Site-specific functionalization of oligodeoxynucleotides for non-radioactive labelling

Disclosed are compounds consisting of a plurality of nucleosides which are covalently linked by at least one aminoalkylphosphoramidate linkage of the formula ##STR1## wherein n=2 to 6 and Nu.sub.1 and Nu.sub.2 represent nucleoside phosphates.

BACKGROUND OF THE INVENTION 
There is at present growing interest in non-radioactively labelled modified 
oligodeoxynucleotides. Biotin (Agrawal. S. et al., Nucleic Acid Research, 
14:6227-6245 (1986); Agrawal, S. Tet. Lett., 30:7025-7028 (1989)), 
florophores (Cardullo, R. A. et al., Proc. Natl. Acad. Sci. USA, 
85:8790-8794 (1988); Agrawal, S. et al., J. Cell Biology, 107:468 (1988); 
Haralambidis, J. et al., Nucleic Acids Res., 18(3):501-505 (1989)), 
intercalating (Helene, C. and J. J. Toulme, 
Oligodeoxynucleotides-Antisense Inhibitors of Gene Expression, Ed. J. S. 
Cohen, Macmillan Press, 137-166 (1989)) and chelating (Oser, A. et al., G. 
Nucleic Acid Research, 16:1181-1196 (1988)) reagents attached to synthetic 
oligonucleotides are becoming important tools of molecular biology. A 
variety of enzymatic and chemical procedures have been developed for their 
synthesis (Matthews, J. S. and L. J. Kricka, Anal. Biochem., 169:1-25 
(1988)). Central to some of these procedures are (a) the introduction of a 
reactive group at either the 3'- or 5'- terminus of the oligonucleotide 
(Agrawal. S. et al., Nucleic Acid Research, 14:6227-6245 (1986); Agrawal, 
S. Tet. Lett., 30:7025-7028 (1989); Fidanza, J. A. and L. W. McLaughlin, 
J. Am. Chem. Soc., 111:9117-9119 (1989); Nelson, P. S. et al., Nucleic 
Acid Research, 17:7187-7194 (1989)) or (b) the synthesis of modified 
nucleosides which contain the masked reactive group and are incorporated 
into the nucleic acid (Fidanza, J. A. and L. W. McLaughlin, J. Am. Chem. 
Soc. 111:9117-9119 (1989)). The presently-available methods are useful, 
but are limited in their usefulness for site specific internal 
non-radioactive labelling of synthetic oligonucleotides possible. 
SUMMARY OF THE INVENTION 
The present invention relates to a method of site specific 
functionalization of oligodeoxynucleotides for non-radioactive labelling, 
as well as to functionalized oligodeoxynucleotides and non-radioactively 
labelled oligodeoxynucleotides produced by the method. This method makes 
it possible to modify one or more selected internucleoside phosphate(s) in 
a synthetic oligodeoxynucleotide in such a manner that it (they) can be 
used to incorporate a non-radioactive material into the synthetic 
oligodeoxynucleotide. In particular, the method of the present invention 
is used to modify one or more selected internucleoside phosphates in a 
synthetic oligodeoxynucleotide , to give aminoalkylphosphoramidate 
residues or aminoalkylphosphotriester residues. The amino group(s) of the 
resulting modified residue(s) is then further reacted with a 
non-radioactive label, such as biotin, fluorescein or rhodamine (e.g., 
N-hydroxysuccinimide ester of biotin, N-caproyl amidobiotin, and a variety 
of fluorophore isothiocyanates), to produce a non-radioactively labelled 
oligodeoxynucleotide in which the label is present at a predetermined 
location or locations. 
In the present method, an H-phosphonate internucleoside linkage is oxidized 
with an appropriately protected diamine, such as 
N-1-trifluoroacetylhexanediamine, (CF.sub.3 CO NH(CH.sub.2).sub.6 
NH.sub.2), in the presence of an appropriate solvent, such as carbon 
tetrachloride, to give a phosphoramidate internucleoside linkage 
(Zwierzak, A., Synthesis, 507-508 (1975); Froehler, B. et al., Nucleic 
Acid Research, 16:4831-4839 (1989); Letsinger, R. L. et al., J. Am Chem. 
Soc., 110:4470-4471 (1988); Agrawal, S. et al., Proc. Natl. Acad. Sci. 
