Achiral, acyclic nucleic acids: Synthesis and biophysical studies of a possible prebiotic polymer

Article abstract of DOI:10.1039/c5ob00898k

The search for prebiotic, nucleic acid precursors is, at its best, a speculative undertaking. Given the complex structure of RNA, it is not very likely that RNA was the first information system in the universe and thus finding possible precursor/s i.e. pre-RNA remains an open challenge. We, in this paper, have tried to construct nucleic acid polymers with a simple acyclic, achiral backbone. Such a linear, achiral backbone may have been formed from simple monomers that may have existed in the "prebiotic soup". We have shown that such polymers are capable of identifying the complementary "other self" and thus forming a potential system for information storage and transmission. This study thus involves investigation of nucleic acid analogues with a modified backbone that are likely to have formed in the prebiotic setting.

Full text of DOI:10.1039/c5ob00898k

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Achiral, acyclic nucleic acids: synthesis and
biophysical studies of a possible prebiotic
Cite this: DOI: 10.1039/c5ob00898k
polymer†
P. Srivastava,a R. Abou El Asrar,a C. Knies,b M. Abramov,a M. Froeyen,a J. Rozenski,a
H. Rosemeyerb and P. Herdewijn*a
The search for prebiotic, nucleic acid precursors is, at its best, a speculative undertaking. Given the
complex structure of RNA, it is not very likely that RNA was the first information system in the universe and
thus finding possible precursor/s i.e. pre-RNA remains an open challenge. We, in this paper, have tried to
construct nucleic acid polymers with a simple acyclic, achiral backbone. Such a linear, achiral backbone
may have been formed from simple monomers that may have existed in the “prebiotic soup”. We have
shown that such polymers are capable of identifying the complementary “other self” and thus forming a
potential system for information storage and transmission. This study thus involves investigation of nucleic
acid analogues with a modified backbone that are likely to have formed in the prebiotic setting.
Received 4th May 2015,
Accepted 21st July 2015
DOI: 10.1039/c5ob00898k
www.rsc.org/obc
Introduction
“Supporters of the hypothesis that RNA was the first genetic
material must explain where the nucleotides came from and how
they were self-organized. Those who believe in a simple precursor
have a difficult task of identifying such a precursor, but they
hope that explaining monomer synthesis will then be simpler.”
L. Orgel1
The space between the classic Urey–Miller experiment on
the origin of biomolecules2,3 and the “RNA-world”4 leaves
much room for possible permutation combinations in the
search for a pre-RNA ancestor/ancestors.5 With the “RNA
world” theory rather firmly established, the puzzle now is the
origin of the RNA world.6–8 What was/were the RNA precursor/
s9 that underwent chemical evolution and the “fittest” of them
came to dominate the biotic landscape?10 This would mean a
longer list of “extinct” molecular species. A variety of organic
moieties have been presented as RNA precursor/s i.e. the Pre-
RNA,11–15 one of them being PNA,16 in the category of non-
sugar nucleobase conjugates (Fig. 1B). It has also been pro-
posed that a pre-RNA may have completely lacked sugar/s17
and even phosphates18 and thus a linear, achiral polymer
conjugated to nitrogen heterocycles might represent a more
Fig. 1 Representative examples of nucleic acid polymers; (A) RNA (B)
PNA, (C) DEANA, (D) TEANA.
realistic prebiotic (pre-RNA) organic molecule capable of
assembling and recognizing a distinct “partner”.19–22 Apart
from their therapeutic potential,23,24 acyclic nucleic acid ana-
logues have been proposed as candidates for the prebiotic,
pre-RNA world.25–30
aMedicinal Chemistry, KU Leuven, Minderbroedersstraat 10, B-3000 Leuven,
Belgium. E-mail: Piet.Herdewijn@rega.kuleuven.be
bOrganic Materials Chemistry and Bioorganic Chemistry, Institute or Chemistry,
University of Osnabrück, Barharastrasse 7, 49069 Osnabrück, Germany
†Electronic supplementary information (ESI) available: General experimental
procedures, details of the primer extension assay, Tm profiles of oligos, the
hairpin model. See DOI: 10.1039/c5ob00898k
Herein, we report the synthesis and biophysical evaluation
of an achiral, acyclic nucleic acid analogue, named DEANA i.e.
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DiethanolAmideNucleicAcid having canonical nucleobases as the nucleobases were protected as benzoyl and isobutanoic
recognition elements with a flexible phosphodiester backbone amides for adenine/cytosine and guanine bases, thus provid-
(Fig. 1C). In this paper, we also introduce another analogue ing 3b, 3c and 3d respectively. One of the hydroxyls was pro-
that we have named TEANA i.e. TriethanolAminoNucleicAcid tected as dimethoxytrityl ether (4a–4d) and the remaining
having phosphorylated triethanolamine as the repeating unit hydroxyl was converted into phosphoramidites with 2-cyano-
engrafted with nucleobases (Fig. 1D). The synthesis of TEANA ethyl N,N-diisopropyl-chlorophosphoramidite, thus providing
was carried out on account of extensive degradation of DEANA the monomer phosphoramidites (5a–5d) for further solid
oligomers during deprotection of the synthesized oligos. We phase oligomer synthesis using the standard, automated solid
are introducing the early studies on TEANA with adenine, and support synthetic protocol on an Expedite® RNA/DNA synthe-
full studies shall be reported in the future. Structurally similar sizer. 5-(Ethylthio)tetrazole was used as the activating agent
analogues31,32 e.g. PPNA33 and PHONA34 have been syn- with 10 min coupling time for all DEANA phosphoramidites.
thesized and the lower pairing affinity of such acyclic phospho- This was followed by de-protection from solid support and
nate oligomers has been reported.
base deprotection in ammonia at 55 °C followed by purifi-
The backbone of DEANA is composed of diethanolamine cation to obtain fully modified (DEANA) or chimeric (DNA-
conjugated to the recognition element via amide bond for- DEANA) oligonucleotides.
mation between N-alkylated nucleobase acetic acids and the
Synthesis of the triethanolamine nucleoside (Scheme 2)
secondary amine. The acetic acid analogues of nucleobases starts with regioselective alkylation of adenine with 1,2-dibro-
may have been present in the primordial soup35,36 and the moethane as reported.40 The 9-(2-bromoethyl)-adenine (6a)
reaction of ammonia with oxirane37 would easily result in the was then refluxed in dioxane with silyl protected 2,2′-imino-
formation of diethanolamine and triethanolamine in the same diethanol and cesium carbonate to obtain 6b. The exocyclic
soup. We have thus attempted to construct oligomeric conju- amino group of compound 6b was then protected as bis-benz-
gates formed by possible prebiotic molecules i.e. nucleobase amide using benzoyl chloride in pyridine, thus providing 6c.
acetic acids and diethanolamine and, through the study of bio-
Triethylamine trihydrofluoride was then used to deprotect
physical properties of such “prebiotic” polymers, tried to the silyl ethers to obtain the bis-hydroxyl product 6d. One of
expand the repertoire of potential pre-RNA constructs.
the hydroxyls was next converted into a dimethoxytrityl ether
(6e) and the other was converted into a phosphoramidite with
2-cyanoethyl N,N-diisopropylchlorophosphoramidite, thus
providing the adenine-containing a TEANA phosphoramidite
monomer 6f for further solid phase oligomer synthesis using
the standard, automated solid support, synthetic protocol on
an Expedite® RNA/DNA synthesizer. 5-(Ethylthio)tetrazole was
used as the activating agent with 10 min of coupling time for
the TEANA phosphoramidite followed by deprotection and
purification to obtain the singly modified, TEANA/DNA mixed
oligonucleotide.
