Inverse Electron Demand Diels-Alder Research Articles

Inverse Electron Demand Diels-Alder Reaction of Pyrazines with 2,5-Norbornadiene as Acetylene Precursor

Herein, we report the reaction of pyrazines with 2,5-norbornadiene, an acetylene precursor, as a cascade of Diels-Alder reactions yielding substituted pyridines. In contrast to the known examples of intermolecular Diels-Alder type reaction of pyrazines with acetylene, which usually leads to a mixture of products, in our case the formation of a single isomer was observed. The proposed approach allows the conversion of pyrazines with different types of substitution, except those containing methyl and amino donor groups, to pyridines. In addition, highly functionalized aminonicotic esters were obtained, which can be used for the synthesis of bioactive compounds. An unusual course of the Diels-Alder reaction was found in the case of tetrasubstituted pyrazines, which cleave organic nitriles instead of HCN. Quantum-chemical modeling of possible transition states showed that this Diels-Alder reaction proceeds via cycloaddition of 2,5-norbornadiene to pyrazine with sequential elimination of HCN and cyclopentadene; the reaction pathway including initial formation of acetylene from norbornadiene and subsequent Diels-Alder reaction with the acetylene was rejected as kinetically unfavorable.

Synthesis of Polyoxygenated 4,5-Diarylpyridazines with Antiproliferative and Antitubulin Activity via Inverse Electron-Demand Diels-Alder Reaction of 1,2,4,5- Tetrazine

: The synthesis of a series of multifunctionalized 4,5-diarylpyridazines via inverse electron-demand Diels-Alder reaction between highly oxygenated diarylacetylenes and unsubstituted 1,2,4,5-tetrazine was developed using polyalkoxybenzenes isolated from industrial essential oils as starting material. The reaction proceeded smoothly to afford combretastatin A-4 analogs with pyridazine linker in consistently high yield. In a phenotypic sea urchin embryo assay, diarylpyridazine with 3,4,5-trimethoxyphenyl and 3-amino-4- methoxyphenyl aryl rings was identified as a potent antimitotic microtubule-destabilizing compound.

Bioorthogonal Activation of Deep Red Photoredox Catalysis Inducing Pyroptosis.

The revolutionary impact of photoredox catalytic processes has ignited novel avenues for exploration, empowering us to delve into nature in unprecedented ways and to pioneer innovative biotechnologies for therapy and diagnosis. However, integrating artificial photoredox catalysis into living systems presents significant challenges, primarily due to concerns over low targetability, low compatibility with complex biological environments, and the safety risks associated with photocatalyst toxicity. To address these challenges, herein, we present a novel bioorthogonally activatable photoredox catalysis approach. In this approach, potent photocatalyst selection via atom replacement of the rhodamine core yielded the bioorthogonally activatable photocatalyst (PC-Tz). The introduction of 1,2,4,5-tetrazine quenched its photocatalytic properties, which were restored upon an intracellular inverse electron-demand Diels-Alder (iEDDA) reaction with trans-cyclooctene (TCO) localized in mitochondria. This reaction led to remarkable photocatalytic oxidation of nicotinamide adenine dinucleotide (NADH), effectively manipulating the mitochondrial electron transport chain (ETC) under hypoxic conditions in cancer cells. Additionally, photocatalytic pyroptotic cell death was observed through a caspase-3/gasdermin E (GSDME) pathway, achieving notable antitumor efficacy and adenosine triphosphate (ATP) reduction in tumor cells. To the best of our knowledge, this represents the first example of bioorthogonally activatable photoredox catalysis, opening new avenues for chemists to spatiotemporally control activity in specific cell organelles without disrupting other native biological processes.

Unveiling the Structure-Fluorogenic Property Relationship of Seoul-Fluor-Derived Bioorthogonal Tetrazine Probes.

