CA1231650A - Defined sequence single strand oligonucleotides incorporating reporter groups, process for the chemical synthesis thereof, and nucleosides useful in such synthesis - Google Patents

Abstract

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, incor-porate 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, are disclosed. Each reporter group is attached to the base of the respective nucleo-tide 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 predeter-mined 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 oligo-nucleotide chain. Novel nucleosides useful in the chemical synthesis of labeled, defined sequence single strand oligonucleotides are also disclosed.

Description

~3~5~

Description Defined Sequence Single Strand Oligonucleotides Incorporating Reporter Groups, Process for the Chemical Synthesis Thereof, and Nucleosides Useful in Such Synthesis conical Field The present invention relates Jo defined sequence single strand oligonucleotides having a length of fewer than 200 base units and which contain one or more nucleotide.unlts to the base of which is attached a substituent group which has bound thereto one or more detectable reporter groups; such oligonucleotides being useful in the identification, localization and detection of complementary nucleic acid sequences 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 into doubles Rand DNA by the nick translation protocol of P. Rugby et at, J. Mol. Blot. 113: 237-251 (1977), or the gap-filling reaction described by G. Bourguignon et at, . Viral. 20: 290-306 (1976). Specifically, a -nick is introduced via DBase, and then translated along the DNA strand using a DNA polymers. During the nick translation procedure, DNA polymers from E. golf pull It will in the presence of added deoxynucleoside troughs-plates, condense knuckleheads to the 3' hydroxyl terminus in a single strand nick region of double-stranded DNA.
Simultaneously the enzyme will remove nucleotides Roy

-2-the 5' end of the nick. I f one or more of the added triphos~hates is labeled, for example with a- P-phosphate, the label will be incorporated into the new strand by Pot I. Following the gap-filling procedure, recessed ends left after restriction ~ndonuclease cutting can be filled in with Clown fragments from Pot I or To DNA Polymers.
Both the nick translation and the ~ap-filling procedures will yield double-stranded labeled DNA.
The length of the product depends upon how much DBase 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 nucleated units in length.
The product length is unpredictably heterogeneous within the 400 to 800 unit range. In order to conserve enzyme as well as labeled nucleated, only one microgram of DNA is usually labeled in each reaction vessel.
Double-stranded polynucleotides which incorporate pyrimidine bases modified by carbon chains at the C-5 position have been prepared enzymatic ally in a similar manner. This has been done by enzymatic elongation of a homopolymeric primer-template as reported by J. Sari et at., Bloc em. BiosPhys. Act. 6~6: 196-201 (1980). or by nick translation/gap filling by DNA polymerizes using 2'-deoxyuridine 5'-triphosphate covalently linked to boo-tin as reported by P. Lunger et at, Pro. Nat. Aged. Sat.
USA 78:6633-6637 (1981), the button being capable of acting as a recognition site for aiding Enzymatic methods described by Rugby, et at, Bour~ui~non, et at. and Lunger, et at, result in products having similar physical characteristics. Such enzymatic-ally prepare polynucleotides are 400-800 units in length, require double-stranded polynucleotides as starting materials, produce double-stranded PolYnucleotides in all cases, and do not allow Labeling at preselected 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-cusses, enzymes replace either all units with modified units or, when provided with a mixture of modified and naturally-occurrin~ nucleoside triphosphates, randomly insert modified units. Furthermore, the envy-matte process described by Lunger, et at, is incapable of producing polynucleotides incorporating such reporter groups as fluorescent, luminescent, or antigenic reporter groups. None of this art is capable of producing oily-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 enzymatic ally. This has been done by incorporating 8-aminohexylamlno-ATP (a ribonucleotide) onto DNA fragments, as reported by C. Vincent et at, Null. Acids Rest 10:6787-6796 ~1982). The method is limited in scope, however, allowing only end labeling with triphosphates of adenine rabbinical-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.

~23~5~:1 The prior art enzymatic methods referred to above require the chemical synthesis of a substituted nucleoside 5'-tripho'sphate, and demand subsequent enzymatic recognition and incorporation of the unnatural 5 substrate into nicked' double stranded DNA. Such methods are incapable of producing polynucleotides of any pro-selected length or sequence, and the polymerizes and DNases used therein are expensive. Moreover, these methods are time consuming, inefficient, demand sub Stan-10 trial enzymatic activity and are limited to double-stranded DNA. Only small amounts, i.e., micrograms, of ill-defined polynucleotides, usually restriction fragments, are produced, and these must be tediously isolated from natural sources. Moreover, the scope of modifications lo obtainable in the polynucleotide product is severely limited, since the DNA polymers cannot recognize or in-corporate potentially useful reporter groups such as fluoresce in or dinitrophenyl.
Attachment of one fluorescent molecule to the 3' end 20 of long polyribonucleotide molecules (RNA) for limited biological application is disclosed by GO Bagman et at, J. Histochem. Cy~ochem. 29:238 (1981~. This approach also used very small amounts (microgram quantities) of RNA tediously isolated prom natural sources using envy-' 25 matte methodology, and cannot be applied to DNA since both 2' and 3' hydroxyls are required therefore The much greater chemical instability of RNA relative to DNA also minimizes the scope ox 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 SPA. Nearing et at, Mesh. Enamel 65:610 (1980), R. Let singer, J.
Chum. 45:2715 (1980), M. Mattocks et at, J. Amer. Chum.

Sock 103:3185 (~982), and G. Alvarado-Urbina et at, Science 214:270 (1981). Such synthesis usually in-valved chain extension by coupling an activated nucleated monomer and a free hydroxyl-bearin~
terminal unit of a growing nucleated chain. The coupling is effected through a phosphorus-containing group, as in the phosphate trimester method reviewed by Nearing et at, or one of the phosphate trimester methods. Of the latter, those of Let singer et at and Alvarado-Urbina et at use phosphochloridite chemistry, and that of Mattocks et at uses phosphor 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 rBigge, et at, J. Curb., 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-silly contaminated with metal ions, and are capable of modifying only citizen residues of a DNA pylon-clouted. The method is incapable of producing defined sequence oligonucleotides of specific lengths, cannot modify thiamine or Purina bases, and cannot modify at previously selected sites.

I I

From the foregoing it will be apparent that labeled, defined sequence polynucIeol:ides have been produced heretofore only by enzymatic methods which have the disadvantages pointed out herein, portico-laxly 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 swooper-lion 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-ductile of renaturation, subjecting the denaturedpolynucleotide 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 I by competition from the other strand.

Lo odif~a~ion of nucleosides has been undertaken, for example, in the synthesis of anti viral C-5 sub-stituted pyrimidine nucleosides disclosed by Bergstrom, et at, J. Amer. Chum. Sock 98:1587-1589 (1976); Ruth, 5 et at, J. Org. Chum. 43:2870-2876 (1978); and Bergstrom, et at, Us S. Patent Nos. 4,247,544 and 4,267,171, or in the synthesis of C-8 substituted adenine derivatives disclosed by Zappelli, et at, U. S. Patent Nos.
4,336,188 and 4,199,498. These nucleosides, and 10 others reported by Lunger, et at, 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 oli~,onucleo~ide synthesis ~,~; chemical 15 methods, would result in undesired side proxy unwon-troll able synthesis, and no desired product. Moreover, such reported nucleosides do not contain wrier sites ; in the substituent group, cannot be modified by the attachment of reporter groups nor do they contain 20 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 2, 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 object of Lo 3 the present invention. This object is accomplished by a chemical,. i.e., non enzymatic., 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 labeled, defined sequence single strand oligonucleo-tides.
The process of the present invention accomplishes the de nova chemical synthesis of labeled, defined sequence oligonucleotides and is superior to prior art enzymatic methods in a number of respects. Gore specifically the process of the present invention makes possible the synthesis of labeled, defined sequence single-stranded oligonucleotides of homogeneous pro-dictably defined length, preferably having fewer than 200 base 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 Lo go methods. The single strand configuration of the product oligonucleotldes avoids the competition from a complementary strand inherent in reburied-ration 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 nucleon tide unit having attached to the base thereof at a starkly tolerant site (such as C-5 of pyrimidines and C-8 of urines) a substi~uent group which has bound thereto one or more detectable reporter groups. The invention Allah comprises a process for the chemical, i.e., non enzymatic, synthesis of a defined sequence single strand oligonucleotide which comprises coupling an activated nucleated monomer and a free hydroxyl-bearing terminal unit of a growing nucleated chain, at least one of the monomer and terminal unit having its base modified at a circle tolerant site by attachment thereto of a substituent group capable of binding one or more detectable reporter groups. The invention additionally comprises novel nucleosides and nucleated 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 ribonucleotides or deoxyribonucleotide, and prior to the synthesis thereof, the reporter groups are preselected, as are the particular nucleated units to which the reporter groups are attached.

s;
. . .

~L~3~6~1 Best Mode of Carrying Out the Invention The chemical process by which the defined sequence single strand oli~onucleotides of the present invention are preferably synthesized comprises coupling an anti-voted nucleated monomer and a free hydroxy-bearing terminal unit of growing knucklehead chain, at least one of said monomer and terminal unit having its base modified at a starkly tolerant site by attachment thereto of a substituent group capable 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 nucleon Philip properties with respect to such reporter groups.
Exemplary of such substituent groups are those which contain primary or aromatic amine, carboxylic acids, hydroxyls and the like.
The bates of the nucleated monomer and terminal unit are selected to provide the predetermined sequence of nucleated units desired in the end product oligo-nucleated. Such bases can take the form of the urines adenine (A), guanine (Go, or hypoxanthine (Ho, or of the pyrimidines Ursula (U), citizen (C), or thiamine (T).
Such bases may also take the form of any other base which can be isolated from natural sources.
A starkly tolerant site on the nucleated 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 whereto of the substituent group without causing significant interference with hybridization of the product oligonucleotide and complementary nucleic acid component, and without starkly preventing the substituent group from binding one or more reporter groups. Circle tolerant sites are found at the C-8 ~L~3~5~

position of porn and at the C-5 position of pyrimi-dines. Since the oligonucleotid~s of the present invention are particularly useful as hybridization probes, modifications which attach substituent and/or 5 reporter groups should not be at sites on the pyrimi-dine or Purina bases which are necessary for specific hybridization Sites which should not be modified include No and ,6 of adenine bases; No, No, and ox of guanine bases; No and No of citizen bases. Goner-ally, substitution a any heteroatom (N or O) should be avoided.
A reporter groups 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 pro-sent invention are readily detectable. Ready detect-ability may be provided by such characteristics as color orange, 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 calorimetric, luminescent, fluorescent, radioactive or ligand recog-notion groups. Among such groups are those suitable for ready detection by conventional detection techniques, such as calorimetric, spectrophotometric, fluorometric or radioactive detection, as well as those which are capable of participating in the formation of specific ligand-ligand complexes which contain groups detectable by such conventional detection procedures.
A reporter group as used herein may be characterized as a label which has physical, chemical or other characteristics which can be readily measured or de-tooted by the use of appropriate measurement or detect lion procedures. A reporter group as defined herein also includes ligand recognition groups which are capable 5 of initiating one or more ligand-ligand 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 appear-private measurement or detection procedures.
Exemplary of the measurable or detectable kirk-teristics which such groups, reaction products or come plexus may exhibit or induce are a color change, luminescence, fluorescence or radioactivity. Such characteristics can be measured or detected by the use 15 of conventional calorimetric, spectrophotometric, fluorometric or radioactivity sensing instrumentation.
The interactions which usefully can be initiated by the reporter group defined herein include appropri-lately specific and selective ligand-ligand interactions productive of groups or complexes which are readily detectable, for example, by calorimetric, spectxophoto-metric, fluorome~ric, or radioactive detection prove-Doria. Such interactions may take the form of protein-ligand, enzyme-substrate, antibody-antigen, carbohydrate-lath protein-cofactor, protein-effector, nucleic acid-nucleic acid or nucleic acid-ligand interactions.
Exemplary of such ligand-ligand interactions are dinitrophenyl-dinitrophenyl antibody, biotin-avîdini oligonu~leotide-complementary oligonucleotide, DNA-DNA, RNA-DNA and NADH-dehydrogenase. Either one of each such ligand-ligand pairs may serve as a ligand recognition type reporter group. Other useful interactions will suggest themselves Jo those skilled in the art.

