Synthesis and hybridization properties of an acyclic achiral phosphonate DNA analogue

Article abstract of DOI:10.1016/S0968-0896(97)10041-4

Protected N-(2-hydroxyethyl)-N-(nucleobase- acetyl)aminomethanephosphonic acids (6a-d) of all four DNA nucleobases have been prepared and oligomerized by solid-phase synthesis. Four DNA decamers containing 1-10 of these 'PPNA' monomers were prepared and evaluated by T(m) measurements (medium salt) for binding to their DNA and RNA complements. One central modification reduced the binding strongly (?T(m) =-10 °C), but contiguous PPNA monomers gave smaller effects, and the all-PPNA decamer bound to RNA with a ?T(m) of -1.2°C per modification. Thus PPNA oligomers are inferior DNA and RNA binders compared to the closely related and strongly binding PNA oligomers.

Full text of DOI:10.1016/S0968-0896(97)10041-4

BIOORGANIC &
MEDICINAL
CHEMISTRY
Bioorganic & Medicinal Chemistry 6 (1998) 315±322
Synthesis and Hybridization Properties of an Acyclic Achiral
Phosphonate DNA Analogue
Jan Kehler, Ulla Henriksen, Helene Vejbjerg and Otto Dahl*
Department of Chemistry, The H. C. érsted Institute, University of Copenhagen, Universitetsparken 5,
DK-2100 Copenhagen, Denmark
Received 28 August 1997; accepted 21 October 1997
AbstractÐProtected N-(2-hydroxyethyl)-N-(nucleobase-acetyl)aminomethanephosphonic acids (6a±d) of all four DNA
nucleobases have been prepared and oligomerized by solid-phase synthesis. Four DNA decamers containing 1±10
of these `PPNA' monomers were prepared and evaluated by Tm measurements (medium salt) for binding to their DNA
and RNA complements. One central modi®cation reduced the binding strongly (ÁTm
ˆ
10 ꢀC), but contiguous
PPNA monomers gave smaller e€ects, and the all-PPNA decamer bound to RNA with a ÁTm of 1.2 ꢀC per mod-
i®cation. Thus PPNA oligomers are inferior DNA and RNA binders compared to the closely related and strongly
binding PNA oligomers. # 1998 Elsevier Science Ltd. All rights reserved.
Introduction
matches, and to form very stable PNA:DNA:PNA tri-
plexes. Here we describe the preparation and properties
of a new acyclic and achiral phosphonate nucleotide
analogue, `PPNA' (Fig. 1), which has a structure similar
to PNA, but has a phosphonate linkage between the
units instead of the carboxamide linkage in PNA.
Compared to PNA, the analogue PPNA has a more
¯exible backbone and is negatively charged. This would
be expected to change such properties as binding to
biomolecules and solubility in water which are relevant
to the use of PPNAs as therapeutic agents. Recently and
independent of us, van der Laan et al.6(a) and Peyman et
al.6(b) have published on the preparation of the same
oligomers. However, their monomers are di€erent from
ours, and they prepared oligomers derived from pyr-
imidines only, by solid-phase6(a) or solution6(b) chemis-
tries.
The synthesis and characterization of new oligonucleo-
tide analogues has for several years been an area of
active research due to the potential of such compounds
to exert therapeutic e€ects via antisense or antigene
mechanisms.1,2 Modi®cation of the oligonucleotide is
necessary in order to confer resistance in vivo to
nucleases which cleave natural DNA outside the
nucleus.2 Compounds with modi®ed phosphate groups
(e.g. phosphorothioates, methylphosphonates, or phos-
phoramidates) have been studied extensively, and more
recently a number of `dephospho' analogues, where the
phosphate linkage has been substituted with, for exam-
ple, amide, hydroxylamine, and formacetal groups, have
been evaluated.3 Radically changed analogues, where
the whole sugar phosphate backbone is substituted,
have shown poor binding to DNA or RNA, with two
exceptions: `Morpholino-DNA'4 and `PNA'5 (Figure 1).
Both analogues bind more strongly than unmodi®ed
DNA to complementary DNA and RNA, and PNA has
been shown to be highly discriminative towards mis-
Results and Discussion
The monomers used to obtain PPNA oligomers were
prepared as shown in Scheme 1. The pixyl protecting
group was chosen to give crystalline intermediates, and
the methyl protecting group on the phosphonate group
was chosen to allow easy removal with thiolate reagents.
