Site-selective Incorporation Research Articles

Devising Biocompatible, NIR-Activated Helical Pyroptosis Agents via 𝛑-Twisting Strategy for Promoting Antitumor Immunity.

Specifically controlling cell pyroptosis is advantageous for oncotherapy as it allows simultaneous ablation of primary tumors and activation of immunogenicity of tumor environment. Herein, a facile and robust strategy is presented to construct efficient NIR-activated helical pyroptosis agents (PyroAs) with negligible dark cytotoxicity. It is demonstrated that the construction of four intramolecular B-X bonds (X = O or N) within the BODIPY chromophore enforces a significant twisting of its π-conjugation, yielding a variety of helical HBD molecules with desired high photosensitivity and negligible dark toxicity. A robust approach is established to extend HBD into the near-infrared (NIR) region through site-selective incorporation of an electron-withdrawing ester moiety. It is also proved that targeted delivery of the NIR-activated HBD-ER to the endoplasmic reticulum (ER) specifically activates pyroptosis pathway by equipping it with an ER-targeting moiety. Finally, the favorable biocompatibility, excellent antitumor efficacy, and remarkable systematic immune response of this unique NIR-activated helical PyroAs are shown in vivo, demonstrating its potential application in solid tumor immunotherapy.

Tuning singlet oxygen generation with caged organic photosensitizers

Controlling the succession of chemical processes with high specificity in complex systems is advantageous for widespread applications, from biomedical research to drug manufacturing. Despite synthetic advances in bioorthogonal and photochemical methodologies, there is a need for generic chemical approaches that can universally modulate photodynamic reactivity in organic photosensitizers. Herein we present a strategy to fine-tune the production of singlet oxygen in multiple photosensitive scaffolds under the activation of bioresponsive and bioorthogonal stimuli. We demonstrate that the photocatalytic activity of nitrobenzoselenadiazoles can be fully blocked by site-selective incorporation of electron-withdrawing carbamate moieties and restored on demand upon uncaging with a wide range of molecular triggers, including abiotic transition-metal catalysts. We also prove that this strategy can be expanded to most photosensitizers, including diverse structures and spectral properties. Finally, we show that such advanced control of singlet oxygen generation can be broadly applied to the photodynamic ablation of human cells as well as to regulate the release of singlet oxygen in the semi-synthesis of natural product drugs.

Genetic Encoding of Fluoro-l-tryptophans for Site-Specific Detection of Conformational Heterogeneity in Proteins by NMR Spectroscopy.

The substitution of a single hydrogen atom in a protein by fluorine yields a site-specific probe for sensitive detection by 19F nuclear magnetic resonance (NMR) spectroscopy, where the absence of background signal from the protein facilitates the detection of minor conformational species. We developed genetic encoding systems for the site-selective incorporation of 4-fluorotryptophan, 5-fluorotryptophan, 6-fluorotryptophan, and 7-fluorotryptophan in response to an amber stop codon and used them to investigate conformational heterogeneity in a designed amino acid binding protein and in flaviviral NS2B-NS3 proteases. These proteases have been shown to present variable conformations in X-ray crystal structures, including flips of the indole side chains of tryptophan residues. The 19F NMR spectra of different fluorotryptophan isomers installed at the conserved site of Trp83 indicate that the indole ring flip is common in flaviviral NS2B-NS3 proteases in the apo state and suppressed by an active-site inhibitor.

Engineering Ultrasoft Interactions in Stiff All-DNA Dendrimers by Site-Specific Control of Scaffold Flexibility.

A combined experimental and theoretical study of the structural correlations in moderately concentrated suspensions of all-DNA dendrimers of the second generation (G2) with controlled scaffold rigidity is reported here. Small-angle X-ray scattering experiments in concentrated aqueous saline solutions of stiff all-DNA G2 dendritic constructs reveal a novel anomalous liquid-like phase behavior which is reflected in the calculated structure factors as a two-step increase at low scattering wave vectors. By developing a new design strategy for adjusting the particle's internal flexibility based on site-selective incorporation of single-stranded DNA linkers into the dendritic scaffold, it is shown that this unconventional type of self-organization is strongly contingent on the dendrimer's stiffness. A comprehensive computer simulation study employing dendritic models with different levels of coarse-graining, and two theoretical approaches based on effective, pair-potential interactions, remarkably confirmed the origin of this unusual liquid-like behavior. The results demonstrate that the precise control of the internal structure of the dendritic scaffold conferred by the DNA can be potentially used to engineer a rich palette of novel ultrasoft interaction potentials that could offer a route for directed self-assembly of intriguing soft matter phases and experimental realizations of a host of unusual phenomena theoretically predicted for ultrasoft interactingsystems.

Photoradical-Mediated Catalyst-Independent Protein Cross-Link with Unusual Fluorescence Properties.

