Click Chemistry in Detecting Protein Modification.

Click chemistry, also known as "link chemistry," is an important molecular connection method that can achieve simple and efficient connections between specific small molecular groups at the molecular level. Click chemistry offers several advantages, including high efficiency, good selectivity, mild conditions, and few side reactions. These features make it a valuable tool for in-depth analysis of various protein posttranslational modifications (PTMs) caused by changes in cell metabolism during viral infection. This chapter considers the palmitoylation, carbonylation, and alkylation of STING and presents detailed information and experimental procedures for measuring PTMs using click chemistry.

Late-Stage Modification of Peptides with Maleimides through Palladium-Catalyzed ß-C(sp3)-H Alkylation.

Transition-metal-catalyzed C-H activation has proven to be a powerful tool for the late-stage modification of peptides. We herein report a method for site-selective alkylation of peptides with maleimides through Pd-catalyzed ß-C(sp3)-H activation. In this protocol, the methionine residues within peptides serve as the directing groups, which circumvented the preinstallation and subsequent removal of the directing groups. This chemistry exhibited broad substrate scope and can be utilized for peptide ligation.

Photoinduced Radical Approach for Desulfurative Alkylation of Cysteine Derivatives to Make Unnatural Amino Acids.

Unnatural amino acids (UAAs) are highly valuable molecules in organic synthesis, pharmaceutical sciences, and material science. Herein, we present a photocatalytic radical approach for desulfurative alkylation of cysteine derivatives with arenethiol as the hydrogen atom transfer catalyst for making UAAs and peptides. The formate salt, acting as the hydrogen atom donor, in situ generates the highly reductive CO2 radical anion species, which is the key to unlocking the C-S bond cleavage process with a simple benzoyl protecting group. No photocatalyst is required for the radical initiation and propagation, which makes such a visible-light-induced process mild, efficient, and sustainable.

Alkylation of Complex Glycine Precursor (CGP) as a Prebiotic Route to 20 Proteinogenic Amino Acids Synthesis.

It is not known why the number of proteinogenic amino acids is limited to 20. Since Miller's experiment, many studies have shown that amino acids could have been generated under prebiotic conditions. However, the amino acid compositions obtained from simulated experiments and exogenous origins are different from those of life. We hypothesized that some simple precursor compounds generated by high-energy reactions were selectively combined by organic reactions to afford a limited number of amino acids. To this direction, we propose two scenarios. One is the reaction of HCN with each side-chain precursor (the aminomalononitrile scenario), and the other is alkylation of the "complex glycine precursor", which is the main product of proton irradiation of the primordial atmosphere (the new polyglycine scenario). Here, selective formation of the 20 amino acids is described focusing on the latter scenario. The structural features of proteinogenic amino acids can be described systematically. The scenario consists of three stages: a high-energy reaction stage (Gly, Ala, Asn, and Asp were established); an alkylation stage (Gln, Glu, Ser, Thr, Val, Ile, Leu, and Pro were generated in considerable amounts); and a peptide formation stage (Phe, Tyr, Trp, His, Lys, Arg, Cys, and Met were selected due to their structural advantages). This scenario is a part of the evolution of Garakuta World, in which many prebiotic materials are contained.

Introducing Chemoselective Peptide Conjugation via N-Alkylation of Pyridyl-alanine: Solution and Solid Phase Applications.

A novel chemoselective peptide conjugation via late-stage N-alkylation of pyridyl-alanine (PAL) in the solution and solid phase, namely, NAP, is demonstrated. The method constructs functionally diverse and highly stable N-alkylated conjugates with various peptides. Notably, conjugations in the solid phase offered a more economical process. The method can provide the opportunity for dual labeling along with a cysteine handle in a peptide chain. Finally, we showcased that the antiproliferative activities of the p53 peptide (MDM2 inhibitor) could be 2-fold enhanced via NAP conjugation with the RGD peptide (selective integrin binder).

Engineered Imine Reductase Catalyzed Enantiodivergent Synthesis of Alkylated Amphetamines.

