97/01060 ;~
X O P~,--O I O-wherein X is a protecting group;
R, is a con~n~ation product of:
(i3 an aliphatic acyclic hydrocarbon diol wherein the diol hydroxyl groups are non-vicinal or are substituted;
(ii) a purine- or pyrimidine-substituted variant of the diols of (i) or of a~iphatic acyclic vicinal diols;
(iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety;
(iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or c~rho~yalkyl substituent;
(v) a hydroxy- or hydroxyalkyl-substituted tetrahydrofuran;
(vi) an indole-substituted acyclic aliphatic diol;
(vii) an aromatic ring or ring system having two substitutions independently selected ~rom the group consisting of hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independently selected from the group consist~ng of hydroxy or hydroxyalkyl; and (ix~ a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, any of which can ~urther include a detectable label.

Another aspect o~ the invention provides a phosphorus ester oligomer of mnnom~ic units, which oligomer has the structure: . .
IC

SVBSTITUTE SHEET ~RlJI E 26) W O 97/28168 PCT~US97/01060 C

B~ R1--~ ---OR1--B2 - A -n -wherein A can be the same or different in each ~onom~ic unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
Bl and B2 can ~e the same or differe~t and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramida~e or a phosphomonoester;
Rl can be the same or different in each ~o~om~ic unit, and is selected from the group of a nucleoside moiety and, in at least one mo~. ~ric unit, R, is indep~n~ntly selected from the group consisting of a cnnd~n.c~tion product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted ~,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic ~unctionality (iv~ a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label; and n is at least one.

Another aspect of the invention provides a combinatorial mixture of the above oliyomers. Pre~erred embodiments o~ combinatorial mixtures of oligomers include those wherein A is 1-20, and particularly preferred 5UB~TE SHEET tRVI E 26) W O 97/28168 PCTrUS97/01060 embodiments include those wherein A is 1-6. Preferred embo~m~ts include comh~n~torial mixtures wherein the ~ oli~omers are labeled with 3~hosphorus, 35sulfur, tritium, fluorescein or biotin.

A large, highly diverse set of structurally unrelated, derivatized m~n~S iS prepared for incorporation into oligomeric libraries. The libraries are ~ynthesized using cn~h;n~torial techni~ues from subsets of available monor~s to generate molecular ensembles from which can be selected those with ~-x;mllm af~inity for the target protein. The number of mnn~mA~S used in the library synthesis is chosen to exploit the full c~r~h; 1; ty o~ the screening technology used to identify the best ligand.

There is no uniform element in the monomer corres~on~ng to the sugar group of an oligonucleotide, or the amide backbone of peptides, PNA's, hydroxyproline or N-substituted glycine derivatives, or s~m;lAr types of oligomers. Therefore, there is a high degree of variability in the molecular geometries that can be introduced into the product oligomers.

The nature o~ the phosphorus l; nki n~ group in these compounds can be varied at any position to create a greater diversity per unit length o~ the oligomer than would otherwise be achieved with one type o~ linkage, such as a phosphodies~er or amide. The ~lexibility of this linkage is also controllable by changing between freely rotatable phosphodiesters, and ro~ationally constrained phosphoramidates or other e~ters with bulky groups directly att~h~A to phosphorus. In addition, the gros physical characteristic~ of the molecules can be varied ~rom anionic to neutral to cationic, with all interm~A;~te cases represented.

Experiments have shown that representative oligomers o~ t~is invention are very stable in serum under conditions \~

SU8STITUTE 51~EET (RULE 26) W O 97t28168 ; PCT~US97tO1060 which rapidly degrade oligonucleotides. Therefore, any lead compound selected from an oligomeric phosphorus ester library is potentially a drug candidate, as compared to oligonucleotide or peptide leads which are readily degraded and there~ore most likely require chemical modification to serve a~ a legitimate drug candidate.
~, The uni~ue and advantageous aspects of the present invention lie pr~nc;rAl-y in the highly variant set of monomers employed for the synthesis o~ combinatorial libraries, and the choice o~ the 1; nk; ng groups in the product oligomers. The mOnOmP~S fu~ill two roles: a structural function whereby the molecular geometry and flexibity o~ the oligomer are controlled, and secondly, they contain appropriate ~unctional groups to bind to protein or other biological targets. As stated above, the mono~~~s do not contAi n the sugar or sugar analog element that is found in most other types o~ oligomers. Instead, any molecule ~ont~; n~ n~ two hy~u~yl functions may be considered a suitable mnn~m~ candidate. By employing such ~ariant compounds to construct oligomers, large ensem~hles of molecules with highly variant geometries can be generated. In addition, control can be excercised over the degree of ~lexibility in these compounds. For example, constraints can be introduced by either using intrinsically in~lexible monomers, or m~n- ~S in which the two hydroxy y-~S are vicinal and the resultant steric congestion limits certain bond rotations. Alternatively, it may be desirable to introduce a ~lexible element into some portion o~ a molecule, and this could be easily achieved by e~ploying a suitably derivatized CY,~ Al iphAtic diol. The importance of being able to modulate the con~ormational freedom o~ potential drug candidates in order to optimize their h~nA;ng and selectivity profiles is a well understood principal in medicinal ~h~mi fitry. Also, coni~ormationally-constrAineA compounds are desirable as lead molecules in any rational drug design plOy- d~

SUBSrlTUTE SHEET (RULE 26) CA 02244924 l998-07-30 W O 97/28168 PCT~US97/01060 In relation to the ability o~ the mnIl~m-~s to present appropiate binding groups to protein targets, essentially all common organic functional groups, when suitably protected, are compatible with the conAf~nF:Ation ch~m;~:try that is employed for synthe~izing the oligomers. The m~n~m~S used for the synthesis of a library directed against a particular bioloyical target are chosen to u contribute to the affinity for the target. To this end, the monomers can be subdivided into a range of different categories describing their principal mode of ~;n~;n~, examples being; hydrophobic, h~dL~yell bond donor, lly~ o~
bond acceptor, electrostatic cation, and electrostatic anion. Obviously, many mo~om~s cont~in multiple ~;nA;ng elPm~nt5 and can be assigned to more than one category.

In this invention, each phosphorus l;nk;ng element connecting the monom~ units can be indep~n~ntly modified to both introduce additional b~nAin~ ~unctionality, and also control the structural fl~ ;l;ty of the oligomers.
These linkages can exist either as phosphates, phosphorothioates or phosphoramidates, dep~n~ng on the oxidation conditions employed ~or their synthesis.
Additional h~ nrl;ng groups can be introduced into the oligomers by the oxidation o~ interm~;Ate ~-phosphonate linkages using a diverse range of pri~ary and se~onA~y, simple or high}y functionalized ~m; n~-~, an obvious example being amino acids. By using secon~Ary and ~-brAnch~
~m; n~s, significant rotational constriction of the phosphorus linkage can be achieved, and this constitutes another me~h;:ln;cm for introducing conformational constraint.

To complement the above methods ~or the introduction of conformational constraints, an extension is the synthesis o~ libraries o~ cyclic analogs o~ oligomeric phosphorus esters. These compounds can be prepared via oxidation reactions of thiol groups to produce cyclic disul~ides, such as is described in c~ o~ly assigned 1~1 SUBSTmlTE SHEET tRULE Z6) _ W O 97128168 PCT~US97/01060 copending U.S. patent applica~ion Serial No. 08/004,284.
This creates structures such as the disulfide-bridged cyclic oligomer as described in Example 45 herein. Other methods for cyclization can be employed, using methods known to those skilled in the art.

A primary utility of oligomeric phosphate ester libraries is the screening o~ such mixtures ~or h; n~;ng activity to particular biological targets, including but not limited to proteins, with the objective o~ identifying therapeutically important molecules. The structures o~ the oligomers o~ the present invention that bind with high af~inity to a biological target can be ~P~nc~ using procedures such as that described in co~monly assigned, cop~n~;ng U.S. patent application Serial No. 08/223,519, or by the use of encoded libraries similar to those described in Gallop et al., 1994, or by other methods obvious to those skilled in the art.

Another application of this invention is the u~e of these compounds a~ glyco~m~noglycan mimetics.
Glyso~mtnoglycans are large sulfated oligosaccarides that bind to a range of important regulatory proteins. Work directed at the synth~is of mimetics of these compounds has focused on the preparation of structurally defined heparin oligosaccarides and sulfated dextrans (T.~n~r 1994).

A particular example of this application is in the area of glycos~m;noglycan mimetics. Glycos~minQglycans are multiply charged, anionic, sulfated polysaccarides that are typically around 250 saccharide units in length. They are known to modulate a series of biochemical processes by h~n~ing to certain polycationic receptor sites on proteins, and play key roles in a conditions as diverse as c~nCpr and Al~he~mPr's disease. The interaction between the protein and the glycosaminoglycan is mostly electrostatic in nature, and consequently the dissociation constant for SUBSTITUTE SHEET ~RULE ~6) W O 97/28168 PCT~US97/01060 these interactions is comparatively large, typically in the range 10-5 to 10-8 M. One approach to the design of mimetics of this class of compound which would have therapeutic application, would re~uire the synthesis of much lower molecular weight anionic molecules, con~;n-n~ certain hydrophobic h;n~ng groups. (T-~n~, 1994). These criteria are met in the oligomeric phosphorus ester libraries of the type discussed in Example 43.

In another aspect, the oligomers of the present invention can be used in a method ~or detecting the presence or absence o$ a target molecule in a sample, or determi n i ng the nllmh~r of such molecules in a sample. In such methods, the oligomer can be labeled, exposed to target, and the amount o$ labeled oligomer which is bound to the target is quantified by measurement of the signal derived $rom the label. The label can be radioactive or non-radioactive. Radioactive labels include phosphorus-32, sulfur-3~, tritium, and heavy metal isotopes which can be trapped by chelating groups att~ch~ to the oligomer. Non-radioactive labels include, but are not limited to, biotin and its analogs, fluorescein, tetramethyl-rhn~m;ne, substrates for enzymes such as alk~l ine phosphatase or horser~ h peroxidaE;e, haptens such as dinitrophenyl or digoxigenin and other labels known to tho~e experienced in the art. Thus, oligomers o$ the present invention can be used for diagnostics in a m~nn~r s;m; 1~ to methods employed ~or antibody-~a~ed diagnostics. The same labeling techni~ues may also be employed in the protocols $or the identification of protein ligands, discussed above.

Figure 1 shows a method $or introduction o$ a triti~m label by reduction of a hydroxyacetorh~nnn~ functionality.

Figure 2 shows a method for the introduction of glycolic amide groups at the terminus of a library (Structure 8), including a method for the introduction of a radioactive label (Structure 10).
1~

SUBSTITUTE SHEET (RULE 26) WO 97128168 PCTrUS97/01060 -Figure 3 shows the h~n~;n~ of~ a combinatorial libraryof non-nucleotide phosphorus ester oligomers to two protein targets.

Figure 4 shows the hi n~; ng of a library of non-nucleotide phosphorus ester oligomers to thrombin.
..
Figure 5 shows the prolongation of clotting time for a library of non-nucleotide phosphorus ester oligomers as compared with a control in the absence of library.

Figure 6 ~hows the clotting time as a function of concentration of library of non-nucleotide phosphorus ester oligomers.

Preferred hydrogen bond donors are functionalities cont~n;ng amine, amide, imide, alcohol and thiol moieties.
Examples of such compounds are those wherein R~ is a 3-substituted di~ly~L~yalkyl indole (e.g., indolyl-dihy~l~y~L~ane), or a bis(hydroxyalkyl)-substituted heterocycle ( e . g., 1,2-di~lyd~xyethylthiazole and pyri~oxin~) or a 2-amino-1,3-propandiol ~e.g.;
thiomicAm;n~)~

Preferred hydrogen bond acceptor c~...~o~lds include those cont~ining amine or ether moieties. Examples of such compounds are those wherein Rl is a purine or pyrimidine substituted 1,2-diol (e.g., theophylline) or a bis (hydroxyalkyl)-substituted heterocycle (e. g., 1,2-dihydroxyethylthiazole and pyridine dimethanol) or a 5-substituted 2-hydroxymethyl 3-~lyd~v~-tetrahydrofuran ( e . g., 1,2 dideoxy-D-ribose).

Preferred hydrophobic groups are alkyl groups and aromatic rings. Examples of such compounds are those wherein Rl is a substituted 1,3-dihydroxyAlkAnp ( e . g. 4 -methoxyrh~noxy-1,3-pro~AnP~;ol), a substituted 1,2-dihy~oxy ~lk~ne (e.g.; 1, 2-dihyd-oxy-3-butene), a 3,3-1~

SUBSTITUTE SH EET (RULE ~6) W O 97t28168 PCTrUS97/01060 disubstituted 3-amino-1,2-propanediol (e.g., 3-benzyl-3-methylamino-l~2-prop~nen;ol and 3,3-diethyl~mi no-l ~ 2--propanediol), a substituted or unsubstituted alicyclic diol wherein the ring size is from 4-12 (e.g. cyclooc~An~ol and cyclopentAn~A;ol), a 3-substituted dihydlo~dlkyl indole (e.g., indolyl-dihydroxypropane) or a substituted or unsubstituted hydroxyalkyl phenol (e. g., tetralin and lly~Lu~benzyl alcohol) or a 1,2-dihydroxy alicyclic, dicarboxylic acid (e. g., tricyclo~onene dicarboxylic acid).

Preferred electrostatically charged functionalities include anionic ~unctionalities, such as cA~ho~ylic, sulfonic and phosphoric acid and tetrazole moieties.
Examples o~ such compounds are those wherein R~ is a dihydroxy-substituted carboxylic acid (e.g., cyclopent~ne~;ol acetic acid) or a 1,2-dihydroxy alicyclic dicarboxylic acid (e.g., tricyclonoJ~f~ne dicArhoxylic acid).

Preferred electrostatically charged functionalities also include cationic functionalities. ~xamples of such compounds are those wherein Rl is a substituted 1,3-dihy~oxydlkane ( e. g., 2-amino-1,3-propAnen;ol, 1-phenyl-2-amino-1,3-propAnediol and thiomi~m;n~), a 3,3-disubstituted 3-amino-1,2-propanediol ~e. g., 3,3-diethyl ~m~ n~propAne~;ol and 3-benzyl-3-methyl Am; no-1,2-propAnen;ol) or abis(hydroxyalkyl)-substituted heterocycle (e. g., 1,2-dihydroxyethyl-thiazole and pyridine dimethanol).

Preferred heterocyclic dihydroxy alcohols are those contA;n;n~ an indole, thiazole, imidazole, purine or pyr~;A;ne riny structure. Examples o~ such compounds are those wherein Rl is a 3-substituted dihydroxyalkyl indole (e.g., indolyl-dihydroxypropane) or a purine or pyrimidine substituted 1,2-diol (e.g., 2,3-dihydroxypropyl-theophylline) or a bis Blyd~u~yalkyl)- substituted 1~

SUBSTITUTF SHEE r (RULE 26) W O97/28168 PCT~US97/01060 heterocycle (e.g., thiazole and pyridine dimethanol) or a 5-su~stituted 2-hydro~ymethyl 3-hydroxy-tetrahydrofuran (e.g., 1, 2 dideoxy-D-rihose).

- Preferred alicyclic dihydroxy- alcohols are those cont~;nin~ a cyclopPnt~n~ or cyclooctane ring structure, including those which are diols and which are substituted with at least one c~rh~xylic acid moiety. Examples of such compounds are those wherein Rl is a substituted or un~ubstituted alicyclic diol wherein the ring size is from 4-12 (e. g. cyclooct~ne~;ol and cyclop~nt~n~;ol~ and those wherein Rl i8 a dihydr~y-substituted carboxylic acid (e. g. ' cyclopentAn~iol acetic acid~.

Pre~erred polycyclic dihydroxy alcohols are those con~A~nin~ a bicyclic or tricyclic ring structure, including alkanes ~uch as a bicycloheptane, ~lk~ne~ such as tricyclonene diol and polycyclic arenes such as diphenyl bicyclooctane diol. Examples of such compounds are those wherein Rl is a dihydroxy substituted polycyclic compound (e.g. tricyclnn~n~ne diol dicarboxylic acid and p;n~ne~;ol).

Examples of non-nucleotidephosphorus oligomers where Rlis a con~n~ation product of an ~lip~Atic acyclic hydro~hon diol wherein the diol hyd~Ox~l groups are non-vicinal or are substituted include those formed ~rom mnn~m~S wherein Rl is 1,3-prop~n~;ol or 2-amino-1,3-pro~n~A;ol.

Examples of non-nucleotide phosphorus oligomers where Rl is a con~n~ation product of a purine or pyrimidine substituted variant of the diols of (i) or of aliphatic acyclic hydror~hnn vicinal diols include those ~onmed from m~no~-~s wherein Rl is a purine su-hstituted 1,2-diol (e.g.
2,3-dil~yd~o~ypropyl theophylline).
~q SUBSTlTlJTE SHEET (RULE 26) W O 97/28168 PCTrUS97/01060 Examples of non-nucleotide phosphorus oligomers where Rl is a con~ns~tion product of an acyclic ~liph~t;c diol having an amino group with at least one ~ly~Loy~-substitution moiety include those formed from mon~m~s wherein Rl is 3-diethyl;~mino-l~3-pror~ne~i ol.

Examples of non-nucleotidephosphorus oligomers where Rl is a co~n~ation product of an alicyclic or polycyclic diol, optionally substituted with a carboxy or c~ho~yalkyl substituent include those formed from mnnom~s wherein Rl is cyclopent~nP~iol~ cyclo-oct~n~;ol, and those formed from m~nom~ns wherein Rl is a dihydroxy-substituted carboxylic acid ( e . g., cyclopentane diol acetic acid) or a 1,2-dihydroxy alicyclic dicarboxylic acid ( e . g., tricyclono~ne diol dicarboxylic acid).

Examples of non-nucleotide phosphorus oligomers where Rl is a con~n~ation product of a hydroxy or hydroxyalkyl substituted tetrahydrofuran include those formed from monomers wherein Rl is 1,2-dideoxy-D-ribose.

Examples of non-nucleotide phosphorus oli~. ~ where R, is a con~ ation product of an indole substituted acyclic ~l;rh~tic diol include those formed from mnnom~s wherein Rl is indolyl-dihydroxypropane.

~ xamples of non-nucleotide phosphorus oligomers where Rl is a çon~n~ation product of an aromatic ring or ring system having two substitutions indep~n~ntly selected from the group consisting of hydroxy or hyd~o~yalkyl include those formed ~rom m~nom~s wherein Rl is 2,6-bis-11YdL o~y-methyl-pyridine~

~ xamples of non-nucleotide phosphorus oligo~ers where ~ is a ~nn~n~tion product of a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or h~dLo~yalkyl include those ~G

SV~ JTE SH EET (RVLE 26) ~ormed ~rom m~no~s wherein Rl is 1,2,3,4-tetrahydro-1,5-dihydLo~-n~rhth~lene.

Examples o~ non-nucleotidephosphorus oligomers where Rl is a con~n~Ation product of a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline include those ~ormed ~rom ~no~rs wherein Rl is 2-amino-4-(1,2-dihydroxyethyl)-1,3-thiazole.

The structures of the oligomers of the present invention that bind with high affinity to a protein target can be deduced using procedures including, but not limited to, those described in ~mon~y assigned, cop~n~;n~ U.S.
patent application Serial No. 08/223,519, or by the use o~
encoded libraries similar to those described in Gallop et al., 1994.

The libraries are typically synthesized ~rom a diverse pool of about 20 to 30 of such monomer units which are chosen to ensure su~ficient diversity with regard to chemical functionality, shape and physical properties, such as charge and ~lyd-~hobicity. However, larger or smaller numbers of such m~nn~rs can be employed. For the purpose o~ synthesizing the libraries, these monomers are prepared as 4,4'-dimethoxytrityl (DMT)-protected phosphoramidites and con~n~ed together using known techniques (see Andrus et al., 1988) to produce oligomers with negative charges on the phosphorus backbone. Phosphodiester or phosphorothioate h~c~kh~n~: can be produced by these methods. Alternatively, the mnnom~s can be prepared as H-phosphonate derivatives which can be coupled together to produce oligomers using st~n~d procedures ~Milligen/Biosearch Inc., Novato C~., H-phosphonate Synthesis In~ormation). The oligomer H-phosphonate inter~ tes can be subsequently oxidized with iodine and water to produce phosphodiester or with a sul~ur reagent to give phosphorothioate linkages.

SUBSTITUTE SHEET(RULE 26) W O 97128168 PCT~US97/01060 An example of a simple oligomeric phosphodiester compound of this type which ha~ already been synthesized is as follows: ~

~0 ~ ~lo r ~ N O r ~--O_p_O A--oPo~2~

3 N ~ > ~

Another aspect o~ the present invention is the introduction of additional diversity into the backbone of the oligomers by oxidation of the H-phosphonate intermediates with a diverse set o~ ~m~ n~ to produce phosphoramidate derivative~. The amines can be selected ~o as to introduce aliphatic groups, aromatic groups, nch~ged polar groups such as hydrogen bond donor or acceptor groups, negati~ely charged groups and/or positively charged groups. Thus the overall ~tructural diver~ity of the oligomer can be greatly increased above that which can be achieved when the backbone is comprised exclusively of phosphodiester groups. Examples of ~m; n which can be reacted with oligomer H-phosrhon~te intermediates to produce oligomer phosphoramidates are as follows: 2-(2-aminoethyl)pyridine; 1-(2-aminoethyl)piperidine; 2-(3, 4-dimethyloxy-phenyl)ethylamine; 4-(2-aminoethyl)-morpholine; 1-(3-aminopropyl)-4-methylpiperazine; 1-(3-aminopropyl)-2-pyrrolidinone; l-(3-aminopropyl)imidazole; 1-(2-aminoethyl)pyrrolidine; 3-aminopropionitrile; Z-(2-aminoethyl)-}-methyl-pyrrolidinei 4-fluorophenethylamine;

4-bromophenethylamine; aminomethyl-cyclopropane; 3,3-~;ph~nylpropylamine; ~ormylpiperazinei trifluoromethyl-phenylpiperazine; thiomorpholine; 1-(2-pyridyl)piperazine;
i~

SVBSTtTVTE SH EET (RULE 26) W O 97/28168 PCTrUS97/01060 homopiperazine; hexamethyleneimine; cis-2, 6-dimethylmorpholine; 2,5-dimethylphenylpiperazine; 3,s-dimethylpiperidine; 1-(4-~luorophenyl) piperazine; N-(3,4-dichlorophenyl)piperazine; 2-t4-chlorophenyl)ethyl ~m~ ne; 4-piperazineacetophenone; 4-piperidinopiperidine; 2-thiophenemethyl ~mi ne; furfurylamine; heptamethyl~n~i mi ne;
and 1-(4-methoxyphenyl)piperazine.

