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LIPOPHILIC OLIGONUCLEOTIDE ANALOGS
Field of the Invention
The invention is directed to oligonucleotide analogs, in particular to
lipophilic oligonucleotide analogs that efficiently enter cell cytoplasm or cross
membranes without the aid of either transfection compounds or other agents
or techniques.
Background of the Invention
The invention relates to oligonucleotide analogs which are capable of
passive permeation of cell membranes and synthetic intermediates therefor as
well as their use in cell staining, diagnostic and therapeutic applications.
Workers have described oligonucleotide analogs, complexes of
oligonucleotide analogs and nucleoside analogs having lipophilic or related
modifications for enhancing their delivery into cells, increasing nuclease
stability or other purposes (see e.g., WO 96/15778; WO 96/07392; WO 96/05298;
WO 96/04788; WO 90/10448; WO 89/12060; EP 462 145 B1; EP 092 574 B1; U.S.
Patent Nos. 5,420,330, 4,958,013, 4,904,582; Agrawal et al., Proc. Natl. Acad. Sci.
(U.S.A.) 85: 7079-7083 1988; Dagle et al., Nucl. Acids Res. 19:1805-1810 1991;
Matteucci et al., Nucl. Acids Res. 16:4831 4839 1988; Huang et al., J. Org. Chem.
56:3869-3882 1991; Huff et al., l. Biol. Chem. 262:12843-12850 1987; Inoue et al.,
Nucl. Acids Res. 15:6131-6148 1987; Itoh et al., Heterocycles 17:305-309 1982; Itoh
et al., Nucleosides ~ Nucleotides 1:179-190 1982; Kabanov et al., FEBS 259:327-
330 1990; Manoharan et al., Tet. Lett. ;~:7171-7174 1991; Montgomery et al., J.
Het. Chem. 14:195-197 1977; Montgomery et al., J. Med. Chem. 10:165-167 1967;
Mizuno et al., J. Org. Chem. 28:3329-3331 1963; Praseuth et al., Proc. Natl. Acad.
Sci. (U.S.A.) ~:1349-1353 1988; Schneider et al., Tet. Lett. 31:335-338 1990;
Severin et al., Adv. Enz. Regul. 31:417-430 1991; Shoji et al., Nucl. Acids Res.19:5543-5550 1991; Singer et al., Biochem. 28:1478-1483 1989; Sproat et al., Nucl.
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Acids Res. 17:3373-3386 1989; Tanaka et al., Tet. Lett. 19:4755 4758 1979;
Uhlmann et al., Chem. Revs. 90:543-585 1990; Wright et al., J. Med. C1~em.
~Q:109-116 1987.
Workers have described permeation of molecules, including
5 oligonucleotides, across lipid membranes or into cells (see e.g., Hansch et al., J.
Pharm. Sci. 61:1-19 1972; Lieb et al., N~ture ~:240-243 1969; Loke et al., Proc.Natl. Acad. Sci. (U.S.A.) 86:3474-3478 1989; W.D. Stein New Comprehensive
Biochemistry, vol 2, Chapter 1: Permeabili~y for Lipophilic Molecules,
Membrane Transport Elsevier/North-Holland Biomedical Press, Amsterdam,
10S.L. Bonting et al. editors, 1981, pages 1-28; Walter et al., J. Membrane Biol.
~Q:207-217 1986; Yakubov et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:6454-6458
1989).
Objects of the Invention
The invention compositions or methods inc}ude one or more
compounds or methods that accomplish one or more of the following objects.
It is an object of the invention to provide lipophilic oligonucleotide
analogs and intermediates for making them.
Another object of the invention is to provide lipophilic oligonucleotide
20 analogs that are suitable for permeation into cell cytoplasm or cell nuclei in
vitro or in vivo in the presence or absence of serum or blood.
Another object is to provide lipophilic oligonucleotide analogs that are
suitable for staining one or more subcellular organelles or compartments.
Another object of the invention is to provide lipophilic oligonucleotide
25 analogs that are suitable for tagging or marking items or compounds.
Another object is to provide methods to deliver lipophilic
oligonucleotide analogs into cells in vitro or in vivo.
Another object is to provide compositions comprising lipophilic
oligonucleotide analogs bonded to a detectable label.
Summary of the Invention
The invention is directed to oligonucleotides capable of passive
diffusion across mammalian cell or organelle membranes or any other cell
35 membrane (plant, parasite, bacterial, yeast, viral, or fungal).
Invention oligonucleotide analogs are characterized by oligonucleotides
comprising internucleotide linkages, bases and sugars wherein the
oligonucleotide has a Log value of the octanol:water partition coefficient of
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-0.3 to +2.5 and a solubility in water of at least 0.001 ,ug/mL, and the and salts,
solvates and hydrates thereof.
Invention embodiments include oligonucleotide analogs having
structure (1)
R--R5 B
~R4~ (1)
_\ _
R~- R5 B
~,R4~
~\J
I
wherein
R is OH, blocked OH, N(R14)2, P(o)(Rl5)2~ or a linker;
R1 is an oligonucleotide, a blocking group, OH, N(R14)2, P(o)(Rl5)2~ a
solid support, or a linker bonded to the 2' or 3' position of a furanose ring (or
its carbocyclic analog), and the remaining 2' or 3' position is substituted withR3;
each R2 independently is an internucleotide linkage bonded to the 2' or
3' position, and the remaining 2' or 3' position is substituted with R3;
each R3 independently is H, OH, F, blocked hydroxyl, N(Rl4)2, -O-alkyl
(C1 8), -O-alkyl (C1 8) where the alkyl group is substituted with halogen,
hydroxyl or oxygen, -O-alkenyl (C3 8), -S-alkyl (Cl 8) or a linker;
each R4 independently is O or CH2;
each R5 independently is CH2, NR6, O, S, SO, SO2;
each R6 independently is H, alkyl (Cl 6) or alkyl (Cl 6) where the alkyl
group is substituted with halogen, hydroxyl or oxygen;
each R14 independently is hydrogen, a protecting group, hydrocarbyl, or
pseudohydrocarbyl;
each Rl5 independently is hydroxyl (OH), blocked hydroxyl, SH, blocked
~ SH, or N(Rl4)2;
n is an integer from 0 to 48; and
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each B independently is a base, wherein the total number of bonded
monomers designated by the variable n plus any oligonucleotide at Rl is 2-50.
The invention oligonucleotide analogs are useful to visualize or stain
cells and are thus useful in a method comprising: contacting cells to be
visualized with the oligonucleotide under conditions wherein diffusion
across the cell membrane can occur so as to internalize said oligonucleotide
within the cells; removing from the cells any oligonucleotide which has not
diffused across the membrane and become internalized; and detecting the
oligonucleotide which has been internalized in the cells to visualize the cells.The invention oligonucleotide analogs are useful as agents to deliver
oligonucleotides into cells and are thus useful in a method comprising:
contacting a cell with an invention oligonucleotide.
Brief Description of the Dr~wing~
Figure 1 shows a standard curve for the determination of partition
coefficient based on retention time in RPLC.
Figure 2 shows the chemical structures of oligonucleotide analogs used
to visualize cells.
Detailed Descri~tion of the Invention
Definitions. Halogen means F (fluorine), Cl (chlorine), Br (bromine) or
I (iodine).
Alkyl means linear, branched or cyclic saturated hydrocarbons.
Alkenyl means linear, branched or cyclic unsaturated hydrocarbons
where one or more double bonds are present.
Alkynyl means linear, branched or cyclic unsaturated hydrocarbons
where one or more triple bonds are present.
As used herein, hydrocarbyl groups contain only carbon and hydrogen
and includes alkyl, alkenyl or alkynyl groups. Hydrocarbyl groups typically
contain 1, 2 ,3 ,4, 5, 6, 7 or 8 carbon atoms, but includes groups having more
than 8 carbon atoms, such as groups containing 9,10,11, 12, 13,14, 15, 16, 17 or18 carbon atoms.
As used herein, psuedohydrocarbyl groups are hydrocarbyl substituents
which contain one or more heteroatoms (including those present as
substituents) representing less than 50% of the total non-hydrogen atoms in
the pseudohydrocarbyl substituent. Typically, pseudohydrocarbyl groups
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bonded to invention oligonucleotide analogs, including structure (1)
oligonucleotides, contain 1-18 carbon atoms, usually 3-12. Pseudohydrocarbyl
moieties optionally contain 1, 2, 3 or 4 heteroatoms. Heteroatoms usually
found in pseudohydrocarbyl groups are O, N, S or halogen. The heteroatoms
may be present as an ether, ketone, hydroxyl, thiol (SH), protected thiol,
primary amine, secondary amine, tertiary amine, protected primary amine,
amide, thioether (-S-), carboxyl, protected carboxyl, nitro (NO2), azido (N3),
ester (-C(O)-ORX where Rx is hydrocarbyl or pseudohydrocarbyl), carbonate (-
O-C(O)-ORX) or carbamate (-o~(o)-NR14RX). When an amine or carboxyl is
present, the group will potentially carry a partial or full charge at physiological
pH, and sufficient carbon atoms, generally about 12-18, will generally need to
be present in the pseudohydrocarbyl group to offset the hydrophilic character
of the charge. Generally, pseudohydrocarbyl substituents that facilitate passivediffusion decrease the polarity or increase the lipophilicity of the parent
oligonucleotide and do not carry any charged atoms or groups, unless more
than about 14 carbon atoms are present.
As used herein "base" means protected and unprotected purine,
pyrimidine heterocycles found in nucleic acids or their modified forms.
Modifications include alkylated purines or pyrimidines, acylated purines or
pyrimidines, or other heterocycles previously described (see, e.g., PCT
US94/10539). Bases suitable for use herein include alkylated purines or
pyrimidines, acyl~ted purines or pyrimidines, or other analogs of purine or
pyrimidine bases and their aza and deaza analogs. Exemplary bases include
N4,N4-ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N6-
methyladenine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl~uracil, 5-
fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-
carboxymethylaminomethyl uracil, inosine, N6-isopentenyl-adenine, 1-
methyladenine, 2-methylguanine, 5-methylcytosine, N6-methyladenine, 7-
methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2-
thiouracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-
(1-propynyl)-4-thiouracil, 5-(1-propynyl)-2-thiouracil, 5-(1-propynyl)-2-
thiocytosine, 5-(1-butynyl)-4-thiouracil, 5-(1-butynyl)-2-thiouracil, 5-(1-
butynyl)-2-thiocytosine, 2-thiothymidine, and 2,6-diaminopurine. In addition
. to these base analogs, one can incorporate pyrimidine analogs including 6-
azacytosine, 6-azathymidine and 5-trifluoromethyluracil described in WO
92/02258 into the invention oligonucleotides. Typically bases are adenine,
guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(1-propynyl)uracil, 5-(1-
propynyl)cytosine, 8-oxo-N6-methyladenine, 7-deaza-7-methylguanine, 7-
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deaza-7-methyladenine, 7-deazaxanthosine, 7-deaza-7-(1-propynyl)adenine, 7-
deaza-7-(1-propynyl)guanine, 7-deaza-7-(1-butynyl)adenine, 7-deaza-7-(1-
butynyl)guanine, 5-(1-butynyl)uracil, 5-(1-butynyl)cytosine.
"Nucleoside," "nucleotide" and "monomer" include those moieties
5 which contain both the common purine and pyrimidine bases adenine,
guanine, cytosine, thymine and uracil, and modified bases or analogs thereof,
particularly either lipophilic analogs or analogs that enhance binding affinity
for complementary nucleic acid sequences. Monomers, nucleosides or
nucleotides are bonded together to form the invention oligonucleotide
10 analogs. The terms "nucleoside," "nucleotide" and "monomer" are generic to
ribonucleosides or ribonucleotides, deoxyribonucleosides or
deoxyribonucleotides, or to any other nucleoside which is an N-glycoside or C-
glycoside of a purine or pyrimidine base, or modified purine or pyrimidine
base. Thus, the stereochemistry of the sugar carbons may be other than that of
15 D-ribose in one or more residues. Also included are oligonucleotide-like
compounds or analogs where the ribose or deoxyribose moiety is replaced by
an alternate structure such as the 6-membered morpholino ring described in
U.S. patent number 5,034,506 or where an acyclic structure serves as a scaffold
that positions the base or base analogs in a manner that permits efficient
20 binding to target nucleic acid sequences or other targets. Oligonucleotide-like
compounds with acyclic structures in place of the sugar residue and/or the
linkage moiety are specifically intended to include both (i) structures that
serve as a scaffold that positions bases or base analogs in a manner that
permits efficient sequence-specific binding to target nucleic acid base sequences
25 and (ii) structures that do not permit efficient binding or hybridization with
complementary base sequences. Elements ordinarily found in
oligonucleotides, such as the furanose ring or the phosphodiester linkage may
be replaced with any suitable functionally equivalent element.
