Novel compounds that mimic and/or modulate the activity of wild-type nucleic acids. In general, the compounds contain a selected sequence of nucleosidic bases or other reactive groups that are covalently bound through nitrogen-containing linear, hairpin, dumbbell, and circular shaped tethers.

FIELD OF THE INVENTION 
This invention relates to the design, synthesis and application of 
oligonucleotide mimics. More particularly, the invention relates to 
linear, duplex, hair-pin, stem-loop and cyclic compounds wherein 
naturally-occurring nucleobases, nucleobase-binding moieties, or other 
chemically active moieties are covalently bound to a somewhat linear, 
heteroatom-containing backbone. 
BACKGROUND OF THE INVENTION 
It is well known that most of the bodily states in mammals, including most 
disease states, are effected by proteins. Proteins, either acting directly 
or through their enzymatic functions, contribute in major proportion to 
many diseases in animals and man. 
Classical therapeutics generally has focused upon interactions with 
proteins in an effort to moderate their disease causing or disease 
potentiating functions. Recently, however, attempts have been made to 
moderate the production of proteins by interactions with the molecules 
(i.e., intracellular RNA) that direct their synthesis. These interactions 
have involved hybridization of complementary "antisense" oligonucleotides 
or certain analogs thereof to RNA. Hybridization is the sequence-specific 
hydrogen bonding of oligonucleotides or oligonucleotide analogs to RNA or 
to single stranded DNA. By interfering with the production of proteins, it 
has been hoped to effect therapeutic results with maximum effect and 
minimal side effects. 
The pharmacological activity of antisense oligonucleotides and 
oligonucleotide analogs, like other therapeutics, depends on a number of 
factors that influence the effective concentration of these agents at 
specific intracellular targets. One important factor for oligonucleotides 
is the stability of the species in the presence of nucleases. It is 
unlikely that unmodified oligonucleotides will be useful therapeutic 
agents because they are rapidly degraded by nucleases. Modification of 
oligonucleotides to render them resistant to nucleases therefore is 
greatly desired. 
Modification of oligonucleotides to enhance nuclease resistance generally 
has taken place on the phosphorus atom of the sugar-phosphate backbone. 
Phosphorothioates, methyl phosphonates, phosphoramidates and 
phosphorotriesters have been reported to confer various levels of nuclease 
resistance. Phosphate-modified oligonucleotides, however, generally have 
suffered from inferior hybridization properties. See, e.g., Crooke, S. T. 
and Lebleu, B., eds., Antisense Research and Applications (CRC Press, 
Inc., Boca Raton, Fla., 1993). 
Another key factor is the ability of antisense compounds to traverse the 
plasma membrane of specific cells involved in the disease process. 
Cellular membranes consist of lipid-protein bilayers that are freely 
permeable to small, nonionic, lipophilic compounds and are inherently 
impermeable to most natural metabolites and therapeutic agents. See, e.g., 
Wilson, Ann. Rev. Biochem. 1978, 47, 933. The biological and antiviral 
effects of natural and modified oligonucleotides in cultured mammalian 
cells have been well documented. It appears that these agents can 
penetrate membranes to reach their intracellular targets. Uptake of 
antisense compounds into a variety of mammalian cells, including HL-60, 
Syrian Hamster fibroblast, U937, L929, CV-1 and ATH8 cells has been 
studied using natural oligonucleotides and certain nuclease resistant 
analogs, such as alkyl triesters and methyl phosphonates. See, e.g., 
Miller, et al., Biochemistry 1977, 16, 1988; Marcus-Sekura, et al., Nuc. 
Acids Res. 1987, 15, 5749; and Loke, et al., Top. Microbiol. Immunol. 
1988, 141, 282. 
Often, modified oligonucleotides and oligonucleotide analogs are 
internalized less readily than their natural counterparts. As a result, 
the activity of many previously available antisense oligonucleotides has 
not been sufficient for practical therapeutic, research or diagnostic 
purposes. Two other serious deficiencies of prior art compounds designed 
for antisense therapeutics are inferior hybridization to intracellular RNA 
and the lack of a defined chemical or enzyme-mediated event to terminate 
essential RNA functions. 
Modifications to enhance the effectiveness of the antisense 
oligonucleotides and overcome these problems have taken many forms. These 
modifications include base ring modifications, sugar moiety modifications 
and sugar-phosphate backbone modifications. Prior sugar-phosphate backbone 
modifications, particularly on the phosphorus atom, have effected various 
levels of resistance to nucleases. However, while the ability of an 
antisense oligonucleotide to bind to specific DNA or RNA with fidelity is 
fundamental to antisense methodology, modified phosphorus oligonucleotides 
have generally suffered from inferior hybridization properties. 
Replacement of the phosphorus atom has been an alternative approach in 
attempting to avoid the problems associated with modification on the 
pro-chiral phosphate moiety. For example, Matteucci, Tetrahedron Letters 
1990, 31, 2385 disclosed the replacement of the phosphorus atom with a 
methylene group. However, this replacement yielded low affinity compounds 
with nonuniform insertion of formacetal linkages throughout their 
backbones. Cormier, et al., Nucleic Acids Research 1988, 16, 4583, 
disclosed replacement of phosphorus with a diisopropylsilyl moiety to 
yield homopolymers having poor solubility and hybridization properties. 
Stirchak, et al., Journal of Organic Chemistry 1987, 52, 4202, disclosed 
that replacement of phosphorus linkages by short homopolymers containing 
carbamate or morpholino linkages to yield compounds having poor solubility 
and hybridization properties. Mazur, et al., Tetrahedron 1984, 40, 3949, 
disclosed replacement of a phosphorus linkage with a phosphonic linkage 
yielded only a homotrimer molecule. Goodchild, Bioconjugate Chemistry 
1990, 1, 165, disclosed ester linkages that are enzymatically degraded by 
esterases and, therefore, are not suitable for antisense applications. 
The limitations of available methods for modification of the phosphorus 
backbone have led to a continuing and long felt need for other 
modifications which provide resistance to nucleases and satisfactory 
hybridization properties for antisense oligonucleotide diagnostics and 
therapeutics. 
Consequently, there is considerable interest in developing oligonucleotide 
surrogates that are capable of maintaining Watson-Crick base pairing to 
native RNA (DNA) or duplex DNA targets (formation of a triplex) but do not 
contain the usual phosphodiester linkages. One of the approaches to this 
problem involves the use of backbones containing peptide type linkages 
which connect the bases required for base pairing. These compounds are 
commonly known as peptide nucleic acids (PNA). Thus, in principle, it 
should be possible to design reagents or molecules that recognize any 
predetermined sequence, simply by connecting nucleosidic bases or other 
ligands with appropriate linear spacer molecules to maintain a desired 
geometry required for recognition of the hydrogen bonding groups in the 
minor or major groove of a nucleic acid. Recently, PNA and related 
molecules have demonstrated high affinity and specificity towards nucleic 
acid targets (Egholm, et. al., J. Am. Chem. Soc. 1992, 114, 1895, 9667; 
Nielsen, et. al., Science 1991, 254, 1497; Hyrup, et. al., J. Chem. Soc. 
Chem. Comm. 1993, 518). 
Molecular recognition plays a key role in the binding step, that is, the 
formation of the stable reactive complex. Much work has been done on the 
so called molecular clefts, where molecules are constructed so that they 
can recognize specific nucleobases by base pairing or base stacking 
(Rebek, Jr. Acc. Chem. Res. 1990, 23, 399; Inouye, et. al., J. Am. Chem. 
Soc. 1992, 114, 778.) It is believed that convergent functionality would 
provide an advantage in that activity could be `focused` in a highly 
localized manner at an active site. The ultimate goal is to merge the 
recognition and reaction steps in space and in time such that maximum 
binding would occur to transition states, as was anticipated by Pauling, 
Chem. Eng. News 1946, 24, 1375. 
There remains a need in the art for molecules which have fixed preorganized 
geometry that matches that of a target such as a nucleic acid or protein. 
The backbone of such molecules should be rigid with some flexibility and 
easy to construct in solution or via automated synthesis on solid support. 
OBJECTS OF THE INVENTION 
It is an object of the invention to provide oligonucleotide mimics for 
diagnostic, research, and therapeutic use. 
It is a further object of the invention to provide oligonucleotide mimics 
capable of forming duplex or triplex structures with, for example, DNA. 
It is a further object to provide oligonucleotide mimics having enhanced 
cellular uptake. 
Another object of the invention is to provide oligonucleotide mimics having 
greater efficacy than unmodified antisense oligonucleotides. 
It is yet another object of the invention to provide methods for synthesis 
of linear, hairpin, dumbbell or circular molecules as oligonucleotide 
mimics. 
These and other objects will become apparent to persons of ordinary skill 
in the art from a review of the present specification and the appended 
claims. 
