United States Patent (19)
`Summerton et al.
`
`73) Assignee:
`
`54 UNCHARGED MORPOLINO-BASED
`POLYMERS HAVING PHOSPHOROUS
`CONTAINING CHIRAL INTERSUBUNIT
`LINKAGES
`75) Inventors: James E. Summerton; Dwight D.
`Weller, both of Corvallis, Oreg.
`Anti-Gene Deveopment Group,
`Corvallis, Oreg.
`The portion of the term of this patent
`subsequent to Jul. 23, 2008 has been
`disclaimed.
`21) Appl. No.: 799,681
`22 Filed:
`Nov. 21, 1991
`
`(*) Notice:
`
`63)
`
`Related U.S. Application Data
`Continuation of Ser. No. 454,057, Dec. 20, 1989, aban
`doned, which is a continuation-in-part of Ser. No.
`100,033, Sep. 23, 1987. Pat. No. 5,142,047, which is a
`continuation-in-part of Ser, No. 944,707, Dec. 18, 1986,
`and a continuation-in-part of Ser. No. 911,258, Sep. 24,
`1986, abandoned, and a continuation-in-part of Ser.
`No. 712,396, Mar. 15, 1985, abandoned.
`51) Int. Cl. .................. C07D 413/12; CO7D 413/14;
`C08G 79/02; C08G 79/04
`(52) U.S. Cl. ........................................ 544/81; 544/82;
`528/398; 528/399; 528/403; 528/405; 528/406
`58 Field of Search .................... 544/81, 82; 528/391,
`528/403, 405, 406,398,399
`References Cited
`U.S. PATENT DOCUMENTS
`4,558,047 12/1985 Takaya et al. ...................... 514/229
`5,034,506 7/1991 Summerton et al. ............... 528/391
`FOREIGN PATENT DOCUMENTS
`WO/86/055
`18 3/1986 World Int. Prop. O. .
`WO/86/055
`19 3/1986 World Int. Prop. O. .
`
`(56)
`
`
`
`III US005185444A
`
`11) Patent Number:
`(45) Date of Patent:
`
`5,185,444
`* Feb. 9, 1993
`
`WO9118898A 6/1990 World Int. Prop. O. .
`OTHER PUBLICATIONS
`Stirchak et al., Nucleic Acids Research, vol. 17, No. 15,
`(1989), pp. 6129-6141.
`Khym, J. X., Biochemistry 2 (2):344 (1963).
`Mungall, W. S. et al., J. Org. Chem. 42 (4): 703 (1977).
`Tittensor, J. R., J. Chem. Soc. (C): 2656 (1971).
`Gait, M. J., et al., J. C. S. Perkins I: 1684 (1974).
`Jones, A. S., et al., Biochem. et Biphys. Acta 365:365
`(1973).
`Blake et al., Biochem, 24:6132 (1985a).
`Blake et al., Biochem, 24:6139 (1985b).
`Froehler, et al., Nucleic Acids Res. 16:4831 (1988).
`Jayaraman, et al., Proc Natl. Acad Sci USA 78:1537
`(1981).
`Miller, et al., Biochemistry 18:5134 (1979).
`Miller, et al., J Biol Chem Chem 255:9659 (1980).
`Miller, et al., Biochimie 67:769 (1985).
`Murakami, et al., Biochemistry 24:4041 (1985).
`Pitha, Biochem Biphys Acta 204:39 (1970a).
`Pitha, Bipolymers 9:965 (1970b).
`Smith, et al., Proc Natl. Acad Sci USA 83:2787 (1986).
`Stirchak, E. P. et al., J. Organic Chem. 52:4202 (1987).
`Primary Examiner-Mukund J. Shah
`Assistant Examiner-Philip I. Datlow
`Attorney, Agent, or Firm-Peter J. Dehlinger; Gary R.
`Fabian
`ABSTRACT
`(57)
`A polymer composition is disclosed composed of mor
`pholino subunit structures linked together by un
`charged, chiral linkages, one-three atoms in length.
`These chiral linkages join the morpholino nitrogen of
`one subunit to the 5’ exocyclic carbon of an adjacent
`subunit. Each subunit contains a purine or pyrimidine
`base-pairing moiety effective to bind by hydrogen
`bonding to a specific base or base-pair in a target poly
`nucleotide.
`
`15 Claims, 15 Drawing Sheets
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 1 of 15
`
`5,185,444
`
`N
`
`S 4
`3
`
`N
`
`7
`
`12
`2
`
`Fig. 1
`
`l.
`
`2.
`
`3.
`
`2N1N
`
`2
`
`N
`
`2 N
`
`{} gy I?
`
`f
`
`N 4.
`
`N
`
`5.
`
`s
`
`6.
`
`.. ) ". ..)
`
`s
`
`s
`
`>
`
`7
`
`8.
`
`O
`
`9
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 2 of 15
`
`5,185,444
`
`AE P
`
`O
`
`i
`
`it
`
`Fig. 3A
`
`z=-
`Y.
`O
`
`P
`
`to
`
`Fig. 3B
`
`-
`z-F-
`". P
`Fig. 3C
`Ea = .
`
`2E P - x
`
`O Ot
`
`.
`rCBR, -O-CER, -S-CHR, -NRRs, F;
`R = H, CBs, or other oieties which do not interfer with target
`binding;
`R & Rs lay be the sale or different and are selected from R or may
`comprise a cyclic aliphatic or aromatic moiety;
`O, S, CH, NR; Ya F O, S, CHs ; Ys = O, S, NR; Z = O or S.
`
`Y
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 3 of 15
`
`5,185,444
`
`t
`t Fig. 4B - B
`
`Fig. 4A-A
`
`'it':
`
`z FP-X
`
`

