United States Patent [J 9]
`Hobbs, Jr. et al.
`
`[54] ALKYl\'YLAMINO-NUCLEOTIDES
`Inventors: Frank W. Hobbs, Jr.; George L.
`[75]
`Trainor, both of Wilmington, Del.
`[73] Assignee: E. I. Du Pont de Nemours and
`Company, Wilmington, Del.
`The portion of the term of this patent
`subsequent to Sep. 10, 2008 has been
`disclaimed.
`[21] Appl. No.: 713,906
`Jun. 12, 1991
`[22] Filed:
`
`[ •] Notice:
`
`Related U.S. Application Data
`[63] Continuation-in-part of Ser. No. 57,565, Jun. 12, 1987,
`Pat. No. 5,047,519, which is a continuation-in-part of
`Ser. No. 881,372, Jul. 2, 1986, abandoned.
`[51]
`Int. CI.5 ............................................... C07H 1/00
`[52] U.S. Cl ......................................... 536/23; 536/24;
`536/26; 53°6/27; 536/29; 544/243; 544/244
`[58] Field of Search ....................... 536/23, 24, 26, 27,
`536/29; 544/243, 244
`References Cited
`U.S. PATENT DOCUMENTS
`5,047,519 9/1991 Hobbs, Jr. et al. ................... 536/29
`
`[56]
`
`OTHER PUBLICATIONS
`Bergstrom et al., J. Am. Chem. Soc., vol. 98; p. 1587
`(1976).
`Langer et al., Proc. Natl. Acad. Sci., U.S.A., vol. 78;
`No. 11, pp. 6633-6637.
`Draper, Nucleic Acid Research; vol. 12, No. 2,
`989-1022 (1984).
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US005151507A
`Patent Number:
`Date of Patent:
`
`5,151,507
`* Sep. 29, 1992
`
`[I I]
`
`[45]
`
`Barr et al., J. Chem. Soc.; Perkins Trans I, pp.
`1263-1267 (1978).
`Bergstrom et al., J. Am. Chem. Soc.; vol. JOO, p 8106
`(1978).
`Vincent et al., Tetrahedron Letters, vol. 22, pp. 945-947
`(1981).
`.
`Robins et al., J. Org. Chem.; vol. 48, 1854-1862 (1983).
`Sonogashira et al., Tetrahedron Letters; No. 50, pp.
`4467-4470 (1975).
`Edo et al., Chem. Pharm. Bull.; vol. 26, No. 12, pp.
`3843-3850 (1978).
`Seda et al., Chem. Bor., vol. 11; 2925-2930 (1978).
`Schram et al., J. Carbohyd., Nucles., Nucleotis, vol. 2,
`No. 2, pp. 177-184 (1975).
`Bergstrom et al., J. Org. Chem., vol. 46, No. 7; pp.
`1423-1431 (1981).
`Bergstrom et al., Nucleic Acid Research, vol. 8; pp.
`6213-6219 (1980).
`Haralambidis et al., Nucleic Acid Research, vol. 15, No.
`12, pp. 4857-4867 (1987).
`Gibson et al., Nucleic Acid Research, vol. 15, No. 16,
`pp. 6455-6467 (1987).
`Primary Examiner-Johnnie R. Brown
`Assistant Examiner-]. Oliver Wilson
`Attorney, Agent, or Firm-George A. Frank
`ABSTRACT
`[57]
`Alkynylamino-nucleotides
`and
`labeled
`al(cid:173)
`kynylaminonucleotides useful, for example, as chain
`terminating substrates for DNA sequencing are pro(cid:173)
`vided along with several key intermediates and pro(cid:173)
`cesses for their preparation. For some applications,
`longer, hydrophilic linkers are provided.
`
`9 Claims, No Drawings
`
`Page 1
`
`Illumina Ex. 1056
`IPR Petition - USP 10,435,742
`
`

`

`ALKYNYLAMINO-NUCLEOTIDES
`
`FIELD OF THE INVENTION
`This invention pertains to alkynylamino-nucleotides
`and especially to their use in preparing fluorescently(cid:173)
`labeled nucleotides as chain-terminating substrates for a
`fluorescence-based DNA sequencing method.
`
`1
`
`5,151,507
`
`REFERENCE TO RELATED APPLICATIONS
`This application is a continuation-in-part of applica(cid:173)
`tion Ser. No. 057,565, filed Jun. 12, 1987, now U.S. Pat.
