A Track-Based Molecular Synthesizer that Builds a Single-Sequence Oligomer through Iterative Carbon-Carbon Bond Formation

Abstract

•A rotaxane ring on a phosphonium ylide track joins monomers through Wittig reactions•Nanomachine makes single-sequence oligomer with continuous backbone of carbon bonds•Utilizes chemistry and reactivity patterns unavailable to biomolecular machines•Future assemblers may provide solutions to challenging problems in synthesis Sequence is crucial in the molecular world. Proteins are built from a common set of 20 amino acids, but different sequences afford materials as diverse as snake venom, muscle, and spider silk. However, the synthesis of artificial sequence polymers remains challenging. Biology uses molecular machines (e.g., ribosomes) for such tasks, inspiring the invention of artificial systems that move along tracks, picking off and joining building blocks in sequence. To date, such small-molecule machines have used amide formation to join building blocks, the same bonds the ribosome uses to make peptides. Here, we report on the design, synthesis, and operation of a track-based molecular machine that assembles a single-sequence oligomer with a continuous backbone of carbon-carbon bonds. This new class of de novo molecular synthesizer utilizes chemistry and reactivity patterns unavailable to biological machines. The long-term goal is for such molecular assemblers to ultimately be able to play significant roles in molecular construction. We report an artificial molecular machine that moves along a track, iteratively joining building blocks to form an oligomer of single sequence with a continuous backbone of carbon-carbon bonds. The rotaxane features a macrocycle bearing an aldehyde-terminated chain and an axle containing different phosphonium ylides separated by rigid spacers. Each ylide is large enough to block the passage of the macrocycle, trapping the ring between the stopper at the terminus of original threading and the next ylide along the track. Once a building block is reachable, it is removed from the track through a Wittig reaction that adds it to the terminus of the growing chain. Operation on a four-barrier tetra(phosphonium salt) track produces a tetra(diphenylpropane) of single sequence linked through alkene bonds. The prototype extends the principle for molecular machines that build polymers by moving along tracks to the synthesis of sequence-encoded chains with continuous carbon backbones. We report an artificial molecular machine that moves along a track, iteratively joining building blocks to form an oligomer of single sequence with a continuous backbone of carbon-carbon bonds. The rotaxane features a macrocycle bearing an aldehyde-terminated chain and an axle containing different phosphonium ylides separated by rigid spacers. Each ylide is large enough to block the passage of the macrocycle, trapping the ring between the stopper at the terminus of original threading and the next ylide along the track. Once a building block is reachable, it is removed from the track through a Wittig reaction that adds it to the terminus of the growing chain. Operation on a four-barrier tetra(phosphonium salt) track produces a tetra(diphenylpropane) of single sequence linked through alkene bonds. The prototype extends the principle for molecular machines that build polymers by moving along tracks to the synthesis of sequence-encoded chains with continuous carbon backbones. Polymers with backbones of continuous carbon-carbon bonds (e.g., polystyrene, polyethylene, polymethyl methacrylate, etc.) are among the most widely used manmade materials.1Geyer R. Jambeck J.R. Law K.L. Production, use, and fate of all plastics ever made.Sci. Adv. 2017; 3: e1700782Crossref PubMed Scopus (4433) Google Scholar, 2Matyjaszewski K. Chemistry. Architecturally complex polymers with controlled heterogeneity.Science. 2011; 333: 1104-1105Crossref PubMed Scopus (233) Google Scholar, 3Lutz J.-F. Lehn J.-M. Meijer E.W. 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Epoxidation of polybutadiene by a topologically linked catalyst.Nature. 