DEFINED SEQUENCE SINGLE STRAND OLIGONUCLEOTIDES INCORPORATING REPORTER GROUPS, PROCESS FOR THE CHEMICAL SYNTHESIS THEREOF, AND NUCLEOSIDES USEFUL IN SUCH SYNTHESIS

Abstract: Defined sequence single strand oligonucleotides which have a length of fewer than 200 units, incorporate modified nucleotide units which are pyrimidine or purine-based, and which may have readily detectable reporter groups attached to substituents on the modified nucleotide units thereof. Each reporter group is attached to the base of the respective nucleotide unit at a sterically tolerant site thereon, exemplified by the C-5 position of pyrimidine-based nucleotides and the C-8 position of purine-based nucleotides. Such oligonucleotides are useful in identification, localization, isolation and/or detection of complementary nucleic acid sequences of interest in cellular or cell-free systems. Also disclosed is a process for the chemical synthesis of single strand oligonucleotides having a predetermined number of modified pyrimidine and/or purine-based nucleotide units in predetermined sequence, to the bases of which readily detectable reporter groups are attached at sterically tolerant sites through substituents thereon, either prior to or after incorporation of such units into the oligonucleotide chain. Novel nucleosides useful in the chemical synthesis of labeled, defined sequence single strand oligonucleotides are also disclosed.

Description: Descri tion Defined Sequence Single Strand Oligonucleotides Incorporating Reporter Groups, Process for the Chemical Synthesis Thereof, and Nucleo ' sides Useful in Such Synthesis 5 Technical Field The present invention relates to defined sequence single strand oligonucleotides having a length of fewer than 200 base units and which contain one or more nucleotide units to the base of which is attached a 10 substituent group capable of functioning as one or more detectable reporter groups, or which has bound thereto one or more detectable reporter groups; such oligonucleo¬ tides being useful in the identification; localization and detection of complementary nucleic acid sequences 15 of interest in cellular or cell-free systems. Background Art The enzymatic production of labelled, double stranded deoxypolynucleotides has been accomplished with prior art techniques involving the incorporation of radioisotopes 20 into double-strand DNA by the nick translation protocol of P. Rigby et al, J. Mol. Biol. 113: 237-251 (1977), or the gap-filling reaction described by G. Bourguignon et al, J. Virol, 20: 290-306 (1976). Specifically, a nick is introduced via DNase, and then translated along the 25 DNA strand using 'DNA polymerase. During the nick translation procedure, DNA polymerase from E. coli (Pol I) will, in the presence of added deoxynucleoside triphos- phates , condense nucleotides to the 3' hydroxyl terminus in a single strand nick region of double-stranded DNA. 30 Simultaneously the enzyme will remove nucleotides from the 5' end of the nick. -I f one or more of the added - - - 32 triphosphates is labelled, for example, with a- P- phosphate the label will be incorporated into the new strand by Pol I. Following the gap-filling procedure, recessed ends left after restriction endonuclease cutting can be filled.in with Klenow fragments from Pol I or 4 DNA Polymerase. Both the nick translation and the gap-filling procedures will yield double-stranded labelled DNA. The length of the product depends upon how much DNase I is added to the reaction. This is usually optimized so that thirty percent of the label is incorporated and the strands are 400 to 800 nucleotide units in length. The product length is unpredictably heterogeneous within the 400 to 800 unit range. In order to -conserve enzyme as well as labelled nucleotide, only one microgram of DNA is usually labelled in each reaction vessel. Double-stranded polynucleotide ' s which incorporate pyrimidine bases modified by carbon chains , at the C-5 position have been prepared enzymatically in a similar manner. This has been done by enzymatic elongation of a homopolymeric primer-template as reported by J. Sagi et al. , Biochem. Biosphys. Acta. 606: 196-201 (1980), or ' by nick translation/gap filling by DNA polymerases using 2'-deoxyuridine 5'-triphosphate covalently linked to bio- tin as reported by P. Langer et al, Proc. Nat. Acad. Sci. USA 78:6633-6637 (1981), the biotin being capable of acting as a recognition site for avidin. Enzymatic methods described by Rigby, et.al, Bourguignon, et al, and Langer, et al, result in products having similar physical characteristics. Such enzymatic- ally-prepared polynuclεotides are 400-800 units in length, require double-stranded polynucleotides as starting materials, produce double-stranded poiynucleotides in all cases, and do not allow labeling at pre-selected sites. • In addition, all enzymatic-methods modify both strands of the polynucleotide, . and such product strands cannot be isolated from one another. During such pro¬ cesses, enzymes replace either all units with modified units or, when provided with a mixture of modified and naturally-occurring nucleoside triphosphates, randomly insert modified units. Furthermore, the enzy¬ matic process described by Langer, et al, is incapable of producing polynucleotides incorporating such reporter groups as fluorescent, luminescent, or antigenic reporter groups. None of this art is capable of producing oligo¬ nucleotides of defined length, defined sequence or single- stranded character, either with or without reporter groups. Moreover, by the prior art methods, modified bases having reporter groups attached thereto cannot be incorporated in a polynucleotide at preselected sites. Double-stranded polynucleotides which incorporate adenine bases modified at the C-8 position have also been prepared enzymatically. This has been done by incorporating 8-aminohexylamino-ATP (a ribonucleotide) • onto DNA fragments, as reported by C. Vincent et al, Nucl. -Acids Res. 10:6787-6796 (1982). The method is. limited in scope, however, allowing only 3'-end labeling with triphosphates of adenine ribonucleo- tides. No modified pyrimidine nucleosides can be incorporated. Furthermore, as with other enzymatic methods, both strands of a double-stranded polynucleo¬ tide are labeled and no short (<100 units) oligonucleo¬ tides of defined sequence can be produced. The prior art enzymatic methods referred to above require the. chemical synthesis of a substituted nucleoside 5'-triphosphate, and demand subsequent enzymatic recognition and incorporation of the unnatural substrate into nicked ' double-stranded DNA. Such methods are incapable of producing polynucleotides .of any pre¬ selected length or sequence, and the polymerases and DNases used therein are expensive. Moreover, these methods are time consuming, inefficient, demand substan- tial enzymatic activity and are limited to double-stranded DNA. Only small amounts, i.e., icrograms, of ill-defined polynucleotides, usually restriction fragments, are produced, and these must be tediously isolated from natural sources. Moreover, the scope of modifications obtainable in the polynucleotide product is severely limited, since the DNA polymerase cannot recognize or in- corporate potentially useful reporter groups such as fluorescein or dinitrophenyl. Attachment of one fluorescent molecule to the 3' end of long polyribonucleotide molecules (RNA) .for .limited biological application is disclosed by J.G.J. Bauman et al, J. Histochem. Cytochem. 29:238 (1981). This approach also used very small amounts (microgram quantities) of RNA tediously isolated from natural sources using enzy- ' matic methodology, and cannot be applied to DNA since both 2' and 3' hydroxyls are required therefor. The much greater chemical instability of RNA relative to DNA also minimizes the scope of application of the polyribonucleo¬ tide produced by this method. The non-enzymatic synthesis of defined sequence oligonucleotides incorporating naturally-occurring nucleic acid bases.has been reported or reviewed by S.A. Narang et al, Meth. Enzymol 65:610 (1980), R. Letsinger, J^ Org. Chem. 45:2715 (1980), M. Matteucci et al, J. Amer. Chem. Soc. 103:3185 0982), and G. Alvar do-Urbina et al, Science 214:270 (1981). Such synthesis usually in¬ volved chain extension by coupling an activated nucleotide monomer and a free hydroxyl-bearing terminal unit of a growing nucleotide chain. The coupling is. effected' through a phosphorus-containing group, as in the phosphate triester method reviewed by Narang et al, or one of the phosphite triester methods. Of the latter, those of Letsinger et al and Alvarado-Urbina et al use phosphochloridite chemistry, and that of Matteucci et al uses phospho- amidite chemistry. The aforementioned chemical synthesis of oligo¬ nucleotides has incorporated only unmodified or naturally-occurring nucleic acid bases, and the end product there of is in short fragments and resembles unmodified or naturally-occurring RNA or DNA. It is also worthy of note that the end product of such synthesis does not incorporate any labels or reporter groups. Direct modification of homopolymeric polynucleo¬ tides has been reported in systems of polyuridylic acid [Bigge, et al, L Carb. , Nucleosides, Nucleotides 8:259 (1981)]. The reported procedure is of limited scope and is not productive of useful products. Treatments described cause extensive polynucleotide cleavage and degradation, result in product irrever-- sibly contaminated with metal ions, and are capable of modifying only cytosine residues of a DNA polynu- cleotide. The method is incapable of producing defined sequence oligonucleotides of specific lengths, cannot modify thymine. or purine bases, and cannot modify at previously selected sites. OMPI W From the foregoing it will be apparent tha . labelled, defined sequence polynucleotides have been produced heretofor only by enzymatic methods which have the disadvantages pointed out herein, .particu- larly those relating to time, cost, product length, sequence and yield. Moreover, such methodology is productive of only double-stranded products. Such double-stranded polynucleotides can be denatured by alkali or heat to cause spontaneous short-term separa- tion of strands in solution. However, the individual single strands cannot be physically isolated from each other, and removal of the denaturing conditions results in rapid renatura ion to double-stranded form. Since conditions productive of hybridization are also pro- ductive of renaturation, subjecting the denatured polynucleotide to hybridization conditions results in return of the polynucleotide to its original double- stranded configuration in which hybridization of either of the strands to a target polynucleotide is limited by competition from the other strand. Modific-ation of nucleosides has been undertaken, for example, in the synthesis of antiviral C-5 sub¬ stituted pyrimidine nucleosides disclosed by Bergstrom, et al, J. Amer. Chem. Soc. 98:1587-1589 (1976); Ruth, et al, J. Or . Chem. 43:2870-2876 (1978); and Bergstrom, et al, U. S. Patent Nos. 4,247,544 and 4,267,171, or in the synthesis of C-8 substituted adenine derivatives disclosed by Zappelli, et al, U. S. Patent Nos. 4,336,188 and 4,199,498. These nucleosides, and others reported by Langer, et al, and D. Ward, European Patent Application No. 0063879, are not useful in ^ the process of the present invention. Such reported nucleosides are highly reactive at undesired sites-, and, if used in oligonucleotide synthesis by chemical methods, wouldresult in undesired,side products, uncon¬ trollable synthesis, and no-desired product. Moreover, such reported nucleosides do not contain proper sites in the substituent group, cannot be modified by the attachment of reporter groups, .nor do they contain masked reactive functionalities. No such nucleosides are useful in the process of the present invention. No ' chemical synthesis of defined sequence oligo¬ nucleotides incorporating modified bases of any kind, either with or without reporter groups, has been disclosed in the prior art. There is an urgent need for high quality labelled, defined sequence single strand oligonucleotides which do not involve hazardous and unstable radioisotopes, and satisfaction of this need is a principal obiect of O PI the present invention. This object is accomplished by a chemical,, i.e., nonenzymatic., process which provides a predictable, superior product in high yield. More particularly, the process of the present invention accomplishes the chemical incorporation into defined sequence oligonucleotides of nucleotides modified with a wide variety of selected detectable reporter groups, such oligonucleotides being useful, for example, for the identification, localization, isolation and/or quantitation of complementary sequences of interest. Another object of the invention is to provide novel nucleosides useful in the chemical synthesis of labelled, defined sequence single strand oligonucleo¬ tides. The process of the present invention accomplishes the de novo chemical synthesis oJ labelled, defined sequence oligonucleotides and is superior to prior art enzymatic methods in a number of respects. .More specifically the process of the present invention makes possible the synthesis of labe.lled, defined sequence single-stranded oligonucleotides of homogeneous pre¬ dictably defined Length, preferably having fewer than 200 bas ' e units, in contrast to the production of heterogeneous unpredictable populations of 400 to 10,000 base units of double-stranded character produced by prior art enzymatic methods. The yield of product produced by the process of the present invention is of the order of hundreds to tens of thousands of micrograms, in contrast to the yield of a few micrograms provided by the prior art enzymatic methods. Moreover, the product oligonucleo¬ tides of the present invention are single-stranded, rather than the double-stranded products of enzymatic - JR£4 _ OMPI __ . ?