Sequence-defined polymers via orthogonal allyl acrylamide building blocks

Article abstract of DOI:10.1021/ja507262t

Biological systems have long recognized the importance of macromolecular diversity and have evolved efficient processes for the rapid synthesis of sequence-defined biopolymers. However, achieving sequence control via synthetic methods has proven to be a difficult challenge. Herein we describe efforts to circumvent this difficulty via the use of orthogonal allyl acrylamide building blocks and a liquid-phase fluorous support for the de novo design and synthesis of sequence-specific polymers. We demonstrate proof-of-concept via synthesis and characterization of two sequence-isomeric 10-mer polymers. 1H NMR and LCMS were used to confirm their chemical structure while tandem MS was used to confirm sequence identity. Further validation of this methodology was provided via the successful synthesis of a sequence-specific 16-mer polymer incorporating nine different monomers. This strategy thus shows promise as an efficient approach for the assembly of sequence-specific functional polymers.

Full text of DOI:10.1021/ja507262t

Communication
pubs.acs.org/JACS
Sequence-Defined Polymers via Orthogonal Allyl Acrylamide
Building Blocks
Mintu Porel and Christopher A. Alabi*
School of Chemical and Biomolecular Engineering, Cornell University, Ithaca, New York 14853, United States
S
* Supporting Information
this approach are templated reactions and sequential living
ABSTRACT: Biological systems have long recognized the
importance of macromolecular diversity and have evolved
efficient processes for the rapid synthesis of sequence-
defined biopolymers. However, achieving sequence control
via synthetic methods has proven to be a difficult
challenge. Herein we describe efforts to circumvent this
difficulty via the use of orthogonal allyl acrylamide building
blocks and a liquid-phase fluorous support for the de novo
design and synthesis of sequence-specific polymers. We
demonstrate proof-of-concept via synthesis and character-
radical polymerization. Templated reactions involve the spatial
arrangement and subsequent ligation of monomers on a
preorganized template2−4 or the sequential use complementary
templates to facilitate monomer ligation.5,6 Sequential living
radical polymerizations involve precise tuning of monomer
reactivity (or relative reactivity) and reaction time to achieve
well-defined multiblock copolymers.7−10 The latter offers
operationally simple conditions, with a trade-off of more limited
sequence control.
In contrast to the one-pot approach, the supported synthesis
approach involves the iterative addition and washing of
monomers to a tethered solid or liquid support. This approach
was first developed for the synthesis of peptides11 and nucleic
acids12 and involves extensive monomer protection and
deprotection chemistries. Recently, alternative strategies that
circumvent the need for protecting groups via the use of
orthogonal chemical reactions have been developed. Notable
examples include the submonomer method for peptoid syn-
thesis,13,14 thiolactone aminolysis,15 and the Passerini three-
component reaction.16 Inspired by the promise of these
1
ization of two sequence-isomeric 10-mer polymers. H
NMR and LCMS were used to confirm their chemical
structure while tandem MS was used to confirm sequence
identity. Further validation of this methodology was
provided via the successful synthesis of a sequence-specific
16-mer polymer incorporating nine different monomers.
This strategy thus shows promise as an efficient approach
for the assembly of sequence-specific functional polymers.
patial control of a monomer sequence along a polymer
Sbackbone is essential to the complex self-assembly of
proteins and nucleic acids. To achieve macromolecular diversity,
biological systems have evolved extremely efficient processes for
the rapid synthesis of sequence-defined biopolymers virtually
error free. Similarly, achieving primary sequence control using
synthetic monomers, for which we have a nearly unlimited
toolbox, should facilitate control over structural properties such
as folding, self-assembly into nanostructures, structural stimuli
response, and formation of catalytic sites. These structural
properties will invariably determine bulk material properties
including solubility, conductivity, elasticity, nonfouling, bio-
compatibility, and catalytic performance.1 Understanding
sequence−structure−material property relationships is of para-
mount importance toward our ability to carry out predictive
bottom-up materials design and fabrication. Progress toward this
goal requires the development of reliable methods for achieving
precise polymeric sequence control.