Other ~m~ne~ can be employed ~or this purpose as will be evident to those skilled in the art, and amino acid derivatives can also be used in the same way. Thus oligomers can be constructed that can be either anionic, neutral or cationic in character. Using this method, a highly diverse library o~ molecules with lower molecular weights can be produced.

An example of an oligomeric phosphoramidate of this type which can be synthesized is as follows:
~ a O ~ -P-O. ~ N
~D NH Nf 1 9--NH2 FLUOR ~ ~ S

~3 NH

Cl Another aspect o~ the invention relates to the use o~ preferred labeling and protecting groups, for example as B~ and/or B2. In one embodiment B, and/or B2 is a group cont~; n; ng at least one tritium atom for the purposes of detection. This approach to tritium labeling employs a ~labeling mono~erl1 which introduces a reactive aldehyde or ketone group at a late stage o~ synthesis, followed by reduction with labeled borohydride to introduce the tritium label. In the resulting compound at least one o~ B~ and B~
is a phosphodiester attached to a tritium-labeled p- or m-1-hydroxy (C~-C6 alkyl) phenyl. An important consideration ~.

SUB51~TUTE SH EET (RUL~ 26) requires that the labeling group should also be stable to the alk~7~ne conditions used to cleave the material from the solid support. Simple glycol groups with flexible hAckhon~s were therefore ruled out since Font~n~l et al.
have shown that oligonucleotides terminated with glycol moieties undergo degradation in concentrated A~on; ~ at 5~~C, presumably by attack of the free hydroxyl group, on the adjacent pho~phate group followed by cyclization and ~l;m;n~tion of the terminal unit. For these reasons hydroxyacetoph~no~ was selected as a r~m~cially available starting material for the labeling monomer approach, since the possibility of attack of the hydroxyl group produced by reduction on the adjacent phosphorus atom was eliminated because of the rigidity of the benzene ring.
A comparison of reductions of the ketone group of both p-and m-hydL~ydcetorhPnone indicated that the meta isomer was more easily reduced using sodium borohydride, this i~omer was selected for further studies. The phosphoramidite derivative 2 wa~ prepared by a conventional procedure (~eaucage et al.) and trial experiments with model reactions indicated that this could be coupled to ~upport-bound thymidine and oxidized to produce the diester 3a. Other methods can be used to prepare 3~. For example, 1 can be converted into an H-phosphonate derivative which can be coupled to a support bound nucleoside using st~n~d chemistry known to those in the art.

The reduction of 3a with sodium ~orohydride was investigated to determine the m; ~ m amount of reagent needed for the tritiation experiment and to confirm the identity of the reduction product. Reduction was studied both in solution and on a solid support. Complete reduction in solution could be ob~ine~ using one equivalent of borohydride in ethanol, and evidence for the structure of the re~llc~ product 4a was obt~ n~ from electrospray MS, which showed a molecular ion at 441 for the reduced compound as expected, ~ersus 439 for the ketone starting material 3a. Upon reduction, proton NMR indicated SUBSl ITUTE SHEET (RULE 26) the disappearance of signals as~igned to the acetorhe methyl group at 2.5 ppm as expected for the conversion of a ketone to an alcohol group.

In another embodiment relating to nucleotides as labeling/protecting groups Bl and B2, libraries of non-nucleotide phosphorus ester oligomers were prepared u~ing a ~ v~lltional thymidine derivatized solid support, which after cleavaye from the support results in each member of the library having a thymidylate protecting group attached to its terminus at B2. This thymidine group plays a role as a protecting group in stabilizing the libraries to degradation by the concentrated ammonia conditions required for cleavage from the support. In addition to thymidine, a wide variety of other nucleosides can be u~ed as protecting groups for this purpose. in the absence of such a protecting group, compounds with flexible terminal groups might be expected to undergo degradation by attack of the terminal hy~yl group on the neighboring phosphoryl group to produce a cyclic phosphate interm~;~te followed by ~l~;nAtion of the r~m~n~e~ of the molecule.
Degradation of this type has been ~m~n~trated with oligonucleotides which terminated in ethylene glycol units (Font~n-~l et al . ) .

Another embo~m~nt provides glycolic amide protecting groups B~ and B2. Methods have been developed for the synthesis of other protecting groups which impart stability to the libraries when subjected to ~lkAl ;nF~ degradation.
One approach was to prepare a support which, when treated with ~ n; ~, generates an amide group, and is similar to a reported method for generating 3'-~n~c~ped oligonucleotides ~Hovinen et al. ) . Amino-derivatized controlled pore glass was treated with O-dimethoxytrityl glycolic acid (5, Figure 2) to give the solid supported amide derivative 6, which was detritylated and coupled to a second molecule of 5 to give the diglycolic ester derivative 7. This support was used as a replacement for SUBSTmJTE SHEET (RULE Z6) W O 97128168 P~lA~7/01060 the thymidine derivatized support ~or the synthe~is of libraries. A$ter completion of the synthesis, the library was cleaved from the support by reaction with ~mmoni ~, which cleaved the ester group to produce compounds which terminated with glycolic amide functionalities 8, where R
= H. Removal o~ the thymidine groups and substitution with glycolic amide groups in this way results in libraries of smaller molecular weight without bulky protecting groups.

This glycolic acid derivatized support as discussed above can be used to introduce another element of diversity into combinatorial libraries by cleavage using a variety o~
primary or secondary ~m~neS to produce substituted amides.
This is achieved by tr~m~nt of the oligomer attached to the solid support with a variety o~ ~mi n~S to produce oligomers of structure 8, (Figure 2), where R = a wide range of substituted or unsubstituted aliphatic or aromatic groups.

In another variation, the glycolic acid derivatized support can be treated with a ~;Am~ne such as ethylen~ min~ to produce a glycolic amide of structure 9, which can be used to introduce a radioactive label by reductive amination with formaldehyde and tritium labeled borohydride to give a labeled library such as 10, where T
is a tritium atom.

The diversity in this type of oligomer is derived ~rom both the monnr~r units as described in Examples 1-41, as well as the ~mi nP~ which are attached to the phosphorus atoms. The library can be prepared by solid-phase synthesis on a solid support, using a pool and divide strategy employing techniques already reported (Furka et al., 1991).

Known chemical and physical properties o~ the target can be used to dictate the choice o~ m~o~rs that are used in the construction o~ the library. The libraries can be assem~led ~rom a monom~ subset which is weighted to have ~(p SUBSTITUTE SH EET (RULE 26) O 97/28168 PCT~US97/01060 -a high likelihood o~ a~finity ~or the b~ n~; n~ site on the target as possible, thereby increasing the probability of identi~ying a suitable ligand. The weighting o~ the subset can be achieved through the use of molecular mo~l; ng techniques. In the absence of such information, a monomer subset is cho~en to be a~ diverse as is considered desirable. The number of monnm~rs that may be employed for the synthesis of a library is limited by the screening technology used in identi~ying potential ligands.

Example 1 SYntheqi-q of 5'-0-(4,4'-dimethoxYtritYl)-1,2-dideoxy-D-ribose-3'-O-(N,N-~ ov~ lamino-2-cYanoethYl)-~hosPho~ ;te 5'-(4,4'-dimethoxytrityl)-1,2-dideoxy-D-ribose(10.7 g) was prepared according to the method of (Grollman et al., 1987). This DMT derivative (10.7 g, 25 mmol) was dried over P2Os under high vacuum, dissolved in dry dichloromethane (60 mL) under nitrogen, then treated with diisopropylethyl~;nP (13.2 g, 102 mmol) and 2-cyanoethoxy-~N,N-diisopropylAm~no) chlorophosphine (9 g, 38 mmol).
A~ter stirring for 2 hours dry methanol (0.5 mL) was A~
and the reaction mixture was stirred ~or 20 minutes, poured into 5~ aqueous sodium bicarbonate (250 mL), and extracted with ethyl acetate (2 x 200 mL). The organic layers were wA~h~ with saturated aqueous sodium chloride (200 mL), dried overnight over anhydlous sodium sul~ate, concentrated to an oil and coevaporated with toluene (20 mL), ~ollowed by methanol (20 mL), hP~Ane (20 mL), and dichloromethane (20 mL). The crude product (13.79 g) was puri~ied by Normal Phase High Pressure Liquid Chromatography (NP-HPLC) eluting with a gradient o~ dichloromethane/hexane (10 to 0~) and then eluting with a gradient o~
dichloromethane/methanol (0 to 2~). The purities of the fractions were monitored by TLC and ~ractions cont~;n~ng pure material were combined and evaporated to dryness to give 5'-0-(4, 4'-dimethoxytrityl)-~,2-dideoxy-D-ribose-3'-SUBSTITUTE SH EET (RUl E 26) W O 97/28168 PCT~US97/01060 O - (N, N- diisopropylamino-2-cyanoethyl)-phosphoramidite (13.8 g) as an oil having the following structure:
OCH~

H, C O ~0 _~

N~ O
J~ ~
~C
IH NMR (500 M~z) DMSO ~ (ppm) 7.38-6.86 (m, 13H, aromatic), 5.74 (s, lH), 4.32-4.26 (m, lH~, 3.94-3.44 (m, 3H~, 3.72 (s, 6H, O~EI3), 3.04-2.93 (m, 2H), ~.75-2.71 (m, 2H, CEI2), ;~.64-2.61 (m, 2H, CH~), 2.09-2.03 (m, 2H, CH), l.g5-1.91 (m, lH, H-~''),1.87-1.83 (m, lH, H2'), 1.15-0.95 (m, 12H, 4 x: CH3). 31p NMR (202 MHz) DMSO ~ (ppm) 147.5, 147.3.
Ex~m~le 2 ~Ynthe~is of 1-(4,4'-dimetho~yL~ityl)-Propanediol-3- (N,N-diiso~roPylA~n;n~-2-cyanoethyl) -PhosPhoramidite 1-(4,4'-dimethoxytrityl)-1,3-propanediol (6.8 g) was prepared by the method of Seela and Kaiser (1987).
This DMT derivative (6.8 g, 18 mmol) was dissolved in dry dichloromethane (35 mL) under nitrogen and treated with diisopropylethylamine (9.3 g, 72 mmol), and 2-cyanoethoxy-(N,N-diisopropylamino)chlorophosphine (6.4 g, 27 mmol). After stirring for 30 minutes, the mixture was concentrated and 5~ aqueous sodium bicarbonate (250 mL) was added. The mixture was extracted with ethyl acetate ~3 x 200 mL), the organic layers were washed with SUBSTITUTE SHEE'r (RULE 26) W O 97/28168 PCT~US97/01060 saturated aqueous sodium chloride (200 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product (12 g) as an oil. This was purified by NP-HPLC eluting with a gradient of dichloromethane/hP~An~
(50 to 2596). The purities of the fractions were monitored by TLC and fractions cont~i n i ng pure material were combined and evaporated to dryness to give l-(4, 4'-dimethoxytrityl3-1,3-propanediol-3-(N,N-diisopropylamino-2-cyanoethyl~-phosphoramidite (10.3 g) as a yellow oil having the following structure:
OCH~

O~--O--C~--OCH~

IH NMR (500 M~z) DMSO ~ (ppm) 7.37-6.83 (m, 13H, aromatic3, 3.72 (s, 6E, O~EI3), 3.69-3.60 (m, 2H, CH2-3), 3.50-3.44 (m, 2H, (~EI2), 3.09-3.01 (m, 2H, (~I2). 2.68 (t, 2E, .:r=5.8 Hz, CE2-l), 1.84-1.79 (m, 2~I, CH2-2), 1.10 (d, 6H, J=6.8 Hz, 2 x CH3), 1.03 (d, 6H, IJ = 6.8 Hz, 2 x C~H3) . 3~P
NMR (202 MHz) DMSO ô (ppm) 146.9.

Exam~le 3 SYnthesis of 1-(4,4'-dimethox~rtrityl)-3-(4-methox~h~n n-ry ) -1,2-pror~Anl~;ol-2-(N,N-diis~ Ylamino-2-cyanoethyl) Phosphoramidite 3-(4-Methoxyphenoxy)-1,2-propanediol (4 g, 20 mmol) in dry pyridine (50 mL) was treated with DMT
chloride (8.2 g, 24 mmol), triethylamine (2.9 g, 28 mmol), and dimethylaminopyridine (120 mg, 1 mmol) and stirred at room temperature for 16 hours. The mixture was poured into 5Y6 a~ueous sodium bicarbonate (250 mL), ~C~

SUBSTITVTE SH ~ RULE 26) W O 97/28168 PCTrUS97/~1060 extracted with dichloromethane (3 x 200 mL). The combined organic layers were washed with saturated agueous sodium ch~oride (25~ mL) and dried over anhydrous sodium sul~ate. The solid was filtered off and the ~iltrate was evaporated to dryness and coevaporated with toluene, methanol, hexane, and dichloromethane to give crude produc~ which was purified by NP-HPLC eluting with a gradient o~ dichloromethane/hP~An~ (90 to 0~). The purities of the frac~ions were monitored by T~C and ~ractions cont~; ni ng pure material were combined and evaporated to dry~ess to give 1-(4,4'-dimethoxytrityl)-3-(4-methoxyrhenoxy)-1,2-pror~ne~;ol (9.7 g) as a pale yellow oil having the following structure:
OCH~

0~0--C:~OCH, OH ~3 OC~1, IH NMK (500 MHz) DMSO ~ (ppm) 7.39-6.80 (m, 13H, aromatic~, 5.09 (d, lH, J=4.7, OH), 3.96-3.87 (m, 3H, CH2 & CH), 3.72 (s, 6H, 2 x OCH3), 3.68 (s, 3H, OCH3), 3.06-3.03 (m, 2H, CH2). TLC R~ 0.16 (9:1 dichloromethane/methanol).

This DMT derivative (3.25 g, 6.5 mmol) was dissolved in dry dichloromethane (20 m~) under nitrogen and treated with diisopropylethylamine (2.5 g, 19.5 mmol), and 2-cyanoethoxy-(N,N-diisopropyl ~m~ no)chlorophosphine (2.3 g, 9.7 mmol). A~ter stirring for 1 hour, the mixture was poured into 5~
aqueous sodium bicarbonate (200 m~). The mixture was extracted with ethyl acetate (3 x 200 mL), the organic layers were washed with saturated aqueous sodium chloride SUBSmUTE SHEET (RULE ~6) W O 97/28168 P~~ 7/01060 (200 mL), dried over anhydrous ~;odium sulfate, and concentrated to give the crude product (4.8 g) as an oil.
This was purified by NP-HPLC eluting with a gradient of dichloromethane/hexane (90 to 0%). The puritie~; o~ the ~ractions were monitored byTLC and ~ractions cont~,ning pure material were combined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-3-(4-methoxyph~noxy-1,2-propanediol-2-(N,N-diisopropyl ;~mi no-2-cyanoethyl)-phosphoramidite (3.4 g, 4.9 mmol) as a colorle~;~ oil having the ~ollowing structure:
OCH, ~CO~

o~O--C~OCH, 1.~ fo ~3 NC
IH NMR (500 MHz) DMSO ~ (ppm~ 7.40-6.77 (m, 13H, aromatic), 4.21-3.95 (m, 4H,), 3.72 ~ 3.71 (s, 6H, OCH3,2 diastereomers), 3.68 & 3.67 (s, 3H, OCH3,2 diastereomers) 3.66-3.46 (m, 4H, CH2-3), 3.22-3.09 (m, 3H, CEI2), 2.72 (t, lH, J=5.8 Hz, ), 2.63 (t, lH, ~=5.6 Hz), 1.22-0.92 (m, 12H, 4 x CH3). 31p NMR (202 MHz) DMSO ~ (ppm) 149.6, 149.2.

ExamPle 4 SYnthe~is Of 1- (4,4'-dimethoxytrityl) -3- (diethylr ;no) -1,2-~ror)~ 1;ol-2-(N,N-diisv,~v~lamino-2-cyanoethyl)-PhosPhoramidite - 3-(Diethylamino)-1,2-propanediol (5 g, 34 mmol) in dry pyridine (100 mL) was treated with DMT chloride (13.8 g, 41 mmol), triethylamine (4.8 g, 48 mmol), and SUBS 111 UTE SHEET (RULE ~6) CA 02244924 l998-07-30 dimethyl;~m~ nopyridine (207 mg, 1.7 n~nol) and stirred at room temperature for 4 hours. The mixture was - concentrated by rotary evaporation, dissolved in dichloromethane (200 mL) and poured into 5~ aqueous sodium bicarbonate (250 mL). The layers were separated and the aqueous layer was extracted with dichloromethane (2 x 200 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (250 mL) and ~ried over anhydrous sodium sulfate. The solid was filtered off and the filtrate was evaporated to dryness and coevaporated with toluene, methanol, h~AnP, and dichloromethane to give crude product (20 g) which was purified by column chromatography on silica gel (300 g) eluting with a gradien~ of dichloromethane/methanol (O to 4%). The purities of the fractions were monitored by TLC
and ~ractions cont~ n~ ng pure material were com~ined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-3-(diethyl2m~no)-1,2-propanediol (14.9 g) as a brown oil having the ~ollowing structure:

--N~--O--C--~30CH~
OH

'H NMR (500 MHz) DMSO ~ (ppm) 7.42-6.50 (m, 13~, aromatic), 4.47 (br s, lH, OH), 3.72 (s, 6H, 2 x OCE~3), 3.65 (br s, lH, CH-2) 2.95-2.88 (m, 2H, CH2- 3), 2.40-2.39 (m, 4H, 2 x N-CH2), 2.35-2.28 (m, 2H, CH2-l), 0.84 (t, 6H, ~= 6.9 Hz, 2 x CH3). TLC Rf 0.4 (9:1 dichloromethane/methanol).

This DMT derivative (0.5 g, 1.1 mmol) was dis~olved in d~y dichloromethane (4 mL) under nitrogen and treated wi~h diisopropylethylamine (0.43 g, 3.3 SVBSTITUTE SHEET (RULE 26) W O 97128168 PCT~US97/01060 mmol), and 2-cyanoethoxy-(N,N~
diisopropylamino)chlorophosphine (0.39 g, 1.7 mmol).
After stirring for 2.5 hours, dry methanol (0.5 mL) was added and the mixture wa~ evaporated to dryness, dissolved in 5~ aqueous sodium bicarbonate, and extracted with ethyl acetate (3 x 75 mL). The organic layers were washed with saturated aqueous sodium chloride (100 m~) and dried over anhydrous sodium sul~ate. The solid was ~iltered o~ and the mixture evaporated to dryness to give crude 1-(4~4~-dimethoxytrityl)-3-(diethyli~mino)-1~2-propanediol-2- (N,N-dii SO~l ~ylamino-2-cyanoethyl)-phosphoramidite ~0.79 g) as an oil having the ~ollowing st~ucture: OCH, N
~ O-~ ~ OCH~

1 N C~ 13 IH NMR (500 MHz) DMSO ~ (ppm) 7.48-6.9~ (m, 13H, aromatic), 4.~7-2.26 (m, llH,), 3.72 (s, 6H, 2 x OCH3), 2.43-2.26 (m, 4H, 2 x CH2) ,1.25-1.06 (m, 12H, 4 x CH3), 0.86-0.73 (m, 6H, 2 x CH3) . 3~P NMR (202 MHz) DMSO ~
(ppm) 148.3, 17.3, 16.7, lQ.3, 3.7. TLC R~ Q.71 (9:1 dichloromethane/methanol).

ExamPle 5 Synthe~ i8 o ~ 4, 4 ~-dimethoxYtri tyl - t~ans- 9,10-- e~h~nsAnthracene-11,12-dimethnnol-(N,N-dii~o~ropylamino-2-CYanoeth~l)-Phos~horamidite SUBSmUTE SHEET ~RUI E Z6) trans-9,10-Ethanoanthracene-11,12-dimethanol (2 g, 7.5 mmol) in dry pyridine (30 mL) was treated with DMT
chloride (2.5 g, 7.4 mmol), triethyl~mtnp (0.9 g, 8.9 mmol), and dimethylaminopyridine (45 mg, 0.4 mmol) and stirred at room temperature for 1 hour. The mixture wa~
poured into 5~ a~ueous sodium bicarbonate (200 mL). The mixture was extracted with dichloromethane (4 x 50 mL) and the combined organic layers were washed with saturated agueous sodium chloride (2 X 50 mL) and dried over anhydrou~ ~odium sulfate. The solid wa~ filtered of~ and the filtrate was evaporated to dryness and coevaporated with toluene to give crude product (4.9 g) which was puri~ied by column chromatography on silica gel (200 g) eluting with a gradient of dichloromethane/methanol (0 to 3~). The purities o~ the fractions were monitored by TLC and fractions con~ning pure material were combined and evaporated to dryness to give 4,4'-dimethoxytrityl-t~ans- 9,10-eth~no~nthracene-11,12-dim ethanol (2.4 g) as foam having the following structure:
H~CO OC~
c~C~a IH NMR (500 MHz) DMSO ~ (ppm1 7.36-6.82 (m, 21H, aromatic), 4.59 (d, lH, J=5.3, OH), 4.40 (d, lH, J=2.1 Hz, H-9 or 10), 4.26 (d, lH, ~-2.0 Hz, H-10 or 9), 3.713 & 3.707 (~, 6H, 2 x OCH3), 3.01 (m, lH), 2.83 (m, lH), 2.68 (m, lH), 2.29 (m, lH), 1.39 (m, lH), 1.16 (m, lH), 0.92 (m, lH). TLC Rf 0.5 (99:1 dichloromethane/methanol).

