Nucleic Acids Research, Vol. 18, No. 8 2109
Octa(thymidine methanephosphonates) of partially defined
stereochemistry: synthesis and effect of chirality at
phosphorus on binding to pentadecadeoxyriboadenylic
acid
Zbigniew J.Lesnikowski, Maria Jaworska and Wojciech J.Stec
Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Department of
Bioorganic Chemistry, Sienkiewicza 112, 90-363 Lodz, Poland
Received November 28, 1989; Revised and Accepted March 19, 1990
ABSTRACT
Block condensation of MePOCI2 or MeP(NEt2)2
with appropriately protected tetra(thymidine
methanephosphonates) of predetermined sense of
chirality at asymmetric phosphonate centres gave
two pairs of diastereomeric mixtures, namely
(SpSpSpSpSpSpSp+SpSpSpRpSpSpSp) 5a and
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp) 5b. A
comparison of the CD spectra of 5a and 5b with those
of octathymidylic acid (7) and a random mixture of
diastereomers of octa(thymidine methanephosphonate)
(6), and also a comparison of the Tm of complexes
formed between 5a, 5b, 6 or 7, and pentadecadeoxyriboadenylic acid (8), indicates that octamer
5b and its complex with its complementary oligonucleotide has a well-ordered structure due to the
'outward' or 'pseudoequatorial' orientation of the
methyl group of each internucleotide methanephosphonate function of Rp configuration. Results
presented in this report clearly indicate that the stability
of hybrids formed between octa(thymidine
methanephosphonate) and pentadecadeoxyriboadenylic acid depends on the stereochemistry of
each internucleotide methanephosphonate function
and strongly suggests that stereoselective synthesis
of P-chiral oligonucleotide analogues is an important
goal.
INTRODUCTION
Oligonucleotide analogues possessing asymmetric centres at
phosphorus atoms involved in modified internucleotide linkages
have found wide application as models for the study of nucleic
acids structure and dynamics( ,2) and as probes for the
elucidation of specific interactions of DNA/RNA with proteins
and enzymes3-6). Their ability to modulate gene expression has
also been extensively explored(7'8). When non-bridging oxygens
at internucleotide phosphorus atoms are replaced by sulphur, alkyl
or alkoxy groups, in many cases the configuration of asymmetric
phosphorus centres drastically influences the interaction between
modified oligonucleotides and their complementary sequences
as well as the conformation of
single- and double-
stranded oligonucleotides. Unfortunately the nature of these
phenomena is not fully understood. Studies on the relationship
between the absolute configuration at phosphorus and
oligonucleotide conformation or binding have so far
concerned dinucleotides(9" 0), oligonucleotides with one
P-chiral centre(1 -14), oligonucleotides possessing alternating
modified/unmodified internucleotide linkages(' l,S16), or
oligonucleotides possessing n P-chiral centres and consisting of
a mixture of all 2n possible diastereomers(14"17).
Recently we have reported on stereospecific synthesis of
phosphorothioate(Il l9) and methanephosphonate(20 -22)
oligonucleotide analogues with defined sense of chirality
at phosphorus. In this paper we wish to present the results of
our studies on the synthesis and binding properties of
two octa(thymidine methanephosphonates) 5a and Sb, each
composed of two diastereomers of known absolute configuration,
namely (SpSpSpSpSpSpSp + SpSpSpRpSpSpSp) and
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp).
EXPERIMENTAL
Materials and methods
Column chromatography and TLC were performed on silica gel
230-400 mesh and on silica gel F 254 plates, respectively, (both
from E.Merck). Reversed phase high performance liquid
chromatography (RP-HPLC) was performed with a LDC/Milton
Roy system, using ODS Hypersil 5At, 4.6 x 300 mm column. The
elution conditions are given below. UV spectra were recorded
and Tm measurements were performed with a Uvikon 860
spectrometer (Kontron Instruments AG). 31P-NMR spectra were
recorded with a Bruker MSL 300 spectrometer operating at
121.47 MHz, with 85% H3PO4 as an external standard. Positive
chemical shift values are assigned for compounds absorbing at
lower field than standards. CD spectra were recorded on
Dichrograf Mark Ill (Jobin-Yvon).
