Solution-phase synthesis of branched DNA hybrids based on dimer phosphoramidites and phenolic or nucleosidic cores

Article abstract of DOI:10.1021/jo202505h

Branched oligonucleotides with CG zippers as DNA arms assemble into materials from micromolar solutions. Their synthesis has been complicated by low yields in solid-phase syntheses. Here we present a solution-phase synthesis based on phosphoramidites of dimers and phenolic cores that produces six-arm or four-arm hybrids in up to 61% yield. On the level of hybrids, only the final product has to be purified by precipitation or chromatography. A total of five different hybrids were prepared via the solution-phase route, including new hybrid (TCG)₄TTPA with a tetrakis(triazolylphenyl)adamantane core and trimer DNA arms. The new method is more readily scaled up than solid-phase syntheses, uses no more than 4 equiv of phosphoramidite per phenolic alcohol, and provides routine access to novel materials that assemble via predictable base-pairing interactions.

Full text of DOI:10.1021/jo202505h

Article
pubs.acs.org/joc
Solution-Phase Synthesis of Branched DNA Hybrids Based on Dimer
Phosphoramidites and Phenolic or Nucleosidic Cores
Helmut Griesser, Mariyan Tolev, Arunoday Singh, Thomas Sabirov, Claudia Gerlach,
and Clemens Richert*
Institute for Organic Chemistry, University of Stuttgart, 70569 Stuttgart, Germany
S
* Supporting Information
ABSTRACT: Branched oligonucleotides with “CG zippers” as DNA arms assemble into materials from micromolar solutions.
Their synthesis has been complicated by low yields in solid-phase syntheses. Here we present a solution-phase synthesis based on
phosphoramidites of dimers and phenolic cores that produces six-arm or four-arm hybrids in up to 61% yield. On the level of
hybrids, only the final product has to be purified by precipitation or chromatography. A total of five different hybrids were
prepared via the solution-phase route, including new hybrid (TCG)4TTPA with a tetrakis(triazolylphenyl)adamantane core and
trimer DNA arms. The new method is more readily scaled up than solid-phase syntheses, uses no more than 4 equiv of
phosphoramidite per phenolic alcohol, and provides routine access to novel materials that assemble via predictable base-pairing
interactions.
dinucleotides of the sequence CG suffice to induce assembly
into a material from dilute aqueous solution upon addition of
divalent cations.17,18 The strong assembly forces observed in
these multivalently binding hybrids have led to the term “CG
zippers” for dinucleotide arms in hybrids, such as 1 and 2
(Figure 1). Diffusion of intercalators into assemblies demon-
strated that the materials are porous and produced fluorescent
solids with interesting optical properties.18
The stability of phenolic phosphodiester linkages in hybrids
of oligodeoxynucleotides to the deprotection conditions used in
standard DNA synthesis protocols had been established prior
to our work.19 But, no more than minute amounts of branched
oligonucleotides with rigid phenolic cores had been available,
hampering an exploration of the interesting materials formed by
hybrids. Compounds like 1 and 2 were synthesized in low yield
on controlled pore glass (cpg), using a combination of 5′- and
3′-phosphoramidite building blocks, phenolic cores, and on-
support phosphitylation, immediately followed by coupling of
alcohols.17
INTRODUCTION
■
Synthesis has been the driving force behind countless advances
in science and technology. This is also true for the synthesis of
nucleic acids. The development of automated solid-phase
synthesis of oligodeoxynucleotides in the late 1970s and 1980s1
enabled decisive advances in biotechnology, genetics, and
diagnostics. Likewise, synthetic methodologies are driving new
developments in sequencing2 and synthetic biology.3,4
Currently, chain assembly on solid support, based on
phosphoramidites of protected nucleosides as monomers, is
the dominating methodology for synthesizing both DNA and
RNA strands.5 This methodology routinely produces linear
oligonucleotides of any given sequence up to 150 nucleotides in
length in fully automated fashion.
The advent of DNA nanotechnology6 has increased the
demand for conventional synthetic oligonucleotides7 but has
also spawned research into branched oligonucleotides not
found in biology.8,9 Among the branched structures are
conjugates between DNA and dendrimers,10 transition-metal
complexes,11 gold nanoparticles,12,13 cross-links,14 or organic
branching elements.15,16 We were recently able to show that
branched oligonucleotide hybrids featuring rigid organic
branching elements as cores and a direct phosphodiester
linkage to the DNA assemble at much higher temperatures than
linear control strands.17 When self-complementary DNA
sequences are appended to the termini of tetrahedral or
pseudo-octahedral cores, such as tetrakis(p-hydroxyphenyl)-
methane (TPM) or hexakis(p-hydroxyphenyl)xylene (HPX),
Optimization of the solid-phase syntheses proved challeng-
ing. The lability of the phosphoramidite-terminated chain, the
simultaneous assembly of three or five oligonucleotide chains
on a nonswellable support, together with issues arising from the
properties of aromatic triester intermediates made it difficult to
generate quantities suitable for systematic crystallization screens
Received: December 8, 2011
Published: February 27, 2012
© 2012 American Chemical Society
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Figure 1. Branched DNA hybrids with six or four DNA chains attached to a phenolic core.
Figure 2. Retrosynthetic approaches to a protected DNA hybrid, starting from a phenolic core and either dinucleotide building blocks or
phosphoramidites of mononucleosides; PG = protecting group.
Scheme 1. Synthesis of 3′-Phosphoramidites of Dimer
or studies on the absorption of gases or guest molecules.
Therefore, we decided to establish a solution-phase synthesis,
well knowing that this approach, classically employed for DNA
chain assembly,20 had become unpopular among most nucleic
acid chemists.21 Here we report an efficient solution-phase
synthesis of hybrids 1−4, based on the coupling of 3′-
phosphoramidites of dimer fragments and unmodified cores,
together with observations on the chemistry of these phenolic
diesters. All intermediates on the hybrid stage were isolated
without chromatography.
RESULTS AND DISCUSSION
■
Two strategies for the assembly of fully protected branched
DNA hybrids were tested: block coupling of dimers to the core,
or stepwise elaboration of the DNA chains from monomer
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a
Scheme 2. Synthesis of the TPM Hybrids (CG)4TPM (2), (TC)4TPM (3), and (GA)4TPM (4)
a
AMA = ammonium hydroxide/methylamine (1:1).
phosphoramidites, as shown in Figure 2 for a TPM hybrid with
DNA arms of the sequence 5′-TC-3′. Initial experiments
focused on the latter approach, as this promised low costs,
being based on inexpensive commercial phosphoramidites.
Attempts to employ the methodology of van der Marel and
colleagues, originally developed for solution-phase synthesis of
short linear DNA chains,21b were not satisfactory. Besides
incomplete coupling to phenolic cores, severe losses during
handling and purification of intermediates that were apparently
caused by the lability of the cyanoethyl groups of the
phosphotriesters limited overall yields to below 5% in the
very best cases and usually to yields ≤1%.
Chain assembly started from commercial phosphoramidites
5−8. In our case, they were prepared by phosphitylation of the
3′-unprotected nucleosides, as detailed in the Supporting
Information (Scheme S1). Unexpectedly, the diastereomeric
mixture of silyl-protected phosphoramidites (8) crystallized
from pentane. Phosphoramidites 5−8 were coupled to 5′-
alcohols 9−11, and the resulting phosphite triesters were
oxidized in situ with TBHP to give fully protected dimers 12−
15 in 71−80% yield, after chromatography. The 3′-terminal
phenoxyacetyl (PAC) groups were then removed via aminolysis
to yield 16−19. Careful monitoring of this reaction is
recommended to avoid the loss of base protecting groups.
Two equivalents of ammonia gave better yields than the 10
equiv reported in ref 23. Phosphitylation of the 3′-alcohols then
gave the desired dimer building blocks 20−24 in 50−80% yield
after column chromatography (31−54% overall yield from the
monomeric starting materials). Lower yields for 23 and 24 may
be due to unoptimized conditions.
We then turned to the assembly of hybrids. First, the less
challenging four-arm hybrids with TPM as core were
synthesized (Scheme 2). Initially, all attempts to obtain 25−
27 in high yield were unsuccessful. Independent of how many
equivalents of phosphoramidite were used and how long the
coupling reaction was allowed to proceed, incomplete
conversions were observed, as monitored by MALDI-TOF
mass spectra of crudes, measured after detritylation (the DMT-
protected species gave too little signal). Invariably, the spectra
also showed peaks for products with just three or two DNA
chains.
A detailed study then demonstrated that over time
decomposition or hydrolysis of the initial phosphite triester
set in, competing with the coupling to the remaining free
hydroxy groups. The modest solubility of the core further
complicated the synthesis. Five steps were taken to overcome
these problems. First, the concentration of the tetrazole catalyst
was reduced, thus reducing the rate of the decomposition
reaction. Second, the core was freeze-dried from a shock-frozen
hot solution in dioxane, producing a fine powder that helped to
bring the TPM rapidly into solution upon reacting with the
phosphoramidites. Third, to remove residual water traces, the
core was dried in vacuo at 130 °C for 2 h, and molecular sieves
(3 Å) were added to the mixture of educts for coupling. Fourth,
an unusual temperature protocol was followed because a
temperature study had shown that decomposition became more
dominant at elevated temperature than in the cold. During the
We then focused on block coupling, based on dimer
phosphoramidites, to reduce the number of steps on the
hybrid level and to facilitate purification. Failure products
lacking entire chains should be easier to separate from products
than failures lacking only a single nucleotide. Several
approaches to the synthesis of dimer or trimer phosphor-
amidites as building blocks in solid-phase DNA synthesis
exist.22 We chose the method of Virnekas et al.,23 originally
̈
developed for trimer phosphoramidites, because it gave
satisfactory yields in exploratory experiments and because it
uses methyl protecting groups for the phosphotriesters. The
methyl groups should be more stable than the cyanoethyl
groups tested first. They should be removable by SN2
displacement with soft nucleophiles.
Three different DNA sequences were tested: the established
self-complementary 5′-CG-3′ “zipper” dimer,17,18 and the two
nonselfcomplementary sequences 5′-TC-3′ and 5′-GA-3′ that
were used as sticky ends in designed nanoporous DNA
crystals.24 Two different 5′-protecting groups were tested: the
conventional dimethoxytrityl (DMT) group, and the tert-
butyldimethylsilyl (TBDMS) group that can be removed under
basic conditions, thus avoiding acidic conditions on the hybrid
level. Tailored silyl-protecting groups have been developed for
solid-phase RNA synthesis,25 where multiple deprotections are
required. However, TBDMS group are well established as 2′-
protecting groups of oligoribonucleotide chains, where a single
deprotection step is employed after assembly of the entire
chain.26 Also, both methyl and cyanoethyl protecting groups
were tested for the 3′-terminal phosphite/phosphate (dimers 20
and 24). The cyanoethyl group can be removed under milder
conditions, reducing the likelihood of cleaving phenolic esters
during the process. Scheme 1 shows the route to the dimer
phosphoramidites.
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Scheme 3. Synthesis of HPX Hybrid (CG)6HPX (1)
initial phase of the reaction, the solution was sonicated at room
temperature until the core had dissolved, and then the reaction
mixture was cooled to allow the coupling to progress to
completion. Finally, two consecutive cycles of coupling with
subsequent oxidation were employed to drive the assembly of
the hybrids to completion, where necessary, with isolation of
the first crude via precipitation. With these measures, hybrids
25−27 were obtained in 85−99% yield.
groups were removed first. For this, thionaphthol was identified
as a less toxic and more selective substitute for thiophenol. This
Deprotection of the hybrids proved challenging. Difficulties
in deprotecting the phenolic triester were expected, based on
the literature.27 Partial loss of DNA chains on one of the three
stages of the deprotection schemes was difficult to suppress.
The acidic step for the removal of the DMT groups was studied
first. For each of the three hybrids 25−27, less than 5% three-
arm product was observed at the end of the reaction, both with
trichloroacetic acid (TCA, 3% in CH2Cl2) followed by
methanol and with 80% AcOH. The same was true for the
detritylation of the analogous six-arm hybrid with HPX core
(Scheme 3). Only when the methyl groups were removed first,
and the resulting diesters were treated with strongly acidic
cation-exchange resin Amberlyst 15 (H+ form) did depurina-
tion set in noticeably, as monitored by MALDI-TOF mass
spectrometry. The final two basic steps (deprotection of the
phosphotriesters to phosphodiesters and removal of nucleobase
protecting groups) caused more side reactions, leading to
partial loss of DNA chains for purine-containing sequences.
The side products lacked one or several DNA chains, usually
featuring phosphate groups instead. These “failures” were not
the result of incomplete couplings, though, because MALDI-
TOF mass spectra of protected intermediates showed little
evidence of such species (Figure 3) and because incomplete
coupling should result in unphosphorylated, free phenols.
For TPM hybrids 25−27, two different protocols were
tested. For purine-containing 25 and 27, the methyl protecting
Figure 3. MALDI-TOF mass spectra of crudes of protected hybrids
after removal of the DMT groups with trichloroacetic acid (TCA, 3%
in CH2Cl2 for 30 s, followed by addition of methanol). (a) Four-arm
hybrid 25 and b) six-arm hybrid 28. In each spectrum, peaks of
residual side products lacking one oligonucleotide chain are labeled,
with m/z = 2843 for (CBzGiBu)3TPM, and m/z = 4761 for
(CBzGiBu)5HPX.
