Mimics of Small Ribozymes Utilizing a Supramolecular Scaffold

For elucidating the mechanism of the general acid/base catalysis of the hydrolysis of RNA phosphodiester bonds, a number of cleaving agents having two cyclen moieties tethered to a 1,3,5-triazine core have been prepared and their ability to bind and cleave UpU studied over a wide pH range. Around neutral pH, the cleaving agents form a highly stable ternary complex with UpU and ZnII through coordination of the uracil N3 and the cyclen nitrogen atoms to the ZnII ions. Under conditions where the triazine core exists in the deprotonated neutral form, hydrolysis of UpU, but not of ApA, is accelerated by approximately two orders of magnitude in the presence of the cleaving agents, suggesting general base rather than metal ion catalysis. The probable mechanism of the observed catalysis and implications to understanding the general acid/base-catalyzed phosphodiester hydrolysis by ribozymes are discussed

Control of structure, stability and catechol oxidase activity of copper(II) complexes by the denticity of tripodal platforms

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Biokomplex – szérum protein kölcsönhatások vizsgálata = Biocomplex – serum protein interactions

Antidiabetikus fémkomplexek Modelszámításokkal igazoltuk, a V(IV) és V(V) számára a transzferrin at elsődleges fémionkötőhely a vérszérumban. Mindkét fémiont képes kiszorítani komplexeiből, és bár az eredeti VO(IV) komplexek kötődnek egy másik szérumfehérjéhez az albuminhoz is, ez a köcsönhatás nem elég erős ahhoz, hogy megakadályozza a komplexek elbomlását. Mindez alátámasztja, hogy a fémion önmagában az aktív metabolit, és komplexképződéssel aktivitását nem lehet befolyásolni, legfeljebb a felszívódás hatékonyságát elősegíteni. Hasonló modellszámítások alapján megállapítottuk, hogy a Humán szérumalbumin a legfontosabb Zn(II) ion kötőhely a vérszérumban. ugyanazen fehérje képes megkötni számos ligandumot önmagában is. Rákellenes tioszemikarbazon komplexek Bár a triapin meglehetősen stabilis komplexek képez Fe(III)-mal, Ga(III) komplexe híg vizes oldatban jelentős mértékben hidrolizál. A stabiliás növekedést sikerül elérni szalicilaldehid alkalmazásával, ebben az esetben azonban a biológiai aktivitás csökken, ennek oka lehet a a hatásmechanizmus szempontjából fontos Fe(II)-komplexek kisebb stabilitása. Ruténium(II/III) komplexek A Ru(III)edta és Ru(II)-p-cimol bidentát ligandumokkal alkot terner komplexeinek stabilitása jelentősen növekszik ha a ligandum (S,O) és nem (O,O) vagy (N,O) donorcsoportokat tartalmaz. Ezt mindenképp érdemes figyelembe venni a gyógyszertervezési és a lehetséges biotranszformációs folyamatok esetében. | Antidiabetic metal complexes Based on model calculations, it can be stated that in the human serum transferrin is the primary V(IV,V) binder. The protein is able to displace the original carrier ligands for both meal ions. The other serum protein, albumin is also able to bind the V(IV,V) complexes, this interaction is not strong enough to prevent the dissociation of the complexes due to binding to transferrin. All these facts confirm that the metal ion itself is the active metabolite. By complex formation it’s activity cannot be tuned only the bioavailability can be increased. Similar model calculations confirmed that in the case of Zn(II) albumin (HSA) is the primary binder in the human serum. The same protein is able to bind the carrier ligands too. Anticancer tiosemicarbazone complexes Triapin forms high stability complexes with both Fe(II, III), but its Ga(III) complexes hydrolyze extensively in dilute water solutions. We were able to increase the stability by using salicilaldehyde-tiosemicarbazone, however in this case the anticancer activity decreased, probably due to the decreased stability of the Fe(II) complex, which has an important role in the mechanism of action. Ruthenium(II/III) complexes Ru(III)edta and Ru(II)p-cimol terner complexes with bidentate ligands are much more stable in the case of (S,O) type ligands as (O,O) or (N,O) type ligands. These facts should be considered during drug design and their potential biotransformation processes

On the possible roles of N-terminal His-rich domains of Cu,Zn SODs of some Gram-negative bacteria

