A Support-Free Strategy to Synthesize Sequence-Defined Macromolecules Using Solvent-Resistant Nanofiltration

Abstract

Sequence-defined macromolecules (SDMs) offer tremendous promise for critical applications like data storage, optoelectronics, or biomimetics, but their synthesis remains particularly challenging. A generic SDM synthesis strategy is reported now using a support-free orthogonal approach starting from single monomers to produce SDMs. The procedure does not require any support, protection, or deprotection steps, nor does it require solvent switches. Crucially, each reaction step is followed by purification using solvent-resistant nanofiltration to ensure product retention from the reaction mixture with the complete removal of impurities (excess reagents, catalyst, byproducts). The sharp molecular weight resolution of well-tuned epoxy-based membranes renders this strategy highly efficient and versatile, as proven here by applying varying reactions, monomer types, and sizes to prepare two different sequence-defined conjugated macromolecules (SDCMs). Demonstrating the effectiveness of these membrane separations to purify SDCMs across very diverse conditions thus highlights the transformative potential of advanced membrane technology for SDM synthesis. Overall, a robust foundation is provided for advancing SDM development from single monomers to complex, high-purity oligomers.

Introduction

Polymers find applications across every sector of life, yet conventional polymerization methods produce materials with broad molar mass and sequence distributions. A major advance came with sequence-defined macromolecules (SDMs), which introduce “primary structure” control into synthetic polymers, enabling precision-encoded functions in, e.g., biomimetic structures, optoelectronics, and information storage. Unfortunately, the precision that makes SDMs attractive renders them difficult to produce. Solid-phase synthesis requires sequential monomer addition, purification by washing, and final cleavage from an insoluble resin. , However, throughput typically collapses as chain length increases, branched architectures are sterically blocked, and the resin itself becomes an expensive waste stream. Liquid-phase strategies avoid such solid supports, yet demand extensive purification steps requiring optimization as molar mass or functionality changes, thus increasing solvent use and cost. ,

Purification is a major bottleneck in the SDM production. Simple methods like precipitation are easy and fast, but may trap impurities and show limitations for the separation of structures with similar solubility profiles. Crystallization reduces solvent use but is unsuitable for amorphous polymers. , Moreover, conformational changes may alter the polymer properties upon the addition of a nonsolvent. Chromatography, although versatile, is time-consuming, hardly scalable, involves high solvent consumption with associated environmental concerns, and it is not well-suited for separating closely related oligomers.

Well-engineered solvent-resistant nanofiltration (SRNF) membranes offer a way to minimize these limitations. This technology is recognized as highly scalable, easy to automate, and sustainable. It offers pressure-driven molecular separations with low energy consumption and cost, not creating extra waste streams. − Membrane-based purification in SDM synthesis was pioneered by Brandstetter et al. in 1973, using a 10 kDa poly­(ethylene glycol) (PEG) soluble support for the aqueous-phase synthesis of nucleotides, employing commercial ultrafiltration membranes for purification. More recently, Livingston et al. introduced a PEG-based support to synthesize sequence-defined polyethers in organic solvents, revealing new challenges linked to membrane stability and inefficient separation due to the linear structure of the support. To address this, a three-arm star-shaped support was also developed, but limitations remained, particularly with regard to monomer diversity, since the methodology primarily targeted hydroxyl groups, excluding the incorporation of monomers without such functionalities. Additionally, the required solvent switches after each deprotection and coupling step resulted in high consumption of solvents, raw materials, and reagents, as well as increased operational complexity, extended processing time, and environmental impact.

In this work, we present a support-free, membrane-guided platform to prepare SDMs, which marks an important milestone in the area of SDM design. Indeed, membranes in SRNF can typically reject substances with molar masses from 200 to 1000 Da, ,, allowing the use of small and single monomers. The separation is mainly based on size exclusion, but mutual interactions between the solute, solvent, and membrane can also play key roles, especially for small molecules, i.e., in the first steps of SDM preparation. Clearly, the more interactions that are possibly involved, the more the membrane must be fine-tuned to be able to separate monomers. Furthermore, most SDM methods generate homopolymers composed of a single monomer type, with interactions that are restricted to van der Waals interactions, H-bonding, and rather weak Coulombic interactions due to the lack of charge separation within the oligomer backbone. In fact, research on SDMs has so far mainly focused on nonconjugated monomers or click-type chemistry, in which the conjugation is disrupted after monomer coupling, which limits versatility.