~'~

SUBSTITVTE SHEET ~RULE 26) W O 97/28168 PCT~US97/01060 This DMT derivative ~2.3 g, 4 mmol) was dissolved in dry dichloromethane (15 mL) under nitrogen and treated with diisopropylethylamine (2.1 g, 16 mmol), and 2-cyanoethoxy-(N,N-diisopropyl~mino)chlorophosphine (1.3 g, 5.6 mmol) After stirring for 1 hour, dry methanol (0.5 mL) was ~ ~ and the mixture evaporated to dryness. The resulting oil was dissolved in 5~ aqueous sodium bicarbonate (300 mL), extracted with ethyl acetate (300 mL, then 100 mL). The organic layers were washed with saturate~ a~ueous sodium chloride (300 mL), dried over anhydrous sodium sulfate, and concentrated to give the crude product as an oil. This was purified by NP-HPLC
eluting with a gradient of dichloromethane/hexane (80 to 0%). The purities of the ~ractions were monitored by TLC
and fractions cont~ining pure material were combined and evaporated to dryness to give 4,4'-dimethoxytrityl-trans-9,10-ethanoanthracene-11,12-dim ethanol-(N,~-diiso~v~ylamino-2-cyanoethyl)-phosphoramidite (2 g) as a colorless foam having the ~ollowing structure:

~Co OC~
~CN

~;.n--O ~

IH NMR (500 MHz) DMSO ~ (ppm) 7.36-6.80 (m, 21H, aromatic), 4.41-4.25 (m, 2H, H-9, 10), 3.71 & 3.70 (s, 6H, 2 x OCH3), 3.69-3.63 (m, 2H) 3.56-3 48 (m, 3H), 3.27-3.25 (m, lH), 3.18-3.16 (m, lH), 2.85-2.80 (m, 2H), 2.76-2.68 (m, 3H), 2.39 (m, lH), 1.42 (m, lH), 1.36 (m, 2H), 3~

SUBSTITUTF S1t EEl- (RULE 26) W O 97/28168 PCT~S97/01060 1.22-0.98 (m, 12H, 4 x ~3). 3~P NnMR ~202 MHz) DMSO

(ppm) 147.1, 146.7.

ExamDle 6 sYm t~esis of 1-(4~4~-dimetho ~ tritYl)-3-(N-benzYl-N-mçth~lamino)-1,2-~roPaIlediol-2-(N,N-diisc"~ Ylamino-2-cYanoethyl)-PhosPhoramidite 3-(N-Benzyl-N-methyl~;no)-1,2-propanediol (5 g, 26 mmol) in dry pyridine (50 mL) was treated with DMT
chloride ~10.4 g, 31 mmol), triethyl~mine (3.6 g, 36 mmol), and dimethylaminopyridine (160 mg, 1.3 mmol) and stirred at room temperature for 2 hours. The mixture was poured into 5~ aqueous ~odium bic~hon~te (300 mL). The layers were separated, the aqueous layer was extracted with dichloromethane (3 x 200 mL), the combined organic layers were washed with saturated aqueous sodium chloride (300 mL), and dried over anhydrous sodium sulfate. The solid was ~iltered off, the filtrate was evaporated to dryness, and coevaporated with toluene and dichloromethane to give crude product (14 g) which was puri~ied by column chromatography on silica gel (300 g) eluting with a gradient of dichloromethane/methanol (0 to 4~). The purities o~ the fractions were monitored by ~LC
and ~ractions cont~;n~ng pure material were combined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-3-(N-benzyl-N-methyl)-1,2- propanediol (12.1 g) as a brown oil having the following structure:

OCH~

----N ~ O--C~ OCH, 3~-SUBSTtTUTE SHEET ~RULE 26) W O 97/28168 PCTnUS97/01060 'H NMR (5Q0 MHz) DMSO ~ (ppm) 7. 4~-6 .85 (m, 18H, aromatic), 4.64 (d, lH, J=4.8 Hz, OH), 3.81 (q, lH, J=5.3 Hz, CH-O), 3.71 & 3.70 (s, 6E, OCH32 diastereomers), 3.72(s, 6H, OCH3), 3 . 46 (AB quartet, lH, J~=13.3 Hz, benzylic ~), 3 . 38 (AB quartet, lH, JAR=13 . 3 Hz, benzylic H), 2.94 (m, 2H, CH~-N), 2 . 36 (m, 2H, CEI2-O), 2 . 08 (S , 3H, N-CH3). TL~ R~ 0.6 (9:1 dichloromethane/methanol).
This DMT derivative (2.4 g, 4.8 mmol) was dissolved in dry acetonitrile (5 mL) under nitrogen and treated with diisopropylamine (0 . 73 g, 7 . 2 mmol), tetrazole (340 mg, 4.8 mmol), and 2-cyanoethoxy-(N,N,N,N-tetraisopropylamino3phosphine (3.63 g, 12.5 mmol). A~ter stirring ~or 16 hours at room temperature, the mixture was evaporated to dryness, dissolved in ethyl acetate (200 mL) and washed with 5~ aqueous sodium bicarbonate (200 mL). The layers were separated and the organic layer was wa~hed with saturated aqueous sodium chloride (200 mL) and dried over anhydrous sodium sul~ate. The solid was ~iltered o~f and the mixture evaporated to dryness to give the crude product (5.4 g) as a yellow oil which was purified by NP-HPLC eluting with a gradient o~
dichloromethane/h~n~ (20 to 0~). The purities of the fractions were monitored by TLC and ~ractions cont~; ni ng pure material were com~;nP~ and evaporated to dryness to give 1-(4,4'-dimethoxytrityl~-3-(N-~enzyl-N-methylamino~-1,2-pror~n~-l;ol -2-(N,N-diisopropy~ no-2-cyanoethyl)- phosphoramidite (3.3 g) as an oil having the ~ollowing structure:
OC

--N~--O--C~OCH~

1 N ' ~

NC
3;~

5UBSTITUTE SHEET (RULE Z6) W O 97/28168 PCTrUS97/01060 'H NMR (5~0 M~z) DMSO ~ (ppm) 7.42-6.77 (m, 18H, aromatic), 4.08-3.06, (m, llH), 3.73 (s, 6H, 2 x OCH3), 2.78-2.42 ~m, 4H, ~ x CH2) ,1.25-1.06 (m, 12H, 4 x CH3), O.86-0.73 (m, 6H, 2 x CH3). 3Ip NMR (202 MHz) DMSO ~
(ppm) 148.6, 148.5, 140.2, 139.~, 124.1 TLC Rf 0.92, 0.82 (9:1 dichloromethane/methanol).

ExamDle 7 S ~ thes i5 0 f 2 - ( 4, 4 ' - dimethox~rtri tYl)- 2, 6 - biE;-r o~ ~methYl-PYridine-6-~N,N-diiQo.. ~ lamino-2-cvanoethYl)-phosPh~ ;dite 2~6-bis-h~dlo~y,..ethylpyridine (2.1 g, lS mmol) in dry pyridine (50 mL) was treated with DMT chloride (1 g, 3 mmol), triethylamine (0.37 g, 3.6 mmol), and dimethyl ~m; noryridine (20 mg, O.15 mmol) and stirred at ~oom temperature for 16 hours. The mixture concentrated by rotary evaporation and poured into 5% a~ueous sodium bicarbonate (200 mL), then extracted with dichloromethane (3 x 200 mL) and the c~mh~ne~ organic layers were w~
with saturated sodium chloride solution (200 mL~ and dried over anhydrous sodium ~ul~ate. The solid was filtered off and the filtrate was evaporated to dryne~s and coevaporated with toluene and dichloromethane to give crude product which was purified by column chromatography on silica gel (300 g) eluting with a gradient of dichloromethane/methanol (O to 4%). The purities of the fractions were monitored by TLC and ~ractions ~ont~i n; n~
pure material were c~i ne~ and evaporated to dryness to give 2-(4,4'-dimethoxytrityl)-2,6-bis-hydroxymethylpyridine (3.8 g) as an oil having the following structure:

3~

SUBSTITUT~ SH EET (RULE 26) W O 97/28168 PCTrUS97/01060 H~CO ~ C- O ~ OH

IH NMR (500 MHz3 DMSO ~ (ppm) 7.87-6.90 (m, 16H, aromatic), 5.29 (s, lH, OH), 4.44 (s, 2H, CH2), 4.06 (s, 2H, CH2), 3.73 (s, 6H, 2 x OCH3). TLC Rf 0.68 (9:1 dichloromethane/methanol).
This DMT derivative (3.3 g, 5.2 mmol) was dissolved in dry acetonitrile (65 mL) under nitrogen and treated with tetrazole (0.33 g, 4.6 mmol), diisopropylamine (0.68 g, 6.7 mmol), and Z-cyanoethoxy-(N,N,N,N-tetraisopropyl ~mi n~) phosphine (3.3 g, 10.9 mmol). A~ter stirring for 30 minutes, additional diiso~ylamine (0.7 g, 6.7 mmol), and 2-cyanoethoxy-(N,N,N,N-tetraisopropyl ~mi nn) -phosphine (1.8 g, 6.2 mmol) was A~A~A and the reaction mixture was stirred for 1 hour. The mixture was poured into 5~ a~ueous sodium bicar~onate (250 ml-), extracted with dichloromethane (2 x ~00 m~, 2 X 2~0 ml-~ 6 X 50 ml,), the organic layers were w~he-l with saturated aqueous sodium chloride (250 mL), dried over anhydrous sodium sul~ate, and concentrated to give the crude prodùct (7 g).as an oil. This was purified by NP-HPLC eluting with a gradient of dichloromethane/h~ne (10 to 0~). The purities of the fractions were monitored ~y TLC and ~ractions con~i n~ ny pure material were cnmh~n~d and evaporated to dryness to give 2-(4,4'-dimethoxytrityl)- 2,6-blS-hydroxymethylpyridine-6-(N,N-diisopropyl ~minQ-2-cyanoethyl)-phosphoramidite (5.4 g) as a pale yellow oil having the ~ollowing structure:

3q SUBSTITUTE SH EFr tRULE 26) W O 97128168 PCTrUS97/01060 H~Co~3C--oJ~N~ o~P~ 1 'H NMR (500 MHz) DMSO ~ (ppm) 7.91-6.20 (m, 16H, aromatic), 4.64 (m, 2~, CH2-6), 4.09 (s, 2H, CE2-2), 3.73 (s, 6H, 2 x OCH3), 3.82-3.46 (m, 6H, CH2& CH), 1.24-1.10 (m, 12H, 4 x CH3) . 31p NMR (202 MHz) DMSO ~ (ppm) 148.6, 139.1, 123.6. TLC R~ 0.77, 0.68 (1:1 ethyl acetate/dichloromethane).

Example 8 Synthe~is of 1-(4,4'-dimetho~Llityl)-2-N-tri~luoroacetyl-2-amino-1,3-propanediol-3-(N,N-dii~oPropYlamino-2-CYanoethYl) ~ho~horami~ite Serinol (1 g, 11 mmol) in dry dichloromethane (10 mL) and dry pyridine (2.7 mL) was cooled to -78 C. on a dry ice/acetone bath and treated with tri~luoroacetic anhydride ~or 1 hour. The reaction mixture was evaporated to dryness and coevaporated with toluene, methanol, hexane, and dichloromethane to give crude 2- N-tri~luoroacetamido-serinol (4.2 g) as an oil with the ~ollowing structure:

HO ~ OH

NH

0~

An analytical sample (100 mg) was puri~ied by column chromatography on neutral alumina eluting with isocratic me~hanol SU85T~TUTE SHEET (RULE 26) WO97/28168 PCT~S97/01060 -'H NMR (500 MHz) DMSO ~ (ppm~ 4.69 (t, 2H, J=s.7 Hz, O~), 3.80 (m, lH, CH), 3.46 (m, 2H, 2 x CH2) 3.17 (d, lH, J=5.2 Hz, NH). TLC R~ 0.44 (7:3 dichloromethane/
methanol3 visualized with ninhydrin spray.

This material (10 3 g, 55 mmol) in dry pyridine (200 mL) was treated with DMT chloride (16.7 g, 49 mmol), triethyl~minP (7.8 g, 77 mmol), and dimethylaminopyridine (335 mg, 2.7 mmol). Additional DMT chloride (3.35, 9.9 mmol), triethyl~mine (1.8 g, 18 mmol), and dimethylaminopyridine ~70 mg, 0.6 mmol) was added a~ter 18 hours and at 26 hours, and the reaction mixture was stirred at room temperature for a total of 90 hours. The mixture was poured into 5~ aqueous sodium ~icarbonate (400 mL) and extracted with dichloromethane (500 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (300 mL) and dried over anhydrous sodium sul~ate. The solid was ~iltered off and the ~iltrate was evaporated to dryness and coevaporated with toluene, methanol, h~ne, and dichloromethane to give crude product (25 g) which was purified ~y column chromatography on silica gel ~280 g) eluting with a gradient o~ dichloromethane/methanol (0 to 3~). The purities of the ~ractions were monitored ~y TLC and fractions cont~ining pure material were combined and evaporated to dryness to give 1-~4,4'-dimethoxytrityl)-2-N-tri~luoroacetamido-serinol (13.5 g) as an having the following structure:

OCH~

H O~ O--C ~} OCH, CH, ~1 SUBS rl~ UTE SHEET (RUI E 26 W O 97/28168 . PCT~US97/01060 IH ~nM~ (500 ~DHz) DMSO ~ (ppm) 9.16 (d, lH, ~= 8.4 Hz, NX), 7.77-6.82 (m, 13H, aromatic), 4.70 (t, lH, J=
5.5 Hz, OHI, 4.08 (m, lH, CH), 3.73 (s, 6H, 2 x OCH3), 3.14-3.11 (m, 2H, CH2), 3.01-2.98 (m, 2H, CH2). Tl,C Rf O.37 (99:1 dichloromethane/ methanol).

This DMT derivative (4.4 g, 9 mmol) was dis801ved in dry dichloromethane (20 mL) under nitrogen and treated with diisopropylethylamine (3.5 g, 27 mmol), and 2-cyanoethoxy-(N,N-diisopropylamino)chlorophosphine (3.2 g, 13.5 mmol). After stirring for 45 minutes the mixture was poured into 5% aqueous sodium bicarbonate (200 mL), and extracted with ethyl acetate (200 mL). The organic layers were washed with saturated a~ueous sodium chloride (200 mL) and dried over anhydrous sodium sul~ate. The solid was ~iltered off and the mixture evaporated to dryne~s to give crude product (7.2 g) which was purified by NP-HPLC using a gradient of dich~oromethane/h~x~n~ (40 to 0%). The purities of the fractions were monitored by TLC and fraction~ cont~; n; ng pure material were com~ined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-2-N-trifluoroacetamido-serinol-3-(N ,N-diisopropyl ~m~ no-2 -cyanoethyl)-pho8phoramidite ~5.2 g) as an oil having the following structure:
NC OCH~

C8~ ~ OCH, IH NMR (500 MHz) DMSO B (ppm) 9.40 (m, lH, NH), 7.36-6.83 (m, 13H, aromatic), 4.22 (m, lH, CH), 3.72 (s, 6H, 2 x OCH3), 3.~7-2.62 (m, 10H, 4 x CH2 2 x CH),1.24-0.85 (m, 12H, 4 x CH3), 0.86-0.73 (m, 6H, 2 x CH3). 31p ' ~

SUBSTITUTE SffEET tRULE ;~6) W O 97128168 PCTnUS97/01060 NMR (202 MHz) DMSO ~ (ppm) 150.3. TLC Rf 0.71 (9:1 trichloroethane/ethyl acetate) -Exam~le 9 Synlthe~is o~ 7-(4,4'-dimetho~rYtrityl)-tra~ls-7~8-dihYdroxy-dimethyl-exo-tric~cl n~ ~n ~n ~ ~ 4, 2 . 1 . 02~5~nona-3-ene-3,4-dicarboxylate-8-(N~N-diis~l~lamino-2-CY ~ oethY

~hosPhor~idite Dimethyl-exo-tricyclo[4.2.1. o2~5] -3,7-diene-3,4-dicarboxylate (1 g, 4.3 mmol) was added dropwise to a mixture of 88~ formic acid (2.6 mL, 59 mmol) and 30~
hydrogen peroxide (0.6 mL, 6 mmol) that had been cooled on an ice bath. The ice bath was removed and the mixture was stirred ~or 18 hours. The mixture was evaporated to dryness, co-evaporated with methanol, toluene, methanol, and dichloromethane. This residue was dissolved in methanol (10 mL), Amberlite~ IR- 120 strongly acidic ion-~C~An~e resin (1 g) was added, and the mixture was refluxed for 2 hours. The solid was filtered o~f and the ~iltrate evaporated to give crude trans-dimethyl-exo-tricyclo[4.2.l.025]-3-ene-7,8-diol-3,4 -dicA~ho~ylate (1.4 g) as a brown oil having the following structure:
H~cO~6~
H~ C~O H

OH
IH NMR (500 MHz) DMSO ~ (ppm) 5.06 (d, lH, J=5.6 Ez, OH), 4.39 (d, lH, J=7.0 Hz, OH~, 3.93 (m, lH, CHO), 3.714 (s, 3H, OCH3), 3.710 (s, 3H, OCH3), 3.65 (m, lH, CHO), 3.02 (m, lH, CH-1 or 6), 2.92 (m, lH, CH-6 or 1), 2.37 (m, lH, CH-2 or 5), 2.21 (m, lH, CH-5 or 2), 1 65 (m, 2H, CH2).

SUBSTITVTE SHEET (RULE 26) W O 97128168 PCT~US97/01060 This material ~1.4 g, 4.3 mmol) in dry pyridine (20 mL) was treated with DMT chloride (1.3 g, 3 9 mmol3, triethyl~m;ne (290 mg, 5.2 mmol), and dimethylamino-pyridine (2 mg, 0.02 mmol) and stirred at room temperature for 4 hours. The mixture was poured into 5 aqueous sodium bicarbona~e (100 mL). The layers were separated and the agueous layer was extracted with dichloromethane ~6 x 30 mL) and the combined organic layers were washed with saturated aqueous sodium chloride (2 x 30 mL) and dried over anhydrous sodium sulfate. The solid was filtered off and the filtrate was evaporated to dryness and coevaporated with toluene, methanol, and dichloromethane to give crude product (2.4 g3 which was purified by column chromatography on silica gel (100 g) eluting with a gradient of dichloromethane/methanol (o to 1%). The purities of the fractions were monitored by TLC
and fractions cont~-ning pure material were combined and evaporated to dryness to give 7~(4,4'-dimethoxytrityl)-trans-7,8-dihydroxy-dimethyl-exo-tricyclononener4.2.1.025]nona-3-ene-3,4-dicarboxylate (1.2 g) as a yellow foam having the following structure:

~Co~O
H~Co~O H

OCH, OC~l~

IH NMR (500 MHz3 DMSO ~ (ppm) 7.36-6.82 (m, 13H, aromatic), 4.65 (d, lH, J=5.0 Hz, OH), 3.76 (m, lH, OCH), 3.724 ~ 3.721 (s, 6H, 2 x OCH3, 2 diastereomers), 3.63 4~

SU~STITUTE SHEET ~RULE 26) (s, 3H, 0~3), 3.47 (m, lH, OCH), 3.38 (s, 3H, OCH3), 2.90 (m, lH, CH-l or 6) 2.77 (m, lH, CH-6 or 1), 2.09 (m, lH, - CH-2 or 5), 2.02 (m, lH, CH-5 or 2), 1.44 (m, lH, CH2), 0.82 (m, lH, CH2) . TLC Rf 0.28 (dichloromethane).
J

This DMT derivative (1.2 g, 2.1 mmol) was dissolved in dry acetonitrile (20 mL) under nitrogen and treated with tetrazole ~150 mg, 2.1 mmol~, diisopropylamine (0.32 g, 3.15 mmol), and 2-cyanoethoxy-~, N, N, N-tetraisopropyl ~mi no~ phosphine (1. 6 g, 5.3 mmol).
A~ter stirring for 21 hours the mixture was evaporated to dryness, dissolved in 5% aqueous sodium bic~hnn~te (125 mL), and extracted with dichloromethane (2 x 100 mL).
The organic layers were washed with water (125 mL) and dried over anhydrous sodium sul~ate. The solid was ~iltered o~ and the mixture evaporated to dryness to give crude product (2.6 g) which was purified by NP-~PLC
eluting with a gradient of dichloromethane/h~ne (60 to o~). The purities o~ the fractions were monitored by TLC
and fractions cont~in;ng pure material were co~h;n~d and evaporated to dryness to give 7-l4,4'-dimethoxytrityl)-trans-7,8-dihydroxy-dimethyl-exo-tricyclot4.2.1.025]nona-3-ene-3,4- dicar~oxylate-8-(N,N- diiso~L~yl~m;n~-2-cyanoethyl)-phosphoramidite (1.4 g) as a colorless ~oam having the ~ollowing structure:
NC

H~C

OCH~

OCHS
~5 SUBSTITUTE SHEET(RULE 26) W O 97/28168 PCT~US97/01060 IH NMR (500 MHz) DMSO ~ (ppm) 7.38-6.80 (m, 13H, aromatic), 3.92 (m, lH, OCH), 3.82 (m, lH, OCH), 3.72 &
3.71 (s, 6H, 2 x OCH3for 2 diastereomers), 3.630 & 3.629 (s, 3H, OCH3for 2 diastereomers), 3.353 fi 3 . 347 (S, 3H, OCH3for 2 diastereomers), 2.96 (m, lH, CH-1 or 6), 2.82 (m, lH, CH-6 or 1), 2.72 (m, 4H, 2 x ~H2), 2.26 (m, lH, H-2 or 5), 2.18 (m, lH, CH-5 or 2), 1.39 (m, lH, CH2), 1.19-1.05 (m, 12H, 4 x CH3), 0.80 (m, lH, CH~). 3'P NMR
(202 MHz) DMSO ~_(ppm) 146.8, 146.4, 123.7. TLC R~ 0.56 (99:1 dichloromethane/methanol).

ExamPle 10 SYnthe3 i8 o~ 1-( 4,4'-dimet~ox~rtritYl~-tra~s-l,2-cYcloPent~n~iol-3-methYlacetate-2-(N,N-diiso~roPylamino-2-csra~oeth~rl) PhosPhosamidite 2-Cyclopentene-1-acetic acid (2.35g, 18.7 mmol) in diethyl ether (5 mL) was cooled on an ice/salt bath and treated with diazomethane (50 mL, 0.46 M) at 0 C. The mixture was concentrated to dryness to give 2-cyclopentene-1-methylacetate (3.1 g) a~ a colorless oil with the following structure:

~_ C~ OClt~

IH NMR (500 MHz) DMSO ~ (ppm) 5.74 (m, lH, vinylic CH), 5.65 (m, lH, vinylic CH), 3.58 (s, 3H, OCH3), 2.95 (m, lH, CH), 2.39-2.18 ~m, 4H, CH~), 1.98 (m, lH, CH~), SU~STITUTE SHEET (RUI E 26) W O 97/28168 P~llu~7lolo6o 1.38 (m, lH, CE2), 1.65 ~m, 2H, CH2). TLC R~ 0.89 ~9:1 - .
dichloromethane/methanol), visualized with ~m~noT~ium molybdate/sul~uric acid.

