Bis(hydroxymethyl)phosphinic acid analogues of acyclic nucleosides; synthesis and incorporation into short DNA oligomers

Article abstract of DOI:10.1016/S0040-4039(02)01067-5

Novel analogues of acyclic nucleosides based on a bis(hydroxymethyl)phosphinic acid (BHPA) backbone were obtained by condensation of the bis(4,4′-dimethoxytrityl) derivative of BHPA with N-1- or N-3-(2-hydroxyethyl)thymine in the presence of MSNT, or by an Appel reaction with N-1- or N-3-(2-aminoethyl)thymine. After selective deprotection and phosphitylation they were used as monomers for automated solid support synthesis of short oligomers by the phosphoramidite approach.

Full text of DOI:10.1016/S0040-4039(02)01067-5

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 43 (2002) 5397–5400
Bis(hydroxymethyl)phosphinic acid analogues of acyclic
nucleosides; synthesis and incorporation into
short DNA oligomers
B. Nawrot,* O. Michalak, M. Nowak, A. Okruszek, M. Dera and W. J. Stec
Centre of Molecular and Macromolecular Studies, Department of Bioorganic Chemistry, Polish Academy of Sciences,
Sienkiewicza 112, 90-363 Lodz, Poland
Received 30 April 2002; revised 27 May 2002; accepted 7 June 2002
Abstract—Novel analogues of acyclic nucleosides based on a bis(hydroxymethyl)phosphinic acid (BHPA) backbone were obtained
by condensation of the bis(4,4%-dimethoxytrityl) derivative of BHPA with N-1- or N-3-(2-hydroxyethyl)thymine in the presence of
MSNT, or by an Appel reaction with N-1- or N-3-(2-aminoethyl)thymine. After selective deprotection and phosphitylation they
were used as monomers for automated solid support synthesis of short oligomers by the phosphoramidite approach. © 2002
Elsevier Science Ltd. All rights reserved.
Acyclic analogues of nucleosides are of interest due to
their attractive chemical and pharmacological properties.
Up to now, several purine and pyrimidine linked
aliphatic derivatives have been found to be effective
antiviral agents.1–3 Acyclic derivatives of nucleosides are
also considered as components of novel DNA analogues.
The aim of synthesizing such analogues, including those
derived from isosteric glyceronucleosides, is to identify
potential antisense or antigene therapeutics with
improved nucleolytic stability and binding affinity
toward complementary DNA or RNA strands.4–7
N-1- or N-3-ethylthymine and the incorporation of such
analogues into a short oligonucleotide chain.
In the presented analogues 1 and 2, bis(hydroxymethyl)-
phosphinic acid (BHPA) replaces the 3%-, 4%- and 5%-car-
bon atoms of the sugar–phosphate backbone of DNA (3)
and provides a site for attachment of nucleobases via a
one- or two-carbon linker. Esters or amides of BHPA (2,
X=O or NH) possess an additional hydrogen bond
acceptor center at the phosphinyl oxygen atom. The
non-ionic nature of such a unit, as in 2, may enhance
cellular uptake, while attached nucleobases (B) may
interact via stacking and/or hydrogen bonds with com-
plementary DNA or RNA.
The synthesis of the monomers is outlined in Scheme 1.
Both OH functions of bis(hydroxymethyl)phosphinic
acid methyl ester8 4 were protected with acid-labile
4,4%-dimethoxytrityl (DMT) groups. Fully protected
derivative 5 was demethylated with tert-butylamine at
100°C (sealed tube). BHPA esters 7a and 7b could be
synthesized in good yields (74 and 75%, respectively) by
condensation of 6 with N-1-9 or N-3-(2-hydroxy-
ethyl)thymine10 in the presence of 1-(2-mesitylensul-
fonyl)-3-nitro-1,2,4-triazole (MSNT) as activator. To
obtain 7c and 7d, phosphinate 6 was subjected to an
Appel reaction11 with N-1- or N-3-(2-aminoethyl)-
thymine.10 The reaction was carried out in pyridine in the
presence of triphenylphosphine (3 equiv.) and carbon
tetrachloride (5 equiv.). The yields of the desired prod-
ucts were rather low (25–30%), as was reported by Appel
for such reactions.11 Selected physicochemical character-
istics of 7a–d are presented in Table 1.
We report here a general method for the synthesis of
bis(hydroxymethyl)phosphinic acid derivatives carrying
Keywords: phosphinic acid derivatives; nucleic acid analogues.
* Corresponding author. Tel.: +48-42-681-6970; fax: +48-42-681-5483;
e-mail: bnawrot@bio.cbmm.lodz.pl
0040-4039/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(02)01067-5