Photo-actively modified natural amino acids have served as lucrative probes for precise mapping of the dynamics, interaction networks, and turnover of cytosolic proteins both in vivo and ex vivo. In our attempts to extend the utility of photoreactive reporters to map the molecular characteristics of vital membrane proteins, we carried out site-selective incorporation of 7-fluoro-indole in the human mitochondrial outer membrane protein VDAC2 (voltage-dependent anion channel isoform 2), with the aim of generating Trp-Phe/Tyr cross-links. Prolonged irradiation at 282 nm provided us with a surprisingly unusual fluorophore that displayed sizably red-shifted excitation (λex-max =280 nm→360 nm) and emission (λem-max =330 nm→430 nm) spectra that was reversible with organic solvents. By measuring the kinetics of the photo-activated cross-linking with a library of hVDAC2 variants, we demonstrate that formation of this unusual fluorophore is kinetically retarded, independent of tryptophan, and is site-specific. Using other membrane (Tom40 and Sam50) and cytosolic (MscR and DNA Pol I) proteins, we additionally show that formation of this fluorophore is protein-independent. Our findings reveal the photoradical-mediated accumulation of reversible tyrosine cross-links, with unusual fluorescent properties. Our findings have immediate applications in protein biochemistry and UV-mediated protein aggregation and cellular damage, opening avenues for formulating therapeutics that prolong cell viability in humans.

Site-Selective Incorporation of a Functional Group into Lys175 in the Vicinity of the Active Site of Chymotrypsin by Using Peptidyl α-Aminoalkylphosphonate Diphenyl Ester-Derivatives.

We previously reported that Lys175 in the region of the active site of chymotrypsin (Csin) could be site-selectively modified by using an N-hydroxy succinimide (NHS) ester of the peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester [NHS-Suc-Ala-Ala-PheP(OPh)2]. In this study, the Lys175-selective modification method was expanded to incorporate functional groups into Lys 175 in Csin. Two types of peptidyl phosphonate derivatives with the dansyl group (Dan) as a functional molecule, Dan-β-Ala-[Asp(NHS) or Glu(NHS)]-Ala-Ala-(R)-PheP(OPh)2 (DanD and DanE, respectively), were synthesized, and their action was evaluated when modifying Lys175 in Csin. Ion-exchange chromatography (IEC), fluorescence spectroscopy, and LC-MS/MS were used to analyze the products from the reaction of Csin with DanD or DanE. By IEC and LC-MS/MS, the results showed that DanE reacted with Csin more effectively than DanD to produce the modified Csin (DanMCsin) bearing Dan at Lys175. DanMCsin exhibited an enzymatic activity corresponding to 1/120 of Csin against Suc-Ala-Ala-Phe-pNA. In addition, an effect of Lys175 modification on the access of the proteinaceous Bowman-Birk inhibitor to the active site of DanMCsin was investigated. In conclusion, by using a peptidyl derivative containing 1-amino-2-ethylphenylphosphonate diphenyl ester, we demonstrated that a functional group could be incorporated into Lys175 in Csin.

Rhodium(III)-Catalyzed Conjugate Addition of β-CF3-Enones with Quinoline N-Oxides.

The site-selective incorporation of a trifluoromethyl group into biologically active molecules and pharmaceuticals has emerged as a central topic in medicinal chemistry and drug discovery. Herein, we demonstrate the rhodium(III)-catalyzed conjugate addition of β-trifluoromethylated enones with quinoline N-oxides, which result in the generation of β-trifluoromethyl-β'-quinolinated ketones. The reaction proceeds under mild conditions with complete functional group tolerance. The synthetic applicability was showcased by successful gram-scale experiments and valuable synthetic transformations of coupling products.

Engineering enzyme activity using an expanded amino acid alphabet.

Enzyme design and engineering strategies are typically constrained by the limited size of nature's genetic alphabet, comprised of only 20 canonical amino acids. In recent years, site-selective incorporation of non-canonical amino acids (ncAAs) via an expanded genetic code has emerged as a powerful means of inserting new functional components into proteins, with hundreds of structurally diverse ncAAs now available. Here, we highlight how the emergence of an expanded repertoire of amino acids has opened new avenues in enzyme design and engineering. ncAAs have been used to probe complex biological mechanisms, augment enzyme function and, most ambitiously, embed new catalytic mechanisms into protein active sites that would be challenging to access within the constraints of nature's genetic code. We predict that the studies reviewed in this article, along with further advances in genetic code expansion technology, will establish ncAA incorporation as an increasingly important tool for biocatalysis in the coming years.

