Phenanthrene-derived DNA hairpin mimics

Article abstract of DOI:10.1002/hlca.200390256

Self-complementary oligodeoxynucleotides containing 3,6-disubstituted phenanthrenes adopt highly stable, hairpin-like structures. The thermodynamic stability of the hairpin mimics depends on the overall length of the phenanthrene building block. Hairpin loops composed of a phenanthrene-3,6-dicarboxamide and ethylene linkers were found to be optimal. The hairpin mimics are more stable than the analogous hairpins containing either a dT₄ or dA₄ tetraloop. Model studies indicate that the thermodynamic stability of the hairpin mimics is primarily due to aromatic stacking of the phenanthrene-3,6-dicarboxamide onto the adjoining base pair of the DNA duplex.

Full text of DOI:10.1002/hlca.200390256

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Phenanthrene-Derived DNA Hairpin Mimics
by Alfred Stutz, Simon M. Langenegger, and Robert H‰ner*
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012 Bern
(phone: ‡ 41316314382; e-mail: robert.haener@ioc.unibe.ch)
Self-complementary oligodeoxynucleotides containing 3,6-disubstituted phenanthrenes adopt highly
stable, hairpin-like structures. The thermodynamic stability of the hairpin mimics depends on the overall
length of the phenanthrene building block. Hairpin loops composed of a phenanthrene-3,6-dicarboxamide and
ethylene linkers were found to be optimal. The hairpin mimics are more stable than the analogous hairpins
containing either a dT4 or dA4 tetraloop. Model studies indicate that the thermodynamic stability of the hairpin
mimics is primarily due to aromatic stacking of the phenanthrene-3,6-dicarboxamide onto the adjoining base
pair of the DNA duplex.
Introduction.
Hairpins belong to the most common and most important
secondary-structure motives found in nucleic acids. [1] In functional RNA, they are
essential elements for the formation of the correct three-dimensional structure [2 5].
In particular, hairpins containing a four-base loop (tetraloop) are emerging as a class of
highly stable and structurally well-defined components of ribonucleic acids [1][6].
Hairpin formation is, although to a lesser extent, also observed in DNA. The
requirements for the formation of DNA hairpins [7] and cruciform structures [8] in
palindromic DNA sequences have been investigated in detail, and the involvement of
such structures in the regulation of gene expression has been discussed [9][10]. Due to
the importance of the hairpin motif, the replacement of the nucleotide loop by
synthetic, non-nucleosidic linkers has been a topic of thorough investigation. Thus, the
hairpin loop has been replaced with flexible, oligo(ethylene glycol) linkers in DNA [11]
and RNA [12][13] as well as with more rigid aromatic derivatives [14 17]. In
particular, (E)-stilbene-derived linkers led to a strong stabilization of the hairpin [15].
Structural analysis revealed a favorable stacking interaction between the stilbene and
the adjacent base pair of he stem [18].
We have previously shown that phenanthrene-3,6-carboxamide-modified oligonu-
cleotides form stable duplexes. Phenanthrene residues positioned in opposite sites can
significantly enhance duplex stability, most likely via interstrand aromatic stacking
[19]. In the course of this work, we found that self-complementary oligonucleotides
connected through a phenanthrene-3,6-carboxamide-derived linker form stable, hair-
pin-like structures. We report here the synthesis of phenanthrene-3,6-dicarboxamide
derivatives and their use as hairpin-loop mimics.
Results and Discussion. Preparation of Phenanthrene-Derived Building Blocks
and Incorporation into Oligonucleotides. Synthesis of the different phenanthrene-3,6-
dicarboxamide phosphoramidite building blocks was carried out in analogy to a

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previously reported procedure [19]. Thus, dimethyl phenanthrene-3,6-dicarboxylate
(1) was converted into the corresponding N,N'-bis(2-hydroxyethyl) and N,N'-bis(3-
hydroxypropyl)phenanthrene-3,6-carboxamides by reaction with the corresponding
amino alcohols (Scheme). Subsequent treatment of the crude intermediates with DMT-
Cl in pyridine resulted in a mixture of mono- and bis-protected derivatives. The desired
mono(4,4'-dimethoxytrityl) compounds 2 and 3 were isolated by column chromatog-
raphy and converted to the corresponding phosphoramidite building blocks 4 and 5,
respectively. Attempts to prepare the corresponding Bu-linked phosphoramidite in an
analogous way failed. Reaction of dimethyl phenanthrene-3,6-dicarboxylate with 4-
aminobutan-1-ol even under forced conditions (1808, 24h) did not afford the desired
N-(4-hydroxybutyl)phenanthrene-3,6-dicarboxamide. Therefore, diester 1 was hydro-
lyzed with aqueous NaOH. The crude sodium phenanthrene-3,6-dicarboxylate was
activated with BOP and further reacted with a 1:1 mixture of 4-[(4,4'-dimethoxy-
trityl)oxy]butylamine (6) and 4-aminobutan-1-ol in DMF. This procedure yielded,
albeit in a modest yield (22%), the desired 4,4'-monomethoxytritylated product 7,
which was converted into the phosphoramidite 8. The three different phenanthrene-
derived building blocks 4, 5, and 8 were incorporated into self-complementary
oligonucleotides according to the standard phosphoramidite procedure [20][21]. To
ensure a high incorporation yield of the modified building blocks, the coupling time was
prolonged to 1.5 min in the respective cycles (see Exper. Part). Deprotection (conc.