(SpSpSp)- and (RpRpRp)-Isomers of 5'-O-monomethoxytrityl-3'-O-acetyl tetra (thymidine methanephosphonate) (la and lb)
The title compounds were prepared as described previously(22).
2110 Nucleic Acids Research, Vol. 18, No. 8
TABLE 1. Yield and 31P-NMR characteristics of 3'-0-acetyl, 5'-O-monomethoxytrityl protected octanucleotide
4a and 4b.
octa(thymidine
methanephosphonate)
Yield
[%]
TLCa)
4a-(SpSpSpSpSpSpSp+SpSpSpRpSpSpSp)
5.0
0.65
4b-(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp)
10.0
0.65
a) Developing solvent system:
b) in C5D5N (4a) or CD30D
0[ppmj
33.88
33.78
33.74
33.55
33.47
35.12
34.91
34.88
34.84
34.62
3lP-NMRb)
integration
2
4
2
4
2
1
1
5
3
4
CH3CI3-CH30H (6:4)
(4b).
(SpSpSp)- and (RpRpRp)-Isomers of 3'-O-acetyl
tetra(thymidine methanephosphonate) (2a and 2b)
The removal of 5'-monomethoxytrityl group was performed
according to the literature method(23). Thus, individual isomers
of 1 (58.5 mg, 0.04 mmol) were treated with 80% aqueous acetic
acid (1.0 mL) at room temperature. The reaction progress was
monitored by means of TLC. After 1-3 h AcOH was removed
by coevaporation with n-BuOH. The oily residue was dissolved
in pyridine and dropped into hexane. The precipitate was washed
with n-pentane and dried under reduced pressure.
2a: Rf 0.04 vs. la:Rf 0.17; 2b: Rf 0.07 vs. Jb:Rf 0.19,
[CHC13-CH30H (9:1) as developing solvent system].
(SpSpSp)- and (RpRpRp)-Isomers of 5'-O-monomethoxytrityl
tetra(thymidine methanephosphonate (3)
To the solution of individual isomers of 1 (50 mg, 0.034 mmol)
in 0.75 mL of methanol, 0.5 mL 25 % aq ammonia solution was
added. The reaction progress was monitored by means of TLC.
After 2-3 h the post-reaction mixture was evaporated to dryness.
The residue was redissolved in 2 mL of chloroform and extracted
with 2 mL of water. The organic layer was separated, dried over
MgSO4 and evaporated to dryness. The residue was redissolved
in chloroform and the product was precipitated from hexane.
3a: Rf 0.05 vs. Ja: Rf 0.17; 3b: Rf 0.08 vs. 1b: Rf 0.19
[CHC13-CH30H (9:1) as developing solvent system].
(SpSpSpSpSpSpSp+SpSpSpRpSpSpSp)-Isomer of 5'-Omonomethoxytrityl-3'-O-acetyl octa(thymidine methanephosphonate) (4a)
Method A.
To the solution of methanephosphonic dichloride (1.9 mg, ca.
0.014 mmol) in dry pyridine (0.027 mL), the solution of3a (19.0
mg, ca. 0.016 mmol) in pyridine (0.023 mL) was added under
dry argon. After 0.5h at room temperature the solution of
intermediate 5'-O-monomethoxytrityl tetra(thymidine
methanephosphonate)-3'-O-methanephosphonic chloride was
filtered for removal of pyridine hydrochloride and the filtrate was
added to the solution of 2a (16.0 mg, 0.016 mmol) in pyridine
(0.020 mL). After 1.5 h, the reaction was quenched by addition
of pyridine/water (1:1, 0.1 mL) and the resultant mixture was
extracted with chloroform (0.2 mL). The chloroform layer was
separated, then reextracted with water (0.2 mL), dried over
MgSO4 and evaporated to dryness. The crude 4a was purified
by means of preparative silica gel TLC using
chloroform/methanol (8:2) as an eluting solvent system. The yield
and 31P-NMR characteristic are presented in Table 1.
