Synthesis of an elongated linear oligo(phenylene ethynylene)-based building block for application in DNA-programmed assembly

Article abstract of DOI:10.1039/b605844b

The synthesis of an elongated linear oligonucleotide-functionalised module (ELOM) is described. The ELOM structure is based on an oligo(phenylene ethynylene) backbone substituted with two decyloxy groups. The two termini constitute two salicylaldehyde moieties acting as chemical cross-linkers. Before incorporation into an oligonucleotide sequence the organic part of the module, the elongated linear module (ELM), is functionalised with a dimethoxytrityl group and a phosphoramidite group. This enables incorporation into the middle of 30-mer oligonucleotide sequences by automated DNA synthesis. The obtained ELOMs were characterised by polyacrylamide gel electrophoresis and MALDI-TOF mass spectrometry. In analogy with previously reported LOM and TOM structures the coupling reactions of the ELOM modules were tested. The Royal Society of Chemistry 2006.

Full text of DOI:10.1039/b605844b

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis of an elongated linear oligo(phenylene ethynylene)-based building
block for application in DNA-programmed assembly†‡
Peter Blakskjæra,b and Kurt V. Gothelf*a
Received 24th April 2006, Accepted 12th June 2006
First published as an Advance Article on the web 3rd July 2006
DOI: 10.1039/b605844b
The synthesis of an elongated linear oligonucleotide-functionalised module (ELOM) is described. The
ELOM structure is based on an oligo(phenylene ethynylene) backbone substituted with two decyloxy
groups. The two termini constitute two salicylaldehyde moieties acting as chemical cross-linkers. Before
incorporation into an oligonucleotide sequence the organic part of the module, the elongated linear
module (ELM), is functionalised with a dimethoxytrityl group and a phosphoramidite group. This
enables incorporation into the middle of 30-mer oligonucleotide sequences by automated DNA
synthesis. The obtained ELOMs were characterised by polyacrylamide gel electrophoresis and
MALDI-TOF mass spectrometry. In analogy with previously reported LOM and TOM structures the
coupling reactions of the ELOM modules were tested.
could alternatively be reduced with NaBH3CN which gave the
Introduction
corresponding amine-linked structures.10
The interest in using molecular building blocks for assembly of ge-
One limitation associated with this procedure is that more than
ometrically well-defined and functional molecular nanostructures
four modules could not be assembled in satisfactory yields. We
has led to the search for new ways to assemble organic molecules
imagine that this may be caused by steric interactions of the very
into supra- or macromolecular structures as an alternative to
large oligonucleotide duplexes. The diameter of the DNA duplex is
conventional organic synthesis. The formation of large and well-
2 nm, and the LOMs are also about 2 nm long. Thus, the multiple
defined molecular assemblies from a relatively simple set of
duplexes formed in these nanostructures are expected to induce
building blocks is of great interest for the field of molecular
steric strain as the structures grow. We therefore rationalised that
electronics.1 During the past few years a number of molecular
if this reason is prevalent, then one way of solving the problem
electronic components and wires have been synthesised and
may be by extending the length of the linear module.
evaluated for their electronic behaviour.2 Reliable methods for
Here we report on the synthesis of an elongated linear module
the controlled assembly of these devices into nanostructures have,
(ELM) designed to be incorporated into oligonucleotides by auto-
however, not yet been developed. In recent years much interest
mated DNA synthesis. We have previously shown that compounds
has been focused on the application of DNA as an encodable
of this type functionalised as a nucleoside phosphoramidite
material for the spatial positioning of molecules and materials.3,4
In addition, DNA-programmed chemistry has proven to be an
efficient tool to assemble and couple organic molecules.5,6
can be efficiently incorporated into an oligonucleotide strand
by automated oligonucleotide synthesis.4,7 This ELM meets the
requirements previously stated for the linear modules (LM’s),
i.e. it is rigid and potentially conducting.11 It is appropriately
functionalised both to be incorporated into oligonucleotides and
to undergo salen-formation. The aldehydes and the phenols have
to be protected since they are not compatible with the oligonu-
cleotide synthesis.1,12 The amide bonds provide a stable linkage to
the oligonucleotides7,8 and the C10 chains have been added to aid
solubility of the oligo(phenylene ethylene) backbone.13
As part of an ongoing programme addressing this problem we
recently published a new bottom-up method for the programmed
assembly and covalent coupling of multiple organic modules.6–10
The building blocks used in these reactions were linear (LOM)
and tripoidal (TOM) oligo(phenylene ethylene)-based structures
containing terminal salicylaldehyde moieties (Scheme 1). They
were encoded with short (15 nt) oligonucleotides at their termini.
