3′-Immobilized probes with 2′-caps: Synthesis of oligonucleotides with 2′-N-methyl-2′-(anthraquinone carboxamido)uridine residues

Article abstract of DOI:10.1021/ol0502432

(Chemical Equation Presented) A synthesis for oligodeoxynucleotides with a 3′-terminal 2′-N-methyl-2′-acylamido-2′-deoxyuridine residue was developed. Unlike their unmethylated counterparts, these oligodeoxynucleotides can be stably immobilized on aldehyd

Full text of DOI:10.1021/ol0502432

ORGANIC
LETTERS
2005
Vol. 7, No. 8
1569-1572
3
′
-Immobilized Probes with 2
Synthesis of Oligonucleotides with
-N-Methyl-2 -(anthraquinone
′-Caps:
2′
′
carboxamido)uridine Residues
Samy Al-Rawi, Carolin Ahlborn, and Clemens Richert*
Institute for Organic Chemistry, UniVersita¨t Karlsruhe (TH),
D-76131 Karlsruhe, Germany
cr@rrg.uka.de
Received February 4, 2005
ABSTRACT
A synthesis for oligodeoxynucleotides with a 3
′
-terminal 2
′
-N-methyl-2
′
-acylamido-2
′
-deoxyuridine residue was developed. Unlike their
unmethylated counterparts, these oligodeoxynucleotides can be stably immobilized on aldehyde-displaying glass surfaces to provide DNA
microarrays. An anthraquinone carboxamido group as a 2 -substituent doubled the capture efficiency of an immobilized tetradecamer.
′
Double helices between complementary DNA strands1 are
exquisitely predictable structures that follow base pairing
rules and play pivotal roles in practical applications.2,3 The
extended, linear structure of duplexes, which differs markedly
from the globular structure of most soluble proteins, causes
different levels of base pairing fidelity in different regions
of the duplex, however. While mismatches in the interior of
duplexes, where neighboring base pairs provide tight con-
straints, significantly lower duplex stability, mismatches at
the termini, where few neighbors are affected and fraying
occurs frequently, have minimal effects. The smallest effect
of mismatches on duplex stability is found for the terminal
base pair, particularly when one of the nucleobases is adenine
or thymine/uracil. Here, mismatches can cause decreases in
free energy of duplex formation of less than 1 kcal/mol.4
Nature, in certain instances, has chosen to accept the pro-
miscuous binding at the termini of duplexes, e.g., by develop-
ing a degenerate code that compensates for mismatches at
the termini of codon:anticodon duplexes.5 Biomedical appli-
cations call for high fidelity, however, to avoid false posi-
tives. Lengthening the probes to make the terminal base pairs
less significant is not promising, as the penalty for single
mismatches will drop, the likelihood of partial complemen-
tarity with other targets will increase, and the kinetics of
duplex formation will be slowed. Instead, one might chemi-
cally modify the probes to increase pairing fidelity.
One known type of modification that increases base pairing
fidelity employs short stretches of minor groove binders
covalently linked to PCR primers.6 These do not significantly
affect the termini, however. To achieve better discrimination
at the termini, nonnucleosidic appendages that bridge the
terminus and create a chemical environment resembling the
interior of duplexes could be employed. Examples of such
bridging appendages or “molecular caps” that bind to
* To whom correspondence should be addressed.
(1) Watson, J. D.; Crick, F. H. Nature 1953, 171, 737-738.
(2) (a) Sanger, F.; Nicklen, S.; Coulson, A. R. Proc. Natl. Acad. Sci.
U.S.A. 1977, 74, 5463-5467. (b) Mullis, K. B.; Faloona, F. A. Methods
Enzymol. 1987, 155, 335-350.
(4) Narayanan, S.; Gall, J.; Richert, C. Nucleic Acids Res. 2004, 32,
2901-2911.
(5) Crick, F. H. C. J. Mol. Biol. 1966, 19, 548-555.
(3) Yan, N. Science 2004, 306, 2048-2049.
10.1021/ol0502432 CCC: $30.25
© 2005 American Chemical Society
Published on Web 03/24/2005