Λ'ATIO^ J methods. The single strand configuration of the product oligonucleotides avoids the competition from a complementary strand inherent in rehybridization of double-stranded ' polynucleotides, . Disclosure of Invention The invention comprises a defined sequence single strand oligonucleotide of fewer than about 200 base units in length which comprises ,at least one nucleo¬ tide unit having attached to the base thereof at a sterically tolerant site (such as C-5 of pyri idines and C-8 of purines) a substituent group which is capable of functioning as one or more detectable reporter groups, or of binding one or more detectable reporter groups. The invention also comprises a process for the chemical, i.e., nonenzymatic, synthesis of a defined sequence single strand oligonucleotide which comprises coupling an activated nucleotide monomer and a free hydroxyl-bearing terminal unit of a growing nucleotide chain, at least one of the monomer and terminal unit having its base modified at a sterically tolerant site by attachment thereto of a substituent group capable of functioning as one or more detectable reporter groups, . or of binding one or more detectable reporter groups. The invention additionally comprises novel nucleosides useful in this synthetic process. The oligonucleotide produced by the process of the present invention may include one or more pyrimidine- based or purine-based units which may be ribonuclεotides or deoxyribonucleotides, and prior to the synthesis thereof, the reporter groups are preselected, as are the particular nucleotide units to which the reporter groups are attached. Best Mode of Carrying Out the Invention The chemical process by which the defined sequence single strand oligonucleotides of the present invention are preferably synthesized comprises coupling an acti- vated nucleotide monomer and a free hydroxyl-bearing terminal unit of a growing nucleotide chain., at least one of said monomer and terminal unit having its base modified at a sterically tolerant site by attachment thereto of a substituent group capable of functioning as one or more detectable reporter groups, or of binding one or more detectable reporter groups. The substituent groups of the present invention which are capable of binding reporter groups can be generally characterized as those which exhibit nucleo- philic properties. Exemplary of such groups are those which contain primary or aromatic amines ' , carboxylic acids , hydroxyls and the like. The bases of the nucleotide monomer and terminal unit are selected to provide the predetermined sequence of nucleotide units desired in the end product oligo¬ nucleotide. Such bases can take the form of the purines adenine (A) , guanine (G) , or hypoxanthine (H) , or of the pyri idines uracil (U) , cytosine (C) , or thymine (T) . Such bases may also take the form of any other base which can be isolated from natural sources. A sterically tolerant site on a nucleotide unit can ' be defined as a position on the nucleic acid base of the unit at which modification of the unit can be effected by attachment thereto of the substituent group without causing significant interference with hybridization of ' the product oligonucleotide and complementary nucleic άeid components, and without sterically preventing the substituent group from functioning as one or more reporter groups, or from binding one or more reporter groups. Sterically tolerant sites are found at the C-8 OMPI position of purines and at the C-5 position of pyrimi- dines. Since the oligonucleotides of the present invention, are particularly useful as hybridization probes, modifications which attach substituent and/or reporter groups should ' not be at: sites on the pyrimi¬ dine or purine bases which are necessary for specific hybridization. Sites which should not be modified include N and N of adenine bases; N , N , .and 0 of guanine bases; N 3 and N4 of cytosine bases. Gener- ally, substitution at any heteroatom (N or 0) should be avoided. A reporter group can be defined as a chemical group which may be aromatic and/or polycyclic and which has a physical or chemical characteristic which can be readily measured or detected by appropriate physical or chemical detector systems or procedures. Reporter groups which are useful in oligonucleotides of the pre¬ sent invention are readily detectable. Ready de ect- ability may be provided by such characteristics as color change, luminescence, fluorescence, or radio¬ activity; or it may be provided by the ability of the reporter group to.serve as a ligand recognition site. Such groups are termed functionally colorimetric, luminescent, fluorescent, radioactive or ligand recog- nition groups. Among such groups are those suitable for ready detection by conventional detection techniques, such as colorimetric, spectrophotometric, fluorometric or radioactive detection, as well as those which are capable of * participating inthe formation of specific ligand-ligand complexes which contain groups detectable by such conventional detection procedures. ' χ A reporter group as used herein is defined as a substituent group which has physical, chemical or other characteristics which can be readily measured or de¬ tected by the use of appropriate measurement or detec¬ tion procedures. A reporter group as defined herein also includes a substituent group which is capable of initiating one or more..interactions which ultimately provide a reaction product or a complex having physical, chemical or other characteristics which can be readily measured or detected by the use of appropriate measure¬ ment or detection procedures. Exemplary of the measurable or detectable charac¬ teristics which such groups, reaction products or com¬ plexes may exhibit or induce are a color change, luminescence, fluorescence or radioactivity. Such characteristics can be measured or detected by the use of conventional colorimetric, spectrophotometric, fluorometric or radioactivity sensing instrumentation. The interactions which usefully can be initiated by the reporter group defined herein include appropri¬ ately specific and selective ligand-ligand interactions productive of groups or complexes which are readily detectable, for example, by colorimetric, spectrophoto¬ metric, fluorometric, or radioactive detection proce¬ dures. Such interactions may take the form of protein- ligand, enzyme-substrate, antibody- ntigen, carbohydr te- lectin, protein-cof ctor, protein-effector, nucleic acid-nucleic acid or nucleic acid-ligand interactions. Exemplary of- such ligand-ligand interactions are dinitrophenyl-dinitrophenyl antibody, biotin-avidin, oligonucleotide-complementary oligonucleotide, DNA-DNA, RNA-DNA and NADH-dehydrogenase. Other useful interac¬ tions will suggest themselves to those skilled in the art. OMPI In the process of the present invention, a selected reporter group or groups can optionally be attached to the nucleotide monomer before coupling of the monomer to the terminal unit of the nucleotide chain, or it can be attached to the"product oligonucleotide after formation thereof. The sequence of the nucleotide units in the product oligonucleotide is preselected to provide such oligonucleotide with precise specificity for its ultimate use. The oligonucleotides of the present invention are useful tools in recombinant DNA and other protocols involving nucleic acid rehybridization tech¬ niques. Among such uses are identification, localiza¬ tion, isolation and/or quantitation of complementary sequences of interest in cellular or cell-free systems. More specifically, such uses may include diagnostic applications or any fundamental biological event in- volving. hybridization of nucleic acid components, or purification of complementary sequences by affinity chromatography when the product oligonucleotide is ' attached to a solid support through the modifications at a sterically tolerant site, with or without sub¬ sequent detection. The nucleotide units in the product oligonucleotide may be purine or pyrimidine-based and may comprise units having naturally-occurring bases intermixed with units having modified bases. Such units may be ribonucleo- tides or deoxyribonucleotides. The coupling step pre¬ ferably involves coupling of a monomer unit activated at the 3 1 position with a free 5' hydroxyl of the ter- minal unit of the growing nucleotide chain. Alterna¬ tively such coupling can involve coupling of a monomer unit activated at the 5' position with a free 3' hydroxyl of the terminal unit of the nucleotide chain. The _. OMPI terminal unit may be the initial or only unit in the growing nucleotide chain at the time of coupling thereto of the nucleotide monomer, or it may be the terminal one of a plurality of nucleotide units. The process of the ' present"invention produces defined sequence oligonucleotides of the following generic formula: FORMULA I Wherein n is 1 to about 199, preferably about 5 to- about 60, and most preferably about 10 to about 40, R' is hydrogen or hydroxy, and B is any of the naturally- occurring purine or pyrimidine bases adenine, guanine, cytosine, uracil., thymine, or any other naturally- occurring base, the nucleotide units having naturally- occurring bases being independently intermixed with one or more nucleotide units having modified bases (B m ) . The modified pyrimidine bases (Py ) are substituted at the C-5 position, and typical examples thereof are the uracil and cytosine bases illustrated by the following generic formulas : modified uracil base modified cytosine base The modified purine bases (Pu TIK) are substituted at the C-8 position, and typical examples thereof are the modified adenine and guanine bases illustrated by the following generic formulas: modified adenine base modified guanine base The substituent group R is characterized by its ability to function as one or more reporter groups, or to bind one or more reporter groups . In the modified pyrimi- ,'dine bases the substituent group R comprises two or more carbon atoms, whereas in the modified purine bases R comprises one or more carbon atoms. In this context, R preferably takes the form of one of the following functionalized carbon chains: R = -CH=CR 1 R 2 , -CH 2 CHR 1 2 . -CHR-^, • ' P - ■ -01=0^- CNHR 2 , or -CH=CR 1 -C-R 2 wherein R- j _ is hydrogen or alkyl; 2 is alkyl, alkenyl, aryl, or functionalized alkyl, alkenyl, aryϊ wherein functional groups include one or more amines, amides , nitriles, carboxylic acids and esters, hydroxy, dini- trophenyl, aminobenzenesulfonates, or the like; and Z is a polyvalent heteroatom such as nitrogen, oxygen or sulfur. In addition, R 2 may be attached to a solid support, or to one or more reporter groups which func¬ tion, for example, ds a colorimetric, fluorescent, luminescent, radioactive, or ligand recognition group. Functionally fluorescent groups include fluoresceins , rhoda ines, and the like or adducts thereof; func¬ tionally luminescent groups include luminols , acridines, luciferins , dioxetanes, dioxamides , and the like or adducts thereof. Ligand recognition groups include vitamins (such as biotin, iminobiotin or desthiobiotin) , antigens such as dinitrop ' henols, carbohydrates and other functional groups or adducts of such groups which can be recognized .by ligand-like interactions with proteins, or from which such ligand-like interactions can be elicited. Another oligonucleotide capable of interaction with nucleic acids is illustrative of a group from which a ligand-like interaction can be elicited. Ligand recognition groups may also serve as functionally colorimetric reporter groups when recog- nition results in dye formation. For example, when dinitrophenyl is used as a reporter group, known detection systems using an antidinitrophenyl antibody coupled to peroxidase can be used as a detection system, resulting in a color change. Functionally radioactive groups incorporate a radioactive element in the chosen reporter