Tetrazine (Tz)-embedded fluorescent probes, known for their fluorogenicity following bioorthogonal inverse electron-demand Diels-Alder (iEDDA) reactions, are extensively used in bioimaging. Despite significant research on fluorogenic Tz probes, there has been limited systematic exploration of their fluorogenic responses with various dienophiles. In this study, we elucidate the structure-fluorogenic property relationship of bioorthogonal Tz probes. We synthesized a series of Seoul-Fluor-Tz (SFTz) probes designed to exhibit differentiated turn-on fluorescence upon iEDDA reactions with three dienophiles: trans-cyclooctene (TCO), bicyclo[6.1.0]nonyne (BCN), and spiro[2.3]hex-1-ene (Sph). Our findings revealed that the fluorogenic properties of the SFTz probes are highly dependent on the structures of dienophile-Tz adducts. By systematically modifying the electronic properties and employing quantum chemical calculations, we developed a series of SFTz probes with optimal dienophile-dependent fluorescence. These probes enabled simultaneous dual-color imaging of different cellular targets using a single probe, providing a robust approach for advanced bioimaging applications that require precise and efficient multicolor labeling strategies.

A Dual‐Purpose Non‐Canonical Amino Acid for the Expanded Genetic Code: Combining Metal‐Binding and Click Chemistry

AbstractA rationally designed dual‐purpose non‐canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold by genetic code expansion. Trz contains a 5‐pyridyl‐1,2,4‐triazine system, which allows for inverse‐electron‐demand Diels–Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site‐specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)‐bound protein blue‐shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual‐purpose non‐canonical amino acid with great potential for myriad bioapplications where metal‐based functionality is required, for example in imaging, catalysis, and photo‐dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual‐modality imaging or therapeutic payloads.

A Dual-Purpose Non-Canonical Amino Acid for the Expanded Genetic Code: Combining Metal-Binding and Click Chemistry.

A rationally designed dual-purpose non-canonical amino acid (Trz) has been synthesised and successfully incorporated into a protein scaffold by genetic code expansion. Trz contains a 5-pyridyl-1,2,4-triazine system, which allows for inverse-electron-demand Diels-Alder (IEDDA) reactions to occur on the triazine ring and for metal ions to be chelated both before and after the click reaction. Trz was successfully incorporated into a protein scaffold and the IEDDA utility of Trz demonstrated through the site-specific labelling of the purified protein with a bicyclononyne. Additionally, Trz was shown to successfully coordinate a cyclometallated iridium(III) centre, providing access to a bioorthogonal luminogenic probe. The luminescent properties of the Ir(III)-bound protein blue-shift upon IEDDA click reaction with bicyclononyne, providing a unique method for monitoring the extent and location of the labelling reaction. In summary, Trz is a new dual-purpose non-canonical amino acid with great potential for myriad bioapplications where metal-based functionality is required, for example in imaging, catalysis, and photo-dynamic therapy, in conjunction with a bioorthogonal reactive handle to impart additional functionalities, such as dual-modality imaging or therapeutic payloads.

Advancements in Inverse-Electron-Demand Diels–Alder Cycloaddition of 2-Pyrones: Mechanisms, Methodologies

The Diels-Alder (DA) cycloaddition is well-known for its effectiveness in synthesizing natural products and multifunctional materials. This article specifically explores the inverse-electron-demand Diels-Alder (IEDDA) cycloaddition involving 2-pyrones, which display ambiphilic properties due to their unique electronic characteristics. We investigate the mechanisms underlying IEDDA, with a focus on how electron-donating and electron-withdrawing substituents influence reactivity and product selectivity. Various methodologies are reviewed, encompassing non-catalytic and catalytic approaches. Special attention is given to advancements in microwave-assisted techniques and high-pressure conditions, which enhance both reaction efficiency and selectivity. Additionally, the synthesis of chiral bridged bicyclic lactones from substituted 2-pyrones is examined, illustrating their versatility in organic synthesis. This review underscores the significance of IEDDA cycloaddition in pioneering new synthetic routes for building complex molecular structures.

Towards a switchable nanoparticle behavior using inverse electron-demand Diels-Alder chemistry and ectoenzyme-based ligand activation

Nanoparticles (NPs) as drug delivery platforms encounter numerous obstacles on their journey from administration to the target site. Often, diametrically opposing particle properties are desirable to overcome biological and physical barriers. Therefore, stimuli-responsive NPs have been developed to allow for specific particle adaptation. In this work, it was demonstrated that NPs can be rendered switchable with respect to their interaction with a receptor through an external chemical stimulus. A combination of the inverse electron-demand Diels-Alder (iEDDA) reaction for subsequent NP functionalization and ectoenzyme-based ligand activation allowed for specific particle tailoring. Building on this, a two-step process for target cell recognition was developed. First, NPs were functionalized with Angiotensin-I (Ang-I) as inactive ligand using iEDDA chemistry. At the target site, the ligand was enzymatically processed to Angiotensin-ll (Ang-II) by cellular ectoenzymes. Ang-ll binds as active ligand to the angiotensin ll type 1 (AT1) receptor on the target cell surface. This enzymatic activation aims to minimize the biological effect of the ligand prior to particle binding, while the NP target cell specificity is increased by a two-step recognition with enzymatic processing and receptor binding.

