High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues

Article abstract of DOI:10.1021/ol027026a

(Matrix Presented) Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and 9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were

Full text of DOI:10.1021/ol027026a

ORGANIC
LETTERS
2002
Vol. 4, No. 25
4395-4398
High-Affinity Peptide Nucleic Acid
Oligomers Containing Tricyclic Cytosine
Analogues†
Kallanthottathil G. Rajeev, Martin A. Maier, Elena A. Lesnik, and
Muthiah Manoharan*
Department of Medicinal Chemistry, Isis Pharmaceuticals, Inc.,
Carlsbad, California 92008
mmanoharan@isisph.com
Received August 6, 2002 (Revised Manuscript Received October 31, 2002)
ABSTRACT
Peptide nucleic acid (PNA) monomers containing the tricyclic cytosine analogues phenoxazine, 9-(2-aminoethoxy)phenoxazine (G-clamp), and
9-(3-aminopropoxy)phenoxazine (propyl-G-clamp) have been synthesized. The modified nucleobases were incorporated into PNA oligomers
using Boc-chemistry for solid-phase synthesis. PNAs containing single G-clamp modifications exhibit significantly enhanced affinity toward
RNA and DNA targets relative to unmodified PNA while maintaining mismatch discrimination. These PNA G-clamp modifications exhibit the
highest increase in affinity toward nucleic acid targets reported so far for PNA modifications.
In peptide nucleic acids (PNAs) the sugar phosphate
backbone of an oligonucleotide is replaced by a pseudopep-
tide backbone derived from N-(2-aminoethyl)glycine.1,2
Unfortunately, certain PNA sequences have poor solubility
and unmodified PNAs have limited cellular uptake and a
tendency for self-aggregation.3 These problems have been
addressed by introducing PNA backbones carrying charges,2,4,5
by preparing chimeras of PNA with nucleic acids,6 peptides,7
and polyamines,8 and by introducing hydrophilic amino
acids9 into the backbone of PNA.
In addition to the natural nucleobases, a number of
heterocyclic modifications on the PNA backbone have been
reported in the literature including 2,6-diaminopurine,10,11
pseudoisocytosine,12 5-bromouracil,13,14 5-iodouracil,15 4-thio-
† Presented in part at the 222nd National Meeting of the American
Chemical Society, August 26-30, 2001 Chicago, IL, 2001; American
Chemical Society: Washington, DC, 2001; Abstracts of Papers (CARB-
015). Rajeev, K. G.; Maier, M. A.; Manoharan, M. Modified nucleobase
containing peptide nucleic acids (PNAs). US patent application filed May
25, 2001, Isis Pharmaceuticals, Inc.
(6) Uhlmann, E.; Will, D. W.; Breiphohl, G.; Langner, D.; Ryte, A.
Angew. Chem., Int. Ed. Engl. 1996, 35, 2632-2635.
(7) Simmons, C. G.; Pitts, A. E.; Mayfield, L. D.; Shay, J. W.; Corey,
D. R. Bioorg. Med. Chem. Lett. 1997, 7, 3001-3006.
(8) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Biochem. Biophys.
Res. Commun. 1997, 240, 778-782.
(1) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
(Washington, D.C.) 1991, 254, 1497-500.
(9) Haaima, G.; Lohse, A.; Buchardt, O.; Nielsen, P. E. Angew. Chem.,
Int. Ed. Engl. 1996, 35, 1939-1941.
(2) Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. 1996, 4, 5-23.
(3) Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem.,
Int. Ed. 1998, 37, 2796-2823.
(10) Gangamani, B. P.; Kumar, V. A.; Ganesh, K. N. Chem. Commun.
(Cambridge) 1997, 1913-1914.
(11) Haaiima, G.; Hansen, H. F.; Christensen, L.; Dahl, O.; Nielsen, P.
E. Nucleic Acids Res. 1997, 25, 4639-4643.
(4) Puschl, A.; Sforza, S.; Haaima, G.; Dahl, O.; Nielsen, P. E.
Tetrahedron Lett. 1998, 39, 4707-4710.
(12) Egholm, M.; Christensen, L.; Dueholm, K. L.; Buchardt, O.; Coull,
J.; Nielsen, P. E. Nucleic Acids Res. 1995, 23, 217-22.