The common backbone structure (3) was obtained by
Key words: DNA analogue; phosphonate; antisense; hybrid-
ization.
*Corresponding author. Tel: (45) 35 32 01 76; Fax: (45) 35 32
02 12; e-mail: dahlo@kiku.dk
0968-0896/98/$19.00 # 1998 Elsevier Science Ltd. All rights reserved
PII: S0968-0896(97)10041-4

316
J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
O
P
O
O
P
EtPri2
N
(CF3SO2)2O
Lutidine
+
CH2O
H
OMe
HOCH
P
OMe
CF SO CH
2
OMe
2
3
3
∆
OMe
OMe
OMe
1
B
NH
2
B-CH2COOH
PixO
O
P
O
H
4a-d
OMe
2
O
N
OMe
PixO
OMe
DCC, DhbtOH
N
P
PixO
OMe
3
5a-d
B
4a-6a : B = 6-N-benzoyl-9-adeninyl
4b-6b : B = 4-N-benzoyl-1-cytosinyl
4c-6c : B = 2-N-isobutyryl-9-guaninyl
4d-6d : B = 1-thyminyl
O
PhSH, Et3N
O
P
Et3NH+
-
O
N
PixO
OMe
6a-d
Scheme 1. Preparation of the PPNA monomers 6a±d.
alkylation of 2-(pixyloxy)ethylamine (2) with dimethyl
tri¯uoromethylsulfonylmethylphosphonate (1). Less
reactive leaving groups than tri¯ate (e.g. p-toluene-
sulfonate or oxytributylphosphonium) gave unac-
ceptable amounts of demethylation of the phosphonate
ester. Other methods to prepare 3 were also investigated
(e.g. reductive alkylation of dimethyl aminomethylpho-
sphonate with pixyloxyacetaldehyde, and Arbuzov
reactions of protected N-chloromethylethanolamines
with trimethyl phosphite), but the method given here
was the easiest and gave a better yield. The backbone (3)
was acylated with the four properly protected nucleo-
base-acetic acids (4a±d)7* to give the protected mono-
mers (5a±d), and one of the methyl groups was ®nally
removed with thiophenolate to give the monomers (6a±
d) in good yields.
eciency of ca. 95%,y but the coupling eciency
decreased for subsequent couplings to reach ca. 65% for
base 9. This was not improved by double couplings. The
reason for the decrease in coupling eciencies after a
few couplings is unknown, but could be caused by
aggregation of the growing oligomers. Accordingly the
low-loaded ABI polystyrene support gave somewhat
better coupling eciencies after base 4. Following
synthesis, the phosphonate methyl groups were removed
with RS , disodium 2-carbamoyl-2-cyanoethylene-1,1-
dithiolate,10 in methanol-acetic acid at rt for 2 h. It was
crucial to bu€er the RS reagent with acetic acid to pH
5 to 7 in order to avoid excessive cleavage of the base-
labile linker to the support at this step. The PPNA oli-
gomers were then cleaved from the support and the base
protecting groups removed with conc. aq ammonia at
55 ꢀC. Four decamers, containing 1 to 10 PPNA units,
were prepared and characterized (Table 1). The PPNA
DNA chimera B and C were puri®ed by ethanol pre-
cipitation and gave yields (30±40%) in reasonable
accordance with the pixyl and DMT eciencies. The 50-
pixyl PPNA oligomers D and E were puri®ed and the
Solid-phase syntheses of PPNA oligomers (Scheme 2)
were performed on 7, a normal T polystyrene support
(7 mmol/g, ABI, or 22 mmol/g, Pharmacia) pretreated
with a reagent (50 Phosphate-ON, Cruachem), which
enabled cleavage of 30-unmodi®ed oligomers from the
support with aq ammonia. The strongly activating
reagent, PyFNOP,9 and NMI were used to enable cou-
pling of the relatively unreactive phosphonate mono-
mers (6a±d). Coupling eciencies, estimated from the
absorbance of the released Pix+ at 375 nm, were depen-
dent on the support type and the dryness of 6a±d. For
freshly dried monomers the ®rst 3 to 4 couplings gave an
H
N
O
O
B
B
B
N
P
N
P
N
O
NMe2
O
O
- O
O
O
O
*The guanine derivative 4c was prepared by a modi®ed proce-
dure of Will et al.8
yThe ABI support gave the best results; CPG-based supports
gave unsatisfactory coupling eciencies (about 70%).