SUMMARY OF THE INVENTION 
The present invention provides novel compounds that mimic and/or modulate 
the activity of wild-type nucleic acids and proteins. In certain 
embodiments, the compounds contain a selected nucleobase sequence which is 
hybridizable with a targeted nucleoside sequence of single stranded or 
double stranded DNA or RNA. At least a portion of the compounds of the 
invention has structure I: 
##STR1## 
wherein: each R.sub.N is, independently, H, -T-L, alkyl having 1 to about 
10 carbon atoms; alkenyl having 2 to about 10 carbon atoms; alkynyl having 
2 to about 10 carbon atoms; aryl having 7 to about 14 carbon atoms; 
heterocyclic; a reporter molecule; an RNA cleaving group; a group for 
improving the pharmacokinetic properties of the compound; or a group for 
improving the pharmacodynamic properties of the compound; 
each Q is, independently, N--R.sub.N, O, S, SO, SO.sub.2, or CH.sub.2 ; k 
is zero or 1; 
each A is, independently, R.sub.S --X(T-L)--R.sub.S ;N--R.sub.N ; C(O); a 
single bond; or (CH.sub.2).sub.m where m is 1-5; 
each R.sub.S is, independently, a single bond or alkyl having 1 to about 12 
carbon atoms; 
each T is, independently, a single bond, a methylene group or a group 
having structure II: 
EQU --CR.sup.1 R.sup.2 !.sub.n --B--CR.sub.1 R.sub.2 !.sub.o 
--D!.sub.p--N(R.sub.N)!.sub.q -- II 
where: 
D is C(O), C(S), C(Se), C(R.sup.1) (NR.sup.3 R.sup.4), CH.sub.2 R.sup.1, 
CHR.sup.1 R.sup.2, or NR.sup.3 R.sup.4 ; 
B is a single bond, CH.dbd.CH, C.tbd.C, O, S or NR.sup.4 ; 
each R.sup.1 and R.sup.2 is independently selected from the group 
consisting of hydrogen, alkyl or alkenyl having 1 to about 12 carbon 
atoms, hydroxy- or alkoxy- or alkylthio-substituted alkyl or alkenyl 
having 1 to about 12 carbon atoms, hydroxy, alkoxy, alkylthio, amino and 
halogen; 
R.sup.3 and R.sup.4, independently, are H, -T-L, alkyl having 1 to about 10 
carbon atoms; alkenyl having 2 to about 10 carbon atoms; alkynyl having 2 
to about 10 carbon atoms; aryl having 7 to about 14 carbon atoms; 
heterocyclic; a reporter molecule; an RNA cleaving group; a group for 
improving the pharmacokinetic properties of the compound; or a group for 
improving the pharmacodynamic properties of the compound; or 
R.sup.3 and R.sup.4, together, are cycloalkyl having 3 to about 10 carbon 
atoms or cycloalkenyl having 4 to about 10 carbon atoms; 
n and o, independently, are zero to 5; 
q is zero or 1; 
p is zero to about 10; 
each L is, independently, a nucleosidic base, an amino acid side chain, an 
aromatic hydrocarbon, a heterocycle moiety containing nitrogen, sulfur, 
and/or oxygen; a carbohydrate, a drug, a reporter molecule; an RNA 
cleaving group; a group for improving the pharmacokinetic properties of 
the compound; or a group for improving the pharmacodynamic properties of 
the compound; or group capable of hydrogen bonding; 
each X is, independently, N or CH, or 
X and T, together, form an aromatic moiety, a pentose, a hexose, or a deoxy 
derivative of a pentose or a hexose. 
The compounds of the invention generally are prepared by coupling 
preselected bifunctional synthons under conditions effective to form the 
above-noted structures. In certain embodiments, the compounds of the 
invention are prepared by intermolecular reductive coupling of, for 
example, a hydrazine moiety on a first synthon with an aldehyde moiety on 
a second synthon. In other embodiments, the compounds of the invention are 
prepared by coupling a carbocentric radical on a first synthon with, for 
example, a radical acceptor moiety on a second synthon. In further 
embodiments, the compounds are prepared through a nucleophilic alkylation 
wherein a nucleophilic moiety on a first synthon displaces a leaving group 
on a second synthon.

DETAILED DESCRIPTION OF THE INVENTION 
In designing novel drugs that recognize specific DNA sequences or protein 
binding sites, it is important to have thermodynamic information on the 
structure of the complexes. In particular, single stranded hairpin-shaped 
or dumbbell-shaped molecules are favorable for these studies because they 
form stable duplexes of defined geometry and secondary structure (see, 
e.g., Ma, et. al., Nucleic Acids Research 1993, 21, 2585; Rentzeperis, et. 
al., Biochemistry 1993, 32, 2564.) The structure and overall physical 
properties of such preorganized oligomeric molecules may be useful both to 
study nucleic acid-protein interactions as well as to provide a means for 
therapeutic inventions as transcription decoys. 
In addition, methods are available to further stabilize the secondary 
structure of synthetic molecules by crosslinking without perturbing their 
native geometries. (see, e.g., PCT/US93/02059, filed Mar. 5, 1993 and 
incorporated herein by reference). We would like to utilize the 
chemistries described herein to trap, isolate, and even circularize the 
oligomer under the given conditions. Thus, it should be possible to target 
single strands of nucleic acids (e.g., m-RNA) using circular oligomers. 
(see, e.g., Perkin, et. al., J. Chem. Soc. Chem. Comm. 1993, 215). 
The term "nucleoside" as used in connection with this invention refers to a 
unit made up of a heterocyclic base and a sugar. The term "nucleotide" 
refers to a nucleoside having a phosphate group on its 3' or 5' sugar 
hydroxyl group. Thus nucleosides, unlike nucleotides, have no phosphate 
group. "Oligonucleotide" refers to a plurality of joined nucleotide units 
formed in a specific sequence from naturally occurring bases and 
pentofuranosyl groups joined through a sugar group by native 
phosphodiester bonds. This term refers to both naturally occurring and 
synthetic species formed from naturally occurring subunits. 
The compounds of the invention generally can be viewed as "oligonucleotide 
mimics", that is, compounds which function like oligonucleotides but which 
have non-naturally occurring portions. Oligonucleotide mimics can have 
altered sugar moieties or no sugar moieties, altered base moieties or 
altered inter-sugar linkages. Representative modified bases include deaza 
or aza purines and pyrimidines used in place of natural purine and 
pyrimidine bases; pyrimidines having substituent groups at the 5 or 6 
position; purines having altered or replacement substituent groups at the 
2, 6 or 8 positions. Representative modified sugars include carbocyclic or 
acyclic sugars, sugars having substituent groups at their 2' position, and 
sugars having substituents in place of one or more hydrogen atoms of the 
sugar. Other altered base moieties and altered sugar moieties are 
disclosed in U.S. Pat. No. 3,687,808 and PCT application PCT/US89/02323. 
Altered base moieties or altered sugar moieties also include other 
modifications consistent with the spirit of this invention. Such compounds 
are best described as moieties that are structurally distinguishable from 
yet functionally interchangeable with naturally occurring or synthetic 
wild type oligonucleotides. All such compounds are comprehended by this 
invention so long as they function effectively to mimic the structure of a 
desired RNA or DNA strand. 
For use in antisense methodology, the oligonucleotide mimics of the 
invention preferably comprise from about 10 to about 30 bases. It is more 
preferred that such mimics comprise from about 15 to about 25 bases. 
It is preferred that the RNA or DNA portion which is to be modulated using 
oligonucleotide mimics of the invention be preselected to comprise that 
portion of DNA or RNA which codes for the protein whose formation or 
activity is to be modulated. The targeting portion of the composition to 
be employed is, thus, selected to be complementary to the preselected 
portion of DNA or RNA, that is, to be an antisense oligonucleotide mimic 
for that portion. 
In accordance with one preferred embodiment of this invention, the 
compounds of the invention hybridize to HIV mRNA encoding the tat protein 
or to the TAR region of HIV mRNA. In another preferred embodiment, the 
compounds mimic the secondary structure of the TAR region of HIV mRNA and 
by doing so bind the tat protein. Other preferred compounds are 
complementary to sequences for herpes, papilloma and other viruses. 
The oligonucleotide mimics of the invention can be used in diagnostics, 
therapeutics and as research reagents and kits. They can be used in 
pharmaceutical compositions by including a suitable pharmaceutically 
acceptable diluent or carrier. They further can be used for treating 
organisms having a disease characterized by the undesired production of a 
protein. The organism should be treated with an oligonucleotide having a 
sequence that is capable of specifically hybridizing with a strand of 
nucleic acid coding for the undesirable protein. Treatments of this type 
can be practiced on a variety of organisms ranging from unicellular 
prokaryotic and eukaryotic organisms to multicellular eukaryotic 
organisms. Any organism that utilizes DNA-RNA transcription or RNA-protein 
translation as a fundamental part of its hereditary, metabolic or cellular 
control is susceptible to therapeutic and/or prophylactic treatment in 
accordance with the invention. Seemingly diverse organisms such as 
bacteria, yeast, protozoa, algae, all plants and all higher animal forms, 
including warm-blooded animals, can be treated. Further, since each cell 
of multicellular eukaryotes can be treated since they include both DNA-RNA 
transcription and RNA-protein translation as integral parts of their 
cellular activity. Furthermore, many of the organelles (e.g., mitochondria 
and chloroplasts) of eukaryotic cells also include transcription and 
translation mechanisms. Thus, single cells, cellular populations or 
organelles can also be included within the definition of organisms that 
can be treated with therapeutic or diagnostic oligonucleotide mimics. As 
used herein, therapeutics is meant to include the eradication of a disease 
state, by killing an organism or by control of erratic or harmful cellular 
growth or expression. 
The oligonucleotide mimics of the invention are believed to exhibit 
increased stability relative to their naturally occurring counterparts. 
Extracellular and intracellular nucleases generally do not recognize--and 
therefore do not degrade--the compounds of the invention. In addition, the 
neutral or positively charged compounds of the present invention can be 
taken into cells by simple passive transport rather than by complex, 
protein-mediated processes. Another advantage of the invention is that the 
absence of a negatively charged backbone facilitates sequence specific 
binding of the oligonucleotide mimics to targeted RNA, which has a 
negatively charged backbone and will repel similarly charged 
oligonucleotides. Still another advantage of the present invention is that 
it presents sites for attaching functional groups that initiate cleavage 
of targeted RNA. 
