NON-NUCLEOTIDE-BASED-LINKER REAGENTS FOR OLIGOMERS

REACTIFS DE LIAISON GENETIQUE NON NUCLEOTIDIQUE POUR INCORPORATION DANS DES OLIGOMERES

English Abstract

2089087 9202532 PCTABS00010
Novel non-nucleotide reagents for incorporation into oligomers
are provided which optionally have a chirally pure skeleton and
non-nucleotide reagents are provided which may be coupled into a
non-nucleotide/nucleotide polymer. These reagents optionally have a
chirally pure skeleton and may include a linker arm to which a
detectable label or cross-linking agent may be conjugated.

Claims

WO 92/02532 PCT/US91/08769
Claims
1. A chirally pure non-nucleotide reagent suitable
for preparing a nucleotide/non-nucleotide polymer and
which remains chirally pure when incorporated into said
polymer which comprises a non-nucleotide monomeric unit
which has a chirally pure non-nucleotide skeleton and
connected to the skeleton has a ligand moiety and first
and second coupling groups, wherein the first coupling
group is capable of coupling the skeleton to a first addi-
tional monomeric unit, while the second coupling group
remains inactivated so as to be substantially incapable of
coupling, but which can thereafter be activated under non-
adverse conditions to couple the skeleton to a second
additional monomeric unit, wherein said nucleotide/non-
nucleotide polymer comprises at least one nucleotide
monomeric unit.
2. A non-nucleotide reagent according to claim 1
wherein at least one of the first and second additional
monomeric units comprises a nucleotide monomeric unit.
3. A non-nucleotide reagent according to claim 1
wherein said nucleotide/non-nucleotide polymer comprises
at least one phosphate diester linkage between monomeric
units.
4. A non-nucleotide reagent according to claim 3
wherein said nucleotide/non-nucleotide polymer comprises
a phosphate diester oligonucleotide.
5. A non-nucleotide reagent according to claim 1
wherein said nucleotide/non-nucleotide polymer comprises
at least one linkage between monomeric units selected from
phosphorothioate, phosphoramidate and neutral phosphate
ester linkages.

WO 92/02532 PCT/US91/08769
36
6. A non-nucleotide reagent according to claim 1
wherein said nucleotide/non-nucleotide polymer comprises
linkages between monomeric units of the following formula:
<IMG>
wherein U is oxygen, sulfur or imino; Y is -CH2-, -S-, -O-,
or -NH-; Z is -S-, -O- or -NH-; and W is alkyl, aryl,
alkoxy, aryloxy, alkylthio, arylthio, S-, O-, amino or
substituted amino.
7. A chirally pure non-nucleotide reagent according
to claim 1 wherein said non-nucleotide monomeric unit com-
prises:
<IMG>
wherein SKEL comprises a chirally pure non-nucleotide
skeleton of about 1 to about 20 carbon atoms, wherein
-NHL, Y, and Z are covalently linked to a carbon atom of
SKEL, L is a ligand; Y is -CH2-, -O-, -S- or -NH-; and
Z is -O-, -S- or -NH-.
8. A non-nucleotide reagent according to claim 7
wherein SKEL comprises a backbone of about 1 to about
10 carbon atoms between Y and Z.
9. A non-nucleotide reagent according to claim 8
wherein L comprises -Pr or a protected linker arm selected
from:

WO 92/02532 PCT/US91/08769
37
<IMG>
and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.
10. A non-nucleotide reagent according to claim 1
wherein said non-nucleotide monomeric unit comprises:
<IMG>
wherein at least one of *C is a carbon atom which com-
prises a chiral center, one of R1 and R2 is hydrogen and
the other is -NH-L wherein L is a ligand, one of R3 and R4
is hydrogen and the other is lower alkyl of about 1 to
about 10 carbon atoms; Y is -CH2-, -O-, -S- or -NH-; and Z
is -O-, -S-, or -NH-.
11. A non-nucleotide reagent according to claim 10
wherein the L comprises -Pr or a protected linker arm
selected from:
<IMG> and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.

WO 92/02532 PCT/US91/08769
38
12. A non-nucleotide reagent according to claim 11
wherein said nucleotide/non-nucleotide polymer comprises
at least one phosphate diester linkage between monomeric
units.
13. A non-nucleotide reagent suitable for preparing
a nucleotide/non-nucleotide polymer having at least one
alkyl- or aryl-phosphonate linkage between monomeric units
which comprises a non-nucleotide monomeric unit which has
a non-nucleotide skeleton and connected to the skeleton
has a ligand moiety and first and second coupling groups,
wherein the first coupling group is capable of forming an
alkyl- or aryl-phosphonate linkage between the skeleton
and a first additional monomeric unit, while the second
coupling group remains inactivated so as to be substan-
tially incapable of coupling but which can thereafter be
activated under non-adverse conditions to couple the
skeleton to a second additional monomeric unit, wherein
said nucleotide/non-nucleotide polymer comprises at least
one nucleotide monomeric unit.
14. A non-nucleotide reagent according to claim 13
wherein at least one of the first and second additional
monomeric units comprises a nucleotide monomeric unit.
15. A non-nucleotide reagent according to claim 13
wherein said alkyl- or aryl-phosphonate linkage comprises
a methylphosphonate linkage.
16. A non-nucleotide reagent according to claim 15
wherein said monomeric units comprises:
<IMG>

WO 92/02532 PCT/US91/08769
39
wherein one of R1 and R2 is hydrogen and the other is -NH-L
wherein L is a ligand; one of R3 and R4 is hydrogen and
the other is lower alkyl of about 1 to about 10 carbon
atoms: Y is -CH2-, -O-, -S-, or -NH-; and Z is -O-, -S-,
or -NH-.
17. A non-nucleotide reagent according to claim 16
wherein L comprises -Pr or a protected linker arm selected
from:
<IMG>
and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.
19. A non-nucleotide reagent suitable for preparing
a nucleotide/non-nucleotide polymer having at least one
alkyl- or aryl-phosphonate linkage between monomeric units
and which remains chirally pure when incorporated into
said polymer which comprises a non-nucleotide monomeric
unit having a chirally specific non-nucleotide skeleton
and connected to the skeleton; a ligand moiety; and first
and second coupling groups wherein the first coupling
group is capable of forming an alkyl- or aryl-phosphonate
linkage between the chirally pure skeleton and a first
additional monomeric unit while the second coupling group
remains inactivated so as to be substantially incapable of
coupling but which can thereafter be activated under non-
adverse conditions to couple the chirally pure skeleton to
a second additional monomeric unit, wherein said nucleo-
tide/non-nucleotide polymer comprises at least one nucleo-
tide monomeric unit.

WO 92/02532 PCT/US91/08769
20. A non-nucleotide reagent according to claim 19
wherein at least one of the first and second additional
monomeric units comprises a nucleoside unit.
21. A non-nucleotide reagent according to claim 19
wherein said alkyl- or aryl-phosphonate linkage comprises
a methylphosphonate linkage.
22. A chirally pure non-nucleotide reagent according
to claim 21 wherein said non-nucleotide monomeric unit
comprises:
<IMG>
wherein SKEL comprises a chirally pure non-nucleotide
skeleton of about 1 to about 20 carbon atoms, wherein
-NHL, Y and Z are covalently linked to a carbon atom of
SKEL, L is a ligand, Y is -CH2-, -O-, -S- or -NH- and
Z is -O-, -S- or -NH-.
23. A non-nucleotide reagent according to claim 22
wherein SKEL comprises a backbone of about 1 to about
10 carbon atoms between Y and Z.
24. A non-nucleotide reagent according to claim 23
wherein L comprises -Pr or a protected linker arm selected
from:
<IMG> and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.

WO 92/02532 PCT/US91/08769
41
25. A non-nucleotide reagent according to claim 19
wherein said non-nucleotide monomeric units comprises:
<IMG>
wherein at least one of *C is a carbon which comprises a
chiral center, one of R1 and R2 is hydrogen and the other
is -NH-L wherein L is a ligand; one of R3 and R4 is
hydrogen and the other is lower alkyl of about 1 to about-
10 carbon atoms; Y is -CH2-, -O-, -S-, or -NH-; and Z is
-O-, -S-, or -NH-.
26. A non-nucleotide reagent according to claim 25
wherein L comprises -Pr or a protected linker arm selected
from:
<IMG> and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.
27. A non-nucleotide reagent according to claim 26
wherein said alkyl- or aryl-phosphonate linkage comprises
a methylphosphonate linkage.
28. A chirally pure non-nucleotide reagent of the
formula:
<IMG>

WO 92/02532 PCT/US91/08769
42
wherein SKEL comprises a chirally pure non-nucleotide
skeleton of about 1 to about 20 carbon atoms, wherein
-NHL, Y and Z are covalently linked to a carbon atom of
SKEL, L is a ligand, -YCp1 is a first coupling group and
-ZCP2 is a blocked second coupling group; where Y is -CH2-,
-O-, -S-, or -NH-; and Z is -O-, -S-, or -NH-.
29. A non-nucleotide reagent according to claim 28
wherein the first coupling group, -YCp1 is selected from
<IMG> , <IMG> and <IMG>
wherein X1 is halogen or substituted amino; X2 is halogen,
substituted amino or 0; R5 is alkyl, optionally substi-
tuted alkoxy or optionally substituted aryloxy; and R6 is
alkyl, optionally substituted alkoxy or optionally substi-
tuted aryloxy; or, if X2 is 0, optionally hydrogen; U is
oxygen, sulfur or imino, V is O, S, or substituted amino;
and W is alkyl, aryl, alkoxy, aryloxy, alkylthio, aryl-
thio, S, O, amino or substituted amino.
30. A non-nucleotide reagent according to claim 29
wherein the blocked second coupling group, -ZCp2, has a
protecting group cleavable under non-adverse deblocking
conditions to recover the second coupling group, -ZH.
31. A non-nucleotide reagent according to claim 30
wherein the first coupling group is
<IMG>
wherein R5 is methyl and X1 is diisopropylamino.