Morpholino-DNA
PNA
PPNA
Figure 1. Structures of morpholino-DNA, PNA, and PPNA.

J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
317
pixyl group removed on an Oligo±Pak column (Milli-
pore), and the products isolated by lyophilization.
However, the isolated yields were only 18% for D and
2% for E for unknown reasons. The purities were esti-
mated by capillary gel electrophoresis to be ca. 90% for
B±D, and ca. 55% for the fully modi®ed E (Fig. 2).
ends of the decamers C and D (ÁTm 2 to 5 ꢀC per
modi®cation). The fully modi®ed decamer E did not
give a de¯ection on the melting curve against DNA (Tm
less than 10 ꢀC), but bound to the RNA complement
with a Tm of 26 ꢀC (ÁTm 1.2 ꢀC per modi®cation).
These results show that although one PPNA unit in a
DNA strand disturbs the helix structure severely, con-
tiguous PPNA units in¯uence the duplex stability to a
lesser degree, and a fully modi®ed PPNA strand is able
to bind to RNA with only a moderate Tm depression per
modi®cation. The PNA analogue of E (H-GTA GAT
CAC T-NH2) binds to its DNA complement with a Tm
of 49 ꢀC and to its RNA complement with a Tm of 54 ꢀC
Thermal melting temperature measurements (Table 1)
against the complementary DNA or RNA decamer at
medium salt concentrations showed that the oligonu-
cleotides modi®ed with PPNA hybridized poorly to both
DNAandRNA. One PPNAunit inthe middleofthe DNA
decamer B gave a Tm depression of ca. 10 ꢀC, whereas
the depression was smaller for PPNA units placed at the
NC
O
(1) CCl 3COOH
(2) 6 + NMI + PyFNOP
O
P
O
B
(3) Ac2O/NMI
O
SO
2
DMTO
O
n times
O
P
7
B
NC
SO
B
O
P
O
O
O
P
O
O
B
O
P
(1) RS -
O
2
-
Pix
O
N
O
O
R
N
O
n
O
(2) aq NH3
-
OMe
O
n
O
P
NO
2
O
N
-
H N
2
S
-
PF6
N
2 Na+
N
O
CF
-
+
P N
3
NC
S
3
PyFNOP
RS -
Scheme 2. Solid-phase synthesis of PPNA oligomers.
Table 1. PPNA oligomers prepared and their characterization by MALDI TOF MS and hybridization data
c
d
Oligomera
MALDI TOF MSb
Tm
Tm
A
B
C
D
E
50-GTA GAT CAC T-30
50-GTA GAT CAC T-30
50-GTA GAT CAC T-30
50-GTA GAT CAC T-30
50-GTA GAT CAC T-30
Ð
39.5
30.0
24.0
32.0
37.5
27.0
27.0
Ð
3022.7 (3023.5)
3104.9 (3101.5)
3020.4 (3021.5)
3095.0 (3094.6)
<10
26.0
aDeoxyribonucleotides modi®ed with PPNA units at the underlined poisition.
bFound (calcd for monoanions with H+ as the counterions).
cMelting temperatures (ꢀC) against the DNA complement, 50=AGT GAT CTA C-30, ca. 5 mM in each strand, bu€er 140 mM MaCl,
10 mM Na2HPO4, 1 mM EDTA, pH 7.2. The values were obtained by increasing the temperature from 5 to 70 ꢀC and were repro-
ducible within 1 ꢀC.
dMelting temperature against the RNA complement, 50-AGU GAU CUA C-30, conditions as above.

318
J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
Figure 2. Capillary gel electrophoresis electropherograms of puri®ed C and E.

J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
319
(at 100 mM NaCl),11 which show that substitution of
the carboxamide groups of the PNA backbone with
phosphonate groups severely compromise the hybridi-
zation to DNA and RNA. Whether the reason for this
poor binding of PPNA oligomers, compared to PNA, is
charge repulsion, a more ¯exible backbone (increased
entropy loss), or a combination of these e€ects, are not
known from the limited data of this work. The only
other data on the binding properties of PPNA oligomers
is Tm measurements of a T9-mer against d(A)9 at high
salt conditions (1 M NaCl, 20 mM MgCl2), where a Tm
of 21 ꢀC was found.6(b) This is close to the Tm of 22 ꢀC
measured for the corresponding DNA±DNA hybrid
and indicate that charge repulsions between PPNA oligo-
mers and DNA, which is of lesser importance at such
high salt conditions, is a major determinant of the poor
hybridization.