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp)-Isomer of 5'-Omonomethoxytrityl-3'-O-acetyl octa(thymidine methanephosphonate) (4b)
Method B.
The (N,N,N',N'-tetraethyl)methanephosphondiamidite(24) (3.8
mg, 0.02 mmol) was added under dry argon to a solution of 3b
(2.80 mg, 0.02 mmol) and N,N-diisopropylammonium tetrazolide
(1.7 mg, 0.01 mmol) in dry methylene chloride (0.2 mL). The
reaction was stirred for 5 h at room temperature, then the solution
of 2b (38.0 mg, 0.032 mmol) and tetrazole (11.2 mg, 0.16 mmol)
in dry acetonitrile (0.2 mL) was added. After 3 h, the resultant
3 '-O-[5 '-O-monomethoxytrityl tetra(thymidine methanephosphonate)-5' -O-(3 '-O-acetyl tetra(thymidine methanephosphonate)] methanephosphonite was oxidized with 0.15 M
12 in H20/2.6 lutidine/THF (1:10:40) yielding 4b.
The post-reaction mixture was concentrated and to the resultant
oily residue water (1.0 mL) was added. The mixture was
extracted with chloroform (3 x 1.0 mL), the combined chloroform
extracts were dried over MgSO4 and evaporated to dryness. The
crude 4b was purified by means of preparative silica gel TLC
using chloroform/methanol (8:2) as an eluting solvent system.
The yield and 31P-NMR characteristic are presented in Table 1.
(SpSpSpSpSpSpSp+ SpSpSpRpSpSpSp)- and
(RpRpRpRpRpRpRp + RpRpRpSpRpRpRp)-Isomers of
octa(thymidine methanephosphonate) (5a) and (Sb)
The removal of 5'-O-monomethoxytrityl and 3'-O-acetyl groups
were performed as described above for 2 and 3, respectively.
Sa: UV: Xmin 236, max 266 (H20); HPLC: Rt 17.45 min.
Sb: UV: Xmin 235, mx 264 (H20); HPLC: Rt 17.03 min.
The HPLC elution conditions: a linear gradient of acetonitrile
in 0.1 M TEAB (pH 7.0) at a flow-rate of 1.5 mL/min, starting
from 1.2% CH3CN, gradient 1.4%/min.
Octa(thymidine methanephosphonate) with random
distribution of P-diastereomers (6)
6 was synthesized using 1 zmol of support-bound nucleoside on
a Biosearch Cyclone DNA Synthesizer that employed 5'-O-
dimethoxytritylthymidine-3'-O-[(2-cyanoethyl)methanephosphonite], according to standard protocol (25). The 5'-DMTprotected oligomer was cleaved from the resin by treatment with
29% NH3aq/C2H5OH (3:1), for lh at room temperature. The
Nucleic Acids Research, Vol. 18, No. 8 2111
MMTTPMeTPMeTpMeTAC
(SpSpSp)-la
or (RpRpRp)-lb
11
Li
HOTPMeTPMeTPMeTAC
MMTTPMeTPmeTpmeTOH
(SpSpSp)-2a
(SpSpSp)-3a
or (RpRpRp)-2b
or (RpRpRp)-3b
iii
iv, v
MMTTPMeTPMeTPMe TP e/MepT T T TAC
(SpSpSpSp/RpSpSpSp)-4a
or (RpRpRpRp/SpRpRpRp)-4b
HP , M 1
34.0
OHTPMeTPMeTPMe TPMe / MepTPMeTpMeTep MeToH
(SpSpSpSp/RpSpSpSp)-5a
or (RpRpRpRp/SpRpRpRp)-5b
33.5
PPM
FIGURE 2. 31P-NMR spectrum (121.47 MHz) of (SpSpSpSpSpSpSp+SpSpSpRpSpSpSp) octa(thymidine methanephosphonate) (4a). Chemical shifts are given
in ppm (6) relative to 85% H3PO4.