`No. 5,047,519 which, in turn, is a continuation-in-part of
`application Ser. No. 07/881,372, filed Jul. 2, 1986, now
`abandoned, and is related to U.S. Pat. No. 4,833,332 and
`application Ser. No. 07/057,566, filed on Jun. 12, 1987, 10
`now abandoned, which is also a continuation-in-part of
`Ser. No. 07/881,372, filed Jul. 2, 1986.
`
`2
`Labeled dNTP is added to afford labeled fragments.) If
`a dNTP is incorporated by the polymerase, chain exten(cid:173)
`sion can continue. If the corresponding ddNTP is se(cid:173)
`lected, the chain is terminated. The ratio of ddNTP to
`5 dNTP's is adjusted to generate DNA fragments of ap(cid:173)
`propriate lengths. Each of the four reaction mixtures
`will, thus, contain a distribution of fragments with the
`same dideoxynucleoside residue at the 3'-terminus and a
`primer-defined 5' -terminus.
`The terms "terminator", "chain terminator" and
`"chain terminating substrate" are used interchangeably
`throughout to denote a substrate which can be incorpo(cid:173)
`rated onto the 3'-end of a DNA or RNA chain by an
`enzyme which replicates nucleic acids in a template-
`15 directed manner but, once incorporated, prevents fur(cid:173)
`ther chain extension. In contrast, the natural deoxynu(cid:173)
`cleotide substrates can be considered to be "chain prop(cid:173)
`agating substrates".
`The term "nucleoside" is used throughout to denote a
`BACKGROUND OF THE INVENTION
`20 heterocyclic base-sugar unit composed of one molecule
`of pyrimidine or purine (or derivatives thereof) and one
`DNA sequencing is one of the cornerstone analytical
`techniques of modern molecular biology. The develop-
`molecule of a ribose sugar (or derivatives or functional
`ment of reliable methods for sequencing has led to great
`equivalents thereof). The term "nucleotide" is used
`advances in the understanding of the organization of
`throughout to denote either a nucleoside or its phos-
`genetic information and has made possible the manipu- 25 phorylated derivative.
`lation of genetic material (i.e. genetic engineering).
`In both the Sanger and Maxam-Gilbert methods, base
`There are currently two general methods for se-
`sequence information which generally cannot be di-
`quencing DNA: the Maxam-Gilbert chemical degrada-
`rectly determined by physical methods has been con-
`tion method [A. M. Maxam et al., Meth. in Enzym.,
`Vol. 65, 499-559 (1980)] and the Sanger dideoxy chain 30 verted into chain-length information which can be de-
`termination method [F. Sanger, et al., Proc. Nat. Acad.
`termined. This determination can be accomplished
`Sci. USA, Vol 74, 5463-5467 (1977)]. A common fea-
`through electrophoretic separation. Under denaturing
`ture of these two techniques is the generation of a set of
`conditions (high temperature, urea present, etc.), short
`DNA fragments which are analyzed by electrophoresis.
`DNA fragments migrate as if they were stiff rods. If a
`The techniques differ in the methods used to prepare 35 gel matrix is employed for the electrophoresis, the
`these fragments.
`DNA fragments will be sorted by size. The single-base
`With the Maxam-Gilbert technique, DNA fragments
`resolution required for sequencing can usually be ob-
`are prepared through base-specific, chemical cleavage
`tained for DNA fragments containing up to several
`of the piece of DNA to be sequenced. The piece of
`hundred bases.
`DNA to be sequenced is first 5'-end-labeled with 32p 40
`To determine a full sequence, the four sets of frag-
`and then divided into four portions. Each portion is
`ments produced by either Maxam-Gilbert or Sanger
`subjected to a different set of chemical treatments de-
`methodology are subjected to electrophoresis in four
`signed to cleave DNA at positions adjacent to a given
`parallel lanes. This results in the fragments being spa-
`base (or bases). The result is that all labeled fragments
`tially resolved along the length of the gel. The pattern
`will have the same 5'-terminus as the original piece of 45 of labeled fragments is typically transferred to photo-
`DNA and will have 3'-termini defined by the positions
`sensitive film by autoradiography (i.e. an exposure is
`of cleavage. This treatment is done under conditions
`produced by sandwiching the gel and the film for a
`which generate DNA fragments which are of conve-
`period of time). The developed film shows a continuum
`nient lengths for separation by gel electrophoresis.
