Modular Strategy to Expand the Chemical Diversity of DNA and Sequence-Controlled Polymers

Article abstract of DOI:10.1021/acs.joc.8b01184

Sequence-defined polymers with customizable sequences, monodispersity, substantial length, and large chemical diversity are of great interest to mimic the efficiency and selectivity of biopolymers. We report an efficient, facile, and scalable synthetic route to introduce many chemical functionalities, such as amino acids and sugars in nucleic acids and sequence-controlled oligophosphodiesters. Through achiral tertiary amine molecules that are perfectly compatible with automated DNA synthesis, readily available amines or azides can be turned into phosphoramidites in two steps only. Individual attachment yields on nucleic acids and artificial oligophosphodiesters using automated solid-phase synthesis (SPS) were >90% in almost all cases. Using this method, multiple water-soluble sequence-defined oligomers bearing a range of functional groups in precise sequences could be synthesized and purified in high yields. The method described herein significantly expands the library of available functionalities for nucleic acids and sequence-controlled polymers.

Full text of DOI:10.1021/acs.joc.8b01184

Article
pubs.acs.org/joc
Cite This: J. Org. Chem. XXXX, XXX, XXX−XXX
Modular Strategy To Expand the Chemical Diversity of DNA and
Sequence-Controlled Polymers
Donatien de Rochambeau,† Yuanye Sun,† Maciej Barlog,‡ Hassan S. Bazzi,‡ and Hanadi F. Sleiman*,†
†
́
́
Department of Chemistry, McGill University, 801 Sherbrooke St. West, Montreal, Quebec H3A 0B8, Canada
‡Department of Chemistry, Texas A&M University at Qatar, P.O. Box 23874, Doha, Qatar
S
* Supporting Information
ABSTRACT: Sequence-defined polymers with customizable
sequences, monodispersity, substantial length, and large
chemical diversity are of great interest to mimic the efficiency
and selectivity of biopolymers. We report an efficient, facile, and
scalable synthetic route to introduce many chemical function-
alities, such as amino acids and sugars in nucleic acids and
sequence-controlled oligophosphodiesters. Through achiral
tertiary amine molecules that are perfectly compatible with
automated DNA synthesis, readily available amines or azides
can be turned into phosphoramidites in two steps only.
Individual attachment yields on nucleic acids and artificial
oligophosphodiesters using automated solid-phase synthesis
(SPS) were >90% in almost all cases. Using this method,
multiple water-soluble sequence-defined oligomers bearing a range of functional groups in precise sequences could be
synthesized and purified in high yields. The method described herein significantly expands the library of available functionalities
for nucleic acids and sequence-controlled polymers.
We have developed an efficient strategy to make sequence-
defined polymers based on automated phosphoramidite solid-
phase synthesis.30−34 This method allowed the formation of
polyphosphodiesters35 that are stable and highly soluble in
water due to their anionic nature. Other groups subsequently
showed the synthesis of very long artificial sequence-controlled
polyphosphodiesters (DP of 100)36 and reported a method for
post-synthesis dual functionalization.37 Phosphoramidite
chemistry has also been used to make “oligopyrenotides”
with novel supramolecular assembly properties.38,39 Thus,
monodispersity, cost-efficiency, length of the oligomers, and
perfect sequence-control have been achieved using automated
solid-phase synthesis with phosphoramidite chemistry. The
next goal for this method is to expand the library of available
compatible phosphoramidites in a straightforward and scalable
manner.
Solid-phase synthesis on a DNA synthesizer requires the
phosphoramidite monomers to stay unaltered to a great
number of chemical conditions (repetitive treatment with
oxidant and mild acid and final deprotection in aqueous base).
For the purpose of versatile and multiple functionalization of
synthetic oligophosphodiesters, even more restrictive con-
ditions apply, making the task arduous. (i) Due to the need for
numerous monomers, their synthesis must be fast, cost-
effective, and scalable. This notably prevents the use of
INTRODUCTION
■
The precise molecular recognition and folding properties of
nucleic acids and proteins derived from their primary sequence
give them central roles in biological systems. Driven by the
goal of mimicking the structures and functions of these
biopolymers,1 interest in sequence-controlled polymers (SCP)
has grown tremendously in the past few years.2 Digital storage
molecules,3,4 foldamers−artificial oligomers with highly
ordered secondary structures,5−8 and single-chain nano-
particles that fold into protein-like structures9−15 are some of
the applications envisaged by these studies. For these purposes,
a strategy allowing the synthesis of monodisperse oligomers
with a large number of chemical monomer variants is of great
interest.
Novel methods, such as iterative exponential growth16 and
other elegant approaches17−20 have been introduced to make
sequence-controlled polymers in a scalable manner. However,
solid-phase synthesis remains the method of choice for the
most precise sequence control.21−25 Automated solid-phase
phosphoramidite chemistry, in particular, has shown excep-
tional coupling yields for the synthesis of DNA and RNA.26,27
Decades of optimization have allowed it to attain the highest
degrees of polymerization (DP) for a solid-phase synthesis. Up
to 200 monomer-long oligonucleotides can be made in good
yields and with simple purification methods.28 Importantly, the
cost of phosphoramidite synthesis has been significantly and
steadily declining, making it a practical as well as powerful
strategy.29
Received: May 22, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.joc.8b01184
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Article
Figure 1. Synthesis of sequence-controlled oligophosphodiesters made of monomers based on the novel tertiary amine backbone. The three
moieties shown on the oligophosphodiester (phenylalanine, β-D-glucose, and alkyne) were used as examples. CEP = cyanoethylphosphoramidite,
DMT = dimethoxytrityl.
a
Scheme 1. Different Backbones Explored
a
Molecules with a prime (e.g., 1′ and 2′) designate the DMT derivatives before conversion to the phosphoramidite.
nucleoside derived phosphoramidites.40 (ii) Very high
coupling yields, leading to high DPs, are essential. As an
example, a 22mer would be obtained in <10% yields if coupling
yields do not exceed 90%. (iii) The monomer should
preferably not contain a chiral center, or it must be
enantiomerically pure. A mixture of enantiomers should be
avoided as the stereochemical complexity of the oligomer
increases exponentially with each monomer addition. Previous
reports of non-nucleosidic phosphoramidites only partially
satisfy these criteria. For example, propanediol-,41,42 threoni-
nol-,43 and serinol-based44,45 phosphoramidites can be
attached on a DNA strand, but their synthesis starts either
with racemic mixtures or costly enantiopure diols. Other
reports show the elegant synthesis and use of hydroxyproli-
nol-46,47 and oxamide-based48,49 phosphoramidites, but these
may lead to stability issues during the final deprotection under
basic conditions.
In this manuscript, we explore the use of a tertiary amine
scaffold to make a range of multifunctional DNA strands and
sequence-controlled polymers via phosphoramidite synthesis.
Two monomers bearing two convenient chemical handles for
the attachment of a variety of side-chains in two steps were
designed: An alkyne-containing unit would allow the
introduction of azides (e.g., sugar molecules), while a
carboxylate-containing molecule would allow functionalization
with amine-containing monomers, including amino acids
(Figure 1). Applicability of this strategy was tested with the
synthesis of a variety of DNA strands and fully artificial
oligophosphodiesters. This tertiary amine backbone is a cost-
effective, efficient, robust, and achiral alternative to commer-
cially available DNA modifications and will open the door to
significantly greater chemical diversity in both oligonucleotide
and sequence-controlled polymer synthesis.
RESULTS AND DISCUSSION
■
1. Tertiary Amine Backbones. The starting point for this
study is the observation that tertiary amines (such as
diisopropylethylamine and triethylamine) are commonly used
as bases and thus compatible with phosphoramidite syn-
thesis.50 We thus reasoned that an achiral tertiary amine-
containing backbone may be a good candidate for the
introduction of functional groups. Initially, diethanolamine
was chosen as starting material due to its ready availability
(Scheme 1).51 Simple nucleophilic substitution with the
moiety of interest on the secondary amine is then required
to functionalize the monomer. As a preliminary proof of
concept, molecules 1′ (Scheme S1) and 2′ were synthesized
and attached to a DNA strand following a standard procedure
that does not require isolation of the phosphoramidite.
Excellent coupling yields were observed for molecule 2′
(>95%),31 whereas molecule 1′ consistently led to unsat-
isfactory coupling yields followed by spontaneous degradation
of the DNA strand in water at 4 °C after a few days. We
hypothesized that the nucleophilic nitrogen atom in the case of
molecule 1′ can attack an electrophilic phosphorus atom
B
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forming a favorable five-membered ring (Figure S1). On the
other hand, the fluorine atoms in 2′ exert an inductive effect
that reduces the nitrogen lone pair nucleophilicity, thus
possibly explaining the good yields obtained with this
molecule. As further evidence for this mechanism, a
phosphoramidite bearing an anthraquinone moiety directly
attached to the diethanolamine nitrogen atom (thus also
reducing its nucleophilicity) was reported elsewhere and led to
good yields.52
This observation led to further monomer optimization.
Molecules 3, 4, and 5 were used to check backbone suitability.
For 3 and 4, the previous degradation mechanism is less likely
to happen because they do not have a nucleophilic nitrogen,
while 5 has a longer spacer separating the nucleophilic nitrogen
from the phosphorus, thus avoiding the five-membered ring
intermediate in this degradation mechanism. 3 was made
following a straightforward synthesis: Myristoyl chloride was
added onto diethanolamine leading to an amide in quantitative
yields, which was transformed to phosphoramidite 3 following
standard procedures (Scheme S2). 4 was made from the
precursor for 1′ using methyliodide, followed by DMT
protection and phosphoramidation using standard procedures
(Scheme S3). Finally, bromohexanol was substituted onto
hexadecylamine, leading to compound 5′ further transformed
to 5 (Scheme S4).
Figure 2. Chemical formula of 3, 4, 5, and HEG after automated
synthesis and deprotection (a). Electrophoretic mobility gel assay for
DNA 19mers singly modified with 3, 4, and 5 at the 5′ end (b).
Reverse-phase (RP) high-performance liquid chromatography
(HPLC) traces (UV detection, 260 nm) from crude mixtures (c)
and of sequence-controlled oligomers containing hexaethylene glycol
HEG and thymidines (T) (d). Full gel is shown on Figure S3.
Gradients are available on Figure S4. DNA sequence: 5′-
TTTTTCAGTTGACCATATA- 3′.