Two-enzyme cascade catalyzed trideuteromethylative modification of natural products

Site-selective incorporation of the trideuterated “magic methyl” group may provide profound pharmacological benefits, which has been considered as an important tool for drug optimization and development in medicinal chemistry. S-adenosyl-L-methionine (SAM) usually acts as the methyl donor under the catalysis of SAM-dependent methyltransferases (MTs), which transfer a methyl group from SAM to substrates, and are widely reported for methylation in natural product biosynthesis. However, it is difficult to introduce other high-value functional groups to substrates in this way since SAM analogs are not easy to obtain in biological methods. Herein we report that trideuterated SAM can be obtained from S-adenosylhomocysterine (SAH) and trideuterated methyl iodide (CD3I) under the participation of halide methyltransferase (HMT). Subsequent one-pot cascades with different MTs achieved the regioselective trideuteromethylation of three natural products, which indicated that trideuterated SAM can be accepted as a co-substrate by C-, N-, O-specific MTs for enzyme-catalyzed trideuteromethylation of natural products.

Chemoenzymatic synthesis of fluorinated polyketides.

Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.

Engineering site-selective incorporation of fluorine into polyketides.

Although natural products and synthetic small molecules both serve important medicinal functions, their structures and chemical properties are relatively distinct. To expand the molecular diversity available for drug discovery, one strategy is to blend the effective attributes of synthetic and natural molecules. A key feature found in synthetic compounds that is rare in nature is the use of fluorine to tune drug behavior. We now report a method to site-selectively incorporate fluorine into complex structures to produce regioselectively fluorinated full-length polyketides. We engineered a fluorine-selective trans-acyltransferase to produce site-selectively fluorinated erythromycin precursors in vitro. We further demonstrated that these analogs could be produced in vivo in Escherichia coli on engineering of the fluorinated extender unit pool. By using engineered microbes, elaborate fluorinated compounds can be produced by fermentation, offering the potential for expanding the identification and development of bioactive fluorinated small molecules.

Frontispiece: The Deuterated “Magic Methyl” Group: A Guide to Site‐Selective Trideuteromethyl Incorporation and Labeling by Using CD3 Reagents

Profound pharmacological benefits can be provided by site-selective incorporation of the deuterated “magic methyl” group, which can be considered an important tool for drug optimization and development. This review highlights site-selective trideuteromethylation strategies according to the trideuteromethylation reagent used and describes the mechanism and scope of the reactions. As such, this review aims to be a guide for researchers, offering them the available C−CD3 formation strategies for the preparation of new or modified drugs or materials. For more details see the Review by M. Rueping et al. on page 11751 ff.

Site-selective incorporation of phosphorylated tyrosine into the p50 subunit of NF-κB and activation of its downstream gene CD40.

The NF-κB family of transcriptional activators is responsible for the expression of numerous genes that control key functions such as cell development and survival. Subunit p50 has been studied extensively and is known to include 13 tyrosines, but the extent and pattern of tyrosine phosphorylation that accompanies p50 function has not been defined in the literature, especially at the level of selectivity of gene expression. In this study, phosphorylated tyrosine (pTyr) was site-selectively incorporated into the p50 subunit using an E. coli in vitro expression system containing a modified ribosome. In human T cells, the NF-κBs containing a pTyr at position 60 or 82 of p50 strongly increased the expression of CD40, which is a potential target for cancer or viral immunotherapy. Promoter DNA binding was studied for CD40 promoters, and verified two pTyr residues in NF-κB p50/p65 heterodimers that facilitated this process, and that support the possible importance of phosphorylation stoichiometry. This study defines a new approach for studying tyrosine residues whose phosphorylation alters protein binding to DNA promoters, and contributes to the facility of DNA expression.

Synthesis of substituted 3,4-dihydroquinazolinones via a metal free Leuckart-Wallach type reaction.

The 3,4-dihydroquinazolinone (DHQ) moiety is a highly valued scaffold in medicinal chemistry due to the vast number of biologically-active compounds based on this core structure. Current synthetic methods to access these compounds are limited in terms of diversity and flexibility and often require the use of toxic reagents or expensive transition-metal catalysts. Herein, we describe the discovery and development of a novel cascade cyclization/Leuckart–Wallach type strategy to prepare substituted DHQs in a modular and efficient process using readily-available starting materials. Notably, the reaction requires only the addition of formic acid or acetic acid/formic acid and produces H2O, CO2 and methanol as the sole reaction byproducts. Overall, the reaction provides an attractive entry point into this important class of compounds and could even be extended to isotopic labelling via the site-selective incorporation of a deuterium atom.

Site-selective protein modification via disulfide rebridging for fast tetrazine/trans-cyclooctene bioconjugation.