WO 92/02532 PCT/US91/08769
43
32. A non-nucleotide reagent according to claim 31
wherein said ligand comprises -Pr or a protected linker
arm selected from:
<IMG> and
<IMG>
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.
33. A non-nucleotide reagent according to claim 32
wherein n and m are independently integers between 1
and 5.
34. A non-nucleotide reagent according to claim 33
wherein Z is oxygen.
35. A chirally pure non-nucleotide reagent of the
formula:
<IMG>
wherein *C is a carbon atom which comprises a chiral cen-
ter, wherein one of R1 and R2 is hydrogen and the other is
-NH-L where L is a ligand moiety; one of R3 and R4 is
hydrogen and the other is lower alkyl of about 1 to about
10 carbon atoms, -YCp1 is a first coupling group and -ZCP2
is a blocked second coupling group; where Y is -CH2-, -O-,
-S- or -NH-; and Z is -O-, -S-, or -NH-.
36. A non-nucleotide reagent according to claim 35
wherein the first coupling group, -YCp1 is selected from

WO 92/02532 PCT/US91/08769
44
<IMG> , <IMG> and <IMG>
wherein X1 is halogen or substituted amino; X2 is halogen,
substituted amino or O; R5 is alkyl, optionally substi-
tuted alkoxy or optionally substituted aryloxy; and R6 is
alkyl, optionally substituted alkoxy or optionally substi-
tuted aryloxy; or, if X2 is O, optionally hydrogen; U is
oxygen, sulfur or imino, V is O, S, or substituted amino;
and W is alkyl, aryl, alkoxy, aryloxy, alkylthio, aryl-
thio, S, O, amino or substituted amino.
37. A non-nucleotide reagent according to claim 36
wherein the blocked second coupling group, -ZCp2, has a
protecting group cleavable under non-adverse deblocking
conditions to recover the second coupling group, -ZH.
38. A non-nucleotide reagent according to claim 37
wherein said protecting group is a dimethoxytrityl group.
39. A non-nucleotide reagent according to claim 37
wherein the first coupling group is
<IMG>
wherein R5 is methyl and X1 is diisopropylamino.
40. A non-nucleotide reagent according to claim 39
wherein said ligand comprises -Pr or a protected linker
arm selected from:
<IMG> and
<IMG>

WO 92/02532 PCT/US91/08769
wherein n and m are independently integers from about 1 to
about 15 and Pr is a protecting group removable under non-
adverse conditions.
41. A non-nucleotide reagent according to claim 40
wherein n and m are independently integers between 1
and 5.
42. A non-nucleotide reagent according to claim 40
wherein Z is oxygen.
43. A non-nucleotide reagent according to claim 35
wherein said ligand comprises -Pr or a protected linker
arm selected from:
<IMG> and
<IMG>
wherein n and m are independently integers between 1
and 15 and Pr is a protecting group removable under non-
adverse conditions.
44. A chirally pure non-nucleotide reagent suitable
for preparing a nucleotide/non-nucleotide polymer and
which remains chirally pure when incorporated into said
polymer which comprises a non-nucleotide monomeric unit
which has a chirally pure non-nucleotide skeleton and
connected to the skeleton has a ligand moiety and first
and second coupling groups, wherein the first coupling
group couples the skeleton to a support by a linkage which
may be cleaved under non-adverse conditions, while the
second coupling group remains inactive, but which can
thereafter be activated under non-adverse conditions to
couple the skeleton to an additional monomeric unit,

WO 92/02532 PCT/US91/08769
46
wherein said nucleotide/non-nucleotide polymer comprises
a nucleotide monomeric unit.
45. A non-nucleotide reagent according to claim 44
wherein said skeleton is coupled to said support by an
ester linkage.
46. A non-nucleotide reagent according to claim 44
wherein said nucleotide/non-nucleotide polymer comprises
at least one alkyl- or aryl-phosphonate linkage between
monomeric units.
47. A non-nucleotide reagent suitable for preparing
a nucleotide/non-nucleotide polymer having at least one
alkyl- or aryl-phosphonate linkage between monomeric units
which comprises a non-nucleotide monomeric unit which has
a non-nucleotide skeleton and connected to the skeleton
has a ligand moiety and first and second coupling groups,
wherein the first coupling group couples the skeleton to
a support by a linkage which may be cleaved under non-
adverse conditions, while the second coupling group
remains inactive, but which can thereafter be activated
under non-adverse conditions to couple the skeleton to an
additional monomeric unit, wherein said nucleotide/non-
nucleotide polymer comprises a nucleotide monomeric unit.
48. A non-nucleotide reagent according to claim 47
wherein said skeleton is coupled to said support by an
ester linkage.
49. An oligomer which comprises at least about
8 monomeric units wherein at least one monomeric unit
comprises a non nucleotide monomeric unit as defined in
claim 1.

WO 92/02532 PCT/US91/08769
47
50. An oligomer according to claim 49 which com-
prises from about 1 to about 5 independently selected non-
nucleotide monomeric units.
51. An oligomer which comprises at least about
8 monomeric units wherein at least one monomeric unit
comprises a non-nucleotide monomeric unit as defined in
claim 13.
52. An oligomer according to claim 51 which com-
prises a methylphosphonate oligomer.
53. An oligomer which comprises at least about
8 monomeric units wherein at least one monomeric unit
comprises a non-nucleotide monomeric unit as defined in
claim 19.
54. An oligomer according to claim 53 which com-
prises a methylphosphonate oligomer.
55. An oligomer according to claim 54 which com-
prises from about 1 to about 5 independently selected non-
nucleotide monomeric units.
56. An oligomer which comprises at least about
8 monomeric units wherein at least one monomeric unit
comprises a non-nucleotide monomeric unit according to
claim 23.
57. An oligomer according to claim 56 which com-
prises a methyl phosphonate oligomer.
58. An oligomer according to claim 57 which com-
prises from about 1 to about 5 independently selected non-
nucleotide monomeric units.

WO 92/02532 PCT/US91/08769
48
59. An oligomer according to claim 58 where n and m
are independently integers from 1 to 5.
60. An oligomer which comprises at least 8 monomeric
units wherein at least one monomeric unit comprises a non-
nucleotide monomeric unit according to claim 26.
61. An oligomer according to claim 60 which com-
prises a methylphosphonate oligomer.
62. An oligomer according to claim 61 which com-
prises from about 1 to about 5 independently selected non-
nucleotide monomeric units.
63. An oligomer according to claim 62 wherein n and
m are independently integers from 1 to 5.

Description

~V092/0~53~ PCT/US91/08769
20~9~387
DESCRIPTION
Improved Non-Nucleotide-Based Linker Reaaents
For Oliqomers
Backaround of the Invention
Non-nucleotide based linking reagents ~or labelling
oligonucleotides have a distinct advantage over nucleotide
based reagents in that they do not adversely affect the
normal base pairing between an oligonucleotide and a
target nucleic acid and allow attachment at any ~ocation
within the oligonucleotide or on its termini. Several
non-nucleotide based linking reagents have already been
described for use in labelling standard oligodeoxy-
ribonucleotides, i.e. having a phosphodiester backbone(Lyle J. Arnold, Jr., et al., "Non-Nucleotide Linking
Reagents for Nucleotide Probes", PCT WO 8902439 assigned
to Gen-Probe, Inc.). However, those non-nucleotlde rea-
gents were of mixed chirality. ~hose reagents are coupled
into the phosphodiester backbone oiE an oligonucleotide by
chemical synthesis as disclosed thl~rein.
~ ecently, other non-nucleotid6~based linking raagents
have been described. For example, one of the reagents
referred to specifically in the Gen-Probe patent applica-
tion has also been described by Paul S. Nelson et al.,(Nucleic Acids Res., 198~, vol. 17, p. 7179). The syn-
thesis o~ polyamide-oligonucleotide probes for use in
attaching nonisotopic labels has also been ~e~cribed by
J. Haralambidis et al. ~Nucleic Acids Res., 1990, vol. 18,
p. 501).
Summary of the Invention
One aspect o~ the present invention provides non-
nucleotide reagents which are suitable ~or preparing
nucleotide/non-nucleotide polymers and which remain
chirally pure when incorporated in a nucleotide/non-
nucleotide polymer. These non-nucleotide reagents
SUBSTITUTE SHEE~T
` ~. "`
` . . . `
. . ~ . " . ` ` -` . ~ . `. ~ `
` . ` ~ . .
` ` . . ~ ~ . : `
.. . ;, ` `

W~9~/0253 PCT/US91/08769
20~90~7
comprise: a non-nucleotide monomeric unit which has an
enantiomerically (or chirally) pure non-nucleotide
skeleton and, connected to the skeleton, has a ligand
moiety and first and second coupling groups. The first
coupling group is capable of coupling the skeleton to a
first additional monomeric unit, while the second coupling
group remains inactivated so as to be substantially
incapable of coupling, but the second coupling group can
thereafter be activated under non-adverse conditions to
couple the skeleton to a second additional monomeric unit,
wherein said nucleotide/non-nucleotide polymer comprises
at least one nucleotide monomeric unit.
In another aspect, the present invention provides
non-nucleotide reagents which are useful in preparing
nucleotide/non-nucleotide polymers which have alkyl- or
aryl- phosphonate diester linkag~as between monomeric
units. The non-nucleotide reagent comprises a non-
nucleotide monomeric unit which has a non-nucleotide
skeleton and connected to the skeleton has a ligand moiety
and first and second coupliny groups, wherein the first
coupling group is capable of forming an alkyl- or aryl-
phosphonate linkage between the skeleton and a first
additional monomeric unit whila the second coupling group
remains inactivated so as to be substantially incapable of
coupling, but which second coupling group can thereafter
be activated under non-adverse conditions to couple the
skeleton to a second additional monomeric unit, wherein
said nucleotide/non-nucleotide polymer comprises at least
one nucleotide monomeric unit.
According to one preferred aspect of the present
invention, novel non-nucleotide based reagen~s are
provided which are useful in preparing nucleotid~/non-
nucleotide polymers which have intermonomeric unit alkyl-
or aryl- phosphonate linkages. These non-nucleotide
reagents comprise enantiomerically pure non-nucleotide
monomeric units which have an enantiomerically (or
chirally) pure non-nucleotide skeleton and connected to
SlJBSrlTUTE SHEET
., . . . ~ . " ~ ` : ,
~` . . . , ,.. , ~ , ,
. . ``, ` `. ` ` . " ,
, . . , " -