Linkage and internucleotide linkage mean an uninterrupted chain of
30 atoms that bond adjacent monomers or nucleotides together. Linkage
includes unmodified phosphodiester linkages, -O-P(O)(OH)-O-, and modified
or substitute linkages. Substitute linkage means a linkage other than a
phosphodiester linkage that links the sugar or sugar analog of adjacent
monomers or nucleotides. Substitute linkages may be used at R2-R5 in
35 oligonucleotides of structure (1). Many substitute linkages are non-ionic andcontribute to the desired ability of the oligomer to diffuse across membranes.
These "substitute" linkages are defined herein as conventional alternative
linkages such as phosphorothioate or phosphoramidate, are synthesized as
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described in the generally available literature. Substitute linkages groups thusinclude, but are not limited to linkages comprising a moiety of the formula -O-
P(O)S-O-, ("thioate"), -O-P(S)S-O- ("dithioate"), -O-P(O)N(R6)2-O-, -o-P(O)R6-O-, -O-P(o)oR7-o-~ -O-CO-O-, or -O-CON(R6)2-O- wherein each R6 independently
is H (or a salt) or alkyl (C1 8) and R7 is alkyl (C1 8). Also included are
alkylphosphonate linkages such as methyl-, ethyl- or propylphosphonates.
Substitute linkages that may be used in the oligonucleotides disclosed herein
also include nonphosphorous-based internucleotide linkages such as the 3'-
thioformacetal (-S-CH2-O-), 5'-thioformacetal (-O-CH2-S-), formacetal (-O-CH2-
10 O-), 5' amine (-CH2-CH2-NR13- where Rl3 is hydrogen, a protecting group or
alkyl C1 6, see, e.g., PCT US91/06855), and 3'-amine (-NR13-CH2-CH2-)
internucleotide linkages.
Substitute linkages suitable for R2 or R2-R5 and for other invention
oligonucleotides have been described, e.g., phosphorodithioates (Marshal,
15 Science ~2:1564, 1993), phosphorothioates and alkylphosphonates (Kibler-
Herzog, Nucleic Acids Research 19:2979, 1991; PCT 92/01020; EP 288,163),
phosphoroamidates (Froehler, Nucleic Acids Research 16:4831, 1988),
phosphotriesters (Marcus-Sekura, Nucleic Acids Resenrch 15:5749, 1987),
boranophosphates (Sood, 1. Am. Chem. Soc. 112:9000, 1991), 3'-0-5'-S-
20 phosphorothioates (Mag, Nucleic Acids Research 19:1437, 1991), 3'-S-5'-O-
phosphorothioates (Kyle, BiochemistnJ 31 :3012, 1992), 3'-CH2-5'-O-
phosphonates (Heinemann, Nucleic Acids Resenrch 19:427, 1991), 3'-NH-5'-O-
phosphonates (Mag, Tct. Lett. 33:7323, 1992), sulfonates and sulfonamides
(Reynolds, J. Org. Chem. 57:2983, 1992), sulfones (Huie, J. Org. Chem. 57:4519,
25 1992), sulfoxides (Huang, ~. Or~. Chcm. ~6:3869, 1991), sulfides (Schneider, Tet
Lett. 30:335, 1989), sulfamates, ketals and formacetals (Matteucci, J. Am. Chem.Soc. 113:7767, 1991, PCT 92/03385 and PCT 90/06110), 3'-thioformacetals (Jones,
J. Org. Chem. 58:2983, 1993), 5'-S-thioethers (Kawai, Nucleosides Nucleo~ides
10:1485, 1991), carbonates (Gait, J. C~lem. Soc. Perkin Trans 1 1389, 1979),
30 carbamates (Stirchak, J. Org. Chem. 52:4202, 1987), hydroxylamines (Vasseur, J.
Am. Chem. Soc. 114:4006, 1992), methylamine (methylimines) and
methyleneoxy (methylimino) (Debart, Bioorg. Med. Chem. Let~. 2:1479, 1992)
and amino (PCT 91/06855), hydrazino and siloxane (U.S. Patent 5,214,134)
linkages.
Substitute linkages also are known for the replacement of the entire
phosphoribosyl group of conventional oligonucleotides. These include for
example morpholino-carbamates (Stirchak, Nucleic Acids Research 17:6129,
1989) and riboacetal linkages (PCT 92/10793).
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Additional substitute linkages suitable for use in the invention
oligonucleotide analogs are disclosed in PCT 91/08213, 90/15065, 91/15500,
92/20702, 92/20822, 92/20823, 92/04294, 89/12060 and 91/03680; Mertes, J. Med.
Chem. 12:154, 1969; Mungall, J. Org. Chem. 42:703, 1977; Wang, Tet Lett
~:7385, 1991; Stirchak, Nucleic Acids Research 17:612g, 1989; Hewitt,
Nucleosides and Nucleotides 11:1661,1992; and U.S. Patents 5,034,506 and
5,142,047.
The phosphodiester or substitute linkages herein are used to bond the 2'
or 3' carbon atoms of ribose or ribose analogs to the 5' carbon atoms of the
10 adjacent ribose or ribose analog. Ordinarily, the linkages in oligonucleotides
are used to bond the 3' atom of the 5' terminal oligonucleotide to the 5' carbonatom of the next 3'-adjacent nucleotide or its analog.
As used herein, "sugar" includes furanose moieties usually found in
nucleic acids and their isomers, e.g., arabinose, as well as other sugars, hexoses
15 such as glucose. Sugar also includes carbocyclic analogs of these sugars. Sugars
optionally comprise modification of the 2' or 3' position by a O-hydrocarbyl,
NH-hydrocarbyl, S-hydrocarbyl group, O-pseudohydrocarbyl, NR14-
pseudohydrocarbyl, or S-pseudohydrocarbyl group, including 2'- or 3'-O-
methyl, O-ethyl, O-propyl, O-isopropyl, O-butyl, O-isobutyl, O-propenyl or O-
20 allyl, which are used due to their increased lipophilicity compared to the 2'-
hydrogen or 2'-hydroxyl found in unmodified DNA or RNA. Corresponding
S-alkyl or NH-alkyl substituents may also be utilized. Modifications such as
2'-O-alkyl C1-4, 2'-O-haloalkyl Cl-4 and 2'-fluoro are generally suitable for
binding competent oligonucleotides and may thus be used to prepare the
25 oligonucleotides. One may modify the 2' or 3' position using
pseudohydrocarbyl groups. Such groups will typically contain about 1-6 carbon
atoms and include substituents such as 2'- or 3'-O-(CH2)l 3-O-(CH2)1 3-Rll
where R11 is a halogen, hydrogen, hydroxyl or NHRl2 and R12 is hydrogen or a
protecting group, and substituents such as 2'- or 3'-O-(CH2)1-2-O-(CH2)l-2-O
30 (CH2)1 2-R1l. Workers have described such modifications (see, e.g., U. S.
patent numbers 5,466,786 and 5,399,676; PCT US91/03680; Cotten, M., et al.,
Nucleic Acids Res 19:2629 26351990; Blencowe, B.J., et al., Cell 59:531-539 1989;
Sproat, B.S., et al., Nucleic Acids Res 17:3373 3386 1989; Inoue, H., et al.,
Nucleic Acids Res 15:6131-6148 1987; Morisawa, H., et al., European Patent
35 Publication No. 0339842; Chavis, C., et al., J Organic Chem 47:202-206 1982;
Sproat, B.S., et al., Nucleic Acids Res 19:733 738 1991). The 2'-modified
oligomers were reported to be relatively nuclease stable compared to
unmodified controls (Guinosso, C.~., et al., Nucleosides and Nucleotides
.
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10:259-262 1991). Synthesis of 2'-fluoro nucleosides and their incorporation
into oligonucleotides has also been described (Codington, J.F., et al., J Org
Chem 22:558-564 1964; Fazakerley, G.V., et al., FEBS Lett 182:365-369 1985).
Synthesis of oligonucleotide analogs containing the modified bases described
5 herein would be based on methods described. Synthesis of 2'-thioalkyl
nucleosides is accomplished as described in U.S. Patent No. 5,484,908.
As used herein "oligonucleotide" or "oligomer" is generic to
polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms
thereof such as arabinose or carbocyclic analogs of ribose), i.e., DNA, to
10 polyribonucleotides (containing D-ribose or modified forms thereof), i.e.,
RNA, and to any other type of polynucleotide which is an N-glycoside or C-
glycoside of a purine or pyrimidine base, or modified purine or pyrimidine
base. Oligonucleotide or oligomer, as used herein, is intended to include (i)
compounds that have one or more furanose moieties that are replaced by
15 furanose derivatives or by ~ny structure, usually cyclic, that may be used as a
point of covalent attachment for the base moiety, (ii) compounds that have
one or more phosphodiester linkages that are either modified, as in the case of
phosphoramidate or thioate linkages, or where the phosphorus atom is
completely replaced by a suit~ble linking moiety as in the case of, e.g.,
20 formacetal linkages, and/or (iii) compounds that have one or more bonded
furanose-phosphodiester linkage moieties replaced by any structure, cyclic or
acyclic, that may be used as a point of covalent attachment for the base.
Invention oligonucleotides are of any convenient length and generally
comprise 2-50 bonded monomers, often 3-20, usually 4-15. Oligonucleotide
25 also includes short molecules such as dimers, trimers and tetramers having a
Log value of the octanol:water partition coefficient of -0.3 to +2.5 and a
solubility in water of at least 0.001 ~g/mL, which are useful as synthetic
intermediates. A standard oligonucleotide dimer with two linkage groups has
a molecular weight of about 650 Daltons. The corresponding invention
30 oligonucleotides optionally have molecular weights of 1,500 or more, e.g.,
1,500-6,000, or 6,000-10,000.
As used herein, the terms "lipophilic oligonucleotide," "lipophilic
linkage," lipophilic base," lipophilic sugar," "lipophilic modification," and
"lipophilic substitution" mean an oligonucleotide, or a linkage, base, sugar,
35 modification or substitution, respectively, that makes the modified or
substituted molecule more lipophilic than the corresponding unmodified
molecule at pH 7.4 in water or low ionic strength buffer. An unmodified
oligonucleotide means one that is composed of ribose, or 2'-deoxyribose,
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phosphodiester linkages and the bases guanine, adenine, cytosine, thymine
and/or uracil, i.e., DNA or mRNA.
In general, each modified linkage, base or sugar optionally comprises a
single lipophilic modification, although more than one may be present,
particularly at a linkage or base. A linkage such as -O-CH2~- has a single
lipophilic modification, i.e., -CH2- replaces -P(O)(OH)- in the O-P(O)(OH)-O-
linkage. A linkage such as -CH2-CH2-CH2- would have 3 lipophilic
modifications compared to the normal phosphodiester linkage in DNA or
RNA. However, as used herein, a -CH2-CH2-CH2- linkage is considered to be a
10 single modification for the purpose of determining the ~/O of modifications in
an oligonucleotide. Thus, an oligonucleotide having 6 bonded monomers
with only -CH2-CH2-CH2- linkages would have 100% of the linkages modified
and not 300%. An invention oligonucleotide, such as a structure (1)
oligonucleotide, containing modifications at each linkage, base and sugar
15 would have modifications that sum to 300'~" i.e., 100'l/., of linkages plus 100'l/.,
of sugars plus 100~/o of bases.
Oli~onucleotide uptake into cells. We have discovered lipophilic
oligonucleotide analogs that enter cells by passive diffusion across cell,
20 endosome and/or organelle membranes. The oligonucleotide analogs may
enter cells by multiple mechanisms, e.g., pinocytosis, receptor-mediated
uptake, phagocytosis as well as by passive diffusion. It has been generally
assumed that oligomers containing the native phosphodiester linkages enter
cells by receptor-mediated endocytosis (Loke, S.L., et al., Proc. NQtl. Ac~d. Sci.
25 (U.S.A.) 86:3474-3478 1989; Yakubov, L.A., et al., Proc. Nntl. Acad. Sci. (U.S.A.)
86:6454-6458 1989). Subsequent studies appear to show that oligomers with
modified internucleotide linkages that may mitigate the presence of negative
charges also enter the cells through specific receptors, rather than by passive
diffusion (Akhtar, S., et al., Nucl. Acids Res. 19:5551 5559 1991; Shoji, Y., et al.,
30 Nucl. Acids Res. 19:5543 5550 1991). Entry of oligomers into cells by either
receptor mediated endocytosis or by other mechanisms results in their
localization into intracellular endosomes or vesicles. Thus, entry of oligomers
into cellular cytoplasm or nucleoplasm is prevented by the membrane barrier
surrounding these subcellular organelles. Because of the low rate of such
35 endocytosis, it has been necessary to attempt to protect the oligonucleotidesfrom degradation in the bloodstream either by inclusion of these materials in
protective transport complexes, for example with LDL or HDL (deSmidt, P., et
al., Nucl. Acids Res. 19:4695 4700 1991) or by capping them with nuclease-
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resistant internucleotide linkages (Hoke, G.D., et al., Nucl. Acids Res. 19:5743-
5748 l9gl).