These monomers were coupled to the 5′ end of a DNA
strand as a first step. Molecule 3 did not lead to good yields
(<30%, Table 1, Figure 2), and the synthesis of T-HEG-HEG-
Table 1. Yields and ESI-MS Characterization of Oligomers
Containing Phosphoramidites 3, 4, and 5
unique
global
calculated
found
incorporation yields exact mass exact mass
a
b
c
strand
yields (%)
(%)
[g mol−1
]
[g mol−1
]
DNA-3
DNA-4
DNA-5
T-HEG-HEG-3-3-T
T- HEG-HEG-4-4-T
T- HEG-HEG-5-5-T
26−44
90−92
90−96
NA
−
−
−
<20
79
6142.23
6171.29
6268.41
1988.84
2047.00
2241.20
6142.28
6171.31
6268.47
1988.69
2046.82
2241.03
Molecule 5 was incorporated in an oligomer and on a DNA
strand with the best yields obtained so far (Figure 2, Table 1).
This result indirectly confirms the potential degradation
mechanism that occurred with 1′. Yields obtained are high
enough to consider using such a backbone for making more
complex sequence-controlled oligomers with a higher number
of units. Moreover, 5 is pH responsive (predicted pKb is 3.2)53
and most probably protonated at neutral pH, which allows
better solubility and possible zwitterionic character, as with
oligomers of 4.
90
97
82
a
Yields were calculated through image analysis of electrophoretic
mobility shift assays and through product peak area by RP-HPLC
(260 nm detection) vs unmodified DNA. For non-DNA oligomers,
only HPLC yields of the second amidite (3, 4, or 5) introduction are
b
c
reported. Product peak area by RP-HPLC over total area. Mass was
found using electrospray ionization-mass spectrometry (ESI-MS)
technique, detecting multiply charged species. See SI for HPLC
traces, more details about yield,s and mass spectrometry. HEG stands
for a commercially available hexaethylene glycol phosphoramidite and
T for a thymidine phosphoramidite.
2. Synthesis of the Optimized Monomer Platform.
Based on the conclusions obtained from the backbone
investigations, we synthesized two molecules to be used as
versatile platforms to attach a variety of moieties. Molecules
PT1 and PT2 have many advantages: They are easy to
synthesize in two steps for PT1 and three for PT2 from readily
available starting materials, need only one chromatography
purification, and have a reactive function for simple and
efficient attachment of the moiety of interest. Furthermore, the
DMT monoprotection being the lowest yielding step, we can
limit material waste by performing it as the second step and
attach the moiety of interest afterward. Compound PT1 has an
alkyne moiety for click chemistry. Many biorelated compounds
are now available as azides, making this platform highly
versatile for possible functionalization with carbohydrates,
dyes, biotin-bearing, or cyclodextrin-containing molecules for
example. As with 5, oligomers from PT1 can possibly have
zwitterionic character, due to protonation of their tertiary
amine moiety. Compound PT2 is of interest as well since its
3-3-T (using commercially available thymidine and hexaethy-
leneglycol (HEG) phosphoramidites) gave low coupling yields
(Table 1). On the other hand, molecule 7 led to high yields
when inserting it at the 5′ end of a DNA strand and on
sequence-controlled oligomers (Figure 2, Table 1). Molecule 4
is of interest because its amphiphilic character can increase its
affinity toward membrane lipids. The positively charged
backbone in 4 can lead to novel sequence-controlled
zwitterionic polymers with positive quaternary ammonium
and negative phosphate groups. The quaternary ammonium
center is chiral in this case but could be made achiral by
condensing twice the same iodide onto diethanolamine.
C
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Scheme 2. Synthetic Route for Platform PT1, Alkyne Phosphoramidite 6, and a β-D-Glucose Containing Phosphoramidite 7
Scheme 3. Synthetic Route for Platform PT2 and a Phenylalanine-Functionalized Phosphoramidite 8
carboxylate allows common amide coupling with readily
available amines such as amino acids or peptides. These two
platforms were synthesized with good yields after double
alkylation of propargylamine (Scheme 2) and glycine t-butyl
ester (Scheme 3), respectively, with bromopropanol, followed
by DMT protection of one hydroxyl group. As before, PT1 and
PT2b were attached to a DNA strand without isolation of the
phosphoramidite. These results confirmed that PT1 and PT2
backbones are suitable for phosphoramidite-based SPS (Figure
3, Table 2).
In addition, PT1 and PT2 were functionalized with model
molecules. We chose a protected sugar azide as well as a
methyl ester amino acid, phenylalanine. Standard click
chemistry procedure was performed to obtain molecule 7′ in
95% yields which was then turned into phosphoramidite 7.
Only one chromatography purification is required here.
Phenylalanine could be coupled successfully to PT2, and the
obtained molecule was turned into monomer 8. Successful
incorporation of 7′ and 8′ on a DNA strand was achieved, and
high incorporation yields were obtained in both cases (Figure
3, Table 2). The deprotection conditions used are suitable for
classic DNA synthesis and are described in the Supporting
Information (SI). Intriguingly, methylamine/ammonium hy-
droxide deprotection conditions allowed the quantitative
conversion of the methyl ester into the secondary methyl
amide derivative, which can be of interest. As a conclusion, we
show the simple and cost-effective incorporation of an azide
Figure 3. Chemical formulas of PT1, PT2b, 7, and 8 after automated
synthesis and deprotection (a). Electrophoretic mobility gel assay for
DNA 19mers modified with PT1, PT2b, 7, and 8 at the 5′ end (b).
RP-HPLC traces (UV detection, 260 nm) from crude mixtures (c).
Full gel is available on Figure S3, and gradients are detailed in Figure
S4. DNA sequence: 5′-TTTTTCAGTTGACCATAT-3′.
molecule or a primary amine bearing compound in DNA
strands. Due to the nonchiral character of the platforms used
and the good to excellent incorporation yields obtained, one
D
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Article
with commercially available monomers (nucleotides and
TEG), hydrophobic 12 carbon long chain phosphoramidite
(10), and a novel naphthalene-containing amidite 9. Each
monomer introduces a new orthogonal interaction to DNA
and sequence-controlled polymers, in a similar manner to
functionalities imparted to peptides and proteins by the 20
amino acids. For example, 10 and TEG can be used to tune the
hydrophobicity and hydrophilicity of the oligomers,30 and 9
was synthesized to allow the introduction of Pi-Pi stacking
interactions. More interestingly, PT1 and PT2 can be
substituted with a wide range of functionalities leading to
sugar-(7) and amino acid-(8) bearing phosphoramidite, for
example. One could imagine using 7 and 8 to impart host−
guest interactions, hydrogen-bonding, and new biological
activity. With such a palette of interactions, one could imagine
making functional polymers with protein-like selectivity and
efficiency.
To further show the versatility of monomer 6, we carried out
post-SPS functionalization on two DNA strands. Indeed, we
synthesized two DNA 19mers containing, respectively, 1
(Figure 3) and 5 alkyne moieties (96% individual coupling
yields, oligomer F). Following a straightforward protocol for
copper-catalyzed cycloaddition on oligonucleotides,55 high
yielding functionalization of these strands with β-D-glycosyl
azide was achieved (>80%, see SI-VI). Interestingly we also
report the synthesis of novel hybrid oligomers containing DNA
and glycosyl modifications as well as amino acids (oligomer E).
Indeed, a DNA 14mer was functionalized twice with
phosphoramidite 7 followed by three times with 8. Yields are
high for such a complex hybrid (see Figure S6 for discussion).
Functional diversity imparted by the strategy described here in
DNA strands could significantly expand the toolbox of DNA
nanotechnology.56
Table 2. Yields and ESI-MS Characterization of Modified
19mers Containing Monomers PT1, PT2b, 7, and 8
incorporation
calculated exact mass found exact mass
b
a
modification
yields (%)
[g mol−1
]
[g mol−1
]
PT1
PT2b
7
93−99
90−92
88−94
90−94
5998.06
6018.05
6203.13
6165.13
5998.00
6018.01
6203.13
6165.01
8
a
Yields were calculated through image analysis of electrophoretic
mobility shift assays and through product peak area by RP-HPLC
b
(260 nm detection) vs unmodified DNA. Mass was found using ESI-
MS technique, detecting multiply charged species. See SI for more
details about yields and mass spectrometry.
could consider these molecules as good candidates for the
efficient preparation of sequence controlled oligomers with a
variety of side chain functions. Finally, the ease of preparation
of PT1, PT2, and their functionalized counterparts illustrates
the great practicality and ready accessibility of this method.
Sequence-specific on-bead functionalization of PT1 in the
synthesizer could also be an efficient method. Such a method
has been reported with an alkyne-containing phosphonate
reagent elsewhere.54 It was shown to be very modular with
individual coupling yields between 63 and 89%.
3. Sequence-Controlled Oligophosphodiesters. The
new tertiary amine platforms allowed the modular and highly
efficient synthesis of sequence-controlled oligomers featuring
different moieties. The phosphoramidites were used in a
standard automated DNA synthesizer along with commercially
available DNA synthesis reagents. Coupling cycles were the
same as for DNA synthesis except that the coupling time was
increased to 10 min and phosphoramidite concentration was
kept about 0.1 M in dichloromethane. After synthesis and
deprotection, isolation could be performed using RP-HPLC,
and the oligomers were further characterized using LC-MS
techniques. Due to the ionic nature of our polymers, they are
perfectly soluble in water leading to great ease of manipulation.
As with nucleic acids, they can be quantified easily using
spectrophotometry with the appropriate absorption coeffi-
cients (see SI-V for details). Yields were measured on the
HPLC traces considering the relative absorbance of each
byproduct. Most sequences were designed to show high
functional diversity on one chain. Oligomers A to D show the
possibility of using up to 6 different phosphoramidites and
highlight single coupling yields over 90% in all cases (Table 3,
Figure 4). We were able to work with the new monomers
based on the tertiary amine backbone (6, 7, and 8) combined
CONCLUSIONS
■
We highlight here a strategy to significantly increase the
chemical diversity of phosphoramidite monomers. These can
be used in an automated synthesizer for the synthesis of
sequence-controlled oligomers and modification of nucleic
acids. We investigated several tertiary amine backbones to be
used as platforms for making a large variety of phosphor-
amidite monomers. This study led to the design of two achiral
molecules that could be obtained in a few simple synthetic
steps from inexpensive starting materials and that bear a
reactive moiety. Their attachment on DNA strands was shown
to be very efficient. As a model study, the first platform was
functionalized with a sugar azide derivative and turned into a
phosphoramidite. This new monomer could be attached to a
Table 3. Yields and ESI-MS Characterization of Sequence-Controlled Oligophosphodiesters
sequence (iterative synthesis from left
monomer)
global yields
average individual coupling yields
calculated exact mass
found exact mass
c
a
b
oligomer
(%)
(%)
[g mol−1
]
[g mol−1
]
A
B
C
D
E
F
T-62-8-TEG-9
T-62-8-TEG-9-82-10-TEG-9
T3-HEG4-7-62
T-6-8-6-8-6-10
DNA 14mer-72-83
DNA 19mer-65
64
36
60
52
34
82
91
90
93
90
81
96
1742.62
3442.28
3130.99
2005.76
6321.47
6930.39
1742.63
3442.30
3130.97
2005.74
6321.44
6930.16
a
Yields were calculated through product peak area by RP-HPLC (260 nm detection) considering the relative absorbance (see SI-V) of byproducts.
b
c
These numbers were obtained by taking the global yields at the power 1/n, where n is the number of couplings after the closest nucleotide. Mass
was found using ESI-MS technique, detecting multiply charged species except for oligomers A and D. All HPLC traces are shown in Figures S4 and
S5. All MS spectra are on Figures S6 and S7.
