Design, synthesis, and hybridisation of water-soluble, peptoid nucleic acid oligomers tagged with thymine

Article abstract of DOI:10.1002/ejoc.200900781

The preparation of a new class of backbone-modified PNA mimetic incorporating thymine is described. Target dipeptoid monomer 21 was synthesised from an N-[2-(thyniin-l-yl)ethyl]glycinate ester and a properly protected iminodiacetic acid. The distinctive s

Full text of DOI:10.1002/ejoc.200900781

FULL PAPER
DOI: 10.1002/ejoc.200900781
Design, Synthesis, and Hybridisation of Water-Soluble, Peptoid Nucleic Acid
Oligomers Tagged with Thymine
Rosa Zarra,[a] Daniela Montesarchio,[b] Cinzia Coppola,[b] Giuseppe Bifulco,[c]
Simone Di Micco,[c] Irene Izzo,*[a] and Francesco De Riccardis*[a]
Keywords: Peptide nucleic acids / Peptoids / DNA recognition
The preparation of a new class of backbone-modified PNA
mimetic incorporating thymine is described. Target dipeptoid
monomer 21 was synthesised from an N-[2-(thymin-1-yl)eth-
yl]glycinate ester and a properly protected iminodiacetic
acid. The distinctive structural motif in the backbone is a
carboxy group, inserted to impart water solubility to the
oligomer. Two achiral oligopeptoid sequences (8-mer and 12-
mer), characterised by the shift of the amide carbonyl group
ation studies showed that the two homopyrimidine oligopep-
toids do not effectively hybridise with complementary se-
quences of DNA and RNA or fully synthetic (2,4-diamino)-
triazin-6-yl-tagged peptoid 22. A possible reason could re-
side in the concurrent unfavourable influence of the anionic
N-(carboxymethyl) moieties and the flexible nucleobase/
backbone ethylene linker.
away from the nucleobase, were efficiently assembled ac- (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
cording to solid-phase synthesis protocols. Thermal denatur- Germany, 2009)
other hand, negatively charged PNA derivatives (hypNA-
Introduction
pPNA)[13] are showing promise in antisense experiments in
Peptide nucleic acid (PNA, 1; Figure 1) is a synthetic nu-
cell culture, demonstrating that anionic backbones do not
cleic acid mimic built on a polyamide backbone.[1] The re-
necessarily deteriorate the hybridisation properties of nucleo-
markable hybridisation and biostability properties make
pseudopeptides.
PNA of considerable interest in several areas of chemistry,
biology and medicine.[2] However, despite its many excellent
properties, PNA has two serious limitations: low water solu-
bility[3] and poor cellular uptake.[4] A significant number
of PNA derivatives have been designed and synthesised to
circumvent these drawbacks, leading to a variety of promis-
ing analogs.[5] Common strategies include conjugation of
PNA termini with polar moieties,[6] elaboration of PNA/
DNA chimeras[7] and addition of polar side chains onto the
pseudopeptide PNA backbone.[1,2,8] In the last approach,
the enantiotopic H atoms of the N-(2-aminoethyl)glycine
are often replaced by charged groups.[9] When -lysine[10a]
or -arginine[5b,10b,10c] is introduced, a stabilizing effect is
Figure 1. Structures of PNA (1), N-methyl peptoid nucleic acid (2),
observed in the PNA-DNA hybridisation, both for confor-
triazine-tagged N-(carboxymethyl) peptoid nucleic acid (3) and thy-
mational preferences [a (P)-helix is formed][11] and for the
mine-based N-(carboxymethyl) peptoid nucleic acid (4). B = nu-
presence of positive charges along the backbone.[12] On the
cleobase.
[a] Dipartimento di Chimica, Università di Salerno,
Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Fax: +39-089-969603
Since no conclusive data are yet available concerning the
relevance of specific molecular features in the PNA struc-
ture, and considering the excellent hybridisation properties
of neutral and polyanionic, pure-α-peptoid[14] backbones
(2[15] and 3,[16] Figure 1), we decided to investigate novel
PNA backbones consisting of N-alkylated glycine oligo-
mers with alternating N1-(2-aminoethyl)thymine and N-
(carboxymethyl) residues (4). We opted for a PNA mimic
made of an integral peptoid backbone, in order to compare
E-mail: dericca@unisa.it
iizzo@unisa.it
[b] Dipartimento di Chimica Organica e Biochimica, Università di
Napoli Federico II, Complesso Universitario di Monte S. An-
gelo,
Via Cintia 4, 80126 Napoli, Italy
[c] Dipartimento di Scienze Farmaceutiche, Università di Salerno,
Via Ponte Don Melillo, 84084 Fisciano (SA), Italy
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.200900781.
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the recognition abilities of 4 (once mixed with the proper their pairing properties, hybridisation assays with comple-
polyadenylated strand), with the known, fully peptoidic 2[15] mentary sequences of DNA, RNA and synthetic (2,4-di-
and 3,[16] both capable of hybridising nucleic acids.
amino)triazin-6-yl-tagged oligopeptoid 3 were carried out.
The exchange of the backbone methylene group with the Preliminary theoretical studies were performed on models
side chain carbonyl group, the elimination of the secondary based on the reported, solution-phase NMR structure of
amide linkage and the anionic nature of the oligomers, con- the antiparallel DNA/PNA duplex carrying the sequence
stitute risky features for successful hybridisation experi- 5Ј-d(GACATAGC)-3Ј/NH2(CTGTATCG)-COOH.[17b]
ments. The first two traits, in particular, abolish the H-bond
donor capacity of the backbone. However, no spectral[17] or
crystallographic evidence[18] has ever supported a supposed
“strong H bond between the side-chain carbonyl oxygen
atom (α to the methylene group carrying the base) and the
backbone amide of the next residue” (5; Figure 2), as re-
ported by Almarsson and co-workers.[14]
Figure 4. Structures of target oligomers 6 and 7.
Results and Discussion
Figure 2. Hypothesised H bonds between the side-chain carbonyl
oxygen atoms and the backbone amides.
Computational Studies
We performed preliminary molecular dynamics (MD)
Furthermore, two enlightening studies on PNAs showing
Nγ alkylation,[19] indicated that the N–H H-bonding was
not crucial for the stabilization of the PNA/nucleic acid
double strand. Thus, we reasoned that N-alkylation of the
backbone with polar groups, in a pure peptoid framework,
could represent a novel strategy for a fine-tuning of PNA
properties and provide a new way to overcome the pressing
water-solubility issue.
In the design of the proposed peptoid oligomer 4, we
chose the highly hydrophilic N-(carboxymethyl) side chain.