Scheme 1
duplexes with correct canonical base pairs exist.4,7,8 Besides
increasing base pairing fidelity, the molecular caps allow a
tuning of duplex stability.
nucleotide probes on DNA microarrays. This residue carries
both a cap at its 2′-position and a phosphoramidite group
reactive toward a linker for immobilization on derivatized
glass surfaces at its 3′-position. The cap employed is an
anthraquinone. Anthraquinones have recently been shown
to be excellent caps for termini when attached as 2′-acyl-
amido groups of 3′-terminal deoxyuridine residues.11 They
give melting point increases of up to 14 °C per residue and
increase base pairing fidelity at the terminus. Other anthra-
quinone derivatives covalently linked to oligonucleotides are
being explored for their charge transport capabilities.12 Dif-
ferent modes of attaching anthraquinone residues to DNA13
or PNA14 exist. Unconjugated anthraquinones can act as
photonucleases,15 and certain anthraquinone derivatives bind
G quadruplex DNA,16 making them a particularly versatile
class of nucleic acid ligands.
Probes on DNA microarrays9 are not commonly employed
with caps, however. This is unfortunate, as the massive
multiplexing of DNA microarray experiments, where up to
2.5 × 105 hybridization equilibria compete with each other,
calls for exquisite selectivity in duplex formation. Recent
studies have led to caps that increase capture efficiency for
hybridization probes 3′-immobilized on glass surfaces.7c,10
The caps tested to date were all attached to the 5′-position
of the probes. For the 3′-termini, similar caps have been
lacking, as these require nucleosides providing both a link
to the surface and a molecular cap. The example of RNA
shows how much a simple 2′-substituent in oligonucleotides
can complicate oligomer syntheses.
Our attempts to generate 2′-capped hybridization probes
for immobilization on microarrays started with the synthesis
of 1 (Scheme 1). This nucleoside features the anthraquinone
carboxamido cap and a free 3′-hydroxyl group for phosphi-
Here we present a synthesis of a deoxyuridine derivative
that functions as a cap-bearing 3′-terminal residue of oligo-
(6) (a) Kutyavin, I. V.; Afonina, I. A.; Mills, A.; Gorn, V. V.; Lukhtanov,
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1570
Org. Lett., Vol. 7, No. 8, 2005