E
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Figure 4. Formula of oligomers A and E (a) and representative RP-HPLC traces (UV detection, 260 nm) from crude mixtures of sequence-
controlled oligophosphodiesters A, B, E, and F (b). Gradients are detailed in Figures S4 and S5. All main peaks were found to be the expected
product except for DNA 14mer-SUG2-PHEN3 for which the second peak is associated with the product. T stands for thymidine, TEG is a
commercially available triethylene glycol monomer, 9 is a novel naphthalene-containing monomer (Scheme S5), and 10 is a 12 carbons long alkyl
chain monomer (Scheme S6).
fine, DNA grade) was purchased from Glen Research. All other
reagents were obtained from Sigma-Aldrich. Triethylammonium
acetate (TEAA) buffer is composed of 50 mM TEA with pH
adjusted to 8.0 using glacial acetic acid. TBE buffer is 90 mM Tris, 90
mM boric acid, and 1.1 mM EDTA with a pH of 8.0.
DNA strand and several sequence-controlled oligomers with
good yields. Phenylalanine was attached to platform 2 through
standard amide coupling and was further attached to DNA
with very good yields. Noticeably, this strategy allowed to
synthesize a new class of zwitterionic sequence-controlled
oligophosphodiesters with average individual coupling yields
rarely under 90%. Thus, the goal of increasing side-chain
diversity in DNA and sequence-controlled oligomers could be
achieved thanks to achiral, easily synthesized, inexpensive
phosphoramidites that are compatible with automated solid-
phase synthesis. This strategy paves the way to the synthesis of
an extensive library of monomers. For example, with a careful
choice of protecting groups on amino acids, many of them
could potentially be attached to platform 2 and used in a DNA
synthesizer. This would be a promising step toward the
synthesis of functional sequence-defined polymers and
ultimately fully artificial enzyme mimics.
Instrumentation. Standard automated solid-phase synthesis was
performed on a Mermade MM6 synthesizer from Bioautomation.
HPLC purification was carried out on an Agilent Infinity 1260. DNA
and oligomers quantification measurements were performed by UV
absorbance with a NanoDrop Lite spectrophotometer from Thermo
Scientific. A Varian Cary 300 Bio spectrophotometer was used for UV
absorbance studies. Polyacrylamide gel electrophoresis (PAGE)
experiments were carried out on a 20 × 20 cm vertical Hoefer 600
electrophoresis unit. Gel images were captured using a ChemiDoc MP
System from Bio-Rad Laboratories. Dry solvents were taken from an
Innovation Technology device. Low-resolution mass determination
was carried out using electron-spray ionization-ion trap-mass
spectrometry (MS) on a Finnigan LCQ Duo device. High-resolution
mass determination was achieved using a Bruker Maxis API
(Atmospheric pressure ionization) QTOF or a THERMO Exactive
Plus Orbitrap-API. Liquid chromatography electrospray ionization
mass spectrometry (LC-ESI-MS) of oligomers was carried out using
Dionex Ultimate 3000 coupled to a Bruker MaXis Impact QTOF.
Some oxygen- and moisture-sensitive experiments were carried out in
a Vacuum Atmospheres Co. glovebox. Column chromatography was
performed using a CombiFlash Rf system from Teledyne Isco. The
NMR spectra were recorded on Bruker 400 MHz, 500 MHz, Varian
300 or 400 MHz for 1H, 13C, and 31P with chloroform-d1 (δ 7.26, 1H;
EXPERIMENTAL SECTION
■
General Information. All starting materials were obtained from
commercial suppliers and used without further purification unless
otherwise noted. Dimethoxytrityl chloride (DMT-Cl) was purchased
from GenScript. (3-Dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride (EDC-Cl) and glycine t-butyl ester hydrochloride were
purchased from AKScientific. 3-Bromo propanol was purchased from
Alfa Aesar. Acetic acid, boric acid, and solvents were purchased from
Fisher Scientific. Choroform-d1 was purchased from Cambridge
Isotope Laboratories. Importantly, it was stored on molecular sieves in
order to keep it neutral. If used as sold, hydrolysis of phosphoramidite
(fast) as well as DMT deprotection (slow) may be observed. GelRed
nucleic acid stain was purchased from Biotium Inc. Concentrated
ammonium hydroxide, ammonium persulfate, acrylamide/bis-acryl-
amide (40% 19:1 solution), and TEMED were obtained from
Bioshop Canada Inc. and used as supplied. One μmol Universal 1000
Å LCAA-CPG supports and standard reagents used for automated
DNA and RNA synthesis were purchased through Bioautomation.
N,N-Diisopropylamino cyanoethyl phosphonamidic-chloride (CEP-
Cl) and DMT-hexaethyloxy glycol (cat. no. CLP-9765) phosphor-
amidites were purchased from Chemgenes. Sephadex G-25 (super
1
δ 77.16, 13C), acetone-d6 (δ 2.04, H; δ 29.8, 13C) or DMSO-d6 (δ
1
2.50, H; δ 39.5, 13C) as internal lock solvents and chemical shift
standard.
General Procedure for Dimethoxytrityl Monoprotection
(except for 9′ and 10′). 3.63 mmol of the diol starting material and
1.47 mL of TEA (10.9 mmol, 3 equiv) were dissolved in 30 mL of dry
DCM in a 100 mL dry round-bottom flask. DMT chloride (1.23g,
3.63 mmol, 1 equiv) was dissolved in 20 mL of dry DCM and added
dropwise to the reaction mixture at 0 °C under vigorous stirring. After
the chloride addition, the reaction was allowed to warm up to room
temperature and left stirring for 2 h 30 min. Purification conditions
are described for each molecule individually. For each purification,
F
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silica gel was pretreated with the least polar solvent containing 0.1%
NEt3.
wash in 95% acetonitrile. Column: Hamilton PRP-C18 5 μm 100 Å
2.1 × 150 mm. For each analytical separation approximately 0.5
OD260 of crude DNA or 0.3 OD260 of crude oligomers was injected as
a 20−50 μL solution in Millipore water. Detection was carried out
using a diode-array detector, monitoring absorbance at 260 nm.
Post-Solid-Phase Synthesis Functionalization of Alkyne-
Containing DNA Strands. Two strands containing 1 and 5 times
phosphoramidite 6 have been modified after solid-phase synthesis
following a standard protocol reported elsewhere.55 With DNA-6: To
20 μL of a DNA solution (directly from post-deprotection crude 6.6
nmol) in water, 10 μL of an azide solution (164 nmol, 25 equiv) and
10 μL of a freshly prepared solution containing CuBr and
tris(benzyltriazolylmethyl)-amine in a 1:1 ratio in 4:3:1 water:-
DMSO:tBuOH were added (66 nmol, 10 equiv). The mixture was
vortexed and shaken at room temperature for 2 h.
With DNA-65 (oligomer F): To 20 μL of a DNA solution (directly
from postdeprotection crude, 9.5 nmol) in water, 10 μL of an azide
solution (475 nmol, 50 equiv) and 10 μL of a freshly prepared
solution containing CuBr and tris(benzyltriazolylmethyl)-amine in a
1:1 ratio in 4:3:1 water:DMSO:tBuOH were added (180 nmol, 20
equiv). The mixture was vortexed and shaken at room temperature for
2 h. In the case of post-SPS click chemistry compounds, purification
was carried out through gel electrophoresis instead of RP-HPLC.
Crude products were purified on 20% polyacrylamide gels,
supplemented with 7 M urea (loading up to 10 OD260 of crude
mixture per gel, 500 V field applied). Electrophoresis was run at lower
voltage for the first 30 min. Following electrophoresis, the gel was
wrapped in plastic and visualized by UV shadowing over a fluorescent
TLC plate. The full-length product was quickly excised, then crushed
and incubated in ∼10 mL of Millipore water. The solution was frozen
in liquid N2 for 3 min and left at 65 °C overnight. The supernatant
was then concentrated to 1.0 mL and desalted using size exclusion
chromatography (Sephadex G-25). The DNA strand was then
quantified (OD260) and converted to micromolar concentrations
using the calculated extinction coefficient.
2,2′-(Hexadecylazanediyl)diethanol, 1′′. Modified protocol from
ref 57. Diethanolamine (3.8 g, 36 mmol) and 1-hexadecylbromide
(9.19 g, 30 mmol) were added to dried round-bottomed flask (RBF)
charged with KHCO3 (6.02 g, 60 mmol) and KI (0.5 g, 3 mmol) then
mixed in dry acetonitrile (80 mL). The reaction mixture was heated
under reflux for 3 h and cooled down, and the solvent removed under
vacuum. Residue was taken in dichloromethane (100 mL) and
washed with water (3 × 100 mL). Organic layer was dried with
MgSO4 and solvent removed under vacuum to obtain a yellow oil
solidifying fast on standing, forming slightly orange waxy solid as a
pure product in quantitative yield (9.82 g). Synthesis of this
compound has been reported elsewhere.58 1H NMR (400 MHz,
CDCl3) δ 0.87 (t, J = 7.0 Hz, 3H), 1.15−1.36 (m, 26H), 1.36−1.54
(br. m, 2H), 2.55 (t, J = 7.6 Hz, 2H), 2.69 (t, J = 5.4 Hz, 4H), 2.74
(br. s, 2H), 3.63 (t, J = 5.4 Hz, 4H). 13C NMR (100 MHz, CDCl3) δ
14.1 (CH3), 22.7−31.9 (multiple CH2), 54.8 (CH2), 56.1 (CH2),
59.4 (CH2).
General Procedure for Converting the Monoprotected
Alcohols into Phosphoramidites (except for 9 and 10).