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B. Nawrot et al. / Tetrahedron Letters 43 (2002) 5397–5400
Scheme 1. Reagents and conditions: (i) 4,4%-dimethoxytrityl (DMT) chloride (2.5 equiv.)/pyridine, 12 h, rt; (ii) tert-butylamine (6
equiv.), 100°C, 10 h; (iii) N-1 or N-3-(2-hydroxyethyl)thymine (1 equiv.), MSNT (1 equiv.) in pyridine, 24 h, rt or (2-
aminoethyl)thymine (1 equiv.), PPh3 (3 equiv.), CCl4 (5 equiv.) in pyridine, rt, 24 h; (iv) toluene-4-sulfonic acid in methanol (0.02
M, 0.8 equiv.), rt, 5 min, then pyridine, (v) O-2-cyanoethyl-N,N,N%,N%-tetraisopropylphosphordiamidite (1.2 equiv.) and
2-ethylthio-1H-tetrazole (2.4 equiv.) in acetonitrile, rt, 1 h.
Table 1. Spectral and chromatographic data for bis(hydroxymethyl)phosphinic acid derivatives 7–9
Comp.
Yield (%)
74
Rf (TLC)a
FAB MS m/z
l
31P NMR (ppm)
7a
0.34
882.6 [M+H]
881.8 [M−H]
881.8 [M−H]
882.8 [M+H]
880.3 [M−H]
880.6 [M−H]
579.3 [M−H]
579.1 [M−H]
578.2 [M−H]
578.3 [M−H]
49.10
7b
7c
75
30
0.22
0.58
48.43
38.55
7d
8a
8b
8c
25
35
33
32
30
70
60
75
50
0.42
0.61
0.50
0.82
0.73
0.32
0.42
0.48
0.50
37.35
48.75
50.65
38.26
8d
37.51
9ac
9bd
9cd
9dd
153.37; 153.0; 46.96; 46.59b
152.69; 152.31; 46.32; 46.16b
152.10; 152.00; 151.96; 151.86; 36.59; 36.53; 36.44; 36.39b
151.92; 151.61; 36.37; 36.00b
a CHCl3/MeOH, 9:1, v/v.
b The presence of multiple resonances (four or eight signals) may be due to the presence of threo–erythro diastereoisomerism and/or PꢀCꢀOꢀP spin
couplings.
c Methyl-protection of phosphoramidite.
d 2-Cyanoethyl-protection of phosphoramidite.
The resulting derivatives 7a–d were subjected to the
selective removal of one DMT group by brief treatment
with toluene-4-sulfonic acid in methanol (0.02 M, 0.8
equiv.). The reaction had to be stopped after 5–6 min
while ca. 60% of substrate was still present in the
reaction mixture. Longer reaction times led to signifi-
cant amounts of fully deprotected BHPA ester or amide
derivatives and to their further decomposition resulting
from PꢀO and PꢀN bond cleavage. Thus, the reactions
were terminated by addition of an excess of pyridine.
The desired products were isolated by means of prepar-
ative TLC (yield ca. 30–35%), and about 60% of start-
ing material was recovered.
Phosphitylation12 of the free OH group in 8a–d by
O-2-cyanoethyl-N,N,N%,N%-tetraisopropylphosphordi-
amidite (1.2 equiv.) was carried out in anhydrous aceto-
nitrile in the presence of 2-ethylthio-1H-tetrazole (2.4
equiv.) providing the novel acyclic nucleoside amidite
synthons 9a–d.

