Inverse Electron Demand Diels-Alder Cycloaddition Research Articles

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.

Abstract 2614: GlycoConnect™ ADC toolbox expansion with high DAR technology

Abstract Recently topoisomerase 1 inhibitors have gained significant traction as a cytotoxic payload for ADCs based on the clinical success of camptothecin-based ADCs Enhertu® (DXd) and Trodelvy® (SN-38). In contrast to the majority of other approved ADCs, both Enhertu® and Trodelvy® are feature an average of 7-8 payloads per antibody. Obviously, such a high drug-to-antibody ratio (DAR) effectively delivers more drug to the tumor per internalization event, which is key in driving the clinical success. Previously, we have shown that the native glycan of monoclonal antibodies is a privileged site for attachment of cytotoxic payloads1, for the generation of site-specific and stable ADCs with DARs varying from 1-6, while the therapeutic index is further elevated by a highly polar spacer technology (HydraSpace™)2. The success of GlycoConnect™/HydraSpace™ ADCs is further driven by the use of exatecan as a potent and highly cell-permeable payload (SYNtecan E™). Here we show that GlycoConnect™ technology can be readily extended to generate efficacious and safe DAR8 ADCs. We have developed a modular, non-genetic method, involving three simple process steps from any antibody: (a) enzymatic glycan remodeling, (b) strain-promoted azide-alkyne cycloaddition of a bifunctional, branched spacer comprising one strained alkyne and two orthogonal click tags (e.g. tetrazines), and (c) inverse electron-demand Diels-Alder cycloaddition with a branched linker-drug construct based on BCN and a two cytotoxic payloads. The resulting conjugates, with DAR 6 or 8, are rapidly generated with high homogeneity and with good stability. In addition, HIC profiles of the resulting ADCs indicate small relative retention time as an indication of good PK profile. Excellent in vivo tolerability is demonstrated, while efficacy studies in mice xenograft models show that the DAR8 technology clearly out-performs currently marketed ADCs (including Enhertu®). 1 (a) van Geel et al. Chemoenzymatic Conjugation of Toxic Payloads to the Globally Conserved N-Glycan of Native mAbs Provides Homogeneous and Highly Efficacious Antibody−Drug Conjugates. Bioconj. Chem. 2015, 26, 2233-2242. (b) Wijdeven et al. Enzymatic glycan remodeling-metal free click (GlycoConnect™) provides homogenous antibody-drug conjugates with improved stability and therapeutic index without sequence engineering. MAbs 2022, DOI: 10.1080/19420862.2022.2078466. 2 Verkade et al. A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates. Antibodies 2018, 7, 12, doi:10.3390/antib7010012. Citation Format: Lianne Lelieveldt, Elias Post, Marie Nassiet, Veronique Hendriks, Mick Verhagen, Remon van Geel, Sander van Berkel, Floris van Delft. GlycoConnect™ ADC toolbox expansion with high DAR technology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2614.

4-Isocyanoindole-2ʹ-deoxyribonucleoside (4ICIN): An isomorphic indole nucleoside suitable for inverse electron demand Diels-Alder reactions

Isomorphic nucleosides are powerful tool compounds for interrogating a variety of biological processes involving nucleosides and nucleic acids. We previously reported a fluorescent isomorphic indole nucleoside called 4CIN. A distinguishing molecular feature of 4CIN is the presence of a 4-cyano moiety on the indole that functions as the nucleobase. Given the known chemical reactivity of isonitriles with tetrazines through [4+1]-cycloaddition chemistry, we investigated whether conversion of 4CIN to the corresponding isonitrile would confer a useful chemical probe. Here we report the synthesis of 4-isocyanoindole-2ʹ-deoxyribonucleoside (4ICIN) and the propensity of 4ICIN to undergo inverse electron demand Diels-Alder cycloaddition with a model tetrazine.

Characterization of Structurally Diverse 18F-Labeled d-TCO Derivatives as a PET Probe for Bioorthogonal Pretargeted Imaging.