In the process of thy present invention, a selected reporter group or groups can optionally be attached to the nucleotlde monomer before coupling of the monomer to the terminal unit of the nucleated chain, or it can be attached to the product oligonucleotide after formation thereof. The sequence of the nucleated units in the product oligonucleotide is preselected to provide such oligonuc1eotide 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-piques. Among such uses are identification, focalize-lion, 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-valving 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 starkly tolerant site, with or without sub-sequent detection.
The nucleated units in the product oligonucleotide may be Purina or pyrimidine-based and may comprise units having naturally~o~curring bases intermixed with units having modified bases. Such units may be rabbinical-tides or deoxyribonucleotide. The coupling step pro-fireball involves coupling of a monomer unit activated at the 3' position with a free 5' hydroxyl of the ton-final unit of the growing nucleated chain. Alterna-lively 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 nucleated chain. The terminal unit may be the initial or only unit in the rowing nucleated chain at the time of coupling thereto of the nucleated monomer or it may be the terminal one of a plurality of nucIeotide units.
The process of the present invention produces defined sequence oligonucleotides of the following generic formula:

5, B
HO - _ B

OPT I

HO R

FORMULA I

Therein 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 it any of the naturally-occurring Purina or pyrimidine bases adenine, guanine, citizen, Ursula, thiamine, or any other naturally-occurring base, the nucleated units having naturally-occurring bases being independently intermixed with owner more nucleated units having modified bases (By).
The modified pyrimidine bases (Pam) are substituted at the C-5 position, and typical examples thereof are the Ursula and citizen bases illustrated by the following generic formulas:

- 15~ I

NH"
p m I Hi Jo or NOR
OWN OWN

modified Ursula base modified citizen base The modified Purina bases (Put) are substituted at the C-8 position, and typical examples thereof are the modified adenine and guanlne base striated by the 5 following ~en~lric formulas:

put ,. No R or NJ~Z

modifies Rdenine base modified Gwen base .

The substituent group R is characterized by its ability to bind one or more reporter groups. In the modified pyrimidine bases the substituent group R comprises two or more carbon atoms, whereas in the modified Purina base R comprises one or more carbon atoms. In this context, '''";~

-16~ 6 R preferably take the form of one of the following unctionalized carbon chains:
R OH ~Rl~2, CH2GHRlR2, Cruller, o '1 I! ' -CH=CRl-~NHR2, or -CH=CRl-~-R~
wherein Al is hydrogen or alkali R2 us alkyd, alkenyl, aureole, or functionalized alkyd, alkenyl, Rowley wherein functional groups include one or more amine 9 asides, nitrites, carboxylic acids and esters,hydroxy, dint-trophenyl, aminobenzenesulfonates, or the like; and Z
is a polyval~nt heteroatom such as nitrogen, oxygen or sulfur. In addition, R2 may be attached to a solid support, or to one or more reporter groups which lung-lion, for example, as a calorimetric, fluorescent, luminescent, radioactive, or ligand recognition group.
Functionally fluorescent groups include fluoresce ins, radiomen, an the like or adduces thereof; function-ally luminescent groups include luminous, assuredness, Lucifer ins, dioxetanes, dioxamides, and the like or adduces thereof. Ligand recognition groups include vitamins (such as button or adduces thereof, including iminobiotin and desthiobiotin), antigens such as donator-phenols, carbohydrates and other functional groups or adduces of such groups which can be recognized by ligand-like interactions with proteins or from which such ligand-like interactions can be elicited. Another oligonucleotidecapable 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 calorimetric reporter groups when recog-notion results in dye formation. For example, whendinitrophenyl is used as a reporter group, known detection systems using an antidinitrophenyl antibody coupled to peroxides can be used as a detection system, resulting in a color change. Functionally radioactive groups incorporate a radioactive element in the chosen reporter group.

. .

~3~5 When reference is made herein to the use of Purina or pyrimidine bases, such expressions are intended to include analogs of such bases. Among such analogs are the analogs of Purina bases, such as the deazaadenosines (tubercidins, formycins, and the like), and the analogs of pyrimidine bases, such as desirously, deazacytosine, azauracils, azacytosines, and the like.
Oligonucleotides of Formula I are best prepared by chemical synthesis from monomer nucleated analog units 10 of the formula:

(~) s PYRE
or ' R joy I = OPERA

O Where:
R6 = methyl, sheller phenol FORMULA II X - sheller, dialkyl-amino, morpholino wherein R3 is tritely (triphenylmethyl), dimethoxytrityl, or other appropriate masking group for the 5'~hydroxyl;
B and R' sure masked, if appropriate; and represents 15 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 are preferably alkyd phosphomonochloridites or alkyd 20 phosphomonoamidites, Alternatively phosphate trimesters may be employed for this purpose. The monomer unit may , .

-18- .

alternatively have R3 attached at the 3' hydroxyl and attached at the 5'-hydroxyl.
Generally, the term "masking group" or "blocking group" it a functional expression referring to the 5 chemical modification our "blocking" of an integral functional group by attachment of a second moiety to disguise the chemical reactivity of the functional group and prevent it from reacting in an undesired manner. Such modification is reversible, and allows 10 subsequent conversion back to the original functional group by suitable treatment. In many cases, such mask-in formally inter converts structural functionality, e.g., a primary amine masked by acetylation becomes a substituted aside which can be later converted back to 15 the primary Camille 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 n~nreactive cations and include, for example, nitrogen-20 containing bases such as ammonium salts, moo-, do-, in- 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 25 be generally described and illustrated diagra~atically.
Thereafter, the.i~ention will be illustrated more specie call and detailed examples thereof provided. Since the invention Ritz Jo oligonucleotides incorporating ought pyrimidine-based and purine-based nucleated units, the 30 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 Purina classes, and it is to be understood that any other member --19- ' 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 under-5 stood that ribonucleotide compounds are also con-template by the invention and can be substituted for the deoxyribonucleotide compounds wherever rabbinical-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 sync the sizing the new oligonucleotides. Such nucleosides each have a base which is modified by a substituent 15 group comprising a functionalized carbon chain and one or more asides, the nitrogen of the asides being attached to a starkly tolerant site on the base through the carbon chain. In the case of pyrimidine-based nucleon sides, the carbon chain it attached at the C-5 position, 20 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 I a group, such as dimethoxytrityl, appropriate for the chemical synthesis of oligonucleotides.

In the new class of nucleosides the substituent group may be chosen from CH2CHRlCn on . 1 n on ' CHRl~nH2nY, OH Curl C NHCnH2n , 1 n Zen wherein Al is hydrogen or Clue lower alkali n is 5 0 to 20 and Y it one or more amino, substituted amino, substituted amino or substituted aminoalkylphenyl groups.
O
More specifically, Y may include one or more -NH~CX3 wherein X it hydrogen, fluorine or chlorine. Synthesis of these nucleosides, as well as of the masked forms 10 thereof described hereinafter in Examples I, II, IV, VI, VII, VOW, X, XII, and XIII. Preferred nucleosides incorporate the substituent group -CH=CHG NCHnH2nY at the C-5 of pyrimidine nucleosides wherein n = 3 to 12 15 and Y is -NHCCX3. Most preferred are such nucleosides wherein the pyrimidine base is Ursula.
The process of thy present invention may be initiated by the preparation of the selected knucklehead. Generally 9 the most preferred nucleoside~ are best prepared in the 20 following manner. methyl 3-acrylyl)-2'-deoxyuridine is prepared from 2'-deoxyuridine by the method of Bergstrom and Ruth [J. Amer. Chum. Sock 96:1587 (1976)].
The nucleoside is then treated with 1.05 equivalents of dimethoxytrityl chloride in pardon for 4 hours to 25 block the 5' hydroxyl with dimethoxytrityl (DOT). The resulting product is purified by silica chromatography eluding a gradient of 0-10% methanol in chloroform containing 2Z triethylamine. The purified 5'-DMT-5-(methyl 3-acrylyl)-2'-deoxyuridine is treated with 30 1 N KOCH for 24 his. at ambient temperature to hydrolyze the methyl ester.

-21~

The resulting 5'-DMT-5-(3~acrylyl)-2'-deoxyuridine is treated with excess dicyclohexylcarbodiimide and hydroxybenztriazole in pardon. After 4 hours, a 2-5 fold excess of,yl,~-diaminoheptane is 5 added, and the reaction stirred overnight. After 12-20 hours, a 10-20 fold excess of trifluoroacetic android is added, and the reaction stirred at room temperature for 4 hours. The product is purified by silica chromatography eluding a gradient of 0-10%
10 methanol in chloroform containing 2% triethylamine, followed by exclusion chromatography using Sephadex LH-20 eluding 1% triethylamine in methanol. Appear-private fractions are combined to yield 51DMT-5-[N-(7-trifluoroacetylaminoheptyl)-l-acrylamido]-2''-15 deoxyuridine; such product is appropriate for oligo-nucleated synthesis by the phosphochloridite prove-dune 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 20 diaminoheptane in this process with other Damon-alikeness (e.g., diaminopropane, diaminohexane, Damon-dodecane) is productive of other compounds of varying substituent length wherein n = 3, 6, Go 12 and P
R = -C~l=CHC NHCnH~nNHCCX3. Two such nucleosides, one 25 pyrimidine (uracil~based and the other Purina (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 3C bouncily group (By) to the amine at the 6 position of the adenine-based nucleoside. Such masking it generally described in "Synthetic Pro-seeders in Nucleic Acid Chemistry", Vol. 1, W. Zorbach and R. Tip son ens. (Wiley - Intrusions, NAY., 1968) Unprotected amine on the substituent group are masked, for example, by attachment thereto of triflu~roacetyl groups (A), a also shown in Reaction 1.
The selected 3' or 5' hydroxyl of the nucleoside is then masked by attachment thereto of a dimetho-10 xytrityl (DOT) 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 Jo an activated nucleated monomer, preferably by attachment to its 3' hydroxyl Off a phosphorus-containing group which includes an activating moiety. When the modified nucleoside is properly blocked, modifications of the procedures de-scribed by Let singer, et at, Mattocks, et at, or as reviewed by Nearing, et at can be utilized for oligo-nucleated synthesis. The use of phosphochloridite chemistry such as that disclosed by Letsingf2r en at, is detailed in Examples XVI-XVIII. In order to. use phosphoamidite chemistry, a modification of the prove-dune of Mattocks, et at, is used, phosphitylatingthe protected modified nucleoside with methyl sheller (N, N-diisopropyl)phosphoamidite or methyl sheller-phosphomorpholidite, a in the improved procedure of Dormer, et at [Nucleic Acids Rest 11:2575(1983)]. Al-fry r i f,' ,:
' of ternatively, the protected modified nucleoside can be phosphorylated with 1~2 en. chlorophenyl dichlorophos~
plate in trimethylphosphate at room temperature lot-lowed by quenching with water to give the sheller-5 phenol phosphate adduce of Lye modified nucleoside, suchadducts being useful in a modification of the phosphor trimester approach as illustratively reviewed by Nearing, et at. The diagram illustrates in Reaction 3 the sync thesis of activated monomer nucleated units of Formula 10 II by attachment to the nucleoside 3' hydroxyl of a pros-phomonochloridite group in which the chlorine functions as an activating moiety.
Coupling or condensation of the selected activated nucleated monomer, i.e. the uracil-based monomer or 15 the adenine-based monomer, to the terminal unit of a growing nucleated chain is illustrated in Reaction 4 in the diagram. The nucleated chain is shown as in-eluding in its right hand end a nucleoside unit having a naturally occurring base and having a solid support o'er masking group R4 attached to its 3' hydroxyl. The illustrated chain also includes one or more (n') nucleon tide units having naturally-occurring bases, said units being coupled to the 5' hydroxyl of the nucleoside unit, the terminal one of the nucleated units having a free 25hydroxyl at the 5' position. In the coupling reaction ~'~ I 5 the chlorine of the monomer reacts with the hydrogen of the free hydroxyl of the terminal unit and is displaced, 50 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 nucleated chain.
The DOT 5' blocking group is then removed to permit further extension of the nucleated chain by sequential coupling thereto of additional activated nucleated monomer units. The nucleated units added to the chain can be preselected and may have either naturally-occur-ring or modified bases. The diagram shows in Reaction pa the further extension of the chain by the addition of one or more (n") nucleated units having naturally-occurring bases.
When an oligonucleotide of the selected length and sequence has been synthesized, the DOT group is removed from the terminal unit thereof, and the masked reactive groups are unmasked. Examples of modified Ursula and adenine bases with their reactive groups unmasked are also shown diagrammatically at Reaction 5. If the initial nucleated unit of the chain is bound to a solid support R4, the chain is then removed from such solid support. The appropriate order of unmasking can be pro-selected.
Reporter groups R5 appropriate for the .
it ! ' ' , i intended use of the product oligonucleo~ide can then be bound to such substituent groups as exemplified in Reaction 6, which illustrates the respective bases with reporter groups I bound to the respective 5 substituent group thereof.