TOF MS data on a HP G2025A LD-TOF spectrometer.
Melting temperature measurements were performed on
a Gilford Response II spectrometer or a Perkin±Elmer
Lambda 20 spectrometer with a PTP 6 controller and
UV TempLab software.
Dimethyl tri¯uoromethylsulfonyloxymethylphosphonate (1).
A mixture of dimethyl phosphite (2.75 g, 2.30 mL,
25 mmol), paraformaldehyde (0.75 g, 25 mmol CH2O),
and ethyldiisopropylamine (0.25 g, 0.34 mL, 1.9 mmol)
was heated under N2 in an oil-bath at 140 ꢀC for ca.
2 min when an exothermic reaction occurred to give
dimethyl hydroxymethylphosphonate (dP 26, ca. 92%
pure). The oil was dissolved under N2 in a mixture of
dry dichloromethane (100 mL) and 2,6-lutidine (4.4 mL,
38 mmol), cooled in an acetone/dry-ice bath, and tri-
¯uoromethanesulfonic anhydride (7.1 g, 25 mmol) in
dichloromethane (100 mL) and pre-cooled to 78 ꢀC
was added in one portion. The rection mixture was stir-
red for 2.5 h at 78 ꢀC and then at rt overnight. The
resulting solution of 1 (dP 14.2, ca. 80% pure) was used
directly in the preparation of dimethyl N-[2-(9-phenyl-9-
xanthenyloxy)ethyl]aminomethylphosphonate (3).
Conclusion
An ecient synthetic route to the PPNA monomers
(6a±d) derived from the four DNA nucleobases has been
developed. Oligomerization by solid phase synthesis was
possible using the strong activator PyFNOP+NMI,
although the coupling yields were low after the ®rst four
to ®ve couplings. Decamers containing 1 to 10 PPNA
monomers were prepared and shown by Tm measure-
ments (medium salt) to bind less strongly than unmodi-
®ed DNA to their DNA and RNA complements.
Compared to the structurally related PNA oligomers,
the negative charge and/or the higher ¯exibility of the
PPNA oligomers thus makes them much poorer DNA
and RNA binders.
2-(9-Phenyl-9-xanthenyloxy)ethylamine (2). N-(2-Hydro-
xyethyl)phthalimide (3.8 g, 20 mmol) was dried by eva-
poration from dry pyridine (20 mL) and redissolved in
dry pyridine (20 mL) under N2. Pixyl chloride (5.3 g,
18 mmol) was added and the reaction mixture stirred at
rt for 4 h. The mixture was poured into ice water
(450 mL), and the gum that precipitated was dissolved
in dichloromethane (100 mL). The dichloromethane
solution was washed with sat aq NaHCO3 (50 mL),
brine (50 mL), dried (Na2SO4), and the solvent removed
in vacuo to give a colourless foam. The foam was dis-
solved in a mixture of dichloromethane (100 mL) and
methanol (20 mL), hydrazine monohydrate (6 mL,
120 mmol) was added, and the reaction mixture was
stirred for 16 h at rt. The phthalazine formed was
removed by ®ltration, the solution was washed with
brine (30 mL), and the solvent removed in vacuo to give
a clear oil. The oil was dissolved in dry ether (50 mL),
®ltered, and the solvent removed in vacuo to give the
product as a colourless powder (5.2 g, 95%), mp 102±
104 ꢀC, pure according to TLC and 1H NMR. TLC
Experimental
Dicyclohexylcarbodiimide (DCC), dimethyl phosphite,
ethyldiisopropylamine, 3-hydroxy-1,2,3-benzotriazin-4-
one (DhbtOH), N-(2-hydroxyethyl)phthalimide, and
tri¯uoromethanesulfonic anhydride were from Aldrich,
9-chloro-9-phenylxanthene (pixyl chloride) and hydra-
zine monohydrate from Fluka, and paraformaldehyde
from Merck. Solvents were HPLC grade from LAB-
SCAN and were dried over molecular sieves (Grace 4
A). 6-N-benzoyl-9-adeninylacetic acid.7 4-N-benzoyl-1-
cytosinylacetic acid,7 2-N-isobutyryl-9-guaninylacetic
acid,8 and 1-thyminylacetic acid7 were prepared accord-
ing to literature methods. TLC was run on Merck 5554
silica 60 aluminium sheets, column chromatography on
Merck 9385 silica 60 (0.040±0.063 mm). 31P NMR spec-
tra were run on a JEOL FX 90 Q spectrometer, 1H
NMR spectra on a Bruker 250 MHz or a Varian Unity
400 MHz spectrometer, FAB MS data were obtained on
a JEOL HX 110/110 Mass Spectrometer, and MALDI
1
Rf=0.15 (CH2Cl2:MeOH 95:5 v/v). H NMR (CDCl3)
dH 7.33±6.92 (m, 13H, pixyl), 2.90 (t, J=5.3, 2H,
OCH2CH2N), 2.70 (t, J=5.3, 2H, OCH2CH2N), 1.2 (br.