Results and discussion
Our scheme for the synthesis of DEANA (Scheme 1) starts with
the regioselective alkylation of nucleobases, i.e. thymine,
adenine, cytosine and guanine by ethyl bromoacetate in DMF
using the established synthetic procedure.38 The appropriately
alkylated ethyl esters of thymine-1-yl acetic acid, cytosine-1-yl
acetic acid, adenine-9-yl acetic acid, and guanine-9-yl acetic
acid were refluxed with 2,2′-iminodiethanol overnight in
ethanol as reported for the synthesis of N,N-bis-(2-hydroxy-
ethyl)-2-(thymin-1-yl)acetamide39 to obtain N,N-bis-(2-hydroxy-
Thermal melting studies
ethyl)-2-(thymin-1-yl)acetamide (2a), N,N-bis-(2-hydroxyethyl)- The base pairing properties of DEANA and TEANA modified
2-(cytosine-1-yl)acetamide (2b), N,N-bis-(2-hydroxyethyl)-2- oligomer sequences were investigated by temperature depen-
(adenine-9-yl)acetamide (2c), and N,N-bis-(2-hydroxyethyl)-2- dent UV absorption at 260 nm wavelength. In our initial
(guanine-1-yl)acetamide (2d) respectively, as white solids in attempt, the fully modified 15-mer thymine containing the
nearly quantitative yields. The exocyclic amino functions of DEANA sequence (ON-1) showed no duplex melting transition
Scheme 1 Reagents and conditions: (i) 2,2’-iminodiethanol, ethanol, reflux, overnight; (ii) TMS-Cl, benzoyl chloride/isobutyryl chloride, dry pyri-
dine, 0 °C to r.t., 3 h; (iii) DMTr-Cl, dry pyridine or dry DMF and Et3N, 0 °C to r.t., overnight; (iv) 2-cyanoethyl N,N-diisopropylchlorophosphoramidite,
DIPEA, dry DCM/THF, 0 °C to r.t. 30 min. B = thymine (1a–5a), cytosine (1b, 2b), adenine (1c, 2c) and guanine (1d, 2d), or CBz (3b–5b), ABz (3c–5c)
and Gibu (3d–5d) respectively.
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Scheme 2 Reagents and conditions: (i) 1,2-dibromoethane, K2CO3, dry DMF; (ii) CsCO3, dry dioxane, reflux, 12 h; (iii) benzoyl chloride, dry pyridine,
0 °C to r.t. overnight; (iv) triethylamine trihydrofluoride, dry THF, 0 °C to r.t. 6–8 h; (v) DMTr-Cl, dry pyridine, 0 °C to r.t., overnight; (vi) 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite, DIPEA, dry THF, 0 °C to r.t. for 30 min.
Table 1 Thermal melting studies: DEANA modificationsa
with a 15-mer-dA sequence. To find out if the analogues show
self-pairing, a fully modified 15-mer sequence was synthesized
with the phosphoramidite monomer 5c (Scheme 1).
Tm
in °C
Mass found/
calculatedb
Oligo.
Sequence
However, the fully modified adenine containing DEANA was
hydrolyzed during the base deprotection step, probably via
trans-esterification, analogous to the mechanism of RNA
degradation. Previously,30 we had observed similar problems
while trying to synthesize other acyclic nucleic acids. In the
case of DEANA, an intranucleotide trans-esterification reaction
in addition to the cleavage of the amide bond between nucleo-
base acetic acid and diethanolamine could have occurred.
Taking clues from other acyclic systems, we next synthesized a
modified Dickerson–Drew dodecamer41 with two flanking
deoxyribo nucleotides at the 3′ and 5′ ends, thus avoiding
decomposition of the DEANA containing oligonucleotide by
trans-esterification in the basic medium. This self-complemen-
tary sequence was chosen since it has been extensively
studied42 for gaining insights into the structure of nucleic
acids.43–47 The temperature-dependent UV absorption study of
the chimeric Dickerson Drew dodecamer sequence at 260 nm
exhibited a sigmoidal melting curve (Fig. SI-3 and SI-4, in the
ESI†) at 0.1 M NaCl concentration thus providing a Tm of
27.5 °C (Table 1) corresponding to an approximately 24 °C
drop in Tm as compared to the all DNA-dodecamer (ON-2).
This accounts to an approximately 1.5 °C drop in Tm per modi-
fication at the same salt and oligo concentration. Realizing
that end capping was necessary, we next synthesized another
self-complementary oligomer (ON-4) with diethyleneglycol
monomethyl ether as the 5′ end cap and a natural dC as the 3′
end cap (also see the discussion on CD studies). The yields of
ON-1
ON-2
ON-3
ON-4
(t)15: d(A)15
d(CGCGAATTCGCG)2
d(CGcgaattcgCG)2
N.A.
51.5
27.5
29.5
4936.3/4936.5
—
3878.8/3878.7
3487.1/3487.4
[CH3O(CH2CH2O)2gcgcgcgcgC]2
a Thermal melting studies of oligonucleotides containing natural/
modified nucleic acid analogues (DEANA). Modifications are
represented by lower case bold letters (a, t, g, c) in the oligonucleotide
sequence. Change in absorbance measured at 260 nm in NaCl (0.1 M)
buffer with KH2PO4 (20 mM, pH 7.5) and EDTA (0.1 mM) at the
concentration of 4 μM for each strand and 8 µM for the self-
complementary Dickerson Drew dodecamer (ON-3) and ON-4. b Mass
of the modified sequence.
oligonucleotide synthesis were better after deprotection as
compared to the results obtained with fully modified oligo-
mers, similar to ON-3. This self-complementary sequence
(ON-4) exhibited a Tm of 29.5 °C with no hysteresis between
heating and annealing curves (Fig. SI-5 in the ESI†). To further
understand the hybridization process, a concentration depen-
dent Tm analysis was done for ON-4. The melting temperature
for ON-4 was found to be constant from 2 μM to 12 μM concen-
trations (Fig. SI-7 in the ESI†) thus indicating a unimolecular
process. Given the acyclic nature of the DEANA, it is not un-
likely that we have a hairpin model in our hands (also see the
Molecular modeling studies section). Thus, we were able to
show that DEANA is capable of self-pairing albeit with low
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Table 2 Thermal melting studies: TEANA modificationa
similar to the experimental PNA structure with 18 base pairs
per turn forming an elongated helix.52 The phosphates on the
outside of the helix are expelled more than the phosphates in
the classical DNA helix (Fig. 2B). However, the pairing pro-
perties of DEANA are markedly inferior to that of PNA,
wherein the PNA duplexes give higher melting temperatures
than DNA duplexes.53 In addition to the previously mentioned
entropic penalty, this may be explained by the lack of phos-
phate groups in PNA (no repulsion of the single strands in the
duplex) and the inter-strand stacking due to the high incli-
nation. The DEANA has charged phosphate groups, so the
enthalpy contribution to the hybridization energy is expected
to be lower due to the electrostatic repulsion. It must however
be noted that the model was created by superposition of the
Watson–Crick base pairs of DEANA onto the PNA bases and
then minimizing the energy of the duplex, resulting in the
creation of the phosphate bulges, and almost not changing the
base positions. It must also be noted that Dickerson–Drew
dodecamer is known to exist in duplex and hairpin forms,
no attempt however was made to dissect the molecularity of
hybridization for ON-3.