`of bands distributed in the four lanes, often referred to
`With Sanger's technique, DNA fragments are pro- 50 as a sequencing ladder. The ladder is read by visually
`duced through partial enzymatic copying (i.e. synthesis)
`scanning the film (starting with the short, faster moving
`of the piece of DNA to be sequenced. In the most com-
`fragments) and determining the lane in which the next
`mon version, the piece of DNA to be sequenced is in-
`band occurs for each step on the ladder. Since each lane
`serted, using standard techniques, into a "sequencing
`is associated with a given base (or combination of bases
`vector'', a large, circular, single-stranded piece of DNA 55 in the Maxam-Gilbert case), the linear progression of
`such as the bacteriophage M13. This becomes the tern-
`lane assignments translates directly into base sequence.
`plate for the copying process. A short piece of DNA
`The Sanger and Maxam-Gilbert methods for DNA
`with its sequence complementary to a region of the
`sequencing are conceptually elegant and efficacious but
`template just upstream from the insert is annealed to the
`they are operationally difficult and time-consuming.
`template to serve as a primer for the synthesis. In the 60 Analysis of these· techniques shows that many of the
`problems stem from the use of a single radioisotopic
`presence of the four natural deoxyribonucleoside tri-
`phosphates (dNTP's), a DNA polymerase will extend
`reporter. [A reporter can be defined as a chemical
`the primer from the 3'-end to produce a complementary
`group which has a physical or chemical characteristic
`copy of the template in the region of the insert. To
`which can be readily measured or detected by appropri-
`produce a complete set of sequencing fragments, four 65 ate physical or chemical detector systems or proce-
`dures. Ready detectability can be provided by such
`reactions are run in parallel, each containing the four
`dNTP's along with a single dideoxyribonucleoside tri-
`characteristics as color change, luminescence, fluores-
`phosphate (ddNTP) terminator, one for each base. (32p_
`cence, or radioactivity; or it may be provided by the
`
`Page 2
`
`

`

`5,151,507
`
`3
`ability of the reporter to serve as a ligand recognition
`site to form specific ligand-ligand complexes which
`contain groups detectable by conventional (e.g., colori(cid:173)
`metric, spectrophotometric, fluorometric or radioac(cid:173)
`tive) detection procedures. The ligand-ligand com- 5
`plexes can be in the form of protein-ligand, enzyme-sub(cid:173)
`strate, antibody-antigen, carbohydrate-lectin, protein(cid:173)
`cofactor, protein-effector, nucleic acid-nucleic acid or
`nucleic acid-ligand complexes.]
`The use of short-lived radioisotopes such as 32p at 10
`high specific activity is problematic from both a logisti(cid:173)
`cal and a health-and-safety point of view. The short
`half-life of 32p necessitates the anticipation of reagent
`requirements several days in advance and prompt use of
`the reagent. Once 32P-labeled DNA sequencing frag- 15
`ments have been generated, they are prone to self(cid:173)
`destruction and must be immediately subjected to elec(cid:173)
`trophoretic analysis. The large electrophoresis gels
`required to achieve single base separation lead to large
`volumes of contaminated buffer leading to waste dis- 20
`posal problems. The autoradiography required for sub(cid:173)
`sequent visualization of the labeled DNA fragments in
`the gel is a slow process (overnight exposures are com(cid:173)
`mon) and adds considerable time to the overall opera(cid:173)
`tion. Finally, there are the possible health risks associ- 25
`ated with use of such potent radioisotopes.
`The use of only a single reporter to analyze the posi(cid:173)
`tion of four bases lends considerable operational com(cid:173)
`plexity to the overall process. The chemical/enzymatic
`steps must be carried out in separate vessels and electro- 30
`phoretic analysis must be carried out in four parallel
`lanes. Thermally induced distortions in mobility result
`in skewed images of labeled DNA fragments (e.g. the
`smile effect) which, in turn, lead to difficulties in com(cid:173)
`paring the four lanes. These distortions often limit the 35
`number of bases that can be read on a single gel.
`The long times required for autoradiographic imag(cid:173)
`ing along with the necessity of using four parallel lanes
`force a "snapshot" mode of visualization. Since simulta(cid:173)
`neous spatial resolution of a large number of bands is 40
`needed, very large gels must be used. This results in
`additional problems: large gels are difficult to handle
`and are slow to run, adding more time to the overall
`process.