group. When reference is made herein to the use of purine or pyrimidine bases, such expressions are intended to include analogs of such bases . Among such analogs are the analogs of purine bases, such as the deazaadenosines (tubercidins, formycihs ' j and the " like) , and the analogs of pyrimidine bases, such as deazauracil, Leazacytos ±ne , azauracils , azacytosines, and the like. Oligonucleotides of Formula I are best prepared by chemical synthesis from monomer nucleotide analog units of the formula: o- X - chloro, dialkyl- FORMULA II amino, morpholino wherein R- is trityl (triphenylmethyl) , dimethoxytrityl, or other appropriate masking group for the 5 '-hydroxyl; B and R' are masked, if appropriate; and (P represents a phosphorus-containing group suitable for internucleo- tide bond formation during chain extension in synthesis of a product oligonucleotide. The phosphorus-containing groups (?) suitable for internucleotide bond formation ar* 2 preferably alkyl phosphomonochloridites or alkyl phosphomonoamidites. Alternatively phosphate triesters may be employed for this purpose. The monomer unit may alternatively have R, attached at the 3' hydroxyl and (^ attached at the 5'-hydroxyl. Generally, the term "masking group" or "blocking group" is a functional expression referring to the chemical modification ' or "blocking" of an integral functional group by attachment of a second jnoiety to disguise the chemical reactivity of the functional group and prevent it from reacting in an undesired manner. Such modification is reversible, and allows subsequent conversion back to the original functional group by suitable treatment. In'many cases, such mask¬ ing formally interconverts structural functionality, e.g., a primary amine masked by acetylation becomes a substituted amide which can be later converted back to the primary amine by appropriate hydrolysis. The compounds of Formula I include the acceptable conjugate acid salts thereof. Conjugate acids which may be used to prepare such salts are those containing •nonreactive cations and include, for example, nitrogen- containing bases such as ammonium salts, mono-, di-, tri- or tetra-substituted amine salts, and the like, or suitable .metal salts such as those of sodium, potassium, and the like. The process steps of the present invention will now be generally described and illustrated diagrammatically. Thereafter, the. invention will be illustrated more speci¬ fically and detailed examples therof provided. ' Since the . invention relates to . oligonucleotides incorporating both pyrimidine-based and purine-based nucleotide units, the use of both pyrimidine and purine-based compounds in the synthetic process will be illustrated. The specific pyrimidine and purine-based compounds illustrated are only exemplary of the respective pyrimidine and purine " classes, and it is to be understood that any other member of the respective class can be substituted therefore in the process and the product oligonucleotide, when¬ ever suitable or desired. While deoxyribonucleotide compounds are shown for the most part, it is unde - stood that ribonucleotide compounds are also con¬ templated by the invention and can be substituted for the deoxyribonucleotide compounds wherever ribonucleo¬ tide compounds are desired in the product oligonucleo¬ tide. One of the more important aspects of the invention is the provision of a new class of nucleosides which are essential as intermediates in the process for syn¬ thesizing the new oligonucleotides. Such nucleosides each have a base which is modifie'd by a substituent group comprising a functionalized carbon chain and one or more amides, the nitrogen of the amides being ' attached to a sterically tolerant site on the base through the carbon chain. In the case of pyrimidine-based nucleo¬ sides, the carbon chain is attached at the C-5 position, and in the case of"the purine-based nucleosides, the carbon chain is attached at the C-8 position through a polyvalent heteroatom, such as nitrogen, oxygen or sulfur. In addition, such nucleosides are chemically blocked at the 5' position (or the 3' position) with a group, such as dimethoxytrityl, appropriate for the chemical synthesis of oligonucleotides. In the new class of nucleosides the substituent hydrogen or C-,_g lower alkyl and Y is one or more amido, substituted amido, substituted amino or substituted aminoalkylphenyl groups. More specifically, Y may include one or more wherein X is hydrogen, fluorine or chlorine. Synthesis of these nucleosides, as well as of the masked forms thereof, is described hereinafter in Examples I, II, IV, VI, VII, VIII, X, XII, and XIII. Preferred nucleosides incorporate 0 the substituent group -CH=CHC'-NHC n H 2n Y at C-5 of pyrimidine nucleosides wherein n = 3 to 12 and Y is -NH 9C.CX . Most preferred are such ' nucleosides wherein the pyrimidine base is uracil. The process of the present invention may be initiated by the preparation of the selected nucleoside. Generally, the most preferred nucleosides are best prepared in the following manner. 5- (Methyl 3-acrylyl) -2'-deoxyuridine is prepared from 2' -deoxyuridine by the method of Bergstrom and Ruth [J\_ Amer. Chem. Soc. 96:1587 (1976)]. The nucleoside is then treated with 1.05 equivalents of dimethoxytrityl chloride in pyridine for 4 hours to block the 5 ' ••hydroxyl with dimethoxytrityl (DMT) . The resulting product is puri¬ fied by silica chro atography eluting a gradient of 0-10% methanol in chloroform containing 27, triethy- lamine. The purified 5'-DMT-5-(methyl 3-acrylyl) - Z[-deoxyuridine is treated with 1 N KOH for 24 hr. at ambient temperature to hydrolyze the methyl ester. . OMPI The resulting 5'-DMT-5-(3-acrylyl)-2'-deoxyuridine is treated with excess dicyclohexylcarbodiimide and hydroxybenztriazole in pyridine. After 4 hours, a 2-5 fold excess of 1,7-diaminoheptane is added, and the reaction stirred overnight. -After 12-20 hours, a 10-20 fold excess of trifluoroacetic anhydride is added, and the reaction stirred at room temperature for 4 hours. The " product is purified by silica chromatography eluting a gradient of 0-10% methanol in chloroform containing 2% triethy1amine, followed by exclusion chromatography using Sephadex LH-20 eluting 1% triethylamine in methanol. Appro¬ priate fractions are combined to yield 5'DMT-5- [N-(7-trifluoroacetyla inoheptyl)-1-acrylamido]-2'- deoxyuridine; such product is appropriate for oligo¬ nucleotide' synthesis by the phosphochloridite proce¬ dure described in Examples XV and XVIII. Alternatively, such a compound can be prepared by the combination of methods described in Examples II and III. Replacing diaminoheptane in this process with other diamino- alkanes (e.g., diaminopropanε, diaminohexane, diamino- dodecane) is productive of other compounds of varying substituent length wherein n = 3, 6, or 12 and (adenine)-based, are shown at the top of the diagram below illustrating the process. Reactive sites on the bases of the nucleosides are then masked, as shown in Reaction 1, by attachment of, for example, a benzoyl group (Bz) to the amine at the 6 position of the adenine-based nucleoside. Such masking is generally described in "Synthetic Pro¬ cedures in Nucleic Acid " Chemistry", Vol. 1, W. Zorbach and R. Tipson eds. (Wiley - Interscience, N.. Y.) 5 1968) . Unprotected amines on the substituent group are masked, for example, by attachment thereto of trifluoroacetyl groups (Ac), as also shown in Reaction 1. The selected 3' or 5' hydroxyl of the nucleoside is then masked by attachment thereto of a dimetho- 0 xytrityl (DMT) group. In Reaction 2 illustrated below, the 5'-hydroxyl is masked, leaving the 3' hydroxyl free or available for reaction. Alternatively, the 3' hydroxyl could be masked, leaving the 5' hydroxyl free. The nucleoside is then converted to' an activated 5 nucleotide monomer, preferably by attachement to its 3' hydroxyl of a phosphorus-containing group which includes an activating moiety. When the modified nucleoside is properly blocked, modifications of the procedures de- 0 scribed by Letsinger, et al, Matteucci, et al, or as reviewed by Narang, et al can be utilized for oligo¬ nucleotide synthesis. The use of phosphochloridite chemistry such as that disclosed by Letsinger, et al, is detailed in Examples XVI-XVIII. In order to use 5 phosphoamidite chemistry, a modification of- the proce¬ dure of Matteucci, et al, is used, phosphitylating the protected modified nucleoside with methyl chloro (N, N-diisopropyl)P,hosphoamidate or methyl chloro- phosphomorpholidate, as in the improved procedure of 0 Dorper, et al [Nucleic Acids Res. 11:2575(1983)]. Al- OMPI ternatively, * the protected modified nucleoside can be phosphoryl ' ated with 1.2 eq. chlorophenyl dichlorophos- phate in trimethylphosphate at room temperature fol¬ lowed by quenching with water to give the 3'-chloro- phenyl phosphate adduct of the modified nucleoside, such adducts being useful in a modification of the phospho- triester approach as illustratively reviewed by Narang, et al. The diagram illustrates in Reaction 3 the syn¬ thesis of activated monomer nucleotide units of Formula II by attachment to the nucleoside 3* hydroxyl of a phos- pho onochloridite group in which the chlorine functions as an activating moiety. Coupling or condensation of the selected activated nucleotide monomer, i.e. the uracil-based monomer or the adenine-based monomer, to the ,terminai unit of a growing nucleotide chain is illustrated in Reaction 4 in the diagram. The nucleotide chain is shown as- .in¬ cluding in its right hand end a nucleoside unit having a naturally occurring base and having a solid support or masking group R, attached to its 3' hydroxyl. The illustrated chain also includes one or more (n') nucleo¬ tide units having naturally-occurring bases, said units being coupled to the 5' hydroxyl of the nucleoside unit, the terminal one of the nucleotide units having a free hydroxyl at the 5' position. In the coupling reaction the chlorine- of the monomer reacts with the hydrogen of the free hydroxyl of the terminal unit and is displaced, so that the oxygen of the terminal unit couples to the phosphorus of the monomer as shown, and the monomer thereby becomes the new terminal unit of the nucleotide chain. The DMT 5' blocking group is then removed to permit further extension of the nucleotide chain by sequential coupling thereto of additional activated nucleotide monomer units. The nucleotide units added to the chain can be preselected and may have either naturally-occur¬ ring or modified bases. The diagram shows in Reaction 4a the .further extension of the chain by the addition of one or more (n") nucleotide units having naturally- occurring bases. , When an oligonucleotide of the selected length and sequence has been synthesized, the DMT group is removed from the terminal unit thereof, and the masked reactive groups are unmasked. Examples .of modified .uracil and adenine bases with their reactive groups unmasked are also shown diagrammatically at Reaction 5. If the initial nucleotide unit of the chain is bound to a solid support R, , the chain is then removed from such solid support. The appropriate order of unmasking can be pre- selected. If the substituent groups of the modified bases are not capable of functioning as reporter groups in the intended use of the product oligonucleotide, appropriate reporter groups R,- can then be bound to such substituent groups as exemplified in Reaction " 6, which illustrates the respective bases with reporter groups R^ bound to the respective substituent groups-thereof. Illustration of Synthetic Process uracil-based adenine-based Reaction 1 \ Masking of reactive sites on nucleoside . bases ^ J!3R£ 7 - OMPI Reaction 2 Masking of 5'-hydroxyl (DMT = dimethoxytrityl) Reaction 3 Activation to phospho- chloridite w ere B modi ie ase see pg 29) where, for example, B in product oligonucleotide (pg 28) is : uracil base adenine base (modified) (modified) To the product oligonucleotide a variety of useful reporter groups (Re) may be attached. For example: PRODUCT OLIGONUCLEOTIDES WITH REPORTER GROUPS (where Re = biotin or fluorescein) JΌ ; OMPI Having discussed the process of the present invention in general terms and illustrated the same diagrammatically, each of the reactions referred to will-now be discussed more specifically. " With reference to Reaction 1 * -, masking of chemically 4 6 reactive amines such as N of cytosine, N of adenine, N 2 of guanine, and alkyl or aryl amines of the modified bases with suitable masking groups can be conveniently accomplished in suitable solvents such as alcohols, pyri- dines, lutidines, chloroform, and' the like, by reaction of the nucleosides with an excess of appropriate acid anhydrides for about 1 to 24 hours at temperatures in the range of 0°C to 110°C, generally 20°C to 80°C. Appropriate acid anhydrides include acetic anhydride, trifluoroacetic anhydride, benzoyl anhydride, anisoyl anhydride, and the