Monoprotected diol was suspended in toluene or acetonitrile, and
solvent was evaporated under reduced pressure (60 °C). The
operation was repeated once, and the dried compound was kept
under high vacuum for at least 5 h. In an oven-dried flask, 0.64 mmol
of alcohol starting material was then dissolved in anhydrous DCM (5
mL), and 0.56 mL of dry DIPEA (3.2 mmol, 5 equiv) was added
while stirring. CEP-Cl (0.14 mL, 0.64 mmol, 1 equiv) was added
slowly, and the reaction was allowed to stir under inert gas at room
temperature for 2 h. Two fast extractions with DCM from a 10%
Na2CO3 aqueous solution were performed. Organic fractions were
combined, dried over MgSO4, and filtered, and the solvent was
evaporated under reduced pressure (40 °C). The crude product was
loaded on Celite and purified using the combiFlash system. For each
purification, silica gel was pretreated with the least polar solvent
containing 1% NEt3.
General Solid-Phase Synthesis Procedure. Standard DNA
synthesis was performed on a 1 μmol scale, starting from a universal
1000 Å LCAA-CPG solid support. Amidites HEG and TEG were
dissolved in dry acetonitrile, and all other amidites were dissolved in
dry DCM to obtain 0.1 M solutions. Extended coupling times of 10
min were used except for standard DNA amidites and for oligomer A
synthesis (4 min couplings). Removal of the DMT protecting group
was carried out using 3% dichloroacetic acid in dichloromethane on
the DNA synthesizer. Figure S2 describes the coupling cycle in more
details. The DNA sequence used along this study is called AT and is
5′- TTTTTCAGTTGACCATATA-3′ except for the oligomer E for
which the sequence used was 5′- CAGTTGACCATATA-3′.
General Procedure for Attaching Moiety without Phos-
phoramidite Isolation. Under a nitrogen atmosphere in a glovebox
(<2.5 ppm trace moisture), in a 20 mL oven-dried round-bottom
flask, monoprotected alcohol 1′ (31.6 mg, 0.050 mmol) is dissolved in
dry DCM (500 μL). Diisopropylethylamine (8.7 μL, 0.050 mmol, 1
equiv) and N,N-diisopropylamino cyanoethyl phosphonamidic-Cl
(10.0 μL, 0.045 mmol, 0.9 equiv) are added. Reaction is allowed to
stir at room temperature for 45 min. Coupling was done using the
“syringe” technique: The crude solution containing the phosphor-
amidite (200 μL, 0.1 M) is mixed with the standard activator solution
(200 μL, 0.25 M) in the presence of the CPG using syringes. After 10
min, the solution was removed from the columns and the strands
underwent capping, oxidation, and deblocking steps in the
synthesizer.
General Deprotection Procedure. Sequences without moiety
PT2b or 8 underwent classical deprotection procedures: Completed
syntheses were cleaved from the solid support and deprotected in 28%
aqueous ammonium hydroxide solution for 16−18 h at 65 °C or in
28% aqueous ammonium hydroxide/30% methylamine solution 1:1
mixture for 3 h at 65 °C. With moiety 8, 1:3 t-butylamine/water
solution during 6 h at 65 °C (recommended with dmf-protected
guanosines) or 0.4 M NaOH 1:4 water/methanol solution at room
temperature during 16 h, followed by quenching with 2.0 M TEAA
buffer (recommended with isobutyryl-protected guanosines) were
used. With moiety PT2b, a solution of 0.4 M NaOH 1:4 water/
methanol solution at 65 °C overnight, followed by quenching with 2.0
M TEAA buffer could fully deprotect the tBu ester. The crude
product solution was separated from the solid support and
concentrated under reduced pressure at 60 °C. This crude solid
was resuspended in 1 mL Millipore water. Filtration with 0.22 μm
centrifugal filter was then performed prior to HPLC purification. The
resulting solution was quantified by absorbance at 260 nm. NB: If 1:3
t-butylamine/water solution during 6 h at 65 °C does not lead to
complete unylinker deprotection, concentrated ammonium hydroxide
at room temperature overnight could be performed as a second step.
General Procedure for RP-HPLC Purification. Solvents (0.22
μm filtered): 50 mM triethylammonium acetate (TEAA) buffer (pH
8.0) and HPLC grade acetonitrile. Elution gradient is described on
Figures S4 and S5. All gradients were followed by a short column
2-((2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)(hexadecyl)-
amino)ethanol, 1′. Reaction was performed from 9.1 mmol of 1′′.
After the reaction, solvent was evaporated, and resulting yellow oily
residue was purified by chromatography on triethylamine pretreated
silica gel with slow gradient of EtOAc/Hexane (0−15%) to obtain the
product as a sticky yellow oil: 1.32 g, 23% (pure fractions). HRMS
(ESI-QTOF) m/z: [M + H]+ calcd for C41H62NO4 632.4673; found
1
632.4671. H NMR (400 MHz, acetone-d6) δ 0.88 (t, J = 7.0 Hz,
3H), 1.29 (br. s, 26H), 1.43−1.48 (br. m, 2H), 2.48 (t, J = 7.4 Hz,
2H), 2.58 (t, J = 6.0 Hz, 2H), 2.71 (t, J = 5.9 Hz, 2H), 3.15 (t, J = 5.9
Hz, 2H), 3.19 (br. s, 1H), 3.51 (t, J = 6 Hz, 2H), 3.78 (s, 6H), 6.86−
7.51 (DMT, 13H). 13C NMR (100 MHz, acetone-d6) δ 14.4 (CH3),
23.3−32.6 (aliphatic CH2), 55.1 (CH2), 55.5 (2 × CH3), 55.9 (CH2),
57.8 (CH2), 60.1 (CH2), 63.2 (CH2), 86.8 (C), 113.8 (4 × CH),
127.4 (CH), 128.5 (2 × CH), 129.0 (2 × CH), 130.9(4 × CH), 137.4
(2 × C), 146.5 (C), 159.5 (2 × C).
N,N-Bis(2-hydroxyethyl)tetradecanamide, 3′′. Modified from
synthesis reported elsewhere.59 Diethanolamine (2g, 9.51 mmol, 4
G
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The Journal of Organic Chemistry
Article
equiv) was mixed with 10 mL of dry dichloromethane and cooled to
−10 °C in a dry round-bottom flask. 1.17g of myristoyl chloride (4.78
mmol) was diluted in 6 mL of dry DCM and added dropwise in the
reaction medium while stirring with temperature kept under 0 °C.
After the chloride addition, reaction mixture was allowed to slowly
come to room temperature and left stirring overnight. The mixture
was transferred in a separatory funnel. Upper layer was removed and
occurred to be the unreacted diethanolamine. Water and DCM were
added in the separatory funnel, and the product was extracted three
times. The organic fractions were combined, washed with brine, dried
over MgSO4, and filtered, and the solvent was evaporated under
reduced pressure (40 °C). The reaction yielded 1.44g of a shiny white
solid (96%). 1H NMR (500 MHz, CDCl3): δ (ppm) = 3.90 (t, J = 5
Hz, 2H), 3.82 (t, J = 5 Hz, 2H), 3.60 (t, J = 5 Hz, 2H), 3.53 (t, J = 5
Hz, 2H), 3.90 (t, J = 5 Hz, 2H), 3.03 (br, 1H), 2.79 (br, 1H), 2.41 (t,
J = 8 Hz, 2H), 1.66 (quin, J = 7 Hz, 2H) 1.28−1.33 (m, 20H), 0.90 (t,
J = 7 Hz, 3H).
N-(2-(bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-N-(2-hy-
droxyethyl)-N-methylhexadecan-1-aminium chloride, 4’. Reaction
was performed from 4.2 mmol of 4’’. After 16 h of reaction, solvents
were removed under vacuum to form yellow dense oil which was
purified by chromatography on triethylamine pretreated silica gel with
CH2Cl2/MeOH with 0.1% of NEt3. Desired material was collected at
3% MeOH. It was found to contain triethylammonium salts that were
removed after a short DCM extraction from 2 M NaOH followed by
brine wash. Organic fractions were combined, dried over MgSO4,
filtered and the solvent was evaporated under reduced pressure (40
°C). An orange semisolid was isolated (24%). HRMS (ESI-QTOF)
m/z: [M]+ calcd for C42H64NO4 646.4830; found 646.4845. 1H NMR
(500 MHz, CDCl3): δ (ppm) = 7.37−7.36 (m, 2H), 7.33−7.22 (m,
7H), 6.86 (d, J = 9 Hz, 4H), 4.23 (br, 1H), 4.09 (br, 2H), 3.88−3.77
(m, 8H), 3.63−3.52 (m, 4H), 3.35−3.32 (m, 2H), 3.20 (s, 3H),
1.70−1.63 (m, 2H), 1.31−1.19 (m, 26H), 0.88 (t, J = 7 Hz, 3H). 13C
NMR (126 MHz, CDCl3): δ (ppm) = 159.0, 143.6, 134.5, 129.9,
128.5, 127.8, 127.5, 113.8, 88.0, 64.5, 64.3, 62.5, 57.3, 55.8, 55.5, 50.8,
32.1, 29.8, 29.8, 29.8, 29.6, 29.5, 29.4, 26.3, 22.9, 22.8, 14.3.
N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-N-(2-
hydroxyethyl)tetradecanamide, 3′. Reaction was performed from
3.63 mmol of 3. At the end of the reaction, product was extracted
twice with DCM from a 10%Na2CO3 aqueous solution, washed once
with a 10%Na2CO3 aqueous solution, dried over MgSO4, and filtered,
and solvent was evaporated under reduced pressure (40 °C). The
crude product was loaded on Celite and purified using the combiFlash
system with a 40 g SiO2 “gold” column. The solvents used were
hexanes/TEA (10:1) and ethyl acetate in a gradient from 0 to 30%
EtOAc in ∼17 CV. A pale yellow oil was isolated (843 mg, 38%).
