Organic & Biomolecular Chemistry (v.11, #44)

Front cover (7633-7633).

Inside front cover (7634-7634).

Contents list (7635-7641).

No washing, less waiting: engineering biomolecular reporters for single-step antibody detection in solution by Sambashiva Banala; Remco Arts; Stijn J. A. Aper; Maarten Merkx (7642-7649).
Detection of antibodies is essential for the diagnosis of many disease states, including infectious diseases, autoimmune diseases and allergies. Most current antibody detection assays involve multistep detection schemes in which molecular recognition and signal generation are separate processes. A well-known example is the enzyme-linked immunosorbent assay (ELISA), which combines high sensitivity and specificity with strong signal amplification. However, ELISA and other heterogeneous methods require multiple, time-consuming washing and incubation steps, which limits their applicability in point-of-care diagnostics and high-throughput applications. In recent years, several new antibody detection strategies have been developed in which antibody binding and signal generation are integrated within a single biomolecular reporter. These strategies aim to rival ELISA in terms of sensitivity and specificity, while decreasing the time and effort required to perform an assay. Here, we review recent developments in this field according to their mechanism of action and discuss their advantages and limitations.

Miltiradiene (1) is the precursor of phenolic diterpenoids such as ferruginol (2), requiring aromatization and hydroxylation. While this has been attributed to a single cytochrome P450 (CYP76AH1), characterization of the rosemary ortholog CYP76AH4 led to the discovery that these CYPs simply hydroxylate the facilely oxidized aromatic intermediate abietatriene (3).

Efficient and regioselective nickel-catalyzed [2 + 2 + 2] cyclotrimerization of ynoates and related alkynes by Sanjeewa K. Rodrigo; Israel V. Powell; Michael G. Coleman; Jeanette A. Krause; Hairong Guan (7653-7657).
A nickel-based catalytic system has been developed for [2 + 2 + 2] cyclotrimerization of various alkynes, especially ynoates. This catalytic system enables facile construction of substituted aromatic compounds in excellent yields with high regioselectivity.

Treatment of allyl-1,1-dichlorovinyl ethers with n-BuLi at −78 °C, followed by quenching with ketones, epoxides, and oxetanes, leads to highly substituted β-, γ-, and δ-lactones in good to excellent yields.

NaH mediated isomerisation–allylation reaction of 1,3-substituted propenols by Adam J. S. Johnston; Mark G. McLaughlin; Jolene P. Reid; Matthew J. Cook (7662-7666).
A base mediated isomerisation–allylation protocol of 1,3-disubstituted propenols has been established. The use of diaryl and aryl-silyl substrates is reported alongside the use of substituted allyl bromides. Mechanistic experiments have also been conducted to elucidate the reaction pathway.

On the role of guests in enforcing the mechanism of action of gated baskets by Yian Ruan; Bao-Yu Wang; Jeremy M. Erb; Shigui Chen; Christopher M. Hadad; Jovica D. Badjić (7667-7675).
We designed and prepared a spacious and gated basket of type 2 (V = 318 Å3) in ten synthetic steps. With the assistance of 1H NMR spectroscopy, we found that the pyridine gates at the rim of 2 form a seam of N–H⋯N hydrogen bonds, thereby adopting right- (P) and left-handed (M) helical arrangements. The recognition characteristics of the smaller basket 1 (V = 226 Å3) and the larger 2 for various solvents as guests were quantified by 1H NMR spectroscopy in CD2Cl2 (61 Å3), CDCl3 (75 Å3), CFCl3 (81 Å3) and CCl4 (89 Å3); the apparent guest binding equilibria Ka were found to be inversely proportional to the affinity of bulk solvents KS for populating each host. The rate of the P/M racemization (krac, s−1) was, for both 1 and 2, studied in all four solvents using dynamic NMR spectroscopy. From these experiments, two isokinetic relationships (ΔS‡P/Mvs. ΔH‡P/M) were identified with each one corresponding to a different mechanism of P/M racemization. A computational study (B3LYP/6-31+G**//PM6) of 1 and 2 in the gas phase indicates two competing racemization pathways: (a) RM1–2 describes a pivoting of a single gate followed by the rotation of the remaining two gates, while (b) RM3 depicts simultaneous (geared) rotation of all three gates. The racemization of the larger basket 2, in all four solvents (packing coefficient, PC = 0.19–0.28), conformed to one isokinetic relationship, which also coincided with the operation of the smaller basket 1 in CD2Cl2 (PC = 0.27). However, in CDCl3, CFCl3 and CCl4 (PC = 0.33–0.39), the mode of action of 1 appears to correlate with a different isokinetic relationship. Thus, we propose that the population of the basket's inner space (PC) determines the mechanism of P/M racemization. When PC < 0.3, the mechanism of operation is RM1–2, whereas, a greater packing, represented when PC > 0.3, enforces the geared RM3 mechanistic alternative.

