Increasing the Antigenicity of Synthetic Tumor‐Associated Carbohydrate Antigens by Targeting Toll‐Like Receptors

Synthetic cancer vaccines : A number of fully synthetic vaccine candidates have been designed, chemically synthesized, and immunologically evaluated to establish a strategy to overcome the poor immunogenicity of tumor‐associated carbohydrates and glycopeptides and to determine the importance of Toll‐like receptor (TLR) engagement for antigenic responses against these compounds.

     

Chemical and Enzymatic Synthesis of Modified Sialyl Lewis X Tetrasaccharides with High Affinity for E and P‐Selectin

A series of sialyl‐Lewis X tetrasaccharide analogs 1a — d was prepared using a combined chemical and enzymatic approach. Sialic acid analogs 5b , c were obtained from 2‐azido mannose 4c or 2‐deoxy mannose 4b and pyruvate by an aldolase reaction and converted to the protected thioglycosides 3b , c that served as sialyl donors for the Lewis X acceptor trisaccharide 2 . The resulting sialyl‐Lewis X tetrasaccharides 8a — c were deprotected by deacylation and saponification of the methyl ester. Debenzylation was achieved by careful transfer hydrogenation in the presence of formic acid or ammonium formate as a hydrogen source. Three sialyl‐Lewis X derivatives 1a — c were thus obtained and the parent compound 1a was further modified by alkaline hydrolysis of the two acetamides to give the lyso sialyl‐Lewis X derivative 1d . The four sialyl‐Lewis X tetrasaccharides 1a — d were tested for their binding affinity to E and P‐selectin with the lyso sialyl‐Lewis X derivative 1d showing the highest inhibitory potency for both lectins.     

From Type I to Type II: Design,Synthesis, and Characterization of Potent Pyrazin‐2‐ones as DFG‐Out Inhibitors of PDGFRβ

Reversible protein kinase inhibitors that bind in the ATP cleft can be classified as type I or type II binders. Of these, type I inhibitors address the active form, whereas type II inhibitors typically lock the kinase in an inactive form. At the molecular level, the conformation of the flexible activation loop holding the key DFG motif controls access to the ATP site, thereby determining an active or inactive kinase state. Accordingly, type I and type II kinase inhibitors bind to so‐called DFG‐in or DFG‐out conformations, respectively. Based on our former study on highly selective platelet‐derived growth factor receptor β (PDGFRβ) pyrazin‐2‐one type I inhibitors, we expanded this scaffold toward the deep pocket, yielding the highly potent and effective type II inhibitor 5 (4‐[(4‐methylpiperazin‐1‐yl)methyl]‐N‐[3‐[[6‐oxo‐5‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐3‐yl]methyl]phenyl]benzamide). In vitro characterization, including selectivity panel data from activity‐based assays (300 kinases) and affinity‐based assays (97 kinases) of these PDGFRβ type I ( 1 ; 5‐(4‐hydroxy‐3‐methoxy‐phenyl)‐3‐(3,4,5‐trimethoxyphenyl)‐1H‐pyrazin‐2‐one) and II ( 5 ) inhibitors showing the same pyrazin‐2‐one chemotype are compared. Implications are discussed regarding the data for selectivity and efficacy of type I and type II ligands.     

Molecular Basis for the Stereoselective Ammoniolysis of N‐Alkyl Aziridine‐2‐Carboxylates Catalyzed by Candida antarctica Lipase B

Candida antarctica lipase B catalyzed the stereoselective ammoniolysis of N‐alkyl aziridine‐2‐carboxylates in tBuOH saturated with ammonia and yielded the (2S)‐aziridine‐2‐carboxamide and unreacted (2R)‐aziridine‐2‐carboxylate. Varying the N‐1 substituent on the aziridine ring changed the rate and stereoselectivity of the reaction. Substrates with a benzyl substituent or a (1′R)‐1‐phenylethyl substituent reacted approximately ten times faster than substrates with a (1′S)‐1‐phenylethyl substituent. Substrates with a benzyl substituent showed little stereoselectivity (E=5–7) while substrates with either a (1′R)‐ or (1′S)‐1‐phenylethyl substituent showed high stereoselectivity (D>50). Molecular modeling by using the current paradigm for enantioselectivity—binding of the slow enantiomer by an exchange‐of‐substituents orientation—could not account for the experimental results. However, modeling an umbrella‐like‐inversion orientation for the slow enantiomer could account for the experimental results. Steric hindrance between the methyl in the (1′S)‐1‐phenylethyl substituent and Thr138 and Ile189 in the acyl‐binding site likely accounts for the slow reaction. Enantioselectivity likely stems from an unfavorable interaction of the methine hydrogen with Thr40 for the slow enantiomer and from subtle differences in the orientations of the other three substituents. This success in rationalizing the enantioselectivity supports the notion that an umbrella‐like‐inversion orientation can contribute to enantioselectivity in lipases.     

