U.S. patent number 4,469,863 [Application Number 06/206,297] was granted by the patent office on 1984-09-04 for nonionic nucleic acid alkyl and aryl phosphonates and processes for manufacture and use thereof.
Invention is credited to Paul S. Miller, Paul O. P. Ts'o.
United States Patent |
4,469,863 |
Ts'o , et al. |
September 4, 1984 |
Nonionic nucleic acid alkyl and aryl phosphonates and processes for
manufacture and use thereof
Abstract
Oligonucleoside alkyl-- or aryl phosphonates are nonionic
analogues of nucleic acid which possess unique physical and
biological properties. These properties enable the analogues to
enter living cells intact and to bind with specifically selected
nucleic acids within the cell. As a result, the analogues can
specifically inhibit the function or expression of a preselected
nucleic acid sequence. Thus the analogues could be used to
specifically inhibit the growth of tumor cells or replication of
viruses in infected cells. Four methods are provided for preparing
oligonucleoside methylphosphonates: (1) Coupling a protected
nucleoside 3'-alkyl or aryl phosphonate with the 5'-hydroxyl group
of a protected nucleoside using a condensing agent; (2) Coupling
protected nucleoside 3'-alkyl or aryl phosphonic acid derivative
with the 5'-hydroxy group of a protected nucleoside with the
activated alkyl or aryl phosphonic acid derivative possessing
functionalities which are good leaving groups; (3) Coupling a
protected nucleoside 3'-alkyl or aryl phosphinate derivative with
the 5'-hydroxyl group of a protected nucleoside with the resulting
phosphinate derivative being then oxidized to the phosphonate; and
(4) Converting a oligonucleoside methoxyphosphite derivative to the
alkyl or aryl phosphonate derivative by reaction with an alkyl or
aryl iodide. It has been demonstrated that procedures (1) and (2)
can be used to prepare oligonucleoside methylphosphonates. Others
have shown that procedure (4) can be used to prepare a
diribonucleoside methylphosphonate.
Inventors: |
Ts'o; Paul O. P. (Lutherville,
MD), Miller; Paul S. (Baltimore, MD) |
Family
ID: |
22765756 |
Appl.
No.: |
06/206,297 |
Filed: |
November 12, 1980 |
Current U.S.
Class: |
536/24.5;
536/25.1; 536/25.34 |
Current CPC
Class: |
C07H
21/00 (20130101) |
Current International
Class: |
C07H
21/00 (20060101); C07H 021/02 (); C07H 021/04 ();
C07H 021/00 () |
Field of
Search: |
;536/27,28,29 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Agarwal, K., and Reftina, F., Nucleic Acids Research, vol. 6, pp.
3009-3024, 1979. .
Miller, P., et al., Biochemistry, vol. 18, pp. 5134-5143, 1979,
vol. 20, pp. 1874-1880, 1981. .
Nemer, M. and Ogilvie, K., Tetrahedron Letters, vol. 21, pp.
4149-4152, 1980. .
Miller, P., et al., Federation Proceedings, Abstract 2231, vol. 36,
1977. .
Miller, P., et al., J. Am. Chem. Soc., vol. 93, pp. 6657-6665,
1971. .
Pless, R., et al., Biochem., vol. 16, pp. 1239-1250, 1977. .
Jayarama, K., et al., Proc. Natl. Acad. Sci., vol. 78, pp.
1537-1541, 1977..
|
Primary Examiner: Hazel; Blondel
Attorney, Agent or Firm: Finch; Walter G.
Claims
What is claimed is:
1. Alkyl or aryl phosphonate nucleic acid analogs, comprising, at
least five nucleosides, and an alkyl or aryl phosphonate group,
said nucleosides being linked together by said alkyl or aryl
phosphonate group to form phosphonate nucleic acid analogs, where
the alkyl phosphonate group or the aryl phosphonate group do not
sterically hinder the phosphonate linkage or interact with each
other, said alkyl or aryl phosphonate nucleic acid analog as
defined by the structural formula: ##STR9## where B is a base; R'
is a hydrogen, hydroxyl, O-alkyl or O-aryl or O-halogen; R is alkyl
or aryl; and where the S and R configurations define the spatial
location of the alkyl or aryl group, with R denoting the alkyl or
aryl group in a pseudoequitorial position and S denoting the alkyl
or aryl group in a pseudoaxial position.
2. The alkyl or aryl phosphonate nucleic acid analog as recited in
claim 1, wherein each said nucleoside includes a 3'-hydroxyl group
and a 5'-hydroxyl group, with a 3'-hydroxyl group of one nucleoside
being linked with a 5'-hydroxyl group of the other nucleoside.
3. The alkyl or aryl phosphonate nucleic acid analog as recited in
claim 1, wherein each said nucleoside has one of the following
sugars: ribose, 2'-deoxyribose, 2'-O alkyl ribose,
2'halogenoribose, or aryl ribose.
4. The alkyl or aryl phosphonate nucleic acid analog as recited in
claim 1, wherein each said nucleoside has a base consisting of at
least one of the following: adenine, thymine, cytosine, guanine,
uracil or hypoxanthine.
5. The alkyl or aryl phosphonate nucleic acid analog as recited in
claim 1, wherein the number of nucleosides can range from at least
five to at least twenty linked together by said alkyl or aryl
phosphonate groups.
6. The alkyl or aryl phosphonate nucleic acid analog as recited in
claim 1, wherein said alkyl or aryl phosphonate group consists of R
and S configurations, wherein R and S define the spatial location
of the alkyl or aryl group, with R denoting the alkyl or aryl group
in a pseudoequitorial position and S denoting the alkyl or aryl
group in a pseudoaxial position.
7. A heptadeoxyribonucleoside methylphosphonate nucleic acid analog
comprising six methylphosphonate groups, and seven nucleosides,
covalently linked together in the following order: deoxyadenosine,
deoxyquanosine, deoxyguanosine, deoxyadenosine, deoxyguanosine,
deoxyguanosine, and thymidine, as defined by the structural
formula: ##STR10## where Ad is adenine, Gu is guanine, and Th is
thymine.
Description
This invention relates generally to biochemical and biological
effects of nonionic nucleic acid methylphosphonates, and more
particularly to nonionic nucleic acid alkyl and aryl
methylphosphonates and processes for the manufacture and use
thereof.
Prior to the present invention, studies on nucleic acid analogs and
derivatives possessing modified internucleoside linkages have made
important contributions to understanding nucleic acid conformation
in solution and have provided materials for various biochemical and
biological studies.
Recent studies have been made on the physical, biochemical and
biological properties of one class of nonionic nucleic acid
derivative, the oligonucleotide alkyl phosphotriesters.
The physical properties of dinucleotide methyl and ethyl
phosphotriesters have been studied by ultraviolet, circular
dichroism, infrared and proton nuclear magnetic resonance
spectroscopy. The interaction of deoxyribooligonucleotide ethyl
phosphotriesters with sequences complementary to the amino acid
accepting stem and anticodon region of transfer RNA have been
characterized and their inhibitory effects on in vitro
aminoacylation have been studied. More recently, the inhibitory
effect of a 2'-O-methylribooligonucleotide triester, G.sub.p.sup.m
(Et) G.sub.p.sup.m (Et)U, on cellular protein synthesis and growth
of mammalian cells in culture has been reported. In addition,
selective binding of an octathymidylate ethyl phosphotriester,
[Tp(Et)].sub.7 T to polydeoxyadenylic acid has been extensively
investigated.
An object of this invention is to provide nonionic nucleic acid
alkyl or aryl phosphonates analogs.
Still another object of this invention is to teach the preparation
of nonionic nucleic acid alkyl or aryl phosphonate analogs by
several novel synthetic processes or methods.
To provide nonionic nucleic acid alkyl or aryl phosphonate analogs
for interacting with complementary cellular or viral nucleic acids
with the objective of controlling or regulating the function or
expression of the cellular or viral nucleic acids, is still another
object of this invention.
To provide a heptadeoxyribonucleoside methyl phosphonate with a
base sequence which is complementary to the 3'-terminus of
bacterial 16S ribosonal ribonucleic-acid with objective of
preventing bacterial protein synthesis, is a further object of this
invention.
Even another object of this invention is to provide alkyl and aryl
phosphonate nucleic acid analogs comprising at least two
nucleosides, a base, and an alkyl or aryl phosphonate group, with
the nucleosides being linked together to form alkyl or aryl
phosphonate nucleic acid analogs for the purpose of controlling or
regulating the function or expression of cellular or viral nucleic
acids.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the general molecular structure of an oligonucleoside
alkyl or aryl phosphonate;
FIG. 2 illustrates the general molecular structure of an
oligonucleoside alkyl or aryl phosphonate;
FIG. 3 illustrates the molecular structure of a
heptadeoxyribonucleoside methylphosphonate;
FIG. 4 is a schematic of a process for the synthesis of a
dinucleoside alkyl or arylphosphonate;
FIG. 5 is a schematic of a process for the synthesis of a
dinucleoside alkyl or arylphosphonate;
FIG. 6 is a schematic of a process for the synthesis of a
dinucleoside alkyl or arylphosphonate;
FIG. 7 is a schematic of a process for the synthesis of a
dinucleoside alkyl or arylphosphonate;
FIG. 8 illustrates the process for the synthesis of a dinucleoside
alkyl or arylphosphonate;
FIG. 9 illustrates the process for the synthesis of a dinucleoside
alkyl or arylphosphonate;
FIG. 10 shows a process for the synthesis of a dinucleoside alkyl
or arylphosphonate;
FIG. 11 illustrates the process for synthesis of a dinucleoside
alkyl or arylphosphonate;
FIG. 12 is spectral diagrams of the 360 MHz .sup.1 H nmr spectra of
(a) d-ApA).sub.1 ; (b) d-ApA).sub.2 ; (c) d-TpT).sub.1 ; and (d)
d-TpT).sub.2 at 25.degree. C. in D.sub.2 O containing 1 mM
ethylenediaminetetracetate-10 mM sodium phosphate, pH 7.0, with the
tentative chemical shift assignments appear above each dimer;
FIG. 13 is spectral diagrams of circular dichroism spectra of (a)
dApA).sub.1 (--); dApA).sub.2 ( - - - ); (b) dApT).sub.1 (--),
dApT.sub.2 ( - - - ); (c) dTpA).sub.1 ( - - - ), dTpA).sub.2 (--);
and (d) dTpT in 10 mM Tris.HCl, 10 mM MgCl.sub.2, pH 7.5 at
27.degree. C.;
FIG. 14 is a sketch showing mixing experiment between polyuridylic
acid and dApA).sub.1 (O) or dApA).sub.2 (o) in 10 mM Tris, 10 mM
MgCl.sub.2, pH 7.5 at 0.degree. C., with the total nucleotide
concentration is 1.times.10.sup.-4 M.
FIG. 15 is a sketch showing melting curves of poly U+dApA).sub.1
(O) and poly U+dApA).sub.2 (o) in 10 mM Tris, 10 mM MgCl.sub.2, pH
7.5, with the stoichiometry of each complex is 2U:1A and the total
nucleotide concentration is 5.times.10.sup.-5 M;
FIG. 16 is molecular diagrams of the diastereoisomers of
dideoxyribonucleoside methyl phosphonates;
FIG. 17 is a schematic of a synthetic of this invention for
preparation of the oligonucleoside methyl phosphonate; and
FIG. 18 illustrates the transport of (O) 100 .mu.M d-GpGp-[.sup.3
H]-T and (o) 100 .mu.M d-Tp).sub.8 -[.sup.3 H]-T into transformed
Syrian hamster fibroblasts growing in monolayer at 37.degree.
C.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The general structure of the oligonucleoside alkyl or aryl
phosphonates are shown in FIGS. 1 and 2.
FIG. 1 shows the general structure of an oligonucleoside alkyl or
aryl phosphonate. The nucleoside units which consists of a base (B)
comprising adenine, thymine, guanine, cytosine, uracil or
hypoxanthine and a sugar where R' can be hydrogen, hydroxyl,
O-alkyl or O-aryl or O-halogeno are linked in a 3'-5' manner by a
phosphosphonate group where R can be alkyl or aryl. The
configuration of the alkyl or aryl phosphonate group is S.
FIG. 2 shows the general structure of an oligonucleoside alkyl or
aryl phosphonate. The nucleoside units which consists of a base (B)
comprising adenine, thymine, guanine, cytosine, uracil or
hypoxanthine and a sugar where R' can be hydrogen, hydroxyl,
O-alkyl or O-aryl or O-halogeno are linked in a 3'.fwdarw.5' manner
by a phosphosphonate group where R can be alkyl or aryl. The
configuration of the alkyl or aryl phosphonate group is R.
A specific example of an oligodeoxyribonucleoside methylphosphonate
is shown in FIG. 3.