i/ 80% CH3COOH
ii/ 25% NH3 aq/CH30H (1:2)
ii1/ CH3P[N(C2H5)2]2/tetrazole-diisopropylamine/CH2C12
iv/ tetrazole/2a or 2b/CH3CN
v/ 0.15M
J2
in H20/2.6-lutidine/THF (1:10:40)
FIGURE 1. Synthesis of (SpSpSpSpSpSpSp+SpSpSpRpSpSpSp) and
(RpRpRpRpRpRpRp + RpRpRpSpRpRpRp) octa(thymidine methanephosphonates).
resin was washed with CH3CN/H20 (1:1, 0.5 mL) and the
combined washings were evaporated to dryness. The purification
of crude 6 was accomplished by means of HPLC using the
following elution conditions: a linear gradient of acetonitrile in
0.1 M TEAB (pH 7.0) at a flow-rate of 1.5 mL/min, starting
from 1.2% CH3CN, gradient 1.4%/min., Rt 15.66 min.
Octa(thymidine phosphate) (7) and pentadeca(deoxyriboadenosine phosphate) (8)
7 And 8 were prepared on a Biosearch Cyclone DNA Synthesizer
that employed phosphoramidite chemistry according to standard
protocol(26). The resultant 7 and 8 were purified by means of
two steps of RP-HPLC using the following eluting conditions:
5'-DMT-protected oligomer: gradient from 5% to 30% CH3CN
in 0. 1 M TEAB, 5'-deprotected oligomer: gradient from 5 % to
15% CH3CN, 1.5 mL/min., 7, Rt 9.83 min. 8 in addition to
RP-HPLC was purified by means of polyacrylamide gel
electrophoresis.
Circular dichroism measurements
The CD spectra of Sa, Sb, 6 and 7 were measured in double
distilled water solution at concentrations of 0.43 g/L, 1.06 g/L,
0.93 g/L and 0.82 g/L, respectively. The following molar
extinction coefficients were used: Sa, e265 = 8500 [as for
d(TpMeTp)4TpT-2)(1)',
Sb,
-265 = 8300
[as
for
d(TpMeTP)4TpT-I(1)], 7, E266 = 8700 [as for d(Tp)9T)()], 6,
-265 = 8450 (average value for Sa and Sb).
Tm measurements
Melting experiments were performed on a Uvikon 860
spectrometer (Kontron Instruments AG). The temperature control
was through a programmable Tischkryostat KT6 (VEB MLW.
Prufgerate-Werk Meadingen). Temperature was monitored by
a Uvikon 860 thermistor unit with the temperature transducer
connected to a thermostated sample cell holder. Cuvettes had
Teflon stoppers and were of 1 cm pathlength. Nitrogen gas was
passed continously through the sample and reference compartment
of the Uvicon 860 during low temperature measurements.
Digitalized absorbance values were stored by the computer and
next plotted as a function of temperature on the Plotter 800
(Kontron Instrument AG). The computer collected 10 absorbance
readings and averaged them for each point on the Tm curve to
improve signal-to-noise ratio. Tm values were established on the
basis of first order derivative of Tm curves. The measurements
were initiated at 0°C or slightly below 0°C and the temperature
ramp was 0.5 °C/min. These experiments were carried out in 0.1
M sodium cacodylate, pH 6.8, at a total nucleotide concentration
of 1 x 10-4M and proportions of ldTldA and 2dTldA of both
oligonucleotide components(l). All samples were pre-melted at
60°C and then cooled slowly (0.5°C/min) to 0°C.
