Synthesis of a 1′-aminomethylthymidine and oligodeoxyribonucleotides with 1′-acylamidomethylthymidine residues

Article abstract of DOI:10.1021/jo049062o

Reported here is a 10-step synthesis of a phosphoramidite building block of 1′-aminomethylthymidine that starts from 2-deoxyribose. The framework of the branched aminonucleoside was elaborated from a known 1-cyano-1-bromo glycosyl donor, whose reaction with the silylated nucleobase furnished the 1′-cyanide, which was reduced to the desired aminomethylnucleoside. The N-allyloxycarbonyl (Alloc)-protected nucleoside was converted to a phosphoramidite building block and incorporated into the oligonucleotides 5′-GCAT*TATTAC-3′, and 5′-GCAT*TAT*TAC- 3′, where T* denotes 1′-acylamidomethylthymidine residues. Removal of the Alloc protecting group and acylation with the residue of pyrene-1-yl-butanoic acid were achieved on support, using microwave irradiation to ensure full conversion. The UV-melting point of the duplex of the singly and doubly modified decamers with their fully complementary target sequence is 0.1-6.9 °C higher than that of the unmodified control duplex, depending on the salt concentration. This suggests that the aminomethyl linker may allow for the placing of a functional "payload" in the minor groove of DNA duplexes without disrupting the helix. Oligonucleotides thus endowed with functional modifications may become useful for biomedical applications.

Full text of DOI:10.1021/jo049062o

Syn th esis of a 1′-Am in om eth ylth ym id in e a n d
Oligod eoxyr ibon u cleotid es w ith 1′-Acyla m id om eth ylth ym id in e
Resid u es
Peter Gru¨nefeld and Clemens Richert*
Institute for Organic Chemistry, University of Karlsruhe (TH), D-76131 Karlsruhe, Germany
cr@rrg.uka.de
Received J une 4, 2004
Reported here is a 10-step synthesis of a phosphoramidite building block of 1′-aminomethylthymidine
that starts from 2-deoxyribose. The framework of the branched aminonucleoside was elaborated
from a known 1-cyano-1-bromo glycosyl donor, whose reaction with the silylated nucleobase
furnished the 1′-cyanide, which was reduced to the desired aminomethylnucleoside. The N-
allyloxycarbonyl (Alloc)-protected nucleoside was converted to a phosphoramidite building block
and incorporated into the oligonucleotides 5′-GCAT*TATTAC-3′, and 5′-GCAT*TAT*TAC-3′, where
T* denotes 1′-acylamidomethylthymidine residues. Removal of the Alloc protecting group and
acylation with the residue of pyrene-1-yl-butanoic acid were achieved on support, using microwave
irradiation to ensure full conversion. The UV-melting point of the duplex of the singly and doubly
modified decamers with their fully complementary target sequence is 0.1-6.9 °C higher than that
of the unmodified control duplex, depending on the salt concentration. This suggests that the
aminomethyl linker may allow for the placing of a functional “payload” in the minor groove of
DNA duplexes without disrupting the helix. Oligonucleotides thus endowed with functional
modifications may become useful for biomedical applications.
In tr od u ction
Labeling oligonucleotides with dyes or radioactive
elements facilitates their detection.10 Most frequently, the
dyes are attached to the termini of the oligonucleotides,
using commercially available phosphoramidites of fluo-
rophores featuring flexible alkyl linkers. Doubly dye-
labeled oligonucleotides can be used as molecular bea-
cons.11 When repeated labeling is required or the termini
have to remain unmodified, labeling has to occur in the
interior of strands. In order not to interfere with the
function of the oligonucleotides, most notably their ability
to form Watson-Crick duplexes, the newly added moi-
eties have to point away from the base pairing surface
of the nucleobases. This can be achieved by labeling
phosphorothioate backbones,12 thus placing the label in
the solvent. Alternatively, one may introduce substitu-
ents at the 5-position of pyrimidines and the 7- or
8-position of purine residues.13 This places the substit-
uents such that they point into the major groove of duplex
DNA. If oligonucleotides were available that place sub-
stituents in both the major and the minor groove without
disrupting duplexes, improved molecular recognition of
Oligodeoxynucleotides can be used as hybridization
probes and biomedical agents. In their unmodified form,
they can be employed in site-directed mutagenesis,1 as
primers for polymerase chain reactions,2 and as immuno-
stimulatory agents,3 to name just three applications. Also
numerous are the applications for modified oligonucleo-
tides. For example, oligonucleotides with chemical modi-
fications are routinely immobilized on surfaces to gener-
ate DNA microarrays (DNA chips).4,5 A chemical modi-
fication can also increase the selectivity of oligonucleotide
probes that bind a target sequence against a background
of other sequences.6 Further, chemical modifications have
been developed that render oligonucleotides more re-
sistant toward nuclease attack,7 increase their affinity
for target strands,8 or both.9
* To whom correspondence should be addressed. Tel: int. -49 (0)
721 608 2091. FAX: int.-49 (0) 721 608 4825.
(1) Zoller, M. J .; Smith, M. Nucleic Acids Res. 1982, 10, 6487-6500.
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Chem., Int. Ed. Engl. 1994, 33, 1209-1213.
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Olsen, C. E.; Wengel, J . J . Am. Chem. Soc. 1998, 120, 5458-5463.
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3747.
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Chem., Int. Ed. 2002, 41, 1277-1289.
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Acids Res. 2001, 29, 5090-5098. (b) Dombi, K. L.; Griesang, N.;
Richert, C. Synthesis 2002, 816-824 and references therein.
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Lukhtanov, E. A.; Belousov, E. S.; Singer, M. J . Walburger, D. K.;
Lokhov, S. G.; Gall, A. A.; Dempcy, R.; Reed, M. W.; Meyer, R. B.;
Hedgpeth, J . Nucleic Acids Res. 2000, 28, 655-661. (b) Dogan, Z.;
Paulini, R.; Rojas Stu¨tz, J . A.; Narayanan, S.; Richert, C. J . Am. Chem.
Soc. 2004, 126, 4762-4763.
(10) (a) Oligonucleotides and Analogues, A Practical Approach;
Eckstein, F., Ed.; IRL Press: Oxford, 1991. (b) Hermanson, G. T.
Bioconjugate Techniques; Academic Press: San Diego, 1995.
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2001, 1480-1481. (c) Fang, X.; Liu, X.; Schuster, S.; Tan, W. J . Am.
Chem. Soc. 1999, 121, 2921-2922.
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10.1021/jo049062o CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/05/2004
J . Org. Chem. 2004, 69, 7543-7551
7543

Gru¨nefeld and Richert
F IGURE 1. Structure of 1′-acetamidomethylthymidine-containing DNA. (a) Two-dimensional drawing of a base pair of the modified
thymidine residue with a deoxyadenosine residue. (b) Structure of the DNA duplex 5′-GCAT*TATTAC-3′:5′-GTAATAATGC-3′,
where T* denotes the 1′-acetamidomethylthymidine residue, as obtained through force field minimization using the program
Macromodel. The nucleotides of the modified base pair are highlighted in black.
target strands could be envisioned, as well as improved
molecular beacons or affinity labels for proteins that bind
in either groove. Fluorophore-labeling at the 2′-position
of oligonucleotides or analogues thereof has been re-
ported,14 though this point of attachment may change the
conformation of duplexes toward the A-type conformation
commonly found for RNA. Molecular modifications that
place chemical appendages in the minor groove of DNA
are less well-known. Minor groove binders have been
appended through linkers originating in the termini of
oligonucleotides15,16 but not the interior of strands.