like. Preferred are acetyl, tri- fluoroacetyl, benzoyl, and isobutyryl anhydride. Masking of the 5 τ -hydroxy in Reaction 2 can be conveniently effected by reaction of the nucleosides with a slight excess of appropriate acid-labile masking reagents, such as tritylchlorides, monomethoxytrityl chloride, dimethoxytrityl chloride (DMTCl), trimethoxy- trityl chloride and the like. Preferred is dimethoxy¬ trityl chloride. Typical reactions are carried out in suitable solvents, such as pyridine, lutidines, tri- alkylamines, and the like, at temperatures in the range of -20°C to 120°C, generally 20°C to 100°C, for about 1 to 48 hours. The preferred reaction utilizes 1.1 equivalents of DMTCl * * in pyridine at room temperature for 2 hours. It is generally preferred that the respective pro¬ ducts of each reaction described hereinabove be separa¬ ted and/or isolated prior to use as a starting material for a subsequent reaction, Separation and isolation can be effected by any suitable purification procedure such as, for example, evaporation, filtration, crystal¬ lization, column chromatography, thin layer chromatog- raphy, etc. Specific • illustrations of typical separa¬ tion and isolation procedures can be had by reference to the appropriate examples described hereinbelow; how¬ ever, other equivalent separation procedures can, of course, also be used. Also, it should be appreciated that, where typical reaction conditions (e.g., tempera¬ tures, mole ratios, reaction times) have been given, conditions both above and below the typical ranges can also be used, though generally less conveniently. Activation to the phosphite analog illustrated in Reaction 3 can be most conveniently effected by treat¬ ment of the nucleoside compounds with suitable phosphi- tylating agents in appropriate solvents at temperatures in the range of -90°C to 60°C for 1 minute to 2 hours. Suitable phosphitylating agents include methylphospho- dichloridite, o-chlorophenylphosphodichloridite, p-chlo- rophenylphosphodichloridite, methylphospho(dialkylamino) monochloridite, and the like. Appropriate solvents in¬ clude pyridine, lutidines, acetonitrile, tetrahydro- furan, dioxane, chloroform and the like containing 0-20% ' appropriate base (generally 1-5 vol %) such as lutidines, collidines, triakylamines and the like. Preferred phos¬ phitylating agents are methylphosphodichloridite, _o- chlorophenylphosphodichloridite,* and methylphospho- (di-isopropylamino)-monochloridite. Preferred phosphy- tilating conditions are with 0.9 equivalents of methyl phosphodichloridite in pyridine or acetonitrile contain¬ ing 5% 2,6-lutidine for 5 to 10 minutes at room tempera¬ ture or below. 5U £ O PI _ ?NATlθ The chemical incorporation of the modified nucleo¬ tide analog monomers into a growing nucleotide chain to produce defined sequence nucleotides is ' illustrated in Reactions 4 and 4a. Typical condensations are in appro- priate solvents at temperatures in the range of -20°C to 50°C, preferably at ambient temperature, for about 1 to 60 minutes. Appropriate solvent mixtures include pyridine, lutidines, acetonitrile, tetrahydrofuran, dioxane, chloroform and the like containing 0-20% appro- priate base (generally 1 to 5 volume %) such as lutidines, collidines, trialkylamines and the like. The growing chain may be soluble, insoluble, or attached to a suit¬ able solid support by appropriate chemical methods known in the art. Preferred is attachment to a solid support. Furthermore, the growing chain may or may not have pre¬ viously incorporated one or more modified nucleoside analogs. After condensation of the activated monomer to the growing chain, in Reaction 4, the initial product ' is treated with suitable reagents to accomplish oxidation of the intermediate phosphitet iester, optional capping to. block unreacted 5'-hydroxyls on the oligonucleotide chain, and removal of the 5'-DMT group. Oxidation of the phosphite triester can be accomplished by treatment * with 0.1-5 w/vol % iodine in suitable solvents, for example, tetrahydrofuran/water/lutidine mixtures. Chemi¬ cal capping of unreacted 5'-hydroxyls can be accomplished by acetylation or acylation with, for example, acetic anhydride and 4-dimethylaminopyridine in tetrahydrofuran/ lutidine mixtures. Removal of the 5*-blocking group, usually DMT, is most conveniently effected by treatment with mild organic acids in nonprotic solvents, such as mild acids including, for example, 1-5 vol % dichloro- acetic or trichloroacetic acid in chloroform or dichloromethane. The growing nucleotide chain, after removal of DMT, can now serve as acceptor for subse¬ quent elongation by sequential reaction with activated monomers to eventually produce the oligonucleotide of desired length and sequence, as shown in Reaction 4a. After an oligonucleotide of desired sequence is produced, Reaction 5 is accomplished to provide the product oligonucleotide. To this end, thiophenol treat¬ ment is used to remove methyl masking groups from phos- phate triesters, and suitable aqueous alkali or ammonia treatment is used to remove benzoyl, acetyl, isobutyl, trifluoroacetyl, or other groups from the protected amines and/or to remove the product from the solid sup¬ port. Removal of DMT from the oligonucleotide product is accomplished by the appropriate treatment with a mild acid, such as aqueous acetic acid at ambient temperature to 40°C for 10 to 60 minutes. Such reactions may be accomplished before or during final purifications. Final purification is accomplished by appropriate methods, such as polyacrylamide gel electrophoresis, high pressure liquid chromatography (HPLC) , reverse phase or anion exchange on DEAE-cellulose, or combinatins of these methods The process described herein for synthesis of oligo¬ nucleotides can utilize modified deoxyribonucleosides (where R' is H) or modified ribonucleosides (where R' is hydroxyl). When ribonucleosides are used, the 2'-hydroxyl is masked by an appropriate masking group such as, for example, that afforded by silylethers. Other ribose OMPI analogs, including arabinose and 3'-deoxyribose, can also be accommodated in the process to produce the desired oligonucleotide. The substituent group modifying a nucleotide base may itself function as one or more reporter groups. An example thereof is a substituent group which incor¬ porates a dinitrophenyl group. Where the siibstituent group is not capable of functioning as a reporter group, it must be capable of binding one or more reporter groups either prior to or after the chain extension coupling .reaction. Selected product oligo¬ nucleotides are reacted with suitable agents to attach such reporter groups. For example, when modified bases are incorporated into the oligonucleotide and R 2 of the substituent group contains one or more primary amines, coupling with amine-reactive groups . such as isocyanate, isothiocyanate, active carboxylic acid conjugates, expoxides or active aromatic com¬ pounds using suitable mild conditions is productive of amide, urea, thiourea, amine or aromatic amine linkages. For example, an oligonucleotide which contains an uracil or adenine base modified by a substituent group having a primary amine, as shown in the Reaction 5 diagram, can be reacted with a suitable reagent, such as fluorescein isothiocyanate, to provide a reporter group Re (fluorescein) bound to the substituent group as shown in Reaction 6. Other reporter groups which can be attached in similar manner include a wide variety of organic moieties such as fluoresceins, rhόdamines, acridinium salts, dini- trophen ls, benzenesulfonyls, luminols, luciferins , biotins, vitamins, carbohydrates and the like. Suitably O PI active reporter groups are available commercially, or can be synthesized, for example, by processes of the type generally described in * "Bioluminescence and Chemiluminescence" [M. DeLuca and W.. McElroy, eds. , Acad. Press, New York (1.981) ] , by . D. Roswell, et al., or H. Schroeder, et al. [Meth. Enzymol. LXII, 1978], and references cited therein. Typically, attachment of reporter groups is conveniently accomplished in predominately aqueous solvents by reaction of the substituent groups of modified bases wherein z~ = Cn Hzn NHz n with excess of the selected reporter group at temperatures in the range of about -20°C to 50°C (preferably 20°C to 40°C) for 1 to 24 hours. Suitable solvents are an aqueous buffer and 0-50% organic solvents such as lower alcohols, tetrahydrofuran, dimethylforma- mide, pyridine, and the like. Preferred reporter group reactants include fluorescein • isothiocyanates, dinitrophenylisothiocyanates, fluorodinitrobenzene, N-hydroxysuccinimidylbiotin, N-hydroxysuccinimidyl dinitrobenzoate, isothiocyanates such as aminobutyl ethyl isoluminol isothiocyanate and the like, active esters of carboxyfluorescein, rhodamine, biotin adducts, dioxεtanes, dioxamides, carboxyacridines, carbohydrates and the like. Additionally, when the product oligonucleotide includes modified bases wherein R contains one or more carboxylic acids, mild condensations with, for example, primary alkylamines is productive of amide linkages. Typically, this is conveniently effected in predo¬ minately aqeuous solvents by reaction of the oligo¬ nucleotide with excess reporter group which contains a primary amine in the presence of suitable condensing agents, such as water-soluble carbodiimides, at temperatures in the range of about -20°C to 50°C (preferably 20°C to 40°C) for 6 to 72 hours. Pre¬ ferred reporter groups ' o this class include (amino- alkyl)-amino-naphthaiene-1, 2-dicarboxylic acid hydrazides, amino-fluoresceins, aminorhodamines, aminoalkyl luminols, aminoalkylaminobenzenesulfonyl adducts, amino sugars, and the like. Furthermore, the chemical synthesis of the initial oligonucleotide product may be accomplished with modified nucleotide monomers wherein the substituent group includes such reporter groups. If such groups require masking, they are appropriately masked during the coupling reactions. On the other hand, certain reporter groups do not require masking. For example, nitrophenyl adducts without masking may be present on the modified nucleo¬ tide monomer during the coupling reaction without adverse effect. Reporter groups useful in the method of this inven- tion generally include aromatic, polyaromatic, cyclic, and polycyclic organic moieties which are further functionalized by inclusion of heteroatoms such as nitrogen, oxygen, sulfur and the like. Product oligonucleotides can include more than one type of modification or more than one modified base. An illustrative example of an oligonucleotide of this type is one of the structure: wherein C m is 5-(3-aminopropyl) cytosine, U is 5-[N-( -aminbbutyl)-l-acrylamido_uracil, and A" is 8-[6-2,4-dinitrophenyl)-aminohexyl]aminoadenine This product is further- modified by reaction with fluorescein isothiocyanate to provide a fluorescein reporter group on C and U . Such a product oligonucleotide illustrates the variety of the selection of modified and unmodified nucleotide units in a product oligonucleotide made , possible by the process of the present invention. More specifically, such oligonucleotide illustrates the use of more than one type of nucleotide unit having its base modified by substituent groups pro¬ viding the ' same or different types of reporter group function. Also illustrated are units whose bases are modified by substituent groups to'which reporter groups are bound, i.e., C and U , whereas A is illustrative of a unit whose base is modified by a substituent group which itself is capable of func- tioning as a reporter group by virtue of incorpora¬ tion therein of a dinitrophenyl group. Such oligo¬ nucleotide additionally illustrates that it can include more than one nucleotide unit of the same type, and that it can include units having unmodified bases intermixed with units having modified bases. Instead of attaching reporter groups to the primary amines of the substituent groups as illu¬ strated in Reaction 6, such amine or other group can alternatively be coupled to suitably activated solid supports. This produces a defined sequence oligo¬ nucleotide which is covalently bound to such supports through the modified bases. Such solid supports are useful in the detection and isolation of complementary nucleic acid components. Alternatively, the modified nucleoside monomers can be coupled to solid supports prior to the chain extension coupling Reaction 4, to thereby provide solid supports for such monomers during the coupling -reaction. The following specific examples are provided to enable those skilled in the art to practice the inven¬ tion. The examples should not be considered limita¬ tions upon the scope of the invention, but merely as being illustrative and representative thereof. To aid in structural clarification, references are made to the reactions illustrated in the aforementioned process diagram. - ΕEX^ OMPI WIPO -VJ EXAMPLE I This example illustrates the synthesis of a modi¬ fied nucleoside precursor 5-(3-trifluoroacetylamino- propenyl)-2'-deoxyuridine. 