Rational Design of a Humanized Glucagon‐Like Peptide‐1 Receptor Agonist Antibody

Bovine antibody BLV1H12 possesses a unique “stalk–knob” architecture in its ultralong heavy chain CDR3, allowing substitutions of the “knob” domain with protein agonists to generate functional antibody chimeras. We have generated a humanized glucagon‐like peptide‐1 (GLP‐1) receptor agonist antibody by first introducing a coiled‐coil “stalk” into CDR3H of the antibody herceptin. Exendin‐4 (Ex‐4), a GLP‐1 receptor agonist, was then fused to the engineered stalk with flexible linkers, and a Factor Xa cleavage site was inserted immediately in front of Ex‐4 to allow release of the N‐terminus of the fused peptide. The resulting clipped herceptin–Ex‐4 fusion protein is more potent in vitro in activating GLP‐1 receptors than the Ex‐4 peptide. The clipped herceptin–Ex‐4 has an extended plasma half‐life of approximately four days and sustained control of blood glucose levels for more than a week in mice. This work provides a novel approach to the development of human or humanized agonist antibodies as therapeutics.     

Synthesis of β‐Cyclodextrin Containing Copolymer via “Click” Chemistry and Its Self‐Assembly in the Presence of Guest Compounds

We report the synthesis of a hydrophilic copolymer with one polyethylene glycol (PEG) block and one β‐cyclodextrin (β‐CD) containing block by a “click” reaction between azido‐substituted β‐CD and propargyl flanking copolymer. ¹H NMR study suggested a highly efficient conjugation of β‐CD units by this approach. The obtained copolymer was used as a host macromolecule to construct assemblies in the presence of hydrophobic guests. For assemblies containing a hydrophobic polymer, their size can be simply adjusted by simply changing the content of hydrophobic component. By serving as a guest molecule, hydrophobic drugs can also be loaded accompanying the formation of nanoparticles, and the drug payload is releasable. Therefore, the copolymer synthesized herein can be employed as a carrier for drug delivery.     

Synthesis of Diacetylene‐Containing Peptide Building Blocks and Amphiphiles,Their Self‐Assembly and Topochemical Polymerization in Organic Solvents

A series of functional iodoacetylenes was prepared and converted into the corresponding diacetylene‐substituted amino acids and peptides via Pd/Cu‐promoted sp–sp carbon cross‐coupling reactions. The unsymmetrically substituted diacetylenes can be incorporated into oligopeptides without a change in the oligopeptide strand's directionality. Thus, a series of oligopeptide‐based, amphiphilic diacetylene model compounds was synthesized, and their self‐organization as well as their UV‐induced topochemical polymerizability was investigated in comparison to related polymer‐substituted macromonomers. Solution‐phase IR spectroscopy, gelation experiments, and UV spectroscopy helped to confirm that a minimum of five N‐H???O?C hydrogen‐bonding sites was required in order to obtain reliable aggregation into stable β‐sheet‐type secondary structures in organic solvents. Furthermore, the non‐equidistant spacing of these hydrogen‐bonding sites was proven to invariably lead to β‐sheets with a parallel β‐strand orientation, and the characteristic IR‐spectroscopic signatures of the latter in organic solution was identified. Scanning force micrographs of the organogels revealed that compounds with six hydrogen‐bonding sites gave rise to high aspect ratio nanoscopic fibrils with helical superstructures but, in contrast to the related macromonomers, did not lead to uniform supramolecular polymers. The UV‐induced topochemical polymerization within the β‐sheet aggregates was successful, proving parallel β‐strand orientation and highlighting the effect of the number and pattern of N‐H???O?C hydrogen‐bonding sites as well as the hydrophobic residue in the molecular structure on the formation of higher structures and reactivity.     

“Reduced” Coumarin Dyes with an O‐Phosphorylated 2,2‐Dimethyl‐4‐(hydroxymethyl)‐1,2,3,4‐tetrahydroquinoline Fragment: Synthesis,Spectra, and STED Microscopy