Less steric ketones exhibited low stereoselectivity toward M5 due to their difficulty in restricting the free rotation of the imine intermediate. An engineered enantio-complementary imine reductase from M5 was obtained with catalytic activity. We identified four key residues that play essential roles in controlling stereoselectivity. Two mutants, I149Y-W234L (up to 99%S ee) and L200M-F260M (up to 99%R ee), were achieved, showing excellent stereoselectivity toward the tested substrates, offering valuable biocatalysts for synthesizing alkylated amphetamines.

Langmuir monolayers provide an effective strategy for studying molecular recognition of nucleobases using alkylated nucleotides.

Molecular Recognition in nucleotides is crucial for medicine, underpinning precise interactions in genetic replication and therapy. Alkylated nucleotides, in particular, play a key role in modifying DNA to inhibit cancer cell growth. In this study, we focused on an alkylated nucleotide, PNM2 (3',4',6'-O-tristearoyl uridine or uridine tri-stearate), to investigate the interaction between adenine molecules in the aqueous subphase and PNM2 Langmuir monolayers. Utilizing techniques such as tensiometry, Brewster angle microscopy, infrared spectroscopy, surface potential measurements, and dilatational surface rheology, we found compelling evidence of molecular Recognition between the polar head of the insoluble amphiphile (uridine) in the monolayer and adenine in the aqueous subphase, attributed to hydrogen bonding. These interactions significantly influenced the physicochemical properties of the air-water interface, including monolayer expansion upon molecular recognition, decreased dilatational modulus, increased tensiometric stability of the monolayer when compressed to relevant surface pressures, and decreased surface potential. These findings are noteworthy for drug development, providing crucial insights into the mechanisms of nucleotide interactions.

Design and Evolution of an Artificial Friedel-Crafts Alkylation Enzyme Featuring an Organoboronic Acid Residue.

Creating artificial enzymes by the genetic incorporation of noncanonical amino acids with catalytic side chains would expand the enzyme chemistries that have not been discovered in nature. Here, we report the design of an artificial enzyme that uses p-boronophenylalanine as the catalytic residue. The artificial enzyme catalyzes Michael-type Friedel-Crafts alkylation through covalent activation. The designer enzyme was further engineered to afford high yields with excellent enantioselectivities. We next developed a practical method for preparative-scale reactions by whole-cell catalysis. This enzymatic C-C bond formation reaction was combined with palladium-catalyzed dearomative arylation to achieve the efficient synthesis of spiroindolenine compounds.

Inhibition of human DNA alkylation damage repair enzyme ALKBH2 by HIV protease inhibitor ritonavir.

The human DNA repair enzyme AlkB homologue-2 (ALKBH2) repairs methyl adducts from genomic DNA and is overexpressed in several cancers. However, there are no known inhibitors available for this crucial DNA repair enzyme. The aim of this study was to examine whether the first-generation HIV protease inhibitors having strong anti-cancer activity can be repurposed as inhibitors of ALKBH2. We selected four such inhibitors and performed in vitro binding analysis against ALKBH2 based on alterations of its intrinsic tryptophan fluorescence and differential scanning fluorimetry. The effect of these HIV protease inhibitors on the DNA repair activity of ALKBH2 was also evaluated. Interestingly, we observed that one of the inhibitors, ritonavir, could inhibit ALKBH2-mediated DNA repair significantly via competitive inhibition and sensitized cancer cells to alkylating agent methylmethane sulfonate (MMS). This work may provide new insights into the possibilities of utilizing HIV protease inhibitor ritonavir as a DNA repair antagonist.

Reductive alkyl-alkyl coupling from isolable nickel-alkyl complexes.

The selective cross-coupling of two alkyl electrophiles to construct complex molecules remains a challenge in organic synthesis1,2. Known reactions are optimized for specific electrophiles and are not amenable to interchangeably varying electrophilic substrates that are sourced from common alkyl building blocks, such as amines, carboxylic acids and halides3-5. These limitations restrict the types of alkyl substrate that can be modified and, ultimately, the chemical space that can be explored6. Here we report a general solution to these limitations that enables a combinatorial approach to alkyl-alkyl cross-coupling reactions. This methodology relies on the discovery of unusually persistent Ni(alkyl) complexes that can be formed directly by oxidative addition of alkyl halides, redox-active esters or pyridinium salts. The resulting alkyl complexes can be isolated or directly telescoped to couple with a second alkyl electrophile, which represent cross-selective reactions that were previously unknown. The utility of this synthetic capability is showcased in the rapid diversification of amino acids, natural products, pharmaceuticals and drug-like building blocks by various combinations of dehalogenative, decarboxylative or deaminative coupling. In addition to a robust scope, this work provides insights into the organometallic chemistry of synthetically relevant Ni(alkyl) complexes through crystallographic analysis, stereochemical probes and spectroscopic studies.