`

`U.S. Patent
`US. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 4 of 15
`Sheet 4 of 15
`
`5,185,444
`5,185,444
`
`no
`
`0
`
`Y T
`
`N
`
`p:
`
`Trityl c1 EOYOTH
`
`4——
`
`i
`
`Sarepta Exhibit 1077, Page 5 of 34
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 5 of 15
`
`5,185,444
`
`/2 %
`
`N. G)
`
`E2
`
`NaCNBE
`
`No
`
`o1
`
`p
`
`

`

`US. Patent
`U.S. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 6 of 15
`Sheet 6 of 15
`
`5,185,444
`5,185,444
`
`no
`
`0
`
`P1
`
`nurc1
`
`———-’
`
`no
`
`on
`
`
`
`
`
`0
`
`o
`
`O
`1’1
`
`no
`
`on
`
`8310‘
`
`Nacxnna
`
`I_
`
`Flg O
`
`7
`
`0/
`
`\
`
`0 ®
`
`*
`
`N
`
`2: P_ 06
`6
`
`l0
`
`TBA
`ax
`ncc
`
`i
`P1
`
`
`
`@
`
`0
`
`“
`
`z==P—-x
`
`I0
`
`0
`
`Sarepta Exhibit 1077, Page 7 of 34
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 7 of 15
`
`5,185,444
`
`D
`
`21 N
`N --"
`N
`H
`O
`
`s
`\ N
`
`N
`O
`HC
`H
`o o H. N.
`3
`7 \ .
`
`Fig. 8A
`
`G
`
`aY
`s - "
`
`N
`
`O
`
`H
`N
`7 N H O O
`s
`\ . .
`. \ N
`ins
`x= g
`
`O o H N
`H
`
`C
`
`5'
`5
`3
`
`GGDDDGDDGucDGDDGGcDDDDD Polymer
`ggaaagaagttcaga aggcaaaaa Target
`cc tittct tcagtc.ttcc.gtttitt Duplex
`=High specificity hydrogen bonding
`: =Low specificity hydrogen bonding
`D=2, 6-diaminopurine or 2-aminopurine
`G= Guanine or Thioguanine
`a = Adenine
`cs Cytosine
`g = Guanine
`t=Thymine
`use Uracil
`
`Fig. 8B
`
`

`

`US. Patent
`U.S. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 8 of 15
`Sheet 8 of 15
`
`5,185,444
`5,185,444
`
`Sarepta Exhibit 1077, Page 9 of 34
`
`

`

`US. Patent
`U.S. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 9 of 15
`Sheet 9 of 15
`
`5,185,444
`5,185,444
`
`BO—t:TP; ®
`
`Ic1
`
`z: p— x
`
`I
`
`[Cl
`
`2: 9-— x
`
`@ c1
`
`Sarepta Exhibit 1077, Page 10 of 34
`
`

`

`US. Patent
`U.S. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 10 of 15
`Sheet 10 of 15
`
`5,185,444
`5,185,444
`
`Sarepta Exhibit 1077, Page 11 of 34
`
`

`

`US. Patent
`U.S. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 11 of 15
`Sheet 11 of 15
`
`5,185,444
`5,185,444
`
`Sarepta Exhibit 1077, Page 12 of 34
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 12 of 15
`
`5,185,444
`
`E.
`)
`
`O
`
`O
`
`Pt
`
`:= - 0
`ky G
`EO.
`O.
`
`s:
`
`7.
`
`O S. 6)
`O - - O N /
`kY
`
`O
`
`raio.
`
`O.
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 13 of 15
`
`5,185,444
`
`Fig. 12 (cont)
`
`

