The synthesis of a thymine containing E-olefinic peptide nucleic acid (OPA) monomer

Article abstract of DOI:10.1055/s-1999-2711

A monomeric unit of a conformationally constrained PNA analog, E- olefinic peptide nucleic acid (E-OPA) containing the base thymine was synthesized in 14 steps. The key step involved a palladium (0) catalyzed cross-coupling reaction utilizing a reformatsky reagent as nucleophile. A protecting group regime that is compatible with solid-phase PNA and DNA chemistries was chosen.

Full text of DOI:10.1055/s-1999-2711

LETTER
819
The Synthesis of a Thymine Containing E-Olefinic Peptide Nucleic Acid (OPA)
Monomer
Christopher D. Roberts, Rolf Schütz, Christian J. Leumann*
Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, CH-3012 Berne, Switzerland
Fax +41 31 631 34 22; E-mail: leumann@ioc.unibe.ch
Received 19 February 1999
Z-OPA monomer has already been reported,4 herein we
Abstract: A monomeric unit of a conformationally constrained
report the synthesis of the thymine containing E-OPA
PNA analog, E-olefinic peptide nucleic acid (E-OPA) containing
monomer.
the base thymine was synthesized in 14 steps. The key step involved
a palladium (0) catalyzed cross-coupling reaction utilizing a Refor-
matsky reagent as nucleophile. A protecting group regime that is
compatible with solid-phase PNA and DNA chemistries was cho-
sen.
Key words: OPA, PNA, Reformatsky, DNA-analog, antisense
Peptide nucleic acid (PNA), reported in 1991 by Nielsen,1
is a nucleic acid analog with remarkable pairing proper-
ties. Much work has since been invested in understanding
and improving this potentially useful analog.2 Oligomers
of PNA are able to form stable duplexes with both DNA
and RNA in both antiparallel and parallel orientations.
Structural characterizations3 show that when PNA is com-
plexed with a natural nucleic acid the internal amide bond
of the PNA units are uniformly aligned so that the carb-
onyl oxygen of the tertiary amide bond points to the carb-
oxy terminus in an antiparallel Watson-Crick duplex. Ole-
finic peptide nucleic acid (OPA) monomers were de-
signed as PNA analogs with an internal olefin of defined
configuration. We reasoned that the replacement of the
conformationally labile amide bond with a conformation-
ally rigid olefin bond would allow an assessment of the
role of this preorganized structural element in the duplex
binding orientation observed in PNA (Scheme 1). From
structural models, we anticipate that the Z-OPA isomer
will bind in a parallel fashion, while the E-OPA isomer
will bind in an anti-parallel orientation. The synthesis of a
The syntheses of OPA monomers were designed to be di-
vergent, with both E and Z-isomers utilizing a common in-
termediate 1 (Scheme 2). The E-isomer is then obtained
using functional group interconversion to reverse the ami-
no acid polarity and introduce the correct nucleobase(s).
The basic OPA skeleton 1 is assembled from the vinyl io-
dide via a palladium(0) catalyzed cross-coupling reaction
utilizing an ethyl a-bromoacetate derived Reformatsky re-
agent as the nucleophile.5 The geometry of the olefin is
defined via a stereospecific conversion of the propargyl
alcohol to the Z-iodo olefin.6 In anticipation of assembly
of this monomer into oligomers utilizing solid-phase syn-
thesis, a strategy incorporating the monomethoxytrityl
amino protecting group and acyl base protection was cho-
sen.7 This system was selected over the standard PNA
(and peptide) chemistries due to the need to avoid strongly
acidic conditions which would scramble the double-bond
position and geometry. Furthermore, this strategy is com-
patible with DNA synthesis chemistry, allowing us to
construct DNA/OPA chimeras.
The synthesis of the thymine E-OPA amino acid 10 was
begun from commercially available 3-butynol 2 (Scheme
3). The alcohol was protected as the THP acetal followed
Scheme 1
Synlett 1999, No. 6, 819–821 ISSN 0936-5214 © Thieme Stuttgart · New York

