Solid-Phase Synthesis of Sequence-Defined Informational Oligomers

Article abstract of DOI:10.1021/acs.joc.0c01977

Genetic biopolymers utilize defined sequences and monomer-specific molecular recognition to store and transfer information. Synthetic polymers that mimic these attributes using reversible covalent chemistry for base-pairing pose unique synthetic challenge

Full text of DOI:10.1021/acs.joc.0c01977

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Solid-Phase Synthesis of Sequence-Defined Informational Oligomers
Kyle R. Strom and Jack W. Szostak*
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ABSTRACT: Genetic biopolymers utilize defined sequences and
monomer-specific molecular recognition to store and transfer
information. Synthetic polymers that mimic these attributes using
reversible covalent chemistry for base-pairing pose unique synthetic
challenges. Here, we describe a solid-phase synthesis methodology
for the efficient construction of ethynyl benzene oligomers with
specific sequences of aniline and benzaldehyde subunits. Handling
these oligomers is complicated by the fact that they often exhibit
multiple conformations because of intra- or intermolecular pairing.
We describe conditions that allow the dynamic behavior of these
oligomers to be controlled so that they may be manipulated and
characterized without needing to mask the recognition units with
protecting groups.
covalent ester linkages (Figure 1b).9 These demonstrations
build on several examples of templated polymerization in
which the length and structure of the daughter polymers are
INTRODUCTION
■
In genetic polymers, molecular recognition between comple-
mentary nucleotides is the chemical basis for self-replication
and self-assembly. In all known living things, purine and
pyrimidine nucleobases function as the recognition units in
base-pairing interactions and allow parent polymers to transfer
information to their daughter polymers.1 The canonical
nucleobases of DNA and RNA (A, G, C, T, and U), and
nearly all synthetic XNAs,2 utilize patterns of hydrogen-bond
donors and acceptors to give specificity to their base-pairing
interactions. Although inefficient, the demonstrated enzyme-
free copying of these biopolymers suggests that the
information transfer function is intrinsic to the polymer
structure and is not entirely dependent on the complex
biomachinery of living systems.3−6 Alternative synthetic
systems that use reversible covalent chemistry to form base
pairs could conceivably share the ability of biological genetic
polymers to store and propagate information and to self-
assemble into higher-order functional structures. Ultimately, it
may be possible to develop such synthetic polymers into
systems that can mimic the greatest aspect of natural
biopolymers, namely, the spontaneous development and
refinement of function through genetic evolution.7
Recently, we described a synthetic system capable of
information storage and self-replication; two dimers with
sequence-defined aniline (A) and benzaldehyde (B) subunits
capable of templating the synthesis of their sequence-
complementary daughters through reversible covalent imine
bonds (Figure 1a).8 Concurrent with our work, Hunter and co-
workers reported a trimer with sequence-defined phenol (P)
and benzoic acid (N) subunits capable of templating the
synthesis of its complementary trimer through reversible
controlled by the length of a parent template.10,11
A
particularly notable example is the templated ring-opening
metathesis polymerization of ester-linked cyclic olefins recently
reported by Zhou and Palermo.12 Although this work
demonstrates the exceptional control of polymerization
products that can be achieved using templates, sequence
information is not transferred in this system as the parent
oligomers are oligothiophene homopolymers and do not
encode sequence information.
To our knowledge, the examples reported by us and by
Hunter et al. are the first examples to demonstrate the transfer
of sequence information through reversible covalent chemistry.
These two systems are markedly distinct in their molecular
structures and in the chemistries they employ: for backbone
synthesis, Sonogashira coupling versus azide−alkyne cyclo-
addition, and for base pair formation, imine bonds versus ester
bonds. These differences may indicate the existence of an
expansive chemical space of genetic polymers that is distant
from the nucleotides of biology and that is nearly unexplored.
The study of sequence-defined polymers functionalized with
subunits amenable to reversible covalent interactions has been
limited by the lack of synthetic methods for their construction
Received: August 16, 2020
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functional groups necessary for sequence recognition has
limited the synthetic accessibility of such oligomers. The
synthesis of sequence-defined oligoarylacetylenes with similar
backbones to those described herein has been well-developed
by Moore and co-workers,15−17 and oligoarylacetylenes with a
slightly different backbone have been built by Lutz and co-
workers.18 Although the synthesis of these hetero-oligomers
represents an important synthetic milestone, these molecules
did not contain the reactive groups necessary to form dynamic
covalent bonds. The Moore group subsequently synthesized
arylacetylene homo-oligomers containing either aniline or
benzaldehyde functional groups and showed that when mixed,
these condensed to form molecular ladders through imine
bonds.19 These homo-oligomers avoid the complications
resulting from the simultaneous presence of coreactive
functional groups within one molecule, but as a consequence,
they do not store information. To our knowledge, no general
method for the synthesis and isolation of arbitrary sequence-
defined polymers bearing reversible covalent recognition units
capable of information storage and reversible bond formation
has been reported. Only single sequences9 and sequences with
protected recognition units have been described.20 Recently,
two heterotrimers bearing boronic acid and catechol
recognition units were described by Ng and co-workers,21
but the synthetic methods for the heteropolymer sequences
were not fully described, and the dynamic behavior of the
individual heteromeric sequences was not addressed.
Figure 1. Sequence information transfer using reversible covalent
chemistry. (a) Template copying of sequence-defined macrocycles
through transimination and linking of monomers. (b) Template
copying of a sequence-defined trimer through ester bond formation,
polymerization, and hydrolysis.
Methods to control and reverse the many potential
intramolecular and intermolecular base-pairing interactions of
these dynamic molecules have not been well-developed.
Sc(OTf)3 has been reported to catalyze transimination
reactions and to affect the equilibration and equilibrium of
aniline and benzaldehyde condensation and hydrolysis.22,23
However, this approach has only been shown to facilitate the
formation of duplexes between homopolymers20,24 and hetero-
and a poor understanding of how to control their dynamic
behavior so that they may be easily manipulated once in hand.
Although great strides have been made in developing methods
to build long sequence-defined informational polymers,13,14 the
complexity resulting from the presence of the coreactive
Figure 2. Solid-phase synthesis of sequence-defined ABOs.
B
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Scheme 1. Synthesis of Monomers ATMS and BTMS
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oligomers with masked recognition units.19,25 For the hetero-
oligomers, Sc(OTf)3 was cleverly employed by Scott and co-
workers to both catalyze the removal of acetal-protecting
groups from benzaldehyde in situ and to facilitate imine
formation. The single-stranded oligomers containing both
aniline and benzaldehyde functional groups could not be
isolated as such but were inferred from duplexes formed
between complementary sequences. They assert that their
strategy of protection of at least one reactive group and in situ
deprotection is inherently necessary for the synthesis and
handling of oligomers containing multiple coreactive groups
capable of forming dynamic covalent bonds. Compared to
genetic biopolymers, which are routinely manipulated as single
strands without chemical modifications, the uncontrolled
formation of intra- and intermolecular imine base pairs
would pose a significant limitation. The effects of temperature,
pH, metal ions, and ionic strength are comparatively well-
understood for biopolymers so that they can be predictably
interconverted between their folded, single-stranded, and
double-stranded states.26−28 Understanding how to manipulate
synthetic aniline benzaldehyde oligoarylacetylenes (ABOs) in
their native unmodified state is critical for an exploration of the
folding and replication of such oligomers, and we have
therefore explored protecting group-free methods for the
synthesis, purification, and characterization of such hetero-
oligomers. Herein, we describe an efficient means of
constructing sequence-defined ABOs on solid supports. We
then discuss how these molecules can be maintained as
kinetically stable single-stranded oligomers or returned to their
single-stranded state following common manipulations such as
concentration which are apt to generate intractable mixtures of
higher-order structures.
benzaldehyde, a double Sonogashira coupling with TMS-
acetylene gave compounds 1a and 1b in acceptable yields.