SRNF is now introduced for the purification of growing SDMs, specifically exemplified here by sequence-defined conjugated macromolecules (SDCMs), a particularly demanding class of SDMs to be combined with SRNF due to their potentially strong intermolecular interactions with the solvent, other SDCM (solute) and polymeric membranes which could lead to fouling , or negative retentions. Conjugated macromolecules are composed of repeating units linked by alternating single and multiple bonds, allowing for the delocalization of electrons. They combine highly appealing electrical and optical properties, which qualify them as active components of devices, but are known to be very sensitive to structural imperfections, thus benefiting from precise sequence-defined structures. As they combine processability and versatility of polymers with electronic characteristics of inorganic semiconductors, current need for innovative energy technologies, sustainable materials, nanoelectronics and quantum technologies, momentum has been added to their research recently.

By eliminating chromatographic columns, precipitation, and supports, this breakthrough membrane technology allows the usage of monomer libraries previously incompatible with anchor-based strategies. Our synthetic approach involves two alternating orthogonal cross-coupling reactions without any intermediate solvent switch. While the Suzuki–Miyaura (SM) reaction creates a single bond between aryl units via a reaction between a bromine and a boronic ester group, the Horner–Wadsworth–Emmons (HWE) olefination introduces a double bond by reacting an aldehyde with a phosphonate. Both reactions were earlier optimized for rapid synthesis with high yields for the monodisperse conjugated oligomers. , The targeted molecules, two tetramers, consist of a sequence of differently conjugated monomeric units, allowing for a range of interactions. This is highly relevant for the final applications of these conjugated systems, but also to prove the versatility of the new strategy, i.e., to link oligomer structure and composition with adequate membrane separation. The SDCMs are constructed to the level of tetramers to stay within the most challenging SRNF range. In later stages, these tetramers could then be coupled into larger macromolecules approaching more polymer-like lengths via a modular approach. Crucial for efficient separation, especially for the first reaction steps, is the creation of a solvent-stable membrane with excellent molecular weight (MW) resolution, i.e., the difference in retention between the two molecules, specifically the short oligomers and the remaining reagents and possible side products. Based on recent developments, an epoxide-based top layer was applied on a polyimide-based support for this purpose via interfacial initiation of polymerization (IIP), creating a thin-film composite (TFC)-membrane with a dense selective layer to enhance separation efficiency and achieve the required purity. − Moreover, the SDCM-monomers employed in this strategy were selected to have molar masses (200–600 Da) that are very close to the molar mass of the growing SDCM, making the separation even more challenging. Additionally, very diverse and strong interactions between the solute, membrane, and solvent were selected to better prove the versatility of the approach and the broad potential of the developed membrane. Indeed, van der Waals interactions are increased in SDCMs through the inevitable presence of side chains. H-bonding can be present, and partial charges can lead to significant dipole moments due to the potentially largely delocalized, more polarizable π-electrons. Finally, π–π interactions, typical for conjugated systems but completely absent in earlier membrane-purified SDMs, also come into play. This makes the use of SRNF as a purification method in the synthesis of SDCMs the ultimate challenge.

Results and Discussion

Membrane Development

The synthesis protocols leading to the targeted SDCMs create significant challenges to find a suitable membrane that simultaneously combines an acceptable flux with the required retention of the growing oligomer while remaining stable in the reaction media. The first purification step is particularly challenging due to the minimal size differences between the monomer and dimer, which historically led to the exclusive use of anchored monomers as starting building blocks. , With this in mind, a series of membranes was first evaluated to design a material that enables the effective separation of the dimer from unreacted monomers, catalysts, and side products.