NH3, 558), followed by standard HPLC purification, yielded oligodeoxynucleotides 9
Scheme
a) 2-Aminoethanol (n ˆ 2) or 3-aminopropan-1-ol (n ˆ 3), 1758, 30 min. b) 4,4'-Dimethoxytrityl chloride
(DMT-Cl), Py, r.t. c) (iPr2N)2P(OCH2CH2CN), N,N-Diisopropylammonium 1H-tetrazolide, CH2Cl2, r.t. 3 h.
d) NaOH (2 equiv.), THF/EtOH/H2O 1 :1:1, reflux, 2 h. e) 4-(4,4'-Dimethoxytrityloxy)butanamine (6)/4-
aminobutan-1-ol 1 :1, iPr2NEt, [(benzotriazol-1-yl)oxy]tris(dimethylamino)phosphonium hexafluorophosphate
(BOP), DMF, r.t., 1 h.

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Table 1. Tm-Values of Hairpin Sequences Containing a Phenanthrene Linker in Comparison with Their
Counterparts Containing Either a T4- or A4-Hairpin Loop. Conditions: oligonucleotides 2.5 mm, 100 mm NaCl,
10 mm Tris¥ HCl pH 7.5.
Hairpin
Tm [8C]
9
10
11
5'-GCA ATT GC -P2- GC AAT TGC-3'
5'-GCA ATT GC -P3- GC AAT TGC-3'
5'-GCA ATT GC -P4- GC AAT TGC-3'
79.2
77.5
76.7
12
13
5'-GCA ATT GC TTTT GC AAT TGC-3'
5'-GCA ATT GC AAAA GC AAT TGC-3'
73.4
72.5
14
5'-ATT GC -P2- GC AAT-3'
65.7
15
16
5'-ATT GC TTTT GC AAT-3'
5'-ATT GC AAAA GC AAT-3'
60.0
57.6
P2 n ˆ 2
P3 n ˆ 3
P4 n ˆ 4
11 and 14 (Table 1), along with the corresponding control oligodeoxynucleotides 12, 13,
15, and 16, containing dT4 and dA4 sequences instead of the phenanthrenes.
Investigation of Phenanthrene-Derived Hairpins. In their pioneering work, Let-
singer and Wu [15], and Lewis et al. have demonstrated that (Z)- and (E)-stilbene
linkers can give rise to stable hairpin analogues. In a series of publications, they showed
that (E)-stilbene derivatives form more stable hairpin structures than the correspond-
ing (Z)-isomers. The higher stability of the (E)-isomers was attributed to the difference
of the spacer lengths between the (Z)- and the (E)-derivative [15][22]. This finding
can, however, also be explained by the fact that (Z)-stilbene derivatives are
considerably deviating from planarity and, thus, do not allow perfect stacking onto
the duplex stem [23][24]. Phenanthrene represents a planar analogue of (Z)-stilbene,
and should, therefore, result in more-favorable stacking effects. To investigate the
suitability of phenanthrene-3,6-dicarboxamide linkers as hairpin mimics, the self-
complementary oligonucleotides bearing Et, Pr, and Bu spacers (9, 10, and 11, resp.; see
Table 1) were prepared, along with the corresponding control oligonucleotides
containing either a dT4 or dA4 loop (12 or 13, resp.). Thermal-denaturation
experiments were carried out at 100 mm NaCl, pH 7.5 (10 mm Tris-HCl) with a
2.5 mm oligonucleotide concentration. As can be seen from the Tm data in Table 1, all
phenanthrene-linked oligonucleotides form a more-stable structure than the control
oligonucleotides. Thermal-denaturation curves revealed a single, sharp transition for all
oligonucleotides under the given experimental conditions (see Fig. 1). Several
observations supported the conclusion that the observed transitions correspond to
the melting of the respective hairpins. First, the experimental Tm values correspond well
with the calculated transition temperatures [25] for the hairpins 12 (Tm calc: 70.18,
found: 73.48) and 13 (Tm calc: 66.68, found: 72.58). Second, the Tm values were
independent from the concentration of the oligonucleotides over a range from 1 to 5 mm
(see Table 2), indicating a mono-molecular process. The hairpin mimic 9 containing the

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Fig. 1. Melting curves of the hairpin mimics 9 11 in comparison with the control hairpins 12 and 13. Conditions:
oligonucleotides 2.5 mm, 100 mm NaCl, 10 mm Tris¥ HCl, pH 7.5.