5-Chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml methanol. N-Allyltrifluoro- acetamide(6.8 ml, 55 mmol) is added, followed by addi¬ tion o " f 41 ml of 0.2 N lithium tetrachloropalladate in methanol. After 18 hours stirring at room temperature, the reaction is gravity filtered to remove the black solid palladium, and the yellow methanolic filtrate is treated with five 200 mg portions of sodium borohydride, then concentrated under reduced pressure to solid resi¬ due. The residue is purified by flash column chroma- tography on silica gel eluting 15 vol % methanol in chloroform. Appropriately pure fractions of product are combined and concentrated under reduced pressure to give crystalline 5-(3-trifluoroacetylaminopropenyl)-2'- deoxyuridine (2.4 g) . UV λ maχ 291 ' ran (€7800), ' min 266 nm, £ 4400) ; TLC (silica eluting 15 vol % methanol in chloroform) R£ = 0.4. EXAMPLE II This example illustrates the synthesis of a modified nucleoside precursor 5-[N-(trifluoroacetylaminoheptyl)-l- acrylamido]-2'-deoxyuridine. 5-Chloromercuri-2'-deoxyuridine (3.6 g, 7.8 mmol) is suspended in 200 ml. methanol. N-(7-trifluoroacetylaird.no- heptyl)-acrylamide,(55 mmol) is added, followed by addi¬ tion of 41 ml of 0.2 N lithium tetrachloropalladate in methanol. After 18 hours stirring at room temperature, the reaction is gravity filtered to remove the black solid palladium, and the yellow methanolic filtrate is treated with five 200 mg portions of sodium borohydride, then concentrated tuider reduced pressure to solid resi¬ due. The residue is purified by flash column chroma¬ tography on silica gel eluting 10 vol % methanol in chloroform. Appropriately pure fractions of product are combined and concentrated under reduced pressure to give crystalline 5-[N-(7-trifluoroacetylaminoheptyl)-1-acryl- amidoJ-2'-d 302 nm (£ 18000) , ca eluting 15 vol % methanol in chloroform) R,_ = 0.3. EXAMPLE III This example illustrates masking of 5'-hydroxyl to produce 5'-dimethoxytrityl-5-(3-trifluoroacetyl- aminopropenyl)-2'-deoxyuridine as illustrated in Reaction 2. 5-(3-trifluoroacetylaminopropenyl)-2'-deoxyuri¬ dine (2.4 g) is thoroughly evaporated twice from pyridine, then stirred in 40 ml pyridine. Dimethoxy¬ trityl(DMT)chloride(2.3 g, 6.6 mmol) is added, and the mixture stirred at room temperature for four hours. After thin layer chromatography (TLC) on silica eluting 10 vol % methanol in chloroform indicates reaction is complete, the reaction is concentrated to a solid residue. This residue is purified by column chromatography on silica eluting chloroform until all faster running impurities have eluted, then bringing off product with 5 vol % methanol in chloroform. The residue is then -con¬ centrated to give 5..'-dimethoxytrityl-5-(3-trifluoroacetyl- aminopropen-1-yl) -.2'-deoxyuridine as a white fluffy solid (4 g) . Product decomposes upon heating; UV maχ 291 nm, →±n 266 nm; TLC R f 0.6 on silica eluting 10 vol % methanol in chloroform. EXAMPLE IV This example illustrates hydrogenation of exocy- clic double bond and 5 ! -hydroxyl masking to produce 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropyl)-2 '- deoxyuridine. Repeating the nucleoside precursor synthesis and 5 '-hydroxyl masking procedures of Examples I and III, but, prior to the addition of the DMT chloride, subject¬ ing the purified 5-(3-trifluoroacetylaminopropenyl)-2- deoxyuridine to two atmospheres of hydrogen while stirring at room temperature in methanol over 10% palladium-on-carbon catalyst is productive of 5'-dime¬ thoxytrityl-5-(3-trifluoroacetylaminopropyl)-2 '-deoxyu¬ ridine. Examples V to VIII illustrate the synthesis of addi¬ tional modified uracil nucleosides, and subsequent masking of 5'-hydroxyls as represented by Reaction 2. EXAMPLE V Repeating the nucleoside precursor synthesis and 5' hydroxyl masking, procedures of Examples I and III, but replacing N-allyltrifluoroacetamide with compounds numbered 1 through 8_ below is productive of the com¬ pounds numbered V through 8 * below, respectively; i.e s bstitution of. ' N-(3-butenyl)trichloroacetamide 2_ N-(5-hexenyl)trifluoroacetamide 3_ N- (2-methyl-2-ρropenyl)trifluoroacetamide 4 N-(4-ethenylphenylmethyl)trifluoroacetamide 5_ N-(l-methyl-3-butenyl)trifluoroacetamide 6_ N-(12-trichloroaminododecyl)acrylamide 1_ N-(pertrifluoroacetyϊpolylysyl)acrylamide 8_ N-(3-trifluoroacetylamidopropyl)acrylamide is productive of !_' 5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten-l- yl)-2'-deoxyuridine 2_' 5 '-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-l- yl)-2'-deoxyuridine 3_' 5'-dimethoxytrityl-5- (3-trifluoroacetylamino-2-methyl- propεn-l-yl)-2'-deoxyuridine 4' 5 '-dimethoxytrityl-5-[2-(4-trifluoroacetylaminomethyl- phenyl)ethen-1-yl]-2'-deoxyuridine 5_' 5'-dimethoxyltrityl-5-(4-triflύoroacetylamino-4-methyl- buten-1-yl)-2'-deoxyuridine 6_' 5'-dime hoxytrityl-5-[N-(12-trichloroacetylaminodo- decyl)-1-acrylamido]-2'-deoxyuridine _' 5 '-dimethoxytrityl-5-[N-(ρertrifluoroacetylpolylysyl)- 1-acrylamido] -2' -deoxyuridine 8_' 5'-dimethoxytrityl-5-[N-(3-trichloroacetylamino- propyl)-acrylamido]-2'-deoxyuridine EXAMPLE VI - Repeating the 5' hydroxyl masking procedure of Example III but replacing 5-(3-trifluoroacetylamino¬ propenyl)-2' -deoxyuridine with the 5-substituted-2'-de- oxyuridines numbered through 18 ^ below is productive of the products numbered 9_' through 1^' below, respectively; i.e. substituting -42a- 9_ 5- (propen-1-yl)-2'-deoxyuridine 10 5-(carbmethoxyethyl)-2 '-deoxyuridine 11 5-(3-carbmethoxylprop-l-yl)-2'-deoxyuridine 12 5-(4-carbmethoxy-2-methylbuten-l-yl)-2 '-deoxyuridine 513 5-(3-cyanopropen-l-yi)-2'-deoxyuridine 14 5-(4-cyano-2-methylbuten-l-yl)-2'-deoxyur.idine 15 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2 '-deoxyuridine 16 5-(4-acetoxybuten-l-yl)-2'-deoxyuridine 17, 5-(4-acetoxybut-l-yi)- '-deoxyuridine 10 _ 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine is productive of the following 5'-dimethoxytrityl-5- alkyl-2 '-deoxyuridines 9 5 '-dimethoxytrityl-5-(proρen-1-yl)-2'-deoxyuridine 10' 5 '-dimethoxytrityl-5-(2-carbmethoxyethyl)-2 ' -deoxy- 15 uridine 11' 5'-dimethoxytrityl-5-(3-carbmethoxyprop-l-yl)-2 '- deoxyuridine 12' 5' -dimethoxytrityl-5-(4-carbmethoxy-2-methylbύten- 1-yl)-2 '-deoxyuridine 2013 5'-dimethoxytrityl-5-(3-cyanopropen-l-yl)-2 '-deoxy¬ uridine 14' 5'-dimethoxytrityl-5-(4-cyano-2-methylbuten-1-yl)- 2'-deoxyuridine 15' 5 '-dimethoxytrityl-5-[2- (4-carbmethoxyphenyl)ethen- 25 1-yl]-2'-deoxyuridine 16 ' 5 '-dimethoxytrityl-5-(4-ace ' toxybuten-l-yl)-2 '- deoxyuridine 17' 5'-dimethoxytrityl-5-(4-acetoxybut-l-yl)-2 '-deoxy¬ uridine 30 18' 5'-dimethoxytrityl-5-[4-(2,4-dinitrophenyl)butyl]- , 2'-deoxyuridine -43- EXAMPLE VII Repeating the nucleoside precursor synthesis and 5 '-hydroxyl masking procedures of Examples I-VI, but replacing 5-chloromercuri-2'-deoxyuridine with 5-chloro- mercuriuridine is productive of the corresponding 5'-dimethoxytrityl-5-substituted uridines . Examples VIII to XI illustrate the syn hesis of modified cytosine nucleosides. Since cytosine nucleo¬ sides, as well as adenosine nucleosides, have reactive groups on their bases unlike the uracil nucleosides, such reactive groups are masked to prevent unwanted reactions therewith. These examples illustrate masking of reactive groups on the cytosine base moiety as in Reaction 1, as well as masking of the 5'-hydroxyl as in Reaction 2. EXAMPLE VIII 5-(3-trifluoroacetylaminopropenyl)- . N -benzoyl-2'-deoxycytidine Repeating the nucleoside precursor synthesis pro- cedure of Example I, but replacing 5-chloromercuri-2'- deoxyuridine with 5-chloromercuri-2'-deoxycytidine is productive of 5-(3-trifluoroacetylaminopropenyl)-2' - deoxycytidine (UV X 287 mn) . Purified 5-(3-tri- fluoroacetylaminopropenyl)-2'-deoxycytidine (1.3 g, 4.6 mmol) is stirred in 80 ml anhydrous ethanol, benzoyl anhydride (1.5 g, 7 mmol) is added, and the reaction -44- refluxed. Five additional 1.5 g portions of benzoyl anhydride are added hourly. After the reaction is judged complete by thin layer chromatography [silica plates eluting n-butanol/methanol/conc (60: 20:1:201 in 6-10 hours, the reaction is cooled and con¬ centrated under reduced pressure to a semisolid. The solid is .triturated with ether three times, decanted and dried. The crude product is crystallized from 4 water to give chromatographically pure N -benzoyl-5- (3-trifluoroacetylaminopropenyl)-2'-deoxycytidine as a white solid. The product decomposes above 120°C; UV max 311 nm. EXAMPLE IX 5 '-Dimethoxytrityl-5-(3-trifluoroacetyl- aminopropenyl)-N -benzoyl-2' -deoxycytidine Repeating the 5'-hydroxyl masking procedure of ExampleIII, but replacing 5-(3-trifluoroacetylamino¬ propenyl)-2' -deoxyuridine with 5-(3-trifluoroacetyl¬ aminopropenyl)-N -benzoyl-2'-deoxycytidine is productive of 5 ' -dime hoxytrifey1-5-(3-trifluoroacetylaminopropenyl)- N -benzoyl-2'-deoxycytidine. EXAMPLE X Repeating the nucleoside precursor synthesis and 5' hydroxyl masking procedures of Examples VIII and IX, but replacing N-allyltrifluoroace ' tamide with the N-alkyltrifluoroacetamides of Example V is productive of the corresponding 5'-dimethoxytrityl-5- (trifluoroacetyl) 4 aminoalkyl)-N -benz,oyl-2'-deoxycytidines , viz; 5'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten-l-yl)- N -benzoyl-2'-deoxycytidine 5''-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1-yl)- 4 N.-benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl- propen-1-yl)-N -benzoyl-2'-deoxycytidine OMPI - • 45- 5 -dιmethσxytrityl-5-[2-(4-trifluoroacetylaminomethyl- phenyl)ethen-1-yl]-N -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-methyl- buten-1-yl)-N -benzoyl-2'-deo.χycytidine 5 '-dimethoxytrityl-5-[N-(12-trifluoroacetylaminodo- decyl)-l-acrylamido]-N -benzoyl-2'-deoxycyti-dine 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylysyl)- l-acrylamido]-N -benzoyl-2'-deoxycytidine EXAMPLE XI Synthesis of 5'-di.mmeetthhooxxyyttrrii1tyl-N -benzoyl-5- (2-carb ethoxyethenyl)-2'-deoxycytidine 5-(2-Carbmethoxyethenyl)-2'-deoxycytidine (0.82 g, 2.6 mmol) is stirred in 50 ml anhydrous ethanol. Ben- zoic anhydride (500 mg, 2.2 mmol) j.s added, and the reaction heated to reflux. Five additional 500 mg portions of benzoic anhydride are added hourly. - After the reaction is judged complete by thin layer chroma¬ tography (usually 6-8 hours) the reaction is cooled, and evaporated under reduced pressure to a yellow semi- solid. Chromatography on silica gel eluting a linear 1:19 to 1:3 methanol/chloroform mixture followed by thorough evaporation of appropriately combined fractions gives N -benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycyti¬ dine as an amorp ~ hous white solid. UV Xmax 296 nm, X' -m,m_ -, * 270 nm. The solid is dried thoroug σ hly J , and dissolved in 20 ml pyridine. Dimethoxytrityl chloride (1.1 eq) is added, and the reaction stirred at ambient temperature for six hours. Concentration to a solid followed by column chromatography on silica gel eluting 10% methanol in chloroform yields 5 '-dimethoxytrityl-N - ben"zoyl-5-(2-carbmethoxyethenyl)-2'-deoxycytidine as a fluffy off-white solid. ξϋRE ; OMPI_ > -46- EXAMPLE XII Repeating the nucleoside precursor synthesis pro¬ cedure of Example XI, but replacing 5-(2-carbmethoxy- ethenyl)-2'-deoxycytidine with the following compounds numbered !L9_ through 2_7 below is productive of the corresponding compounds numbered 19 through 27' below, respectively, i.e., substituting: 19 5-(2-carbmethoxyethyl)-2'-deoxycytidine . 