A cross-linked polyimide (XL-PI) membrane, resembling commercial SRNF membranes, was synthesized first. This membrane retained only 60% of the dimer (Figure a), which is insufficient to achieve the desired yield of >95%. To enhance selectivity, a TFC membrane was developed by coating the XL-PI with a poly­(epoxy ether) layer via IIP (Figure b). The TFC membrane was prepared through ring-opening polymerization of a tetrafunctional epoxide at the water–toluene interface, initiated by a bifunctional tertiary amine, i.e., N,N,N′,N′-tetramethylhexane-1,6-diamine (Figure b). The resulting selective layer was further tuned by reapplying the epoxide and amine solutions in a post-treatment to create extra cross-links and introduce quaternary ammonium groups (NR₄ ⁺). As improvements were limited, the IIP solvent was then changed from immiscible toluene to partially miscible anisole, which improved layer uniformity by optimizing the interfacial reaction kinetics and monomer diffusion. ,, When using 1.5 w/v% epoxide in anisole (‘Anisole-1.5′ in Figure a), dimer retention increased to 88%, with a permeance of 5.1 L m^–2 h^–1 bar^–1. Further optimization by raising the epoxide concentration to 2.5 w/v% boosted dimer retention to 98%, with only a slight drop in permeance to 4.9 L m^–2 h^–1 bar^–1, which is still a great value compared to reported SRNF membranes with comparable selectivity. Increasing the concentration to 3 w/v% did not further enhance dimer retention but adversely caused the retention of the first monomer to rise from 0% to an unacceptable 40% (Figure a). This lowered MW resolution can be ascribed to the overdensification of the selective layer. These results underscore the importance of precisely controlling the conditions to prepare high-performance membranes.

1.

(a) Influence of membrane preparation conditions (IIP solvent and epoxide concentration) on permeance and retention of dimer and first monomer in tetrahydrofuran (THF). (b) PEE-based TFC membrane prepared via IIP and membrane cross-section morphology characterization via SEM.

As previously reported, , the exact thickness of the selective layer could not be directly measured, as there is insufficient contrast with the support layer or even below the limits of conventional SEM, TEM, and AFM. As a result, no absolute thickness value could be determined. Surface roughness measurements by AFM consistently confirmed the formation of a continuous and uniform selective layer across the membrane surface.

Synthesis of SDCMs

The iterative synthesis and purification of two SDCMs, varying in their sequence, were carried out using the above-described TFC membrane (referred to as Anisole-2.5 in Figure a). For all filtration and diafiltration experiments, the same membrane was used throughout the full SDCM synthesis. Using an orthogonal approach, key parameters, including product recovery and the removal of undesired side products, were evaluated. The synthesis alternated SM cross-coupling with HWE olefination as described earlier (Figure ). , Both reactions were first optimized to proceed rapidly and in high yields, achieving complete conversion within 30 min in the presence of a slight excess of monomer. The SM reaction used palladium­(II) acetate (Pd­(OAc)₂) as a catalyst (0.05 equiv) with 2-dicyclohexylphosphino-2′,6′-diisopropoxybiphenyl (RuPhos) (0.25 equiv) as a ligand, and 1.2 equiv of the boronic ester monomer, relative to the oligomer. Potassium phosphate served as a base, and the reaction was carried out in tetrahydrofuran (THF) with the addition of water (THF:H₂O = 50:1, v/v) to enhance the reaction rate at 65 °C under nitrogen. The HWE olefination was performed using the phosphonate monomer (1.1 equiv) and potassium tert-butoxide as base in THF at room temperature under nitrogen. After each coupling step, SRNF was applied to purify the intermediate oligomer. Filtration was stopped after about 80% volume permeation to prevent membrane dry-out, leaving up to 20% of nonpurified retentate. Therefore, diafiltration (Figure S1) was realized to increase product purity, i.e., the retentate was diluted with THF to its original volume, and filtration was repeated. Because all reactions and filtrations were conducted in THF, no solvent exchange was needed between reaction steps, allowing for continuous iterative synthesis. To fully prove the potential of this strategy and assess the versatility of this precisely engineered membrane purification system, monomers were selected to vary in topology, partial charge, hydrogen bonding, and π–π stacking, introducing as diverse as possible interactions to challenge the SRNF separation. Such a variety of interactions would also render proper selection and optimization of conventional separation techniques highly challenging.

2.

Synthesis of two different SDCMs ((a) SDCM 1 and (b) SDCM 2) using orthogonal Suzuki–Miyaura (SM) cross-coupling and Horner–Wadsworth–Emmons (HWE) reactions. Both reactions were performed sequentially to produce well-defined dimers, trimers, and tetramers. The different monomers are shown in different colors.