Table 2. Tm-Valuesa) of 9 13 Measured at Different Oligomer Concentrations
Hairpin
Tm [8C]
1 mm
2.5 mm
5 mm
9 (P2)
10 (P3)
11 (P4)
12 (dT4)
13 (dA4)
78.8
77.6
76.1
73.0
79.3
77.2
77.2
73.2
79.4
77.0
76.7
73.8
72.472.6
72.6
a) Conditions: 100 mm NaCl, 10 mm Tris¥ HCl pH 7.5.
Et-linked phenanthrene derivative P2 showed the highest Tm (79.28). The analogues 10
and 11 containing longer spacers had slightly lower Tm values (77.58 and 76.78 for P3
and P4, resp.). A similar dependence of the stability of hairpin mimics on the linker
length has been reported for stilbene-derived linkers. [18] The optimal phenanthrene
building block, P2, was further incorporated into a shorter, self-complementary
oligonucleotide (Table 1, oligonucleotide 14) and compared to its dT4 and dA4
analogues (oligodeoxynucleotides 15 and 16). Again, the phenanthrene-derived
hairpin mimic showed a significantly higher thermodynamic stability (DTm ˆ 5.78 and
8.18, resp.). Circular dichroism (CD) spectral analysis of the hairpin mimic 14 is in full
agreement with an overall B-conformation (data not shown).
Structural Model of the Phenanthrene Hairpin Mimic. A model of the P2-containing
hairpin 9 is shown in Fig. 2. The conformation represents a local-minimum structure

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obtained with Hyperchem starting from B form DNA by using the amber force-field.
According to this model, the concave side of the phenanthrene moiety is directed
towards the minor groove of the B form helix. The distance between the phenanthrene
and the adjacent GC base pair is ca. 3.3 3.4ä, i.e., well within the distance normally
observed for base stacking in B-DNA. The increase of the transition temperature of the
phenanthrene-modified hairpin analogues compared with their natural dA4- or dT4-
containing counterparts can, therefore, be attributed to the optimal stacking interaction
with the first base pair of the stem. The distance between the two phenanthrene-linked
phosphates in the modelled structure of hairpin 9 is ca. 16.0 ä. This distance is shorter
than the 17.8 ä found in a (E)-stilbene-derived hairpin structure [18], but lies well
within the range of diametrical PÀP distances (15.0 to 16.9 ä) observed in other,
reported DNA hairpin structures [26][27]. This relatively large range of PÀP distances
can also serve as an explanation for the observation that the steady increase in the
linker length from oligomers 9 11 (Table 1) does not affect the stability in a dramatic
way. All these factors point towards the stacking effects as the major contribution to the
stability of the hairpin-like structures.
Fig. 2. Amber-Minimized structure of hairpin 9 containing P2 (dark blue). Left: view along the helical axis;
right: view perpendicular to the helical axis. The GC base pair adjacent to the phenanthrene is shown in light
blue.
Conclusions. Self-complementary oligodeoxynucleotides containing 3,6-disubsti-
tuted phenanthrenes adopt highly stable, hairpin-like structures. The thermodynamic
stability of these hairpin mimics is higher than that of comparable hairpins with a dT4 or
dA4 tetraloop. Spectroscopic analysis of the phenanthrene-modified hairpins shows no
structural deviation from canonical B form DNA. Model studies, as well as the relative
insensitivity of the structural stability towards the linker length, indicate that the
thermodynamic stability of the hairpin mimics is primarily due to aromatic stacking of
the phenanthrene-3,6-dicarboxamide onto the adjoining base pair of the DNA duplex.
Financial support by the Swiss National Science Foundation is gratefully acknowledged.