RESULTS
P-Homochiral 5 '-O-monomethoxytrityl-3 '-O-acetyl tetra
(thymidine methanephosphonates) (1) were prepared, as described
previously (22), by stepwise, stereospecific coupling of a
5'-hydroxyl activated growing oligonucleotide chain with (Sp)-
2112 Nucleic Acids Research, Vol. 18, No. 8
or (Rp)-5'-O-monomethoxytritylthymidylyl[3'-O-(4-nitrophenyl)
35.0
PPM
of
(121.47
3. 31P-NMR
MHz)
FIGURE
spectrum
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp) octa(thymidine methanephosphonate)
(4b). Chemical shifts are given in ppm (0) relative to 85% H3PO4.
1.0
I
I
5
I
0~~~~~~~
-0.5
220
240
X
300
Th0
260
32o
mm
4. Circular
of
spectra
FIGURE
dichroism
d(TpMe)7T
(0
),
(SpSpSpSpSpSpSp + SpSpSpRpSpSpSp)-(Sa)
d(TPme)7T
), d(TPMe)7T with
(RpRpRpRpRpRpRp + RpRpRpSpRpRpRp)-(Sb) (0
random distribution of P-diastereomers (6) (0--0I) and d(Tp)7 (7) (E--E)in
H20. The molecular ellipticity [6], is given per base residue.
-0
-0
methanephosphonate].
The mixtures of 5'- and 3'-protected diastereomers
and
4a
(SpSpSpSpSpSpSp + SpSpSpRpSpSpSp)
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp) 4b of octa(thymidine
methanephosphonate) (4) were prepared via block condensation
of suitably deprotected tetramers 2 and 3. Deprotection of the
5'-hydroxyl function of la and lb was achieved by the treatment
of fully protected tetranucleotides I with 80% acetic acid. After
standard work up, chromatographically homogeneous byproducts
2 suitable for the coupling reaction were obtained and used for
synthesis of 4 without further purification.
Removal of the 3'-O-acetyl group in la and lb was performed
under alkaline conditions by treatment of 1 with a mixture of
concentrated aqueous ammonia and methanol (1:1.5 v/v) at room
temperature. No degradation products were observed by means
of TLC and additional purification of deprotected byproducts 3
was not necessary for further condensation.
Octamer Sa was prepared by condensation of appropriately
protected tetramer blocks 2a and 3a according to the methodology
described previously by Miller for the synthesis of di- and
tri(nucleotide methanephosphonates)(Th.
Thus, the 3'-hydroxyl group of 3a was phosphonylated with
methanephosphonic dichloride and then 2a was added. The
preparative yield of fully protected octamer 4a was low (see Table
I) (ca. 5%) which may be explained by the fact that relatively
long oligonucleotides were coupled together.
Whereas octamer Sb could not be synthesised according to the
method described, an alternative procedure applying
phosphonamidite chemistry was successful. Phosphonylation of
the 3'-OH function of 3b was performed by means of
(N,N,N',N'-tetraethyl)methanephosphondiamidite. Intermediate
5 '-O-MMT tetra(thymidine methanephosphonate)-3'-O-(N,Ndiethyl methanephosphonamidite) was reacted, without isolation,
with 2b and the central methanephosphonite group of the resultant
octamer was oxidized with 12 in H20/2,6-lutidine/THF. The
preparative yield of Sb was 10%.
The resultant octanucleotides 4a and 4b were characterized by
means of 31P-NMR. After deprotection, resulting Sa and 5b
were analysed by means of UV and HPLC. Since in the course
of the first step of the coupling reaction the formation of a
symmetrical product is possible, the presence of 3'-O-acetyl and
5'-O-monomethoxytrityl protecting groups in 4 was proved by
means of selective deprotection and analysis by TLC (for MMT
removal) or HPLC (acetyl removal from 5'-deprotected 4).