Synthetic approaches should enable large-scale production of
sequence-controlled polymers with massive structural diversity.
Synthetic efforts toward controlling polymer sequence, i.e. the
placement of different monomeric groups in a predefined order
along a polymer chain, can be grouped into two categories: (i)
one-pot synthesis approach and (ii) supported synthesis
approach. In the one-pot approach, different monomers are
introduced and allowed to react to near full conversion, with no
purification steps. Two strategies that fall under the umbrella of
Scheme 1. Fluorous Assisted Sequence Control via Allyl
Acrylamides and Dithiols
approaches and the need for increased backbone diversity, we
report an efficient strategy for the assembly of sequence-defined
polymers via unique allyl acrylamide building blocks with
orthogonal reactive sites coupled with a powerful fluorous
separation technology (Scheme 1). The latter allows us to
decouple monomer reaction from purification by performing
Received: July 21, 2014
Published: September 10, 2014
© 2014 American Chemical Society
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monomer addition in solution and polymer purification on a
supported solid phase.
Our approach for achieving synthetic sequence control
involves the design of a unique monomer with two orthogonal
reactive sites to the same nucleophile. The monomer framework
includes a reactive acrylamide group that can undergo
phosphine-catalyzed Michael addition with thiols, the desired
functional group and a reactive allyl group that can undergo
photoinitiated thiol−ene “click” addition. These reactions were
chosen due to their rapid solution kinetics at room temperature
and pressure.17 Since both reactive ends of the monomer
undergo orthogonal reactions with thiols, we reasoned that a
dithiol molecule could be used as a comonomer without the need
for protective groups. The allyl acrylamide monomer can be
synthesized in two steps from a plethora of primary amines or
halides (Figure 1) and tolerates many functional groups. For this
Figure 1. (i) For X = NH2; K2CO3, allyl bromide (0.2 equiv). For X =
Br/Cl; K2CO3, allyl amine (5 equiv). (ii) Acryloyl chloride (1 equiv),
Et3N, CH2Cl2.
Figure 2. Assembly of a test oligomer. (i) fluorous polymer: 1,3-
propanedithiol (1:5), 2,2-dimethoxy-2-phenyl acetophenone (DMPA),
hv (20 mW/cm2), MeOH; (ii) fluorous polymer: 2a (1:2), Me2PhP,
MeOH; (iii) 50% trifluoroacetic acid (TFA) in CH2Cl2. Fluorous tag is
highlighted in green. Blue dots, olefin protons; green dots, CH2CH2C-
(CH3)2 protons on fluorous tag; and red dots, SH proton. Full spectra
(with omitted sections 2.5−3.5 ppm) are available in Figure S5.
proof-of-concept study, we synthesized eight different allyl
acrylamide monomers 2a−h and utilized 1,3-propanedithiol as
the comonomer. In our hands, the phosphine-catalyzed Michael
addition of N-allyl-N-methylacrylamide, 2a (Figure 1), with 1,3-
propanedithiol is complete in 540 s (Figure S1), and the
photoinitiated thiol−ene reaction of N-allyl-N-methyl-3-(octyl-
thio)acrylamide (SI, page 8) with 1,3-propanedithiol is complete
in 90 s (Figure S2).