LRMS: Calc.exact mass: 617.41 g/mol. Measured (positive mode):
N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-N-(2-(((2-
cyanoethoxy)(diisopropylamino)phosphanyl)oxy)ethyl)-N-methyl-
hexadecan-1-aminium chloride, 4. Reaction was performed from
0.57 mmol of 4′. Purification was achieved with a 24g neutral Al2O3
column. The solvents used were DCM and methanol in a gradient
from 0 to 2% MeOH in ∼10 CV. A clear transparent oil was isolated
(207 mg, 241%). NB: silica-based chromatography was tried many
times without success. HRMS (ESI-QTOF) m/z: [M]+ calcd for
C51H81N3O5P 846.5908; found 846.5902. 1H NMR (500 MHz,
CDCl3): δ (ppm) = 7.37−7.36 (m, 2H), 7.32−7.23 (m, 7H), 6.86 (d,
J = 9 Hz, 4H), 4.14−4.05 (m, 2H), 4.01−3.85 (m, 5H), 3.80 (s, 6H),
3.76−3.70 (m, 1H), 3.62 (br, 2H), 3.58−3.52 (m, 2H), 3.46−3.39
(m, 2H), 3.26 (s, 3H), 2.67−2.64 (m, 2H), 1.68 (br, 2H), 1.31−1.22
(m, 26H), 1.17 (dd, J = 7, 13 Hz, 12H), 0.88 (t, J = 7 Hz, 3H). 13C
NMR (126 MHz, CDCl3): δ (ppm) = 159.0, 143.7, 134.6, 130.0,
128.3, 127.8, 127.4, 117.8, 113.7, 87.9, 63.5, 63.3, 63.1, 62.3, 62.2,
58.5, 58.3, 58.2, 57.6, 55.4, 49.7, 43.5, 43.4, 32.0, 29.8, 29.8, 29.7,
29.6, 29.6, 29.5, 29.4, 26.4, 24.8, 24.8, 24.7, 24.7, 23.0, 22.8, 20.6,
20.6, 14.2. 31P NMR (203 MHz, CDCl3): δ (ppm) = 149.9, 149.9.
6,6′-(Hexadecylazanediyl)bis(hexan-1-ol), 5′′. To a solution of
hexadecylamine (121 mg, 0.5 mmol) in acetonitrile were added
KHCO3 (200 mg, 2 mmol, 3 equiv), KI (17 mg, 0.1 mmol, 0.2 equiv),
and 6-bromo-1-hexanol (0.131 mL, 1.0 mmol, 2 equiv) while stirring.
Temperature was raised until a reflux of acetonitrile was reached and
left stirring overnight. The reaction was followed by TLC. After
completion, the reaction was worked up with saturated NaHCO3
solution and DCM. The organic layer was collected, dried over
MgSO4, and filtered, and solvent was evaporated under reduced
pressure (40 °C). 197 mg of a yellow oil were obtained (89%).
Compound obtained was found to be pure up to 90%(NMR) and was
1
640.2 (M + 23), 303.3 (DMT+). H NMR (500 MHz, CDCl3): δ
(ppm) = 7.39−7.37 (m, 2H), 7.30−7.21 (m, 7H), 6.83 (d, J = 9 Hz,
4H), 3.79 (s, 6H), 3.69 (q, J = 5 Hz, 2H), 3.54−3.46 (m, 10H), 3.26
(t, J = 6 Hz, 2H), 2.40 (t, J = 8 Hz, 2H), 1.65−1.58 (m, 2H), 1.30−
1.25 (m, 20H), 0.88 (t, J = 7 Hz, 3H). 13C NMR (126 MHz, CDCl3):
δ (ppm) = 176.2, 158.8, 135.8, 130.1, 128.2, 128.1, 127.1, 113.3,
113.3, 86.9, 62.9, 61.7, 55.4, 50.6, 49.4, 33.5, 32.1, 29.8, 29.7, 29.7,
29.6, 29.5, 25.4, 22.8, 14.3.
2-(N-(2-(Bis(4-methoxyphenyl)(phenyl)methoxy)ethyl)-
tetradecanamido)ethyl(2-cyanoethyl)diisopropylphosphoramidite,
3. Reaction was performed from 0.64 mmol of 3′. Purification was
achieved on a 12 g SiO2 “gold” column. The solvents used were
hexanes/TEA (10:1) and ethyl acetate in a gradient from 0 to 20%
EtOAc in ∼10 CV. A clear transparent oil was isolated (366 mg,
70%). HRMS (ESI-QTOF) m/z: [M
+
Na]+ calcd for
1
C48H72N3O6PNa 840.5051; found 840.5040. H NMR (500 MHz,
CDCl3): δ (ppm) = 7.39−7.37 (m, 2H), 7.29−7.19 (m, 7H), 6.83−
6.79 (m, 4H), 3.85−3.68 (m, 11H), 3.60−3.52 (m, 5H), 3.26 (t, J = 5
Hz, 1H), 3.22 (t, J = 5 Hz, 1H), 2.59 (t, J = 6 Hz, 2H), 2.37−2.33 (m,
2H), 1.63−1.57 (m, 2H), 1.32−1.23 (m, 20H), 1.17 (ddd, J = 7, 9, 12
Hz, 12H), 0.88 (t, J = 7 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ
(ppm) = 173.7, 173.5, 158.7, 158.6, 145.1, 144.8, 136.3, 136.0, 135.9,
130.1, 128.2, 128.2, 128.0, 127.9, 127.0, 126.9, 117.7, 117.7, 113.3,
113.2, 86.8, 86.7, 62.6, 61.8, 61.7, 58.6, 58.5, 58.4, 58.4, 55.4, 55.3,
53.6, 49.1, 47.3, 46.3, 43.3, 43.2, 43.1, 33.5, 33.4, 32.1, 29.8, 29.8,
29.7, 29.5, 25.6, 25.5, 24.8, 24.8, 24.7, 24.7, 22.8, 22.7, 20.5, 20.5,
14.3. 31P NMR (203 MHz, CDCl3): δ (ppm) = 148.4, 147.9.
N,N-Bis(2-hydroxyethyl)-N-methylhexadecan-1-aminium iodide,
4′′. Anhydrous acetonitrile (15 mL) was added to RBF charged with
1′′ (1.5 g, 4.55 mmol) and heated to 50 °C until material dissolved.
CH3I (840 mg, 5.91 mmol) was added slowly, and the reaction
mixture was stirred at this temperature for another 1 h. Hot reaction
mixture was added to EtOAc (100 mL) at room temperature and left
to cool down. Shiny off-white crystalline solid was filtered off and
dried to obtain the pure product: 1.94g (91%). Synthesis of this
compound has been reported elsewhere.60 1H NMR (600 MHz,
CDCl3) δ (ppm) 0.85−0.88 (m, 3H), 1.24(br. s, 22H), 1.35 (br. s,
4H), 1.76 (br. s, 2H), 3.33 (br. s, 3H), 3.53−3.56(m, 2H), 3.72−3.80
(m, 4H), 4.07 (br. s, 2H), 4.14 (br. s, 4H). 13C NMR (125 MHz,
CDCl3) δ 14.1 (CH3), 22.6−31.9 (aliphatic CH2), 50.8 (CH3−N),
55.7 (2 × CH2−N), 64.0 (2 × CH2OH), 64.5 (CH2−N).
1
used as is for the next step. H NMR (500 MHz, CDCl3): δ (ppm)
3.67 (m, 4H), 2.40 (m, 5H), 1.63−1.23 (m, 45H), 0.91 (t, J = 5 Hz,
3H).
6-((6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)(hexadecyl)-
amino)hexan-1-ol, 5′. Reaction was performed from 1.54 mmol of
5′′. Product was extracted twice with DCM from a 10%Na2CO3
aqueous solution, washed once with a 10%Na2CO3 aqueous solution,
dried over MgSO4, and filtered, and solvent was evaporated under
reduced pressure (40 °C). The crude product was loaded on Celite
and purified using the combiFlash system with a 4 g SiO2 gold
column. Hexanes/TEA (10:1) and ethyl acetate were used in a
gradient from 0 to 50% EtOAc. A clear yellow oil was isolated (204
mg, 18%). LRMS: Calcd exact mass: 743.6 g/mol. Measured (positive
mode): 744.4 (M + 1), 303.3 (DMT+). 1H NMR (400 MHz,
CDCl3): δ (ppm) 7.45 (d, J = 7 Hz, 2H), 7.35−7.29 (m, 6H), 7.23 (t,
J = 7 Hz, 1H), 6.85 (d, J = 9 Hz, 4H), 3.65 (t, J = 4 Hz, 2H), 3.05 (t, J
= 4 Hz, 2H), 2.43−2.35 (m, 6H), 1.67−1.55 (m, 7H), 1.49−1.22 (m,
41H), 0.91 (t, J = 4 Hz, 3H).
6-((6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)(hexadecyl)-
amino)hexyl(2-cyanoethyl) diisopropylphosphoramidite, 5. Reac-
tion was performed from 0.20 mmol of 5′. Purification was achieved
H
DOI: 10.1021/acs.joc.8b01184
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The Journal of Organic Chemistry
Article
with a 12 g SiO2 “gold” column. The solvents used were hexanes/TEA
(10:1) and ethyl acetate in a gradient from 0 to 10% EtOAc in ∼10
CV. A clear transparent oil was isolated (121 mg, 64%). HRMS (ESI-
QTOF) m/z: [M + H]+ calcd for C58H95O5N3P 944.7004; found
glucopyranoside tetraacetate (379 mg, 0.80 mmol) and platform 1
(300 mg, 0.80 mmol, 1 equiv) were suspended in 1 mL CHCl3, and a
tBuOH/water (1:1) mixture (12 mL) was added. Freshly prepared
solution of sodium ascorbate (12.1 mg, 0.061 mmol, 0.2 equiv) in 0.2
mL of water and another of copper sulfate (7.6 mg, 0.030 mmol, 0.1
equiv) in 0.1 mL of water were sequentially added. Reaction was
allowed to stir overnight at room temperature. Product was extracted
twice with DCM from 10% Na2CO3 aqueous solution, washed once
with a 10% Na2CO3 aqueous solution, dried over MgSO4, and filtered,
and solvent was evaporated under reduced pressure (40 °C). The
crude obtained was resuspended in toluene to evaporate remaining
tBuOH. 643 mg of a pale golden powder was obtained (95%). LRMS:
Calcd exact mass: 846.4 g/mol. Measured (positive mode): 869.3 (M
1
944.6990. H NMR (500 MHz, CDCl3): δ (ppm) = 7.43−7.44 (m,
2H), 7.33−7.26 (m, 6H), 7.21−7.8 (m, 1H), 6.82 (d, J = 9 Hz, 4H),
3.87−3.76 (m, 8H), 3.69−3.54 (m, 4H), 3.03 (t, J = 7 Hz, 2H), 2.62
(t, J = 7 Hz, 2H), 2.39−2.35 (m, 6H), 1.61 (quin., J = 7 Hz, 2H),
1.45−1.34 (m, 10H), 1.31−1.22 (m, 32H), 1.18 (t, J = 6 Hz, 12H),
0.88 (t, J = 7 Hz, 3H). 13C NMR (126 MHz, CDCl3): δ (ppm) =
158.4, 145.6, 136.9, 130.1, 128.3, 127.8, 126.6, 117.8, 113.0, 85.7,
63.9, 63.7, 63.6, 58.5, 58.3, 55.3, 54.4, 54.3, 54.3, 43.1, 43.0, 32.0,
31.4, 30.3, 29.8, 29.8, 29.5, 27.8, 27.7, 27.5, 27.2, 27.1, 26.5, 26.1,
24.8, 24.7, 24.7, 24.7, 22.8, 20.5, 20.4, 14.2. 31P NMR (203 MHz,
CDCl3): δ (ppm) = 147.3.