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Experimental Part
General. Chemicals, solvents, and reagents for reactions were from Acros, Aldrich, or Fluka, and were of the
highest quality available. Compound 1, 3, and 5 were prepared as described in [19]. (Cyanoethoxy)bis(dime-
thylamino)phosphine was prepared according to [28]. Solvents for extraction and chromatography were of
technical grade and distilled prior to use. TLC: silica-gel 60 F254 glass plates (Merck); visualization by UVand/or
A) by dipping in a soln. of anisaldehyde (10 ml), conc. H2SO4 (10 ml), and AcOH (2 ml) in EtOH (180 ml) or
B) cerium(IV) sulfate (3 mm)/ammonium molybdate (250 mm) in aq. H2SO4 (10%), followed by heating. Flash
column chromatography (CC): silica gel 60 (40 63 mm, 230 400 mesh, Fluka) at low pressure. The
chromatography of acid-sensitive compounds was carried out with eluent containing 2% Et3N. 1H- and
13C-NMR: Bruker AC-300 or Bruker AMX-400, d values in ppm (solvent signals as internal standards), J [Hz];
31P-NMR: Bruker AMX-400, d values in ppm (85% H3PO4 as external standard). ESI-MS: VG Platform single
quadrupole ESI mass spectrometer. HR-MALDI-MS: IonSpec Ultima FTMS mass spectrometer, 3,5-
dihydroxybenzoic acid as matrix, pos. mode; Abbreviations: DMT: 4,4'-dimethoxytrityl; BOP: [(Benzotria-
zol-1-yl)oxy]tris-(dimethylamino)phosphonium hexafluorophosphate.
Synthesis of Phenanthrene-Derived Phosphoramidite Building Blocks. N-{2-[(4,4'-Dimethoxytrityl)oxy]eth-
yl}-N'-(2-hydroxyethyl)phenanthrene-3,6-carboxamide (2). Dimethyl phenanthrene-3,6-dicarboxylate (1; 1.38 g,
4.69 mmol) was dissolved in 2-aminoethanol (6.9 ml, 0.11 mol) and heated to 1758 for 30 min. Then, the excess
of 2-aminoethanol was removed under reduced pressure to give the crude N,N'-bis(2-hydroxyethyl)phenan-
threne-3,6-dicarboxylate as a yellow solid (TLC (AcOEt/MeOH 9 :1): Rf 0.24). At r.t., this intermediate was
dissolved in dry pyridine (12 ml). Then, a soln. of DMT-Cl (1.52 g, 4.5 mmol) in pyridine (6 ml) was added over
1 h. The mixture was stirred for another 2 h, and then the solvent was removed under reduced pressure. The
residue was taken up in CH2Cl2 and washed first with 10% citric acid and then with sat. aq. NaHCO3 soln., dried
(MgSO4), and evaporated to give a highly viscous oil. The purification by CC (silica gel; AcOEt/hexane 8 :2 !
AcOEt (‡1% NEt3)) furnished 2 (1.391 g, 2.12 mmol, 45%) as colorless foam. TLC (AcOEt/hexane): Rf 0.29.
UV (MeCN): 315 (15900), 309 (13800), 303 (16400), 288 (10100), 278 (sh, 13600), 258 (54500), 254 (52900), 251
(53800), 242 (49800), 237 (50000), 220 (36200). 1H-NMR (400 MHz, CDCl3): 2.43 (br. s, OH); 3.42 (t, J ˆ 5.3,
ROCH2); 3.68 (m, CH2OH); 3.73 (s, 2 MeO); 3.74( t, J ˆ 4.8, (partially hidden), CH2N); 3.91 (t, J ˆ 4.9, CH2N');
6.80 (d, J ˆ 8.8, 4arom. H); 7.12 7.82 ( m, 17 arom. H); 8.80, 8.85 (2s, 2 CONH). 13C-NMR (100 MHz, CDCl3):
41.0, 43.7 (2t, CH2N, CH2N'); 55.6 (q, MeO); 62.5 (t, CH2OH); 67.3 (t, CH2OCAr3); 86.7 (s, Ar3C); 113.6, 122.0,
122.7, 124.8, 125.8, 127.3, 128.1, 128.2, 128.3, 128.5, 128.9, 129.0 (12d, arom. C); 129.7, 130.0 (2s, arom. C); 130.4
(d, arom. C); 132.65, 132.71, 134.01, 134.02 136.4, 145.2, 158.9 (7s, arom. C); 168.4, 169.2 (2s, CONH). HR-
‡
MALDI-MS: 677.2630 ([M ‡ Na] ; calc. 677.2622).