`Finally, there is a problem of manual interpretation. 4S
`Conversion of a sequencing ladder into a base sequence
`is a time-intensive, error-prone process requiring the
`full attention of a highly skilled scientist. Numerous
`attempts have been made to automate the reading and
`some mechanical aids do exist, but the process of inter- SO
`preting a sequence gel is still painstaking and slow.
`To address
`these problems,
`replacement of
`32P/autoradiography with an alternative, non-radi(cid:173)
`oisotopic reporter/detection system has been consid(cid:173)
`ered. Such a detection system would have to be excep- SS
`tionally sensitive to achieve a sensitivity comparable to
`32p; each band on a sequencing gel contains on the
`order of lQ-16 mole of DNA. One method of detection
`which is capable of reaching this level of sensitivity is
`fluorescence. DNA fragments could be labeled with 60
`one or more fluorescent labels (fluorescent dyes). Exci(cid:173)
`tation with an appropriate light source would result in a
`characteristic emission from the label thus identifying
`the band.
`The use of fluorescent labels, as opposed to radioiso- 65
`topic labels, would allow easier tailoring of the detec(cid:173)
`tion system to this particular application. For example,
`the use of four different fluorescent labels distinguish-
`
`4
`able on the basis of some emission characteristic (e.g.
`spectral distribution,
`life-time, polarization) would
`allow linking a given label uniquely with the sequencing
`fragments associated with a given base. With this link(cid:173)
`age established, the fragments could be combined and
`resolved in a single lane and the base assignment could
`be made directly on the basis of the chosen emission
`characteristic.
`So far two attempts to develop a fluorescence-based
`DNA sequencing system have been described. The first
`system, developed at the California Institute of Tech(cid:173)
`nology, has been disclosed in L. M. Smith, West Ger(cid:173)
`man Pat. Appl. #DE 3446635 Al (1984); L. E. Hood et
`al., West German Pat. Appl. #DE 3501306 Al (1985);
`L. M. Smith et al., Nucleic Acids Research, Vol. 13,
`2399-2412 (1985); and L. M. Smith et al., Nature, Vol.
`321, 674-679 (1986). This system conceptually ad(cid:173)
`dresses the problems described in the previous section
`but the specifics of the implementation render Smith's
`approach only partially successful. For example, the
`large wavelength range of the emission maxima of the
`fluorescently-labeled DNA sequencing fragments used
`in this system make it difficult to excite all four dyes
`efficiently with a single monochromatic source. More
`importantly, the significant differential perturbations in
`electrophoretic mobility arising from dyes with differ(cid:173)
`ent net charges make it difficult or impossible to per(cid:173)
`form single-lane sequencing with the set of dyes used in
`this system. These difficulties are explicitly pointed out
`by Smith et al.
`In general, the methodology used to prepare the
`fluorescence-labeled sequencing fragments creates diffi(cid:173)
`culties. For Maxam-Gilbert sequencing, 5'-labeled oli(cid:173)
`gonucleotides are enzymatically ligated to "sticky
`ended'', double-stranded fragments of DNA produced
`through restriction cleavage. This limits one to se(cid:173)
`quencing fragments produced in this fashion. For
`Sanger sequencing, 5' -labeled oligonucleotides are used
`as primers. Four special primers are required. To use a
`new vector system one has to go through the complex
`process of synthesizing and purifying four new dye(cid:173)
`labeled primers. The same thing will be true whenever
`a special primer is needed.
`The use of labeled primers is inferior in other respects
`as well. The polymerization reactions must still be car(cid:173)
`ried out in separate vessels. As in the Maxam-Gilbert
`and Sanger sequencing systems, effectively all frag(cid:173)
`ments derived from the labeled primer will be fluores(cid:173)
`cently labeled. Thus, the resulting sequencing pattern
`will retain most of the common artifacts (e.g. false or
`shadow bands, pile-ups) which arise when enzymatic
`chain extension is interrupted by processes other than
`incorporation of a chain terminator.
`In a second approach, W. Ansorge et al., J. Biochem.