Because the coupling of the two tetramers 2 and 3, leading
to octamers 4, is not a stereospecific reaction, compounds 4a
and 4b consist of a mixture of two diastereomers epimeric at the
phosphorus atom of central methanephosphonate intemucleotide
linkage. However, it should be pointed out that the analogous
octamers (i.e. 6) obtained by non-stereospecific synthesis consists
of a mixture of 128 diastereomers.
Attempts at separation of diastereomers constituting
pairs (SpSpSpSpSpSpSp + SpSpSpRpSpSpSp)-5a and
(RpRpRpRpRpRpRp+RpRpRpSpRpRpRp)-Sb by means of RPHPLC were unsuccessful. This is in contrast to the described
fractionation of decathymidylic acid bearing only one, centrally
located methanephosphonate internucleotide linkage(28). The
reasons for this difference in chromatographic behavior are not
known at the present. Perhaps, the higher lipophilicity of the
molecule, Sa and Sb, compared to the monomethanephosphonate
analogue of decathymidylic acid, may be responsible.
Nucleic Acids Research, Vol. 18, No. 8 2113
A0
260
TABLE 2. Tm values of complexes formed between octathymidylic acid 7, and
its analogues S and 6, and pentadecadeoxyriboadenylic acid (8)
1.40I
2,60
00
Oligomer
0
Ratio dT:dAa)
(TpMe)7T, Sa
0
1. 30
0
(TPMe)7T, Sb
0
*
0
* m
1: 1
2: 1
0
1.20 -*
Tmb)
[°C]
00
(TpMe)7T random, 6
u
(TP)7T, 7
0
1: 1
2:1
1: 1
2: 1
1: 1
2:1
< 2 (nd.)
< 2 (n.d.)
38.2
39.3
ca. 12.9
19.5
13.4
13.1
0
D
1.10
a) The experiments were carried out in 0.1 M sodium cacodylate, pH 6.8, at
a total nucleotide concentration of 1 x 10-4 M.
b) The Tm values are the transition midpoints of IT: IdA or 2T: IdA mixtures
of suitable oligonucleotides.
0
0
0
0
,
1.00 *.
:
10
0
*.@0000
0
50
20
30
40
0 **.
60 tOC
FIGURE 5. Melting curves of d(TpMe)7T (SpSpSpSpSpSpSp+
(0), d(TpMe)7T (RpRpRpRpRpRpRp +
SpSpSpRpSpSpSp)-(Sa)
RpRpRpSpRpRpRp)-(Sb) (0), d(TpMe)7T with random distribution of Pdiastereomers (6) (O), d(Tp)7T (7) (e), and d(Ap)14A in 0.1 M sodium
cacodylate, pH 6.8 at proportions ldT- IdA and a total nucleotide concentration
I 10-4M.
Tm measurements of the duplexes between octanucleotides Sa
Sb and pentadecadeoxyriboadenylic acid matrix were
performed and compared with those formed between unmodified
octathymidylic acids (7) or octa(thymidine methanephosphonate)
consisting of the random mixture of 128 diastereoisomers (6).
Tm studies and CD measurements on Sa, Sb, 6 and 7 are
discussed below.
or
DISCUSSION
The key step in the synthesis of octanucleotides Sa and Sb was
the condensation of two suitably protected tetranucleotide blocks:
2a-3a and 2b-3b, respectively. This was achieved according to
phosphotriester (Method A) or phosphonamidite (Method B)
chemistries. Unfortunately, in both cases the efficiencies of the
coupling steps were low. Similarly, low yields (7-50%)
were
reported by Miller(29) for coupling of
d[DMTTPMe(TpOPhCI4TpMe)3TPOphCI-] and d[TP0PhCp4)nTAc]
(n= 1-4). One of the possible ways to overcome this difficulty
may be via the recently reported chemical ligation of
oligonucleotides annealed to complementary sequences(30) or by
efficient stepwise and stereospecific solid phase synthesis.
