|||||||||||||III US005391785A United States Patent 19 11) Patent Number: 5,391,785 Jones Et Al

|||||||||||||III US005391785A United States Patent 19 11) Patent Number: 5,391,785 Jones et al. 45) Date of Patent: Feb. 21, 1995

54 INTERMEDIATES FOR PROVIDING FOREIGN PATENT DOCUMENTS FUNCTIONAL GROUPS ON THE 5' END OF 0147768 2/1989 European Pat. Off. . OLGONUCLEOTDES 0354323 2/1990 European Pat. Off. . 75 Inventors: David S. Jones; John P. Hachmann; 0399330 11/1990 European Pat. Off. . Michael J. Conrad, all of San Diego; 3937116 5/1991 Germany . o gO; 4-312541 11/1992 Japan ...... 568/672 StephenDouglas Coutts,A. Livingston, Rancho San Santa Diego, Fe; all wo85/03704110434 5/19648/1985 wiPo.Norway ...... 568/672 of Calif. WO86/04093 7/1986 WIPO . 73) Assignee: La Jolla Pharmaceutial Company, WO87/02777 5/1987 WIPO : San Diego, Calif. OTHER PUBLICATIONS 21) Appl. No.: 915,589 IMitsuo et al., "Preparation of lecithin-type of phos pholipid analogues and mesomorphic states' Chem. 22 Filed: Jul. 15, 1992 Pharm. Bull. (1978) 26(5):1493-1500. Gupta et al., “A general method for the synthesis of Related U.S. Application Data (List continued on next page.) 63 Continuation-in-part of Ser. No. 731,055, Jul. 15, 1991, abandoned, which is a continuation-in-part of Ser. No. Primary Examiner-John W. Rollins 494,118, Mar. 13, 1990, Pat. No. 5,162,515, which is a Assistant Examiner-L. Eric Crane continuation-in-part of Ser. No. 466,138, Jan. 16, 1990, Attorney, Agent, or Firm-Morrison & Foerster abandoned. 51) Int. Cl.' ...... CoTB 39/00 ABSTRACT 52 U.S. C...... 552/105:536/25.32; Phosphoramidites of the formula 549/214; 549/388 58) Field of Search ...... 536/25, 32; 560/112, R-O-P-O-G-Z 560/180; 549/453,214, 388; 568/648, 672; 552/105 R11 NR2 56 References Cited U.S. PATENT DOCUMENTS T. 909,012 4/1973 McCollum .. ... 549/453 where R is a base-labile protecting group, R and R2 are 2,028,403 1/1936 Mares ...... i06/177 individually alkyl of 1 to 6 carbon atoms, cycloalkyl of 2,362,326 11/1944 Thurston et al...... 560/188 3 to 8 carbon atoms, or aryl of 6 to 20 carbon atoms or 2,585,884 2/1952 Whetstone et al. 2,619,493 11/1952 Norris...... -- 27. are joined together to form with the nitrogen atom a 2,946,806 7/1960 Nentwig et al. ... . 549/333 cyclic structure of 4-7 carbon atoms and 0 to 1 annular 3,004,894 10/1961 Johnson et al...... 514/152 chalcogen atoms of atomic number 8 to 16, G is a hy 3,201,420 8/1965 Fuzesi et al. ... - - - - - 549/372 drocarbylene group of 1 to 20 carbon atoms and Z is a 3,225,063 12/1965 D'Alelio ...... ------549/229 hydroxy-protected vicinal diol group bound to G by 3,308,064 3/1967 Schlight et al...... 252/46.6 one of the vicinal diol carbon atoms or a disulfide group 3,926,912 12/1975 Mayer-Mader et al. 4,058,550 1 1/1977 Shepard et al...... - - - - 526/20 and bound to G by one of the sulfur atoms of the disul 4,191,668 3/1980 Katz ...... : fide group, with the proviso that G is of at least 4 car 4,220,565 9/1980 Katz ...... 525/591 bon atoms when Z is said disulfide group are used in 4,465,869 8/1984 Takaishi et al. - we a 568/672 conventional automated oligonucleotide synthesis to 4,575,558 3/1986 Mai et al...... 549/453 introduce a functional aldehyde or thiol group on the 5' 4,588,824 5/1986 Baldwin et al. ... 549/453 end of the oligonucleotide to thereby provide a reactive 4,650,675 3/1987 Borel et al. ... " .2/85 site on the oligonucleotide that may be used to conju 4,751,181 6/1988 Keene ...... 435/70 gate the oligonucleotide to molecules that contain a free 4,845,219 7/1989 Inoue et al. ... - - - - 10 o 4,877,891 10/1989 Becker et al. .. ------...,S. amino group or an electrophilic center reactive with a 4,983,759 1/1991 Inoue et al...... 500/174 thiol group. 4,997,928 3/1991 Hobbs ...... 536/027 5,252,760 10/1993 Urdea et al...... 552/105 6 Claims, 5 Drawing Sheets 5,391,785 Page 2

OTHER PUBLICATIONS Jackson et al., “Synthetic Glycerides. IV. Esters of 3'-sulfhydryl and phosphate group containing oligonu Aromatic and Aliphatic Acids,’ J. Am. Chem. Soc., 55, cleotides' Chem. Abstracts (1991) 115:976 (abstract No. 678-680 (1933). 183758p). Fréchet et al., “Use of Polymers as Protecting Groups in Organic Synthesis. III. Selective Functionalization of Agrawal S., et al., Nucl. Acids Res. (1986) Polyhydroxy Alcohols, Can. J. Chem, 54, 926-934 14(15):6227-6245. (1976). Kremsky J., et al., Nucl. Acids Res. (1987) Ichikawa et al., “An Asymmetric Synthesis of Glycerol 15(7):2891-2909. Derivatives by the Enantioselective Acylation of Pro Eshhar Z., et al., J. Immunol. (1975) 114(2):872-876. chiral Glycerol,” Chem. Lett, 1984, 949-952. Borel Y., et al., Science (1973) 182:76-78. Sedarevich, "Glyceride Isomerizations in Lipid Chem Parker L., et al., J. Immunol. (1974) 113(1):292-297. istry,” J. Am. Oil Chem. Soc., 44(7), 381--393 (1967); Borel H., et al., Ann. N.Y. Acad. Sci (1986) 475:296-306. Chem. Abstr., 67, p. 10965, Abstr. No. 116524m (1967), Papalian M., et al., J. Clin. Invest. (1980) 65:469-477. Only abstract supplied. Stollar B., et al., J. Clin. Invest. (1980) 66:210-219. Shvets et al., “Lipids. LIX. Determination of the Con Borel Y., et al., J. Clin. Invest. (1988) 82:1901-1907. figuration of a, g-Diglycerides Prepared from D-Man Williams et al., “Stereocontrolled Transformations of nitol,” Zh. Org. Kihm., 4(4), 597-603 (1968); Chem. Orthoester Intermediates into Substituted Tetrahydro Abstr., 69, p. 231, Abstr. No. 2454z, (1968); Only ab furans,” J. Am. Chem. Soc., 106(9), 2641-2644 (1984). stract supplied. Verkade et al., “Synthesen von Glyceriden mit Hilfe Hedgley et al., “Glycerol Esters of Thiobenzoic O-A- von Tritylverbindungen. IV., Recl. Tray. Pays-Bas, 59, cids and Thioglycerol Esters of Benzoic Acid, J. 1123-1140 (1940). Chem. Soc. C, 1970, 467-471. Baggett et al., “Aspects of Stereochemistry. Part XXI. Barker et al., "Herbicidal (Benzyloxy)dioxanes and Absolute Configuration of Benzylidene Derivatives of (Benzyloxy)methyldioxolanes,” Ger. Offen. 2,326,586, Some Acyclic Polyhydric Alcohols,” J. Chen. Soc., 06 Dec. 1973; Chem. Abstr., 80, p. 360, Abstr. No. 1965, 3394-3400. 59946t (1974); Only abstract supplied. U.S. Patent Feb. 21, 1995 Sheet 1 of 5 5,391,785

