M.Ya. Karpeisky

Condensation of oxazoles with dienophiles : Synthesis of vitamin B6 analogues

Abstract Pyridoxine analogues have been synthesized via a Diels—Alder reaction of 5-ethoxyoxazoles with dimethyl maleate followed by reduction. The products on oxidation with manganese dioxide yielded pyridoxal analogues. Pyridoxamine derivatives were obtained by reduction of the pyridoxal oximes. Phosphorylation of the pyridoxamine analogues and the Schiff bases of pyridoxal compounds followed by hydrolysis afforded pyridoxamine phosphate and pyridoxal phosphate analogues, respectively. The mechanism of the Diels—Alder condensation of 5-ethoxyoxazoles is discussed on the basis of calculation of the π-electron charge distributions for diene and dienophile molecules according to the Huckel MO method. PMR, IR and UV spectra of the compounds synthesized and their TLC chromatography have been studied.

The structure of the complex of ribonuclease s with fluoride analogue of UpA at 2.5 Å resolution

Different physico-chemical methods are used to estimate the mutual position of substrate and enzyme in the course of the catalytic act. A widely exploited approach involves the study of complexes with substrate-like molecules, taking into account their individual inhibitory properties. Such information for pancreatic ribonuclease, obtained mainly by X-ray and NMR methods, is most thoroughly surveyed in [ 1,2] . Much of that information was deduced from studies of enzyme-product complexes with nucleoside-3’-phosphates. Atomic coordinates have been published both for the enzyme (RNase S) [3,4] and the enzyme-bound UpA analogue (UpcA) in which the 5’-oxygen of adenosine is replaced by a CH2 group [3] . The present report describes the results of an X-ray study of RNase S in complex with a non-hydrolysed analogue of UpA, 2’-deoxy-2’-fluoro uridilyl-3’,5’adenosine, abbreviated further as 2’-FidUpA. This analogue is assumed to take up, on the enzyme, a position very close to that of unmodified UpA because replacement of 2’-OH by 2’-F produces no significant changes in the conformational state of the’ribose, as follows from the practically identical physico-chemical properties and conformation of 2’-F-dUpA and UpA [31. From the difference maps of electron density at 3-2.5 A resolution it has been possible to establish the position of the analogue in the active site of the enzyme, to deduce the conformation of 2’-F:dUpA bound to RNase S, and to reveal specific conformational changes of the protein caused by ligand binding.

Formation and properties of S-protein complex with S-peptide-containing fusion protein

A fusion protein (FP) comprised of the RNase A S‐peptide and human epidermal growth factor was shown to form a stable noncovalent catalytically active complex with the RNase A S‐protein at a stoichiometric ratio 1:1 with K diss = 5.0 × 10−7M. The S‐protein complex with FP exhibits the pyrimidine specificity toward substrates in both reactions catalyzed by RNase S, transesterification and hydrolysis. The fusion protein can be determined specifically and quantitatively in the presence of S‐protein by RNase activity assays. The possibility of effective purification of S‐peptide‐containing proteins by affinity chromatography on an S‐protein‐Sepharose column has been demonstrated.

Three-dimensional structure of ribonuclease from Bacillus intermedius 7P at 3.2 Å resolution

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Proton magnetic resonance studies of DES‐(121–124)‐ribonuclease A

Bovine pancreatic ribonuclease A (RNAase A) has been extensively investigated from the point of view of structure and function [l] . NMR spectroscopy has been successful in providing information about the state of RNAase A in aqueous solution, estimates of the pK values for each histidine residue and an evaluation of the enzyme-inhibitor complex structure 12-51. In this paper we report the PMR data for des-( 121124, Asp-Ala-Ser-Val-)-RNAase A, which was obtained by a limited hydrolysis of RNAase A and was designated as PIR [6-81. The data presented here on the pH-dependence of PMR spectra of PIR and its complexes with cytidine3’-phosphate (3’CMP) were used to estimate the individual pK’s (apparent) for the four histidines in PIR as well as for a comparison of the conformations of both proteins. The experimental data thus obtained suggest the role of Asp-121 in maintaining the precise orientation of the essential functional groups in the active site of RNAase A.

Analysis of non-polar regions in proteins

A new method for finding hydrophobic nuclei and microclusters in protein structure is proposed. The method uses simple and clear-cut criteria based on an analysis of distances between the hydrocarbon groups of all residues. A detailed analysis of the composition and properties of hydrophobic nucleic and microclusters for proteins of different types has been carried out. This approach reveals that a hydrophobic nucleus can be composed not merely of classical hydrophobic amino acids, but also of dicarboxylic acids, their amides, arginine, lysine, histidine and tyrosine. The hydrophobic nucleus defined by this method should be considered as an individual structural unit along with such elements of the secondary structure as alpha-helices, beta-turns and beta-sheets.

A route to 2',5'-oligoadenylates with increased stability towards phosphodiesterases.

The rates of enzymatic hydrolysis of 2′,5′‐oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3′‐terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2′‐5′)adenylyl(2′‐5′)adenosine.

Interaction of HIV-1 Reverse Transcriptase and T7 RNA Polymerase with Phosphonate Analogs of NTP and Inorganic Pyrophosphate

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPianalogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

Analogues of vitamin B6: Synthesis and properties of 3-deoxypyridoxal phosphate and 3-O-methylpyridoxal phosphate

Abstract 3-Deoxypyridoxal phosphate and 3-O-methylpyridoxal phosphate have been synthesized by transamination of the corresponding pyridoxamine phosphate analogues. The amine phosphates were obtained by reduction of oximes of pyridoxal analogues followed by phosphorylation with polyphosphoric acid. PMR, UV spectra and TLC have been studied.

Synthesis and Properties of Novel NTP Derivatives

Abstract Simple method for the preparation of anhydrides of nucleoside-5′-monophosphoric acid and with 1-hydroxyethane-1,1-diylbis(phosphonic acid) has been developed.