Total synthesis of carbazoquinocin C: application of the o-benzannulation of Fischer carbene complexes to carbazole-3,4-quinone alkaloids

[reaction: see text] The photoinduced o-benzannulation of 3-(2-vinyl)indolylcarbene complexes provides a direct route to carbazole derivatives that are oxygenated in the 3- and 4-positions. This reaction is quite efficient and provides for a unique synthesis of the lipid peroxidation inhibitor carbazoquinocin C.     

CuI/Oxalamide Catalyzed Couplings of (Hetero)aryl Chlorides and Phenols for Diaryl Ether Formation

Couplings between (hetero)aryl chlorides and phenols can be effectively promoted by CuI in combination with an N‐aryl‐N′‐alkyl‐substituted oxalamide ligand to proceed smoothly at 120 °C. For this process, N‐aryl‐N′‐alkyl‐substituted oxalamides are more effective ligands than bis(N‐aryl)‐substituted oxalamides. A wide range of electron‐rich and electron‐poor aryl and heteroaryl chlorides gave the corresponding coupling products in good yields. Satisfactory conversions were achieved with electron‐rich phenols as well as a limited range of electron‐poor phenols. Catalyst and ligand loadings as low as 1.5 mol % are sufficient for the scaled‐up variants of some of these reactions.     

Charge profiling and stability testing of biosimilar by capillary isoelectric focusing

CIEF was developed for the rapid analysis of charge heterogeneity of trastuzumab biosimilar using commercially available fluorocarbon‐coated capillary. The CIEF master mix was composed of 0.30% w/v methyl cellulose, 2.3 M urea, 56.8 mM l ‐arginine, 1.52 mM iminodiacetic acid, 4.5% v/v carrier ampholytes (broad‐range pI 3–10 and narrow‐range pI 8–10.5 with ratio of 3:1), and 0.45% v/v 10.0, 9.5, 7.0, 5.5, 4.1 pI markers. To get a robust method to analyze charge heterogeneity, some separation parameters, including focusing time and separation temperature, were investigated and optimized. The optimized method gave good precision in estimated pI values of charge variants with RSDs of not more than 0.16% intraday analysis (n = 6) and < 0.18% interday analysis (n = 9). In addition, the applications of this method including purity, stability, lot consistency, peptide N‐glycosidase F digest, and C‐terminal lysine variants characterization were also investigated.     

Iterative Suzuki‐Miyaura Cross‐coupling/Bromo‐desilylation Reaction Sequences for the Assembly of Chemically Well‐defined,Acyclic Oligopyrrole/Benzenoid Hybrids Embodying Mixed Modes of Connectivity

Syntheses of a range of chemically well‐defined oligopyrrole/benzenoid hybrids are described using tandem Suzuki‐Miyaura cross‐coupling/bromo‐desilyation reaction sequences for linking borylated pyrroles, halogenated pyrroles and/or dibromobenzenes to one another. By such means, including iterative variants, a range of all α‐linked, all β‐linked oligopyrroles as well as certain combinations thereof have been assembled, some of them for the first time. The conductivities of iodine‐treated thin films formed from certain such systems have been determined.     

Directed Evolution of RebH for Site‐Selective Halogenation of Large Biologically Active Molecules

We recently characterized the substrate scope of wild‐type RebH and proceeded to evolve variants of this enzyme with improved stability for biocatalysis. The substrate scopes of both RebH and the stabilized variants, however, are limited primarily to compounds similar in size to tryptophan. A substrate walking approach was used to further evolve RebH variants with expanded substrate scope. Two particularly notable variants were identified: 3‐SS, which provides high conversion of tricyclic tryptoline derivatives; and 4‐V, which accepts a broad range of large indoles and carbazoles. This constitutes the first reported use of directed evolution to enable the functionalization of substrates not accepted by wild‐type RebH and demonstrates the utility of RebH variants for the site‐selective halogenation of biologically active compounds.     