No clearly documented progress has been reported in designing
oligonucleotides which are capable of passive cell membrane diffusion and
5 enter cells rapidly across cellular membranes to interact with intracellular
targets. Those factors related to molecular characteristics which determine the
diffusion coefficients of molecules in general have, however, been extensively
studied. See, for example, Stein, W.D., in "New Comprehensive
Biochemistry", Vol. 2 (Membrane Transport), Elsevier/North Holland
Biomedical Press (1981), pp. 1-28; Eieb, W.R., et al., Nnture ~2~:240-243 1969. It
has been concluded that the distribution constant for a particular substance
between the lipophilic membrane and an external aqueous phase is a direct
function of the partition coefficient of the material between octanol and water
times the molecular weight of the material of interest raised to an appropriate
negative power characteristic of the membrane. As the appropriate negative
power for, for example, red blood cells is about -4, it appears that high
molecular weight substances must have hopelessly low distribution
coefficients between cellular membrane and the external environment, even
if their partition coefficients for octanol:water are quite high. The validity of
this relationship for various small molecules, however, appears to be
substantiated by experiment (Hansch, C., et al., J. Phnrm. Sci. 61:1-19 1972;
Walter, A., et al., J. MembrQne Biol. 90:207-217 1986).
The partition coefficient for native DNA or RNA is relatively low with
a log value of the octanol:water partition coefficient being less than -4 (Dagle,
J.M., et al., Nucl. Acids Res. 19:1805-1810 1991). DNA modified by synthesis of
2-methoxyethylphosphoramidite internucleoside linkages in place of the
phosphodiester linkage eliminates the negative charge associated with the
internucleotide linkage, which increases the hydrophobicity of DNA.
However, the log value of the octanol:water partition coefficient (Log Poct)
remains less than -2 (Dagle, supra). Increased Log Poct values for 2-
methoxyethylphosphoramidite-modified DNA were assayed by measuring the
partitioning of radiolabeled DNA in an octanol-aqueous buffer system.
Increased Log Poct was correlated with increased retention time on reversed-
phase HPLC columns (Dagle, supra). Other DNA analogs, such as
methylphosphonates or phosphorothioates, or DNA with lipophilic adducts
(Severin, E.S., et al., Adv. Enz~me l~egulation 31:417-430 1991) that are
described in the literature are similarly expected to have Log Poct values less
than 0Ø
11
.. .... , ~.. ,~ . .. . ..
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WO 98/04575 - PCI/US96/12530
We have found that oligonucleotides can be modified by appropriate
design of their molecular features so as to permit their passive diffusion across
cellular membranes, despite the high molecular weights inherent in these
molecules. Because of the high molecular weights of the invention
oligonucleotide analogs, the relevant factor generated in determining
distribution between membrane and aqueous medium is very small, which
indicates that such a molecule is essentially impermeable to cell membranes.
Despite this, the dimers and higher molecular weight oligonucleotides of this
invention are, however, capable of passive diffusion into cells, and are thus
10 not sequestered exclusively in endosomes in the same manner as described for
previously known oligonucleotide analogs (Fisher et al., Nuel. Acids Res.
~:3857-3865 1993).
An aspect of the invention oligonucleotide analogs is our finding that
when one introduces lipophilic modifications into multiple sites on the
15 oligonucleotide, the oligonucleotide usualiy will passively diffuse into cells.
We believe this may be due, at least in part, to the use of multiple, relativelysmall lipophilic modifications, such as hydrocarbyl or pseudohydrocarbyl
moieties containing about 1-8 carbon atoms, at several locations on the
oligonucleotide, rather than one or several large lipophilic moieties such as
20 cholesteryl groups (see, e.g., Kabanov et al., FEBS ~:327-330 1990, Severin et
al., Adv. Enz. Regul. 31:417-430 1991, EP 0 462 145 B1, U. S. Patent Nos. 4,958,013
and 5,420,330). The modifications of this invention that render an
oligonucleotide analog permeation competent and soluble in aqueous media,
as hereinbelow defined, are thus located at more than one location on the
25 oligonucleotide which alters its overall lipophilicity character, making the
molecule capable of efficient partitioning across membranes.
Oligonucleotide synthesis. Oligonucleotides and the nucleotide
synthons therefor are conventionally synthesized. Methods for such synthesis
30 are found, for example, in Froehler, B., et al., Nucleic Acids Res. 14:5399 5467
1986; Nucleic Acids Res. 16:4831-4839 1988; Nucleosides and Nucleotides
6:287-291 1987; Froehler, B., Tetrahedron Letters 27:5575-5578 1986. Amine,
carboxyl and hydroxyl groups present anywhere on the molecule may be
protected during oligonucleotide synthesis using standard protecting groups.
35 Other conventional methods may be used to synthesize the oligomers or
segments thereof, including methods employing phosphoramidite chemistry
and/or methods that utilize solution phase synthesis. For oligonucleotide
synthesis, R or R1 optionally are oligonucleotide coupling groups. "Coupling
12
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group" as used herein means any group suitable for generating a linkage or
phosphodiester substitute linkage between nucleotide bases or their analogs.
These coupling groups are conventional and well-known for the preparation
of oligonucleotides, and are prepared and used in the same fashion here. In
general, each compound of structure (1) will contain two blocking groups: R or
R1, but with only one of them being a coupling group. The coupling groups
are used as intermediates in the preparation of 3'-5' 5'-3', 5'-2' and 2'-5'
internucleotide linkages in accord with known methods.
As used herein, the "blocking group" of R or Rl refers to a substituent
10 other than OH that is conventionally coupled to oligomers or nucleosides,
either as a protecting group, an activated group for synthesis or other
conventional conjugate partner such as a solid support, label, immunological
carrier and the like. Suitable protecting groups are, for example, hydroxyl
protecting groups such as DMT, MMT or FMOC; suitable activated groups are,
15 for example, H-phosphon~te, methyl phosphonate, methylphosphoramidite
or ~-cyanoethylphosphoramidite. R or Rl may also comprise a solid support.
In general, the nucleosides and oligomers of the invention may be derivatized
to such "blocking groups" ~s indicated in the relev~nt formulas.
Suitable coupling gro~lps for phosphodiester link~ges include OH, H-
20 phosphonate; (for amidite chemistries) alkylphosphonamidites or
phosphoramidites such as beta-cyanoethylphosphoramidite, N, N-
diisopropylamino-beta-cyanoethoxyphosphine, N,N-diisopropylamino-
methoxyphosphine, N,N-diethylamino-methoxyphosphine, N,N-
diethylamino-beta-cyanoethoxyphosphine, N-morpholino-beta-
25 cyanoethoxyphosphine, N-morpholino methoxyphosphine, bis-morpholino-
phosphine, N,N-dimethylamino-beta-cyanoethylmercapto-phosphine, N,N-
dimethylamino-2,4-dichlorobenzylmercapto-phosphine, and bis(N,N-
diisopropylamino)-phosphine; and (for triester chemistries) 2-, or 4-
chlorophenyl phosphate, 2,4-dichlorophenyl phosphate, or 2,4-dibromophenyl
30 phosphate. See for example U.S. patents 4,725,677; 4,41S,732; 4,458,066; and
4,959,463; and PCT 92/07864. If R1 is a coupling group then R typically will be
hydroxyl blocked with a group suitable for ensuring that the monomer is
added to the oligomer rather than dimerizing. Such groups are well known
and include DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PAC
35 (phenoxyacetyl), a silyl ether such as TBDMS (t-butyldiphenylsilyl) and TMS
(trimethylsilyl). Obviously, the opposite will apply when one desires to
synthesize an oligomer in the opposite direction (5'~3'). Ordinarily, R is
13
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WO 98/04575 PCT/US96/12S30
DMT, R1 is located on the 3' carbon, the remaining R1 is H and the R1 groups
are in the alpha anomer conformation.
In addition to solid phase synthesis techniques, oligonucleotides may
also be partially or fully synthesized using solution phase methods such as
5 triester synthesis. These methods are workable, but in general, less efficient for oligonucleotides of any substantial length.
T inkage synthesis. Table A below sets forth various examples of
suitable substitute linl<ages for use with the oligonucleotide analogs of this
10 invention. The columns designated RA (5~) and RB (3~ or 2') describe
substituents used to produce the R2-R5 linkage of structure (1), shown in the
right column, using methods known per se in the art and described in PCT
US93/05202 and other citations above. The starting materials in Table A, or
those used to prepare the starting materials of Table A, generally possess a
15 ribose or a ribose analog comprising a 5' hydroxyl group and a 3' or 2' hydroxyl
group, prepared as described herein or in the citations, with the substitute
linkage being substituted for the phosphodiester linkages in unmodified
nucleic acids. Sequentially useful starting materials are designated by an
arrow. Bracketed monomers are reacted to form dinucleotide analogs having
20 the R2-R5 substitute linkage. The reactions are repeated or ganged with
phosphodiester linkages in order to produce trimers, tetramers or larger
oligomers.
Bl in Table A means a blocking group. As used herein, "blocking
group" refers to a substituent other than H that is conventionally attached to
25 oligomers or nucleotide monomers, either as a protecting group, a coupling
group for synthesis, po~,-2, or other conventional conjug~te such as a solid
support. As used herein, "blocking group" is not intended to be construed
solely as a nucleotide protecting group, but also includes, for example,
coupling groups such as hydrogen phosphonate, phosphoramidite and others
30 as set forth herein. Accordingly, blocking groups are species of the genus of "protecting groups" which as used herein means any group capable of
preventing the O-atom or N-atom to which it is attached from participating in
a reaction involving an intermediate compound of structure (1) or otherwise
forming an undesired covalent bond. Such protecting groups for O- and N-
35 atoms in nucleotide monomers or nucleoside monomers are described andmethods for their introduction are conventionally known in the art.
Protecting groups also are useful to prevent reactions and bonding at
14
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wO 98/04575 PCT/US96/12530
carboxylic acids, thiols and the like as will be appreciated by those skilled in the
art.
Table A
Substitute T.ink~es
R (5') RA (3~ or 2') 2'/3'-RB -5'
OH I DMTO -CH2CH=CH2~CH2CHO ~ -(CH2)2-NHcH
NH2 OBl }
OH ~ DMTO N3 ~ NH2 ) -NH(CH2)2-
CH2C(OEt)2 -OBl
OH I DMTO -CH2CH-CH2~-CH2CHO- ) -CH2NH(CH2)2-
-cH2NH2 -OBl
OH I DMTO OH~-ocH2cH=cH2 } -O(CH2)2NHCH2-
-CH2NH2 -OBl
OH~-OCH2CH= OBI ) -NH(CH2)2OcH2
CH2
OH-~ DMTO NH2
DMTO CHO ~ -CH2NHCH2-
2~ -NH2 OBl
CH2CN~-CH2 OBl ) -NH(CH2)2-
CHO
DMTO NH2
(cH2)2oH ~ OBl ~ -S(CH2)3-;
(CH2)2OTs ) -S(O)(CH2)3-; or
DMTO SH ) -S(O)(O)(CH2)3-
CH20H~-CH2Br OBl ) -S(CH2)2-;
-S(O)(CH2)2-; or
DMTO SH ) -S(O)(O)(CH2)2-
DMTO CH2O ~ CH2OH ~ CH2OTS) -CH2SCH2-;
) -CH2S(O)CH2-; or
SH OBl ) -CH2S(O)(O)CH2-
TsOCH2 OBl ~ -O(CH2)2-
DMTO OH
..... . . .