Design, synthesis, biological evaluation, and molecular modeling study of 4-alkoxyquinazoline derivatives as potential VEGFR2 kinase inhibitors by Jian Sun; Dong-Dong Li; Jing-Ran Li; Fei Fang; Qian-Ru Du; Yong Qian; Hai-Liang Zhu (7676-7686).
A series of novel 4-alkoxyquinazoline derivatives were prepared and synthesized and their biological activities were evaluated as potential inhibitors of vascular endothelial growth factor receptor 2 (VEGFR2). Of these compounds, compound 3j demonstrated the most potent inhibitory activities against VEGFR2 tyrosine kinase and cell proliferation, the IC50 values of this compound reaching up to 2.72 nM and 0.35 μM, respectively, compared with Tivozanib (3.40 nM and 0.38 μM). The obtained results, along with a 3D-QSAR study and molecular docking that was used for investigating the probable binding mode, could provide an important basis for further optimization of compound 3j as a potential tyrosine kinase inhibitor.

Using N-acetyl-d-glucosamine as a starting material, the anti-influenza drugs oseltamivir and tamiphosphor were synthesized via a pivotal intermediate of aldehyde 8. An intramolecular Horner–Wadsworth–Emmons reaction was utilized to construct the highly functionalized cyclohexene ring. The existing N-acetyl group was transformed into an azido group for the subsequent aziridination, followed by implantation of a 3-pentoxy group of the desired stereochemistry.

Triazole biotin: a tight-binding biotinidase-resistant conjugate by Anne I. Germeroth; Jill R. Hanna; Rehana Karim; Franziska Kundel; Jonathan Lowther; Peter G. N. Neate; Elizabeth A. Blackburn; Martin A. Wear; Dominic J. Campopiano; Alison N. Hulme (7700-7704).
The natural amide bond found in all biotinylated proteins has been replaced with a triazole through CuAAC reaction of an alkynyl biotin derivative. The resultant triazole-linked adducts are shown to be highly resistant to the ubiquitous hydrolytic enzyme biotinidase and to bind avidin with dissociation constants in the low pM range. Application of this strategy to the production of a series of biotinidase-resistant biotin-Gd-DOTA contrast agents is demonstrated.

Asymmetric organocatalytic 1,4-additions provide access to a large number of biologically relevant compounds. Chiral squaramides efficiently catalyse enantioselective Michael addition of 1,3-dicarbonyl compounds to aliphatic nitroalkenes. The resulting γ-nitro carboxylic derivatives were obtained in high yields and in high enantiomeric purities. Quantum chemical calculations helped us to devise a transition state model, which explains the observed stereochemical course of the addition. The best results were obtained with Meldrum's acid as a donor, with which enantiomeric purity of the Michael adduct was 97 : 3 e.r. Using this methodology pregabalin was synthesized in three steps in overall 52% yield.