B. Nawrot et al. / Tetrahedron Letters 43 (2002) 5397–5400
5399
Table 2. Characteristics of short DNA analogues possess-
of the phosphate protecting methyl groups was
achieved by treatment of solid support-bound
oligomers with thiophenol/dioxane/triethylamine mix-
ture (2:1:2, v/v) for 5 min at rt followed by the cleavage
of oligomers form the solid support with 1% aqueous
triethylamine for 7–10 min at rt.
ing bis(hydroxymethyl)phosphonic acid units (Y)
Comp.
no.
Compound
HPLCc Rt
(min)
MALDI TOF
Calc.
Found
10ca
10d
11
12a
12b
5%-dApYpdC-3%
16.15
17.10
13.49
20.06
20.63
878
878
725
1828
1828
879
878
727
1835
1833
These oligomers were also obtained by using oxalyl-
LCAA CPG solid support and protection of the phos-
phate function with a 2-cyanoethyl group. In this case,
treatment of solid support-bound oligomers with 1%
aqueous triethylamine for 10 min at room temperature
resulted in simultaneous cleavage of the oligomer from
the solid support and removal of the 2-cyanoethyl
protecting groups. Hexamers 12a and 12b were purified
by HPLC and their structure was confirmed by
MALDI TOF analysis (Table 2).
5%-dApYpdC-3%
5%-dApY-pdC-3%b
TpTpYpYpTpT-3%
TpTpYpYpTpT-3%
a a–d are according to Scheme 1.
b Y− is ionic bis(hydroxymethyl)phosphinic acid residue as in 1.
c Analytical HPLC was performed on a C18 (4.6×250 mm, Thermo-
Quest) column with a linear gradient of acetonitrile in 0.1 M
triethylammonium bicarbonate pH 7.5 buffer (0–20 min/0–20%
CH3CN, 20–25 min/20–40% CH3CN, flow 1 mL/min).
Anionic analogues of DNA, such as 11, were also
obtained by selective protection of one OH function
with the DMT group and subsequent phosphitylation
of the second OH group of BHPA methyl ester 4,
followed by introduction of such monomer into the
oligonucleotide by the solid phase methodology with
two-step deprotection/purification.15
The phosphoramidite monomers 9 were used for the
synthesis of modified trinucleotides 5%-dApYpdC-3%
(where Y is a modified BHPA unit) by automated solid
phase methodology.13 The synthesis of oligomers 10–12
was performed on an ABI 394 synthesizer (Applied
Biosystem Inc., Foster City, CA) using succinyl-linked
LCAA-CPG solid support. The only difference in the
manufacturer recommended procedure was a prolonged
coupling time (up to 600 s). The coupling efficiency of
9a–d, determined by DMT-ion assay, was in the range
of 95–97%. The 5%-terminal DMT group was removed
before cleavage of the product from the solid support.
A standard ammonium deprotection and one-step RP
HPLC purification led to the desired trimers 10c and
10d for which HPLC showed no separation of dia-
stereomeric trimers (P-epimers, Table 2). Unfortunately,
this approach did not yield the desired trimers 10a and
10b, derived from esterified BHPA. As revealed by
MALDI-TOF analysis, both isolated products were
in fact the same compound 11, possessing no alkyl-
nucleobase moiety. Hydrolytic deprotection conditions
(concentrated aqueous ammonia, 16 h, 55°C) resulted
in cleavage of the ester bond of the phosphinic acid
derivatives 10a and 10b, giving rise to anionic DNA
analogues with an abasic site (as in the structure 1).
This instability of the BHPA ester bond of 10a and 10b
was also observed under less harsh conditions, routinely
used for cleavage of oligomers from the solid support
(aq. NH4OH, 1 h, 20°C). Despite the observed stability
of the ester bond of 7a and 7b in concentrated aqueous
ammonia for 2 h at rt (data not shown), it was expected
that such compounds, as base-labile phosphotriester
analogues, may not survive the deprotection conditions.
In summary, acyclic nucleoside analogues 7a–d derived
from bis(hydroxymethyl)phosphinic acid linked via an
amide or ester bond to an alkylnucleobase moiety were
synthesized and then introduced into an oligonucleotide
chain using the phosphoramidite methodology. The
routine synthetic strategy has proved to be successful
for 10c and 10d, and oxalyl-LCAA CPG afforded the
homo-thymidylate oligomers 12a and 12b. Studies on
the application of the aforementioned methodology for
the synthesis of longer oligonucleotide analogues, their
hybridization properties as well as nuclease resistance
are underway.
Experimental
Typical procedures for the synthesis of compounds 7
Preparation of 7a (MSNT procedure): MSNT (0.44 g,
1.5 mmol) was added to a solution of 6 (0.80 g, 1.0
mmol) and N-1-(2-hydroxyethyl)thymine (0.17 g, 1
mmol) in anhydrous pyridine (8 mL) and the mixture
was stirred at rt for 24 h. The solvent was evaporated
and the crude product was isolated by column chro-
matography on silica gel with a gradient of 0–5%
MeOH in CHCl3 to give 0.66 g (75%) of pure 7a in the
form of a white foam.
Preparation of 7c (Appel procedure): CCl4 (0.54 mL, 5.6
mmol) was added with stirring under argon at rt to a
solution of 6 (0.90 g, 1.1 mmol) and PPh3 (0.88 g, 3.4
mmol) in anhydrous pyridine (8 mL). Stirring was
continued for 30 min and a solution of N-1-(2-
aminoethyl)thymine (0.19 g, 1.1 mmol) in anhydrous
pyridine (4 mL) was added. The resulting solution was
stirred for 24 h at rt and the solvent was removed under
Thus we could not use the routine phosphoramidite
methodology for the synthesis of the modified oligonu-
cleotides 10a,b with protected nucleobases, which
demand the use of basic deprotection conditions. How-
ever, using oxalyl-LCAA CPG solid support14 and
methyl-phosphoramidite methodology13 we obtained
hexamers 5%-TpTpYpYpTpT-3% (12a and 12b). Removal