The excellent pairing properties showed by the (2,4-di-
amino)triazin-6-yl/N-methylencarboxy-tagged peptoid 3[16]
encouraged us to insert anionic residues. The thymine resi-
dues were attached to the oligoglycine backbone through
an ethyl linker, so that they were separated by the same
number of bonds found in nucleic acids (Figure 3, bold
black bonds). The spacing between the recognition units on
the peptoid framework was the same number of bonds as
that present in DNA (Figure 3, bold grey bonds).
studies in order to evaluate the ability of the thymine-tagged
oligoglycine strand to form a stable duplex with polyadenyl-
ated DNA strands. We used the solution-phase NMR struc-
ture of the 5Ј-d(GACATAGC)-3Ј/NH2(CTGTATCG)-
COOH[17b] duplex as a reference model to estimate the per-
formance of calculation parameters (simulation time, sol-
vation model) and correctly interpret the computational
outcomes. On the double-stranded octanucleotide under in-
vestigation, we performed MD calculations (10 ns, 300 K)
in the presence of water [generalized Born/surface area
(GB/SA) model].[21] The inspection of the MD trajectory
revealed that the duplex was stable throughout the simula-
tion, even though we used no distance restraints in the cal-
culations. We observed, at 10 ns, a little perturbation at the
edge of the duplex, consisting in a deviation from the plan-
arity of two base pairs (Figure 5, NMR model). The MD
results showed that, in the NMR model, the duplex pre-
served its arrangement with respect to the set of H bonds
between the base pairs. This outcome confirmed the sta-
bility of the reference structure and the reliability of our
computational methodology.
At this point, we turned our attention to models pres-
enting an oligoglycine backbone and exchanged the side-
chain amide carbonyl groups with the methylene groups of
the polyamide framework, obtaining a peptoid skeleton. In
a first model, we substituted the Nγ with a methyl group
(Figure 6, model C, similar to the Xu oligomer,[15] except
for the nucleobases). In a second model, we linked the same
nitrogen atom to an N-(carboxymethyl) side chain, giving
the backbone present in our target oligomers 6 and 7 (Fig-
ure 6, model D).
Figure 3. Backbone thymine positioning in the designed peptoid
oligomer (A) and A-type DNA (B).[20]
In the present work, we report the synthesis, the solid-
The results of the MD calculations (10 ns, 300 K) showed
phase oligomerisation and the base-pairing behaviour of that the two models preserved the gross double-helix ar-
two oligomeric peptoids 6 and 7 (Figure 4), carrying eight rangement (Figure 5, models C and D). However, the pres-
and twelve repetitive units, respectively. In order to evaluate ence, in the repetitive, six-atom, peptoid framework of four
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Water-Soluble, Peptoid Nucleic Acid Oligomers Tagged with Thymine
simulation (Figure 5, models E and F). In particular, we
considered the fact that the computational studies on model
E (coincident with the Xu thyminated peptoid oligomer,
which was known to hybridise DNA with a Tm of
42 °C)[15] predicted a stable duplex as a propitious auspice
for the correct annealing of the designed, carboxylated,
congener F, which, in silico, behaved in a similar way. Being
encouraged by the theoretical outcomes, we promptly
moved towards the chemical synthesis of oligomers 6 and
7.[22]
Chemistry
From a retrosynthetic point of view, we traced target 6
and 7 back to the properly protected dipeptoid unit 8 (Fig-
ure 7). The synthon 8 could be easily assembled from an
iminodiacetic acid derivative (9a) and a 1Ј-thyminylated
ethylglycine residue (9b).
Figure 5. View of hybrid duplex structures for the solution-phase
NMR conformation (NMR model), C, D, E and F models, after
MD calculation (10 ns, 300 K). The models are represented by
tubes and coloured by atom type (C, gray; polar H, white; N, dark
blue; O, red; P, purple). The nonpolar H atoms have been omitted
for clarity.
Figure 7. Retrosynthetic scheme for oligomers 6 and 7. T =
thymine. Pg = protecting group.
The synthesis of the monomer started with the elabora-
tion of the required N-[2-(thymin-1-yl)ethyl]glycine (16), as
shown in Scheme 1. We prepared the N-deprotected build-
ing block in five steps and 26% overall yield. The Mitsu-
nobu reaction[23] of 14 – easily assembled through the re-
duction of a diprotected iminodiacetic acid – with the
known N3-benzoylthymine[24] gave thyminated adduct 15.
This we deprotected at the two nitrogen atoms in the pres-
ence of trifluoroacetic acid to yield α-amino ester 16.
Figure 6. Models used in the MD calculations. For the nucleobase
(B) in C and D, see the solution-phase NMR structure solved by
Eriksson and Nielsen.[17b] T = thymine.
sp2-hybridised atoms, instead of three in the related PNA,
the addition of N-methyl or N-acetate groups and, above
all, the fact that there were conformationally demanding
ethylene linkers separating the nucleobase from the back-
bone, induced in models C and D a slightly larger helical
pitch in the peptoid counterpart of the double helix. We
note that the anionic N-(carboxymethyl) side chain of
model D, establishing favourable electrostatic interactions
with the solvent, positively contributed to duplex stability
(when compared with model C, see the Supporting Infor-
mation). Similarly to the DNA phosphates, the carboxylates
projected the nucleobases towards the hydrophobic core of
the duplex, thus reinforcing double-strand stability.
The last step in the preliminary computational studies
was to substitute all the nucleobases of models C and D
with thymine units. In order to compensate the loss of stabi-
lizing G–C H-bonds, we decided to elongate the duplex
with a couple of extra base-pairing units, obtaining a de-
camer of thymine/adenine pairs. Once again, the integrity
Scheme 1. Synthesis of N-[2-(thymin-1-yl)ethyl]glycine ethyl ester
(16). Reagents and conditions: (a) (i) DIPEA, DMF, (ii) (Boc)2O,
Et3N, CH2Cl2, 88%; (b) H2, Pd/C, AcOH, quantitative; (c)
BH3·SMe2, THF, 69%; (d) N3-benzoylthymine, DEAD, PPh3,
of the duplexes was maintained throughout the whole 10 ns THF; (e) TFA, CH2Cl2, 43%, two steps. T = thymine.
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The
2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetrameth- the investigated cases, the CD profiles for the mixtures
yluronium hexafluorophosphate (HATU) promoted con- simply resembled the spectra of the polyadenylated, single-
densation of 16 with the Fmoc/tBu-protected acid counter- stranded DNA or RNA counterparts, indicating no rel-
part 19 (Scheme 2) gave the expected dipeptoid 20. Careful evant interaction. Additionally, we did not observe the typi-
LiOH-mediated hydrolysis preserved the base-labile Fmoc cal diagnostic curve for a duplex Ǟ single-strand transition
group, affording target dipeptoid monomer 21.
in the CD-monitored thermal denaturation experiments
carried out at λ = 260 nm (see the Supporting Information).
In addition, we could not observe any signals in the imino
region (δ = 10–15 ppm) diagnostic of Watson–Crick base
1
pairings in the H NMR spectra of a 1:1 7/d(A)16 mixture
(0.1 m in each strand), even at low temperatures (see the
Supporting Information).