`Biophys. Methods, Vol. 13, 315-323 (1986), have dis(cid:173)
`closed a non-radioisotopic DNA sequencing technique
`in which a single 5'-tetramethylrhodamine fluorescent
`label is covalently attached to the 5' -end of a 17-base
`oligonucleotide primer. This primer is enzymatically
`extended in four vessels through the standard dideox(cid:173)
`ynucleotide sequencing chemistry to produce a series of
`enzymatically copied DNA fragments of varying
`length. Each of the four vessels contains a dideoxynu(cid:173)
`cleotide chain terminator corresponding to one of the
`four DNA bases which allows terminal base assignment
`from conventional electrophoretic separation in four
`gel lanes. The 5' -tetramethylrhodamine fluorescent
`label is excited by an argon ion laser beam passing
`
`Page 3
`
`

`

`5,151,507
`
`5
`through the width of the entire gel. Although this sys(cid:173)
`tem has the advantage that a fluorescent reporter is used
`in place of a radioactive reporter, all of the disadvan(cid:173)
`tages associated with conventional sequencing and with
`preparing labeled primers still remain.
`Until now, no one has created a DNA sequencing
`system which combines the advantages of fluorescence
`detection with terminator labeling. If appropriate
`fluorescently-labeled chain terminators could be de(cid:173)
`vised, labeled sequencing fragments would be produced
`only when a labeled chain terminator is enzymatically
`incorporated into a sequencing fragment, eliminating
`many of the artifacts associated with other- labeling
`methods. If each of the four chain terminators needed to
`sequence DNA were covalently attached to a different 15
`distinguishable fluorescent reporter, it should be possi(cid:173)
`ble, in principle, to incorporate all four terminators
`during a single primer extension reaction and then to
`analyze the resulting sequencing fragments in a single
`gel lane. If such fluorescently-labeled chain terminators 20
`could be devised, these compounds would probably
`also be useful for other types of enzymatic labeling of
`nucleic acids. In particular, analogs of fluorescently(cid:173)
`labeled chain terminators could be designed to use
`other, non-fluorescent, reporters or to serve as chain- 25
`propagating substrates for enzymes which replicate
`nucleic acids in a template-directed manner (e.g., re(cid:173)
`verse transcriptase, RNA polymerase or DNA poly(cid:173)
`merase). Introducing a reporter into DNA in a manner
`useful for sequencing is one of the most difficult nucleic 30
`acid labeling problems. Compounds and/or strategies
`developed for DNA sequencing are also likely to be
`applicable to many·other labeling problems.
`To be useful as a chain-terminating substrate for
`fluorescence-based DNA sequencing, a substrate must 35
`contain a fluorescent label and it must be accepted by an
`enzyme useful for sequencing DNA. Suitable substrate
`candidates are expected to be derivatives or analogs of
`the naturally-occurring nucleotides. Because of the
`expectation that a fluorescent label and a nucleotide will 40
`not fit into the active site of a replication enzyme at the
`same time, a well-designed substrate must have the
`fluorescent label separated from the nucleotide by a
`connecting group of sufficient length and appropriate
`geometry to position the fluorescent label away from 45
`the active site of the enzyme. The nature of the connect(cid:173)
`ing group can vary with both the label and the enzyme
`used. For ease of synthesis and adaptability to variations
`in label and/or enzyme requirements, however, it is
`most convenient to consider the connecting group as SO
`consisting of a linker which is attached to the nucleotide
`and to the fluorescent label.
`In the design of fluorescently-labeled chain termina(cid:173)
`tors for DNA sequencing, the linker must satisfy several
`requirements:
`1) one must be able to attach the same or a function(cid:173)
`ally equivalent linker to all four bases found in DNA;
`2) the linker must not prevent the labeled nucleotide
`from being utilized effectively as a chain terminating
`substrate for an enzyme useful for DNA sequencing;
`3) the linker (plus optional spacer and label) must
`perturb the electrophoresis of oligonucleotides to
`which it is attached in a manner which is independent of
`the base to which it is attached;
`4) the attachment of the linker to the base and the 65
`spacer or label must be stereoselective and regioselec(cid:173)
`tive to produce a single, well-defined nucleotide sub(cid:173)
`strate; and
`
`6
`5) the linker should preferably contain a primary or
`secondary amine for coupling with the label.
`Although five different types of amine linkers have
`been disclosed for attaching labels to nucleotides and
`5 oligonucleotides (see below), none of these linkers meet
`all five of the requirements listed above for use in a
`chain terminating substrate useful in DNA sequencing.