W~)~)2/~)~53~ 2 0 ~ 9 0 8 7 PCT/~S91/08769
the skeleton have a ligand moiety and first and second
coupling groups wherein the first coupling group is
capable of forming an alkyl- or aryl- phosphonate linkage
between the enantiomerically pure skeleton and a first
additional monomeric unit, while the second coupling group
remains inactivated so as to be substantially incapable of
coupling, but which second coupling group can thereafter
be activated under non-adverse conditions to couple the
chirally specific skeleton to a second additional mono-
meric unit, wherein said nucleotide/non-nucleotide polymer
comprises at least one nucleotide monomeric unit.
The-ligand moiety may comprise a linker-arm group
which may participate in conjugation reactions upon its
activation or deprotection, a detectable chemical moiety
or label or a side arm to which a detectable chemical
moiety or label has been attached prior to inltiating
synthesis of the polymer. Suitable chemical moieties
include detectable labels, chelators, catalysts, nucleo-
lytic moieties, drug carriers, hormone receptors, sub-
stances which enhance oligomer uptake by cells, haptensfor hormone receptors, and the like. Suitable chemical
moieties include psoralen and analogs thereof, acridines
and analogs thereof, porphyrins and porphyrin analogs,
cyclic chelators and the like.
Definitions
As used herein, the following terms have the follow-
ing meanings, unless expressly stated to the contrary:
The term "nucleotide" refers to a subunit of a
nucleic acid consisting of a phosphate group, a 5 carbon
sugar and a nitrogen containing base. In RNA the 5 carbon
sugar is ribose. In DNA, it is a 2-deoxyribose. The term
also includes analogs of such subunits.
The term "nucleotide multimer" refers to a chain of
nucleotides linked by phosphodiester bonds, or analogs
thereof.
SU~I-ITUTE ~IEET
,, -. :, ,
~ . , , ~ ' .
'- : : `

W~2/1)2S3~ PCT/US9l/08769
2~9087
An "oligonucleotide" is a nucleotide multimer gen-
erally about 10 to about loo nucleotides in length, but
which may be greater than 100 nucleotides in length. They
are usually considered to be synthesized from nucleotide
monomers, but may also be obtained by enzymatic means
An "deoxyribooligonucleotide" is an oligonucleotide
consisting of deoxyribonucleotide monomers.
A "polynucleotide" refers to a nucleotide multimer
generally about 100 nucleotides or more in length. These
are usually of biological origin or are obtained by enzy-
matic means.
A "nucleotide multimer probe" is a nucleotide multi-
mer having a nucleotide sequence complementary with a
target nucleotide se~uence contained within a second
nucleotide multimer, usually a polynucleotide. Usually
the probe is selected to be perfect].y complementary to the
corresponding base in the target sequence. However, in
some cases it may be ade~uate or e~en desirable that one
or more nucleotides in the probe not be complementary to
the corresponding base in the target sequence.
A "non-nucleotide monomeric u~it" refers to a mono-
meric unit which does not signifi.cantly participate in
hybridization of a polymer. Such monomeric units must
not, for example, participate in any significant hydrogen
bonding with a nucleotide, and would exclude monomeric
units having as a component, one of the 5 nucleotide bases
or analogs thereof.
A "nucleotide/non-nucleotide polymer" refers to a
polymer comprised of nucleotide and non-nucleotide mono-
meric units.
An "oligonucleotide/non-nucleotide multimer" is a
multimer generally of syntnetic origin having less than
100 nucleotides, but which may contain in excess of 200
nucleotides and which contains one or more non-nucleotide
monomeric units.
A "monomeric unit" refers to a unit of either a
nucleotide reagent or a non-nucleotide reagent of the
SUBSTITUTE ~HEET
. . . ..
. . .'
- . .~ . ` . . .
- ~ . . . . .
- . . . .' .
- ..
' . ~ .- . . .
.

W~2/0253~ PCT/US91/087h9
20g90~7
present invention, which the reagent contributes to a
polymer.
A "hybrid" is the complex formed between two nucleo-
tide multimers by Watson-Crick base pairings between the
complementary bases.
The term "oligomer" refers to oligonucleotides, non-
ionic oligonucleoside alkyl- and aryl-phosphonate analogs,
phosphorothioate analogs of oligonucleotides, phosphor-
amidate analogs of oligonucleotides, neutral phosphate
ester oligonucleotide analogs such as phosphotriesters,
and other oligonucleotide analogs and modified oligo-
nucleotides, and also includes nucleotide/non-nucleotide
polymers. The term also includes nucleotide/non-nucleo-
tide polymers wherein one or more of the phosphorous group
linkages between monomeric units has been replaced by a
non-phosphorous group linkage such 2lS a formacetal linkage
or a carbamate linkage.
The term "alkyl- or aryl-phosphonate oligomer" refers
to nucleotide oligomers (or nucleotide/non-nucleotide
polymers) having internucleoside (or intermonomer) phos-
phorus group linkages wherein at le~st one alkyl- or aryl-
phosphonate linkage replaces a phosphodiester linkage.
The term "methylphosphonate oligomer" (or "MP-oli-
gomer") refers to nucleotide oligomers (or nucleotide/non-
nucleotide polymer) having internucleoside (or intermono-
mer) phosphorus group linkages wherein at least one
methylphosphonate internucleoside linkage replaces a
phosphate diester internucleoside linkage.
The term "nucleoside" includes a nucleosidyl unit and
is used interchangeably therewith.
In some of the various oligomer sequences listed
herein "P" in, e.g., as in ApA represents a phosphate
diester linkage, and ''Pll in, e.g., as in C~G represents a
methylphosphonate linkage. Certain other sequences are
depicted without the use of p or ~ to indicate the type of
phosphorus diester linkage. In such occurrances, A as in
ATC indicates a phosphate diester linkage between the
8UBSTITUTE SHEET
.~ " ' ~ .'
.,
, r

W~2/0253' 2 ~ PCT/US91/08769
3'-carbon of A and the 5' carbon of T, whereas A, ~TC or
ATC indicates a methylphosphonate linkage between the
3'-carbon of A and the 5'-carbon of T or T.
The term "non-adverse conditions" describes condi-
tions (of reaction or synthesis) which do not substan-
tially adversely affect the polymer skPleton and its
sugar, base, linker-arm and label components' nor the
monomeric reagents. One skilled in the art can readily
identify functionalities, coupling methods, deprotection
procedures and cleavage conditions which meet these
criteria.
The term "deblocking conditions" describes the
conditions used to remove the blocking (or protecting)
group from the 5'-OH group on a ribose or deoxyribose
group.
The term "deprotecting conditions" describes the
conditions used to remove the prot.ecting groups from the
nucleoside bases.
ri~ Description of the Drawinqs
Figures lA, lB and lC depict: the ~ormulas of non-
nucleotide reagents of the present invention having Fmoc-
protected linker arms.
Figure 2 depicts a synthetic scheme for preparing
non-nucleotide reagents of the present invention having
C2, C4 and C6 linker arms.
Figure 3 depicts a synthetic scheme for preparing
non-nucleotide reagents of the present invention having
C8, ClO and C12 linker arms.
Figure 4 depicts a synthetic scheme for a psoralen
reagant which may form a psoralen conjuyate with the
linXer arm of one of the non-nucleotide reagents of the
present invention.
Detailed DescriPtion of the Invention
According to the present invention, we have invented
certain novel non-nucleotide based linking reagents which
SU8~1TUTE SHEET
.. . ..
. ` ... ; . .... .. . . .

W0~2l0253~ PCT/US9t/0~769
` 2~ 7
are especially useful in incorporating ligands such as
cross-linking agents in oligomers, especially alkyl- or
aryl- phosphonate oligomers. These reagents may be
coupled into an oligomer using the automated coupling
chemistry used for coupling the nucleotide based phos-
phonamidite monomers. The resulting modified oligomers,
including alkyl- or aryl- phosphonate oligomers, contain
nucleophilic primary amines through which a variety of
secondary compounds may be attached by standard aqueous
chemistries already known in the art. Examples of secon-
dary compouncls include intercalators, alkylators, photo-
activated reactive moieties such as psoralens, chelating
agents, etc. We believe that by applying such chemis-
tries, we will be able to increase the potency of methyl-
phosphonate oligomers as therapeutic agents.
In a preferred aspect of the present invention, thenon-nucleotide based linking reagents are prepared in a
chirally pure form. ~oreover, these non-nucleotide rea-
gents remain chirally pure when incorporated in oligomers
~non-nucleotide/nucleotide polymers). This advantage may
be critical when it is desired or desirable to direct a
label to a particular location when the oligomer is hybri-
dized to a corresponding target nucleic acid. In a parti-
cularly preferred aspect, the hydrocarbon skeleton of
these reagents comprises a reduction product of threonine.
Since the four enantiomers of threonine are commercially
available, non-nucleotide reagents having a chirally pure
skeletons derived from any one of the four stereoisomers
of threonine may be prepared.
The choice of threonine as the starting material to
supply the chirally pure skeleton for some of these rea-
gents has additional advantages. First, a three carbon
skeleton is available for insertion into the phosphorus
backbone of the oligomer, which closely resembles the
three carbon spacing of traditional deoxyribose groups.
Second, reduced threonine has a primary hydroxyl and a
secondary hydroxyl which enables the subsequent
~:UBgl-ITUTE SHEET i.
,
.: , ` `, ` . . . `
, . . . .
. : . .

W092/0253' PCT/US91/08769
2~087
protection, deprotection, blocking, deblocking steps and
derivatization steps to proceed in improved yields.
According to one aspect of the present invention, we
have functionalized these non-nucleotide reagents in such
a way that an alkyl- or aryl- phosphonate diester linkage
results when these non-nucleotide reagents are inserted
into the phosphorus backbone of the oligomer. Such
reagents couple to nucleotides or other non-nucleotide
reagents in high yield. Furthermore, we have demonstrated
that these non-nucleotide reagents can be used for the
attachment of psoralen analogs to the oligomers. These
psoralen analogs are attached to a non-nucleotide reagent
using a novel chemistry (See Example 13).
General Pro~erties of Non-Nucleotide Reaqents
Thus, in general, the present invention provides a
non-nucleotide reagent, with a non-nucleotide monomeric
unit which can be coupled synthetically with specific
nucleotide monomeric units from nucleotide reagents, to
produce a defined sequence polymer which is comprised of
nucleotide and non-nucleotide mono~neric units. Said non-
nucleotide reagent also possesses a ligand which may
comprise a linker-arm moiety which may participate in
conjugation reactions once the lin~er-arm has been depro-
tected, or may comprise a side-arm to which a useful
desired chemical moiety has been attached prior to incor-
porating the non-nucleotide reagent in the polymer. In
general, the techniques for linking moieties to the linker
arm may be similar to the techniques for linking labels to
groups on proteins. However, modifications of such tech-
niques may be required. Examples of useful chemistriesinclude the reaction of alkylamines with active esters,
active imines, arylhalides, or isothiocyanates, and the
reaction of thiols with maleimides, haloacetyls, etc. (for
further potential techniques see G.M. Means and R.E.
Feeney, "Chemical Modification of Proteins", Holden-Day
Inc., 1971; R.E. Feeney, Int. J. Peptide Protein Res.,
~lJBSTlTUTE SHEET
`` ~ ; :. ! ~ ` ,
: ' ' ": ' ~ ' : ' ; :