In order to check for intrasystem pairing, we performed
annealing experiments by mixing 7 with the (2,4-diamino)-
triazin-6-yl-tagged dodecamer 22 (Figure 8).[16] As before,
UV melting experiments showed no clear transition from
double to single strands upon heating, indicating that no
effective binding occurred between the thyminylated strand
7 and triazine-tagged oligomer 22.
Scheme 2. Synthesis of N-[N-(fluoren-9-ylmethoxycarbonyl)-N-
(tert-butoxycarbonylmethyl)glycyl]-N-[2-(thymin-1-yl)ethyl]glycine
(21). Reagents and conditions: (a) (i) DIPEA, DMF, (ii) LiOH,
1,4-dioxane, (iii) FmocCl, NaHCO3, 88%; (b) 16, HATU, DIPEA,
DMF, 95%; (c) LiOH, 1,4-dioxane, 0 °C, 69 %.
We manually assembled peptoid oligomers 6 and 7 in a
stepwise fashion on a Rink amide solid support using
HATU as the coupling agent. We monitored the coupling
efficiency by measuring the absorbance of the dibenzoful-
vene–piperidine adduct (spectroscopically evidenced after
every deprotection step). The average coupling yield was
higher than 97% (after a double coupling). Once we had
condensed all the monomers, we detached the oligomers
from the solid support using trifluoroacetic acid. The acid
efficiently cleaved also the tBu protecting groups from the
carboxy groups. We purified the water-soluble oligomers by
HPLC on an anion-exchange column, followed by gel-fil-
tration chromatography on a Sephadex G25 column, yield-
ing desired 6 and 7 as pure compounds (see the Supporting
Information). We determined the purity of the synthesised
peptoids by HPLC (anion-exchange column) and their
identity by MALDI-TOF mass spectrometry.
Figure 8. Structure of the dodecamer 22.
Even though we did not obtain positive evidence of
hybridisation to nucleic acids, the current investigation enu-
cleates the limits of the peptoid backbone in the search for
novel, biologically active nucleopeptides. The favourable
theoretical premise did not consider the loss of entropy due
to duplex formation. The delicate balance between enthalpy
gain (due to interstrand H-bond formation) and reduction
of conformational freedom probably plays against the inter-
and intrasystem base pairing. The preliminary theoretical
studies were, in fact, executed on a preformed duplex and
lacked the dynamic information on possible double-strand
formation. The presence of a peptoid backbone is compati-
ble with nucleic-acid hybridisation. This is clearly asserted
by the Xu[15] and Eschenmoser[16] contributions. Nonethe-
less, small backbone/nucleobase variations may have large
Hybridisation Studies
In order to verify the ability of peptoids 6 and 7 to bind and unpredictable effects on the nucleosidated peptoid con-
complementary DNA and/or RNA, we performed UV- formation and on binding to nucleic acids. A similar effect
monitored melting experiments by mixing the water-solu- has been recently evidenced in the intriguing (and not fully
ble, thyminylated oligomers with either d(A)16 or poly-r(A), understood) Nγ-ε-aminoalkyl-PNA, length-dependent,
(5 µ peptoid concentration, λ = 260 nm). Our preliminary DNA hybridisation.[19b] In this systematic study, it was
thermal denaturation analysis of 6 or 7 in the presence of found that short, polar side chains (protruding from the
complementary strands of d(A)16 or poly-r(A) gave no Nγ atom of peptoid-based PNAs) negatively influenced the
characteristic S-shaped curve, even after varying the salt hybridisation properties of modified PNAs, whereas longer,
conditions and the mixing ratios, demonstrating that no polar side chains positively modulated nucleic-acid binding.
hybridisation occurred in any case (see the Supporting In- The reported data did not clarify the reason for this effect,
formation). We further confirmed the absence of efficient but it seems that factors different from electrostatic interac-
hybridisation to nucleic acids by CD analysis. In fact, in all tion are at play.
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Water-Soluble, Peptoid Nucleic Acid Oligomers Tagged with Thymine
vent peak (CHCl3: δ = 7.26 ppm; 13CDCl3: δ: = 77.0 ppm;
CD2HOD: δ = 3.34 ppm; 13CD3OD: δ = 49.0 ppm; 13CCD2Cl4: δ
=
Conclusions
72.1 ppm; C2DHCl4: δ = δ =
5.80 ppm, or 13CCD2Cl4:
The present work indicates that the annealing properties
of oligoglycine backbones can be sensitive to small struc- 72.1 ppm), and the multiplicity of each signal is designated by the
tural variations. The substitution of an Nγ-methyl group
following abbreviations: s, singlet; d, doublet; t, triplet; q, quartet;
quint, quintuplet; m, multiplet and br., broad. Coupling constants
with an Nγ-(carboxymethyl) or an Nγ-H[14] suppresses nu-
cleic-acid binding, as demonstrated herein [Nγ-(carboxy-
(J) are given in Hz. Homonuclear decoupling, COSY-45 and DEPT
experiments completed the full assignment of each signal. High-
methyl)] and by the work of Almarsson and co-workers
resolution ESI mass spectra were obtained with a Q-Star Applied
(Nγ-H).[14] In particular, in our case, we believe that restric-
Biosystem mass spectrometer. ESI-MS analysis in positive-ion
tions imposed by a charged oligoglycine backbone and the
higher conformational freedom of the nucleobases nega-
tively contributed to the pairing properties of the synthetic
nucleopeptoids.
mode was performed by using a Finnigan LCQ Deca ion trap mass
spectrometer (ThermoFinnigan, San José, CA, USA), and the mass
spectra were acquired and processed by using the Xcalibur software
provided by Thermo Finnigan. Samples were dissolved in 1:1
In conclusion, in this paper we have chemically built an CH3OH/H2O, 0.1% formic acid and infused into the ESI source
by using a syringe pump; the flow rate was 5 µL/min. The capillary
voltage was set at 4.0 V, the spray voltage at 5 kV and the tube lens
offset at –40 V. The capillary temperature was 220 °C. MALDI-
TOF mass spectrometric analyses were performed with a PerSep-
tive Biosystems Voyager-De Pro MALDI mass spectrometer in the
linear mode by using α-cyano-4-hydroxycinnamic acid, picolinic/2-
hydroxypicolinic acid or sinapinic acid as the matrix. HPLC analy-
ses were performed with a Beckman System Gold instrument
equipped with a UV detector module 166 and a Shimadzu Chro-
matopac C-R6A integrator. UV measurements were carried out
orthogonally protected N-(carboxymethyl)-N-[2-(thymin-1-
yl)ethyl] dipeptoid unit. It was efficiently assembled accord-
ing to standard solid-phase synthesis protocols to give the
corresponding octa- and dodecamers. The hybridisation
studies indicated that the Nγ substitution in a pure peptoid
scaffold can play an essential role in the overall annealing
process. Whereas thyminylated oligomers with a methyl
group linked to the Nγ-position bind to their complemen-
tary target, other substituents can prevent the double-
strand formation. A previous study by Nielsen,[12] reporting with a Jasco V-530 UV spectrophotometer equipped with a Jasco
ETC-505T temperature controller unit. Circular dichroism (CD)
experiments were recorded with a JASCO J-715 spectrophotometer
equipped with a thermostatically controlled cuvette holder (JASCO
PTC-348) in a 0.1 cm path length cuvette. CD spectra were col-
lected from 220 to 300 nm, at 20 nm/min, with a response time
of 16 s and at a 1 nm bandwidth. The oligonucleotide d(A)16 was
assembled with a Millipore Cyclone Plus DNA synthesiser by using
commercially available 3Ј-O-(2-cyanoethyl)-N,N-diisopropylphos-
phoramidite 2Ј-deoxyribonucleosides as building blocks and puri-
fied through sequential anion-exchange HPLC/gel filtration
chromatography. Polyadenylated ribonucleic acid (60 µ in base)
was purchased from Sigma–Aldrich and used as received.