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U.S. Patent Feb. 21, 1995 Sheet 3 of 5 5,391,785

VETRITTOIHIL Iwao):~~JLVdIIGIIwao):JºJº U.S. Patent Feb. 21, 1995 Sheet 4 of 5 5,391,785

U.S. Patent Feb. 21, 1995 Sheet 5 of 5 5,391,785

5,391,785 1. 2 A principal object of the present invention is to pro NTERMEDIATES FOR PROVIDING vide novel modified phosphorous intermediates that FUNCTIONAL GROUPS ON THE 5' END OF may be employed in the various types of oligonucleo OLGONUCLEOTDES tide synthesis methods and which have activatable groups that may be converted to a free aldehyde or CROSS-REFERENCE TO RELATED sulfhydryl group once they have been added onto the 5' APPLICATIONS of an oligonucleotide. The free aldehyde/sulfhydryl This application is a continuation-in-part of applica group is useful for coupling or conjugating the oligonu tion Ser. No. 731,055, filed Jul. 15, 1991, now abandoned, cleotide to labels, ligands, polymers or solid surfaces. which in turnis a continuation-in-part of application Ser. 10 These new intermediates meet the following criteria: 1) No. 494,118, filed Mar. 13, 1990, now U.S. Pat. No. the activatable group is compatible with all steps of 5,162,515, which in turn is a continuation-in-part of conventional oligonucleotide synthesis procedures; 2) application Ser. No. 466,138, filed Jan. 16, 1990, now the activation is effected under conditions that do not abandoned. These parent applications are incorporated damage the oligonucleotide; 3) the coupling is effected herein by reference. 15 under conditions that do not damage the oligonucleo tide or the moiety to which the oligonucleotide is cou DESCRIPTION pled. 1. Technical Field This invention is in the field of organophosphate DISCLOSURE OF THE INVENTION chemistry and solid state oligonucleotide synthesis. 20 The novel phosphorus-containing compounds of the More particularly, it concerns reactive phosphorous invention include intermediates that are useful in the intermediates that may be stably attached to the 5' end H-phosphonate, phosphotriester, phosphorchloridite of an oligonucleotide and which have an activatable and phosphoramidite methods of oligonucleotide syn moiety which, when activated, provides a functional thesis as well as intermediates that result in 5' modifica aldehyde or sulfhydryl group that may be used to con 25 tions that involve phosphodiester analogs such as jugate the oligonucleotide to any molecule having a free methyl phosphonates, methyl phosphates, phosphoro amino group. thioates and phosphoramidates. 2. Background These compounds may be defined generically by the It is necessary to provide oligonucleotides with a free following formula functional group in order to couple the oligonucleotide 30 to labels, ligands, solid surfaces, polymers or other mol (1) ecules or surfaces. One technique for providing oligonucleotides with a terminal functional group involves synthesizing the 35 desired oligonucleotide by conventional solid-state au tomated synthesis procedures and incorporating the where X is: functional group at the 5' end of the oligonucleotide via (i) oxygen when X1 is O and X2 is hydrogen or a modified phosphoramidite. RO- where R is a protecting group; Agrawal, S., et al., Nucl. Acids Res. (1986) (ii) not present when 14:6227-6245, describes a modified phosphoramidite (a) X1 is chlorine and X2 is methyl or RO-, or that may be introduced on the 5' of an oligonucleotide when that has an activatable group that may be activated (b) X2 is RO- and X1 is NRR2 where R1 and R2 through deprotection to provide a free amino group on are individually alkyl of 1 to 6 carbon atoms, the 5' terminus of the oligonucleotide. The linker (VIII 45 cycloalkyl of 3 to 8 carbon atoms, or aryl of 6 to on page 6236), O-(2-(9-fluorenylmethoxycarbonyl) 20 carbon atoms or are joined together to form aminoethyl)-O-(2-cyanoethyl)-N-N-diisopropyl phos with the nitrogen atom a cyclic structure of 4-7 phoramidite, is added to the end of the desired oligonu carbon atoms and 0 to 1 annular chalcogen atoms cleotide on an automated DNA synthesizer using deox of atomic number 8 to 16 inclusive (O or S); ynucleoside-2-cyanoethyl-N-N-diisopropyl phos 50 G is a hydrocarbylene group of 1 to 20 carbon atoms; phoramidites. The adduct is deprotected (the 9 and fluorenylmethoxycarbonyl group is removed with am Z is a hydroxy-protected vicinal diol group bound to G monia) to provide a free amino group. by one of the vicinal diol carbon atoms or a disulfide Kremsky, J. N., et al., Nucl. Acids Res. (1987) group bound to G by one of the sulfur atoms of the 15:2891-2909, describes a functionalized phosphorami 55 disulfide group, with the proviso that G is of at least 4 dite (1 on page 2893) that is introduced onto the 5' end carbon atoms when Z is said disulfide group. of an oligonucleotide and then modified to provide a 5' The above compounds where X is oxygen, X1 is O-, carboxy or aldehyde group that is used to immobilize and X2 is hydrogen are H-phosphonates and are em the oligonucleotide. ployed in the H-phosphonate method of oligonucleo Another functionalized phosphoramidite, O-6-(4,4'- tide synthesis (Sinha and Cook, NAR (1988) dimethoxytriphenylmethylthio)hexyl-O-(2-cyanoe 16:2659-2669). H-phosphonates may be converted to thyl)-N,N-diisopropylphosphoramidite, is available phosphite diesters, phosphorothioates, or phosphorami commercially from Clontech Laboratories. This mole dates once they are incorporated onto the 5' end of the cule