Breaking the symmetry of these molecules proved somewhat
difficult. Standard methods using TBAF or K2CO3 gave
complete deprotection of both alkynes, whereas more mild
fluoride sources such as HF·Pyridine or TEA·3HF gave no
detectible reaction even after heating at 50 °C overnight. We
then considered that a single equivalent of a moderately
available fluoride and a mild proton source would be ideal to
maximize the yield of the singly deprotected product. A
mixture of KF (1 equiv), a small amount of 18-crown-6 (0.25
equiv), and acetic acid (2 equiv) in THF gave clean conversion
to the expected stoichiometric product distribution, approx-
imately, 25% unreacted starting materials 1a and 1b, 50% of
the desired singly deprotected products 2a and 2b, and 25% of
the doubly deprotected diacetylenes, as assessed by LCMS
(Figures S2 and S3). The desired products could be isolated in
31% (2a) and 42% (2b) yields, the unreacted starting materials
recovered in 18% (1a) and 24% (1b) yields, and the doubly
deprotected diacetylenes isolated in 13 and 12% yield. The
monoprotected diynes 2 were then coupled to diiodide 3 using
standard Sonogashira coupling conditions. In order to disfavor
double coupling to the diiodides, the use of 10 equiv of 3 was
necessary. This excess could be recovered nearly quantitatively
and reused. The desired monomers ATMS and BTMS were
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obtained in excellent yields, and none of the double coupling
product was observed. The initial monomer for solid-phase
synthesis CBH was functionalized with a carboxylic acid for
attachment to the resin bead. Sonogashira coupling of BTMS
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with 5-hexynoic acid, followed by TMS deprotection with
TBAF gave CBH in acceptable yield (38% over two steps,
Scheme S1).
Following the scheme outlined in Figure 2, CBH was
attached to chlorotrityl-functionalized polystyrene beads under
mildly basic conditions. To make handling of the resin-bound
material easier and to avoid site−site interactions, resin loading
was reduced by a factor of 10 during the addition of the first
monomer by the addition of a 10-fold excess of acetic acid
RESULTS AND DISCUSSION
■
Solid-Phase Synthesis of ABOs. In order to study the
informational properties of aniline benzaldehyde oligoarylace-
tylenes, we developed a method for the solid-phase synthesis of
sequence-defined ABOs. Our synthetic strategy relied on
access to two monomers functionalized with an aryl iodide on
one side and a TMS-protected alkyne on the other, IATMS and
IBTMS, and the carboxylic acid-functionalized monomer CBH
(Figure 2a). We imagined that CBH could be easily attached to
a chlorotrityl-functionalized polystyrene solid support and then
oligomers of any desired sequence could be built through
iterative rounds of chain extension via Pd(0)-catalyzed
Sonogashira coupling29 and fluoride-mediated TMS depro-
tection (Figure 2b).30 Once the desired sequence was
constructed, acid-catalyzed cleavage from the resin would
afford the desired sequence-defined oligomers.
relative to BH. Coupling steps were accomplished in toluene
C
under copper-free Sonogashira coupling conditions using the
AsPh3 ligand, Pd2(dba)3 as a catalyst, and DIPEA as a base.31
With an excess of monomers ATMS or BTMS (6 equiv),
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coupling was completed cleanly in 1−3 h at 50 °C. After
coupling, the unreacted monomer could be recovered from the
organic solvent used to wash the resin beads and reused in
subsequent couplings after repurification. Under the Sonoga-
shira coupling conditions, no imine bonds between reacting
monomers and complementary sites on the growing oligomer
were observed. TMS deprotection was accomplished in 10 min
at 25 °C using TBAF (20 mM) buffered with AcOH (20 mM)
and stabilized with BHT (20 mM) in degassed THF.
The synthesis of the appropriately functionalized monomers
IATMS and BTMS is depicted in Scheme 1. Starting from
Following the protocol described above, we synthesized a set
of eight oligomers ranging in length from dimers to tetramers
I
commercially available 2,5-dibromoaniline and 2,5-dibromo-
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(Table 1). The yields presented in Table 1 are isolated yields
following a short normal-phase silica column. LCMS
Table 1. Isolated Yields of Synthesized ABOs
entry
sequence
yield (%)
1
2
3
4
5
6
7
8
CBBH
90
71
83
68
59
66
37
56
CBAH
CBBBH
CBBAH
CBAAH
CBABH
CBBBAH
CBABBH
chromatograms of the syntheses are shown in Figure S1. For
the homo-oligomers CBBH and CBBBH, purification and
characterization required no special handling; however, for
hetero-oligomers, special care needed to be taken to prevent
imine base-pairing between B and A subunits, which could lead
to the formation of dynamic mixtures. Our efforts to
understand and control these dynamic equilibria are presented
in the next section, as well as our strategy to isolate and
characterize the hetero-oligomers.
In addition to furnishing sequence-defined oligomers, our
method differs from the impressive solid-phase synthesis of
similar aniline benzaldehyde m-phenylene ethynylene homo-
polymers reported by Moore and co-workers19 in a few
noteworthy details. For our approach, protecting groups on the
aniline or benzaldehyde monomers were not required. In
addition, we simplified the synthesis of the requisite monomers
and used an air-stable palladium/ligand system requiring only
that the head space of the reaction vessel be purged with inert
gas prior to coupling. Finally, the addition of each monomer to
the growing oligomer required only a single coupling and
deprotection step, opposed to two.
Dynamic Behavior of ABOs. The equilibrium distribution
of imine bonds and their hydrolyzed precursors is known to
depend on many factors, such as the solvent, pH, temperature,
available water for hydrolysis, available amines for transimina-
tion reactions, and presence of metal catalysts.32−35 Disequi-
librium states are also possible as the kinetics of imine bond
formation can change drastically under different conditions,
and populations can become kinetically trapped if imine
formation or exchange is sufficiently slowed. Generally, under
mildly acidic conditions, the rate of imine formation and
hydrolysis is fast and favors the hydrolyzed amine and
aldehyde products, while under more basic conditions, imine
formation and hydrolysis is slower but imine formation is
favored at equilibrium.
Figure 3. Dynamic behavior of CBAH. Equilibrium between single-
stranded and duplex structure (top). (a) 1H NMR spectrum of crude
CBAH (2 mM) in CDCl3 with 2% TFA (v/v). (b) Solution from a
after 5 μL of D2O was added and then 30 μL of TEA was added. (c)
Solution from a after 30 μL of TEA was added.
stranded forms appeared to be quite stable, in that when TEA
was added to the water-quenched solution, no change in the
amount of ss-CBAH was observed in the NMR spectrum after
two days and similarly, when water was added to the TEA-
quenched solution, no change in the amount of ds-CBAH was
observed in the NMR spectrum after two days. However, when
the excess TEA was removed from the solution of ds-CBAH in
vacuo and the dimer was redissolved in CDCl3 with TFA and
H2O, the NMR spectrum showed complete conversion to
ss-CBAH.