In FIG. 3, there is shown the structure of a
heptadeoxyribonucleoside methylphosphonate. The deoxyribonucleoside
units which occur in the order
deoxadenosine-deoxyguanosine-deoxyquanosine-deoxyadenosine-deoxyguanosine-
deoxyquanosinethymidine are linked in a 3'.fwdarw.5' manner by
methylphosphonate groups. The configuraitons of the
methylphosphonate groups are not specified.
The following description will now be given of the synthesis of a
particular series of oligodeoxyribonucleoside methyl phosphonates
and their physical properties.
The synthetic procedure resulted in the separation of two
diastereoisomers of each dimer analog. This synthetic scheme also
allowed the preparation of analogs containing a .sup.13 C-enriched
phosphonate methyl group. The influence of backbone configuration
on overall dimer conformation was studied by ultraviolet, circular
dichroism and .sup.1 H, .sup.13 C and .sup.31 P nuclear magnetic
resonance techniques and the results were compared to the
conformations of their parent dideoxyribonucleoside monophosphates.
Furthermore, the effects of backbone configuration and the removal
of the negative charge on the interaction of
deoxyadenosine-containing dimers with polyuridylic acid and
polythymidylic acid were assessed.
Several processes or methods will now be described for synthesis of
the various materials of this invention.
FIG. 4 shows the synthesis of a dinucleoside alkyl or
arylphosphonate. This process consists of esterification of a
5'-O-protected nucleoside having 3'-hydroxyl group with an alkyl or
arylphosphonic acid in the presence of an activating agent to form
a 5'-O-protected nucleoside-3'-O-alkyl or arylphosphonate.
The latter compound is then esterified with a 3'-O-protected
nucleoside having a 5'-hydroxyl group in the presence of an
activating agent to form a fully protected dinucleoside alkyl or
arylphosphonate. The protecting groups are then removed from the
fully protected dinucleoside alkyl or arylphosphonate to form the
dinucleoside alkyl or arylphosphonate.
Referring now to FIG. 5, there is illustated the synthesis of a
dinucleoside alkyl or arylphosphonate. This process consists of
esterification of a 3'-O-protected nucleoside having a 5'-hydroxyl
group with an alkyl or arylphosphonic acid in the presence of an
activating agent to form a 3'-O-protected nucleoside-5'-O-alkyl or
arylphosphonate.
The latter compound is then esterified with a 5'-O-protected
nucleoside having a 3'-hydroxyl group in the presence of an
activating agent to form a fully protected dinucleoside alkyl or
arylphosphonate. The protecting groups are then removed from the
fully protected dinucleoside alkyl or arylphosphonate to form the
dinucleoside alkyl or arylphosphonate.
FIG. 6 shows the synthesis of a dinucleoside alkyl or
arylphosphonate. This process consists of esterification of a
5'-O-protected nucleoside having a 3'-hydroxyl group with
disubstituted alkyl or arylphosphonate. The substitutents (R"") can
be chloride, imidazolide, triazolide or tetrazolide.
A 5'-O-protected nucleoside-3'-O-monosubstituted alkyl or
arylphosphonate is formed in this reaction. This compound is then
esterified with a 3'-protected nucleoside having a 5'-hydroxyl
group to give the fully protected dinucleoside alkyl or
arylphosphonate. The protecting groups are then removed from the
fully protected dinucleoside alkyl or arylphosphonate to form the
dinucleoside alkyl or arylphosphonate.
The synthesis of a dinucleoside alkyl or arylphosphonate is shown
in FIG. 7. This process consists in the esterification of a
3'-O-protected nucleoside having a 5'-hydroxyl group with
disubstituted alkyl or arylphosphonate. The substituents (R"") can
be chloride, imidazolide, triazolide or tetrazolide. A
3'-O-protected nucleoside-5'-O-monosubstituted alkyl or
arylphosphonate is formed in this reaction.
This compound is then esterified with a 5'-protected nucleoside
having a 3'-hydroxyl group to give the fully protected dinucleoside
alkyl or arylphosphonate. The protecting groups are then removed
from the fully protected dinucleoside alkyl or arylphosphonate to
form the dinucleoside alkyl or arylphosphonate.
FIG. 8 illustrates the synthesis of a dinucleoside alkyl or
arylphosphonate. In this process a 5'-O-protected nucleoside having
a 3'-hydroxyl group is reacted with a dichloro alkyl or
arylphosphine to form a 5'-O-protected nucleoside-3'-O-monochloro
alkyl or arylphosphonate.
This compound is then reacted with a 3'-O-protected nucleoside
having a 5'-hydroxyl group to give a fully protected dinucleoside
alkyl or arylphosphonate.
The latter compound is then oxidized to form a fully protected
dinucleoside alkyl or arylphosphonate. The protecting groups are
then removed from the fully protected dinucleoside alkyl or
arylphosphonate to give the dinucleoside alkyl or
arylphosphonate.
FIG. 9 shows the synthesis of a dinucleoside alkyl or
arylphosphonate. In this process, a 3'-O-protected nucleoside
having a 5'-hydroxyl group is first reacted with a dichloro alkyl
or arylphosphine to form a 3'-O-protected
nucleoside-5'-O-monochloro alkyl or arylphosphonate.
This compound is next reacted with a 5'-O-protected nucleoside
having a 3'-hydroxyl group to give a fully protected dinucleoside
alkyl or arylphosphinate. The latter compound is then oxidized to
form a fully protected dinucleoside alkyl or arylphosphonate.
The protecting groups are then removed from the fully protected
dinucleoside alkyl or arylphosphonate to give the dinucleoside
alkyl or arylphosphonate.
FIG. 10 shows the synthesis of a dinucleoside alkyl or
arylphosphonate. In this process, a 5'-O-protected nucleoside
having a 3'-hydroxyl group is first reacted with
ethyldichlorophosphite to give a 5'-O-protected
nucleoside-3'-O-ethyl monochlorophosphite.
This compound is then reacted with a 3'-O-protected nucleoside
having a 5'-hydroxyl group to give a fully protected dinucleoside
ethylphosphite. The latter compound is then reacted with an alkyl
or aryliodide to give a fully protected dinucleoside alkyl or
arylphosphonate. The protecting groups are then removed from the
fully protected dinucleoside alkyl or arylphosphonate to give the
dinucleoside alkyl or arylphosphonate.
FIG. 11 shows the synthesis of a dinucleoside alkyl or
arylphosphonate. In this process, a 3'-O-protected nucleoside
having a 5'-hydroxyl group is reacted with ethyldichlorophosphite
to give a 3'-O-protected nucleoside-5'-O-ethyl monochlorophosphite.
This compound is reacted with a 5'-O-protected nucleoside having a
3'-hydroxyl group to give a fully protected dinucleoside
ethylphosphite. The latter compound is then reacted with an alkyl
or aryliodide to give a fully protected dinucleoside alkyl or
arylphosphonate. The protecting groups are then removed from the
fully protected dinucleoside alkyl or arylphosphonate to give the
dinucleoside alkyl or arylphosphonate.
In previous processes (FIGS. 4 to 11), there was described several
methods for joining nucleosides to form dinucleoside alkyl or aryl
phosphonates. The chain length can be extended from at least 2 to a
finite number of greater than 2, for example up to 20. For rapid
and efficient synthesis oligonucleoside alkyl or aryl phosphonates
can be joined by processes analogous to there previously described
for the preparation of dinucleoside alkyl or aryl phosphonates.
Materials and Methods
Thymidine and 2'-deoxyadenosine were checked for purity by paper
chromatography before use. 5'-Mono-p-methoxytrityl thymidine,
5'-di-p-methoxy-trityl-N-benzoyldeoxyadenosine,
3'-O-acetylthymidine and 3'-O-acetyl-N-benzoyldeoxyadenosine were
prepared according to published procedures.
Diethyl [.sup.13 C]-methyl phosphonate was prepared by reaction of
[.sup.13 C]-methyl iodide (96% enriched) with triethyl phosphate
followed by vacuum distillation of the product (b.p.
64.degree.-66.degree./2 mm/Hg. The pyridinium salt of methyl
phosphonic acid was prepared by hydrolysis of dimethyl
methylphosphonate or diethyl [.sup.13 C]-methyl phosphonate in 4N
hydrochloric acid followed by isolation of the product as the
barium salt.
The barium salt was converted to the pyridinium salt by passage
through a Dowex 50X pyridinium ion-exchange column.
Mesitylenesulfonyl chloride was treated with activated charcoal and
recrystallized from pentane immediately before use. 1-H-Tetrazole
was then prepared. All solvents and reagents were purified.
Silica gel column chromatography was performed using Baker 3405
silica gel (60-200 mesh). Thin layer silica gel chromatography and
thin layer cellulose chromatography were done. Paper chromatography
was carried out on Whatmann 3 MM paper using the following solvent
systems: solvent A, 2-propanol-concd. ammonium hydroxide-water
(7:1:2 v/v); solvent C, 1M ammonium acetate-95% ethanol (3:7, v/v);
solvent F, 1-propanol-concd. ammonium hydroxide-water (50:10:35,
v/v) or solvent I, 2-propanol-water (7:3, v/v).
High pressure liquid chromatography was performed using columns
(2.1 mm.times.1 m) packed with DuPont Permaphase ODS reverse phase
material. Linear gradients (40 ml) from 0% to 75% methanol in water
were used at a flow rate of 1 ml/min. The HPLC mobility refers to
the percentage of methanol in water required to elute the compound
from the column.
For reactions carried out in pyridine, the reactants were dried by
repeated evaporation with anhydrous pyridine and were then
dissolved in anhydrous pyridine. Unless otherwise noted, all
reactions and operations were performed at room temperature.
Preparation of Mesitylenesulfonyl Tetrazolide
Although the preparation of MST has been published, a modified
procedure was used. A solution of 1-H-tetrazole (3.36 g: 48 mmoles)
in 40 ml of dry methylene chloride containing 5.6 ml (40 mmoles) of
triethylamine was added dropwise to 40 ml of anhydrous ether
containing mesitylenesulfonyl chloride (8.76 g, 40 mmoles) at room
temperature. After 2 hrs, the precipitated triethylammonium
chloride was removed by filtration, and washed with 50 ml of
methylene chloride-ethyl ether (1:1 v/v).
The filtrate was cooled to 0.degree. C. and pentane was added until
the solution became cloudy. This procedure was repeated until a
total volume of 25 ml had been added over a 4 hour period. After
storage overnight at 4.degree., the resulting white solid was
removed by filtration on a sintered glass filter. The solid was
dissolved in 500 ml of anhydrous ethyl ether.
The solution was then filtered to remove a small amount of
insoluble material. The filtrate was evaporated to dryness and the
resulting mestilylene sulfonyl tetrazolide (4.46 g) was obtained in
44% yield. The MST was pure as indicated by silica gel tlc, Rf
(C.sub.6 H.sub.6) 0.11 (m.p. 109.degree.-110.degree. C.) and was
stored in a desicator at -80.degree. C. Under these storage
conditions, the MST maintained its condensing activity for at least
one month.
Preparation of [MeOTr]TpCE
[MeOTr]T (10.3 g, 20 mmoles), the pyridinium salt of methyl
phosphonic acid (40 mmoles) and Dowex pyridinium resin (0.5 g) were
treated with dicyclohexyl-carbodiimide (41.2 g, 200 mmoles) in 100
ml of pyridine at 37.degree. C. for 3 days. The resulting
[MeOTr]Tp, Rf silica gel tlc 0.00 (EtOAc/THF 1:1), appeared to be
formed in approximately 86% yield as determined by HPLC. The
material is eluted from the HPLC column with 37%
methanol/water.
Hydracrylonitrile (100 ml) was added to the reaction mixture, which
was kept at 37.degree. C. for 2 days. Aqueous pyridine (200 ml) was
then added and the resulting dicyclohexylurea was removed by
filtration. The filtrate was evaporated, dissolved in 250 ml of
ethyl acetate and the solution extracted with 3 (250 ml) portions
of water. The ethyl acetate solution was dried over anhydrous
sodium sulfate. After filtration and evaporation, the mixture was
chromatographed on a silica gel column (5.4.times.37 cm) which was
eluted with ether (IL), ethyl acetate (1.2 L) and tetrahydrofuran
(1.6 L). Pure [MeOTr]TpCE (7.5 g) was isolated in 55% yield after
precipitation from tetrahydrofuran by addition of hexane. The
monomer has Rf values of 0.32 (EtOAc/THF 1:1) and 0.66 (20%
MeOH-CHCl.sub.3) on silica gel tlc and is eluted from the HPLC
column with 54% methanol/water. The UV spectrum gave .lambda. max
267 nm, sh 230 nm, .lambda. min 250 nm, .epsilon.260/.epsilon.280
1.44 in 95% ethanol.