and 1:0.5 demonstrated that, for fluorous-tagged substrates, the
use of more than a 2-fold excess of dithiol is necessary to push the
reaction to completion in 90 s (Figure S6A). In addition, the
monosubstituted product appears to be the dominant species
even at a low ratio of 1:0.5 (Figure S6B). The progress of the
thiol−ene reaction was followed via the appearance of the thiol
peak (red dot) at ∼1.33 ppm and the disappearance of the olefin
peaks (blue dots) at ∼5.14 and 5.75 ppm (Figure 2). The
phosphine-catalyzed Michael addition proceeded slightly faster
on fluorous-tagged substrates and was complete in just 300 s
(Figure S7). The Michael addition reaction progress was
monitored via the disappearance of the thiol peak (red dot)
and the reappearance of the olefin peaks (blue dot). The wash
and elute steps, which take place between each monomer
addition, were performed over a prepacked mini-fluorous silica
column in ∼5 min. These prepacked columns can be regenerated
and reused up to 10 times. As such, the total time for monomer or
comonomer addition is roughly 10−15 min. After this first round
of monomer addition, we continued oligomer synthesis with
another round of dithiol and N-allyl-N-methylacrylamide
addition. Again, both reactions proceeded smoothly as
We employed fluorous tags as a liquid phase reaction support
in order to combine the advantages of both solution-phase and
solid-phase iterative syntheses. Fluorous tags are removable
perfluorocarbon alkyl chains that are soluble in common organic
solvents, yet selectively partition onto a fluorous solid phase.18
Assembling our sequence-defined polymers on fluorous tags
allows us to perform monomer addition in solution while
simultaneously benefiting from rapid fluorous solid phase
extraction (FSPE) for purification. The advantages of using a
fluorous liquid support include homogeneous reaction con-
ditions, fast solution phase kinetics, and reaction monitoring via
common spectroscopic techniques. Fluorous tags are also inert
to most common reaction conditions and are commercially
available. We employed a fluorous tag with an acid cleavable Boc
functionality to initiate our proof-of-concept studies.
To verify that the reaction kinetics remain rapid in the
presence of bulky fluorous tags and test the efficiency of FSPE,
we initiated the synthesis of a short oligomer with N-allyl-N-
methylacrylamide, 2a, and 1,3-propanedithiol (Figures 2 and
S3−S5). The thiol−ene reaction of 1,3-propanedithiol (5-fold
excess) with a fluorous Boc protected allyl amine was complete in
90 s and provided the monosubstituted product exclusively, as
determined via 1H NMR (Figure 2). Additional experiments that
employed a fluorous tagged allyl amine to dithiol ratio of 1:2, 1:1,
1
determined by H NMR (Figure 2) and mass spectroscopy
(Figure S8). Finally, we performed an acid deprotection to cleave
the desired oligomer off the fluorous support. The structure of
the final product was confirmed by 1H NMR via the
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disappearance of the fluorous Boc groups (green) at 1.46, 1.98,
and 2.11 ppm respectively (Figure 2) and by liquid
chromatography/mass spectrometry (LCMS) (Figure S8).
After verification of the reaction kinetics in the presence of
fluorous tags, we proceeded with the synthesis of two 10-mer
sequence isomeric polymers, isomer 1 and isomer 2 (ISO1 and
ISO2, Figure 3a). Both polymers were synthesized with the same
1386.62 Da. The multiply charged [M + 2H]2+ and [M + 3H]3+
ions for both isomers were also identical.
Sequence identity was elucidated via a tandem MS (MS/MS)
experiment on one isotope of the [M + 2H]2+ ion. Cleavage of
the carbon−sulfur bond β to the carbonyl to yield a thiol-
containing [M + 2]+ fragment ion was the most prominent and
consistent fragmentation mechanism. All expected M ions, with
the exception of M1 due to its mass being below the detection
limit, were detected in the MS/MS fragmentation spectra
(Figure 3c; see Figures S18, S19 and Tables S1, S2 for full
fragmentation analysis). Moreover, none of the M ions of ISO1
could be detected in the fragmentation profile of ISO2, and vice
versa. Identification of the desired M ions as well as differences in
the fragmentation pattern of the two sequence isomers confirms
their chemical identity and sequence specificity.
To further test the durability and scope of this synthetic
method for attaining sequence control, we synthesized a 16-mer
polymer (Figure 4a) consisting of all eight allyl acrylamide
Figure 3. (a) Chemical structure of two polymeric sequence isomers
(ISO1-green and ISO2-red). (b) MS spectra of ISO1 and ISO2. (c)
Tandem MS spectra of parent ion [M + 2H]2+ showing sequence
specific fragmentation patterns. M ions are annotated.
allyl acrylamide monomers (2a, 2c, 2e, 2f, and 2h) and 1,3-
propanedithiol as the comonomer but have different sequences
(Figure 3a). The synthesis of ISO1 was followed and confirmed
at each step with 1H NMR and LCMS (Figures S9−S15). ISO2
on the other hand was synthesized in one setting without
spectroscopic stepwise confirmation in less than a day with pre-
and postcleavage yields of 72% and 68% respectively. As shown
Figure 4. (a) Schematic representation of 16-mer polymer. (b) LCMS
and MALDI (inset) of 16-mer polymer. (c) Tandem MS spectra of 16-
mer polymer. Inset shows M2 fragment. M ions are annotated. a m/z
ratio for monoisotopic species.
