Hexafluoroisopropanol as a Bioconjugation Medium of Ultrafast, Tryptophan-Selective Catalysis.

The past decade has seen a remarkable growth in the number of bioconjugation techniques in chemistry, biology, material science, and biomedical fields. A core design element in bioconjugation technology is a chemical reaction that can form a covalent bond between the protein of interest and the labeling reagent. Achieving chemoselective protein bioconjugation in aqueous media is challenging, especially for generally less reactive amino acid residues, such as tryptophan. We present here the development of tryptophan-selective bioconjugation methods through ultrafast Lewis acid-catalyzed reactions in hexafluoroisopropanol (HFIP). Structure-reactivity relationship studies have revealed a combination of thiophene and ethanol moieties to give a suitable labeling reagent for this bioconjugation process, which enables modification of peptides and proteins in an extremely rapid reaction unencumbered by noticeable side reactions. The capability of the labeling method also facilitated radiofluorination application as well as antibody functionalization. Enhancement of an α-helix by HFIP leads to its compatibility with a certain protein, and this report also demonstrates a further stabilization strategy achieved by the addition of an ionic liquid to the HFIP medium. The nonaqueous bioconjugation approaches allow access to numerous chemical reactions that are unavailable in traditional aqueous processes and will further advance the chemistry of proteins.

Friedel-Crafts reactions for biomolecular chemistry.

Chemical tools and principles have become central to biological and medical research/applications by leveraging a range of classical organic chemistry reactions. Friedel-Crafts alkylation and acylation are arguably some of the most well-known and used synthetic methods for the preparation of small molecules but their use in biological and medical fields is relatively less frequent than the other reactions, possibly owing to the notion of their plausible incompatibility with biological systems. This review demonstrates advances in Friedel-Crafts alkylation and acylation reactions in a variety of biomolecular chemistry fields. With the discoveries and applications of numerous biomolecule-catalyzed or -assisted processes, these reactions have garnered considerable interest in biochemistry, enzymology, and biocatalysis. Despite the challenges of reactivity and selectivity of biomolecular reactions, the alkylation and acylation reactions demonstrated their utility for the construction and functionalization of all the four major biomolecules (i.e., nucleosides, carbohydrates/saccharides, lipids/fatty acids, and amino acids/peptides/proteins), and their diverse applications in biological, medical, and material fields are discussed. As the alkylation and acylation reactions are often fundamental educational components of organic chemistry courses, this review is intended for both experts and nonexperts by discussing their basic reaction patterns (with the depiction of each reaction mechanism in the ESI) and relevant real-world impacts in order to enrich chemical research and education. The significant growth of biomolecular Friedel-Crafts reactions described here is a testament to their broad importance and utility, and further development and investigations of the reactions will surely be the focus in the organic biomolecular chemistry fields.

An Ionic Liquid Medium Enables Development of a Phosphine-Mediated Amine-Azide Bioconjugation Method.

While a diverse set of design strategies have produced various chemical tools for biomolecule labeling in aqueous media, the development of nonaqueous, biomolecule-compatible media for bioconjugation has significantly lagged behind. In this report, we demonstrate that an aprotic ionic liquid serves as a novel reaction solvent for protein bioconjugation without noticeable loss of the biomolecule functions. The ionic liquid bioconjugation approach led to discovery of a novel triphenylphosphine-mediated amine-azide coupling reaction that forges a stable tetrazene linkage on unprotected peptides and proteins. This strategy of using untraditional media would provide untapped opportunities for expanding the scope of chemical approaches for bioconjugation.

Linear Hydrocarbon Chain Growth from a Molecular Diruthenium Carbide Platform.

The formation of linear hydrocarbon chains by sequential coupling of C1 units on the metal surface is the central part of the Fischer-Tropsch (F-T) synthesis. Organometallic complexes have provided numerous models of relevant individual C-C coupling events but have failed to reproduce the complete chain lengthening sequence that transforms a linear Cn hydrocarbon chain into its Cn+1 homologue in an iterative fashion. In this work, we demonstrate stepwise growth of linear Cn hydrocarbon chains and their conversion to their Cn+1 homologues via consecutive addition of CH2 units on a molecular diruthenium carbide platform. The chain growth sequence is initiated by the formation of a µ-η1:η1-C═CH2 ligand from a C + CH2 coupling between the µ-carbido complex [(Cp*Ru)2(η-NPh)(µ-C)] (1; Cp* = η5-C5Me5) and Ph2SCH2. Then, the chain propagates via a general C═CHR + CH2 coupling and subsequent hydrogen-assisted isomerization of the resulting allene ligand µ-η1:η3-H2C═C═CHR to a higher vinylidene homologue µ-η1:η1-C═CH(CH2)R. By repeating this reaction sequence, up to C6 chains have been synthesized in a stepwise fashion. The key step of this chain homologation sequence is the selective hydrogenation of the µ-η1:η3-allene unit to the corresponding µ-alkylidene ligand. Isotope labeling and computational studies indicate that this transformation proceeds via the hydrogenation of the allene ligand to a terminal alkene form and its isomerization to the µ-alkylidene ligand facilitated by the coordinatively unsaturated diruthenium platform.