Preparation of [MeOTr]Tp Pyridinium Salt
[MeOTr]TpCE (2.17 g, 3.36 mmoles) was treated with 16.8 ml of 1N
sodium hydroxide in a solution containing 126 ml of dioxane and 25
ml of water for 15 min. The solution was neutralized by addition of
Dowex 50X pyridinium resin. After filtration, the solution was
evaporated and the residue was rendered anhydrous by evaporation
with pyridine. The [MeOTr]Tp (1.90 g; 2.83 mmoles) was obtained in
84% yield after precipitation from pyridine by addition to
anhydrous ether. The material has Rf values of 0.00 (EtOAc/THF 1:1)
and 0.04 (20% MeOH/CHCl.sub.3) on silica gel tlc. The UV spectrum
gave .lambda. max 267 nm, sh 230 nm, .lambda. min 250 nm,
.epsilon.230/.epsilon.267 1.68, .epsilon.260/.epsilon.280 1.44 in
95% ethanol.
The monomethoxytrityl group was removed from 70 mg (0.1 mmole) of
d-[(MeO)Tr]Tp by treatment with 80% aqueous acetic acid. The
resulting Tp (874 A.sub.267 units, 0.095 mmole) was isolated in 95%
yield by chromatography on a DEAE Sephadex A25 column (3.times.8.5
cm) using a linear gradient of ammonium bicarbonate (0.01M to
0.20M, 500 ml). The monomer has the following Rf values on
cellulose TLC:0.41 (solvent A), 0.77 (solvent C) and 0.69 (solvent
F). The UV spectrum gave .lambda. max 267 nm, .lambda. min 235 nm
in water pH 7.0. The pmr spectrum was consistent with the structure
of the monomer.
Preparation of TpCE
[MeOTr]TpCE (3.26 g, 5.04 mmoles) dissolved in 20 ml of methanol
was treated with 80 ml of 80% acetic acid solution for 5 hr. at
37.degree. C. The solvents were removed by evaporation and the
residue was evaporated repeatedly with 50% toluenetetrahydrofuran
to remove the acetic acid. TpCE (1.80 g, 4.8 mmoles) was obtained
in 96% yield after precipitation from tetrahydrofuran (10 ml) by
addition of hexane (200 ml). The material has Rf values of 0.08
(EtOAc/THF, 1:1) and 0.16 (15% MeOH/CHCl.sub.3) on silica gel tlc.
The UV spectrum gave .lambda. max 265 nm, .lambda. min 233 nm,
.epsilon.260/.epsilon.280 1.61 in absolute ethanol.
Preparation of d-[(MeO).sub.2 Tr]bzApCE
A solution containing d-[MeO).sub.2 Tr]bzA (10.5 g; 16 mmoles),
methyl phosphonic acid (32 mmoles) and Dowex 50X pyridinium resin
(0.5 g) in 80 ml of anhydrous pyridine was treated with
dicyclohexylcarbodiimide (25 g; 121 mmoles) for 3 days at
37.degree. C. Examination of the reaction mixture by HPLC showed
essentially quantitative conversion of d-[(MeO).sub.2 Tr]bzA to
d-[(MeO).sub.2 Tr]bzAp, which has HPLC retention time of 22.8 min.
The reaction mixture was treated with 80 ml of hydracrylonitrile
for 2 days at 37.degree. C.
After filtration and evaporation of the solvents, the residue was
dissolved in 200 ml of ethyl acetate and the solution was extracted
with three (200 ml) portions of water. The ethyl acetate solution
was dried over anhydrous sodium sulfate, concentrated to 50 ml and
chromatographed on a silica gel column (5.4.times.37 cm).
The column was eluted with ether (1.5 L), ethyl acetate (1.5 L) and
tetrahydrofuran (1.5 L). The resulting d-[(MeO).sub.2 Tr]bzApCE
weighed 5.4 g (6.84 mmoles, 43%) after precipitation from
tetrahydrofuran (100 ml) with hexane (500 ml). The material elutes
from the HPLC column with 68% methanol/water, and has silica gel
TLC Rf values of 0.13 (EtOAc/THF, 1:1 v/v) and 0.27 (THF). The UV
spectrum shows .lambda. max 279 nm and 234 nm, .lambda. min 258 nm
and 223 nm; .epsilon.234/.epsilon.279 1.44,
.epsilon.260/.epsilon.280 0.67, in 95% ethanol.
Preparation of d-[(MeO).sub.2 Tr]bzAp
A solution containing d-[(MeO).sub.2 Tr]bzApCE (3.79 g, 4.8 mmoles)
in 180 ml of dioxane and 36 ml of water was treated with 24 ml of
1N sodium hydroxide for 7 min. The solution was neutralized with
Dowex 50X pyridinium resin and then was passed through a Dowex 50X
pyridinium ion exchange column (3.times.30 cm). The eluate was
evaporated and the residue was rendered anhydrous by evaporation
with pyridine. The resulting d-[(MeO).sub.2 Tr]bzAp (2.9 g; 3.56
mmoles) was obtained in 74% yield after precipitation from
anhydrous ether. The material has Rf values of 0.00 (THF) and 0.36
(50% MeOH/CHCl.sub.3) on silica gel tlc. The UV spectrum showed
.lambda. max 280 nm and 233 nm, .lambda. min 255 nm and 225 nm
.epsilon.233/.epsilon.280 1.37, .epsilon.260/.epsilon.280 0.67 in
95% ethanol.
The protecting groups were removed from a small sample of
d-[(MeO).sub.2 Tr]bzAp (80 mg, 0.1 mmole) by sequential treatment
with concentrated ammonium hydroxide in pyridine and 80% acetic
acid. The monomer dAp (1400 A.sub.260 units, 0.09 mmole) was
isolated by chromatography on a DEAE Sephadex A-25 column
(3.times.8.5 cm) using a linear gradient of ammonium bicarbonate
(0.01 to 0.2M, 600 ml). The monomer has the following Rf values on
cellulose TLC:0.45 (solvent A), 0.56 (solvent C) 0.67 (solvent F)
and 0.44 (solvent I). The UV spectrum showed .lambda. max 259 nm,
.lambda. min 227 nm 260/280 6.13, in water pH 7.0. The pmr spectrum
was consistent with the structure of the monomer.
Preparation of d-bzApCE
A solution of d-[(MeO).sub.2 Tr]bzApCE (1.58 g, 2 mmoles) in 6.3 ml
of methanol was treated with 25 ml of 80% acetic acid for 1.5 hrs.
The solvents were evaporated and the residue was repeatedly
evaporated with toluene and tetrahydrofuran to remove acetic acid.
The residue was precipitated from 20 ml of tetrahydrofuran by
dropwise addition to 250 ml of hexane to give 0.95 g (1.95 mmoles)
of d-bzApCE in 98% yield. The material has Rf values of 0.09 (THF)
and 0.25 (20% MeOH/CHCl.sub.3) on silica gel TLC and is eluted from
the HPLC column with 12% methanol/water. The UV spectrum shows
.lambda. max 280 nm, sh 233 nm, .lambda. min 247 nm
.epsilon.233/.epsilon.280 0.65 .epsilon.260/.epsilon.280 0.60, in
95% ethanol.
Preparation of Dinucleoside Methyl Phosphonates
The general procedure for the preparation of protected dinucleoside
methyl phosphonates is given in this section. Table 1 shows the
specific reaction conditions and yields for each dimer. The
protected nucleoside 3'-methyl phosphonate and protected nucleoside
or nucleoside 3'-methyl phosphonate cyanoethyl ester were dried by
evaporation with anhydrous pyridine. The condensing agent was added
and the reactants were taken up in anhydrous pyridine to give a
0.2M solution. After completion of the reaction as indicated by TLC
and/or HPLC, an equal volume of water was added and the solution
was kept at room temperature for 30 min. The solvents were then
evaporated and the residue dissolved in ethyl acetate or
chloroform. The organic solution was extracted with water and then
dried over anhydrous sodium sulfate. After concentration, the
organic solution was applied to a silica get column (3.times.28 cm
for a 1 mmole scale reaction). The column was eluted with ethyl
acetate, ethyl acetate/tetrahydrofuran (1:1 v/v) and
tetrahydrofuran. The progress of the elution was monitored by
silica get TLC. Dimers terminating with a 3'-acetyl group separated
into their individual diastereoisomers on the column and were
eluted as pure isomer 1, a mixture of isomer 1 and 2 and pure
isomer 2. The dimers were isolated as white solids, by
precipitation from tetrahydrofuran solution upon addition of
hexane. The Rf values on silica get tlc, the mobilities on the HPLC
column and the ultraviolet spectral characteristics of the
protected dimers are given in Table 2.
TABLE 1
__________________________________________________________________________
Preparation of Protected Dideoxyribonucleoside Methyl Phosphonates
Condensing Agent.sup.(a) Reaction Dimer Monomers (mmole) (mmole)
Time (mmole) Yield
__________________________________________________________________________
d-[(MeO)Tr]Tp (0.20) DCC 3 days d-[(MeO)Tr]TpTOAc 16% + d-TOAc
(0.22) (0.73) 37.degree. C. (0.031) d-[(MeO)Tr]Tp (2.40) MST 3 hrs.
d-[(MeO)Tr]TpTpCE 55% + d-TpCE (3.60) (9.60) r.t. (1.32)
d-[(MeO).sub.2 Tr]bzAp (1.26) TPSCl 4 days d-[(MeO).sub.2
Tr]bzApbzAOAc 39% + d-bzAOAc (1.50) (2.0) 37.degree. C. (0.50)
d-[(MeO).sub.2 Tr]bzA- (0.70) MST 4 hrs. d-[(MeO).sub.2
Tr]bzA-[.sup.13 C]--pbzAOAc 41% [.sup.13 C]--p + d-bzAOAc (1.05)
(2.8) r.t. (0.29) d-[(MeO).sub.2 Tr]bzAp (0.85) MST 6 hrs.
d-[(MeO).sub.2 Tr]bzApbzApCE 46% + d-bzApCE (1.28) (4.0) r.t.
(0.39) d-[(MeO)Tr]Tp (1.0) TPSCl 16 hrs. d-[(MeO)tr]TpbzAOAc 35% +
d-bzAOAc (1.0) (3.0) 37.degree. C. (0.35) d-[(MeO).sub.2 Tr]bzAp
(1.0) TPSCl 46 hrs. d-[(MeO).sub.2 Tr]bzApTOAc 38% + d-TOAc (1.3)
(1.5) 37.degree. C. (0.38)
__________________________________________________________________________
.sup.(a) DCC -- dicyclohexylcarbodiimide TPSCl --
triisopropylbenzenesulfonyl chloride MST -- mesitylenesulfonyl
tetrazolide
The base labile protecting groups were removed from the dimers by
treatment with 50% concentrated ammonium hydroxidepyridine solution
for 3 days at 4.degree. C. Alternatively, the N-benzoyl protecting
groups of dimers containing deoxyadenosine could be removed by
treatment with 85% hydrazine hydrate in 20% acetic acid-pyridine
buffer overnight at room temperature (Letsinger et al., 1968). This
treatment also partially removed the 3'-O-acetyl group. The acetyl
group was completely removed by further treatment with 50%
concentrated ammonium hydroxide-pyridine solution for 2 hours at
4.degree. C. After complete removal of solvents, the trityl
protecting groups were removed by treatment with 80% acetic
acid-methanol (8:2 v/v) solution at room temperature. The solvents
were then removed and the dimers were chromatographed on Whatmann 3
MM paper using solvent A. The dimers were eluted from the paper
with 50% aqueous ethanol. For dimers terminating with 3'-OH groups,
the ethanol solutions were passed through small (0.5.times.1 cm)
DEAD cellulose columns to remove trace impurities eluted from the
paper chromatogram.
Dimers terminating with 3'-methyl phosphonate groups were absorbed
to small DEAE cellulose columns and then eluted with 0.5M ammonium
bicarbonate solution. The dimers were stored as standard solutions
in 50% ethanol at 0.degree. C., and were found to be complete
stable under these conditions for at least 9 months. For physical
and nmr studies, aliquots containing the required amount of dimer
were evaporated to remove the ethanol and then lyophilized from
water or D.sub.2 O before use. The Rf values and UV spectral
characteristics of the dimers are given in Table 3. The pmr spectra
and tentative chemical shift assignments of the two
diastereoisomers of d-ApA and d-TpT are shown in FIG. 12.