USA, 85:7070-7083 (1988); Jager, A. et al., Biochemistry, 27:7237-7246 
(1988)). The resulting phosphoramidate internucleoside linkage is stable 
under oligonucleotide assembly conditions using phosphoramidite chemistry 
(Caruthers, M. H. et al., Methods in Enzymology, 154:287-313 (1987)) and 
to subsequent deprotection steps. Alternatively, the H-phosphonate 
internucleoside can be oxidized, to give a phosphotriester internucleoside 
linkage, with an appropriately protected aminoalkyl alcohol in 
N-methylimidazole-triethylamine-carbon tetrachloride (e.g., 5:5:90). 
The present method can be used to produce non-radioactively labelled 
oligodeoxynucleotides which include a non-radioactive material at one or 
more sites and are useful in research and in the diagnosis and treatment 
of diseases and conditions of interest.

DETAILED DESCRIPTION OF THE INVENTION 
The present invention relates to a method of producing 
oligodeoxynucleotides which have a desired (selected) nucleotide sequence 
and which are labelled internally with a non-radioactive material or 
reporter group at one or more internucleoside linkages. In the method of 
the present invention, one or more selected internucleoside phosphate 
residues are modified to produce aminoalkylphosphoramidate residues or 
aminoalkylphosphotriester residues which are present in an 
oligodeoxynucleotide at selected positions. The amino group(s) in such 
modified (functionalized) residues is further reacted with a label or 
reporter group, resulting in production of a non-radioactively labelled 
oligodeoxynucleotide labelled internally at selected location(s). 
Briefly, the present method is carried out by oxidizing an H-phosphonate 
internucleoside linkage using an appropriately protected diamine, 
represented by the formula XCONH(CH.sub.2).sub.n NH.sub.2, in which X is a 
base labile protecting group and n can be 2 or more. For example, an 
H-phosphate internucleoside linkage is oxidized using 
N-1-trifluoroacetylhexanediamine (CF.sub.3 CONH(CH.sub.2).sub.6 NH.sub.2) 
in the presence of an appropriate solvent, such as anhydrous carbon 
tetrachloride. As a result, a primary aliphatic amine is incorporated at 
the internucleoside phosphate as phosphoramidate. In the case of 
phosphotriester linkages, oxidation is carried out using a suitably 
protected amino alcohol, represented by the formula XNH(CH.sub.2).sub.n 
OH, in which X is a base labile protecting group and n can be 2 or more. 
For example, an H-phosphonate internucleoside linkage is oxidized using 
N-1-fluoroenylmethyoxcarbonylaminohexanol F-MOC-NH(CH.sub.2).sub.6 OH in 
the presence of N-methylimidazole-triethylamine-carbon tetrachloride. The 
remaining nucleotides needed to produce the desired nucleotide sequence 
are added using art-recognized techniques, such as phosphoramidate, 
H-phosphonate chemistry or methyl phosphoramidate. (Caruthers, M. H. et 
al., Methods in Enzymology, 154:287-313 (1987); co-pending U.S. patent 
application Ser. No. 07/334,679 (Method of Synthesizing Oligonucleotides 
and Their Analogs Adaptable to Large Scale Synthesis, by S. Agrawal and P. 
Zamecnik, filed Apr. 6, 1989, the teachings of which are incorporated 
herein by reference; Agrawal, S. and J. Goodchild Tet. Lett., 
28(31):3539-3542 (1987)). After the desired oligodeoxynucleotide is 
produced, the protecting group present on the primary aliphatic amine is 
removed. The unmasked amino group can now react with one or more selected 
labels or reporter groups. As a result, the oligodeoxynucleotide is 
labelled, non-radioactively, at one or more selected internal locations. 
One or both amino groups present in the diamine react with the selected 
label. 
The method of the present invention is represented in a series of steps 
below. The following is an explanation of those steps, with reference to 
the respective reactants and steps represented below. 
##STR2## 
1. Initial coupling of two nucleotides (designated Nu.sub.1 and Nu.sub.2) 
is carried out, using H-phosphonate chemistry. Generally, Nu.sub.1 is 
bound to a solid support, such as CPG and terminates in a diemthoxytrityl 
residue (designated X). As a result, a support-bound dinucleoside 
H-phsophonate is produced (designated (II)). 