Illustration of Synthetic Process a ~N~CH2~7N~2 SHEEHAN

Ho 0 ¦ Starting Ho 0 Nucleosides Ho Ho Ursula based adenine-based . Reaction 1:
Mooney of reactive sites , on nucleoside base 8 H~a~CH2~NHC:C: FJ I N H C C I
HO HO

Jo HO ( By bouncily so Reaction 2 Masking of 5'-hydroxyl . I (DOT = d.~methoxytrityl) r HN~a~cH2~NHCC lFJ No\> N~CH2~NHCC FJ

DMT-0~ DMT-0 HO HO

reaction 3 Activation to phosphor ~chloridite r HN~H~CH2~NHCCF3 ~N~CH2~NHCCF, DMT-O DMT-O

O Monomer nucleated O
SHOP- units of Formula II SHOP:

Reaction 4 Condensation to terminal unit of growing oligonucleo~ide chain COO _ / OR

Growing oligonucleotide chain \ , (x = amine masking groups . on A, C, G, or sub-\ stituent grow \ ' Box --r DMT-O~

o ox 1 X
O
Lo H3 _ I

Reaction pa ¦ Further chain eon-gushiness r BY r DMT-O--~'~J
of O Box lo H O

Pi 1 BY
O Pow OR

. Reaction 5 HO - P Deb locking, unmasking, r removal from R4 . I
O By OPT

OWE I

PRODUCT B
POW
OLIGONUCLEOTIDE _ _ ,~" Y
n ' + n" = O to about 198 Jo where By modified base (see pug 29) where, for example, By in product oligonucleotide (pug 28) is:
C3 No Hl.J~N~C~12~NH2 ~N~CH2~NH2 Ursula base adenine base (modified) (modified) To the product oligonucleotide a variety of useful reporter groups (R5) may be attached. For example:
I
Product oligonucleotide Product oligonucleotide with above modified with above modified . Ursula be, ye adenine base Reaction 6 N-hydroxy- Attachment of FIT
; succinimidyl reporter groups (R5) buttonhole-aminocaproic NH2 acid ~cH2~6NHcNH-fluor0~cein r '.
O O `
HNJ~N~cH2~NH~cH2 ~NH-biotin I

PRODUCT OLIGONUCLEOTIDES WITH REPORTER GROUPS (where R5 = blown or fluoresce in) Having discussed the process of the present invention in general terms and illustrated the same diagrammatically 9 each of the reactions referred to will now be discussed more specifically.
I With reference Jo reaction 1, masking ox chemically reactive amine such as No of citizen, No of adenine, N of guanine, and alkyd or aureole amine of the modified bases with suitable masking groups can be conveniently accomplished in suitable solvents such as alcohols, pyre-10 dines, letdowns, chloroform, and the like, by reaction of the nucleosides with an excess of appropriate acid androids for about 1 to 24 hours at temperatures in the range of OKAY to 110C, generally 20C to 80C.
Appropriate acid androids include acetic android, 15 trifluoroacetic android, bouncily android, anisoyl android, and the like. Preferred are acutely, in-fluoroacetyl, bouncily, and isobutyryl android.
Masking of the 5'-hydroxy in Reaction 2 can be conveniently effected by reaction of the nucleosides 20 with a slight excess of appropriate acid-labile masking reagents, such as tritylchlorides, monomethoxytrityl chloride, dimethoxytrityl chloride (DMTCl),trimethoxy-tritely chloride and the like. Preferred is dimethoxy-tritely chloride. Typical reactions are carried out in 25 suitable solvents, such as pardon, letdowns, in-alkylamines, and the like, at temperatures in the range of -20C to 120C, generally 20C to 100C, for about 1 to 48 hours. The preferred reaction utilizes 1.1 equivalents of DMTCl in pardon at room temperature 30 for 2 hours.
It is generally preferred that the respective pro-ducts of each recline described hereinabove be swooper-ted and/or isolated prior to use as a starting material -31~

for a subsequent reaction. Separation and isolation can be effected by any suitable purification procedure such as, for example, evaporation, filtration, crystal-ligation, column chromatography, thin layer chromatog-5 rough, etc. Specific illustrations-of typical swooper-Zion 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 10 that, where typical reaction conditions (e.g., tempera-lures, 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 phosphate analog illustrated in 15 Reaction 3 can be most conveniently effected by treat-mint of the nucleoside compounds with suitable phosphi-tilting agents in appropriate solvents at temperatures in the range of -90C to 60C for 1 minute to 2 hours.
Suitable phosphitylating agents include methylphospho-20 dichloridite, o-chlorophenylphosphodichloridite, p-chlo-rophenylphosphodichloridite, methylphospho(dialkylamino~
monochloridite 9 and the like. Appropriate solvents in-elude pyridlne, letdowns, acetonitrile, tetrahydro-Furman, Dixon 9 chloroform and the like containing 0-20%
25 appropriate base generally 1-5 vow %) such as letdowns, colliding triakylamines and the like. Preferred pros-phitylating agents are methylphosphodichloridite, o-chlorophenylphosphodichloridite, and methylphospho-(di-isopropylamino)-monochloridite. Preferred phosphy-30 tilting conditions are with 0.9 equivalents of methylphosphodichloridite in pardon or acetonitrile contain-in 5% letdown for 5 to 10 minutes at room tempera-lure or below.

The chemical incorporation of the modified nucleon tide analog monomers into a growing nucleated chain to produce defined sequence nucleotides is illustrated in Reactions 4 and pa. Typical condensations are in appear-5 private solvents at temperatures in the range of -20~C
to 50C~ preferably at ambient temperature, for about 1 to 60 minutes. Appropriate solvent mixtures include pardon, letdowns, acetonitrile, tetrahydrofuran, Dixon, chloroform and the like containing 0-20% appear-lo private base (generally 1 to 5 volume %) such as lutidines,collidines, trialkylamines and the like. The growing chain may be Swahili, insoluble, or attached to a suit-able solid support by appropriate chemical methods known in the art. Preferred is attachment to a solid support.
15 Furthermore, the growing chain may or may not have pro-piously incorporated one or more modified nucleoside analogs.
After condensation of the activated monomer to the growing chain, in Reaction 4, the initial product is 20 treated with suitable reagents to accomplish oxidation of the intermediate phosphitetriester, optional capping to block unrequited 5'-hydroxyls on the oligonucleotide chain, and removal of the 5'-DMT group. Oxidation of the phosphate trimester can be accomplished by treatment 25 with 0.1-5 wavily % iodine in suitable solvents, for example, tetrahydrofuran/water/lutidine mixtures. Comma-eel capping of unrequited 5'-hydroxyls can be accomplished by acetylation or acylation with, for example, acetic android and ~dimethylaminopyridine in tetrahydrofuran/
I letdown mixtures. Removal of the blocking group, usually DOT, is most conveniently effected by treatment with mild organic acids in nonerotic solvents, such as mild acids including, for example 9 1-5 vow % dichloro-acetic or trichloroace~ic acid in chloroform or dichloromethane. The growing nucleated chain, after removal of DOT, can now serve as acceptor for subset quint elongation by sequential reaction with activated monomers to eventually produce the oligonucleotide of 5 desired length and sequence, as shown in Reaction pa.
After an oligonucleotide of desired sequence is produced, Reaction 5 is accomplished to provide the product oligonucleotlde. To this end, thiophenol treat-mint is used to remove methyl masking groups from pros-10 plate trimesters, and suitable aqueous alkali or ammonia treatment is used to remove bouncily, acutely, isobutyl, trifluoroacetyl, or other groups from the protected amine and/or to remove the product from the solid sup-port. Removal ox DOT from the oligonucleotide product 15 is accomplished by the appropriate treatment with a mild acid, such as aqueous acetic acid at ambient temperature to 40C for 10 to 60 minutes. Such reactions may be accomplished before or during final purifications. Final purification is accomplished by appropriate methods, such 20 as polyacrylamide gel electrophoresis, high pressure liquid chromatography PLUCK), reverse phase or anion exchange on DEAE-cellulose, or combinations of these methods.
The process described herein for synthesis of oligo-nucleotides can utilize modified deoxyribonucleosides worry 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 rubs analogs, including Arabians and 3'-deoxyribose, can also be accommodated in the process to produce the desired oligonucleotide.
The substituent group modifying a nucleated 5 base must be capable of binding one or more reporter groups either prior to or after the chain extension coupling reaction. In the latter case, selected product oligonucleo~ides are reacted with suitable agents to attach such reporter group. For example, when modified 10 bases are incorporated into the oligonucleotide and R2 of the substltuent group contains one or more primary amine, coupling with amine-reactive groups such as isocyanate, isothiocyanate, active carboxylic acid conjugates, epoxies or active aromatic compounds 15 using suitable conditions is productive of aside, urea, Thor, amine or aromatic amine linkages. For example, an oligonucleotide which contains an Ursula or adenine base modified by a substituent group having a primary amine, as shown in the Reaction 5 diagram, can be 20 reacted with a suitable reagent, such as fluoresce in isoth~ocyanate or N-hydroxysuccinimidyl 6-biotinylamino-caproic acid to provide a reporter group R5 (fluoresce in or button, respectively) bound to the subs~i~uent group as shown in Reaction 6. Other reporter groups which 25 can be attached in similar manner include a wide variety of organic moieties such as fluoresce ins, Rhoda mines, acridinium salts, dinitrophenyls, benzenesulfonyls, luminous, Lucifer ins, buttons, vitamins, carboxyhydrates and the like. Suitably -35~