W~2/0253' PCT/US91/08769
20~3~7
Vol. 29, 1987, p 145-161). Suitable protecting groups
which can be used to protect the linker arm functional
group during formation of a polymer are also similar to
those used in protein chemistry (see for example, "The
5 Peptides: Analysis and Synthesis, Biology," Vol. 3, ed.
E. Gross and J. Meienhofer, Academic Press, 1971). Due to
the chemical nature of the non-nucleotide reagent, it may
be placed at any desired position within the nucleotide
monomer sequence. This makes it possible to design a wide
10 variety of properties into polymers which contain nucleo-
tide monomers. These include: (1) attachment of specific
chemical moieties at any desired location within the poly-
mer, such moieties can include (but are not limited to)
detectable labels, intercalating agents, chelators, drugs,
15 hormones, proteins, peptides, haptens, radical generators,
nucleolytic agents, proteolytic agents, catalysts, recep-
tor binding substances, and other binding substances of
biological interest, and agents which modify DNA transport
across a biological barrier tsuch as a membrane), and sub-
20 stances which alter solubility of a nucleotide multimer.
This means that it is possible to position such l~bels and
intercalating agents adjacent to any desired nucleotide;
(2) the ability to immobilize the defined sequence to a
solid support employing its linker-arm for conjunction to
25 a chemical moiety of said support in order to construct,
for example, nucleotide affinity supports; (3) the ability
to attach multiple chemical moieties to the polymer
through linker-arms by incorporating multiple non-
nucleotide monomeric units into the polymers; (4) the
30 ability to construct polymers which differ from naturally
occurring polynucleotides in that they have altered
activities with proteins and enzymes which act on poly-
nucleotides. For exampla, the placement of the non-
nucleotide monomeric unit on the 3' terminus of an other- ;~
35 wise pure polynucleotide imparts resistance to degradation
by snake venom phosphodiesterase. Such non-nucleotide r
monomeric units may create specific cleavag2 sites for
8UBSTITUTE SHEE~
` :
.
.. .;,
.
. ~ ` ; . ,. ` .:
- . ~.,; .
,

WO~l/n253~ PCT~US91/08769
2~90&7
other nucleases; (5) the ability to construct hybridiza-
tion probes by interspersing hybridizable nucleotide and
non-nucleotide monomeric units. For example, a mixed
bloc~ synthesis of nucleotide and non-nucleotide monomers
5 can be produced, whereby a defined sequence of nucleotide
monomers are synthesized followed by a stretch of the one
or more non-nucleotide monomeric units followed by second
block of defined sequence nucleotide monomers; (6) the
ability to construct synthetic probes which simultaneously
lO detect target nucleotide multimers which differ by one or
more base pairs. This is accomplished by using the non-
nucleotide reagent described herein to replace the nucleo-
tides in the probe with non-nucleotide monomeric units at
sites where differences occur in the nucleotide sequence
lS of the vario~ls target nucleotide multimers.
In a preferred form of the invention, labelled oligo-
mers are constructed with a defined sequence comprised of
nucleotide and non-nucleotide monomers. In another pre-
ferred form of the invention, the non-nucleotide monomeric
20 units are used to connect two or more defined sequence
nucleotide multimers, and the non-nucleotide monomeric
units are chemically labelled for use in cross-linking
reactions.
In yet another preferred embodiment, the non-nucleo-
25 tide reagent is constructed in a manner to permit it to be
àdded in a step-wise fashion to produce a mixed nucleo-
tide/non-nucleotide polymer employing one of the current
DNA synthesis methods. Such nucleotide and non-nucleo-
tide reagents normally add in a step-wise fashion to
30 attach their corresponding monomeric units to a growing
oligomer chain which is covalently immobilized to a solid
support. Typically, the first nucleotide is attached to
the support through a cleavable es~er linkage prior to the
initiation of synthesis. Step-wise extension of the
35 oligomer chain is normally carried out in the 3' to 5'
direction. For standard DNA and RNA synthesis methods,
see ~or exampIe, "Synthesis and Applications of DNA and r
~;UBSTITUTE SHEET
` `, , ;
.. . . ~. ` . .
~ . . ` . ` ` .` ::- : ` `

wo ~2/n2s3~ Pcr/us~l/n8769
2~9~7
RNA" ed. S.A. Narang, Academic Press, 1987, and M.J. Gait,
"Oligonucleotide Synthesis", IRL Press, Wash. D.C. U.S.A.,
1984. When synthesis is complete, the polymer is cleaved
from the support by hydrolyzing the ester linkage men-
tioned above and the nucleotide originally attached to thesupport becomes the 3' terminus of the resulting Oligomer.
By analogy, an alternative way to introduce a non-nucleo-
tide monomeric unit is to similarly attach it to a DNA
synthesis support prior to initiation of DNA synthesis.
In a preferred embodiment the non-nucleotide monomeric
unit is attached to a DN~ synthesis support through an
ester linXage formed using the free alcohol form of the
non-nucleotide monomer.
Accordingly, the present invention provides a non-
nucleotide reagent for preparing polymers which contain amixture of nucleotide and non-nucl~otide monomeric units.
Said non-nucleotide monomers additionally may contain one
or more protected linker-arms or one or more linker-arms
con~ugated to a desired chemical moiety such as a label,
a cross-linking agent or an intercalating agent.
Such a non-nucleotide monomer additionally possesses
two côupling groups so as to permit its step-wise inclu-
sion into a polymer of nucleotide and non-nucleotide
monomeric units. A first one of said coupling groups has
the property that it can couple efficiently to the termi-
nus of a growing chain of monomeric units. The second of
said coupling groups is capable of further extending, in
a step-wise fashion, the growing chain of mixed nucleotide
and non-nucleotide monomers. This requires that the
second coupling group be inactivated while the first coup-
ling group is coupling, so ~s not to substantially couple
` at that time, but can thereafter be activated so as to
then couple the non-nucleotide monomeric unit. The "inac-
tivation" is preferably accomplished with a blocking group
on the second coupling group, which can be removed to
"activate" the second coupling group. However, it is
within the scope of the invention that such "inactivation"
SUBSTITUTE SHE~T
`. ,~, .
`;:` -.
' '::
. ,
', . ` ; ,:: -

w~ ~2/n2s3~ 9 ~ ~ 7 PCT/US91/~769
and "activation" might be accomplished simply by changing
reaction conditions (e.g., pH, temperature, altering the
concantration of some other component in the reaction
system) with second coupling groups of a suitable chemical
structure, which also lend themselves to inactivation and
activation by such techniques. Said coupling groups per-
mit the adjacent attachment of either nucleotide or non-
nucleotide monomeric units. In a preferred embodiment
said coupling groups operate through coupling and deblock-
ing and deprotection steps which are compatible with oneof the standard DNA synthesis methods.
Such methods require that synthesis occur undirec-
tionally and that all coupling cleavage and deblocking or
deprotection steps occur under "non-adverse" conditions,
that is they do not substantially adversely affect the
polymer skeleton and its sugar, base, linker-arm and label
components nor the monomeric reagent:s. One skilled in the
art can readily identify funct:ionalities, coupling
methods, deblocking and deprotection procedures, and
cleavage conditions which meet these criteria (see, for
example, the Gait reference, supra).
The non-nucleotide monomer prel.`erably has a skeleton,
to the ends of which the coupling groups are linked. The
skeleton is preferably an acyclic one to twenty atom
chain, and preferably an acylic hydrocarbon chain of from
one to twenty carbon atoms.
Preferred Non-Nucleotide Reaqents
Preferred non-nucleotide reagents comprise non-
nucleotide monomeric units in which the skeleton has a
backbone of about 2 to about l0 carbon atoms in which said
backbone comprises at least one asymmetric carbon which
remains chirally pure upon being coupled into a nucleo-
tide/non-nucleotide polymer. Skeletons ha~ing backbones
of about three carbons are preferred, in part, because
such backbones resemble the three-carbon spacing of deoxy-
ribose groups.
SUE~STITUTE SHEET
.; . ............... . .. : :. , - .
... .... ~ . ......... .

W0~2/0253' PCT/US9t/08769
20~30~7
13
One preferred aspect of the present invention is
directed to chirally pure non-nucleotide reagents which
when incorporated in an oligomer comprise a chirally pure
non-nucleotide monomeric unit of the formula:
- Z i ~ HL
wherein SKEL comprises a chirally pure non-nucleotide
skeleton of from about 1 to about 20 car~on atoms, wherein
-NHL, Y and Z are covalently linked to a carbon atom of
SKEL, L is a ligand, Y is -CH2-, -o-, -S- or -NH- and Z is
-O-, -S- or -NH-. Pre~erably SKEL further comprises a
backbone of about 1 to about lO carbon atoms separating
Y and Z. E'xamples of non-nucleotide monomeric units
lncorporating these preferred SKEL groups include:
_ I X ~ HL _~ ~ CH Z__L--XCsXs l
~ ~ Y- ~ L _ ~~--_ y_ , LXf _,NHL
25 _ ___. /X~ I -Z-- L f ~
Xs-C---- ----NHL (Xs~f~xS)q
X-f---- y x5 c -- ---NHL
s ~ _ _ X - f - - - - - - - - y - - r
~ ~Z__- ~_____¢Xs _ Xs _
I s
(Xs~f~~s)q
Xs-C------ --NHL
(Xs-f--xs) r
Xs_f______ __y__
: 45 ~ 5 _ i
8UBSTITUTE SHEET
`, ~ .~'` .
....
:,
. ` . .
. - " . - - `, . .. - ~ . ` ` .
.- ' , . . ; ` , .
.
.