Table 3 shows the remarkable similarities in the helical
parameters of PNA and a DEANA (ON-3) duplex, obtained from
the superposition of the DEANA model onto the PNA structure
(Fig. 3) despite the fact that we have two extra atoms (or 2 extra
bonds) in the backbone. Starting from the left handed PNA
NMR structure (pdb structure 2K4G),52 an attempt was made
to create a left handed DEANA helix as well.
However, while optimizing the final structure in Amber, the
amide connections from the main chain to the base showed
high torsion energy with non-planar amide bonds. It must be
noted that DEANA has no chiral center and hence left and
right handed helixes with the same geometry can be formed
with equal probability, and DEANA (nucleosides or oligo-
nucleotides) by itself cannot contribute to symmetry breaking.
Since the concentration dependent Tm study on ON-4 indi-
cated a unimolecular pairing process (Fig. SI-7†), and coupled
with the absence of classical sigmoidal Tm response, a hairpin
model of 10mer g·c repeat sequence was constructed with
DEANA using a PNA double helix from the 3MBS pdb entry,54
deleting all pairs except 3 GC pairs and adding a GCGC tetra-
Sequence-duplex
Tm in °C
47.0 °C
40.0 °C
Mass found/calculatedb
5′-d(CTTCATTTTTTCTTC)-3′
3′-d(GAAGTAAAAAAGAAG)-5′
5′-d(CTTCaTTTTTTCTTC)-3′
3′-d(GAAGTAAAAAAGAAG)-5′
—
4462.9/4462.8
a Thermal melting studies of oligonucleotides containing natural/
modified nucleic acid analogues. Modification is represented by a
lower case bold letter (a) in the oligonucleotide sequence. Change in
absorbance measured at 260 nm in NaCl (0.1 m) buffer with KH2PO4
(20 mM, pH 7.5) and EDTA (0.1 mM) at the concentration of 4 μM for
each strand. b Mass of the modified sequence.
thermal stability. The lability of the carboxamide bond guided
us towards the synthesis of TEANA and thus a stable N-alkyl
linker was substituted for a labile amide linker in DEANA. In a
preliminary study, a single TEANA modification was intro-
duced in a DNA sequence. The thermal melting study (Fig. SI-6
in the ESI†) of this modified oligo with the complementary
DNA sequence exhibited a drop of 7.0 °C in Tm as compared to
the natural all DNA:DNA duplex of the same sequence
(Table 2).
Since the bis-hydroxyethanol-phosphodiester backbone in
DEANA has a higher degree of torsional flexibility, we had
hoped that a stable duplex formation would be possible by
flexing to maximize stacking interactions and hydrogen
bonding. The decrease in stability when comparing DEANA to
DNA can be explained by the entropic penalty48,49 for the intra
system base pairing. The thermal melting study of homo-
duplexes with our acyclic analogue (DEANA) is in contrast with
a previous study,50 wherein the homoduplexes of other acyclic
analogues exhibited higher thermal stability. It is known that
pairing (self or cross) of oligonucleotides is determined by the
backbone inclination.51 Hence, to gain insights into the struc-
tural features of DEANA oligonucleotides, molecular modeling
studies were conducted.
Molecular modeling studies
In order to gain insight into the DEANA duplex structure and loop (conformation not defined). The molecular mechanics
to answer the question as to how a nucleic acid analogue with energy of the loop part was optimized in Amber12. A DEANA
8-bonds between the repeating nucleotides could be formed hairpin was then constructed based on this PNA hairpin and
instead of 6 bonds in the natural DNA, we constructed a static energy minimized in Amber12, restraining the base atoms.
model of a right-handed DEANA double stranded helix with With only three base pairs in the stem of a 10-mer hairpin
the sequence CGTACG:CGTACG as described in the Experi- sequence (Fig. SI-8 in the ESI†), it is not surprising that we see
mental section. It should be noted that the DEANA nucleotide only a slight change in the absorbance during melting.
has no 5′ end different from the 3′ end, so duplexes cannot be A single strand melting of a non-hairpin structure, however,
labeled parallel or anti-parallel (Fig. 2).
cannot be ruled out.
The static model shows that two strands with DEANA resi-
dues can be combined to form double stranded self-pairing
Circular dichroism study
duplexes with Watson Crick base pairing geometry (Fig. 2A) The self-complementary dodecamer sequence containing the
which is also supported by the CD spectra of the dodecamer achiral acyclic DEANA (ON-3) synthesized with flanking
flanked by natural deoxy residues (see further in the CD natural deoxy residues (Table 1) was used to study if such
studies). The modeled DEANA duplex adopts a structure very chimeric oligomer will adopt a discernible helical structure.
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Fig. 2 Static DEANA 18-mer duplex model structure. (A) Front view, (B) top view shows the characteristic popping out of the phosphates, (C) view
from the minor groove, (D) close-up showing hydrogen bonding and nucleobase stacking. Carbons of one strand are colored in yellow, in the other
strand in green. Hydrogen bonds are shown as cyan lines.
Table 3 Comparison of helical parameters for PNA, DEANA and DNA double helicesa
Handedness
Disp.
Rise [Å]
Incl. [°]
Tilt
Twist [°]
Bp per turn
P1–P1 dist.
P1–P2 dist.
P1–P3 dist.
PNA
DEANA
B-DNA
Right
Right
Right
8.3
8.3
0.0
3.2
3.2
3.4
0.3
0.3
2.4
1.0
1.0
0
19.8
19.8
36
18
18
10
—
15.01
18.14
—
11.12
16.69
—
9.55
14.93
a Helical parameters for PNA and B-DNA double helices.52 Values for DEANA are copied from the PNA structure, except the P–P distances. P1–P1
is the distance in Å units between 2 phosphates of complementary nucleotides in the anti-parallel duplex, P1–P2 is the distance of the next P in
the chain etc.