Inspection of the structure of B-form DNA duplexes
indicates that a substituent replacing the hydrogen at
the 1′-position of nucleotides should point into the minor
groove. Duplexes with such a substituent, e.g., in the
form of an acylamidomethyl group attached to a thymi-
dine residue in the interior of one strand, do not show
major distortions when subjected to force field mini-
mization (Figure 1). This led us to pursue the synthesis
of oligonucleotides that contain 1′-acylamidomethyl-
thymidines. Thymidine as the nucleoside to be modified
was chosen, because thymidine forms weaker base pairs
with deoxyadenosine than deoxycytidines pairing with
deoxyguanosines. Eventually, the weaker base pair may
be stabilized through duplex-bridging interactions pro-
vided by a substituent introduced at the 1′-position. This
might lead to isostable DNA,17 i.e., DNA where the
affinity for complementary strands does not depend on
the sequence and where universally stringent hybridiza-
tion conditions can be developed, even when complex
mixtures of probes and target strands are present.
Further, the thymidine residue has the advantage of
lending itself more readily to synthetic transformations,
as the nucleobase does not require a protecting group
when generating building blocks for automatic DNA
synthesis.
Here we report the synthesis of a 1′-aminomethyl-
thymidine, together with that of oligonucleotides contain-
ing this nucleoside, prepared via the 3′-phosphoramidite
of the aminomethylnucleoside. The synthesis of the
oligonucleotides included the conversion of residues in
the interior of protected oligonucleotides on solid supports
in microwave-assisted conversions.18
Resu lts
As mentioned above, 1′-aminomethylnucleosides of
general structure 1 (Figure 2) were the key intermediate
of the synthetic study. Nucleoside 1 should lead to 2 in
no more than three steps, involving N-protection, 5′-
protection, and phosphitylation. Several possible routes
to aminomethylnucleoside 1 with either uracil or thymine
as nucleobase were evaluated, as indicated in Figure 2
on the level of abridged retrosynthetic analyses. Route
A employed the known19,20 elaboration of tricyclic 3 from
fructofuranose (4). This route has been used extensively
for the synthesis of 1′-modified nucleic acids.21-24 Al-
(13) Selected references: (a) Sando, S.; Abe, H.; Kool, E. T. J . Am.
Chem. Soc. 2004, 126, 1081-1087. (b) Brakmann, S.; Lo¨bermann, S.
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41, 3215-3217. (c) Rosenblum, B. B.; Lee, L. G.; Spurgeon, S. L.; Khan,
S. H.; Menchen, S. M.; Heiner, C. R.; Chen, S. M. Nucleic Acids Res.
1997, 25, 4500-4504. (d) Khan, S. I.; Grinstaff, M. W. J . Am. Chem.
Soc. 1999, 121, 4704-4705.
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J . W.; Baird, E. E.; Dervan, P. B. Angew. Chem., Int. Ed. Engl. 1996,
35, 1487-1489. (c) Robles, J .; Rajur, S. B.; McLaughlin, L. W. J . Am.
Chem. Soc. 1996, 118, 5820-5821.
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N. T. Nucleic Acids Res. 1999, 27, 1492-1498.
(18) Berlan, J . Radiat. Phys. Chem. 1995, 45, 581-589.
(19) Holy, A. Nucleic Acids Res. 1974, 1, 289-298.
(20) Ono, A.; Dan, A.; Matsuda, A. Bioconjugate Chem. 1993, 4, 499-
508.
(16) Kutyavin, I. V.; Afonina, I. A.; Mills, A.; Gorn, V. V.; Lukhtanov,
E. A.; Belousov, E. S.; Singer, M. J . Walburger, D. K.; Lokhov, S. G.;
Gall, A. A.; Dempcy, R.; Reed, M. W.; Meyer, R. B.; Hedgpeth, J . Nucleic
Acids Res. 2000, 28, 655-661.
7544 J . Org. Chem., Vol. 69, No. 22, 2004

1′-Aminomethylthymidine and Acylamidomethylthymidine Residues in DNA
F IGURE 2.
though syntheses for 1′-hydroxymethyl-2′-deoxyuridine
and 1′-hydroxymethylthymidine have been reported, to
the best of our knowledge, no synthesis of 1′-amino-
methylthymidine is known. In our hands, the first step
of Route A (addition of cyanamide to fructose) gave no
more than 40% conversion, and purification of 3 required
benzoylation followed by debenzoylation.19 The total yield
for 3 from 4 was thus lowered to 11%. Further, Mit-
sunobu reaction of 3 with phthalimide gave a difficult-
to-separate mixture of regioisomers, with the 5′-substi-
tuted regioisomer of 5 as the predominant species. Route
A was therefore not pursued further.
Route B is based on transformations reported by
Miyasaka and co-workers.25-27 We employed a 3′,5′-TIPS
protecting group scheme rather than one TBDMS group
each for the 3′- and 5′-hydroxyl groups, hoping to avoid
a low-yielding deprotection step.27 Silyl protection of
anhydrouridine 6, opening of the tricyclic structure with
phenylselenide to 7, and oxidation with MCPBA followed
by elimination proceeded uneventfully, following the
protocols published by Miyasaka and co-workers. The
mixture of diastereomers obtained upon addition of a
bromine and a pivalate moiety26 proved difficult to
handle, however, and all attempts to generate 8 by
treatment with cyanides and various activating agents
failed.
followed by a base introduction reaction to give 11 as a
mixture of anomers. The details of this synthesis, which
led to the thymidine form of 2, are given in Scheme 1.
Starting from deoxyribose, chlorofuranoside 12 was
prepared in three steps, following a literature protocol29
that is an optimized version of Hoffer’s original protocol.30
Cyanide 13 could be prepared from 12 by treatment with
NaCN in DME,31 but this method occasionally suffered
from partial conversion and some formation of the
2-benzyloxymethylfuran elimination product. The use of
Et2AlCN in THF28 was considered, but the use of TMS-
CN/BF3‚OEt2 was pursued, as it had proven higher
yielding in reactions with a similar chlorosugar.32 In the
event, cyanide 13 was obtained in 75% yield and was
smoothly converted to bromofuranoside 14, using the
conditions of a literature protocol.28 Bromosugar 14 was
directly employed in the subsequent base introduction
with silylated nucleobase 15 and Hg(II) cyanide as Lewis
acid.33 As expected for the 2-deoxyglycosyl donor, a
mixture of the R- and â-anomers 16a /b was obtained. The
combined yield of the anomers was 69%, and either
diastereomer was found at approximately the same
percentage. Separation of approximately 30% of the
anomers by flash chromatography required 50 g of silica
per gram of crude, making it advisable to achieve full
separation by rechromatographing mixed fractions. As-
signment of the two anomers was chiefly based on
ROESY NMR spectra (see Supporting Information). For
example, the H6 proton of the nucleobase gave stronger
NOEs to H5′/5′′ in the case of 16b, whereas the same
proton gave a stronger NOE to H4′ in the case of 16a .