20 5-(3-carbmethoxyprop-l-yl)-2'-deoxycytidine 2_1 5-(4-carbmethoxy-2-methylbuten-l-yl)-2'-deoxycyti¬ dine 22 5-(3-cyanoproρen-l-yl)-2'-deoxycytidine 23 5-(4-cyano-2-methylbuten-l-yl)-2'-deoxycytidine 24 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2' -deoxycy- tidine 25 5-(4-acetoxybuten-l-yl)-2' -deoxycytidine 26 5-(4-acetoxybut-l-yl)-2'-deoxycytidine 27 5- [4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine is productive of the following 5 '-dimethoxytrityl-N - benzoyl-5-alkyl-2'-deoxycytidines : 19/ 5'-DMT-N 4 -benzoyl-5-(2-carbmethoxyethen-l-yl)-2'- deoxycytidine 20' 5 '-DMT-N -benzoyl-5-(3-carbmethoxyprop-l-yl) -2'- deoxycytidine 21/ 5'-DMT-N -benzoyl-5-(4-carbmethoxy-2-methylbuten- 1-yl)-2'-deoxycytidine 22' 5 '-DMT-N -benzoyl-5-(3-cyanopropen-l-yl)-2'-deoxy¬ cytidine 23' 5 '-DMT-N 4 -benzoyl-5-(4-cyano-2-methylbuten-l-yl)-2' - deoxycytidine 4' 5 '-DMT-N -benzoyl-5-[2-(4-carbmethoxyphenyl)ethen- \ 1-yl]-2'-deoxycytidine 5' 5 *-DMT-N -benzoyl-5-(4-acetoxybuten-l-yl)-2'-deoxy¬ cytidine OMPI $ I?0 -47- 26' 5'-DMT-N -benzoyl-5-(4-acetoxybut-l-yl)-2'-deoxy¬ cytidine 27/ 5'-DMT-N 4 -benzoyl-5-[4-(2,4-dinitrophenyl)butyl]- 2'-deoxycytidine Similarly, the use of the other acid anhydrides, e.g., acetic anhydride, anisoyl anhydride, or tolyl anhydride, is productive of the corresponding N 4-acyl or N4-acetyl 5-alkyl-2'-deox cytidines of Examples X and XI wherein benzoyl is replaced by acetyl or acyl. EXAMPLE XIII Repeating the nucleoside precursor synthesis and 5 '-hydroxyl masking procedures of Examples VIII to X, but replacing 5-chloromercuri-2'-deoxycytidine with 5-chloromercuricytidine is productive of the corres¬ ppoonnddiinngg 55 '-dimethoxytrityl-N -benzoyl-5-substituted cytidines EXAMPLE XIV This example typifies the masking of reactive base moieties and the ma.sking of 5'hydroxyl of adenine nucleosides. N —benzoyl-8-(6-aminohexyl)amino-2'-deoxyadenosine (4 mmol) is stirred in 60 ml anhydrous ethanol. Tri- fluoroacetic anhydride (6 mmol) is added, and the reac- * X tion stirred at room temperature. Two additional por- tions of trifluoroace ic anhydride are added hourly. -48- After four hours , the reaction is concentrated to a solid residue, and lyophilized overnight. The crude N -benzoyl-8- (6-trifluoroacetylaminohexyl)amino-2 '- deoxyadenosine is dried thoroughly and concentrated to a solid residue twice from pyridine. The solid is stirred in 40 ml of pyridine, and dimethoxy.trityl chloride (6.5 mmol) is added. After four hours, the reaction is concentrated to leave a solid residue. Purification by column chromatography on silica gel eluting a multi-step gradient of 0 to 15% methanol in chloroform gives 5' -dimethoxytrityl-N -benzoyl-8- (6-trifluoroacetylaminohexyl) mino-2'-deoxyadenosine as an off-white solid. Examples XV to XVII typify the activation of 5 '- masked 5-substituted, and naturally occurring nucleo¬ sides, to their respective phosphomonochloridites , as illustrated in Reaction 3 of the diagram. EXAMPLE XV Preparation of 3'-phosphomonochloridite of 5'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)- 2' -deoxyuridine Dry 5-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2 ' - deoxyuridine (1.54 g, 2.2 mmol) is lyophilized from 2.0 ml benzene three times for more than twelve hours each to remove residual water and solvents. The result¬ ing very fluffy white power is transferred to a nitrogen -49- atmosphere while still under vacuum and dissolved in anhydrous acetonitrile containing 5 vol % 2,6-lutidiήe to a final nucleoside concentration of 30 mM. While swirling vigorously under nitrogen, one rapid bolus of methylphosphodichloridite (1.0 eq) is added by syringe. The reaction is swirled for about one minute under nitrogen. The resulting crude 5 '-DMT-5-(3-trifluoro- acetylaminoprop-l-yl)-2'-deoxyuridine 3'-methylphospho- monochloridite reaction solution is then used directly for deoxyoligonucleotide synthesis (Example XVIII) with no further purification, [ 31 P-NMR(CH 3 CN/CDC1 3 ) generally indicates 40-70 mol % desired product (167.5 ppm); remainder is composed of bis-3' ,3'-[5'DMT-5-(3-trifluoro- acetyla inoprop-1-yl)-2'-deoxyuridylyl] methylphosphite (140 ppm) and 5'-DMT-5-(3-trifluoroacetylaminoprop-l-yl)- 2'-deoxyuridine 3'- ethylphosphonate (9.-5 ppm), the latter product being formed in amounts reflecting the presence of water in the reaction.] EXAMPLE XVI Preparation of 3'-phosphomonochloridites of the naturally-occurring 2'-deoxynucleosides Repeating the procedure of Example XV, but replac- ' ing 5' -DMT-5-(3-trifluoroacetylaminoprop-l-yl)-2'- deoxyuridine with:. is productive of the corresponding phosphomonochlori- elites, viz. : 5'-DMT-2'-deoxythymidine 3'-me hylphosphomonochloridite 5'-DMT-N -benzoyl-2'-deoxycytidine 3'-meth lphosphomono¬ chloridite OMPϊ -50 - ' 5 ' -DMT-N -benzoyl-2 ' - deoxyadenosine 3 ' -methylphospho- monochloridite 2 5 5*'--DDMMTT--NN 2 --iissoobbutyryl-2'-deoxyguanosine 3'-methylphos- phomonochloridite EXAMPLE XVII Repeating the phosphomonochloridite synthesis procedures of Examples XV and XVI, but replacing methyl¬ phosphodichloridite with o-chlorophenylphosphodichlori- dite is productive of the corresponding 5' -DMT-nucleoside 3'-phosphomonochloridites, viz.: 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3'-o-chlorophenylphosphomonochloridite 5'-DMT-2'-deoxythymidine 3'-o-chlorophenylphosphomono- chloridite 5'-DMT-N -benzoyl-2'-deoxycytidine 3'-o_-chlorophenyl- phosphomonochloridite 5'-DMT-N -benzoyl-2' -deoxyadenosine 3'-£-chlorophenyl- phosphomonochloridite 2 5'-DMT-N -isobutyryl-2 '-deoxyguanosine 3'-o-chloro- phenylphosphomonochloridite Similarly, the use of p_-chlorophenylphosphodichloridite is productive of the analogous 3'-p-chlorophenylphos- 32 phomonochloridite adducts. [ PjNMR (CH 3 CN CDC^) of o-chlorophenylphosphomonochloridite products 160.7, 160.5 ppm (diasteriomers) . Examples XVIII - XXIV typify the chemical synthesis of oligonucleotides which incorporate modified bases, as illustrated by Reactions 4 and 5 in the diagram. OV.PI -51- EXAMPLE XVIII Synthesis of deoxyoligonucleo ides containing 5- (3-aminopropyl)-uracil and naturally- occurring nucleotide units The phosphomonochloriditε synthesis procedures of Examples XV and XVI are accomplished immediately before deoxyoligonucleotide synthesis, and the result¬ ing products are used directly as 30 mM crude 3 '-methyl- phosphomonochloridites in anhydrous acetonitrile/5 vol % 2,6-lutidine. Solid support (5-DMT-N -benzoyl-2'-deoxyadenosine 3'-succinamidepropyl silica, 250 mg, 20 jueq) is put into a suitable reaction flow vessel (glass or TefIon (R—)' column or funnel) . The solid support is preconditioned by successive treatments with acetonitrile/5 vol % luti- dine, 2 w/v % iodine in tetrahydrofuran/water/lutidine for 2 minutes, acetonitrile/5% lutidine, chloroform, 4 vol % dichloroacetic acid in chloroform for 2.5 min¬ utes, and acetonitrile/5% lutidine, where treatments are total volumes of 5-15 ml in either 2 or 3 portions or by constant flow, as desired. The deoxyoligonucleotide is synthesized in accord¬ ance with Reaction 4 by sequential addition of the desired activated " 5'-DMT-nucleoside 3'-methylphospho- monochloridite monomer and coupling thereof to the free 5'-hydroxyl of the terminal unit of the growing nucleo¬ tide chain, which unit is initially the only unit of the chain, i.e., the deoxyadenosine based unit compris¬ ing the solid support. Additions are by reaction of 10 ml of the crude 30 mM monochloridites chosen from Examples XV and XVI with the now-unprotected 5' hydroxyl of the chain in either 2 or 3 portions or by constant flow, for 2 to 6 minutes. The first phosphomonochlori- dite addition followed by one complete reagent cycle consists of sequential treatments with: -52- -5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3'-methylphosphomonochloridite -acetronitrile/lutidine wash -capping for 5 minutes with 0.3 M 4-dimethylaminopyridine in acetic anhydride/lutidine/tetrahydrofuran (1:3:2) -acetonitrile/5% lutidine wash -oxidation with 2% iodine in tetrahydrofuran/water/ lutidine (6:2:1) for 2 minutes -acetronitrile/5% lutidine wash -chloroform wash -removal of DMT by 2.5 minute treatment with 4 vol % dichloroacetic acid in chloroform -chloroform wash -acetonitrile/lutidine wash The above cycle is repeated thirteen times , each time replacing 5' -DMT-5-(3-trifluoroacetylaminopropyl)-2'- deoxyuridine 3'-methylphosphomonochloridite with a different one of the following 3'-methylphosphomono- chloridites : 5 '-DMT-2 '-dεoxythymidine 3' -methylphosphomonochloridite 5 '-DMT-5-(3-trifluoroacetylaminopropyl)-2 '-deoxyuridine 3'-methylphosphomonochloridite . 5'-DMT-N -benzoyl-2'-deoxyadenosine 3'-methylphosphomono¬ chloridite 5'-DMT-N -benzoyl-2'-deoxycytidine 3'-methylphosphomono¬ chloridite 5 '-DMT-N 2 - isobutyryl-2'-deoxyguanosine 3'-methylphospho¬ monochloridite '-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3'-methylphosphomonochloridite '-DMT-2 '-deoxythymidine 3'-methylphosphomonochloridite \'-DMT-5-(3-trifluoroacetylaminopropyl)-2 '-deoxyuridine 3'-methylphosphomonochloridite QMPI JV TIO -53- 5 '-DMT-deoxyth idine 3'-methylphosphomonochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine 3 τ -methylphosphomonochloridite 2 5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-methylphos- phomonochloridite 5'-DM -N -benzoyl-2'-deoxyadenosine 3'-methylphospho¬ monochloridite 5'-DMT-N -benzoyl-2'-deoxycytidine 3'-methylphosphomono¬ chloridite in respective order, deleting dichloroacetic acid treat¬ ment during the last reagent cycle. The support is transferred and treated with 2 ml concentrated ammonium hydroxide for 4 hours at ambient temperature to release the product from the support. The supernatant is removed, the solid washed three times with 0.5 ml con¬ centrated ammonium hydroxide, and the combined super- natants are sealed and heated at 50°C overnight. The clear yellow supernatant is lyophilized thoroughly. Initial purification is accomplished by reverse phase high pressure liquid chromatography (HPLC) on an RP-8 (C-8) column eluting a 60 minute linear gradient of 0 to 30% vol % acetonitrile in 25 mM ammonium acetate, pH 6.8. The 5 '-DMT- erminated product, eluting as a sharp peak at about 40 minutes, is collected; all shorter chains, both capped and uncapped, elute before 25 minutes. The collected product is evaporated to a solid residue, treated with 80% acetic acid at ambient temperature for 20 minutes (to remove DMT) , lyophilized to a solid residue, and dissolved in a small amount of aqueous buffer. The product, generally greater than 90% homo¬ geneous after HPLC, is further purified by conventional e.ectrophoresis on 20% polyacrylamide gels (1 to 6 mm thick) by excision and extraction of the appropriate product band (product generally migrates slower than -54- unmodified deoxyoligonucleodites of similar length) . The purified 5-aminoproρyl-uracil-containing pentadeca- deoxyoligonucleotide product illustrated diagrammati¬ cally below, wherein U ra = 5-(3 aminopropyl)uracil, is thereby produced. Note the conventional deprotection of the oligo¬ nucleotide with ammonia has also removed the trifluoro- .acetyl masking group on the substituent. The length and sequence of this oligonucleotide may then be determined by 32P-kinasing and sequencing using suitable protocols, for example the protocols hereto- . fore used to determine the length and sequence of the prior art oligonucleotides in which the bases of the * nucleotide units therein are unmodified. Similarly, intentional variation of the order . and number of methylphosphomonochloridite additions employed here is productive of other 5-(modified)uracil-containing deoxyoligonucleotides which vary in selected length and base sequence. In addition, replacement of the nucleo- side 3'-methylphosphomonochloridite adducts of Examples XV and XVI with the corresponding 3'-o- or p_-chlorophenylphosphomonochloridite adducts of Example \ XVII and inclusion of pyridinium oximate treatment to remove chlorophenyl blocking groups (at the end of the deoxyoligonucleotide synthesis and before concentrated • "gfREXT O PI -55- ammonium hydroxide treatment) is productive of the same deoxyoligonucleotide products . EXAMPLE XIX Repeating the phosphomonochloridite and deoxyoligo- nucleotide synthesis procedures of Examples- XV to XVIII, but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'- deoxyuridine with the 5'-DMT-5-alkyl-2'-deoxyuridines numbered 2j3 through 36_ below is productive of the corresponding oligonucleotides having uracil bases U numbered 28/ through 36/ below, respectively, i.e., subs ituting: 28 5'-dimethoxytrityl-5-(3-trifluoroacetylaminopropen- 1-yl)-deoxyuridine 29 5' -dimethoxytrityl-5-(4-trifluoroacetylaminobut- 1-yl)- '-deoxyuridine < 30 5 '-dimethoxytrityl-5-(4-trifluoroacetylaminobuten- 1-yl)-2'-deoxyuridine 31 5' -dimethoxytrityl-5- (6-trifluoroacetylaminohex-1- yl)-2'-deoxyuridine 3_2_ 5 '-dimethoxytrityl-5-(6-trifluoroacetylaminohexen-1- yl)-2'-deoxyuridine 33 5'-dimethoxytrityl-5-(2-trifluoroacetylaminoprop-2- yl)-2'-deoxyuridine 34 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2- me hyl-propen-1-yl)-2'-deoxyuridine 35 5 '-dimethoxytrityl-5-(3-trifluoroacetylamino-2- ' methyl-prop-l- ' yl)-2 '-deoxyuridine 36 5'-dimethoxytrityl-5-[2- (4-trichluoroacetylamino- methylphenyl)ethen-l-yl]-2'-deoxyuridine 37 5'-dime hoxytrityl-5-[N- (pertrifluoroacetylpoly- λ lysyl)-l-acrylamido]-2'-deoxyuridine 38 5'-DMT-5-[N-(7-trifluoroacetyla inoheptyl)-1- acrylamido]-2'-deoxyuridine O PI -56- is productive of the deoxynucleotides corresponding to the product of Example XVIII, wherein U m is: 28' 5- (3-aminopropen-l-yl) uracil 29 ' 5-(4-aminobut-l-yl) uracil 5 30/ 5- (4-aminobuten-l-yl) uracil 31 ' 5-(6-aminohex-l-yl) uracil 32 ' 5- (6-aminohe'xen-l-yl) uracil 33' 5-(3-aminoprop-2-yl) uracil 34' 5- (3-amino-2-methylpropen-l-yl) uracil 0 35/ 5-(3-amino-2-methylprop-l-yl) uracil 36 ' 5- [2- (4-aminoethylphenyl)ethen-l-yl] uracil 37' 5-[N-(polylysyl)-1-acrylamido] uracil 38' 5- [N-(7-aminoheptyl)-1-acrylamido] uracil 5 Similarly, by employing other 5 '-DMT-5-(acylaminoalkyl) * - 2'-deoxyuridines , the analogous deoxyoligonucleotides are produced. EXAMPLE XX Repeating the phosphomonochloridite and deoxyoligo- nucleotide synthesis procedures of Examples XV to XVIII, but replacing 5 ' -DMT-5-(3-acetylaminopropyl)-2'-deoxy¬ uridine with 5-substituted-2' -deoxyuridines numbered 37 through 4_6_ below is productive of the corresponding oligonucleotides having the U uracil bases numbered 37/ through 46/ , below respectively; i.e., substituting: 37_ 5 ' -DMT-5- (proρen-1-yl) -2 ' -deoxyuridine 38 5 ' -DMT-5- (2-carbmethoxyethyl) -2 ' -deoxyuridine 39 5 ' -DMT-5- (3-carbmethoxyprop-l-yl) -2 ' -deoxyuridine 40 5 ' -DMT-5- (4-carbmethoxy-2-methylbuten-l-yl) -2 ' - deoxyuridine 41 5 ' -DMT-5-(3-cyanopropen-l-yl) -2' -deoxyuridine 42 5 f -DMT-5-(4-cyano-2-methylbuten-l-yl) -2 '-deoxy¬ uridine -57- 43 5 * -DMT-5-[2-(4-carbmethoxyphenyl)ethen-1-yl)-2'- deoxyuridine 44 5'-DMT-5-(4-acεtoxybuten-l-yl)-2'-deoxyuridine 5 5 » -DMT-5-(4-acetoxybut-l-vl)-2'-deoxyridine 46 5'-DMT-5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxy¬ uridine is productive of the products wherein, U is.