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R (5') RA (3~ or2') 2'/3'-RB-5'
DMTO CH2CHO I (CH2)20H } -(CH2)2OCH2-
OH ~ MsO OBl
DMTO N~alk(C1-6) ~ -N(alk)(CH2)3
CH2CHO OBl
DMTO NH(COOEt) I
N(COOEt)(CH2SCH3) ~ -N(COOEt)CH20CH2-
OH OBl
(CH2)2I OBl ~ -S(CH2)3-
DMTO SH
TolO NH2 } NHC(O)OcH2-
pNPhOC(O)O OBI
TolO OCH2Cl ~ ~CH2SCH2-
SH OBI
TolO OC(O)OpNPh ~ -OC(O)N(R)CH2-
-NHR OBl
(R=H or lower alkyl)
TolO OCH2SMe ~ OCH2ocH2
OH OBl
DMTO SH ~ -SCH20CH2-
OCH2Cl OBl
DMTO OH ~ OCH2CH=CH-
BrCH2CH= -OBl
DMTO SH ~ -SCH2CH=CH-
BrCH2CH= -OBl
Invention embodiments. The invention oligonucleotides are
characterized by a log value of the partition coefficient between octanol and
water of about -0.3 to +2.5 and a solubility in water of at least 0.001 ~g/mL,
usually at least 0.01 ~lg/mL. In general, the invention oligonucleotides will
have at least 60%, often at least 80"/o, of their internucleotide linkages as
lipophilic modifications or at least 60%, often at least 80(~o, of their bases will
contain a lipophilic substitution, or at least 60~/0, often at least 80%~ of their
16
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wO 98/04s75 ~ PCT/us96ll2s3o
sugars will contain a lipophilic substitution, or wherein the percent non-ionic
nucleotide linkages, the percent bases containing a lipophilic substitution and
the percent sugars containing a lipophilic substitution sums to at least 60~/..,often at least 80"/.,. The linkages in the invention oligonucleotides are present
as ionic linkages, non-ionic linkages or as a mixture of ionic and non-ionic
linkages. Lipophilic modifications for the oligonucleotides are independently
chosen for each modification of the molecule. Generally, the log value of the
partition coefficient between octanol and water will be 0.0-2.5, typically 0.2-2.3,
usually 0.6-2.1.
Invention embodiments include structure (1) oligonucleotide analogs
wherein the linker at R, R1 or R3 optionally has 1-10 carbon, oxygen, sulfur
and/or nitrogen atoms bonded together in an uninterrupted chain. The
linker, which may be bonded to any invention oligonucleotide analog,
including structure (1) oligonucleotides, will either connect the
oligonucleotide analog to a detectable moiety or is bonded to the
oligonucleotide analog at one end of the linker and, at the other end, has a
reactive group available for bonding with a detectable moiety, e.g., amine,
carboxyl or hydroxyl group. The carbon or other atoms that comprise the
uninterrupted linker chain may be substituted with one or more substituents
that do not interfere with the function of the linker or the oligonucleotide
analog, e.g., hydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or
pseudohydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms and one or more of
the substitutions described above. Invention oligonucleotide analogs, such as
structure (1) oligonucleotides optionally have no linkers, but when a linker is
present there is usually only 1 linker which, for structure (1) is located at R, R
R3 or a base, e.g., at the 5 position of pyrimidines. Occasionally, the
oligonucleotides, including structure (1) oligonucleotides, contain 2, 3 or morelinkers, which are usually linkers having 1-10 bonded atoms that form an
uninterrupted chain.
For structure (1) oligonucleotides, each R2 independently is a
phosphodiester linkage or another linkage bonded to the 2' or 3' position and
usually any linkage containing a phosphorus atom is bonded to the 3'
position. For structure (1) oligonucleotides, each R3 independently is in the
ribo or ara configuration and independently is H, OH, F, blocked hydroxyl,
N(R14)2, O-hydrocarbyl including -O-alkyl (C1 8) and -O-alkenyl (C3 8), and O-
pseudohydrocarbyl including -O-alkyl (Cl 8) where the alkyl group is
substituted with halogen, hydroxyl, NO2, N3, carboxyl, ester, amide or oxygen
as a keto or ether moiety, -S-alkyl (C1 8) or a linker, wherein the linker
17
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W O 98/04575 PCT~US96/12530
optionally has 1-10 carbon atoms, usually each R3 independently is H or -O-
alkyl (C1 3). When R3 is in the ara configuration it is usually H or F. Usually
R4 is O. Usually R5 is CH2. Usually R6 is H or CH3. Generally one R14 is
hydrogen, C1~ hydrocarbyl or C1 8 pseudohydrocarbyl, and usually the other
5 R14 is hydrogen or a protecting group. Usually n is 2-13.
In invention oligonucleotide analogs, including structure (1)
oligonucleotides, each B independently is a base or an analog thereof in the a
or ,~ anomeric configuration, usually in the ~ configuration, wherein base
analogs are optionally lipophilic analogs that comprise a Cl-8 alkyl, Cs g
cycloalkyl, C6 8 aryl, C3 g heteroaryl, C2 8 alkenyl or C2 8 alkynyl moiety or the
analog contains a linker having 1-10 carbon atoms, wherein the
oligonucleotide has a log value of the octanol:water partition coefficient of
about -0.3 to +2.5, generally 0.0-2.5, typically 0.2-2.3, usually 0.6-2.1 and having a
solubility in water of at least 0.001 ~Lg/mL, usually at least 0.01 llg/mL.
Oligonucleotides having nucleosides containing bases in the oc anomeric
configuration binds to duplexes in a manner similar to that for the ~ anomers,
and one or more nucleosides may contain a base in the ~c anomeric
configuration, or more typically a domain thereof. (Praseuth, D., et al., Proc
Natl Acad Sci (USA) 85:1349-1353 1988).
For invention oligonucleotide analogs, or for structure (1)
oligonucleotides, the linkage (R2-R5) will generally be a linkage moiety that is3-5 atoms in length, usually 4. The linkages typically comprise bonded carbon,
oxygen, nitrogen, sulfur and/or phosphorus atoms in an uninterrupted chain.
Invention embodiments include oligonucleotide analogs of structure
(1) wherein each lipophilic substitution at substituted bases independently is ahydrocarbyl group, typically a C1 8 hydrocarbyl group or a pseudohydrocarbyl
group that is substituted with one or more heteroatoms selected from the
group consisting of nitrogen, oxygen and sulfur, typically a Cl 8
pseudohydrocarbyl group. Exemplary lipophilic substitutions include ones
wherein the Cl 8 hydrocarbyl or C1 8 pseudohydrocarbyl group is bonded to a
purine or pyrimidine base position selected from the group consisting of a C5
position of pyrimidines, the 04 position of thymine, the N6 position of
adenine, the C8 position of adenine, the N2 position of guanine, the C8
position of guanine, the N4 position of cytosine and the C7 position of 7-
deazapurines.
Any phosphate present at the 5' or 3' terminus of the oligonucleotide is
optionally derivatized, for example, by further esterification to a lipophilic
18
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WO 98/04575 PCT/US96112530
group containing about 3-8 carbon atoms. A particularly useful derivatizing
group may contain a linker and a label, for example, a fluorescent label such asfluorescein, rhodamine, or dansyl. Thus, useful derivatizing groups include
Fl-CONH (C~I2)2 8- and Rh-CONH (CH2)2 8-, wherein Fl and Rh signify
fluorescein and rhodamine, respectively.
Invention oligonucleotides are optionally coupled to a label such as a
fluorescent moiety and usually the label is coupled through a linker. A linker
is a moiety that contains an uninterrupted chain of atoms, e.g., 1-25 bonded (orlinked) atoms, that connect the label and the oligonucleotide analog.
10 Generally the linker contains 1-10 bonded atoms in such a chain. The
invention oligonucleotides are optionally able to bind single or double-
stranded nucleic acid in a sequence-specific manner when the lipophilic base,
sugar and linkage modifications do not completely destroy the
oligonucleotide's binding capacity. Such modifications are known and
15 include, e.g., (a) an alkynyl group such as 1-propynyl or 1-butynyl present at
the 5 position of pyrimidine bases or pyrimidine base analogs, (b) an alkynyl
group such as 1-propynyl or 1-butynyl present at the 7 position of 7-
deazapurine bases or 7-deazapurine base analogs, (c) non-ionic linkages such
as formacetal linkages or (d) sugar analogs where R3 is -O-alkyl (C1 3) or F.
One introduces the invention oligonucleotide analogs into cells by
contacting the cells an invention oligonucleotide. The oligonucleotide may be
delivered into cells in tissue culture medium containing serum or lacking
serum, or the oligonucleotide may be in an aqueous buffer or solution such as
PBS. One uses this method to deliver invention oligonucleotides into cells in
25 vitro or in vivo.
Invention embodiments include a method to visualize cells, usually
viable cells which are optionally mammalian cells in tissue culture or in vivo,
which method comprises: (a) contacting the cells to be visualized with an
invention oligonucleotide containing a detectable moiety, e.g., a fluorescent
30 moiety or radioactive moiety, under conditions wherein passive diffusion
across the cell membrane can occur so as to internalize the oligonucleotide; (b)washing the cells to remove any oligonucleotide which has not passively
diffused across the membrane and become internalized; (c) and detecting the
internalized oligonucleotide to visualize the cells. One optionally performs
35 the method using an invention oligonucleotide having a solubility of at leastabout 10 ~LM, usually at least 50 ,uM in water, to facilitate visualizing the
stained cells using fluorescence methods. The inventors have found that
some of the invention oligonucleotides are suitable for staining or visualizing
19
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a subcellular compartment of the viable cell. One can stain or visualize
subcelIular compartments including endoplasmic reticulum, nuclear
envelope, cell nuclei and mitochondria.
Using oligonucleotides of structure (1) as a reference, when at least 80%
5 of the linkages are modified, trimers have 2 modified internucleotide linkagesand tetramers have 3, and so forth. Thus, all internucleotide linkages must be
converted to lipophilic forms for oligomers which are less than hexamers. For
hexamers, having 5 internucleotide linkages, only 4 of these need to be
modified if only linkages, but not any bases or sugars, are modified.
One optionally includes in the invention oligonucleotide analogs
relatively large lipophilic moieties, e.g., 1, 2, 3 or 4 Cg 16 hydrocarbyl
substituents, or 1, 2, 3, or 4 Cl4-l8 pseudohydrocarbyl substituents. However,
such oligonucleotide analogs will generally contain either (1) a domain or
structural feature in the large hydrocarbyl or pseudohydrocarbyl substituent
15 that limits membrane binding, e.g., branched, polar or charged groups or
regions or (2) there will usually only be 1 or 2 of such large groups, if they are
compatible with membrane binding. Usually, such large hydrocarbyl or
pseudohydrocarbyl groups, particularly if they are compatible with membrane
binding, will not be located on adjacent monomers, i.e., there will be 1, 2, 3 or
20 more monomers that contain a smaller lipophilic group or no lipophilic
group interspersed between the monomers containing the larger groups.
To possess the desired diffusion properties, the invention
oligonucleotide analogs will generally contain a lipophilic modification at
least at 60%, often at least 80"/." of either their internucleotide linkages, sugars
25 and/or their bases. Invention embodiments include oligonucleotide analogs
where no sugar contains a lipophilic modification and at least 60'1/o, often at
least 80%, of the internucleotide linkages are non-ionic or at least 60"/o, often at
least 80%, of the bases contain a lipophilic modification. Other embodiments
include oligonucleotide analogs where no base contains a lipophilic
30 nlo~ifi~tion and at least 60~/." often at least 80%, of the internucleotide
linkages are non-ionic or at least 60%, often at least 80%, of the sugars contain
a lipophi~ic modification. Other embodiments include oligonucleotide
analogs where no linkage is non-ionic and at least 60~/u, often at least 80%, ofthe bases contain a lipophilic modification or at least 60%, often at least 80%, of
35 the sugars contain a lipophilic modification. Thus, for a 10-mer
oligonucleotide, the oligonucleotide will have 6 modified or substituted
linkages, bases or sugars, or two each may be modified or substituted or the
CA 02261704 1999-01-29
WO 98/04~75 PCT~US96/12530
oligonucleotide will have some combination that results in 6 modifications or
substitutions at the linkages, sugars or bases.
Larger hydrocarbyl groups, e.g., ones containing 9-18 carbon atoms will
generally contribute more lipophilic character to a nucleoside, i.e., a base, sugar
5 or linkage, than is required for permeation competence. In addition, the
presence of several of the larger groups such as ones containing 16-20 carbon
atoms may result in anchoring of the oligonucleotide in cell membranes. One
will thus generally use Cl 8 hydrocarbyl groups and/or Cl 8 pseudohydrocarbyl
groups to obtain a sufficiently lipophilic monomer. For example, replacement
10 of the -P(O)(O~)- group in a phosphodiester linkage with the hydrocarbyl
moiety -CH2- confers a significant amount of lipophilic character on the
linkage, due to the loss of the negative charge associated with the
phosphodiester moiety. The use of Cl 8 hydrocarbyl groups generally confers
sufficient non-ionic character and lipophilicity on the oligonucleotide to result
15 in a compound that conforms to the required solubility and distribution
coefficient values. One confirms this by assays.
By the use of appropriate larger lipophilic substituents, such as moieties
containing about 10-16 carbon atoms, the proportion of either bases, sugars
and/or linkages that must be modified for permeation competence can be
20 reduced to about 40-60'~or less. When designing such oligonucleotides, one
will generally use at least one structure such as a branched alkyl group or
pseudohydrocarbyl group having a polar moiety (e.g., a hydroxyl group) or a
polar domain to decrease the tendency of the lipophilic substituent to anchor
the oligonucleotide in a membrane.