N-{2-[(Diisopropylamino)(2-cyanoethyl)phosphinoxy]ethyl}-N'-{2-[(4,4'-dimethoxytrityl)oxy]ethyl}-phen-
anthrene-3,6-carboxamide (4). At r.t., a soln. of 2 (1.29 g, 1.97 mmol) in CH2Cl2 (4ml) was added slowly (over
30 min) to a suspension of diisopropylammonium 1H-tetrazolide (472 mg, 2.76 mmol) and (2-cyanoethyl)bis-
(diisopropylamino)phosphine (713 mg, 2.36 mmol) in CH2Cl2 (6 ml). After 4h at r.t., the mixture was diluted
with CH2Cl2 and washed with sat. aq. NaHCO3 soln. The org. layer was dried (K2CO3) and evaporated under
reduced pressure. Purification of the resulting oil by CC (silica gel; AcOEt/hexane 2 :3 ! 3 :2 (‡2% Et3N))
furnished 4 (1.284g, 1.502 mmol, 76%) as colorless foam. TLC (AcOEt/hexane/NEt 3 5 :4:1): Rf 0.36. UV
(MeCN): 316 (15800), 310 (13600), 303 (16500), 288 (10000), 279 (sh., 13600), 258 (54600), 255 (52800), 251
(53700), 242 (49500), 237 (49900), 220 (36800). 1H-NMR (400 MHz, CDCl3): 1.19 (d, J ˆ 6.5, 2 Me2CHN); 2.62
(t, J ˆ 6.0, CH2CN); 3.4 4.0 ( m, OCH2CH2CN, 2 Me2CHN, NCH2CH2, N'CH2CH2); 3.75 (s, 2 MeO); 6.91 (d,
J ˆ 8.3, 4arom. H); 6.9 8.1 ( m, 17 arom. H); 9.23 (s, NH, N'H). 13C-NMR (100 MHz, CDCl3): 21.0 (t, J(C,P) ˆ
5, CH2CN); 25.0, 25.1 (2q, J(C,P) ˆ 4, Me2CH); 40.9 (t, CH2N); 41.6 (t, J(C,P) ˆ 7.0, POCH2CH2N'); 43.5 (d,
J(C,P) ˆ 12, Me2CH); 55.6 (q, MeO); 58.9 (t, J(C,P) ˆ 20, POCH2); 62.7 (t, J(C,P) ˆ 15, POCH2); 62.9 (t,
DMTOCH2); 86.6 (s, Ar3C); 113.6 (d, arom. C); 118.8 (s, CN); 122.5, 122.7, 125.3, 125.7, 127.2, 127.3, 128.3, 128.5,
128.6, 129.4, 129.5, 130.4 (12d, arom. C); 133.2, 133.3, 134.38, 134.43, 136.4, 145.3, 158.9 (7s, arom. C); 167.88,
‡
168.00 (2 s, CONH). 31P-NMR (216 MHz, CDCl3): 149.53. HR-ESI-MS (pos. mode): 873.4220 ([M ‡ NH4] ;
calc. 873.4230).
4-[(4,4'-Dimethoxytrityl)oxy]butanamine (6). A mixture of 4-aminobutan-1-ol (1 g, 11.22 mmol) and
phthalic anhydride (1.662 g, 11.22 mmol) was kept at 1458 for 2 h. After cooling to r.t., the resulting yellow oil
was dissolved in pyridine (20 ml), then DMT-Cl (4.06 g, 12 mmol) was added. After 2 h at r.t., the soln. was
diluted with AcOEt/hexane 1:1 (100 ml), washed twice with sat. aq. NaHCO3 soln., dried (MgSO4), and
evaporated. The remaining solid was dissolved in MeOH (40 ml) together with NH2NH2 ¥ H2O (2.5 g, 50 mmol).
This soln. was stirred at 408 for 4h. Then, the precipitate was filtered off, and the filtrate was reduced to ca.