`Bergstrom et al., J. Am. Chem. Soc., Vol. 98, 1587
`(1976), disclose a method for attaching alkene-amino
`10 and acrylate side-chains to nucleosides by Pd(II)-cat(cid:173)
`alyzed coupling of 5-mercurio-uridines to olefins. Ruth,
`PCT /US84/00279, discloses the use of the above side(cid:173)
`chains as linkers for the attachment of reporters to non-
`enzymatically synthesized oligonucleotides. Langer et
`al., Proc. Nat. Acad. Sci. USA, Vol. 78, 6633 (1981),
`disclose the use of allylamino linkers for the attachment
`of reporters to nucleotides. The disadvantages of these
`linkers include the difficulty of preparing regioselec(cid:173)
`tively the appropriate mercurial nucleotide precursors,
`the difficulty of separating the mixture of products
`generated by some of these nucleotide/olefin coupling
`reactions, and the potential }ability of vinyl substituted
`nucleosides. Furthermore, the only reporters which
`have been incorporated with this linker are biotin and
`digoxigenin, Schmitz et al., Analytical Biochemistry,
`Vol. 192, 222-231 (1991). These reporters have the
`disadvantage that they must be detected via a complex
`with avidin, streptavidin, or anti-digoxigenin antibodies,
`proteins which bind tightly to them. These proteins, and
`thus indirectly biotin and digoxigenin, are detected by
`attaching fluorescent or enzymatic reporters to them.
`For some applications, such as fluorescent in situ hy(cid:173)
`bridization, direct fluorescent tagging would provide a
`superior method for tagging DNA. Klevan et al., WO
`86/02929, disclose a method for attaching linkers to the
`N4 position of cytidine and the N6 position of adeno-
`sine. The disadvantage of this method is that there is no
`analogous site in uridine and guanosine for attaching a
`linker.
`Another potential linker which might satisfy the five
`requirements listed above is an alkynylamino linker, in
`which one end of the triple bond is attached to the
`nucleoside and the other end of the triple bond is at(cid:173)
`tached to a group which contains a primary or second(cid:173)
`ary amine. To insure chemical stability, the amine
`should not be directly attached to the triple bond. Some
`methods of attaching alkyne groups to nucleosides have
`been disclosed (see below).
`Barr et al., J. Chem. Soc., Perkins Trans. I, 1263-1267
`(1978), disclose the syntheses of 5-ethynyluridine, 2'(cid:173)
`deoxy-5-ethynyluridine, 5-ethynylcytosine, 5-ethynyl(cid:173)
`cytidine, 2' -deoxy-5-ethynylcytidine and the a-anomers
`of the 2'-deoxyribonucleosides. The 2'-deoxyribonu(cid:173)
`cleosides ;were prepared by constructing the heterocy(cid:173)
`cles, coupling with a functionalized 2-deoxy sugar, sep(cid:173)
`arating the anomeric mixtures, and removing the pro-
`tecting groups on the sugars.
`Bergstrom et al., J. Am. Chem. Soc., Vol. 100, 8106
`(1978), disclose the palladium-catalyzed coupling of
`alkenes with 5-mercuri or 5-iodo derivatives of uracil
`nucleosides. This method was reported to fail in analo-
`gous reactions of alkynes with uracil nucleoside deriva(cid:173)
`tives.
`Vincent et al., Tetrahedron Letters, Vol. 22, 945-947
`(1981), disclose the synthesis of 5-alkynyl-2'-deoxyuri(cid:173)
`dines by the reaction of 0-3',5'-bis(trimethylsilyl)deox(cid:173)
`yuridine with alkynylzinc reagents in the presence of
`palladium or nickel catalysts [dichloro-bis(triphenyl-
`
`SS
`
`60
`
`Page 4
`
`

`

`7
`phosphine)palladium(II), dichloro-bis(benzonitrile)pal(cid:173)
`ladium(II) or dichloro(ethylene-(bis(diphenylphos(cid:173)
`phine))nickel(II)].