The term heterocyclic is intended to denote moieties wherein a heteroatoms 
is inserted into the carbon backbone of an aromatic or alicyclic moiety. 
Representative heteroatoms include N, O, S, Se, and Te. The terms alkyl, 
aryl, alkenyl, and alkynyl are intended to include straight-chain, 
branched, and cyclic moieties, including those substituted with, for 
example, hydroxyl, alkoxy, alcohol, benzyl, phenyl, nitro, thiol, halogen, 
or alkyl, aryl, alkenyl, or alkynyl groups. Groups that enhance 
pharmaco-dynamic properties improve oligonucleotide uptake, enhance 
oligonucleotide resistance to degradation, and/or strengthen 
sequence-specific hybridization with RNA. Groups that enhance 
pharmacokinetic properties improve oligonucleotide transport, uptake, 
distribution, metabolism or excretion. 
The compounds of the invention generally are prepared by coupling 
preselected bifunctional synthons under conditions effective to form 
compounds having structure I. In certain embodiments, the compounds of the 
invention are prepared by intermolecular reductive coupling. In other 
embodiments, the compounds of the invention are prepared by intermolecular 
radical addition reactions. In further embodiments, the compounds are 
prepared by nucleophilic displacement. In each of these embodiments, free 
amino groups in the resulting linkage can be further functionalized. For 
example, the nucleophilic amino group can be reacted with a group having 
structure R.sub.L -T-L, thereby displacing the R.sub.L leaving group and 
forming a covalent -N-T-L linkage. 
In the reductive coupling methods, compounds having structure I are formed 
by coupling synthons having structures III and IV: 
##STR2## 
wherein: R.sub.N1 and R.sub.N2 are, independently, amine protecting 
groups, or a group comprising: N(R.sub.N)-Q-A-CH.sub.2 !.sub.r where r is 
1-100, a nucleoside, a nucleotide, an oligonucleotide, an oligonucleotide 
analog, an oligonucleoside, a PNA or a hydroxyl-protected and/or 
amine-protected derivative thereof, or R.sub.N1 and R.sub.N2, together, 
form an amine protecting group; and 
R.sub.A1 and R.sub.A2 are, independently, carbonyl protecting groups, or a 
group comprising: N(R.sub.N)-Q-A-CH.sub.2 !.sub.r where r is 1-100, a 
nucleoside, a nucleotide, an oligonucleotide, an oligonucleotide analog, 
an oligonucleoside, a PNA or a hydroxyl-protected and/or amine-protected 
derivative thereof, or R.sub.A1 and R.sub.A2, together, form a carbonyl 
protecting group. 
The radical addition reactions can be divided into two steps. The first 
step involves generation of an initial radical, which undergoes the 
desired reaction. The second step involves removal of the radical from the 
reaction before the occurrence of an intervening, undesired reaction such 
as cross coupling. In certain embodiments, the compounds of the invention 
are prepared by providing a donor synthon having structure V and an 
acceptor synthon having structure VI where R.sub.B is a radical generating 
group, generating a carbocentric radical at the --CH.sub.2 --R.sub.B 
position, and then forming an intermolecular linkage by reacting 
radical-bearing donor synthon V with acceptor synthon VI. Radical 
generating groups according to the invention include I, OC(S)O--C.sub.6 
H.sub.5, Se--C.sub.6 H.sub.5, OC(S)O--C.sub.6 F.sub.5, OC(S)O--C.sub.6 
Cl.sub.5, OC(S)O--(2,4,6--C.sub.6 Cl.sub.3), Br, NO.sub.2, Cl, OC(S)S-Me, 
OC(S)O--(p-CH.sub.4 F), bis-dimethylglyoximato-pyridine cobalt, 
OC(S)C.sub.6 H.sub.5, OC(S)SCH.sub.3, OC(S)-imidazole, and 
OC(O)O-pyridin-2-thione. 
##STR3## 
The nucleophilic displacement (alkylation) reactions involve reacting a 
first synthon VII bearing a leaving group, R.sub.L, with a second synthon 
VIII bearing a nucleophilic nitrogen moiety under conditions effective to 
displace the leaving group and form the above-identified linkages. 
##STR4## 
Leaving groups according to the invention include chloro, fluoro, bromo, 
iodo, p-(2,4-dinitroanilino)benzenesulfonyl, benzenesulfonyl, 
methylsulfonyl (mesylate), p-methylbenzenesulfonyl (tosylate), 
p-bromobenzenesulfonyl, trifluoromethylsulfonyl (triflate), 
trichloroacetimidate, acyloxy, 2,2,2-trifluoroethanesulfonyl, 
imidazolesulfonyl, and 2,4,6-trichlorophenyl groups. 
Reductive Coupling 
The linkages of the invention can be formed by selecting a 
formyl-derivatized compound (e.g., structure III) as an upstream synthon 
and an amino-derivatized compound (e.g., structure IV) as a downstream 
synthon. 
Formyl-terminated compounds such as structure III can be formed via several 
synthetic pathways. One preferred method utilizes a radical reaction of 
the corresponding xanthate-terminated compound. The xanthate compound is 
treated with 2,2'-azobisisobutrylonitrile (AIBN), and tributyltin styrene 
in toluene. Subsequently, the styrene derivative is hydroxylated and 
cleaved to furnish a formyl group. Alternately, formyl-terminated 
compounds can be synthesized from a cyano-terminated compound by 
techniques well known in the art. Terminal formyl groups can be blocked in 
a facile manner, for example, utilizing o-methylamino-benzenthiol as a 
blocking group. The formyl blocking group can be deblocked with silver 
nitrate oxidation. 
An alternate method of preparing formyl-terminated compounds employs 
tosylation of a terminal hydroxyl group, which on iodination followed by 
cyanation with KCN in DMSO will furnish a nitrile. Reduction with DIBAL-H 
gives the desired formyl-terminated compound. In yet another method, a 
terminal C.dbd.C bond is oxidized with OsO.sub.4 and cleavage of the 
resulting diol with NaIO.sub.4 gives the desired formyl functionality. 
Hydroxylamino terminated compounds such as those having structure IV (Q=O) 
can be prepared by treating the corresponding hydroxyl compound with 
N-hydroxyphthalimide, triphenylphosphine and diethylazodicarboxylate under 
Mitsunobu conditions to provide an O-phthalimido derivative. The free 
hydroxylamino compound can be generated in quantitative yield by 
hydrazinolysis of the O-phthalimido derivative. 
Hydrazino-terminated compounds such as those having structure IV (Q=NH) can 
be prepared by treating hydroxyl-terminated compounds with tosyl chloride 
in pyridine to give an O-tosylate derivative. Treatment of benzylcabazide 
with O-tosylate will furnish a benzylcarbazide derivative, which on 
hydrogenation provides the free hydrazino moiety for reductive coupling. 
Amino-terminated compounds such as those having structure IV (Q=CH.sub.2) 
can be synthesized by treating the corresponding hydroxyl-terminated 
compound with Ph.sub.3 P, CBr.sub.4 and LiN.sub.3 according to the 
procedure of Hata, et al., J. Chem. Soc. Perkin 1 1980, 306, to furnish a 
terminal azide. Reduction of the azido group with tributyltin hydride 
provides the desired amino functionality. 
Coupling of structures III and IV then is effected to furnish a dimeric 
unit having an imine or oxime linkage. This linkage then is reduced in 
situ with NaCNBH.sub.3 to furnish a --C--N-- linked unit. In certain 
embodiments these --C--N-- linked units contain two hydroxyl groups, one 
placed at the upstream end and the other placed at downstream end. One of 
these groups can be protected with a dimethoxytrityl group, and the other 
group can be protected as a cyanoethyldiisopropyl-phosphite. These units 
can be inserted into any desired sequence by standard, solid phase, 
automated DNA synthesis utilizing standard phosphoramidite chemistry. 
(see, e.g., Protocols For Oligonucleotides And Analogs, Agrawal, S., ed., 
Humana Press, Totowa, N.J., 1993.) 
Thus, one or more of such oligomeric units can be attached, as in Example 
1, at the ends of a DNA sequence or placed internally connected by 
phosphodiester linkages. The resulting oligonucleotide analog or oligomer 
has a "mixed" backbone containing more than one type of linkages of this 
invention. In a similar manner, an oligomer containing alternating 
phosphodiester linkages can be prepared. Such a structure should provide 
enhanced affinity and base-pair specificity towards the nucleic acid 
targets and proteins. Furthermore, these structures should have increased 
hydrophilicity compared to an oligomer without phosphate linkages. 
Oligomers containing a uniform backbone linkage can be synthesized using 
CPG-solid support and standard nucleic acid synthesizing machines such as 
Applied Biosystems Inc. 380B and 394 and Milligen/Biosearch 7500 and 
8800s. The initial monomer (number 1 at the 3'-terminus) is attached, via 
an appropriate linker, to a solid support such as controlled pore glass or 
polystyrene beads. In sequence specific order, each new monomer (e.g., 
structure III or IV) is attached either by manual manipulation or by the 
automated synthesizer system. In the case of a methylenehydrazine linkage 
(Q=N), the repeating nucleoside unit can be of two general types: a linear 
structure with a protected aldehydic function at one end and a 
C-hydrazinomethyl group at the opposite end, or a structure bearing a 
terminal hydrazino group and a protected C-formyl group. In each case, the 
conditions that are repeated for each cycle to add the subsequent base 
include: acid washing to remove the terminal aldehydo protecting group; 
addition of the next molecule with a methylenehydrazino group to form the 
respective hydrazone connection; and reduction with any of a variety of 
agents to afford the desired methylene-hydrazine linked CPG- or 
polystyrene-bound structure. One such useful reducing agent is sodium 
cyanoborohydride. 