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B. Nawrot et al. / Tetrahedron Letters 43 (2002) 5397–5400
reduced pressure. The crude product was purified by
silica gel column chromatography with a gradient of
0–5% MeOH in CHCl3 to give 0.27 g (30%) of pure 7c
as white crystals.
8c: 7.92–6.93 (m, 14H), 4.98 (br. s, OH), 4.55 (d, J=3.0
Hz, 2H), 4.45–4.25 (m, 2H), 3.97–3.67 (m, 4H), 3.65 (s,
6H), 1.88 (d, J=1.0 Hz, 3H).
8d: 7.81–6.86 (m, 14H), 5.19 (br. s, OH), 4.57 (m, 2H),
4.45 (t, J=6.0 Hz, 2H), 3.98–3.67 (m, 4H), 3.64 (s, 3H),
3.63 (s, 3H), 1.87 (d, J=1.0 Hz, 3H).
1H NMR characterization of products 7a–d (200 MHz,
CDCl3)
7a: 7.38–6.71 (m, 26H), 7.32 (q, J=1.1 Hz, 1H), 4.04
(m, 4H), 3.78 (s, 6H), 3.77 (s, 6H), 3.69 (dd, J=9.5 Hz,
J=12.5 Hz, 2H), 3.41 (dd, J=9.7 Hz, J=12.5 Hz, 2H),
1.64 (d, J=1.1 Hz, 3 H).
Acknowledgements
The results presented here were obtained within the
project funded by the State Committee for Scientific
Research (KBN Grant 3T09A 03917 for B.N.).
7b: 7.37–6.71 (m, 27H), 4.13 (m, 4H), 3.77 (s, 6H), 3.76
(s, 6H), 3.60 (dd, J=9.0 Hz, J=12.5 Hz, 2H), 3.42 (dd,
J=9.0 Hz, J=12.5 Hz, 2H), 1.77 (d, J=1.1 Hz, 3H).
7c: 7.40–6.70 (m, 26H), 7.08 (q, J=1.0 Hz, 1H), 3.77 (s,
6H), 3.76 (s, 6H), 3.73–3.58 (m, 4H), 3.28 (dd, J=9.6
Hz, J=11.5 Hz, 2H), 3.14 (dd, J=9.6 Hz, J=11.5 Hz,
2H), 1.76 (d, J=1.0 Hz, 3H).
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J
PCH=5.7 Hz, 4H), 3.55 (dd, J=9.3 Hz, J=12.0 Hz,
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Preparation of 8c: To a solution of compound 7c (0.13
g, 0.22 mmol) in MeOH (4 mL), a methanolic solution
of toluene-4-sulfonic acid (0.13 mL, 0.02 M) was added,
with stirring. After 5 min the reaction was terminated
by addition of pyridine (0.4 mL). The product and
unreacted substrate were isolated by means of prepara-
tive TLC on silica gel with CHCl3/MeOH (9:1, v/v).
The recovered substrate was again reacted as above.
After two-fold repetition of the procedure, pure
product 8c was obtained in 35% yield as a white solid.
1H NMR characterization of products 8a–d (d5-Py):
12. Adams, S. P.; Kavka, K. S.; Wykes, E. J.; Holder, S. B.;
Galluppi, G. R. J. Am. Chem. Soc. 1983, 105, 661–663.
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14. Alul, R. H.; Singman, Ch. N.; Zhang, G.; Letsinger, L.
Nucleic Acids Res. 1991, 19, 1527–1532.
8a: 7.77–6.80 (m, 14H), 4.98 (br. s, OH), 4.69–4.52 (m,
4H), 4.18–4.01 (m, 2H), 3.84 (t, J=3.3 Hz, 2H), 3.65 (s,
3H), 3.64 (s, 3H), 1.85 (d, J=1.0 Hz, 3H).
15. Zon, G.; Stec, W. J. In Oligonucleotides and Analogues. A
Practical Approach; Eckstein, F., Ed.; IRL Press at
Oxford University Press: Oxford, 1991; pp. 87–100.
8b: 7.78–6.74 (m, 14H), 4.90 (br. s, OH), 4.46–4.20 (m,
2H), 4.17–4.03 (m, 4H), 3.79 (s, 3H), 3.78 (s, 3H),
3.61–3.23 (m, 2H), 1.87 (d, J=1.0 Hz, 3H).