The Cu,Zn superoxide dismutases (Cu,Zn SOD) isolated from some Gram-negative bacteria possess a His-rich N-terminal metal binding extension. The N-terminal domain of Haemophilus ducreyi Cu,Zn SOD has been previously proposed to play a copper(II)-, and may be a zinc(II)- chaperoning role under metal ion starvation, and to behave as a temporary (low activity) superoxide dismutating center if copper(II) is available. The N-terminal extension of Cu,Zn SOD from Actinobacillus pleuropneumoniae starts with an analogous sequence (HxDHxH), but contains considerably fewer metal binding sites. In order to study the possibility of the generalization of the above mentioned functions over all Gram-negative bacteria possessing His-rich N-terminal extension, here we report thermodynamic and solution structural analysis of the copper(II) and zinc(II) complexes of a peptide corresponding to the first eight amino acids (HADHDHKK-NH2, L) of the enzyme isolated from A. pleuropneumoniae. In equimolar solutions of Cu(II)/Zn(II) and the peptide the MH2L complexes are dominant in the neutral pH-range. L has extraordinary copper(II) sequestering capacity (KD,Cu = 7.4×10–13 M at pH 7.4), which is provided only by non-amide (side chain) donors. The central ion in CuH2L is coordinated by four nitrogens {NH2,3Nim} in the equatorial plane. In ZnH2L the peptide binds to zinc(II) through a {NH2,2Nim,COO–} donor set, and its zinc binding affinity is relatively modest (KD,Zn = 4.8×10–7 M at pH 7.4). Consequently, the presented data do support a general chaperoning role of the N-terminal His-rich region of Gram-negative bacteria in copper(II) uptake, but do not confirm similar function for zinc(II). Interestingly, the complex CuH2L has very high SOD-like activity, which may further support the multifunctional role of the copper(II)-bound N-terminal His-rich domain of Cu,Zn SODs of Gram-negative bacteria. The proposed structure for the MH2L complexes have been verified by semiempirical quantum chemical calculations (PM6), too

Bio-ihletésű fémkötő peptidek alkalmazása a metalloenzimek funkcionális modellezésénél = Bioinspired metallopeptides for the functional mimicking of metalloenzymes

Munkánk során számos metalloprotein/enzim fémkötő szekvenciájával azonos oligopeptidet állítottunk elő és vizsgáltuk azok fémkötő és enzimutánzó sajátságait. Az eredmények hozzájárultak a vizsgált fehérjék (humán endostatin, humán hisztidin-gazdag glikoprotein (HRG), két Gram-negatív patogén baktérium Cu,Zn-SOD-ja) tulajdonságainak, működésének, fémion-felvételi folyamatainak jobb megértéséhez. Az általunk korábban és a jelen projektben vizsgált fémkötő oligopeptidek sajátságainak ismeretében több peptidalapú, biomimetikus enzim modellt is terveztünk, melyek utánozni képesek a metalloproteinek aktív centrumában kialakuló fémion környezetet. E modellek közül több is hatékonynak mutatkozott mind hidrolitikus, mind oxidatív folyamatok katalízisére. Fentiek mellett több, nem-peptid típusú többmagvú fémkomplexek kialakítására alkalmas ligandumot vizsgáltunk, melyek hatékonyan és (bázis)szelektíven képesek foszforsavmonoészterek és az RNS hidrolízisét elősegíteni. | During our work we prepared a number of oligopeptides with sequences identical to the meta binding sites of some selected metalloproteines/enzymes, and we studied their interactions with metal ions, as well as the enzyme mimicking ability of their metal complexes. Our results contributed to the understanding the properties, the catalytic mechanisms and metal uptake processes of the mimicked proteins (human endostatin, human histidine-rich glycoprotein (HRG), two Gram-negative patogen bacterial Cu,Zn-SOD). Based on the metal binding properties of the peptides studied by us (earlier or in this project) we designed some peptide based, biomimetic enzyme models, which are able to mimic the metal ion environment in the active sites of metalloenzymes. Some of these models have notable activity in promoting both hydrolytic and oxidative reactions. Besides, we designed several non-peptide based, dinucleating ligands, too. The Cu(II) and Zn(II) complexes of these ligands are able to promote (base)selective hydrolysis of both phosphomonoesters and RNAs

Increasing the histidine 'density' in tripodal peptides by gradual N-functionalization of tris(2-aminoethyl)amine (tren) with l-histidyl units: The effect on zinc(II) complexes