Background: The pretargeted imaging strategy using inverse electron demand Diels-Alder (IEDDA) cycloaddition between a trans-cyclooctene (TCO) and tetrazine (Tz) has emerged and rapidly grown as a promising concept to improve radionuclide imaging and therapy in oncology. This strategy has mostly relied on the use of radiolabeled Tz together with TCO-modified targeting vectors leading to a rapid growth of the number of available radiolabeled tetrazines, while only a few radiolabeled TCOs are currently reported. Here, we aim to develop novel and structurally diverse 18F-labeled cis-dioxolane-fused TCO (d-TCO) derivatives to further expand the bioorthogonal toolbox for in vivo ligation and evaluate their potential for positron emission tomography (PET) pretargeted imaging. Results: A small series of d-TCO derivatives were synthesized and tested for their reactivity against tetrazines, with all compounds showing fast reaction kinetics with tetrazines. A fluorescence-based pretargeted blocking study was developed to investigate the in vivo ligation of these compounds without labor-intensive prior radiochemical development. Two compounds showed excellent in vivo ligation results with blocking efficiencies of 95 and 97%. Two novel 18F-labeled d-TCO radiotracers were developed, from which [18F]MICA-214 showed good in vitro stability, favorable pharmacokinetics, and moderate in vivo stability. Micro-PET pretargeted imaging with [18F]MICA-214 in mice bearing LS174T tumors treated with tetrazine-modified CC49 monoclonal antibody (mAb) (CC49-Tz) showed significantly higher uptake in tumor tissue in the pretargeted group (CC49-Tz 2.16 ± 0.08% ID/mL) when compared to the control group with nonmodified mAb (CC49 1.34 ± 0.07% ID/mL). Conclusions: A diverse series of fast-reacting fluorinated d-TCOs were synthesized. A pretargeted blocking approach in tumor-bearing mice allowed the choice of a lead compound with fast reaction kinetics with Tz. A novel 18F-labeled d-TCO tracer was developed and used in a pretargeted PET imaging approach, allowing specific tumor visualization in a mouse model of colorectal cancer. Although further optimization of the radiotracer is needed to enhance the tumor-to-background ratios for pretargeted imaging, we anticipate that the 18F-labeled d-TCO will find use in studies where increased hydrophilicity and fast bioconjugation are required.

Transferring Substituents from Alkynes to Furans and Pyrroles through Heteronorbornadienes as Intermediates: Synthesis of β-Substituted Pyrroles/Furans.

The use of 7-oxa/azanorbornadienes as synthetic intermediates for the preparation of 3/4-substituted (β-substituted) furans/pyrroles is presented. The method lies in the inverse electron demand Diels-Alder (iEDDA) cycloaddition between a substituted heteronorbornadiene and an electron-poor tetrazine followed by spontaneous fragmentation of the resulting cycloadduct via two retro-Diels-Alder (rDA) reactions affording a β-substituted furan/pyrrole. The scope of this tandem iEDDA/rDA/rDA reaction was explored in the preparation of 29 heterocycles. A one-pot procedure starting directly from the alkyne precursors of the heteronorbornadiene intermediates is also described.

Site Reversal in Nucleophilic Addition to 1,2,3-Triazine 1-Oxides.

One of the most important reactions of 1,2,3-triazines with a dienophile is inverse electron demand Diels-Alder (IEDDA) cycloaddition, which occurs through nucleophilic addition to the triazine followed by N2 loss and cyclization to generate a heterocycle. The site of addition is either at the 4- or 6-position of the symmetrically substituted triazine core. Although specific examples of the addition of nucleophiles to triazines are known, a comprehensive understanding has not been reported, and the preferred site for nucleophilic addition is unknown and unexplored. With access to unsymmetrical 1,2,3-triazine-1-oxides and their deoxygenated 1,2,3-triazine compounds, we report C-, N-, H-, O-, and S-nucleophilic additions on 1,2,3-triazine and 1,2,3-triazine-1-oxide frameworks where the 4- and 6-positions could be differentiated. In the IEDDA cycloadditions using C- and N-nucleophiles, the site of addition is at C-6 for both heterocyclic systems, but product formation with 1,2,3-triazine-1-oxides is faster. Other N-nucleophile reactions with triazine 1-oxides show addition at either the 4- or 6-position of the triazine 1-oxide ring, but nucleophilic attack only occurs at the 6-position on the triazine. Hydride from NaBH4 undergoes addition at the 6-position on the triazine and the triazine 1-oxide core. Alkoxides show a high nucleophilic selectivity for the 4-position of the triazine 1-oxide. Thiophenoxide, cysteine, and glutathione undergo nucleophilic addition on the triazine core at the 6-position, while addition occurs at the 4-position of the triazine 1-oxide. These nucleophilic additions proceed under mild reaction conditions and show high functional group tolerance. Computational studies clarified the roles of the nucleophilic addition and nitrogen extrusion steps and the influence of steric and electronic factors in determining the outcomes of the reactions with different nucleophiles.