A preferred method is shown in FIG. 1. This method utilizes a solid support 
to which a linear molecule having a protected aldehyde or an aldehyde 
precursor at its terminal end is attached. The terminal aldehyde can be 
suitably protected with various groups, such as described by Greene and 
Wuts in Protective Groups in Organic Synthesis, John Wiley & Sons, Inc., 
1991, pp 175-223. In one preferred method, the aldehyde group is protected 
with N,N'-diphenyl imidazolidine, which can be cleaved with aqueous HCl as 
described by Giannis, et al. Tetrahedron 1988, 44, 7177. 
2,3-Dihydro-1,3-benzo-thiazole is yet another preferred protecting group 
for aldehyde functionality and is cleaved by AgNO.sub.3 at neutral pH 
(see, e.g., Trapani, et. al., Synthesis 1988, 84). More preferably, a 
terminal vinyl group is oxidized with OsO.sub.4 and cleaved with 
NaIO.sub.4 to yield a free aldehydo group. 
A bifunctional synthon having a protected aldehydo group at one end (the 
masked coupling end) and a hydrazino group at the opposite end (the 
reactive coupling end) can be coupled under acidic conditions with a 
linear aldehyde attached to the solid support. The intermediate hydrazone 
then is reduced with NaBH.sub.3 CN to furnish a hydrazino linkage attached 
to the solid support. 
Subsequently, bisalkylation of the hydrazino moiety via an appropriate 
halide or aldehyde provides a N,N-substituted hydrazine linked to the 
solid support. Thereafter, the cycle can be repeated by the addition of 
bifunctional synthon under acidic conditions, reduction, and alkylation of 
hydrazine moiety to create a polymeric molecule of a desired sequence 
connected by one or more substituted hydrazino linkages. In some preferred 
embodiments of this invention, the final unit utilized for coupling can 
bear a phosphate or a phosphonate linkage to provide water solubility for 
such molecules. 
One preferred process employees an aldehyde-protected synthon attached to 
the solid support. Attachments can be effected via standard procedures as 
described by R. T. Pon in Protocols For Oligonucleotides And Analogs, 
Chapter 24, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993. 
As an alternative, a solution phase synthesis of substituted hydrazino 
linked linear molecules can be accomplished via hydroxyl protected 
synthons, such as shown in FIG. 1 (R.sub.Z =hydroxyl protecting group or 
solid support) utilizing a t-butyl diphenylsilyl group. 
A further method of synthesizing N-substituted hydroxylamine linked linear 
molecules is depicted in FIG. 4 (L.sub.S =a linker attached to solid 
support, or a protecting group, such as t-butyldiphenylsilyl). This method 
also employs a solid support to which a linear molecule having an 
O-phthalimido group at its terminal end is attached. A further 
bifunctional unit that has an aldehyde functionality at the coupling end 
and an O-phthalimido group at the growing end is utilized as the middle 
block via repeating cycles. The synthesis of polymeric structures can be 
stopped by use of a terminating unit that bears a hydroxyl protecting 
group rather than a phthalimido group. A wide variety of hydroxyl 
protecting groups can be employed in the methods of the invention. In 
general, protecting groups render chemical functionality inert to specific 
reaction conditions, and can be appended to and removed from such 
functionality in a molecule without substantially damaging the remainder 
of the molecule. Representative protecting groups are disclosed by 
Beaucage, et al., Tetrahedron 1992, 48, 2223. 
The O-phthalimido group attached to the support is hydrazinolyzed with 
methyl-hydrazine to generate a reactive O-amino group. Acid catalyzed 
coupling of the resulting bifunctional unit provides an oxime linked 
support. The oxime linkage can be reduced with NaBH.sub.3 CN/acetic acid 
to yield a hydroxyl amino linkage, which is then alkylated with 
appropriate functionality. Alternately, the coupled unit can be treated 
with methyl hydrazine and the coupling with bifunctional unit repeated 
until an oligomer of desired length is obtained. The multiple oxime 
linkages thus created can be reduced in one step utilizing NaBH.sub.3 
CN/AcOH to create free O-amino groups, which can be further substituted 
uniformly with appropriate functionality. 
In a similar manner, a solution phase synthesis of such polymeric molecules 
connected via substituted hydroxylamino linkages utilizes the 
coupling/reduction/alkylation hydrazinolysis steps in a sequential order, 
starting with a hydroxyl protected molecule. 
Radical Coupling 
The radical-based methods of the invention generally involve "nonchain" 
processes. In nonchain processes, radicals are generated by stoichiometric 
bond homolysis and quenched by selective radical-radical coupling. It has 
been found that bis(trimethylstannyl)benzopinacolate and 
bis(tributylstannyl)benzopinacolate (see, e.g., Comprehensive Organic 
Synthesis: Ed. by B. M. Trost & J. Fleming, Vol. 4, pp 760)--persistent 
radicals--can be used to enhance the radical-radical coupling and reduce 
cross-coupling. It will be recognized that a persistent radical is one 
that does not react with itself at a diffusion-controlled rate. 
Hillgartner, et al., Liebigs. Ann. Chem. 1975, 586, disclosed that on 
thermolysis (about 80.degree. C.) pinacolate undergoes homolytic cleavage 
to give the suspected persistent radical (Ph.sub.2 C.sup.. OSnMe.sub.3), 
which stays in equilibrium with benzophenone and the trimethylstannyl 
radical (Me.sub.3 Sn.sup..). It is believed that the Me.sub.3 Sn.sup.. 
radical abstracts iodine from radical precursors such as iodo-terminated 
compounds having structure V to give radical-terminated intermediates. The 
radicals then add to immino acceptors such as structure VI to yield a 
--C--C--N-- linkage. 
At high concentrations the initial radical can be trapped by coupling prior 
to addition, and at low concentrations the adduct radical can begin to 
telomerize. It is believed that a three molar equivalent excess of 
pinacolate provides satisfactory results for such couplings. The 
efficiency of radical reactions is highly dependent on the concentration 
of the reagents in an appropriate solvent. Preferably, the reaction 
mixture contains benzene, dichlorobenzene, t-butylbenzene, t-butyl 
alcohol, water, acetic acid, chloroform, dichloro methane, carbon 
tetrachloride, or mixtures thereof. The solvent should contain a combined 
concentration of about 0.1 to about 0.4 moles/liter of radical precursor 
and acceptor, preferably about 0.1 to about 0.2 moles/liter. It has been 
found that best results are obtained using benzene solutions containing 
about 0.2 moles/liter of radical precursor and acceptor. 
As exemplified in FIG. 5, the radical coupling of an oxime ether 31 as an 
acceptor with radical precursor 33 occurs in the presence of 
bis(trimethylstannyl)benzopinacolate in refluxing benzene. The reaction is 
carried out under argon and a 35-50% isolated yield of the product is 
obtained after purification. The hydroxylamino linkages thus obtained can 
be alkylated with an appropriate functionality. Subsequently, the hydroxyl 
group is deblocked and treated with N-hydroxyphthalimide under Mitsunobu 
conditions to yield an O-phthalimido derivative. Hydrazinolysis and 
formylation of the latter compound gives an oxime ether functionality at 
the reactive end of the molecule. Therefore, a radical coupling cycle can 
be repeated with high chemoselectivity to yield an oligomer or polymeric 
unit linked via one or more substituted hydroxylamino linkages. The chain 
elongation can be terminated at any point during the described method by 
avoiding the Mitsunobu reaction at the hydroxyl function. 
The desired method essentially can be transferred from solution to solid 
phase systems by utilizing an oxime unit linked to a support via a linker. 
The radical coupling methodology also can employ a bifunctional unit, as 
depicted in FIG. 5. Thus, coupling between an oxime linked to a support 
and the bifunctional unit under the described conditions will provide a 
hydroxylamino linked molecule. This compound can be alkylated in a 
standard manner to yield a N-substituted molecule. Subsequently, 
deblocking of the phthalimido group with methyl hydrazine liberates a free 
O-amino group, which on treatment with formaldehyde gives a terminal 
oxime. The oxime can be used in another round of coupling with an iodo 
derivative. In this manner the synthesis is more convenient, due to the 
Mitsunobu reaction prior to coupling. Radical coupling cycles can be 
repeated as often as needed until a polymer of desired length is obtained. 
The elongation usually is terminated by using a last unit, as shown in 
FIG. 5, that bears a protected hydroxyl group. The foregoing procedure is 
highly adaptable to solution phase chemistry in a similar manner. 
EXAMPLE 1 
Reductive Coupling 
I. Solution phase Synthesis of an Oligomeric Molecule Linked Via Hydrazino 
Linkages (FIG. 1) 
A. Synthesis of a `First Unit`, 
1-O-(t-butyldiphenylsilyl)-butyraldehyde-1-ol, 3 (R.sub.Z 
=t-butyldiphenylsilyl (TBDPS), r=1) 
A mixture of 4-penten-1-ol (10 mmol), t-butyldiphenylsilylchloride (12 
mmol), imidazole (25 mmol) and dry DMF (50 ml) is stirred at room 
temperature for 16 h under argon. The reaction mixture is poured into 
ice-water (200 ml) and the solution extracted with CH.sub.2 Cl.sub.2 
(2.times.200 ml). The organic layer is washed with water (2.times.200 ml) 
and dried (MgSO.sub.4). The CH.sub.2 Cl.sub.2 layer is concentrated to 
furnish a gummy residue, which on purification by silica gel 
chromatography gives silylated 4-penten-1-ol. The silylated compound is 
oxidized with OsO.sub.4 (1 mmol) and N-methylmorpholine oxide (20 mmol) in 
diethyl ether (40 ml) and water (20 ml) at room temperature for 18 h. 
