Preparations and Biological Properties of Chiral Compounds

Enantiomeri nekih kiralnih spojeva mogu pokazivati različita biološka svojstva kao što su farmakološka učinkovitost ili toksičnost. Stoga priprava enantiomerno čistih spojeva sve više dobiva na važnosti, posebice u farmaceutskoj industriji. Laboratorijskom sintezom u pravilu nastaje racemična smjesa nekog kiralnog spoja (enantiomerni par), dok u biološkim sustavima nastaje samo jedan enantiomer, kao kod aminokiselina, šećera ili lipida. U radu su opisani osnovni pojmovi o kiralnosti molekula. Prikazane su metode za pripravu enantiomerno čistih spojeva koje se temelje na kiralnim svojstvima enzima i proteina. Pobliže su opisane sinteze enantiomera enzimski kataliziranim reakcijama visoke stereoselektivnosti, i to hidrolitičkim, oksidacijskim i redukcijskim biopretvorbama. Naveden je primjer poboljšanja stereoselektivnosti enzimske reakcije promjenama pojedinih aminokiselina u aktivnom centru lipaze. Opisane su također kromatografske metode, kapilarna elektrokromatografija i tekućinska kromatografija visoke učinkovitosti, s proteinima kao kiralnim izbornicima kojima se razdvajaju enantiomeri iz racemične smjese. U radu su navedeni primjeri različitog biološkog djelovanja enantiomera nekih kiralnih spojeva.Enantiomers of chiral compounds may express various biological activities and also different toxicities. Examples of different pharmacological effects of some chiral drugs such as fluoxetine, penicillamine, ibuprofen and albuterol are provided in this paper. Due to possible differences in activity, the chiral drugs are required to be pure enantiomeric compounds in order to be more effective and safer to use. In the laboratory, enantiomers are mainly synthesized as racemates (an equimolar mixture of enantiomers) while in biological pathways only one enantiomeric form is produced, such as amino acids, sugars and lipids. This paper presents the principles of chirality, general information about enantiomers and their biological aspects. It gives an outline of stereoselective methods for chromatographic resolution of enantiomers with stereoselective protein stationary phases, i.e. capillary electrochromatography (CEC) and high performance liquid chromatography (HPLC). The use of enzyme biotransformations (hydrolysis, oxidation and reduction) in chiral syntheses of carboxyl-, phosphoryl- or β-hydroxy esters, alcohols, epoxides and cis-carboxyl sulphoxide is described. This article also includes an example of lipase stereoselectivity improvement by amino acid mutations within the enzyme active site

50. godišnjica osnutka baze struktura bioloških makromolekula

Baze podataka važne su zbog objedinjenog prikaza informacija prema određenom ključu. Javno dostupne baze podataka imaju dodatnu vrijednost zbog olakšanog pristupa znanju i informacijama. U članku je opisana baza proteinskih struktura, Protein Data Bank – PDB, njezino osnivanje, skromni početak te vrlo važan status koji ima danas. Posebno je naglašen dio baze posvećen profesorima i učenicima odnosno studentima da bi lakše pristupali podatcima u bazi te ih primjenjivali u svojem radu ili obrazovanju

Exploring the Active Sites of Cholinesterases by Inhibition with Bambuterol and Haloxon

The paper describes the inhibition of mouse acetylcholinesterase (AChE; EC 3.1.1.7) and mouse, human, and horse butyrylcholinesterase (BChE; EC 3.1.1.8) by 5-[2-(tert-butylamino)-1-hydroxyethyl]-m-phenylene-bis(dimethylcarbamate) hydrochloride (bambuterol) and by O,O-bis-(2-chloroethyl)-O-(3-chloro-4-methylcoumarin-7-yl) phosphate (haloxon). The haloxon inhibition rate constant (ki) for mouse BChE was 3.7 × 107 min–1 mol–1 dm3, which was 40-fold higher than the rate constant for mouse AChE. Bambuterol inhibition of horse BChE (ki = 2.1 × 105 min–1 mol–1 dm3) was about 25-fold slower than that of human or mouse BChE, whereas the respective haloxon inhibition of horse BChE (ki = 1.2 × 107 min–1 mol–1 dm3) was about 2-3-fold slower. Sequence alignments and the computational model of the three-dimensional structure of horse BChE suggest that residues inside the active site at positions 69, 277 and 285 are important for the differences in the inhibition of these three BChE species

Usporedba protokola za mjerenje aktivnosti kolinesteraza u ljudskoj krvi Ellmanovom metodom