is incorporated into oligonucleotides using conven oligonucleotide (Miller et al., NAR (1983) tional phosphoramidite protocols. The dimethoxytrity 65 11:5189-5204, Eckstein, Ann Rey Biochem (1985) protected sulfhydryl group may be deprotected with 54:367-402, and Froehler and Matteucci, NAR (1988) silver nitrate to yield a free sulfhydryl at the 5' of the 16:4831-4839). Correspondingly, the above compounds oligonucleotide chain. where X is oxygen, X is O and X2 is RO- are used 5,391,785 3 in the phosphotriester approach to synthesizing oligo nucleotides (Garegg, et al., Chemica Scripta (1985) A- a (4) 26:5). When X is not present and X1 is chlorine and X2 a Y is RO-, the resulting compound is a phosphochloridite and it is used in the phosphochloridite technique for Y- -y oligonucleotide synthesis (Wada et al., J Org Chem H---a-oh (1991) 56: 1243–1250). The phosphoramidites of the H. H. above formula are preferred. The preferred phosphoramidites of the invention may where Yl and Y2 are individual hydroxyl protecting be represented by the formula: 10 groups or are joined by a single-atom bridge to form a five-membered ring protecting group, and G is de scribed as above. Preferably G is alkylene of 4 to 20 R-O-P-O-G-Z (2) carbon atoms. Another aspect of the invention is a disulfide of the 15 formula V N s 1 Y3-O-G-S-S-G-OH (5) where R is a base-labile protecting group, R and R2 are wherein Y3 is a hydroxyl protecting group and G is as individually alkyl of 1 to 6 carbon atoms, cycloalkyl of 20 described above. The two divalent groups represented 3 to 8 carbon atoms, or aryl of 6 to 20 carbon atoms or by G may be the same or different. Preferably they are are joined together to form with the nitrogen atom a the same, making the disulfide symmetrical. Preferably cyclic structure of 4-7 carbon atoms and 0 to 1 annular Y3 is base stable. Preferably G is alkylene of 4 to 20 chalcogen atoms of atomic number 8 to 16 inclusive (O carbon atoms. or S), G is a hydrocarbylene group of 1 to 20 carbon 25 atoms and Z is a hydroxy-protected vicinal diol group BRIEF DESCRIPTION OF THE DRAWINGS bound to G by one of the vicinal diol carbon atoms or FIGS. 1-3 are schematic diagrams of the synthesis a disulfide group bound to G by one of the sulfur atoms schemes described in Examples 1-3. of the disulfide group, with the proviso that G is of at FIGS. 4 and 5 are autoradiograms of the gels de least 4 carbon atoms when Z is said disulfide group. 30 scribed in Examples 5 and 6. Another aspect of the invention is a 5' modified oligo MODES FOR CARRYING OUT THE nucleotide of the formula: INVENTION As indicated above, the phosphoramidites of the in X1 (3) 35 vention may be represented by the formula:

X R-O-P-O-G-Z (2) where (O.N.) represents an oligonucleotide chain, X is a chalcogen atom of atomic number 8 to 16, inclusive (O or S), X1 is O-, methyl, --OCH3 or NR1R2 where R1 W. s, 1 and R2 are individually hydrogen or alkyl of 1 to 6 carbon atoms, G is a hydrocarbylene group of 1 to 20 where R is a methyl or a base-labile protective group, carbon atoms and Z is a hydroxy-protected vicinal diol 45 R1 and R2 are alkyl of 1 to 6 carbon atoms, cycloalkyl of group bound to G by one of the vicinal diol carbon 3 to 8 carbon atoms, or aryl of 6 to 20 carbon atoms or atoms or a disulfide group bound to G by one of the are joined together to form with the nitrogen atom a sulfur atoms of the disulfide group, with the proviso cyclic structure of 4-7 carbon atoms and 0 to 1 annular that G is of at least 4 carbon atoms when Z is said disul chalcogen atoms of atomic number 8 to 16 inclusive (O fide group. 50 or S), G is a hydrocarbylene group of 1 to 20 carbon A further aspect of the invention is the above atoms and Z is a hydroxy-protected vicinal diol group described modified oligonucleotides where the hydroxy covalently bound to G via one of the vicinal carbon protecting groups have been removed to leave free atoms or a disulfide group that is covalently bound to G hydroxyl groups. via one of the sulfur atoms of the disulfide, provided 55 that G is of at least 4 carbon atoms when Z is said disul Yet another aspect of the invention is the above fide group. described 5'-modified oligonucleotide in which Z repre Preferably R is g-cyanoethyl, R1 and R2 are both sents a deprotected vicinal diol group which has been isopropyl, and G is -(CH2)- where n is an integer oxidized to form a terminal aldehyde group on the oli from 4 to 6, inclusive. Examples of other protecting gonucleotide. groups represented by R are 6-nitroethyl, 2,2,2-tri Another aspect of the invention is a conjugate of the chloroethyl, methyl, 1,1-dimethyl-2,2,2-trichloroethyl, above-described oligonucleotide having a terminal al 2,2,2-tribromoethyl, benzyl, o-chlorophenyl, p-nitro dehyde group and a free amino group-containing car phenylethyl, 2-methylsulfonylethyl, and 1,1-dimethyl rier molecule wherein the conjugate is formed by reac 2-cyanoethyl. Examples of other groups which R and tion between the aldehyde group and the free amino 65 R2 may represent are other alkyl groups such as butyl, group. hexyl, nonyl, dodecyl, and hexadecyl, cycloalkyl A further aspect of the invention is a partially pro groups such as cyclopropyl, cyclobutyl, cyclohexyl and tected triol of the formula: cyclooctyl, aryl groups such as phenyl, tolyl, benzyl, 5,391,785 5 6 xylyl and naphthyl, and when joined together heterocy tide. Suitable groups R6 and R7 include aryl and substi clic groups such as morpholino, piperidinyl and thi tuted aryl groups of 6-30 carbon atoms, C1-C20 alkyl omorpholino. Examples of other hydrocarbylene radi groups, and aromatic substituted alkyl groups of less cals which G may represent are branched alkylene, and than 30 carbon atoms. Preferred is phenyl and phenyl groups containing cycloalkylene (e.g., cyclohexylene) substituted with C1-C8 alkyl, C1-8 alkoxy; 1 to 4 atoms of or phenylene. It will be appreciated that G functions fluorine, chlorine, bromine, nitro- or phenyl. Most pre primarily as an inert spacer moiety and that it may have ferred are acetal structures wherein R6 is phenyl, p substituents and/or heteroatoms (e.g., O, S, ND in its butylphenyl, p-methoxyphenyl, p-tertbutylphenyl, and structure that do not affect its ability to act as an inert biphenyl. It will be known to those skilled in the art that spacer. 