TABLE 2
__________________________________________________________________________
Chromatographic Mobilities and Ultraviolet Spectral Properties of
Protected Dideoxyribonucleoside Methyl Phosphonates ##STR1## HPLC
Mobility.sup.(c) 10% MeOH/ 15% MeOH/ (% methanol Ultraviolet
Spectral Properties.sup.(b) Dimer THF CHCl.sub.3 CHCl.sub.3 water)
.lambda. max (nm) .lambda. min
__________________________________________________________________________
(nm) d-[(MeO)Tr]TpTOAc 0.44 0.63 0.53 -- -- 267sh235 246 ##STR2##
d-[(MeO)Tr]TpTpCE -- -- 0.28 0.22 -- 265sh235 245 ##STR3##
d-[(MeO).sub.2 Tr]bzApbzAOAc 0.34 0.29 -- 0.51 0.43 68% 281sh233
256 ##STR4## d-[(MeO).sub.2 Tr]bzApbzApCE 0.09 0.32 0.28 0.39 0.35
-- 281sh230 256 ##STR5## d-[(MeO)Tr]TpbzAOAc 0.47 0.24 0.19 0.59
62% 276;230, sh260 247;227 ##STR6## d-[(MeO).sub.2 Tr]bzApTOAc 0.49
0.41 -- 0.52 0.48 66% 277;235, sh263 255,227 ##STR7##
__________________________________________________________________________
.sup.(a) Two Rf values refer to the mobilities of the individual
diastereoisomers. .sup.(b) Ultraviolet spectra were measured in 95%
ethanol at room temperature. .sup.(c) Percentage of methanol in
water required to elute compound from HPLC column (DuPont
Phermaphase ODS)
TABLE 3 ______________________________________ Chromatographic
Mobilities and Ultraviolet Spectral Properties of
Dideoxyribonucleoside Methyl Phosphonates Ultraviolet Spectral
Mobility Paper Properties.sup.(a) Dimer Chromatography
Rf(A)Rf(C)Rf(I) .lambda. max (nm) .lambda. min (nm) ##STR8##
______________________________________ d-TpT -- 0.67 0.73 267 234
1.91 d-ApA -- 0.48 0.62 258 223 6.65 d-ApAp 0.30 -- 0.38 258 227
4.52 d-TpA 0.42 0.57 0.63 262 230 2.80 d-ApT 0.33 0.55 0.61 261 233
3.22 ______________________________________
TABLE 4 ______________________________________ Hypochromicity of
Dideoxyadenosine Methyl Phosphonate Analogs Compound
.epsilon.(molar).sup.(a) % Hypochromicity
______________________________________ dpA 15.3 .times. 10.sup.3 --
d-ApA 12.7 .times. 10.sup.3 17% (d-ApA).sub.1 13.7 .times. 10.sup.3
11.0% (d-ApA).sub.2 14.3 .times. 10.sup.3 7.1% (d-ApAp).sub.1 13.0
.times. 10.sup.3 13.3% (d-ApAp).sub.2 13.3 .times. 10.sup.3 11.3%
______________________________________ .sup.(a) Measured in 1 mM
Tris HCl pH 7.4 at 27.degree. C.
Similar pmr spectra were obtained for d-ApT and d-TpA (data not
shown). The spectra are consistent with the structures of the
dimers. The complete characterization of all these dimers by pmr
spectroscopy will be described in a subsequent paper (Kan et al.,
manuscript in preparation).
Physical Studies and Interaction with Polynucleotides
Ultraviolet and circular dichroism spectra were recorded
respectively on a Cary 15 spectrophotometer and a Cary 60
spectropolarimeter with CD attachment. The continuous variation
experiments, melting experiments and circular dichroism experiments
were carried out as previously described. The molar extinction
coefficient of poly U is 9.2.times.10.sup.3 (265 nm) and poly dT is
8.52.times.10.sup.3 (264 nm). The molar extinction coefficients of
the dideoxyadenosine methyl phosphonates were determined by
comparing the absorption of a solution of the dimer at pH 7.4 with
the absorption of the same solution at pH 1.0. The dimer extinction
coefficient was then calculated from the observed hyperchromicity
of the dimer at pH 1.0 using an extinction coefficient for
deoxyadenosine at pH 1.0 of 14.1.times.10.sup.3.
Preparation of Dinucleoside Methyl Phosphonates
The synthetic route used to prepare the dinucleoside methyl
phosphonates has previously been described. 5'-Mono-p-methoxytrityl
thymidine and 5'-di-p-methoxytrityl-N-benzoyl deoxyadenosine were
converted to the corresponding 3'-methyl phophonate
.beta.-cyanoethyl esters (2) by sequential reaction of (1) with
methyl phosphonic acid and .beta.-cyanoethanol in the presence of
dicyclohexylcarbodiimide.
The preparation of nucleoside 5'-methyl phosphonates by reaction of
a suitably protected nucleoside with methyl phosphonic acid has
been previously known. Direct conversion of the protected
nucleoside-3'-methyl phosphonate to its .beta.-cyanoethyl ester
allows purification of this intermediate on a large scale by silica
gel column chromatography, thus avoiding the use of ion exchange
chromatography. They trityl or .beta.-cyanoethyl protecting groups
can be selectively removed from 2 by treatment with either 80%
acetic acid or 0.1N sodium hydroxide solution, respectively, at
room temperature.
Protected nucleoside-3'-methyl phosphonate (4) was condensed with
either 3'-O-acetyl thymidine or 3'-O-acetyl-N-benzoyldeoxyadenosine
to give fully protected dinucleoside methyl phosphonate 6.
Alternatively, 4 was condensed with the .beta.-cyanoethyl ester of
thymidine-3'-methyl phosphonate or
N-benzoyldeoxyadenosine-3'-methyl phosphonate to give 7. The
condensing agents used in these reactions were
dicyclohexylcarbodiimide, triisopropylbenzenesulfonyl chloride or
mesitylenesulfonyl tetrazolide. The reaction conditions and yields
are given in Table 1.
The fully protected dimers were readily purified by silica gel
column chromatography. For dimers terminating with 3'-O-acetyl
groups, the two diastereoisomers were sufficiently separated on the
silica gel column that fractions containing each pure
diastereoisomer were obtained. These isomers were designated isomer
1 and isomer 2 in reference to their order of elution from the
column. The diastereoisomers were generally formed in a 4:6 ratio
of isomer 1 to isomer 2.
Alternatively, the diastereoisomers could be obtained in pure form
by thick layer chromatography on silica gel plates. The dimers
terminating in a 3'-O-.beta.-cyanoethyl methyl phosphosphonate
group (7) consists of four diastereoisomers, although only two
separate bands were observed on silica gel thin layer
chromatography (see Table 2). For the deoxyadenosine-containing
dimer, these two bands turned out to the two isomers with opposite
configuration (axial and equatorial, see FIG. 16) about the methyl
phosphonyl internucleoside linkage.
Removal of the protecting groups from 6 and 7 was accomplished by
sequential treatment with concentrated ammonium hydroxide in
pyridine for 3 days at 4.degree. C. followed by treatment with 80%
acetic acid. In the case of the dideoxyadenosine methyl
phosphonates, some hydrolysis of the phosphonate linkage was noted
when the ammonium hydroxide treatment was carried out at room
temperature. However, the hydrolysis was suppressed at low
temperature. Alternatively, the N-benzoyl protecting groups of
these dimers could be removed by treatment with hydrazine hydrate.
The dimers were then purified by paper chromatography. The
individual diastereoisomers of each deprotected dimer had the same
chromatographic mobilities on paper chromatography in all solvent
systems tested (See Table 3).
Ultraviolet and Hypochromicity Measurements
The ultraviolet spectral properties of the dinucleoside methyl
phosphonates are recorded in Table 3. Qualitatively, the spectra
are similar to those of (3'-5')-linked dinucleoside monophosphates.
The spectra of the individual diastereoisomers are qualitatively
similar to each other.
Hypochromicity measurements for the dideoxyadenosine methyl
phosphonates were carried out in water at pH 7.4 and are shown in
Table 4. The percent hypochromicity of the methyl phosphonate
dimers is from 4% to 10% lower than the percent hypochromicity of
d-ApA. Each diastereoisomer has an unique molar extinction
coefficient. The hypochromicity of isomer 1, the isomer eluted
first from the silica gel column, is greater that that of isomer 2,
reflecting differences in the extent of base-base overlap in these
dimers.
Circular Dichroism Spectra
Differences in the extent and mode of base stacking interactions
are observed for individual diastereoisomers within a given dimer
sequence as reflected by the CD spectra of the dimers. The profile
of the CD spectrum of d-ApA).sub.1 (FIG. 13a) is qualitatively
similar to that of the parent dinucleoside monophosphate, d-ApA
(Miller et al., 1971). However, the magnitudes of the molecular
ellipticity (.theta.) at 267 nm and 270 nm of d-ApA).sub.1 are
approximately one-half of those found for d-ApA. A very dramatic
difference in the CD spectrum of d-ApA).sub.2 is observed. Only
negative [.theta.] is found at 250 nm and the amplitude of the
molecular ellipticity is approximately three-fold less than that of
d-ApA).sub.1. Similar results were observed for d-ApAp).sub.1 and
d-ApAp).sub.2 (data not shown).
In the case of d-ApT (FIG. 13b), the profiles of the CD spectra of
both isomers 1 and 2 are qualitatively similar to that of d-ApT
(Cantor et al., 1970). However, the magnitude of the ellipticity of
the peak (272 nm) and trough (253 nm) of the dinucleoside methyl
phosphonate are less than those in the dinucleoside monophosphate.
For d-ApT.sub.1, the peak is reduced 1.8-fold and the trough is
reduced 1.3-fold compared to d-ApT while for d-ApT).sub.2 the
reductions are 8.1 and 5.0-fold.
The CD spectra of d-TpA).sub.1 and d-TpA).sub.2 (FIG. 13c) show
differences in both the magnitude of the molecular ellipticity and
in the position of the positive and negative bands. Isomer 2 has a
CD spectrum which is virtually identical to that observed for
d-TpAp (Cantor et al., 1970). Isomer 1, on the other hand, has a
lower magnitude of the (.theta.) value, while the positions of the
peak and trough are shifted to shorter wavelengths.
The CD results for dApT).sub.1 and 2 and dTpA).sub.1 and 2 are
qualitatively similar to those obtained by others on the CD spectra
of the separated disastereoisomers of the dinucleoside ethyl
phosphotriesters, dAp(Et)T and dTp(Et)A. In the case of these
triesters, one isomer has a spectrum which is almost identical to
that of the corresponding dinucleoside monophosphate. The other
isomer shows significant reductions in the magnitudes of both the
positive and negative CD absorbtion bands. It is not possible at
this time to make detailed comparisons between the present results
and those on the triesters, since the absolute configurations of
the modified phosphate groups in the triesters are not known.
FIG. 13d shows the CD spectrum of a 1:1 mixture of the
diastereoisomers of d-TpT. The spectrum of this mixture is clearly
different than the spectrum of d-TpT (Cantor et al., 1970). For
d-TpT positive (.theta.) occurs at 280 nm with a magnitude
approximately 1.8-fold greater than the 275 nm band of d-TpT.
Similarly, d-TpT shows negative (.theta.) at 250 nm which is
approximately 1.8-fold greater than the negative band at 245 nm in
d-TpT.
Interaction of Dideoxyadenosine Methyl Phosphonates with Poly (U)
and Poly (dT)
Both diastereoisomers of d-ApA form complexes with poly U at
0.degree. C. The mixing curves for d-ApA.sub.1 and d-ApA).sub.2
with poly U (FIG. 14) show that complex formation occurs with a
base stoichiometry of 2U:1A. Similar results were obtained for the
interaction of d-ApAp).sub.1 and d-ApAp).sub.2 with poly U and for
the interaction of d-ApA with poly (dT).
As shown in FIG. 15, the methyl phosphonate-polynucleotide
complexes exhibit a cooperative thermal transition with a
well-defined melting temperature. The melting temperature of the
d-ApA).sub.1 -poly U complex is 4.4.degree. higher than the
d-ApA).sub.2 -poly U complex. A similar difference in melting
temperatures for the d-ApAp-poly U complexes was also observed
(Table 5). Essentially no difference is observed between the Tm
values of the two d-ApA-poly(dT) complexes, however.
Significant increases are observed in the thermal stabilities of
the dinucleoside methyl phosphonate-polynucleotide complexes as
compared to similar complexes formed between d-ApA and poly U or
poly dT. The non-ionic d-Apa forms complexes with Tm values
8.4.degree. and 12.4.degree. higher that that of d-Apa-poly U,
while the singly-charged d-ApAp from complexes with Tm values
6.5.degree. and 10.4.degree. higher than d-Apa-poly U. Similarly,
the complexes formed between d-ApA and poly (dT) each melt
approximately 10.degree. higher than the d-ApA-poly(dt)
complex.
