Exchanging and Releasing Information in Synthetic Digital Polymers Using a Strand-Displacement Strategy.

Toehold-mediated strand displacement (TMSD) was tested as a tool to edit information in synthetic digital polymers. Uniform DNA-polymer biohybrid macromolecules were first synthesized by automated phosphoramidite chemistry and characterized by HPLC, mass spectrometry, and polyacrylamide gel electrophoresis (PAGE). These precursors were diblock structures containing a synthetic poly(phosphodiester) (PPDE) segment covalently attached to a single-stranded DNA sequence. Three types of biohybrids were prepared herein: a substrate containing an accessible toehold as well as input and output macromolecules. The substrate and the input macromolecules contained noncoded PPDE homopolymers, whereas the output macromolecule contained a digitally encoded segment. After hybridization of the substrate with the output, incubation in the presence of the input led to efficient TMSD and the release of the digital segment. TMSD can therefore be used to erase or rewrite information in self-assembled biohybrid superstructures. Furthermore, it was found in this work that the conjugation of DNA single strands to synthetic segments of chosen lengths greatly facilitates the characterization and PAGE visualization of the TMSD process.

Using ion mobility spectrometry to understand signal dilution during tandem mass spectrometry sequencing of digital polymers: Experimental evidence of intramolecular cyclization.

RATIONALE: Optimizing the structure of digital polymers is an efficient strategy to ensure their tandem mass spectrometry (MS/MS) readability. In block-truncated poly(phosphodiester)s, homolysis of C-ON bonds in long chains permits the release of smaller blocks amenable to sequencing. Yet the dissociation behavior of diradical blocks was observed to strongly depend on their charge state. METHODS: Polymers were ionized in negative mode electrospray and activated in-source so that blocks released as primary fragments can be investigated using ion mobility spectrometry (IMS) or sequenced in the post-IMS collision cell. Collision cross sections (CCS) were derived from arrival times using a calibration procedure developed for polyanions using the IMSCal software. A multistep protocol based on quantum methods and classical molecular dynamics was implemented for molecular modeling and calculation of theoretical CCS. RESULTS: Unlike their triply charged homologues, dissociation of diradical blocks at the 2- charge state produces additional fragments, with +1 m/z shift for those holding the nitroxide α-termination and -1 m/z for those containing the carbon-centered radical ω-end. These results suggest cyclization of these diradical species, followed by H• transfer on activated reopening of this cycle. This assumption was validated using IMS resolution of the cyclic/linear isomers and supported by molecular modeling. CONCLUSIONS: Combining IMS with molecular modeling provided new insights into how the charge state of digital blocks influences their dissociation. These results permit to define new guidelines to improve either ionization conditions or the structural design of these digital polymers for best MS/MS readability.

Complex Sequence-Defined Heteropolymers Enable Controlled Film Growth in Layer-By-Layer Assembly.

Digitally-encoded poly(phosphodiesters) (d-PPDE) with highly complex primary structures are evaluated for layer-by-layer (LbL) assembly. To be easily decoded by mass spectrometry (MS), these digital polymers contain many different monomers: 2 coding units allowing binary encryption, 1 cleavable spacer allowing controlled MS fragmentation, and 3 mass tags allowing fragment identification. These complex heteropolymers are therefore composed of 6 different motifs. Despite this strong sequence heterogeneity, it is found that they enable a highly controlled LbL film formation. For instance, a regular growth is observed when alternating the deposition of negatively-charged d-PPDE and positively-charged poly(allyl amine hydrochloride) (PAH). Yet, in this approach, the interdistance between consecutive coded d-PPDE layers remains relatively small, which may be an issue for data storage applications, especially for the selective decoding of the stored information. Using poly(sodium 4-styrene sulfonate) (PSS) as an intermediate non-coded polyanion, it is shown that a controlled interdistance between d-PPDE layers can be easily achieved, while still maintaining a regular LbL growth. Last but not least, it is found in this work that d-PPDE of relatively small molecular weight (i.e., significantly smaller than those of PAH and PSS) still enables a controlled LbL assembly.

