Synthesis and incorporation into PNA of fluorinated olefinic PNA (F-OPA) monomers.

Article abstract of DOI:10.1021/ol034579s

[structure: see text] A fluorinated OPA monomer containing the base thymine ((Z)-t-F-OPA) was synthesized in 12 steps, featuring a highly selective allylic over homoallylic Mitsunobu substitution for the introduction of the nucleobase. F-OPA modified PNA

Full text of DOI:10.1021/ol034579s

ORGANIC
LETTERS
2003
Vol. 5, No. 11
1987-1990
Synthesis and Incorporation into PNA of
Fluorinated Olefinic PNA (F-OPA)
Monomers
Marcel Hollenstein and Christian J. Leumann*
Department of Chemistry and Biochemistry, UniVersity of Bern, Freiestrasse 3,
CH-3012 Bern, Switzerland
leumann@ioc.unibe.ch
Received April 3, 2003
ABSTRACT
A fluorinated OPA monomer containing the base thymine ((Z)-t-F-OPA) was synthesized in 12 steps, featuring a highly selective allylic over
homoallylic Mitsunobu substitution for the introduction of the nucleobase. F-OPA modified PNA decamers were prepared by the MMTr/acyl
protection strategy. The thermal stability of duplexes of PNA decamers containing (Z)-t-F-OPA units with antiparallel complementary DNA was
measured. We found a strong dependence of stability from the sequential position of the (Z)-t-F-OPA units, ranging from ∆Tm of +2.4 to −8.1
°C/modification relative to unmodified PNA.
The peptide nucleic acid (PNA) is a DNA analogue entirely
based on an achiral polyamide backbone.1 PNA readily
undergoes sequence-specific Watson-Crick base-pairing
with complementary DNA and RNA.2 The relatively high
binding affinity of PNA toward natural oligonucleotides is
attributed to the lack of electrostatic repulsion between the
uncharged PNA backbone and the negatively charged sugar-
phosphate backbone of DNA and RNA.3
PNA/RNA,6 and PNA/PNA7 complexes, whereas both rota-
meric forms coexist in the free monomer. Furthermore, in
the crystal structures of a 50% N-methylated PNA hexamer8
and of a hexameric PNA containing the 1,8-naphthyridin-
2(1H)-one base,9 the same situation was encountered, reveal-
ing that structural alterations in the base and in the inter-
residue amide bond have no influence on the orientation of
the linker-carbonyl unit.
An interesting structural feature of PNA is the central
amide linker, connecting the nucleobases with the amide
backbone (Figure 1). The carbonyl oxygen of these units
uniformly points toward the carboxy termini in PNA/DNA,4,5
To structurally constrain the base-linker unit in PNA and
to explore its electrostatic contribution to complementary
DNA binding, we recently designed and investigated the
olefinic peptide nucleic acids (OPA), built from units of 2
and 310,11 (Figure 1).
(1) Recent reviews: (a) Kumar, V. A. Eur. J. Org. Chem. 2002, 2021-
32; (b) Nielsen, P. E. Curr. Med. Chem. 2001, 8, 545-550; (c) Nielsen, P.
E. Curr. Opin. Biotechnol. 2001, 12, 16-20; (d) Uhlmann, E.; Peyman,
A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. 1998, 37, 2796-
2823; (e) Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624-30.
(2) Egholm, M.; Burchardt, O.; Christensen, L.; Behrens, C.; Freier, S.
M.; Driver, D. A.; Berg, R. H.; Kim, S. K.; Norden, B.; Nielsen, P. E.
Nature 1993, 365, 566-568.
(5) Leijon, M.; Gra¨slund, A.; Nielsen, P. E.; Burchardt, O.; Norde´n, B.;
Kristensen, S. M.; Eriksson, M. Biochemistry 1994, 33, 9820-25.
(6) Brown, S. C.; Thomson, S. A.; Veal, J. M.; Davis, D. G. Science
1994, 265, 777-780.
(7) Rasmussen, H.; Kastrup, J. S.; Nielsen, J. N.; Nielsen, J. M.; Nielsen,
P. E. Nat. Struct. Biol. 1997, 4, 98-101.
(8) Haaima, G.; Rasmussen, H.; Schmidt, G.; Jensen, D. K.; Kastrup, J.
S.; Stafshede, P. N.; Norde´n, B.; Burchardt, O.; Nielsen, P. E. New J. Chem.
1999, 23, 833-40.
(9) Eldrup, A. B.; Nielsen, B. B.; Haaima, G.; Rasmussen, H.; Kastrup,
J. S.; Christensen, C.; Nielsen, P. E. Eur. J. Org. Chem. 2001, 1781-1790.
(3) Nielsen, P. E.; Egholm, M.; Burchardt, O. Bioconjugate Chem. 1994,
5, 3-7.
(4) Betts, L.; Josey, J. A.; Veal, J. M.; Jordan, S. R. Science 1995, 270,
1838-41.
10.1021/ol034579s CCC: $25.00 © 2003 American Chemical Society
Published on Web 05/08/2003