Invention oligonucleotides have a log value of the partition coefficient
between octanol and water of -0.3 to +2.5, generally 0.0-2.5, typically 0.2-2.3,usually 0.6-2.1. These oligonucleotides optionally contain C1 8 hydrocarbyl
substituents, generally C1 ~, hydrocarbyl substituents, or Cl l4
pseudohydrocarbyl substituents, generally C2 8 psudohydrocarbyl substituents.
Such oligonucleotides optionally contain 1 or 2 Cg 12 hydrocarbyl substituents
or they optionally contain 1 or 2 C1s 18 pseudohydrocarbyl substituents. Such
oligonucleotides typically contain 3-30 monomers, usually 4-15. The
hydrocarbyl or pseudohydrocarbyl substituents are generally located at least at
60%, usually at least at 80'~o of the linkages, bases or sugars, or the
oligonucleotides have these substituents at a combination of the linkages,
bases and sugars that sums to at least 60~/O or at least 80~/ . Exemplary
embodiments include oligonucleotides wherein the hydrocarbyl or
pseudohydrocarbyl substituents are located on 60-90~/.., usually at 80-120% of
21
. .
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WO 98/04575 PCT/US96/12530
the linkages, bases or sugars. Alternatively, the oligonucleotides have these
substituents on a combination of the linkages, bases and sugars that sum to 60-
90% or 80-120~/.,. Exemplary embodiments include oligonucleotides having
branched or unbranched alkyl, alkenyl or alkynyl hydrocarbyl groups (Cl 8) or
5 branched or unbranched alkyl, alkenyl or alkynyl pseudohydrocarbyl groups
(Cl 14) at the linkages, bases and/or sugars. Other embodiments include
oligonucleotides having cyclic alkyl or alkenyl hydrocarbyl groups (Cs 8) or
cyclic alkyl or alkenyl pseudohydrocarbyl groups (C3 g) at the linkages, bases
and/or sugars.
In some embodiments, it may be desirable to substitute oligonucleotides
of the invention with base, linkage and/or sugar modifications that do not
significantly interfere with the capacity of the oligonucleotide to bind to a
complementary nucleic acid target. The target may be a single-chain or duplex
nucleic acid. Appropriate substitutions for binding competent modified
15 oligomers with target nucleic acids refer to substitutions at base, linkage
and/or sugar positions that do not completely disrupt the oligonucleotide's
capacity to hydrogen bond with complementary nucleic acids. Those positions
on bases include the N6 or C8 of adenine, the N2 or C8 of guanine, the C5 of
pyrimidines, N4 of cytosine and C7 of 7-deazapurines. Synthesis of such
20 modified bases is described in the art, as are methods for incorporation of such
bases into oligonucleotides by solid-phase or solution-phase methods
(Uhlmann, E., et al., C11emical ~eviews, 90:543-584 1990, and references cited
therein, U. S. Patent No. 5,484,908, PCT US91/06855 and PCT US92/09195).
Thus, for invention oligonucleotides designed to bind single-stranded or
25 double-stranded nucleic acid targets, care must be taken to place the lipophilic
substituents in such a way so as to avoid disruption of binding to the target.
However, for uses of the invention oligonucleotide analogs that do not
involve binding to complementary sequences by base pairing, e.g., staining
cells or subcellular components, the bases, sugars or linkages may have
30 modifications that are not compatible with oligonucleotide binding
competence or with base pairing.
Representative lipophilic substituents at the base residues include
saturated and unsaturated straight-chain, branched-chain, or cyclic hydrocarbyl
groups, such as an alkane C1 8 (usually C2-4), alkene Cl 8 (usually C2-4), or an35 alkyne Cl 8 (usually C2-4), including ethynyl, vinyl, isopropyl, isobutyl,
butynyl, butenyl, pentyl, pentenyl, isopentyl, phenethyl, methyl, ethyl, propyl,propynyl, phenyl, phenylvinyl, propenyl, butyl, pentynyl and their
CA 02261704 1999-01-29
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stereoisomers and positional isomers substituted at appropriate positions on
the base.
Exemplary base substituted nucleosides include 5-ethynyl-dU (5-
ethynyl-2'-deoxyuridine), 5-ethynyl-dC, 8-ethynyl-dG, 5-vinyl-dU, 5-ethyl-dU,
- 5 8-ethynyl-dA, 8-propynyl-dG, 8-propynyl-dA, 5-pentyl-dU, 5-pentynyl-dU, 5-
phenethyl-dU, 5-pentyl-U, 5-pentynyl-U, 5-benzyl-dC, N6-methyl-8-oxo-2'-
deoxy-A, 4~-butyl-T, 5-propynyl- dC and 5-propynyl-dU.
Invention oligonucleotide analogs are optionally labeled using a
detectable moiety, such as a fluorescent label, radiolabel or enzyme label. One
links detectable moieties to the oligonucleotide analog using a linker. A wide
range of linkers are known and may be used by known methods. Exemplary
linkers include linkers that having 2-10 carbon atoms and contain reactive
groups that are convenient for linking to oligonucleotides, e.g., amines,
hydroxyls, thiols or carboxylic acids. Exemplary linkers include ones having a
structure such as H2N-(CH~)2 l(l-NH2 or H2N-(cH2)2-s-R5-(cH2)2-s-NH2~
where R5 is O, C(O), NRl~, S, SO or SO2 where R10 is hydrogen, alkyl Cl 4 or a
protecting group. Such linkers would link an invention oligonucleotide, at
e.g., the R, R1 or R3 position of structure (1) oligonucleotides, to a detectable
moiety. Many linkers are available commercially. Linkers can also comprise a
chelating agent that binds to a detectable atom (see, e.g., U.S. Patent No.
5,534,497)
Therapeutic methods which utilize oligonucleotides as active agents are
based on a number of end strategies. One method, the "antisense" approach
wherein the oligonucleotide is designed to be the antisense counterpart of an
mRNA transcript and is thus expected to interrupt translation of a gene which
has an undesired effect in the cell. More recently, it has been found possible to
utilize the polymerase chain reaction (PCR) to amplify selectively
oligonucleotides that empirically preferentially bind to targets of diverse
molecular structure, including proteins and lipids (see, e.g., PCT US92/01383).
The antisense approach permits targeting of any desired nucleic acid sequence,
e.g., mRNA, by the properly selected oligonucleotide. The ability to obtain
specifically binding oligonucleotides in this way has expanded the possibilitiesfor oligonucleotide therapy because one can design oligonucleotides to target
substances that reside at the cellular surface or at intracellular locations such as
in the cytoplasm or nucleus.
Numerous publications have appeared that describe inhibition of gene
expression by exogenously added oligomers in various cell types (Agrawal, S.,
et al., Proc. Natl. Acad. Sci. (U.S.A.) ~:7079-7083 1988; Uhlmann, E., et al.,
23
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WO 98/04575 PCT/US96/12530
Chem. Revs. ~:583-584 1990). However, oligomers added directly to cells enter
the cellular cytoplasm at a low efficiency, at best. Many of the apparent
sequence-specific effects that have been described are likely to be due to effects
on cellular activity that do not arise from binding of the oligomer to target
5 nucleic acid sequences in cytoplasm or nucleoplasm. Gene specific effects do
appear to occur by binding of the oligomer to the target sequence for RNA
antisense sequences generated in situ that are complementary to a target
sequence or in cell-free in vitro systems with exogenously added oligomers
(Oeller, P.W., et al., Science ;~:437-439 1991; Joshi, S., et al., J. Virol. 65:5524-
10 5530 1991; Haeuptle, M-T., et al., Nucl. Acids Res. 14:1427-1448 1986).
The oligonucleotides of the invention, especially when fluorescently
labeled and utilized to visualize cells or subcellular structures, are
characterized by having a minimum solubility in water or aqueous media of at
least 10 nM, usually 50 nM. The minimum solubility requirement is based on
15 the minimum concentration of fluor required by current fluorescent
microscopes for visualizing the label. The oligonucleotides of the invention,
when utilized as (i) diagnostic or therapeutic agents that bind to intracellularor extracellular structures such as proteins or nucleic acids, or (ii) labeled
compounds to detect or visualize complementary nucleic acid sequences, or
20 cells, cell membranes or subcellular components in tissue samples, intact cells
or in cell lysates or fractions, are characterized by a minimum solubility in
water or aqueous media of at least about 0.001 llg/mL.
Some of the oligonucleotides of the invention were found to bind to
specific subcellular components such as endoplasmic reticulum or
25 mitochondria. Because of this, permeation-competent oligonucleotides that
are fluorescently labeled can be used to directly visualize live cells or cell
components in cell lysates. The aspects of the compounds that confer
subcellular component-specific binding on the oligonucleotides of the
invention are believed not to reside in the fluorescent moiety that is attached
30 to the compound. However, the same oligonucleotides, either containing the
fluorescent label or without the label can be synthesized utilizing, say, 32p or14C instead of the normal nonradioactive phosphorus or carbon isotope. Any
other appropriate radiolabel can also be utilized according to conventional
methods. Such radiolabeled oligonucleotides would retain their cell
35 component-specific binding properties, but need not be directly visualized. In
this case, cells or cell lysates can be specifically bound by the oligonucleotide
followed by detection of bound oligonucleotide as a means to measure the
presence or amount of bound material. Radiolabeled oligonucleotides used in
24
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WO 98/04575 PCT/US96/12530
this manner would have a minimum solubility requirement in water or
aqueous media of about 0.001 ~lg/mL in order to be conveniently detected or
quantitated by conventional methods such as scintillation counting.
The distribution coefficient need not be determined directly; that is, the
5 distribution of the material obtained by mixing it with octanol and water and
then effecting equilibrium distribution need not be evaluated, see e.g., Dagle et
al., Nucl Acids Res. 19:1805-1810 1991. Alternate ways to measure these values
take advantage of simpler techniques such as reverse-phase liquid
chromatography, wherein retention times can be correlated to partition
coefficient (Veith, G.D., et al., Water Research 13:43-471979), as described in
Example 1 below.
One measures the partition coefficient and solubility characteristics of
the invention oligonucleotide analogs by procedures known in the art and
exemplified below. The presence of these properties provides characterization
of oligomers that are capable of efficient passive diffusion across cell
membranes.
Utility and Administration
Since the oligonucleotides of the invention are capable of passive
diffusion across cell membranes they can be used to visualize and label cells
and intracellular organelles or other structures. For this use, the
oligonucleotides of the invention are provided with a detectable label, such as
a radiolabel, fluorescent label, chromogenic label, or enzyme label, and are
contacted with cells to be visualized. After a suitable incubation period of
about 15 minutes to 2 hours, usually at about 25 to 35~C, the solution
containing the labeled oligonucleotides is removed and the cells are washed to
remove any unincorporated oligonucleotide. The cells are then prepared for
visualization by fluorescence microscopy and detected by visualization of the
labeled oligonucleotide. For example, for a fluorescent labeled
oligonucleotide, the cells can be plated on a microscope slide and visualized
directly.
In addition to employing the oligonucleotides of the invention to
visualize cells, the oligonucleotides of the invention are useful in therapy anddiagnosis.
Those oligonucleotides that are capable of significant single-stranded or
double-stranded target nucleic acid binding activity to form duplexes, triplexesor other forms of stable association, or which bind specific target substances,
such as proteins, are useful in diagnosis and therapy of conditions that are
.. .... . . . .. . .
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associated with these targets. For example, one or more genes associated with
viral infections due to say, HIV, HCMV, HSV or HPV may be targeted. Other
therapeutic applications may employ the oligomers to specifically inhibit the
expression of genes that are associated with the establishment or maintenance
5 of a pathological condition, such as those for adhesion molecules, receptor
molecules or oncogenes that may be associated with inflammatory conditions,
immune reactions or cancer respectively. Diagnostic applications for the
oligomers include their use as probes for detection of specific sequences by anystandard method.
In therapeutic applications, the oligomers are utilized in a manner
a~ropriate for treatment of, for example, viral infections or malignant
conditions. For such therapy, the oligomers can be formulated for a variety of
modes of administration, including systemic, topical or localized
administration. Techniques and formulations generally may be found in
15 Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest
edition. The oligomer active ingredient is generally combined with a carrier
such as a diluent or excipient which may include fillers, extenders, binders,
wetting agents, disintegrants, surface-active agents, or lubricants, depending
on the nature of the mode of administration and dosage forms. Typical dosage
20 forms include tablets, powders, liquid preparations including suspensions,
emulsions and solutions, granules, capsules and suppositories, as well as
liquid preparations for injections, including liposome preparations.