the synthesis of a PNA analogue devoid of the tertiary
amide carbonyl group, emphasized the importance of the
amide group(s) in the PNA for their contribution to back-
bone rigidity. The present study suggests that overly rigid
polar frameworks can hamper duplex formation.
Finally, on the basis of the acquired information, new
oligomers with the more dependable N-(2-aminoethyl)gly-
cyl backbone (aegPNA), bearing Nγ polar chains, will be
synthesised and studied with the aim of discovering novel
PNA mimics with improved pharmacological traits.
Computational Details: All calculations were performed by using
the MACROMODEL 8.5 software package[25] and the AMBER
force field.[26] In all geometry-optimization and MD calculations,
the GB/SA[21] solvent treatment was used, mimicking the presence
of H2O, for reducing the artifacts derived from the absence of the
solvent. The solution-phase NMR structure of 5Ј-d(GACATAGC)-
3Ј-NH2-(CTGTATCG)-COOH[17b] (PDB archive code 1PDT) was
used as a template to build the models C–F (Figure 6); the acetyl
linker between the base and nitrogen atom of the backbone was
replaced by an ethyl linker, and the N-(2-aminoethyl) group was
converted into a glycine residue. For model E and F, the sequence
was converted into a hybrid (A-T)10 double strand. In particular,
the new bases were inserted by maintaining the planarity of the
substituted aromatic rings and the H-bond distances for A–T base
pairs in the solution-phase NMR structure. The geometries of the
peptoid bonds in the strands were built as all-trans and used in
the subsequent simulations of 10 ns (see below). All built peptoid
backbone atoms of models C–F were optimized by using Polak-
Ribiere Conjugate Gradient (PRCG, 9ϫ106 steps, convergence
threshold 0.001 kJmol–1 Å–1) by fixing the atoms of the DNA
strand and the purine/pyrimide bases. The geometries so obtained
Experimental Section
General Methods: All reactions involving air- or moisture-sensitive
reagents were carried out under dry argon or nitrogen by using
freshly distilled solvents. Tetrahydrofuran (THF) was distilled from
LiAlH4 under argon. Toluene and CH2Cl2 were distilled from
CaH2. Glassware was flame-dried (0.05 Torr) prior to use. When
necessary, compounds were dried in vacuo over P2O5 or by the
azeotropic removal of water with toluene under reduced pressure.
Starting materials and reagents purchased from commercial suppli-
ers were generally used without purification unless otherwise men-
tioned. Reaction temperatures were measured externally; reactions
were monitored by TLC with Merck silica gel plates (0.25 mm) and
visualized by UV light, I2 or by spraying with H2SO4/Ce(SO4)2,
phosphomolybdic acid or ninhydrin solutions and drying. Flash
chromatography was performed with Merck silica gel 60 (particle
size: 0.040–0.063 mm), and the solvents employed were of analyti-
cal grade. Yields refer to chromatographically and spectroscopically
(1H and 13C NMR) pure materials. The NMR spectra were re-
corded with Bruker DRX 400, (1H at 400.13 MHz, 13C at
100.03 MHz), Bruker DRX 250 (1H at 250.13 MHz, 13C at
62.89 MHz) and Bruker DRX 300 (1H at 300.10 MHz, 13C at for the newly built strands underwent MD (100 ps, 300 K) to relax
75.50 MHz) spectrometers. 1H NMR spectra of the duplex
7/d(A)16 were recorded with a Bruker Avance 600 spectrometer.
Chemical shifts (δ) are reported in ppm relative to the residual sol-
the double helix. After 1 ps of equilibration at 300 K, a 1.5 fs inte-
gration time step was used, and 20 structures were collected. Dif-
ferent conformers for structures C–F were obtained and used for
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MD calculations without distance restraints to evaluate the sta-
bility of the hybrid duplexes. A temperature of 300 K was used
during the dynamics simulations over a period of 10 ns, and a stan-
dard constant temperature velocity Verlet algorithm was used to
integrate the equation of motions. The SHAKE algorithm[27] was
used and applied to all bonds. A 1.5 fs integration time step was
used. After 1 ps of equilibration at 300 K, the MD trajectories were
obtained by sampling every 40 ps.
16: To a solution of 14 (0.08 g, 0.32 mmol), N3-benzoylthymine
(0.11 g, 0.48 mmol) and PPh3 (0.21 g, 0.81 mmol) in dry THF
(3 mL) at 0 °C, DEAD (0.13 mL, 0.81 mmol) was added. The reac-
tion mixture was stirred overnight. The solvent was evaporated un-
der reduced pressure, and the crude 15 was dissolved in CH2Cl2/
TFA (1:3, 2.4 mL) and stirred overnight. Volatiles were removed in
vacuo, and the oily residue was precipitated in cold diethyl ether.
The white amorphous precipitate was washed again with diethyl
ether, dried (high vacuum, room temp., overnight) and used with-
out further purification (36 mg, 43% over two steps). 1H NMR
(300 MHz, CD3OD): δ = 1.35 (t, J = 7.1 Hz, 3 H, CH3), 1.93 (s, 3
H, CH3), 3.42 (t, J = 5.8 Hz, 2 H, HNCH2CH2), 4.03 (s, 2 H,
OCCH2NH), 4.11 (t, J = 5.8 Hz, 2 H, HNCH2CH2), 4.34 (q, J =
7.1 Hz, 2 H, CH2CH3), 7.44 (s, 1 H, HC=) ppm. 13C NMR
(75 MHz, CD3OD): δ = 12.2, 14.3, 46.0, 48.1, 49.0 (CD3OD, over-
lapped), 63.6, 112.1, 118.2 (q, J = 290 Hz, CF3COO–), 142.4, 153.9,
163.0, (q, J = 38.3 Hz, CF3COO–), 166.7, 167.6 ppm. MS (ESI):
m/z (%) = 278.1 (100) [M + Na]+. HRMS (ESI): calcd. for
C11H17N3NaO4 278.1117; found 278.1115.
