Labeling Procedure Research Articles

Al18F-labeled alpha-melanocyte-stimulating hormone (α-MSH) peptide derivative for the early detection of melanoma.

Early detection plays a role in the prognosis of melanoma, the most aggressive skin cancer. 64Cu- and 68Ga-labeled alpha-melanocyte-stimulating hormone (α-MSH) analogs targeting the melanocortin-1 receptor are promising positron emission tomography (PET) tracers for detecting melanoma, and the use of 18F-labeling will further contribute to the detectability and availability. However, the high radiochemistry demand related to the conventional 18F-labeling methods has restricted the development of 18F-labeled α-MSH analogs. A recently developed radiofluorination method using aluminum-fluoride (Al18F) offers a simple, efficient, and time-saving labeling procedure compared to the conventional 18F-labeling methods. Herein, we sought to establish a simple preparation method for an 18F-labeled α-MSH analog using Al18F, and we examined its potential for the early detection of melanoma. A 1,4,7-triazacyclononane-N,N',N″-triacetic acid (NOTA)-conjugated α-MSH analog (NOTA-GGNle-CycMSHhex) was prepared by the Fmoc solid-phase strategy. NOTA-GGNle-CycMSHhex was labeled with Al18F by heating at 105°C using a microwave synthesizer for 15min. Biodistribution study was conducted on B16/F10-luc melanoma-bearing mice at 30min, 1h and 3h after injection of Al18F-NOTA-GGNle-CycMSHhex. PET imaging was conducted on melanoma-bearing mice at 1h post-injection. One day prior to the PET imaging, bioluminescence imaging was also performed. Al18F-NOTA-GGNle-CycMSHhex was readily prepared with a high radiochemical yield (94.0 ± 2.8%). The biodistribution study showed a high accumulation of Al18F-NOTA-GGNle-CycMSHhex in the tumor at 30min and 1h post-injection (6.69 ± 1.49 and 7.70 ± 1.71%ID/g, respectively). The tumor-to-blood ratio increased with time: 3.46 ± 0.89, 12.67 ± 1.29, and 35.27 ± 9.12 at 30min, 1h, and 3h post-injection, respectively. In the PET imaging, Al18F-NOTA-GGNle-CycMSHhex clearly visualized the tumors and depicted very small tumors (< 3mm). We successfully prepared Al18F-NOTA-GGNle-CycMSHhex in a simple and efficient manner. Al18F-NOTA-GGNle-CycMSHhex showed high tumor accumulation and clearly visualized very small tumors in melanoma-bearing mice. These findings suggest that Al18F-NOTA-GGNle-CycMSHhex will be a promising PET tracer for melanoma imaging at an earlier stage.

Indirect Aminoallyl-dUTP Labeling of RNA for Microarrays.

The labeling procedure described here is called indirect because the fluorescent moiety is not incorporated during the reverse transcription reaction. Instead, a reactive nucleotide analog (aminoallyl-dUTP) is incorporated during reverse transcription, the cDNA is isolated, and then the cyanine dyes are incorporated by binding with the aminoallyl group to produce the desired fluorescent cDNA.

TMT Labeling for the Masses: A Robust and Cost-efficient, In-solution Labeling Approach

Isobaric stable isotope labeling using, for example, tandem mass tags (TMTs) is increasingly being applied for large-scale proteomic studies. Experiments focusing on proteoform analysis in drug time course or perturbation studies or in large patient cohorts greatly benefit from the reproducible quantification of single peptides across samples. However, such studies often require labeling of hundreds of micrograms of peptides such that the cost for labeling reagents represents a major contribution to the overall cost of an experiment. Here, we describe and evaluate a robust and cost-effective protocol for TMT labeling that reduces the quantity of required labeling reagent by a factor of eight and achieves complete labeling. Under- and overlabeling of peptides derived from complex digests of tissues and cell lines were systematically evaluated using peptide quantities of between 12.5 and 800 μg and TMT-to-peptide ratios (wt/wt) ranging from 8:1 to 1:2 at different TMT and peptide concentrations. When reaction volumes were reduced to maintain TMT and peptide concentrations of at least 10 mm and 2 g/l, respectively, TMT-to-peptide ratios as low as 1:1 (wt/wt) resulted in labeling efficiencies of > 99% and excellent intra- and interlaboratory reproducibility. The utility of the optimized protocol was further demonstrated in a deep-scale proteome and phosphoproteome analysis of patient-derived xenograft tumor tissue benchmarked against the labeling procedure recommended by the TMT vendor. Finally, we discuss the impact of labeling reaction parameters for N-hydroxysuccinimide ester-based chemistry and provide guidance on adopting efficient labeling protocols for different peptide quantities.