The inverse electron demand Diels-Alder cycloaddition with carbon-11 and fluorine-18: A gateway to pretargeted imaging across the blood-brain barrier.

There is increased focus on developing tools to image large biomolecules, such as antibodies, within the brain using positron emission tomography (PET). The inverse electron demand Diels-Alder cycloaddition (IEDDA) reaction has offered the greatest prospect of achieving such a feat and has gained much interest over the past decade. The fast reaction kinetics of the IEDDA reaction opens up the possibility of utilising a pretargeted approach, whereby the subject is pretreated with a biomolecule that has high specificity for its target. A radiolabelled second component is then administered to the subject, enabling the biomolecule to be visualised by PET. However, for this to become common practice, there is a need for the development of either radiolabelled trans-cyclooctenes (TCOs) or tetrazines that can cross the blood-brain barrier (BBB). This review highlights the advancements in the development of both radiolabelled TCOs and tetrazines, which have been radiolabelled with either carbon-11 or fluorine-18 and show promise or have been evaluated for use in pretargeted PET imaging across the BBB.

Dynamic bioorthogonal imaging using a tetrazine NIR-AZA fluorogenic probe

Tetrazine fluorogenic probes are of high value due to their ability to modulate emission intensity from low to high in response to inverse electron demand Diels-Alder cycloadditions with dienophiles. In this study, a tetrazine substituted BF2-azadipyrromethene probe and a related model tetrazine were investigated for their reactivity with strained alkyne and alkene dienophiles to identify the optimal probe for cellular use. Specifically, the reactivity of 2-deoxy-d-glucose substituted 1-methyl cyclopropene and bicyclononyne was tested, with the latter demonstrating superior reactivity with complete reactions observed within 15 min. Moreover, the tetrazine substituted BF2-azadipyrromethene showed a strong fluorogenic response at a λmax of 715 nm in aqueous solution as the cycloaddition proceeded. The high reaction efficiency and significant 43-fold fluorescence enhancement factor encouraged its translation to bioorthogonal cell imaging. Pre-incubating HeLa Kyoto cells with 2-deoxy-glucose-alkyne dienophile followed by treatment with the BF2-azadipyrromethene fluorogenic probe, allowed dynamic imaging of the bioorthogonal reaction in live cells. Pre-incubation of 2-deoxy-glucose alkyne for 24 h with subsequent fluorogenic cycloaddition indicated that bioorthogonal reaction occurred in subcellular regions such as the mitochondria. This study showed the potential for real-time observation of intra-cellular fluorogenic cycloadditions in the near-infrared spectral region.

Visualizing ion channels in lipid nanodomains using live-cell fluorescence lifetime imaging.

Ion channels function in a lipid-membrane nano-environment where the lipid composition and membrane compartmentalization can undergo rapid changes. How transmembrane potential activates voltage-gated ion channels differently in liquid-ordered, cholesterol-rich (Lo, or called “lipid raft”) versus in liquid-disordered, cholesterol-poor membrane (Ld) nanodomains remains less explored in intact cells. Using phasor plot fluorescence-lifetime imaging microscopy (phasor FLIM), combined with Förster resonance energy transfer (FRET) between donor- and acceptor-fluorophores conjugated to cholera toxin subunit B (CTxB) or to short lipidated peptides that specifically recognize Lo and Ld domains, we visualized the membrane domains in different cells, including Xenopus oocytes, cultured primary hepatocytes and dorsal root ganglion (DRG) sensory neurons. We postulate that a large percentage of the plasma membrane could be Lo domain. Extracting cholesterols using β-cyclodextrin was able to decrease the FRET efficiency indicating a reduced size of Lo domains. Interestingly, the measured apparent FRET was low in DRG neurons, consistent with the unique lipid organization of neuronal membranes, in comparison to other cell types. Furthermore, the localization of voltage-gated ion channels in proximity to lipid nanodomains could be examined by either FLIM-FRET or spectral FRET using these fluorescent markers. Moreover, fluorescent noncanonical amino acids (ncAAs) incorporated near the hydrophobic constriction site of voltage sensors of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels showed a similar lifetime as when their free forms were dissolved in hydrophobic solvents; lifetime of the incorporated ncAAs changed upon membrane hyperpolarization. In addition, using the stop-codon suppression strategies and click chemistry via the inverse-electron demand Diels-Alder cycloaddition, a photostable dye was tagged site-specifically to the extracellular region of the HCN voltage sensor. These labeling strategies will facilitate measuring the voltage-sensor conformational dynamics of ion channels in distinct lipid nanodomains using time-resolved phasor FLIM-FRET or single-molecule FRET.