Annealing of the structures followed by imine formation of the
aldehyde moieties using ethylenediamine in the presence of a
metal salt such as Mn(OAc)2 led to structures consisting of up
to four segments linked by metal–salen complexes.7 The imines
Results and discussion
The synthesis of the salicylaldehyde building block 3 for prepa-
ration of the functional end groups in ELM has previously
been reported by us (Scheme 2).8 The most obvious way to
build up the ELM oligo(phenylene ethylene) backbone is by a
series of Sonogashira couplings and the synthesis was carried
out in a straightforward manner according to Scheme 3.14 First,
the TMS-protected 4-(ethynyl)-1-iodobenzene building block 4
was prepared from (4-bromophenylethynyl)-trimethylsilane. The
(4-bromophenylethynyl)-trimethylsilane is commercially available
from Aldrich, but it was easily synthesised in high yield (85%)
aCenter for Catalysis and Interdisciplinary Nanoscience Center (iNANO),
Department of Chemistry, University of Aarhus, Langelandsgade, 140, Den-
mark. E-mail: kvg@chem.au.dk; Fax: +45 86196199; Tel: +45 89423907
bVipergen, Fruebjergvej 3, DK-2100, Copenhagen, OE, Denmark
† This paper was published as part of a themed issue on DNA-Based
Nano-Architectures and Nano-Machines.
‡ Electronic supplementary information (ESI) available: HPLC analyses
and UV spectra of ELOM 11, 12 and 13. See DOI: 10.1039/b605844b
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Scheme 1 Chemical structures of the linear oligonucleotide-functionalised module (LOM), the tripoidal oligonucleotide-functionalised module (TOM)
and the elongated linear oligonucleotide-functionalised module (ELOM).
Scheme 2 Synthesis of the protected salicylaldehyde head group.
by a Sonogashira coupling between 4-bromo-1-iodobenzene and
trimethyl-silylacetylene catalysed by bis(triphenyl-phosphine)-
palladium(II) chloride and copper(I) iodide in the presence of
diethylamine.15 Lithiation of the bromide facilitated by treatment
◦
with two equivalents of tert-butyllithium at −78 C followed by
addition to molecular iodine gave the corresponding iodide 4
Scheme 3 Preparation of the linear backbone by Sonogashira reactions.
in essentially quantitative yield.16 The central 1,4-bis-decyloxy-
2,5-diethynylbenzene (5) was synthesised according to a known
literature procedure.13c The conjugated backbone was assembled
by a Sonogashira coupling, using almost the same conditions as
in the first coupling, of the diacetylene 5 with two equivalents of
the iodide 4. The appearance of a strongly fluorescent solution
indicated the formation of the conjugated backbone and com-
pound 6 was isolated in an excellent yield of 95%. Removal of
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Scheme 4 Preparation of the elongated linear module (ELM) 9, functionalised as a phosphoramadite.
the trimethylsilyl groups in aqueous base gave the oligo(phenylene
ethylene) 7 in 87% yield.
chromatography on silica gel.8 The precipitated material was
analysed by 31P NMR and showed only minor impurities and
the sample was therefore not further purified but used directly for
automated oligonucleotide synthesis.