Combining inverse photogrammetry and BIM for automated labeling of construction site images for machine learning

Abstract Image-based object detection provides a valuable basis for site information retrieval and construction progress monitoring. Machine learning approaches, such as neural networks, are able to provide reliable detection rates. However, labeling of training data is a tedious and time-consuming process, as it must be performed manually for a substantial number of images. The paper presents a novel method for automatically labeling construction images based on the combination of 4D Building Information Models and an inverse photogrammetry approach. For the reconstruction of point clouds, which are often used for progress monitoring, a large number of pictures are taken from the site. By aligning the Building Information Model and the resulting point cloud, it is possible to project any building element of the BIM model into the acquired pictures. This allows for automated labeling as the semantic information of the element type is provided by the BIM model and can be associated with the respective regions. The labeled data can subsequently be used to train an image-based neural network. Since the exact regions for all elements are defined, labels can be generated for basic tasks like classification as well as more complex tasks like semantic segmentation. To prove the feasibility of the developed methods, the labeling procedure is applied to several real-world construction sites, providing over 30,000 automatically labeled elements. The correctness of the assigned labels has been validated by pixel based area comparison against manual labels.

Label-free and immobilization-free photoelectrochemical biosensing strategy using methylene blue in homogeneous solution as signal probe for facile DNA methyltransferase activity assay

Photoelectrochemical (PEC) methods have recently witnessed ever expanding application in bioanalysis, but it is still desirable to further simplify the sensing procedures and develop simple and reliable PEC biosensing approaches. Herein, we proposed a truly label-free and immobilization-free PEC sensing platform, utilizing solution-phase methylene blue (MB) as the signal probe, and bare indium tin oxide (ITO) glass as the photoelectrode. Based on the diffusivity difference between free MB molecules and MB intercalated in DNA G-quadruplex, the activity and inhibition of DNA adenine methyltransferase (Dam), a proof-of-concept methyltransferase (MTase), is quantitatively analyzed. By taking advantage of the endonuclease-catalyzed cleavage of the Dam-methylated hairpin DNA probe, as well as the KF polymerase/Nt.AlwI endonuclease-aided signal amplification, highly sensitive and specific PEC detection of Dam activity has been achieved. Moreover, this approach can be easily extended to assay other types of MTase by choosing the appropriate methylation-sensitive endonucleases. The as-proposed strategy has also been successfully applied to analyze Dam spiked in human serum samples and to assess the inhibitory effects of antibiotics on Dam activity. More importantly, this label-free and truly immobilization-free PEC biosensing strategy shows additional merits of simplicity and satisfactory repeatability, due to the elimination of both labelling and immobilization procedures, making it a promising candidate for the application in highly sensitive, facile and reliable bioanalysis and drug screening.

Minimizing the Influence of Fluorescent Tags on IgG Partition in PEG-Salt Aqueous Two-Phase Systems for Rapid Screening Applications.

Aqueous two-phase extraction (ATPE) has been showing significant potential in the biopharmaceutical industry, allowing the selective separation of high-value proteins directly from unclarified cell culture supernatants. In this context, effective high-throughput screening tools are critical to perform a rapid empirical optimization of operating conditions. In particular, microfluidic ATPE screening devices, coupled with fluorescence microscopy to continuously monitor the partition of fluorophore-labeled proteins, have been recently demonstrated to provide short diffusion distances and rapid partition, using minimal reagent volumes. Nevertheless, the currently overlooked influence of the labeling procedure on partition must be carefully evaluated to validate the extrapolation of results to the unlabeled molecule. Here, three fluorophores with different global charge and reactivity selected to label immunoglobulin G (IgG) at degrees of labeling (DoL) ranging from 0.5 to 7.6. Labeling with BODIPY FL maleimide (DoL = 0.5), combined with tris(2-carboxyethyl) phosphine (TCEP) to generate free thiol groups, is the most promising strategy to minimize the influence of the fluorophore on partition. In particular, the partition coefficient (Kp ) measured in polyethylene glycol (PEG) 3350-phosphate systems with and without the addition of NaCl using microtubes (batch) or microfluidic devices (continuous) is comparable to those quantified for the native protein.