This material (1.5 g) was added dropwise to a mix~ure of 9696 formic acid (5.6 mL, 150 mmol) and 30~
hydrogen peroxide (O.5 mL, 15 mmol) that had been cooled on an ice/salt bath. The ice bath was removed and the mixture was stirred for 16 hours. The mixture was evaporated to dryness, co-evaporated with toluene, methanol, and hexane. This residue was dissolved in methanol (30 mI~), Amberlite~' IR-120 strongly acidic ion-exchange resin (3 g) was added, and the mixture was re~luxed ~or 2 hours. The solid was filtered o~ and the filtrate evaporated to give crude trans-1,2-cyclopent~n-~-1iol-3-methylacetate (1 g) as an oil having the following ~;tructure:

O~ ~ C- OCH, 1/ \1 OH

1H NMR (500 MHz) DMSO ~ (ppm) 4.90 (br s, 2H, OH), 4.57 (m, lH), 4.04 (m, lH), 3.57 (s, 3H, OCH3), 2.91 (m, lH), 2.78 (m, lH) 2.22 (m, lH), 2.03 (m, lH, CH2), 1.68 (m, lH, ~I2), 1.53 (m, lH, CH2), 1.42 (m, lH, CH2).

This material (1 g, 6 mmol) in dry pyridine (20 mL~ was treated with DMT chloride (2.4 g, 7.2 ~nol), triethyl~m;n~ (0.85 g, 8.4 mmol), and dimethyl;~minopyridine (37 mg, 0.3 mmol) and stirred at ~ room temperature for 4 hours. The mixture was concentrated by rotary evaporation, dissolved in dichloromethane (200 mL) and poured into 596 aqueous sodium bicarbonate (200 mL). The layers were separated and the aqueous layer was extracted with dichloromethane SUBSTITUTE SHEET (RULE 26) W O 97/28168 PCT~US97/01060 (2 x 200 mL) and che combined organic layers were washed with saturated a~ueous sodium chloride (250 mL) and drled over anhydrou~ sodium sulfate. The solid was filtered o~ and the filtrate was evaporated to dryness and coevaporated with toluene, methanol, hexane, and dichloromethane to give crude product (3.3 g) which was puri~ied by column chromatography on silica gel (150 g) eluting with a gradient of dichloromethane/methanol (0 to 2~) The purities of the ~ractions were monitored by TLC
and f ractions cont~ini ng the de~ired material (~20 mg) were combined, evaporated to dryness and repuri~ied ~y NP-HPLC eluting with i~ocratic dichloromethane. The purities of the fractions were monitored by TLC and fractions cont~ining the pure material were combined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-trans-l,2-cyclopentanediol-3-methyl acetate ~260 mg) as a pale yellow oil having the following structure:

O H ~ C--OCH~
V ~' ~3C~ C)CHs OCH~
IH NMR (500 MEz3 DMSO ~ (ppm) 7.47-6.83 (m, 13H, aromatic), 4.65 (d, lH, J=5.5 Hz, OH), 3.73 (s, 6H, 2 x OCEI3), 3.58 (m, 2H), 3.55 (s, 3H, OC~H3), 3.49 (m, 2H), 2.54 (m, 2H) 2.20 (m, 2H), 1.81 (m, 2H), 1.50 (m, 2H), 1.14 (m, 2H), 0.87 (m, 2H), 0.77 (m, 2H). TLC Rf 0.32 (9:1 ~richloroethane/ethylacetate).

T~i~ DMT derivati~e (260 mg, 0.6 mmol) was dissol~ed in dry dichloromethane (~ mL) under nitrogen and treated with diisopropylethyl~mine (0.23 g, 1.8 mmol), and 2-cyanoethoxy-(N,N-SUBSTITUTE SH EET (RULE 26) ~ r WO 97/28168 PCT~US97/01060 diisopropylamino)chlorophosphine (0.21 g, 0.9 mmol).
A~ter stirring for 15 minutes the mixture was evaporated to dryness, dissolved in ethylaceta~e (75 mL), washed with 5~ aqueous sodium bicarbonate (100 mL). The aqueous layer was extracted with ethylacetate (2 x 75 mL). The organic layers were washed with saturated a~ueous sodium chloride (100 mL) and dried over anhydrous sodium ~ulfate. The solid was filtered of~ and the mixture evaporated to dryness to give crude product (540 mg) which was purified by NP-HPLC eluting with isocratic dichloromethane/h~nP (6:4). The purities of the ~ractions were monitored by TLC and ~ractions cont~ini n~
pure material were combined and evaporated to dryness to give 1-(4,4'-dimethoxytrityl)-trans-l,Z-cyclop~nt~n~ol-3- methyl acetate-2-(N,N-diisopropyl~mino-2-cyanoethyl)-phosphoramidite (105 mg/fast diastereomer & 144 mg/slow diastereomer) as a colorless oil ha~ing the ~ollowing CN
structure: ~
1ol C- OCH~

C ~ OCH, OCH~
IH NMR (500 MHz) DMS0 fast ~ (ppm) 7.43-6.86 (m, 13H, aromatic), 3.84 (m, 2H), 3.76 (m, lH), 3.75 (m, lH), 3.73 (s , 6H, 2 x OCH3), 3.57 (s , 3H, OCH3), 3.56 -3.45 (m, 5H), 2.63 -2.62 (m, lH), 2.61-2. S8 (m, 2H), 2.56-2.53 (m, lH), 2.39 (d, lH, J=9.5 Hz), 2.38 (d, lH, J=9.5 Hz), 2.14 (m, 2H), 1.69 (m, 3H), 1.32 (m, 3H, CH2), 1.23 (m, 3H), 1.11 (d, 6H, J=6.8 Hz, 2 x CH3), 1.06 (d, 6H, J=6.8 Hz, 2 x CH3) . 3~P NMR (202 MHz) DMSO ~ (ppm) 148.8 ~ast.

SUBSTmJTE SHEFr (RULE 2fi) W O 97/28168 PCTrUS97/01060 IH ~nMR (500 ~DHz) DMSO slow ~ (ppm) 7.43-6.84 (m, -13H, aromatic), 3.8~3-3.75 (m, 3H), 3.72 (s, 6H, 2 x OCH3), 3.65-3.59 (m, 2H), 3.57 (s, 3H, OCH3), 3.56-3.45 (m, 4H), 2.72 (m, 2H), 2.69 (m, lH), 2.64-2.52 ~m, 3H), 2.41-2.34 (m, 2H), 2.15 ~m, 2H), 1.66 (m, 3H), 1.24 (m, 5H), 1.14-0.98 (m, 12H, 4 x CH3). 3~P NMR (202 MHz) DMSO
(ppm) 148.3. TLC R~ 0.61 (9:1 trichloroethane/ethylacetate).

Exam~le 11 Synthesis of 2-(4,4'-dimethoxYtritYl)-hY~y ~thyl-phenol-1-(N,N-diisG~ lamino-2-cYanoethyl)-PhosPhoramidite 2-Hydroxybenzyl alcohol ~5 g, 40.3 mmol) in dry pyridine (100 mL) was treated with DMT chloride (16.4 g, 48.3 mmol3, triethy-~m~ne (5.5 g, 54.5 mmol), and dimethylAminopyridine (244 mg, 2 mmol) and stirred at room temperature for 19 hours. The mixture was poured into 5~ aqueous sodium bicarbonate (500 mL), extracted with dichloromethane (4 x 100 mL). The combined organic layers were washed with saturated a~ueous sodium chloride (100 mL) and dried over anhydrous sodium sulfate. The solid was filtered o~f and the filtrate was evaporated to dr~ness to give the crude product (20 g) which was purified by column chromatography on silica gel (300 g) eluting with a gradient or 1,1,1-trichloroethane/ethyl acetate (0 to 2%). The purities o~ the ~ractions were monitored by TLC and fractions cont~ining the desired material were combined and evaporated to dryness to give 2-(4,4'- dimethoxytrityl)-hydroxymethylphenol (25.7 g) as a pale yellow oil having the following structure:
oc-~, OH 1~j3 O--C~OCH, 5C~
SUBSTITVTE SHEET ~RULE 26) W O 97/28168 PCTnUS97/01060 IH NMR (5Q0 MHz) DMSO ~ (ppm) 9.39 (s, lH, phenoiic OH), 7.59~6.72 (m, 17H, aromatic~, 4.01 (s, 2H, benzylic CH2), 3.72 (s, 6H, 2 x OCH3), 3.68 (s, 3H, OCH3). TLC R~
0.68 (1,1,1-trichloroethane).

This DMT derivative (4 g, 10.5 mmol) was dissolved in dry dichloromethane (25 mL) under nitrogen and treated with diisopropylethyl~min~ (6.7 g, 52 mmol), and 2-cyanoethoxy-(N,N-diisopropylamino)chlorophosphine (3.2 g, 13.5 mmol). After stirring for 1 hour, additional 2-cyanoethoxy-(N,N-diisopropyl ~m; n~) - chlorophosphine (1 1 g, 4.2 mmol) wa~ added and the mixture was stirred ~or 2 hours. Ethyl acetate (300 mL) and methanol (1 mL) were added and the mixture was wa~hed with 10 ~ aqueous sodium carbonate (2 x 200 mh), agueous sodium chloride (2 x 200 mL), add the organic layers were dried over anhydrous sodium sul~ate. The solid was ~iltered o~ and the ~iltrate was evaporated to dryness and coevaporated with toluene to give the crude product. This was purified column chromatography on silica gel (200 g) eluting with dichloromethane. The purities of the ~ractions were monitored by TLC and fractions contAintng pure material were combined and evaporated to dryness to give 2-(4,4'-dimethoxytrityl)- l~Lu~methylph~nol-l-(N,N-diisopropylamino-2-cyanoethyl)-phosphoramidite (4.2 g, 6.8 mmol) as an oil having the ~ollowing structure:
HC
OCH~
~P~O ~ -~--O--C ~ OCH, ~3 IH NMR (500 MHz) DMSO ~ (ppm) 7.75-6.75 (m, 17H, aromatic), 4.10 (m, 2H), 4.01 (s, lH, benzylic CH2), 3.72 SUBS 11 l UTE SHEET (RULE ~

W O97/28168 PCT~US97/01060 (s, 6H, OCH3), 3.71-3.60 ~m, 2H), 3.49 (m, 2H), 2.69 (m, 2H), 1 . 09 ~d, 6H, J=6 . 7 Hz, 2 x CH3), 0 . 90 (d, 6H, J=6 . 7 - Hz, 2 x CH3) . 31p WMR (202 MHz) DMSO a (ppm) 145.2. TLC
R~ 0.57, 0.49 (99~ Cl2/MeOH) .

ExamPle 12 Q~- (2-CYanoethYl-N,N-DiisG,~o~yl-Pho~phoramidite~ -o2- (4, 4 ' -D~methox~trityl~ -2 -HYdroxYethanol 2-Cyanoethyl-N,N-Diisopropylchlorophosphoramidite (O.68 mL, O.04 mmol~ was added dropwise to a stirred solution of o2- ~4,4' -dimethoxytrityl)-2- hydroxyethanol (1.3 g, 2.76 mmol) in anhydrous THF (15 mL~ contAin~ng triethyl ~mi n~ ~0.84 ml., 6.1 mmol). On addition, the mixture was stirred at RT ~or 30 min, and then ~iltered through a sintered glass ~unnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dissolved in anhyrous benzene and then filtered. The ~iltrate was evaporated in vacuo, and the re~idue dissolved in an ethyl acetate-he~ne mixture (3:7) and applied to a flash column. The product was eluted using 30~ ethyl acetate in he~ne ~ont~;ning 1~ triethyl~m;n~, and homogenous ~ractions were combined and evaporated to give the product as an almost colorless oil, (1.53 g, 82~) having the ~ollowing structure:
b NC~-- 'P' ~~--~ O
N(lPr)2 Ç3 octl, 31p NMR ~ 145 . IH NMR ~ 7.~0-6.80(13H, m aromtic), 3.70 (6H, s, OCH3), 3 . 85- 3 .60 (4H, m, CH?ODMT~CH,?OP+CH2CH2CN) , 3 . 16 ( lH, m, CH?CN~, 3.06( lH, m, SUBSTITVTE SHEET (RULE 26) W O 97/28168 PCT~US97/01060 C~I2CN), 2.74(2H, m, C~I(C~I3)2), 1.13(12H, m, C~I3). Rf =
0.35 30~ Ethyl acetate in h~An~

ExamPle 13 ol-(2-CYs~oetb-yl-N~N-Diiso~ rl-Dhos~hosamidite)-(4~4~-D~methoxytrityl)-l~2-Dihydroxycyclopentane Dimethoxytrityl chloride t3.38 g, lOmmol) was in portions to a ~tirred solution of the cyclop~nt~n~l (5 g, 49 mmol) in anhydrous pyridine (300mL) con~n~ng DMAP (20 mg~. On addition, the mixture was le~t to stir at RT overnight a~ter which the pyridine was removed under vacuum, and the residue adsorbed onto silica gel. This mixture was then applied to a ~lash silica gel column and the product eluted using 25~ ethyl acetate in hP~n~. Homogenous ~ractions were co~h;n~d and evaporated in vacuo, to give (+/_)_o2_4,4,_ dimethoxytrityl-1,2- dih~Lu~yclop~nt~ne as a yellow solid. R~ = 0.29 (25~ Ethyl acetate in h~An~).

2-Cyanoethyl-N,N-Diis~Lu~ylchlorophosphoramidite (1.4 mL, 6.24 mmol) was added d~ ise to a stirred solution o~ the above DMT derivative (2.29 g, 5.67 mmol) in anhy-~lous ~HF ~20 mL) csnt~n;ng triethyl~mtne (1.56 mL, 11.34 mmol). On addition, the mixture was stirred at RT for 60 min, and then fi~tered through a scintered glass ~unnel under nitrogen. The ~iltrate was evaporated under vacuum, and the residue dis~olved in anhydlous benzene andthen ~iltered. The filtrate was evaporated in ~acuo, and the residue dissolved in an ethyl acetate/h~nP mixture (3:7) and applied to a flash column. The product was then eluted using 30% ethyl acetate in h~Ane con~n~ng 1% triethylamine, and h~...~y~llous ~ractions were combined and evaporated to give the product as an almost colorless oil, (1.84 g, 57%) having the ~ollowing structure:
rj~

SU~STITUTE SHEFr ~RULE 26) W O 97/28168 PCTrUS97/01060 O ''' O Q ~ (~ -N(lPr)2 ~ ~

31P NMR ~ 147. 'H NMR ~ 7.50-6.80(13H, m, aromatic), 3 .93 (lH, m, CEIOP), 3. 82(lH, m, ~OL.1T), 3 .72(6H, m, OCH3), 3.58(2H, m, CH2OP), 3.49(2H, m, CH2~), 2.72(1H, m, NCH), 2.66(lH, m, NCH), 1.88(1II, m, alicyclic~, 1.52(lH, m, alicyclic), 1.41(1~I, m, alicyclic), 1.21(lH, m, alicyclic), 1.06(12H, m, CH(5~3) 2)~ 0 .92(lH, m, alicyclic), 0.82(1~I, m, alicyclic). Rf = 0.47 (30~ Ethyl acetate in hexane).

ExamPle 14 Q1-(2-C~fanoethYl-N~N-Dii~G~ ~Yl~ho~n~horamidite)-o2- (4,4'-Dimeth~ yL~itYl) -1,2-DihYdroxs~cYclo-oct~ e Dimethoxytrityl chloride (3.76 g, 11.1 mmol) was l to a stirred solution o~ cis-1,2-dihydroxycyclooctane (8 g, 55.5 Imnol) in anhydrous pyridine (300 mI,) cont~;n;ng DMAP (20 mg). The reaction was left to stir at RT under nitrogen overnight. The reaction mixture was then evaporated under vacuum, and the residue adsorhed onto silica gel and applied to a silica gel :Elash column. The product was eluted using 30%
ethyl acetate in hexane, and homogenous ~ractions comh~n~d and evaporated to give o2-(4~4~-dimethoxytrityl) -cis-l, 2-dihydroxycyclooctane as an almost colorless oil, (3.30 g, 67~).

SU~SllTUTE SH EET (RU~E 26) W O 97/28168 pcTrus97lolo6o IH NMR ~ 7 50-6.80 (13H, m, aromatic), 4.22 (~H, m, - .
OH), 3.72 (6H, m, OCH3), 3.46 (2H, m, CEOH+CHODMT), 1.60-0.90 (12H, m, alicyclic). Rf = Q.38 (30~ ~thyl acetate in hexane).

2-Cyanoethyl-N,N-Diisopropylchlorophosphoramidite (o.54 mL, 2.4 mmol) was added dropwise to a stirred solution of the above DMT derivati~e (0.97 g, 2.18 mmol) in anhydrous THF (20 mL) containing triethyl~mine (0.63 mL, 4.59 mmol). On addition, the mixture was stirred at RT for 60 min, and then filtered through a sintered glass funnel under nitrogen The ~iltrate was evaporated under vacuum, and the residue dissolved in anhyrous benzene and then filtered. The filtrate was evaporated in vacuo, and the residue dissolved in an ethyl acetate/hexane mixture (3:7) and applied to a ~lash column. The product was then eluted using 25~ ethyl acetate in h~x~n~ co~t~n;n~ 1~
triethyl ~m; ne, and homogenous fractions were cornh; n~ and evaporated to give the title compound as an almo~t colorless oil, (1 07 g, 80%) with the following structure: OCH
b NC~--O~p~O ~ O
N~IPr)20 Ç3 OCH~
31p NMR_~ 145. ~H NMR ~ 7.50-6.80 (13H, m, aromatic), 3.72 (6H, s, OCE3), 3.63 (2E, m, CH20P), 3.48 (2E, m, CE2CN), 2.68 (2H, m, NCE), 1.70-1.20 (12H, m, alicyclic), 1.10 (6H, m, CH(CE3)2~, 1.00 (6E, m, CE(CH3) 2) . R~ = 0.27 (25% Ethyl acetate in h~nP) Ex ~ ple 15 SUBSTlTtJTE SH EET (RULE 26) W O 97/28168 PCTrUS97/01060 o2- (2-CYanoethyl-N~N-Diisc~ ",Yl~ho~~horamidite~ -o3- (4,4'-DimethoxYtritYl)-7- (2,3-DihYd~,~Y~ro~Yl) - ~h~orhylline Dimethoxytrityl chloride ~22.7 g, 67 mmol) was ~ in portions to a stirred solution of 7-(2,3-Dihyd~oxy~ropyl)theophylline (10 g, 61 mmol) in anhydrous pyridine (200 mL) con~in;ng DMAP (20 mg). The reaction was left to stir at RT under nitrogen overnight. The reaction mixture was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column. The product was eluted using 3%
methanol in dichloromethane, and hG,.,oyellous ~ractions combined and evaporated to give 7-(03-(4,4'-dimethoxytrityl)-2,3-dih~dLoxypropyl)theophylline as an almost colorless oil (14.4 g, 51%).

IH NMR ~ 7.91(1H, s, H8), 7.50-6.80(13H, m, aromatic), 5.28(lH, d, CHOH), 4.47(lH, d of d, N5~
4.16(lH, d of d, N5~ .04(lH, m, CHOH), 3.72~6H, s, OCH~), 3.40(3H, s, NCH3), 3.22(3H, s, NCH3), 3.00(lH, m, CH20DMT), 2.87(1H, m, ~ ODMT). Rf = 0.29 (3% Methanol in dichloromethane).

2-Cyanoethyl-N,N-Diiso~L~ylchlorophosphoramidite (4.6 mL, 20 mmol) was ~ ru~.ise to a stirred solution of the above DMT derivative (5.0 g, 9 mmol) in anhydrous THF (40 mL) cont~n;ng triethylamine (2.7 mL, 19 mmol). On addition, the mixture was stirred at RT for 60 min, and then ~iltered throu~h a sintered glass funnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dissolved in anhydrous benzene and then filtered. The filtrate was evaporated in vacuo, and the residue dissolved in an ethyl acetate/h~np mixture (3:7) and applied to a flash column. The product was then eluted using 35~ ethyl acetate in he~ne cont~ n~ ng 1~
triethyl ~m; ne, and hu~-~o~ellous fractions were com~ined and SUBSTITVTE SHEET (RULE 26) r W O 97128168 PCTrUS97101060 evaporated to give the title compound a~ a powder, (3.9 g, 57~) with the following structure:
OCJ1~ -~ k ~
C o~

$
CH~ OCH~
3~P NMR ~ 149. 'H NMR ~ 7.99 ~lH, s, H8), 7.50-6.80 (13H, m, aromatic), 4.50 (2H, m, N,CH2), 4.38 (lH, m, CHOP), 3.72 (6H, s, OCH3), 3.51 (2H, m, OCH2), 3.42 (2H, m, CH2CN), 3.40 (3H, s, NCH3), 3.22 (3H, s, NCH3), 3.18 (lH, m, CH20DMT), 3.10 (lH, m, CH20DMT), 2.55 (2H, m, NCH), 1.05 (6H, d, CHCH3), 0.97 (6H, d, CHCH3). Rf =
0.34, 0.16 (50~ Ethyl acetate in h~An~) ExamDle 16 (+/-)-O2-(2-Cyanoethyl-N,N-DiisG~ v~YlPho~Phoramidite) 0~-(4,4'-DimethoxytritYl)-1,2-.2-DihYd ~L~tane 4,4'-Dimethoxytrityl chloride (10.33 g, 30.5 mmol) was A~ in portions to a stirred solution of 1,2-but~n~;ol (2.5 g, 27.7 mmol) in anhydrous pyridine (200 mL) co~t~in;ng DMAP (1.7 g). The mixture was stirred at RT under nitrogen overnight. The solution was then evaporated under vacuum, and the residue adsor~ed onto silica gel and applied to a silica gel f lash column which was eluted using 10% ethyl acetate in hexane. Ho..loyellous ~ractions were combined and evaporated to give (+/-)-O'-(4,4'-dimethoxytrityl)-1,2-dihydroxy~utane as an almost colorless oil, (8.15 g, 75~).

'H NMR ~ 7.50-6.80 (13H, m, aromatic), 4.59 (lH, d, OH), 3.73 (6H, s, OCH3), 3.53 (lH, d of d, CHOH), 2.91 (lH, t, CH~ODMT), 2.78 (lH, t, CH20DMT), 1.55 (lH, m, SUBST~VTE SHEET (RULE 26) CA 02244924 l998-07-30 W O 97/28168 PCTrUS97/01060 I3~, 1.29 (lH, m, CH2CH3), 0.8 (3H, t, CH3). Rf~ = 0.2 (10~ Ethyl acetate in hexane).