A final Sonogashira coupling with the 5-iodosalicylic aldehyde
derivative 3 constructed the ELM backbone 8a in good yield
(Scheme 4). One of the DMTr groups was removed by 50% acetic
acid in dichloromethane for 20 min. This reaction was carefully
monitored by thin layer chromatography and stopped as soon
as the product in which both DMTr groups were removed was
observed. Chromatography gave the desired mono-deprotected
derivative 8b in a moderate yield of 31%. However, 60% of the
starting material could easily be recovered and applied in repeated
deprotections. A following treatment with the required phos-
phoramidite chloride and Hu¨nigs base provide phosporamidite
9 which was isolated quantitatively by precipitation in pentane
(overall yield for two steps of 31%). We had previously shown
that phosporamidites of this type would partly decompose during
Phosphoramidite 9 was used as precursor for the elongated
linear oligonucleotide-functionalised module ELOM. The phos-
phoramidites were incorporated by standard automated oligonu-
cleotide synthesis into the middle of an oligonucleotide containing
30 nucleotides. After basic removal of the base protecting groups
the conjugates were purified by FPLC. The 5ꢀ-terminal DMTr
group was removed, the phenol moiety was deprotected in 25%
aqueous ammonia at 50 ◦C and finally the acetal protecting group
was removed in NaOAc buffer at pH 4. In this manner we have
prepared the three ELOM modules 11–13 listed in Table 1. This
table also includes LOM compound 14, which has been described
previously.7,8
Table 1 DNA-sequences and MALDI-TOF mass spectrometry characterization of ELOM and LOM structures
Module
DNA sequences
Calc. mass (Da)a
Observed mass (Da)b
ELOM 11
ELOM 12
ELOM 13
LOM 14c
a-ELM-bꢀ
b-ELM-cꢀ
c-ELM-dꢀ
b-LM-cꢀ
5ꢀ-ATTGATCTAGTTGAT-ELM-TGTACATCTACACTT-3ꢀ
5ꢀ-AAGTGTAGATGTACA-ELM-ACTTCAGTTGGTCGT-3ꢀ
5ꢀ-ACGACCAACTGAAGT-ELM-CTGTAGACATATGTT-3ꢀ
5ꢀ-AAGTGTAGATGTACA-LM-ACTTCAGTTGGTCGT-3ꢀ
10413.11
10528.13
10466.14
10480
10616
10552
a Mass spectra were recorded before removal of the acetal protecting groups. b The reported values represents the max. height of the peak; the peaks are
broad with a width at half max. height of approximately 400 Da. c This linear module is described in ref. 7.
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The resulting ELOM modules were characterised by MALDI-
TOF mass spectrometry and by denaturing polyacrylamide gel
electrophoresis.
The mass peaks are relatively broad with a width at half
max. height of around 400 Da, as it appears from the example
on the MALDI-TOF spectrum of ELOM 11 in Fig. 1A. Thus
the calculated masses fall well within the observed mass peaks.
The ELOMs were also analysed by denaturing polyacrylamide
gel electrophoresis (PAGE) in a 8 M urea buffer (Fig. 1B). The
mobility of the ELOM modules are shown in lanes 1–3 and 9 and
they show well-defined bands with a slightly lower mobility than
the previously reported LOM modules (lane 8).7 Additionally, the
purity and UV absorption of the three ELOMSs were determined
by HPLC (see: electronic supplementary information‡).
The coupling reaction between ELOM modules by a DNA-
programmed reaction between the salicylaldehyde moieties was
attempted in the presence of ethylenediamine and Mn(OAc)2
(Scheme 5). This approach was very efficient for the coupling
of up to four LOM modules.6,7,9,10 Our attempts to couple two or
three ELOM modules were unfortunately unsuccessful (Fig. 1B,
lanes 4–7), whereas the coupling between ELOM 13 and LOM
14 was successful and resulted in a well-defined product band
(lane 10). For the ELOM dimerisations and trimerisations in lanes
4 and 6, no well-defined product bands result from the PAGE
analysis. A smear is observed at the top of the gel close to the
ELOM monomer. Native PAGE did not improve the appearance
of the gels. We speculate that the lacking ability of the ELOM
structures to undergo dimerisation is due to aggregation of the
ELOM moieties. In addition to the length, a major difference
between the LOM/TOM structures and the ELOM structure
is the two decyloxy substituents in the ELOM structure. The
decyloxy groups were added to improve solubility of the ELM
in organic solvents. As a consequence, however, the ELOM
structure may behave as an amphiphile due to the hydrophilic
DNA-sequences and the hydrophobic decyloxy groups. Hence,
the ELOM structures may aggregate by hydrophobic interactions
during the coupling reactions leading to formation of higher
order oligomeric structures. To suppress the aggregation it was
attempted to perform the dimerisation and trimerisation reactions
in the presence of sodium dodecyl sulfate (lanes 5 and 7). However,
this did not improve the coupling reactions and the smearing was
only slightly reduced or the mobility of smeared bands changed.