2. The support-bound dinucleoside H-phosphonate (II) is subsequently 
oxidized by being combined with an appropriately protected diamine in the 
presence of a suitable solvent, resulting in formation of a 
phosphoramidate internucleoside linkage or a phosphotriester 
internucleoside linkage (and linking of the protected diamine to the 
dinucleoside through the unprotected amino group of the diamine). The 
resulting product is designated (III). 
3. The dimethoxytrityl residue present on the unbound end of (I) is removed 
and the remaining deoxynucleotides of the desired oligodeoxynucleotide to 
be produced are added at the now free end, using phosphoramidate chemistry 
or H-phosphonate chemistry, producing a support-bound oligodeoxynucleotide 
(IV) which includes the phosphoramidate linkage produced in step (2). 
4. The protecting group Y present on the diamine is removed and the 
compound is removed from the solid support. This results in production of 
an unbound functionalized oligodeoxynucleotide (i.e., an aminoaliphatic 
oligomer or an oligodeoxynucleotide having a desired nucleotide sequence 
and an alkyl amino group present at the selected internucleoside 
phosphate(s) as a phosphoramidate or phosphotriester). 
5. The unbound functionalized oligodeoxynucleotide is reacted with an 
appropriate form of a non-radioactive material, which becomes bound to the 
amino group and serves as a label or reporter group on the 
oligodeoxynucleotide. This results in production of an 
oligodeoxynucleotide labelled site specifically with a non-radioactive 
material. The non-radioactive material can be a fluorophore, a spin label, 
an enzyme, a chelator, a heterocyclic molecule, a protein, a lipid, a drug 
derivative, an antigen, an intercalator or other organic or inorganic 
moiety. 
In a specific embodiment of the present invention, which has been used to 
produce non-radioactively labelled oligodeoxynucleotides, the following 
steps were carried out to produce a non-radioactively labelled 
oligodeoxynucleotide: 
1. Initially, (I) and Nu.sub.2 were coupled, using art-recognized 
H-phosphonate chemistry, resulting in production of a support-bound 
dinucleoside H-phosphonate. 
2. The support-bound dinucleoside H-phosphonate was oxidized, using 
N-1-trifluoroacetyldiaminohexane (NH(CH.sub.2).sub.6 NH-CO-CF.sub.3) in 
carbon tetrachloride-dioxane, resulting in formation of a phosphoramidate 
internucleoside linkage between Nu.sub.2 and Nu.sub.1. 
3. The remainder of the nucleotide sequence of the oligodeoxynucleotide was 
produced in a two-step procedure in which the dimethoxytrityl residue 
[DMTrO] was removed from the nucleotide now bound to the solid support 
(Nu.sub.2 in the reaction scheme above) and the desired nucleotides were 
added stepwise (i.e., to the now free end of the dinucleoside which, for 
convenience, can be referred to as the 5' end). 
4. The protecting group (-CO-CF.sub.3) present on NH(CH.sub.2).sub.6 
NH-CO-CF.sub.3 was removed during deprotection of oligonucleotides in 
aqueous ammonia, resulting in production of a functionalized 
oligodeoxynucleotide (one in which the previously protected amino group is 
unprotected) of the desired sequence, in which there is an 
aminoalkylphosphoramidate residue of the formula 
##STR3## 
present at the desired internucleoside phosphate linkage(s). 
5. The unbound modified oligodeoxynucleotide with the 
aminoalkylphosphoramidate residue was reacted with a non-radioactive 
material, such as biotin, fluorescein or rhodamine in appropriate form 
(e.g., N-hydroxysuccinimide ester of biotin, N-caproyl amidobiotin, 
fluorophore isothiocyanates), which becomes bound to the amino group of 
the aminoalkylphosphoramidate internucleoside linkage. 
Alternatively, a functionalized oligodeoxynucleotide of the desired 
sequence, in which there is an aminoalkylphosphotriester residue present 
at the desired internucleoside phosphate linkage(s) can be produced. In 
this case, the non-radioactive material becomes bound to the amino group 
of the aminoalkylphosphotriester internucleoside linkage. 
The production of oligodeoxynucleotides labelled at a selected site or 
sites by the present method is described in greater detail in the 
Exemplification. 