The 31P-NMR spectra of both 4a and 4b reveal five signals
(Figures 2 and 3). Their relative integrations 2:4:2:4:2 and
1:1:5:3:4, respectively, corresponding to fourteen phosphorus
atoms, are consistent with the fact that both Sa and Sb consist
of a mixture of two diastereomeric octa(thymidine
methanephosphonates), epimeric at the phosphorus of the central
methanephosphonate internucleotide linkage and each contaning
seven phosphorus centres. The different compositions of
overlapping signals observed for Sa and Sb reflect the perturbation
introduced by the central methanephosphonate function of
'doubled' sense of chirality in both pairs of diastereomers.
CD measurements have shown a strong influence of
configuration at internucleotide methanephosphonate functions
on the CD spectra of Sa, Sb, 6 and 7 which exhibit the same
shape but drastically different molecular ellipticity values.
Octa(thymidine methanephosphonates): Sb, 6 and 7, all exhibit
a positive CD signal between 295 and 265 nm, while the CD
spectrum of octa(thymidine methanephosphonate) Sa is entirely
negative in the wavelengh range 220-320 nm. The molecular
ellipticity of Sa, Sb, 6 and 7 decrease in the order 7> Sb >6> Sa
and is reduced by 26%, 60% and 108% (negative sign of CD
signal) for Sa, 6 and Sb, respectively, as compared with 7. It
should be mentioned that these observations are in agreement
with similar measurements reported previously(22) for
homochiral tetra(thymidine methanephosphonates), but it should
be pointed out however, that this dependence is more strongly
pronounced in octanucleotides than in tetranucleotides, which may
suggest that the effect of absolute configuration of
methanephosphonate groups upon oligonucleotide conformation
is additive.
Taking into account that molecular ellipticity is sensitive to
the extent and mode of base stacking and that the nonionic
methanephosphonate group of intemucleotide linkage perturbs
the stacking interactions between the bases of modified
oligonucleotides(31), the conclusion can be drawn that the
stacking perturbation is much stronger for the (Sp)-configuration
of internucleotide methanephosphonates than that observed for
the (Rp)-configuration. This correlates with NMR studies of
duplexes of complementary oligonucleotides bearing
intemucleotide methanephosphonate function with defined sense
of chirality(32) as well as with results of Tm measurements.
Dependence of Tm upon the stereochemistry of the modified
phosphate group is a well known phenomenonO"9"3'33).
However, it has not been studied so far for longer oligomers
possesing adjacent P-chiral centres with defined sense of chirality.
Our results of Tm measurements for duplexes of octa(thymidine
methanephosphonates)/pentadecadeoxyriboadenylic acid illustrate
very dramatically the relationship between 'tacticity' of
oligo(thymidine methanephosphonates) and Tm values. The Tm
values decrease in the order: Sb > > 7 > 6 > > Sa (Table 2 and
Figure 5).
Since according to MillerO 29), oligo(thymidine methanephosphonates) can form triplex structure with complementary
poly(oligo)deoxy(ribo)adenylic acid, Tm measurements of
ldT ldA and 2dT ldA mixtures were also performed for all
oligonucleotide studied Sa, Sb, 6 and 7. No substantial differences
of Tm were observed.
2114 Nucleic Acids Research, Vol. 18, No. 8
Tm values for 7/(dA)15 and 6/(dA)15 are quite similar: 13.4°C
and ca. 12.9°C (for ldT: IdA mixture), respectively. It should
be pointed out, however, that the melting curve observed for
6/(dA)15 is markedly broader and is characterized by lower
hypochromicity: 17% vs. 22.4%. The broad melting curve for
6/(dA)15 might result from the superimposition of a multitude
of octa(thymidine methanephosphonates)/pentadecadeoxyriboadenylic acid transitions with different stabilities(34). This
supposition is strongly supported by the comparison of melting
curves obtained for complexes ofSa/(dA)15 and 5b1(dA)15 which
are completely different in their character. The curve for
5a/(dA)15 is flat, with hypochromicity of 3.8% and no shoulder
point. By contrast, the melting curve for complex Sb/(dA)15 is
sigmoidal with a distinct shoulder at 38.2°C and hypochromicity
of 35.2%.