Macromolecular Information Transfer.

Macromolecular information transfer can be defined as the process by which a coded monomer sequence is communicated from one macromolecule to another. In such a transfer process, the information sequence can be kept identical, transformed into a complementary sequence or even translated into a different molecular language. Such mechanisms are crucial in biology and take place in DNA→DNA replication, DNA→RNA transcription and RNA→protein translation. In fact, there would be no life on Earth without macromolecular information transfer. Mimicking such processes with synthetic macromolecules would also be of major scientific relevance because it would open up new avenues for technological applications (e.g. data storage and processing) but also for the creation of artificial life. In this important context, this minireview summarizes recent research about information transfer in synthetic oligomers and polymers. Medium- and long-term perspectives are also discussed.

Conception and Evaluation of a Library of Cleavable Mass Tags for Digital Polymers Sequencing.

A library of phosphoramidite monomers containing a main-chain cleavable alkoxyamine and a side-chain substituent of variable molar mass (i.e. mass tag) was prepared in this work. These monomers can be used in automated solid-phase phosphoramidite chemistry and therefore incorporated periodically as spacers inside digitally-encoded poly(phosphodiester) chains. Consequently, the formed polymers contain tagged cleavable sites that guide their fragmentation in mass spectrometry sequencing and enhance their digital readability. The spacers were all prepared via a seven steps synthetic procedure. They were afterwards tested for the synthesis and sequencing of model digital polymers. Uniform digitally-encoded polymers were obtained as major species in all cases, even though some minor defects were sometimes detected. Furthermore, the polymers were decoded in pseudo-MS3 conditions, thus confirming the reliability and versatility of the spacers library.

Design of Abiological Digital Poly(phosphodiester)s.

In biological systems, the storage and transfer of genetic information rely on sequence-controlled nucleic acids such as DNA and RNA. It has been realized for quite some time that this property is not only crucial for life but could also be very useful in human applications. For instance, DNA has been actively investigated as a digital storage medium over the past decade. Indeed, the "hard-disk of life" is an obvious choice and a highly optimized material for storing data. Through decades of nucleic acids research, technological tools for parallel synthesis and sequencing of DNA have been readily available. Consequently, it has already been demonstrated that different types of documents (e.g., texts, images, videos, and industrial data) can be stored in chemically synthesized DNA libraries. However, DNA is subject to biological constraints, and its molecular structure cannot be easily varied to match technological needs. In fact, DNA is not the only macromolecule that enables data storage. In recent years, it has been demonstrated that a wide variety of synthetic polymers can also be used for such a purpose. Indeed, modern polymer synthesis allows the preparation of synthetic macromolecules with precisely controlled monomer sequences. Altogether, about a dozens of synthetic digital polymers have already been described, and many more can be foreseen. Among them, sequence-defined poly(phosphodiester)s are one of the most promising options. These polymers are prepared by stepwise phosphoramidite chemistry like chemically synthesized oligonucleotides. However, they are constructed with non-natural building blocks and therefore share almost no structural characteristics with nucleic acids, except phosphate repeat units. Still, they contain readable digital messages that can be deciphered by nanopore sequencing or mass spectrometry sequencing. In this Account, we describe our recent research efforts in synthesizing and sequencing optimal abiological digital poly(phosphodiester)s. A major advantage of these polymers over DNA is that their molecular structure can easily be varied to tune their properties. During the last 5 years, we have engineered the molecular structure of these polymers to adjust crucial parameters such as the storage density, storage capacity, erasability, and readability. Consequently, high-capacity PPDE chains, containing hundreds of bits per chains, can now be synthesized and efficiently sequenced using a routine mass spectrometer. Furthermore, sequencing data can be automatically decrypted with the help of decoding software. This new type of coded matter can also be edited using practical physical triggers such as light and organized in space by programmed self-assembly. All of these recent improvements are summarized and discussed herein.