- 37' 5-(propen-1-yl)uracil 38' 5-(2-carboxyethyl)uracil 39/ 5-(3-carboxyprop-l-yl)uracil 40' 5-(4-carboxy-2-methylbuten-l-yl)uracil 41' 5-(3-cyanopropen-l-yl)uracil 42' 5-(4-cyano-2-mεthylbuten-l-yl)uracil 43' 5-[2-(4-carboxyρhenyl)ethen-l-yl]uracil 44' 5-(4-hydroxybuten-l-yl)uracil 45' 5-(4-hydroxbut-l-yl)uracil ' 46' 5-[4-(2,4-dinitrophεnyl)butyl]uracil Note: 46/can function directly as a reportεr group, i.ε. usε of antidinitrophεnyl antibody as ligand. Similarly, by employing other appropriate 5'-DMT-5-alkyl -2'-deoxyuridinεs, the analogous deoxyoligonuclεotidεs arε produced. EXAMPLE XXI Repeating the procedurεs of Examples XV to XVIII, -but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)- 2-'deoxyuridinε with 5'-DMT-N -benzoyl-5-(3-trichloro- acetylaminopropyl) -2 f -deoxycytidine is productive of deoxyoligonucleotidεs as in Examplε XX whεrεin U [5-(3-aminopropyl)-uracil] is replaced by 5-(3-amino- propyl)cytosines. For example, -58- EXAMPLE XXII Repeating the deoxyoligonucleo ' tide synthesis pro¬ cedure of Example XXI but replacing 5'-DMT-N -benzoyl- 5-(3-trichloroacetylaminopropyl)-2'-deoxycytidine with compounds numbered 47_ through 5_7 below is productive of the corresponding oligonucleotides having the C cy¬ tosine bases numbered 47' through 57' below, respec¬ tively, i.e., substituting: 47 5-DMT-N 4-benzoyl-5-(3-trifluoro'acetylaminopropεn,- 1-yl)-2'-deoxycytidine 48 5 '-DMT-N -benzoyl-5-(4-trifluoroacetylaminobut-l- yl)-2'-dεocycytidine 49 55 ''--DDMMTT--NN 4 --bbeennzzooyyll--55--(4-trifluoroacetylaminobuten- 1-yl)-2'--dceoxycytidine 50_ 55 ''--DDMMTT--NN 4 --bbeennzzooyyll--55--(6-trifluoroacetylaminohex-1- yl)-2'-dde£oxycytidine 51 55''DDMMTT--NN --bbeennzzooyyll--55--((6-trifluoroacεtylaminohexεn-l- yl)-2' -d leeo<xycytidine 52 5 ''--DDMMTT--NN 4 --bbeennzzooyyll--55-- (3-trif luoroacetylaminoprop-2- yl)- * 2'-dde«oxycytidine 5^ 55''--DDMMTT--NN 4 --bbeennzzooyyll--55--((3-trif luoroace tylamino- 2- methylproopiεn-1-yl)-2'-deoxycytidine 54 55 ''--DDMMTT--NN --bbeennzzooyyll--55--((33--ttrriifflluuoorrooaace tylamino- 2- me hylpr •o°pI-1-yl)-2'-deoxycytidine 55_ . '• 55*'--DDMMTT--NN 4 --bbeennzzooyyll--5S[t2Z--(C4--ttrriifflluuoorroacetylaminomethyl- phenyl)ethen-1-yl]-2'-deoxycytidine -59- 56 5'-DMT-N -benzoyl-5-[N-(pertrifluoroacetylpoly- lysyl)-1-acrylamido)-2'-deoxycytidine 57 55''--DDMMTT--NN 4 --bbeennzzooyyll--55--[[NN--((ttrriiffluoroacetylaminoheptyl) acrylamido]-2'-deoxycytidine is productive of the products wherein C m is; 47' 5-(3-aminopropen-l-yl)cytosinε 48' 5-(4-aminobut-l-yl)cytosine 49' 5-(4-aminobuten-l-yl)cytosinε 50' 5-(6-aminohεx-1-yl)cytosinε 51' 5-(6-aminohexen-l-yl)cytosine 52' 5-(3-aminoprop-2-yl)cytosine 53* 5-(3-amino-2-mεthylproρen-l-yl)cytosine 54' 5-(3-amino-2-mεthylprop-l-yl)cytosine 55' 5-[2-(4-aminomethylphεnyl)εthen-l-yl]cytosme 5_6' 5-[N-(polylysyl)-1-acrylamido]cytosine 57' 5-[N-(7-aminoheptyl)-l-acrylamido]cytosinε Similarly, by employing other N -acyl-5-(acylaminoalkyl) 2' -dεoxycytidines thε analogous dεoxyoligonuclεotidεs arε producεd. EXAMPLE XXIII Repeating the,dεoxyoligonuclεotide synthesis pro¬ cedure of Example XXI, but replacing 5'-DMT-N -benzoyl- 5-(3-trifluoroacetylaminopropyl)-2'-deoxycytidinε with the compounds numberεd ^8_ through _68 below is productive of the corresponding oligonucleotides having the C cytosine bases numbrered 5j3' through S8' below, respec¬ tively, i.e., substituting: - 60- 58 5'-DMT-N -benzoyl-5-(propen-l-yl)-2'-deoxycytidinε 52 5'-DMT-N -benzoyl-5-(2-carbmethoxyethyl)-2' dεoxy- cytidinε 6>0 5 '-DMT-N -bεnzoyl-5- (2-carbmethoxyethen-l-yl)-2- deoxycytidine 61 5'-DMT-N -benzoyl-5-(3-carbmεthoxyρrop-l-yl)-2'- dεoxycytidinε 62 5'-DMT-N -bεnzoyl-5-(4-carbmethoxy-2-mεthylbuten- 1-yl)-2'-deoxycytidine 63_ 5'-DMT-N -benzoyl-5-(3-cyanopropεn-l-yl)-2'-deoxy¬ cytidine 64 5'-DMT-N -benzoyl-5-(4-cyano-2-methylbuten-l-yl)- 2'-deoxycytidine 65 5 '-DMT-N -benzoyl-5-[2-(4-carbmεthoxyphenyl)ethεn- 1-yl]-2'-deoxycytidine , 66 55''--DDMMTT--NN --bbeennzoyl-5-(4-acatoxybutεn-l-yl)-2'- deoxycytiidcine 67 55''--DDMMTT--NN --bbeennzoyl-5-(4-acetoxybut-l-yl)-2'- deoxycyt:iid<ine 68 55''--DDMMTT--NN 44 --bbeennzoyl-5-[4-(2,4-dinitrophenyl)butyl]- 2-deoxycytidine is productive of the products wherεin C is; 58' 5-(propen-1-yl)cytosine 59' 5-(2-carboxyethyl)cytosine _60' 5-(2-carboxyethen-l-yl)cytosine 61' 5-(3-carboxyρrop-1-yl)cytosine 62' 5-(4-carboxy-2-methylbutεn-l-yl)cytosinε 63' 5-(3-cyanopropεn-l-yl)cytosinε ,64' 5-(4-cyano-2-methylbuten-l-yl)cytosinε -61- 65' 5-[2-(4-carboxyphenyl)ethen-l-yl]cytosine 66' 5-(4-hydroxybutεn-l-yl)cytosine 67' 5-(4-hydroxybut-l-yl)cytosine 68' 5-[4-(2,4-dinitrophenyl)butyl]cytosine Similarly, by employing other appropriate 5'-DMT-N ■ acyl-5-alkyl-2'-dεoxycytidinεs thε analogous deoxy¬ oligonucleotides are producεd. EXAMPLE XXIV Repeating the phosphomonochloridite and deoxyoligo- nuclεotidε synthesis procεdurεs of Examplεs XV-XXIII, but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)- 2'-dεoxyuridinewith 5'-DMT-N -benzoyl-8-(6-trifluoro- acetylaminohexyl)amino-2'-dεoxyadεnosinε is productive of deoxyoligonucleotides as in Example XVIII, except that the U is replaced by A , and A = 8-(6-aminohexyl) amino-2'-deoxyadenosine. Examples XXV to XXVIII typify the binding of rεpor- ter groups to oligonucleotides containing appropriately modified bases, as illustrated in Reaction 6. EXAMPLE XXV Fluorescεinatεd dεoxyoligonuclεotides A purified pentadeca-nucleotide (from Example XVIII) of the structure OMPI - -62- [whεrε U m is 5-[N-(7-aminoheptyl)-1-acrylamido]uracil is dissolved at25 A 260 units per ml in aqueous 300mM sodium borate or sodium carbonatε buffer, pH 9.5, con¬ taining 30 mM sodium chloride. Solid fluorescein iso- thiocyanate (0.5 mg per ml) iso added,oand the mixture sealεd and shaken gently at 4 C to 25 C overnight. The reaction is chromatographed directly on a column of G-50 Sεphadεx*^ to separate unbound fluorescein adducts which are retained; the fluoresceinated deoxyoligo- nucleotide adducts elute near the void volume. Early fractions containing significant A 260 units are combined and lyophilized to solid product of structure similar to the starting pentadecadεoxyoligonucleotidε wherε U is now either or unreacted 5-[N-(7-aminoheptyl)-l-acrylamido]uracil. A max (H 2 0) 262 nm, 498 nm. Repeating the procedure on compounds recited in Examples XIX, XXI, XXII, and XXIV is productive of thε corrεsponding fluoresceinated or polyfluoresceinated deoxyoligonuclεoti ' des in like manner. EXAMPLE XXVI Attachment of reporter groups other than fluorescein an be accomplished by repeating the procedurε of Examplε -63- XXV, but rεplacing fluorescein isothiocyanate with, for example: 2,4-dinitrophenyl isothiocyanate l-fluoro-2,4-dinitrobenzεne aminoεthyl isoluminol isothiocyanate aminoεthylaminonaphthalεne-1,2-carboxylic hydrazide isothiocyanatε N,N'-bis (alkylsulfonyl)-N-aryl-N'-isothiocyanatoaryl- dioxamidε m-sulfonyl anilinε isothiocyanatε 9-(N-hydroxysuccinimidyl) biotin 9-(N-hydroxysuccinimidyl carboxy)-N-mεthylacridine, or cyanogenbromidε-activated Sepharose^ is productive of the correspondi g adducts wherein the attached group is other than fluorescεin. EXAMPLE XXVII Attachmεnt of isoluminol and freε primary amine-containing rεporter groups A purified pentadecanuclεotide from Example XVIII of the structure: [where U is 5-(2-carboxyεthenyl)uracil] is dissolved in watεr at30 A 26Q units per ml. , and diluted with one volume pyridine. Aminobutyl ethyl isoluminol is added -64- to a final concentration of 1 mg/ml, followed by addi¬ tion of a five-fold molar excess of l-ethyl-3-(3- dimεthylaminopropyl) carbodiimidε. The reaction is sεalεd and shakεn gently in the dark for 12 to 48 hours. The reaction mixture is concentrated under reduced pressurε to a solid residue, and chromatographed dirεctly on a column of G-50 Sephadex * - * *; thε isoluminol- dεoxyoligonucleotidε conjugates elute near the void volume. Early fractions containing significant A 2 ^ Q units are combined and lyophilized to solid product of structure similar to the starting deoxyoligonucleotide where U is now either isoluminol Or unreacted 5-(2-carboxyεthεnyl) racil. Repeating the procedure on compounds from Examples XV and XXIII wherein R 2 contains carboxy is productive of the corresponding deoxyoligonucleotidε-isoluminol adducts in likε manner. Repeating the procedure, but replacing aminobutyl isoluminol with other reporter groups containing a freε primary aminε is productivε of thε corresponding deoxy- oligonuclεotidε-rεportεr adducts in likε mannεr. -65- EXAMPLE XXVIII Attachment of dinitrophenyl reporter groups A purified nonanucleotide of the structurε: where A = 8-(6aminohεxyl)aminoadeninε is dissolved at 20 A 2f - Q units per ml in 250 mM sodium carbonatε buffεr, pH 9, and l-fluoro-2,4-dinitrobεnzεnε is added. The reaction solution is shaken at ambient temperature overnight, then chromatogra hed 'directly on a column of Sephadεx-^ G-50. Early fractions containing signifi- cant A 2 £ 0 units are combined and concentratεd to give an oligonucleotidε product similar to thε starting dεcanucleotide whεrεin A is now either Λ X i{CHANH V or unreactεd 8-(6-aminohexyl)aminoadeninε. Rεpεating the procedure, but rεplacing 8-(6-amino- hexyDaminoadeninewith other modified bases containing a free primary amine is similarly productive of the • >corrεsponding dinitrophenylated oligonucleotide adducts

Claims: - 66- Claims 1. The process for the chemical synthesis of a de¬ fined sequence single strand oligonucleotide which comprises coupling an activated nucleotide monomer and a freε hydroxyl-bεaring tεrminal unit of a growing nucleotidε chain, at least one of said monomer and terminal unit having its base modified at a sterically tolerant sitε by attachmεnt thereto of a substituent group capable of functioning as at least one reporter group, or of binding at least one reporter group. 2. The process of Claim 1 wherein the ' base of said nucleotide monomer is modifiεd. 3. The procεss of Claim 1 whεrein thε basε of said terminal unit is modified. 4. The process of Claim 1 wherein at least one of said monomer and nucleotide chain is attached to a solid support prior to the coupling step. 5. The process of Claim 1 wherein said terminal unit is attached to a solid support prior to the coupling step. 6. The process of Claim 1 wherein said substituent group is aromatic, polycyclic or aromatic and poly- ,'cyclic. -67- 7. The process of Claim 1 wherεin the substituent group functions as a colorimetric, fluorescent, lumi¬ nescent, radioactive, or ligand recognition reporter group. • 8. The process of Claim 1 wherεin, prior to thε coupling step, a least one reporter group is bound to the substituent group. 9. The process of Claim 1 wherεin, aftεr thε coupling step, at least one reportεr group is bound to the. substituent group. 10. The process of Claim 8 or 9 wherein said at least one reportεr group is aromatic, polycyclic or aromatic and polycyclic. ' 11. Thε procεss of Claim 8 or 9 whεrεin said at ' ' lεast one reportεr group is a functionally colorimetric, fluorescent, luminescent, radioactive or ligand 'recog¬ nition group. -12. The process of Claim 11 wherεin said at least one reporter group is a fluorescεin, rhodamine, acri- dinium salt, nitrophenyl, dinitrophenyl, benzene- sulfonyl, vitamin, luminol, isoluminol, luciferin, dioxetane, dioxamide, or an adduct thereof. 13. The procεss of Claim 1 whεrεin said nuclεotide monomer has an activated phosphorus-containing group at its 3' position, and the free hydroxyl of the terminal unit is at the 5' position of the latter. OMPI 14. The process of Claim 1 wherein said nucleotidε monomεr has an activatεd phosphorus-containing group at its 5' position, and the free hydroxyl of the terminal unit is at the 3' position of the latter. 15. The process of Claim 1 wherein thε nuclεotidε monomεr is pyrimidine-based and the substituent group is attached thereto at the C-5 position. 16. The process of Claim 15 wherein thε substituent group comprises a chain of two or more carbon atoms. 