(5) Nielsen, P. E. Methods Enzymol. 2000, 313, 156-164.
10.1021/ol027026a CCC: $22.00 © 2002 American Chemical Society
Published on Web 11/15/2002

thymine,16 2-thiouracil,17,18 5-methyl-4-(3H-(1,2,4)triazol-1-
yl)-3,4-dihydro-1H-pyrimidine-2-one,19 and 5-methyl-
cytosine.12 Incorporation of a tricyclic 3,5-diaza-4-oxo-
phenothiazine into PNA showed an affinity increase of 3
°C per modification with complementary DNA or RNA as
compared to unmodified PNA.20 PNAs containing 1,8-
naphthyridin-2(1H)-one and benzo[b]-1,8-naphthyridin-
2(1H)-one as substitutes for thymine have also been re-
ported.20,21 For each of these modifications, the increase in
thermal binding was rather small to modest. We became
interested in the design of heterocyclic modifications that
could enhance the binding affinity to mRNA, increase
solubility, and increase the cellular uptake of PNA.
the tethered amino group formed an additional hydrogen
bond with O6 in the Hoogsteen face of a complementary
guanine.22 It has been shown by X-ray crystal structural
analysis of a oligonucleotide decamer duplex containing a
guanidino G-clamp, an analogue of G-clamp, that a Hoog-
steen-type hydrogen bond does exist between O6 and N7 of
G and the tethered guanidino group.24 This suggests that the
increased affinity of G-clamp is mediated by the combination
of extended base stacking and at least one additional specific
hydrogen bond between O6 of G and the tethered amino
group. Single incorporation of the G-clamp at the 3′-terminus
of an ODN resulted in a significant increase in nuclease
stability as well.25 Biological studies on G-clamp-containing
phosphorothioate ODNs for specific antisense targets such
as cyclin-dependent kinase inhibitor, p27kip1, and c-raf
showed enhanced potency relative to unmodified ODN.23
Given the benefits the G-clamp modification offers for
antisense phosphorothioate oligonucleotides, we envisioned
that introduction of the G-clamp and its derivatives into PNA
would improve the molecular recognition properties of PNA.
We reasoned that the additional positive charge on the
modified base might have a positive impact on solubility
and cellular uptake properties of the modified PNAs.
In the present Letter we describe convenient syntheses of
suitably protected tricyclic phenoxazine (2a), G-clamp (2b),
and 9-(3-aminopropoxy)phenoxazine (propyl G-clamp, 2c)
PNA monomers shown in Figure 1. Each monomer has been
incorporated into PNA using Boc-chemistry and solid-phase
synthesis and deprotection protocols. Also reported are
preliminary accounts of the hybridization behavior of these
analogues with complementary DNA and RNA, and mis-
matched RNA strands.
Several tricyclic cytosine analogues have been synthesized.
These include 9-(2-aminoethoxy)phenoxazine, called the
G-clamp (1, Figure 1), that carries an aminoethoxy moiety
Figure 1. Structure of monomeric DNA and PNA G-clamp.
An orthogonally protected G-clamp PNA monomer (10a)
was synthesized from 5-bromouracil (3) as shown in Scheme
1. Trimethylsilylation of 5-bromouracil in HMDS under
reflux followed by treatment of the silylated derivative with
ethyl bromoacetate in refluxing acetonitrile yielded 5-bromo-
3,4-dihydro-2,4-dioxo-1(2H)-pyrimidine-acetic acid ethyl
ester (4) in quantitative yield. Activation at C4 of compound
4 by treatment with POCl3 and triazole in the presence of
triethylamine and subsequent substitution of the triazole
moiety at C4 with 2-aminoresorcinol in the presence of
diisopropylethylamine yielded compound 5 in 76% yield.
Compound 5 was then subjected to monoalkylation with
benzyl N-(2-hydroxyethyl)carbamate under Mitsunobu alky-
lation conditions in acetonitrile to obtain compound 6a in
80% yield.22 Treatment of compound 6a with a 10 molar
excess of cesium fluoride26 and one molar equiv of Cs2CO3
in absolute ethanol under reflux for 36 h gave a mixture of
ethyl ester 7a and its corresponding carboxylic acid 8a.
