Synthesis of amino- and guanidino-G-clamp PNA monomers

Article abstract of DOI:10.1021/ol026815p

(matrix presented) Syntheses of the protected amino- and guanidino-G-clamp PNA monomers, 9a and 9b, respectively, have been accomplished in eight steps from 5-bromouracil. Enhanced stacking interactions and additional hydrogen bonds with guanine should in

Full text of DOI:10.1021/ol026815p

ORGANIC
LETTERS
2002
Vol. 4, No. 23
4073-4075
Synthesis of Amino- and
Guanidino-G-Clamp PNA Monomers
Cristina Aus´ın, Jose´-Antonio Ortega, Jordi Robles, Anna Grandas, and
Enrique Pedroso*
Departament de Qu´ımica Orga`nica, Facultat de Qu´ımica, UniVersitat de Barcelona,
Mart´ı i Franque`s 1, 08028 Barcelona, Spain
epedroso@qo.ub.es
Received August 29, 2002
ABSTRACT
Syntheses of the protected amino- and guanidino-G-clamp PNA monomers, 9a and 9b, respectively, have been accomplished in eight steps
from 5-bromouracil. Enhanced stacking interactions and additional hydrogen bonds with guanine should increase the affinity of PNAs
incorporating these cytosine analogues for their complementary strands.
The therapeutic potential of antisense oligonucleotides
(ODNs)1 has stimulated studies aiming at chemically modi-
fying the nucleic acids structure in order to increase their
affinity toward their complementary strands and to improve
their nuclease resistance and cell uptake.2 Increase in affinity
between nucleic acids strands is mainly achieved by improv-
ing stacking interactions and hydrogen bonding.3 Enhanced
stacking can be accomplished by introducing polycyclic base
analogues, and an increase in the number of hydrogen bonds
achieved by the simultaneous recognition of both the
Watson-Crick and Hoogsteen binding faces of guanine and
adenine bases. For this purpose, a tricyclic cytosine analogue,
having the structure of an aminoethoxy-derivatized phenox-
azine ring, was designed to bind to the guanine residue like
a clamp (amino-G-clamp, Figure 1a), and incorporated into
(1) Abbreviations: ACN ) acetonitrile, DIEA ) N,N-diisopropylethyl-
amine, EDC ) 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro-
chloride, Fmoc ) 9-fluorenylmethoxycarbonyl, ODN ) oligodeoxynucle-
otide, PNA ) peptide nucleic acid, TEA ) triethylamine, Z ) benzyloxycarb-
onyl.
(2) (a) Crooke, S. T. Biochim. Biophys. Acta 1999, 1489, 31-44. (b)
Figure 1. Hydrogen bonding in base-pairs of guanine with cytosine
Crooke, S. T. Methods Enzymol. 2000, 313, 3-45. (c) Uhlmann, E.; Peyman,
analogues: (a) amino-G-clamp, (b) guanidino-G-clamp, and (c) the
A. Chem. ReV. 1990, 90, 543-584.
fluorescent phenothiazine PNA monomer.
(3) Saenger, W. Principles of Nucleic Acid Structure; Springer-Verlag:
New York, 1984.
10.1021/ol026815p CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/16/2002