Accidental Encounter of Repair Intermediates in Alkylated DNA May Lead to Double-Strand Breaks in Resting Cells.

In clinics, chemotherapy is often combined with surgery and radiation to increase the chances of curing cancers. In the case of glioblastoma (GBM), patients are treated with a combination of radiotherapy and TMZ over several weeks. Despite its common use, the mechanism of action of the alkylating agent TMZ has not been well understood when it comes to its cytotoxic effects in tumor cells that are mostly non-dividing. The cellular response to alkylating DNA damage is operated by an intricate protein network involving multiple DNA repair pathways and numerous checkpoint proteins that are dependent on the type of DNA lesion, the cell type, and the cellular proliferation state. Among the various alkylating damages, researchers have placed a special on O6-methylguanine (O6-mG). Indeed, this lesion is efficiently removed via direct reversal by O6-methylguanine-DNA methyltransferase (MGMT). As the level of MGMT expression was found to be directly correlated with TMZ efficiency, O6-mG was identified as the critical lesion for TMZ mode of action. Initially, the mode of action of TMZ was proposed as follows: when left on the genome, O6-mG lesions form O6-mG: T mispairs during replication as T is preferentially mis-inserted across O6-mG. These O6-mG: T mispairs are recognized and tentatively repaired by a post-replicative mismatched DNA correction system (i.e., the MMR system). There are two models (futile cycle and direct signaling models) to account for the cytotoxic effects of the O6-mG lesions, both depending upon the functional MMR system in replicating cells. Alternatively, to explain the cytotoxic effects of alkylating agents in non-replicating cells, we have proposed a "repair accident model" whose molecular mechanism is dependent upon crosstalk between the MMR and the base excision repair (BER) systems. The accidental encounter between these two repair systems will cause the formation of cytotoxic DNA double-strand breaks (DSBs). In this review, we summarize these non-exclusive models to explain the cytotoxic effects of alkylating agents and discuss potential strategies to improve the clinical use of alkylating agents.

Site-specific N-alkylation of DNA oligonucleotide nucleobases by DNAzyme-catalyzed reductive amination.

DNA and RNA nucleobase modifications are biologically relevant and valuable in fundamental biochemical and biophysical investigations of nucleic acids. However, directly introducing site-specific nucleobase modifications into long unprotected oligonucleotides is a substantial challenge. In this study, we used in vitro selection to identify DNAzymes that site-specifically N-alkylate the exocyclic nucleobase amines of particular cytidine, guanosine, and adenosine (C, G and A) nucleotides in DNA substrates, by reductive amination using a 5'-benzaldehyde oligonucleotide as the reaction partner. The new DNAzymes each require one or more of Mg2+, Mn2+, and Zn2+ as metal ion cofactors and have kobs from 0.04 to 0.3 h-1, with rate enhancement as high as ∼104 above the splinted background reaction. Several of the new DNAzymes are catalytically active when an RNA substrate is provided in place of DNA. Similarly, several new DNAzymes function when a small-molecule benzaldehyde compound replaces the 5'-benzaldehyde oligonucleotide. These findings expand the scope of DNAzyme catalysis to include nucleobase N-alkylation by reductive amination. Further development of this new class of DNAzymes is anticipated to facilitate practical covalent modification and labeling of DNA and RNA substrates.

Alkoxyalkylation of Electron-Rich Aromatic Compounds.

Alkoxyalkylation and hydroxyalkylation methods utilizing oxo-compound derivatives such as aldehydes, acetals or acetylenes and various alcohols or water are widely used tools in preparative organic chemistry to synthesize bioactive compounds, biosensors, supramolecular compounds and petrochemicals. The syntheses of such molecules of broad relevance are facilitated by acid, base or heterogenous catalysis. However, degradation of the N-analogous Mannich bases are reported to yield alkoxyalkyl derivatives via the retro-Mannich reaction. The mutual derivative of all mentioned species are quinone methides, which are reported to form under both alkoxy- and aminoalkylative conditions and via the degradation of the Mannich-products. The aim of this review is to summarize the alkoxyalkylation (most commonly alkoxymethylation) of electron-rich arenes sorted by the methods of alkoxyalkylation (direct or via retro-Mannich reaction) and the substrate arenes, such as phenolic and derived carbocycles, heterocycles and the widely examined indole derivatives.