Nuclear Overhauser effects found for later intermediates
of the synthesis confirmed the assignment.
Accordingly, Route C was evaluated next. It starts from
deoxyribose (9) and involves the oxidation of a suitably
protected 1-cyano-2-deoxyribose to a derivative of 10,28
(21) Tatsuda, T.; Imao, K.; Suzuki, K. Heterocycles 1986, 24, 2133-
2136.
(22) Yoshimura, Y.; Otter, B. A.; Ueda, T.; Matsuda, A. Chem.
Pharm. Bull. 1992, 40, 1761-1769.
(23) Kværnø, L.; Nielsen C.; Wightman, R. H. J . Chem. Soc., Perkin
Trans. 1 2000, 2903-2906.
(29) Eger, K.; J alalian, M.; Schmidt, M. Tetrahedron 1994, 50,
8371-8380.
(30) Fox, J . J .; Yung, N. C.; Wempen, I.; Hoffer, M. J . Am. Chem.
Soc. 1961, 83, 4066-4068.
(31) (a) Bergstrom, D. E.; Zhang, P. Tetrahedron Lett. 1991, 32,
6485-6488. (b) Miller, T. J .; Farquar, H. D.; Sheybani, A.; Tallini, C.
E.; Saurage, A. S.; Fronczek, F. R.; Hammer, R. P. Org. Lett. 2002, 4,
877-880.
(32) J azouli, M.; Guianvarc’h, D. M.; Soufiaoui, K.; Bougrin, P.;
Vierling, R.; Benhida, A. Tetrahedron Lett. 2003, 44, 5807-5810.
(33) Uteza, V.; Chen, G.-R.; Le Quan Tuoi, J .; Descotes, G.; Fenet,
B.; Grouiller, A. Tetrahedron 1993, 49, 8579-8588.
(24) Kværnø, L.; Wightman, R. H.; Wengel, J . J . Org. Chem. 2001,
66, 5106-5112.
(25) Haraguchi, K.; Tanaka, H.; Hayakawa, H.; Miyasaka, T. Chem.
Lett. 1988, 931-934.
(26) Itoh, Y.; Haraguchi, K.; Tanaka, H.; Gen, E.; Miyasaka, T. J .
Org. Chem. 1995, 60, 656-662.
(27) Yoshimura, Y.; Kano, F.; Miyasaki, S.; Ashida, N.; Sakata, S.;
Haraguchi, K.; Ito, Y.; Tanaka, H.; Miyasaka, T. Nucleosides Nucleo-
tides 1996, 15, 305-324.
(28) Lenox, H. J .; McCoy, C. P.; Sheppard, T. L. Org. Lett. 2001, 3,
2415-2418.
J . Org. Chem, Vol. 69, No. 22, 2004 7545

Gru¨nefeld and Richert
SCHEME 1a
a
Reagents and conditions: (a) (1) HCl, MeOH, (2) pClBzCl, pyridine, (3) HCl, AcOH (80% over 3 steps); (b) TMSCN, BF3‚OEt2 (75%);
(c) NBS, benzoyl peroxide, CCl4 (quant); (d) 15, Hg(CN)2, MeCN (69% for anomeric mixture, 27% 16a , 30% 16b); (e) NaBH4, TFA, THF;
(f) Alloc-Cl, THF (70% over 2 steps); (g) NaOMe, MeOH (97%); (h) DMT-Cl, DMAP, TEA, THF (74%); (i) Cl-P(NiPr2)OC2H4CN, DIEA,
MeCN (71%).
SCHEME 2a
a
Reagents and conditions: (a) Standard DNA synthesis, including extension cycle(s) with 21; (b) Pd(PPh3)4, [NEt2H2][HCO3], DMF,
microwave irradiation 200 W, 80 °C, 10 min; (c) HOBT, HBTU, DIEA, pyrene butyric acid, DMF, microwave irradiation 150-200 W, 80
°C, 10 min; (d) NH4OH, rt; (e) HOBT, HBTU, DIEA, mixture of carboxylic acids, DMF, microwave irradiation 150-200 W, 80 °C, 10 min.
Amine 17 was obtained through reduction of 16b with
sodium trifluoroacetoxy borohydride,34 prepared in situ
from NaBH4 and TFA. The free amine was Alloc-
protected in situ to avoid handling losses of this very
polar compound during purification. An Fmoc group for
N-protection proved inferior, as it was partially lost
during the subsequent steps, even after careful optimiza-
tion of the reaction conditions. Alloc-protected 18, ob-
tained in 70% over two steps, was readily debenzoylated
to 19, and 5′-re-protected with a dimethoxytrityl group
to give 20. Phosphitylation of the latter provided key
building block 21 in 6% overall yield from 9.
starting from support 22 proceeded in high yield, the on-
support removal of the Alloc group of 23 initially proved
difficult. Exposing the fully assembled strand on solid
support to Pd(PPh3)4 and diethylammonium bicarbonate
in CH2Cl2, conditions successfully employed for Alloc
removal on cpg in earlier cases,35 led to little 24, as
determined by MALDI-TOF mass spectra of crudes
obtained after treatment with saturated aqueous am-
monia. Heating to 40 °C for up to 3 d, combined with
repeated sonications for 30 min, did not lead to clean
conversions either. Subsequent acylation attempts with
HBTU/HOBT-activated pyrene butyric acid, a lipophilic
acid that can readily be detected in the UV-vis range,
did not give satisfactory quantities of 25, as only small
peaks for 26 were observed in MALDI mass spectra of
With phosphoramidite 21 in hand, oligodeoxyribo-
nucleotides with one or two modified thymidine residues
could be prepared (Scheme 2). Whereas chain assembly
(34) Iyer, R. P.; Phillips, L. R.; Egan, W. Synth. Commun. 1991, 20,
2053-2063.
(35) Mokhir, A. A.; Connors, W. H.; Richert, C. Nucleic Acids Res.
2001, 29, 3674-3684.
7546 J . Org. Chem., Vol. 69, No. 22, 2004

1′-Aminomethylthymidine and Acylamidomethylthymidine Residues in DNA
groups may have migrated from the exocyclic amino
groups of adenine and cytosine nucleobases.
When performed with microwave irradiation using an
instrument designed for organic synthesis (Discover,
CEM Inc.), the acylation of amine 24 with the active ester
of pyrene butyric acid gave surprisingly pure 25, how-
ever. The MALDI-TOF mass spectrum of a representative
crude of 26, obtained after deprotection and removal from
the cpg, is shown in Figure 3b. High-yielding conversions
were confirmed when crude 26 was analyzed by HPLC,
where the peak of the product dominated. An exploratory
synthesis of doubly modified 27 was performed, using the
same conditions for on-support modification as for 26.
Again, MALDI-TOF mass spectra of crudes showed
successful double Alloc removal and doubly acylation,
even though one of the thymdines to be modified was only
the fourth residue from the surface of the solid support.