Tripodal peptidomimetics have received increasing interest among others as efficient metal ion chelators. Most of these studies have focused on symmetrical, tri-substituted ligands. Our aim was to establish how the increasing donor group 'density', i.e. the gradual N-histidyl substitution, alters the coordination chemical properties of the tripodal platform. To this end we synthesized mono-, bis- and tris(l-histidyl)-functionalized tren derivatives (L1, L2 and L3, respectively), and studied their zinc(II) complexes by pH potentiometry, 1H NMR and MS spectroscopy. The three ligands provide a variety of donor sites, and consequently different stability and structure for their zinc(II) complexes depending on the pH and metal-to-ligand ratios. In the neutral pH range histamine-like coordination is operating in all cases. Due to the formation of macrochelate between the two/three (Nim,NH2) binding sites, L2 and L3 have considerably higher zinc(II) binding ability than histamine, or any other simple peptide with N-terminal His unit. The situation is fundamentally different at higher pH. The tren-like subunit in L1 acts as an anchoring site for amide deprotonation, and the (3NH2,N-,Ntert) type coordination, a rare example where zinc(II)-amide N- coordination takes place, results in outstanding stability. Although L1 provides tight binding above pH 7, it forms only mononuclear species. However, the increasing level of functionalization in L2 and L3 allows the formation of oligonuclear complexes, and at threefold zinc(II) excess the three ligands share nearly the same amount of zinc(II). Moreover, the high histidine 'density' in L2 and L3 also provides the formation of imidazolato-bridged structures, which has never been observed before in zinc(II) complexes of simple linear peptides. © 2017

Fémionok és fémkomplexek kölcsönhatása makromolekuláris bioligandumokkal. = Interactions of metal ions with macromolecular bioligands.

I.Meghatároztuk a VO(IVés V) kölcsönhatását jellemző kötési állandókat a szérum transzferrinhez és albuminhoz és megállapítottuk, hogy a különböző antidiabetikus komplexek vérben való szállításában a transzferrin az elsődleges transzporter. Megállapításainkat humán szérum mintákon végzett ex vivo HPLC-ICP-MS mérésekkel kísérletileg is igazoltuk. Biszpiridin-, és biszdipeptid-származékok között sikerült az Alzheimer kórban alkalmazható potenciális fémkelátor típusú gyógyszermolekulákat találni, melyek képesek voltak a ?-amiloid oligomerizációját gátolni. Vizsgáltuk Al(III) komplexek hatását az alkalikus foszfatáz enzimre. Vizsgálataink arra utalnak, hogy a hordozó ligandumok képesek kiszorítani az aktív centrum fémionjait, míg az Al(III) képes helyettesíteni az egyik fémiont. II.Szerkezeti és funkcionális modellezés céljából vizsgáltuk a Cu,ZnSOD, NiSOD és endostatin N-terminális részletét, Zn-transzporter fehérjék fémkötő szekvenciáját,HRG fehérje His-gazdag régióját és az MMP13 enzim aktív centrumát modellező, illetve prolint és/vagy lizint tartalmazó peptidek fémkomplexeit. A modellkomplexek közül néhányat szilárd hordozón rögzítettünk. Nukleáz hatású modelljeink hatékonyaknak bizonyultak modell-szubsztrátok, DNS és RNS hasítása során. A specifikus mesterséges metalloenzimek vizsgálatának első lépéseként molekulafelismerési folyamatok és a lehetséges nukleáz domén mélyebb megismerésére törekedtünk. Fúziós fehérjék kölcsönhatását vizsgáltuk fémionokkal és DNS-sel. | I.Binding constants of VO(IV and V) to the serum proteins transferrin and albumin were determined and found that in transport of the different antidiabetic complexes in the blood transferrin is the primary transporter. This finding was confirmed by ex vivo HPLC-ICP-MS measurements made in real human samples. Chelators of bispyridyl and bis-dipeptide derivatives were synthetised against AD, which were able to inhibit oligomerization of ?-amyloids. The effect of various Al(III) complexes were studied on alkalaine phosphatase. It was assumed that the carrier ligands were able to displace the metal ions from the active center of the enzyme, while the metal ion was able to displace one of the metal ions in the active center. II.For structural and functional modelling we studied metal complexes of N-terminal fragments of a Cu,Zn-SOD, Ni-SOD and human endostatin, of the metal binding site of Zn-transporter, of the His-rich region of HRG, the acive centre of MMP13 proteins, as well as of proline and/or lysine containing peptides. Some model complexes were immobilized on a solid carrier. The models with nuclease activity proved to be efficient in cleavage of model-substrates, DNA and RNA. As a first step in the studies of specific artificial metalloenzymes we intended to deeply understand the molecular recognition processes and the properties of the possible nuclease domain. Fusion proteins have been expressed and their interaction with metal ions and DNA was studied