active reporter groups are available commercially, or can be synthesized, for example, by processes of the type generally described in "Bioluminescence and Chemiluminescence" [M. Delco and W. McElroy, ens., 5 Aged. Press, New York (1981)~, by D. Russell, et at., or H. Schroeder, eta [Moth. Enzymol. LXII, 1978], and references cited wherein.
Typically, attachment of reporter groups is conveniently accomplished in predominately aqueous lo solvents by reaction of the substituent groups of modified bases wherein R2 = On Hen NH2 wit of the selected reporter group at temperatures in the range of about -20C to 50C (preferably 20C
to 40C) for 1 to 24 hours. Suitable solvents are 15 an aqueous buffer and 0-50% organic solvents such as lower alcohols, tetrahydrofuran, dimethylforma-mode, pardon, and the like. Preferred reporter group reactants include fluoresce in iso~hiocyanates, dinitrophenylisothiocyanates, fluorodinitrobenzene, 20 N-hydroxysuccinimidylbiotin, N-hydroxysuccinimidyl dinitrobenzoate, isothiocyanates such as aminobutyl ethyl isoluminol isothiocyanate and the like, active esters of carboxyfluorescein, rhodamlne, button adduces, dioxetanes, dioxamides, carboxyacridines, 25 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 aside linkages.
30 Typically, this is conveniently effected in Prado-minutely aqueous solvents by reaction of the oligo-knucklehead with excess reporter group which contains a primary amine in the presence of suitable condensing I

agents, such as wa~er-soluble carbodiimides, at temperatures in the range of about -20 C to 50 C
(preferably 20C to 40C) for 6 to 72 hours. Pro-furred reporter groups of this class include (amino-5 alkyl)-amino-naphthalene-1,2-dicarboxylic acid hydra-wide amino-fluoresceins, aminorhodamines, aminoal~yl luminous, aminoslkylaminobenzenesulfonyl adduces, amino sugar and the like. Furthermore, the chemical synthesis of the initial oligonucleotide product may be accomplished 10 with modified nucleated monomers wherein, prior to the coupling reaction, such reporter groups are attached to the substituent group. If any such reporter groups would adversely affect the coupling reaction, they are appropriately masked to forestall any such adverse 15 effect. On the other hand, certain other reporter groups are substantially unreactive with respect to the coupling reaction and therefore do not require masking. For example, nitrophenyl adduces may be attached to the substituent group prior to the coupling reaction, and 20 without masking, may be present on the modified nucleon tide monomer during the coupling reaction without adverse effect.
Reporter group useful in the method of this invention generally include aromatic 9 polyaromatic, cyclic, and 25 polycycl~c organic moieties which are further function-alized 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 30 base. An illustrative example of an oligonucleotide of this type is one of the structure:
m G T U Us An . Am A
f o o o o o o o o I I I I I o \ OWE

':', wherein Cm is 5-(3-aminopropyl) citizen, Us is 5-rN-(4-aminobutyl)-1-acrylamido3uracil, and Am is 8-[6-2,4-dlnitrophenyl)-aminohexyl]aminoadenine.
This product is further modified by reaction with 5 floweriest isothioeyanate to provide a fluoresce in reporter group on Cm and us.
Such a product oligonucleotide illustrates the variety of the selection of modified and unmodified nucleated 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 nucleated unit having it base modified by substituent groups having bound thereto reporter groups providing the same or different types of reporter groups function. Also illustrated are units whose bases are modified by substituent groups to which reporter groups are bound after the coupling reaction, i.e., Cm and us, whereas Am is illustrative of a unit whose base is modified by a substituent group to which a dinitrophenyl reporter group was attached prior to the coupling reaction.
Such oligonucleotide additionally illustrates that it can include more than one nucleated 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 amine of the substituent groups as ill-striated in Reaction 6, such amine or other group can alternatively be coupled to suitably activated solid supports. This produces a defined sequence oligo-nucleated which is covalently bound to such supports through the modified bases. Such solid supports are useful in the detection and isolation of complementary I I

nucleic cold components. Alternatively, the modified nucleoside monomers can be coupled Jo solid supports prior to the chain extension coupling Reaction 4, to thereby provide solid supports for such monomers 5 during the coupling reaction.
The following specific examples are provided to enable those skilled in the art to practice the invent lion. The examples should not be considered limit-lions upon the scope of the invention, but merely as 10 being illustrative and representative thereof. To aid in structural clarification, references are made to the reactions illustrated in the aforementioned process diagram.

EXAMPLE I
This example illustrates the synthesis of a mod-fled nucleoside precursor 5-(3-trifluoroacetylamino-propenyl)-2'-cleoxyuridine.
5 Chloromercuri-2'-deoxyuridine I g, 7.8 Molly) is suspended in 200 ml methanol. N-Allyltrifluoro-acetamide(6.8 ml, 55 Molly) is added, followed by add-lion 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 my portions of sodium bordered, then concentrated under reduced prowar to solid nest-due. The residue is purified by flash column chrome-tography on silica gel eluding 15 vow % methanol in chloroform. Appropriately pure fractions of product are combined and concentrated under reduced pressure to give crystalline 5-(3-trifluoroacetylaminopropenyl)-~'-deoxyuridine (2.4 g). US Max 291 no (~7800), A mix 266 no, (I 4400); TLC (silica eluding 15 vow % methanol in chloroform) I = 0.4.

EXAMPLE I
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 Molly) is suspended in 200 ml methanol. N-(7-trifluoroacetylamino-heptyl)-acrylamide(55 Molly) is added, followed by add-lion of 41 ml of 0.2 N lithium tetrachloropalladate in 30 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 my portions of sodium bordered, I $

then concentrated under reduced pressure to solid nest-due. The residue is purified by flash column chrome-tography on silica gel eluding 10 vow % methanol in chloroform. Appropriately pure fractions of product are 5 combined and concentrated under reduced pressure to give crystalline 5-[N-(7-trifluoroacetylaminoheptyl)-1-acryl-amidoJ-2'-deoxyuridine (2.8 g). US Max 302 no (I 18000), Mooney 230 no, 280 no; TLC (silica eluding 15 vow % methanol in chloroform) Of = 0.3.

EXAMPLE III
This example illustrates masking of 5'-hydroxyl to produce 5' dimethoxytrityl-5-(3-trifluoroacetyl-arninopropenyl)-2'-deoxyuridine as illustrated in Reaction 2.
5-(3-tri~luoroacetylaminopropenyl)-2'-deoxyuri-dine (2.4 g) is thoroughly evaporated twice from pardon, then stirred in 40 ml pardon. Dimethoxy-trityl(DMT)chloride(2.3 g, 6.6 Molly) is added, and the mixture stirred at room temperature for four hours.
; 2Q After thin layer chromatography (TLC) on silica eluding 10 vow % methanol in chloroform indicates reaction is complete, the reaction is concentrated to a solid residue.
This residue is purified by column chromatography on silica eluding chloroform until all faster running US impurities have eluded, then bringing off product with 5 vow % methanol in chloroform. The residue is then con-cent rated to give 5'-dimethoxytrityl-5-(3-trifluoroacetyl-aminopropen-l-yl) -2'-deoxyuridine as a white fluffy solid (4 g). Product decomposes upon heating; US Max 3Q 291 no, 'I in 266 no; TLC Of 0.6 on silica eluding 10 vow % methanol in chloroform.

EXAMPLE IV
This example illustrates hydrogenation of excuse-die double bond and 5'-hydroxyl masking to produce 5'-dimethoxytri..tyl-5-(3-trifluoroacetylaminoproppull'-5 deoxyurîdine.
Repeating the nucleoside precursor synthesis and 5'-hydroxyl masking procedures of Examples I and III, but, prior to the addition of the DOT chloride, subject-in 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 dummy-thoxytrityl-5-(3-trifluoroacetylaminopropyl)-~'-deeoxyu-riding.

Examples V to VIII illustrate the synthesis of add-tonal modified Ursula 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 Andy, but replacing N-allyltrifluoroacetamide with compounds numbered 1 through 8 below is productive of the come pounds numbered 1' through 8' below, respectively; i.e.
substitution of.
1 N-(3-butenyl~trichloroacetamide 2 N-(5-hexenyl)trifluoroacetamide -42~

3 N-(2-methyl-2-propenyl)tri~luoroacetamide

4 N-(4-ethenylphenylmethyl)trifluoroacetamide N-(l-methyl-3-butenyl)trifluoroacetamide 6 N-(12-trichloroaminododecyl)acrylamide 7 N (pertrifluoroacetylpolylysyl)acrylamide 8 N-(3-trifluoroacetylamidopropyl)acrylamide is productive of 1'5'-dimethoxytrityl-5-(4-trichloroacetylaminobuten--1-yl)-2'-deoxyuridine 2'5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen--1-yl)-2'-deoxyuridine 3'5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-metthy-propen-l-yl)-2'-deoxyuridine 4'5'-dimethoxvtrityl-5-[2-(4-trifluoroacetylaminometthy-phenyl)etherl-1-yl]-2'-deoxyuridine

5'5'-dimethoxyltrityl-5-(4-trifluoroacetylamino-4-meethyl-buten-l-yl)-2'-deoxyuridine

6'5'-dimethoxytrityl-5-[N-(12-trichloroacetylaminodoo-decyl)-l-acrylamido]-2'-deoxyuridine I 7'5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpolylyssol)-l-acrylamido]-2'-deoxyuridine 8' 5'-dimethoxytrityl-5-[N-~3-trichloroacetylamino-~ro~yl)-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 9 through 18 below is productive of the products numbered 9' through 18' below, respectively;
i.e. substituting -aye- I So 9 5-(propen-1-yl)-2'-deoxyuridine 5-(carbmethoxyethyl)-2'-deoxyuridine '1 5-(3-carbmethoxylprop-1-yl)-2'-deoxyuridine 12 5-(4~carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridiire 5 13 5-(3-cyanopropen-1-yl)-2'-deoxyuridine 14 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxyuridine 15 5-~2-(4-carbmethoxyphenyl)ethen-1-yl~-2'-deoxyuriddine 16 5-(4-acetoxybu~en-1-yl)-2'-deoxyuridine _ 5-(4-acetoxybut-1-yl)-2'-deoxyuridine 10 18 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxyuridine is productive of the following 5'-dimethoxytrityl-5-alkyl-2'-deoxyuridines 9'5'-dimethoxytrityl-5-(propen-l-yl)-2'-deoxyuridinee 10'5'-dimethoxytrityl-5-(2-carbmethoxyethyl)-2'-deoxyy-uridine 11' 5'-dimethoxytrityl-5-(3-carbmethoxyprop-l-yl)-2'-deoxyuridine 12'5'-dimethoxytrityl-5-(4-carbmethoxy-2-methylbuten--l-yl3-2'-deoxyuridine 20 13'5'-dimethoxytrityl-5-(3-cyanopropen-1-yl~-2'-deoxyy-uridine 14'5'-dimethoxytrityl-5-(4-cyano-2-methylbuten-l-yl)--2'-deoxyuridine 15'5'-dimethoxytrityl-5-[2-(4-carbmethoxyphenyl)ethenn-l-yl]-2'-deoxyuridine 16' S'-dimethoxytrityl-5-(4-acetoxybuten-1-yl)-2'-deoxyuridine 17'5'-dimethoxytrityl-5-(4-acetoxybut-l-yl)-2'-deoxy--uridine 30 18'5'-dimethoxytrityl-5-[4-(2,~-dinitrophenyl)butyl]--2'-deoxyuridine 3~.'6~3 I
EXAMPLE VII
Repeating the nucleoside precursor synthesis and 5'-hydroxyl masking procedures of Examples I-VI, but replacing 5-cnloromercuri-2'-deoxyuridine with sheller-5 mercuriuridine is productive of the corresponding 5'-dimethoxytrityl-5-substituted uridines.