A novel route to synthesize the biologically active benzimidazolopyrazine core is outlined. The reaction proceeds via tandem benzimidazole formation/annulations of indole, pyrrole and aliphatic δ-alkynyl aldehydes in the presence of copper salts and green solvents with moderate to good yields.

Lighting up cysteine and homocysteine in sequence based on the kinetic difference of the cyclization/addition reaction by Fuqiang Guo; Minggang Tian; Fang Miao; Weijia Zhang; Guofen Song; Yong Liu; Xiaoqiang Yu; Jing Zhi Sun; Wai-Yeung Wong (7721-7728).
A novel one- and two-photon fluorescent probe CB1 has been developed for discriminating Cys and Hcy in a successive manner with high selectivity. The discrete time-dependent fluorescent responses enable us to sequentially detect Cys and Hcy in different time windows. Two-step reaction and kinetic modes were used to explain the sensing mechanism. As a promising biosensor for cell imaging, CB1 has been confirmed to exhibit membrane permeability to intact cells, low cytotoxicity to viable cells and photostability to ultraviolet light excitation. Furthermore, the results from the control assay have shown that the one- and two-photon fluorescence of CB1 within cells is associated with intracellular mercapto biomolecules but yet there is little interference with physiological pH value, viscosity and common bioanalytes. Finally one- and two-photon fluorescent images of CB1 within living SiHa cells have been presented.

Boron functionalization of BODIPY by various alcohols and phenols by Bertrand Brizet; Claire Bernhard; Yulia Volkova; Yoann Rousselin; Pierre D. Harvey; Christine Goze; Franck Denat (7729-7737).
The synthesis of new B–O BODIPY derivatives functionalized with different alkoxy or diarylalkoxy derivatives is described. These compounds were synthesized from the reaction of different B–F BODIPY precursors with various alcohols and phenols, in the presence of AlCl3. Water-soluble dyes could be synthesized as well with this method, specifically by the introduction of polyethyleneglycol (PEG) groups. A photophysical study of the different compounds was performed, and showed that the B–O BODIPY derivatives exhibit rich fluorescence properties. Finally, the conjugation of the BODIPY core has been extended using two distyryl groups, hence providing NIR emitting BODIPY derivatives, in which one or two PEG groups have been anchored, making these systems very promising for future medical imaging applications.

Chemically triggered C–ON bond homolysis in alkoxyamines: regioselectivity and chemoselectivity by Gérard Audran; Paul Brémond; Matisse Bim Batsiandzy Ibanou; Sylvain R. A. Marque; Valérie Roubaud; Didier Siri (7738-7750).
Recently, we examplified the activation of the C–ON bond homolysis by protonation, alkylation, benzylation, acylation, oxidation and complexation with a Lewis acid of the nitrogen atom of the 1-(pyridin-4-yl)ethyl fragment (Chem. Commun., 2011, 4291 and Org. Lett., 2012, 358) and of the 1-(pyridin-2-yl)ethyl fragment (J. Org. Chem. ASAP Doi:10.1021/jo401674v) of (N-(2-methylpropyl)-N-(1-diethylphosphono-2,2-dimethylpropyl)-N-oxyl) SG1-based alkoxyamines. The quaternization of the 1-(pyridin-3-yl)ethyl fragment by the aforementioned reactions was investigated for the corresponding SG1-based alkoxyamines. In sharp contrast to the quaternization at ortho and para positions of the pyridyl moiety, the effect of the quaternization at the meta position was weak. The effects of quaternization at ortho, meta and para positions were investigated through natural bond orbital and Mulliken charges, HOMO–LUMO interactions in the starting materials and the radical stabilization energy of the released 1-puridylmethyl radicals using DFT calculations with the B3LYP/6-31G(d) and UBMK/6-311+G(3df,2p)//R(O)B3LYP/6-31G(d) methods, respectively.