Temperature dependent CD spectra were recorded in the have steered the acyclic DEANA to this structure in a well-
neutral cacodylate buffer solution. As evident from the CD known phenomenon of chiral induction55 also observed in the
spectra (Fig. 4), the dodecamer adopts a right handed helix case of PNA, wherein a terminal amino acid residue or deoxy-
with maxima around 280 nm and minima around 250 nm. nucleotides residue/s drive(s) the polymer to adopt a right
This signature ellipticity is analogous to that shown by the handed or left handed form.56–60 Since we had in our hand,
B-type DNA duplex. The Tm of the dodecamer, as determined ON-4 with only a single natural residue at the 3′ end, a CD
by the UV melting study, was also confirmed with this temp- study was warranted to show that the signature CD shown by
erature dependant CD measurement.
the ON-3 was indeed a result of chiral induction. The ON-4 did
An achiral, acyclic DEANA polymer was not expected to not exhibit any ellipticity in the temperature dependent CD
show this CD spectra and the flanking chiral deoxy residues study (Fig. 5).
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Fig. 6 (A) DEANA triphosphate used in the study; (B) template (T) and
primer (P) sequence used in the Therminator™ DNA polymerase cata-
lysed primer extension assay.
phosphate analogues on deoxynucleotide templates.61,62 A fact
that a deoxy ribose backbone primer can be extended by using
non-natural nucleoside triphosphates and a non-natural oligo-
nucleotide can be extended by using natural nucleoside tripho-
sphates, even in the absence of stable duplex formation
between the two, is of great significance with regard to the
evolution of nucleic acids and its progenitors.63–66 Our next
attempt thus was to study if the achiral acyclic adenine con-
taining DEANA-triphosphate (Fig. 6A) is recognized by DNA
polymerases in a template assisted, enzymatic, primer exten-
sion assay (Fig. SI-1†). This study was undertaken less in view
of the potential prebiotic occurrence of DEANA, but because of
its potential adaptability to contemporary biology. We however
were not able to detect the template directed primer extension
by our acyclic DEANA triphosphate. The results and discussion
are provided in the ESI (Fig. SI-1 and SI-2†).
Fig. 3 Superposition of the static DEANA model structure onto the PNA
X-ray structure 1PUP, highlighting the popping out of the phosphate
groups.
Fig. 4 Temperature dependent CD spectra of the dodecamer duplex
sequence ON-3 recorded in cacodylate buffer.
Conclusions
We have constructed two alternative base pairing systems i.e.
DEANA and TEANA which are acyclic and achiral, lack sugar
units but, have a phosphodiester backbone linking the mono-
meric units. The polymers contain eight bonds between
repeating units instead of six bonds found in natural DNA/
RNA backbone. The oligonucleotides formed by the DEANA
were found to be labile under the standard base deprotection
conditions. However, we did manage to obtain deoxynucleo-
tide end capped oligos in low yields for some initial structural
studies. The CD study of the dodecamer and modeling of the
acyclic nucleotide polymers indicate that a right handed
B-type helix could (theoretically) be formed by the mechanism
of chiral induction. Further, the model of DEANA oligo-
nucleotide showed that the helical parameters of a DEANA
duplex are similar to the PNA parameters. In the model, the
phosphate groups are stretched outside, away from the hydro-
phobic core. We also subjected DEANA triphosphate to a tem-
plate assisted, polymerase catalyzed incorporation assay. At
least, with a Therminator™ DNA polymerase, we were not able
to detect the incorporation of DEANA nucleotides in DNA.
Further assays with other mutant polymerases might provide
for the potential use of our analogues in future. The synthesis
Fig. 5 Temperature dependent CD spectra of the dodecamer duplex
sequence ON-4 recorded in cacodylate buffer.
This was though, in contrast to a previous study with PNA
wherein a single amino acid was able to transfer the chirality
to the macromolecule. We could also infer that the hybridi-
zation of ON-3 was not solely due to the presence of 4 flanking
deoxy residues.
It is well known that some DNA polymerases are capable of
catalyzing template directed primer extension using acyclic tri-
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of monomeric units of both DEANA and TEANA are plausible ether to obtain N,N-bis-(2-hydroxyethyl)-2-(thymin-1-yl)aceta-
under prebiotic conditions.
mide (2a); N,N-bis-(2-hydroxyethyl)-2-(cytosine-1-yl)acetamide
(2b); N,N-bis-(2-hydroxyethyl)-2-(adenine-9-yl)acetamide (2c);
and N,N-bis-(2-hydroxyethyl)-2-(guanine-1-yl)acetamide (2d);
respectively as white solids, analogous to the synthesis of N,N-
bis-(2-hydroxyethyl)-2-(thymin-1-yl)acetamide39 reported in the
literature.
Experimental
General experimental procedures
All moisture-sensitive reactions were performed under a nitro-
gen atmosphere by using oven-dried glassware. Dry solvents
were purchased from commercial sources and used as such.
Reactions were monitored by TLC (a pre-coated silica gel plate
F254, Merck). Column chromatography: Merck Kieselgel 60
(70–230 mesh); flash chromatography: Merck Kieselgel 60
(230–400 mesh). 1H and 13C NMR spectra were recorded on
NMR spectrometers operating at 300 or 500 MHz and 75 or
125 MHz, respectively. DEANA triphosphate (Fig. 6A) was syn-
thesized according to the one-pot synthesis described by
Ludwig for natural triphosphates67 and purified on a DEAE
Sephadex-A25 column using a triethylammonium bicarbonate
gradient and lyophilized for further use.
N,N-Bis(2-hydroxyethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydro-
pyrimidin-1(2H)-yl)acetamide (2a). Yield = 95%. 1H NMR
(500 MHz; DMSO-d6) δ: 7.27 (1H, d, J = 1.1 Hz), 4.59 (2H, s),
3.56 (2H, appt, J = 6 Hz), 3.46 (2H, appt, 6 Hz), 3.40 (2H, appt,
J = 6.0 Hz), 3.33 (2H, appt, J = 6 Hz), 1.73 (3H, d, J = 1.1 Hz);
13C (125 MHz; DMSO-d6) δ: 167.4, 164.9, 151.4, 142.8, 108.5,
59.1, 58.8, 50.0, 48.9, 48.5, 12.2; HRMS (ESI+): calcd for
C11H18N3O5 [M + H]+ 272.1240, found 272.1241.
N,N-Bis(2-hydroxyethyl)-2-(4-amino-2-oxopyrimidin-1(2H)-
yl)-acetamide (2b). Yield = 92%. 1H NMR (300 MHz; DMSO-d6)
δ: 7.4 (1H, d, J = 7.26 Hz), 5.64 (1H, d, J = 7.26 Hz), 4.58 (2H,
bs), 3.65–3.27 (8H, m); 13C (75 MHz; DMSO-d6) δ: 167.7, 166.2,
156.0, 147.1, 93.0, 59.1, 58.7, 49.9, 49.1, 48.8; HRMS (ESI+):
calcd for C10H16N4O4Na [M + Na]+ 279.1063, found 279.1062.
2-(6-Amino-9H-purin-9-yl)-N,N-bis(2-hydroxyethyl)acetamide
Molecular modeling (creation of a static model of a DEANA
double stranded helix)
1
(2c). Yield = 96%. H NMR (300 MHz; DMSO-d6) δ: 8.23 (1H,
The peptide nucleic acid structure (1PUP, 1.7Ang)68 was used
as a template to construct a 3D model of a DEANA duplex.