Examples VIII to XI illustrate the synthesis of modified citizen nucleosides. Since citizen nucleon sides, as well as adenosine nucleosides, have reactive 10 groups on their bases unlike the Ursula nucleosides, such reactive groups are masked to prevent unwanted reactions therewith. These examples illustrate masking of reactive groups on the citizen base moiety as in Reaction 1, as well as masking of the 5'-hydroxyl as in Reaction 2.

EXAMPLE VIII
5-t3-trifluoroacetylaminopropenyl)-N4-benzoyl-2'-deoxycytidine Repeating the nucleoside precursor synthesis pro-seedier of Example I, but replacing 5-chloromercuri-2'-deoxyuridine with 5-chloromercuri-2'-deoxycytidine is productive of 5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine (US Max 287 my). Purified try-fluoroacetylaminopropenyl~-2'-deoxycytidine (1.3 g, 4.6 Molly) is stirred in 80 ml an hydrous ethanol, bouncily android (1.5 g, 7 Molly) is added, and the reaction reflexed. Five additional 1.5 g portions of bouncily android are added hourly. After the reaction is judged complete by thin layer chromatography [silica plates eluding n-butanol/methanol/conc NH40H/H~ (60:
inn 6-10 hours, the reaction is cooled and con-cent rated under reduced pressure to a semisolid. The solid is ,tritura~ed with ether three times, decanted and dried. The crude product is crystallized from water to give chromatographically pure N4-benzoyl-5-(3-trifluoroacetylaminopropenyl)-2'-deoxycytidine as a white solid. the product decomposes above 120~C; W Max 311 no.

EXAMPLE IX
5'-Dimethoxytrityl-5-(3-trifluoroacetyl-aminopropenyl)-N4-benzoyl-2'-deoxycytidine Repeating the 5'-hydroxyl masking procedure of Example, but replacing 5-(3-trifluoroacetylamino-propenyl)-2'-deoxyuridine with 5-(3-trifluoroacetyl-aminopropenyl)-N4-benzoyl-2'-deoxycytidine is productive of5'-dime~hoxytrityl-5-(3-trifluoroacetylaminopropennil)-N4-benzoyl-2'-deoxycytidine.

EXAMPLE X
Repeating the nucleoside precursor synthesis and S' hydroxyl masking procedures of Examples VIII and IX, but replacing N-allyltrifluoroacetamide with the N-alkyl~rifluoroace~amides of Example V is productive of the corresponding 5'-dimethoxytrityl-5-,(trifluoroacetyl) aminoalkyl)-N4-benzoyl-2'-deoxycytidines, viz;
S'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten--yule)-N4-benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(6-trifluoroacetylaminohexen--yule)-N4-benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-metthy-propen-l-yl)-N4-benzoyl-2'-deoxycytidine 5'-dimethoxytrityl~5-[2-(4-trifluoroacetylaminometthy-phenyl)ethen-l-yl]-N4-benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-(4-trifluoroacetylamino-4-metthy-buten-l-yl)-N -benzoyl-2'-deoxycytidine 5'-dimethoxytrityl-5-[N-~12 trifluoroacetylaminodo-decyl)-l-acrylamido]-N4-benzoyl-2'-deoxyeytidine 5'-dimethoxytrityl-5-[N-(pertri~luoroacetylpolylyssol)-l-acrylamido]-N4-benzoyl-2'-deoxycytidine EXAMPLE XI
Synthesis of 5'-dimethoxytrityl-N4-benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycytidine 5-(2-Carbmethoxyethenyl)-2'-deoxycytidine (0.82 g, 2.6 Molly) is stirred in 50 ml an hydrous ethanol. Ben-zoic android (500 my, 2.2 Molly) is added, and the reaction heated to reflex. Five additional 500 my portions of benzoic android are added hourly. After the reaction is judged complete by thin layer chrome-tography (usually 6-8 hours) the reaction is cooled, and evaporated under reduced pressure to a yellow semi-solid. Chromatography on silica gel eluding a lunar to 1:3 methanollchloroform mixture followed by thorough evaporation of appropriately combined fractions gives N -benzoyl-5-(2-carbmethoxyethenyl)-2'-deoxycyti-dine as an amorphous white solid. US Max 296 no, mix 270 no. The solid is dried thoroughly, and dissolved in 20 ml pardon. Dimethoxytrityl chloride (1.1 en) is added, and the reaction stirred at ambient temperature for six hours. Concentration to a solid followed by column chromatography on silica gel eluding 10% methanol in chloroform yields 5'-dimethoxytrityl-N -benzoyl-~(2-carbmethoxyethenyl)-2'-deoxycytidine as a fluffy oft solid.

EXAMPLE XII
Repeating the nucleoside precursor synthesis pro-seedier of Example XI, but replacing 5-(2-carbmethoxy-ethenyl)-2'-deoxycytidine with the following compounds 5 numbered 19 through 27 below is productive of the corresponding compounds numbered 19' through 27' below, respectively, i.e., substituting:
19 5-(2-carbmethoxyethyl)-2'-deoxycytidine 5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine 10 21 5-(4-oarbmethoxy-2-methylbuten-1-yl)-2'-deoxycyti--dine 22 5-~3-cyanopropen-1-yl)-2'-deoxycy~idine 23 5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytidine 24 5-[2-(4-carbmethoxyphenyl)ethen-1-yl]-2'-deoxycy-tiding 5-(4-acetoxybuten-1-yl)-2'-deoxycytidine 26 5-(4-acetoxybut-1-yl)-2'-deoxycytidine 27 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxycytidine is productive of the following 5'-dimethoxytrityl-N4-20 benzoyl-5-alkyl-2'-deoxycytidines:
19' 5'-DMT-N4-benzoyl-5 (2-carbmethoxyethen-1-yl)-Z'-deoxycytidine 20' 5'-DMT-N4-benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine 25 21' 5'-~MT-N4-benzoyl-5-(4-carbmethoxy-2-methylbu~en-l-yl~-2'-deoxycytidine 22' 5'-DMT-N4-benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxy--cytidine 23' 5'-DMT-N4-benzoyl-5-(4-cyano-2-methylbuten-1-yl)-22'-deoxycy~idine _' 5'-DMT-N4-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen--l-yl]-2'-deoxycytidine 25' 5l-DMT-N4-benzoyl-5-(6-acetoxybuten-l-yl)-2l-de cytidine 26' 5'-DMT-N4-benzoyl-5-(4-acetoxybut-1-yl)-2'-deoxy-cytidine 27' 5'-DMl'-N4-benzoyl-S-[4-(2,4-dinitrophenyl)butyl]--2'-deoxycytidine .

S Similarly, the use of the other acid androids, e.g., acetic android, anisoyl android, or oilily android, is productive of the corresponding Nuzzle or No acutely alkali 2'-deoxycytidines of Examples X and XI wherein bouncily is replaced by acutely or azalea.

EXAMPLE XIII
Repeating the nucleoside precursor synthesis and 5'-hydroxyl masking procedures of Examples VlII to X, but replacing 5-chloromercuri-2'-deoxycytidine with 5-chloromercuricytidine is productive of the cores-15 pounding 5'-dimethoxy~rityl-N4-benzoyl-5-substituted cytidines.

EXAMPLE XIV
This example typifies the masking of reactive base moieties and the masking of 5'hydroxyl of adenine 20 nucleosides.
N6-benzoyl-8-(6-aminohexyl)amino-2'-deoxyadenosinee (4 Molly) is stirred in 60 ml an hydrous ethanol. Trip fluoroacetic android (6 Molly) is added, and the react lion stirred at room temperature. Two additional port I lions of trifluoroacetic android are added hourly.

After four hours, the reaction is concentrated to a solid residue, and lyophilized overnight. The crude N6-benzoyl-8-(6-~rifluoroacetylaminohexyl)amino-2''-deoxyadenosine is dried thoroughly and concentrated to 5 a solid residue twice prom pardon. The solid is stirred in 40 ml of pardon, and dimethoxytrityl chloride (6.5 Molly) is added. After four hours, the reaction is concentrated to leave a solid residue.
Purification by column chromatography on silica gel 10 eluding a multi-step gradient of 0 to 15% methanol in chloroform gives 5'-dimethoxytrityl-N6-benzoyl-8-(6-trifluoroacetylaminohexyl)amino-2'-deoxyadenosiire as an off-white solid.

Examples XV to XVII typify the activation of 5'-masked 5-substituted, and neutral occurring nucleon sides, to their respective phosphomonochloridites, as illustrated in Reaction 3 of the diagram.

. EXAMPLE XV
Preparation of 3'-phosphomonochloridite of 5'-DMT-5-(3-t~ifluoroacetylaminoprop-l-yl)-2'-deoxyuridine Dry 5-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-d~oxyuridine (1.54 g, 2.2 Molly) is lyophilized from 20 ml Bunsen three times for more than twelve hours each to remove residual water and solvents. The result-in very fluffy white power is transferred to a nitrogen Jo atmosphere while still under vacuum and dissolved in anhydro~s acetonitrile containing 5 vow % letdown to a final nucleo.side concentration of 30 my. While swirling vigorously under nitrogen, one rapid bonus ox S me~hylphosphodichloridite (1.0 en) 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, [3lP-NMR(CH3CN/CDCl3) generally indicates 40-70 mow % desired product (167.5 Pam);
remainder is composed of bis-3',~'-[5'DMT-5-(3-trifluoro-acetylamino~rop-l-yl)-2'-deoxyuridylyl] methylphosphite (140 Pam) and S'-DMT-5-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyurLdine 3'-methylphosphonate (9~5 Pam), the latter product being formed in amounts reflecting the presence of water in the reaction.

EXAMPLE XVI
Preparation of 3'-phosphombnochloridites of the naturally-occurring 2'-deoxynucleosides Repeating the procedure of Example XV, but replace in 5'~DMT-S-(3-trifluoroacetylaminoprop-1-yl)-2'-deoxyuridine with:
5'-DMT-2'-deoxythymidinc 5'-DMT-N4-benzoyl-2'-deoxycytidine S'-DMT-N6-benzoyl-2'-deoxyadenosine S'-DMT-N2-isobutyryl-2'-deoxyguanosine is productive of the corresponding phosphomonochlori-dotes, vows 5' DMT-2'-deoxythymidine 3'-methylphosphomonochloridite 5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-methylphosphomono-chloridite 5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphospho-monochloridite 5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-methylphos-phomonochloridite .. . ..
EXAMPLE XVII
Repeating the phosphomonochloridite synthesis procedures of Examples XV and XVI, but replacing methyl-phosphodichloridite with o-chlorophenylphosphodichlori-dote is productive of the corresponding 5'-DMT-nucleoside 3l-phosphomonochloridites~ viz.:
5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding 3'-o-chlorophenylphosphomonochloridite 5'-DMT-2'-deoxythymidine 3'-o-chlorophenylphosphomono-chloridite 5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-o-chlorophenyl-phosphomonochloridite 5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-o-chlorophenyl-phosphomonochloridite 5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-o-chloro-phenylphosphomonochloridite Similarly, the use of ~-chlorophenylphosphodichloridite is productive ox the analogous 3'-p-chlorophenylphos-phomonochloridite adduces. [32P]NMR (CHICANO CDCl3) of o-chlorophenylphosphomonochloridite products 160.7, 160.5 Pam ~diasteriomers).

. . .
Examples XVIII - XXIV typify the chemical synthesis of oligonucleotides which incorporate modified bases, as illustrated by Reactions 4 and 5 in the diagram.

