Improved nucleic acid triggered probe activation through the use of a 5-thiomethyluracil peptide nucleic acid building block

Article abstract of DOI:10.1021/ol0478382

(Chemical Equation Presented) To improve the efficiency of a nucleic acid triggered probe activation (NATPA) system a 5-thiomethyluracil peptide nucleic acid (PNA) building block has been synthesized. Attachment of imidazole and a coumarin ester to uracils at the ends of two PNAs resulted in a 550 000-fold acceleration of DNA-triggered coumarin release relative to imidazole and a 6-fold increase in k_cat relative to a system which had these groups attached to the amino and carboxy ends of PNAs.

Full text of DOI:10.1021/ol0478382

ORGANIC
LETTERS
2005
Vol. 7, No. 5
751-754
Improved Nucleic Acid Triggered Probe
Activation through the Use of a
5-Thiomethyluracil Peptide Nucleic Acid
Building Block
Jianfeng Cai, Xiaoxu Li, and John Stephen Taylor*
Department of Chemistry, Washington UniVersity, One Brookings DriVe,
St. Louis, Missouri 63130
taylor@wustl.edu
Received October 19, 2004 (Revised Manuscript Received January 12, 2005)
ABSTRACT
To improve the efficiency of a nucleic acid triggered probe activation (NATPA) system a 5-thiomethyluracil peptide nucleic acid (PNA) building
block has been synthesized. Attachment of imidazole and a coumarin ester to uracils at the ends of two PNAs resulted in a 550 000-fold
acceleration of DNA-triggered coumarin release relative to imidazole and a 6-fold increase in kcat relative to a system which had these groups
attached to the amino and carboxy ends of PNAs.
Recently, we described a new idea for the design of
chemotherapeutic agents and diagnostic probes that makes
use of a unique or overexpressed nucleic acid sequence
specific to the disease state to trigger the activation of a
cytotoxic drug or reporter molecule.1 In one recent formula-
tion of this idea, a disease-specific mRNA is used to template
the association of a PNA that is linked to a prodrug or probe
via an ester (the prodrug or probe component), with another
PNA bearing an imidazole group (the catalytic component),
which then catalyzes the release of the drug.2 Because of
the base lability of the ester, the prodrug or probe was linked
to an amino terminal cysteine on the PNA following
automated solid-phase synthesis via addition of the thiol
group to a maleimide substituent at neutral pH and ambient
temperature. The imidazole was introduced into the catalytic
component via a carboxy terminal histidine. While we were
able to show that both DNA and E. coli 5S rRNA could
trigger the release of hydroxycoumarin from a pair of
complementary PNAs, we found that the rate of coumarin
release was low, presumably due in part to the lack of
sufficient preorganization of the catalyst and the ester due
to attachment to the ends of the PNAs by long flexible linkers
(Figure 1). We reasoned that greater preorganization, and
(1) (a) Ma, Z.; Taylor, J.-S. Proc. Natl. Acad. Sci. U.S.A. 2000, 97,
11159-63. (b) Ma, Z.; Taylor, J. S. Bioorg. Med. Chem. 2001, 9, 2501-
2510.
Figure 1. Previous PNA-based NATPA system.
(2) Ma, Z.; Taylor, J. S. Bioconjugate Chem. 2003, 14, 679-683.
10.1021/ol0478382 CCC: $30.25
© 2005 American Chemical Society
Published on Web 02/02/2005