The simplest explanation for these results is that the acidic
and relatively anhydrous environment of the resin cleavage
conditions favored the fast exchange of imine bonds on the
time scale of the NMR such that only broad peaks
corresponding to the time average of many different
equilibrating species and their various protonation states
were observed. Adding water to these acidic solutions likely
shifted the equilibrium nearly entirely to hydrolysis of the
imine bonds and the spectrum of ss-CBAH emerged. Adding
TEA sequestered the acidic protons and prevented imine
exchange reactions such that the spectrum of the thermody-
namically favored ds-CBAH emerged. Once formed, solutions
of ss-CBAH and ds-CBAH did not interconvert under identical
conditions with added water and TEA, strongly suggesting that
in the absence of a proton source, these solutions were
kinetically stable. This insight proved vital for handling and
purifying both these heterodimers and the longer hetero-
oligomers. Basified solutions of such oligomers were therefore
purified by chromatography on base-treated silica gel using
THF with a 0−20% water gradient. Attempts to purify hetero-
oligomers without quenching the acid with TEA failed
As illustrated in Figure S1, LCMS analysis of the crude
products suggested that the solid-phase synthesis had been
quite successful. However, for even the simplest heteromeric
1
dimer BAH, H NMR spectroscopy of the product following
C
cleavage from the resin with TFA showed uninterpretable
broad resonances (Figure 3a). Only after the addition of a
small amount of H2O or D2O (3−10 μL to a sample in 600 μL
of CDCl3) did the expected spectrum of the single-stranded
CBAH appear (Figure 3b). Surprisingly, when TEA was added
instead of water, an entirely different spectrum was observed,
which was confirmed by HRMS36 to be the self-comple-
mentary duplex, ds-CBAH (Figure 3c). The single- and double-
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presumably because dynamic mixtures were generated during
washing or on the column.
to the appearance of several new sets of peaks, including small
peaks consistent with an imine functional group (e.g., BBAH
C
Figure 4c, δ 8.61 ppm).
The formation of duplexes between fully self-complementary
dimers was expected, based on our previous work,8 other work
with longer homopolymeric ABOs,19 and recent work with
structurally similar aniline- and benzaldehyde-functionalized
peptoids.20 However, we expected the behavior of the
heteropolymer trimers synthesized here to be more complex
than simple duplex formation as these sequences are only
partially self-complementary. They could potentially form
several different self-complementary duplexes, higher-order
oligomers, or folded structures. For example, a duplex
ds-CBBAH formed from the sequence CBBAH would leave
sticky ends of unpaired monomers, while out of register base
pairing could create poly-CBBAH and intramolecular base-
pairing could potentially create a macrocyclic folded structure
(Scheme 2).
To better understand the effect of added water on these
1
systems, we compared the area of the two aldehyde H NMR
resonances of ss-CBBAH (δ 10.05 ppm) and the peak area of
the presumed imine proton resonance (δ 8.61 ppm) as D2O
was added to a 5 mM solution of CBBAH in 500 μL of CDCl3
with 2% TFA (v/v) (Figure 5).37 Only in a narrow range of
Scheme 2. Potential Dynamic Behavior of BBAH
C
1
Figure 5. H NMR signal of CBBAH in CDCl3 with 2% TFA (v/v)
and added D2O.
conditions were solutions found to be well-behaved and to give
sharp peaks consistent with the expected spectrum of
ss-CBBAH. With a ratio of 1:2 D2O to TFA, the spectra
showed only ss-CBBAH.
We searched for conditions that would allow us to effectively
control the equilibria of the more complicated trimer and
tetramer sequences so that we could routinely handle and
characterize such molecules. Analogous to our observations of
This trend was observed for all heteromeric-ABOs shown in
Table 1. Hetero-oligomers were thus characterized by 1H
NMR under conditions which assured the predominately
single-stranded state [CDCl3 with 5% TFA and 1% H2O (v/
v)]. Heating the samples to 50 °C moved the H2O/TFA
resonance upfield so as to not overlap with the analyte peaks.
At oligomer concentrations above 10 mM, significant peak
1
CBAH, the crude H NMR spectra of the heteromeric trimers
and tetramers under the acidic resin cleavage conditions
(CDCl3 with 2% TFA v/v) gave only broad resonances (e.g.,
CBBAH, Figure 4a). Only after the addition of a small amount
of D2O (3−10 μL) did the expected spectrum of the single-
stranded trimer appear (Figure 4b). Unlike the CBAH
sequence, these equilibria were found to be quite sensitive to
the ratio of TFA to D2O, and adding more than a few
microliters of H2O or D2O to samples in 500 μL of CDCl3 led
1
broadening was observed by H NMR which could not be
completely reduced by adjusting the ratio of TFA and H2O.
Such higher concentrations may favor the intermolecular
formation of imine bonds which cannot be entirely overcome.
For homopolymeric-ABOs (CBBH and BBBH), no dynamic
C
behavior was observed as expected because these molecules
lack the anilines necessary for imine formation. We are
currently working to fully describe the dynamic behavior of the
trimers and tetramers we have synthesized under conditions
which favor imine bonds.
Understanding the sensitivity of ABOs to the ratio of TFA
and water in a solvent was vital for effective handling. For
example, when pure ss-CBBAH in CDCl3 was concentrated to
dryness, up to 30% of the starting mass was lost as insoluble
white solids, and the material that was resolubilized was found
1
by H NMR to be about 60% ss-CBBAH and 40% complex
heterogeneous materials. We have previously reported that
wet/dry cycles of aniline- and benzaldehyde-containing
solutions are effective at forming imine bonds,8 so we
suspected that the hetero-oligomers were forming higher-
order structures through condensation of B and A subunits,
with some of these structures being insoluble in CDCl3. We
were initially unable to recover these insoluble products as they
were insoluble in all pure solvents tested as well as with various
mixtures of solvents along with the added acid and water
expected to favor imine hydrolysis. Only with the precise ratio
of TFA and water corresponding to the peak in Figure 5 (5:1
1
Figure 4. H NMR spectra of the heterotrimer CBBAH. (a) Crude
CBBAH (approx. 5 mM) in 600 μL of CDCl3 with 2% (v/v) TFA. (b)
Solution from a immediately after 5 μL of D2O was added. (c)
Solution from b immediately after 200 μL of D2O was added.
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equiv, 135 mg/g resin, 0.29 mmol/g resin) was added and dissolved
in DCE (5 mL/g resin) and DIPEA (1.4 mL/g resin). Acetic acid (1.5
equiv, 114 μL/g resin, 2 mmol/g resin) was added to the CBH
solution and then this mixture was added to the vial containing the
swelled resin beads. After the beads were shaken gently overnight,
they were transferred to a sintered glass washing vessel using DCM
and washed with 5 portions of DCM (50 mL/g resin). The washed
beads were then transferred to a storage vial and dried under high
vacuum (approx. 0.2 mbar) for several hours to remove the residual
solvent. The resin loading was determined by quantitative 1H NMR as
follows: An aliquot (10.0 mg) of dry resin was added to an NMR tube
TFA/H2O v/v) were the materials resolubilized and the
mixture of products observed in the H NMR resolved to
ss-CBBAH. These conditions proved very robust, and CHCl3
with 5% TFA and 1% H2O (v/v) was used routinely to return
hetero-ABOs to their single-stranded form following purifica-
tion and concentration.
1
CONCLUSIONS
■
The “holy grail” of Orgel38 and many subsequent polymer
chemists has been the synthesis of sequence-defined polymers
with the ability to undergo Darwinian evolution, but with all
the diversity in structure that chemistry and creativity will
allow. Realizing this ambitious goal requires ready access to
sequence-defined polymers with reversible recognition be-
tween subunits. Chemical methods for the synthesis,
purification, concentration, annealing, denaturation, and
folding of RNA and DNA have been fine-tuned by chemists
and biochemists for the better part of a century.39,40 Our
understanding of how to manipulate these and closely related
polymers and to control their dynamic behaviors is accordingly
quite well-developed. For synthetic oligomers very different in
structure from nucleotide-based oligomers, developing con-
ditions to control their dynamic behavior is an integral part of
their synthesis. For oligomers with imine-based reversible
covalent recognition units, precisely controlling the amount of
TFA and water proved a straightforward and effective way to
ensure that solutions of these molecules were in their fully
hydrolyzed state. This allowed for single-stranded oligomers to
1
and swelled in CDCl3 (600 μL). TFA (5 μL) was added and the H
NMR spectrum was recorded immediately. Cleavage was found to be
complete after 5 min and NMR spectra recorded at subsequent times
(10 min and 1 h) showed no change. To the NMR tube, 50 μL of a
2.0% solution of DMF in CDCl3 was added and the 1H spectrum with
a d1 relaxation delay of 10 s and 45 deg pulse angle was acquired. The
normalized area of the DMF standard was then used to calculate the
quantity of CBH from which the resin loading could also be calculated.