Catalytic asymmetric inverse-electron-demand Diels–Alder reaction of 2-pyrones with aryl enol ethers

Chiral aryl cyclohex-3-en ether scaffold is widely present in bioactive natural products and drugs. The exploitation of efficient and enantioselective methods for the construction of aryl cyclohex-3-en ether scaffold is significant. Herein we disclose a chiral N,N’-dioxide/Lewis acid complex-catalyzed asymmetric inverse-electron-demand Diels–Alder (IEDDA) reaction using electron-deficient 3-carboalkoxyl-2-pyrones and less electron-enriched aryl enol ethers as reactants. A wide range of non- and 1,2-disubstituted acyclic aryl enol ethers are applicable to deliver diverse chiral bridged bicyclic lactones in high yields and stereoselectivities (up to 95% yield, >20:1 dr, 97:3 er). The bridged bicyclic lactone core can be easily converted into chiral aryl cyclohex-3-en ether scaffold. Notably, DFT calculations revealed a stepwise and endo mechanism to explain the high enantioselectivity controlled by the cooperative effect of the steric factors and the dispersion interactions between ligands and enol ethers.

Innovative Peptide Bioconjugation Chemistry with Radionuclides: Beyond Classical Click Chemistry.

Background: The incorporation of radionuclides into peptides and larger biomolecules requires efficient and sometimes biorthogonal reaction conditions, to which click chemistry provides a convenient approach. Methods: Traditionally, click-based radiolabeling techniques have focused on classical click chemistry, such as copper(I)-catalyzed alkyne-azide [3+2] cycloaddition (CuAAC), strain-promoted azide-alkyne [3+2] cycloaddition (SPAAC), traceless Staudinger ligation, and inverse electron demand Diels-Alder (IEDDA). Results: However, newly emerging click-based radiolabeling techniques, including tyrosine-click, sulfo-click, sulfur(VI) fluoride exchange (SuFEx), thiol-ene click, azo coupling, hydrazone formations, oxime formations, and RIKEN click offer valuable alternatives to classical click chemistry. Conclusions: This review will discuss the applications of these techniques in peptide radiochemistry.

Exploring the reactivity and interactions of a poly(fluorene‐co‐tetrazine)‐conjugated polymer with Single‐walled Carbon Nanotubes

AbstractConjugated tetrazine‐containing polymers that undergo inverse electron demand Diels‐Alder (IEDDA) reactions with trans‐cyclooctenes are interesting not only for their intrinsic optoelectronic properties, but also their interactions with π‐conjugated surfaces. Here, we prepared a series of poly(fluorene‐co‐tetrazine) polymers and carried out IEDDA reactions to decorate them with hydroxyl, hexadecyl, or triethylene glycol side chains. The polymers were investigated pre‐ and post‐IEDDA coupling in terms of their ability to disperse single‐walled carbon nanotubes (SWNTs) in organic solvent. It was found that polymer molecular weight, side chain structure, and degree of conjugation all impacted the quality of SWNT dispersions. While the starting poly(fluorene‐co‐tetrazine) polymer produced concentrated dispersions, the post‐IEDDA polymer containing dihydropyridazine groups did not produce dispersions of equal concentration. However, upon oxidation to the fully aromatic pyridazines, the polymers regained their ability to form concentrated dispersions. Furthermore, the post‐IEDDA polymers exhibited increased selectivity toward metallic SWNTs relative to the starting polymer. Due to the efficiency of the IEDDA reaction, it was also possible to use this chemistry to derivatize the nanotube complex with poly(fluorene‐co‐tetrazine) post‐dispersion. Overall, this work demonstrates the first use of reactive polytetrazines to disperse SWNTs, allowing rapid modification of polymer‐nanotube complexes.

Modular Semisynthetic Approach to Generate T Cell-Dependent Bispecific Constructs from Recombinant IgG1 Antibodies.