Retro-[4+2]/Intramolecular Diels–Alder Cascade Allows a Concise Total Synthesis of Lucidumone

AbstractLucidumone is a recently isolated meroterpenoid displaying interesting biological activity. This natural product possesses a complex structure, including a bicyclo[2.2.2]octane possessing 6 contiguous stereogenic centers. Herein, we discuss strategies to solve this synthetic challenge. In particular, we developed a new method for the inverse electron-demand Diels–Alder cycloaddition between 2-pyrones and acyclic enol ethers, as a mean to obtain a ‘masked’ cyclohexadiene. This method allowed an expeditious enantioselective synthesis of (+)-lucidumone through a retro-[4+2]/intramolecular Diels–Alder reaction cascade.1 Introduction2 Retrosynthetic Considerations on the Bicyclo[2.2.2]octane3 Development of a Methodology for Enantioselective IEDDA Cycloadditions4 Enantioselective Total Synthesis of (+)-Lucidumone5 Conclusion

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels-Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels-Alder cycloaddition, which serves as the first example of a bifunctional Diels-Alderase that utilizes this mechanism.

Synthesis of tetrazine-tetracyanobutadienes and their transformation into pyridazines via inverse-electron demand Diels-Alder cycloaddition (IEDDA).

We report the synthesis of novel triphenylamine-tetrazine-tetracyanobutadiene based asymmetric and symmetric push-pull chromophores (1T and 1DT) by [2 + 2] cycloaddition-retroelectrocyclization of tetrazine connected electron-rich ethynyl triphenylamine with tetracyanoethene (TCNE). The electron-deficient tetrazine and tetracyanobutadiene (TCBD) moieties in 1T and 1DT create strong intramolecular charge transfer (ICT) interactions with TPA units, resulting in strong visible absorption with red edges reaching up to 700 nm (bandgaps of 1.79-1.89 eV). Furthermore, the structural, optical, and electronic properties of 1T and 1DT have been further tuned by transforming tetrazine units into pyridazines (1T-P and 1DT-P) via inverse-electron demand Diels-Alder cycloaddition (IEDDA). The relatively electron-donating character of pyridazine raised the HOMO and LUMO energies and widened the bandgap by ∼0.2 eV. This is the first synthetic strategy which allows two levels of tuning of the properties. 1DT acts as a selective colorimetric sensor for CN-via a nucleophilic attack on the dicyanovinyl unit of TCBD. The transformation results in a distinct colour change from orange to brown, while no change was observed for a range of anions tested (F-, Br-, HSO4-, NO3-, BF4-, and ClO4-).

Quantitative Analysis and Optimization of Site-Specific Protein Bioconjugation in Mammalian Cells.

Despite a range of covalent protein modifications, few techniques exist for quantification of protein bioconjugation in cells. Here, we describe a novel method for quantifying in cellulo protein bioconjugation through covalent bond formation with HaloTag. This approach utilizes unnatural amino acid (UAA) mutagenesis to selectively install a small and bioorthogonally reactive handle onto the surface of a protein. We utilized the fast kinetics and high selectivity of inverse electron-demand Diels-Alder cycloadditions to evaluate reactions of tetrazine phenylalanine (TetF) with strained trans-cyclooctene-chloroalkane (sTCO-CA) and trans-cyclooctene lysine (TCOK) with tetrazine-chloroalkane (Tet-CA). Following bioconjugation, the chloroalkane ligand is exposed for labeling by the HaloTag enzyme, allowing for straightforward quantification of bioconjugation via simple western blot analysis. We demonstrate the versatility of this tool for quickly and accurately determining the bioconjugation efficiency of different UAA/chloroalkane pairs and for different sites on different proteins of interest, including EGFP and the estrogen-related receptor ERRα.