WO~2/0253' PCT/US91/08769
20~0~7
14
wherein the Xs groups are independently selected from
hydrogen or alkyl and may be the same or different, and
q and r are independently selected integers from 0 to 10.
Thus, in one embodiement, these preferred non-
nucleotide reagents may be represented by the generalformula: _ _ .
CP2~`` __--NHL
-`SKEL~
- ---YCpl
wherein -Y-Cpl is a first coupling group, -Z-CP2 is a
blocked second coupling group, wherein L, Y and Z are as
.... .defined above and
(a) the first coupling group, -YCp~ is selected from:
Xl O U
lS
-OP , -OP-X2 and -YP-W
R5 R6 . V
wherein Xl is halogen or substituted amino; X2 is halogen,
amino, or substituted amino, or O ; R~ is alkyl, optionally
substituted alkoxy or optionally substituted aryloxy; and
R6 is alkyl, optionally substituted alkoxy or optlonally
substituted aryloxy, or if Xz is o , optionally hydrogen;
U is oxygen, sulfur or imino, W is alkyl, aryl, alkoxy,
aryloxy, alXylthio, arylthio, S , o , amino or substituted
amino, and V is alkoxy, alkylthio, amino or substituted
: amino.
: (b) blocked second coupling group, -ZCp2, wherein
Cp2, is a blocking group cleavable under deblocking
`30 conditions to recover the second coupling group -ZH
~ wherein Z is -O-, -NH- or -S-.
Since preferred are non-nucleotide reagants which are
capable of forming alkyl- or aryl-phosphonate, and in
particular methylphosphonate, diester linkages between
` 35 monomeric units, especially preferred non-nucleotide
SUB~TITUTE SHEET
: : ~ . - -, : :`:., ,; : ` -. .` . `
.' ~ ' ` `. `:, `, ~, "`:

WO~/n253~ PCT/US91/08769
2~9~87
reagents include those wherein the first coupling group,
-YCp1, is selected from
1 1 o
-O-P or -O-P-X2
Rs R6
wherein Xl is chloro or secondary amino and Rs is alkyl; X2
is substituted amino, halogen or O and R6 is alkyl.
The ligand moiety, L is preferably selected from a
functional moiety or from a protected linking arm which
can be deprotected under non adverse conditions so as to
be capable of then linking with a functional moiety (under
non-adverse conditions).
In one pre~erred aspect of the present invention,
L comprises a protecting group, Pr, or protected linker
arm which can be deprotected under non-adverse conditions
so as to be capable of then linking with a functional
moiety, including a cross linking aS~ent such as psoralen,
or a drug carrier molecule. Preferred linker arms include
those havinq one of the followin~ formulas:
O .,.. ~
11
(a) -C-(C~2)~-NH-Pr or
0 0 r
Il 11
(b) C (CH2)m-NH-C-(CH2)n-NH-Pr
wherein n and m are independently integers between 1 and
15, preferably between 1 and 5, and ~r is a protecting
group removable under non-adverse conditions.
One group of particularly preferred non-nucleotide
reagents has a skeleton derived from the amino acid threo-
nine. These preferred reagents comprise a 3-carbon back-
bone having two asymmetric carbons, each of which remains
chirally pure when incorporated in a nucleotide/non-
nucleotide polymer. In addition, these reagents having
threonine-derived backbones advantageously have a primary
hydroxyl and a secondary hydroxyl, which due to their
differing re`activities allow selectivity and high yields
SUBSTITUTE SHEET
`
: .

WO !)2/n253~ Pcr~uss!/og7~s
20'390~7 `
16
in the subsequent protection, deprotection, blocking,
deblocking and derivatization steps. In one preferred
embodiment of the present invention, the first coupling
group is associated with the secondary hydroxyl group and
the second coupling group is associated with the primary
hydroxyl.
Thus, according to an especially preferred aspect of
the present invention, the threonine-based non-nucleotide
reagents have the following formula:
Cp2-Z-lH2
R ~C R
R3- C-R4
Cpl
wherein C denotes an asymmetric carbon which is chirally
pure, and wherein one of Rl and 1~2 is hydrogen and the
other is -NH-L where L is a ligancl moiety as hereinafter
defined; one of R3 and R4 is hydrogen and the other is
lowor alkyl of about 1 to about 10 carbon atoms, -Y-Cp1 is
a first couplin~ group, and -ZCP2 is a blocked second coup-
ling group, wherein:
(a) The first coupling group, -YCp1, wherein Y is
-CH2-, -S-, -NH-, or -0- is selected from
Xl o U
.,~. 1 11 11 .
30 1 -OP-X2 and -YP-W
R5 R6 V
wherein X1 is halogen or substituted amino; X2 is halogen,
: amino, or substituted amino, or 0 ; Rs is alkyl, optionally
substituted alkoxy or optionally substituted aryloxy; and
R6 is alkyl, optionally substituted alkoxy or optionally
substituted aryloxy, or if X2 is o , optionally hydrogen;
U is oxygen or sulfur, W is alkyl, aryl, alXoxy, alkyl-
thio, aryloxy, arylthio, 0 , S , amino or substituted
amino; and V is alkoxy, alkylthio, amino or substituted
amino; and
SUBSrITl)TE SHEET
.. ~ .
.,. ... - . ... ..
.. ... .. . ` . ..
. . ,. ~ . ;.
- '' ~ 'i . :.,, !. ~ ` ` :