With 26 and 27, a set of melting curve analyses were
performed to test the effect of the 1′-modification on the
duplex with complementary strand 5′-GTAATAATGC-
3′ (28). The results of the UV-melting experiments are
compiled in Table 1. It can be discerned that the
modification increases the melting points and that this
effect is stronger for the doubly modified strand, with a
∆Tm of up to 6.9 °C over the control (3.4 °C per modifica-
tion). This suggests that the acylamidomethyl modifica-
tion at the 1′-position of the thymidine residues is
tolerated by the helix, as also suggested by the un-
changed sigmoidal appearance of the melting curve
(Figure 4). The thermodynamic signatures (Table 1) show
that duplexes carrying the 1′-acylamidomethyl substitu-
ent experience a decreased entropic penalty for duplex
formation. This suggests that the 1′-substituent may help
to preorganize 26 and 27 toward duplex formation. The
Alloc-protected strand, 30, on the other hand, leads to
lower UV-melting points. The same is true for oligo-
nucleotides with 1′-carbamoylmethylthymidine residues
that are known from the literature.20
F IGURE 3. MALDI-TOF mass spectra of crude 26 as obtained
after deprotection with NH4OH. (a) Deprotection of 23 and
acylation of 24 without microwave irradiation (3 d reaction
time for each reaction and sonication of 5 × 30 min at 40 °C).
(b) Deprotection and acylation for 10 min with microwave
irradiation (200 W, 80 °C max). Parentheses show peak
assignments, with side products resulting from incomplete
acylation of 24, indicated as {-TNH2-} and from blocking of 24
through benzoyl migration, presumably from neighboring
residues, as {-TNHBz-}.
Finally, we have tested the suitability of our method
for preparing oligonucleotides with other 1′-acylamido-
methylthymidines. For this, solid support 24 was acyl-
ated with hippuric acid, 3-indolepropionic acid, NR-Boc-
and â-O-benzyl-protected L-serine, the quinolone cinox-
acin, and dehydrocholic acid. This diverse set of carbox-
ylic acid building blocks was successfully used in a
microwave-assisted coupling reaction. The ratio of the
acids was chosen such that it creates a reactivity-adjusted
mixture.37 Figure 5 shows that even the mixed coupling,
with its much higher propensity for side reactions, gives
a clean conversion, resulting in a combinatorial library
directly suitable for spectrometrically monitored selection
assays (SMOSE).38
deprotected crudes (Figure 3). This seemed to suggest
that the steric hindrance caused by the tightly packed
strands on the non-swellable support was overwhelming.
Rather than reverting to discontinuous syntheses,
where chain assembly is interrupted after coupling of
each modified residue, followed by individual Alloc
removal and acylation prior to continuation of the chain
assembly, we chose to employ irradiation with micro-
waves to assist the reactions in the interior of the strands.
Although the effect of microwave irradiation on the
heterogeneous reaction mixture was small with CH2Cl2,
probably because of the low boiling point, 150- to 200-W
irradiation (limited by a maximum temperature of 80 °C
in the flask) for 10 min was sufficient for full Alloc
removal when DMF was employed as solvent. Without
subsequent acylation, MALDI spectra of crudes showed
contamination of the deprotected amine with products
carrying an acetyl or benzoyl group. These were most
likely the result of the migration of protecting groups to
the primary amine of the modified thymidine residue in
the presence of base. Labile acetyl groups may have been
introduced through the capping steps, and the benzoyl
Discu ssion
Reactions in the interior of fully assembled and pro-
tected oligonucleotides on nonswellable supports are
difficult. This is why we35 and others13d have employed
(36) McDowell, J . A.; Turner, D. H. Biochemistry 1996, 35, 14077-
14089.
(37) Dombi, K. L.; Steiner, U. E.; Richert, C. J . Comb. Chem. 2003,
5, 45-60.
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Chem. Biol. 1997, 4, 63-77.
J . Org. Chem, Vol. 69, No. 22, 2004 7547

Gru¨nefeld and Richert
TABLE 1. UV-Meltin g P oin ts a n d Th er m od yn a m ic Va lu es for DNA Du p lex F or m a tion
duplex
[NaCl]/[MgCl2]a
Tm (°C)b
∆H° (kcal/mol)
∆S° (cal/K‚mo)l
∆G° (kcal/mol)c
29:28 (control)
-/-
16.6 ( 1.4
33.5 ( 0.5
37.5 ( 0.3
9.4 ( 0.3
21.2 ( 0.4
23.8 ( 0.2
17.8 ( 0.8
34.2 ( 0.8
37.6 ( 1.4
23.5 ( 1.0
39.2 ( 0.8
41.5 ( 0.7
-77.8 ( 8.4
-81.4 ( 1.9
-76.0 ( 1.6
-129.0 ( 10.2
-72.0 ( 5.4
-69.0 ( 4.0
-76.0 ( 5.1
-76.6 ( 2.0
-67.3 ( 10.9
-65.8 ( 0.6
-73.3 ( 0.7
-68.8 ( 1.1
-239.3 ( 28.0
-236.2 ( 6.1
-215.5 ( 5.2
-427.4 ( 36.5
-215.5 ( 18.7
-203.3 ( 13.5
-232.1 ( 17.5
-220.0 ( 7.3
-187.2 ( 34.0
-192 ( 1.0
-3.6 ( 0.4
-8.2 ( 0.2
-9.2 ( 0.1
3.6 ( 1.1
-5.2 ( 0.4
-6.0 ( 0.2
-4.1 ( 0.4
-8.4 ( 0.3
-9.2 ( 0.4
-6.1 ( 0.3
-9.6 ( 0.2
-10.1 ( 0.2
100 mM/10 mM
1 M/10 mM
-/-
100 mM/10 mM
1 M/10 mM
-/-
100 mM/10 mM
1 M/10 mM
-/-
100 mM/10 mM
1 M/10 mM
30:28
26:28
27:28
-205 ( 3.0
-189 ( 4.0
a
b
In 10 mM PIPES (pH 7) + salt concentrations given. Mean of four values ( SD at 1.6 µM strand concentration. c Calculated on the
basis of curve fitting with Meltwin36 for 37 °C.
F IGURE 4. Representative UV-melting curves of the duplexes
formed by doubly modified oligonucleotide 27 (9) or unmodified
5′-GCATTATTAC-3′ (O) with their fully complementary strands
at 1.6 µM strand concentration, 100 mM NaCl, 10 mM MgCl2,
and 10 mM PIPES buffer, pH 7.
interrupted syntheses for internal residues to be modified
in the past. Initially, we were hesitant to apply micro-
wave irradiation, because it was not clear why it may be
more successful than conventional heating.39 In the
present case, microwave irradiation not only accelerated
a Pd(0)-catalyzed transallylation and an amide-forming
reaction to an extent that made them synthetically
useful, but also led to cleaner reactions (Figure 3). The
microwave-assisted heating apparently breaks up the
inaccessibility of the reaction sites, so that the desired
intermolecular reactions prevail and competing reactions,
such as protecting group migrations from neighboring
residues, are suppressed. Similar effects may be observed
for other conversions that would otherwise be difficult
to perform with partially protected oligonucleotides on
solid support. Other reports on successful microwave-
assisted transformations of nucleic acids on solid supports
are beginning to emerge,40 and so are reports on micro-
wave-assisted Pd-mediated reactions.41
F IGUR E 5. MALDI-TOF mass spectrum of the pseudo-
molecular ions ([M - H]-) of the members of ch em set 1, as
obtained after acylation with a reactivity-adjusted mixture of
activated caboxylic acids and deprotection.
will be tolerated when introduced after completion of
chain assembly but lead to side reactions with phos-
phoramidites in approaches that involve interrupted
chain assembly. One may argue that introducing acyl
groups during interrupted chain assembly has the ad-
vantage that different acyl groups can be introduced at
different sites of an oligomer, but the same may be
achieved by employing orthogonal protecting groups for
the different modified nucleosides.