Activity-Based Sensing Methods for Monitoring the Reactive Carbon Species Carbon Monoxide and Formaldehyde in Living Systems.

Carbon is central to the chemistry of life, and in addition to its fundamental roles as a static component of all major biomolecules spanning proteins, nucleic acids, sugars, and lipids, emerging evidence shows that small and transient carbon-based metabolites, termed reactive carbon species (RCS), are dynamic signaling/stress agents that can influence a variety of biological pathways. Recent examples include the identification of carbon monoxide (CO) as an ion channel blocker and endogenous formaldehyde (FA) as a one-carbon metabolic unit formed from the spontaneous degradation of dietary folate metabolites. These findings motivate the development of analytical tools for transient carbon species that can achieve high specificity and sensitivity to further investigate RCS signaling and stress pathways at the cell, tissue, and whole-organism levels. This Account summarizes work from our laboratory on the development of new chemical tools to monitor two important one-carbon RCS, CO and FA, through activity-based sensing (ABS), where we leverage the unique chemical reactivities of these small and transient analytes, rather than lock-and-key binding considerations, for selective detection. Classic inorganic/organometallic and organic transformations form the basis for this approach. For example, to distinguish CO from other biological diatomics of similar shape and size (e.g., nitric oxide and oxygen), we exploit palladium-mediated carbonylation as a synthetic method for CO sensing. The high selectivity of this carbonylation approach successfully enables imaging of dynamic changes in intracellular CO levels in live cells. Likewise, we apply the aza-Cope reaction for FA detection to provide high selectivity for this one-carbon unit over other larger biological aldehydes that are reactive electrophiles, such as acetaldehyde and methylglyoxal. By relying on an activity-based trigger as a design principle for small-molecule detection, this approach can be generalized to create a toolbox of selective FA imaging reagents, as illustrated by a broad range of FA probes spanning turn-on and ratiometric fluorescence imaging, positron emission tomography imaging, and chemiluminescence imaging modalities. Moreover, these chemical tools have revealed new one-carbon biology through the identification of folate as a dietary source of FA and alcohol dehydrogenase 5 as a target for FA metabolism. Indeed, these selective RCS detection methods have been expanded to a wider array of imaging platforms, such as metal-complex-based time-gated luminescence and materials-based imaging scaffolds (e.g., nanotubes, nanoparticles, and carbon dots), with modalities extending to Raman and Rayleigh scattering readouts. This pursuit of leveraging selective chemical reactivity to develop highly specific ABS probes for imaging of RCS provides not only practical tools for deciphering RCS-dependent biology but also a general design platform for developing ABS probes for a broader range of biological analytes encompassing elements across the periodic table.

Metal-Mediated Functionalization of Natural Peptides and Proteins: Panning for Bioconjugation Gold.

Selective modification of natural proteins is a daunting methodological challenge and a stringent test of selectivity and reaction scope. There is a continued need for new reactivity and new selectivity concepts. Transition metals exhibit a wealth of unique reactivity that is orthogonal to biological reactions and processes. As such, metal-based methods play an increasingly important role in bioconjugation. This Review examines metal-based methods as well as their reactivity and selectivity for the functionalization of natural proteins and peptides.

Safety and efficacy of noncardiac surgical procedures in the management of patients with trisomy 13: A single institution-based detailed clinical observation.