Acyclic and Heterocyclic Azadiene Diels-Alder Reactions Promoted by Perfluoroalcohol Solvent Hydrogen Bonding: Comprehensive Examination of Scope.

Herein, the first use of perfluoroalcohol H-bonding in accelerating acyclic azadiene inverse electron demand cycloaddition reactions is described, and its use in the promotion of heterocyclic azadiene cycloaddition reactions is generalized through examination of a complete range of azadienes. The scope of dienophiles was comprehensively explored; relative reactivity trends and solvent compatibilities were established with respect to the dienophile as well as azadiene; H-bonding solvent effects that lead to rate enhancements, yield improvements, and their impact on regioselectivity and mode of cycloaddition are defined; new viable diene/dienophile reaction partners in the cycloaddition reactions are disclosed; and key comparison rate constants are reported. The perfluoroalcohol effectiveness at accelerating an inverse electron demand Diels-Alder cycloaddition is directly correlated with its H-bond potential (pKa). Not only are the reactions of electron-rich dienophiles accelerated but those of strained and even unactivated alkenes and alkynes are improved, including representative bioorthogonal click reactions.

Nanoparticular Carriers As Objects to Study Intentional and Unintentional Bioconjugation.

Synthetic nanoparticles are interesting to use in the study of ligation with natural biorelevant structures. That is because they present an intermediate situation between reactions onto soluble polymers or onto solid surfaces. In addition, differently functionalized nanoparticles can be separated and studied independently thereafter. So what would be a "patchy functionalization" on a macroscopic surface results in differently functionalized nanoparticles, which can be separated after the interaction with body fluids. This paper will review bioconjugation of such nanoparticles with a special focus on recent results concerning the formation of a protein corona by unspecific adsorption (lower lines of TOC), which presents an unintentional bioconjugation, and on new aspects of intentionally performed bioconjugation by covalent chemistry (upper line). For this purpose, it is important that polymeric nanoparticles without a protein corona can be prepared. This opens, e.g., the possibility to look for special proteins adsorbed as a result of the natural compound ligated to the nanoparticle by covalent chemistry, like the Fc part of antibodies. At the same time, the use of highly reactive, bioorthogonal functional groups (inverse electron demand Diels-Alder cycloaddition) on the nanoparticles allows an efficient ligation after administration inside the body, i.e., in vivo.

Triazines: Syntheses and Inverse Electron-demand Diels-Alder Reactions.

Triazines are an important class of six-membered aromatic heterocycles possessing three nitrogen atoms, resulting in three types of regio-isomers: 1,2,4-triazines (a-triazines), 1,2,3-triazines (v-triazines), and 1,3,5-triazines (s-triazines). Notably, the application of triazines as cyclic aza-dienes in inverse electron-demand Diels-Alder (IEDDA) cycloaddition reactions has been established as a unique and powerful method in N-heterocycle synthesis, natural product preparation, and bioorthogonal chemistry. In this review, we comprehensively summarize the advances in the construction of these triazines via annulation and ring-expansion reactions, especially emphasizing recent developments and challenges. The synthetic transformations of triazines are focused on IEDDA cycloaddition reactions, which have allowed access to a wide scope of heterocycles, including pyridines, carbolines, azepines, pyridazines, pyrazines, and pyrimidines. The utilization of triazine IEDDA reactions as key steps in natural product synthesis is also discussed. More importantly, a particular attention is paid on the bioorthogonal application of triazines in fast click ligation with various strained alkenes and alkynes, which opens a new opportunity for studying biomolecules in chemical biology.

Synthesis of dibenzosuberenone-based novel polycyclic π-conjugated dihydropyridazines, pyridazines and pyrroles.