Future studies will show if it is possible to circumvent these
problems by substitution of the decyloxy side chains with e.g.
ethyleneglycol oligomers. However, it will also be investigated
if we can take advantage of the amphiphilic nature of the
ELOM. For example, by testing the assembly and coupling of
ELOM structures in the interface between water and a hydropho-
bic solvent. It has been reported that oligo(phenylene ethynylene)s
with alkyl side chains absorb and align on the surface of carbon
nanotubes.17 We will explore the possible DNA-programmed
Fig. 1 Analysis of ELOM and LOM and their coupling reactions. (A)
MALDI-TOF mass spectrum of ELOM 11 with a calc. mass of 10413
Da and an obs. mass of 10480 Da. (B) Denaturing polyacrylamide gel
electrophoresis in 8 M urea of the coupling products. Reactions were
performed in the presence of 1.0 mM EDA and 0.5 mM Mn(OAc)2. Lanes
1–3: ELOM monomers, lanes 4–7 coupling reactions of ELOM modules in
the absence and presence of SDS as indicated, lanes 8–9 LOM and ELOM
monomers, coupling between LOM 14 and ELOM 13.
Scheme 5 DNA-programmed coupling between two salicylaldehyde head groups by manganese salen formation (left) and the results of basic couplings
between ELOMs and LOM (right).
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assembly of the ELOM structures in the presence of carbon
nanotubes.
MALDI-TOF C52H70O2Si2 [M+]; calculated: 782.49 found: 799.26.
UV/VIS (abs. ethanol) kmax/nm 256, 323, 385.
1,4-Bisdecyloxy-2,5-bis(4-ethynylphenylethynyl)-benzene
(7).
Conclusion
1,4-Bisdecyloxy-2,5-bis(4-trimethylsilanylethynyl-phenylethynyl)-
benzene (6) (162.2 mg, 0.207 mmol) dissolved in a mixture of
THF (5.0 mL) and methanol (3.0 mL) and was cooled to 0 ◦C
and treated with 5 M NaOH(aq) (0.25 mL, 1.25 mmol). This
mixture was warmed to room temperature and stirred for 16 h.
Then water and CH2Cl2 were added and the aqueous phase was
extracted with CH2Cl2 (3 × 10 mL). The organic phase was dried
(MgSO4) and evaporated which gave the desired product as a
In conclusion, an efficient synthesis of a highly functionalised
elongated linear module containing an oligo(phenylene ethyny-
lene) backbone has been developed. This ELM is functionalised
as a nucleoside phosporamidite which can be incorporated
into oligonucleotides by automated synthesis. The assembly and
coupling between a LOM structure and an ELOM structure
was successful, however, it was not possible to induce DNA-
programmed ELOM dimerisation or trimerisation. It is proposed
that this is due to the amphiphilic nature of the ELOMs. Future
studies may reveal if the amphiphilic nature of the ELOM can be
used for assembly and coupling at interfaces.
1
yellow fluorescent solid (114.5 mg, 87%). H NMR (CDCl3) d
(ppm) 7.48 (s, 8H), 7.01 (s, 2H), 4.02 (t, 4H, J = 6.4 Hz), 3.19 (s,
2H), 1.85 (quint, 4H, J = 7.6 Hz), 1.54 (quint, 4H, J = 7.6 Hz),
1.40–1.22 (m, 24H), 0.88 (t, 6H, J = 7.2 Hz). 13C NMR (CDCl3)
d (ppm) 153.9, 132.3, 131.7, 124.1, 122.1, 117.0, 114.1, 94.7, 88.2,
83.5, 79.2, 69.8, 32.1, 29.9, 29.8, 29.7, 29.6, 29.5, 26.3, 22.9, 14.4.
MALDI-TOF C46H54O2 [M+]; calculated: 638.41 found: 634.82.