1
+ 23), 303.3 (DMT+). H NMR (500 MHz, CDCl3): δ (ppm) 7.69
(s, 1H), 7.41 (d, J = 7 Hz, 2H), 7.32−7.26 (m, 6H), 7.21−7.19 (m,
1H), 6.82 (d, J = 9 Hz, 4H), 5.83−5.79 (m, 1H), 5.43−5.37 (m, 2H),
5.28−5.22 (m, 1H), 4.30 (dd, J = 5, 13 Hz, 1H), 4.14 (dd, J = 2, 13
Hz, 1H), 3.99−3.95 (m, 1H), 3.82−3.75 (m, 2H), 3.79 (s, 6H), 3.71
(t, J = 5 Hz, 2H), 3.09 (t, J = 6 Hz, 2H), 2.68−2.60 (m, 2H), 2.60−
2.51 (m, 2H), 2.07 (s, 6H), 2.03 (s, 3H), 1.83 (quin, J = 6 Hz, 2H),
1.79 (s, 3H), 1.75−1.63 (m, 2H). 13C NMR (126 MHz, CDCl3): δ
(ppm) 170.7, 170.0, 169.5, 169.0, 158.5, 145.6, 145.4, 136.6, 130.2,
128.3, 127.9, 126.8, 121.1, 113.2, 86.0, 75.4, 72.7, 70.5, 67.9, 64.0,
61.6, 55.3, 53.6, 51.1, 48.7, 28.3, 27.8, 20.8, 20.7, 20.7, 20.2.
3,3′-(Prop-2-yn-1-ylazanediyl)bis(propan-1-ol), PT1a. To a sol-
ution of propargylamine (551 mg, 10.0 mmol) in acetonitrile were
added KHCO3 (3.00g, 30.0 mmol, 3 equiv), KI (166 mg, 1 mmol, 0.1
equiv), and 3-bromo-1-propanol (1.82 mL, 20.00 mmol, 2 equiv)
while stirring. Temperature was raised until a reflux of acetonitrile was
reached and left stirring overnight. The reaction was followed by
TLC. After completion, solid was filtered out, and solvent was
evaporated under reduced pressure (40 °C). Crude obtained was
resuspended in DCM, filtered, dried over MgSO4, and filtered, and
solvent was evaporated under reduced pressure (40 °C). 1.68 g of a
dark brown red oil were obtained (98%). Compound obtained was
found to be pure up to 90% (NMR) and was used as is for the next
step. NB: in order to get good characterization data, some compound
could be further purified using the CombiFlash with a SiO2 gold
column, (80:20:2 DCM:EtOH:NH4OH). 1H NMR (500 MHz,
CDCl3): δ (ppm) 3.76 (t, J = 6 Hz, 4H), 3.50 (d, J = 2 Hz, 2H), 2.76
(t, J = 6 Hz, 4H), 2.23 (t, J = 2.4 Hz, 1H), 1.79−1.69 (m, 4H).
3-((3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)(prop-2-yn-
1-yl)amino)propan-1-ol, PT1. Reaction was performed from 9.8
mmol of PT1a. Product was extracted twice with DCM from a 10%
Na2CO3 aqueous solution, washed once with a 10%Na2CO3 aqueous
solution, dried over MgSO4, and filtered, and solvent was evaporated
under reduced pressure (40 °C). The crude product was loaded on
Celite and purified using the combiFlash system with a 80 g SiO2 gold
column. Hexanes/TEA (100:1) and ethyl acetate were used in a
gradient from 0 to 50% EtOAc. A clear greenish oil was isolated (1.77
g, 38%). HRMS (ESI-QTOF) m/z: [M + Na]+ calcd for
C30H35O4NNa 496.2458; found 496.2448. 1H NMR (500 MHz,
CDCl3): δ (ppm) 7.45 (d, J = 7 Hz, 2H), 7.35−7.29 (m, 6H), 7.23 (t,
J = 7 Hz, 1H), 6.85 (d, J = 9 Hz, 4H), 4.33 (br, 1H), 3.82 (s, 6H),
3.77 (t, J = 5 Hz, 2H), 3.47 (s, 2H), 3.13 (t, J = 6 Hz, 2H), 2.75 (t, J =
6 Hz, 2H), 2.64 (t, J = 7 Hz, 2H), 2.23 (s, 1H), 1.80 (quin, J = 7 Hz,
2H), 1.69 (quin, J = 5 Hz, 2H). 13C NMR (126 MHz, CDCl3): δ
(ppm) 158.4, 145.2, 136.5, 130.0, 128.2, 127.7, 126.6, 113.0, 85.9,
77.9, 73.3, 64.2, 61.5, 55.2, 53.6, 51.1, 41.7, 28.2, 28.0.
(2S,3R,4R,5S,6S)-2-(Acetoxymethyl)-6-(4-(((3-(bis(4-
methoxyphenyl)(phenyl)methoxy)propyl)(3-(((2-cyanoethoxy)-
(diisopropylamino)phosphanyl)oxy)propyl)amino)methyl)-1H-
1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-triyl triacetate, 7. Re-
action was performed from 0.22 mmol of 7′. Purification was achieved
with a 12 g SiO2 “gold” column. Hexanes/TEA (10:1) and ethyl
acetate were used in a gradient from 0 to 50% EtOAc in ∼10 CV. A
white crystalline powder was isolated (162 mg, 70%). HRMS (ESI-
QTOF) m/z: [M + H]+ calcd for C53H72N6O14P 1047.4839; found
1047.4866. 1H NMR (500 MHz, CDCl3): δ (ppm) 7.60 (s, 1H), 7.42
(d, J = 8 Hz, 2H), 7.32−7.26 (m, 6H), 7.22−7.18 (m, 1H), 6.82 (d, J
= 9 Hz, 4H), 5.82−5.77 (m, 1H), 5.44−5.37 (m, 2H), 5.27−5.23 (m,
1H), 4.30 (dd, J = 5, 13 Hz, 1H), 4.12 (dd, J = 2, 13 Hz, 1H), 3.99−
3.95 (m, 1H), 3.85−3.72 (m, 10H), 3.70−3.63 (m, 1H), 3.62−6.54
(m, 2H), 3.08 (t, J = 6 Hz, 2H), 2.59 (t, J = 7 Hz, 2H), 2.53−2.48 (m,
4H), 2.07 (s, 3H), 2.06 (s, 3H), 2.03 (s, 3H), 1.82−1.73 (m, 7H),
1.16 (dd, J = 7, 10 Hz, 12H). 13C NMR (126 MHz, CDCl3): δ (ppm)
170.7, 170.1, 169.5, 168.8, 158.5, 146.3, 145.5, 136.7, 130.2, 128.3,
127.9, 126.7, 120.9, 117.9, 113.1, 85.9, 85.9, 75.3, 72.9, 70.4, 67.9,
62.1, 62.0, 61.7, 58.5, 58.4, 55.4, 50.8, 50.4, 48.7, 43.2, 43.1, 29.1,
28.0, 24.8, 24.8, 24.8, 24.7, 20.8, 20.7, 20.7, 20.5, 20.5, 20.2. 31P NMR
(203 MHz, CDCl3): δ (ppm) 147.3, 147.3 (two diasteroisomers).
tert-Butyl Bis(3-hydroxypropyl)glycinate, PT2a. To a solution of
glycine t-butyl ester hydrochloride (2.57g, 15.3 mmol) in acetonitrile
were added KHCO3 (4.60g, 45.9 mmol, 3 equiv), KI (254 mg, 1.53
mmol, 0.1 equiv), and 3-bromo-1-propanol (2.77 mL, 30.6 mmol, 2
equiv) while stirring. Temperature was raised until a reflux of
acetonitrile was reached and left stirring overnight. The reaction was
followed by TLC. After completion, solvent was evaporated under
reduced pressure (40 °C). Product was extracted twice with ethyl
acetate from 10% Na2CO3 aqueous solution, washed once with a 10%
Na2CO3 aqueous solution, dried over MgSO4, and filtered, and
solvent was evaporated under reduced pressure (40 °C). Compound
obtained was a pale yellow oil found to be pure up to 90% (NMR)
and was used as is for the next step (3.56g, 94%). LRMS calcd exact
mass: 247.2 g/mol. Measured (positive mode):248.1 (M + 1), 270.1
(M + 23). 1H NMR (500 MHz, CDCl3): δ (ppm) 3.77 (t, J = 6 Hz,
4H), 3.18 (s, 2H), 2.36 (t, J = 6 Hz, 4H), 1.72 (quin., J = 6 Hz, 4H),
1.47 (s, 9H).
3-((3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)(prop-2-yn-
1-yl)amino)propyl(2-cyanoethyl)diisopropylphosphoramidite, 6.
Reaction was performed from 0.48 mmol of PT1 and purification
was achieved with a 12 g SiO2 gold column. Hexanes/TEA (10:1) and
ethyl acetate were used in a gradient from 0 to 40% EtOAc in ∼10
CV. A clear transparent oil was isolated (249 mg, 70%). HRMS (ESI-
QTOF) m/z: [M + K]+ calcd for C39H52N3O5PK 712.3276; found
1
712.3296. H NMR (500 MHz, CDCl3): δ (ppm) 7.43 (d, J = 8 Hz,
2H), 7.33−7.26 (m, 6H), 7.20 (t, J = 9 Hz, 1H), 6.82 (d, J = 9 Hz,
4H), 3.87−3.74 (m, 2H), 3.79 (s, 6H), 3.71−3.55 (m, 4H), 3.37 (d, J
= 2 Hz, 2H), 3.09 (t, J = 6 Hz, 2H), 2.61−2.54 (m, 6H), 2.14 (t, J = 2
Hz, 1H), 1.74 (sex, J = 7 Hz, 4H), 1.17 (dd, J = 7, 9 Hz, 12H). 13C
NMR (126 MHz, CDCl3): δ (ppm) 158.3, 145.3, 136.6, 130.0, 128.2,
127.7, 126.6, 113.0, 85.9, 78.7, 72.7, 61.9, 61.8, 61.6, 58.4, 58.2, 55.2,
50.8, 50.2, 43.1, 43.0, 41.9, 29.3, 29.3, 28.3, 24.7, 24.6, 24.6, 20.4,
20.3. 31P NMR (203 MHz, CDCl3): δ (ppm) 147.5.