The current approach of modifying aminomethyl nu-
cleosides on solid support lends itself readily to combi-
natorial chemistry. Therefore, it may lead to an efficient
search for new modified oligonucleotides with increased
affinity for target strands, particularly when combined
with mass spectrometrically monitored selection experi-
ments (SMOSE). Such searches have been performed
with oligonucleotides acylated at one of the termini.42 To
allow generation of a wide range of modified residues, it
The advantage of the post-assembly derivatization of
oligonucleotides on solid support is not just a more
convenient procedure for the assembly of oligomers but
also the fact that the residues introduced do not have to
be compatible with subsequent extension steps. For
example, alcohol groups on the newly attached moieties
(39) Adam, D. Nature 2003, 421, 571-572.
(40) Andrzejewska, M., Kaminski, J .; Kazimierczuk, Z. Nucleosides
Nucleotides Nucleic Acids 2002, 21, 73-78.
(41) Fu¨rstner, A.; Seidel, G. Org. Lett. 2002, 4, 541-543.
7548 J . Org. Chem., Vol. 69, No. 22, 2004

1′-Aminomethylthymidine and Acylamidomethylthymidine Residues in DNA
3,5-Bis-O-(p -ch lor ob en zoyl)-2-d eoxy-D-r ib ofu r a n osyl
is desirable to append diversity elements via amide
bonds, as carboxylic acids form a large structure space
of commercially available building blocks. The diversity
will increase if other nucleobases than thymidine are
being furnished with 1′-acylamidomethyl substituents.
Deoxycytidine residues with such substituents may be
readily prepared by treating 21 with 1,2,4-1H-triazole,
POCl3, and triethylamine43 and liberating the cytosine
from the triazolide thus prepared during the deprotection
with aqueous ammonia.44
Besides the synthetic issues, one may ask how well the
1′-aminomethyl group fulfills the role of a tether that
allows presenting substituents to the minor groove of a
regular Watson-Crick helix without introducing a steric
conflict. The data available thus far show good cooper-
ativity in the thermal dissociation, as evidenced by
melting curves of modified and unmodified duplex that
are almost identical (Figure 4). Further, there is no drop
in the melting point for the modified duplex 26:28, whose
acyl group is not expected to stabilize the duplex through
binding in the minor groove, though one cannot rule out
intercalation. Pyrene is a chromophore that is readily
detected and that does not require protecting groups. As
a result of its lipophilicity, it facilitates purification of
modified strands carrying it. Other oligonucleotides
containing 1′-acylamidomethylthymidines may carry pen-
dant groups for electron transfer or Fo¨rster fluorescence
resonance energy transfer studies.45 Thus, oligonucleo-
tides containing 1′-acylamidomethylthymidine residues
may provide an entry into richly functionalized nucleic
acids that act as functional hybridization probes.
Cya n id e (13). To a stirred solution of 12 (6 g, 14 mmol) in
anhydrous CH2Cl2 (80 mL) were added TMS-CN (2.1 g, 2.8
mL, 21 mmol, 1.5 equiv) and BF3‚OEt2 (3.97 g, 3.5 mL, 28
mmol, 2 equiv). The reaction mixture was stirred for 12 h.
After complete conversion, the reaction was quenched with
saturated ammonium bicarbonate solution (80 mL) and stirred
for additional 30 min. The slurry was extracted with CH2Cl2
(5 × 300 mL). The combined organic phases were dried over
Na2SO4 and evaporated to dryness. The residue was purified
by column chromatography (silica, hexane/EtOAc 4:1) to give
4.36 g (10.4 mmol, 75%) of 13 as a pale yellow solid (mixture
of anomers). An analytical sample was rechromatographed,
yielding a pure fraction of the fast eluting anomer. TLC
1
(hexane/EtOAc 4:1) Rf 0.42, 0.34. H NMR (500 MHz, CDCl3,
fast eluting anomer) δ 8.02 (d, J ) 8.5 Hz, 2H, Ar-H), 7.94 (d,
J ) 8.5 Hz, 2H, Ar-H), 7.43 (dd, J ) 7.2 Hz, J ) 1.3 Hz, 2H,
Ar-H), 5.62-5.61 (m, 1H, H3), 4.94 (dd, J ) 9.1 Hz, J ) 6.9
Hz, 1H, H1), 4.62-4.58 (m, 1H, H4), 4.56-4.52 (m, 2H, H5),
2.79-2.65 (m, 2H, H2). 13C NMR (126 MHz, CDCl3, fast eluting
anomer) δ 165.3, 164.8, 140.3, 139.9, 131.1, 131.1, 129.0, 128.9,
127.8, 127.3, 117.9, 83.6, 77.4, 77.1, 76.9, 75.6, 65.9, 64.0, 37.6.
MALDI-TOF MS m/z 443.2 ([M + Na]+).
1-Br om o-3,5-b is-O-(p -ch lor ob en zoyl)-2-d eoxy-D-r ib o-
fu r a n osyl Cya n id e (14). Compound 14 was prepared from
13 following a literature protocol and was obtained in quan-
titative yield. It was used in the subsequent reaction without
recrystallization.28
1′-Cya n o-3′,5′-bis-O-(p-ch lor oben zoyl)th ym id in e (16).
To a stirred solution of thymine (568 mg, 4.5 mmol) in CH3CN
(6 mL) were added HMDS (1.5 g, 2.0 mL, 9.4 mmol, 2.1 equiv)
and TMS-Cl (1.0 g, 1.2 mL, 9.4 mmol, 2.1 equiv). The resulting
slurry was stirred for 5 min. TMS-OTf (244 mg, 213 µL, 1.1
mmol, 0.25 equiv) was added under stirring, and the reaction
mixture was heated to reflux for 6 h to give a clear solution of
5-methyl-2,4-bis(trimethylsilyloxy)pyrimidine (15). The solu-
tion was evaporated to dryness, and the residue was redis-
solved in CH3CN (10 mL). A solution of previously vacuum-
dried 14 (1.5 g, 3 mmol) in anhydrous CH3CN (15 mL) was
stirred under presence of 4Å molecular sieves (50 mg) for 30
min. Then Hg(CN)2 (1.1 g, 1.4 equiv) and the freshly prepared
solution of 15 in CH3CN were added, and the reaction mixture
was stirred for 3 d at room temperature. The solids were
filtered off and washed with CH3CN. The filtrate was evapo-
rated, and the residue was redissolved in CHCl3 (30 mL). The
resulting solution was washed once with 50% KI in half-
saturated sodium chloride solution and once with brine. The
organic phase was dried over Na2SO4, and the solvent was
evaporated. The residue was purified by column chromatog-
raphy (100 g silica, CH2Cl2/EtOAc 85:15) yielding 1.1 g (2.1
mmol, 69%) total of the of the title compound as off-white solid.