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Helvetica Chimica Acta Vol. 86 (2003)
10 ml. This soln. was diluted with CH2CH2 (150 ml), washed with 2m Na2CO3, dried (MgSO4), and evaporated
to give the crude product as a yellow oil. After purification by CC (20 g silica gel; CH2Cl2 ! CH2Cl2 ‡ 3%
MeOH (‡ 5% NEt3)), 6 (2.89 g, 7.4mmol, 66%) was obtained as highly viscous, colorless oil. TLC (CH 2Cl2/
MeOH/NEt3 8 :1:1): Rf 0.6. UV (MeOH): 272 (8300), 256 (6700); 235 (17100), 221 (13900). 1H-NMR
(300 MHz, CDCl3): 1.46 (br. s, NH2); 1.55, 1.64(2 m, NCH2CH2CH2CH2O); 2.68 (t, J ˆ 6.6, CH2N); 3.07 (t, J ˆ
6.5 CH2O); 3.80 (s, 2 MeO); 6.83 (d, J ˆ 8.8, 4arom. H); 7.1 7.5 ( m, 9 arom. H). 13C-NMR (100 MHz, CDCl3):
26.5, 27.7 (2t, CH2CH2CH2CH2); 40.4 (t, CH2N); 54.3 (q, MeO); 62.1 (t, CH2ODMT); 84.9 (s, Ar3C); 112.1,
125.8, 126.9, 127.3, 127.5, 129.1 (6d, arom. C); 135.7, 144.4, 157.5 (3s, arom. C). HR-ESI-MS (pos. mode):
‡
783.4390 ([2M ‡ H] ; calc. 783.4373).
N-{4-[(4,4'-Dimethoxytrityl)oxy]butyl}-N'-(4-hydroxybutyl))phenanthrene-3,6-carboxamid (7). At r.t., a
suspension of 1 (0.5 g, 1.7 mmol) in EtOH/THF 1:1 (10 ml) was treated with 1m aq. NaOH (3.4ml) and heated
to reflux for 2 h. The solvent was then removed under reduced pressure, and the remaining solid was dried under
high vacuum. The crude intermediate was suspended in DMF (8.5 ml)/iPr2NEt (0.6 ml) and stirred for 30 min
after the addition of BOP (827 mg, 1.87 mmol). Then, a soln. of 6 (800 mg, 2.04mmol) and 4-aminobutan-1-ol
(182 mg, 2.04mmol) in CH 2Cl2 (3 ml) was added, and the suspension was stirred for another h. The mixture was
then diluted with AcOEt/hexane 1:1 (100 ml), washed with sat. aq. NaHCO3 soln., dried (MgSO4), and
evaporated. The purification by CC (AcOEt) afforded 7 (269 mg, 0.378 mmol, 22%) as colorless foam. TLC
(AcOEt/MeOH 95 :5): Rf 0.54. UV (MeCN): 315 (14900), 309 (12700), 302 (15400), 292 (9600), 258 (51200),
254 (49900), 250 (51100), 241 (47800), 237 (48400) 220 (35900), 205 (58000). 1H-NMR (300 MHz, CDCl3): 1.75
(m, 2 CH2CH2CH2CH2); 3.13 (t, J ˆ 5.5, DMTOCH2); 3.52 (m, CH2N, CH2N', OH); 3.75 (s, 2 MeO); 3.76 (m,
CH2OH); 6.80 (d, J ˆ 8.8, 4arom. H); 7.00 7.92 ( m, 17 arom. H); 8.87, 8.88 (2s, NH, N'H). 13C-NMR(100 MHz,
CDCl3): 26.2, 26.7, 27.8, 30.1 (4t, NCH2CH2CH2, N'CH2CH2CH2); 40.3, 40.4 (2t, CH2N, CH2N'); 55.2 (q, 2 MeO);
62.5, 63.1 (2t, CH2ODMT, CH2OH); 85.9 (s, Ar3C); 113.0, 121.4, 121.8, 124.7, 125.3, 126.6, 126.7, 127.5, 127.8,
128.1, 128.2, 128.5 (12d, arom. C); 129.4, 129.5 (2s, arom. C); 130.0 (d, arom. C); 132.3, 132.5, 133.4, 133.5, 136.5,
‡
145.2, 158.3 (7s, arom. C); 167.9, 168.0 (2s, 2 CONH). HR-MALDI-MS: 733.3256 ([M ‡ Na] ; calc. 733.3248).