therefore a greater rate of release, could be achieved by
linking the catalyst and ester to the bases via short tethers
and that 5-thiomethyluracil would be an ideal choice for
coupling to a maleimido ester. Herein we describe an eight-
step synthesis of a 5-thiomethyluracil PNA building block
6 (Scheme 1) for Fmoc synthesis from commercially
modification of nucleic acids and analogues with sensitive
functional groups, such as aryl esters, is best accomplished
following automated synthesis and deprotection, with chemose-
lective reactions that can proceed under near neutral condi-
tions at ambient temperature. Thiol groups have been
particularly useful in this regard and have been used to link
oligonucleotides to such molecules as biotin and fluorescent
probes7 as well as to peptides, proteins, and gold particles.8
Scheme 1. Synthesis of the PNA Building Block
C5-(Thioalkyl)uridines are particularly useful for both
internal and terminal modifications of DNA. Substituents at
the C5 position of pyrimidine nucleosides do not interfere
with Watson-Crick base pairing, and project out into the
major groove of duplex DNA thus allowing for more precise
conformational control compared to substituents linked to
the ends of DNA. Another advantage of linking to the bases
is that a modification can be introduced anywhere in a
sequence since the number and position of thiol-bearing
building blocks can be controlled by oligonucleotide syn-
thesis. While the synthesis of a series of 5-(thioalkyl)uridines
and 5-(thioalkyl)-2′-deoxyuridines has been described,9 the
preparation of 5-(thioalkyl)uracil PNA building blocks has
not yet been reported. A p-methoxybenzyl-protected 5-thio-
propynyl PNA monomer has been reported, but its incor-
poration into PNA has not.10 To reduce the conformational
degrees of freedom of the probe and catalyst in the nucleic
acid triggered probe activating system, we decided to
synthesize the 5-thiomethyluracil PNA building block 6
(Scheme 1).
Thiomethyluracil 1 was obtained in three steps from
hydroxymethyluracil according to a known procedure.11
Treatment of 1 with di-tert-butyl-1-(tert-butylthio)-1,2-
hydrazine dicarboxylate12 in DMF at room temperature for
12 h gave 5-(tert-butyldithio)methyluracil 2 in 91% yield
and in 51% overall yield from 5-hydroxymethyluracil.
Compound 2 has also been synthesized in 58% overall yield
in three steps from 5′-hydroxymethyluracil via the in situ
preparation of 5-thiomethyluracil.9b Compound 2 was alky-
lated with ethyl bromoacetate in the presence of potassium
carbonate in DMF to give ester 3 in 93% yield. Ester 3 was
hydrolyzed with 4% NaOH in methanol followed by
acidification to give acid 4 in 98% yield. EDC-mediated
available 5-hydroxymethyluracil and show how it can be used
to synthesize both the catalytic and probe components of a
DNA-triggered probe-releasing system. We also show that
this C5-linked system is more efficient in releasing hydroxy
coumarin than our previous system.
We previously chose peptide nucleic acid (PNA) to
recognize the triggering mRNA sequence because of a
number of features that make it ideal for in vivo use. PNA
is a oligonucleotide analogue in which the nucleobases are
linked to an N-(2-aminoethyl)glycine backbone instead of a
sugar-phosphate backbone.3 PNA has higher affinity for
complementary RNA sequences than natural ODNs and can
invade duplex regions, presumably because of its lack of
charge.4 PNA also has much higher biological stability than
ODNs in serum and in cells.5 For these reasons, PNA has
shown great promise for the design of nucleic acid-targeted
therapeutic agents, probes, and tools, either by itself or when
modified by other molecules, some of which are chemically
sensitive.3,6
(6) (a) Hyrup, B.; Nielsen, P. E. Bioorg. Med. Chem. 1996, 4, 5-23.
(b) Nielsen, P. E. Curr. Opin. Struct. Biol. 1999, 9, 353-357. (c) Pooga,
M.; Land, T.; Bartfai, T.; Langel, U. Biomol. Eng. 2001, 17, 183-192. (d)
de Koning, M. C.; van der Marel, G. A.; Overhand, M. Curr. Opin. Chem.
Biol. 2003, 7, 734-740. (e) Nielsen, P. E. Mol. Biotechnol. 2004, 26, 233-
248.
(7) (a) Connolly, B. A.; Rider, P. Nucleic Acids Res. 1985, 13, 4485-
502. (b) Beaucage, S. L.; Iyer, R. P. Tetrahedron 1993, 49, 1925-1963.
(8) (a) Eritja, R.; Pons, A.; Escarceller, M.; Giralt, E.; Albericio, F.
Tetrahedron 1991, 47, 4113-4120. (b) Zuckermann, R. N.; Schultz, P. G.
J. Am. Chem. Soc. 1988, 110, 6592-6594. (c) Mirkin, C. A.; Letsinger, R.
L.; Mucic, R. C.; Storhoff, J. J. Nature 1996, 382, 607-609.
(9) (a) Goodwin, J. T.; Glick, G. D. Tetrahedron Lett. 1993, 34, 5549-
5552. (b) Sun, S.; Tang, X.-Q.; Merchant, A.; Anjaneyulu, P. S. R.; Piccirilli,
J. A. J. Org. Chem. 1996, 61, 5708-5709.
Because of the basic and/or acidic conditions used in
various steps in solid-phase synthesis and final deprotection,
(3) Nielsen, P. E.; Egholm, M.; Berg, R. H.; Buchardt, O. Science 1991,
254, 1497-1500.
(4) (a) Egholm, M.; Buchardt, 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. (b) Peffer, N. J.; Hanvey, J. C.; Bisi, J. E.;
Thomson, S. A.; Hassman, C. F.; Noble, S. A.; Babiss, L. E. Proc. Natl.
Acad. Sci. U.S.A. 1993, 90, 10648-10652.
(10) Hudson, R. H. E.; Li, G.; Tse, J. Tetrahedron Lett. 2002, 43, 1381-
1386.
(11) Giner-Sorolla, A.; Medrek, L. J. Med. Chem. 1966, 9, 97-101.
(12) Wunsch, E.; Moroder, L.; Romani, S. Hoppe-Seyler’s Z. Physiol.
Chem. 1982, 363, 1461-1464.
(5) Uhlmann, E.; Peyman, A. Chem. ReV. 1990, 90, 543-584.
752
Org. Lett., Vol. 7, No. 5, 2005