This paper presents a protocol for routine assays of human blood cholinesterase activities which separates erythrocytes from plasma by centrifugation and measures acetylcholinesterase activity in unwashed erythrocytes and butyrylcholinesterase activity in the plasma. The recommended substrate for both enzymes is 1.0 mM acetylthiocholine. The protocol is compared with other two recommended protocols for the activity measurements of the two enzymes using the Ellman method. The paper discusses the advantages and disadvantages of each and concludes with a proposal for an international agreement between laboratories for the evaluation of a standardized protocol.Mjerenjem aktivnosti acetilkolinesteraze i butirilkolinesteraze u krvi može se u osoba izloženih organofosfornim spojevima i karbamatima odrediti apsorpcija tih spojeva. Za mjerenje aktivnosti butirilkolinesteraze u ljudskoj plazmi postoji standardizirani protokol usvojen u velikom broju kliničkih i toksikoloških laboratorija, dok za mjerenje aktivnosti eritrocitne acetilkolinesteraze ima nekoliko predloženih protokola, ali ni jedan još nije opće privaćen. Zajednički princip svih predloženih protokola je spektrofotometrijska Ellmanova metoda, koja za mjerenje aktivosti kolinesteraza rabi tiokolinske supstrate. Autori ovoga rada predlažu jednostavni protokol u kojem se zasebno mjere aktivnosti u plazmi i u eritrocitima, koji nisu prani, a koji se odvoje centrifugiranjem. Aktivnost u eritrocitima mjeri se nakon hemolize eritrocita smrzavanjem. MjerI se samo s 1,0 mM acetiltiokolinom. Drugi autori mjere aktivnost u punoj krvi koristeći se dvama supstratima (acetiltiokolinom i butiriltiokolinom), koji se u hemoliziranu krv dodaju sukcesivno i nakon svakog dodavanja supstrata mjeri se aktivnost. Neki autori predlažu uporabu etopropazina, specifičnog inhibitora butirilkolinesteraze, pa se mjerenje vrši u prisutnosti i odsutnosti toga inhibitora. U ovome su radu opisane prednosti i nedostaci pojedinog protokola te izvori mogućih pogrešaka u predloženim postupcima. Za usvajanje standardiziranog protokola za mjerenje aktivnosti eritrocitne kolinesteraze potreban je dogovor autora predloženih protokola nakon čega bi trebala uslijediti međulaboratorijska provjera toga protokola

Resorcinol-, catechol- and saligenin-based bronchodilating beta2-agonists as inhibitors of human cholinesterase activity

We investigated the influence of bronchodilating 2-agonists on the activity of human acetylcholinesterase (AChE) and usual, atypical and fluoride-resistant butyrylcholinesterase (BChE). We determined the inhibition potency of racemate and enantiomers of fenoterol as a resorcinol derivative, isoetharine and epinephrine as catechol derivatives, and salbutamol and salmeterol as saligenin derivatives. All of the tested compounds reversibly inhibited cholinesterases with Ki constants ranging from 9.8 µM to 6.4 mM, and had the highest inhibition potency toward usual BChE, but generally none of the cholinesterases displayed any stereoselectivity. Kinetic and docking results revealed that the inhibition potency of the studied compounds could be related to the size of the hydroxyaminoethyl chain on the benzene ring. The additional - interaction of salmeterol’s benzene ring and Trp286 and hydrogen bond with His447 probably enhanced inhibition by salmeterol which was singled out as the most potent inhibitor of all the cholinesterases

4-Aminoqionolines as reversible inhibitors of human cholinesterase activity

We synthesised eight derivatives of 4-aminoquinolines differing in the substituents attached to the C(4)-amino group and C(7) carbon of 4-aminoquinoline, and tested their potency to inhibit human AChE and BChE. All of the compounds reversibly inhibited both enzymes with dissociation inhibition (Ki) constants from 0.50 to 50 µM exhibiting selectivity. In other words, for all compounds, AChE exhibited higher affinity than BChE. The most potent inhibitors of AChE were compounds with an octyl chain or adamantane, regardless of the group in position C(7). The shortening of the chain length caused the AChE inhibition decrease by 5-20 times. Docking studies made it clear that the high AChE affinity resulted from simultaneous interactions of the quinoline group with aromatic residues of both the catalytic active site and the peripheral site. In conclusion, the inhibition potency and selectivity classify several novel compounds as leads for further modification and optimization towards the development of new inhibitors of AChE and potential drugs for treatment of neurodegenerative diseases.13th International Meeting on Cholinesterases and and the 7th International Conference on Paraoxonases, 9th to 14th of September 2018 Hradec Králové, Czech Republi

Inhibition of Human Blood Acetylcholinesterase and Butyrylcholinesterase by Ethopropazine

Inhibition of human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and serum butyrylcholinesterase (BChE; EC 3.1.1.8) by ethopropazine, 10-(2-diethylaminopropyl)phenothiazine hydrochloride, was measured with acetylthiocholine (ATCh) as substrate. Dissociation constants for the enzyme-inhibitor complexes were calculated from the effect of ATCh concentration on the apparent dissociation constants by applying non-linear regression to fit the model to experimental data. Inhibition of AChE revealed a competitive inhibition for two binding sites (Ka = 161 and Ki = 393 μmol dm-3), inhibition of the atypical BChE was non-competitive (Ki = 7.5 μmol dm-3) while that of the usual BChE was competitive (K(I) = 0.16 μmol dm-3). At the ethopropazine concentration of 20 μmol dm-3 and the acetyl-thiocholine concentration of 1.0 mmol dm-3 (conditions used for differentiation between AChE and BChE activities), the erythrocyte AChE was 8% inhibited and the BChE phenotypes UU, UA, FF/FS, AF, AJ/AK and AA/AS between 98% and 74%