10 the stability of the protecting group can be adjusted for Preferred hydroxy-protected vicinal diol groups rep a particular use by a suitable choice of substituent(s). resented by Z are those of the formula: The above-described acetals and ketals are easily prepared directly from the corresponding triols in one wer' - mean W W step. It is an important and unexpected feature of this w 15 embodiment of the present invention that the vicinal Yli-O O-Y2 diol is selectively protected in the presence of another -C-C-R free alcohol in the molecule. Thus, the triol wherein Y1 and Y2 are H is simply contacted with an aldehyde to H R3 20 yield the acetal or a ketone to yield the ketal in the where R3 and Rare individually hydrogen, alkyl of 1 presence of an acid catalyst. It is preferred that the to 20 carbon atoms or monocyclic arylene of 6 to 20 contacting take place under conditions where the water carbon atoms and Y and Y2 are individual hydroxy formed during the reaction is removed during the reac protecting groups or may be joined (designated by the tion, either by the application of vacuum or by solvent dashed line) by a single-atom (C, S or Si) bridge to form 25 azeotrope. Alternatively, acetals or ketals of lower-boil a five-membered ring protecting group. Yland Yare of ing alcohols can be similarly employed in place of the a nature that they are stable during the addition of the aldehyde or ketone in an acetal exchange reaction. molecule to the 5' of an oligonucleotide chain during The phosphoramidites of the above-described acetals chemical synthesis (i.e., conventional automated phos and ketals are prepared by the conventional methods phoramidite synthesis) and can be removed thereafter 30 described herein, and they are coupled to the oligonu without damaging the oligonucleotide chain. Further, cleotide during the synthesis, as is also described herein. as discussed below, the vicinal diol structure of the Following the synthesis and purification of the free, deprotected group permits it to be "activated' by oxi coupled oligonucleotide, mild acid hydrolysis of the dation to convert it from a diol to a functional aldehyde protecting group generates the diol that is the substrate group. Yland Y2 may be the same or different and may 35 for the oxidation reaction that produced the aldehyde be any of the individual hydroxy protecting groups that used for the conjugation reaction. Typical mild hydro are compatible with conventional automated solid state lysis conditions are 80% acetic acid/water at 25 C. for oligonucleotide chemistry using phosphoramidite 30 minutes, similar to those used to remove a dimethox chemistry. Examples of such blocking groups are dime ytrityl group in conventional oligonucleotide synthesis. thoxytrityl (DMT), trityl, pixyl, benzoyl, acetyl, isobu Preferred disulfide groups represented by have the tyryl, p-bromobenzoyl, t-butyldimethylsilyl, and pival formula: oyl. The protecting groups may be removed with the same or different treatments. Such vicinal diol groups in which R3 and Rare hydrogen and Yl and Y2 are ben zoyl or DMT are particularly preferred. where R is an alkylene group of 1 to 20 carbon atoms As indicated, Yi and Y2 may be linked by a one-atom 45 or a monocyclic arylene group of 6 to 20 carbon atoms bridge, thus forming a five-membered ring. Suitable and Y3 is a hydroxy protecting group (as described bridging atoms include silicon, sulfur and carbon. It is above). preferred that the one-atom bridge be a carbon bridge. Most preferably R5 is alkylene of 4 to 6 carbon atoms, Thus, the diol group is preferred to be protected as an 50 -OY is bound to the a) carbon atom of the alkylene acetal or ketal, i.e., group and Y is trityl. As discussed below, the disulfide structure of the group permits it to be “activated' by reduction to cleave the disulfide bond and produce a N N / C-H C free sulfhydryl group. / N / N 55 The phosphoramidites wherein Z represents a vicinal O O O O diol may be prepared from an alcohol of the formula HC-CH-G-OH. The hydroxyl group of the alcohol H--- H--- is protected and the double bond is oxidized to form the H H H H diol group. The hydroxyls of the diol are then protected Acetal Ketal with an orthogonally removable protecting group (Y2 and Y), i.e., the protecting group on the original hy It is important that the bridging atom and its substitu droxy can be removed without removing the protecting ents be stable to the subsequent reactions in the se groups on the vicinal diol. The protecting group on the quence used to add the linker to the oligonucleotide. original hydroxy is then removed and the resulting The diol protecting group must also be capable of being 65 deprotected hydroxy is reacted with an appropriate removed under mild conditions that do not substantially phosphitylating agent. degrade the oligonucleotide. For example, very acidic The phosphoramidites wherein Z represents a disul conditions will lead to depurination of the oligonucleo fide may be prepared from symmetrical or asymmetri 5,391,785 7 8 cal disulfides. The general reaction scheme employing been derivatized to include such groups as recited symmetrical disulfides is shown in FIG.3 and exempli above. fied by Example 3, infra. Asymmetrical disulfides may be prepared as described by Mannervik, B., and Larson, EXAMPLES K., Meth, in