NaIO.sub.4 (30 mmol) solution in water (2 ml) is added to the above 
solution and stirring is continued for 12 h. The aqueous layer is 
extracted with diethyl ether (2.times.200 ml) and evaporation of combined 
organic layers gives crude aldehyde 3. 
B. Synthesis of a `Bifunctional Units`, 4-Penten-1-hydrazine hydrochloride, 
8, and Imidazolidine Derivative, 5 
Treatment of 4-Penten-1-ol with tosylchloride in pyridine will furnish 
tosylated 6, which on treatment with benzylcarbazate in dimethylacetamide 
as described in Example 1 of Ser. No. 08/039,979, filed Mar. 30, 1993, 
provides the carbazyl derivative 7. Hydrogenation with Pd/C in MeOH/HCl 
provides the title compound 8 as hydrochloride salt. 
The aldehyde group of 3 is protected as N,N'-diphenylimidazolidine 
derivative utilizing the procedure of Giannis, et. al., Tetrahedron 1988, 
44, 7177, to furnish 4. Subsequently, 4 is treated with Bu.sub.4 NF/THF to 
deblock the silyl protecting group. The hydroxyl group of the latter 
compound is transformed into a hydrazino group via the two step procedure 
described above to yield title compound 5. 
C. Synthesis of a `Terminal Unit`, 
3-O-(t-butyldiphenylsilyl)-1-(hydrazine)-propanol hydrochloride (11, 
R.sub.Z =TBDPS, r=1). 
The title compound is prepared from propane-1,3-diol, via selective 
silylation with t-butyl-diphenylsilylchloride, followed by treatment with 
benzylcarbazate and hydrogenation as described above in Example 1(I)(B). 
D. Solution Phase Coupling of a `First Unit` and a `Bifunctional Unit` 
Aldehyde 3 and hydrazino derivative 8 are coupled in dry CH.sub.2 Cl.sub.2 
/MeOH/AcOH as described in Example 3 of Ser. No. 08/039,979, filed Mar. 
30, 1993, to furnish an intermediate hydrazone 9 (L.sub.S =CHO). The 
latter product is reduced with NaBH.sub.3 CN/AcOH to furnish a hydrazino 
linked molecule 10 (L.sub.S =N,N'-diphenylimidazolidino). Subsequently, 10 
is bis-alkylated with N1-methylformylthymine to yield 12 (R.sub.Z =H, 
L.sub.S =CH.sub.2 OH, R.sub.Y =N1-ethylthymine). 
The reactive aldehyde moiety of 12 can be regenerated by acid treatment to 
deblock the N,N-diphenyl imidazolidine. If compound 5 is used in place of 
compound 8, the aldehyde moiety can be regenerated by OsO.sub.4 
oxidation/NaIO.sub.4 cleavage of the terminal vinyl moiety (i.e., L.sub.S 
=CH.dbd.CH.sub.2) . Thus, another round of coupling is carried out 
followed by reduction and alkylation with tether or tether plus a new 
ligand. In this manner, one can place a variety of ligands on a single 
molecule, separated by an appropriate linear chain, an important feature 
for recognition of macromolecules. 
The coupling may be terminated at any point by utilizing a terminal unit, 
such as molecule 11. This compound provides a hydrazino end to couple with 
an aldehyde but bears a protected hydroxyl group, which will be deblocked 
to provide an hydroxyl moiety. 
In addition, one may choose to attach a phosphate or phosphonate group via 
terminal hydroxyl group in order to provide higher solubility to 
oligomeric unit. 
II. Automated Solid Support Synthesis of an Oligomeric Molecule Linked Via 
Hydrazino Linkages (FIG. 1) 
A. Synthesis of a 4-Penten-1-ol Attached to Solid Support 
4-Penten-1-ol is attached via a succinyl linker onto CPG following standard 
protocol (e.g., R. T. Pon in Protocols For Oligonucleotides And Analogs, 
Chapter 24, Agrawal, S., ed., Humana Press, Totowa, N.J., 1993.). The CPG 
bound 4-penten-1-ol 2 (R.sub.Z =CPG, r=1) is oxidized with OsO.sub.4, and 
the product treated with NaIO.sub.4 to yield 3 with a free aldehydo group. 
Next, a reductive coupling with bifunctional unit such as 5 furnishes 10 
bound on CPG. Subsequent alkylation with a tether such as chloroethane 
furnishes 12. In a similar manner, the deblocking of imidazolide with acid 
and repeated coupling with another bifunctional unit allows the linear 
growth of the hydrazino linked oligomer, until a desired length of the 
molecule is obtained. 
The foregoing solid support synthesis can be transferred to a robotic or 
automated synthesis technology as, for example, in the generation and 
rapid screening of libraries of molecules (see, e.g., Zuckermann, et. al., 
J. Am. Chem. Soc. 1992, 116, 10646). 
EXAMPLE 2 
Reductive Coupling 
Solution Phase Synthesis of Oligomeric Molecule Linked Via Amino Linkages 
(FIG. 2) 
The `first unit` for this synthesis is the same as used in Example 1, 
above. 
A. Synthesis of Bifunctional Units, 4-Pentenyl-1-amine, 15, and 
3-(N,N.sup.1 -diphenyl imidazolidine)-butyl-1-amine, 13. 
Treatment of 4-penten-1-ol 1 (r=1) with methlysulfonyl chloride in pyridine 
at 0.degree. C. affords the sulfonate, which on treatment with lithium 
azide in DMF gives azido derivative 14. Reduction of 14 with tributyltin 
hydride in dimethyl acetamide furnishes title compound 15. 
Yet another bifunctional unit 13 is prepared in five steps, starting from 
1. The hydroxyl group initially is protected with t-butyldiphenyl silyl 
group and the product, on oxidative cleavage using OsO.sub.4 /NaIO.sub.4, 
gives aldehyde 3 (R.sub.Z =TBDPS). The latter compound is further 
transformed to the imidazolidine derivative 4, which on desilylation 
followed by conversion of the hydroxyl group to an amino group via an 
azide, furnishes 13 (see, e.g., Lin, et. al., J. Med. Chem. 1978, 21, 
109). 
B. Coupling of First and Bifunctional Units 
To a stirred solution of aldehyde 3, amine 13 and acetic acid in CH.sub.2 
Cl.sub.2 is added NaBH(OAc).sub.3 under argon. Alternatively, amine 15 is 
used in place of amine 13. The suspension is stirred for 3 h and the 
reaction mixture, on work up as described in Example 17 of Ser. No. 
08/039,979, filed Mar. 30, 1993, gives the dimeric 17 (L.sub.S =CHO or 
CH.dbd.CH.sub.2) . Reductive amination is performed thereon generally in 
accordance with Tet. Lett., 1990, 31, 5595. Subsequently, the amino 
functionality is reductively alkylated with N1-methylformylthymine to 
provide 19 (R.sub.Z =H, L.sub.S =CH.sub.2 OH, R.sub.Y =N1-ethylthymine). 
Coupling can be repeated to obtain compounds of formula 19 with varying 
length (e.g. r=1-20). 
EXAMPLE 3 
Nucleophilic Coupling 
Oligomeric Molecules Linked Via Amino Linkages (FIG. 3) 
FIG. 3 describes a general method for assembly of amino linked linear 
molecules. Further methods are described by Niitsu, et. al., Chem. Pharm. 
Bull. 1986, 31, 1032. 
A. Synthesis of First Unit, 23 
The title compound is prepared from commercial 1, 3-propanediol, which on 
monosilylation with t-butyldiphenylsilylchloride gives protected 20 
(R.sub.Z =TBDPS, r=1). The free hydroxyl group of 20 is then converted 
into a tosyl leaving group as described by J. March in Advanced Organic 
Chemistry, Reactions, Mechanisms, and Structure, page 352, John Wiley & 
Sons, New York, 1992 to furnish 23 (R.sub.L =O-tosyl). Other suitable 
leaving groups include brosylates, nosylates, mesylates or halides. 
B. Synthesis of Bifunctional Unit, 22 
Treatment of 3-bromo-1-propanol with lithium azide in DMF furnishes 
3-azido-1-propanol, which on silylation provides 21 (L.sub.S =TBDPS, r=1). 
The azido group of 21 is reduced to provide the bifunctional unit 22. The 
nitrogen nucleophile at the reactive end of compound 22 is blocked with a 
9-fluorenylmethoxycarbonyl (FMOC) group, and the hydroxyl group at the 
dormant end is deblocked and transformed into a reactive ester as in 
Example 3(A), above to provide 23 (L.sub.S =tosyl). 
C. Coupling of First Unit and Bifunctional Unit 
Compounds 22 and 23 are reacted in presence of an appropriate base to 
furnish a secondary amine 24 as the product. Subsequently, amino group of 
24 is reductively alkylated with N1-methylformylthymine to yield compound 
19 (R.sub.Z =H, L.sub.S =H, R.sub.Y =N1-ethylthymine). In order to 
continue with coupling, the blocking group from hydroxyl moiety is removed 
and the resulting hydroxyl group connected to an active ester moiety. 
Another round of coupling takes place, followed by 
alkylation/deblocking/esterification steps until a molecule of desired 
length is obtained. 