`Robins et al., J. Org. Chem., Vol. 48, 1854-1862 5
`(1983), disclose a method for coupling terminal alkynes,
`HC==CR (R=H, alkyT, phenyl, trialkylsilyl, hydroxyal(cid:173)
`to 5-iodo-1-
`kyl or protected hydroxyalkyl),
`methyluracil and 5-iodouracil nucleosides (protected as
`their p-toluyl esters) in the presence of bis(triphenyl- 10
`phosphine)palladium(II) chloride and copper(I) iodide
`in warm triethylamine. When 3',5'-di-O-acetyl-5-iodo-
`2'-deoxyuridine was
`reacted with hexyne, 4-(p(cid:173)
`toluyloxy)butyne, 4-(tetrahydropyranyloxy) or 4- 15
`(trityloxy)butyne, the major products were the cyclized
`furano[2,3-d]pyrimidin-2-ones rather than the desired
`alkynyluridines.
`None of the above references discloses a method for
`attaching an alkynylamino linker to nucleosides. The
`methodology of Bergstrom fails, and that of Barr is not
`directly applicable. The catalysts used by Robins et al.
`and Vincent et al. have the potential to promote numer(cid:173)
`ous undesirable side reactions (e.g., cyclization or inter- 25
`molecular nucleophilic addition of the amine to an al(cid:173)
`kyne) when the alkyne contains an amino group. Cou(cid:173)
`pling reactions have been reported only with iodonu(cid:173)
`cleosides which contain an electron-deficient uracil 30
`base. Since Pd-catalyzed coupling reactions generally
`work best with electron-deficient aryl iodides, problems
`may be anticipated in coupling alkynes to any of the
`other three bases (which are all more electron-rich than
`uracil).
`There remains a need for alkynylamino nucleotides
`and for methods permitting their preparation.
`
`20
`
`35
`
`SUMMARY OF THE INVENTION
`The compounds of this invention are alkynylamino(cid:173)
`nucleotides having the structure:
`
`40
`
`(I),
`
`45
`
`wherein Rt is a substituted or unsubstituted diradical
`moiety of 1-20 atoms. Rt can be straight-chained alkyl(cid:173)
`ene, Ct-C20. optionally containing within the chain
`double bonds, triple bonds, aryl groups or heteroatoms 50
`such as N, 0 or S. The heteroatoms can be part of such
`functional groups as ethers, thioethers, esters, amines or
`amides. For DNA sequencing, Rt is preferably straight(cid:173)
`chained alkylene, Ct-C10; most preferably Rt is
`-CH2-. For enzymatic incorporation of multiple re- 55
`porters Oabels) into DNA, Rt can be a diradical moiety
`of an unsubstituted chain of 10-20 carbon atoms option(cid:173)
`ally containing oxygen, nitrogen and/or sulfur atoms in
`place of one or more carbon atoms. Most preferably, 60
`Rt is -CH20CH2(CH20CH2)nCH2-, where n=2-5.
`Substitutents on Rt can include Ct-C6 alkyl, aryl, ester,
`ether, amine, amide or chloro groups;
`Ri and R3 are independently H, Ct-C4 alkyl, or a 65
`protecting group such as acyl, alkoxycarbonyl or sulfo(cid:173)
`nyl. Preferably R1 is H, and R3 is H or trifluoroacetyl;
`Nuc (nucleotide) is ~-Het (heterocyclic base):
`
`5,151,507
`
`8
`
`JlA
`or ~ .l )
`
`Z
`
`N
`
`N
`\
`R.i
`
`Z is H or NH2: and
`R.i is a sugar or sugar-like moiety:
`
`RsO...---......._.o....._..r
`
`and wherein Rs is H, P03H2, P206H3, P309H4 or salts
`thereof, and when R1=Rs=H, then R6=H, OH, F, N3
`or NH2; or when R1=H and Rs=OH, then R6=H or
`OH; or when R1=0H and Rs=H, then R6=0H.
`The labeled alkynylamino-nucleotides of this inven(cid:173)
`tion are structure I where R3 is a reporter (label), prefer(cid:173)
`ably comprising biotin.
`
`DETAILED DESCRIPTION OF THE
`INVENTION
`The strategy used to incorporate reporters in the
`DNA sequencing fragments in a base-specific fashion is
`a critical feature of any DNA sequencing system. The
`use of the alkynylamino-nucleotides of this invention
`permit the modification of the Sanger methodology
`most advantageously by attaching a reporter Oabel) to
`an alkynylamino-nucleotide chain terminator. Although
`the reporter can be chosen from a wide variety of
`readily detectable groups Oabels), for convenience, the
`preferred approach is illustrated below using fluores(cid:173)
`cent reporters.