2-Cyanoethyl-N,N-~iisopropylchlorophosphoramidite (0.94 mL, 4.2 mmol) was added dropwise to a stirred solution o~ the abo~e DMT derivative (1.5 g, 3.8 mmol) in anhydrous THF (20 mL) cont;~in~ng triethylAm~ne (1.12 mL, 8.4 mmol). On addition, the mixture was stirred at RT
for 7 h., and was then filtered through a sintered glass funnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dissolved in anhyrous benzene and then filtered. The filtrate was evaporated in vacuo, and the residue dissolved in an ethyl acetate h~ne mixture (3:7) and applied to a flash column. The product was then eluted using 20% ethyl acetate in hexane cont~;ning 1%
triethyl ~m~ n~, and homogenous ~ractions were combined and evaporated to give the product as a viscous oil, (1.0 g, 44~) with the following structure:

OCH~

7(1Pr) NC ~P~ ~J
~~ O
n OC11~

31P NMR ~i 148.5 'H NMR ~ 7.50-6.80(13H, m, aromatic), 3.82~lH, m, OCH), 3.71(6H, s, OCH3), 3.53(2H, m, C~EI2CN), 2.97(lH, m, CH~ODMT), 2.80(lH, m, cH~oDMr)~
2.60(lH, m, NCH), 1.67(lH, m, CH3CH2), 1.51(IH, m, CH3CH?), 1.12(12H, m, CH(CH3)2), 0.78(3H, m, CH2CH3). Rf = 0.16 20~ Ethyl acetate in hexane.

SUBSTITVTE 5HEET(RULE 26) Exam~le 17 (+/-~-~-(2-CYanoethYl-N,N-Diiso~v~lPho~PhOramidite)-0l-(4 ,4'- Dimetho~LlitYl)-3,3-DimethYl-1,2-~ Di~y~ ~L~tane 4,4'-Dimethoxytrityl chloride (9.4 g, 27.7 mmol) was ~e~ in portions to a stirred solution of freshly distilled 3,3 dimethyl-1,2-dihydroxybl~t~n~A;ol (3 g, 25.4 mmol.) in anhydrous pyridine (200 mL) ~nt~;n;ng DMAP
(0.31 g). The mixture was stirred at RT under nitrogen overnight. The solution was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel short path column. The product was eluted using 2L of 10% ethyl acetate followed by lL of 20~ ethyl acetate in h~x~n~, and ~inally lL o~ 30~ ethyl acetate in h~x~n~. Hc..l~e~lous fraction~ were combined and evaporated to give (+/_)_ol- (4,4'-dimethoxytrityl)-3,3-dimethyl-1,2-dihydro xyhllt~n~ as an almost colorless oil, (6.03 g, 56.~%).

IH NMR_~ 7.50-6.70(13H, m aromatic), 4.70(lH, d, OH), 3.72(6H, s, O~EI3), 3.32(lH, t, C~HOH), 2.93(2H, m, C~EI2), 0.72(9H, s, CH3). Rf = 0.37 (2096 Ethyl acetate in hP~;~ne ) .

2-Cyanoethyl-N,N-diiso~l~ylchlorophosphoramidite (1.81 mL, 8.1 mmol) was added dlu~ise to a stirred 801utiûn of the above DMT derivative (2.27 g, 5.4 mmol) in anhydrous THF (50 mL) cont~ntng triethyl~m~ne (1.6 mL, 11.3 mmol). On addition, the mixture was stirred at RT overnight, and was then filtered through a sintered glass funnel under nitrogen. The ~iltrate was evaporated under vacuum, and the residue dissolved in anhydrous benzene and then filtered. The filtrate was then evaporated in vacuo, and the residue dissolved in an ethyl acetate/h~x~n~ mixture (3:7) and applied to a ~lash column. The product was eluted using 30~ ethyl acetate in 5C~

SUBSTITUT~ SH EET (RULE 26) W O 97/28168 PCTrUS97/01060 hexane containing 1% triethylamine, and homogenous fractions were combined and evaporated to give the title - compound as a viscous oil, (1.89 g, 56~) having the following structure:

N(lPr)2 Ib NC o~P~O
>~~ ~

31P NMR ~ 149. IH NMR ~ 7.60-6.80 (13H, m, aromatic), 3.82 (lH, m, CEIOP), 3.65 ~2H, m, OCH7), 3.63 (2H, m, CH~CN), 3.19 (lH, d of d, CH20DMT), 3.08 (lH, d of d, CH20DMT), 2.79 (lH, m, NCH), 2.67 (lH, m, NCH), 1.15 (12H, m, CH(5~E3)2), 0.78 (9H, m, (5~3)3). Rf = 0.13 (20%
Ethyl acetate in h~x~ne) EXamD1e 18 )-O2-(2-Cya~oethyl-N,N-DiisG~ ~lphos~horamidite)-0~-(4,4t- Dimethoxytrityl)-1,2-Dih~droxypropA~e 4,4'-Dimethoxytrityl chloride (7.42 g, 21.9 mmol) was A~e~ in portions to a stirred solution of 1,2-propanediol (1.52 g, 20.0 mmol) in anhydrous pyridine (200 mL) cont~n~ng DMAP (0.28 g). The mixture was stirred at RT under nitrogen overnight. The solution was then evaporated under vacuum, and the residue adsorbed onto silica gel and applied to a silica gel flash column.
The product was eluted using 20~ ethyl acetate in h~n~, and homogenous fractions were combined and evaporated to give (~/-)-0'-(4,4'-dimethoxytrityl)-1,2-dihydroxypropane as an almost colorless oil(3.1 g, 41~) . ~

C~

SUB!;TITVTE SHEET ~RULE 26) W O 97/28168 PCTrUS97/01060 - -IH NMR ~ s 7.50-6.80 (13H, m, aromatic), 4.6 (lH, d, CHOH), 3 78 (lH, m, CHOH), 3.66 (6H, s, OC~I3), 2.91 - (lH, t, CH2), 2.68 (lH, t, CH2). Rf = 0.25 (20% Ethyl acetate in hexane).

2-Cyanoethyl-N,N-Diisopropylchlorophosphoramidite (1.36 mL, 6.1 mmol) was added dropwise to a stirred solution o~ the above DMT derivati~e (2.1 g, 5.6 mmol) in anhydrous THF (50 mL) cont~;n;ng triethyl~m;n~ (1.63 mL, 11.7 mmol). On addition, the mixture was stirred at RT overnight., and was then filtered through a sintered glass ~unnel under nitrogen. The ~iltrate was evaporated under vacuum, and the residue dissolved in anhyrous benzene and then ~iltered The filtrate was evaporated 7n vacuo, and the residue dis~olved in an ethyl acetate/h~x~ne mixture (3:7) and applied to a flash column. The product was then elute~ using 20~ ethyl acetate in h~n~ cont~n~ng 1~ triethylamine, and hu~n~yellous fractions were combined and evaporated to give the title compound as a viscous oil, ~1.0 g, 31~) having the ~ollowing structure:

oa tlPlh b ~~ o ç3 o~

31p NMR ~ 147. 'H NMR ~ 7.50-6.70 (13H, m, aromatic), 4.03 (lH, m, CHOP), 3.72 (6H, s, OCH3), 3.65 (2H, m, OCHz), 3.58 (2H, m, CH2CN), 3.05 tlE, m, CH20DMT), (~'1 SUBSTITUTE SHEET ~RULE 26) W O 97/28168 PCT~US97/01060 2.85 (lH, m, ~ODMT), 2.77 (lH, m, N ~ ~, 2.64 (lH, m, NCH), l.lS (12H, m, NCH~CH3~2). Rf = 0.28, 0.33 (20 Ethyl acetate in h~ne) Exam~le 19 (~/ ) o2-(2-CYanoethY~ N-Diisoprop~lrl~hosphoramidite~-01-(4,4'- Dimet~o~nrtritYl) -3-(3-IndolY~ 2-Dihydroary Pro~alle 4,4'-Dimethoxytrityl chloride (1.7 g, 5.~ Imnol) was ~P~ in portions to a stirred solution of (+/-)-3-(3-indolyl)-1,2 dihy~lLo~y~ropane (0.87 g, 4. 6 mmol) in anhydrous pyridine (150 mL) contA;n;n~ DM~P (0.28 g.). On addition, the solution was left to stir at RT overnight under nitrogen, after which the mixture was evaporated under vacuum. The residue was then adsorbed onto silica gel, and this mixture applied to a silica gel short path column and the product eluted using 1~ methanol in dichloromethane. Hc...J~e~lous fraction~ were combined, and evaporated in vacuo to give the (~ 0l-(4,4'-dimethoxytrityl)-3-~3-indolyl~-1,2 dihy~lo~y~ropane as an oil(0.5 g, 22%).

'H NMR ~ 7.60-6.75 (18H, m, aromatic), 4.64 (lH, d, ex., OH), 3.92 (lH, m, CHOH), 3.72 (6H, s, OCH~), 2.94 (3H, m, CH?-ind.+CH2-OH), 2.72 (lH, m, CH~OH). Rf = 0.25 (1~ Methanol in dichloromethane).

2-Cyanoethyl-N,N-diiso~l~ylchlorophosphoramidite (O.18 ~L, 0.81 mmol) was added ~Lu~.~iSe to a ~tirred ~olution oE the above DMT derivative (O.4 g, 0.74 mmol) in anllydluus THF (25 mL) cont~in;n~ triethyl~m;ne (0.22 mL, 1.5~ mmol). On addition, the mixture was stirred at RT overnight., and then ~iltered through a sintered glass ~unnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dis~olved in anhyd~o~s benzene and then ~iltered. The ~iltrate was evaporated in vacuo, and the residue dissolved in an ethyl acetate/h~ne (~

SUBSTITUTE SHEET (RULE 26) W O 97/28168 . PCT~US97/OlQ60 mixture (3:7) and applied to a flash column. The product --was eluted using 50~ ethyl acetate in hexane con~ining - 1% triethylamine, and homogenous fractions were combinèd and evaporated to give the product as a viscous oil, (0.224g, 41%) having the following structure:

OCH~

NC

OCH~

31p NMR ~ 148. 'E NMR_~ 7.50-6.70 (18H, m, aromatic), 4.24 (lH, m, CHOH), 3.72 ~6H, m, 05~), 3.70-3.42 (4~, m, 5~2CN+CH7OP~, 3.18-2.90 (CH70DMT+CH2-ind), 1.28-0.93 (12H, m, CH~5~3)2). Rf = 0.47 (2096 Ethyl acetate in h~n~).

Exam~le 20 ,/-)_ol- (2-Cy~Lnoethyl-N,N-Dii~ }Phos~ho ;Ai te3 o2-(4,4~ -Dimethoxytrityl)-N-AcetY1-2-Amino-4-(1,2-DihYroxYethYl)-1,3-Thiazole A solution o~ ethyl 2-amino-4-thiazolglyoxylate (lOg, 50mmol) in anhydrous THF (150 mL) was added dropwise to a suspension of lithium al-~mi nl~m hydride (1.9 g) in anhydrous THF ~200 mL), and the mixture stirred -under nitrogen at RT ~or 1 h. Excess ethyl acetate was SUE~STITUTE SHEET tRULE 26) then added to destroy residual reductant, a~ter which an exces~ o~ Glauber's Salt was added and the resultant suspension stirred at RT for 40min. The slurry was then filtered, and the filtrate evaporated in vacuo, and then dissolved in methanol and adsorbed onto silica gel. This mixture was then applied to a flash silica co~umn, and the product eluted using 10~ methanol in dichloromethane.
Homogenous fractions were combined and evaporated under vacuum to ~ive (+/-)-2-amino-4-(1,2-dihydroxyethyl)-1,3-thiazole product as a pale orange colored solid, (2.6 g, 32.5%) with the following structure:

~H
, OH

It2N S

IH NMR ~ 6.74 (2H, s, NH2), 6.28 (lH, s, aromatic), 4.92 (1~, broad, OH), 4.48 (lH, broad, OH), 4.34 (lH, m, CHOH), 3.60 (lH, d of d, CH70H), 3.38 (lH, m, CH20H3. ~f = 0.43 (30 ~ Methanol in dichloromethane).

Acetic anhydride (4.5 mL, 47.4 mmol) was added to a solution of the above aminothiazole (2.3 g, 14.4 mmol) in anhydrous pyridine (30 mL~ and the solution stirred under nitrogen at room temperature overnight. The mixture was then evaporated under vacuum, and the residue dissolved in ethly acetate. This solution wa3s washed successively with 2M hydrochloric acid, saturated sodium bicarbonate solution, and then brine. The mixture was then dried over anhydrous sodium sulfate, filtered and the filtrate evaporated under reduced pressure. The residue was triturated with ether, after which the product separated as a tan colored solid which was collected by filtration, giving (+/-)-N2,0,0-triacetyl-2-SV8STITUTE SHEFI~ (RULE 26) W O 97128168 PCTrUS97/01060 a~ino-4-(1,2-dihydroxyethyl)-1,3-thiazole as a powder, (1.08 g, 37~).

lH NMR ~ 7.14 (lH, s, aromatic), 5.96 (lH, m, CHQAc), 4.42 (lH, m, CH2OAc), 4.33 (lH, m, CH~QAc), 2.12 (3H, s, CH~CO), 2.07 (3H, s, CH3CO), 1.98 (3H, s, Ç~CONH). Rf _ O.48 (10% Methanol in dichloromethane).

The above triacetylthiazole derivative (1.0 g, 3.7 mmol.) was suspended in methanol and 0.1 M sodium hyd~ide solution (20 mL) was A~ in a single portion.
The reaction mixture was stirred at room temperature and the reaction carefully monitored by TLC (10~ methanol in dichloromethane). The reaction was stopped after 40 min by neutralizing the mixture using 2M hydrochloric acid in an ice bath. The resultant mixture was evaporated to dryness under vAc~ m, and the residue dissolved in a mixture of dichloromethane and methanol and then adsorbed onto silica gel. This material was applied to a flash silica column, and the product eluted using 15~ methanol in dichloromethane. Homoyenous fractions were combined and evaporated in vacuo to give (+/-)-N-acetyl-2-amino-4-(1,2-dihyroxyethyl)-1,3-thiazole as a tan colored powder (0.475 g, 69~).

IH NMR ~ 6.88 (lH, s, aromatic), 5.12 (lH, d, OH), 4.53 (lH, m, CHOH), 3.66 (lH, m, CH2), 3.47 (lH, m, CH2), 2.11 (3H, s, ~CO). Rf = O.22 (10~ Methanol in dichloromethane).

The N-acetyl-derivatized thiazole derivative from the above reaction (0.91~ g, 2.7 mmol.) was ~P~ in a single portion to a stirred solution of N-acetyl- 2-amlno-4-(1,2-dihydlo~yethyl)-l~3-thiazole in an~lyd-o~s pyridine (20 mL~ ron~i ni ng DMAP (20 mg). The resultant mixture was then le~t to stir at room temperature under nitrogen for 3 h, and was then evaporated under vacuum to give an orange colored oil. This material was dissolved SUBSTITUTE SHEET tRULE 26) W O 97/28168 PCTrUS97/01060 in d~chloromethane and adsorbed onto silica gel and then applied to a silica flash column. The product was eluted using 3~ methanol in dichloromethane, and homogenous ~ractions combined and evaporated in vacuo to give an off-white foam of (+/-)-02-(4,4'-dimethoYytrityl)-N-acetyl-2-amino-4-(1,2- dihyroxyethyl)-1,3-thiazole (800 mg, 66~).

IH NMR ~ 7.44-6.80 (14H, m, aromatic), 5.42 (lH, d, OH), 4.79 (lH, m, CHOH), 3.73 (6H, s, O ~ ), 3.18 (lH, m, CH~ODMT), 3.12 (lH, m, 5~ODMT). Rf = 0.34 (5~ Methanol in dichloromethane).

2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite (1.0 mL, 4.48 mmol) was added dLu~ise to a stirred solution of the above DMT derivative (1.0 g, 2.04 mmol) in anhydrous THF (45 mL) cont~ining triethylamine (1.2 mL, 8.56 mmol). On addition, the mixture was stirred at RT for 4 h, and then filtered through a sintered glass funnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dissolved in anhyrous benzene and then filtered. The filtrate was evaporated in vacuo, and the residue dissolved in an ethyl acetate h~An~
mixture (3:7) and applied to a flash column. The product was then eluted using 50~ ethyl acetate in h~Y~n~
cont~ining 1% triethylamlne, and ho..-~e~ous fractions were combined and evaporated to give the title compound as a viscous oil, (0.224g, 41%) with the ~ollowing structure:
OCH~
N(lPr)2 Ib NC

H~C~
OCH~
~,~

SUBSTlTtJTE SHEET (RULE 26 W O 97/28168 PCT~US97/01060 31p NMR ~ 149. IH NMR ~ 12.09 (lH, s, NH), 7.50-6.7 (14H, m, aromatic), 4.96 (lH, m, CHOP), 3.72 (6H, s, 05~), 3.62 (4H, m, CH?OP+CH2CN), 3.30 (lH, m, CH20DMT), 3.09 (lH, m, CH?ODMT), 2.06 (3H, s, 5~CO), 1.25-1.04 (12H, m, CH(5~)2). Rf = O.52, 0.58(50~ Ethyl acetate in h~X~ n e).

Exam~le 21 Q5-(2-CYanoethYl-N,N-Diis~l~Yl~hosPho~ ;te)-OI-(4,4~-Dimeth~A~ritYl~-1,2,3~4-TetrahYdro-1,5-DihYdroxyna~hthalene Dimethoxytrityl chloride (11.35 g, 33.5 mmol) was added to a stirred solution of 1,5-dih~dlu~y-1,2,3,4-tetrahydrnn~rhth~lene (5 g, 30.5 mmol) in pyridine (100 mL) c~t~in~ng DM~P (200 mg), and the resultant mixture was stirred ~or 2 h at room temperature under nitrogen.
The reaction mixture was then poured into water and the product extracted with ethyl acetate. The extracts were w~he~ with brine, dried over anhydrous sodium ~ul~ate, ~iltered, and the filtrate evaporated in vacuo. The residue was then adsorbed onto silica gel, and the mixture applied to a silica gel short path column and the product eluted using 30% ethyl acetate in h~x~n~, HG.l.oyellous fractions were combined and evaporated in vacuo to give 0'-(4,4'-dimethoxytrityl)-1,2,3,4-tetrahydro-1,5-dihy~o~y nA~hth~lene as an o~f-white foam, (5.52 g, 38?~).

IH NMR ~ 9.01(1H, s, OH), 7.50-6.50 (16H, m, aromatic), 4.21 (lH, m, CHOH), 3.72 (6H, s, OCH~), 2.60 (lH, m, CH?-aromatic), 2.34 (lH, m, CH?- aromatic), 1.88 (lH, m, alicyclic), 1.37 (lH, m, alicyclic), 1 28 (lH, m, alicyclic), 1.23 (lH, m, alicyclic). R~ = 0.17 (30 Ethyl acetate in ~eX~ne)~

(~

SUBSTITUTE SHEE7- (RULE 263 W O 97/28168 PCTrUS97/01060 2-Cyanoethyl-N,N-diisopropylchlorophosphoramidite ~0.56 mL, 2.5 mmol~ was added dropwi~e to a stirred solution of the above DMT derivative (1.0 g, 2.3 mmol) in anhydrous THF (10 mL) cont~ining diisopropylethylamine (0.88 mL, 5.1 mmol). On addition, the mixture was stirred at RT ~or 1 h, and was then filtered through a sintered glass funnel under nitrogen. The filtrate was evaporated under vacuum, and the residue dissolved in anhydrous benzene and then filtered. The filtrate was evaporated in vacuo, and the residue di~solved in an ethyl acetate~h~ne mixture (3:7) and applied to a flash column. The product was then eluted using 30~ ethyl acetate in h~ne cnntAi ni ng 1~ triethy~i n~, and h~ll~yellous ~ractions were com~ined and evaporated to give the title compound as a viscou~ oil (0.92 g, 63~), having the following structure: o~, O

oa~, NC~O ~ , O
N~lPr)2 31p NMR ~ 145.6. IH NMR ~ 7.50-6.80 (16H, m, aromatic), 4.23 (lH, m, CHOP), 3.93-3.60 (lOH, m, 05~+CH~OP+CH2CN), 2.80 (2H, m, NCH), 2.70 (lH, m, benzylic CH), 2.42 (lH, m, henzylic CH), 1.88 (lH, m, alicyclic), 1.42 (1~, m, alicyclic), 1.31 (lH, m, alicyclic), 1.09 (13H, m, alicyclic + CH(5~)2). Rf =
0.42(30~ Ethyl acetate in hexane).

Exsm~le 22 SUBSTITUTE SH EET (RULE 263 W O 97128168 PCT~US97/01060 SYnthesis of 5'-0-(4,4'-dimetho~L itYl)-diol-triethylammonium-H-Pho~onate~

The DMT derivatives from examples 1 to 21 are dis~olved in dry dichloromethane and added a ~lask over 10 minutes co~t~ning imi~7ole (15 eguivalents), A phosphorous trichloride (4.3 equivalents), triethyl~m;ne (29 equivalents), and dry dichloromethane cooled in an ice bath. The reaction mixture is stirred ~or 30 minutes at O C, the ice bath is removed and the mixture i~
stirred ~or an additional 30 minutes. Water is ~ A and this mixture was stirred for 10 minutes. The layers are separated and the the aqueous layer is extracted with chlorofonm. The comhine~ organic layers are evaporated and then co-evaporated twice with toluene. The crude material is puri~ied by column chromatography on silica gel eluting with a gradient of dichloromethane/methanol (O to 30%). The purities o~ the ~ractions are monitored by thin layer chromatography (TLC) and ~ractions ~ont~ln~ng pure material are c~mh~ne~ and evaporated to dryness. The resulting material is dissolved in dichloromethane and w~h~ with 0.1 M triethyl~ ~ onium bic;~hon~te. The aqueous layer is bach- I hf~ once with dichloromethane and the comh~n~A organic layers are evaporated to dryness to give the H-pho~phonate mono~.

Examole 23 SYnthe~is of 5'-0-(4,4'-dimethoxYtritYl)-1,2-dideoxy-D-ribo~e-3'-~-~hos~honate 5'-(4,4'-dimethoxytrityl)-1,2-dideoxy-D-ribose (5.6 g, 13.4 mmol) was treated according to the protocol used in example 22 to give 5~-0-(4, 4'-dimethoxytrityl)-1,2-dideoxy-D-ribose-3'-H-phosphonate (6.3 g) as a colorless ~oam having the ~ollowing structure:

~'~

SUBSmUTE SH EET (RULE 26) W O 97/28168 PCT~US97/01060 oct~, ¢~
H, C O

O
E~NH~
31p NMR (202 MHz~ DMSO ~ (ppm) 0.61 ~doublet of doublets, JP~i= 578 Hz, JP-O-C-II= 9.1 Hz~. TLC Rf = 0.37 (8:2 dichloromethane/methanolJ0.1% triethylamine).