It is possible, using the present method, to produce oligodeoxynucleotides 
of desired sequence which are labelled internally at one or more 
nucleosides. The oligodeoxynucleotide backbone can be unmodified (e.g., as 
it occurs in nature) or modified (e.g., amidate, methylphosphate, 
phosphothioate, phosphotriester backbones). The label present at two or 
more sites can be the same (e.g., biotin) or different and can be present 
at as many sites as desired. As described in the Exemplification, a 17-met 
of the sequence shown has been produced, functionalized at a selected site 
or sites and labelled at the site(s) with a non-radioactive material. 
In the above description of the present method, two single nucleotides 
(designated Nu.sub.1 and Nu.sub.2) are initially joined using 
H-phosphonate chemistry and the resulting oligodeoxynucleotide is 
functionalized at the internucleoside phosphate linkage formed between 
Nu.sub.1 and Nu.sub.2. However, any number of nucleotides can be joined, 
using art-recognized techniques such as H-phosphonate chemistry, before 
modification of a selected internucleoside phosphate linkage is carried 
out. This has been carried out, as described in the Exemplification, to 
produce a 17-mer functionalized at a central internal site (oligomer 3) 
and a 17-mer functionalized at two internal phosphate linkages (oligomer 
5). Using H-phosphonate chemistry, for example, an internal nucleotide 
(e.g., a support-bound nucleotide such as Nu.sub.1) can be added to, 
resulting in production of a longer sequence (e.g. Nu.sub.10 Nu.sub.9. . . 
Nu.sub.1). The longer sequence can then be functionalized, by the method 
described above, resulting in production of a functionalized 
oligodeoxynucleotide (e.g., Nu.sub.10 .dwnarw.Nu.sub.9 . . . Nu.sub.1, in 
which the internucleoside phosphate linkage between Nu.sub.9 and Nu.sub.10 
is modified). The modified oligodeoxynucleotide can then be further 
elongated by addition of selected nucleotides to produce a modified 
oligodeoxynucleotide of desired sequence. The protecting group present can 
be removed, as described above. Alternatively, the functionalized 
oligodeoxynucleotide initially produced can be extended (by addition of 
selected nucleotides), one or more additional internucleoside phosphate 
linkages can be modified and a functionalized oligodeoxynucleotide which 
has two or more sites at which non-radioactive material can be added is, 
thus, produced. 
Protecting groups other than trifluoroacetyl (--CO--CF.sub.3), as described 
above, can also be used to protect the diamine. Other base labile 
protecting groups, such as F-moc and t-boc, may also be used. The linker 
present between the two amino groups in the diamine used can be of any 
suitable length (e.g., --(CH.sub.2).sub.2 -- to --(CH.sub.2).sub.n --); 
the length used in a particular case can be determined empirically. The 
diamine can be branched or unbranched and bi-functional or 
multifunctional. 
EXEMPLIFICATION 
Preparation of N-1-trifluoroacetylhexanediamine and Its Use for 
Site-Specific Introduction of Amino Groups into Oligodeoxynucleotides 
N-1-trifluoroacetylhexanediamine, CF.sub.3 CONH(CH.sub.2).sub.6 NH.sub.2, 
was prepared by adding ethyltrifluoroacetate (1.2 ml, 10 mmol) dropwise 
over one hour to a stirred mixture of hexanediamine (1.16g; 10 mmol) and 
triethylamine (1 ml; 7 mmol) in 20 ml methanol. The solution was stirred 
overnight. After removal of solvents, the reaction mixture was flash 
chromatographed on silica using 0-25% methanol in dichloromethane. The 
fractions containing the desired product were pooled and concentrated to 
give a colorless powder (1.1 gm, yield-42.6%) , M.pt. 52.degree.. .sup.1 
NMR (CDCl.sub.3, d, TMS=0.00) 7.1-7.2 (m 3H, NH.sub.2, NH) 3.2-3.3 (m 2H 
CO-NH-CH.sub.2) 2.8-2.9 (m 2H CH.sub.2 -NH.sub.2) 1.2-1.6 (m 8H - CH.sub.2 
- (CH.sub.2 ).sub.2 -CH.sub.2). 