Novel pyrrolidine-alkylamino-substituted dicyanoisophorone derivatives as near-infrared fluorescence probe for imaging ß-amyloid in vitro and in vivo.

BACKGROUND: The formation of amyloid-ß (Aß) plaques is one of the key neuropathological hallmarks of Alzheimer's disease (AD). Near-infrared (NIR) probes show great potential for imaging of Aß plaques in vivo and in vitro. Dicyanoisophorone (DCIP) based Aß probes have attracted considerable attention due to their exceptional properties. However, DCIP probes still has some drawbacks, such as short emission wavelength (<650 nm) and low fluorescence intensity after binding to Aß. It is clear that further modification is needed to improve their luminescence efficiency and sensitivity. RESULTS: We designed and synthesize four novel pyrrolidine-alkylamino-substituted DCIP derivatives (6a-d) as imaging agents for ß-amyloid (Aß) aggregates. Compound 6c responds better to Aß aggregates than the other three compounds (6a, 6b and 6d) and its precursor DCIP. The calculated detection limit is to be as low as 0.23 µM. Compound 6c shows no cytotoxicity in the tested concentration for SH-SY5Y and HL-7702 cells. Additionally, compound 6c is successfully applied to monitor Aß aggregates in live SH-SY5Y cells and APP/PS1 transgenic mice. The retention time in the transgenic mice brain is much longer than that of age-matched wild-type mice. SIGNIFICANCE: The results indicates that compound 6c had an excellent ability to penetrate the blood-brain barrier and it could effectively distinguish APP/PS1 transgenic mice and wide-type mice. This represents its promising applications for Aß detection in basic and biomedical research.

Asymmetric photoenzymatic incorporation of fluorinated motifs into olefins.

Enzymes capable of assimilating fluorinated feedstocks are scarce. This situation poses a challenge for the biosynthesis of fluorinated compounds used in pharmaceuticals, agrochemicals, and materials. We developed a photoenzymatic hydrofluoroalkylation that integrates fluorinated motifs into olefins. The photoinduced promiscuity of flavin-dependent ene-reductases enables the generation of carbon-centered radicals from iodinated fluoroalkanes, which are directed by the photoenzyme to engage enantioselectively with olefins. This approach facilitates stereocontrol through interaction between a singular fluorinated unit and the enzyme, securing high enantioselectivity at ß, γ, or δ positions of fluorinated groups through enzymatic hydrogen atom transfer-a process that is notably challenging with conventional chemocatalysis. This work advances enzymatic strategies for integrating fluorinated chemical feedstocks and opens avenues for asymmetric synthesis of fluorinated compounds.

REV1 coordinates a multi-faceted tolerance response to DNA alkylation damage and prevents chromosome shattering in Drosophila melanogaster.

When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.

Concise Total Syntheses of (-)-Crinipellins A and B Enabled by a Controlled Cargill Rearrangement.

Herein, we report concise total syntheses of diterpene natural products (-)-crinipellins A and B with a tetraquinane skeleton, three adjacent all-carbon quaternary centers, and multiple oxygenated and labile functional groups. Our synthesis features a convergent Kozikowski ß-alkylation to unite two readily available building blocks with all the required carbon atoms, an intramolecular photochemical [2 + 2] cycloaddition to install three challenging and adjacent all-carbon quaternary centers and a 5-6-4-5 tetracyclic skeleton, and a controlled Cargill rearrangement to rearrange the 5-6-4-5 tetracyclic skeleton to the desired tetraquinane skeleton. These strategically enabling transformations allowed us to complete total syntheses of (-)-crinipellins A and B in 12 and 13 steps, respectively. The results of quantum chemical computations revealed that the Bronsted acid-catalyzed Cargill rearrangements likely involve stepwise paths to products and the AlR3-catalyzed Cargill rearrangements likely involve a concerted path with asynchronous alkyl shifting events to form the desired product.