An inverse electron demand Diels-Alder reaction between tetrazine and trans-cyclooctene (TCO) holds great promise for protein modification and manipulation. Herein, we report the design and synthesis of a tetrazine-based disulfide rebridging reagent, which allows the site-selective installation of a tetrazine group into disulfide-containing peptides and proteins such as the hormone somatostatin (SST) and the antigen binding fragment (Fab) of human immunoglobulin G (IgG). The fast and efficient conjugation of the tetrazine modified proteins with three different TCO-containing substrates to form a set of bioconjugates in a site-selective manner was successfully demonstrated for the first time. Homogeneous, well-defined bioconjugates were obtained underlining the great potential of our method for fast bioconjugation in emerging protein therapeutics. The formed bioconjugates were stable against glutathione and in serum, and they maintained their secondary structure. With this work, we broaden the scope of tetrazine chemistry for site-selective protein modification to prepare well-defined SST and Fab conjugates with preserved structures and good stability under biologically relevant conditions.

Recent Advancements in Transition-Metal-Catalyzed One-Pot Twofold Unsymmetrical Difunctionalization of Arenes.

Transition-metal-catalyzed direct C-H bond activation reactions have been embraced as a powerful synthetic tool to access diverse functionalized arenes. However, site-selective incorporation of multiple distinct functionalities in an arene has always been a formidable challenge. Recent efforts from the synthetic community have disclosed a few dynamic synthetic approaches to fabricate multifunctionalized arenes in one-pot using a single catalytic system. These reports manifested the immense potential of such approaches to expedite contemporary organic synthesis towards building molecular complexity. In this minireview, we have illustrated the recent progress in this area, highlighting the contribution from several synthetic chemists including our group.

Biosynthesis and Chemical Applications of Thioamides.

Thioamidation as a posttranslational modification is exceptionally rare, with only a few reported natural products and exactly one known protein example (methyl-coenzyme M reductase from methane-metabolizing archaea). Recently, there has been significant progress in elucidating the biosynthesis and function of several thioamide-containing natural compounds. Separate developments in the chemical installation of thioamides into peptides and proteins have enabled cell biology and biophysical studies to advance the current understanding of natural thioamides. This review highlights the various strategies used by Nature to install thioamides in peptidic scaffolds and the potential functions of this rare but important modification. We also discuss synthetic methods used for the site-selective incorporation of thioamides into polypeptides with a brief discussion of the physicochemical implications. This account will serve as a foundation for the further study of thioamides in natural products and their various applications.

Site-Selective C–H Functionalization of (Hetero)Arenes via Transient, Non-symmetric Iodanes

A strategy for C-H functionalization of arenes and heteroarenes has been developed to allow site-selective incorporation of various anions, including Cl, Br, OMs, OTs, and OTf. This approach is enabled by in situ generation of reactive, non-symmetric iodanes by combining anions and bench-stable PhI(OAc)2. The utility of this mechanism is demonstrated via para-selective chlorination of medicinally relevant arenes, as well as site-selective C-H chlorination of heteroarenes. Spectroscopic, computational, and competition experiments describe the unique nature, reactivity, and selectivity of these transient, unsymmetrical iodanes.

Efficient and irreversible antibody-cysteine bioconjugation using carbonylacrylic reagents.

There is considerable interest in the development of chemical methods for the precise, site-selective modification of antibodies for therapeutic applications. In this protocol, we describe a strategy for the irreversible and selective modification of cysteine residues on antibodies, using functionalized carbonylacrylic reagents. This protocol is based on a thiol-Michael-type addition of native or engineered cysteine residues to carbonylacrylic reagents equipped with functional compounds such as cytotoxic drugs. This approach is a robust alternative to the conventional maleimide technique; the reaction is irreversible and uses synthetically accessible reagents. Complete conversion to the conjugates, with improved quality and homogeneity, is often achieved using a minimal excess (typically between 5 and 10 equiv.) of the carbonylacrylic reagent. Potential applications of this method cover a broad scope of cysteine-tagged antibodies in various formats (full-length IgGs, nanobodies) for the site-selective incorporation of cytotoxic drugs without loss of antigen-binding affinity. Both the synthesis of the carbonylacrylic reagent armed with a synthetic molecule of interest and the subsequent preparation of the chemically defined, homogeneous antibody conjugate can be achieved within 48 h and can be easily performed by nonspecialists. Importantly, the conjugates formed are stable in human plasma. The use of liquid chromatography-mass spectrometry (LC-MS) analysis is recommended for monitoring the progression of the bioconjugation reactions on protein and antibody substrates with accurate resolution.

Synthesis and Spectroscopic Properties of Fluorinated Coumarin Lysine Derivatives.

The site-selective incorporation of fluorescent amino acids into proteins has emerged as a valuable alternative to expressible protein reporters. For successful application, a robust and scalable, yet flexible, route to non-natural amino acids is required. This work describes an improved synthesis of coumarin-conjugated lysine derivatives where fluorinated variants are accessed. These analogues can be utilized at low pH and should find application probing biological processes that operate under acidic conditions.