Substitution-Induced Mechanistic Switching in SNAr-Warheads for Cysteine Proteases.

The aim of this study was to investigate the transition from non-covalent reversible over covalent reversible to covalent irreversible inhibition of cysteine proteases by making delicate structural changes to the warhead scaffold. To this end, dipeptidic rhodesain inhibitors with different N-terminal electrophilic arenes as warheads relying on the SNAr mechanism were synthesized and investigated. Strong structure-activity relationships of the inhibition potency, the degree of covalency, and the reversibility of binding on the arene substitution pattern were found. The studies were complemented and substantiated by molecular docking and quantum-mechanical calculations of model systems. Furthermore, the improvement in the membrane permeability of peptide esters in comparison to their corresponding carboxylic acids was exemplified.

Reversible Polymer-Protein Functionalization by Stepwise Introduction of Amine-Reactive, Reductive-Responsive Self-Immolative End Groups onto RAFT-Derived Polymers.

Many promising therapeutic protein or peptide drug candidates are rapidly excreted from an organism due to their small size or their inherent immunogenicity. One way to counteract these effects is PEGylation, in which the biopolymer is shielded by synthetic polymers exploiting their stealth properties. However, these modifications are often accompanied by a reduction in the biological function of the protein. By using responsive moieties that bridge the polymer to the protein, a reversible character is provided to this type of conjugation. In this regard, the reductive-responsive nature of disulfides can be exploited via self-immolative structures for reversible linkage to aminic lysine residues and the N-terminus on the protein surface. They enable a traceless release of the intact protein without any further modification and thus preserve the protein's bioactivity. In this study, we demonstrate how this chemistry can be made broadly accessible to RAFT-derived water-soluble polymers like poly(N,N-dimethylacrylamide) (pDMA) as a relevant PEG alternative. A terminal reactive imidazole carbamate with an adjacent self-immolative motif was generated in a gradual manner onto the trithiocarbonate chain transfer moiety of the polymer by first substituting it with a disulfide-bridged alcohol and subsequently converting it into an amine reactive imidazole carbamate. Successful synthesis and complete characterization were demonstrated by NMR, size exclusion chromatography, and mass spectrometry. Finally, two model proteins, lysozyme and a therapeutically relevant nanobody, were functionalized with the generated polymer, which was found to be fully reversible under reductive conditions in the presence of free thiols. This strategy has the potential to extend the generation of reversible reductive-responsive polymer-protein hybrids to the broad field of available functional RAFT-derived polymers.

Efficient Self-Immolative RAFT End Group Modification for Macromolecular Immunodrug Delivery.

The reversible addition-fragmentation chain-transfer (RAFT) polymerization provides access to a broad variety of biocompatible and functional macromolecules for diverse polymer-drug conjugates. Due to thiocarbonylthio groups at the ends of each growing polymer chain, they can straightforwardly be converted into disufilde-containing self-immolative motives for reversible drug conjugation by traceless linkers. This may be relevant for RAFT-polymerized poly(N,N-dimethylacrylamide) (pDMA), which has been demonstrated to provide similar properties as poly(ethylene glycol) (PEG) in terms of improving the drug's poor pharmacokinetic profile or enhancing its bioavailability. For that purpose, we established a highly efficient one-pot reaction procedure for introducing various functionalities including both primary and secondary amines and primary alcohols and demonstrated their reversible conjugation and traceless release from pDMA's polymer chain end. Next, a first polymer-drug conjugate with a Toll-like receptor agonist exhibited significantly increased activity in vitro compared to conventional irreversibly covalently fixed variants. Finally, α-ω-bifunctional dye or drug conjugates could be generated by a cholesterol-modified RAFT chain-transfer agent. It facilitated the polymer-drug conjugate's internalization at the cellular level monitored by flow cytometry and confocal imaging. This approach provides the basis for a variety of potentially impactful polymer-drug conjugates by combining versatile small molecular drugs with a plethora of available RAFT polymers through reductive-responsive self-immolative linkers.

Photoinduced Amyloid Fibril Degradation for Controlled Cell Patterning.