Raman Spectroscopy and Aptamers for a Label-Free Approach: Diagnostic and Application Tools.

Raman spectroscopy is a powerful optical technique based on the inelastic scattering of incident light to assess the chemical composition of a sample, including biological ones. Medical diagnostic applications of Raman spectroscopy are constantly increasing to provide biochemical and structural information on several specimens, being not affected by water interference, and potentially avoiding the constraint of additional labelling procedures. New strategies have been recently developed to overcome some Raman limitations related, for instance, to the need to deal with an adequate quantity of the sample to perform a reliable analysis. In this regard, the use of metallic nanoparticles, the optimization of fiber optic probes, and other approaches can actually enhance the signal intensity compared to spontaneous Raman scattering. Moreover, to further increase the potential of this investigation technique, aptamers can be considered as a valuable means, being synthetic, short, single, or double-stranded oligonucleotides (RNAs or DNAs) that fold up into unique 3D structures to specifically bind to selected molecules, even at very low concentrations, and thus allowing an early diagnosis of a possible disease. Due to the paramount relevance of the topic, this review focuses on the main Raman spectroscopy techniques combined with aptamer arrays in the label-free mode, providing an overview on different applications to support healthcare management.

Image-Based Quantification of Cell Debris as a Measure of Apoptosis.

Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis-detection methods rely on fluorescent markers, which necessitate the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic-cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field-microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture-media samples followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death.

Visualization of Germinosomes and the Inner Membrane in Bacillus subtilis Spores.

The small size of spores and the relatively low abundance of germination proteins, cause difficulties in their microscopic analyses using epifluorescence microscopy. Super-resolution three-dimensional Structured Illumination Microscopy (3D-SIM) is a promising tool to overcome this hurdle and reveal the molecular details of the process of germination of Bacillus subtilis (B. subtilis) spores. Here, we describe the use of a modified SIMcheck (ImageJ)-assistant 3D imaging process and fluorescent reporter proteins for SIM microscopy of B. subtilis spores' germinosomes, cluster(s) of germination proteins. We also present a (standard)3D-SIM imaging procedure for FM4-64 staining of B. subtilis spore membranes. By using these procedures, we obtained unsurpassed resolution for germinosome localization and show that >80% of B. subtilis KGB80 dormant spores obtained after sporulation on defined minimal MOPS medium have one or two GerD-GFP and GerKB-mCherry foci. Bright foci were also observed in FM4-64 stained spores' 3D-SIM images suggesting that inner membrane lipid domains of different fluidity likely exist. Further studies that use double labeling procedures with membrane dyes and germinosome reporter proteins to assess co-localization and thus get an optimal overview of the organization of Bacillus germination proteins in the inner spore membrane are possible.

Labeling of Nanofiber-Forming Peptides by Site-Directed Bioconjugation: Effect of Spacer Length on Self-Assembly.

The conjugation of small organic molecules to self-assembling peptides is a versatile tool to decorate nanostructures with original functionalities. Labeling with chromophores or fluorophores, for example, creates optically active fibers with potential interest in photonic devices. In this work, we present a rapid and effective labeling procedure for a self-assembling peptide able to form nanofibers. Rapid periodate oxidation of the N-terminal serine residue of the peptide and subsequent conjugation with dansyl moiety generated fluorophore-decorated peptides. Three dansyl-conjugated self-assembling peptides with variable spacer-length were synthesized and characterized and the role of the size of the linker between fluorophore and peptide in self-assembling was investigated. Our results show that a short linker can alter the self-assembly in nanofibers of the peptide. Herein we report on an alternative strategy for creating functionalized nanofibrils, able to expand the toolkit of chemoselective bioconjugation strategies to be used in site-specific decoration of self-assembling peptides.