I I I

EXAMPLE XVIII
Synthesis of deoxyoligonucleotides containing 5-(3-aminopropyl)-uracil and naturally-occurring nucleated units The phosphomonochloridite synthesis procedures of Examples XV and XVI are accomplishes immediately before deoxyoli~onucleotide synthesis, and the result-in products are used directly as 30 my crude methyl-phosphomonochloridites in an hydrous acetonitrile/5 10 vow % letdown.
Solid support (5-DMT-N6-benzoyl-2'-deoxyadenosine 3'-succinamidepropyl silica, 250 my, 20 eke) is put into a suitable reaction flow vessel (glass or Teflon column or formula). The solid support is preconditioned 15 by successive treatments with acetonitrile/5 vow % lull-dine, 2 w/v % iodine in tetrahydrofuran/water/lutidine for 2 minutes, acetonitrile/5% letdown, chloroform, 4 vow % dichloroacetic acid in chloroform for 2.5 mint vies, and acetonitrile/5% letdown, 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-ante with Reaction 4 by sequential addition of the desired activated 5'-DMT-nucleoside 3'-methyl~hospho-monochloridite monomer and coupling thereof to the free5'-hydroxyl of the terminal unit of the growing nucleon tide chain, which unit is initially the only unit of the chain, i.e., the deoxyadenosine based unit comprise in the solid support. Additions are by reaction of 10 ml of the crude 30 my 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-dote addition followed by one complete reagent cycle 35 consists of sequential treatments with:

-52~

-5'-DMT-5-(3-trifluoroace~ylaminopropyl)-2'-deoxyuuridine 3'-methylphosphomonochloridite -acetronitrile/lutidine wash -capping for S minutes with 0.3 M 4-dimethylaminopyridine in acetic anhydridellutidine/tetrahydro~uran (1:3:2) -acetonitrile/5% letdown wash oxidation with 2% iodine in te~rahydrofuran/water/
letdown (6:2:1) for 2 minutes -acetronitrile/5% letdown wash -chloroform wash removal of DOT by 2.5 minute treatment with 4 vow %
dichloroacetic acid in chloroform -chloroform wash -acetonitrile/lutidine wash The above cycle is repeated thirteen times, each time replacing 5'-DMT-5-(3-tri1uoroacetylaminopropyl)-2'-deoxyuridine 3'-methylphosphomonochloridite with a different one of the following 3'-methylphosphomono-chloridites:
5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite 5'-DMT-5-(3-tri~luoroacetylaminopropyl)-2'-deoxyurriding 3i-methylphosphomonochloridite .
5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphosphomono-chloridite 5'-DMT-N4-benzoyl-~'-deoxycytidine 3'-methylphosphomono-chloridite 5l-DMT-N2-isobutyryl-2l-deoxyguanosine 3'-methylphospho-monochloridite 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding 3'-methylphosphomonochloridite 5'-DMT-2'-deoxythymidine 3'-methylphosphomonochloridite S'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyurriding 3'-methylphosphomonochloridite _53_ I

S'-DMT-deoxythmidine 3'-methylphosphomonochloridite 5'-DMT-5-~3-trifluoroace~ylaminopropyl)-2'-deoxyurriding 3'-methylphosphomonochloridite 5'-DMT-N -isobutyryl-2'-deoxyguanosine 3'-methylphos-phomonoch`loridite 5'-DMT-N6-benzoyl-2'-deoxyadenosine 3'-methylphospho-monochloridite 5'-DMT-N4-benzoyl-2'-deoxycytidine 3'-methylphosphomono-chloridite in respective order, deleting dichloroacetic acid treat-mint 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 prom the support. The supernatant is removed, the solid washed three times with 0.5 ml con-cent rated autonomy hydroxide, and the combined super-Nat ants are sealed and heated at 50C overnight. The clear yellow supernatant is lyophilized thoroughly Initial purification is accomplished by reverse phase 20 high pressure liquid chromatography (HPLC) on on RP-8 (C-8) column eluding a 60 minute linear gradient of 0 to 30% vow % acetonitrile in 25 my ammonium acetate, pi 6.8.
The 5'-DMT-terminated product, eluding as a sharp peak at about 40 minutes J is collected; all shorter chains, 25 both capped and uncapped, elude 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 DOT), lyophilized to a solid residue, and dissolved in a small amount of aqueous buffer. The product, generally greater than OWE home-generous after HPLC, is further purified by conventional electrophoresis on owe 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-aminopropyl-uracil-containing pentadeca-deoxyoligonucleotide product illustrated diagrammatic gaily below, wherein Us = 5-(3 aminopropyl)uracil,is thereby produced.
CA G U T U T Us G
O O' O O O O O O
HOPE O-,P-O~--S:~-P-O~ r P top o, JO p O\r~P~-o~--o Pow -ox owe P-o¦ o P ox I I¦-- I¦-Note the conventional deprotection of the oligo-nucleated with ammonia has also removed the trifler-acutely masking group on the substituent.

The length and sequence of this oligonucleotide may then be determined by P-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 nucleated units therein are unmodified.
15Similarly,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 nucleon side 3'-methylphosphomonochloridite adduces of Examples XV and XVI with the corresponding owe- or ~-chlorophenylphosphomonochloridite adduces of Example XVII and inclusion of pyridinium oximate treatment to remove chlorophenyl blocking groups (at the end of the deoxyoligonucleotlde synthesis and before concentrated ammonium hydroxide treatment) is produc~ivè of the same deoxyoligonucleotide products.

EXAMPLE XIX
Repeating the phosphomonochloridite and deoxyoligo-5 nucleated 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 28 through 36 below is productive of the corresponding oligonucleotides having Ursula bases Us lo numbered 28' through 36' below, respectively, i.e., substituting:
285'-dimethoxytrityl-5-(3-trifluoroacetylaminopropenn-l-yl)-deoxyuridine 29 5'-dimethoxytrityl-5-(4-tri~luoroacetylaminobut-1-yl)-2'-deoxyuridine 305'-dimethoxytrityl-5-(4-trifluoroacetylaminobuten--l-yl)-2'-deoxyuridine 315'-dimethoxytrityl-5-(6-trifluoroacetylaminohex-1--yl)-2'-deoxyuridine 20 32S'-dimethoxytrityl-5-(5-tri~luoroacetylaminohexen--1-yl)-2'-deoxyuridine 335'-dimethoxytrityl-5-(2-trifluoroacetylaminoprop-22-yl)-2'-deoxyuridine - 34 5'-dimethoxytrityl-5-(3-trlfluoroace~ylamino-2-methyl-propen-1-yl)-2'-deoxyuridine 35 5'-dimethoxytrityl-5-(3-trifluoroacetylamino-2-methyl-prop-l-yl)-2'-deoxyuridine 36 5'-dimethoxytrityl-5-[2- (~-trichluoroacetylamino-methylphenyl~ethen-l-yl]-2'-deoxyuridine _ 5'-dimethoxytrityl-5-[N-(pertrifluoroacetylpoly-lysyl)-l-acrylamido]-2'~deoxyuridine _ 5'-DMT-5-[N-(7-trifluoroacetylaminoheptyl)-l-acrylamido]-2'-deoxyuridine -56~
is productive of the deoxynucleotides corresponding to the product of Example XVIII, wherein Us is:
28' 5-(3-aminopropen-1-yl) Ursula 29' 5-(4-aminobut-1-yl) Ursula 30' 5-(4-aminobuten-1-yl) Ursula 31' 5-(6-aminohex-1-yl) Ursula 32' 5-(6-aminohexen-1-yl) Ursula 33' 5-(3-aminoprop-2-yl) Ursula 34' 5-(3-amino-2-methylpropen-1-yl) Ursula 35' 5-(3-amino-2-methylprop-1-yl) Ursula 36' 5-[2-(4-aminoethylphenyl)ethen-1-yl] Ursula 37' 5-[N-(polylysyl)-l-acrylamido] Ursula 38' 5-[N-(7-aminoheptyl)-1-acrylamido] Ursula Similarly, by employing other 5'-DMT-5-(acylaminoalkyl)-2'-~eoxyuridine~, the analogous ~eoxyoligonucleoti~es are produced.
. .
EXAMPLE XX
Repeating the phosphomonochloridite and deoxyoligo-20 nucleated synthesis procedures of Examples XV to XVIII, buy replacing 5'-DMT-5-(3-acetylaminopropyl)-2'-deoxy-Rodney with 5-substituted-2'-deoxyuridines numbered 37 through 46 below is productive of the corresponding oligonucleotides having the Us Ursula bases numbered 37' through 46', below respectively; i.e., substituting;
37 5'-DMT-5-~propen-1-yl)-2'-deoxyuridine 38 5'-DMT-5-(2-earbmethoxyethyl)-2'-deoxyuridine 39 5'-DMT-5-(3-carbmethoxyprop-1-yl)-2'-deoxyuridine 5'-DMT-5-(4-carbmethoxy-2-methylbuten-1-yl)-2'-deoxyuridine 41 5'-DMT-5-(3-cyanopropen-1-yl)-2'-deoxyuridine 42 5'-DMT-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxy-uridine ~57~

43 5'DMT-5-12-(4-carbmethoxyphenyl)ethen-l-yl)-2'-deoxyuridine.
44 5'-DMT-5-(4-acetoxybuten-l-yl)-2'-deoxyuridine . 45 5'-DMT-S-(4-ace~oxybut-l-yl~-2'-deoxyridine 46 5'-DMT 5-[4-(2,4-dinitrophenyl)butyl]-2'-deoxy-uridine is productive of the products wherein, Us is:
37' 5-(propen-l-yl)uracil 38' 5 (2~carboxyethyl)uracil lo 39' 5-(3-carboxyprop-l-yl)uracil 40' 5-(4-carboxy-2-methylbuten-l-yl)uracil Al' 5-(3-cyanopropen-l-yl~uracil 42' 5-(4-cyano-2-methylbuten-l-yl)uracil 43' 5-12-(4-carboxyphenyl)ethen-l-yl]uracil 44' 5-(4-hydroxybuten-l-yl)uracil 45' 5-(4-hydroxbut-l-yl)uracil 46' 5-[4-(2,4-dinitrophenyl)butyl~uracil Note In 46' the dinitrophenyl is a ligand recognition type reporter group, i.e. use of antidinitrophenyl antibody as the ligand. Similarly, by employing other appropriate 5'-DMT-5-alkyl-2'-deoxyuridines, the analog gout deoxyoligonucleotides are produced.

EXAMPLE XXI
Repeating the procedures of Examples XV to XVIII, but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with 5'-DMT-N4-benzoyl-5-(3-trichloro-acetylaminoproply)-2'-deoxycytidine is productive of deoxyoligonueleotides as in Example TV ereLn Us [5-(3-aminopropyl)-uracil] is replace by Amman-propyl)cytosines. For example, ; : , -58~
C A G Cm T Cm T em I;
O O C) O O O O O
HO OPT OPT -0-1~-0\ -POW\ -0\ -O-PI-O\ r I- \ POW I

Jo ~o~g-oio o owe where Cm = 5-(3-aminopropyl)cytosine.