oligodeoxynucleotides.4 A single incorporation of an amino-
G-clamp was found to increase the Tm of a DNA decamer
by 18 °C, presumably by the formation of four hydrogen
bonds and enhanced stacking interactions. The amino-G-
clamp conferred enhanced potency to a 15-mer phosphoro-
thioate (PS) antisense oligonucleotide that showed sequence-
specificity and RNase H activation.5 Additionally, unaided
cellular permeation was observed for a 7-mer PS-ODN
incorporating the naked phenoxazine nucleobase analogue
(without the aminoethoxy arm).6 Furthermore, a single
incorporation of an amino G-clamp at the 3′-end completely
protects the oligonucleotides against 3′-exonuclease attack.7
Recently, the X-ray diffraction analysis of a modified DNA
decamer containing a nucleoside analogue, the guanidino-
ethoxyphenoxazine derivative (Figure 1b), has shown that
this guanidino-G-clamp forms five hydrogen bonds with
guanine.8
The phenothiazine cytosine analogue has been incorporated
into both oligonucleotides4a and peptide nucleic acids9
(PNAs; the structure of the PNA monomer is shown in Figure
1c). The phenothiazine-containing oligomers formed more
stable duplexes than the unmodified ones, and exhibited high
fluorescent quantum yields.9b PNAs are nucleic acid ana-
logues that contain a pseudo-peptide backbone to which the
nucleobases are attached.10 PNAs, which form sequence-
specific and stable duplexes with DNA and RNA and are
resistant to nuclease degradation, have been used for numer-
ous therapeutic and biological applications.11
Scheme 1a
a Reaction conditions: (i) BrCH2CO2tBu, K2CO3/DMF, rt, 21
h; (ii) Ph3P, CCl4/CH2Cl2, reflux, 3 h, and then 2-aminoresorcinol,
DBU, rt, 13 h; (iii) 1,2,4-triazole, POCl3, TEA/ACN, 0 °C, 30 min,
and then addition of 2, rt, 21 h; (iv) 2-aminoresorcinol, DBU/ACN,
rt, 20 h.
However, attempts to directly introduce 2-aminoresorcinol
at the C-4 position of bromouracil by the described method4b
(activation of C-4 with Ph3P-CCl4 and then reaction with
the aromatic amine in the presence of DBU) failed in our
hands. Similarly, discouraging results were obtained when
the starting material was N1-trityl-5-bromouracil or N1-
methoxycarbonylmethyl-5-bromouracil. Only when N1-tert-
butoxycarbonylmethyl-5-bromouracil 2 was used were we
able to obtain the N4-dihydroxyphenyl derivative of 5-bro-
mocytosine 4, but in a low yield (12%).
The success came through the activation of the C-4
position of the uracil ring of 2 with POCl3/1,2,4-triazole,12
which allowed the isolation of the triazole derivative 3.
Subsequent reaction with 2-aminoresorcinol/DBU afforded
the key intermediate 4 in 72% yield from 2. It is worth
mentioning that most of the difficulties found were associated
with the low solubility of 2-aminoresorcinol, 5-bromouracil,
and most of its derivatives. For instance, reactions iii and iv
(Scheme 1) on N1-methoxycarbonylmethyl-5-bromouracil,
instead of 2, provided the 2-aminoresorcinol-substituted
derivative in low yield (8%).
In this context, we were prompted to prepare the suitably
protected amino- and guanidino-G-clamp PNA monomers,
to introduce these base modifications into PNAs and examine
their hybridizaton properties and other potential applications.
Herein, for the first time we describe the synthesis of such
PNA monomers via an independent route that is significantly
different from the originally employed synthesis of the
amino-G-clamp nucleoside derivative.4b
Since 5-bromodeoxyuridine had been employed for the
synthesis of the nucleoside derivative,4b we chose 5-bromo-
uracil 1 as the starting material for our route (Scheme 1).
(4) (a) Lin, K.-Y.; Jones, R. J.; Matteucci, M. J. Am. Chem. Soc. 1995,
117, 3873-3874. (b) Lin, K.-Y.; Matteucci, M. J. Am. Chem. Soc. 1998,
120, 8531-8532.
(5) Flanagan, W. M.; Wolf, J. J.; Olson, P.; Grant, D.; Lin, K.-Y.;
Wagner, R. W.; Matteucci, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 3513-
3518.
The next two synthetic steps are the cyclization reaction
to form the phenoxazine derivative 5 and the introduction
of the Z-protected amino- or guanidino-arms by a Mitsunobu
alkylation to afford either 6a or 6b (Scheme 2). Matteucci
and co-workers4b employed a saturated solution of NH3 in
MeOH for the anchimerically assisted cyclization of the
nucleoside derivative, but extremely low yields were obtained
in our case using this reagent. Instead, a dilute solution of
10 equiv of KF in EtOH allowed us to isolate the desired
phenoxazine 5 in reasonably good yield (76%). With respect
to the Mitsunobu alkylation reaction, it was carried out using
either N-Z-ethanolamine or N,N′-bis-Z-N′′-(2-hydroxyethyl)-
(6) Flanagan, W. M.; Wagner, R. W.; Grant, D.; Lin, K.-Y.; Matteucci,
M. Nat. Biotechnol. 1999, 17, 48-52.
(7) Maier, M. A.; Leeds, J. M.; Ballow, G.; Springer, R. H.; Bharadwaj,
R.; Manoharan, M. Biochemistry 2002, 41, 1323-1327.
(8) Wilds, C. J.; Maier, M. A.; Tereshko V.; Manoharan, M.; Egli. M.
Angew. Chem., Int. Ed. 2002, 41, 115-117.
(9) (a) 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.
(b) Wilhelmsson, L. M.; Holme´n, A.; Lincoln, P.; Nielsen, P. E.; Norde´n,
B. J. Am. Chem. Soc. 2001, 123, 2434-2435.
(10) (a) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science
1991, 254, 1497-1500. (b) Egholm, M.; Buchardt, O.; Nielsen, P. E.; Berg,
R. H. J. Am. Chem. Soc. 1992, 114, 1895-1897. (c) Egholm, M.; Buchardt,
O.; Christensen, L.; Behrens, C.; Freier, S. M.; Driver, D. A.; Berg, R. H.;
Kim, S. K.; Norde´n, B.; Nielsen, P. E. Nature 1993, 365, 566-568. (d)
Uhlmann, E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int.
Ed. 1998, 37, 2796-2823.
(11) (a) Corey, D. R. Trends Biotechnol. 1997, 224-229. (b) Larsen,
H. J.; Bentin, T.; Nielsen, P. E. Biochim. Biophys. Acta 1999, 1489, 159-
166. (c) Ray, A.; Norde´n, B. FASEB J. 2000, 14, 1041-1060.
(12) Robles, J.; Grandas, A.; Pedroso, E. Tetrahedron 2001, 57, 179-
194.
4074
Org. Lett., Vol. 4, No. 23, 2002