N-{4-[(Diisopropylamino)(2-cyanoethyl)phosphinoxy]butyl}-N'-{4-[(4,4'-dimethoxytrityl)oxy]butyl}phen-
anthrene-3,6-carboxamide (8). A soln. of 7 (370 mg, 0.52 mmol) in CH2Cl2 (2.5 ml) was added at r.t. over 30 min
to a suspension of diisopropylammonium 1H-tetrazolide (125 mg, 0.73 mmol) and (2-cyanoethyl)bis(diisopro-
pylamino)phosphine (188 mg, 0.63 mmol) in CH2Cl2 (3 ml). The mixture was stirred at r.t. for 4h, then diluted
with CH2Cl2 and washed with sat. aq. NaHCO3 soln. The org. layer was dried (K2CO3), and the solvent was
removed under reduced pressure. After CC (silica gel; AcOEt/hexane 2 :3 ! 3 :2 (‡2% Et3N)), 8 (1.284g,
1.502 mmol, 76%) was obtained as colorless foam. TLC (AcOEt/hexane/Et3N 5 :4:1): Rf 0.32. UV (MeCN):
315 (15700), 309 (13300), 303 (16200), 286 (10400), 278 (sh, 13600), 259 (55400), 255 (53500), 250 (54500), 241
(49500), 237 (49800) 220 (37100). 1H-NMR (400 MHz, CDCl3): 1.08, 1.09 (2d, J ˆ 6.8, Me2CH); 1.6 1.8 (m,
CH2CH2CH2CH2); 2.54( t, J ˆ 6.3, CH2CN); 3.06 (t, J ˆ 6.0, CH2ODMT); 3.4 3.8 ( m, P(OCH2)2, CH2N, CH2N',
NCHMe2); 3.67 (s, 2 MeO); 6.6 8.0 (m, 21 arom. H); 5.05 (s, NH, N'H). 13C-NMR (100 MHz, CDCl3): 20.9 (t,
J(C,P) ˆ 8, CH2CN); 25.0, 25.1 (2q, J(C,P) ˆ 4, Me2CH); 26.7, 27.2, 28.2 (3t, CH2CH2CH2CH2); 29.1 (t, J(C,P) ˆ
7, POCH2CH2); 40.4, 40.7 (2t, CH2N, CH2N'); 43.4 (d, J(C,P) ˆ 12, PNCH); 55.6 (q, MeO); 58.6 (t, J(C,P) ˆ 20,
POCH2CH2CN); 63.5 (t, CH2ODMT); 63.6 (t, J(C,P) ˆ 17, POCH2CH2CH2); 86.3 (s, Ar3C); 113.4( d, arom. C);
118.5 (s, CN); 122.4, 122.5, 125.4, 125.6, 127.0, 128.2, 128.4, 128.5, 129.3, 130.4 (10d, arom. C); 133.3, 133.4, 134.2,
134.3, 136.9, 145.6, 158.7 (7s, arom. C); 167.96, 167.99 (2s, CONH). 31P-NMR (216 MHz, CDCl3): 148.68. HR-
‡
MALDI-MS: 933.4338 ([M ‡ Na] ; calc. 933.4327).
Synthesis and Analysis of Oligonucleotides. Standard nucleoside phosphoramidites were from Chemgenes
(Ashland, MA). Oligonucleotides were synthesized on a 392 DNA/RNA Synthesizer (Applied Biosystems)
according to the standard phosphoramidite chemistry [20][21] on a 1.0-mmol scale (−trityl-off× mode). The
coupling time was extended to 1.5 min for 4, 5, and 8. Coupling efficiencies were > 98% for all building blocks,
including 4, 5, and 8. The oligomers were detached and deprotected under standard conditions (conc. aq. NH3,
558, 16 h). The crude oligomers were purified by anion-exchange HPLC: MonoQ HR 5/5 (Pharmacia); flow
1 ml/min; eluent A: 20 mm Na3PO4 in H2O (pH 11.5); eluent B: 20 mm Na3PO4, 2m NaCl in H2O (pH 11.5);
elution at r.t.; detection at 260, 280, and 320 nm and desalted over SepPak cartridges (Waters, Milford, USA).
All oligonucleotides were analyzed by ESI-MS. The masses were found to be within 0.0005% of the expected
mass. The extinction coefficients at 260 nm (e260) were calculated with −Biopolymer Calculator× (http://
paris.chem.yale.edu/extinct.html). For the analogues P2, P3, and P4, resp., an e260 value of 49300 was estimated,
according to the UV spectra of N,N'-bis(2-hydroxyethyl)phenanthrene-3,6-carboxamide (data not shown,
measured in H2O, 0.1m NaCl, 10 mm Tris¥ HCl pH 7.5.

Helvetica Chimica Acta Vol. 86 (2003)
3163
Thermal-Denaturation Experiments. UV Melting curves were determined at 260 nm on a Varian Cary 3e
spectrophotometer equipped with a Peltier block and Varian WinUV software. Unless otherwise indicated, the
melting curves were measured at an oligomer concentration of 2.5 mm in 10 mm Tris¥ HCl, 100 mm NaCl, pH 7.5.
A heating/cooling cycle in the temp. range of 08 958 or 208 958 was applied with a temp. gradient of 0.58/min.