2003; 424: 915-918Crossref PubMed Scopus (346) Google Scholar or to thread precise numbers of macrocycles onto polymeric threads,17Fuller A.M. Leigh D.A. Lusby P.J. One template, multiple rings: controlled iterative addition of macrocycles onto a single binding site rotaxane thread.Angew. Chem. Int. Ed. Engl. 2007; 46: 5015-5019Crossref PubMed Scopus (68) Google Scholar, 18Fuller A.M. Leigh D.A. Lusby P.J. Sequence isomerism in [3]rotaxanes.J. Am. Chem. Soc. 2010; 132: 4954-4959Crossref PubMed Scopus (71) Google Scholar, 19Erbas-Cakmak S. Fielden S.D.P. Karaca U. Leigh D.A. McTernan C.T. Tetlow D.J. Wilson M.R. Rotary and linear molecular motors driven by pulses of a chemical fuel.Science. 2017; 358: 340-343Crossref PubMed Scopus (185) Google Scholar, 20Qiu Y. Song B. Pezzato C. Shen D. Liu W. Zhang L. Feng Y. Guo Q.H. Cai K. 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Nanotechnology: a molecular assembler.Nature. 2017; 549: 336-337Crossref PubMed Scopus (9) Google Scholar that moves along a track iteratively joining together building blocks to form an oligomer of single sequence with a continuous backbone of carbon-carbon bonds. Molecular machine-track conjugate 1 (Figure 1) was designed to use iterative Wittig reactions to form carbon-carbon double bonds between a macrocycle and building blocks abstracted one at a time and in sequence from a track. The Wittig reaction24Wittig G. Schöllkopf U. Über triphenyl-phosphin-methylene als olefinbildende reagenzien (I. Mitteil).Chem. Ber. 1954; 87: 1318-1330Crossref Scopus (761) Google Scholar, 25McDonald R.N. Campbell T.W. The Wittig reaction as a polymerization method 1a.J. Am. Chem. Soc. 1960; 82: 4669-4671Crossref Scopus (107) Google Scholar, 26Anuragudom P. Newaz S.S. Phanichphant S. Lee T.R. Horner-Emmons F. Facile Horner−Emmons synthesis of defect-free poly(9,9-dialkylfluorenyl-2,7-vinylene).Macromolecules. 2006; 39: 3494-3499Crossref Scopus (64) Google Scholar was chosen as it is robust and structurally tolerant, lending itself to exploitation in a range of contexts, including dynamic DNA-template synthesis.9Meng W. Muscat R.A. McKee M.L. Milnes P.J. El-Sagheer A.H. Bath J. Davis B.G. Brown T. O'Reilly R.K. Turberfield A.J. An autonomous molecular assembler for programmable chemical synthesis.Nat. Chem. 2016; 8: 542-548Crossref PubMed Scopus (99) Google Scholar Our machine is based on a rotaxane architecture, in which the macrocycle has a reactive aldehyde attachment and the axle has the building-block sequence encoded as phosphonium salts during its synthesis. The 2,2-diphenylpropane phosphonium units act both to restrict the position of the ring on the track and, upon deprotonation, as reactive ylide functionalities. Each ylide is large enough to block the passage of the macrocycle, trapping the ring within a compartment defined by the bulky stopper at the terminus of original threading and the next ylide along the track. Once a reactive building block can be reached by the macrocycle-appended aldehyde, it can be removed from the track through a Wittig reaction that adds it to the terminus of the growing chain. Each barrier also contains an aldehyde unit, so that once the building block is added to the end of the chain, it is able to react with the next barrier on the track that the macrocycle can access, enabling the alkene-connected oligomer to grow through successive Wittig reactions. The specific size and constitution of the 2,2-diphenylpropane motif of the building blocks proved important for successful machine operation. Early track designs in which the ylide and aldehyde were attached to the same aromatic ring or extended conjugated system proved insufficiently reactive (see Section S7 for a brief discussion of initial designs). Embedding the phosphorus atoms within the vector of the track allowed synthetically accessible triaryl phosphines to be the basis of the track design, expediting the synthesis (see Sections S2 and S3). The phenyl substituent at each phosphorus center (e.g., 4a–4d) also proved important: when a tolyl (4-methylphenyl) linking group was investigated, it