For systemic administration, injection is preferred, including
intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection,
25 the oligomers of the invention are formulated in liquid solutions, preferablyin physiologically compatible buffers such as Hank's solution or Ringer's
solution. In addition, the oligomers may be formulated in solid form and
redissolved or suspended immediately prior to use. Lyophilized forms are
also included. Dosages that may be used for systemic administration
30 ~re~rably range from about 0.01 mg/Kg to 50 mg/Kg administered once or
twice per day. However, different dosing schedules may be utilized depending
on (i) the potency of an individual oligomer at inhibiting the activity of its
target gene, (ii) the severity or extent of a pathological disease state associated
with a given target gene, or (iii) the pharmacokinetic behavior of a given
35 oligomer.
Systemic administration can also be by transmucosal or transdermal
means, or the compounds can be administered orally. For transmucosal or
transdermal administration, penetrants appropriate to the barrier to be
26
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WO 98/04575 PCT/US96/12530
permeated are used in the formulation. Such penetrants are generally kno~n
in the art, and include, for example, bile salts and fusidic acid derivatives for
transmucosal administration. In addition, detergents may be used to facilitate
permeation. Transmucosal administration may be through use of nasal
sprays, for example, or suppositories. For oral administration, the oligomers
are formulated into conventional oral administration forms such as capsules,
tablets, and tonics.
For topical administration, the oligomers of the invention are
formulated into ointments, salves, gels, or creams, as is generally known in
the art.
In addition to use in therapy, the oligomers of the invention may be
used as diagnostic reagents to detect the presence or absence of the target
substances to which they specifically bind. Such diagnostic tests are conducted
by complexation with the target which complex is then detected by
conventional means. For example, the oligomers may be labeled using
radioactive, fluorescent, or chromogenic labels and the presence of label bound
to solid support detected. Alternatively, the presence of complexes may be
detected by antibodies which specifically recognize them. Means for
conducting assays using such oligomers as probes are generally known.
The invention oligonucleotides may be used to mark or "tag" various
items such as plastics, chemicals (e.g., fertilizers, explosives, gunpowders andfuels), oils and emulsions (e.g., crude or refined oils, mineral oils and
cosmetics), and fibers (e.g., synthetic or natural clothing fabrics). In these
applications, one optionally mixes the invention oligonucleotides with the
item or substance one wants to tag or one covalently links the oligonucleotide
to the tagged item or substance. One can detect the presence invention
oligonucleotides in various products using known direct detection means
such as by detecting the presence of radioactive atoms in the oligonucleotides
that are present in unique proportions that are easily identified (e.g., 10 atoms
of 14C per 1 atom of 3H). One can detect the invention oligonucleotides using
indirect means, e.g., techniques for amplifying small amounts of nucleic acids,
i.e. by polymerase chain reaction (PCR) techniques. PCR techniques are
known for amplifying nucleic acid analogs, or their derivatives, that one
normally can not amplify by standard techniques (see, e.g., PCT US93/07130).
When one elects to use PCR to detect invention oligonucleotides using PCR,
one will generally use base analogs that retain at least some of their base
pairing capacity, i.e., base pairing capacity sufficient to facilitate performing the
PCR procedure.
27
....... , ~ ... . .
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In addition to the foregoing uses, the ability some of the oligomers to
inhibit gene expression can be verified in in vitro systems by measuring the
levels of expression in recombinant systems using described methods (see, e.g.,
Lewis et al., Proc. Natl. Acad. Sci. (U.S.A.) 93:3176-3181 1996; Wagner et al.,
Science 260:1510-1513 1993).
The following examples are intended to further illustrate but not to
limit the invention. Applicants incorporate all citations herein with
specificity.
EXAMPLE 1
Evaluation of Distribution Coefficient. When a compound is allowed
to partition between octanol and water, the concentration of the compound in
the octanol divided by the concentration of the compound in the water is
15 commonly referred to as the octanol/water partition coefficient (Poct). This
number and the logarithm of the partition coefficient (Log Poct) are useful
parameters when describing the permeability of a compound towards its
membrane. A modification of the procedure of Veith, G.D., Austin, N.M., and
Morris, R.T., Wafer Resear~h 13:43-47 1979 using the HPLC retention time of
20 compounds was used to determine the log of the partition coefficients of
oligonucleotides (Log Poct). Essentially, the partition coefficients for
compounds with unknown Log Poct may be determined by comparison of
retention times of the desired compounds with compounds of known Log
Poct. The HPLC retention times of a set of standard compounds having
25 known Log Poct values was used to generate a plot of Log Poct versus Log k',
where k'=[tr-to]/to. (Here tr = retention time, and to = void time). The
resulting plot was fit to a third degree polynomial curve using Cricket Graph
software. A typical equation for the curve was y=-32.376 + 102.51x -
107.01x2+37.810x3. Typical R2 values were R2=o.999.
The column used was a Hamilton PRP-1, 10 micron, 150 x 4.6 mm ID
column. Solvent buffers used were: Solution A: 5 mM potassium phosphate
in 2% CH3CN in H2O, pH=7.4, and Solution B: 85~/.. CH3CN in H2O. A flow
rate of 1 mL/min was used. A linear gradient was used that went from 0~/O to
100% solution B in 30 min. Detection was monitored at 254 and 500
nanometers. Stock solutions of five standards were made up as 100 OD (A260
units)/mL solutions in 50'~., aqueous CH3CN. Then 10 OD (A260)/mL
solutions were made from the stock solutions by dilution with H2O. Void
28
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WO 98/0457~ PCT/US96/12530
times (to) were calculated by injecting MeOH and monitoring for the first
baseline disturbance. These values were typically k'=1.45 min.
The five standard compounds used for the determination of the curve
were 3-aminophenol (Log Poct=0.17), 2-aminophenol (Log Poct=0.62), aniline
5 (Log Poct=0.9), o-nitroaniline (Log Poct=1.44), and benzophenone (Log
Poct=3.18). The Log Poct values for these compounds were discussed in the
paper of Veith et al. above. The five samples were mixed in a 1:1:1:1:3
proportion. Aliquots of 20-50 microliters of this mixture were injected. Their
resulting retention times and known Log Poct values were used to generate a
10 curve as described above. Samples with an unknown Log Poct were made up
as solutions of 5 OD (A260)/mL in MeOH. Aliquots of 20-40 microliters were
injected. A typical standard curve is shown for the five standard reference
compounds in Figure 1. The retention time was used to calculate a k' value.
This k' value and the standard curve were then used to determine the Log
15 Poct value for unknown compounds.
EXAMPLE 2
Determination of Solubility of Oligonucleotide Analogs.
Oligonucleotides were resuspended in water at a stock concentration of 10 IlM
20 to 10 mM. The solution was then diluted in the aqueous media such as
DMEM tissue culture medium at decreasing concentrations. The microscope
was then used to analyze the solution for fine particles, micelles, etc.
Solubility was detected at a minimum oligomer concentration of 50 nM. This
lower solubility limit was determined by the sensitivity of the fluorescent
25 microscope. This value can be extended to a 10 nM concentration using more
sensitive apparatus.
EXAMPLE 3
Cell staining protocol. Oligonucleotides with various base or backbone
30 modifications were synthesized with one of a variety of amino-linkers. These
linkers included "5'-amino-modifier C6" (Glen Research; cat. no. 10-1906),
"amino-modifier dT" (Glen Research; cat. no. 10-1039), and
"3'-amino-modifier CPG" (Glen Research; cat. no. 20-2950). The following
fluors were bonded to the oligomers to monitor uptake:
35 tetramethylrhodamine, resorufin, fluorescein, BODIPY (Molecular Probes) and
acridine. A number of other fluors including dansyl, various coumarins,
bimane, and pyrene have been evaluated as potential fluorescent probes,
however these did not have a bright enough signal (relative quantum yield) to
29
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enable further investigation. A ~,e~lled fluor is fluorescein. This dye is itself
permeant to most of the cell types tested, giving a total cellular fluorescence.Within 15 min after washing the dye away from the exterior of the cells, the
intrac~ r pool of the dye is pumped out, either by an organic anion pump
mechanism or by diffusion. Fluorescein was conjugated to all of the linkers
(without oligonucleotide) used and these conjugates were shown to retain the
same biological properties. This fluor is very fluorescent, it does however
quench rapidly. It is also pH sensitive, being greater than an order of
magnitude less fluorescent at pH 5.0 than at pH 7.5. At pH 5.0 the molecule
has a net neutral charge, at pH 7.5 it has a net negative charge. BODIPY, which
has desirable molecular characteristics such as a neutral charge at cellular pH
ranges, lower molecular weight than fluorescein and a greater quantum yield
than fluorescein is also a preferred fluor.
Fluorescent measurements were made using a Zeiss Axiovert 10
microscope equipped with a 50W mercury arc lamp and outfitted with a set of
fluorescent filters available from Omega Optical (Burlingtion, VT, USA).
Observations were made from live cells with a 63x or 100x objective (culture
chamber and conditions described below). Photographs were taken with Tri-X
/ ASA 400 Kodak film and developed with Diafine developer (ASA rating
1600). Exposure time was fixed at 15 to 60s to enable direct comparison.
Fluorescent measurements were also made using a Nikon Diaphot
inverted microscope equipped with a phase 4 long working distance
condensor, 100W mercury arc lamp, Omega optical fluorescent filters, 40x, 60x
and 100x PlanApochromat phase/oil-immersion objectives, and 100~/l,
transmission to the video port. A Quantex high-intensity/intensified CCD
camera was used to digitize the fluorescent information. This information
was sent to a Data Translations FrameGrabber board mounted on a Macintosh
II CPU. The Macintosh II was equipped with 8MB RAM and had attached to it
a 330MB hard drive. Images were recorded using public domain NIH software
"IMAGE". Linearity of information was established using a series of neutral
density filters. Relative fluorescent intensity was compared between samples
using the same camera settings and variable neutral density filters.
Optimal fluorescence measurements were made using a confocal
microscope imaging system which optically slices "sections" through a cell. A
Noran real-time confocal imaging optical path equipped with a 3-line (457nm,
488nm, 529nm) laser which is hooked up to the Zeiss Axiovert 10 inverted
microscope described above was used. The imaging system was the Macintosh
II system described above.
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The cell stainhlg assay utilized various cell lines and included P388D1
(mouse macrophage), HEPG2 (human liver), CV1 (monkey epithelial), ccd50sk
(untransformed human fibroblast), Rat2 (rat fibroblast), MDCK (kidney cells),
L6 (rat myoblast), L ceIls (mouse fibroblast), HeLa (human adenocarcinoma),
5 skov3 (human ovarian adenocarcinoma), and slcbr3 (human breast
adenocarcinoma) cells. Other cell lines that were used included Jurkat
(hurnan T cell), H9 (human T cell), NIH3T3 (mouse fibroblast), HL60 (human
T cell), and H4 (rat liver). All cell lines are commercially available from the
American Type Culture Collection, Rockville, MD.
Cells were grown on 25mm~1 coverslips in media containing 25mM
HEPES, pH 7.3, (which helps maintain pH on the microscope) without phenol
red (which can lead to high background fluorescence when working with
living cells). Coverslips were used so that the high numerical aperture
oil-immersion lenses on the microscope could be used. The coverslips were
15 mounted onto "viewing chambers": 6-well petri dishes which have 22mm
holes drilled into the bottom. The slides were mounted with silicon vacuum
grease which was shown to be non-toxic to the cells. 12x12 mm glass rasching
rings (Stanford Glassblowing Laboratory, Stanford, CA) were mounted directly
onto the coverslip using paraffin wax. The chamber permitted the use of
20 incubation volumes less than 200 IlL. Fluorescent oligonucleotide conjugates
were added at concentrations ranging from 0.1 to 150 IlM. Stock
concentrations of oligonucleotides were prepared in 25mM HEPES, pH 7.3.
Oligonucleotides were added to media with or without 10'~.. 4hr-heat
inactivated (567C) fetal bovine serum.
Incubation times ranged from 15 minutes to 24 hours. 2 hour
incubations were generally utilized for cell staining. Cells were then
extensively washed to remove extracellular oligomer using media and
observed at room temperature. Slides were optionally replaced in the
incubator and were observed over the following 48-72 hours.
EXAMPLE 4
Subcellular Compartment Staining. Fluorescent oligomer compounds
were placed on fibroblasts, hepatocytes, muscle and carcinoma cell lines at 50
~M for 2 hours at 37~C; the cells were washed with cell media and live cells
were visualized for cellular staining using fluorescent confocal microscopy.