Amyloid-like fibrils are a special class of self-assembling peptides that emerge as a promising nanomaterial with rich bioactivity for applications such as cell adhesion and growth. Unlike the extracellular matrix, the intrinsically stable amyloid-like fibrils do not respond nor adapt to stimuli of their natural environment. Here, a self-assembling motif (CKFKFQF), in which a photosensitive o-nitrobenzyl linker (PCL) is inserted, is designed. This peptide (CKFK-PCL-FQF) assembles into amyloid-like fibrils comparable to the unsubstituted CKFKFQF and reveals a strong response to UV-light. After UV irradiation, the secondary structure of the fibrils, fibril morphology, and bioactivity are lost. Thus, coating surfaces with the pre-formed fibrils and exposing them to UV-light through a photomask generate well-defined areas with patterns of intact and destroyed fibrillar morphology. The unexposed, fibril-coated surface areas retain their ability to support cell adhesion in culture, in contrast to the light-exposed regions, where the cell-supportive fibril morphology is destroyed. Consequently, the photoresponsive peptide nanofibrils provide a facile and efficient way of cell patterning, exemplarily demonstrated for A549, Chinese Hamster Ovary, and Raw Dual type cells. This study introduces photoresponsive amyloid-like fibrils as adaptive functional materials to precisely arrange cells on surfaces.

pH-degradable, bisphosphonate-loaded nanogels attenuate liver fibrosis by repolarization of M2-type macrophages.

Immune-suppressive (M2-type) macrophages can contribute to the progression of cancer and fibrosis. In chronic liver diseases, M2-type macrophages promote the replacement of functional parenchyma by collagen-rich scar tissue. Here, we aim to prevent liver fibrosis progression by repolarizing liver M2-type macrophages toward a nonfibrotic phenotype by applying a pH-degradable, squaric ester­based nanogel carrier system. This nanotechnology platform enables a selective conjugation of the highly water-soluble bisphosphonate alendronate, a macrophage-repolarizing agent that intrinsically targets bone tissue. The covalent delivery system, however, promotes the drug's safe and efficient delivery to nonparenchymal cells of fibrotic livers after intravenous administration. The bisphosphonate payload does not eliminate but instead reprograms profibrotic M2- toward antifibrotic M1-type macrophages in vitro and potently prevents liver fibrosis progression in vivo, mainly via induction of a fibrolytic phenotype, as demonstrated by transcriptomic and proteomic analyses. Therefore, the alendronate-loaded squaric ester­based nanogels represent an attractive approach for nanotherapeutic interventions in fibrosis and other diseases driven by M2-type macrophages, including cancer.

End Group Dye-Labeled Polycarbonate Block Copolymers for Micellar (Immuno-)Drug Delivery.

Defined conjugation of functional molecules to block copolymer end groups is a powerful strategy to enhance the scope of micellar carriers for drug delivery. In this study, an approach to access well-defined polycarbonate-based block copolymers by labeling their end groups with single fluorescent dye molecules is established. Following controlled polymerization conditions, the block copolymers' primary hydroxy end group can be converted into activated pentafluorophenyl ester carbonates and subsequently aminolyzed with fluorescent dyes that are equipped with primary amines. During a solvent-evaporation process, the resulting end group dye-labeled block copolymers self-assemble into narrowly dispersed ∼25 nm-sized micelles and simultaneously encapsulate hydrophobic (immuno-)drugs. The covalently attached fluorescent tracer can be used to monitor both uptake into cells and stability under biologically relevant conditions, including incubation with blood plasma or during blood circulation in zebrafish embryos. By encapsulation of the toll-like receptor 7/8 (TLR7/8) agonist CL075, immune stimulatory polymeric micelles are generated that get internalized by various antigen-presenting dendritic cells and promote their maturation. Generally, such end group dye-labeled polycarbonate block copolymers display ideal features to permit targeted delivery of hydrophobic drugs to key immune cells for vaccination and cancer immunotherapy.

Nontoxic N-Heterocyclic Olefin Catalyst Systems for Well-Defined Polymerization of Biocompatible Aliphatic Polycarbonates.