For both SDCMs, the iterative synthesis started with an SM reaction, coupling a brominated starting monomer (S) to a phenyl-based boronic ester functionalized monomer. Such phenyl derivatives are well-established building blocks in conjugated polymer synthesis. ,

For SDCM 1, the starting monomer bore amide functions and dodecyl side chains, improving the solubility of the growing oligomers and allowing hydrogen bond formation while still bringing conjugation in its backbone. The dimer was obtained upon coupling via an aryl bond to a phenyl group, equipped with a linear octyl side chain. During the first step, all reagents were introduced to the reaction mixture simultaneously, leading to an undesired bis-coupled monomer. Due to the similar molar mass of the dimer (1013 Da) and this side product (723 Da), multiple diafiltration steps were required to achieve sufficient dimer purity. Permeance values (Figure a) decreased slightly with each subsequent diafiltration, ascribed to potential membrane fouling or initial compaction, yet, the dimer could be obtained in high purity (see NMR in Figure S2a). Moreover, with a dimer retention exceeding 96%, the product yield remained very high. The need for multiple diafiltrations reflects the challenge posed by the molecular size similarity between the dimer and the impurities in this first step of the protocol.

3.

Membrane performance in the purification of SDCMs. (a) SDCM 1: Diafiltration performance for dimer, trimer, and tetramer purification was illustrated by changes in permeance over multiple filtration cycles. (b) SDCM 2: Membrane performance for dimer, trimer, and tetramer purification, highlighting diafiltration efficiency across different polymer sizes and removal steps. (c) SEC analysis of SDCMs 1 and 2, with elution profiles of dimer (black), trimer (red), and tetramer (blue).

The dimer was then converted to a trimer using a HWE reaction (Figure a). The size difference between the trimer and the impurities was much more pronounced than in the dimer synthesis, enabling effective separation in even a single diafiltration step. For the trimer synthesis, the choice for a pyridine-derivative without any side chains as a monomer was motivated by the simplicity of its synthesis requiring only two steps to obtain the phosphonate functionality. , Furthermore, this monomer suppresses the undesired conversion of an E-double bond into an Z-double bond, highly relevant in conjugated systems where rigidity of the oligomer backbone is crucial. The permeance during this diafiltration was 3 L m^–2 h^–1 bar^–1 (Figure a), and a highly pure trimer was obtained (Figure S2b).

The final SDCM 1 tetramer was synthesized from the purified trimer by using an optimized SM reaction. However, in contrast to the dimer synthesis, the boronic ester monomer was now introduced dropwise to the reaction mixture to prevent unwanted dimerization of this monomer and simplify the purification process. This illustrates how clever reaction design can significantly facilitate the subsequent filtration. The additional building block was a small, commercially available, and inexpensive thiophene monomer without any side chain, leading to a high molar mass oligomer with a rigid backbone. Thanks to the incorporation of this small conjugated, electron-rich monomer, a push–pull system was integrated into the structure, which rendered the backbone planar and led to partial charge separation. The permeance decreased slightly from an initial 3.75 to 2.6 L m^–2 h^–1 bar^–1 by the end of the diafiltration (Figure a). However, the tetramer retention consistently remained at 100% throughout the process, and high purity was again achieved (Figure S2c).

The sequence of monomers for SDCM 1 was chosen based on interactions that determine which specific conventional purification methods could potentially be used. The resulting tetramer had a linear, more compact size in which the separation of partial charges gave rise to a strong polarity within the structure. van der Waals interactions between the octyloxy side chains allowed sufficient stacking, while the oligomer remained soluble. Moreover, the absence of side chains on the pyridine and thiophene monomers enhanced the π–π stacking ability. Still, SRNF was capable of properly separating all of these molecules.

To further validate the effectiveness of our strategy, an even more challenging SDCM 2 was synthesized (Figure b). A dimer was obtained via the optimized SM reaction with the gradual addition of the boronic ester monomer. While for the synthesis of SDCM 1, a monomer with linear octyloxy side chain was chosen, which could also potentially have allowed crystallization of the dimer to purify the compound, here a branched, racemic side chain (i.e., 2-methylbutyloxy) was chosen for SDCM 2, which hindered stacking and therefore rendered crystallization impossible to isolate the dimer. The dimer was efficiently purified with 100% retention using only two diafiltrations. Permeance remained quite stable during both steps (Figure b). This efficient filtration highlights the membrane’s ability to purify compounds that cannot be isolated via crystallization and to manage the separation of components with small molecular size differences while maintaining acceptable permeances. NMR spectroscopy showed that the dimer was completely retained in the retentate with no coupling of the monomer detected in the permeates (Figure S3a). The large size difference between the dimer and impurities facilitated the filtration.