Redirecting T cells to tumor cells by bispecific antibodies is an effective approach to treat cancer, and T cell-dependent bispecific antibodies (TDBAs) are an emerging class of potent immunotherapeutic agents. By simultaneously targeting antigens on tumor cells and T cells, T cells are activated to kill tumor cells. Herein, we report a platform to generate a novel class of 2:1 structure of T cell-dependent bispecific antibody with bivalency for HER2 receptors on tumor cells and monovalency for CD3 receptors on T cells. For this, we use a biogenic inverse electron-demand Diels-Alder (IEDDA) click reaction on genetically encoded tyrosine residues to install one TCO handle on therapeutically approved antibody trastuzumab. Subsequent TCO-tetrazine click with a tetrazine-functionalized CD3-binding Fab yields a 2:1 HER2 × CD3 TDBA that exhibits a tumor-killing capability at picomolar concentrations. Monovalency toward the CD3 receptor on T cells can lower the chances of cytokine release syndrome, which is a common side effect of such agents. Our semisynthetic approach can generate highly potent TDBA constructs in a few chemoenzymatic and synthetic steps.

7-Deazapurine and Pyrimidine Nucleoside and Oligonucleotide Cycloadducts Formed by Inverse Diels-Alder Reactions with 3,6-Di(pyrid-2-yl)-1,2,4,5-tetrazine: Ethynylated and Vinylated Nucleobases for Functionalization and Impact of Pyridazine Adducts on DNA Base Pair Stability and Mismatch Discrimination.

The manuscript reports on 7-deazapurine and pyrimidine nucleoside and oligonucleotide cycloadducts formed by the inverse electron demand Diels-Alder (iEDDA) reaction with 3,6-di(pyrid-2-yl)-1,2,4,5-tetrazine. Cycloadducts were constructed from ethynylated and vinylated nucleobases. Oligonucleotides were synthesized containing iEDDA modifications, and the impact on duplex stability was investigated. iEDDA reactions were performed on nucleoside triple bond side chains. Oxidation was not required in these cases as dihydropyridazine intermediates are not formed. In contrast, oxidation is necessary for reactions performed on alkenyl compounds. This was verified on 5-vinyl-2'-deoxyuridine. A diastereomeric mixture of 1,2-dihydropyridazine cycloadduct intermediates was isolated, characterized, and later oxidized. 12-mer oligonucleotides containing 1,2-pyridazine inverse Diels-Alder cycloadducts and their precursors were hybridized to short DNA duplexes. For that, a series of phosphoramidites was prepared. DNA duplexes with 7-functionalized 7-deazaadenines and 5-functionalized pyrimidines display high duplex stability when spacer units are present between nucleobases and pyridazine cycloadducts. A direct connectivity of the pyridazine moiety to nucleobases as reported for metabolic labeling of vinyl nucleosides reduced duplex stability strongly. Oligonucleotides bearing linkers with and without pyridazine cycloadducts attached to the 7-deazaadenine nucleobase significantly reduced mismatch formation with dC and dG.

Click Chemistry Enables [89Zr]Zr-DOTA Radioimmunoconjugation for Theranostic 89Zr-immunoPET.

There have been predictions that the use of the macrocyclic chelating agent 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) in zirconium-89 (89Zr) immuno-positron emission tomography (89Zr-immunoPET) could enhance the in vivo stability of 89Zr radioimmunoconjugates. However, conjugating [89Zr]Zr-DOTA to a monoclonal antibody (mAb) remains a challenge as the heat treatment required for [89Zr]Zr-DOTA chelation can lead to thermal denaturation of the mAb moieties. We developed a method for synthesizing [89Zr]Zr-DOTA-mAb based on a tetrazine (Tz)-conjugated bifunctional DOTA derivative 2,2',2″-(10-(1-(4-(1,2,4,5-tetrazin-3-yl)phenyl)-3,21,26-trioxo-6,9,12,15,18-pentaoxa-29-carboxy-2,22,25-triazanonacosane-29-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTAGA-Tz) and the inverse electron-demand Diels-Alder (IEDDA) click chemistry reaction where trans-cyclooctene-modified mAbs are conjugated to [89Zr]Zr-DOTAGA without being exposed to heat. The stability of IEDDA-derived [89Zr]Zr-DOTAGA-trastuzumab was confirmed by in vitro, ex vivo, and in vivo testing and comparative analysis against the conventional deferoxamine (DFO) counterpart [89Zr]Zr-DFO-trastuzumab. The in vivo immunoPET imaging using [89Zr]Zr-DOTAGA-trastuzumab clearly visualized human epidermal growth factor receptor 2-positive malignancies in murine xenograft models. Greater tumor contrast was observed from [89Zr]Zr-DOTAGA-trastuzumab at a 72-h delayed scan compared with [89Zr]Zr-DFO-trastuzumab. These findings suggest that our IEDDA ligation approach can be an effective means of synthesizing [89Zr]Zr-DOTA-mAb and can enhance the theranostic potential of 89Zr-immunoPET in DOTA-mediated radioimmunotherapy.