Enzym. (1981) 77:420-424, or Mukuiyama, The following examples further illustrate the inven T., and Takahashi, K., Tet Lett (1968) 5907-5908. By tion. These examples are not intended to limit the inven way of example, a symmetrical disulfide (HO-G-S- tion in any manner. In the examples, Et=ethyl, Ac=a- S-G-OH) is oxidized with hydrogen peroxide and cetyl, and THF = tetrahydrofuran. formic acid to provide the corresponding thiolsulfinate. Treatment of the thiolsulfinate with a mercaptain (e.g., 10 Example 1 HS-G'-OY3 where Y3 is as described above and G' is a different G than in the starting symmetrical disulfide) Preparation of at a pH greater than 3 yields an asymmetrical disulfide O-(5,6-(bis-O-benzoyloxy)-hexyl)-O-(2-cyanoethyl)- (HO-G-SS-G'-OY3). This disulfide may be re N,N-diisopropylphosphoramidite acted with a phosphitylating agent to yield the phos 15 FIG. 1 schematically depicts the invention scheme phoramidate. used to make this phosphoramidite. The details of this The phosphoramidites of the invention may be added scheme are described below. to the 5' of an oligonucleotide chain using the conven O-(tert-butyldimethylsilyl)-5-hexenol, 5. To a solu tional automated phosphoramidite method used to pre tion of 12.47 mL (10.4g, 104 mmol) of 5-hexene-1-ol in pare oligonucleotides. See Matteucci, M. D., and 20 104 mL of DMF was added 15.66 g (230 mmol) of Caruthers, M. H., Tet Lett (1980) 521:719, and U.S. Pat. imidazole and 20.0 g (130 mmol) of tert-butyldimethyl No. 4,500,707. The oligonucleotide chain itself may be silyl chloride (TBDMSC). The mixture was stirred at made by the same method. The length and sequence of ambient temperature for 4 hours and partitioned be the oligonucleotide to which the phosphoramidite of tween 200 mL of EtOAc and 100 mL of saturated the invention is added will depend upon the use of the 25 NaHCO3 solution. The EtOAc layer was washed with resulting 5'-functionalized oligonucleotide. For in 100 mL of saturated NaHCO3 solution, 100 mL of satu stance, if the oligonucleotide is to be used for the pur rated NaCl solution, dried (MgSO4), filtered, and con poses described in parent application Ser. No. 494,118 centrated to a volume of approximately 100 mL. Distil (i.e., systemic lupus erythematosus (SLE) treatment), lation under vacuum provided 70.07 g of 5: bp then the oligonucleotide will have the ability of bind 30 SLE antibodies. If the oligonucleotide is to be used as a 130-143° C. Q 100 mmHg, 1H NMR (CDCl3) 0.11 (s, labeled probe then the length and sequence will be such 6H), 0.95 (s, 9H), 1.48 (m, 2H), 1.57 (m, 2H), 2.11 (dt, as to be capable of hybridizing to a nucleotide sequence 2H), 3.66 (t, 2H), 5.03 (m, 2H), 5.86 (m, 1H); 13C NMR of interest. (CDCl3) -5.25, 18.40, 25.21, 26.01, 32.35, 33.60, 63.09, 114.40, 138.92. As indicated above, the resulting modified oligonu 35 cleotide may be represented by the formula: 1-O-(tert-butylidimethylsilyl)-1,5,6-hexanetriol, 6. To a solution of 9.86 g (46.0 mmol) of 5 in 92 mL of acetone was added a solution of 6.46 g (55.2 mmol) of N-methyl morpholine oxide (NMMO) in 23 mL of H2O. To the mixture was added 443 u of a 2.5% solution of OsO4 in tert-butyl alcohol (360 mg of solution, 9.0 mg of OsO4, X 35 umol) and 50 uL of 30% H2O2. The mixture was where (O.N.) represents an oligonucleotide chain and stirred for 16 hours and a solution of 474 mg of sodium X, X, G and Z are as defined previously. The designa dithionite in 14 mL of H2O was added. After another tion '5' indicates that the modifying group is attached 45 0.5 hour the mixture was filtered through celite. The to the 5' end of the oligonucleotide chain. The chain filtrate was dried with MgSO4 and filtered through 1" will typically be 10 to 200 nucleotides in length, more of silica gel in a 150 mL Buchner funnel using 250 mL. usually 20 to 60 nucleotides in length. portions of EtOAc to elute. Fractions containing prod Once the phosphoramidite has been added to the 5’ uct were concentrated to provide 11.0 g of 6 as a vis end of an oligonucleotide chain, the protecting groups cous oil: TLC R? 0.2 (1:1 hexane/EtOAc); 1H NMR (Y1, Y2, Y) may be removed by appropriate treatment (CDCl3) 0.05 (s, 6H), 0.89 (s, 9H), 1.25 (m, 4H), 1.55 (m, (e.g., base or acid treatment) to yield free hydroxy 2H), 3.41 (dd, 2H), 3.62 (t, 2H), 3.71 (m, 1H); 13C NMR groups. In the case of the vicinal diol, the diol group is (CDCl3) -5.23, 18.42, 21.91, 26.02, 32.68, 32.81, 63.16, oxidized, e.g., with periodate, to form a terminal alde 66.74, 72.24. hyde group. In the case of the disulfide group, the disul 55 5,6-(bis-O-benzoyl)-1-O-(tert-butyldimethylsilyl)- fide is reduced with an appropriate reducing agent, e.g., 1,5,6-hexanetriol, 7. To a solution of 5.29 g (21.3 mmol) a mercaptain such as dithiothreitol or 2-mercaptoethanol of 6 in 106 mL of pyridine was added 6.18 mL (7.48 g, or borohydride to cleave the disulfide bond to form a 53.2 mmol) of benzoyl chloride. The mixture was terminal sulfhydryl group. stirred for 18 hours and concentrated on the rotary The resulting 5' modified oligonucleotide may be 60 evaporator. The mixture was partitioned between 100 coupled via the aldehyde group to labels, carriers, or mL of cold 1N HCl and 100 mL of EtOAc. The pH of other molecules having a free amino group or via the the aqueous layer was checked to made sure it was sulfhydryl group to an electrophilic center such as acidic. The EtOAc layer was washed successively with maleimide or a-haloacetyl groups or other appropriate 100 mL of H2O and 100 mL of saturated NaCl, dried Michael acceptors such as acrylates or acrylamides. 