(2S,3R,4R,5S,6S)-2-(Acetoxymethyl)-6-(4-(((3-(bis(4-
methoxyphenyl)(phenyl)methoxy)propyl)(3-hydroxypropyl)-
amino)methyl)-1H-1,2,3-triazol-1-yl)tetrahydro-2H-pyran-3,4,5-
triyl triacetate, 7′. Adapted from ref 61. 1-Azido-1-deoxy-β-D-
tert-Butyl N-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-
N-(3-hydroxypropyl)glycinate, PT2b. Reaction was performed from
14.4 mmol of PT2a. Product was extracted twice with DCM from a
10% Na2CO3 aqueous solution, washed once with a 10% Na2CO3
aqueous solution, dried over MgSO4, and filtered, and solvent was
evaporated under reduced pressure (40 °C). The crude product was
loaded on Celite and purified using the combiFlash system with a 220
I
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The Journal of Organic Chemistry
Article
g SiO2 gold column. Hexanes/TEA (10:1) and ethyl acetate were
used in a gradient from 0 to 35% EtOAc (∼25CV). A clear yellow oil
was isolated (2.96 g, 37%). HRMS (ESI-QTOF) m/z: [M + Na]+
mg, 68%). HRMS (ESI-QTOF) m/z: [M + Na]+ calcd for
C48H63N4O8PNa 877.4276; found 877.4257. H NMR (500 MHz,
1
CDCl3): δ (ppm) 7.64 (d, J = 8 Hz, 1H), 7.40 (d, J = 7 Hz, 2H),
7.31−7.15 (m, 10H), 7.07 (d, J = 7 Hz, 2H), 6.82 (d, J = 9 Hz, 4H),
4.83 (q, J = 8 Hz, 1H), 3.84−3.76 (m, 8H), 3.68 (s, 3H), 3.60−3.50
(m, 2H), 3.16−3.12 (m, 1H), 3.05−2.97 (m, 5H), 2.58 (td, J = 2, 7
Hz, 2H) 2.55−2.47 (m, 4H), 1.67−1.59 (m, 4H), 1.17 (dd, J = 7, 9
Hz, 12H). 13C NMR (126 MHz, CDCl3): δ (ppm) 171.9, 11.5, 158.2,
145.3, 136.6, 136.2, 130.1, 129.3, 128.7, 128.3, 127.9, 127.2, 126.8,
113.2, 86.0, 61.5, 58.5, 55.3, 53.0, 52.7, 52.5, 52.3, 43.2, 43.1, 38.0,
29.3, 28.2, 24.8, 24.7, 20.5, 20.5. 31P NMR (203 MHz, CDCl3): δ
(ppm) 147.6.
1
calcd for C33H43NO6Na 572.2983; found 572.2978. H NMR (500
MHz, CDCl3): δ (ppm) 7.42 (d, J = 7 Hz, 2H), 7.32−7.26 (m, 6H),
7.21−7.18 (m 1H), 6.82 (d, J = 9 Hz, 4H), 4.54 (br, 1H), 3.79 (s,
6H), 3.74 (t, J = 5 Hz, 2H), 3.19 (s, 2H), 3.09 (t, J = 6 Hz, 2H), 2.70
(t, J = 6 Hz, 2H), 2.64−2.61 (m, 2H), 1.79 (quin, J = 8 Hz, 2H), 1.65
(quin, J = 5 Hz, 2H), 1.46 (s, 9H). 13C NMR (126 MHz, CDCl3): δ
(ppm) 170.7, 158.5, 145.3, 136.6, 130.1, 128.3, 127.9, 126.8, 113.2,
86.0, 81.5, 63.5, 61.8, 56.6, 55.3, 54.0, 51.8, 28.6, 28.2, 27.9.
Sodium N-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-N-
(3-hydroxypropyl)glycinate, PT2. Compound PT2b (260 mg, 0.47
mmol) was dissolved in about 2 mL of methanol. To the mixture was
added 25 mL of 0.4 M NaOH in MeOH/water 4:1. The reaction
mixture was left stirring for 3 h at 65 °C. Reaction was monitored by
TLC. When higher mobility spot disappeared, methanol was
evaporated under reduced pressure (60 °C) until a precipitate
appears but a small amount of water remains. Precipitate was filtered
and quickly washed four times with cold water. The solid was
suspended in MeOH, and solvent was evaporated under reduced
pressure (40 °C). The obtained solid was resuspended in DCM and
dried over MgSO4, solution was filtered, and solvent was evaporated
under reduced pressure (40 °C) to obtain a white to pale yellow solid
(206 mg, 85%). NB: an alternate protocol involved a workup with 2
equiv of tetrabutylammonium chloride and sodium carbonate
solutions. The obtained tetrabutylammonium salt started to degrade
after a few days under high vacuum. HRMS (ESI-QTOF) m/z: [M]−
calcd for C29H34O6N 492.2392; found 492.2405. 1H NMR (400
MHz, DMSO-d6): δ (ppm) 7.36−7.28 (m, 4H), 7.24−7.19 (m, 5H),
6.88 (d, J = 9 Hz, 4H), 3.73 (s, 6H), 3.37 (t, J = 6 Hz, 2H), 2.96 (t, J
= 6 Hz, 2H), 2.71 (s, 2H), 2.43−2.37 (m, 4H), 1.61 (quin, J = 7 Hz,
2H), 1.38 (quin, J = 6 Hz, 2H). 13C NMR (126 MHz, DMSO-d6): δ
(ppm) 174.4, 157.9, 145.3, 136.1, 129.6, 127.8, 127.6, 126.5, 113.1,
85.2, 61.6, 59.3, 58.2, 55.0, 50.5, 50.1, 29.5, 27.3.
Methyl N-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-N-
(3-hydroxypropyl)glycyl-L-phenylalaninate, 8′. To a solution of
PT2 (206 mg, 0.40 mmol) in DMF (5 mL) were successively added
anhydrous 1-hydroxybenzotriazole (HOBt) (81 mg, 0.60 mmol, 1.5
equiv) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydro-
chloride (EDC-Cl) (115 mg, 0.60 mmol, 1.5 equiv). The pale yellow
solution was left stirring for 5 min until complete dissolution of EDC-
Cl. Phenylalanine methyl ester hydrochloride (95 mg, 0.44 mmol, 1.1
equiv) and triethylamine (0.15 mL, 1.04 mmol, 2.6 equiv) were then
successively added. The reaction mixture slowly turned cloudy and
was left under vigorous stirring overnight. Solvent was then
evaporated under reduced pressure (60 °C). The crude product
was loaded on Celite and purified using the combiFlash system with a
12 g SiO2 “gold” column. Hexanes/TEA (10:1) and ethyl acetate
were used in a gradient from 0 to 70% EtOAc in ∼12 CV. A clear pale
yellow oil was isolated (173 mg, 66%). NB: Interestingly, when the
crude in DMF is analyzed using RP-HPLC, conversion yields reached
90%, but isolated yields never exceeded 66%. This may be due to
issues with column chromatography or to the presence of undesired
salts at the end of the previous step. LRMS: Calcd exact mass: 654.33
g/mol. Measured (positive mode): 303.3 (DMT+), 677.3 (M + 23).
1H NMR (500 MHz, CDCl3): δ (ppm) 7.60 (d, J = 8 Hz, 1H), 7.41
6,6′-(Naphthalene-2,7-diylbis(oxy))bis(hexan-1-ol), 9′′. An oven-
dried round-bottom flask (RBF) was charged with 2,7-dihydrox-
ynaphthalene (2 g, 12.5 mmol), K2CO3 (5.18 g, 37.5 mmol), and
Bu4NI (461 mg, 1.25 mmol), atmosphere exchanged to argon, and
anhydrous DMF (20 mL) was added followed by 6-bromohexanol
(5.65g, 31.25 mmol). Reaction mixture was stirred at room
temperature for 48 h until TLC analysis confirmed consumption of
starting material, quenched with water (100 mL), and product
extracted with CH2Cl2 (3 × 50 mL), dried over MgSO4, and solvent
evaporated under vacuum. Crude product was purified by
chromatography with n-hexane/EtOAc eluent, collecting desired
material at 40−60% of EtOAc. Product was isolated as a white
solid: 2.2 g, 64%. Synthesis of this compound has been reported
elsewhere.62 1H NMR (400 MHz, CDCl3) δ 1.23 (t, J = 5.3 Hz, 2H),
1.44−1.90 (m, 16H), 3.68 (dt, J = 6.3, 5.4 Hz, 4H), 4.07 (t, J = 6.5
Hz, 4H), 6.98 (dd, J = 8.8, 2.4 Hz, 2H), 7.03 (d, J = 2.3 Hz, 2H), 7.64
(d, J = 8.8 Hz, 2H). 13C NMR (100 MHz, CDCl3) δ (25.5, CH2),
25.9 (CH2), 29.2 (CH2), 32.6 (CH2), 62.9 (CH2OH), 67.8 (CH2O),
106.0 (2 × CH), 116.2 (2 × CH), 124.1 (2 × C), 129.0 (2 × CH),
135.9 (2 × C), 157.6 (2 × C−O).
6-(7-(6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexyloxy)-
naphthalen-2-yloxy)hexan-1-ol, 9′. 9′′ (2.0 g, 5.55 mmol) was
dissolved in anhydrous CH2Cl2 (50 mL). Freshly distilled triethyl-
amine (2.5 mL) was added, followed by DMTCl (2.1 g, 5.55 mmol)
added portionwise over 30 min. Reaction mixture was stirred at room
temperature for 16 h. Solvents were removed under vacuum to form
yellow dense oil which was purified by chromatography on
triethylamine pretreated silica gel with n-hexane/EtOAc with 1% of
NEt3. Desired material was collected at 40−50% EtOAc/n-hexane as
very viscous yellow oil: 1.1 g (30%). HRMS (ESI-QTOF) m/z: [M +
1
K]+ calcd for C43H50O6K 701.3239; found 701.3254. H NMR (400
MHz, acetone-d6) δ 1.42−1.87 (m, 16H), 3.08 (t, J = 6.4 Hz, 2H),
3.42 (t, J = 5.2 Hz, 1H), 3.54−3.59 (m, 4H), 3.76 (s, 6H), 4.05−4.09
(m, 4H). 6.86 (d, J = 8.9 Hz, 4H), 6.96 (ddd, J = 8.6. 6.0, 2.4 Hz,
2H), 7.16−7.35 (m, 9H), 7.47 (d, J = 7.5 Hz, 2H), 7.68 (dd, J = 8.9,
1.2 Hz, 2H). 13C NMR (100 MHz, acetone-d6) δ 26.5−33.7 (8 ×
CH2), 55.5 (2 × CH3O), 62.4 (CH2O), 63.8 (CH2OH), 68.4
(CH2O), 68.5 (CH2O), 86.5 (C), 106.9 (2 × CH), 113.8 (4 × CH),
117.0 (2 × CH), 125.1 (2 × C), 127.4 (CH), 128.5 (2 × CH), 129.0
(2 × CH), 129.8 (2 × CH),130.8 (4 × CH), 137.3 (2 × C), 137.4 (2
× C),146.6 (C), 158.6 (2 × C−O), 159.5 (2 × C−O).