Scheme 2
tylation and subsequent coupling to linker-bearing support
employed for an N-ethyl nucleoside, formed by reacting the
3′-hydroxyl group with alkyl isocyanates.21 For our substrate,
we wished to avoid both methylation on the nucleoside level
and the use of highly toxic methyl isocyanate. Our first
attempt to prepare 6 therefore involved N-methyl carbamic
acid NHS ester (7) as reagent, but no conversion of 5 was
observed under several conditions. When the more reactive
bis(p-nitrophenyl) carbonate22 or its monochloro derivate23
were used, a carbonate was successfully generated. The in
situ reaction with methylamine gave 6 in 87% overall yield.
To induce isomerization via intramolecular ring opening,
carbamate 6 was treated with NaH, which led to the
formation of oxazolidinone 8 in 88% isolated yield. Com-
pound 8 was hydrolyzed to N-methylated amine 9 using
conditions similar to those employed for other nucleosides.24
2.10 All attempts to convert 1 to phosphoramidite 3 in high
yield and to generate 3′-capped oligonucleotides for covalent
immobilization failed. Probably the amido functionality at
the 2′-position, particularly in the presence of base, was
reactive toward the neighboring 3′-phosphoramidite or phos-
photriester. An attack of the oxygen of the 2′-amido group
on the phosphorus can occur via a six-membered ring, and
an attack of the (deprotonated) amido nitrogen can occur
via a five-membered ring. Other base-induced nucleophilic
attacks of amido groups on 3′-esters of nucleosides are
known.17
To eliminate the high nucleophilicity of the amido groups
in the presence of bases, we decided to prepare 2′-N-methyl
derivative 4 (Scheme 1). The synthesis of 4 started with the
conversion of uridine to 5′-DMT-protected anhydro derivate
5 (Scheme 1) in 51% yield, following literature protocols.18
Two routes were considered for introducing the methylamino
functionality at the 2′-position. One would involve opening
of 5 with an azide,19 reduction to a 2′-amine, and N-
methylation, possibly under Eschweiler-Clarke conditions,
as described for an aminodeoxyadenosine derivative.20 The
other involves an intramolecular attack of the nitrogen of a
3′-attached urethane moiety. The latter route has been
Several methods were tested for coupling of anthraquinone
carboxylic acid to the secondary amine of 9. Activation of
(19) (a) Verheyden, J. P. H.; Wagner, D.; Moffatt, J. G. J. Org. Chem.
1971, 36, 250-54. (b) McGee, D. P. C.; Sebesta, D. P.; O’Rourke, S. S.;
Martinez, R. L.; Jung, M. E.; Pieken, W. Tetrahedron Lett. 1996, 37, 1995-
1998.
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Y.; Williams, L. D.; Bottomley, L. A.; Schuster, G. B. J. Am. Chem. Soc.
1997, 119, 5043-5044.
(22) (a) Stirchak, E. P.; Summerton, J. E.; Weller, D. D. J. Org. Chem.
1987, 52, 4202-4206. (b) Aguilera, B.; Wolf, L. B.; Nieczypor, P.; Rutjes,
F. P.; Overkleeft, H. S.; van Hest, J. C.; Schoemaker, H. E.; Wang, B.;
Mol, J. C.; Fu¨rstner, A.; Overhand, M.; van der Marel, G. A.; van Boom,
J. H. J. Org. Chem. 2001, 66, 3584-3589. (c) Ruttens, B.; Van der Eycken,
J. Tetrahedron Lett. 2002, 43, 2215-2221.
(16) Sun, D. Y.; Thompson, B.; Cathers, B. E.; Salazar, M.; Kerwin, S.
M.; Trent, J. O.; Jenkins, T. C.; Neidle, S.; Hurley, L. H. J. Med. Chem.
1997, 40, 2113-2116.
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17, 1177-1182.
(23) Schmalhofer, W. A.; Bao, J.; McManus, O. B.; Green, B.;
Matyskiela, M.; Wunderler, D.; Bugianesi, R. M.; Felix, J. P.; Hanner, M.;
Linde-Arias, A.-R.; Ponte, C. G.; Velasco, L.; Koo, G.; Staruch, M. J.;
Miao, S.; Parsons, W. H.; Rupprecht, K.; Slaughter, R. S.; Kaczorowski,
G. J.; Garcia, M. L. Biochemistry 2002, 41, 7781-7794.
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Chem. 1996, 61, 781-785. (b) Ho¨bartner, C.; Micura, R. J. Am. Chem.
Soc. 2004, 126, 1141-1149.
Org. Lett., Vol. 7, No. 8, 2005
1571