Experimental
General methods
DMTrO-ELM-ODMTr (8a). To a Schlenk flask charged with
Pd(PPh3)2Cl2 (2.2 mg, 3.1 lmol) and CuI (1.2 mg, 6.2 lmol) was
added 1,4-bisdecyloxy-2,5-bis-(4-ethynyl-phenylethynyl)-benzene
(7) (109.7 mg, 0.172 mmol) dissolved in THF (4.0 mL)
and 1-benzoyloxy-2-(N-(3-dimethoxytrityloxyprop-1-yl)-amino-
carbonyl)-6-(1,3-dioxan-2-yl)-4-iodobenzene (3) (279.9 mg,
0.344 mmol) dissolved in THF (4.0 mL). To this solution was
added triethylamine (4.0 mL) and the mixture was stirred for
24 h at room temperature. The solvents were evaporated and
a 1 : 1 mixture of EtOAc and CH2Cl2 was added. Filtration
through a plug of silica gel and evaporation gave a residue that
was purified by flash chromatography on silica gel using EtOAc–
CH2Cl2–pentane 1 : 1 : 3 → 5 : 4 : 11 as eluents. This gave the
desired product as a yellow fluorescent foamy solid (255.4 mg,
74%). 1H NMR (CDCl3) d (ppm) 8.15 (dd, 4H, J = 7.2, 1.2 Hz),
7.95 (d, 2H, J = 2 Hz), 7.74 (d, 2H, J = 2), 7.67 (tt, 2H, J =
7.6, 1.2 Hz), 7.53–7.47 (m, 12H), 7.36 (dd, 4H, J = 7.2, 1.2 Hz),
7.30–7.21 (m, 12H), 7.19 (tt, 2H, J = 7.2, 1.2 Hz), 7.03 (s, 2H),
6.77 (d, 8H, J = 8.8 Hz), 6.49 (t, 2H, J = 6.0 Hz), 5.59 (s, 2H),
4.14–4.09 (m, 4H), 4.05 (t, 4H, J = 7.0 Hz) 3.81–3.72 (m, 4H),
3.76 (s, 12H), 3.40 (q, 4H, J = 6.0 Hz), 3.09 (t, 4H, J = 6.0 Hz),
2.18–2.06 (m, 2H), 1.87 (quint, 4H, J = 7.0 Hz), 1.66 (quint, 4H,
J = 6.0 Hz), 1.56 (quint, 4H, J = 7.0 Hz), 1.40–1.24 (m, 26H),
0.88 (t, 6H, J = 7.0 Hz). 13C NMR (CDCl3) d (ppm) 165.2, 164.7,
158.4, 153.7, 145.4, 144.7, 136.2, 133.9, 132.6, 132.5, 132.3, 131.61,
131.55, 131.2, 130.3, 129.9, 129.2, 128.8, 128.1, 127.9, 126.8, 123.5,
122.7, 121.6, 116.8, 113.9, 113.1, 97.6, 94.7, 90.2, 89.9, 88.1, 86.2,
69.6, 67.4, 61.6, 55.2, 38.2, 32.0, 29.7, 29.6, 29.5, 29.41, 29.37, 29.3,
26.1, 25.5, 22.8, 14.2. MALDI-TOF C130H132N2O18 [M + Na+];
calculated: 2031.94 found: 2032.58 UV/VIS (CH2Cl2): kmax/nm
(log e): 237 (4.83), 277 (4.32), 332 (4.37), 389 (4.40)
1
The H NMR, 31P NMR and 13C NMR were recorded at 400,
160 and 100 MHz, respectively. Chemical shifts are reported in
1
ppm downfield to TMS (d = 0) for H NMR, relative to the
central CDCl3 resonance (d = 77.16) for 13C NMR and relative to
85% H3PO4 (d = 0) for 31P NMR. All reactions were carried out
under an argon atmosphere using standard Schlenk equipment
and vacuum line techniques unless otherwise stated.
Materials
Solvents were dried according to standard procedures and
distilled under an atmosphere of argon or nitrogen prior to
use. (4-Bromophenylethynyl)-trimethylsilane14 is commercially
available from Aldrich but was synthesised from 1-bromo-4-
iodobenzene (Aldrich). (4-iodophenylethynyl)trimethylsilane,16
1,4-bis-decyloxy-2,5-diethynylbenzene13c and 1-benzoyloxy-2-(N-
(3-dimethoxytrityloxyprop-1-yl)-aminocarbonyl)-6-(1,3-dioxan-
2-yl)-4-iodobenzene (3)8 were synthesised according to literature
procedures.