Large Stokes‐shift coumarin dyes with an O‐phosphorylated 4‐(hydroxymethyl)‐2,2‐dimethyl‐1,2,3,4‐tetrahydroquinoline fragment emitting in the blue, green, and red regions of the visible spectrum were synthesized. For this purpose, N‐substituted and O‐protected 1,2‐dihydro‐7‐hydroxy‐2,2,4‐trimethylquinoline was oxidized with SeO₂ to the corresponding α,β‐unsaturated aldehyde and then reduced with NaBH₄ in a “one‐pot” fashion to yield N‐substituted and 7‐O‐protected 4‐(hydroxymethyl)‐7‐hydroxy‐2,2‐dimethyl‐1,2,3,4‐tetrahydroquinoline as a common precursor to all the coumarin dyes reported here. The photophysical properties of the new dyes (“reduced coumarins”) and 1,2‐dihydroquinoline analogues (formal precursors) with a trisubstituted C=C bond were compared. The “reduced coumarins” were found to be more photoresistant and brighter than their 1,2‐dihydroquinoline counterparts. Free carboxylate analogues, as well as their antibody conjugates (obtained from N‐hydroxysuccinimidyl esters) were also prepared. All studied conjugates with secondary antibodies afforded high specificity and were suitable for fluorescence microscopy. The red‐emitting coumarin dye bearing a betaine fragment at the C‐3‐position showed excellent performance in stimulation emission depletion (STED) microscopy.     

Heat‐Induced Supramolecular Organogels Composed of α‐Cyclodextrin and “Jellyfish‐Like” β‐Cyclodextrin–Poly(ε‐caprolactone)

In general, the complexation and gelation behavior between biocompatible poly(ε‐caprolactone) (PCL) derivatives and α‐cyclodextrin (α‐CD) is extensively studied in water, but not in organic solvents. In this article, the complexation and gelation behavior between α‐CD and multi‐arm polymer β‐cyclodextrin‐PCL (β‐CD‐PCL) with a unique “jellyfish‐like” structure are thoroughly investigated in organic solvent N,N‐dimethylformamide and a new heat‐induced organogel is obtained. However, PCL linear polymers cannot form organogels under the same condition. The complexation is characterized by rheological measurements, DSC, XRD, and SEM. The SEM images reveal that the complexes between β‐CD‐PCL and α‐CD present a novel topological helix porous structure which is distinctly different from the lamellar structure formed by PCL linear polymers and α‐CD, suggesting the unique “jellyfish‐like” structure of β‐CD‐PCL is crucial for the formation of the organogels. This research may provide insight into constructing new supramolecular organogels and potential for designing new functional biomaterials. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part B: Polym. Phys. 2013 , 51, 1598–1606     

Experimental and Theoretical DFT Study on Synthesis of Sterically Crowded 2,3,3,(4)5‐Tetrasubstituted‐4‐nitroisoxazolidines via 1,3‐Dipolar Cycloaddition Reactions Between Ketonitrones and Conjugated Nitroalkenes

1,3‐Dipolar cycloaddition reactions between ketonitrones ( 1a , 1b , 1c , 1d , 1e ) and β‐substituted nitroalkenes ( 2a , 2b , 2c , 2d ) proceed under mild conditions, with complete regioselectivity, and lead with high yields to sterically crowded 2,3,3,5‐tetrasubstituted‐4‐nitroisoxazolidines ( 3a , 3b , 3c , 3d , 3e , 3f ). Reaction course may be interpreted on the basis of nature of local, nucleophile–electrophile interactions. Moreover, density functional theory (DFT) simulations of reaction paths suggest that these reactions should be considered as polar, “one‐step two‐stage” cycloadditions. Subsequently, it has been found that similar reactions between ketonitrones and α‐substituted nitroalkenes do not allow to obtain the expected 2,3,3,4‐tetrasubstituted‐4‐nitroisoxazolidines.     

Anti‐Electrostatic Hydrogen Bonds

Ab initio and hybrid density functional techniques were employed to characterize a surprising new class of H‐bonded complexes between ions of like charge. Representative H‐bonded complexes of both anion–anion and cation–cation type exhibit appreciable kinetic stability and the characteristic theoretical, structural, and spectroscopic signatures of hydrogen bonding, despite the powerful opposition of Coulomb electrostatic forces. All such “anti‐electrostatic” H‐bond (AEHB) species confirm the dominance of resonance‐type covalency (“charge transfer”) interactions over the inessential (secondary or opposing) “ionic” or “dipole–dipole” forces that are often presumed to be essential for numerical modeling or conceptual explanation of the H‐bonding phenomenon.     

pH‐Controlled Formation of a Stable β‐Sheet and Amyloid‐like Fibers from an Amphiphilic Peptide: The Importance of a Tailor‐Made Binding Motif for Secondary Structure Formation