The pyridine-derivative, also used for SDCM 1, was added to obtain the trimer, and a 94% retention was obtained after one diafiltration. Although the same pyridine monomer was used to synthesize the trimer in both oligomerization approaches, a small percentage of the trimer was now lost into the permeate (Figure S3b), likely due to the smaller size of this SDCM 2-trimer carrying shorter 2-metylbutyloxy side chains.

Purifying the tetramer was particularly challenging due to the molecular size, shape, and chemical structure of the unreacted monomer involved in this step. The phenanthrene monomer is a large molecule (around 600 Da) (Figure S3c), which renders separation very difficult. Therefore, a total of eight diafiltrations were required to achieve the desired purity, yet the tetramer retention consistently remained at 100%.

The successful purification of the more challenging SDCM 2 further demonstrates the robustness of SRNF for purifying SDCMs, even if separations are difficult when sizes, MW, or polarities of solutes become very similar. An overview of the results for the purification of the two SDCMs is given in Table .

1. Overview of the Number of Diafiltrations Required to Purify Each Oligomer and Retention for Both Reaction Pathways.

	SDCM 1			SDCM 2
	dimer	trimer	tetramer	dimer	trimer	tetramer
number of diafiltrations	4	1	1	2	1	8
retention (%)	>96	100	100	100	>94	100

To validate the efficacy of membrane purification in isolating oligomers, size-exclusion chromatography (SEC) was used to follow the evolution of the synthesis of these uniform SDCMs (Figure c). First, the absence of shoulders confirmed the successful purification of each product, as no residual impurities or lower/higher molar mass oligomers were observed within the detection limits of the SEC (estimated to be around 10%). The sharpness and symmetry of the peaks further confirmed the high purity of the SDCMs. Table shows the peak average molar mass (M _p), which corresponds to the actual molar mass of the oligomers for the monodisperse samples. The deviation from the real molar mass values arises from the inherent dispersion on the SEC column. Clearly, SDCM 1 shows moderate increase in the molar mass of the consecutive oligomers, whereas the large increase in molar mass for the tetramer of SDCM 2 corresponds to the addition of the large phenanthrene monomer. In addition, mass spectrometry provided independent confirmation of the macromolecule’s purity (Figure S4), serving as a third orthogonal characterization technique.

2. Peak Average Molar Mass (M _p) Obtained by SEC Following the Iterative Steps Leading to SDCM 1 and 2.

M p (kDa)	SDCM 1	SDCM 2
dimer	1.6	1.4
trimer	1.7	1.5
tetramer	1.8	2.3

Comparison of SRNF with Conventional Purification Techniques

To further prove the strength of SDCM synthesis and purification by well-designed SRNF, a comparison was made with column chromatography using silica for the SDCM 2 dimer, for which crystallization was deliberately excluded by the introduction of two branched racemic alkyl side chains. This structure contains short side chains rendering purification with the membrane approach difficult, given the minimal size difference between the unreacted monomer and the dimer. Even for this most challenging sequence step, the dimer retention was 100%. In addition, column chromatography showed a 71% efficiency (number of moles in crude divided by those in the purified sample) compared to 77% for filtration (see NMR spectroscopy, SI).

Although NMR spectroscopy reliably confirms the controlled presentation of the functional groups after each elongation step and the uniformity of the molecular structure across oligomers, its sensitivity remains limited with a detection threshold of 3%–5%. To substantiate the monodispersity of the samples beyond this limitation, complementary techniques such as MALDI-ToF and GPC were employed, each independently verifying the purity and uniformity of the synthesized oligomers. However, to draw exclusive conclusions regarding molecular purity, it is essential to utilize highly sensitive techniques that probe distinct physicochemical processes.