The resin loading was found to be 90−115 μmol/g BH and around
C
1.2 mmol/g acetic acid, which is in accordance with the commercially
reported 1.3 g/mol loading of the beads.
Solid-Phase Synthesis, Extension of the Chain via Sonoga-
shira Cross Coupling. To a 20 mL scintillation vial, AsPh3 (8 mol/
10 mol resin load) and Pd2(dba)3 (1 mol/10 mol resin load) were
added and dissolved in anhydrous toluene (1 mL/5 μmol Pd2(dba)3).
The head space of the vial was purged with argon and then the vial
was sealed with a screw on cap. The purple solution was gently
swirled until the Pd2(dba)3 dissolved and the color changed to bright
yellow (approx. 10 min). To a separate 40 mL vial equipped with a
stir bar, the resin (100 mg to 2 g, with a loading from 90 to 115 μmol/
g) to be extended was added and swelled in toluene (5 mL/g resin)
and DIPEA (2 mL/g resin). To this solution, the desired TMS-
protected monomer ATMS or BTMS (6 equiv) was added and stirred
1
be observed by H NMR and ESI−MS, to be returned to
solution following concentration, and to be purified without
the dynamic behavior of their subunits generating mixtures.
The solid-phase synthesis developed herein is concise and high
yielding. We imagine that these synthetic efforts will provide a
solid foundation on which to build oligomers of any desired
sequence and a starting point to more fully describe their
dynamic behavior. Our observations and methods may prove
useful to other chemists using imine condensation and
hydrolysis in dynamic covalent chemistry contexts.
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gently to dissolve (approx. 5 min). Once the monomer had dissolved,
the catalyst solution was added through a small cotton filter to the vial
containing the resin. The head space of the reaction vessel was purged
with argon and then the vial was sealed with a screw on cap. The
reaction mixture was heated in an oil bath to 50 °C and stirred very
gently for 3 h or until LCMS analysis of an aliquot of the reaction
showed extension to be complete. The reaction mixture was allowed
to cool to room temperature and then transferred to a sintered glass
washing vessel using DCM and washed with 5 portions of DCM (50
mL/g resin). The DCM washes were combined and the unreacted
monomers could be recovered and reused following a normal-phase
flash column. The washed beads were transferred to a storage vial and
dried under high vacuum (approx. 0.2 mbar) for several hours to
remove the residual solvent. LCMS analysis of the resin beads was
accomplished as follows: 10 mg of the resin was added to a 2 mL
HPLC vial and swelled in 500 μL of DCM. To this, 30 μL of TFA was
added, followed by 5 μL of H2O. This solution was then diluted, 50
μL into 1 mL of 9:1 THF/H2O, and the diluted solution was analyzed
by LCMS as described in the General section (above).
Solid-Phase Synthesis, TBAF Cleavage of the TMS-Protect-
ing Group. To a 20 mL scintillation vial, TBAF·3H2O (0.4 mmol/g
resin) was added and dissolved in dry degassed THF (10 mL/g resin)
stabilized with 250 ppm BHT. To this solution, BHT (0.4 mmol/g
resin) and AcOH (0.4 mmol/g resin) were added. The head space of
the vial was purged with argon and then the vial was sealed with a
screw on cap until ready for use. To a separate vial equipped with a
stir bar, the resin to be deprotected was added (100 mg to 2 g, with a
loading from 90 to 115 μmol/g) and swelled with dry degassed THF
(10 mL/g resin) stabilized with 250 ppm BHT. The resin solution
was stirred gently at 25 °C and the TBAF/BHT/AcOH solution was
added in one portion. After stirring for 10 min, the reaction was
quenched by adding ice (approx. 50 g/g resin). The reaction mixture
was then transferred to a sintered glass washing vessel using stabilized
THF and washed with 2 portions of THF (50 mL/g resin), then 2
portions of water (50 mL/g resin), and finally 5 portions of DCM (50
mL/g resin). The washed beads were transferred to a storage vial and
EXPERIMENTAL SECTION
■
1
General. The H NMR and 13C NMR spectra were recorded at
93.94 kG (1H 400 MHz, 13C 100 MHz) at ambient temperature or 50
°C, as noted. Hydrogen chemical shifts are expressed in parts per
million (ppm) relative to the residual protio-solvent resonance:
CDCl3: δ 7.26. For 13C spectra, the centerline of the solvent signal
was used as the internal reference: CDCl3: δ 77.00. Unless otherwise
noted, each carbon resonance represents a single carbon (relative
intensity). LCMS spectra were obtained on an Agilent Technologies
system HPLC with a phenyl hexyl reverse-phase column. Mobile
phases were water with 0.1% formic acid and THF using a THF
gradient from 55 to 75%. Mass-to-charge ratios were determined from
an inline electrospray (ESI) source in the positive ion mode and a
Thermo Scientific quadrupolar mass analyzer. The MS settings were a
capillary voltage of 3.5 kV and a desolvation temperature of 325 °C.
High-resolution mass spectrometric data were obtained on a ToF
(time-of-flight) Agilent Technologies system by electrospray (ESI) in
the positive ion mode. Samples were injected as 10 μM solutions in
8:2 THF/H2O. The MS settings were a capillary voltage of 4000 V, a
desolvation temperature of 300 °C, and a fragmentor voltage of 500
V.
Solid-Phase Synthesis, Attachment of the First Monomer,
CBH. Polystyrene resin beads (0.5−2 g) functionalized with 2-
chlorotrityl chloride (1% DVB, 1.3 mmol/g loading) were added to a
40 mL scintillation vial. Anhydrous DCE (5 mL/g resin) was added
and the resin allowed to swell for 5 min. To a separate vial, CBH (0.22
F
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dried under high vacuum (approx. 0.2 mbar) for several hours to
remove the residual solvent. Success of the cleavage was verified by
LCMS analysis following the same protocol outlined in the previous
section.
solution was sparged with argon for 30 min and then TMS-acetylene
(4 equiv, 32 mmol, 4.64 mL) was added, followed by Pd(PPh3)4 (0.02
equiv, 0.16 mmol, 184 mg) and CuI (0.05 equiv, 0.4 mmol, 72 mg).
The head space of the reaction vessel was purged with argon and the
flask was sealed with a glass stopper. After stirring overnight at 50 °C,
the reaction mixture was cooled to room temperature and diluted
with DCM (approx. 100 mL), and dry silica (approx. 10 g) was
added. The mixture was then concentrated onto the silica gel using
rotoevaporation and the resulting white powder was used to dry load
a silica gel column. Normal-phase silica gel flash chromatography (80
g silica column, hexane with a 0−10% gradient of EtOAc over 25
min) gave a mixture of doubly alkynylated product 1b and singly
alkynylated side product 3-bromo-5-((trimethylsilyl)ethynyl)-
benzaldehyde. The mixture of products was concentrated to a light
yellow oil and then subjected to a C18 reverse-phase flash column (50
g column loaded using 5 mL DMF, then run with water and a 30−
100% ACN gradient over 15 min). The reverse-phase column
afforded pure 1b (Rf = 2 min 100% ACN, 2.07 g, 6.93 mmol, 87%
yield) as a colorless oil. 1H NMR (400 MHz, CDCl3): δ 9.94 (s, 1H),
7.87 (d, J = 1.6 Hz, 2H), 7.79 (t, J = 1.6 Hz, 1H), 0.25 (s, 18H);
13C{1H} NMR (100 MHz, CDCl3): δ 190.7, 140.3 (2C), 136.4, 132.4
(2C), 124.6, 102.5 (2C), 97.0 (2C), −0.2 (6C). HRMS (ESI) m/z:
[M + Na]+ calcd for C17H22OSi2Na, 321.1101; found, 321.1126.