The new amphiphilic peptide 1 is composed of alternating cyclohexyl side chains and guanidiniocarbonyl pyrrole (GCP) groups. In contrast to analogue 2 , which contains lysine instead of the GCP groups and only exists as a random coil owing to charge repulsion, peptide 1 forms a stable β‐sheet at neutral pH in aqueous medium. The weakly basic GCP groups (pK_a≈7) are key for secondary structure formation as they stabilize the β‐sheet through mutual interactions (formation of a “GCP zipper”). The β‐sheets further aggregate into left‐handed helically twisted fibers. However, β‐sheet formation is completely reversible as a function of pH. At low pH (ca. 4), peptide 1 is unstructured (random coil) as all GCP units are protonated. Only round colloidal particles are observed. The amyloid nature of the fibers formed at neutral pH was confirmed by staining experiments with Congo Red and thioflavin T. Furthermore, at millimolar concentrations, peptide 1 forms a stable hydrogel.     

Water‐Enabled Catalytic Asymmetric Michael Reactions of Unreactive Nitroalkenes: One‐Pot Synthesis of Chiral GABA‐Analogs with All‐Carbon Quaternary Stereogenic Centers

Water enables new catalytic reactions for otherwise unreactive substrate systems. Under the “on water” reaction conditions, extremely unreactive β,β‐disubstituted nitroalkenes smoothly underwent enantioselective Michael addition reactions with dithiomalonates using a chiral squaramide catalyst, affording both enantiomers of highly enantioenriched Michael adducts with all‐carbon‐substituted quaternary centers. The developed “on water” protocol was successfully applied for the scalable one‐pot syntheses of chiral GABA analogs with all‐carbon quaternary stereogenic centers at the β‐position, which might show highly interesting pharmaceutical properties.     

Enhancing the Sensitivity of Aptameric Detection of Lysozyme with a “Feed‐Forward” Network of DNA‐Related Reaction Cycles

In this study, a network of DNA‐related reaction cycles was established to enhance the sensitivity of lysozyme detection with dual signal amplification, and aptamer‐based reactions were integrated into this system to provide high specificity. The network was organized in a feed‐forward manner: the “upstream cycles” recognized the lysozyme (the target) and released the “messenger strands” from probe A (a DNA construct); the “downstream cycles” received them and then released the “signal strands” from another DNA construct, probe B, in multiplied quantities to that of the original inputted lysozyme. The upstream cycles centered on “target‐displacement polymerization”, which circulates the lysozyme to provide primary amplification; the downstream cycles centered on “strand‐displacement polymerization”, which circulates the messenger strand to provide further amplification. There were also several “nicking–polymerization” cycles in both reaction groups that provide extra signal amplification. In total, the network enclosed eight interconnected and autonomic reaction cycles, with only two probes, two primers, and two enzymes needed as raw feeds, and the network can be operated simply in one‐pot mode. With this network, lysozyme could be quantified at lysozyme concentrations as low as 2.0×10^?14 M , with a detection limit of 3.6×10^?15 M (3σ rule), which was seven orders of magnitude lower than that obtained without any amplification(1.8×10^?8 M ). Detection of lysozyme in real serum samples confirmed the reliability and practicality of the assay based on this reported reaction network.     

Structural investigation of 3,5‐disubstituted isoxazoles by 1H‐nuclear magnetic resonance

HIV‐1 integrase (IN) is a very promising and validated target for the development of therapeutic agents against AIDS. In an effort to design and synthesize biological isosteric analogs of β‐diketoacid‐containing inhibitors of IN, we prepared a series of substituted isoxazole carboxylic acids. Several of these compounds inhibited catalytic activities of purified IN at micromolar concentration range. With an aim to prepare a large number of analogues based on the isoxazole pharmacophore we focused our study on a series of 3,5‐disubstituted isoxazole isomers. For a rapid structural analysis we discovered a convenient ¹H‐nmr method for distinguishing between isomeric structures based on their H‐4 assignments. This “finger print” approach to isomer identification will be useful in combinatorial chemistry settings where a mixture can be further derivatized.     

A new strategy for synthesis of “umbrella‐like” poly(ethylene glycol) with monofunctional end group for bioconjugation

A novel strategy was used to synthesize poly(ethylene glycol) (PEG) with “umbrella‐like” structure containing a single reactive group at the “handle” of the “umbrella”. 1‐(Bis(2‐hydroxyethyl)amino)‐3‐(1‐ethoxyethoxy)propan‐2‐ol was used to initiate the ring‐opening polymerization (ROP) of ethylene oxide (EO) in the presence of diphenylmethylpotassium (DPMK) to obtain three‐arm PEG (PEG3), then terminated by benzyl bromide or ethyl bromide. The resultant PEG3 was hydrolyzed to generate hydroxyl group at the conjunction point, and the second step ROP of EO was carried out using PEG3‐OH as macroinitiator in the presence of DPMK. The obtained four‐arm PEG (PEG4) contained a functional hydroxyl group at the end of the fourth arm, which could be easily modified to bioactive groups such as carboxyl, active ester, amino, etc. The well‐defined structure of “umbrella‐like” PEG was characterized by GPC, ¹H NMR, and MALDI‐TOF MS in detail. Propionic acid succinimidyl ester of PEG4 (10 kDa) was utilized for protein conjugation with interferon α‐2b. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2010     