Covalent Attachment and Detachment by Reactive DESI of Sequence-Coded Polymer Taggants.

The use of sequence-defined polymers is an interesting emerging solution for materials identification and traceability. Indeed, a very large amount of identification sequences can be created using a limited alphabet of coded monomers. However, in all reported studies, sequence-defined taggants are usually included in a host material by noncovalent adsorption or entrapment, which may lead to leakage, aggregation, or degradation. To avoid these problems, sequence-defined polymers are covalently attached in the present work to the mesh of model materials, namely acrylamide hydrogels. To do so, sequence-coded polyurethanes containing a disulfide linker and a terminal methacrylamide moiety are synthesized by stepwise solid-phase synthesis. These methacrylamide macromonomers are afterward copolymerized with acrylamide and bisacrylamide in order to achieve cross-linked hydrogels containing covalently-bound polyurethane taggants. It is shown herein that these taggants can be selectively detached from the hydrogel mesh by reactive desorption electrospray ionization. Using dithiothreitol the disulfide linker that links the taggant to the gel can be selectively cleaved. Ultimately, the released taggants can be decoded by tandem mass spectrometry.

Chain Entropy Beats Hydrogen Bonds to Unfold and Thread Dialcohol Phosphates inside Cyanostar Macrocycles To Form [3]Pseudorotaxanes.

The recognition of substituted phosphates underpins many processes including DNA binding, enantioselective catalysis, and recently template-directed rotaxane synthesis. Beyond ATP and a few commercial substrates, however, little is known about how substituents effect organophosphate recognition. Here, we examined alcohol substituents and their impact on recognition by cyanostar macrocycles. The organophosphates were disubstituted by alcohols of various chain lengths, dipropanol, dihexanol, and didecanol phosphate, each accessed using modular solid-phases syntheses. Based on the known size-selective binding of phosphates by π-stacked dimers of cyanostars, threaded [3]pseudorotaxanes were anticipated. While seen with butyl substituents, pseudorotaxane formation was disrupted by competitive OH···O- hydrogen bonding between both terminal hydroxyls and the anionic phosphate unit. Crystallography also showed formation of a backfolded propanol conformation resulting in an 8-membered ring and a perched cyanostar assembly. Motivated by established entropic penalties accompanying ring formation, we reinstated [3]pseudorotaxanes by extending the size of the substituent to hexanol and decanol. Chain entropy overcomes the enthalpically favored OH···O- contacts to favor random-coil conformations required for seamless, high-fidelity threading of dihexanol and didecanol phosphates inside cyanostars. These studies highlight how chain length and functional groups on phosphate's substituents can be powerful design tools to regulate binding and control assembly formation during phosphate recognition.

Precise Alkoxyamine Design to Enable Automated Tandem Mass Spectrometry Sequencing of Digital Poly(phosphodiester)s.

A major step towards reliable reading of information coded in the sequence of long poly(phosphodiester)s was previously achieved by introducing an alkoxyamine spacer between information sub-segments. However, MS/MS decoding had to be performed manually to safely identify useful fragments of low abundance compared to side-products from the amide-based alkoxyamine used. Here, alternative alkoxyamines were designed to prevent side-reactions and enable automated MS/MS sequencing. Different styryl-TEMPO spacers were prepared to increase radical delocalization and stiffness of the structure. Their dissociation behavior was investigated by EPR and best results were obtained with spacers containing in-chain benzyl ring, with no side-reaction during synthesis or sequencing. Automated decoding of these polymers was performed using the MS-DECODER software, which interprets fragmentation data recorded for each sub-segment and re-align them in their original order based on location tags.

Optimal conditions for tandem mass spectrometric sequencing of information-containing nitrogen-substituted polyurethanes.