An OFF–ON chemosensor for biological and environmental applications: sensing Cd2+ in water using catanionic vesicles and in living cells by Andrea Bencini; Francesco Caddeo; Claudia Caltagirone; Alessandra Garau; Mike B. Hurstouse; Francesco Isaia; Sandrina Lampis; Vito Lippolis; Francesco Lopez; Valeria Meli; Maura Monduzzi; Maria Cristina Mostallino; Sergio Murgia; Stefano Puccioni; Judith Schmidt; Pietro Paolo Secci; Yeshayahu Talmon (7751-7759).
A new OFF–ON fluorescent chemosensor (L1) for Cd2+ recognition based on a 5-chloro-8-hydroxyquinoline pendant arm derivative of 1,4,7-triazacyclononane ([9]aneN3) will be presented and its photochemical features in an MeCN–H2O 1 : 1 (v/v) mixture, in pure water, after inclusion within catanionic vesicles, and in living cells will be discussed. The coordination properties of L1 both in solution and in the solid state were preliminarily studied and its selectivity towards Cd2+versus a set of different metal ions (Cu2+, Zn2+, Cd2+, Pb2+, Al3+, Hg2+, Co2+, Ni2+, Mn2+, Mg2+, K+, Ca2+, Ag+, and Na+) was verified in MeCN–H2O 1 : 1 (v/v). In water, upon addition of increasing amounts of Cd2+ to L1 an enhancement of the fluorescence emission was detected. To overcome this serious drawback, L1 was dissolved in an innovative catanionic vesicular solution based on sodium bis(2-ethylhexyl) sulfosuccinate, a traditional surfactant, and 1-dodecyl-3-methylimidazolium bromide, an ionic liquid. When enclosed within the vesicle bilayers in water, L1 restored its fluorescence emission property upon addition of Cd2+. Remarkably, L1 enters the cellular membrane of living cells thus allowing the detection of intracellular Cd2+. These findings encourage the application of this new fluorescent chemosensor in real samples for histological and environmental analyses.

Synthesis of integerrimide A by an on-resin tandem Fmoc-deprotection–macrocyclisation approach by Sirirat Kumarn; Nitirat Chimnoi; Somsak Ruchirawat (7760-7767).
A solid-phase total synthesis of integerrimide A (1) is reported. This work employs a safety-catch linker which enables head-to-tail cyclisation of the required linear peptide 6 as a method of cleaving the peptide from the solid support, and highlights a new tandem approach to direct macrocyclisation. It provides access to useful quantities of 1 in 16 steps and 19% overall yield, based on the manufacturer's stated resin substitution from commercially available materials, and also verifies the absolute stereochemistry of the natural product.

Enantioselective total synthesis of macrolide (+)-neopeltolide by Arun K. Ghosh; Khriesto A. Shurrush; Zachary L. Dawson (7768-7777).
The asymmetric total synthesis of the anti-proliferative macrolide (+)-neopeltolide has been completed. The stereochemically defined trisubstituted tetrahydropyran ring was constructed via a catalytic hetero-Diels–Alder reaction creating two new chiral centers in a highly diastereoselective manner. The other key features of this synthesis included Brown's asymmetric allylation to install the requisite C-11 and C-13 stereocenters. The synthesis of the oxazole side chain consisted of a hydrozirconation of an alkynyl stannane to establish the Z stereochemistry, followed by a palladium catalyzed cross coupling to introduce the desired Z olefin in the oxazole side chain.