TABLE 5 ______________________________________ Melting Temperatures
of Complexes Formed Between Dideoxyadenosine Methyl Phosphonate
Analogs and Polyuridylic Acid or Polythymidylic Acid
Complex.sup.(a) Tm .degree.C. (poly U).sup.(b) Tm .degree.C. (poly
dT) ______________________________________ d-ApA 7.0 9.2
(d-ApA).sub.1 15.4 18.7 (d-ApA).sub.2 19.8 18.4 (d-ApAp).sub.1 13.5
-- (d-ApAp).sub.2 17.4 -- ______________________________________
.sup.(a) Complex stoichiometry: 2U:1A or 2T:1A .sup.(b) 10 mM
Tris.HCl 10 mM MgCl.sub.2 pH 7.5
Discussion
Dinucleoside methyl phosphonates are novel nucleic acid analogs in
which the phosphodiester internucleoside linkage is replaced by a
3'-5' linked internucleoside methyl phosphonyl group. Unlike the
dinucleoside methylene phosphonates prepared by others, the methyl
phosphonate analogs do not contain a negatively charged backbone
and are nonionic molecules at pH 7. The methyl phosphonate group is
isosteric with respect to the phosphate group of dinucleoside
monophosphates. Thus, these analogs should present minimal steric
restrictions to interaction with complementary polynucleotides or
single-stranded regions of nucleic acid molecules. Since the methyl
phosphonyl group is not found in naturally occuring nucleic acid
molecules, this internucleoside linkage may be resistant to
hydrolysis by various nuclease and esterase activities and this has
in fact been observed (Miller, unpublished data). These properties
make analogs of this type potentially useful as vehicles for
exploring the interactions of selected oligonucleotide sequences
with nucleic acids and nucleic acid-related enzymes within the
living cell (Miller et al., 1977).
The preparation of the oligonucleoside methyl phosphonates follows
the basic strategy used for the preparation of protected
oligonucleotide phosphotriesters. The synthetic scheme which has
been adopted first involves preparation of a protected nucleoside
3'-methyl phosphonate .beta.-cyanoethyl ester (FIG. 17). This
two-step preparation can be carried out in a one-flask reaction and
proceeds in high overall yield. Since the product is readily
purified by silica gel column chromatography, multigram quantities
of this key intermediate can be prepared. By selective removal of
the 5'-trityl group or the .beta.-cyanoethyl group, chain extension
can proceed in either direction. Thus, compound 2 in FIG. 17 serves
as a basic building block for the preparation of longer oligomers.
This type of synthetic scheme was originally developed by others
for the preparation of oligonucloetide
.beta..beta..beta.-trichloroethyl phosphotriesters and has more
recently been used by others for the preparation of oligonucleotide
p-chlorophenyl phosphotriesters. This procedure also allows the
preparation of specifically [.sup.13 C]enriched dimers by use of
[.sup.13 C]-methyl phosphonic acid in the synthesis of 1. Dimers
and oligomers containing [.sup.13 C]-methyl phosphonate groups
could be very useful for probing the physical and biological
properties of oligonucleoside methyl phosphonates by nuclear
magnetic resonance spectroscopic techniques (Cheng et al.,
manuscript in preparation).
In the present study (FIG. 17), the -cyanoethyl group was removed
from 1 and chain extension was continued in the 3'-direction. Two
types of condensation reactions were carried out: (1) condensation
with a 3'-O-acetylated nucleoside to give dimers with the general
structure 6 and (2) condensation with a -ucleoside 3'-methyl
phosphonate .beta.-cyanoethyl ester to give dimers with general
structure 7. The latter type of dimer can be further extended by
removal of the .beta.-cyanoethyl group followed by condensation
with other oligonucleoside methyl phosphonate blocks. In this way,
oligonucleoside methyl phosphonates containing up to four
deoxyadenosine residues and up to nine thymidine residues have been
prepared.
Different condensing agents were used in these reactions, including
dicyclohexylcarbodiimide (DCC), triisopropylbenzenesulfonyl
chloride (TPSCl) and mesitylenesulfonyl tetrazolide (MST). The
order of condensing efficiency was found to be MST>TPSCl>DCC.
Although DCC did bring about condensation, several days at elevated
temperatures were required and the yields were quite low.
Considerable improvement in reaction yield was obtained when TPSCl
was used. However, again prolonged reaction periods were required
and noticeable buildup of side products was observed. The reagent
of choice for these reactions in MST. The reaction occurs within a
period of several hours, with little or no side products. The
efficiency of a particular condensing agent depends not only upon
its structure but also upon the nature of the
phosphorous-containing substituent which is activated. Thus, when
MST was used as a condensing agent, we observed that reactions
involving nucleoside 3'-methyl phosphonates or nucleoside 3'-ethyl
phosphates usually proceed in lower yield than those involving
nucleoside 3'-p-chlorophenyl phosphates.
The ability to separate the individual diastereoisomers of each
dimer sequence allowed examination of effect of the configuration
of the phosphonyl methyl group on the overall dimer conformation.
As shown in FIG. 16, the isomers differ in configuration at the
internucleoside linkage with the methyl group assuming either a
pseudo-axial or pseudoequitorial position when the dimers are drawn
in a stacked conformation. The unique conformational properties of
each diastereoisomer of d-ApA and d-ApAp are most readily seen by
examining the percent hypochromicity of each diastereoisomer (Table
4). Isomer 1 of both d-ApA and d-ApAp exhibits a greater percent
hypochromicity than does isomer 2 of this series. Since the percent
hypochromicity is related to the extent of base-base overlap in
dimers of this type (Ts'o, 1974), the result suggests that
d-ApA).sub.1 and d-ApAp).sub.1 and more highly stacked in solution
than are d-ApA).sub.2 and d-ApAp).sub.2. Comparison of the percent
hypochromicities of the methyl phosphonate dimers with that of
d-ApA shows that these dimers are less stacked than the parent
dinucleoside monophosphate. A similar result was observed for the
methyl and ethyl phosphotriesters of d-ApA. Thus, non-ionic methyl
or ethyl phosphotriester or methyl phosphonate internucleoside
linkages appear to perturb the stacking interactions between the
bases in these dimers.
The circular dichroism spectra of dinucleoside monophosphates are
indicators of both the extent and mode of base stacking, as well as
the population of right-handed versus left-handed stacks. The CD
spectra of each diastereoisomer for the methyl phosphonate dimer
sequences d-ApA, d-ApAp, d-ApT and d-TpA suggest that each
diastereoisomer has an unique stacking mode in solution. The
profiles of the CD spectra of d-ApA).sub.1 and d-ApAp).sub.1 are
very similar to those of d-Apa and r-ApA, and differ only in the
magnitude of the molecular ellipticity. This result and the results
of the hypochromicity measurements suggest that the stacking modes
of the bases in these dimers are similar to those of d-ApA and
r-ApA. On the other hand, the profiles of the DC spectra of
d-ApA).sub.2 and d-ApAp).sub.2 are quite different. The magnitudes
of the molecular ellipticities of dApA).sub.2 and dApAp).sub.2 are
greatly diminished, with complete loss of [.theta.] at 270 nm.
Since the hypochromicity measurements suggest that the bases in
these dimers have substantial overlap, the mode of stacking in
these dimers must be quite different from that found for isomer 1
or for d-ApA. The magnitude of the molecular ellipticity in dimers
of this type is sensitive to the angle, .theta., between the
transition dipoles of the bases. The value of the molecular
ellipticity is greatest when .theta. is 45.degree. and diminishes
to 0 when .theta. is 0.degree., 90.degree. or 180.degree.. Thus,
the most reasonable interpretation of the CD results is that in
d-ApA).sub.1 and d-ApAp).sub.1, the bases tend to orient in an
oblique manner, while in d-ApA).sub.2 and d-ApAp).sub.2, the bases
tend to orient in a parallel or perpendicular manner. This
interpretation is supported by the base-base stacking patterns as
determined by pmr spectroscopy. The substantial change in the CD
profile of d-ApA).sub.2 rather than a simple diminution of the
amplitude of the [.theta.] values suggests that variation of the
population of right-handed versus left-handed stacks would not
provide an adequate explanation of the CD results.
The CD spectra of d-ApT isomers 1 and 2 have the same shape as the
CD spectrum of d-ApT, but with diminished molecular ellipticity.
For d-TpA, the spectrum of isomer 2 is identical to that of d-TpAp,
while the spectrum of isomer 1 shows diminished [.theta.] values of
the peak and trough regions. Thus, the stacking modes in these
methyl phosphonate dimers are expected to be basically similar to
the stacking modes of the parent dinucleoside monophosphates, but
with perhaps different degrees of base-base overlap or different
populations of right- and left-handed stacks.
The dimer, d-ApA, forms stable complexes with both polyribo- and
polydeoxyribonucleotides. These poly U and poly dT complexes have
greater stability than similar complexes formed by the parent
dinucleoside monophosphate, d-ApA. Similar observations have
previously been made for triple helix formation between the alkyl
phosphotriesters d-Ap(Me)A or d-Ap(Et)A and poly U, for duplex
formation between oligonucleotide triesters and tRNA and for
helical duplex formation between the octathymidylate ethyl
phosphotriester, d-[Tp(Et)].sub.7 T, and poly dA. It should be
noted, however, that d-[Tp(Et)].sub.7 T, in contrast to d-ApA,
exhibits selective binding to polydeoxyribonucleotides versus
polyribonucleotides in duplex formation.
Previous analyses indicate that the increased stability of the
complexes formed between nonionic oligomers and complementary
polynucleotides results from the reduction in charge repulsion
between the nonionic backbone of the oligomer and the negatively
charged sugar-phosphate backbone of the polynucleotide. Although
both d-ApAp and d-ApA possess a formal negative charge, the
d-ApAp.poly U complexes are more stable than the d-ApA.poly U
complex. The 3'-terminal methyl phosphonate group of dApAp is free
to rotate away from the negatively charged phosphate backbone of
poly U without disrupting the base-pairing and base-stacking
interactions in the complex. In contrast, repulsion between the
negative charge of the phosphodiester linkage in d-ApA and the
polymer backbone directly opposes base-pairing and stacking. Thus,
the presence of a negative charge at the internucleotide linkage
contributes much more effectively to the charge repulsion effect
between the dimers and polynucleotides.
Under the conditions of the present experiments, the Tm values of
d-Ap(Me)A.poly U and d-Ap(Et)A.poly U are 13.degree. C. and
12.degree. C. respectively. These Tm values are lower than those of
d-ApA and d-ApAp complexes with poly U. These results suggest that
the increasing size of the methyl and ethyl side chains in the
phosphotriester dimers may provide a greater steric hindrance to
complex formation. The methyl group of the phosphonate dimers
should be only slightly larger in size than the oxygen of the
phosphate group, and thus would be expected to have the least
steric effect. A similar phenonenon has been observed when the
stabilities of poly U complexes with the ethyl phosphotriester and
methyl phosphonate analogs of d-ApApApA are compared.
The differences in the conformations of the individual
diastereoisomers of d-ApA and d-ApAp are reflected in their
interactions with poly U. For each dimer, the diastereoisomer with
greater base-base overlap (isomer 1) forms a complex of lower
stability with poly U. In a previous analysis of the influence of
C-2' substituents of adenine polynucleotides on the Tm values of
the helices, it can be reasoned that the conformation free-energy
difference (F.sub.D -F.sub.S) at the melting temperature is
directly related to the Tm value, where F.sub.D represents the free
energy of the double-stranded duplex, and F.sub.S represents the
free energy of the base-stacked single strand. The values of
F.sub.D -F.sub.S reflect the conformation of the duplex state and
the single-stranded state. The data indicates that (F.sub.D
-F.sub.S) for isomer 1 of dApA or dApAp is slighty less than
(F.sub.D -F.sub.S) for isomer 2 of dApA or dApAp. This reduction
may reflect a higher F.sub.S value of isomer 1 since this isomer
indeed has a greater degree of stacking, assuming that F.sub. D for
isomer 1 and isomer 2 remains the same. In contrast to the behavior
with poly U, both diastereoisomers of d-ApA form complexes with
poly(dT) which have similar Tm values. Since the geometry of the
triple helix of dApA.2 poly U is likely to be different than the
geometry of the dApA.2 poly dT triple helix, the difference in
F.sub.S of isomer 1 versus F.sub.S of isomer 2 may be compensated
by a difference in F.sub.D of isomer 1 versus F.sub.D of isomer
2.
The studies reported here have shown that dideoxyribonucleotide
analogs containing nonionic 3'-5' internucleoside methyl
phosphonate linkages can be readily synthesized. The configuration
of the methyl group in the backbone of these dimers influences
their conformation in solution and their ability to form complexes
with complementary polyribonucleotides.
In addition, preliminary studies have shown that
oligodeoxyribonucleoside methyl phosphonates are resistant to
nuclease hydrolysis, are taken up in intack form by mammalian cells
in culture and can exert specific inhibitory effects on cellular
DNA and protein synthesis. Unlike 2'-O-methyl oligonucleotide ethyl
phosphotriesters, the methyl phosphonates appear to have relatively
long half-lives within the cells. Thus, oligonucleoside methyl
phosphonates of specific sequence could complement oligonucleotide
phosphotriesters as probes and regulators of nucleic acid function
within living cells.