Herein, N-heterocyclic olefins (NHOs) are utilized as catalysts for the ring-opening polymerization (ROP) of functional aliphatic carbonates. This emerging class of catalysts provides high reactivity and rapid conversion. Aiming for the polymerization of monomers with high side chain functionality, six-membered carbonates derived from 2,2-bis(hydroxymethyl)propionic acid (bis-MPA) served as model compounds. Tuning the reactivity of NHO from predominant side chain transesterification at room temperature toward ring-opening at lowered temperatures (-40 °C) enables controlled ROP. These refined conditions give narrowly distributed polymers of the hydrophobic carbonate 5-methyl-5-benzyloxycarbonyl-1,3-dioxan-2-one (MTC-OBn) (D < 1.30) at (pseudo)first-order kinetic polymerization progression. End group definition of these polymers demonstrated by mass spectrometry underlines the absence of side reactions. For the active ester monomer 5-methyl-5-pentafluorophenyloxycarbonyl-1,3-dioxane-2-one (MTC-PFP) with elevated side chain reactivity, a cocatalysis system consisting of NHO and the Lewis acid magnesium iodide is required to retune the reactivity from side chains toward controlled ROP. Excellent definition of the products (D < 1.30) and mass spectrometry data demonstrate the feasibility of this cocatalyst approach, since MTC-PFP has thus far only been polymerized successfully using acidic catalysts with moderate control. The broad feasibility of our findings was further demonstrated by the synthesis of block copolymers for bioapplications and their successful nanoparticular assembly. High tolerability of NHO in vitro with concentrations ranging up to 400 µM (equivalent to 0.056 mg/mL) further emphasize the suitability as a catalyst for the synthesis of bioapplicable materials. The polycarbonate block copolymer mPEG44-b-poly(MTC-OBn) enables physical entrapment of hydrophobic dyes in sub-20 nm micelles, whereas the active ester block copolymer mPEG44-b-poly(MTC-PFP) is postfunctionalizable by covalent dye attachment. Both block copolymers thereby serve as platforms for physical or covalent modification of nanocarriers for drug delivery.

Cell-Instructive Surface Gradients of Photoresponsive Amyloid-like Fibrils.

Gradients of bioactive molecules play a crucial role in various biological processes like vascularization, tissue regeneration, or cell migration. To study these complex biological systems, it is necessary to control the concentration of bioactive molecules on their substrates. Here, we created a photochemical strategy to generate gradients using amyloid-like fibrils as scaffolds functionalized with a model epitope, that is, the integrin-binding peptide RGD, to modulate cell adhesion. The self-assembling ß-sheet forming peptide (CKFKFQF) was connected to the RGD epitope via a photosensitive nitrobenzyl linker and assembled into photoresponsive nanofibrils. The fibrils were spray-coated on glass substrates and macroscopic gradients were generated by UV-light over a centimeter-scale. We confirmed the gradient formation using matrix-assisted laser desorption ionization mass spectroscopy imaging (MALDI-MSI), which directly visualizes the molecular species on the surface. The RGD gradient was used to instruct cells. In consequence, A549 adapted their adhesion properties in dependence of the RGD-epitope density.

Fluorovinylsulfones and -Sulfonates as Potent Covalent Reversible Inhibitors of the Trypanosomal Cysteine Protease Rhodesain: Structure-Activity Relationship, Inhibition Mechanism, Metabolism, and In Vivo Studies.

Rhodesain is a major cysteine protease of Trypanosoma brucei rhodesiense, a pathogen causing Human African Trypanosomiasis, and a validated drug target. Recently, we reported the development of α-halovinylsulfones as a new class of covalent reversible cysteine protease inhibitors. Here, α-fluorovinylsulfones/-sulfonates were optimized for rhodesain based on molecular modeling approaches. 2d, the most potent and selective inhibitor in the series, shows a single-digit nanomolar affinity and high selectivity toward mammalian cathepsins B and L. Enzymatic dilution assays and MS experiments indicate that 2d is a slow-tight binder (Ki = 3 nM). Furthermore, the nonfluorinated 2d-(H) shows favorable metabolism and biodistribution by accumulation in mice brain tissue after intraperitoneal and oral administration. The highest antitrypanosomal activity was observed for inhibitors with an N-terminal 2,3-dihydrobenzo[b][1,4]dioxine group and a 4-Me-Phe residue in P2 (2e/4e) with nanomolar EC50 values (0.14/0.80 µM). The different mechanisms of reversible and irreversible inhibitors were explained using QM/MM calculations and MD simulations.

Targeted Repolarization of Tumor-Associated Macrophages via Imidazoquinoline-Linked Nanobodies.