Eluting first was a fraction of 16a (351 mg, 0.64 mmol, 21%),
followed by a mixture of anomers (295 mg, 0.54 mmol, 18%)
and a fraction of 16b (469 mg, 0.9 mmol, 30%). Analytical data
for 16a : TLC (CH2Cl2/EtOAc 85:15) Rf 0.45. 1H NMR (500
MHz, CDCl3) δ 9.94 (bs, 1H, NH), 8.04 (d, J ) 8.8 Hz, 2H,
Ar-H), 7.69 (d, J ) 8.8 Hz, 2H, Ar-H), 7.47 (s, 1H, H6), 7.47
(d, J ) 8.8 Hz, 2H, Ar-H), 7.37 (d, J ) 8.8 Hz, 2H, Ar-H), 5.70
(d, J ) 5.7 Hz, 1H, H3′), 4.95 (m, 1H, H4′), 4.76 (dd, J ) 12.6
Hz, J ) 3.5 Hz, 1H, H5′), 4.65 (dd, J ) 12.6 Hz, J ) 4.4 Hz,
1H, H5′), 3.46 (d, J ) 15.7 Hz, 1H, H2′), 3.28 (dd, J ) 15.7
Hz, J ) 6.0 Hz, 1H, H2′), 1.91 (s, 3H, H7). 13C NMR (126 MHz,
CDCl3) δ 165.2, 164.4, 163.8, 149.6, 140.8, 140.4, 132.6, 131.2,
129.2, 129.1, 127.3, 126.6, 115.0, 111.5, 87.7, 87.7, 74.2, 63.4,
44.8, 12.7. MS (MALDI-TOF) m/z 566.2 ([M + Na]+). Analytical
data for 16b: TLC (CH2Cl2/EtOAc 85:15) Rf 0.39. 1H NMR (500
MHz, CDCl3) δ 8.34 (bs, 1H, NH), 8.08 (d, J ) 8.8 Hz, 2H,
Ar-H), 7.87(d, J ) 8.8 Hz, 2H, Ar-H), 7.49 (d, J ) 8.8 Hz, 2H,
Ar-H), 7.48 (s, 1H, H6), 7.45 (d, J ) 8.8 Hz, 2H, Ar-H), 5.72
(d, J ) 8.0 Hz, 1H, H3′), 4.97 (m, 1H, H4′), 4.91 (dd, J ) 12.9
Hz, J ) 3.5 Hz, 1H, H5′), 4.59 (dd, J ) 12.9 Hz, J ) 3.8 Hz,
1H, H5′), 3.75 (d, J ) 15.4 Hz, 1H, H2′), 2.76 (dd, J ) 15.4
Exp er im en ta l Section
Gen er a l. Anhydrous solvents were purchased over molec-
ular sieves and used without further purification. HOBT
(1-hydroxybenzotriazole) and HBTU (O-benzotriazol-1-yl-
N,N,N ′,N ′-tetramethyluroniumhexafluorophosphate) were from
Advanced ChemTech (Louisville, KY). Phosphoramidites (dABz,
dCBz, dCBzcpg, dGdmf, dT), as well as other reagents for DNA
synthesis, were from Proligo (Hamburg, Germany). Reactions
involving controlled pore glass (cpg) were performed in polypro-
pylene reaction chambers for DNA synthesis (Prime Synthesis,
Aston, PA). Oligonucleotides were purified by HPLC on 250
mm × 4.6 mm Nucleosil C4 or 250 mm × 4.6 mm Nucleosil
C18 columns, using a gradient of CH3CN in 0.1 M triethyl-
ammonium acetate, pH 7, and detection at 260 nm. MALDI-
TOF spectra were recorded in positive, linear mode in the case
of small molecules, and in negative, linear mode in the case
of oligonucleotides. MALDI matrixes were 2,5-dihydroxy-
benzoic acid (0.1 M in CH3CN) for small molecules and a
mixture of 2,4,6-trihydroxyacetophenone (0.3 M in EtOH) and
diammonium citrate (0.1 M in H2O) (2:1 v/v) for oligonucleo-
tides.
3,5-Bis-O-(p -ch lor ob en zoyl)-2-d eoxy-D-r ib ofu r a n osyl
Ch lor id e (12). Compound 12 was prepared following a
literature protocol29 on a 0.15 mol scale and was obtained in
80% yield over three steps.
(42) (a) Altman, R. K.; Schwope, I.; Sarracino, D. A.; Tetzlaff, C. N.;
Bleczinski, C. F.; Richert, C. J . Comb. Chem. 1999, 1, 493-508. (b)
Mokhir, A. A.; Tetzlaff, C. N.; Herzberger, S.; Mosbacher, A.; Richert,
C., J . Comb. Chem. 2001, 3, 374-386. (c) Connors, W. H.; Narayanan,
S.; Kryatova, O. P.; Richert, C. Org. Lett. 2003, 5, 247-250. (d)
Narayanan, S.; Gall, J .; Richert, C. Nucleic Acids Res. 2004, 32, 2901-
2911.
(43) Webb, T. R.; Matteucci, M. D. J . Am. Chem. Soc. 1986, 108,
2764-2765.
(44) Martin, P. Helv. Chim. Acta 1995, 78, 486-504.
(45) Clegg, R. M. Methods Enzymol. 1992, 211, 353-388.
J . Org. Chem, Vol. 69, No. 22, 2004 7549

Gru¨nefeld and Richert
Hz, J ) 6.0 Hz, 1H, H2′), 1.72 (s, 3H, H7). 13C NMR (150 MHz,
CDCl3) δ 165.1, 164.8, 162.7, 149.0, 140.7, 140.1, 132.8, 131.4,
130.7, 129.3, 129.2, 127.1, 126.9, 115.7, 111.6, 87.5, 86.8, 74.8,
63.0, 46.7, 12.6. MS (MALDI-TOF) m/z 566.2 ([M + Na]+).
CH), 5.65 (bs, 1H, NH-Alloc), 5.23 (d, J ) 17.3 Hz, 1H, CHd
CH2), 5.15 (dd, J ) 10.4, J ) 1.3 Hz, 1H, CHdCH2), 4.40 (dd,
J ) 13.5, J ) 5.7 Hz, 1H, O-CH2), 4.48 (dd, J ) 13.5, J ) 5.6
Hz, 1H, O-CH2), 4.36 (t, J ) 4.7, 1H, H3′), 4.32 (d, J ) 4.7,
1H, H2′), 4.04 (dd, J ) 14.5 Hz, J ) 8.2 Hz, 1H, -CH2-NH-),
3.76 (d, J ) 1.3 Hz, 6H, DMT-OMe), 3.54 (dd, J ) 14.5 Hz, J
) 7.2 Hz, 1H, -CH2-NH-), 3.20 (dd, J ) 10.3 Hz, J ) 4.7 Hz,
1H, H5′), 3.04 (dd, J ) 10.3 Hz, J ) 3.8 Hz, 1H, H5′), 2.61
(dd, J ) 14.4, J ) 6.0, 1H, H2′), 2.28 (dd, J ) 14.4 Hz, J ) 3.1
Hz, 1H, H2′), 1.70 (s, 3H, H7). MS (MALDI-TOF) m/z 680.5
([M + Na]+). 13C NMR (126 MHz, CDCl3) δ 164.5, 158.6, 150.4,
144.4, 137.1, 135.5, 135.4, 132.7, 129.9, 127.89, 127.85, 127.0,
117.8, 113.2, 108.5, 98.8, 88.1, 86.7, 72.7, 65.8, 63.4, 55.2, 45.9,
45.1, 12.6, 10.7.