Nonionic Nucleic Acid Alkyl and Aryl Methylphosphonates
There will now be described the synthesis of a series of
oligonucleoside methylphosphonates whose base sequences are
complementary to the anticodon loops of tRNA.sup.lys and to the
--ACCAOH amino acid accepting stem of tRNA. The effects of these
analogues on cell-free aminoacylation and cell-free protein
synthesis will be considered. The uptake of selected analogues by
mammalian cells in culture and the effects of these compounds in
bacterial and mammalian cell growth are also discussed.
Materials
Nucleosides were checked for purity by paper chromatography before
use. N-Benzoyldeoxyadenosine, N-isobutyryldeoxyguanosine, their
5'-O-dimethoxytrityl derivatives and 5'-O-monomethoxytrityl
thymidine were prepared according to published procedures.
d-[(Meo).sub.2 Tr]bzApbzApCE, d-[(Meo).sub.2 Tr]bzApbzAOAC,
d-[(Meo)Tr]TpTpCE, d-ApT, d-Ap-[.sup.3 H]-T, d-TpT and d-Tp-[.sup.3
H]T were also synthesized by standard procedures.
Dimethylmethylphosphonate and benzenesulfonic acid were used
without further purification. Hydracrylonitrite was dried over 4
.ANG. molecular sieves. Methylphosphonic acid dipyridinium salt and
mesitylenesulfonyl tetrazolide were prepared.
Anhydrous pyridine was prepared by refluxing reagent grade pyridine
(3L) with chlorosulfonic acid (40 ml) for 7 hrs followed by
distillation onto sodium hydroxide pellets (40 g). After refluxing
for 7 hrs, the pyridine was distilled onto 4 .ANG. molecular sieves
and stored in the dark.
Silica gel column chromatography was carried out using Baker 3405
silica gel (60-200 mesh). Thin layer silica gel chromatography
(TLC) was performed on E. Merck Silica Gel 60F 254 plastic backed
TLC sheets (0.2 mm thick).
High pressure liquid chromatography (HPLC) was carried out using a
Laboratory Data Control instrument on columns (2.1 mm.times.1 m)
packed with HC Pellosil. The columns were eluted with a linear
gradient (40 ml total) of chloroform to 20% (V/V) methanol in
chloroform at a flow rate of 1 ml/min. Ultraviolet spectra were
recorded on a Cary 14 or a Varian 219 ultraviolet spectrophotometer
with a thermostatted cell compartment.
The following extinction coefficients (260 nm) were used: d-T,
9,100; d-[(Meo)Tr]T, 10,200; d-[(Meo).sub.2 Tr]bzA,12,500; d-bzA,
10,600; d-[(Meo).sub.2 Tr]ibuG, 17,400; and d-ibuG, 16,700. Paper
chromatography was carried out on Whatman 3 mm paper using solvent
A: 2-propanol-concentrated ammonium hydroxidewater (7:1:2 V/V).
Preparation of d-[(Meo).sub.2 Tr]ibuGpCE:
d-[(Meo).sub.2 Tr]ibuG (12 g; 18.7 mmoles) and the pyridinium salt
of methyl-phosphonic acid (21 mmoles) were dried by evaporation
with anhydrous pyridine (4.times.20 ml) and the residue in 40 ml of
pyridine was treated with 2,4,6-triisopropylbenzenesulfonyl
chloride (12.7 g, 42 mmoles) for 8 hrs at room temperature.
Hydracrylonitrile (4.5 g, 63 mmoles) and
2,4,6-triisopropylbenzenesulfonyl chloride (0.61 g, 2 mmoles) were
added and the reaction mixture was kept at room temperature. After
2 days the reaction mixture was poured into 500 ml of ice-cold 5%
NaHCO.sub.3 solution.
The solution was extracted with ethyl acetate (2.times.250 ml) and
the combined extracts were dried over anhydrous Na.sub.2 SO.sub.4.
Examination of the extract by TLC showed the presence of both
d-[(Meo).sub.2 Tr]ibuGpCE (Rf-0.31 silica gel tlc, 10%
MeOH/CHCl.sub.3) and d-ibuGpCE (Rf-0.14, silica gel tlc, 10%
MeOH/CHCl.sub.3).
After concentration, the ethyl acetate extract was chromatographed
on silica gel (4.times.35 cm) using ether (1L ) and a 0 to 20%
linear gradient of methanol in chloroform (1.6L total) as solvents.
d-[(Meo).sub.2 Tr]ibuGpCE (2.75 mmoles) was obtained in 15% yield
while d-ibuGpCE (2.46 mmoles) was obtained in 13% yield.
Additional d-[(Meo).sub.2 Tr]ibuGp (3.69 mmoles, 20%) was obtained
from the aqueous bicarbonate solution after extraction with
chloroform (2.times.200 ml).
Preparation of Protected Oligonucleoside Methylphosphonates:
The same general procedures were used for the preparation of
dinucleoside methylphosphonates. The specific conditions used in
the condensation reactions and the yields obtained after silica gel
column chromatography are given in Table VI. The ultraviolet
spectroscopic characteristics and the mobilities of the protected
oligonucleotides on silica gel TLC and silica gel HPLC are given in
Table VII.
Preparation of Oligonucleoside Methylphosphonates:
The protecting groups were removed from the blocked oligonucleoside
methylphosphonates using conditions described previously. In the
case of the dA-containing oligomers, the N-benzoyl groups were
removed by treatment with hydrazine. The oligomers were purified by
preparative paper chromatography using solvent A. For the [.sup.3
H] -labeled oligothymidine methylphosphonates, d-Tp).sub.n -[.sup.3
H]-T, the condensation reactions containing d-[(Meo).sub.2
Tr]Tp).sub.n +[.sup.3 H]TOAC were run on 0.01 (n=1) and 0.005
(n=4,8) mmole scales while d-GpGp-[.sup.3 H]-T was prepared on a
0.012 mmole scale.
After completion of the reaction, the protecting groups were
removed and the entire reaction mixture was chromatographed on
paper. The oligonucleoside methylphosphonates were eluted from the
paper with 50% aqueous ethanol. The ethanol solutions were passed
through DEAE cellulose columns (0.5.times.1 cm) and stored at
0.degree. C.
The UV spectral properties and chromatographic mobilities of the
oligonucleoside methylphosphonates are given in Table VII. For use
in the physical, biochemical, and biological experiments described
below, aliquots containing the required amount of oligomer were
evaporated to dryness and the oligomer was dissolved in the buffer
used in the particular experiment.
TABLE VI
__________________________________________________________________________
Preparation of Protected Oligodeoxyribonucleoside
Methylphosphonates 3'-Methylphosphonate 5'-OH MST Product Yield
Components (mmoles) Component (mmoles) (mmoles) (mmoles) %
__________________________________________________________________________
d-[(MeO).sub.2 Tr]ibuGp (0.50) d-ibuGpCE (0.50) 2.0 d-[(MeO).sub.2
Tr]ibuGpibuGpCE (.082) 16 d-[(MeO).sub.2 Tr]ibuGp (1.0) d-bzAOAC
(1.5) 4.0 d-[(MeO).sub.2 Tr]ibuGpbzAOAC 42.42) d-[(MeO)Tr]TpTp
(0.33) d-TpTpCE (0.50) 1.6 d-[(MeO)Tr]TpTpTpTpCE 50.168)
d-[(MeO)Tr]Tp(Tp).sub.2 TpCE (0.0324) d-Tp(Tp).sub.2 TpCE (.0524)
0.16 d-[(MeO)Tr]Tp(Tp).sub.6 TpCE 430138) d-[(MeO).sub.2
Tr]ibuGpibuGp (.07) d-TOAC (.15) 0.28 d-[(MeO).sub.2
Tr]ibuGpibuGpTOAC (0.0153) 22 d-[(MeO).sub.2 Tr]bzApbzAp (0.065)
d-bzAOAC (0.043) 0.163 d-[(MeO).sub.2 Tr]bzApbzApbzAOAC (0.023) 53
d-[(MeO).sub.2 Tr]bzApbzAp (0.13) d-bzApbzAOAC (0.20) 0.52
d-[(MeO).sub.2 Tr]bzApbzApbzApbzAOAC (0.031) 24 d-[(MeO).sub.2
Tr]bzApbzAp (0.0168) d-ibuGpbzAOAC (0.0168) 0.0735 d-[(MeO).sub.2
Tr]ApbzApibuGpbzAOAC (0.0029) 1
__________________________________________________________________________
TABLE VII
__________________________________________________________________________
Ultraviolet Spectral Properties and Chromatographic Mobilities of
Protected Oligodeoxyribonucleoside Methylphosphonates Silica UV
Spectra.sup.a Gel HPLC.sup.c .lambda. max. .lambda. min.
.epsilon..sub.260/235 .epsilon..sub.260/280 Silica Gel TLC
Retention time Oligomer nm nm calcd. obsvd. calcd. obsvd. 5% 10%
15% 20% (min)
__________________________________________________________________________
d-[(MeO)Tr]TpTpTpTpCE 265 243 1.34 1.31 1.55 1.64 -- -- .08 .29 --
235 sh d-[(MeO)Tr]Tp(Tp).sub.6 TpCE 265 243 1.75 0.92 1.57 1.56 --
0.00 -- .13 -- d-[(MeO).sub.2 Tr]ibuGpibuGpCE 238 225 1.19 1.05
1.33 1.32 -- 0.16 -- -- 19.2 253 245 260 256 280 270 d-[(MeO).sub.2
Tr]ibuGpbzAOAC 235 256 0.82 0.75 0.88 0.87 -- 0.29 -- -- 12.3 278
d-ibuGpbzAOAC 260 239 1.63 1.27 0.90 0.90 -- 0.18 -- -- 15.5 280
267 0.14 17.6 d-[(MeO).sub.2 Tr]ibuGpibuGpTOAC 240 sh 228 1.34 1.51
1.38 1.45 -- 0.18 -- -- 16.0 260 275 sh d-[(MeO).sub.2
Tr]bzApbzApbzAOAC 234 227 0.66 0.61 0.59 0.59 -- 0.41 0.55 -- 13.4
280 255 0.38 0.53 14.3 d-[(MeO).sub.2 Tr]bzApbzApbzApbzAOAC 233 sh
253 0.71 0.60 0.59 0.60 -- -- 0.31 -- 19.3 280 d-[(MeO).sub.2
Tr]bzApbzApibuGpbzAOAC 235 sh 255 0.89 0.74 0.73 0.75 -- 0.15 0.44
-- 23.8 280
__________________________________________________________________________
.sup.a Measured in 95% ETOH .sup.b EM silica gel 60 F.sub.254
sheets, 0.2 mm thick. .sup.c HC Pellosil (2.1 mm .times. 1 m) 0% to
20% methanol in chloroform ml/min., 40 ml total volume
Interaction of Oligodeoxyadenylate Methylphosphonates With
Polynucleotides
The continuous variation experiments and melting experiments were
carried out. The extinction coefficients of the oligomers were
determined by comparing the absorption of a solution of the
oligomer in water at pH 7.0 to the absorption of the same solution
at pH 1.0. The oligomer extinction coefficient was calculated from
the observed hyperchromicity of the oligomer at pH 1.0 by using the
following extinction coefficients: d-A pH 1.0, 14.1.times.10.sup.3
and d-G pH 1.0, 12.3.times.10.sup.3. The molar extinction
coefficient of poly(U) is 9.2.times.10.sup.3 (265 nm) and of
poly(dT) is 8.52.times.10.sup.3 (264 nm).
Cell-Free Aminoacylation
(1) E. coli system: Unfractionated tRNA E. coli was purchased from
Schwarz Mann and unfractionated E. coli aminoacyl synthetase was
purchased from Miles Laboratories, Inc. Reactions were run in 60
.mu.l buffer containing 100 nM Tris, HCl, pH 7.4, 10 mM
Mg(OAC).sub.2, 5 mM KCl, 2 mM ATP, 4 .mu.M [.sup.3 H]-amino acid,
1.8 .mu.M tRNA coli and 0 to 100 .mu.M oligonucleotide.
Reactions were initiated by addition of 4 .mu.g of aminoacyl
synthetase. Aliquots (10 .mu.l) were removed at various times,
added to 1 ml of cold 10% trichloroacetic acid and the resulting
precipitate filtered on Whatman G/F filters.
After washing with 4 (1 ml) portions of 2N HCl and 4 (1 ml)
portions of 95% ETOH, the filters were dried and counted in 7 ml
New England Nuclear 949 scintillation mixture.
(2) Rabbit Reticulocyte System
A rabbit reticulocyte cell-free translation system was obtained
from New England Nuclear. Reactions were run in 12.5 .mu.l of
buffer containing 1 .mu.l translation mixture, 79 mM potassium
acetate, 0.6 mM magnesium acetate, 57 .mu.M [.sup.3 H]-Lysine, and
50 .mu.M oligomer. The reactions were initiated by addition of 5
.mu.l of reticulocyte lysate and were assayed as described for the
E. coli system.
Cell-Free Protein Synthesis
(1) E. coli system
A cell-free protein synthesizing system was isolated from E. coli B
cells (S-30). The system incorporates 300 pmoles of [.sup.3
H]-phenylalanine/mg of S-30 protein after 15 min incubation at
37.degree. C. when poly U is used as a message.