the base thymine, its incorporation into PNA via the MMTr/
acyl protecting group strategy, and first pairing experiments
with complementary antiparallel DNA.
We started the synthesis of the tetrasubstituted olefin with
the commercially available 3-hydroxy glutarate 5 and fol-
lowed a Wittig strategy for the introduction of the vinylic
fluorine. Desymmetrization of the double bond was con-
trolled via lactone formation (Scheme 1).
Scheme 1a
Figure 1. Two rotameric forms of the PNA monomers (top) and
chemical structures of the (E)- and (Z)-OPA monomers (center)
and of the F-OPA monomer (bottom).
a Reagents and conditions: (a) LiAlH4, THF, rt, 4 h; (b)
MMTrCl, DMAP, pyridine, rt, 8 h; (c) IBX, DMF, rt, 3 h; (d)
n-BuLi, (EtO)2P(O)CHFCO2Et, THF, -78 °C, 1.5 h, then 8, THF,
-78 °C f rt, 5 h; (e) BCl3, CH2Cl2, rt, 2 h.
So far, we have investigated fully modified OPA-oligo-
amides containing the bases adenine and thymine. We found
that (E)-OPA forms preferentially parallel duplexes with
DNA of markedly lower affinity compared to unmodified
PNA. Furthermore, oligothymine-OPA was unable to form
triplexes with DNA. Thus, it became clear that the amide
function in the base-linker unit in PNA significantly deter-
mines affinity and strand orientation in PNA/DNA duplexes.
We reasoned that the differences in the recognition properties
of OPA are mainly related not to conformational differences
relative to PNA but rather to changes in the H-bonding
capacity, electrostatic properties, or solvation.
Consequently, the intriguing question arose as to what
extent the dipole moment of the linker-carbonyl group in
PNA influences its binding properties. The OPA scaffold is
ideally suited to address this question. Since a C-F bond is
structurally and electrostatically a decent mimic of a CdO
bond without hydrogen bonding capacity12 and was often
used in that respect,13 we decided to synthesize and inves-
tigate the pairing properties of the (Z)-F-OPA system 4
(please note that the (Z)-F-OPA system has the same
geometric configuration as the (E)-OPA series). Introduction
of the fluorine atom at the vinylic position thus mimicks the
geometry and in part the dipole moment of the carbonyl
group in bound PNA.
Reduction of 5 with LiAlH4 yielded triol 6 (65%), which
was then bis-MMTr protected (82%). Oxidation of alcohol
7 under mild conditions afforded ketone 8 (79%) which was
subsequently converted to the R-fluoroester 9 by means of
a Wittig-Wadsworth-Emmons14 reaction in 67% yield.
â-Elimination of the MMTrO moieties as a side reaction
during olefination was observed but could be controlled.
To chemically differentiate between the two alcohol
moieties, ester 9 was converted to intermediate 10 by means
of a deprotection-lactonization step mediated by BCl3 in
77% yield.15
Lactone 10 was then TBDMS protected (99%) and reduced
to diol 12 (94%) under Luche conditions with NaBH4/CeCl3
in methanol (Scheme 2).16 In our plan, we had hoped to be
able to chemically differentiate between the two primary
hydroxyl functions in 12, reasoning that the allylic nature
of one of the two leads to an advantage in reactivity during
substitution. Indeed, introduction of the N3-benzoyl-protected
thymine17 under Mitsunobu conditions led to a rewarding
72% yield of 13. No isomeric substitution product could be
isolated (Scheme 2).
Herein, we report on a highly stereoselective synthesis of
the novel fluorinated (Z)-F-OPA building block 17 containing
(10) (a) Roberts, C. D.; Schu¨tz, R.; Leumann, C. J. Synlett 1999, 6, 819-
821. (b) Cantin, M.; Schu¨tz, R.; Leumann, C. J. Tetrahedron Lett. 1997,
38, 4211-4214.
(14) Patrick, T. B.; Lanahan, M. V.; Yang, C.; Walker, J. K.; Hutchinson,
C. L.; Neal, B. E. J. Org. Chem. 1994, 59, 1210-1212.
(15) Evans, D. A.; Carter, P. H.; Carreira, E. M.; Charette, A. B.; Prunet,
J. A.; Lautens, M. J. Am. Chem. Soc. 1999, 121, 7540-7552.
(16) (a) Hale, K. J.; Hummersone, M. G.; Bhatia, G. S. Org. Lett. 2000,
2, 2189-2192. (b) Luche, J.-L.; Rodriguez-Hahn, L.; Crabbe, P. J. Chem.
Soc., Chem. Commun. 1978, 601-602.
(11) Schu¨tz, R.; Cantin, M.; Roberts, C.; Greiner, B.; Uhlmann, E.;
Leumann, C. J. Angew. Chem., Int. Ed. 2000, 39, 1250-1253.
(12) (a) Smart, B. E. J. Fluorine Chem. 2001, 109, 3-11. (b) Wang, X.,
Houk, K. N. Chem. Commun. 1998, 2631-2632.
(13) O’Neill, B. M.; Ratto, J. E.; Good, K. L.; Tahmassebi, D. C.;
Helquist, S. A.; Morales, J. C.; Kool, E. T. J. Org. Chem. 2002, 67, 5869-
5875.
(17) Cruickshank, K. A.; Jiricny, J.; Reese, C. B. Tetrahedron Lett. 1984,
25, 681-684.
1988
Org. Lett., Vol. 5, No. 11, 2003