â-1′-[N-Allyloxyca r bon yl(a m in om eth yl)]-3′,5′-bis-O-(p-
ch lor oben zoyl)th ym id in e (18). To a stirred solution of 16b
(527 mg, 1 mmol) in anhydrous THF (25 mL) was added
NaBH4 (113 mg, 3 mmol, 3 equiv). To the resulting slurry was
added TFA (177 mg, 155 µL, 1.5 mmol, 1.5 equiv). After brief
gas evolution, a clear solution formed, which was stirred at
room temperature for 12 h. After complete conversion, excess
NaBH4 was hydrolyzed with water (5 mL), and Alloc-Cl (241
mg, 198 µL, 2 mmol, 2 equiv) was added. (Attention: older
samples of Alloc-Cl may be under substantial pressure and
may contain highly toxic decomposition compounds.) The
resulting solution was stirred for 24 h at room temperature.
The solution was diluted with water (5 mL) and extracted trice
with CHCl3 (3 × 30 mL). The combined organic phases were
dried over Na2SO4, and the solvent was evaporated. The
residue was purified by column chromatography (silica, CH2Cl2/
MeOH 99:1), yielding 442 mg of 18 (0.70 mmol, 70%). TLC
(CHCl3/EtOH 95:5) Rf 0.48. 1H NMR (400 MHz, CDCl3) δ 10.10
(bs, 1H), 7.98 (d, J ) 8.6 Hz, 2H), 7.82 (d, J ) 8.6 Hz, 2H),
7.52 (s, 1H), 7.43 (d, J ) 8.3 Hz, 2H), 7.36 (d, J ) 8.3 Hz, 2H),
6.07 (bs, 1H), 5.80 (m, 1H), 5.56 (d, J ) 6.6 Hz, 1H), 5.22 (d,
J ) 17.2 Hz, 1H), 5.15 (d, J ) 10.4, 1H), 4.81-4.70 (m, 2H),
4.52-4.41 (m, 3H), 4.07-3.93 (m, 1H), 3.73 (dd, J ) 14.2 Hz,
J ) 6.6 Hz, 1H), 3.02 (d, J ) 15.6 Hz, 1H), 2.92 (dd, J ) 15.6
Hz, J ) 6.6 Hz, 1H), 1.67 (s, 3H). 13C NMR (100 MHz, CDCl3)
δ 165.2, 165.0, 164.7, 156.8, 150.3, 140.22, 140.16, 139.6, 136.5,
133.0, 132.7, 131.2, 130.7, 129.04, 129.01, 128.5, 128.2, 127.5,
117.8, 108.9, 107.6, 99.0, 84.3, 76.0, 67.4, 65.8, 46.2, 42.5, 29.5,
23.8, 12.5. MS (MALDI-TOF) m/z 655.4 ([M + Na]+).
â-1′-[N-Allyloxycar bon yl(am in om eth yl)]-5′-(dim eth oxy-
trityl)thymidin-3′-O-yl-cyanoethyl-N,N-diisopropylphophor-
a m id ite (21). To a stirred solution of 20 (90 mg, 0.14 mmol)
and DIEA (74 µL, 0.42 mmol, 3 equiv) in anhydrous CH3CN
(1.5 mL) was added 2-cyanoethyl-N,N-diisopropyl-chloro-
phophoramidite (41 µL, 0.19 mmol, 1.33 equiv). The solution
was stirred for 1.5 h. After complete conversion, CH2Cl2 (60
mL) was added, and the solution was washed twice with a
saturated NaHCO3 solution (2 × 50 mL) and once with brine
(50 mL). The organic phase was dried over Na2SO4, and the
solvent was evaporated to an approximate volume of 0.5 mL.
This solution was added dropwise to pentane (35 mL) and
stored at 7 °C overnight. The supernatant was aspired, and
the oily residue was dried in vacuo. The title compound was
obtained as pale yellow foam (83 mg, 0.10 mmol, 71%). 31P
NMR (202 MHz, CDCl3) 148.3, 147.8. MALDI-TOF MS m/z
897.2 [M + K]+.
DNA Syn th esis (Com p ou n d 23). Oligodeoxynucleotides
were prepared on
a 1-µmol scale, following the protocol
â-1′-[N-Allyloxycar bon yl(am in om eth yl)]th ym idin e (19).
To a stirred solution of 18 (220 mg, 0.35 mmol) in methanol
(5 mL) was added a solution of NaOMe in methanol (1 M, 163
µL, 0.19 mmol, 0.6 equiv), and the resulting solution was
stirred at room temperature for 6 h. The solution was neutral-
ized by adding Dowex 50 (H+ form), and the resulting mixture
was stirred for 30 min. The ion-exchange resin was filtered
off and washed with methanol, and the filtrates were combined
and evaporated. The residue was chromatographed (silica,
CH2Cl2/MeOH 85:15) to give 115 mg (0.32 mmol, 91%) of 19.
recommended by the manufacturer of the synthesizer (8909
Expedite DNA synthesizer, system software 2.01). The cou-
pling of modified phosphoramidite 21 to solid-support-bound
DNA was carried out in a polypropylene vessel using a 0.12
M solution of the phosohoramidite in activator solution (250
µL). The oligodeoxynucleotides were cleaved from the solid
support and deprotected with 30% aqueous ammonia for 12
h.
Rem ova l of Alloc Gr ou p s (23 to 24). Solid support 23 (5
mg, approximately 0.14 µmol loading) was treated with a
mixture of Pd[PPh3]4 (7.5 mg, 20.5 µmol) and diethylammo-
nium bicarbonate (7.5 mg, 56 µmol) in DMF (0.6 mL) in a
tapered Pyrex vial. This mixture was irradiated in a micro-
wave synthesis system (Discover, CEM) for 10 min with up to
200 W and an upper temperature limit of 80 °C. The super-
natant was removed, and the solid support was washed with
DMF (3 × 1 mL) and once with CH2Cl2 (1 mL), followed by
drying at 0.1 Torr.