`

`U.S. Patent
`
`Feb. 9, 1993
`
`Sheet 14 of 15
`
`5,185,444
`
`
`
`3O
`
`6O
`5O
`4O
`TEMPERATURE (C)
`
`7O
`
`

`

`
`
`oéwsgéiisués}2953..
`
`
`
`C
`
`??.5%???£5ä5%!
`!!!!!!!!!!!!!!!!\/$$
`
`3..nononoaoaoaoan...nonoa»a...nonoon?
`
`U.S. Patent
`US. Patent
`
`Feb. 9, 1993
`Feb. 9, 1993
`
`Sheet 15 of 15
`Sheet 15 of 15
`
`5,185,444
`5,185,444
`
`[fig’
`If.
`.5
`531.,
`
`dd.udh
`
`?T ° 6?T, BI
`
`Sarepta Exhibit 1077, Page 16 of 34
`
`
`

`

`1.
`
`5,185,444
`
`UNCHARGED MORPOLINO-BASED POLYMERS
`HAVING PHOSPHOROUS CONTAINING CHIRAL
`INTERSUBUNIT LINKAGES
`
`This application is a continuation of application Ser.
`No. 454,057, filed Dec. 20, 1989 now abandoned, which
`is a continuation-in-part (CIP) of co-owned U.S. patent
`application Ser. No. 07/100,033 filed Sep. 23, 1987, now
`U.S. Pat. No. 5,142,047, application Ser. No. 07/100,033
`is a CIP of pending U.S. patent application Ser. No.
`06/944,707, filed Dec. 18, 1986, and a CIP of Ser. No.
`06/911,258, filed Sep. 24, 1986, now abandoned, and a
`CIP of Ser. No. 06/712,396 filed Mar. 15, 1985, now
`abandoned.
`This application was filed on even date with co-pend
`ing U.S. patent applications Ser. Nos. 07/454,056 and
`07/454,055, now issued as U.S. Pat. No. 5,034,506.
`FIELD OF THE INVENTION
`The present invention relates to morpholino-based
`polymers.
`
`5
`
`25
`
`2
`BACKGROUND OF THE INVENTION
`Polymers which are designed for base-specific bind
`ing to polynucleotides have significant potential both
`for in vitro detection of specific genetic sequences char
`acteristic of pathogens and for in vivo inactivation of
`genetic sequences causing many diseases-particularly
`viral diseases.
`Standard ribo- and deoxyribonucleotide polymers
`have been widely used both for detection of comple
`10
`mentary genetic sequences, and more recently, for inac
`, tivating targeted genetic sequences. However, standard
`polynucleotides suffer from a number of limitations
`when used for base-specific binding to target oligonu
`cleotides. These limitations include (i) restricted pas
`sage across biological membranes, (ii) nuclease sensitiv
`ity, (ii) target binding which is sensitive to ionic concen
`tration, and (iv) susceptibility to cellular strand-separat
`ing mechanisms.
`In principle, the above limitations can be overcome
`or minimized by designing polynucleic acid analogs in
`which the bases are linked along an uncharged back
`bone. Examples of uncharged nucleic acid analogs have
`been reported. Pitha et al (1970a, b) have disclosed a
`variety of homopolymeric polynucleotide analogs in
`which the normal sugar-phosphate backbone of nucleic
`acids is replaced by a polyvinyl backbone. These nu
`cleic acid analogs were reported to have the expected
`Watson/Crick pairing specificities with complementary
`polynucleotides, but with substantially reduced Tm
`values (Pitha, 1970a). One serious limitation of this ap
`proach is the inability to construct polymers by sequen
`tial subunit addition, for producing polymers with a
`desired base sequence. Thus the polymers cannot be
`used for base-specific binding to selected target sequen
`CCS.
`Polynucleotide analogs containing uncharged, but
`stereoisomeric, methylphosphonate linkages between
`the deoxyribonucleoside subunits have also been re
`ported (Miller, 1979, 1980; Jayaraman; Murakami;
`Blake, 1985a, 1985b; Smith). More recently a variety of
`analogous uncharged phosphoramidate-linked oligonu