1
in Figure S16A and B, the H NMR of fluorous tagged and
cleaved ISO1 and ISO2 are nearly identical. Minor differences in
peak positions are possibly due to the differences in the relative
positions of the functional groups. ISO1 and ISO2 were analyzed
and purified via reversed phase chromatography and eluted at
17.8 and 17.1 min respectively (Figure S17). Further structural
confirmation of both isomers was obtained via LCMS. The
observed parent ions of the two sequence isomers were identical
at 1386.66 Da (Figure 3b) and matched the theoretical value of
monomers (Figure 1) and 1,3-propanedithiol as the comonomer.
LCMS of the final cleaved product only showed the multiply
charged [M + 2H]2+, [M + 3H]3+, and [M + 4H]4+ ions as the
parent [M + H]+ ion (2198.02 Da) was beyond the instrument
detection limit. The total parent mass of the polymer was
confirmed by MALDI (Figure 4b inset, [M + Na]+ and [M + K]+
1
ions). The H NMR of the full product (Figure S20) is also in
agreement with the proposed structure. The observed peaks in
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the MS/MS fragmentation pattern of the [M + 2H]2+ ion
matched those of the assigned M ions in the polymer structure
(Figure 4c; see Figure S21 and Table S3 for full fragmentation
analysis) thus confirming the molecular sequence of the 16-mer
product. Although the C9F19 fluorous tag employed in this work
was capable of separating the 16-mer polymer from its
byproducts, we do anticipate a decrease in recovery as the
organic to fluorine ratio increases. However, the latter can be
circumvented with the use of larger fluorous tags.
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In conclusion, we have described the development of new
functional allyl acrylamide monomers coupled with an innovative
fluorous-mediated methodology for the rapid and efficient
assembly of sequence-defined polymers. We have shown
1
evidence of sequential polymer assembly via H NMR and
LCMS. The sequences of our prepared polymers were confirmed
via tandem MS fragmentation analysis. The ease of our monomer
synthesis along with the rapid polymer assembly should facilitate
exploration of a wide variety of monomer combinations and
functional polymer structures that may lead to the discovery of
advanced materials. Additionally, all the polymers generated by
our coupling strategy have an amine and alkene as orthogonal
terminal functionalities, which make them attractive for
postsynthetic modification of proteins, nucleic acids, nano-
particles, and other biomaterials. Precise control over the
polymer sequence holds great potential for mediating a high
level of control over the chemical and physical properties of
materials, which can span the molecular to the macromolecular
scale. As such, our future studies will focus on expanding the
number and types of available building blocks as well as the
synthesis of longer functional sequence-specific polymeric
structures. We anticipate these polymers will find use in
sequence-specific self-assembly, stimuli-responsive materials,
controlled drug delivery, and much more.
ASSOCIATED CONTENT
■
S
* Supporting Information
Experimental procedures, details on the synthesis of monomers
1
and polymers, and additional spectroscopic data such as H
NMR and LCMS are included in the Supporting Information.
This material is available free of charge via the Internet at http://
pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
caa238@cornell.edu.
Funding
No competing financial interests have been declared.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by startup research funds from Cornell
University and the Nancy and Peter Meinig Investigator
Fellowship. The authors also acknowledge Jonathan Shrimp
and Professor Hening Lin for access to and assistance with
acquiring LCMS and tandem MS data. This work made use of the
Cornell Center for Materials Research Shared Facilities
supported by the National Science Foundation under Award
Number DMR-1120296.
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