3-Ethynyl-5-((trimethylsilyl)ethynyl)aniline (2a). To a 40 mL
scintillation vial equipped with a stir bar, 1a (330 mg, 1.16 mmol) was
added and then dissolved in dry degassed THF stabilized with 250
ppm BHT (5.8 mL). To this solution, 18-crown-6 (76.4 mg, 0.25
equiv, 0.29 mmol), AcOH (2 equiv, 2.31 mmol, 132.4 μL), and KF
(1.0 equiv, 1.16 mmol, 67 mg) were added. After stirring at room
temperature overnight, the reaction was quenched by the addition of
silica (approx. 2 g). The mixture was then concentrated onto the silica
gel using rotoevaporation and the resulting white powder was used to
dry load a silica gel column. Silica gel flash chromatography (40 g
silica column, hexane with a 10−50% gradient of EtOAc over 18 min)
gave the pure product (76 mg, 0.356 mmol, 31% yield) as a white
solid. Starting material 1a and doubly deprotected 3,5-diethynylanilne
were also isolated in 18% and 13% yields, respectively. 1H NMR (400
MHz, CDCl3): δ 7.01 (t, J = 1.4 Hz, 1H), 6.78−6.72 (m, 2H), 3.67 (s,
2H), 3.00 (s, 1H), 0.23 (s, 9H); 13C{1H} NMR (100 MHz,
chloroform-d): δ 146.1, 125.8, 123.9, 122.9, 118.5, 118.4, 104.3, 94.2,
83.0, 77.0, −0.2 (3C). HRMS (ESI) m/z: [M + H]+ calcd for
C13H16NSi, 214.1047; found, 214.1031.
Using THF free of residual peroxides was vital to the success of this
reaction as compounds consistent with oxidation products were
otherwise observed. Dissolving aldehyde-containing oligomers in
THF with any detectable peroxides was detrimental, and detectable
oxidation products were observed after sitting a few hours. Oxidation
was greatly exacerbated by concentrating the material in vacuo, giving
products consistent with multiple oxidation events corresponding to
the number of aldehyde functional groups (Figure S4). We suggest
that peroxy radicals generated from exposure of THF to air initiated
auto-oxidation of the aldehyde functional groups to carboxylic acids.41
No oxidation side products were observed when using freshly distilled
THF or THF stabilized with 250 ppm BHT and stored under an inert
atmosphere. No oxidation was observed in air for non-THF solvents.
Solid-Phase Synthesis, Cleavage from the Resin Beads and
Purification. The resin (20−100 mg) was added to a resin-washing
vessel and treated for 5 min with CDCl3 or DCM with 0.5% TFA and
0.1% H2O (10 mL/g resin). The organic solution was collected by
filtration and the procedure was repeated three more times. The resin
was then washed with a single portion of CDCl3 or DCM (10 mL/g).
To the combined organic washes, TEA (20 mL/g) was then added in
a single portion and the solution was allowed to stand for 10 min. The
organic solution was then washed with three portions of water (25
mL/g) and one portion of brine (25 mL/g). The aqueous washes
were discarded and the organic phase was loaded directly onto a short
normal-phase silica gel column which had been packed with DCM
containing 0.5% TEA. The column was run with freshly distilled THF
buffered with 0.1% TEA and a water gradient from 0 to 20% over 30
min with a flow rate of 2 mL/min. The fractions containing the
desired products were concentrated to a white solid on a speedvac
rotary concentrator. The pure materials were dissolved in DCM or
CDCl3 for NMR, LCMS, or HRMS analysis. For heteropolymers,
0.5% TFA and 0.1% H2O were added to the solvent. Following
analysis, the TFA could be removed by washing the organic phase
with several portions of water and then the materials could be
concentrated to a white powder for storage.
3,5-Bis((trimethylsilyl)ethynyl)aniline (1a). To a 200 mL round-
bottom flask equipped with a stir bar, 3,5-dibromoaniline (1.0 g, 4
mmol) was added and then dissolved in anhydrous toluene (20 mL)
and DIPEA (10 equiv, 20 mmol, 7 mL). The solution was sparged
with argon for 30 min and then TMS-acetylene (4 equiv, 16 mmol,
2.32 mL) was added, followed by Pd(PPh3)4 (0.02 equiv, 0.08 mmol,
92 mg) and CuI (0.05 equiv, 0.2 mmol, 36 mg). The head space of
the reaction vessel was purged with argon and the flask was sealed
with a glass stopper. After stirring overnight at 55 °C, the reaction
mixture was cooled to room temperature and diluted with DCM
(approx. 100 mL), and dry silica (approx. 10 g) was added. The
mixture was then concentrated onto the silica gel using rotoevapora-
tion and the resulting white powder was used to dry load a silica gel
column. Normal-phase silica gel flash chromatography (80 g silica
column, hexane with a 5−50% gradient of EtOAc over 25 min) gave a
1:1 mixture of doubly alkynylated product 1a and singly alkynylated
side product 3-bromo-5-((trimethylsilyl)ethynyl)aniline. The mixture
of products was concentrated to a light yellow oil and then subjected
to a C18 reverse-phase flash column (50 g column loaded using 5 mL
DMF, then run with water and a 45−95% ACN gradient over 15
min). The reverse-phase column afforded pure 1a (eluted from 85 to
90% ACN, 340 mg, 1.19 mmol, 30% yield) as a colorless oil. 1H NMR
(400 MHz, CDCl3): δ 7.00 (t, J = 1.4 Hz, 1H), 6.71 (d, J = 1.4 Hz,
2H), 3.64 (s, 2H), 0.22 (s, 18H); 13C{1H} NMR (100 MHz, CDCl3):
δ 146.0, 126.1, 123.9 (2C), 118.3 (2C), 104.4 (2C), 94.1 (2C), −0.1
(6C). HRMS (ESI) m/z: [M + H]+ calcd for C16H24NSi2, 286.1442;
found, 286.1448.
3-Ethynyl-5-((trimethylsilyl)ethynyl)benzaldehyde (2b). To a 40
mL scintillation vial equipped with a stir bar, 1b (313 mg, 1.05 mmol)
was added and then dissolved in dry degassed THF stabilized with
250 ppm BHT (5.6 mL). To this solution, 18-crown-6 (69.3 mg, 0.25
equiv, 0.26 mmol), AcOH (2 equiv, 2.1 mmol, 120 μL), and KF (1.0
equiv, 1.05 mmol, 61 mg) were added. After stirring at room
temperature overnight, the reaction was quenched by the addition of
silica (approx. 2 g). The mixture was then concentrated onto the silica
gel using rotoevaporation and the resulting white powder was used to
dry load a silica gel column. Silica gel flash chromatography (40 g
silica column, hexane with a 0−10% gradient of EtOAc over 18 min)
gave a mixture of product 2b, starting material 1b, and doubly
deprotected product 3,5-diethynylbenzaldehyde. Reverse-phase Com-
biFlash chromatography (50 g C18 column, water with 30% ACN
increasing to 100% ACN over 20 min) gave pure 2b as a white solid
(99.8 mg, 0.44 mmol, 42% yield). Starting material 1b and doubly
deprotected 3,5-diethynylbenzaldehyde were also isolated in 24% and
1
12% yields, respectively. H NMR (400 MHz, CDCl3): δ 9.95 (s,
1H), 7.92 (t, J = 1.6 Hz, 1H), 7.90 (t, J = 1.6 Hz, 1H), 7.80 (t, J = 1.6
Hz, 1H), 3.16 (s, 1H), 0.26 (s, 9H); 13C{1H} NMR (100 MHz,
CDCl3): δ 190.3, 140.2, 136.4, 132.7, 132.4, 124.6, 123.5, 102.2, 97.2,
81.3, 79.4, −0.3 (3C). HRMS (ESI) m/z: [M + Na]+ calcd for
C14H14OSiNa, 249.0706; found, 249.0697.