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

Capillary isoelectric focusing (cIEF) was online coupled to a Q‐TOF MS by a flow‐through microvial interface for the analysis of therapeutic mAb. Intact molecular weights obtained from the mass spectrum deconvolution of separated charge variants provided information on the structural heterogeneity of therapeutic mAbs. A sandwich cIEF–MS configuration composed of anolyte, sample, and catholyte segments sequentially injected into a neutrally coated capillary was used for the charge heterogeneity separation of four mAbs. Acetic acid and ammonium hydroxide were used in places of the non‐volatile acids and bases commonly used for IEF but are incompatible with online MS detection. Glycerol was added as the anti‐convective reagent. A chemical modifier was mixed with the cIEF effluent in the flow‐throw microvial to maintain the ESI stability and to mitigate ion suppression from the co‐eluted carrier ampholytes and glycerol. Analysis of mAb samples have shown relative populations of two basic variants originating from C‐terminal lysine process and acidic variant of deamidation. The lysine clippings, deamidation, and sialic acid modification in oligosaccharide chains were revealed in infliximab. Two lysine clipping variants and a deamidated variant were observed in adalimumab. The duplicate analyses of a reference mAb demonstrated five charge variants separated by cIEF due to some unidentified modifications, as their mass spectra shared close similarities. The mAb analyses demonstrated the feasibility of the cIEF–MS method, and they demonstrated how charge and structural variants and minor differences in therapeutic mAbs are observed with this technology. Online cIEF–MS is an information rich technology with high throughput, demonstrated by the initial data presented here.     

Determination of genetic transferrin variants in human serum by high‐resolution capillary zone electrophoresis†

High‐resolution capillary zone electrophoresis in the routine arena with stringent quality assurance is employed for the determination of carbohydrate‐deficient transferrin in human serum. The assay comprises mixing of human serum with a Fe^III‐containing solution prior to analysis of the iron‐saturated mixture in a dynamically double‐coated capillary using a commercial buffer at alkaline pH. In contrast to other assays, it provides sufficient resolution for proper recognition of genetic transferrin variants. Analysis of 7290 patient sera revealed 166 isoform patterns that could be assigned to genetic variants, namely, 109 BC, 53 CD, one BD and three CC variants. Several subtypes of transferrin D can be distinguished as they have large enough differences in pI values. Subtypes of transferrin C and B cannot be resolved. However, analysis of the detection time ratios of tetrasialo isoforms of transferrin BC and transferrin CD variants revealed multimodal frequency histograms, indicating the presence of subtypes of transferrin C, B and D. The data gathered over 11 years demonstrate the robustness of the high‐resolution capillary zone electrophoresis assay. This is the first account of a capillary zone electrophoresis based carbohydrate‐deficient transferrin assay with a broad overview on transferrin isoform patterns associated with genetic transferrin variants.     

Myoglobin‐Catalyzed C−H Functionalization of Unprotected Indoles

Functionalized indoles are recurrent motifs in bioactive natural products and pharmaceuticals. While transition metal‐catalyzed carbene transfer has provided an attractive route to afford C3‐functionalized indoles, these protocols are viable only in the presence of N‐protected indoles, owing to competition from the more facile N−H insertion reaction. Herein, a biocatalytic strategy for enabling the direct C−H functionalization of unprotected indoles is reported. Engineered variants of myoglobin provide efficient biocatalysts for this reaction, which has no precedents in the biological world, enabling the transformation of a broad range of indoles in the presence of ethyl α‐diazoacetate to give the corresponding C3‐functionalized derivatives in high conversion yields and excellent chemoselectivity. This strategy could be exploited to develop a concise chemoenzymatic route to afford the nonsteroidal anti‐inflammatory drug indomethacin.     

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

Synthetic folded insulin variants containing an ornithine‐hydroxylamine residue are readily modified in aqueous buffers by amide‐forming ligations with potassium acyltrifluoroborates (KATs). The synthetic insulin analogs were prepared by Fmoc‐SPPS, α‐ketoacid‐hydroxylamine (KAHA) ligation, and a prosthetic C‐peptide that delivers the correct disulfide pattern and allows facile incorporation at the B0 position of Glargine M2 of a new ornithine hydroxylamine protected with a photolabile group. The folded insulin is readily modified by photo‐deprotection followed by amide‐forming KAT ligation to give insulin variants labeled with dyes, lipids, and PEGs, as well as the formation of a covalent dimer.     

Chiral Cp‐Rhodium(III)‐Catalyzed Asymmetric Hydroarylations of 1,1‐Disubstituted Alkenes

Metal‐catalyzed functionalizations at the ortho position of a directing group have become an efficient bond‐forming strategy. A wide range of transformations that employ Cp*Rh^III catalysts have been described, but despite their synthetic potential, enantioselective variants that use chiral versions of the Cp* ligand remain scarce (Cp*=pentamethyl cyclopentadienyl). Cyclopentadienyl compounds with an atropchiral biaryl backbone are shown to be suitable ligands for the efficient intramolecular enantioselective hydroarylation of aryl hydroxamates. Dihydrofurans that bear methyl‐substituted quaternary stereocenters are thus obtained by C? H functionalization under mild conditions.     