Chemistry
12: To a solution of glycine ethyl ester (1.00 g, 7.19 mmol) in DMF
(6 mL), DIPEA (2.50 mL, 14.4 mmol) was added, followed by ben-
zyl bromoacetate (0.82 g, 3.60 mmol) 10 min later. The reaction
mixture was stirred at room temp. overnight, concentrated in
vacuo, dissolved in AcOEt (10 mL) and washed with NaCl/H2O
(1:1). The aqueous phase was extracted with AcOEt (2ϫ10 mL).
The combined organic phases were washed with brine, dried
(MgSO4) and concentrated in vacuo, affording a crude material,
which was used in the next step without purification. To a solution
of the crude material in CH2Cl2 (10 mL), Et3N (0.650 mL,
4.68 mmol) and (Boc)2O (1.02 g, 4.68 mmol) were added. The reac-
tion mixture was stirred at room temp. overnight, concentrated in
vacuo and flash-chromatographed (10–60% diethyl ether in petro-
leum ether) to give pure 12 as a viscous oil (1.11 g, 88%). 1H NMR
(250 MHz, CDCl3, mixture of rotamers): δ = 1.25 and 1.26 (t, J =
19: To a solution of glycine tBu ester (6.57, 39.2 mmol) in DMF,
(35 mL) DIPEA was added (78.4 mmol, 13.6 mL). The reaction
mixture was stirred for 10 min, and methyl bromoacetate (1.86 mL,
19.6 mmol) was added dropwise over 20 min. The reaction mixture
was stirred at room temp. for 24 h, concentrated in vacuo, dissolved
in AcOEt (10 mL) and washed with NaCl/H2O (1:1). The aqueous
7.1 Hz, 3 H, CH3), 1.37 and 1.42 [s, 9 H, C(CH3)3], 3.98 and 4.03 phase was extracted with AcOEt (2ϫ10 mL). The combined or-
(s, 2 H, CH2NBoc), 4.09 and 4.15 (s, 4 H, CH2NBoc, CH2CH3,
overlapped), 5.14 and 5.15 (s, 2 H, CH2Ph), 7.33 (m, 5 H, Ar-H)
ppm. 13C NMR (62.5 MHz, CDCl3): δ = 14.1 (br. s), 28.0 (3 C),
49.0, 49.6, 61.0, 66.7, 81.0, 128.3 (2 C), 128.5 (3 C), 135.3, 155.0,
ganic phases were washed with brine, dried (MgSO4) and concen-
trated in vacuo, affording crude 19, which was used in the next step
without purification. To a solution of 19 in 1,4-dioxane at 0 °C,
LiOH·H2O (0.91 g, 21.6 mmol) was added. After the reaction mix-
169.6 (2 C) ppm. MS (ESI): m/z (%) = 352.2 (100) [M + H]+. ture was stirred at 0 °C for 3 h, NaHCO3 (1.8 g, 21.6 mmol) and
HRMS (ESI): calcd. for C18H26NO6 352.1760; found 352.1754.
Fmoc-Cl (5.58 g, 21.6 mmol) were added. The reaction mixture was
stirred at room temp. for 24 h, acidified to pH = 3–4 with saturated
aq. NaHSO4 and extracted with AcOEt (200 mLϫ3). The com-
bined organic phases were washed with H2O (100 mLϫ2) and
brine (100 mL), dried (Na2SO4), concentrated in vacuo and flash-
chromatographed (40% diethyl ether in petroleum ether to 10%
methanol in AcOEt, all containing 1% of AcOH) to give 19 as a
13: To a solution of 12 (1.86 g, 5.29 mmol) in AcOEt (30 mL), a
catalytic amount of 10% Pd/C (50 mg) and AcOH (0.1 mL) were
added. The flask was evacuated and flushed with H2 three times.
The reaction mixture was stirred vigorously under H2 overnight.
The mixture was filtered through a short pad of Celite® and con-
centrated, affording 13 as a viscous oil (1.40 g, quantitative). 1H
NMR (400 MHz, CDCl3, mixture of rotamers): δ = 1.32 (t, J =
7.1 Hz, 3 H, CH3), 1.44 and 1.46 [s, 9 H, C(CH3)3], 3.96 and 4.00
(s, 2 H, CH2NBoc), 4.10 and 4.11 (s, 2 H, CH2NBoc), 4.27 (q, J =
7.1 Hz, 2 H, CH2CH3) ppm. MS (ESI): m/z (%) = 262.1 (100) [M
+ H]+. HRMS (ESI): calcd. for C11H20NO6 262.1291; found
262.1297.
1
viscous oil (7.08 g, 88%). H NMR (400 MHz, CDCl3, mixture of
rotamers): δ = 1.48 and 1.49 [s, 9 H, C(CH3)3], 4.02 and 4.06 (s, 2
H, CH2NFmoc), 4.08 and 4.19 (s, 2 H, CH2NFmoc), 4.23 (m, 1
H, CHFmoc), 4.43 (m, 2 H, CH2Fmoc), 7.31 (m, 2 H, Ar-H), 7.37
(m, 2 H, Ar-H), 7.56 (m, 2 H, Ar-H), 7.75 (m, 2 H, Ar-H) ppm.
13C NMR (75 MHz, CDCl3, mixture of rotamers): δ = 28.0 (3 C),
46.9 and 47.0 (CHFmoc), 50.4, 50.6 and 51.1 (CH2NFmoc), 68.5,
68.7 (CH2Fmoc), 83.2 and 83.7 [C(CH3)3], 120.0, 125.0, 127.1,
127.8, 141.2, 143.6, 155.9, 169.4, 170.6, 172.4, 173.1 ppm. MS
(ESI): m/z (%) = 412.2 (100) [M + H]+. HRMS (ESI): calcd. for
C23H26NO6 412.1760; found 412.1758.
14: To a solution of 13 (1.81 g, 6.93 mmol) in dry THF (35 mL) at
0 °C, BH3·SMe2 (13.8 mL, 27.7 mmol, 2  in THF) was added. The
reaction mixture was stirred overnight. The solvent was evaporated
under reduced pressure, saturated aq. NH4Cl was added, and the
aqueous layer was extracted three times with CH2Cl2 and one time
with AcOEt. The combined organic phases were washed with brine,
dried (MgSO4) and concentrated in vacuo, affording a crude mate-
rial, which was flash-chromatographed (50–90% diethyl ether in
petroleum ether) to give 14 as a viscous oil (1.18 g, 69%). 1H NMR
(300 MHz, CDCl3, mixture of rotamers): δ = 1.26 and 1.28 (t, J =
7.1 Hz, 3 H, CH3), 1.45 and 1.46 [s, 9 H, C(CH3)3], 3.43 (m, 2 H,
20: To a solution of 16 (0.15 g, 0.40 mmol) in DMF (3 mL), a solu-
tion of 20 (0.32 g, 0.78 mmol), DIPEA (0.260 mL, 1.52 mmol) and
HATU (0.29 g, 0.76 mmol) in DMF (3 mL) was added. The reac-
tion mixture was stirred at room temp. overnight, concentrated in
vacuo, dissolved in CH2Cl2 (16 mL) and washed with saturated aq.