An Efficient Synthesis of 4‐Arylmethylidene‐3‐substituted‐Isoxazol‐5(4H)‐ones in Aqueous Medium

A series of 4‐arylmethylidene‐3‐substituted‐isoxazol‐5(4H)‐ones were efficiently synthesized by eco‐benign, one pot uncatalyzed reaction of β‐keto‐ester, hydroxylamine hydrochloride, and aromatic aldehyde with electron donating substituent in water.     

Synthesis of Novel Core Cross‐Linked Star‐Based Polyrotaxane End‐Capped via “CuAAC” Click Chemistry

The first example of core cross‐linked star (CCS) polyrotaxane was prepared using the poly(ϵ‐caprolactone) (PCL) CCS three‐dimensional (3D) scaffold. The 3D CCS polymer was firstly prepared through the “arm‐first” approach. Then, the “arms” of the resultant PCL CCS polymer were threaded with α‐cyclodextrins (α‐CDs). The threaded α‐CDs were permanently locked by the “click” reaction of terminal alkyne functionalities of the star polymers with the azide‐functionalized end caps to afford the CCS polyrotaxanes. All analytical results confirm the formation of the CCS polyrotaxanes and reveal their characteristics, including fluorescence under UV, a channel‐type crystalline structure, a two‐step thermal decomposition, and a unique core‐shell structure in great contrast to the polymer precursors.     

Chemical Synthesis of Burkholderia Lipid A Modified with Glycosyl Phosphodiester‐Linked 4‐Amino‐4‐deoxy‐β‐L‐arabinose and Its Immunomodulatory Potential

Modification of the Lipid A phosphates by positively charged appendages is a part of the survival strategy of numerous opportunistic Gram‐negative bacteria. The phosphate groups of the cystic fibrosis adapted Burkholderia Lipid A are abundantly esterified by 4‐amino‐4‐deoxy‐β‐L ‐arabinose (β‐L ‐Ara4N), which imposes resistance to antibiotic treatment and contributes to bacterial virulence. To establish structural features accounting for the unique pro‐inflammatory activity of Burkholderia LPS we have synthesised Lipid A substituted by β‐L ‐Ara4N at the anomeric phosphate and its Ara4N‐free counterpart. The double glycosyl phosphodiester was assembled by triazolyl‐tris‐(pyrrolidinyl)phosphonium‐assisted coupling of the β‐L ‐Ara4N H‐phosphonate to α‐lactol of β(1→6) diglucosamine, pentaacylated with (R)‐(3)‐acyloxyacyl‐ and Alloc‐protected (R)‐(3)‐hydroxyacyl residues. The intermediate 1,1′‐glycosyl‐H‐phosphonate diester was oxidised in anhydrous conditions to provide, after total deprotection, β‐L ‐Ara4N‐substituted Burkholderia Lipid A. The β‐L ‐Ara4N modification significantly enhanced the pro‐inflammatory innate immune signaling of otherwise non‐endotoxic Burkholderia Lipid A.     

Antiparallel Self‐Association of a γ,α‐Hybrid Peptide: More Relevance of Weak Interactions

To learn how a preorganized peptide‐based molecular template, together with diverse weak non‐covalent interactions, leads to an effective self‐association, we investigated the conformational characteristics of a simple γ,α‐hybrid model peptide, Boc‐γ‐Abz‐Gly‐OMe. The single‐crystal X‐ray diffraction analysis revealed the existence of a fully extended β‐strand‐like structure stabilized by two non‐conventional C?H???O=C intramolecular H‐bonds. The 2D ¹H NMR ROESY experiment led us to propose that the flat topology of the urethane‐γ‐Abz‐amide moiety is predominantly preserved in a non‐polar environment. The self‐association of the energetically more favorable antiparallel β‐strand‐mimic in solid‐state engenders an unusual ‘flight of stairs’ fabricated through face‐to‐face and edge‐to‐edge Ar???Ar interactions. In conjunction with FT‐IR spectroscopic analysis in chloroform, we highlight that conformationally semi‐rigid γ‐Abz foldamer in appositely designed peptides may encourage unusual β‐strand or β‐sheet‐like self‐association and supramolecular organization stabilized via weak attractive forces.     