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Figure 4. MALDI-TOF mass spectra of (a) crude (TC)4TPM (3), (b) crude (CG)6HPX (1), and (c) crude (TCG)4TTPA (34).
reagent also gave higher yields than 2-carbamoyl-2-cyano-
ethylene-1,1-dithiolate, a reagent developed specifically for the
demethylation step.28 A solution in THF/water with
diisopropylethylamine (DIEA) as base gave the best results.
Subsequently, the DMT groups were removed by treatment
with acidic cation-exchange resin in a mixture of water and
ethyl acetate. Finally, the base protecting groups were cleaved
using ammonia and methylamine. For purine-free 26, the
demethylation step was also performed with thionaphthol in
THF, but with triethylamine as base, followed by detritylation
with 80% acetic acid. The final deprotection step proceeded in
high yield with just aqueous ammonia. Hybrid 3 was obtained
in sufficiently pure form for assembly studies, without
chromatography, after precipitation from water/ethanol
(Figures 4a and 5).
As expected, on the basis of statistics, establishing a high-
yielding synthesis for six-arm hybrid (CG)6HPX was more
difficult than for the TPM hybrids (Scheme 3). High-yielding
coupling to all six phenolic hydroxy groups of HPX was
achieved when the strategy described above was applied (finely
dispersed core, “inverse” temperature regime, two coupling
cycles) for each of the different CG dimers employed (20, 23,
and 24). With fully protected hybrids 28−30, a range of
conditions was tested in order to optimize the deprotection to
hybrid 1. For this, crudes were again analyzed by MALDI-TOF
mass spectrometry, and relative peak heights were compared
without an attempt to compensate for differences in desorption
and ionization efficiency. Since longer sequences fly more
poorly,29 the intensity of product peaks should underrepresent
the true concentration of the fully assembled hybrids, compared
to the failure sequences. As with the TPM hybrids, sufficient
MALDI signal was observed only after removal of DMT
groups.
For 28 with its conventional protection of 5′-termini and
phosphodiesters, crudes of 1 contained 25−30% of the five-arm
product with a phosphoryl group on one of the phenolic arms,
when thionaphthol was used first, followed by removal of base
protecting groups with NH4OH and methylamine (a mixture
commonly referred to as “AMA”) and subsequent detritylation.
When the DMT groups were removed first, significantly more
truncation products were observed in MALDI spectra of crude
1, with up to 10% four-arm products and up to 40% five-arm
fragments. A similar result (35% terminally phosphorylated
five-arm side product) was obtained when silyl-protected 29
was demethylated with thionaphthol and DIEA, followed by
desilylation with TBAF and deacylation of the bases with AMA.
Changing the order of desilylation with TBAF and deacylation
Figure 5. 1H NMR spectrum of (TC)4TPM (3) in D2O at 40 °C (500
MHz). Assignments, based on 2D spectra, are given above the peaks.
Table 1. Yield and Composition of Crudes and Isolated Yields of Purified Hybrids
c
d
entry
no.
yield of crude
(%)
product ratio by
MS Pr/phosph/−arm
product ratio by
HPLC Pr/phosph/−arm
yield UV /grav
a
b
compd
(%)
1
2
3
4
5
6
7
(CG)6HPX (1) from 28
(CG)6HPX (1) from 29
(CG)6HPX (1) from 30
(CG)4TPM (2)
98
72:25:3
57:35:8
74:12:14
60:29:11
96:<1:4
35:59:6
88:4:8
62:27:11
13/n.d.
95
−/−
25/50
e
88
72:13:15
61:28:11
94:<1:6
51:40:9
94
18/n.d.
f
(TC)4TPM (3)
87
40/61
(GA)4TPM (4)
94
5/n.d.
e
(TCG)4TTPA (34)
quant
−/24
a
Ratio of product (Pr) to phosphorylated side product lacking one DNA arm (phosph) and hybrid lacking one arm (−arm), as determined by
b
MALDI-TOF MS. Also compare Scheme 4 for the structure of fragments formed during deprotection. Ratio of product and fragments, as
determined by HPLC, corrected for differences in extinction coefficient. Yield of hybrid determined by UV absorption at λ = 260 nm. Yield of
purified, isolated hybrid, determined gravimetrically. Purified by HPLC. Purified by precipitation.
c
d
e
f
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with AMA did not affect the compositions of crudes
significantly.
strand cleavage, with the phosphoryl group remaining on the
phenolic core. Scheme 4 shows possible side products resulting
from cleavage of phosphodiester linkages. In order to better
understand the side products observed in the crudes of hybrids,
we studied the resistance of hybrid 3 toward a number of
different bases (Table 2, hybrid 1 gave similar results). Under
conventional deprotection conditions (NH4OH), little frag-
mentation was found (entries 1−3). The same was true for
exposure to DBU, a base commonly used to induce β-
eliminations (entries 4 and 5). When the hybrid was exposed to
methylamine plus NH4OH (AMA) at 60 °C, strand cleavage
did occur, with a phosphorylated phenolic or aliphatic alcohol
as the most common terminal group of the chain suffering
cleavage. Exposure to potassium hydroxide in the heat gave the
free phenolic chain, however, as expected for hydrolysis of a
mixed alkyl/aryl ester, where the phenolate is the best leaving
group.
Since the latter route avoids acidic steps, this, again,
confirmed that the detritylation step is not the cause of the
presumed strand cleavage reaction producing the phosphory-
lated five-arm product. In order to set the phenolic
phosphodiester free early, 30 with its cyanoethyl protecting
groups at the proximal linkages was first treated with tertiary
amine, followed by demethylation of the distal methyl triesters,
detritylation and final removal of the base protecting groups
with AMA. Removal of methyl and cyanoethyl protecting
groups was induced by a one-pot procedure, starting with a
solution of DIEA, to which thionaphthol was added after 1 h.
The four-step protocol gave less phosphorylated five-arm
product, but some 14% five-arm hybrid with free phenolic
hydroxy group, possibly due to incomplete coupling early on
(Figure 4b).
Table 1 gives an overview of the results of the syntheses with
the different hybrids. The comparison of ratios between full
chain products and fragments determined by HPLC validate
the results obtained by MALDI-TOF mass spectrometry. Only
for (GA)4TPM (4) did MALDI analysis show considerably less
favorable product composition, most probably due to
depurination during ionization and/or desorption in the
MALDI, overlaying the fragmentation occurring during
deprotection in solution. It is interesting to note that UV-
determined yields are lower than those determined gravimetri-
cally. This may be due, at least in part, to hypochromicity,
caused by assembly (UV measurement), encapsulated water
(gravimetry), and possibly incomplete dissolution of hybrids in
stock solutions for absorption measurements.
Next, phenyl 2′-deoxyguanosine-3′-monophosphate was
prepared as a model compound that contains the phenolic
ester moiety but lacks the dendrimeric structure of hybrids
(Scheme S2, Supporting Information). Using phosphoramidite
6 and phenol, the methyl phosphite triester intermediate was
synthesized, mimicking fully protected hybrids with a 3′-
terminal guanosine residue, such as 28. When this intermediate
was demethylated by treatment with thionaphthol, detritylated
with TCA, and finally base-deprotected with AMA, the crude
did not show signs of chain cleavage or other kinds of
fragmentation (Figure S50, Supporting Information). Together,
these results suggest that the lability toward basic deprotection
under forced basic conditions is caused by the dendritic
structure, not by the fact that the linkage to the core is a
phenolic ester. The results also suggest that more labile
protecting groups for the exocyclic amines of the nucleobases
could reduce what remains a low-level side reaction for purine-
containing hybrids.
Scheme 4. Base-Induced Fragmentation of Side Chains of
DNA Hybrids
Finally, we performed a hybrid synthesis starting from
(GiBu)4TTPA (33) as core, where TPPA stands for tetrakis-
(triazolylphenyl)adamantane. Adamantane-based DNA hybrids
with a triazole linkage to the 3′-terminal nucleoside were
recently shown to assemble into materials from micromolar
aqueous solutions at temperatures as high as 95 °C.18 Starting
from 33, a hybrid with four trimer arms was assembled by
coupling it to dimer phosphoramidite 22, followed by oxidation
and three-stage deprotection. Figure 4c shows the MALDI
spectrum of the crude, confirming that the methodology
established for TPM and HPX cores is also suitable for chain
extension on cores terminating in (less reactive) aliphatic
We suspected that the protected hybrids show an increased
propensity to undergo an elimination reaction that leads to
Table 2. Extent of Fragmentation of DNA Hybrid (TC)4TPM (3) under Different Basic Conditions
a
a
a
entry
base
time (d)
T (°C)
PO3H-C-(TC)3TPM (%)
PO3H-(TC)3TPM (%)
H-(TC)3TPM (%)
1
2
3
4
5
6
7
8
9
−
−
3
20
60
60
20
60
20
60
20
60
−
−
4
1
2
5
b
b
NH4OH
2
NH4OH
10
1
8
10
3
c
DBU
−
1
1
c
DBU
4
6
8
d
AMA
3
−
28
−
1
1
4
d
AMA
4
37
3
e
KOH
1
2
7
e
KOH
1
approx 10−20
65
a
As determined by MALDI-TOF MS, based on and relative peak heights, not corrected for differences in desorption and ionization efficiency.
NH4OH 25% aqueous ammonia. DBU/H2O (1:4, v/v). AMA = NH4OH/NH2Me (1:1, v/v). KOH, 2 M aqueous solution.
b
c
d
e
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Scheme 5. Synthesis of Adamantane-Based Hybrid 34 with Four Trimer Arms
alcohols. As is common for branched oligonucleotides with a
strong propensity to assemble, conventional HPLC conditions
gave modest yields (24%). We are currently establishing
chromatography under denaturing conditions to overcome this
limitation. Still, one-stage HPLC purification gave spectroscopi-
cally pure 34 (Figure S37 and S49, Supporting Information).
With 34 in hand, we studied the assembly properties of this
hybrid, whose dangling terminal nucleotide was designed to
temper the very strong propensity of (CG)4TTPA to form
materials.18 Figure 6 shows the binding curves, acquired under
UV-melting curve conditions. Under low-salt conditions, a
melting point of 18 °C was found. Thus, the reduction in
melting point compared to (CG)4TTPA, which shows a Tm of
61 °C at low salt18 is very significant. Addition of NaCl (150
mM) increased this value to 35 °C. The strong dependence of
the Tm on the salt concentration is expected for tightly packed
three-dimensional lattices.17 Further addition of MgCl2 (10
mM) led to precipitation. At this magnesium concentration, the
precipitate redissolved upon heating. At 100 mM MgCl2 the
material resisted thermal denaturation, even at 85 °C (Figure
6b). The material is porous enough to take up approximately 2
equiv of ethidium bromide per hybrid (Figure S51, Supporting
Information), with strong fluorescence of the intercalator under
UV light.
CONCLUSIONS
■
Here we present a methodology for solution-phase synthesis of
branched oligonucleotide hybrids with up to three nucleosides
per oligonucleotide chain. The methodology allows for the
scalable preparation of compounds that assemble into a new
type of material, based on programmable base-pairing
interactions. The results show that high yields can be obtained
for such hybrids, despite the congested steric situation, the
lability of phenolic esters, low solubility of the cores, and the
need to grow several chains simultaneously in dendrimer-like
fashion. On the level of molecular recognition and material
design, the properties of (TCG)4TTPA (34) with its dangling
5′-terminal nucleotide demonstrate how the sequence and
length of the DNA chain can be used to fine-tune the assembly
process.
The phenolic nature of the linkages to the cores was initially
considered the most problematic feature of the design. Certain
aryl diesters of nucleotides are “active esters” that undergo
rapid reactions with nucleophiles in aqueous solution, at least
when nucleophile and ester are aligned on a template.30
Classical DNA syntheses, such as the “phosphotriester
method”, employ chlorophenyl groups as protecting groups
for the backbone linkages.31 Other oligonucleotide constructs
with phenolic ester linkages did exist prior to our study,
though,19 confirming that such esters can survive the
deprotection conditions of conventional solid-phase DNA
synthesis. Our experiments on the lability of hybrids (Table 2
and Figure S50, Supporting Information) indicate that the low-
level fragmentation observed in crudes of purine-containing
sequences (Figure 4) is not the consequence of the leaving
group qualities of the phenolates. Rather, it appears that the
Figure 6. Assembly of (TCG)4TTPA (34), as monitored by UV
absorbance of 5 μM solutions in phosphate buffer (10 mM, pH 7) at
λdet = 260 nm and a cooling rate of 0.5 °C/min: (a) filled circles, buffer
only; filled triangles, buffer plus 150 mM NaCl; (b) buffer plus 150
mM NaCl, plus 100 mM MgCl2. Note the different expansion of the y-
axis. The massive drop in absorbance is caused by precipitation. See
Figure S51 in the Supporting Information for photographs of
precipitates.
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pretreated with CH2Cl2/NEt3 0.25%) and a step gradient of 0−5%
CH3OH in CH2Cl2. Product-containing fractions were combined and
dried in vacuo (40 °C, 0.001 mbar) to yield the 3′-PAC-protected
dimer nucleotide as colorless or slightly colored solid.
more crowded steric situation in the (partially protected)
hybrids amplifies an intrinsic lability of the DNA chain. Due to
their greater steric demand, purines appear to favor this
fragmentation more than pyrimidines. A proper choice of
protecting groups and of the order or steps minimizes the
fragmentation (Scheme 3). More extended cores and more
labile protecting groups on the nucleobases may all but
eliminate it under optimized conditions.