First, a DEANA dinucleotide ApT was drawn using Chemdraw
10 and 3D coordinates were generated and optimized by
Chem3D.69 This structure was then superimposed onto a PNA
dinucleotide unit (with A and T bases), using a flexible super-
position program where the torsion angles are optimized while
superimposing the conformations onto the rigid PNA di-
nucleotide. The scoring was done in a similar way as in the SEAL
program for the steric and electronic alignment.70 Despite the
fact that the DEANA backbone has one more atom than the
PNA backbone, a very good superposition of the bases was
obtained. This is possible because the phosphate groups are
pushed outside. Using this new conformation a DEANA
hexamer duplex model with the sequence CGTACG·CGTACG
was constructed by combining, editing the model pdb file, and
eventually changing some bases by superposition to get the
correct sequence using Quatfit.71 The model was subjected to
a vacuum energy minimization in the ff99bsc0 force field
using the Amber 12 software.72 The force field had to be
patched with some gaff force field parameters because of the
non-conventional DEANA residues.73 Charges on atoms of the
phosphate groups were adjusted to preserve correct the total
charges of the nucleotides.
s), 8.11 (1H, s), 5.31 (2H, s), 3.64 (2H, appt, J = 6 Hz), 3.54 (2H,
appt, J = 6 Hz), 3.47 (2H, appt, J = 6 Hz), 3.36 (2H, appt, J = 6
Hz); 13C (75 MHz; DMSO-d6) δ: 167.1, 155.9, 152.7, 150.2,
142.6, 118.3, 59.1, 58.7, 50.2, 48.9, 44.4; HRMS (ESI+): calcd for
C11H17N6O3 [M + H]+ 281.13565 found 281.1355.
2-(2-Amino-6-oxo-1H-purin-9(6H)-yl)-N,N-bis(2-hydroxyethyl)-
1
acetamide (2d). Yield = 85%. H NMR (300 MHz; DMSO-d6) δ:
7.52 (1H, s), 4.95 (2H, s), 3.6–3.3 (8H, m); 13C (75 MHz; DMSO-
d6) δ: 166.6, 156.8, 153.4, 151.6, 138.3, 115.9, 58.8, 58.5, 49.8,
48.5, 43.8; HRMS (ESI+): calcd for C11H17N6O3 [M + H]+
281.13565, found 281.1355.
N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-N-(2-hydroxy-
ethyl)-2-(5-methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)-
acetamide (4a). In a dry round bottom flask was suspended,
2b (2.7 g, 10 mmol) in dry pyridine (5 ml), and co-evaporated
twice. The residue was suspended again in dry pyridine
(50 ml) and cooled in an ice bath. To this pre-cooled solution
was added 4,4′-dimethoxytriphenylmethyl chloride (3.38 g,
10 mmol) and the reaction mixture was stirred overnight
at room temperature. After completion, the reaction was
quenched with saturated aqueous NaHCO3 and extracted
thrice with DCM (50 mL). The organic layers were pooled,
dried, evaporated and chromatographed over silica (0–5%
MeOH/DCM, v/v: 5% pyridine) to obtain the product 4a as a
white foam (3.78 g, 6.6 mmol; yield = 66%).
Synthesis of the DEANA monomers with A, G, C and T bases
1H NMR: mix. of rotamers; (300 MHz; CDCl3/D2O) δ:
The ethyl esters of thymine-1-yl acetic acid (1a) (2.2 g, 7.39–7.15 (20H, m), 6.93 (d, H-6, min, J = 1.27 Hz), 6.87–6.80
10 mmol), cytosine-1-yl acetic acid (1b) (1.9 g, 10 mmol), (6H, m), 6.43 (d, H-6, maj, J = 1.3 Hz), 4.73 (s, CH2, maj ), 4.62
adenine-9-yl acetic acid (1c) (2.2 g, 10 mmol) and guanine-9-yl (s, CH2, min), 3.79 (s, 2 × OCH3, maj ), 3.7 (s, 2 × OMe, min),
acetic acid (1d) (2.3 g, 10 mmol) were refluxed with 2,2′-imino- 3.67 (appt, CH2, J = 5.83 Hz, 4.73 Hz), 3.60 (appt, CH2, J = 6.74
diethanol (10 eq.) overnight in 50 ml of dry ethanol. After over- Hz, 4.73 Hz), 3.40 (appt, CH2, J = 5.83 Hz, 4.73 Hz), 3.32 (appt,
night reflux, the reaction mixture was cooled and filtered. The CH2, J = 5.83 Hz, 4.73 Hz), 1.88 (d, T-CH3, min, J = 1.2 Hz), 1.76
white precipitate was washed with cold ethanol and diethyl (d, T-CH3, maj, J = 1.2 Hz); 13C (75 MHz; DMSO-d6) δ: 167.0,
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166.7, 164.4, 164.3, 158.1, 158.0, 151.0, 144.9, 144.6, 142.2, to obtain the product 4b (3.0 g, 4.55 mmol; yield = 65%) as a
141.9, 135.6, 135.5, 129.7, 128.8, 128.2, 127.9, 127.8, 127.6, white foam.
126.8, 126.6, 125.3, 113.2, 107.9, 86.2, 85.5, 61.2, 58.8, 58.5,
55.0, 49.8, 48.1, 47.9, 46.0, 21.0, 11.9; HRMS (ESI+): calcd for d, J = 7.6 Hz), 7.94 (d, J = 6.4 Hz), 7.62 (appt, J = 8.3 Hz,
C32H35N3O7Na 596.2367, found 596.2375. 7.2 Hz), 7.51 (appt, J = 8.3 Hz, 7.5 Hz), 7.41–7.21 (m), 6.90 (d,
1H NMR mix. of rotamers; (500 MHz, DMSO-d6) δ: 8.02 (1H,
2-(N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(5- J = 8.6 Hz), 4.88 (s, CH2, maj ), 4.74 (s, CH2, min), 3.73 (s, 2 ×
methyl-2,4-dioxo-3,4-dihydropyrimidin-1(2H)-yl)acetamido)ethyl OCH3, maj ), 3.66–3.54 (m), 3.49 (appt, J = 6.71 Hz, 5.12 Hz),
2-cyanoethyl diisopropylphosphoramidite (5a). 4a (5.0 g, 3.43 (m), 3.36 (s, 2 × OCH3, min), 3.34–3.27 (m), 3.09 (appt, J =
8.7 mmol) was dissolved in dry DCM (40 ml). DIPEA (2.3 ml, 7.22 Hz, 5.46 Hz); 13C (125 MHz, DMSO-d6) δ: 167.3, 167.0,
13 mmol) was added and the reaction was cooled to 0 °C. 166.6, 163.4, 158.2, 158.1, 155.4, 151.4, 145.0, 144.8, 135.7,
To this was added 2-cyanoethyl N,N diisopropyl- 135.6, 133.3, 132.7, 129.8, 129.7, 128.5, 128.0, 127.87, 127.7,
phosphoramidochloridite (2.3 mL, 10.4 mmol), and the 126.7, 113.3, 95.7, 86.3, 85.6, 61.4, 61.3, 59.0, 58.5, 55.1, 50.1,
mixture was stirred for 15–30 min at room temperature. After 49.9, 48.5, 47.5, 46.3; HRMS (ESI+): calcd for C38H39N4O7
completion, the reaction was quenched by addition of MeOH [M + H]+ 663.2813, found 663.2813.