EXAMPLE 4 
Reductive Coupling 
Solution Phase Synthesis of an Oligomeric Molecule Linked Via Hydroxylamine 
Linkages (FIG. 4) 
A. Synthesis of a `First Unit`, Amino-O-benzylalcohol, 27 
Title compound 27 is prepared in two steps starting from commercial benzyl 
alcohol 25 (L.sub.S =phenyl). In the first step, Mitsunobu reaction of 25 
with N-hydroxyphthalimide/triphenylphosphine/diethylazodicarboxylate gives 
an o-phthalimido derivative 26. Treatment of 26 with methylhydrazine gives 
27. 
B. Synthesis of a `Bifunctional Unit`, 3 
Title compound 3 is prepared in a manner described in Example 1, above. 
C. Coupling of a `First Unit` and a `Bifunctional Unit` 
A mixture of 27, 3, and acetic acid is stirred in CH.sub.2 Cl.sub.2 for 1 h 
at room temperature. The solvent is evaporated to furnish the crude oxime 
28, which on reduction with NaBH.sub.3 CN/AcOH (as described in Example 11 
of Ser. No. 08/039,979, filed Mar. 30, 1993) furnishes 29. The amino group 
of 29 is further reductively alkylated with N1-methylformylthymine to 
yield 30 (R.sub.Z =H, L.sub.S =phenyl, R.sub.Y =N1-ethylthymine). 
Alternatively, the terminal phthalimido group of 28 is deblocked with 3% 
methylhydrazine in CH.sub.2 Cl.sub.2 and the o-amino group is coupled with 
another bifunctional unit under acidic conditions. This cycle of treatment 
can be repeated with methylhydrazine and coupling until an oligomer of 
desired length is formed. All oxime linkages can be reduced in one step 
using NaBH.sub.3 CN/AcOH treatment, as described above. A common tether or 
a tether and ligand then can be attached in a single alkylation step to 
yield 30. However, this methodology provides a means to obtain an 
oligomeric unit with similar tether or tether and ligand placed onto amino 
group. 
In another method, the oxime linkage is reduced immediately after coupling 
and attachment of the tether or tether and ligand is effected. This 
modification in the procedure allows placement a tether or tether and 
ligand of choice at a preselected position within an oligomer. 
EXAMPLE 5 
Radical Coupling 
I. Solution Phase Radical Coupling Methodology for Linear Hydroxylamino 
Linked Oligomers (FIG. 5) 
A. Synthesis of a `First Unit`, O-Benzylformaldoxime, 31 (L.sub.S =phenyl) 
The title compound is prepared from benzyl alcohol following a procedure 
generally in accordance with Hart, et. al., J. Am. Chem. Soc. 1988, 110, 
1631. 
B. Synthesis of Bifunctional Unit, 2-Iodo-1-O-phthalimidoethanol, 33 
Ethyleneglycol is selectively protected with t-butyldiphenylsilyl group 
generally in accordance with Nair, et. al., Org. Prep. Procedures Int. 
1990, 22, 57. A Mitsunobu reaction of the monosilylated ethyleneglycol 
with N-hydroxyphthalimide in a manner described by Debart, et. al., Tet. 
Lett. 1992, 33, 2645, furnishes 
2-O-tert-butyldiphenylsilyl-1-O-phthalimidoethanol. Deblocking of the 
silyl group of this compound with Bu.sub.4 NF/THF, followed by iodination 
provides the desired bifunctional molecule 33. 
C. Coupling of a `First Unit` and a `Bifunctional Unit` 
Bis(trimethyltstannyl)benzopinacolate mediated intermolecular free-radical 
carbon-carbon bond-forming reaction is carried out in benzene generally in 
accordance with Example 85 of Ser. No. 08/039,979, filed Mar. 30, 1993, 
with 31 as a radical acceptor and 33 as a radical precursor to yield a 
linear hydroxylamine 29 (R.sub.Z =Phth.). 
The amino group of hydroxylamine 29 is reductively alkylated with 
N1-methylformylthymine to yield 30 (R.sub.Z =Phth., R.sub.Y 
=N1-ethylthymine). Treatment of 30 with 3% methylhydrazine/CH.sub.2 
Cl.sub.2 provides a terminal O-amino group, which on formylation with 1 
mol equivalent of HCHO/MeOH provides an oxime functionality at the 
reactive end of 30 (R.sub.Z =N.dbd.CH.sub.2) for the next round of 
coupling. Thus, the chain length is extended by reacting 30 with 33 in a 
similar manner, followed by alkylation, hydrazinolysis and formylation to 
obtain the desired length of the oligomer. The final, terminal unit 32 is 
employed when no more chain elongation is required. Deblocking with 
Bu.sub.4 NF will furnish a terminal hydroxyl group in oligomeric 30. 
II. Solid Support Synthesis 
As described in Example 1(II) (A), above, oligomeric molecules are prepared 
by attaching 31 (L.sub.S =CH.sub.2 OH) to a solid support such as CPG or 
polystyrene via an appropriate linker. Once the oligomer of desired length 
is obtained, the product is cleaved from the support to furnish fully 
deblocked product, 30. 
EXAMPLE 6 
Reductive Coupling 
Solid Support Synthesis of Covalently Linked Duplex Structures as 
Hairpins/Stem-Loops and Cyclic Oligomeric Structures Via Hydroxylamino 
Linkages (FIG. 6) 
A. Cyclic Oligomers 
An appropriate solid support, such as 35 (Y=phenyl) is prepared from 
trisubstituted benzene following a double Mitsunobu reaction described in 
Tet. Lett. 1992, 33, 2645 and loading of the product via succinyl linker 
(Z) onto a CPG support (see, e.g., R. T. Pon in Protocols For 
Oligonucleotides And Analogs, Chapter 24, Agrawal, S., ed., Humana Press, 
Totowa, N.J., 1993.). The CPG bound material is packed into a 1 .mu.M 
column and attached to an ABI DNA synthesizer 380 B model. Bis-phthalimido 
groups are deblocked with 3% N-methyl hydrazine/CH.sub.2 Cl.sub.2 solution 
to liberate desired bis-O-amino moiety, 35. Then, bifunctional reagent 3 
(R.sub.Z =TBDPS) is employed with 5% AcOH/CH.sub.2 Cl.sub.2 to give 
bis-oxime 38 (r=1). Deblocking with N-methyl hydrazine and coupling with 3 
is repeated until an oligomeric bis-oxime of desired length is obtained. 
The CPG loaded 40 is removed from the synthesizer and treated with 
ACOH/NaCNBH.sub.3 to yield reduced hydroxyl amine 39. Subsequently, all 
amines are reductively alkylated with N1-methylformylthymine to provide 40 
(R.sub.Y =N1-ethylthymine). The terminal bis-phthalimido groups of 40 are 
deblocked with N-methyl hydrazine and final conjugation with bis-aldehyde 
37 provides circularized 41, which can be further reduced, alkylated and 
removed from CPG to yield appropriate circular oligomers, such as 42. 
B. Circular/Dumbbelled Oligomers 
The method set forth in Example 6(I) (A), above, can be further modified to 
produce molecules that are constructed as linear strands but that on 
partial self-hybridization assume defined secondary structures. 
Heterobifunctional solid support 36 (Y=phenyl, Z=succinyl, L.sub.S 
=N,N'-diphenyl imidazolidino) is prepared from trisubstituted benzene 
according to the procedures of Examples 1(I)(A) and 6(I)A). The support 
bears a protected aldehydo group on one end, a succinyl linker attached to 
the CPG support on a second end, and an O-amino functionality on a third 
end. Coupling of 3 with 36 provides oxime 43. The product 43 is reduced 
with NaCNBH.sub.3 /EtOH solution, followed by alkylation with 
N1-methylformylthymine to provide a ligand 40 (R.sub.Y =N1-ethylthymine) 
with hydrogen bonding capacity. Similarly, deblocking with N-methyl 
hydrazine, followed by coupling with 3, and reductive alkylation provides 
a linear sequence bearing nucleic acid bases (A,C,G,T) in a defined order. 
Elongation of this oligomer is terminated when an appropriate length is 
achieved. The oligomer is detached from the CPG and purified by HPLC. The 
pure oligomer is able to self-hybridize to provide either circular or 
dumbbell structures of any length. 
C. Hairpin/Stem-Loop Duplexes 
In order to prepare partially or fully self-complementary molecules, 
synthesis is commenced with a molecule bearing two functionalities. One of 
these functionalities is the reactive end of the molecule and the other 
remains dormant/protected. Therefore, a heterobifunctional molecule is 
attached to the CPG to give protected 36, which is deblocked with N-methyl 
hydrazine to yield 36 with a free O-amino group. As in Example 6(I) (B), 
above, coupling with 3 in presence of acetic acid provides oxime 43. In 
two steps, the oxime is reduced and alkylated with an appropriate nucleic 
acid base (such as A,C,G,T) via a tether to furnish 44. The chain is 
elongated utilizing a three step process (deblocking, then coupling, then 
reductive alkylation) until an oligomer of desired length is obtained. 
Finally, the linear molecule is deblocked from CPG and dissolved in 
salt-buffer to provide a self complementary secondary structure as per the 
preorganized nucleic acid bases. 
The protected end of the molecule is deblocked and utilized for a 
site-specific cross-linking on the complementary strand. Such cross-linked 
molecules are expected to provide additional conformational and structural 
stability to maintain a duplex hairpin or stem-loop or dumbbelled shape. 
EXAMPLE 7 
Solid Support Synthesis of Covalently Linked Duplex/Hairpins/Stem-Loops and 
Cyclic Oligomers Via Amino Linkage 
FIG. 7 describes one method for assembly of amino linked duplexes or 
circular oligomers. Tuladhar, et. al., Tet. Lett. 1992, 33, 2203, 
describes a synthetic route for the preparation of poly-N-N.sub.1 
-dimethylethylenediamines, which method can be adapted for preparation of 
the title oligomers. 