Tumor-associated macrophages (TAMs) promote the immune suppressive microenvironment inside tumors and are, therefore, considered as a promising target for the next generation of cancer immunotherapies. To repolarize their phenotype into a tumoricidal state, the Toll-like receptor 7/8 agonist imidazoquinoline IMDQ is site-specifically and quantitatively coupled to single chain antibody fragments, so-called nanobodies, targeting the macrophage mannose receptor (MMR) on TAMs. Intravenous injection of these conjugates result in a tumor- and cell-specific delivery of IMDQ into MMRhigh TAMs, causing a significant decline in tumor growth. This is accompanied by a repolarization of TAMs towards a pro-inflammatory phenotype and an increase in anti-tumor T cell responses. Therefore, the therapeutic benefit of such nanobody-drug conjugates may pave the road towards effective macrophage re-educating cancer immunotherapies.

Self-Immolative RAFT-Polymer End Group Modification.

Reversible modifications of reversible addition-fragmentation chain transfer (RAFT)-polymerization derived end groups are usually limited to reductive degradable disulfide conjugates. However, self-immolative linkers can promote ligation and traceless release of primary and secondary amines as well as alcohols via carbonates or carbamates in ß-position to disulfides. In this study, these two strategies are combined and the concept of self-immolative RAFT-polymer end group modifications is introduced: As model compounds, benzylamine, dibenzylamine, and benzyl alcohol are first attached as carbamates or carbonates to a symmetrical disulfide, and in a straightforward one-pot reaction these groups are reversibly attached to aminolyzed trithiocarbonate end groups of RAFT-polymerized poly(N,N-dimethylacrylamide). Quantitative end group modification is confirmed by 1 H NMR spectroscopy, size exclusion chromatography, and mass spectrometry, while reversible release of attached compounds under physiological reductive conditions is successfully monitored by diffusion ordered NMR spectroscopy and thin layer chromatography. Additionally, this concept is further expanded to protein-reactive, self-immolative carbonate species that enable reversible bioconjugation of lysozyme and α-macrophage mannose receptor (MMR) nanobodies as model proteins. Altogether, self-immolative RAFT end group modifications can form the new basis for reversible introduction of various functionalities to polymer chain ends including protein bioconjugates and, thus, opening novel opportunities for stimuli-responsive polymer hybrids.

Beyond p-Hexaphenylenes: Synthesis of Unsubstituted p-Nonaphenylene by a Precursor Protocol.

The synthesis of unsubstituted oligo-para-phenylenes (OPP) exceeding para-hexaphenylene-in the literature often referred to as p-sexiphenyl-has long remained elusive due to their insolubility. We report the first preparation of unsubstituted para-nonaphenylenes (9PPs) by extending our precursor route to poly-para-phenylenes (PPP) to a discrete oligomer. Two geometric isomers of methoxylated syn- and anti-cyclohexadienylenes were synthesized, from which 9PP was obtained via thermal aromatization in thin films. 9PP was characterized via optical, infrared and solid-state 13 C NMR spectroscopy as well as atomic force microscopy and mass spectrometry, and compared to polymeric analogues. Due to the lack of substitution, para-nonaphenylene, irrespective of the precursor isomer employed, displays pronounced aggregation in the solid state. Intermolecular excitonic coupling leads to formation of H-type aggregates, red-shifting emission of the films to greenish. 9PP allows to study the structure-property relationship of para-phenylene oligomers and polymers, especially since the optical properties of PPP depend on the molecular shape of the precursor.

2-Sulfonylpyrimidines as Privileged Warheads for the Development of S. aureus Sortase A Inhibitors.

Staphylococcus aureus is one of the most frequent causes of nosocomial and community-acquired infections, with emerging multiresistant isolates causing a significant burden to public health systems. We identified 2-sulfonylpyrimidines as a new class of potent inhibitors against S. aureus sortase A acting by covalent modification of the active site cysteine 184. Series of derivatives were synthesized to derive structure-activity relationship (SAR) with the most potent compounds displaying low micromolar KI values. Studies on the inhibition selectivity of homologous cysteine proteases showed that 2-sulfonylpyrimidines reacted efficiently with protonated cysteine residues as found in sortase A, though surprisingly, no reaction occurred with the more nucleophilic cysteine residue from imidazolinium-thiolate dyads of cathepsin-like proteases. By means of enzymatic and chemical kinetics as well as quantum chemical calculations, it could be rationalized that the S N Ar reaction between protonated cysteine residues and 2-sulfonylpyrimidines proceeds in a concerted fashion, and the mechanism involves a ternary transition state with a conjugated base. Molecular docking and enzyme inhibition at variable pH values allowed us to hypothesize that in sortase A this base is represented by the catalytic histidine 120, which could be substantiated by QM model calculation with 4-methylimidazole as histidine analog.