Compound 8a was precipitated from the aqueous extract of
the reaction by the addition of KHSO4. The ethyl ester 7a
was stirred with LiOH at a temperature below 10 °C and
attached to the rigid phenoxazine scaffold.22,23 Incorporated
into oligonucleotides, these cytosine modifications hybridize
with guanine and enhance duplex stability through extended
stacking interactions. Binding studies demonstrated that a
single incorporation of the G-clamp enhanced the binding
affinity of an oligodeoxynucleotide (ODN) to its comple-
mentary target DNA or RNA with a ∆Tm of up to 18 °C
relative to 5-methylcytosine (dC5Me). It was suggested that
(13) Ferrer, E.; Shevchenko, A.; Eritja, R. Bioorg. Med. Chem. 2000, 8,
291-7.
(14) Rasmussen, H.; Kastrup, J. S.; Nielsen, J. N.; Nielsen, J. M.; Nielsen,
P. E. Nat. Struct. Biol. 1997, 4, 98-101.
(15) Betts, L.; Josey, J. A.; Veal, J. M.; Jordan, S. R. Science
(Washington, D.C.) 1995, 270, 1838-41.
(16) Clivio, P.; Guillaume, D.; Adeline, M.-T.; Hamon, J.; Riche, C.;
Fourrey, J.-L. J. Am. Chem. Soc. 1998, 120, 1157-1166.
(17) Izvolsky, K. I.; Demidov, V. V.; Nielsen, P. E.; Frank-Kamenetskii,
M. D. Biochemistry 2000, 39, 10908-10913.
(18) Lohse, J.; Dahl, O.; Nielsen, P. E. Proc. Natl. Acad. Sci. U.S.A.
1999, 96, 11804-11808.
(19) Breipohl, G.; Knolle, J.; Langner, D.; O’Malley, G.; Uhlmann, E.
Bioorg. Med. Chem. Lett. 1996, 6, 665-670.
(20) 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.
(21) Eldrup, A. B.; Christensen, C.; Haaima, G.; Nielsen, P. E. J. Am.
Chem. Soc. 2002, 124, 3254-3262.
(22) Lin, K.-Y.; Matteucci, M. D. J. Am. Chem. Soc. 1998, 120, 8531-
8532.
(24) Wilds, C. J.; Maier, M. A.; Tereshko, V.; Manoharan, M.; Egli, M.
Angew. Chem., Int. Ed. 2002, 41, 115-117.
(25) Maier, M. A.; Leeds, J. M.; Balow, G.; Springer, R. H.; Bharadwaj,
R.; Manoharan, M. Biochemistry 2002, 41, 1323-1327.
(23) Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.;
Wagner, R. W.; Matteucci, M. D. Proc. Natl. Acad. Sci. U.S.A. 1999, 96,
3513-3518.
(26) Kurchavov, N. A.; Stetsenko, D. A.; Skaptsova, N. V.; Potapov, V.
K.; Sverdlov, E. D. Nucleosides Nucleotides 1997, 16, 1837-1846.
4396
Org. Lett., Vol. 4, No. 25, 2002

Scheme 1a
Scheme 2a
a Reagents and conditions: (a) (i) POCl3, triazole, TEA/MeCN,
-10 °C, (ii) 2-aminophenol, DIEA/DCM, rt, 6 h (51.7%); (b) CsF
(10 equiv), Cs2CO3 (1 equiv)/EtOH, reflux 36 h; (c) (i) LiOH, (ii)
KHSO4; (d) (Boc)HNCH2CH2NH(CH2COOEt), DCC, DhbhOH,
DMAP/DMF, 12 h (6%, over all yield from 5c).
aminoethyl)glycinate as described for the synthesis of
compound 9a to obtain the fully protected monomer 9c in
6% over all yield. Alkaline hydrolysis of compound 9c gave
the desired phenoxazine PNA monomer 10c in quantitative
yield.
a Reagents and conditions: (a) (i) HMDS, reflux, (ii) ethylbro-
moacetate, MeCN reflux (94%); (b). (i) POCl3, triazole, TEA/
MeCN, -10 °C, (ii) 2-aminoresorcinol, DIEA/DCM, rt, 6 h (76%);
(c) Ph3P, DEAD, ZHN(CH2)nOH (n ) 2 or 3)/MeCN, 24 h (80%);
(d) CsF (10 equiv), Cs2CO3 (1 equiv)/EtOH, reflux 36 h (83%);
(e) (i) LiOH, (ii) KHSO4 (>95%); (f) (Boc)HNCH2CH2NH(CH2-
COOEt), DCC, DhbhOH, DMAP/DMF, 12 h (50%).