RATIONALE: To prevent solubility issues faced with sequence-defined polyurethanes, a new family of digital polyurethanes was conceived with the alkyl coding chain held by the carbamate nitrogen (N) atom and CH3 instead of OH as the ϖ termination. This led to different dissociation mechanisms that were explored prior to optimizing tandem mass spectrometric (MS/MS) sequencing. METHODS: N-Substituted polyurethanes (N-R PUs) were dissolved in methanol and subjected to collision-induced dissociation (CID) as deprotonated chains in the negative ion mode, and as ammonium and sodium adducts in the positive ion mode, using electrospray ionization (ESI) as the ionization technique. Their dissociation behavior was thoroughly investigated using a quadrupole time-of-flight (QTOF) instrument in order to provide accurate mass measurements to support proposed fragmentation mechanisms. RESULTS: While O-(CO) bonds were broken in N-H PUs, the CH2 -O linkage between repeating units was cleaved upon CID of N-R PUs. This main process occurred either from deprotonated molecules or from cationized chains but was followed by different rearrangements, producing up to four product ion series. Yet, MS/MS spectra could be drastically simplified for precursor ions having their acidic α group methylated, as was found to spontaneously occur upon storage in methanol. CONCLUSIONS: Using experimental conditions aimed at avoiding any reactive proton in precursor ions (no acidic end-groups and alkali adduction), full coverage sequence of N-R PUs was successfully achieved with the single ion series observed in MS/MS, opening a promising perspective for reading large amounts of information stored in these dyad-encoded polymers.

Selective Bond Cleavage in Informational Poly(Alkoxyamine Phosphodiester)s.

The collision-induced dissociation (CID) of sequence-defined poly(alkoxyamine phosphodiester)s is studied by electrospray ionization mass spectrometry. These informational polymers are synthesized using three different nitroxide building blocks, namely proxyl-, SG1-, and TEMPO-derivatives. For a polymer containing TEMPO- and SG1-based main chain alkoxyamines, it is found that both types of alkoxyamines break in CID tandem mass spectrometry (MS/MS). However, SG1-sites are preferentially cleaved and this predominance can be increased by reducing collision energy, even though selective bond fragmentation is not observed. On the other hand, for a polymer containing proxyl- and SG1-alkoxyamines, selective bond cleavage is observed at all studied collision energies. The SG1-alkoxyamines can be first cleaved in MS/MS conditions and secondly the proxyl-alkoxyamines in pseudo-MS3 conditions. These results open up interesting new avenues for the design of readable, erasable or programmable informational polymers.

Damage and Repair in Informational Poly(N-substituted urethane)s.

The degradation and repair of uniform sequence-defined poly(N-substituted urethane)s was studied. Polymers containing an ω-OH end-group and only ethyl carbamate main-chain repeat units rapidly degrade in NaOH solution through an ω→α depolymerization mechanism with no apparent sign of random chain cleavage. The degradation mechanism is not notably affected by the nature of the side-chain N-substituents and took place for all studied sequences. On the other hand, depolymerization is significantly influenced by the molecular structure of the main-chain repeat units. For instance, hexyl carbamate main-chain motifs block unzipping and can therefore be used to control the degradation of specific sequence sections. Interestingly, the partially degraded polymers can also be repaired; for example by using a combination of N,N'-disuccinimidyl carbonate with a secondary amine building-block. Overall, these findings open up interesting new avenues for chain-healing and sequence editing.

Revealing Data Encrypted in Sequence-Controlled Poly(Alkoxyamine Phosphodiester)s by Combining Ion Mobility with Tandem Mass Spectrometry.