NH4Cl (8 mL). The aqueous phase was extracted with CH2Cl2
(2ϫ16 mL). The combined organic phases were washed with aq.
HOCH2CH2NBoc), 3.68 and 3.73 (m, 2 H, HOCH2CH2NBoc), NaHCO3 and brine, dried (MgSO4) and concentrated in vacuo,
3.90 and 3.95 (s, 2 H, OCCH2NBoc), 4.21 (q, J = 7.1 Hz, 2 H, affording a crude mixture, which was flash-chromatographed (0–
CH2CH3) ppm. 13C NMR (100 MHz, CDCl3, mixture of rota-
mers): δ = 13.9, 14.1, 28.0 and 28.1 [C(CH3)3], 50.4, 50.9, 51.9,
52.1, 60.8, 61.0, 61.3, 61.4, 80.4 [C(CH3)3], 155.3, 155.5, 171.6,
171.9 ppm. MS (ESI): m/z (%) = 270.1 (100) [M + Na]+. HRMS
(ESI): calcd. for C11H21NNaO5 270.1317; found 270.1321.
10% methanol in CH2Cl2, containing 1% of AcOH) to give 20 as
a viscous oil (0.25 g, 95%). 1H NMR (250 MHz, C2D2Cl4, 360 K):
δ = 1.13 (t, J = 7.1 Hz, 3 H, CH3), 1.33 [s, 9 H, C(CH3)3], 1.72 (s,
3 H, CH3), 3.46 (t, J = 5.8 Hz, 2 H, NCH2CH2N), 3.70 (t, J =
5.8 Hz, 2 H, NCH2CH2N), 3.85–4.11 [m, 9 H, CH2NFmoc (3 C),
6118
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6113–6120

Water-Soluble, Peptoid Nucleic Acid Oligomers Tagged with Thymine
CHFmoc, CH2CH3, overlapped], 4.28 (d, J = 7.0 Hz, 2 H,
CH2Fmoc), 6.90 (s, 1 H, HC=), 7.12–7.27 (m, 4 H, Ar-H), 7.41 (m,
2 H, Ar-H), 7.59 (m, 2 H, Ar-H), 7.82 (br. s, 1 H, NH) ppm. 13C
NMR (75 MHz, C2D2Cl4, 360 K): δ = 10.1, 12.3, 26.4 (3 C), 44.5,
45.6, 45.9, 46.9, 48.2, 49.2, 60.2, 66.6, 80.3, 109.0, 118.2 (2 C), 123.2
(2 C), 125.4 (2 C), 126.0 (2 C), 139.0, 139.5 (2 C), 142.1 (2 C),
148.8, 154.3, 162.3, 167.0 (3 C) ppm. MS (ESI): m/z (%) = 671.2
was precipitated in cold diethyl ether (30 mL). The white precipitate
was washed again with diethyl ether and dried (high vacuum, room
temp., overnight).
Purification and Characterization of Oligomers 6 and 7: The crude
detached oligomers were analyzed and then purified by HPLC (an-
alytical anion-exchange column; NUCLEOGEN SAX 1000–8/46,
5 µm; linear gradient from 0 to 100% of B solution in A solution
over 60 min for the 8-mer 6 and over 30 min for the 12-mer 7;
flowrate of 0.8 mL/min; detection at λ = 260 nm; solution A =
1 m KH2PO4, 20% CH3CN, pH = 7.0; solution B = 350 m
KH2PO4, 1  KCl, 20% CH3CN, pH = 7.0). In both cases, the
HPLC profiles showed a single major peak at 13.5 min (for 6) and
11.2 min (for 7). The corresponding peaks were collected, concen-
trated and then desalted by size-exclusion chromatography on a
Sephadex G25 column eluted with water/ethanol (4:1, v/v). After
HPLC purification, 58 OD of pure 6 and 61 OD of 7 could be
isolated (OD: optical density unit, equal to the amount of material,
which – dissolved in 1.0 mL – gives an absorbance of 1.0 at λ =
260 nm), as determined from UV measurements at λ = 260 nm of
the samples dissolved in H2O at 90 °C. By HPLC analysis (Nucleo-
sil 100-5 C18 Supelco analytical column, 250ϫ4.6 mm, 5 µm; lin-
ear gradient from 0 to 100% CH3CN over 30 min in H2O; flowrate
of 0.8 mL/min; detection at λ = 260 nm), the isolated oligomers
were Ͼ98% pure. MALDI-TOF MS analysis confirmed the ex-
pected structures for the two samples, with unique signals at the
following m/z values: 6: MALDI-TOF MS (positive mode): m/z (%)
= 2610.95 (100) [M + H]+, (70) [M + Na]+. 7: MALDI-TOF MS
(positive mode): m/z (%) = 3907.30 (100) [M + H]+, (70) [M +
Na]+, (40) [M + K]+.
(100) [M
671.2693; found 671.2691.
+
Na]+. HRMS (ESI): calcd. for C34H40N4NaO9
21: To a solution of 20 (0.50 g, 0.77 mmol) in 1,4-dioxane (6.5 mL)
at 0 °C, LiOH (0.071 g, 1.69 mmol) in H2O (6.5 mL) was added.
The reaction mixture was stirred for 50 min, acidified to pH = 3–
4
with saturated aq. NaHSO4 and extracted with CH2Cl2
(50 mLϫ3) and AcOEt (50 mLϫ1). The combined organic phases
were washed with H2O (100 mLϫ2) and brine (100 mL), dried
(Na2SO4), concentrated in vacuo and flash-chromatographed (2–
20% methanol in CH2Cl2, containing 1% of AcOH) to give 21
1
as an amorphous white solid (0.33 g, 69%). H NMR (250 MHz,
C2D2Cl4, 360 K): δ = 1.27 [s, 2 H, C(CH3)3], 1.68 (s, 3 H, CH3),
3.45–4.28 (m,
7 H NCH2CH2N, NCH2CH2N, CH2NFmoc,
CHFmoc, overlapped), 6.90 (s, 1 H, HC=), 7.12–7.27 (m, 4 H, Ar-
H), 7.41 (m, 2 H, Ar-H), 7.59 (m, 2 H, Ar-H), 7.81 (br. s, 1 H,
NH) ppm. 13C NMR (75 MHz, C2D2Cl4, 360 K): δ = 10.0, 26.4 (3
C), 44.7, 44.8, 45.6, 47.2 (2 C), 48.3, 66.6, 80.4, 109.3, 118.2 (2 C),
123.2 (2 C), 125.4 (2 C), 126.4 (2 C), 139.1, 139.4 (2 C), 142.1 (2
C), 149.3, 154.4, 162.2, 166.8, 168.0, 168.6 ppm. MS (ESI): m/z (%)
= 643.2 (100) [M + Na]+. HRMS (ESI): calcd. for C32H36N4NaO9
643.2380; found 643.2383.