s, 2H, NH).
Dimethyl
N-[2-(9-phenyl-9-xanthenyloxy)ethyl]amino-
methylphosphonate (3). A solution of 2-(9-phenyl-9-xan-
thenyloxy)ethylamine (2) (6.3 g, 21 mmol) in dry
dichloromethane (45 mL) and ethyldiisopropylamine
(7.8 mL, 45 mmol), pre-cooled to 10 ꢀC, was added to
the above solution of dimethyl tri¯uoromethylsulfonyl-

320
J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
oxymethylphosphonate (1) at 10 ꢀC under N2. The
reaction mixture was stirred at 0 ꢀC for 2 h, washed with
sat aq NaHCO3 (2Â50 mL), brine (50 mL), dried
(Na2SO4), and evaporated in vacuo to a yellow oil
which was puri®ed on a silica column (7.5Â14 cm) eluted
with CH2Cl2:EtOAc:MeOH:Et3N 60:33.7:6:0.3 v/v/v/v.
The fractions containing the product were evaporated in
vacuo and the residue recrystallized from ethyl acetate:
pentane 1:1 (32 mL) to give pure 3 (6.6 g, 71%), colour-
less crystals, mp 81±85 ꢀC. TLC Rf=0.22 (CH2Cl2:
MeOH, 95:5). NMR (CDCl3) dP 28.0, dH 7.32±6.93 (m,
13H, pixyl), 3.71 (d, J=10.6, 6H, POCH3), 2.99 (t,
J=5.2, 2H, OCH2CH2N), 2.84 (d, J=12.6, 2H, PCH2),
2.73 (t, J=5.2, 2H, OCH2CH2N). FAB MS 424.2
(M CH3+, calcd 424.1). Anal. (C24H26NO5P), calcd C:
65.60, H: 5.96, N: 3.19; found C: 65.36, H: 5.98, N: 3.14%.
under N2 in a mixture of dry pyridine (2 mL), and dry
triethylamine (1.4 mL), the solution degassed in vacuo,
and thiphenol (0.50 mL, 5 mmol) added. After 1 h at rt
the solution was added dropwise to vigorously stirred
dry ether (100 mL). The precipitate was isolated by ®l-
tration, washed several times with dry ether, and dried
by lyophilization twice from dry acetonitrile to give the
product (6a) as a colourless powder (0.35 g, 87%).
NMR (CDCl3) dP 15.1 (16.1). NMR (DMSO-d6) dP 12.8
(13.3), dH 11.1 (s, 1H, NH), 10.7 (s, 1H, NH), 8.63 (8.57)
(s, 1H, H2), 8.13 (8.16) (s, 1H, H8), 8.05 (d, J=7.3, 2H,
Bz), 7.65±7.52 (m, 3H, Bz), 7.37±7.08 (m, 13H, pixyl),
5.49 (5.35) (s, 2H, CH2CO), 3.58 (3.93) (t, J=5.2, 2H,
OCH2CH2N), 3.48 (d, J=9.7, 2H, PCH2), 3.44 (3.25)
(d, J=10.3 (9.9), 3H, POCH3), ca. 3.02 (3.21) (m, 2H,
OCH2CH2N, partly hidden), 3.00 (q, J=7.3, 6H,
Et3NH+), 1.15 (t, J=7.3, 9H, Et3NH+). FAB MS
703.4 (M Et3NH+, calcd 703.2).
Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]-N-(6-N-
benzoyl-9-adeninylacetyl)aminomethylphosphonate (5a).
Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]amino-
phosphonate (3) (0.44 g, 1 mmol) was dissolved in a
mixture of dry DMF (6 mL) and dry dichloromethane
(6 mL) and cooled to 0 ꢀC under N2. Dry triethylamine
(0.28 mL, 2 mmol), DCC (0.29 g, 1.4 mmol), and
DhbtOH (0.18 g, 1.1 mmol) were added, followed by
Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]-N-(4-N-
benzoyl-1-cytosinylacetyl)aminomethylphosphonate (5b).
This compound was prepared in the same way as 5a
from 4-N-benzoyl-1-cytosinylacetic acid (4b) (0.30 g,
1.1 mmol), yield 0.40 g (58%), pure according to TLC
and NMR. NMR (CDCl3) dP 23.4 (23.2). NMR
(DMSO-d6) dH 11.19 (11.21) (s, 1H, NH), 9.02 (d,
J=8.1, 2H, bz), 7.82 (7.85) (d, J=7.1, 1H, H6), 7.65±
7.50 (m, 3H, Bz), 7.41±7.11 (m, 14H, pix+H5), 4.82
(4.88) (s, 2H, CH2CO), 3.72 (4.08) (d, J=8.0 (9.8),
PCH2), 3.59 (3.74) (d, J=10.6 (10.8), 6H, POCH3), 3.7
(3.6) (partly hidden, 2H, OCH2CH2N), 3.24 (3.09) (t, J
ca. 4, OCH2CH2N). FAB+MS 695.2 (M+H+, calcd
695.7), 717.2 (M+Na+, calcd 717.7).
6-N-benzoyl-9-adeninylacetic
acid
(4a)
(0.33 g,
1.1 mmol). The reaction mixture was stirred under N2
for 1 h at 0 ꢀC followed by 20 h at rt. The reaction mix-
ture was diluted with dichloromethane (75 mL), ®ltered,
and the ®ltrate washed with sat aq NaHCO3 (2Â25 mL),
brine (25 mL), dried (MgSO4), and evaporated in vacuo.
The residue was puri®ed on a silica column (4Â14 cm)
eluted with CH2Cl2:EtOAc:MeOH:Et3N 60:33:7:0.1 v/v/
v/v. The fractions containing the product were evapo-
rated in vacuo and lyophilized from dry acetonitrile to
give 5a as a colourless powder (0.54 g, 75%). The pro-
duct was pure according to TLC and NMR. TLC
Rf=0.16 (EtOAc:MeOH:Et3N 9:1:0.1 v/v/v). NMR
(DMSO-d6; this and the following compounds exist at rt
as two C(O)N rotamers; chemical shifts and coupling
constants for the minor rotamer are given in brackets)
dP 23.9 (23.7), dH 11.15 (s, 1H, NH), 8.58 (8.67) (s, 1H,
H2), 8.20 (8.23) (s, 1H, H8), 8.07 (d, J=7.1, 2H, bz),
7.67±7.54 (m, 3H, bz), 7.41±7.10 (m, 13H, pixyl), 5.45
(5.40) (s, 2H, CH2CO), 3.66 (4.17) (d, J=11.2 (9.7), 2H,
PCH2), 3.58 (3.80) (d, J=10.8 (10.7), 8H,
POCH3+OCH2CH2N (hidden under the POCH3
doublets)), 3.29 (3.08) (t, J=4.8 (5.2), 2H,
OCH2CH2N). FAB+MS 719.1 (M+H+, calcd 719.2),
741.0 (M+Na+, calcd 741.2).
Methyl triethylammonium N-[2-(9-phenyl-9-xanthenyloxy)-
ethyl] -N-(4-N-benzoyl -1-cytosinylacetyl)aminomethyl-
phosphonate (6b). Dimethyl N-[2-(9-phenyl-9-xanthenyl-
oxy)ethyl] - N - (4 - N - benzoyl - 1 - cytosinylacetyl)amino
methylphosphonate (5b) (0.35 g, 0.5mmol) was demethyl-
ated as described for 6a. Yield 0.35 g (90%). NMR
(DMSO-d6) dP 12.6 (13.0), dH 11.2, 10.8 (2 x s, br, NH),
8.02 (d, J ca. 8, 2H, bz), 7.80±7.75 (m, 1H, H6), 7.64±
7.50 (m, 3H, bz), 7.40±7.10 (m, 14H, pixyl+H5), 4.99
(4.79) (s, 2H, CH2CO), 3.84±3.17 (m, 9H, POCH3+P-
CH2+OCH2CH2N), 3.00 (q, J ca. 7, 6H, Et3NH+),
1.16 (t, J ca. 7, 9H, Et3NH+). FAB MS 679.4
(M Et3NH+, calcd 679.2).
Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]-N-(2-N-
isobutyryl-9-guaninylacetyl)aminomethylphosphonate (5c).
This compound was prepared in the same way as 5a
from 2-N-isobutyryl-9-guaninylacetic acid (4c) (0.31 g,
1.1 mmol), but with increased reaction time (42 h), yield
0.44 g (63%), pure according to TLC and NMR. TLC
(CH2Cl2:EtOAc:MeOH:Et3N, 55:30:15:0.1) Rf=0.43.
NMR (DMSO-d6) dP 24.0 (23.9), dH 12.06 (12.08) (s,
Methyl triethylammonium N-[2-(9-phenyl-9-xanthenyloxy)-
ethyl]-N-(6-N-benzoyl-9-adeninylacetyl)aminomethyl-
phosphonate (6a). Dimethyl N-[2-(9-phenyl-9-xanthenyl-
oxy)ethyl]-N-(6-N-benzoyl-9-adeninylacetyl)aminome
thylphosphonate (5a) (0.36 g, 0.5 mmol) was dissolved

J. Kehler et al./Bioorg. Med. Chem. 6 (1998) 315±322
321
1H, NH), 11.56 (11.51) (s, 1H, NH), 7.55 (7.69) (s, 1H,
H8), 7.42±7.11 (m, 13H, pixyl), 5.12 (5.15) (s, 2H,
CH2CO), 3.74 (4.12) (d, J=11.0 (9.9), 2H, PCH2), 3.58
(3.75) (d, J=10.8 (10.8), 6H, POCH3), ca. 3.75 (3.53) (t,
J=(5.6), 2H, OCH2CH2N (the signals from the major
rotamer were hidden)), 3.26 (3.07) (t, J=5.1 (5.6), 2H,
OCH2CH2N), 2.76 (sept, J=6.8, 1H, i-butyryl), 1.10 (t,
J=6.8, 6H, i-butyryl). FAB+MS 701.2 (M+H+, calcd
701.2), 723.2 (M+Na+, calcd 723.2).
Preparation of oligomers. Couplings were performed in
0.6 to 0.7 mmol scale (Pharmacia Primer Support T, ca.
22 mmol/g) or 0.2 mmol scale (ABI polystyrene T sup-
port, ca. 7 mmol/g). The DNA segments of the chimera
were prepared by standard phosphoramidite chemistry.
For E and the chimera C the support was ®rst modi®ed
to give 7 by one coupling with a phosphoramidite
reagent (50 Phosphate-ON, Cruachem). The couplings
with 6a±d were performed manually by injecting (pr
mmol of support) a freshly made solution of 20 mmol of
6, 120 mmol of PyFNOP, and 300 mmol of NMI in
CH3CN:CH2Cl2 (1:1, 100 mL). After 15 min coupling the
column was drained for the coupling mixture and con-
nected to a DNA synthesizer (ABI 380), and washed
with acetonitrile (2Â 0.4 mL) and methanol (3Â0.4 mL),
followed by capping and removal of the pixyl group by
standard 1 mmol cycle procedures. Following synthesis
the support in the column was dried in a stream of air,
and treated in the column for 2 h at rt with a solution of
disodium 2-cyanoethylene-1,1-dithiolate trihydrate
(130 mg) in methanol (0.5 mL), bu€ered with conc.