PNA synthesis was performed using Boc chemistry-based
automated synthesis (Applied Biosystems, model 433A) and
synthesis protocols based on previously published proce-
dures.27,28 The C-terminal lysine was introduced by using a
MBHA polystyrene resin (NovaBiochem) pre-loaded with
BOC-Lys(2-Cl-Z)-OH. Using a preactivation time of 5 min
and a coupling time of 1 h, all novel monomers 10a-c
coupled with efficiencies comparable to the standard Boc-
PNA monomers. The coupling efficiency was monitored by
qualitative Kaiser test.29 After cleavage and deprotection
according to standard low/high TFMSA protocols, the PNA
oligomers were purified by RP-C18 HPLC, analyzed by ESI-
MS, lyophilized, and stored at -20 °C. HPLC buffer: A )
0.1% TFA in water; B ) MeCN; 0 to 35% B in 35 min.
PNA monomer 10a was incorporated into sequences 13
and 17. Monomer 10b was incorporated in sequences 14 and
18, and monomer 10c was incorporated into sequences 12
and 16. All sequences are listed in Table 1. Sequence
complementarity and stability of PNA-DNA and PNA-RNA
hybrids were determined by thermal denaturation studies and
subsequent addition of KHSO4 gave the free carboxylic acid
8a in quantitative yield.
The carboxylic acid 8a was coupled to ethyl N-(2-tert-
butyloxycarbonylaminoethyl)glycinate via prior activation of
the carboxylic group with DCC and DhbhOH to obtain the
fully protected G-clamp monomer 9a in 52% yield.11
Saponification of 9a at low temperature using LiOH yielded
the desired PNA monomer 10a. The propyl G-clamp
monomer (10b) was obtained from compound 5 via monoalky-
lation with benzyl N-(3-hydroxypropyl)carbamate under
Mitsunobu alkylation conditions in acetonitrile followed by
subsequent reactions as described for the synthesis of
compound 10a.
The phenoxazine PNA monomer (10c) was synthesized
from compound 4 as shown in Scheme 2. Unlike the ring
closure reaction in the tricyclic systems that have the
aminoalkyl tethers (10a and 10b), the ring closure of
compound 5c with cesium fluoride and cesium carbonate to
obtain compound 7c or 8c was very sluggish. For this reason,
without purifying the ester 7c, it was saponified to 8c and
subsequently coupled to ethyl N-(2-tert-butyloxycarbonyl-
(27) Christensen, L.; Fitzpatrick, R.; Gildea, B.; Petersen, K. H.; Hansen,
H. F.; Koch, T.; Egholm, M.; Buchardt, O.; Nielsen, P. E.; et al. J. Pept.
Sci. 1995, 1, 185-83.
(28) Koch, T.; Hansen, H. F.; Andersen, P.; Larsen, T.; Batz, H. G.;
Otteson, K.; Oerum, H. J. Pept. Res. 1997, 49, 80-88.
(29) Kaiser, E.; Colescott, R. L.; Bossinger, C. D.; Cook, P. I. Anal.
Biochem. 1970, 34, 595-8.
Org. Lett., Vol. 4, No. 25, 2002
4397

30
30
Table 1.
Tm Data for Duplexes of 10-mer PNA Containing
Table 2.
Base Recognition of 10-mer PNA Containing
Cytosine (control), Phenoxazine (X ) 2a), G-clamp (Y ) 2b),
and Propyl-G-clamp (Z ) 2c) with Complementary DNA and
RNA
Cytosine (control), Phenoxazine (X ) 2a), G-clamp (Y ) 2b),
and Propyl-G-clamp (Z ) 2c) with Complementary G, A, C,
and U of RNA
(Table 2). With G-clamp (PNA 13) and propyl G-clamp
(PNA 14), significantly less stable duplexes were formed
against oligonucleotides with mismatched A, C, and T as
compared to the target strand containing a matching G (Table
2). The results indicate that all tricyclic modifications exhibit
excellent sequence specificity.