(Invited) Reactivity on Endohedral Metallofullerenes

Endohedral metallofullerenes (EMFs) are fullerene molecules with unique host-guest structures that allow chemists to manipulate encapsulated metal ions with the arsenal of organic chemistry, for potential applications including spin-quantum computing, single-molecule magnets, dynamic nuclear polarization, artificial photosynthesis, magnetic resonance imaging contrast agents, and therapeutic agents. To unleash the power to do so, the development of new reactions for EMFs has been an ongoing task.A key step to improve the solubility of EMFs as well as to obtain new materials for multiple applications is via exohedral functionalization. In medicine, for instance, water-soluble gadofullerenols are used as powerful contrast agents.1 Besides, the external groups might impart complementary features to EMFs that tune their physical properties for use as acceptors in polymer solar cells or as donor-acceptor dyad conjugates.2 Among the most used methods to obtain EMF monoadducts are cycloaddition reactions and radical additions, including the Diels-Alder cycloaddition, Bingel-Hirsch cyclopropanation, Prato 1,3-dipolar addition, carbene addition, etc. In these reactions, fullerenes typically play a similar electron-accepting role, thus their reactivity and regioselectivity are highly consistent. However, the reactivity of EMFs is far more complicated than of empty fullerenes due to the relatively low molecular symmetry and the influence of the encapsulated species. To understand the role of metal-cage interactions on the reactivity of EMFs is desirable for tuning their chemical reactivity.Here, we report the functionalization of actinide endofullerenes, in particular, the mono-EMFs U@C s(4)-C82 and U@C 2v(9)-C82, which their chemical reactivities were investigated through the Bingel-Hirsch (nucleophilic addition) and carbene addition (electrophilic addition) reactions. Interestingly, only for U@C 2v(9)-C82, the Bingel-Hirsch reaction leads to mono-adduct and multi-adduct compounds. Another subgroup of EMF that we study its reactivity here is the nitride clusterfullerenes, especially the Lu3N@Ih-C80, with high stability and high production yields, that results its chemical modification to be challenging. In this case, the reaction studied is the inverse-electron demand Diels-Alder (IEDDA) reaction and it will be compared to the corresponding reactivity on C60 fullerene. We show that Lu3N@Ih-C80presents higher reactivity than for C60, as well as, different features. 1Bolskar, R. D.; Benedetto, A. F.; Wilson, L. J.; Alford, J. M. et al. J. Am. Chem. Soc. 2003, 125, 5471-5478. 2Ross, R. B.; Cardona, C. M.; Guldi, D. M. et al. Nature Mater. 2009, 8, 208-212. Figure 1

Bioorthogonal Cycloadditions of C3-Trifluoromethylated 1,2,4-Triazines with trans-Cyclooctenes.

1,2,4-triazines are a valuable class of heterodienes that can be employed in inverse electron-demand Diels-Alder reactions. However, their broader application in bioorthogonal chemistry is limited due to their low reactivity. This article focuses on 3-(trifluoromethyl)-1,2,4-triazines, which can be efficiently prepared in a one-pot reaction from NH-1,2,3-triazoles. These triazines are highly reactive in reactions with strained cyclooctenes, giving second-order rate constants as high as 230 M-1 s-1. Despite their high reactivity, the compounds remain sufficiently stable under biologically relevant conditions. We show that some of the compounds are fluorogenic, a property of potential use in bioimaging. In addition, we demonstrate the successful application of the triazines in labeling model biomolecules. Our work shows that the reactivity of 1,2,4-triazines can be enhanced by the 3-CF3-substitution, which we consider an important step toward the wider use of this promising class of reagents.