The defined sequence of two comonomers in sequence-controlled macromolecules can be used to store binary information which is further decoded by MS/MS sequencing. In order to achieve the full sequence coverage requested for reliable decoding, the structure of these polymers can be optimized to minimize their dissociation extent, as shown for poly(alkoxyamine phosphodiester)s (PAPs) where weak alkoxyamine bonds were introduced in each repeating unit to make all phosphate groups MS/MS silent. However, for secret communications, a too high MS/MS readability could be a drawback. In this context, the design of PAPs was further optimized in this work to also include a decrypting key based on slight variation of a fragment collision cross section. This was achieved by employing two different nitroxides to build the alkoxyamine moiety, each containing a coding alkyl segment of the same mass but different architectures. As a result, the digital sequence determined from primary fragments observed in MS/MS had to be decrypted according to appropriate rules that depend on the drift times measured by ion mobility spectrometry for repeating units released as secondary product ions.

Synthesis of Macromolecules Containing Phenylalanine and Aliphatic Building Blocks.

Aiming at developing efficient interfacial agents for fiber-reinforced composite materials, macromolecules are designed to have different components able to stick to the fiber and be compatible with the polymer matrix, respectively. Herein, macromolecules are prepared by solid-phase synthesis considering phenylalanine residues to promote adsorption of the macromolecule on aramid fibers and aliphatic building blocks to interact with a hydrophobic polymer matrix. Using phenylalanine as building block for the preparation of macromolecules by iterative synthesis has been shown to be challenging. Thus, the screening of various parameters for the optimization of the synthesis of these macromolecules is discussed in this communication. A preliminary thermal study by thermal gravimetric analysis is conducted to evaluate their thermal stability.

Sequences of Sequences: Spatial Organization of Coded Matter through Layer-by-Layer Assembly of Digital Polymers.

A library of 16 digitally encoded polyanions was used in a layer-by-layer (LbL) polyelectrolyte assembly to nanofabricate thin films containing digitally coded strata. The polyanions were digital polyphosphodiesters (d-PPDE) prepared via an automated phosphoramidite process. Each component of the library contained 10 bytes of ASCII-encoded text (i.e. 80 coded monomers); thus the entire library allows the writing of a full sentence, which can be stored in a multilayer film as a sequence of sequences. To prepare fully segregated digital domains, non-coded layers composed of poly(allylamine hydrochloride) (PAH)/poly(sodium 4-styrenesulfonate) (PSS) were included between the d-PPDE coded layers as an intermediate barrier. Detailed analysis of the film homogeneity indicated formation of 70 nm-thick films in which digital layers are kept apart from another by non-coded interlayers. As a result, the sequence-coded polymer library could be piled-up in a defined sequence of layers.

Cleavable Binary Dyads: Simplifying Data Extraction and Increasing Storage Density in Digital Polymers.

Digital polymers are uniform macromolecules that store monomer-based binary sequences. Molecularly stored information is usually extracted from the polymer by a tandem mass spectrometry (MS/MS) measurement, in which the coded chains are fragmented to reveal each bit (i.e. basic coded monomer unit) of the sequence. Here, we show that data-extraction can be greatly simplified by favoring the formation of MS/MS fragments containing two bits instead of one. In order to do so, digital poly(alkoxyamine phosphodiester)s, containing binary dyads in each repeat unit, were prepared by an orthogonal solid-phase approach involving successive phosphoramidite and radical-radical coupling steps. Three different sets of monomers were considered to build these polymers. In all cases, four coded building blocks-two hydroxy-nitroxides and two phosphoramidite monomers-were required to build the dyads. Among the three studied monomer sets, one combination allowed synthesis of uniform sequence-coded polymers. The resulting polymers led to clear dyad-containing fragments in MS/MS and could therefore be efficiently decoded. Additionally, an algorithm was created to detect specific dyad fragments, thus enabling automated sequencing.

Abiotic Sequence-Coded Oligomers as Efficient In†Vivo Taggants for the Identification of Implanted Materials.