A structural and dynamic investigation of the inhibition of catalase by nitric oxide by Marco Candelaresi; Andrea Gumiero; Katrin Adamczyk; Kirsty Robb; César Bellota-Antón; Vartul Sangal; John Munnoch; Gregory M. Greetham; Michael Towrie; Paul A. Hoskisson; Anthony W. Parker; Nicholas P. Tucker; Martin A. Walsh; Neil T. Hunt (7778-7788).
Determining the chemical and structural modifications occurring within a protein during fundamental processes such as ligand or substrate binding is essential to building up a complete picture of biological function. Currently, significant unanswered questions relate to the way in which protein structural dynamics fit within the structure–function relationship and to the functional role, if any, of bound water molecules in the active site. Addressing these questions requires a multidisciplinary approach and complementary experimental techniques that, in combination, enhance our understanding of the complexities of protein chemistry. We exemplify this philosophy by applying both physical and biological approaches to investigate the active site chemistry that contributes to the inhibition of the Corynebacterium glutamicum catalase enzyme by nitric oxide. Ultrafast two-dimensional infrared spectroscopy (2D-IR) experiments exploit the NO ligand as a local probe of the active site molecular environment and shows that catalase displays a dynamically-restricted, ‘tight,’ structure. X-ray crystallography studies of C. glutamicum catalase confirm the presence of a conserved chain of hydrogen-bonded bound water molecules that link the NO ligand and the protein scaffold. This combination of bound water and restricted dynamics stands in stark contrast to other haem proteins, such as myoglobin, that exhibit ligand transport functionality despite the presence of a similar distal architecture in close proximity to the ligand. We conclude not only that the bound water molecules in the catalase active site play an important role in molecular recognition of NO but also may be part of the mechanistic operation of this important enzyme.

Evaluating minimalist mimics by exploring key orientations on secondary structures (EKOS) by Dongyue Xin; Eunhwa Ko; Lisa M. Perez; Thomas R. Ioerger; Kevin Burgess (7789-7801).
Peptide mimics that display amino acid side-chains on semi-rigid scaffolds (not peptide polyamides) can be referred to as minimalist mimics. Accessible conformations of these scaffolds may overlay with secondary structures giving, for example, “minimalist helical mimics”. It is difficult for researchers who want to apply minimalist mimics to decide which one to use because there is no widely accepted protocol for calibrating how closely these compounds mimic secondary structures. Moreover, it is also difficult for potential practitioners to evaluate which ideal minimalist helical mimics are preferred for a particular set of side-chains. For instance, what mimic presents i, i + 4, i + 7 side-chains in orientations that best resemble an ideal α-helix, and is a different mimic required for a i, i + 3, i + 7 helical combination? This article describes a protocol for fitting each member of an array of accessible scaffold conformations on secondary structures. The protocol involves: (i) use quenched molecular dynamics (QMD) to generate an ensemble consisting of hundreds of accessible, low energy conformers of the mimics; (ii) representation of each of these as a set of Cα and Cβ coordinates corresponding to three amino acid side-chains displayed by the scaffolds; (iii) similar representation of each combination of three side-chains in each ideal secondary structure as a set of Cα and Cβ coordinates corresponding to three amino acid side-chains displayed by the scaffolds; and, (iv) overlay Cα and Cβ coordinates of all the conformers on all the sets of side-chain “triads” in the ideal secondary structures and express the goodness of fit in terms of root mean squared deviation (RMSD, Å) for each overlay. We refer to this process as Exploring Key Orientations on Secondary structures (EKOS). Application of this procedure reveals the relative bias of a scaffold to overlay on different secondary structures, the “side-chain correspondences” (e.g. i, i + 4, i + 7 or i, i + 3, i + 4) of those overlays, and the energy of this state relative to the minimum located. This protocol was tested on some of the most widely cited minimalist α-helical mimics (1–8 in the text). The data obtained indicates several of these compounds preferentially exist in conformations that resemble other secondary structures as well as α-helices, and many of the α-helical conformations have unexpected side-chain correspondences. These observations imply the featured minimalist mimics have more scope for disrupting PPI interfaces than previously anticipated. Finally, the same simulation method was used to match preferred conformations of minimalist mimics with actual protein/peptide structures at interfaces providing quantitative comparisons of predicted fits of the test mimics at protein–protein interaction sites.

Back cover (7803-7804).