w~ ~2tn253~ PCl~ S91/08769
2G~9S~7
17
(b) blocked second coupling group, -ZCp2, wherein
Cp2, is a blocking group cleavable under deblocking condi-
tions to recover the second coupling group -ZH wherein
Z is -O-, -NH- or -S-.
The ligand moiety, L is preferably selected from a
functional moiety or from a protected linking arm which
can be deprotected under non-adverse conditions so as to
be capable of then linking with a functional moiety (under
non-adverse conditions).
Since non-nucleotide reagents which are capable of
forming alkyl- or aryl-phosphonate, and in particular
methylphosphonate, diester linkages between monomeric _
units, are preferred especially preferred non-nucleotide
reagents include those wherein the first coupling group,
-YCp1, is selected from
Il O ""~
-o-p or -O-IP-X2
R5 R6
wherein Xl is chloro or secondary amino and R5 is alkyl,
X2 is substituted amino, halogen or 0~ and R~ is alkyl.
In one preferred aspect of the present invention,
L comprises a protecting group, Pr, or a protected linker
arm which can be deprotected under non-adverse conditions
so as to be capable of then linking with a functional
moiety, including a cross linking agent such as psoralen,
or a drug carrier molecule. Preferred linker arms include
those having one of the following formulas:
0
Il
(a) -C-(CH2)n-NH Pr or
O` O
Il 11
(b) -c-(cH2)~-NH-c-(cH2)n-NH Pr
wherein n and m are independently integers between 1 and
15, preferably between 1 and 5, and Pr is a protecting
group removable under non-adverse conditions.
SUB~TITUTE SHEET
- ``` ` ." .'` `:; ` ; ';
`

W0~2/0253~ PCT/US91/08769
20g~7
18
Suitable protecting groups, Pr, include 9-fluorenyl-
methoxycarbonyl ~"Fmoc"), trifluoroacetyl, phenoxyacetyl
and the like. See, e.g., Chapter 7 of Greene, Theodore
W., "Protective Groups in Organic Synthesis", John Wiley
& Son, New York, 1981. These linker arms may be conveni-
ently prepared according to the reaction schemes outlined
in Figures 2 and 3 and described the Examples herein.
Utilitv
These non-nucleotide reagents are useful in preparing
oligomers having ligand moieties conjugated to the oligo-
mer without adversely affecting the normal base pairing
associated with hybridization to a target nucleic acid.
These ligand moieties may comprise functional groups or
protected linker arms which may later (after synthesis of
the oligonucleotide) be deprotected and react with a
labelling reagent to give a linker arm--labelling reagent
complex. Functional groups of particular utility may
include detectable labels, agentls which react with a
target nucleic acid such as cross~linking agents, agents
which cleave the target nucleic acid, agents which
~ncreasQ the uptake of oligomers into cells or skin and
agents which slow the excretion of oligomers from the
body.
In certain instances, where these non-nucleotide
reagents are incoproated into oligomers having alkyl- or
aryl-phosphonate linkages between monomeric units, it may
be advantageous to incorporate nucleotide monomeric units
having modified ribosyl moieties. The incorporation of
nucleotide units having 2'-O-alkyl-, in particular 2'-O-
methyl, ribosyl moieties into alkyl- or aryl-phosphonate
oligomers advantageously may improve hybridization of the
oligomer to its complimentary target sequence.
To assist in understanding the present invention, the
following examples follow, which include the results of a
series of experiments. The following examples relating to
this invention are illustrative and should not, of course,
SUBSTIT~ITE SH~ET
.... . ... .. .
- . . . . . ' . .
- . . . : . ....
., . : . .. . . . . .
: .; .. . - . . :
~.. .. : .. ` :

W~'~2/~253' 2 9 ~ ~ 9 ~ I ~CTtUS91/08769
be construed as specifically limiting the invention.
Moreover, such variations of the invention, now known or
later developed, which would be within the purview of one
skilled in the art are to be considered to fall within the
scope of the present invention hereinafter claimed.
Examples
ExamPle 1
Reduction of L-Threonine MethYl Ester
L-Threonine methyl ester (purchased from Sigma) was
reduced according to the procedure of Stanfield et al.
(J. org.--Chem. (1981), 46, 4799): in a 500 ml three
necked flask, 5 g of L-threonine methyl ester and 200 ml
dry THF were mixed and 150 ml of 1 M solution of LiAlHL was
added dropwise with stirring while under argon. The reac-
tion mixture was then warmed up to the boiling temperatureof THF and refluxed under argon overnight. The completion
of the reaction was monitored by TLC on Silica Gel which
wa~ visualized with ninhydrin. The reaction mixture was
cool~d to 5-10- C and quenched wil~h dropwise addition of
0.~5 M NaOH ~100 ml). The mixl:ure was evaporated to
remove over 90~ of THF and the residue was diluted with
100 ml of dimethylformamide which facilitates the filtra-
tion. The mixture was then filtered through a Whatman #1
paper using aspirator vacuum. The filtrate was P~aporated
to dryness and the residue was purified on a flash Silica
Gel column. The column was packed with dichloromethane
and the product was eluted with 50% methanol in dichloro-
methane.
ExamPle 2
Svnthesis of 4-n-(9-FluorenYlmethoxYcarbonvlL~4-Amino-n-
Butyric Acid
Fmoc-aminobutyric acid (for C4 linker arm) was pre-
pared according to the following procedure. (Note: other
FMOC-aminocarboxylic acids are commercially available.
For example, Fmoc-aminocaproic acid (for C6 linker arm)
SUBgTlTUTE SHEET
- ` , .
. :

WO92/0253' PCT/US91/08769
2 ~ 7
and Fmoc-glycine (for C2 linker a~m) are commercially
available from Bachem, Inc., Torrance, California.)
A mixture of 1.8 g 4-aminobutyric acid and 1.24 g
sodium hydrogen carbonate in 35 ml water/acetone (50:50)
was prepared and 5 g Fmoc-succinimidyl carbonate (N-Fluor-
enylmethyl-succinimidylcarbonate) (Bachem) was added. The
reaction mixture was stirred overnight at room tempera-
ture. The pH of the reaction mixture was adjusted to 2 by
lN HCl and the solvent was removed under reduced pressure
and the residue was dissolved in 20 ml ethanol and fil-
tered. The filtrate was evaporated to dryness and the
residue was taken up in dichloromethane and filtered to
give 4.8 g of pure product.
lH NMR in DMSO-d6, 1.61 (CH2), 2.22 (CH2), 3.01
(CH2-N), 4.32 (CH2-C=0), 4.22 (NH), ~.25-7.95 (8 aromatic
protons)~
~m~.
~lockin~ o~ the Amine MoietY o~ Red~!ced L-Threonine
The amine moiety o~ the reduced L-threonine was coup-
led with a 9~fluoxenylmethoxycarbony:L ("Fmoc") group usingwith a procedure similar to the ~noc-aminobutyric acid
preparation described above. After the overnight reac-
tion, adjustment o~ the pH was not necessary. The solvent
was removed and the residue was dissolved in 40 ml
dichloromethane and extracted with water (2 x 50 ml). The
organic phase was then dried and purified on a flash
Silica Gel column. The product was eluted with 2% meth-
anol in dichloromethane to give 3.85 g of the product.
lH NMR 1.20 (CH3), 2.85 (NH), 3.~6 (CH), 3.48 (CH),
3.72 (OH), 7.3-7.9 (8 aromatic protons).
SUBSTITUTE SHEET
. . ~ . -` ,: ; .
, . ~
- .
:, . . .

W()')~/~253' PCT/US91/08769
2~9~7
21
Exam~le 4
Preparation of FMOC-Blocked Linker Arms:FMOC-Glycylamido-
Ca~roic Acid (C8). FMOC-4-Aminobutrylamido-Ca~roic Acid
fC10) and FMOC-Caproylamido-Ca~roic Acid (Cl2)
Fmoc-glycine, Fmoc-4-aminobutyric acid and Fmoc-
aminocaproic acid were coupled to the aminocaproic acid in
order to synthesize the above-identified C8, C10 and C12 ;`
linker arm. ~he desired Fmoc-amino acid (17 mmol) was
dried with co-evaporation with dry pyridine (3 x 30 ml).
The dried material was then dissolved in 30 ml of dry
dimethylformamide and 30 ml dry tetrahydrofuran was added.
The solution was cooled to 0C-and 1 equivalent (17 mmol)~
of N,N-diisopropylethylamine was added. While stirring,
1 equivalent of trimethylacetyl chloride was added drop-
wise at 0'C and s~irred for 45 min. 1.2 equivalent of dry
aminocaproic acid was then added and the reaction mixture
was warmed up to room temperature and stirred overnight.
The progress of the reaction was monitored by TLC. After
the completion, the solvents were evaporated under reduced
20 pressure. Tho residue was reconstLtuted with 50 ml water
and the pH was adjusted to 2 by lN HCl. The mixture was
extracted with 100 ml of ethyl acetate and the organic
phase was washed with 20 ml of water and dried (MgSO4).
The mixture was then filtered and the solvent was evap-
orated under reduced pressure to a volume of about 40 ml.Hexane was added dropwise to this solution until cloudi-
ness and cleared by heating. The product was then cry-
stallized overnight.
C8 1H NMR in DMSO-d6, 1.30 (CH2), 1.39 (CH2), 1.48
(CH2), 2.20 (CH2-N), 3.06 (CH2 of FMOC), 3.58 (CH2-COOH),
4.24 (2NH), 4.34 (CH of FMOC and CH2 of Glycine), 7.3-7.9
(8-Aromatic protons).
C10 lH NMR in DMSO-d6, 1.30-1.70 (5CH2's), 2.07
(CH2), 2.20 (CH-N), 3.0-3.1 (CH2-COOH and CH2 of FMOC), 4.26
~2NH), 4.31 (CH of FM0C), 7.3-7.9 (8-Aromatic proton~).
SUBSTlTUTE SHEET
' ~'1'~
.

W(~ ~2/02S3' PCr~US91/08769
2~ 0~7 ``
22
C12 1H N~ in DMSO-d6, 1.2-1.5 (6CH2's), 2.00 (CH2-N),
2.18 (CH2-N), 2.9-3.0 (2CH2-C=O), 4.23 (2NH), 4.31 (CH2 of
E'MOC), 7 . 3-7 . 9 ~8-Aromatic protons).
Example 5
5 Cou~lina of Reduced L-Threonine to Linker Arms
The desired linker arm (11 mmol), which was made
according to Examples 2 or 4 above [~moc-glycine (C2),
Fmoc-4-aminobutyric acid (C4), Fmoc-caproic (C6), Fmoc-
glycyclamido-caproic acid (C8), Fmoc-4-aminobutyrylamido-
10 caproic acid (C10), and Fmoc-aminocaproylamidocaproic acid
(C12)], was-dried with-co-evaporation with pyridine (3 x
20 ml). The dry residue was dissolved in 40 ml of a mix-
ture of anhydrous dimethylformamide and anhydrous tetra-
hydrofuran (1:1). The solution was cooled in an ice bath
15 and 1 equivalent of diisopropylethylamine was added.
While stirring, 1.1 e~uivalent of trimethylacetyl chloride
was added dropwise and stirred for 45 min at 0~C. A solu-
tion of 1. 5 equivalent of reduced L-threonine (Example
above) was added and the reaction mixture was allowed to
20 warm to room temperature and stirred for one hour. The
progress of the reaction was monitored by TLC on Silica
Gel which was developed by CHzCl2~Cl~3OH/CH3COOH (lo:l:O.l)
solvent system. After the completion of the reaction, the
solvent was removed under reduced pressure and the residue
25 was mixed with 50 ml ethyl acetate. The water soluble
materials were removed by extraction with 40 ml saturated
sodium bicarbonate. The organic phase was washed with
20 ml of water and dried (MgSO4). The product was cr~-