synthesis developed earlier.19 A CPG support, carrying a
glycine ester unit as the anchoring point was used as a solid
phase,11 and the N-termini of the oligoamides were equipped
with lysine units in order to enhance water solubility and
ease purification. The oligomers were isolated by reversed-
phase HPLC using typical procedures for PNA purification.
The composition of the purified oligomers was confirmed
by ESI+-TOF mass spectrometry as shown in Table 1. The
Scheme 2a
Table 1. Mass Spectrometric and Tm Data from UV-Melting
Curves of PNA Sequences Containing (E)-t-OPA, (Z)-t-OPA, or
(Z)-t-F-OPA Units with Complementary DNA
m/z
m/z
Tm
sequencea
(calcd) (found)b (°C)c
18
19
20
21
22
23
24
25
Lys-TTTTAATATA-Gly-NH2
2900.9
2900.1
2883.3
2883.3
2901.3
2883.2
2901.6
2883.2
2901.6
33.2
36.7
28.0
35.6
30.0
27.0
28.1
25.1
Lys-TTTTAAtEATA-Gly-NH2 2883.9
Lys-TTTTAAtZATA-Gly-NH2
Lys-TTTTAAtF ATA-Gly-NH2
Lys-TTTtEAATATA-Gly-NH2 2883.9
Lys-TTTtF AATATA-Gly-NH2
Lys-TTtETAATATA-Gly-NH2 2883.9
Lys-TTtF TAATATA-Gly-NH2
2901.9
2883.9
2901.9
2901.9
a tZ ) (Z)-t-OPA, tE ) (E)-t-OPA, and tF ) (Z)-t-F-OPA. b ESI+-MS.
c Tm data are from UV-melting curves (260 nm) at 4 µM duplex concen-
tration in 100 mM NaCl, 10 mM Na2HPO4, pH 7.0; with antiparallel DNA
complement d(TATATTAAAA).
coupling yields for the OPA and (Z)-t-F-OPA building blocks
were in the range of 90-95%, as measured by the trityl
assay.
The oligomers were hybridized with the corresponding
antiparallel DNA complement, and the thermal stability of
the duplexes was examined by UV-melting curve analysis.
Representative melting curves (260 nm) are reproduced in
Figure 2. The corresponding Tm data are summarized in Table
1.
a Reagents and conditions: (a) TBDMSCl, imidazole, DMF, rt,
12 h; (b) NaBH4, CeCl3‚7H2O, methanol, 0 °Cf rt, 4.5 h; (c) N3-
benzoyl thymine, PPh3, DIAD, THF, rt, 12 h; (d) LiN3, CBr4, PPh3,
DMF, 0 °Cf rt, 12 h; (e) TBAF, THF, rt, 5 h; (f) Dess-Martin
periodinane, CH2Cl2, rt, 2 h; (g) NaClO2, NaH2PO4, 2-methyl-2-
butene, tBuOH, rt, 12 h; (h) PPh3, pyridine, concentrated NH3, rt,
3 h; (i) MMTrCl, pyridine, rt, 12 h.
PNA oligomer 21, containing the (Z)-t-F-OPA unit in a
central position of the sequence, showed a stabilization of
the duplex (∆Tm ) +2.4 °C) compared to the unmodified
PNA oligomer 18. Introduction of an (E)-OPA unit at the
same location in the sequence (19) also led to a stabilization
(∆Tm ) +3.5 °C), while a (Z)-OPA unit (20) strongly
destabilized the duplex, due to the structural mismatch
imposed by the (Z)-configuration of the double bond.
A marked sequence dependence that had already been
observed previously,11 but to a lesser extent, appeared upon
introducing (E)-t-OPA units in various locations of the strand.
Indeed, insertion of an (E)-t-OPA unit between two adenine
PNA units, as in 19, leads to stabilization, whereas position-
ing of a (E)-t-OPA between two thymine PNA units, as in
24, leads to a significant decrease in the Tm (∆Tm ) -5.1
°C). Positioning between an adenine and a thymine PNA
unit, as in 22, leads to an intermediate Tm (∆Tm ) -3.2 °C
compared to 18). Substitution by (Z)-t-F-OPA, as in 21, 23,
and 25, essentially follows the same stability pattern as in
Azidation of 13, again by Mitsunobu reaction,18 provided
14 in excellent yield (92%). The N3-benzoyl protecting group
of the nucleobase neatly departed under these conditions.
TBDMS deprotection to alcohol 15 (75%) was followed by
a two-step oxidation of the hydroxy function. In the first
step, 15 was oxidized to the corresponding aldehyde using
the Dess-Martin periodinane and was subsequently further
oxidized with sodium chlorite in tBuOH, with 2-methyl-2-
butene as a Cl+ scavenger,10 to the acid 16 (68% for the
two steps). Conversion of the azide to the amine under
Staudinger conditions, followed by MMTr protection, af-
forded the monomer building block 17 in 35% yield (two
steps).
PNA oligoamides containing the (Z)-t-F-OPA unit and the
corresponding (Z)- or (E)-OPA units for comparison were
synthesized according to the MMTr/acyl solid-phase peptide
(18) Yamamoto, I.; Sekine, M.; Hata, T. J. Chem. Soc., Perkin Trans. 1
1978, 1, 306-310.
(19) Will, D. W.; Breipohl, G.; Langner, D.; Knolle, J.; Uhlmann, E.
Tetrahedron 1995, 51, 12069-12082.
Org. Lett., Vol. 5, No. 11, 2003
1989