Figure shows the absorbance and fluorescence spectra of each SDCM 2 oligomer, purified via either column chromatography or membrane filtration. After column chromatography, the sample solely contained the target oligomer, whereas membrane filtration retained trace amounts of BHT and RuPhosresiduals from the synthetic procedure. Importantly, these residual compounds do not perturb the optical characteristics of the samples. UV–vis absorbance measurements for both purification methods demonstrate electronic equivalence, indicating that no bonds are broken or formed during the purification nor the formation of any defects. Given the intrinsic error margin of UV–vis spectroscopy (1–3%), minor spectral shiftssuch as the small deviation of a few nanometers observed for the trimerremain within acceptable experimental uncertainty.

4.

UV–vis absorption (top) and fluorescence spectra (bottom) of the SDCM 2 dimer, trimer, and tetramer, each purified by column chromatography (black) and membrane filtration (red). For all oligomers, the spectra from both purification methods are fully overlapping, with no observable shifts nor additional emission bands. This demonstrates that both techniques yield electronically and optically pure samples, with even the highly sensitive fluorescence measurements revealing no traces of impurities.

To reach even more definitive conclusions regarding the sample purity, fluorescence spectroscopy was employed to achieve its superior sensitivity, capable of detecting impurities at concentrations far below 1%. Since this is based on different steps compared to the absorbance, from the excited state to the ground state, it is sensitive for different pathways of emission. Unlike absorbance, which probes ground-state electronic transitions, fluorescence arises from excited-state relaxation pathways and is, thus, sensitive to alternative emission mechanisms. The presence of structural defects or impurities can introduce nonradiative decay channels or charge-transfer energy states, manifesting as red-shifted emission bands. The complete absence of such red-shifts in fluorescence spectra of membrane-filtered samples, when compared to those purified via column chromatography, unequivocally confirms the absence of any contaminants across all oligomers.

The versatility of the SRNF-based purification strategy comes from the possibility to tune the MWCO of the membranes by adjusting the membrane preparation conditions. Such tuning properties have also been reported already for many other types of polymer-based SRNF membranes, based on polyimide, PVDF, and PSf. This allows the selective separation of intermediates, monomers, or side products with very similar molecular weights and makes the approach applicable to different types of SDMs, as long as the membrane remains stable under the conditions required for purification and as long as the size of the molecules falls within the tunable separation range of the membrane.

Conclusions

This study establishes a robust, support-free platform that combines orthogonal synthesis with purification using SRNF membranes. This approach enables the efficient, iterative preparation of monodisperse oligomers, demonstrating high purity and structural variety. The selected oligomers cover a wide range of molar masses, topologies, and interaction profiles. The demonstrated membrane properties ensure solvent resistance, fast filtrations, and excellent SDM purity. Moreover, the compatibility with conjugated systems, exhibiting challenging π–π interactions, hydrogen bonding, and Coulombic forces, further highlights the method’s broad applicability.

Beyond offering a more versatile, cleaner, and more sustainable alternative to techniques like chromatography, crystallization, and precipitation, this SRNF approach reduces solvent consumption, avoids solvent switches that are linked to membrane applicability, enables rapid iteration, and opens, like any membrane process, the way for upscaling and automated processing. The strategic variation of side chains, backbone geometry, and monomer functionality further demonstrated that this method is adaptable to diverse structural motifs, including linear and nonlinear oligomers.

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/prechem.5c00409.

• Materials and methods, diafiltration process, NMR spectroscopy, and MALDI-ToF mass spectrometry. (PDF)

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Z.B. and J.P. contributed equally to this work. Z.B.: Conceptualization, methodology, validation, investigation, writingoriginal draft, visualization. J.P.: Conceptualization, methodology, validation, investigation, writingoriginal draft, visualization. W.M.: Conceptualization, methodology, validation, investigation, writingreview and editing. L.A.J.R.: Methodology, writingreview and editing. N.B.: Conceptualization, writingreview and editing. X.T.: Methodology, writingreview and editing. C.V.G.: Supervision, conceptualization, methodology. S.R.H.: Conceptualization, writingreview and editing. F.E.D.P.: Conceptualization, supervision, writingreview and editing. G.K.: Supervision, writingreview and editing, project administration. I.F.J.V.: Supervision, writingreview and editing, project administration, funding acquisition, corresponding author.

The authors declare no competing financial interest.