2-(2-Methoxyethoxy)ethyl 3-((3-amino-5-((trimethylsilyl)-
ethynyl)phenyl)ethynyl)-5-iodobenzoate (IATMS). To a 200 mL
round-bottom flask equipped with a stir bar, 2a (111 mg, 0.52
mmol) was added and then dissolved in anhydrous toluene (5.2 mL)
and DIPEA (3 equiv, 1.56 mmol, 271 μL). Diiodide 3 was added (10
3,5-Bis((trimethylsilyl)ethynyl)benzaldehyde (1b). To a 200 mL
round-bottom flask equipped with a stir bar, 3,5-dibromobenzalde-
hyde (2.1 g, 8 mmol) was added and then dissolved in anhydrous
toluene (40 mL) and DIPEA (10 equiv, 40 mmol, 14 mL). The
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equiv, 5.2 mmol, 2.48 g) and the solution was stirred until
homogeneous (approx. 15 min). To the stirred solution, Pd(PPh3)4
(0.05 equiv, 0.026 mmol, 30 mg) and CuI (0.1 equiv, 0.052 mmol, 9.9
mg) were then added. The head space of the reaction vessel was
purged with argon and the flask was sealed with a glass stopper. After
stirring for 5 h at room temperature, dry silica was added. The mixture
was then concentrated onto the silica gel using rotoevaporation and
the resulting white powder was used to dry load a silica gel column.
Normal-phase silica gel flash chromatography (40 g silica column,
hexane with a 10−50% gradient of EtOAc over 18 min) gave a
head space of the vial was purged with argon, and the vessel was
sealed with a screw on cap. After stirring at room temperature
overnight, the reaction mixture was transferred to a round-bottom
flask and diluted with DCM (approx. 50 mL). Silica gel was added to
the vessel and the crude reaction mixture was concentrated onto the
silica gel. Silica gel flash chromatography (hexane with a 30−100%
gradient of EtOAc, each with 0.5% AcOH) gave the pure product (75
mg, 0.13 mmol, 45%) as a colorless oil. 1H NMR (400 MHz, CDCl3):
δ 9.94 (s, 1H), 8.06 (s, 1H), 8.01 (s, 1H), 7.91 (s, 1H), 7.88 (s, 1H),
7.82 (s, 1H), 7.67 (s, 1H), 4.51−4.43 (m, 2H), 3.86−3.78 (m, 2H),
3.71−3.64 (m, 2H), 3.59−3.52 (m, 2H), 3.36 (s, 3H), 2.54 (t, J = 7.3
Hz, 2H), 2.50 (t, J = 6.9 Hz, 2H), 1.92 (tt, J = 7.3, 6.9 Hz, 2H), 0.24
(s, 9H); 13C{1H} NMR (100 MHz, CDCl3): δ 190.6, 178.4, 165.1,
139.9, 138.3, 136.5, 132.8, 132.5, 132.0, 131.8, 130.7, 124.8, 124.6,
124.1, 123.0, 102.3, 97.2, 90.8, 89.6, 88.2, 79.8, 71.8, 70.4, 69.1, 64.4,
59.0, 32.7, 23.3, 18.7, −0.3 (3C). HRMS (ESI) m/z: [M + Na]+ calcd
for C32H34O7SiNa, 581.1966; found, 581.1982.
6-(3-((3-Ethynyl-5-formylphenyl)ethynyl)-5-((2-(2-
methoxyethoxy)ethoxy)carbonyl)phenyl)hex-5-ynoic Acid (CBH).
To a 20 mL scintillation vial, TBAF·3H2O (0.28 mmol, 88 mg)
was added and dissolved in dry degassed THF stabilized with 250
ppm BHT. To this solution, BHT (0.28 mmol, 62 mg) and AcOH
(0.28 mmol, 16 μL) were added. In a separate vial equipped with a
stir bar, CBTMS (75 mg, 0.13 mmol) was dissolved in dry degassed
THF stabilized with 250 ppm BHT (2.8 mL). The TBAF solution
was then added to the CBTMS solution. The head space was purged
with argon and the mixture was stirred at room temperature. After 5
min, the reaction was quenched by adding ice (approx. 5 g). After
stirring for 1 min, silica (approx. 1 g) was added and the reaction
mixture was concentrated in vacuo. Silica gel flash chromatography
(hexane with a 30−100% gradient of EtOAc, each with 0.5% AcOH)
gave the pure product (53 mg, 0.11 mmol, 85% yield) as a colorless
oil which solidified to a white amorphous solid on refrigeration
overnight. 1H NMR (400 MHz, CDCl3): δ 9.99 (s, 1H), 8.11 (s, 1H),
8.06 (s, 1H), 7.99 (s, 1H), 7.95 (s, 1H), 7.87 (s, 1H), 7.71 (s, 1H),
4.54−4.46 (m, 2H), 3.89−3.82 (m, 2H), 3.74−3.68 (m, 2H), 3.62−
3.56 (m, 2H), 3.40 (s, 3H), 3.20 (s, 1H), 2.58 (t, J = 7.2 Hz, 2H),
2.54 (t, J = 6.9 Hz, 2H), 1.96 (tt, J = 7.2, 6.9 Hz, 2H); 13C{1H} NMR
(100 MHz, CDCl3): δ 193.1, 181.1, 167.8, 142.7, 141.1, 139.3, 135.5,
135.3, 135.2, 134.5, 133.4, 127.3, 126.9, 126.5, 125.6, 93.5, 92.5, 90.8,
83.9, 82.5, 82.3, 74.5, 73.1, 71.8, 67.1, 61.7, 35.4, 26.0, 21.4. HRMS
(ESI) m/z: [M + Na] calcd for C29H26O7Na, 509.1571; found,
509.1605.
CBBH. The resin (100 mg with 95 μmol/g loading) functionalized
with CBBH was cleaved and purified according to the procedure
outlined above, and CBBH (7.4 mg, 8.6 μmol, 90% yield) was afforded
as a white solid. 1H NMR (400 MHz, CDCl3): δ 10.03 (s, 1H), 10.01
(s, 1H), 8.26−8.17 (overlap, 2H), 8.13 (t, J = 1.6 Hz, 1H), 8.07 (t, J =
1.7 Hz, 1H), 8.04−7.99 (overlap, 3H), 7.97 (t, J = 1.6 Hz, 1H), 7.95
(t, J = 1.7 Hz, 1H), 7.91−7.87 (overlap, 2H), 7.74 (t, J = 1.7 Hz, 1H),
4.60−4.45 (overlap, 4H), 3.94−3.81 (overlap, 4H), 3.78−3.67
(overlap, 4H), 3.65−3.54 (overlap, 4H), 3.41 (s, 3H), 3.41 (s, 3H),
3.22 (s, 1H), 2.59 (t, J = 7.3 Hz, 2H), 2.55 (t, J = 7.0 Hz, 2H), 1.97
(tt, J = 7.3, 7.0 Hz, 2H). HRMS (ESI) m/z: [M + Na]+ calcd for
C52H44O12Na, 883.2725; found, 883.2879.
CBAH. The resin (20 mg with 100 μmol/g resin loading)
functionalized with CBAH was cleaved and purified according to the
procedure outlined above, and CBAH (1.2 mg, 1.4 μmol, 71% yield)
was afforded as a white solid. 1H NMR (400 MHz, CDCl3 with 0.5%
TFA and 0.1% H2O): δ 10.00 (s, 1H), 8.20 (s, 1H), 8.16 (s, 1H), 8.10
(s, 1H), 8.06−8.00 (overlap, 3H), 7.97 (s, 1H), 7.90 (s, 1H), 7.76 (s,
1H), 7.74 (s, 1H), 7.53 (s, 1H), 7.48 (s, 1H), 4.63−4.47 (overlap,
4H), 3.97−3.86 (overlap, 4H), 3.84−3.75 (overlap, 4H), 3.75−3.68
(overlap, 4H), 3.48 (s, 3H), 3.47 (s, 3H), 3.26 (s, 1H), 2.64 (t, J = 7.3
Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 1.98 (tt, J = 7.3, 6.8 Hz, 2H).