6-((7-((6-(Bis(4-methoxyphenyl)(phenyl)methoxy)hexyl)oxy)-
naphthalen-2-yl)oxy)hexyl (2-cyanoethyl) diisopropylphosphora-
midite, 9. Monoprotected diol 9′ was suspended in toluene, and
solvent was evaporated under reduced pressure (60 °C). The
operation was repeated once, and the dried compound was kept
under high vacuum for at least 5 h. In the glovebox, 416 mg of 9′
(0.63 mmol) was then dissolved in anhydrous THF (1 mL), and 0.23
mL of ETT activator, 0.25 M in dry acetonitrile, (0.94 mmol, 1.5
equiv, 4.8 mL) was added to the reaction medium while stirring. 2-
Cyanoethyl N,N,N′,N′-tetraisopropyl phosphoramidite (0.50 mL,
1.57 mmol, 2.5 equiv) was added slowly, and the reaction was
allowed to stir under inert gas at room temperature overnight. Solvent
was evaporated under reduced pressure (40 °C). The crude product
was loaded on a 25 g SiO2 column and purified using a 90/10/1
hexanes/ethyl acetate/triethylamine mixture in ∼3 column volumes
(CV). A clear transparent oil was isolated (424 mg, 78%). NMR
purity >95%. HRMS (ESI-QTOF) m/z: [M + H]+ calcd for
(d, J = 7 Hz, 2H), 7.31−7.18 (m, 10H), 7.08 (d, J = 7 Hz, 2H), 6.83
(d, J = 9 Hz, 4H), 4.85 (q, J = 8 Hz, 1H), 3.79 (s, 6H), 3.71 (s, 3H),
3.53 (t, J = 6 Hz, 2H), 3.15−2.94 (m, 6H), 2.61−2.53 (m, 2H),
2.49−2.43 (m, 2H), 1.68−1.61 (m, 2H), 1.60−1.55 (m, 2H). 13C
NMR (126 MHz, CDCl3): δ (ppm) 172.5, 171.5, 158.5, 145.3, 136.6,
136.2, 130.1, 129.3, 128.7, 128.3, 127.9, 127.2, 126.8, 113.2, 86.0,
61.5, 60.8, 58.8, 55.3, 52.7, 525, 52.4, 52.0, 37.9, 30.2, 28.03.
Methyl N-(3-(Bis(4-methoxyphenyl)(phenyl)methoxy)propyl)-N-
(3-(((2-cyanoethoxy)(diisopropylamino)phosphanyl)oxy)propyl)-
glycyl-L-phenylalaninate, 8. Reaction was performed from 0.333 mol
of 8′. Purification was achieved with a 12 g SiO2 “gold” column.
Hexanes/TEA (10:1) and ethyl acetate were used in a gradient from 0
to 40% EtOAc in ∼12 CV. A clear transparent oil was isolated (195
J
DOI: 10.1021/acs.joc.8b01184
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The Journal of Organic Chemistry
Article
C52H68N2O7P 863.4759; found 863.4745. 1H NMR (500 MHz,
CDCl3): δ (ppm) 7.63 (dd, J = 2, 9 Hz, 2H), 7.45−7.43 (m, 2H),
7.33 (d, J = 9 Hz, 4H), 7.29−7.26 (m, 2H), 7.21−7.18 (m, 1H), 7.02
(s, 1H), 7.01 (s, 1H), 6.98−6.95 (m, 2H), 6.81 (d, J = 9 Hz, 4H),
4.04 (q, J = 7 Hz, 4H), 3.88−3.76 (m, 8H), 3.71−3.66 (m, 1H),
3.63−3.57 (m, 3H), 3.06 (t, J = 7 Hz, 2H), 2.62 (t, J = 7 Hz, 2H),
1.88−1.80 (m, 4H), 1.70−1.63 (m, 4H), 1.54−1.45 (m, 8H), 1.18
(dd, J = 3, 7 Hz, 12H). 13C NMR (126 MHz, CDCl3): δ (ppm)
158.4, 157.8, 157.7, 145.6, 139.8, 136.9, 136.1, 130.2, 129.2, 128.3,
127.8, 126.7, 124.3, 117.8, 116.4, 116.3, 113.1, 106.1, 85.8, 68.0, 67.9,
63.8, 63.6, 63.4, 58.5, 58.3, 55.3, 43.2, 43.1, 31.3, 31.3, 30.2, 29.4,
29.4, 26.3, 26.2, 26.0, 25.9, 24.8, 24.8, 24.7, 20.5, 20.5. 31P NMR (203
MHz, CDCl3): δ (ppm) 147.3.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: hanadi.sleiman@mcgill.ca.
ORCID
Hanadi F. Sleiman: 0000-0002-5100-0532
Notes
The authors declare the following competing financial
interest(s): A US Provisional Patent Application has been
filed for the tertiary amine-based phosphoramidites and their
precursors.
12-(Bis(4-methoxyphenyl)(phenyl)methoxy)dodecan-1-ol, 10′.
1,12-Dodecanediol (6g, 29.65 mmol) was dissolved in anhydrous
pyridine (30 mL), and DMTCl (10.04g, 29.65 mmol) was added in
small portions over 30 min. Reaction mixture was stirred for 2 h at
room temperature, and pyridine was removed in vacuo, leaving dark
orange dense oil. Crude material was purified by chromatography on
triethylamine pretreated silica gel with slow gradient of EtOAc/n-
hexane with 1% of NEt3. Target material was collected at 15−20%
EtOAc/Hexane, solvent evaporated, and product obtained as a pale
yellow very viscous oil (4.17g, 31%). Synthesis of this compound has
been reported elsewhere.63 HRMS (ESI-QTOF) m/z: [M + Na]+
calcd for C33H44O4Na 527.3132; found 527.3124. 1H NMR (400
MHz, acetone-d6) δ 1.35−1.42 (m, 16H), 1.48−1.55 (m, 2H), 1.58−
1.65 (m, 2H), 3.06 (t, J = 6.5 Hz, 2H), 3.42 (br. s, 1H), 3.53 (t, J =
6.5 Hz, 2H), 3.77 (s, 6H), 6.87 (d, J = 8.9 Hz, 4H), 7.18−7.22 (m,
1H), 7.25−7.37 (m, 6H), 7.45−7.48 (m, 2H). 13C NMR (100 MHz,
acetone-d6) δ 26.7−33.8 (multiple aliphatic CH2), 55.4 (2 × CH3O),
62.5 (CH2OC), 63.9 (CH2OH), 86.4 (C), 113.7 (4 × CH), 127.3
(CH), 128.4 (2 × CH), 128.9 (2 × CH), 130.8 (4 × CH), 137.4 (2 ×
C), 146.6 (C), 159.4 (2 × C).
ACKNOWLEDGMENTS
■
The authors thank NSERC, CIHR, FRQNT, the Canada
Research Chairs program, the Qatar National Research Fund
(QNRF, project number NPRP 5-1505-1-250) for support. D.
de Rochambeau thanks NSERC for a CREATE training
program in Bionanomachines (CTPB) scholarship. We thank
Dr. Alexander Wahba for his help with MS characterization.
REFERENCES
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12-(Bis(4-methoxyphenyl)(phenyl)methoxy)dodecyl(2-
cyanoethyl)diisopropylphosphoramidite, 10. Monoprotected diol
10′ was suspended in toluene, and solvent was evaporated under
reduced pressure (60 °C). The operation was repeated once, and the
dried compound was kept under high vacuum for at least 5 h. In a 50
mL oven-dried round-bottom flask, molecule 10′ (191 mg, 0.38
mmol, 1 equiv) is dissolved in 1.2 mL of THF. 5-(Ethylthiotriazole)
(ETT, 2.25 mL, 0.25 M in acetonitrile, 0.94 mmol, 1.5 equiv) is added
under vigorous stirring. 3-((Bis(diisopropylamino)phosphanyl)oxy)-
propanenitrile (210 μL, 0.84 mmol, 2.2 equiv) is added on the
reaction medium. The reaction was left stirring for 5 h. Solvent is
evaporated under reduced pressure. Molecule 10 is purified by
column chromatography. The mix of solvents used was hexanes/ethyl
acetate/TEA (90:10:2) and was degassed before use. We obtained
226.1 mg of a colorless oil. Yield: 85%. Synthesis of this compound
has been reported elsewhere.62 HRMS (ESI-QTOF) m/z: [M + H]+
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́
1
calcd for C42H62O5N2P 705.4391; found 705.4382. H NMR (500
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MHz, acetone-d6) δ 7.47−7.44 (m, 2H), 7.34−7.28 (m, 6H), 7.23−
7.19 (m, 1H), 6.89−6.86 (m, 4H), 3.89−3.80 (m, 2H), 3.78 (s, 6H),
3.70−3.59 (m, 4H), 3.05 (t, J = 7 Hz, 2H), 2.74 (t, J = 6 Hz, 2H),
1.64−1.58 (m, 4H), 1.41−1.36 (m, 4H), 1.35−1.28 (m, 12H), 1.19
(dd, J = 4, 7 Hz, 12H). 13C NMR (126 MHz, acetone-d6) δ 159.5,
146.7, 137.5, 130.9, 129.0, 128.5, 127.4, 113.8, 86.5, 64.4, 64.2, 63.9,
59.5, 59.3, 55.5, 43.8, 43.7, 32.1, 32.0, 30.8, 30.4, 30.3, 30.2, 27.1,
26.7, 24.9, 24.9, 20.8, 20.7. 31P NMR (203 MHz, acetone-d6): δ
(ppm) 147.0.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.joc.8b01184.
Supplementary synthetic schemes, NMR spectra, HPLC
chromatograms, MS spectra, detailed analytical proce-
dures (PDF)
K
DOI: 10.1021/acs.joc.8b01184
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