Example 24 Srnthe~is o~ 1-(4,4'-dimethoxytrityl~-3-(4-methoxv~h~nnYv)-1,2-propanediol-2-E-Pho~phonate 1-(4,4'-Dimethoxytrityl)-3-(4-methoxyphenoxy)-1,2-propanediol (3 g, 6 mmol) was treated according to the protocol used in example 22 to ~ive 1-(4,4'-dimethoxytrityl)-3-(4-methoxy~h~n~y-1,2-prop~ne~;ol-2-H-phosphonate l0.65 g) as a colorless oil ha~ing the ~ollowing structure:
OC~

H,CO~ ~3 OCH~
0~ ~
H-- --O

Et~NH
3'P NMR ~202 M~Iz) DMSO ~ (ppm) 1.5 (doublet o~
doublets, JP-H= 586 Hz, ~pOcll= 10.7 Hz). TLC R~ = 0.26 (9/1 dichloromethane/methanol).

Ex~m~le 25 ~'~
SUBS 111 UTE SHEET (RUl E 26) W O 97128168 pcTruss7lolo6o SYnthesis of 1-(4,4'-dimethoxYtrityl)-3-(diethylamino) 1~2-propanediol-2-~-~hosphonate 1-~4,4'-Dimethoxytrityl)-3-(diethyl ~m; nQ) -1, 2 -propanediol (2.7 g, 6 mmol) was treated according to the protocol used in example 22 to give 1-(4,4'-dimethoxytrityl)-3-(diethylamino)-1,2-propanediol-2-H-phosphonate (4.1 g) as an oil having the ~ollowing structure:
Ot:H

N 1~3 ~~O¢~ ~}OCH~

It--P=O
I _ ~
E~NH~

3~P NMR (202 MHz) DMSO ~ ~ppm) 5.2 (dd, JP-H= 601 HZ ~ JP~-C-H = 10 . 7 Hz). TLC R~ 0.26 (9:1 dichloromethane/methanol).

Example 26 Synthesis of 4,4'-dimethoxYtritYl-tran~-9,10-ethanoa~thracene-11,12-dimethanol-~-phosPhonate 4,4'-Dimethoxytrityl-trans-9,10-ethanoanthracene-- 11,12-dimethanol (1.64 g, 2.9 mmol) was treated according to the protocol used in example 22 to give 4,4'-dimethoxytrityl-trans-9,10-ethanoanthracene-11,12-~I

SUBSTITUTE SHEET~RULE 26) W O 97/28168 PCTrUS97/0106~ -dimethanol-H-phosphona~e (1.8 g) as a white foam having the following structure:
H~co E~NH~ ~ C ~ OCH~

~ = 'PG--~ ~

3'P NMR (202 MHz) DMSO ~ (ppm) 1.6 (JP-H = 613 Hz).
TLC Rf = 0.09 (dichloromethane/0.5~ triethyl ~mi ne), ExamPle 27 SYnthesi~ of 2-(4,4'-dimethoxvtrityl)-2,6-bi~-hYdroxYmethYlPYridine-6-~-~ho~Phonate 2-(4,4'-Dimethoxytrityl)- 2,6-bis-hydroxymethylpyridine (7.0 g, 15.8 mmol) was treated according to the protocol used in example 22 to give 2-(4,4'-dimethoxytrityl)- 2,6-bis-hydroxymethylpyridine-6-H-phosphonate (8 g) as a yellow oil having the following structure: OCH~

H~CO ~ -O ~ O-PI-H

~3 Et3NH ~

3~P NMR (202 MHz) DMSO ~ (ppm) 1.65 (doublet of triplets, JP-H= 584 Hz, JP-O-CH2= 9.1 Hz~. TLC Rf 0.08 (9:1 dichloromethane/methanol/0.1% t~iethyl~m;ne)~

I

SUBSTmlTE SHEET(RULE 26) W O 97/28168 PCTrUS97/01060 EX ~ P1e Z8 SYnthesis of 7- (4,4'-dimethox~tritYl) -trans-7,8-- dihydroxy-dimeth~l-exo-tricycl~sn~n~[4.2.1. o2~5~ nona-3-ene-3,4-dic~h~YYlate-8-~-Pho~Phonate 7-(4,4'-Dimethoxytrityl)-~rans-7,8-dihydroxy-dimethyl-exo-tricyclononene t4-2.1.o25]nona-3-ene-3~4-dicarboxylate (3.5 g, 6.1 mmol) was treated according to the protocol used in example 22 to give 7-~4,4'-dimethoxytrityl)-trans-7,~-dihydroxy-dimethyl-exo-tricyclononenel4.2.1.025]nona-3-ene-3,4-diCarbOXylate-8-H-phosphonate (4.8 g) as a white foam having the following structure: o o H,C0 ~ ll H~CO~ O- E~NH~

~:~OCH~
~' OC~
31p NMR (202 MHz) DMS0 ~ ~ppm) 0 .66 (JP-H-- 613 HZ)-TLC RfØ12 (9:1 dichloromethane/methanol/0.1 triethylamine).

ExamPle 29 SYnthesi-Q of (4,4'-dimethoxYtritYl)-hYdro~in~n~-biQ-~hydroxyethyl)-ether-~-~hosPhonate (4,4'-Dimethoxytrityl)-hydroquinone-bis-(hydroxyethyl)-ether (2.2 g, 4.4 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4~-dimethoxytrityl)-hydroquinone-bis-(hydroxyethyl)-SUBSTITUT~ SH EET ~RULE 26) W O 97/28168 PCT~US97/01060 ether-H-phosphonate (2.5 g) as a white sticky solid --having the followi~g structure: oc~, b ~o c~oc~ .

o O
~--r--o o E~NH~
3tP NMR (202 MHz) DMS0 ~ (ppm) 1.9 (JP-H =
578 HZ).
TLC Rf 0.09 (9:1 dichloromethane/methanol/0.1%
triethylamine).

ExamDle 30 SYnthesi.~ of (4,4~-dimethoxytrityl)-N,N-bis-(2-h~droxyethyl)-isonico~; n: ide-H-PhosPhonate (4,4'-dimethoxytrityl)-N,N-bis-(2-hydroxyethyl)-isonicotinamide (10.2 g, 19.9 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4'-dimethoxytrityl)-N,N-bis-(2-hydroxyethyl)-isonicotinamide-H-phosphonate (10.5 g) as a white foam having the following structure:
OCIl~

~ o-c~oct~, I~ J f N H ~ =O

E~NH~

SUBSTITUTE SH EET ~RULE 26) W O 97/28168 PCTrUS97/01060 3~P NMR (202 MHz) DMSO ~ (ppm) 1.55 (lH, doublet - -.
o~ triplets, JP-H= 583 HZ, JP-O-CI~2= 7.7 Hz), 1.98 (lH, -doublet of triplets, JP-II= 583 Ez, JP-O-CI~2= 9.1 HZ). TLC
Rf Q.06 (9:1 dichloromethane/methanol/0.1 triethyl ~mi ne), Example 31 Synthesi.~ o~ 3-~4,4'-~imethoxYtritYl)-2-amino-1-phenyl-1,3-prop~n~iol-H-phosphonate 3-(4,4'-dimethoxytrityl)-2-amino-1-phenyl-1,3-propanediol (8.0 y, 14.1 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give 3-(4,4~-dimethoxytrityl)-2-amino-1-phenyl-1,3-propanediol-H-phosphonate (7.1 g) as a white foam ha~ing the following structure: OCH~

Et3NH 11 ~ ~}oc~, H N H

C~
3~P NMR (202 MHz) DMSO ~ (ppm) 0.66 (doublet of doublets, JP-H = 594 Hz, JP-O~H2= 12.1 Hz). TLC Rf 0.11 (9:1 dichloromethane/methanol/0.1% triethyl ~mi ne ) .

Example 32 ~ SYnthesis of (4,4'-d~methoxYtritYl)-1,4-bis-(hydrox~ethyl)-~iperazine-~-~hosPhonate SUBSTITUTE SHEET (RULE 26) W O 97/28168 PCTrUS97/01060 (4,4'-dimethoxytrityl)-1,4-bis-(hydroxyethyl)- - .
pipera~ine (10.2 g, 21.3 mmol~, prepared according to the - protocol used in example 4, was treated a~cording to the protocol used in example 22 to give (4,4'-dimethoxytrityl)-1,4-bis-(hydroxyethyl)-piperazine-H-phosphonate (g.1 g) as a white foam having the following structure:

OCI~, .
~3 f o-c~oc~t o_.. o EI~NH~

31p NMR (202 MHz) DMSO ~ (ppm) 4.3 ~doublet o~
triplets, JP~= 595 HZ, JP-O-CH2= 13.7 Hz). TLC R~ 0.11 (8:2 dichloromethane/methanol/0.1~ triethyl ~m~ n~), Exam~le 33 Synthesi~ o~ (4,4~-dimethoxytritYl)-ethYlene qlYcol-~-~hosPhonate ~ 4,4'-dimethoxytrityl)-ethylene glycol (2.9 g, 7.96 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4~-dimethoxytrityl)-e~hylene glycol-H-phosphonate (3.8 g) as a yellow oil having the following structure:

SUBSTITUTE SH EET (RULE 26) =

W O 97/28168 PCTrUS97/01060 OCH~

~,C~C--O--~ t \J o-E~NH~

3~P NMR (202 MHz) DMSO ~ (ppm) 2.0 (J,,"= 580 Hz).
TLC R~ 0.09 (95:5 dichloromethane/methanol/0.5 triethylamine).

Example 34 Synthesis of 1-(4,4'-dimethoxytritYl)-1-5-1,2-propa~ediol-~-~hosphonate 1-(4,4'-dimethoxytrityl)-1,2-propanediOl (2.3 g, 6.0 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give 1-(4,4'-dimethoxytrityl)-1,2-propanediol-H-phosphonate 11.75 g~ as a yellow gum ha~ing the ~ollowing structure:
OCH, o H~CO~C--o~~
~ 3 CH~ Et3NH ~

31P NMR (202 MHz) DMSO O Ippm) 1.7 (JP-H= 616 HZ).
TLC R~ 0.04 (95:5 dichloromethane/methanol/0.5 triethylaminel.

Example 35 Synthe~i~ of 1-(4~4~-dimethoxytritv~ 2-dihydroxy-3 butene-H-pho~phonate SUBS 111 UTE SHEEl~ (RULE 26 8 PCTrUS97/01060 1-(4,4~-dimethoxytrityl)-1,2-dihydroxy-3-butene (4.4 g, 11.2 mmol)~, prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give 1-(4,4'-dimethoxytrityl)-1,2-dihydroxy-3-butene-H-phosphonate (4.2 g) as a yellow gum having the following structure:
OCH~

~CO~-O~ 1, ~ E~NH+

31P NMR ~202 MHz) DMSO ~ (ppm) 8.17 (JP-H= 703 HZ) TLC Rf 0.13 (3:7 ethylacetate/hexane/0.5~ triethylamine).

ExamPle 36 SYnthe~is of 3-(4,4'-dimetho~Llit~1)-1-R-~henYl-1~3-propanediol-~-~hosphonate 3-(4,4'-dimethoxytrityl)-1-phenyl-1,3-propanediol (2.5 g, 5.5 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to gi~e 3-(4,4~-dimethoxytrityl)-1-phenyl-1,3-propanediol-~-phosphonate (2.3 g) as a yellow foam ha~ing the ~ollowing structure:

OCH~

E~N~+

~= I--~"' ~ ~ ~--C~3 OCH~

H ~3 13 - SUBSTITUTE 5HEET (RULE 26) W O 97/28168 PCTrUS97/01060 3~P Nn~R 1202 ~nHz) DMSO ~ (ppm) -1.7 (Jp~= 585 Hz).
TLC Rf 0.084 (94:6 dichloromethane/methanol/0.5%
triethylamine).

Exam~le 37 SYntheRis of (4,4'-dimethoxytrityl)-2,3-dihYdroxypropyl-theophylline-H-phosphonate (4,4'-dimethoxytrityl)-2,3-dihydroxypropyl-~heophylline (1.9 g, 3.5 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4~-dimethoxytrityl)-theophylline-H-phosphonate (2.6 ~3 as a white foam having the following structure:
E t3 NH ~ ~ CH, ~~
G--~--H

C ~3 OCH~

o~ N
C~1~
3Ip NMR (202 MHz) DMSO ~ (ppm) 0.76 (doublet of doublets, JP-H = 584 HZ, JPO~H = 8 . 5 HZ) ) . TLC Rf O . 34 (94:6 dichloromethane/methanol/0.5~ triethylamine).

ExamDle 38 SYnthesis of (4~4~-d~methoxytrit~l)-~ilocarpine-H
Phosphonate (4,4'-dimethoxytrityl)-pilocarpine (1.2 g, 1. 8 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example - 22 ~o give (4,4'-dimethoxytrityl)-pilocarpine-H-phosphonate (1.5 g) as a pale yellow foam having the ~ollowing structure:
lq - SlJBSllTUTE 5HEET (RULE Z6) W O 97/28168 PCTrUS97/01060 OCH~

HICO--~O ~CH~

E~NH+
31P NMR (202 MHz) DMSO ~ (ppm) 1.8 (Ip-H= 573 Hz). TLC Rf 0 122 (95:5 dichloromethane/methanol/1% triethyl ~m; n~), ExamPle 39 SYn~he~is of (4,4'-dimethoxYtritYl)-l S, 2S, 3R, 55- ( ~ ) - PinAn~Aiol-H-phosphonate ~ 4,4'-dimethoxytrityl)-lS,2S,3R,5S-(+)-pinanediol (2.51 g, 5.36 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4~-dimethoxytrityl)-l S, 2S, 3R, 55- ( + ) - p; n~n ~; ol-H-phosphonate (1.8 g) as a pale yellow foam having the following structure:
fC~, o-C ~ OCH, H-ll'-O
E~NH+
3'P NMR (202 MHz) DMSO ~ (ppm~ / (JP-H = 585 Hz).
TLC Rf 0.234 (95:5 dichloromethane/methanol/0.5%
triethylamine).
~, - SUBST~TUTE SHEET ~RULE 26) W O 97/28168 PCTrUS97/01060 EX ~ P1e 40 SYnnthe8i8 Of (4,4'-dimethO ~ trit~1)-thiOmi~; ;ne-trif1UO~OaCet ~ ide-E-PhOSPhOnate (4,4'-dimethoxytrityl)-thiomic2minP-tri~luoroacetamide (3.6 g, 6.0 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4~-dimethoxytrityl)-thiomicamine-tri~luoroacetamide-H-phosphonate (3.0 g) as a pale yellow foam having the ~ollowing structure: OCH~

HJCO~C--0~ 0--~= O

3¢3 Et3NH
SCH~
- 3~P NMR (202MHz) DMSO ~ (ppm) -1. 7 (JP-H = 585 HZ).
TLC R~ 0.234 (95:5 dichloromethane/methanol/0.5%
triethylamine).

ExamPle 41 Synthe-Qis of (4,4'-dimethoxytrityl)-~Yri~oYin~-H-PhosPhonate (4,4'-dimethOXYtritY1)-PYridOXine (4.57 g, 9.69 mmol), prepared according to the protocol used in example 4, was treated according to the protocol used in example 22 to give (4,4'-dimethoxytrityl)-pyridoxine-H-phosphonate (3.9 g) as a white foam having the ~ollowing structure:

~1 SUBSTITVTE SHEE'r (RULE 26) W O97/28168 PCTrUS97/01060 E~N~
1- OCH, ~--I = o ~
~~ ~
~~,X~ ~-C~ OC~

31p NMR ~202 MHz) DMSO ~ (ppm) 1.9 (Jp.~,= 587 Hz).
TLC Rf O 33 ~8:2 dichloromethane~methanol/0 1%
trlethylamine).

ExamPle 42 Synthesi~ o~ a Tetrameric Phosphoruq Ester Samples of the mnno~rS f rom examples 15, 7, 12 and 5 were separately diluted in anhydrous acetonitrile to give a 0.2 M solution Of each. These solutions were used in a stAn~d amidite syntheses on an automated DNA
synthesizer using the 1 ~mol phosphoramidite cycle with an extended coupling time o~ 5 min. The oligomer was synthesized on a thymidine-derivatized solid support that had been initially derivatized with the chemical phosphory~ation reagent (2-cyanoethoxy)-2-(2'-0-4,4'-dimethoxytrityloxyethyl-sul~onyl)ethoxy-N,N'-diisopropylaminophosphine. After the addition of each of the mnno~rS to the column, added in the order speci~ied above, the e~iciency of the coupling was checked by moni~oring of the trityl colors ~rom each cycle, and was determined to be in excess o~ g5% throughout all of the coupling steps. A~ter the addition of the monomers, deoxycytidine phos~horamidite was added ~or labeling purposes. The column was then treated with concentrated ~mmnn ium hydroxide for 4 h at 55 C and the ~mmon ia removed by bubbling the solution with nitrogen f or 20 min. The solution was lyophilized, and the residue dlssolved in water and puri~ied on a C18 HPLC column SUBSTITUTE SHEET ~RUL~ 2~;) W O 97/28168 PCTrUS97/01060 using a gradient o~ acetonitrile (solvent -B)/triethylammonium acetate, pH 7 (solvent A). Gradient:
8-20% B over 24 min, then 20-40~ B over 10 min. The material eluting at 14.75 min was lyophilized to give an oligomer with the ~ollowing structure:

HO
~ ~ O ~ O
H C ~ O -P- O ~ O -P- O O p O ~ po32 CH~

Example 43 SyntheRis of a Fluorescein-Labeled Pentamer LibrarY

Samples of the monomPrs from examples 1-10 were separately diluted in anhydrous acetonitrile to give 2 mL
of a 0.2 M solution of each. These solutions were used in a st~n~d amidite syntheses on three Applied Biosystems 394 Automated DNA synthesizers using the 1 ~mol phosphoramidite cycle with an extended coupling time of 5 min ~or each. The library was synthesized on ten columns each loaded with 10 mg of 100 ~m thymidine-derivatized controlled pore glass support that had been derivatized with the chemical phosphorylation reagent (2-cyanoethoxy)-2-(2~-0-4,4'-dimethoxytrityl-oxyethylsulfonyl)ethoxy-N,N'-diisopropyl-aminophosphine.
After the addition of each of the mnn~mers to their specified columns the efficiency of the coupling was checked by monitoring of the trityl colors from each synthesis, and was determined to be in excess of 90 throughout all of the coupling steps. After each synthesis cycle, the resins were removed from the columns and pooled and divided using the isopycnic slurry method.
The synthesis columns were then weighed to ensure an even division of the resin between the columns, the variation of which was determined to be less than 5~ throughout the SUBSTITVT~ SHEET (RULE 26) W O 97/28168 rCTAUS97/01060 -four pooling and dividing steps. After the addition o~the fifth amidite, the oligomers in each of the ten ~ columns were fluoresceinated using fluorescein 6-FAM
amidite ~Applied Biosystems, Foster City, CA). The resin from each column was then removed, and treated with concentrated ~mmnn~um hydroxide for 4 h at 55 C. The resultant suspensions were filtered and the ~mmon~
removed from the filtrate by bubbling the solutions with nitrogen for 20 min. The ten solutions were then lyophilyzed and the residues dissolved in water and purified over an OPC cartridge ~Applied Biosystems) using the st~n~d procedure as provided by the manufacturer.
The products from the purification were then analyzed by electrophoresis on a non-denaturing polyacrylamide gel.
Each of the ten products ran as a single band, with almost the same mobility as a pentameric deoxyoligonucleotide. The library was then assembled by ~;~;ng all of these solutions and lyophilizing the resultant mixture.

~ xam~le 44 SYnthesis of a 32P-Labeled Trimer Phos~hor~midate LibrarY.

Samples of the m~no~ers from examples 23, 27, 29, 32-37 and 39 were separately diluted in 1:1 anhydrous acetonitrile/pyridine to give a 0.12 M solution of each.
These solutions were used in two automated DNA
synthesizers using a modified H-phosphonate cycle with an extended coupling time of 5 minutes for each ~n~ The library was synthesized on 10 columns each lo~ with 60 mg of long chain alkyl~mine controlled pore glass (37-70 ~m, 44 umol/g), that had been derivatized with dimethoxytrityl-thymidine. Each column was initially reacted with the chemical phosphorylating reagent (2-cyanoethoxy)-2-(2'-0-4,4'-dimethoxytri~yloxyethylsulfonyl)ethoxy-N, N'-diisopropyl ~m; nophosphine using ~he st~n~d phosphoramidite procedure. Each of the 10 mono~e~s was 3~

SUBSTITUTE S~EET (RULE :~6) W O 97/28168 PCTrUS97/01060 then coupled to its specified column, m~)nOm~r one to column one, mnnoTne~ two to column two, etc., up to mor~ ~ 10 to column 10. The efficiency of each coupling was checked by monitoring the trityl colors ~rom each synthesis, and was determined to be in excess of 9o~
throughout all of the coupling steps. A~ter addition of the 10 m~no~s, the resins were then removed ~rom the columns and pooled and divided using the isopycnic slurry method. The synthesis columns were weighed to ensure an even division of the resin among the columns. A~ter a second addition of all 10 monQmers as previously described, followed by a second pool-and-divide procedure, the non-nucleotide H-phosphonate linkages were converted to phosphoramidate linkages using the ~ollowing oxidatlon procedure with each column being oxidized with one of the ~m; ne~ listed below:

1. 2-(2-Aminoethyl)pyridine, 2. 1-~2-~n~ethyl)piperidine, 3. 2-(3, 4-Dimethyloxyphenyl)ethy-~i n~, 4. 4-~2-Aminoethyl)-morpholine, 5. 1-(3-Aminopropyl)-2-pyrroli~inone, 6. 2-(3-chlorophenyl~ethyl~mi n~, 7. 4-Bromophenethyl ~mi n~, 8. N- (3- trifluoromethyl)phenylpiperazine, 9 . 3, 3-Diphenylpropyl ~m~ n~, 10. Thiomorpholine, 11. 1-(2-Pyridyl)piperazine, 12. Hexamethyl~n~i mi n~, 13. cis-2, 6 -Dimethylmorpholine, 14. 1-(4-fluorophenyl) piperazine, 15. 4-Fluorophenethyl ~mine, 16 . 3,5-Dimethylpiperidine.