820
C. D. Roberts et al.
LETTER
by hydroxymethylation with paraformaldehyde to afford
the protected propargylic alcohol 3. The formation of the
Z-iodo olefin was accomplished in high yield by ste-
reospecific reduction of the propargyl system with Red-Al
followed by addition of N-iodosuccinimide. Attempts to
use the significantly cheaper iodine as the I+ source were
only successful on small scale reactions. Scale up howev-
er, resulted in dramatic drops in yield (<30% total) due to
scrambling of THP groups, probably due to traces of HI in
solution. The allylic alcohol was then protected to afford
the tert.-butyl-diphenyl-silyl ether 4 in high yield.
With large amounts of ester 1 in hand the protected E-
OPA amino acid was ready for assembly (Scheme 5).
Azide 6 was produced by reduction of 1 with LiAlH4 in
good yield followed by azidation using modified Mit-
sunobu conditions with the more soluble zinc diazide
dipyridine complex and diisopropyl azodicarboxylate
(DIAD) in toluene.10 The silyl ether was very efficiently
cleaved using standard tetrabutylammonium fluoride
treatment. The N3-benzoyl protected thymine
nucleobase11 was subsequently introduced in high yield
under Mitsunobu conditions to afford 7.
The construction of the common intermediate 1 using the
palladium(0) mediated Reformatsky reaction initially
turned out to be problematic and unreliable. Eventually,
several crucial factors in this reaction were elucidated, re-
sulting in consistent and high yields of 1 on a range of re-
action scales (Scheme 4).8 We found that the activation of
the zinc prior to formation of the Reformatsky reagent and
the quality of the palladium(0) tetrakis-triphenylphos-
phine were absolutely crucial.9 The ratio of DMPU to
dimethoxymethane was also important (roughly 4:1 is op-
timal). And finally, the relationship between reaction time
and reaction scale was important as well. As the reaction
progresses, product ester 1 becomes a substrate for the Re-
formatsky reagent yielding the "bis-coupled" b-keto-ester
5. As the scale of the reaction increases, the ratio of 1 to 5
decreases (Scheme 4 entries A vs. B vs. C). And with large
scale reactions (Scheme 4 entries C vs. D), decreasing the
reaction time increases the ratio of 1 to 5 while the abso-
lute amount of 1 formed remains constant.
The THP acetal of 7 was cleanly removed by acidic treat-
ment to afford the free alcohol. Initial attempts to oxidize
the alcohol directly to the acid with PDC were unsuccess-
ful due to the inability to separate the polar acid 8 from the
chromate salt by-products. Instead, a two-step oxidation
was utilized. The alcohol was oxidized to the aldehyde us-
ing the Dess-Martin periodinane.12 The crude aldehyde
was then oxidized with sodium chlorite in tert-butyl alco-
hol with 2-methyl-2-butene as a Cl+ scavenger to afford
the acid 8 in good yield.13 The next step was the reduction
of the azide 8 to the primary amine. Hydrogenation using
Lindlar conditions repeatedly failed in our hands. This re-
sult is interesting in view of the fact that the corresponding
transformation in the Z-OPA series proceeds successfully
and in high yield. Successful reduction to the amino acid
was accomplished by treatment of the azide 8 with tri-
phenylphosphine in pyridine,14 followed by hydrolysis of
the phosphinimine (and thymine benzoyl protecting
group) with ammonium hydroxide to give the free amino
Synlett 1999, No. 6, 819–821 ISSN 0936-5214 © Thieme Stuttgart · New York