DNAzyme-based Y-shaped label-free electrochemiluminescent biosensor for lead using electrically heated indium-tin-oxide electrode for in situ temperature control

The temperature control in the traditional DNAzyme-based electrochemiluminescent (ECL) biosensors for Pb2+ is realized through bulk solution heating, which needs tedious procedures, relative large equipment and this limits their application. Moreover, the ECL indicators had been modified on the DNA through chemical reaction, which has the limitation of low efficiency and high cost. In this study, electrically heated indium-tin-oxide (ITO) electrode is applied to adjust the temperature instead of the whole bulk solution heating. Y-shaped double strand DNA (dsDNA) was formed through the hybridization of DNAzyme, substrate and capture DNA, then Ru(phen)32+ was embedded into the groove of dsDNA and acted as the ECL indicator, which avoided the costly labeling procedure of ECL indicator on DNA. The results indicated that the two different ways of heating had the same effects on the DNA hybridization and DNAzyme action, but the temperature controlled by the heated electrode is simpler and quicker than that with bulk solution heating. Furthermore, the performance of the biosensor had been further improved at an elevated electrode surface temperature because temperature affected the performance of the ECL of Ru(phen)32+ greatly. Under the optimized conditions, the ECL signal has a linear relationship with logarithm concentration of Pb2+ in the range of 0.25 ˜ 500 pM with a detection limit of 0.2 pM at the electrode temperature of 45 °C, and this outcome was approximately 5 times lower than that at 25 °C of electrode surface temperature. The proposed biosensor has been applied to detect Pb2+ in soil samples with satisfactory results.

Study of a Miniaturizable System for Optical Sensing Application to Human Cells

Conventional approaches to human intracellular optical sensing, generally, require dedicated laboratories with bulky detection systems. They are performed by cell labeling procedures based on the use of fluorophores that are, mostly, phototoxic, invasive, bleached in case of prolonged light exposures, which require carriers and/or structural modifications for the cellular uptake. These issues, together with the sensitivity of the eukaryotic cell model, could be problematic towards the development of a robust sensing system suitable for biomedical screening. In this work, we studied a sensing system resulting from the combination of the commercial tris(2,2’bipyridyl)ruthenium(II) fluorophore, for cell labeling, with a potentially miniaturizable optical system composed by a laser source and a photomultiplier tube, for the fluorescence analysis.

Experimental Strategies of Mesenchymal Stem Cell Propagation: Adverse Events and Potential Risk of Functional Changes.

Mesenchymal stem cells (MSCs) are attractive candidates for cell-based tissue repair approaches. Hundreds of clinical trials using MSCs have been completed and many others are still being investigated. For most therapeutic applications, MSC propagation in vitro is often required. However, ex vivo culture condition is not fully physiological and may affect biological properties of MSCs including their regenerative potential. Moreover, both cell cryopreservation and labelling procedure prior to infusion may have the negative impact on their expected effect in vivo. The incidence of MSC transformation during in vitro culture should be also taken into consideration before using cells in stem cell therapy. In our review, we focused on different aspects of MSC propagation that might influence their regenerative properties of MSC. We also discussed the influence of different factors that might abolish MSC proliferation and differentiation as well as potential impact of stem cell senescence and aging. Despite of many positive therapeutic effects of MSC therapy, one has to be conscious about potential cell changes that could appear during manufacturing of MSCs.

Clonal Composition of Human Multipotent Mesenchymal Stromal Cells: Application of Genetic Barcodes in Research.

Clonal composition of human multipotent mesenchymal stromal cells (MMSCs) labeled with lentiviral vectors carrying genetic barcodes was studied. MMSCs were transduced with a cloned library of self-inactivating lentiviral vectors carrying 667 unique barcodes. At each cell culture passage, 120 cells were plated one cell per well in 96-well plates. The efficiency of cloning and labeling of the clonogenic cells was determined. DNA was extracted from the cell-derived colonies, and the barcodes were identified by Sanger sequencing. Also, DNA was extracted from the total MMSC population at each passage to analyze the diversity and representation of barcodes by deep sequencing using the Illumina platform. It was shown that the portion of MMSCs labeled with the lentiviral vectors remained stable in the passaged cells. Because of the high multiplicity of infection, the labeling procedure could decrease the proliferative potential of MMSCs. Identification of barcodes in individual cell clones confirmed the polyclonal character of the MMSC population. Clonal composition of MMSCs changed significantly with the passages due to the depletion of proliferative potential of most cells. Large clones were found at the first passage; at later passages, many small clones with a limited proliferative potential were detected in the population. The results of deep sequencing confirmed changes in the clonal composition of MMSCs. The polyclonal MMSC population contained only a small number of cells with a high proliferative potential, some of which could be stem cells. MMSCs with a high proliferative potential were detected more often in the earliest passages. In this regard, we would recommend to use MMSCs of early passages for regenerative medicine applications based on cell proliferation.