A 10 ~ solution of each amine was prepared individually in ~hon tetrachloride and the H-phosphonate linkages of the oligomers were then oxidized to phosphoramidates using the freshly prepared solution ~5 SUBSTITUTE SHEET(RULE 26) W O 97/28168 PCTrUS97/01060 of the required amine for 10 minutes. The amine solutions were then flushed from the columns and the supports were washed extensively with acetonitrile. The supports were removed from the columns and pooled and di~ided as described above. A second round of m~n~r addition followed by pooling, dividing and amine addition was carried out, ~ollowed by addition of thymidine-H-phosphonate at the last step to provide an att~cl -nt site ~or the 32p label. The supports were then remo~ed from the columns and treated with concentrated ~mmon ~ um hydroxide ~or 5.5 hours at 55~C. The resultant suspensions were filtered and the ~mmnni ~ removed from the filtrates by bubbling the solutions with nitrogen for 20 minutes. The sixteen solutions then were lyophilized, redissolved in water and purified by reversed-phase ~PLC
using a gradien~ of 5-100~ acetonitrile in TEAA, 0.1 M, pH 7. The products from these purifications were c~mh;
and resultant mixture was lyophilized to give the oligomeric phosphoramidate li~rary which was then labeled with 32p by a con~entional method using T4 polynucleotide kinase.

~xam~le 45 SY~the-~is of a Disulfide-Brid~ed PhoQ~horu-a Ester Oli~omer The 3~,5'-dithiol oligomer was synthesized on an Applied Biosystems Model 394 DNA synthesizer on a 1 ~mol scale using O.Q2 M solution of iodine ~or all of the oxidation steps. 1-O-Dimethoxytrityl-hexyl-disulfide-l~-r (2-cyanoethyl)-N,N-diisopropyl-phosphoramidite] (thiol modifier C6 S-S, Glen Research, Stirling, VA) was directly coup}ed to a nucleoside residue anchored to a controlled pore glass solid support (i.e. a thymidine column) and the coupling time was extended to 15 min in this cycle and the following two phosphoramidite coupling cycles. The ~on~m~r phosphoramidites as specified in SUBSTITIJTE SHEET (RULE 26) W O 97128168 PCT~US97/01060 examples 2, 1, 3, 2, 3 and 1 were then incorporated sequentially in the exact order specified using a coupling time of 5 min for each. Fluorescein p~osphoramidite (ClonTech Co.) was then added followed by the thiol modi~ier C6 S-S using a 15 min coupling time.
Coupling efficiencies for the monomPrs averaged 88%, as detenmined by trityl assay.

After the synthesis and cleavage from the solid support, the solution was concentrated to a small volume and the tritylated oligomer was isolated by HPLC on a preparative Cl8 column using a gradient of acetonitrile in 0.1 M triethyl-~m~n~um acetate (TEAA) buffer (5-30~ over 15 min then 30-55~ over 30 min, then at 55~ for 20 min).
Fractions eluting at 47-51 min were evaporated to dryne8s and redis~olved in 0.1 M TEAA buffer (pH 8.0, O.5 mL).
Af~er addition of dithiothreitol (DTT, 10 mg), the resulting mixture was stirred under an argon blanket at room temperature for 4 hr to cleave the disulfide bonds and liberate free thiol groups at both ends of the oligomer. The disulfide bond cleavage reaction was monitored by HPLC using a Cl8 analytical column. The byproducts were removed by extraction with ethyl acetate (3 X) and ether (2 X) and the lntenmediate oligomer dithiol was purified by HPLC on a preparative Cl8 column using a gradient of 2-20~ acetonitrile in 0.1 M TEAA
buffer over lS min followed by 20-50% over 30 min, then 50-65% over 15 min. The fraction eluting between 30-35 min was e~d~dted to dryness, dissolved in triethyl ~mm~n~ um acetate buffer pH 8.0 (1 mL) and oxidized with oxygen with stirring at room temperature ~or 1 day. The reaction mixture was analyzed by HPLC on a Cl8 analytical column using a gradient of acetonitrile in O.1 M TEAA buffer (5-30~ over 10 min, then 15-50~ over 23 min, then 50-80% over 7 min. The appropriate fractions were lyophilized to give a disulfide-bridged oligomer (HPLC retention time 24.0 min) with the following structure:
~ ' SUBSTITUTE SHEET ~RlJLE 26) W O 97/28168 PCTrUS97/01060 ~~0~~
o--~
~~o'~o~~o'~o~O-~o,~ ~ ~
~~ ~

ou~ o~ p~~r EX ~ Ple 46 Serum Stability of a Non-nucleotide Oliqomeric Phosphorus Ester The H-phosphonate synthesis method was used to prepare a fluorescein-labeled pentamer with the following structure:

rLuo..~N o~o~_~

OCH~

Portions (9.6 nmol) of this material in separate tubes were each dissolved in water (105 ~L), added to h~ n serum (500 ~h) and incubated at 37~. At various time points, samples were ~iltered through a 0.45 ~m ~ilter and analyzed by ion exchange HPLC using a Dionex PA-100 column (4 x 250 mm). The column was eluted with a gradient o~ 25 mM Tris chloride, lM ~ ~n ium chloride, pH
8 (buffer B) in 25 mM Tri~ chloride, pH 8 (buf~er A), using 15-65% B over 19 min, then 65~ B ~or 2 min, then 65-75% B over 2 min. The area under the peak corresponding to starting material was measured versus time to determine the degradation rate. By this method the t"2 was determined to be greater than 1 month. A 20-g~

SUBSTITVTE SHEET ~RUl E ~6) W O 97/28168 PCT~US97/01~60 base oligodeoxynucleotide was used as a control, and under the same conditions, the tl~2 for this oligonucleotide was determi n~ to be 7 min.

Exam~le 47 B;~;~ of a Library of PhosPhorus Ester Oli~omers to Protein Tar~et~

The oligomeric library o~ Example 43 wa~ screened against the target proteins IL-4 and IFN~ for high affinity ligands, using the COMPILE method as outlined in copending patent application "Determination and Identification of Active Compounds in a Compound Library," U. S. patent application serial number 08/223,519. Binding reactions consi~ting of 2.5 ~M
oligomeric library and 1 ~M target protein as well as a protein-minus control (data not shown) were prepared in a buf~er cont~;n;ng 150 mM NaCl, 3 mM MgCl2 and 25 mM Tris-~Cl (pH 7.53. The reactions were eq~ ;hrated for 30 min at room temperature prior to centrifugation through Microcon-10 spin filtration units (M.Wt.cutoff = 10,000).
The M.Wt. cutoff of the filtration unit was selected to allow free passage of the oligomeric library members but not the target protein or the oligomeric molecules bound to it.

The centrifugation was adjusted to allow d~ro~imately 270 ~L of the total reaction volume of 400 ~L to flow through the membrane, resulting in the lo~s of a~Lu~imately 67% of the unbound oligomeric library from the ret~;nPA sample. The volume of the ret~;n~A sample was ~rouyllt up to the original 400 ~L with buffer (ront~in;ng 20% of the original target conc~nt~ation to compensate for lo~ses/inactivation of the target ~uring the experiment) and the selection was repeated.

5c~

SUBST1TVTE SHEET (RULE 26) W O 97/28168 PCTrUS97/0106U

The results of this serial selection are presented in Figure 1. The concentration o~ the oligomeric library present in the ret~ n~ fraction (as measured by fluorescence o~ the ~luorescein tag incorporated into the library members) is plotted as a function o~ the rounds of selection - i.e. the number o~ dialysis-based separations. Tt should be noted here that any method of separation of target-bound library members from unbound library members, even separation not based on size, could have been chosen. Data are not presented for the first two rounds of selection since the library was in excess over target at that point, and we would not observe protein dependent retention of the library.

As shown in Figure 3, the oligome~ic library members are lost at approximately the same rate for both IL-4 and IFN~ (and for the protein-minus control - data not shown) over the ~ir~t seven rounds of selection. At this point, however, the IFN~ reaction begins to retain a progressively larger fraction of the library whereas the IL-4 reaction ~ails to show such increased proportion o~
retention.

The progressive increase in the ~raction of compounds retained at each round, as observed with the IFN~ reaction, is evidence of a subpopulation of the original library that binds to the target. This subpopulation is enriched with each successive round of selection due to selective retention of compounds in the library that bind to the target protein. From the target protein concentration and the curve shown in Figure 1, it i8 possible to calculate that this subpopulation of~
~inding oligomeric species is about 1% o~ the original mixture, and that it binds with Kd of approximate-y 250 nM.

SVBSTITUTE St~EET(RULE 26) W O 97/28168 PCT~US97/01060 Example 48 Synthesis of 1-O-(4,4'-dimet~oxYtritYl)-1,2,3,4-tetrahydro-1,5-dihydroxynaPht~alene-S-~-~hosPhonate 1-O-~4,4'-dimethoxytrityl)-1,2,3,4-tetrahydro-1,5-dihydro xynaphthalene (4.77 g, 10.2 mmol) was treated according to the protocol used in Example 22 to give 1-O-(4, 4~-dimethoxytrityl)-1,2,3,4-tetrahydro-1,5-dihydroxyn~rhth~lene-5-H-phosphona te (4.7 g) as a colorless foam ha~ing the ~ollowing structure:

¢~
O C~

E~NH + .o - ~ - o OCH3 3~P NMR (202 MHz) DMSO d (ppm) -2.39 (doublet, JP-H = 597 Ez).
TLC Rf = O.19 (99:1 dichloromethane/methanol/0.1 triethyl Ami n~), EXAn~1e 49 32p habelin~ of a ~ibrarv of Non-Nucleotide PhosPhosus Ester Oli~omers This label was introduced onto the terminal thymidine residue of a compound or library using ~_32p _ labeled adenosine-5'-triphosphate a~ the donor source and bacteriophage T4 polynucleotide kinase as the enzyme.
Conditions for labeling approximated those for labeling of an oligonucleotide (Sambrook et al .). Purification was ql SV~STITUTE SHEET(RULE26) W O 97/281C8 PCT~US97101060 achieved by preparative polyacrylamide gel electrophoresis.

Ex~mDle 50 Pre~aration of 3-AcetYlPhenYl 2-CY~noethYl N,N-d~ 1 PhosPhoramidite (2) To a stirred solution of 3'-hyd~ aceto~h~none (1, Figure 1, 3g, 22 mmol) in anhydrous CH2CI2 (120 mL) and N,N-diiso~.o~ylethylAmin~ (11.4 g, 88.1 mmol) was ~ 0-(2-cyanoethyl)-N,N-diisopropyl~m;no- chloro-phosphoramidite (7.3 g, 30.9 mmol) ~lo~Jise over 8 min. The reaction mixture was stirred ~or 2 h at room temp and evaporated to dryness.
The crude oil was partitioned between ethyl acetate (150 mL) and 5~ a~ueous sodium bi~hon~te (150 mL) and the aqueous layer was extracted twice with ethyl acetate (100 mL). The combined organic layers were w~~h~ with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and the solid ~ ~ved by filtration. The solvents were removed by rotary evaporation and the crude yellow oil was adsorbed onto silica gel and applied to a silica gel ~lash column (16 x 10 cm) which was eluted from the column with hp~n~/eth acetate (5:1). Fractions ront~; n~ ng pure material were combined and the solvents evaporated to give 2 as a clear liquid (4.8 g, 64~ H NMR 7.64 (lH, m), 7.52 (lH, m), 7.45 (lH, m), 7.28 (lH, m), 3.87 (2H, m), 3.71 (2H, m), 2.83 (2H, t,~ = 5.7 Hz), 2.55 (3H, s), 1.19 (6H, d, J =
6.7 Hz3, 1.11 (6H, d, J = 6.7 Hz). Rf = 0.19 (Hexane/~thyl Acetate 5:1).

Ex D le 51 Tritium Labelinq of a Model ComPound In this examp}e the l~h~l;n~ group is att~rh~ to a thymidine residue att~che~ to a solid support.
Thymidine-5~-(m-acetylphenyl)phosphate (3a) was prepared q~

SUBSTITUTE SHE~T (RULE 26) W O 97/28168 PCT~US97101060 on a DNA synthesizer using ~our 10 ~mol cartridges with a modified lO~mol pho~phoramidite coupling cycle and 2 x-1 mi nllte coupling times. A solution o~ 2 (0.2 M) in anhydrous acetonitrile was used as the coupling reagent.
A portion of the ~olid support (25 ~mol) wa~ cleaved with 29~ NH40H to give crude 3a (559 OD2~0). This material was puri~ied in 4 portions using a C4 reversed-phase ~PLC
column with a gradient o~ 5-35~ acetonitrile in O.lM TEAA
pH 7 over 6 min and then to 45~ acetonitrile over 20 min.
Pure fractions were combined and evaporated to give pure 3a (424 OD26o) as white powder. lH NMR 7.72 (lH, m), 7.64 (lH, m), 7.44 (lH, m), 7.40 (lH, m), 7.38 (lH, m, H-4), 6~33 (lH, m), 4.57 (lH, m), 4.32 (lH, m), 4.16 (2H, m), 3.18 (6~, q), 2.56 (3H, 8), 2.33 (2H, m), 1.47 (3H, s), 1.26 (9H, t). Electrospray MS: M- 439 (theory 439).

Sodium Borotr~tiide ~eduction o~ 3a: To o.5 ~mol o~ 3a as a lyorh;lized ~oam in a screw-cap glass test tube was a~ded sodium ~orotritiide (20 mCi, 0.31 ~mol, American Radiolabeled Chemicals, Inc., St. Louis, Missouri, ~pecific activity 65 Ci/mmol) in 0.01 N
a~ueou~ sodium hydroxide solution (200 4L) and the mixture wa~ allowed to react for 2 h. TJn- ~h~l ed sodium borohydride (0.024 mg, 0.63 ~mol) in 0.01 N agueous sodium hydroxide solution (200 ~L of stock solution 2.4 mg/20 mL) was A~e~ and the mixture was allowed to 2.5react an additional 2 h. The reaction mixture was lo~ onto a Sep-Pak C18 plus cartridge previously primed with acetonitrile (10 mL) and water (10 mL). The cartridge was w~he~ with water (5 x 10 mL), then eluted with 6:4 methanol/water (v:v) (4 x 1 mL) followed by methanol (1 mL) and the methanolic fractions were evaporated to give the tritium labeled compound 4 (20 OD260, 73 nmol, 86 ~Ci, 4~ radiochemical yield). Specific activity: 1.18 mCi/~mol.

E~amDle 52 Tritium Labelin~ of a Librars~ Of q3 SUBSTITUTE SHEET (RULE 26) W O 97/28168 PCTrUS97/01060 NQn-NUC1eOtide PhO8~hO ~ 8 ESter O1i~ - A
.
A library of non-nucleotide phosphorus ester oligomers was prepared and coupled to 3-acetylphenyl 2-cyanoethyl N,N-diis~l~yl phosphoramidite (2) to produce a library 3b, possessing 3-acetylphenyl moiety at it~ terminus. To 0.5 ~mol o~ this library of non-nucleotide phosphorus ester oligomers in a ~crew-cap glass test tube was added sodium borotritiide (20 mCi, 0.29 ~mol, American Radiolabeled Chemicals, Inc., St.
Louis, specific activity 70 Ci/mmol) in 0.01 N aqueous sodium hydroxide solution (200 ~L) and the mixture wa~
allowed to react ~or 2 hours. Sodium borohydride (0.024 mg, 0.63 ~mol) in 0.01 N aqueous sodium h~dloxide solution (200 gL of stock solution 2.4 mg/20 mL) was ~eA and the mixture was allowed to react an additional 2 hours. The reaction mixture was loaded onto a Sep-Pak~
C18 plus cartridge previously primed with acetonitrile (10 mL) and water (10 mL). The cartridge was w~h~ with water (5 x 10 mL), then eluted with 6:4 methanol/water (v:v) (4 x 1 mL), methanol (1 mL) and the methanolic fractions were evaporated to give tritium labeled library 4b, 0.82 mCi, 5.7 ~ radiochemical yield, specific activity 1.65 mCi/~mol.

ExamDle 53 SYnt~esis o~ a Library of Non-Nucleotide Pho~Dhorus Ester Oliqomers with G1YCO1iC P i~ Protectin~
Grou~s A. Synthesis of Dimet~G~L ityl-glycolic acid (5) Glycolic acid (1.27 g, 16.57 mmol) was treated with pyridine (50 mL) and dimethoxytrityl chloride (6.20 g, 18.22 mmol) and the resulting mixture was stirred at room temperature ~or 4 hours. The reaction mixture was concentrated, treated with 200 mL ethyl acetate and then extracted with water (3 X 150 mL). The organic layer was concentrated, redissolved in methanol/water and pa~sed Cl~

SVBSmVTE SHEET (RUI_~ 26) __ CA 02244924 l998-07-30 W O 97/28168 PCTrUS97/01060 through a column of of Dowex-AG50X8 resin, pyridinium form (60 mL). The filtrate was collected and concentrated to yield 6.0 g of dimethoxytrityl-glycolic acid (5) as the pyridinium salt. This m~terial was used for the next reaction without any further purification.

B. SynthesiQ of the solid su~lL 7.
Long chain alkyl~ine controlled pore glaQs (1.0 g, 200-400 mesh, 88.6 umol/g) was treated with 10~
triethyl ~mi ne in 80~ aqueous ethanol (100 mL) and then washed extensively with acetonitrile and dried under high vaccum. The solid was suspended in anhydLo~s pyridine (5 mL) cont~inin~ diisopropylcar~odiimide (150 ul, 1.0 mmol) and N-hydlo~y~uccinimide (58 mg, 0.5 mmol). The resulting mixture wa~ ~ken vigorously overnight at room temperature, and then ~iltered. The solid was w~hP~
with pyridine, dried under high vacuum and treated with acetic anhydride/pyridine/N-methylimidazole (1:5:1,7.0 mL, v/v) for 30 minutes at room temperature to acetylate unreacted amino groups. T.OAA; ng of the DMT-glycolic acid (5) according to the dimethoxytrityl cation assay was 48 umol/g. The material obtA;nPrl was w;~:h~l with 5~
dichloroacetic acid in dichloromethane (40 mL) on a glass filter funnel to remove the dimethoxytrityl group and then w~h~d with CH2Cl2 and dried under v~c~ m. A
suspension of the dry solid support (2 g) in pyridine (2.5 mL) was treated with a solution of dimethoxytrityl-glycolic acid, (0.73g, 1.6 mmol~, triiso~o~yl ~ulfonyl chloride (0-44 g, 1.44 mmol) and N-methyl imidazole (1~5 ~1, 1.44 mmol) in 1.3 mL of pyridine (1.3 m~) and the mixture was shaken at room temperature overnight and then filtered through sintered glass funnel. The support was treated with a mixture of acetic anhydride/pyridine/ N-methyl ; mi ~ ~ole (1:5:1, 7.0 mL, v/v) for 30 minutes at room temperature. After filtration, the solid support was w~hP~ with pyridine, CH3CN, CH2Cl2 and ~inally with diethyl ether and then q 5 SUBSTITUTE SHEET (RULE 26) W O 97/28168 PCT~US97/01060 dried under high ~acuum. Loading o~ the DMT-glycolic acid according to the DMT-cation assay was 50 ~mol/g.
-C. Library Synthesis The library is synthesized on 10 columns eachloaded with 60 mg of modified solid support that had been derivatized with dimethoxytrityl-glycolic acid (50 umol/g). Each o~ the 10 ~nn~~~ H-phosphonates is coupled to its speci~ied support, ~on~m~ one to column one, m~n~ 2 to column 2 , etc., up to monom~ 10 to column 10. The e~iciency o~ each coupling can be checked ~y monitoring the intensity of the trityl cation released from each coupling step. After addition o~ the m~n~m~s, the supports are ,~,.,o~ed from the columns and pooled and divided using the isopycnic slurry method, with acetonitrile as the solvent. The synthesis columns are weighed to ensure an even division of the resin among the columns. After addition of all 10 m~no~s as pre~iously described, followed by a second pool-and-divide procedure, the non-nucleotide H-phosp~on~te linkages are converted to phosphoramidate linkages using the following oxidation procedure with each column being oxidized with one of the following A~; ~e~ 2-(2-aminoethyl)pyridine, 1-(2-aminoethyl)piperidine, 2-~3,4--dimethoxyphenyl )ethylAm; nf:~, 4-(2-aminoethyl)morpholine, 1-(3-aminopropyl)-2-pyrol;~;none, 2-(3-chlorophenyl)ethylAm;ne, N-isopropyl-l-piperazine acetamide, N-(3-tri~luoromethyl)phenylpiperazine, 3,3-diphenylpropylAm;n~, thiomorpholine, 1-(2--pyridyl)piperazine, hF~yAm~thylPn e~ m;n~, CiS- 2,6-dimethylmorpholine, 1-(4-fluorophenyl)piperazine, 4--~luororh~nethyl ~; ne, 3,5-dimethylpiperidine. A 10~
solution o~ each amine is prepared indi~idually in carbon tetrachloride and the H-phosphonate moieties are oxidized to phosphoramidate on the ~NA synthesizer using a ~resh solution o~ the required amine ~or 14 minutes. The amine solutions are then flushed from the column and the q~

SUBSTIT-JTE St~EET~RULE 26 W O 97/28168 PCTnUS97/01060 ~upports are washed extensively with acetonitrile. The supports are removed from the columns and pooled and divided as described above. A second round of monom~
addition followed by pooling, dividing and amine addition is carried out, followed by addition of the synthon required for introduction of the label (either fluorescein phosphoramidite, thymidine H-phosphonate or m-hydroxyacetorh~nnn~ phosphoramidite). The supports are then removed from the columns and treated with c~nc~nt~ated ammonium hydL~ide for 5.5 h at 55~C. The resultant suspensions are filtered and the Amm~n; A
~e-"~v~d ~rom the ~iltrates by bubbling nitrogen through the solutions for 20 minutes. The sixteen sol7ltiQn~ are then lyophilized, redissolved in water, purified by reversed-phase HPLC using a gradient of 5-100%
acetonitrile in TEAA, 0.1 M, pH 7.0 and pooled to give the library.

Ex~mPle 54 Tritium Labelina of a Phos~L~ ;~te LibrarY via Reducti~e MethYlation A phosphoramidate library is prepared as synthe~ized above and after the last pool and divide step all 16 columns are opened and ~he solid supports are transfered to 16 screw capped glass tubes and suspended in ethyl~ne~i~mine/CH3CN (500 ~l, 2~ or 2 hours at 55~C. After two hours the su~lL i~ ~iltered and washed with absolute ethanol (2 X 500 ~l). The filtrates are c~mh;n~d, evaporated to dryne~s and purified by HP~C
using a C4 reversed column to yield the desired sub-libraries. Each sub-library is suspended in 0.1 M
HEPES, pH 7.5 (1 mL) and treated with tritium labeled NaG~3H3 (3 equiv, 0.57 mg) and formaldehyde (3 equiv, 0.27 mg). A~ter 3 hours at room temperature the reaction mixtures from each sub-library are loaded on Sep-Pak cartridges (Cl8, Waters). Each cartridge is wA~h~d initially with water (15 mL) and then with 50:50 H2O/CH3OH
q~

SUB5TITUTE SHEET (RIJLE :Z6) W O 97/28168 PCTrUS97/01060 (5 mL). The H20/methanol solutions are collected and ~reeze dried to yield labeled libraries having stucture 10 which are further purified by HPLC using a C4 column with 5-80~ acetonitrile in TEAA, 0.1 M, pH 7.0 as the eluant. Alternatively the libraries can be reduced using sodium borohydride instead of sodium cyanoborohydride.
In this case each sub-library is ~uspended in 25 mM
Na2B407, pH 9.0 (1 mL) and initially treated with formaldehyde (10 equiv, 0.9 mg) for 30 minutes followed by NaBH4 (3 equiv, 0.34 mg). A~ter 5 hours the reaction mixture is loaded on Sep-Pak cartridge as is described above and the desired material is eluted using 50:so H20/CH30H (5 mL).