EXAMPLE XXII
Repeating the deoxyoligonucleotide synthesis pro-seedier of Example XXI but replacing 5'-DMT-N4-benzoyl-5-(3-trichloroacetylaminopropyl)-2~-deoxycytidine with compounds numbered 47 through 57 below is productive of the corresponding oligonucleotides having the Cm cry-cosine bases numbered 47' through 57' below, respect lively, i.e., substituting:
47 5-D~T-N -benzoyl-5-~3-trifluoroacetylaminopropen-1-yl)-2'-deoxycytidine 48 5'-DMT-N -benzoyl-5- (4-tri~luoroacetylaminobut-1-yl~-2'-deocycytidine 49 5'-DMT-N4-benzoyl-5-~4-trifiuoroacetylaminobuten-l-yl)-2'-deoxycytidine 5'-DMT-N4-benzoyl-5-(6-trifluoroacetylaminohex-1-yl)-2'-deoxycytidine 51 5'D~r-N4-benzoyl-5-(6-trifluoroacetylaminohexen-1--yl)-2'-deoxycytidine 52 5'-DMT~N4-benzoyl-5-(3-trifluoroace~ylaminoprop-2--ye)- 2'-deoxycytidine 53 5'-DMT-N4-benzoyl-5-(3-trifluoroacetylamino-2-methylpropen-l-yl)-2'-deoxycytidine 54 5'-DMT-N4-~enzoyl-5-(3-trifluoroacetylamino~2-methylprop-l-yl)-2'-deoxycytidine 55 5'-DMT-N4-benzoyl-5[2-(4-trifluoroacetylaminomethyye-phenyl)e~hen-l-yl]-2'-deoxycytidine _59_ I 3 56 5'-DMT-N4-benzoyl-5-[N-(pertrifluoroacetylpoly-lysyl)-l-acrylamido)-2'-debxycytidine 57 5'-DMT-N4-benzoyl~-[N-~trifluoroacetylaminoheptyl))-acrylamido]-2'-deoxycytidine is productive of the products wherein Cm is:

47' 5-(3-aminopropen-l-yl)cytosine 48' 5-(4-aminobu~ yl~cytosine 49' 5-;4-aminobuten-l-yl)cytosine 50' 5-(6-aminohex-l-yl)cytosine lo Al' 5-(6-aminohexen-l-yl)cytosine 52' 5-(3-aminoprop-2-yl)cytosine 53' 5-(3-amino-2-methylpropen-l-yl~cytosine 54' 5-(3-amino-2-methylprop-l-yl)cytosine 55' 5-~2-(4-aminomethylphenyl)ethen-l-yl]cytosine I 5-[N-(polylysyl)-l-acrylamido]cytosine 57' 5-[N-(7-aminoheptyl)-l-acrylamido]cytosine Similarly, by employing other N4-acyl-5-(acylaminoalkyl)-2'-deoxycytidines the analogous deoxyoligonucleotides are produced.

EXAMPLE XXIII

: Repeating the deoxyoligonucleotide synthesis pro-seedier of Example XXI, but replacing 5'-DMT-N4-benzoyl-5-(3-trifluoroacetylaminopropyl)-2'-deoxycytidine with the compounds numbered 58 through 68 below is productive of the corresponding oligonucleo~ides having the Cm citizen bases numbered 58' through 68' below, respect lively, i.e., substituting:

58 5'-DMT-N4-benzoyl-5-(propen-1-yl)-2'-deoxycytidinee 59 5'-DMT-N4-benzoyl-5-(2-carbmethoxyethyl)-2' Dixie-cytidine 60 5'-DMT--N4-benzoyl-5-(2-carbmethoxyethen-1-yl)-2'--deoxycytidine 61 5'-DMT-N4-benzoyl-5-(3-carbmethoxyprop-1-yl)-2'-deoxycytidine 62 5'-DMT-N4-benzoyl-5-(4-carbmethoxy-2-methylbuten-l-yl)-2'-deoxycytidine 63 5'-DMT-N4-benzoyl-5-(3-cyanopropen-1-yl)-2'-deoxy--cytidine 64 5'-DMT-N4-benzoyl-5-(4-cyano-2-methylbuten-1-yl)-2'-deoxycytîdine 65 5l-DMT-N4-benzoyl-5-[2-(4-carbmethoxyphenyl)ethen--1-yl]-2'-deoxycytidine 66 5'-DMT-N4-benzoyl-5-(4-acetoxybuten-1-yl)-2'-deoxycytidine 67 5'-DMT-N4-benzoyl-5-(4-acetoxybut-1-y1)-2'-deoxycytidine 68 5'-DMT-N4-benzoyl-5-~4-(2,4-~initrophenyl)butyl]-2-deoxycytidine .
is productive of the products wherein Cm is;
58' 5-(propen-1-yl)cytosine 59' 5-(2-carboxyethyl)cytosine 60' 5-(2-carboxyethen-1-yl)cytosine 61' 5-(3-carboxyprop-1-yl)cytosine 62' 5-(4-carboxy-2-methylbuten-1-yl)cytosine 63' 5-(3-cyanopropen-1-yl)cytosine 64' 5 (4-cyano-2-methylbuten-1-yl)cytosine 9.~3~6~11 65'. 5-[2-(4-carboxyphenyl)ethen-1-yl]cytosine 66' 5-~4-hydroxybuten-1-yl)cytosine 67' 5-(4-hydroxybut-1-yl)cytosine 68' 5-[4-(2,4-dinitrophenyl)butyl]cytosine Similarly, by employing other appropriate 5'-DMT-N4-acyl-5-alkyl-2'-deoxycy~idines the analogous Dixie-oligonucleotides are produced.

EXAMPLE XXIV
Repeating the phosphomonochloridite and deoxyoligo-nucleated synthesis procedures of Examples XV-XXIII, but replacing 5'-DMT-5-(3-trifluoroacetylaminopropyl)-2'-deoxyuridine with 5'-DMT-N5-benzoyl-8-(6-trifluoro-acetylaminohexyl)amino-2'-deoxyadenosine is productive of deoxyoligonucleotides as in Example XVIII, except that the Us is replaced by Am, and Am = 8-(6-aminohexyl) amino-2'-deoxyadenosine.
Examples XXV to XXVIII typify the binding of report ton groups to oligonucleotides containing appropriately modified bases, as illustrated in Reaction 6.

EXAMPLE XXV
Fluoresceinated deoxyoligonucleotides A purified pentadeca-nucleotide (from Example XVIII) of the structure C A G Us T us T us G
O O O O O O O
~10\ -O-P O\ OPT -0-~-0\ -O-P-O\ -POW\ OPT 'I\ POW
r Jo I_ PUP P ~1' [where Us is 5~[N-(7-aminoheptyl)-l-zcrylamido]uracil is dissolved await Aye units per ml in aqueous 300mM
sodium borate or sodium carbonate buffer, pi 9.5, con-twining 30 sodium chloride. Solid fluoresce in is-thiocyanate (OHS my per ml) is added, and the mixture sealed and shaken gently at 4 C to 25 C overnight. The reaction is chromatographed directly on a column of G-50 Seafood to separate unbound fluoresce in adduces which are retained; the fluoresceinated deoxyoligo-nucleated adduces elude near the void volume. Early fractions containing significant Aye units are combined and lyophilized to solid product of structure similar to the starting pentadecadeoxyoligonucleotide where us is now either O o S
HNJ~N~CH2~NHCNH-Fluorescein OWN

or unrequited 5-[N-(7-aminoheptyl)-l-acrylamido]uracil.
I Max (H20) 262 no, 498 no.
Repeating the procedure on compounds recited in Examples XIX, XXI, XXII, and XXIV is productive of the corresponding fluoresceinated or polyfluoresceinated I deoxyoligonucleotides in like manner.

EXAMPLE XXVI
Attachment of reporter groups other than fluoresce in can be accomplished by repeating the procedure of Example XXVD but replacing fluore5cein isothiocyanate with, for example:
2,4-dinitrophenyl ~sothiocyanate l-fluoro-2,4-dinitrobenzene amino ethyl isoluminol lsothiocyanate aminoethylaminonaphthalene-1,2-carboxylic hydrazide isothiocyanate N,N'-bi~ talkyl~ulfonyl)-N~aryl-N'-isothiocyanatoaryl-dioxamide . 10 m-sulfonyl aniline isothiocyanate N-hydroxysuccinimidyl button 9- (N-hydroxysuccinimidyl carboxy)-N-methylacridine, or cyanogenbromide-activated Suffer is productive no the corresponding.adducts wherein the attached group is other than fluoresce in.
EXAMPLE XXVII
Attachment of isoluminol and free primary amine-containing reporter group purified pentadecanucleotide from Example XVIII
20 of the structure: .
C A G U T Us T em G
O O O O ' O O ' O O
. 1~0~,~ OX I I O\ -O-P-O\ I I I\ -UP Ox -Ox "I

O O- ox ox O-where TV it 5-(2-carboxyeth~nyl)urac~l] it dissolved in water await Aye units per my and diluted with one volume pardon. Aminobutyl ethyl lsoluminol is added I

to a final concentration of 1 mg/ml,followed by add-lion of a five-fold molar excess of loathly-dimethylaminopropyl) carbodiimide. The reaction is sealed and shaken gently in the dark for 12 to 48 hours. The reaction mixture is concentrated under reduced pressure to a solid residue, and chroma~ographed directly on a column of G-50 Seafood I; the isoluminol-deoxyoligonucleotide conjugates elude near the void volume. Early fractions containing significant Aye lo units are combined and lyophilized to solid product of structure similar to the starting deoxyoligonucleotide where Us is now either HO N-isoluminol or unworked 5-(2-carboxyethenyl)uracil.
Repeating the procedure on compounds from Examples XV and XXIII wherein R2 contains car boxy is productive of the corresponding deoxyoligonucleotide-isoluminol adduces in like manner.
Repeating the procedure, but replacing aminobutyl isGluminol with other reporter groups containing a free primary amine is productive of the corresponding Dixie-oligonucleotide-reporter adduces in like manner.

-65~

EXAMPLE XXVIII
Attachment of dinitrophenyl reporter groups A purifiefl nonanucleotide of the structure:

HO i O-P-O~O-P-O~¦--o_P-O¦--o-P-O~o-P~O O-P-Oi 0- too where Am = 8-(6aminohexyl)aminoadenine is dissolved at 20 Aye units per ml in 250 my sodium carbonate buffer, pi 9, and 1-fluoro~2,4-dinitrobenzene is added. The reaction solution is shaken at ambient temperature overnight, then chromatographed directly on a column of Seafood G-50. Early fractions containing signify-cant Aye units are combined and concentrated to given oligonucleotide product similar to the starting decanucleotide wherein Am is now either NH ~N2 ,.1 N2 or unrequited 8-(6-aminohexyl)aminoadenine.
Repeating the procedure, but replacing Amman-hexyl)~minoadenine with other modified bases containing a free primary amine is similarly productive of the corresponding dinitrophenylated oligonucleotide adduces.

Claims (38)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A defined sequence single strand oligonucleotide of fewer than about 200 base units in length incorporating at least one ribonucleotide or deoxyribonucleotide unit having a pyrimidine or purine base modified at a sterically tolerant site by attachment thereto of a substituent group which incorporates a chain of one or more carbon atoms and at least one functional group including a nitrogen, oxygen, or sulfur atom, said at least one functional group having bound thereto or being capable of binding at least one reporter group or solid support, said substituent group being attached to said base through a nitrogen, oxygen or sulfur atom when said base is a purine.

2. The oligonucleotide of Claim 1 wherein, when said modified base is pyrimidine, said substituent group is attached thereto at the C-5 position, and when said modified base is a purine, said substituent group is attached thereto at the C-8 position, there being at least one functionally colorimetric, fluorescent, luminescent, radioactive or ligand recognition type reporter group bound to said at least one functional group.

3. The oligonucleotide of Claim 1 wherein said at least one functional group includes at least one amine, carboxy, hydroxy or sulfonate group or an adduct thereof.

4. The oligonucleotide of Claim 1 wherein at least one reporter group which is fluorescein, rhodamine, an acridinium salt, a protein, a nucleic acid, a carbohydrate, nitrophenyl, dinitrophenyl, benzenesulfonyl, biotin, imino-biotin, desthiobiotin, luminol, isoluminol, luciferin, dioxetane, dioxamide or an adduct thereof is bound to said at least one functional group.