65 (MgSO4), filtered, and concentrated to provide 10.33g Examples of such carriers are amino acid polymers such of 7 as a viscous yellow oil; TLC Rf0.45 (1:4 EtOAc/- as copolymers of D-lysine and D-glutamic acid, or im hexanes); 1H NMR (CDCl3) 0.05 (s, 6H), 0.88 (s, 9H), munoglobulin, or other polymers that inherently have 1.59 (m, 4H), 1.85 (m, 2H), 3.14 (t, 2H), 4.49 (dd, 1H) 5,391,785 10 4.59 (dd, 1H), 5.54 (m, 1H), 7.45 (m, 4H), 7.58 (m, 2H), C. The mixture was partitioned between 50 mL of 8.05 (m, 4H). EtOAc and 50 mL of saturated NaHCO3 solution. The 5,6-(bis-O-benzoyl)-1,5,6-hexanetriol, 8. To a solution EtOAc layer was washed with 50 mL of saturated of 2.62 g (5.36 mmol) of 7 in 10.9 mL of THF was added NaHCO3 solution, 25 mL of saturated NaCl solution, 10.7 mL (10.7 mmol) of a 1N solution of tetrabutylam dried (Na2SO4), filtered, and concentrated. Purification monium fluoride (CTBAF) in THF. The mixture was by silica gel chromatography (10:89:1 EtOAc/hex allowed to stir for 16 hours. The mixture was parti ane/Et3N) provided 1.66 g of 11 as a viscous oil: TLC tioned between 25 mL of saturated NaHCO3 solution R?.27 (1:9 EtOAc/hexane); 1H NMR (CDCl3) 0.5 (s, and 3X25 mL of EtOAc. The combined EtOAc ex 6H), 0.87 (s, 9H), 1.40 (m, 2H), 1.56 (m, 2H), 1.82 (m, tracts were washed with saturated NaCl solution, dried 10 2H), 3.28 (dd, 2H), 3.60 (t, 2H), 3.80 (s, 6H), 5.38 (m, (MgSO4), filtered and concentrated to a viscous oil 1H), 6.79 (m, 4H), 7.17–7.65 (m, 12H), 8.11 (d, 2H). which was purified by silica gel chromatography (1:1 5-O-benzoyl-6-O-(4,4'-dimethoxytriphenylmethyl)- hexane/EtOAc) to provide 823 mg of 8 as a viscous oil; 1,5,6-hexanetriol, 12. To a solution of 1.66 g (2.56 mmol) Rf.14 (1:1 hexane/EtOAc); H NMR (CDCl3) 1.58 (m, of 11 in 5.2 mL of THF under N2 atmosphere was added 2H), 1.68 (m, 2H), 1.88 (m, 2H), 3.68 (t, 2H), 4.52 (dd, 15 5.12 mL (5.12 mmol) of a 1M solution of tetrabutylam 1H), 4.62 (dd, 1H), 5.56 (m, 1H), 7.46 (m, 4H), 7.58 (m, monium fluoride in THF. The mixture was stirred for 3 2H), 8.05 (m, 4H); 13C NMR (CDCl3). 22.08, 31.20, hours at ambient temperature and concentrated on the 31.30, 32.88, 62.92, 66.17, 72.63, 128.93, 130.19, 130.57, rotary evaporator. Purification by silica gel chromatog 133.62, 166.72, 166.86. raphy (1:1 EtOAc/hexane) provided 1.18 g (86%) of 12 O-(5-6-(bis-O-benzoyloxy)-hexyl)-O-(2-cyanoethyl)- 20 as a viscous oil. Further purification was possible by N,N-diisopropylphosphoramidite, 9. To a solution of preparative HPLC (12 mL/min, 9:1 MeOH/H2O, 22.4 1.02 g (2.98 mmol) of 8 and 255 mg (1.49 mg) of diiso mm C18): TLC R?.14 (1:1 hexane/EtOAc); 1H NMR propylammonium tetrazolide (DIPAT, prepared by (CDCl3) 1.37 (m, 2H), 1.57 (m, 2H), 1.79 (m, 2H), 3.29 mixing acetonitrile solutions of diisopropylamine and (dd, 2H), 3.60 (t, 2H), 3.75 (s, 6H), 5.36 (m, 1H), 6.80 (m, tetrazole in a one-to-one mole ratio and concentrating 25 4H), 7.17-7.60 (m, 12H), 8.12 (d, 2H). to a white solid) in 14.9 mL of CH2Cl2 was added a O-5-benzoyloxy-6-O-(4',4'-dimethoxytriphenylme solution of 989 mg (3.28 mmol) of O-cyanoethyl thyl)hexyl-O-(2'-cyanoethyl)-N,N-diisopropylphos N,N,N',N'-tetraisopropylphosphorodiamidite in 2.0 mL phoramidite, 13. To a solution of 681 mg (1.26 mmol) of of CH2Cl2. The mixture was stirred for 4 hours and 12 and 111 mg (0.65 mmol) of diisopropylammonium partitioned between 25 mL of CH2Cl2 and 25 mL of 30 tetrazolide in 6.5 mL of CH2Cl2 was added a solution of chilled saturated NaHCO3 solution. The CH2Cl2 layer 417 mg (1.38 mmol) of O-cyanoethyl-N,N,N',N'-tet was washed with saturated NaCl solution, dried (Na2 raisopropylphosphorodiamidite in 1.0 mL of CH2Cl2. SO4), filtered, and concentrated. Purification by filtra The mixture was stirred for 2 hours and partitioned tion through a 2" plug of basic alumina in a 25 mm between 25 mL of CH2Cl2 and 25 mL of chilled satu column, eluting with 9:1 EtOAc/Et3N provided 1.5g 35 (93%) of 9 as a viscous oil: 1H NMR (CDCl3) 1.19 (m, rated NaHCO3 solution. The CH2Cl2 layer was washed 12H), 1.62 (m, 2H), 1.73 (m, 2H), 1.90 (m, 2H), 2.62 (dd, with saturated NaCl solution, dried (Na2SO4), filtered, 2H), 3.53-3.92 (m, 6H), 4.53 (dd, 1H), 4.62 (dd, 1H), and concentrated. Purification by filtration through a 2" 5.58 (m, 1H), 7.48 (m, 4H), 7.60 (m, 2H), 8.09 (m, 4H); plug of basic alumina in a 25 mm column, eluting with 31P NMR (CDCl3 with 15% H3PO4 internal standard) 9:1 CH2Cl2/Et3N provided 798 mg of 13 as a viscous 148.2; HRMS (FAB, MH+), calculated for oil: H NMR (CDCl3) 1.19 (m, 12H), 1.42 (m, 2H), 1.65 C29H40O6N2P1543.2624, found 543.2619. (m, 2H), 1.81 (m, 2H), 2.69 (m, 2H), 3.28 (dd, 2H), 3.57 (m, 4H), 3.78 (s, 6H) (underlying m, 2H), 5.40 (m, 1H), Example 2 6.79 (dd, 4H), 7.27-7.64 (m, 12H), 8.17 (d, 2H); 31P Preparation of NMR (CDCl3, 15% H3PO4 internal standard) 148.0; O-5-benzyloxy-6-O-(4,4'-dimethoxytrity) 45 HRMS (FAB, MH), calc'd for C3H54O7N2P1 hexyl-O-(2-cyanoethyl)-N,N-diisopropylphosphorami 741.3669, found 741.3678. dite Example 3 FIG. 2 schematically depicts the reaction scheme for Preparation of making this phosphoramidite. The details of the scheme 50 O-(14-(4,4'-dimethoxytriphenylmethoxy)-7,8-dithi are described below. 