The method presented here employs no more than 4 equiv
of dimer phosphoramidites, overcomes the low solubility of
cores, and simplifies purification due to the block coupling
strategy. These advances also facilitated implementation of an
alternative approach, based on H-phosphonates, that is
presented in the accompanying paper.32 Irrespective of what
type of building block is being used, syntheses that avoid solid
supports should facilitate scale-up beyond the 10 mg batches
prepared in this work, opening avenues to larger quantities of
new chiral materials, assembled via programmable DNA:DNA
interactions.
5′-DMT-CBz-PO(OMe)-GiBu-3′-PAC (12). The reaction was
performed following general protocol A, starting from N2-isobutyryl-
3′-O-phenoxyacetyl-2′-deoxyguanosine (9, 640 mg, 1.36 mmol) and
[N4-benzoyl-5′-O-(4,4′-dimethoxytrityl)-2′-deoxycytidine-3′-yl]-(N,N′-
diisopropyl)methylphosphoramidite (5, 1.19 g, 1.5 mmol) in dioxane
(7 mL). Yield 1.29 g of 12 (1.09 mmol, 80%) as a pale yellow foam:
TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.38; 31P NMR (203 MHz,
1
DMSO-d6) δ −0.94, −1.10 (ratio = 56:44); H NMR (500 MHz,
DMSO-d6) δ 12.08 (s, 1H), 11.61 and 11.60 (2 brs, 1H), 11.27 (m,
1H), 8.23, 8.22 (2 s, 1H), 8.14 (d, J = 7.5 Hz, 1H), 8.00 (d, J = 7.6 Hz,
2H), 7.63 (m, 1H), 7.52 (t, J = 7.6 Hz, 2H), 7.40−7.19 (m, 12H),
7.00−6.85 (m, 7H), 6.25 and 6.13 (2 m, 2H), 5.45 and 4.95 (2 m,
2H), 4.86, 4.85 (2 s, 2H), 4.33−4.19 (m, 4H), 3.74, 3.72 (2 s, 6H),
3.61, 3.58 (2 d, J = 11.4 Hz, 3H), 3.31−3.26 (m, 2H), 2.96−2.80 (m,
1H), 2.76 (sept, J = 7.0 Hz, 1H), 2.70−2.15 (m, 3H), 1.11 (m, 6H);
13C NMR (126 MHz, DMSO-d6) δ 180.1, 168.4, 167.3 163.2, 158.1,
157.5, 154.7, 148.5, 148.2, 144.5, 144.3, 137.3, 135.3,135.2, 133.0,
132.8, 130.0, 129.7, 129.4, 128.4, 127.9, 127.7, 126.8, 121.3, 120.4,
120.1, 114.5, 113.2; 96.1, 86.1, 85.8, 84.1, 83.2, 82.2, 77.6, 75.2, 66.9,
64.6, 62.4, 54.9, 54.5, 54.4, 39.0, 36.0, 35.4, 18.8; HRMS (ESI-TOF)
m/z calcd for C60H61N8O16P [M + Na]+ 1203.384, m/z obsd
1203.383.
EXPERIMENTAL SECTION
■
N4-Benzoyl-5′-O-tert-butyldimethylsilyl-3′-O-[(N,N-
diisopropylamino)methoxyphosphino]-2′-deoxycytidine (8).
N4-Benzoyl-5′-O-tert-butyldimethylsilyl-2′-deoxycytidine33 (940 mg,
2.11 mmol), previously dried at 0.001 mbar and 50 °C for 4 h, was
suspended in acetonitrile (5 mL) at room temperature. After addition
of molecular sieves (3 Å, 10 beads), DIEA (0.75 mL, 4.28 mmol), and
chloro-N,N-diisopropylmethylphosphoramidite (0.45 mL, 2.35 mmol),
the clear yellowish solution was shaken at room temperature for 4 h.
The solution was then poured into a mixture of ethyl acetate (30 mL)
and NaHCO3 (30 mL, saturated aqueous solution) at 0 °C with
vigorous stirring. The aqueous layer was separated and extracted twice
with ethyl acetate (2 × 30 mL). The combined organic layers were
washed with brine (20 mL), dried over MgSO4, and concentrated in
vacuo. The crude was purified by flash chromatography (15 g silica,
pretreated with hexane/ethyl acetate/triethylamine 74:25:1 v/v/v;
eluted with a step gradient of 25−50% ethyl acetate). Product-
containing fractions were pooled and concentrated in vacuo, followed
by recrystallization from pentane to yield 800 mg (1.32 mmol, 63%) of
the title compound (8, mixture of diastereomers) as colorless needles:
mp = 120−121 °C; TLC (hexane/ethyl acetate/triethylamine 49:50:1
v/v/v) Rf = 0.31/0.37; 31P NMR (122 MHz, CD3CN) δ 148.98,
5′-DMT-GiBu-PO(OMe)-ABz-3′-PAC (13). The reaction was
performed following general protocol A, starting from N6-benzoyl-3′-
O-phenoxyacetyl-2′-deoxyadenosine (11, 735 mg, 1.5 mmol) and [5′-
O-(4,4′-dimethoxytrityl)-N2-isobutyryl-2′-deoxyguanosine-3′-yl]-
(N,N′-diisopropyl)methylphosphoramidite (6, 1.32 g, 1.65 mmol) in
dioxane (7 mL). Yield 1.30 g of 13 (1.08 mmol, 72%) as a colorless
foam: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.43; 31P NMR (122
MHz, DMSO-d6) δ −0.96, −1.21 (ratio =44:56); 1H NMR (500 MHz,
DMSO-d6) δ 12.05 (s, 1H), 11.61 and 11.60 (2 s, 1H), 11.22, 11.20 (2
s, 1H), 8.75, 8.73, 8.67 and 8.66, (4 s, 2H), 8.10 and 8.09 (2s, 1H),
8.01 (t, J = 7.1 Hz, 2H), 7.65 (t, J = 7.4 Hz, 1H), 7.54 (m, 2H), 7.32−
7.27 (m, 4H), 7.23−7.15 (m, 7H), 6.98−6.95 (m, 3H), 6.81−6.77 (m,
4H), 6.52 and 6.21 (2 m, 2H), 5.58 and 5.00 (2 m, 2H), 4.88 and 4.87
(2 s, 2H), 4.36−4.26 (m, 3H), 4.14 (m, 1H), 3.70 and 3.69 (2 s, 6H),
3.59and 3.58 (2d, J = 11.2 Hz, 3H), 3.25−3.12 (m, 3H), 2.92 (m, 1H),
2.73 (sept, J = 7.0 Hz, 1H), 2.69−2.58 (m, 2H), 1.11 (d, J = 7.0 Hz,
6H); 13C-NMR (126 MHz, DMSO-d6) δ 180.0, 168.5, 165.6, 158.0,
157.5, 154.8, 151.9, 151.7, 150.5, 148.6, 148.0, 144.6, 143.0, 137.3,
135.3, 133.2, 132.5, 129.7, 129.6, 129.5, 128.5, 128.4, 127.7, 127.6,
126.7, 125.8, 121.3, 120.5, 120.4, 114.5, 113.1; 85.7, 84.1, 83.8, 82.7,
82.3, 78.1, 74.6, 66.7, 64.6, 63.3, 54.9, 54.4, 54.3, 36.6, 35.4, 34.8, 18.8;
HRMS (ESI-TOF) m/z calcd for C65H65N10O15P [M + Na]+
1227.395, m/z obsd 1227.395.
1
148.54 (ratio =53:47); H NMR (300 MHz, CD3CN) δ 9.13 (brs,
1H), 8.34 and 8.31 (2d, J = 7.5 Hz, 1H), 7.96 (m, 2H), 7.63 (m, 1H),
7.52 (m, 2H), 7.38 (br, 1H), 6.15 (t, J = 6.1 Hz, 1H), 4.56−4.46 (m,
1H), 4.16−4.10 (m, 1H), 3.98−3.80 (m, 2H), 3.67−3.54 (m, 2H),
3.36 (2 d, J = 13.2 Hz, 3H), 2.64−2.51 (m,1H), 2.25−2.13 (m, 1H),
1.17 (d, J = 6.8 Hz, 12H), 0.93 and 0.92 (2 s, 9H), 0.13 (2 s, 3H), 0.12
(2 s, 3H).
5′-DMT-T-PO(OMe)-CBz-3′-PAC (14). The reaction was per-
formed following the general protocol A, starting from N4-benzoyl-
3′-O-phenoxyacetyl-2′-deoxycytidine (10, 700 mg, 1.5 mmol) and [5′-
O-(4,4′-dimethoxytrityl)thymidine-3′-yl](N,N′-diisopropyl)-
methylphosphoramidite (7, 1.17 g, 1.65 mmol) in dioxane (8 mL).
Yield 1.15 g of 14 (1.06 mmol, 71%) as a colorless foam: TLC
(CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.64; 31P NMR (122 MHz, DMSO-
d6) δ −0.84, −0.98 (ratio = 57:43); 1H NMR (500 MHz, DMSO-d6) δ
11.38 and 11.29 (2 brs, 2H), 8.17 (2d, J = 7.8 Hz, 1H), 8.00 (d, J = 7.8
Hz, 2H), 7.63 (m, 1H), 7.53−7.49 (m, 3H), 7.39−7.19 (m, 12H),
6.98−6.94 (m, 3H), 6.89−6.86 (m, 4H), 6.22 and 6.16 (2 m, 2H),
5.34 and 5.04 (2 m, 2H), 4.86 (m, 2H), 4.34−4.25 (m, 3H), 4.17 (m,
1H), 3.72 and 3.71 (2 s, 6H), 3.68 and 3.63 (2d, J = 11.2 Hz, 3H),
3.29−3.23 (m, 2H), 2.60−2.34 (m, 4H), 1.45 (2 s, 3H); 13C NMR
(126 MHz, DMSO-d6) δ 168.3, 167.2, 163.5, 163.2, 158.1, 157.4,
154.2, 150.2, 144.9, 144.4, 135.5, 135.1, 135.0, 132.9, 132.7, 129.6,
129.4, 128. 4, 128.3, 127.8, 127.5, 126.7, 121.2, 114.4, 113.1, 109.8,
96.3, 86.8, 86.0, 83.7, 83.2, 82.4, 77.6, 64.3, 66.8, 64.4, 63.0, 54.9, 54.5,
54.4, 37.3, 37.0, 11.6; HRMS (ESI-TOF) m/z calcd for C56H56N5O16P
[M + Na]+ 1108.335, m/z obsd 1108.335.
General Protocol A (Synthesis of 3′-PAC-protected Dimers).
The protocol is a modification of that given in ref 23. To a solution of
the 3′-phenoxyacetyl-2′-deoxynucleoside (0.2 M, previously coevapo-
rated from toluene, 2 × 10 mL and dried at 0.001 mbar, 50 °C) in
dioxane or CH3CN were added molecular sieves (3 Å, 10 beads), the
(N,N-diisopropylamino)methoxyphosphoramidite of a 5′-DMT-pro-
tected 2′-deoxynucleoside (1.1 equiv, previously dried at 0.001 mbar,
40 °C), and 1-H-tetrazole (1.1 equiv of a 0.45 M solution in CH3CN).
The reaction mixture was shaken at this temperature for 20 h with
diisopropylammonium tetrazolide (DIPAT) beginning to precipitate
after approximately 30 min. The mixture was cooled to 0 °C, and tert-
butyl hydroperoxide (2.7 equiv of a 5.5 M solution in decane) was
added. After the mixture was stired at 0 °C for 15 min, ethyl acetate
(50 mL) was added, and the solution was washed with phosphate
buffer (25 mL, 0.2 M, pH 7). The aqueous layer was extracted with
ethyl acetate (2 × 20 mL). The combined organic layers were washed
with brine (10 mL), dried over Na2SO4, and concentrated in vacuo.
The resulting crude was purified by chromatography using silica (50 g,
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5′-TBDMS-CBz-PO(OMe)-GiBu-3′-PAC (15). The reaction was
performed following general protocol A, starting from N2-isobutyryl-
3′-O-phenoxyacetyl-2′-deoxyguanosine (9, 500 mg, 1.06 mmol) and
[N4-benzoyl-5′-O-(tert-butyldimethylsilyl)-2′-deoxycytidine-3′-yl]-
(N,N′-diisopropyl)methylphosphoramidite (8, 705 mg, 1.16 mmol;
previously prepared via phosphitylation from the 3′-unprotected
precursor as detailed in the Supporting Information) in CH3CN (5
mL). Yield 800 mg of 15 (0.81 mmol, 76%) as a colorless solid: TLC
(CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.55; 31P NMR (122 MHz, DMSO-
d6) δ −1.10, −1.13 (ratio = 54:46); 1H NMR (500 MHz, DMSO-d6) δ
12.09 (s, 1H), 11.64 (s, 1H), 11.27 and 11.26 (2 s, 1H), 8.26 and 8.25
(2 s, 1H), 8.21 and 8.19 (2 d, J = 7.5 Hz, 1H), 8.01 (d, J = 7.7 Hz,
2H), 7.63 (t, J = 7.5 Hz, 1H), 7.51 (t, J = 7.6 Hz, 2H), 7.37 (d, J = 7.4
Hz, 1H), 7.30 (m, 2H), 6.97 (m, 3H), 6.27 (2dd, J = 5.9 Hz, J = 8.6
Hz, 1H), 6.13 (t, J = 6.4 Hz, 1H), 5.48 and 4.86 (2 m, 2H), 4.86 (s,
2H), 4.35−4.19 (m, 4H), 3.80−3.70 (m, 2H), 3.67 (d, J = 11.3 Hz,
3H), 3.00 (m, 1H), 2.76 (sept, J = 6.9 Hz, 1H), 2.69, 2.60, 2.24 (3 m,
3H), 1.12, 1.11 (2 d, J = 6.9 Hz, 6H), 0.83, 0.82 (2 s, 9H), 0.05, 0.04
(2 s, 3H), 0.03, 0.02 (2 s, 3H); 13C NMR (126 MHz, DMSO-d6) δ
180.1, 168.4, 167.3, 163.2, 157.5, 154.8, 154.2, 148.5, 148.2; 144.3,
137.5, 133.0, 132.8, 129.5, 128.4, 121.3, 120.5, 120.4, 114.6; 96.0, 86.4,
85.6, 83.2, 82.2, 77.7, 74.6, 67.0, 64.6, 62.3, 54.5, 54.4, 39.4, 35.5, 34.8,
25.6, 18.8, 17.8, −5.7, −5.8; HRMS (ESI-TOF) m/z calcd for
C45H57N8O14PSi [M + H]+ 993.357, m/z obsd 993.358.