`cleotide analogs have also been reported (Froehler,
`1988). These polymers comprise deoxynucleosides
`linked by the 3'OH group of one subunit and the 5'OH
`group of another subunit via an uncharged chiral phos
`phorous-containing group. These compounds have
`been shown to bind to and selectively block single
`strand polynucleotide target sequences. However, un
`charged phosphorous-linked polynucleotide analogs
`using deoxyribonucleoside subunits are particularly
`costly and difficult to prepare; the subunit starting mate
`rial is quite costly and of limited availability.
`More recently, deoxyribonucleotide analogs having
`uncharged and achiral subunit linkages have been con
`structed (Stirchak 1987). These uncharged, achiral
`deoxyribonucleoside-derived analogs are, as mentioned
`above, limited by relatively high cost of starting materi
`als.
`
`30
`
`REFERENCES
`Agarwal, Proc Nat Acad Sci USA, 85:7079 (1988)
`Balgobin, N., et al., Tetrahedron Lett, 22:3667 (1981).
`Belikova, Tetrahedron Lett, 37:3557 (1967). Blake et al.,
`Biochem, 24:6132 (1985a). Blake et al., Biochem 24:6139
`(1985b). Bower et al., Nucleic Acids Res. 15:4915
`(1987). Dikshit et al., Canadian J Chem, 66:2989 (1988).
`Froehler, et al., Nucleic Acids Res. 16:4831 (1988). Fox,
`J. J., et al., J. Am Chem Soc, 80: 1669 (1958). Gait, "Oli
`gonucleotide Synthesis A Practical "Approach," pp.
`31-33, IRL Press (Oxford, England) (1984). Goldberg,
`M. L. et al; Methods in Enzymology 68:206 (1979).
`Greenlee, J Org Chem, 492632 (1984). Grunstein, M. et
`al; Methods in Enzymology 68:379 (1979). Himmels
`bach, F., and W. Pfleiderer, Tetrahedron Lett, 24:3583
`(1983). Jayaraman, et al., Proc Natl Acad Sci USA
`78:1537 (1981). Kamimura et al., Chem Lett (The
`Chem. Soc. of Japan) pg. 1051 (1983) LaPlanche et al.,
`Nucleic Acids Res, 14: 9081 (1986). Lerman, L. S.,
`"DNA Probes: Applications in Genetic and Infectious
`Disease and Cancer,' Current Comm in Molec Biol
`(Cold Spring Harbor Laboratory) (1986). Letsinger and
`Miller, J Amer Chem Soc, 91:3356 (1969). McBride et
`al., J Amer Chem Soc 108:2040 (1986). Miller, et al.,
`50
`Biochemistry 18:5134 (1979). Miller, et al., J Biol Chem
`255.6959 (1980). Miller, et al., Biochimie 67:769 (1985).
`Murakami, et al., Biochemistry 24:4041 (1985). Nied
`balla, U., and H. Vorbruggen, J Org Chem, 39:3668
`(1974). Pitha, Biochem Biophys Acta 204:39 (1970a).
`Pitha, Biopolymers 9:965 (1970b). Reese, C. B., and R.
`S. Saffhill, J Chem Soc PerkinTrans, 1:2937 (1972).
`Smith, et al., J.A.C.S. 80:6204 (1958). Smith, et al., Proc
`Natl Acad Sci USA 83:2787 (1986). Southern, E.; Meth
`ods in Enzymology 68:152 (1979) Stirchak E. P. et al.,
`Organic Chem. 52:4202 (1987). Summerton, J., et al., J
`Molec Biol 122:145 (1978) Summerton, J., et al., J
`Molec Biol 78:61 (1979a). Summerton, J., J Molec Biol
`78:77 (1979b). Szostak, J. W. et al; Methods in Enzy
`mology 68:419 (1979). Thomas, P.; Methods in Enzy
`65
`mology 100:255 (1983). Toulme et al., Proc Nat Acad
`Sci USA, 83:1227 (1986). Trichtinger et al., Tetrahe
`dron Lett 24:711 (1983).
`
`35
`
`45
`
`55
`
`SUMMARY OF THE INVENTION
`It is one general object of the invention to provide a
`polymer capable of sequence-specific binding to
`polynucleotides and which overcomes or minimizes
`many of the problems and limitations associated with
`polynucleotide analog polymers noted above.
`The invention includes a polymer composition con
`taining morpholino ring structures of the form:
`
`