proved difficult to develop macrocycles that could both thread during the rotaxane-forming reaction and, subsequently, pass over the phosphine oxide in the track formed from the Wittig reaction. Each phosphorus center is attached to a methylene group bearing a diarylpropane building block derivatized with a different pair of substituents (H, Ph, C6H4CH2CHMe2, or C6H4OMe). These provide different sidechains in the machine product, the same role that different amino acids play in proteins. However, two (identical) sidechains are present per monomer using this artificial molecular machine design compared with one sidechain per amino acid in proteins. This was chosen partly to illustrate how artificial machines and their products are not subject to the same constraints as biomolecular synthesizers but, conveniently, the symmetry of the building blocks also makes their synthesis more straightforward. Each phosphonium moiety is separated from the next by rigid spacers that prevent folding of the track and so ensure that the phosphonium salts can only react with the aldehyde group at the end of the chain attached to the macrocycle rather than others on the track. Rotaxane systems with a sufficiently rigid track such that the axle cannot fold are rare and making such a long rigid track presents particular challenges.27De Juan A. Pouillon Y. Ruiz-González L. Torres-Pardo A. Casado S. Martín N. Rubio Á. Pérez E.M. Mechanically interlocked single-wall carbon nanotubes.Angew. Chem. Int. Ed. Engl. 2014; 53: 5394-5400Crossref PubMed Scopus (48) Google Scholar,28Balakrishna B. Menon A. Cao K. Gsänger S. Beil S.B. Villalva J. Shyshov O. Martin O. Hirsch A. Meyer B. et al.Dynamic covalent formation of concave disulfide macrocycles mechanically interlocked with single-walled carbon nanotubes.Angew. Chem. Int. Ed. Engl. 2020; https://doi.org/10.1002/anie.202005081Crossref PubMed Scopus (9) Google Scholar The inter-residue spacers in the track of 1 contain methyl groups adjacent to the biphenyl connections to break the planarity and conjugation of the track and improve the solubility of the machine-track conjugate. Macrocycle 2 features an endotopic pyridine group in order to direct an active metal template29Aucagne V. Hänni K.D. Leigh D.A. Lusby P.J. Walker D.B. Catalytic “click” rotaxanes: a substoichiometric metal-template pathway to mechanically interlocked architectures.J. Am. Chem. Soc. 2006; 128: 2186-2187Crossref PubMed Scopus (299) Google Scholar, 30Crowley J.D. Goldup S.M. Lee A.L. Leigh D.A. McBurney R.T. Active metal template synthesis of rotaxanes, catenanes and molecular shuttles.Chem. Soc. Rev. 2009; 38: 1530-1541Crossref PubMed Scopus (490) Google Scholar, 31Denis M. Goldup S.M. The active template approach to interlocked molecules.Nat. Rev. Chem. 2017; 1: 61Crossref Scopus (126) Google Scholar copper-catalyzed azide-alkyne cycloaddition (CuAAC)32Meldal M. Tornøe C.W. Cu-catalyzed azide-alkyne cycloaddition.Chem. Rev. 2008; 108: 2952-3015Crossref PubMed Scopus (3725) Google Scholar of azide stopper 3 and dialkyne building block 4 through the macrocycle cavity. This was used to assemble a one-barrier rotaxane building block, 5, which was isolated in modest yield (23%), in part because the rotaxane-forming step also involves desymmetrization of the phosphonium bis-alkyne 4 (Figure 1, step i) and also because 5 was somewhat unstable with respect to purification by chromatography on silica gel, as it is likely incompatible with silica OH groups. However, the short route to 5 proved more effective than desymmetrization at later stages of the synthesis, and 5 proved a convenient intermediate for constructing the rest of the track. The active metal template strategy avoids the requirement for persistent attractive interactions between the macrocycle and thread that could slow component dynamics in the fully assembled machine. In this way, the macrocycle is restricted in its movements along the track only by the bulky building blocks. Rotaxane 5 was elongated through a series of further CuAAC reactions (Figure 1, steps ii–vii) to assemble the four-building-block