1,4 - Bis - decyloxy - 2,5 - bis - (4 - trimethylsilanylethynyl - phenyl-
ethynyl)-benzene (6). To a Schlenk flask charged with 1,4-
bisdecyloxy-2,5-diethynylbenzene (5) (87.7 mg, 0.20 mmol), (4-
iodophenylethynyl)-trimethylsilane (4) (132.1 mg, 0.44 mmol),
Pd(PPh3)2Cl2 (2.8 mg, 0.004 mmol) and CuI (1.5 mg, 0.008 mol)
was added THF (3.0 mL) and diethylamine (1.0 mL). This solution
was stirred at room temperature for 19 h. The solvents were
evaporated and the residue diluted with CH2Cl2 and satd. NaCl
solution. The aqueous solution was extracted with CH2Cl2 (3 ×
10 mL), dried (MgSO4) and evaporated. The crude material was
purified by flash chromatography on silica gel using CH2Cl2–
pentane 1 : 9 → 1 : 4 as eluents to yield the title compound as
DMTrO-ELM-OH (8b). To a 50 mL roundbottomed flask
charged with DMTrO-ELM-ODMTr (8a) (109.7 mg, 54.6 lmol)
was added a 1 : 1 solution of acetic acid and dichloromethane
(7.5 mL). This solution was stirred for 20 min. at room temper-
ature and then poured into satd. NaHCO3 and extracted with
dichloromethane (3 × 20 mL), dried over Na2SO4, filtered and
evaporated. The residue was purified by flash chromatography on
silica gel using EtOAc–pentane 1 : 1 → 4 : 1 as eluents. This
1
a yellow fluorescent crystalline solid (149.0 mg, 95%). H NMR
(CDCl3) d (ppm) 7.44 (center of an AB spin system, 8H, JAB
=
8.4 Hz), 6.99 (s, 2H), 4.02 (t, 4H, J = 6.4 Hz), 1.84 (quint, 4H, J =
6.8 Hz), 1.53 (quint, 4H, J = 6.8 Hz), 1.38–1.10 (m, 24H), 0.88 (t,
6H, J = 6.8 Hz), 0.25 (s, 18H). 13C NMR (CDCl3) d (ppm) 153.8,
132.0, 131.4, 123.6, 123.0, 116.9, 114.1, 104.8, 96.4, 94.7, 88.0,
69.7, 32.0, 29.8, 29.7, 29.5, 29.4 (2 signals), 26.2, 22.8, 14.2, 0.0.
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gave the desired alcohol 8b as a yellow fluorescent solid (28.8 mg,
31%) together with the starting material (66 mg, 60%). 1H NMR
(CD2Cl2) d (ppm) 8.12 (d, 2H, J = 7.6 Hz), 8.04 (d, 2H, J = 7.6 Hz),
7.86 (s, 2H), 7.78 (d, 1H, J = 2.0 Hz), 7.67 (d, 1H, J = 2.0 Hz), 7.63
(t, 1H, J = 7.5 Hz), 7.61 (t, 1H, J = 7.5), 7.49 (t, 4H, J = 7.5 Hz),
7.45 (s, 8H), 7.28 (d, 2H, J = 7.5 Hz), 7.19–7.11 (m, 2H), 7.16 (d,
4H, J = 8.4 Hz), 6.97 (s, 2H), 6.69 (d, 4H, J = 8.4 Hz), 6.50 (br m,
1H), 6.40 (br m, 1H), 5.53 (s, 2H), 4.04–3.94 (m, 8H), 3.73–3.65
(m, 4H), 3.66 (s, 6H), 3.39 (m, 2H), 3.31 (q, 2H, J = 6.2 Hz),
3.26 (q, 2H, J = 6.2 Hz), 2.98 (t, 2H, J = 5.6 Hz), 2.57 (br s,
1H), 2.06–1.94 (m, 2H), 1.78 (quint, 4H, J = 7.2 Hz), 1.56–1.38
(m, 8H), 1.34–1.12 (m, 26H), 0.80 (t, 6H, J = 6.8 Hz). 13C NMR
was not obtained of this compound. MALDI-TOF C109H114N2O16
[M + Na+]; calculated: 1729.81 found: 1730.24.
recovered by precipitation overnight with 3 volumes of ethanol at
◦
−20 C and centrifugation (15 min at 10 000g). Resulting pellets
were washed and centrifuged three times with 75% (v/v) ethanol
and stored in 10 mM EPPS (pH 8.0) at −20 ◦C.