The synthesis of novel polycyclic π-conjugated dihydropyridazines, pyridazines, and pyrroles was studied. Dihydropyridazine dyes were synthesized by inverse electron-demand Diels–Alder cycloaddition reactions between a dibenzosuberenone and tetrazines that bear various substituents. The pyridazines were synthesized in high yields by oxidation of dihydropyridazine-appended dibenzosuberenones with PIFA or NO. p-Quinone derivatives of pyridazines were also obtained by H-shift isomerization following the inverse electron-demand Diels–Alder reaction of tetrazines with p-quinone dibenzosuberenone. Then these pyridazines were converted to the corresponding pyrroles by reductive treatment with zinc. It was observed that all the dihydropyridazines obtained gave absorbance and emission at long wavelengths.

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells.

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels–Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)‐a method that allows to incorporate noncanonical amino acids (ncAAs) site‐specifically into proteins in vivo. These reactions enable residue‐specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to β‐elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo β‐elimination and b) a fluorescence flow cytometry based FRET‐assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE‐specific experimental conditions, such as long‐term exposure of the ring‐strained ncAA to living cells, that are not taken into account in other assays.

Site-Specific and Residualizing Linker for 18F Labeling with Enhanced Renal Clearance: Application to an Anti-HER2 Single-Domain Antibody Fragment.

Single-domain antibody fragments (sdAbs) are promising vectors for immuno-PET; however, better methods for labeling sdAbs with 18F are needed. Herein, we evaluate a site-specific strategy using an 18F residualizing motif and the anti-epidermal growth factor receptor 2 (HER2) sdAb 5F7 bearing an engineered C-terminal GGC tail (5F7GGC). Methods: 5F7GGC was site-specifically attached with a tetrazine-bearing agent via thiol-maleimide reaction. The resultant conjugate was labeled with 18F by inverse electron demand Diels-Alder cycloaddition with a trans-cyclooctene attached to 6-18F-fluoronicotinoyl moiety via a renal brush border enzyme-cleavable linker and a PEG4 chain (18F-5F7GGC). For comparisons, 5F7 sdAb was labeled using the prototypical residualizing agent, N-succinimidyl 3-(guanidinomethyl)-5-125I-iodobenzoate (iso-125I-SGMIB). The 2 labeled sdAbs were compared in paired-label studies performed in the HER2-expressing BT474M1 breast carcinoma cell line and athymic mice bearing BT474M1 subcutaneous xenografts. Small-animal PET/CT imaging after administration of 18F-5F7GGC in the above mouse model was also performed. Results:18F-5F7GGC was synthesized in an overall radiochemical yield of 8.9% ± 3.2% with retention of HER2 binding affinity and immunoreactivity. The total cell-associated and intracellular activity for 18F-5F7GGC was similar to that for coincubated iso-125I-SGMIB-5F7. Likewise, the uptake of 18F-5F7GGC in BT474M1 xenografts in mice was similar to that for iso-125I-SGMIB-5F7; however, 18F-5F7GGC exhibited significantly more rapid clearance from the kidney. Small-animal PET/CT imaging confirmed high uptake and retention in the tumor with very little background activity at 3 h except in the bladder. Conclusion: This site-specific and residualizing 18F-labeling strategy could facilitate clinical translation of 5F7 anti-HER2 sdAb as well as other sdAbs for immuno-PET.

Optimization of IEDDA bioorthogonal system: Efficient process to improve trans-cyclooctene/tetrazine interaction

The antibody pretargeting approach for radioimmunotherapy (RIT) using inverse electron demand Diels-Alder cycloaddition (IEDDA) constitutes an emerging theranostic approach for solid cancers. However, IEDDA pretargeting has not reached clinical trial. The major limitation of the IEDDA strategy depends largely on trans-cyclooctene (TCO) stability. Indeed, TCO may isomerize into the more stable but unreactive cis-cyclooctene (CCO), leading to a drastic decrease of IEDDA efficiency. We have thus developed both efficient and reproducible synthetic pathways and analytical follow up for (PEGylated) TCO derivatives, providing high TCO isomeric purity for antibody modification. We have set up an original process to limit the isomerization of TCO to CCO before the mAbs’ functionalization to allow high TCO/tetrazine cycloaddition.