Chemo‐Enzymatic Synthesis of Position‐Specifically Modified RNA for Biophysical Studies including Light Control and NMR Spectroscopy

The investigation of non‐coding RNAs requires RNAs containing modifications at every possible position within the oligonucleotide. Here, we present the chemo‐enzymatic RNA synthesis containing photoactivatable or ¹³C,¹⁵N‐labelled nucleosides. All four ribonucleotides containing ortho‐nitrophenylethyl (NPE) photocages, photoswitchable azobenzene C‐nucleotides and ¹³C,¹⁵N‐labelled nucleotides were incorporated position‐specifically in high yields. We applied this approach for the synthesis of light‐inducible 2′dG‐sensing riboswitch variants and detected ligand‐induced structural reorganization upon irradiation by NMR spectroscopy. This chemo‐enzymatic method opens the possibility to incorporate a wide range of modifications at any desired position of RNAs of any lengths beyond the limits of solid‐phase synthesis.     

Spectral Properties of Fluorogenic Thiophene‐derived Triarylmethane Dyes

Spectral properties and fluorogenic behaviors of five novel thiophene variants of malachite green (MG), termed MGTs, were determined. Appreciable changes as a function of homologation and substitution pattern, including absorption band positions and intensities and fluorescence quantum yields were observed. In particular, the shorter wavelength y‐band absorption was found to shift over a nearly 200 nm range based on aryl group variation, allowing fine‐tuning of the excitation wavelength for these dyes. In addition, the fluorescence intensity of some MGTs increased significantly (up to 4600‐fold) when the dye was bound to a cognate protein partner, which is potentially useful for cell imaging studies.     

Drug Oxidation by Cytochrome P450BM3: Metabolite Synthesis and Discovering New P450 Reaction Types

There is intense interest in late‐stage catalytic C?H bond functionalization as an integral part of synthesis. Effective catalysts must have a broad substrate range and tolerate diverse functional groups. Drug molecules provide a good test of these attributes of a catalyst. A library of P450_BM3 mutants developed from four base mutants with high activity for hydrocarbon oxidation produced human metabolites of a panel of drugs that included neutral (chlorzoxazone, testosterone), cationic (amitriptyline, lidocaine) and anionic (diclofenac, naproxen) compounds. No single mutant was active for all the tested drugs but multiple variants in the library showed high activity with each compound. The high conversions enabled full product characterization that led to the discovery of the new P450 reaction type of oxidative decarboxylation of an α‐hydroxy carboxylic acid and the formation a protected imine from an amine, offering a novel route to α‐functionalization of amines. The substrate range and varied product profiles suggest that this library of enzymes is a good basis for developing late‐stage C?H activation catalysts.     

Kinetic Resolution of sec‐Thiols by Enantioselective Oxidation with Rationally Engineered 5‐(Hydroxymethyl)furfural Oxidase

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec‐thiols by using structure‐guided engineering of 5‐(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec‐thiols, thus yielding the corresponding thioketones and nonreacted R‐configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen‐bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec‐thiols.     

Centromeric Alpha‐Satellite DNA Adopts Dimeric i‐Motif Structures Capped by AT Hoogsteen Base Pairs

Human centromeric alpha‐satellite DNA is composed of tandem arrays of two types of 171 bp monomers; type A and type B. The differences between these types are concentrated in a 17 bp region of the monomer called the A/B box. Here, we have determined the solution structure of the C‐rich strand of the two main variants of the human alpha‐satellite A box. We show that, under acidic conditions, the C‐rich strands of two A boxes self‐recognize and form a head‐to‐tail dimeric i‐motif stabilized by four intercalated hemi‐protonated C:C⁺ base pairs. Interestingly, the stack of C:C⁺ base pairs is capped by T:T and Hoogsteen A:T base pairs. The two main variants of the A box adopt a similar three‐dimensional structure, although the residues involved in the formation of the i‐motif core are different in each case. Together with previous studies showing that the B box (known as the CENP‐B box) also forms dimeric i‐motif structures, our finding of this non‐canonical structure in the A box shows that centromeric alpha satellites in all human chromosomes are able to form i‐motifs, which consequently raises the possibility that these structures may play a role in the structural organization of the centromere.     