6-O-(4,4'-dimethoxytriphenylmethyl)-1-O-(tert otetradecyl)-O-(2-cyanoethyl)-N-N-diisopropylphos butyldimethylsilyl)-1,5,6-hexanetriol, 10. To a solution phoramidite of 1.11 g (4.47 mmol) of 6 and 891 uL (638 mg, 6.30 FIG. 3 schematically shows the reaction scheme for mmol) of Et3N in 22 mL of pyridine was added 1.81 g 55 this phosphoramidite. The details of the scheme are (5.33 mmol) of 4,4'-dimethoxytriphenylmethyl chloride. described below. The mixture was stirred at ambient temperature for 16 S-(6-hydroxyhexyl)isothiuronium chloride, 14. To a hours, concentrated, and purified by silica gel chroma solution of 16.6 mL (20.0 g, 146 mmol) of 6-chlorohex tography (29:70:1 EtOAc/hexane/Et3N) to provide anol in 49 mL of ethanol was added 11.1 g (146 mmol) 2.06 g (85%) of 10 as a viscous oil; TLC R?.35 (39:60:1 of thiourea, and the mixture was refluxed for 24 hours. EtOAc/hexane/Et3N). The mixture was cooled to 0° C., and the product crys 5-O-benzoyl-6-O-(4,4'-dimethoxytriphenylmethyl)- tallized. The crystals were collected by vacuum filtra 1-O-(tert-butyldimethylsilyl)-1,5,6-hexanetriol, 11. To a tion and dried to give 28.4 g (92%) of 14 as a white solution of 2.06 g (3.8 mmol) of 10 in 19 mL of pyridine solid: mp 122-124° C.; 1H NMR (DMSO) 1.40 (m, 4H), was added 532 mL (644 mg, 4.58 mmol) of benzoyl 65 1.65 (m, 2H), 3.21 (t, 2H) 3.41 (t, 2H), 9.27 and 9.33 chloride, and the mixture was stirred for 20 hours and (overlapping broad singlets, 4H). concentrated on the rotary evaporator to remove most 6-Mercaptohexan-1-ol, 15. To a solution of 17.8 mg of the pyridine keeping the bath temperature below 30 (83.6 mmol) of 14 in 120 mL of H2O and 120 mL of 5,391,785 11 12 EtOH was added 9.25 g of NaOH pellets. The mixture performed on a Milligen 8800 DNA synthesizer using was refluxed for 4 hours. The mixture was carefully the manufacturer's protocols for DNA synthesis. concentrated to approximately 75 mL, and the concen In a separate instance, in a 1 umole scale reaction on trate was purified by vacuum distillation to provide 7.4 a Pharmacia Gene-Assembler DNA synthesizer, the g (66%) of 15: bp 95°-105 C. G5 mmHg, 1H NMR coupling efficiency was determined to 96% by trityl (CDCl3) 1.41 (m, 9H) 2.59 (dt, 2H), 3.69 (t with under release. For this determination, the phosphoramidite lying brds, 3H). from Example 3 was used. Bis-(6-hydroxyhexyl)disulfide, 16. To a solution of After the reaction, the CPG was suspended in 100 ml 4.26 g (31.7 mmol) of 15 in 10 mL of MeOH and 13.7 concentrated ammonia and kept at 55 C. overnight. mL (9.97g, 98.5 mmol) of Et3N under N2 atmosphere 10 After filtration, the deprotected oligonucleotide was and cooled in an ice bath was added dropwise over 10 purified by sodium chloride gradient and ion-exchange min a solution of 4.02 g (15.8 mmol) of I2 in 90 mL of chromatography. MeOH. The cooling bath was removed, and the mixture The fractions were analyzed by polyacrylamide gel was stirred at ambient temperature for 4 hours. The electrophoresis and the product containing fractions mixture was concentrated on the rotary evaporator and 15 pooled, adjusted to 0.3M NaCl with 3M NaCl solution purified by silica gel chromatography (1:1 hex and precipitated by the addition of an equal volume of ane/EtOAc) to provide 3.12 g (73%) of 16 as a pale cold isopropanol. The product was collected by centrif. yellow solid: TLC Rf .18 (1:1 hexane/EtOAc); mp ugation and dried in vacuo. 38-48 C.; 1H NMR (CDCl3) 1.15-2.20 (m, 16H), 2.73 The pellet was then dissolved in 40 ml water and (t, 4H), 3.70 (t, 4H). oxidized by treatment with a fivefold molar excess of Mono-O-(4,4'-dimethoxytriphenylmethyl)-bis-(6- sodium metaperiodate (83.6 mg for 2g purified oligonu cleotide in this example) at 0° C. for 30 min. The solu hydroxyhexyl)disulfide, 17. To a solution of 3.12 g (11.7 tion was again adjusted to 0.3M NaCl and precipitated mmol) of 16 and 45 mL of pyridine was added 3.97g as above to remove the formaldehyde produced in this (11.7 mmol of 4,4'-dimethoxytriphenylmethyl chloride, 25 and the mixture was stirred at ambient temperature for reaction. After centrifugation and drying, this material 16 hours. Most of the pyridine was removed on the wa used in the next step. rotary evaporator, and the residue was partitioned be Example 5 tween 100 mL of saturated NaHCO3 solution and 100 Conjugation of Oligonucleotide of Example 4 to mL of EtOAc. The EtOAc layer was washed with 50 30 D-glutamic acid, D-lysine (DEK) polymer mL of saturated NaCl solution, dried (Na2SO4), filtered and concentrated to an oil. Purification by silica gel 100 mg of oxidized oligonucleotide (2.5 pmoles) was chromatography (9:1 CH2Cl2/EtOAc) yielded 2.84 g dissolved in 1.33 ml of 100 mM NaBO3, pH 8.0. Then, (43%) of 17 as a viscous oil: TLC Rf .35 (9:1 2.5 mg of DEK (0.25 pmoles, MWt 10,000, 60:40 CH2Cl2/EtOAc); H NMR (CDCl3) 1.41 (m, 8H), 1.65 35 weight ratio of D-glutamic acid to D-lysine) and 0.79 (m, 8H), 2.70 (two overlapping triplets, 4H), 3.08 (t, mg NaCNBH3 (12.5 umoles) was added. The mixture 2H), 3.65 (t, 2H), 3.81 (s, 6H), 6.85 (d, 4H), 7.32 (m, 7H), (2.0 ml) was incubated at 37 C. for 3 days. The conden 7.47 (d, 2H). sation product was purified by S-200 (Pharmacia, Upp O-(14-(4,4'-Dimethoxytriphenylmethoxy)-7,8-dithi sala, Sweden) chromatography. otetradecyl)-O-(2-cyanoethyl)-N,N-diisopropylphos 40 The fractions were labeled with alpha 32P ddATP and terminal transferase for viewing on a standard 8% phoramidite, 18. To a solution of 771 mg (1.36 mmol) of DNA sequencing polyacrylamide gel. 17 and 116 mg (0.68 mmol) of diisopropylammonium The various radiolabeled fractions were visualized by tetrazolide in 6.8 mL of CH2Cl2 under N2 atmosphere electrophoresis and autoradiography as presented in was added a solution of 458 mg (1.52 mmol) of O FIG. 4. The lanes labeled "2' contain unconjugated full cyanoethyl-N,N,N',N'-tetraisopropylphosphorodiami 45 length oligonucleotide and the arrow indicates the posi dite in 0.5 mL of CH2Cl2. The mixture was stirred for 4 tion of the 50-mer. Lanes labeled “1” contain conju hand partitioned between 25 mL of NaHCO3 and 3X25 gates of decreasing molecular weight. Fractions which mL of CH2Cl2. The combined CH2Cl2 layers were contain the higher substitute (region A) oligo-DEK washed with saturated NaCl solution, dried (Na2CO3), conjugate were pooled for subsequent annealing to the filtered and concentrated to an oil. Purification by filtra 50 complementary oligonucleotide strand to construct a tion through a 2' plug of basic alumina in a 25 mm double stranded DNA-DEK conjugate. column, eluting with 9:1 CH2Cl2/Et3N provided 831 mg (80%) of 18 as a viscous oil; 1H NMR (CDCl3) 1.25 Example 6 (m, 12H), 1.45 (m, 8H), 