(2) Rabbit Reticulocyte
The reticulocyte translation system prepared by New England Nuclear
was used. For the translation of globin mRNA the reactions were run
in 12.5 .mu.l of buffer containing: 1 .mu.l of translation mixture,
0.10 .mu.g of globin mRNA (Miles Laboratories), 79 mM, potassium
acetate, 0.2 mM magnesium acetate 0 to 50M oligomer and 20.5 .mu.M
[.sup.3 H]-leucine.
For the translation of poly(U) the reactions were run in 12.5 .mu.l
buffer containing: 1 .mu.l of translation mixture, 120 mM potassium
acetate, 0.8 mM magnesium acetate, 367 .mu.M poly(U), 0 to 200
.mu.M oligomer (base concentration) and 32 .mu.M [.sup.3
H]-phenylalanine. Reactions were initiated by addition of 5 .mu.l
of reticulocyte lysate.
Aliquots (2 .mu.l) were removed at various times and added to 1.0
ml of bovine serum albumin (100 .mu.g) solution. The protein was
precipitated by heating with 1 ml of 10% trichloroacetic acid at
70.degree. C.; filtered on G/F filters and counted in 7 ml of
Betaflour.
Uptake of Oligodeoxyribonucleoside Methylphosphonates
The uptake of d-Ap-[.sup.3 H]-T, d-GpGp-[.sup.3 H]-T and
d-Tp).sub.n -[.sup.3 H]-T by transformed Syrian hamster fibroblasts
were determined.
Effects of Oligodeoxyribonucleoside Methylphosphonates On Colony
Formation
(1) E. coli
E. coli B was grown in M-9 medium supplemented with glucose (36
g/l) and 1% Casamino acids. The cells were harvested in midlog
phase and resuspended in 50 .mu.l of fresh medium containing 0 to
160 .mu.M oligomer at a final cell density of 1.times.10.sup.4
cells/ml.
The cells were incubated for 1 hr. at 37.degree. C. and then
diluted with 0.9 ml of medium. A 0.8 ml aliquot was added to 2.5 ml
of 0.8% Bactoagar at 45.degree. C. This solution was quickly poured
onto a 100 mm plate containing solid 1.2% Bactoagar. After
solidification, the plates were incubated overnight at 37.degree.
C. and the resulting colonies were counted.
(2) Transformed Syrian Hamster Embryonic Fibroblasts (BP-6) and
Transformed Human Fibroblasts (HTB1080)
Colony formation by the fibroblasts in the presence of the
methylphosphonate analogues was carried out.
RESULTS
Synthesis of Oligodeoxyribonucleoside Methylphosphonates
The synthetic scheme used for preparing the oligonucleoside
methylphosphonates followed the basic approach used to synthesize
dideoxyribonucleoside methylphosphonates. Suitably protected
monomers or oligomer blocks carrying a 3'-terminal
methylphosphonate group were condensed with protected mono- or
oligonucleotides bearing a free 5'-hyroxyl group.
Mesitylenesulfonyl tetrazolide was used as the condensing agent.
The fully protected oligomers were purified by silica gel column
chromatography. The reaction conditions used and the yields
obtained are given in Table VI. The oligomers were characterized by
ultraviolet spectroscopy, thin layer chromatography and high
pressure liquid chromatography as indicated in Table VII.
The protecting groups were removed as previously described. In the
case of the deoxyadenosine-containing oligomers, the N-benzoyl
groups were first removed by treatment with hydrazine hydrate. The
remaining 3'-O acetyl and 5'-O dimethoxytrityl groups were removed
by sequential treatment with ammonium hydroxide and 80% acetic
acid. The oligomers were purified by preparative paper
chromatography and were characterized by UV spectroscopy (Table
VIII).
Interaction of Oligodeoxyribonucleoside Methylphosphonates With
Complementary Polynucleotides
Table IX summarizes the melting temperatures of complexes formed
between oligodeoxyadenosine methylphosphonates and poly (U) or poly
(dT). For comparison, the melting temperatures of complexes formed
by oligodeoxyribo- and oligoriboadenosines are included. Each
oligomer forms a triple-stranded complex with a stoichoimetry of
2U:1A or 2T:1A.
The melting temperatures increase as the chainlength of the
oligonucleotide increases. For a given chain length, the complexes
formed by the methylphosphonate analogues melt at higher
temperatures than those formed by the natural diester
oligomers.
TABLE VIII
__________________________________________________________________________
Spectral Properties and Chromatographic Mobilities of
Oligodeoxyribonucleo side Methylphosphonates UV Spectra.sup.a Paper
Chromatography.sup.b .lambda. max. .lambda. min. .epsilon. Rf
Oligomer nm nm .epsilon..sub.260 /.epsilon..sub.280 .lambda. max.
Solvent A
__________________________________________________________________________
d-GpGpT.sup.c 257 230 1.45 33.4 .times. 10.sup.3 0.31 270 sh
d-ApApA 258 232 4.27 39.0 .times. 10.sup.3 0.29 d-ApApApA 258 230
3.77 50.4 .times. 10.sup.3 0.11 d-ApApGpA 258 227 3.03 50.3 .times.
10.sup.3 0.11 d-Tp-[.sup.3 H]--T 267 235 1.53 -- 0.59 (d-Tp).sub.4
-[.sup.3 H]--T 266 235 1.49 -- 0.21 (d-Tp).sub.8 -[.sup.3 H]--T 266
235 1.56 -- 0.17
__________________________________________________________________________
.sup.a Measured in water, pH 7.0 .sup.b Rf.sup.A pT = 0.11 .sup.c
The UV spectrum is similar to that of dGpGpT (Miller et. al.,
1974).
TABLE IX ______________________________________ Interaction of
Oligonucleoside Methylphosphonates with Complementary
Polynucleotides.sup.a Oligomer Tm Poly U (2U:1A) Tm Poly dT (2T:1A)
______________________________________ d-ApA isomer 1 15.4 18.7
.sup. isomer 2 19.8 18.4 d-ApApA 33.0 36.8 d-ApApApA 43.0 44.5
d-ApA 7.0 9.2 d-ApApApA 32.0 35.5 r-ApApApA 36.2.degree. C.
2.4.degree. C. ______________________________________ .sup.a 5
.times. 10.sup.-5 M total [nucleotide], 10 mM Tris, 10 mM
MgCl.sub.2, pH 7.5
With the exception of r-ApApApA, the complexes formed by the
oligomers with poly (dT) have slightly higher melting temperatures
than the corresponding complexes formed with poly (U).
The interaction of d-GpGp-[.sup.3 H]-T with unfractionated tRNA
E.coli was measured by equilibrium dialysis. The apparent
association constants at 0.degree., 22.degree., and 37.degree. C.
are 1,100M.sup.-1, 200M.sup.-1, and 100M.sup.-1 respectively. These
binding constants are much lower than those of the
2'-O-methylribooligonucleotide ethyl phosphotriester, G.sup.m p
(Et)-G.sup.m p(Et)-[.sup.3 H]-U, which are: 9,300M.sup.-1
(0.degree. C.), 1,900M.sup.-1, (22.degree. C.) and 2,000M.sup.-1
(37.degree. C.).
Effect of Oligodeoxyribonucleoside Methylphosphonates on Cell-Free
Aminoacylation to tRNA
The effects of selected oligodeoxyribonucleoside methylphosphonates
on aminoacylation of unfractionated tRNA E. coli are shown in Table
X. Three amino acids were tested at various temperatures. The
deoxyadenosine-containing analogs which are complementary to the
--UUUU-- sequence of the anticodon of tRNA.sub.E.coli.sup.lys have
the largest inhibitory effect on aminoacylation of
tRNA.sub.E.coli.sup.lys.
The percent inhibition increases with increasing chain length and
decreases with increasing temperature. Inhibition by d-ApApGpA and
by the diesters dApApApA is less than that exhibited by d-ApApApA.
In contrast to their behavior with TRNA.sub.E.coli.sup.lys, neither
the methylphosphonates, d-ApApApA, d-ApApGpA nor the
phosphodiesters, d-ApApApA, r-ApApApA, had any inhibitory effect on
tRNA.sub.rabbit.sup.lys in the rabbit reticulocyte cell-free system
(data not shown).
TABLE X ______________________________________ Effects of
Oligonucleoside Methylphosphonates on Aminoacylation in an E. coli
Cell-Free System % Inhibition.sup.(b) Phe Leu Lysine
Oligomer.sup.(a) 0.degree. C. 0.degree. C. 0.degree. C. 22.degree.
C. 37.degree. C. ______________________________________ d-ApA 6 0 7
-- -- d-ApApA 9 0 62 15 0 d-ApApApA 9 12 88 40 16 d-ApApGpA 12 12
35 0 -- d-GpGpT 31 5 34 9 15 dGpGpT (400 .mu.M) 23 -- -- -- --
d-ApApApA 0 7 .sup. 71.sup.(c) .sup. --5.sup.(c) r-ApApApA -- --
.sup. 78.sup.(d) .sup. --7.sup.(d)
______________________________________ .sup.(a) [oligomer] = 50
.mu.M .sup.(b) [tRNA.sub.coli ] = 2 .mu.M .sup.(c) [oligomer] = 100
.mu.M .sup.(d) [oligomer] = 125 .mu.M
Effects of Oligodeoxyribonucleoside Methylphosphonates On Cell-Free
Protein Synthesis
The ability of deoxyadenosine containing oligonucleoside
methylphosphonates to inhibit polypeptide synthesis in cell-free
systems directed by synthetic and natural messages was tested. The
results of these experiments are given in Table XI. Poly (U)
directed phenylalanine incorporation and poly (A) directed lysine
incorporation are both inhibited by oligodeoxyadenosine
methylphosphonates and diesters in the E.coli system at 22.degree.
C. The percent inhibition increases with oligomer chain length and
is greater for polyphenylalanine synthesis. The methylphosphonate
analogues are more effective inhibitors than either d-ApApApA or
r-ApApApA at the same concentration. Although both the
oligodeoxyadenosine methylphosphonates and the phosphodiesters
inhibit translation of poly (U) in the rabbit reticulocyte system,
no effect on the translation of globin message was observed.
As in the case of the E.coli system, inhibition of phenylalanine
incorporation increased with oligomer chain length and was greater
for the methylphosphonate analogues than for the diesters.
Uptake of Oligodeoxyribonucleoside Methylphosphonates By Mammalian
Cells
FIG. 18 shows the incorporation of radioactive 100 .mu.M
d-GpGp-[.sup.3 H]-T with time into transformed Syrian hamster
embryonic fibroblasts growing in monolayer. The incorporation is
approximately linear for the first hour and begins to level off
after 1.5 hours. The concentration of radioactivity inside the cell
is approximately 117 .mu.M after 1.5 hours assuming a cell volume
of 1.5 .mu.l/10.sup.6 cells.
TABLE XI ______________________________________ Effects of
Oligonucleoside Methylphosphonates on Bacterial and Mammalian
Cell-Free Protein Synthesis at 22.degree. C. E. coli Rabbit
Reticulocyte Poly U Poly A Poly U Globin mRNA Oligomer
directed.sup.(a) directed.sup.(b) directed.sup.(a) directed.sup.(c)
______________________________________ d-ApA 20 10 -- -- d-ApApA 84
30 81 -- d-ApApApA 100 65 77 0 d-ApApGpA 22 -- -- 0 d-ApApApA 13 19
18 0 r-ApApApA 18 17 85 0 ______________________________________
.sup.(a) [Poly U] = 360 .mu.M in U [oligomer] = 175-200 .mu.M in
base .sup.(b) [Poly A] = 300 .mu.M in A [oligomer] = 175-200 .mu.M
in base .sup.(c) [oligomer] = 200 .mu.M in base
Cells were incubated with 25 .mu.M d-GpGp-[.sup.3 H]-T for 18
hours. The medium was removed, the cells were washed with phosphate
buffer and then lysed with SDS. Approximately 30% of the total
radioactivity from the lysate was found in TCA precipitable
material. The DNA was precipitated from the lysate and digested
with deoxyribonuclease and snake venom phosphodiesterade. The
culture medium, the DNA-free lysate and the DNA digest were each
examined by paper chromatography. Only intact d-GpGp-[.sup.3 H]-T
was found in the medium. Radioactivity corresponding to [.sup.3
H]-TTP (6%) and to d-GpGp-[.sup.3 H]-T (94%) was found in the
lysate, while the DNA digest gave [.sup.3 H]-dpT and [.sup.3 H]-dT
as products.