molecular machine 1 in 35% overall yield from 5 (Figure 1; Section S3). Two shorter two- and three-barrier machines, 12 and 14, were elaborated from 5 in similar fashion in 54% and 39% overall yields, respectively (Figure 3; Section S5). Distinctive upfield shifts of triazole proton t1 (from 8.90 ppm in 6 to 8.37 ppm in 5 and 1, see Section S3 for full assignment) and A2 in the proximal alkyl chain (from 2.34 ppm in 6 to 1.93 ppm in 5 and 1) in the 1H NMR spectrum of rotaxane 5, compared with thread 6, confirm the localization of the macrocycle in the left-hand compartment of rotaxanes 1 (Figure 2), 12, and 14 (Section S5). Effective procedures for machine operation were first established for the two-building-block rotaxane, 12 (Figure 3; Section S6). Under optimized conditions, machine 12 was activated by deprotonation of the bis(phosphonium salt) using a polymer-bound phosphazene base (BEMP resin) in CH2Cl2. A resin-bound base was found to facilitate reaction monitoring by mass spectrometry. The resin was added to the solution of 12 in CH2Cl2 at −78°C, and the reaction mixture was allowed to warm to room temperature, forming the corresponding bis(phosphonium ylide) of the machine. The subsequent intra-machine reaction was allowed to proceed until the product distribution no longer changed (as evidenced by mass spectrometry): 1 day for machine 12, 3 days for machine 14, and 5 days for machine 1. The machine products, the sequence oligomer and the phosphine oxide track, were then isolated after a short work-up procedure followed by extensive chromatography (see Sections S4 and S6). Phosphine oxides are often difficult to remove by chromatography and, indeed, this proved to be the case with several of the molecular machine products. In addition to the fully elongated oligomeric product, small amounts of byproducts were detected by mass spectrometry that correspond to products missing one diarylpropane monomer from the chain. As the pristine molecular machine-track conjugates showed no evidence of missing barriers prior to operation (e.g., by mass spectrometry), these byproducts apparently arise from hydrolysis of the building blocks from the track under the machine’s operating conditions. In the case of the two-building-block machine operation, barrier loss corresponds to ∼5% of the isolated products, i.e., ∼2.5% loss per barrier. This rises to approximately 4% barrier loss during the operation of four-building-block machine 1. When non-interlocked thread 9 was reacted with macrocycle 2 (Figure 3A, for synthesis and operation see Sections S4 and S5) the stilbene product (10) was formed in a 58:42 E:Z ratio of alkene isomers.33Likhtenshtein G. Stilbenes. Applications in Chemistry, Life Sciences and Materials Science. Wiley-VCH Verlag GmbH & Co. KGaA, 2010Google Scholar However, when the same components were interlocked as rotaxane 11, the E:Z ratio of 10 formed was 88:12, indicating a significant stereochemical effect of the restricted vector of the macrocycle-appended aldehyde’s approach to the phosphonium ylide in the rotaxane, favoring the E-alkene-forming oxaphosphetane. The operation of the two-building-block machine 12 formed stilbene product 13 and had an 87:13 E:Z ratio for the first formed alkene, while the second formed in a 55:45 ratio, apparently as a consequence of the greater flexibility of the extended oligomer chain (Figure 3B; Section S6). Operation of three-building-block machine 14 under the same reaction conditions afforded the anticipated tri-stilbene product 15 (Figure 3C; Section S6). The sequence of 15 was confirmed by tandem MS/MS spectroscopy (Figure S22), confirming that the machine selectively extracts the building blocks embedded in the track in sequence. Although we were unable to fully assign the stereochemistry of all of the alkenes in 15 due to a lack of clearly resolved signals in the 1H NMR, we were able to determine that the right-hand-terminus alkene of 15 is formed in a 73:27 E:Z ratio. If the stereochemistry of the first two alkenes mirrors that of the one- and two-building-block machines 11 and 12, then the