1.70 (m, 8H), 2.72 (m, 6H), 3.09 Conjugation of Oligonucleotide of Example 4 to (t, 2H), 3.65 (m, 4H), 3.87 (s, 6H), 3.91 (m, 2H), 6.89 (d, 55 Keyhole Limpet Hemocyanin (KLH) 4H), 7.35 (m, 7H), 7.49 (d, 2H); 31P NMR (CDCl3 with 100 mg crude oxidized oligonucleotide (2.5 umoles) 15% H3PO4 internal standard) 147.69; HRMS (FAB, was dissolved in 1.33 ml of 50 mM NaBO3, pH 8.0. MH+) calc'd for C42H62N2O5PS2 769.3839, found Then, 31.3 mg of KLH (0.208 umoles) and 2.0 mg 769.3853. NaCNBH3 (31.8 moles) was added. The mixture (2.0 Example 4 ml) was incubated at 37 C. for 3 days. The condensa tion product was purified by S-200 chromatography. Addition of Phosphoramidite of Example 1 to The various fractions were radiolabeled using the same Oligonucleotide process as described above for D-EK and were then A fivefold molar excess (760mg) of the phosphorami 65 visualized after electrophoresis and autoradiography as dite of Example 1 was coupled to the 5' end of an oligo presented in FIG. 5. Lanes labeled “1” are high molecu nucleotide which was attached to 10 g (300 pmoles) lar weight conjugates, lanes labeled ''2''' contain mostly CPG (control pore glass) support. This synthesis was unconjugated oligo and the arrow indicates the position 5,391,785 13 14 of the 50-mer. Modifications of the above-describes In a similar manner, the following phosphoramidites modes for carrying out the invention that are obvious to are prepared: those of ordinary skill in the fields of organic chemistry, (4-(2-methoxyphenyl-1,3-dioxol-4-yl) butyl)-O-(2- and particularly oligonucleotide synthesis and derivati cyanoethyl)-N-N-diisopropylphosphoramidite; 5 (4-(2-p-butylphenyl-1,3-dioxol-4-yl) butyl)-O-(2-cyano zation are intended to be within the scope of the follow ethyl)-N-N-diisopropylphosphoramidite; ing claims. The fractions which contained the oligo (4-(2-biphenyl-1,3-dioxol-4-yl) butyl)-O-(2-cyanoethyl)- KLH conjugate were pooled for subsequent annealing N-N-diisopropylphosphoramidite; to the complimentary oligonucleotide strand to con (4-(2-methyl-2-phenyl-1,3-dioxol-4-yl)butyl)-O-(2- struct a double-stranded DNA-KLH conjugate. 10 cyanoethyl)-N-N-diisopropylphosphoramidite. Example 7 Example 8 Preparation of Acetal-Protected Diol Phosphoramidite Addition of Phosphoramidite of Example 7 to 4-(4-hydroxy-1-butyl)-2-phenyl-1,3 dioxolane. Oligonucleotide A mixture of 1,2,6-trihydroxyhexane (2.58 g) and 15 In the manner of Example 4, the phosphoramidite of benzaldehyde dimethyl acetal (3.18 g) is treated with Example 7 is coupled to the oligonucleotide. Following toluene sulfonic acid hydrate (2.08 g). The mixture is purification, the acetal protecting group is removed allowed to stir at room temperature for 60 hours, and is with 80% acetic acid/water for 40 minutes. The then partitioned between saturated aqueous sodium 20 progress of the reaction is monitored by HPLC using a bicarbonate (50 ml) and methylene chloride (20 ml). Gen Pak Fax column (Waters Associates), using 0.5M sodium phosphate at pH 7.5, with a 1.0M sodium chlori The layers are separated, the aqueous layer is re de/10% methanol gradient. The starting acetal elutes at extracted with methylene chloride, the organic layers 20.1 minutes, and the hydrolyzed diol elutes at 18.9 are dried over anhydrous sodium sulfate, filtered, and minutes. concentrated to an oil (2.66 g), which is purified by 25 Modifications of the above-described modes for car column chromatography (silica gel, 1:1 ethyl acetate/- rying out the invention that are obvious to those of skill hexanes). Pooling and concentrating the appropriate in the fields of organophosphorous chemistry, nucleo fractions give the title compound as an oil (1.19 g): TLC tide chemistry, oligonucleotide synthesis, or related Rf=0.18 (silica, 1:1 ethyl acetate/hexanes); 1H NMR fields are intended to be within the scope of the follow (CDCl3), 8, 1.62 (m, 6H), 3.67 (m, 3H), 3.25 (m, 2H), 30 ing claims. 6.37 (s, 0.6H), 6.50 (s, 0.4H), 8.04 (br. s, 5H). We claim: In a similar manner, but beginning with benzaldehyde 1. A partially protected alcohol of the formula: a- - - - - in place of benzaldehyde dimethyl acetal, the title com M w pound is also obtained. Yl-o o-y (4-(2-phenyl-1,3-dioxol-4-yl)butyl)-O-(2-cyanoethyl)- 35 N-N-diisopropylphosphoramidite. A solution of the H---g-oh above dioxolane (1.19 g), and diisopropylamine (2.0 ml) h h in methylene chloride (22 ml) is treated with cyanoe thyldiisopropylchlorophosphoramidite (0.92 ml) and 40 wherein Y1 is selected from dimethoxytrity, allowed to stir at 24 C. for 1.5 hours. The mixture is monomethoxytrityl, trityl or pixyl, Y2 is selected partitioned between saturated aqueous sodium bicar from benzoyl, acetyl, isobutyryl, p-bromobenzoyl, bonate (25 ml) and methylene chloride (25 ml). The t-butyldimethylsilyl, pivaloyl or other base hydro layers are separated, the aqueous layer is re-extracted lyzable acyl groups and G is selected from alkyl of 45 1 to 20 carbon atoms or monocyclic arylene of 6 to with methylene chloride, the organic layers are dried 20 carbon atoms. over anhydrous sodium sulfate, filtered, and concen 2. The alcohol of claim 1, wherein Yl is dimethoxytri trated to an oil (2.13 g), which is purified by column tyl, Y2 is benzoyl and G is butylene. chromatography (basic alumina, 1:1 methylene chlori 3. The alcohol of claim 1, wherein G is alkylene of 4 de/hexanes, 1% triethylamine). Pooling and concen to 20 carbons. trating the appropriate fractions gives the title com 50 4. The alcohol of claim 1, wherein G is butylene. pound as an oil (1.28 g): 1H NMR (CDCl3), 8, 1.13 5. The alcohol of claim 1, wherein Y1 is dimethoxy (12H), 1.5-1.9 (m, 8H), 2.58 (q, 2H), 3.5-3.8 (m, 8H), trityl (DMT). 4.0-4.3 (m, 2H), 5.8 (s, 0.6H), 5.92 (s, 0.4H), 7.3–7.5 (m, 6. The alcohol of claim 1, wherein Y2 is benzoyl. sk sk ck 2k Ek 5H). 55