To test the efficacy of (CF.sub.3 CONH(CH.sub.2).sub.6 NH.sub.2) for amino 
group introduction at specific sites of oligonucleotides, a 17-mer 
sequence, GTA AAA CGA CGG CCA GT, (oligomer 1) was made. Oligomers 
(designated 2-5 below) carrying aminohexyl residues at different sites, as 
shown by (.dwnarw.), were also made. 
______________________________________ 
Oligomer # Sequence 
______________________________________ 
1 GTA AAA CGA CGG CCA GT 
2 GTA AAA CGA CGG CCA G.sup..dwnarw. T 
3 GTA AAA CG.sup..dwnarw. A CGG CCA GT 
4 G.sup..dwnarw. TA AAA CGA CGG CCA GT 
5 G.sup..dwnarw. TA AAA CGA CGG CCA G.sup..dwnarw. T 
______________________________________ 
The steps involved for labelling, for sequence 2, are shown below: 
##STR4## 
The first coupling (represented by (a) in the above reaction) was carried 
out using H-phosphonate chemistry. This resulted in a production of a 
support-bound dinucleoside H-phosphonate (II), which was then oxidized 
with 4% N-1-trifluoroacetyldiaminohexane (I) in carbon 
tetrachloride-dioxane (8:2, v/v) for 30 minutes, resulting in production 
of (III). After oxidation with CF.sub.3 CONH(CH.sub.2).sub.6 NH.sub.2 
(step (b) of the above reaction), the assembly of the rest of the 
oligodeoxynucleotide sequence was carried out (step (c)) using 
phosphoramidite or H-phosphonate chemistry. This resulted in production of 
the oligonucleotide (IV), which was deprotected in aqueous ammonia for 6 
hours at 55.degree. C. (step (d)), resulting in formation of the 
aminohexyl oligomer (V). 
Assessment of the oligomers 1-5 was carried out. Analytical ion exchange 
HPLC of oligomer 2 showed the major peak eluting earlier than that of 
oligomer 1 with the same gradient (FIG. 1a and 1b), confirming that in 
oligomer2, one of the internucleoside linkages is a phosphoramidate 
linkage, which is non-ionic at phosphores. Sequences 3 and 4, which are 
functionalized at different sites, also showed a HPLC profile similar to 
that of oligomer 2. Oligomer 5, which is functionalized at two sites, was 
eluted even earlier (FIG. 1A, chromatograph (c)). 
When ion exchange HPLC purified oligomer 2 was checked on reversed phase 
HPLC, it gave a doublet peak in ratio of 1:2 (FIG. 1A, chromatograph (e)) 
compared to 1 (FIG. 1A, chromatograph (d)). This results from the 
diastereoisomeric nature of phosphoramidate internucleoside linkage. 
Similarly, oligomer 5 eluted as a broad peak because of two such 
diastereoisomeric linkages (FIG. 1f). Both oligomers 2 and 5 had retention 
times longer than that of oligomer 1 because of the hydrophobic nature of 
the alkyl chain present in oligomers 2 and 5. 
Reaction of oligomer 2 was carried out with biotin N-hydroxysuccinimide 
using reported conditions (Agrawal, S. et al., Nucleic Acid Research, 
14:6227-6245 (1986)). The reaction mixture after gel filtration (Sephadex 
G-25) showed two new peaks of the diastereomeric biotin adducts (FIG. 2A, 
chromatograph (b)). Similarly, reaction of oligomer 5 gave a broad peak 
ass a doublet eluting later than the unreacted material (FIG. 2B, 
chromatographs (c) and (d)). 
The method described herein provides a way for functionalizing 
oligonucleotides at one or more selected or specified sites. As described 
above, the subject method of introducing reporter groups has been carried 
out by reaction of the functionalized oligodeoxynucleotide with biotin 
active ester, resulting in production of an oligodeoxynucleotide labelled 
at the selected site(s) with biotin. In addition, the aminohexyl residue 
present was reacted in high yield with fluorescein and rhodamine 
isothiocyanate, to produce a fluorescent hybridization probe. Multiple 
labelling may increase the sensitivity of detection in diagnostic assays. 
Equivalents 
Those skilled in the art will recognize, or be able to ascertain using no 
more than routine experimentation, many equivalents to the specific 
embodiments of the invention described specifically herein. Such 
equivalents are intended to be encompassed in the scope of the following 
claims.