A Bioorthogonal Dual Fluorogenic Probe for the Live-Cell Monitoring of Nutrient Uptake by Mammalian Cells.

Cells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron-Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein-diacetate-succinimidyl esters (CFSE)-a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity-with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron-rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile-containing nutrients in live primary immune cell populations using flow cytometry and live-cell microscopy.

A Bioorthogonal Dual Fluorogenic Probe for the Live‐Cell Monitoring of Nutrient Uptake by Mammalian Cells

AbstractCells rely heavily on the uptake of exogenous nutrients for survival, growth, and differentiation. Yet quantifying the uptake of small molecule nutrients at the single cell level is difficult. Here we present a new approach to studying the nutrient uptake in live single cells using Inverse Electron‐Demand Diels Alder (IEDDA) chemistry. We have modified carboxyfluorescein‐diacetate‐succinimidyl esters (CFSE)—a quenched fluorophore that can covalently react with proteins and is only turned on in the cytosol of a cell following esterase activity—with a tetrazine. This tetrazine serves as a second quencher for the pendant fluorophore. Upon reaction with nutrients modified with an electron‐rich or strained dienophile in an IEDDA reaction, this quenching group is destroyed, thereby enabling the probe to fluoresce. This has allowed us to monitor the uptake of a variety of dienophile‐containing nutrients in live primary immune cell populations using flow cytometry and live‐cell microscopy.

First Proof of Concept of a Click Inverse Electron Demand Diels-Alder Reaction for Assigning the Regiochemistry of Carbon-Carbon Double Bonds in Untargeted Lipidomics.

Lipidomics by high-resolution mass spectrometry (HRMS) has become a prominent tool in clinical chemistry due to the proven connections between lipid dysregulation and the insurgence of pathologies. However, it is difficult to achieve structural characterization beyond the fatty acid level by HRMS, especially when it comes to the regiochemistry of carbon-carbon double bonds, which play a major role in determining the properties of cell membranes. Several approaches have been proposed for elucidating the regiochemistry of double bonds, such as derivatization before MS analysis by photochemical reactions, which have shown great potential for their versatility but have the unavoidable drawback of splitting the MS signal. Among other possible approaches for derivatizing electron-rich double bonds, the emerging inverse-electron-demand Diels-Alder (IEDDA) reaction with tetrazines stands out for its unmatchable kinetics and has found several applications in basic biology and protein imaging. In this study, a catalyst-free click IEDDA reaction was employed for the first time to pinpoint carbon-carbon double bonds in free and conjugated fatty acids. Fatty acid and glycerophospholipid regioisomers were analyzed alone and in combination, demonstrating that the IEDDA reaction had click character and allowed the obtention of diagnostic product ions following MS/MS fragmentation as well as the possibility of performing relative quantitation of lipid regioisomers. The IEDDA protocol was later employed in an untargeted lipidomics study on plasma samples of patients suffering from prostate cancer and benign prostatic conditions, confirming the applicability of the proposed reaction to complex matrices of clinical interest.

Data-Efficient, Chemistry-Aware Machine Learning Predictions of Diels-Alder Reaction Outcomes.

The application of machine learning models to the prediction of reaction outcomes currently needs large and/or highly featurized data sets. We show that a chemistry-aware model, NERF, which mimics the bonding changes that occur during reactions, allows for highly accurate predictions of the outcomes of Diels-Alder reactions using a relatively small training set, with no pretraining and no additional features. We establish a diverse data set of 9537 intramolecular, hetero-, aromatic, and inverse electron demand Diels-Alder reactions. This data set is used to train a NERF model, and the performance is compared against state-of-the-art classification and generative machine learning models across low- and high-data regimes, with and without pretraining. The predictive accuracy (regio- and site selectivity in the major product) achieved by NERF exceeds 90% when as little as 40% of the data set is used for training. Another high-performing model, Chemformer, requires a larger training data set (>45%) and pretraining to reach 90% Top-1 accuracy. Accurate predictions of less-represented reaction subclasses, such as those involving heteroatomic or aromatic substrates, require higher percentages of training data. We also show how NERF can use small amounts of additional training data to quickly learn new systems and improve its overall understanding of reactivity. Synthetic chemists stand to benefit as this model can be rapidly expanded and tailored to areas of chemistry corresponding to the low-data regime.