A. Circular Polyamine, 55 
A bis-N-alkylated phenyl amine bearing a tether, T, and a ligand, L, is 
conjugated to CPG via standard procedures (see, e.g., R. T. Pon in 
Protocols For Oligonucleotides And Analogs, Chapter 24, Agrawal, S., ed., 
Humana Press, Totowa, N.J., 1993.) to provide 48 (Y=phenyl, Z=succinyl, 
R.sub.Y =N.sub.1 -alkylated pyrimidine bases or N9-alkylated purine 
bases). A complete set of appropriately alkylated amine building blocks 50 
(R.sub.L =O-tosyl, R.sub.Y =N1-ethylthymine, R.sub.Z =FMOC) next are 
prepared with a leaving group and protected secondary amine at opposite 
ends. Nucleophilic displacement of the leaving group of 50 by 
bis-N-alkylated 48 in presence of an appropriate base, such as K.sub.2 
CO.sub.3 or triethylamine results in formation of branched 51. The 
protecting group of the bis-amino function is removed and yet another 
round of base catalyzed coupling furnishes a longer oligomer. Thus, 
repetition of deblocking and coupling provides a molecule of desired 
length. To close the loop or tie the two amino branched, compound 53 
having bis-leaving groups (R.sub.L) are employed to provide a circularized 
oligomer 54. The oligomer is then deblocked from the support in a standard 
manner (see, e.g., Oligonucleotide Synthesis, Gait, M. J., ed., IRL Press, 
Oxford, 1984.) 
Alternatively, 51 is deblocked after a desired length is achieved to 
provide a linear oligomer. This oligomer is circularized by 
template-directed coupling, wherein a short complementary oligomer is 
employed to hybridize the loose ends and then carry out the coupling with 
53 to provide compound 55. Kool, et. al., J. Chem. Soc. Chem. Commun. 
1991, 1161, have reported similar ligation of reactive ends (utilizing a 
template) to yield circularized products. 
B. Hairpin and Stem-Loops Linked Via Polyamines 
As described in Example 6(I) (C), above, self-complementary hairpin and 
stem-loop structures are prepared in accordance with FIG. 7. Synthesis is 
accomplished by alkylation of N-alkyl amine 50 (R.sub.L =I) with monoamine 
49 (L.sub.S =N,N'-diphenyl imidazolidino) to furnish 52. Use of an iodo 
leaving group in 50 is preferred, due to high coupling efficiency. Also 
preferred is use of a bifunctional reagent 50 which already bears a 
nucleic acid residue attached via a tether. Thus, it is possible to 
incorporate appropriate ligands (heterocyclic bases) one at a time to 
introduce the desired recognition element into the growing oligomer. Once 
an oligomer of expected length is obtained, it is removed from the support 
by standard methods. 
The oligomer is allowed to anneal under appropriate salt concentrations to 
provide a hairpin or stem-loop structure. The development of these methods 
for cationic polyamine synthesis are attractive because their unique 
interaction with anionic DNA and presence of an active uptake system in a 
variety of cell types. 
EVALUATION 
PROCEDURE 1--Nuclease Resistance 
A. Evaluation of the resistance of oligonucleotide-mimicking macromolecules 
to serum and cytoplasmic nucleases. 
Compounds of the invention can be assessed for their resistance to serum 
nucleases by incubation of the oligonucleotide-mimicking macromolecules in 
media containing various concentrations of fetal calf serum or adult human 
serum. Labelled compounds are incubated for various times, treated with 
protease K and then analyzed by gel electrophoresis on 20% 
polyacrylamine-urea denaturing gels and subsequent autoradiography. 
Autoradiograms are quantitated by laser densitometry. Based upon the 
location of the modified linkage and the known length of the 
oligonucleotide-mimicking macromolecules it is possible to determine the 
effect on nuclease degradation by the particular modification. For the 
cytoplasmic nucleases, an HL 60 cell line can be used. A 
post-mitochondrial supernatant is prepared by differential centrifugation 
and the labelled compounds are incubated in this supernatant for various 
times. Following the incubation, compounds are assessed for degradation as 
outlined above for serum nucleolytic degradation. Autoradiography results 
are quantitated for evaluation of the macromolecules of the invention. It 
is expected that the compounds of the invention will be completely 
resistant to serum and cytoplasmic nucleases. 
B. Evaluation of the resistance of oligonucleotide-mimicking macromolecules 
to specific endo- and exo-nucleases. 
Evaluation of the resistance of natural oligonucleotides and compounds of 
the invention to specific nucleases (i.e., endonucleases, 3',5'-exo-, and 
5',3'-exonucleases) can be done to determine the exact effect of the 
macromolecule linkage on degradation. The compounds are incubated in 
defined reaction buffers specific for various selected nucleases. 
Following treatment of the products with protease K, urea is added and 
analysis on 20% polyacrylamide gels containing urea is done. Gel products 
are visualized by staining with Stains All reagent (Sigma Chemical Co.). 
Laser densitometry is used to quantitate the extent of degradation. The 
effects of the compound's linkage are determined for specific nucleases 
and compared with the results obtained from the serum and cytoplasmic 
systems. As with the serum and cytoplasmic nucleases, it is expected that 
the compounds of the invention will be completely resistant to endo- and 
exo-nucleases. 
PROCEDURE 2-5-Lipoxygenase Analysis and Assays 
A. Therapeutics 
For therapeutic use, an animal suspected of having a disease characterized 
by excessive or abnormal supply of 5-lipoxygenase is treated by 
administering a compounds of the invention. Persons of ordinary skill can 
easily determine optimum dosages, dosing methodologies and repetition 
rates. Such treatment is generally continued until either a cure is 
effected or a diminution in the diseased state is achieved. Long term 
treatment is likely for some diseases. 
B. Research Reagents 
The compounds of this invention will also be useful as research reagents 
when used to cleave or otherwise modulate 5-lipoxygenase mRNA in crude 
cell lysates or in partially purified or wholly purified RNA preparations. 
This application of the invention is accomplished, for example, by lysing 
cells by standard methods, optimally extracting the RNA and then treating 
it with a composition at concentrations ranging, for instance, from about 
100 to about 500 ng per 10 Mg of total RNA in a buffer consisting, for 
example, of 50 mm phosphate, pH ranging from about 4-10 at a temperature 
from about 30.degree. to about 50.degree. C. The cleaved 5-lipoxygenase 
RNA can be analyzed by agarose gel electrophoresis and hybridization with 
radiolabeled DNA probes or by other standard methods. 
C. Diagnostics 
The compounds of the invention will also be useful in diagnostic 
applications, particularly for the determination of the expression of 
specific mRNA species in various tissues or the expression of abnormal or 
mutant RNA species. In this example, while the compounds target a abnormal 
mRNA by being designed complementary to the abnormal sequence, they would 
not hybridize to normal mRNA. 
Tissue samples can be homogenized, and RNA extracted by standard methods. 
The crude homogenate or extract can be treated for example to effect 
cleavage of the target RNA. The product can then be hybridized to a solid 
support which contains a bound oligonucleotide complementary to a region 
on the 5' side of the cleavage site. Both the normal and abnormal 5' 
region of the mRNA would bind to the solid support. The 3' region of the 
abnormal RNA, which is cleaved, would not be bound to the support and 
therefore would be separated from the normal mRNA. 
Targeted mRNA species for modulation relates to 5-lipoxygenase; however, 
persons of ordinary skill in the art will appreciate that the present 
invention is not so limited and it is generally applicable. The inhibition 
or modulation of production of the enzyme 5-lipoxygenase is expected to 
have significant therapeutic benefits in the treatment of disease. In 
order to assess the effectiveness of the compositions, an assay or series 
of assays is required. 
D. In Vitro Assays 
The cellular assays for 5-lipoxygenase preferably use the human 
promyelocytic leukemia cell line HL-60. These cells can be induced to 
differentiate into either a monocyte like cell or neutrophil like cell by 
various known agents. Treatment of the cells with 1.3% dimethyl sulfoxide, 
DMSO, is known to promote differentiation of the cells into neutrophils. 
It has now been found that basal HL-60 cells do not synthesize detectable 
levels of 5-lipoxygenase protein or secrete leukotrienes (a downstream 
product of 5-lipoxygenase). Differentiation of the cells with DMSO causes 
an appearance of 5-lipoxygenase protein and leukotriene biosynthesis 48 
hours after addition of DMSO. Thus induction of 5-lipoxygenase protein 
synthesis can be utilized as a test system for analysis of 
oligonucleotide-mimicking compounds which interfere with 5-lipoxygenase 
synthesis in these cells. 
A second test system for oligonucleotide-mimicking compounds makes use of 
the fact that 5-lipoxygenase is a "suicide" enzyme in that it inactivates 
itself upon reacting with substrate. Treatment of differentiated HL-60 or 
other cells expressing 5 lipoxygenase, with 10 .mu.M A23187, a calcium 
ionophore, promotes translocation of 5-lipoxygenase from the cytosol to 
the membrane with subsequent activation of the enzyme. Following 
activation and several rounds of catalysis, the enzyme becomes 
catalytically inactive. Thus, treatment of the cells with calcium 
ionophore inactivates endogenous 5-lipoxygenase. It takes the cells 
approximately 24 hours to recover from A23187 treatment as measured by 
their ability to synthesize leukotriene B.sub.4. Compounds directed 
against 5-lipoxygenase can be tested for activity in two HL-60 model 
systems using the following quantitative assays. The assays are described 
from the most direct measurement of inhibition of 5-lipoxygenase protein 
synthesis in intact cells to more downstream events such as measurement of 
5-lipoxygenase activity in intact cells. 