the acid to an acid chloride with oxalyldichloride and addition
to the aminonucleoside gave higher yields than preactivation
with “uronium salt” agents such as HBTU.25 With the former
method, amide 4 was obtained in 73% yield. Despite the
steric bulk of the 2′-substituent, 4 could be converted to
phosphoramidite 10 using 2-cyanoethyl-N,N-diisopropyl-
chlorophosphoramidite in 58% yield after chromatography.
These results show that an oligonucleotide probe enforced
with 3′-terminal anthraquinone carboxamido cap can be
successfully prepared and employed in microarray-based
hybridization experiments. Since the anthraquinone cap gives
the strongest melting point increase among all caps tested
in our work, with a ∆Tm of up to 14 °C per residue,11 it is
not surprising that the increase in signal for spots featuring
13 over those with unmodified control (14) is so substantial
for a tetradecamer. It was previously shown that the
2′-anthraquinone carboxamido cap does not require blunt
With key building block 10 in hand, 3′-capped oligonucle-
otides were prepared using standard phosphoramidite chem-
istry. A tetradecamer sequence containing all four nucleotides
that has been well studied in the context of microarrays7c,10
was prepared starting from lysine-bearing, controlled pore
glass 1126 (Scheme 2). One-step HPLC purification of 13
gave a sample suitable for immobilization on aldehyde-bear-
ing glass slides via reductive amination.10 Unmodified control
strand 5′-TGGTTGACTGCGAT-DP-Lys-3′ (14), where DP
denotes the dimethylhydroxypropionic acid linker,26 was
spotted alongside 13, and the remaining free surface sites
were reacted with a triethyleneglycol amine, following a
protocol reported earlier.7c Incubation with target strand 5′-
Cy3-ATCGCAGTCAACCA-3′ (15), washing with buffer
solutions (1 × SSC/0.2% SDS and 1 × SSC), and scanning
with a microarray reader gave fluorescence images such as
the one shown in Figure 1. It can be discerned that the cap
increases the affinity for the target strand, as evidenced by
the high fluorescence signal from the spots displaying 3′-cap-
ped 14. This effect was observed reproducibly and over a
range of hybridization temperatures (Table SI, Supporting
Information).
Both modified probe 13 and control strand 14 feature the
same 3′-substituent for immobilization. The amino group for
immobilization is 15 atoms away from the position at which
the two oligonucleotides differ in structure. Further, modified
probe 13 is slightly more sterically hindered. The increase
in fluorescence signal was observed in four independent
experiments, making it unlikely that local changes in surface
coating are responsible for the effect observed. Taken
together, this leads us to believe that 13 does indeed capture
its target strand more efficiently than 14.
An independent hybridization experiment, performed on-
chip under the same experimental conditions, employed 3′-
labeled target strand 5′-ATCGCAGTCAACCA-Cy3-3′ (16).
This strand was prepared using 5′-phosphoramidites so that
the fluorophore could be incorporated in the last step of the
solid-phase synthesis. When a slide displaying 13 and 14
on its surface was incubated with 16 at 70 °C, followed by
washing, and scanning, a fluorescence image was obtained
that closely resembles the one shown in Figure 1 (see Figure
S7, Supporting Information). Again, 13 captured the target
significantly more strongly, while unmodified 14 gave only
41% as much signal. The slightly greater capture efficiency
in this case might be due to the absence of low-level
fluorescence quenching that could occur when Cy3 label and
anthraquinone are at the same terminus of the duplex.
Figure 1. Fluorescence of spots of a DNA microarray after
incubation at 70 °C with fluorescent complementary strand Cy3-
ATCGCAGTCAACCA (15) and washing (a) Fluorescence image,
with spots featuring control strand 14 (left) and 3′-capped strand
13 (right). Spotting was in duplicate to demonstrate reproducibility.
(b) Integration of fluorescence over the area vertically above.
ends to exert its duplex-stabilizing effect;4 therefore, captur-
ing longer target strands with overhangs at either terminus
can be expected to occur with equally high efficiency. The
2′-appended cap may now be combined with 5′-caps7 and
possibly other fidelity- and affinity-enhancing27 modifica-
tions. Thus, some of the weaknesses of DNA microarrays
such as the low target affinity of A/T-rich sequences, low
base pairing fidelity at the termini, lability toward exonu-
clease attack, and difficult-to-obtain electric signals for
binding may be alleviated. The synthetic route described here
is short, as it avoids protecting group changes, and may
provide a blueprint for the synthesis of other 2′-modified
oligonucleotides to be employed in functional nucleic acid
constructs immobilized via a 3′-phosphodiester.
Acknowledgment. This work was supported by DFG
grants RI 1063/4-3 and FOR 434.
Supporting Information Available: Experimental pro-
cedures, Table SI, and NMR or MALDI spectra. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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OL0502432
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