(1 ml) and the solvent was evaporated. The residue was then
2-(2-(4-Benzamido-2-oxopyrimidin-1(2H)-yl)-N-(2-(bis(4-methoxy-
treated with 10% aq. NaHCO3 solution and extracted thrice phenyl)(phenyl)methoxy)ethyl)acetamido)ethyl 2-cyanoethyl diiso-
with DCM (100 ml). The organic layers were pooled, dried, propyl-phosphoramidite (5b). 4b (3.0 g, 4.55 mmol) was
evaporated and chromatographed over silica (0–50% acetone/ dissolved in dry THF (25 ml). DIPEA (4.0 ml, 22.75 mmol) was
hexane, v/v: 2% Et3N) to obtain the product 5a (4.7 g, added and the reaction was cooled to 0 °C. To this was
6.0 mmol; yield = 70%) as a white foam.
31P NMR (121 MHz; CDCl3) δ: 148.5, 147.9; HRMS (ESI+): (1.5 mL, 6.8 mmol), and the mixture was stirred for 15–30 min
calcd for C41H53N5O8P [M + H]+ 774.3639, found 774.3626.
at room temperature. After completion, the reaction was
added 2-cyanoethyl N,N diisopropyl-phosphoramido-chloridite
N-(1-(2-(Bis(2-hydroxyethyl)amino)-2-oxoethyl)-2-oxo-1,2-dihydro- quenched by addition of MeOH (1 ml) and the solvent was
pyrimidin-4-yl)benzamide (3b). In a round bottom flask, 2b evaporated. The residue was chromatographed over silica
(2.56 g, 10 mmol) was suspended in dry pyridine (5 ml) and (0–50% acetone/hexane, v/v: 2% Et3N) to obtain the product 5b
co-evaporated twice. The residue was suspended again in dry (2.4 g, 2.8 mmol; yield = 62%) as a white foam.
pyridine (50 ml) and cooled to 0 °C in an ice bath. To this pre-
cooled suspension was added trimethylsilyl chloride (5.0 ml, calcd for C47H55N6O8P Na [M + Na]+ 885.3711, found 885.3719.
40 mmol) and the reaction mixture was stirred at 0 °C for N-Benzoyl-N-(9-(2-(bis(2-hydroxyethyl)amino)-2-oxoethyl)-
31P-NMR (121 MHz; CDCl3) δ: 148.7, 147.8; HRMS (ESI+):
30 min and then at room temperature for 1 h. The reaction 9H-purin-6-yl)benzamide (3c). In a round bottom flask was
was cooled again to 0 °C and freshly distilled benzoyl chloride suspended 2c (2.8 g, 10 mmol) in dry pyridine and co-evapo-
(3.0 ml, 25 mmol) was added drop-wise to this turbid solution. rated twice. The residue was suspended again in dry pyridine
The reaction mixture was stirred at 0 °C for 1 h and then (50 ml) and cooled to 0 °C in an ice bath. To this pre-cooled
stirred at room temperature for 3 h. After completion, the reac- suspension was added trimethylsilyl chloride (5.0 ml,
tion was cooled and quenched by addition of 25 ml of a 50 : 40 mmol) and the reaction mixture was stirred at 0 °C for
50 methanol : water mixture and the mixture was stirred for 30 min and then at room temperature for 1 h. The reaction
1 h. After the evaporation of the solvent, the gummy residue was cooled again to 0 °C and freshly distilled benzoyl chloride
was chromatographed over silica (0–10% MeOH/DCM, v/v: 5% (3.0 ml, 25 mmol) was added drop-wise to this turbid solution.
pyridine) to obtain the product 3b (2.8 g, 7.7 mmol, yield = The reaction mixture was stirred at 0 °C for 1 h and then
77%) as a white powder.
stirred at room temperature for 3 h. After completion, the reac-
1H NMR (300 MHz; DMSO-d6) δ: 8.0 (3H, m), 7.62 (1H, d, tion was cooled and quenched by addition of 10 ml of a 50 :
J = 7.0 Hz), 7.51 (2H, m), 7.29 (1H, d, J = 7.0 Hz), 4.83 (2H, s), 50 methanol : water mixture and the mixture was stirred for
3.62 (2H, m), 3.50 (4H, m), 3.38 (2H, m); 13C (75 MHz; DMSO- 1 h. The solvent was evaporated and the gummy residue was
d6) δ: 166.8, 163.3, 155.3, 151.4, 133.4, 132.8, 128.5, 95.7, 59.1, chromatographed over silica (0–10% MeOH/DCM, v/v: 5%
58.7, 50.1, 48.9; HRMS (ESI+): calcd for C17H21N4O5 [M + H]+ pyridine) to obtain the product 3c (4.0 g, 8.2 mmol; yield =
361.1506, found 361.1505.
N-(1-(2-((2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(2-
82%) as a white powder.
1H NMR (300 MHz; CDCl3) δ: 8.55 (1H, s), 8.24 (1H, s), 8.05
hydroxyethyl)amino)-2-oxoethyl)-2-oxo-1,2-dihydropyrimidin-4- (2H, m), 7.82 (4H, m),7.44 (3H, m), 7.31 (5H, m), 5.20 (2H, s),
yl)benzamide (4b). 3b (2.5 g, 7.0 mmol) was dissolved in dry 3.62 (4H, bs), 3.41 (4H, bs); 13C (75 MHz; CDCl3) δ: 172.6,
DMF (40 ml). Et3N (3.0 ml, 21 mmol) was added and the reac- 167.4, 153.6, 152.0, 151.3, 147.2, 134.0, 133.2, 129.4, 128.8,
tion was cooled to 0 °C in an ice bath. To this pre-cooled solu- 59.9, 59.7, 51.1, 50.1, 44.9.
tion was added 4,4′-dimethoxytriphenylmethyl chloride (2.3 g,
7.0 mmol) and the reaction mixture was stirred overnight at found 489.1881.
HRMS (ESI+): calcd for C25H25N6O5 [M + H]+ 489.1880,
room temperature. The reaction was quenched with methanol
N-Benzoyl-N-(9-(2-((2-(bis(4-methoxyphenyl)(phenyl) methoxy)-
(1 ml) and the solvent was evaporated. The residue was chro- ethyl)(2-hydroxyethyl)amino)-2-oxoethyl)-9H-purin-6-yl)benz-
matographed over silica (0–10% MeOH/DCM, v/v; 5% pyridine) amide (4c). 3c (4.0 g, 8.2 mmol) was dissolved in dry DMF
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(40 ml). Et3N (3.5 ml, 25 mmol) was added and the reaction 166.3, 154.9, 149.3, 147.8, 140.7, 119.5, 58.8, 58.5, 49.8, 48.5,
was cooled to 0 °C in an ice bath. To this pre-cooled solution 44.5, 34.6, 18.8; HRMS (ESI+): calcd for C15H23N6O5 [M + H]+
was added 4,4′-dimethoxytriphenylmethyl chloride (3.3 g, 367.1724 found 367.1723.