In conclusion, we have synthesized a novel, fluorinated
olefinic peptide nucleic acid monomer (Z)-t-F-OPA in 12
steps, the key steps being a lactonization as a stereodiffer-
entiating step in the elaboration of the double bond and a
highly selective Mitsunobu substitution of an allylic over a
homoallylic hydroxy function by the thymine base. Incor-
poration of (Z)-t-F-OPA into PNA was achieved by the
MMTr/acyl strategy. It was found that duplex stability
strongly depends on the position of the modification within
the strand. The reason for this is still unknown, but might
likely be caused by the differential stacking interactions or
solvation properties of the OPA units relative to those of
PNA. Dipole effects of the base-linker unit could be
excluded.
With the synthetic access to F-OPA accomplished, it will
now certainly be of interest to investigate the pure F-OPA
system. Differential analysis of the pairing properties with
PNA and OPA will shed light on dipolar vs solvation or
H-bonding effects of the respective structural elements on
duplex stability and will thus advance our understanding of
fundamental aspects of supramolecular folding and as-
sembly.20
Figure 2. UV-melting curves (260 nm) of duplexes of oligoamides
21, 23, and 25 with the corresponding antiparallel oligodeoxy-
nucleotide d(TATATTAAAA). Experimental conditions were the
same as those in Table 1.
the case of the (E)-t-OPA units, but with more pronounced
sequence-stability effects. Here the differences in Tm, relative
to PNA, vary between +2.4 and -8.1 °C.
Acknowledgment. Financial support from the Swiss
National Science Foundation (Grant 20-63582.00) is grate-
fully acknowledged. Dr. J. Hunziker, Dr. T. J. Ryan, D.-R.
Ahn, and D. Gautschi are thanked for their helpful advice
and fruitful discussions.
Various effects can be responsible for the observed
sequence dependence in DNA affinity. It is certainly possible
that OPA units intrinsically may prefer a base-linker
conformation that is different from that in PNA. Assuming
that this preferred conformation is independent of nearest
neighbor influence, this would not, however, explain the
sequence effects. Replacing a nonpolar H-atom at the double
bond by a more polar F-atom does not change the sequence-
affinity pattern of the OPA system, ruling out dipole
differences in the base-linker units as being mainly respon-
sible for this. Thus, there remain differential stacking
interactions of the OPA-base-linker units and/or differential
solvation patterns of this subunit as likely sources for the
differential behavior relative to PNA.
Supporting Information Available: Experimental pro-
cedures and analytical data of compouds 7-17 as well as a
general procedure for the synthesis and purification of
oligomers 18-25. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL034579S
(20) Hill, D. J.; Mio, M. J.; Prince, R. B.; Hughes, T. S.; Moore, J. S.
Chem. ReV. 2001, 101, 3893-4011.
1990
Org. Lett., Vol. 5, No. 11, 2003