(sept, J = 6.9 Hz, 1H), 2.57 and 2.43 (2 m, 2H), 1.11 (d, J = 6.9 Hz,
6H); 13C NMR (126 MHz, DMSO-d6) δ 180.0, 158.0, 154.8, 150.4,
148.6, 144.6, 143.1, 135.2, 129.7, 129.6, 128.5, 127.7, 127.6, 113.1,
85.7, 84.8, 84.0, 83.7, 82.7, 77.9, 70.1, 67.3, 63.4, 54.9, 54.4, 38.4, 36.7,
34.8, 18.8; HRMS (ESI-TOF) m/z calcd for C53H55N10O13P [M +
Na]+ 1093.358, m/z obsd 1093.358.
5′-DMT-T-PO(OMe)-CBz (18). The reaction was performed
following the General Protocol B, starting from 14 (1.1 g, 1.01
mmol) and ammonia (0.3 mL, 7 M solution in CH3OH) in CH3OH
(8 mL) and CH2Cl2 (2 mL). The reaction was complete after 30 min.
Yield 920 mg of 18 (0.96 mmol, 95%) as a colorless solid: TLC
(CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.46; 31P NMR (122 MHz, DMSO-
d6) δ −0.73, −0.84 (ratio = 57:43); 1H NMR (500 MHz, DMSO-d6) δ
11.36 and 11.24 (2 brs, 2H), 8.14 (2d, J = 7.7 Hz, 1H), 8.00 (d, J = 7.7
Hz, 2H), 7.62 (m, 1H), 7.52−7.47 (m, 3H), 7.38−7.20 (m, 10H),
6.89−6.86 (m, 4H), 6.22 and 6.15 (2 m, 2H), 5.50 (d, J = 4.4 Hz, 1H),
5.05 (m, 1H), 4.28−4.16 (m, 4H), 4.03 (m, 1H), 3.72 and 3.71 (2 s,
6H), 3.69 and 3.65 (2d, J = 11.2 Hz, 3H), 3.31−3.23 (m, 2H), 2.54−
2.47 (m, 2H), 2.36−2.31 (m, 1H), 2.15−2.08 (m, 1H), 1.46 and 1.45
(2 s, 3H); 13C NMR (126 MHz, DMSO-d6) δ 167.4, 163.6, 163.5,
158.2, 154.2, 150.3, 144.6, 144.5, 135.6, 135.2, 135.1, 133.1, 132.7,
129.7, 128. 4, 127.9, 127.6, 126.8, 113.2, 109.8, 96.2, 86.4, 86.1, 85.0,
83.7, 83.3, 77.6, 69.7, 67.2, 63.1, 55.0, 54.5, 54.4, 40.0, 37.4, 11.7;
HRMS (ESI-TOF) m/z calcd for C48H50N5O14P [M + Na]+ 974.298,
m/z obsd 974.298.
General Protocol B (Cleavage of the 3′-PAC Protecting
Group of Dimers). The protocol is a modification of that given in ref
23. To a solution of the 3′-phenoxyacetyl protected dimer (0.1 M) in
CH3OH (and 20% CH2Cl2 in case of poorly soluble educt) was added
ammonia (7 M solution in CH3OH, 2 equiv), and the mixture was
stirred at 20 °C. After TLC analysis (CH2Cl2/CH3OH = 9:1 v/v)
showed complete conversion (30−60 min), excess ammonia was
removed with a gentle stream of nitrogen, and the solution was
concentrated in vacuo at 20 °C. The resulting crude was purified by
chromatography, using silica (20 g, pretreated with CH2Cl2/NEt3
0.25%) and a step gradient of 0−10% CH3OH in CH2Cl2. Product-
containing fractions were combined, concentrated, and dried in vacuo
(40 °C, < 0.001 mbar) to yield the 3′-OH dimer nucleotide as a
colorless solid.
5′-TBDMS-CBz-PO(OMe)-GiBu (19). The reaction was performed
following the General Protocol B, starting from 15 (700 mg, 0.7
mmol) and ammonia in CH3OH (0.22 mL, 7 M solution) in CH3OH
(7 mL) with a reaction time of 30 min. Yield 490 mg of 19 (0.57
mmol, 81%) as a colorless foam: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf
= 0.33; 31P NMR (122 MHz, DMSO-d6) δ −0.94, −1.03 (ratio
1
=55:45); H NMR (500 MHz, DMSO-d6) δ 12.07 (brs, 1H), 11.63
(brs, 1H), 11.26 (brs, 1H), 8.21 (m, 2H), 8.00 (d, J = 7.6 Hz, 2H),
7.63 (m, 1H), 7.51 (t, J = 7.7 Hz, 2H), 7.37 (d, J = 7.5 Hz, 1H), 6.26
(t, J = 6.7 Hz, 1H), 6.13 (q, J = 6.4 Hz, 1H), 5.53 (br, 1H), 4.86 and
4.43 (2 m, 2H), 4.28−4.13 (m, 3H), 4.03 (m, 1H), 3.80−3.70 (m,
2H), 3.69 and 3.68 (2 d, J = 11.4 Hz, 3H), 2.76 (sept, J = 7.0 Hz, 1H),
2.72−2.63 (2 m, 2H), 2.36−2.22 (2 m, 2H), 1.11 (m, 6H), 0.83 and
0.82 (2 s, 9H), 0.05 and 0.04 (2 s, 3H), 0.03 (s, 3H); 13C NMR (126
MHz, DMSO-d6) δ 180.1, 167.3, 163.2, 154.8, 154.2, 148.3, 148.0,
144.3, 137.5, 133.0, 132.8, 128.4, 120.3, 96.0, 86.4, 85.6, 84.9, 83.1,
77.5, 70.1, 67.4, 62.3, 54.4, 54.3, 39.3, 38.8, 34.8, 25.6, 18.8, 17.8, −5.7,
−5.8; HRMS (ESI-TOF) m/z calcd for C37H51N8O12PSi [M + H]+
859.321, m/z obsd 859.321.
5′-DMT-CBz-PO(OMe)-GiBu (16). The reaction was performed
following the general protocol B, starting from 12 (1.2 g, 1.02 mmol)
and ammonia (0.3 mL, 7 M solution in CH3OH) in CH3OH (8 mL)
and CH2Cl2 (2 mL) for 45 min. Yield 900 mg of 16 (0.86 mmol, 84%)
as a colorless solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.27; 31P
1
NMR (203 MHz, DMSO-d6) δ −0.82, −0.98 (ratio = 55:45); H
NMR (500 MHz, DMSO-d6) δ 12.06 (brs, 1H), 11.62 (brs, 1H),
11.30 (brs, 1H), 8.18 and 8.17 (2 s, 1H), 8.16 and 8.15 (2 d, J = 7.6
Hz, 1H), 8.00 (d, J = 7.7 Hz, 2H), 7.63 (m, 1H), 7.52 (t, J = 7.7 Hz,
2H), 7.39−7.20 (m, 10H), 6.90−6.87 (m, 4H), 6.24 and 6.14 (2 m,
2H), 5.54 (d, J = 4.1 Hz, 1H), 4.95 and 4.41 (2 m, 2H), 4.27−4.09 (m,
3H), 4.01 (m, 1H), 3.73 and 3.72 (2 s, 6H), 3.63 and 3.58 (2 d, J =
11.5 Hz, 3H), 3.31−3.28 (m, 2H), 2.75 (sept, J = 7.0 Hz, 1H), 2.70−
2.61 (2 m, 2H), 2.43−2.30 (2 m, 2H), 1.11 (m, 6H); 13C NMR (126
MHz, DMSO-d6) δ 180.0, 163.1, 158.1, 154.7, 148.3, 148.0, 144.3,
137.4, 135.1, 135.0, 133.1, 132.7, 129.7, 128.4, 127.9, 127.7, 126.8,
120.3, 113.2, 86.3, 86.1, 84.8, 84.0, 83.1, 76.7, 70.1, 67.3, 62.4, 55.0,
54.4, 54.3, 45.6, 38.8, 34.8, 18.8; HRMS (ESI-TOF) m/z calcd for
C52H55N8O14P [M + Na]+ 1069.347, m/z obsd 1069.347.
General Protocol C (Synthesis of 3′-Phosphoramidites of
Dimers). The protocol is a modification of that given in ref 21. The 5′-
O-protected dinucleotide previously coevaporated from CH3CN (10
mL) and dried (<0.001 mbar, 50 °C) was suspended in CH3CN (0.2
M) at room temperature. After addition of molecular sieves (3 Å, 10
beads), diisopropylethylamine (DIEA, 2 equiv), and chloro-N,N-
diisopropylmethylphosphoramidite or chloro-cyanoethyl-N,N-diiso-
propylphosphoramidite (1.1 equiv), the slurry turned into a clear,
yellowish solution within a few minutes and was shaken at 20 °C for 3
h. The reaction mixture was then poured into a vigorously stirred
mixture of ethyl acetate (20 mL) and NaHCO3 (20 mL, saturated
aqueous solution) at 0 °C. The aqueous layer was separated and
extracted twice with ethyl acetate (2 × 10 mL). The combined organic
layers were washed with brine (10 mL), dried over Na2SO4, and
concentrated in vacuo. The resulting crude was purified by flash
chromatography, using silica (3 g, pretreated with ethyl acetate with
2% triethylamine) and a step gradient of CH3CN (0−30%) in ethyl
acetate with 1% triethylamine. Product-containing fractions were
combined and concentrated in vacuo, followed by precipitation
(twice) from ethyl acetate (0.5 mL) treated with hexane (4 mL). The
precipitate was separated by centrifugation (3500 rpm, 5 min) and
dried in vacuo (<0.001 mbar, 40 °C) to yield the 5′ protected
dinucleotide phosphoramidite as a colorless solid.
5′-DMT-GiBu-PO(OMe)-ABz (17). The reaction was performed
following the general protocol B, starting from 13 (1.15 g, 1.02 mmol)
and ammonia (0.27 mL, 7 M solution in CH3OH) in CH3OH (8 mL)
and CH2Cl2 (2 mL) for 60 min. Yield 820 mg of 17 (0.77 mmol, 81%)
as a colorless solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.25, 0.27;
1
31P NMR (122 MHz, DMSO-d6) δ −0.87, −1.09 (ratio = 55:45); H
NMR (500 MHz, DMSO-d6) δ 12.05 (s, 1H), 11.62, 11.61 (2 s, 1H),
11.19 and 11.17 (2 s, 1H), 8.72, 8.70, 8.63, and 8.62 (4s, 2H), 8.11 and
8.10 (2s, 1H), 8.02 (t, J = 7.3 Hz, 2H), 7.65 (m, 1H), 7.54 (m, 2H),
7.30 (d, J = 7.6 Hz, 2H), 7.24−7.16 (m, 7H), 6.82−6.77 (m, 4H), 6.50
and 6.22 (2 m, 2H), 5.59 (2 d, J = 4.8 Hz, 1H), 5.00 and 4.52 (2 m,
2H), 4.29−4.13 (m, 3H), 4.05 (m, 1H), 3.70 (s, 6H), 3.60, 3.58 (2d, J
= 11.2 Hz, 3H), 3.23 and 3.15 (2 m, 2H), 2.98−2.87 (m, 2H), 2.74
5′-DMT-CBz-PO(OMe)-GiBu-3′-PN(iPr)2(OMe) (20). The reaction
was performed following general protocol C, starting from 16 (750
mg, 0.72 mmol), DIEA (185 mg, 1.44 mmol), and chloro-N,N-
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diisopropylmethylphosphoramidite (155 μL, 0.8 mmol) in CH3CN
(3.5 mL). Yield 650 mg of 20 (0.54 mmol, 75%) as a colorless solid:
TLC (ethyl acetate/CH3CN/NEt3 = 7:3:0.1 v/v) Rf = 0.34; 31P NMR
(122 MHz, CD3CN) δ 149.12, 149.02, 148,85 (P(III)), −1.05, −1.10,
(m, 1H), 7.52 (m, 2H), 7.42−7.16 (m, 10H), 6.89−6.84 (m, 4H),
6.26−6.02 (m, 2H), 5.11−4.68, (m, 1H), 4.52−4.21 (m, 4H), 3.91−
3.75 (m, 2H), 3.76, 3.75, and 3.73 (3 s, 6H), 3.73−3.58, (m, 5H),
3.35−2.20 (m, 9H), 1.23−1.06 (m, 18H); HRMS (ESI-TOF) m/z
calcd for C61H72N10O15P2 [M + Na]+ 1269.455, m/z obsd 1269.455.