Role of Surface Chemistry in the Superhydrophobicity of the Springtail Orchesella cincta (Insecta:Collembola).

Collembola are ancient arthropods living in soil with extensive exposure to dirt, bacteria, and fungi. To protect from the harsh environmental conditions and to retain a layer of air for breathing when submerged in water, they have evolved a superhydrophobic, liquid-repelling cuticle surface. The nonfouling and self-cleaning properties of springtail cuticle make it an interesting target of biomimetic materials design. Recent research has mainly focused on the intricate microstructures at the cuticle surface. Here we study the role of the cuticle chemistry for the Collembola species Orchesella cincta (Collembola, Entomobryidae). O. cincta uses a relatively simple cuticle structure with primary granules arranged to function as plastrons. In contrast to the Collembolan cuticle featuring structures on multiple length scales that is functional irrespective of surface chemistry, we found that the O. cincta cuticle loses its hydrophobic properties after being rinsed with dichloromethane. Sum frequency generation spectroscopy and time-of-flight secondary ion mass spectrometry in combination with high-resolution mass spectrometry show that a nanometer thin triacylglycerol-containing wax layer at the cuticle surface is essential for maintaining the antiwetting properties. Removal of the wax layer exposes chitin, terpenes, and lipid layers in the cuticle. With respect to biomimetic applications, the results show that, combined with a carefully chosen surface chemistry, superhydrophobicity may be achieved using a relatively unsophisticated surface structure rather than a complex, re-entrant surface structure alone.

Site-Specific DBCO Modification of DEC205 Antibody for Polymer Conjugation.

The design of multifunctional polymer-based vectors, forming pDNA vaccines, offers great potential in cancer immune therapy. The transfection of dendritic immune cells (DCs) with tumour antigen-encoding pDNA leads to an activation of the immune system to combat tumour cells. In this work, we investigated the chemical attachment of DEC205 antibodies (aDEC205) as DC-targeting structures to polyplexes of P(Lys)-b-P(HPMA). The conjugation of a synthetic block copolymer and a biomacromolecule with various functionalities (aDEC205) requires bioorthogonal techniques to avoid side reactions. Click chemistry and in particular the strain-promoted alkyne-azide cycloaddition (SPAAC) can provide the required bioorthogonality. With regard to a SPAAC of both components, we firstly synthesized two different azide-containing block copolymers, P(Lys)-b-P(HPMA)-N3(stat) and P(Lys)-b-P(HPMA)-N3(end), for pDNA complexation. In addition, the site-specific incorporation of ring-strained dibenzocyclooctyne (DBCO) moieties to the DEC205 antibody was achieved by an enzymatic strategy using bacterial transglutaminase (BTG). The chemical accessibility of DBCO molecules within aDEC205 as well as the accessibility of azide-functionalities on the polyplex' surface were investigated by various SPAAC experiments and characterized by fluorescence correlation spectroscopy (FCS).

Electron Transfer from Bi-Isonicotinic Acid Emerges upon Photodegradation of N3-Sensitized TiO2 Electrodes.

The long-term stability of dye-sensitized solar cells (DSSCs) is determined to a large extent by the photodegradation of their sensitizers. Understanding the mechanism of light-induced decomposition of dyes sensitizing a mesoporous oxide matrix may therefore contribute to solutions to increase the life span of DSSCs. Here, we investigate, using ultrafast terahertz photoconductivity measurements, the evolution of interfacial electron-transfer (ET) dynamics in Ru(4,4'-dicarboxylic acid-2,2'-bipyridine)2(NCS)2 (N3) dye-sensitized mesoporous TiO2 electrodes upon dye photodegradation. Under inert environment, interfacial ET dynamics do not change over time, indicating that the dye is stable and photodegradation is absent; the associated ET dynamics are characterized by a sub-100 fs rise of the photoconductivity, followed by long-lived (≫1 ns) electrons in the oxide electrode. When the N3-TiO2 sample is exposed to air under identical illumination conditions, dye photodegradation is evident from the disappearance of the optical absorption associated with the dye. Remarkably, approximately half of the sub-100 fs ET is observed to still occur but is followed by very rapid (∼10 ps) electron-hole recombination. Laser desorption/ionization mass spectrometry, attenuated total reflection-Fourier transform infrared, and terahertz photoconductivity analyses reveal that the photodegraded ET signal originates from the N3 dye photodegradation product as bi-isonicotinic acid (4,4'-dicarboxylic acid-2,2'-bipyridine), which remains bonded to the TiO2 surface via either bidentate chelation or bridging-type geometry.