Sequence-defined oligourethanes were tested as in vivo taggants for implant identification. The oligomers were prepared in an orthogonal solid-phase iterative approach and thus contained a coded monomer sequence that can be unequivocally identified by tandem mass spectrometry (MS/MS). The oligomers were then included in small amounts (1 wt %) in square-centimeter-sized crosslinked poly(vinyl alcohol) (PVA) model films, which were intramuscularly and subcutaneously implanted in the abdomen of rats. After one week, one month, or three months of implantation, the PVA films were explanted. The rat tissues exposed to the implants did not exhibit any adverse reactions, which suggested that the taggants are not harmful and probably not leaching out from the films. Furthermore, the explanted films were immersed in methanol, as a solvent for oligourethanes, and the liquid extract was analyzed by mass spectrometry. In all cases, the oligourethane taggant was detected, and its sequence was identified by MS/MS.

Defining the Field of Sequence-Controlled Polymers.

Over the last ten years, the development of synthetic polymers containing controlled monomer sequences has become a prominent topic in fundamental and applied polymer science. This emerging area is particularly broad and combines classical polymer chemistry tools with techniques imported from other domains such as biology, biochemistry, organic synthesis, engineering, and bioanalytics. Consequently, it also generates new structures, terminologies, and applications that are not within the traditional scope of polymer science. The term "sequence-controlled polymers" (SCPs) was recently proposed as a generic name to describe all these recent trends. However, since the field of SCPs has been growing very rapidly in recent literature, it is urgent to accurately define its scientific frontiers. In this important context, this review is an attempt to define, rationalize, and classify the field of SCPs. In particular, all synthetic approaches that have been reported for the synthesis of SCPs are discussed and categorized. In addition, the characterization tools, properties, and potential applications of these new polymers are described herein. Overall, this review serves as a reference guide for understanding the burgeoning field of SCPs.

2D Sequence-Coded Oligourethane Barcodes for Plastic Materials Labeling.

Mixtures of uniform sequence-defined oligourethanes are evaluated as 2D molecular barcodes for labeling three different commodity polymers, namely polystyrene, polyvinylchloride and polyethylene terephthalate. Six different oligourethanes are synthesized by solid-phase iterative synthesis and are coded using a binary monomer alphabet. High-resolution mass spectrometry studies indicate that all oligomers are uniform and sequence-defined. However, instead of using them as individual coded chains, oligomers with different chain-length, mass and sequence are mixed into intentionally polydispersed libraries. In particular, a three-component library and a four-component library are created to encode a 2-bytes model binary sequence. These 2D-coded libraries are incorporated in all commodity plastics via a simple solvent casting procedure. Furthermore, in all cases, the oligomer mixtures can be extracted from the host polymer films and deciphered by mass spectrometry, thus opening interesting avenues for anti-counterfeiting and traceability applications.

Photocontrolled Synthesis of Abiotic Sequence-Defined Oligo(Phosphodiester)s.

A photoregulated phosphoramidite iterative process is studied for the synthesis of non-natural, digitally encoded oligo(phosphodiester)s. The oligomers are prepared using two reactive phosphoramidite monomers containing a 2-(2-nitrophenyl)propoxycarbonyl (NPPOC) protected OH group. The stepwise synthesis is performed on an OH-functional soluble polystyrene support, which allows recycling by precipitation in a nonsolvent. Repeating cycles involving phosphoramidite coupling, oxidation of phosphite to phosphate, and NPPOC deprotection by light irradiation at λ = 365 nm are performed in order to prepare oligomers with different lengths and sequences. Synthesis is conducted on a micromolar scale and good recycling yields are obtained in all cases. The use of a soluble polymer support allows an in-depth characterization of the NPPOC photo-deprotection step by 1 H NMR, UV spectroscopy, and size exclusion chromatography, and thus identification of optimal synthesis conditions. After cleavage from the support, the oligo(phosphodiester)s are characterized by tandem mass spectrometry, which confirms preparation of uniform sequence-coded oligomers.