Investigations of the reactivity of “kick‐started” oxetanes in photoinitiated cationic polymerization

The ability of certain alkyl substituted epoxides to accelerate the photoinitiated cationic ring‐opening polymerizations of oxetane monomers by substantially reducing or eliminating the induction period altogether has been termed by us “kick‐starting.” In this communication, the rates of photopolymerization of several model “kick‐started” oxetane systems were quantified and compared with the analogous biscycloaliphatic epoxide monomer, 3,4‐epoxycyclohexylmethyl 3′,4′‐epoxycyclohexanecarboxylate (ERL). It has been found that the “kick‐started” systems undergo photopolymerization at rates that are at least two‐fold faster than ERL. These results suggest that “kick‐started” oxetanes could replace ERL in many applications in which high speed ultraviolet induced crosslinking photopolymerizations are carried out. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2015 , 53, 586–593     

Tetraphenylethylene‐based Glycoconjugate as a Fluorescence “Turn‐On” Sensor for Cholera Toxin

Tetraphenylethylene (TPE)‐based glycoconjugates were easily synthesized by copper(I)‐catalyzed “click reactions” between propargyl‐attached TPE and azido‐functionalized sugars. The TPE compound bearing lactosyl moieties ( Lac‐TPE ) was found to be a fluorescence “turn‐on” sensor for cholera toxin by virtue of aggregation‐induced emission characteristics of the TPE motif owing to the specific interaction of lactose with the cholera toxin B subunit, whilst a cellobiose‐functionalized TPE derivative did not show any response to the toxin. Therefore, Lac‐TPE shows promising applications in the detection of cholera toxin, as well as in the investigation of carbohydrate–protein interaction.     

Computational Insights into the Charge Relaying Properties of β‐Turn Peptides in Protein Charge Transfers

Density functional theory calculations suggest that β‐turn peptide segments can act as a novel dual‐relay elements to facilitate long‐range charge hopping transport in proteins, with the N terminus relaying electron hopping transfer and the C terminus relaying hole hopping migration. The electron‐ or hole‐binding ability of such a β‐turn is subject to the conformations of oligopeptides and lengths of its linking strands. On the one hand, strand extension at the C‐terminal end of a β‐turn considerably enhances the electron‐binding of the β‐turn N terminus, due to its unique electropositivity in the macro‐dipole, but does not enhance hole‐forming of the β‐turn C terminus because of competition from other sites within the β‐strand. On the other hand, strand extension at the N terminal end of the β‐turn greatly enhances hole‐binding of the β‐turn C terminus, due to its distinct electronegativity in the macro‐dipole, but does not considerably enhance electron‐binding ability of the N terminus because of the shared responsibility of other sites in the β‐strand. Thus, in the β‐hairpin structures, electron‐ or hole‐binding abilities of both termini of the β‐turn motif degenerate compared with those of the two hook structures, due to the decreased macro‐dipole polarity caused by the extending the two terminal strands. In general, the high polarity of a macro‐dipole always plays a principal role in determining charge‐relay properties through modifying the components and energies of the highest occupied and lowest unoccupied molecular orbitals of the β‐turn motif, whereas local dipoles with low polarity only play a cooperative assisting role. Further exploration is needed to identify other factors that influence relay properties in these protein motifs.     

Site‐Selective Approach to β‐Fluorination: Photocatalyzed Ring Opening of Cyclopropanols

To expand upon the recent pioneering reports of catalyzed sp³ C?H fluorination methods, the next rational step is to focus on directing “radical‐based fluorination” more effectively. One potential solution entails selective C?C bond activation as a prelude to selective fluorination. Herein, we report the tandem photocatalyzed ring‐opening/fluorination reactions of cyclopropanols by 1,2,4,5‐tetracyanobenzene (TCB) and Selectfluor to afford a process tantamount to site‐selective β‐fluorination of carbonyl‐containing compounds. This new approach provides a synthetically mild and operationally simple route to otherwise difficult‐to‐prepare β‐fluorinated products in good yields and with good‐to‐excellent regioselectivity. Remarkably, substrates that contain other usually reactive (e.g., benzylic) sites undergo ring‐opening fluorination preferably. The versatility of this method to give cyclic β‐fluorides from tertiary cyclopropanols and γ‐fluoro alcohols is also highlighted.