Similar uptake studies were carried out with d-Ap[.sup.3 H]-T and
with a series of oligothymidylate analogues, d-(Tp)n-[.sup.3 H]-T
(n=1,4,8). The rates and extents of uptake of these analogues were
very similar to that of d-GpGp-[.sup.3 H]-T (FIG. 1). Examination
of the culture medium and cell lysate after overnight incubation
with these oligonucleotides gave results similar to those found for
d-GpGp-[.sup.3 H]-T.
Effects of Oligodeoxyribonucleoside Methylphosphonates On Colony
Formation By Bacterial and Mammalian Cells
The effects of selected oligodeoxyribonucleoside methylphosphonates
on colony formation of E. coli B, transformed Syrian hamster
fibroblast (BP-6) and transformed human fibroblast (HTB 1080) cells
are summarized in Table XII. The d-Ap)nA analogues appear to
inhibit E. coli colony formation at high concentrations (160
.mu.M). However, no inhibitory effects on cellular protein or DNA
synthesis could be detected in the presence of these compounds by
the present assay procedures.
TABLE XII ______________________________________ Effects of
Oligonucleoside Methylphosphonates on Colony Formation by Bacterial
and Mammalian Cells in Culture % Inhibition.sup.(a) E. coli B HTB
1080 Oligomer 50 .mu.M 160 .mu.M BP-6 (50 .mu.M) (50 .mu.M)
______________________________________ d-ApT 4 5 5, 16.sup.(b) 12
d-ApA 8 58 6, <1.sup.(b) 5 d-ApApA 3 44 29 31 d-ApApApA 19 78 36
19 d-GpGpT 7 11 7 9 ______________________________________ .sup.(a)
The results are the average of two or three experiments. Each
experiment consisted of 2 plates (bacterial cells) or 3 plates
(Mammalian cells). The average variation is: .+-.3% in %
inhibition. The cells were treated with and grown in the presence
of the oligomer at 37.degree. C. .sup.(b) The % inhibition of
isomer 1 and 2 respectively.
Colony formation of both transformed hamster and human cells are
inhibited to various extents by the oligonucleoside
methylphosphonates. Both the hamster and human cells appear to be
affected to a similar extent by a given analogue. It appears in the
case of dApA, that each diastereoisomer exerts a different
inhibitory effect on the growth of the hamster cells. As in the
case of E. coli, no inhibition of cellular protein synthesis could
be detected.
DISCUSSION
Oligodeoxyribonucleoside methylphosphonates with sequences
complementary to the anticodon loop of tRNA.sup.lys and to the
--ACCA--OH amino acid accepting stem of tRNA were prepared in a
manner similar to that used to prepare dideoxyribonucleoside
methylphosphonates.
The present studies demonstrate the ability to join blocks of
protected methyl-phosphonates to give oligomers with chain lengths
up to nine nucleotidyl units. The yields in these condensation
reactions are acceptable, although reactions involving
deoxyguanosine residues appear to proceed in low yield.
Similar difficulties have been encountered in the syntheses of
oligonucleotide phosphotriesters. Unlike the dideoxyribonucleoside
methyl-phosphonates previously reported, the
oligodeoxyribonucleoside methylphosphonates prepared for this study
were not resolved into their individual diasteroisomers.
The oligodeoxyadenosine analogues form triple stranded complexes
with both poly(U) and poly(dT). These complexes are more stable
than similar complexes formed by either oligoribo- or
oligodeoxyribonucleotides. As previously suggested for
oligonucleotide ethyl phosphotriesters, and dideoxyribonucleoside
methyl-phosphonates, this increased stability is attributed to the
decreased charge repulsion between the nonionic backbone of the
analogue and the negatively charged complementary polynucleotide
backbone. With the exception of r-ApApApA (Table IX), the stability
of the complexes formed with poly (dT) are slightly higher than
those formed with poly(U), a situation which is also observed for
the interaction of poly(dA) with poly(dT) and with poly(U). The
lower stability of the (r-ApApApA).2 poly(dT) complex is also
reflected at the polymer level.
Thus, under the conditions of the experiments described in Table
IX, it was found that the Tm of poly(rA).2 poly(rU) is 83.degree.
C. while the Tm of poly(rA).2 poly(dT) is 59.degree. C. It was
observed that formation of the poly(rA).2 poly(dT) complex occurs
only at a sodium ion concentration of 2.5M in the absence of
magnesium, while poly(rA).2 poly(rU) forms in 0.1M sodium phosphate
buffer.
The oligodeoxyadenosine methylphosphonates and their parent dieters
selectively inhibit cell-free aminoacylation of
tRNA.sub.E.coli.sup.lys. The extent of inhibition is temperature
dependent and parallels the ability of the oligomers to bind to
poly(U). These observations and the previously demonstrated
interaction or r-ApApApA with tRNA.sub.E.coli.sup.lys suggest that
inhibition occurs as a result of oligomer binding to the --UUUU--
anticodon loop of the tRNA. The reduced inhibition observed with
d-ApApGpA is consistent with this explanation, since interaction of
this oligomer with the anticodon loop would involve formation of a
less stable G.U base pair.
Recent studies by others have shown that the rate of aminoacylation
of tRNA.sub.E.col.sup.lys substituted with 5-fluorouracil is
considerably lower than that of non-substituted
tRNA.sub.E.coli.sup.lys. The increased Km of the 5-fluorouracil
substituted tRNA suggested a decreased interaction with the lysyl
aminoacyl synthetase.
These results and those of others suggest that the anticodon loop
of tRNA.sub.E.coli.sup.lys is part of the synthetase recognition
site. Thus, inhibition of aminoacylation by the
oligodeoxyribonucleoside methylphosphonates could result from the
reduction in the affinity of the synthetase for tRNA.sup.lys
-oligonucleotide complexes.
The greater inhibition observed with d-ApApApA versus the diesters,
d-ApApApA or r-ApApApA may result from greater binding of the
analogue to the anticodon loop or to the decreased ability of the
synthetase to displace the nonionic oligonucleotide analogue from
the anticodon loop.
Alternatively, oligomer binding to the anticodon loop could induce
a conformational change in the tRNA, leading to a lower rate and
extent of aminoacylation. Such conformational changes have been
detected when r-ApApApA binds to tRNA.sub.E.coli.sup.lys.
None of the oligomers have any effect on the aminoacylation of
tRNA.sub.rabbit.sup.lys in a cell free system. Since the anticodon
regions of tRNAs from bacterial and mammalian sources probably are
similar, the oligo A analogues are expected to interact with the
anticodon region of both tRNA.sup.lys s. The failure to observe
inhibition of aminoacylation of tRNA.sub.rabbit.sup.lys in the
presence of these oligo d-A analogs suggestes that there may be a
difference between the interaction of the lysine aminoacyl
synthetase with tRNA.sup.lys from E. coli and from rabbit systems,
or a difference between the structure of these two tRNA.sup.lys s
in response to the binding of oligo A analogues.
The trimer, dGpGpT, inhibits both phenylalanine and lysine
aminoacylation at 0.degree., but has little effect on leucine
aminoacylation. The aminoacyl stems of both tRNA.sub.E.coli.sup.lys
; and tRNA.sub.E.coli.sup.Phe terminate in a G-C base pair between
nucleotides 1 and 72, while a less stable G-U base pair is found at
this position in tRNA.sub.E.coli.sup.leu. Thus the observed
differences in inhibition of aminoacylation by d-GpGpT may reflect
differences in the ability of this oligomer to bind to the
different --ACC-- ends of the various tRNAs.
Inhibition of lysine aminoacylation by dGpGpT is very temperature
sensitive and parallels the decrease in binding to tRNA with
increasing temperature. This behavior of d-GpGpT contrasts that of
G.sub.p.sup.m (ET)G.sub.p.sup.m (ET)U. Although both oligomers can
potentially interact with the same sequences in tRNA, the
2'-O-methylribotrinucleotide ethyl phosphotriester binds more
strongly and more effectively inhibits aminoacylation. The
differences in binding ability may be due to overall differences in
the conformation of the deoxyribo- versus 2'-O-methylribo backbones
of these oligomers.
The oligodeoxyribonucleoside methylphosphonates effectively inhibit
polyphenylalanine synthesis in cell-free systems derived from both
E. coli and rabbit reticulocytes. In the E. coli system, the extent
of inhibition by the oligodeoxyadenosine analogures parallels the
Tm values of the oligomers wiht poly(U), The tetramer, d-ApApGpA
which would have to form a G.U base pair with polyU, was 4.5-fold
less effective than d-ApApApA.
These results suggests that the oligomers inhibit polypeptide
synthesis as a consequence of forming complexes with the poly(U)
message. A similar inhibitory effect by poly(dA) on the translation
of poly(U) has been observed by others.
It is unlikely that inhibition results from non-specific
interaction of the methylphosphonates with protein components of
the translation systems.
In the E. coli system, poly(A) translation is inhibited to a lesser
extent than is translation of poly(U), while in the reticulocyte
system, no inhibition of globin mRNA translation is observed.
The data suggest that the magnitude of inhibition of
poly(U)-directed polypeptide synthesis in the E. coli system does
not reflect proportionally the ability of the oligomer to bind to
poly(U). Although the oligomer pairs d-ApApA/d-ApApApA and
d-ApApApA/r-ApApApA form complexes with poly(U) which have very
similar Tm's (see Table IX), in each case the methylphosphonate
analogues inhibit 5.5 to 6.5 times better than do the diesters. The
stronger inhibitory effect could result from a decreased ability of
the ribosome to displace the nonionic oligodeoxyribonucleoside
methylphosphonates form the poly(U) message, or alternatively,
there may be a degradation of the oligonucleotides
(phosphodiesters) by nucleases in the cellfree translation systems,
but not the corresponding phosphonate analogues.
Experiments with radioactively labeled oligonucleotide
methylphosphonates show that these analogues are taken up by
mammalian cells growing in culture. The extent of uptake is
consistent with passive diffusion of the oligomer across the cell
membrane. Both d-Tp-[.sup.3 H]-T and d-(Tp).sub.8 -[.sup.3 H]-T are
taken up to approximately the same extent which suggests that there
is no size restriction to uptake over this chain length range. This
behavior is in contrast to results obtained with E. coli cells.
Examination of lysates of mammalian cells exposed to labeled
oligomers for 18 hours showed that approximately 70% of the labeled
thymidine was associated with intact oligomer with the remainder
found in thymidine triphosphate and in cellular DNA.
These observations indicate that the oligodeoxyribonucleoside
methylphosphonates, which are recovered intact from the culture
medium, are slowly degraded within the cell. Failure to observe
shorter oligonucleotides and the known resistance of the
methylphosphonate linkage to nuclease hydrolysis suggests that
degradation may result from cleavage of the 3'-terminal [.sup.3
H]-thymidine N-glycosyl bond with subsequent reutilization of the
thymine base.
The uptake process of the oligonucleoside methylphosphonates is
quite different from that of previously studied oligonucleotide
ethyl phosphotriesters.
In the case of G.sub.p.sup.m (Et)G.sub.p.sup.m (Et)-[.sup.3 H]-U,
the oligomer is rapidly taken up by the cells and is subsequently
deethylated. Further degradation to smaller oligomers is then
observed, presumably as a result of nuclease-catalyzed hydrolysis
of the resulting phosphodiester linkages.
Approximately 80% of the oligomer is metabolized within 24 hours.
Although the rate of uptake of d-Gp(Et) Gp(ET)-[.sup.3 H]-T is
similar to that of d-GpGp-[.sup.3 H]-T, examination of the cell
lysate showed extensive degradation of the phosphotriester
analogue. The relatively long half lives of the
oligodeoxyribonucleoside methylphosphonates may be of value in
potential pharmacological applications of these oligonucleotide
analogues.
The effects of these analogues on cell colony formation confirmed
that the methylphosphonates are taken up by both mammalian and
bacterial cells. All the oligomers tested inhibited colony
formation of both cell types of various extents. The mechanism(s)
by which these compounds exert their inhibitory effects is
currently under investigation.
No decrease in either overall short term cellular protein synthesis
or DNA synthesis was detected by the present procedure in the
presence of these compounds. This does not rule out the possibility
that the syntheses of certain critical proteins are perturbed by
these oligomers. Currently, studies are being made of this
possibility by examination of the cellular proteins using
2-dimensional gel electrophoresis.
The experiments described hereinbefore extend these studies on the
use of nonionic oligonucleotides as sequence/function probles of
nucleic acids both in biochemical experiments and in living cells.
In a future, there will be described the effects of an
oligodeoxyribonucleoside methylphosphonate complementary to the
3'-terminus of 16S rRNA on bacterial protein synthesis and growth.
The results here, however, suggest that sequence specific
oligonucleoside methylphosphonates may find important applications
in probing and regulating nucleic acid function within living
cells.
* * * * *