17. The process of Claim 15 wherein the substituent 0 0 group is -CH=CR 1 C-R 2 , -O-K^C- HR^ -CH^HR^, or -CH=CR 1 R 2 , wherεin R χ is hydrogεn or C 1-6 lower alkyl and R 2 is C Y wherein n is 0 to ' 20 and Y is hydrogen or at least one amido, substituted amido, nitrophenyl, substituted nitrophenyl, substitutεd amino, ester, substituted carboxyphenyl, substitutεd aminoalkylphεnyl, or substitutεd aminobεnzεnεsulfonic acid. 18. The process of Claim 17 wherein Y is at least one 0 ester, nitrophenyl adduct or NHCCX., , and X is hydrogen or halogen. 19. The process of Claim 18 wherein X is fluorine or chlorine. 20. The process of Claim 17 wherein R 2 is C H Y, and prior to the coupling step, at least one functionally colorimetric, fluorescent, luminescent, radioactive or ligand recognition reporter group is bound to Y. O PI -69- 21. The process of Claim 15 wherεin thε substituεnt 0 0 group is -CH 2 CHR 1 2 , -CH=CR 1 CR * 2 , --CH=CR 1 CNHR 2 , or -CH * =CR 1 R 2 , whεrein R^ is hydrogen or C 1 _ 6 lowεr alkyl and R is C H„ Y whεrein n is ' 0 to 20 and Y is hydrogen z n Zn or at least one amido, substituted amido, nitrophenyl, substituted nitrophenyl; or a masked a ino, substituted amino, carboxy, hydroxy, carboxyphenyl, substitutεd carboxyphenyl, aminoalkylphεny1,. substitutεd amino- alkylphεnyl, aminobεnzenesulfonic acid, or substitutεd aminobenzenesulfonic acid. 22. Thε p c rocεss of Claim 21 whεrein RZ is CnH2nY, ' and aftεr coupling step, Y, if masked, is unmasked, and a solid support is bound to Y. 23. The process of Claim 21 wherein R Z.-, is CnHZnY* , and after the coupling step, Y, if maskεd, is unmaskεd, and at lεast onε functionally colorimetric, fluorescent, luminescent, radioactive or ligand recognition reporter group is bound to Y. 24. The process of any of Claims 1 to 9 and 12 to 23 wherein the nucleotidε monomεr is uracil-based or cytosine-basεd. 25. The process of Claim 1 wherein the nucleotide monomer is purine-based and the substituent group > is attached therεto at thε C-8 position. 26. Thε process of Claim 25 wherein the substituent group comprises at least one carbon atom attached to the purine base through a polyvalent hetεroatom. -70- 27. The process of Claim 25 whεrein the substituent group is representεd by the formula -ZCHR,R 2 , wherεin Z is a polyvalent hetεroatom, R, is hydrogen or C-^_g lower alkyl and R« is C H Y wherein n is 0 to 20 and Y is at least one ainido, substituted amido, nitro¬ phenyl, substituted nitrophenyl, substituted amino, ester, substituted carboxyphenyl, substituted amino- alkylphenyl, or substituted aminobenzenesulfonic acid. 28. The process of Claim 27 wherein Y is at least one P NHCCX^ and X is hydrogen or halogen. 29. The process of Claim 28 wherei * X is fluorine or chlorine. • 30. The process of Claim 27 wherεin Rz is CnHZ n nY, and prior to the coupling step, at least one func¬ tionally colorimetric, fluorescent, luminescent, ' ' radioactive, or ligand recognition reporter group is bound to Y. 31. The process of Claim 25 wherein the substitutent group is represented by the formula -ZCHR-,R 2 , wherein Z is a polyvalent heteroatom, R, is hydrogen or C-, g lower alkyl and R 2 is C H« Y wherεin -n is 0 to 20 and Y is at lεast onε amido, substituted amido, nitrophenyl, substituted nitrophenyl; or a masked amino, substituted amino, carboxy, hydroxy, carboxy¬ phenyl, substituted carboxyphenyl, aminoalkylphenyl, substituted aminoalkylphenyl, aminobenzenεsulfonic acid, or substitutεd aminobenzenεsulfonic acid. - 71- 32. Thε procεss of Claim 31 wherein R 2 is c n H 2n Y ' and after the coupling step, Y, if maskέd, is * unmaskεd, and_ a solid support is bound to Y. 33. Thε procεss of Claim 31 wherein R 2 is C H 2 Y, and after the coupling step, Y, if masked, is unmasked, and at lεast one functionally colorimetric, fluorescεnt, luminescent, radioactive, or ligand recognition reporter group is bound to Y. 34. The process of Claim 20, 23, 30, or 33.,- wherein said at least one reporter group is a fluorescein, rhodamine, acridinium salt, nitrophεnyl, dinitro¬ phenyl, benzenεsulfonyl, vitamin, luminol, isoluminol, lucife in, dioxetane, dioxamide, or an adduct thereof. 35. The process of any of Claims 26 to 33, wherein the heteroatom is N, 0, or S. 36. The process of any of Claims 1 to 9, 12 to 14, and 25 to 33, wherein the nucleotidε monomεr is adεnine-based. 37. The process of any of Claims 1 to 9, 12 to 23, and 25 to 33, wherein said nucleotide monomer is a ribonucleotidε. 38. Thε procεss of any of Claims 1 to 9, 12 to 23, and 25 to 33, whεrεin said nuclεotidε monomer is a deoxyribonuclεotid . ■ 72 - 39. Thε pr.ocεss of any of Claims 1 to 9, 12 to 23 and 25. to 33, wherein the ' nucleotidε monomεr becomes a new terminal unit of said nucleotidε chain upon coupling thεrεto, and aftεr the coupling stεp, thε nuclεotidε chain is extended by coupling another nuclεotidε.monomer to said nεw tεrminal unit thereof. 40. An oligonucleotide produced by the process of any of the preceding claims. 41. An oligonucleotidε produced by the process of any of Claims 1 through39 and which is fewer than about 200 base units in length. 42. A defined sequεncε single strand ol'igonucleotidε of fewer than about 200 base units in length comprising at least one nucleotide unit having attached to the.base thereof at a sterically tolerant site a substituent group which is capable of binding, or of functioning as, at least one reporter group. 43. The oligonucleotide of Claim 42 which additionally comprises at least one nucleotide unit having a natur¬ ally occurring nucleotidε basε. 44. The oligonucleotidε of Claim 42 or 43 wherein the substituent group is aromatic, polycyclic or aromatic and polycyclic. -73- 45. The oligonucleotide of Claim 42 wherein the substituent/group functions at " least one colorimetric, fluorescent, luminescεnt, radioactivε or ligand rεcognition rεportεr group. 46. The oligonucleotide of Claim 42 or 43 wherεin at lεast onε rεportεr group is bound to said sub¬ stituent group. 47. The oligonucleotidε of Claim 46 whεrein said at least one reporter group is aromatic, polycyclic or aromatic and polycyclic. 48. The oligonucleotidε of Claim 46 whεrεin said at least one reporter group is a functionally colori¬ metric, fluorescent, luminscent, 'radioactive, or ligand recognition reportεr group. 49. The oligonucleotide of Claim 46 wherein said at least one rεportεr group is a fluorεscεin, rhodaminε, acridinium salt, nitrophεnyl, dinitrophenyl, bεnzεne- sulfonyl, vitamin, " luminol, isoluminol, luciferin, dioxεtanε, dioxamidε, or an adduct thεrεof. 50. The oligonucleotidε of any of Claims 42, 43 ' , and* 5 to 49 wherein the nucleotidε unit to which the substituent group is attached is pyrimidine-based, and the sub¬ stituent group is attached therεto at thε C-5 position. 51. Thε oligonuclεotidε of Claim 50 whεrein thε sub¬ stituent group comprises a chain of two or more carbon atoms. 52. The oligonucleotide of Claim 50 wherein the substituent groups is -CH^HR.^, -CH=CR- L CR 2 , -CH=CR 1 CNHR 2 *, or -CH=CR 1 R 2 wherein R χ is hydrogen or . C ι 6 lower alkyl and R 2 is C H 2n Y ' wherεin n is 5 0 to 20 and Y is hydrogεn or at least one amido, substituted amido, nitrophenyl, substituted jiitro- phεnyl, amino, substituted amino, carboxy, hydroxy, carboxyphenyl, substitutεd carboxyphenyl, amino¬ alkylphenyl, substituted aminoalkylphenyl, amino- 10. bεπzenesulfonic acid, or substituted aminobenzene¬ sulfonic acid. 53. The oligonucleotide of Claim 52 wherεin Y is at lεast one amide, amine, carboxyl, ester, nitrophenyl, or an adduct thereof. 54. The oligonucleotide of Claim 52 wherein R 2 is C H 2 Y, and at least one functionally colorimetric, fluorescent, luminescent, radioactive or ligand recog¬ nition reporter group is bound to Y. 55. The oligonucleotide of any of Claims 42, 43, 45, 47 to 49, and 51 to 54 wherein the nucleotide unit to which the substituent group is attached is uracil-based or cytosine-basεd. 56. The oligonucleotide of any of Claims 42, 43 and 45 through 49 wherein the nucleotidε unit to which the sub¬ stituent group is attached is purinε-basεd, and the sub¬ stituent group is attached thereto at the C-8 position. 57. The oligonucleotide of Claim 56 wherein the sub- ' X stituent group comprises at least one carbon atom attached to the purine base through a polyvalent . hetεroatom. OMPI -75- 58. The oligonucleotide of Claim 56 wherein the sub¬ stituent group is represented by the formula -ZCHR-.R-, wherein Z is a polyvalent heteroatom, R-, is hydrogen, or C-^_g lower alkyl and R 2 is c n H 2n- l or C n H 2n Y wherein n is 0 to 20 and Y is at least one amino, sub¬ stitutεd amido, nitrophenyl, substituted nitrophenyl, amino, substituted amino, hydroxy, carboxy, carboxy¬ phenyl, substituted carboxyphenyl, aminoalkylphenyl, substituted aminoalkylphenyl, aminobenzenesulfonic acid, or substituted aminobenzenεsul oriic acid. 59. Thε oligonuclεotide of Claim 58 wherein R is C H Y', and at least one functionally colorimetric, fluorescεnt, luminεscεnt, radioactive, or ligand recognition reporter group -is bound to Y. 60. The oligonucleotidε of any of Claims 54 and 57 to 59 whεrein said at least one reportεr group is a fluorescein, rhodamine, acridinium salt, nitrophenyl, dinitrophenyl, benzεnesulfonyl, vitamin, luminol, iso- luminol, luciferin, dioxetane, dioxamide, or an ' adduct therεof. ' 61. Thε oligonucleotide of Claim 59 wherein Y is amino and said at least one reporter group is fluores¬ cein, fluorescamine, rhodamine, luminol, isoluminol,. dioxetane, dioxamide, biotin, iminobiotin, desthio- biotin or an adduct thereof. 62. The oligonucleotide of any of Claims 57, 58 or 59 wherein the heteroatom is N, 0, or S. 63- The oligonucleotide of any of Claims 42, 43, 45 to 49, 57 to 59, and 61, wherein the nucleotide unit to which the substituent group is attached is adenine- basεd. OMPI -76- 64. Thε oligonucleotide of any of Claims 42, 43, 45, 47 to 49, 51 to 54, 57 to 59 and 61, wherein said nucleotidε is a ribonucleotide. 65. The oligonucleotide- of any of Claims 42, 43, 45, 47 to 49, 51 to 54, 57 to 59, and 61, wherεin said r nucleotide is a deoxyribonucleotide. 66. The oligonucleotide of any of Claims 42, 43, 45, 47 to 49, 51 to 54, 57 to 59, and-61, which is from about 5 to about 60 base units in length. 67. The oligonucleotide of any of Claims 42, 43, 45, 47 to 49, 51 to 54, 57 to 59, and 61, which is from about 10 to about 40 base units in length. 68. A nucleoside useful as an int'ermediatε in the chemical synthesis of an oligonucleotide, said nucleo¬ side having its base modified by a substituent group comprising a carbon chain and at least one amide, the nitrogen of said at least one amide being attached to a sterically tolerant site on said base through said carbon chai . 69. The nucleoside of Claim 68 wherein the substituent 0 0 group is -CH 2 CHR 1 C n H 2n Y, -CH-CR^^I^Y, -CH^C H^H^Y, or -CH=CR-,C H- 2 Y, wherεin R, is hydrogen or ~-t _ c lower alkyl and Y is at least one amido, substituted amido, substituted amino or substituted aminoalkylphenyl. 70. The nucleoside of Claim 69 wherein Y is at least \ 0 one NHGCXO, and wherein X is hydrogen or halogen. CMPI -77- 71. The nucleoside of Claim 70 wherein said halogen is fluorine or chlorine. 72. The nucleoside of Claim 71 wherεin the substi- 0 fl tuent group is -CH=CR- L CC H 2 Y or -CH=CR ;L CNH n H 2n Y. 73. A nuclεosidε as in any of Claims 68 to 72 which is pyrimidinε-bas d and wherein the carbon chain is attached to the C-5 position. 74. A nucleosidε as in Claim 73 which is uracil or cytosinε-basεd. 75. The nucleosidε of Claim 73 whεrεin thε 5'-hydroxyl is maskεd with a masking group. 76. Thε nuclεosidε of Claim 75 wherein said masking group is monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl. 77. Thε nuclεoside of Claim 76 wherein said masking group is dimethoxytrityl. 78. The nuclεoside of Claim 73 wherein the 3'-hydroxyl is masked. 79. Anuclεoside as in any of Claims 68 to 72 which is purine-basεd and. whεrεin the carbon chain is attached at the C-8 position through a nitrogen, sulfur or oxygεn atom. /. 80-. A nuclεosidε as in Claim 79 which is adeninε- based. OMPI IO -78- 81. The nucleoside of Claim 80 wherein the 5'-hydroxyl is masked with a masking group. 82. The nucleoside of Claim 81 wherεin said masking group is monomεthoxytrityl, dimethoxytrityl, or trimethoxytrityl. 83. Thε nucleoside of Claim 82 wherεin said ' masking group is dimethoxytrityl. 84. The nucleoside of Claim 80 wherein the 3'-hydroxyl is masked. 85. A nucleosidε as in any of Claims 68 to 72, 74 to 78, and 80 to 84 which is a ribonuclεoside or a deoxyribonuclεoside. 86. The process of Claim 12 or 3 'wherein the vitamin is biotin, iminobiotin or desthiobiotin. 87. The oligonucleotidε of Claim 49 whεrein the vitamin is biotin, iminobiotin or desthiobiotin. • OMPI

Inventors: RUTH JERRY L, RUTH, JERRY, L.
Applicants: MOLECULAR BIOSYSTEMS INC, MOLECULAR BIOSYSTEMS, INC.
Application year: 1984
Main classification: C07H17
IPC codes: C07H19, C07H21, C12Q1