Monitoring the On-Surface Synthesis of Graphene Nanoribbons by Mass Spectrometry.

We present a mass spectrometric approach to characterize and monitor the intermediates of graphene nanoribbon (GNR) formation by chemical vapor deposition (CVD) on top of Au(111) surfaces. Information regarding the repeating units, lengths, and termini can be obtained directly from the surface sample by a modified matrix-assisted laser desorption/ionization (MALDI) method. The mass spectrometric results reveal ample oxidative side reactions under CVD conditions that can be drastically diminished by the introduction of protective H2 gas at ambient pressure. Simultaneously, the addition of hydrogen extends the lengths of the oligophenylenes and thus the final GNRs. Moreover, the prematurely formed cyclodehydrogenation products during the oligomer growth can be assigned by the mass spectrometric technique. The obtained mechanistic insights provide valuable information for optimizing and upscaling the bottom-up fabrication of GNRs. Given the important role of GNRs as semiconductors, the mass spectrometric analysis provides a readily available tool to characterize and improve their structural perfection.

Chemical Vapor Deposition Synthesis and Terahertz Photoconductivity of Low-Band-Gap N = 9 Armchair Graphene Nanoribbons.

Recent advances in bottom-up synthesis of atomically defined graphene nanoribbons (GNRs) with various microstructures and properties have demonstrated their promise in electronic and optoelectronic devices. Here we synthesized N = 9 armchair graphene nanoribbons (9-AGNRs) with a low optical band gap of ∼1.0 eV and extended absorption into the infrared range by an efficient chemical vapor deposition process. Time-resolved terahertz spectroscopy was employed to characterize the photoconductivity in 9-AGNRs and revealed their high intrinsic charge-carrier mobility of approximately 350 cm2·V-1·s-1.

Cycloparaphenylenes and Their Catenanes: Complex Macrocycles Unveiled by Ion Mobility Mass Spectrometry.

The insoluble product mixture obtained from cycloparaphenylene (CPP) synthesis from Suzuki coupling and reductive aromatization was analyzed. Traditional mass spectrometry suggests a homologous series of macrocycles with 12 to 84 phenylene units. Ion-mobility mass spectrometry, however, unravels an unexpected complexity of isomers with identical chemical formula, but different topologies. Whereas macrocycles containing up to 30 phenylene units show only one structure, the homologue with 36 phenylene units forms at least four different isomers with significant molecular size differences. They can be assigned to catenanes composed of CPPs with 2×18 and 12+24 phenylene units together with the ordinary [36]CPP macrocycle. Most likely, a trefoil knot of the CPP with 36 moieties is also present. For the first time, catenanes can be elucidated in a simple reaction mixture by analyzing their ions in the gas phase, an analysis which lies beyond the scope of traditional analytical methods.

Synthesis of Graphene Nanoribbons by Ambient-Pressure Chemical Vapor Deposition and Device Integration.

Graphene nanoribbons (GNRs), quasi-one-dimensional graphene strips, have shown great potential for nanoscale electronics, optoelectronics, and photonics. Atomically precise GNRs can be "bottom-up" synthesized by surface-assisted assembly of molecular building blocks under ultra-high-vacuum conditions. However, large-scale and efficient synthesis of such GNRs at low cost remains a significant challenge. Here we report an efficient "bottom-up" chemical vapor deposition (CVD) process for inexpensive and high-throughput growth of structurally defined GNRs with varying structures under ambient-pressure conditions. The high quality of our CVD-grown GNRs is validated by a combination of different spectroscopic and microscopic characterizations. Facile, large-area transfer of GNRs onto insulating substrates and subsequent device fabrication demonstrate their promising potential as semiconducting materials, exhibiting high current on/off ratios up to 6000 in field-effect transistor devices. This value is 3 orders of magnitude higher than values reported so far for other thin-film transistors of structurally defined GNRs. Notably, on-surface mass spectrometry analyses of polymer precursors provide unprecedented evidence for the chemical structures of the resulting GNRs, especially the heteroatom doping and heterojunctions. These results pave the way toward the scalable and controllable growth of GNRs for future applications.