General Protocol D (Synthesis of DNA Hybrids from Cores
and Dimer Phosphoramidites). The core (200 mg of tetrakis(4-
hydroxyphenyl)methane17 or of 1,4-phenylenebis[tris(4′-
hydroxyphenyl)methane])18 was dissolved in dioxane (10 mL) by
heating, and the solution then immediately frozen by cooling with
liquid nitrogen, followed by drying in vacuo (0.001 mbar) in a process
resembling lyophilization. The resulting voluminous material was
further dried in vacuo (<0.001 mbar) at 130 °C for 2 h. The core (5−
10 μmol) and the 3′-phosphoramidite of the 5′-protected dinucleoside
phosphate (20, 21, 22, 23, or 24, 1.5−3 equiv per OH group of the
core) were transferred to a 5 mL vial, and the mixture was dried in
vacuo at 40 °C for 1 h. The flask was flushed with argon and sealed
with a septum, after addition of molecular sieves (3 Å, 5 beads).
Dioxane (freshly distilled over sodium/benzophenone) and 1H-
tetrazole (0.45 M solution in CH3CN, 2−3 equiv for each OH
group of the core) was added at room temperature, and the slurry was
put in an ultrasonic bath for 2 min at 20 °C until the core was fully
dissolved. The reaction mixture was kept at 20 °C for 30 min and then
at 5 °C for 3 h. (The reaction was usually monitored by drawing
samples, oxidizing with tert-butylhydroperoxide in CH2Cl2 and
precipitation with hexane. After the DMT groups were removed
with trichloroacetic acid (3% TCA in CH2Cl2) and CH3OH, the
analytical samples were analyzed by MALDI-TOF MS.) After 3 h, tert-
butyl hydroperoxide (5.5 M solution in decane, 5 equiv for each OH
group of the core) was added, and the mixture was again kept at 5 °C
for 15 min. The solution was then diluted with CH2Cl2 (20 mL) and
washed with phosphate buffer (15 mL, 0.2 M, pH 7). The aqueous
phase was back-extracted with CH2Cl2 (2 × 10 mL), and the
combined organic layers were dried over NaSO4, filtered, and
concentrated in vacuo. To remove excess dimer, the residue was
dissolved in CH2Cl2 (0.5 mL) and precipitated with hexane (3 mL).
The precipitate was treated three times with ethyl acetate and 0−20%
hexane (3 × 3 mL) in an ultrasonic bath for 1 min and separated by
centrifugation. In isolated cases, it was purified by chromatography
using silica (5 g, pretreated with CH2Cl2/NEt3 0.2%) and a step
gradient of CH3OH in CH2Cl2. The resulting product was dried
(<0.001 mbar, 40 °C) to yield the protected hybrid as a colorless or a
pale yellow solid. The completeness of the reaction was checked again
by MALDI-TOF MS after DMT deprotection in the analytical sample.
In the case of incomplete conversion (<90% of the last phenole of the
core remained unreacted), the hybrid was coupled again, as described
above, using the 3′-phosphoramidite of 5′-protected dinucleotide (0.5
equiv per OH group of the starting amount of the core), 1H-tetrazole
(0.5−1 equiv), and tert-butyl hydroperoxide (3 equiv) for the
oxidation. After workup, as described above, the resulting product
was dried (<0.001 mbar, 40 °C) to yield the protected DNA hybrid
(>98% of all OH groups reacted) as a colorless solid.
1
−1.20, −1.25 (P(V)); H NMR (300 MHz, CD3CN) δ 10.25 (br,
1H), 8.20−7.94 (m, 3H), 7.76 (m, 1H), 7.64 (m, 1H), 7.53 (t, J = 7.7
Hz, 2H), 7.42−7.15 (m, 10H), 6.88−6.83 (m, 4H), 6.23−6.02 (m,
2H), 5.07 and 4.95 (2 m, 1H), 4.66 (m, 1H), 4.53−4.21 (m, 4H), 3.76,
3.75, and 3.73 (3 s, 6H), 3.73, 3.72, 3.68, and 3.67 (4d, J = 11.1 Hz,
3H), 3.65−3.56 (m, 2H), 3.38 and 3.35 (2 d, J = 13.3, 3H), 3.30−2.20
(m, 7H), 1.22−1.06 (m, 18H); HRMS (ESI-TOF) m/z calcd for
C59H71N9O15P2 [M + H]+ 1208.462, m/z obsd 1208.460.
5′-DMT-GiBu-PO(OMe)-ABz-3′-PN(iPr)2(OMe) (21). The reaction
was performed following general protocol C, starting from 17 (535
mg, 0.5 mmol), DIEA (130 mg, 1 mmol), and chloro-N,N-
diisopropylmethylphosphoramidite (106 μL, 0.55 mmol) in CH3CN
(2.5 mL). Yield 415 mg of 21 (0.34 mmol, 68%) as a colorless foam:
TLC (ethyl acetate/CH3CN/NEt3 = 7:3:0.1 v/v) Rf = 0.29; 31P NMR
(122 MHz, CD3CN) δ 149.36, 149.34, 149.06, 149,01 (P(III)), −0.80,
1
−0.84, −0.98, −1.01 (P(V)); H NMR (500 MHz, CD3CN) δ 9.95
(br), 8.66 (m, 1H), 8.29 (s, 1H); 7.96−7.91 (m, 2H), 7.76 and 7.75 (2
s, 1H), 7.61 (m, 1H), 7.52−7.46 (m, 2H), 7.31 (m, 2H), 7.21−7.15
(m, 7H), 6.76−6.73 (m, 4H), 6.43 (m, 1H), 6.07 and 6.03 (2 m, 1H),
5.20 and 5.11 (m, 1H), 4.96−4.82 (m, 1H), 4.32−4.21 (m, 3H), 4.09
(m, 1H), 3.72 and 3.71 (2 s, 6H), 3.67, 3.66, 3.58, and 3.57 (4 d, J =
11.3 Hz, 3H), 3.65−3.60 (m, 2H), 3.39 and 3.37 (2 d, J =13.3, 3H),
3.29−3.02, 2.92−2.78, and 2.68−2.39 (m, 7H), 1.22−1.09 (m, 18H);
HRMS (ESI-TOF) m/z calcd for C60H71N11O14P2 [M + Na]+
1254.455, m/z obsd 1254.455.
5′-DMT-T-PO(OMe)-CBz-3′-PN(iPr)2(OMe) (22). The reaction
was performed following general protocol C, starting from 18 (475
mg, 0.5 mmol), DIEA (130 mg, 1 mmol) and chloro-N,N-
diisopropylmethylphosphoramidite (106 μL, 0.55 mmol) in CH3CN
(2.5 mL). Chromatography was performed using ethyl acetate with
triethylamine (1%) as eluant. Yield 440 mg of 22 (0.4 mmol, 80%) as a
colorless solid: TLC (ethyl acetate/NEt3 = 10:0.1 v/v) Rf = 0.30; 31P
NMR (122 MHz, CD3CN) δ 149.52, 149.15, (P(III)), −0.89, −0.92,
−0.93, −0.96 (P(V)); 1H NMR (500 MHz, CD3CN) δ 9.10 (br, 2H),
8.08 and 8.03 (2d, J = 7.6 Hz, 1H), 7.94 (d, J = 7.4 Hz, 2H), 7.63 (m,
1H), 7.51(m, 2H), 7.43−7.21 (m, 11H), 6.87−6.84 (m, 4H), 6.25 and
6.12 (2 m, 2H), 5.10, 4.47 (2 m, 2H), 4.31−4.20 (m, 4H), 3.74 (2 s,
6H), 3.72−3.68 (m, 3H), 3.63−3.56 (m, 2H), 3.37−3.01 (m, 5H),
2.61−2.52 (m, 2H), 2.48−2.41 (m, 1H); 2.23−2.18 (m, 1H), 1.45 and
1.44 (2 s, 3H), 1.17−1.15 (m, 12H); HRMS (ESI-TOF) m/z calcd for
C55H66N6O15P2 [M + Na]+ 1135.395, m/z obsd 1135.396.
5′-TBDMS-CBz-PO(OMe)-GiBu-3′-PN(iPr)2(OMe) (23). The reac-
tion was performed following general protocol C, starting from 19
(430 mg, 0.50 mmol), DIEA (130 mg, 1 mmol), and chloro-N,N-
diisopropylmethylphosphoramidite (106 μL, 0.55 mmol) in CH3CN
(2.5 mL). Yield: 250 mg of 23 (0.25 mmol, 50%) as a colorless solid:
TLC (ethyl acetate/CH3CN/NEt3 = 7:3:0.1 v/v) Rf = 0.40; 31P NMR
(122 MHz, CD3CN) δ 149.21, 149.18, 148.93 (P(III)), −1.32, −1.37,
−1.39 (P(V)); 1H NMR (300 MHz, CD3CN) δ 12.09, 10.53, and 9.70
(3 brs, 2H), 8.21−8.15 (m, 1H), 7.99 (m, 2H), 7.80 and 7.79 (2 d, J =
6.8 Hz, 1H), 7.63 (m, 1H), 7.53 (m, 2H), 7.44 and 7.38 (2 br, 1H),
6.26−6.08 (m, 2H), 4.98, 4.88, and 4.69 (3 m, 2H), 4.53−4.19 (m,
4H), 3.78, 3.77, 3.72, and 3.71 (4d, J = 11.4 Hz, 3H), 3.72−3.55 (m,
2H), 3.40, 3.39, 3.38, and 3.37 (4 d, J = 13.3, 3H), 3.04−2.13 (m, 7H),
1.22−1.16 (m, 18H), 0.86 (s, 9H), 0.08−0.05 (m, 6H); HRMS (ESI-
TOF) m/z calcd for C44H67N9O13P2Si [M + H]+ 1020.418, m/z obsd
1020.417.
[5′-DMT-CBz-PO(OMe)-GiBu-3′-PO(OMe)]4TPM (25). The reac-
tion was performed following general protocol D, starting from TPM
(3.8 mg, 10 μmol) and 20 (145 mg, 120 μmol, 3 equiv per OH group)
in dioxane (0.7 mL), and 1H-tetrazole (0.27 mL of a 0.45 M solution
in CH3CN, 120 μmol) and then tert-butyl hydroperoxide (36 μL of a
5.5 M solution in decane, 200 μmol). For removal of excess dimer,
ethyl acetate/hexane (9:1 v/v) was used. Yield 55 mg of crude 25
(quant). MALDI-TOF MS showed 80−85% conversion of the fourth
phenolic OH group. In a second coupling cycle, the hybrid was reacted
again with 20 (24 mg, 20 μmol, 0.5 equiv/OH) in dioxane (0.7 mL)
and 1H-tetrazole (45 μL, 0.45 M solution in CH3CN, 20 μmol) and
then tert-butyl hydroperoxide (22 μL of a 5.5 M solution in decane,
120 μmol). The crude was washed with ethyl acetate (3 × 3 mL),
followed by dissolving in CH2Cl2 (0.1 mL) and precipitation with
ethyl acetate. Yield 44 mg of 25 (9.0 μmol, 90%), after centrifugation,
as a colorless solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.43; 31P
NMR (203 MHz, CD2Cl2) δ −1.51, −1.67 (POCH2), −6.18, −6.35
5′-DMT-CBz-PO(OMe)-GiBu-3′-PN(iPr)2(OCET) (24). The reaction
was performed following general protocol C, starting from 16 (550
mg, 0.52 mmol), DIEA (140 mg, 1.08 mmol), and chloro-2-
cyanoethyl-N,N-diisopropylphosphoramidite (130 μL, 0.58 mmol) in
CH3CN (3 mL). Yield 340 mg of 24 (0.27 mmol, 52%) as a colorless
solid: TLC (ethyl acetate/CH3CN/NEt3 = 7:3:0.1 v/v) Rf = 0.43; 31
P
NMR (122 MHz, CD3CN) δ 148.56, 148.37, 148.33 148.08 (P(III)),
1
−1.26, −1.27, −1.29 −1.33 (P(V)); H NMR (300 MHz, CD3CN) δ
1
11.90 and 10.42 (2 brs, 2H), 8.12−7.95 (m, 3H), 7.76 (m, 1H), 7.63
(POAr); H NMR (500 MHz, CD2Cl2) δ 12.13 (br, 4H), 10.70 (br,
2712
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4H), 9.52 (br, 4H), 8.12−7.77 (m, 4 × 4H), 7.71−6.90 (m, 4 × 17H),
6.75 (m, 4 × 4H), 6.25−5.91 (m, 4 × 2H), 5.37 and 5.04−4.80 (2 m, 4
× 2H), 4.63−4.00 (m, 4× 4H), 3.85−3.50 (m, 4 × 12H), 3.40−2.10
(m, 4 × 7H), 1.15−0.85 (m, 4 × 6H); MALDI-TOF MS (DMT off)
calcd for C153H172N32O60P8 [M − H]− 3665, obsd 3663 plus 2843
(1%, [(CG)3TPM − H]−).
12H), 3.48−1.90 (m, 6 × 7H), 1.20−0.95 (m, 6 × 6H); MALDI-TOF
MS (DMT off) calcd for C236H262N48O90P12 [M − H]− 5580, obsd
5578 plus 4761 (3%, [(CG)5HPX − H]−).