coupling of 4 to tert-butyl-N-Fmoc-aminoethylglycinate13
afforded 5 in 84% yield. Finally, deprotection of the tert-
butyl ester with TFA/DCM gave the desired PNA monomer
6 in 90% yield and in 35% overall yield from 5-hydroxy-
methyluracil.
DIPEA in DMF for 1 day. Final deprotection and cleavage
from the resin, followed by reversed-phase HPLC purifica-
tion, provided PNA 11.
To determine the efficiency of fluorescent probe release,
4 µM PNA 9 was incubated with and without 4 µM PNA
11 and/or the fully complementary template 12a in pH )
7.0 sodium phosphate buffer (Scheme 4). Efficient coumarin
For the synthesis of the probe component 9 (Scheme 2),
monomer 6 was used to introduce 5-(tert-butyldithio)-
Scheme 4. Kinetic Scheme for DNA-Triggered Coumarin
Scheme 2. Synthesis of the Probe Component
Release
methyluracil into the carboxy terminus of the 9-mer PNA 7
by standard solid-phase Fmoc synthesis on an ABI 8909
PNA synthesis machine. Following synthesis, deprotection,
and purification, the disulfide bond of the PNA was reduced
with excess TCEP to give 8 and then incubated with excess
N-maleoyl-D-valine coumarin ester1 to give the PNA probe
component 9 in 59% yield.
release only occurred when the catalytic and probe compo-
nents were in the presence of the complementary DNA
(Figure 2). The system was also found to be sensitive to
mismatches, dropping 23- and 30-fold in rate with a
For the synthesis of the catalytic component 11 (Scheme
3), monomer 6 was used to introduce 5-(tert-butyldithio)-
Scheme 3. Synthesis of the Catalytic Component
methyluracil into the amino terminus of the 9-mer PNA 10.
The disulfide bond was reduced with TCEP on the column
and then treated with 4-(2′-chloroethyl)imidazole14 in 10%
Figure 2. Kinetics of coumarin release with matched and
mismatched DNA triggers. The prodrug component 9 (4 µM) was
incubated in the presence or absence of the catalytic component
11 (4 µM), fully complementary DNA 12a or mismatched DNAs
12b and 12c in 10 mM phosphate pH 7 buffer. Coumarin release
was assayed by fluorescence (Ex ) 350 nm, Em ) 452 nm).
(13) 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.
(14) Zoeteman, M.; Bouwman, E.; de Graaff, R. A. G.; Driessen, W.
L.; Reedijk, J.; Zanello, P. Inorg. Chem. 1990, 29, 3487-3492.
Org. Lett., Vol. 7, No. 5, 2005
753