Labelling halophilic Archaea using 13C and 15N stable isotopes: a potential tool to investigate haloarchaea consumption by metazoans.

The use of stable isotope (SI) labelling and tracing of live diets is currently considered one of the most comprehensive tools to detect their uptake and assimilation by aquatic organisms. These techniques are indeed widely used in nutritional studies to follow the fate of specific microbial dietary components, unraveling trophic interactions. Nevertheless, to the current date our understanding of aquatic trophic relationships has yet to include a whole domain of life, the Archaea. The aim of the present research was, therefore, to describe a halophilic Archaea (haloarchaea) labelling procedure, using the SI 13C and 15N, to enable the application of SI tracing in future studies of haloarchaea consumption by aquatic metazoans. To this end, three 13C enriched carbon sources and two 15N enriched nitrogen sources were tested as potential labels to enrich cells of three haloarchaea strains when supplemented to the culture medium. Our overall results indicate 13C-glycerol as the most effective carbon source to achieve an efficient 13C enrichment in haloarchaea cells, with Δδ13C values above 5000‰ in all tested haloarchaea strains. As for 15N enriched nitrogen sources, both (15NH4)2SO4 and 15NH4Cl seem to be readily assimilated, also resulting in efficient 15N enrichment in haloarchaea cells, with Δδ15N values higher than 20,000‰. We believe that the proposed methodology will allow for the use of SI labelled haloarchaea biomass in feeding tests, potentially providing unambiguous confirmation of the assimilation of haloarchaea biomass by aquatic metazoans.

Nanobody: outstanding features for diagnostic and therapeutic applications.

Nanobodies (Nbs) have arisen as an alternative to conventional antibodies (Abs) and show great potential when used as tools in different biotechnology fields such as diagnostics and therapy. Different approaches have been described for the production of Nbs and these methods face new challenges focused on improving yield, affinity, and reducing production costs. This review summarizes these challenges, and also the latest advances in the detection of different kinds of molecules, such as proteins and small molecules, and describes their potential use for noninvasive in vivo imaging and for in vitro assays. Moreover, the unique properties of Nbs are outlined like internalization, size, thermal and chemical stability, affinity, blood clearance, and labeling procedures. Concerning therapeutic applications, we highlight some already reported examples about Nbs being used for the treatment of several diseases such as cancer, neurodegenerative or infectious diseases among others. Finally, future trends, opportunities, and disadvantages are also discussed.

Automated production of [68 Ga]Ga-DOTANOC and [68 Ga]Ga-PSMA-11 using a TRACERlab FXFN synthesis module.

The interest in gallium-68 labelled positron-emission tomography probes continues to increase around the world. However, one of the barriers for routine clinical use is the cost of the automated synthesis units for relatively simple labelling procedures. Herein, we describe the adaptation of a TRACERlab FXFN synthesis module for the automated production of gallium-68 radiopharmaceuticals using a cation-exchange cartridge for postprocessing of the 68 Ge/68 Ga generator eluate. The recovery of activity from the cartridge was 95.6% to 98.9% using solutions of acidified sodium chloride (5M with pH=1-3). The radiosyntheses of [68 Ga]Ga-DOTANOC and [68 Ga]Ga-PSMA-11 were performed using acetate sodium buffer or 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid, with a total duration of 21 and 23minutes, respectively, including generator elution and radiopharmaceutical dispensing. Activity yields were 77%±2% for [68 Ga]Ga-PSMA-11 and 68%±3% for [68 Ga]Ga-DOTANOC (n>100). The labelled peptides had a radiochemical purity exceeding 97%, and all quality control parameters were in conformity with the limits prescribed by the European Pharmacopoeia.