[5′-TBDMS-CBz-PO(OMe)-GiBu-3′-PO(OMe)]6HPX (29). The re-
action was performed following general protocol D, starting from HPX
(3.3 mg, 5.0 μmol) and 23 (61 mg, 60 μmol, 2.0 equiv per OH group)
in dioxane (0.4 mL) and 1H-tetrazole (0.14 mL of a 0.45 M solution in
CH3CN, 60 μmol) and then tert-butyl hydroperoxide (27 μL of a 5.5
M solution in decane, 150 μmol). The crude was treated with ethyl
acetate (3 × 3 mL) to give 26 mg of 29 (4.2 μmol, 83%) as a colorless
solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.27; 31P NMR (122
[5′-DMT-T-PO(OMe)-CBz-3′-PO(OMe)]4TPM (26). The reaction
was performed following general protocol D, starting from TPM (3.8
mg, 10.0 μmol) and 22 (133 mg, 120 μmol, 3 equiv/OH) in dioxane
(0.7 mL), using 1H-tetrazole (0.27 mL of a 0.45 M solution in
CH3CN, 120 μmol) and then tert-butyl hydroperoxide (36 μL of a 5.5
M solution in decane, 200 μmol). Excess dimer was removed by
chromatography, using silica (5 g, pretreated with CH2Cl2/NEt3 0.2%)
and a step gradient of 0−5% CH3OH in CH2Cl2. Product-containing
fractions were combined and concentrated in vacuo, followed by
dissolving in CH2Cl2 (0.2 mL) and precipitation with hexane (3 mL).
Centrifugation gave 38 mg of 26 (8.5 μmol, 85%) as a colorless solid:
TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.49; 31P NMR (122 MHz,
CD2Cl2) δ −0.61, −0.71, −0.96, −1.00 (POCH2), −6.15, −6.23,
1
MHz, CD2Cl2) δ −1.73 (POCH2), −6.23, −6.41 (POAr); H NMR
(300 MHz, CD2Cl2) δ 12.45−12.16 (m, 6H), 11.37−10.82 (m, 6H),
10.30−9.48 (m, 6H), 8.22−8.13 (m, 6H), 8.09−7.93 (m, 6 × 3H),
7.84−7.42 (m, 6 × 5H), 7.28−7.00 (m, 28H), 6.32−6.13 (m, 6 × 2H),
5.84−5.43 and 5.04−4.83 (m, 6 × 2H), 4.73−4.13 (m, 6 × 4H), 3.87
and 3.77 (2 m, 6 × 6H), 3.72−3.58 (m, 6 × 2H), 3.29, 2.86−3.15 and
2.09 (3 m, 6 × 5H), 1.22−1.08 (m, 6 × 6H), 0.85 (s, 6 × 9H), 0.07
and 0.06 (2s
6 × 6H); MALDI-TOF MS calcd for
1
−6.31, −6.73 (POAr); H NMR (500 MHz, CD2Cl2) δ 10.24−9.90
C272H346N48O90P12Si6 [M − H]− 6266, obsd 6268 plus 5330 (6%,
[(CG)5HPX − H]−.
(m, 4 × 2H), 8.11−7.91 (m, 4 × 3H), 7.61−7.37 (m, 4 × 7H), 7.33−
7.13 (m, 4 × 11H), 6.86−6.81 (m, 4 × 4H), 6.34 and 6.19 (2 m, 4 ×
2H), 5.20−5.10 (m, 4 × 2H), 4.43−4.18 (m, 4 × 4H), 3.84 (m, 4 ×
3H), 3.75, (brs, 4 × 6H), 3.73−3.64 (m, 4 × 3H), 3.48−3.34 (m, 4 ×
2H), 2.82, 2.62, 2.39, and 2.21 (4 m, 4 × 4H), 1.41 (brs, 4 × 3H);
MALDI-TOF MS (DMT off) calcd for C137H152N20O60P8 [M − H]−
3284, obsd 3281 plus 2556 (3%, [(TC)3TPM − H]−).
[5′-DMT-CBz-PO(OMe)-GiBu-3′-PO(OCET)]6HPX (30). The reac-
tion was performed following general protocol D, starting from HPX
(3.3 mg, 5.0 μmol) and 24 (112 mg, 90 μmol, 3 equiv per OH group)
in dioxane (0.4 mL) and 1H-tetrazole (0.2 mL of a 0.45 M solution in
CH3CN, 90 μmol) and then tert-butyl hydroperoxide (27 μL of a 5.5
M solution in decane, 150 μmol). The crude was washed with ethyl
acetate (3 × 3 mL) to give 33 mg of 30 (4.3 μmol, 85%). The hybrid
was treated again with 24 (37 mg, 30 μmol, 1 equiv per OH group) in
dioxane (0.4 mL), 1H-tetrazole (67 μL of a 0.45 M solution in
CH3CN, 30 μmol), and then tert-butyl hydroperoxide (27 μL of a 5.5
M solution in decane, 150 μmol). The crude was again washed with
ethyl acetate (3 × 3 mL). Yield 31 mg 30 (82%, 4.1 μmol) of a pale
yellow solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.35; 31P NMR
(122 MHz, CD2Cl2) δ −1.46, −1.84 (POCH2), −7.84, −8.07, −8.17
(POAr); 1H NMR (300 MHz, CD2Cl2) δ 12.32−12.10 (m, 6H),
11.02−10.60 (m, 6H), 10.30−9.40 (m, 6H), 8.16−7.88 (m, 6 × 3H),
7.80−6.97 (m, 112H), 6.91−6.78 (m, 6 × 4H), 6.28−6.06 (m, 6 ×
2H), 5.50 and 5.10−4.92 (2 m, 6 × 2H), 4.70−4.19 (m, 6 × 6H),
3.82−3.57 (m, 6 × 9H), 3.48−2.15 (m, 6 × 9H), 1.20−0.95 (m, 6 ×
6H).
[5′-DMT-GiBu-PO(OMe)-ABz-3′-PO(OMe)]4TPM (27). The reac-
tion was performed following general protocol D, starting from TPM
(3.8 mg, 10.0 μmol) and 21 (148 mg, 120 μmol, 3 equiv/OH group)
in dioxane (0.7 mL), and 1H-tetrazole (0.27 mL of a 0.45 M solution
in CH3CN, 120 μmol) and then tert-butyl hydroperoxide (36 μL of a
5.5 M solution in decane, 200 μmol). To remove excess dimer, the
crude chromatographed on silica (5 g, pretreated with CH2Cl2/NEt3
0.2%) with a step gradient of 0−8% CH3OH in CH2Cl2. Product-
containing fractions were combined and concentrated in vacuo,
followed by dissolving in CH2Cl2 (0.2 mL) and precipitation with
hexane (3 mL). Centrifugation gave50 mg of 27 (10 μmol, quant) as a
colorless solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.44; 31P NMR
(203 MHz, CD2Cl2) δ −0.47, −0.75, −1.08, −1.17 (POCH2), −6.32,
−6.58, −7.60 (POAr); 1H NMR (500 MHz, CD2Cl2) δ 12.03 (br, 4 ×
1H), 10.93−10.30 (m, 4 × 1H), 9.7−9.23 (m, 4 × 1H), 8.66 and 8.30,
(2 m, 4 × 2H), 7.94 (m, 4 × 2H), 7.70 (m, 4 × 1H), 7.59−7.05 (m, 4
× 16H), 6.80−6.73 (m, 4 × 4H), 6.50−6.07 (m, 4 × 2H), 5.01−4.75
(m, 4 × 2H), 4.49−4.16 (m, 4 × 4H), 3.88 (m, 4 × 3H), 3.73 and 3.72
(2s, 4 × 6H), 3.66 and 3.58 (2d, J = 11.3 Hz, 4 × 3H), 3.32−3.20 (m,
4 × 2H), 2.83−2.00 (m, 4 × 5H), 1.13−0.99 (m, 4 × 6H); MALDI-
TOF MS (DMT off) calcd for C157H172N40O56P8 [M − H]− 3761,
obsd 3759 plus 2916 (4%, [(GA)3TPM − H]−).
(CG)4TPM (2). (Caution: The 1-thionaphthol used for cleavage of
the POMe groups is toxic. The resulting waste should be oxidized with
an aqueous KMnO4 solution.) To a solution of protected hybrid
(CG)4TPM (25, 12 mg, 2.5 μmol) in THF/H2O (0.15 mL, 2:1 v/v)
was added a solution of diisopropylethylamine (DIEA, 25 mg, 180
μmol) and 1-thionaphthol (0.07 mL, 500 μmol) in THF (0.2 mL),
and the clear solution was stirred at 20 °C for 2 h. Ethyl acetate (0.5
mL) and hexane (3 mL) were added, and the precipitate was separated
by centrifugation (3500 rpm, 5 min). The resulting viscous oil was
treated with ethyl acetate (3 × 3 mL) and again isolated by
centrifugation (3500 rpm, 5 min). The resulting pale yellow solid was
dissolved in H2O (2 mL) and dried by freeze-drying to yield 12.0 mg
of (5′-DMT-CBzGiBu)4TPM as a pale yellow solid. TLC (n-PrOH/
H2O/NH4OH (25%) 55:10:35 v/v/v) Rf = 0.66. For removal of the
DMT groups, the solid was taken up in water (3 mL). After addition of
ethyl acetate (2 mL) and a weakly acidic cation exchange resin
DOWEX MAC3 (70 mg wet, approximately 250 μmol H+ according
the specification of suppliers), and the mixture was shaken for 12 h at
20 °C. Ethyl acetate was aspired, and the reaction mixture was washed
with ethyl acetate (3 × 2 mL). The resin was filtered off and washed
with THF/H2O (3 × 1 mL, 1:1 v/v). After addition of NH4OH (1 M,
50 μL), THF was removed in vacuo, and the remaining solution was
evaporated to dryness by lyophilization to yield 9.0 mg of
(CBzGiBu)4TPM as a colorless solid. TLC (n-PrOH/H2O/NH4OH
(25%) 55:10:35 v/v/v) Rf = 0.65. For removal of the base protecting
groups, the solid was treated with NH4OH (250 μL, 25% aqueous
solution) at 5 °C for 1 h and at room temperature for 2 h. After
addition of MeNH2 (250 μL, 40% aqueous solution), the reaction
mixture was again stored at 5 °C for 1 h and at room temperature for 2
[5′-DMT-CBz-PO(OMe)-GiBu-3′-PO(OMe)]6HPX (28). The reac-
tion was performed following General Protocol D, starting from HPX
(7.0 mg, 10.6 μmol) and 20 (115 mg, 95 μmol, 1.5 equiv/OH group)
in dioxane (0.7 mL) and 1H-tetrazole (0.21 mL of a 0.45 M solution in
CH3CN, 95 μmol) and then tert-butyl hydroperoxide (58 μL of a 5.5
M solution in decane, 320 μmol). The crude was treated with ethyl
acetate (3 × 3 mL). Yield 73 mg of 28 (9.9 μmol, 93%); MALDI-TOF
MS showed 70−75% conversion of the fifth phenolic OH group. In a
second cycle the hybrid was coupled again with 20 (36 mg, 30 μmol,
0.5 equiv per OH group) in dioxane (0.7 mL), 1H-tetrazole (67 μL of
a 0.45 M solution in CH3CN, 30 μmol), and then tert-butyl
hydroperoxide (33 μL of a 5.5 M solution in decane, 180 μmol).
The resulting crude was treated with ethyl acetate (2 × 3 mL),
dissolved in CH2Cl2 (0.3 mL) and precipitated with ethyl acetate (3
mL). Centrifugation gave 64 mg of 28 (82%, 8.7 μmol) as a colorless
solid: TLC (CH2Cl2/CH3OH = 9:1 v/v) Rf = 0.37; 31P NMR (122
MHz, CD2Cl2) δ −1.45, −1.51, −1.72 (POCH2), −6.16, −6.27, −6.42
(POAr); 1H NMR (500 MHz, CD2Cl2) δ 12.25 (m, 6H), 11.03−10.70
(m, 6H), 10.30−9.30 (m, 6H), 8.17−7.88 (m, 6 × 3H), 7.80−6.93 (m,
112H), 6.89−6.76 (m, 6 × 4H), 6.30−6.04 (m, 6 × 2H), 5.46 and
5.10−4.92 (2 m, 6 × 2H), 4.70−4.10 (m, 6 × 4H), 3.88−3.55 (m, 6 ×
2713
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h. Excess ammonia and methylamine were removed with a gentle
stream of nitrogen, and the remaining solution was evaporated to
dryness by lyophilization. The solid was treated twice with CH2Cl2/
ethyl acetate (3 mL, 1:2 v/v) in an ultrasonic bath to yield 7.0 mg
(approximately 94%, 2.3 μmol crude) of the deprotected hybrid 2 after
centrifugation (3000 rpm, 5 min) as a colorless solid. TLC (n-PrOH/
H2O/NH4OH (25%) 55:10:35 v/v/v) Rf = 0.43. The crude was
purified by chromatography using a cartridge (Chromabond C18ec)
and a manual step gradient of 5−30% CH3CN in 10 mM TEAA
buffer, with elution at 10−15% CH3CN, followed by HPLC
chromatography, using a C8 column and a gradient of MeCN in 10
mM TEAA buffer (5% for 5 min, 5−8% in 5 min, 8−18% in 55 min)
at 55 °C. Hybrid 2 eluted at tR = 18.2 min, yield 18%. MALDI-TOF
MS calcd for C101H116N32O52P8 [M − H]− 2857, obsd 2856,
reinjection gave HPLC tR = 19.1 min (95% of integration).
resin DOWEX MAC3 (70 mg wet, approximately 250 μmol H+
according the specification of suppliers), 6.8 mg of (GiBuABz)4TPM
was obtained as a colorless solid. TLC (n-PrOH/H2O/NH4OH (25%)
55:10:35 v/v/v) Rf = 0.70. Removal of the base protecting groups with
NH4OH (250 μL, 25% aqueous solution) and MeNH2 (250 μL, 40%
aqueous solution) yielded 6.0 mg of the title hybrid 4 (1.94 μmol,
94%) as a colorless solid; TLC (n-PrOH/H2O/NH4OH (25%)
55:10:35 v/v/v) Rf = 0.5. The crude was purified by chromatography
using a cartridge (Chromabond C18ec) and a manual step gradient of
5−30% CH3CN in 10 mM TEAA buffer, with elution at 10−15%
CH3CN, followed by HPLC chromatography, using a C8 column and
a gradient of MeCN in 10 mM TEAA buffer (5% for 5 min, 5−8% in 5
min, 8−18% in 55 min) at 55 °C. Hybrid 4 eluted at tR = 23.7 min,
yield 5% (2% from 11); MALDI-TOF MS calcd for C105H116N32O52P8
[M − H]− 2953, obsd 2953; reinjection gave HPLC tR = 32.3 (90%).