compared with the corresponding unmodified control se-
quences. The modifications were evaluated in two different
sequences with the heterocylic modification at different
positions within the oligomer and with different flanking
bases. In the first set of oligomers (sequences 11-14, Table
1), the flanking bases are T and A. In the second set
(sequences 15-18, Table 1) they are both C. While a single
incorporation of the phenoxazine monomer (10c) in PNA
12 resulted in a Tm increase of only 1.4 °C when hybridized
to complementary RNA, the PNA containing the G-clamp
(PNA 13) showed an affinity enhancement of 10.7 °C. PNA
containing the propyl G-clamp, with the increased tether
length at position 9 of the heterocycle (monomer 10b, PNA
14), resulted in further Tm advantage with an increase of 13.5
°C relative to unmodified PNA. When the heterocylic
modification was flanked by two Cs in the second set of
sequences, the Tm enhancement was even higher (Table 1).
The phenoxazine modification (PNA 16) resulted in a Tm
increase of 4.1 °C toward complementary RNA. The G-
clamp (PNA 17) resulted in an increase of 18.2 °C. This Tm
increase corresponds well to the data reported for a G-clamp-
containing ODN of a similar sequence.22 The enhancement
of melting temperature for the G-clamp modification in
ODNs also has significant sequence dependence.
Hybridization of the modified PNAs with complementary
DNA gave results similar to those observed with comple-
mentary RNA (Table 1). The Tm enhancement was greater
when the modified base was flanked by two Cs than when
the flanking bases were T and A. A Tm enhancement of 23.7
°C was observed when the G-clamp PNA (PNA 17) was
hybridized to complementary DNA (Table 1). This Tm
enhancement is the highest known increase in affinity for a
PNA-DNA hybrid. The propyl G-clamp (PNA 18) gave a
Tm advantage of 19.7 °C when hybridized with complemen-
tary DNA.
In summary, tricyclic cytosine analogues based on the
phenoxazine were incorporated into PNA oligomers. A
convenient route for the facile synthesis of suitably protected
phenoxazine, G-clamp, and propyl G-clamp PNA monomers
has been developed. Single incorporation of these tricyclic
moieties into to PNA showed sequence-dependent affinity
enhancement of up to 18 °C when hybridized to RNA and
up to 24 °C when hybridized to DNA. These are the highest
Tm advantages for heterocyclic modifications of PNA
reported so far. Furthermore, the magnitude depends on the
length of the aminotether employed. Recently, we have also
demonstrated the conversion of the G-clamp to the corre-
sponding guanidinium group in oligonucleotide analogues
and the same process can be extended to the PNAs reported
here.24,32 Further evaluation of these high-affinity modifica-
tions with respect to their effect on the cellular uptake and
pharmacology of PNA antisense oligomers and on the strand
invasion potential of PNA are in progress.
Acknowledgment. We thank our colleagues Drs. Ramesh
Bharadwaj and Bruce S. Ross for 2-aminoresorcinol synthesis
and Svetlana Doronina for earlier synthesis contributions in
initiating this project. This work was supported in part by
an SBIR grant (GM-60087).
Supporting Information Available: ESI-MS character-
ization of PNAs 11-18. This material is available free of
charge via the Internet at http://pubs.acs.org.
OL027026A
(30) The melting experiments were performed at 8 µM total strand
concentration at pH 7.0 in 10 mM phosphate buffer containing 100 mM
Na+ and 0.1 mM EDTA. The Tm values presented are the average of three
measurements and reproducibility of the measurements was within (0.5
°C. [Lesnik, E. A.; Risen, L. M.; Driver, D. A.; Griffith, M. C.; Sprankle,
K.; Freier, S. M. Nucleic Acids Res. 1997, 25, 568-574.]
(31) Noncooperative melting.
For the control PNA20 (11) and the phenoxazine modifica-
tion (PNA 12), incorporation of a base mismatch (A, T, or
C) in the target strand opposite to the modified site resulted
in noncooperative melting due to a lack of base specificity
(32) Maier, M. A.; Barber-Peoc’h, I.; Manoharan, M. Tetrahedron Lett.
2002, 43, 7613-7616.
4398
Org. Lett., Vol. 4, No. 25, 2002