The results obtained for representative compounds were:
31
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Compound Log Poct* Cellular Compartment Stained
223-19C ND Mitochondria
183-53 0.26 Cytoplasmic/nucleus
223~4D 1.61 Endoplasmic reticulum/nuclear envelope
156-71A 2.09 Cytoplasmic/nucleus
156-31F ND Outer membrane
22~98E 1.14 Cytoplasmic/nucleus
273-21D 1.86 Cytoplasmic/nucleus
273-22D 2.18 Cytoplasmic/nuclear stain
10 ~ Log Poct at pH 7.4; ND, not determined
The structures of the listed compounds are given in Figure 2. All of the
- listed compounds were soluble in aqueous solution to the extent that they
could be visualized by fluorescence microscopy. Each compound entered
15 cellular cytoplasm rapidly after addition to cells in tissue culture. As indicated
in Figure 2, the molecular weight of the compounds ranged from 846 daltons
to 3484 daltons and, in the case of compound 273-22D, carried a negative
charge. These results are the first examples known by the present inventors of
efficient passive diffusion by oligonucleotide analogs into cells.
EXAMPLE 5
Synthesis of Monomers. The following compounds of the formula
R2
R30/~
R40
2~
are shown in Table 1 and synthesized as described below.
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Table 1
Compound R1 R2 R3 R4
OH -C-C-CH2CH2CH3 H H
2 OH -C_C-CH2CH2CH3 DMT H
3 OH -C_C-CH2CH2CH3 DMT HP02- HTEA+
4 OH -CH2CH2CH2CH2CH3 H H
OH -CH2CH2CH2CH2CH3 DMT H
6 OH -CH2CH2CH2CH2CH3 DMT HP02- HTEA+
7 OH -CH2CH2CH2CH2CH3 DMT -CH2S-CH3
8 OH -CH2CH2CH2CH2CH3 H TBS
9 OCH2CH2CH2CH3 -CH3 DMl~ H
OCH2CH2CH2CH3 -CH3 DMT HP02- HTEA+
DMT = 4,4'-dimethoxytrityl
TBS = t-butyldimethylsilyl
HTEA+ = hydrogentriethylammonium
5-(1-Pentynyl)-2'-deoxyuridine (1). This compound was prepared by the same
procedure that Hobbs, F.W.~., J. Org. Chem. 54:3420-3422 1989, used for the
preparation of other alkynyl substituted nucleosides. A mixture of 30.0 tg (84.7mmol) of 5-iodo-2'deoxyuridine (purchased from Sigma), 23.6 mL of
1-pentyne (Aldrich), 9.79 g of tetrakis (triphenylphosphine) palladium (0)
(Aldrich), and 3.23 g of copper (I) iodide were stirred at room temperature for
26 h. To the reaction was added 250 mL of MeOH and 250 mL of CH2Cl2. The
mixture was neutralized with Dowex 1 x 8-200 (bicarbonate form) ion exchange
resin. The mixture was filtered and concentrated. The residue was partitioned
between H2O and CH2Cl2. The aqueous layer was extracted three times with
CH2Cl2 and then concentrated. Purification of the crude product by column
chromatography afforded 20.4 g (81.9~/., yield) of product.
5'-0-(4,4'-Dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridine (2). To 20.4 g (69.3
mmol) of 5-(1-pentynyl)-2'-deoxyuridine in 300 mL of dry pyridine was added
22.8 g of 4,4'-dimethoxytrityl chloride. The reaction was stirred for 17 h at
room temperature and then concentrated. The residue was taken up in
CH2Cl2 and washed twice with 0.5'l/o aqueous NaHCO3, dried (Na2SO4),
CA 02261704 1999-01-29
WO 9B/04575 PCTtUS96/12530
filtered, and concentrated. Purification of the crude product by column
chromatography afforded 21.4 g (52.9~/l) yield) of product.
5'-0-(4,4'-Dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridin-3'-0-yl-
5 hydrogenphosphonate hydrogentriethylammonium salt (3). To an ice-cold
solution of 1.36 g (2.28 mmol) of 5'-0-(4,4'-dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridine in 7.39 mL of dry pyridine and 17.8 mL of dry CH2Cl2 was added
9.30 mL of a 1.00 M solution of 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one
(purchased from Aldrich as a solid) in CH2cl2~ dropwise over two minutes.
10 The reaction was stirred at 0~C for 45 minutes and then poured onto a
rapidly-stirred, ice-cooled mixture of 62 mL of 1 M aqueous
triethylammonium bicar~onate (TEAB, pH = 8.2) and 31 mL of CH2Cl2. The
mixture was stirred for 15 minutes and the layers were separated. The organic
layer was washed with 1 M aqueous TEAB, dried (Na2so4)~ filtered, and
15 concentrated. After isolation of the product by column chromatography on
silica gel, the product was taken up in CH2cl2/ washed with 1 M aqueous
TEAB, dried (Na2SO4), filtered, and concentrated. This procedure afforded 938
mg (53.9~/0 yield) of product.
5-Pentyl-2'-deoxyuridine (4). To a solution of 1.03 g (3.50 mmol) of
5-(1-pentynyl)-2'-deoxyuridine in 25 mL of MeOH was added a catalytic
amount of 10% Pd on charcoal. The mixture was hydrogenated under 300 psi
of H2 for 14 hours at room temperature. The mixture was filtered through
Celite and concentrated, affording a quantitative yield of product.
5'-0-(4,4'-Dimethoxytrityl)-5-pentyl-2'-deoxyuridine (5). This compound was
prepared from 1.04 g (3.49 mmol) of 5-pentyl-2'-deoxyuridine by the same
procedure used for the preparation of 5'-0-(4,4'-dimethoxytrityl)-5-
(1-pentynyl)-2'-deoxyuridine. Column chromatography of the crude residue
30 on silica gel afforded 1.70 g (81.0~/yield) of product.
5'-0-(4,4'-Dimethoxytrityl)-5-pentyl-2 '-deoxyuridin-3'-0-yl-
hydrogenphosphonate hydrogentriethylammonium salt (6). This compound
was prepared from 1.48 g (2.46 mmol) of 5'-0-(4,4'-dimethoxytrityl)-5-
35 pentyl-2'-deoxyuridine by the same procedure used for the preparation of 5'-0-
(4,4'-dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridine-
3'-yl-hydrogenphosphonate hydrogentriethylammonium salt. This procedure
afforded 1.35 g (71.8"/l. yield) of product.
34
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5'-0-(4,4'-Dimethoxytrityl)-3'-0-methylthiomethyl-5-pentyl-2'-deoxyuridine
(7). To a solution of 3.50 g (5.83 mmol) of 5'-0-(4,4'-dimethoxytrityl)-5-pentyl-
2'-deoxyuridine in 148 mL of dry THF was carefully (hydrogen evolution!)
added 835 mg of sodium hydride (97%) in small portions at room temperature.
After stirring the mixture for 30 minutes, 959 mg of sodium iodide (NaI) was
added, followed by 0.557 mL of chloromethyl methyl sulfide (Aldrich). The
reaction was stirred for 4 h and then carefully quenched with MeOH. The
mixture was concentrated. The residue was partitioned between CH2Cl2 and
H20, shaken, and separated. The organic layer was washed with sat.
10 aqueous NaHCO3, H20, dried (Na2SO4), filtered, and concentrated. The crude
residue was purified by column chromatography on silica gel affording 2.76 g
(71.7% yield) of product.
3'-0-t-Butyldimethylsilyl-5-pentyl-2'-deoxyuridine (8). To a mixture of 3.50 g
15 (5.83 mmol) of 5'-0-(4,4'-dimethoxytrityl)-5-pentyl-2'-deoxyuridine and 1.91 g
of im~ 7ole in 23.3 mL of dry DMF was added 1.05 g of t-butyldimethylsilyl
chloride ~purchased from Petrarch). The reaction was stirred at room
temperature for 20 h and then concentrated. The residue was partitioned
between CH2Cl2 and H20, shaken, and separated. The organic layer was
20 washed with H20, and concentrated. The crude material was stirred in 150 mL
of 80% HOAc in H20 for 3 h and then concentrated. The residue was taken up
in CH2Cl2 washed with H20, saturated aqueous NaHCO3, dried (Na2SO4),
filtered, and concentrated. Column chromatography of the crude residue
afforded 1.88 g (78.3% yield) of product.
4~-Butyl-5'-0-(4,4'-dimethoxytrityl)-thymidine (9). To an ice-cold solution of
5'~-(4,4'-dimethoxytrityl)-thymidine (2.0 g; 3.67 mmole) in 20 mL of CH2Cl2
was added 6 mL of N,N-dimethylaminotrimethylsilane. After stirring 30 min.
at O C, the reaction mixture was concentrated to dryness. The crude residue
30 was dissolved in 50 mL of acetonitrile. To this was added triethylamine (11 g;
110 rnmoles) and 1,2,4-triazole (1.52 g; 22 mmoles), and the mixture cooled to
0~C. To this ice-cold mixture was added POCl3 (1.10 g; 7.3 mmole). The
reaction mixture was stirred at 0~C for 3 h, then at room temperature
overnight. The reaction was then concentrated. The residue was dissolved in
35 CH2Cl2, and washed twice with saturated aqueous NaHCO3. The organic
phase was dried over Na2SO4, filtered, and concentrated. The residue was
purified by column chromatography on silica gel, affording 2.20 g of triazole
intermediate. The triazole intermediate (2.1 g; 3.5 mmole) was dissolved in
CA 02261704 1999-01-29
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anhydrous n-butanol (12 mL) and treated with DBU (1.0 g; 7.0 mmole). After
one h, the reaction mixture was concentrated to dryness. The residue was
dissolved in CH2cl2~ washed with 10~/n aqueous citric acid, dried over Na2S04,
and filtered. The residue was purified by column chromatography on silica
5 gel, affording 1.0 g of product.
4-0-Butyl-5'-0-(4,4 '-dimethoxytrityl)-thymidin-3 '-0-yl-
hydrogenphosphonate hydrogentriethylammonium salt (10). This compound
was prepared from 4-0-butyl-5'-0- (4,4'-dimethoxytrityl)-thymidine in the
10 same manner as described for the preparation of 5'-0-(4,4'-
dimethoxytrityl)-5-(1 -pentynyl)-2' -deoxyuridin-3'-0-yl-hydrogenphosphonate
hydrogentriethylammonium salt.
EXAMPLE 6
Synthesis of Dimer Synthons Containing Formacetal Linkages. The
following dimers of the formula
R~N
N~o ~7
\¢~N
0 ~' 0
ORl~
are shown in Table 2 and synthesized as described below.
36
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WO 98/0457~ PCT/US96/12530
Table 2
Compound R5 R6 R7
11 OH -CH2CH2CH2CH2CH3 OH
12 OH -CH2CH2CH2CH2CH3 OH
13 OH -CH3 OH
14 OCH2CH2CH2CH3 -CH3 OCH2CH2CH2CH3
OCH2CH2CH2CH3 -CH3 OCH2CH2CH2CH3
Compound R8 R9 Rl~
11 -CH~CH2CH2CH2CH3 DMT H
12 -C~2CH2CH2CH2CH3 DMT HPO2-HTEA+
13 CH3 DMT H
14 CH3 DMT H
CH3 DMT HPO2-HTEA+
For abbreviations, see Table 1
5'-0-([5'-0-(4,4'-Dimethoxytrityl~-5-pentyl-2'-deoxyuridin-3'-0-yl]-methyl-5-
25 pentyl-2'-deoxyuridine (11). This compound was prepared from compounds 7
and 8 in the same manner as that previously described for the preparation of
5'-0-([5'-0-(4,4'-dimethoxytrityl)-thymidin-3'-0-yl~-methyl)-thymidine in U.S.
Patent No. 5,264,562 in 86'~. yield.
5'-0-([5'-0-(4,4'-Dimethoxytrityl)-5-pentyl-2'-deoxyuridin-3'-0-yl]-methyl)-5-
pentyl-2'-deoxyuridin-3'-0-yl-hydrogenphosphonate
hydrogentriethylammonium salt (12). This compound was prepared from 5'-
0-([5'-0-(4,4'-dimethoxytrityl)-5-pentyl-2'-deoxyuridin-3'-0-yl]-methyl-5-
pentyl-2'-deoxyuridine using the same procedure described for the preparation
of 5'-0-(4,4'-dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridin-3'-0-yl-
hydrogenphosphonate hydrogentriethylammonium salt.
5'-0-([5'-0-(4,4'-Dimethoxytrityl)-thymidin-3'-0-yl]-methyl)-thymidine (13).
This compound was prepared as described in U.S. Patent No. 5,264,562.
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5'~-([4-0-Butyl-5'-0-(4,4'-dimethoxytrityl)-thymidin-3' -0-yl] -methyl)-4-0-
butylthymidine (14). This compound was prepared from 5'-0-([5'-0-(4,4'-
dimethoxytrityl)-thymidin-3'-0-yl]-methyl)-thymidine by the same procedure
used for the preparation of 4-0-butyl-5'-0-(4,4'-dimethoxytrityl)-thymidine.