The shape of the curve is consistent with the assumption that
Sb forms, in the range of temperatures studied, a well ordered,
stable complex and that Sa does not form a complex with (dA)15
at all, and that the binding properties of Sa and 5b depend upon
the absolute configuration of internucleotide methanephosphonate
functions.
The presence of a methyl group in the pro-S position decreases
the duplex stability. The possible factors influencing the binding
properties, related to the configuration of intermediate
methanephosphonate groups, are discussed below.
If the complexes of octa(thymidine methanephosphonate)/pentadecadeoxyriboadenylic acid have B-type
geometry, the P-CH3 bond of the methyl group of
intemucleotide methanephosphonate function of (Sp)-isomer is
oriented 'inward' towards the DNA double-helix, near the
hydrophobic base-stacking region of the complex, and that of
the (Rp)-isomer is oriented 'outward' away from the DNA double
helix (into the solvent)( l). The geometry of oligothymidine/oligodeoxyriboadenosine may, however, deviate from
classical B-DNA since such deviations were observed for
poly(dA):poly(dT) complexes(35'36).
At present it is not possible to discuss the exact features of
perturbations due to the introduction of methanephosphonate
functions into oligonucleotide chains without further NMR
studies. However, both CD and Tm data seem to indicate that
the interaction between oligo(thymidine methanephosphonates)
and pentadecadeoxyriboadenylic acid is an entropy driven
process, and binding of oligo(thymidine methanephosphonate)
of exclusive Sp configuration suffers from constraints, as
indicated by molecular mechanics calculations(37), due to
interactions between P-methyl groups and the 5-CH3 groups of
thymines.
It has been proposed that the effect of the methylation of
phosphates on the stability of duplexes results from three main
factors(32): 1) elimination of the phosphate charge, 2) electronic
and other substituent effects, 3) the steric effect of the substituent
group. At least one more factor can be added, namely, 4) the
differences in hydration of the analogue/phosphodiester backbone
of the oligomer in the duplex compared to that in the singlestranded formsO"29).
From our and other observations, the hypothesis emerges that
the influence of factors mentioned above is potentiated by the
desired 'tacticity' of oligomers, which results from the
predetermined sense of chirality at each internucleotide
methanephosphonate function.
Thus, comparison of the Tm of octa(thymidine methanephosphonate) Sa and Sb suggests that the destabilizing effect of
factors 2 and 3 is successfully neutralized if internucleotide
methanephosphonate groups exist in the (Rp)-configuration (PCH3 bond pointing towards the solvent); on the other hand it
seems that (Sp)-configuration is especially unfavourable. A
similar relationship was also observed for triester analogues(' ).
It is true for methanephosphonates as well as for triester
oligonucleotide analogues(33) that the isomer with a
'pseudoequatorial configuration' of methanephosphonates (Rpconfiguration) forms more stable complexes than their
counterparts with 'pseudoaxial' orientation of the P-methyl group.
Our studies suggest that although the duplex stabilization or
destabilization results from the balance between many factors,
the balance itself can be shifted into a desirable direction by a
carefully designed sequence of absolute configurations of
modified P-chiral internucleotide functions.
ACKNOWLEDGMENTS
This project was financially assisted by the Polish Academy of
Sciences, Grant No. CPBR 3.13.4. Authors are indebted to Mr.
Andrzej Wilk for the synthesis of oligomers 6, 7 and 8.
This paper is dedicated to Professor J.Michalski on the occasion
of his 70th birthday.
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