`

`
`
`(A)
`
`5,185,444
`4.
`target sequence of a polynucleotide. The polymer is
`composed of morpholino-based ring structures which
`are linked together by uncharged, chiral linkages, one
`to three atoms long, joining the morpholino nitrogen of
`one structure to the 5'exocyclic carbon of an adjacent
`Structure.
`
`O
`
`15
`
`25
`
`A. Morpholino-Based Subunits
`FIG. 1 shows the g-morpholino ring structures on
`which the polymer subunits are based, where the mor
`pholino carbon atoms are numbered as in the parent
`ribose. As seen in FIG. 1, the ring structure contains a
`5'methylene attached to the 4'carbon in the g-orienta
`tion.
`Each ring structure includes a purine or pyrimidine
`or related hydrogen-bonding moiety, Pi, attached to the
`backbone morpholine moiety through a linkage in the
`As-orientation.
`The purine hydrogen-bonding moieties or bases in
`clude purines as well as purine-like planar ring struc
`tures having a 5-6 fused ring in which one or more of
`the atoms, such as N3, N7, or N9 is replaced by a suit
`able atom, such as carbon. The pyrimidine moieties
`likewise include pyrimidines as well as pyrimidine-like
`planar 6-membered rings in which one or more of the
`atoms, such as N1, is replaced by a suitable atom, such
`as carbon. Preferred hydrogen-bonding moieties in the
`invention include the set of purines and pyrimidines
`shown in FIG. 2. Each base includes at least two hydro
`gen-bonding sites specific for a polynucleotide base or
`base-pair. Where the polymers are used for sequence
`specific binding to single-stranded polynucleotides, the
`purine structures 1-3 are designed to bind to thymine or
`uracil bases; structures 7-8, to guanine bases; structures
`4-6, to cytosine bases; and structure 9, to adenine bases.
`The polymers of the invention are also effective to
`bind to hydrogen-bonding sites accessible through the
`major-groove in duplex polynucleotides having mostly
`purine bases in one strand and mostly pyrimidine bases
`in the complementary strand, as discussed below.
`Because of the similar type and positioning of the two
`central polar major-groove sites among the different
`base-pairs of duplex nucleic acids (i.e., the NH4 and O6
`of a CG base-pair present the same H-bonding array as
`the NH6 and O4 of an AT base-pair), the H-bonding
`moiety of a duplex-binding polymer must hydrogen
`bond to the N7 of its target base-pair in order to
`uniquely recognize a given base-pair in a target genetic
`duplex. Thus, in the polymers of the present invention,
`which are targeted against duplex genetic sequences
`containing predominantly purines in one strand and
`predominantly pyrimidines in the other strand, the hy
`drogen-bonding moieties of the polymer preferably
`contain purines having an amine at the 2 position since
`that amine is suitably positioned for H-bonding to the
`N7 of the target base-pair. More specifically, Structures
`2 and 3 of FIG. 2 provide for specific binding to a TA
`or UA base-pair while Structures 4 and 6 provide for
`specific binding to a CG base-pair. Two bases which are
`particularly useful in a duplex-binding polymer are
`2,6-diaminopurine (structure 3) and guanine (structure
`4). FIG. 8A illustrates the binding of these two bases to
`the polar major-groove sites of their respective target
`base-pairs in duplex nucleic acids. FIG. 8B illustrates a
`representative base sequence of a polymer designed for
`binding a target genetic sequence in the duplex state.
`
`The ring structures are linked together by uncharged,
`chiral linkages, one to three atoms long, joining the
`morpholino nitrogen of one ring structure to the 5'exo
`cyclic carbon of an adjacent ring structure.
`Each ring structure includes a purine or pyrimidine
`base-pairing moiety P which is effective to bind by
`base-specific hydrogen bonding to a base in a target
`sequence in a polynucleotide.
`These and other objects and features of the invention
`will become more fully apparent when the following
`detailed description of the invention is read in conjunc
`20
`tion with the accompanying examples and figures.
`BRIEF DESCRIPTION OF THE FIGURES
`FIG. 1 shows a basic (3-morpholino ring structure
`which is linked through uncharged, chiral linkages to
`form the polymer of the present invention;
`FIG. 2 shows several exemplary purine and pyrimi
`dine base-pairing moieties (represented as Pi of the ring
`structures shown in FIG. 1);
`FIG. 3 shows several preferred subunits suitable for
`forming polymers having 5-atom (subunit A), 6-atom
`(subunit B), and 7-atom (subunits C-E) unit-length
`backbones;
`FIG. 4 shows a repeating subunit segment of exem
`plary morpholino-based polymers, designated A-A
`35
`through E-E, constructed using subunits A-E, respec
`tively, of FIG. 3;
`FIG. 5 shows the steps in the synthesis of several
`types of morpholino subunits from a ribonucleoside;
`FIG. 6 shows an alternative synthesis of the basic
`morpholino subunit;
`FIG. 7 shows the steps in the synthesis of a morpho
`lino subunit designed for construction of polymers with
`seven-atom repeating-unit backbones;
`FIG. 8 shows the binding mode for 2-amine-contain
`ing purines to polar major-groove sites of respective
`target base-pairs and a representative base sequence of a
`duplex-binding polymer;
`FIG. 9 shows the steps in linking two morpholino
`subunits through a phosphonamide linkage;
`FIG. 10 shows two methods for linking morpholino
`subunits through a phosphoramidate linkage;
`FIG. 11 shows the linking of morpholino subunits
`through a phosphonoester linkage;
`FIG. 12 illustrates a subunit coupling procedure
`55
`which concurrently generates the morpholino ring
`structure;
`FIG. 13 shows the thermal denaturation plot for a
`(morpholino-C)6/poly(G) complex, where the (mor
`pholinoC)6 polymer was constructed according to the
`60
`present invention; and
`FIG. 14 illustrates the use of a morpholino polymer in
`a probe-diagnostic system.
`DETAILED DESCRIPTION OF THE
`INVENTION
`The present invention includes a morpholino-based
`polymer which is designed for base-specific binding to a
`
`30
`
`45
`
`SO
`
`65
`
`