stallized from ethyl acetate.
C2 Linker 1H N~R in DMSQ-d6, 1.03 (CH3 of reduced
L-threonine), 3.35 (OH), 3.3-3.45 (2CH), 3.91 (NH), 4 . 27
(other NH), 4 . 31 (OH), 4.34 (CH2), 4.63 (CH2 and CH of
FMOC), 7.3-7.9 t8-Aromatic protons).
C4 Linker 1H NMR in DMSC~d6, 1.03 tCH3 of reduced
35 L~threonine), 1.62 (CH2), 2.14 (CH2), 2 . 91 (CH), 2. 97 (CH2),
3.3-3.5 (2CH), 3.63 (OH), 3.84 (OH), 4.23 (CH), 4.33 (CH
8UBSTITUTE SHEFI'
- ~ .. . . ` , :
.. , ` . . . ............ ~ ..
, ... . .

W~2/0253~ PCT/US91/0876q
2a~30~7
and CH2 o FMOC), 4.60 (NH), 6.32 (NH), 7.3-7.9 (8-Aromatic
protons).
C6 Linker 1H NMR in DMSO-d6, 1.03 (CH3 of reduced
L-threonine), 1.3-1.7 (3 CH2's), 2.52 (CH2-N), 3.12
(CH-C=O), 3.8-3.9 (2 OH), 4.1-4.2 (2CH), 4.41 (C~2 of
FMOC), 5.22 (NH), 6.48 (NH), 7.3-7.9 (8-Aromatic protons).
C8 Linker lH NMR in ~MSO-d6 major proton signals are
as follows: 1.01 (CH3 o~ reduced L-threonine), 1.22-1.52
(3 CH2 of caproate), 3.62 and 3.84 (2 OH), 5.35 (NH), 6.18
(NH), 7.3-7.9 t8-Aromatic protons).
C10 Linker lH NMR in DMSO-d6, 1.02 (CH3 of reduced
- L-threonine), 1.-3-1.50- (4- CH2's), 3.64 (OH), 3.82 (OH),
4.64 (NH), 6.33 (NH), 6.62 (NH), 7.3-7.9 (8-Aromatic
protons)~
15C12 ~ink~ lH NMR in DMSO-d6, major proton signals
for identification 1.01 (CH~ of reduced L-threoni~e), 1.30-
1.50 (6CH2's), 3.63 (OH), 3.82 (OH), 4.62 (NH), 6;31 (NH),
6.63 (NH), 7.3-7.9 (8-Aromatic protons).
~amPle 6
20 ~;lm~thoxY T~lation of the PrimarY H~d~l MoietY
of the Non-Nucleotide Rea~ent
The desired non-nucleotide reagent (6 mmol), which
was made according to Examples 3 ancl 5 above, was dried by
co-evaporation with dry pyridine and dissolved in 15 ml of
dry pyridine. A solution of 2.2 g of dimethoxytrityl
chloride in 20 ml of CH2Cl2/pyridine (1:1) was added
dropwise with stirring. The reaction continued at room
temperature for 45 min. The progress of the reaction was
monitored by TLC. After the completion of the reaction it
was quenched by the addition of ~ ml methanol which was
stixred for 10 min. The solvents were removed under
reduced pressure and the residue was dissolved in 50 ml of
dichloromethane and extracted with saturated sodium hydro-
gen carbonate (2 x 50 ml) followed by water (30 ml). The
organic phase was dried (MgSO~) and filtered. After the
evaporation of the solvent, the residue was purified with
SUBSTiTUTE SHEET
.
. . . .
. . . . . . .. . .

w~ ~2/n253 2 ~ ~ 9 0 '~ ~ PCT/US91/087fi9
24
a flash column chromatography. The product was eluted
with 2% methanol in dichloromethane containing 0.5%
triethylamine.
Co Linker 1H NMR, CDCl3, 1.18 (CH3 of reduced L-threo-
nine), 1.63 (CH), 2.83 (NH), 3.77 (2 CH3 of DMT), 3.82 (CH2of FMOC), 5.48 (CH2-0-DMT), 6.82-7.90 (21 aromatic
protons).
C2 Linker lH NMR, CDCl3, 1.18 (CH~ of reduced L-threo-
nine), 3.78 (2 CH3's of DMT), 4.35 (CH2-0-DMT), 5.98 (NH)
10 6.80-7.78 (21 aromatic protons).
C4 Linker lH NMR, CDCl3 major signals 1.18 (CH3 of
reduced L-threonine), 1.83 (CH2), 2.28 (CH2), 3.74 (2 CH3
of DMT), 4.21 (OH), 4.38 (CH2 of FMOC), 5.22 and 6.42
(2 N~), 6.80-7.65 (21 aromatic protons).
C6 Linker lH NM~, CDC13 major peaks 1.12 (CH3 of
reduced L-threonine), 1.3-1.6 (3 CH2's), 3.75 (2 CH3 of
DMT), 4 .38 (CH2 of FMOC), 6.80-7.90 (21 aromatic protons).
C8 Linker 1H NMR, CDC13 Major identifying signals
were 1.12 ~CH3 of reduced L-threonine), 3.80 (2 CH3 of
20 DMT), 5.~2 (CH2 of FMOC), 6.18 and ~.321 (2 NH), 6.82-7.80
(21 aromatic protons).
C12 Linker lH NMR, CDC13, major identifying signals
were 1.12 (CH3 of reduced L-threonine), 3.78 (2 CH3 of
DMT), 4.59 (CH2 of FMOC), 6.8-7.8 (21 aromatic protons).
C10 Linker 1H NMR, CDCl3 1.18 (CH3 of reduced
L-threonine), 3.78 (2 CH3 of DMT), 4.40 (CH2 of FMOC), 6.8-
7.8 (21 aromatic protons) all the CH2 and CH (non-aromatics
were also accounted for but not assigned).
Example 7
30 MethvlphosPhinvlation of the_Secondary Hvdroxyl Moietv
f the Non-Nucleotide Rea~ents
A DMT blocked linker arm made according to the proce-
dure described in Example 6 above (~ mmol) was dried by
co-evaporation with dry pyridine and the residue was
35 dissolvad in 20 ml of anhydrous dichloromethane. Under
closed argon atmosphere, 1.5 equivalent of diisopropyl-
~;UBS~ITUTE SHEET
!
` `' ' ' ~

W()')2/(~2531 PCT/US91/08769
~0~9~7
;`
2 5 r
ethylamine was added and 1.2 equivalent of N,N-diisopro-
pylmethyphosphinamidic chloride [(CH3)2CH]2NP(CH3)Cl was
added dropwise. The reaction was completed in 45 min.
The solvent was removed under reduced pressure and the
residue was purified on a flash Silica Gel column. The
column was packed with ethyl acetate/hexane (1:) contain-
ing 5% triethylamine and washed with the ethyl acetate/
hexane containing 1% triethylamine. The reaction mixture
was then loaded on the column and the product was eluted
with ethyl acetate/hexane (1:1) containing 1% triethyl-
amine.
Other non-nucleotide reagents are prepared by coup-
ling of the linker arm-modified reagents made according to
the methods described in Example 6 with other phosphory-
lating agents such as N,N-diisopropylmethyl phosphonamidic
chloride, ~(CH3)2CH]2NP(OCH3)Cl, and 2-cyano-ethyl N,N-
diisopropylchloro-phosphoramidite, ~(cH3)2c~l]2Np(cl)ocHz
CH2CN. Such reagents are useful in the synthesis of
phosphate diester coupled non-nucleotide-oligomers.
CO 1H NMR, CDC13, 0.9-1.3 (18 protons of 6 C~13's),
3.11 (C~l2 of FMOC), 3.78 (2 CH~Is of DM'~), 4.42 (CH2-O-
DMT), 4.98 (NH), 6.8-7.8 (21 aromatic protons).
C4 1H NMR, CDCl3, 0.9-1.2 (18 protons of 6 CH3's),
1-88 (CHz)~ 2-21 (CH2), 3.08 (CR2 of FMOC), 3.80 (2 CH3's of
25 DMT), 4.36 (CH2-O-DMT), 5.16 (NH), 5.75 (NH), 6.8-7.8 (21
aromatic protons).
C6 1H NMR, CDCl3, 0.9-1.2 (18 protons of 6 CH3's),
1.18-2.2 (4 CH2's), 3.07 (CH2 of FMOC), 3.78 ~2 CH3's of
DMT), 4.42 (CH2-O-DMT), 5.6 and 6.21 (2 NH), 6.8-7.8 (21
aromatic protons).
Example 8
Meth~lphos~hinvlation of the Secondary Hydroxv Moiety
of a Non-Nucleotide Reaqent Havinq a C6-Linker Arm
A 4 mmol portion of a dimethoxytrityl(DMT)-blocked
non-nucleotide reagent having a C6 linker arm (prepared
according to the methods described in Example 6 herein)
SUBSrlTUTE S~IEET
.; .
. . ,~ .:
. .. ....
: ,; : . ~ - :
- .

WO()~/()253~ PCT/US91/08769
2 0 ~ 9 0 ~ ~
26
was dried by co-evaporation with dry pyridine. The resi-
due was dissolved in 20 ml of anhydrous dichloromethane.
Under a closed argon atmosphere, 1.5 equivalents of N,N-
diisopropylethylamine was added; then 1.2 equivalent of
N,N-diisopropylmethylphosphonamidic chloride [(C~3)2CH]2NP
(Cl)OCH3] was added dropwise. The reaction mixture was
then worked up using the procedures described in Example 7
to give 3.2 mM of the above-identified product.
1H NMR in CDCl3, ~ ppm: 1-1.5 (5 methyl and 1 methy-
lene), 1.42 (CH2), 1.73 and 1.73 (2 CH2), 2.21 (CH2-N), 3.15
(CH2-C=O), 3.78 (2 CH3 of DMT), 6.80-7.85 (21 aromatic pro-
tons). Other proton signals present were not assigned.
Exam~le g
Pre~aration of a PhosPhate Diester Oliqomer Which Incor-
Porates a MethoxyE~hos~horamidite Non-Nucleotide Rea~ent
Havinq a C8 Linker Arm
A phosphate diester oligodeoxyribonucleotide was syn-
thesized which incorporated a C8 methoxyphosphoramidite
non-nucleotide reagent in the following sequence:
5'-TTT-AAG-CAG-AGT-TCA-AAA-GCC-CTT-CAG-CG-~C8-Linker)-
T-3' was prepared according to the following procedure.
The C8 methoxyphosphoramidite non-nucleotide reagent
(l-O-dimethoxytrityl-2-N[N'-(N"-fluorenyl-methoxycarbo-
nyl-6-aminohexanoyl)-2-aminoacetyl]-3-O-[N,N-diisopropyl-
methoxy-phosphinyl]-2-amino-1,2-dihydroxybutane) was
dissolved in dry acetonitrile at a concentration of 100 mM
and coupled into the oligonucleotide sequence using a Bio-
search Model 8750 DNA synthesizer by standard phosphorami-
dite chemistry (M.H. Caruthers, et al., Methods of Enzy-
mol. 154:287-313 (1985~) according to the manufacturer's
recommendations. The 5'dimethoxytrityl protecting group
was left on at the end of the synthesis to permit purifi-
cation on a Sep-PakTH C18 cartridge (Millipore/Waters,
Bedford, MA) as described by K.M. Lo et al. (1984, Proc.
Natl. Acad. Sci. USA, 81, pp. 2285-2289). During this
SUBSTITUTE Sl IEET
`~ . .
. .. .

W092/0253~ 2 ~ ~ 3 ~ ~ ~ PCT~US91/08769
procedure, the dimethoxytrityl protecting group was
removed.
ExamDle lo
Preparation of MethYl~hos~honate Oliqomers Which Incor-
~orate Non-Nucleotide Reaaents
(a) PreParatlon of MethvlPhosPhonate Oliqomers
Methylphosphonate oligomers which incorporated non-
nucleotide reagents of the present invention were synthe-
sized using methylphosphonamidite monomers and non-
nucleotide methylphosphonamidite non-nucleotide reagents,
according to chemical methods described by P.S. Miller
et al. (1983, Nucleic Acids Res., 11, pp. 6225-6242),
A. Jager and J. Engels (1984, Tetrahedron Lett., 25,
pp. 1437-1440), and M.A. Dorman et al. (1~84, Tetra-
hedron, 40, pp. 95-102). Solid-phase synthesis was
performed on a Biosearch Model 8750 DNA Synthesizer
according to the manufacturer's recommendations with the
following modifications: 'IG" and ll'C" monomers were dis-
solved in 1:1 acetonitrile~dichloromethane at a concentra-
tion of 100 mM. "A" and "T" monomers were dissolved inacetonitrile at a concentration of :L00 mN. Non-nucleotide
linker reagents were dissolved in acetonitrile at a con-
centration of 120 mM. DEBLOCK reagent = 2.5~ dichloro-
acetic acid in dichloromethane. OXIDIZER reagent = 25 g/L
~5 iodine in 2.5~ water, 25% 2,6-lutidine, 72.5% tetrahydro-
furan. CAP A = 10% acetic anhydride in acetonitrile. CAP
B = 0.625% N,N-dimethylaminopyridine in pyridine. The
5'-dimethoxytrityl protecting group was left on at the end
of the synthesis to facilitate purification of the oligo-
mers, as described below.
The crude, protected non-nucleotide reagent incorpor-
ating methylphosphonate oligomers were removed from the
solid support by mixing with concentrated ammonium hydrox-
ide for two hours at room temperature. The solution was
drained from the support using an Econo-ColumnTH (Bio-Rad,
Richmond, CA) and the support was washed five times with
SUB~TITUTE SHEET

W(~/0253' PCT~US91/08769
~0~9~3'~
28
1:1 acetonitrile/water. The eluted oligomer was then
evaporated to dryness under vacuum at room temperature.
Next, the protecting groups were removed from the bases
with a solution of ethylenediamine/ethanol/acetoni-trile/
water (50:23.5:23.5:2.5) for 6 hours at room temperature.
The resulting solutions were then ~vaporated to dryness
under vacuum.
(b) Purification of linker-modified methvl~hosPhonate
oliaomers.
The 5'-dimethoxytrityl (trityl) containing oligomers