9.8 mmol) and the reaction mixture was stirred overnight at
2-(N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-2-(2-
room temperature. After completion, the reaction was isobutyramido-6-oxo-1H-purin-9(6H)-yl)acetamido)ethyl 2-cya-
quenched with methanol (1 ml) and the solvent was evapo- noethyl diisopropylphosphoramidite (5d). In a round bottom
rated. The residue was chromatographed over silica (5% pyri- flask was suspended 3d (3.0 g, 8.0 mmol) in dry DMF (40 ml).
dine/0–10% MeOH/DCM, v/v) to obtain the product 4c (4.0 g, Et3N (3.5 ml, 24 mmol) was added and the reaction was cooled
5.0 mmol; yield = 62%) as a yellowish foam.
to 0 °C in an ice bath. To this pre-cooled solution was added
1H NMR (300 MHz; CDCl3) δ: 8.56 (s, min), 8.54, s, maj ), 4,4′-dimethoxytriphenylmethyl chloride (3.25 g, 9.6 mmol) and
8.14 (s, min), 7.95 (s, maj ), 7.47–7.17 (several multiplets), the reaction mixture was stirred overnight at room tempera-
6.87–6.77 (several multiplets), 5.26 (s, CH2, maj ), 5.21 (s, CH2, ture. After completion, the reaction was quenched with metha-
min), 3.76 (s, OMe, maj ), 3.75 (s, OMe, min), 3.70–3.59 (several nol and the solvent was evaporated. The residue was collected
multiplets), 3.55–3.38 (several multiplets), 3.33 (appt, J = for the next reaction. This crude 4d was dissolved in dry THF.
5.83 Hz, 4.77 Hz); 13C (75 MHz; CDCl3) δ: 172.4, 167.2, 166.3, DIPEA (4.0 ml, 22.5 mmol) was added and the reaction was
158.8, 158.6, 153.6, 153.5, 152.0, 151.5, 146.6, 146.2, 144.7, cooled to 0 °C. To this was added 2-cyanoethyl N,N-diiso-
144.4, 135.9, 135.4, 134.2, 134.1, 133.0, 130.2, 130.0, 129.5, propylphosphoramidochloridite (1.20 mL, 5.4 mmol) drop-
128.8, 128.2, 128.1, 127.9, 127.3, 126.9, 126.8, 113.4, 113.3, wise, and the reaction mixture was stirred for 15–30 min at
87.5, 86.7, 61.8, 61.0, 59.9, 55.4, 55.3, 53.5, 50.7, 49.6, 49.0, room temperature. After completion, the reaction was
46.8, 44.6; HRMS (ESI+): calcd for C46H43N6O7 [M + H]+ quenched by addition of MeOH (1 ml) and the solvent was
791.3187, found 791.3201.
evaporated. The residue was chromatographed over silica
2-(2-(6-(N-Benzoylbenzamido)-9H-purin-9-yl)-N-(2-(bis(4-methoxy- (0–60% acetone/hexane–2% Et3N, v/v) to give the product 5d
phenyl)(phenyl)methoxy)ethyl)acetamido)ethyl 2-cyanoethyl diiso- (2.5 g, 3.0 mmol, yield = 66%) as a yellowish foam.
propyl phosphoramidite (5c). In a dry round bottom flask, 4c
31P-NMR (121 MHz; CDCl3) δ: 148.7, 147.0; HRMS (ESI+):
(3.0 g, 3.8 mmol) was dissolved in dry THF (20 ml). DIPEA calcd for C45H58N8O8P [M + H]+ 869.4109, found 869.4114.
(3.3 ml, 19 mmol) was added and the reaction was cooled to
0 °C. 2-Cyanoethyl N,N-diisopropylphosphor-amidochloridite
(1.0 mL, 4.56 mmol) was added and the reaction mixture was
Synthesis of the TEANA monomer with adenine nucleobase
stirred for 15–30 min at room temperature. After completion,
9-(2-Bromoethyl)-9H-purin-6-amine (6a). Adenine (10 g,
the reaction was quenched by addition of 1 ml of MeOH and 74 mmol) and K2CO3 (20 g, 148 mmol) were suspended in dry
the solvent was evaporated. The residue was chromatographed DMF (500 ml) and to this was added dibromoethane (12 ml,
over silica (0–60% acetone/hexane, v/v; 2% Et3N) to obtain the 148 mmol) drop-wise and stirred at room temperature over-
product 5c (2.82 g, 2.85 mmol; yield = 75%) as a yellowish night. After completion, the reaction was filtered and the
foam.
31P-NMR (121 MHz; CDCl3) δ: 149.1, 148.0; HRMS (ESI+): evaporation of the solvent was washed with water and ether,
calcd for C55H60N8O8P [M + H]+ 991.4266, found 991.4266.
and dried to obtain the product 6a (13 g, 55 mmol, yield =
N-(9-(2-(Bis(2-hydroxyethyl)amino)-2-oxoethyl)-6-oxo-6,9- 74%) as a yellow orange solid.
solvent was evaporated in vacuo. The solid residue left after
dihydro-1H-purin-2-yl)isobutyramide (3d). In a round bottom
1H NMR (300 MHz; DMSO-d6) δ: 8.16 (1H, s), 8.14 (1H, s),
flask, 2d (3.0 g, 11 mmol) was suspended in dry pyridine 4.56 (2H, appt, J = 7.37 Hz, 6.03 Hz), 3.93 (2H, appt, J =
(1 ml) and co-evaporated twice. The residue was suspended 7.37 Hz, 6.03 Hz); 13C (75 MHz; DMSO-d6) δ: 156.0, 152.5,
again in dry pyridine (50 ml) and cooled to 0 °C in an ice bath. 149.5, 118.7, 44.7, 31.6; HRMS (ESI+): calcd for C7H9BrN5
To this pre-cooled suspension was added trimethylsilyl chlor- [M + H]+ 242.0036, found 242.0036.
ide (5.0 ml, 40 mmol) and the reaction mixture was stirred at
9-(2-(Bis(2-(tert-butyldimethylsilyloxy)ethyl)amino)ethyl)-9H-
0 °C for 30 min and then at room temperature for 1 h. The purin-6-amine (6b). Compound 6a (10 g, 41 mmol) was sus-
reaction was cooled again to 0 °C and freshly distilled iso- pended in 100 ml dry dioxane. To this was added bis-silyl pro-
butyryl chloride (2.6 ml, 25 mmol) was added drop-wise to this tected ethanolamine (13 g, 41 mmol). Next CsCO3 (13.35 g,
turbid solution. The reaction mixture was stirred at 0 °C for 41 mmol) was added and the reaction mixture was refluxed
1 h and then stirred at room temperature for 3 h. After com- overnight. After completion, the reaction mixture was filtered
pletion, the reaction was cooled and quenched by addition of and the filtrate was evaporated. The residue was chromato-
10 ml of a 50 : 50 methanol : water mixture. The solvent was graphed on a silica column (0–60% acetone/hexane–2% Et3N,
evaporated and the residue was chromatographed over silica v/v) to obtain the product 6b (12 g, 26 mmol, yield = 64%).