HRMS (ESI) m/z: [M + Na] calcd for C51H45NO11Na, 870.2885;
found, 870.2859.
mixture of product ATMS and 3. The mixture of the product and
I
reactant was concentrated to a light yellow oil and then subjected to a
C18 reverse-phase flash column (50 g column loaded using 5 mL
DMF, then run with water and a 50−100% ACN gradient over 5 min,
followed by 100% ACN for 10 min). The reverse-phase column
afforded pure 3 (eluted from 85 to 90% ACN, 4.63 mmol, 2.21 g, 99%
recovery) as a white solid and desired product IATMS (Rf = 5 min with
100% ACN, 295 mg, 0.52 mmol, 99% yield) as a waxy solid. 1H NMR
(400 MHz, CDCl3): δ 8.32 (s, 1H), 8.12 (s, 1H), 8.00 (s, 1H), 7.06
(s, 1H), 6.77 (s, 2H), 4.53−4.46 (m, 2H), 3.86−3.82 (m, 2H), 3.76−
3.66 (overlap, 4H), 3.61−3.55 (m, 2H), 3.40 (s, 3H), 0.24 (s, 9H);
13C{1H} NMR (100 MHz, CDCl3): δ 164.3, 146.3, 143.9, 137.9,
131.8, 131.7, 125.5, 125.3, 124.1, 123.1, 118.5, 117.8, 104.2, 94.3,
93.2, 91.0, 86.4, 71.8, 70.5, 69.0, 64.5, 59.0, −0.2 (3C). HRMS (ESI)
m/z: [M + Na]+ calcd for C25H28INO4SiNa, 584.0724; found,
584.0746.
2-(2-Methoxyethoxy)ethyl 3-((3-formyl-5-((trimethylsilyl)-
ethynyl)phenyl)ethynyl)-5-iodobenzoate (IBTMS). To a 200 mL
round-bottom flask equipped with a stir bar, 2b (165 mg, 0.73
mmol) was added and then dissolved in anhydrous toluene (7.3 mL)
and DIPEA (3 equiv 2.19 mmol, 384 μL). Diiodide 3 was added (10
equiv, 7.3 mmol, 3.49 g) and the solution was stirred until
homogeneous (approx. 15 min). To the stirred solution, Pd(PPh3)4
(0.05 equiv, 0.037 mmol, 42 mg) and CuI (0.1 equiv, 0.073 mmol,
13.9 mg) were then added. The head space of the reaction vessel was
purged with argon and the flask was sealed with a glass stopper. After
stirring for 2.5 h at room temperature, dry silica was added. The
mixture was then concentrated onto the silica gel using rotoevapora-
tion and the resulting white powder was used to dry load a silica gel
column. Normal-phase silica gel flash chromatography (40 g silica
column, hexane with a 10−50% gradient of EtOAc over 18 min) gave
a mixture of product BTMS and 3. The mixture of the product and
I
reactant was concentrated to a light yellow oil and then subjected to a
C18 reverse-phase flash column (50 g column loaded using 5 mL
DMF, then run with water and a 50−100% ACN gradient over 5 min,
followed by 100% ACN for 10 min). The reverse-phase column
afforded pure 3 (eluted from 85 to 90% ACN, 6.24 mmol, 2.97 g, 95%
recovery) as a white solid and desired product IBTMS (Rf = 5 min with
100% ACN, 418 mg, 0.73 mmol, 99% yield) as a waxy solid. 1H NMR
(400 MHz, CDCl3): δ 9.98 (s, 1H), 8.37 (t, J = 1.6 Hz, 1H), 8.16 (t, J
= 1.6 Hz, 1H), 8.05 (t, J = 1.6 Hz, 1H), 7.95 (t, J = 1.6 Hz, 1H), 7.93
(t, J = 1.6 Hz, 1H), 7.86 (t, J = 1.6 Hz, 1H), 4.53−4.48 (m, 2H),
3.88−3.82 (m, 2H), 3.73−3.68 (m, 2H), 3.61−3.56 (m, 2H), 3.40 (s,
3H), 0.27 (s, 9H); 13C{1H} NMR (100 MHz, CDCl3): δ 190.2,
164.0, 143.8, 139.7, 138.4, 136.4, 132.6, 131.83, 131.80, 131.77, 124.7,
124.4, 123.7, 102.2, 97.2, 93.2, 89.0, 88.5, 71.7, 70.4, 68.9, 64.5, 58.9,
−0.4 (3C). HRMS (ESI) m/z: [M + Na]+ calcd for C26H27IO5SiNa,
597.0565; found, 597.0582.
6-(3-((3-Formyl-5-((trimethylsilyl)ethynyl)phenyl)ethynyl)-5-((2-
(2-methoxyethoxy) ethoxy)carbonyl)phenyl)hex-5-ynoic Acid
(CBTMS). To a 20 mL scintillation vial, AsPh3 (0.12 mmol, 37.2 mg)
and Pd2(dba)3 (0.015 mmol, 14 mg) were added and dissolved in
anhydrous DCE (1 mL). The head space of the vial was purged with
argon and then the vial was sealed with a screw on cap. The purple
solution was gently swirled until the Pd2(dba)3 dissolved and the
color changed to bright yellow (approx. 10 min). In a separate vial
equipped with a stir bar, IBTMS (175 mg, 0.30 mmol) was dissolved in
anhydrous DCE (1 mL). To this solution, DIPEA (1 mL) and 5-
hexynoic acid (0.46 mmol, 50 μL) were added. While stirring, a
portion of the catalyst solution was added dropwise (0.6 mL). The
CBBBH. The resin (100 mg with 88 μmol/g resin loading)
functionalized with CBBBH was cleaved and purified according to
the procedure outlined above, and CBBBH (9.0 mg, 7.3 μmol, 83%
H
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1
yield) was afforded as a white solid. H NMR (400 MHz, CDCl3): δ
(overlap, 6H), 8.09 (t, J = 1.6 Hz, 1H), 8.07−7.95 (overlap, 9H),
7.95−7.90 (overlap, 4H), 7.83 (s, 1H), 7.77 (t, J = 1.5 Hz, 1H), 7.58−
7.54 (overlap, 2H), 4.62−4.51 (overlap, 8H), 3.97−3.88 (overlap,
8H), 3.85−3.76 (overlap, 8H), 3.76−3.69 (overlap, 8H), 3.49−3.47
(overlap, 9H), 3.47 (s, 3H), 3.22 (s, 1H), 2.63 (t, J = 7.3 Hz, 2H),
2.55 (t, J = 6.9 Hz, 2H), 1.99 (tt, J = 7.3, 6.9 Hz, 2H). HRMS (ESI)
m/z: [M + Na]+ calcd for C97H81NO21Na, 1619.5227; found,
1619.5234.
10.05 (s, 1H), 10.04 (s, 1H), 10.01 (s, 1H), 8.25−8.19 (overlap, 4H),
8.13 (t, J = 1.5 Hz, 1H), 8.07 (t, J = 1.3 Hz, 1H), 8.05−7.99 (overlap,
5H), 7.98−7.94 (overlap, 3H), 7.91 (t, J = 1.4 Hz, 1H), 7.90−7.88
(overlap, 2H), 7.74 (t, J = 1.4 Hz, 1H), 4.58−4.48 (overlap, 6H),
3.93−3.83 (overlap, 6H), 3.76−3.68 (overlap, 6H), 3.64−3.57
(overlap, 6H), 3.42 (s, 3H), 3.41−3.40 (overlap, 6H), 3.22 (s, 1H),
2.59 (t, J = 7.3 Hz, 2H), 2.55 (t, J = 7.0 Hz, 2H), 1.97 (tt, J = 7.3, 7.0
Hz, 2H). HRMS (ESI) m/z: [M + Na]+ calcd for C75H62O17Na,
1257.3879; found, 1257.3888.