Intensive treatment including surgery for patients with trisomy 13 (T13) remains controversial. This study aimed to evaluate the safety and efficacy of noncardiac surgical intervention for T13 patients. Medical records of patients with karyotypically confirmed T13 treated in the neonatal intensive care unit in Nagano Children's Hospital from January 2000 to October 2016 were retrospectively reviewed, and data from patients who underwent noncardiac surgery were analyzed. Of the 20 patients with T13, 15 (75%) underwent a total of 31 surgical procedures comprising 15 types, including tracheostomy in 10 patients and gastrostomy in 4. Operative time, anesthesia time, and amount of bleeding are described for the first time in a group of children with T13. All the procedures were completed safely with no anesthetic complications or surgery-related death. The overall rate of postoperative complications was 19.3%. Patients receiving tracheostomy had stable or improved respiratory condition. Six of them were discharged home and were alive at the time of this study. These results suggest at least short-term safety and efficacy of major noncardiac surgical procedures, and long-term efficacy of tracheostomy on survival or respiratory stabilization for home medical care of children with T13. Noncardiac surgical intervention is a reasonable choice for patients with T13.

A Naturally Encoded Dipeptide Handle for Bioorthogonal Chan-Lam Coupling.

Manipulation of biomacromolecules is ideally achieved through unique and bioorthogonal chemical reactions of genetically encoded, naturally occurring functional groups. The toolkit of methods for site-specific conjugation is limited by selectivity concerns and a dearth of naturally occurring functional groups with orthogonal reactivity. We report that pyroglutamate amide N-H bonds exhibit bioorthogonal copper-catalyzed Chan-Lam coupling at pyroglutamate-histidine dipeptide sequences. The pyroglutamate residue is readily incorporated into proteins of interest by natural enzymatic pathways, allowing specific bioconjugation at a minimalist dipeptide tag.

A Three-Component Organometallic Tyrosine Bioconjugation.

Metal-based bioconjugation linkages represent a little-studied approach to protein functionalization that provides novel reactivity, stability, and function. Described is an organometallic bioconjugation, employing rhodium(III) salts, to link boronic acids with tyrosine residues by an arene complex. Both peptides and proteins are amenable to the mild bioconjugation in aqueous media, allowing incorporation of useful functionalities, such as affinity handles or fluorophores. Because of the metastability of the inorganic linkage, the conjugates are susceptible to cleavage by nucleophilic redox mediators but are stable toward typical biological conditions.

A Hexa-rhodium Metallopeptide Catalyst for Site-Specific Functionalization of Natural Antibodies.

Preparation of antibody-drug conjugates (ADCs), an emerging novel class of highly targeted biological hybrid agents, necessitates precise control of conjugation reactivity. Antibodies have complex multistranded architectures, and specific modification of natural antibodies has proven quite challenging. Here, we demonstrate that cooperative activity of a multimetallic metallopeptide enables efficient site-specific antibody functionalization, based on molecular recognition of the constant Fc region. This interplay of multiple metal centers enables introduction of an orthogonal alkyne handle into monoclonal or polyclonal antibodies from different species in an Fc-specific fashion. Elaboration of this simple functionalization allows preparation of conjugates with fluorophore, affinity handle, and pharmacological agents. This method opens a new opportunity for quick and easy production of well-defined antibody conjugates from a variety of antibody sequence and species of origin.

Histidine-Directed Arylation/Alkenylation of Backbone N-H Bonds Mediated by Copper(II).

Chemical modification of proteins and peptides represents a challenge of reaction design as well as an important biological tool. In contrast to side-chain modification, synthetic methods to alter backbone structure are extremely limited. In this communication, copper-mediated backbone N-alkenylation or N-arylation of peptides and proteins by direct modification of natural sequences is described. Histidine residues direct oxidative coupling of boronic acids at the backbone NH of a neighboring amino acid. The mild reaction conditions in common physiological buffers, at ambient temperature, are compatible with proteins and biological systems. This simple reaction demonstrates the potential for directed reactions in complex systems to allow modification of N-H bonds that directly affect polypeptide structure, stability, and function.

A diruthenium µ-carbido complex that shows singlet-carbene-like reactivity.

Low-temperature deprotonation of the cationic µ-methylidyne complex [(Cp*Ru)2(µ-NPh)(µ-CH)][BF4] (Cp* = η(5)-C5Me5) with KN(SiMe3)2 affords a thermally unstable µ-carbido complex [(Cp*Ru)2(µ-NPh)(µ-C)] (2), as evidenced by trapping experiments with elemental S or Se and (13)C NMR spectroscopic observation. The reactivity of 2 toward CO2, Ph2S(+)CH2(-), EtOH, and an intramolecular C-H bond indicates that the µ-carbido carbon in 2 has an ambiphilic (nucleophilic and electrophilic) nature consistent with the formulation of 2 as the first example of a transition-metal-substituted singlet carbene. DFT study suggests that the Ru substituents in 2 are stronger σ-donor and weaker π-donor to the carbene center than amino substituents in N-heterocyclic carbenes.