1
TLC (CHCl3/EtOH 85:15) Rf 0.5. H NMR (500 MHz, CDCl3)
δ 7.68 (s, 1H, H6), 7.36 (t, J ) 6.6 Hz, 1H,), 5.83-5.75 (m, 1H,
CH2dCH), 5.65 (bs, 1H, NH-Alloc), 5.21 (dd, J ) 17.3 Hz, J )
1.6 Hz, 1H, CHdCH2), 5.15 (dd, J ) 10.4, J ) 1.6 Hz, 1H,
CHdCH2), 4.41 (m, 1H, O-CH2), 4.29 (dd, J ) 13.8, J ) 5.6
Hz, 1H, O-CH2), 4.10 (m, 1H, H3′), 3.97 (m, 1H, H4′), 3.80 (dd,
J ) Hz, J ) 6.6 Hz, 1H, -CH2-NH-), 3.46-3.34 (m, 2H, H5′),
3.32-3.35 (m, 1H, -CH2-NH-), 2.61 (dd, J ) 14.5, J ) 6.0, 1H,
H2′), 2.28 (dd, J ) 14.5 Hz, J ) 3.1 Hz, 1H, H2′), 1.70 (s, 3H,
H7). 13C (126 MHz, CDCl3) δ 168.0, 164.4, 164.3, 150.9, 150.1,
134.3, 134.0, 131.5, 117.8, 109.4, 109.3, 93.2, 91.8, 88.2, 87.4,
75.3, 70.6, 70.5, 67.3, 63.8, 61.4, 60.5, 59.0, 53.4, 48.6, 47.1,
42.6, 12.9. MS (MALDI-TOF) m/z 680.5 ([M + Na]+).
Cou p lin g of P yr en e Bu tyr ic Acid (24 to 25). A mixture
of HOBT (15.3 mg, 100 µmol), HBTU (34.1 mg, 90 µmol), and
pyrene butyric acid (29 mg, 100 µmol) was dissolved in DMF
(600 µL). To this was added DIEA (80 µL, 486 µmol), and the
resulting solution was shaken for 10 min. The solution was
added to a tapered Pyrex vial together with support 24 (2.0
mg, 0.05 µmol DNA loading) and irradiated in a microwave
synthesis system for 10 min at 200 W maximum and 80 °C
upper temperature limit. After removal of the supernatant,
the solid support was washed with DMF (3 × 1 mL) and then
with CH2Cl2 (2 × 1 mL), followed by drying at 0.1 Torr.
â-1′-[N-Allyloxycar bon yl(am in om eth yl)]-5′-(dim eth oxy-
tr ityl)th ym id in e (20). A mixture of DMT-Cl (265 mg, 0.78
mmol, 2 equiv) and DMAP (4.8 mg, 0.04 mmol, 0.1 equiv) was
dried at 0.1 Torr and then added to a stirred solution of 19
(141 mg, 0.39 mmol) and NEt3 (108 µL, 0.78 mmol, 2 equiv)
in anhydrous THF (5 mL). The solution was stirred at room
temperature for 24 h. The reaction mixture was diluted with
CH2Cl2 and washed twice with saturated NaHCO3 solution and
once with brine. The organic phase was dried over Na2SO4,
and the solvent was evaporated. The residue was purified by
column chromatography (silica, pretreated with CH2Cl2/MeOH/
TEA 96:1:3, elution with CH2Cl2/MeOH 99:1) to give 188 mg
20 (0.29 mmol, 74%) as a solid. TLC (CHCl3/EtOH 95:5) Rf
Cou p lin g of P yr en e Bu tyr ic Acid (to give 27). Pyrene
butyric acid (29 mg, 100 µmol) was activated with a mixture
of HOBT (15.3 mg, 100 µmol), HBTU (34.1 mg, 90 µmol) and
DIEA (80 µL, 486 µmol) in DMF (600 µL) and was coupled to
24 (5.0 mg, 0.14 µmol DNA loading) as described for 25.
GCATAM-P yBATATTAC (26). HPLC: CH3CN gradient 0%
for 5 min to 27% in 55 min, tR ) 54.5 min. MALDI-TOF MS
calcd for [M - H]- 3301.4, found 3300.9. Yield 3.0 nmol (6%).
1
0.5. H NMR (500 MHz, CDCl3) δ 7.65 (s, 1H, H6), 7.32-7.17
(m, 5H, Ar-H), 7.20 (dt, J ) 8.8 Hz, J ) 2.8, 4H, Ar-H), 6.78
(dt, J ) 8.8 Hz, J ) 2.8, 4H, Ar-H), 5.85-5.77 (m, 1H, CH2d
7550 J . Org. Chem., Vol. 69, No. 22, 2004

1′-Aminomethylthymidine and Acylamidomethylthymidine Residues in DNA
GCATAM-P yBATATAM-P yBATAC (27). HPLC: CH3CN gradi-
ent 0% for 5 min to 37% in 45 min, tR ) 39.0 min. MALDI-
TOF MS calcd for [M - H]- 3600.8, found 3599.4. Yield 14.8
nmol (12%).
heating and cooling rates of 1 °C/min, using solutions of
equimolar concentrations of the DNA strands in 10 mM PIPES
(piperazine-N,N ′-bis(ethane sulfonic acid)) buffer, pH 7, in
deionized water and the salt concentrations given in Table 1.
The melting temperatures and the thermodynamic data were
determined with the program MeltWin 3.0.
P r ep a r a tion of 30. A sample of 23 (5.0 mg, 0.14 µmol DNA
loading) was treated with 30% aqueous ammonia for 12 h to
yield the Alloc-protected oligonucleotide, which was purified
by HPLC analogously to 26.
Ack n ow led gm en t. The authors are grateful to C.
Ahlborn for helpful discussions and Prof. J a¨ger (Uni-
versity of Stuttgart) for access to his microwave ap-
paratus for exploratory experiments. P.G. is a recipient
of a predoctoral fellowship from the State of Baden-
Wu¨rttemberg. This work was supported by DFG (RI
1063/1-2 and FOR 434) and Fonds der Chemischen
Industrie (grant 164431).
GCATAM-AllocTATTAC (30). HPLC: CH3CN gradient 0%
for 5 min to 20% in 40 min, tR ) 34.0 min. MALDI-TOF MS
calcd for [M - H]- 3115.7, found 3113.9. Yield: 54.8 nmol
(38%).
Ch em set 1. A mixture of hippuric acid (0.7 mg, 3.6 µmol),
3-indolepropionic acid (0.2 mg, 1 µmol), Boc-Ser(Bzl)-OH (1.4
mg, 4.8 µmol), dehydrocholic acid (2.37 mg, 5.9 µmol), and
cinoxacin (0.5 mg, 1.7 µmol) in DMF (300 µL) was prepared
from stock solutions and activated with HOBT (2.7 mg, 20
µmol), HBTU (6.8 mg, 18 µmol), and DIEA (8 µL, 46 µmol)
and coupled to 24 (1.0 mg, 0.03 µmol DNA loading) as
described for 25. The coupling step was repeated once. MALDI-
TOF MS calcd for ([M - H]-) 3192.4/3202.2/3275.4/3308.5/
3415.9, found 3191.2/3201.0/3274.3/3307.2/3414.6.
UV-Meltin g Exp er im en ts. UV-melting experiments were
acquired at 260 nm wavelength and 1 cm path length at
Su p p or tin g In for m a tion Ava ila ble: Proton NMR spectra
of 13, 16a /b, 18, 19, and 20; ROESY NMR spectra of 16a /b;
and MALDI-TOF mass spectra of 26, 27, and 30. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O049062O
J . Org. Chem, Vol. 69, No. 22, 2004 7551