5. The oligonucleotide of Claim 1 wherein said modified base is uracil or cytosine and said substituent group is -CH=CR1CnH2nY, -CH2CHR1CnH2nY, -CHR1CnH2nY, , or , wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is at least one amine or masked amine, carboxy or masked carboxy, hydroxy or masked hydroxy, carboxyphenyl or masked carboxyphenyl, aminoalkylphenyl or masked aminoalkylphenyl, or an adduct thereof.

6. The oligonucleotide of Claim 1 wherein said modified base is adenine and said substituent group is -CHR1CnH2nY
and is attached to the C-8 position of said adenine base, R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is at least one amine or masked amine, carboxy or masked carboxy, hydroxy or masked hydroxy, carboxyphenyl or masked carboxyphenyl, aminoalkylphenyl, or masked aminoalkylphenyl, or an adduct thereof.

7. The oligonucleotide of Claim 5 or 6 wherein Y is not masked, and a solid support or at least one functionally colorimetric, luminescent, fluorescent, radioactive or ligand recognition type reporter group is bound to Y.

8. The oligonucleotide of Claim 5 or 6 wherein Y is not masked and a solid support or at least one reporter group which is fluorescein, rhodamine, an acridinium salt, a protein, a nucleic acid, a carbohydrate, nitrophenyl, dinitrophenyl, benzenesulfonyl, biotin, iminobiotin, desthiobiotin, luminol, isoluminol, luciferin, dioxetane, dioxamide or an adduct thereof is bound to Y.

9. The oligonucleotide of Claims 1, 5 or 6 which is from about 5 to about 60 base units in length.

10. The process for the chemical synthesis of a defined sequence single strand oligonucleotide incorporating at least one nucleotide unit having a modified base, comprising condensing to an unprotected hydroxyl-bearing terminal unit of a nucleotide chain having fewer than about 200 base units, a nucleotide monomer having attached thereto an activated phosphorus-containing group, whereby said chain is extended by coupling of said monomer thereto through said active phosphorus-containing group at the site of said unprotected hydroxyl, at least one of said monomer and terminal unit being a ribonucleotide or deoxyribo-nucleotide having a pyrimidine or purine base modified at a sterically tolerant site by attachment thereto of a substituent group whch incorporates a chain of one or more carbon atoms and at least one functional group including a nitrogen, oxygen or sulfur atom, said at least one functional group having bound thereto or being capable of binding at least one reporter group or solid support, said substituent group being attached to said base through a nitrogen, oxygen or sulfur atom when said base is a purine, said at least one functional group or any reporter group bound thereto being masked during said coupling step if it would otherwise have a substantial adverse effect on said condensation reaction.

11. The process of Claim 10 wherein, prior to or after the coupling step, at least one of said monomer and nucleotide chain is attached to a solid support.

12. The process of Claim 10 wherein said at least one functional group includes at least one amine, carboxy, hydroxy or sulfonate group or an adduct thereof.

13. The process of Claim 10 wherein, prior to or after the coupling step, at least one functionally colorimetric, fluorescent, luminescent, radioactive or ligand recognition type reporter group is bound to said functional group.

14. The process of Claim 10 wherein prior to or after the coupling step, at least one reporter group which is fluorescein, rhodamine, an acridinium salt, a protein, a nucleic acid, a carbohydrate, nitrophenyl, dinitrophenyl, benzenesulfonyl, biotin, iminobiotin, desthiobiotin, luminol, isoluminol, luciferin, dioxetane, dioxamide or an adduct thereof is bound to said functional group.

15. The process of Claim 10 wherein said phosphorus-containing group is at the 3' or 5' position of the nucleotide monomer and said unprotected hydroxyl of the terminal unit is at the 5' position of the latter when the phosphorus-containing group is at the 3' position of the monomer and is at the 3' position of the terminal unit when the phosphorus-containing group is at the 5' position of the monomer.

16. The process of Claim 10 wherein said modified base is uracil or cytosine, said substituent group is -CH=CR1CnH2nY, -CH2CHR1CnH2nY, -CHR1CnH2nY, , or , wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, Y is at least one amino, carboxy, hydroxy, carboxyphenyl, aminoalkylphenyl or an adduct thereof, and said reporter group or solid support is bound to Y.

17. The process Claim of 13 or 16 wherein said reporter group is bound to said functional group or to Y prior to the coupling step and is nitrophenyl, dinitrophenyl, benzenesulfonyl, desthiobiotin or an adduct thereof.

18. The process of Claim 10 wherein said modified base is uracil or cytosine, and prior to the coupling step, said substituent group has no solid support or reporter group bound thereto and is -CH=CR1CnH2nY, -CH2CHR1CnH2nY, -CHR1CnH2nY, or , wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is at least one masked amine, masked carboxy, masked hydroxy, masked carboxyphenyl, masked aminoalkylphenyl or an adduct thereof.

19. The process of Claim 10 wherein said modified base is adenine and said substituent group is -CHR1CnH2nY
wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is amino, carboxy, hydroxy, carboxyphenyl, aminoalkylphenyl or an adduct thereof, said substituent group being attached to the C-8 position of said base, and prior to or after said coupling step, there is bound to said substituent group a functionally colorimetric, luminescent, fluorescent, radioactive or ligand recognition type reporter group.

20. The process of Claim 19 wherein said reporter group is bound to said substituent group prior to the coupling step and is nitrophenyl, dinitrophenyl, benzenesulfonyl, desthiobiotin or an adduct thereof.

21. The process of Claim 10 wherein said modified base is adenine, and prior to the coupling step, said substituent group has no solid support or reporter group bound thereto and is -CHR1CnH2nY wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is at least one masked amine, masked carboxy, masked hydroxy, masked carboxyphenyl, masked aminoalkylphenyl or an adduct thereof, said substituent group being attached to the C-8 position of said base.

22. The process of Claim 18 or 21 wherein Y is at least one in which X is hydrogen, fluorine or chlorine.

23. The process of Claim 18 or 21 wherein, after the coupling step, Y is unmasked, and a solid support or at least one functionally colorimetric, luminescent, fluorescent, radioactive or ligand recognition type reporter group is bound to Y.

24. The process of Claim 18 or 21 wherein, after the coupling step, Y is unmasked and a solid support or at least one reporter group which is fluorescein, rhodamine, an acridinium salt, a protein, a nucleic acid, a carbohydrate, nitrophenyl, dinitrophenyl, benzenesulfonyl, biotin, iminobiotin, desthiobiotin, luminol, isoluminol, luciferin, dioxetane, dioxamide or an adduct thereof is bound to Y.

25. The process of Claim 10 wherein the nucleotide monomer becomes a new terminal unit of said growing nucleotide chain upon coupling thereto, and after the coupling step, the nucleotide chain is extended by sequentially coupling one or more additional nucleo-tide monomers to the nucleotide chain until a product oligonucleotide having a preselected number of base units fewer than about 60 is produced.

26. A compound useful as an intermediate in the chemical synthesis of an oligonucleotide and having the structure wherein B is a pyrimidine base, R is a substituent group attached to said base at a sterically tolerant site and incorporating a chain of one or more carbon atoms and at least one functional group including a nitrogen, oxygen, or sulfur atom, at least one masking group, reporter group or solid support being attached to said at least one functional group, R4 is hydrogen or a masking group R5 is hydrogen, or a masking group when R4 is hydrogen, and is hydrogen when R4 is a masking group, and R8 is hydrogen or hydroxy.

27. A compound useful as an intermediate in the chemical synthesis of an oligonucleotide and having the structure wherein B is uracil or cytosine R is -CH=CR1CnH2nY, -CH2CHR1CnH2nY, -CHR1CnH2nY, , or , wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20, and Y is at least one amine, masked amine, aminoalkylphenyl or masked aminoalkylphenyl, R being attached to said base at the C-5 position, R4 is hydrogen or a masking group, R5 is hydrogen or a masking group when R4 is hydrogen, and is hydrogen when R4 is a masking group R8 is a hydrogen or hydroxy, and said masking group is monomethoxytrityl, dimethoxytrityl or trimethoxytrityl.

28. The compound of Claim 27 wherein Y is not masked, and a reporter group which is nitrophenyl, dinitrophenyl, benzenesulfonyl, desthiobiotin or an adduct thereof is attached to Y.

29. A compound useful as an intermediate in the chemical synthesis of an oligonucleotide and having the structure wherein B is a pyrimdine base, R is a substituent group attached to said base at a sterically tolerant site and incorporating chain of one or more carbon atoms and at least one functional group including a nitrogen, oxygen or sulfur atom, R4 is a masking group or or , R5 is or when R4 is a masking group, and is a masking group when R4 is or , wherein R6 is methyl or chlorophenyl, and R7 is chloro, diaklyamino or morpholino, and R8 is hydrogen or masked hydroxy

30. The compound of Claim 29 wherein at least one masking group, reporter group or solid support is attached to said at least one functional group.

31. The compound of Claim 29 wherein at least one functionally colorimetric, fluorescent, luminescent, radioactive or ligand recognition type reporter group is attached to said at least one functional group.

32. The compound of Claim 29 wherein at least one reporter group which is fluorescein, rhodamine, an acridinium salt, a protein, a nucleic acid, a carbohydrate, nitrophenyl, dinitrophenyl, benzenesulfonyl, biotin, iminobiotin, desthiobiotin, luminol, isoluminol, luciferin, dioxetane, dioxamide or an adduct thereof is attached to said at least one functional group.

33. A compound useful as an intermediate in the chemical synthesis of an oligonucleotide and having the structure wherein B is a uracil or cytosine base R is -CH=CR1CnH2nY, -CH2CHR1CnH2nY, , , or -CHR1CR1CCnH2nY, wherein R1 is hydrogen or C1-6 lower alkyl, n is 0 to 20 and Y is at least one masked amine or masked aminoalkyl-phenyl or is at least one amine or aminoalkyl-phenyl to which a reporter group is attached, which reporter group comprises nitrophenyl, dinitrophenyl, benzenesulfonyl, desthiobiotin or an adduct thereof, R being attached to said base at the C-5 position, R4 is a masking group or or R5 is or when R4 is a masking group, and is a masking group when R4 is or R6 is methyl or chlorophenyl, R7 is chloro, diaklyamino or morpholino, R8 is hydrogen or masked hydroxy, and said masking group is monomethoxytrityl, dimethoxytrityl, or trimethoxytrityl.

34. A compound useful as an intermediate in the chemical synthesis of an oligonucleotide and having the structure wherein B is an adenine base R is a substituent group attached to said base at a sterically tolerant site through a nitrogen, oxygen or sulfur atom and incorporating a chain of one or more carbon atoms and at least one functional group including a nitrogen, oxygen or sulfur atom, R4 is a masking group or or R5 is or when R4 is a masking group, and is a masking group when R4 is or R6 is methyl or chlorophenyl, and R7 is chloro, dialkylamino or morpholino R8 is hydrogen or masked hydroxy

35. The compound of Claim 34 wherein said at least one functional group includes an amine, carboxy, hydroxy or sulfonate group or an adduct thereof.

36. The compound of Claim 34 wherein R is attached to said base at the C-8 position and is -CHR1CnH2nY, in which R1 is hydrogen or C1-6 lower alkyl, n is 6, and Y is at least one masked amine or masked aminoalkylphenyl or at least one amine or aminalkylphenyl to which a reporter group is attached, which reporter group is nitrophenyl, dinitro-phenyl, benzenesulfonyl, desthiobiotin or an adduct thereof.

37. The compound of Claim 27, 33 or 36 wherein Y is at one , in which X is hydrogen, fluorine or chlorine.

38. The compound of Claim 34 wherein a solid support or functionally colorimetric, fluorescent, luminescent, radio-active, or ligand recognition type reporter group is attached to said at least one functional group.