Synthesis,Pharmacological Evaluation,and Docking Studies of Novel Pyridazinone‐Based Cannabinoid Receptor Type 2 Ligands

In recent years, cannabinoid type 2 receptors (CB₂R) have emerged as promising therapeutic targets in a wide variety of diseases. Selective ligands of CB₂R are devoid of the psychoactive effects typically observed for CB₁R ligands. Based on our recent studies on a class of pyridazinone 4‐carboxamides, further structural modifications of the pyridazinone core were made to better investigate the structure–activity relationships for this promising scaffold with the aim to develop potent CB₂R ligands. In binding assays, two of the new synthesized compounds [6‐(3,4‐dichlorophenyl)‐2‐(4‐fluorobenzyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2,3‐dihydropyridazine‐4‐carboxamide ( 2 ) and 6‐(4‐chloro‐3‐methylphenyl)‐cis‐N‐(4‐methylcyclohexyl)‐3‐oxo‐2‐pentyl‐2,3‐dihydropyridazine‐4‐carboxamide ( 22 )] showed high CB₂R affinity, with K_i values of 2.1 and 1.6 nm , respectively. In addition, functional assays of these compounds and other new active related derivatives revealed their pharmacological profiles as CB₂R inverse agonists. Compound 22 displayed the highest CB₂R selectivity and potency, presenting a favorable in silico pharmacokinetic profile. Furthermore, a molecular modeling study revealed how 22 produces inverse agonism through blocking the movement of the toggle‐switch residue, W6.48.     

Synthesis of Functionalized Indolizines by Lewis Acid‐Mediated Cyclocondensation of 3‐(Pyridin‐2‐yl)‐propiolates with Enones

Functionalized indolizines were prepared by Lewis acid‐catalyzed cyclizations of 3‐(pyridin‐2‐yl)‐propiolates with enones. This new type of reaction provides a convenient and regioselective approach to ester‐substituted indolizine derivatives which are not readily available by other methods. Based on density function theory (DFT) calculations, a mechanism of the reaction has been suggested.     

Phenylalanine Ammonia‐Lyase‐Catalyzed Deamination of an Acyclic Amino Acid: Enzyme Mechanistic Studies Aided by a Novel Microreactor Filled with Magnetic Nanoparticles

Phenylalanine ammonia‐lyase (PAL), found in many organisms, catalyzes the deamination of l ‐phenylalanine (Phe) to (E)‐cinnamate by the aid of its MIO prosthetic group. By using PAL immobilized on magnetic nanoparticles and fixed in a microfluidic reactor with an in‐line UV detector, we demonstrated that PAL can catalyze ammonia elimination from the acyclic propargylglycine (PG) to yield (E)‐pent‐2‐ene‐4‐ynoate. This highlights new opportunities to extend MIO enzymes towards acyclic substrates. As PG is acyclic, its deamination cannot involve a Friedel–Crafts‐type attack at an aromatic ring. The reversibility of the PAL reaction, demonstrated by the ammonia addition to (E)‐pent‐2‐ene‐4‐ynoate yielding enantiopure l ‐PG, contradicts the proposed highly exothermic single‐step mechanism. Computations with the QM/MM models of the N‐MIO intermediates from l ‐PG and l ‐Phe in PAL show similar arrangements within the active site, thus supporting a mechanism via the N‐MIO intermediate.     

Understanding the Key Factors that Control the Inhibition of Type II Dehydroquinase by (2R)‐2‐Benzyl‐3‐dehydroquinic Acids

The binding mode of several substrate analogues, (2R)‐2‐benzyl‐3‐dehydroquinic acids 4 , which are potent reversible competitive inhibitors of type II dehydroquinase (DHQ2), the third enzyme of the shikimic acid pathway, has been investigated by structural and computational studies. The crystal structures of Mycobacterium tuberculosis and Helicobacter pylori DHQ2 in complex with one of the most potent inhibitor, p‐methoxybenzyl derivative 4 a , have been solved at 2.40 Å and 2.75 Å, respectively. This has allowed the resolution of the M. tuberculosis DHQ2 loop containing residues 20–25 for the first time. These structures show the key interactions of the aromatic ring in the active site of both enzymes and additionally reveal an important change in the conformation and flexibility of the loop that closes over substrate binding. The loop conformation and the binding mode of compounds 4 b – d has been also studied by molecular dynamics simulations, which suggest that the benzyl group of inhibitors 4 prevent appropriate orientation of the catalytic tyrosine of the loop for proton abstraction and disrupts its basicity.     

Synthesis of 2H‐Thiopyrans by Rhodium(II) Acetate‐Catalyzed Reaction of 4‐Amino‐2,5‐dihydro‐3‐thiophenecarbonitriles with α‐Diazocarbonyl Compounds. Part 1

4‐Amino‐2,5‐dihydro‐3‐thiophenecarbonitriles 1 reacted with dimethyl diazomalonate in the presence of rhodium(II) acetate to give regioselectively 4‐cyano‐2H‐thio‐pyrans 2 (C ₂— S insertion), and 5‐cyano‐2H‐thiopyrans (C ₅— S insertion) were not isolated. Similar insertion was also observed in the reaction of 1 with methyl diazoacetoacetate and ethyl diazobenzoylacetate. The starting compounds 1 were synthesized by the reaction of tetrahydro‐4‐oxo‐3‐thiophene‐carbonitrile with morpholine, piperidine, and pyrrolidine in the presence of formic acid in ethanol.