Iron-sensitive protein conjugates formed with a Wittig reaction precursor in ionic liquid.

In this report, formation of protein conjugates with an iron-sensitive enamine linkage is demonstrated through the ionic liquid-based bioconjugation method.

Correction: Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids.

[This corrects the article DOI: 10.1039/D1SC05204G.].

Site-specific DNA functionalization through the tetrazene-forming reaction in ionic liquids.

Development of multiple chemical tools for deoxyribonucleic acid (DNA) labeling has facilitated wide use of their functionalized conjugates, but significant practical and methodological challenges remain to achievement of site-specific chemical modification of the biomacromolecule. As covalent labeling processes are more challenging in aqueous solution, use of nonaqueous, biomolecule-compatible solvents such as an ionic liquid consisting of a salt with organic molecule architecture, could be remarkably helpful in this connection. Herein, we demonstrate site-specific chemical modification of unprotected DNAs through a tetrazene-forming amine-azide coupling reaction using an ionic liquid. This ionic liquid-enhanced reaction process has good functional group tolerance and precise chemoselectivity, and enables incorporation of various useful functionalities such as biotin, cholesterol, and fluorophores. A site-specifically labeled oligonucleotide, or aptamer interacting with a growth factor receptor (Her2) was successfully used in the fluorescence imaging of breast cancer cell lines. The non-traditional medium-promoted labeling strategy described here provides an alternative design paradigm for future development of chemical tools for applications involving DNA functionalization.

A puromycin-dependent activity-based sensing probe for histochemical staining of hydrogen peroxide in cells and animal tissues.

Hydrogen peroxide (H2O2) is a key member of the reactive oxygen species family of transient small molecules that has broad contributions to oxidative stress and redox signaling. The development of selective and sensitive chemical probes can enable the study of H2O2 biology in cell, tissue and animal models. Peroxymycin-1 is a histochemical activity-based sensing probe that responds to H2O2 via chemoselective boronate oxidation to release puromycin, which is then covalently incorporated into nascent proteins by the ribosome and can be detected by antibody staining. Here, we describe an optimized two-step, one-pot protocol for synthesizing Peroxymycin-1 with improved yields over our originally reported procedure. We also present detailed procedures for applying Peroxymycin-1 to a broad range of biological samples spanning cells to animal tissues for profiling H2O2 levels through histochemical detection by using commercially available anti-puromycin antibodies. The preparation of Peroxymycin-1 takes 9 h, the confocal imaging experiments of endogenous H2O2 levels across different cancer cell lines take 1 d, the dot blot analysis of mouse liver tissues takes 1 d and the confocal imaging of mouse liver tissues takes 3-4 d.

Phosphine-mediated three-component bioconjugation of amino- and azidosaccharides in ionic liquids.

Bioconjugation of carbohydrates has been a challenging task because of their chemical, functional, and structural diversities, and no single chemical modification tool can be universally applicable to all the target substrates in different environments. In this report, we have developed a bioconjugation strategy for labeling of carbohydrate derivatives through a phosphine-mediated three-component coupling reaction in an ionic liquid medium.

[Anesthetic management of bilateral PA banding in seven patients with HLHS].

Regarding the established phased operation for hypoplastic left heart syndrome (HLHS), the recent increase in cases involving Fontan operation has led to concomitant problems that seem to be caused by invasive open-heart surgery during the newborn period. In the new method to overcome this situation, one starts with the non-open-heart pulmonic artery banding (bil. PAB) during the newborn period, followed by the Norwood-Glenn operation, and finally with the Fontan operation. This is a report on seven cases treated with the pulmonic artery banding on HLHS at the Nagano Children's Hospital, in which we were personally involved as anesthesiologists.

[Anesthetic management for PDA in two patients with HLHS stent after bilateral PA banding].

Hypoplastic left heart syndrome (HLHS) is one of the most serious conditions among congenital cardiac disorders. In the course of the newly developed phased operation, we experienced some cases in which adverse side effects developed due to PGE1 preparation. I gave anesthesia in two cases that had to be treated with PDA stenting because of the above mentioned side effects.