`

`10
`
`15
`
`30
`
`20
`
`5,185,444
`6
`5
`appreciated that a polymer may contain more than one
`Polymers comprising predominantly 2-amine-con
`linkage type.
`taining purines, thus suitable for high-specificity bind
`Subunit A in FIG. 3 contains a 1-atom phosphorous
`ing to polar major-groove sites of duplex genetic Se
`containing linkage which forms the five atom repeating
`quences, can provide effective binding to their targeted
`unit backbone shown at A-A in FIG. 4, where the
`genetic duplexes using alternative backbones, in addi
`morpholino rings are linked by a 1-atom phosphona
`tion to the morpholino-based backbone. Examples of
`mide linkage. It is noted here that the corresponding
`such alternative backbones include phosphodiester
`chiral thionyl-containing linkage (substituting an S=O
`linked deoxyribonucleosides where a pendant group on
`moiety for the phosphorous-containing group) was
`the phosphorous is one of the following: a negatively
`found to have inadequate stability in aqueous solution.
`charged oxygen (i.e., the natural DNA backbone); a
`Subunit B in FIG. 3 is designed for 6-atom repeating
`methyl or other alkyl group (referred to as an alkyl
`unit backbones, as shown at B-B, in FIG. 4. In struc
`phosphonate); a methoxy or other alkoxy group (re
`ture B, the atom Y linking the 5'morpholino carbon to
`ferred to as a phosphotriester); or a mono- or dialkyl
`the phosphorous group may be sulfur, nitrogen, carbon
`amine (referred to as a phosphoramidate).
`or, preferably, oxygen. The X moiety pendant from the
`The morpholino subunits of the instant invention are
`phosphorous may be any of the following: fluorine; an
`combined to form polymers by linking the subunits
`alkyl or substituted alkyl; an alkoxy or substituted alk
`through stable, chiral, uncharged linkages. The linking
`oxy; a thioalkoxy or substituted thioalkoxy; or, an un
`group of a subunit includes a phosphorous-containing
`substituted, monosubstituted, or disubstituted nitrogen,
`electrophile which is usually reacted with a nucleophile
`including cyclic structures. Several cyclic disubstituted
`of the subunit to which it is to be linked.
`nitrogen moieties which are suitable for the X moiety
`The selection of subunit linking groups for use in
`are morpholine, pyrrole, and pyrazole.
`polymer synthesis is guided by several considerations.
`Subunits C-E in FIG.3 are designed for 7-atom unit
`Initial screening of promising intersubunit linkages (i.e.,
`length backbones as shown for C-C through E-E in
`those linkages which are predicted to not be unstable
`FIG. 4. In Structure C, the X moiety is as in Structure
`25
`and which allow either free rotation about the linkage
`B and the moiety Y may be a methylene, sulfur, or
`or which exist in a single conformation) typically in
`preferably oxygen. In Structure D the X and Y moieties
`volves the use of space-filling CPK or computer molec
`are as in Structure B. In Structure E, X is as in Structure
`ular models of duplex DNA or RNA. The DNA and
`B and Y is O, S, or NR.
`RNA duplexes are constructed according to parameters
`B. Subunit Synthesis
`determined by x-ray diffraction of oligodeoxyribonu
`cleotides in the B-form and oligoribonucleotidecontain
`The most economical starting materials for the syn
`ing duplexes in the A-form.
`thesis of morpholino-subunits are generally ribonucleo
`In each of these constructed duplexes, one of the two
`sides. Typically, ribonucleosides containing hydrogen
`sugar phosphate backbones is removed, and the pro