CBBAH. The resin (50 mg with 111 μmol/g resin loading)
functionalized with CBBAH was cleaved and purified according to
the procedure outlined above, and CBBAH (4.6 mg, 3.8 μmol, 68%
yield) was afforded as a white solid. 1H NMR (400 MHz, CDCl3 with
0.5% TFA and 0.1% H2O): δ 9.99 (s, 1H), 9.97 (s, 1H), 8.23−8.17
(overlap, 3H), 8.15 (s, 1H), 8.09 (s, 1H), 8.07−8.02 (overlap, 4H),
8.01 (s, 1H), 8.00 (s, 1H), 7.98 (s, 1H), 7.94 (s, 1H), 7.91 (s, 1H),
7.77 (s, 1H), 7.75 (s, 1H), 7.53 (s, 1H), 7.48 (s, 1H), 4.62−4.51
(overlap, 6H), 3.98−3.89 (overlap, 6H), 3.85−3.77 (overlap, 6H),
3.77−3.70 (overlap, 6H), 3.49−3.47 (overlap, 6H), 3.47 (s, 3H), 3.24
(s, 1H), 2.63 (t, J = 7.3 Hz, 2H), 2.54 (t, J = 6.9 Hz, 2H), 1.98 (tt, J =
7.3, 6.9 Hz, 2H). HRMS (ESI) m/z: [M + Na]+ calcd for
C74H63NO16Na, 1244.4039; found, 1244.4162.
CBAAH. The resin (60 mg with 95 μmol/g resin loading)
functionalized with CBAAH was cleaved and purified according to
the procedure outlined above, and CBAAH (4.1 mg, 3.4 μmol, 59%
yield) was afforded as a white solid. 1H NMR (400 MHz, CDCl3 with
0.5% TFA and 0.1% H2O): δ 9.97 (s, 1H), 8.18 (t, J = 1.5 Hz, 1H),
8.16−8.12 (overlap, 3H), 8.08 (t, J = 1.6 Hz, 1H), 8.03−7.99
(overlap, 3H), 7.96 (t, J = 1.5 Hz, 1H), 7.90 (t, J = 1.5 Hz, 1H), 7.86
(t, J = 1.5 Hz, 1H), 7.79 (s, 1H), 7.76 (t, J = 1.5 Hz, 1H), 7.72 (s,
1H), 7.56−7.51 (overlap, 3H), 7.47 (s, 1H), 4.60−4.50 (overlap,
6H), 3.97−3.87 (overlap, 6H), 3.83−3.76 (overlap, 6H), 3.75−3.69
(overlap, 6H), 3.48−3.45 (overlap, 9H), 3.24 (s, 1H), 2.63 (t, J = 7.3
Hz, 2H), 2.54 (t, J = 6.9 Hz, 2H), 1.98 (tt, J = 7.3, 6.9 Hz, 2H).
HRMS (ESI) m/z: [M + Na]+ calcd for C73H64N2O15Na, 1231.4199;
found, 1231.4198.
ASSOCIATED CONTENT
* Supporting Information
■
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.joc.0c01977.
NMR spectra and LCMS chromatograms (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Jack W. Szostak − Howard Hughes Medical Institute,
Department of Molecular Biology and Center for
Computational and Integrative Biology, Massachusetts General
Hospital, Boston, Massachusetts 02114, United States;
Department of Genetics, Harvard Medical School, Boston,
Massachusetts 02115, United States; Department of Chemistry
and Chemical Biology, Harvard University, Cambridge,
Massachusetts 02138, United States; orcid.org/0000-0003-
4131-1203; Email: szostak@molbio.mgh.harvard.edu
Author
Kyle R. Strom − Howard Hughes Medical Institute, Department
of Molecular Biology and Center for Computational and
Integrative Biology, Massachusetts General Hospital, Boston,
Massachusetts 02114, United States; Department of Genetics,
Harvard Medical School, Boston, Massachusetts 02115, United
States; orcid.org/0000-0002-0567-347X
CBABH. The resin (80 mg, 95 μmol/g loading) functionalized with
CBABH was cleaved and purified according to the procedure outlined
above, and CBABH (6.1 mg, 5.0 μmol, 66% yield) was afforded as a
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.joc.0c01977
1
white solid. H NMR (400 MHz, CDCl3 with 0.5% TFA and 0.1%
H2O): δ 9.98 (s, 1H), 9.95 (s, 1H), 8.20 (t, J = 1.6 Hz, 1H), 8.19 (t, J
= 1.6 Hz, 1H), 8.18−8.16 (overlap, 2H), 8.09 (t, J = 1.6 Hz, 1H),
8.06−8.00 (overlap, 4H), 8.00−7.96 (overlap, 2H), 7.94−7.91
(overlap, 2H), 7.91 (t, J = 1.4 Hz, 1H), 7.83 (t, J = 1.4 Hz, 1H),
7.77 (t, J = 1.5 Hz, 1H), 7.58−7.54 (overlap, 2H), 4.61−4.51
(overlap, 6H), 3.97−3.89 (overlap, 6H), 3.84−3.77 (overlap, 6H),
3.76−3.70 (overlap, 6H), 3.49−3.47 (overlap, 6H), 3.47 (s, 3H), 3.22
(s, 1H), 2.64 (t, J = 7.3 Hz, 2H), 2.55 (t, J = 6.9 Hz, 2H), 1.99 (tt, J =
7.3, 6.9 Hz, 2H). HRMS (ESI) m/z: [M + Na]+ calcd for
C74H63NO16Na, 1244.4039; found, 1244.4133.
CBBBAH. The resin (62 mg, 81 μmol/g loading) functionalized with
CBBBAH was cleaved and purified according to the procedure
outlined above, and CBBBAH (2.9 mg, 1.8 μmol, 37% yield) was
afforded as a white solid. 1H NMR (400 MHz, CDCl3 with 0.5% TFA
and 0.1% H2O): δ 10.00 (s, 1H), 9.99 (s, 1H), 9.98 (s, 1H), 8.22−
8.18 (overlap, 5H), 8.15 (t, J = 1.6 Hz, 1H), 8.09 (t, J = 1.6 Hz, 1H),
8.08−7.99 (overlap, 9H), 7.97 (t, J = 1.5 Hz, 1H), 7.95 (t, J = 1.6 Hz,
1H), 7.94 (t, J = 1.6 Hz, 1H), 7.90 (t, J = 1.5 Hz, 1H), 7.76 (t, J = 1.6
Hz, 1H), 7.75 (s, 1H), 7.54 (s, 1H), 7.48 (s, 1H), 4.62−4.51 (overlap,
8H), 3.98−3.88 (overlap, 8H), 3.85−3.77 (overlap, 8H), 3.77−3.70
(overlap, 8H), 3.49−3.48 (overlap, 9H), 3.47 (s, 3H), 3.24 (s, 1H),
2.64 (t, J = 7.3 Hz, 2H), 2.55 (t, J = 6.8 Hz, 2H), 1.99 (tt, J = 7.3, 6.8
Hz, 2H). HRMS (ESI) m/z: [M + H]+ calcd for C97H82NO21,
1597.5408; found, 1597.5484.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors thank Dr. Noam Prywes for his thoughts and
helpful comments on this manuscript, as well as the Szostak
laboratory members for useful discussions. This work was
supported by a grant (290363) from the Simons Foundation to
J.W.S. J.W.S. is an Investigator of the Howard Hughes Medical
Institute.
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