Radiochemical and analytical aspects of inter-institutional quality control measurements on radiopharmaceuticals

BackgroundClinically applied radiopharmaceuticals have to meet quality release criteria like a high radiochemical yield and radiochemical purity. Many radiopharmaceuticals do not have marketing authorization and have no dedicated monograph within the European pharmacopeia, therefore general monographs on quality control have to be applied for clinical applications. These criteria require standardization and validation in labeling and preparation, including QC measurements according to well-defined standard operation procedures. QC measurements however, are often based on detection techniques specific for a certain LC-system. Multi-institutional research and development of new radiopharmaceuticals lead to an increase in multicenter trials. Although all institutes’ radiopharmacies are using the same standardized labeling and operation procedures, they often use different LC and radiodetection systems. Here we present a comparison of QC assessments for 3 radiopharmaceuticals with focus on the interpretation of chromatograms, data-output and potential differences in local practical performances of QC on (U)HPLC.MethodsQC assessments for [111In]In-CCK, [68Ga]Ga-Bombesin and [177Lu]Lu-PSMA analogs were compared. Two of the radiopharmaceutical QC assessments were also applied in other institutes using their own HPLC-systems and concordant software. Data from the HPLC-injections and measurements is processed and summarized in chromatograms, based on a variety of smoothing algorithms for which different software programs are applied. Described radiopeptides were labeled and analyzed according their standardized labeling and operation procedures.ResultsIntegration of main peaks on chromatograms resulted in a range of RCP, depending on the smoothing algorithm used. [111In]In-CCK(A), 68Ga-Bombesin(B) and [177Lu]Lu-PSMA(C) analogs had a RCP range of 88%–96%(A), 89–95%(B) and 92–99%(C) respectively. Important factors affecting final RCP value were site specific background radiation-levels, intrinsic system properties such as noise and sensitivity, personal interpretation e.g. peak-tailing and smoothing algorithms.ConclusionMeasurement of RCP shows a strong method- and system-dependency, even when parameters are validated, standardized and SOP are followed. Release criteria are frequently based on RCP data from one central location. The lack of inter inter institutional validation and standardization in RCP determination makes the results therefore rather arbitrary. For multicenter trials, we recommend to compare locally determined RCP under validated and standardized conditions of in-line activity detection between institutes for each radiopharmaceutical.

15N–1H Transfer of Light-Induced Nuclear Hyperpolarization in Frozen Photosynthetic Reaction Centers

Recently, we have demonstrated the possibility to transfer the light-induced hyperpolarization occurring on select 13C nuclei of photosynthetic cofactors due to the solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) effect into the proton pool via inverse 13C–1H cross-polarization Bielytskyi et al. (J Magn Reson 293: 82–91, 2018). Such approach allowed us to observe the selective response from the protons belonging to photochemically active cofactors in their native protein environment. In the present study, we demonstrate that also 15N nuclei can be used as a source of polarization in a similar type of experiments. We present the 2D photo-CIDNP 15N–1H heteronuclear correlation (HETCOR) experiment acquired on uniformly 15N-labeled quinone-blocked reaction center (RC) from Rhodobacter sphaeroides R26. Obtained 1H chemical shifts match with previously observed ones on selectively 13C-labeled RC from Rhodobacter sphaeroides WT. We expect that using 15N as a source of polarization in potential heteronuclear spin-torch experiments could be advantageous for systems where the procedure for selective 13C labeling is not yet established.

Navigating around Patented Routes by Preserving Specific Motifs along Computer-Planned Retrosynthetic Pathways

Summary By keeping track of lists of specific bonds one wishes to preserve, a computer program is able to identify the key disconnections used in the patented syntheses and design synthetic routes that circumvent these approaches. Here, we provide examples of computer-designed syntheses relevant to medicinal chemistry, in which the machine avoids “strategic” disconnections common to industrial patents and is forced to use different starting materials. The ability of modern retrosynthetic planners to navigate around patented solutions may have significant implications for the ways in which intellectual property related to multistep syntheses is protected and/or challenged.