Scheme 2a
Scheme 3a
a Reaction conditions: (v) KF/EtOH, reflux, 20 h; (vi) R1-OH,
DEAD, Ph2PC6H4-polymer/CH2Cl2, rt, 4 h.
a Reaction conditions: (vii) 4 N HCl (g)/dioxane, rt, 16 h; (viii)
FmocNHCH2CH2NHCH2CO2tBu, EDC/DMF, 0 °C to room tem-
perature; (ix) TFA/CH2Cl2, 0 °C, 30 min, and room temperature,
30-60 min.
guanidine. The former is commercially available and the
latter was prepared by reaction of N,N′-bis-Z-S-methyl-
isothiourea13 and ethanolamine following literature proce-
dures.14 The alkylation reaction was carried out using
polymer-bound triphenylphosphine15 because it allows an
easy separation of 6a and 6b from triphenylphosphine oxide.
In the synthesis of the amino-G-clamp nucleoside,4b these
last two steps were reversed, that is the alkylation was
followed by the cyclization. We have also performed the
transformation of 4 into 6 in this order but slightly lower
overall reaction yields were obtained (25% instead of 30%).
When Mitsunobu reaction was performed first with 4, it
furnished undesirable dialkylated products lowering the
overall yields. More importantly in our scheme we utilize a
common intermediate 5 for the synthesis of both the amino-
and the guanidino-G-clamp PNA monomers.
In summary, the protected amino-G-clamp and guanidino-
G-clamp PNA monomers 9a and 9b have been obtained from
5-bromouracil in 5% and 10% overall yields, respectively.
Work is in progress to incorporate these two cytosine
analogues into PNA oligomers in order to exploit their
remarkable capacity of recognition of guanine in the PNA
series, and to enlarge their field of applications. It is also
expected that the solubility of these modified PNAs will
benefit from the charged amino- and guanidino-arms and
improve with respect to that of the cytosine-containing
oligomers.
The remaining steps of the synthesis scheme are straight-
forward, but laborious (Scheme 3). Acid treatment of 6a,b
deprotected the carboxylic acid to give 7a,b. EDC-mediated
coupling of 7a,b to tert-butyl N-Fmoc-aminoethylglycinate16
afforded 8a,b. Finally, anhydrous acid deprotection of the
tert-butyl esters allowed the formation of the desired PNA
monomer synthons 9a and 9b.
Acknowledgment. We thank support by funds from the
Spanish Ministerio de Ciencia y Tecnolog´ıa (MCYT, grant
BQU2001-3693-C02-01) and the Generalitat de Catalunya
(GC, 2000SGR18 and 2001SGR49 and Centre de Refere`ncia
de Biotecnologia). C.A. and J.-A.O. are recipients of fel-
lowships from the MCYT and the GC, respectively. We are
thankful to Dr. Yogesh S. Sanghvi (Isis Pharmaceuticals)
for helpful discussions and supply of certain chemicals.
(13) Bansi Lal; Gangopadhyay, A. K.; Tetrahedron Lett. 1996, 37, 2483-
2486.
(14) Ramakrishna, N. V. S.; More, T. S.; Khandelwal, Y.; Naik, R. G.;
Bansi Lal; Gupte, R. D.; Vadlamudi, R. V. S. V. Indian J. Chem. 1999,
38B, 1331-1337.
Supporting Information Available: Procedures for the
preparation of compounds 2 to 9a,b and their relevant
spectroscopic (1H and 13C NMR, MS) data. This material is
available free of charge via the Internet at http://pubs.acs.org.
(15) Tunoori, A. R.; Dutta, D.; Georg, G. I. Tetrahedron Lett. 1998, 39,
8751-8754.
(16) Thomson S. A.; Josey, J. A.; Cadilla, R.; Gaul, M. D.; Hassman C.
F.; Luzzio, M. J.; Pipe, A. J.; Reed, K. L.; Ricca, D. J.; Wiethe, R. W.;
Noble, S. A. Tetrahedron 1995, 51, 6179-6194.
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