A direct effect which oligonucleotide-mimicking compounds can exert on 
intact cells and which can be easily be quantitated is specific inhibition 
of 5-lipoxygenase protein synthesis. To perform this technique, cells can 
be labelled with .sup.35 S-methionine (50 .mu.Ci/mL) for 2 hours at 
37.degree. C. to label newly synthesized protein. Cells are extracted to 
solubilize total cellular proteins and 5-lipoxygenase is 
immunoprecipitated with 5-lipoxygenase antibody followed by elution from 
protein A Sepharose beads. The immunoprecipitated proteins are resolved by 
SDS-polyacrylamide gel electrophoresis and exposed for autoradiography. 
The amount of immunoprecipitated 5-lipoxygenase is quantitated by scanning 
densitometry. 
A predicted result from these experiments would be as follows. The amount 
of 5-lipoxygenase protein immunoprecipitated from control cells would be 
normalized to 100%. Treatment of the cells with 1 .mu.m, 10 .mu.m, and 30 
.mu.m of the compounds of the invention for 48 hours would reduce 
immunoprecipitated 5-lipoxygenase by 5%, 25% and 75% of control, 
respectively. 
Measurement of 5-lipoxygenase enzyme activity in cellular homogenates could 
also be used to quantitate the amount of enzyme present which is capable 
of synthesizing leukotrienes. A radiometric assay has now been developed 
for quantitating 5-lipoxygenase enzyme activity in cell homogenates using 
reverse phase HPLC. Cells are broken by sonication in a buffer containing 
protease inhibitors and EDTA. The cell homogenate is centrifuged at 
10,000.times.g for 30 min and the supernatants analyzed for 5-lipoxygenase 
activity. Cytosolic proteins are incubated with 10 .mu.m .sup.14 
C-arachidonic acid, 2 mM ATP, 50 .mu.m free calcium, 100 .mu.g/ml 
phosphatidylcholine, and 50 mM bis-Tris buffer, pH 7.0, for 5 min at 
37.degree. C. The reactions are quenched by the addition of an equal 
volume of acetone and the fatty acids extracted with ethyl acetate. The 
substrate and reaction products are separated by reverse phase HPLC on a 
Novapak C18 column (Waters Inc., Millford, Mass.). Radioactive peaks are 
detected by a Beckman model 171 radiochromatography detector. The amount 
of arachidonic acid converted into di-HETE's and mono-HETE's is used as a 
measure of 5-lipoxygenase activity. 
A predicted result for treatment of DMSO differentiated HL-60 cells for 72 
hours with effective the macromolecules of the invention at 1 .mu.m, 10 
.mu.m, and 30 .mu.m would be as follows. Control cells oxidize 200 pmol 
arachidonic acid/ 5 min/ 10.sup.6 cells. Cells treated with 1 .mu.m, 10 
.mu.m, and 30 .mu.m of an effective oligonucleotide-mimicking compound 
would oxidize 195 pmol, 140 pmol, and 60 pmol of arachidonic acid/ 5 min/ 
10.sup.6 cells respectively. 
A quantitative competitive enzyme linked immuno-sorbant assay (ELISA) for 
the measurement of total 5-lipoxygenase protein in cells has been 
developed. Human 5-lipoxygenase expressed in E. coli and purified by 
extraction, Q-Sepharose, hydroxyapatite, and reverse phase HPLC is used as 
a standard and as the primary antigen to coat microtiter plates. Purified 
5-lipoxygenase (25 ng) is bound to the microtiter plates overnight at 
4.degree. C. The wells are blocked for 90 min with 5% goat serum diluted 
in 20 mM Tris.cndot.HCL buffer, pH 7.4, in the presence of 150 mM NaCl 
(TBS). Cell extracts (0.2% Triton X-100, 12,000.times.g for 30 min.) or 
purified 5-lipoxygenase were incubated with a 1:4000 dilution of 
5-lipoxygenase polyclonal antibody in a total volume of 100 .mu.L in the 
microtiter wells for 90 min. The antibodies are prepared by immunizing 
rabbits with purified human recombinant 5-lipoxygenase. The wells are 
washed with TBS containing 0.05% tween 20 (TBST), then incubated with 100 
.mu.L of a 1:1000 dilution of peroxidase conjugated goat anti-rabbit IgG 
(Cappel Laboratories, Malvern, Pa.) for 60 min at 25.degree. C. The wells 
are washed with TBST and the amount of peroxidase labelled second antibody 
determined by development with tetramethylbenzidine. 
Predicted results from such an assay using a 30 mer 
oligonucleotide-mimicking compound at 1 .mu.m, 10 .mu.m, and 30 .mu.m 
would be 30 ng, 18 ng and 5 ng of 5-lipoxygenase per 10.sup.6 cells, 
respectively with untreated cells containing about 34 ng 5-lipoxygenase. 
A net effect of inhibition of 5-lipoxygenase biosynthesis is a diminution 
in the quantities of leukotrienes released from stimulated cells. 
DMSO-differentiated HL-60 cells release leukotriene B4 upon stimulation 
with the calcium ionophore A23187. Leukotriene B4 released into the cell 
medium can be quantitated by radioimmunoassay using commercially available 
diagnostic kits (New England Nuclear, Boston, Mass.). Leukotriene B4 
production can be detected in HL-60 cells 48 hours following addition of 
DMSO to differentiate the cells into a neutrophil-like cell. Cells 
(2.times.10.sup.5 cells/mL) will be treated with increasing concentrations 
of the macromolecule for 48-72 hours in the presence of 1.3% DMSO. The 
cells are washed and resuspended at a concentration of 2.times.10.sup.6 
cell/mL in Dulbecco's phosphate buffered saline containing 1% delipidated 
bovine serum albumin. Cells are stimulated with 10 .mu.m calcium ionophore 
A23187 for 15 min and the quantity of LTB4 produced from 5.times.10.sup.5 
cell determined by radioimmunoassay as described by the manufacturer. 
Using this assay the following results would likely be obtained with an 
oligonucleotide-mimicking compound directed to the 5-LO mRNA. Cells will 
be treated for 72 hours with either 1 .mu.m, 10 .mu.m or 30 .mu.m of the 
macromolecule in the presence of 1.3% DMSO. The quantity of LTB.sub.4 
produced from 5.times.10 .sup..tbd. cells would be expected to be about 75 
pg, 50 pg, and 35 pg, respectively with untreated differentiated cells 
producing 75 pg LTB.sub.4. 
E. In Vivo Assay 
Inhibition of the production of 5-lipoxygenase in the mouse can be 
demonstrated in accordance with the following protocol. Topical 
application of arachidonic acid results in the rapid production of 
leukotriene B.sub.4, leukotriene C.sub.4 and prostaglandin E.sub.2 in the 
skin followed by edema and cellular infiltration. Certain inhibitors of 
5-lipoxygenase have been known to exhibit activity in this assay. For the 
assay, 2 mg of arachidonic acid is applied to a mouse ear with the 
contralateral ear serving as a control. The polymorphonuclear cell 
infiltrate is assayed by myeloperoxidase activity in homogenates taken 
from a biopsy 1 hour following the administration of arachidonic acid. The 
edematous response is quantitated by measurement of ear thickness and wet 
weight of a punch biopsy. Measurement of leukotriene B.sub.4 produced in 
biopsy specimens is performed as a direct measurement of 5-lipoxygenase 
activity in the tissue. Compounds of the invention are applied topically 
to both ears 12 to 24 hours prior to administration of arachidonic acid to 
allow optimal activity of the compounds. Both ears are pretreated for 24 
hours with either 0.1 .mu.mol, 0.3 .mu.mol, or 1.0 .mu.mol of the 
macromolecule prior to challenge with arachidonic acid. Values are 
expressed as the mean for three animals per concentration. Inhibition of 
polymorphonuclear cell infiltration for 0.1 .mu.mol, 0.3 .mu.mol, and 1 
.mu.mol is expected to be about 10%, 75% and 92% of control activity, 
respectively. Inhibition of edema is expected to be about 3%, 58% and 90%, 
respectively while inhibition of leukotriene B.sub.4 production would be 
expected to be about 15%, 79% and 99%, respectively. 
F. Hybridization Probes. 
Multiple varieties of mRNA can be quantitated without the need to purify 
the mRNA from cellular components by first a compound of the invention 
that hybridizes to the mRNA. The compound then is immobilized on an 
insoluble solid support such as CPG (see, e.g., R. T. Pon in Protocols for 
Oligonucleotides and Analogs, pages 465-496, S. Agrawal, ed., Humana 
Press, Totowa, N.J., 1993). The sample under investigation then is 
incubated with the insoluble CPG support so that the mRNA present in the 
sample will hybridize to and become immobilized on the CPG support. 
Non-immobilized materials and components are washed off with suitable 
media and mRNA on the support then is labelled with ethidium bromide, 
biotin or a commercial radionucleotide. Measurement of the amount of label 
immobilized on the CPG support will indicate the amount of mRNA present in 
the starting sample. Such measurement will provide an indication of the 
pathophysiology of a disease state associated with the mRNA. 
Those skilled in the art will appreciate that numerous changes and 
modifications may be made to the preferred embodiments of the invention 
and that such changes and modifications may be made without departing from 
the spirit of the invention. It is therefore intended that the appended 
claims cover all such equivalent variations as fall within the true spirit 
and scope of the invention.