stereochemistry of the machine product from 14 is approximately E(87%),E(55%),E(73%)-15. Molecular machine 1 was activated by deprotonation of the tetra(phosphonium salt) using the polymer-bound phosphazene base (BEMP resin) in CH2Cl2, forming the corresponding tetra(phosphonium ylide) (Figure 1, step viii). Initially, macrocycle 2 is constrained to the left-most compartment by the first barrier. However, after a Wittig reaction between the aldehyde of the macrocycle and the first phosphonium ylide, the resulting phosphine oxide is not large enough to prevent the macrocycle from passing. The macrocycle now accesses the second compartment as well as the first, shuttling at random over the length of the increased amount of accessible thread until it reacts with the second barrier, forming another carbon-carbon double bond. This process is repeated twice more with barriers three and four. Once the fourth alkene connection is generated, all of the phosphonium ylides have been removed from the track, leaving the smaller phosphine oxides in their place, and the macrocycle bearing the oligomeric product, 7, dethreads from the residual track, 8. After 5 days, mass spectrometry indicated complete consumption of the starting material. Size-exclusion chromatography followed by preparative thin-layer chromatography led to the isolation of two major products: the tetra(phosphine oxide) thread 8 (69%) and oligomer 7 (21% isolated yield; the low yield is a reflection of the material lost during chromatography), obtained as a mixture of stereoisomers (E/Z for each of the four alkenes). The sequence of the building blocks in 7 was unambiguously determined by tandem mass spectrometry (MS/MS). Fragmentation along the chain of 7 shows sequential loss of fragments from each monomer strictly in the anticipated order, demonstrating the sequence integrity of the molecular machine operation (Figures 4A–4C ). The regulation of the building-block sequence in 7 corresponds to control over the primary structure of a sequence polymer, a degree of order critical in proteins, where a single monomer missing or positioned out of sequence can cause loss or change of function.34Fersht A. Structure and mechanism in protein science. Freeman, 1998Google Scholar The next level of structural order in proteins, secondary structure, corresponds to the 3D shape of local segments (e.g., motifs such as α-helices).34Fersht A. Structure and mechanism in protein science. Freeman, 1998Google Scholar The E- or Z- configuration of the stilbene units offers at least the potential to control this in carbon backbone polymers produced by molecular machines such as 1.35Nakano T. Okamoto Y. Synthetic helical polymers: conformation and function.Chem. Rev. 2001; 101: 4013-4038Crossref PubMed Scopus (1254) Google Scholar For example, molecular modeling indicates that the all-Z- stereoisomer of 7 should adopt a helical conformation with the two side chains per residue directed outward with a pitch of ∼7 Å and two monomers per turn (Figures 4D and 4E; in comparison, a peptide α-helix has one side chain per residue, a pitch of 5.4 Å, and 3.6 monomers per turn). However, although we were able to determine that the right-hand terminus alkene of 7 forms in a 70:30 E:Z ratio (similar to the E:Z ratio obtained with the operation of the three-barrier machine), suggesting that the E:Z distribution is approximately E(87%),E(55%),E(73%),E(70%)-7 by extrapolating from the shorter machine products,36It is not clear whether the geometry formed in the first Wittig reaction affects the geometries of subsequent Wittig reactions. The stereochemistry indicated are averaged values based on the outcome of the shorter machine outputs. the complete distribution of the alkene stereochemistry in 7 could not be established unambiguously. Furthermore, attempts to isomerize all of the alkenes in 7, 13, or 15 to a single stereoisomer (for example, by extended heating or treatment with I2 to try to form the all-E isomer or photochemistry to try to favor the all-Z isomer)37Yu Z. Hecht S. 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