`bonding moieties or bases (e.g., A, U, G, C) are synthe
`35
`spective backbone, including the morpholino ring and
`sized to provide a complete set of subunits for polymer
`intersubunit linkage, is replaced, if possible, on the sites
`synthesis. Where a suitable ribonucleoside is not avail
`of the bases from which the original sugar-phosphate
`able, a 1-haloribose or, preferably, a la-bromoglucose
`backbone has been removed. Each resulting polynu
`derivative, can be linked to a suitable base and this
`cleotide/polymer duplex is then examined for coplanar
`nucleoside analog then converted to the desired (3-mor
`ity of the Watson/Crick base pairs, torsional and angle
`pholino structure via periodate cleavage, and closing
`strain in the prospective binding polymer backbone,
`the resultant dialdehyde on a suitable amine.
`degree of distortion imposed on the nucleic acid strand,
`Because of the reactivity of the compounds used for
`subunit synthesis, activation, and/or coupling, it is gen
`and interstrand and intrastrand nonbonded interactions.
`Initial studies of this type carried out in support of the
`erally desirable, and often necessary, to protect the
`invention show that a morpholino-based polymer has a
`exocyclic ring nitrogens of the bases. Selection of these
`preferred unit backbone length (i.e., the number of
`protective groups is determined by (i) the relative reac
`atoms in a repeating backbone chain in the polymer) of
`tivity of the nitrogen to be protected, (ii) the type of
`reactions involved in subunit synthesis and coupling,
`6 atoms. However, the modeling studies also show that
`and (iii) the stability of the completed polymer prior to
`certain 5-atom and 7-atom repeating-unit morpholino
`50
`based backbones meet the requirements for binding to
`base deprotection.
`targeted genetic sequences.
`Methods for base protecting a number of the more
`Since the morpholino structure itself contributes 4
`common ribonucleosides are given in Example 1. The
`methods detailed in the example are generally applica
`atoms to each repeating backbone unit, the linkages in
`ble for forming nucleosides with amine-protective
`the five-atom, six-atom, and seven-atom repeating-unit
`groups. Standard base-protective groups used for nu
`backbone contributes one, two, and three atoms to the
`cleic acid chemistry are often suitable including the
`backbone length, respectively. In all cases, the linkage
`following groups: benzoyl for the N4 of C; benzoyl or
`between the ring structures is (a) uncharged, (b) chiral,
`p-nitrobenzoyl for the N6 of adenine (A); acetyl,
`(c) stable, and (d) must permit adoption of a conforma
`phenylacetyl or isobutyryl for the N2 of guanine (G);
`tion suitable for binding to the target polynucleotide.
`and N2,N6-bis(isobutyryl) for 2,6-diaminopurine resi
`Subunit backbone structures judged acceptable in the
`dues. These protective groups can be removed after
`above modeling studies are then assessed for feasibility
`polymer assembly by treatment with ammonium hy
`of synthesis. The actual chemical stability of the inter
`subunit linkage is often assessed with model compounds
`droxide.
`It is sometimes desirable to protect the base portion of
`or dimers.
`65
`the morpholino subunit with a group which can be
`FIG.3 shows several preferred 6-morpholino subunit
`readily removed by other than a nucleophilic base.
`types, including linkage groups, which meet the con
`Suitable base protective groups removable by a strong
`straints and requirements outlined above. It will be
`
`45
`
`55
`
`