(0–10% MeOH/DCM, v/v: 5% pyridine) to obtain the product
3d (3.6 g, 9.9 mmol; yield = 90%) as a white powder.
1H NMR (500 MHz; DMSO-d6) δ: 8.10 (1H, s), 8.0 (1H, s),
4.12 (2H, appt, J = 6.5 Hz, 5.9 Hz), 3.41 (4H, , appt, J = 6.9 Hz,
1H NMR (300 MHz; DMSO-d6) δ: 7.83 (s, 1H), 5.11 (2H, s), 6.4 Hz), 2.89 (2H, appt, J = 6.5 Hz, 5.9 Hz), 2.61 (4H, appt, J =
3.64 (2H, bs), 3.53–3.47 (4H, m), 3.38–3.34 (2H, m), 2.77 (1H, 7.1 Hz, 6.3 Hz), 0.8 (18H, s), 0.07 (12H); 13C (125 MHz; DMSO-
m), 1.10 (6H, d, J = 6.7 Hz); 13C (75 MHz; DMSO-d6) δ: 180.1, d6) δ: 155.9, 152.2, 149.5, 141.4, 118.7, 61.5, 56.5, 54.3, 41.7,
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25.8, 17.9, −5.4; HRMS (ESI+): calcd for C23H47N6O2Si2 (75 MHz; CDCl3) δ: 171.9, 157.9, 153.0, 151.3, 150.4, 147.5,
[M + H]+ 495.3293, found 495.3289.
145.0, 135.8, 133.5, 133.2, 129.5, 129.1, 128.8, 128.6, 128.4,
N-Benzoyl-N-(9-(2-(bis(2-(tert-butyldimethylsilyloxy)ethyl)- 127.7, 127.6, 126.6, 126.5, 113.1, 85.6, 61.7, 59.2, 56.3, 54.9,
amino)ethyl)-9H-purin-6-yl)benzamide (6c). Compound 6b 45.7, 42.1; HRMS (ESI+): calcd for C46H44N6O6Na [M + Na]+
(10 g, 20 mmol) was co-evaporated thrice with 5 ml dry pyri- 799.3214, found 799.3214.
dine and dissolved in the same (50 ml) and cooled in an ice
2-((2-(6-(N-Benzoylbenzamido)-9H-purin-9-yl)ethyl)(2-(bis(4-
bath to 0 °C. Freshly distilled benzoyl chloride (5.0 ml, methoxyphenyl)(phenyl)methoxy)ethyl)amino)ethyl 2-cyano-
45 mmol) was added drop-wise to this cooled solution and the ethyl diisopropyl phosphoramidite (6f). In a round bottom
reaction mixture was stirred for 6 h. After the disappearance of flask, 6d (1.0 g, 1.28 mmol) was dissolved in dry THF and
the starting material, the reaction was quenched with NaHCO3 cooled to 0 °C in an ice bath. 2-Cyanoethyl N,N-diisopropyl-
in ice. The aqueous layer was extracted with DCM. The organic phosphoramidochloridite (0.3 mL, 1.28 mmol) was then
layers were pooled, dried, evaporated and the residue was chro- added drop-wise to this pre-cooled solution and stirred in the
matographed on silica (0–50% EtOAc/hexane v/v) to obtain the ice bath for 15 min and then the ice bath was removed. After
product 6c (11 g, 16 mmol, yield = 82%).
completion, the reaction was quenched by addition of metha-
1H NMR (300 MHz; CDCl3) δ: 8.65 (1H, s), 8.36 (1H, s), 7.85 nol and the solvent was evaporated. The residue was chromato-
(4H, m), 7.45–7.33 (6H, m), 4.29 (2H, appt, J = 6.7 Hz, 5.5 Hz), graphed on silica to obtain the product 6f (0.65 g, 0.66 mmol,
3.57 (4H, appt, J = 6.7 Hz, 5.9 Hz), 3.03 (2H, appt, J = 6.7 Hz, yield = 52%) as a white foam.
5.6 Hz), 2.70 (4H, appt, J = 6.8 Hz, 6.0 Hz), 0.86 (18H, s), 0.02
31P-NMR (121 MHz; CDCl3) δ: 147.7; HRMS (ESI+): calcd for
(12H, s); 13C (75 MHz; CDCl3) δ: 172.4, 153.4, 151.9, 151.5, C55H62N8O7P [M + H]+ 977.4473, found 977.4471.
146.4, 134.4, 132.8, 129.5, 128.7, 127.5, 61.9, 57.3, 55.2, 42.9,
26.0, 18.3, −5.2; HRMS (ESI+): calcd for C37H55N6O4Si2
[M + H]+ 703.3817, found 703.3808.
N-Benzoyl-N-(9-(2-(bis(2-hydroxyethyl)amino)ethyl)-9H-purin-
Acknowledgements
6-yl)benzamide (6d). In
a plastic falcon tube, 6c (7 g, Mass spectrometry was made possible with the support of the
10 mmol) was dissolved in 10 ml dry THF and cooled. To this Hercules Foundation of the Flemish Government (grant
was added triethylamine trihydrofluoride (3.2 ml, 20 mmol) 20100225-7). We thank FWO (Vlaanderen) (G.078014N) for
and the reaction mixture was stirred at room temperature over- financial support. The research leading to these results has
night. The reaction was quenched by addition of solid silica received funding from the European Research Council
and chromatographed over silica (10–60% acetone/hexane–2% under the European Union’s Seventh Framework Programme
Et3N, v/v) to obtain the product 6d (4.0 g, 8.5 mmol, yield = (FP7/2007–2013)/ERC Grant agreement no ERC-2012-
85%).
ADG_20120216/320683. We thank Guy Schepers for synthesis
1H NMR (300 MHz; DMSO-d6) δ: 8.65 (1H, s), 8.62 (1H, s), and purification of oligonucleotides and Tm measurement and
7.78 (5H, m), 7.47 (5H, m), 4.33 (2H, appt, J = 6.8 Hz, 5.6 Hz), Chantal Biernaux for editorial support.
3.22 (4H, appt, J = 6.8 Hz, 6.2 Hz), 2.91 (2H, appt, J = 6.5 Hz,
5.9 Hz), 2.54 (4H, m); 13C (75 MHz; CDCl3) δ: 172.7, 153.6,
151.8, 151.0, 149.8, 148.6, 137.2, 134.0, 133.9, 129.5, 129.4,
Notes and references
128.8, 127.4, 124.7, 59.4, 56.7, 54.2, 42.8; HRMS (ESI+): calcd
for C25H27N6O4 [M + H]+ 475.2088, found 475.2086.
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