All ramps were indicating equilibrium melting processes. Tm Values were defined as the maximum of the first
derivative of the melting curve.
RNA-Folding Prediction and Modelling. Secondary-structure predictions and Tm calculations of 12 and 13
were obtained by using RNA mfold ([25], http://www.bioinfo.rpi.edu/applications/mfold/). The conformation of
hairpin 9 (see Fig. 2) was optimized by the amber force field (Hyperchem 7.0, Hypercube, Waterloo, Ontario).
REFERENCES
[1] R. T. Batey, R. P. Rambo, J. A. Doudna, Angew. Chem., Int. Ed. 1999, 38, 2326.
[2] H. W. Pley, K. M. Flaherty, D. B. Mckay, Nature 1994, 372, 111.
[3] J. H. Gate, A. R. Gooding, E. Podell, K. H. Zhou, B. L. Golden, A. A. Szewczak, C. E. Kundrot, T. R.
Cech, J. A. Doudna, Science 1996, 273, 1696.
[4] J. H. Cate, A. R. Gooding, E. Podell, K. H. Zhou, B. L. Golden, C. E. Kundrot, T. R. Cech, J. A. Doudna,
Science 1996, 273, 1678.
[5] M. Perbandt, A. Nolte, S. Lorenz, R. Bald, C. Betzel, V. A. Erdmann, FEBS Lett. 1998, 429, 211.
[6] P. B. Moore, Annu. Rev. Biochem. 1999, 68, 287.
[7] C. W. Hilbers, C. A. G. Haasnoot, S. H. Debruin, J. J. M. Joordens, G. A. Vandermarel, J. H. Vanboom,
Biochimie 1985, 67, 685.
[8] A. I. H. Murchie, D. M. J. Lilley, Methods Enzymol. 1992, 211, 158.
[9] D. T. Weaver, M. L. Depamphilis, J. Mol. Biol. 1984, 180, 961.
[10] U. R. Muller, W. M. Fitch, Nature 1982, 298, 582.
[11] M. Durand, K. Chevrie, M. Chassignol, N. T. Thuong, J. C. Maurizot, Nucleic Acids Res. 1990, 18, 6353.
[12] M. Y. X. Ma, L. S. Reid, S. C. Climie, W. C. Lin, R. Kuperman, M. Sumnersmith, R. W. Barnett,
Biochemistry 1993, 32, 1751.
[13] W. Pils, R. Micura, Nucleic Acids Res. 2000, 28, 1859.
[14] M. Salunkhe, T. F. Wu, R. L. Letsinger, J. Am. Chem. Soc. 1992, 114, 8768.
[15] R. L. Letsinger, T. F. Wu, J. Am. Chem. Soc. 1995, 117, 7323.
[16] F. D. Lewis, R. S. Kalgutkar, Y. S. Wu, X. Y. Liu, J. Q. Liu, R. T. Hayes, S. E. Miller, M. R. Wasielewski, J.
Am. Chem. Soc. 2000, 122, 12346.
[17] K. Yamana, A. Yoshikawa, H. Nakano, Tetrahedron Lett. 1996 , 37, 637.
[18] F. D. Lewis, X. Liu, Y. Wu, S. E. Miller, M. R. Wasielewski, R. L. Letsinger, R. Sanishvili, A. Joachimiak, V.
Tereshko, M. Egli, J. Am. Chem. Soc. 1999, 121, 9905.
[19] S. M. Langenegger, R. H‰ner, Helv. Chim. Acta 2002, 85, 3414.
[20] S. L. Beaucage, M. H. Caruthers, Tetrahedron Lett. 1981, 22, 1859.
[21] N. D. Sinha, J. Biernat, J. Mcmanus, H. Koster, Nucleic Acids Res. 1984, 12, 4539.
[22] F. D. Lewis, X. Y. Liu, J. Am. Chem. Soc. 1999, 121, 11928.
[23] M. Traetteberg, E. B. Frantsen, J. Mol. Struct. 1975, 26, 69.
[24] C. H. Choi, M. Kertesz, J. Phys. Chem. A 1997, 101, 3823.
[25] M. Zuker, Methods Enzymol. 1989, 180, 262.
[26] M. Ghosh, N. Rumpal, U. Varshney, K. V. Chary, Eur. J. Biochem. 2002, 269, 1886.
[27] N. B. Ulyanov, W. R. Bauer, T. L. James, J. Biomol. NMR 2002, 22, 265.
[28] W. Bannwarth, A. Treciak, Helv. Chim. Acta 1987, 70, 175.
Received May 6, 2003