ExamDle 55 SYnthesis of a Library of Non-Nucleotide Pho-RPhorus E~ter Olic~omer~ in which A i-Q a~l N-T-i nkQ~ Amino Acid Deri~ative m e library i~ synthesized on 10 columns each loaded with 60 mg o~ modified solid s~oLL that had been derivatized with glycolic acid. Each o~ the 10 ~n~m~s is coupled to its speci~ied column, monomer one to column one, mnn~m~r 2 to column 2, etc., up to monomer 10 to column 10. The e~iciency of each coupling is checked by monitoring the intensity o~ the trityl cation released from each coupling step to be in exces~ o~ 90 ~
throughout all of the coupling steps. After addition of the mon~m~s, the resins are then removed from the columns and pooled and di~ided using the isopycnic slurry method, with acetonitrile as the solvent. The synth~sis columns are weighed to ensure an even division of the resin among the columns. After addition of all 10 monomers as previously described, followed by a second pool-and-divide procedure, the ~Qnnllcleotide H-phosrhnn~te linkages are converted to phosphoramidate linkage using the ~ollowing oxidation procedure with each SVBST1TVTE SH EET ~RULE 26) __ _ _ W O 97/28168 PCT~US97/01060 column being oxidized with one of the following ~ino~cid derivatives: L-methionine ethyl e~ter HCl, L~ ni ne methyl ester, L-valine ethyl ester HCl, L-phenyl~l~nin~
methyl ester HCl, L-glutamic acid diethyl ester, L-tyrosine ethyl ester HCl. A 12~ solution o~ each aminoacid derivative is prepared in DMF/CC14 (1:1, 5 mL) and the solution is trea~ed wi~h 1 equiv of triethyl ~mi ne, ~iltered and the solution is used directly ~or oxidation of the H-phosphonate moieties. Oxidation is carried out on the DNA ~ynthesizer using a 30 minute reaction time. The amino acid solutions are then ~lushed ~rom the columns and the supports are wa~hed extensively with acetonitrile. The supports are removed ~rom the columns and pooled and divided as descri~ed above. A
second round of mnnom~ addition ~ollowed by pooling, dividing and addition of amino acid derivative is carried out, ~ollowed by addition of the synthon required for introduction of the label (either fluorescein phosphoramidite, dimethoxytritylthymidine H-phosphonate or m-hydroxyacetoph~no~e phosphoramidite). The supports are then l_...oved ~rom the columns and treated with ethyl~ne~i~;n~ in acetonitrile (1-5 mL, 2:1) for 1 hour at 55~C. The resultant suspensions are ~iltered and washed with ethanol (2 X ~00 ~1). The filtrate~ are combined, concentrated and then redissolved in water and purified by reversed-phase ~PLC using a gradient of 5--100~ acetonitrile in TEAA, O.1 M, pH 7Ø The puri~ied ~ractions are c~h; n~ to give a library with the structure as ~ollows:

c~
B1 Rl--O I ORl--B2 - A -n where B, is a labeling group, B2 is a glycolic ethylene~i~min~ amide, A is an N-linked amino acid ester, ~q SUBSlTI UTE SH EET (RULE 26) W O 97/28168 PCTrUS97tO1060 and Rl and ~ are derived from diols as previously de~cribed.

ExamDle 56 B;n~in~ of a LibrarY of Pho~Dhorus Ester Oliqomer~ to Thrombin A library of non-nucleotide phosphorus ester oligomers was screened again~t the target protein thrombin for high affinity ligands, using the COMPILB
method as outlined in copendiny patent application ~Determination and Identification of Active Compounds in a Compound Library," U. S. patent application serial number 08/223,519. Ri n~; ng reactions consisting of 100 ~M oligomeric library and 1 ~M target protein as well as a protein-minus control were prepared in a buffer c~nt~;n~ng 14~ mM NaCl, 1.75 mM MgCl2, 4 mM Na2HPO4, 1.35 mM KCl, 0.45 mM CaCl2, O.75 mM KH2PO4 and lZ.5 mM Tris-HCl (pH 7.5). The reactions were e~l;l;h~ated $or 15-30 min at room temperature prior to centri$ugation through Microcon-10 ~pin filtration units (M.Wt. cutof~ =
10,000). The M.Wt. cuto$f of the $iltration unit wa~
selected to allow $ree passage o~ the oligomeric library members but not the target protein or the oligomeric molecules bound to it.

The centrifugation was adjusted to allow d~L~imately 360 ~L of the total reaction volume o~ 400 ~L to flow through the ~ ..~ ~ne, resulting in the loss of approximately 90~ of the unbound oligomeric library from the ret~;n~A sample. The volume of the ret~;n~A sample was ~l~yllt up to the original 400 ~h with buffer to which was ~ A 20~ of the original target concentration to compen~ate for losses/inactivation O$ the target during the experiment. After four rounds of selection the library was labeled with 32p using T4 kina~e followed by puri$ication of the labeled library by $orced 1~' SUBSllTUTE SHEET ~RULE 26) W O 97/28168 P~~ 7/01060 dialysis Approximately 38% of the 32p counts were incorporated into the library. During subse~uent rounds of ~election the unincorporated counts ~lowed freely through the Microcon-10 ...e~ ne. To control for unincorporated counts ret~tn~A, a "no library" sample was carried along with the library sample. The ~no library~
sample was identical to the library sample with the exception that no library wa~ added to the reaction at the beg~ nn; n~ of the selection.

The results of this serial selection are presented in Figure 4. The concentration o~ the oligome~ic library present in the re~ n~ fraction (as measured by counts due to the 32p tag incorporated into the library members) is plotted as a ~unction of the rounds of selection -i.e. the number of dialy~is-based separations. Data are not presented for the ~irst few rounds of selection since the library was labeled after four rounds of selection.

The progressive increase in the fraction of compounds ret~i n~ at each round is evidence of a subpopulation of the original library that binds to the target. This subpopulation is enriched with each successive round of selection due to selective retention of compound~ in the library that bind to the target protein. From the target protein concentration and the curve shown in Figure 3, it is possible to estimate that this subpopulation of thro ~hin-h; n~; ng oligomeric species i~ between 0.001 and 0.01% of the original mixture, and that it bind~ an apparent Kd below 100-500 nM.

lOI

SU~S 1 1 1 UTE SH EET (RVLE 26) W O 97128168 PCTrUS97/01060 ExamDle 57 Prolonqation of the Thrombin Tn~c~ Clottinq Time bY a Li~rarY of Non-Nucleotide Phos~horus E ter Oli~omer~

Libraries of non-nucleotide phosphorus ester oligomers were prepared as previously described and tested for their ability to ~nh;h; t throTnbin activity as measured by an extension of the time for thrombin-catalyzed fibrin clot formation. The assay was perfonmed as previou~ly de~cribed by Bock et al. ~llm~n fi~rinogen in 140 mM NaCl, 5 mM K~l, 1 mM MgCl2, 1 mM
CaCl2, 20 mM Tris acetate, pH 7.4 ~100 uL) was eguilibrated at 37~C for 1 min in the presence of the library of non-nucleotide phosphorus ester oligomers.
Clotting reactions were initiated by the addition of h~ n thromh~n (100 uL in the same buf~er preeguilibrated to 37 ~C ~or 1 min) to ~inal concentrations of 2 mg/mL
~ibrinogen and 3 nM thrombin. The ~irst indication of the clot was ~aken as the clotting time. Clot formation was usually complete within 5 sec o~ the first appearence of the clot. As shown in Figure 5, the library substantially prolonged the thrombin induced clotting time as c~r~ed with control reactions without addition o~ library.

The inhibition o~ clotting was furthe~
inve~tigated by carrying out the same clotting reaction using various concentrations o~ the li~rary of non-nucleotide pho phorus e~ter oligomers. As shown in Figure 6, clotting wa~ strongly dose depPn~Pnt.

Literature Cited Andrus et al., Tetrahedron ~ett., 29: 861 (1988).
Beaucage et al.,Tetrahedron Letts., 22:1859-1862, (1981).
Bock et al.,Nature, 355:564-566, (1992).
Bohacek et al., JACS, 116:5560-5571 (1994).
Cload and S~r~tz, JACS, 113:6324-6326 (1991).
Durand et al., Nucl. Acids Res., 18:6353-6359 (1990).
1~

SUBSTITUTE SHEET (RULE 26) W O 97/28168 PCTrUS97/01060 ~cker, Meeting, "Exploiting Mo~ecular Diversity." San Diego, CA, ~an 12-14 (1994).
Ecker et al., W0 93/04204 (1993).
Fon~nel et al., Nucleic. Acids Res., 22:2022-2027, 1994.
Furdon et al., Nuc. Ac. Res., 17:9193-9204 (1989).
Furka et al., Int. J. Protein Res., 37:487-493 (1991).
Gallop et al., Proc. Natl. Acad. Sci. USA, 90:10700-10704 (1994).
Gallop et al ~. Med. C~em., 37:1233-1251 (1994).
Gordon et al., J. Med. Chem., 37:1385-1401 (1994).
Grollman et al., J. Biol. Chem. 262:10171 (1987).
Hardy and ~affe, C~emical Abs~racts, 92:42894 (1980).
Hata et al., Tetrahedron Lett., 26:935 (1986).
Hovinen et al., Tetrahedron, 50:7203-7218, 1994.
T.~n~, Chem. & Biol., 1: 73-78 (1994).
Leumann et al., Helvetica Chemica Acta, 79:481-510 (1993).
Matteucci et al., JACS, 11~:9816-9817 (1993).
Ogilvie et al., Tetrahedron Lett., 21:4149 (1980).
Richardson and S~h~tZ, JACS, 113:5109-5111 (1991).
Salunkhe et al., JACS, 114:8768-8772 (1992).
Sambrook et al., Molecular Cloning, a Laboratory M;7n 2nd edition; p. 5.68, Cold Spring Harbor Laboratory Press: Cold Spring Harbor, NY, 1989.
Sase et al., Chen7ical Abstracts, 108: 6181 (1988).
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Zuckerman et al., J. Med. Chem., 37:2678-2685 (1994).

~'3 SUBSTlTlJTE SH EE'r tRUI E Z6)

Claims (50)

What Is Claimed Is:

1. A phosphorus ester oligomer of monomeric units, which oligomer has the structure:

wherein A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
B1 and B2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
R1 can be the same or different in each monomeric unit, and in at least one of the non-nucleotide monomeric units, R1 is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic functionality (iv) a diol attached to a ring-substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label; and n is at least one.

2. The oligomer of claim 1 wherein the hydrogen bond donor contains a hydroxyl, amide, imide or thiol group.

3. The oligomer of claim 1 wherein the hydrogen bond donor is a basic compound.

4. The oligomer of claim 3 wherein the basic compound contains an amine moiety.

5. The oligomer of claim 3 wherein the basic compound is a substituted or unsubstituted thiazole.

6. The oligomer of claim 1 wherein R1 is a hydrogen bond acceptor which further includes an amine or ether moiety.

7. The oligomer of claim 1 wherein the hydrogen bond acceptor is selected from a 5-substituted 2-hydroxymethyl-3-hydroxy-tetrahydrofuran and a bis (hydroxyalkyl)-substituted heterocycle.

8. The oligomer of claim 1 wherein the hydrogen bond acceptor is a substituted or unsubstituted theophylline.

9. The oligomer of claim 1 wherein R1 is a hydrophobic functionality selected from the group consisting of aromatic rings, alkanes, cycloalkanes and aromatic rings fused to cycloalkanes.

10. The oligomer of claim 9 wherein R1 is a hydrophobic functionality selected from a substituted alkane, a 3,3-disubstituted 3-amino-1,2-propanediol, a substituted or unsubstituted alicyclic ring wherein the ring size is from 4-12, a 3-substituted indole, a substituted or unsubstituted hydroxyalkyl phenol and an alicyclic dicarboxylic acid.

11. The oligomer of claim 1 wherein the anionic functionality is an alicyclic dicarboxylic acid moiety.

12. The oligomer of claim 11 wherein the anionic functionality is a tricyclononene dicarboxylic acid moiety.

13. The oligomer of claim 1 wherein the anionic functionality is a cyclopentane acetic acid moiety.

14. The oligomer of claim 1 wherein the cationic functionality is a bis(hydroxyalkyl) -substituted nitrogen-containing heterocycle.

15. The oligomer of claim 14 wherein the cationic functionality is selected from a substituted alkane and a 3,3-disubstituted 3-amino-1,2-propanediol.

16. The oligomer of claim 1 wherein in at least one of the monomeric units R1 is a heterocyclic, an alicyclic or a polycyclic ring system.

17. The oligomer of claim 16 wherein the heterocyclic ring system contains an indole, thiazole, imidazole, pyridine, purine or pyrimidine ring.

18. The oligomer of claim 16 wherein the alicyclic ring system contains a cyclopentane or cyclooctane ring.

19. The oligomer of claim 18 wherein the cyclopentane or cyclooctane ring system is substituted with at least one carboxylic acid moiety.

20. The oligomer of claim 16 wherein the polycyclic ring system contains a bicyclic or tricyclic ring or is a polycyclic arene.

21. The oligomer of claim 20 wherein the bicyclic ring is a bicyclic alkane.

22. The oligomer of claim 21 wherein the bicyclic ring is bicycloheptane.

23. The oligomer of claim 20 wherein the tricyclic ring is an alkene.

24. The oligomer of claim 23 wherein the alkene is tricyclononene.

25. The oligomer of claim 20 wherein the polycyclic arene is diphenyl bicyclooctane.

26. The oligomer of claim 1 wherein A is selected from the group consisting of 2-(2-aminoethyl)pyridine, 1-(2-aminoethyl)piperidine, 2-(3,4-dimethyloxy-phenyl) ethylamine, 4-(2-aminoethyl)-morpholine, 1-(3-aminopropyl) -4-methylpiperazine, 1-(3-aminopropyl)-2-pyrrolidinone, 1-(3-aminopropyl)imidazole, 1-(2-aminoethyl)pyrrolidine, 3-aminopropionitrile, 2-(2-aminoethyl)-1-methyl-pyrrolidine, 4-fluorophenethylamine, 4-bromophenethylamine, aminomethyl-cyclopropane, 3,3-diphenylpropylamine, formylpiperazine, trifluoromethyl-phenylpiperazine, thiomorpholine, 1-(2-pyridyl)piperazine, homopiperazine, hexamethyleneimine, cis-2,6-dimethylmorpholine, 2,5-dimethylphenylpiperazine, 3,5-dimethylpiperidine, 1-(4-fluorophenyl) piperazine, N-(3, 4-dichlorophenyl)piperazine, 2-(4-chlorophenyl)ethylamine, 4-piperazineacetophenone, 4-piperidinopiperidine, 2-thiophenemethylamine, furfurylamine, heptamethyleneimine, and 1-(4-methoxyphenyl)piperazine.

27. The oligomer of claim 1 wherein A is an N-linked amino acid.

28. A phosphorus ester oligomer of monomeric units, which oligomer has the structure:

wherein A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
B1 and B2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
R1 can be the same or different in each monomeric unit and in at least one of the monomeric units is independently selected from a condensation product of:
(i) an aliphatic acyclic diol wherein the diol hydroxyl groups are non-vicinal or are substituted;
(ii) a purine- or pyrimidine-substituted variant of the diols of (i) or of aliphatic acyclic vicinal diols;
(iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety;
(iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;
(v) a hydroxy- or hydroxyalkyl-substituted tetrahydrofuran;
(vi) an indole-substituted acyclic aliphatic diol;
(vii) an aromatic ring or ring system having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl; and (ix) a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, any of which can further include a detectable label; and n is at least one.

29. The oligomer of claim 28 wherein n is 1-20.

30. The oligomer of claim 28 wherein A is selected from the group consisting of 2-(2-aminoethyl)pyridine, 1-(2-aminoethyl)piperidine, 2-(3, 4-dimethyloxyphenyl)ethylamine, 4-(2-aminoethyl)-morpholine, 1-(3-aminopropyl)-4-methylpiperazine, 1-(3-aminopropyl)-2-pyrrolidinone, 1-(3-aminopropyl)imidazole, 1-(2-aminoethyl)pyrrolidine, 3-aminopropionitrile, 2-(2-aminoethyl)-1-methyl-pyrrolidine, 4-fluorophenethylamine, 4-bromophenethylamine, aminomethyl-cyclopropane, 3,3-diphenylpropylamine, formylpiperazine, trifluoromethylphenylpiperazine, thiomorpholine, 1-(2-pyridyl)piperazine, homopiperazine, hexamethyleneimine, cis-2,6-dimethylmorpholine, 2,5-dimethylphenylpiperazine, 3,5-dimethylpiperidine, 1-(4-fluorophenyl) piperazine, N-(3, 4-dichlorophenyl)piperazine, 2-(4-chlorophenyl)ethylamine, 4-piperazineacetophenone, 4-piperidinopiperidine, 2-thiophenemethylamine, furfurylamine, heptamethyleneimine and 1-(4-methoxyphenyl)piperazine.

31. The oligomer of claim 28 wherein A is an N-linked amino acid.

32. A non-nucleotide monomeric unit having the structure:

wherein X1 is a protecting group;
X2 is a branched or unbranched lower alkyl group or a substituted or unsubstituted alkoxy group;
Y is a branched or unbranched lower alkyl group;
and R1 is selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label.

33. The non-nucleotide monomeric unit of claim 32 wherein the anionic functionality is a cyclopentane acetic acid moiety.

34. The non-nucleotide monomeric unit of claim 32 wherein the cationic functionality is a bis(hydroxyalkyl)-substituted nitrogen-containing heterocycle.

35. The non-nucleotide monomeric unit of claim 32 wherein the cationic functionality is a 3,3-disubstituted 3-amino-1,2-propanediol.

36. The non-nucleotide monomeric unit of claim 32 wherein R1 is a condensation product of:
(i) an aliphatic acyclic diol wherein the diol hydroxyl groups are non-vicinal or are substituted;

(ii) a purine or pyrimidine substituted variant of the diols of (i) or of aliphatic acyclic hydrocarbon vicinal diols;
(iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety;
(iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;
(v) a hydroxy or hydroxyalkyl substituted tetrahydrofuran;
(vi) an indole substituted acyclic aliphatic diol;
(vii) an aromatic ring or ring system having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl; and (ix) a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, and in which R1 can optionally be substituted with a detectable label.

37. A non-nucleotide monomeric unit having the structure:

wherein X is a protecting group;
R1 is selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle; (iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophobic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label.

38. The non-nucleotide monomeric unit of claim 37 wherein the anionic functionality is a cyclopentane acetic acid moiety.

39. The non-nucleotide monomeric unit of claim 37 wherein the cationic functionality is a bis(hydroxyalkyl)-substituted nitrogen-containing heterocycle.

40. The non-nucleotide monomeric unit of claim 37 wherein the cationic functionality is a 3,3-disubstituted 3-amino-1,2-propanediol.

41. The non-nucleotide monomeric unit of claim 37 wherein R1 is a condensation product of:
(i) an aliphatic acyclic hydrocarbon diol wherein the diol hydroxyl groups are non-vicinal or are substituted;
(ii) a purine- or pyrimidine-substituted variant of the diols of (i) or of aliphatic acyclic vicinal diols;
(iii) an acyclic aliphatic diol having an amino group with at least one hydrogen substitution moiety;
(iv) an alicyclic or polycyclic diol, optionally substituted with a carboxy or carboxyalkyl substituent;

(v) a hydroxy- or hydroxyalkyl-substituted tetrahydrofuran;
(vi) an indole-substituted acyclic aliphatic diol;
(vii) an aromatic ring or ring system having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl;
(viii) a heterocyclic compound having two substitutions independently selected from the group consisting of hydroxy or hydroxyalkyl; and (ix) a dihydroxyalkyl thiazole or dihydroxyalkyl oxazoline, and in which R1 can optionally be substituted with a detectable label.

42. A combinatorial library comprising a mixture of the oligomers of claim 1.

43. A combinatorial library comprising a mixture of the oligomers of claim 16.

44. A combinatorial library comprising a mixture of the oligomers of claim 28.

45. A phosphorus ester oligomer of monomeric units, which oligomer has the structure:

wherein A can be the same or different in each monomeric unit and each is independently selected from the group consisting of oxygen, sulfur, lower alkyl, substituted or unsubstituted alkylamino, substituted or unsubstituted arylamino and aminoalkyl;
B1 and B2 can be the same or different and each is independently selected from hydrogen, lower alkyl, a labeling group, a protecting group, a phosphoramidate or a phosphomonoester;
R1 can be the same or different in each monomeric unit, and is a nucleoside moiety or, in at least one of the non-nucleotide monomeric units, R1 is independently selected from the group consisting of a condensation product of (i) a non-vicinal diol attached to a hydrogen bond donor functionality; (ii) a hydrogen bond acceptor selected from an ether, a purine or pyrimidine substituted 1,2-diol or a disubstituted heterocycle;
(iii) a non-vicinal diol attached to a hydrophobic functionality or a vicinal diol attached to an aliphatic or alicyclic hydrophohic functionality (iv) a diol attached to a ring substituted anionic functionality and (v) a cationic moiety attached to a non-vicinal or alicyclic diol, any of which can further include a detectable label; and n is at least one.

46. The oligomer of claim 45 wherein the nucleoside moiety is a ribonucleoside, 2'-O-methyl- ribonucleoside or a deoxyribonucleoside moiety.

47. The oligomer of claim 1 wherein n is 1-20.

48. The phosphorous ester oligomer of claim 45 wherein at least one of B1 and B2 is a nucleotide.

49. The phosphorus ester oligomer of claim 45 wherein at least one of B1 and B2 is a phosphodiester attached to a glycolic amide moiety.

50. The phosphorous ester oligomer of claim 45 wherein at least one of B1 and B2 is a phosphodiester attached to a tritium-labeled p- or m-1-hydroxy (C1-C6 alkyl) phenyl.