Solid-Phase Synthesis of Oligomers 6 and 7: Linear peptide se-
quences were synthesised by using standard, manual, Fmoc-based,
solid-phase, peptide synthesis protocols. Rink-amide resin (0.075 g,
Aldrich, crosslinked with 1% DVB, 100–200 mesh, 0.5–1.5 mmol/
g) was swelled in dry N-methylpyrrolidinone (NMP, 1 mL) for
20 min and washed once with dry NMP (1 mL). The resin was
incubated twice with 20% piperidine/NMP (1 mL) on a shaker
platform for 3 min and 7 min respectively, followed by extensive
washes with NMP (9ϫ3 mL). Compound 21 (0.014 mmol) in dry
NMP (0.6 mL), DIPEA (0.028 mmol, 5 µL, 2 equiv.) and HATU
(0.014 mmol) were added, and the mixture was agitated on a shaker
platform at room temperature for 2 h and then washed with dry
NMP (2ϫ1 mL). The resin loaded with the first monomer was
incubated with a mixture of NMP, 2,6-lutidine and Ac2O (1.78 mL,
120 µL, 100 µL) on a shaker platform for 30 min and then exten-
sively washed with NMP (6ϫ1 mL). The deprotection of the sec-
ond amino acid was effected as described above. The yields of load-
ing and of the following coupling steps were evaluated by interpol-
ating the absorption of the dibenzofulvene–piperidine adduct (λmax
= 301 nm, ε = 7800 –1 cm–1), obtained in the Fmoc deprotection
step. After loading of the first monomer, all subsequent N-Fmoc
monomer-addition and Fmoc-deprotection steps were performed
as follows, until the desired oligomer length was obtained. The
resin was incubated twice with a solution of N-Fmoc-protected
monomer (0.052 mmol), HATU (0.050 mmol) and DIPEA (18 µL,
0.010 mmol) in dry NMP (0.6 mL) on a shaker platform for 1 h
and then extensively washed with NMP (9ϫ1 mL). The chloranil
test was performed, and once the coupling was complete, the Fmoc
group was deprotected with piperidine as described above, and the
resin was washed again to prepare it for the next coupling (the
average coupling yield was Ͼ97% with double coupling). The
oligomer was cleaved from the resin in 1 mL of TFA/m-cresol
(95:5). The cleavage was performed twice on a shaker platform at
room temp. for 30 min The resin was then filtered off, and the fil-
trates were concentrated under a nitrogen flux. The oily residue
Hybridisation Experiments: Hybridisation experiments were carried
out by recording UV- and CD-monitored melting curves at λ =
260 nm of the appropriate solutions of peptoid 6 or 7 mixed with
either polyadenyl DNA 16-mer, polyadenyl RNA or the triazine-
tagged peptoid 22 (no CD-monitored melting curves were recorded
for the last mixture). The UV melting curves were recorded in 1 mL
of the test solution in Teflon-stoppered quartz cuvettes of 1 cm
optical path length. The concentration of the oligomers was deter-
mined spectrophotometrically at λ = 260 nm and 90 °C, by using
the following molar extinction coefficients, calculated for the un-
stacked oligomers: 15400 (A),[28] 8800 (T)[28] and 4200 (2,4-diami-
notriazine) cm–1 –1.[16] The melting experiments were performed
in two different aq. solutions: solution A: 140 m NaCl, 10 m
NaH2PO4, pH = 7.4 and solution B: 1.0  NaCl, pH = 7.0. Mix-
tures of the peptoid 6 or 7 were prepared and analyzed in the fol-
lowing ratios with the complementary DNA, RNA or triazine-
tagged oligomer 22: 1:1 (at approximately 5 µ for each strand),
1:2 (at approximately 5 µ:10 µ) and 2:1 (at approximately
5 µ:2.5 µ), respectively. The resulting solutions were then heated
at 85 °C for 10 min and then slowly cooled and maintained at 5 °C
overnight. After thermal equilibration at 10 °C, the UV absorption
at λ = 260 nm was monitored as a function of the temperature,
which increased at a rate of 0.5 °C/min over the range 20–80 °C.
For all the mixtures prepared as described above, CD spectra were
then recorded at 20 °C in a 0.1 cm path length cuvette. The wave-
length was varied from 220 to 340 nm at 20 nmmin–1. CD spectra
were recorded with a response of 16 s at a 2.0 nm bandwidth and
normalised by subtraction of the background scan of buffer alone.
The molar ellipticity was calculated from the equation [ϑ] = ϑ/cl
where ϑ is the relative intensity, c the concentration of the oligomer
and l is the path length of the cell in cm.
Supporting Information (see footnote on the first page of this arti-
cle): Computational details, purification of oligomer 6 and 7,
Eur. J. Org. Chem. 2009, 6113–6120
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
6119

I. Izzo, F. De Riccardis et al.
FULL PAPER
Natl. Acad. Sci. USA 2003, 100, 12021–12026; b) P. Zhou, M.
Wang, L. Du, G. W. Fisher, A. Waggoner, D. H. Ly, J. Am.
Chem. Soc. 2003, 125, 6878–6879; c) A. Dragulescu-Andrasi,
P. Zhou, G. He, D. H. Ly, Chem. Commun. 2005, 244–246.
[11] a) T. Tedeschi, S. Sforza, R. Corradini, A. Dossena, R. March-
elli, Chirality 2005, 17(S1), S196–S204; b) T. Tedeschi, S.
Sforza, R. Corradini, R. Marchelli, Tetrahedron Lett. 2005, 46,
8395–8399.
UV-monitored melting curves of 7 mixed with d(A)16, poly-r(A)
and (2,4-diamino)triazin-6-yl-tagged dodecamer 22, CD spectra for
7 mixed with d(A)16 and H NMR spectra (600 MHz) of 7 mixed
1
with d(A)16 in a 1:1 ratio at 0.1 m in each strand.
Acknowledgments
[12] B. Hyrup, M. Egholm, O. Buchardt, P. E. Nielsen, Bioorg.
Med. Chem. Lett. 1996, 6, 1083–1088. Recent work suggests
that the duplex stability is independent of the positive charges
on the side chain. See ref.[5a,19b]
[13] V. A. Efimov, M. Choob, A. A. Buryakova, D. Phelan, O. G.
Chakhmakhcheva, Nucleosides Nucleotides Nucleic Acids 2001,
20, 419–428.
Financial support from the University of Salerno is appreciated.
The authors wish to thank Prof. A. Eschenmoser and Dr. R. Krish-
namurthy (The Scripps Research Institute) for the generous gift of
22 and Ms. Graziella Ianniello and Mr. Davide Farina for experi-
mental work.
[14] O. Almarsson, T. C. Bruice, J. Kerr, R. N. Zuckermann, Proc.
Natl. Acad. Sci. USA 1993, 90, 7518–7522. This paper de-
scribed the first example of a mixed peptide/peptoid oligomer.