Comprehensive Profiling for Histone H4of Human Liver Cells Using High Resolution LTQ-Orbitrap Mass Spectrometry

The post translational modifications of histone variants are playing an important role in the structure of chro‐ matin, the regulation of gene activities and the diagnosis of diseases, and conducting in‐depth researches and discovering new sites depend on new and rational analytical methods to some extent. In this work, the combinatorial method of high resolution LTQ‐Orbitrap mass spectrometry and multiple enzymes was employed to identify the post translational modifications (PTMs) of histone H4 of human liver cells. The novel methylation site, argnine 67 (R 67), was observed besides some sites reported previously such as lysine 31 (K 31), lysine 44 (K 44), argnine 55 (R 55) and lysine 59 (K 59) in the global domain. Meanwhile, various combinations of acetylation of lysine 5 (K 5), lysine 8 (K 8), lysine 12 (K 12), lysine 16 (K 16) and methylation of lysine 20 (K 20) in the NH₂‐terminal tails were also identified after the LC‐MS/MS analysis of trypsin, Arg‐C, Glu‐C and chymotrypsin digests.     

Enzymatic Chemoselective Aldehyde–Ketone Cross‐Couplings through the Polarity Reversal of Methylacetoin

The thiamine diphosphate (ThDP) dependent enzyme acetoin:dichlorophenolindophenol oxidoreductase (Ao:DCPIP OR) from Bacillus licheniformis was cloned and overexpressed in Escherichia coli. The recombinant enzyme shared close similarities with the acetylacetoin synthase (AAS) partially purified from Bacillus licheniformis suggesting that they could be the same enzyme. The product scope of the recombinant Ao:DCPIP OR was expanded to chiral tertiary α‐hydroxy ketones through the rare aldehyde–ketone cross‐carboligation reaction. Unprecedented is the use of methylacetoin as the acetyl anion donor in combination with a range of strongly to weakly activated ketones. In some cases, Ao:DCPIP OR produced the desired tertiary alcohols with stereochemistry opposite to that obtained with other ThDP‐dependent enzymes. The combination of methylacetoin as acyl anion synthon and novel ThDP‐dependent enzymes considerably expands the available range of C? C bond formations in asymmetric synthesis.     

High‐resolution separation of monoclonal antibodies mixtures and their charge variants by an alternative and generic CZE method

The determination of mAb critical quality attributes (CQA) is crucial for their successful application in health diseases. A generic CZE method was developed for the high‐resolution separation of various mAb charge variants, which are often recognized as important CQA. A dynamic coating of the capillary was obtained with polyethylene oxide (PEO), whereas Bis‐Tris allowed the analysis of mAbs under native conditions at pH 7.0. The effect of PEO and Bis‐Tris concentrations, as well as the nature of the acidic counter ion on the method performance was systematically studied. The %RSD on migration times was below 5% on three different CE instruments using the optimized method. Additional charge variants (in particular acidic variants) were resolved for 10 out of 17 mAbs compared to a reference CZE approach involving the use of ε‐amino‐caproic acid (EACA), triethylenetetramine (TETA), and hydroxypropylmethyl cellulose (HPMC). The amount of basic and acidic charge variants of 17 Food and Drug Administration (FDA) approved mAbs covering a wide range of physico‐chemical properties, e.g., pI between 8.0 and 9.4 and different hydrophobicity, were mainly comprised between 5–15% and 15–30%, respectively. It is noteworthy that applications for the quality control in hospitals as well as for the combination of the immune checkpoint inhibitors nivolumab and ipilimumab were presented.     

Stereoselective synthesis of (2E) 3‐amino‐2‐(1H‐benzimidazol‐2‐yl)acrylate and symmetric bisacrylates by transamination reactions

A range of various amines 2(a–i) was tested in transamination reactions using ethyl 2‐(1H‐benzimidazol‐2‐yl)‐3‐dimethylamino‐acrylate 1a. The (E)‐s‐cis/trans conformation of some representative products 4 was analyzed by ¹H and ¹³C NMR spectra. The C‐2/C‐3 bond of the compounds 3(a–i) is strongly polarized by a push‐pull effect. In the same manner, reactions of ethyl 2‐(benzoxazol‐2‐yl)‐3‐dimethylamino‐acrylate 1c with 1,4‐diaminobenzene 2i, ethylenediamine 2i, and 1,5‐diaminomaphthalene 2k have been investigated and gave directly the corresponding symmetric bis‐acrylates 4(a–c) in good yields. © 1999 John Wiley & Sons, Inc. Heteroatom Chem 10: 446–454, 1999     

Intra‐ and Intermolecular Nickel‐Catalyzed Reductive Cross‐Electrophile Coupling Reactions of Benzylic Esters with Aryl Halides

Nickel‐catalyzed cross‐electrophile coupling reactions of benzylic esters and aryl halides have been developed. Both inter‐ and intramolecular variants proceed under mild reaction conditions. A range of heterocycles and functional groups are tolerated under the reaction conditions. Additionally, the first example of a stereospecific cross‐electrophile coupling of a secondary benzylic ester is described.