(CG)6HPX (1) from 28. To a solution of protected hybrid 28 (17.3
mg, 2.34 μmol) in THF (0.3 mL) and H2O (0.05 mL) were added a
mixture of DIEA (25 mg, 180 μmol) and 1-thionaphthol (0.07 mL,
500 μmol) in THF (0.2 mL). The clear solution was stirred at 20 °C
for 2 h. Ethyl acetate (0.5 mL) and hexane (3 mL) were added, and
the precipitate was separated by centrifugation (3500 rpm, 5 min).
The resulting viscous oil was treated with ethyl acetate (3 × 3 mL) and
isolated by centrifugation (3500 rpm, 5 min). The residue was
dissolved in H2O (2 mL) and dried by lyophilization to yield 19.8 mg
of (5′-DMT-CBzGiBu)6HPX as a colorless foam. TLC (n-PrOH/H2O/
NH4OH (25%) 55:10:35 v/v/v) Rf = 0.61. For removal of the base
protecting groups, the solid was treated with NH4OH (250 μL, 25%
aqueous solution) at 5 °C for 1 h and at room temperature for 2 h.
After addition of MeNH2 (250 μL, 40% aqueous solution), the
reaction mixture was again stored at 5 °C for 1 h and at room
temperature for 2 h. Excess ammonia and methylamine were removed
with a gentle stream of nitrogen, and the remaining solution was
evaporated to dryness by lyophilization. The solid was treated twice in
an ultrasonic bath with CH2Cl2/ethyl acetate (3 mL, 1:1 v/v) to yield
15.0 mg of 32 after centrifugation (3000 rpm, 5 min) as a colorless
solid: TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35 v/v/v) Rf = 057.
Finally, for removal of the DMT groups, the product was taken up in
H2O (5 mL) at room temperature. After addition of ethyl acetate (3
mL) and weakly acidic cation exchange resin DOWEX MAC3 (70 mg,
approximately 250 μmol H+ according the specification of suppliers),
the mixture was shaken at 20 °C for 12 h. The organic layer was
aspired, and the aqueous layer was washed with ethyl acetate (2 × 2
mL). The resin was filtered off and washed three times with H2O (3 x
1 mL). NH4OH (1 M, 50 μL) was added, and the solution was
evaporated to dryness by lyophilization. The resulting residue was
treated twice with CH2Cl2/ethanol (1:1, 2 × 2 mL) to yield 10.5 mg
crude 1 as a colorless solid after centrifugation (3500 rpm, 5 min).
TLC (n-PrOH/H2O/NH3 (25%) 55:10:35 v/v/v) Rf = 0.24. The
crude was purified by HPLC, using a C8 column and a gradient of
MeCN in 10 mM TEAA buffer (5% for 5 min, 5−8% in 5 min, 8−18%
in 55 min) at 55 °C. Hybrid 1 eluted at tR = 28.4 min: yield 13%;
MALDI-TOF MS calcd for C158H178N48O78P12 [M − H]− 4367, obsd
4367.
(TC)4TPM (3). To a solution of protected (TC)4TPM hybrid 26
(8.4 mg, 1.87 μmol) in THF (0.1 mL) and H2O (0.05 mL) was added
a solution of triethylamine (0.1 mL, 0.72 mmol) and 1-thionaphthol
(0.07 mL, 500 μmol) in THF (0.1 mL), and the mixture was stirred at
20 °C for 1 h. The reaction mixture was diluted with H2O (5 mL) and
extracted twice with ethyl acetate (2 × 10 mL). The aqueous phase
was concentrated in vacuo, and the residual viscous oil was treated
twice with ethyl acetate/hexane (2 × 4 mL, 1:1 v/v). After
centrifugation (3500 rpm, 5 min), the residue was dissolved in water
(2 mL) and evaporated to dryness by lyophilization to yield 7.7 mg of
(5′-DMT-TCBz)4TPM as a colorless solid. For removal of the DMT
groups, the solid was stirred with acetic acid (1 mL, 80% in water) at
20 °C for 1 h. The reaction mixture was diluted with H2O (4 mL) and
evaporated to dryness by lyophilization. The residual crude was
washed with ethyl acetate (2 × 3 mL), followed by centrifugation
(3500 rpm, 5 min). NH4OH (1M, 50 μL) was added, and the solvent
was evaporated by lyophilization to yield 6.0 mg of (TCBz)4TPM as a
colorless solid. TLC (i-PrOH/H2O/NH3 (25%) 7:2:1 v/v/v) Rf =
0.68. For removal of the benzoyl protecting groups, the solid was
treated with aqueous ammonia (1 mL, 25%) at 20 °C for 3 h. Excess
ammonia was removed with a gentle stream of nitrogen, and the
remaining solution was evaporated to dryness by lyophilization,
followed by washing in an ultrasonic bath with CH2Cl2/ethyl acetate
(2 mL, 1:1 v/v) to yield 4.7 mg (“1.62 μmol, 87%”, from 26) of the
deprotected hybrid 3 after centrifugation (3000 rpm, 5 min) as a
colorless solid . TLC (n-PrOH/H2O/NH3 (25%) 55:10:35 v/v/v) Rf
= 0.50; A sample of the crude hybrid 3 was purified by HPLC
chromatography, using a C18 phase and a gradient of MeCN in 0.1 M
TEAA buffer (0% for 3 min; 0−5% in 10 min and 5−15% in 50 min)
at 60 °C. Hybrid 3 eluted at tR = 32.6 min, yield 40%; MS MALDI-
TOF MS of 3 calcd for C101H120N20O56P8 [M − H]− 2757, obsd 2756.
The residual crude (4.5 mg) was then solved in water (0.5 mL) and
precipitated by vapor diffusion of ethanol for 5 days. The precipitate
was separated by centrifugation and washed with ethanol (1 mL) to
yield 3.3 mg (1.14 μmol, 61%; 28% from 10) of a colorless but
amorphous solid: 31P NMR (203 MHz, D2O) δ −0.92, (POCH2),
1
−5.88, (POAr); H NMR (500 MHz, D2O, suppression of the excess
solvent peak was achieved by presaturation during the recycle delay,34
40 °C) δ 8.14 (d, J = 7.9 Hz, 4 × 1H, H6-C), 7.69 (s, 4 × 1H, H6-T);
7.16 (brs, 4 × 4H, Ar-Hcore), 6.36 (d, J = 7.9 Hz, 4 × 1H, H5-C), 6.29
(dd, J = 6.4 Hz, J = 7.5 Hz, 4 × 1H, H1′-T), 6.16 (dd, J = 6.0 Hz, J =
7.7 Hz, 4 × 1H, H1′-C), 5.01 (m, 4 × 1H, H3′-C), 4.90 (m, 4 × 1H,
H3′-T), 4.43 (m, 4 × 1H, H4′-C), 4.27 (m, 4 × 1H, H4′-T), 4.23 (m, 4
× 1H, H5′-C), 4.15 (m, 4 × 1H, H5″-C), 3.91 (dd, J5′−4′ = 3.5 Hz, J5′−5″
= 12.5 Hz, 4 × 1H, H5′-T), 3.87 (dd, J5″−4′ = 4.6 Hz, J5″−5′ = 12.5 Hz, 4
× 1H, H5″-T), 2.55 (m, 4 × 1H, H2′-T), 2.41 (m, 4 × 1H, H2″-T),
2.35 (m, 4 × 1H, H2′-C), 2.22 (m, 4 × 1H, H2″-C), 1.94 (m, 4 × 3H,
CH3-T).
(CG)6HPX (1) from 29. Protected hybrid 29 (2.0 mg, 0.32 μmol)
was treated with DIEA (25 mg, 180 μmol) and 1-thionaphthol (0.07
mL, 500 μmol) in THF (0.5 mL) and H2O (0.05 mL) as described for
compound 28 to yield 2.0 mg of (5′-TBDMS-CBzGiBu)6HPX as a pale
yellow solid. TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35 v/v/v) Rf
= 0.66. The resulting solid was taken up in THF (100 μL) and H2O
(50 μL) at 20 °C, and the clear solution was treated with TBAF (30
μL, 30 μmol, 1 M solution in THF) for 12 h. After addition of H2O (1
mL) and NH4OH (0.1 mL, 25%), THF was removed with a gentle
stream of nitrogen, and the remaining solution was evaporated to
dryness by lyophilization. The resulting residue was washed twice with
ethyl acetate (2 × 3 mL) to yield 2 mg of 31 after centrifugation (3000
rpm, 5 min) as a colorless solid: TLC (n-PrOH/H2O/NH4OH (25%)
55:10:35 v/v/v) Rf = 0.62. Finally, hybrid 31 was successively treated
with NH4OH (250 μL, 25% aqueous solution) and MeNH2 (250 μL,
40% aqueous solution) at 5 °C for 1 h and at room temperature for 2
h, as described above. After excess ammonia and methylamine were
(GA)4TPM (4). The cleavage of the fully protected (GA)4TPM (27)
was performed as described for (CG)4TPM (2). Starting from 27
(10.3 mg, 2.07 μmol), treatment with 1-thionaphthol (0.07 mL, 500
μmol) and DIEA (25 mg, 180 μmol) in THF/H2O (0.35 mL, 6:1 v/v)
yielded 10.2 mg of (5′-DMT-GiBuABz)4TPM as a pale yellow solid.
TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35 v/v/v) Rf = 0.72. After
removal of the DMT groups with the weakly acidic cation-exchange
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removed with a gentle stream of nitrogen, and the remaining solution
was evaporated to dryness by lyophilization. The crude was taken up
in H2O/THF (1 mL, 1:1 v/v) and treated with DOWEX MAC3
cation exchange resin (50 mg) for 5 min. The resin was filtered off and
washed with water (2 × 1 mL). NH4OH (0.1 mL, 25%) was added to
the filtrate, and the excess ammonia was again removed with a gentle
stream of nitrogen. The remaining solution was evaporated to dryness
by lyophilization, and the solid was treated twice in an ultrasonic bath
with CH2Cl2/ethyl acetate (3 mL, 1:1 v/v) to yield, after
centrifugation, 1.0 mg (0.2 μmol, approximately 95%) of 1 as a
colorless solid: TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35 v/v/v)
Rf = 0.25; MALDI-TOF MS calcd for C158H178N48O78P12 [M − H]−
4367, obsd 4363.
decane, 275 μmol) was added, and the mixture was stirred for 15 min
at room temperature. Finally, the product was precipitated by slowly
adding the solution to ethyl acetate (1 mL). The resulting solid was
filtered off, washed with ethyl acetate (6 × 1 mL), and dried in vacuo
to yield 7.8 mg of crude. For MALDI-TOF MS analysis, an analytical
sample was fully deprotected. When the spectrum indicated an
incomplete conversion of the core, the product was coupled again to
22 (32.8 mg, 29.5 μmol) in propylencarbonate (300 μL) and THF
(100 μL), 1-H-tetrazole (0.1 mL of a 0.45 M solution in CH3CN, 45
μmol) and tert-butyl hydroperoxide (60 μL of a 5.5 M solution in
decane, 330 μmol), as described above. The product was then
precipitated again from ethyl acetate (1 mL). The solid was filtered off,
washed with ethyl acetate (6 × 1 mL), and dried in vacuo, to yield 14.4
mg (2.36 mmol, 81%) of protected hybrid 34. To a solution of
protected hybrid 34 (10.5 mg, 1.72 μmol) in THF (0.13 mL) and
H2O (0.07 mL) was added a mixture of DIEA (25 μL) and 1-
thionaphthol (50 μL) in THF (1.25 mL). The clear solution was
shaken at 20 °C for 2.5 h. Ethyl acetate (0.5 mL) and hexane (2 mL)
was added, the aqueous layer was separated and washed with hexane/
ethyl acetate (3 × 1 mL, 4:1 v/v). After addition of water (200 μL),
the mixture was evaporated to dryness by lyophilization. For removal
of the DMT groups, acetic acid (0.2 mL, 80% in water) was added at
20 °C, and the solution was stirred for 1 h, while the mixture was
washed six times with hexane (6 × 1 mL) during this time. The
reaction mixture was then diluted with H2O (0.6 mL) and evaporated
to dryness by lyophilization. The resulting solid was treated with
NH4OH (250 μL, 25% aqueous solution) at room temperature for 2 h.