`This approach offers a number of operational advan(cid:173)
`tages. Most importantly, terminator labeling firmly
`links the attached reporter with the base-specific termi(cid:173)
`nation event. Only DNA sequencing fragments result(cid:173)
`ing from bona fide termination events will carry a re(cid:173)
`porter. This eliminates many of the artifacts observed in
`conventional sequencing. This approach also affords
`complete flexibility in the choice of sequencing vector
`since no special primers are involved. Automation is
`facilitated by the fact that the reporters are carried by
`four low molecular-weight reagents which can be selec(cid:173)
`tively introduced in a single reaction.
`There are no inherent operational disadvantages; the
`problems with this approach are encountered in the
`design stage. In general, the enzymes used for sequenc(cid:173)
`ing DNA are highly substrate selective and there is no
`reason a priori to expect to be able to make a nucleoside
`triphosphate with a covalently attached reporter that is
`an efficient chain-terminating substrate for a sequen.cing
`
`Page 5
`
`

`

`5,151,507
`
`10
`2',3'-dideoxy-3'-fluoro-,B-D-ribofuranosyl [(e), Z. G.
`Chidgeavadze et al., FEBS Lett., Vol. 183, 275-278
`(1985)], and
`2' ,3' -dideoxy-2' ,3' -didehydro-,B-D-ribofuranosyl [ (f),
`Atkinson et al., Biochem., Vol. 8, 4897-4904 (1969)].
`
`9
`enzyme. It might be thought that attachment of a re(cid:173)
`porter to a substrate would cause sufficiently large
`changes in the steric and electronic character of the
`substrate to make it unacceptable to the enzyme or,
`even if accepted, it would not be incorporated in the 5
`DNA chain. It has bec;n found, however, that the small
`size of the alkynylamino linker of this invention and the
`ability to attach the alkynylamino linker to the 5-posi(cid:173)
`tion of the pyrimidine nucleotides and the 7-position of
`the purine nucleotides provide labeled chain-terminat- 10
`ing substrates that do not interfere excessively with the
`degree or fidelity of substrate incorporation.
`The alkynylamino-nucleotides of this invention will
`be illustrated through the description of fluorescently(cid:173)
`labeled alkynylamino nucleotide chain terminators. To 15
`delineate the structural scope and rationale of fluores(cid:173)
`cently-labeled alkynylamino-nucleotides of this inven(cid:173)
`tion, it is useful to break the labeled structure (I) into
`five components:
`
`20
`
`0
`"
`~o"''\_Y
`/
`~
`HO
`OH
`
`(b)
`
`(d)
`
`(I)
`
`(a)
`
`OH
`
`(c)
`
`F
`
`(e)
`
`~o~o~
`
`(g)
`
`triphosphate-sugar-Hei-c= CR 1-NR2-label (fluorescent)
`
`(i)
`
`(ii)
`
`(iii)
`
`(iv)
`
`(v)
`
`25
`
`30
`
`(i) a triphosphate moiety, Rs
`(ii) a "sugar", R4
`(iii) a heterocyclic base (Het),
`(iv) a linker (-C==CR1NR2-), and
`(v) a fluorescent label, R3.
`(i) Triphosphate Moiety (Rs)
`The triphosphate moiety or a close analog (e.g., a-thi-
`otriphosphate) is an obligate functionality for any en(cid:173)
`zyme substrate, chain terminating or otherwise. This
`functionality provides much of the binding energy for 35
`the substrate and is the actual site of the enzyme-sub-
`strate reaction.
`
`0
`0
`0
`II
`II
`II
`-o-P-O-P-0-P~
`I
`I
`I
`-o
`-o
`-o
`
`(ii) Sugar CR4)
`The "sugar" portion corresponds to the 2'-deox- 45
`yribofuranose structural fragment in the natural enzyme
`substrates. This portion of the molecule contributes to
`enzyme recognition and is essential for maintaining the
`proper spatial relationship between the triphosphate
`moiety and the heterocyclic base. To be useful for so
`DNA sequencing, when the "sugar" is a ribofuranose,
`the 3'-a-position must not have a hydroxyl group capa(cid:173)
`ble of being subsequently used by the enzyme. The
`hydroxyl group must either be absent, replaced by an- SS
`other group or otherwise rendered unusa