mismatch in either the catalytic (12b) or probe (12c) binding
sites of the triggering DNA.
As in previous studies,1,2 the initial rate of DNA triggered
coumarin release exhibited saturation kinetics (Figure 3)
kinetics,15 appearing to be fast and substrate concentration
dependent during the first turnover (pre-steady-state regime),
but slow and concentration independent during the second
turnover (steady-state regime). This type of kinetics is
indicative of a rate-limiting step following coumarin release.
Indeed, the rate of coumarin release in the presence of
saturating substrate (8 µM 9) could be fit to the burst equation
catt
[coumarin] ) [11‚12a]o((1 - e-k ) + k2t) from which the
steady-state rate constant (k2) was determined to be (6.46 (
0.02) × 10-5 s-1 (see the Supporting Information). The value
of k2 is remarkably similar to that of 6.2 × 10-5 s-1 calculated
for the dissociation rate constant (kd) of an 8-mer PNA‚DNA
duplex from published data.16 This similarity suggests that
dissociation of the hydrolyzed 9-mer PNA product 13 from
the catalytic complex 11‚12a (kd-13, Scheme 4) is limiting
the turnover rate of the reaction. If so, it should be possible
to increase the turnover rate by decreasing the length of PNA
9 and hence the dissociation rate of its hydrolysis product,
PNA 13. Future studies will be aimed at increasing the
overall catalytic efficiency of both DNA- and RNA-triggered
coumarin release by optimizing the length of the substrate
PNA, and the lengths of the linkers to the ester and imidazole
subunits.
In conclusion, we have developed an efficient route for
the synthesis of a 5-thiomethyluracil PNA monomer for
solid-phase Fmoc synthesis that allows for postsynthetic
modification of PNA in the major groove. Most importantly,
we have shown that attachment of imidazole and a coumarin
ester to the C5 of uracil substantially increases the efficiency
of DNA-triggered prodrug activation over those systems in
which these groups are attached to the chain termini,2 thereby
paving the way to better RNA-triggered systems.
Figure 3. Kinetics of coumarin release as a function of probe
component concentration. The catalytic component 11 (2 µM) and
DNA trigger 12a (1 µM) were incubated with probe component 9
in 10 mM phosphate pH 7 buffer. Coumarin release was assayed
by fluorescence (Ex ) 350 nm, Em ) 452 nm).
indicative of reversible binding of substrate 9 to the DNA
trigger sequence (Scheme 4). A plot of the initial rates as a
function of the concentration of 9 could be fit to a Michaelis-
Menten equation with a kcat of 5.0 ( 0.2 × 10-4 s-1 and a
Km of 0.8 ( 0.1 µM (see the Supporting Information). The
kcat value is 6 times that of 8.4 × 10-5 s-1 for the DNA-
triggered PNA-based system in which the ester and imidazole
were tethered to the amino and carboxy ends of the PNA
(Figure 1)2 and 11 times that of 4.4 × 10-5 s-1 observed for
coumarin release from a DNA-triggered DNA-based system.1b
Based on (kcat/Km)/kIm, the C5-linked DNA-triggered probe
activating system shows the greatest acceleration of coumarin
release (550 000-fold based on kIm ) 1.13 × 10-3 M-1 s-1
for imidazole-catalyzed hydrolysis of N-malemido-D-valine
coumarin ester).1b
Acknowledgment. This work was supported by the NIH
(RO1-CA92477) with the assistance of the Washington
University Mass Spectrometry Resource (Grant No.
P41RR0954). We also thank Xuan Yue for obtaining the
MALDI spectra.
Supporting Information Available: Abbreviations used,
experimental procedures and analytical data for compounds
2-9 and 11, and analyses of the kinetic data. This material
is available free of charge via the Internet at http://pubs.acs.org.
OL0478382
The reaction also exhibited catalysis, as evidenced by the
release of more moles of coumarin than the number of moles
of the catalytic complex 11‚12a in the presence of excess
substrate 9 (Figure 2). The reaction showed biphasic or burst
(15) Bender, M. L.; Kezdy, F. J.; Wedler, F. C., Jr. J. Chem. Educ. 1967,
44, 84-88.
(16) Kushon, S. A.; Jordan, J. P.; Seifert, J. L.; Nielsen, H.; Nielsen, P.
E.; Armitage, B. A. J. Am. Chem. Soc. 2001, 123, 10805-10813.
754
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