were purified from non-tritylated failure sequences using
a Sep-PakTM C13 cartridge (Millipore/Waters, Bedford, MA)
as follows: The cartridge was washed with acetonitrile,
50~ acetonitrile in 100 mM triethylammonium bicarbonate
(TEAB, pH 7.5~, and 25 mM TEAB. Next, the crude methyl-
phosphonate oligomer was dissolved in a small volume of
1:1 acetonitrile/water and then diluted with 25 mM TEAB
to a final concentration of 5% acetonitrile. This solu-
tion was then passed through the cartridge. Next, the
cartridge was washed with 15-20% acetonitrile in 25 mM
TEAB to elute failure sequences from the cartridge. The
trityl-on oligomer remaining bouncl to the cartridge was
then detritylated by washing with 25 mM TEAB, 2% tri-
fluoroacetic acid, and 25 mM TEAB, in that order.
Finally, the trityl-selected oligomer was eluted from the
cartridge with 50% acetonitrile/water and evaporated to
dryness under vacuum at room temperature.
The lin~er-modified methylphosphonate oligomers
obtained from the previous step, above, were further
purified by reverse-phase HPLC chromatography as follows:
A Beckman System Gold HPLC, described in a previous
example, was used with a Hamilton PRP-l column (Reno, NV,
10 ~, 7 mm i.d. x 305 mm long). Buffer A - 50 mM tri-
ethylammonium acetate (pH 7); Buffer B = S0% acetonitrile
in 50 mM triethylammonium acetate (pH 1). The sample,
dissolved in a sma~l volume of 10-50~ acetonitrile/water,
~UB~TITiJTE ~:HE~ET
.. .

W~2/0253~ PCTJ~S91/08769
20~90~7
29
was loaded onto the column while flowing at 2.5-3
ml/minute with 100% Buffer A. Next, a linear gradient of
0-70% Buffer B was run over 30-50 minu~es at a flow rate
of 2.5-3 ml/minute. Fractions containing full-length non-
nucleotide reagent incorporating methylphosphonate oligo-
mer were evaporated under vacuum and resuspended in 50
a~etonitrile/water.
Exam~le 11
Labellinq of Phosphate Diester Oliaomers Incor~oratinq
a C8 Linker Arm Non-Nucleotide Monomer With Biotin
The phosphate diester oligonucleotide of Example 9
(19 nmoles) was suspended in 115 ~il of 0.15 M HEPES buffer
(pH 8.0). Next, NHS-LC-biotin (Pierce Chemical Co.,
Rockford, IL) was added (10 ~il of a 100 mM solution in
dimethylsulfoxide). The solution was heated at 37C for
30 minutes. Additional NHS-LC-biotin solution was added
(10 ~l) and the reaction was continued for 30 minutes at
37-C. Next, the biotinylated oligonucleotide was precipi-
tated by addition of 3 M sodium acetate buffer (pH 5.5,
15 ~il) and absolute ethanol (500 ~l); the resulting solu-
tion was chilled in dry ica for 30 minutes. Ths product
was recovered by centri~ugation for 15 minutes at 4C in
a microcentrifugs and the supernatant was discarded.
The product was then dissolved in water (lO0 ~l) and
purified by reverse-phase HPLC chromatography according to
the following method. The ~PLC apparatus consisted of a
Beckman System Gold Model 126 Solvent Module and ~odal 167
Detector interfaced to an IBM compatible computer. A
PLRP-S column was used (Polymer Laboratories, 8 ~i, 300 A
pore size, ~.6 mm i.d. x 250 mm long). Buffer A = 50 mM
triethylammonium acetate (pH 7); Buffer B = 50% acetoni-
trile in 50 mM triethylammonium acetate (pH 7). A linear
gradient of 20-60% B was run over 30 minutes at a flow
rater of 1.5 ml/minute. Under these conditions, the bio-
tinylated product oligonucleotide eluted at 10.3 minutes.
~;UBSrlTUTE SHEET
..... .. `.. `--, -` .. `.. ,. , ~ ~ . .
:
. ....... : . . . ..
, ;....... - : . . .: . , ,:

W0~2/02~3~ PCT/US91/08769
~93~7 `
xamPle 12
Bindinq of a Biotinylated C8 Non-Nucleotide ~onomer Modl-
fied Phos~hate Diester Oliaonucleotide to a Stre~tavidin-
Modified Solid Su~ort
The biotinylated oligonucleotide of Example 11 was
labelled with 32p at the 5'-terminus using [ -32P]-ATP
(3000 Ci/mmol) and T4 polynucleotide kinase as follows:
10 pmol of the oligonucleotide was dissolved in 10 ~1 of
50 mM Tris (pH 7.8), 10 mM MgCl2, 5 mM DTT, 0.1 mM EDTA,
O.1 mM spermidine containing 50 ~Ci of [ -32P]-ATP. T4
polynucleotide kinase (4 units) was added, and the solu-
tion was incubated for 90 minutes at room temperature.
The radiolabeled product was purified on a NensorbT~-20
column (New England Nuclear/DuPont) according to the
manufacturer's instructions.
The 32P-labelled biotinylated oligonucleotide (10,000
cpm) was dissolved in 0.5 ml of 50 m~ sodium phosphate (pH
6.8), 0.5 M sodium chloride, 2 mM El)TA in a 1.5 ml micro-
centrifuge tube. Controls were also prepared containing
the same components plus 1 mg/ml biotin ~Calbiochem Corp.,
San ~Diego, CA). Next, 50 ~1 oE steptavidin-agarose
(Pierce Chemical Co., Rockford, IL) was added to each tube
and the contents were mixed on a vor.teXer for 15 minutes.
The tubes were then centrifuged for 2 minutes in a micro-
centrifuge to pellet the streptavidin-agarose; and the
supernatants were transferred to fresh tubes. The pellets
wera then washed twice with 0.5 ml of buffer (see above)
and the washes were likewise separated by centrifugation
and transferred to fresh tubes. The tubes were counted
for radioactivity in a scintillation counter. Samples
prepared without the addition of free biotin bound to the
streptavidin-agarose support at greater than 85%. Samples
prepared with added free biotin (controls) bound to the
support at less than Q.5%.
SVE3ST~TlJTF SHEET
. ` . ~ ` . . ::
.. : .
..`. .
` , ~ . ` , .
. ~ ..

wo 92/n2s~ 2 0 g 9 ~ ~ 7 PCT/US91/0~769
31
Exam~le 13
Labellinq of a Methvlphosphonate Oligomer Incorporatinq a
C4 Linker Non-Nucleotide Monomer With Psoralen
A C4 linker-modified methylphosphonate oligomer was
prepared having the following se~uence:
5~GGC-TTT-TGA-~C4-lin~er)-AC~-CTG-CTT-37
where the bold letters (including C4-linker) refer to
bases or non-nucleotide monomeric units connected by
methylphosphonate linkages and the upper case letters
refer to bases connected by diester linkages. The metllod
of synthesis and pl~rification of this oligonucleotide is
described in a previous example,_above.
This oligomer was labelled with a psoralen-NHS label-
ling reagent as follows:
The following coupling reaction of NHS-psoralen rea-
gent to linker arm (present in the oligomer) was carried
out in a 1.5 ml polypropylene microfuge tube. Approxi-
mately 3.4 mg ~98 OD260 units) of the oligomer was dissolved
in 100~1 of 1:1 acetonitrile/water. Next, the following
reagents were added in order, with vortexing at each addi-
tion to avoid precipitation of the oligomer: dimethylsul-
foxide (170~1), water (100 ~lj, 1 M HEPES buffer, pH 8~0
(50 ~1), and 50 mM psoralen-NHS reagent in dimethylsul-
foxide (80 ~1). Total volume: 500 ~1. The mixture was
reacted for 2.5 hours at room temperature in the absence
of light. Ethanol (1 ml) was then added, and the result-
ing solution was chilled at -20C overnight. The tube was
then spun in a microcentrifuge for 5 minutes and the
supernatant was aspirated and discarded. The resulting
pellet was resuspended in 500 ~1 of 1:1 acetonitrile/water
and filtered through a 0.22 ~ DuraporeT~ membrane to remove
particulate material.
HPLC purification of the solution of crude psoralen-
oligomer conjugate described above was conducted as fol-
lows: A Beckman System Gold analytical HPLC system wasused with a Hamilton PRP-l column (4.1 x 250 mm). Buffers
used for elution were: Buffer ~ - 50 mM triethylammonium
SUBSl ITUTE SHEEr
. .
:: ` . . . . . ~
. .. . ; .
` .
` , .. .

W092/0253 PCT/US9!/08769
2 ~ ~ 9 ~
32
acetate (pH 7); Buffer B - 50% acetonitrile in 50 mM
triethylammonium acetate (pH 7). The sample was loaded
onto the column in five 100 ~1 portions at two minute
intervals with a 500 ~1 sample loop while the column was
flowing at 1.5 ml/min with 10% Buffer B. Next, a linear
gradient from 10 - 70% Buffer B was run over 30 minutes.
Fractions were collected at 0.5 minute intervals. Under
these conditions, unmodified oligomer and psoralen-modi-
fied oligomer eluted at 17.9 minutes and 21.7 minutes,
respectively. Fractions containing the psoralen-modified
oligomer were pooled and evaporated. The overall yield
was 16%.
Example 14
Cross-Linkinq of Psoralen-Labelled MethYlphosPhonate
Ol~qomer to a ComPlementary Phos~hate Diester Oliqo-
nucleotide Tarqet
A phosphate diester oligonucleotide complementary to
the methylphosphonate oligomer o~ ~Sxample 13 was synthe-
sized on a Biosearch Model 8750 DNA Synthesizer according
to the manufacturer's recommendati~ns; this oligonucleo-
tide has the following sequence:
5'-AAG-CAG-AGT-TCA-AAA-GCC-3l
The psoralen-modi~ied methylphosphonate oligomer, prepared
according to the procedure of Example 13, was labeled at
its 5'-end with 32p as described above (See Example 12).
The 32P-labeled, psoralen modified oligomer (10-50,000 cpm)
was hybridized to its diester oligonucleotide target
(1 pmol, above), in a borosilicate glass vial containing
10 ~1 of 10 mM Tris (pH 7.2), 0.1 mM EDTA, 0.03% potassium
sarkosylate at 37C for 30 minutes. Control reactions
were also run without the diester oligonucleotide target.
The vials were then irradiated at 365 nm on crushed ice
using a Model B-lOOA long wavelength ultraviolet lamp
(W P, Inc., San Gabriel, CA) at a dis~ance of 15 centi-
meters. Intensity of irradiation at this distance wasgreater than or equal to 60 ~W/100 cm2. After 30 minutes,
S~JBSTITUTE SHEET
. .
'' '~
-
- : . :.: : :

W092/0253' PCT/US91108769
20~9~ ~
90% formamide c~ntaining 0.1% bromphenol blue, 0.1 M tris-
borate-EDTA buffer (pH 8.2) was added (5 ~1) and the sam-
ples were loaded onto a 15% polyacrylamide gel containing
7 M urea (0.5 mm). The gel was electrophoresed at 900 V
for 2 hours. The wet gel was then placed between two
sheets of Saran Wrap7M and exposed to XAR-5 film (Eastman-
Kodak, Rochester, NY) for 30-60 minutes. The resulting
autoradiograph revealed upper bands for samples containing
diester target oligonucleotide which migrated slower in
the gel than the bands corresponding to psoralen-modified
methylphosphonate oligomer alone. Using the autoradio-
graph as a template, the bands were then excised from the
wet gel with a scalpel and counted in a scintillation
counter. Based on this method, it was determined that
cross-linking of psoralen-modified methylphosphonate
oligomer to its complementary diest:er target was greater
than 95%.
8UBSTITUTE SHEET
.. . . . .
:~ ,, , -. .. . -. : ~
. . :: : , ~ .
:: . ` ~. .; . .. `, ...... ..
, .. ; . .. . ` . , . . .. ~ ,
: . .: . , ;, `:, ~

WO 92/0253~ PCI`/US9!/087b9
20~9~734
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