5 Column chromatography afforded a 52% yield of product.
5'~-([4-0-Butyl-5'-0-(4,4'-dimethoxytrityl)-thymidin-3'-0-yl]-methyl)-4-0-
butylthymidin-3'-0-yl-hydrogenphosphonate hydrogentriethylammonium
salt (15). This compound was prepared from 5'-0-([4-0-butyl-5'-0-(4,4'-
10 dimethoxytrityl)-thymidin-3'-0-yl]-methyl-4-0-butylthymidine by the same
procedure used for the preparation of 5'-0-(4,4'-dimethoxytrityl)-5-(1-pentynyl-2'-deoxyurdin-3'-0-yl-hydrogenphosphonate hydrogentriethylammonium
salt.
EXAMPLE 7
Synthesis of Oligonucleotides - General Procedures. The pivaloyl
chloride (trimethylacetylchloride) was purified by distill~tion at atmospheric
pressure and stored under argon. The solvents (pyridine, dichloromethane,
acetonitrile) were dried over activated molecular sieves (3A). The solvents
20 used in the coupling cycle should be as anhydrous as possible to avoid any
undesirable hydrolysis reactions. The starting dimethoxytrityl protected
deoxynucleoside H-phosphonates were dried by co-evaporation from
anhydrous acetonitrile and subsequently reconstituted in 1:1 anhydrous
pyridine and acetonitrile. Synthesis was performed with the aid of a Biosearch
25 Model 8700 DNA synthesizer employing solid support, preferably CPG
(controlled pore glass).
Functionalization of Solid Support. To a solution of an appropriate
nucleoside (such as 5'-0-(4,4'-dimethoxytrityl)-5-(1-pentynyl)-2'-deoxyuridine,
5'-0-(4,4'-dimethoxytrityl)-5-pentyl-2'-deoxyuridine, 4-0-butyl-5'-0-(4,4'-
30 dimethoxytrityl)- thymidine, 5'-0-([5'-0-(4,4'-dimethoxytrityl)
-5-pentyl-2' -deoxyuridin-3'-0-yl] -methyl) -5-pentyl-2'-deoxyuridine,
5'-0-( [4-0-Butyl-5'-0-(4,4'-dimethoxytrityl)-thymidin-3'-0-yl]-methyl)-4-0-butylthymidine, or 5'-0-([5'-0-(4,4'-dimethoxytrityl)-thymidin-3'-0-yl]-
methyl)-thymidine) in 12 mL of anhydrous pyridine containing triethylamine
(TEA, 80 ,uL) was added 384 mg of DEC [1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide hydrochloridel, 12 mg of DMAP (N,N-dimethylaminopyridine),
and 1 g of CPG LCAA succinic acid (LCAA, long chain alkyl amine). The
resulting mixture was sealed under argon, wrapped in foil, and shaken for 14
38
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hours. The amount of nucleoside loading was determined by the
dimethoxytrityl cation assay described below.
Dimethoxytrityl Cation Ass~y for the Determin~tion of Nucleoside
Loadin.g on Solid Support. To 1 mg of functionalized CPG was added 1 mL of
5 0.1 M p-toluenesulfonic acid monohydrate (TSA) in dichloromethane. The
UV absorption of the solution using a standard cell was then measured at 498
nm. The degree of substitution (loading) was calculated using the following
formula: substitution (llmole/g) = A498 x 14.3, where A = absorbance.
Nucleoside substitutions (loadings) achieved were typically between 20
10 and 40 ,umole of nucleoside per gram of functionalized support. The
unreacted succinic acid sites on the solid support were capped by adding 134
mg of pentachlorophenol and shaking the mixture for 16 h. This formed the
corresponding ester. The mixture was filtered and the support was
sequentially washed with pyridine, dichloromethane, and then diethylether.
15 The support was then shaken with 10 mL of anhydrous piperidine in a 25 mL
round bottomed flask for 5 min. The mixture was filtered and the support
washed with dichloromethane and then diethylether. The support was then
added to an anhydrous solution containing 2.5 mL of acetic anhydride, 10.0 mL
of pyridine, and 10 mg of DMAP. The solution was placed under argon,
20 capped, and shaken for 4 h. The mixture was filtered, and the functionalized
CPG was washed sequentially with pyridine, dichloromethane, methanol and
diethylether. The CPG was dried in vacuum and was then ready for solid
phase oligonucleotide synthesis.
Preparation of DNA H-phosphonate. The oligonucleotide ~-
25 phosphonate having the following structures were prepared according to thefollowing procedure. First, the functionalized (A for sequence A, B for
sequence B) solid support was placed in a reactor vessel (column) and was
washed with dichloromethane. Then, a 2.5~/.. solution of dichloroacetic acid
(DCA) in dichloromethane was introduced to remove the 5' protecting group
30 of the support-bound nucleoside. After the deprotection step, the solid
support was washed with dichloromethane, and then anhydrous
pyridine/acetonitrile (1/1, by volume).
The first coupling cycle was initiated by the addition of a 1.5% solution
of pivaloyl chloride in anhydrous pyridine/acetonitrile, 1/1) and ten
35 equivalents (based on the amount of loading of the support bound nucleotide)
of the appropriate protected nucleoside hydrogenphosphonate in anhydrous
pyridine/acetonitrile (1/1) in alternating pulses. The reagents were allowed to
react for 3.5 min.
39
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WO 98/04575 PCT/US96/12530
At this point the oligonucleotide could be further extended by repeating
the sequence of DCA deprotection and pivaloyl chloride coupling until the
desired length and sequence of bases was attained. Alternatively, the linkage
or linkages could be oxidized to the thiophosphate, phosphodiester or the
5 phosphoramidate.
The final coupling for fluorescent labelling utilizes coupling of 6-N-(4-
methoxytrityl)-aminohexan-1-O-yl)-hydrogenphosphonate hydrogen-
triethylammonium salt. The coupling of this hydrogenphosphonate was
identical to the other hydrogenphosphonate couplings. After coupling and
10 desired oxidation, the monomethoxytrityl protecting group was removed
from the amine in a similar fashion as described above.
Conjugation of 5'Amino Linker Oligonucleotide with ~ Fluorescein
. A 10 llmole reaction (calculated from the loading of the CPG in llm/g
and the mass of the support bound nucleoside) was placed in 3.6 mL of
15 anhydrous N,N-dimethylforamide (DMF) and 0.4 mL of
disopropylethylamine. To this solution was added 24 mg of 5- (and 6-)
carboxyfluorescein, succinimidyl ester. The reaction was capped and shaken in
the dark for 10 h, and then filtered. The solid support was then sequentially
washed with dichloromethane, DMF, water, methanol and then diethylether.
20 The support-bound, fluorescently-labelled oligonucleotide was then washed to
remove unconjugated carboxyfluorescein.
Oxidation of the Oligonucleotide H-Phosphon~te to the Thiophosphate.
The DNA H-phosphonate, prepared above, was converted directly to the
thiophosphate, preferably while the DNA was still bound to the solid support,
25 by the addition to the reactor vessel of 1 mL of an oxidizing mixture comprised
of a 2.5% solution (by weight) of elemental sulfur (sublimed sulfur powder
available from Aldrich Chemical Company, Milwaukee, Wisconsin, USA, Cat
No. 21,523-6) in anhydrous pyridine/carbon disulfide (1/1, v/v). The contents
of the reactor were mixed for 20 min., and then the reagents were removed.
30 This oxidation cycle was carried out a second time using 1 mL of an oxidizingsolution comprising equal volumes of a 2.5 wt'~.. solution of elemental sulfur
in anhydrous pyridine/carbon disulfide (1/1, v/v) and 10% by volume
diisopropylethylamine in anhydrous pyridine. Finally, the oxidized
copolymer-bound oligonucleotide was washed with anhydrous
35 pyridine/acetonitrile (1/1, v/v), followed by anhydrous dichloromethane.
Oxidation of the Oligonucleotide H-Phosphonate to the Phosphodiester
and the Phosphoramid?lte Analog. The oligonucleotide H-phosphonate was
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WO 98/04~75 PCT/US96/12530
oxidized, when desired, to the phosphodiester derivative by the following
procedure:
Method A: To the solid support, obtained from the process outlined
above, was added 1 mL of an oxidizing solvent mixture comprised of 0.1 M I2
5 in water/pyridine (2/98, v/v). The resulting mixture was agitated for 15 min.,and then the reagents were removed. Afterwards, 1 mL of a second oxidizing
solvent mixture made from equal volumes of 0.1 M I2 in water/pyridine (2/98,
v/v) and 0.1 M triethyl ammonium bicarbonate in water/pyridine (1/9, v/v)
was added to the solid support. After mixing the contents of the reactor for 5
10 min., the reagents were removed. ~inally, the oxidized copolymer-bound
product was washed with anhydrous pyridine/acetonitrile (1/1, v/v) and then
anhydrous dichloromethane.
Method B: Alternatively, the oligonucleotide H-phosphonate was
oxidized to the phosphoramidate analog by the following procedure: To the
15 solid support, obtained from the procedure outlined above, was added 18 mL
of an oxidizing solvent mixture made from 10"/., by volume of the desired
amine in anhydrous/pyridine/carbon tetrachloride (1/1, v/v). The resulting
mixture was agitated for 15 min., after which time the spent oxidizing solvent
mixture was discarded. Finally, the oxidized copolymer-bound product was
20 washed with anhydrous pyridine/acetonitrile (1/1, by volume), and then
anhydrous dichloromethane.
The oligonucleotide H-phosphonates could be oxidized or converted to
a number of other linkage derivatives, such as phosphoric acid triesters,
dithiophosphoric acids, their corresponding esters and amidates, and other
25 which are desirable to and which are within the skill of those knowledgeable
in the art. Related oxidation procedures are described, for example, in
application no. EP 0 219 342, the complete disclosure of which is incorporated
herein by reference. Thus, oligonucleotides having a variety of linkages
derived from phosphoric acid, such as phosphoric acid diesters, phosphoric
30 acid triesters, thiophosphoric acid, dithiophosphoric acid, phosphoric acid
thioesters, phosphoric acid dithioesters, phosphoric acid amidates, or
thiophosphoric acid amidates, can be readily obtained from the methods
described above.
Cleavage of the Oligonucleotide From the Copolymer Support. Once
35 the synthesis of the oligonucleotide was complete, the DNA was cleaved from
the solid support, with the concurrent removal of any base protecting groups,
by the addition of concentrated aqueous ammonium hydroxide and heating
41
... ... . . . . . .......
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WO 98/04575 PCT/US96/12530
the resulting mixture at 45~C for 24 h. The product oligonucleotide was
washed from the solid support with methanol/water.
Purification was effected by reverse-phase HPLC, under the conditions
described further below.
HPLC Purification of the Fluorescently Labeled Oligonucleotide. A
crude sample containing approximately 10 Tmole of the fluorescently labeled
oligonucleotide, prepared by the methods described above, and dissolved in a
solvent mixture of 1/1 (v/v) methanol/water (10 mL) was concentrated under
vacuum. The oligonucleotide was resuspended in 1 mL of methanol and then
10 diluted with 100 mM aqueous triethylammonium acetate (TEAA, pH 7.0) and
5% (by volume) aqueous acetonitrile to a final volume of 10 mL. This dilute
oligonucleotide solution was then loaded, at a flow rate of 2 mL/min, on a
Septech A/E 160 cm x 10 cm i.d., column packed with Hamilton PRP-l
(polystyrene stationary phase, 12-20 llm), which had been preconditioned with
60 mL of a buffer solution comprised of 5'~'.. by volume of acetonitrile in 100
mM TEAA (Buffer C, pH=7.0) at a flow rate of 3 mL/min. After the completion
of the sample loading a 45 min. linear gradient to 100'~, of Buffer D (75~/O by
volume acetonitrile in 100 mM TEAA, pH=7.0) was initiated. After 45 min., a
linear gradient of 100~/., Buffer B in 15 min. (100"~. acetonitrile) was initiated.
20 The product was eluted and collected. The collected fractions were then driedin vacuum, and the excess TEAA salt was removed by co-evaporation 3x with
1 mL 90~/O ethanol, 10~/., water. The counter ion (if present) was then
exchanged by passing the oligonucleotide (in 0.5 mL) over a Poly-Prep, Bio-Rad
column (packing AG 50 w x 8 Na form) and eluting with 3 mL of water to yield
25 a highly pure fluorescently labeled oligonucleotide.
Applicants incorporate all citations herein with specificity. Applicants
incorporate all citations herein by reference.
42