After addition of MeNH2 (250 μL, 40% aqueous solution), the
reaction mixture was again reacted at room temperature for 2 h. Excess
ammonia and methylamine were removed with a gentle stream of
nitrogen, and the remaining solution was evaporated to dryness by
lyophilization to yield 8.0 mg of crude 34 as a colorless solid. The
crude was purified by HPLC, using a Nucleosil C8 column and a
gradient of MeCN in 10 mM TEAA buffer (10% for 5 min, 10−15% in
10 min, 15−18% in 15 min and 18−22% in 30 min) at 20 °C. Hybrid
34 eluted at tR = 46.0 min, yield 24%: 31P NMR (202 MHz, D2O, 40
(CG)6HPX (1) from 30. To a solution of protected hybrid 30 (17
mg, 2.23 μmol) in THF (0.6 mL) and H2O (0.1 mL) was added DIEA
25 mg (180 μmol), and the mixture was shaken for 1 h at 20 °C. Then,
1-thionaphthol (0.07 mL, 500 μmol) was added, and the solution was
again shaken for 1 h at 20 °C. Ethyl acetate (0.5 mL) and hexane (3
mL) was added, and the precipitate was separated by centrifugation
(3500 rpm, 5 min). The resulting viscous oil was treated with ethyl
acetate (3 × 3 mL) and isolated by centrifugation (3500 rpm, 5 min).
The resulting solid was dissolved in H2O (2 mL) and dried by
lyophilization to yield 20.0 mg of (5′-DMT-CBzGiBu)6HPX as colorless
foam. TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35 v/v/v) Rf = 0.65.
For DMT removal, the solid was taken up in water (3 mL) at 20 °C.
After addition of ethyl acetate (2 mL) and cation-exchange resin
DOWEX MAC3 (70 mg, approximately 250 μmol H+), the mixture
was shaken at 20 °C for 12 h. The resin was filtered off and washed
with THF/H2O (1:1, 3 × 1 mL). The organic layer of the filtrate was
aspirated, and the aqueous layer was washed three times with ethyl
acetate (3 × 1 mL). After addition of NH4OH (50 μL, 1 M), the THF
was removed in vacuo, and the remaining aqueous solution was
evaporated to dryness by lyophilization to yield 11 mg of 31 as a
colorless solid. TLC (n-PrOH/H2O/NH4OH (25%) 55:10:35): Rf =
0.63. Finally, hybrid 31 was treated with NH4OH (250 μL, 25%
aqueous solution) and MeNH2 (250 μL, 40% aqueous solution), as
described above for conversion of 28 to 1. The resulting solid was
treated twice with CH2Cl2/ ethanol (1:1, 2 × 2 mL) to yield 9.0 mg (2
μmol, approximately 88%) of the title hybrid 1, after centrifugation
(3500 rpm, 5 min), as a colorless solid. TLC (n-PrOH/H2O/NH3
(25%) 55:10:35 v/v/v) Rf = 0.24. A sample of the crude was then
purified by HPLC, using a C8 column and a gradient of MeCN in 10
mM TEAA buffer (5% for 5 min, 5−8% in 5 min, 8−18% in 55 min)
at 55 °C. Hybrid 1 eluted at tR = 29.2 min, yield 25%. The residual
crude (8 mg) was then washed with methanol (2 × 1 mL), and a
portion (2.6 mg, 0.57 μmol) was purified by HPLC, using a
semipreparative Nucleosil C8 column and a gradient of MeCN in
10 mM TEAA buffer (5% for 5 min, 5−10% in 5 min, 10−17% in 50
min) to yield 1.3 mg (0.28 μmol, 50%) of 1: 31P NMR (122 MHz,
1
°C) δ −0.72; H NMR (500 MHz, D2O, suppression of solvent peak
by presaturation,34 40 °C) δ 8.50 (s, 4 × 1H, H5-triazole), 7.92 (s, 4 ×
1H, H8-G), 7.72 (d, J = 6.5 Hz, 4 × 2H, Ar-H), 7.49 (d, J = 7.4 Hz, 4 ×
1H, H6-C), 7.31 (s, 4 × 1H, H6-T), 7.18 (bs, 4 × 2H, Ar-H), 6.32 (bs,
4 × 1H, H1′-G), 5.92 (m, 4 × 2H, H1′-C, H1′-T), 5.75 (d, J = 7.4 Hz, 4
× 1H, H5-C), 5.66 (bs, 4 × 1H, H3′-G), 4.67 (m, 4 × 1H, H3′-C), 4.60
(m, 4 × 1H, H3′-T), 4.47 (suppression of H4′-G), 4.04 (m, 4 × 2H,
H5′-G and H5″-G), 3.98 (m, 4 × 1H, H4′-C), 3.92 (m, 4 × 1H, H4′-T),
3.83 (m, 4 × 2H, H5′-C and H5″-C), 3.60 (m, 4 × 2H, H5′-T and H5″-
T), 3.03 (m, 4 × 2H, H2′-G, H2″-G), 2.27 (m, 4 × 2H, H2′-C, H2′-T),
2.08 (m, 4 × 1H, H2″-T), 1.89 (m, 4 × 1H, H2″-C), 1.58 (s, 4 × 3H,
CH3), 1.55 (brs, 12H, ad-H); MS (MALDI-TOF) calcd for
−
C158H179N52O64P8 4077 [M − H]−, obsd 4075.
1
CD3CN/D2O 2:1 v/v) δ −1.0, (POCH2), −5.34, (POAr); H NMR
Phenyl 2′-Deoxyguanosine-3′-monophosphate. This model
compound was synthesized purely to study basic deprotection of a
phenolic phosphodiester via MALDI-TOF MS. To a solution of 3′-
phosphoramidite 6 (280 mg, 0.35 mmol) and phenol (30.0 mg, 0.32
mmol) in dioxane (1 mL) were added molecular sieves (3 Å, 5 beads).
After addition of 1H-tetrazole (0.71 mL, 0.45 M solution in CH3CN),
the mixture was put in an ultrasonic bath for 2 min at 20 °C. The
reaction mixture was kept at 20 °C for 5 min and then in a refrigerator
at 5 °C for 3 h. Then, tert-butyl hydroperoxide (0.2 mL, 5.5 M solution
in decane) was added, and the mixture was kept at 5 °C again for 15
min. The solution was diluted with CH2Cl2 (20 mL) and washed with
phosphate buffer (15 mL, 0.2 M, pH 7). The aqueous phase was back-
extracted with CH2Cl2 (10 mL), and the combined organic layers were
dried over NaSO4, filtered, and concentrated in vacuo. The residue was
dissolved in ethyl acetate (1 mL) and precipitated with hexane (4 mL).
The mixture was centrifuged, and the supernatant solution was
discarded. This process was repeated, and the resulting solid was
purified by chromatography using silica (10 g) and a step gradient of
2-propanol (0−4%) in ethyl acetate. Product-containing fractions were
combined, concentrated, and dried in vacuo to yield 220 mg (0.27
mmol, 85%) of methyl phenyl N2-isobutyryl-2′-deoxy-5′-O-dimethox-
(500 MHz, NaOH (0.1 M)/D2O 9:1 v/v, suppression of solvent peak
with the WATERGATE gradient pulse sequence,34 90 °C) δ 8.28 (s, 6
× 1H, H8-G), 7.79 (d, J = 7.9 Hz, 6 × 1H, H6−C), 7.46−7.40 (m,
28H, Ar-Hcore), 6.62 (m, 6 × 1H, H1′-G), 6.44 (m, 6 × 1H, H1′-C),
6.14 (d, J = 7.9 Hz, 6 × 1H, H5−C), 5.46 (m, 6 × 1H, H3′-G), 3.99
(dd, J5′−4′ = 2.6 Hz, J5′−5″ = 11.9 Hz, 6 × 1H, H5′-C), 3.92 (dd, J5″−4′
=
4.6 Hz, J5″−5′ = 11.9 Hz, 6 × 1H, H5″-C), 3.13 (m, 6 × 1H, H2′-G),
3.03 (m, 6 × 1H, H2″-G), 2.74 (m, 6 × 1H, H2′-C), 2.24 (m, 6 × 1H,
H2″-C); MALDI-TOF MS calcd for C158H178N48O78P12 [M − H]−
4367, obsd 4365.
(TCG)4TTPA (34). To a sample of (G)4TTPA (33, 5.8 mg, 2.9
μmol, previously coevaporated from CH3CN, 3 × 1 mL) was added
phosphoramidite 22 (19.5 mg, 17.5 μmol), and the mixture was dried
at <0.001 mbar and 60 °C for 1.5 h. After addition of molecular sieves
(3 Å, 5 beads), the flask was flushed with argon and sealed with a
septum. Propylenecarbonate (500 μL) and THF (100 μL) were
added, and the suspension was heated to 55 °C. After addition of 1H-
tetrazole (0.1 mL of a 0.45 M solution in CH3CN, 45 μmol), the
reaction mixture was stirred for 4 h at 55 °C. The heating bath was
removed, tert-butyl hydroperoxide (50 μL of a 5.5 M solution in
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ytritylguanosine-3′-monophosphate: TLC (ethyl acetate/2-propanol =
95:5 v/v) Rf = 0.16, 0.25; 31P NMR (122 MHz, CD2Cl2) δ −5.65,
−5.69 (POAr); 1H NMR (300 MHz, CD2Cl2) δ 11.84 (brs, 1H), 8.83
and 8.71 (2s, 1H), 7.75 and 7.73 (2s, 1H), 7.38−7.05 (m, 14H), 6.79−
6.72 (m, 4H), 6.16 and 6.11 (2t, J = 6.1 Hz, 1H), 5.88 and 5.79 (2 m,
1H), 4.22 (m, 1H), 3.84 and 3.75 (2d, J = 11.4 Hz, 3H), 3.75 and 3.74
(2s, 6H), 3.39, (m, 1H), 3.26−3.10 (m, 2H), 2.72−2.54 (m, 1H),
2.27−2.09 (m, 1H), 1.11−0.96 (m, 6H); MALDI-TOF MS (DMT
off) calcd for C21H26N5O8P [M − H]− 506, obsd 506. To a solution of
methyl phenyl N2-isobutyryl-2′-deoxy-5′-O-dimethoxytritylguanosine-
3′-monophosphate (10 mg, 12.3 μmol) in THF (0.1 mL) and H2O
(0.05 mL) was added a solution of N,N-diisopropylethylamine (DIEA,
13 mg, 0.1 mmol) and 1-thionaphthol (0.07 mL, 0.5 mmol) in THF
(0.1 mL). The mixture was stirred at 20 °C for 15 min. A mixture of
ethyl acetate (0.5 mL) and hexane (3 mL) was added, and the
precipitate was isolated by centrifugation (3500 rpm, 5 min). This
process was repeated twice, and the residual viscous oil was dried in
vacuo. For removal of the DMT group, the product was dissolved in
CH2Cl2 (0.2 mL) and was treated with trichloroacetic acid (0.1 mL,
3% TCA in CH2Cl2) for 30 s at room temperature, followed by
addition of CH3OH. After 30 s, hexane (3 mL) was added, and the
precipitate was isolated by centrifugation. The detritylation procedure
was repeated once. [MALDI-TOF MS calcd for C20H24N5O8P [M −
H]− 492, obsd 493.] For the base-induced removal of the isobutyryl
protecting group (compare Figure S50, Supporting Information), the
solid was treated with a mixture of NH4OH (150 μL, 25 aqueous
solution) and MeNH2 (150 μL, 40% aqueous solution) at 5 °C for 2 h
and at room temperature for 2 h. Excess ammonia and methylamine
were removed with a gentle stream of nitrogen, and the remaining
solution was evaporated to dryness by lyophilization to yield 4.9 mg
(93%, 11.6 μmol) of phenyl 2′-deoxyguanosine-3′-monophosphate as a
colorless solid. MALDI-TOF MS calcd for C16H18N5O7P [M − H]−
422, obsd 422.
NMR of Hybrids. For the assignment of the NMR spectra of
hybrids, samples were taken up in 99.9% D2O (200 μL) and
transferred to a Bruker Match System NMR tube to give a 3 mM
solution. Two-dimensional spectra were recorded using a Bruker
Avance 500 MHz spectrometer at 313 K. Suppression of the excess
solvent peak was achieved by presaturation during the recycle delay.
The repetition delay was set to 2 s. ROESY spectra were acquired at a
mixing time of 300 ms, and TOCSY spectra at a mixing time of 60 ms.
All two-dimensional spectra were recorded with 256 increments in F1
and 2048 in F2, at 8 scans per increment and were processed with zero
filling to 512 data points in F1. Assignment of H1′ and H6 protons of
the nucleotides was based on conventional assignment strategies for
DNA.35 The starting point for the assignment of thymidine residues
was the resonance of the methyl group, which was identified on the
basis of the ROESY crosspeak to its H6 neighbor. The deoxycytidine
residues were identified based on the ROESY crosspeak of H5 to H6.
Starting from the H1′ resonance of each nucleotide, chemical shifts of
the deoxyribose protons were assigned based on TOCSY crosspeaks
and were confirmed via the ROESY spectrum. The resonances of the
core were assigned based on chemical shifts and coupling constants.
ACKNOWLEDGMENTS
We thank Martin Meng for sharing results of syntheses with
commercial β-cyanoethyl phosphoramidites, Melanie Eisele,
■
Daniela Gohringer, Derbora Schamel, and Martin Trautmann
̈
for contributing to the synthesis of intermediates, and Dr. B.
Claasen for help with the acquisition and interpretation of
NMR spectra. This work was supported by DFG (Grant No. RI
1063/13-1 to C.R.).
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ASSOCIATED CONTENT
* Supporting Information
■
S
Synthesis of nucleoside derivatives, NMR spectra, HPLC
chromatograms, MALDI-TOF mass spectra, and photographs
of material formed by 34 prior to and after addition of ethidium
bromide. This material is available free of charge via the
Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Fax: 49 (0) 711 608 64321. Tel: 49 (0) 711 608 64311. E-
mail: lehrstuhl-2@oc.uni-stuttgart.de.
■
Notes
The authors declare no competing financial interest.
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