Hydrogen peroxide responsive theranostics for cancer-selective activation of DNA alkylators and real-time fluorescence monitoring in living cells.

Triple negative breast cancer (TNBC) is a notoriously difficult disease to treat, and many of the existing TNBC chemotherapeutics lack tumor selectivity and the capability for simultaneously visualizing and monitoring their own activity in the biological context. However, TNBC cells have been known to generate high levels of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2). To this end, three novel small molecule theranostics 1a, 1c, and 2 consisting of both H2O2-responsive nitrogen mustard prodrug and profluorophore character have been designed, synthesized, and evaluated as targeted cancer therapeutics and bioimaging agents. The three theranostics comprise of boronate esters that deactivate nitrogen mustard functional groups and fluorophores but allow their selective activation through H2O2-specific oxidative deboronation for the release of the active drug and fluorophore. The three theranostics demonstrated H2O2-inducible DNA-alkylating capability and fluorescence turn-on properties in addition to selective anticancer activity. They are particularly effective in killing TNBC MDA-MB-468 cells with high H2O2 level while safe to normal epithelial MCF-10A cell. The conjugated boron-masked fluorophores in 1c and 2 are highly responsive towards H2O2, which enabled tracking of the theranostics in living cellular mitochondria and nucleus organelles. The three theranostics 1a, 1c, and 2 are capable of both selective release of the active drug to take effect in H2O2-rich cancer sites and simultaneously monitoring its activity. This single molecule system is of utmost importance to understand the function, efficacy, and mechanism of the H2O2-activated prodrugs and theranostics within the living recipient.

Threonine Aldolase-Catalyzed Enantioselective α-Alkylation of Amino Acids through Unconventional Photoinduced Radical Initiation.

Visible light-driven pyridoxal radical biocatalysis has emerged as a promising strategy for the stereoselective synthesis of valuable noncanonical amino acids (ncAAs). Previously, the use of well-tailored photoredox catalysts represented the key to enable efficient pyridoxal phosphate (PLP) enzyme-catalyzed radical reactions. Here, we report a PLP-dependent threonine aldolase-catalyzed asymmetric α-C-H alkylation of abundant amino acids using Katritzky pyridinium salts as alkylating agents. The use of engineered threonine aldolases allowed for this redox-neutral radical alkylation to proceed efficiently, giving rise to challenging α-trisubstituted and -tetrasubstituted ncAA products in a protecting-group-free fashion with excellent enantiocontrol. Mechanistically, this enantioselective α-alkylation capitalizes on the unique reactivity of the persistent enzymatic quinonoid intermediate derived from the PLP cofactor and the amino acid substrate to allow for novel radical C-C coupling. Surprisingly, this photobiocatalytic process does not require the use of well-established photoredox catalysts and operates through an unconventional photoinduced radical generation involving a PLP-derived aldimine. The ability to develop photobiocatalytic reactions without relying on classic photocatalysts or photoenzymes opens up new avenues for advancing stereoselective intermolecular radical reactions that are not known in either organic chemistry or enzymology.

Strand-resolved mutagenicity of DNA damage and repair.

DNA base damage is a major source of oncogenic mutations1. Such damage can produce strand-phased mutation patterns and multiallelic variation through the process of lesion segregation2. Here we exploited these properties to reveal how strand-asymmetric processes, such as replication and transcription, shape DNA damage and repair. Despite distinct mechanisms of leading and lagging strand replication3,4, we observe identical fidelity and damage tolerance for both strands. For small alkylation adducts of DNA, our results support a model in which the same translesion polymerase is recruited on-the-fly to both replication strands, starkly contrasting the strand asymmetric tolerance of bulky UV-induced adducts5. The accumulation of multiple distinct mutations at the site of persistent lesions provides the means to quantify the relative efficiency of repair processes genome wide and at single-base resolution. At multiple scales, we show DNA damage-induced mutations are largely shaped by the influence of DNA accessibility on repair efficiency, rather than gradients of DNA damage. Finally, we reveal specific genomic conditions that can actively drive oncogenic mutagenesis by corrupting the fidelity of nucleotide excision repair. These results provide insight into how strand-asymmetric mechanisms underlie the formation, tolerance and repair of DNA damage, thereby shaping cancer genome evolution.