`

`O
`
`5
`
`5,185,444
`8
`7
`coupled to a 5' moiety of a second morpholino subunit
`non-nucleophilic base via a £3-elimination mechanism
`(FIG. 7). Subunits of this type are suitable for construct
`include: 2-(4-nitrophenyl)ethoxy carbonyl or 2-(phenyl
`ing morpholino polymers with 7-atom repeating-unit
`sulfonyl)ethoxycarbonyl for both the N4 of C and the
`N6 of A; and the 9-fluorenyl methoxycarbonyl for the
`backbones.
`An example of the synthesis of a subunit suitable for
`N2 of G and the N2 and N6 of 2,6-diaminopurine. These
`7-atom unit-length backbones is detailed in Example 5
`groups can be removed after polymer assembly by
`(with reference to FIG. 7).
`treatment with the strong nonnucleophilic base 1,8-
`Example 7 describes, with reference to Structure E of
`diazabicyclo5.4.0]undec-7-ene (DBU), under strin
`FIG. 3, the preparation of non-morpholino subunits
`gently anhydrous conditions.
`which are converted into morpholino structures during
`The syntheses of representative morpholino subunits
`polymer assembly.
`are described particularly in Examples 2-7. With refer.
`ence to the synthesis scheme depicted in FIG. 5, a base
`C. Activation and Coupling Reactions
`protected ribonucleoside is reacted with sodium perio
`The subunits prepared as above are coupled, in a
`date to form a transient 2", 3'-dialdehyde which then
`controlled, sequential manner, often by activating the
`closes upon ammonia to form a morpholino-ring having
`5'hydroxyl of one subunit (having a protected morpho
`2" and 3'hydroxyl groups (numbered as in the parent
`lino nitrogen) and contacting this activated subunit with
`ribose, see FIG. 1). The compound is then treated with
`another subunit having an unprotected morpholino
`sodium cyanoborohydride to reduce the ring hydroxyl
`nitrogen as described in Example 9. It will be recog
`groups. The ring nitrogen is preferably protected by
`nized that different types of linkages, such as those
`trityl derivatization or by a benzhydraloxycarbonyl
`20
`illustrated below, may be employed in the construction
`group for subsequent subunit coupling. The protective
`of a single polymer.
`group can be added by reacting the morpholino subunit
`The simplest morpholino-type binding polymers are
`with trityl chloride or with nitrophenyl benzhydrl car
`carbamate-linked polymers where the morpholino ni
`bonate or by reacting the dialdehyde with a primary
`trogen is linked through a carbonyl to the 5' oxygen of
`amine, as illustrated in FIG. 6 and described in Exam
`25
`another subunit. Experiments conducted in support of
`ples 3 and 5. The stereochemistry of the nucleoside
`the present invention demonstrate that such a polymer
`starting material is retained as long as the pH of the
`effectively binds to a single-stranded DNA target se
`reaction mixture at the iminium stage is not allowed to
`quence. However, in binding studies with an RNA
`go above about 10.
`target, the polymer exhibited unusual binding, as evi
`The above synthesis results in a morpholino-ring with
`30
`denced by a highly atypical hypochromicity profile in
`an available 5'-hydroxyl. The 5'-hydroxyl can be con
`the 320 to 230 nm spectral range and lack of a normal
`verted to other active groups including 5' amine and
`sulfhydral (Example 6) or 5'phosphonate (Example 4).
`thermal denaturation.
`Early modeling studies indicated that in a carbamate
`In the above morpholino synthesis a variety of nitro
`linked polymer bound to DNA existing in a B confor
`gen sources can be used-including particularly ammo
`35
`mation, the backbone of the polymer provides adequate
`nia, ammonium hydroxide, ammonium carbonate, and
`length for binding and the carbamate moieties of the
`ammonium bicarbonate. Best results are obtained when
`polymer backbone can assume a nearly planar confor
`the reaction solution is maintained near neutrality dur
`mation. This modeling result was in good accord with
`ing the oxidation and morpholino ring closure reac
`the effective binding of the carbamate-linked polmers to
`tions. This can be accomplished by continually titrating
`DNA. In contrast, similar modeling studies suggested
`the reaction mix or, more conveniently, by using ammo
`that binding of the carbamate-linked polymer to an
`nium biborate as the ammonia source. When the solu
`RNA target requires one of the following: (i) the carba
`tion is too acidic the yield of product is low and when
`mate linkage of the polymer adopt a substantially non
`it is too basic, side products (possibly due to epimeriza
`planar conformation, or (ii) the RNA target sequence
`tion of the 1" and/or 4' carbons) are produced which are
`45
`adopt a strained conformation in which base-stacking
`difficult to separate from the desired product. It is also
`interactions are quite different from that in a normal A
`noted that the reducing agent can be added before,
`conformation. This observation may explain the atypi
`during, or after the oxidation step with little noticeable
`cal binding of a carbamate-linked polymer to an RNA
`effect on product yield.
`target sequence.
`Ribonucleosides lacking base protection groups can
`The modeling work further indicated that replacing
`also be successfully oxidized, ring closed, and reduced
`the carbonyl intersubunit linking moiety with either an
`in aqueous solution to generate the morpholino ring.
`achiral sulfonyl-containing intersubunit linkage or with
`However, without base protection the number and
`a chiral phosphorous-containing linkage would provide
`quantity of undesired side products frequently in
`added length of about 0.32 angstrom per intersubunit
`creases, particularly in the case of cytidine.
`55
`linkage. Such linkages would also provide greater rota
`The subunits formed by the above methods contain a
`tional freedom about key bonds, and bond angles of the
`5'-OH, SH, or amine which is modified, reacted with,
`intersubunit linkage compatible with an oligomer back
`and/or activated, as described below, to be suitable for
`bone conformation suitable for pairing to both RNA
`coupling to a second morpholino subunit. For example,
`and DNA target sequences in their standard conforma
`FIG. 5 shows the conversion of a 5'-OH of a morpho
`lino subunit to a phosphonyl linking moiety to form a
`tions.
`The linkage in structure A-A in FIG. 4 (five-atom
`subunit (Structure 10) which is linked to form a 5-atom
`backbone) can be formed according to the reaction
`unit-length backbone polymer. Details of the subunit
`scheme shown in FIG. 9, and detailed in Example 8.
`synthesis are given in Example 4; modification to the
`Briefly, the 5'-OH of a morpholino subunit is converted
`thiophosphonyl linking moiety is also described.
`to a phosphorous-containing moiety as desc