DNA-programmed coupling between the modules
A solution (10 ll) of the two or three ELOM or LOM modules
(5 lM each) in 100 mM KCl, 50 mM EPPS (pH 8.0) were
heated to 60 ◦C for 5 min and cooled slowly to rt in a water
bath. The coupling reactions were performed by addition of
0.25 mM ethylenediamine, 1 mM Mn(OAc)2 and incubation for
2 h at 30 ◦C. Analysis of the reaction products was performed by
electrophoresis at 90 V in 7.5% polyacrylamide gels (30 : 1.6) in
50 mM Tricine (pH 8.1) and 8 M urea. Samples were loaded in 8 M
urea without heating and addition of dyes. Gels were fixed in 50%
(v/v) ethanol, stained with ethidium bromide and photographed
in UV light.
DMTrO-ELM-OP(NiPr2)O(CH2)2CN (9). DMTrO-ELM-
OH (8b) (104.0 mg, 0.061 mmol) was dissolved in CH2Cl2 (10 mL)
in a Schlenk flask and cooled to 0 ◦C then diisopropylethylamine
(74.2 lL, 0.43 mmol) was added followed by dropwise treatment
with diisopropyl-chlorophosphoramidite (40.8 lL, 0.18 mmol).
This mixture was stirred at this temperature for 10 min then
allowed to reach room temperature and stirred for another 1 h
followed by dilution with CH2Cl2 (20 mL) and washed twice with
satd. NaHCO3 and once with water. Drying over Na2SO4 and
concentration gave a yellow oil that was precipitated into pentane
to give the desired phosphoramidite as a yellow fluorescent
chunky solid (116.1 mg, 100%) after removal of the liquid and
a wash with pentane followed by drying in vacuo. 1H NMR
(CD2Cl2) d (ppm) 8.13 (dd, 2H, J = 8.0, 1.2 Hz), 8.04 (dd, 2H, J =
8.0 Hz), 7.87 (d, 1H, J = 2.0 Hz), 7.86 (d, 1H, J = 2.0 Hz), 7.75
(d, 1H, J = 2.0 Hz), 7.67 (d, 1H, J = 2.0 Hz), 7.65–7.60 (m, 2H),
7.50 (t, 4H, J = 8.0 Hz), 7.46 (centre of an AB spin system, 8H,
JAB = 7.2 Hz), 7.28 (d, 2H, J = 8.0 Hz), 7.19–7.10 (m, 2H), 7.17
(d, 4H, J = 8.8 Hz), 6.97 (s, 2H), 6.69 (d, 4H, J = 8.8 Hz), 6.45 (t,
1H, J = 5.6 Hz), 6.39 (t, 1H, J = 5.6 Hz), 5.53 (s, 2H), 4.20–4.00
(m, 10H), 3.82–3.70 (m, 4H), 3.72 (s, 6H), 3.70–3.24 (m, 6H), 3.05
(t, 2H, J = 5.6 Hz), 2.55 (t, 2H, J = 6.5 Hz), 2.18–2.01 (m, 2H),
1.85 (quint, 4H, J = 7.2 Hz), 1.68–1.38 (m, 8H), 1.44–1.10 (m,
26H), 1.15 (d, 6H, J = 7.0 Hz), 1.10 (d, 6H, J = 7.0 Hz), 0.85 (t,
6H, J = 6.8 Hz). 31P NMR (CD2Cl2) d (ppm) 148.8.
Acknowledgements
We are indebted to the Danish National Science Foundation and
the Carlsberg Foundation for financial support.
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This journal is
The Royal Society of Chemistry 2006
Org. Biomol. Chem., 2006, 4, 3442–3447 | 3447
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