[15] Y. Wu, J.-C. Xu, Chin. Chem. Lett. 2000, 11, 771–774.
[16] G. K. Mittapalli, R. R. Kondireddi, H. Xiong, O. Munoz, B.
Han, F. De Riccardis, R. Krishnamurthy, A. Eschenmoser, An-
gew. Chem. Int. Ed. 2007, 46, 2470–2477.
[17] a) S. C. Brown, S. A. Thomson, J. M. Veal, D. G. Davis, Sci-
ence 1994, 265, 777–780; b) M. Eriksson, P. E. Nielsen, Nat.
Struct. Biol. 1996, 3, 410–413.
[1] P. E. Nielsen, U. Koppelhus, F. Beck in Pseudo-Peptides in
Drug Discovery (Ed.: P. E. Nielsen), Wiley-VCH, Weinheim,
2004, pp. 153–191.
[2] a) A. Porcheddu, G. Giacomelli, Curr. Med. Chem. 2005, 12,
2561–2599; b) P. E. Nielsen, Curr. Opin. Biotechnol. 2001, 12,
16–20; c) E. Uhlmann, A. Peyman, G. Breipohl, D. W. Will,
Angew. Chem. Int. Ed. 1998, 37, 2796–2823; d) P. E. Nielsen,
G. Haaima, Chem. Soc. Rev. 1997, 26, 73–78.
[3] J. P. Vernille, L. C. Kovell, J. W. Schneider, Bioconjugate Chem.
2004, 15, 1314–1321.
[4] a) U. Koppelhus, P. E. Nielsen, Adv. Drug Delivery Rev. 2003,
55, 267–280; b) P. Wittung, J. Kajanus, K. Edwards, P. E. Niel-
sen, B. Nordén, B. G. Malmstrom, FEBS Lett. 1995, 365, 27–
29.
[5] a) E. A. Englund, D. H. Appella, Angew. Chem. Int. Ed. 2007,
46, 1414–1418; b) A. Dragulescu-Andrasi, S. Rapireddy, G. He,
B. Bhattacharya, J. J. Hyldig-Nielsen, G. Zon, D. H. Ly, J. Am.
Chem. Soc. 2006, 128, 16104–16112; c) P. E. Nielsen, Q. Rev.
Biophys. 2006, 39, 1–6; d) A. Abibi, E. Protozanova, V. V. De-
midov, M. D. Frank-Kamenetskii, Biophys. J. 2004, 86, 3070–
3078.
[6] a) M. G. Svahn, K. E. Lundin, R. Ge, E. Tornquist, E. O. Si-
monson, S. Oscarsson, M. Leijon, L. J. Branden, C. I. E.
Smith, J. Gene Med. 2004, 6, S36–S44; b) T. Ljungstrom, H.
Knudsen, P. E. Nielsen, Bioconjugate Chem. 1999, 10, 965–972;
c) B. P. Gangamani, V. A. Kumar, K. N. Ganesh, Biochem. Bio-
phys. Res. Commun. 1997, 240, 778–782; d) T. Shiraishi, R.
Hamzavi, P. E. Nielsen, Bioconjugate Chem. 2005, 16, 1112–
1116.
[7] Some examples are: a) E. Uhlmann, D. W. Will, G. Breipohl,
D. Langner, A. Ryte, Angew. Chem. Int. Ed. Engl. 1996, 35,
2632–2635; b) D. A. Stetsenko, E. N. Lubyako, V. K. Potapov,
T. L. Azhikina, E. D. Sverdlov, Tetrahedron Lett. 1996, 37,
3571–3574; c) K. H. Petersen, D. K. Jensen, M. Egholm, P. E.
Nielsen, O. Buchardt, Bioorg. Med. Chem. Lett. 1995, 5, 1119–
1124; d) F. Bergmann, W. Bannwarth, S. Tam, Tetrahedron
Lett. 1995, 36, 6823–6826.
[18] L. Betts, J. A. Josey, J. M. Veal, S. R. Jordan, Science 1995, 270,
1838–1841.
[19] a) G. Haaima, H. Rasmussen, G. Schmidt, D. K. Jensen, J.
Sandholm Kastrup, P. Wittung Stafshede, B. Nordén, O. Buch-
ardt, P. E. Nielsen, New J. Chem. 1999, 23, 833–840; b) X.-W.
Lu, Y. Zeng, C.-F. Liu, Org. Lett. 2009, 11, 2329–2332.
[20] a) M. Egli, Angew. Chem. Int. Ed. Engl. 1996, 35, 1894–1909;
b) A. Eschenmoser, Chimia 2005, 59, 836–850.
[21] W. C. Still, A. Tempczyk, R. C. Hawley, T. Hendrickson, J. Am.
Chem. Soc. 1990, 112, 6127–6129.
[22] The oligomers were synthesised in different lengths in order to
correlate Tm values with the number of nucleobases.
[23] a) G. Lowe, T. Vilaivan, J. Chem. Soc. Perkin Trans. 1 1997,
539–546; b) Y. Wu, J.-C. Xu, J. Liu, Y.-X. Jin, Tetrahedron
2001, 57, 3373–3381.
[24] O. R. Ludek, C. Meier, Eur. J. Org. Chem. 2006, 941–946.
[25] MACROMODEL, version 8.5, Schrödinger LLC, New York,
NY, 2003.
[26] a) D. M. Ferguson, P. A. Kollman, J. Comput. Chem. 1991, 12,
620–626; b) D. Q. McDonald, W. C. Still, Tetrahedron Lett.
1992, 33, 7743–7746; c) P. K. Weiner, P. A. Kollman, J. Comput.
Chem. 1981, 2, 287–303; d) S. J. Weiner, P. A. Kollman, D. A.
Case, U. C. Singh, C. Ghio, G. Alagona, S. Profeta Jr, P.
Weiner, J. Am. Chem. Soc. 1984, 106, 765–784; e) S. J. Weiner,
P. A. Kollman, D. T. Nguyen, D. A. Case, J. Comput. Chem.
1986, 7, 230–252.
[27] J. P. Ryckaert, G. Ciccotti, H. J. C. Berendsen, J. Comput. Phys.
1977, 23, 327–341.
[8] V. A. Kumar, K. N. Ganesh, Acc. Chem. Res. 2005, 38, 404–
412.
[9] G. Haaima, A. Lohse, O. Buchardt, P. E. Nielsen, Angew.
Chem. Int. Ed. Engl. 1996, 35, 1939–1942.
[10] a) V. Menchise, G. De Simone, T. Tedeschi, R. Corradini, S.
Sforza, R. Marchelli, D. Capasso, M. Saviano, C. Pedone, Proc.
[28]
Handbook of Biochemistry and Molecular Biology, vol. I (“Nu-
cleic Acids”) (Ed.: G. D. Fasman), 3rd ed., CRC Press, Cleve-
land, Ohio, 1975, p. 589.
Received: July 14, 2009
Published Online: October 26, 2009
6120
www.eurjoc.org
© 2009 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2009, 6113–6120