acetic acid (14 mL). After repeated washing with metha-
nol and drying the column was opened and the support
treated with conc. aq NH3:EtOH (3:1) for 6 to 18 h at
55 ꢀC. The supernatant, after centrifugation, was col-
lected with a syringe and the support washed twice with
H2O:EtOH (3:1). The combined solutions were evapo-
rated to a small volume to remove EtOH, diluted with
H2O, and the oligomer puri®ed by standard EtOH-pre-
cipitation for B and C, and standard reverse-phase pur-
i®cation with removal of the Pix groups (Oligo±Pak
columns, Milligen) for D and E. The yield of puri®ed
oligomer was 26 OD (40%) for B, 22 OD (32%) for C,
12 OD (18%) for D, and 1.5 OD (2%) for E.
Methyl triethylammonium N-[2-(9-phenyl-9-xanthenyl-
oxy)ethyl]-N-(2-N-isobutyryl-9-guaninylacetyl)amino-
methylphosphonate (6c). Dimethyl N-[2-(9-phenyl-9-
xanthenyloxy)ethyl]-N-(2-N-isobutyryl-9-guaninylacetyl)-
aminomethylphosphonate (5c) (0.35 g, 0.5 mmol) was
demethylated as described for 6a. Yield 0.37 g (93%) as
a colourless powder. NMR (DMSO-d6) dP 14.1 (13.1),
dH 12.06 (12.04) (s, 1H, NH), 11.88 (11.85) (s, 1H, NH),
10.05 (br s, 1H, NH+), 7.65 (7.52) (s, 1H, H8), 7.39±
7.08 (m, 13H, pixyl), 5.23 (5.06) (s, 2H, CH2CO), 3.9±
3.0 (m, POCH3+PCH2+OCH2CH2N+HOD), 3.04 (q,
J=7.2, 6H, Et3NH+), 2.76 (sept, J=6.8, 1H, i-butyryl),
1.17 (t, J=7.2, 9H, Et3NH+), 1.09 (t, J=6.8, 6H,
i-butyryl). FAB MS 685.3 (M Et3NH+, calcd 685.2).
Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]-N-(1-thy-
minylacetyl)aminomethylphosphonate (5d). This com-
pound was prepared in the same way as 5a from 1-thy-
midylacetic acid (4d) (0.20 g, 1.1 mmol), yield 0.42 g
(70%), pure according to TLC and NMR. Recrystalli-
zation from EtOAc gave white crystals (0.33 g, 55%).
TLC Rf=0.2 (CH2Cl2:MeOH 95:5 v/v). NMR (CDCl3)
dP 23.5 (22.9). NMR (DMSO-d6) dH 11.29 (s, 1H, NH),
7.41±7.03 (m, 14H, pixyl+H6), 4.69 (4.67) (s, 2H,
CH2CO), 3.58 (3.72) (d, J=10.8, 6H, POCH3), 3.65
(3.5) (partly hidden, 2H, OCH2CH2N), 3.63 (4.02) (d,
J=11.3 (9.7), 2H, PCH2), 3.19 (3.06) (t, J ca. 5, 2H,
OCH2CH2N), 1.70 (1.76) (s, 3H, CH3). FAB+MS 606.5
(M+H+, calcd 606.2), 628.5 (M+Na+, calcd 628.2).
Anal. (C31H32N3O8P), calcd C: 61.48, H: 5.33, N: 6.94;
found C: 61.42, H: 5.24, N: 6.84%.
Acknowledgements
Mrs Anette W. Jùrgensen is thanked for performing
some of the Tm measurements, Britta M. Dahl for the
DNA syntheses, and Thomas Kofoed (PNA Diag-
nostics, Copenhagen) for the MALDI TOF MS mea-
surements. This work was supported by The Danish
Natural Science Research Council.
Methyl triethylammonium N-[2-(9-phenyl-9-xanthenyl-
oxy)ethyl]-N-(1-thyminylacetyl)aminomethylphosphonate
(6d). Dimethyl N-[2-(9-phenyl-9-xanthenyloxy)ethyl]-N-
(1-thyminylacetyl)aminomethylphosphonate (5d) (0.30 g,
0.5 mmol) was demethylated as described for 6a. Yield
0.28 g (80%) as a colourless powder, pure according
to TLC and NMR. TLC Rf=0.19 (CH2Cl2:EtOAc:
MeOH:Et3N, 49:38:12:1, v/v/v/v). NMR (DMSO-d6) dP
13.4 (14.4), dH 11.22 (11.26) (s, 1H, T-NH), 10.7 (s, br,
1H, NH), 7.39±7.01 (m, 14H, pixyl+H6), 4.77 (4.66) (s,
2H, CH2CO), 3.76±3.12 (m, 9H, OCH2CH2N+
POCH3+PCH2), 3.00 (q, J ca. 7, 6H, Et3NH+), 1.74
(1.69) (s, CH3), 1.16 (t, J ca. 7, 9H, Et3NH+). FAB MS
590.2 (M Et3NH+, calcd 590.2).
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