LETTER
Synthesis of an E-Olefinic Peptide Nucleic Acid (OPA) Monomer
821
acid 9,15 which was subsequently protected using
monomethoxytrityl chloride in DMSO with triethylamine
to afford 10 as the triethylammonium salt.16
freshly distilled ethyl a-bromoacetate (3.0 ml). The flask was
placed in a 50° C oilbath with vigorous stirring until initiation
had occurred, at which point the reaction was removed from
heat, and with continuous stirring, ethyl a-bromoacetate (12.0
ml) dissolved in dimethoxymethane (80 ml) was added at such
a rate that a reflux was maintained. After addition was
complete the mixture was refluxed for an additional 20 min.
The resulting green mixture was then removed from the heat
and stirring was stopped. After 20 min the green solution was
carefully removed from the deposited zinc and added to the
solution of 4 in one batch. After 1 h at 70° C the reaction was
quenched by the addition of 100 ml of saturated aqueous
NH4Cl. After standard aqueous work-up, the resulting crude
mixture was separated by flash chromatography (SiO2, 5%
ether/hexane to 25% ether/hexane) to give 1 (11.39 g, 22.3
mmol) in 76% yield and the bis-coupled product 5 (1.07 g, 1.9
mmol) in 7% yield.
The herein described divergent synthesis allows incorpo-
ration of a trisubstituted olefin of defined geometry which
is then efficiently assembled into the final OPA skeleton
by a palladium(0) catalyzed Reformatsky reaction. Re-
moval of the silyl protecting group from 6 will allow the
introduction of any nucleobase and work in this direction
is currently under way. Furthermore, the final protected
amino acid salt 10 is suitable for use in solid-phase pep-
tide synthesis and work towards incorporation into oligo-
mers is currently being investigated.
(9) Activation of the zinc as described in Perrin, D. W.;
Armarego, W. L. F.; Perrin, D. W. Purification of Laboratory
Chemicals, 2nd ed.; Pergamon: Oxford, 1980; p 547 and use of
freshly prepared tetrakis(triphenylphosphine)palladium(0),
Coulson, D. R. in Inorganic Syntheses; Vol. 28; Angelici, R.
J., Ed.; Wiley: New York, 1990; p 107. rather than the use of
commercially available sources were necessary for
satisfactory and reproducible results.
(10) Viaud, M. C.; Rollin, P. Synthesis 1990, 130.
(11) Cruickshank, K. A.; Jiricny, J.; Reese, C. B. Terahedron Lett.
1984, 25, 681.
(12) Dess, D. B.; Martin, J. C. J. Am. Chem. Soc. 1991, 113, 7277.
(13) Balkrishna, S. B.; Childers, W. E.; Pinnick, H. W.
Tetrahedron 1981, 37, 2091.
Acknowledgement
Financial support for this work by the Swiss National Science Foun-
dation (Grant No. 20-49191.96) is gratefully acknowledged.
References and notes
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497.
(2) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew.
Chem. Int. Ed. Engl. 1998, 37, 2796; Angew. Chem. 1998,
110, 2954.
(3) Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G.
Science 1994, 265, 777. Betts, L.; Josey, J. A.; Veal, J. M.;
Jordan, S. R. Science 1995, 270, 1838. Eriksson, M.; Nielsen,
P. E. Nature Struct. Biol. 1996, 3, 410.
(4) Cantin, M.; Schütz, R.; Leumann, C. J. Tetrahedron Lett.
1997, 38, 4211.
(5) Fauvarque, J. F.; Jutand, A. J. Organomet. Chem. 1981, 209,
109. Curé, J.; Gaudemar, M. Bull. Soc. Chim. Fr. 1968, 3244.
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1987, 52, 3860. Marshall, J. A.; Lebreton, J.; DeHoff, B. S.;
Jenson, T. M. J. Org. Chem. 1987, 52, 3883.
(7) Will, D. W.; Breipohl, G.; Langner, D.; Knolle, J.; Uhlmann,
E. Tetrahedron 1995, 51, 12069.
(8) Synthesis of 1: In a dry atmosphere 4 (16.13g, 29.3 mmol) was
dissolved in dry DMPU (390 ml), then
(14) Mungall, W. S.; Greene, G. L.; Heavner, G. A.; Letsinger, R.
L. J. Org. Chem. 1975, 40, 1659.
(15) Spectral characterization of amino acid 9: 1H NMR D2O (d):
1.73 (s, 3H), 2.47 (t, J = 7.35 Hz, 2H), 2.87 (s, 2H), 2.99 (t, J
= 7.35 Hz, 2 H), 4.29 (d, J = 6.98 Hz, 2H), 5.37 (t, J = 6.98 Hz,
1H), 7.36 (s, 1H). 13C NMR D2O (with DMSO reference) (d):
12.81, 29.75, 38.73, 46.48, 47.45, 112.3, 127.08, 136.64,
144.18, 153.68, 168.39, 181.15. Anal. calcd. for C12H17N3O4:
C, 53.92; H, 6.41; N, 15.72. Found: C, 53.54; H, 6.39; N,
15.63.
(16) Breipohl, G.; Will, D. W.; Peyman, A.; Uhlmann, E.
Tetrahedron 1997, 53, 14671.
tetrakis(triphenylphosphine) palladium(0) (6.82 g, 5.86
mmol) was added and the mixture heated to 70° C. In a
separate flask, freshly activated zinc powder (10.0 g) was
suspended in dry dimethoxymethane (15.0 ml) containing
Article Identifier:
1437-2096,E;1999,0,06,0819,0821,ftx,en;G08499ST.pdf
Synlett 1999, No. 6, 819–821 ISSN 0936-5214 © Thieme Stuttgart · New York